Immunoendocrinology in Health and Disease

Immunoendocrinology in

Health and Disease

Immunoendocrinology in

Health and Disease edited by

Vincent Geenen University of Liege Liege-Sart Tilman, Belgium

George Chrousos National Institute of Child Health and Human Development National Institutes of Health Bethesda, Maryland, U.S.A.

M ARCEL D EKKER

N EW Y ORK

This edition published in the Taylor & Francis e-Library, 2005. “To purchase your own copy of this or any of Taylor & Francis or Routledge’s collection of thousands of eBooks please go to www.eBookstore.tandf.co.uk.” Although great care has been taken to provide accurate and current information, neither the author(s) nor the publisher, nor anyone else associated with this publication, shall be liable for any loss, damage, or liability directly or indirectly caused or alleged to be caused by this book. The material contained herein is not intended to provide specific advice or recommendations for any specific situation. Trademark notice: Product or corporate names may be trademarks or registered trademarks and are used only for identification and explanation without intent to infringe. Library of Congress Cataloging-in-Publication Data A catalog record for this book is available from the Library of Congress.

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Preface

Immuno(neuro)endocrinology was recognized as a scientific field early in the 20th century, soon after immunology was identified as a specific domain of investigation. In the 1930s, Hans Selye provided experimental evidence for and introduced the concept of stress–induced, adrenal cortex–mediated immunosuppression. In the 1940s and 1950s the discovery and chemical synthesis of glucocorticoids, and their introduction in the treatment of rheumatoid arthritis, represented a major advance in modern medicine recognized by the 1950 Nobel prize in medicine or physiology. However, the deciphering of the intricate cellular and molecular interactions between the neural, endocrine, and immune systems was only initiated in the 1970s and has received acceptance by immunologists and other scientists gradually and somewhat reluctantly. Recently, the field of immunoendocrinology has been expanding exponentially. This book covers some of the fundamental aspects of immunoendocrinology and its numerous and diverse clinical implications. Interestingly, it is endocrinology, the science of cell-to-cell signaling and the medical specialty dealing with the pathological consequences of disturbances in intercellular communication, that performed the first convincing studies in immunoendocrinology. Endocrinologists did not hesitate to open the door to this field and provide the first robust experimental evidence of its importance as a legitimate area of physiology. In retrospect, it should have been evident and utterly logical to have hypothesized such an intricate communication between the three major systems—the immune, nervous, and endocrine systems—that are responsible for maintaining resting and stress-related homeostasis within multicellular, complex organisms. Both the stability of the internal milieu and the adaptation to forces of the environment depend on these systems. It is also interesting to see this in the context of the evolutionary histories of the neuroendocrine and immune systems. In invertebrate species, the foundations of both the neuroendocrine system and innate immunity have coexisted seamlessly over the eons. Some 300 million years ago, adaptive immunity iii

iv

Preface

emerged in the first cartilaginous agnathan fish. Since then, a progressively evolving somatic gene recombination machinery appeared that is responsible for an increasingly complex generation of diverse immune receptors (B- and T-cell receptors) able to recognize and react to an almost infinite number of nonself antigens. The emergence of an extremely sophisticated adaptive immunity defense system exerted potent pressures for the creation of structures and mechanisms necessary to impose self-tolerance, the inability of the immune system to attack the host organism. Together with diversity and memory, self-tolerance is a fundamental intrinsic property of the immune system. The progressive rise in the level of immune diversity and complexity may explain why so many failures in self-tolerance (systemic and organ-specific autoimmune diseases) are increasingly detected during evolution, with the maximum observed in the human species. The first thymus appeared in cartilaginous fish (e.g., sharks and rays), concomitantly with rudimentary forms of adaptive immunity. This organ stands at the crucial intersection between the immune and neuroendocrine systems. Among all lymphoid tissues, the thymus is unique in that it is the place where there is a constant confrontation between ancient immune mechanisms and neuroendocrine principles, as well as a more recently evolved system equipped with a machinery that stochastically generates adaptive immune diversity. Because the program of intrathymic T-cell education is complex, mistakes easily occur, leading to thymic output and enrichment of peripheral self-reactive T-cells oriented against molecular and/or cellular components of the neuroendocrine system and other systems. Thus, autoimmunity appears to be the tribute paid by mankind for the complexity and efficiency of its immune defenses. The immunological self-tolerance of the neuroendocrine system was an obvious necessity, as explained in the first part of this volume. Many hormones and neuropeptides influence the immune and inflammatory response through binding with and activation of neuroendocrine receptors expressed on target immunocompetent cells. Physiologically, neuroimmunomodulation plays a central role in the adaptation of immune defenses in infectious, inflammatory, allergic, and neoplastic diseases, as well as during stress and the process of aging. If self-tolerance to neuroendocrine ligands and receptors were not firmly established, then the risk of developing autoimmune phenomena would be very high and the integrity of the organism and the survival of the species would be seriously compromised. The importance of coordinated neuroendocrine–immune interactions for the harmonious development and function of the endocrine glands is thoroughly documented in the first part of this book. Our goal is to illustrate the clinical importance of immunoendocrinology in the understanding of the pathogenesis and treatment of autoimmune endocrine disorders, as well as of infectious, allergic, inflammatory, and neoplastic diseases, and the process of natural senescence. In the second part of the book, prominent experts have written up-todate chapters dealing with chronic diseases and conditions that have a tremendous emotional and economic impact on human society. It is our sincere hope that the fundamental and clinical perspectives of immunoendocrinology offered in this volume will convince readers that this is a field of science useful to both basic scientists and clinicians of most specialties. Indeed, endocrinology and immunology have greatly contributed to better knowledge of the physiology of living beings and the pathophysiology of many endocrine, immune, and other diseases. Endocrinologists should consider their specialty as embracing various fundamental aspects of human life, including the immune and inflammatory reaction and its aberrations. Even if they are not in charge of patients seen by experts of other specialized fields of medicine

Preface

v

or surgery—for instance, rheumatologists or intensivists—they should continue to play an important integrative role by bringing new rational ideas and therapeutic options for the final choice of their clinical management. The time for integrative science and medicine has arrived and is here to stay. This volume is only a first glimpse of what is coming! Vincent Geenen George Chrousos

Contents

Preface Contributors

iii xi

Fundamental Aspects 1. A Global View of Immunological Development and Biology Rachel Allen and Anne Cooke 2. Common Signaling in the Neuroendocrine and Immune Systems Nithya Krishnan and Arthur R. Buckley 3. Hypothalamic-Pituitary-Adrenal Axis Effects on Innate and Adaptive Immunity Emmanouil Zoumakis, Ilia Elenkov, and George Chrousos 4. Glucocorticoids and the Immune System G. Jan Wiegers, Ilona E.M. Stec, Pia U. Mu¨ller, and Georg Wick 5. Cytokines and Leptin as Mediators of the Hypothalamo-Pituitary-Adrenal Axis Rolf-Christian Gaillard

1

21

51

65

83

vii

viii

6. Role of Pro-Inflammatory Cytokines in Regulating the HypothalamicPituitary-Gonadal Axis of the Male Rat Catherine Rivier

Contents

107

7. Sex Hormones and B Cells Christine M. Grimaldi, Elena Peeva, and Betty Diamond

127

8. Vitamin D3 in Control of Immune Response Chantal Mathieu, Evelyne van Etten, Lut Overbergh, and Roger Bouillon

145

9. The Growth Hormone/Insulin-like Growth Factor-I Axis and the Immune System Ron Kooijman

163

10. Somatostatin Control of Immune Functions P. M. van Hagen, V. Dalm, L. J. Hofland, D. Ferone, and S. W. J. Lamberts

193

11. Prolactin and the Immune System Robert Hooghe, Nele Martens, and Elisabeth L. Hooghe-Peters

207

12. VIP and PACAP Immune Mediators Involved in Homeostasis and Disease Rosa P. Gomariz, Carmen Martinez, Catalina Abad, Mario Delgado, Maria Guillerma Juarranz, and Javier Leceta 13. Neurotransmitters Talk to T Cells in a Direct, Powerful, and Contextual Manner Affecting Key Immune Functions Mia Levite

241

263

14. Role of Neuropeptides in T-Cell Differentiation Doina Ganea and Mario Delgado

289

15. Natriuretic Peptides and Inflammation Angelika M. Vollmar and Alexandra K. Kiemer

305

16. Neuroendocrinology of the Thymus Wilson Savino and Mireille Dardenne

319

17. The Central Role of the Thymus in the Development of Self-Tolerance and Autoimmunity in the Neuroendocrine System Vincent Geenen, Fabienne Brilot, Isabelle Hansenne, Celine Louis, Chantal Charlet-Renard and Henri Martens 18. Two-Way Communication Between Mast Cells and the Nervous System Hanneke P. M. van der Kleij, Michael Blennerhassett, and John Bienenstock

337

357

Contents

19. Peripheral Nervous System Programming of Dendritic Cell Function Georges J. M. Maestroni 20. Neuroendocrine Host Factors in Susceptibility and Resistance to Autoimmune/Inflammatory Disease Jeanette I. Webster and Esther M. Sternberg

ix

381

393

Clinical Aspects 21. Autoimmune Type 1 Diabetes Edwin A. M. Gale and Polly J. Bingley

417

22. Autoimmune Central Diabetes Insipidus Annamaria De Bellis, Antonio Bizzarro, and Antonio Bellastella

439

23. Autoimmune Thyroid Disease Marian Elizabeth Ludgate and Gherardo Mazziotti

461

24. Addison’s Disease and Autoimmune Polyglandular Syndromes Corrado Betterle

491

25. Premature Ovarian Failure Annemieke Hoek and Hemmo A. Drexhage

537

26. Myasthenia Gravis Alexander Marx, Philipp Stro¨bel, and Hans Konrad Mu¨ller-Hermelink

571

27. Rheumatoid Arthritis Maurizio Cutolo and Rainer Straub

593

28. Aging and Neuroimmunoendocrinology Rainer Straub and Maurizio Cutolo

607

29. Quality of Life in Asthma and Rhinitis Jean Bousquet, Philippe J. Bousquet, and Pascal Demoly

619

30. Atopic Dermatitis Markus Bo¨hm and Thomas Luger

631

31. Neuroendocrine Control of Th1 and Th2 Responses: Clinical Implications Ilia Elenkov 32. HIV Infection and the Central Nervous System Marcus Kaul and Stuart A. Lipton

647

673

x

Contents

33. Opioid Receptors and HIV Infection Burt M. Sharp

693

34. Mechanisms of Cytokine-Induced Sickness Behavior Robert Dantzer, Jan-Pieter Konsman and Patricia Parnet

707

35. Immunotherapy of Neuroendocrine Tumors Matthias Schott, Jochen Seissler, and Werner A. Scherbaum

721

36. Glucocorticoid Resistance in Inflammatory Diseases Denis Franchimont and George Chrousos

737

Index

747

Contributors

Catalina Abad, Ph.D. Department of Cell Biology, Complutense University, Madrid, Spain Rachel Allen, D.Phil. Department of Pathology, Cambridge University, Cambridge, United Kingdom Antonio Bellastella, Ph.D. Departments of Clinical and Experimental Medicine and Surgery, Second University of Naples, Naples, Italy Corrado Betterle, M.D. Department of Medical and Surgical Sciences, University of Padova, Padova, Italy John Bienenstock, C.M., F.R.C.P., F.R.C.P.C., F.R.S.C. Departrments of Pathology and Molecular Medicine, McMaster University, Hamilton, Ontario, Canada Polly J. Bingley, M.D., F.R.C.P. Department of Diabetes and Metabolism, University of Bristol, Bristol, United Kingdom Antonio Bizzarro, Ph.D. Departments of Clinical and Experimental Medicine and Surgery, Second University of Naples, Naples, Italy Michael Blennerhassett, B.Sc., Ph.D. Division of Gastroenterology, Department of Medicine, Queen’s University, Kingston, Ontario, Canada xi

xii

Contributors

Markus Bo¨hm, M.D. Department of Dermatology, University of Mu¨nster, Mu¨nster, Germany Roger Bouillon, M.D., Ph.D. Katholieke Universiteit Leuven, Leuven, Belgium Jean Bousquet, M.D., Ph.D. Department of Medicine and Allergology, Montpellier University, Montpellier, France Philippe J. Bousquet Department of Medical Information, Montpellier University, Nimes, France Fabienne Brilot, Ph.D. Center of Immunology, University of Liege, Liege-Sart Tilman, Belgium Arthur R. Buckley, Ph.D. Department of Molecular and Cell Physiology, University of Cincinnati, Cincinnati, Ohio, U.S.A. Chantal Charlet-Renard Center of Immunology, University of Liege, Liege-Sart Tilman, Belgium George Chrousos, M.D., Sc.D. Pediatric and Reproductive Endocrinology Branch, National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, Maryland, U.S.A. Anne Cooke, D.Phil. Department of Pathology, Cambridge University, Cambridge, United Kingdom Maurizio Cutolo, M.D. Department of Internal Medicine, University of Genoa, Genoa, Italy V. Dalm, M.D. Department of Internal Medicine, Erasmus Medical Center, Rotterdam, The Netherlands Robert Dantzer, D.V.M., Ph.D. Department of Integrative Neurobiology, INRA-INSERM U394, Bordeaux, France Mireille Dardenne, M.D. CNRS FRE 2444, Hoˆpital Necker, Paris, France Annamaria De Bellis, M.D. Departments of Clinical and Experimental Medicine and Surgery, Second University of Naples, Naples, Italy Mario Delgado, Ph.D. Department of Cell Biology and Immunology, Instituto de Parasitologia y Biomedicina, Granada, Spain Pascal Demoly Department of Respiratory Medicine, Montpellier University, Montpellier, France

Contributors

xiii

Betty Diamond, M.D. Department of Microbiology and Immunology and Department of Medicine, Albert Einstein College of Medicine, Bronx, New York, U.S.A. Hemmo A. Drexhage, M.D., Ph.D. Department of Immunology, Erasmus Medical Center, Rotterdam, The Netherlands Ilia Elenkov, M.D., Ph.D. Clinical Neuroendocrinology Branch, National Institute of Mental Health, National Institutes of Health, Bethesda, Maryland, U.S.A. Sonita Claire K. Estrada, M.D. Department of Microbiology and Immunology and Department of Medicine, Albert Einstein College of Medicine, Bronx, New York, U.S.A. D. Ferone, M.D. Department of Endocrinological and Metabolic Sciences, University of Genoa, Genoa, Italy Denis Franchimont, M.D., Ph.D. Department of Gastroenterology, Erasme University Hospital, Brussels, Belgium Rolf-Christian Gaillard, M.D. Department of Endocrinology, Diabetes and Metabolism, CHUV University Hospital, Lausanne, Switzerland Edwin A. M. Gale, M.B., F.R.C.P. Department of Diabetes and Metabolism, University of Bristol, Bristol, United Kingdom Doina Ganea, Ph.D. Department of Biological Sciences, Rutgers University, Newark, New Jersey, U.S.A. Vincent Geenen, M.D., Ph.D. Center of Immunology, University of Liege, Liege-Sart Tilman, Belgium Rosa P. Gomariz, Ph.D. Department of Cell Biology, Complutense University, Madrid, Spain Christine M. Grimaldi, Ph.D. Department of Microbiology and Immunology, Albert Einstein College of Medicine, Bronx, New York, U.S.A. Isabelle Hansenne, B.Sc. Center of Immunology, University of Liege, Liege-Sart Tilman, Belgium Annemieke Hoek, M.D., Ph.D. Department of Obstetrics and Gynecology, Academic Hospital Groningen, Groningen, The Netherlands L. J. Hofland, M.D. Department of Internal Medicine, Erasmus Medical Center, Rotterdam, The Netherlands Robert Hooghe, M.D., Ph.D. Department of Neuroendocrine Immunology, The Free University of Brussels, Brussels, Belgium

xiv

Contributors

Elisabeth L. Hooghe-Peters, Ph.D. The Free University of Brussels, Brussels, Belgium Maria Guillerma Juarranz, Ph.D. Department of Cell Biology, Complutense University, Madrid, Spain Marcus Kaul, Ph.D. Del E. Webb Center for Neuroscience and Aging, The Burnham Institute, La Jolla, California, U.S.A. Alexandra Kiemer, Ph.D. Department of Pharmacy, University of Munich, Munich, Germany Jan-Pieter Konsman, Ph.D. Department of Neurobiology, INRA-INSERM U394, Bordeaux, France Ron Kooijman, Ph.D. Department of Endocrine Immunology, Free University of Brussels, Brussels, Belgium Nithya Krishnan, Ph.D. Department of Experimental Hematology, Cincinnati Children’s Research Foundation, Cincinnati, Ohio, U.S.A. S. W. J. Lamberts, M.D., Ph.D. Department of Internal Medicine, Erasmus Medical Center, Rotterdam, The Netherlands Javier Leceta, Ph.D. Department of Cell Bioogy, Complutense University, Madrid, Spain Mia Levite, Ph.D. Department of Neurobiology, The Weizmann Institute of Science, Rehovot, Israel Stuart A. Lipton, M.D., Ph.D. Del E. Webb Center for Neuroscience and Aging, The Burnham Institute, La Jolla, California, U.S.A. Celine Louis, B.Sc. Center of Immunology, University of Liege, Liege-Sart Tilman, Belgium Marian Elizabeth Ludgate, Ph.D. Department of Medicine, University of Wales College of Medicine, Cardiff, Wales, United Kingdom Thomas Luger, M.D. Department of Dermatology, University of Mu¨nster, Mu¨nster, Germany Georges J. M. Maestroni, Ph.D. Department of Experimental Pathology, Istituto Cantonale di Patologia, Locarno, Switzerland Henri Martens, Ph.D. Center of Immunology, University of Liege, Liege-Sart Tilman, Belgium

Contributors

xv

Nele Martens, M.Sc. Department of Neuroendocrine Immunology, The Free University of Brussels, Brussels, Belgium Carmen Martinez, Ph.D. Department of Cell Biology, Complutense University, Madrid, Spain Alexander Marx, M.D. Department of Pathology, University of Wu¨rzburg, Wu¨rzburg, Germany Chantal Mathieu, M.D., Ph.D. Katholieke Universiteit Leuven, Leuven, Belgium Gherardo Mazziotti, M.D. Department of Clinical and Experimental Medicine, Second University of Naples, Naples, Italy Pia U. Mu¨ller Institute of Pathophysiology, Innsbruck Medical University, Innsbruck, Austria Hans Konrad Mu¨ller-Hermelink, M.D. Department of Pathology, University of Wu¨rzburg, Wu¨rzburg, Germany Lut Overbergh, Ph.D. Katholieke Universiteit Leuven, Leuven, Belgium Patricia Parnet INRA-INSERM U394, Bordeaux, France Elena Peeva, M.D. Department of Microbiology and Immunology, Albert Einstein College of Medicine, Bronx, New York, U.S.A. Catherine Rivier, Ph.D. Peptide Biology Laboratory, The Salk Institute, La Jolla, California, U.S.A. Wilson Savino, Ph.D. Department of Immunology, Oswaldo Cruz Foundation, Rio de Janeiro, Brazil Werner A. Scherbaum, M.D. Department of Endocrinology, Diabetes, and Rheumatology, Heinrich-Heine University of Duesseldorf, Duesseldorf, Germany Matthias Schott, M.D. Department of Endocrinology, Diabetes, and Rheumatology, Heinrich-Heine University of Duesseldorf, Duesseldorf, Germany Jochen Seissler, M.D. German Diabetes Research Institute, Heinrich-Heine University of Duesseldorf, Duesseldorf, Germany Burt M. Sharp, M.D. Department of Pharmacology, University of Tennessee Health Science Center, Memphis, Tennessee, U.S.A. Ilona E. M. Stec, M.D. Institute of Pathophysiology, Innsbruck Medical University, Innsbruck, Austria

xvi

Contributors

Esther M. Sternberg, M.D. Department of Neuroendocrine Immunology and Behavior, National Institute of Mental Health, National Institutes of Health, Bethesda, Maryland, U.S.A. Rainer Straub, M.D. Department of Internal Medicine, University Hospital Regensburg, Regensburg, Germany Philipp Stro¨bel, M.D. Institute of Pathology, University of Wu¨rzburg, Wu¨rzburg, Germany Hanneke van der Kleij, Ph.D. Department of Pathology and Molecular Medicine, McMaster University, Hamiltion, Ontario, Canada Evelyne van Etten Katholieke Universiteit Leuven, Leuven, Belgium P. M. van Hagen, M.D., Ph.D. Department of Internal Medicine, Erasmus Medical Center, Rotterdam, The Netherlands Angelika M. Vollmar, Ph.D. Department of Pharmacy, University of Munich, Munich, Germany Jeanette I. Webster National Institute of Mental Health, National Institutes of Health, Bethesda, Maryland, U.S.A. Georg Wick, M.D. Institute of Pathophysiology, Innsbruck Medical University, Innsbruck, Austria G. Jan Wiegers, Ph.D. Institute of Pathophysiology, Innsbruck Medical University, Innsbruck, Austria Emmanouil Zoumakis, Ph.D. Department of Peptide and Reproductive Endocrinology, Branch, National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, Maryland, U.S.A.

1 A Global View of Immunological Development and Biology RACHEL ALLEN and ANNE COOKE Cambridge University, Cambridge, United Kingdom

An effective immune response coordinates the action of organs, circulating cells, and soluble components as an integrated system to eradicate foreign material encountered within the body. Many of these agents are already present within the circulation, under tight regulation yet able to migrate to the source of infection to mount a rapid response upon challenge. Others are stationed within lymphoid organs, awaiting stimulation to direct a targeted immune response. This chapter aims to provide a general overview of how our immune system develops, encompassing innate and adaptive (or acquired) aspects of immunity, their complex interactions, and the regulatory mechanisms that control their actions. I. KEY PLAYERS OF THE IMMUNE SYSTEM A defining characteristic of vertebrate immune systems is the production of a pathogenspecific response that can be swiftly recalled with an increased potency upon repeated challenge. By directing these specificity and memory functions, B and T lymphocytes compose the main cellular agents of the adaptive immune response. A. Key Cell Types 1. T Lymphocytes T lymphocytes provide adaptive immunity at a cellular level. The antigen-specific T-cell receptor (TCR) recognizes short peptide fragments displayed on the surface of potential target cells by specialized antigen presenting protein complexes (see Sec. I.C). Distinct 1

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Allen and Cooke

populations of T lymphocytes are responsible for various effector functions (Fig. 1). Cytotoxic T lymphocytes (CTL) (also known as CD8Ⳮ T cells due to their characteristic expression of the CD8 co-receptor) are responsible for the lysis of infected or transformed cells. To enable their lytic role, the cytoplasm of a cytotoxic T lymphocyte contains enzyme-rich granules, whose components are released to form pores in the target cell membrane and trigger programmed cell death (apoptosis). Cytotoxic T lymphocytes can also induce lysis through activation of death receptors such as Fas or TNFR on their target cell surface. The primary functions of helper T (TH) lymphocytes (also known as CD4Ⳮ T cells) are mediated through the action of secreted agents known as cytokines and surface molecules such as CD40L. The TH cell population may be further divided into TH0, TH1, TH2, TH3, or Tr1 subsets on the basis of their cytokine secretion profiles. Cytokines produced by TH cells provide stimuli that are essential to support cytotoxic T-cell responses, delayed hypersensitivity reactions, B-cell responses, and monocyte activity. Recent years have seen a growing interest in some unusual T-cell populations that express an invariant set of T-cell receptors, recognize nonpeptide antigens, and/or display a limited tissue distribution. The earliest of these to be identified were T-cell subsets expressing a ␥␦ TCR. More recently, NKT cells expressing a restricted ␣␤ TCR have been shown to recognize glycolipid antigens presented by the MHC-like protein CD1. 2. B Lymphocytes B lymphocytes are the source of antibodies (or immunoglobulins), the soluble effectors of acquired immunity (Fig. 2). Antigen specificity is conferred by the B-cell receptor (BCR), a membrane-bound immunoglobulin that recognizes soluble antigens or extracellu-

Figure 1 T-lymphocyte subsets.

Global View of Immunological Development and Biology

3

Figure 2 B-lymphocyte subsets.

lar pathogens encountered in body fluids. Upon activation, B cells differentiate to become plasma cells, with the majority of their cytoplasm taken up by protein expression machinery in order to produce secreted antibodies. Antibodies are capable of performing a range of effector functions designed to inactivate or remove foreign antigens from the body (see Sec. I.B). Another key function of B lymphocytes is an ability to present their cognate antigen to T lymphocytes; upon engagement, B-cell receptors internalize their antigen ligand for processing and presentation to T cells. TH cells can then in turn supply the cytokine stimuli necessary to drive B-cell differentiation. Some antigens, however, do not require TH-secreted cytokines to stimulate an effective B-cell response. These Tcell–independent (TI) responses, triggered by antigens with multiple repeating haptens (microbial polysaccharides, for example), can generate IgM antibody without affinity maturation to allow a rapid response in the early stages of infection. TI antigens are generally responsible for activation of the B1 lymphocyte subset, which are characterized by expression of the CD5 marker on their surface. Expression of the pattern recognizing Toll-like receptors (TLRs) on B cells provides another avenue of activation. Naı¨ve B cells only proliferate and differentiate into Ig-secreting cells in response to the TLR9 binding antigen CpG if they are also triggered through the BCR. Memory B cells, however, can proliferate and differentiate in response to CpG directly. 3. Polymorphonuclear Leukocytes Other immune cells support the function of antigen-specific lymphocytes, while performing essential mechanisms of their own. Infectious agents can be eliminated in a variety of ways; alternative means of pathogen destruction are suited to different types of challenge and different phases of the immune response. Phagocytes have developed specialized functions to destroy ingested material and secrete inflammatory mediators that recruit and direct the action of other immune cells. Phagocytosis therefore provides an important frontline of innate immune defense. Although capable of phagocytosis, the primary functions of polymorphonuclear leukocytes are generally secretory. Polymorphonuclear leukocytes can be identified as neutrophils, eosinophils, or basophils on their basis of their dyestaining tendencies. Representing 50–70% of circulating leukocytes, neutrophils are the dominant polymorphonuclear population and are recruited to sites of infection or injury where they play a central role in the inflammatory process. Bacteria ingested by neutrophils succumb to a range of bactericidal proteins stored within cytoplasmic granules. Neutrophils also produce reactive oxygen intermediates (ROI) in a respiratory burst following phagocytosis. In contrast, eosinophils are involved in the elimination of parasitic infection, but

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Allen and Cooke

are also associated with allergic reactions. Eosinophils comprise roughly 5% of circulating leukocytes and are characterized by their acidophilic granules. Upon stimulation, eosinophils degranulate, releasing the contents of their acidic granules into the extracellular environment to combat pathogens that are too large to be phagocytosed. Basophils and related mast cells represent only a small population of circulating leukocytes; their granules contain histamine, heparin, and other vasoactive amines that are released in response to antibody-antigen interactions. The influence of these secreted agents on vasodilation and vascular permeability enhances an inflammatory response. This same process also mediates the symptoms of allergy when basophils and mast cells release histamine in response to IgE sensitization. 4. Monocytes/Macrophages Monocytes circulate through the blood, undergoing differentiation to become macrophages upon migration into tissues. Tissue-specific macrophages include microglial cells, which become activated in response to pathological events, providing the central nervous system (CNS) with an immune defense system [1]. Specialized receptors on the monocyte/macrophage surface recognize microbial components (see Sec. IV) or agents of the immune response (see Sec. I.B) as signals for phagocytosis. Macrophages/monocytes not only function in pathogen clearance, but also play a pivotal role in the adaptive immune response through their action as professional antigen-presenting cells (APC). High-level expression of MHC class 1I allows professional antigen presenting cells to become potent stimulators of T lymphocytes and thus elicit an adaptive immune response. During an inflammatory response, macrophages can become activated to enhance their phagocytic and secretory functions. 5. Dendritic Cells Dendritic cells (DCs) form the most potent population of antigen-presenting cells. Dendritic cells are found in every tissue. Immature dendritic cells patrolling the periphery ingest fluid by macropinocytosis and are actively phagocytic, allowing them to acquire potential antigens. Following antigen uptake, the dendritic cell loses its ability to acquire antigen and differentiates into a professional antigen-presenting cell, upregulating the production of MHC class 1I proteins in order to display newly acquired peptide antigens on the cell surface. Subsequent peptide/MHC complexes display a longer half-life and do not recirculate from the cell membrane. Dendritic cells returning through the lymphatic system carry their antigens to lymph nodes, where they deliver an activation signal to stimulate resting T-cell differentiation, thus triggering an adaptive immune response. 6. Natural Killer Cells Natural killer (NK) cells are large, granular lymphocytes with cytotoxic activity and can destroy tumor or virally infected cells with no requirement for prior immunization or activation. NK cells display many features in common with cytotoxic T cells, carrying lytic granules within their cytoplasm and expressing certain protein markers on their surface. Although these leukocytes were first identified and named for their cytolytic properties, NK cells provide two other important functions; they can lyse IgG-coated targets in a process known as antibody-dependent cell-mediated cytotoxicity (ADCC) or secrete cytokines that influence lymphocyte function. NK cells appear to play a role in the early phase of the immune response to various intracellular pathogens [2]. Cytokine stimulation can enhance the activity of natural killer cells by up to 100-fold.

Global View of Immunological Development and Biology

5

B. Soluble Factors 1. Antibodies Antibodies provide specific immunity in a soluble form. A complex of four protein chains (two heavy and two light) held together by disulfide bonds form the basic structural unit of an antibody (or immunoglobulin). Antibodies are functionally subgrouped on the basis of their heavy chain determinants (known as isotypes); humans have five classes of heavy chains, each designed to evoke a different set of effector mechanisms (Table 1). Each Blymphocyte clone can express only a single specificity of immunoglobulin (see Sec. I.C). However, a B-cell clone can switch heavy chain isotypes during the course of an immune response in order to associate an immunoglobulin specific for a single antigen with a series of heavy chains. IgM is the first immunoglobulin to be produced during B-cell differentiation, and its membrane-bound form functions as the B-cell receptor (BCR). Soluble IgM molecules form pentamers in serum. IgD can also be found as a membrane-bound BCR, which may be involved in B-cell activation. IgG is the most abundant immunoglobulin in serum and represents the only antibody isotype that can cross the placenta from mother to child. Soluble IgA, found in the form of monomers and dimers, can cross epithelial membranes to become the dominant antibody isotype in secretions. Secretory IgA, found in saliva and milk, associates with the secretory component (a 70 kDa protein synthesized by epithelial cells) to facilitate IgA transport across epithelia [3]. IgE attaches to the surface of basophils and mast cells, ready to trigger their rapid degranulation upon contact with antigen. Immunoglobulins mediate a number of immune mechanisms. At the simplest level, antibodies may neutralize a pathogenic agent by binding to its surface to block interaction with host cells. Target cell lysis can occur when antibodies on the surface of a pathogen activate complement or are recognized by Fc receptors on the surface of immune leukocytes to elicit antibody-dependendent cellular cytotoxicity (ADCC) (see Sec. IV). Fc receptors specific for different heavy-chain isotypes determine the fine specificity of these processes.

Table 1 Antibody Isotypes Isotype

Structure

IgG

Monomer

IgM

Cell-associated: Monomer Serum: Pentamer

IgA IgE

Serum: Monomer Secretions: Dimer Monomer

IgD

Monomer

Function Predominant immunoglobulin in plasma, can also cross placenta. IgG can activate complement, bind Fc receptors and act as an efficient opsonin. First antibody produced during an adaptive immune response, also functions as B-cell receptor. Can activate complement. Binds some Fc receptors. Provides local protection in secretions such as milk, saliva, tears and in digestive tract. Binds Fc receptors on basophils and mast cells before interacting with antigen. Recognition of allergens will trigger Fc receptor-mediated degranulation of mast cells to release histamines. Functions as B-cell receptor, found at low levels in serum, does not fix complement.

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2. Cytokines Like hormones, this diverse family of soluble mediators may act locally upon the cell of their origin (autocrine), on nearby cells (paracrine), or exert long-range effects (endocrine). Because of their potential for wide-ranging immune modulation, the expression of cytokines and their receptors must be kept under tight regulation. For example, interleukin 1 (IL-1) is a pro-inflammatory cytokine which, due to its effects on a wide variety of cells, must be tightly regulated in vivo. Antagonists that block the interaction between IL-1 and its receptor provide a potential treatment for certain inflammatory conditions [4]. The pleiotropic effects of cytokines acting alone or in concert on a variety of cell types can make it difficult to determine the complex functions of any individual cytokine. However, knockout mice and immunodeficient patients lacking a particular cytokine or its receptor have provided some clues to the primary roles of certain molecules. One of the best characterized examples is provided by patients with inherited disorders of IFN␥, who are particularly vulnerable to intracellular infections from viruses and mycobacteria. IL-12 plays an important role in the development of TH1 responses. Thus, patients lacking a function IL-12 receptor are susceptible to intracellular bacterial infections such as Salmonella and mycobacteria [5]. Cytokines have been subgrouped into families on the basis of their source, their range of influence, or structural similarities. For example, hematopoietins such as GMCSF exert their influence upon cells of haemopoetic lineage, while interferons were originally named for their role in antiviral defence. Structural similarities are evident between members of the tumor necrosis factor (TNF) family, which exist as homotrimers and trigger a similar multimerization of their receptors upon engagement. The classic example of a T-cell–derived cytokine or interleukin is IL-2, a small soluble mediator expressed by T lymphocytes as a means to stimulate T-cell activation, proliferation, and further cytokine production. CD4Ⳮ T cells in particular express characteristic cytokine profiles which establish their helper functions (Table 2); cytokines that favor a cellular (cytotoxic) immune response to intracellular infections such a leishmania or m. leprae are classified as TH1, and include IFN-␥, IL-2, TNF, and GM-CSF. TH2 cytokines direct a humoral (antibody) response against extracellular pathogens and include IL-4 and IL-10, which stimulate B cells and macrophages, respectively. Antigen-specific T cells are themselves subject to counterregulation by cytokines from other key sources such as regulatory T cells (see Sec. IV), macrophages, and natural killer cells. 3. Chemokines Chemotactic cytokines or chemokines are intimately involved in the migration of leukocytes through the periphery and their homing to specific tissues. Chemokines are characterized by the presence of a conserved motif of cysteine residues (C). The pattern of these cysteines defines four distinct chemokine families; C, CC, CXC, and CX3C, terminology for chemokine receptors reflects that of their ligands (i.e., CR, CCR, CXCR, CX3CR). Chemokines generated by inflamed tissue recruit circulating immune cells by initiating leukocyte migration across the endothelium. T cell homing can also be determined by chemokine receptor profiles; this can be seen in lymphoid organs, while in the periphery TH1 cells expressing CXCR3 and CCR5 and TH2 cells expressing CCR3, CCR4 and CCR8 localize to different areas of inflammation. Secondary production of further chemokines can then recruit other leukocytes.

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Table 2 Cytokines Cytokine

Major source(s)

GM-CSF

T-cells, macrophages

IL-1 IL-2

Myelomonocytic cells, lymphocytes, NK cells etc T-cells

IL-3

T-cells

IL-4 IL-5 IL-6

T-cells, mast cells T-cells, mast cells Lymphocytes, macrophages etc

IL-8 IL-10

Monocytes, lymphocytes etc T-cells

IL-13

T-cells, mast cells, NK cells

IFNα/IFNβ

Macrophages, lymphocytes

IFNγ

T-cells, NK cells

TGFβ

T-cells, macrophages

TNFα

Macrophages, T-cells

Effects include Growth and survival factor for cells of the haematopoietic lineage. T-cell activation. Also acts as pyrogen. Growth and differentiation factor for lymphocytes, NK cells, macrophages etc. Colony formation for erythroid and myelomonocytic lineages. Lymphocyte growth and differentiation factor. Eosinophil activation and colony formation. Regulates lymphocyte function and haematopoiesis. Attracts and activates neutrophils. Proliferation of B cells, thymocytes and mast cells, Blocks activation of cytokine production by T-cells, NK cells and monocytes. Proliferation of B cells, suppression of inflammatory responses. Inhibition of cellular proliferation, regulation of MHC expression. Activates lymphocytes, macrophages, NK cells etc. Enhanced MHC expression. Acts on lymphocytes, macrophages and dendritic cells. Inhibits cell growth and acts as a switch factor for IgA and IgG2b. Inflammatory mediator, regulates growth/differentiation of various immune cells.

4. Complement Proteins The complement system of proteins is involved in inflammation, opsonization of antibodycoated antigens for phagocytosis, lysis of pathogens or infected cells, and immune complex solubilization. These soluble acute phase proteins circulate within plasma as inactive proenzymes. Activation of the complement system triggers a cascade of proteolytic actions whereby proenzymes are cleaved into multiple fragments, which in turn act as soluble immune mediators. Nomenclature for the complement system of proteins can be complex. Factors of the classical complement system are designated by numbers (e.g., C1, C2, C4, C5). Following proteolytic cleavage, lowercase letters are used to designate the multiple fragments (e.g., C5a, C5b), while an overbar indicates an enzymatically active factor. Many components of the alternative pathway are designated by letters (e.g., factor B, factor D). Complement proteins can follow three main pathways of activation: classical, alternative, and mannin-binding lectin (MBL) (Fig. 3). These routes intersect with the generation of a C3 convertase, which binds pathogens, targeting them for destruction. The classical complement pathway plays a role in both innate and acquired immunity and becomes

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Figure 3 Complement pathways. The classical complement pathway is initiated by binding of C1 to antibody:antigen complexes or pathogen surfaces. The catalytic subunit of C1 in turn activates C2 and C4, the next two components of this pathway. Activated C4 and C2 together form the classical C3 convertase. The mannan-binding lectin pathway is triggered by lectin to carbohydrate components on a pathogen surface. Mannan-binding lectin (MBL) associates with MASP, the MBPassociated serum protease, to cleave C4 and C2. Both MBL and alternative complement pathways can proceed in the absence of specific antibodies.

triggered by the binding of C1q to antibody-antigen complexes or to pathogen surfaces. The serum concentration of acute phase proteins increases during infection. Of these, Creactive protein binds bacterial cell walls to become an efficient activator of the classical complement pathway. The mannan-binding lectin pathway represents an ancient pathway of immune defense. Mannan-binding lectin (MBL) is a serum protein similar to C1q, and initiates the mannan-binding lectin pathway upon binding to microbial carbohydrate structures [6]. The alternative pathway allows complement to become directly activated by the spontaneous hydrolysis of C3 to form C3i. Like C3b (generated by the classical or lectin pathways), C3i binds to the surface of microbes, where it combines with factor B to trigger the alternative pathway. By proceeding in the absence of antibodies, the alternative pathway provides an important first line of innate defense. C3 convertase complexes on the pathogen surface are cleaved to produce C3b, a central effector of the complement system, and C3a, an inflammatory mediator. C3b fragments target the pathogen for phagocytosis and help trigger subsequent complement reactions to induce the formation of a membrane attack complex (MAC). The MAC can disrupt plasma membranes, destroying the proton gradient to trigger lysis of the target cell. Soluble inhibitors are required to regulate this powerful system and act to prevent inappropriate complement binding, which would lead to destruction of host tissue. Complement deficiencies provide clues to the importance of this system in particular types of infection. For

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example, individuals lacking the central C3 molecule are highly susceptible to bacterial infection. Defiencies in C1 complement components may predispose an individual to systemic lupus erythematosus (SLE). C. Cell-Associated Proteins While B-cell and T-cell receptors mediate antigen specificity, other proteins on the leukocyte cell surface play essential roles as co-receptors, signal transduction proteins, and adhesion molecules (Fig. 4). Many of these proteins are members of the Ig (immunoglobulin) superfamily and share a characteristic immunoglobulin domain of 110 amino acids that resembles either a constant or variable Ig segment. Lineage, activation, and maturation markers are arranged into a systematic nomenclature, each designated by a CD (cluster of differentiation) number. Some common CD molecules are described in Table 3. 1. Antigen-Presenting Proteins Specialized proteins are required to present antigens in an appropriate form for recognition by the T-cell receptor. The classical antigen presenting proteins are encoded within the MHC genes, along with various cytokines, complement proteins, and antigen processing factors. Two protein families, MHC class 1 and MHC class 2, present peptides for recognition by CD4Ⳮ helper T cells and CD8Ⳮ cytotoxic T lymphocytes, respectively. These antigen-presenting proteins exhibit high levels of gene polymorphism, particularly for residues located within the peptide binding groove. The chemical and physical nature of the residues that line the peptide binding groove of an MHC protein exert a direct influence on the properties of the peptides it can bind. Thus, MHC polymorphisms are thought to

Figure 4 The immune synapse. In addition to the recognition of MHC presented peptide, the immune synapse between a T lymphocyte and an antigen-presenting cell requires molecules fulfilling adhesion and co-stimulatory functions.

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Table 3 Leukocyte Antigens Marker

Ig superfamily

Expressed on Cortical thymocytes, dendritic cells, activated monocytes Thymocytes, T-lymphocytes, NK cells

CD1

Yes

CD2

Yes

CD3

Yes

T-lymphocytes

CD4

Yes

CD5

No

CD8

Yes

CD11a

No

CD11b

No

Thymocytes, Subset of T-lymphocytes Mature T-lymphocytes, subset of B-lymphocytes Thymocytes, Subset of T-lymphocytes Lymphocytes, granulocytes, monocytes, macrophages Myeloid cells, NK cells

CD11c

No

CD14

No

CD28 CD34

Yes No

CD40

No

CD45

No

CD56

Yes

Myeloid cells (particularly tissue macrophages) Myelomonocytic cells T-lineage cells, plasma cells Bone marrow cells including hematopoietic stem cells Mature B-lymphocytes, but not plasma cells All cells of haematopoietic origin (except erythrocytes) NK cells, subset of T-lymphocytes

Description Presents glycolipids for recognition by NKT cells Enhances adhesion between T-cells and antigen presenting cells Signal transduction following engagement of the T-cell receptor Binds MHC class II proteins to act as a co-receptor Thought to play a role in signal transduction Binds MHC class I proteins to act as a co-receptor Mediates cell/cell adhesion Complement receptor, also potential roles in cell migration and signalling Possible role in cell adhesion Receptor for bacterial lipopolysaccharide Co-stimulatory protein Adhesion protein Required for secondary immune responses and germinal centre formation Required for signalling through T-cell receptor, Remains controversial

Source: From Ref. 15.

have been selected by evolutionary pressure to generate a wide range of specificities [7]. MHC diversity is useful at both the population level and the level of the individual. Each individual expresses up to six different MHC class 1 and six different class 2 proteins from haplotypes of three encoded on each chromosome (Fig. 5). A primary difference between class 1 and class 2 MHC proteins is the source of their antigenic peptides. With the exception of dendritic cells, peptides bound by class 1 are generally derived from intracellular proteins and are presented on the surface of most nucleated cells. This enables cytotoxic T cells to identify transformed or virally infected

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HLA-DP␣␤ HLA-DQ␣␤ HLA-DR␣␤

HLA-B

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HLA-C

HLA-A

Figure 5 MHC haplotypes. The two copies of chromosome 6 (maternal and paternal) each encode three different MHC class 2 and three different MHC class 1 genes. Therefore, with a different MHC haplotype from each parent, an individual can express up to six different MHC class 1 and six different MHC class 2 proteins.

cells. In contrast, helper T cells recognize soluble antigens, which are usually acquired from outside the antigen-presenting cell via the endocytic pathway, then processed and bound by MHC class 2 proteins. Cell surface expression of MHC class 2 is usually confined to specialized antigen-presenting cells. MHC expression is generally absent from the central nervous system but can be seen during inflammation, accompanied by migration of lymphocytes across the blood-brain barrier [8]. 2. B-Cell and T-Cell Receptors T and B lymphocytes with antigen receptors corresponding to a huge range of specificities are found circulating through the periphery. The receptor repertoire of these populations is achieved by combinatorial rearrangement of multiple gene segments. Each T- or B-cell receptor molecule can be divided into variable and constant immunoglobulin domains (Fig. 6). Antigen specificity is conferred by the variable region, which forms an antigen binding site, while constant regions form a supporting structural scaffold. For antibodies, the antigen binding site is often referred to as the Fab fragment, which can be separated from the constant (Fc) region by enzymatic digestion. The Fc region mediates effector mechanisms by binding complement factors or Fc receptors. Each antibody unit has two antigen-binding sites and may be expressed as a membrane-bound receptor or as soluble antibody. In contrast, the T-cell receptor exists only in a membrane-bound form with a single antigen-binding site. Each TCR chain has one variable and one constant domain with the paired variable domains forming an antigen binding site. Approximately 95% of circulating T cells express an ␣␤ TCR heterodimer, but a subpopulation of specialized T cells uses an alternative receptor composed of ␥ and ␦ chains. 3. Signaling Components Following receptor engagement, a dedicated complex of associated proteins transmits the signal from a surface B- or T-cell receptor to the interior of the cell, leading to gene transcription for lymphocyte activation, differentiation, and function. The CD3 protein complex associates with the T-cell receptor and, following antigen recognition, triggers an intracellular phosphorylation pathway. Immunotyrosine activation motifs (ITAMs) in the cytoplasmic domains of CD3 components become phosphorylated to trigger an intracellular signaling cascade, which may also control endocytosis of the entire T-cell receptor complex. An equivalent role is performed in B cells by association of Ig␣ and Ig␤ with

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Figure 6 Antibody and T-cell receptor structures. Antibody is the secreted version of the B-cell receptor. Antibodies are formed from pairs of heavy and light chains. The antigen binding site is formed by the variable domains of both heavy and light chains.

the B-cell receptor. Once the receptor has been cross-linked by antigen, ITAMs in the cytoplasmic tails of Ig␣ and Ig␤ become phosphorylated to trigger a signaling cascade. 4. Co-receptors The classical ␣␤ T-cell receptor can recognize either MHC class 1 or MHC class 2 protein complexes. A functional distinction between helper and cytotoxic T lymphocytes results from the influence of the CD4 and CD8 co-receptors, which confer specificity for MHC class 2 and class 1 proteins, respectively. CD4 and CD8 co-receptors, stabilize the interaction between a TCR and its cognate MHC and introduce their associated lck kinase required for phosphorylation of the CD3 signaling machinery 5. Co-stimulatory Proteins In addition to co-receptors, which stabilize the recognition event, a variety of costimulator proteins are required to fully activate an effective T cell response. Alternative co-stimulation systems are employed by naı¨ve and memory lymphocytes. CD28, a member of the Ig superfamily, is expressed on both naı¨ve and memory T cells. The interaction between CD28 and the co-stimulatory molecules B7.1 (CD80) and B7.2 (CD86) expressed on dendritic cells is essential to trigger an immune response from naı¨ve T cells. On activated T cells, B7 molecules are recognized by the CTLA-4 receptor, which acts to regulate lymphocyte responses. Additional interactions involved in lymphocyte activation include recognition of LFA-3 (CD58) expressed on hemopoietic and epithelial cells by the T-cell CD2 receptor.

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6. Adhesion Proteins The ability of leukocytes to migrate into particular tissues is important both for their function and for immune homeostasis. Circulating cells must first adhere to blood vessels at an appropriate site before they can pass into the tissue. Similarly, T cells and NK cells must adhere to their targets in order to allow efficient recognition. These functions are provided by specific adhesion proteins including members of the Ig superfamily (ICAM1, VCAM-1, integrins) and the selectin family of carbohydrate recognition receptors (also known as the leukocyte endothelial cell cell-adhesion molecules, or (LEC-CAMS) (Table 4). Selectins are particularly important for lymphocyte homing while integrins such as the lymphocyte function–associated antigen-1 (LFA-1) play a primary role in adhesion. During the T-cell recognition process, LFA-1 attaches to ICAM-1 on the antigen-presenting cell surface to prolong cell-cell contact. II. LYMPHOID ORGANS Lymphoid organs generate a specialized microenvironment where lymphocytes are provided with all the necessary lymphoid- and non–lymphoid-derived factors essential for their survival and development or activation. Primary lymphoid organs provide a site for lymphocyte development, where a framework of non-lymphoid cells secrete cytokines and growth factors to sustain lymphocyte development. Circulating lymphocytes congregate in secondary lymphoid organs, where they first encounter antigens and undergo activation to generate an active immune response. A. Primary Lymphoid Organs 1. Thymus The thymus is formed of several discrete lobules, each comprising an inner medulla of mature thymocytes surrounded by a cortex of proliferating cells. In the outer cortex, immature thymocytes committed to the T-cell lineage are surrounded by epithelial nurse cells. The corticomedullary junction is colonized by interdigitating dendritic cells and macrophages expressing MHC proteins. During embryonic development, a thymic stroma is generated from the third pharyngeal pouch and branchial cleft. This early thymic struc-

Table 4 Adhesion Proteins VCAM-1 (CD106)

ICAM-1 (CD54)

E-Selectin (CD62E) P-Selection (CD62P) L-Selectin (CD62L) Integrins

Members of the Ig superfamily, calcium independent. Promotes adhesion of leukocytes. Expressed at high levels during chronic lymphocytic inflammation. Contributes to extravasation. Contributes to extravasation of leukocytes, particularly during inflammation. Enhances the interaction between T-lymphocytes and antigen presenting cells Calcium-dependent, bind carbohydrate structures to mediate cell-cell adhesion. Selectins are involved in the initial (tether and rolling) stages of extravasation. Integrins are heterodimeric transmembrane glyocproteins that bind extracellular matrix proteins or ligands on other cells.

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ture is colonized by cells of the hematopoietic lineage, which in turn generate thymocytes and intrathymic dendritic cells. The murine thymus continues to develop 3–4 weeks after birth, whereas for humans it has already matured by the time of birth. The size of the thymus reduces after puberty once the mature T-cell repertoire been established. 2. Bone Marrow Hematopoiesis occurs within the bone marrow during adult life. Bone marrow does not show the same defined structure as the thymus. B-cell lymphopoiesis takes place in the outer cavities of the bone. Rapidly multiplying B cells progress towards the mantle zone and eventually reach the central venous sinus ready for entry into the circulation. Between these are areas of proliferating erythrocytes, granulocytes, and monocytes. B. Secondary Lymphoid Organs 1. Lymph Nodes Lymph nodes are located at lymphatic vessel junctions, where they provide an organized site for the cellular interactions between B cells, T cells, and antigen-presenting cells that are required to activate an immune response. Professional antigen-presenting cells returning from the periphery enter the lymph node through afferent lymph vessels. An outer cortex of B cells surrounds a paracortex of T cells. The inner medulla contains both T and B lymphocytes with medullary cords of macrophages and plasma cells. Dendritic cells are present as interdigitating cells (IDCs) and follicular dendritic cells (FDCs). During an active immune response, growth in germinal centers can manifest as swollen glands. 2. Spleen Lying within the abdomen, the spleen collects antigens directly from the blood. The majority of this organ is composed of red pulp, where senescent red blood cells are phagocytosed by macrophages. Arterioles carrying blood into the spleen are surrounded by lymphoid tissue in the form of white pulp. An inner sheath of T cells surrounds the central arteriole. The B-cell area contains primary and secondary follicles of unstimulated and stimulated B cells. Unlike dendritic cells found in the periphery, follicular DCs in the germinal center of secondary B-cell follicles capture immune complexes on their surface. Interdigitating dendritic cells from both myeloid and lymphoid lineages are found within T-cell zones. III. LYMPHOCYTE DEVELOPMENT Cells of the immune system are hematopoietic, deriving from a single precursor in the bone marrow during early development. This hematopoietic stem cell gives rise to a variety of lineages. The various hematopoietic cell populations then migrate around the body as appropriate lymphoid organs develop. Early during embryogenesis, precursors of both myeloid and lymphoid lineages are established from the hematopoietic stem cell in the yolk sac. Hematopoietic stem cells colonize the developing liver, which becomes the source of B cells until birth. Newly established bone marrow then takes over as the primary source of lymphoid cells. The common lymphoid progenitor (expressing CD34ⳭCD10ⳭCD45RAⳭ markers) in the bone marrow is capable of giving rise to T and B lymphocytes, NK cells, and dendritic cells according to the influence of exogenous factors. Other immune cells develop from a myeloid progenitor, which gives rise to granulocytes as well as macrophages, mast cells, and dendritic cells. Initial development of

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myelomonocytic cells takes place within the bone marrow and is driven by the combined action of colony-stimulating factors (CSFs) and various cytokines. The final differentiation of cells derived from the common myeloid progenitor occurs within the tissues where these leukocytes mature according to their local environment. Lymphocytes develop and mature within lymphoid organs before migrating to the periphery where they can exert their immune functions. The antigen specificity of a B or T lymphocyte will be determined early in its development by combinatorial gene rearrangement. Differentiation takes place in ordered stages, marked by sequential rearrangement of the receptor genes, and can also be visualized by expression of various cell surface markers. Productive rearrangement of receptor segments is required to generate a functional B-cell or T-cell receptor before the lymphocyte can undergo further development. Signals from surrounding stromal cells provide the necessary environmental and developmental stimuli. A. B-Cell Development Following an initial receptor rearrangement, adhesion to bone marrow stromal cells through molecules such as VLA-4/VCAM-1 and CAMs allows efficient stem cell factor stimulation of pro-B cells. IL-7 is then required for proliferation of pre-B-cells upon completion of heavy-chain rearrangement. As B cells progress through the differentiation process, they migrate from the subendosteum towards the central axis of the bone marrow cavity. Immature B cells are then selected for self-tolerance and proceed to the periphery, where they differentiate into mature naı¨ve B cells. Several mechanisms ensure that the majority of autoreactive B cells do not reach the periphery. B cells that encounter a strong response to multivalent ligands (on a self cell surface, for example) during development in the bone marrow will undergo clonal deletion. Receptor editing or further gene rearrangements may also act to remove the autoreactive receptor. In contrast, a weak reaction to self antigen will render developing B cells anergic. Most of the B cells that do reach the periphery will not survive, possibly as a result of restricted access to peripheral lymph nodes. Any self-reactive B cells surviving within the periphery are subject to a further means of control imposed by their dependence on T-cell help. In the absence of appropriate TH cells, self-reactive B cells remain ignorant in the periphery. B. T-Cell Development Following their generation in the bone marrow, T cells migrate to the thymus, where they undergo their differentiation process under the influence of appropriate exogenous stimuli. Stages of T-cell differentiation can be followed by tracking the expression of various cell surface markers. Under the influence of thymic stromal factors, progenitor T cells (CD3–CD4–CD8–) arriving in the thymus are stimulated to proliferate and differentiate. Development proceeds from CD4ⳮCD8ⳮ (double negative) to CD4ⳭCD8Ⳮ (double positive) cells. Small resting double positive cells expressing the IL-2 receptor (CD25) recombine a preliminary T-cell receptor and undergo positive selection. This selection at the double positive stage allows TCR specificity to be harmonized with the appropriate CD4 or CD8 co-receptor. Most of the T-cell developmental pathway takes place within the thymic cortex, where neighboring epithelial cells express high levels of MHC class 1 and class 2 to aid the process of positive selection. T cells with a mature phenotype are

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found in the thymic medulla, where they subsequently undergo negative selection through interactions with professional antigen-presenting cells. The ultimate fate of a developing T cell is determined by the specificity and affinity of its T-cell receptor for self antigens that it encounters within the thymus. T-cell receptors are selected to recognize self MHC but not self peptide. Thus, the process of positive selection allows cells whose receptors show a weak interaction with self MHC proteins to survive. However, TCRs that bind strongly to self MHC-peptide complexes will trigger negative selection in order to maintain self tolerance. As a result of positive and negative selection processes, only a small fraction of T cells will survive to enter the periphery. Peripheral immune tolerance is maintained by programmed cell death of autoreactive T cells following repeated recognition of self antigens (see Sec. V). Mature T and B lymphocytes circulate through blood and peripheral lymph organs until they encounter appropriate antigen. Chemokine receptors determine the homing ability of lymphocytes to particular locations. For example, cell-surface expression of CXCR5 directs B cells to lymph node follicles. Similarly, CCR7 directs T cells to an appropriate T-cell zone where they can encounter professional antigen-presenting cells. IV. THE INNATE IMMUNE RESPONSE Agents of the innate response can act immediately upon infection to generate their own immune defense, while providing the necessary stimuli to initiate and direct an adaptive response. Macrophages are found throughout the periphery and at potential sites of infection such as the lungs and gastrointestinal tract. Along with neutrophils, they can recognize, phagocytose, and destroy foreign pathogens. Following pathogen encounter, macrophages and neutrophils undergo a respiratory burst, releasing cytokines and inflammatory mediators. A major function of inflammatory mediators is to trigger vasodilation and vascular permeablization, leading to increased blood flow accompanied by leukocyte extravasation and migration to the site of distress. Polymorphonuclear cells are the first leukocytes to reach a site of inflammation. Neutrophils dominate the cellular infiltrate during initial stages of inflammation, followed by monocytes, which release cytokines and can in turn act as antigen-presenting cells to stimulate a T-cell response. Integrating with immune and adaptive pathways, phagocytes express receptors for soluble immune components of both systems. For example, complement receptors such as CD11b, CD11c are found on macrophages and granulocytes. While complement receptors bind complement-coated particles to stimulate phagocytosis, soluble antibody/antigen complexes are similarly primed for phagocytic uptake following their recognition by receptors for the antibody Fc region. Complement reactions can also contribute directly to inflammation. In addition to their role in smooth muscle contraction and vascular permeability, C3a, C4a, and C5a induce the expression of adhesion molecules, thus recruiting soluble and cellular effectors to the site of inflammation. The importance of innate immune agents in response to infection is underlined by the clinical symptoms observed for immunodeficient patients. Patients with primary neutrophil decificiencies are susceptible to recurrent bacterial infections, skin abcesses, and chronic granulomatous disease. Complement deficiencies are also associated with recurrent bacterial infection. A. Pattern Recognition Receptors Although they lack the fine antigen specificity of B and T lymphocytes, cells of the innate response are capable of responding to ‘‘microbial nonself,’’ ‘‘missing self,’’ or ‘‘induced/

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altered self’’ [9]. Pattern recognition receptors (PRRs) on the surface of macrophages and dendritic cells recognize conserved pathogen-associated molecular patterns (PAMPS), alerting phagocytes to the presence of infection. Such highly conserved microbial structures include lipopolysaccharides (LPS), lipids, and CpG DNA. Distinct classes of pattern recognition receptor have evolved to induce different host response pathways in response to various stimuli. Secreted pattern recognition receptors act in a similar manner to antibodies by opsonizing their target for phagocytosis or complement destruction. Toll-like receptors (TLRs), a family of cell-surface PRR that are defined by their homology to the IL-1 receptor, are activated in response to a range of microbial stimuli. For example, TLR4 is involved in the CD14-mediated response to bacterial lipopolysaccharide, while TLR9 recognizes bacterial DNA [10]. Signaling through Toll-like receptors can trigger cytokine release and microbial killing mechanisms such as the production of reactive nitrogen species. Toll-like receptor signaling can also influence responses of the adaptive immune system by modifying the nature of an antigen-presenting cell; Toll signaling triggers cytokine expression accompanied by an upregulation of the costimulatory proteins B7.1 and B7.2 to enable the antigen-presenting cell to stimulate lymphocyte effector functions. Recognition of different structures by alternative TLR receptors elicits unique cytokine secretion patterns as an apparent means to polarize the subsequent adaptive response [10]. B. NK Cells and Receptors Missing self is detected by MHC class 1-specific receptors on the surface of natural killer cells. These ‘‘NK receptors’’ include members of the immunoglobulin superfamily known as killer cell inhibitory receptors (KIRs) and the CD94 family of lectins. KIRs recognize particular MHC allotypes while CD94-NKG2A and CD93-NKG2C engage the non-classical MHC protein HLA-E. KIR and CD94-NKG2 proteins exist in both inhibitory and activating isoforms, and the final outcome of NK recognition is thought to depend upon a balance between the two conflicting signals [11]). Another member of the NKG2 family, NKG2D recognizes induced self in the form of MHC-like proteins expressed on the surface of virally infected or transformed cells. Recognition of these structures by NKG2D will trigger mechanisms that induce programmed cell death of the target cell. Two populations of natural killer cells can be defined by expression levels of the CD56 marker. Both CD56bright and CD56dim NK subsets are capable of lymphokine-activated killing (LAK). CD56dim NK cells express CD16 and granule components and are thought to compose the major cytotoxic NK cell population. In contrast, the CD56bright subset are potent producers of cytokines such as IFN-␥ and tumor necrosis factors and are therefore more likely to play an immunoregulatory role. C. Unusual Lymphocyte Subsets Unusual B- and T-lymphocyte populations may be more closely aligned with the innate than with the adaptive immune response. NKT cells and those expressing a ␥␦ receptor display restricted TCR usage and recognize alternative antigens such as glycolipids presented by the class 1–like molecule CD1. Similarly, B-1 cells (originally distinguished by their expression of CD5) show a limited ability for isotype switching and are known to secrete polyreactive IgM in the absence of antigen stimulation. Immunoglobulin molecules expressed by B-1 cells tend to recognize common bacterial and self antigens, and these cells have been implicated in some autoimmune situations [12].

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V. THE ADAPTIVE IMMUNE RESPONSE The first requirement of an adaptive immune response is for activation of naı¨ve lymphocytes by professional antigen-presenting cells. This function is usually performed by dendritic cells, which act as the primary agent in the adaptive immune response. Dendritic cells are found throughout the periphery (for example, as Langerhans cells in the skin) and, upon pathogen encounter, become activated to express costimulatory molecules and present antigen on their surface. Activated dendritic cells express high levels of MHC molecules, adhesion proteins, and costimulatory molecules and are the only cell type to present endocytosed protein fragments through class 1 MHC. Following antigen stimulation, dendritic cells lose their phagocytic ability and reenter the lymphatic system where they migrate to peripheral lymph nodes in order to initiate a lymphocyte response. Lymphocyte activation and proliferation occurs within peripheral lymph nodes where naı¨ve T cells encounter mature dendritic cells providing an appropriate combination of antigen and costimulation. Antigen recognition in the absence of costimulation will lead to T-cell tolerance. The interdigitating dendritic cells found in the lymph node are the most potent stimulators of naı¨ve T lymphocytes. Macrophages can also stimulate naı¨ve T cells if they express appropriate MHC and costimulatory molecules, but B cells can present antigen only to primed and not naı¨ve T cells. Upon successful priming, a primary response is triggered and T lymphocytes undergo clonal expansion to generate a large population of cells with a single specificity. This expansion is driven under the influence of autocrine IL-2. Activated (or primed) T cells express an alternative repertoire of cell surface molecules, which provide them with the ability to home to sites of inflammation rather than continuing to circulate between blood and lymph nodes. Similarly, upregulation of specific adhesion molecules enables activated T cells to engage antigen-presenting cells for TCR recognition. The cytokine milieu at the time of naı¨ve T-cell activation can influence the outcome of the immune response. Production of IL-12 and IFN-␥ promote the outgrowth of Th1 cells, whereas IL-10 and IL-4 promote the differentiation into Th2 cells. These cytokines may have been produced by NK cells, NKT cells, and DCs, the innate immune system. In the periphery, contact between T cells and their cognate antigen/MHC complexes triggers a functional response. In the case of cytotoxic T cells, actions may be mediated via perforin/granzymes or Fas/FasL interactions to trigger target cytolysis, or through expression of IFN-␥ to block viral replication. The primary effector molecules of helper T cells are cytokines. The adaptive immune system must be able to focus its response on either humoral or cell-mediated systems, depending on the type of pathogen encountered. A combination of input signals including the cytokine secretion patterns of innate system cells, presence of co-stimulators, PRR signaling, and the nature of MHC-peptide will determine which of the two profiles is eventually favored. For example, high densities of MHC class 2/peptide complexes indicate intracellular infection and consequently favor a TH1 response. Low-affinity low-density complexes push the response in the TH2 direction. Dominance of the TH1 response favors a cell-mediated immune response to fight intracellular bacteria, accompanied by appropriate B-cell isotype switching, whereas a predominantly TH2 profile provides humoral immunity via B-cell stimulation. Various regulatory mechanisms are enforced in the periphery. TH subsets can regulate one another; IL-4 produced by TH2 cells inhibits TH1 cell development and differentiation, and in the inverse situation IFN-␥ influences some control over the development of cells secreting TH2 cytokines. Other T-cell populations can also regulate the immune

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response via cytokine production. Regulatory T-cell subsets include TH3 cells, whose cytokine profile is dominated by high levels of TGF-␤ expression. TGF-␤ acts as an immunosuppressor inhibiting T cells, macrophages and NK cells. TH3 lymphocytes may provide some protection from systemic autoimmune diseases such as multiple sclerosis. A second regulatory subset, termed Tr1, expresses high levels of IL-10, an inhibitory cytokine that induces the downregulation of MHC and costimulatory molecules on antigenpresenting cells in addition to inhibiting various other cytokines. The hypothalamic-pituitary-adrenal axis can modulate expression of various cell surface and soluble immune factors and can influence immune cell maturation and differentiation; glucocorticoids suppress pro-inflammatory cytokines while upregulating anti-inflammatory cytokines, thus pushing the immune response from a Th1 to a Th2 profile. By suppressing pro-inflammatory cytokines, an excess of glucocorticoids can lead to an increased risk of infection. Conversely, a deficit of glucocorticoids can predispose towards inflammation [13]. Programmed cell death provides an important mechanism for the maintenance of peripheral immune tolerance. Apoptosis clears activated lymphocytes from the circulation following a specific immune response. The importance of programmed cell death in immune regulation can be seen when defects in the apoptotic mechanism lead to autoimmune disease [14] and have been implicated in systemic lupus erythematosus, Type 1 diabetes, Canale Smith, and autoimmune lymphoproliferative (ALPS) syndrome. Similarly, mutations of Fas or its ligand (FasL) lead to systemic autoimmunity in mice and humans. In addition to its function as a signaling molecule, the B-cell antigen receptor internalizes antigen for presentation to the helper T cells that will subsequently provide cytokine help. In particular, IL-4 produced by TH2 cells drives expansion of an antigen-reactive B-cell clone. This interaction takes place within peripheral lymph tissue. Heavy-chain switching is an important facet of the humoral immune response. A B-cell clone can produce antibodies with the same specificity but a whole range of functions. The first antibodies produced in the early phase of an adaptive B-cell response are IgM, which exhibit low affinity but high valency and are effective activators of the complement system. IgG is the most common antibody isotype in the blood. Of its subsets, IgG1 can elicit a variety of immune processes including pathogen neutralization, opsonization, antibodydependent cellular toxicity by NK cells, and complement activation. IgG2 provides an effective route for pathogen neutralization but cannot elicit ADCC, whereas IgG3 is best suited for complement activation. Fc receptors are intimately involved in the transport and activation functions of antibodies. Mast cells express high-affinity receptors for IgE, which becomes bound to their surface. Cross-linking of these IgE receptors by antigen leads to histamine release as seen in an allergic response. In response to antigenic stimulation, the adaptive response generates large populations of lymphocytes of a given specificity. Some of these will eventually persist as memory cells, which can mount a rapid and intense response in the case of reexposure. Memory lymphocytes require smaller doses of antigen and produce a more vigorous response than those of the primary response. Memory B cells circulate through peripheral lymph glands and can present antigen at low concentrations. Memory T lymphocytes are distinguished from naı¨ve ones by expressing a different repertoire of cell surface markers such as alternative CD45 isoforms and homing receptors. VI. CONCLUSION This chapter describes the key cell types and wide array of soluble mediators that compose the innate and adaptive immune response. The integrated interaction between the innate

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and adaptive immune responses ensures that an optimal response to infectious agents is achieved. Any dysregulation of this response can result in pathology. REFERENCES 1. Kreutzberg GW. Microglia, a sensor for pathological events in the CNS. Trends Neurosci 1996; 19:312–318. 2. Scalzo AA. Successful control of viruses by NK cells—a balance of opposing forces?. Trends Microbiol 2002; 10:470–474. 3. Mestecky J, McGhee JR. Immunoglobulin A (IgA): molecular and cellular interactions involved in IgA biosynthesis and immune response. Adv Immunol 1987; 40:153–245. 4. Dinarello CA. Interleukin-1, interleukin-1 receptors and interleukin-1 receptor antagonist. Int Rev Immunol 1998; 16:457–499. 5. Hunter CA, Reiner SL. Cytokines and T cells in host defense. Curr Opin Immunol 2000; 12: 413–418. 6. Gadjeva M, Thiel S, Jensenius JC. The mannan-binding-lectin pathway of the innate immune response. Curr Opin Immunol 2000; 13:74–78. 7. Trowsdale J. Genetic and functional relationships between MHC and NK receptor genes. Immunity 2001; 15:363–374. 8. Sehgal A, Berger MS. Basic concepts of immunology and neuroimmunology. Neurosurg Focus 2000; 9:1–6. 9. Medzhitov R, Janeway CR. Decoding the patterns of self and nonself by the innate immune system. Science 2002; 296:298–300. 10. Akira S. Mammalian Toll-like receptors. Curr Opin Immunol 2003; 15:5–11. 11. Lanier LL. Face off—the interplay between activating and inhibitory immune receptors. Curr Opin Immunol 2001; 13:326–331. 12. Berland R, Wortis HH. Origins and functions of B-1 cells with notes on the role of CD5. Annu Rev Immunol 2002; 20:253–300. 13. Webster JI, Tonelli L, Sternberg EM. Neuroendocrine regulation of immunity. Annu Rev Immunol 2002; 20:125–163. 14. Grodzicky T, Elkon KB. Apoptosis: a case where too much or too little can lead to autoimmunity. Mount Sinai J Med 2002; 69:208–219. 15. Barclay AN, Brown MH, Law SKA, McKnight AJ, Tomlinson MG, van der Merwe PA. The Leukocyte Antigen Facts Book. 2d ed.. San Diego: Academic Press, 1997.

2 Common Signaling in the Neuroendocrine and Immune Systems NITHYA KRISHNAN Cincinnati Children’s Research Foundation, Cincinnati, Ohio, U.S.A. ARTHUR R. BUCKLEY University of Cincinnati, Cincinnati, Ohio, U.S.A.

I. INTRODUCTION The neuroendocrine and immune systems are linked through a regulatory loop that permits a bi-directional communication between them [1,2]. These interactions are mediated by hormones produced by neuroendocrine organs (hypothalamus, pituitary gland) acting on immune cells or cytokines produced by the hematopoietic tissues that exert regulatory influences on neuroendocrine structures. The accumulated evidence suggests that these interactions are central to the maintenance of organism homeostasis during stress, inflammation, or infection [3–5]. For example, interleukin-1 (IL-1), derived from immune cells, acts on the hypothalamus and pituitary gland to stimulate secretion of adrenocorticotropin (ACTH) [6,7]. On the other hand, hormones secreted by the pituitary gland such as growth hormone (GH) and prolactin (PRL) modulate immune cell responses via specific hormone receptors present on lymphocytes [8–10] (Fig. 1). In fact, recent evidence suggests that several neuroendocrine hormones and their receptors are expressed by immune cells and that cytokines and their receptors are produced by neuroendocrine and certain peripheral organs. Thus, secretion of these substances provides a milieu that facilitates local (intracrine/autocrine/paracrine) interactions [8]. These hormones or cytokines stimulate specific receptors and signaling intermediates to regulate immune or neuroendocrine function. The focus of this chapter is with the various aspects of signaling that are common among the neuroendocrine and immune systems. We will address the issue of hormones and receptors common between the systems, effects of hormones on immune cells, effects of cytokines on neuroendocrine tissues (primarily the hypothalamus and pituitary gland), 21

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Figure 1 Bidirectional communication between the neuroendocrine system (hypothalamus, pituitary) and the immune system. Hormones released by the hypothalamus stimulate or inhibit pituitary hormone release. The hypothalamic or pituitary hormones affect the functioning of the immune system through specific receptors. In addition to cytokines, immune cells also release neurohormones, which affect the functioning of immune system. See text for additional details.

and common signaling pathways, which form a molecular basis for this bi-directional communication. II. EFFECT OF IMMUNE SYSTEM COMMUNICATION TO NEUROENDOCRINE CELLS The concept that the immune system affects neuroendocrine responses was first suggested by Wexler et al. [11,12]. These workers showed that administration of a bacterial toxin to rats caused elevation of plasma corticosterone levels, which paralleled the immune response [13]. Results reported by Besedovsky et al. [14] demonstrating increased corticosterone levels in rats treated with conditioned medium obtained from concanavalin A (ConA)–treated splenocytes also confirmed that activation of the neuroendocrine system could be stimulated by an immunogenic mechanism. Interleukin-1 (IL-1) was subsequently identified as the splenocyte product that activated the neuroendocrine responses reported in these experiments. Interleukins are immune cell–regulating cytokines that are synthesized and secreted by lymphocytes, macrophages, monocytes, and other cells in response to tissue injury, inflammation, or infection. Understanding the mechanisms by which these substances alter hypothalamic-pituitary function was made possible by identification of several neurotransmitters and hypothalamic peptides that stimulate release of hormones from the anterior pituitary gland. Neurohormones released into the hypophyseal portal system circulate

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to the anterior pituitary gland, where they stimulate or inhibit hormone release [15,16]. Corticotrophin-releasing hormone (CRH), luteinizing hormone–releasing hormone (LHRH), follicle-stimulating hormone–releasing hormone (FSH-RH), growth hormonereleasing hormone (GHRH), growth hormone release–inhibiting hormone (somatostatin), prolactin (PRL)-inhibiting and releasing factors, and thyrotropin-releasing hormone (TRH) represent a family of substances that regulate pituitary hormone release. Several other neurohypophyseal hormones such as vasopressin, oxytocin, and atrial natriuretic peptide have also been linked to modulation of anterior pituitary hormones by directly acting on the gland; catecholamines such as dopamine appear to directly affect hormone (PRL) synthesis. During infection, the pattern of pituitary hormone secretion is thought to reflect contributions from cytokine-induced release of hypothalamic peptides that further inhibit or stimulate hormone release. Furthermore, cytokines also directly alter pituitary hormone release and, as a result, the response of the gland to hypothalamic peptides [17]. An example of an effect of immune cell–derived cytokines on the neuroendocrine system occurs during the induction of the acute phase response (APR). The APR refers to an immediate set of reactions, triggered by the host subsequent to infection, which prevents tissue damage, destroys infecting agents, or activates repair processes to maintain tissue homeostasis. In addition to several other events, APR is characterized by fever, altered synthesis or release of cytokines and hormones of the hypothalamic-pituitaryadrenal axis (HPA), and de novo synthesis of acute phase proteins (APP). Immune cell–derived cytokines such as IL-1, IL-6, and TNF-␣ initiate the APR. These cytokines activate leukocytes, affect the central nervous system and HPA axis in vivo alone or synergistically when present in combination (reviewed in Ref. 18). IL-1 (-␣ and-␤) is thought to be the major mediator of fever and inducer of APPs. In addition, TNF-␣, IL6, and ␥-interferon (␥-IFN) are also pyrogenic. During the APR, IL-1 and TNF-␣ stimulate glucocorticoid (GC) production by the adrenal glands by activating the HPA axis, resulting in the synthesis of APPs by the liver. This effect coincides with the inhibitory influence of GCs on IL-1 synthesis in macrophages, which contributes to the suppression of the immune response. Intraperitoneal injection of lipopolysaccharide (LPS) induces the APR in laboratory animals. LPS is a potent stimulator of the synthesis and secretion of several cytokines from immune cells such as IL-1, IL-2, IL-6, TNF-␣, and ␥-IFN [17]. Due to the absence of an arterial blood supply to the anterior pituitary gland, cytokines released into the circulation from immune cells can only enter hypophyseal portal capillaries by diffusion into the median eminence (ME) where the blood-brain barrier is minimal [19]. Banks et al. [20] reported the presence of a transport system that delivers IL-1 and other cytokines into the brain. There is also evidence to indicate that IL-1, IL-2, and IL-6 are produced within the brain by glia [21]. In addition to LPS, a variety of other agents stimulate IL6 release, including IL-1-␣ and -␤, vasoactive intestinal peptide (VIP), pituitary adenylate cyclase activating peptide (PACAP), and calcitonin-gene related peptide [22–24]. Recent evidence also indicates that LPS-stimulated pituitary secretion of adrenocorticotrophic hormone (ACTH) is mediated by IL-6 that is released from pituitary folliculostellate cells through a mechanism involving the p38 MAPK/ NF-␬B pathway [25]. In addition, LPS increases IL-1 production by the anterior pituitary [26]. IL-10, a modulator of proinflammatory cytokines such as IL-1 and TNF, also appears to be produced in the pituitary and hypothalamus [27]. It enhances CRH secretion and ACTH production in hypothalamic and pituitary tissues, which stimulates downstream GC production [28].

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McCann et al. [29] elucidated the mechanisms by which cytokines influence hypothalamic and pituitary release of hormones during infection. These workers demonstrated that IL-2 activated neural nitric oxide synthase (nNOS), which catalyzes nitric oxide (NO) synthesis. Released NO was shown to diffuse into CRH-secreting neurons to stimulate CRH release, thereby augmenting ACTH release. This group also demonstrated a direct effect of IL-2 in the pituitary gland to stimulate ACTH secretion. In contrast, IL-1␣ blocked NO-induced release of LHRH from neurons and, thus, blocked pulsatile LH but not FSH secretion. IL-1␣ also inhibited GHRH release, blocking GH secretion by stimulating somatostatin secretion through a NO-dependent mechanism. However, IL-1␣–dependent PRL release was stimulated by a mechanism involving NO suppression of dopamine (PRLinhibiting factor) release [29]. Leukemia inhibitory factor (LIF) is an additional pleiotropic IL-6 family–related cytokine [30] that activates the HPA axis to induce ACTH expression in vivo [31,32]. Results reported by Kim and Melmed [33] indicate that LIF enhanced ACTH secretion from rat pituitary cells in vitro. The presence of LIF mRNA in normal rat pituitary suggests that it may act in an autocrine/paracrine manner to regulate neuroendocrine function in the pituitary [33]. Other inflammatory cytokines such as IL-6 and TNF-␣ also affect hypothalamic or pituitary hormone secretion. For example, IL-6 stimulates ACTH, PRL, GH, LH, and FSH secretion by rat anterior pituitary cells in vitro (reviewed in Ref. 34) and inhibits adenylate cyclase activity stimulated by VIP. It also suppresses TRH-induced inositol phosphate generation and CaⳭ2 release from intracellular stores. The effect of TNF-␣ on pituitary cells is controversial. It was reported to stimulate PRL, ACTH, GH, and TSH secretion from rat pituitary cells in vitro [35,36]. However, results from another study indicated that TNF-␣ inhibited secretion of ACTH and other hormones through a direct action on pituitary cells [37]. A similar controversy exists for the effect of ␥-IFN on PRL secretion in rat anterior pituitary cells. Miyake and colleagues [38] suggested that ␥-IFN–stimulated PRL release is coupled to IL-6 secretion by the pituitary folliculostellate cells. Denef and coworkers [39–41] demonstrated that ␥-IFN inhibited PRL secretion by a mechanism involving NO release also by folliculostellate cells. PRL is a neuroendocrine hormone that is produced by pituitary lactotrophs that also functions as a cytokine. Rodent and human lymphocytes synthesize and secrete PRL [42–46] and express PRL receptors (PRLR) on their cell surface [47,48]. In addition to PRL, GH is also produced by cells of the immune system [49–51]. The physiological consequences of these immune cell–derived hormones on neuroendocrine responses remains to be elucidated. However, PRL appears to regulate the expression of its own gene in lactotrophs at the level of the hypothalamus and pituitary [52]. PRL also regulates its own secretion by a dopamine-mediated negative feedback mechanism. In 2001 Torner and colleagues [53] demonstrated anxiolytic and antistress effects of brain PRL. In this study, it was shown that intracerebral administration of PRL in rats attenuated the responsiveness of the HPA axis. Moreover, antisense targeting of the brain PRLR caused increased ACTH secretion that suggests an inhibitory role exerted by PRL on activation of the HPA axis during lactation [54]. Thus, it is possible that PRL secreted by the extrapituitary tissues, such as immune cells, may function to dampen HPA axis activation during stress, pregnancy, or lactation to reduce anxiety. In addition to synthesis and secretion of cytokines, cells within the thymus also secrete several peptides that affect hormone release by the hypothalamus or pituitary gland. For example, thymosin-␤4 and thymulin stimulate pituitary LH and hypothalamic LHRH

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release. Thymulin stimulates pituitary ACTH release in vitro and inhibits PRL release while GH was unaffected [55]. In contrast, thymosin-␣1 reduced pituitary ACTH, TSH, and PRL secretion in vivo by directly acting on the HPA axis. Together, these observations indicate that a complex relationship between the neuroendocrine and immune system exists. To discuss common signaling in each system, we will consider cytokines, such as interleukins, and LIF as components of the immune system, and GH and PRL as examples of neuroendocrine hormones. These cytokines/hormones all signal through the class I cytokine family of receptors [56,57]. Ligand-receptor interactions are initially thought to first cause homo-dimerization of receptors prior to activating downstream signaling mechanisms. The salient features of these signaling pathways are discussed below. III. NEUROENDOCRINE HORMONES AND THEIR RECEPTORS ON IMMUNE CELLS Several in vivo observations suggested that the neuroendocrine system may influence functioning of immune cells. The Pavlovian conditioning of the immune response, positive and negative effects of brain and pituitary lesions on the immune system, and the adverse effects of stress on immune regulation strongly support this concept [58–61]. The molecular basis for these interactions appears to reflect the actions of neuroendocrine hormones on their respective receptors present on immune cells. A. Pro-opiomelanocortin–Derived Peptides ACTH, derived from Pro-opiomelanocortin (POMC), is generally considered a steroidogenic hormone. It is clear that lymphocytes recognize and respond to several peptide hormones [62] produced by differential proteolysis of POMC including ACTH, ␣-melanocyte–stimulating hormone (␣-MSH), and ␤-endorphin. The immune actions of these peptides were originally identified in ␣-IFN–producing human leukocytes that also expressed a peptide that was antigenically similar to pituitary-derived ACTH [63]. Other studies demonstrated that leukocyte–derived endorphins and ACTH were identical to their pituitary-produced counterparts in bioactivity, antigenicity, molecular mass, and retention time evaluated by reverse phase HPLC [64–66]. In B lymphocytes, ACTH acts as a growth factor [67]. It also stimulates the production of immunoglobulins [68,69] and inhibits IFN generation [70]. These actions of ACTH are thought to be mediated by specific highaffinity ACTH receptors on lymphocytes [71]. Results from studies employing an athymic nude mouse model demonstrated that CD4Ⳮ T lymphocytes play an important role in the release of ACTH from splenocytes [72]. Wermerskirchen et al. [73] showed that ACTH acts on activated T cells in a biphasic manner. Lower concentrations stimulated proliferation, while higher levels suppressed it. ␣-MSH, also a POMC-derived peptide, inhibited IL-1–induced mouse thymocyte proliferation [74]. It also inhibited chemotaxis stimulated by cytokine chemoattractants in mouse neutrophils [75], modified mouse T and B lymphocytes, and altered natural killer (NK) cytotoxicity in response to shock [76]. These observations suggest that the POMC-derived peptides, which were originally identified as neuroendocrine hormones of pituitary origin, are synthesized by immune cells and regulate their function. Other POMC-derived peptides, such as melanocortins (MC), modulate immune cell functioning by interactions with specific MC receptors (MCR) [77]. MCRs share features

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common to the seven transmembrane G protein–coupled receptor (GPCR) family. MCR5 is widely expressed in hematopoietic tissues including spleen, thymus, and bone marrow [78]. Interestingly, ␣-MSH binding to the MCR-5 receptor was shown to activate Jak/Stat signaling leading to B-cell proliferation [79]. This observation was the first to demonstrate that GPCR stimulation transduced signals common to those activated by cytokine receptors. However, the mechanism by which MCR-5 is coupled to Jak/Stat activation is presently unclear. B. TSH/TRH In addition to POMC-related peptides, TSH is synthesized by immune cells. TSH is a member of the glycoprotein family of hormones that includes LH, FSH, and chorionic gonadotropin (CG). The biosynthesis of TSH by pituitary thyrotrophs is strictly regulated by TRH produced in the hypothalamus. Synthesis of TSH by human peripheral mononuclear cells was first observed in response to staphylococcal enterotoxin A, a T-cell mitogen [80]. It has also been shown to be constitutively produced by human T leukemia cells [81]. Lymphocyte-generated TSH is similar to the pituitary-derived hormone with respect to glycosylation, immunogenicity, molecular mass, and subunit structure. Cellular effects of TSH are stimulated by its interaction with TSH receptors, a 84.5 kDa protein member of the GPCR family [82]. TRH has been reported to have both direct and indirect effects within the immune system. TRH receptor mRNA is expressed in human lymphocytes and rat splenocytes [83]. Its administration enhanced proliferation of rat splenic and thymic lymphocytes and blocked GC-induced suppression [83,84]. TRH was also reported to augment IL-2 secretion in humans [85]. C. Gonadotropins The T-cell mitogenic response that occurs in mixed lymphocyte reactions was shown to be accompanied by synthesis of immunoreactive CG. Immunoreactive LH was also shown to be secreted by porcine, human, and mouse lymphocytes [86–88]. Other studies demonstrated that FSH was produced by ConA-stimulated rat lymphocytes [89]. Rat splenocytes and lymphocytes express LHRH and its mRNA [90,91]. Finally, hypothalamic regulatory peptides (LHRH, TRH), thought to directly act on immune cell function or indirectly by stimulating release of pituitary hormone (LH, FSH, and TSH), stimulated ␥-IFN production [92]. D. VIP Vasoactive intestinal peptide (VIP) and PACAP are neuroendocrine peptides that affect innate and adaptive immunity [93–95]. These peptides inhibit iNOS expression and its stimulation of proinflammatory cytokine secretion in stimulated macrophages which downregulates the innate response [96–101]. VIP and PACAP also inhibit IL-2 production and cell proliferation in activated T cells [102]. Inhibition of IL-2 transcription by these peptides appears to involve interference with c-Jun N-terminal kinase (JNK) signaling causing decreased c-Jun in T cells [103]. Moreover, VIP and PACAP have also been shown to inhibit activation-induced cell death in T cells [104] by downregulating Fas ligand (FasL) [105]. VIP is thought to provoke cellular effects by stimulation of type 2 VIP receptors, which belong to the GPCR family. Activated VIP receptors stimulate cAMP generation,

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which activates protein kinase A (PKA) (reviewed in Ref. 106). VIP/PACAP also inhibited expression and/or the DNA-binding activity of transcription factors required for FasL expression in activated T lymphocytes including c-myc, NF-Atp (nuclear factor of activated T cells), NF-␬B, and early growth factors (Egr) 2/3 [107]. VIP also stimulated activation of the T-cell–specific tyrosine kinase p59fyn in resting murine thymocytes by cAMP-mediated mechanism [108]. E. PRL/GH PRL was originally identified as a neuroendocrine hormone of pituitary origin [109,110]. The hypothesis that PRL may function as an immunomodulatory hormone was derived from studies conducted in hypophysectomized rats or animals treated with bromocriptine, both exhibiting suppressed humoral or cell-mediated immunity [111]. These effects were reversed by PRL administration. The first observation that lymphocytes produced PRL was provided by Montgomery et al. [42]. These workers reported increased bioactive PRL in conditioned medium obtained from ConA-stimulated mouse splenocytes. It was demonstrated in these studies that PRL antiserum nullified the PRL activity present in the conditioned medium [42]. These observations were confirmed by others, who showed that PRL was synthesized and secreted from normal [45] and malignant human mononuclear cells [112,113]. Growth hormone was classically defined as a polypeptide hormone synthesized and secreted by anterior pituitary somatotrophs. GH has also been shown to be synthesized by immune cells [114,115]. Prolactin and GH receptors belong to the class I cytokine hematopoietic receptor family. The PRLR was cloned and sequenced from both rat liver [116] and Nb2 cells, a PRL-dependent, T-lymphoma cell line [117]. Russell et al. [47] were the first to demonstrate the expression of the PRLR on human T and B lymphocytes. Gagnerault and colleagues [118] showed that 90% of thymus- and bone marrow–derived lymphoid cells expressed the PRLR. They also found that thymocytes expressed a lower density of PRLRs compared to bone marrow lymphocytes. In the latter, it was shown that T lymphocyte subsets expressed the PRLR more frequently (⬎85%) compared to those present in the periphery (50–65%). Closer scrutiny revealed that while thymocytes were frequently PRLR positive, receptor densities were generally low. However, a small population of CD4ⳮ/CD8ⳮ and CD4Ⳮ T cells exhibited high receptor densities. In peripheral blood, B cells, all monocytes, and 75% of T lymphocytes were found to express the PRLR [118]. In addition to pituitary hormones, somatostatin has been detected in platelets, mononuclear leukocytes, mast cells, and polymorphonuclear leukocytes [119,120]. In addition, parathyroid hormone–related protein [121,122] and insulin-like growth factor-1 (IGF-1) [123–126] are also thought to be synthesized by and/or associated with immune cells. F. Opioid and Nonopioid ␤-Endorphins There is considerable evidence in support of an immunomodulatory role for endogenous opioids during acute and chronic inflammatory processes in situ [127–130]. ␤-Endorphin is synthesized by splenocytes, peripheral blood lymphocytes, monocytes, and peripheral blood mononuclear cells [131]. It stimulated lymphocyte proliferation, increased IL-2 production [132], and augmented IL-2 receptor levels on human T cells [133]. Furthermore, endorphins, dynorphins, and enkephalins bind to opioid and nonopioid, ␤-endorphin receptors on leukocytes. Based upon similarities between the lymphocyte ␤-endorphin and other

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opioid receptors, the receptors are thought to represent splice variants of opioid receptors expressed centrally [134]. Cloning and characterization of brain ␦, ␬, and ␮ opioid receptors facilitated subsequent identification of immune cell opioid receptors. These receptors were found to belong to the rhodopsin superfamily of receptors characterized by seven membrane spanning hydrophobic regions. ␬ and ␦ opioid receptors have been identified in T-cell lymphoma cell lines and in thymocytes, respectively [134]. In addition, other opioid receptors appear to be expressed on activated murine and human lymphocytes [134]. It is evident that the neuroendocrine and immune systems share similar ligands and receptors, which establish intra- and intersystem communication pathways to maintain homeostasis. There is increasing evidence that numerous hormones and neuropeptides function as immunomodulators by regulating immune function during health and disease [135]. Since PRL/GH are neuroendocrine hormones with important immune functions, we will employ PRL-PRLR coupling to illustrate signaling pathways common to the neuroendocrine and immune systems. IV. CLASS I CYTOKINE RECEPTOR SUPERFAMILY The cytokine receptor superfamily consists of two distinct subclasses: class 1 and class 2. Class 1 family members include receptors for GH, PRL, IL 2–7, granulocyte colonystimulating factor (G-CSF), granulocyte-macrophage colony-stimulating factor (GMCSF), LIF, oncostatin (OM), erythropoietin (Epo), thrombopoietin (TPO), gp130, and the obesity factor leptin [136,137]. The class 2 cytokine family is represented by IFNs and IL-10 receptors [136,137]. Three isoforms of the PRLR have been cloned in the rat: the short form (PRLR-S, 291 amino acids, 49 kDa), the long form (PRLR-L, 592–598 amino acids, 80–85 kDa), and an intermediate isotype (PRLR-I, 393 amino acids, 65 kDa) [138], (Fig. 2). The PRLI, primarily expressed in the PRL-dependent Nb2 T lymphoma cell line, is thought to represent a deletion mutant of PRLR-L that lacks amino acids 323–520 [139]. Long and intermediate forms of the receptor have also been identified in humans [138,139]. All PRLR isoforms are homologous with respect to their extracellular and transmembrane domains. However, they differ in their cytoplasmic domains. This is due to alternative mRNA splicing or, in the case of the mutant Nb2 isoform, a mutation in the exon encoding the rat PRLR-L intracellular domain [138]. The PRLR shares several common motifs with those of the other cytokine 1 receptor family members including conserved extra- and intracellular features. For example, the extracellular domains exhibit two pairs of disulfide-linked cysteines (Cys) in the N-terminal subdomain and a tryptophan-serine box motif (WSXWS) present in the membrane proximal region of the C-terminal subdomain. The intracellular domain of the PRLR receptor contains four highly conserved structural motifs: box 1, a variable box (V box), box 2, and an extended box (X box) [140]. Box 1 consists of a hydrophobic proline-rich region, which is similar to Src homology 3 (SH3) binding sites. It is thought to associate with Jak2 during hormonal signaling (Fig. 2). Since the receptor lacks intrinsic enzymatic activity, ligand-induced receptor dimerization activates signal transduction. The V-box exhibits minimal homology among the cytokine receptors, whereas box 2, composed of acidic and hydrophobic residues, is highly conserved [141–143]. Mutational analysis of the GHR also revealed the importance of box 1 and box 2 motifs as key participants in Jak2 association and activation [144,145].

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Figure 2 Class 1 cytokine family of receptors. The cytokines are represented by LIF and IL-2 and the neuroendocrine hormones by GH and PRL. See text for additional details.

V. IMMUNOREGULATORY EFFECTS OF PRL/GH Early evidence that PRL/GH were required for the development and maintenance of the immune system was obtained from studies that demonstrated restoration of the immune system in immunodeficient Snell dwarf mice by administration of exogenous hormones. Immunodeficiency in these animals reflects a lack of the Pit-1 transcription factor [146,147]. PRL also restored humoral and cell-mediated immune responses in rats in which endogenous PRL release was blocked by hypophysectomy or administration of bromocriptine [148–150]. Numerous laboratories investigated the immunoregulatory actions of PRL and its mechanisms of action. PRL in combination with interleukin-2 (IL2), Staphylococcus aurieus cowan, or phytohemaglutinin increased proliferation in natural killer and T and B lymphocytes [151–153]. Immunoneutralization of PRL blocked IL-2–

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and IL-4–stimulated mitogenesis in cytokine-responsive cell lines [154]. Treatment of bone marrow cells or splenocytes with physiological concentrations of PRL stimulated signaling mechanisms leading to the transcriptional activation of an immune regulator gene, interferon regulatory factor (IRF-1) [155]. A study conducted in vivo by Yang et al. [156] demonstrated that the thymi of pups born to rats continuously infused with PRL were smaller in size, indicating reduced thymic input of progenitor cells from the bone marrow, enhanced T-cell maturation, and delivery to the periphery. However, thymi from animals treated with a PRL antagonist were three-fold larger and composed of apoptotic thymocytes. Rodent and human lymphocytes secrete and synthesize PRL [42–46] and express PRLR on their cell surface [47,48]. PRL has also been shown to stimulate mitogenesis in an in vitro T-cell model, Nb2 lymphoma cells [157]. However, despite the extensive evidence demonstrating an immunomodulatory role for PRL, its precise physiological role in maintenance of immune homeostasis has remained elusive. Recently, two laboratories generated genetic models of PRL (PRL-KO), [158] and PRLR deficiency (PRLR, KO), [159] and investigated whether PRL was required for development or functioning of the immune system. PRL deficiency caused marked deficits in mammary gland development and in reproductive function, but did not adversely affect the hematopoietic system. These mice were capable of normal humoral and cell-mediated immune responses following exposure to T-independent and -dependent antigens and challenges with Listeria monocytogenes [158,160]. These observations were consistent with those obtained using the PRLR-KO mice. These animals similarly exhibited normal immune responses to antigenic challenges [159]. Together, these observations indicated that PRL was most likely not required for the development or functioning of the immune system under basal conditions. However, the clear capacity of PRL to affect immune cell functions in vivo and in cell culture suggested that it may be critical under physiological conditions that deviate from steady state. Physiological and psychological stressors are known to evoke a neuroendocrine response termed the ‘‘general adaptive syndrome’’ that is, in part, characterized by stimulation of the HPA axis resulting in increased production of GCs and catecholamines [161]. The immunosuppressive effects of GCs have been extensively investigated. For example, increased GCs during restraint stress induce thymic involution, decrease CD4Ⳮ/CD8Ⳮ thymocytes, and induce apoptosis [162,163]. In contrast to these negative effects, low GC concentration is required for proper cytokine functioning [164]. In addition to GCs, stress also elevates other hormones that may affect the immune system, including GH, PRL, and IGF-1, which have been shown to counteract certain effects of GCs and other stress mediators [165]. To investigate whether PRL functioned to maintain immune cell integrity, we conducted experiments using the PRL-KO mice. Administration of GCs (dexamethasone, DEX) to mimic stress-induced GC elevation significantly increased thymocyte apoptosis, which was blocked in mice with constitutively elevated PRL [166]. We also identified a survival gene, XIAP, as a possible mediator of the survival effects of PRL. In other studies, we demonstrated that PRL regulated the expression of survival genes such as pim-1, bcl-2, bcl-xL, bax, and XIAP in Nb2 T cells, a model in which PRL also exhibits an antiapoptotic action in cells challenged with GCs [167–171]. Moreover, recent data from our laboratory indicate that PRL regulates expression of bcl-2 family members in primary cultures of murine thymocytes exposed to GCs [244]. A summary of survival genes that are regulated by PRL in T cells is presented in Table 1.

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Table 1 Survival Genes Regulated by PRL in T Cells Gene

Family/Characterization

Ref.

XIAP bcl-2 pim-1 bag-1 bcl-XL

IAP family Bcl-2 family Serine/threonine kinase Bcl-2 binding protein Bcl-2 family

167 166 155 168 244

Similarly, the effect of GH as a survival factor in immune cells was investigated based on observations made in dwarf mice. Like PRL, GH treatment reversed the apoptotic effects of GCs in T lymphocytes [172] and promoted T-cell proliferation in response to mitogenic factors. Together, these observations suggest that PRL and GH, which do not appear to affect the immune system under steady state conditions, are most likely important under conditions of stress or during other challenges to homeostasis. Several laboratories have investigated the mechanism of the immunomodulatory effects of these neuroendocrine hormones, focusing primarily on activation of signaling pathways leading to proliferation or apoptosis suppression. Some of these pathways, as discussed below, are activated by cytokines, thereby providing an additional level of neuroendocrine-immune interactions.

VI. SIGNALING PATHWAYS A. Jak Family of Tyrosine Kinases The Janus-associated family of tyrosine kinases (Jak) consists of four members, Jaks 1, 2, 3, and Tyk 2 [48,173]. The members of the cytokine receptor superfamily appear to rapidly activate one or more of these signaling intermediates following stimulation by ligand. Activation of Jak2 by PRL [174,175], GH [176], and erythropoeitin [177] occurs rapidly after ligand binding. The important role of Jak2 is evident from studies that showed that its genetic disruption was embryonically lethal and that the embryonic tissues were unresponsive to IL-3, erythropoeitin, G-CSF, thrombopoietin, and interferon [178]. Structurally, the Jak kinases demonstrate molecular masses of 120–140 kDa and contain several tyrosine residues, which rapidly undergo phosphorylation/dephosphorylation during activation by cytokines or hormones. This is thought to regulate Jak enzymatic activity and its downstream signal transduction events. Jak2 is constitutively associated with the PRLR [173,179,180], whereas GH binding to its receptor is required for the association of Jak2 with the GHR [176]. Many reports indicate that two Jak2 molecules must associate with the PRL-PRLR complex for functional activation to occur. This is supported by studies demonstrating that loss of either member of the kinase pair in heterodimeric cytokine receptor complexes (i.e., IFN-␣-Jak1/Tyk2, IFN-␥-Jak1/2, IL2-Jak2/3) caused functional inactivation of the receptor [181,182]. Use of chimeras encompassing the extracellular domain of GMSCF and the intracellular portion of the PRLR showed that two intact Jak2 association domains were required for PRL-stimulated proliferation

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[142]. The proline-rich box I motif, present in the intracellular domain of PRLR (Fig. 2), is required for Jak2 association [183]. Ligand-induced receptor dimerization activates Jak2 by autophosphorylation. Subsequently, tyrosine residues within the PRLR and Stat 5 transcription factors are phosphorylated. Stat 5a and, in turn, Stat 5b then hetero- or homodimerize, translocate to the nucleus [184], and activate transcription of hormone/ cytokine responsive genes such as IRF-1, cyclin B, and histone in T lymphocytes [155,185]. Several negative regulators of Jak2 signaling have been identified. Suppressors of cytokine signaling (SOCS) and cytokine-inducible SH2-containing protein (CIS) directly bind and inactivate the kinases [186]. In addition to Jak2 activation and subsequent Stat phosphorylation, cytokines also increase SOCS levels, thereby inhibiting Jak2 signaling and providing a negative feedback mechanism to regulate cytokine action. In PRL-responsive cell lines, SOCS-1-7 and CIS were induced by the hormone [187]. Functionally, SOCS proteins differ in their effects on PRL signaling. Most studies report inhibitory effects for SOCS 1 and 3 on PRLR signaling by inhibition of the Jak/Stat pathway. SOCS 2 and CIS appear to block the inhibitory effects of SOCS 1 and 3 [188,189]. The precise role of Jak2 in the immunoregulatory effects of PRLR remains to be elucidated. Although there is evidence to indicate that PRL-activated Jak2 is required for its actions [190], it is not clear whether its activation is sufficient for all of the observed effects of PRL. In fact, we recently demonstrated that PRL-activated Jak2 was not required for hormonal signaling to expression of survival genes such as pim-1 and bcl-xL in Nb2 T cells [191,192]. However, other studies indicate that PRL-activated Jak2 appears to suppress GC-induced degradation of XIAP (Krishnan and Buckley, unpublished). We and others have demonstrated that PRL-activated signaling pathways, in addition to Jak2, may mediate some of its effects in the immune system. LIF, produced by immune, pituitary, or hypothalamic cells, which also acts through a class I cytokine family receptor, causes hetero- or homodimerization of the signal transducer, gp130 [193], activation of Jak2 kinase [194], and subsequent phosphorylation of Stat 1 and Stat 3 [195]. Phosphorylated Stat 3 dimers translocated to the nucleus and activated transcription of the POMC gene to enhance ACTH synthesis and subsequently increase GC levels. This interaction provides a molecular basis for HPA activation by cytokines under conditions of stress and/or inflammation [196]. During inflammation, gp130-coupled cytokines, including LIF and IL-1, released by immune cells, and CRH from the hypothalamus, stimulate SOCS 3 induction via the Jak/Stat and cAMP/PKA pathways, respectively, to increase POMC mRNA expression [197]. SOCS 3 inhibits Jak/ Stat signaling activated by the gp130 cytokines and subsequent POMC and ACTH induction, which suppresses the inflammatory response to cytokines. Both LIF and IL-1 induce SOCS 3 in the murine hypothalamus and pituitary, providing a mechanism for suppression of ACTH release, and, as a consequence, GC secretion by the adrenal gland [198]. Based on the evidence discussed above, the Jak/Stat pathway appears to be a common signaling mechanism in the neuroendocrine and immune systems, which regulates gene transcription and ultimately affects cell survival or the inflammatory response. B. Ras/Raf/MAPK Pathways Although the Jak/Stat pathway has been viewed as central to the actions of cytokines, other signaling mechanisms, activated by PRL and cytokines, also appear to be important. One such pathway is the Ras/Raf/MAPK cascade.

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Mitogen-activated protein kinases (MAPK) were originally identified as low molecular weight proteins that were rapidly activated by mitogens [199]. In recent years this family of kinases has expanded and now includes a variety of kinases that are activated by cellular stressors, in addition to mitogens. The original members of this family were identified as MAPK-1 and -2, also referred to as extracellular related kinase-1 and -2 (Erk-1, -2). These signaling molecules were activated and tyrosinephosphorylated upon receptor stimulation by insulin or nerve growth factor [200]. MAPKs share similarities with other family member kinases including JNK/SAPK (cjun N terminal kinase/stress activated protein kinase) [201,202] and p38 MAPK [203,204]. All known family members are proline-directed serine/threonine protein kinases. Several family members are activated downstream of PRLR stimulation in response to ligand activation [205,206]. In addition to MAPK1/2, PRL activates p38 MAPK in human mononuclear and polymorphonuclear leukocytes by a mechanism that is independent of Jak/Stat signaling [207]. Hormone binding to the receptor leads to the activation of this cascade, resulting in the recruitment of two adaptor proteins Shc and Grb2, a guanine nucleotide exchange factor (Sos), and a small G protein (Ras). These, in turn, signal to activation of downstream Raf, MEK, and MAPK1/2, resulting in the nuclear translocation of MAPK. In the nucleus, MAPK phosphorylates trans-activating proteins that regulate transcription of genes associated with proliferation or survival in T cells [208]. It is currently unknown whether activation of the MAPK cascade requires Jak2 or other signaling pathways. Recent evidence suggests that these two pathways may be interlinked. Phosphorylation of the PRLR by Jak2 could provide a docking site for the adaptor protein, Shc, which in turn, may activate Ras/MAPK to alter expression of growth-associated genes [209]. An involvement of MAPK in TRH signaling to regulation of PRL gene transcription in the pituitary has been reported. TRH was shown to activate MAPK through a protein kinase C (PKC)- and Ca2Ⳮ-dependent mechanism, resulting in phosphorylation of the Ets transcription factor and subsequent PRL transcription [210]. Similarly, the PRL-releasing peptide (PrRP) activated Ets-dependent transcription of PRL gene through Ras/MAPK and JNK signaling pathways [211]. Thus, one signaling pathway (MAPK) appears to mediate the action of hormones (PRL, TRH, and PrRP) in both the immune and neuroendocrine systems. The JNK family of MAPKs is defined by their capacity to phosphorylate serine residues 63 and 73 in the N-terminal region of c-jun [212], an immediate early gene that is a component of the AP-1 transcription factor complex [213]. JNKs have also been described as stress-activated SAPKs since they are stimulated by cellular stressors and inflammatory cytokines such as IL-1 and TNF [214,215]. Recent evidence obtained in the Nb2 T-cell model indicated that JNK may be involved in PRL-mediated suppression of apoptosis in immune cells [216]. Recently, we observed that transfection of a dominant negative mutant of JNK blocked PRL inhibition of GC-induced degradation of survival proteins. This observation suggests that PRL-activated JNK participates in the immunoregulatory effects of PRL during stress [217]. Thus, in addition to Jak2, activation of JNK by hormones of the neuroendocrine system, such as PRL, and cytokines such as IL-1 and TNF demonstrates signaling common to both the neuroendocrine and immune systems.

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C. Phosphatidylinositol-3-Kinase Phosphatidylinositol-3-kinase (PI3K) was first identified as an enzyme, the activity of which was associated with oncoproteins and tyrosine kinase growth factor receptors that regulate survival and proliferation [218]. PI3K is a dimer composed of 110 kDa catalytic and 85 kDa regulatory subunits. Tyrosyl phosphorylation of p85 by activated growth factor receptors enhances the activity of PI3K [218]. Moreover, p85 has been shown to associate with the PRLR complex following ligand stimulation [219,220]. Gene disruption studies have revealed that the p85␣ subunit may be required for IL-4–dependent survival of mouse splenic B cells. However, p85␣ appears to be dispensable for IL-2– or IL-3–dependent T- or mast cell proliferation, respectively (reviewed in Ref. 221). Results from numerous pharmacological inhibitor and transfection experiments have established that PI3K and its downstream target, Akt, link multiple cytokine receptors to gene regulation, associated with entry into cell cycle or survival. As indicated above, cytokine receptors are generally thought to initiate signaling by Jak/Stat pathway. However, activation of PI3K downstream of these receptors has also been described and appears to proceed by a complex mechanism involving recruitment of adaptor proteins Shc, Grb2, and Gab2, which is tyrosyl phosphorylated to facilitate binding to PI3K (reviewed in Ref. 221). This appears to be the case for IL-1 and IL-15 receptors. Ligand activation of the IL-4 and -7 receptors causes Jak2-mediated receptor phosphorylation allowing recruitment and phosphorylation of IRS-1/2 docking proteins. This interaction promotes association with PI3K, indicating further complexity in regulation of its signaling (reviewed in Ref. 221). We have recently demonstrated in an in vitro T-cell model that PRL activates PI3K and its downstream target, Akt [222], which was required for expression of Pim-1, an antiapoptotic protein. This suggests that PRL-mediated Akt activation most likely contributes to the survival action of the hormone [191,222]. Akt, a 57–60 kDa protein serine/threonine kinase, was isolated as a transforming gene encoding three highly homologous isoforms (reviewed in Ref. 223). PI3K-induced Akt activation in T lymphocytes was first observed in IL-2–regulated proliferation of peripheral blood–derived T lymphocytes; IL-2 rapidly stimulated and sustained Akt activity [224]. IL-3 inhibition of T cell apoptosis required activation of the PI3K/Akt cascade. Activated Akt phosphorylated the pro-apoptotic Bcl-2 family member protein, Bad [225,226]. Phosphorylated Bad associates with the 14.3.3 protein, which prevents its dimerization with Bcl-xL, thereby favoring T-cell survival [227]. Since PRL also activates the PI3K/Akt cascade and increases Bcl-xL levels, a similar mechanism may account for certain survival effects of PRL in T cells. PRLR-associated Fyn or Ras may contribute to the activation of PI3K [219,228]. Interactions of PRLR with the adaptor protein Cbl through a Fyn-dependent mechanism [229] or insulin receptor substrate 1 (IRS-1) [220] may couple the receptor to PI3K/Akt signaling. IL-2 family cytokines appear to promote T-cell survival by activation of Akt in the presence of an apoptotic stimulus such as growth factor deprivation [224]. Recently, Hayakawa et al. [230] demonstrated that PrRP activated the PI3K/Akt cascade through GPCR-coupling mechanism, which stimulated PRL gene transcription in rat pituitary cells. D. Other Signaling Mechanisms Activated by PRL Several components of other signaling pathways have been shown to be associated with and/or activated by PRL. These include the tyrosine kinase ZAP-70 [231], Tec [232],

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p59fyn [233], a guanine nucleotide exchange factor, vav [234], and a SH2-containing phosphatase SHP2 [235]. PRL also activated nuclear protein kinase C (PKC) in purified rat splenocytes [236]. Activated PKC particularly, the novel isoform PKC ␪ and ε, promote T-cell survival by the Rsk-dependent phosphorylation of Bad [237]. As indicated above, phosphorylated Bad can associate with 14.3.3, which inhibited its dimerization with BclxL. Since PRL is a survival hormone in T cells, it will be important to determine whether PRL also activates the novel isoforms of PKC. Coupling of PRL to activation of PKC also appears to occur in neuroendocrine tissues. Tyrosine hydroxylase (TH), the rate-limiting enzyme in dopamine biosynthesis, is activated by PRL, in the median eminence, by the PKC pathway [238]. The molecular mechanism for the activation of PKC by PRL and whether this pathway contributes to the immunoregulatory effects of PRL remain to be determined. A summary of the signal transduction pathways activated by PRL are represented in Fig. 3.

Figure 3 PRLR signaling in immune cells. PRL binding to its receptor results in the activation of Jak/Stat pathway, which leads to transcription of an immunoregulatory gene, IRF-1. Activation of Jak/Stat pathway by PRL also increases levels of SOCS, which inhibits further activation of Jak2. In addition to Jak2, ligand activation of the PRLR also activates ZAP-70, a tyrosine kinase, which in addition to Jak2 may facilitate the association of PRLR with the adaptor proteins (Shc/Grb2), resulting in the activation of the ras/MAPK pathway. PRLR-associated Fyn or Vav may contribute to hormone activation of PI3K/Akt cascade. Intermediate components from each of these signaling pathways phosphorylate transcription factors to activate transcription of genes associated with proliferation or survival. TF, transcription factor.

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VII. MOLECULAR BASIS FOR THE NEUROENDOCRINE-IMMUNE INTERACTIONS A reciprocal relationship exists between the neuroendocrine and immune systems in which the CNS affects the immune system through hormonal and neuronal pathways and the immune system signals to the CNS through cytokines. The HPA axis is the primary hormonal pathway by which the CNS regulates the immune system. This requires hormones of the neuroendocrine system provoked during a stress response and the production of GCs. Sympathetic pathways regulate the immune response through adrenergic neurotransmitters that are released by neuronal routes. Neuroendocrine regulation of immunity is essential for survival during stress or to modulate the immune response during inflammation. During stress or inflammation, the HPA axis is stimulated. Its overstimulation, which may produce GCs, can suppress the immune response and, thus, increase susceptibility to infection. It is well known that GCs inhibit the production of several cytokines and affect multiple other components of an immune response, which contribute to suppressed immunity. For example, as indicated above, suppression of the immune response by restraint stress–induced GCs induced T-cell apoptosis. PRL, generally viewed as a neuroendocrine mammotrophic hormone of pituitary origin, also exerts cytokine-like effects. However, little is known regarding the effects of PRL secreted by immune cells on neuroendocrine tissues. During inflammation or stress, LIF and other cytokines stimulate ACTH release through POMC induction by activating the Jak/Stat pathway, subsequently leading to increased GC synthesis and release. PRL, which is also secreted by the anterior pituitary gland during stress, may function to counteract the immunosuppressive actions of GCs on cells of the immune system, thus promoting their survival and responsiveness. Signaling through Jak/Stat, Ras/MAPK, PI3K, and PKC pathways may mediate a spectrum of interactions among components of the neuroendocrine and immune systems. A. Jak/STAT Pathway 1. Immune System As described above, PRL secreted by pituitary lactotrophs or extrapituitary tissues binds to its receptor on T lymphocytes to activate Jak2 leading to phosphorylation of Stat 5. Stat5 dimers bind Stat-specific promoter elements (GAS, ␥-IFN–activating sequences) to induce transcription of several genes, including IRF-1 [155]. IRF-1 is an immunoregulatory transcription factor that activates the production of ␥-IFN. However, the effect of ␥-IFN on pituitary hormone release is controversial. Miyake and coworkers [38] demonstrated that ␥-IFN stimulated PRL release from rat pituitary cells in vitro via IL-6 production by folliculostellate cells. In contradistinction, Denef and colleagues [39–41] reported that ␥IFN inhibited PRL secretion through a NO-dependent mechanism. Thus, PRL is a neuroendocrine hormone that acts on immune cells to stimulate the transcription of the immunoregulatory machinery. IRF-1, in turn, provokes the production of cytokines, which act on the HPA axis to either increase or decrease PRL secretion (Fig. 4). 2. Neuroendocrine System Dopamine is the principal regulator of PRL secretion by pituitary lactotrophs. Hormone secretion is inhibited by dopamine produced by neurosecretory neurons within the hypothalamus. Dopamine, in turn, acts on D2 receptors present in pituitary lactotrophs to inhibit PRL secretion (reviewed in Ref. 239). In addition to tuberoinfundibular dopamine neurons

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Figure 4 Common signaling in the neuroendocrine (pituitary lactotrophs) and immune systems activated by PRL. PRL released by the pituitary gland or immune cells acts via receptors present on the hypothalamic dopaminergic neurons to stimulate dopamine (DA) synthesis through the Jak2/ Stat pathway possibly by increasing tyrosine hydroxylase (TH, rate-limiting enzyme in dopamine synthesis). Released DA inhibits pituitary PRL release through D2 receptors present on the pituitary lactotrophs. In the immune system, pituitary PRL or immune cell–derived PRL activates the Jak2/ Stat pathway to stimulate expression of IRF-1, which in turn stimulates transcription of ␥-IFN that affects pituitary or hypothalamic hormone release. See text for additional details.

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located in the ME, a primary source of dopamine in the hypophyseal circulation, periventricular and tuberohypophyseal neurons also release dopamine into the intermediate and posterior pituitary lobes, respectively. It has been suggested that dopamine from these structures contributes to regulation of PRL secretion (reviewed in Ref. 239). It is well established that PRL activates each of these dopaminergic neuron populations [240] to regulate its own secretion by a PRLR-mediated negative feedback mechanism [241,242]. Grattan et al. [243], using Stat 5b–deficient mice, demonstrated that in the absence of Stat 5b, PRL signal transduction in tuberoinfundibular dopaminergic neurons was defective. These workers concluded that Stat 5b and hence the PRL-activated Jak/Stat pathway was required for the negative feedback regulation of PRL secretion. Since PRL, secreted by both the pituitary and immune cells, has been shown to activate the Jak/Stat pathway, it is possible that PRL secreted by the immune cells may also affect pituitary secretion of the hormone (Fig. 4). The focus of this chapter has been with similarities between the neuroendocrine and immune systems with respect to polypeptide mediators and their intracellular signaling mechanisms. VIII. CONCLUSION The discussion above suggests that the Jak/STAT pathway is a common signaling mechanism activated by PRL in both the neuroendocrine and immune systems but with differing effects. In this chapter we reviewed shared signaling mechanisms between the neuroendocrine and immune systems. The HPA axis is considered the most important aspect of neuroendocrine-immune interaction. Its activation during stress or inflammation is mediated by cytokines released from immune cells and centrally, both triggering the release of hypothalamic and pituitary hormones. Moreover, numerous neuroendocrine hormones and their receptors are expressed on immune cells, thereby providing a mechanism for bi-directional interaction between the systems. Signaling through the Jak/Stat pathway appears to be a common feature among cytokines and neuroendocrine hormones. Cytokine and hormonal activation of this pathway may provide a potential mechanism for the interactions between the two systems. In addition to the Jak/Stat pathway, several other mechanisms including Ras/MAPK and PI3K signaling are also activated by pituitary hormones such as PRL and inflammatory cytokines including several interleukins and TNF. Although these pathways have been identified and extensively studied in immune cells, further investigation into potential cross-talk interactions between these and other pathways with respect to cytokines and hormones will provide a more complete understanding of neuroendocrine-immune system interactions. ACKNOWLEDGMENTS Supported in part by grants from the NIH (DK53452) and the American Institute for Cancer Research. REFERENCES 1. Blalock JE. The syntax of immune-neuroendocrine communication. Immunol Today 1994; 15:504–511. 2. Weigent DA, Blalock JE. Neuroendocrine-immune interactions. In: Keane RW, Hickey WF, Eds. Immunology of the Nervous System. New York: Oxford University Press, 1996: 359–377.

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3 Hypothalamic-Pituitary-Adrenal Axis Effects on Innate and Adaptive Immunity EMMANOUIL ZOUMAKIS and GEORGE CHROUSOS National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, Maryland, U.S.A. ILIA ELENKOV National Institute of Mental Health, National Institutes of Health, Bethesda, Maryland, U.S.A.

I. INTRODUCTION The nervous, endocrine, and immune systems secrete and respond to various regulatory molecules including steroids, neuropeptides, cytokines, prostanoids, and neurotransmitters, which provide the molecular basis for neuroendocrine-immune responses during disturbances of homeostasis. The central effectors of these responses include the locus ceruleusnoradrenaline (LC-NA)/autonomic (sympathetic) neurons of the hypothalamus and brain stem and the corticotropin-releasing hormone (CRH)/arginine-vasopressin (AVP) neurons of the paraventricular nucleus of the hypothalamus, which respectively regulate the peripheral activities of the systemic/adrenomedullary sympathetic nervous systems (SNS) and the hypothalamic-pituitary-adrenal (HPA) axis. Activation of the LC-NA/autonomic system and HPA axis results in systemic elevations of catecholamines (CAs) and glucocorticoids (GCs), respectively, that act in concert to maintain a steady state (Chrousos, 1995). Stress hormones, and especially glucocorticoids, have been known to shrink the thymus and lymph nodes, to inhibit lymphocyte proliferation, migration and cytotoxicity, and to suppress the secretion of certain cytokines, such as interleukin (IL)-2 and interferon␥ (IFN-␥) (Selye, 1936; Chrousos, 1995). Early observations and the broad use of glucocorticoids as potent anti-inflammatory/immunosuppressant agents led to the initial conclusion that stress was, in general, immunosuppressive. However, there has been convincing evidence that glucocorticoids, at levels that can be achieved during stress, influence the immune response in a less monochromatic way (Franchimont et al., 2003). Recently, CRH 51

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secreted by sympathetic postganglionic peripheral nerves and sensory afferent fibers was identified in inflammatory sites (‘‘immune’’ CRH) and shown to possess inflammatory properties (Karalis et al. 1991; Chrousos, 1995). This new understanding helps explain some well-known, but often contradictory, effects of stress on the immune system and on the onset and course of certain infectious, autoimmune/inflammatory, allergic, and neoplastic diseases. Immune cytokines are a large group of pleiotropic hormones. They are induced in response to tissue injury, infection, or inflammation or in the presence of neoplastic tissue (Chrousos, 1995). They also behave as paracrine or autocrine cell regulators mediating adjacent cell functions. Complex interactions have been described for cytokine actions, including overlapping, synergistic, and antagonistic activities. The terms ‘‘proinflammatory’’ and ‘‘anti-inflammatory’’ cytokines reflect the basis of their peripheral action. This, however, does not necessarily translate directly to their central nervous system actions and central regulation of the HPA axis (Allan and Rothwell, 2001). II. THE HPA AXIS The HPA axis and the systemic sympathetic and adrenomedullary (sympathetic) systems are the peripheral limbs of the stress system, whose main function is to maintain basal and stress-related homeostasis (Lamberts et al., 1984; Burns et al., 1989). The central components of this system are located in the hypothalamus and the brain stem. The stress system is active when the body is at rest, responding to many distinct circadian, neurosensory, bloodborne, and limbic signals. These signals include cytokines produced by immune-mediated inflammatory reactions, such as tumor necrosis factor-alpha (TNF-␣), interleukin-1 (IL-1), and IL-6. Activation of the stress system heightens arousal, accelerates motor reflexes, improves attention and cognitive function, decreases appetite and sexual arousal, and increases the tolerance of pain. The activated stress system also changes cardiovascular function and intermediary metabolism and inhibits immune-mediated inflammation. Corticotropin-releasing hormone and noradrenergic neurons of the central stress system innervate and stimulate each other (Elkabir et al., 1990). Thus, CRH stimulates the secretion of norepinephrine through specific receptors, and norepinephrine stimulates the secretion of CRH primarily through ␣1-noradrenergic receptors. By means of autoregulatory, ultrashort negative-feedback loops, CRH and norepinephrine collateral fibers inhibit presynaptic CRH and ␣2-noradrenergic receptors, respectively. CRH, AVP, and noradrenergic neurons are stimulated by the serotonergic and cholinergic systems and inhibited by the ␥aminobutyric acid–benzodiazepine and opioid-peptide systems of the brain. Centrally secreted substance P inhibits hypothalamic CRH neurons but not AVP neurons and stimulates the central noradrenergic system (Engler et al., 1986; Ixart et al., 1987; Redekopp et al., 1989). Each of the paraventricular nuclei has three parvicellular divisions: a medial group that mostly produces CRH and secretes it into the hypophysial portal system, an intermediate group that secretes AVP into the hypophysial portal system, and a lateral group that primarily produces CRH and innervates noradrenergic and other neurons of the stress system in the brain stem. Some parvicellular neurons contain and secrete both CRH and AVP (Whitnall, 1989; De Goeij et al., 1991). Other paraventricular CRH neurons project to and innervate pro-opiomelanocortin–containing neurons of the central stress system in the arcuate nucleus of the hypothalamus, as well as neurons in pain-control areas of the hind brain and spinal cord.

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Activation of the stress system causes CRH-induced secretion of pro-opiomelanocortin–derived and other opioid peptides that enhance analgesia. These peptides simultaneously inhibit the stress system by suppressing the secretion of CRH and norepinephrine. CRH also stimulates the secretion of corticotropin through the corticotrophs of the anterior pituitary. When CRH is absent, very little corticotropin is secreted. AVP alone has little effect on corticotropin secretion but acts synergistically with CRH (Rittmaster et al., 1987). Every hour, the parvicellular neurons secrete two or three mostly synchronous pulses of CRH and AVP into the hypophysial portal system (Carnes et al., 1990; Calogero et al., 1992). Early in the morning, when these pulses are at their peak, they increase the magnitude of corticotropin and cortisol pulses. The amplitude of these pulses also increases during acute stress, but under these conditions the stress system recruits additional secretagogues of CRH, AVP, or corticotropin, such as magnicellular AVP and angiotensin II (Holmes et al., 1986; Phillips, 1987). Corticotropin is the key regulator of glucocorticoid secretion by the adrenal gland. Other hormones, including those from the adrenal medulla, and autonomic neural input to the adrenal cortex can also regulate cortisol secretion (Ottenweller and Meier 1982; Vinson et al., 1988; Hinson, 1990; Andreis et al., 1992).

III. CYTOKINES AND THE HPA AXIS RESPONSE TO STRESS The neuroimmune-endocrine interface is mediated by cytokines, such as IL-1 and TNF␣, acting as auto/paracrine or endocrine factors regulating pituitary development, cell proliferation, hormone secretion, and feedback control of the HPA axis (Reichlin, 1999; Ericsson et al., 1997; Rivest, 2001). Cytokines participate as mediators of the complex HPA axis response to stress or inflammation. During inflammatory stress, cytokines that stimulate corticotroph POMC expression and ACTH secretion are produced both peripherally as well as within the hypothalamus and pituitary, and by stimulating the HPA axis they antagonize their own peripheral proinflammatory action (Elenkov et al., 1999). Several cytokines affect the release of anterior pituitary hormones by an action on the hypothalamus, the pituitary gland and/or the adrenal cortex (Fig. 1). The major cytokines involved are IL-1, IL-2, IL-6, TNF-␣, and interferon-gamma (IFN-␥). The predominant effects of these cytokines are to stimulate the HPA axis and to suppress the hypothalamic-pituitarythyroid (HPT) and gonadal axes and growth hormone (GH) release. Further HPA axis stimulation leads to immunosuppression and increased susceptibility to infection. Inflammatory cytokines also induce the production of ACTH secretagogues and other cytokines. Pituitary corticotroph POMC gene expression is regulated by CRH as well as several gp130 receptor–dependent cytokines, such as IL-6 and leukemia inhibitory factor (LIF). Effects of gp130-dependent cytokines are mediated by the JAK-STAT signaling cascade. This cross-talk of different signaling cascades enables the HPA axis to respond rapidly to inflammatory and stress stimuli. gp130-dependent cytokines have the ability to activate the HPA axis even in the absence of CRH (Bethin et al., 2000). Both IL-6 and LIF mediate HPA responses to stress and inflammation by inducing CRH/AVP and ACTH in the course of the inflammatory reaction, and experiments with CRH-deficient mice demonstrate ample activation of the HPA axis by IL-6. Moreover, IL-6 directly induces the release of adrenal corticosteroids. IL-6 is known to be important for continued CRH expression during late phases of inflammation. Similarly, hypothalamic LIF induced in the course of chronic inflammatory process is important for maintaining an HPA axis inflammatory stress response. LIF administered before lethal septic shock

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Figure 1 Interactions between the inflammatory reaction and the hypothalamic-pituitary-adrenal (HPA) axis. Inflammation-induced activation of the HPA axis participates in a feedback loop, through which the end products of this axis, glucocorticoids, inhibit inflammation. Proinflammatory molecules, such as bacterial products (LPS), released intracellular products (HSP70 or HMG-2 proteins), oxygen radicals, and/or oxidized molecules, activate the transcription factor NF-␬B (see Fig. 2 legend), leading to elevation of proinflammatory cytokines, which further stimulate this transcription factor. Note involvement of the neural afferent system and glia cells. M␾: macrophage; TLR: Toll-like receptors; CVO: circum-ventricular organs.

has a protective effect, preventing sepsis-induced tissue damage. LIF is also protective for neurons and peripheral tissues during injury. IV. PROINFLAMMATORY EFFECTS OF PERIPHERAL, IMMUNE CRH CRH profoundly influences the function of the immune system indirectly through activation of the global stress response and directly through local modulatory actions on inflammatory responses. Immune CRH is secreted peripherally and plays a direct immunomodulatory role as an autocrine or paracrine mediator of inflammation (Karalis et al., 1991; Chrousos, 1995). Our laboratory localized immunoreactive CRH (irCRH) in local immune accessory cells in various experimental models of inflammation, including carrageenininduced aseptic inflammation in Sprague-Dawley rats, acute and chronic streptococcal cell wall– and adjuvant-induced arthritis in Lewis rats (Crofford et al., 1992), and RP-16 induced uveitis in Lewis rats and B10.A mice (Mastorakos et al., 1995). Immunocytochemistry and HPLC also have verified irCRH in human tissues undergoing inflammatory processes, including joints of patients with rheumatoid arthritis, thyroid glands of patients with Hashimoto thyroiditis, and colonic mucosa of patients with ulcerative colitis (Crofford

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et al., 1993; Scopa et al., 1994; Kawahito et al., 1995). While CRH mRNA was detected in chronically inflamed synovia in rats, generally in early stages of inflammation there is a major discrepancy between the abundance of the peptide and the paucity of its mRNA that is undetectable or present in minute quantities. The demonstration of CRH-like immunoreactivity in the dorsal horn of the spinal cord and dorsal root ganglia and in sympathetic nerve cell bodies and sympathetic ganglia, including postganglionic neurons and nerve fibers in spleen after IL-2 administration, support the hypothesis that the majority of immune CRH in early inflammation is of peripheral nerve rather than immune cell origin (Merchenthaler et al., 1986; Suda et al., 1984; Skofitsch et al., 1985; Udelsman et al., 1986a). Immune CRH appears to have pro-inflammatory and vasomotor actions, since systemic administration of rabbit anti-CRH sera caused suppression of both inflammatory exudate volume and cell number in carrageenin-induced inflammatory sites in rats and, when administered in the early stages of the disease, ameliorated the severity of experimentally induced uveitis in mice. In addition, CRH administration to humans or nonhuman primates caused major peripheral vasodilation manifested as flushing and increased blood flow and hypotension secondary to third spacing of fluids (Schurmeyer et al., 1984; Udelsman et al., 1986b). An intradermal CRH injection induced stronger vascular permeability and mast cell degranulation than an equimolar concentration of C48/80, a potent mast cell secretagog (Boucher et al., 1995; Theoharides et al., 1998). Vascular permeability was inhibited by a specific CRH antiserum or the CRH receptor type 1 antagonist antalarmin. Stress-induced intracranial mast cell degranulation was completely inhibited by pretreating rats with specific CRH antiserum (Theoharides et al., 1995). Thus, as a target of immune CRH, the mast cell appears to mediate some of its proinflammatory properties. The high concentrations of CRH in experimental animal models of inflammation and in disease states associated with inflammation suggest that CRH plays an important role in initiating, propagating, and/or regulating inflammatory responses, including but not limited to those mediated by mast cells. Indeed, CRH-binding sites have been found on a number of immune and immune accessory cells, including human peripheral blood leukocytes, resident splenic mouse macrophages, and inflamed synovia from arthritic rats (Singh et al., 1998; Audhya et al., 1991). CRH stimulates secretion of IL-1 from monocytes, IL-2 from lymphocytes, and IL-6 from mononuclear cells, promotes lymphocyte proliferation and IL-2 receptor expression and chemotaxis by mononuclear leukocytes, and enhances production of oxygen radicals by macrophages. Recently, immune CRH was shown also to participate in a major fashion in local, opioid-mediated analgesia (Singh, 1989; McGillis et al., 1989; Singh and Leu, 1990; Genedani et al., 1992; Angioni et al., 1993; Koshida and Kotake, 1994; Schafer et al., 1996). Thus, the presence of CRH and CRH receptors at local inflammatory sites and in peripheral nerves led us to hypothesize that CRH released from peripheral nerves participates in an axon reflex loop with immune cells. Upon release from peripheral sensory afferent and/or postganglionic sympathetic nerves, CRH could then act on local immune cells to elicit proinflammatory responses. The inflammatory mediators released could recruit circulating immune and immune accessory cells to the inflammatory site, activate local immune accessory cells, and act on the nerve endings to release more inflammatory peptides. This could be of particular importance in allergic inflammatory states triggered by acute stress, such as asthma or eczema, in which activation of the sympathetic system and local secretion of CRH could lead to mast cell degranulation and initiation of a new episode or exacerbation of chronic disease. We propose a similar mechanism in stress-

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induced vasokinetic phenomena, for instance, migraine headaches (Theoharides et al., 1995). V. EFFECTS OF GLUCOCORTICOIDS ON THE IMMUNE RESPONSE Glucocorticoids are pleiotropic hormones that at stress levels or pharmacological doses prevent or suppress inflammation and other immunologically mediated processes (Franchimont et al., 2003). At the molecular level, glucocorticoids form complexes with specific receptors that migrate to the nucleus, where they interact with selective regulatory sites within DNA; this results in positive and negative modulation of several genes involved in inflammatory and immune responses. At the cellular level, glucocorticoids inhibit the access of leukocytes to inflammatory sites, interfere with the functions of leukocytes, endothelial cells, and fibroblasts, and suppress the production and the effects of humoral factors involved in the inflammatory response. Glucocorticoids exert many of their antiinflammatory effects through protein-protein interactions with a set of transcription factors known to stimulate the inflammation, such as the nuclear factor-␬B (NF-␬B), several signal transducers and activators of transcription (STATs), and the activator protein-1 (AP1). There is a very good example of how glucocorticoids suppress inflammation by blocking NF-␬B activity (Fig. 2). Clinically, several modes of glucocorticoid administration are used, depending on the disease process, the organ involved, and the extent of involvement. High doses of daily glucocorticoids are usually required in patients with severe diseases involving major organs, whereas alternate-day regimens may be used in patients with less aggressive diseases. Intravenous glucocorticoids (pulse therapy) are frequently used to initiate therapy in patients with rapidly progressive, immunologically mediated diseases. The benefits of glucocorticoid therapy can easily be offset by severe side effects; even with the greatest care, side effects may occur. Moreover, for certain complications (e.g., infection diathesis, peptic ulcer, osteoporosis, avascular necrosis, and atherosclerosis), other drug toxicities and pathogenic factors overlap with glucocorticoid effects. Minimizing the incidence and severity of glucocorticoid-related side effects requires carefully decreasing the dose, using adjunctive disease-modifying immunosuppressive and anti-inflammatory agents, and taking general preventive measures (Chrousos, 2001). A. Antigen Presentation For T cells to be optimally activated, recognition of antigen/major histocompatibility complexes (MHC) by the T-cell receptor (TCR) must be accompanied by a second costimulatory signal. This costimulatory signal is predominantly generated by B7.1 and/or B7.2 molecules, expressed on APCs, when engaged to their counterreceptor, CD28, present on T cells. GCs inhibit the expression of B7.1 and B7.2 in human monocytes and dendritic cells (DCs) respectively, and downregulate MHC II expression in APCs. The downregulation of B7 and MHC II molecules may contribute to the inhibitory effects of GCs on APCdependent T-cell activation (Fox et al., 1997; Girndt et al., 1998; Ganea and Delgado, 2001; Pan et al., 2001). A key aspect of the innate immune response is the ability to discriminate between self and nonself molecules. This is achieved through pattern recognition receptors, which directly recognize molecular epitopes expressed by microbes. Scavenger receptors (SRs) are also involved in the innate immune response for the removal of damaged tissue and debris and recognize a wide variety of pathogens. CD163 expression is downregulated by

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Figure 2 Interaction between nuclear factor-␬B and the activated glucocorticoid receptor. CR: cytokine receptor; TLR: toll-like receptor; LBP: ligand binding protein. GR␣: glucorticoid receptor. PG5, p5O: components of NF-␬B. GRE: glucocorticoid responsive element. IKB: inhibitor of NF␬B. (From Franchimont et al., 2003.)

proinflammatory and upregulated by anti-inflammatory mediators and GC (Norris and Benveniste, 1993; Maimone et al., 1993). CD163, as well as eight other scavenger receptors, were strongly upregulated by GC. Finally, the IL-1R/Toll-like receptor (TLR) superfamily has emerged as an expanding family of receptors whose function is to respond rapidly to infection and injury (Sanders et al., 1997). TLR-4 and TLR-2 mediate the host response to gram-negative and grampositive bacteria, respectively, and the finding that TLR-4 is important for responses to LPS may allow for novel means to intervene therapeutically during sepsis (Borger et al., 1998). Three members of the Toll family were regulated by GC; TLR-4 and TLR-2 were upregulated, and TLR-3 downregulated. That GCs regulate this superfamily could be critical for many aspects of inflammation and host defense, since TLRs control innate immune responses in vivo. The main receptor for lipopolysaccharide (LPS) on the mouse monocyte is TLR-4. To further demonstrate the role of TLR-4 ligation in providing the second signal, splenocytes were stimulated with lipid A, a specific mitogen for TLR-4. Lipid A stimulation of SDR cells from an endotoxin-responsive strain resulted in high levels of cytokines and reduced sensitivity to corticosterone. In C3H/HeJ mice, lipid A did not affect cell function significantly, indicating that LPS-induced GC resistance can be signaled via TLR-4. B. Immune Cell Traffic, Proliferation, and Activity After a single dose of a short-acting glucocorticoid, the concentration of neutrophils increases, whereas the lymphocytes (T and B cells), monocytes, eosinophils, and basophils

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in the circulation decrease in number. The increase of neutrophils is due both to the increased influx from the bone marrow and to the demargination and impaired extravasation of neutrophils. The decreased migration of neutrophils from the blood vessels combined with diminished chemotaxis and adherence to vascular endothelium of neutrophils and monocytes results in inhibition of the accumulation of these cells at the site of inflammation. These effects underlie the potent anti-inflammatory properties of GCs. The reduction in circulating lymphocytes, monocytes, eosinophils, and basophils is the result of their movement from the vascular bed to lymphoid tissue. Glucocorticoids antagonize macrophage differentiation and inhibit many of their functions. These agents (a) depress myelopoiesis and inhibit expression of class II major histocompatibility complex antigens induced by IFN-␥, (b) block the release of numerous cytokines, such as IL-1, IL-6, and TNF-␣; (c) depress production and release of proinflammatory prostaglandins, and leukotrienes, and (d) depress tumoricidal and microbicidal activities of activated macrophages. The major effect of glucocorticoids on neutrophils appears to be the inhibition of neutrophil adhesion to endothelial cells. This effect diminishes trapping of neutrophils in the inflamed site and probably is responsible for the characteristic neutrophilia. At pharmacological doses, glucocorticoids only modestly impair important neutrophil functions, such as lysosomal enzyme release, the respiratory burst, and chemotaxis to the inflamed site. Lower doses do not affect these neutrophil functions. Just as they affect macrophages, glucocorticoids decrease circulating eosinophil and basophil counts. They also decrease the accumulation of eosinophils and mast cells at sites of allergic reactions. Functionally, glucocorticoids inhibit IgE-dependent release of histamine and leukotriene C (Andersson and Persson, 1988) from basophils, and they also inhibit degranulation of mast cells. By inhibiting IL-1 production by monocytes and IL2 and IFN-␥ production by lymphocytes, GCs may also contribute to decreased lymphocyte proliferation. C. Pro- and Anti-inflammatory Cytokine Production Studies in the 1970s and the 1980s revealed that GCs inhibit lymphocyte proliferation and cytotoxicity and the secretion of TNF-␣, IL-2, and IFN-␥ (Beutler et al., 1986; Boumpas et al., 1993). These observations, in the context of the broad clinical use of GCs, initially led to the conclusion that GCs are, in general, immunosuppressive. Recent evidence indicates, however, that systemically, GCs cause selective suppression of pro-inflammatory cytokine production and cellular immunity and a shift towards Th2-mediated humoral immunity, rather than generalized immunosuppression. This new concept is briefly outlined below. GCs act through their classic cytoplasmic/nuclear receptors on APCs to suppress the production of IL-12, the main inducer of Th1 responses (Blotta et al., 1997; Elenkov et al., 1996). Since IL-12 is extremely potent in enhancing IFN-␥ and inhibiting IL-4 synthesis by T cells, the inhibition of IL-12 production by APCs may represent a major mechanism by which GCs affect the Th1/Th2 balance. Thus, GC-treated monocytes/macrophages produce significantly less IL-12, leading to their decreased capacity to induce IFN␥ production by antigen-primed CD4Ⳮ T cells. This is also associated with a downregulation of the expression of IL-12 receptors on T and NK cells and an increased production of IL-4 by T cells (DeKruyff et al., 1998; Wu et al., 1998). GCs also have a direct effect on Th2 cells by upregulating their IL-4, IL-10, and IL-13 production (Ramierz et al., 1996). GCs do not affect the production of IL-10 by

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monocytes; yet, lymphocyte-derived IL-10 production is upregulated by GCs (Van der Poll et al., 1996; Elenkov et al., 1996). This could be the result of a direct stimulatory effect of GCs on T-cell IL-10 production and/or a block on the restraining inputs of IL12 and IFN-␥ on lymphocyte IL-10 production. The peripheral levels of IL-6 and, to a greater extent, those of TNF-␣ and IL-1␤ are tonically inhibited by basal levels of glucocorticoids. The increased IL-6 production that occurs when cortisol levels fall might explain the symptomatology of acute glucocorticoid deficiency. The adipose tissue is a major determinant of circulating IL-6 in states of obesity. In human adipocytes dexamethasone suppresses the release of IL-6 in a dosedependent manner (Vicennati et al., 2002). The effect of dexamethasone is inhibited in the presence of RU 486, suggesting that it is mediated via the glucocorticoid receptor. REFERENCES 1. Allan SM, Rothwell NJ. Cytokines and acute neurodegeneration. Nat Rev Neurosci 2001; 10: 734–744. 2. Andersson PT, Persson CG. Developments in anti-asthma glucocorticoids. Agents Actions Suppl. 1988; 23:239–260. 3. Andreis PG, Neri G, Mazzocchi G, Musajo F, Nussdorfer GG. Direct secretagogue effect of corticotropin-releasing factor on the rat adrenal cortex: the involvement of the zona medullaris. Endocrinology 1992; 131:69–72. 4. Angioni S, Petraglia F, Gallinelli A, Cossarizza A, Franceschi C, Muscettola M, Genazzani AD, Surico N, Genazzani AR. Corticotropin-releasing hormone modulates cytokines release in cultured human peripheral blood mononuclear cells. Life Sci. 1993; 53:1735–1742. 5. Audhya T, Jain R, Hollander CS. Receptor-mediated immuno-modulation by corticotropinreleasing factor. Cell. Immunol. 1991; 134:77–84. 6. Bethin KE, Vogt SK, Muglia LJ. Interleukin 6 is a essential, corticotropin-releasing hormoneindependent stimulator of the adrenal axis during immune system activation. Proc Natl Acad Sci USA 2000; 97:9317–9322. 7. Beutler B, Krochin N, Milsark IW, Luedke C, Cerami A. Control of cachectin (tumor necrosis factor) synthesis: mechanisms of endotoxin resistance. Science 1986; 232:977–980. 8. Blotta MH, DeKruyff RH, Umetsu DT. Corticosteroids inhibit IL-12 production in human monocytes and enhance their capacity to induce IL-4 synthesis in CD4Ⳮ lymphocytes. J. Immunol. 1997; 158:5589–5595. 9. Borger P, Hoekstra Y, Esselink MT, Postma DS, Zaagsma J, Vellenga E, Kauffman HF. Betaadrenoceptor-mediated inhibition of IFN-gamma, IL-3, and GM-CSF mRNA accumulation in activated human T lymphocytes is solely mediated by the beta2-adrenoceptor subtype. Am. J. Respir. Cell Mol. Biol. 1998; 19:400–407. 10. Boucher W, Pang X, Chrousos G, Papanicolaou D, Theoharides TC. Corticotropin-releasing hormone (CRH) induces rat mast cell (MC) secretion (abstr). FASEB J. 1995:A919. 11. Boumpas DT, Chrousos GP, Wilder RL, Cupps TR, Balow JE. Glucocorticoid therapy for immune-mediated diseases: basic and clinical correlates. Ann. Intern. Med 1993; 119: 1198–1208. 12. Burns G, Almeida OF, Passarelli F, Herz A. A two-step mechanism by which corticotropinreleasing hormone releases hypothalamic-endorphin: the role of vasopressin and G-proteins. Endocrinology 1989; 125:1365–1372. 13. Calogero AE, Norton JA, Sheppard BC, et al. Pulsatile activation of the hypothalamic-pituitaryadrenal axis during major surgery. Metabolism 1992; 41:839–845. 14. Carnes M, Lent SJ, Goodman B, Mueller C, Saydoff J, Erisman S. Effect of immunoneutralization of corticotropin-releasing hormone on ultradian rhythms of plasma adrenocorticotropin. Endocrinology 1990; 126:1904–1913.

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4 Glucocorticoids and the Immune System ¨ LLER, and G. JAN WIEGERS, ILONA E. M. STEC, PIA U. MU GEORG WICK Innsbruck Medical University, Innsbruck, Austria

I. HISTORICAL OVERVIEW The term glucocorticoid derives from early observations that these hormones were involved in glucose metabolism. They stimulate several processes that collectively serve to increase and maintain normal concentrations of glucose in the blood. The influence of glucocorticoids (GC) on the immune system has been recognized for more than half a century. Before the discovery of GC as hormonal substances produced by the adrenal cortex, morphological manifestations of the regulation of lymphoid tissues were extensively studied. Experimental manipulations such as adrenalectomy led to thymic hypertrophy in rats, as reported by Jaffe in 1924 [1]. Conversely, exposure of rats to various types of stress induced, apart from adrenal enlargement, thymus involution. The magnitude of these effects was much less pronounced in adrenalectomized or hypophysectomized animals [2], which demonstrated that the pituitary-adrenal axis can act as a functional link between the central nervous and immune systems. Going one step further towards identification of potential agents responsible for these effects, it was subsequently shown that administration of steroid containing extracts of the adrenal cortex produced thymus involution as well [3–5]. The availability of pure steroids finally confirmed that GC were the active substances of the adrenal cortex in this respect. Although the findings described above were straightforward, the interpretation of the results for the functional regulation of the immune system by GC was quite different from today’s view of the field. At that time, most researchers were convinced that GC released upon stress in general served to stimulate host defense mechanisms, such as the immune system. In 1945, White and Dougherty reported that lymphocyte breakdown 65

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after injection of adrenocorticotropin (ACTH) caused increased blood antibody titers by releasing antibodies stored in the lymphocytes. The authors proposed that the pituitaryadrenal axis thus enhanced immune defense mechanisms [6]. The concept that stress strengthened immune and other defense mechanisms was mainly introduced by Hans Selye, who is called the father of stress research. He defined his general adaption syndrome (GAS) as follows: The sum of all nonspecific, systemic reactions of the body that develop upon long exposure to stress [7]. In close connection to the GAS was Selye’s view on the pathophysiology of certain diseases, the so-called diseases of adaptation. Selye hypothesized that such diseases, among which he listed diffuse collagen disease, allergy, and rheumatic diseases, were provoked by excessive adaptive responses to stress [7]. Although the results of White and colleagues correlated well in Selye’s concept, they could not be reproduced by others [8]. At odds with Selye’s theory of diseases of adaptation was the landmark discovery of the anti-inflammatory effects of GC in the late 1940s. Fascinated by the ameliorating effect of pregnancy on rheumatoid arthritis, Hench and colleagues, searching for the endogenous compound responsible for this effect, administered cortisone to a patient with rheumatoid arthritis and so discovered the therapeutic effects of GC. Hench published this observation together with Kendall, Slocumb, and Polley in 1949 [9], and together with the biochemists Reichstein and Kendall he received the Nobel Prize for medicine in 1950. They rejected the idea that steroids were of etiological significance for rheumatoid arthritis and instead emphasized their unique potential as a tool for pathophysiological research. Their results on GC were not restricted to rheumatoid arthritis, and it appeared that GC potently inhibited all kinds of inflammatory responses, which was unexpected in the scientific community. These remarkable findings almost overnight established GC as the miracle drugs of the 1950s. However, in an attempt to reconcile Selye’s concept to the newly discovered effects of GC, most researchers, with the notable exception of Tausk [10], designated the anti-inflammatory properties of GC ‘‘pharmacological,’’ without any physiological significance [8]. Consequently, few researchers devoted their work to the physiological role of GC in the immune system, while research on the pharmacological effects of GC rapidly expanded because of their therapeutic potential. The discovery of the GC receptor (GR) [11,12], its basic molecular characteristics, and the fact that GR are present in virtually every nucleated cell type reinforced scientific interest in the field of GC physiology. Ironically, Munck and coworkers found no indication in the literature until 1976 that GC enhanced the body’s defense mechanisms, which was still the leading concept. Numerous studies testing GC effects in isolated in vitro systems made it clear that GC had not only anti-inflammatory, but also immunosuppressive properties. Munck and colleagues argued that there was no justification for separating anti-inflammatory (pharmacological) from physiological effects. In 1984, these investigators published a new hypothesis stating that (1) ‘‘the physiological function of stress-induced increases in GC levels is to protect not against the source of stress itself, but against the normal defense reactions that are activated by stress’’ and (2) ‘‘the GC accomplish this function by turning off those defense reactions, thus preventing them from overshooting and themselves threatening homeostasis’’ [13]. A similar concept was previously proposed in 1951 by Tausk in a review on the clinical use of GC, wherein he argued that ‘‘cortisone treatment is appropriate where the defense reactions of the organism cause more damage than the agent against which they defend’’ and used the following metaphor: ‘‘GC protect against the water damage caused by the fire brigade’’ [10]. This remarkable review was published in a rather unknown pharmaceutical company journal and was thus unavailable to Munck and coworkers in 1984.

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In the mid-1970s, Besedovsky and colleagues proposed that the immunosuppressive effects of GC serve as a physiological regulatory system by preventing overreaction of the immune system and preserving the antigenic specificity of the immune response by preventing unrestricted proliferation of lymphocytes with little affinity for the antigen that could otherwise lead to autoimmunity [14–16]. Despite the pioneering studies of Besedovsky and coworkers, the general physiological role of GC in the immune system was only accepted after publication of the above-mentioned review of Munck et al. in 1984. At the time Selye developed his concept of GAS, evidence was also found that GC, at basal concentrations, often functioned in a ‘‘permissive’’ way. According to Ingle, GC, while ineffective alone, are necessary to permit normal expression of the effects of other agents [17]. In Selye’s concept, high levels of GC were needed to enhance defense mechanisms. Although both Selye and Ingle pointed out that permissive effects at low levels of GC could not account for the resistance against stress conferred by high levels of GC, the view that GC could exert permissive effects became an alternative to Selye’s theory. This development, together with the discovery of the anti-inflammatory and immunosuppressive effects of GC, further complicated concepts of the role of GC in the immune system. Ironically, the alternative belief that the effects of GC on immunity were stimulatory rather than inhibitory has been renewed in current thinking in this field, since research over the last two decades had made it clear that endogenous GC can either stimulate or inhibit antigen-specific immunity depending on the dose and duration of GC exposure. Our intent in this chapter is to make it clear that GC action should not be polarized in one or the other direction. In contrast, the effects of GC on the immune system seem to be better characterized by the term ‘‘regulatory,’’ which can be both stimulatory or inhibitory depending on (experimental) parameters such as GC dose, timing, and type of immune response. II. SUPPRESSIVE EFFECTS OF GLUCOCORTICOIDS ON THE IMMUNE SYSTEM Studies of Besedovsky and colleagues in the mid-1970s showed that injection of antigens in rats or mice was associated with increased GC blood levels [14]. The highest GC concentration was reached at approximately the same time (about 6 days postinjection) at which the immune response, measured by antibody production, reached a maximum. They subsequently found that GC are instrumental for the ‘‘antigenic competition’’ phenomenon described in textbooks until that time as follows: injection of one antigen inhibits the immune response to a noncrossreactive antigen administered together or later. Antigenic competition was almost abolished when adrenalectomized animals were used [16]. The authors proposed that GC prevent overreaction of the immune system and preserve the specificity of the immune response [15,16]. If this idea and the hypothesis of Munck and colleagues were correct, inflammatory responses should then be exacerbated in the absence of endogenous GC, which appeared to be the case. Carrageenan, a sulfated cellwall polysaccharide found in certain red algae, induced nonspecific inflammation in adrenalectomized rats that was substantially stronger than in normal animals [18]. Treatment with RU 486, a GC antagonist, also resulted in an increased inflammatory response compared to control animals [19]. These experiments showed that endogenous GC are sufficient to mediate an anti-inflammatory effect. Recently, it was demonstrated that adrenalectomized mice, in contrast to normal controls, did not survive infection with murine cytomegalovirus (MCMV) [20]. Interestingly, viral load in the liver of adrenalectomized mice 36–48

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hours after infection was not increased. Moreover, viral load was even slightly reduced 60 hours post-infection, indicating that adrenalectomized mice did not die as a direct consequence of an augmented viral replication. Serum cytokine levels (TNF-␣ and IFN␥) were clearly increased in adrenalectomized mice and pretreatment with neutralizing antibodies against TNF-␣ protected these animals against MCMV-induced lethality [20]. Earlier evidence of a pathological role of cytokines came from experiments with adrenalectomized mice, which are susceptible to TNF-␣ and IL-1 in terms of mortality [21]. Taken together, these studies conclusively demonstrate that endogenous GC protect against unnecessary strong inflammatory and immune responses. The observation that immune activation led to an increase in blood GC levels created an important basis for the further development of neuroimmunoendocrinology. Again, Besedovsky and coworkers were pioneers in showing that products of activated immune cells (so-called GC-increasing factors, or GIF) can activate the hypothalamo-pituitaryadrenal (HPA) axis [22]. The most extensively studied substance in this respect is IL-1, which can stimulate ACTH and corticosterone output in mice and rats and is also a mediator of GC changes induced by viruses, such as MCMV [23–26]. Other cytokines, including IL-2, IL-6, TNF, and IFN-␥, have subsequently been shown to activate the HPA axis [26–28]; this is discussed in more detail elsewhere in this book. Evidence that normal HPA activity is needed for a physiological immune response came from studies by Wick and colleagues investigating animal models of autoimmunity. Antigen (sheep red blood cell) injection of obese strain (OS) chickens, which spontaneously develop autoimmune thyroiditis resembling human Hashimoto’s disease, leads to a decreased serum GC surge compared to normal control chickens [29]. It was subsequently shown that administration of IL-1 to OS chickens also induces a deficient activation of the HPA axis [30]. Thus, these animals appear to have a hyporesponsive HPA axis upon immune stimulation [31]. In addition, basal corticosterone serum levels of OS chickens are normal, but the concentration of serum corticosteroid-binding globulin (CBG) is twice that of control chickens such that these animals also have decreased levels of free, biologically active corticosterone levels [32]. The reduced GC release after IL-1 injection is associated with a hyperreactive response of peripheral blood T cells to mitogenic activation with either concanavalin A (ConA) or phytohemagglutinin (PHA) [29]. Finally, treatment of OS chickens with GC prevents the development of spontaneous autoimmune thyroiditis [32]. The finding of a hyporeactive HPA axis upon cytokine injection was subsequently confirmed in several spontaneously autoimmune-prone strains of mice, such as MRL/MP-faslpr or (NZB/ NZW)F1, two animal models for systemic lupus erythematosus (SLE) [33,34]. Interestingly, although the responsiveness of the HPA axis was diminished in all animal models studied, the molecular mechanisms underlying this altered immune-endocrine communication via the HPA axis appear to differ from one animal model to the other [32,35,36]. For example, a significant IL-1 receptor deficiency is present in the dentate gyrus of NZB and (NZB/NZW)F1 autoimmune lupus mice, but not in NZW, MRL/MP-faslpr, C3H/He, or outbred Swiss mice [35]. The results of these experiments with animal models that spontaneously develop autoimmune diseases were confirmed by several other groups using various models of experimentally induced autoimmunity in susceptible Lewis rats, i.e., streptococcal cell wall polysaccharide (SCW)–induced arthritis and experimental allergic encephalomyelitis (EAE) after injection of guinea pig myelin basic protein (MBP), which is an animal model for human multiple sclerosis. Lewis rats, which develop much more severe arthritis after challenge with SCW than Fischer control rats, were found to have a defect in the biosyn-

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thesis of corticotropin-releasing hormone (CRH) that accounts for the decreased GC response found in these animals [37,38]. Injection of dexamethasone protects against the reaction to SCW, whereas Fischer rats become more sensitive to SCW after treatment with RU 486 [38]. Administration of MBP to Lewis rats induces EAE, from which the animals spontaneously recover. Development of EAE can also be prevented by GC treatment, but this is critically dependent on the dose of GC used [39]. First, adrenalectomy abrogates the recovery phase and the disease becomes fatal. Second, when adrenalectomized rats received subcutaneous implants of corticosterone to maintain basal steroid levels, these animals died when EAE was induced. However, if the steroid replacement therapy was adjusted to mimic the physiological hormone levels in rats developing EAE, then the disease followed a nonfatal course closely resembling that in the nonadrenalectomized controls. Finally, replacement therapy that achieved serum corticosterone levels slightly higher than those found in intact rats with EAE completely suppressed the disease [39]. From this and other evidence, the authors concluded that endogenous corticosterone release in rats with EAE plays an essential role in spontaneous recovery. The results of these experiments support the speculation that the sensitivity of a given animal to EAE would be predicted by the presence of a hyporesponsive HPA axis. This idea was recently challenged by Reul and coworkers, who showed that dark agouti (DA) rats developed MBP-induced EAE despite the fact that their HPA axis responded in the same way as that of resistant Fischer rats [40]. Moreover, administration of myelinoligodendrocyte glycoprotein (MOG), which induces a chronic relapsing, inflammatorydemyelinating variant of EAE closely resembling human multiple sclerosis, to DA or brown Norway (BN) rats (both robust HPA responder strains) resulted in severe EAE in both strains, whereas the Lewis rat developed a late onset, milder, and more slowly progressive disease than DA and BN rats. Reul and colleagues concluded that HPA axis characteristics, while playing an important role in the course of the disease, do not predict EAE disease susceptibility [40]. Interestingly, a subsequent study found that relapse and onset of chronic progressive disease in MOG-induced EAE in DA rats was associated with a failure of the disease process to sufficiently stimulate endogenous corticosterone production. Indeed, exogenous GC treatment reduced the severity of the disease [41]. The findings gained in both spontaneous and experimentally induced animal models of autoimmunity have clinical significance as well. One-sided adrenalectomy in patients with Cushing’s disease caused by adrenal tumors gave rise to the development of autoimmune thyroiditis [42]. In addition, patients with Addison’s disease also present with bronchial asthma and several allergic diseases [43–45]. It is clear that cytokines, released after activation of the immune system, stimulate the HPA axis and thus increase peripheral levels of GC. But what effects do GC have on these cytokines? Most evidence points to a negative regulatory feedback system in which GC suppresses synthesis and release of a great number of cytokines. These actions of GC are thought to underlie their potent therapeutical efficacy. Initially, most of the observations that GC suppresses cytokine production came from in vitro studies that have since been extended to whole organisms (see Ref. 13). To date, GC have been shown to inhibit IL-1␣ , IL-1␤, IL-2, IL-3, IL-5, IL-6, IL-8, IL-12, IL-13, IFN-␥, TNF-␣, granulocytemacrophage colony-stimulating factor (GM-CSF), RANTES (regulated on activation, normal T-cell expressed and secreted) and macrophage inflammatory protein 1-␣ (MIP1-␣) in various cell types [46–50]. Molecular mechanisms by which GC inhibit cytokines are reported to act at the level of transcription, translation, mRNA stability or secretion, and combinations of these mechanisms also occur (see Ref. 47). However, not all cytokines

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are suppressed by GC. Controversy exists in case of IL-4, which has been reported on the one hand to be enhanced by GC in murine T-cells both in vitro [51] as well as in vivo [52,53], and on the other hand to be either enhanced [54] or inhibited in human lymphocytes in vitro [55]. Recent data may shed some light on this issue; GC were shown to inhibit the production of IL-12, which may indirectly lead to enhanced IL-4 synthesis, since IL12 has been shown to potently inhibit the production of IL-4 and, conversely, to stimulate IFN-␥. This mechanism was shown to be present in murine [56] as well as human systems [54]. Cytokines that are not suppressed by GC are macrophage colony-stimulating factor (M-CSF) and transforming growth factor-␤ (TGF-␤) [57]. GC increase the activity of TGF-␤ by activating a latent form of this cytokine [58], an effect that may indirectly suppress some parameters of the immune response, since this cytokine inhibits activation of T cells and macrophages [59,60]. Production of the IL-10 cytokine is increased by GC [48,54], which, parallel to TGF-␤, may lead to immunosuppression, since IL-10 inhibits antigen presentation and T-cell activation [61]. Taken together, GC seem to inhibit proinflammatory cytokine synthesis or to induce cytokines that have immunosuppressive potential. This fits well with the view that GC protect against overshooting immune defense mechanisms, as is also illustrated by the fact that a number of cytokines (TNF-␣, IFN-␥, GM-CSF, IL-1, IL-2, and IL-6) are themselves toxic at higher concentrations [57]. III. STIMULATORY EFFECTS OF GLUCOCORTICOIDS ON THE IMMUNE SYSTEM Since GC inhibit the production of a broad spectrum of cytokines, one might speculate that cytokine signaling is another level at which GC suppress immune responsiveness. However, this is not uniformly the case. In various experimental settings, GC have been shown to act synergistically with exogenously added cytokines. Thus, in cultures of hepatic cells, GC strongly potentiate IL-1– and/or IL-6–induced expression of acute phase proteins [62]. In the rat, GC and IL-6 synergistically induce the acute phase protein ␣1-acid glycoprotein involving a direct protein-protein interaction between the ligand-activated GC receptor (GR) and the IL-6–induced nuclear factor-IL-6 (NF-IL-6) [63]. Synergistic effects between GC and IL-1 and IL-6 have also been observed in human B cells. The combination of these agents potently induce the production of IgM and IgG by these cells [64]. The production of another class of antibodies, IgE, by IL-4–stimulated human PBMC is also enhanced in the presence of GC [65], a fact that may crucially impinge on the therapeutic effects of GC in patients with type I allergies. Recently, the effect of GC on IL-7-induced IL-2R␣ expression on human CD4Ⳮ cord blood T cells was investigated. While GC alone had little effect on IL-2R␣ expression, IL-7 alone induced a moderate increase, and GC greatly enhanced IL-7–induced IL-2R␣ expression [66]. Since IL-7 inhibits T-cell apoptosis in vitro [67,68], a potential consequence of enhanced IL-7 responsiveness in the presence of GC may be enhanced T-cell survival. While GC alone induced CD4Ⳮ T-cell apoptosis, the same dose of GC indeed inhibited apoptosis in the presence of IL-7 to a larger extent than IL-7 alone [66]. Human eosinophils express increased levels of MHC-II molecules in the presence of GC and either IL-3, IL-5, or GM-CSF, leading to functionally enhanced antigen presentation [69]. Other biological responses to a variety of cytokines (IL-2 [70], IFN-␥ [71], granulocyte colony-stimulating factor (G-CSF) [72], GM-CSF [72], and oncostatin M [73]) are enhanced in the presence of GC as well. Thus, the observations that GC

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inhibit the production of many cytokines, but can also act synergistically with various cytokines on several cell types, creates a paradox that is poorly understood, both in terms of molecular mechanism and physiological significance. Importantly, these synergistic effects cannot be experimentally induced artifacts (in view of the inhibitory effects of GC on cytokine production), because infection-induced cytokine production precedes a surge of GC (a number of cytokines activate the HPA-axis as mentioned above), possibly potentiating the effect of cytokines that are already present. Subsequently, any additional cytokine production is expected to be inhibited by GC. Over the last decade, evidence has accumulated that GC induce the expression of a number of cytokine receptors, which is in marked contrast to their reported effects on the production of cytokines. These include receptors for IL-1, IL-2, IL-4, IL-6, IL-8, IFN␥ , TNF-␣, GM-CSF, and CSF-1, as well as the common signal transducer gp130 (CD130) (reviewed in Ref. 46). However, it should be noted that contradictory results have been obtained with the IL-2 receptor, as discussed [74]. Recently, IL-7R␣ has been shown to be induced by GC in T cells [66] and in T-lymphocytic leukemia cell lines [75]. The observation that the common signal transducer gp130 [76,77] is augmented by GC is of considerable interest, as this subunit is shared by the IL-6 receptor (IL-6R), IL-11R, leukemia inhibitory factor receptor (LIFR), ciliary neurotrophic factor receptor (CNTFR), and oncostatin M-R [78]. GC thus have the potential to augment the action of several cytokines by increasing the expression of a single, common subunit. With respect to the IL-6 receptor complex, both the IL-6 binding subunit (gp80) and the signal transduction subunit (gp130) are induced by GC. Of great interest is whether GC affect expression of the common ␥chain (CD132, shared by IL-2, IL-4, IL-7, IL-9 and IL-15), which remains to be clarified. A very important issue is, of course, whether functional consequences of GC-evoked cytokine receptor expression can be measured in view of the fact that GC inhibit the production of many ligands of the same cytokine receptors induced by GC. Unfortunately, in many cases where GC-evoked induction of cytokine receptors was observed, whether the elevated cytokine receptor density had any functional consequences was not determined. Thus, very few studies simultaneously addressed the effects of GC on the production of an endogenous cytokine, the expression of its receptor, and the biological effects resulting from their interaction in the same biological test system. When these parameters were studied simultaneously in the IL-2/IL-2R system, it was found that GC accelerate the de novo appearance of IL-2R␣ on anti-T-cell receptor (TCR)–activated T cells, i.e., the hormone shifted the peak of IL-2R␣ expression to a time point about 2 days earlier than in the control cultures [79]. The consequence of a high expression of IL-2R is a greatly accelerated cell progression through the cell cycle [80]. Indeed, the GC-evoked shift in IL-2R␣ expression was paralleled by a shift in the T-cell proliferative response. As expected, IL-2 production was inhibited by GC, but was not rate-limiting for T-cell proliferation during the phase of enhanced proliferation [79]. In this system, GC seemed to increase the sensitivity of T cells for IL-2, resulting in an enhanced responsiveness to this cytokine. In the same test system, GC inhibited T-cell proliferation after 4–5 days, a time point where IL-2 became rate-limiting. It was concluded that GC act to optimize the course of the T-cell proliferative response by increasing the density of IL-2R␣. With respect to the IL-6/IL-6R system, it was reported that GC and IL-6 synergistically induce acute phase protein production by hepatic cells. Indeed, a GC-evoked induction of IL-6R expression was associated with an enhanced biological response to IL-6 in this system [81]. In a human glioblastoma cell line, GC treatment resulted in an increase in IL-1R binding accompanied by an increased capacity of IL-1␤ to induce IL-6 production [82]. Thus, by

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increasing the density of cytokine receptors, GC can, at least in some cell types, increase the sensitivity of the cell to the respective cytokine, leading to potentiation of the biological response. IV. GLUCOCORTICOIDS AND T-HELPER-CELL DIFFERENTIATION Naive CD4Ⳮ T cells can differentiate upon activation into two subsets, called Th1 or Th2, which carry out different functions in host defense mechanisms. These subsets can be distinguished by the spectrum of cytokines they secrete. Th1 cells produce IL-2, IFN␥, and TNF-␤ and contribute largely to T-cell–mediated responses such as delayed-type hypersensitivity. The Th2 subset produces IL-4, IL-5, IL-6, IL-10, and IL-13, which help B cells proliferate, differentiate and participate in humoral, e.g., allergic responses. Selective differentiation of either subset is established during priming of naive CD4Ⳮ T cells and can be significantly influenced by a variety of factors, including the cytokine environment in the early phase of the response and the nature and amount of antigenic peptides presented to CD4Ⳮ T cells [83]. There is increasing evidence that GC also participate in guiding helper T-cell differentiation to a shift towards the Th2 phenotype. Daynes and Araneo reported that in vivo treatment of mice with GC during immunization led to substantially lower in vitro production of IL-2 upon antigenic restimulation [52]. Under these experimental conditions, IL-4 production appeared to be increased. A shift from IL-2 toward IL-4 was also observed when primed T cells from immunized mice were rechallenged in vitro with antigen in the presence of physiological concentrations of GC [52]. When rat CD4Ⳮ T cells were activated in vitro in the presence of GC, then expanded in exogenous IL-2 and restimulated in the absence of GC, cytokine production shifted to a Th2 phenotype [51]. In contrast, IL-4 production has been shown to be either enhanced [54] or inhibited by GC in human lymphocytes in vitro [55]. Moreover, GC also inhibit the production of other Th2 cytokines, such as IL-5 in human lymphocytes [84], which initially seemed to make it unlikely that GC can induce a similar shift toward Th2 in human cells. Recent data, however, show that GC inhibit the production of IL-12 [54], the expression of the ␤1 and ␤2 chains of the IL-12R and responsiveness to IL-12 in human cells [85]. The reduced production of IL-12 resulted in a decreased capacity of the monocytes to induce IFN-␥ and an increased ability to induce IL-4 in T cells, a mechanism shown to be similar in murine cells [56]. Thus, inhibiting production and responsiveness to IL-12 may be a major mechanism by which GC affect the balance between Th1 and Th2 in favor of Th2. Although GC are still the most effective drugs for the treatment of allergic diseases (probably by inhibiting leukocyte infiltration and proinflammatory cytokine synthesis), in the long run they may indirectly exacerbate the course of these disorders due to the capacity of GC to induce IL-4. Supporting such a potentially detrimental role of GC is the finding that these hormones synergize with IL-4 to induce the production of IgE, a principal mediator of allergic diseases, by human peripheral blood mononuclear cells [65]. This finding is intriguing with respect to the frequently observed resistance of allergic patients to GC-based therapy. V. GLUCOCORTICOIDS AND T-CELL DEVELOPMENT/SELECTION A. Glucocorticoid Production by the Thymus Complex effects of GC have been found in studies addressing T-cell development and selection. It is well known that GC induce apoptosis in (immature) thymocytes, an effect

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observed in vitro [86–89] and either after injection of (synthetic) GC [90–92] or by endogenous GC [93]. Conversely, removal of endogenous GC by adrenalectomy leads to hypertrophy of the thymus [1] and spleen [94]. Interestingly, a recent unexpected perspective emerged with the demonstration that, in addition to the adrenals, the murine thymus was able to produce some metabolites (pregnenolone, 11-deoxy-corticosterone) of the GC synthetic pathway [95]. By using steroidogenic enzyme assays with radiolabeled precursors followed by separation of the products by thin layer chromatography, we subsequently showed that the thymus definitively contains all enzymes and cofactors needed to generate each intermediary steroid of the GC synthetic pathway, including the end products corticosterone and cortisol [96]. The capacity of the thymus to produce GC is not limited to the mouse. The chicken thymus also has the capacity to produce GC, and in contrast to the mouse, the chicken thymus also expressed P450-c17␣- hydroxylase (CYP17) activity, suggesting that cortisol is a major GC produced by the chicken thymus [97]. Avian species provide the unique opportunity to study B-cell development and differentiation in a separate primary lymphoid organ, the bursa of Fabricius. Surprisingly, all GC synthetic enzyme activities appeared to be present in the bursa, too, suggesting that bursa-derived endogenous cortisol influences B-cell development and differentiation [97]. How the production of thymus-derived GC is regulated remains to be elucidated. Analogous to the adrenals, ACTH enhanced intrathymic production of pregnenolone [95]. Irradiation of mice, a procedure to selectively eliminate thymocytes, but not epithelial cells, in the thymus resulted in complete abolishment of the activity of P450 c11␤-hydroxylase (CYP11B1), the final enzyme of the GC pathway. P450 c11 ␤-Hydroxylase activity was also undetectable in experiments with a thymic epithelial cell (TEC) line. Both experimental procedures have in common an absence of contact between TEC and developing thymocytes, suggesting that an intact thymic architecture is necessary for complete GC production [96]. Controversial data have been published as to which thymic cellular subset(s) produce GC hormones. At the mRNA level, TEC, but not thymocytes, were shown to produce P450scc (CYP11A1), P450 c21-hydroxylase (CYP21), and P450 c11␤-hydroxylase [98]. In contrast, another group recently reported that CD4ⳭCD8ⳭCD69Ⳮ and CD4ⳭCD8CD69Ⳮ thymocytes, but not TEC, produce P450scc mRNA [99]. At the protein level, immunohistochemical studies revealed that P450scc and P450 c11␤-hydroxylase are expressed mainly in cortical epithelium [95]. By double immunofluorescence analyses, we found that both cortical and medullary TEC stain with an antibody against P450scc, although staining of cortical TEC clearly was more prominent. Thymocytes did not stain with this antibody. In addition, it was found that thymic nurse cells, a subset of TEC that can engulf thymocytes, stained with the anti-P450scc antibody [96]. At the level of enzyme activity, TEC, but not thymocytes, were shown to produce the GC intermediary product of P450scc, pregnenolone and that of P450 c21-hydroxylase, 11-deoxy-corticosterone [95]. A TEC line was shown to produce the GC intermediary product of P450scc, pregnenolone, as well as the product of 3␤-hydroxy-steroid dehydrogenase (3␤-HSD), progesterone [96]. In addition, TEC were reported to produce GC activity, although this was indirectly measured [98]. Thus, TEC appear to be the primary producers of GC, although a potential role of CD4ⳭCD8ⳭCD69Ⳮ and CD4ⳭCD8-CD69Ⳮ thymocytes cannot presently be excluded.

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B. Interaction Between GR- and TCR-Mediated Signaling A physiological role of thymus-derived GC in T-cell development and selection is implied by the observation that TCR- and GR-signaling demonstrate crosstalk. The GR and the TCR independently induce apoptosis in T-hybridoma cells, yet together they promote Tcell survival [100,101]. According to this so-called ‘‘mutual antagonism’’ model, GC would, by preventing TCR-induced apoptosis, set the TCR avidity window for thymocyte selection [102]. This model predicts that any decrease in GR signaling leads to deletion of thymocytes that would otherwise be positively selected. Evidence in favor of this model has been presented by two independent experimental approaches. First, the addition of metyrapone, a GC biosynthesis inhibitor, to fetal thymic organ cultures (FTOC) induced apoptosis of Ag-specific TCR transgenic CD4ⳭCD8Ⳮ thymocytes that normally undergo positive selection [103]. Second, thymocytes of transgenic mice bearing GR antisense transgenes selectively in thymocytes displayed a decreased viability at the CD4ⳭCD8Ⳮ stage, and their number was profoundly reduced [104]. At the functional level, these mice responded normally to the complex Ag purified protein derivative (PPD), but were nonresponders to pigeon cytochrome c 81–104 (PCC), indicating the presence of an Agspecific ‘‘hole’’ in the T-cell repertoire [105]. However, a recent study described experiments with two different transgenic mouse strains that either overexpress GR twofold selectively in thymocytes and peripheral T cells, or present with reduced GR expression in both thymocytes and T cells. Whereas both thymocyte and peripheral T cell number were decreased in the former, they were increased in the latter mice [106]. Contributing to this complex and inconsistent picture of GR physiology in T cells is the finding that fetal thymocytes of mice carrying a GR hypomorph allele develop normally up to embryonic day 18 and T-cell selection appears grossly normal [107]. Recently, it was reported that reconstitution of adult, lethally irradiated wild-type mice with GR-deficient fetal liver cells did not affect thymocyte numbers or subset distribution [108]. Collectively, in some experimental systems GC modulate T-cell development and selection, but their precise role remains unclear. An experimental system allowing the study of T-cell development and selection is FTOC. This experimental system, in contrast to cells in suspension culture, has been shown to support the full program of T-cell development. Moreover, it has been shown that removal of cells from their microenvironment perturbs normal signaling. For example, thymocytes start to upregulate expression of the ␣␤-TCR once separated from thymic stromal cells. Using FTOC, we studied the effects of both exogenous and endogenous GC on TCR-mediated apoptosis. Endogenous GC production can be inhibited with the selective P450 c11␤-hydroxylase inhibitor metyrapone, which does not influence T-cell development at low concentrations (150 ␮g/mL), but does enhance apoptosis of CD4ⳭCD8Ⳮ thymocytes in thymic lobes induced by anti-TCR antibodies. Addition of low concentrations of exogenous GC alone potently enhanced the number of CD4ⳭCD8Ⳮ cells recovered from fetal thymic lobes, whereas higher concentrations were inhibitory (see Fig. 1). Thus, in this experimental system, GC have a finely balanced effect, i.e., permissive at low versus suppressive at high concentrations on T-cell differentiation. The ‘‘mutual antagonism’’ hypothesis assumes that the signal mediated by GR must be relatively constant, while the TCR-mediated signal is variable. In addition, TCR expression increases during maturation of immature CD4ⳭCD8ⳭTCRlow thymocytes so that TCR signaling is also quantitatively variable. Until recently, however, no data were available addressing GR expression in individual thymocyte subsets. Ligand-binding assays

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Figure 1 Effect of exogenous GC on cell recovery in FTOC. Day 15 fetal BALB/c thymi were cultured in the absence or presence of various concentrations of GC for 72 h, harvested, and stained for CD4 and CD8. Total CD4ⳭCD8Ⳮ cell recovery was determined by microscopic enumeration of cells excluding trypan blue, and fractional recovery of thymocyte subsets by flow cytometry.

have the disadvantage of requiring that the endogenous ligand be removed by adrenalectomy, a procedure that in itself modulates GR expression [109]. Moreover, this type of assay cannot determine whether GR expression is homogeneously distributed within each thymocyte subset. A technique to study GR expression in different thymocyte subsets is to combine immunofluorescence cell surface staining for CD4, CD8, and TCR with intracellular staining of GR in four-color cytometry [89]. The highest GR expression was observed in CD4-CD8-TCR-thymocytes, whereas the lowest expression was found in the CD4ⳭCD8ⳭTCRlow subset (see Fig. 2). In each thymocyte subset, GR was homogeneously distributed. Upregulation of TCR expression by the CD4ⳭCD8ⳭTCRlow subset to CD4ⳭCD8ⳭTCRhigh cells was accompanied by a parallel increase of GR expression in this latter subset. Since the GR appeared to be homogeneously distributed among CD4ⳭCD8ⳭTCRlow cells and its expression increased along with the TCR in the CD4ⳭCD8ⳭTCRhigh subset, our data support the view that the GR can, at least on the basis of its concentration, produce a constant signal relative to that of the TCR. Unclear is why CD4ⳭCD8ⳭTCRlow cells, the most sensitive subset to GC-induced apoptosis, express the least GR. A potential explanation is that the expression of a major antiapoptotic protein, bcl-2, is almost absent in CD4ⳭCD8ⳭTCRlow cells (Fig. 2). Thus, the substantial downregulation of antiapoptotic proteins, such as bcl-2, in CD4ⳭCD8ⳭTCRlow cells may have a greater impact on their high sensitivity to GC-induced apoptosis than downregulation of the GR would have on their survival. CONCLUDING REMARKS Recent evidence convincingly shows that GC at levels that can be reached physiologically affect the immune response in a more differentiated way than previously thought. Although

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Figure 2 Expression of GR vs bcl-2 in various thymocyte subsets. Freshly prepared BALB/c thymocytes were analyzed for cell surface markers (TCR, CD4, CD8) as well as intracellular determinants (GR, bcl-2) by flow cytometry. Both GR and bcl-2 staining are expressed as percent median fluorescence intensity of CD4-CD8-TCR-cells. (Adapted from Ref. 89.)

the anti-inflammatory and immunosuppressive properties of high doses of GC prevail in clinical settings, this should not distract our efforts to understand their physiological role. Important in this respect is the dose of GC administered and the duration of treatment. For example, T-cell–mediated responses such as delayed-type hypersensitivity (DTH) can be stimulated by low doses of GC, whereas high doses are suppressive [110]. In the same experimental model, acute (2h) restraint stress enhanced DTH, whereas chronic restraint stress (several weeks) inhibited DTH [111]. It will also be interesting to elucidate any role of so-called preparative effects of GC [47]. When GC are administered from 6 hours before until 6 hours after LPS challenge, they inhibit a subsequent surge of various cytokines (TNF-␣ and IL-6) in the plasma [112]. In contrast, administration of GC relatively long before LPS challenge leads to substantially higher plasma levels of cytokines. Clearly, we do not yet know exactly how endogenous GC modulate the immune system. Perhaps these hormones act to generate a more efficient immune response that is only suppressed by GC when this immune reaction may cause damage to the host. ACKNOWLEDGMENTS The authors’ work reported herein was supported by the Jubila¨umsfonds of the Austrian Nationalbank (Grant no. P8131), the Austrian Science Fund (FWF; Grant No. 14466), and the European Union (Grant no. QLG1-CT-2001-01574). REFERENCES 1. Jaffe HL. The influence of the suprarenal gland on the thymus. III. Stimulation of the growth of the thymus gland following double suprarenalectomy in young rats. J Exp Med 1924; 40: 753–760.

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2. Selye H. Thymus and adrenals in the response of the organism to injuries and intoxications. Br J Exp Pathol 1936; 17:234–248. 3. Ingle DJ. Effect of two steroid compounds on thymus weight of adrenalectomized rats. Proc Soc Exp Biol Med 1940; 44:174–175. 4. Ingle DJ. Problems relating to the adrenal cortex. Endocrinology 1942; 31:419–438. 5. Wells BB, Kendall A. A qualitative difference in the effect of compounds separated from the adrenal cortex on distribution of electrolytes and on atrophy of the adrenal and thymus glands of rats. Mayo Clinic Proc 1940; 15:133–139. 6. White A, Dougherty TF. The pituitary adrenotrophic hormone control of the rate of release of serum globulins from lymphoid tissue. Endocrinology 1945; 36:207–217. 7. Selye H. The general adaptation syndrome and the diseases of adaptation. J Clin Endocrinol Metab 1946; 6:117–230. 8. Sayers G. The adrenal cortex and homeostasis. J Clin Invest 1950; 76:1755–1764. 9. Hench PS, Kendall EC, Slocumb CH, Polley HF. The effect of a hormone of the adrenal cortex (17-hydroxycorticosterone: compound E) and of pituitary adrenocorticotropic hormone on rheumatoid arthritis. Mayo Clinic Proc 1949; 24:181–197. 10. Tausk M. Hat die Nebenniere tatsa¨chlich eine Verteidigungsfunction. Das Hormon Organon Oss 1951:1–24. 11. Munck A, Brinck-Johnsen T. Specific and nonspecific physicochemical interactions of glucocorticoids and related steroids with rat thymus cells in vitro. J Biol Chem 1968; 243: 5556–5565. 12. Schaumburg BP, Bojesen E. Specificity and thermodynamic properties of the corticosteroid binding to a receptor of rat thymocytes in vitro. Biochim Biophys Acta 1968; 170:172–188. 13. Munck A, Guyre PM, Holbrook NJ. Physiological functions of glucocorticoids in stress and their relation to pharmacological actions. Endocr Rev 1984; 5:25–44. 14. Besedovsky HO, Sorkin E, Keller M, Muller J. Changes in blood hormone levels during the immune response. Proc Soc Exp Biol Med 1975; 150:466–470. 15. Besedovsky HO, Sorkin E. Network of immune-neuroendocrine interactions. Clin Exp Immunol 1977; 27:1–12. 16. Besedovsky HO, Del Rey A, Sorkin E. Antigenic competition between horse and sheep red blood cells as a hormone-dependent phenomenon. Clin Exp Immunol 1979; 37:106–113. 17. Ingle DJ. Permissibility of hormone action. A review. Acta Endocrinol 1954; 17:172–186. 18. Flower RJ, Parente L, Persico P, Salmon JA. A comparison of the acute inflammatory response in adrenalectomised and sham-operated rats. Br J Pharmacol 1986; 87:57–62. 19. Laue L, Kawai S, Brandon DD, Brightwell D, Barnes K, Knazek RA, Loriaux DL, Chrousos GP. Receptor-mediated effects of glucocorticoids on inflammation: enhancement of the inflammatory response with a glucocorticoid antagonist. J Steroid Biochem Mol Biol 1988; 29:591–598. 20. Ruzek MC, Pearce BD, Miller AH, Biron CA. Endogenous glucocorticoids protect against cytokine-mediated lethality during viral infection. J Immunol 1999; 162:3527–3533. 21. Bertini R, Bianchi M, Ghezzi P. Adrenalectomy sensitizes mice to the lethal effects of interleukin 1 and tumor necrosis factor. J Exp Med 1988; 167:1708–1712. 22. Besedovsky HO, Del Rey A, Sorkin E. Lymphokine-containing supernatants from con Astimulated cells increase corticosterone blood levels. J Immunol 1981; 126:385–387. 23. Besedovsky HO, Del Rey A, Sorkin E, Dinarello CA. Immunoregulatory feedback between interleukin-1 and glucocorticoid hormones. Science 1986; 233:652–654. 24. Sapolsky R, Rivier C, Yamamoto G, Plotsky P, Vale W. Interleukin-1 stimulates the secretion of hypothalamic corticotropin-releasing factor. Science 1987; 238:522–524. 25. Berkenbosch F, van Oers J, Del Rey A, Tilders F, Besedovsky H. Corticotropin-releasing factor-producing neurons in the rat activated by interleukin-1. Science 1987; 238:524–526. 26. Ruzek MC, Miller AH, Opal SM, Pearce BD, Biron CA. Characterization of early cytokine responses and an interleukin (IL)-6-dependent pathway of endogenous glucocorticoid induction during murine cytomegalovirus infection. J Exp Med 1997; 185:1185–1192.

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5 Cytokines and Leptin as Mediators of the Hypothalamo-Pituitary-Adrenal Axis ROLF-CHRISTIAN GAILLARD CHUV University Hospital, Lausanne, Switzerland

I. INTRODUCTION The endocrine and immune systems are closely linked via an elaborate communication system constituted by an array of cytokines and neuropeptides which interact to modulate the integrated response of an organism to infection [1,2]. This bi-directional network between the two systems allows hormones and neuropeptides to affect immune function and, in turn, cytokines to induce neuroendocrine changes [3,4]. This tight communication is made possible, because both systems ‘‘speak’’ a common language by sharing a common set of ligands and receptors of classical hormones, neuropeptides, and immunoregulatory mediators [5]. Indeed, in addition to possessing numerous hormonal receptors classically associated with endocrine tissues, the cells of the immune system produce numerous hormones and neuropeptides [6–10]. Thus, in addition to the wellknown immunosuppressive effects of adrenal glucocorticoids, many neuropeptides and hormones are involved in the modulations of the immune processes. For example, hypothalamic hormones such as corticotropin-releasing-hormone (CRH) and somatostatin as well as pituitary hormones such as growth hormone (GH) and prolactin (PRL) have been shown to modulate the immune system function. CRH, the major stressintegrating peptide, does act centrally as an immunosuppressant agents [11–13]. This central immunosuppressive effect is possibly linked to an effect of CRH on the central sympathetic nervous system, but is independent of circulating glucocorticoids since it is still active in adrenalectomized animals. CRH has also been shown to be produced peripherally at inflammatory sites by cells of the immune system. However, in contrast 83

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to its immunosuppressive effect at central level, CRH produced peripherally has proinflammatory effects. Interestingly, by blocking CRH action at inflammatory sites, administration of neutralizing antibodies against CRH or of Antalarmin, a CRH antagonist, diminishes or prevents the inflammatory process [11]. Somatostatin, another hypothalamic hormone, has also been found to be produced in the periphery, at inflammatory sites, by cells of the immune system [10]. Somatostatin exerts anti-inflammatory actions and may probably participate in the anti-inflammatory action of glucocorticoids [14]. The two pituitary hormones, GH and PRL, are also produced by cells of the immune system and have been shown to be potent immunoenhancers [15–19]. Thus, hormones produced by the immune system do locally modulate numerous immune functions and participate in the regulation of inflammation. Being produced in relatively small amounts, these hormones produce essentially autocrine and paracrine actions; exceptionally, they can also function in an endocrine fashion by acting on other cells at a distance, causing some clinical endocrine syndromes. One case of Cushing’s syndrome due to an ectopic secretion of ACTH by a granulamatous mass has been reported [20] as well as one case of acromegaly due to a non-Hodgkin’s lymphoma producing GH [21]. The reciprocal arm of this bi-directional relationship between the immune and endocrine systems is the modulation of the neuroendocrine responses by the immune system through messengers released by the activated immune cells, the cytokines. These substances, which were initially thought to be exclusively produced by the immune system, are also produced by most endocrine tissues [22] and by the brain [23]. Furthermore, as for hormone and neurotransmitter receptors on immune cells, cytokine receptors are also expressed in endocrine tissues and in the brain. All these observations demonstrate that the immune and endocrine systems communicate via a common ‘‘chemical language’’ which allows them to exert on one another biologically relevant effects. Since the discovery in 1994 of leptin, an important satiety factor, it has become evident that the adipose tissue is a true endocrine organ [24,25]. It is now quite obvious that the adipose tissue is also an important partner in the immune-endocrine network [26]. Indeed, weight loss and anorexia frequently accompany infection, probably secondary to cytokine release, but leptin, the product of the ob gene, may also play an important role. Indeed, the leptin system seems quite likely to be ancestrally related to the cytokines. Like cytokines, leptin is a peripheral messenger that conveys signals to the brain, and the leptin receptor (Ob-Rb) is closely related to the gp-130 family of receptor, a member of the class I cytokine receptor group [27]. However, signal transduction by the Ob-Rb is distinct, as it does not oligomerize with gp 130 or leukemia inhibitory factor receptor [28]. It is, therefore, not a surprise that endotoxins as well as numerous cytokines can influence leptin levels [29]. Endotoxins and cytokines induce leptin expression even in face of anorexia, suggesting that this induction of the ob gene may contribute to the decreased food intake observed during infection [30]. The endotoxin-induced surge of leptin seems to be mediated by two pro-inflammatory cytokines: TNF-␣ and IL-1␤ [31]. TNF-␣ stimulates leptin expression and secretion from adipocytes in vitro and increases circulating leptin in vivo [32]. Adipocytes from TNF-␣ knock-out mice have increased levels of leptin expression, but reduced circulating concentrations of the protein, implying that TNF-␣ actively stimulates leptin secretion [33]. The importance of IL-1␤ in mediating LPS-induced leptin secretion has been demonstrated in vivo using turpentine inflammation, which induces secretion of IL1␤ and IL-6, but not TNF-␣ or IL-1␣. Turpentine and lipopolysaccharide (LPS) induce

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leptin expression in adipose tissue and increase circulating leptin concentrations in wild-type and IL-6 knock-out mice but have no effect in IL-1␤ knockouts [34]. These data suggest that TNF-␣ is an intermediary, promoting IL-1␤, which is responsible for the stimulation of leptin. As between the endocrine and immune systems, the communication between the immune and adipose systems is also bi-directional. Indeed, cytokines modulate leptin production, and the adipocytes through leptin secretion are involved in the regulation of immune responses. Thus, leptin regulates pro-inflammatory immune responses by upregulating both phagocytosis and the production of proinflammatory cytokines [35]. The importance of leptin for the immune function is also illustrated by the impaired T-cell immunity observed in ob/ob and db/db mice [36,37]. In both genotypes there is a diminished in vivo response to cell-mediated and humoral challenges. In addition, Faggioni et al. demonstrated that leptin deficiency in ob/ob mice is accompanied by an increased susceptibility to endotoxin-induced lethality and by a decreased induction of anti-inflammatory cytokines [38]. All these observations concur with an important role of leptin in the cytokine cascade activated during infection and injury. They suggest that leptin may provide a link between immunosuppression and malnutrition and that disorders with decreased leptin levels, such as cachexia, malnutrition, and starvation, might present impaired host defenses. These speculations have been confirmed in recent animal and human studies. Leptin reversed starvationinduced suppression of cellular immune responses [39] and starvation-induced thymic involution [40]. In addition, in ob/ob mice leptin administration reduced thymocyte apoptosis and increased both thymic cellularity and the CD4ⳭCD8Ⳮ/CD4ⳮCD8ⳮ ratio [40]. In humans, nutritional deprivation does also affect immune function [41], and a recent study has confirmed the protective effect of leptin by showing a clear association between the increase in leptin and the immunological recovery observed after refeeding of malnourished infants [42]. Such a protective effect is also suggested by the observation that the survivors in patients with acute sepsis have significantly higher average leptin levels that those who do not survive [43]. A bi-directional communication also connects the hypothalamo-pituitary-adrenal (HPA) axis and the adipose tissue system, since the expression of leptin is modulated by glucocorticoids and ACTH, while in turn leptin inhibits the activation of the HPA axis [44–47]. Thus, the adipose tissue, the immune system, and the HPA axis constitute an important network through which the three systems are linked in a classical endocrine feedback loop, and each system is able to modulate the function of the other two (Fig. 1). In this chapter we shall focus our review on the effects of cytokines and leptin—respectively messengers of the immune system and of the adipose tissue—discussing their role, as mediators of the hypothalamo-pituitary-adrenal axis. II. CYTOKINES AS MEDIATORS OF THE HPA AXIS Cytokines modulate the HPA axis by acting at all three levels—the hypothalamus, the pituitary gland and the adrenal glands—inducing the production of, respectively, CRH, ACTH, and glucocorticoids. A. Hypothalamic Level Intravenous and intraperitoneal administration of cytokines cause a prompt rise (within minutes) in plasma ACTH [48,49]. This acute effect of cytokines is mainly exerted

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Figure 1 Bi-directional communication between the immune system, the hypothalamo-pituitary-adrenal axis, and the adipose tissue through cytokines, ACTH, glucocorticoids, and leptin.

at the hypothalamic level by inducing release of the corticotropin-releasing hormone (CRH) [48–51] into the hypophysial portal vessels. Indeed, cytokines have been shown in vitro to induce a rapid (within 10–20 min) CRH release directly from incubated hypothalamic fragments [51,52]. The paraventricular nucleus (PVN) is an important hypothalamic site for the action of the cytokines. Indeed, lesioning the rat PVN by electrolytic obliteration markedly reduces the rise in plasma ACTH levels produced by a number of cytokines in vivo [53,54]. IL-1 and IL-6 not only stimulate CRH secretion, but also its biosynthesis, as demonstrated by increases in CRH mRNA levels [55–57]. Further evidence for a direct hypothalamic effect is the capability of the hypothalamus to synthesize numerous cytokines, such as IL-1, IL-6, and TNF-␣ [58–61]. The mechanisms by which cytokines initiate the release of CRH involve catecholamines, prostaglandins, and NO [62–65]. NO restrains the response of the HPA axis to i.v. administration of cytokines, whereas L-NAME, by blocking NO formation, increases the ACTH released by circulating IL-1␤. According to Rivier and Shen NO could either influence the synthesis of various neurosecretagogues such as prostaglandins and catecholamines or modulate the effects of CRH/AVP on the corticotrophs [65]. The rapid effects of cytokines on hypothalamic CRH secretion in vivo as well as in vitro strongly favor the brain as the primary site of cytokine action in the acute stimulation of the HPA axis. However, being water-soluble proteins of relatively large molecular weight, cytokines are not expected to cross the blood-brain barrier (BBB). The BBB consists primarily of unfenestrated endothelial cells that are connected by tight junctions and thus form a continuous cell layer that has the permeability properties of a continuous plasma membrane [66]. Many mechanisms have been proposed by which peripheral cytokines may gain access to the brain: 1. Loss of BBB Integrity Loss of BBB integrity may occur during inflammatory insults to the brain. Such disruption can increase BBB permeability and not only enables the passage of large peptides such as cytokines, but also augments the rate of entry of cells, such as macrophages, monocytes, lymphocytes, and neutrophils, which are capable of cytokine synthesis and secretion, but whose passage into the normal healthy brain is very limited.

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However, it is clear that the initial neuroendocrine effects of peripherally administered cytokines or LPS can be observed more quickly and at lower doses than can be accounted for by damage to the BBB. 2. Saturable Transport System It has been suggested that cytokines cross the BBB by a saturable transport system [67]. Such a transport mechanism has been described for IL-1␣, IL-1␤, IL-1 Ra, IL6, and TNF-␣ [68]. This mechanism affords a means for cytokine entry into the brain, even when BBB integrity is not compromised. It should, however, be stressed that the passage of cytokines across the BBB is still a matter of controversy [69,70]. Many authors have questioned whether the small amount of cytokines entering the brain via a saturable transport mechanism is physiologically relevant and can exert rapid effects, such as those observed after peripheral injection of cytokines. Nevertheless, it seems plausible that such a mechanism may play a significant role when peripheral blood levels of cytokines are elevated for longer periods of time, for example, during chronic inflammation. 3. Circumventricular Organs The BBB is absent or defective in small areas of the brain, the so-called circumventricular organs, which are located at various sites within the walls of the cerebral ventricles. These include the median eminence, the organum vasculosum of the laminae terminalis (OVLT), the subfornical organ, the choroid plexus, the neural lobe of the pituitary, and the area postrema. Being an area of the hypothalamus that is richly supplied by vasculature devoid of a functional BBB, the median eminence is a potential target for activation of the HPA by cytokines [71,72]. Circulating cytokines can enhance CRH secretion by interacting with the CRH nerve terminals and stimulating CRH release without directly stimulating CRH cell bodies in the PVN [73,74]. There is, however, some controversy [75]. Another possible site of cytokine action is the OVLT [76]. Instead of being a portal of entry of cytokines into the CNS, the OVLT could be a kind of interface where the chemical messages of bloodborne cytokines are transformed into neuronal signals, so that secondary messengers might be evoked that transmit original signals to the preoptic area [77]. The second mediator released in the OVLT and acting on surrounding neurons with efferent projections to the PVN could be a cytokine [78] or prostaglandins [79], but it could also be other neuroregulators or neurotransmitters such as serotonin or norepinephrine [80,81]. 4. Endothelial Cells of the Vasculature Circulating IL-1 may also interact with IL-1 receptors on endothelial cells of the vasculature and thereby stimulate secondary molecules such as IL-1, nitric oxide, and/ or prostaglandins, which can act locally to influence neurons [82–85]. 5. Activation of Vagal Afferent Fibers Recent studies have suggested that the neuronal pathway may convey these peripheral messages to the brain and may represent another route through which cytokines may influence the CNS without gaining access to brain parenchyma, the BBB interface, or even the circulation. Indeed, the vagus nerve has been shown to be implicated in mediating the LPS- and cytokine-induced corticosterone secretion following systemic administration [86–92]. The vagus nerve appears to be important in signalling the

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brain specifically during intra-abdominal/peritoneal infection. This is evidenced by the fact that surgical subdiaphragmatic transection of the vagus (SDVX) attenuates the rise in plasma ACTH and corticosterone concentrations produced by intraperitoneal IL-1␤ [89–93]. However, when either LPS or IL-1 is administered via routes other than into the abdomen/peritoneum, SDVX has no effect on the acute activation of the HPA axis [94,95]. 6. Cytokine Synthesis Within the Brain Cytokines are also generated within the brain, thus raising the possibility that such brain-derived cytokines may influence HPA axis activity [96–98]. B. Pituitary Gland Level Since the first observation that IL-1 induced ACTH release from the corticotroph tumor cell line AtT 20 [99], many studies have provided evidence for a direct effect of cytokines on the pituitary [1,21,100,101]. The majority of the observations demonstrate that in contrast to their rapid effect observed at hypothalamic level, cytokines effects at pituitary level are much slower. Indeed, cytokines stimulate ACTH release from pituitary cells only after a prolonged incubation time of over 8–10 hours. However, several studies were unable to show any direct effect of cytokines on ACTH release [52,102]. The reason for this discrepancy is not clear, but it is possible that the presence of folliculo-stellate cells may be mandatory. Indeed, it has been demonstrated that these cells are essential to allow pituitary effects of the cytokines [103]. This may concord with the demonstration of gap junction-mediated exchanges between endocrine and folliculo-stellate cells [104]. The folliculo-stellate cells therefore appear to constitute a kind of interface through which the pituitary gland perceives changes in the state of activation of the immune system. The presence of pituitary binding sites for cytokines as well as the intrapituitary production of cytokines is consistent with their direct pituitary effect [96]. Indeed, a number of cytokine receptors or their corresponding mRNAs have been localized in the pituitary (e.g., IL-1, IL-2, IL-6, TNF-␣, LIF). In addition, the pituitary has been shown to produce many cytokines, some of which have been localized to corticotrophs (e.g., IL-2, IL-10, LIF, and MIF), to thyrotrophs (e.g., IL-1, MIF), and to folliculostellate cells (IL-6) [96]. We shall discuss in some details the macrophage migration inhibitory factor (MIF) and the leukemia inhibitory factor (LIF), since both have recently been added to the list of cytokines produced within the pituitary. Macrophage migration inhibitory factor, a protein produced by lymphocytes and discovered more than 30 years ago, has recently been shown to be a pituitary-derived cytokine [105] and has even been proposed to serve as a pituitary hormone [105–107]. Indeed, MIF has been identified as a protein released by the corticotroph tumor cell line AtT 20 as well as by anterior pituitary cells in response to LPS stimulation. Immunocytochemical studies show that resting pituitary cells contain a significant amount of preformed MIF. MIF secretory granules are located within corticotroph and thyreotroph cells of mice pituitary glands [108]. The pituitary intracellular pools of MIF are released by the direct action of LPS or by a specific hypothalamic-releasing factor secreted during endotoxemia. Our recent finding that CRH upregulates transcriptional activity of the MIF promoter in both AtT20 and isolated anterior pituitary cells corroborates that MIF synthesis takes place at the pituitary level and that the hypothalamic factor CRH regulates pituitary MIF synthesis [109].

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Studies on the role of MIF during endotoxin shock revealed an important role of this substance since it potentiates LPS lethality, while administration of anti-MIF antibodies confers full protection against lethal endotoxemia [105,110]. Interestingly and unexpected, glucocorticoids were found to stimulate rather than inhibit MIF production [111]. Therefore, MIF possesses a unique property among cytokines in that its release is induced by glucocorticoids. In addition, MIF has been shown to counterregulate or override the suppressive effects of glucocorticoids on the production of inflammatory cytokines, as well as to block the protective effect of the glucocorticoids against LPSinduced lethality. Altogether, these results indicate that MIF plays a pivotal role in the immuneneuroendocrine interactions [106]. Together with ACTH and the glucocorticoids, MIF may modulate the systemic inflammatory responses. MIF is unique in being released under glucocorticoid stimulation and in antagonizing certain effects of glucocorticoids. The observation that endotoxemia leads to the production of both anti-inflammatory (glucocorticoids) and pro-inflammatory (MIF) mediators presumes that the organism possesses potent counterregulatory mechanisms, allowing a fine-tuning of the immune and endocrine responses to severe stressors. However, a very recent study suggests that in humans the regulation of circulating MIF is different from that in rodents because stimuli of the HPA axis, such as CRH of hypoglycemia, were ineffective in stimulating circulating levels [112]. The authors propose that if pituitary MIF is released into the circulation and contributes significantly to circulating levels, it may do so only in response to severe stressors such as endotoxemia, inflammation, or tissue invasion. In other clinical situations, pituitary-derived MIF has more likely an autocrine or paracrine role [112]. Leukemia inhibitory factor, a protein originally isolated as a factor inducing differentiation and suppressing proliferation of a monocytic leukemia murine cell line M1 [113], has also been found in the pituitary gland. This cytokine is secreted by bovine pituitary cells in culture [114] as well as by human pituitaries. Indeed, its gene expression has been demonstrated in the developing human fetal pituitary (predominantly in corticotroph and somatotroph cells) and in normal as well as in adenomatous adult pituitary tissue [115,116]. LIF mRNA was also detected in mouse and rat adenohypophysis [117] as well as in mouse hypothalamus [118]. Specific LIF receptors are present in murine AtT20 pituicytes, in human fetal pituitary cells (in corticotrophs and somatotrophs) and in other functional hormoneproducing cells [115]. These binding sites consist of heterodimers between the specific low-affinity LIF receptor and the shared affinity converter gp130 common to IL-6, LIF, oncostatin, and ciliary nerve neurotrophic factor [119]. In mice, pituitary and hypothalamic LIF receptors have been shown to be significantly induced by LPS in vivo [118]. LIF action occurs principally at the level of the pituitary corticotrophs, where it stimulates the secretion of ACTH and the expression of POMC [115,120–122]. It shows strong transcriptional synergy with CRH on POMC mRNA expression mediated by a common binding element in the POMC promoter region [123] and potentiates the CRH-induced ACTH secretion [122]. Most interestingly, transgenic mice expressing pituitary-directed LIF were found to have an increased number of ACTH-positive pituitary cells, resulting in pituitary corticotroph hyperplasia [124]. Recent studies clearly demonstrate that LIF contributes to the regulation of HPA axis secretion under basal as well as under stress conditions. Indeed, LIF knock-out

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mice not only have basal plasma concentrations of ACTH lower than those seen in wild-type littermates, but present a defect in the activation of the HPA axis in response to stress, to IL-1, and to inflammation or infection [125–127]. These data implicate LIF in the HPA response during inflammatory stress and suggest that the absence of this cytokine may lead to impaired physiological communication between the immune and endocrine systems. In summary, LIF is an immuno-neuroendocrine modulator which has an important function in the maintenance and regulation of the HPA axis. C. Adrenal Gland Level Several cytokines are expressed in the adrenal glands, and some studies demonstrate the presence of cytokine receptors within the adrenal gland. IL-6 receptor mRNA has been detected mainly in the zona glomerulosa and fasciculata in human adrenals [128] and in the adrenal medulla [129]. IL-1, IL-6, and TNF-␣ have direct actions on glucocorticoids secretion [130,131]. However, these effects are only observed after prolonged incubation time [132]. Thus, as with the direct effects of cytokines on the pituitary, it seems unlikely that a direct action of the cytokines on the adrenal gland can account for the rapid in vivo effects of cytokines on plasma glucocorticoid levels, except in circumstances involving prolonged increases in cytokines, such as in chronic inflammation. III. INTERACTIONS BETWEEN THE IMMUNE SYSTEM AND THE HPA AXIS What is the purpose of such an elaborate control system? Why during an inflammatory process does the immune system promote the production of cytokines, which in turn activate the HPA axis leading to the production of glucocorticoids? As suggested by Munck et al. [133], the inflammation-induced activation of the HPA axis may represent a potent negative feedback mechanism through which the immune system, by stimulating the HPA axis and therefore the production of the immunosuppressive glucocorticoids, avoids an overshoot of the inflammatory and febrile effect during the acute-phase response (Fig. 2). The production of glucocorticoids allows the body to have tight control of the local immune response, inhibiting this defense mechanism from endangering the body’s integrity. Because virtually all the components of the immune response are inhibited by the glucocorticoids, the consequence of the activation of the HPA axis will be a reduction or modulation of inflammatory responses to toxins or invading organisms. Therefore, any dysfunction or disruption of this network preventing increased glucocorticoid secretion might result in an inflammatory disease. This has indeed been illustrated by studies on Lewis rats, a strain of rats that are, because of a genetic defect in the synthesis of CRH, unable to respond to inflammation with an increased secretion of glucocorticoids. The susceptibility of these rats to the development of arthritis is clearly associated with the inability of their HPA axis to respond adequately to inflammatory stimuli [134,135]. Acute arthritis develops when these rats are injected with a suspension of streptococcal cell wall polysaccharides, whereas it does not develop in Fisher rats, in which the response of the HPA axis is normal (Fig. 3). The important involvement of glucocorticoids in this process is confirmed by the observation that the administration of glucocorticoids to Lewis rats suppresses the inflammation, whereas suppression of adrenal function in Fisher rats by adrenalectomy or by the

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Figure 2 Interactions between the immune system and the hypothalamo-pituitary-adrenal (HPA) axis during an inflammatory reaction (IR). The activation of the immune system by aggression induces the production of cytokines that stimulate the HPA axis, provoking an elevation of the immunosuppressive glucocorticoids (GLC). (From Ref. 191.)

glucocorticoid receptor antagonist RU 486 produces an enhanced susceptibility to inflammatory processes similar to that of susceptible Lewis rats [135]. Recent studies have shown that these abnormalities of the Lewis rats have parallels in humans. Patients with rheumatoid arthritis present a defective hypothalamic response to immune inflammatory stimuli and indeed have an inadequate cortisol production [136]. IV. LEPTIN AS MEDIATOR OF THE HPA AXIS Leptin, a 167-amino-acid peptide, the product of the obesity gene [24], is a cytokinelike circulating protein acting principally as a peripheral satiety signal to the hypothalamus. Initially described as a product of white adipose cells, leptin is also expressed in endocrine and neuroendocrine tissues like the ovary [137], the placenta [138], and the hypothalamus [139] as well as the anterior pituitary gland [139–145]. The leptin receptor has multiple isoforms, but only the subtype with a long intracellular domain (Ob-Rb) is currently considered to be capable of signal transduction [146]. Although leptin is certainly a potent satiety signal afferent to the hypothalamus, it is now quite clear that human obesity generally does not result from leptin deficiency [147] except in very rare cases of human leptin deficiency [148,149] or leptin receptor deficiency [150] in patients bearing inactivating mutations in the leptin gene and its receptor. Very early on, neuropeptide Y (NPY), an abundant hypothalamic neurotransmitter involved in numerous metabolic as well as neuroendocrine regulations [151–155] was

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Figure 3 Illustration of the concept that the susceptibility of Lewis rats to streptococcal cell well (SCW)–induced arthritis is related to defective HPA axis responsiveness to inflammatory stress and that resistance of Fischer rats is due to an intact HPA axis–immune system feedback loop leading to an increase in the immunosuppressive glucocorticoids. (From Ref. 191.)

identified as one of the hypothalamic targets of leptin. NPY is a potent stimulator of food intake [156,157], and it also participates in the central adaptations of corticotrope, thyreotrope, gonadotrope, and somatotrope axes to poor metabolic conditions [46,158,159]. Therefore, in addition to its modulation of food intake and energy expenditure, leptin participates to the modulation of the activity of the pituitary endocrine axes [46]. All these endocrine effects of leptin have progressively been shown to be important functions of this molecule, demonstrating that leptin is much more than a satiety signal [160]. In this part of the chapter, we shall review the effects of leptin on the HPA axis, demonstrating that leptin contributes to the fine-tuning of the corticotrope axis. Recently, it has indeed become apparent that the adipose tissue, by way of leptin secretion, is an important partner in the crosstalk modulating the stress response [161,162]. Indeed, glucocorticoids [44,163] and ACTH [46,164] regulate leptin biosynthesis and secretion, and in turn, leptin exerts a down-regulation upon adrenal gland activity [165–168]. The first clue suggesting that leptin might be involved in the regulation of the stress axis came with the demonstration that it could partially counteract the regulation of the HPA axis induced by an acute fast [46]. This initial observation was entirely consistent with older work performed in the genetically obese and leptin-deficient ob/ ob mouse. Indeed, these mice exhibit increased basal production of glucocorticoids [169] as well as an exaggerated glucocorticoid secretion in response to an ether stress

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[170]. Therefore, these data concur to suggest that leptin has a predominantly inhibitory action upon the HPA axis. Such an inhibition has now been confirmed by several studies. Thus, Heiman et al. were able to demonstrate that leptin could block corticosterone secretion in response to an immobilization stress [171]. Furthermore, these authors generated in vitro data demonstrating a direct inhibition of hypothalamic CRH secretion by leptin, suggesting that at least part of the effects of leptin observed in vivo are mediated via central mechanisms [171]. We have recently made a similar observation in vivo, consistent with an inhibitory action of leptin occurring at central level [172]. In fasted mice, we found that the insulin-induced stimulation of corticosterone secretion could be prevented by re-feeding the animals immediately prior to insulin injection. This effect could not be ascribed only to the achievement of a blunted hypoglycemia in refed mice, but was mainly dependent upon the rising endogenous leptin levels induced by refeeding [172]. However, there is some controversy in the literature, since other data diverge markedly from these results. Indeed, a stimulation of corticosterone secretion by leptin infused directly in the cerebrospinal fluid has also been reported in the rat [173]. Such a stimulatory effect of centrally administered leptin would be consistent with the leptin-induced increase in CRH and AVP observed by some authors [173,174]. There is at present no satisfactory explanation to reconcile these conflicting results. A. Leptin Effect at the Adrenal Gland Level In contrast, there is now a general agreement with a direct inhibitory effect of leptin on adrenal glucocorticoid production. Leptin has been found to downregulate ACTHstimulated glucocorticoid biosynthesis and secretion in bovine [166], rat [165], and human [165,175] adrenocortical cells in vitro. Such an inhibition has also recently been reported in vivo after chronic leptin administration [176]. In addition, these in vivo data [176] also suggested that a leptin-mediated downregulation of pituitary ACTH could also participate in this effect. The cellular and molecular mechanisms underlying the adrenal effects of leptin are partially elucidated. In the bovine adrenal gland, leptin induces inhibition of the expression of the steroidogenic enzymes cytochrome P450 C21-hydroxylase (P450C21), side-chain cleavage (P450SCC), and C17␣-hydroxylase (P45017␣) [166,167]). In the rat we were able to demonstrate that leptin significantly inhibits the adrenal expression of steroidogenic acute regulatory protein (StAR) [177]. This effect is apparent at both the mRNA and protein levels. StAR is a key element in the rate-limiting step of steroid hormone biosynthesis, it regulates cholesterol delivery to the P450SCC enzyme located in the inner mitochondrial membrane [178,179]. Thus, leptin inhibits glucocorticoid production by affecting various steps in the adrenal biosynthesis. More recently, a leptin-induced downregulation of the expression of the leptin receptor in the adrenal gland was demonstrated [163]. In this study, the researchers [163] confirmed that leptin inhibits corticosterone secretion from rat adrenal slices. In addition, they were able to demonstrate that leptin can desensitize the adrenal gland to is own effects by decreasing the expression of the leptin receptor Ob-R. Interestingly, in parallel with these effects of leptin, ACTH itself was found to reduce the adrenal expression of all isoforms of Ob-R, thus providing a limitation to the inhibition of its own effect induced by leptin [168].

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Overall, the effect of leptin on the adrenal gland could be relevant to human pathophysiology. Indeed, the sometimes markedly elevated leptin levels observed in human obesity [147] could induce significant alterations in the adrenal responsiveness to ACTH stimulation. It could therefore lead to abnormal feedback of glucocorticoids on the hypothalamo-pituitary unit, eventually resulting in a disruption of the circadian rhythm. Such an abnormal regulation of the HPA axis is indeed seen in obesity. This adrenal effect of leptin could also be important in critically ill septic patients. In these patients, elevated leptin levels and, incidentally, a loss of diurnal rhythm of cortisol have been associated with a better clinical outcome [43]. It could therefore be hypothesized that these elevated leptin levels act to decrease the stress response, thus allowing a more efficient immune response to take place. B. Leptin at the Pituitary Level Leptin as well as its active receptor isoform are expressed by most anterior pituitary cell types, both in animals [139–141] and in human tissues [141,142,144,145]. The corticotroph cells in particular exhibit strong leptin receptor expression [145]. This observation probably indicates that leptin may well play a role in modulating directly pituitary function, in addition to its effects on the hypothalamus and the adrenal glands. However, this evidence remains circumstantial. To our knowledge there are at present no published data demonstrating a direct effect of leptin to alter pituitary ACTH secretion. In our laboratory we did not observe any effect of leptin upon ACTH secretion from cultured rat anterior pituitary cells. Taken together, all these data demonstrate the existence of a ‘‘classical’’ endocrine loop between the HPA axis and adipose tissue: glucocorticoids can stimulate leptin expression and secretion from the adipocytes, whereas rising circulating leptin levels can modulate the stress response at all levels of the HPA axis [180]. In this bi-directional relationship between the HPA axis and adipose tissue, ACTH seems to play a role, since it has been shown to modulate leptin levels in vivo [46] and to directly inhibit leptin gene expression as well as leptin secretion by cultured primary adipocytes [164]. V. TOLERANCE TO ENDOTOXINS The occurrence of a tolerance phenomenon to bacterial endotoxin represents a hallmark of chronic endotoxin stimulation. Tolerance can be described as the hyporesponsiveness of an organism to a repetitive stimulus, such as repeated administration of LPS [181,182]. The mechanism underlying endotoxin tolerance is not well understood, but it seems that glucocorticoid-dependent and -independent mechanisms are involved [183]. The glucocorticoid-independent mechanism(s) may include blockade of the LPS receptors by minimal, inactive LPS structures [184], blockade of LPS itself by a lipoprotein [185], or blockade of LPS by neutralizing antibodies induced as a consequence of repeated stimulation [186]. It is also possible that the effect of cytokines released from LPS-activated macrophages could be blocked by soluble receptors [187] or receptor antagonists [188]. On the other hand, endogenous glucocorticoids stimulated by the endotoxin-induced HPA activation could also be involved [183,189]. In a recent study [190], we demonstrated that repeated LPS administration induces a tolerance of the corticotroph axis, the immune and the adipose tissue responses to endotoxin. Indeed the ACTH, corticosterone, TNF␣, and leptin responses to endotoxin were completely blocked after repeated LPS administration (Fig. 4).

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Figure 4 Daily injection of LPS (25 ␮g/kg) for 5 days induces a tolerance of ACTH-corticosterone, TNF-␣, and leptin responses to endotoxin. Indeed, the response to LPS on day 1 (one LPS injection) is significantly more pronounced than the responses after days 3 and 5. (Adapted from Ref. 190.)

In addition, using adrenalectomized animals implanted with a subcutaneous corticosterone pellet, we demonstrated that a normal glucocorticoid response to endotoxemia is necessary for the development of full tolerance of the HPA axis, but not for tolerance of the immune and adipose tissue responses (Fig. 5). Indeed, tolerance of the corticotroph response to repeated LPS administration in adrenalectomized rats, substituted with corticosterone, was only partial and delayed, suggesting that an inappropriate adrenal response may delay tolerance. In contrast, tolerance of the immune system in releasing TNF-␣ remained normal in this animal model, suggesting that, unlike the corticotroph axis, tolerance of the immune response does not depend upon stimulated corticosterone levels. Finally, if glucocorticoids do modulate the adipose tissue response during repeated endotoxemia, they do not play a crucial role in the occurrence of the tolerance phenomenon at the adipose tissue level.

VI. CONCLUSION The HPA axis, the immune system, and adipose tissue are closely interrelated via tight bi-directional communication, in which cytokines, leptin, and glucocorticoids play a crucial role. The crosstalk between these three systems may be of prime importance to homeostasis in pathophysiological events occurring during infection.

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Figure 5 Tolerance phenomenon to repeated LPS administration (see Fig. 4) in sham (left panel) and in adrenalectomized rats implanted with s.c. corticosterone pellet (right panel). The tolerance of the corticotroph (ACTH) response was significantly reduced, whereas that of the immune system (TNF-␣) was not affected by the absence of an appropriate adrenal response. (From Ref. 190.)

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157. Clark JT, Kalra PS, Crowley WR, Kalra SP. Neuropeptide Y and human pancreatic polypeptide stimulate feeding behavior in rats. Endocrinology 1984; 115:427–429. 158. Gruaz NM, Pierroz DD, Rohner-Jeanrenaud F, Sizonenko PC, Aubert ML. Evidence that neuropeptide Y could represent a neuroendocrine inhibitor of sexual maturation in unfavourable metabolic conditions in the rat. Endocrinology 1993; 133:1891–1894. 159. Catzeflis C, Pierroz DD, Rohner-Jeanrenaud F, Rivier JE, Sizonenko PC, Aubert ML. Neuropeptide Y administered chronically into the lateral ventricle profoundly inhibits both the gonadotropic and the somatotropic axis in intact adult female rats. Endocrinology 1993; 132: 224–234. 160. Harris RBS. Leptin, much more than a satiety signal. Annu Rev Nutr 2000; 20:45–75. 161. Bornstein SR. Is leptin a stress related peptide?. Nat Med 1997; 3:937. 162. Licino J, Mantzoros C, Negrao AB, Cizza G, Wong ML, Bongiorno PB, Chrousos GP, Karp B, Allen C, Flier JS, Gold PW. Human leptin levels are pulsatile and inversely related to pituitary-adrenal function. Nat Med 1997; 3:575–579. 163. Slieker LJ, Sloop KW, Surface PL, Kriauciunas A, LaQuier F, Manetta J, Bue-Valleskey J, Stephens TW. Regulation of expression of ObmRNA and protein by glucocorticoids and camp. J Biol Chem 1996; 271:5301–5304. 164. Renz M, Tomlinson E, Hultgren B, Levin N, Gu Q, Shimkets RA, Levin DA, Stewart TA. Quantitative expression analysis of genes regulated by both obesity and leptin reveals a regulatory loop between leptin and pituitary-derived ACTH. J Biol Chem 2000; 275: 10429–10436. 165. Pralong FP, Roduit R, Waeber G, Castillo E, Mosimann F, Thorens B, Gaillard RC. Leptin inhibits directly glucocorticoid secretion by normal human and rat adrenal gland. Endocrinology 1998; 139:4264–4268. 166. Bornstein RS, Uhlmann K, Haidan A, Ehrhart-Bornstein M, Scherbaum WA. Evidence for a novel peripheral action of leptin as a metabolic signal to the adrenal gland. Leptin inhibits cortisol release directly. Diabetes 1997; 46:1235–1238. 167. Kruse M, Bornstein SR, Uhlmann K, Paeth G, Scherbaum WA. Leptin down-regulates the steroid producing system in the adrenal. Endocr Res 1998; 24:587–590. 168. Tena-Sempere M, Pinilla L, Gonzalez LC, Casanueva F, Dieguez C, Aguilar E. Homologous and heterologous down regulation of leptin receptor messenger ribonucleic acid in rat adrenal gland. J Endocrinol 2000; 167:479–486. 169. Garthwaite TL, Martinson DR, Tseng LF, Hagen TC, Menahan LA. A longitudinal hormonal profile of the genetically obese mouse. Endocrinology 1980; 107:671–676. 170. Edwardson JA, Hough CA. The pituitary-adrenal system of the genetically obese (ob/ob) mouse. J Endocrinol 1975; 65:99–107. 171. Heiman ML, Ahima RS, Craft LS, Schoner B, Stephens TW, Flier JS. Leptin inhibition of the hypothalamic-pituitary-adrenal axis in response to stress. Endocrinology 1997; 138: 3859–3863. 172. Giovambattista A, Chisari AN, Gaillard RC, Spinedi E. Food intake-Induced leptin secretion modultes hypothalamo-pituitary-adrenal axis response and hypothalamic Ob-Rb expression of insulin administration. Neuroendocrinology 2000; 72:341–349. 173. Morimoto I, Yamamoto S, Kai K, Fujihira T, Morita E, Eto S. Centrally administered murineleptin stimulates the hypothalamus-pituitary-adrenal axis through arginine-vasopressin. Neuroendocrinology 2000; 71:366–374. 174. Costa A, Poma A, Martignoni E, Nappi G, Ur E, Grossman A. Stimulation of corticotrophinreleasing hormone release by the obese (ob) gene product, leptin, from hypothalamic explants. Neuroreport 1997; 8:1131–1134. 175. Pralong FP, Gomez F, Guillou L, Mosimann F, Franscella S, Gaillard RC. Food-dependent Cushing’s syndrome: possible involvement of leptin in cortisol hypersecretion. J Clin Endocrinol Metab 1999; 84:3817–3822.

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6 Role of Pro-Inflammatory Cytokines in Regulating the Hypothalamic-PituitaryGonadal Axis of the Male Rat CATHERINE RIVIER The Salk Institute, La Jolla, California, U.S.A.

I. INTRODUCTION The presence of infectious and inflammatory diseases, critical illness, and a variety of trauma often leads to low sex steroid levels and decreased reproductive activity [1–7]. Numerous mechanisms have been invoked to explain this phenomenon, and an overview of presently available information suggests that two types of neurosecretagogues play a critical role: corticotropin-releasing factor (CRF), a peptide released in the brain during stress that inhibits luteinizing hormone (LH)–releasing hormone (LHRH) and LH synthesis and/or release [8], and pro-inflammatory cytokines, which can inhibit both the synthesis/ secretion of hypothalamic LHRH [9,10] and testicular steroidogenesis [11]. Pro-inflammatory cytokines such as tumor necrosis factor-␣ (TNF-␣), interleukin-1 (IL-1) and IL-6 are proteins that were originally thought to be released only by activated immune cells during infection or inflammation. We now know that these proteins may also be present under basal, nonpathological conditions in the brain [12] and can be synthesized in (and released from?) nonimmune cells such as glial and neural cells [13–17]. In addition, there is convincing evidence that TNF-␣, IL-1␤, and IL-6 secretion is stimulated by nonimmune (neurogenic) stressors such as footshocks, restraint and, in humans, mental stress and strenuous exercise [18–23]. Not surprisingly, the array of circumstances during which proinflammatory cytokines are now thought to modulate reproductive functions has increased accordingly [2]. This chapter is not meant as an inclusive overview of all of the effects of CRF and cytokines on the hypothalamic-pituitary-gonadal (HPG) axis, nor does it 107

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describe all of the secretagogues that are released by these peptide/proteins and therefore potentially participate in their influence. The role of CRF on LHRH/LH release has already been addressed in detail [9], and the conclusions discussed in this review remain valid. Rather, we will first focus on what we consider the most salient facts regarding the sites at which pro-inflammatory cytokines modify the release of LHRH, LH, and testosterone (T) in males and some of the mechanisms involved in this influence. Second, we will discuss recent findings, mostly obtained in our laboratory, that point to a neural pathway through which the brain exerts a very rapid inhibitory influence on Leydig cell activity that is independent of the pituitary, but is activated by CRF and specific cytokines. The literature that we cite should be regarded as illustrative rather than comprehensive.

II. INFLUENCE OF CYTOKINES ON THE HYPOTHALAMICPITUITARY-GONADAL AXIS A. Hypothalamus and Pituitary Endotoxemia, induced experimentally by the systemic injection of lipopolysaccharide (LPS), is accompanied by the release into the circulation of pro-inflammatory cytokines [13,24,25]. It is often used to mimic the acute-phase response, a primary host response to infectious and inflammatory stimuli [26], because it stimulates many of the immune, endocrine, and behavioral responses that are part of this phenomenon. The intravenous (iv) injection of LPS is a potent inhibitor of LH release in rodents, sheep, and primates [27–30]. Because peripheral LPS administration influences brain functions in areas protected by the blood-brain barrier (BBB), it is difficult to use this approach in order to distinguish between the peripheral circuits (described below) and the brain areas beyond the BBB within which immune signals act to inhibit HPG axis activity. Consequently, many investigators have used the injection of single cytokines to investigate the site of action of immune stimulation in influencing LHRH and LH secretion. The finding that, in the rat, even large doses of single cytokines such as TNF-␣ or IL-1␤, injected iv, do not alter plasma LH level [9], suggested that endotoxemia modifies the activity of the rat LHRH-LH axis primarily by augmenting brain levels of these cytokines. (It should, however, be mentioned that a recent report suggested that TNF-␣ and IL-6 were able to block LHRH-induced LH release by isolated pituitary cells [31].) Consequently, the overall consensus is that the intracerebroventricular (icv) administration of these cytokines represents the most valid model to study the influence of immune signals on the hypothalamic-gonadotrop axis. Several laboratories have reported that the icv injection of single pro-inflammatory cytokines leads to a significant decrease in plasma LH levels in castrated or intact rats (Fig. 1) and that this phenomenon is primarily due to the inhibition of neurons that produce LHRH [28,32–35] (Fig. 2). The concept that icv-injected pro-inflammatory cytokines primarily act within the brain was further supported by the finding that they inhibit LHRH secretion by isolated hypothalamic fragments [36,37]. In the intact animal, decreased LHRH release caused by the icv injection of single pro-inflammatory cytokines lowers LH levels through pathways that depend, at least in part, on opiates and prostaglandins [38–41]. What do these results tell us about the mechanisms through which systemically administered LPS inhibits the HPG axis? Differences in the strain and gender of the rats used, the length of exposure to cytokines, and the different experimental paradigms make it difficult to satisfactorily determine whether the well-documented decrease in LHRH gene expression of rats injected with LPS in the periphery [28] represents the

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Figure 1 The icv injection of recombinant human (rh) IL-1␤ (80 ng) decreases plasma LH levels in both intact and castrated male rats. Each point represents the mean Ⳳ SEM of 5–7 male rats. **p ⬍ 0.01 vs. vehicle.

dominant mechanism, or whether a direct influence of LPS on pituitary gonadotrophs might also play an important role. The situation is different in other species, particularly sheep and primates, in which single cytokines appear able to act on the pituitary gonadotrophs following their systemic injection [42] Another species difference is that while CRF does not mediate the influence of icv-injected pro-inflammatory cytokines or LPS on the HPG axis of the rat [9], this peptide is important in the primate model [43]. Additionally, LPS-induced decreases in pituitary responsiveness to LHRH play a role in sheep, though changes in LHRH release are also present [29,44,45]. Finally prostaglandins, which, as mentioned above, mediate IL-1–induced decreases in LH levels in rats [34], also play a role in the endotoxemic sheep [46], while opiates are reported to modulate the influence of LPS on LH release by primates [30]. In conclusion, most studies done in rats point to hypothalamic LHRH neurons as the primary site at which LPS, injected systemically or icv, inhibits LH release, while in other species the pituitary appears to also be an important site of action of this polysaccharide. B. Testis The systemic injection of LPS, which, as discussed above, decreases LH release, also lowers plasma T levels (Fig. 3), and this response can be very long-lasting [47] Does the change in Leydig cell activity only result from low LHRH/LH secretion, or does it also involve a direct gonadal influence of immune signals? We have already mentioned that the iv injection of LPS causes a massive release of TNF-␣ and IL-6 in the circulation. LPS can also induce cytokines production by testicular macrophages [48,49]. It is well known that virtually all steps of sex steroid synthesis can be inhibited by cytokines, includ-

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Figure 2 The icv injection of rh IL-1␤ (80 ng) decreases LHRH release from the median eminence in a representative proestrus female rat. (Adapted from Refs. 9 and 34.)

Figure 3 The iv injection of LPS (5 ␮g/kg) decreases plasma LH and T levels. Each bar represents hormone levels measured 45 minutes after LPS or its vehicle (mean Ⳳ SEM of 5–6 intact male rats). **p ⬍ 0.01 vs. vehicle.

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ing adenylate cyclase second messenger systems, steroidogenic acute regulatory protein (StAR), the conversion of cholesterol to pregnenolone, and/or levels of cytochrome P450 enzymes [11]. This influence may be exerted at least in part by inducing testicular synthesis of secretagogues such as opiates and POMC- or CRF-like peptides, which are present in the testis where they inhibit T synthesis [50–52]. Leydig and Sertoli cells, which express receptors for pro-inflammatory cytokines [53,54], represent, along with macrophages [55,56], potential sources of these proteins as well as their targets [57,58]. It is therefore reasonable to assume that a large part of the effect of LPS on T secretion is mediated by the influence of TNF-␣ and/or IL-6 on steroidogenesis, regardless of whether these cytokines are released in the circulation or manufactured within the testes themselves [59]. Taken together, these observations suggest that in as much as it induces production within the brain of pro-inflammatory cytokines and/or their mediators, endotoxemia is likely to influence testicular activity in part via changes in LHRH/LH secretion. It most probably also alters steroidogenesis through direct testicular effects, which are secondary to increased circulating cytokine levels and/or testicular synthesis. On the other hand, inflammatory conditions such as tissue injury (experimentally induced, for example, by turpentine injection [60]), which are thought to only induce the release of IL-6 into the blood [61], probably alter T release mostly by acting on testicular steroidogenesis, rather than on hypothalamic LHRH neurons. In general, however, infection and inflammation are accompanied by the presence of multiple pro-inflammatory cytokines in many compartments of the body. It is therefore reasonable to assume that both the hypothalamus and the gonads (and, in some species other than the rat, the pituitary) represent potential targets for these proteins. The known ability of infectious and inflammatory diseases to compromise reproductive functions thus probably relies on an array of mechanisms operative at different steps of LHRH, LH, and sex steroid synthesis and release. III. INFLUENCE OF CYTOKINES ON HYPOTHALAMIC-TESTICULAR PATHWAY Most of the work investigating the influence of centrally injected cytokines on LH release was carried out in castrated or ovariectomized rats, providing an elevated LH baseline that made it easier to measure subsequent declines. As mentioned earlier, this model was used to mimic the influence of immune infectious/inflammatory processes that lead to increased cytokines levels in the brain. When we investigated the effect of icv-injected IL-1␤ in intact male rats, we also observed that it decreased plasma LH (Fig. 1) and T levels (Fig. 4). However, in this model, the very low basal LH levels made it difficult to precisely correlate changes in LH and T values. We have briefly outlined above evidence for a direct influence of LPS and other inflammatory signals on testicular activity. We also know, of course, that decreased LH levels will influence T secretion. However, the question of the role played by specific changes in LH levels on the subsequent inhibition of gonadal activity under any given experimental condition remains interestingly unsettled. For example, experiments in which IL-1 was injected icv did not, for the most part, focus on the respective influence (if any) of decreased LH pulse amplitude versus frequency on altered Leydig cell activity. One way to approach this question was to obtain frequent measurements of plasma LH levels and compare them to changes in circulating T values. This approach requires the removal of relatively large volumes of blood. While replacement with either homologous or heterologous red blood cells has been reported, we found over the years that the damage to these cells, which is inherent to the procedure, as well as

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Figure 4 The icv injection of rh IL-1␤ (80 ng) decreases T levels. Each point represents the mean Ⳳ SEM of 5–7 male rats. **p ⬍ 0.01 vs. vehicle.

stressful antigenic responses that we observed when we used red blood cells from donor rats, yielded unsatisfactory results in rodents. We therefore reasoned that an alternate approach would be to determine the influence of exogenous gonadotropin treatment: if the decreased T release of rats injected with IL-1␤ icv was caused by low LH levels (whether accompanied or not by altered pulse amplitude/frequency), exogenous administration of the LH-like compound human chorionic gonadotropin (hCG) should restore normal Leydig cell function. Much of our surprise, this was not the case, and as illustrated in Fig. 5, the icv injection of IL-1␤ significantly interfered with the ability of exogenously administered hCG to release T. This was a very unexpected finding, and we started to search for the mechanisms that could account for it. We first ascertained that the blunted T response we had observed was not modulated by altered LH secretion. While it was unlikely that the acute absence of LH drive would decrease Leydig cells’ responsiveness to gonadotropin, we tested this hypothesis by comparing the ability of icv IL-1␤ to decrease the T response to hCG in rats pretreated with the vehicle or a potent LHRH antagonist that completely abolished LH release. We showed that IL-1␤ was equally effective in both models [62] (Fig. 6), which suggested that altered activity of the pituitary gonadotrops was not involved. We then examined the role of TNF-␣ and/or IL-6 in the circulation (which, as mentioned above, act on the testis to block steroidogenesis [11]), of corticosteroids (which also interfere with androgen synthesis/release [63], though this influence is usually only observed following long-term treatment [64,65]), and of increased prolactin levels or prostaglandin release. Of all these putative mediators, only prostaglandins were found to play a partial role, as illustrated by the ability of ibuprofen to modestly restore the T response to hCG (Fig. 7A) [62,66]. The lack of importance of corticosteroids was illustrated by the ability of icv IL-1␤ to blunt the T response to hCG in both intact and adrenalectomized rats (Fig. 7B). The importance of nitric oxide or opiates could not be readily investigated because the inherent ability of their antagonists to alter the T response to hCG can interfere with the interpretation of the

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Figure 5 The icv injection of rh IL-1␤ (80 ng) blunts the T response to hCG (1 U/kg, iv). Icv vehicle or IL-1␤ were injected 60 minutes prior to hCG. Each point represents the mean Ⳳ SEM of 5–6 intact male rats.

data in the presence of IL-1␤ (Rivier, unpublished). Another critical mechanism that we considered was that the inhibitory effect of icv-injected IL-1␤ was due to sympathetically mediated vasoconstriction, which would decrease T release into the circulation and/or interfere with hCG delivery to Leydig cells. However, the following observations argue against this possibility. First, histological evaluation of the testes of rats injected with IL-1␤ icv failed to indicate signs of decreased vascularization [67]. Second, reductions in blood flow induced by testicular nerve stimulation, for example, are under the

Figure 6 Comparison between the ability of icv rh IL-1␤ (80 ng) to inhibit the T response to hCG in male rats pretreated with the vehicle or the LHRH antagonist Azaline B (40 ␮g/kg, iv—30 min). Each bar represents cumulative T levels measured 20, 45, and 90 minutes after hCG (mean Ⳳ SEM of 5–6 animals). **p ⬍ 0.01 vs. corresponding vehicle.

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Figure 7 (A) The prostaglandin inhibitor ibuprofen (10 mg/kg, iv—15 min) partially reverses the inhibitory effect of rh IL-1␤ (80 ng/icv) on the T response to hCG (1 U/kg). (B) Comparison between the ability of IL-1␤ (80 ng/icv) to blunt the T response to hCG (1 U/kg) in intact or adrenalectomized (adx) rats. Each bar represents cumulative T levels measured 20, 45, and 90 minutes after hCG (mean Ⳳ SEM of 5–7 male rats). **p ⬍ 0.01 vs. corresponding vehicle.

control of ␣-, not ␤-adrenergic pathways [68]. In our model, however, only the ␤adrenergic antagonist propranolol, whether injected systemically or icv, reversed the effect of icv IL-1␤ on the T response to hCG [66]. This finding is not consistent with the importance, in our model, of vascular events as they are presently thought to be mediated. Also, as mentioned above, the icv injection of IL-1␤ decreased testicular levels of the StAR protein, but not of other steroidogenic enzymes [67]. The presence of a specific enzymatic defect at the exclusion of others, and its persistence once Leydig cells had been removed from the animals, points to a mechanism that is likely distinct from a nonspecific influence of vasoconstriction. Finally, we recently provided direct evidence through direct measurement of testicular blood flow that this parameter is not allowed by ICV- injected IL-1␤ [96]. The next step was to determine whether other secretagogues also inhibited the T response to hCG. We found that CRF and related compounds, such as urocortin and urocortin-2, all markedly interfered with the effect of hCG (Fig. 8). As urocortin2 binds selectively to CRF receptors type 2 (CRFR2) [69] while CRF and urocortin bind to both CRFR1 and CRFR2 [70], our results suggest that activation of CRFR2 plays an important role in the influence of CRF-like peptide on the hypothalamictesticular pathway. Vasopressin exerted a lesser though still significant inhibitory influence. In contrast, the icv injection of a scrambled 8- or 41-amino-acid peptide was without effect. In view of our earlier observation that IL-1␤ released CRF [71], we first thought that the influence of this cytokine might be mediated by CRF, but this was not the case because the icv injection of the CRF antagonist ␣-hel CRF9–41, prior to IL-1␤, at doses known to provide widespread inhibition of CRF receptors (see, e.g., Refs. 72 and 73), did not restore T release (Fig. 9) [66]. We therefore concluded that IL-1␤ and CRF acted through a common mechanism. The known ability of IL-1␤ and CRF to increase brain levels of catecholamines [14,74], coupled with the extreme

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Figure 8 The icv injection of CRF (3 ␮g), urocortin (Ucn, 3 ␮g), urocortin-2 (Ucn-2, 3 ␮g) or vasopressin (VP, 0.8 ␮g) significantly decreases the T response to hCG (1 U/kg, iv). In contrast, the icv injection of an 8- or 41-amino-acid scrambled peptide did not. (In view of the similar effect of these two scrambled peptides, their results have been combined.) HCG was injected 60 minutes prior to icv treatments. Each point represents the mean Ⳳ SEM of 5–7 rats.* p ⬍ 0.05; **p ⬍ 0.01 vs. vehicle/hCG.

Figure 9 Blockade of central CRF receptors by the icv injection of the CRF antagonist ␣-

hel CRF9–41 (10 ␮g, icv—30 min) does not restore the T response to hCG (1 U/kg). Each bar represents cumulative T levels measured 20, 45, and 90 minutes after hCG (mean Ⳳ SEM of 5–6 animals). **p ⬍ 0.01 vs. rats without IL-1␤.

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rapidity with which this cytokine interfered with hCG-induced T release (Fig. 10), pointed to a possible role of the brain adrenergic system as an important component of the proposed neural brain-testicular pathway. However, investigating the putative ability of adrenergic agonists to mimic the inhibitory influence of IL-1␤ or CRF had to take into account the fact that in the rat, testicular responsiveness to hCG is regulated by circulating and intratesticular catecholamines [75]. Indeed, the systemic injection of adrenergic agonists or antagonists (particularly those that act on alpha receptors) significantly influences Leydig cell activity by, at least in part, altering LH receptors [66,75]. Consequently, we had to ensure that icv-injected adrenergic agonists did not reach the circulation. We observed that the icv injection of norepinephrine or isoproterenol significantly blunted the T response to hCG (Fig. 11), but that equivalent systemic doses of these compounds did not alter the effect of hCG on Leydig cell activity [66]. On the other hand, blockade of ␤-adrenergic receptors in the brain reversed the inhibitory effect of icv IL-1␤ (Fig. 12) [66]. As we had done for IL-1␤ or CRF, we showed that the influence of isoproterenol was also observed in rats pretreated with the GnRH antagonist Azaline B. On the basis of these observations, we proposed the existence of a neural pathway between the brain and the testis, independent of the pituitary and of sympathetically mediated vasoconstriction, that rapidly regulates testicular responsiveness to gonadotropin. IV. FUNCTIONAL RELATIONSHIP BETWEEN THE KNOWN HORMONAL HYPOTHALAMIC-PITUITARY-TESTICULAR AXIS AND PROPOSED NEURAL HYPOTHALAMIC-TESTICULAR AXIS If Leydig cell activity is controlled not only through LH by also via a neural hypothalamic-testicular pathway, it will be of great interest to know how these two

Figure 10 Time course of the influence of rh IL-1␤ (80 ng, injected icv 5–90 min prior to hCG) on the T response to hCG (1 U/kg, iv). Each bar represents cumulative T levels measured 20, 45, and 90 minutes after hCG (mean Ⳳ SEM of 5–7 animals). **p ⬍ 0.01 vs. vehicle.

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Figure 11 The icv injection of isoproterenol or norepinephrine decreases the T response to hCG (1 U/kg, iv). Each bar represents cumulative T levels measured 20, 45, and 90 minutes after hCG (mean Ⳳ SEM of 5–7 male rats). Dashed line: T levels of rats injected with the vehicle in the absence of hCG. **p ⬍ 0.01 vs. vehicle.

mechanisms interact to promote adequate T release. The first question that comes to mind is what happens to LH secretion when the T response to hCG is blunted. While the resulting low T levels should lead to enhanced LH release through decreased negative feedback, this well-founded hypothesis is not easy to test. First, we could not use models in which rats received icv injection of IL-1␤ or CRF because these treatments not only lower T levels [62,66], they also interfere with LH release [9].

Figure 12 The icv injection of the ␤-adrenergic antagonist propranolol reverses the inhibitory effect of rh IL-1␤ (80 ng, icv) in a dose-related manner. Each bar represents cumulative T levels measured 20, 45, and 90 minutes after hCG (mean Ⳳ SEM of 5–7 male rats).* p ⬍ 0.05; **p ⬍ 0.01 vs. corresponding vehicle.

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The influence of catecholamines on LH secretion in male rats remains somewhat controversial and has been less well studied than in females. This influence is also known to depend on the presence of sex steroids [76]. The very low basal LH levels of intact males did not permit us to observe any significant changes after the icv injection of isoproterenol or norepinephrine. In castrated rats we observed a slight but not significant inhibitory influence of isoproterenol (cumulative ng/mL LH released over a 60-min period after icv injections: vehicle, 22.00 Ⳳ 2.15; 5 ␮g isoproterenol, 19.83 Ⳳ 1.96; p ⬎ 0.05), which agrees with the role of ␤-adrenergic receptors in controlling LH release in this model [77]. The icv injection of norepinephrine to these animals resulted in a significant decrease in LH levels (ng LH/mL, measured 30 min after icv injection: vehicle: 7.42 Ⳳ 0.5; 10 ␮g noradrenaline, 2.97 Ⳳ 0.24; p ⬍ 0.01). While these results correspond to previously published results [78], they also mean that we will not be able to use norepinephrine to investigate functional relationships between LH and T levels in our model. Other experimental avenues that are presently being pursued therefore include investigating the physiological importance of the proposed pathway in regulating the normal oscillatory pattern of T secretion, the onset of puberty, and/or adult male fertility. V. ANATOMICAL AND FUNCTIONAL CHARACTERIZATION OF A NEURAL HYPOTHALAMIC-TESTICULAR PATHWAY Postulating the presence of a direct circuit between the brain and the testes was not novel. Indeed, many years ago hemi-orchidectomy was reported to induce biochemical alterations that were only evident on one side of the hypothalamus [79,80]. Also, hemi-deafferentiation of the hypothalamus was found to interfere with the hemicastration–induced FSH rise if the two interventions were made on the same side [81]. These observations suggested the existence of a purely neuronal central mechanism in the control of T release [82]. However, to our knowledge this hypothesis had been laid dormant. The question then became, how do we provide functional evidence for our proposed pathway? Sympathetic testicular nerves that have cell bodies in the prevertebral nerve plexus around the major arteries provide the so-called ‘‘classical’’ sympathetic testicular innervation. At first view, an obvious way to determine whether this innervation was part of a neural hypothalamic-testicular circuit was to determine what happened if we disrupted it. However, as stated earlier, sympathetic fibers are critical for maintaining testicular responsiveness to LH-like molecules in the rat, and surgical transection of the superior spermatic nerves or their anesthesia by the local application of lidocaine lower Leydig cell LH receptors and interfere with hCG-induced T release [66,83]. Consequently, severing these fibers did not represent a viable approach for determining whether they were important for the inhibitory effect of icv IL-1␤, CRF, or adrenergic agonists. As the pathway that our results suggested was undoubtedly multisynaptic, we could not use classical markers such as FluoroGold or horseradish peroxidase. We therefore turned to the transneuronal labeling technique based on pseudorabies virus (PRV), an extremely powerful neuroanatomical tool used for the identification of hierarchical chains of central neurons innervating a central nervous system (CNS) cell group or a specific end-organ. This technique, which has been used to delineate the brain areas involved in the somatic and/or motor control of the adrenals, bladder, heart, ovary, and pancreas [84–91], among others, relies on the injection of PRV into the peripheral structure of interest. This is followed by replication of viruses

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which are transported retrogradely through ascending chains of synaptically connected neurons [92]. Immunohistochemical procedures are then used to label brain neurons that have become infected and are part of a particular autonomic outflow to the periphery. In our studies, PRV was injected into one testis and labeling was found in the spinal cord, the brain stem, and the hypothalamus [93], which agrees with results published by others [94]. The time-course of appearance of the virus in the CNS was significantly slower than that usually observed with other visceral organs, which corresponds to the finding of very sparse innervation of the rat testis and low number of nerve terminals present in its testicular capsule [75]. Probably for the same reason, brain labeling remained relatively modest. We therefore decided to examine the functional importance of the proposed pathway by determining whether spinal cord injury (SCI) would prevent PRV migration from the testis to the CNS. This was indeed the case with one notable exception: a few PRV-positive cells remained detectable in the PVN of SCI rats injected with the virus [93]. The presence in spine-intact rats, during very early stages of infection, of virus-positive neurons in the nucleus of the solitary tract (NTS), when other labeled perikarya could only be observed in the A5 cell group and the raphe obscurus, led Gerendai et al. to propose that infection had reached the NTS along afferent vagal fibers [94]. As these fibers remained intact in SCI rats, it was theoretically possible that PVN labeling found in this report [94] was indeed at least in part due to passage of the virus from the vagus to the PVN via the NTS. However, our finding that there were no PRV-labeled cells in the NTS following cord severance argues against this hypothesis. It must be noted that the use of the classical PRV protocol (i.e., injection of ⬍5–10 ␮L PRV into the end organ) had not allowed us [93] or others [94] to detect any CNS labeling. In contrast, the intratesticular injection of 100–150 ␮L PRV caused detectable viral staining in the cord, the brain stem, and the diencephalon. However, even though we used stringent conditions to prevent PRV leakage into the abdominal cavity, we could not rule out that some particles might have escaped. As the hypothalamus represents an important component of the vascular and/or sympathetic control of visceral organs and is an area that is commonly reported to contain PRV-labeled cells following virus injection into these organs (see, e.g., Refs. 84–91), it therefore remained possible that PRV-positive cells in the PVN of rats with spinal cord injury may have resulted from particles leaking from the testis to the abdominal cavity. In other words, while our results clearly indicated that spine transection interfered with passage of the virus from the testis to the brain, they did not provide unequivocal anatomical information regarding the presence of a specific brain-testicular pathway. In view of these findings, we thought it critically important to establish the physiological relevance of the proposed pathway. As indicated above, we had reported that the icv injection of CRF or IL-1␤ inhibited the T response to hCG independently of the pituitary. We reasoned that if this inhibitory effect depended on the integrity of the neural circuit we had uncovered, it would not be present in SCI rats. Despite the possibility that cutting the cord might disrupt not only spinal pathways connecting the brain to the testes, but also the pelvic innervation of the gonads, which is important for testicular LH receptors (see above), SCI rats retained a significant T response to hCG [93]. While indicating that these rats represented a valid model in which to test our hypothesis, our results also supported the concept that the neural pathway we propose is different from the known sympathetic testicular innervation that controls

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Leydig cell responsiveness to gonadotropin. Indeed, if these pathways were identical or significantly related, severing the connection between the brain and the testes should have had the same consequence as surgical transection of the superior spermatic nerves or their anesthesia, which, as described above, completely abolished the T response to hCG [66,83]. However, Fig. 13 illustrates the fact that though SCI rats showed a shift of this response, compared to intact animals, they remained quite capable of responding to hCG. We then showed that the icv injection of IL-1␤ or CRF did not significantly alter the hCG-induced T response of SCI rats, which indicates that these secretagogues lost their ability to decrease testicular responsiveness to hCG in the absence of an intact neural circuit that connected the brain and the testes. We therefore concluded that they exerted their influence by stimulating a descending pathway that travels through the spinal cord. Our findings also ruled out a participation of vagal fibers in the ability of icv CRF or IL-1␤ to blunt the T response to hCG. As indicated above, these fibers, which innervate the testis, had been suggested by Gerendai et al. as providing a route through which PRV traveled to the brain [94]. VI. MAIN COMPONENTS OF THE PROPOSED NEURAL BRAINTESTICULAR PATHWAY The possibility, suggested by our results [93] and those of others [94], that the PVN was part (represented the origin?) of a neural brain-testicular pathway suggested two hypotheses. First, lesions of this area should prevent icv-injected IL-1␤, CRF, or isoproterenol from inhibiting the T response to hCG; second, microinfusion of these treatments into the PVN should mimic the effect of their icv injection. Recent studies in our laboratory indicate that this was indeed the case [95]. While these results do

Figure 13 Comparison between the ability of icv injected rh IL-1␤ (80 ng) or CRF (3 ␮g) to blunt the T response to hCG in intact (sham-operated) or rats with spinal cord injury (SCI). Intact rats were injected with IU hCG/kg, SCI rats with SU hCG/kg. Each bar represents cumulative T levels measured 20, 45, and 90 minutes after hCG (mean Ⳳ SEM of 5–7 male rats). **p ⬍ 0.01 vs. corresponding vehicle. (From Ref. 93, copyright 2002, The Endocrine Society.)

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not rule out the contributing influence of other areas, such as the locus coerulus, they point to the PVN as a critical component of the circuit we had uncovered. Another area of further study will be the identification of the Leydig cells components and/or steroidogenic steps that mediate the influence of icv treatments other than IL-1␤. Our earlier work had indicated that testicular StAR protein levels were significantly decreased in rats injected with IL-1␤ icv, and that treatment of Leydig cells with a waterpermeable form of cholesterol that bypassed the requirement for StAR restored hCGinduced T release [66]. This suggested that StAR played a role in the suppression of testicular function evoked by the central administration of IL-1␤, and we need to extend these results to CRF and catecholamines. We also need to identify the mediators through which icv-injected IL-1␤ or other treatments exert their inhibitory influence. CRF, opiates, nitric oxide, and testicular catecholamines, among others, represent potential candidates that are presently under investigation. Finally, an essential step will be the demonstration of the role played by the neural pathway in mediating T release in response to endogenous LH under physiological conditions. VII. CONCLUSION Appropriate testosterone is not only crucial for reproductive purposes, it is central to protein metabolism and plays a role in many other functions such as the activity of immune cells and specific brain functions. It therefore comes as no surprise that its regulation would be both multifaceted and involve several redundant mechanisms. We discussed here the fact that Leydig cell activity is controlled not only by the classical LHRH-LH axis, but also by a neural pathway between the hypothalamus and the testes, which is independent of the pituitary. We propose that while LH represents an important overall drive for androgen synthesis and release, the neural pathway provides a fine-tuned and minute-by-minute regulation of Leydig cell responsiveness to this gonadotropin, which is responsive to changes in brain levels of secretagogues released during stress. It seems likely that both mechanisms are indispensable for appropriate testicular function and that their disregulation will lead to abnormal T release and impaired reproductive functions. A future challenge will be to identify the circumstances under which the activity of either or both pathways is altered and the mechanisms responsible for these changes. This may be particularly important for the possible treatment of reproductive disorders that have hitherto remained elusive. ACKNOWLEDGMENT Research in the author’s laboratory was supported by NIH grant AA 12810. REFERENCES 1. Spratt DI, Cox P, Orav J, Moloney J, Bigos T. Reproductive axis suppression in acute illness is related to disease severity. J Clin Endocrinol Metab 1993; 76:1548–1554. 2. Marchetti B, Morale MC, Gallo F, Batticane N, Farinella Z, Cioni M. Neuroendocrineimmunology (NEI) at the turn of the century: towards a molecular understanding of basic mechanisms and implications for reproductive physiopathology. Endocrine 1995; 3:845–861. 3. van Steenbergen W, Naert J, Lambrecht S, Scheys I, Lesaffre E, Pelemans W. Suppression of gonadotropin secretion in the hospitalized postmenopausal female as an effect of acute critical illness. Neuroendocrinology 1994; 60:165–172.

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7 Sex Hormones and B Cells CHRISTINE M. GRIMALDI, ELENA PEEVA, and BETTY DIAMOND Albert Einstein College of Medicine, Bronx, New York, U.S.A.

I. INTRODUCTION Sex hormones such as estrogen and prolactin are best known for their effects on reproductive tissue. It is now widely accepted, however, that there is a complex interplay between sex hormones and numerous organ systems and that sex hormones exert pleiotropic effects on multiple cell types. Of particular importance to the field of autoimmunity is the mounting evidence that sex hormones contribute to the sexual dimorphism observed in the immune system. It is well established that females typically mount a more vigorous immune response than males [1–3]. A consequence of this enhanced immunoresponsiveness may be the increased susceptibility of females to autoimmune diseases [4,5]. Despite emerging data implicating sex hormones in autoimmunity, the effects of sex hormones on the immune system are not well understood. There is some evidence that sex hormones can contribute to the progression of systemic lupus erythematosus (SLE). The gender bias in this disease is striking with a female to male ratio of 9:1 [6,7]. Disease occurrence is most prevalent in females following menarche and prior to menopause. There are also some reports that SLE flares are more common during pregnancy, perhaps related to a prolonged elevation in estrogen levels [8–10], although this topic is controversial. Alterations in estrogen metabolism and prolactin concentrations have been reported in some patients with SLE [11–14], suggesting that a cohort of patients may have a disease that is hormonally regulated. Finally, anti-hormonal therapies such as bromocriptine and dehydroepiandrosterone (DHEA) have been shown to have some clinical benefits [15,16]. Thus, modulation of sex hormones in patients with SLE may be a potential therapeutic strategy. While it is reasonable to hypothesize that increased exposure to estrogen may exacerbate disease in some patients with SLE, it is clear that not all autoimmune diseases are 127

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exacerbated by sex hormones. In fact, elevations in estrogen during pregnancy alleviate disease in patients with rheumatoid arthritis [17,18] and multiple sclerosis [19,20]. Since there are increasing data supporting a role for estrogen and prolactin in the modulation of B-cell development, activation, and survival, this chapter will focus on the effects of these hormones on B cells and describe models for hormone-induced autoreactivity, especially SLE-like autoreactivity. II. OVERVIEW OF B-CELL DEVELOPMENT, SELECTION, AND ACTIVATION Early B-cell development occurs through a series of orchestrated steps in the bone marrow [21]. The critical events that regulate B-cell development include the successful rearrangement of immunoglobulin genes and the subsequent surface expression of membrane immunoglobulin. The rearrangement events that form the heavy- and light-chain variable regions and the random association of heavy and light chains generate a diverse repertoire of immunoglobulin molecules that is capable of recognizing numerous antigens. Because this repertoire is generated without any prior knowledge of beneficial or pathogenic specificities, both protective and autoreactive receptors are generated. The stages of B-cell development are defined not only by the absence or presence of surface immunoglobulin, but also by an array of developmentally regulated cell surface and intracellular molecules that play key roles in the differentiation pathway. Communication between developing B cells and stromal cells and expression of soluble factors such as cytokines and chemokines are critical for successful maturation. The first step in B-cell lymphopoiesis is the differentiation of pluripotent stem cells into pro-B cells [22]. At this stage of development, the gene products that participate in heavy-chain gene rearrangement are expressed. Failure to express an intact heavy chain blocks further development. Following the production of a functional rearranged heavy chain, cells progress to the pre-B-cell stage where light-chain gene rearrangement takes place. At the immature stage of B-cell development, heavy chains and light chains are assembled intracellularly and then expressed on the cell surface. The immature B-cell stage represents a critical developmental checkpoint for B cells. Immature B cells are particularly prone to undergo apoptosis, in part, due to the downregulation of the Bcl-2 antiapoptotic molecule [23,24]. Surface immunoglobulin composes a major component of the B-cell receptor (BCR). When bound by antigen, the BCR mediates signal transduction events that regulate B-cell activity. A low degree of binding of antigen (or autoantigen) to the BCR is insufficient to mediate an activation or tolerization signal on immature B cells, but extensive cross linking of surface immunoglobulin on naı¨ve B cells mediates a tolerization signal. These immature B cells that bear self-reactive immunoglobulin and are engaged by self-antigen can mature to immunocompetence if there is little engagement of surface immunoglobulin by antigen. Alternatively, these cells are removed from the naı¨ve B-cell pool when there is sufficient engagement of surface immunoglobulin by antigen. As a result, only a small percentage of developing B cells succeeds in exiting the bone marrow and populating the periphery. Immature B cells that survive selection processes in the bone marrow transit to the spleen to become transitional type 1 B cells [25]. Similar to immature B cells in the bone marrow, transitional type 1 B cells are subject to negative selection and undergo apoptosis following ligation by self-antigen [26–28]. Therefore, self-reactive immature B cells that are not eliminated in the bone marrow have a second opportunity to be eliminated as

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transitional type 1 B cells if they encounter self-antigen in the spleen. Transitional type 1 B cells that are not eliminated by negative selection will differentiate into transitional type 2 B cells, which are the precursors for conventional (also known as B-2) mature Bcell subsets, marginal zone B cells, and follicular B cells. Marginal zone B cells are found exclusively in the spleen at the marginal sinuses, and follicular B cells are found in B-cell follicles of all secondary lymphoid tissue [29]. While there are data to suggest that both marginal zone B cells and follicular B cells derive from transitional type 2 B cells [25,30,31], these mature B-cell subsets differ in anatomical location, requirements for T-cell help, and responses to antigenic stimulation. Marginal zone B cells participate in T-cell–independent B-cell responses [32]. The antibodies produced by this B-cell subset are typically IgM, and it is yet uncertain whether they undergo somatic mutation. Conversely, follicular B cells respond to antigen in a Tcell–dependent fashion, and the antibodies produced by this B-cell subset undergo isotype class switching and somatic hypermutation [33,34]. Following antigenic challenge and T-cell costimulation, activated B cells can form discrete structures in peripheral lymphoid tissue known as germinal centers. Germinal centers are sites where antigen-activated B cells undergo isotype class switching. In addition, the process known as affinity maturation occurs in germinal centers. This process involves mutations in the variable region of immunoglobulin genes, which affect the affinity of antibody for antigen. Affinity maturation means that B cells with the highest affinity for antigen have preferential survival potential. In some instances the antigenic specificity of the molecule is altered, and reactivity for self-antigen may be acquired. Bcl2 is downregulated in germinal center B cells, rendering germinal center B cells susceptible to apoptosis and facilitating the removal of autoreactivity acquired as a result of somatic hypermutation [35,36]. Following B-cell selection in germinal centers, some of the surviving B cells undergo terminal differentiation into long-lived memory cells. These cells remain quiescent in the circulation pending a second encounter with antigen. Others differentiate into plasma cells that secrete large quantities of immunoglobulin.

III. IMMUNOMODULATORY EFFECTS OF ESTROGEN ON B CELLS A. Effects of Estrogen on B-Cell Development Experiments performed on mice provided the initial evidence that estrogen regulates Bcell development in the bone marrow. Analysis of pregnant mice revealed a dramatic decrease in the number of B-cell precursors in the bone marrow [37]. Treatment of female mice with exogenous estrogen had a similar effect on the number of B-cell progenitors in the bone marrow with the pre-B-cell population being most affected by estrogen treatment [38,39]. Conversely, B-cell lymphopoiesis is enhanced in ovariectomized female mice and in mice that harbor a mutation in the gonadotrophin-releasing hormone gene that results in negligible concentrations of sex hormones [40]. These data provided clear evidence that estrogen can act as a negative regulator in B-cell development. Kincade and colleagues have performed a series of experiments to gain a better understanding of the impact of estrogen on the early stages of B-cell development. When pro-B cells were cultured in the presence of IL-7 and stromal cells, addition of estrogen to the cultures inhibited lymphopoiesis. Examination of bone marrow cells treated in vitro with estrogen revealed that estrogen impairs development of early B-cell precursors

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[39,41,42]. Similarly, studies performed on mice treated in vivo with estrogen demonstrated that an elevation in estrogen levels diminishes the number of IL-7 responsive cells and decreases proliferation and survival of bone marrow B-cell precursors [43]. The mechanism by which estrogen negatively regulates B-cell lymphopoiesis is not completely understood. However, results from these studies suggest that estrogen receptors expressed in both B-cell precursors and stromal cells in the bone marrow mediate this effect [42,44]. The inhibitory effects of estrogen on B-cell development results also in a decrease in the number of immature B cells that emigrate to the spleen for further maturation [38,45]. Kincade and colleagues reported a decrease in the percentage of splenic B cells with a phenotype consistent with immature (transitional) B cells [38]. In our laboratory, we have shown that both transitional type 1 and transitional type 2 B-cell populations are significantly reduced in mice treated with doses of estrogen that raise the serum concentration to levels that occur at the peak of estrus cycle [45]. In normal mice, the number of transitional type 1 B cells is approximately twofold greater than the number of transitional type 2 B cells. The reduction in the number of the more mature transitional type 2 B cells that usually occurs appears to be due to the elimination of autoreactive B cells at the transitional type 1 B-cell stage. Interestingly, in estrogen-treated mice there is no significant reduction in the transitional type 2 population compared to the type 1 population. As will be discussed in Section V, this shift in the transitional B-cell populations may reflect defective negative selection of autoreactive B cells. As described in Section II, transitional type 2 B cells differentiate into the mature follicular and marginal zone B-cell subsets. Approximately 50–60% of the splenic B-cell pool is composed of follicular cells and 5–8% is composed of marginal zone B cells. The percentage of follicular B cells in estrogen-treated mice is similar to that in placebo-treated mice. However, estrogen-treated mice display a significant expansion of marginal zone B cells, indicating that estrogen selectively modulates the development of this T-cell–independent B-cell subset [45]. B. SLE and Estrogen Autoimmune disorders are complex, and it is clear that multiple genes are involved in susceptibility and that disease can be precipitated by encounter with environmental triggers such as microorganisms. SLE is an autoimmune disease of unknown etiology. The disease is mediated by the generation of autoreactive B cells and requires T cells [46]. The hallmark of SLE is the production of antinuclear antibodies. Deposition of these autoantibodies as immune complexes leads to inflammation and tissue destruction. One of the most clinically relevant autospecificities is antibody to double-stranded (ds) DNA [47]. Titers of serum anti-dsDNA antibodies correlate with disease severity and with the occurrence of glomerulonephritis. Based on the observations that the pathogenic autoantibodies present in lupus patients and in lupus-prone mice have undergone isotype class switching and somatic hypermutation, it is evident that these antibodies are produced by T-cell–dependent follicular B cells [48–50]. In mouse models of lupus, it appears that the marginal zone B-cell population, which requires T-cell–derived factors but not cognate T-cell help, can also secrete anti-DNA antibodies [51,52]. However, there is little evidence linking the autoantibodies secreted by the marginal zone B-cell subset to tissue destruction. C. Autoimmunity in Estrogen-Treated Mice Studies performed several decades ago demonstrated that increases in the level of estrogen can augment or accelerate disease in the NZB ⳯ NZW F1 (B/W) mouse strain that

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spontaneously develops lupus by 5 months of age. These observations were consistent with the fact that both female and male mice succumb to disease, but disease onset occurs earlier in female mice [53]. Investigators showed also that castration of female mice delayed disease onset, whereas castration of male mice accelerated disease onset [53]. Administration of testosterone had an opposing affect, as female mice given testosterone displayed a delay in disease onset and an increase in life span [54]. Thus, the results from these studies provided a plausible explanation for the gender bias seen in patients with SLE; estrogen can exacerbate disease, while androgens can exert a protective effect. Similar results were obtained in the lupus-prone MRL/lpr mouse strain [55] and in a graftversus-host model of lupus in C57BL/6 ⳯ DBA/2 F1 mice in which disease was exacerbated by estrogen as well [56]. We have been studying the regulation of anti-DNA B cells in mice that do not spontaneously develop lupus. The R4A mouse expresses a transgene that encodes the heavy chain of a nephritogenic antibody [57,58]. The transgenic heavy chain is expressed as an IgG2b molecule, which permits the detection of transgene-expressing B cells among the naı¨ve IgMⳭ B-cell pool. The R4A heavy chain can associate with a multitude of light molecules to generate antibodies with varying affinities for DNA, as well as non-DNA reactive antibodies [59–61]. In the R4A transgenic mouse, 5–10% of B cells are positive for surface expression of the transgene when the transgene is expressed on a nonautoimmune background, such as the BALB/c mouse strain [57]. Based on the specificity and affinity for self-antigen conferred by the light chain, we have characterized four distinct B-cell populations in R4A transgenic mice. The first is a population with no affinity for DNA. The second is a population in which the affinity for DNA is too low to mediate signals that would trigger tolerance; these low-affinity ‘‘ignorant’’ B cells remain in the periphery and mature to immunocompetence [59]. The third is a population of transgene-expressing B cells with high affinity for DNA [61]. This population possesses light chains with unmutated variable regions, demonstrating that these autoreactive B cells derive from the naı¨ve repertoire. In BALB/c mice, this population is deleted unless the R4A transgene is expressed in mice that are also transgenic for overexpression of Bcl-2 in the B-cell compartment [62]. Alternatively, this population can be detected when the transgene is expressed in the lupusprone B/W mouse strain [61]. The fourth population also produces high-affinity anti-DNA antibody. This population, however, possesses mutated light chains, demonstrating that these B cells are derived from an antigen-activated pool that has matured in germinal centers [60]. Even though these B cells are present in peripheral lymphoid tissue of R4A mice, they exist in an anergic state and cannot be activated through engagement of their surface immunoglobulin. We have shown that estrogen treatment of R4A-BALB/c mice impairs tolerance induction of anti-DNA B cells. Following several weeks of estrogen treatment, serum IgG2b anti-DNA titers increase [63]. Consistent with the increase in anti-DNA antibody levels, estrogen-treated R4A-BALB/c mice display an increase in the percentage of transgene-positive B cells in the spleen and in the number of B cells spontaneously secreting IgG2b anti-DNA antibody [63]. The secreted anti-DNA antibodies are potentially pathogenic, as IgG2b antibody deposits are observed in the glomeruli of estrogen-treated R4A-BALB/c mice. Thus, without additional manipulation, estrogen treatment is sufficient to abrogate B-cell tolerance and induce a lupus-like phenotype in non–autoimmune-prone mice.

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Examination of the light chains utilized by the DNA-reactive B cells that are activated in estrogen-treated R4A-BALB/c mice show little evidence of somatic mutation [63]. This suggests that the B cells rescued by estrogen treatment are derived from the naı¨ve B-cell pool that arises in the bone marrow and would normally be deleted at an immature stage of development. The escape of naı¨ve autoreactive B cells prior to their differentiation into either of the mature subsets is consistent with the alterations observed in the transitional B-cell pool of estrogen-treated mice described in Section II. As stated above, negative selection occurring at the transitional type 1 stage results in a reduced number of transitional type 2 B cells [25,30]. Since the transitional type 2 B-cell population is not significantly decreased in estrogen-treated mice, it appears that the autoreactive transitional type 1 R4A B cells are escaping negative selection and moving into the transitional type 2 compartment where they can differentiate into mature marginal zone or follicular B cells. Consistent with this hypothesis is the observation that transitional B cells isolated from estrogen-treated BALB/c mice are more resistant to anti-immunoglobulin treatment, which simulates binding of self-antigen and triggers BCR-mediated signal transduction events [64]. Thus, an elevation in estrogen levels appears to block the elimination of autoreactive B cells at the transitional B-cell stage. It is widely held that the follicular B-cell population is predominantly responsible for the secretion of pathogenic autoantibodies seen in patients and mice with lupus. Analysis of the mature subsets generated in R4A-BALB/c mice demonstrated that the transgenic B cells differentiate into both follicular and marginal zone B cells [45]. To ascertain which population is spontaneously activated in vivo to secrete anti-DNA antibody in estrogentreated mice, both populations were isolated, and the number of IgG2b anti-DNA-secreting B cells in each mature population was enumerated. While both populations display higher numbers of B cells secreting anti-DNA antibody compared with control mice, the bulk of these B cells belongs to the marginal zone subset [45]. This provides the best evidence that marginal zone B cells can produce autoantibodies that are directly pathogenic. The marginal zone B-cell pool is also expanded in young B/W mice [65,66] and has been shown to secrete anti-DNA antibodies [51], yet the antibodies produced by this subset have not been tested for deposition in glomeruli. A similar phenomenon is observed in mice that overexpress the TNF family member BAFF and also display a lupus phenotype [67]. Again, however, it is not clear that the autoantibodies produced by marginal zone B cells in these mice are nephritogenic. Clearly, the role of marginal zone B cells in lupus requires further investigation. Examination of the marginal zone B-cell population in estrogen-treated mice may provide important clues to the role of this B-cell subset in lupus. The effects of estrogen on the regulation of autoreactive B cells appear to be strain specific. Unlike R4A-BALB/c mice, we have found that R4A-C57BL/6 mice treated with estrogen do not display an increase in serum anti-DNA antibody titers. This observation suggests that there are additional genetic factors that contribute to the estrogen-induced loss of B-cell tolerance. The difference in susceptibility to estrogen-induced perturbations in B cells among different mouse strains may, in fact, mimic genetic differences in patients with SLE. Thus, there may be patients with a disease that is estrogen responsive and others with a non–hormonally responsive disease. D. Estrogen Receptors in B Cells Estrogen responsiveness is conferred by intracellular estrogen receptors. Estrogen receptor complexes regulate gene expression by binding to DNA elements known as estrogen-

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responsive elements present in the promoter region of various genes [68]. Transactivation of target genes by estrogen-estrogen receptor complexes also requires specific coactivators proteins [69]. Alternatively, ligand-bound estrogen receptors can act in a nongenomic manner to activate intracellular pathways such as mitogen activated kinases [70,71]. Two estrogen receptors have been identified thus far and are designated estrogen receptor alpha (ER␣) and estrogen receptor beta (ER␤). The functional distinctions between ER␣ and ER␤ remain to be elucidated, but increasingly the data suggest they differ in gene activation [72,73]. ER␣ has two activation domains, AF-1 and AF-2, while ER␤ contains only AF-2 [74,75]. It has been demonstrated that in some cell types, the interaction of both AF-1 and AF-2 of ER␣ with a coactivator is required for the estrogen receptor to cause transcriptional activation, while in other cell types the interaction with AF-1 coactivator is sufficient for transcriptional activation. Studies examining the expression pattern of ER␣ and ER␤ in a variety of different tissues have revealed that some cell lineages express one type of estrogen receptor, while others can express both [76,77]. Both receptors appear to bind estrogen with a similar affinity and in many instances can activate the same estrogen-responsive genes. In general, ER␣ is a better transactivator of estrogen responsive genes than ER␤ [78]. However, when both estrogen receptors are expressed in the same cell type, ER␤ can inhibit transcriptional activation by ER␣ [79]. Recently, data from our laboratory and from others investigators have demonstrated the presence of estrogen receptors in B cells. Transcripts for both ER␣ and ER␤ have been detected in both human and murine B cells [44,80]. We have examined ER␣ and ER␤ expression in all stages of murine B-cell development [64]. Both ER␣ and ER␤ are present in B cells. It appears that expression of estrogen receptors is developmentally regulated, with ER␣ levels increasing as the B-cell matures [64]. While it is apparent that both estrogen receptors are expressed in B cells, the distinct roles of ER␣ and ER␤ will require further examination. It is likely that they mediate different effects and regulate different genes and functions at different stages of B-cell development. The role of estrogen receptors in SLE also needs to be addressed. Analyses performed thus far of small numbers of patients have revealed no abnormalities in estrogen receptor transcripts or function [80–82]. Interestingly, however, the estrogen receptors expressed in the lupus-prone B/ W and MRL/lpr mice display a higher affinity for estrogen than the receptors expressed in BALB/c mice [83,84]. To learn more about the pathways regulated by estrogen receptors in B cells, we performed subtractive hybridization and microarray screening of B cells from estrogenand placebo-treated mice. Nineteen genes were identified as upregulated in the B cells of estrogen-treated mice, 11 of which appeared to be downregulated [64]. We focused our attention on CD22 and SHP-1, which act as negative regulators of BCR signaling, and Bcl-2, which is an antiapoptotic molecule. Levels of these molecules were measured by flow cytometry and were found to be upregulated significantly in all peripheral B-cell subsets of estrogen-treated mice [64]. Bcl-2 expression is increased following estrogen treatment in the estrogen receptor-positive MCF-7 breast cancer cell line [85,86] and there is evidence of the existence of estrogen-responsive elements in the bcl-2 promoter [87,88]. However, no estrogen-responsive elements have been reported in the cd22 or shp-1 genes. To determine if the increases in gene expression occur through a direct effect of ligandactivated estrogen receptors, B-cell lines were transfected with a cDNA construct expressing a constitutively active ER␣. Similar to the B cells of estrogen-treated mice, cells transfected with an activated ER␣ display increases in CD22, SHP-1, and Bcl-2 levels,

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suggesting that increased expression is B-cell autonomous and occurs through activated estrogen receptors [64]. While it remains to be determined whether the increased expression of CD22, SHP-1, and Bcl-2 plays a significant role in the lupus phenotype observed in estrogen-treated mice, overexpression of Bcl-2 has been shown to block the deletion of autoreactive B cells in several mouse models [23,36,89–91]. Overexpression of CD22 or SHP-1 increases the threshold required for anti-immunoglobulin-induced BCR activation, as demonstrated by a reduction in calcium influx following BCR engagement by anti-IgM treatment [64]. Since both CD22 and SHP-1 function as inhibitors of BCR signaling [92,93], the decrease in BCR signaling observed in the B cells of estrogen-treated mice may increase the signaling strength required for the elimination of autoreactive cells. E. Selective Estrogen Receptor Modulators and B-Cell Autoreactivity The hypothesis that female sex hormones may be a risk factor for the development of SLE led to the speculation that antiestrogen treatments may be beneficial in lupus and that selective estrogen receptor modulators (SERMs) might counteract the effects of estrogen on the immune system. SERMs are agents that bind estrogen receptors and demonstrate estrogen agonist or antagonist activity in a tissue-specific manner. For example, tamoxifen and raloxifen, which are SERMs widely used in clinical practice, manifest estrogen antagonistic effects in the breast, but estrogen agonistic effects in the bone [94,95]. The biological effects of estrogen and SERMs are mediated through binding to estrogen receptors, ER␣ and ER␤. Tamoxifen is an AF-1 [96] agonist and AF-2 antagonist [97]. Crystallographic studies have helped explain the antagonist activity of both tamoxifen [97] and raloxifen [98] by demonstrating distinct conformational changes in the complexes they form with ER␣. These complexes prevent the interaction of AF2 with its coactivator. It has been shown that the estrogen–ER␣ complex binds to steroid receptor coactivator (SRC)-1, while the ER␣–tamoxifen complex binds to a different coactivator, which has not yet been identified [99]. Its existence has been shown, however, by fingerprinting studies of the surface changes of the receptor induced by different molecular interactions. These data demonstrate that the agonist activities of estrogen and tamoxifen do not occur through an identical pathway. There are also differences in tissue effects among the SERMs themselves. For example, tamoxifen causes endometrial proliferation, but raloxifen does not [100]. This tissue selectivity among SERMs cannot be explained by the differential activation of the ER␣ or ER␤; rather it seems that conformational differences of the ligand–estrogen receptor complexes play a crucial role in determining their functional activity [96,101]. The conformation of the receptor changes depending on the ligand bound; therefore, complexes of ER␣ with tamoxifen and raloxifen are not identical, just like complexes of ER␣ with estrogen and tamoxifen are not identical. The distinct ER/ligand conformations lead to the binding of particular coactivators [102]. Thus, the transcriptional profiles of tamoxifen and raloxifen are different. Studies performed in mouse models of lupus suggest that tamoxifen is capable of acting on immune cells. In B/W mice, tamoxifen decreased anti-DNA antibody titers, proteinuria, and kidney damage [103]. In MRL/lpr mice, tamoxifen resulted in a decline in the number of T cells and in CD4ⳮ/CD8ⳮ T cells and a significant increase in IL-2 production by lymph node cells [104]. In 16/6 Id-induced SLE, treatment with tamoxifen decreased the levels of IL-1, IL-2, IL-4, IFN-␥, and TNF-␥ to normal levels [105]. Despite these encouraging results in murine models of lupus, treatment with tamoxifen showed

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no beneficial effect on disease activity in a small clinical trial that included 11 SLE patients [106]. The reason for the discrepancy between the murine lupus studies and the human lupus study is not known, although it may reflect the fact that only some patients with lupus have a hormonally responsive disease. Studies investigating the mechanism of action of tamoxifen have demonstrated that it inhibits the maturation of dendritic cells and diminishes their ability to activate T cells [107], decreases CD5Ⳮ (B-1) B cells [103], and affects cytokine production [105]. In addition, tamoxifen can influence B-cell development. In estrogen-treated R4A-BALB/c mice we found that tamoxifen was able to prevent the development of a lupus-like phenotype. Tamoxifen abrogated the development of serum anti-DNA antibody titers and immunoglobulin deposition in the glomeruli blocked the expansion of marginal zone B cells observed in estrogen-treated mice. Since anti-DNA antibody titers were absent in mice treated with estrogen and tamoxifen together, it appears that inhibition of the estrogenmediated expansion of marginal zone B cells may be crucial for maintaining tolerance in estrogen and tamoxifen-treated mice [108]. Furthermore, we found that mice given both estrogen and tamoxifen continued to display increased expression of Bcl-2. This alteration in gene expression would favor survival of autoreactive cells. Estrogen- and tamoxifentreated B cells display normal levels of CD22. It is reasonable, therefore, to assume that signaling through the BCR of B cells from mice given estrogen and tamoxifen would be similar to the BCR signaling in wild-type mice. These observations support a model in which there is prolonged survival of autoreactive B cells, yet the cells receive a tolerization signal. Thus, anergy of autoreactive cells appears to maintain tolerance in mice given both estrogen and tamoxifen. Effects on the immune system of raloxifen are less well documented. One study demonstrated that the raloxifen analogue LY117018 induces less prominent thymic atrophy than estradiol in mice and causes no alterations in the ratio of CD4 to CD8 cells in the thymus [109]. There are no published data on the effects of raloxifen on B cells. The mechanisms by which raloxifen may exert immunomodulating effects in autoimmune disease have not been investigated yet. Future studies will be needed to investigate in detail the cellular and molecular mechanisms by which SERMs affect the immune system. Understanding that conformational changes of the estrogen receptors and their interaction with an array of coactivators provides the basis for developing new SERMs that can selectively block the estrogeninduced survival and activation of autoreactive cells. This raises the possibility that novel SERMs can be developed to serve as therapeutic tools for a subset of SLE patients with a hormonally responsive disease. Furthermore, it appears that estrogen acts on at least two distinct stages to disrupt tolerance in autoreactive B cells. In the first stage, estrogen increases the survival of autoreactive B cells, and in the second, autoreactive B cells are activated. Different antagonists may be necessary to block each step.

IV. IMMUNOMODULATORY EFFECTS OF PROLACTIN ON B CELLS A. Effects of Prolactin on B-Cell Development Compared to estrogen, the effects of prolactin on lymphopoiesis have been studied to a lesser extent. Both prolactin-deficient mice [110] and prolactin receptor–deficient mice [111] display normal hematopoiesis, suggesting either that prolactin does not play a crucial role in this process or that its effects can be compensated by other factors. However,

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treatment with recombinant human prolactin in mice causes an increase in hematopoiesis in the spleen and, to a lesser degree, in the bone marrow [112,113]. B. Prolactin Receptor Prolactin exerts its effects on the immune system by binding to prolactin receptors, which are expressed on stem cells, T cells, B cells, NK cells, monocytes, granulocytes, and thymic epithelial cells. Prolactin receptors are transmembrane proteins that belong to the type I cytokine receptor superfamily. Prolactin binds to the extracellular domain of the prolactin receptor, causes dimerization, and induces a cascade of intracellular events that involve the Jak/Stat signaling pathway. In rat bone marrow and spleen cells, prolactin receptors coupled to ligand signal through Janus tyrosine kinase (JAK) 2 and transcription factor Stat5 [114] with subsequent activation of interferon regulatory factor-1 (IRF-1) gene [115]. In human peripheral blood mononuclear cells, prolactin induces phosphorylation of Jak2 and Stat1/5 transcriptional factors, leading to induction of several genes including IRF-1 and suppressors of cytokine signaling (SOCS) 2, 3, and 7 [116]. C. Autoimmunity in Hyperprolactinemic Mice Pioneering experiments demonstrated that prolactin acts not only as a lactogenic hormone, but also as an immunomodulator, which can contribute to the development of SLE [117]. Increased serum prolactin levels induced by transplantation of pituitary glands in B/W lupus-prone mice lead to an exacerbation of disease activity [118,119]. Hyperprolactinemia causes increased IgG levels and anti-DNA antibody titers and premature glomerulonephritis leading to early mortality. Interestingly, there was no difference in mortality between mice with moderate and severe hyperprolactinemia, suggesting that the immunostimulatory effect of prolactin does not correlate linearly with serum concentration. On the other hand, treatment with bromocriptine, an inhibitor of prolactin secretion, decreased disease activity and improved survival in B/W mice [118]. Although there is the possibility that bromocriptine has a direct impact on the immune cells, its effects are mostly mediated through the inhibition of prolactin secretion [107]. This model is supported by the observation that prolactin can overcome bromocriptine-induced immunosuppression [120]. Our laboratory has demonstrated that prolactin can affect the development of splenic B cells. Treatment with prolactin in BALB/c mice caused a significant reduction in the more immature transitional type 1 B-cell subset, and there was no concomitant reduction in the transitional type 2 B cells. Since negative selection takes place at the type 1 to type 2 transition, this observation suggests that, like estrogen, prolactin may impair negative selection of immature B cells. Prolactin also caused an increase in mature subsets, mainly follicular B cells. We have also demonstrated that prolactin, like estrogen, can induce a lupus-like phenotype in nonspontaneously autoimmune mice. Treatment of R4A-BALB/c mice with prolactin for 4 weeks induced an expansion of transgene-expressing B cells with increase in anti-DNA antibody titers and deposition of IgG in the glomeruli. The lupus-like phenotype did not develop in prolactin-treated R4A-BALB/c mice deficient in CD4Ⳮ T cells, demonstrating that the prolactin-induced effects on B cells require the presence of T cells. The expanded population of transgene-expressing B cells mature as follicular B cells, consistent with their requirement for CD4Ⳮ T cells. Enumeration of B cells that spontaneously secrete anti-dsDNA antibodies demonstrate that the follicular B-cell subset was responsible for anti-DNA antibody production [121]. This is in contrast to estrogen treat-

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ment, which leads to escape of autoreactive cells and activation of these cells as marginal zone B cells. Although it appears that both estrogen and prolactin may rescue the same population of naı¨ve autoreactive B cells, estrogen directs them to develop as marginal zone B cells, and prolactin directs them towards a follicular phenotype. The distinct mechanisms employed by estrogen and prolactin in the induction of autoreactivity result from different patterns of gene expression. Estrogen upregulates CD22 and SHP-1 in R4A-BALB/c mice, but these molecules are not affected by prolactin. In estrogen-treated mice, increased CD22 and SHP-l lead to a reduction in BCR signaling, which has been shown to skew B-cell development towards a marginal zone phenotype, while in prolactin-treated mice, unchanged levels of CD22 and SHP-1 are associated with stronger B-cell signaling and a predominance of follicular B cells. Interestingly, both bromocriptine and tamoxifen inhibit the activation of autoreactive B cells, but through different mechanisms. Tamoxifen modulates autoreactive B cells by interfering directly with estrogen effects on B cells, while bromocriptine prevents estrogen effects by depriving the immune system of the baseline physiological levels of prolactin. Without prolactin, there is no activation of an immune response, probably because T-cell activation requires prolactin. Prolactin upregulates CD40 expression on B cells, providing the opportunity for enhanced T-cell costimulation of B cells. Incubation of dendritic cells with prolactin strongly increases their antigen-presenting activity. It is speculated that prolactin leads to increased CD40 on the surface of dendritic cells, which facilitates T-cell activation [122]. CD40 engagement will increase expression of the anti-apoptotic gene bcl-2 in B cells [123]; thus, CD40 engagement can rescue transitional B cells from BCR-mediated apoptosis [28]. These observations suggest how prolactin may permit autoreactive B cells to survive and reach maturation. The effects of prolactin, like estrogen, on immune cells are genetically determined. R4A-C57BL/6 mice treated with prolactin did not develop an increased number of transgene-expressing B cells, anti-DNA titers, or IgG deposits in the glomeruli, showing that prolactin can induce a lupus-like phenotype only in a susceptible genetic background.

V. PROPOSED MODELS FOR THE CONTRIBUTION OF SEX HORMONES AND SLE A. Estrogen-Induced Lupus There are data demonstrating that estrogen can exacerbate lupus in both human and in animal models. An elevation in estrogen has profound effects on B-cell development, survival, and activation. Based on the data described above, we propose a model describing how estrogen-induced changes in B-cell gene expression lead to a lupus-like phenotype in mice. Either an increase in estrogen levels or enhanced estrogen responsiveness due to quantitative or qualitative alterations in ER␣ and ER␤ lead to augmented expression of certain genes important in B-cell development, survival, and activation. Increased expression of the antiapoptotic molecule Bcl-2 may impair elimination of autoreactivity that arises at the immature stage of B-cell development. In addition, increased expression of CD22 and SHP-1 will diminish the intensity of the signal mediated by the BCR. Augmented expression of CD22 and SHP-1 causes a weaker signal on a BCR engagement, making it more difficult to delete autoreactive B cells. There is also evidence that the weaker BCR signal would favor the maturation of marginal zone B cells [25,124]. Once the autoreactive

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marginal zone B cells have escaped tolerance induction, bloodborne foreign or self-antigen can directly activate this population to become autoantibody-secreting plasma cells. Tamoxifen appears to block some but not all of the effects of estrogen on B cells. The estrogen-induced upregulation of Bcl-2 in B cells is not antagonized by tamoxifen, but tamoxifen does block the increased expression of CD22. These observations can explain increased survival of autoreactive B cells, without the activation of these cells in mice treated with both estrogen and tamoxifen. Since B-cell tolerance is maintained in these treated mice, modulation of BCR signaling pathways seems crucial for the estrogen-induced loss of B-cell tolerance. B. Prolactin-Induced Lupus Human and murine studies have implicated prolactin in the development of lupus. Increased serum levels of prolactin affect B-cell survival, maturation, and activation leading to the development of a lupus-like phenotype in mice with a susceptible genetic background. CD4Ⳮ T cells are required for prolactin to exert its immunostimulatory effects on autoreactive B cells. Upregulation of Bcl-2 contributes to the survival of autoreactive B cells, while upregulation of CD40 on B cells may facilitate T-cell help and thus rescue B cells triggered for apoptosis. The B cells will develop as mature follicular B cells. C. Summary SLE-like serology can be hormonally regulated in some mouse strains and not in others. This suggests that only some patients are likely to have a hormonally regulated disease. The study of hormones and B-cell tolerance can, however, identify pathways in which dysregulation leads to autoreactivity. Thus, these studies will help identify targets for new therapies in autoimmune disease. REFERENCES 1. Butterworth M, McClellan B, Allansmith M. Influence of sex in immunoglobulin levels. Nature 1967; 214:1224–1225. 2. Sandborg C. Expression of autoimmunity in the transition from childhood to adulthood: role of cytokines and gender. J Adolesc Health 2002; 30:76–80. 3. Ansar AS, Penhale WJ, Talal N. Sex hormones, immune responses, and autoimmune diseases. Mechanisms of sex hormone action. Am J Pathol 1985; 121:531–551. 4. Whitacre CC. Sex differences in autoimmune disease. Nat Immunol 2001; 2:777–780. 5. Ansar AS, Talal N. Sex hormones and autoimmune rheumatic disorders. Scand J Rheumatol 1989; 18:69–76. 6. Kotzin BL. Systemic lupus erythematosus. Cell 1996; 85:303–306. 7. Grossman CJ, Roselle GA, Mendenhall CL. Sex steroid regulation of autoimmunity. J Steroid Biochem Mol Biol 1991; 40:649–659. 8. Petri M, Howard D, Repke J. Frequency of lupus flare in pregnancy. The Hopkins Lupus Pregnancy Center experience. Arthritis Rheum 1991; 34:1538–1545. 9. Wong KL, Chan FY, Lee CP. Outcome of pregnancy in patients with systemic lupus erythematosus. A prospective study. Arch Intern Med 1991; 151:269–273. 10. Urowitz MB, Gladman DD, Farewell VT, Stewart J, McDonald J. Lupus and pregnancy studies. Arthritis Rheum 1993; 36:1392–1397. 11. Lahita RG, Bradlow HL. Klinefelter’s syndrome: hormone metabolism in hypogonadal males with systemic lupus erythematosus. J Rheumatol 1987; 13:154–157.

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8 Vitamin D3 in Control of Immune Response CHANTAL MATHIEU, EVELYNE VAN ETTEN, LUT OVERBERGH, and ROGER BOUILLON Katholieke Universiteit Leuven, Leuven, Belgium

I. 1,25-DIHYDROXYVITAMIN D3 1,25-Dihydroxyvitamin D3 [1,25(OH)2D3] is the biologically active form of the secosteroid vitamin D3 and was originally found to be important for bone and mineral homeostasis. Vitamin D3 can be derived from nutrients (e.g., fortified diary products, fatty fish, and fish liver oils) but is mainly derived from photosynthesis in the skin [1]. The first activation step in the synthesis of active 1,25(OH)2D3 (reviewed in Ref. 2), the 25-hydroxylation of vitamin D3 to 25(OH)D3, occurs predominantly in the liver. This 25-hydroxylated form of vitamin D3 is quite stable (half-life of 3 weeks), and it is mainly in this form that the hormone is transported, bound to vitamin D3 –binding protein (DBP), in the serum. Further hydroxylation by the enzyme 25(OH)D3-1␣-hydroxylase is required to obtain the biologically active form of vitamin D3, 1,25(OH)2D3. This second hydroxylation step occurs mainly in the convoluted tubule cells of the kidney. In addition to its central function in mineral homeostasis and bone metabolism, 1,25(OH)2D3, also has important effects on the differentiation, growth, and function of many other target tissues outside this system, including normal (e.g., keratinocytes of the skin, islet cells of the pancreas) as well as malignant cell types (e.g., breast, colon, and prostate cancer cells). These biological effects of 1,25(OH)2D3 are mediated through the vitamin D3 receptor (VDR), a member of the steroid-nuclear receptor superfamily, whose members include receptors that bind glucocorticoids, retinoids, thyroid hormones, sex steroids, fatty acids, and eicosanoids [3]. Ligand binding induces conformational changes in the VDR, subsequently promoting heterodimerization with the retinoid X receptor 145

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(RXR) and binding of this complex to different types of vitamin D3 response elements (VDREs) within the promotor region of target genes. The classical DR3-type VDRE is composed of a direct hexanucleotide repeat separated by three interspacing nucleotides [4]. The existance of other natural VDREs has also been reported such as direct repeats with four or six interpacing nucleotides or an inverted palindromic arrangement of two hexameric binding motifs referred to as the IP9-type VDRE [5]. After binding of the ligand–VDR–RXR complex to a VDRE, a number of nuclear receptor coactivator proteins, including steroid receptor coactivator family (SRC) members and a novel coactivator complex, D receptor interacting proteins (DRIP), are recruited, inducing chromatin remodeling through intrinsic histone-modifying activities and direct recruitment of key components of the transcription initiation complex at the regulated promoters. Thus, the VDR functions as a ligand-activated transcription factor that binds to specific DNA sequence elements (VDRE) in vitamin D3 –responsive genes and ultimately influences the rate of RNA polymerase II–mediated transcription [6]. II. METABOLISM OF 1,25-DIHYDROXYVITAMIN D3 IN THE IMMUNE SYSTEM The detection of VDR in almost all cells of the immune system [7], especially in antigenpresenting cells (APC) (macrophages and dendritic cells) and activated T lymphocytes, has led to the investigation of a potential role for 1,25(OH)2D3 as immunomodulator. Application of the molecule in vitro and in vivo in a multitude of settings confirmed the importance of the role of 1,25(OH)2D3 in the immune system [8,9]. Not only is the VDR present in a broad range of cells of the immune system, making them responsive for the actions of 1,25(OH)2D3, but activated macrophages are also able to synthesize and secrete 1,25(OH)2D3 in a regulated fashion. These cells indeed express 25(OH)D3-1-␣-hydroxylase, the enzyme responsible for the ultimate and rate-limiting hydroxylation step in the synthesis of active 1,25(OH)2D3, as could recently be demonstrated on the molecular level by RT-PCR in activated macrophages [10]. Although cloning and sequencing of the mRNA clearly demonstrated this enzyme to be identical to the known renal form, its regulation seems to be under completely different control. The renal enzyme is predominantly under the control of mediators of calcium and bone homeostasis (such as parathyroid hormone and 1,25(OH)2D3 itself) while macrophages are mainly sensitive to immune signals, with IFN-␥ being one of the most powerful. Moreover, in macrophages no clear downregulation of the enzyme by the end product, 1,25(OH)2D3, could be observed, explaining the hypercalcemia occurring in situations of macrophage over-activation such as in tuberculosis or sarcoidosis [11]. The secretion of classical macrophage products, such as the cytokines IL-1, TNF-␣, and IL-12, precedes the transcription of the enzyme and thus the secretion of 1,25(OH)2D3. Therefore, the timing of 1-␣-hydroxylase is compatible with that of an autocrine-suppressive feedback signal, first allowing recruitment and activation of other immune cells, followed by a downtapering of the immune reaction. Additionally, the activation stage of the macrophage seems to be a prerequisite for induction of 1-␣-hydroxylase, since induction is only evident in differentiated macrophages, reached by, e.g., a combined incubation of IFN-␥ with phorbol-12-myristate-13-acetate (PMA), lipopolysaccharide, (LPS), or TNF-␣ (unpublished results). Next to the presence of 1-␣-hydroxylase, 1,25(OH)2D3-24-hydroxylase, the major 1,25(OH)2D3-degrading enzyme, is also expressed in monocytes/macrophages. 24-Hydroxylase is highly inducible by 1,25(OH)2D3 itself. This induction is mediated by binding of

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ligand–VDR–RXR complexes to specific VDREs in the promoter region of 24-hydroxylase [12]. In the monocyte/macrophage lineage, however, the 1,25(OH)2D3-mediated induction of 24-hydroxylase is highly dependent on the activation/differentiation stage of the cell. Undifferentiated monocytes are especially susceptible to 1,25(OH)2D3-mediated induction of 24-hydroxylase. In activated/differentiated macrophages, a resistance to 1,25(OH)2D3 in the control of its own production and degradation is observed. This is mainly due to antagonistic effects of IFN-␥ on 1,25(OH)2D3 action, mediated by IFN-␥/ 1,25(OH)2D3 cross-talk. Indeed, nuclear accumulation of Stat-1 and VDR by IFN-␥ and 1,25(OH)2D3 was shown to induce protein-protein interactions between Stat-1 and the DNA binding domain of the VDR. Thereby binding of ligand-bound VDR-RXR to the VDRE in the 24-hydroxylase promoter is prevented, and 1,25(OH)2D3 activation of 24hydroxylase is inhibited [13,14]. The almost universal presence of VDR in the different cells of the immune system combined with the presence of 1-␣-hydroxylase activity, regulated by specific immune signals, suggests a paracrine role for 1,25(OH)2D3 in the immune system. III. MOLECULAR PATHWAYS OF IMMUNOMODULATION BY 1,25(OH)2D3 Due to the expression of VDR in activated T lymphocytes, 1,25(OH)2D3 can act directly on these immune cells. In this way the T-helper 1 cytokines IFN-␥ and IL-2 are directly inhibited. By inhibiting IFN-␥ transcription, a positive feedback signal for antigen-presenting cells, 1,25(OH)2D3 prevents further antigen presentation and recruitment of T lymphocytes. This inhibition occurs directly through interaction of the ligand–VDR–RXR complex with a negative VDRE in the promotor region of the cytokine gene. Moreover, progressive deletion analysis of the IFN-␥ promotor revealed that negative regulation by 1,25(OH)2D3 is also exerted at the level of an upstream region containing an enhancer element [15]. On the other hand, 1,25(OH)2D3-mediated inhibition of IL-2 transcription occurs through impairment of nuclear factor of activated T cells (NF-AT)/AP-1 protein complex formation and subsequent association of the ligand–VDR–RXR complex with the NF-AT binding site in the IL-2 promotor [16,17]. As a consequence of this inhibited IL-2 expression, further T-helper 1 activation and proliferation is blocked by 1,25(OH)2D3. Not only is the expression of T-helper 1 cytokines directly inhibited, but T-helper 2 cytokines stand under the direct influence of 1,25(OH)2D3. Direct induction of a T-helper 2 phenotype by 1,25(OH)2D3 occurs through the increased expression of the T-helper 2–specific transcription factors GATA-3 and c-maf and results in the production of the cytokines IL-4, IL-5, and IL-10 [18]. On the other hand, Staeva-Vieira and Freedman demonstrated that the ligand-bound VDR directly inhibits IL-4 transcription by interaction with the IL4 promotor, probably at the level of the NF-AT–binding sites [19]. Most immunomodulators that presently are used in the clinic focus their action on the T lymphocytes. Because of this major overlap in mechanism of action between the different immunomodulators, the benefit that can be obtained from a combination of several of these drugs is limited. A major asset of 1,25(OH)2D3 as an immunomodulator is the fact that it not only interacts with T lymphocytes but primarily targets the pivotal cell in the immune cascade, the antigen-presenting cell. Chemotactic and phagocytotic capacity of monocytes/macrophages (necessary for the tumor cell cytotoxicity and mycobactericidal activity of these cells) are enhanced by exposure to 1,25(OH)2D3, whereas their antigenpresenting cell function is decreased [20]. Moreover, it has been demonstrated by different

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research groups that 1,25(OH)2D3 also inhibits the differentiation and maturation of the most potent among the antigen-presenting cells, the dendritic cell (DC) [21–25]. In vitro differentiation of DC from its precursors (human peripheral blood monocytes or murine bone marrow precursors) is inhibited by 1,25(OH)2D3 and in some cases even deviated, giving rise to a cell type still positive for CD14, a non-DC surface marker. Moreover, the APC function of DC is profoundly altered by 1,25(OH)2D3. Surface expression of MHC class II complexes and of costimulatory molecules, such as CD86, CD80, and CD40, is inhibited by treatment with 1,25(OH)2D3 during in vitro or in vivo maturation. The secretion of cytokines by APC, which are crucial for recruitment and activation of T lymphocytes, is influenced by 1,25(OH)2D3. IL-12, the major cytokine determining in which direction the immune system will be activated, is clearly inhibited by 1,25(OH)2D3 [26,27]. IL-12 stimulates the development of CD4Ⳮ T-helper 1 lymphocytes, secreting mainly IL-2 and IFN-␥, and inhibits the development of CD4Ⳮ T-helper 2 lymphocytes, secreting cytokines such as IL-4, IL-5, and IL-10. T-helper 1 lymphocytes are considered to be the most important cells in graft rejection and autoimmunity, while T-helper 2 lymphocytes have a more regulatory function in these disorders. Due to this inhibition of IL-12, observed in vitro (by ELISA and by intracellular FACS analysis) as well as in vivo (by RT-PCR), 1,25(OH)2D3 interferes directly with the heart of the immune cascade shifting the ongoing reaction towards a T-helper 2 profile [28]. In addition, expression of the immunoregulatory cytokine IL-10, opposing the T-helper 1–driving effects of IL-12, is increased by treatment with 1,25(OH)2D3 [21]. Other cytokines secreted by APC are under the influence of 1,25(OH)2D3: the suppressive prostaglandin E2 is stimulated, whereas the monocyte recruiter granulocyte-macrophage colony-stimulating factor (GM-CSF) is suppressed [29,30]. The different molecular mechanisms that 1,25(OH)2D3 utilizes to regulate production of the various cytokines under its influence are intriguing. Whereas IL-2 secretion is inhibited through direct interference with binding of NF-AT to the promotor region of the cytokine gene and IFN-␥ is directly downregulated through interaction of the ligandbound VDR-RXR complex with an inhibitory VDRE, IL-12 secretion is inhibited through interference with the NF-␬B pathway [27]. Suppression of GM-CSF is achieved in yet another, rather exceptional, manner: whereas the ligand-VDR typically forms heterodimers with RXR and thus exerts its effects at DNA level, GM-CSF secretion is inhibited by binding of ligand-bound VDR monomers to functional repressive complexes in the promotor region of the cytokine gene [30]. All these changes (antigen presentation, costimulation, cytokine production) induced by 1,25(OH)2D3 result in an APC with decreased T-lymphocyte–activation capacities. T lymphocytes are, besides the direct effects of 1,25(OH)2D3 on the T-lymphocyte level, particularly in this indirect way influenced by 1,25(OH)2D3. A complete hypo-responsiveness (proliferation as well as cytokine production is inhibited) of naı¨ve T lymphocytes can be observed when cultured in vitro in the presence of 1,25(OH)2D3-modified DC but in the absence of 1,25(OH)2D3 itself [22]. One study even shows that committed human autoreactive T lymphocytes can be influenced by 1,25(OH)2D3-modified DC [21]. After incubation with 1,25(OH)2D3-modified DC but again without 1,25(OH)2D3 itself, IFN-␥ production in these autoreactive T lymphocytes was decreased, suggesting a redirection towards a more T-helper 2 profile. Also in vivo, 1,25(OH)2D3 can induce DC with tolerogenic properties, as demonstrated in models of allograft rejection. These in vivo generated tolerogenic DC are probably responsible for the induction of CD4ⳭCD25Ⳮ regulatory T lymphocytes mediating tolerance towards insulin-producing islet allografts [31,32].

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Moreover, in nonobese diabetic (NOD) mice (having a defect in these regulatory T lymphocytes) a restoration of the regulatory T-lymphocyte population was observed after treatment with 1,25(OH)2D3 [33]. When this regulatory T-lymphocyte population was eliminated by cyclophosphamide, however, 1,25(OH)2D3-treated mice were still protected from diabetes development [34]. These data confirm that the effects of 1,25(OH)2D3 are not solely mediated through induction of regulatory T lymphocytes, but also through elimination of effector T lymphocytes. Indeed, restoration of the apoptosis sensitivity centrally in the thymus of NOD mice could also be observed by treatment with 1,25(OH)2D3, resulting in a better elimination of autoreactive diabetogenic T lymphocytes [34,35]. IV. SYNTHETIC ANALOGS OF 1,25(OH)2D3 The major drawback related to the widespread use of 1,25(OH)2D3 in clinical application for immunomodulatory purposes is the dose-limiting calcemic side effect (such as hypercalcemia, hypercalciuria, and increased bone resorption). Intensive efforts have led to the development of synthetic 1,25(OH)2D3 analogs that maintain or amplify its nonclassical effects while decreasing its hypercalcemic potential [36–38]. The secosteroid 1,25(OH)2D3 with its open B-ring and side chain is indeed a very flexible molecule. Different parts of the molecule have been modified (A, seco-B, C, and D ring and side chain) by addition or transposition of hydroxyl groups, introduction of unsaturated bonds and hetero atoms, inversion of the stereochemistry, and alterations in the length of the side chain. Moreover, nonsteroidal analogs have been created that lack the full CD region of the parent molecule [39,40]. The recent elucidation of the crystal structure of the VDR bound to its natural ligand [41] will further facilitate the rational design of future 1,25(OH)2D3 analogs with improved immunomodulatory potency, reduced calcemic effects, and increased tissue specificity. Several mechanisms may contribute to the altered biological profile of these analogs when compared to 1,25(OH)2D3. Differences in binding affinity to DBP may result in altered availability and clearance rates in the various target tissue [42]. Metabolization of analogs of 1,25(OH)2D3 is probably different than for the parent compound. It is therefore conseivable that the metabolization rate and the occurence of analog-specific metabolites contribute to the altered biological profile and tissue specificity [43]. Altered affinity to the VDR and modifications in the VDR conformation and stability due to interaction with an analog may affect the heterodimerization with RXR, the binding of the analog-VDRRXR complex to a VDRE, and the interaction with coactivators and factors of the preinitiation complex and thus lead to differences in gene transcription [44,45]. Moreover, the existence of tissue-specific forms of VDR has been postulated to explain the apparent tissue specificity of certain analogs [46]. V. POTENTIAL OF 1,25(OH)2D3 AND ITS ANALOGS AS IMMUNOMODULATORS IN VIVO The fact that 1,25(OH)2D3 influences the immune system, not by plain suppression, but by modulation through induction of immune shifts and regulator cells, makes this compound very appealing for clinical use, especially in the treatment and prevention of autoimmune diseases and in the prevention of graft rejection. The structural analogs of 1,25(OH)2D3 that have been tested in in vivo models of autoimmunity and transplantation are summarized in Table 1. It has indeed been demonstrated that autoimmune type 1

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Table 1 1,25(OH)2D3 and Its Analogs in Animal Models of Autoimmune Diseases and Transplantation Animal model Autoimmunity Arthritis Collagen-induced Lyme Diabetes Type 1 Low-dose streptozotocin-induced Experimental autoimmune encephalomyelitis Inflammatory bowel disease Systemic lupus erythematosus Nephritis Heyman Lupus Mercuric chloride–induced Experimental autoimmune thyroiditis Transplantation Aorta Bone marrow Heart Liver Pancreatic islets

Skin Small bowel

Major effects

Ref.

Prevention of disease, suppression of severity Prevention of disease, amelioration of symptoms

54,55 55

Prevention of disease, inhibition of insulitis and diabetes Decreased incidence of insulitis and diabetes

33,34,47,48, 58 86

Prevention of disease, attenuation of severity, delay of onset, inhibition of relapse Amelioration of symptoms, block of disease progression Prevention of disease

51–53, 67–69, 87, 88 57

Reduction of proteinuria and autoantibodies Reduction of proteinuria, prevention of skin lesions Prevention of proteinuria, downregulation of serum antibody levels Reduction of histological lesions and severity

56 50,89

Reduced allograft rejection Decreased graft-versus-host disease Prolongation of vascularized and nonvascularized allograft susrvival Decreased severity of acute allograft rejection Induction of allo-transplantation tolerance, prevention of autoimmune recurrence after syngeneic transplantation, prevention of early xenograft failure Prolonged allograft survival Prolonged allograft survival

66 93 60–62

50

90,91 92

63 31,59,71,94

64,65 62

diabetes can be prevented in NOD mice by 1,25(OH)2D3 and its analogs [33,47]. Not only did the treatment of NOD mice from weaning until old age prevent clinical diabetes, but the histological lesion, insulitis, became less prevalent [48]. In this model of autoimmune diabetes, upregulation of regulatory immune cells and a shift from T-helper 1 towards Thelper 2 lymphocytes locally in the pancreases of treated mice was observed. This protective T-helper 2 population is induced not only at the site of the ␤-cell attack, but also in the peripheral immune system [49]. After immunization of 1,25(OH)2D3-treated NOD

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mice with a diabetes-specific auto-antigen (a peptide of GAD65), lymphocytes of the draining lymph nodes showed increased IL-4 and decreased IFN-␥ production in vitro (ELISA) and in vivo (RT-PCR). Strikingly, this immune deviation induced by 1,25(OH)2D3 is limited to pancreatic auto-antigens and was not seen after immunization with the ␤-cell–irrelevant protein ovalbumin. Other effects on the immune system have been described, the most important being a restoration of the defective apoptosis sensitivity of lymphocytes, leading to a more efficient elimination of potentially dangerous autoimmune effector cells [34,35]. This increased apoptosis, induced by 1,25(OH)2D3 and its analogs in DC and T lymphocytes of NOD mice, has been described after different apoptosis-inducing signals (e.g., corticosteroids) and could help to explain why early short-term treatment with these agents, before the onset of autoimmunity, confers long-term protection and promotes tolerance restoration. 1,25(OH)2D3 and its analogs have been applied in other spontaneous and experimental models of autoimmune diseases. They can prevent systemic lupus erythematosus in lpr/ lpr mice [50], experimental allergic encephalomyelitis (EAE) [51–53], collagen-induced arthritis [54,55], Heymann nephritis [56], and inflammatory bowel disease [57]. 1,25(OH)2D3 and its analogs are able to not only prevent the onset of but also treat ongoing autoimmune diseases. Treatment of NOD mice with analogs of 1,25(OH)2D3 can prevent the progression of an initial ␤-cell attack (reflected by the presence of insulitis) to clinical overt diabetes [58]. Interestingly, in this model of secondary prevention, no suppressor cells could be demonstrated. Within the pancreases of protected mice, again a shift from T-helper 1 towards T-helper 2 cytokines is noted. Moreover, analogs of 1,25(OH)2D3 are able to inhibit the recurrence of autoimmune diabetes after syngeneic islet transplantation in NOD mice, suggesting that a completely established autoimmune disease can be overcome and thus reinduction of self-tolerance can be achieved [59]. Analysis of the cytokine profile in the surviving grafts themselves again demonstrated the induction of the same immune shift from T-helper 1 to T-helper 2. In addition, 1,25(OH)2D3 and its analogs prolong the survival of heart [60–62], liver [63], skin [64,65], small bowel [62], and pancreatic islet allografts [31] and inhibit, in association with CsA, not only acute but also chronic allograft rejection [66]. VI. IMMUNOMODULATORY SYNERGISM BETWEEN 1,25(OH)2D3 OR ITS ANALOGS AND CLASSICAL IMMUNOSUPPRESSANTS An additional way to avoid hypercalcemic side effects of 1,25(OH)2D3 is by exploiting the additive and even synergistic effects that can be observed between 1,25(OH)2D3 or its analogs and more classical immunosuppressants, such as cyclosporin A, and by using these combinations at individual subtherapeutic levels [67]. These effects are observed in in vitro phytothemagglutinin-stimulated T-lymphocyte proliferation or mixed lymphocyte reaction (MLR) and can be confirmed in vivo in different models of autoimmune diseases and in graft rejection (summarized in Table 2). In vitro, varying levels of synergism with 1,25(OH)2D3 can be observed for different immunosuppressants, with cyclosporin A having one of the strongest synergisms with 1,25(OH)2D3 and mycophenolate mofetil belonging to the category of weaker (but still) synergistic agents [67]. In experimental autoimmune encephalomyelitis, 1,25(OH)2D3 itself [68,69], as well as its analogs [67], are found to work synergistically with the different immunosuppressants cyclosporin A, rapamycin and mycophenolate mofetil, leading to near-total disease protec-

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Table 2 1,25(OH)2D3 and Its Analogs Combined In Vitro and In Vivo with Other Immunosuppressants Drug Cyclosporin A

Rapamycin FK506 Mofetil Leflunomide Dexamethasone

Cyclosporin A

Rapamycin Mofetil IFN-␤

Model In vitro models Proliferation and IL-2 production of PHA-stimulated PBMC Proliferation and cytokine production in MLR Proliferation of PHA-stimulated PBMC Proliferation of PHA-stimulated PBMC Proliferation and cytokine production in MLR Proliferation of PHA-stimulated PBMC Proliferation of PHA-stimulated PBMC Proliferation and cytokine production of anti-CD3stimulated PBMC Proliferation, cytokine, and chemokine production and T-cell activation of dendritic cells Induction of regulatory IL-10 producing T lymphocytes In vivo models Experimental autoimmune encephalomyelitis Type 1 diabetes Mercuric chloride induced autoimmunity Adjuvant arthritis Thyroiditis Transplantation of syngeneic islets Transplantation of xenogeneic islets Transplantation of vascularized renal allografts Transplantation of liver allografts Bone marrow transplantation Transplantation of aorta allografts Transplantation of heart allografts Transplantation of skin allografts Experimental autoimmune encephalomyelitis Experimental autoimmune encephalomyelitis Transplantation of allogeneic islets Transplantation of syngeneic islets

Ref. 67,95,96 70,101 67,69 67 70 67 67 98 99 100 67,68 58,101 90,91 102 92 59,97,103,104 94 105,106 63 93 66 107 108 69 67 31 71,109

PHA, PBMC, peripheral blood mononuclear cells; MLR.

tion accompanied by fewer side effects compared to monotherapy. Moreover, in syngeneic islet transplantation in NOD mice, a combination of analogs of 1,25(OH)2D3 with the classical immunosuppressants cyclosporin A and FK506 resulted in complete prevention of autoimmune recurrence, even after withdrawal of therapy, suggesting a reinduction of self-tolerance [70]. The immunosuppressants mentioned above all interact at different levels with the T-lymphocyte activation and proliferation cascade (Fig. 1). Cyclosporin A and FK506, after binding to their binding proteins, interact with calcineurin, inhibiting its dephosphorylating activity and consequently preventing the passage of the transcription factor NF-AT to the nucleus. In this way, activation of T lymphocytes is blocked in a very early phase. Rapamycin blocks a step in the intracellular protein kinase cascade after

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Figure 1 Schematic overview of the molecular mechanisms of action of 1,25(OH)2D3, cyclosporin A, Rpamycin, FK506, and mycophenolate mofetil in T-helper lymphocytes and antigenpresenting cells. Cyclosporin A (CsA) and FK506 interact with the Ca2Ⳮ/calmodulin/calcineurin pathway in T lymphocytes inhibiting the dephosphorylating activity of calcineurin and consequently preventing the passage of the transcription factor NF-AT to the nucleus. Rapamycin (Rap) blocks a step in the intracellular protein kinase cascade after binding of IL-2 to its receptor, thus preventing further autocrine-stimulated proliferation of the T lymphocytes. Also Ca2Ⳮ-independent pathways of T-lymphocyte activation are inhibited by rapamycin. Mycophenolate mophetil (MMF) interferes with the de novo nucleotide synthesis in T lymphocytes, leading to abrogation of T-lymphocyte proliferation. 1,25(OH)2D3 (D3) also acts directly on T lymphocytes. It inhibits the transcription of the T-helper 1 cytokines IL-2 (by interference with NF-AT/AP-1 complex formation and with binding of NF-AT to the IL-2 promotor region) and IFN-␥ (through direct interaction with an inhibitory VDRE located in the IFN-␥ promotor). Moreover, it is the only drug shown here that has direct effects on the level of the antigen-presenting cell (APC). 1,25(OH)2D3 inhibits the production of the T-helper 1–inducing cytokine IL-12 (by interference with the NF-␬B pathway). Also, antigen presentation and costimulation are inhibited by 1,25(OH)2D3, leading to downregulation of the T-lymphocyte activation capacities of the APC. Transcription of GM-CSF in the APC is inhibited by binding of ligand-bound VDR monomers to functional repressive complexes in the GM-CSF promotor.

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binding of IL-2 to its receptor, preventing further auto-activation and proliferation of T lymphocytes. Mycophenolate mofetil inhibits de novo nucleotide synthesis, leading to loss of DNA synthesis and eventually to abrogation of T-lymphocyte proliferation. More recently, it was found that interferon-␤ acts synergistically with analogs of 1,25(OH)2D3 in the model of syngeneic islet transplantation [71]. VII. IMMUNE EFFECTS OF VITAMIN D3 DEFICIENCY Considering the fact that these immune effects of 1,25(OH)2D3 are mediated through the VDR, the immune system of VDR knockout (VDR-KO) mice was investigated. VDRKO mice are characterized by severe hypocalcemia, impaired bone formation, and alopecia as in humans with vitamin D3 –dependent rickets type II [72]. In these VDR-KO mice, mild immune defects can be observed in vitro at the level of macrophage chemotaxis and T-lymphocyte proliferation after anti-CD3 stimulation (a calcium dependent pathway). In vivo these defects are reflected by a near-complete resistance to experimentally induced diabetes. However, correcting for the hypocalcemia observed in these vitamin D3 –resistant mice using dietary intervention restores completely the immune abnormalities [73]. This failure of VDR-KO mice to display major immune abnormalities, which moreover can be restored by normalizing calcium levels, probably suggests that 1,25(OH)2D3 serves a redundant function in the immune system. When not the function but the synthesis of 1,25(OH)2D3 is interrupted by targeted ablation of the 25(OH)D3-1-␣-hydroxylase enzyme, a significant reduction in CD4Ⳮ and CD8Ⳮ peripheral T lymphocytes can be observed [74]. VIII. EPIDEMIOLOGY OF IMMUNE EFFECTS OF VITAMIN D3 Several data point towards a possible physiological role for vitamin D3 in the immune system. Epidemiological studies have for many years shown a risk of impaired glucose tolerance and reduced insulin secretion in vitamin D3 –deficient states, such as rickets. Restoration of vitamin D3 status also corrected glucose metabolism [75,76]. Furthermore, correlations have been described between the risk of type 1 diabetes and the presence of certain VDR polymorphisms [77,78]. From the four major VDR variants that have been identified (FokI, BsmI, ApaI, and TaqI), FokI is the only one resulting in an alternative transcription initiation site and thus in the expression of an alternative VDR protein. Recently, a positive correlation between this FokI allele of VDR and type 1 diabetes has been described [79]. Dahlquist et al. have demonstrated a correlation between supplements of vitamin D3 in early life and the risk of type 1 diabetes later on [80]. Children exposed to vitamin D3 supplements in early infancy were better protected from the development of type 1 diabetes. A major problem of this study is, however, the fact that no data are available on the initial vitamin D3 status of the children or the doses of vitamin D3 administered. Therefore, in this study no distinction between the effect of restoring normal vitamin D3 levels in deficient children and the effect of supplementing vitamin D3 to children with a normal vitamin D3 status can be made. Moreover, the authors were not clear about the form of vitamin D3 administered (regular vitamin D3 or its activated form, 1,25(OH)2D3). These issues have to be solved before embarking on large-scale clinical trials investigating the effect of vitamin D3 supplements in early life on type 1 diabetes incidence later in life.

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Another epidemiological study demonstrated not only a correlation between vitamin D3 supplements in early life and type 1 diabetes, but more convincingly that children with vitamin D3 deficiency or rickets in early life have a threefold increase in diabetes incidence later in life [81]. Preliminary data in NOD mice confirm that the rachitic state predisposes to the development of type 1 diabetes. However, a small study in NOD mice shows that treating NOD mice with normal vitamin D3 levels for a short period of time, corresponding to neonatal life and childhood in humans, with physiological supplements of regular vitamin D3 [82] or one of the structural analogs of 1,25(OH)2D3 [83] is not sufficient to prevent type 1 diabetes. Treated mice did show a clear conservation of their ␤-cell potential. Not only the diabetic but also the normo-glycemic mice have clearly higher levels of insulin in their pancreas. In vitro ␤-cell protective effects of 1,25(OH)2D3 have been shown, protecting ␤-cells against cytokine-mediated cell death and preserving insulin synthesis and secretion [84,85]. At present, the physiological role of vitamin D3 in autoimmune diseases such as type 1 diabetes is not clear. It might be that vitamin D3 supplements do not alter the disease appearance, but that the vitamin D3 –deficient state specifically predisposes to the development of the disease. At present there are not enough data to support intervention trials with daily vitamin D3 in large populations. Individuals with normal levels of vitamin D3 should not use regular vitamin D3 but pharmacologically high levels of 1,25(OH)2D3 or an analog in order to observe deviations in the immune system and protection against disease.

IX. CONCLUSIONS The widespread presence of VDR in the immune system and the regulated expression of 1-␣-hydroxylase by specific immune signals suggest a paracrine immunomodulatory role for 1,25(OH)2D3 and its analogs. Moreover, a pharmacological potential for 1,25(OH)2D3 and its analogs to modulate the immune system has been demonstrated, making them interesting to use in the prevention and treatment of autoimmune diseases and the prevention of graft rejection. The immune effects of these VDR ligands are primarily mediated via actions on the APC, although direct effects at the T-lymphocyte level are also present, resulting in inhibition of pathogenic T lymphocytes and enhancement of the frequency of T lymphocytes with regulatory properties. This unique capacity of 1,25(OH)2D3 to directly interfere with APC leads to strong synergistic effects with classical immunosuppressants in multiple models of autoimmunity and transplantation. A physiological role for 1,25(OH)2D3 in the immune system has also been postulated, based on a correlation between vitamin D3 levels and the occurrence of autoimmune diseases.

REFERENCES 1. Holick MF. Photobiology of vitamin D In: Feldman D, Glorieux F, Pike J, Eds. Vitamin D. San Diego: Academic Press, 1997:33–39. 2. Jones G, Strugnell SA, DeLuca HF. Current understanding of the molecular actions of vitamin D. Physiol Rev 1998; 78:1193–1231. 3. Haussler MR, Whitfield GK, Haussler CA, Hsieh JC, Thompson PD, Selznick SH, Dominguez CE, Jurutka PW. The nuclear vitamin D receptor: biological and molecular regulatory properties revealed. J Bone Miner Res 1998; 13:325–349.

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9 The Growth Hormone/Insulin-like Growth Factor-I Axis and the Immune System RON KOOIJMAN Free University of Brussels, Brussels, Belgium

I. INTRODUCTION Bidirectional communication between the neuroendocrine system and the immune system is made possible by the expression of a common set of receptors. This review will deal with several aspects of the interaction between the growth hormone (GH)/insulin-like growth factor-I (IGF-I) axis and the immune system. This interaction is quite complicated, because the immune system does not only express GH and IGF-I receptors, but also produces GH and IGF-I. In addition, whereas endocrine IGF-I in the liver is mainly regulated by GH, IGF-I in the immune system is differentially regulated, mainly by cytokines. Therefore, this review will address possible autocrine and paracrine effects of GH and IGF-I and the regulation of their expression in the immune system compared to the endocrine system. In vitro and in vivo effects of GH and IGF-I have been demonstrated by many investigators. This opens up possibilities for therapeutic intervention, but does not indicate that GH and IGF-I are obligate factors in the immune system. This chapter will mainly address the above-mentioned points of interest and will not cover all the literature. A more complete overview of the literature can be found in other publications [1–5]. II. THE GH/IGF-I AXIS A. Growth Hormone and the Somatomedin Hypothesis GH is a 22 kDa polypeptide hormone that is mainly released from somatrope cells in the anterior pituitary. The pituitary also produces minor quantities of GH variants with molecu163

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lar weights of 20 or 44 kDa by means of alternative splicing or dimerization, respectively [6]. GH release is stimulated by GH-releasing hormone (GHRH) and inhibited by somatostatin, both of which are produced by the hypothalamus. The presence of a third player regulating GH release has emerged from recent studies on GH secretagogues, which are potent synthetic compounds acting through the GH secretagogue receptor on somatotrope cells. A recently discovered endogenous ligand for the GH secretagogue receptor ghrelin is expressed in the stomach and the hypothalamus and induces a potent GH release from the pituitary. Whereas GH is not required for intrauterine growth, it is essential for postnatal growth, especially for the growth spurt during the peripubertal period. This appears from studies with GH receptor null mice that do not show any growth retardation until 10 days after birth [7]. Also, children with a congenital GH deficiency do not exhibit any growth retardation at birth. According to the somatomedin hypothesis of Salmon and Daughaday [8], the growthpromoting effects of GH are mediated by a serum factor that was absent in hypophysectomized rats, but could be induced by GH treatment. Subsequently, this serum factor has been identified as IGF-I. It is mainly produced in the liver under the control of GH. It was assumed that liver-derived, endocrine IGF-I mediates the growth-promoting effects of GH. The finding of D’Ercole and coworkers [9,10] that many nonhepatic tissues also produce IGF-I, often under the control of GH, led to the hypothesis that autocrine or paracrine IGF-I could also mediate the effects of GH. Local production of IGF-I in the growth plate chondrocytes has been disputed, but the expression of IGF-I in the periosteum, perichondrium, fat cells, or muscle has been implicated in the mediation of longitudinal bone growth [11,12]. It should, however, be emphasized that GH also exerts direct (IGFI–independent) effects, for instance, on longitudinal growth through stimulation of germinal zone cell proliferation [13]. In addition to growth-stimulating effects, GH exerts metabolic actions on many tissues. It has lipolytic actions and stimulates protein synthesis in muscle. As a result, GH treatment in adults reduces fat mass and increases lean body weight [14]. B. The Insulin-like Growth Factor System The known components of the IGF system include three ligands that belong to the insulinrelated peptide family (insulin, IGF-I, and IGF-II), their cognate receptors, and six IGFbinding proteins (IGFBPs), which are not structurally related to the receptors of the IGF system. The IGF-I receptor has a high affinity for both IGF-I and IGF-II and mediates the effects of both growth factors. The IGF-II receptor is identical to the cation-independent mannose-6-phosphate receptor. The signal transduction pathway of this receptor is not clear, and it has been postulated that this receptor serves as a sink for excess IGF-II. In order to address the in vivo role of different partners of the IGF system, gene-targeting strategies have been used to produce mice with defects in IGF-I, IGF-II, or their receptors. Targeted disruption of the IGF-I and -II genes revealed that IGF-II is an intrauterine growth factor [15], whereas IGF-I stimulates both intrauterine and postnatal growth [16,17]. Experiments in which the various knockout mice were crossed (reviewed by Nakae et al. [18]) showed that the growth-promoting effects of IGF-I were mediated exclusively by the IGFI receptor, whereas the effects of IGF-II were mediated by the IGF-I and insulin receptors. It was hypothesized that a splice-variant of the insulin receptor with a high affinity for IGF-II, which is highly expressed in fetal tissues, mediates in part the intrauterine growthpromoting effects of IGF-II. Remarkably, disruption of the IGF-II receptor gene resulted

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in a fetal overgrowth syndrome. This lethal phenotype was less severe when IGF-II receptor knockout mice were crossed into an IGF-II–deficient background. These results indicate that high levels of IGF-II are detrimental to fetal growth and that the IGF-II receptor is involved in down-regulation of IGF-II levels. In addition to its role in longitudinal growth, IGF-I also serves as a growth or differentiation factor for tissues other than bone and exerts anabolic effects. The stimulating effects of IGF-I on cell cycling and the inhibitory effects on apoptosis play a pivotal role in tissue homeostasis. Anabolic effects of IGF-I include stimulation of protein synthesis and inhibition of protein degradation and its insulin-like effect: stimulation of glucose uptake. The existence of at least six IGFBPs (reviewed in Ref. 19) renders the IGF system much more complex. IGFBPs are not homologous to IGF receptors, but they do bind IGFI and IGF-II with an affinity comparable to the affinity of the IGF-I receptor for IGF-I and IGF-II. Thereby IGFBPs regulate the amount of free IGF-I in serum, prolong the halflife of IGFs, and modulate tissue distribution. In serum, about 80% of the IGFs exist as a 150 kDa complex comprising IGF, IGFBP-3, and the acid-labile subunit (ALS). In tissues, IGFBPs may inhibit IGF actions by preventing IGF binding to their receptors. Alternatively, IGFBPs prolong the actions of IGFs by ensuring a slow release in the vicinity of target cells. Proteolytic cleavage and phosphorylation of IGFBPs are likely to be involved in the regulation of IGF release. In addition, there is emerging evidence that IGFBPs can exert direct cellular effects in an IGF-independent way, most likely through interaction with cell surface receptors such as integrins. New insights in the role of liver-derived IGF-I were obtained by using the Cre-loxP technique to create mice with IGF-I null mutations in liver [20,21]. The liver IGF-I deficiency resulted in a 75% reduction in circulating IGF-I levels, indicating that the liver is the main source of circulating IGF-I. Remarkably, these mice did not show any decrease in somatic growth, emphasizing the importance of nonhepatic IGF-I for body growth. The absence of a compensatory increase in IGF-I transcripts in other tissues suggests that nonhepatic IGF-I is also important for body growth under physiological conditions. A role for serum IGF-I in body growth became apparent in mice with a simultaneous disruption of ALS and liver IGF-I. As ALS is crucial in the formation of a stable 150 kDa complex, these mice show a further decrease in serum IGF-I to 10–15% of normal levels, which led to a significant reduction in linear growth [22]. The postulated roles in body growth for GH and paracrine or endocrine IGF-I are depicted in Fig. 1. Since local IGF-I production occurs in many cell types, including leukocytes, the discussion on the role of local IGF-I versus liver-derived IGF-I is also relevant with respect to the modulating effects of IGF-I on the immune system.

III. EXPRESSION GH, IGF-I, AND THEIR RECEPTORS IN THE IMMUNE SYSTEM A. GH Receptor Expression in the Immune System GH receptors are ubiquitously expressed. In addition to the liver, many cell types including neurons, adipocytes, muscle cells, kidney cells, mammary epithelium, and leukocytes have been found to express GH receptors [23,24]. The presence of GH receptors on the transformed human B-cell line IM-9 was first detected by radioligand binding assays [25] and later confirmed by affinity cross-linking studies [26]. Radioligand binding studies also revealed the presence of GH receptors on bovine and murine thymocytes [27] and human

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Figure 1 Possible endocrine and autocrine/paracrine effects of GH and IGF-I on body growth and the immune system. Thin arrows represent endocrine pathways, whereas thick arrows indicate autocrine or paracrine effects of GH or IGF-I. Functions for autocrine or paracrine loops for GH and IGF-I in the immune system are suggested by in vitro experiments using antisense oligonucleotides, antibodies against GH, IGF-I, or the IGF-I receptor. Although not shown in this figure, it is not excluded that bone and immune cells also contribute to serum IGF-I.

peripheral blood mononuclear cells (PBMC) [28]. In addition, binding sites for human GH have been shown on rat liver macrophages [29] and on a murine thymic epithelial cell line [30]. To identify the primary targets for GH, several groups investigated the distribution of GH receptors on mononuclear cell subsets by two-color flow cytometry (Table 1). Three different groups reported high levels of GH receptor expression on circulating B cells and monocytes compared to T cells using a GH receptor–specific mAb or labeled GH as a ligand (see Table 1). Human tonsillar lymphoid cells also expressed GH receptors, which were mainly present on B cells [34]. In addition to these studies, Badolato and coworkers [37] quantified the binding of fluorescein-labeled anti-GH receptor and fluorescein-labeled human GH on different subsets of human PBMC. Their results were in agreement with those in Table 1 and showed that higher amounts of both anti-GH receptor and human GH bound to B cells compared to T cells and natural killer (NK) cells. Relatively high expression levels of GH receptor transcripts on B cells were observed by the same workers and, more recently, by Hattori et al. [38]. GH receptor expression on different developmental stages of human thymocytes was assessed by flow cytometry [39]. Less than 5% of the single positive (CD4ⳮ/CD8Ⳮ or CD4Ⳮ/CD8ⳮ) and double positive (CD4ⳭCD8Ⳮ) populations expresses GH receptors, whereas 26% of the immature double negative (CD4ⳮ/CD8ⳮ) thymocytes were GH receptor positive. Further analysis revealed that 90% of the most immature thymocytes, which are CD2Ⳮ/CD3ⳮ/CD4ⳮ/CD8ⳮ/

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Table 1 Expression of GH Receptors on Leukocyte Subsets as Assessed by Flow Cytometry Leukocytes from: Human blood Human blood Human blood Human tonsils Mouse blood Mouse spleen Mouse lymph nodes Rat spleen Rat lymph nodes

Probe

T cells

Anti-GH-R hGH Anti-GH-R Anti-GH-R Bovine GH

5 2–20 2 ⬍5 7 16 15 7

Bovine GH

CD4⫹ T cells

2 ⬍5 8 20 18 10 7 11

CD8⫹ T cells

4 ⬍5 5 5 10 4 6 7

B cells Monocytes Ref. 85 ⬎90 96 77 ⫾50

20 20

95 ⬎90 99 ⫾50

31 32 33 34 35

36

Numbers indicate the percentage of cells stained positive with either anti-GH receptor (GH-R) or labeled GH.

CD19ⳮ/CD34Ⳮ, express GH receptors. In mice, GH receptors were also detected in 24% of the bone marrow cells and 1.6% of the thymocytes [35]. After overnight culture, the proportion of GH receptor–bearing thymocytes increased to 26%. Importantly, concanavalin A (Con A) induced a transient increase in the percentage of GH receptor expressing splenocytes and thymocytes. Similar effects were observed by Thellin et al. [34], who showed that the mitogens phytohemagglutinin (PHA) and Staphylococcus aureus Cowan induced a marked increase in the proportion of GH receptor bearing human tonsillar T cells. Taken together, the data suggest that B cells, monocytes, and T-cell precursors are the primary GH targets among mononuclear cells. However, further studies on the regulation of GH receptor expression are needed to gain more insight into the proposed increase in GHresponsive T-cell subsets as a result of T-cell activation during immune responses. Since human GH can also bind to the prolactin (PRL) receptor (i.e., the lactogenic receptor), the possibility that GH exerts some of its actions through this receptor should also be taken into account. Indeed, PRL receptors are also expressed on leukocytes. B. GH Expression in the Immune System GH expression in the immune system is today well established. Many tissues, including bone marrow, thymus, spleen, and lymph nodes, have been shown to express GH. GH expression in different populations of mononuclear cells has been reviewed in detail by Weigent [40]. Leukocyte-derived GH has been characterized by several groups, and it appears that leukocytes produce bioactive GH. Weigent et al. [41] showed that affinity purified GH from human PBMC has an apparent molecular weight of 22 kDa, as assessed by gel filtration analysis. The biological activity of this leukocyte-derived GH was tested in an Nb2 cell assay. Nb2 cells are rat lymphoma cells, the proliferation of which depends on PRL receptor engagement and activation. The finding that human leukocyte–derived GH stimulates DNA synthesis in Nb2 cells indicates that this molecule, like pituitary GH, has lactogenic activity. Gel filtration assays also revealed the secretion of 22 kDa GH by the human leukemic B-cell lines Raji and Daudi [38]. Using Western blot analysis, it was shown that human thymocytes contain 23 kDa GH [42] and that the human HL-60 cell line expresses 20, 22, and 44 kDa immunoreactive (ir) GH [43].

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Several in vitro studies indicate that leukocyte-derived GH exerts autocrine or paracrine effects in the immune system. An autocrine/paracrine role for GH in splenocyte proliferation is suggested by inhibition studies with antisense deoxyoligonucleotides [44]. Inhibition of GH synthesis in cultured rat splenocytes in the absence of mitogens was shown to result in a 87% decrease in DNA synthesis, which could be reversed by addition of exogenous GH. Experiments by Malarkey and coworkers [45] indicate a possible autocrine of paracrine function for GH produced by human PBMC: the recall antigen Candida stimulated the expression of GH and IFN-␥ in human PBMC. The stimulation of IFN-␥ expression was inhibited by a GH antagonist. Also, exogenous GH induced significant, however limited (Ⳳ15%), stimulating effect on IFN-␥ expression in human PBMC [46]. Another indication for a function of local GH production in the immune system was obtained from human thymocyte cultures. Addition of an affinity-purified anti-GH polyclonal antibody to PHA-stimulated thymocytes inhibited the proliferation of these cells [42]. In contrast to the regulation of nonhepatic IGF-I, the control of GH expression in extra-pituitary tissues is not very clear. There are several indications from the literature that GH regulation in the immune system is different from that in the pituitary, but other reports suggest that a similar GH regulation mechanism exists in leukocytes. In the human, two genes code for GH. The GH-N gene codes for the classical pituitary hormone, and the GH-V gene codes for the placental form. As GH-V is released from the placenta into the maternal circulation, GH-N production is gradually suppressed. The two corresponding proteins are highly homologous and differ by 13 out of 191 amino acids. GH-V binds to the human GH receptor with a similar affinity as GH-N, but it has a lower affinity than GH-N for lactogenic receptors [47]. GH-N has also been shown to stimulate growth in rodents [48,49]. With respect to molecular GH regulation in human leukocytes compared to that in the pituitary, it is of importance to assess which GH gene is actually expressed in the immune system. Hattori et al. [50] showed that human PBMC express GH-N transcripts but no GH-V transcripts. Selective expression of GH-N transcripts was also observed in human cell lines of lymphoid (Hut-78) and of myelomonocytic origin (U937) [51]. We also found selective expression of GH-N in human granulocytes [52] and PBMC (unpublished observations). In contrast, Melen et al. [53] described the expression of both GH-N and GH-V in PBMC from men and women. The regulation of GH-N in the pituitary depends on the transcription factor Pit-1 [54], whereas Pit-1, which is expressed in the human placenta [55], is not obligatory for GH-V gene expression during pregnancy [56]. In mice, pituitary GH expression also depends on Pit-1, but the existence of a GH-V gene has not been shown. The role of Pit-1 in the regulation of the GH genes in leukocytes can be addressed by investigating GH expression in organisms with a genetic defect in Pit-1. Melen and colleagues [53] showed that GH-V is expressed in PBMC from a woman with a Pit-1 deficiency, whereas GH-N gene transcripts were absent. This result suggests that the regulation of GH-N expression in human PBMC depends on a functional transcription factor Pit-1. Snell dwarf mice are deficient in pituitary GH, PRL and thyroid-stimulating hormone (TSH) expression as a result of a mutation in the transcription factor Pit-1 [57]. Although Pit-1 is also expressed in cells of the immune system of normal mice [52,58], two different groups observed expression of GH in the immune system of Snell dwarf mice [59,60]. This would imply that GH expression in murine leukocytes is, in contrast to its expression in the pituitary, Pit-1–independent. Further indications for an alternative regulation mechanism for GH in leukocytes were obtained from rat GH promoter studies in the transfected mouse macrophage cell line P-388 [61]. According to

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these studies, overexpression of Pit-1 resulted in a decrease in GH promoter activity. Although an upstream transcription start site for GH has been indicated by analysis of the 5′-untranslated region of lymphocyte mRNA in bovine lymphocytes [62], there are no indications for alternative promoter usage in dogs [63] or rodents [61]. A pituitary-like regulation for GH in leukocytes is conceivable, because GHRH, somatostatin, ghrelin, and their cognate receptors are expressed in the immune system [38,64–67]. However, although one study demonstrated a stimulatory effect of GHRH on GH expression in rat mononuclear leukocytes [64], two other studies [68,69] showed that GHRH and somatostatin do not affect the spontaneous and PHA-induced secretion of GH by human PBMC. IGF-I is assumed to play a role in the systemic feedback control of serum GH by inhibition of pituitary GH release. In the immune system, IGF-I reduced the number of rat splenocytes that expressed irGH [70], but this effect was not observed in PHA-stimulated human PBMC [68]. In conclusion, the effects in the immune system of hormones that regulate pituitary GH expression are not clear, and it remains to be established whether the observed differences in GH regulation are species-dependent. Interestingly, there is more consensus on the increase in GH expression during leukocyte activation. Varma et al. [71] assessed the number of GH-secreting cells in human PBMC by the enzyme-linked immunoplaque assay. They found that 1% of unstimulated PBMC secreted GH. The GH secretion was increased by at least 100% by stimulation with the T-cell mitogen PHA or interleukin (IL)-2, but not by lipopolysaccharide (LPS), a B-cell mitogen. Additionally, the authors showed that PHA increased the number of GH-secreting cells by about 50%. Hattori et al. [72] studied the regulation of GH secretion by human PBMC using an enzyme-linked immunoassay. The secretion of irGH was increased by in vitro stimulation with PHA or pokeweed mitogen (PWM), but not by LPS. Secreted irGH eluted at the position of a 22 kDa protein on gel chromatography. As mentioned above, GH expression in PBMC was also upregulated by Candida antigen [45]. In vivo observations of Baxter et al. [73] revealed that intraperitoneal injection of LPS enhanced GH expression in the immune system of rats. C. IGF-I Receptor Expression in the Immune System Radioligand binding studies revealed the presence of IGF-I receptors on human peripheral T cells [74–77], monocytes [77], thymocytes [78,79], and neutrophils [80]. Multicolor flow cytometric analysis, using ␣IR3 as an IGF-I receptor–specific mAb, demonstrated that IGF-I receptors are expressed on all major subpopulations of human PBMC: CD4Ⳮ T cells, CD8Ⳮ T cells, monocytes, NK cells, and B cells [77,81]. In addition, thymocytes of all developmental stages (CD4ⳮ/CD8ⳮ, CD4Ⳮ/CD8Ⳮ, CD4ⳮ/CD8Ⳮ, and CD4Ⳮ/CD8ⳮ) express IGF-I receptors [79,82]. The differential expression of IGF-I–binding sites in the rat has been studied by two-color flowcytometry using biotin-labeled des(1–3)IGF-I, which binds poorly to IGFBPs but binds to IGF-I receptors with an affinity comparable to that of IGF-I. The binding capacity for des(1–3)IGF-I was high on monocytes, intermediate on B cells, and low on T cells [83]. More detailed information on IGF-I receptor expression in leukocytes is presented in a number of reviews [1,84]. Several studies indicate that IGF-I receptors are modulated during lymphocyte activation. In rats, the binding of des(1–3)IGF-I on CD4Ⳮ and CD8Ⳮ T cells was increased after stimulation with Con A [83]. Kozak and coworkers [74] showed a transient increase in the amount of 125I-IGF-I bound to human T cells after activation with PHA, and the same effect was observed by Johnson et al. [76] after stimulation with anti-CD3. T-cell

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stimulation with anti-CD3 also resulted in an increase in IGF-I receptor transcripts [85]. High levels of IGF-I receptor expression during the culture period coincided with high rates of DNA-synthesis [74,76]. Earlier reports showed that the proportion of ␣IR3Ⳮ cells within in vivo activated (HLA-DRⳭ) T cells from the synovial fluid of patients with rheumatoid arthritis was markedly lower than in nonactivated (HLAⳮDRⳮ) cells. Additionally, after a 6-day culture period, PHA-activated T cells and CD4Ⳮ/CD45R0Ⳮ cells activated with recall antigens contained only 1–6% ␣IR3Ⳮ cells, whereas nonactivated cells contained 30–54% ␣IR3Ⳮ cells [86]. These results seem to be in contrast with the upregulation of IGF-I receptors described above. However, Walsh and O’Connor [87] recently confirmed the upregulation of IGF-I receptors in response to T-cell activation, but also showed that this was a transient phenomenon. They assessed IGF-I receptor expression by flow cytometry on human T cells that were cultured in the presence of ConA and IL-2 and observed an increase of IGF-I receptor expression by 24 and 48 hours of culture. Remarkably, no IGF-I receptors were detected on day 6. The same workers also showed that IGF-I receptor expression could be augmented through stimulation of CD28 by either a cross-linking antibody or the natural ligand CD80, which is normally expressed on antigen-presenting cells. Since stimulation through CD28 generates an accessory intracellular signal that acts in synergism with T-cell receptor signaling, CD28-mediated upregulation of IGF-I receptor expression might be of physiological importance during T-cell activation in immune responses. D. IGF-I Expression in the Immune System Bioactive IGF-I is produced in lymphoid organs and by several leukocyte subsets. Bone marrow stromal cells, macrophages, and in vitro activated monocytes produce large amounts of IGF-I, whereas activated lymphocytes produce minor quantities. Detailed information on the expression and secretion of IGF-I by distinct leukocyte subsets is presented elsewhere [1,40]. The use of two distinct tissue- and development-specific promoters leads to two classes of IGF-I mRNAs that differ in their 5′ ends due to alternate splicing of exon 1 and 2 (class 1 and class 2 IGF-I mRNA, respectively). Class 1 transcripts are relatively abundant in extrahepatic tissues and thought to represent paracrine IGF-I, whereas class 2 transcripts are expressed in liver and regulated by GH. Indeed, the expression of class 1 transcripts has been found in murine macrophages [88]. However, several reports indicate that GH regulates IGF-I expression also in the immune system. For instance, Baxter et al. [70] demonstrated the presence of irIGF-I in a fraction (4%) of rat splenocytes, and the secretion by these cells of a 7.6 kDa irIGF-I. The amount of both cytoplasmic and secreted IGF-I was enhanced by incubation with GH. Since irIGF-I and irGH were found in the same splenocyte subpopulations [89] and an anti-GH antiserum decreased the number of IGF-I containing splenocytes [70], the authors suggested that irGH produced in the immune system might be involved in IGF-I regulation. In hypophysectomized rats, reduced IGF-I mRNA levels in spleen and thymus were increased after GH treatment, but the GH dependency was lower than in liver [90]. Mathews et al. [91], however, demonstrated the presence of IGF-I transcripts in spleen extracts from normal mice and from dwarf mice with a mutation in the GHRH receptor (lit/lit) that leads to reduced serum GH levels. As in most nonhepatic tissues, IGF-I expression in thymus and spleen was not affected and thus seems to be GH-independent. Also, IGF-I expression in human PBMC does not seem to be affected by GH in vivo, because IGF-I levels in PBMC from GH-deficient patients

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are not different from that in normal individuals [92]. Remarkably, in vitro GH expression on cultured PBMC was stimulated by GH. These results indicate that, although IGF-I expression in the immune system can under certain conditions be influenced by GH, it is less GH-dependent than hepatic IGF-I. In addition, in vitro studies indicate that the regulation of IGF-I in the immune system is mainly regulated by factors other than GH. The regulation of IGF-I in monocytes and macrophages has been studied in detail by several groups, and it appears that inflammatory mediators are important regulatory factors for IGF-I expression in these cells. The inflammatory cytokine tumor necrosis factor (TNF)-␣ induced a transient increase in IGF-I transcripts in murine bone marrow–derived macrophages. TNF-␣ also stimulated the synthesis of a 16–17 kDa IGF-I peptide [93,94]. Furthermore, IL-1␤, which did not exert a direct effect on IGF-I secretion, enhanced the stimulating effect of TNF-␣. Noble and coworkers [93] also demonstrated that hyaluronate, a glycosaminoglycan present in the extracellular matrix, induces IGF-I synthesis in murine bone marrow–derived macrophages via the cell surface adhesion molecule CD44. This effect was mediated by TNF-␣, because anti-TNF-␣ antiserum blocked hyaluronate-stimulated IGF-I synthesis. Another major inflammatory mediator, PGE2, stimulated IGF-I synthesis (15–16 kDa), although it reduced IGF-I mRNA levels [94]. Posttranscriptional processes such as mRNA stability and translation might thus be involved in PGE2 regulation of IGF-I. In human monocytes, IGF-I transcripts could be induced by advanced glycosylation end products (AGEs) [95]. Stimulation experiments with AGE-BSA in the presence of antisera against these factors indicate that IL-1␤ and PDGF can mediate induction of IGF-I. The induction of IGF-I transcripts by AGE-BSA was completely inhibited by IFN-␥. In addition, IFN-␥ also inhibited the TNF-␣–induced IGF-I expression in bone marrow–derived macrophages [96] and IGF-I expression in CSF-1–differentiated murine bone marrow macrophages [97]. IGF-I transcripts were also found in human and murine bone marrow cells [88,98]. In stromal cell lines, Abboud et al. [99] reported IGF-I secretion, whereas Landreth et al. [100] detected IGF-I mRNA. Thus, next to macrophages, stromal cells are likely candidates as IGF-I–producing cells in bone marrow. In addition, IGF-I mRNA is expressed in a murine pro-B-cell line [101], and Arkins et al. [98] showed that IGF-I transcripts are expressed in a development-dependent manner, being expressed during differentiation of hemopoietic cells into multiple myeloid lineages. The expression of IGF-I in lymphocytes is less well studied. By RT-PCR it was shown that PHA-activated human PBMC express IGF-I mRNA [102], whereas non-activated cells were negative [98,102]. The effects of cytokines and physiological T-cell stimulation on IGF-I expression remain to be established. E. IGF-Binding Proteins in the Immune System IGFBPs are expressed by several types of cells in the immune system. IGFBP-2 and -3 transcripts are present in freshly isolated human PBMC [102,103]. PHA-activated PBMC express the same level of IGFBP-3, but increased levels of IGFBP-2, -4, and -5 [102]. Thymic epithelial cells and murine stromal bone marrow cells express IGFBP-2 to -6 [99,104,105]. Murine thymic macrophages secrete IGFBP-4 [106], and des(1–3)IGF-I, a truncated IGF-I with a reduced affinity for IGFBPs, which is a more potent stimulator of DNA synthesis in thymic macrophages than IGF-I. Since this effect was observed in serum-free medium, it was concluded that autocrine or paracrine IGFBP-4 inhibits the proliferative effects of IGF-I on thymic macrophages or other cell types. Similar experiments were done by Long et al. [107] using murine bone marrow–derived macrophages.

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They showed that these cells express IGFBP-4 and that IGF-I is a less effective stimulator of differentiation compared to des(1–3)IGF-I. Furthermore, IGFBP-1 has been shown to inhibit the stimulatory effects of IGFs on the proliferation of human T cells [108]. Thus, leukocytes may orchestrate the effects of local IGF-I in lymphoid organs through the secretion of IGFBPs. Moreover, the capacity of the neutrophil proteolytic enzymes elastase and cathepsin G to degrade IGFBPs [109] suggests that degranulation of neutrophils may increase the local concentration of IGF-I through the release of IGFBP-bound IGF-I in the extracellular compartment. IV. EFFECTS OF GH AND IGF-I ON THE IMMUNE SYSTEM A. Development of Granulocytes and PBMC There is a large body of evidence that GH and IGF-I can stimulate the development of myeloid cells. Snell dwarf mice, which are deficient in pituitary PRL, GH, and TSH, have decreased peripheral blood counts that affect all lineages. All hematological parameters, including the white blood cell count, were improved after 2 weeks of treatment with recombinant human (rh) GH [110]. GH also increased the number of hemopoietic progenitors [colony-forming units–granulocyte/macrophage (CFU-GM)] in normal mice and in mice with the severe combined immunodeficiency (SCID) syndrome [111]. The effect of GH in SCID mice, which are T- and B-cell deficient due to their inability to rearrange immune receptor genes, indicates that lymphocyte-derived cytokines were not involved. The same investigators also demonstrated that GH could partially reverse the myelosuppressive effect of azidothymidine (AZT). These results suggest that GH treatment may be of use in AIDS patients undergoing AZT therapy. In mice that underwent syngeneic bone marrow transplantation after total body irradiation, GH augmented the number of granulocytes in bone marrow, spleen, and peripheral blood [112]. More recently, French et al. [113] showed that the GH enhanced the number of myeloid progenitor cells in bone marrow and spleen in aged rats. Like GH, IGF-I has been shown to exert growth-promoting hematopoietic effects in normal and AZT-treated mice [114]. On the contrary, Jardieu and colleagues [115] did not find any effect of IGF-I on myelopoiesis in normal mice. In fact, the mean number of CFU-GM were slightly reduced by IGF-I treatment. In vitro, GH and IGF-I stimulate granulopoiesis as indicated by the increase in the number of granulocyte precursor cells after culture of bone marrow–derived progenitors in the presence of GMCSF [116]. The effects of GH were dependent on the presence of bone marrow–adherent cells and inhibited by a monoclonal antibody against the IGF-I receptor, so paracrine IGFI may mediate the effects of GH on myelopoiesis. Several mechanisms may be responsible for the effects of IGF-I on hematopoiesis: 1. The effect of IGF-I on myelopoiesis is a consequence of inhibition of apoptosis [117–119]. 2. IGF-I stimulates the differentiation of adherent bone marrow cells into macrophages through local production of hematopoietic growth factors such as GMCSF [120]. 3. Although there are no experimental data that support this hypothesis, IGF-I may promote cell cycle progression in myeloid precursor cells. Clark et al. [121] showed that IGF-I treatment of normal mice increased the number of splenic B cells. The effect of IGF-I on B-cell development in mice was examined by IGF-

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I treatment of irradiated mice after reconstitution with syngeneic bone marrow [115]. IGFI treatment increased the number of splenic B cells, and this effect coincided with an increase in numbers of B-cell progenitors (B220Ⳮ/sIgMⳮ) and mature B cells (sIgMⳭ) in bone marrow. The increase in the frequency of splenic B220Ⳮ cells in the S-phase of the cell cycle in IGF-I–treated mice indicates that IGF-I may exert its effects on the Bcell compartment through stimulation of cell cycling. This hypothesis is supported by in vitro studies showing that IGF-I potentiates the effect of IL-7 on DNA synthesis in proB-cell lines [101]. In addition, IGF-I may also promote B-cell development through stimulation of differentiation, as it was shown that IGF-I promotes the differentiation of proB cells into pre-B cells [122]. It remains to be established whether IGF-I may affect the B-cell compartment through modulation of apoptosis. Although these studies indicate that IGF-I can exert lymphopoietic effect on the B-cell lineage, Dorshkind et al. [123], who carefully addressed the B-cell compartment in IGF-I knockout mice, found no aberrations in the frequency of B lineage cells and their absolute number corrected for body size. Taken together, results indicate that although overexpression of IGF-I may enhance Bcell development in normal and immune-deficient mice, it is not required for normal Bcell development. This hypothesis is supported by the finding that the reduced frequency of B lineage cells in bone marrow of Snell dwarf mice, originally observed by Murphy et al. [110], could not be corrected by IGF-I treatment [123]. Further studies revealed the importance of thyroid hormones in B-cell development. In contrast to IGF-I and the pituitary hormones GH and prolactin, thyroxin was able to restore the defect in the B-cell lineage in Snell dwarf mice [124]. Furthermore, B-cell development was also impaired in hypothyroid mice, but not in lit/lit mice, with a defect in the GHRH receptor [123], or PRL knockout mice [125]. The absence of B-cell lineage defects in lit/lit mice indicates that pituitary-derived GH, which is under the control of GHRH, is not an obligate factor in B-cell development. The results from Dorshkind and coworkers [123] also show that IGF-I and pituitaryderived GH are not required for murine T-cell development under normal conditions. However, the idea that GH and IGF-I may be useful therapeutic agents in immunosuppressed conditions and during immune reconstitution has prompted several laboratories to assess the effects of GH and IGF-I on thymopoiesis. The effects of IGF-I on normal mice has been addressed by Clark et al. [121], who showed that IGF-I treatment enhanced the number of thymocytes and splenic CD4Ⳮ T cells [121]. Since thymic atrophy coincides with the decline in GH and IGF-I levels during aging, several laboratories have investigated the effects of GH and IGF-I on thymopoiesis. The age-dependent loss of CD4Ⳮ/CD8Ⳮ thymocytes in Snell dwarf mice observed by Cross et al. [126] was normalized by treatment with rhGH, and thymic hypoplasia was partially corrected [127,128]. Regeneration of thymic tissue in aged rats was obtained by implantation of GH3 pituitary adenoma cells which produce GH and small quantities of PRL [129]. Further experiments showed that administration of rhGH restored thymic morphology and increased the number of medullary and cortical thymocytes [113]. In addition, thymopoietic effects of GH have also been observed in dogs and mice [130,131]. In rats that were immune suppressed by cyclosporine treatment [132], both GH and IGF-I enhanced thymic recovery. Treatment resulted in an enlargement of the thymus, an increase in CD4Ⳮ/CD8ⳮ cells, the regeneration of Hassall’s corpuscles, and an increased expression of class II MHC. In addition, thymopoietic effects of IGF-I were found in aged mice [82] and diabetic rats [133]. How do GH and IGF-I exert their thymopoietic activities? They may influence different processes such as DNA synthesis, cell differentiation, and apoptosis and operate

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at the level of bone marrow or thymus. For instance, stimulation by IGF-I of DNA synthesis in thymocytes [79,133] or inhibition of apoptosis [87,134] may lead to increased numbers of thymocytes and also peripheral T cells. GH has been shown to inhibit Fas-induced apoptosis in B- and T-cell lines [135] and glucocorticoid-induced apoptosis in CD4Ⳮ T cells [136]. The in vitro effects of GH and IGF-I on thymopoiesis were addressed in detail by different in vitro experiments using fetal thymic organ cultures (FTOC). It was shown that addition of neither GH nor IGF-I influenced in vitro thymopoiesis in intact fetal thymic lobes [82,137]. However, both GH and IGF-I stimulated thymopoiesis in irradiated and bone marrow–reconstituted fetal thymic lobes. These observations suggest that GH and IGF-I increase the colonization of bone marrow–derived precursors to the thymus. The putative effects of GH at the level of bone marrow are in line with other studies which show that administration of GH to rats stimulates DNA synthesis in bone marrow [138] and reverses bone marrow hypocellularity [82]. In addition, GH promoted the engraftment of syngeneic thymocytes and of human PBMC in SCID mice which lack mature lymphocytes [139,140], and IGF-I–stimulated T-cell reconstitution in irradiated mice after syngeneic bone marrow grafting [115]. Furthermore, studies of Eren and coworkers [141,142] showed that aged animals exhibit a reduced number of T-cell progenitors in bone marrow. These observations raise the possibility that the decrease in GH and IGFI levels during aging are responsible for the reduced capacity of bone marrow progenitors to colonize the thymus. The absence of IGF-I effects on thymopoiesis in intact fetal thymic lobes is in accordance with the observation of Kecha et al. [143], who addressed the role of autocrine/ paracrine IGF-I and IGF-II in thymopoiesis. They showed that fetal thymic lobes express IGF-I, IGF-II, and their cognate receptors and that addition of an anti-IGF-I antibody did not affect thymopoiesis, whereas antibodies against IGF-II, the IGF-I receptor, or the IGFII receptor all blocked T-cell development at the CD4ⳮ/CD8ⳮ stage. This finding and the thymopoietic effects of IGF-II overexpression in a transgenic model [144] suggest that IGF-II rather than IGF-I is an important local factor for thymopoiesis. B. Innate Immunity Effects of GH and IGF-I have been well addressed in the innate immune system. Innate immunity is not influenced by prior contact with the infectious agent, so it acts as a first line of defense against foreign organisms. Phagocytic neutrophils, macrophages, and natural killer cells are the most prominent cellular effectors of innate immunity, and the generation of reactive oxygen intermediates (oxidative burst) by neutrophils and macrophages is an important element of this defense system. Hypophysectomized rats showed an increased susceptibility to lethal effects of Salmonella infection [145]. In both intact and hypophysectomized rats, porcine GH increased the survival rate by 200–400%. Additionally, macrophages from hypophysectomized rats exhibited a 50% reduced capacity to kill extracellular Salmonella as compared to those derived from intact rats. In vivo treatment with GH increased the ability of macrophages isolated from intact and hypophysectomized rats to kill extracellular bacteria in vitro by Ⳳ85%. Furthermore, the increased resistance to Salmonella infection after GH treatment correlated with an increased release of reactive oxygen metabolites [146]. In vitro experiments have shown that GH primes rat macrophages [147] for superoxide anion production. These results indicate that GH can enhance innate immune responses in rats, possibly through oxidative burst stimulation. Indirect effects of GH through augmentation of cyto-

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kine secretion may also be involved. GH and IFN-␥ partially corrected the reduction of TNF-␣ synthesis by rat macrophages [148]. A mediating role for IGF-I in enhancing inflammatory cytokine expression is also conceivable, because IGF-I stimulates the in vitro production of IFN-␥ [149], IL-8 [80], and TNF-␣ [150] in human leukocytes. In GH-deficient children, plasma levels of TNF-␣ were normal and GH treatment evoked a transient increase plasma TNF-␣ levels [151]. However, in GH-deficient adults, basal TNF-␣ levels were increased and prolonged GH administration restored TNF-␣ levels [152]. A role for pituitary hormones in innate immunity was suggested by the finding that bacterial clearance 2 days after Listeria monocytogenes infection was hampered in Snell dwarf mice compared to their normal littermates [153]. However, the observation by the same group that the clearance was normal in lit/lit mice with a mutation in the GRHR indicates that the GH/IGF-I axis is not required for normal function of the innate immune system in mice. Although a role for local GH or IGF-I produced by cells of the immune system cannot be excluded, it is more likely that thyroid hormones are important for innate immunity, because hyt/hyt mice with a mutation in the TSH receptor also exhibit an impaired innate immune response. These results are in line with observations in GHdeficient humans, which are not considered to be immune deficient, although GH and IGF-I affect certain parameters of the innate immune system. For instance, GH augments the oxidative burst in human monocytes and neutrophils [154–156]. Fu et al. [156] revealed that although IGF-I could also stimulate the oxidative burst, it did not mediate the effect of GH. Remarkably, the effect of GH was mediated by the PRL-R [157]. The reduced phagocytic capacity of monocytes and neutrophils in GH-deficient children [158] and the reduced oxidative burst in GH-deficient adults [159] could be restored by GH treatment. In acromegalic patients, pronounced elevation of GH was related to an enhanced phagocytic activity of granulocytes [160]. Since GH receptors have not been detected on human neutrophils [38], in vivo effects of GH may take place through the PRL receptor or via stimulation of IGF-I production. The effects of IGF-I on human peripheral leukocytes were further investigated by Bjerknes and Aarskog [161]. Physiological concentrations of IGF-I stimulated the phagocytosis of IgG-opsonized microorganisms and PMA- or fMLPinduced oxidative burst. Higher concentrations were necessary to potentiate fMLP-induced degranulation and to stimulate membrane expression of receptors for complement on stimulated and unstimulated cells. In addition to direct stimulation of granulocyte function, GH and IGF-I may also support innate immunity through regulation of neutrophil recruitment and apoptosis. For instance, neutrophil recruitment may be enhanced through stimulation by IGF-I of monocytes to produce the principal neutrophil chemoattractant IL-8 [80] or through stimulation by GH of neutrophil adhesion [162]. The inhibitory effect of IGFI on apoptosis of neutrophils may delay the resolution of granulocyte-mediated inflammatory reactions [80]. Inhibition by GH of Fas-mediated apoptosis in the promonocytic cell line U937 [163] indicate that GH may exert similar actions. There is also evidence that NK cell activity can be stimulated by the GH/IGF-I axis. IGF-I stimulates NK cell activity in vitro [77,164], and GH-deficient children showed a decreased cytotoxic activity of NK cells, which could be corrected by GH treatment [165]. GH treatment of adult women with an impaired GH secretion also resulted in an increased natural killer cell activity [166]. The effects of GH therapy have also been studied in girls with Turner’s syndrome [167]. Endogenous GH secretion is normal in quantitative terms in those patients, although some small abnormalities in the profile have been reported. GH treatment resulted in

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normalization of height in most individuals [168] and restoration of decreased numbers of NK cells. In conclusion, GH and IGF-I may affect the innate immune response through effects on neutrophils and cells of the monocyte/macrophage lineage, resulting in an increased production of reactive oxygen metabolites, or through activation of NK cells. There is, however, no evidence for an obligate role of GH or IGF-I in innate immunity, because GH-deficient patients have no signs of immune deficiency [169]. C. Humoral Immunity In vitro studies with human B cells indicate that GH and IGF-I can modulate humoral immune responses. Both GH and IGF-I stimulated B-cell proliferation and the production of antibodies in human B-cell lines and Staphylococcus aureus Cowan-activated tonsillar B cells [170–172], and GH has been shown to inhibit Fas-induced apoptosis in the human B-cell line IM-9 [135]. Furthermore, GH and IGF-I induced class switching in human tonsillar B cells [173]. The stimulation by IGF-I of IL-6 expression [174], which plays a role in the final maturation of B cells into immunoglobulin-secreting plasma cells, opens up the possibility that IL-6 mediates the effects of IGF-I. The in vivo antibody response to tetanus toxoid was recently addressed in Ames dwarf mice that are deficient in pituitary GH, PRL, and TSH due to a mutation in the transcription factor Prophet of Pit-1. It appeared that the specific antibody response was not impaired in the dwarfs compared to their control littermates [175]. The same group addressed the effects of GH in transgenic mice and found that the generation of tetanus toxoid specific antibodies was markedly impaired in GH transgenic mice. The authors speculated that high levels of corticosterone of nonadrenal origin or early aging in GH transgenic mice could be responsible for this result. The primary and secondary antibody responses to ovalbumin-conjugated dinitrophenyl (DNP-OVA) in normal mice with implanted minipumps were increased by IGF-I as compared to excipient-injected mice [176]. Furthermore, ex vivo IgG production by cultured splenocytes from IGF-I treated mice was markedly enhanced [121]. B-cell function was also tested in normal mice by immunization with DNP-OVA 2 weeks after bone marrow reconstitution. IGF-I stimulated antigen-specific IgG synthesis by 60–80% [115]. In contrast Binz et al. [133] did not show any improved antibody response to BSA in IGF-I–treated diabetic rats. The role of normal GH and IGF-I levels in the humoral immune response was assessed by Foster et al. [153]. Their finding that the primary and secondary antibody response against DNP was not affected in GH/IGF-I–deficient lit/lit mice and Snell dwarf mice indicates that the central GH/IGF-I axis is not required for normal humoral immunity. The results for GH are in agreement with the above-mentioned observations of Hall and coworkers [175], but in contrast to the early results of Nagy et al. [177], who showed that the antibody response to sheep red blood cells in hypophysectomized rats was reduced to 15% of the response in control rats. Moreover, they found that rat, bovine, and human GH corrected the antibody response to 60–75% of control values. Since recombinant GH was not available at the time the experiments were performed, the GH preparations may have been contaminated with other pituitary hormones, e.g., PRL. Additional explanations for the discrepancy have been provided by Clark [2] and Dorshkind [3], who hypothesized that GH and IGF-I are of importance during stress. This will be discussed in Section VI. Studies on the effects of GH replacement therapy in GH-deficient children yielded contradicting results with respect to the number of circulating B cells (reviewed by Wit

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et al. [169]). Decreased B-cell numbers during GH therapy were observed in some but not all studies. Moreover, reduced in vitro IgM synthesis in GH-deficient children was partially corrected by GH therapy [178]. However, the reduction in B-cell counts observed in these studies is generally not indicative for immunodeficiency, and in two studies the effects were transient. D. Cell-Mediated Immunity The effects of GH and IGF-I on cell-mediated immune responses have not been extensively investigated. Early studies of Fabris et al. [179] showed that GH promotes skin graft rejection in Snell dwarf mice. In addition, impaired contact sensitivity in hypophysectomized rats could be corrected by GH treatment [177]. In vitro, human GH stimulated the generation of cytotoxic T cells [180]. The role of the central GH/IGF-I axis under normal physiological conditions has been addressed in lit/lit mice [153]. Cell-mediated immunity involving CD4Ⳮ and CD8Ⳮ T cells can addressed by assessing the control of Listeria monocytogenes infection 6 days after infection. It appeared that the response against the bacteria in lit/lit mice was not impaired. Thus, although there are indications that GH can stimulate cell-mediated immune responses, pituitary-derived GH is not required for cellmediated immunity in mice. V. MODULATION OF GH/IGF-I AXIS BY THE IMMUNE SYSTEM The inflammatory cytokines IL-1␤, IL-6, and TNF-␣ are readily detectable in blood and act as endocrine factors that communicate to the brain and the endocrine system. These cytokines are involved in the induction of fever, sickness behavior, and hormonal changes, including modulation of the hypothalamus-pituitary-adrenal axis and GH/IGF-I axis. Effects of inflammatory cytokines on the GH/IGF-I axis have been implicated in several clinical conditions. Stunted growth is a major complication of recurrent infections and chronic inflammation in children. For instance, children with systemic juvenile rheumatoid arthritis exhibit growth impairment during periods of disease activity, but not during remission [181]. These patients show reduced plasma IGF-I levels [182–184], which has also been observed in children with Crohn’s disease [185] or with recurrent infections due to AIDS [186]. The results on GH secretion in patients with juvenile rheumatoid arthritis are controversial, but reduced IGF-I levels have been observed in patients with normal or near normal GH levels [184]. This indicates that reduced IGF-I levels are not always due to decreased GH concentrations. Reduced IGF-I levels have also been implicated in the catabolic state in critically ill patients. In rats, IGF-I deficiency in catabolic states induced by sepsis has been related to GH insensitivity [187]. Administration of endotoxin in rats resulted not only in a decline of plasma IGF-I levels but also in a decreased expression of GH receptors in liver [187], indicating that GH insensitivity in sepsis may be due to downregulation of the GH receptor. A role for IL-1␤ and TNF-␣ in this process is supported by the fact that they induce GH insensitivity in hepatocytes through downregulation of the GH receptor [188,189]. In addition, GH insensitivity can also be induced by stimulation of the expression of suppressors of cytokine signaling (SOCS), which inhibit cytokine and GH receptor signaling [190,191]. These observations may be relevant for the cytokine-induced decline in IGF-I levels as indicated by the findings that IL-6 stimulates SOCS-3 expression in cultured rat hepatocytes [192,193] and that IL-1␤ and TNF␣ potentiate the stimulating effect of GH on SOCS-3 expression [193]. Additionally, in

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vivo induction by endotoxin and IL-6 of SOCS-3 mRNA expression in rat hepatocytes was recently demonstrated by Wang et al. [194]. IL-6 has been hypothesized to mediate inhibition of growth through induction of GH insensitivity. Indeed, IL-6 transgenic mice are small and exhibit normal GH and decreased IGF-I levels [195]. However, although IL-6 induces SOCS-3 expression, it does not affect liver IGF-I levels in IL-6 transgenic mice or GH responsiveness in cultured hepatocytes [188]. It was postulated that an IL6–induced decrease in serum IGFBP-3, which stabilizes serum IGF-I, causes a decline in serum IGF-I levels [195]. Taken together, these results indicate that the inflammatory cytokines IL-1␤ and TNF-␣ mediate inhibiting effects of the immune system on liver IGF-I secretion through the regulation of GH receptors and inhibitors of GH receptor signal transduction. The possible mechanisms by which inflammatory cytokines may affect serum IGF-I levels without modulating GH secretion are depicted in Fig. 2. However, since endotoxin and inflammatory cytokines also exert effects on pituitary GH secretion in vitro [196] and in vivo [197], GH-mediated effects on IGF-I regulation cannot be excluded. VI. CONCLUSIONS GH and IGF-I have profound, usually stimulating, effects on the development and function of the immune system. Since GH and IGF-I are not only produced in the pituitary and liver, respectively, but also in leukocytes and stromal cells of lymphoid organs, it is important to consider possible autocrine or paracrine effects. A role for extrahepatic IGFI in growth has been demonstrated by studies of Sjo¨gren et al. [21]. They showed that

Figure 2 Inflammatory cytokines may affect serum IGF-I levels by modulating GH receptor (GH-R) and SOCS-3 levels in hepatocytes and IGFBP-3 in serum. Reduced levels of IGFBP-3 may be due to either reduced production in Kupffer cells or increased degradation in serum. A reduction in IGFBP-3 will lead to a decreased formation of the relatively stable 150 kDa complex. More details are discussed in Sec. V.

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liver-specific disruption of the IGF-I gene hardly affects body growth. The absence of a function for liver-derived IGF-I in growth does not automatically imply that it is also dispensable for immune function. Experiments by the group of Dorshkind using lit/lit mice with a genetic defect in the GHRH receptor addressed the role of GH and IGF-I in immune function and lymphopoiesis. Although the regulation of extrapituitary GH has not been addressed extensively, there are indications that it is less dependent on GHRH than pituitary GH. Moreover, as described in section III.D, IGF-I expression in leukocytes is much less dependent on GH than liver IGF-I. Thus, the defect in the GHRH receptor will mainly affect the central GH/IGF-I axis comprising pituitary GH and liver IGF-I. Therefore, the finding of Foster et al. [153] that lit/lit mice show normal lymphopoiesis and do not exhibit any defect in innate or acquired immune function indicate that pituitary GH and liver-derived IGF-I are not essential for lymphopoiesis and immune function. However, low levels of GH and IGF-I which are still expressed in lit/lit mice could be of extrapituitary origin and contribute to normal immunity (see also Fig. 1). The same holds true for Snell dwarf mice, which exhibit normal lymphopoiesis and immune function. Since the genetic defect in the transcription factor pit-1 does not affect extra-pituitary GH expression [59,60], autocrine/paracrine GH may be involved. Further studies using IGFI knockout mice revealed that IGF-I is not an obligate factor in lymphopoiesis. The possible roles of autocrine/paracrine GH and IGF-I in immune function may be further addressed by leukocyte-specific disruption of the GH and IGF-I genes. However, the possibility that GH and IGF-I are absolutely dispensable for normal immune function should be taken into account. If that were the case, then why do leukocytes express GH and IGF-I receptors which mediate GH and IGF-I effects in vitro and in vivo? The interesting hypothesis that GH and IGF-I, like prolactin, are important immune-stimulatory factors that counteract negative immunoregulatory signals has been put forward by several investigators [2,3]. According to this hypothesis, physiological levels of GH of IGF-I may exert immunestimulatory actions under stressful situations. This hypothesis can also explain the contradictory results from different laboratories with regard to the immune status of hormonedeficient animals. It was postulated that, for instance, Snell dwarf mice were only found to be immune deficient when they were housed under stressful conditions. Indeed, recent experiments of Welniak et al. [4] showed that Snell dwarf mice exhibited decreased thymic cellularity when they were housed with their normal-sized heterozygous littermates, but not when they were separated. Furthermore, impaired thymopoiesis could be restored by GH administration. In addition to longitudinal growth, GH and IGF-I also promote the growth of multiple organs. Especially IGF-I has been shown to be an important homeostatic factor in many tissues, by influencing cell cycling, differentiation, and apoptosis. As such, GH and IGFI have been considered as proportional growth factors for the immune system [2,3]. With regard to this hypothesis it is tempting to speculate that IGF-I also controls homeostasis of immune responses, including inflammatory reactions. Indeed, IGF-I stimulates DNA synthesis in T cells [75,108,198,199] and B cells [170,171], the synthesis of the T-cell growth factor IL-2 [200,201] and inhibits apoptosis in T cells [87,134]. Furthermore, IGF-I stimulates the in vitro production of inflammatory cytokines, including the major neutrophil chemoattractant IL-8 [80] and inhibits neutrophil apoptosis [80]. Also, the recruitment of monocytes to sites of inflammation may be enhanced by IGF-I, because IGF-I has been shown to increase the expression of cellular adhesion molecules on endothelial cells and monocyte adhesion to these cells [202–204]. Thereby IGF-I may act as a proinflammatory factor by enhancing T-cell proliferation, the recruitment of neutrophils

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and monocytes, and counteracting the resolution of inflammation by inhibiting neutrophil apoptosis (see Fig. 3). This idea is consistent with the upregulation of both IGF-I and IGFI receptor expression in leukocytes during activation (see Secs. III.C and III.D), leading to increased immune responses as a result of increased proliferation of specific T- and B-cell lineages and upregulation of cytokine secretion. Definitive proof awaits the development of lymphoid-specific disruption of the IGF-I gene. VII. THERAPEUTIC POTENTIAL The presence of functional receptors for GH and IGF-I on leukocytes allows the therapeutic use of GH and IGF-I to stimulate immune function, either directly or through stimulation of immune reconstitution. For instance, impaired recovery of lymphopoiesis is a major problem after bone marrow transplantation or chemotherapy leading to opportunistic infections. The thymopoietic effects of GH or IGF-I may be of benefit to restore the lymphopoietic capacity. Plasma levels of GH and IGF-I decline during aging with a concomitant decline in immune functions [205]. GH therapy in elderly has already been shown to increase lean body mass and reduce fat [14]. GH or IGF-I therapy may also be useful as therapeutic agents to restore immune functions in the elderly. In a preliminary clinical study, the potential application of GH for immune reconstitution in HIV-infected adults was tested. It appeared that GH treatment of HIV-1–infected men resulted in an increase in thymic mass and circulating naı¨ve CD4Ⳮ cells [206]. However, as indicated by the authors, a randomized controlled study with more patients should be done to confirm the therapeutic potential of GH in AIDS. In another study in which HIV-infected individuals were treated with GH for a shorter period [207], such effects were not found. Detailed knowledge of the effects of GH and IGF-I on the immune system is also of value with respect to therapeutic interventions that are not aimed at the immune system. GH replacement therapy has been offered to GH-deficient children for many years, and GH effects have been well addressed. GH-deficient children are not immune deficient, and GH therapy has been shown to have no adverse effects on immunity. However, the effects of GH or IGF-I on other patients may be different. Due to their anabolic properties,

Figure 3 Putative mechanisms by which IGF-I acts as a local proinflammatory factor, based on results from in vitro experiments.

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GH and IGF-I have been used to reverse the catabolic state of intensive care patients with critical illness. During a first major multicenter clinical trial, it appeared that GH worsened morbidity and doubled mortality [208]. Although many explanations are possible, it is conceivable that immune stimulatory effects including stimulation by IGF-I of inflammatory cytokines and the acute phase response are also involved. Other examples of patients that may benefit from GH treatment are children with stunted growth due to renal failure or chronic inflammatory diseases including juvenile chronic rheumatoid arthritis or Crohn’s disease. Although immune disorders have not been shown in acromegalic patients, overexpression of GH in mice provoked the development of autoimmune antibodies and osteoarthritis [209]. Taken together, possible GH effects on the immune system should be taken into account in certain patients undergoing GH therapy, especially during high-dose GH therapy.

ACKNOWLEDGMENTS Robert and Elisabeth Hooghe are thanked for their fruitful discussion of the manuscript. This work has been funded by the Flemish Government (GOA 97-02-4), the Fund for Scientific Research-Flanders (FWO G0167.98N), and institutional grants from the VUB.

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10 Somatostatin Control of Immune Functions P. M. VAN HAGEN, V. DALM, L. J. HOFLAND, and S. W. J. LAMBERTS Erasmus Medical Center, Rotterdam, The Netherlands D. FERONE University of Genoa, Genoa, Italy

I. INTRODUCTION Somatostatin is a neuropeptide that is widely distributed throughout the body. It was first identified as a growth hormone release–inhibiting factor synthesized in the hypothalamus. Outside the central nervous system (CNS), the peptide is present in endocrine as well as nonendocrine tissues. Somatostatin functions as a peptide with a generally inhibitory action in the central nervous and endocrine system. In the CNS it can act as a neurotransmitter, while in peripheral tissues it regulates endocrine and exocrine secretion and acts as a modulator of motor activity in the gastrointestinal tract (Reichlin, 1983, 1995; Brazeau, 1986). Somatostatin produced in the hypothalamus is transported through the portal circulation to the anterior pituitary, where it inhibits the secretion of growth hormone and other pituitary hormones such as thyroid-stimulating hormone. Somatostatin has also been shown to have antiproliferative effects in vitro (Schally, 1988). Somatostatin is secreted in two biologically active forms: a 14-amino-acid form (somatostatin-14) and an amino-terminally extended 28-amino-acid form (somatostatin-28). Native somatostatin has a plasma halflife of less than 3 minutes. Therefore, synthetic, metabolically stable analogues have been developed for clinical use. Structure-function analysis of native somatostatin and peptide analogues has shown that the amino acid residues Phe7, Trp8, Lys9, and Thr10 are necessary for receptor binding. A 14-amino-acid somatostatin-like neuropeptide, cortistatin, has been discovered in mouse brain tissue (de Lecea et al., 1996, 1997). The receptor-binding 193

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site of cortistatin shares the same amino acid residues as somastostatin. Up to now, no specific cortistatin receptor has been identified, but differential effects in the CNS have been reported. Structurally, somatostatin receptors are so-called seven transmembrane domain (TMD) glycoproteins, comprised of seven membrane-spanning ␣-helical domains connected by short loops, an N-terminal extracellular domain, and a C-terminal intracellular domain. The genes for a family of five somatostatin receptors (sst1–5 by IUPHAR convention) have been cloned in recent years. Although the sst genes do not have introns, there is a cryptic intron in the sst2 gene, and alternatively spliced forms of sst2 (named sst2A for the unspliced form and sst2B for the spliced variant) have been identified in humans, mice, and rats (Vanetti, 1992; Patel et al., 1993, 1996; Schindler et al., 1998). The sst genes are all located on different chromosomes, but have a high degree of sequence homology (Patel et al., 1996). A number of short synthetic, metabolically stable peptide analogues have been studied for their relative affinities at the human recombinant receptors in radioligand-binding studies (Rohrer et al., 1998). The proclaimed selectivity of such ligands may, however, vary, depending on the assay conditions and radioligands used. The clinically used somatostatin analogues octreotide and lanreotide bind with high affinity to sst2 and sst5, whereas the newly developed synthetic somatostatin analog SOM-230 binds with high affinity to sst1, 2, 3, and 5. The latter compound is currently under investigation (Bruns et al., 2002; Weckbecker et al., 2002). The signal transduction pathways coupled to activation of ssts have been studied in transfected cell systems. All five subtypes of ssts have been found to be linked to adenylyl cyclase via pertussis toxin sensitive, i.e., inhibitory, guanine nucleotide–binding proteins (G-proteins). Many cellular effector proteins such as phospholipase C, calcium channels, potassium channels, NaⳭ/HⳭ exchanger, protein tyrosine phosphatases, phospholipase A2, mitogen-activated protein kinase (MAPK), or p53 were reported to be modulated by sst subtypes (Patel, 1997). The antiproliferative effects of somatostatin are thought to be due to activation of a subclass of protein tyrosine phosphatase enzymes (Lopez et al., 1996; Reardon et al., 1997). Depending on the cell type, the various ssts have been shown to couple to a number of transduction systems. II. SOMATOSTATIN RECEPTORS IN THE IMMUNE AND HEMATOPOIETIC SYSTEMS Somatostatin receptor expression has been described in the immune system of various species including humans. Bathena et al. (1981) demonstrated the presence of somatostatinbinding sites on human peripheral blood mononuclear cells (PBMC). Using radionuclidelabeled somatostatin and employing Scatchard analysis, they showed that both lymphocyteenriched cell populations and monocyte-enriched populations contained several hundred binding sites per cell. Since then, somatostatin-binding sites have been demonstrated on several immune and hematopoietic cells and tissues using various techniques. Radioligand binding demonstrated somatostatin receptors to be present on human lymphoid and myeloma cell lines (Nakamura et al., 1987; Sreedharan et al., 1989). Using radioligand binding as well as fluorescence-labeled somatostatin and FACS analysis, somatostatin-binding sites were detected on mitogen-activated human peripheral lymphocytes (Hiruma et al., 1990). On resting PBMC, red blood cells, and granulocytes, no binding sites were detectable by the radioligand assay. In the same study human leukemic cells were shown to

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contain somatostatin-binding sites. It is therefore a long-known fact that human immune cells and their progenitors can express somatostatin receptors (Fig. 1; Table 1). As a consequence, ssts have been described in primary and secondary human immune organs. The human thymus was studied extensively by Ferone (Ferone et al., 1999). The thymus is a central lymphoid organ in mammals, which plays a pivotal role in the control of the immune system. The presence of the thymus and its functionality are required for a full establishment of immunocompetence. Binding studies and RT-PCR analysis have been used to characterize somatostatin and sst expression in the normal human thymus. Somatostatin and three subtype receptors (sst1, sst2a, sst3) have been found in thymic tissue (Ferone et al., 1999). The expression of these different subtypes appeared heterogeneous within the tissue and on different cell subsets. A selective expression of sst1 and sst2a has been found on cultured thymic epithelium cells (TEC), which is in line with [125ITyr3]-octreotide binding in the thymic medulla, where TEC is the prevalent cell type. TEC were found to be a potential source of somatostatin production within the thymus, since somatostatin mRNA has been detected in these cells (Ferone et al., 1999). In freshly isolated thymocytes, sst2a and sst3 mRNA expression has been found. The human spleen has not been studied extensively, but PCR studies showed sst2 and sst3 expression, whereas human bone marrow expresses only sst2. Somatostatin receptor expression in mice and rat immune system differs significantly from the human immune system. Murine B and T lymphocytes from spleen and Peyer’s patches were also shown to express ssts, as were lymphocytes isolated from the granulomas of mice infected with the parasitic helminth Schistosoma mansoni (Stanisz et al., 1986; Weinstock, 1991; Stanisz, 1994) Most of these studies were carried out using native somatostatin analogues, thus giving no additional information about which of the five ssts were expressed. Only recently a number of these early studies were revisited using molecu-

Figure 1 Somatostatin receptor subtype expression during differentiation of hematopoietic cells in humans.

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Table 1 Expression of Somatostatin Receptor Subtypes in the Human Immune System Including Human Leukemic Cell Lines Cell CD34⫹ stem cells Thymocyte T lymphocytes B lymphocytes T lymphoid cell lines B lymphoid cell lines Monocytes Macrophages Dendritic cells Myeloid leukemia

Som

CST

Sst1

Sst2a

Sst3

Sst4

Sst5

– – – – – – – – – –

⫹ ⫹ ⫹ ⫹ ⫹ ⫹ ⫹ ⫹ ⫹ ⫹

– – – – – – – – – –

⫹ – – – ⫹ ⫹ – ⫹ ⫹ ⫹

– ⫹ ⫹ ⫹ ⫹ ⫹ – – – –

– – – – – – – – – –

– – – – – – – – – –

Source: Ferone et al., 1999 a, b; ten Bokum et al., 1999 a, c; van Hagen et al., 1999; Lichtenauer-Kaligis et al., 2000; Oomen et al., 2000.

lar biological techniques in order to answer this question. Murine splenocytes, thymocytes, CD4Ⳮ T-cell lines, and CD4Ⳮ T lymphocytes isolated from schistosome granulomas were also shown by RT-PCR to express sst2 mRNA, although expression of sst4 and sst5 was not investigated in this study (Elliott et al., 1994). In our studies we found that sst3 and sst4 were the main subtypes to be expressed at the mRNA level in rat splenocytes, thymocytes, and lymph node cells (ten Bokum et al., 1999b), whereas others showed that cultured rat thymocytes expressed mRNA for sst1 and sst2 (Sedqi et al., 1996). Rat and mice sst-mRNA expression in the immune system is summarized in Table 2. III. SOURCES OF SOMATOSTATINS WITHIN THE IMMUNE ENVIRONMENT The presence of ssts on cells of the immune system implies that these cells are capable of showing a functional response to somatostatin. As somatostatin is rapidly degraded in

Table 2 Somatostatin Subtype Receptor Expression in Mouse and Rat Tissue or cells Lymph nodes Spleen Thymus Bone marrow Peripheral blood cells Splenocytes T lymphocytes Thymocytes

Mouse

Rat

sst1 sst1, sst4 sst1, sst2 sst2 sst4 sst2 (MRL mouse) sst2 sst2

sst3, sst4 sst1, sst3, sst4 sst3, sst4

Source: ten Bokum et al., 2000; Weinstock and Elliott, 2000.

sst3, sst4

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the circulation, it was expected that somatostatin to which these cells respond is produced locally, in the vicinity of sst-bearing cells. Lymphoid organs are innervated by nerves of the sympathetic nervous system and by sensory nerves containing neuropeptides such as somatostatin, which have been shown to be released during local inflammatory responses (Goetzl et al., 1985; Stevens-Felten and Bellinger, 1997). In endocrine organs and gastrointestinal tract, somatostatin is produced by specialized neuroendocrine cells, where it may reach high concentrations locally (Dimaline, 1996). Neuropeptides that function as hormones in the gastrointestinal tract may regulate in a paracrine fashion the function of cells of the gut-associated lymphoid tissue and other immune cells present in this compartment (O’Dorisio, 1986; Ottaway, 1991). In addition (nonhuman) immune cells and lymphoid organs have been shown to contain somatostatin and to present mRNA for somatostatin (Fuller and Verity, 1989; Weinstock et al., 1990; Aguila, 1991; Weinstock, 1991; Dardenne and Savino, 1994; Teitelbaum et al., 1996). Immune cells from dispersed schistosome granulomas secrete somatostatin (Weinstock, 1991). They contain the 117-amino-acid precursor peptide preprosomatostatin (ppSS) but only make the 14-amino-acid peptide. The sequence of granuloma ppSS mRNA is identical to that predicted by the sequence obtained from murine genomic DNA (Elliott et al., 1998). Granuloma cells from at least three mouse stains (C57BL/6, CBA, and B129) express somatostatin mRNA (Weinstock and Elliott, 2000). Thus, production of somatostatin at sites of chronic inflammation is a property universal to murine species. Adherent granuloma and spleen cells from infected mice, comprised mostly of macrophages, express ppSS mRNA. Three macrophage cells lines were shown to express ppSS mRNA by RT-PCR (Elliott et al., 1998). Various Bcell and T-cell lines do not express ppSS transcripts. These observations, in addition to immunohistochemical and RIA evidence, strongly suggest that macrophages synthesize and secrete somatostatin and are the predominant source of this molecule within murine granulomas. The importance of endogenously produced somatostatin in regulating the proliferation of immune cells was shown in a very elegant set of experiments by Aguila et al. (1996). These authors showed that an antisense oligonucleotide designed to block translation of somatostatin mRNA stimulated the spontaneous proliferation of rat splenocytes in vitro. The induction of somatostatin gene transcription was believed to be due primarily to activation of a protein tyrosine phosphatase. Induction of somatostatin mRNA by proinflammatory stimuli has been demonstrated in murine splenocytes (Blum et al., 1998). Somatostatin produced by immune cells may act as an autocrine or paracrine regulator within the local immune microenvironment in mice. In humans, synovial cells isolated from inflamed synovium of patients with rheumatoid arthritis and thymic epithelium were shown to produce mRNA for somatostatin cells (Takeba et al., 1997; Ferone et al., 2000). The presence of somatostatin, however, has not been demonstrated in human immune cells. In a recent study the expression of the somatostatin-like peptide cortistatin-17 (CST) was investigated in human lymphoid tissues, immune cells, and lymphoid cell lines (2003 Dalm et al.). SS mRNA were detected in the human thymus, but not in thymocytes. On the other hand, CST mRNA was clearly expressed in all immune cells and immune tissues studied. Moreover, two CST iso forms were found, and both forms were expressed in the majority of immune cells and immune tissues. Differential expression was shown of the two iso forms in B- and T-cell lines. Whereas both fragments were detected in nearly all T-cell lines, most of the B-cell lines expressed the short fragment only. Autoradiographic studies showed that CST was able to displace [125I-Tyr3]octreotide binding on cryostat sections of human thymic tissue and

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of sst2-expressing cells. On the basis of these observations, a role for CST as an endogenous ligand for sst in the human immune system, rather than SS itself, was hypothesized. IV. FUNCTIONAL ROLE OF SOMATOSTATIN RECEPTORS IN IMMUNE CELLS Numerous effects of somatostatin on immune cell function have been described in vitro as well as in vivo. It is difficult to compare all these studies, as experiments were carried out with cells from different animal species, under different experimental conditions, and with different analogues. Inhibition of lymphoid cell proliferation is the best-documented effect of somatostatin on immune function. We have summarized the results of in vitro studies on the effect of somatostatin on the spontaneous or mitogen-induced proliferation of lymphoid cell lines and on cells isolated from lymphoid tissues of several animal species (van Hagen et al., 1994). Most of these studies involved mixed cell populations under Tcell–stimulating conditions. The clearest inhibitory or biphasic effects of somatostatin were observed when purified T lymphocytes or T-cell lines were used (Payan et al., 1984). Most of the studies reported were carried out using native somatostatin, but studies using the octapeptide analogues octreotide and lanreotide indicated that the effect was mediated by sst2, sst5, or possibly sst3. A recent study showed that PHA-stimulated proliferation of human PBMC was inhibited by octreotide and also by an analogue with enhanced selectivity for sst5, in contrast to the recent observtion that human lymphocytes express sst3 (Atiya et al., 1997; Lichtenauer-Kaligis et al., 2000). Sst2 is the sst subtype that is most often implicated in inhibition of cell proliferation by somatostatin, although inhibition of proliferation of human Jurkat T lymphocytes was shown to be mediated by sst3 (Cardoso et al., 1998). Activation of the sst2 receptor has been shown to increase transcription of the somatostatin gene, thereby inducing an antiproliferative autocrine feedback loop (Rauly et al., 1996; Delesque et al., 1997), although this mechanism has not yet been shown to operate in cells of the immune system. Besides the inhibitory effect on proliferation, somatostatin has been shown to affect the production of cytokines by T lymphocytes. Somatostatin reduced the secretion of interferon-gamma (IFN-␥) from human PBMC (Muscettola and Grasso, 1990). Production of IFN-␥ by splenocytes and T lymphocytes isolated from murine schistosome granulomas was decreased by somatostatin and octreotide in vivo and in vitro (Blum et al., 1992). This effect was probably mediated by sst2, as mRNA for this receptor subtype was shown to be expressed by granuloma T lymphocytes (Elliott et al., 1994), and the effect was blocked by an antiserum directed against sst2. In contrast, somatostatin enhanced the production of IL-2 by an ovalbumin-specific mouse hybridoma T-cell line (Nio et al., 1993) and the production of IL-2 by a murine Th1-cell line and of IFN-␥ by a number of murine Th2-cell lines (Levite et al., 1998). IL-2 secretion from mononuclear cells isolated from the human intestine was also enhanced by somatostatin in a dose-dependent manner. The expression of activation markers on T lymphocytes can be modulated by somatostatin: spontaneous IL-2 receptor expression was shown to be increased after in vivo administration of octreotide in humans (Malec et al., 1989). There is, however, some conflict with the results of a more recent study by Casnici et al. (1997), who reported that the mitogen-induced expression of the IL-2 receptor ␣ chain (CD25) and the activation marker CD69 by human PBMC was decreased by somatostatin in vitro. Controversial reports have been published concerning the possible enhancement of lymphocyte cytotoxicity by somatostatin, but this is not thought to be an important mecha-

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nism in immune regulation by somatostatin (van Hagen et al., 1994). An exciting new finding is that somatostatin stimulates adhesion of resting human T lymphocytes to fibronectin (Levite, 1998). This may indicate a role for neuropeptides in the regulation of lymphocyte migration and recirculation. Somatostatin at physiological concentrations inhibits T-lymphocyte proliferation but may also change a number of effector functions such as cytokine secretion and receptor expression. Somatostatin has been shown to inhibit the production of immunoglobulins by B lymphocytes in a number of experimental settings. Somatostatin dose-dependently reduced the secretion of IgA from lymphocytes from spleen, Peyer’s patches, and mesenteric lymph nodes (Stanisz et al., 1986). Immunoglobulin E (IgE) production by B lymphocytes from atopic patients was inhibited by somatostatin, however, only when B lymphocytes were cultured together with T lymphocytes and a murine plasmacytoma cell line and from murine ConA-stimulated monocytes (Scicchitano et al., 1988; Kimata et al., 1993). The latter observation suggests an indirect mode of action of SS on B lymphocytes. On the other hand, the synthetic somatostatin analogue octreotide inhibited pokeweed mitogen (PWM)–induced B-cell differentiation to plasma cells in PBMC from normal human donors. It is therefore not clear whether somatostatin exerts a direct effect on human B lymphocytes. In vivo treatment of rats diminished the number of antigen-specific plasma cells formed during a primary immune response (Eglezos et al., 1993), again pointing to an effect on B-cell activation, proliferation, and/or differentiation. In vitro differentiation of human peripheral blood B lymphocytes to plasma cells was shown to be reduced by octreotide, indicating again the involvement of sst2, sst3, or sst5. These findings correlate with the presence of binding sites for octreotide in the germinal centers of secondary lymphoid follicles as they are sites of the generation of B memory cells (Reubi et al., 1993, 1998). Somatostatin has been shown to influence a number of activities of cells of the monocyte-macrophage lineage. In human monocytes opposing effects of somatostatin have been described as well. Komorowski and Stepien (1995) reported that somatostatin stimulates the release of IL-6 from lipopolysaccharide (LPS)-activated adherence-selected monocytes whereas Peluso et al. (1996) demonstrated inhibition of IL-6 (and of TNF-␣ and IL-1␤) by somatostatin. Monocyte activation, as measured by HLA-DR expression and chemotaxis of neutrophils to IL-8 produced by monocytes, was also reduced. Chemotaxis of human peripheral blood monocytes and of a murine macrophage cell line were blocked by somatostatin, although a strong dose-dependency of the effect was not observed (Wiedermann et al., 1993; Ahmed et al., 1998). Enhancement of TNF-␣ production by rat peritoneal macrophages was shown at low concentrations (10ⳮ11 M) of somatostatin or octreotide, whereas production was reduced in the presence of higher concentrations (10ⳮ9 –10ⳮ5 M) of these peptides (Chao et al., 1995). Somatostatin antagonized the substance P–induced enhancement of the secretion of IL-1, IL-6, and TNF-␣ from LPSstimulated murine peritoneal macrophages (Berman et al., 1996). The effect of somatostatin on local inflammatory processes has been studied in a subcutaneous air pouch in experimental animals. Aseptic inflammation in a rat air pouch was shown to be modulated by local or systemic administration of octreotide and lanreotide. Both somatostatin analogues dose-dependently reduced the volume of the exudate, the number of leukocytes, and the levels of inflammatory mediators such as TNF-␣ and the neuropeptide substance P in the inflamed air pouch via either route of administration (Karalis et al., 1994). Both somatostatin and octreotide are also able to inhibit the respiratory burst in human monocytes (Niedermuhlbichler and Wiedermann, 1992). Somatostatin also suppressed mediator re-

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lease from rat Kupffer cells, i.e., hepatic macrophages (Chao et al., 1997). So despite contradictory results, somatostatin in general appears to suppress monocyte and macrophage effector functions. Recently, interesting effects were found on murine and human bone marrow stem cells. In a myeloid cell line (mouse 32D cells) stably transfected with sst2 and G-CSF receptors, somatostatin responses were studied. In this model, somatostatin and octreotide reduced G-CSF–induced proliferation by approximately 50%. In view of the observation that sst2 is expressed on CD34Ⳮ bone marrow cells and because other G␣i-coupled receptors have been implicated in the control of hematopoietic cell migration, such as CXCR4 and IL-8 receptor (Aiuti et al., 1997; Luster, 1998; Mohle et al., 2001), the effect of somatostatin on migration of primitive hematopoietic cells was studied. Octreotide acts as a potent pro-migratory stimilus for CD34Ⳮ stem cells (Oomen et al., 2000). In the 32D cell line model referred to above, it was shown that octreotide acts predominantly as a chemoattractant but also has some chemokinetic activity. These data suggest that somatostatin may have an effect on the homing and migration stem cells with possible implications for the clinical application of somatostatin and its analogues. V. SOMATOSTATIN AND ITS RECEPTORS IN CLINICAL PERSPECTIVE Somatostatin receptor expression can be detected by in vivo somatostatin receptor scintigraphy after injection of 111In-labeled octreotide, a somatostatin analogue. This technique is used extensively for the localization of neuroendocrine tumors and other malignancies that express high levels of sst (Krenning et al., 1993). Among the non-neuroendocrine tumours that can be visualised by octreotide scintigraphy are malignant lymphomas: both T and B non-Hodgkin lymphomas and Hodgkin disease lymphomas (van Hagen et al., 1993). In a number of infectious diseases (e.g., tuberculosis), autoimmune diseases (e.g., Graves’ ophthalmopathy), and other immune-mediated diseases (e.g., sarcoidosis and rheumatoid arthritis), the sites of inflammation can also be visualized (van Hagen 1994, 1994c). In addition, octreotide scintigraphy labels the spleen, and this labeling is reduced in patients pretreated with unlabeled octreotide, indicating that the observed competitive binding of the radioligand is receptor-mediated (Krenning et al., 1993; Reubi et al., 1993). In patients with rheumatoid arthritis, the uptake of radioactivity in the affected joints showed a high correlation with clinical activity. Immunohistochemical and RT-PCR studies confirmed the presence of sst2a receptors. In biopsies of rheumatoid synovium (van Hagen et al., 1994c; Takeba et al., 1997; ten Bokum et al., 1999) sst2A expression was detected in endothelial cells of venules (especially high endothelial venules), fibroblast-like synovial cells, and a subset of cells of the monocyte/macrophage lineage. Intra-articular injection of somatostatin in patients with rheumatoid arthritis has been shown to reduce pain and synovial swelling (Coari et al., 1995; Matucci-Cerinic et al., 1995). The analgesic effect of somatostatin may have been mediated by reduction of the hypersensitivity of afferent sensory nerves in the joint (Heppelmann and Pawlak, 1997). Somatostatin decreases the extravasation of plasma and leukocytes and may have reduced synovial swelling through this mechanism (Karalis et al., 1994). Through effects on vasodilatation, somatostatin might influence the local blood flow and the migration of leukocytes into the inflamed tissue. Somatostatin has also been shown to inhibit angiogenesis and thus may have an influence on tissue remodeling (Danesi and Del Tacca, 1996; Danesi et al., 1997). In addition, there is anecdotal evidence that octreotide may have an effect on the circulation

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of lymph fluid (Bac et al., 1995; Ballinger and Farthing, 1998). Reduction of joint swelling by intra-articular injection of somatostatin was also observed in a rabbit model of chronic arthritis. In addition, histological studies in this model revealed reduction of lymphocyte infiltration of the synovium and reduced vasculitis in somatostatin-treated animals (Matucci-Cerinic et al., 1995). Lanreotide was shown to reduce the severity of adjuvantinduced arthritis in rats, although we did not observe a reduction in the severity of rat adjuvant arthritis using the somatostatin analogue octreotide, which is in concordance with the receptor expression pattern (ten Bokum et al., 1999b). Recently, Takeba et al. (1997) showed that somatostatin at physiological concentrations inhibited the in vitro proliferation of fibroblast-like synovial cells from rheumatoid arthritis patients. The stimulation-induced production of IL-6, IL-8, and matrix metalloproteinases by these cells was also reduced by somatostatin treatment in vitro. Sst2 mRNA in cultured synovial cells was sensitive to upregulation by proinflammatory stimuli. Uncontrolled studies suggest an important role in the treatment of rheumatoid arthritis with somatostatin analogues (Paran et al., 2001; Koseoglu and Koseoglu, 2002). Based on subtype receptor expression in rheumatoid synovium (sst1–3), the recently developed long-acting somatostatin analogue SOM230, which binds to all subtype receptors except sst4, is the most promising candidate. In biopsies from patients suffering from granulomatous diseases, such as sarcoidosis and Wegener’s granulomatosis, sst2a expression was also associated with cells of the mononuclear phagocyte lineage, including epithelioid cells and multinucleated giant cells within the granulomas (ten Bokum et al., 1999a). In none of the biopsies was sst2a expression observed, which is in agreement with the sst3 expression pattern in humans (Table 1). On basis of classical ligand-binding studies, it had previously been assumed that lymphocytes were the major cell types expressing sst2 in human immune-mediated diseases (van Hagen, 1994; Tsutsumi et al., 1997). Based on present studies, this does not appear to be the case (Lichtenauer-Kaligis et al., 2000). Besides a direct role of somatostatins on immune cells, important effects were reported on blood vessel smooth musle cells (SMC). In transplantation, graft-vessel disease (GVD) is a major cause of graft loss after the first year following transplantation. GVD is a complex, multifunctional process that involves immunological as well as nonimmunological events such as ischemia/reperfusion injury. An important target cell to interfere with the development of GVD is the smooth muscle cell. Somatostatin analogues have been shown to inhibit the proliferation of SMC in vitro and in vivo. Bruns et al. (2002) provided evidence that octreotide, known to have antiproliferative effects on SMC proliferation, inhibited vascular remodeling in a rat angioplasty model. Furthermore, in two allotransplantation models, octreotide effectively interfered with the development of signs of chronic rejection/GVD. The role of the different ssts in chronic graft rejection is currently under investigation. VI. CONCLUSION Differential Expression of ssts are found on immune cells from the various species studied. This differential expression has important consequences for conclusions that are made about the functional role of ssts and its ligands in the immune system. In humans, sst2 and sst3 are the main subtypes expressed in the immune system. Cortistatin seems to be the immunoligand in the human immune system and binds to all five subtype receptors; no specific receptor has been identified. Based on the receptor pattern in autoimmune diseases, controlled studies are needed to investigate the efficacy of somatostatin analogues in the treatment of autoimmune diseases like rheumatoid arthritis. In rheumatoid arthritis

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11 Prolactin and the Immune System ROBERT HOOGHE, NELE MARTENS, and ELISABETH L. HOOGHE-PETERS The Free University of Brussels, Brussels, Belgium

I. INTRODUCTION Prolactin (PRL) is secreted mainly by the anterior pituitary gland and is a major vehicle in the neuroendocrine communication system. It relays messages from the brain, through the hypothalamus, in response to various endogenous or exogenous stimuli, to a large number of target tissues, including the immune system. After hypophysectomy in the rat, PRL levels are very low. At the same time, profound immune deficiencies are present. These deficiencies can be prevented by various protocols of hormone replacement, such as treatment with PRL only. Updated information on PRL can be retrieved via Refs. 1 and 2. PRL was thus proposed to be a link between the nervous and the immune systems: in contrast to the glucocorticoids, which have mainly immunosuppressive properties, PRL exerts immunostimulatory activity. Why is it then that PRL is not even mentioned in the fattest textbooks of immunology? The PRL-receptor (PRL-R) was the second member of the cytokine-hemopoietin receptor family to be cloned. Although PRL and growth hormone (GH) probably represent the oldest members of the cytokine-hemopoietin family in evolution, this cannot be sufficient to make them bona fide cytokines. Ectopic expression of PRL has been known for some time. PRL is indeed produced, usually in small amounts, by various normal and malignant extrapituitary tissues, including the immune system. The importance of paracrine PRL is being appreciated in the decidua and in several other tissues. Though a wealth of solid data have been obtained using the rat Nb2 T-cell lymphoma line, which is dependent on PRL for growth, much fewer data on possible effects of PRL were generated with untransformed leukocytes. Most important, the immune system develops normally in transgenic mice overexpressing PRL or carrying only null alleles of 207

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either the PRL or the PRL-R gene, and immune responses are normal in both PRLⳮ/ⳮ and in PRL-Rⳮ/ⳮ mice. Children with a severe PRL deficiency develop normally. In adults, PRL deficiency is compatible with normal life, with the exception of lactation. However, the total absence of either PRL or PRL-R has not been documented in humans. Although few novel experimental data showing effects of PRL and GH in the immune system have been produced, several reviews and hypotheses have been published on the immunobiology of PRL [2–5] (see also full issues of NeuroImmune Biology [6], Lupus [7], the Journal of Neuroimmunology [8], Cell and Molecular Life Science [9], and Ref. 10). Therefore, in the present chapter we will review mainly recent experimental data relating to a possible role of PRL in the mammalian immune system. The hope that PRL will have therapeutic value in the treatment of hematological failure and in some forms of immune deficiencies is one major incentive for ongoing studies [11]. Also, testing the hypothesis that PRL plays a deleterious role in some autoimmune conditions such as systemic lupus erythematosus (SLE) has clinical implications [7]. We will briefly consider the nature and the sources of lactogenic hormones: their receptors, their biochemical and molecular effects in leukocytes, their in vivo and in vitro effects on the development of the immune system and on immune responses, their possible role in hemopoietic malignancies and in tumor immunology. Signaling through the PRL-R has been covered in detail in Chapter 2. We will critically review evidence that PRL is the oldest cytokine and, more important, that it is a hemapoietic growth and differentiation factor in mammals. II. PROLACTIN: A FAMILY OF MOLECULES A. Pituitary Prolactin PRL is the prototype of lactogenic hormones. In mammals, its main actions are related to the development of the mammary gland and milk production [1,2]. Several other hormones and growth factors are involved in these processes, but only agonists acting through the PRL receptor (PRL-R or lactogenic receptor) are called lactogens. In addition to PRL, the lactogenic hormones include GH (from primates only) and placental lactogens. There is only one gene for PRL in human, mouse, and rat, mapping close to the MHC in the human (on chromosome 6, 6p22.2–p21.3). In the mouse in particular, many PRL-related proteins (PLP) are coded for by related genes, but, having no lactogenic activity, they are not considered here [2]. One of these, the murine PRL-like protein PLP-E, is a true hemopoietic growth factor [12]. PRL is the most abundant pituitary hormone in humans. It is a 4-helix-bundle polypeptide with a strong homology to GH and placental lactogen and a weak homology to other cytokines such as interleukin (IL)-6. The major form of PRL is 23 kDa PRL in the human, mouse, and rat. Posttranslational modifications yield a large array of PRL variants. PRL can undergo glycosylation, phosphorylation, dimerization (big PRL), or proteolytic cleavage. Some features of PRL are summarized in Table 1. The PRL variants differ in their specific activity. Immunoreactive forms with an apparent molecular weight of 45 kDa or more in reduced gels have been detected, but in most cases compelling evidence that they are indeed products of the PRL gene was missing. High molecular weight complexes containing PRL [‘‘big-big PRL,’’ consisting of PRL multimers or PRL-immunoglobulin (PRL-Ig) complexes, possibly PRL-antiPRL complexes] are also found in normal and pathological sera. In some cases, the presence of a covalent linkage between PRL and Ig has been ascertained [13–15]. Big-big PRL is active in vitro (Nb2 assay) but not in vivo (due to poor crossing of the endothelial barrier). A 16 kDa N-terminal proteolytic fragment

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Table 1 Human Prolactin: Gene, mRNA, and Protein Gene Size Location Polymorphism mRNA Pituitary PRL Extra pituitary PRL (alternative promotor usage, alternative initiation codon) Differences do not affect coding sequence. Protein Size Disulfide bridges N-Linked glycosylation site Variants arise through

Binding partners

⬃10 kb, 5 exons, 4 introns Chromosome 6p22.2–21.3, telomeric to HLA-A In noncoding sequences or silent 1.38 kb 1.55 kb

Preprolactin, 227 AA; Prolactin, 199 AA AA 4–11, 58–174, 191–199 AA 31 Proteolysis, glycosylation, phosphorylation, acetylation, dimerization, polymerization Prolactin itself, heparin, proteoglycan, immunoglobulin (anti-PRL?)

Source: Refs. 2, 6, 19, and 34.

of rat PRL does not bind the PRL-R but has biological (e.g., antiangiogenic) activity [16,17]. The 6 kDa C-terminal fragment has some residual lactogenic activity [18]. Recombinant PRL is the golden standard for experimental studies. Consideration should be given to the glycosylation level (absent in prokaryotic, variable in eukaryotic protein) and possible contamination by endotoxin. B. Other Sources of PRL The expression and secretion of PRL are under negative (and to a lesser extent positive) control from the hypothalamus, but in contrast to the situation with other hormones from the anterior pituitary, there is no known hormone (or factor) induced by PRL in a peripheral tissue that would reduce PRL secretion by a feedback mechanism. PRL itself downregulates its secretion through direct action on the pituitary cell or through indirect action via hypothalamic dopaminergic neurons. After hypophysectomy, PRL serum concentration declines to undetectable levels. When this is not the case, it is considered that hypophysectomy was not complete. Thus, extrapituitary tissues produce PRL that may result in high local concentrations leading to paracrine or autocrine effects without contributing significantly to serum levels, except during pregnancy (Fig. 1). The myometrium and the decidualized endometrium are the main sources of extrapituitary PRL, which accumulates to high levels in the amniotic fluid [19]. The expression of PRL (messenger RNA, protein) by leukocytes has been thoroughly documented also. Quantitative data are scarce, however.

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Figure 1 Endocrine, paracrine, and autocrine PRL. PRL (black dots) secreted by PRL-secreting cells, e.g., leukocytes can enter the blood stream to reach target cells throughout the body (endocrine). PRL can also act locally on a cell close to the PRL-secreting cell (paracrine) or on the PRL-secreting cell itself (autocrine). Autocrine mechanisms without secretion have also been postulated (intracrine). : PRL-R, •: PRL, 䡩: A similar situation exists for GH (see review by Kooijman, Chapter 9). ( secretory granule).

The expression is higher in human T cells than in monocytes and lowest in B cells. Thymocytes and bone marrow (CD34Ⳮ but also stromal) cells express PRL [21–23]. Some human hemopoietic cell lines expressing PRL are listed in Table 2. Remarkably, when monocytic differentiation is induced in U937 cells, PRL gene transcription is upregulated by a factor of 60; this differential expression was confirmed in CD14Ⳮ monocytes obtained from CD34Ⳮ precursor cells [24]. A proximal promoter directs PRL expression by the lactotroph cells from the pituitary gland. The expression of PRL in extrapituitary tissues can be regulated in two different ways, depending on whether the proximal (‘‘pituitary’’) or the distal (‘‘extrapituitary’’ or ‘‘decidual,’’ or ‘‘lymphocytic’’) promoter is used (use of the pituitary promoter is thus not restricted to the pituitary). Expression directed from the extrapituitary promoter results in a larger mRNA, but the coding sequence and the protein are not different [24]. The use of the extrapituitary promoter implies transcription of noncoding exon 1a at ⳮ5.8 kb. Pituitary PRL production is positively regulated by estrogen, IGF-I, insulin, T3, thyrotropin-releasing hormone (TRH), VIP, EGF, insulin, and prostaglandin F2␣, and negatively by dopamine and dexamethasone. Secretion is strongly inhibited by dopamine and stimulated by TRH. The neural control of PRL secretion has been reviewed recently [34]. While the regulation of the pituitary promoter has been studied extensively, much less is known about the extrapituitary promoter [19,25,29–33]. Cyclic AMP and insulin positively regulate the expression of PRL via the extrapituitary promoter [25,30–33; S. Gerlo, in press]. Dexamethasone, IL-4, tumor necrosis factor (TNF)-␣, and retinoic acid have an inhibitory action [33]. III. THE PROLACTIN RECEPTOR A. Structure Only one gene codes for lactogenic receptors. In the human, mouse, and rat, this gene is found in a cluster of genes coding for cytokine receptors also containing the IL-

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Table 2 Expression of PRL and PRL-R in Human Lymphoid and Myeloid Cell Lines PRL Cell line Myeloid K562 Eol-1 HL60 U937 Lymphoid B Daudi Ramos 1M9 1M9 P-3 (c) U266 RPMI-8226 Lymphoid T Jurkat Molt4 HUT78 NK YT

mRNA

Protein

nd ⫹ nd ⫹

nd ⫹ nd nd

⫹ ⫹ ⫺ ⫹ ⫹ nd

⫹ ⫹ ⫺ ⫹ ⫹ nd

⫹/⫺ ⫺ ⫹ nd

PRL-R Ref.

mRNA

Protein

Ref.

⫹ ⫺ ⫹ ⫹

⫹ ⫺ ⫹ ⫹

21,42 a 21,42 21

26 26 25 25 169,b

nd nd ⫺ nd ⫹ ⫹

⫹ nd nd nd ⫹ nd

26

169, b b

⫹/⫺ nd nd

21,27 21 21

⫹/⫺ ⫹ ⫹

⫹/⫺ nd nd

21,27 21 21

⫹

28

⫹

⫹

21,42

28 24

b

a

From S. Gerlo, personal communication. Authors’ unpublished results. c After mutagenesis of IM9 cells, a PRL-producing clone was isolated. In certain cases (e.g., Jurkat), expression of PRL or PRL-R is seen in some sublines only. (⫹, expression detectable; ⫺, expression not detectable; nd, not determined. b

7-R, GH-R, and LIF-R [in humans, more than 100 kb long (11 exons, 10 introns) on chromosome 5, 5p13–p12) [35]. The PRL-R belongs to the class I cytokine receptors (hemopoietin receptors). These share a general structure, with an extracellular domain (ECD), a single transmembrane domain, and an intracellular domain without intrinsic enzymatic activity and several structural motifs, including a cytokine receptor homology (CRH) domain with two pairs of conserved cysteine residues and a WSXWS sequence motif in the ECD. Although GH-R and PRL-R were the first genes cloned among the members of this family, variants of human PRL-R have been described only recently (Fig. 2), and all variants may not yet have been identified [1,2,36–39]. Detailed information is available for rat PRL-R variants [1,2]. For the known variants of human PRL-R, little is known about their tissue distribution. In addition to the full-length (bona fide, classical) form, they include the ⌬S1 form (missing about one half of the ECD), the intermediate, the S1a, and the S1b forms, all three missing a large part of the intracellular domain and the PRL-binding protein, corresponding to ECD only [36–38]. Some variants have a reduced ligand-binding activity compared to the long form. Other variants have reduced or no signaling capacity. When such variants form homodimers or nonsignaling heterodimers with a long form, they can act as dominant negative forms. Though homodimerization is the rule, combinations between different PRL-R variants are also possible. Moreover, bovine placental lactogen heterodimerizes the bovine GH-R and PRL-R [40], but it is not known whether similar rules also

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Figure 2 The human PRL-R isoforms. The extracellular domain is nearly entirely present in the soluble PRL-binding protein, but has lost its N-terminal half in the ⌬S1 form, which is generated by alternative splicing, resulting in the deletion of exons 4 and 5 of the PRL-R gene. All other receptor variants have complete extracellular and transmembrane domains. The intermediate form mRNA results from alternative splicing within exon 10, leading to a large deletion and, as a result of a frameshift, a different C-terminal sequence with premature termination (grey box). The S1a and S1b forms result from alternative splicing of exons 9, 10, and 11. They differ from the long form by having shorter intracellular domains with 39 (S1a) and 3 (S1b), unique, C-terminal AA. (CRH, cytokine receptor homology domain; S1, subdomain 1; S2, subdomain 2; C-C, disulfide bridge; WSXWS, tryptophan-serine motif. (For references for full-length receptor see Refs. 1 and 2; for the intermediate form see Refs. 2 and 37; for S1a and S1b see Ref. 36; for PRL-binding protein see Ref. 38.)

apply for instance to human placental lactogen. The biological significance of variants with different signaling capacity still awaits experimental study. In mammals, short forms that are unable to signal could act as decoy, to prevent excessive stimulation in situations where lactogenic hormone levels are high (lactation, pregnancy) [39]. B. Cell Types Expressing PRL-R So far, no tissue has been identified that does not express PRL-R. This does not mean that all cell types express PRL-R at all developmental stages. Ovary, mammary gland, liver, and kidney express the highest amounts of PRL-R. In reproductive tissues, PRLR expression is hormone-dependent [2]. Even on classical target cells, such as the mammary epithelium, only low numbers of PRL-R (about 350 receptors/cell) are present [4]. Such low receptor numbers allow for the strong effects seen in the mammary gland, and it can be assumed that the same holds true for other targets such as leukocytes. In the lymphohemopoietic system, mRNA expression of the PRL-R has been documented [2,5,6,21,36–39,41,42]. In the murine spleen, labeling with anti PRL-R antibody was highest on Mac1Ⳮ cells, intermediate on B-220Ⳮ cells, and lowest on Thy-1Ⳮ cells [43,44]. PRL-R were also found on mouse bone marrow pro-B and pre-B cells [45]. We have documented high PRL-R expression in rat spleen and thymus cells and weaker expression levels in bone marrow (RT-PCR) [46]. Quality reagents for human PRL-R protein are not widely available. Older studies suggesting that a large proportion of leukocytes express low numbers of PRL-R should be repeated, according to the authors of the initial study [41]. With biotinylated PRL as a probe, 22–34% of human PBMC are found to express PRL-R [41].

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No recent data are available for human leukocyte subsets. Clevenger and Kline found that human bone marrow, thymus, spleen, lymph nodes, and peripheral blood leukocytes all express relatively high amounts of full-length PRL-R mRNA (about 50% of the amount present in the pituitary gland). Expression of the ⌬S1 and the intermediate forms is more variable [2,37,38]. The shorter forms identified by Hu et al. are also expressed in human blood cells, but expression levels in various leukocyte populations have not been determined [36]. In human peripheral blood leukocytes, we consistently documented the expression of PRL-R mRNA in the mononuclear fraction (lymphocytes Ⳮ monocytes). However, we have not been able to demonstrate the expression of any form of PRL-R in the granulocyte fraction [47]. Yet, effects of PRL were seen in granulocytes [47,48]. In T-cell clones, PRL-R mRNA was detected in one CTL clone but not in four T-helper clones [49]. The presence of PRL-R on NK cells has been well documented [50,51]. Taken together, the characterization of PRLR variants is still underway, and data for human leukocytes, in particular expression levels in subsets are still fragmentary. The PRL-R is also expressed in some hemopoietic cell lines (myeloid, B, T, NK) (see Table 2). C. Signal Transduction Through PRL-R Signaling through PRL-R, with emphasis on data gathered in rat Nb2 lymphoma Tcells, is reviewed in detail by Buckley et al. (see Chapter 2). Important data have also been obtained in normal and transformed mammary or liver cells. After binding to its receptor, PRL induces receptor dimerization. This in turn activates signaling cascades, in particular the JAK-Stat and the MAP-kinase (MAPK) pathways [52] (Fig. 3). Of physiological relevance is the well-known bell-shaped doseresponse curve for PRL. In different experimental systems, optimal stimulation was obtained when a maximum number of receptors binds PRL in a 2:1 stoechiometry (i.e., one hormone molecule for two receptor molecules). At high hormone concentrations, dimerization cannot occur [1,2]. Only one JAK kinase, JAK2, is activated, but three different Stat factors, Stat1, Stat3, and Stat5, can be activated through the PRL-R, with a strong preference for Stat5. Recent progress in the field of Stat5 signaling concerns interactions of Stat5 with nuclear receptors and with NF-␬B [3,53–61]. Positive enhancement of the Stat5 transactivation potential of PRL on the ␤casein promoter is provided by the glucocorticoid receptor [53,54]. Also, simultaneous presence of activated Stat5 and the androgen receptor is required for expression of the PRL-inducible protein/gross cystic disease fluid-15 (PIP/GCDFP-15, a CD4-binding protein normally found in seminal plasma) in breast carcinoma cells [56]. As an example of antagonistic interactions, estrogen receptors (ER) ␣ and ␤ were found to potently repress PRL-induced Stat5 transcriptional activity on a ␤-casein promoter construct in a ligand-dependent manner in 293 HEK cells (transfected with a ␤-casein reporter gene together with plasmids encoding the PRL-R, Stat5a or Stat5b, and ER␣ or ER␤). This downregulation was found to rely on direct physical interaction between the ERs and Stat5, mediated via the ER DNA-binding domain [57]. The progesterone receptor can, depending on the promoter context, either repress (␤-casein) or enhance [3 ␤-hydroxysteroid dehydrogenase (HSD)] Stat5-mediated gene activation [55]. NF-␬B inhibits Stat5 signaling. Thus, in HC11 cells, TNF-␣ inhibited PRLinduced expression of the endogenous ␤-casein gene and of a ␤-casein reporter construct

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Figure 3 PRL signal transduction in human leukocytes. PRL signals through the MAPK and JAK/Stat signal transduction pathways (only JAK2/Stat5 represented here). Among the genes induced are iNOS, IRF-1, CIS, SOCS-2, and -3. CIS inhibits the binding of Stat5 to the activated PRL-R. SOCS-2 also associates with the PRL-R, whereas SOCS-3 associates with JAK2 and inhibits PRL-R tyrosyl phosphorylation. PRL-induced SOCS factors may also act on signal transduction through other cytokine receptors, e.g., IL3-R or IL6-R. (TF, transcription factors.)

in a dose-dependent fashion, and this inhibition is mediated by NF-␬B [59]. It should be noted that Stat5 can also inhibit signaling by NF-␬B [3,60]. Also, liganded thyroid hormone receptor ␤-1 in the presence of its heterodimeric partner, retinoid X receptor ␥(RXR␥), inhibited PRL-induced Stat5a- and Stat5b-dependent reporter gene expression by up to 60% [61]. Finally, PRL interacts with cyclophilin B, a member of the immunophilin family of proteins. The PRL/cyclophilin B complex translocates into the nucleus, where it interacts directly with Stat5. As a result, the Stat-repressor protein inhibitor of activated Stat3 (PIAS3) is removed, thereby enhancing Stat5 DNA-binding activity and PRLinduced, Stat5-mediated gene expression [62]. Thus, signaling through the PRL-R activates several cascades, which are themselves intertwined in complex networks of signaling molecules and transcription factors, and the consequences of activation cannot easily be predicted [1–3,37]. The experiments discussed below are intended to document the mode of action of PRL in leukocytes rather than contribute to the knowledge on PRL-R signaling. We have shown that a physiological concentration of PRL stimulates the phosphorylation of JAK-2 and Stat5 in rat bone marrow and spleen cells, with subsequent activation of the interferon regulatory factor-1 (IRF-1) gene [46]. Signaling studies were also done on human leukocytes. In PBMC, PRL increased the tyrosine phosphorylation of JAK-2 and Stat5. In granulocytes, no phosphorylation of JAK-2 or Stat5 could be detected after stimulation with PRL. However, tyrosine phosphorylation of JAK-2 and Stat5 was observed in

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GM-CSF–treated granulocytes. In contrast, in granulocytes, PRL induced Stat1 phosphorylation. PRL also caused a significant increase in the phosphorylation level of p38 MAPK in PBMC and granulocytes [47]. Gubbay et al. have shown that PRL induced the activation of the ERK MAPK in human CD56Ⳮ natural killer (NK) cells from secretory phase endometrial stroma and first trimester decidua [51]. D. PRL-Induced Gene Expression 1. Gene Expression in Normal Lymphohemopoietic Tissues We have analyzed gene expression in PBMC, granulocytes, bone marrow cells, tonsillar cells, and CD34Ⳮ cells, with particular attention to members of the recently described SOCS family (suppressors of cytokine signaling). Also, in CD34Ⳮ cells purified from cord blood, PRL activated Stat5 and induced CIS and SOCS-2, as did IL-3 [63]. PRL induced SOCS-3 and inducible nitric oxide synthase (iNOS) gene expression in PBMC, IRF-1, CIS, and iNOS gene expression in granulocytes; CIS and SOCS-2 in bone marrow cells and SOCS-2 and SOCS-7 in tonsillar cells [47]. We have reproduced several of the findings reported above (rapid activation of Stat, induction of gene expression) in human T-cell clones [49]. This suggests that T cells contribute to the effects observed in PBMC. Recently, Kochendoerfer et al. found that PRL induces the expression of X-linked inhibitor of apoptosis protein and Bcl-xL, two survival genes, in thymocytes from PRL-KO and from normal mice respectively [205; unpublished results]. A more complete picture will emerge when the PRL-responsive cell populations is identified and when microarray gene expression studies become available. PRLmediated activation of Stat5 allows the expression of IRF-1, CIS, and Bcl-xL [47,49,64,203]. In turn, the upregulation of iNOS expression is best explained by the increase in IRF-1 generated by PRL. 2. Gene Expression in Other Systems Considerable work has been performed to identify genes induced by PRL in Nb2 lymphoma cells, with special interest for cell-cycle and apoptosis-related genes [1,2,65]. There is little overlap between results obtained with PRL treatment in nontransformed leukocytes and in the Nb2 cell line. Upregulation of IRF-1, X-linked inhibitor of apoptosis protein, and Bcl-xL, however, has been documented in both systems. In the mammary gland, PRL favors the expression of milk proteins (which all have GAS elements that bind Stat5 in their promoters) [1,2]. Is this relevant for immunology? Probably not, though ␤-casein is expressed in cytotoxic lymphocytes [66]. In the mouse mammary gland, PRL also stimulates the expression of GlyCAM1; the significance of this finding is unknown [67]. More to the point, PRL stimulated ICAM-1 expression in the corpus luteum [68] and in thyrocytes [69], and these observations may be relevant for leukocyte trafficking in PRL-target tissues: indeed, the expression of ICAM-1 could be correlated with the recruitment of macrophages in the rat corpus luteum. In thyrocytes, PRL treatment resulted in increased expression of B7.1, which may be relevant for induction of autoimmunity [69]. In the rat corpus luteum, gene array studies indicate that PRL stimulates several genes involved in the maintenance of the corpus luteum, such as the LH receptor, 11 ␤-hydroxysteroid dehydrogenase2, sterol carrier protein2, and inhibits the expression

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of, among others, 20␣-hydroxysteroid dehydrogenase (which metabolizes progesterone), PGF(2␣)-receptor (which mediates luteolysis), phospholipase C ␦(1), TGF-␤(1), and c-jun. [70].

IV. IN VITRO EFFECTS OF PRL IN THE IMMUNE SYSTEM Cellular uptake of lactogenic hormones has been thoroughly documented, and the hormone can thereafter be released again [71]. Thorough washing and long preculture in serum-free medium or in the presence of horse serum (with very low lactogenic activity) are prerequisites for meaningful experiments with cells that have been exposed to fetal bovine serum in vitro and with cells that have been obtained from subjects or animals that are not hypoprolactinemic. See the next section for experiments performed with cells obtained from hypoprolactinemic mice. A. Myeloid Lineage 1. Macrophages, Granulocytes Stimulation through the PRL-R increases the oxidative burst of macrophages and granulocytes (H2O2 production, superoxide anion secretion) [5,48]. Recently, it was shown that PRL induced the release of IL-12 from murine macrophages. Through IL12, PRL might induce the secretion of IFN-␥ and favor a shift of T-helper (TH) responses towards TH1 [172]. 2. Dendritic Cells Recently, two research groups have demonstrated potent effects of PRL on the differentiation and maturation of dendritic cells. Matera et al. have shown that physiological concentrations of PRL added to GM-CSF drive monocytes into the DC differentiation pathway. At supra-physiological concentrations, PRL alone can increase the differentiation of monocytes into immature DC and further into mature DC [72,73]. PRL also increased the expression of MHC class II by dendritic cells cultured from monocytes in the presence of GM-CSF and IL-4, leading to more efficient antigen presentation [74]. B. Lymphoid Lineage 1. B cells The expansion of murine bone marrow–derived B-cell precursors was only slightly increased by ovine PRL (100 ng/mL) [75]. The proliferation and plasmocytic differentiation of human B cells and Ig production were significantly increased by human PRL (10 or 100 ng/mL). PRL at 10 ng/mL also increased IgM and IgG secretion. A synergistic effect between IL-2 and PRL was demonstrated [76]. In other studies, PRL stimulated human B-cell proliferation in the presence of submitogenic concentrations of Staphylococcus aureus Cowan [77] and enhanced IgA secretion when added to LPS-activated B-cell cultures [78]. In some studies, however, no effect of PRL could be detected [79]. PRL, IL-2, and IL-7 could have additive effects on Stat5-dependent transcription [80].

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2. T cells PRL (12–50 ng/mL) stimulates IFN-␥ release by T cells and increases IFN-␥ release by IL-12 [81]. This is in agreement with data showing that activation of Stat4 alone is not sufficient for IL-12–induced IFN-␥ production and proliferation, suggesting that other Stats play a role in these responses to IL-12 [82]. Stat5, activated in this case by PRL, could contribute to the expression of IFN-␥. In PBMC treated with PRL (1–100 ng/mL), we found no effect on IFN-␥ expression, but there was a modest inhibition of IL-5 expression [83]. 3. NK and LAK Cells The stimulatory effect of PRL on NK cells has been studied in great detail [6,8,77,84]. PRL favors the development of LAK cells [85]. Although a less potent activator than IL-2 or IL-12, PRL (25–50 ng/mL) stimulated IFN-␥ release by human NK cells much more than by T cells [81]. The increase of IFN-␥ production seen in the total PBMC fraction should thus probably be ascribed to a large extent to NK cells [86]. In this system, too, PRL probably collaborates with other cytokines through Stat5 activation. In particular, PRL induces IRF-1 expression, which is essential for NK cell function in vivo and in vitro [87]. In the human uterus, the coincidence of increased PRL secretion from the stroma and the accumulation of CD56Ⳮ NK cells within mid to late secretory phase endometrium is consistent with promotion of CD56Ⳮ NK-cell growth by PRL. In addition to cell growth, PRL may also stimulate the maturation of uterine CD56Ⳮ NK cells. This maturation process may be dependent on PRL-induced IRF-1 [51]. The role of decidual NK cells at the time of implantatation and the mechanisms involved are not fully understood, but PRL—possibly together with IL-15 produced at the same site and also activating Stat5 [88]—is a likely modulator of uterine NK function (for a review on uterine NK cells, see Ref. 89). C. Conclusion from In Vitro Data Effects of in vitro treatment with PRL (usually 10–100 ng/mL) have been documented in different populations of leukocytes. Most of these effects can be explained by the activation of Stat5. For many of these data, however, confirmation is still needed.

V. IMMUNOMODULATORY ACTIVITY OF PRL IN VIVO A. Prolactin: Still in Search of a Function? As the name indicates, PRL promotes lactation. Targeted deletions of PRL or PRLR genes in mice demonstrate an absolute requirement for PRL during pregnancy and lactation only. In the male, PRL acts on the prostate and other reproductive tissues. (The major symptom in PRL transgenic mice overexpressing PRL is prostate hypertrophy [105].) PRL-R are ubiquitously distributed, and, indeed, there are many PRL targets outside the reproductive sphere. Recent investigations focused on brain, behavior, pancreatic islets, cartilage, and bone [1,2,90,91]. Interest for direct effects of PRL on the immune system was growing until PRL and PRL-R knockout mice were generated.

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B. Animal Data 1. PRL-Deficient Rodents Genetic Defects. DWARF MICE. Dorshkind and Horseman have reviewed studies performed in mutant mouse strains with low PRL levels and in PRLⳮ/ⳮ mice [4]. Snell (dw/dw) and Ames (df/df) pituitary dwarf mice have reduced PRL, GH, and TSH levels, although neither of these hormones is totally absent. In particular, there has been no search for extra-pituitary PRL expression. Extrapituitary GH is expressed in bone marrow cells from Snell dwarfs [175]. As a result of GH and TSH deficiencies, these mice also have low IGF-I and T3/T4 levels. B-cell development is impaired in dw/dw dwarfs, but this is accounted for by the thyroid hormone deficiency. T-cell numbers are normal after correction for the reduced size of the mice. Most aspects of immune function seem normal, at least when the mice are housed under optimal conditions. Innate immunity maybe deficient in dw/dw mice: indeed, 2 days after the injection of Listeria monocytogenes, bacterial load was higher in dwarf than in control mice. The interpretation of the above-mentioned studies is hampered to a certain extent by the presence of low amounts of pituitary and extrapituitary PRL and, more importantly, by other hormone deficiencies. It should also be remembered that animals were exposed to lactogenic hormones including PRL during pregnancy and lactation. There is evidence that intact PRL can be absorbed from milk [92]. PRLⳮ/ⳮ MICE. Targeted disruption of the PRL gene has no noticeable effect on the development of the immune system or the cellular or humoral response to antigen challenges [4,93]. This is similar to observations made in PRL receptor–deficient mice (see below). As is the case for pituitary dwarf mice, knockout mice are exposed to PRL during gestation and lactation. Immune responses have been assessed carefully and found to be normal. After burn injury, however, bone marrow cells from PRLⳮ/ⳮ mice have a greater number of granulocyte-macrophage colony-forming units (GMCFU) when stimulated by GM-CSF, whereas the proliferative response of splenic cells to PHA was reduced [94]. The higher response to GM-CSF in bone marrow cells could be due to a lack of (PRL-induced) SOCS factors (in particular, SOCS-1 and SOCS-3). The lower proliferation rate in splenocytes points to additive effects of PRL and other factors in normal mice. Activation of the cyclin D1 promoter by Stat5 is but one of the many possible targets of PRL [95]. Acquired Defects. HYPOHYSECTOMIZED RATS. One month after adult hypophysectomy, rats have moderate anemia and thrombopenia and either slightly elevated leukocytosis or leukopenia. When such hypohysectomized rats were further treated daily with anti-PRL serum (to neutralize residual PRL), they became leukopenic and died within 4 weeks. Unfortunately, the cause of death was not clearly established [96]. Widespread immune deficiency has been documented in hypohysectomized rats. This includes poor antibody response to sheep red blood cells, reduced skin reactions to dinitrochlorobenzene, delayed rejection of allogeneic skin, and suppression of adjuvant arthritis (reviewed in Refs. 5, 6, and 10). Hypohysectomized rats are also more susceptible to infection with Salmonella typhimurium as a result of lower functional

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capacity of macrophages [97–99]. These immune deficiencies are readily compensated by pituitary hormones, either PRL, GH, placental lactogen, or an ectopic syngeneic pituitary graft. The latter is known to secrete mainly PRL. Unfortunately, these experiments have not been repeated with recombinant hormone preparations. BROMOCRIPTINE TREATED MICE. Dopaminergic agonists such as bromocriptine (Bcr) have been widely used to lower PRL levels in patients (with a pituitary adenoma) and in experimental animals. Bcr-treated mice had an increased susceptibility to Listeria monocytogenes infection due to reduced phagocytic capacity of macrophages, resulting most likely from a lack of T-cell–generated IFN-␥ [100]. The fact that PRL reversed the action of Bcr is a clear indication that PRL deficiency accounts for most of the effects of Bcr. Dopaminergic agonists such as dopamine and Bcr, however, can act directly on leukocytes [23,101–103]. Thus, in contrast to genetic defects, where no clear-cut immune deficiency can be observed, acute deprivation of PRL leads to overt deficits. 2. PRL-R–Deficient Mice The development of the immune system, as well as B-, T-, macrophage, and NK-cell function, appear normal in PRL-Rⳮ/ⳮ mice [1,2,4,104]. It can thus only be concluded that lactogenic hormones are dispensable for the development and the function of the murine immune system when they are absent from the time of conception. 3. Hyperprolactinemic Rodents PRL Transgenic Mice. No immune pathology has been reported in transgenic mice overexpressing PRL (with either slightly elevated (15 ng/mL) or very high PRL (up to 250 ng/mg), but no detailed investigation of the immune status has been reported [105]. PRL Administration. Yang et al. have administered recombinant human PRL to pregnant mice via minipumps to reach maternal serum levels of 50 ng/mL (moderate hyperprolactinemia). This led to erythrocytosis, lymphopenia, and small thymi in the pups [106]. Also in PRL-deficient dw/dw dwarf mice, treatment with PRL depressed thymic cellularity [107]. In contrast, the administration of Bcr or anti-PRL serum increased thymic cellularity in newborn Balb/c mice) [108]. Ovine PRL administration resulted in a significant increase in the number and function of antigen-specific peripheral T cells in both immunized dwarf (dw/dw) and control (Ⳮ/?) mice [107]. Similarly, grafting a pituitary gland into newborn mice increased B- and T-cell responsiveness at day 45 [108]. Also, when normal mice were treated with 0, 1, 10, or 100 ␮g of recombinant human (rh) PRL) for 4 days, both frequencies and absolute numbers of splenic colony-forming unit granulocyte-macrophage (CFU-GM) and burstforming unit-erythroid (BFU-e) were significantly increased in mice receiving rhPRL. Though bone marrow cellularity was not significantly affected by any dose of rhPRL, the absolute numbers and frequencies of bone marrow CFU-GM and BFU-e were augmented by rhPRL. In addition, PRL counteracted the anemia and myelosuppression induced by azidothymidine (AZT, 2.5 mg/mL in drinking water). rhPRL partially restored the hematocrit in the animals after 2 weeks of treatment and increased CFUGM and BFU-e in both spleen and bone marrow [109]. Recently, Buckley and coworkers obtained evidence that PRL protects normal lymphocytes against glucocorticoid-induced apoptosis. Dexamethasone induced

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apoptosis in thymocytes from adrenalectomized normal (PRLⳭ/ⳮ) and PRLⳮ/ⳮ mice, but not from adrenalectomized hyperprolactinemic mice (PRLⳮ/ⳮ carrying a pituitary graft). Protection could be correlated with the level of the X-linked inhibitor of apoptosis protein [110,111]. Chaudry and coworkers conducted a large series of experiments on the immunoprotective role of PRL after experimental hemorrhage. The ability of murine macrophages and Kupfer cells, harvested 2 hours after acute hemorrhage, to release IL-1, IL-6, and TNF-␣ is markedly decreased. If such mice were treated with PRL (100 ␮g/25 g body weight, s.c.) immediately before resuscitation, the cytokine release capacity was restored and was thus comparable to the values in sham animals. PRL also improved the survival of animals subjected to sepsis after hemorrhage. Similar results were obtained when hyperprolactinemia was induced with the dopamine antagonist metoclopramide. This compound was inactive in vitro, suggesting that it stimulates PRL secretion by pituitary lactotroph cells. After hemorrhagic shock, murine splenocytes secrete less IL3 and IL-10. Again, this defect was corrected with PRL or metoclopramide [112–114]. The administration of GH and, to a lesser extent, of PRL had stimulatory effects on the number and size of testicular macrophages in long-term hypophysectomized rats [115]. Similarly, in the corpora lutea from hypophysectomized rats, PRL induced the accumulation of macrophages [68]. 4. Conclusion from In Vivo Animal Data In rodents, acute PRL-deprivation in adult life by hypophysectomy or treatment with Bcr leads to immune deficiency, which can be reversed with PRL. Evidence is particularly strong for innate immunity. Hypoprolactinemia was correlated with impaired clearance of injected Listeria in several experimental systems. In mice, PRL is dispensable for the developing immune system and for most immune responses when absent from the time of conception. Recent evidence supports a protective role for PRL in hematopoietic or immunological failure. Indeed, effects of PRL were seen in immunocompromized animals (as a result of treatment with glucocorticoids or AZT or submitted to experimental hemorrhage or burn injury) [4,11,109,112–114]. Dorshkind and Horseman were among the first to propose that, whereas PRL has little effect on the immune system in intact rodents, it is able to compensate immunosuppressive mechanisms operating in various stress situations [4]. Indeed, experiments documenting immunopotentiating effects of PRL were often performed in rodents that had been operated (e.g., hypophysectomy, ovariectomy), bled, submitted to burn injury, or treated with glucocorticoids or AZT. The mechanisms by which PRL acts in these different systems are still under investigation. Antagonizing NF-␬B may play a role in inflammatory conditions [3,60]. Protection against dexamethasone-induced apoptosis could rely on the activation of Akt/PI3 by PRL and the subsequent inhibition of caspase 3 or caspase 9 [116,117]. Redundancy is a key feature of the immune system and in particular of the cytokine network. PRL signals mainly through Stat5 and only when both Stat5a and Stat5b genes are missing is an immunological phenotype observed [118–126]. When signaling through the PRL-R is absent, only a fraction of Stat5 activation in leukocytes is lost, and major deficiencies in the immune system are not expected (see Sec. VII. B).

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It remains possible that PRL plays a role that can be taken over by other factors, certainly when signaling through the PRL-R is absent from conception. There is ample experimental evidence that acute deprivation of PRL is responsible for immune defects. Accordingly, conditional PRL knockout mice (using, e.g., the cre-lox system) should have similar defects. Alternatively, double knockouts (where signaling through both the PRL-R and the receptor for the factor redundant with PRL would be simultaneously deleted) would show immune deficits greater than in single knockouts. Efforts are underway to disrupt both PRL-R and GH-R, which use closely related signaling pathways. Receptors for both GH and PRL are probably widely distributed in leukocytes, although only fragmentary data are available, in particular in humans. Compensation of PRL by GH would reconcile data from mouse knockouts and rat hypophysectomy but cannot account for data with Bcr-treated mice (although Bcr does more than preventing PRL secretion by the pituitary gland). PRL is probably redundant with one or more other cytokines signaling in part through Stat5 such as GM-CSF, IL-2, IL3, IL-5, IL-7, IL-9, or IL-15. In view of the numerous known targets of PRL and the distribution of receptors for these cytokines, we would suggest possible redundancy with IL-2, IL-3, or GM-CSF as a likely explanation for the lack of strong immunological or hematological effects in PRL and PRL-Rⳮ/ⳮ mice. C. Human Data 1. Hypoprolactinemia in Humans The sensitivity of the currently used assays for PRL in serum only extends to the lower limit of the normal range, and discrimination in the lower range would be interesting. In cases of so-called ‘‘aprolactinemia’’, how low is actually serum PRL? To what extent does extra-pituitary PRL production compensate the effects of hypophysectomy? Genetic Defects. There is no hematological or immune deficit in children lacking PRL, and in their adult life no immune deficiency was mentioned [127–129]. Combined deficiencies (PRL, GH, and TRH) have no known consequences for the immune system. It should be recalled that GH also binds the PRL-R. As is the case for PRL, GH is expressed in extrapituitary sites, a fact that has hardly been discussed in the context of immune responses. Hypophysectomy. As a rule, hypophysectomy does not lead to hematological or immunological impairment in humans, in contrast with the animal model. In hypopituitarism resulting from Sheehan’s syndrome, immune up-regulation at both cellular and humoral levels was seen [130]. Possibly, the immunosuppressive effects of the pituitaryadrenal axis are more important than immunostimulatory actions from other pituitary hormones in humans, the reverse being true in the rat. Dopaminergic Agents (Bcr, Cabergoline). These PRL-lowering drugs have not been held responsible for any change in immunological parameters in normal subjects. Beneficial effects have been occasionally reported in autoimmune diseases (see below and Refs. 131, 132). 2. Hyperprolactinemia in Humans Moderate hyperpolactinemia is induced by a large number of antipsychotic drugs, and such treatments have not been correlated with changes in the hematological or immune

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systems. Moderate or severe hyperprolactinemia is seen in pituitary tumors of the lactotroph cells (prolactinomas). Clinical symptoms are related to hyperprolactinaemia directly (galactorrhoea) or indirectly (oligomenorrhoea and amenorrhoea, erratic or absent ovulation, sexual dysfunction and reduced bone mineral density) [7]. Hyperprolactinemia, either moderate or severe, has not been correlated with changes in the hematological or immune systems. Changes in the immune system are well known during pregnancy and lactation, two circumstances where PRL, and other hormones, are increased. The inhibition of B-lymphopoiesis seen in mice during pregnancy and lactation has been ascribed mainly to high estrogen levels [190]. In women, minimal changes are seen. For instance, during the lactation period, there is a negative correlation between CD19Ⳮ percentages and serum PRL levels [133]. Also, IFN-␥ production by blood leukocytes is increased during lactation, and this increase correlates with PRL serum levels [134]. 3. Conclusions from In Vivo Data in Humans As a rule, hypo- or hyperprolactinemia is not associated with hemato- or immunological signs in humans, suggesting that PRL has very little impact on the immune system. It is possible that a baseline stimulation by PRL is effective and that higher levels or more frequent surges are useful in various stress situations. Nearly nothing is known about local PRL production in the immune system. The pulsatile nature of pituitary secretion of PRL and GH makes these issues very complex indeed. Also, the different bioactivity of PRL variants has led to speculation, but only few data are available. Finally, it should be remembered that higher concentrations of PRL lead to less efficient signaling. VI. ROLE OF PRL IN AUTOIMMUNE DISEASE A. Autoimmune Reactivity The incidence of several autoimmune diseases is higher in females, and this points to a role for sex hormones, in particular estrogen, and thus for PRL (as the expression and secretion of PRL are stimulated by estrogen). PRL has immunostimulatory activity in several systems. Could hyperprolactinemia result in overstimulation of the immune system and favor the development or the progression of autoimmune processes? Diamond and coworkers have provided strong indications that a sustained increase in serum estradiol favors the survival and activation of autoreactive cells and thus the rupture of tolerance. Several genes involved in B-cell activation and survival, including cd22, shp-1, bcl-2, and vcam-1, were upregulated in B cells from estrogen-treated mice. As the PRL-lowering drug Bcr prevents these events, a role for PRL was suggested but not further investigated. Anti-IgM-induced apoptosis was, however, reduced after a 5-hour treatment with estradiol of splenic B cells isolated from ovariectomized mice. It is very unlikely that these in vitro effects are mediated by PRL [135–137]. Whereas the contribution of PRL to the pathogenesis of autoimmune disease in humans remains largely speculative, the role of PRL has been clearly established in a few animal models of autoimmunity. B. Systemic Lupus Erythematosus Only recent data pertaining to SLE will be reviewed, as the subject has been covered in a recent issue of Lupus [7]. SLE is considered to be essentially TH-2–mediated.

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Whereas an aggravating role of PRL has been established in several murine models of SLE, the evidence is only circumstantial in human SLE: pregnancy and lactation are associated with SLE flares, and in some (but not in all) studies, patients had higher PRL levels than controls, in particular during flares [138–143]. Some studies suggested that PRL levels correlate with the severity of the disease. However, among adult and pediatric SLE patients with idiopathic hyperprolactinemia, 20–40% had PRL-Ig complexes. These complexes have low in vivo bioactivity (see Sec. II.A). Patients with idiopathic hyperprolactinemia and PRL-Ig complexes had no clinical signs of hyperprolactinemia and relatively low SLE disease activity [144,145]. Supporting a role for PRL in SLE is the fact that Bcr and cabergoline have improved symptoms, with relapse at the arrest of the treatment in a few cases [7,131]. Interestingly, genetic studies have identified a linkage between polymorphism in the extrapituitary PRL promoter and SLE in a group of UK patients. The ⳮ1149 extrapituitary promoter region polymorphism was demonstrated to affect PRL mRNA production. The G allele leads to increased levels of PRL mRNA compared to the T allele, probably as a result of differential binding of the transcription factor GATA-3. There was a preponderance of the G allele of the ⳮ1149 extrapituitary promoter region polymorphism in SLE patients from the United Kingdom but not from Spain, relative to the matched controls [146,147; A. Stevens, personal communication]. C. Scleroderma or Systemic Sclerosis Mild hyperprolactinemia was also reported in scleroderma. In a recent study, serum levels of PRL measured in the morning (8–10 a.m.) were significantly higher in patients with scleroderma (17.9 Ⳳ 7.7 ng/mL), compared with controls (9.3 Ⳳ 4.2 ng/mL). In patients, peaks of secretion were detected between 6 and 11 a.m., instead of 2–6 a.m., but there was also a sustained increase over 24 hours [148]. D. Diabetes Although the pathogenesis of diabetes is far from clear, TH1 cells are considered pathogenic and TH2 cells protective in type I diabetes. PRL is a growth factor for the endocrine pancreas [90]. The role, if any, of PRL in experimental diabetes has received little attention so far, and there are no data for humans. PRL has hyperglycemic activity on its own, and this complicates the interpretation of studies addressing its possible role in the pathogenesis of diabetes. Moreover, SOCS1 and -3 (induced by many cytokines, including PRL) inhibit signal transduction by insulin [149]. The incidence of diabetes was significantly lower in female mice receiving the PRL-lowering drug Bcr [150], but in a more recent study pregnancy hormones had a strong protective effect, and PRL given alone had a partially protective effect on the development of diabetes [151]. E. Rheumatoid Arthritis Rheumatoid arthritis (RA) is regarded as a T-cell–mediated and TH1 immune response–driven disease. Pregnancy induces a shift from TH1 to TH2 immune response, increasing the anti-inflammatory cytokines IL-4 and IL-10, which may contribute to gestational amelioration of RA.

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The serum levels of both PRL and the chemokine MIP-1␣ were increased in relation to the duration and the severity of RA in women [152]. In male patients with RA, serum PRL levels were significantly higher than in controls, and the increase was related to the duration and the activity of the disease [153]. Nagafuchi et al. [23] have characterized primary synovial cells from RA patients: CD14Ⳮ, 35%; CD4Ⳮ: 26%; CD8Ⳮ: 20%; fibroblast-like: 19%. By immunocytochemistry, the production of PRL could be localized to both T cells and, to a lesser extent, fibroblasts. In these cells, Bcr reduced the production of PRL and of inflammatory factors [IL-6, TNF-␣, and matrix metalloproteinases (MMP)] and PRL (100–1000 ng/ mL) restored IL-6 secretion to control levels. (To our knowledge, this is the only report showing an inhibition by Bcr of extrapituitary PRL production.) PRL-R was expressed in fibroblast-like cells and in lymphocytes. PRL (500 ng/mL) induced the rapid translocation of Stat5 from the cytoplasm into the nucleus and enhanced proliferation of the fibroblast-like cells. The production of MMP-3 was augmented and the production of tissue inhibitor of metalloproteinases (TIMP)-1 was inhibited by PRL, suggesting that PRL enhances total collagenase activity in the joints. F. Multiple Sclerosis The link between MS and PRL, if any, is very tenuous indeed. Serum PRL levels are normal, but TRH-induced secretion is higher in MS patients than in control subjects [131,132,154,155]. Hyperprolactinemia may, however, be one of the characteristic features of Asian MS patients with preferential involvement of the optic nerve. Moreover, hyperprolactinemia was significantly associated with acute relapse involving the optic nerves [156]. G. Autoimmune Thyroid Diseases Thyrotropin-releasing hormone is a PRL-releasing factor, and this accounts for the rise in PRL in patients with hypothyroidy [9,157]. For instance, patients with Hashimoto’s thyroiditis exhibited significantly higher PRL values (14.0 Ⳳ 3.8 ng/mL) than did control subjects (6.5 Ⳳ 1.3 ng/mL) [158]. Hypothyroidy is frequent in Hashimoto’s disease. In such cases, the hyperprolactinemia is thus not responsible for the thyroid disease. More recently, however, experimental studies suggested that PRL may contribute to the progression of autoimmune thyroiditis. Indeed, when exposed in vitro to PRL, thyrocytes from normal subjects expressed higher levels of ICAM-1, B7.1, and thyroid peroxidase [69]. Also, ovine PRL could antagonize the stimulation by IFN-␥ and the inhibition by IL-4 of CD40 expression on thyrocytes from both normal and Graves’ disease patients. PRL had no effect on the expression of HLA-DR in this system. The authors speculate that PRL could play a role also in the development of Graves’ disease [159]. H. Conclusion Data in experimental and human autoimmunity fail to convincingly establish a role for PRL. Yet hyperprolactinemia has been associated with several autoimmune diseases, but there is very limited information about causal relationships. The hyperprolactinemia could result from cerebral or pituitary involvement by the disease process in some but certainly not in all cases. The incidence of several autoimmune diseases is higher

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in women than in men, and both pregnancy and lactation affect the course of SLE (flares of pregnancy and lactation), RA (ameliorating effect of pregnancy on disease activity, relapse and flares postpartum; breast-feeding increases the risk of RA), Graves’ disease (remission during pregnancy), and MS (the rate of relapse declines during pregnancy, especially in the third trimester, and increases during the first 3 months postpartum before returning to the prepregnancy rate) [160–162]. The exact role of PRL, in combination of other hormones, obviously different in individual autoimmune diseases, has yet to be defined [163].

VII. PRL AND LEUKEMIA/LYMPHOMA A. Effect of PRL on Hemopoietic Cell Lines There is no known human equivalent of the PRL-dependent rat Nb2 T-cell lymphoma or the 2779 rat lymphoma where the PRL-R gene acts as an oncogene [164]. Hyperprolactinemia is uncommon in leukemic patients but has been reported in individual cases and in small cohorts of acute myeloid leukemia and in myeloma patients with advanced disease [165–167]. Production of PRL by the leukemic or lymphoma cells themselves was documented in a limited number of cases (Table 2) [9,10,13,19,166–168]. Many leukemic cells express PRL-R (see Table 2). They include myeloid, T, NK, and B cells and myeloma lines. Theoretically, PRL could act as an autocrine growth factor for leukemic cells, but this has been shown in one Jurkat subline only [27]. Actually, the significance of PRL-R expression in human leukemic cells has received little attention so far. Gado et at. have studied extensively the effects of PRL in U266 myeloma cells [169]. Pharmacological doses of PRL inhibited etoposide-induced apoptosis, whereas lower, near-physiological doses were pro-apoptotic. In addition, PRL was able to stimulate proliferation of murine B-cell hybridomas in a dosedependent manner and enhanced their proliferation in response to IL-4, IL-5, and IL6. This increase in proliferation resulted in an overall increase in antibody production. Also, in hybridoma cell lines, the addition of rhPRL to the cultures reversed the antiproliferative effects of transforming growth factor (TGF)-␤ [170]. B. Effect of PRL on Antitumor Immunity In the context of tumor immunology, PRL could also act on the immune response to cancer cells. Since many tumor cells and tumor stromal cells also express PRL receptors, effects on the tumor, in particular at the level of antigen presentation, should also be considered. With the exception of studies on NK and LAK activity, few papers deal with the role of PRL in antitumor responses [171]. There is very limited evidence that PRL can play a significant role in antitumor immunity by improving immune defenses. Effects on the tumor cell have been considered on a few occasions: PRL-treated target cells are more susceptible to NK lysis. The proposed explanation was cell cycle recruitment by PRL and increased expression of cycle-related cell surface target molecules [172]. Obviously, therapy with PRL should not be considered in hormone-responsive tumors (such as breast and prostate carcinoma) where PRL is a growth factor [173].

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C. Conclusions Only in exceptional cases of experimental or human hematological or lymphoid malignancy did PRL show significant growth factor activity. In vitro, PRL is a growth factor for a very limited number of leukemic clones. Whenever PRL appears to be a growth factor for tumor cells, therapeutic intervention with dopaminergic agents seems indicated. In view of immunostimulatory properties of PRL, its use as adjuvant in immunotherapy has been considered. Experimental studies indicate that PRL can modify NK cell activity and cell surface antigen expression by tumor cells. The use of PRL has also been considered in hemopoietic reconstitution after bone marrow failure following, e.g., chemotherapy or radiotherapy [11,74,109,174]. The ubiquitous distribution of PRL-R should lead to great caution, as PRL could act on both the tumor target cells and the immune cells, and this has indeed been documented. Whereas PRL is mainly seen as a hemato-immunostimulatory factor, it should be remembered that PRL is a growth factor for many breast, prostate, and possibly also other tumors [1,174]. VIII. CONCLUSIONS AND PROSPECTS A. PRL as a Product of the Neuroendocrine System PRL is produced by various cell types, including some myeloid and lymphoid cells. The bulk of PRL being produced by the pituitary gland, PRL remains a major vehicle in the neuroendocrine communication system. In response to various endogenous or exogenous stimuli, PRL relays messages from the brain to a large number of target tissues, including the immune system. PRL secretion is related to the development and function of the mammary gland and reproductive tissues. Estrogen is a major inducer of PRL. In addition, several hormones and various forms of stress induce the secretion of PRL. The immune system is thus exposed to pituitary (endocrine) PRL and extra-pituitary PRL produced by cells from tissues hosting leukocytes (e.g., decidua, paracrine) or cells from the immune system themselves (autocrine or paracrine). The relative contribution of endocrine and paracrine/autocrine to physiological and pathological responses is not known. Whereas the importance of paracrine PRL is being increasingly recognized, only the modulation of pituitary PRL secretion is presently well understood. Xenobiotics, with special concern for psychotropic drugs, on the one hand, and chemical pollutants—in particular those with estrogenic activity—on the other hand, have received particular attention as modulators of PRL secretion [176–178]. B. PRL Activation of Stat5 Although PRL is dispensable for the development and the function of the murine immune system, most cell types in the immune system are targets for PRL. Adult hypophysectomy results in immune deficiency in the rat but not in humans, which suggests either differences in the functions of PRL in the immune system or different relative contributions of the immunosuppressive glucocorticoids and the mainly immunostimulatory PRL. Whatever the origin of lactogenic substances (endocrine, paracrine, autocrine), their effects in the immune system have been demonstrated in vivo and in vitro. In cells expressing receptors for PRL and for cytokines, the effects of PRL and other activators of Stat5 could thus be additive: convergent signaling has been experimentally demonstrated [179].

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Stat transcription factors mediate to a large extent signaling through hemopoietin receptors, and Statⳮ/ⳮ mice illustrate the importance of Stat factors in immunology. Stat4 and Stat6 are the most important Stat factors determining the TH1 versus TH2 fate of CD4Ⳮ lymphocytes, but central to the control of IFN-␥ gene expression, the hallmark of TH 1 cells, is the transcription factor T-bet, whose induction is Stat1dependent. IL-12 is dominant in directing TH1 development through Stat4 activation, while synergizing with IL-18 for IFN-␥ production from differentiated TH1 cells [180–182]. Stat6-deficient animals do not generate TH2 cells, producing, e.g., IL-4, IL-5, and expressing the transcription factors GATA3 and NFATc2 [182]. At the basis of B-cell differentiation lies the expression of IL-7R, which requires the PU.1 transcription factor [183]. IL-7 signals mainly through Stat5. As far as monocytes and macrophages are concerned, the differentiation of resident macrophages by IFN-␥ occurs through activation of Stat1. In contrast, IL-10 inactivates the activated macrophages through activation of SOCS-3 (see below) [184]. Assuming that PRL acts mainly through Stat5 activation, PRL is expected to act in leukocytes in the same way, and in collaboration with other Stat5-activating cytokines. The absence of PRL would have, at most, the same consequences as the absence of Stat5 factors. In lymphoid cells, targeted deletion of Stat5a Ⳮ Stat5b abrogates the development of NK cells. T cells do develop, but they fail to proliferate in response to IL-2 and, like cells with targeted deletions of the IL-2 and IL-2-R genes, they develop lymphoproliferative disease. Observations in Stat5a-deficient mice indicate that Stat5a favors T-helper cell differentiation towards the TH2, rather than the TH1 type [119]. This limited phenotype is in contrast to the marked effects seen in Stat4 and Stat6ⳮ/ⳮ mice. Yet Stat5 is required for normal immune responses. A decrease in Stat5 has been associated with immune deficiency: HIV infection leads to a decrease in the Stat5b protein in T cells. Decrease in Stat5 was also seen in purified T cells from HIV patients and in purified B and T cells from tumor-bearing mice [185,186]. Ablating PRL or PRL-R should have fewer consequences on the immune system than ablating Stat5 genes, as all other Stat5-activating systems would be conserved. This prediction was verified: (1) absence of signaling through the PRL-R has only very limited consequences on the immune system; (2) most reported effects of PRL in the immune system can be explained by Stat5 activation and are often observed in combination with Stat5-activating cytokines (IL-2, GM-CSF); (3) NK cells are major targets for PRL. The Stat5-additive model, though most likely to account for the effects of PRL in the immune system, still awaits confirmation. As Stat5a favors TH2 differentiation, one can ask whether PRL also favors a shift towards TH2 responses. As long as the distribution of PRL-R in leukocyte subpopulations is not known, only speculations can be made. Stronger TH2 activity may favor allergic responses and be detrimental to some patients with SLE and allergy and beneficial to patients with various autoimmune diseases (other than SLE). As PRL could play an aggravating role in SLE, the use of PRL-lowering drugs in SLE has been advocated. What role can be ascribed to PRL produced under the control of the ‘‘extrapituitary’’ promoter should first be established. Whereas Stat5 is widely expressed in leukocytes and can be activated by several cytokines, little is known about the cellular distribution of nuclear receptors (with the exception of the glucocorticoid receptor) now known to interact with Stat factors, though

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all the nuclear receptors discussed in Sec. III.C have been identified in leukocytes [136,187–192]. C. Negative Signaling PRL, like other cytokines, induces inhibitors of cytokine signaling that prevent the activation of any Stat factor, not only Stat5. For instance, after its induction by interferons, SOCS1 inhibited the activation of Stat6. PRL could in principle have the same effect [193,194]. The induction of SOCS factors by PRL is very well documented, as is the inhibition of PRL signal transduction when SOCS factors are overexpressed [46,47,49,195,196]. A recent paper suggests that SOCS factors play a major role in the TH1-TH2 dichotomy and would be instrumental in the mutually exclusive use of Stat4 and Stat6. Thus, SOCS-3 may mediate repression of IL-12/Stat4 signaling in TH2 cells, and SOCS-1 may account for silencing of the IL-4/Sta6 signaling in TH1 cells. SOCS-1 expression is five fold higher in TH1 than in TH2 cells, and TH2 cells contain 23 times more SOCS-3 than TH1 cells [197]. Accordingly, abnormal expression of SOCS factors could also be critical for the development and the progression of autoimmune diseases [198]. So far, the inhibition by PRL-induced SOCS factors of heterologous signal transduction (signaling by other cytokines or growth factors) has not been reported, though it is very likely to occur. The situation can be quite complex, however, in view of the pulsatile production of many factors, the short half-life of many components of signaling cascades, and the existence of feedback mechanisms [199]. D. PRL Activation of Other Stats and the MAP-Kinase pathway PRL can activate Stat1 and Stat3. The activation of Stat1 in leukocytes has been documented, and possible implications have been discussed as for Stat5 [47]. ERK and p38 activation are critical events in determining the fate of T lymphocytes. In early mouse thymocyte development there is a shift from high p38 activation to high ERK activation. Sustained ERK activation leads to the CD4Ⳮ cells, whereas other signals lead to the CD8Ⳮ phenotype. At that stage, JNK and p38 may lead to negative selection and death. In naı¨ve CD4Ⳮ cells, Stat4 together with sustained ERK activation leads to the TH2 pathway, whereas Stat6 together with p38 is critically involved in TH1 differentiation [200–202]. E. PRL in the Immune System: Many Targets, Few Effects? Lactogenic hormones (PRL, GH, placental lactogens) bind to the PRL-R on leukocytes and activate mainly Stat5. How important are the lactogenic signals in immunobiology? Tools are now available to study their contribution to the immune system in animals and humans. DNA microarray technology will indicate which genes are activated by PRL and in which cell populations. We favor the idea that PRL is involved in fine-tuning of the lympho-hemopoietic system, for instance, at the level of the TH1-TH2 balance. It could also contribute to sex-related differences in the immune system and account in part for higher antibody responses and increased prevalence of autoimmune diseases in females. More important functions—but purely speculative so far—would relate to the immunology of pregnancy [204]. In a minimalist view, PRL protects the immune system against various insults, such as, immunosuppressive drugs (glucocorticoids, cyclosporine, AZT), surgery

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(aiming at hormone depletion, such as hypophysectomy, ovariectomy), hemorrhage, or burn injury [4,11]. Taken together, the evidence for a direct immunomodulating role of PRL is quite tenuous indeed. Yet, indications that PRL can favor hematopoietic reconstitution should be explored in depth as this hormone, which has a very low toxicity, could be of value for the expansion of certain cell populations. ACKNOWLEDGMENTS We thank A. Buckley, S. Gerlo, and A. Stevens for permission to quote unpublished results and J. R. Davis, S. Gerlo, H. Heinen, N. Horseman, R. Kooijman, O. Thellin, and B. Velkeniers for discussion. REFERENCES 1. Goffin V, Binart N, Touraine P, Kelly PA. Prolactin: the new biology of an old hormone. Annu Rev Physiol 2002; 64:47–67. 2. Horseman ND, Ed. Prolactin. Boston: Kluwer Academic Publishers, 2001. 3. Yu-Lee L-y. Prolactin modulation of immune and inflammatory responses. Recent Prog Horm Res 2002; 57:435–455. 4. Dorshkind K, Horseman ND. The roles of prolactin, growth hormone, insulin-like growth factor-I, and thyroid hormones in lymphocyte development and function: insights from genetic models of hormone and hormone receptor deficiency. Endocr Rev 2000; 21:292–312. 5. Kooijman R, Hooghe-Peters EL, Hooghe R. Prolactin, growth hormone, and insulin-like growth factor-I in the immune system. Adv Immunol 1996; 63:377–454. 6. Matera L, Rapaport R, Eds. Growth and lactogenic hormones. NeuroImmune Biol. Vol. 2, 2002:1–306. 7. Walker SE, Yu-Lee L-y. Prolactin and systemic lupus erythematosus: mechanisms and clinical studies. Lupus 2001; 10:659–768. 8. Matera L, Davis JR. Hormones and cytokines: signaling molecules in immune system-neuroendocrine cross-talk. J Neuroimmunol 2000; 109:1–62. 9. Hooghe-Peters EL, Hooghe R. Hormones, blood cells and immunity. Cell Mol Life Sci 1998; 54:1057–1108. 10. Hooghe-Peters EL, Hooghe R. Growth Hormone, Prolactin and IGF-I as Lymphohematopoietic Cytokines. Austin: Springer-Verlag, 1995. 11. Welniak LA, Richards SM, Murphy WJ. Effects of prolactin on hematopoiesis. Lupus 2001; 10:700–705. 12. Bhattacharyya S, Lin J, Linzer DI. Reactivation of a hematopoietic endocrine program of pregnancy contributes to recovery from thrombocytopenia. Mol Endocrinol 2002; 16: 1386–1393. 13. Walker AM, Montgomery DW, Saraiya S, Ho TW, Garewal HS, Wilson J, Lorand L. Prolactin-immunoglobulin G complexes from human serum act as costimulatory ligands causing proliferation of malignant B lymphocytes. Proc Natl Acad Sci USA 19951; 92:3278–3282. 14. Bonhoff A, Vuille JC, Gomez F, Gellersen B. Identification of macroprolactin in a patient with asymptomatic hyperprolactinemia as a stable PRL-IgG complex. Exp Clin Endocrinol Diabetes 1995; 103:252–255. 15. Heffner LJ, Gramates LS, Yuan RW. A glycosylated prolactin species is covalently bound to immunoglobulin in human amniotic fluid. Biochem Biophys Res Commun 1989; 165: 299–305. 16. Clapp C, Weiner RI. A specific, high affinity, saturable binding site for the 16-kilodalton fragment of prolactin on capillary endothelial cells. Endocrinology 1992; 130:1380–1386.

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12 VIP and PACAP Immune Mediators Involved in Homeostasis and Disease ROSA P. GOMARIZ, CARMEN MARTINEZ, CATALINA ABAD, MARIA GUILLERMA JUARRANZ, and JAVIER LECETA Complutense University, Madrid, Spain MARIO DELGADO Instituto de Parasitologia y Biomedicina, Granada, Spain

I. INTRODUCTION The health of vertebrates depends on numerous regulatory interactions between the basic framework constituted by the three systems involved in homeostasis: the nervous, the endocrine, and the immune systems. An important factor in the behavior of the circuit is the presence of common mediators with three different origins: innervation, endocrine release from the hypothalamus-pituitary-adrenal (HPA) axis, and the in situ cellular origin. The fact that the nervous and endocrine systems produce similar mediators was soon established, and it was later when the immune system was involved in this circuit in two ways: glia cells [1] and neurons [2] are able to synthesize traditional immune factors as interleukin (IL)-1, and immune cells produce neuroendocrine mediators. Since 1980, when Blalock and Smith [3] demonstrated that immune cells could produce adrenocorticotropic hormone (ACTH) and endorphins, and in spite of years of reticence about this endogenous production of neuroendocrine mediators by cells of the immune system, it has been well established at that the nervous, endocrine, and immune systems speak a common biochemical language, sharing ligands and receptors. Although the important functional role played by these common mediators in the immune system is well accepted by the scientific community, it should not be ignored that their source is not only the innervation of the lymphoid organs but also the immune cellular origin. At least 27 traditional neuroendocrine mediators are also produced by cells of the immune system, including lymphoid stroma cells, antigen-presenting cells, granulocytes, and lymphocytes. The most recent mediators are glucocorticoids synthesized by thymic epithelial cells [4], procalcitonin produced by 241

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various types of human leukocytes [5], calcitonin gene–related peptide (CGRP) secreted by T lymphocytes [6,7], and the hypothalamic decapeptide gonadotropin-releasing hormone (GnRH), which has been reported to be produced by human peripheral lymphocytes [8]. The study of these common neuroendocine-immune mediators has recently experienced explosive growth, not only in basic research, but also expanding to the point that prospective clinical research could soon be a reality. In this chapter we will consider certain basic aspects of vasoactive intestinal peptide (VIP) and pituitary adenylate cyclase–activating polypeptide (PACAP), which have opened the way to suggest potential therapeutic roles for these peptides, we will cite some of the strongest evidences that actually support such role in inflammatory and autoimmune pathogenesis, and we will finally raise some conclusions and future research possibilities.

II. VIP AND PACAP IN THE NERVOUS, ENDOCRINE, AND IMMUNE SYSTEMS A. Ligands VIP and PACAP belong to the glucagon/growth hormone–releasing factor/secretin superfamily of peptides found in nervous, endocrine, and immune systems of complex living organisms showing a highly conserved sequence, a similar gene structural organization and a great degree of homology between the peptides and their precursors. These facts support that both PACAP and VIP genes originated from a common ancestral sequence after gene duplication [9]. In 1969 Said and Mutt [10] reported for the first time the presence in lung of a 28amino-acid peptide, which was later isolated from the small intestine [11] as a gastrointestinal hormone. It was later described in the central and peripheral nervous system, acting as a neurotransmitter and neuromodulator, and it has since been ‘‘rediscovered’’ as an important immunomodulator. The first data about of the role of VIP in immunity appeared in 1990 with the demonstration of VIP immunoreactivity in the cytoplasm of lymphocytes [12]. This finding was later corroborated by biochemical characterization at both protein and mRNA levels of VIP by reverse-phase high-performance liquid chromatography (HPLC), radioimmunoassay (RIA), in situ hybridization, and reverse transcriptase polymerase chain reaction (RT-PCR) [13–15]. Using these technique approaches, we demonstrated VIP production in T and B lymphocytes and in double- and single-positive thymocytes. We also developed a specific enzyme-linked immunoabsorbent assay (ELISA) demonstrating that agents that mediate important immune functions, such as proliferation and antigenic stimulation [concanavalin A, lipopolysaccharide (LPS), and anti-T-cell receptor (TCR) antibody], inflammation (LPS, TNF-␣, IL-6, and IL-1␤), or apoptosis (dexamethasone) induced the production and release of VIP to the lymphoid microenvironment [16]. Moreover, it has been recently described that VIP is preferentially produced by type 2 T cells following antigenic stimulation [17]. Twenty years later than VIP, PACAP was discovered [18], being the most recent member of the VIP/glucagon/growth hormone–releasing factor/secretin superfamily. First isolated from ovine hypothalamic extracts based on its ability to activate cAMP formation in anterior pituitary cells, it exists in two amidated forms, PACAP27 and PACAP38, with 27 and 38 amino acid residues, respectively. At present, PACAP is a peptide with a wide distribution throughout the organism, being located in nerve cell bodies and nerve fibers of the central and peripheral nervous system, being especially abundant in the central

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nervous system, adrenal medulla, and testis [19], performing diverse biological activities (see Refs. 20 and 21). Similar to VIP, PACAP was also ‘‘rediscovered’’ in the immune system, demonstrating the first report that this neuropeptide inhibited mitogen-stimulated proliferation of splenocytes [22]. Recently, we also demonstrated PACAP production in the immune system [23] in lymphocytes from lymphoid organs and peritoneum using immunohistochemistry, immunocytochemical staining, Western blot, and RT-PCR and cDNA sequencing methods. Our study revealed PACAP immunoreactivity in thymocytes and in lymphocytes and plasma cells from spleen and lymph nodes. Western blot analysis showed a band corresponding to PACAP for all lymphoid organs studied. mRNA appeared in both double- (CD4ⳭCD8Ⳮ) and single-positive (CD4ⳭCD8ⳮ, CD4ⳮCD8Ⳮ) thymocytes, T subsets, and B cells, and in spleen and lymph nodes. Curiously, our studies demonstrated that PACAP storage and gene expression appear in exactly the same cell populations as VIP. In addition, VIP and PACAP mRNA were expressed in lymphocytes, but not in macrophages from peritoneal suspensions. As we discuss below, in the immune system there are few functional differences between VIP and PACAP, since most of the actions exerted by VIP are also shared by PACAP. Moreover, the two peptides act in the immune system through the binding to a common family of cell surface receptors. Based on these observations, VIP and PACAP could be considered as lymphocyte-derived cytokines in the central and peripheral lymphoid acting on lymphocytes and microenvironmental cells (Fig. 1).

Figure 1 VIP and PACAP production in lymphocytes: (A) VIP immunoreactivity in thymic cortex; (B) in situ hybridization showing VIP expression in thymocytes; (C) cytocentrifuge preparation showing immunoreactive PACAP in spleen lymphocytes; (D) Western blot analysis of PACAP in lymphocyte membranes from thymus (2), spleen (3), and lymph nodes (4). Brain membranes were used as positive control (1).

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B. Receptors VIP and PACAP regulate numerous biological activities through binding to specific plasma membrane receptors, which together with the receptors for VIP-related peptides clearly constitute an original subfamily within the superfamily of G-protein–coupled receptors [24]. This subfamily, referred to as class II, also comprises receptors for parathyroid hormone, calcitonin, corticotropin-releasing factor, and the so-called EGF-TM7 receptors [25]. Class II subfamily receptors display several common properties, including large Nterminal extracellular domains containing highly conserved cystein residues, N-terminal leader sequences, and complex gene organization with many introns [26]. The N-terminal domain plays an important role in the binding of the ligand [26–28], although both extracellular and transmembrane domains are also involved [29–31]. To date, three receptors have been described in vertebrates with a wide distribution in different cell types of the organism including cells of nervous, endocrine, and immune systems: VPAC1, VPAC2 receptors that bind VIP and PACAP with equal affinity, and the PAC1 receptor that is PACAP selective [21]. Our group has widely studied the expression and distribution of the three receptors in cells of central and peripheral lymphoid organs. 1. VPAC1 Receptor This was the first VIP receptor described and cloned [32]. It binds VIP and PACAP with equal affinity, and to date no splice variants of this receptor have been described. In spite of the differences between species in the pharmacology of VPAC1 receptors that have been described [24], two conserved hydrophobic residues, Y239 and L240, are implicated in the coupling to G-protein [33]. This receptor is coupled mainly to the adenylate cyclase (AC) pathway. In the immune system, the VPAC1 receptor was identified for the first time in 1981 using binding techniques in human peripheral blood lymphocytes [34]. Later, it was reported in human monocytes [35], murine lymphocytes [36,37], and rat alveolar macrophages [38,39]. Furthermore, gene expression of VPAC1 receptor has been demonstrated in T and B murine lymphocyte subpopulations from spleen and lymph nodes, in double- and single-positive CD4CD8 murine thymocyte subsets and peritoneal macrophages [40–42]. As will be discussed below, it is the main receptor involved in the therapeutic role exerted by VIP and PACAP in the immune system. 2. VPAC2 Receptor The presence of this receptor was reported for the first time in the human lymphoma cell line SUPT1 [43] as a VIP/helodermin-preferring receptor and first cloned from the rat olfactory bulb [44]. No splice variants of this receptor have been described to date. Like the VPAC1 receptor, the VPAC2 receptor binds VIP and PACAP with comparable affinity, but this receptor does not bind secretin with high affinity as the VPAC1 receptor does. The AC pathway mediates the VIP/PACAP effects exerted through interaction with VPAC2 receptor. In the immune system, the expression of this receptor has been reported in lymphocytes and macrophages, but its expression is inducible, being detected in lymphocytes only after stimulation through the T-cell receptor (TCR)–associated CD3 molecule and in macrophages after LPS stimulation [45,46]. Furthermore, VPAC2 receptor is detected in mononuclear cells by immunohistochemical techniques 2 days after the detection of VPAC1 receptor at sites of inflammation and antigen recognition [47]. Nevertheless, VPAC2 is the only receptor of the VIP/PACAP receptor family expressed in some murine

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T-cell lines [48], and its constitutive expression has been reported in human lymphoid cell lines [43,49]. The AC pathway mediates the best characterized VIP/PACAP effects on immune cells through interaction with VPAC2 receptor. 3. PAC1 Receptor PAC1 is a PACAP selective receptor, but in micromolar concentration VIP is a heterologous ligand [24]. To date, it has eight variants produced by alternative splicing of the transcript, codifying to N-terminal (involved in ligand binding) [50,51], IC3 [implicated in both adenylate cyclase (AC) and inositol triphosphate/phospholipase C (IP/PLC) pathways] [52,53], and TM-2 and TM-4 (involved in the activation of an L-type calcium channel) [54]. Recently, a novel splice variant in the C-terminal domain of frog PAC1 receptor has been described [55]. With the wide range of splice variants described, several possibilities for ligand binding or coupling to second messengers remain to be established. In immune cells, this receptor is expressed only in macrophages, as lymphocytes lack PAC1 receptor expression. The PAC1 receptor in macrophages binds VIP and PACAP with the same affinity and activates the IP/PLC pathway [56] Although the splice variant of PAC1 present in macrophages is still unknown, it seems clear that this receptor plays a pivotal role in the effect of VIP and PACAP regulation of the several crucial agents involved in inflammation as IL-6 [57]. Thus, the redundancy between VIP and PACAP in terms of synthesis and effects in the immune system is also observed at the receptor level. This could explain in part the important role attributed to both neuropeptides in the control of immune homeostasis and the inflammatory response, mainly under pathological conditions. Therefore, the time course and the factors that control the synthesis of both neuropeptides should be crucial to understanding their physiology in the immune system. III. THE VIP/PACAP SYSTEM IN HOST DEFENSE MECHANISMS Host defense mechanisms indicate that several cell types are responsible for functions that result in the elimination of pathogens as well as transformed or altered self cells. Inflammation is a vital process that involves both non–antigen-specific and antigen-specific mechanisms. The two main cell types involved in these mechanisms are macrophages and lymphocytes. Macrophages play a crucial role in the fight against pathogens by contributing to the integration of both non–antigen-specific and antigen-specific defense mechanisms. Phagocytosis of pathogens is the main characteristic of macrophages, leading to their activation in terms of cytokine production and antigen presentation and to the reduction of the pathogen load. Macrophages initiate the inflammatory response through the secretion of inflammatory cytokines and production of reactive oxygen and nitrogen intermediates. Antigen-specific mechanisms are based on clonal activation of T and B lymphocytes, which is triggered in cooperation with both antigen-specific and non–antigen-specific cell populations. Signals from accessory populations include antigen presentation and mediators such as cytokines that lead to activation, proliferation, and differentiation of lymphocytes. A large body of recent literature demonstrates that VIP and PACAP are two potent anti-inflammatory factors, which regulate the production of both anti- and pro-inflammatory mediators. In addition, VIP and PACAP regulate the expression of costimulatory molecules, an action that may be related to the modulation of Th1/Th2 differentiation.

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A. Counterbalancing the Generation of Proinflammatory Factors One of the ways in which VIP and PACAP modulate the immune response is by influencing many macrophage functions. Both neuropeptides have been shown to stimulate migration, adherence, phagocytosis, and superoxide production, as well as the synthesis of IL-6 in resting macrophages (see Refs. 58–60). Microbial products such as the endotoxin LPS induce macrophages to secrete several pro-inflammatory products, such as cytotoxic oxygen and nitrogen intermediates and TNF␣, and the release of chemokines and cytokines that attract and activate other immune cells. Although necessary in innate immunity for the elimination of pathogens, macrophage activation leads to serious deleterious effects in the host if left unchecked. Therefore, in the course of a normal immune response, several inhibitory mechanisms mediated by endogenous macrophage deactivating factors come into play. In the last few years, numerous reports have identified VIP and PACAP as endogenous anti-inflammatory factors (see Refs. 58–62). Briefly, VIP and PACAP inhibit the expression and release of pro-inflammatory cytokines and chemokines (IL-12 TNF-␣, MIP-1␣, MIP-1␤, MCP-1, MIP-2, KC, and RANTES), inhibit the expression of inducible nitric oxide synthase (iNOS) and subsequent release of NO, and enhance the production of the anti-inflammatory cytokine IL-10. These effects were reported both in vitro and in vivo, are mediated by the presence of PAC1, VPAC1, and VPAC2 receptors on activated macrophages, and, as we will discuss below, are at least partially responsible for the protective effects of VIP/PACAP in models of inflammatory/autoimmune disorders. Several of the mechanisms involved in the effects of VIP/PACAP on macrophages have been elucidated (see Refs. 59–62). NF-␬B is a major transcriptional factor involved in the expression of several macrophage genes (TNF-␣, IL-12 p40, iNOS, MIP-1␣, MIP1␤, RANTES). VIP/PACAP decrease NF-␬B binding through a complex mechanism [63]. A cAMP-independent pathway initiated through the VPAC1 receptor results in the inhibition of I␬B phosphorylation and its subsequent stabilization. Due to the prolonged presence of the inhibitor I␬B, p65 (an essential component of the NF-␬B transactivating complex) is retained in the cytoplasm. For maximal activation, NF-cob has to bind to co-activators such as CBP (the CREB-binding protein) and phosphorylated TBP (the TATA-box binding protein). VIP/PACAP affect both CBP and TBP in a cAMP-dependent manner. First, VIP/ PACAP increase CREB phosphorylation and, subsequently, CBP is sequestered by nuclear phosphorylated CREB, becoming unavailable for binding to p65. Second, VIP/PACAP inhibit the MEKK1/MEK3–6/p38 pathway, and subsequently TBP phosphorylation, leading to a decrease in TBP binding to both DNA and p65. In addition, VIP/PACAP induce changes in the CRE-binding complex from highclow Jun/ CREB to lowc-Jun/highCREB for the TNF-␣ promoter [64], inhibit IRF-1 binding to the iNOS and IL-12p40 promoters [65–67], and induce changes in the composition of the AP-1 complexes from c-Jun/c-Fos to JunB/c-Fos with subsequent reduction in AP-1 binding to the TNF-␣ promoter [68]. Reduction in IRF-1 binding and synthesis is mediated through the inhibition of IFN-␥–induced Jak1/2-STAT1 phosphorylation [69], and changes in the composition of the AP-1 complexes are mediated through the inhibition of the MEKK1/MEK4/JNK pathway leading to a reduction in c-Jun phosphorylation and a direct, positive effect on JunB synthesis [68] (Fig. 2). Of particular interest is the dual effect of VIP and PACAP on IL-6 production by macrophages, depending on the nature and dose of the inflammatory stimuli [70,71].

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Figure 2 Mechanisms of action of VIP and PACAP on activated macrophages. For legends see text.

Whereas VIP/PACAP inhibit the release of IL-6 from macrophages stimulated with a medium-high dose of LPS, they enhance IL-6 secretion in unstimulated macrophages or macrophages stimulated with very low concentrations of endotoxin. Both stimulation and inhibition of IL-6 by VIP/PACAP are mediated by different receptors and intracellular signals. Whereas the inhibition of LPS-induced IL-6 production is mediated through PAC1 binding and PKC activation, stimulation of IL-6 release is VPAC1/cAMP-dependent [70,71]. A recent report has suggested that the inhibitory effect of VIP/PACAP on IL-6 production could be mediated through the inhibition of LPS binding to its receptor CD14 in macrophages by inducing the shedding of membrane-bound CD14 [72].

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B. Favoring Th2 Versus Th1 Pathways Macrophages also contribute to antigen-specific responses acting as antigen-presenting cells, providing T lymphocytes with costimulatory molecules and a cytokine environment that influences the proliferation and differentiation of the T cells. B7.1 and B7.2 are costimulatory molecules in macrophages that are expressed only after activation, B7.2 being induced earlier and at higher levels than B7.1 [73]. VIP and PACAP are among the endogenous factors that regulate B7 expression in macrophages. Of interest is that both neuropeptides affect B7 expression in resting and activated macrophages in an opposite way. In resting macrophages, VIP and PACAP upregulate B7.2, but not B7.1, at both mRNA and protein level. However, in LPS/IFN-␥–activated macrophages, both peptides inhibit both B7.1 and B7.2 expression. The actions of VIP and PACAP on B7 expression correlate with effects on the stimulatory function for T cells. The inhibition of B7.1/B7.2 expression in activated macrophages is in agreement with the described role of both peptides as endogenous anti-inflammatory factors. Because VIP and PACAP affect B7 but not MHC class II expression [74,75], these neuropeptides could contribute to peripheral tolerance by inducing T-cell anergy. Nevertheless, the upregulation of B7.2 in unstimulated macrophages could represent one of the mechanisms by which VIP and PACAP promote Th2 differentiation. When an immune response is initiated, naive helper T cells (Th) secrete IL-2 and proliferate, and the progeny differentiates into two functionally subsets producing distinct effectors cytokines: Th1 cells secreting IFN-␥ and Th2 cells secreting IL-4, IL-5, and IL10. VIP and PACAP administration induce Th2 responses both in vivo and in vitro [75]. Thus, macrophages treated in vitro with VIP and PACAP induce the production of Th2type cytokines (IL-4 and IL-5) and inhibit Th1-type cytokines (IFN-␥, IL-2) in Ag-primed CD4 T cells. In vivo administration of both peptides in Ag-immunized mice results in a decreased number of IFN-␥–secreting cells and an increased number of IL-4–secreting cells [75]. There are several possible nonexcluding mechanisms for VIP and PACAP induction towards Th2. Inhibition of macrophage IL-12 production by both peptides is one possibility. Since IL-4 dominates IL-12, driving naı¨ve CD4 T cells toward the Th2 phenotype [76], a reduction in IL-12 by VIP and PACAP, even in the absence of an effect on IL-4, will result in Th2 differentiation. A second possibility is upregulation of B7.2 expression on macrophages by VIP and PACAP. Finally, VIP and PACAP may support Th2 but not Th1 proliferation in vitro, promoting the generation of memory Th2 cells in vivo. In view of these data, it can be concluded that VIP and PACAP modulation of several crucial steps of natural and acquired immunity could lead to the amelioration or prevention of several acute and chronic inflammatory and autoimmune disorders.

IV. VIP AND PACAP AS CURATIVE AGENTS IN INFLAMMATORY AND AUTOIMMUNE DISEASES To date, different animal models have been used to study the potential therapeutic effect of VIP and PACAP in human diseases such as septic shock and others disorders such as rheumatoid arthritis and Crohn’s disease, which share characteristics in terms of participation of pro-inflammatory cytokines, of oligoclonal expansion, and of activation of CD4Ⳮ T cells and Th1 production of cytokines.

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A. Septic Shock Although the inflammatory process is a localized protective response, the sustained production of inflammatory mediators can lead to serious pathological conditions such as endotoxic shock. Septic shock, a common cause of death in the intensive care unit, is a systemic response to severe bacterial infections, generally caused by gram-negative bacterial endotoxins that induce the generation of proinflammatory factors [77]. Because of their antiinflammatory properties, VIP and PACAP have been reported to protect against endotoxic shock syndrome [78]. TNF-␣ and IL-6 are overproduced in this state, and VIP and PACAP have been shown to reduce the circulating levels of these cytokines in an animal model of such pathology [79]. Exogenous administration of VIP and PACAP protects mice from the lethal effects of high endotoxemia, presumably by down-regulating pro-inflammatory mediators such as TNF-␣, IFN-␥, IL-6, IL-12, and NO [78]. Although our in vitro and in vivo studies using specific VIP agonists have indicated that the VPAC1 receptor is the main agent of the VIP and PACAP anti-inflammatory action [80], we have recently demonstrated PAC1 receptor involvement using knockout mice for the PACAP receptor [57]. Our results indicated that PAC1 receptor acts in vivo as an anti-inflammatory receptor, at least in part, by attenuating LPS-induced production of pro-inflammatory IL-6, which appears to be the main cytokine regulating the expression of the majority of the acute phase protein genes, which are important deleterious components of septic shock. Moreover, we have shown PAC1 receptor involvement in the inhibition of neutrophil infiltration measured by both myeloperoxidase activity and microscopic analysis [81] in different key organs affected by endotoxemia such as liver, lung, and bowel. Probably due to the pleiotropic effects inhibiting pro-inflammatory mediators that appear later during the inflammatory response, VIP and PACAP protect from endotoxemia if given 2 hours after endotoxin injection [79]. B. Rheumatoid Arthritis In light of the involvement of VIP and PACAP in the complex network formed by chemokines and cytokines involved in the regulation of inflammatory/Th1 diseases, we next evaluate the potential therapeutic role of both peptides in a collagen-induced model of arthritis. Rheumatoid arthritis (RA) is a chronic and debilitating autoimmune disease of unknown etiology that leads to chronic, progressive inflammation in the joints and subsequent erosive destruction of the cartilage and bone. The main symptoms of this disease result from massive infiltration of immune cells into the synovial membrane and fluid as neutrophils, macrophages, and T cells. These immune cells, together with activated synoviocytes, release (1) high amounts of chemokines, which recruit cells to the site of inflammation, (2) matrix metalloproteinases, which destroy the joint tissues, and (3) pro-inflammatory mediators (e.g., TNF-␣, IL-1, IL-6, and IFN-␥), which contribute to generate joint damage [82]. Although the contribution of Th1 and Th2 responses to RA is not completely understood, several studies in animal models revealed that the Th1 cytokine profile predominates at the induction and acute phases of the disease, whereas Th2-mediated responses are associated with the remission phase of the disease [83–87], thus suggesting a pathogenic role of Th1-derived cytokines. As the result of all these facts, the synovium, which in normal conditions is a fragile bilayer membrane covering the cartilage and bone in the joint, is transformed into a thick

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invasive one that destroys the joint structure, producing a deformation of the tissue that explains the rigidity and paralysis characteristic of the last stages of the disease. By using the experimental murine model of collagen-induced arthritis produced by immunization with type II collagen (CII), which shares common clinical, histological, and immunological features with human RA, we have shown that treatment with VIP and PACAP produce general amelioration of the disease [88,89]. Treatment of arthritic mice with VIP and PACAP decreases the frequency of arthritis, delaying the onset, reducing the severity of symptoms, and preventing joint damage (Fig. 3). The therapeutic effect of both peptides is due to a reduction of the two deleterious components of the disease, i.e., the inflammatory and the autoimmune. Thus, VIP and PACAP not only produce a reduction in the levels of pro-inflammatory agents such as TNF-␣, IL-6, IL-1␤, IL-12, iNOS, and IL-18, but also increase the levels of anti-inflammatory cytokines like IL-10 and IL-1Ra. VIP and PACAP also downregulate the levels of chemokines like RANTES, MCP-1, MIP-1␣, and MIP-2, responsible for the infiltration and activation of various leukocyte populations in joint tissue contributing to the pathology of rheumatoid arthritis. Moreover, VIP and PACAP also reduce the expression and activity of some matrix metalloproteinases (MMP), which have a crucial role in the depletion of proteoglycan, contributing to the destruction of both cartilage and bone. Because of the decreasing levels of all of these harmful soluble factors in RA, there is a patent remission in the chronic inflammation of the joints of affected mice. The effects of both peptides are not restricted to the mediators produced by synoviocytes, but also affects the cytokines released by the infiltrated T cells. There is strong evidence that the majority of the T cells in the inflamed tissues in RA have a Th1 cytokine pattern [83]. The Ig isotype switching that is directed by Th1 or Th2 cytokines in a different way (i.e., IFN-␥ and IL-4 induce IgG2a and IgG1 synthesis, respectively) is another marker of this disease that shows high circulating levels of anti-CII IgG2a [90]. VIP and PACAP treatments produce a reduction of IFN-␥ levels (Th1 cytokine) and an increase in IL-4 level (Th2 cytokine) together with a reduction of IgG2a and an increase in IgG1. These two effects confirm the VIP- and PACAP-inducing Th2 response, possibly contributing to remission of the illness, blocking the autoimmune component (Fig. 4). Of biological significance is the fact that VIP levels, like those of other recently described antiarthritic neuropeptides and hormones, such as CGRP and melanocyte-stimulating hormone (␣-MSH) [91,92], are specifically increased in serum and joints of arthritic mice during development of the disease (Fig. 3). This fact suggests that endogenous neuroimmune mediators represent a natural antiarthritic mechanism activated in response to autoimmune/inflammatory conditions, such as arthritis, in an attempt to counterbalance the effects of inflammatory mediators. The fact that VIP and PACAP affect both the inflammatory and autoimmune components of the disease represents an alternative approach to already existing treatments of RA. Extending the use of both peptides to the human system will depend on the dosage and manner of administration. In addition, VIP and PACAP gene transfer could be attractive as a potential means to deliver consistent, prolonged therapeutic titers of this anti-inflammatory factor with fewer side effects and without the need for repeated administration. C. Crohn’s Disease Human inflammatory bowel disease is a worldwide, chronic, idiopathic, inflammatory disease of the distal small intestine and the colon mucosa, clinically characterized by two

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Figure 3 VIP and PACAP are antiarthritic factors. Histological sections: (A) Arthritic mice with massive inflammatory infiltration in the joint, showing an almost complete destruction of bone and cartilage; (B) VIP-treated mice showing a normal morphology of the joint; (C) PACAP-treated mice with similar results.

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VIP/PACAP Th1 IFN-␥ Th2 B cell

IL-4

Induce Th2 response

IgG2a IgG1 TNF IL-1 IL-6 IL-12 NO

Decrease proinflammatory factors

IL-10 IL-1 Ra

Increase anti-inflammatory factors

Metalloproteinases

Prevent destruction of joint tissue

Figure 4 VIP and PACAP prevent arthritis by downregulating both components of the disease, i.e., inflammation and autoimmunity.

overlapping phenotypes: ulcerative colitis and Crohn’s disease. Crohn’s disease is an incurable autoimmune disease that leads to chronic transmural inflammation resulting in abdominal pain, diarrhea, and weight loss. Although its etiology is unknown, Crohn’s disease has been shown to be marked by an exaggerated gut-associated lymphoid tissue–developed immune response, giving rise to a prolonged severe inflammatory status of the intestinal mucus, characterized by uncontrolled production of pro-inflammatory cytokines and oligoclonal expansion and activation of CD4 T cells, specifically associated with a Th1 response [93]. Therapeutic agents currently used for Crohn’s disease are not completely effective, are nonspecific, and have multiple adverse side effects, with surgical resection being the sole alternative. Therefore, the present therapeutic strategy is to find drugs or agents that specifically modulate both components of the disease, i.e., inflammatory and Th1-driven responses. We have investigated the therapeutic effect of VIP and PACAP on the pathogenesis of Crohn’s disease by using a murine experimental model, which resembles many of the clinical, histopathological, and immunological characteristics of Crohn’s disease in humans [94]. Chronic intestinal inflammation was induced by intrarectal administration of 2,4,6trinitrobenzene sulfonic acid (TNBS) that haptenates colonic protein with trinitrophenil, diluted in 50% ethanol. VIP was administered intraperitoneally at different doses 12 hours

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after TNBS administration, in a single injection (pulse) or in five injections on alternate days. Mice were monitored for the occurrence of clinical signs of colitis. Mice treated with TNBS developed a chronic colitis characterized by a fast and dramatic decrease in body weight, accompanied by a severe bloody diarrhea and rectal prolapse and an extensive wasting disease. Macroscopic analysis of colons obtained 3 and 7 days after TNBS administration showed striking hyperemia, necrosis, and inflammation. In contrast, control mice treated with vehicle (50% ethanol) alone showed a healthy appearance with minor colitis signs. Treatment with VIP or PACAP drastically prevented the signs of TNBS-induced colitis. Although a single administration of VIP or PACAP at the onset of the disease was very effective in ameliorating the pathological signs of colitis, VIP administered on alternate days was the most effective treatment, with recovery of the initial body weight in only 8 days. Histopathological analysis of the colon of mice treated with TNBS in the acute phase of the disease showed a transmural inflammation, characterized by a massive neutrophill infiltration into the mucosa and submucosa, associated with a thickening of the colon wall, ulcerations, loss of globet cells, and fibrosis throughout the colon. This was followed by a chronic inflammation, marked by a massive infiltration of lymphocytes. VIP or PACAP administration significantly ameliorated the histopathological signs of the disease, restoring the normal mucosa and submucosa histological appearance, as compared to untreated mice (Fig. 5). We next investigated the mechanisms through which VIP mediates its therapeutic effects in TNBS-induced colitis. Crohn’s disease is characterized by a dysregulated immune response, with two well-known components: an unbalanced inflammatory process mediated by pro-inflammatory factors shared by both innate and acquired immune responses, and a T-cell–mediated immune response. Initiation of the inflammatory cascade occurs primarily through activated leukocytes at the site of inflammatory stimulus. Upon activation, leukocytes, epithelial cells, and endothelial cells in the inflamed intestine secrete large amounts of inflammatory mediators, including chemokines, cytokines, acute phase proteins, and myeloperoxidase, which are involved in the development of bowel inflammation. Because VIP acts as a potent anti-inflammatory agent, we studied the effect of VIP on the local (extracts of inflamed colons) and systemic (blood serum) production of these inflammatory mediators in TNBS-treated mice. Mice injected with TNBS alone showed increased myeloperoxidase activity, a measure of neutrophil infiltration, which is correlated with an augmented local production of pro-inflammatory cytokines in intestine extracts, including MIP-1␣, MCP-1, MIP-1, and Gro-␣/KC. In addition, in the acute phase of TNBS-induced colitis, pro-inflammatory cytokines such as TNF-␣, IL-6, IL-1␤, and IL-12, involved in inflammation and tissue damage, were significantly increased. VIP treatment, in a single pulse and in alternate days, significantly decreased TNBS-induced myeloperoxidase activity, chemokine levels, and proinflammatory cytokine levels in colon extracts. These results were confirmed at the systemic level in the serum. In addition, VIP drastically inhibited TNBS-induced levels of serum amyloid A (SAA), a hepatic acute phase protein that is synthesized in response to a systemic acute inflammatory stimulus. Although macrophages and neutrophils are the primary sources of pro-inflammatory mediators, CD4 T cells, especially Th1 cells, have a key role in the initiation and perpetuation of the disease [95]. Therefore, we next investigated the involvement of VIP in the modulation of CD4 T-cell response. TNBS administration resulted in a twofold increase in the percentage of CD4 T cells in spleen and lamina propria, in comparison with control mice treated with vehicle alone. These effectors CD4 T cells are predominantly Th1 cells,

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Figure 5 Histological sections of colons: (A) normal morphology in control; (B) inflammation with massive leukocyte infiltration and fibrosis in TNBS-induced colitis; (C) VIP-treated mice recovering the normal morphology of the colon.

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Colitis and weight loss Transmural inflammation, fibrosis and leukocyte infiltration VIP/PACAP

Proinflammatory factors (chemokines and cytokines) Acute phase proteins Prevention of Th1 and induction of Th2 responses

Figure 6 VIP ameliorate TNBS-induced colitis, decreasing both the inflammatory and the Tcell–mediated components of the disease.

because they produced IFN-␥ following stimulation. VIP treatment partially prevented TNBS-induced generation of CD4 T cells and of Th1 response. In addition, spleen and lamina propria CD4 T cells from mice treated with VIP preferentially showed a Th2 pattern, because they have an increased production of IL-4 and IL-10 after stimulation, a response beneficial for Crohn’s disease [96]. Our results also demonstrate that the therapeutic effect of VIP is through mediated mainly VPAC1. The capacity of VIP to regulate a wide spectrum of inflammatory and Th1/Th2 mediators in TNBS-induced colitis represents a therapeutic advantage over current treatments directed against a single mediator. Therefore, VIP could represent a possible multistep therapeutic agent for Crohn’s disease (Fig. 6). V. CONCLUSIONS AND PERSPECTIVES It is well established that VIP and PACAP are important immune mediators sharing cellular origin, receptors, and functions affecting both innate and acquired immunity. Their antiinflammatory and T-cell–mediated properties favoring Th1 versus Th2 responses confer on both peptides promising effects in the treatment of inflammatory and autoimmune diseases. Thus, VIP and PACAP represent potential multistep therapeutic agents to act at different levels in the cascade of the complex network formed by chemokines and cytokines involved in the regulation of inflammatory/Th1 diseases. The knowledge from basic and

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71. Martinez C, Delgado M, Pozo D, Leceta J, Calvo JR, Ganea D, Gomariz RP. Vasoactive intestinal peptide and pituitary adenylate cyclase-activating polypeptide modulate endotoxininduced IL-6 production by murine peritoneal macrophages. J Leuk Biol 1998; 63:591–601. 72. Delgado M, Leceta J, Abad C, Martinez C, Ganea D, Gomariz RP. Shedding of membranebound CD14 from lipopolysaccharide-stimulated macrophages by Vasoactive intestinal peptide and pituitary adenylate cyclase-activating polypeptide. J Neuroimmunol 1999; 99:61–71. 73. Lenschow DJ, Herold KC, Rhee L, Patel B, Koons A, Oin HY, Fuchs E, Singh B, Thompson CB, Bluestone JA. CD28/B7 regulation of Th1 and Th2 subsets in the development of autoimmune diabetes. Immunity 1996; 5:285–293. 74. Delgado M, Sun W, Leceta J, Ganea D. VIP and PACAP differentially regulate the costimulatory activity of resting and activated macrophages through the modulation of B7.1 and B7.2 expression. J Immunol 1999; 163:4213–4223. 75. Delgado M, Leceta J, Gomariz RP, Ganea D. VIP and PACAP stimulate the induction of Th2 responses by upregulating B7.2 expression. J Immunol 1999; 163:3629–3635. 76. O’Garra AO. Cytokines induce the development of functionally heterogeneous T helper cell subsets. Immunity 1998; 8:275–283. 77. Karima R, Matsumoto S, Higashi H, Matsushima K. The molecular pathogenesis of endotoxic shock and organ failfure. Mol Med Today 1999; 5:123–132. 78. Delgado M, Pozo D, Martinez C, Leceta J, Calvo JR, Ganea D, Gomariz RP. Vasoactive intestinal peptide and pituitary adenylate cyclase-activating polypeptide inhibit endotoxininduced TNF-alpha production by macrophages: in vitro and in vivo studies. J Immunol 1999f; 162:2358–2367. 79. Delgado M, Martinez C, Pozo D, Calvo JR, Leceta J, Ganea D, Gomariz RP. Vasoactive intestinal peptide (VIP) and pituitary adenylate cyclase-activating polypeptide (PACAP) protect mice from lethal endotoxemia through the inhibition of TNF-alpha and IL-6. J Immunol 1999; 162:1200–1205. 80. Delgado M, Gomariz RP, Martinez C, Abad C, Leceta J. Anti-inflammatory properties of the type 1 and 2 vasoactive intestinal peptide receptors: role in lethal endotoxic shock. Eur J Immunol 2000; 30:3236–3246. 81. Martinez C, Abad C, Juarranz G, Delgado M, Leceta J, Gomariz RP, Involvement of PACAP receptor on markers of septic shock: myeloperoxidase, serum amyloid A and nitric oxide. 5th International Congress of the International Society for Neuroimmunomodulation, Sept. 9–11, 2002, Montpellier, France. 82. Feldmann M, Brennan FM, Maini RN. Role of cytokines in rheumatoid arthritis. Ann Rev Immunol 1996; 14:397–440. 83. Mauri C, Williams RO, Walmsley M, Feldmann M. Relationship between Th1/Th2 cytokine patterns and the arthritogenic response in collagen-induced arthritis. Eur J Immunol 1996; 26: 1511–1520. 84. Doncarli A, Stasiuk LM, Fournier C, Abehsira-Amar O. Conversion in vivo from an early dominant Th0/Th1 response to a Th2 phenotype during the development of collagen-induced arthritis. Eur J Immunol 1997; 27:1451–1460. 85. Joosten LA, Lubberts E, Durez P, Helsen MM, Jacobs MJ, Goldman M, Van denn Berg WB. Role in interleukin-4 and interleukin-10 in murine collagen-induced arthritis. Arthritis Rheum 1997; 40:249–260. 86. Walmsley M, Katsikis PD, Abney E, Parry S, Williams RO, Maini RN, Feldman M. Interleukin 10 inhibition of the progression of established collagen-induced arthritis. Arthritis Rheum 1996; 39:495–503. 87. Horsfall AC, Butler DM, Marinova L, Warden PJ, Williams RO, Maini RN, Feldman M. Suppression of collagen-induced arthritis by continuous administration of interleukin-4. J Immunol 1997; 159:5687–5696. 88. Delgado M, Abad C, Martinez C, Leceta J, Gomariz RP. Vasoactive intestinal peptide prevents experimental arthritis by down-regulating both autoimmune and inflammatory components of the disease. Nat Med 2001; 7:563–568.

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89. Abad C, Martinez C, Leceta J, Gomariz RP, Delgado M. Pituitary adenylate cyclase-activating polypeptide inhibits collagen-induced artritis: an experimental immunomodulatory therapy. J Immunol 2001; 167:3182–3189. 90. Seki N, Sudo Y, Yoshioka T, Sugihara S, Fujitsu T, Sakuma S, Ogawa T, Hamaoka T, Senoh H, Fujiwara H. Type II collagen-induced murine arthritis induction and perpetuation of arthritis require synergy between humoral and cell-mediated immunity. J Immunol 1988; 140: 1477–1484. 91. Takeba Y, Suzuki N, Kaneko A, Asai T, Sakane T. Evidence for neural regulation of inflammatory synovial cell functions by secreting calcitonin gene-related peptide and vasoactive intestinal peptide in patients with rheumatoid arthritis. Arthritis Rheum 1999; 42:2418–2429. 92. Lipton JM, Catania A. Anti-inflammatory actions of the neuroimmunomodulator ␣-MSH. Immunol Today 1997; 18:140–145. 93. Blumberg RS, Strober W. Prospect for research in inflammatory bowel disease. JAMA 2001; 285:643–647. 94. Neurath MF, Fuss I, Kelsall BL, Stuber E, Strober W. Antibodies to IL-12 abrogate established experimental colitis in mice. J Exp Med 1995; 182:1280–1289. 95. Neurath MF, Finotto S, Glimcher LH. The role of Th1/Th2 polarization in mucosal immunity. Nat Med 2002; 8:567–573. 96. Schreiber S. Experimental immunomodulatory therapy of inflammatory bowel disease. Neth J Med 1998; 53:S24–S31.

13 Neurotransmitters Talk to T Cells in a Direct, Powerful, and Contextual Manner Affecting Key Immune Functions MIA LEVITE The Weizmann Institute of Science, Rehovot, Israel

For decades, neurotransmitters have traditionally been viewed as nerve-secreted molecules which interact primarily with specific receptors expressed on cells of the nervous system (peripheral and central). Accordingly, the study of neurotransmitter function has been considered of primary importance for the understanding of synaptic transmission and of neuronal development and function. As stated in an authoritative neuroscience textbook: ‘‘The identification of neurotransmitters mediating synaptic interactions in the brain and spinal cord is of fundamental importance for understanding the function of the nervous system.’’ This chapter will present a collection of evidence calling for a reassessment of the traditional view and suggesting that neurotransmitters may be as important for the immune system as they are for the nervous system. I. WHAT IS A NEUROTRANSMITTER? A substance is conventionally viewed as a neurotransmitter if the following criteria are met: 1. It is synthesized in neurons. 2. It is present in the presynaptic nerve terminal and is released in amounts sufficient to exert a defined action on the postsynaptic neuron or effector organs. 263

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3. When administered exogenously (as a drug) in reasonable concentrations, it precisely mimics the action of the endogenously released neurotransmitter (e.g., it activates the same ion channels or second messenger pathway in the postsynaptic cell). 4. A specific mechanism exists for its removal from its site of action (the synaptic cleft). In general, neurotransmitters fall into one of three chemical categories: 1. Amino acids, among them glutamate, glycine, and ␥-aminobutyric acid (GABA). 2. Biogenic amines, among them dopamine, norepinephrine, epinephrine, and serotonin. 3. Neuropeptides, among them somatostatin, substance P, vasoactive intestinal polypeptide (VIP), neuropeptide Y, NPY opioids, gonadotropin releasing hormone (GnRH), TRH, neurotensin, CRH, bombasin, prolactin, galanin, motilin, and many others. Neurotransmitters exert either excitatory or inhibitory effects on their targets within the nervous system and have a very wide spectrum of activities via which they affect a kaleidoscope of body functions.

II. WHERE CAN T CELLS ‘‘MEET’’ NEUROTRANSMITTERS? In principle, T cells may encounter neurotransmitters at sites where they patrol or reside under physiological and pathological conditions. Specifically, T cells may be exposed to neurotransmitters in the brain, lymphoid organs, other innervated peripheral organs, and the blood. Recent data indicate that the cellular sources of the neurotransmitters are clearly not only neuronal but also immune, since lymphocytes produce and secrete various neurotransmitters although in very low concentrations. This suggests that neurotransmitters may have the ability to stimulate T cells in a paracrine or autocrine fashion. The different T cellular sources for neurotransmitters and their relevance to neurotransmitters–T-cellinteractions will be further discussed in the following sections. A. Neuronal Sources in the Brain T cells may be exposed to neurotransmitters in the brain since activated T (and B) lymphocytes, in contrast to nonactivated immune cells, have the property to transmigrate into the central nervous system (CNS) across the blood-brain barrier (BBB), even in physiological conditions (and not only under pathological conditions, as was previously believed). Thus, one may envision that such T-cell–neurotransmitter interactions in the brain occur in everyday life, perhaps as part of an immune surveillance mechanism, protection, and routine coordination of various immune and neuronal activities. Moreover, such neurotransmitter–T-cell dialogues in the brain may occur in various pathological conditions in which T cells cause a disease, such as in T-cell–mediated autoimmune multiple sclerosis [1], or combat it, as, for example, in T-cell–mediated clearance of the encephalomyelitisinducing virus from brain [2], and in T-cell–dependent neuroprotection after neuronal injury [3]. Direct interactions between neurotransmitters and T cells in the brain may turn

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out to be of importance even in certain neuropsychiatric diseases shown recently to be associated with inflammatory processes in which T cells are probably involved. Since the factors responsible for regulating the beneficial or detrimental T-cell activities within the brain are to a large extent still unknown, the elucidation of the direct influence of neurotransmitters on T-cell functions can have far-reaching implications for the understanding and treatment of various CNS pathologies. B. Neuronal Sources in Lymphoid Tissues It has been established that all the primary and secondary lymphoid organs, among them the thymus, spleen, lymph nodes, bone marrow, and gut, are massively innervated by nerves which release a variety of neurotransmitters and neuropeptides [4,5]. Within these lymphoid organs, double immunocytochemistry with antibodies directed against immunespecific antigens and against enzymes involved in biosynthesis of various neurotransmitters has revealed the existence of direct contacts between nerve terminals and individual immune cells, among them T and B lymphocytes and mast cells [4–6]. This indicates that T cells within the lymphoid organs are likely to be exposed to neurotransmitters secreted in their vacinity. C. Neuronal Sources in the Blood Direct encounters between T cells and neurotransmitters probably take place in the blood, since both fenestrated and nonfenestrated blood capillaries are intensely innervated by nerve-secreting neurotransmitters. The direct contacts between neurotransmitter and bloodborne T cells may take place both under physiological and pathological conditions. The latter include: 1. Pathophysiological conditions causing an elevation of the plasma levels of certain neurotransmitters. Stress, for instance, is believed to increase very significantly the plasma levels of dopamine, noradrenaline, adrenaline, cortisol, and neuropeptide Y [7–9]. 2. Emergency conditions, among them hypertension and brain trauma, in which neurotransmitters such as dopamine and noradrenaline, or their specific receptor agonists, are administered intravenously for therapeutic purposes [10–13]. D. Immunological Sources in Other Organs Where Lymphocytes Reside or Patrol It has been established that T cells (and other types of lymphocytes) actively produce and secrete low concentrations of a variety of neurotransmitters and neuropeptides (the latter reviewed in Ref. 14). These include dopamine [15,16], norepinephrine [15,16], acetylcholine [17], serotonin [18], somatostatin [19,20], vasoactive intestinal peptide (VIP) [21,22], calcitonin gene–related peptide (CGRP) [23], gonadotropin-releasing hormone I and II (GnRH-I and -II) [24], and growth hormone–releasing hormone (GHRH) [25]. In addition, mRNA and protein corresponding presumably to CRH, the major hypothalamic peptide regulating the hypothalamic-pituitary-adrenal (HPA) axis, have been identified in human T and B lymphocytes, although neither is identical to the authentic hypothalamic CRH [26].

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In some cases the production and/or release of neurotransmitters by T cell are enhanced by T-cell stimulation with cytokines, mitogens, or others. Examples of such phenomena are: 1. Acetylcholine: The stimulation of T cells with the nonspecific human T-cell mitogen phytohemagglutinin (PHA) activates the lymphoid cholinergic system, as evidenced by increased synthesis and release of acetylcholine (Ach) [17]. 2. VIP: The proinflammatory cytokines IL-1, IL-6, and TNF-␣, but not IL-2, stimulate in a similar time-dependent manner the VIP production by lymphocytes [22]. 3. Calcitonin gene-related peptide (CGRP) [21,22]: While ␣- and ␤-CGRP mRNA are constitutively expressed in unstimulated human peripheral blood mononuclear cells (PBMC), PHA induces, in a time- and dose-dependent manner, the synthesis of ␤-CGRP and its secretion by human lymphocytes. Human IL-2 alone does not seem to affect hCGRP secretion but potentiates the PHAevoked hCGRP secretion from human spleen lymphocytes. Can T cells be affected in vivo by the neurotransmitters they produce? This intriguing question is still open and therefore awaits further in-depth investigation. Meanwhile, examples that this can indeed occur is presented by the stimulation of splenic T-lymphocyte function by endogenous serotonin and by low-dose exogenous serotonin [18]. Thus, the pretreatment of mice with p-chlorophenylalanine (PCPA) to deplete intracellular stores of serotonin reduced the capacity of the splenic T cells to proliferate and express the interleukin-2 receptor (IL-2R) in response to the nonspecific mitogen concanavalin A (ConA). These responses were restored by the addition of serotonin to spleen cell cultures [18]. The studies of Benguist et al., discover endogenous catecholamines, among them dopamine, in T-cells, and provide evidence for catecholamine regulation of lymphocyde function via an autocrine loop (62).

III. T CELLS EXPRESS SPECIFIC RECEPTORS FOR VARIOUS NEUROTRANSMITTERS T cells (as well as other immune cells) express specific receptors for various classical neurotransmitters and neuropeptides. These include: 1. Dopamine: We recently showed that purified normal human T cells express functional dopamine receptors of the D2, D3 subtypes that upon activation by dopamine trigger T cell functions [27]. Peripheral blood lymphocytes express dopamine D3, D4, and D5 receptor mRNA and/or binding sites [28,29]. 2. Noradrenaline: ␤2-Adrenergic receptors (␤2AR) are expressed by CD4Ⳮ T and B cells (reviewed in Ref. 30). Interestingly, ␤2AR-binding sites are present on Th1 cells, but not on Th2 cells [31]. 3. Glutamate: We recently found that normal, cancer, and antigen-specific human T cells express high levels of functional ionotropic glutamate receptors (ionchannel receptors) of the AMPA subtype 3 (GluR3) [32] which, in response to glutamate binding leads to triggering of T-cell functions. In addition, mouse thymocytes and thymic stromal cell lines express metabotropic (G protein–coupled) glutamate receptors [33].

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4. Acetylcholine immature T cells express the ␣3, ␣5, ␣7, ␤2, and ␤4 subunits, while peripheral T cells seem to express an unusual profile of ␣2, ␣5, and ␣7 subunits [34]. 5. Serotonin: Specific receptors or binding site for serotonin, primarily 5HT1a receptors, have been shown in mitogen-activated, but not in resting human T cells [35], human peripheral T cells [36], mouse splenic T cells [18], and human T cell leukemia line (Jurkat) [37]. 6. Receptors for somatostatin [19,20], VIP [21,22], CGRP [23], gonadotropinreleasing hormone I and II (GnRH-I and II) [24], and growth hormone–releasing hormone (GHRH) [25] have also been described (reviewed in Ref. 14). Interestingly, the expression of certain neurotransmitter receptors on T cells can be regulated either by classical immunological stimulating molecules (such as cytokines and mitogens) or by the neurotransmitter itself. For example, the density of ␤2R on purified CD4Ⳮ and CD8Ⳮ T cells was found to decrease upon incubation in vitro with epinephrine, but was not modified by IL-1␤. In contrast, an upregulation in the number of ␤2R found on purified CD8Ⳮ T-cells was observed following incubation with IL-2. These findings point out to a differential regulation of ␤2R on T-cell subpopulations, with CD8Ⳮ T cells being more susceptible to stimulating agents than CD4Ⳮ T cells [38]. The presence of neurotransmitters in nerves innervating the lymphoid organs and other tissues in which T cells reside or migrate among them the brain, and the existence of various receptors on the surface of T cells, are highly suggestive of functional links. Can neurotransmitters by themselves indeed trigger T-cell function? Recently, several studies from our lab revealed that neurotransmitters by themselves (in the absence of any additional stimulating molecules), acting via their specific receptors on T cells, can indeed activate or inhibit various T-cell functions [27,39–43]. In addition, neurotransmitters were found to modulate (primarily suppress) T-cell activities that are triggered by other stimulating molecules such as mitogens, cytokines, and anti-CD3 monoclonal antibodies. Examples of all these neurotransmitter effects will be discussed in Sec. VI.

IV. WHAT CAN BE GAINED BY DIRECT DIALOGUES BETWEEN T CELLS AND NEUROTRANSMITTERS? In principal, neurotransmitter-mediated signaling has unique characteristics that distinguish it from ‘‘classical’’ immunological signaling (mediated by antigens, cytokines, chemokines, etc). In addition, neurotransmitters may also differ by the context in which they are conveyed to their specific target tissues and the type of information they deliver. Some of the major differences between classical immunological signals and neurotransmitters are as follows: 1. Neurotransmitters are typically released from nerve terminals in transient bursts 2. Following their release, neurotransmitters activate their target receptors in a very rapid time scale of milliseconds and cause signals that can last from milliseconds to minutes. 3. Neurotransmitters are inactivated by specific degradation and/or uptake mechanisms. 4. The neurotransmitter receptors may narrow the time-window of their own activity by undergoing a process of desensitization shortly after activation.

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5. Neurotransmitters, along with electrical signaling, serve the bi-directional transfer of information from and to the nervous system, allowing thereby a control of the peripheral organs by the nervous system. Descending efferent nerves release neurotransmitters at a given location to affect its inner or outer milieu, from which, in turn, a report is conveyed to the brain via afferent ascending sensory nerves. 6. Peptidergic neurotransmitters (i.e., neuropeptides) are well known to exert physiological effects at substantial distances from their sites of release. 7. Since neuronal networks are highly branched and widespread, neurotransmitters can convey ‘‘messages’’ either simultaneously or gradually to several cell populations within the same organ, or even to another organ. Such a network can clearly contribute to a wide, rapid, and efficient spread of information, which may be important for an orchestrated operation of various body systems. Why do T cells need to be stimulated by neurotransmitters? I would agree that all the above properties of neurotransmitter-mediated signaling clearly indicate that the stimulation of T cells by neurotransmitters can be very different from that produced by classical immunological signals [42]. I would further argue that the repertoire of classical immunological signals seems somewhat insufficient and inadequate to account for the fact that at any given moment, T-cell populations throughout the body have to carry out a myriad of different activities. T cells are continuously proliferating and differentiating, being educated and updating their repertoire; they patrol and survey, combat and kill, help certain cells, suppress and tolerate others, memorize crucial past events, and even realize when it is time to die and undergo apoptosis. All these T-cell tasks should be tightly regulated and coordinated with the activities of other cell types. It is hard to imagine how the regulation of all these activities can be mediated through the antigen–T-cell receptor (TCR) interactions, even if assisted by other classical immunological molecules. This point is even strengthened by the fact that under physiologically healthy conditions, only a very small minority of T cells in the body can be genuinely considered as antigen-specific (in terms of their T-cell receptor), and by the assumption that under such conditions, only a small fraction of T-cell activities and duties are in fact dictated by specific antigens. Moreover, it is likely that some T-cell activities need to be triggered very rapidly and in a site-specific manner in response to the rapid changes in the milieu. Antigens and the other immunocyte-derived molecules can hardly initiate such rapid responses, as the cellular responses they elicit are usually observed after hours-days. Accordingly, I would argue that the direct stimulation of T cells by neurotransmitters, acting on behalf of the nervous system as ‘‘door-to-door express postman’’ can endow the immune system with tools that are quantitatively, qualitatively, kinetically, and contextually different from those conveyed by the classical immunological signaling, and can assist the immune system to meet its wide spectrum of needs [42]. In fact, neuroimmune contacts may serve not only for passing information from and to the nervous system, but also for communication within the immune system compartments and for keeping track of which immune cells are doing what, where, and why.

V. IT IS A MATTER OF CONTEXT A clear conclusion that emerges from our studies is that responses of T cells to direct stimulation by a given neurotransmitter are highly context-dependent (Fig. 1). The context

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appears to dictate whether a neurotransmitter will trigger, suppress, or be neutral to a given T-cell function. Variations in the parameters that define the context are most probably the basis of some of the contradictory results reported, for example, where the term ‘‘inhibitory neuropeptide’’ was assigned to neuropeptides that, in physiological concentrations, can, in fact, markedly activate T-cell function. A careful review of the literature reveals that most, if of not all, inhibitory effects of neurotransmitters on immune functions were observed only when the neurotransmitter was added after the cells had already been prestimulated by potent stimulating molecules such as mitogens, cytokines, or activating monoclonal antibodies (Fig. 1, outcome 4). Thus, when studying the effects of neurotransmitter on T cells, one must pay careful attention to the context particularities, as discussed below. A. Stimulation of T-Cells with Only Neurotransmitters in Contrast to Costimulation by Neurotransmitters and Additional Activating Molecules As mentioned (Fig. 1), the effect of a given neurotransmitter on resting T cells (often stimulatory) outcome can differ from its effect (often inhibitory) on T cells preactivated by an antigen, mitogen, anti-CD3 antibodies, or phorbol esters (outcome 4). Moreover, the simultaneous stimulation of T cells with an antigen and a neurotransmitter (Fig. 1, outcome 3) may result in yet a different outcome (often synergistic), differing from the effects of the individual stimuli (Fig. 1, outcomes 1 and 2). Thus, it is absolutely crucial

Figure 1 The importance of context in determining the effect of a particular neurotransmitter on a particular T-cell function. The effect of a given neurotransmitter on resting T cells (often stimulatory) can differ from its effect (often inhibitory) on T cells preactivated by an antigen, mitogen, anti-CD3 antibodies, or phorbol esters (outcome 4). Moreover, the simultaneous stimulation of T cells with an antigen and a neurotransmitter (outcome 3) may result in yet a different outcome (often synergistic), differing from the effects of the individual stimuli (outcome 1 and outcome 2). Thus, it is absolutely crucial to document whether T cells are exposed to a given neurotransmitter in the complete absence of any additional molecule or incubated with other molecules, such as antigens, mitogens, cytokine, chemokines, growth factors, antigens, mitogens, phorbol esters, serum factors, etc.

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to document whether T cells are exposed to a given neurotransmitter in the complete absence of any additional molecule or incubated with other molecules, such as antigens, mitogens, cytokine, chemokines, growth factors, antigens, mitogens, phorbol esters, serum factors, etc. Bearing this in mind, the conclusions reached in many studies are flawed because the contributions of the latter mentioned factors were not taken into account. For example, several studies concluded that neuropeptides suppress T-cell proliferation and cytokine secretion when the T cells were in fact pre- or co-stimulated with additional molecules, such as IL-2, PHA, Con A, LPS, etc. However, a different conclusion might have been reached if the T cells had been exposed to the neurotransmitter only, in which case the neurotransmitter might have revealed its triggering/activating potential. Thus, neurotransmitters, depending on the context, can exert either stimulatory or inhibitory effects on Tcell functions. Two examples supporting this claim emerge from own studies on the effects of dopamine and several neuropeptides on T-cell functions. When IL-2–activated [44] or LPS-activated [45,46] CD4Ⳮ and CD8Ⳮ T cells and monocytes were exposed to dopamine, a marked suppression of the in vitro cell proliferation, differentiation and cytotoxicity was observed [8,15,44. However, when in our own studies untreated resting normal human and mouse T cells, in the absence of any additional molecule, were exposed to dopamine, it triggered, in a potent and specific manner, several key T-cell functions, among which the activation of integrin-mediated T-cell adhesion to the extracellular matrix glycoprotein fibronectin (Fig. 2, Table 1) [27] and cytokine secretion (paper submitted). Accordingly, it is clear that dopamine cannot be considered as an immunosuppressive neurotransmitter, as this may be true only for preactivated T cells, but not for resting T cells. An additional example is the effects of CGRP on the secretion of interferon gamma (IFN-␥) and interleukin-10 (IL-10): stimulation of resting anti-myelin basic protein 87–99 T-cell lines with CGRP only triggered the secretion of IFN-␥ (from a Th0 line) and IL10 (from a Th2 line) [40]. In contrast, the exposure of these T-cell lines simultaneously to an antigen (MBP 87–99 peptide) and to CGRP decreased the antigen-induced secretion of IFN-␥ and IL-10 secretion (unpublished data). In another case, substance P was found to trigger the ‘‘forbidden’’ IL-4 secretion from a Th1 cell line, but only if the T cells are were preactivated by their antigen [40], while exerting no effect on resting T cells [40] (Table 2). B. The Nature of T-Cell Type/Subpopulation It now emerges that a neuropeptide may be either stimulatory or inhibitory, depending on the nature or phenotype of the T-cell subpopulation being stimulated: whether it displays a Th0, Th1, or Th2 phenotype or whether the cells are CD4(Ⳮ) or CD8(Ⳮ), etc. An example of the above is our own observation of the different patterns of cytokine secretion from Th0, Th1, or Th2 cells in response to the direct stimulation by a particular neuropeptide [40]. Another example is that of the effects of noradrenaline on T cells, where ␤2-adrenergic receptors were detected on resting and activated Th1 cell clones, but not on resting or activated Th2 cells [31]. Interestingly, the stimulation of the ␤2ARs on naive CD4(Ⳮ) cells generated Th1 cells that produced two- to fourfold more IFN-␥ [47].

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A Figure 2 Dopamine and a dopamine D3 receptor agonist induce T-cell adhesion to fibronectin. (A) The scheme represents the key molecules and events involved in T-cell adhesion and extravasation across extracellular matrix barriers. Adhesion of T cells to the major glycoproteins of the extracellular matrix (ECM), among them fibronectin and laminin, is a crucial event in the migration and extravasation of T cells from the blood stream into various tissues and organs in general and into inflamed tissues in particular. The adhesion of T cells to the ECM glycoproteins takes place via specific adhesion receptors, belonging primarily to the integrin family. The integrins must become activated to adhere to their ECM ligands and to allow the subsequent T-cell adhesion. Integrin functions were shown to play a key role in a broad spectrum of normal and diseased conditions in general and in inflammation and injury in particular. (B) Both dopamine itself (10 nM) and 7hydroxy DPAT, a dopamine D3 ⬎⬎⬎ D2 receptor agonist, trigger adhesion of purified normal human T cells to fibronectin, and these effects are blocked by a dopamine receptor antagonist selective for the D3 receptor subtype. (C) The activating effects of dopamine and of the dopamine receptor agonist 7-hydroxy DPAT on T-cell adhesion to fibronectin are dose-dependent. (D) Dopamine induces T-cell adhesion to fibronectin by activation of the specific ␣4␤1- and ␣5␤1-integrins. Normal human T cells were pretreated with relevant anti-CD29, VLA-4, and VLA-5 monoclonal antibodies directed against the ␤1, ␣4, and ␣5 integrin chains, respectively, with the nonrelevant anti-LFA-1 monoclonal antibody, with the relevant RGD peptide mediating in the integrins-FN interactions or with the nonrelevant RGE peptide. The T cells were then exposed to either dopamine (10 nM) and their adhesion to fibronectin determined. The results are presented as the mean Ⳳ SD CPM of bound T cells from quadruplicate wells. One experiment, representative of two, is presented. (Adapted from Ref. 27.)

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B

C Figure 2 Continued.

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D Figure 2 Continued.

C. Importance of the Specific T-Cell Function or Secreted Molecule Our studies of the effects of neuropeptides on cytokine secretion revealed that a given neuropeptide can augment the secretion of a particular cytokine, while suppressing that of another [40,48]. For example, NPY greatly enhances IL-4 production but inhibits IFN␥ from antigen-activated helper T-cell clones [48]. In contrast, CGRP inhibits IFN-␥ production, but has no effects on IL-4 production [48]. D. Neurotransmitter Dose As expected, the concentration of the neurotransmitter seems to be critical in determining whether it will affect a given T-cell function, and how. Furthermore, dose-response curves

Table 1 Highly Selective Dopamine Receptor Agonists and Antagonists Used for Studying the Effects of Dopamine on T-Cell Integrin Function Dopamine analog

Agonist/antagonist

SKF 38393 Quinpirole 7-Hydroxy-DPAT PD 168077 L-741,626 U-99194A maleate L-741,741

Agonist Agonist Agonist Antagonist Antagonist Antagonist Antagonist

Selectivity D1 D2 D3⬎⬎⬎D2 D4 D2 D3 D4

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Table 2 Neuropeptides by Themselves Trigger Typical and “Forbidden” Cytokine Secretion from T-Cell Lines and Clones

Neuropeptide Somatostatin

Calcitonin gene–related peptide (CGRP)

Neuropeptide Y

Substance P

Th0

Th1

Th2

Anti-MBP 87–99 line

Anti-MBP 87–99 line

Anti-MBP 87–99 line

Anti-Cop-1 clone

Anti-Hsp 65 clone

IL-2 IFN-␥ IL-4 IL-10 IL-2 IFN-␥ TNF-␣ IL-4 IL-10 0 0 0 0 0 0 0 0

IL-2 0 0 0 0 0 ND IL-4 0 IL-2 IFN-␥ IL-4 0 0 0 0 0

IL-2 IFN-␥ IL-4 IL-10 IL-2 IFN-␥ ND ND IL-10 0 IFN-␥ 0 0 0 IFN-␥ 0 0

0 IFN-␥ 0 IL-10 IL-2 0 ND 0 IL-10 IL-2 (?) IFN-␥ 0 0 IL-2 IFN-␥ 0 0

0 IFN-␥ ND ND 0 0 ND ND ND IL-2 0 ND ND 0 0 ND ND

ND ⫽ not done; MBP ⫽ myelin basic protein. a Shaded boxed ⫽ Considered “forbidden” cytokine secretion for Th1 or Th2 cells. Neuropeptide concentration: 10 nM.

may differ from those displayed by the same neurotransmitter in the nervous system. They also may differ for different T-cell functions, or even reveal opposite effects at a different concentration range of a given neurotransmitter. One example is the dose-response curve of dopamine in the activation of T-cell ␤1-, integrins. Dopamine triggers T-cell adhesion to fibronectin at the optimal dose of 10ⳮ8 M (Fig. 2A–C; Table 1) (the effective range being ⬃10ⳮ10 to 10ⳮ4 M) [27]. Interestingly, the concentration of dopamine in the brains extracellular fluid is ⬃ 10ⳮ8M, while in the plasma under physiological conditions it is much lower (⬃ 10ⳮ11M) (44). In contrast to dopamine, serotonin optimally stimulates IL-16 secretion from CD8Ⳮ T cells at 10ⳮ4 M (the effective range being ⬃10ⳮ7 to 10ⳮ4 M) [36]. Neuropeptides yet act within a wider range of concentrations from 10ⳮ14 to 10ⳮ6 M, encompassing what is regarded as their physiological range. For example, Nio et al. studied a homogeneous line of T cell (AO40.1 hybrid), whose activation is driven by a specific Ag (OVA) and where the endpoint (IL2 release) could not be contributed to by accessory or other cells. They found that VIP stimulated IL-2 release at very low concentrations with a marked effect at 10ⳮ14 M that gradually returned to control levels by 10ⳮ7 M. Somatostatin induced a substantial augmentation of AO40.1 IL-2 release at 10ⳮ10 to 10ⳮ8 M concentrations, whereas substance P demonstrated a stimulatory effect at higher concentrations of 10ⳮ9 to 10ⳮ6 M [49].

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E. Identity of the Neurotransmitter Receptor Subtype Being Stimulated The existence of large families of neurotransmitter receptor subtypes, which is a well known fact to neurobiologists, should be acknowledged by immunologists as well. Each neurotransmitter has several receptor subtypes which differ one from the other in various structural and functional features and respond differently to stimulation. A partial list emphasizing this point includes: Dopamine, with its five receptor subtypes, D1–D5, classically grouped into two families: D1&D5, and D2,D3,D4 receptors; Noradrenaline, with its three subfamilies of receptors: ␣1, ␣2, and ␤; Glutamate, with two large receptor families: the ionotropic receptors (ligand gated ion channel receptors), further subdivided according to the activating glutamate receptor agonist: NMDA, AMPA or Kainate, each with yet additional splice variants and subtypes, and the metabotropic (G-protein-linked) receptors (groups I–VII), each with several subtypes and splice-variants Acetylcholine: the nicotic and the muscarinic receptor families, each with several subtypes Serotonin: three receptor families, 5-HT1, 5-HT2, and 5-HT3, each with several subtypes Somatostatin: five cloned receptors: sst1–sst5 CGRP: two receptor subtypes, CGRP1 and CGRP2 Substance P, with three cloned tachykinin receptors, NK1–NK3 (NK1 was previously named substance P receptor) Neuropeptide Y, with two pharmacologically well-defined receptor subtypes, Y1 and Y2 VI. NEUROTRANSMITTERS AND NEUROPEPTIDES THAT TRIGGER OR SUPPRESS T-CELL FUNCTION A. Dopamine Dopamine is one of the principal neurotransmitters in the central nervous system, and its neuronal pathways are involved in several key functions such as behavior, control of movement, endocrine regulation, and cardiovascular function. Dopamine has at least five G-protein–coupled receptor subtypes, D1–D5, each arising from a different gene. Traditionally, these receptors have been classified into D1-like (the D1 and D5), and D2-like (D2, D3, and D4) receptor subtypes, primarily according to their ability to stimulate or inhibit adenylate cyclase, respectively, and to their pharmacological characteristics. Receptors for dopamine (particularly of the D2 subclass) represent the primary therapeutic target in a number of neuropathological disorders, including schizophrenia, Parkinson’s disease and Huntington’s chorea. 1. Effect of Dopamine on Resting Human T Cells in the Absence of Any Additional Stimulating Agent 1. Example 1: Dopamine activates ␤1-integrin function and triggers T-cell adhesion. While the existence of dopamine receptors on lymphocytes was suggested by ligand-binding studies and RT-PCR assays, it is only recently that

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evidence from our lab was presented for the existence of functional dopamine specific receptors on human T cells [27]. This was demonstrated by studying the effects of dopamine on integrin-mediated T-cell adhesion. The ability of T cells to adhere to and interact with components of the blood vessel walls and the extracellular matrix (ECM), such as fibronectin, laminin, and collagen, is essential for their extravasation and migration into inflamed sites. Such adhesive interactions take place via adhesion receptors, belonging primarily to the integrin family. The integrins ought to be activated to bind their ECM ligands (illustrated schematically in Fig. 2A). We found that dopamine by itself, at an optimum concentration of 10 nM, triggers the adhesion of T cells to fibronectin, a major glycoprotein of the extracellular matrix (ECM) (Fig. 2, Table 1). Since monoclonal antibodies directed against the relevant ␣4␤1 ␣5␤1 integrin moieties inhibit the dopamine-induced T-cell adhesion (Fig. 2D), it was concluded that dopamine activates these integrin moieties and thereby endows the T cells with an ability to adhere to fibronectin [27]. The dopamine-induced effect on the integrin-mediated adhesion to fibronectin was mimicked by selective D3 and D2 receptor agonists and blocked by the respective dopaminergic receptor antagonists [27] (Table 1). 2. Example 2: Dopamine triggers cytokine secretion. Upon interacting with its specific receptors on T cells, we recently found that dopamine by itself triggers secretion of cytotokines, primarily tumor necrosis alpha (TNF␣) and IL-10, from normal and cancer human T cells (M. Levite, submitted). 2. Effects of Dopamine on T Cells Concomitantly Stimulated with Additional Agents. 1. Example 1: Dopamine inhibits proliferation and cytotoxicity. Plasma dopamine levels were recently reported to be elevated in lung cancer patients in comparison to normal controls (48.6 Ⳳ 5.1 vs 10.2 Ⳳ 0.9 pg/mL) [8]. In vitro, domamine at comparable concentrations, the IL-2–induced proliferation and the cytotoxicity of T cells from these lung cancer patients, and those of normal volunteers, measured in presence of the respective sera. This effect has been attributed to the dopamine-induced elevation of intracellular cAMP mediated by the D1 dopamine receptor in these cell populations [8,44]. 2. Example 2: Dopamine induces apoptosis of lymphocytes. Mouse splenocytes and several mouse B- and T-cell hybridomas were reported to contain endogenously produced dopamine at levels ranging from 7 ⳯ 10ⳮ20 to 2 ⳯ 10ⳮ18 mol/cell [15]. The mitogen-induced proliferation and differentiation of mouse lymphocytes were found to be dose-dependently suppressed by dopamine and norepinephrine [15]. Even short-time pretreatment of lymphocytes with LDOPA and dopamine strongly suppressed lymphocyte proliferation and cytokine production. Incubation of lymphoid cells with L-DOPA, dopamine, and norepinephrine dose-dependently induced apoptosis, which, at least partly, explains the suppressive effects of catecholamines on lymphocyte function [15]. B. Noradrenaline The studies by V. Sanders and her team showing that the ␤2-adrenergic receptor stimulation by noradrenaline affects the level of cytokine and antibody produced by CD4Ⳮ T cells

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both in vitro and in vivo, the evidence in support of the expression of the ␤2-adrenergic receptor also by B lymphocytes, and the release of norepinephrine within lymphoid organs are discussed in a recent review [30]. C. Serotonin Serotonin (5-hydroxytryptamine, 5-HT), a potent neurotransmitter and vasoactive amine, is also an immunomodulator with stimulatory or inhibitory effects on T cells, B cells, natural killer (NK) cells, and macrophages. 1. Effect of Serotonin on Resting T Cells in the Absence of Any Additional Stimulating Agent. 1. Example 1: Serotonin and chemoattraction. Serotonin, at a concentration range of 10ⳮ7 –10ⳮ4 M, induces the secretion of IL-16, a chemoattractant factor of T-cell origin and a competent growth factor with selective activity for CD4Ⳮ T-cells (reviewed in Ref. 36). IL-16 is preformed and stored in a biologically active form in CD8Ⳮ T cells, from which it can be secreted following stimulation with histamine via H2-type receptors. Serotonin induces IL-16 secretion from CD8Ⳮ but not from CD4Ⳮ T cells, without requiring de novo protein synthesis [36]. On the basis of the findings of this study, it was suggested that serotonin, via its type 2 receptors, promotes the recruitment of CD4Ⳮ T cells into an inflammatory focus [36]. 2. Example 2: Serotonin ion currents and cell cycle. Serotonin and 2-methyl5HT, two agonists of 5-HT3 receptor channels, evoked NaⳭ ion influx into human leukemia T-cell line (Jurkat), which apparently facilitated the progression of the cells from the S to G2/M phase of the cell cycle [50]. In these cells, 5-HT3 receptors were present at a density (Bmax) of 300 Ⳳ 20 fmol/106 cells and exhibited a Kd value of 30 nM for serotonin. The T-cell 5-HT3 receptor channels were not regulated by either protein kinase C or free intracellular calcium concentrations, as the latter failed to influence the free intracellular NaⳭ concentration [NaⳭ]. 3. Example 3: Serotonin and proliferation. Serotonin alone stimulates the proliferation of human T lymphoblastic leukemia cell line (CCRF-CEM cells), and the effect is mimicked by two 5-HT1B/1D receptor agonists (L-694,247 and GR 46611) and inhibited by the selective 5-HT1B receptor antagonist, SB224289. These results establish the existence of a direct serotonergic control of the T-cell proliferation mediated through 5-HT1B receptors [51]. 2. Effect of Serotonin on T Cells Concomitantly Stimulated with Additional Agents. 1. Example 1: Serotonin and lectin-induced T-cell proliferation. Serotonin upregulates T-(and B-)lymphocyte proliferation by activating mitogen-induced cell surface 5-HT(1A) receptors [52]. 2. Serotonin also inhibits the PHA-stimulated proliferation of human peripheral blood lymphocytes (not shown directly to be T cells) via a mechanism independent of IL-2 production and causes a decrease in the expression and distribution of IL-2 receptors on the surface of responder cells [53]. 3. Example 2: Serotonin and PHA-induced T-cell ion currents. 5-HT and 2-

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methyl-5-HT, two agonists of the 5-HT(3) receptor channels in T cells, induced increases in [NaⳭ]i and potentiated the PHA-induced T-cell activation [54]. D. Glutamate T cells are likely to encounter glutamate, the major excitatory neurotransmitter in the central nervous system, when patrolling the brain and in glutamate-rich peripheral organs. Moreover, CNS glutamate levels increase in various pathological conditions, among them traumatic brain injury, acute brain anoxia/ischemia (i.e., stroke), epilepsy, glaucoma, meningitis, brain neurodegeneration associated with different chronic diseases such as AIDSassociated dementia , multiple sclerosis (MS), amyotrophic lateral sclerosis, and Alzheimer’s disease, in which T cells may play a beneficial or detrimental role. 1. Effect of Glutamate on Resting T Cells in the Absence of Any Additional Stimulating Agent We recently found that normal human T cells, human T-leukemia line, and mouse antigenspecific autoimmune T cells express high levels of specific ionotropic glutamate receptor of the AMPA subtype 3 (GluR3) [32]. The evidence for GluR3 expression in T cells includes GluR3-specific RT-PCR and sequencing establishing identity to authentic brain GluR3, Western blot, and GluR3 cell surface expression, as revealed by immunofluorescence staining and flow cytometry. Glutamate (10 nM), in the absence of any additional molecule, activated specific T-cell functions, among them integrin-dependent adhesion to laminin and fibronectin and chemotactic migration towards the chemokine SDF-1. The effects of glutamate were mimicked by AMPA (receptor agonist specific for the glutamate/ AMPA receptor subtype) and blocked by CNQX and NBQX (receptor antagonists specific for the glutamate/AMPA receptors). On the basis of these findings, we suggest that the expression of GluR3 in T cells and the ability of glutamate to activate T-cell functions could be of importance in physiological and pathological situations, which include: (1) T-cell transmigration into the CNS across laminin-containing capillary endothelial cells that form the blood-brain barrier; (2) T-cell patroling and activity within the CNS; (3) T-cell–mediated multiple sclerosis; (4) specific human epilepsies in which anti-GluR3 antibodies (directed against the so-called GluR3B epitope) are found and suspected to play a neurotoxic role [55]. E. Acetylcholine Acetylcholine (ACh) is a major neurotransmitter in both the central and peripheral nervous systems. Both muscarinic and nicotinic ACh receptors have been identified on lymphocytes isolated from thymus, lymph node, spleen, and peripheral blood, and their stimulation by muscarinic and nicotinic agonists elicits a variety of functional and biochemical effects [17]. On the basis of these findings, it has been postulated that the parasympathetic nervous system may play a role in immune-neurohumoral crosstalks. Moreover, ACh has been detected within blood lymphocytes using radioimmunoassay; Northern blots and reverse transcription-polymerase chain reactions have further shown the expression of choline acetyltransferase, the ACh-synthesizing enzyme, in human blood mononuclear leukocytes, human leukemic T-cell lines, and rat lymphocytes [17]. Stimulation of T cells with PHA activates the lymphoid cholinergic system, as evidenced by the increased synthesis and release of ACh and the increased acetylcholinesterase activity and expression of mRNAs encoding choline acetyltransferase and ACh receptors [17]. The observation that muscar-

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inic receptor stimulation by ACh or its agonists increases the intracellular [Ca2Ⳮ] concentration and upregulates c-fos expression argues that the ACh, synthesized and released from T cells, acts as an autocrine and/or paracrine factor to regulate immune functions. The data present a compelling picture in which immune functions are not only regulated by the cytokine system, but are also under the control of an independent lymphoid cholinergic system [17]. F. Somatostatin The somatostatin neuropeptide family comprises of several peptides derived from a different posttranslational processing of presomatostatin mRNA encoded by a single gene (reviewed in Ref. 14). Two of these peptides, somatostatin-14 and somatostatin-28, are biologically active and are the major somatostatin peptides found in the nervous system. Somatostatin inhibits numerous endocrine secretions, acting either as neurohormone (e.g., in the pituitary where it inhibits growth hormone, prolactin, and TRH) or hormone (e.g., in the pancreas where it inhibits glucagon and insulin release). In the CNS, somatostatin acts as a neurotransmitter in discrete somatostatinergic neuronal pathways [56]. Most of the currently known effects of somatostatin on immune functions are reviewed extensively by Krantic [14]. 1. Effect of Somatostatin on Resting T Cells in the Absence of Any Additional Stimulating Agent 1. Example 1: Somatostatin triggers the secretion of cytokines from T cells. We recently found that somatostatin induces the release of IL-2 from Th0-type anti-MBP 87–99 T-cell line, Th1-type anti-MBP 87–99 T cell, and even from Th2-type anti-MBP 87–99 T cell [40]. The latter secretion of IL-2 from a Th2type T cell is considered ‘‘forbidden’’ since such cells normally and characteristically produce only IL-4 and IL-10, the ‘‘classical’’ Th2 cytokine (Fig. 2A,B; Table 1). Somatostatin also induces, on its own, the release of IFN-␥ [40] (Fig. 3; Table 2). Based on these findings, we suggest that somatostatin (as well as additional neuropeptides) may have the ability to induce both typical and atypical T-cell cytokine secretion [40] (Fig. 3B; Table 2). 2. Example 2: Somatostatin triggers the activation of ␤1-integrin function. We further found that somatostatin triggers on its own the adhesion of normal human T cells to fibronectin, via activation of ␤1-integrins [57]. The optimal pro-adhesive effect was obtained with somatostatin at concentrations of 10ⳮ11, 10ⳮ8, and 10ⳮ5 M. Cyclo-[7-aminoheptanoyl-Phe-Trp-Lys-Thr(bzl)], an antagonist of somatostatin receptor, inhibited somatostatin-induced T-cell adhesion to fibronectin, while control antagonists failed to do so, demonstrating that somatostatin stimulates T-cell adhesion via binding to its specific G-protein–coupled receptors on T cells. The somatostatin-induced T-cell adhesion to fibronectin is mediated by the ␣4␤1 and ␣5␤1 integrins, as specific monoclonal antibodies to these integrin moieties abrogate the effect [57]. Finally, the interaction between somatostatin and its specific T-cell–expressed receptors, which leads to upregulation of ␣4␤1 and ␣5␤1 affinities and to subsequent binding to fibronectin, is mediated through diverse intracellular signaling pathways involving characteristic G-protein, PTK, PKC, and PI-3 kinase signaling, since all the relevant inhibitors blocked the effect [57].

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Th1 cells are associated with cell-mediated immune responses (e.g., antiviral immunity, inflammation, etc.)

Th2 cells are associated with humoral immune responses (e.g., allergic responses) inhibit Th1 responses

A

B

Figure 3 Findings and suggestions regarding neuropeptide-induced versus antigen-induced cytokine secretion. (A) The classical antigenic stimulation of mouse antigen-specific T cells triggers the characteristic secretion of IL-2 and IFN-␥ from Th1 cells, IL-4 and IL-10 from Th2 cells, and all the four cytokines from Th0 precursor cells. (B) The direct stimulation of the Th0, Th1, and Th2 cells by neuropeptides triggers both typical and atypical cytokine secretion. Based on these observations, we postulate that particular neuropeptides can induce the reversal of commitment of Th1 and Th2 cells into their precursor Th0-like cells, which secrete the entire repertoire of T-cell cytokines. (Adapted from Ref. 40.)

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2. Effect of Somatostatin on T Cells Concomitantly Stimulated with Additional Agents 1. Example 1: Somatostatin enhances mitogen-induced proliferation. Somatostatin modulates the proliferation of mitogen-stimulated normal human and mouse T cells in a biphasic manner: an inhibition occurs at subnanomolar concentrations, while stimulation takes place at low micromolar concentration [14]. Somatostatin increases mitogen-induced proliferation of human Jurkat T cells via the sst3 receptor isotype [58]. The controversies surrounding the effects of somatostatin are reviewed by Krantic [14]. 2. Example 2: Somatostatin modulated mitogen or antigen-driven cytokine secretion. Somatostatin increases the mitogen-induced IL-2 secretion from normal mouse [49] and cancer (Jurkat) human T-cell lines [58]. Furthermore, when applied to an antigen (OVA)-specific T-cell line, somatostatin (at 10ⳮ10 – 10ⳮ8 M concentrations) caused a substantial increase of antigen-driven IL-2 release [49]. 3. Somatostatin receptors were also identified on a subset of granuloma CD4Ⳮ T cells, and both somatostatin and octreotide (a Stable somatostatin derivative), at concentrations as low as 10ⳮ10 M, substantially decreased IFN-␥ secretion from antigen- or mitogen-stimulated cells, while higher concentrations of 10ⳮ6 M did not affect IL-5 production [59]. G. Substance P Tachykinins are neuropeptides that are widely distributed in the body and function as neurotransmitters and neuromodulators. Five tachykinin subtypes—substance P, neurokinin A, neurokinin B, neuropeptide K, and neuropeptide gamma—and three receptor subtypes—neurokinin-1, -2, and -3 receptors—have been identified. Substance P was the first peptide of the tachykinin family to be identified. It is considered to be an important neuropeptide in the nervous system and in the intestine, which possesses pleiotropic properties, e.g., neurotransmission and immune/hematopoietic modulation. NK1 receptors were recently shown not only in neurons and immune cells, but also in other peripheral cells, including bone cells. 1. Effect of Substance P on Resting T Cells in the Absence of Any Additional Stimulating Agent We found that substance P by itself causes an atypical secretion of IFN-␥ (a Th1 cytokine) from Th2 antigen-specific T cells, such as the anti-myelin basic protein 87–99 Th2 line, and anti-copolymer 1 (Cop-1) Th2 clone [40] (Fig. 3B; Table 2). These Th2 lines/clones normally secrete only IL-4 and IL-10, the typical Th2 cytokines. This suggests that substance P, like some other neuropeptides (Fig. 2B), has the ability to break the commitment of Th2 cells to secrete an unique set of cytokines [40]. 2. Effect of Substance P on T Cells Concomitantly Stimulated with Additional Agents 1. Example 1: Substance P enhances T-cell cytokine secretion. The capacity of substance P to regulate IL-2 production by Jurkat and HUT 78 T-cell lines and by peripheral blood human T cells activated with PHA plus PMA was investigated by Calvo et al using Northern blot analysis and dosage of the IL-

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2 release in cell supernatants. Substance P was found to act as cosignal with PHA Ⳮ PMA to enhance the expression of specific IL-2 mRNA and IL-2 secretion in T cells. Using the N-terminal substance P (1–4) or C-terminal substance P (4–11) fragments, the cosignal activity was attributed to the Cterminal fragment. The substance P and SP (4–11) optimal effects were observed [60] at 10ⳮ12 and 10ⳮ10 M. 2. Example 2: Substance P enhances Con A–induced proliferation, IL-2 production, and the number of CD4ⴐCD25ⴐ and CD4ⴐRT1Bⴐ T cells. Substance P administration stimulates Con A–induced proliferation of spleen and peripheral blood lymphocytes (PBL) from normal and neonatally capsaicin-treated rats, in correlation with an enhanced IL-2 production and expression of activation antigens, such as the IL-2 receptor alpha chain (CD25) and the RT1B MHC class II molecule. Moreover, substance P markedly increased the percentage of CD5Ⳮ and CD4Ⳮ T cells in the peripheral blood of capsaicin-treated rats. Concomitant administration of substance P with the nonpeptide neurokinin-1 receptor (NK1R) antagonist SR140333 completely inhibited the substance P–mediated augmentation of Con A–induced PBL proliferation and IL-2 production as well as of CD4ⳭCD25Ⳮ and CD4ⳭRT1BⳭ T-cell numbers in normal and capsaicintreated rats [61]. 3. Example 3: Substance P inhibits ␤1-integrins and T-cell adhesion to fibronectin. In our own studies substance P was found to block the ␤1-integrin–mediated T-cell adhesion to fibronectin induced by CGRP, neuropeptide Y, somatostatin, macrophage inflammatory protein-1beta (MIP-1␤), or by the potent and nonspecific T-cell activator PMA [40]. Inhibition of T-cell adhesion was ob䉴 Figure 4 A proposed scheme of events incorporating our recent findings and speculations regarding the direct effects of GnRH-II and GnRH-I on T-cells. (A) Cellular sources for GnRH-II and GnRH-I. T cells migrating across fenestrated blood vessels, mainly in the brain, may encounter GnRH-II and GnRH-I released from nerve terminals (black arrows). In addition, normal human T cells produce GnRH-II and GnRH-I, which may act either in an autocrine (represented by the left cell) and/or in a paracrine fashion on other T cells (right cell) or other cell types. (B) The direct effects of GnRH-II and GnRH-I on T cells. Upon binding of GnRH-II and/or GnRH-I to their postulated receptors, T cells are activated, causing the synthesis and surface expression of a 67 kDa nonintegrin laminin receptor. (C) The postulated physiological consequences of T-cell stimulation by GnRH. The GnRH-induced laminin receptor expression leads to T-cell adhesion to laminin within the endothelial basement membrane, a mesh-like structure composed of numerous components including collagen and proteoglycan. Of note, the basement membrane surrounding the endothelial cells normally prevents the entry of plasma proteins and cells into the tissues. The GnRH-stimulated T cells, via a lamininbinding mediated process, extravasate across the blood vessel and basement membrane towards a chemokine secreted within a ‘‘restful’’ or inflamed tissue. We speculate that during desired T-cell migration and function, the effect of GnRH is beneficial and may be boosted, while in conditions of undesired T-cell migration, such as in T-cell–mediated malignancies, autoimmune diseases (i.e., multiple sclerosis), graft-versus-host disease, graft rejection, etc., the direct effects of GnRH-II and GnRH-I on T cells may be detrimental and should thus be arrested. (A and B adapted from Ref. 24.)

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Figure 4 Continued.

tained both by the intact substance P peptide and by its (4–11), (5–11), and (6–11) C-terminal fragments, at similar nanomolar concentrations, and was abrogated by an substance P NK-1 receptor antagonist, suggesting that the inhibitory effect of substance P was mediated by its NK-1 receptor [57]. The physiological ability of substance P to inhibit T-cell adhesion was in correlation with its ability to inhibit Kv1.3, a T-cell voltage-gated potassium channel [41] found in our studies to be functionally and physically associated with ␤1-integrins: opening of the channel is sufficient to activate the ␤1-integrins and permit adhesion, while its blockade by substance P or by highly selective channel blockers inhibits ␤1-integrin function [41]. Further effects of substance P on lymphocytes in vitro and in vivo are reviewed by Krantic [14]. H. GnRH-II and GnRH-I Gonadotropin-releasing hormone-II (GnRH-II), a unique 10-amino-acid neuropeptide conserved through 500 million years of evolution and recently identified in mammals, shares 70% homology with the mammalian hypothalamic neurohormone GnRH (GnRH-I), the primary regulator of reproduction, but is encoded by a different gene. Although both neuropeptides are produced mainly in the brain, their localization and promoter regulation differ markedly, suggesting distinct functions. Indeed, GnRH-II barely affects reproduction, and its role(s) in mammalian physiology is still an enigma.

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We recently found that normal human T-cells and leukemia T cells produce GnRHII and GnRH-I [24], suggesting that the secreted GnRH-II and GnRH-I may affect the T cell autocrine or paracrine mode (shown schematically in Fig. 4A). The exposure of normal or cancer human or mouse T cells to GnRH-II or GnRH-I, in the absence of any additional molecules, was found to elicit de novo gene transcription and the cell surface expression of a 67 kDa nonintegrin-laminin receptor (67 kDa LR) (shown schematically in Fig. 3B), which plays a key role in cellular adhesion/migration, in tumor invasion/metastasis, and in prion infections [24]. GnRH-II or GnRH-I also induced in vitro adhesion of T cells to laminin and their chemotactic migration towards the potent chemokine SDF-1 (shown schematically in Fig. 3B). In addition, GnRH-II or GnRH-I also augmented the entry in vivo of metastatic EL-4 T-lymphoma into the spleen and bone marrow, the two organs known to be invaded by this highly metastatic T-lymphoma line. Finally, GnRH seems to be required for the normal T-cell entry in vivo into specific organs, since the homing of normal mouse T cells into the spleen and kidney was reduced in GnRH-I knockout mice. A specific GnRH-I receptor antagonist blocked the GnRH-I–but not GnRH-II–induced effects, suggesting signaling through distinct receptors [24]. Taken together we speculate that the direct effects on T cells of GnRH-II and GnRH-I, secreted from nerves or from autocrine/paracrine sources, are likely to be of relevance to various physiological and pathological conditions in which T cells play a beneficial or detrimental role. The findings revealed in this study [24], and the speculations they raise, are shown schematically in Fig. 4. The effects on T cells of the neuropeptides CGRP, NPY, AVP (arginine vasopressin), TRH, CRH, and GHRH (the latter five being hypothalamic-releasing hormones) are summarized in a recent review [14]. VII. CONCLUDING REMARKS The large body of evidence outlined in this chapter show that some of the classical neurotransmitters and numerous neuropeptides have the ability to elicit several key T-cell functions, as well as to either augment or suppress antigen-, cytokine-, or mitogen-induced Tcell functions. Neurotransmitters and neuropeptides, operating in an autocrine or paracrine mode, are thus important factors that ought to be taken into account for a comprehensive understanding of the role of T cells in different physiological and pathological settings. They must be further investigated in depth, as probably only the tip of the iceberg has been revealed thus far. ACKNOWLEDGMENTS I thank Prof. Vivian Teichberg for critical reading of the chapter manuscript and for his knowledgeable comments. I also thank Ph.D. student Yonatan Ganor for helping with the figures and tables. All the studies by M. Levite et al. reported in this chapter were supported by a Volkswagen Stiftung grant to M. L.

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14 Role of Neuropeptides in T-Cell Differentiation DOINA GANEA Rutgers University, Newark, New Jersey, U.S.A MARIO DELGADO Instituto de Parasitologia y Biomedicina, Granada, Spain

I. INTRODUCTION Neuroendocrine-immune interactions occur primarily through the hypothalamus-pituitaryadrenal (HPA) axis, the vagus nerve, and the innervation of the lymphoid organs. Lymphoid tissues are innervated by nerve fibers containing both neurotransmitters and neuropeptides. Some neuropeptides, such as vasoactive intestinal peptide (VIP), neuropeptide Y (NPY), somatostatin (SOM), and galanin (Gal), are present primarily in the noradrenergic autonomic and cholinergic innervation, whereas substance P (SP), neurokinin A (NKA), and calcitonin gene–related peptide (CGRP) reside in the sensory innervation [1–7]. In addition, the immune cells express and release neuropeptides such as opioid peptides, ␣-melanocyte–stimulating hormone (␣-MSH), SP, SOM, NPY, and VIP [8–19]. Although the factors that control neuronal or immune release of neuropeptides during an inflammatory response remain to be identified and characterized, neuropeptides such as VIP, SP, and CGRP were shown to be released in functionally relevant amounts during an immune response, both in patients and in animal models [20–24]. Neuropeptides released within the lymphoid organs bind to receptors expressed by immune cells and act as immunomodulators. VIP and the structurally related neuropeptide pituitary adenylate cyclase–activating polypeptide (PACAP) are two of the best-studied immunoregulatory neuropeptides. Their role as ‘‘macrophage-deactivating factors’’ and potential therapeutic agents in inflammatory and autoimmune conditions has been described in a number of recent reviews [25–30] (see also Chapter 12). In addition, the 289

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downregulation of proinflammatory cytokine and chemokine expression by VIP/PACAP was described recently for brain microglia [31–34]. In addition to their anti-inflammatory effects on innate immunity, VIP and PACAP also affect adaptive immunity, particularly the development, activation, and differentiation of T lymphocytes, which represents the focus of this chapter.

II. EFFECTS OF VIP/PACAP ON THYMIC T-CELL DEVELOPMENT A. VIP Sources in Thymus Several VIP sources are present in all areas of the thymus, i.e., the capsular/septal system, the thymic cortex, and medulla. In addition to VIP-ergic nerve fibers, the cells of the reticulo-epithelial network and thymocytes themselves function as VIP sources [17,35–37]. In vitro studies indicate that thymocytes release VIP following inflammatory signals delivered by mitogens and antigens or by proinflammatory cytokines [17]. However, at the present time the in vivo signals resulting in intrathymic VIP release from any of the three sources mentioned above remain unknown. B. VIP Receptors in Thymocytes Three VIP/PACAP receptors have been cloned: VPAC1 and VPAC2, which bind VIP and PACAP with equal affinity and activate primarily adenylate cyclase, and PAC1, also called the PACAP-preferring receptor, which binds PACAP with high affinity and activates both adenylate cyclase and phospholipase C [38,39]. In human thymus, VIP-binding sites were identified in both cortex and medulla [40]. This was confirmed with both human and mouse thymocytes, where VPAC2 expression was found in all four thymocyte subsets (CD4ⳮCD8ⳮ, CD4ⳭCD8Ⳮ, and the most mature CD4ⳭCD8ⳮ and CD4ⳮCD8Ⳮ cells) [37,41]. Real-time polymerase chain reaction (PCR) studies indicate that although VPAC1 is also expressed in certain thymocyte subsets, VPAC2 expression is prevalent, and that, similar to mature T cells in the periphery, expression of VPAC1, but not VPAC2, is reduced upon thymocyte activation [41]. C. Effects of VIP on Thymocytes The role of VIP/PACAP in thymocyte maturation and function is not clear. In terms of thymocyte proliferation, studies using fetal thymic organ cultures established a marginally positive effect for VIP on thymocyte proliferation [42], whereas in vivo administration of VIP receptor antagonists enhanced the mitotic index, suggesting that endogenous VIP inhibits the basal proliferative activity of thymocytes [43]. Actually, the VIP role in vivo might be more complex, since VIP was shown to protect thymocytes from glucocorticoidinduced apoptosis [44,45], which apparently plays a role in thymocyte selection [46]. In addition, VIP and PACAP were shown to inhibit IL-2 and IL-4 production in thymocytes stimulated through the T-cell receptor (TCR) and to enhance differentiation of CD4ⳭCD8Ⳮ thymocytes into mature CD4Ⳮ T cells [37,47]. Whether these effects are indeed significant for thymocyte proliferation, survival, and differentiation remains to be established by using VPAC1-, VPAC2-, or double-receptor–deficient mice.

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III. EFFECTS OF VIP/PACAP ON THE ACTIVATION, SURVIVAL, AND DIFFERENTIATION OF T CELLS IN THE PERIPHERY A. VIP Sources in the Secondary Lymphoid Organs Similar to the thymus, the sources of VIP in the peripheral immune organs consist of both innervation and immune cells. VIP-ergic nerve fibers of an origin other than the sympathetic nervous system were found in spleen and lymph nodes [35]. In addition, immune cells have been shown to express and secrete VIP [17,19,48,49]. Macrophages express VIP constitutively, whereas T cells, specifically Th2 effectors, express and secrete VIP only following antigen activation [19,49]. With the exception of IL-4, which has been shown to downregulate VIP expression in macrophages [49], the role of other cytokines, and particularly of the proinflammatory cytokines such as TNF-␣, IL-12, and IFN-␥ on VIP production by either macrophages or TCR-stimulated Th2 cells is not known. B. VIP/PACAP Receptors on Peripheral T Cells Binding studies in a number of peripheral lymphoid organs, such as spleen, lymph nodes, tonsils, appendix, and Peyer’s patches, identified VIP receptors in the T-cell–rich areas [40]. VPAC1 expression was confirmed in both murine and human peripheral T cells, including intraepithelial and lamina propria T cells [50–55]. VPAC1 and -2 expression in T cells appears to be differentially regulated during activation. Following T-cell activation, VPAC1 expression is downregulated and VPAC2 expression upregulated [51–53], with VPAC2 becoming the predominant or sole receptor in several T-cell lines [37,56,57]. The factors and mechanisms involved in the differential regulation of VPAC1 and -2 during T-cell activation are not understood at the present time. In murine schistosomiasis, expression of VPAC2 in granuloma CD4Ⳮ T cells is modulated by IL-4 [58]. Whether IL-4 or other cytokines affect VPAC2 and possibly VPAC1 expression in T cells, other than those found in granulomas, remains to be established. Modulation of VPAC1/2 receptor expression during T-cell activation and differentiation raises the question whether the two receptors mediate different immune functions. In vitro studies showed that inhibition of IL-2 production and survival of CD4Ⳮ T cells is mediated by both VPAC1 and VPAC2 [57,59]. However, in vivo studies in mice with Tcell–targeted overexpression of VPAC2 and studies in VPAC2-deficient mice suggest that VPAC2, and not VPAC1, mediates immediate allergic responses [54,60]. Also, VPAC2 and not VPAC1 transduces human T-cell chemotaxis [56,61] and mediates the VIP-enhanced conversion of thymocytes into CD4Ⳮ8ⳮ T cells [47]. In view of these results, and of the fact that VPAC2 expression is upregulated during T-cell activation, VPAC2 appears to function as the essential T-cell receptor. C. Effects of VIP/PACAP on T-Cell Activation, Differentiation, Function, and Survival 1. T-Cell Activation The adaptive immune response against a specific antigen is initiated by the cellular contact between an antigen-presenting cell (APC) and a naı¨ve T cell bearing the appropriate TCR. Both stimulatory (MHC/Ag on the APC – TCR on the T cell) and costimulatory (B7.1/ B7.2 and CD40 on the APC and CD28 and CD40L on the T cell) interactions are required

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for the activation of naı¨ve T cells. Following activation, antigen-specific T cells express high-affinity IL-2 receptors and proliferate in response to autocrine or paracrine IL-2. Neuropeptides, particularly VIP and PACAP, affect T-cell activation and proliferation by acting on both APCs and newly activated T cells. Several cell types, including dendritic cells, macrophages, and B cells in the periphery and microglia in the central nervous system (CNS), function as APCs. We have focused previously on the effects of VIP/PACAP on lipopolysaccharide (LPS)-stimulated macrophages and microglia. VIP and PACAP downregulate B7.1 and B7.2 expression in macrophages and B7.2 and CD40 expression in microglia [32,62]. The inhibition of B7 expression in macrophages by VIP/PACAP correlates with an inhibitory effect on the stimulation of T cells [62] and is in agreement with the role of VIP and PACAP as endogenous antiinflammatory agents (discussed in detail in Chapter 12). Recent studies indicate a similar effect of VIP/PACAP on B7.1 and B7.2 expression in LPS-stimulated dendritic cells derived from murine bone marrow (results not published), suggesting a common mechanism for the VIP/PACAP inhibition of T-cell activation through the downregulation of costimulatory molecules on APCs. In addition, VIP/PACAP also affect the proliferation of newly activated T cells directly by inhibiting IL-2 production. Several authors reported inhibitory effects on Tcell proliferation and IL-2 production [29,63]. Mechanistic studies indicated that VIP/ PACAP inhibit the expression of the IL-2 gene in newly activated T cells by affecting the transcriptional factors NFAT and AP-1 [63,64]. 2. Differentiation of CD4Ⳮ T Cells Following antigenic stimulation and proliferation, CD4Ⳮ T cells differentiate into Th1 and Th2 effector cells, characterized by specific cytokine profiles and functions. Determining factors for differentiation into Th1 or Th2 effectors include the nature of the APCs, the nature and amount of antigen, the genetic background of the host, and particularly the cytokine microenvironment [65]. Other endogenous factors such as progesterone and glucocorticoids have been reported to favor Th2 differentiation [66]. The possible role of neuropeptides in the Th1/Th2 differentiation process is just beginning to be investigated. Several lines of investigation indicate that VIP/PACAP promote Th2-type immune responses. We reported previously that macrophages treated in vitro with VIP or PACAP gain the ability to induce Th2-type cytokines (IL-4 and IL-5) and inhibit Th1-type cytokines (IFN-␥, IL-2) in Ag-primed CD4Ⳮ T cells [67]. Similar results were obtained recently with dendritic cells generated from bone marrow (unpublished results). In agreement with the in vitro results, in vivo administration of VIP or PACAP in immunized mice results in a decreased number of IFN-␥–secreting cells and an increased number of IL-4–secreting cells [67]. Two recent studies confirmed the prevalence of Th2-type responses in mice overexpressing the human VPAC2 receptor in CD4Ⳮ T cells [54] and of Th1-type responses in mice deficient in VPAC2 [60]. These studies confirm and extend the concept that VIP/PACAP affect the Th1/Th2 balance in vivo, and open the possibility of using VIP/PACAP analogs or receptor agonists in the treatment of autoimmune diseases with a prevalent Th1 background. D. Possible Mechanisms by Which VIP/PACAP Promote Th2-Type Immune Responses Several nonexcluding mechanisms (Fig. 1) could contribute to VIP/PACAP promoting Th2-type responses. The neuropeptides could act at the level of Th1/Th2 generation, either

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Figure 1 Effects of VIP/PACAP on the differentiation of CD4Ⳮ T cells. VIP or PACAP released from the peptidergic innervation, antigen-stimulated Th2 cells, or macrophages act on both CD4Ⳮ T cells and macrophages. (1) Action on activated macrophages: VIP/PACAP inhibit IL-12 production by activated macrophages, reducing differentiation into Th1 effectors; (2) action on APCs: differentiation into Th2 effectors depends on the expression of B7 to a much larger degree than differentiation into Th1 effectors. VIP/PACAP induce B7.2 expression on resting macrophages and promote Th2type responses. The Th2-type response is significantly reduced in the presence of neutralizing antiB7.2 antibodies; (3) action on Th1/Th2 effectors: the majority of Th1 and Th2 effectors are eliminated through apoptosis, with a small number surviving as memory T cells. VIP/PACAP promote the survival of Th2, but not Th1 effectors in vivo and in vitro; (4) the possibility exists (indicated by a question mark) that VIP/PACAP also affect Th1/Th2 differentiation directly by acting on the master-switch transcription factors GATA-3 and/or T-bet.

directly or through effects on APCs, and/or at the level of the already generated effectors, by preferentially promoting Th2 proliferation and/or survival. There is evidence that VIP and PACAP affect APCs in at least two ways that are relevant to the generation of Th1/Th2 effectors. First, VIP and PACAP inhibit IL-12 production from activated macrophages [68,69]. Since differentiation into Th1 cells is controlled primarily by the availability of IL-12, the Th1/Th2 balance will be altered in favor of Th2 in the presence of suboptimal doses of IL-12. Second, the presence of costimulatory molecules on APCs seems to be significantly more important for the development of Th2 compared to Th1 cells [70]. We demonstrated previously that VIP and PACAP induce B7.2 expression in resting macrophages [62], and recently we observed a similar phenomenon with immature dendritic cells (unpublished results). The induction of B7.2 in both macrophages and dendritic cells correlates with an increase in the stimulatory

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activity for primed T cells. In addition, resting macrophages treated with VIP or PACAP induce Th2-type cytokines in antigen-primed T cells, and this effect is abolished in the presence of neutralizing anti-B7.2 Abs, supporting the role of VIP/PACAP-induced B7.2 in promoting Th2-type responses [67]. VIP/PACAP could also affect Th1/Th2 differentiation by acting directly on T cells. Indeed, addition of VIP or PACAP to TCR-transgenic T cells cultured with irradiated APCs and antigenic peptide in the presence or absence of polarizing cytokines (IL-12 for Th1 and IL-4 for Th2 differentiation) leads to increased levels of IL-4 and decreased levels of IFN-␥, suggesting that VIP and PACAP promote the development of Th2 cells directly by acting on the differentiating CD4Ⳮ T cells (results not published). Whether VIP and PACAP affect the expression of the master transcriptional factors T-bet and GATA-3, required for Th1 and Th2 differentiation, respectively, remains to be established. Previous studies indicated that VIP and PACAP induce expression of JunB, one of the transcriptional factors required for Th2 differentiation, in both macrophages and newly stimulated T cells [64,71]. Finally, VIP/PACAP could act on the already generated Th1/Th2 effectors (Fig. 1). Both in vitro and in vivo experiments indicate that VIP and PACAP support the survival and possibly the proliferation of Th2, but not Th1 effectors [72]. In addition, the Th2 cells that survive in vivo in mice inoculated with antigen in the presence of VIP or PACAP develop markers characteristic of memory cells [72]. The reasons why VIP and PACAP specifically affect Th2 cells are not clear. Differences between Th1 and Th2 cells in VIP/ PACAP receptor density, or in the nature of transcriptional factors affecting antiapoptotic molecules, might be the answer. IV. FUNCTION OF CD4ⴐ AND CD8ⴐ T CELLS A. T-Cell Traffic and Adhesion Studies on lymphocyte migration following chronic in vivo administration of VIP established a significant inhibitory effect particularly on CD4Ⳮ T-cell migration through lymph nodes and Peyer’s patches [73–75]. The effects of VIP on T-cell adhesion and chemotaxis could play an important role in respect to the traffic patterns. A dual role for VIP in Tcell chemotaxis has been reported. In some systems, VIP can act as a T-cell chemoattractant [76–78]. However, in T cells expressing solely VPAC2, VIP can inhibit cytokine-induced chemotaxis [77]. A similar dual effect was observed for dendritic cells. VIP acts as a chemoattractant for immature DCs and inhibits chemotaxis in CCR7-expressing, mature dendritic cells (DCs) [79]. The dual effect observed both in T cells and (DCs) might result from the differential expression of VPAC1 and 2 during T-cell differentiation and DC maturation. B. CD4ⴐ T-Cell Function: Production of Cytokines 1. Th1 Cytokines: IL-2 and IFN-␥ Th1 and Th2 effectors differ in their cytokine profile and subsequently in their main functions in the immune response. IL-2 and IFN-␥ are considered typical Th1 cytokines, produced by antigen-stimulated Th1 cells. A number of studies established that VIP and PACAP inhibit IL-2 production from mitogen- and antigen-stimulated CD4Ⳮ T cells, primarily through a cAMP-dependent pathway [59,80–85]. VIP and PACAP were shown

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to reduce IL-2 gene transcription through inhibitory effects on NFAT and changes in the conmposition of the AP-1 complex, from c-Jun/c-Fos to JunB/c-Fos, with subsequent decreases in AP-1 binding and transactivating activity [64,83]. The inhibition of IL-2 production by VIP/PACAP correlates with the inhibitory effect on T-cell proliferation. In contrast to IL-2, where VIP/PACAP were unanimously reported to be inhibitory, there is less agreement regarding the VIP/PACAP effects on IFN-␥ production. Muscettola and Grasso [86] reported inhibition of IFN-␥ production by VIP in human peripheral blood mononuclear cells stimulated with a polyclonal T-cell activator, and Taylor et al. [87] showed a similar inhibitory effect in murine Ag-primed T cells reexposed to the antigen. In contrast, other in vitro studies reported a lack of VIP effect on IFN-␥ production by naı¨ve T cells stimulated through the TCR [82,84] or an increase in IFN-␥ production by antigen-specific Th1 cell lines [88]. Since these contradictory results were obtained with T cells in different stages of differentiation, from naı¨ve CD4Ⳮ T-cells to Th1 cell lines, the differential expression of VPAC1 and 2 during T-cell activation and differentiation might be the reason for the disagreement. However, what is probably more important is that in vivo administration of VIP or PACAP together with LPS results in a definite reduction in IFN-␥ in both serum and peritoneal fluid [89]. 2. Th2 Cytokines: IL-4 and IL-5 Our initial studies with naı¨ve T cells stimulated through the TCR indicate that VIP and PACAP inhibit IL-4 production at a posttranscriptional level. The VIP inhibitory effect was mediated through a reduction in IL-2, which led to a destabilization of the newly synthesized IL-4 protein [90]. Studies with established Th2 cell lines cultured in the presence of exogenous IL-2 showed no inhibitory effect of VIP or PACAP on IL-4 production (results not published). However, subsequent studies focused on T-cell differentiation into Th1/Th2 effectors demonstrated that VIP and PACAP promote Th2 differentiation, and subsequent IL-4 production [67,72]. Also, transgenic mice overexpressing the human VPAC2 receptor in CD4Ⳮ T cells have higher serum levels of IL-4 and IL-5, indicative of a prevalent Th2 differentiation [54]. Therefore, although there is no apparent effect on IL-4 production by already established Th2 cells, VIP/PACAP promote Th2 differentiation and subsequently lead to an increase in IL-4 production. VIP and IL-4 appear to be involved in an autoregulatory feedback loop (Fig. 2). In the positive feedback, IL-4–producing antigen-stimulated Th2 cells secrete VIP [19], which then promotes Th2 differentiation and survival, leading to more IL-4 production [54,72]. On the other hand, in a negative feedback, IL-4 inhibits constitutive VIP production by macrophages and reduces the levels of VPAC2 receptors on Th2, but not Th1, cells [49,58]. IL-5, an essential factor for the generation and differentiation of eosinophils, is secreted by Th2 cells. Although VIP does not affect IL-5 secretion by resting or freshly activated CD4Ⳮ T cells, it stimulates IL-5 release from activated splenic or granuloma T cells in a model of murine schistosomiasis [91,92]. Since VIP promotes Th2 differentation, the expection was that, similar to IL-4, VIP will induce an increase in IL-5 in vivo, and indeed, in VPAC2 transgenic mice there is an increase in IL-4, IL-5, and eosinophilia [54]. 3. In Vivo Consequences of the VIP Alteration of the Th1/Th2 Balance Two recent studies contributed significantly to our understanding of the in vivo effects of VIP. Transgenic mice overexpressing the human VPAC2 receptor in CD4Ⳮ T cells

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Figure 2 Positive and negative feedback in the relationship between VIP and Th2 effectors. (1) Positive feedback: Th2 effectors activated by specific antigen release VIP, which in turn increases Th2 survival and/or proliferation. (2) Negative feedback: Th2 effectors activated by specific antigen release IL-4, which inhibits VIP production by macrophages and reduces VPAC2 expression on Th2 effectors.

show increased blood IgE, IgG1, eosinophilia, and the VPAC2 overexpressing CD4Ⳮ T cells produce high levels of IL-4 and IL-5. These mice develop more severe cutaneous allergic reactions and have depressed delayed-type hypersensitivity [54]. In contrast, VPAC2-deficient mice have lower than normal IgE levels, less IL-4, and a 70% decrease in cutaneous anaphylaxis; however, these mice express enhanced delayed-type hypersensitivity [60]. These results position VPAC2 as the functionally predominant VIP/PACAP receptor in CD4Ⳮ T-cell differentiation and indicate that VPAC2 plays an essential role in vivo in tilting the Th1/Th2 balance in favor of Th2. C. Functions of Cytotoxic T Cells Although cytotoxic T cells are primarily CD8Ⳮ, a minority of the CD4Ⳮ T cells also function as cytotoxic cells. There are major differences in the mechanisms for cytotoxicity employed by the two cell types. Cytotoxic CD8Ⳮ T cells kill targets through a major calcium-dependent, perforin/granzyme-mediated mechanism, with a minor contribution from a calcium-independent, FasL-mediated pathway [93]. In contrast, CD4Ⳮ cytotoxic T cells kill targets only through FasL/Fas-mediated interactions [94,95]. In CD8Ⳮ cytotoxic T cells, VIP and PACAP do not affect the perforin/granzymemediated cytotoxicity, but inhibit drastically the FasL-mediated lysis of both allogeneic and syngeneic Fas-bearing targets. This is accomplished through the VIP/PACAP inhibition of FasL expression on the CD8Ⳮ cytotoxic T cells both in vivo and in vitro [96]. Since VIP and PACAP affect only the minor FasL/Fas-mediated lysis in CD8Ⳮ cytotoxic T cells, their overall effect on CD8Ⳮ T-cell cytotoxicity does not have major consequences. However, since cytotoxic CD4Ⳮ T cells kill solely through the FasL/Fas-mediated pathway, VIP and PACAP have a major impact on CD4Ⳮ T-cell cytotoxicity. APCs activate CD4Ⳮ T cells in a MHC II–restricted manner, leading to the upregulation of FasL, which then enables the CD4Ⳮ T cells to lyse both cognate APCs (direct

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targets) and neighboring Fas-bearing cells (bystander targets) in an antigen-independent and MHC-nonrestricted manner [97,98]. VIP and PACAP inhibit FasL expression in allogeneic and antigen-specific CD4 effectors generated in vivo and their cytotoxicity for both direct and bystander targets [99]. Elimination of activated APCs removes potentially harmful cells after the completion of an immune response, and therefore represents a required mechanism to maintain immune homeostasis. However, bystander killing results in collateral damage, particularly relevant for tissues with limited MHC II expression, such as brain. In experimental allergic encephalomyelitis (EAE), myelin-specific CD4Ⳮ T effectors contribute to the pathology through lysis of antigen-nonspecific targets [100]. Similar mechanisms may be responsible for tissue damage in other autoimmune diseases as well [101]. In this respect, endogenous agents such as VIP and PACAP capable to downregulate FasL expression on T cells may be important in the control of FasL/Fasmediated lysis of innocent bystanders, particularly in immune privileged organs such as brain and the anterior chamber of the eye, where these neuropeptides are abundant.

V. SURVIVAL AND DIFFERENTIATION OF CD4ⴐ T CELLS INTO MEMORY CELLS The majority of Th1/Th2 effectors are eliminated through apoptosis following a relatively short period of intense activity. The relatively few surviving T cells become antigenspecific memory cells, which differ from naı¨ve T cells in terms of homing patterns and activation requirements. The elimination of effector T cells occurs through either active or passive apoptosis, depending on whether the antigen persists or is eliminated. In the CNS, VIP and PACAP act as neuronal survival factors [102–106]. In the immune system however, based on the VIP/PACAP anti-inflammatory role, the expectation was that they will promote T-cell apoptosis. Contrary to the expected outcome, VIP and PACAP protect activated CD4Ⳮ T cells against active (antigen-induced) cell death (AICD), both in vitro and in vivo, through the inhibition of FasL ligand (FasL) expression [57]. This inhibition is mediated through effects on the transcription factors NF-␬B, NFATp, and Egr2,3. VIP/PACAP stabilize I␬B and subsequently prevent p65 nuclear translocation in T cells. In addition, VIP/PACAP inhibit NF-ATp nuclear translocation in a calcineurin-independent manner, affecting the expression of Egr2 and 3, which is NFATp dependent [107]. How can the VIP/PACAP anti-inflammatory effects be reconciled with the protective effect against AICD? At first glance, a higher number of surviving effector CD4Ⳮ T cells should result in higher levels of activity, hence a more intense inflammatory response. However, following the proliferative stage, CD4Ⳮ T cells differentiate into Th1 effectors primarily involved in cell-mediated immunity and Th2 effectors involved in humoral immunity. From a functional point of view, Th1 and not Th2 cells mediate acute inflammation through the mobilization and activation of potent inflammatory cells such as neutrophils and macrophages within the inflammatory site. In addition to their known functional differences, Th1 and Th2 effectors also differ in terms of susceptibility to AICD, with Th2 cells being more resistant to apoptosis [108,109]. The Th1/Th2-specific factors responsible for the different susceptibility to apoptosis are not known. VIP and PACAP might function as Th2 survival factors. This would be in agreement with the observed in vivo Th2-type immune responses upon administration of exogenous VIP/PACAP [67] or in mice overexpressing the VPAC2 receptor in CD4Ⳮ T cells [54].

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The preferential effect of VIP/PACAP on the survival of Th2 effectors in vivo and in vitro was indeed confirmed in a recent study from our laboratory [72]. Th1 and Th2 cell lines derived from TCR transgenic mice were transferred into nontransgenic thymectomized hosts, followed by immunization with the specific antigen and VIP or PACAP. Transgenic CD4Ⳮ T cells were recovered 62 days later only from hosts that received Th2 cells and VIP or PACAP. When the hosts received transgenic CD4Ⳮ T cells instead of Th1 or Th2 cell lines, the recovered transgenic T cells exhibited a phenotype typical of memory Th2 cells (CD44hi, L-selectinlo, CD45RBlo, and IL-4 and IL-5, but not IFN-␥ or IL-2 producers). The preferential survival of Th2 cells in the presence of VIP or PACAP was also confirmed in vitro, where VIP/PACAP supported the survival of Th2, but not Th1 TCR-transgenic cell lines by inhibiting antigen-induced apoptosis [72].

VI. CONCLUSIONS AND SIGNIFICANCE VIP and the structurally related neuropeptide PACAP, released in the lymphoid organs by the innervation and by activated immune cells, modulate the function of inflammatory cells through specific receptors, affecting both innate and adaptive immunity. A major player in both innate and adaptive immunity is the macrophage. Activated macrophages decrease the pathogen load through the release of cytotoxic cytokines, oxygen radicals, and nitric oxide, and through the mobilization of additional immune cells in response to macrophage-derived inflammatory chemokines. Responding to stimulatory and costimulatory signals delivered by APCs (including dendritic cells, B cells, and macrophages), CD4Ⳮ T cells proliferate and differentiate into effector Th1 and Th2 cells. At the conclusion of an immune response, both activated APCs and T cells have to be deactivated and/ or eliminated, to avoid excessive tissue and organ damage. A number of endogenous factors, particularly anti-inflammatory cytokines, contribute to the downregulation of the immune response. Neuropeptides such as VIP and PACAP can be added to the list of endogenous anti-inflammatory molecules. VIP and PACAP exert their anti-inflammatory function in several ways: (1) direct inhibition of pro-inflammatory cytokine production (TNF-␣, IL-6, IL-12) by activated macrophages; (2) upregulation of IL-10 production (a potent anti-inflammatory cytokine); (3) inhibition of B7.1/B7.2 expression in activated macrophages and dendritic cells, and subsequent inhibition of their stimulatory activity for antigen-specific T cells; (4) inhibition of IL-2 production and T-cell proliferation; (5) inhibition of Th1 responses (reduction in both the amounts of Th1 cytokines and the number of cytokine-producing Th1 cells); and (6) inhibition of FasL/Fas-mediated cytotoxicity of CD8Ⳮ and CD4Ⳮ T cells against direct and bystander targets. In contrast to these well-defined anti-inflammatory functions, VIP and PACAP support the generation and long-term survival of Th2 cells, representing the first neuropeptides with a possible role in the generation of memory Th2 cells. In immune privileged sites such as brain and the anterior chamber of the eye, this might be one of the most important physiological functions of VIP and PACAP, since the state of immune deviation depends on regulatory cells whose development requires Th2 cytokines [110,111]. In the eye the immune privilege depends on the presence of intact corneal nerves, being lost upon nerve severing and regained once the nerves regrow [112]. Therefore, it is tempting to speculate that neuropeptides such as VIP and PACAP, which are abundant in the immune privileged sites, play an essential role in maintaining immune deviation through their capacity to inhibit Th1type, and to promote Th2-type immune responses.

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15 Natriuretic Peptides and Inflammation ANGELIKA M. VOLLMARand ALEXANDRA KIEMER University of Munich, Munich, Germany

I. INTRODUCTION The discovery of the natriuretic peptide family was a breakthrough in modern cardiovascular physiology as it provided a direct link between the heart and the kidneys in the regulation of natriuresis. The atrial natriuretic peptide (ANP) has been particularly established to play an important role in the regulation of salt and water balance as well as of blood pressure homeostasis. More recently, a direct contributory role for ANP in the pathophysiology of various cardiovascular disorders became evident and changed the understanding of the physiological relevance of ANP. ANP interferes with complex intracellular signaling networks that are responsible for growth, differentiation, ion flux, or transcriptional activation. In this context the following review will focus on a rather novel aspect: ANP will be presented as an endogenous player in vascular inflammation, and the underlying signalling cascades will be communicated. A. Natriuretic Peptides In 1981 de Bold showed that intravenous injection of atrial myocardial extract causes a rapid and potent natriuretic reponse in rats [1]. This experiment lead to the discovery of the first natriuretic peptide, called atrial natriuretic peptide [2,3]. ANP is a 28-residue peptide that contains an intrachain-disulfide bond, which is critical for the biological activity. Peptides homologous to ANP have been found (Fig. 1). Brain natriuretic peptide (BNP), originally isolated from porcine brain, is produced in cardiac ventricles and shows activities similar to ANP. Both cardiac hormones counterbalance the renin-angiotensinaldosterone system [4–7]. Furthermore, ANP inhibits the hypertrophy of cardiomyocytes [8], whereas BNP inhibits pressure-induced ventricular fibrosis [9]. C-type natriuretic 305

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Figure 1 Primary structure of ANP, BNP, and CNP. Conserved residues are shaded.

peptide (CNP) is differentially expressed mainly in the nervous system and vasculature and is involved in vascular control [5]. B. Natriuretic Peptide Receptors The actions of the natriuretic peptides are modulated through their cognate receptors (Fig. 2). Three receptors have been cloned to date, known as NPR-A (GC-A), NPR-B (GC-B), and NPR-C (ANP-C) [10,11]. NPR-A exhibits ligand selectivity in the order ANP ⬎ BNP ⬎⬎ CNP and mediates most of the known actions of the natriuretic peptides. On the other hand, NPR-B shows binding selectivity in the order CNP ⬎⬎ ANP ⬎ BNP. NPR-B is thought to mediate central control of volume homeostasis. Both receptors consist of an extracellular binding domain, a single hydrophobic membrane-spanning region, an intracellular kinase homology domain (KHD), and a carboxy-terminal guanylyl cyclase domain. Binding of ANP, respectively CNP, to the extracellular binding domain stimulates the catalytic activity of the guanylyl cyclase by an as yet unknown mechanism. The NPR-C receptor consists of an extracellular domain, a single membrane-spanning region, and only 37 intracellular amino acids. It binds all three NP and controls the local concentration of

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Figure 2 Schematic illustration of the natriuretic peptide receptors. NPR-A and -B consist of an extracellular binding domain, an intracellular protein kinase domain, and a carboxy terminal guanylyl cyclase domain. NPR-C contains an extracellular binding domain and a short intracellular domain. Activation of NPR-A and NPR-B results in increased production of cGMP, whereas NPR-C can signal through a decrease of cAMP.

NP through receptor-mediated internalization and degradation and may signal through the heterotrimeric G proteins Gi and G0 and a decrease of cAMP. C. Link of ANP to the Immune System The function of ANP and the other NP in cardiovascular physiology and pathophysiology has widely been reviewed [4–7]. Evidence for an involvement of ANP in the immune system came from work demonstrating that immune organs are the site of synthesis of ANP and bear ANP receptors [12,13] (Fig. 3).

Figure 3 ANP and ANP receptors are present in various parts of the immune system.

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Importantly, expression of ANP and ANP receptors are regulated by various stimuli affecting the immune system [13,14]. For instance, involution of the thymus after dexamethasone administration results in a tremendous increase in ANP in apoptosis-activated thymic macrophages [15] (Fig. 4). Bone marrow–derived and peritoneal macrophages as well as human monocytes also respond to inflammatory stimuli by increasing synthesis and release of ANP and CNP [16–18], suggesting a role for natriuretic peptides in inflammatory situations. Besides the anti-inflammatory potential focused on in Sec. II.A, ANP has been documented to influence specifically the thymus. Thymocytes as well as thymic stromal cells possess ANP receptors, which are regulated by various agents [19,20]. ANP has been demonstrated to inhibit thymocyte development in a model of mouse fetal thymus organ culture [21]. In mature thymocytes that have been stimulated with mitogens, ANP reduces proliferation [19]. II. ANTIINFLAMMATORY ACTION OF ANP A. Effects of ANP on Inflammatory Mediators As described above, macrophages produce ANP [16], and activated macrophages show a markedly increased expression of ANP [17]. Macrophages play a crucial role in inflammatory processes. Macrophage mediators of host defense and inflammation are induced by inflammatory stimuli, such as bacterial endotoxin (lipopolysaccharide, LPS) [22]. These mediators comprise inflammatory enzymes, such as iNOS [23] and COX-2 [24], and

Figure 4 Macrophages in involuted rat thymic tissue express ANP (see Ref. 15). Rats were injected with a single dose of dexamethasone (1.5 mg/kg), resulting in a vast apoptosis of thymocytes after 4 days. Macrophages in such involuted apoptotic thymus tissue show a strong increase in expression of ANP (arrows).

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cytotoxic cytokines, such as TNF-␣ [25]. Since macrophages were shown to express all three types of NPR [26], it was suggested that they are target cells for natriuretic peptides. In fact, ANP represents a regulator of the LPS-mediated induction of inflammatory mediators in murine macrophages. This action of ANP as a regulator of macrophage activation has recently been extensively reviewed [14,27], therefore, only a brief overview of these actions of ANP is given here. ANP was shown to markedly inhibit the LPS-induced production of nitric oxide (NO). NO represents an important mediator of host defense, but excessive NO produced in high amounts by activated macrophages contributes to the pathogenesis of several inflammatory diseases [28,29]. The ANP-mediated inhibition of NO production is due to a reduced expression of inducible nitric oxide synthase (iNOS). This action is mediated via activation of the guanylyl cyclase–coupled NPR-A [26] and comprises both transcriptional as well as post-transcriptional processes [30]. The posttranscriptional regulation of iNOS involves a destabilization of iNOS mRNA [30]. Since elevated intracellular calcium levels are known to decrease iNOS mRNA stability [31], it seems interesting to know that ANP increases intracellular calcium levels in macrophages [32] and that elevated calcium was shown to contribute to the inhibitory action of ANP on NO production [32]. Importantly, ANP also reduces availability of the iNOS substrate L-arginine [33]. Besides these posttranscriptional regulatory actions, ANP was shown to markedly inhibit the activation of the transcription factor NF-␬B [30,34] which is crucial for the induction of iNOS in murine macrophages [35]. In addition to influencing induction of iNOS, ANP regulates another NF-␬B–regulated gene, i.e., the pro-inflammatory cytokine TNF-␣ [34,36]. Again, this effect is mediated via the NPR-A [36]. In contrast to these cGMP-mediated actions of ANP on macrophage activation, ANP influences the expression of the inducible cyclooxygenase (COX2) independent of cGMP [37]. An NPR-C–mediated reduction of cAMP is suggested to be involved in this regulatory action. ANP represents an endogenous compound regulating the production of inflammatory mediators in macrophages. Due to the induction of ANP production upon macrophage activation, an autocrine mechanism is suggested. The ability of ANP to inhibit the induction of iNOS, COX-2, and TNF-␣ may represent aspects supporting an anti-inflammatory action of this cardiovascular hormone (Fig. 5). B. Effects of ANP on Endothelial Inflammatory Actions Besides the action of ANP on the production of inflammatory mediators, ANP was shown to regulate inflammatory processes by controlling the action of inflammatory mediators. Endothelial cells represent central target cells for inflammatory processes, such as atherosclerosis, wherein the pro-inflammatory cytokine TNF-␣ plays a pivotal role. TNF-␣ exerts several effects that facilitate the formation of an atheromatous plaque: it increases the expression of endothelial cell adhesion molecules and chemokines, induces proliferation of smooth muscle cells, and increases endothelial cell leakiness. The following sections will describe how ANP influences key events in human endothelial cell activation induced by the pro-inflammatory cytokine TNF-␣. 1. Effect of ANP on TNF-␣–Induced Endothelial Permeability Formation of intercellular gaps in vascular endothelium is regarded as one of the initial conditions contributing to the development of an atheromatous plaque [38]. Increased

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Figure 5 ANP inhibits the LPS-induced production of inflammatory mediators in macrophages. LPS-activated macrophages produce and secrete ANP. ANP inhibits iNOS as well as TNF-␣ production via the NPR-A and cGMP, leading to a reduction of NO and TNF-␣ secreted by LPS-activated macrophages. In contrast, ANP reduces LPS-induced COX-2 expression and secretion of PGE2 via the NPR-C, probably linked to a decrease of cAMP.

vascular permeability is commonly attributed to the reorganization of F-actin filaments followed by contraction of cells and formation of intercellular gaps [39–42]. Activation of endothelial cells with TNF-␣ leads to a significant increase of macromolecule permeability. When human umbilical vein endothelial cells (HUVEC) are pretreated with ANP for 30 minutes, this TNF-␣–induced elevated permeability is significantly reduced [43] (Fig. 6). ANP also reduces the formation of stress fibers, typical bundles of F-actin occurring after TNF-␣ activation [43]. This TNF-␣–induced formation of stress fibers is associated with the polymerization of monomeric G-actin into F-actin fibers. Other studies have reported the ability of ANP to reduce thrombin- and oxidantinduced increases in endothelial permeability and to abrogate cytoskeletal changes [44,45]. The molecular mechanisms involved, however, are essentially unknown. Referring to the inhibitory action of ANP on VEGF-induced permeability increase and actin polymerization, ANP was suggested to regulate a pathway sequentially involving Src, ERK, JNK, and PI3-kinase/Akt [46]. Referring to mechanisms responsible for the influence of ANP on endothelial cytoskeleton upon TNF-␣ activation, ANP was shown to reduce the phosphorylation of the small heat shock protein HSP27 [43]. HSP27 has been closely associated with the regulation of actin polymerization [47]: phosphorylated HSP27 has been shown to significantly stabilize F-actin and thereby to increase fiber formation [47,48]. This phosphorylation of HSP27 is exerted by the mitogen-activated kinase–activated protein kinase-2 (MAPKAPK-2) and thereby represents a downstream target of the p38 mitogen activated protein kinase (MAPK) [49]. The action of ANP on HSP27 phosphorylation is associated with a reduced activation of p38 MAPK. Interestingly, this inhibition is not due to a reduced activation of MAPK kinase 3/6 (MKK3/6) upstream of p38 MAPK [49], but is exerted

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Figure 6 ANP inhibits TNF-␣–induced hyperpermeability and stress fiber formation (see Ref. 43). (A) ANP inhibits macromolecule permeability of endothelial cells induced by TNF-␣. (B) ANP pretreatment abrogates the TNF-␣–induced F-actin stress fiber formation. F-actin in quiescent HUVEC (Co) is concentrated in fine F-actin filaments transversing the cells. TNF-␣ treatment induces formation of stress fibers and contraction of stress fibers into dense microfilamentous masses. Intercellular gaps and cell retraction are indicated by large unstained areas of the image. Co-treatment of the cells with ANP inhibited TNF-␣–induced alterations in actin distribution.

via an induction of MAPK phosphatase-1 (MKP-1). This phosphatase represents a member of dual-specificity phosphatases, which target the two critical phosphorylation sites in the activation loop of MAPK [50]. Antisense experiments against MKP-1 revealed the causal involvement of MKP-1 induction in ANP-mediated inhibition of p38 MAPK (Fig. 7). 2. Effect of ANP on Leukocyte Attraction and Adhesion Activation of p38 MAPK represents a central step in endothelial activation. Therefore, effects of ANP on multiple targets downstream of p38 MAPK were suggested. Among these, the induction of chemokines leading to the recruitment of leukocytes into inflamed tissue represents a crucial pathophysiological feature of inflammatory conditions. TNF␣–induced expression of chemokines, such as monocyte chemoattractant protein-1 (MCP-

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Figure 7 Proposed pathway of ANP inhibition of TNF-␣–induced actin polymerization. ANP induces the expression of MKP-1, which leads to an increased dephosphorylation of p38 MAPK activated upon TNF-␣ treatment. The lower p38 MAPK activation results in reduced phosphorylation of the actin-capping protein HSP27 and therefore to reduced actin polymerization and macromolecule permeability.

1), has been shown to lead to progressed leukocyte extravasation to sites of inflammation [51,52]. Excessive expression of chemokines has therefore been recognized to mediate the initial steps in leukocyte recruitment in several pathophysiological conditions, such as atherosclerosis [53]. Experiments assessing the effects of ANP on TNF-␣–induced MCP-1 in fact revealed that pretreatment of HUVEC with ANP significantly reduced TNF-␣–induced expression of MCP-1 protein and mRNA (Fig. 8). These effects of ANP were shown to be mediated via the guanylyl-cyclase–coupled A-receptor since a cGMP analogue mimicked the effect of ANP on MCP-1 induction. Activation of the other guanylyl-cyclase–coupled receptor (NPR-B) by CNP as well as activation of soluble guanylyl-cyclase with GSNO exerted similar effects like ANP, supporting a role for cGMP in the signal transduction pathway. The critical involvement of p38 MAPK inhibition in MCP-1 induction was shown by antisense experiments: antisense oligonucleotides against MKP-1 completely abrogated the ANP-mediated inhibition of TNF-␣–induced expression of MCP-1. Besides recruitment of leukocytes to sites of inflammation and infection by chemokines, the adhesive properties of the endothelium play a central role in leukocyte trafficking [54]. In health, the luminal endothelial cell surface is a relatively nonadhesive conduit for the cellular and macromolecular constituents of the blood. In inflammatory processes various adhesive interactions between endothelial cells and the constituents of the blood are changed in order to recruit circulating leukocytes to sites of inflammation. During these processes, cellular adhesion molecules (CAM), such as intercellular adhesion molecule 1 (ICAM-1), vascular cell adhesion molecule 1 (VCAM-1), and E-selectin, are induced [55].

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Figure 8 ANP inhibits TNF-␣–inducible MCP-1 secretion. Upon exposure of HUVEC to TNF␣, MCP-1 secretion is induced. Thirty-minute pretreatment of cells with ANP significantly inhibits MCP-1 induction.

The regulation of CAM expression occurs at the transcriptional level and is mediated, especially for ICAM-1 and E-selectin, via the proinflammatory transcription factor NF␬B [55]. Pretreatment of TNF-␣–activated human endothelial cells with ANP markedly reduces the induction of ICAM-1 and E-selectin adhesion molecules [56] (Fig. 9A). This inhibition is associated with an attenuation of TNF-␣–induced endothelial NF-␬B activation and nuclear translocation (Fig. 9B) and seems to be mediated via cGMP and therefore via NPR-A. Interestingly, however, coronary endothelium of NPR-A knockout mice displayed a decreased P-selectin expression and reduced NF-␬B activation after myocardial infarction [57]. This suggests a stimulatory role for NPR-A referring to NF-␬B activation and induction of adhesion molecules. These diverging observations may either represent stimulus or species specific differences in the regulatory action of ANP and NPR-A, respectively. NF-␬B activation in endothelial cells is not solely linked to increased expression of cell adhesion molecules. It is known to also markedly contribute to TNF-␣–induced MCP1 expression [58]. Therefore, a dual regulatory role for ANP may account for its effects on MCP-1. In fact, the inhibition of NF-␬B activation by ANP might represent a central antiinflammatory effect exerted by the cardiovascular hormone. ANP was also shown to attenuate NF-␬B activation in other cell systems. As mentioned in Sec. II.A, ANP regulates macrophage activation by inhibiting NF-␬B activation [30,34]. Also, the protective action of ANP on hepatic ischemia reperfusion injury [59] has been associated with its inhibitory action on NF-␬B activity [60]. Referring to mechanisms involved in the inhibitory action of ANP on NF-␬B inhibition, data obtained from TNF-␣–activated endothelial cells point to the transcriptional induction of I␬B being responsible for this effect (Fig. 10). Under quiescent conditions, NF-␬B is retained in the cytosol by an inhibitory protein, I␬B. Upon stimulation, I␬B is phosphorylated, ubiquitinylated, and degraded. This process leads to liberation of the

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Figure 9 ANP reduces expression of E-selectin and ICAM-1 and attenuates nuclear translocation of NF-␬B (see Ref. 56). (A) Immunocytochemical analysis shows an abrogation of TNF-␣–induced E-selectin and ICAM-1 expression in endothelial cells by ANP. (B) Translocation of NF-␬B is visualized by immunostaining of its p65 subunit. TNF-␣–treated cells show a distinct nuclear fluorescence compared to cytosolic staining in untreated cells (Co). Co-treatment of cells with ANP reduces the number of nuclear-stained cells.

nuclear localization site of NF-␬B, which can then translocate into the nucleus (see Ref. 61 for a recent review on mechanisms of NF-␬B activation). Upregulation of I␬B by NF␬B itself is known as part of a central feedback loop pathway controlling NF-␬B activation [62,63]. In addition to this established pathway of I␬B upregulation, it has recently been shown that induction of I␬B occurs as a response to heat shock [64]. In this context it is interesting to note that ANP treatment of liver can exert a heat shock response–like state in liver [65].

Figure 10 ANP induces I␬B mRNA expression (see Ref. 56). HUVEC treated with ANP show elevated levels of I␬B-␣ mRNA as assessed by RT-PCR.

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III. SUMMARY In summary, ANP inhibits both the release as well as the action of inflammatory mediators in several in vitro cell culture models. Figure 11 shows the hypothesized action of ANP in the inflamed vasculature: ANP reduces the LPS-induced production of the inflammatory mediators iNOS, COX-2, and TNF-␣. This action is exerted in an autocrine fashion since macrophages themselves secrete ANP and LPS-activated macrophages show highly increased ANP production. Due to its inhibitory role on endothelial activation, ANP attenuates the expression of chemokines and cell adhesion molecules and thereby reduces leukocyte infiltration into inflamed tissue. Its inhibitory role on TNF-␣–induced cytoskeletal changes and macromolecule permeability increase sustains the integrity of endothelial barrier function. IV. CONCLUSION Due to its unique ability to inhibit both the production as well as the action of inflammatory mediators, the vascular peptide ANP might represent an important endogenous regulator of the inflammatory response. An antiatherogenic activity as well as a potential role of ANP in counteracting septic shock is therefore hypothesized. In order to determine such a potential physiological role of ANP in inflammatory conditions, the impact of ANP deficiency or ANP supplementation will be examined under in vivo conditions in ongoing studies.

Figure 11 Hypothesized anti-inflammatory action of ANP. Activated macrophages secrete increased amount of ANP, which has been shown to inhibit gene expression of three pivotal proinflammatory mediators, i.e., iNOS, COX-2, and TNF-␣, leading to a reduction of NO, PGE2, and TNF␣ secreted into the circulation. Furthermore, ANP inhibits endothelial cell activation by counteracting TNF-␣–induced formation of stress fibers and increase in endothelial permeability. ANP abrogates TNF-a–induced leukocyte attraction and adhesion via attenuated adhesion molecule expression and reduced production of the chemokine MCP-1.

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46. Pedram A, Razandi M, Levin ER. Deciphering VEGF/VPF signaling to vascular permeability: Inhibition by atrial natriuretic peptide. J Biol Chem:In press. 47. Landry J, Huot J. Modulation of actin dynamics during stress and physiological stimulation by a signaling pathway involving p38 MAP kinase and heat- shock protein 27. Biochem Cell Biol 1995; 73:703–707. 48. Razandi M, Pedram A, Levin ER. Estrogen signals to the preservation of endothelial cell form and function. J Biol Chem 2000; 275:38540–38546. 49. Obata T, Brown GE, Yaffe MB. MAP kinase pathways activated by stress: the p38 MAPK pathway. Crit Care Med 2000; 28:N67–N77. 50. Chen P, Hutter D, Yang X, Gorospe M, Davis RJ, Liu Y. Discordance between the binding affinity of mitogen-activated protein kinase subfamily members for MKP-2 and their ability to catalytically activate the phosphatase. J Biol Chem 2001; 276:29440–29449. 51. Robinson EA, Yoshimura T, Leonard EJ, Tanaka S, Griffin PR, Shabanowitz J, Hunt DF, Appella E. Complete amino acid sequence of a human monocyte chemoattractant, a putative mediator of cellular immune reactions. Proc Natl Acad Sci USA 1989; 86:1850–1854. 52. Goebeler M, Gillitzer R, Kilian K, Utzel K, Brocker EB, Rapp UR, Ludwig S. Multiple signaling pathways regulate NF-kappaB-dependent transcription of the monocyte chemoattractant protein-1 gene in primary endothelial cells. Blood 2001; 97:46–55. 53. Reape TJ, Groot PH. Chemokines and atherosclerosis. Atherosclerosis 1999; 147:213–225. 54. Gimbrone MA, Nagel T, Topper JN. Biomechanical activation: an emerging paradigm in endothelial adhesion biology. J Clin Invest 1997; 100:S61–S65. 55. Collins T, Read MA, Neish AS, Whitley MZ, Thanos D, Maniatis T. Transcriptional regulation of endothelial cell adhesion molecules: NF-kappa B and cytokine-inducible enhancers. FASEB J 1995; 9:899–909. 56. Kiemer AK, Weber NC, Vollmar AM. Induction of IkappaB: atrial natriuretic peptide as a regulator of the NF-kappaB pathway. Biochem Biophys Res Commun 2002; 295:1068–1076. 57. Izumi T, Saito Y, Kishimoto I, Harada M, Kuwahara K, Hamanaka I, Takahashi N, Kawakami R, Li Y, Takemura G, Fujiwara H, Garbers DL, Mochizuki S, Nakao K. Blockade of the natriuretic peptide receptor guanylyl cyclase-A inhibits NF-kappaB activation and alleviates myocardial ischemia/reperfusion injury. J Clin Invest 2001; 108:203–213. 58. Denk A, Goebeler M, Schmid S, Berberich I, Ritz O, Lindemann D, Ludwig S, Wirth T. Activation of NF-kappa B via the Ikappa B kinase complex is both essential and sufficient for proinflammatory gene expression in primary endothelial cells. J Biol Chem 2001; 276: 28451–28458. 59. Gerbes AL, Vollmar AM, Kiemer AK, Bilzer M. The guanylate cyclase-coupled natriuretic peptide receptor: a new target for prevention of cold ischemia-reperfusion damage of the rat liver. Hepatology 1998; 28:1309–1317. 60. Kiemer AK, Vollmar AM, Bilzer M, Gerwig T, Gerbes AL. Atrial natriuretic peptide reduces expression of TNF-alpha mRNA during reperfusion of the rat liver upon decreased activation of NF-kappaB and AP-1. J Hepatol 2000; 33:236–246. 61. Ghosh S, Karin M. Missing pieces in the NF-kappaB puzzle. Cell 2002; 109(Suppl):S81–S96. 62. Bourke E, Kennedy EJ, Moynagh PN. Loss of Ikappa B-beta is associated with prolonged NF-kappa B activity in human glial cells. J Biol Chem 2000; 275:39996–40002. 63. Auwardt RB, Mudge SJ, Chen C, Power DA. Inhibition with antisense oligonucleotide suggests that IkappaB-alpha does not form a negative autoregulatory loop for NF-kappaB in mesangial cells. Exp Nephrol 2000; 8:144–151. 64. DeMeester SL, Buchman TG, Qiu Y, Jacob AK, Dunnigan K, Hotchkiss RS, Karl I, Cobb JP. Heat shock induces IkappaB-alpha and prevents stress-induced endothelial cell apoptosis. Arch Surg 1997; 132:1283–1287. 65. Kiemer AK, Gerbes AL, Bilzer M, Vollmar AM. The atrial natriuretic peptide and cGMP: novel activators of the heat shock response in rat livers. Hepatology 2002; 35:88–94.

16 Neuroendocrinology of the Thymus WILSON SAVINO Oswaldo Cruz Foundation, Rio de Janeiro, Brazil MIREILLE DARDENNE CNRS FRE 2444, Hoˆpital Necker, Paris, France

In the context of the cross-talk between the neuroendocrine and immune systems, increasing evidence shows that the physiology of the thymus is modulated by hormones and neuropeptides. Herein, we discuss examples of such control in terms of neuroendocrine influence upon distinct cell types of the organ. Before discussing these data, it is worthwhile to briefly comment on the process of intrathymic T-cell differentiation in the context of the thymic microenvironment. I. THE THYMIC MICROENVIRONMENT AND ITS ROLE IN T-CELL DIFFERENTIATION The thymus gland is a central lymphoid organ in which bone marrow–derived T-cell precursors undergo differentiation, eventually leading to migration of mature thymocytes to the T-cell–dependent areas of peripheral lymphoid organs [1]. This process involves sequential expression of various membrane proteins and rearrangements of the T-cell receptor (TCR) genes. Accordingly, the most immature thymocytes do not express the TCR/CD3 complex nor the accessory molecules CD4 or CD8, being called double-negative thymocytes. Thymocyte maturation progresses with acquisition of both CD4 and CD8 markers, generating the CD4ⳭCD8Ⳮ double-positive thymocytes. These cells are the most common in the thymus, comprising 80% of total thymocytes. In this stage, TCR genes are rearranged, and the resulting TCR heterodimer is expressed in low density on the cell membrane. Thymocytes that do not succeed to yield a productive TCR gene 319

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rearrangement die by default, through apoptosis. By contrast, those expressing productive TCR will interact with peptides presented by molecules of the major histocompatibility complex (MHC), expressed on the membranes of microenvironmental cells. This interaction will determine the positive and negative selection events, crucial for normal thymocyte differentiation. Positively selected thymocytes progress to the mature CD4ⳭCD8ⳮ or CD4ⳮCD8Ⳮ single positive stage, express high densities of the TCR/CD3 complex, and will ultimately leave the organ to form the large majority of the T-cell repertoire in the periphery of the immune system [1,2]. Thymocyte differentiation (Fig. 1) occurs as cells migrate within the thymic lobules: immature thymocytes (CD3ⳮCD4ⳮCD8ⳮ and CD3ⳭCD4ⳭCD8Ⳮ cells) are cortically located, whereas mature CD3ⳭCD4ⳭCD8ⳮ and CD3ⳭCD4ⳮCD8Ⳮ thymocytes are found in the medulla [1]. Along their journey within the organ, thymocytes interact with components of the thymic microenvironment, a tridimensional network formed of epithelial cells (TEC), the major cell type, macrophages, dendritic cells, fibroblasts,as well as extracellular matrix (ECM) [1].

Figure 1 Intrathymic T-cell development and the thymic microenvironment. This scheme is a simplified model of intrathymic differentiation and migration, also seen in the context of thymic microenvironment. The left panel depicts a typical intrathymic differentiation process. Bone marrow–derived T-cell precursors enter the thymus through blood vessels and migrate towards the subcapsular cortex. At this stage they begin their differentiation, being cells that do not express the T-cell receptor (TCR) or the accessory molecules CD4 and CD8. As differentiation progresses, they expand, and those that suceed in expressing productive TCR rearrangements become TCRⳭCD4ⳭCD8Ⳮ thymocytes, which are located in the cortex of the lobules. These cells are exposed to selection events through interaction with the thymic microenvironment. Positively selected thymocytes migrate to the medulla and change to the mature phenotypes TCRⳭCD4ⳭCD8ⳮ or TCRⳭCD4ⳮCD8Ⳮ thymocytes, which will eventually leave the organ. The right panel schematically depicts a thymic lobule, showing thymocytes intermingled with a heterogeneous cellular network, the thymic microenvironment, composed of epithelial cells, dendritic cells, macrophages, and fibroblasts. The epithelial tissue shows morphological heterogeneity that can be seen in subseptal/ subcapsullary, cortical, and medullary regions. In the cortex, we note a particular lymphoepithelial complex, the thymic nurse cell. (Modified from Refs. 3 and 6.)

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The thymic epithelium is a heterogeneous tissue, and one cortically located lymphoepithelial complex, the thymic nurse cell (TNC), can be isolated in vitro. TNCs are formed by one TEC, which in mice can harbor 20–200 thymocytes. In culture, TNCs spontaneously release thymocytes, and TNC-derived epithelial cells can reconstitute lymphoepithelial complexes after being co-cultured with fetal thymocytes, thus placing TNCs as an in vitro model of thymocyte migration within the TEC context [3]. Several interactions occur between thymocytes and microenvironmental cells. One involves the TCR/CD3 complex, expressed by differentiating thymocytes, with class I or class II MHC products on the microenvironmental cell membranes, complexed with a given endogenous peptide to be recognized, in the context of CD8 or CD4 molecules, respectively. Such interaction is crucial for defining negative and positive selection of developing thymocytes [1]. Thymic microenvironmental cells also modulate thymocyte behavior by means of soluble polypeptides, including typical cytokines, chemokines, typical thymic hormones, and a number of other ‘‘classical’’ hormones, including hypothalamic and pituitary hormones. Interleukin-7 (IL-7), for example, is crucial for thymocyte differentiation: both IL-7ⳮ/ⳮ as well as IL-7 receptor–deficient mice show severe reduction in lymphoid development [4]. Among chemokines, CXC12 (stromal cell derived factor-1␣ or SDF1␣) is highly expressed in the thymus, being produced by epithelial cells, particularly in the cortex [5]. In keeping with this topography, CXCL12 preferentially attracts immature CD4ⳮCD8ⳮ and CD4ⳭCD8Ⳮ thymocytes. Together with ECM-mediated interactions (see below), chemokines drive thymocyte migration [6]. Besides producing cytokines/chemokines, TEC secrete thymic hormones, including thymosin-␣1, thymopoietin, and thymulin, that can also act upon the general process of thymocyte maturation. Thymulin, for instance, is a zinc-coupled nonapeptide able to enhance thymocyte proliferation and to induce several T-cell markers and functions [6]. In addition, the thymic microenvironment (particularly TEC) produces a variety of ‘‘classical’’ hormones, including those of the hypothalamus/pituitary axis, such as corticortropinreleasing hormone (CRH), corticotropin (ACTH), and growth hormone (GH), among others [6]. Yet, as discussed below, the role of these intrathymically produced hormones is not fully understood at the moment. A list of thymic epithelial cell–derived soluble peptides can be seen in Table 1. Moreover, TEC/thymocyte interactions, particularly those involved in cell migration–related events, can be mediated by ECM ligands and receptors. TEC/adhesion is enhanced by ECM proteins, such as laminin and fibronectin, and can be abrogated by antibodies specific to these molecules or to their corresponding integrin-type receptors [7,8]. Similarly, intra-TNC lymphocyte traffic can be blocked by anti-ECM or anti-ECM receptor reagents [9,10]. In fact, we recently proposed that a combined action of chemokines and ECM would drive intrathymic T-cell migration [3]. Having established this background, we present in the following sections some aspects of the thymus physiology that are under neuroendocrine control. II. EXPRESSION OF RECEPTORS FOR HORMONES AND NEUROPEPTIDES BY THYMIC CELLS In the context of neuroendocrine control of the thymus, the very first point to be established is the expression of receptors for hormones and neuropeptides by thymic cells. A nonexaustive list of these is provided in Table 2, and includes receptors for adrenal and sex

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Table 1 Production of Soluble Peptides Produced by Thymic Epithelial Cells Molecules Thymic hormones Thymulin Thymopoietin Thymic humoral factor-␥2 Thymosin-␣1 “Classical” hormones Growth hormone Prolactin Oxytocin Vasopressin Corticotropin Somatostatin Glucagon Neuropeptides Growth hormone–releasing hormone Corticotropin–releasing hormone Thyrotropin–releasing hormone Luteinizing hormone–releasing hormone Vasoactive intestinal peptide Cytokines and growth factors Interleukin-1 Interleukin-3 Interleukin-7 Interleukin-8 Granulocyte colony-stimulating factor Granulocyte-macrophage colony-stimulating factor Transforming growth factor-␣ Transforming growth factor-␤ Leukemia inhibitory factor Stem cell factor Insulin-like growth factor-I Insulin-like growth factor-II Chemokinesb CXCL9 (monokine induced by interferon-␥) CXCL10 (interferon-inducible protein 10) CXCL11 (interferon-inducible T-cell ␣-chemoattractant) CXCL12 (stromal cell–derived factor-1␣) CCL19 (macrophage inflammatory protein-3␤) CCL21 (secondary lymphoid tissue chemokine) CCL22 (macrophage-derived chemokine) CCL25 (thymus-expressed chemokine) a

Distributiona C-M C-M C-M C-M C-M nd C-M C-M C M M nd nd nd nd C-M C-M C-M C nd C-M C-M nd C-M C-M C C-M C-M C-M M C-M C-M M M M C-M

C: cortex; M: medulla; C-M: cortex and medulla; nd: not determined. Chemokines are named according to the international nomenclature, with the corresponding common names in parentheses.

b

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Table 2 Expression of Receptors for Hormones and Neuropeptides in Thymic Cells Molecules Steroid and thyroid hormones Glucocorticoids Estradiol Progesterone Testosterone Triiodothyronine Hormones of the hypothalamus-pituitary axis Growth hormone Prolactin Corticotropin Oxytocin Vasopressin Growth hormone–releasing hormone Corticotropin-releasing hormone Pancreatic hormones and neuropeptides Insulin Somatostatin Glucagon Vasoactive intestinal peptide ␤-Endorphin Insulin-like growth factor-I Insulin-like growth factor-II

Thymocytes

Thymic epithelial cells

⫹ ⫹ – ⫹ ⫹

⫹ ⫹ ⫹ ⫹ ⫹

⫹ ⫹ nd ⫹ ⫹ ⫹ –

⫹ ⫹ ⫹ nd nd nd ⫹

– ⫹ nd ⫹ ⫹ ⫹ ⫹

⫹/⫺ ⫹ nd ⫹ ⫹ ⫹ ⫹

nd: not determined; ⫹/⫺: conflicting results.

steroids, thyroid and pituitary hormones, as well as neuropeptides, which have been detected not only in thymocytes but also in TEC. For example, the expression of glucocorticoid receptors by thymocytes has been defined by ligand binding, immunocytochemistry, cytofluorometry, immunoblotting, and molecular biology [11–15]. Interestingly, the recent availability of green fluorescent protein–glucocorticoid receptor knockin mice revealed that the level of glucocorticoid receptor varies along with thymocyte differentiation, being higher in double CD4ⳮCD8ⳮ cells [16]. In addition to thymocytes, this receptor has been also detected in TEC [17,18]. Accordingly, in mice bearing a partial knockout of the glucocorticoid receptor, the thymic microenvironment is altered, and large TEC-free areas were seen in adult animals [19,20]. We identified nuclear receptors for triiodothyronine (T3) in murine TEC and thymocytes. More recently, this notion was enlarged to human TEC, as well nonepithelial phagocytic cells of the thymic microenvironment [21]. Prolactin (PRL) and GH receptors have also been identified in both TEC and thymocytes [22–27]. Additionally, intrathymic expression of receptors for various neuropeptides has been shown in thymocytes, with fewer studies carried out in TEC and other components of the thymic microenvironment. This is the case for oxytocin, vasopressin, and GH-RH receptors [28–30]. By contrast, the somatostatin receptor family has been identified in mouse thymocytes as well as in human TEC [31–34]. In this respect, both somatostatin and its analog octreotide inhibited human TEC proliferation in vitro [33].

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The expression of receptors for neuropeptides has also been demonstrated in the thymus. For example, the mRNA coding for the VIP1 receptor was detected in thymocytes [35], particularly those bearing the most immature phenotype, the CD4ⳮCD8ⳮ cells [36]. More recently, it was also identified in human TEC [37,38]. Given the constitutive expression of various hormone receptors in thymic cells, one can expect that diverse cell functions can be hormonally influenced, as regards both lymphoid and microenvironmental compartments. III. HORMONES AND NEUROPEPTIDES IN INTRATHYMIC CELL PROLIFERATION AND DEATH As mentioned above, cell proliferation and death are necessary for normal thymocyte development. A relatively extensive literature indicates that both events can be influenced by hormones and neuropeptides [6]. Herein, we will develop this concept through some examples. It has been showed that GH exhibits an in vitro co-mitogenic activity when applied in conjunction with concanavalin A or anti-CD3 antibodies [39]. In addition, injections of GH3 cells (a pituitary adenoma able to produce GH and PRL) to old rats could reverse the age-dependent thymic atrophy with a consequent increase in thymocyte numbers [40]. In the same vein, patients treated with GH exhibited an increase in thymus size, as revealed by computer tomography [41]. Furthermore, transgenic mice overexpressing GH or GH-releasing hormone (GHRH) show an overgrowth of the thymus [42]. Interestingly, that thymocyte-derived GH-induced proliferation in thymocyte suspensions seems to be IGF-I dependent [43], which is in keeping with the fact that IGF-I per se increases total thymocyte numbers [44]. Recent data derived from studies on fetal thymus organ cultures revealed that somatostatin was able to enhance thymocyte proliferation in this in vitro system. By contrast, this neuropeptide diminished proliferation of isolated thymocytes after mitogenic stimulation with concanavalin A or IL-2 [34]. This apparent paradox remains to be understood, but it may be due to the presence of the thymic microenvironment in organ cultures, thus allowing a cross-talk of the lymphoid and nonlymphoid compartments during the biological response to somatostatin. In addition to potentiating thymocyte growth, hormones such as PRL, GH, and IGFI stimulate TEC proliferation, as ascertained in vitro [45–47]. Interestingly, metaclopramide, a substance that promotes hyperprolactinemia, increases the number of solid epithelial islands in adult rat thymuses [48]. Intrathymic cell death is also hormonally controlled. It is known since long time that glucocorticoids are able to induce apoptosis in developing thymocytes [49]. These hormones activate calcium-dependent endonucleases that eventually cleave DNA, with the formation of oligo-nucleosomes, an event preceded by enzymatic degradation of the nuclear skeleton protein lamin B1 [50]. Accordingly, thymocytes from animals whose glucocorticoid receptor was deleted or point-mutated do not undergo apoptosis after the respective hormonal treatment [51,52]. In addition to glucocorticoids, sexual steroids can induce apoptosis in thymocytes, and one natural example is the thymic atrophy occurring along with pregnancy [6]. Although less studied, protection from apoptosis may also be under neuroendocrine control. Using the rat lymphoma Nb2 cell line, it was demonstrated that the apoptotic effect of dexamethasone was inhibited by PRL or GH [53], raising the hypothesis that a similar effect may occur with normal thymocytes. Additionally, the vasoactive intestinal peptide (VIP) appears to protect thymocytes from apoptosis induced by dexamethasone

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in vitro by inhibiting DNA fragmentation [54]. Such protective effect was also seen for other neuropeptides, substance P, and somatostatin [34,55]. Overall, the data discussed briefly above clearly point to a neuroendocrine regulation of thymocyte expansion and death, with obvious consequences upon intrathymic T-cell differentiation. IV. HORMONAL MODULATION OF INTRATHYMIC T-CELL DIFFERENTIATION One example of hormonal influence upon thymocyte differentiation is GH. Implants of GH3 pituitary cells in aging rats increased total thymocyte numbers and the percentage of CD3-bearing cells, with a parallel decrease in the CD4ⳮCD8ⳮ double-negative thymocytes, which normally accumulate in the aging rat thymus [40,56]. Furthermore, in GHdeficient dwarf mice, there is a progressive thymic hypoplasia with decreased numbers of CD4ⳭCD8Ⳮ double-positive thymocytes; a defect that could be restored by prolonged treatment with GH [57]. Recently, it has been shown that addition of exogenous somatostatin in fetal thymus organ culture enhanced the progression from CD4ⳮCD8ⳮ to CD4ⳭCD8Ⳮ stage of thymocyte differentiation [34]. In a second vein, mice treated with estradiol exhibited an increase in the percentages of CD4ⳮCD8ⳮTCRⳭ thymocytes expressing V␤6, V␤8, or V␤11 but not V␤3 gene products [58], thus promoting an imbalance in the generation of the TCR repertoire of the double-negative TCRⳭ cell lineage. More recently, release of autoreactive T cells bearing the V␤3 or V␤11 phenotypes, with autoreactivity to hepatocytes, was seen in estradiol-treated mice [59], suggesting that this sexual steroid can modulate the shaping of the T-cell repertoire. Lastly, a series of data strongly suggests that intrathymically produced glucocorticoids influence the generation of the T-cell repertoire in the thymus. Since low or moderate doses of glucocorticoids prevent anti-CD3-induced apoptosis, it was hypothesized that thymus-derived glucocorticoids might play a physiological role in positive selection of the T-cell repertoire. Addition of metirapone (a selective inhibitor of corticosteroid biosynthesis) to fetal thymus organ cultures enhanced TCR-mediated thymocyte deletion, and this effect could be reversed by addition of corticosterone to the cultures [60]. Using fetal thymus organ cultures from mice bearing transgenic ␣␤TCRs, it was shown that thymusderived glucocorticoid hormones prevent thymocyte apoptosis only when the TCR is capable of recognizing the self antigen/MHC complex with sufficient avidity to normally undergo positive selection [61]. These findings indicate that endogenous glucocorticoids prevent thymocyte apoptosis after TCR/peptide-MHC interaction. Nonetheless, this idea was challenged by data showing that thymocyte development and selection occur normally in glucocorticoid receptor–deficient mice [62]. V. NEUROENDOCRINE CONTROL OF CYTOKINE AND THYMIC HORMONE PRODUCTION Hormones and neuropeptides can modulate the production of cytokines and hormones by thymic microenvironmental cells. For example, it has been shown that the in vitro production of IL-1␣ and IL-1␤ by bovine thymic microenvironmental cells is increased by exogenous GH and by PRL [63]. These hormones also upregulate IL-6 secretion in vivo. In a second vein, Martens and colleagues [64] showed that the production of IL-6 and LIF by cultured human TEC was enhanced when monoclonal antibodies to oxytocin were added

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to the cultures, suggesting that the secretion of these TEC-derived cytokines is partially under the control of oxytocin. Although data are still lacking in terms of thymic cells, it has been recently demonstrated that IL-7 receptor expression by peripheral lymphocytes is enhanced following glucocorticoid treatment, with consequent enhancement of IL-7 driven signaling and function [65]. The concept of neuroendocrine control of thymic hormone secretion has been particularly developed with regard to thymulin, a zinc-coupled nonapeptide strictly produced by TEC [6]. Table 2 summarizes the data revealing that a variety of hormones and neuropeptides can modulate thymulin secretion in vitro and/or in vivo. Such an influence can be exemplified by the data showing that fluctuations in GH levels modulate thymulin secretion (reviewed in Refs. 6 and 66): dwarf mice as well as GH-deficient children exhibit low thymulin serum levels, as compared to age-matched controls, whereas GH treatment consistently restored this thymic endocrine function. On the other hand, GH transgenic animals exhibit higher thymulin serum levels as compared to age-matched wild offsprings (unpublished). In the same vein, abnormally high thymulin serum titers are observed in acromegalic patients and can also be restored to normal values after appropriate treatment. VI. EXTRACELLULAR MATRIX–MEDIATED TEC/THYMOCYTE INTERACTIONS In addition to modulating TEC-derived secretory soluble peptides, hormones can alter ECM production and expression of corresponding receptors by TEC, affecting ECMmediated TEC/thymocyte interactions. During the 1990s we showed that the intrathymic deposition of ECM proteins as well as their production could be upregulated by a variety of hormones including glucocorticoids [67], triiodothyronine [68], as well as PRL, GH, and IGF-I [69]. In vitro we showed a hormone-dependent increase in ECM receptors by TEC, including VLA-5 and VLA-6 [68,69]. More recently we demonstrated that long-term in vivo treatment of mice with T3 resulted in an increase in the expression of fibronectin and laminin receptors by thymocytes [21]. Since thymocyte/TEC adhesion is at least partially mediated by ECM ligands and receptors, we tested various hormones for their ability to modulate such heterotypic cellular interaction. All enhanced the degree of thymocyte adhesion to cultured TEC. Furthermore, regarding pituitary hormones, the hormone-induced enhancement of TEC/thymocyte adhesion was abrogated by monoclonal antibodies specific for each hormone or its corresponding receptor, and also by various anti-ECM or anti-ECM receptor antibodies [68]. VII. NEUROENDOCRINE CONTROL OF THYMOCYTE TRAFFIC Very few studies have been published with respect of a hormonal control T-cell precursor entrance into the thymus. Nevertheless, it was showed that human GH increases human T-cell engraftment into the thymus of SCID mice, an effect that seems to be partially ECM-mediated, since it can be abrogated with anti-␤1-integrin antibodies, and GH-treated cells exhibit an increase in adhesion to fibronectin [70]. In the same vein, IGF-I potentiates the colonization of fetal thymus organ cultures with T-cell precursors, indicating that IGFI favors the entrance of T-cell precursors into the organ [71]. Concerning intrathymic T-cell transit, we showed that thyroid and pituitary hormones enhanced TEC/thymocyte interactions related to cell migration, as, for example, TEC/ thymocyte adhesion. As mentioned above, the involvement of ECM-mediated interactions

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was demonstrated since the hormonal effects on TEC/thymocyte adhesion was abrogated by anti-ECM and anti-ECM receptor antibodies [21,68,69]. Interestingly, we further showed that thymocyte adhesion to nonepithelial microenvironmental cells could be enhanced by T3, also via cell-matrix interactions [21]. We studied thymocyte migration by evaluating the entrance of thymocytes into and their exit from TNCs. Thymocyte release from TNC complexes was accelerated in the presence of T3, PRL, GH, or IGF-I [68,69]. Furthermore, thymocyte release was faster when TNCs were harvested from T3-treated mice [68]. In vitro studies also revealed that TNC reconstitution was increased if cultures were subjected to PRL, GH, or IGF-I [69]. A further strategy to study thymocyte migration in vitro is the use of transwell chambers, which allow cells to migrate through a filter towards a given stimulus. Using this methodology, it was showed that thymocyte migration was stimulated by somatostatin, in levels comparable to those resulting from CXCL12 stimulation, a chemokine well known for its high ability to induce migrations of these cells [34]. One direct way to evaluate thymocyte export in adult animals is analysis of the socalled recent thymic emigrants. Intrathymic injection of fluorescein isothiocyanate randomly labels the cell membranes of many thymocytes, allowing recovery of the FITCⳭ

Figure 2 Endocrine, paracrine/autocrine intrathymic circuits involving growth hormone and insulin-like growth factor-1. Pituitary gland–derived GH can stimulate thymocytes and thymic epithelial cells, triggering the production of IGF-I, which in turn can modulate various cell functions. Circulating IGF-I also affects thymic cells. In both cases, an endocrine pathway (E) is involved. Since both cell types can produce GH, it is conceivable that paracrine (P) and/or autocrine (A) circuits involving intrathymically produced GH and IGF-I play a role in thymus physiology. BV: blood vessel; GHR: growth hormone receptor; IGF-I-R: insulin-like growth factor-I receptor.

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cells that have been recently exported from the thymus [72]. We showed that 16 hours following a single intrathymic injection of T3 there was a consistent increase in the numbers of FITCⳭ cells in lymph nodes [73]. Using a similar protocol, we found that GH could modulate the homing of recent thymic emigrants, enhancing the numbers of FITCⳭ cells in the lymph nodes and diminishing them in the spleen [74].

VIII. GROWTH HORMONE/IGF-I ENDOCRINE/PARACRINE CIRCUITRY: AN EXAMPLE OF COMPLEXITY IN THE NEUROENDOCRINOLOGY OF THE THYMUS Throughout this chapter, we provided examples for the hormonal influence upon distinct aspects of thymus physiology, a notion that is validated by the discovery of corresponding specific receptors expressed by thymic cells. However, the demonstration of intrathymic production of ‘‘classical hormones’’ clearly raises the possibility that the neuroendocrine control of the thymus is actually a resultant vector of simultaneous and/or sequential paracrine/endocrine action of hormones upon thymic cells. This rather ‘‘complicated’’ notion can be exemplified by the GH/IGF-I circuitry that occurs within the organ. As discussed above, a series of data indicates that IGF-I is involved in the effects of GH upon the thymus: thymulin secretion, ECM production, and TEC/thymocyte adhesion can be enhanced by GH and IGF-I, and the GH effect can be blocked by adding antiIGF-I or anti-IGF-I receptor antibodies [46,69]. Additionally, exogenous as well as thymusderived GH promote thymocyte proliferation via IGF-I production [43,75]. Moreover, the enhanced concanavalin A mitogenic response and IL-6 production by thymocytes observed in GH-treated aging animals [76] can be detected in animals treated with IGF-I [77]. This is in keeping with the demonstration of IGF-I receptors in thymocytes [78,79]. In a second vein, we confirmed the constitutive expression of IGF-I and IGF-I receptor by murine and human TEC, as ascertained by immunocytochemistry, immunoblot, immunodot, and RTPCR analyses. Interestingly, their densities can be increased following GH treatment [80]. 䉴 Figure 3 Pleiotropic nature of the neuroendocrine control of the thymus. In this schematic representation, neuroendocrine stimuli are provided by spinal cord–derived nerve endings, hypothalamus-pituitary axis, as well as corresponding target endocrine glands (seen in the upper part of the scheme). In the thymic tissue (botton part of the scheme), neurotransmitters, hormones, and neuropeptides can act directly on thymocytes, modulating their proliferation rate and differentiation degree. Such effects can also occur indirectly via changes in the behavior of the thymic microenvironment, exemplified herein by TEC. Accordingly, thymic hormone and cytokine production, as well as expression of ECM ligands and receptors, can be hormonally regulated, with consequent effects on proliferation, differentiation, and migration of thymocytes. Additionally, the neuroendocrine control of cell migration in the thymus might also occur through the modulation of chemokine secretion by microenvironmental cells (dashed lines). ACTH: corticotropin; AVP: arginine-vasopressin; CRH: corticotropin-releasing hormone; ECM: extracellular matrix; ECM-R: extracellular matrix receptor; FSH: folicle-stimulating hormone; GH: growth hormone; GH-RH: growth hormon–releasing hormone; LH: luteinizing hormone; LH-RH: luteining hormone–releasing hormone; NH: neurohypophysis; OT: oxytocin; PRL: prolactin; RG: dorsal root ganglion; T3: triiodothyronine; T4: thyroxin; TEC: thymic epithelial cells; TNC: thymic nurse cells; TRH: thyrotropinreleasing hormone: TSH: thyrotropin. (Modified from Refs. 6 and 86.)

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Taken together, these data indicate that, at least part of the GH effects in the thymus occurs through an IGF-I–mediated circuitry, as illustrated in Fig. 2. IX. IS NEUROENDOCRINE CONTROL MANDATORY FOR NORMAL THYMUS DEVELOPMENT? The various aspects discussed above quite strongly point to a role for hormones and neuropeptides in regulating various aspects of thymic cell biology, both in vitro and in normal adult life. Yet, a putative role in thymus development has been much less investigated. The possibility of building up genetically engineered mice, either for the hormone biosynthesis or the expression of the corresponding receptor allows a direct in vivo approach to this issue. For example, it has been demonstratred that, T3-receptor knockout mice do not exhibit the typical increase in thymocyte numbers that occurs physiologically in the first month of life. Yet, thymocyte differentiation in these animals does occur, with the generation of mature CD4 and CD8 single-positive cells and export to the peripehry. Nevertheless, in the periphery, the total numbers of T cells is reduced as compared to the age-matched wild-type controls [81]. In a second vein, deletion of estrogen receptor-alpha led to hypoplasia of both thymus and spleen, with a higher frequency of immature double CD4ⳭCD8Ⳮ thymocytes [82]. Very few data are available on the thymus from GH transgenic mice or GH receptor knockout mice. Yet, preliminary findings suggest that, although in both cases thymopoiesis progresses from CD4ⳮCD8ⳮ to CD4 and CD8 simple positive cells, the thymic microenvironment is altered (Smaniotto et al., manuscript in preparation). Overall, these data illustrate the notion that, if hormones are not essential for the thymus to develop, they seem to be mandatory for normal thymus development. Nevertheless, this concept may be not universal for all hormones known to be effective in modulating thymic functions. Studies conducted in prolactin receptorⳮ/ⳮ as well as IGF-1ⳮ/ⳮ mice revealed that the relative numbers of CD4/CD8-defined thymocyte subsets appear normal, thus suggesting that thymocyte development can occur in the absence of prolactinmediated interactions and of IGF-1 [83,84]. Yet, in these studies several other parameters regarding both the lymphoid and the microenvironmental compartments were not dissected. Therefore, we cannot disregard the possibility that some thymic functions are altered in these animals. X. CONCLUDING REMARKS The findings discussed herein clearly indicate that the thymus is physiologically under neuroendocrine control (see Fig. 3). However, such control seems to be far more complex, with possible in situ production of these mediators, as well as the influence of neurotransmitters [85], an aspect that was not discussed herein. Another still unresolved question is how hormone-mediated paracrine circuits are regulated. Yet, independent of which pathway(s) is triggered, the neuroendocrine control of the thymus comprises modulation of genes in different cell types. In this respect, whether or not the neuroendocrine control of thymocyte migration involves modulation of chemokine-mediated interactions remains unknown and certainly represents a promising field for investigation. One major function of the thymus is to generate a T-cell repertoire, bearing simultaneously diversity but not autoreactivity. Thus, precise knowledge as to what extent such a process is under neuroendocrine control is crucial. Phenotypic analysis

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of the V␤ and V␦ gene rearrangements in thymocytes derived from fetal thymus organ cultures subjected to distinct hormones or neuropeptides will be of interest. Lastly, the increasing use of knockout mice as well as animals in which hormonespecific transgenes are coupled to thymus-specific promoters may also be useful in further determining the relative role of each hormone or neuropeptide in the general process of shaping the T-cell repertoire.

ACKNOWLEDGMENTS The authors thank Mrs. Martine Netter for computer drawings. This work was partially supported by Capes (Brazil), as well as Fiocruz/Inserm and Capes/Cofecub Brazil/France conjoint Programs.

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17 The Central Role of the Thymus in the Development of Self-Tolerance and Autoimmunity in the Neuroendocrine System VINCENT GEENEN, FABIENNE BRILOT, ISABELLE HANSENNE, CELINE LOUIS, CHANTAL CHARLET-RENARD, and HENRI MARTENS University of Liege, Liege-Sart Tilman, Belgium

I. INTRODUCTION In distant species and invertebrates, the foundations of the neuroendocrine system and innate immunity have coexisted until now without any apparent problem. Some 300 million years ago, a relatively short time period after jawless fishes (agnathans), adaptive immunity emerged in the first cartilaginous fishes. Somatic recombination machinery characterizes adaptive immunity and is responsible for the random generation of the huge diversity of immune receptors able to recognize nonself antigens. The emergence of this novel form of immune defense exerted so potent an evolutive pressure that structures and mechanisms developed along the paths of lymphocyte traffic to impose immunological self-tolerance, that is, the inability of the immune system to attack the host organism. Together with the generation of diversity and memory, self-tolerance constitutes a fundamental property of the immune system. The progressive rise in the level of immune diversity and complexity also explains why self-tolerance failures (i.e., autoimmune diseases) were increasingly detected during evolution, the maximum being currently observed in the human species´. The first thymus appeared in cartilaginous fishes, concomitantly with the emergence of rudimental forms of adaptive immunity [1]. Though some forms of tolerance induction take place in primary hematopoietic sites (fetal liver and bone marrow), antigen-dependent B-cell tolerance is predominantly due to an absence of T-cell help [2]. Among all lymphoid 337

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structures, the thymus is the only organ specialized in the establishment of central selftolerance. As this chapter will illustrate, the thymus is a crucial meeting place between the neuroendocrine and immune systems. In this organ responsible for thymopoiesis— T-cell generation—the neuroendocrine system regulates the process of T-cell differentiation from the very early stages. In addition, and more specifically, T lymphocytes inside the thymus undergo a complex process that establishes central T-cell self-tolerance of neuroendocrine principles. The thymus is a unique organ wherein occurs a permanent confrontation between ancient neuroendocrine principles and a recent system equipped with recombination machinery promoting the stochastic generation of immune diversity. Contrary to a previous, rather dogmatic view, the thymus functions throughout life and plays a fundamental role in the recovery of a competent T-cell repertoire after intensive chemotherapy [3]. II. DEVELOPMENTAL BIOLOGY OF THE THYMUS The term ‘‘thymus’’ derives from the ancient Greek word ␪␯␮␴´ ς, meaning both ‘‘courage’’ and ‘‘thyme.’’ Indeed, Galen suspected that, because of its proximity to the heart, this mediastinal organ could be the seat of ‘‘courage’’ and ‘‘affection.’’ Galen and Rufus of Ephesus were probably the first to describe the thymus in a superficial manner, but with an accurate anatomical description. The general morphology of the thymus resembles the leaves of the thyme plant. Epithelial cells (TECs) constitute the major part of the thymic parenchyme. This epithelium expands, together with the inferior parathyroid epithelial rudiment, from the endoderm of the third pharyngeal pouch on each side into the surrounding mesenchyme. On embryonic day (E) 10.5, this endoderm already contains epithelial stem cells that are able to form a thymus with normal phenotype and function in vivo [4,5]. Auerbach first illustrated the importance of neural crest–derived ectomesenchyme in thymus development [6]. The interactions of the endodermal rudiment with mesenchymal cells from the cephalic neural crest are absolutely required for proper development of the thymus [7,8]. Following these interactions, the primordial thymus becomes competent to attract lymphoid stem cells. The chemotactic mechanisms for the recruitment of hematopoietic precursors into the thymus might involve chemokines produced by thymic stromal such as the thymic expressed chemokine, or TECK [9]. Some human diseases (and animal models) include defective thymus development leading to primary immunodeficiencies. DiGeorge syndrome associates total or partial agenesis of the derivatives of the third and fourth pharyngeal pouches (thymus and parathyroid glands), as well as cardiovascular anomalies, including interrupted aortic arch. This syndrome may result from abnormalities in the migration of cephalic neural crest cells [10,11]. Most patients with DiGeorge syndrome have a chromosomal deletion of 22q11, and the transcription factor gene Tbx1 is a candidate for the main clinical manifestations of the syndrome [12]. Hoxa3ⳮ/ⳮ mice present thymic aplasia, parathyroid hypoplasia, and frequent defects in heart and basal vessels [13,14]. Wild animals with immune deficiencies related to DiGeorge syndrome are nude (hairless) mice with a marked defect in TEC development. The nude phenotype results from mutations in the nude gene located on murine chromosome 11 that encodes the transcription factor winged-helix nude (whn or Foxn1). In the absence of whn, the thymus rudiment still develops, but is filled with primitive TECs that do not specialize into subtypes [15,16]. Wnt glycoproteins expressed by TECs and thymic T cells (thymocytes) regulate the expression of Foxn1 and thus constitute critical regulatory signals for a normal thymic function [17]. Other transcription

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factors like Pax1 and Pax9 are expressed in the third pharyngeal pouch, and their transcription level is diminished in Hoxa3ⳮ/ⳮ mice [18,19]. The phenotype of the Splotch mouse includes thymic dysplasia and a defective migration of neural crest cells due to a mutation in the Pax3 gene [20]. The functional interaction between TECs and mesenchyme is also reflected at the molecular level since the absence of fibroblast growth factor-10 or its receptor (FGFR-2IIIb) is associated with severe thymus hypoplasia [8,21]. The thymus is classically described as a lympho-epithelial organ. However, it is important to understand that the lymphoid compartment (thymocytes) is a mobile cell population that originates as lymphocyte progenitors in primitive hematopoietic sites, migrates into the thymus, and differentiates by close contact with a unique environment specialized for T-cell differentiation. TECs represent Ⳳ85% of this environment, while bone marrow–derived cells, dendritic cells (DCs), and macrophages constitute the remaining cells. According to their profile in the expression of some markers, TECs are distributed as subcapsular, cortical, and medullary TECs, deriving from a common precursor [4,5,22]. Thymic nurse cells (TNCs) are very large epithelial cells (diameter up to 50 ␮m) in the subcapsular and outer cortex. They contain a number of immature T cells that are engulfed within caveoles delimited by TNC plasma membrane, a process called ‘‘emperipolesis.’’ TNCs express neuroendocrine markers, synthesize neuropeptides, and contain the machinery necessary for antigen processing and presentation [23,24]. The thymus also receives autonomic and sensory innervation, and a number of neuropeptides/neurotransmitters have been identified in thymic nerve fibers [25,26]. III. GENERATION OF T-CELL DIVERSITY AND CENTRAL SELFTOLERANCE: A BROAD OVERVIEW A mature CD8Ⳮ or CD4Ⳮ T lymphocyte bears a receptor (TCR) specific for an antigenic peptide sequence presented by a major histocompatibility complex class I (MHC I) or class II (MHC II) molecule, respectively (see Chapter 1). The prominent role of the thymus in T-cell generation was demonstrated by pioneering studies conducted by Miller in early 1960s [27], and the previously assumed function of the thymus as an endocrine gland was less and less considered. From the primary sites of hematopoiesis (embryonic yolk sac, fetal liver, and then bone marrow), T-cell progenitors migrate into the thymus, possibly under the influence of chemokines, and intensively proliferate in thymic outer cortex. A role for the Notch pathway has been evidenced in the induction of T-lineage restriction and commitment [28]. Then, intimate contacts with TECs/TNCs promote interleukin-7 (IL-7) synthesis and stimulate recombination-activating genes (RAG1 and RAG2) [29,30]. In order to respond to any particular antigen likely to be encountered in the foreign milieu, the peripheral T-cell population must contain a highly diverse repertoire of different TCRs. This huge heterogeneity, which is not encoded in the germline, is generated during intrathymic T-cell differentiation (extensively reviewed in, Ref. 29). Within the thymus, T-cell progenitors undergo a series of developmental events that can be monitored by membrane expression of cluster differentiation (CD) antigens. In a very simplistic view, while early T cells are double negative (CD4ⳮCD8ⳮ), they become double positive (CD4ⳭCD8Ⳮ) before final commitment to either CD4Ⳮ lineage or CD8Ⳮ lineage. During ␣␤ (and ␥␦) T-cell maturation, the juxtaposition of various genes segments leads to the generation of TCRA and TCRB chains (TCRG and TCRD). The antigen-recognizing variable domains of TCR ␣ and ␤ chains are encoded by combinations of variable (V), diversity (D), and joining (J) gene segments (TCR ␤ chains) or V and J gene segments (TCR ␣ chains).

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V(D)J recombination is initiated by the recognition of recombination signal sequences (RSSs) that flank the coding segments. RSSs consist of conserved heptamer and nonamer sequences separated by a less conserved spacer of 12 or 23 bp. During maturation, preT cells transiently express RAG1 and RAG2 so that V, D, and J segments are fused together. Two successive waves of RAG1 and RAG2 expression coincide with TCRB and TCRA chain rearrangements. RAG-1 and RAG-2 cooperate to recognize the RSSs and introduce hair-pinned site-specific cleavages at the signal-coding borders of a given pair of coding segments. Fusion of distinct V, D, and J segments leads to a vast array of different TCR ␤-chain nucleotide sequences. During any rearrangement process, the DNA located between the two RSSs is circularized, resulting in the formation of an extra-chromosomal circular excision product containing the two ligated RSSs. These TCR rearrangement excision circles (TRECs) are stable, are not duplicated during further mitosis, and are thus diluted-out with each cell division. A common requirement for productive rearrangement of the TCRA locus is the deletion of the TCRD locus that it encompasses. Deletion of TCRD is an important step during T-cell differentiation and is a sign of the definitive commitment of thymocytes to the ␣␤ T lineage. This deletion mainly occurs through specific rearrangement of ␦Rec and (␺J␣, leading to the generation of a specific TREC —the signal joint (sj) TREC or ␦Rec-(␺J␣ TREC—that can be observed in Ⳳ70% of ␣␤ T cells [31,32] (Fig. 1). TRECs are stable for a long time; they have been detected in 41-year thymectomized patients [33]. A maximum of two sjTRECs can be present within one ␣␤ T cell if the corresponding rearrangement event occurs in both alleles and if the cell does not proliferate following this rearrangement. TRECs are exported from the thymus to the periphery within recent thymus emigrants (RTEs). However, it has been reported that the TCR ␦ locus can be excised through other recombination events that will not generate a sjTREC molecule. Therefore, peripheral blood quantification of sjTREC frequencies leads to an underestimation of the real frequency of RTEs. Nonetheless, it provides an unequivocal way to estimate the blood concentration of RTEs and, thus, evaluate the magnitude of thymic function. Therefore, TREC level in the periphery reflects RTE numbers and is largely accepted as a surrogate marker for thymic function [34]. Using TREC quantification, thymopoiesis was demonstrated to occur in advanced life (Fig. 2) and to play a crucial role in the regeneration of a peripheral T-cell repertoire after highly active anti-retroviral therapy [35–38]. TCR recombination by pre-T cells constitutes a pivotal event because, among the huge number of possible combinations (⬎108), many of them are able to recognize selfantigens presented by thymic MHC proteins. The so-called negative selection is a massive deletion of T-cell clones expressing self-reactive TCR. This process is extremely powerful since, of 100 T-cell progenitors, only 1–2 T cells will leave the thymus in a state of selftolerance, competence, and potential activity against nonself-antigens [39]. Thus, through a continuous presentation of constant self-antigens to thymocytes that are stochastically rearranging TCR gene segments, the physiological function of the thymus is to ensure the generation of a diverse repertoire of TCRs that are nevertheless self-tolerant. Molecular mechanisms implicated in thymic clonal deletion of self-reactive T cells have been extensively described [40] and will not be reviewed here. However, very little attention has been paid to the nature of self-antigens that are effectively presented by the thymic MHC machinery. This question constitutes the major focus of the present chapter. The concomitant occurrence within the same organ of completely opposite events, such as negative selection of self-reactive T cells and generation of T-cell life/diversity, represents a prominent problem in modern biology. A number of models have been pro-

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Figure 1 Late genetic recombination leading to sjTREC formation. The ␦Rec-⌿J␣ rearrangement deletes the TCRD locus and forms a sjTREC (also called ␦Rec-⌿J␣ TREC) as it is generated from RAG action by ligation of RSS sequences flanking ␦Rec-⌿J␣ gene segements. The dRec-⌿Ja sequences remaining in the genome are excised later in most of the ␣␤ cells during the TCR␣ chain recombination and will be found in a V␣-J␣ coding joint (cj) TREC. (From Ref. 37).

Figure 2 Age-dependent sjTREC number per 105T cells in normal subjects (n ⳱ 41). The concentration of sjTREC decreases with aging in an exponential way (p ⳱ 0.0014.) (From Ref. 37.)

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posed to explain the paradox of thymus physiology. A first model attributed different functional roles to the cells of thymic parenchyme: TECs/TNCs would be mainly responsible for inducing T-cell proliferation and development, whereas thymic DCs and macrophages, acting as dedicated antigen-presenting cells (APCs), would mediate T-cell–negative selection. Although DCs and macrophages are very efficient in inducing T-cell deletion, TECs and TNCs are also able to present self-antigens and to efficiently provoke T-cell–negative selection [41]. Medullary TECs, in particular, are highly competent in achieving self-tolerance [42–44]. A second model is called the ‘‘avidity’’ model [45,46]: T cells bearing a TCR with a high affinity for self-antigen presented at high density will undergo clonal deletion, while those with a low-affinity TCR and/or in presence of lowdensity self antigen will survive and further develop. However, the affinity of a given TCR for its cognate antigen is rather low (ⱕ10ⳮ7 M), and one may thus question the physiological meaning of lower affinities. In addition to deletion of self-reactive T cells, the thymus is also a site for the generation of regulatory T cells (TREG), which play an important role in maintaining selftolerance. TREG are enriched in CD4ⳭCD25Ⳮ T cells that are generated intrathymically and that comprise Ⳳ5–10% of the peripheral CD4Ⳮ T cells. These CD4ⳭCD25Ⳮ T cells require TCR stimulation to mediate their suppressive activities in vitro and regulate various organ-specific autoimmune diseases in vivo. Thus, it is likely that these TREG are specific for peripheral autoantigens and are selected by distinct pathways during thymic T-cell differentiation [47,48].

IV. DUAL ROLE OF THYMIC NEUROENDOCRINE PRECURSORS AND THE NATURE OF NEUROENDOCRINE SELF Intrathymic synthesis of the neurohypophysial peptide oxytocin (OT) was reported in 1986 [49]. Subsequent thorough studies to elucidate the physiological role of thymic OT have shown that thymic OT does not behave as a classic secreted neurohormone, but as the self-antigen of the neurohypophysial peptide family (extensively reviewed in Ref. 50). Thymic MHC presentation of OT to immature T cells involves a 55 kDa protein, which is expressed in TEC plasma membrane and is labeled both by an antibody to neurophysin (OT-binding 10 kDa protein) and one directed to the 45 kDa heavy chain of MHC I proteins [51]. Mechanisms responsible for the synthesis of this protein remain to be elucidated, but a similar neurophysin–MHC I protein has been identified in cell membranes of small-cell lung carcinoma [52,53]. OT targeting to the outer surface of TEC plasma membrane and OT behavior as the self antigen of the neurohypophysial family were confirmed in further experiments. Treatment of human TEC primary cultures with monoclonal antibodies directed to different OT epitopes promoted their secretion of IL-6 and leukemia inhibitory factor (LIF). By contrast, treatment of TEC cultures with monoclonal antibody to vasopressin (VP) did not modify their profile in IL-6 or LIF secretion [54]. Those data suggest that, in the thymus network, the immune recognition of self may be associated both with deletion of self-reactive T cells and delivery of survival and growth signals for other developing T cells. Neurotensin (NT) and somatostatin (SS) were the first neuropeptides detected by immunocytochemistry in the thymic parenchyme [55]. Further studies conclusively demonstrated that NT (and NT-derived peptides) are presented by MHC I proteins purified from human TEC plasma membranes [56].

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Genes encoding specific neurohypophysial receptors are transcribed by thymic Tcell subsets: the OT receptor gene (OTR) is expressed by CD4ⳮCD8ⳮ (double negative), CD4ⳭCD8Ⳮ (double positive), CD4Ⳮ and CD8Ⳮ single positive thymic T cells, whereas V3R expression is restricted to CD4ⳭCD8Ⳮ and CD8Ⳮ thymic T cells [57]. Neither V1R nor V2R expression can be detected in the thymus. Thymocyte OTR and V3R are able to transduce neurohypophysial peptides, in particular OT, according to the rules established for those receptors in other cellular systems (increase of phosphonositide turnover coupled to mitogenic action) [58]. In addition, OT markedly increases the phosphorylation of kinases implicated in focal adhesion (p125FAK, p130Cas) [59]. Such early events in the process of T cell differentiation could be crucial for the establishment of ‘immunological synapses’ between immature T cells and thymic APCs, namely TECs, DCs, and macrophages. During ontogeny in Balb/c mice, OT transcripts are detected on E 13 in both brain and thymus, whereas VP transcription starts on E 14 in the brain and is clearly detected in the thymus only on E 15 (unpublished data). OT precocious transcription in the thymus further concords with the role of thymic OT in the induction of central selftolerance of the neurohypophysial peptides before their expression by hypothalamic neurons. Based on these two roles of thymic OT acting both as a self-antigen and as a ligand for neurohypophysial receptors expressed by pre-T cells, a model was elaborated transposing at the peptide level the dual role of the thymus in T-cell differentiation [60,61] (Fig. 3). This model applies to other neuroendocrine-related self-antigen precursors expressed in the thymic stroma and a definition of neuroendocrine self may be proposed as follows: 1. Neuroendocrine self-antigens correspond to peptide sequences highly conserved throughout evolution of one given family. 2. A hierarchy characterizes their expression pattern in the thymus. 3. Intrathymic processing of neuroendocrine self-antigen precursors is not coupled to the classic model of (neuro)secretion but rather to pathways of antigen presentation by MHC proteins. 4. Some differences exist in the processing between thymic APC and dedicated peripheral APC. For some neuroendocrine self-antigens (OT and NT), these differences imply that their presentation by thymic APCs is not restricted by MHC alleles. With regard to the family of tachykinins, neurokinin A (NKA) is the peptide generated from the processing in TEC of the preprotachykinin A (PPT-A) gene product [62]. Thymic PPT-A expression appears to be glucocorticoid dependent since it is markedly enhanced after rat adrenalectomy (Ericsson A and Geenen V, unpublished observations). NKA exerts IL-1–like mitogenic effects on murine thymocytes, suggesting that tachykinin receptors expressed by immature T cells could also be implicated in another accessory pathway for T-cell development [63]. NKA amino acid sequence shares the same C-terminal epitope with other members of the tachykinin family, and the leucine residue in position 9 could be used in the binding to MHC I alleles, thus making NKA the thymic self-antigen of the tachykinin family. The other tachykinin encoded by PPT-A, substance P (SP), is not detected in TEC but is present in sensory nerve fibers of the thymus [64]. This also supports the existence of a specific posttranslational processing of the precursor encoded by PPTA in TEC. Specific receptors for SP are associated with the vessels in the thymic medulla, where they could mediate control of local blood flow and vascular permeability.

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Figure 3 The dual role of thymic neuroendocrine-related self-peptide genes. Neuroendocrine gene-encoded precursors are the source of two different types of interactions with pre-T cells: (1) They deliver ligands that are not secreted but targeted to the outer surface of thymic cell membranes. Those ligands bind to T-cell neuroendocrine receptors, leading to activation of transduction pathways such as increase of inositol triphosphate (IP3) and phosphorylation of focal adhesion kinases (FAK). FAK phosphorylation may be crucial in promoting the establishment of ‘‘synapses’’ between thymic APC and immature T cells (insert). (2) They are also processed into self-antigens presented by— or in association with—thymic MHC molecules. This presentation will lead to clonal deletion and/or developmental arrest of T cells bearing a randomly rearranged TCR oriented against neuroendocrine families.

V. THYMIC EXPRESSION OF INSULIN-RELATED GENES While searching for a precursor able to represent the whole insulin peptide family in front of T cells during their differentiation in the thymus, insulin-like growth factor 2 (IGF-2) was found to be the dominant insulin-related peptide expressed by TECs from different species [65]. Immunoreactive (Ir) IGF-1 was also detected in thymic cells with a macrophage-like morphology and topography, while Ir insulin could not be clearly identified within human thymic lobules. Again, IGF-2 secretion was not evidenced in human TEC primary cultures, though Ir IGF-2 could be detected by confocal microscopy at the outer surface of TEC plasma membranes [66]. By RT-PCR and in situ hybridization, IGF2 transcription by human TEC was further demonstrated and found to be controlled by the same promoters as in other extrahepatic fetal and adult tissues. IGF1R was found to be expressed in Jurkat T cells, and IGF1 transcripts were also identified by in situ hybridiza-

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tion in thymic macrophages [67,68]. In transgenic mice, overexpression of Igf2 under the control of a MHC promoter leads to high expression of the transgene in TECs and marked thymus hypertrophy [69]. Also in the Igf2 transgenic mice, the increased thymic cellularity is associated with a stimulated generation of normal T cells, in particular CD4Ⳮ [70]. Functional significance of the thymic IGF axis was evaluated by using murine fetal thymic organ cultures (FTOCs). FTOC treatment with an anti-IGF-2 monoclonal antibody induced an inhibition of the transition from CD4ⳮCD8ⳮ T cells to CD4ⳭCD8Ⳮ T cells, as well as an increase in CD8Ⳮ cells. A similar inhibition of early T-cell differentiation was observed when FTOCs were treated with anti-IGF-1R monoclonal antibody or with antiIGF-2R polyclonal antibody. In addition, anti-IGF-1R and anti-IGF-2R induced an 81% and a 34% decrease in FTOC total T-cell number, respectively. In the same model, treatment with a specific antibody directed to (pro)insulin did not exert any significant effect on either T-cell cellularity or T-cell differentiation [71]. A number of studies in 1994 reported that the insulin gene (INS) is also transcribed in the thymus [72–77]. A debate recently arose about the precise nature of the thymic cell type responsible for INS transcription, i.e., medullary TEC or DC [78,79]. Compared to IGF-2 (Ⳳ100 ng/g wet weight), the thymic content in insulin protein is very low (⬍0.01 ng/g wet weight). The insulin thymic content ranged from 98–1200 fmol/g wet weight in one study to 0.44 Ⳳ 0.22 pmol/mg protein in another. Those studies did not investigate the functional effect of thymic insulin on T-cell differentiation, but evidenced an interesting correlation between thymic INS mRNA levels and the presence of alleles conferring genetic susceptibility to type 1 diabetes. VI. THYMIC INSULIN-RELATED GENES, CENTRAL TOLERANCE OF ␤ CELLS, AND TYPE 1 DIABETES Type 1 (juvenile or insulin-dependent) diabetes is a chronic, devastating disease resulting from an autoimmune response specifically oriented against pancreatic islet ␤ cells, the only cells secreting insulin according to the endocrine model (see Chapter 21) [80]. It increasingly appears that loss or absence of tolerance to insulin is potentially central to ␤-cell–specific autoimmune destruction. In 1992, a defect in the process of T-cell education to recognize and tolerate self antigen was hypothesized to play a pivotal role in the development of diabetes insipidus secondary to hypothalamus-specific autoimmunity [81]. As already proposed by Burnet, the pathogenesis of autoimmune diseases could result from the appearance of ‘‘forbidden’’ self-reactive clones in the peripheral lymphocyte repertoire [82]. Since the thymus is the primary site for induction of self-tolerance, thorough investigation of a defective thymic censorship should provide the scientific community with important keys to understand the mechanisms underlying the development of autoimmune responses. A number of abnormalities of thymic morphology and cytoarchitecture have been described for several autoimmune disorders [83–85]. As discussed above, a precise hierarchy and cell topography exist in the intrathymic expression of INSrelated genes: IGF2 (TECs) ⬎ IGF1 (macrophages) ⬎⬎ INS (thymic DC and/or medullary TECs). This hierarchy is important since it is known that immune tolerance of a protein or a protein family primarily involves dominant epitopes of this protein or this family [86,87]. According to this observation, IGF-2 should be more tolerated than IGF-1, and much more than insulin. This assumption is indirectly verified by immunization—i.e., active experimental breakdown of immune tolerance—with insulin-related peptides. Following immunization, the titers and frequency of antibodies to insulin are higher than

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those against IGF-1 and much higher than those against IGF-2. Also, the high prevalence of autoantibodies against insulin (Ⳳ40%) in the normal population [88] could be related to the very low expression of INS in the thymus. Finally, a number of studies recently failed to show any significant tolerogenic effect of insulin administered either orally or subcutaneously [89–91]. The negative results of those trials, however, do not preclude the fact that restoration of specific ␤-cell tolerance remains the most attractive and most promising way to prevent and/or cure type 1 diabetes [92]. To gain further insight into the central tolerance mediated by thymic insulin-related proteins, we investigated the hypothesis that a thymus dysfunction could be involved in the pathophysiology of type 1 diabetes. This was performed through analysis of Ins, Igf1, and Igf2 expression in the thymus of bio-breeding (BB) rats, an animal model of human type 1 diabetes. Ins, Igf1, and Igf2 transcripts were detected in all thymi from diabetesresistant BB rats (BBDR). By contrast, while Ins and Igf1 transcripts were detected in all thymi from diabetes-prone BB rats (BBDP), a defect in Igf2 transcription was observed in 11 of 15 thymi from BBDP rats, in close accordance with diabetes incidence in this strain (86%). This defect was thymus-specific since Igf2 transcription was detected in the brain and liver of BBDP rats [93]. The defect in Igf2 expression evidenced in the thymus of BBDP rats can explain both lymphopenia (including a lack of IGF-2–specific TREG cells) as well as absence of central self-tolerance of insulin family in those rats. A number of genetic loci conferring susceptibility or resistance to type 1 diabetes have been identified [94,95]. Recent studies support the hypothesis that genetically determined thymic INS levels play a critical role in insulin-specific autoreactive T-cell selection. Using an elegant model of graded INS transcription in the thymus, Chentoufi and Polychronakos have found that specific T-cell reactivity to insulin was inversely correlated with INS intrathymic expression [96]. The expression of the transcription factor autoimmune regulator Aire is maximal in murine medullary TECs and is absent in the thymus epithelium of diabetic NOD mice [97,98]. The use of Aireⳮ/ⳮ mice helped in demonstrating that the Aire protein promotes thymic transcription of tissue-specific genes, in particular genes that are known to be expressed in the thymus and to intervene in central self-tolerance such as OT, IGF2, INS, and NPY [99]. These findings are important in view of the fact that thymic expression of autoantigens correlates with a higher level of self-tolerance and resistance to autoimmune diseases [100]. Finally, a defect in central self-tolerance of NOD mice has been demonstrated since Fas-dependent and independent apoptosis pathways are defective in the NOD thymus [101]. Thus, more and more studies give credit to the idea that a defect in thymus central self-tolerance could be involved as a crucial factor in development of organ-specific autoimmune diseases such as type 1 diabetes. Thymic hyperplasia is associated with autoimmune type 3 thyroiditis (Graves’ disease), and TEC expression of the major autoantigen tackled in this disease, the thyrotropin receptor, has been reported [102,103]. The origin of systemic (non–organ-specific) autoimmunity is still a matter of debate but could be elucidated through investigations of the nonspecific, innate immune system. Through thymus dysfunction, self-reactive T cells will continuously leave the thymus and the peripheral T-cell pool will be gradually enriched with T cells equipped with a TCR directed toward epitopes of peripheral tissue antigens. The hypothesis that thymic dysfunction could follow an infection by the diabetogenic strain B4 of coxsackie virus (CVB4) is currently being explored in our laboratory. A persistent and productive infection by CVB4 of cultured human TECs has been demonstrated [104]. The immunological effects induced by CVB4 thymus infection are currently being investigated in depth [105]. A thymus dysfunction

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resulting from CVB4 infection intervened in intimate conjunction with the bystander effect evidenced from CVB4 infection of islet ␤ cells [106]. The presence of self-reactive lymphocytes is not a sufficient condition to develop type 1 diabetes. A bridge must be installed between self-reactive T cells and target ␤-cell autoantigens depending on the influence of some external factors and a genetic background determined by the balance between susceptible and protective alleles.

VII. PHARMACOLOGICAL PERSPECTIVES: FROM THYMUS TO VACCINE The study of neuroendocrine gene expression and precursor processing in the thymus led to the identification of thymic neuroendocrine self-peptide precursors. With regard to insulin-related gene expression in the thymus, IGF-2—a prominent fetal growth factor—was identified as the dominant self-peptide precursor of the insulin family expressed in the thymus from different species, including humans. This observation is in close accordance with the theory of self-recognition which, according to F. M. Burnet, is not an inherited property but is gradually acquired in the course of fetal life. The tolerogenic properties of thymic neuroendocrine self-peptides may already be suspected from what is known about the immunological tolerance of classic hormones. The development of specific antibodies by active immunization (i.e., experimental breakdown of self-tolerance) revealed that OT is more tolerated than VP, and that IGF-2 is also more tolerated than IGF-1, and much more than insulin. Some cases of diabetes insipidus result from an autoimmune process against VP-producing hypothalamic neurons (see Chapter 22). Insulin is the primary autoantigen tackled by the autoimmune response observed in type 1 diabetes, and this might result from its very low expression in the thymus. In contrast, autoimmunity has never been observed against OT and IGF-2. The strong tolerance of these peptides, resulting from high expression of their genes in the thymus, may be considered as the consequence of evolutive pressure to protect fundamental processes such as species reproduction and individual ontogeny. The putative pathogenic role of ‘‘forbidden’’ self-reactive T cells against IGF-2 is currently analyzed through immunization of Igf2ⳮ/ⳮ mice. More and more studies are documenting the highly immunogenic properties of insulin, in particular the sequence INS B:9–23, which is the dominant autoantigenic epitope of the insulin protein. INS B:9–23 can be presented and has been co-crystallized with DQ8, a MHC class II allele conferring major genetic susceptibility to type 1 diabetes [107]. A cellular response to INS B:9–23 occurs in patients with type 1 diabetes. This CD4 response is MHC II restricted and exhibits a proinflammatory profile with a high induction of interferon (IFN)-␥ in response to the presentation of INS B:9–23 [108]. According to the altered peptide ligand (APL) strategy [109], the sequence B:9–23 of INS was modified by alanine substitutions introduced at different sites. The peptide with alanine substitutions in positions 16 and 19 (NBI-6024) induced Th2 T-cell responses in NBI-6024–derived T-cell lines from NOD mice. Also, after administration to female NOD mice, NBI-6024 significantly delays but does not prevent the onset of autoimmune diabetes [110]. However, this strategy is based on INS B:9–23, and two recent studies have shown that, instead of tolerance induction, administration of insulin-derived peptides to NOD mice can prime the autoimmune diabetogenic process or even promote a fatal anaphylactic reaction [111,112]. This type of hypersensitivity was also observed in a phase II trial of an APL of myelin basic protein, a major autoantigen of multiple sclerosis [113].

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Figure 4 Number of cells secreting IFN-␥ and IL-10 in PBMCs isolated from 10 type 1 diabetic DQ8Ⳮ adolescents after presentation of insulin B:9–23 or the homologous self-peptide derived from IGF-2 at two concentrations (10 and 50 ␮M). *p ⬍ 0.05; **p ⳱ 0.002.

On the basis that IGF-2 is the dominant thymic peptide of the insulin family and the fact that IGF-2 is much better tolerated than insulin, a novel type of vaccine could be developed for the prevention and/or cure of type 1 diabetes. The immunogenic response in classic vaccination induces activated/memory T cells that are specific to antigen(s) shared by infectious agents. Analogously in type 1 diabetes, the presentation in periphery of insulin-derived autoantigen(s) activates specific CD4/CD8 T cells, as well as memory T cells directed against these autoantigenic epitopes. In the novel type of tolerogenic vaccination, administration of a self-peptide derived from IGF-2 could anergize or delete self-reactive T cells that have escaped the thymus censorship because of a thymic dysfunction in the establishment of ␤-cell–central self-tolerance. We are currently performing such studies with IGF-2 peptides that exhibit the same affinity and compete with INS B: 9–23 for binding to DQ8. Preliminary data in DQ8Ⳮ type 1 diabetic adolescents show this hypothesis to be correct and that DQ8 presentation of INS B:9–23 and an IGF2–derived peptide drives opposite immune responses with a dominant IL-10 response after DQ8 presentation of an IGF-2 self-antigen (Fig. 4). Two mechanisms may be considered to explain those findings. On the one hand, the peripheral T-cell pool of diabetic patients could contain only CD4 directed against insulin-derived epitopes and the IGF-2 selfantigen would be recognized by those cells as a natural APL, eliciting a different response because of different transduction after binding to TCR specific of DQ8-INS B:9–23 complex. On the other hand, the IGF-2 self-antigen could stimulate IGF-2–specific CD4ⳭCD25Ⳮ TREG cells. These data confirm that the immunodominant epitope of insulin drives an inflammatory response with production of IFN-␥, which is essential for destruction of islet ␤ cells and development of type 1 diabetes [114]. They also show that IGF2 is a potent inducer of the secretion of IL-10, a major regulatory cytokine with powerful immunosuppressive and anti-inflammatory properties [115]. They document the potent

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tolerogenic properties of an IGF-2–derived peptide and may explain why it is so difficult to obtain antibodies directed to IGF-2 at high titers after active immunization with this peptide. Further studies of the immune responses to epitopes specific of the insulin family will be central for our understanding of both pathogenesis and protection against the autoimmune response directed to islet ␤ cells. It may now, be time to distinguish an autoantigen from a self-antigen, even if these terms are not used separately in the field of immunology. Though thymic self-antigens and their corresponding peripheral autoantigens are highly homologous and belong to the same family, they are not identical, and this biochemical difference drives opposite immune responses (i.e., immunogenic vs. tolerogenic, respectively). The powerful physiological role of the thymus in self-tolerance induction should be further exploited to prevent and/ or cure severe debilitating autoimmune diseases (such as type 1 diabetes). ACKNOWLEDGMENTS Vincent Geenen is research director of Belgian NFSR; Fabienne Brilot and Isabelle Hansenne are research assistants at Belgian FRIA. These studies were supported by the Leon Fredericq Foundation (Liege Medical School and Liege University Hospital), NFSR, FRIA, the Belgian Federation Against Cancer, the Belgian Association Against Diabetes, the Vaugrenier Foundation for Tolerance Research (Geneva Switzerland), and the European Association for the Study of Diabetes (EASD) (Du¨sse´ldorf, Germany). REFERENCES 1. Du Pasquier L, Flajnik M. Origin and evolution of the vertebrate immune system. In: Paul WE, Ed. Fundamental Immunology. 4th ed.. Philadelphia: Lippincott-Raven, 1999:605–650. 2. Kamradt T, Mitchison NA. Tolerance and autoimmunity. N Engl J Med 2001; 344:655–664. 3. Mackall CL, Fleisher TA, Brown MR, Andrich MP, Chen CC, Feuerstein IM, Horowitz SM, Magrath IT, Shad AT, Steinberg SM, Wexler LH, Gress RE. Age, thymopoiesis, and CD4Ⳮ T-lymphocyte regeneration after intensive chemotherapy. N Engl J Med 1995; 332:143–149. 4. Bennett AR, Farley A, Blair NF, Gordon J, Sharp L, Blackburn CC. Identification and characterization of thymic epithelial progenitor cells. Immunity 2002; 16:803–814. 5. Gill J, Malin M, Holla¨nder GA, Boyd R. Generation of a complete thymic microenvironment by MTS24Ⳮ thymic epithelial cells. Nat Immunol 2002; 3:635–642. 6. Auerbach R. Morphogenetic interactions in the development of the mouse thymus gland. Dev Biol 1960; 2:271–284. 7. Bockman DE, Kirby ML. Dependence of thymus development on derivatives of the neural crest. Science 1984; 223:498–500. 8. Anderson G, Jenkinson EJ. Lymphostromal interactions in thymic development and function. Nat Rev Immunol 2001; 1:31–40. 9. Wurbel MA, Philippe JM, Nguyen C, Victorero G, Freeman T, Wooding P, Miazek A, Mattei MG, Malissen M, Jordan BR, Malissen B, Carrier A, Naquet P. The chemokine TECK is expressed by thymic and intestinal epithelial cells and attracts double- and single-positive thymocytes expressing the TECK receptor CCR9. Eur J Immunol 2000; 30:262–271. 10. Lischer HW, DiGeorge AM. Role of the thymus in humoral immunity. Lancet 1969; 2: 1044–1049. 11. Couly G, Lagrue A, Griscelli C. Le syndrome de DiGeorge, neurocristopathie rhomboencephalique exemplaire. Rev Stomatol Chir Maxillofac 1983; 84:103–108. 12. Jerome LA, Papaioannou VE. DiGeorge syndrome phenotype in mice mutant for the T-box gene, Txb1. Nat Genet 2001; 27:286–291.

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18 Two-Way Communication Between Mast Cells and the Nervous System HANNEKE P. M. VAN DER KLEIJ Utrecht University, Utrecht, The Netherlands MICHAEL BLENNERHASSETT Queens University, Kingston, Ontario, Canada JOHN BIENENSTOCK McMaster University, Hamilton, Ontario, Canada

I. INTRODUCTION Morphological studies reveal an intimate association between mast cells and neurons in both the peripheral and central nervous system [1–4]. In virtually all tissues of the body, mast cells can be found in close proximity to nerve fibers. This proximity also represents a functional link between the immune and nervous systems, whereby mast cells appear to act as bi-directional carriers of information [5,6]. Mast cells influence their local environment and in turn are influenced by it. They are sensitized to antigens by the binding of antibodies to specific receptors on their surface, but can also be caused to secrete by other types of molecules because of their electrical charge or their chemical nature. Association with the nervous system allows mast cells to act as sensory receptors for a variety of newly encountered or potentially noxious agents. They are therefore an ideal cellular transducer, which act to pass information on through afferent nerves to local tissues by axon reflexes, as well as to the spinal cord and, thence, the brain. Tissue mast cells invariably show ultrastructural evidence of activation in normal healthy conditions, suggesting that these cells are constantly providing information to the nervous system. Since they are located at sites under constant exposure to the external 357

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environment, such as the skin, respiratory system, and gastrointestinal tract, the significance of these associations is evident. Mast cells are motile and may be viewed as sensory receptors with the unique capacity to migrate to and from sites where nervous tissue exists or may be undergoing developmental or regenerative changes. Once in place, they may give information about the local environment, injury, and potentially injurious substances to the nervous system and promote appropriate efferent action. Little is known about their traffic or whether they will remain sessile or dissociate from nerves, once associated. As opposed to sensory receptors, mast cells can also act as efferent targets and can equally be associated with local effector function through release of their potent preformed and newly synthesized mediator molecules. It is in this way that they are involved in the regulation of vascular tone through effects on small blood vessels. In turn, they can be stimulated to synthesize and secrete a variety of mediators through neuronal activity. Sensory neurons play a role in neurogenic inflammation involving changes in functioning due to inflammatory mediators, which results in an enhanced release of neuropeptides from the sensory nerve endings [7,8]. The classical role of the mast cell in hypersensitivity reactions is well known and extensively studied, involving the interaction of allergens with IgE [9]. The mast cell and its mediators play an important role in neurogenic inflammation by affecting neuronal functioning. Neurogenic inflammation has been shown to occur in different tissues, including the skin, airways, urinary tract, and the digestive system. Furthermore, the role of mast cells and the nervous system is becoming apparent in delayed-type hypersensitivity reactions as well as in non–IgE-mediated reaction, e.g., in the airways and intestine [10,11]. In this chapter, while we will only discuss nerve–mast cell communication, it should be mentioned that this is just a small part of the neuroimmune interactions that have been extensively documented in both rodents and humans. Besides mast cells, neural contact can also occur between nerves and eosinophils or plasma cells [1,12]. In addition, neuropeptides released from sensory nerves can directly modulate the function of Langerhans cells. Among these neuropeptides, the tachykinins have been shown to modulate immune cell functions such as cytokine production, antigen presentation, and cell proliferation [13,14]. Therefore, mast cell–nerve exchanges are a most interesting example of a complex network of neuroimmune communication in the body. We have to assume that these communication pathways only add to other basic sensory and efferent methods of nervous communication and are inessential to survival. Nerve–mast cell association is preserved in phylogeny, since even frogs retain these structural and even functional adaptions [15]. In mutant animals in which mast cells are deficient (e.g., W/Wv or Sl/Sld mice), there are various minor physiological aberrations, including delayed responses to parasite infections, but in general, transgenic knockout animals and mutants without mast cells both have similar life spans to their background littermates under laboratory conditions. It is therefore reasonable to conclude that mast cell–nerve communication has evolved as an important nonessential regulatory mechanism that allows for an adaptable set of reactive effector responses. II. MAST CELLS Mast cells are widely distributed throughout the body in connective tissues [2], particularly around blood vessels and nerves. They are abundant in the submucosa of the digestive

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tract [16], oral and nasal mucosa [17], respiratory mucosal surfaces [18], and skin [13]. Mast cells are detected even in the brain [19,20]; no tissue, in fact, has been shown to be devoid of the presence of mast cells. Mast cells are involved in the regulation of their own overall tissue cell mass, since mast cell degranulation leads to an overall increase in mast cells [21]. The generation and secretion of diverse mast cell–derived factors are involved, including granulocytemacrophage–colony-stimulating factor (GM-CSF), stem cell factor (SCF), and nerve growth factor (NGF), all of which are known to promote mast cell growth [22,23]. Mast cells are increased in tissues undergoing inflammation, where they may have an intimate involvement in repair processes. Mast cells also have a phagocytic function, which might contribute to host defense [24], especially where tissue repair and fibrosis is occurring [25,26]. Mast cells have various cytokines stored in their granules that are stimulatory to fibroblasts [27]. These also support immunological defense strategies against parasites [28]. In addition, they contain serine proteases that may be involved in remodeling of the extracellular matrix during healing [26]. Mast cells are most often found in association with blood vessels everywhere and are a major source of glycosoaminoglycans, such as heparin, which have major effects on coagulation and other physiological systems [29]. In addition, many mediators have profound direct effects on vascular tone and permeability, e.g., histamine, serotonin, and many products of arachidonic acid metabolism. Mast cells can be divided into various subpopulations with distinct phenotypes. Two main subsets, connective tissue–type mast cells (CTMC) and mucosal mast cells (MMC), are recognized as distinct mast cell populations with different phenotypical and functional characteristics [30,31]. Many more phenotypically different subsets have been described in rodents [32]. In spite of their differences, both CTMC and MMC are considered to be derived from a common precursor in the bone marrow. Mast cell progenitor cells translocate from bone marrow to mucosal and connective tissues to locally undergo differentiation into mature forms. They possess a remarkable degree of plasticity, so that even apparently fully differentiated CTMC will transform their phenotype to MMC if transplanted into an intestinal mucosal environment [33]. In contrast to many other cell types, mast cells are absent from the blood, and their final maturation takes place in the tissue [34]. Their development and survival essentially depends on SCF and its receptor c-kit [35]. Besides SCF, cytokines such as interleukin (IL)-3, IL-4, and IL-10 influence mast cell growth and differentiation [36], as does NGF [37,38]. Mast cells are versatile cells capable of synthesis of a large number of pro- and antiinflammatory mediators, including cytokines, products of arachidonic acid metabolism, growth factors, including NGF and SCF, serotonin, histamine, etc. These rich sources of mediators can be prestored or newly synthesized upon stimulation. Prestored mediators, such as histamine, serine proteases, proteoglycans, sulfatases, and tumor necrosis factoralpha (TNF-␣), are released within minutes after degranulation of the cell [39]. After this primary response, a second wave of newly synthesized mediators are released and include PGD2, LTC4, LTD4, and LTE4. In the late phase allergic response, cytokines (IL-4, IL5, IL-6, IL-8, IL-13, and TNF-␣) are induced and secreted [39]. Expression of this host of cytokines supports the logical proposal for a role for mast cells in host defense. Stimulation of the enteric nervous system by mast cell activation is also likely to play an important role in mast-cell–mediated host defense [40,41], and in general, mast cell–nerve interactions have been interpreted as important neuronal tissue repair mechanisms following injury [42,43].

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III. MAST CELL/NERVE ASSOCIATION Nerve–mast cell associations have been reported within peripheral nerves, myelinated nerves, unmyelinated nerves, neurofibromata, and neuromata. A morphometric study in infected and in healthy rat intestine showed that mast cells and nerves were closely and invariably approximated in rat intestinal villi [44]. Electron microscopy showed evident membrane-membrane association between mucosal mast cells and nerves with dense core vesicles at the points of contact. The nerves in contact with mast cells contained either substance P (SP), CGRP, or both. The association appeared not to be random [45], and was also described in the human gastrointestinal tract [4]. Similar observations have been made in a variety of different tissues in many species. Other than the intestine, nerve–mast cell associations are also found in rat trachea and peripheral lung tissue [3], skin [46], urinary bladder [47], brain [48], and several other tissues [49,50]. Rozniecki et al. [51] provided evidence for morphological, anatomical, and functional interactions of dura mast cells with cholinergic and peptidergic neurons containing substance P and CGRP. Mast cells have been shown to be abundant in the dura, and they contain a substantial proportion of total brain histamine.

IV. MAST CELL ACTIVATION Mast cells can be activated by IgE-dependent and independent mechanisms. Classically, they are associated with hypersensitivity reactions, involving interaction with IgE [9]. However, mast cells also play a prominent role in non–IgE-mediated hypersensitivity reactions [52,53]. The sensitivity of mast cells to activation by nonimmunological stimuli such as polycationic compounds, complement proteins, superoxide anions, or neuropeptides is dependent on the population of the mast cells examined [54]. Tachykinins can induce mast cell activation via a receptor-dependent mechanism. Activation of the neurokinin receptors is dependent on the C-terminal domain of the tachykinins [55]. C-terminal fragments of substance P cause histamine release from the mouse mast cell line MC/9 via an NK-2 receptor–mediated pathway [56]. Cooke and coworkers [57] demonstrated that RBL-2H3 cells, a mast cell line homologous with MMC, express the high-affinity NK-1–binding sites for substance P on their surface. While it is widely accepted that NK-1 receptors are not generally expressed on mast cells, little is known about their expression in inflammation. Mantyh and coworkers [58,59] have shown that NK-1 receptors were significantly upregulated in inflamed tissues, on epithelium, blood vessels, and in lymphoid accumulations. Karimi et al. [60,61] showed that SP (in the micromolar range) causes dose-dependent degranulation in bone marrow–derived murine mast cells (BMMC) primed with IL-4 and SCF. We ourselves have recently found that murine BMMC cultured with IL-4 and SCF express NK-1 receptors (H. P. M. van der Kleij et al., unpublished). The tachykinins substance P and neurokinin A can, on the other hand, induce mast cell activation via a receptorindependent mechanism. Nonimmunological stimuli such as the basic secretagogues trigger mast cell exocytosis though a mechanism called the peptidergic pathway. This family of polycationic compounds includes positively charged peptides such as substance P, various amines such as compound 48/80, and naturally occuring amines [28]. Instead of interacting with a membrane-bound receptor, the basic secretagogues appear to directly activate and bind to pertussis toxin–sensitive GTP-binding pro-

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teins (G-proteins) through the N-terminal domain located in the inner surface of the plasma membrane [62,63]. Stimulation of G-proteins will activate a signal transduction pathway (the peptidergic pathway), eventually leading to mast cell mediator production and release. There is significant potential for this pathway of mast cell activation to influence the net contribution of mast cells to both tissue physiology and pathology. For example, Janiszewski and coworkers [64] used patch clamp electrophysiology to show that mast cells did not respond to an initial application of very low concentrations of substance P (in the picomolar range), but that both activation and delayed degranulation occurred after a second exposure. Therefore, mast cells can be primed when exposed to physiologically relevant low concentrations of substance P and lower their thresholds to subsequent activation. A. TNF-␣ TNF-␣ is one of the main preformed mediators immediately released upon mast cell degranulation. In addition, newly synthesized TNF-␣ can be secreted by mast cells within 30 minutes following certain stimuli [65]. Furthermore, TNF-␣ is also able itself to induce mast cell degranulation. This, and the fact that TNF-␣ has been shown to affect sensory neurons, makes it an interesting potential mediator in nerve–mast cell communication. TNF-␣ may play a major beneficial role in host defense by mast cells against bacterial infections. Malaviya et al. [40] showed that mast cells may be essential for defense against bacteria in a cecal puncture model and that they mediate bacterial clearance by initiating neutrophil influx. They propose that the recruitment of circulating leukocytes is dependent on the mast cell mediator TNF-␣. Echtenacher et al. [41] showed that reconstitution of mast cell deficient W/Wv with mast cells prevented death from bacterial peritonitis, as did the injection of TNF-␣. Maier et al. [66] showed that subdiaphragmatic vagotomy prevented the pyrexia induced by intraperitoneal injection of endotoxin and that this was associated with the generation of TNF-␣. It is tempting to put this information together with the role of mast cells in innate defense against bacteria, but the crucial experiments have yet to be performed. TNF-␣ is involved in changing neuronal cell function as it can modulate the susceptibility of neurons to an electrical stimulus [67]. The sensitizing effect of TNF-␣ seems to primarily target C-fibers [68]. In vitro incubation of rat sensory nerves with TNF-␣ enhanced the response of C-fibers to capsaicin [69]. According to Junger and Sorkin [68], TNF-␣ causes a subpopulation of C fibers to develop spontaneous activity, which also results in local release of neuropeptides, like substance P and CGRP, from afferent fibers. This is the first time that TNF-␣ has been shown to be directly capable of releasing neuropeptides from C fibers, in addition to their priming ability, proposed by others. These authors suggest that this acute sensitization is due to a fast mechanism such as binding to TNF receptors or activation of a constitutive COX enzyme leading to eicosanoid synthesis. TNF-␣ can enhance the release of arachidonic acid and the synthesis of eicosanoids, in particular prostaglandin E2 (PGE2), which acts directly on the sensory neuron [70]. Nicol et al. [69] also observed that TNF promoted the induction of COX-2 in sensory neurons. Their results demonstrate that selective inhibition of COX-2 prevents the TNFinduced lowering of threshold to activation by capsaicin. However, these authors found that TNF-␣ had a delayed time course of action. The discrepancy in findings suggests that there might be more than one process involved.

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B. Tryptase The major mast cell protease is tryptase, a serine protease that is abundantly present in all mast cells and is stored in a fully active form in the granules. Among the subsets of human mast cells, tryptase can comprise up to 25% of total cellular protein. The functions of tryptase include mitogenic actions on fibroblasts, smooth muscle cells, and epithelial cells and stimulation of ICAM-1 expression by epithelial cells [71]. A recent finding of great importance is that proteases such as mast cell tryptase-act on protease activated receptors (PAR), in which the peptide ligand is physically part of the receptor molecule [72]. Protease activity activates the PAR in an irreversible manner by cleaving within the extracellular N-terminus a tethered ligand domain that then binds to the receptor (e.g., Refs. 73–75). Mast cell tryptase has inflammatory effects on many cells, mediated by the cleavage and activation of PAR2. These include neurons and glia in the central nervous system and in the enteric nervous system, where myenteric neuron PAR2 expression has been detected by RT-PCR. Tryptase has recently been shown to cleave PAR2 on primary spinal afferent neurons, causing the release of substance P, activation of the NK-1 receptor, and amplification of inflammation and thermal and mechanical hyperalgesia [76]. Corvera et al. [77] demonstrated that purified tryptase stimulated calcium mobilization in myenteric neurons. They hypothesized that tryptase excites neurons through PAR2, because activation of PAR2 with trypsin or peptide agonists strongly desensitized the response to tryptase. In addition, a tryptase inhibitor suppressed calcium mobilization in response to degranulation of mast cells. Recent investigations with the use of tryptase inhibitors have implicated tryptase as a mediator in the pathology of various allergic and inflammatory conditions, including arthritis, rhinitis, and most notably asthma. Krishna et al. [78] showed that the selective tryptase inhibitor APC366 inhibited antigen-induced early and late asthmatic responses and bronchial hyperresponsiveness in a sheep model of allergic asthma. Again in the respiratory system, the intratracheal administration of AMG12637, a potent inhibitor of human mast cell tryptase, inhibited the development of airway hyperresponsiveness in allergen-challenged guinea pigs. In both proximal and distal bronchi of nonsensitized humans, the reactivity to histamine was significantly increased by previous incubation with tryptase (1 ␮/mL) [79]. This effect was completely abrogated in the presence of the protease inhibitor benzamidine. The inhibitor APC366 has furthermore been shown to inhibit IgE-dependent histamine release in a dose-dependent manner, with about 70% inhibition being achieved at a dose of 300 ␮M. This study was performed with human synovial mast cells, showing that inhibitors of tryptase could be of therapeutic value in arthritis [80].

V. THE CONCEPT AND SIGNIFICANCE OF PRIMING Priming is a process that increases cellular responsiveness to subsequent stimulation. In other words, an initial event lowers the threshold for response to a subsequent event, such as (re)exposure to a given agonist or mode of stimulation. Priming appears to be a broadbased biological process and has been reported in many cell types. Basophils and mast cells have been reported to be primed in this sense by many different cytokine growth factors for subsequent different agonists [81]. SCF, for instance, can act as a priming agent in some circumstances [82]. Coleman et al. [83] observed that SCF and IL-3 upregulated

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responses to IgE-dependent stimuli. Since the expression of the IgE receptors was not altered, the mechanism behind this remains unclear. Priming may be a prominent aspect of nerve–mast cell interactions. Karimi et al. [61] showed that SCF- and IL-4–primed bone marrow–derived murine mast cells to show increased responsiveness to subsequent challenge by substance P. Although relatively high levels of substance P are necessary on a single challenge to induce mast cell degranulation (⬎10ⳮ5 M), it is possible for repeated doses of very low (picomolar) concentrations of substance P to act via the priming process to cause mast cell degranulation. Janiszewski and coworkers [64] reported that mast cells responded electrophysiologically to very low concentrations of substance P but without degranulation. However, degranulation occurred with subsequent exposure to the same low dose. This raises the question as to whether substance P is more a priming substance than a substance causing direct degranulation. It is interesting that cellular activation followed by mediator release is not the same as degranulation of the cell. Exocytosis is the most obvious event associated with secretion of the mediator molecules contained in granules. However, secretion can occur without evidence of degranulation, and even molecules stored within the same granules can be released and secreted in a discriminatory pattern. For example, serotonin can be released separately from histamine [84]. Moreover, low doses of substance P can cause synthesis and secretion of TNF-␣ from mast cells in the absence of degranulation [85,86] Both stimulatory and inhibitory effects are seen with priming. Thus, NGF caused the synthesis and secretion of IL-6 and PGE2 by rat peritoneal mast cells but inhibited the release of TNF-␣ [86]. Further, differential synthesis and release of arachidonic acid metabolites, prostaglandins, and leukotrienes as a result have been reported [87]. Structural evidence from intact tissue supports a physiological role for the differential release of mast cell mediators. An ultrastructural study by Ratliff et al. [88] showed that mast cells in close proximity to unmyelinated nerve fibers had granules showing ultrastructural features of activation or piecemeal degranulation, a process associated with differential secretion [84]. A subtle role for this process in regulation of inflammation is supported by evidence in vitro that peritoneal mast cells can synthesize and release IL-6 without mast cell degranulation, as monitored by histamine release [89]. In fact, the presence of nerve-associated activated mast cells that do not display anaphylactic degranulation, while found routinely in all tissues, has been suggested as a characteristic feature of interstitial cystitis [47,84]. The concept of priming also applies to neurons. Nicol et al. [69] demonstrated that prior exposure to TNF-␣ can enhance the sensitivity of sensory neurons to the effects of capsaicin. However, Sorkin et al. [90] showed that TNF-␣ is not able to evoke peptide release from peripheral afferent terminals, but enhances capsaicin-evoked release of neuropeptides. Thus, TNF-␣ may exert a priming, rather than a direct stimulatory effect on sensory activity. Considering that mast cells closely approximated to nerves will be exposed to locally released neuropeptides, we propose that priming enhances the sensory effectiveness of the mast cell–nerve physiological unit. VI. STIMULATION OF NERVES CAUSES ACTIVATION OF MAST CELLS Electrical stimulation of nerves has been shown to cause either ultrastructural changes in associated mast cell granules or actual degranulation, supporting the idea that mast cells

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are indeed in direct communication with nerves. MacDonald et al. [91] showed that vagal stimulation in the rat caused neurogenic inflammation in the trachea. Leff [92] have shown that vagal stimulation caused an enhancement of the secretion of histamine from mast cells after challenge of allergic dog lungs. Dimitriadou et al. [93] showed that electrical stimulation of the ipsilateral trigeminal nerve caused activation of the dura mater mast cells, as evidenced by piece meal degranulation. Recently, electrical stimulation of the frog hypoglossal nerve was shown to cause mast cells to undergo progressive time-dependent activation [15]. Gottwald et al. [94] reported that electrical stimulation of the vagus caused moderate to marked edema in the jejunum of stimulated rats in comparison to control rats. The four fold increase in tissue histamine levels in the jejunum was suggested to reflect mast cell activation. Bani-Sacchi and coworkers [95] observed that field stimulation of rat ileum resulted in histamine release and an attenuation in mast cell granularity. This effect was decreased by atropine or tetrodotoxin, a nerve cell toxin. In the brain, granule changes consistent with activation of dura mast cells were observed after sensory afferent stimulation, and increased levels of tissue serotonin were recorded after sympathetic stimulation [93] On the other hand, Miura et al. [96] showed that antigen challenge to sensitized cats caused increased bronchial resistance and an increase in plasma histamine levels. However, in animals pretreated with cholinergic and adrenergic blockers, bilateral electrical stimulation of the vagus nerve caused complete inhibition both of the generation of bronchial resistance and elevation of plasma histamine levels. These data show that the nonadrenergic noncholinergic (NANC) system in cats is able to inhibit mast cell degranulation induced by antigen. Many of the experiments that have shown mast cell degranulation upon electrical stimulation have also shown that these effects were inhibited by atropine or prior treatment with capsaicin, a substance that permanently depletes sensory nerves of substance P and destroys unmyelinated sensory axons. For instance, vagal stimulation does not cause neurogenic inflammation in the airways of rats treated at birth with capsaicin. Furthermore, antidromic nerve stimulation causes mast cell degranulation in the skin, but is absent following neonatal capsaicin treatment [97]. In contrast, Baraniuk et al. [98] proposed that electrically induced neurogenic inflammation in the superficial dermis of the rat skin is a direct response to neuronal release of neuropeptides and that mast cell degranulation is not involved. Their study showed that mast cell activation did not occur except on prolonged stimulation. Another study performed in the airways indicated that both neurogenic increased vascular permeability and plasma exudation into the airway lumen resulted from activation of capsaicin-sensitive sensory nerves without the association of mast cell activation [97]. However, Yano and colleagues [99] demonstrated that mast cell–deficient mice did not develop ear edema or inflammation after substance P injection; in contrast, mice reconstituted with mast cells did show edema. This suggests that substance P needs a mast cell target to cause vascular permeability. In conclusion, there is some disagreement among available studies on the participation of mast cells in neurogenic inflammation. This may reflect differences in the duration of stimulation, the time schedule that was used, the various tissues, and species-specific factors. Overall, there is good evidence supporting the involvement of blood vessels, nerves, and mast cells in neurogenic inflammation, and the direct regulatory effect of nerves upon mast cells.

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VII. NERVE GROWTH FACTOR NGF was the first discovered member of the family of neurotrophins in the 1950s, now including brain-derived neurotrophic factor (BDNF) and neurotrophins 3–5. Nerve growth factor is the best characterized neurotrophic protein and is required for survival and differentiation of neuronal cell types in both the peripheral and central nervous system [100]. For example, removal of circulating NGF has been shown to result in the death of sympathetic neurons [101]. The biological activities of NGF are mediated by binding to two receptors: trkA, a tyrosine kinase receptor, and p75, a low-affinity receptor. In addition to neurons, nonneuronal cells such as mast cells [102], T cells [103], B cells [104], eosinophils, lymphocytes [105], fibroblasts [106], and epithelial cells [107] can synthesize NGF. Many of these inflammatory cells express the high-affinity NGF receptor, which allows NGF to promote inflammatory mediator release. Several of these inflammatory mediators such as IL-1, IL-4, IL-5, TNF-␣, and IFN can, in turn, induce the release of NGF [106,108]. Therefore, NGF seems to be a mediator with functions on both immune and nerve cells and is likely an important factor integrating communication between the nervous and immune systems. NGF acts as a chemoattractant and thereby causes an increase in the number of mast cells as well their degranulation [21,86,109]. NGF receptors on mast cells act as autoreceptors, regulating mast cell NGF synthesis and release, while at the same time being sensitive to NGF from the environment. Inflammation can lead to an enhanced production and release of NGF. In turn, NGF induced the expression of neuropeptides and lowered the threshold of neurons for firing [110] In mice, Braun et al. [111] have shown that nasal treatment of mice with NGF induced airway hyperresponsiveness to subsequent electrical field stimulation. In an earlier study, Braun et al. [112] showed that nasal treatment of mice with anti-NGF prevented the development of airway hyperresponsiveness. Also, NGF-transgenic mice that overexpress NGF in Clara cells showed bronchial hyperreactivity in comparison to wild-type mice [113]. These data suggest that NGF by itself can induce airway hyperresponsiveness in the absence of airway inflammation in mice. Neurogenic inflammation involves a change in function of sensory neurons due to inflammatory mediators, thereby inducing an enhanced release of peptides from sensory nerves. NGF is able to augment neurogenic inflammation and can upregulate the synthesis of products of the PTT gene [114], which codes for several tachykinins such as substance P and NKA. Furthermore, NGF changes the properties of peripheral sensory nerve endings by inducing an accumulation of second messengers or by sensitizing nerve terminals [115]. In vivo administration of NGF into neonatal rats caused a great increase in the number and size of mast cells in the peripheral tissues [116]. Studies have provided evidence that mast cells, similar to nerve cells, express the trkA NGF receptor, suggesting that mast cells are receptive to NGF [117,118]. Furthermore, NGF has been shown to induce degranulation and histamine release from mast cells [119,120]. To complete the circle, mast cells are capable of producing NGF [102] and mRNA for NGF is expressed in adult rat peritoneal mast cells. Medium conditioned by peritoneal mast cells has been shown to contain biologically active NGF. Therefore, it is not surprising that injection of NGF causes mast cell proliferation in part by mast cell degranulation [21]. A study by Bonini and colleagues [121] showed an increase in serum NGF in humans with allergic diseases. The more severe the disease, the higher the NGF values found in the tissues of allergic patients. In asthma patients, a significant increase in the neurotrophins

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NGF, BDNF, and NT-3 has been found in the BAL fluid 18 hours after allergen challenge [122]. NGF levels are increased in the nasal secretions within 10 minutes after allergen challenge in allergic rhinitis patients [123]. It could be hypothesized that mast cells are a major source for NGF in allergic diseases, although a variety of other cells including T and B cells, eosinophils, lymphocytes, and epithelial cells are also capable of synthesizing NGF. The case in support of mast cell involvement is supported by evidence from several allergic animal models in which substance P synthesis is increased [124]: the increase seems to be mimicked by the administration of NGF 24 hours after application, implying that mast cell activation induces NGF release, thereby inducing the increase in substance P.

VIII. THE BRAIN, STRESS, AND THE IMMUNE SYSTEM The nervous and immune systems are the major adaptive systems of the body [125]. Teleological arguments suggest that they are in contact with each other to maintain homeostasis. Several pathways have been shown to link the brain and the immune system, such as the autonomic nervous system acting via direct neural influences, and second, the neuroendocrine humoral outflow via the pituitary. Furthermore, the sympathetic nervous system provides another important regulatory pathway between brain and immune systems [125]. The sympathetic nervous system and the hypothalamic-pituitary-adrenal (HPA) axis are the peripheral and central limbs of the stress-response system, whose main function is to maintain basal and stress-related homeostasis [126]. The key components of this system are located in the brain stem. The stress system is even active when the body is at rest, responding to many signals, including those from cytokines produced by immunemediated inflammatory reactions, such as TNF-␣, IL-1, and IL-6 [127]. Activation of the system changes cardiovascular function, accelerates motor reflexes, increases the tolerance to pain, and affects immune function (e.g., Ref. 128). The adaptive changes to stressors are both behavioral and physical. Initially, the body responds with an adaptive response to the stressor. For example, acute stress actually stimulates immune responsiveness, whereas chronic stress inhibits it [129]. Once a certain threshold has been exceeded, a reaction takes place that involves the brain, the HPA axis, and the sympathetic nervous system [130]. Corticotropin-releasing hormone (CRH), secreted by the pituitary gland, is a major regulator of the HPA axis and cortisone synthesis and acts as a coordinator of the stress response. Mast cells in the central nervous system may participate in the regulation of inflammatory responses through interactions with the HPA axis. Matsumoto et al. [19] showed that in the dog, degranulation of mast cells evoked HPA activation in response to histamine release. In this study, dogs were passively sensitized with IgE and challenged with specific antigen centrally or peripherally. Both routes resulted in cortisone release from the adrenal glands. The effect could be mimicked by intracranial injection of the mast cell secretagogue compound 48/80 and blocked by CRH antibodies or histamine H1 blockers but not H2 blockers. These results suggest that intracranial mast cells may act as allergen sensors and that the activated adrenocortical response may represent a host defense reaction to prevent anaphylaxis. CRH is also thought to be involved peripherally in tissue responses to stress in the skin, respiratory tract, and intestine. Many, if not all the recorded changes have involved

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mast cells and neuronal activation, the latter being often mediated by neurotensin and/or substance P. Theoharides and coworkers were the first to show that CRH could act as a mast cell degranulating agent in the dura mater [131], the skin [132], and the bladder [132,133]. Others have extended these observations. Pothoulakis and coworkers have studied the intestinal responses to stress [134]. They concluded that increased intestinal motility, mucus hypersecretion, and intestinal chloride ion secretion, as judged by increased short circuit current generation by intestinal tissue in ussing chambers, was mediated by CRH. They inhibited these mostly by intracerebral or systemic injection of a specific CRH inhibitor. Furthermore, both mast cells and nerves were involved, since the effects were inhibited by NK-1 antagonists and mast cell stabilizers. Santos and coworkers have shown that ganglionic blockers and inhibition of cholinergic as well as sympathetic activity also blocked these effects [135]. They did not occur in mast cell–deficient rats, and they occurred equally in a chronic stress model [136]. Moreover, increased intestinal permeability to macromolecules occurred after stress and was also abrogated with these pharmacological inhibitors. The sequence of events that occurs and how these various systemic effects are mediated are just beginning to be explored. These studies, taken together, show that the physiological effects of psychological stress are often largely mediated by CRH, released either centrally or peripherally, and that mast cell–nerve interactions are important components of this response. IX. THE MAST CELL IN THE NERVOUS SYSTEM The mast cell–nerve communication pathway has been implicated as an important element in the pathophysiology of various diseases. Stimuli can affect the primary afferent sensory nerve response, the integrative response into the CNS, the response within the autonomic ganglion, and the response at the postganglionic autonomic site within the tissue. For instance, allergen challenge and mast cell degranulation lead to substantial changes in neuronal function [137]. Changes in neuronal activity lead to symptoms such as sneezing, coughing, irritation, itching, and cutaneous flare reactions, key symptoms of allergy, providing direct evidence that neurons are involved in allergic diseases such as asthma and gastroenteritis [138,139]. Thus, allergen challenge can lead to a simple reflex that can affect neuronal activity on a variety of levels in this pathway. The most evident neuronal response to mast cell activation is the stimulation of sensory nerves, releasing tachykinins such as substance P, CGRP, and neurokinin A to cause neurogenic inflammation [140,141]. The stimulation of C-fibers by a range of chemical and physical factors results in afferent neuronal conduction eliciting parasympathetic reflexes and antidromic impulses traveling along the peripheral nerve terminal. Such communication from one nerve to another, without passing through a cell body, is called the axon reflex and results in local release of tachykinin and CGRP from C-fiber terminals [142]. Axon reflexes account for many of the local physiological responses to antigen in sensitized lung [143,144] and gut tissues [145,146] and have long been recognized to be involved in local vasodilatation in the skin. Antidromic stimulation of guinea pig vagal sensory fibers results in contractions of the airway smooth muscle and is mediated by tachykinins [147]. Further studies indicate that neuropeptide release can also be induced via direct depolarization of the terminal and, possibly, via a chemically mediated mechanism independent of electrical excitation [148].

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Mast cells have been shown to be capable of affecting nerve function [1,149]. Arachidonic acid metabolites, serotonin, nitric oxide, nerve growth factor, and histamine have been suggested to affect neuronal function [1,20]. Furthermore, NANC nerve endings express receptors for histamine (H1 and H3) and serotonin (5-HT2a) [150–152], and histamine H1 receptor expression at least is upregulated in primary NANC nerves in inflammation [153]. Mediator release from mast cells can be induced by neurotrophic factors such as NGF [154] and substance P [51,155]. Stimulation of nerve fibers of humans and mammals induces mast cells to degranulate and release histamine and other mediators such as serotonin [156] and TNF-␣ [85]. Thus, mast cell mediators can, on the one hand, sensitize afferent C fibers by lowering their threshold [157] and, on the other hand, cause release of substance P and CGRP from unmyelinated fibers [158]. X. IN VITRO STUDIES Because mast cells are often found in the proximity of nerve endings and may be activated by a number of neuropeptides, including tachykinins, mast cells are strongly implicated in neurogenic inflammation. The tissue site as well as the local environment affects the state of relative activation. Close proximity to nerves of all kinds allows mast cells to communicate with and be communicated with by the nervous system. Burnstock has shown that cells within 200 nm of a nerve can be affected by an action potential moving down a nerve fiber [159]. This lends more meaning to the likelihood of two-way communication. Morphometric analysis has shown mast cells associated with nerves along their axial path, for example, in the intestine [4,160]. A tissue culture model of murine sympathetic neurons cultured with rat basophilic leukemia cells (RBL-2H3; a model of mucosal mast cells) was developed to study functional interactions between mast cells and peripheral nerves. Time-lapse microscopy showed that 60–100% of the mast cells acquired neurite contact within 17 hours of coculture [161], a process facilitated by development of lasting contact between nerves and mast cells when the intervening distance was less than 36 Ⳳ 4 ␮m. NGF synthesized by mast cells is likely to contribute to this trophic effect [102]. Further studies showed that activation of mast cells in vitro had the capacity of altering neuronal physiology by inducing depolarization and decreasing membrane resistance [162]. Contact between nerves and mast cells also had developmental consequences in vitro, since attached RBL-2H3 cells ceased to divide and showed an increase in granules compared to control cells without contact, an indication of maturation of the mast cell [163]. Thus, sympathetic neurons have the ability to change the state of RBL-2H3 cells in coculture. Contact between mast cells and neurites, once formed, was maintained up to 120 hours, while the associated neurites often branched after contact. Electron microscopic examination of the sites of contact showed on average ⬍20 nm distance between apposed membranes. While no specialized structures were seen either in the neurite or the closely approximated mast cell, dense cored neurosecretory vesicles accumulated in the neurite endings apposed to the mast cell membrane [161]. Suzuki and coworkers have shown direct communication between mast cells and superior cervical neurons in coculture [164,165]. Calcium-binding dyes were used to show activation and signaling with confocal microscopy. Scorpion venom and bradykinin were used to activate the neurites, which in turn led to calcium increases in the attached RBL2H3 cells. The ability to block with an NK-1 receptor antagonist showed that the signaling was dependent on substance P. Furthermore, application of an anti-IgE antibody showed

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initial RBL-2H3 activation followed by neurite activation of the associated nerve fiber, providing evidence for bi-directional communication. XI. THE CENTRAL NERVOUS SYSTEM Mast cells are resident in the brain of many species [20] and can also move though the brain in the absence of inflammation, apparently entering via penetrating blood vessels. Brain mast cells are mainly perivascular but are abundant in the thalamus and hypothalamus, only a small number of mast cells occur in the cerebral cortex and basal ganglia [1]. Large numbers of tryptase-containing mast cells have been described surrounding the pituitary gland [166]. These mast cells can respond to antigen and regulate CRH secretion via histamine effects [167]. There is, however, considerable variability in the number and distribution of mast cells among individuals, species, strains between the same species, and sexes [20]. Moreover, the localization of mast cells in the developing rat brain differs from that of the adult animal [168]. The physiological significance of mast cells in brain function and/or metabolism is unclear. However, they can modulate neuroendocrine control systems [1], and they may play a role in the regulation of meningeal blood flow and vessel permeability [169]. A functional interaction between brain mast cells and neurons may be an important neurobiological process, since mast cells have been found in close association with neurons containing substance P and CGRP, neuropeptides that can in turn activate dura mast cells [48,170,171]. Histamine secretion from brain mast cells, in response to local neurotrophic factors and neuropeptides, may contribute to the etiology of neuroinflammatory conditions, such as multiple sclerosis and Alzheimer’s disease [172]. Mast cells and CRH have been to play a significant role in migraine [131]. Substance P and compound 48/80 each have been shown to induce histamine secretion from rat brain mast cells in a concentrationdependent manner [1]. Also, the growth factors NGF and BDNF trigger histamine release from rat brain mast cells, suggesting that these cells contain the TrkA and TrkB receptors [154]. The ability of brain mast cells to secrete histamine and possibly other mediators following exposure to these neurotrophic factors supports their role in the neuroimmune cross-talk. One of the most striking examples of the intimate relationship and functional importance of mast cells and the brain occurs as a result of studies of the courting behavior of doves. Silverman and coworkers [20] have demonstrated that a population of mast cells is present in large numbers in the medial habenula (MH) of the ring dove after a brief period of courtship. There were fewer mast cells in birds housed in isolation, and mast cell numbers were further reduced in long-term castrates [173]. This suggests that the appearance of mast cells is related to the behavioral state of the animal and proposes a novel mechanism for interactions between the nervous and the immune systems. XII. THE GASTROINTESTINAL TRACT The gastrointestinal lamina propria is densely innervated, and mast cells are commonly observed near these nerves. Careful analysis showed that 50–75% of mast cells are closely apposed to nerves in both rodents and humans [174]. Electron microscopy showed nerve terminals containing predominantly small clear neurosecretory vesicles in direct contact with the plasma membrane of mucosal mast cells in the rat ileum [49]. Increased numbers

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of mast cells are a hallmark of inflammatory or allergic conditions, and increased numbers of mast cell–nerve contacts appear in the infected/inflamed condition. For instance, nematode infection of the rat intestine causes the frequency of mast cell–nerve associations in the intestinal mucosa to increase above that of the normal counterparts [26]. In the course of inflammation induced by a nematode (Nippostrongylus brasiliensis) in rat intestine, imaging and histochemistry we used to quantitate changes in nerves. New, small-diameter neurites staining for B50, found in new growth axons, increased in number and persisted well beyond the time that inflammation could be detected. There was a very strong correlation between mast cell numbers and the number of small-diameter neurites [175]. Nerve stimulation can directly cause mast cell degranulation in the intestine, and release of peptidergic neurotransmitters is the most likely mechanism. Electrical stimulation of the vagus has been shown to cause either intestinal mast cell degranulation [176] or an increase in mast cell histamine content, which did not occur after subdiaphragmatic vagotomy [177]. Shananan et al. [178] showed that substance P caused mediator release from intestinal mucosal mast cells. Mast cell mediators also appear to have an effect on the nerves in the intestine. For example, Jiang et al. [179] showed that serotonin and histamine, released from the mast cells after intestinal anaphylaxis, stimulated mesenteric afferents via 5-HT(3) and histamine H(1) receptors. Mesenteric afferent nerve discharge increased approximately 1 minute after luminal antigen challenge and was attenuated by serotonin and histamine receptor antagonists. As already mentioned, tryptase, the major serine protease in mast cells, activates nerves via the PAR2 receptor [77]. Overall, it can reasonably be concluded that nerves and mast cells form a physiological unit, which maintains and regulates homeostasis of diverse aspects of intestinal function, in health, in response to stress and in response to injuries and environmental pathogens. We present some examples below of functional nerve–mast cell interactions in the gastrotestinal tract. Perdue et al. [180] determined the existence of an integral nerve–to–mast cell and mast cell–to–nerve connection during intestinal anaphylaxis. A role for the mast cell–to–nerve connection was established by an increase in the epithelial short-circuit current (Isc) after antigen challenge, which was inhibited by antagonists to histamine, serotonin, cyclooxygenase, and nerve conduction. The intestinal secretory response to antigenic stimulation was reduced in mast cell–deficient (W/Wv) mice compared to their littermates and could be inhibited by different mast cell antagonists in Ⳮ/Ⳮ but not in W/Wv mice, pointing to functional mast cell–to–nerve connections. Importantly, they also showed that electrical field stimulation causing neural activation induced changes in Isc dependent on the presence of mast cells. In further study of this communication using sensitized guinea pig intestine, Cooke et al. showed [57] that the exposure to specific antigen caused acetylcholine release at the same time as the secretory response and that this was blocked by atropine. These data, then, provided evidence for a bi-directional communication between nerves and mast cells in the regulation of ion transport in the gastrointestinal tract, similar to results obtained in the rat lung by Sestini et al. [143]. The diarrhea caused by overgrowth of Clostridium difficile is an intriguing example of the importance of nerve–mast cell communication in the intestine. This enteritis is a significant cause of morbidity after antibiotic therapy and involves a secretory diarrhea due to the production of bacterial toxin A. Castagliuolo et al. showed that toxin A activated capsaicin-sensitive intestinal afferent nerves, which in turn led to mast cell activation, and that this could be blocked by NK1 receptor antagonists [181]. This effect was quite distinct

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from the actions of the non-inflammatory cholera toxin, illustrating the pathological consequences of dysregulation of the nerve–mast cell interaction. XIII. CONCLUSION Topographical associations between mast cells and nerves have now been recorded in most tissues in the body. These now include the stomach, the small and large intestine, blood vessels, mesentery, gall bladder, diaphragm, pleura, skin, urinary bladder, and myocardium. Many of these morphological associations have been shown to occur between substance P and CGRP containing neurons and mast cells of all subspecies. These associations have even been recorded in the frog tongue. The cross-talk between mast cells and nerves is therefore extensive, and it is not surprising that electrical stimulation of nerves can result in both activation/degranulation as well as inhibitory signals, apparently depending on the type of nerve found in association with particular mast cells. The role of this bi-directional communication between the nervous and the immune systems appears to be multifactorial. The maintenance of local homeostasis in skin, blood vessels, and mucosal tissue seems well established. Mast cells are situated in a particularly appropriate position to act as sensors of environmental change, and in this respect their positioning in skin and mucosal tissues make them sentinels for detection of antigens to which the host has been exposed or noxious chemicals and other substances. The communication with the nervous system allows the peripheral and central nervous system to become involved in the regulation of defense mechanisms and inflammation and in response to infection. The further involvement of mast cell–nerve communication in the regulation of blood flow, tissue remodeling, and repair is a natural extrapolation from this information. Clearly, the involvement of mast cells with nerves can affect nerve growth, conduction, transmission, and the central nervous system, the regulation of neuroendocrine systems, such as those involved in the regulation of adrenal hormone secretion (e.g., cortisol). The surprising involvement of nerve–mast cell communication in responses to stress, the neuroendocrine involvement referred to before, the role of mast cells in the courting behavior of doves, all point to extensive development of communication development between nervous and immune systems. Many of these pathways are redundant. How many of them are essential for life remains to be determined, since it is not apparent that mast cell–deficient mice or rats with mutations in the c-kit pathway have shorter-than-normal life spans. However, the absence of mast cells does render these animals more sensitive to infections in experimental models. Despite this observation, it is reasonable to assume that mast cell–nerve bi-directional communication has arisen and developed in evolution as a complementary set of pathways to others that also exist. While not essential for life, they may be important in adding to the diversity of responses by individuals to environmental change. Lastly, the reader should recognize that, while mast cell–nerve interactions have been focused on in this chapter as an example of neuroimmune interaction, similar pathways may, and likely do, exist in which other cells of the immune system may have analogous modes of communication. REFERENCES 1. Purcell WM, Atterwill CK. Mast cells in neuroimmune function: neurotoxicological and neuropharmacological perspectives. Neurochem Res 1995; 20(5):521–532.

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19 Peripheral Nervous System Programming of Dendritic Cell Function GEORGES J. M. MAESTRONI Istituto Cantonale di Patologia, Locarno, Switzerland

I. ROLE OF DENDRITIC CELLS Dendritic cells (DC) are a sparse population of bone marrow–derived antigen-presenting cells, irregular in shape and widely distributed in both lymphoid and nonlymphoid tissues [1,2]. Those having migrated to nonlymphoid tissues such as the epidermal layer of the skin, the respiratory and gastrointestinal systems, and interstitial regions of solid organs are considered immature. Fully mature DC are located in lymphoid organs. After antigen internalization and inflammation, DC leave the tissues interfacing with the external environment and enter the lymphatic vessels to reach the lymphoid organs and undergo maturation [1–3]. While still immature, the primary function of DC is to phagocytize and process antigens, then to present the antigenic peptides and activate specific T cells [1,2]. Activation of naive T cells requires two signals. The first signal is delivered when the TCR engages the MHC/antigen complex, and the second, costimulatory signal is delivered by costimulatory molecules on DC [4]. Failure to deliver a costimulatory signal with antigen presentation induces a state of T-cell anergy [5]. At least two potent costimulatory signal pathways are essential for normal development and maintenance of immunity. In one system CD40 on DC interact with CD40 ligand (CD154) proteins expressed on activated T cells. In the B7/CD28 pathway, DC surface B7.1 (CD80) and B7.2 (CD86) molecules deliver a costimulatory signal by interacting with the CD28 cell surface protein expressed on resting T cells [4,6]. Activation of naive T-helper (Th) cells results in their polarization toward the Th1 and/or Th2 type, which orchestrates the immune effector mechanism that is more appropriate for the invading pathogen. Th1 cells promote cellular immunity, 381

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protecting against intracellular infection and cancer, but carry the risk of organ-specific autoimmunity. Th2 cells promote humoral immunity, highly effective against extracellular pathogens, and are involved in tolerance mechanisms and allergic diseases. The initiation and type of adaptive immune responses is controlled by innate immune recognition, which is mediated by DC that produce interleukin-12 (IL-12), a prerequisite for both the activation of innate immunity and the development of Th1 responses [7,8]. II. SYMPATHETIC NERVOUS SYSTEM–IMMUNE SYSTEM CROSSTALK A continuos dialogue between the brain and the immune system is a prerequisite for homeostasis. A major pathway involved in this cross-talk is the sympathetic nervous system. Primary and secondary lymphoid organs are extensively innervated by sympathetic fibers that release norepinephrine (NE), which in turn acts on adrenoceptors (AR) expressed on target immune cells. Furthermore, activation of antigen-specific CD4Ⳮ cells and B cells was shown to increase NE release in the spleen and bone marrow [9]. The AR mediate the functional effects of epinephrine and norepinephrine by coupling to several of the major signaling pathways modulated by G proteins. The AR family includes nine different gene products: three ␤ (␤1, ␤2, ␤3), three ␣2 (␣2A, ␣2B, ␣2C), and three ␣1 (␣1A, ␣1B, ␣1D) receptor subtypes. Thus, locally released NE or circulating epinephrine affects lymphocyte circulation and proliferation and modulates cytokine production and the functional activity of lymphocytes [10]. For example, a most recent report shows that this interaction is relevant in determining resistance to Listeria monocytogenes infection and the level of IL-12 and interferon-gamma (IFN-␥) production in mice [11]. In this study, chemical sympathectomy resulted in a 20-fold increase in the resistance to lethal doses of the intracellular pathogen [11]. Consistently, ␤-adrenergic agonists were found to preferentially impair IL-12 production and Th1 development [12,13]. NE seems to exert opposite effects on the innate and adaptive immune responses [14], with an enhancing activity especially on cell-mediated immunity [15]. As DC are important players in both the innate and adaptive immune responses, studies on the possible influence of NE on DC function might well be crucial in our understanding of the sympathetic nervous system influence on the immune response. A. Adrenergic Regulation of Langerhans Cell Migration and AntigenPresenting Capacity In contrast to the neural effect on lymphocytes, the adrenergic influence on antigen-presenting cells has only begun to be studied. Langerhans cells (LC) are DC residing within the epidermis and often lie in apposition with epidermal nerves, including both sensory and sympathetic fibers [16,17]. Once activated, LC migrate from the site of antigen deposition to the regional, draining lymph nodes, where they induce a specific immune response. Chemokines expressed by endothelial cells in lymphatics and lymph node venules and chemokine receptors in DC seem to contribute to DC migration as chemoattractants and by triggering integrin-dependent adhesive interactions [7,18,19]. However, the mechanisms that drive LC migration are not completely understood. We investigated whether NE could affect LC migration and/or exert a chemotactic/chemokinetic effect on bone marrow–derived DC in vitro. We used the fluorescent molecule FITC as an antigen to induce migration of Langerhans cells to regional lymph nodes. Mice were painted with FITC on the back after shaving, and the effect of adrenergic agents was evaluated in terms

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of number of cells positive for both FITC and CD86 (B7-2) found 24 hours later in the draining lymph nodes. The results obtained showed that topical application of the ␣1-adrenergic antagonist inhibited migration of LC to the draining lymph nodes. In contrast, LC mobilization was increased by systemic treatment with the ␣2-adrenergic antagonist yohimbine (Fig. 1). Furthermore, strong stimulation of DC emigration from dorsal halves of ear skin was noted when NE was added in organ cultures. On average a threefold increase of migrated DC was found in presence of NE and a fourfold increase was found using the chemokine 6Ckine as positive control (Table 1). As expected, the ␣1-AR antagonist prazosin inhibited the NE effect (Table 1). These results confirmed that NE can mobilize skin DC via ␣1-AR. To investigate whether the adrenergic inhibition of skin LC migration in vivo resulted in an altered development of LC-dependent immune response, we measured the contact hypersensitivity (CHS) response to FITC after sensitization in presence of prazosin. Prazosin treatment during sensitization inhibited the CHS response expressed as net ear swelling after FITC challenge 6 days later [20]. This indicated that the prazosin-induced inhibition of DC migration resulted in a reduced sensitization to FITC. Other experiments performed in vitro revealed that NE is indeed a chemotactic and chemokinetic factor in bone marrow–derived DC via ␣1-AR. We investigated the expression of mRNA coding for the ␣1-AR subtypes in DC and found the expression of ␣1B-AR mRNA [18]. Other authors have confirmed the expression of mRNA coding for various AR in LC. Purified LC as well as LC-like cell lines expressed mRNA for ␤1, ␤2, and ␣1A-AR

Figure 1 Effect of adrenergic antagonists on Langerhans cell migration. The mean values plus the standard deviation of migrated epidermal Langerhans cells after FITC painting of the skin and treatment with adrenergic antagonists are shown. Data are from five experiments (15 mice per group). a: p⬍ 0.01 (ANOVA). (From Ref. 20.)

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Table 1 Effect of NE on Emigration of DC from Skin Explants

Medium 6Ckine (120 ng/mL) NE (10–6 M) NE (10–6 M) ⫹ PRA (10–8 M) PRA (10–8 M)

No. of ears

Emigrated DC (⫻103)

12 12 14 14 12

39 ⫾ 21.8 167.8 ⫾ 47.7a 131.3 ⫾ 14.8a 56.1 ⫾ 18 36.2 ⫾ 16.3

100% 430% 336% 143% 93%

The values represent the mean number of cells emigrated from one ear skin dorsal half ⫾ the standard deviation during 24-hour incubation and the percentage of migration taking the control group as 100%. 6Ckine was used as positive control. PRA: prazosin A. p ⬍ 0.001 vs. Medium; NE ⫹ PRA, PRA (ANOVA). Source: Ref. 20.

(Fig. 2). Furthermore, pretreatment of LC with NE or epinephrine suppressed their antigenpresenting ability and inhibited their capacity to elicit a CHS response. This effect was neutralized by the ␤2-AR blocker ICI 118,551, suggesting the involvement of ␤2-AR [21]. More recently, we found that bone marrow–derived DC indeed express the mRNA coding for two ␤-AR (␤1, ␤2) and two ␣2-AR (␣2A, ␣2C) (G.J.M. Maestroni, unpublished results). In regard to ␣-AR mRNA expression, LC seem thus to differ from bone marrow–derived DC. Therefore, we thought to take advantage of ␤2-AR gene knockout mice to study the role of the ␤2-AR on LC migration and the consequent CHS response. In addition, we used the specific ␤2-AR antagonist ICI 118,551 in normal mice. Figure 3 shows the percentage of migrated LC in regional lymph nodes 24 hours after skin sensitization with FITC in ␤2-AR gene knockout mice and in normal wild-type mice. It seems evident that lymph nodes from ␤2-AR–deficient mice contained more CD11c/FITC-positive cells than normal mice. This indicated that absence of the ␤2-AR resulted in an increased migration of epidermal LC. A similar effect was obtained when FITC was applied on the skin of normal animals together with the ␤2-AR antagonist ICI 118,551 (data not shown). These results indicate that ␤2-AR are involved in modulating LC migration in vivo. The reported adrenergic stimulation of LC migration via ␣1-AR is seemingly in contrast with the present finding. A reasonable explanation may be that, physiologically, the final NE effect on LC migration results from two opposing effects: chemotaxis/chemokinesis mediated by ␣1AR and inhibition mediated by ␤2-AR. The selective blockade of these two AR results, in fact, in divergent effects on both LC migration and CHS response [20,22]. 䉴 Figure 2 Murine LC express mRNA for ARs. Murine LCs were enriched to ⬎98% purity and poly(A)Ⳮ RNA was extracted. Poly(A)Ⳮ RNA was also extracted from the LC-like cell lines XS106 and XS52-4D and from murine brain tissue, as a positive control. RT-PCR was performed using primers designed from sequences in GenBank. Purified LC expressed mRNA of the expected size for the ARs 1 (441 bp; A), 2 (600 bp; B), and 1A (119 bp; C). The LC-like cell line XS106 expressed mRNA for 1-, 2-, and 1A-AR as well. XS52-4D expressed mRNA for 2- and 1A-AR, but not for 1-AR. Expression of mRNA for 3-AR (B), 1B-AR (C), 1D-AR (D), and 2-AR (D) was not found in LCs or the LC-like cell lines. Receptor expression was verified with three separate RNA isolations. (From Ref. 21.)

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Figure 3 LC migration in ␤2-AR gene knockout mice and normal mice during skin sensitization. The mice were sensitized with FITC, and 24 hours later, a CD11cⳭ enriched cell population isolated from the draining lymph nodes was analyzed by flow cytometry. The upper panels relate to the mAb isotype control. The lower ones report the net percentage (minus the isotype value) of single CD11cⳭ, FITC-, and double-labeled CD11cⳭ, FITCⳭ cells. A representative experiment is shown out of three performed with four mice per group. WT: wild-type mice; ␤2-KO: ␤2-AR gene knockout mice.

Taken together, these results show that epidermal LC as well as bone marrow–derived DC express functional AR, which may influence both migration from the site of antigen deposition and antigen-presenting ability in the draining lymph nodes. This finding might be relevant in studies concerning skin diseases, allergy, and autoimmune disorders. B. Adrenergic Programming of Cytokine Production in Maturing DC To investigate in more detail the adrenergic influence on DC functions, we exposed bone marrow–derived DC to NE during the early phase of their activation and showed that this may irreversibly hinder IL-12 and stimulate IL-10 production (Fig. 4). The effect of NE was exerted on the cytokines gene expression, as demonstrated by real-time RT-PCR

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(a) Figure 4 Effect of short-term NE exposure on the kinetic of cytokine production. The effect of short-term (3 h) exposure to NE in DC stimulated with LPS (a) or KLH (b) is shown. The figures are relative to one representative experiment of three. Insets show the mean values relative to the effect of NE and that of NE Ⳮ propranolol (PRO) and yohimbine (YOH) of the three experiments expressed as a percentage, taking the LPS or KLH value as 100%. The white bars represent the effect of NE, and the black ones are relative to the effect of NE Ⳮ adrenergic antagonists. (From Ref. 22.)

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(b) Figure 4 Continued.

quantitation of IL-12 and IL-10 mRNA (Fig. 5). The effect on IL-12 was mediated by both ␤-AR and ␣2-AR, while that on IL-10 was apparently a ␤-AR phenomenon only [22]. More precisely, the AR subtypes involved in modulating cytokine production are the ␤2-AR and the ␣2A-AR (G. J. M. Maestroni, unpublished results). These findings seem relevant, especially in light of a recent report indicating that stimulated DC show a strict temporal regulation in the secretion of IL-12, production of which peaked after 10 hours of incubation and ceased by 24 hours [23]. Delivery of a second stimulus at 24 hours via CD40 ligation failed to stimulate further release of IL-12. These cells were termed ‘‘ex-

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Figure 5 Kinetics of cytokine mRNA expression in NE-exposed DC as determined by real-time RT-PCR. LPS-stimulated DC were exposed to NE (10ⳮ6 M) in the presence or absence of the adrenergic antagonists propranolol (PRO, 10ⳮ6 M) and yohimbine ( YOH, 10ⳮ7 M). One representative experiment of three is shown. (From Ref. 22.)

hausted DC,’’ while the IL-12–producing DC were referred to as ‘‘active DC.’’ Active DC promoted differentiation of Th1 cells, while exhausted DC stimulated differentiation of Th memory cells and Th2 cells [23]. Thus, our finding suggests that the presence of NE in the early phase of DC activation accelerates the transition of DC from the ‘‘active,’’ Th1-inducing state to the ‘‘exhausted,’’ Th2 and Th memory–inducing state. As a matter of fact, we found that short NE exposure of maturing DC not only programmed the kinetic of cytokine production but also influenced their capacity to stimulate T cells. A primary role of activated Th cells in the generation of CD4-dependent cell-mediated immunity is

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to provide CD40 ligation on DC, presumably to increase B7 expression and IL-12 secretion. This activation step then empowers the DC to successfully activate CD8Ⳮ cells and drive their differentiation into cytotoxic effectors [24]. We investigated whether CD40 ligation could restore IL-12 production in maturing DC exposed to NE for 3 hours. We also evaluated the stimulatory capacity of NE-exposed DC to stimulate allogeneic T cells. We found that delivery of a second stimulus by CD40 failed to further stimulate IL-12 production. In addition, when NE-exposed DC were incubated in presence of naive, allogeneic T cells, their stimulatory capacity was reduced as was the concentration of IFN-␥ in the culture supernatants [22]. We then showed that adoptive transfer of NE-exposed DC resulted in an immune response shifted toward the Th2 type [22]. Most interesting, we also found that an in vivo pharmacological, short-term inhibition of NE release at the beginning of skin sensitization with FITC resulted, 6 days later, in a sixfold increase in IFN-␥ production in the draining lymph nodes and in a higher CHS response [22]. III. CONCLUSION AND PERSPECTIVES The first defense mechanism against pathogenic microorganisms is the innate immune system, which in turn activates the adaptive immune response with the appropriate Th-cell differentiation required to eliminate the invading pathogen. The type of microorganism, its route of entry, and its niche in the host also determine the type of immune response. In this context, recognition of structural components of infectious agents by DC alerts the innate immune system to the presence of pathogens so that an immediate response can be mounted to contain the infection. However, how the nature of infection determines the choice between Th1 and Th2 effector responses is not well understood. Altogether, the findings presented suggest that the sympathetic nervous system plays an important role in this choice. In particular, the presence of NE during the early phase of DC activation seems to exert important effects on their migration ability and kinetic of cytokine production. These effects may have an immediate consequence on the innate response by decreasing the inflammatory response and a later effect on the adaptive response by shifting the Th response toward the Th2 type. Thus, the role of the sympathetic nervous system might well be that of providing microenvironmental information suitable to shape the appropriate response to an invading pathogen. This might also be relevant in understanding how the route of entry may determine the type of immune response. For example, it is well known that activation of the mucosal immune system often results in Th2-type immune responses or in tolerance [25]. Beside its physiopathological implications, this phenomenon hinders the use of the oral route for effective vaccination. It has been reported that DC play a crucial role in the phenomena of oral tolerance and tolerance to parenchymal self-antigens [26,27]. On the basis of the present findings, the possible involvement of the sympathetic nervous system in these important aspects of DC function should be investigated. Also within the cutaneous immune system, LC are able to present haptens, immunogenic peptides, and tumor antigens for T-dependent immune responses. Alterations of sympathetic outflow or regulation by emotional disorders might thus be relevant in a variety of skin diseases, including atopic eczema and psoriasis, which are known to be exacerbated by anxiety or depression. The sympathetic regulation of DC functions might be exploited for improving the effectiveness of various vaccines, including cancer vaccines. Protective cell-mediated tumor immunity depends on the appropriate presentation of tumor-associated antigens by

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professional antigen-presenting cells such as DC. DC may present tumor-associated antigens to T cells in MHC I or MHC II–restricted pathways. However, to date, the results of various clinical trial with DC-based cancer vaccines, although provocative, are somewhat disappointing. Emotional and psychological factors in cancer patients may well influence the outcome and strength of the immune reactivity elicited by vaccination. A complete understanding of the influence of the sympathetic nervous system on DC functions will improve our knowledge of the physiology of the immune system and make possible new selective pharmacological approaches in a variety of diseases.

REFERENCES 1. Shortman K, Caux C. Dendritic cell development: multiple pathways to nature’s adjuvants. Stem Cells. Vol. 15, 1997:409–419. 2. Sallgaller ML, Lodge PA. Use of cellular and cytokine adjuvants in the immunotherapy of cancer. J Surg Oncol. Vol. 68, 1998:122–138. 3. Weinlich G, Heine M, Sto¨ssel H, Zanella M, Stoizner P, Ortnet U, Smolle J, Koch F, Sepp NT, Schuler G, Romani N. Entry into lymphatics and maturation in situ of migrating murine cutaneous dendritic cells. J Invest Dermatol. Vol. 110, 1998:441–448. 4. June CH, Bluestone JA, Nadler LM, Thompson CB. The B7 and CD28 receptorfamilies. Immunol Today. Vol. 15, 1994:321–327. 5. Gimmi CD, Freeman GJ, Gribben JG, Gray G, Nadler LM. Human T-cell clonal anergy is induced by antigen presentation in the absence of B7 costimulation. Proc Natl Acad Sci USA. Vol. 90, 1993:6586–6570. 6. Foy TM, Aruffo A, Bajorath J, Buhlman JE, Noelle RJ. Immune regulation by CD40 and its ligand gp39. Ann Rev Immunol. Vol. 14, 1996:591–617. 7. Bancherau J, Steinman RM. Dendritic cells and the control of immunity. Nature. Vol. 392, 1998:245–252. 8. Schnare M, Barton GM, Holt AC, Takeda K, Akira S, Medzhitov M. Toll-like receptors control activation of adaptive immune responses. Nat Immunol. Vol. 2, 2001:947–950. 9. Kohm A, Tang Y, Sanders VM, Jones SB. Activation of antigen-specific CD4Ⳮ Th2 cells and B cells in vivo increases norepinephrine release in the spleen and bone marrow. J Immunol. Vol. 165, 2000:725–733. 10. Elenkov IJ, Wilder RL, Chrousos GP, Vizi S. The sympathetic nerve-An integrative interface between two supersystems: the brain and the immune system. Pharmacol Rev. Vol. 52, 2000: 595–638. 11. Miura K, Kudo T, Matsuki A, Sekikawa K, Tagawa Y, Iwakura Y, Nakane A. Effect of 6hydroxydopamine on host resistance against Listeria monocytogenes infection. Infect Immun. Vol. 69, 2001:7234–7241. 12. Panina-Bordignon P, Mazzeo D, Di Lucia P, D’Ambrosio D, Lang R, Fabbri L, Self C, Sinigaglia F. b2-Agonists prevent Th1 development by selective inhibition of interleukin-12. J Clin Invest. Vol. 100, 1997:1513–1519. 13. Hasko G, Szabo C, Nemeth ZH, Salzman AL, Vizi E. Stimulation of beta-adrenoceptors inhibits endotoxin-induced IL-12 production in normal and IL-10 deficient mice. J Neuroimmunol. Vol. 88, 1998:57–61. 14. Rice PA, Boehm GW, Moynihan JA, Bellinger DL, Stevens SY. Chemical sympathectomy increases the innate immune response and decreases the specific immune response in the spleen to infection with Listeria monocytogenes. J Neuroimmunol. Vol. 114, 2001:19–27. 15. Alaniz RC, Thomas SA, Perez-Melgosa M, Mueller K, Farr AG, Palmiter RD, Wilson CB. Dopamine beta-hydroxylase deficiency impairs cellular immunity. Proc Natl Acad Sci USA. Vol. 96, 1999:2274–2278.

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16. Torii H, Yan Z, Hosoi J, Granstein RD. Expression of neurotrophic factors and neuropeptide receptors by Langerhans cells and the Langerhans cell-like cell line XS52: further support for a functional relationship between Langerhans cells and epidermal nerves. J Invest Dermatol. Vol. 109, 1997:586–591. 17. Botchkarev VA, Peters EM, Botchkareva NV, Maurer M, Paus R. Hair cycle-dependent changes in adrenergic skin innervation, and hair growth modulation by adrenergic drugs. J Invest Dermatol. Vol. 113, 1999:878–887. 18. Kellermann S-A, Hudak S, Oldham ER, Liu Y-J, McEvoy LM. The CC chemokine receptor7 ligands 6Ckine and macrophage inflammatory protein-3b are potent chemoattractants for in vitro and in vivo-derived dendritic cells. J Immunol. Vol. 162, 1999:3859–3864. 19. Warnock RA, Campbell JJ, Dorf ME, Matsuzawa A, McEvoy LM, Butcher EC. The role of chemokines in the microenvironmental control of T versus B cell arrest in Peyer’s patch high endothelial venules. J Exp Med. Vol. 191, 2000:77–88. 20. Maestroni GJM. Dendritic cells migration controlled by a1b-adrenergic receptors. J Immunol. Vol. 165, 2000:6743–6747. 21. Seiffert C, Hosoi J, Torii H, Ozawa H, Ding W, Campton K, Wagner JA, Granstein RD. Catecholamines inhibit the antigen-presenting capability of epidermal Langerhans cells. J Immunol. Vol. 168, 2002:6128–6135. 22. Maestroni GJM. Short exposure of antigen-stimulated dendritic cells to norepinephrine: impact on kinetic of cytokine production and Th polarization. J Neuroimmunol. Vol. 129, 2002: 106–114. 23. Langenkamp A, Messi M, Lanzavecchia A, Sallusto F. Kinetics of dendritic cell activation: impact on priming of Th1, Th2 and nonpolarized T cells. Nature Immunol. Vol. 1, 2000: 311–316. 24. van Kooten C. Immune regulation by CD40-CD40-l interactions—2; Y2K update. Front Biosci. Vol. 5, 2000:D880–693. 25. Akbari O, DeKruyff RH, Umetsu DT. Pulmonary dendritic cells producing IL-10 mediate tolerance induced by respiratory exposure to antigen. Nat Immunol. Vol. 2, 2001:725–732. 26. Adler AJ, Marsh DW, Yochum GS, Guzzo JL, Nigam A, Nelson WG, Pardoll DM. CD4Ⳮ T cell tolerance to parenchimal self antigens requires presentation by bone marrow-derived antigen-presenting cells. J Exp Med. Vol. 187, 1998:1555–1564. 27. Alpan O, Rudomen G, Matzinger P. The role of dendritic cells, B cells, and M cells in gutoriented immune responses. J Immunol. Vol. 166, 2001:4843–4852.

20 Neuroendocrine Host Factors in Susceptibility and Resistance to Autoimmune/Inflammatory Disease JEANETTE I. WEBSTERand ESTHER M. STERNBERG National Institute of Mental Health, National Institutes of Health, Bethesda, Maryland, U.S.A.

I. INTRODUCTION The neuroendocrine and immune systems are controlled by a finely tuned bi-directional regulatory system that is essential for health. Imbalances in this system result in enhanced susceptibility to infection or inflammatory/autoimmune disease. The central nervous system (CNS) regulates the immune system at a systemic and regional level by (1) glucocorticoid production by the hormonal stress response and (2) noradrenalin and acetylcholine release by the autonomic nervous system. Conversely, the immune system regulates the neuroendocrine system through cytokine feedback to the brain. Adrenal glucocorticoid production is regulated by the hypothalamic-pituitary-adrenal (HPA) axis and is a major regulator of the immune system (Fig. 1). The paraventricular nucleus (PVN) of the hypothalamus, the anterior pituitary gland located at the base of the brain, and the adrenal glands constitute the HPA axis. Upon stimulation, corticotropinreleasing hormone (CRH) is secreted from the PVN into the hypophyseal portal blood supply and subsequently stimulates the secretion of adrenocorticotropin hormone (ACTH) from the anterior pituitary gland into the systemic circulation. This, in turn, induces the adrenal glands to synthesize and release glucocorticoids. Regulation of the HPA axis occurs both from within the central nervous system and from the periphery. The HPA axis end product, glucocorticoids, negatively regulate the axis at the hypothalamic and pituitary levels. Other factors, such as neurotransmitters and neuropeptides of the sympathetic nervous system and other neuropeptides [e.g., arginine vasopressin (AVP) and cytokines] also regulate the HPA axis [1–3]. CRH is negatively regulated by itself and by 393

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ACTH, as well as by other neuropeptides and neurotransmitters in the brain (e.g., ␥aminobutyric acid–benzodiazopines (GABA-BDZ) and opioid peptide systems), whereas it is positively regulated by the serotonergic, cholinergic, and histaminergic systems [4]. For many years it has been known that the immune system is influenced by glucocorticoids. These hormones have been used in the treatment of inflammatory diseases since the 1940s, and Kendall, Reichstein, and Hench were awarded the Nobel Prize for the discovery of this effect in 1950 [5]. Extensive research has been carried out into the pharmacological effects of glucocorticoids on many aspects of immune cell function [6,7], although only recently has the essential physiological role of glucocorticoids in the regulation of the immune system in health and disease [8] been fully appreciated. Knowledge

Figure 1 HPA axis. Schematic diagram of the HPA axis showing regulation of the immune system by glucocorticoids and the sympathetic nervous system (SNS) and peripheral nervous system (PNS). Inhibitory loops are shown as a dotted line.

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of the mechanisms involved in glucocorticoid secretion and their regulation of the immune system under both normal and disease conditions is fundamental to our understanding of the pathogenesis of inflammatory/autoimmune disease and ultimately for the development of effective therapies for such diseases. Glucocorticoid regulation of the immune system is, in fact, only one portion of an extensive regulatory network that exists between the central nervous, neuroendocrine, and immune systems. The CNS regulates the immune system locally, regionally, and systemically through a network of connections of nerve pathways, hormonal cascades, and cellular interactions. The immune system likewise regulates the CNS in a similar fashion. During inflammation, cytokines produced at the inflammatory site signal the brain, resulting in the symptoms of sickness behavior and fever [9,10]. Cytokine production in other cells of the brain, such as glia, neurons, and macrophages, have been shown to be involved in neuronal cell death [11,12] and survival [13]. In fact, cytokine-mediated neuronal cell death is thought to play an important role in several neurodegenerative diseases, such as neuroAIDS, Alzheimer’s disease, multiple sclerosis, stroke, and nerve trauma. For a comprehensive review of the regulation of the immune system by the sympathetic nervous system, see the recent review by Elenkov et al. [14]. Several mechanisms exist by which the CNS can also be stimulated by peripherally produced cytokines functioning as hormones. These can cross the blood-brain barrier (BBB) at leaky points, for example, at the organum vasculosum lamina terminalis (OVLT) or median eminence, or can be actively transported in small amounts across the BBB [15]. Additionally, they can rapidly signal the CNS through the vagus nerve [16,17], or by second messengers, such as nitric oxide and prostaglandins, which are activated after cytokines bind to receptors on brain endothelial cells [18,19]. Although a full understanding of this communication between the CNS and immune systems is important, it will not be the main focus of this chapter, and for further information on this regulatory network, the review by Mulla and Buckingham [20] is recommended. Locally, the CNS can regulate the immune system by release of neuropeptides such as substance P and locally produced CRH from the peripheral nerves. In general, these locally released neuropeptides are thought to be pro-inflammatory. This chapter will focus only on the regulation of the immune system by the CNS through the neuroendocrine system. (For information on local CNS regulation of the immune system, see Refs. 21 and 22.) Imbalances can arise from disturbances at any level of the HPA axis. Thus, overstimulation of the HPA axis may lead to excessive circulating glucocorticoids and an overall suppression of the immune system, generally leading to enhanced susceptibility to or severity of infection. On the other hand, understimulation of the HPA axis may lead to lower circulating glucocorticoid levels, resulting in increased susceptibility to inflammation due to ineffective regulation of the immune system by glucocorticoids. Dysregulation at a molecular level such as at the level of the glucocorticoid receptor would result in glucocorticoid resistance and enhanced inflammation. Understanding the mechanisms by which these CNS and neuroendocrine systems regulate the immune system at the systemic, anatomical, and molecular levels will, therefore, enhance our understanding of the pathogenesis of inflammatory/autoimmune and infectious diseases. This knowledge may also lead to improvement of treatment and diagnosis of predisposition to such illnesses.

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II. GLUCOCORTICOID MODULATION OF THE IMMUNE SYSTEM Immunosuppressive glucocorticoids elicit their effects on immune cells and molecules through their cellular receptors. There are two receptors for glucocorticoids, the higheraffinity type I receptor, the mineralocorticoid receptor (MR), and the lower-affinity type II receptor, the glucocorticoid receptor (GR). At low concentrations glucocorticoids bind preferentially to MR, and only at high stress levels do glucocorticoids bind to GR [23]. It has been suggested that in the brain, MR functions in a proactive capacity in the maintenance of homeostasis, whereas GR functions in a reactive role in the recovery from disturbance [24,25]. However, the primary receptor for glucocorticoids in immune cells is GR. The availability of glucocorticoids can also be regulated by other factors such as the expression of 11␤-hydroxysteroid dehydrogenase, an enzyme that converts the active form of steroids, e.g., cortisol and corticosterone, into the inactive form, e.g., cortisone and 11dehydrocortisone [26]. A. The Glucocorticoid Receptor The glucocorticoid receptor (NR3C1), together with the receptors for progesterone, estrogen, mineralocorticoid, and thyroid hormones, is a member of the steroid and thyroid hormone receptor superfamily. This family of nuclear receptor hormones can essentially be thought of as ligand-dependent transcription factors that mediate the endpoint tissue effects of such hormones [27]. Members of this group of nuclear hormone receptors all share a common three-domain structure (Fig. 2) consisting of an N-terminal transactivation domain, a central DNA-binding domain (DBD), and a C-terminal ligand-binding domain (LDB) [6,28,29]. Glucocorticoids circulate in the peripheral blood stream associated with cortisol binding globulin (CBG) or albumin. The small hydrophobic glucocorticoids enter the cell by passive diffusion, although evidence exists for some active transport processes. GR is located in the cytoplasm in a multiprotein complex containing hsp90 and immunophilins that holds the receptor in a conformation accessible to ligand. Upon ligand binding, the activated GR dissociates from the hsp complex, dimerizes, and translocates to the nucleus,

Figure 2 Structure of the glucocorticoid receptor (GR). Diagrammatic structure of the glucocorticoid receptor showing the three domains: the N-terminal transactivation domain, the DNA-binding domain (DBD) and the ligand-binding domain (LBD). The areas of GR involved in aspects of GR function are shown.

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where it binds as a homodimer to its hormone response element (HRE) or glucocorticoid response element (GRE). GR can then modulate gene expression, either upregulating or downregulating target genes, by interacting with many co-activators or co-repressors (Fig. 3) [28]. Repression of target genes by GR can occur via a negative glucocorticoid response element (nGRE), e.g., the bovine prolactin gene [30], but mostly occurs by interaction with other transcription factors, such as AP-1 and NF-␬B [6,31–35]. It should be pointed out that the reverse can also occur, i.e., GR gene activation can be repressed by AP-1 and NF-␬B [36,37]. Glucocorticoid responses can also be affected by the presence of other receptors that also bind glucocorticoids, including the mineralocorticoid receptor and an inactive splice variant of the glucocorticoid receptor (GR␤), which differs in the C-terminal ligand binding domain [38]. GR␤ is found in the cytoplasm complexed to hsp90 but is also located in the nucleus regardless of ligand status, and because of a unique 15-amino-acid C-terminus is unable to bind ligand or activate gene transcription. It has been suggested that GR␤ forms transcriptionally inactive heterodimers with GR␣, thereby acting as a

Figure 3 Molecular mechanism of glucocorticoid receptor (GR) action. Molecular mechanism of GR action showing direct activation of gene transcription and inhibition of NF-␬B and AP-1 pathways. Inhibitory routes are depicted as dotted lines.

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dominant negative receptor or, effectively, as a GR antagonist in vitro [39–43]. However, this mechanism is still under dispute, as other studies have shown no effect of GR␤ on GR␣-mediated transactivation or transrepression [44–47]. GR␤ has also been shown to repress MR [48]. B. Effects of Glucocorticoids—Pharmacological Versus Physiological It is important to recognize that there are different effects of pharmacological and physiological doses or forms of glucocorticoids. Physiological doses of glucocorticoids modulate transcription of inflammatory genes, whereas pharmacological doses suppress the inflammatory response. Different immune responses are elicited by synthetic glucocorticoids, such as dexamethasone, compared to natural glucocorticoids, such as hydrocortisone. For example, dexamethasone exerts a greater suppression on IL-12 than hydrocortisone, consistent with the greater affinity of dexamethasone for GR than hydrocortisone [24]. Physiological doses of the physiological glucocorticoid corticosterone have been shown to have immuno enhancing effects in skin delayed-type hypersensitivity in rats. However, if the dose of corticosterone is increased to pharmacological concentrations or the pharmacological glucocorticoid dexamethasone is used, immunosuppressive effects are observed [49]. This suggests that acute administration of a physiological glucocorticoid is immunoenhancing, whereas chronic administration of a physiological glucocorticoid or administration of a pharmacological glucocorticoid preparation is immunosuppressive. This is in agreement with the observation that acute stress can be beneficial, for example, in enhancement of immune function, whereas chronic stress is detrimental to health, for example, in the exacerbation of autoimmune diseases, susceptibility to infections, and impaired wound healing [50–53]. C. Glucocorticoid Effects on Immune Molecules Glucocorticoids, through the molecular mechanisms described above, regulate a wide variety of immune genes and immune cell functions. For example, glucocorticoids modulate gene expression of cytokines, adhesion molecules, chemoattractants, inflammatory mediators, and other inflammatory molecules (Table 1) (For comprehensive reviews see Refs. 6,7 and 54.) Table 1 Immune-Related Genes Regulated by Glucocorticoids Increase Cytokines IL-4, IL-10, IL-RI

Inflammatory mediators Lipocortin 1 (annexin 1)

Receptors ␤2-Adrenoceptor, I␬B␣

Decrease Cytokines IL-1, IL-2, IL-3, IL-4, I1-5, I1-6, I1-8, IL-11, IL-13, TNF-␣, GM-CSF, IFN-␥ Chemokines RANTES, eotaxin, MIP␣, MCP-1, MCP-3, CINC/gro Inflammatory mediators NOS II, iNOS, COX-2, cPLA2 Adhesion molecules ICAM-1, ELAM-1, VCAM-1, E-selectin, L-selectin Receptors IL-2R, NK1R, NK2R, IL-4R␣

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1. Cytokines In general, glucocorticoids repress pro-inflammatory cytokine gene expression, such as IL-1 [55–57], IL-2 [58], IL-6 [59–61], IL-8 [62], IL-11 [63], IL-12 [64–66], TNF-␣ [67,68], IFN-␥ [58,69], and GM-CSF [70], while they upregulate the expression of antiinflammatory cytokines, such as IL-4 [71–73] and IL-10 [24,69,74]. Glucocorticoid regulation of cytokine expression can also be achieved by modulation of mRNA stabilization, e.g., IL-1␤ [55–57], IL-8 [62], and IL-11 [63]; modulation of receptors and decoy receptor expression, e.g., IL-1R II [75] and IL-12R [64–66]; regulation of translation, e.g., TNF␣ [67]; or modulation of cytokine signaling pathways, e.g., dexamethasone inhibition of IL-2 signaling via the Jak-STAT cascade [76]. As mentioned, glucocorticoids are generally thought to upregulate anti-inflammatory cytokines, but there are some specific cell- and dose-dependent effects. For example, pharmacological doses of glucocorticoids upregulate IL-10, whereas physiological doses suppress IL-10 [24,69,74]. In the lymph nodes and spleen of mice, IL-4 is induced by physiological concentrations of glucocorticoids [77,78], whereas in T cells IL-4 is down-regulated by high stress levels of dexamethasone [58]. Such modulation of cytokine expression by glucocorticoids causes a shift in the pattern of immunity, from Th1 (cellular immunity) to Th2 (humoral immunity). This glucocorticoid modulation of immunity is evident in some autoimmune diseases, such as rheumatoid arthritis, multiple sclerosis (MS), and type I diabetes mellitus, where such a shift towards Th1 immunity has been observed, whereas in systemic lupus erythematosus (SLE) there is a shift towards Th2 [66]. Likewise, a relative resistance to Th1-associated autoimmune diseases has been described in cases of excessive glucocorticoid production, e.g., in animal models with a hyperactive HPA axis (F344/N rats) or in women in the third trimester of pregnancy [66,79], whereas in cases of low glucocorticoid production, e.g., animals lacking glucocorticoids or exhibiting a hypoactive HPA axis (LEW/N rats), susceptibility to Th1-associated autoimmune diseases has been described [80]. 2. Cell Adhesion Molecules and Chemoattractants Glucocorticoids down-regulate the proteins involved in the attraction and adhesion of leukocytes to areas of inflammation. These include intracellular adhesion molecule 1 (ICAM-1) [37,81,82], endothelial-leukocyte adhesion molecule 1 (ELAM-1) [83], vascular adhesion molecule 1 (VCAM-1) [84], E-selectin [85], L-selectin [86], cytokine-induced neutrophil chemoattractant (CINC)/gro [87], IL-5 [72,88], eotaxin [89], RANTES (regulated upon activation normal T-cell expressed and secreted) [90,91], and monocyte chemoattractant protein 1 (MCP-1), MCP-2, and MCP-3 [91]. 3. Inflammatory Mediators The production of inflammatory mediators, such as prostaglandins and nitric oxide (NO), are suppressed by glucocorticoids. Prostaglandin synthesis is inhibited by glucocorticoids at multiple stages. They repress expression of the key enzymes, cytosolic PLA2 and COX2 [92–94], and induce lipocortin 1 (or annexin 1), an inhibitor of arachadonic acid release [95]. Glucocorticoids suppress NO by repression of cytokine-induction of NOS II [96] and by inhibition of iNOS gene transcription [97]. 4. Other Inflammatory Response Factors Several receptors involved in the regulation of the immune system are also regulated by glucocorticoids. These include glucocorticoid-mediated upregulation of the ␤2-adrenore-

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ceptor, which is involved in the adrenergic control of the immune system [98,99], and glucocorticoid-mediated downregulation of the NK1-receptor, the receptor for substance P [100], and the NK2-receptor [101]. III. ROLE OF ENDOGENOUS GLUCOCORTICOIDS IN PROTECTION FROM INFLAMMATION AND REGULATION OF IMMUNE FUNCTION Fluctuations in glucocorticoid levels, such as during exercise and with circadian rhythm, have been shown to be associated with changes in cytokine levels and production by leukocytes [102–106], suggesting a role of physiological glucocorticoids in regulation of the immune system. However, animal models have provided the strongest evidence that endogenous glucocorticoids are essential physiological regulators of the immune response and that disruption of this regulation plays a role in inflammatory/autoimmune disease. The course and severity of inducible autoimmune/inflammatory disease in rodents can be altered by HPA axis intervention, either surgically or pharmacologically. Development of arthritis and high mortality in F344/N rats have been observed following injection of streptococcal cell walls together with administration of the glucocorticoid receptor antagonist RU486 [107]. Increased mortality rates following Salmonella typhimurium infection were observed in hypophysectomized rats compared to nonhypophysectomized animals [108]. Similarly, adrenalectomy resulted in increased lethality following MCMV virus infection [109] and also increased mortality in myelin basic protein (MBP)–induced experimental allergic encephalomyelitis (EAE) in LEW/N rats [80] (Table 2). These animal models provide evidence for the critical importance of an intact HPA axis in protection against septic shock following exposure to a wide range of antigenic, pro-inflammatory, or infectious stimuli. Further evidence of the necessity of an intact HPA axis is demonstrated by the attenuation of the inflammatory disease in these animal models by reconstitution of the HPA axis, pharmacologically with glucocorticoids, or surgically by intracerebral fetal hypothalamic tissue transplantation. Low-dose dexamethasone treatment [107] or intracerebroventricular transplantation of F344/N hypothalamic tissue [110] in LEW/N rats significantly attenuated arthritis and carrageenan inflammation. Likewise, glucocorticoid replacement protected against lethality from MCMV virus [109]. In MBP-induced EAE,

Table 2 Increased Mortality Rates Following Intervention of HPA Axis and Subsequent Survival after Glucocorticoid Replacement

Infection/Disease Streptococcal cell wall (SCW)–induced arthritis Myelin basic protein (MBP)–induced EAE Salmonella typhimurium MCMV

HPA axis intervention

Mortality rate (%)

Mortality rate after corticosterone replacement (%)

Ref.

GR antagonist RU486 Adrenalectomy

100

13

107

80

22

80

Hypophysectomy Adrenalectomy

100 100

5 20

108 109

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replacement of basal corticosterone levels by a subcutaneous steroid implant did not reduce mortality. However, replacement of a dose equivalent to the EAE-induced corticosterone levels resulted in increased survival rates and similar EAE development as in control animals. Complete remission of the disease was achieved if higher corticosterone levels were replaced [80] (Table 2). Thus, in these models, interruption of the HPA axis predisposes to enhanced inflammation and septic shock, while reconstitution attenuates autoimmune/inflammatory disease. This suggests a role of the HPA axis in intensity of autoimmune/inflammatory diseases. IV. ANIMAL MODELS OF INFLAMMATORY DISEASE Animal models have greatly aided the understanding the pathogenesis of autoimmune/ inflammatory disease. Blunted HPA axis responses have been shown in animal species that are predisposed to autoimmune disease, such as the obese strain (OS) chicken (a model for autoimmune thyroiditis) [111]; certain mouse lupus (SLE) models (MRL strain but not NZB/NZW) [112,113]; and the inbred rat strain, LEW/N rats (Table 3). For a review of animal models of inflammatory diseases, see Jafarian-Tehrani and Sternberg [114] and Tonelli et al. [115]. To examine this relationship between HPA axis responses and autoimmune/inflammatory disease, two histocompatible inbred rat strains, Lewis (LEW/N) and Fischer (F344/ N) rats, which exhibit differential neuroendocrine responsiveness and differential susceptibility and resistance to autoimmune/inflammatory disease, have been extensively used. LEW/N rats, which exhibit a blunted HPA response upon stimulation, are highly susceptible to development of a wide range of autoimmune/inflammatory diseases in response to a variety of antigenic or pro-inflammatory stimuli, whereas the hyperactive HPA axis F344/N rats are relatively resistant to such disease upon exposure to the same stimuli [107,116–119]. For example, LEW/N rats, but not F344/N rats, develop experimental allergic encephalomyelitis (EAE) upon immunization with myelin basic protein [120] and

Table 3 Inflammatory/Autoimmune Diseases Correlated with Dysfunctional HPA Axis in Humans and Some Animal Models Model Chicken Mice Rat

Human

Inflammatory/Autoimmune disease

Ref.

Thyroiditis Scleroderma Systemic lupus erythematosus (SLE) Arthritis Experimental allergic encephalomyelitis (EAE) Inflammation Rheumatoid arthritis Systemic lupus erythematosus (SLE) Sjögren’s syndrome Dermatitis Fibromyalgia Chronic fatigue syndrome Multiple sclerosis Inflammatory bowel disease

111 126 112, 113 107, 117, 121, 122 120 110 130–136 131, 137 131, 138 139, 140 131, 142, 145 141–144 146, 147 148, 149

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develop arthritis in response to heat-killed Mycobacterium tuberculosis in adjuvant or streptococcal cell walls and inflammation in response to carrageenan, [107,117,121,122]. Differences in the expression of molecules involved in the HPA axis regulation and glucocorticoid action have been shown in these two rat strains, for example, hypothalamic CRH [117], pro-opiomelanocortin (POMC) [118], corticosterone-binding globulin (CBG) [119], and GR expression and activation [119,123,124]. Blunted HPA axis OS chickens spontaneous develop thyroiditis, a model for Hashimoto’s thyroiditis [111]. These chickens also have increased CBG, which results in decreased free corticosterone levels [125]. An ACTH-hyporesponsive adrenal gland has been shown in UCD-200 chickens, a model for human scleroderma [126]. Blunted HPA axis responses have also been shown in MRP lupus-prone mice [112,113] but not other strains, such as NZB. These associations between a blunted HPA axis and susceptibility to inflammatory/autoimmune disease do not prove cause and effect. Intervention studies, such as those described in section III, must be performed in order to do this. It must be emphasized that HPA axis responsiveness is just one variable amongst many factors that contribute to overall susceptibility and resistance to such complex autoimmune diseases. Genetic linkage studies in rat models have identified genes in over 20 regions on 15 chromosomes involved in susceptibility and resistance to inflammatory arthritis [127–129]. These include many genes that are related to immunity and some neuroendocrine genes, but many that are unknown. Likewise, in humans there are likely to be many factors that contribute to susceptibility/resistance to autoimmune/inflammatory disease. V. DISRUPTIONS OF THE HPA AXIS OR GLUCOCORTICOID RESPONSE IN HUMAN AUTOIMMUNE/INFLAMMATORY DISEASE While enhanced susceptibility to inflammatory/autoimmune disease has been associated with a blunted HPA axis, it is possible and indeed probable that abnormalities in any component of the HPA axis responsiveness could lead to enhanced inflammatory susceptibility. Impaired function of any component of the HPA axis or any hormone, hormone receptor, or signal transduction pathway involved in maintaining an intact HPA axis response might, according to this hypothesis, potentially account for enhanced inflammatory susceptibility in humans with such illnesses. We will now consider some of the possible stages at which a disruption or defect could effectively cause impaired glucocorticoid signaling. A. HPA Axis In humans, a blunted HPA axis with resultant low glucocorticoid levels or low glucocorticoid responses has been associated with a number of inflammatory diseases, such as rheumatoid arthritis [130–136], SLE [131,137], Sjo¨gren’s syndrome [131,138], allergic asthma and atopic skin disease [139,140], chronic fatigue syndrome (CFS) [141–144], fibromyalgia [131,142,145], multiple sclerosis [146,147], and inflammatory and irritable bowel syndrome [148,149] (Table 3). Conversely, excessive stimulation of the HPA axis and chronically elevated glucocorticoid levels that result from chronic stress situations, such as those experienced by care-givers of Alzheimer’s patients, students taking exams, couples during marital conflict, and Army Rangers undergoing extreme exercise, have been associated with enhanced

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susceptibility to viral infection, prolonged wound healing, or decreased antibody production after vaccination [150–153]. HPA axis responses can be measured in patients by stimulation of the HPA axis using, for example, exogenous hormones (ovine CRH, AVP, ACTH) [141,146], physical stress (exercise) [102,144], psychosocial stress [the Trier Social Stress Test (TSST), which involves public speaking and mental arithmetic] [139,140,144], or insulin hypoglycemia [137,144]. B. Glucocorticoid Receptor Several mutations and polymorphisms have been found in the glucocorticoid receptor associated with defective glucocorticoid signaling and glucocorticoid resistance. In the hereditary disease, familial glucocorticoid resistance, mutations of GR that result in decreased number, stability, or nuclear translocation of GR or decreased affinity for the ligand have been identified. Currently, in families with familial glucocorticoid resistance three different point mutations in the ligand-binding domain of GR, one in the hinge region, and a deletion in the ligand binding domain have been identified (for review, see Refs. 154 and 155). Recently, in a French family with familial glucocorticoid resistance, a novel C-terminal mutation that interferes with GR-p160 coactivator interactions has been described [43]. In other diseases and glucocorticoid resistance states, some GR mutations and polymorphisms have been found. For example, in patients with lupus nephritis, a phase shift mutation in GR has been described [156], and in a heterozygotic patient with severe glucocorticoid resistance, a mutant GR (amino acid 559 Ile-Asn) cannot translocate to the nucleus and prevents translocation of wild-type GR [157]. However, not all GR polymorphisms result in glucocorticoid resistance. For example, a polymorphism in GR (codon 363) has been associated with increased sensitivity to glucocorticoids [158], and five polymorphisms in GR (including the one at codon 363) have been described in a normal population [159]. Although the majority of GR mutations found in glucocorticoid resistance patients affect ligand binding, other mutations in GR could affect GR function. Recently, a mutation in the DNA-binding domain of GR has been described in a patient with primary cortisol resistance [160]. Also, mutation of phosphorylation sites in GR results in reduced GR transactivation and reduced stability of the receptor [161]. Although no mutation has been identified, phosphorylation of GR has been implicated in a group of steroid-resistant asthma patients. In this case, production of IL-2 and IL-4 has been suggested to activate p38 mitogen activated protein kinase (MAPK) phosphorylation of GR, resulting in reduced nuclear GR activity and glucocorticoid resistance in these patients [162]. Some patients with glucocorticoid resistance have no detectable mutation in GR, and it is plausible that in these patients there is a defect in another component requires for GR function [163]. One way in which glucocorticoid resistance can occur without a mutation in wild-type GR is by differing the expression of the splice variant and proposed dominant repressor GR␤. In an initial study we identified a functionally relevant glucocorticoid receptor polymorphism in the 3′ untranslated region of exon 9 in an ATTTA motif located at position 3669. This A/G substitution was associated with enhanced stability of the GR␤ receptor when a similar mutated GR␤ was transfected into Cos-1 cells. This polymorphism is more prevalent in human subjects with rheumatoid arthritis (RA) than in controls or patients with SLE [164] (allele frequency of 0.2, p ⬍ 0.035, in RA patients, and 0.18, p ⬍ 0.057,

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in SLE patients compared to 0.06 in controls). In subsequent studies using in vitro mutagenesis, similar mutations in AUUUA sequences throughout the GR␣ and GR␤ have also been associated with enhanced stability of both these receptors [165]. Since the GR␤ is an inactive form of the GR that some postulate may act as an endogenous antagonist under certain circumstances [40–42], this polymorphism could potentially contribute to enhanced inflammatory susceptibility in these subjects through relative antagonism of the GR and resultant relative glucocorticoid resistance. However, the physiological significance of GR␤ as an endogenous GR antagonist is not clear, since other in vitro studies show no antagonism of GR␣ by GR [44–47] and in vivo GR␤ exists in a very low ratio compared to the fully active GR [166], leading some to question whether small amounts of antagonism by GR␤ are physiologically relevant in vivo. Nonetheless, supporting the physiological relevance of GR␤ abnormalities in disease is the finding of increased expression of GR␤ relative to GR␣, which has been described in other diseases, including asthma [167–170], ulcerative colitis [171,172], chronic lymphocytic leukemia [173], and nasal polyposis disease [174]. C. Cortisol-Binding Globulin CBG limits the amount of free cortisol available in the blood. Therefore, theoretically, changes in the expression of this protein or in its binding capacity can also affect the availability of cortisol and glucocorticoid responses. Changes in CBG have not been overly implicated in patients with glucocorticoid resistance. However, increased expression of CBG has been suggested to be responsible for the partial or complete resistance to steroids described in some patients with long-standing Crohn’s disease [175]. In chronic fatigue syndrome patients, a similar increase in CBG levels has also been described [145]. D. 11␤-Hydroxysteroid Dehydrogenase Glucocorticoid availability can also be regulated by other factors such as the expression of 11␤-hydroxysteroid dehydrogenase, an enzyme that converts the active form of steroids, e.g., cortisol and corticosterone, into the inactive form, e.g., cortisone and 11-dehydrocortisone [26]. Changes in the levels of this enzyme could, therefore, cause differences in circulating or tissue glucocorticoid concentrations. For example, decreased plasma cortisol levels were noted in obese men as a result of type I 11␤-hydroxysteroid dehydrogenase impairment [176], and decreased 11␤-hydroxysteroid dehydrogenase mRNA was shown in ulcerative colitis [177]. E. Cofactors GR is able to influence gene transcription by interacting with the basal transcription machinery through many cofactors, coactivators, or corepressors. Thus, defects in these many cofactors could also affect GR function. However, to date no mutations in any cofactors have been found to be associated with glucocorticoid resistance. However, one interesting recent study has shown that a viral protein acting as a cofactor can alter GR responses. The HIV-1 accessory protein, virion-associated protein (Vpr), binds directly to GR and p300/CBP cofactors, thereby functioning as an adapter protein between these components and enhancing GR-mediated gene transaction. This explains the observed glucocorticoid hypersensitive state associated with HIV-1 infection [178,179]. Vpr has been shown to enhance the endogenous glucocorticoid suppression of IL-12, but not IL-10, in human

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peripheral blood monocytes [180]. There is one example where a cofactor defect is thought to be involved in glucocorticoid resistance in two sisters with resistance to multiple steroids [181]. F. Transport Proteins Intracellular ligand concentration is yet another factor that, if altered, could affect GR function. Although glucocorticoids are believed to passively diffuse into cells, evidence for an active transport process out of cells exists. Multidrug-resistance (MDR) proteins, members of the ABC family of transporters [182], have been shown to transport glucocorticoids out of cells [183–187], and their involvement in glucocorticoid unresponsive disease states has begun to be investigated. Increased expression of the human MDR1 (ABCB1) has been shown in patients with inflammatory bowel disease who have failed medical therapy [188] and also in patients with rheumatoid arthritis [189] and SLE [190]. The novel orphan receptor pregnane X receptor (PXR) (also known as SXR), or PXR ligands, have been shown to be regulate these transporter proteins [191–194]. This receptor is activated by many ligands including glucocorticoids [195], suggesting a possible mechanism by which acquired glucocorticoid resistance during long-term therapy might develop. VI. CONCLUSION In summary, the CNS regulates immune function by multiple neuroanatomical, hormonal, and molecular mechanisms. This CNS regulation of the immune system plays a role in susceptibility to and pathogenesis of many autoimmune/inflammatory diseases. The major focus of this chapter has been the HPA axis and its final effector hormones, the glucocorticoids, and their regulation of immunity and severity of immune-mediated and inflammatory disease. Dysregulation of the HPA axis or glucocorticoid responses occurring at the level of the gene, protein, receptor, signaling cascade, and cell function could result in disease. As there are many different hormonal and nerve pathways involved in the regulation of immunity, the potential mechanisms for pathogenesis of autoimmune disease(s) resulting from disruptions in these interactions is large. Nonetheless, a thorough understanding of all levels by which the CNS and immune systems communicate and the disruptions in these communications that lead to disease will ultimately provide new avenues of therapy. REFERENCES 1. Scott LV, Dinan TG. Vasopressin and the regulation of hypothalamic-pituitary-adrenal axis function: Implications for the pathophysiology of depression. Life Sci 1998; 62:1985–1998. 2. Haddad JJ, Saade NE, Safieh-Garabedian B. Cytokines and neuro-immune-endocrine interactions: a role for the hypothalamic-pituitary-adrenal revolving axis. J. Neuroimmunol 2002; 133:1–19. 3. Rivest S. How circulating cytokines trigger the neural circuits that control the hypothalamicpituitary-adrenal axis. Psychoneuroendocrinology 2001; 26:761–788. 4. Calogero AE, Gallucci WT, Gold PW, Chrousos GP. Multiple feedback regulatory loops upon rat hypothalamic corticotropin-releasing hormone secretion. Potential clinical implications. J. Clin. Invest 1988; 82:767–774. 5. Hench PS, Kendall EC, Slocumb CH, Polley HF. Effects of cortisone acetate and primary ACTH on rheumatoid arthritis, rheumatic fever and certain other conditions. Arch. Intern. Med 1950; 85:545–666.

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107. Sternberg EM, Hill JM, Chrousos GP, Kamilaris T, Listwak SJ, Gold PW, Wilder RL. Inflammatory mediator-induced hypothalamic-pituitary-adrenal axis activation is defective in streptococcal cell wall arthritis-susceptible Lewis rats. Proc. Natl. Acad. Sci. U.S.A 1989; 86: 2374–2378. 108. Edwards CKI, Yunger LM, Lorence RM, Dantzer R, Kelley KW. The pituitary gland is required for protection against lethal effects of Salmonella typhimurium. Proc. Natl. Acad. Sci. U.S.A 1991; 88:2274–2277. 109. Ruzek MC, Pearce BD, Miller AH, Biron CA. Endogenous glucocorticoids protect against cytokine-mediated lethality during viral infection. J. Immunol 1999; 162:3527–3533. 110. Misiewicz B, Poltorak M, Raybourne RB, Gomez M, Listwak S, Sternberg EM. Intracerebroventricular transplantation of embryonic neuronal tissue from inflammatory resistant into inflammatory susceptible rats suppresses specific components of inflammation. Exp. Neurol 1997; 146:305–314. 111. Wick G, Sgonc R, Lechner O. Neuroendocrine-immune disturbances in animal models with spontaneous autoimmune diseases. Ann. N.Y. Acad. Sci 1998; 840:591–598. 112. Hu Y, Dietrich H, Herold M, Heinrich PC, Wick G. Disturbed immuno-endocrine communication via the hypothalamo-pituitary-adrenal axis in autoimmune disease. Int. Arch. Allergy Immunol 1993; 102:232–241. 113. Lechner O, Hu Y, Jafarian Tehrani M, Dietrich H, Schwartz S, Herold M, Haour F, Wick G. Disturbed immunoendocrine communication via the hypothalamo-pituitary-adrenal axis in murine lupus. Brain Behav. Immun 1996; 10:337–350. 114. Jafarian-Tehrani M, Sternberg EM. Animal models of neuroimmune interaction in inflammatory diseases. J. Neuroimmunol 1999; 100:13–20. 115. Tonelli L, Webster JI, Rapp KL, Sternberg E. Neuroendocrine responses regulating susceptibility and resistance to autoimmune/inflammatory disease in inbred rat strains. Immunol. Rev 2001; 184:203–211. 116. Wilder RL, Calandra GB, Garvin AJ, Wright KD, Hansen CT. Strain and sex variation in the susceptibility to streptococcal cell wall-induced polyarthritis in the rat. Arthritis Rheum 1982; 25:1064–1072. 117. Sternberg EM, Young WSD, Bernardini R, Calogero AE, Chrousos GP, Gold PW, Wilder RL. A central nervous system defect in biosynthesis of corticotropin-releasing hormone is associated with susceptibility to streptococcal cell wall-induced arthritis in Lewis rats. Proc. Natl. Acad. Sci. U.S.A 1989; 86:4771–4775. 118. Moncek F, Kvetnansky R, Jezova D. Differential responses to stress stimuli of Lewis and Fischer rats at the pituitary and adrenocortical level. Endocr. Reg 2001; 35:35–41. 119. Dhabhar FS, McEwen BS, Spencer RL. Stress response, adrenal steroid receptor levels and corticosteroid-binding globulin levels—a comparison between Sprague-Dawley, Fischer 344 and Lewis rats. Brain Res 1993; 616:89–98. 120. Stefferl A, Linington C, Holsboer F, Reul JM. Susceptibility and resistance to experimental allergic encephalomyelitis: Relationship with hypothalamic-pituitary-adrenocortical axis responsiveness in the rat. Endocrinology 1999; 140:4932–4938. 121. Aksentijevich S, Whitfield HJJ, Young WSI, Wilder RL, Chrousos GP, Gold PW, Sternberg EM. Arthritis-susceptible Lewis rats fail to emerge from the stress hyporesponsive period. Dev Brain Res 1992; 65:115–118. 122. Harbuz MS, Rooney C, Jones M, Ingram CD. Hypothalamo-pituitary-adrenal axis responses to lipopolysaccharide in male and female rats with adjuvant-induced arthritis. Brain Behav. Immun 1999; 13:335–347. 123. Smith CC, Omelijaniuk RJ, Whitfield HJJ, Aksentijevich S, Fellows MQ, Zelazowski E, Gold PW, Sternberg EM. Differential mineralocorticoid (type I) and glucocorticoid (type II) receptor expression in Lewis and Fischer rats. Neuroimmunomodulation 1994; 1:66–73. 124. Dhabhar FS, Miller AH, McEwen BS, Spencer RL. Differential activation of adrenal steroid receptors in neural and immune tissues of Sprague Dawley, Fischer 344, and Lewis rats. J. Neuroimmunol 1995; 56:77–90.

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21 Autoimmune Type 1 Diabetes EDWIN A. M. GALEand POLLY J. BINGLEY University of Bristol, Bristol, United Kingdom

I. INTRODUCTION Type 1 diabetes is often thought of as a disease of children, but the majority of cases present in adult life. The condition is less well characterized in older patients due to overlap with other forms of diabetes and dilution of some of its most distinctive clinical features. The major limiting factor in the study of autoimmune diabetes in humans has been the inaccessibility of its target organ, the pancreatic ␤ cells, and relatively little is known about the histopathology of the condition. To some extent this limitation has been remedied by investigation of the main animal model of autoimmune diabetes, the nonobese diabetic (NOD) mouse [1]. It should, however, be noted that standard descriptions often extrapolate information available only in the mouse to humans; this chapter will focus on evidence concerning the human form of the disease. The classic childhood form of type 1 diabetes affects 1 in 250–350 people in western countries by the age of 20 years. Presentation is typically acute in children, with a history of thirst, polyuria, and weight loss extending over several weeks; a proportion (now ⬍25% in most countries) will present with diabetic ketoacidosis, and occasional deaths still occur when diagnosis has been delayed, particularly in the very young. Clinical presentation in adults is typically less acute, presentation in ketoacidosis is unusual, and the distinction between immune-mediated and non–immune-mediated diabetes may become blurred. Replacement therapy with insulin sustains many millions of people but fails to restore normal glucose homeostasis. It therefore reduces but does not abolish the risk of late microvascular and macrovascular complications of diabetes. There has been steady improvement in the prognosis of childhood-onset type 1 diabetes over recent decades [2], but results from specialized centers merely emphasize the extent of our failure elsewhere. The benefits of optimized therapy have yet to reach the majority of affected children worldwide, and as 417

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a result some 20–30% will still die of or with diabetic nephropathy, and 50% will at some stage require laser therapy to protect their vision. II. HISTORICAL BACKGROUND A. Heterogeneity of Diabetes The clinical heterogeneity of diabetes has been recognized for well over a hundred years, but its etiological heterogeneity was not clearly established until the 1970s. In the preinsulin era, children invariably died of ketoacidosis, tuberculosis, or starvation within months or years of diagnosis, whereas overweight adults often survived reasonably well with dietary restriction. In the 1930s Himsworth correctly inferred that there were two main forms of the disease: an insulin-deficient form in lean young people, and an insulin-resistant form in overweight adults. Bioassay of the insulin content of postmortem pancreas in the 1950s added support to this concept, since the amount of insulin was reduced to below 10% of normal in early-onset patients, but remained at around 50% in late-onset diabetes. Lister first proposed the terms type 1 and type 2 diabetes in 1951 [3], much as they are used today, but did not influence the ruling view that diabetes was a single disease. In this view early-onset diabetes represented the homozygous form of the disorder and adultonset disease the heterozygous form. As a result, the available evidence suggesting that there were two forms of the disease was largely ignored, and Lister’s terminology was forgotten until reintroduced by Cudworth in 1976 [4]. By this time the concept of autoimmune disease had become well established, and it had become clear that juvenile diabetes qualified for inclusion in this category. B. Evidence That Type 1 Diabetes Is Immune-Mediated One of the earliest pointers to an autoimmune etiology was the observation that other autoimmune conditions, for example, affecting the thyroid or adrenal cortex, were overrepresented in juvenile cases of diabetes and their relatives, but that there was no excess in late-onset cases. Another important pointer was lymphocytic infiltration of the islets at diagnosis. This had previously been noted as an occasional finding at autopsy, but it was Gepts who showed that insulitis was largely confined to recently diagnosed cases in the young and suggested its significance [5]. Nerup had in 1971 observed that leukocyte migration was inhibited by the presence of islet tissue in juvenile but not late-onset cases, but 1974 was the annus mirabilis of autoimmune diabetes. In that year two groups reported the first HLA associations with juvenile-onset diabetes, and two groups reported the existence of islet cell antibodies. In the face of this evidence, the heterogeneity of diabetes was rapidly accepted. By this time the tissue tropism of viruses was also well established, mouse models of virally induced diabetes were available, and the phenomenon of MHC restriction was well described. Most investigators therefore conceived of type 1 diabetes as an acute disease in which ␤ cells were destroyed by viral invasion, thus exposing their contents to the immune system and provoking a secondary immune-mediated response resulting in diabetes [6]. It was on this basis that Cudworth and other investigators pioneered the prospective family study in which siblings and parents of affected children provided an accessible and motivated high-risk population for prospective study. It was hoped that repeated screening of the same families would catch the causative virus in passage and thus pave the way to preventive therapy by means of vaccination. In the event, prospective family studies failed

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to catch a virus but took an entirely new turn with the introduction of testing for islet cell antibodies. These antibodies, measured by indirect immunofluorescence, were present in some 70–80% of newly diagnosed cases. The unexpected findings were that they were also present in some 5% of relatives, that risk of progression to diabetes was largely concentrated within this subgroup—in whom the antibodies were therefore predictive of disease—and that, far from being acute, the disease has a very long prodrome [7]. Further evidence of autoimmunity in type 1 diabetes has accumulated over the past 25 years. The establishment of animal models represented a major step forward. The Wistar-derived rat from the Bio Breeding Laboratories of Canada Ltd—the BB rat—was first described in 1976. These nonobese animals developed a spontaneous insulin-deficient, ketosis-prone form of diabetes associated with histological features of insulitis. The most widely used model of type 1 diabetes, the nonobese diabetic (NOD) mouse, was described in 1980 and remains the most widely used disease model. Cell transfer experiments in the mouse have established beyond doubt that autoimmune diabetes is a T-cell–mediated disease requiring the presence of both CD4 and CD8 cells. Evidence in humans is more anecdotal and comes from bone marrow transplantation from donors with type 1 diabetes to nondiabetic recipients. Analysis of the International Bone Marrow Transplant Registry showed 9 cases in which this is known to have occurred; 6 recipients died within 2 years of marrow donation without developing diabetes, but 2 of 3 long-term survivors developed the disease [8]. Further support comes from twin-to-twin pancreas transplants. Unaffected monozygotic twins in long-term discordant pairs are considered unlikely to develop type 1 diabetes, and can therefore donate the tail of their pancreas to their affected co-twin. Attempts to graft from one twin to another were, however, foiled by recrudescence of the type 1 disease process in the transplanted organ. Insulitis developed within weeks of transplantation and progressed rapidly to diabetes, presumably because of reactivation of memory T cells and reenactment of autoimmune destruction of the islets [9]. Final proof that immune processes mediate human type 1 diabetes comes from trials of immune intervention. Most convincing among these were the trials of cyclosporin A in the newly diagnosed. Two major trials showed that immune intervention in recently diagnosed patients prolonged ␤-cell survival and allowed a proportion of cases to survive without insulin for more than a year [10,11]. The effect is lost as soon as the drug is withdrawn, however, and the risk:benefit ratio for this form of intervention is unfavorable. C. Classification of Diabetes Earlier attempts at classification were based upon treatment and relied upon the incorrect assumption that type 1 diabetes was consistently associated with an early need for insulin therapy. To complicate matters further, many people with type 2 diabetes also manifest a need for insulin over the course of time. For these reasons the familiar subdivision into insulin-dependent and non–insulin-dependent diabetes mellitus (IDDM and NIDDM) has been abandoned. An etiological classification is clearly preferable, but can only be as good as the tools available, and the current classification should therefore be considered interim rather than definitive [12]. In essence it relies upon the concept that (known causes of diabetes having been excluded) there are two major independent disease pathways resulting in ␤-cell destruction. Since both are dynamic processes, it is more relevant to define the underlying etiology than the prevailing level of glycemia at any particular stage in the disease process. For example, a hyperglycemic individual may not initially require insulin, but might in the course of time come to depend upon insulin for well-being, and

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ultimately for survival. A child with type 1 diabetes moves very rapidly from normoglycemia to absolute insulin deficiency, whereas the older person progresses towards insulin deficiency much more slowly and may never reach the stage of requiring insulin at all. Insulin-deficient individuals are at risk of diabetic ketoacidosis. This acute complication is often considered the hallmark of type 1 diabetes, although the majority of susceptible individuals within well-organized health care systems will never develop the condition because of early diagnosis and treatment. In people of European descent almost all those who present acutely and are considered ketosis-prone have markers of immune activation and are classed as having type 1A (immune-mediated) diabetes. In other ethnic groups, however, young individuals have been described who present in ketoacidosis in the absence of any immune markers and are therefore classed as having type 1B. The underlying etiology remains unknown. Most people who develop diabetes in adult life do so gradually. A ‘‘Starling curve’’ of insulin production has been described, whereby those in the early stages of the disease secrete quite high levels of insulin, reach a peak of insulin production, and then decompensate with falling insulin levels and progressive hyperglycemia. This sequence of events takes place against a spectrum of insulin sensitivity, and we may picture a range extending from lean insulin-deficient individuals with near-normal insulin sensitivity to overweight individuals with insulin resistance and high circulating levels of insulin. The clinician will have no difficulty in identifying those at either end of the spectrum as having typical type 1 and 2 diabetes, respectively. The majority of cases will, however, show a mixture of insulin deficiency and insulin resistance, and the two disorders can interact in complex ways [13]. Most patients will show declining insulin secretion over time; the rate of ␤-cell loss will depend both upon disease processes intrinsic to the pancreas, such as autoimmunity, and upon the burden of insulin resistance which the surviving ␤-cell mass is required to carry. It has been shown that measurement of islet autoantibodies can identify a subgroup of individuals who differ in phenotype from typical type 2 diabetes: they have a lower body mass, features of the metabolic syndrome are absent, progression to ␤-cell deficiency is more rapid, as judged by loss of residual C-peptide secretion and clinical requirement for insulin, and HLA alleles conferring susceptibility to type 1 diabetes are more likely to be present [14]. This category has been referred to as latent autoimmune diabetes of adults (LADA), and represents some 5–10% of new cases with a clinical diagnosis of type 2 diabetes. GAD autoantibodies (GADA) are the markers most widely used to identify LADA, because they are prevalent, relatively simple to measure, and persist after diagnosis. Autoantibodies directed against insulin and IA-2 (IAA and IA-2Ab) are rarely found at diagnosis in older people, whereas islet cell antibodies (ICA) are useful but difficult to measure. GADA were found in 10% of patients with clinically diagnosed type 2 diabetes recruited to the United Kingdom Prospective Diabetes Study (UKPDS), and ICA were present in 6%; 12% had at least one antibody, and both were found in 4% [15]. The main limitation of this approach is that it relies upon the cut-off value of the GAD assay being used. Workshop analyses show that most assays can identify high antibody levels reliably, but that—as might be expected—the greatest degree of intra- and interassay variation is present around the lower levels used to define the cut-off point. The cut-off point for a radiobinding assay is clearly not ideal as the basis for an etiological classification, and more reliable means of discrimination are badly needed.

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III. GENETICS OF TYPE 1 DIABETES A. Empirical Risks By the age of 20 years, type 1 diabetes will have affected some 0.3–0.4% of children in the background population in western countries, and around 6% of siblings of childhood onset cases, giving a ratio (␭S) of 15. Familial risk is increased in relatives of children diagnosed under the age of 5 [16]. It is sometimes forgotten that the estimates given above do not represent lifetime risk of type 1 diabetes, which may be as high as 1% in the background population and 15% in siblings. Siblings HLA-identical to the proband have a 12–15% risk of developing diabetes by age 20, compared to an approximate 30% risk in monozygotic twins; lifetime estimates for twin risk range up to 70%. For unknown reasons, affected fathers are more likely to transmit type 1 diabetes to their offspring than affected mothers, with risks ranging between 6–9% and 1–3%, respectively [17]. B. The HLA System The HLA complex contains over 200 genes. HLA class I and II genes are highly polymorphic heterodimers made up of ␣ and ␤ chains; the ␤ chain of HLA class I molecules is encoded on chromosome 15 and is not polymorphic, while the ␣ chains of the major class I molecules are encoded by three genes designated HLA-A, -B, and -C, respectively. Of the class II molecules, only the ␤ chain of HLA-DR is polymorphic, compared to both ␣ and ␤ chains of DQ molecules. HLA molecules function by presenting antigens that have been processed into short peptides to antigen-specific receptors on CD4 and CD8 T lymphocytes. Class I molecules are present on most nucleated cells. Peptides derived from pathogens that multiply within the cytoplasm are carried to the cell surface by MHC class I molecules and presented to CD8 cells, which differentiate into cytotoxic T cells in response to this stimulus and kill the infected host cells. In contrast, class II molecules are present on antigen-presenting cells such as dendritic cells and macrophages and are encoded by the HLA DR, DQ, and DP loci. Peptide antigens derived from ingested extracellular bacteria or toxins, or from pathogens multiplying in intracellular vesicles, are carried to the cell surface by MHC class II antigens and presented to CD4 cells, which then differentiate into Th1/Th2 subclasses. The most powerful disease associations with type 1 diabetes involve genes in the class II region. For example, the HLA haplotype DQB1*0302-DQA1*0301 carries increased disease susceptibility, as does DQB1*0201-DQA1*0501; heterozygotes combining both of these haplotypes carry the highest risk of all, implying that each makes an independent contribution to disease susceptibility. Other haplotypes such as DQB1*0602DQA1*0102 confer dominant protection against disease development; thus, 20% of Europeans carry this allele, but it is found in less than 1% of children with type 1 diabetes. Disease susceptibility or resistance does not reside entirely within the class II region, however, since the influence of genes in this region is modified by the presence or absence of class I genes within the extended haplotype [17]. The HLA region is a major player in type 1 diabetes, as in other autoimmune diseases. Despite the intense study that has been lavished on this region, however, its role in disease susceptibility remains unclear. For example, it has been known for some 15 years that single amino acid substitutions within the binding groove of the HLA class II molecule that yield a noncharged amino acid at position 57 of the HLA-DQB1 allele confer susceptibility,

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whereas an aspartate residue (which carries a charge) confers resistance in this position, but even this potent clue has failed to uncover the underlying molecular mechanism. A more recent analysis of the crystal structure of IA—the murine orthologue of DQ in humans—identified conserved similarities between the structural features associated with diabetes resistance and susceptibility in both species. This suggests that similar target antigens and disease pathways may be involved [18]. C. Other Genes Involved in Susceptibility to Type 1 Diabetes The HLA region contributes some 50% of genetic susceptibility to type 1 diabetes and is referred to as IDDM1; other loci tentatively identified as conferring susceptibility are identified as IDDM2 through to IDDM17. IDDM1 and IDDM2 were originally identified as candidate genes based on case-control studies; the remainder have been located by linkage studies in affected sib-pair families using genome scans. A number of these loci have proved spurious, but a consensus genome scan suggests that five regions other than IDDM1 and IDDM2 show evidence of linkage to type 1 diabetes [19]. Of these, IDDM2, located within the regulatory region of the insulin (INS) gene on chromosome 11p15.5, is a minisatellite (VNTR) formed by tandem repeats of a 14–15 bp oligonucleotide sequence. These repeats vary in number from 26 to 209 and fall into three size classes referred to as class I (26–63 repeats), class II, and class III (⬎140 repeats). Class II is generally absent in populations of European origin, and the frequencies of the class I and III alleles are 0.71 and 0.29, respectively. Homozygosity for the class I allele is associated with a two- to fivefold increase in the risk of type 1 diabetes, whereas class III appears to confer dominant protection. The mechanism underlying this susceptibility is conjectural, but it has been proposed that the VNTR modulates insulin transcription in the thymus and pancreas and that greater INS expression in the thymus associated with the class III allele might confer greater tolerance to insulin precursor molecules [20]. A number of loci have attracted particular attention because they are also associated with other autoimmune conditions, suggesting the existence of common pathways predisposing to loss of self-tolerance [21]. IDDM12 maps to chromosome 2q33 in a region that includes the CTLA4 gene. CTLA4 (cytotoxic T-lymphocyte–associated protein 4) is expressed only on activated T lymphocytes and downregulates T-cell function, limiting both activation and expansion. Deletion of this gene results in lethal autoimmune disease in a knockout mouse model. Polymorphisms that impair this function might therefore result in unwanted persistence of lymphocyte activation and failure of immune tolerance. Many studies have shown association between the CTLA4 region and Graves’ disease, one of very few genetic associations in common disease that is considered to be beyond doubt [22]. Associations with autoimmune hypothyroidism have also been reported, and association and linkage to type 1 diabetes have also been described, although less consistently [23]. At present three candidate SNPs in CTLA4 are under investigation and promise intriguing insights into potential disease mechanisms, even though their contribution to genetic susceptibility in type 1 diabetes is likely to be small. IV. IMMUNE CHANGES ASSOCIATED WITH TYPE 1 DIABETES A. Islet Autoantibodies The autoimmune nature of type 1 diabetes was confirmed when islet cell antibodies (ICA) were demonstrated by indirect immunofluorescence in 1974, but the nature of the antigens involved was unknown. Insulin itself was identified as an autoantigen in 1984, followed

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by proinsulin, but autoantibodies directed against these molecules do not participate in the ICA staining reaction. In 1992 glutamic acid decarboxylase (GAD) was identified in the serum of patients with type 1 diabetes and of patients with ‘‘stiff man syndrome’’ (now known as stiff person syndrome) [24]. The isoform involved in diabetes, GAD65, is a protein of 585 amino acids encoded by a gene on chromosome 10p11 and located in small vesicles within neuroendocrine cells; its function is unknown. Antibodies directed against GAD recognize conformational epitopes in the central and C-terminal portions of the molecule. A further major antigen known as islet-autoantigen 2 (IA-2), also known as ICA-512, was identified soon after GAD. This is a member of the transmembrane protein tyrosine phosphatase (PTP) family, composed of 979 amino acids and coded for by a gene on chromosome 2q35. It is present in the secretory vesicles of endocrine and neuronal cells and is enzymatically inactive due to an amino acid substitution at position 911. Autoantibodies recognize predominantly conformational epitopes within the intracellular portion of the molecule [25]. The catalogue of islet autoantigens extends well beyond those listed above and is still far from complete. For example, the ICA staining reaction is not wholly accounted for by antibodies to GAD and IA-2. ICA are tedious and difficult to measure but remain of value in diabetes prediction, whereas the ability to synthesize insulin, GAD65, and IA2 in recombinant form has led to the development of sensitive high-performance radiobinding assays. Insulin autoantibodies are found in up to 50% of young children with newly diagnosed diabetes, but become progressively uncommon with increasing age. IA-2 antibodies are present in around 70% of children and young adults, but are less frequent in late-onset autoimmune diabetes. GADA and ICA are found in patients of all ages; ICA titres decline following diagnosis, presumably due to loss of target molecules as the ␤ cells are destroyed, but GADA are persistent and are therefore useful late markers of autoimmunity. B. Diabetes Prediction Patients and relatives are often characterized in the literature as ‘‘antibody positive.’’ This remains useful shorthand but should be interpreted with caution, since antibody levels are distributed as a continuous variable above the detection limit of the assay, whereas positivity is defined by the cut-off point of the assay used. One approach is to use centiles derived from a healthy childhood population to identify those threshold levels that achieve the best discrimination between health and disease [26]. On this basis one or more islet autoantibodies can be identified in ⬎90% of children with newly presenting diabetes. Antibodies directed against a single antigen have little prognostic significance, but autoantibodies against multiple antigenic determinants confer a very high risk of progression to diabetes [27,28]. C. Cellular Markers of Autoimmune Diabetes It remains a paradox that knowledge of the humoral response to islet autoantigens has advanced to the point that highly specific prediction is possible, yet most investigators believe that autoimmune diabetes is a cell-mediated disease to which humoral autoimmunity contributes little. In humans, as discussed earlier (Sec. II.C), evidence comes from twin-to-twin transplants, marrow donation, and the response to immunotherapy directed towards the cellular immune response. In mice T cells have been shown to transfer diabetes, the process is thymus dependent, and both CD4 and CD8 cells are necessary. Human

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studies have largely depended upon analysis of peripheral T-cell populations. Where human data are available from pancreatic specimens, they suggest a diverse rather than oligoclonal T-cell repertoire. A huge effort has been expended on identification of potential targets of autoreactive T cells, responsive to antigens including insulin, GAD, and hsp60. As with the humoral response, multiple islet autoantigens appear to be implicated in the disease process, whether causally or coincidentally, and are in most cases not tissue-specific. Antigen presentation will of course be influenced by HLA type. This degree of heterogeneity remains an important obstacle to implementation of therapies targeted to autoantigen recognition by T cells, especially while actual disease mechanisms remain to be elucidated. Finally, problems with standardization of assays has greatly limited studies of T-cell responsiveness to antigens, and this has placed a major limitation on their application to diabetes prediction [29]. D. Mechanisms of ␤-Cell Destruction As mentioned earlier, the inaccessibility of the pancreatic islets has been a major bar to progress in understanding the disease. Until recently, knowledge has been acquired either by inference from the NOD mouse or by analysis of autopsy specimens. The insulitis lesion is so elusive that it evaded detection for many years; it is transient, meaning that it can only be detected in those who die shortly after diagnosis, and even then not consistently. Insulitis is typically present in children who die of diabetes under the age of 14, although not in all islets. It is less common in adolescents and is rare in adults [30]. When present, it is characterized by mononuclear cell infiltration and reduction in the number of cells staining for insulin. Pancreatic biopsy has recently been reported from Japan, and biopsy specimens with insulitis are characterized by the presence of both CD8 and CD4 T cells, B lymphocytes and macrophages; CD8 cells predominate. CD4 cells are predominantly Th1 in type, consistent with findings in the NOD mouse. Islet cells show hyperexpression of HLA class I molecules [31]. Fas-mediated apoptosis appears to be an important mechanism of ␤-cell destruction, and biopsy samples show that Fas is expressed on ␤ cells within inflamed islets, while Fas ligand is present on infiltrating mononuclear cells [32]. Functioning ␤ cells are invariably present at the time of diagnosis, but their survival beyond this point is age-dependent. Adults almost invariably show long-term persistence of ␤ cells, whereas almost all are lost within one year in children diagnosed under the age of 7 [30]. This observation, supported by clinical data, makes it clear that there is a strong inverse relation between age at onset and rate of ␤-cell destruction. Equally, it underlines the point that the process is typically indolent in the great majority of instances, both before and after the onset of hyperglycemia. One possible reason for this is that the ␤-cell population is heterogeneous; some cells secrete insulin at low glucose levels, whereas others are only triggered by high levels. The susceptibility of ␤ cells to experimental insult also varies according to their level of metabolic activity, which can be controlled by modifying the ambient concentration of glucose. Finally, ␤ cells exist within a community of cells, which provide regulatory signals and offer a range of support functions, a milieu that is undoubtedly disrupted by the proinflammatory environment generated by cytokines produced by invading immune cells [33,34]. V. DESCRIPTIVE EPIDEMIOLOGY OF TYPE 1 DIABETES A. Initiation of the Disease Process There is some evidence that prenatal influences may affect subsequent development of diabetes. These include reports that intrauterine infection with viruses including rubella

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and coxsackievirus may predispose to later development of diabetes in the child (see below). Increasing maternal age at delivery increases the risk of diabetes in the child 1.5to 3-fold, for reasons that are not understood. Women have tended to have their babies later over recent decades, and this trend accounts in small part for the observed increase in incidence [35,36]. Further evidence that influences encountered early in life play an important role in the development of diabetes comes from prospective studies in newborn children with a family history of type 1 diabetes. Transplacental antibodies disappear over the first 6–9 months of life, and native autoantibodies directed against islet constituents are most likely to appear within the first 1–3 years of life. IAA are generally the first to be detected, but this is not invariable. The immune response spreads to involve additional antigens in those destined to develop diabetes later in life [37], and several patterns of antibody or epitope spreading have been described. Candidate environmental influences will be described later. B. Evidence of a Long Preclinical Prodrome The observation that islet autoantibodies appear early in life is consistent with the observation from prospective studies in relatives that islet autoantibodies can be detected in the circulation long before the development of diabetes. We ourselves have followed one individual with islet autoantibodies for 18 years before he eventually developed diabetes, and have argued that almost all those with multiple islet autoantibodies will eventually develop diabetes if followed for long enough [38]. Does this mean that all cases of type 1 diabetes are initiated in early childhood, whatever the age at clinical diagnosis? This may not be the case, since we have observed that the prevalence of islet autoantibodies in first degree relatives is roughly equal in each gender, but that a strong male bias appears in the second decade of life, consistent with the male excess seen in adults with type 1 diabetes [39]. Studies from Finland support the concept that islet autoantibodies are not necessarily fixed, and continue to appear for the first time into adolescence [40]. C. Capacity to Secrete Useful Amounts of Insulin in Newly Diagnosed Patients In health, pancreatic ␤ cells store quantities of insulin well in excess of the average daily requirement. Early studies showed that the insulin content of the pancreas is reduced to around 10% of normal at postmortem in young patients, and this figure has been quoted somewhat uncritically ever since. Histological studies of specimens taken shortly after disease onset show that some 5–20% of islet cells stain for insulin, but such patients typically died with uncontrolled diabetes, an agonal state in which functioning ␤ cells may have exhausted their reserves of insulin. Animal studies have confirmed that ␤ cells that do not stain for insulin are present at diagnosis, and that these can subsequently function effectively in a noninflammatory environment. Endogenous insulin secretion may be judged by measurement of circulating connecting (C) peptide, a peptide segment that becomes detached as proinsulin splits to form insulin in the course of insulin secretion. Stimulation tests show that residual ␤-cell function improves over the first months of insulin treatment, sometimes to the extent that insulin is no longer needed, but that this ability is lost over time in all save a small minority of patients. Partial preservation of ␤cell function is a justifiable aim of immune intervention [41], even at the advanced stage of the disease process that exists at clinical diagnosis, since patients with residual insulin secretion achieve better metabolic control with less hypoglycemia over the first 6 years of diabetes [42].

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D. Gender It has been estimated that 6.7 million women are affected by autoimmune diseases in the United States, compared to 1.8 million men [43]. The common statement that there are no gender differences in type 1 diabetes is incorrect. An international survey of gender ratios in children presenting under the age of 15 noted a minor male excess in Europe and populations of European origin, while a female excess could be noted in populations of African or Asian origin. Very high-incidence populations are characterized by male excess, and vice versa [44]. In contrast, clear male preponderance has emerged from most studies of patients with type 1 diabetes diagnosed between 15 and 40 years [45]. Adult type 1 diabetes therefore appears to differ from other common autoimmune diseases, which typically show a strong female excess, as does diabetes in the NOD mouse. E. Geography of Type 1 Diabetes Type 1 diabetes has historically been most prevalent in populations of European origin, but is becoming more frequent in other ethnic groups. Within Europe the highest rate of childhood diabetes is found in Finland, and high rates of the disease are found in Scandinavia and northwest Europe. Large differences do, however, exist between genetically related populations: type 1 diabetes in more common in Norway than in Iceland, while Finnish children have a threefold risk compared to Estonians. The incidence of type 1 diabetes is lower in southern or eastern Europe; a child in Macedonia is, for example, 10 times less likely to develop diabetes than a child in Finland [46]. Sardinia is the exception to this general rule, and has the second highest rate of type 1 diabetes in the world. As with Finland, founder effects have played a major role in determining the genetic make-up of the population, and it has been estimated that modern Sardinians are descended from 3000–5000 settlers distantly related to the modern Berbers who colonized the island 5000 or more years ago. The Sardinian population has distinctive HLA characteristics and very high rates of autoimmune conditions, including type 1 diabetes, celiac disease, multiple sclerosis, and autoimmune thyroid disease. On present evidence, geographical differences in the incidence of type 1 diabetes are partly, but incompletely, explained by the distribution of high-risk HLA alleles. The incidence of type 1 diabetes remains relatively low in most other ethnic groups, but it should not be assumed that non-Europeans have some form of genetic protection from the disease, since many non-European populations now report a rising incidence of the disease. Kuwait now has the seventh highest rate of childhood-onset type 1 diabetes in the world. Migrant studies are of particular interest, and suggest that Samoan children moving to New Zealand or Indians or Pakistanis moving to the United Kingdom assume the local risk of childhood diabetes [47]. F. Evidence of a Rising Incidence There are indications that childhood-onset diabetes was a rare condition at the start of the twentieth century. Although a proportion of children undoubtedly died undiagnosed at presentation of the disease, early reports are consistent in suggesting that western populations had a low and relatively constant rate of the disease over the first half of the century, at levels equivalent to those seen today in parts of southern Asia. From the middle of the twentieth century, or soon after, a number of populations showed an upturn in incidence which has continued in more or less linear fashion to the present day. Those who remain

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sceptical as to the value of earlier reports may note that backwards extrapolation of the rising incidence of the disease will reach zero values in the 1930s, thus providing additional support for the concept of an upwards inflection around the middle of the last century. The current overall rate of increase in Europe is around 3–4% per annum, although there are important qualitative differences, with some evidence of a rapid increase in parts of eastern Europe and of a leveling off in high-incidence countries such as Norway [48,49]. G. The ‘‘Spring Harvest’’ Hypothesis Kurtz first suggested in 1988 [50] that the rising incidence of childhood diabetes might represent a left shift in age of onset rather than an absolute increase in the lifetime risk of the disease within a population, and others have noted that this effect would be consistent with increased penetrance of susceptibility genes in the face of a more permissive environment [48]. Consistent with this there is some evidence that the increase in the 0–14 age group has been partially compensated by a decrease in young adults, so that the cumulative incidence by age 30 remains unchanged [51,52], and there is also evidence that the proportion of the highest-risk HLA haplotypes has declined over time in children with earlyonset diabetes, with emergence of haplotypes lower in the risk hierarchy [53]. VI. ETIOLOGICAL FACTORS A. General Principles Harrison has proposed criteria that should be satisfied when assessing the role of an environmental agent in causation of an autoimmune disease: Immune responses to the antigen are disease specific. Immune responses to the antigen precede the onset of disease. Immune responses to the antigen reflect disease pathology; i.e., are surrogate markers. Antigen specific antibodies or T cells mediate disease. Immunization with antigen reproduces disease. Antigen peptides co-purify with disease associated MHC molecules. Manipulation of antigen expression modifies disease expression. Administration of antigen via tolerizing mode or route prevents disease. At present none of the environmental agents proposed for type 1 diabetes fulfills all these criteria, and the self-antigens proinsulin and insulin come closest. Problems that limit progress in this area are the lack of standardization of T-cell proliferation studies, and the need for HLA matching in both immunological and epidemiological analyses [54]. B. Viruses and Type 1 Diabetes Early in the last century, physicians noted an apparent link between mumps infections and the onset of diabetes, but scientific investigation did not really take off until it was shown in the 1960s that diabetes could be induced in mice by the encephalomyocarditis virus and by coxsackie B4. This was followed by the observation that congenital rubella predisposed to childhood diabetes, offering proof that a virus could cause autoimmune diabetes in humans [6]. The literature on this subject predated modern methods of analysis, however, and it remains unclear whether children with congenital rubella had typical type

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1 diabetes: some at least had an insulin-resistant form of the disease. A large number of epidemiological studies followed and appeared on balance to implicate coxsackievirus in recently diagnosed patients. Recent systematic review of 26 case-control studies published between 1966 and 2001 found little evidence of a consistent association [55], however, and it seems unlikely that the question will ever be resolved by retrospective analysis of circulating antibodies. Viruses are attractive etiological candidates. They penetrate cells and alter the way in which these are recognized by the immune system; they show tropism for tissues including the pancreatic islets, and animal models provide good evidence that viruses can provoke autoimmune responses. In addition, susceptibility is often determined by HLA type, in common with a whole range of autoimmune conditions. The difficulties of pinpointing a viral etiology for an autoimmune disease have been discussed by Rose [56]. Most virus infections are asymptomatic or trivial, and causal exposures might precede clinical disease by years or even decades. Common viruses are ubiquitous and frequently exchange genetic material by recombination; the timing or frequency of exposure or the particular strain of virus involved may be critical. There are in addition many potential mechanisms by which viral infection might promote autoimmunity. At one end of the spectrum these may cause acute cell rupture with exposure of intracellular antigens to the immune system; at the other, persistent or latent infection might modify the affected cell in ways that lead to altered recognition by the immune system, followed in turn by chronic low-grade damage. Finally, viruses may simply precipitate clinical presentation when superimposed upon an established chronic autoimmune process involving the islets. Enteroviral infection has attracted the most interest in recent years, with particular emphasis on coxsackievirus. Sequence homology with an identical PEVKEK motif exists between coxsackie B4 and the islet antigen GAD65 [57], but reports of immune crossreactivity are conflicting. As noted above, there are many case-control studies in the recently diagnosed, but these contribute little to the argument. Antibody studies may not discriminate between diabetogenic and neutral strains of virus, and there has been concern that differences between antibody responses or viral persistence in people with diabetes and controls may simply reflect patterns of immune responsiveness related to HLA type [58]. Antibodies represent the footprints of departed viruses, and the actual presence of the virus in the circulation is best detected by PCR techniques. A number of studies have reported detection of viral RNA in newly diagnosed or prediabetic children, most commonly coxsackie B4. PCR techniques have the limitation that the RNA sequences detected come from the untranslated part of the genome and do not indicate the precise serotype, since this is based on variations in capsid proteins [59]. The problem thrown up by these reports is that autoimmune diabetes has a long prodrome, which implies that recent acute infection cannot actually cause the disease (although this possibility cannot be ruled out in very young children). Evidence of recent infection in older individuals might suggest that acute viral infection simply precipitates clinical onset in preclinical cases of autoimmune diabetes, thus excluding any fundamental role in causation of the disease. There is, therefore, a need to establish whether the viremia reported by these studies is acute or persistent, and—if the latter—the age at which the virus was acquired. Further suggestive evidence of acute vs persistent viral infection comes from cytokine studies in the newly diagnosed. The most potent antiviral cytokines are interferons␣ and -␤. These form part of the early innate response to viral infection, and high levels therefore suggest recent infection. Autopsy reports have shown that interferon-␣/␤ was

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expressed on the surface of ␤ cells, but not other islet endocrine cells, in patients who died soon after diagnosis of diabetes [60]. Raised circulating levels of interferon-␣ have also been reported in the serum of newly diagnosed patients, but might simply reflect the proinflammatory milieu that exists at that stage of the disease. An etiological role for viruses was also suggested by case-control studies of mothers whose children subsequently developed type 1 diabetes. A Swedish study showed that such mothers were more likely to show serological evidence of enteroviral infection in pregnancy, although the fraction of cases with maternal infection was small [61]. A smaller study using a combination of PCR and IgM antibodies found evidence that enterovirus RNA was more common in the serum of mothers whose children later developed diabetes, but a subsequent study in Finland failed to confirm this [62]. There is at present conflicting evidence as to the role of early viral exposure in the pathogenesis of type 1 diabetes. Despite a mass of circumstantial evidence relating to serological changes suggestive of recent viral exposure in the newly diagnosed, no reliable conclusions can be drawn at this stage. PCR evidence of viremia before or around the time of diagnosis continues to accumulate, but is hard to interpret. Large-scale prospective studies from birth using improved methods of investigation should eventually resolve some of these issues [59,62]. On present evidence, viral infection can only be implicated in a minority of cases of type 1 diabetes and cannot explain the relentless linear rise in incidence noted from many parts of the world.

C. Milk and Other Early Nutritional Influences Celiac disease and type 1 diabetes have common features, including a degree of overlap between HLA haplotypes conferring susceptibility. The environmental agent responsible for celiac disease is of course well known, but the condition may nonetheless develop years or even decades following first exposure to dietary gluten. Could dietary constituents encountered early in life (and the immune responses these provoke) play a similar role in the pathogenesis of type 1 diabetes? There has been controversy for 15 years as to whether early exposure to cow’s milk predisposes to childhood diabetes; similar arguments have taken place in regard to other diseases, including asthma and multiple sclerosis, but remain equally inconclusive. Animal studies have shown that elimination of milk proteins (present in standard laboratory chow) from the early diet greatly reduces the risk of diabetes in the BB rat, with similar results in the NOD mouse. Other dietary constituents, including wheat and soybeans, have also been implicated, however, and cow’s milk is only one among a number of possible dietary candidates [63]. The only systematic review of the epidemiological evidence in humans found a weak (odds ratio 1.5) but detectable positive effect of early exposure to cow’s milk proteins [64], but subsequent reports have yielded conflicting results, and the area remains controversial [54,65]. Serological responses to milk constituents have also been studied in infants, but once again no consistent pattern has emerged; as with analysis of antibody responses to viruses, case-control studies need to be corrected for HLA susceptibility. One study did, however, suggest that some species of milk antibody are more common in cases than in siblings sharing the same HLA alleles [66]. As with virus exposure, prospective analysis of high-risk populations from birth has great advantages over other means of analysis and will allow exposure to milk and other dietary constituents to be correlated with formation of islet autoantibodies. Improved and standardized methods of analysis of cellular immune responses to dietary constituents are

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also badly needed and should also be corrected for HLA type [54], but the issue will only be resolved beyond doubt by intervention studies (see below). D. The Hygiene Hypothesis We have seen that the role of proposed etiological agents remains controversial, and it would certainly be difficult to account for the steadily rising global incidence of childhood type 1 diabetes in terms of viral exposure or exposure to dietary factors. The alternative possibility is that protective factors have been lost from the early childhood environment [67]. The most widely accepted version of this concept is the hygiene hypothesis. This evolved from epidemiological observations suggesting that atopic disorders were more common in affluent than in traditional societies, that their prevalence was increasing worldwide in parallel with the adoption of a western lifestyle, and that both features might be related to reduced exposure to infections and other immune challenges in early life. These observations were subsequently linked to the proposal that healthy maturation and diversification of the neonatal immune repertoire is impaired by the relative absence of infectious and other environmental immune stimuli [68,69]. The hygiene hypothesis, as originally stated, rested on the proposal that fetal patterns of immune response (conventionally stated to have a Th2 bias) persist into later childhood and potentially into adult life; it is not intuitively obvious how this might account for a Th1-orientated disease such as type 1 diabetes [70]. The prevalence of a range of immune disorders characterized by both Th1 and Th2 deviation is increasing in western countries. Epidemiological evidence suggesting that early infections protect against susceptibility to atopic disorders includes protective effects of higher birth order and family size, protection conferred by early social mixing of children, and other influences such as pet ownership. None of these have been convincingly demonstrated in type 1 diabetes. There is, however, unequivocal evidence that a sterile environment promotes development of diabetes in the NOD mouse. Pinworms are common pathogens of mice colonies and are noted for their ability to protect against NOD diabetes and other experimental models of immune disease; it is therefore of interest that pinworms appear to have been ubiquitous in the childhood population in the middle of the twentieth century and have subsequently become much less prevalent [70]. Several authors have now proposed immune mechanisms, based around maturation of regulatory T- cell responses, that might account for the increasing frequency of immune disorders at both ends of the spectrum of Th-cell response within our community [71,72]. VII. PREVENTING TYPE 1 DIABETES A. General Principles A reasoned approach to prevention of type 1 diabetes might follow a series of questions as follows: What is the time course of the disease process? Are there confirmed etiological factors? At what stage of development are these operative? What are the markers of disease progression? What mechanisms are involved in the target organ? How might these be forestalled or interrupted?

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Material presented earlier in this chapter will have made it clear that there are still wide gaps in our understanding of many of these issues. Considerable progress has, however, been made in answering the third and fourth questions: it is clear that most cases of childhood diabetes are initiated early in life, and reliable markers of ongoing disease have become available. Any form of intervention must at present be largely speculative in nature, given that etiological factors remain contentious and that it is far from clear which of the wide range of proposed disease mechanisms is responsible in humans. There are standard criteria for a condition in which screening would be justified. The disease must represent an important burden for the individual and the community, the natural history of the condition should leave a window of opportunity for screening and intervention, reliable and cost-effective means of screening should be available, and effective forms of intervention should be on hand. Type 1 diabetes satisfies the first three criteria without difficulty, but we still lack an effective form of intervention. The potential for successful intervention does, however, appear to exist. More than 100 interventions have been reported to delay the onset of diabetes in the NOD mouse. The great majority of these are effective only if given within the first week of life, before or around the time at which insulitis becomes apparent, but a few interventions are effective even when given at the time of disease onset. Notable among these are antibodies to CD3 [73], and experience with this therapy in humans will be described below. Intervention trials aimed at ␤cell rescue may be considered at three distinct stages in the disease process. The first level of intervention is before detectable abnormalities are present (primary intervention), the second level is after development of immune markers of progression but before the onset of hyperglycemia (secondary prevention), and the third is after presentation with overt hyperglycemia (tertiary prevention). These will be considered in the following sections. B. Primary Prevention Primary prevention should be offered as early in life as possible, in practice from soon after birth. Children born to a family affected by diabetes, especially those who carry high-risk HLA genotypes, are most appropriate for trial interventions, and affected families are often highly motivated to participate. Safety is the major criterion for any form of primary prevention, since this will inevitably have to be offered to many who would not in any case have developed the disease. The most safe and rational form of primary prevention would be modification of environmental determinants of disease. As we have seen, associations between enteroviral exposure in utero and subsequent development of diabetes in the child are conflicting. If more convincing evidence comes to light, women might potentially be vaccinated against a range of viruses in addition to rubella. The cow’s milk hypothesis is equally controversial, but can be tested. A major multinational trial known as TRIGR (Trial to Reduce IDDM in the Genetically at Risk) is currently underway [74]. More direct forms of immune intervention in the newborn would appear unjustified at our present level of understanding of the disease process, although various forms of vaccination are under consideration. C. Secondary Prevention This is based on identification of individuals at increased risk of progression to diabetes, generally based on combinations of islet autoantibodies, plus measurement of first phase insulin secretion (see above). The latter is technically demanding in large studies, but

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identifies those at most imminent risk of progression and thus helps to stage the disease process. Two large studies have reported to date. In the United States, the Diabetes Prevention Trial—Type 1 (DPT-1) has reported on the use of parenteral insulin therapy. The logistics of this unblinded trial presented a major challenge, since 84,000 relatives required screening—250 for each entrant to the trial. Risk was estimated by measurement of ICA, followed by performance of two intravenous glucose tolerance tests, allowing identification of an estimated ⬎50% risk of progression to diabetes within 5 years. Parenteral insulin proved ineffective in the prevention of type 1 diabetes, but a second trial based on oral insulin administration is still underway at the time of this writing [75]. Meanwhile, and in parallel, a multinational group carried out the European Nicotinamide Diabetes Intervention Trial (ENDIT), based on regular administration of high-dose nicotinamide or placebo [76]. This study required screening of ⬎30,000 first-degree relatives in 21 countries, and recruitment was based upon high-titer ICA, followed by an intravenous glucose tolerance test to allow disease staging. Subsequent analysis showed that screening the same population with multiple antibodies would have allowed recruitment of an enriched sample of high-risk individuals and could potentially have allowed two intervention trials to be conducted within the same screening population [77]. Nicotinamide also proved ineffective in the prevention of diabetes (unpublished results). These two major studies have demonstrated the feasibility of large-scale controlled trials in antibody-positive first-degree relatives, but the logistics of these trails are daunting and the number of interventions that can be tested is very limited. For this reason the preferred strategy at present is to test potential interventions for evidence of efficacy in recently diagnosed patients as a means of identifying the most promising for use in future trials of secondary intervention. D. Tertiary Prevention As noted earlier in this chapter, useful numbers of ␤ cells are present at the time of diagnosis of type 1 diabetes, and the functional mass may be sufficient to permit a patient to maintain normoglycemia without insulin for a period, usually within the first year of treatment. The potential benefits of a functional ␤-cell unit are many, and include better glucose control with less hypoglycemia [41]. Trials with cyclosporin demonstrated that ␤-cell survival can be improved by immunotherapy [10,11], although it should be appreciated that the effects of intervention at this stage of the disease are likely to prove transient, and that not all patients will benefit. Children in particular appear to be on a fast track to insulin deficiency. The main rationale for intervention at diagnosis in type 1 diabetes is to identify potential therapies for use in secondary prevention, and one concern about this approach is that therapies that prove ineffective at this late stage could be effective if administered earlier in the disease process. A further concern is that good glyaemic control from the onset of hyperglycemia prolongs ␤-cell survival [42], and therefore forms an essential element of any intervention protocol. Interventions are typically chosen for consideration because they influence diabetes onset in the NOD mouse, but many of these are unsafe for human use, or else are only effective when given in the early stages of the disease process. In general, proposed interventions fall into two main classes: standard immunosuppressive therapy, which aims to override immune responses of the host by blockade of selected pathways, or antigen-based therapies, which aim to recruit intrinsic pathways to tolerance induction.

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Given the youth of potential participants in such studies and the fact that type 1 diabetes is compatible with many decades of healthy existence, safety becomes of paramount concern in the evaluation of new forms of treatment. The most appealing approach is therefore that of induction of tolerance [78]. This is based on the inbuilt capacity of the immune system to switch off responses to one set of antigens while leaving the remainder unchanged, meaning that unwanted antigenic responses could in theory be modified by appropriate presentation of the target antigen. Animal experiments suggest that large doses of antigen induce anergy or deletion of antigen-specific T cells, whereas low doses invoke regulatory T cells. The latter are primed against a single antigen and migrate to the tissue in which this is present. Once activated by the presence of the target antigen, however, they secrete cytokines that produce nonspecific downregulation of all proinflammatory (Th1) immune processes in the immediate vicinity, including those directed against other antigenic determinants. This characteristic, known as bystander suppression, could be particularly valuable in the treatment of autoimmune disease in which responses are directed against multiple epitopes of multiple autoantigens, some of which are still unknown. Oral tolerance is effective in mouse models of autoimmunity, in which prior exposure to an antigen abolishes or limits the harmful consequences of later exposure to the same antigen. Tolerance can also be achieved in the presence of an established autoimmune process, although with less benefit. Delayed-type hypersensitivity and other cellular responses to keyhole limpet hemocyanin can be suppressed in humans by oral feeding of the antigen, although antibody responses are enhanced [79]. A series of trials of oral tolerance in human autoimmune disease were launched in the 1990s, but they proved disappointing [80]. Promising results have been reported from two recent studies, but both should be interpreted with some caution. The first is based on use of antibodies directed against the CD3 complex, which is located on the surface of lymphocytes in stable association with the T-cell receptor, and plays a central role in antigen-specific activation of T cells. OKT3, a murine anti-CD3 antibody, has been widely used to suppress T-cell responses involved in organ rejection, but is highly antigenic and stimulates production of neutralising antibodies. A further constraint is that the invariant Fc region of this antibody binds to the Fc receptor on macrophages and lymphocytes, causing massive cytokine release. Anti-CD3 molecules have therefore been engineered by substitution of rodent sequences within the antigen-binding regions of the molecule and by use of a human Fc tail modified to prevent binding with the Fc receptor. The result is an antibody that is nonmitogenic, only weakly antigenic, and does not produce the full cytokine release syndrome. Very promising results have been reported from the NOD mouse, which—in marked contrast to all other interventions—responds preferentially at the stage of overt diabetes and enters lasting remission, provided that treatment is given within 7 days of clinical onset [73]. Preliminary trials in humans are promising, although results fall short of the dramatic results observed in the NOD mouse [81,82]. Another antigen that has undergone pilot testing in humans is the 60 kDa heat-shock protein (hsp60), which has been implicated in a variety of human autoimmune diseases. A peptide fraction known as p277 has proved effective in inducing tolerance in NOD mice associated with a shift towards the anti-inflammatory Th2 phenotype, although this effect has not been confirmed independently [83]. A pilot study of subcutaneous injection of p277 in newly diagnosed patients has been reported to delay the decline of C-peptide in treated patients as compared with controls at 10 months [84]. Further follow-up of this group and larger well-controlled trials are clearly necessary. One encouraging development

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in this field has been the agreement of groups within the United States and world-wide to work together in developing and performing future intervention trials, but there is still a long way to go.

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22 Autoimmune Central Diabetes Insipidus ANNAMARIA DE BELLIS, ANTONIO BIZZARRO, and ANTONIO BELLASTELLA Second University of Naples, Naples, Italy

I. INTRODUCTION Central diabetes insipidus (CDI), also called neurogenic, hypothalamic, or cranial diabetes insipidus, is caused by a failure of secretion and/or release of antidiuretic hormone, or vasopressin, by supraoptic and paraventricular nuclei, in which it is synthesized, or from the posterior pituitary, in which it is stored. This failure induces in affected patients a lowest gravity polyuria with polydipsia, which prevents an increase in plasma osmolality. In 1794 Frank first defined diabetes insipidus, or spurius, a diabetes without glycosuria, separating it from diabetes verus or mellitus [1]. About a century ago, Oliver and Scha¨fer [2], injecting animals intravenously with a pituitary gland extract, elicited a significant hypertensive response, which was subsequently attributed to a substance residing in the posterior side of the gland [3]. The effects at the renal level [4] of the postpituitary extract and its ability to reverse polyuria in patients with diabetes insipidus [5] were subsequently described. In most mammals the antidiuretic factor has been chemically identified as arginine vasopressin (AVP) [6], a nonapeptide closely related to other nonapeptides with the same action in the pig family [7] and in other mammalian species, encoded by a single ancestral gene [8,9] or, as more recently demonstrated, by different multiple genes [10]. II. PHYSIOLOGY OF AVP SECRETION AND ACTION AVP, also called antidiuretic hormone (ADH), is synthesized in the supraoptic and paraventricular nuclei of the hypothalamus as part of larger precursor molecules. These nuclei 439

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project to the median eminence and posterior pituitary. The signal peptide, located just before vasopressin on the preprovasopressin peptide, is responsible for proper packaging of the peptide into the secretory granules, which are transported to the posterior pituitary. The mature, active peptide is cleaved from the prepropeptide during this transport process and is stored in the posterior pituitary axon terminals prior to be released. Stores are sufficient for 5–10 days of maximum antidiuresis or one month of normal antidiuresis. AVP is released in free form (not bound to neurophysin at blood pH) and is degraded in the brain, liver, and kidney. AVP exerts antidiuretic activity by binding V2 receptors in the kidney, then stimulating cAMP production by adenylate cyclase, which leads to synthesis and insertion of aquaporin 2 (AQP2) water channels in cells of the collecting tubules, allowing water reabsorption in the hypertonic medulla. Water exits basolaterally via constitutively expressed AQP3 and AQP4, which are the water channels in the basolateral membranes of the renal medullary collecting ducts. These two aquaporins are assumed to function as an exit pathway for water and urea in the basolateral membrane during antidiuresis. AQP3 has a more prominent role in the urinary concentrating mechanism, and its expression is upregulated by dehydration, in contrast to AQP4 [11–13]. Absence of AVP leads to excretion of large volumes of diluted urine. Moreover, AVP acts as a pressor agent at supraphysiological levels (as in severe hypovolemia) by binding V1a receptors on vascular smooth muscles causing their contraction. V1b receptors instead mediate ADH action at the pituitary corticotroph level [14]. Both V1a and V1b receptors are coupled to phospholipase C and thus increase the turnover of inositol phosphates and diacylglycerol, allowing an influx of Ca2Ⳮ through the cell membrane, thus raising intracellular Ca2Ⳮ concentrations. The rat V1a AVP receptor, recently cloned in hepatocytes, has seven transmembrane domains [15]. The gene encoding both human and rat V2 receptors is located on the long arm of the X chromosome; it encodes a 370-amino-acid protein with transmembrane topography characteristic of G-protein–coupled receptors [16]. The regulation of AVP release is based on the integration of AVP secretion and thirst to maintain plasma osmolality at 280–290 mOsm/kg. Two classes of stimuli cause ADH release: osmotic and pressure stimuli. Thus, ADH release can be triggered by an increase in osmolality, which activates osmoreceptors, or by a decrease in pressure in the arterial or venous side of the circulation, which activates baroreceptors. In particular, anterior hypothalamic osmoreceptors (anterior to the 3rd ventricle) are very sensitive to changes in plasma osmolality (Posm), responding to change as low as 1% with suppression of AVP release when Posm is ⬍280 mOsm/kg. Posm values less than those necessary to turn off AVP will not result in an increase in water excretion (18–20 L/d maximum), unless intake is extreme. As Posm increases, there is a linear increase in AVP. When Posm exceeds 292–295 mOsm/ kg, plasma AVP reaches levels (⬃6 pg/mL) sufficient for maximum reduction of diuresis (urinary osmolality ⬎ 800 mOsm/kg, urinary volume ⬍ 2 L/d), which does not decrease further with further Posm increase. Thirst is stimulated at 290 mOsm/kg. Baroreceptors (stimulated by hypervolemia, inhibited by hypovolemia) inhibit AVP release via cranial nerves IX and X. The atrial cardiopulmonary low-pressure baroreceptors are less sensitive than osmoreceptors, requiring a 5–10% decrease in blood volume before inducing AVP release. Severe hypovolemia, however, triggers the sino-aortic high-pressure baroreceptors, causing an exponential increase in AVP, which may be high enough to exert a pressor effect. There is an interaction of osmo- and baroreceptors: a decrease in left atrial pressure (as in hypovolemia, hypotension) leads to a reduction of the osmotic threshold and increases the sensitivity to osmotic AVP release. Volume expansion dampens the sensitivity to osmotic AVP release. This release can be also induced by some cytokines [tumor

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Figure 1 Reciprocal interrelationships among AVP, CRH-ACTH-cortisol axis, and cytokines released from immune inflammatory site. AVP, together with CRH, induces the release of pituitary ACTH, which stimulates the corticosteroid secretion from adrenal gland. AVP can also directly induce cortisol release from the adrenal gland, which dampens the inflammatory response by inhibiting cytokine production. VCRH ⳱ corticotropin-releasing hormone; PV ⳱ Paraventricular nucleus; SO ⳱ supraoptic nucleus; AVP ⳱ arginine-vasopressin; AVPs ⳱ AVP storage in postpituitary; IL-6 ⳱ interleukin-6; IL-1␤ ⳱ interleukin-1␤; TNF-␣ ⳱ tumor necrosis factor ␣.

necrosis factor-␣ (TNF-␣), interleukin-1␤ (IL-1␤), IL-6] released from sites of inflammatory and/or immunological processes [17]. Another physiological effect of AVP is, in synergy with CRH, to stimulate pituitary ACTH production and then adrenal cortisol production [18]. This effect is mediated via V1b receptor. Moreover, AVP may directly stimulate adrenal cortisol production via V3 receptors [19]. It has been shown that cortisol reduces inflammatory and immunological processes. For this reason AVP should be considered a neuroendocrine hormone that influences both immune and inflammatory processes [20] (Fig. 1). Finally, AVP can alter behavior and learning as well as cognitive function via V3 receptors [21]. III. ETIOLOGY The overall incidence of CDI is 1 in 10,000. CDI can be the outcome of a number of diseases that affect the hypothalamic-neurohypophyseal axis. It may be complete or partial, permanent or temporary. Moreover, it is defined as primary when it is due to a marked

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decrease in ADH secretion by the hypothalamic nuclei of the neurohypophyseal system, or secondary (acquired) when the impaired AVP secretion and/or release is caused by a variety of pathological lesions, including hypophysectomy, cranial injuries (particularly basal skull fractures), suprasellar and intrasellar neoplasms (primary or metastatic), Langerhans cell–type histiocytosis (Hand-Schu¨ller-Christian disease), granulomatous disease (sarcoidosis or tuberculosis), vascular lesions (aneurysm and thrombosis), infectious diseases (encephalitis or meningitis), hypoxic insults, or autoimmune disorders (Table 1). All of the pathological lesions associated with CDI involve the supraoptic and paraventricular nuclei of the hypothalamus or a major portion of the pituitary stalk. Most commonly CDI results from damage caused by neurological surgery or head trauma involving the hypothalamus and/or the posterior lobe of the pituitary gland. Usually, the damage limited to the posterior lobe of the pituitary leads to temporary CDI; in fact, the posterior lobe is the major site of AVP storage and release, but, since AVP is synthesized within the hypothalamus, newly synthesized hormone can still be released into the circulation as long as the hypothalamic nuclei and part of the neurohypophyseal tract are intact. It is sufficient that about 10–20% of neurosecretory neurons remain intact to prevent CDI. In fact, destruction of over 80% of AVP-synthesizing hypothalamic neurons is required to determine CDI, although injury to the neurohypophysis may cause transient CDI. Genetic abnormalities are responsible for autosomal dominant forms of primary CDI occurring within the first year of life; these are caused by mutations in the gene encoding for AVPneurophysin II, the polypeptide precursor of AVP, located on chromosome 20 [10]. Heterogeneous point mutations result in selective postnatal death of AVP-producing neurons, possibly by interfering with folding of the protein, which inhibits processing and release leading to toxic accumulation in the endoplasmic reticulum [22]. Age of onset ranges from 1 to 28 years, and a posterior pituitary bright spot on magnetic resonance imaging (MRI) (usually absent in central DI) may still be visible (probable due to accumulated

Table 1 Etiology of Central Diabetes Insipidus Primary Genetic Developmental syndromes Secondary/acquired Trauma postsurgery Tumors

Granulomatous diseases Infectious diseases Vascular

Autoimmune Idiopathic

Autosomal dominant (AVP-neurophysin II gene mutations) DIDMOAD (Wolfram) syndrome Septo-optic dysplasia, Laurence-Moon and Bardet-Biedl syndromes Head injury transcranial/transphenoidal Craniopharyngioma, pinealoma, germinoma, leukemia, lymphoma metastases, pituitary macroadenoma with suprasellar extension Sarcoidosis, histiocytosis, tuberculosis Meningitis, encephalitis Aneurysm Infarction Sheehan’s syndrome Sickle cell disease When none of the above-reported causes can be evidenced

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precursor). Moreover, an autosomal recessive form with incomplete penetrance CDI is part of Wolfram or DIDMOAD syndrome, characterized by CDI, diabetes mellitus, optic atrophy, and deafness. In this case the mutation occurs in the transmembrane protein wolframin [23]. In approximately 30% of patients, CDI occurs without apparent cause and is considered idiopathic (Table 1). IV. DIAGNOSIS The diagnosis of CDI often is made clinically and confirmed by laboratory tests that investigate serum electrolytes, urine specific gravity, and simultaneous plasma and urine osmolality after water deprivation plus desmospressin tests. In particular, an urine osmolality of ⬍300 mOsm/kg and a plasma osmolality of ⬎295 mOsm/kg after dehydration and an increase in urine osmolality of ⬎750 mOsm/kg following desmopressin administration are the hallmark of complete CDI [24]. V. AUTOIMMUNE CENTRAL DIABETES INSIPIDUS An autoimmune involvement has been recognized in most endocrine diseases previously thought as being idiopathic in nature. In 1957 Witebsky et al. established criteria that are still used to define autoimmune diseases [25]. In 1993 these criteria were revised and integrated by Rose and Bona [26]. In subsequent years, on the basis of these criteria, many diseases previously considered as idiopathic were included in the group of autoimmune diseases; consequently, more than 60 diseases are now included in this group [27]. Autoimmune diseases are divided into three categories: (1) non–organ specific, (2) intermediated, and (3) organ specific, characterized by lympho-monocytic infiltration localized in the target organ and by presence of autoantibodies recognizing specific autoantigens of the same organ [27]. Many autoantibodies detected in autoimmune organ-specific diseases are directed against cytoplasmatic microsomal antigens recognizing particular enzymes as targets, whereas other antibodies directed to cytoplasmatic antigens have several target hormones [28]. Moreover, autoantibodies against cell-membrane targets recognizing specific receptors of these cells have been also evidenced. Some forms of CDI previously considered idiopathic can be included in the category of organ-specific autoimmune diseases [29,30]. In fact, in some patients with idiopathic CDI of recent onset, a chronic inflammatory process characterized by T-lymphocyte and plasma cell infiltration (lymphocytic infundibulo-neurohypophysitis) has been evidenced by biopsy of the neurohypophysis and of the pituitary stalk [31]. Moreover, lymphocytic infundibulo-neurohypophysitis can be also suggested by the presence of isolated pituitary stalk thickening (PST) on MRI, which can disappear over time after corticosteroid therapy or spontaneously [31]. This sequence supports the autoimmune hypothesis in some cases of idiopathic CDI. About 20 years ago, antibodies against hypothalamic (supraoptic and paraventricular) vasopressin cells (AVPcAb) were described in patients with idiopathic CDI [32]. AVPcAb were directed against cytoplasmatic [32] and cell surface [33] antigens, suggesting autoimmune involvement in these cases. The frequent finding of other autoimmune diseases [34–36] and the possible association of AVPcAb with other organ-specific autoantibodies [36–38] (Table 2) support this assumption. As described for other autoimmune diseases, also for idiopathic CDI the occurrence of other organ specific autoimmune diseases, as Hashimoto’s thyroiditis (HT), Addison’s

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Table 2 Organ-Specific Autoantibodies Possibly in Apparently Idiopathic Central Diabetes Insipidus Steroid-secreting cell antibodies (StCA) 17-Hydroxylase antibodies (17-OHAb) 21-Hydroxylase antibodies (21-OHAb) Adrenocortical antibodies (ACA) Antipituitary antibodies (APA) Islet cell antibodies (ICA) Glutamic acid decarboxylase antibodies (GADAb) Thyroperoxidase antibodies (TPOAb) Thyroglobulin antibodies (TgAb) TSH receptor antibodies (TRAb) Parietal cell antibodies (PCA) Tissue transglutaminase antibodies (TTGA) Endomysial antibodies (EMA)

disease, lymphocytic hypophysitis, premature ovarian failure and/or not-organ specific diseases can be observed in their first degree relatives [36]. Table 3 summarizes the criteria suggested to single out autoimmune factors in apparently idiopathic CDI. With respect to sex and age of onset, some organ-specific autoimmune diseases (HT, Graves’ disease) affect adult women in particular; other ones, such as pernicious anemia, older women. Addison’s disease can show a double incidence peak: one in pediatric age and another, more frequently, in adult age. Similarly, in CDI the incidence of lymphocytic infundibulo-neurohypophisitis [31,39], the association with other autoimmune endocrine diseases, the presence of AVPcAb and of other organ-specific autoantibodies is less marked in children than in adults [40]. The occurrence of autoimmune CDI in pediatric patients is independent from sex, while that in adult patients is prevalently correlated to female sex. VI. VASOPRESSIN-CELL ANTIBODIES A. Detection and Immunological Characteristics of AVP Cell Antibodies AVPcAb are usually detected by immunofluorescence (IFL) methods using unfixed frozen sections of supraoptic and paraventricular hypothalamus [32]. The choice of substrate

Table 3 Criteria That Indicate Autoimmune Involvement in Apparently Idiopathic Central Diabetes Insipidus Presence of AVPcAb Presence of lymphocytic infundibulo-neurohypophysitis Presence of reversible pituitary stalk thickening on MRI Association with other autoimmune diseases Presence of other organ-specific antibodies Occurrence in first-degree relatives of other autoimmune diseases and/or of some organ-specific autoantibodies AVPcAb ⫽ Antibodies to arginine-vasopressin–secreting cells; MRI ⫽ magnetic resonance imaging.

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seems to have an important role in identifying these antibodies. Ideal tissues, according to Scherbaum et al., are constituted by human fresh fetal hypothalamus immediately frozen in order to avoid cytoplasma antigen alterations; however, fresh baboon hypothalamus can also be used [41]. Human hypothalamic tissues from adult subjects aged ⬎50 years or old baboon tissues are not considered suitable because the presence of lipofuscin pigments in secreting cells produce an autofluorescence that could interfere with the evaluation of antibodies. At the present time, only young baboon hypothalamus sections are used for evaluating AVPcAb due to the difficulty of finding human tissue and the ethical and legal impediments to using human fetal hypothalamus [42]. When patient sera are positive for antibodies against hypothalamic cells, precise characterization of these cells (AVPcAb and not) can be determined by a four-layer double fluorochrome IFL test in which antivasopressin rabbit serum and rhodaminated anti-rabbit IgG serum are applied in the second sandwich [43]. If these cells are AVP-secreting cells, rabbit anti-AVP serum stains the same cells in the serum of patients. AVPcAb can consist mainly of class IgG and IgA and less frequently of class IgM; moreover, about the half are complement fixing [32]. In our patients with preclinical and clinical autoimmune CDI they have been shown to be mostly of class IgG and, more rarely, IgA [37,38]. In analogy to other organ-specific antibodies, i.e., islet cell, adrenocortical and steroid-secreting cell antibodies (ICA, ACA, StCA), which react with well-known cytoplasmatic antigens distinct from the hormone [44–46], AVPcAb also react with cytoplasmatic antigens (in this case not known) distinct from the vasopressin hormone. In CDI patients receiving vasopressin or its analogues it must to be ascertained that AVPcAb are not corresponding to vasopressin antibodies. This can be done verifying that by antibody reactivity is not diminished after preabsorption of positive sera with an excess of vasopressin. B. Role of AVPcAb in the Pathogenesis of Autoimmune CDI Autoimmune mechanisms characterizing other organ-specific autoimmune diseases, such as autoimmune thyroid disease and type 1 diabetes mellitus, are well known because of the ability to study animal models and of the availability of target organ tissues from patients at the onset or before the clinical onset of the disease. At present, the mechanisms triggering the development and the progression of autoimmune CDI are not known. In fact, the absence of animal models of autoimmune hypothalamitis hampers the study of the events initiating autoimmune CDI. Other major obstacles in understanding autoimmune mechanisms are include difficulties in obtaining hypothalamic tissue specimens with infiltrating lymphocytic-macrophagic cells from patients with subclinical or clinical autoimmune CDI at onset. However, hystological studies of biopsy specimens of the pituitary stalk and of neurohypophysis from some idiopathic CDI patients of recent onset [31] revealed inflammatory infiltrates with characteristics similar to those observed in lymphocytic infiltrates of patients with autoimmune chronic thyroiditis or Graves’ disease or autoimmune diabetes mellitus at active and initial phases. In fact, these infiltrates of the pituitary stalk and of neurohypophysis are characterized above all by T lymphocytes and plasma cells (polyclonal in terms of immunoglobulin light-chain types). Moreover, it is possible that in autoimmune CDI the damage resulting from infiltrating T cells could be the most probable perpetuating cause of hypothalamic impairment. In particular, the activity of T-helper cells may lead to the production of IL-2 and other lymphokines, which induce activation of both T-cytotoxic lymphocytes and B lymphocytes able to secrete specific AVPcAb. As a consequence of lymphocytic infiltration, AVPcAb become detectable in

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the serum and could be considered markers of T-cell–mediated aggression to the hypothalamic–pituitary stalk–neurohypophysis tract, initially silent (preclinical stage and at onset of disease) and subsequently markedly evident in late-stage disease (see Sec. III). In fact, a significant correlation between PST (possible finding of lymphocytic-infundibulo neurohypophysitis) and AVPcAb titer can be found in patients in late stages of autoimmune CDI [38]. C. Prevalence and Clinical Significance of AVPcAb The presence of AVPcAb in patients with clinical CDI allows an etiological diagnosis of autoimmune CDI. However, AVPcAb have also been detected in sera of patients with CDI secondary to histiocytosis X. In fact, Scherbaum et al. showed that more than 50% of sera from patients with CDI due to histiocytosis X contain AVPcAb [36]. They explained this result, suggesting that it was caused by the invasion of the hypothalamus by histiocytosis X cells overcoming the blood-brain barrier following a local inflammatory reaction [47,48]. In fact, class II HLA molecules are expressed on membrane of histiocytosis X cells [49]; these cells are able to present antigens to T lymphocytes with subsequent activation of T-helper lymphocytes, which may trigger an autoimmune response (activation of both T-cytotoxic lymphocytes and B lymphocytes) against the surrounding cells [50]. For this reason AVPcAb became detectable in serum. In sera of patients with other secondary forms of CDI, AVPcAb have been found only rarely [40]. In an AVPcAb screening of a large cohort of patients with different secondary forms of complete CDI, we showed that AVPcAb were positive not only in 66.6% of patients with histiocytosis X but also in 19.2% of those who had had surgery for pituitary adenomas; they were negative in all the remaining cases [51] (Table 4). In postsurgery positive patients AVPcAb were present at low titers in the first phase of the observation and disappeared subsequently during follow-up. In these cases their presence could be considered an epiphenomenon, probably due to a transitory and reversible hypothalamic inflammatory process mediated by lymphocyte migration from the barrier to the hypothalamus favored by increased endothelial adhesion to cerebral circulation [52,54]. This adhesion could be due to a stimulation of endothelial cells by interferon (IFN)-␥, TNF, and IL-1 [55,56]. AVPcAb can be also observed in subjects without clinical CDI. This was described in histiocytosis X patients before the onset of clinical CDI [43], suggestive of a subclinical

Table 4 Frequency of AVPcAb in 85 Secondary and Familial Central Diabetes Insipidus Cases Secondary to:

No. of patients tested

No. of AVPcAb-positive patients

9 3 5 3 4 4 52

6 (66.6%) 0 0 0 0 0 8 (19.2%)

Histiocytosis X Sarcoidosis Metastatic tumors Craniophayngioma Germinoma Trauma Postsurgical pituitary adenomas AVPcAb ⫽ Antibodies to AVP-secreting cells. Source: Ref. 51.

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stage of the disease [57]. These results could be important in therapeutically preventing further progression towards overt clinical CDI in such patients [58]. In our experience, AVPcAb can sometimes also be detected in first-degree relatives of patients with autoimmune complete CDI. These subjects are usually positive for AVPcAb at low titers and show a normal postpituitary function (unpublished data). AVPcAb can be also found in patients with other autoimmune diseases without clinical CDI [42]. Some organ-specific autoimmune diseases, such as HT, Graves’ disease, type 1 diabetes mellitus, and atrophic gastritis, are more frequent than others, such as autoimmune Addison’s disease and autoimmune CDI. However, the real incidence of these latter autoimmune diseases is certainly underestimated because they are recognized only when clinically apparent, whereas they are usually misdiagnosed, the in potential or subclinical phases [59]. The potential phase is characterized by the presence of organ-specific autoantibodies with normal function of related target organs; the subclinical phase is characterized by the presence of organ-specific autoantibodies but with subclinical impairment of the target organ function [60]. In this connection, organ-specific autoimmune diseases can be considered an iceberg in which the emerging (minimal) part is constituted by the clinical forms and the immersed portion by the potential and subclinical forms [61–64]. Among patients with autoimmune organ-specific diseases positive for AVPcAb but without clinical CDI, some have a normal postpituitary function (potential autoimmune CDI), and others have a partial impairment of postpituitary function (sub-clinical autoimmune CDI) [42]. During a revision of our collected cases including 3300 patients with organ-specific autoimmune diseases without CDI, we found AVPcAb present in 58 cases (1.7%), 16 (27.6%) of whom presented with functional alterations of postpituitary gland (subclinical forms). Most of the 58 patients suffered from autoimmune thyroid diseases, some from type I diabetes mellitus and Addison’s disease and from other organ-specific endocrine (autoimmune hypophysitis, premature ovarian failure) or nonendocrine (atrophic gastritis, vitiligo, alopecia, celiac disease) autoimmune diseases; one case suffered from a non–organ-specific disease—systemic lupus erythematosus (SLE) [42] (Table 5).

Table 5 Autoimmune Diseases in 58 AVPcAb-Positive Patients Without Clinical Central Diabetes Insipidus Autoimmune diseases

No. of AVPcAb-positive patients

%

18 9 5 10 4 4 7 6 2 1 1

31.03 15.51 8.62 17.24 6.89 6.89 12.06 10.34 3.44 1.72 1.72

Hashimoto’s thyroiditis Graves’ diseases Addison’s disease Type 1 diabetes mellitus Premature ovarian failure Autoimmune hypophysitis Atrophic gastritis Vitiligo Celiac disease Systemic lupus erythematosus Alopecia areata AVPcAb ⫽ Antibodies to arginine-vasopressin secreting cells

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Table 6 Autoimmune Polyglandular Syndromes AVPcAb-Positive in 58 Patients Without Clinical Central Diabetes Insipidus APS Type 2 Incomplete Complete Type 3 Incomplete Complete Type 4 Incomplete Complete

No. of AVPcAb-positive patients

%

14 12 2 31 25 6 13 13 0

24.13 85.71 14.28 53.44 84.64 19.35 22.41 100 0

AVPcAb ⫽ Antibodies to AVP-secreting cells. Source: Refs. 42 and 77.

Autoimmune diseases are characterized by frequent association in the same patient; the coexistence of two or more organ-specific and/or non–organ-specific autoimmune diseases indicates a complete autoimmune polyendocrine syndrome (APS) [65,66]. More frequently in patients with an isolated autoimmune disease, a complete autoantibody screening can evidence other antibodies apart from those of the respective specific disease [67]. On the basis of their characteristics, all of our 58 above-mentioned AVPcAb-positive patients were considered as having a complete or incomplete APS. Taking into account the association with other autoimmune diseases, 14 of 58 patients (24%) fell within the type 2 APS; of these only 2 (14%) presented with a complete form characterized by clinical Addison’s disease, clinical autoimmune thyroid disease, and/or type 1 diabetes mellitus, whereas the remaining 12 (85.7%) presented with an incomplete form characterized by autoimmune thyroid disease and/or type 1 diabetes mellitus with presence of ACA and 21-OHAb but no clinical Addison’ disease. Instead, 31 of 58 patients (53%) fell within type 3 APS: only 6 (19%) had a complete form characterized by autoimmune thyroid disease and other autoimmune diseases (premature ovarian failure, type I diabetes mellitus, autoimmune hypophysitis, atrophic gastritis, vitiligo) in the absence of preclinical and clinical Addison’s disease; the remaining 25 of 31 (84.6%) presented with an isolated autoimmune thyroid disease with other organ-specific autoantibodies in the absence of the respective clinical diseases and therefore had an incomplete form. Following the criteria identifying type 3 APS, we suggest that patients positive for AVPcAb without clinical CDI but with isolated autoimmune thyroid diseases be considered as falling within this incomplete type of APS. Finally, 13 patients positive for AVPcAb without CDI, presenting with isolated autoimmune diseases but without thyroid diseases, could be classified as incomplete type 4 APS (Table 6). VII. ASSOCIATION WITH OTHER AUTOIMMUNE ENDOCRINE DISEASES Before the demonstration of AVPcAb [32,68,69], the coexistence of an established autoimmune disease with CDI had been described by Smith in 1926 [70] and, subsequently,

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confirmed by other authors [35,71,72]. Scherbaum et al. observed an idiopathic CDI in 13 patients presenting with one or more associated autoimmune diseases [36], which would now be included in APS. In particular, one case with mucocutaneous candidosis, hypoparathyroidism, Addison’s disease, total alopecia, and pernicious anemia would fall within a complete type 1 APS; most cases showed characteristics of complete type 3 APS (autoimmune thyroid diseases with or without other autoimmune disease but no Addison’s disease), and some cases showed findings of a type 2 APS (HT with or without type 2 diabetes mellitus, now classified as LADA in the presence of Addison’s disease). Another case presenting with total alopecia and ocular miastenia could be included in type 4 APS. From 1993 to 2002 we carried out an AVPcAb screening both in a large cohort of adult patients with APS and clinical CDI [38] and in another large cohort of patients with only clinical CDI of known and unknown etiology [51]. Considering both cohorts of patients, we found that 56 of 126 CDI patients (44.4%) qualified as autoimmune (Fig. 2). Some of them presented with an isolated form of CDI without other potential, subclinical, or clinical autoimmune diseases. The other patients, in analogy to criteria used for those with potential and subclinical CDI, have been included in type 2, type 3, and type 4 APS according to the respective associations with other autoimmune diseases. In particular, as regards to type 2 APS, we suggest including autoimmune CDI among the minor autoimmune diseases associated with the major components of this syndrome. As regards to type 3 APS, taking into account the new classification criteria recently proposed by Betterle et al., including different and multiple clinical autoimmune conditions [67], we suggest including autoimmune CDI among the possible autoimmune endocrine diseases usually associated with autoimmune thyroid disease in this APS (Fig. 3). Instead, when autoimmune CDI does not fall within the above-mentioned autoimmune combinations, it can be included in type 4 APS.

Figure 2 Prevalence of autoimmune and nonautoimmune forms in 126 patients with clinical central diabetes insipidus. (From Refs. 38 and 51.)

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Figure 3 Autoimmune central diabetes insipidus in the context of autoimmune endocrine diseases characterizing type 3A APS.

VIII. NATURAL HISTORY OF AUTOIMMUNE CDI It is thought that the clinical state of autoimmune endocrine diseases is usually preceded by a long preclinical stage characterized by the presence of the organ-specific antibodies without (potential stage) or with (subclinical stage) findings of altered function of the respective target gland [73,74]. Thus, a periodic screening of the autoantibodies in patients at risk of developing autoimmune diseases can be useful because organ-specific autoantibodies are considered good preventive markers of a future clinical phase of respective diseases [75,76]. In the course of a longitudinal screening of AVPcAb in patients with

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APS without clinical CDI it was demonstrated that, as in other autoimmune endocrine diseases, the overt clinical phase of autoimmune CDI is preceded by a long pre-clinical period [77], characterized by the presence of AVPcAb with or without findings of posterior pituitary function impairment. This suggests that the presence of these antibodies could indicate a high risk for developing overt CDI. In particular, AVPcAb persist over time in potential and subclinical autoimmune CDI and are associated in most cases with a progressive worsening of posterior pituitary function. This seems to differentiate CDI from some other autoimmune endocrine diseases, such as the Addison’s disease, in which a possible spontaneous disappearance of ACA and 21-OHAb (especially when present at low titers and levels, respectively) and a remission of subclinical adrenocortical failure at early preclinical functional stages can occur [45,78]. The natural history of autoimmune CDI in patients with APS involves three functional stages during which AVPcAb are always present (Fig. 4): Stage 1 is characterized by the presence of AVPcAb but with normal posterior pituitary function, as demonstrated by normal response to prolonged water deprivation/desmopressin tests [24,79] and normal MRI of the hypothalamic-pituitary region. MRI is able to identify normal posterior pituitary lobe evidencing the characteristic hyperhyntense signal on T1-weighted image [80], which appears to be a hallmark of the functional integrity of the hypothalamic neurohypophyseal tract [81]. The clear correlation between MRI findings and postpituitary function

Figure 4 Natural history of autoimmune central diabetes insipidus evolving through three functional stages. AVPcAb antibodies to arginine-vasopressin secreting cells; MRI ⳱ magnetic resonance imaging; PST ⳱ pituitary stalk thickening.

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integrity suggests that the neurosecretory granules still contain a sufficient quantity of AVP [82]. In fact, MRI is considered an important tool to study CDI patients because it is able not only to clarify the diagnosis of secondary clinical CDI but also to perform a direct evaluation of the neurohypophyseal functional state [83–86]. In particular, a reduction in or absence of posterior pituitary hyperhintense signal is usually observed in the course of CDI. Stage 2 is characterized by the presence of AVPcAb and posterior pituitary functional findings suggestive of partial CDI [77,79,87,88], subnormal response of plasma AVP after prolonged deprivation test, and further increase in urinary osmolality (9–50%) after DDAVP injection with respect to values observed after water deprivation test. Moreover, in this stage the normal posterior pituitary hyperhintense signal on MRI is also observed. Subclinical organ-specific autoimmune diseases are usually characterized not only by the presence of the respective organ-specific autoantibodies and subclinical functional impairment of the target organ but also by the presence of diffuse or multifocal lymphocytic infiltrations in the target organ, sometimes revealed by fine needle aspiration biopsy or suggested by tomographic scans [89,90]. In contrast, the presence of lymphocytic infiltrations along the hypothalamic-pituitary neurohypophyseal tract cannot be seen in potential or subclinical phases of CDI. In fact, MRI studies carried out serially during a follow-up of patients with potential and subclinical autoimmune CDI showed not only the persistence of the posterior pituitary hyperhintense signal but also the absence of pituitary stalk tickening (PST) on MRI during these preclinical stages of the disease. Stage 3 is characterized by an increase of AVPcAb titers with respect to the values observed in previous stages and by the development of clinical CDI diagnosed by water-deprivation DDAVP tests according to the accepted criteria [24] and by low plasma AVP levels, if still detectable [77]. The pituitary stalk hyperhintense MRI signal can persist in an early phase of stage 3 (stage 3a); the presence of the posterior pituitary hyperhintense signal at the onset of the clinical CDI can be ascribed to the persistence of a residual amount of AVP granules [77,91]. Moreover, PST on MRI in early stages of CDI is also absent. In the late phase of stage 3 (stage 3b), plasma AVP is not detectable and the hyperhintense signal disappears. In stage 3b AVPcAb are present always at high titers, and PST on MRI can sometimes appear [38], suggesting the presence of lymphocytic neurohypophysitis. However, the persistence of the hyperintense signal, not only in preclinical but also in clinically overt states of CDI, suggests that MRI of posterior pituitary cannot be considered an useful tool for the prediction of the progression towards complete CDI in AVPcAb-positive patients. Some organ-specific autoantibodies such as ICA are present in an early phase of the disease but subsequently can disappear [92,93]. AVPcAb, such as ACA, 21-OHAb, and glutamic decarboxylase antibodies (GAD-65Ab) in Addison’s disease and in type 1 diabetes mellitus [94–96], persist over time during the clinical course of the disease in patients with autoimmune CDI with or without other autoimmune diseases [38]. In particular, in patients with APS AVPcAb are present in 94% of cases when tested at the onset of clinical CDI, in 80% when tested from the first to the sixth year after the onset of CDI, and in 50% when tested in a late phase of the disease. In fact, these antibodies can persist more than 20 years after the onset of the disease even if at lower titers [37,38]. In patients with

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an isolated autoimmune CDI, AVPcAb can persist over time. The persistence of AVPcAb in the absence of functional activity of hypothalamic cells could depend on the presence of residual cells, unable to function but still able to present antigens to the immune system. By longitudinal MRI evaluation it has been shown that PST may be present both in CDI patients with APS and in those with isolated CDI, but only in early phases of the disease and persisting for a short period. In fact, PST spontaneously disappear during follow-up, suggesting a self-limiting process [31,39,97]. Finally, in autoimmune CDI a reversal of PST is observed only in AVPcAb-positive patients; for this reason, in patients with idiopathic CDI the presence of PST without AVPcAb does not justify a diagnosis of autoimmune disease [38]. In conclusion, AVPcAb evaluation is of fundamental importance for the diagnosis of autoimmune CDI from the potential phase to the latest phases of the disease, whereas the presence of PST on MRI for a short period (from the first to fourth year after the onset of the disease) only supports an autoimmune diagnosis when accompanied by the presence of AVPcAb. After the diagnosis of clinical isolated autoimmune CDI, periodic screening for other organ-specific or non–organ-specific autoantibodies should be performed. This strategy, pursued in patients with isolated autoimmune CDI, could identify over time one or more serological markers of other autoimmune diseases (more frequently autoimmune thyroid diseases) [38]. In such patients, specific functional tests might show subclinical impairment of the respective gland and could prevent development of clinical APS. IX. THERAPEUTIC STRATEGY Replacement therapy is required in overt CDI; in particular, a number of medications can be given to decrease the quantity of fluid passed out into the urine. These include L-arginine vasopressin (Pitressin, aqueous vasopressin) (injected) and Desmopressin (DDAVP, 1desamino-8-D-arginine-vasopressin). Most widely used, intranasal DDAVP provides antidiuretic activity for 6–24 (usually 8–12) hours 2000 times more specific for antidiuresis than natural AVP, with onset of action 1–2 hours after administration. Doses range from 5 to 20 ␮g. A parenteral form, more potent than the intranasal one (sc, im, or iv), is available for those unable to take it intranasally (e.g., children, postoperative patients) or for use in acute DI (1–4 ␮g iv, dose frequency and magnitude based on desired hourly urine flow). An oral form (0.1–0.8 mg/d) is also available, with the benefit of not needing refrigeration as does the intranasal form. Although several predictive studies have been performed in subclinical autimmune endocrine diseases, only a few therapeutic trials to prevent overt disease have been carried out [59]. A strategy of immuno-modulatory therapy is ‘‘isohormonal therapy,’’ the results of which are still unclear. Such therapy utilizes hormonal products of the target organ to influence autoimmunity in the preclinical stage when the target gland is not still completely and irreversibly destroyed, and it may act by feed-back inhibition of glandular function or by determining suppression of autoimmunity, or by a combination of both mechanisms [98]. The U.S. Diabetes Prevention Trial-1 (DPT-1) was based on promising pilot data for the prevention of progression towards diabetes mellitus in at-risk individuals treated with insulin. However, DPT-1 was abandoned in 2001 because there were no differences in the rates of developing diabetes between the placebo and insulin-treated groups [99–101]. Such isohormonal therapy may be applicable to other endocrine disorders, such as Addison’s disease. In preclinical autoimmune Addison’s disease, a short course of

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glucocorticoids appeared to suppress the expression of adrenal autoantibodies and prevent progressive adrenal destruction [37,102]. The effect of short-term glucocorticoid therapy can be attributed above all to the well-known immunosuppressive effect of steroids [103,104]; this therapy may also act (with minor probability) as an isohormonal therapy in determining the disappearance of ACA and in preventing progressive adrenal destruction with restoration to the normal state of adrenal function in subclinical autoimmune Addison’s disease. We carried out preventive desmopressin therapy in patients with autoimmune partial CDI in order to interrupt or delay the autoimmune damage and the progression towards clinically overt CDI [77]. In fact, DDAVP therapy suppressed the expression of AVPcAb and induced the remission of subclinical posterior pituitary function impairment together with disappearance of AVPcAb. In untreated patients with autoimmune partial CDI, AVPcAb were present over time with increasing titers and final progression of partial CDI towards the clinical stage of CDI. Early desmopressin therapy in subclinical autoimmune CDI delayed hypothalamic cell damage; the effect may have been a result of isohormonal therapy or less probably of the well-known AVP stimuli on pituitary ACTH secretion with a consequent adrenal steroid increase and secondary immunosuppressive action [20,21,105]. This could theoretically lead to a reduction in lymphocytic infiltrate in the hypothalamus, with subsequent AVPcAb disappearance and neurohypophyseal function restoration.

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23 Autoimmune Thyroid Disease MARIAN ELIZABETH LUDGATE University of Wales College of Medicine, Cardiff, Wales, United Kingdom GHERARDO MAZZIOTTI Second University of Naples, Naples, Italy

I. INTRODUCTION The thyroid is the target of the most common autoimmune diseases [1], which produce the full spectrum of thyroid dysfunction from hyperthyroid Graves’ disease (GD) to hypothyroid Hashimoto’s thyroiditis (HT). In common with other autoimmune conditions, autoimmune thyroid diseases (AITD) are more prevalent in women, especially in the 5th and 6th decades of life. Furthermore, AITD may arise in individuals or families suffering with other organ-specific autoimmune disorders, particularly pernicious anemia and type 1 diabetes. Autoimmune thyroid diseases appear to be primarily a disorder of immunoregulation, with organ dysfunction resulting from an antigen-specific attack from inadequately suppressed lymphocytes [1]. The main mechanisms operating to discriminate between self and nonself are the induction of central and peripheral tolerance and anergy [2]. Defects in all three of these have been implicated in AITD, with much of the information being derived from animal models of AITD. Mature CD4Ⳮ thymocytes contain a high frequency of cells with the potential to differentiate into regulatory T cells in the periphery [3] in sufficient numbers to prevent disease. The importance of this mechanism in preventing autoimmune reactions is demonstrated by the prevention of the radiation- and thymectomyinduced thyroiditis model by reconstituting animals with peripheral CD4Ⳮ cells from syngeneic donors [4]. These cells have a ‘‘memory’’ phenotype (CD45RCⳮ), suggesting that recognition of specific antigen in the periphery is an essential development step [4]. Peripheral regulatory T cells that prevent thyroiditis are not found in rats whose thyroids were ablated by radioiodine treatment in utero, although the thymus maintains the capacity to generate them [5]. These experiments suggest an involvement of antigen-specific immunosuppression in the pathogenesis of AITD [6]. 461

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In addition to GD and HT, the AITD also include idiopathic myxedema (IM), Riedel’s ‘‘silent’’ thyroiditis, and postpartum thyroiditis [7]. The phenotype of the immune infiltrate and thus the clinical outcome varies from one condition to another, but there are a number of common features, including the autoantigens implicated. Cell-mediated and humoral responses are directed principally against three thyroidspecific proteins which are central to the function of the thyroid [8], i.e., the production of thyroxine (T4) and triiodothyronine [T3]. These are the thyrotropin receptor (TSHR), thyroid peroxidase (TPO), and thyroglobulin (TG). TSHR is a G-protein–coupled receptor [9] that is activated by the pituitary glycoprotein hormone, thyrotropin (TSH). The growth and function of the thyroid gland are controlled by TSH [10], mainly via the intracellular second messenger cAMP. The TSHR is comprised of two subunits, a 398-residue extracellular domain (ECD) of ⬃55 kDa, which provides the high-affinity TSH-binding domain [11]. The ECD is attached by disulfide bonds to the 346-residue membrane-spanning region (MSR) of ⬃40 kDa, which has the characteristic serpentine portion responsible for signal transduction. TSHR shares significant sequence homology with the receptors for luteinizing and follicle-stimulating hormones [12]. The TSHR gene is more than 60 kb, maps to chromosome 14q31 [13], and has major transcripts of 3.9 and 4.3 kb in addition to a number of alternatively spliced transcripts with the capacity to produce truncated TSHR [14]. Full-length transcripts comprise 10 exons, with 1–9 encoding the ECD, while exon 10 encodes the entire transmembrane domain. The mature 660 kDa TG protein is a glycosylated homodimer of two identical 300 kDa subunits that functions as the substrate and storage protein for thyroid hormones in the follicular lumen [15]. The 2748-amino-acid protein contains 4 hormonogenic tyrosine residues located at the amino and carboxyl termini, the latter region of which shares significant sequence similarity with acetylcholinesterase [16]. The TG gene is more than 300 kb, maps to chromosome 8q24.2–q24.3, contains 48 exons ranging in size from 63 to 1101 nucleotides, and yields a mRNA transcript of ⬃8.3 kb [17,18]. A possible relationship between TPO and the thyroid microsomal antigen was suggested on the basis of experiments using monoclonal antibodies [19]. The separate cloning of cDNAs for TPO and the microsomal antigen and subsequent sequence analysis confirmed that they were identical [20]. TPO is a glycosylated hemoprotein that catalyzes the iodination and coupling of tyrosyl residues in TG to produce T3/T4 [21]. It shares significant sequence similarity with myeloperoxidase. The membrane-bound enzyme of 110 kDa is located mainly at the apex of the thyroid follicle, with its catalytic site facing into the lumen. The TPO gene is 150 kb long, comprises 17 exons, and is located on chromosome 2p25 [22]. TPO has two major transcripts of approximately 3 kb encoding a protein of 933 amino acids and a shorter inactive form of 876 residues. Both forms are expressed in normal and GD thyroids [23]. More recently a fourth thyroid protein, the sodium iodide symporter (NIS), has been suggested to be an autoantigen, although this is highly controversial. The first study reporting NIS as a possible autoantigen found NIS antibodies in 1 patient of 148 tested [24]. Subsequent studies have reported a much higher incidence of NIS antibodies, particularly in Japan [25], although a low frequency in Europe has been confirmed [26]. The NIS protein has 13 putative transmembrane domains [27], is located on the basolateral surface of the thyroid, and is responsible for transporting iodide into the cell using the NaⳭ gradient generated by an associated NaⳭ/KⳭ ATPase. The NIS gene is located on chromosome 19p13.2–p12 and has 15 exons [28]. It has an open reading frame of 1929

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nucleotides encoding a glycoprotein of 643 amino acids with a predicted molecular mass of 68.7 kDa [29]. Apart from the proteins described above, several other candidate autoantigens in AITD have been identified, usually by screening expression libraries using sera from patients, including ATRA1 (whose identity remains unknown) and D1 (a tropomodulin). II. PATHOGENIC MECHANISMS This section will describe the predominating mechanisms operating across the spectrum of AITD, which result in disease. Patients with GD are hyperthyroid, i.e., they have elevated circulating T4/T3 and suppressed TSH. GD is caused by autoantibodies to TSHR that mimic the action of TSH [30]. These TSH agonists are termed thyroid-stimulating antibodies (TSAB), and since TSH controls thyroid proliferation and hormone production, TSAB produce a diffuse goiter and hyperthyroidism. Other TSHR autoantibodies may bind the receptor but not activate adenylate cyclase. These are the thyroid-blocking antibodies responsible for some cases of IM, a condition characterized by a normal or atrophic thyroid and hypothyroidism, i.e., elevated TSH and reduced T4/T3 [31]. Both TSAB and TBAB also function as TSHbinding inhibiting immunoglobulins (TBII), and this has formed the basis of diagnostic assays for GD and IM [32], although bioassays that distinguish TSAB from TBAB are preferable [33,34]. Autoantibodies of IgG, IgA, and IgE isotype, which simply bind TSHR but do not affect its function, and which may be present in normal controls, have also been described [35]. TSAB are predominantly of the IgG1 isotype, and there is some evidence for ␭ light chain restriction [36]. These two features have suggested that TSAB are oligoclonal; they are certainly present at low titer, although this has recently been shown to be in the microgram rather than the nanogram range, as previously reported [37,38]. Antibodies to thyroid autoantigens, including TSAB, are produced both within the gland itself and in lymphoid tissue [39], as exemplified by the persistence of TSAB in the circulation of patients following total ablation of the thyroid [40]. Some immunoglobulin isotypes, including IgG1, are able to cross the placenta. The offspring of mothers with GD can be born with hyperthyroidism, while mothers with IM give birth to hypothyroid infants [41,42]. In each case the condition resolves within a few weeks of birth, consistent with the half-life of immunoglobulins in the circulation. This ‘‘experiment of nature’’ has been observed even in mothers who have undergone total thyroidectomy [43], further proof that TSAB are produced outside the thyroid gland. TSAB and TBAB activity can also be demonstrated in breast milk [44,45], but whether they survive the digestive process intact and contribute to thyroid dysfunction in the child is not clear. Very different mechanisms operate in HT, in which the disruption of thyroid follicular structure with progressive destruction of the gland leads to hypothyroidism [46,47]. CD8Ⳮ T lymphocytes play an important role in mediating the cytotoxic processes [48,49] that culminate in the destruction of the cells by either necrosis or apoptosis [47,50]. In the necrotic process perforin plays a central role [51], as demonstrated by the correlation between perforin expression in infiltrating lymphocytes and T-cell–mediated cytotoxicity [48,52]. Increasing evidence suggests that apoptosis, or programmed cell death, has a role in HT [50] and could be mediated either by the release of granzymes from the cytotoxic lymphocytes into the target cells or the activation of the Fas-dependent pathway [52,53].

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Normal thyroids show a low level of apoptosis [54], but thyroid cells from HT patients display an increased frequency of apoptotic cells [55–58], often in close proximity to infiltrating lymphocytes. The Fas-FasL system is the most studied apoptotic pathway [59], and the general consensus is that thyrocytes can express the death receptor Fas, but little is known about how this expression is regulated. Giordano et al. demonstrated that Fas was not expressed on thyrocytes unless interleukin-1␤ (IL-1␤) was present [60], while others reported the constitutive expression of this protein on thyroid cells [61]. It has been proposed that Fas-mediated apoptosis is normally blocked in thyroid cells, but inflammatory cytokines IFN-␥ and TNF-␣ can activate this pathway by modifying the glycosylation of Fas [62]. By the same token, some reports suggest that thyrocyte expression of antiapoptotic proteins renders them resistant to Fas-mediated cell death [63]. The cognate of Fas, FasL [61], is expressed on activated T lymphocytes. Based on the classic view of thyroid autoimmunity, Fas-mediated thyroid destruction may be induced by cytotoxic T-lymphocyte–expressed FasL (in particular CD4Ⳮ cells), which interacts with Fas-bearing thyrocyte targets [61]. An alternative hypothesis is that the increased apoptosis observed in patients with HT is the consequence of simultaneous expression of Fas and FasL by thyrocytes, resulting in fratricide [50,60]. The data demonstrating FasL on thyroid cells are controversial, perhaps because the expression is transient and regulated by an unknown mechanism [61]. If thyrocyte FasL expression is confirmed, it would suggest that the thyroid is a site of immune privilege and could drive the apoptosis of infiltrating T lymphocytes expressing Fas [64,65]. In experimental models of AITD it has been demonstrated that the expression of FasL on thyroid cells strongly inhibited lymphocytic infiltration, with simultaneous reduction of the antithyroglobulin proliferative and cytotoxic T-cell responses, as well as autoantibody production [66]. The effects were dependent on the level of FasL expression, since only high-level expression was protective, while low-level expression exacerbated the disease by attracting inflammatory cells. In the same model, the Fas/FasL pathway may contribute to resolution of thyroiditis by the activity of CD8Ⳮ lymphocytes [67], perhaps by upregulating FasL on thyrocytes, which in turn kill FasⳭ inflammatory CD4Ⳮ cells and thus limit thyrocyte destruction. Apart from the cell-mediated mechanisms of thyroid destruction, there is some evidence that humoral factors may also be involved. Despite the central importance of Tg in induced animal models of HT [68], a direct role for TgAb in disease pathogenesis is debatable since they do not fix complement but can mediate antibody-dependent cellular cytotoxicity. Furthermore, the autoantibodies can form immune complexes with circulating Tg and activate the complement cascade, although this occurs only with high antibody titer and following release of Tg by destruction of the gland [69]. TPOAb are more common in HT than TgAb and are more closely correlated with thyroid dysfunction and abnormal histology [70]. In contrast to TgAb, TPOAb may produce complement-mediated cytotoxicity by binding to TPO expressed on the thyroid cell surface [1,71]. In HT, TPOAb are polycolonal, being found in all four IgG subclasses with higher percentages of IgG1 and IgG4 than IgG2 and IgG3 [72–75]. IgG3 has a greater capacity to fix complement than the other IgG subclasses, while the highest functional affinity is shown by IgG2 [1]. In addition to complement-mediated cytotoxicity, TPO of the IgG1 subclass [76] may be implicated in ADCC, although this is open to debate [1]. Given the location of TPO on the thyroid apex, it is poorly accessible, and thus autoimmune mechanisms dependent on TPOAB may occur only in advanced stages of disease when thyroid destruction is well progressed [71,76–78]. This constraint applies not only to TPOAB involved in cytotoxic-

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ity, but also those suggested to inhibit the enzyme activity of TPO [79] despite the catalytic region being outside the immunodominant region of molecule [75]. III. AUTOIMMUNE RESPONSE: ROLE OF CYTOKINES AND TH1 VERSUS TH2 BALANCE IN AITD As in other models of T-cell–mediated autoimmune diseases, cytokines play a pivotal role in the pathogenesis of AITD. Infiltrating lymphocytes are the main source, although thyrocytes may also contribute to the inflammatory process through the production of various cytokines [80]. On the basis of the cytokine secretion profile, CD4Ⳮ and CD8Ⳮ lymphocytes can be subdivided into functionally distinct subsets [81–83]. In the mouse, type-1 (Th1 and Tc1) cells produce IL-2 and IFN-␥ whereas type-2 (Th2 and Tc2) cells secrete IL-4, IL-5, IL-6, IL-9, IL-10, and IL-13. Cells with a mixed pattern (Th0 or Tc0) have also been described, but their origin is not clear. In humans, Th(Tc)1 and Th(Tc)2 cells produce similar patterns, although the synthesis of IL-2, IL-6, IL-10, and IL-13 is less tightly restricted to a single subset. It has been suggested that different modes of antigen presentation influence the differentiation of uncommitted T-cell precursors, with macrophages and dendritic cells evoking type 1 responses, while antigens targeted for presentation by B cells tend to promote type 2 responses [84,85]. The functions of the two cell subsets correlate well with their distinctive cytokines: type 1 cells are involved in cell-mediated inflammatory reactions, while type 2 cells are primarily involved in Bcell maturation. Furthermore, the characteristic cytokine products of Th(Tc)1 and Th(Tc)2 cells are mutually inhibitory for the differentiation and effector functions of the reciprocal phenotype [81–83]. Organ-specific autoimmune diseases are generally considered to result from an imbalance between the Th1 and Th2 responses, with type 1 cells generally mediating tissue damage and disease progression and Th2 cells inducing regulation of the autoimmune response and disease remission [86] (Fig. 1). Graves’ disease may be the exception to this paradigm, and perhaps the most convincing evidence for this is an inducible human model of GD happened on by chance [87]. In patients with multiple sclerosis (MS) treated in vivo with a humanized monoclonal to the panlymphocyte antigen CD52, ⬎95% of their circulating T lymphocytes were eliminated and there was considerable amelioration of their disease. Eighteen months after this treatment, T-cell numbers had returned to 35% (mostly CD8Ⳮ, and in the CD4Ⳮ population the RO:RA ratio was low) and B cells to 180% of pretreatment values. Furthermore, less interferon was produced upon in vitro activation of the T cells posttreatment. Of 47 MS patients treated (30 female), 14 (11 female) developed GD with TSAB. The deviation from Th1 to Th2, although beneficial for MS, was permissive for GD and stresses the importance of balance in maintaining appropriate immune responsiveness. Of interest, the same treatment has been applied to more than 600 patients with other Th1 type autoimmune diseases but without this complication. Recently, it has been suggested that GD might go through two different immunological phases [88]. The Th1-dependent immune response might occur in the early phase of the disease, but as it progresses, the Th2 immune response seems to predominate (Fig. 1). This shift could reflect a counterregulatory mechanism against inflammation as observed in other autoimmune diseases [89]. From an immunological point of view, GD is a controlled organ-specific autoimmune disease, since immune-mediated destruction of the gland is minimal or absent and the clinical expression is only the consequence of the activity of

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Figure 1 Th1 and Th2 immune responses in autoimmune thyroid diseases.

TSAb. The Th2 cytokines have a critical role because they block the cell-mediated immune response and limit the apoptotic process in the thyroid gland, favoring the expression of intracellular specific inhibitors [63]. Hashimoto’s thyroiditis is more compliant with the paradigm, since in animal models of HT type 1 responses play a critical role in the development of disease, although the role of individual cytokines in different phases of the disease process remains controversial [90–97]. For example, IFN-␥ can either enhance or suppress autoimmune thyroiditis, depending on the experimental conditions. In contrast, IL-10 [98–101] and IL-4 [102–104] appear to exert protective rather than harmful effects in experimental models of AITD. In patients with HT a Th1 dominance has been demonstrated and found to correlate with the destructive process characterizing the disease [105,106] (Fig. 1), as demonstrated by the fact that cells producing IFN-␥ predominantly express CD8Ⳮ and perforin [105] Furthermore, the type 1 immune response seems to exert a central role in favoring the apoptotic process in the thyroid gland [53,62]. An interesting point is that cytokines produced by infiltrating lymphocytes, or by the thyrocytes themselves, are able to influence thyroid function directly in favor of hypothyroidism. Studies in vitro demonstrated that IFN-␥ inhibits TSH induced transcription of Tg [107], TPO [108], NIS [109], and TSHR [110] genes. In vivo the inhibition seems to be limited to the expression of the NIS [109]. In addition to these effects, the locally produced cytokines may induce morphological changes of the epithelial junction complex, thus reducing the follicular barrier [111]. In this manner, despite the absence of cytotoxicity, local inflammation may promote the exposure of hidden antigens to the immune system.

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The role of Th2 cytokines in HT is more controversial. In AITD patients, elevated thyroidal expression of IL-10 was demonstrated in close proximity to lymphoid cells, suggesting that IL-10 could be involved in leukocyte recruitment [112]. However, in HT thyroids showing end-stage destruction of thyrocytes little IL-10 expression was demonstrated, even in areas with intense lymphocytic infiltration [113]. Thus, IL-10 may be deleterious in early stages of the disease by directing intrathyroidal B-cell stimulation but subsequently beneficial by favoring the apoptosis of infiltrating T cells and inhibiting the Th1 response [114]. This dichotomy highlights the complex nature of the disease process. IV. T AND B EPITOPES IN AITD By now it will be becoming clear that the concept of spectrum in AITD applies not only to the impact on thyroid function but also to the prevailing immune skew. To illustrate this, a Th2 response to the TSHR can produce both hyper- and hypothyroidism, depending on the agonist versus antagonist properties of the antibodies present. The varying biological activity of antibodies to the TSHR suggested that it could be important to try to identify the T- and B-cell epitopes, and a variety of approaches has been applied. These include (1) production of chimeric receptors to identify regions of the TSHR essential for TSAB/ TBAB activity, (2) screening TSHR peptide fragments, (3) neutralizing antibody bioactivity with portions of the TSHR, and (4) measuring T-cell proliferation and cytokine production in response to synthetic peptides. The TSHR is unique among the glycoprotein hormone receptors in being the target of autoantibodies having bioactivity, which has been attributed to its two-subunit nature. The process of generating the two subunits [115] may release a highly immunogenic portion of the TSHR into the circulation, which has the capacity to stimulate affinity maturation of receptor antibodies, some of which will be TSAB or TBAB. The structure also permits the ECD to act as a tethered inverse agonist, which switches to a true agonist in the presence of TSH [116]. Other posttranslational modifications of the TSHR required for TSH activation include tyrosine sulfation [117] and glycosylation on four of the six potential N-linked sites [37]. Whether TSAB and TBAB functions also depend on these has yet to be clarified. The production of TSHR/LHR chimeras has revealed that TSH binds multiple discontinuous residues in the ECD and that the TSH-, TSAB-, and TBAB-binding sites are overlapping but not identical [118,119]. TSAB bind residues located in the N and TBAB in the C termini of the ECD, respectively. Absorption studies, in which synthetic peptides or fragments of the TSHR are used to neutralize biological activity of TSAB or TBAB, have largely confirmed these findings [120,121]. In an attempt to define discontinuous residues in the relevant epitopes, the use of peptides from combinatorial libraries has demonstrated the possible importance of EEFDDA, ETFDDA, and EHFDDA for TSAB [120]. The expected similarity between the TSH- and TSAB-binding sites is inferred from recent studies using the ECD tethered to the cell surface by a glycophosphatidyl inositol (GPI) link. The affinity of TSH for ECD-GPI is much higher than for the holoreceptor on intact cells [37], and TSAB (but not TBAB) preferentially recognize ECD-GPI, compared with the holoreceptor [122]. This suggests that TSH-binding sites and TSAB epitopes are constrained in the holoreceptor but freely accessible in ECD-GPI. The observation may also explain the nonphysiological conditions required to assay TSAB in vitro, using cells expressing the intact TSHR. Sensitivity and specificity are increased in salt-free

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buffer and in the presence of PEG, conditions that presumably reduce the steric hindrance in the TSAB epitopes. Synthetic peptides have been widely applied to the study of T-cell responses to the TSHR. In the earliest studies a battery of peptides covering the ECD elicited a proliferative response in 50–60% of normal controls and GD patients. Even though some patients responded well to certain peptides, the results indicated that there was no dominant T-cell epitope in the ECD of the TSHR [123]. Others have confirmed the reactivity of normal T cells [124] to the receptor, but the lack of consistent response to defined regions of the ECD has been confirmed by some but not all investigators. In two separate studies, four major T-cell epitopes have been described, but only one of the epitopes was common to both [125,126]. More recently an ELISPOT assay has been applied to define the phenotype of TSHR-responsive T cells. Receptor peptides increased the number of IL-4– and decreased the number of IFN-␥–producing T cells in patients with GD [127]. An alternative approach used TSHR-transfected EBV transformed autologous B cells to identify T cells in GD. A small number of clones responded vigorously but produced relatively less IFN-␥ compared with other non–TSHR-responsive thyroid-specific clones. For example, the median IL-4/IFN-␥ was 0.8 in the TSHR but 0.06 in TPO-responsive clones, indicating the Th0/Th2 phenotype of the former and Th1 characteristics of the latter, respectively [128]. The first autoantigen, Tg, was discovered in 1956 and shown to play a role in HT [129], with TgAb present in about 55% of patients [130]. TgAb are also present in healthy subjects but are more polyreactive and recognize mainly thyroxine-containing determinants compared with those found in HT patients in which autoantibody responses are generally against species-specific epitopes [131–133]. Although the TgAb response in HT is somewhat heterogeneous, it is restricted to two conformational epitopes [131]. TgAB are not restricted to any particular isotype, but comprise all four IgG subclasses [72–74,134], with a preferential usage of IgG2, probably in accordance with the predominant Th1 response in this disease [134]. The second thyroid antigen, originally named the microsomal antigen, was described in 1959 but identified as TPO almost 30 years later [19,20]. In recent years, many groups have focused on the identification of B-cell epitopes within TPO [75]. There is controversy as to whether autoantibodies to TPO are able to bind sequential epitopes or recognize the 3-D conformation of this complex, globular molecule [77]. The availability of a panel of monoclonal murine and human autoAbs to TPO has permitted the definition of the conformational antigenic structure of TPO [135–139]. The immunodominant region is localized on the extracellular part of the molecule and overlaps the region of TPO with homology to complement control protein [139]. In addition, TPOAb, unlike TgAb and TSAb, are able to bind short peptide fragments, which may be parts of a larger discontinuous conformational epitope, but nevertheless are recognized in their own right [140–143]. Indeed, it was on this basis that TPO was cloned and shown to be the microsomal antigen [20]. In common with the TSHR, T-cell epitopes of TPO have been identified using synthetic peptides [144] or short recombinant fragments [145]. Even when employing proliferation as an index of stimulation, different authors report diverse T-cell epitopes with virtually no overlap between the various studies, but did not use the more relevant ‘‘thyroid-derived’’ T cells [126,144,145].

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V. PHENOTYPIC CHARACTERIZATION OF THE LOCALIZED AUTOIMMUNE RESPONSE IN THE THYROID Given the differences in immunopathogenesis between GD and HT, with the former an antibody-mediated Th2 disease and the latter due to Th1 immune destruction, we would anticipate a unique phenotype for the thyroid infiltrate in each condition. Some (but not all) experimental approaches have confirmed this hypothesis since single-cell analysis of intrathyroidal lymphocytes reveals that the larger (presumably activated) T cells predominantly produce IL-4 in GD and IFN-␥ in HT [105]. In humans it is difficult to obtain a clear view of the initiating immune events, since patients may present with symptoms months and even years after this occurs. Inevitably much of the information is derived from animal models, predominantly those resembling HT, but some ex vivo analysis of human glands has also been informative. In AITD the autoimmune process is initiated by the activation of autoreactive T cells, which in turn depends on antigen-presenting cells (APC). In fact, in BB rats the first sign of a developing autoimmune reaction is an increase in the number of dendritic cells and macrophages in the thyroid gland [146–148]. In human disease, although dendritic cells are present in patients with goiter as well as GD, they were significantly more abundant and of more mature phenotype in the latter [149]. The mechanisms of attraction of immune cells to the thyroid gland are not completely clear, but the expression of chemokine receptors, including CxCR3, CCR2, and CCR5, is upregulated and has been suggested to participate in recruiting T cells in AITD [150]. Alternatively, a nonspecific inflammation could be the first event eliciting the autoimmune process [146]. Nevertheless, dendritic cells and macrophages are normal constituents of the thyroid, and they have been shown to regulate the growth and function of thyrocytes via cytokines [151]. Therefore, the recognition of the monocyte-derived cells, on the one hand, induces both the initiation of the autoimmune disease, but on the other hand is also involved in the regulation of organ homeostasis (i.e., thyroid cell apoptosis). The presentation of autoantigens to T cells requires the expression of class I and II HLA antigens on the APC associated with costimulatory signals [152]. The thyroid cells also express HLA antigens, resulting in the ability to function as APCs and thus present antigen [153]. Experimental evidence suggests that this abnormal expression occurs after the initiation of the autoimmune process as direct effects of the cytokines produced by the infiltrating lymphocytes [154–156]. Nevertheless, thyroid cells could not provide adequate costimulatory signals, favoring T-cell anergy rather than stimulation [152,157,158]. In this view, the expression of HLA antigens on thyrocytes could prevent the occurrence of autoimmune disease if adequate costimulatory signals are not provided by cooperation with infiltrating ‘‘professional’’ APCs. An interesting point is that the thyroid cells, as ‘‘nonprofessional’’ APC, could display a self-epitope profile different from the one displayed during the antigen presentation by the peripheral blood mononuclear cells, suggesting epitope spreading during the progression of the autoimmune disease [159]. In particular, it was demonstrated that the thyroid cells could present cryptic self-epitopes, which were hidden during thymic education, and thus they are not recognized as self in the periphery [160]. The initial phase of the accumulation of dendritic cells and macrophages in the thyroid is followed by a phase of clonal expansion and maturation of autoreactive T and B cells in the draining lymph node, initially, and then in the thyroid itself [146,161]. In experimental models of HT, CD4Ⳮ T cells and B cells are required for the initiation of the autoimmune thyroiditis, whereas CD4Ⳮ T cells are required not only for the initiation

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but also for the maintenance of the autoimmune process [162–165]. In the TSHR cDNAinduced model in outbred mice, which most closely resembles human GD, the infiltrate is comprised of CD4Ⳮ T cells, B220Ⳮ B cells, and mast cells [166]. In other TSHRinduced models, thyroiditis in both BALBc and NOD mice contained large numbers of activated T cells while macrophages and dendritic cells were plentiful, particularly in the BALBc mice, in which B cells and IL-10–positive cells were also present. The most abundant infiltrates, containing CD8Ⳮ T cells and follicular destruction, were observed in the NOD mice [167]. The recruitment of lymphocytes in the thyroid gland is a multistep process involving adherence and migration across the endothelium, trafficking through the interstitium, and finally movement into the thyroid follicular cells [168]. Several adhesion molecules and chemoattractant cytokines (chemokines) mediate these processes. In the obese strain of chickens developing spontaneous thyroiditis, the intrathyroidal expression of IL-15 has been shown to exert a critical role in driving the lymphoid infiltration of the thyroid [169]. The expression of adhesion molecules and chemokines has been demonstrated both on infiltrating lymphocytes and follicular thyroid cells, suggesting an important cross-talk between the two compartments [88,147,170]. Since the degree of lymphocytic infiltration has been shown to correlate with TPO but not TSAB autoantibody titers, it is not surprising that HT thyroids have been the main focus of attention [171]. Two types of thyroidal lymphoid cell infiltrates are normally observed in established HT. The first are focal accumulations of lymphoid cells with a high degree of histological organization (focal thyroiditis) [172]. These infiltrates are not destructive and are involved in the generation of thyroid autoantibodies [173]. In particular, these structures are crucial sites in the development of anamnestic immune responses because they are sites where cells undergo somatic hypermutation and affinity maturation, with generation of highaffinity autoantibodies [174]. The presence of well-organized B-cell structures in AITD glands may be relevant to the pathogenesis, not only for the production of autoantibodies but also for the development and maintenance of the autoimmune response. In this context, the high concentrations of autoantigen in the thyroid parenchyma are critical for maintaining activation of lymphoid follicle structures. Indeed, focal infiltrates are present in both HT and GD, although in the latter condition they are less active probably due to the lower local concentration of autoantigens [174]. The second kind of infiltrate is diffuse without a recognizable pattern of topological organization and consists of mixtures of CD4Ⳮ and CD8Ⳮ T lymphocytes, macrophages, natural killer cells, and just a few B lymphocytes [175]. The infiltrating cells are found in areas of destroyed thyrocytes, suggesting a cytotoxic function for this cellular compartment [176]. T lymphocytes play a central role in development of HT, as well as GD [70]. Much effort has been focused on characterization of immunodominant self-epitopes and T-cell receptor usage [70,75]. In particular, many attempts have been made to determine whether self-reactive T cells have a restricted V␣ or V␤ usage. Restriction of T cell-receptor V␣, but not V␤, has been observed in some, but not all, thyroids from patients with thyroid autoimunity. Indeed, it is possible that restricted T-cell receptor V-region usage may not always correspond to identical epitopic recognition. Receptors with the same V␣ and V␤ genes, differing only in their joining region, recognize different nonoverlapping epitopes of TPO [160]. Despite the specific immune reaction that triggers HT, the autoreactive T cells are only a small component of the thyroidal infiltrate that comprises cells able to

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respond to a variety of nonthyroidal antigens [177]. Although the few autoreactive cells have a critical role in driving the autoimmune process, it is difficult to induce autoimmune disease without a contribution from the nonspecific infiltrate [177–179]. VI. GENETIC PREDISPOSITION IN AITD AITDs run in families, a first indicator that genetic factors may be involved, and this is further supported by the higher concordance rate in monozygotic (30–60%) compared with dizygotic (3–9%) twins [180,181]. In common with other autoimmune conditions, AITD is polygenic and multifactorial, thus individuals with a genetic susceptibility may develop disease when they encounter an external trigger present in the environment such as stress or infection (discussed below). Furthermore, the incidence of AITD has not changed significantly in recent decades, which also illustrates the strong genetic component. One of the first clues as to the possible genes involved came from the seminal experiments of Vladitui and Rose, who found varying susceptibility to the induction of thyroiditis in mice of differing H-2 haplotype [68]. The equivalent MHC region on human chromosome 6 was subsequently shown to be associated with the majority of autoimmune conditions including AITD. AITD haplotypes vary with the ethnic background of the patients, and in Caucasians the A1;B8;DR3 extended haplotype confers a relative risk of approximately 3 [182], while in Japanese [183] and Chinese [184] an increased frequency of HLA-B35 and HLA-Bw46, respectively, has been reported. However, when the MHC was evaluated by linkage analysis, results were largely negative [185] and at most demonstrate a modest effect on susceptibility to AITD [186]. Reasoning that genes implicated in the control of the immune response were most likely to be associated with autoimmunity, several studies have examined the influence of T-cell receptor ␣ and ␤ gene complexes, the immunoglobulin heavy chain, and the IL1 receptor antagonist genes etc. on the development of AITD [187]. As in the case of the MHC, the results were either contradictory or negative [188]. The one exception was CTLA-4, a T-cell surface protein that interacts with B7 present on APC with the net result being negative regulation of the T cell [189]. Polymorphisms in CTLA-4 have been both associated and linked with AITD [190,191]. One study proposed an association with the production of thyroid autoantibodies rather than the disease process itself [192], and the mechanism is thought to involve a reduction in the inhibitory function of CTLA-4 [193]. Apart from molecules involved in the immune process, other possible candidates include the thyroid autoantigens themselves. Despite the fact that GD thyroids show increased expression of the TSHR [194] and the P52T polymorphism was found at higher frequency in GD patients [195], there is only a weak association between AITD and the TSHR [196], which has not been confirmed by linkage studies [197]. More recently the Tg locus at 8q23–24 has been associated with HT [198], and this has been confirmed by linkage in families with AITD [199]. Since genetic susceptibility to AITD can be only partly explained by the effects of the MHC, Tg, and CTLA-4 loci, considerable effort has gone into performing genomewide scans to identify other susceptibility genes. There are a number of possible pitfalls with such an approach, including the heterogeneity of AITD, as exemplified by linkage analysis of a large Chinese family with AITD in which more than one locus produced a maximum LOD score of ⬎2 [200]. Furthermore, given the variation in age of onset, it is not possible to be certain whether healthy family members will develop AITD in the

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future. Finally, since the diagnosis of GD is usually made on clinical grounds, it is possible that families with activating germline TSHR mutations may be included in the AITD cohort, since the signs and symptoms are very similar [201]. Despite these constraints, some progress has been made [202,203] in characterizing AITD loci (chromosome 6, close to but distinct from the HLA region), GD (chromosomes 14, 20, and X), and HT (chromosomes 12 and 13).

VII. ROLE OF ENVIRONMENTAL FACTORS IN AITD Studies over the past 20 years have culminated in awareness that autoimmune diseases are polygenic disorders in which the penetrance is strongly influenced by environmental factors; (Fig. 2). Probably the most important environmental factor is gender, since, as described in the introduction, all AITD are considerably more prevalent in females, especially after the onset of puberty. Again, animal models have provided useful information, e.g., in the spontaneous obese strain chicken, treatment with testosterone reduces the severity of the disease [204], and in the spontaneous Buffalo rat model, the highest incidence is seen in multiparous females [205]. Similar findings have been reported in thyroglobulin-induced models of thyroiditis in which castration of male rats increases the incidence and severity of disease [206]. Possible mechanisms have been investigated, and it has been demonstrated that a number of genes of immune relevance are regulated by estrogens/androgens. An example is the expression of the Fc␥RIIB2 (an Fc receptor that mediates transport and uptake of

Figure 2 Autoimmune thyroid diseases result from a complex interplay between the environment and the genetic predisposition of an individual. AITD, autoimmune thyroid disease; TSHR, thyrotropin receptor; IgG, immunoglobulin; H & L, heavy and light chains of IgG; MHC, major histocompatability complex; TG, thyroglobulin; TCR, T-cell receptor; TPO, thyroid peroxidase; C, complement; AG. SPEC., antigen specific; GD, Graves’ disease; HT, Hashimoto’s thyroiditis.

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antibodies) on thyrocytes, which is repressed by testosterone [207]. The role of pregnancy has also been examined following the observation that the frequency of women developing GD and HT increased in the 12 months following delivery [208]. In common with several other autoimmune conditions, GD can ameliorate in pregnancy but then exacerbate after delivery. Application of an ELISA-PCR to detect DNA for a male-specific gene revealed fetal microchimerism in the majority of GD thyroids but in only 10% from adenoma. This led the authors to suggest that fetal male (and presumably female—although their presence cannot be easily demonstrated) cells are valid candidates for modulating AITD in pregnancy and postpartum [209]. The mechanism proposed is expression of MHC II by the fetal cells, which allows them to persist in the allogeneic host environment. However, when immunosuppression by the placenta is lifted following delivery, their presence could activate the host immune system. Among the environmental agents implicated in the induction of autoimmune disease, infectious organisms have historically been the most prominent [210]. Although several organisms have been suggested, the greatest focus has been on Yersinia enterocolitica [1,211]. Indeed, evidence of cross-reactivity between antigens from these microorganisms and thyroid cell membrane antigens has been reported [212], but this would not automatically lead to the appearance of AITD. Experimental evidence shows that immunization with Y. enterocolitica leads to the appearance of TRAb, but the histology of the thyroid gland remains normal [1]. Therefore, molecular mimicry does not appear to be sufficient to induce thyroid autoimmunity. Similarly, in subacute thyroiditis the excessive release of thyroid antigens by the disrupted parenchyma induces a transient increase of thyroid autoantibodies, but thyroid autoimmune disease will follow only in subjects who are immunologically predisposed [213]. Epidemiological, clinical, and experimental studies suggest a link between iodine intake and the development of AITD [214]. The introduction of dietary iodine as a public health measure in the early twentieth century eliminated endemic goiter in the United States, as well as in other western countries, but with a significant increase of cases affected by thyroid autoimmunity [215–219]. This link between iodine intake and the development of AITD is further substantiated by the development of autoimmune thyroiditis in animal models of susceptible mouse, rat, and chicken strains given increased dietary iodine [146,220]. The mechanism by which dietary iodine might influence the development of autoimmune thyroid disease is unclear but several possibilities exist, with an increased immunogenicity of Tg being considered the most important [221,222]. Experimental studies demonstrated that the proliferation of T cells in response to Tg was dependent upon the degree of iodination of the molecule [223]. In the absence of iodination, Tg is unable to induce T-cell proliferation even in the presence of IL-2. The insertion of iodine induces stereochemical changes in the Tg molecule, resulting in the loss of some epitopes and gain of others [214]. Recently, it has been demonstrated that the increased iodination of normal Tg creates new antigenic determinants on the molecule facilitating the selective processing and presentation of cryptic pathogenic peptides by APC to the immune system [224]. Probably any central tolerance (and there is evidence for expression of TPO, Tg, TSHR, and NIS in the thymus, at both the transcript and protein levels) would be to noniodinated epitopes of Tg. The Tg found in the serum is poorly iodinated, and there is no evidence to suggest that any Tg produced in the thymus is iodinated [224]. The high TgAb levels in iodine-induced EAT confirms the predominant role of the immune response against Tg in the pathogenesis of disease [225], although Tg antibody titers did not correlate with the severity of thyroiditis. Similarly, a high prevalence of TgAb positivity has been

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demonstrated in the course of iodized salt prophylaxis but associated with a low incidence of thyroid dysfunction, indicating their minimal pathogenic importance [219]. However, an autoimmune reponse to Tg may evolve, by epitope spreading, to generate pathogenic TPO antibodies and thus thyroid dysfunction, once again in genetically prediposed individuals [226]. In addition to the increased immunogenicity of Tg, the iodine may facilitate the occurrence of thyroid autoimmunity by a direct toxic effect on the thyroid gland, leading to the exposure of other autoantigens [146]. The organification of iodine in the thyroid gland induces the production of oxidative elements that damage the thyroid cell membranes by oxidation of lipid and proteins. This initial thyroid cell injury may be an important prerequisite for the subsequent development of AITD [227,228]. Furthermore, the oxidative stress caused by the iodine organification leads to the fragmentation of Tg into small immunoreactive peptides capable of triggering the autoimmune process [229]. The previous iodine-deficient state of the thyroid gland is another important factor favoring the appearance of iodine-induced thyroid autoimmunity. Furthermore, it has been argued that both iodine deficiency and excess may lead to AITD [146]. Adaptive mechanisms occurring in iodine deficiency, including the accumulation of dendritic cells, may be relevant to disease progression, although the resulting AITD is usually mild [146]. In humans, the presence of iodine deficiency–induced nontoxic goiter is associated with a number of autoimmune phenomena such as increased levels of TgAb and/or TPOAb and immunologically competent cells [230,231]. In addition, the thyroid gland is predisposed to the oxidative damage. Excessive amounts of iodine are rapidly oxidized in the hyperplastic thyrocytes by TPO, thereby generating excessive amounts of reactive intermediates that are toxic for the thyroid [146]. Iodine deficiency is often accompanied by selenium deficiency, which in turn compromises the activity of glutathione (which contains a selenocysteine and is an enzyme that ‘‘mops up’’ free radicals) and thus favors oxidative damage of the gland [232]. The possibility that radioactivity might induce thyroid autoimmunity has been widely suggested. An increased incidence of thyroid antibodies was reported in individuals who had received irradiation to the neck and head during childhood for benign and malignant diseases [233,234]. Moreover, an increased incidence of thyroid autoimmunity has been reported in populations exposed to radioactive contamination [235]. Recently, an increased frequency of circulating thyroid antibodies has been described in children and adolescents exposed to Chernobyl radioactive fallout [236,237]. The prevalence was higher in girls than in boys and in adolescents compared to children, in accordance with the known effects of puberty and sex hormones on the autoimmune process. The release of thyroid antigens from the damaged parenchyma could be a mechanism favoring the appearance of thyroid autoimmunity in radio-contaminated subjects. The iodine deficiency could also exert an additive effect favoring the entrance of iodine isotopes in the thyroid cell [237]. Drugs, especially immunomodulators, are important environmental factors known to act as triggers of human autoimmune disease [238]. Following the original description of the first case of IFN-related hypothyroidism [239], thyroid autoimmune disorders have been reported as common side effects of IFN-␣ therapy, although with controversial data regarding the incidence, severity, and physiopathological mechanisms [240]. In most cases the positivity for thyroid autoantibodies is the only clinical manifestation of the thyroid autoimmune process, whereas thyroid dysfunction occurs in a minority of the patients [240]. Indeed, the IFN-related AITD is a heterogeneous condition with different clinical expression and variable long-term outcome [241,242]. This heterogenity probably derives

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from the different underlying genotypes, although there are few data on genetic predisposition to IFN-related AITD [243,244]. The hypothesis is supported by the observation that subjects who do not develop IFN-related AITD remain disease-free for many years after cytokine withdrawal [241], even if a second treatment is performed [245]. The mechanisms responsible for the occurrence of thyroid disease in the course of IFN-␣ treatment have been not completely clarified. IFN-␣ has been shown to activate the innate response [246], as well as to induce a Th1 shift that is considered critical for the antiviral efficacy [247]. The same immunological mechanisms involved in therapeutic activity could be responsible for the occurrence of thyroid disease, although the two events (response to the treatment and appearance of thyroid disease) do not seem to be correlated [248,249]. Cell-mediated immune responses seem to exert an important role in the pathogenesis of IFN-related AITD, since destruction of the thyroid parenchyma was described as an early event, having a temporal relationship with the appearance of thyroid autoantibodies [250]. The mechanisms responsible for this early destructive process have not been clarified. The low thyroid autoantibody levels, as well as the predominant positivity for TgAb [250,251] at this time, could be suggestive of an involvement of antigen-nonspecific and/or antibody-independent mechanisms in the pathogenesis of the destructive process. This hypothesis agrees with experimental evidence describing activation of innate immunity by IFN-␣ [246]. The Th1dependent immune response also has an important pathogenetic role, mainly when the IFN-␣ is associated with ribavirin, which is a Th1-inducing factor [245]. Thus, innate and acquired immunity may act synergistically in the pathogenesis of IFN-related AITD [252], as has been described in the early stages of EAT [253]. In our opinion future characterisation of these aspects could improve our understanding of many of the immunological mechanisms involved in the early phase of AITD development. Since in humans, AITDs often have a long prodromal phase devoid of clinical signs and symptoms, they are not the subject of study. Consequently, IFN-related thyroid disease provides a valuable opportunity to investigate the autoimmune process from the earliest phases of its development. VIII. FUTURE DIRECTIONS Despite progress in the field of autoimmunity over the past 45 years, the treatments for human AITD remain primitive. Present therapies for AITD are relatively efficient but not curative since they do not resolve the underlying immune process. This deficiency stems from our limited understanding of the mechanisms that trigger the loss of tolerance and thereby precipitate an autoimmune reaction. We have already discussed the apparent contradiction in the role of Tg, which is central to the majority of induced (and spontaneous) animal models but of apparent limited importance in human HT, apart from mediating the autoimmunogenic effects of increased iodine intake. A feature of Tg that may warrant further study is its similarity [254] to the MHC class II invariant chain (Ii), whose gene is located on chromosome 5q32 [255]. The Ii CLIP peptide trimerizes with the ␣ and ␤ chains of the MHC class II molecule until it is transported to endosomal compartments, where Ii is proteolytically degraded, allowing class II molecules to separate into dimers and bind exogenous peptides [256]. A sulfated form of Ii, found in association with class II molecules on the cell surface, is found to participate in the T-cell interaction through CD44 molecules on their surface [257]. Individuals in whom the degradation of the CLIP peptide is suboptimal or sulfation is supraoptimal could have APC expressing a Tg-like peptide in association with MHC class II at the cell surface. Of interest, a susceptibility locus (maximum LOD score 3.14) for AITD has been found on 5q31–33 in a study of

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123 Japanese sibling pairs [198]. Although highly speculative, this mechanism would reconcile the apparent difference in the relevance of Tg in eliciting human and animal thyroiditis. The studies in animal models have highlighted the use of cytokine agonists or antagonists in the prevention or cure of AITD [258,259], e.g., antagonists of TNF␣ [258] or IL10 [101] in the development of EAT. Unfortunately in humans, systemic administration of cytokines is rendered difficult by their short half-life. Moreover, the high doses required to produce a therapeutic effect might induce numerous side effects in patients. For these reasons, new approaches based on gene therapy to deliver cDNAs encoding the desired cytokines locally have been proposed [260]. In particular, local IL-10 gene therapy has been shown to cure EAT in CBA/J mice by the abrogation of autoreactive T-cell responses, diminution of IFN-␥ production and a possible deviation of the B-cell response towards a Th2-mediated antibody response [259]. However, a word of caution is necessary in view of the experience in MS in which immune deviation ameliorated one autoimmune condition but induced a second. Furthermore, the contention that the use of nonviral cDNA vectors in humans would reduce the risks of host response [260] must be balanced against the fact that cDNA immunization can be used to induce a model of GD [166]. An alternative approach to cytokine treatment is antigen-specific therapy, inducing oral tolerance or using recombinant vaccines to inactivate specifically the autoimmune response [261–263]. The former method has been applied to a small group of AITD patients diagnosed several years previously. Patients received normal T4 replacement and one half of the group was supplemented with desiccated thyroid extract. There was no difference in parameters of humoral immunity between the groups but some evidence of suppressed cell-mediated responses in the patients receiving thyroid extract. The timing of this trial was not optimal, since immune-tolerance therapies are designed to reprogram immune cells in a highly specific fashion to eliminate pathogenic responses while preserving protective immunity. In this context, knowledge of the mechanisms regulating T-cell recognition and activation, as well as the characterization of autoantibody profiles, is critical for identifying the target of the immune response and thus for developing and selecting DNA tolerizing antigen-specific therapy [261–263].

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216. Doufas AG, Mastorakos G, Chatziioannou S, Tseleni-Balafouta S, Piperingos G, Boukis MA, Mantzos E, Caraiskos CS, Mantzos J, Alevizaki M, Koutras DA. The predominant form of non-toxic goiter in Greece is now autoimmune thyroiditis. Eur J Endocrinol 1999; 140: 505–511. 217. Tsatsoulis A, Johnson EO, Andricula M, Kalogera C, Svarna E, Spyroy P, Seferiadis K, Tsolas O. Thyroid autoimmunity is associated with higher urinary iodine concentrations in an iodine-deficient area of northwestern Greece. Thyroid 1999; 9:279–283. 218. Marwaha RK, Tandon N, Karak AK, Gupta N, Verma K, Kochupillai N. Hashimoto’s thyroiditis: countrywide screening of goitrous healthy young girls in postiodization phase in India. J Clin Endocrinol Metab 2000; 85:3798–3802. 219. Premawardhana LD, Parkes AB, Smyth PP, Wijeyaratne CN, Jayasinghe A, de Silva DG, Lazarus JH. Increased prevalence of thyroglobulin antibodies in Sri Lankan schoolgirls—Is iodine the cause. Eur J Endocrinol 2000; 143:185–188. 220. Sundick RS, Bagchi N, Brown TR. The role of iodine in thyroid autoimmunity: from chickens to humans—a review. Autoimmunity 1992; 13:61–68. 221. Saboori AM, Rose NR, Bresler HS, Vladut-Talor M, Burek CL. Iodination of human thyroglobulin alters its immunoreactivity. I: Iodination alters multiple epitopes of human thyroglobulin. Clin Exp Immunol 1998; 113:297–302. 222. Saboori AM, Rose NR, Burek CL. Iodination of human thyroglobulin alters its immunoreactivity. II: Fine specificity of a monoclonal antibody that recognizes iodinated thyroglobulin. Clin Exp Immunol 1998; 113:303–308. 223. Rasooly L, Rose NR, Saboori AM, Ladenson PW, Burek CL. Iodine is essential for human T cell recognition of human thyroglobulin. Autoimmunity 1998; 27:213–219. 224. Dai YD, Rao VP, Carayanniotis G. Enhanced iodination of thyroglobulin facilitates processing and presentation of a cryptic pathogenic peptide. J Immunol 2002; 168:5907–5911. 225. Rasooly L, Burek CL, Rose NR. Iodine-induced autoimmune thyroiditis in NOD-H-2h4 mice. Clin Immunol Immunopathol 1996; 81:287–292. 226. Mazziotti G, Premawardhana LDKE, Parkes A, De Silva DGH, Wijeyaratne CN, Jayasinghe A, Smyth P, J Lazarus. Evaluation of thyroid autoimmunity during the course of iodine prophylaxis: experience in Srilanka. 52th Meeting of the British Thyroid Association London, Nov. 21, 2002. 227. Bagchi N, Brown TR, Sundick RS. Thyroid cell injury is an initial event in the induction of autoimmune thyroiditis by iodine in obese strain chickens. Endocrinology 1995; 136: 5054–5060. 228. Many MC, Maniratunga S, Varis I, Dardenne M, Drexhage HA, Denef JF. Two-step development of Hashimoto-like thyroiditis in genetically autoimmune prone non-obese diabetic mice: effects of iodine-induced cell necrosis. J Endocrinol 1995; 147:311–320. 229. Duthoit C, Estienne V, Giraud A, Durand-Gorde JM, Rasmussen AK, Feldt-Rasmussen U, Carayon P, Ruf J. Hydrogen peroxide-induced production of a 40 kDa immunoreactive thyroglobulin fragment in human thyroid cells: the onset of thyroid autoimmunity. Biochem J 2001; 360:557–562. 230. Zonenberg A, Kinalska I, Zarzycki W, Telejko B. Incidence of thyroid autoantibodies in the endemic goiter. Horm Metab Res 1994; 26:238–242. 231. Corrales JJ, Orfao A, Miralles JM, Lopez-Berges MC, Garcia LC, Gonzalez M, Mories MT, San Miguel J. Immunological features of sporadic multinodular goiter. Clin Invest 1993; 71: 552–558. 232. Contempre B, Le Moine O, Dumont JE, Denef JF, Many MC. Selenium deficiency and thyroid fibrosis. A key role for macrophages and transforming growth factor beta (TGF-beta). Mol Cell Endocrinol 1996; 124:7–15. 233. DeGroot LJ, Reilly M, Pinnameneni K, Refetoff S. Retrospective and prospective study of radiation-induced thyroid disease. Am J Med 1983; 74:852–862.

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24 Addison’s Disease and Autoimmune Polyglandular Syndromes CORRADO BETTERLE University of Padova, Padova, Italy

I. HISTORICAL INTRODUCTION TO ADRENOCORTICAL INSUFFICIENCY The adrenal glands were first recognized as distinct organs by Bartolomeus Eustachius in 1563 [1]. Almost 300 years later, Thomas Addison, working at Guy’s Hospital in London, described for the first time the signs and symptoms of a disease correlated to histological alterations in the suprarenal capsules, characterized by ‘‘anemia, general languor and debility, remarkable feebleness of the heart’s action, irritability of the stomach and a peculiar change of color of the skin’’ [2]. Following the postmortem examination of 11 patients, Addison found 6 cases with adrenal tuberculosis, 3 cases of adrenal malignancies, 1 case of adrenal hemorrhage, and 1 case of adrenal fibrosis of an unknown origin. Subsequently, Brown-Sequard demonstrated that, in animals, bilateral adrenalectomy induces death [3]. Following these reports, in 1856 Trousseau referred to adrenocortical insufficiency as Addison’s disease (AD) [4]. Many years later it was discovered that adrenal function is regulated by the pituitary gland [5] and that the hypothalamus regulates pituitary function [6]. Between 1920 and 1930, adrenocortical extracts were obtained from adrenal glands [7–9], and their use made possible the survival of both adrenalectomized animals and patients with AD [7,10]. In 1937, both corticosterone and deoxycorticosterone were synthesized [11,12], but it was only in 1938 that they were employed in the treatment of AD in humans [13]. II. CLASSIFICATION OF ADRENOCORTICAL INSUFFICIENCY Primary adrenocortical insufficiency, or Addison’s disease, results from a bilateral involvement of the adrenal cortex, with a deficiency in the production of glucocorticoid and 491

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Figure 1 Primary adrenocortical insufficiency: different clinical presentations in a group of Italian patients (n ⳱ 317), 1969–1999. (Modified from Ref. 15.)

mineralcorticoids, which is associated with high levels of both ACTH and plasmatic renin activity. Secondary adrenocortical insufficiency results from dysfunctional pituitary ACTH secretion, with a predominant insufficiency in adrenal cortisol incretion. Tertiary adrenocortical insufficiency is due to a reduced secretion of the corticotropin-releasing hormone (CRH) by the hypothalamus, a pituitary ACTH deficiency, and a low glucocorticoid production rate. In general, the production of mineralocorticoids is normal in both secondary and tertiary adrenal insufficiencies [14], in contrast to primary AD. Over the last 30 years, 322 Italian patients with AD were screened: 317 were affected by primary and 5 by secondary AD. Eighty-three percent had an autoimmune form of AD, 12% a tuberculosis-based form, and 5% resulted from other rare causes (Fig. 1). III. PRIMARY ADRENOCORTICAL INSUFFICIENCY, OR ADDISON’S DISEASE A. Etiology Primary adrenocortical insufficiency can result from many different causes, which are listed in Table 1. B. Prevalence Primary AD is a very rare disease in New Zealand, where the prevalence is about 4.5 cases per million inhabitants [16]; in the United States 50 cases per million have been estimated [17], whereas in European countries evidence of the disease varies from between 40 to 140 cases per million inhabitants [18–23]. In the past, tuberculosis was the most common cause of primary AD world-wide. In Addison’s original description, 6 out of 11 cases (54%) were suffering from tuberculosis and only 1 (9%) from idiopathic atrophy. In 1930, during post-mortem examinations, adrenal tuberculosis was found in 70% of the patients with AD, and only 17% showed signs of ‘‘idiopathic adrenal atrophy’’ [24]. A clinical evaluation of 1557 patients with primary AD from 1974 to 2002 demonstrated that autoimmune AD was present in 44.5–94% of these cases, as compared with tuberculosis, which was present in only 0–33%

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Table 1 Causes and Frequency of Primary Addison’s Disease in Developed Countries Cause Autoimmune Tuberculosis Other infections

Metastatic malignancy

Infiltrative disorders

Adrenal hemorrhage or thrombosis

Adrenalectomy Drugs

Genetic

Etiological Agents Unknown TBC infection Histoplasmosis Coccidioidomycosis Blastomycosis Cryptococcosis Syphilis Cytomegalovirus HIV Breast, lung, stomach, renal, colon cancer Malignant melanoma Lymphomas Adenocarcinoma of the adrenals Amyloidosis Hemochromatosis Hemosiderosis Histiocytosis X Sarcoidosis Niemann-Pick disease Wolman disease Anticoagulant therapy Antiphospholipid syndrome Trauma LES Periarteritis nodosa Cushing’s syndrome Cancer Fluconazole Ketoconazole Mitotane Aminoglutethimide Metopyrone Trilostane Etomidate Cyproterone acetate Phenytoin sodium Rifampin Barbiturate Adrenoleukodystrophy Congenital adrenal hypoplasia Familial glucocorticoid deficiency Congenital unresponsivness to ACTH Kearns-Sayre syndrome Smith-Lemli-Opitz syndrome Congenital adrenal hyperplasia

Frequency (%) 75–80% 15–20% ⬍1%

⬍1%

⬍1%

⬍1%

⬍1% ⬍1%

1%

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Table 2 Etiological Forms of Primary AD in Europe, 1972–2002 Year 1972 1974 1967/79 1987 1989 1990 1991 1994 1995 1996 2002 Total

Country U.K. Denmark U.K. Italy Italy Sweden Poland U.K. Holland Norway Italy

No. of cases

Autoimmune (%)

Tuberculosis (%)

Other (%)

33 108 434 54 75 62 180 86 91 117 317 1557

81.0 65.7 83.8 44.5 68.0 71.0 69.0 94.0 91.2 83.0 83.0 44.5–94

19.0 17.6 15.1 33.3 21.2 19.4 28.9 0.0 6.6 2.6 12.6 0–33.3

n.d. 16.7 n.d. 22.2 2.6 9.7 1.7 6.0 2.2 14.5 4.4 1–22.2

Ref. 25 18 26,27 28 29 30 31 20 32 33 15

n.d. ⫽ not determined. Source: Modified from Ref. 15.

of them (Table 2). In recent years, in keeping with some other organ-specific autoimmune diseases [34], the prevalence of autoimmune AD seems to have increased [22,23]. C. Autoimmune AD The era of autoimmune diseases began in 1957, when Witebsky et al. [35] described the criteria for defining a disease as autoimmune (Table 3). On the basis of these criteria, more than 60 autoimmune diseases have now been identified [15]. With regard to AD, the majority of these cases, which are initially thought to be affected by idiopathic AD, might also be considered to have an autoimmune form, as they satisfied many of the criteria proposed by Witebsky et al. [35]. 1. Histopathology of the Adrenals This clinical condition, initially called idiopathic adrenal insufficiency, was described by Addison as follows: ‘‘the two adrenals together weighed 49 grains, they appeared exceedingly small and atrophied, so that the diseased condition did not result as usual from a

Table 3 Criteria for Defining a Disease as Autoimmune Demonstration of circulating autoantibodies and/or cellular immuno-mediated events Demonstration of lymphocytic infiltration in the target organs Identification and characterization of autoantigens Induction of the disease in animals with autoantigens Passive transfer of the disease by serum or lymphocytes Benefit from immunosuppressive therapy Source: Ref. 35.

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deposit either of a strumous or malignant character, but appears to have been occasioned by an actual inflammation, that inflammation having destroyed the integrity of the organs, which finally led to their contraction and atrophy’’ [2]. In cases of autoimmune AD, the adrenals are small, often weighing only about 1–2 g during the end stage of the disease (normal weight about 4 g each) [36]. During the active phase of the disease, there is a variable infiltration of lymphocytes, plasma cells, and macrophages. Many years after the commencement of clinical AD, fibrous tissue replaces the three layers of the adrenal cortex, and only the adrenal medulla remains normal [36]. A focal infiltration, predominantly of T lymphocytes and rarely of B cells, can also be present in the adrenals in a normal population, but the significance of this finding has yet to be clarified [37,38]. 2. Humoral Autoimmunity Adrenal Cortex Autoantibodies and 21-Hydroxylase Autoantibodies. IN PATIENTS WITH CLINICAL AD. Adrenal cortex autoantibodies (ACAs) were first detected in patients with AD in 1957 [39], using a complement fixation technique: 36% of the patients with idiopathic AD and 9% with tuberculosis AD had a positive test (Table 4). An indirect immunofluorescence (IIF) test using normal adrenal tissues to detect ACAs was described in 1963 [40]: 61% of the patients with idiopathic AD and 6.7% with tuberculosis type AD tested positive (Table 5). These differences were probably due to variations in technical procedures, patient selection, duration of the disease and specificity of the diagnosis. In 1992 it was discovered that the steroidogenic enzyme 21-hydroxylase (21-OH) was the major adrenal autoantigen in patients with autoimmune AD [58–61]. Subsequently, 21-OH autoantibodies (21-OHAbs) were measured by means of an immunoprecipitation assay (IPA), using 35S-labeled [53,54,56,57,61–63] or 125I-labeled recombinant human 21-OHs [65] Using these methods, the frequency of 21-OHAbs was 78% in patients with autoimmune AD but only 1.9% in patients with tuberculosis and AD (Table 6). In general, a good correlation existed between the ACAs detected using the IIF test and the 21-OHAbs found by means of radioimmunoassay (RIA) (Fig. 2), with the exception of a few studies [53,57]. Both ACAs and 21-OHAbs are good markers of adrenal cortex autoimmunity, not only in patients with clinical autoimmune adrenal failure, but also in subjects at risk of adrenal insufficiency (see below).

Table 4 Frequencies of ACAs by Complement-Fixation Test in Patients with Various Forms of AD Idiopathic AD Year 1957 1963 1967 1970 Total cases Range

Tuberculous AD

Positive/Tested

%

Positive/Tested

%

Ref.

2/8 24/71 15/35 16/45 57/159

25 34 43 36 Mean 36 25–43

0/2 —/— 0/16 2/5 2/23

0 — 0 40 Mean 9 0–40

39 40 26 41

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Table 5 Frequencies of ACAs by Immunofluorescence Test in Patients with Various Forms of AD Autoimmune AD Year 1963 1966 1966 1967 1967 1968 1969 1970 1970 1974 1974 1980 1981 1985 1986 1990 1996 1998 1999 1999 2000 2002 Total cases Range

Tuberculous AD

Positive/Tested

%

Positive/Tested

%

Ref.

36/71 18/35 31/48 22/35 57/118 17/45 14/22 8/15 17/45 161/246 49/70 194/307 27/37 27/38 11/21 15/25 81/97 17/42 116/143 52/65 15/18 28/94 1013/1637

51 51 65 63 48 38 64 53 38 65 70 63 73 71 52 60 84 40 81 80 83 30 Mean 61 30–83

—/— 5/27 2/15 0/16 0/16 3/5 1/9 3/41 3/5 0/62 0/18 —/— —/— 0/7 0/9 —/— —/— 0/14 0/22 —/— 1/1 —/— 18/267

— 18 13 0 0 60 11 7 60 0 0 — — 0 0 — —

40 42 43 26 44 45 46 47 41 48 18 49 50 51 52 30 33 53 54 55 56 57

0 0 — 9 Mean 6.7 0–60

Table 6 Frequencies of 21-OHAbs by RIA in Patients with Various Forms of AD Autoimmune AD Year 1995 1995 1997 1998 1999 1999 2000 2002 Total cases Range

Tuberculous AD

Positive/Tested

%

Positive/Tested

%

Ref.

56/83 24/28 81/99 35/42 116/143 43/65 14/18 77/94 446/572

67 86 82 83 81 66 78 82 Mean 78 66–86

0/9 0/5 0/9 —/— 0/22 —/— 1/11 —/— 1/56

0 0 0 0 0 0 9 — Mean 1.9 0–9

61 62 65 53 54 55 56 57

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Figure 2 Relationship between ACAs titers and 21-OH Abs levels. (From Ref. 15.)

THE NATURAL HISTORY OF AUTOIMMUNE AD IN PATIENTS WITHOUT CLINICAL AD. ACAs have been described in patients without clinical AD, where they were found to be present in 0.2% of the normal controls, in 1.3% of the patients with organ-specific autoimmune diseases, in 4% of the first-degree relatives of AD patients, and in 4% of hospitalized patients (Table 7). 21-OHAb determination has also been used for the screening of the normal population, and, in general, a good correlation existed between ACAs and 21OHAbs [56,66,75]. The significance of ACA positivity in patients without clinical AD initially was unclear [67–69], but subsequent studies revealed that they can be considered to be the best markers for the prediction of autoimmune AD. In the course of the natural history of AD, five main phases of the disease can be identified: one potential, three subclinical, and one clinical [15,70,72,73] (see Table 8 and Fig. 3). Stage 0 is characterized by ACA positivity in a normal ACTH test (Fig. 3, Table 8). The evidence that the zona glomerulosa (which produces the mineralocorticoids) is the first area to be affected and that the zona fasciculata (which produces glucocorticoids) may be involved only in a second phase suggests that either the zona glomerulosa is the first area to be attacked by the immunosystem or that it is more sensitive to a generalized autoimmune attack to the adrenal cortex, as the zona fasciculata is protected from lymphocytic infiltration by locally produced corticosteroid hormones for a longer period of time. Thus, it has become evident that the classical clinical signs of AD appear only in the later stages of the disease. In particular, hyperpigmentation appears many months after the increase in ACTH levels (Table 8). During the subclinical phases of AD, any type of stressful situation (e.g., physical trauma, infection, surgery, pregnancy, or other stress-related events) requiring an increase in cortisol secretion may precipitate adrenocortical failure (Fig. 3).

0/30 4/27 5/374 —/— 1/563 44/628 5/464 8/505 31/1036 —/— 30/1675 23/2571 20/2153 67/8840 14/808 4/289 5/302 2/18 263/20.282

Positive/Tested 0 15 1.3 — 0.2 7.0 1.0 1.6 3.0 — 1.8 0.9 0.9 0.8 1.7 1.4 1.6 11.0 Mean 1.3 0–15

%

Patients with autoimmune diseases

ACAs in Patients Without Clinical Addison’s Disease

1957 1963 1967 1969 1969 1980 1980 1981 1982 1982 1984 1988 1993 1997 1997 1997 1997 2000 Total cases Range

Year

Table 7

0/55 —/— —/— —/— —/— —/— —/— —/— 0/168 49/975 —/— —/— —/— —/— —/— —/— —/— —/— 49/1198

Positive/Tested

Hospitalized patients

0 — — — — — — — 0 5 — — — — — — — — 4 0–5

% —/— —/— —/— 4/49 0/47 —/— —/— —/— —/— —/— —/— —/— —/— —/— —/— —/— —/— —/— 4/96

Positive/Tested

— — — 8 0 — — — — — — — — — — — — — 4 0–8

%

First-degree relatives of AD patients

39 40 26 64 46 48 65 49 66 16 67 68 69 72 73 66 74 56

Ref.

498 Betterle

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Table 8 Stages of Adrenal Function in Patients with Adrenal-Cortex Autoantibodies During Rapid ACTH Test ACTH

Stage 0 (potential) Stage 1 (subclinical) Stage 2 (subclinical) Stage 3 (subclinical) Stage 4 (clinical)

Cortisol

PRA

Aldosterone

0⬘

0⬘

60⬘

0⬘

0⬘

Clinical signs of AD

N N N N/↑ ↑↑

N N N ↓ ↓↓

N N ↓ ↓ ↓

N ↑ ↑ ↑ ↑↑

N N/↓ N/↓ ↓ ↓

Absent Absent Absent Absent Present

PRA ⫽ plasma renin activity; N ⫽ normal value; time 0⬘ ⫽ basal; time 60⬘ ⫽ 60 minutes after i.v. injection of 0.25 mg of synthetic ACTH. Source: Ref. 15.

Figure 3 Natural history of autoimmune adrenalitis with the various stages of potential, subclinical, and clinical hypoadrenalism (see text for details). N ⳱ normal. (From Ref. 15.)

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In the natural history of AD, the prognostic value of these autoantibodies differed greatly between children and adults [72,73]. The cumulative risk of AD in children with respect to adults is shown in Fig. 4. Computed tomography (CT) or nuclear magnetic resonance (MNR) of the adrenals in these subjects revealed, in general, normal adrenal glands (Fig. 5d). Patients who progressed to clinical AD were in general persistently positive for both ACAs and 21-OHAbs [70,72,73,77]. Some of these positive patients, treated with high doses of corticosteroid therapy for Graves’ opthalmopathy, became negative for specific antibodies with the recovery of adrenal function [71,78]. If the above data are confirmed, strategies that could lead to the prediction and prevention of ongoing AD have now become a real possibility. During interferon therapy, a seroconversion for 21-OHAbs has been detected in patients with chronic hepatitis C. Up to now, cases of clinical adrenocortical insufficiency have not occurred [79]. It is our opinion that these patients should be followed up with particular attention in the future, because they could be at high risk of developing clinical adrenocortical insufficiency. This has already been seen in patients who developed islet cell antibodies or glutamic acid decarboxylase autoantibodies during interferon therapy with a high risk of developing type 1 diabetes mellitus [80–82]. Furthermore, as certain episodes associated with AD may aggravate a pre-existing endocrine defect (e.g., in patients with type 1 diabetes mellitus, thyroid autoimmune diseases, hypoparathyroidism, or premature ovarian failure), screening for ACA/21-OHAbs is recommended. Early detection of AD should allow for the diagnosis of a potentially lethal disease [83,84]. ACTH Receptor–Blocking Antibodies. ACTH receptor–blocking antibodies have been described in more than 90% of the patients affected by autoimmune AD [85,86]. Although a pathogenic role has been hypothesized in the development of AD, the presence of these autoantibodies has not been confirmed [87]. Steroid-Producing Cell Autoantibodies. Steroid-producing cell autoantibodies (StCAs) were first described using the IIF technique in males affected by autoimmune AD without gonadal failure [88]. The presence of StCAs in females was generally confirmed by the presence of primary gonadal failure (hypergonadotrophic hypogonadism) associated with a normal chromosomal pattern and lymphocytic oophoritis (reviewed in Refs. 15,89, and 90). Conversely, the majority of female patients with documented lymphocytic oophoritis

Figure 4 Cumulative risk for Addison’s disease in 17 ACAⳭve children (left) and in 74 ACAⳭve adult patients (right) with organ-specific disorders.

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b

c

d

e

f

g

h

i

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Figure 5 CT scan (a–g) or NMR (h) of adrenal glands in patients with primary AD. Minuscule adrenal glands (arrows) in a patient with autoimmune AD in the context of APS Type 1 (a) and in one with APS Type 2 (b). Normal adrenal glands (arrows) in a patient with isolated autoimmune AD (c) and in a patient with potential AD (two years before the onset of clinical AD) (d). Minuscule adrenal glands in a patient with long standing AD (10 years after diagnosis) in the context of APS type 2 (e). Adrenal bilateral calcifications with enlarged left adrenal gland (arrow) in a patient with AD caused by tuberculosis (f). Bilateral adrenal masses (arrows) in a patient with AD caused by bilateral adrenal adenocarcinoma (g) (courtesy of Dr. L. Benedetti from the Dept. of Imaging. Padova General Hospital, Padova, Italy). NMR of adrenals in a patient with AD due to bilateral adrenal massive hemorrhage (h) (courtesy of Dr. F. Presotto, Dept. of Medical and Surgical Sciences, University of Padova, Italy). NMR of adrenals in a patient with AD and congenital adrenal hyperplasia (arrows) (i) (courtesy of Dr. M. Cappa, Ospedale Pediatrico Bambin Gesu`, Rome, Italy). (Modified from Ref. 15.)

at biopsy were found to be StCA-positive [89]. StCAs are uncommon in addisonian males, although one study found that 3 of 79 males with autoimmune AD were positive for StCAs, and that one had autoimmune testicular failure [91,92]. StCAs are present in 60–80% of patients with APS-1, in 25–40% of those with APS-2, and in 18% of patients with isolated AD [15,49,89,93,94]. The different levels of prevalence of gonadal failure among these groups of patients probably accounts for the different levels of prevalence of StCAs [95,96]. In general, in patients with APS-1, hypogonadism follows upon the appearance of AD, whereas it precedes it in those with APS-2 and APS-4 [96].

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It has been discovered that the majority of StCAs react to steroidogenic antigens, 17␣-hydroxylase (17␣-OH), or a P450 side-chain cleavage (P450scc). Using an immunoprecipitation assay, 25S-labeled cloned antigens were used to test this reactivity [64], and good correlation was reported between StCAs and the autoantibodies to these specific enzymes [53,64,97,98]. 3. Animal Models Experimental autoimmune AD has been produced in animals using either autologous or heterologous adrenal extracts [15,99,100]. The adrenal infiltrations were more severe when the heterologous homogenates were used [100], but in general the disease remained at a subclinical level. In some experiments the disease was transferred by the immunocytes [101,102,100], suggesting that cell-mediated immunity may have a role in experimental adrenalitis. 4. Cellular Immunity The data concerning cellular immunity are conflicting, because the initial studies carried out by means of a migration inhibition assay, using different adrenal antigens, revealed a specific reaction [103,104]; a reduction in the suppressor function was subsequently demonstrated [106,107]. The activated T lymphocytes were increased in the peripheral blood in patients with AD of recent onset [108], and a proliferative response to 18–24 kDa adrenal antigen was documented in the majority of patients with AD [109]. 5. Clinical Features of Autoimmune AD Following the initial description of idiopathic AD, multiple insufficiencies in the endocrine glands, sometimes associated with other autoimmune and non-autoimmune diseases, have

Table 9 Prevalence of Clinical Autoimmune Diseases in a Population of 1240 Patients with Autoimmune AD Disease

Range, %

Hashimoto’s thyroiditis Graves’ disease Atrophic gastritis Chronic candidiasis Diabetes mellitus (type 1) Hypoparathyroidism Hypergonadotropic hypogonadism Vitiligo Alopecia Celiac disease Pernicious anemia Multiple sclerosis Inflammatory bowel diseases Sjögren’s syndrome Chronic hepatitis Lymphocytic hypophysitis

3.7–32 2–22.7 25 0.8–21 1.2–20.4 1.2–20 4.5–17.6 0.8–16 0.8–12 1.2–8 0.8–6 3.7 2.4 2.4 1.6–3 0.8

Source: Ref. 15.

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been observed in patients with autoimmune AD, and in their families. Table 9 summarizes the prevalence of clinical autoimmune diseases in an unselected population of patients with autoimmune AD. D. History and Classification of Autoimmune Polyglandular Syndromes As early as 1908 it was noted that multiple endocrine defects can occur in one individual [111]. The associations between AD and various other diseases do not manifest themselves at random, but in particular combinations (see above). In 1926 Schmidt first documented the association between nontuberculous AD and chronic thyroiditis [112], and in 1931 the first case of AD associated with diabetes mellitus and hyperthyroidism was described [114]. In 1964 Carpenter et al. reported that patients affected by Schmidt’s syndrome can develop type 1 diabetes mellitus [113]. In the following years the occurrence of this cluster of autoimmune diseases was reported with increasing frequency: in 1959 63 cases were reported [115] in 1964 more than 100 [113], and in 1981 there were 224 cases [110]. The first association between chronic hypoparathyroidism and chronic candidiasis was described by Torpe and Handley in 1929 [116]. However, only in 1943 was the case of a 12 year-old girl with tuberculous AD associated with both chronic hypoparathyroidism and chronic candidiasis [117]. In 1956 Whitaker et al. [118] added AD to the syndrome first described by Torpe. In 1958 50 patients were found to have developed Whitaker’s syndrome [119], and in 1981 71 cases were discovered [110]. Based on the above-mentioned clinical associations, in 1980–81 Neufeld and Blizzard classified APS into four main types (Table 10). According to this classification, autoimmune AD constitutes one of the major components in APS-1, APS-2, and APS-4. Spontaneous animal models of complete APS are very rare occurrences [121], and, in general, they remain at a subclinical level [122] On the basis of Neufeld’s classification, of 263 patients affected by clinical autoimmune AD who were subsequently followed up, we identified 35 patients with APS-1, 107 with APS-2, 13 with APS-4, and 108 with isolated AD (see Fig. 1, Table 11), which indicated an elevated probability in those patients affected by autoimmune AD of also developing an APS. 1. Autoimmune Polyglandular Syndromes in Autoimmune AD APS-1. MAIN CLINICAL FEATURES. APS-1 is almost always characterized by the presence of two of the following three diseases: chronic candidiasis, chronic hypoparathyroidism,

Table 10 Classification of APS According to Neufeld and Blizzard APS-1 APS-2 APS-3 APS-4

Chronic candidiasis, chronic hypoparathyroidism, autoimmune AD (at least two present) Autoimmune AD ⫹ autoimmune thyroid diseases and/or type 1 diabetes mellitus (AD must always be present) Thyroid autoimmune diseases ⫹ other autoimmune diseases (excluding autoimmune AD, hypoparathyroidism, chronic candidiasis) Two or more organ-specific autoimmune diseases (that do not fall into Type 1, 2, or 3)

Source: Ref. 15.

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Table 11 Mean Features of Autoimmune AD in an Italian Population Forms of autoimmune AD

No. of cases (%)

F/M

Adults/Children

Mean age at onset of AD (years)

APS-1 APS-2 APS-4 Isolated AD Total cases

35 (13) 107 (41) 13 (5) 108 (41) 263 (100)

1.8 3.6 3.3 0.8 1.7

0.08 7.6 13 8.7 3/1

14 36 36 30 30

and autoimmune AD [15,110,120,123–125]. This condition is also called autoimmune polyendocrinopathy, candidiasis, ectodermal dystrophy (APECED) [124]. The estimated prevalence of APS-1 worldwide is very low, ranging from 1/9,000 of the inhabitants among the Iranian Jewish community [126], 1/14,400 in Finland [124], 1/25,000 in Sardinia [127], 1/80,000 in Norway [128], and 1/200,000 in Italy [15]. The female:male ratio is 0.8: 2.4 [15,110,124,129]. In general, the first disease to appear is candidiasis, followed by hypoparathyroidism and lastly by the development of AD [15,124,130]. Chronic candidiasis is the first manifestation of the disease, often occurring before or during the first months of life. Candidiasis is present in 17–100% of patients (Table 12), but it appears to be less prevalent among the Iranian Jewish people (17%) [126] than in other populations [124,125,128]. Candidiasis may affect both the nails and the mucous membranes. Chronic candidiasis is the clinical manifestation of an immunological T-cell defect to Candida albicans antigens [110,120,124] with a normal B-cell response [133]. APS-1 has also been included in the group of immunodeficiencies by the World Health Organization (WHO) [134]. In general, candidiasis is followed by chronic hypoparathyroidism, which affects 76–100% of patients, and usually appears before the age of 10 (Table 12). The histopathology of the parathyroid can reveal an infiltration by mononuclear cells, but in some cases parathyroid tissue proved to be undetectable [118,129,135]. When hypoparathyroidism develops in the neonatal period, it is important to differentiate between it and other genetic forms of the disease [136–139]. Autoantibodies to the cytoplasm of the parathyroid cells have been reported in patients with idiopathic hypoparathyroidism alone, or when associated with APS-1 [140,141], but these autoantibodies also often appear to react towards the human mitochondria [142,143]. Cytotoxic autoantibodies reacting to the surface of the human parathyroid cells have also been described in these patients using cultured bovine parathyroid cells [144,145], but these antibodies lost their reactivity after absorption by nonspecific antigens [146]. The extracellular domain of the calcium-sensing receptor is the candidate autoantigen of 120–140 kDa, which is recognized by the autoantibodies present in patients with hypoparathyroidism [147]. However, the presence of these autoantibodies was not confirmed in a recent study [129]. These data suggest that chronic hypoparathyroidism is a disease that is probably mediated by cytotoxic T lymphocytes, without the presence of any circulating autoantibodies [148]. AD is the third disease that appears in this syndrome. It usually affects 22–100% of patients and develops before the age of 15 (Table 12). CT or NMR of the adrenals reveals normal/atrophic adrenals (Fig. 5a), but at autopsy, evidence of adrenal atrophy together with a lymphocytic infiltration was demonstrated [135].

76 73 100 32 17 nd 13 8 22 13 11 4 1

Hypoparathyroidism Chronic candidiasis Addison’s disease Alopecia areata Hypogonadism Kerathopathy Autoimmune hepatitis Vitiligo Malabsorption Pernicious anemia Chronic thyroiditis Type 1 diabetes Neoplasias

b

From Ref. 110. From Ref. 124. c From Ref. 126. d From Ref. 131. e From Ref. 132. f From Ref. 128. g From Ref. 125 plus personal observation.

a

USA, n ⫽ 71a

79 100 72 72 60 35 12 13 18 13 4 12 nd

Finland, n ⫽ 68b 96 18 22 13 38 0 nd nd nd 9 4 4 nd

Iranians, n ⫽ 23c 100 75 93 50 24 nd 31 12 6 nd 31 nd nd

USA, n ⫽ 16d

Frequency (%) of Clinical Diseases in APS-1 in Different Populations

Clinical manifestation

Table 12

100 100 82 nd 18 nd 27 0 18 9 36 0 nd

South Italians, n ⫽ 11e 85 85 80 40 31 10 5 25 10 0 10 0 nd

Norway, n ⫽ 20f 89 83 77 36 40 6 19 15 11 15 11 4 7

North Italians, n ⫽ 47g

76–100 18–100 22–100 13–72 17–40 0–35 5–31 0–25 6–22 0–15 4–36 0–12 1–7

Total cases, n ⫽ 256 (range)

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Frequently, children with chronic candidiasis and/or hypoparathyroidism are positive for ACAs, and the presence of these antibodies represents an absolute risk of developing AD in this population (see Fig. 9). MINOR CLINICAL COMPONENTS. Other immune- and non–immune-mediated diseases are often present in patients with APS-1, including: 1. Autoimmune endocrinopathies: (a) hypergonadotropic hypogonadism (17–40%) (Table 12) marked by the presence of StCAs, (b) type 1 diabetes mellitus (0–12%) (Table 12) characterized by islet cell and glutamic acid decarboxylase autoantibodies (GADAbs) [125,129,149,150], (c) chronic thyroiditis (4–36%) (Table 12) marked by the presence of thyroid autoantibodies [125,129,132], (d) lymphocytic hypophysitis (7%) 2. Autoimmune gastrointestinal diseases: (a) chronic atrophic gastritis (13–27%) marked by the presence of parietal cell autoantibodies (PCAs), (b) pernicious anemia (0–15%) (Table 12) marked by evidence of both PCA and intrinsic factor autoantibodies [125,132], (c) celiac disease, marked by the presence of reticulin or endomysium autoantibodies 3. Malabsorption (6–22%), the expression of many different disorders: (a) intestinal lymphangiectasia [151], (b) exocrine pancreatic insufficiency [152,153], (c) cystic fibrosis [154], (d) intestinal infections [153], (e) autoimmune gastrointestinal dysfunctions [155,156] marked by evidence of either tryptophan hydroxylase autoantibodies [157] or histidine decarboxylase autoantibodies [158], (f) deficiency in cholecystokinin [159] 4. Liver disease: (a) chronic active hepatitis (5–31%) (Table 12) marked by the presence of liver-kidney autoantibodies (LKM) [160], which recognize the cytochromes P450 CYPIA2 and CYP2A6 as autoantigens [127,161], or aromatic L-amino acid decarboxylase [162]; (b) cholelithiasis (44%) [163] 5. Autoimmune skin diseases: (a) vitiligo (0–25%) (Table 12), which is correlated to the complement-fixing melanocyte autoantibodies [164,165], aromatic Lamino acid decarboxylase autoantibodies [162], or antibodies to SOX9 and SOX10 [166], (b) alopecia areata (13–72%) marked by the presence of tyrosine hydroxylase antibodies [167,168] (Table 12) 6. Autoimmune exocrinopathies: (a) Sjo¨gren’s syndrome (12–18%), which is associated with the antibodies to extractable nuclear antigens (ENA) 7. Rheumatic diseases 8. Ectodermal dystrophy (0–35%) (Table 12) characterized by keratoconjunctivitis, nail dystrophy, defective dental enamel formation, and faulty teeth [129,169] 9. Immunological deficiencies: (a) T-cell defect to Candida albicans, (b) IgA deficiency, (c) polyclonal hypergammaglobulinemia [129] 10. Acquired asplenia [163,170,171] 11. Neoplasias: carcinoma of the oral mucosa and the esophagus and adenocarcinoma of the stomach [124,125] 12. Calcifications of: (a) the basal ganglia, (b) The tympanic membranes, (c) The subcapsular lens opacities 13. Vasculitis (3%) 14. Nephrocalcinosis

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Specific autoantibodies could be used as markers to both facilitate the diagnosis of the minor autoimmune component diseases involved and predict them (Table 13). APS-1 represents the human autoimmune syndrome with the highest concentration of diseases related to a specific genetic pattern (see below), as has been demonstrated by the 150 clinical manifestations observed among our 35 patients with this syndrome. TREATMENT. In the past, amphotericin B, in conjunction with the transfer factor, yielded the best results for the treatment of candidiasis. At present, periodical treatment with fluconazole, ketoconazole, or itraconazole is effective, although this last drug is more effective in patients with nail infections and does not seem to work in patients with mucosal infections. They must also be treated with topic anti-candidal agents. The oral mucosa in these patients needs to be controlled every 6 months, and special dental care and oral hygiene are also necessary [172].

Table 13 APS-1: Autoantibodies Correlated with Minor Autoimmune Diseases Minor disease Hypogonadotropic Hypogonadism Vitiligo

Autoimmune hepatitis

Celiac Disease

Diabetes mellitus (type 1)

Thyroid autoimmune diseases Autoimmune castritis Pernicious anemia Autoimmune malabsorption Alopecia areata Adenohypophysitis

Markers Steroid-producing cell autoantibodies 17␣-Hydroxylase autoantibodies P450 side-chain cleavage autoantibodies Complement-fixing melanocyte autoantibodies Autoantibodies to SOX9 and SOX10 51 kDa autoantibodies (L-amino-acid decarboxylase) Liver-kidney microsomal autoantibodies CYT IA2 autoantibodies CYT 2A6 autoantibodies 51 kDa autoantibodies Reticulin autoantibodies Endomysium autoantibodies Tranglutaminase autoantibodies Islet-cell autoantibodies (ICA) Glutamic acid decarboxylase autoantibodies (GADAbs) Autoantibodies to second islet antigen (IA2Abs) Thyroid microsomal autoantibodies, thyroglobulin autoantibodies Parietal cell autoantibodies (PCA) PCA ⫹ Intrinsic factor autoantibodies Tryptophan hydroxylase autoantibodies Histidine decarboxylase autoantibodies Tyrosine hydroxylase autoantibodies Pituitary autoantibodies (?)

Described before disease onset? Yes Yes Yes Yes ? ? Yes ? ? ? Yes Yes ? Yes Yes Yes Yes Yes Yes ? ? ? ?

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The treatment of hypoparathyroidism is carried out by the continuous administration of both calcium and vitamin D. Acute hypocalcemia must be treated by administering calcium gluconate (about 10–20 mg calcium/mL) rapidly intravenously. Chronic treatment is required with vitamin D and calcium per os in order to maintain the calcium levels in the blood at low levels of normality. Preparations of position 1 hydroxylated vitamin D such as calcitriol, colecalciferol, and dihydrotachysterol are the best forms of treatment [172]. Immunosuppressive therapy has been used in APS-1 only in cases of autoimmune hepatitis and, sometimes, for intestinal dysfunctions [172]. A patient with APS-1 plus other minor clinical manifestations was treated with cyclosporin A. Initially, the clinical manifestations regressed, but they reappeared when the therapy was reduced because of the side effects [156,173]. The associated clinical endocrine deficiencies are controlled by means of conventional substitutive therapy. GENETIC PATTERN. APS-1 is an inherited autosomal recessive condition [124,125]. In 1994 a genetic link between the clinical presentation of this syndrome and the genes located on the long arm of chromosome 21 was identified [174]. The gene responsible for this condition was identified as AIRE (AutoImmune REgulator) [175,176]. To date, more than 40 separate mutations associated with APS-1 have been identified in the AIRE gene [177], four of which appear to be very important. The first and most important mutation is R257X, which is present in exon 6 [175–178] and is found in 82% of the alleles of Finnish people with APS-1, but is also the most frequent mutation found among other ethnic groups, including northern Italian patients [131,179,180]. The second mutation described is del13, which is present in exon 8[175–178] and is the most common mutation found in Caucasian American and British patients [128,131,176,179–181], but also appears in northern Italian patients (Table 14). R139X is a mutation present in exon 3 that represents the most common mutation in Sardinian APS-1 patients, being found in 18 out of 20 independent alleles [182]. A Y85C mutation in exon 2 was the typical mutation detected in the Iranian Jewish patients [183]. Table 14 summarizes the genetic study carried out on 11 northern Italian patients.

Table 14 Haplotype Analysis in 11 Italian Patients with APS Type 1 Patients 1. F. T. 2. F. L. 3. A. E. 4. C. A. 5. C. G. 6. C. G. 7. C. E. 8. T. P. 9. DG. F. 10. H. K. 11. S. M.

Mutations, Allele 1/Allele 2

Sex

Major clinical diseases

Age at onset

R257X/R257X R257X/R257X R257X/R257X R257X/R257X R257X/R257X del 13/R257X del 13/R257X del 13/del 13 del 13/del 13 del 13/del 13 R139X/R139X

F F M M M F F F M F F

CC ⫹ CHP ⫹ AD CC ⫹ CHP ⫹ AD CC ⫹ CHP ⫹ AD CC ⫹ CHP ⫹ AD CC ⫹ CHP ⫹ AD CC ⫹ CHP ⫹ AD CC ⫹ CHP ⫹ AD CC ⫹ CHP ⫹ AD CC ⫹ CHP ⫹ AD CC ⫹ CHP ⫹ AD CC ⫹ CHP ⫹ AD

adulthood childhood childhood childhood childhood childhood childhood adulthood adulthood adulthood childhood

Patients 4 and 5 are brothers, and patients 6 and 7 are sisters. CC ⫽ chronic candidiasis; HP ⫽ chronic hypoparathyroidism; AD ⫽ Addison’s disease. Source: Modified from Ref. 15.

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APS-1 is the first autoimmune disease that is caused by mutations in a gene. The mutation in both alleles of the AIRE gene is usually associated with a clinical expression of the syndrome. Parents and other first-degree relatives of patients with APS-1 who carry only one mutant AIRE allele are not affected by this syndrome. Thus, the genetic mutations observed in APS-1 may be responsible for the breakdown in immunotolerance in humans [178]. The identification of the main AIRE gene mutations should help with the identification of the normal carriers in high-risk communities and the screening of unaffected family members of APS-1 patients. The typical clinical immunological and genetic profile for APS-2 is summarized in Table 15. APS-2. MAIN CLINICAL MANIFESTATIONS. APS-2, or Schmidt’s syndrome [112], is characterized by the presence of autoimmune AD in association with either autoimmune thyroid diseases and/or type 1 diabetes mellitus. AD is present in all the patients: autoimmune thyroid diseases in about two thirds and type 1 diabetes mellitus in less than 50% [30,93,110,112,120]. APS-2 is rarely present, the incidence being about 1.4–2.0 per 100,000 inhabitants [184]. It may occur at any age, but it is more prevalent in middleaged females [93,110,120,185]. In the natural history of APS-2, type 1 diabetes mellitus and Graves’ disease generally precede the onset of AD, but chronic thyroiditis develops either in conjunction with or after AD [93]. The mean age at onset of AD was 36 in our group of 107 patients with APS-2, diabetes occurred at 28 years of age, Graves’ disease at 31 years, and chronic thyroiditis at 40 years. AD, together with another main component disease, was present in 89% of the patients, but only 11% of the patients manifested the complete triad of diseases. CT or NMR of the adrenals in patients with APS-2 at onset revealed normal/ atrophic glands (Fig. 5b,e). The rare autoptic studies of the adrenal glands in APS-2 patients affected by AD revealed evidence of adrenal atrophy, together with a lymphocytic infiltration [15,93]. The specific organ-specific autoantibodies are present in the majority of cases at the onset of autoimmune AD, chronic thyroiditis, or type 1 diabetes mellitus [15]. In the case of Graves’ disease, the majority of patients have thyroid-stimulating antibodies, which are reactive to the TSH receptor [93]. We think that the appearance of the complete form of APS-2 could represent merely the tip of the iceberg. For this reason, patients with one of the fundamental diseases characteristic of APS-2 must be screened for other autoantibodies in order to individuate those with the incomplete form of APS-2. For example, a patient with type 1 diabetes mellitus or with thyroid autoimmune disease showing evidence of ACAs in the serum, or a patient with AD showing evidence of thyroid and/or pancreatic autoantibodies, should be classified as an incomplete APS-2 and followed up with subsequent specific functional tests. A summary of the different combinations of the incomplete form of APS-2 is given in Table 16. MINOR CLINICAL MANIFESTATIONS. Minor autoimmune diseases can develop along with APS-2: (1) vitiligo (4.5–11%), (2) chronic atrophic gastritis, with or without pernicious anemia (4.5–11%), (3) hypergonadotrophic hypogonadism (4–9%), (4) alopecia areata (1–4%), (5) chronic hepatitis (4%), and (6) hypophysitis. However, these autoimmune diseases are present with a lower frequency than in cases of APS-1 [93,123]. In general, these diseases are associated with the presence of respective organ-specific autoan-

(30%) associated with hypogonadism or preceding it (0–11%) Gonadal failure, vitiligo, alopecia, atrophic gastritis, pernicious anemia, celiac disease, chronic hepatitis, hypophysitis, etc. ⬃ 50%

Normal/atrophic glands Lymphocytic adrenalitis

Present 15% 100%

(62%) associated with hypogonadism or preceding it (11–60%) Gonadal failure, vitiligo, alopecia, atrophic gastritis, pernicious anemia, celiac disease, chronic hepatitis, hypophysitis, autoimmune malabsorption ⬃ 65%

Normal/atrophic glands Lymphocytic adrenalitis

Ectodermal dystrophy Cancer ACA and/or 21-OHAbs (at onset of AD) StCA and/or antibodies to other steroidogenic enzymes Minor autoimmune diseases

NMR ⫽ Nuclear magnetic resonance; CT ⫽ computed tomography. Source: Ref. 15.

AutoAbs in the absence of respective minor clinical disease NMR or CT of adrenals Histopathology of adrenals

Absent 3% 100%

AIRE gene mutations Candidiasis, hypoparathyroidism, AD (almost two present)

Normal/atrophic glands Lymphocytic adrenalitis

(38%) associated with hypogonadism or preceding it (100%) Gonadal failure, vitiligo, alopecia, atrophic gastritis, pernicious anemia, celiac disease, chronic hepatitis, hypophysitis, etc. ⬃ 50%

Absent Rare 100%

36 AD (rare) Other autoimmune diseases (frequent) HLA-DR3, DR4 AD (always present)

36 AD (rare) Other autoimmune diseases (frequent) HLA-DR3, DR4 AD (always present) and/02 thyroid autoimmune diseases and/02 type 1 DM

13 APS type 1 (25%) Other autoimmune diseases (rare)

Genetic Major component diseases

F⬎M 30

APS type 4

F⬎M 24

APS type 2

F⬎M 2

APS type 1

Main Features of Four Clinical Presentations of Autoimmune AD in Humans

Gender Mean age at onset of the first manifestation (years) Mean age at onset of AD (years) Family history for

Table 15

Normal/atrophic glands Lymphocytic adrenalitis

⬃ 50%

(13%) preceding hypogonadism (0%)

Absent Rare 80%

30 AD (rare) Other autoimmune diseases (frequent) HLA-DR3, DR4 AD (always present)

M⬎F 30

Isolated AD

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Table 16 Hypothetical Combinations of Incomplete APS-2 Clinical disease Addison’s disease Thyroid autoimmune disease Type 1 diabetes mellitus Thyroid autoimmune disease and type 1 diabetes mellitus None

Serology ⫹ ⫹ ⫹ ⫹

thyroid Abs and/or ICA and/or GADA ACAs/21-OHAbs ACAs/21-OHAbs ACAs/21-OHAbs

⫹

ACAs ⫹ thyroid Abs and/or ICA and/or GADA

ACA ⫽ adrenal cortex autoantibodies; 21-OHAbs ⫽ 21-hydroxylase autoantibodies; ICA ⫽ islet-cell autoantibodies; GADA ⫽ glutamic acid decarboxylase autoantibodies.

tibodies, but sometimes the appearance of a specific autoantibody can precede the clinical onset of the corresponding disease [93]. TREATMENT. The therapies regarding the different components of APS-2 are similar, whether they occur in isolation or in association with other autoimmune diseases. However, it is important to remember that thyroid hormone replacement therapy in patients with both hypothyroidism and AD can precipitate a misdiagnosed adrenal insufficiency, or a reduction in insulin dosage may be the first sign of AD in a patient with type 1 diabetes mellitus. An outline of the therapy for AD is given below. With regard to the therapy for both type 1 diabetes mellitus and the thyroid autoimmune diseases, see the Chapters 21 and 23. Premature ovarian failure should be treated with estrogen and progesterone replacement therapy. In certain cases, patients undergoing immunosuppressive therapy can resume menstruation. Pernicious anemia must be treated by means of a cycle of parenteral injections of cyanocobalamin (1000 ␮g/d), followed by one low dose of this drug (100 ␮g) per month. A periodical gastroscopy should be performed on these patients in order to precociously diagnose a gastric cancer. Celiac disease should be treated by means of a gluten-free diet [186]. GENETIC PATTERN. APS-2 may occur in the same family, in an autosomal dominant fashion, with an incomplete penetrance pattern of inheritance [15,187]. The human leukocyte antigens (HLAs) play a key role in determining the T-cell responses to these antigens, and various HLA alleles have been shown to be associated with autoimmune disorders [188,189]. An increased prevalence of HLA-DR3 and/or DR4 has been found in patients with autoimmune AD, except when the disease occurs as a component of APS-1 [190]. The relative risk of developing autoimmune AD, which was calculated in Caucasian subjects carrying both HLA-DR3 and HLA-DR4 alleles, was high—46.8 [191]. Several studies have confirmed the association of autoimmune AD in APS-2 patients with various alleles within the HLA-DR3 – carrying haplotype, including DRB1*0301, DQA1*0501, and DQB1*0201 [93,191–196]. In contrast, the association of HLA-DR4 with autoimmune AD appeared less evident [93,192–194]. When studying American patients with APS-2, Huang et al. demonstrated that the HLA-DR3 DQB1*0201 subtype and the HLA-DR4 DQB1*0302 subtype had increased in patients with both autoimmune AD and type 1 diabetes mellitus [196]. The frequency of the appearance of HLA-DR3-DQ2 and HLADR4-DQ8 haplotypes among a population of patients with APS-2 from Norway was recently found to be significantly increased, while the frequency of the DR1-DQ5 aplotype

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was found to be significantly reduced [57]. The linkage disequilibrium between the genes does not allow determination of the independent role of other individual genes in conferring susceptibility to the disease [195,197]. The cytotoxic T-lymphocyte antigen-4 (CTLA-4) gene encodes a costimulatory molecule, which is an important negative regulator for T-cell activation [198]. Conflicting results were found in relation to this gene in patients with AD (with either isolated or in the context of APS-2) [199–201]. In a recent study patients with either isolated AD or in the context of APS-2 were evaluated for the presence of del13 in the AIRE gene, but these mutations had not increased. The typical clinical, immunological, and genetic profile for APS-2 is summarized in Table 15. APS-4. APS-4 is a rare syndrome characterized by an association of autoimmune diseases that do not fit into any of the above categories [120]. Autoimmune AD in APS-4 manifests itself when associated with hypogonadism, atrophic gastritis, pernicious anemia, celiac disease, myasthenia gravis, vitiligo, alopecia, hypophysitis, etc., in the absence of chronic candidiasis, chronic hypoparathyroidism, thyroid autoimmune diseases, and type 1 diabetes mellitus. Both ACAs and/or 21-OHAbs are present in the majority of these patients at the onset of AD (Table 15), and the associated clinical autoimmune diseases are, in general, marked by their respective autoantibodies. A study of 13 APS-4 patients was carried out by our team; the clinical, genetic, immunological, and serological features relating to this study are summarized in Table 15. Incomplete APS-4 can also be found in an individul; the different combinations of the latter situation are summarized in Table 17. 2. Isolated AD Isolated AD is characterized by autoimmune adrenal insufficiency in the absence of any other clinical autoimmune disease. Isolated AD is represent in 41% of our autoimmune

Table 17 Hypothetical Combinations of Incomplete APS-4 Clinical disease Addison’s disease Addison’s disease Addison’s disease Alopecia areata Vitiligo Autoimmune gastritis Pernicious anaemia Celiac disease Autoimmune hepatitis Primary biliary cirrhosis Adeno- or neurohypophysitis

Serology ⫹ ⫹ ⫹ ⫹ ⫹ ⫹ ⫹ ⫹ ⫹ ⫹ ⫹

PCA and/or IFA EMA and/or t-TGA LKMA and/or AMA ACAs/21-OHAbs ACAs/21-OHAbs ACAs/21-OHAbs ACAs/21-OHAbs ACAs/21-OHAbs ACAs/21-OHAbs ACAs/21-OHAbs ACAs/21-OHAbs

ACA ⫽ adrenal cortex autoantibodies; 21-OHAbs ⫽ 21-hydroxylase autoantibodies; PCA ⫽ parietal cell autoantibodies; IFA ⫽ intrinsic factor autoantibodies; EMA ⫽ endomysium autoantibodies; t-TGA ⫽ tissue transglutaminase autoantibodies; LKMA ⫽ liver-kidney microsomal autoantibodies; AMA ⫽ mitochondrial autoantibodies.

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AD patients. The female:male ratio and the mean age at onset are different from those in patients with APS-2, APS-4, and APS-1 (Tables 11 and 15). NMR or CT imaging revealed either normal or atrophic adrenal glands in patients with isolated AD (see Fig. 5c). ACAs and/or 21-OHAbs were present in about 80% of the patients at the onset of isolated AD (see Table 15). Following the diagnosis of clinically isolated autoimmune AD, it was suggested that an autoantibody screening should be performed. This strategy made it possible to identify whether these patients were cases of truly isolated AD or whether they could be considered as potential or subclinical APSs. An increased frequency of HLA-DR3 was found in some patients with isolated AD [193,195]. Recently, the frequency of HLA-DR3-DQ2 and DR4-DQ8 haplotypes was found to be significantly increased in a population of Norwegian patients with isolated AD, while the frequency of DR1-DQ5 was significantly reduced (57). The clinical, immunological and genetic profile of isolated AD is summarized in Table 15. 3. Autoimmune AD: Four Clinical Entities The four main types of autoimmune AD are summarized in Table 15. They have some common biochemical immunological and anatomopathological findings, but they differ as regards the clinical (age at onset, female:male ratio, adult:child ratio, type of associated autoimmune disease, presence of candidiasis, presence of hypogonadism, presence of other minor diseases) and genetic patterns (i.e., mutations in the AIRE gene, HLA-DR, CTLA4). 4. Pathogenesis of Autoimmune AD In cases of APS-1, the genetic susceptibility due to the AIRE gene mutations associated with immunodeficiency and chronic candidiasis may be essential elements in inducing both a lack of tolerance to self antigens and a chronic inflammation, which may lead to the occurrence of multiple autoimmunity at a young age and to cancer in adult life. In cases of APS-2, the class II HLA genes play an important role, but are not in themselves enough, as regards the development of autoimmune AD [202]. Endogenous or exogeneous agents, such as infections, drugs, pregnancy, food, or stress (events widely believed to be relevant here) may act as important precipitating cofactors (Fig. 3). But it is not clear what the environmental factor is that induces the initial modification, at a molecular level, of the target cell, or the first event that induces the recruitment of lymphocytes in the adrenal glands. Furthermore, it is still unknown whether the initial autoaggression by T lymphocytes takes place against all three layers or against a particular cell layer of the adrenal cortex. The subsequent activation of the autoreactive T-helper lymphocytes (Th) can lead to the production of various lymphokines, which can induce an activation of both the T-cytotoxic lymphocytes (Tc) and the B lymphocytes, which produce the ACAs and 21-OHAbs (Fig. 6). A localized cytokine released from the infiltrating T cells seems to be the most probable perpetuating cause of the destruction of the adrenal cortex, because the ACAs and/or the 21-OHAbs are not able to induce adrenal cortex damage in vivo and ACTH-receptor blocking autoantibodies were not confirmed (Fig. 6). The main obstacles to a better understanding of the events leading to the occurrence of adrenal cortex autoimmunity are the lack of availability of adrenal tissue from patients with either subclinical or clinical autoimmune AD at onset plus the absence of animal models that spontaneously develop autoimmune AD.

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Figure 6 Pathogenic mechanisms in autoimmune AD (see text for details). (From Ref. 15.)

E. AD Due to Tuberculosis The onset of adrenal insufficiency resulting from tuberculosis is the second cause of AD in developed countries. Patients with tuberculous AD are prevalently male, with a F:M ratio of 0.4. The mean age of these individuals at the onset of this disease is 52 years. Children do not develop this form of AD, as it is a long-standing complication in cases of disseminated tuberculosis. The youngest of the 40 patients we observed with AD due to tuberculosis was a 22-year-old male. There is usually a past history of tuberculosis in these individuals. An infection of certain other organs (skeletal or genitourinary) is often found in patients with AD due to tuberculosis, but active pulmonary disease is not present. Although about half of patients with disseminated tuberculosis have adrenal lesions, only 2% suffer from adrenal insufficiency [36]. The adrenal medulla as well as the cortex can be involved in the process. Calcifications can be identified in 50% of cases by means of an Rx scan. In contrast to cases of autoimmune adrenalitis, the glands are usually enlarged and have calcifications detected by means of CT or NMR (Fig. 5f). Some years after the diagnosis of adrenal insufficiency, the adrenals can become either normal or atrophic [36,203].

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The histology of the adrenals indicates the presence of fibrocaseous tissue with a chronic inflammatory infiltrate plus evidence of a number of giant cells. Mycobacterium tuberculosis can be individuated in the adrenals by using specific types of stains [36]. Patients with tuberculous AD are, in general, negative for ACAs/21-OHAbs (see Tables 4–6). It is our opinion that many of the previously reported ACA-positive findings must have been due either to incorrect diagnoses or to laboratory errors, rather than having been truly positive. In about 12% of patients with tuberculous AD, another autoimmune disease can also be present [92]. F. AD Due to Other Infectious Agents Histoplasmosis, paracoccidioidomycosis, cryptococcosis, coccidioidomycosis, and North American blastomycosis can involve the adrenal glands and cause adrenal insufficiency. In these cases the adrenal glands are enlarged, caseous necrosis may be evident, and they may reveal subcapsular calcifications. Prolonged antifungal treatment may help in the recovery of adrenal function. Moreover, syphilis can sometimes induce adrenal insufficiency; in these cases the adrenals are sclerotic in appearance and have gumma formations [204]. Infections due to cytomegalovirus or the human immunodeficiency virus (HIV) may also produce chronic adrenal damage leading to clinical AD [204,205]. Under certain conditions that induce a drop in immune surveillance, the cytomegalovirus can reactivate itself and cause adrenalitis [206]. Serological tests can help to identify the antibodies to this agent in individuals who are thought to be infected [206]. From a clinical point of view, adrenal insufficiency is rare in HIV patients and usually involves those patients in the advanced stages of the infection who have superimposed infections or who are undergoing treatment with drugs that interfere with adrenolcortical function [207]. G. AD Due to Tumors or Metastases Primary or secondary lymphomas and plasmocytomas are very rarely localized in the adrenals (except for Burkitt’s lymphoma) [36,208]. Many other cancers (breast, lung, gastrointestinal tract, kidney, and melanomas) can spread to the adrenals. Even though at autopsy adrenal metastases are found in about a third of the patients who have died of cancer, this type of infiltration is, in general, asymptomatic [36,204,209]. In all of these conditions CT or NMR can help to individuate the presence of bilateral adrenal masses (Fig. 5g) [203]. A fine-needle agobiopsy using a CT guide can be performed in order to confirm the diagnosis. H. AD Due to Adrenal Hemorrhage or Thrombosis In the past, bilateral massive adrenal hemorrhage (BMAE) was estimated to have been present in up to 1% of unselected autopsies, but with the arrival of new imaging techniques, BMAE is now being reported with increasing frequency in seriously ill patients who undergo abdominal CT scans for different reasons [210]. Subsequent to the description of the first case, in 1857 [211] 430 additional cases of BMAE have been reported in the literature so far [210]. The clinical features of BMAE are, in general, cryptic; they include abdominal pain, fever, aspecific abdominal signs, neuropsychiatric manifestations, and hypotension [210]. Laboratory diagnosis includes the presence of electrolyte changes (see laboratory diagnosis of AD) and signs of occult hemorrhage [210].

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BMAE has been documented in patients suffering from a great variety of conditions, including septicemia, cardiovascular diseases, alcoholism, cancer, chronic disease, surgery, burns, pregnancy, external traumas, invasive procedures, thromboembolic disease, and anticoagulant therapy [210,212]. The first case of the association between coagulation disorders due to the presence of antiphospolipid antibodies and AD was reported in 1988 [213,214]. In 1994 Asherson, in a review of 41 patients, demonstrated that the M:F ratio was 2:1, with a mean age of 42 years. In half of the patients the onset of adrenal vascular occlusion was acute, with severe pain, and sometimes accompanied by ileus, fever, anorexia, lethargy, and nausea. In some cases the onset of the above occurred without abdominal pain but with signs of hypoadrenalism; other patients presented with the typical features of chronic AD, and in three patients the adrenal involvement was part of a multiorgan failure, defined as the ‘‘catastrophic anti-phospholipid syndrome’’ [212]. The typical changes identified by means of CT are a marked enlargement of the adrenals, which appear hyperdense. This is then followed by a reduction in the size of the glands in subsequent scans [212]. Figure 5h shows adrenals in a patient with AD and bilateral massive adrenal hemorrhage in the presence of antiphospholipid antibodies. The Waterhouse-Friderichsen syndrome is an acute adrenal hemorrhage resulting from septicemic shock caused by the presence of Neisseria meningitidis or other microorganisms (Haemophilus influenzae, Pseudomonas aeruginosa, Escherichia coli, pneumococci), which can induce an acute adrenal insufficiency. The shock that occurs in the course of cases of endotoxiemia seems to be the cause of this adrenal hemorrhage [209]. I. AD Induced by Drugs A number of drugs (aminoglutethimide, etomidate, ketoconazole, metyrapone, and suramin) may cause AD by inhibiting cortisol biosynthesis [204] while others (phenytoin, barbiturates, and rifampin) can accelerate cortisol metabolism [215]. These agents do not usually produce clinical adrenal insufficiency in subjects with normal hypothalamicpituitary-adrenal function. J. AD Due to Infiltrative Diseases Chronic adrenal failure may also result from certain metabolic disorders: 1. Systemic amyloidosis induces the infiltration of every organ and can also affect the adrenals. The glands are either of a normal size or are enlarged. The demonstration of amyloid deposition in the mucosa of the gastrointestinal tract can be important as regards the diagnosis of this disorder [209]. 2. Hemochromatosis is a disease related to iron storage. Many organs, including the adrenals, can be affected by chronic iron deposition. The clinical manifestations, in addition to adrenocortical insufficiency, include cirrhosis, a bronze skin pigmentation, diabetes mellitus, and cardiomyopathy. The serum levels of both iron and ferritin are elevated, and urinary iron escretion is markedly increased [209,216]. Adrenal imaging demonstrates hyperdense glands of a normal or reduced size [203]. 3. Sarcoidosis of the adrenal gland has been reported as a possible cause of AD. However, from a review of 10 cases in the literature it emerges that a causal relationship between sarcoidosis and AD has not been clearly established, since

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adrenal failure is more likely to be a result of either autoimmunity or tuberculosis in these patients [217]. 4. Adrenal enlargement has also been noted in Niemann-Pick disease, having large vacuolated cells and a reduction in the number of compact cells and, in some cases, of the mucopolysaccharides [36]. 5. In lysosomal storage disease (or Wolman disease), which is a rare abnormality of lipid metabolism due to a deficiency in lysosomal lipase activity, there is an accumulation of cholesterol esters and triglycerides, together with cellular damage, particularly within the inner zones of the cortex, plus evidence of calcification. The disease affects a variety of organs and is usually fatal in the first 6 months of life [36]. K. AD Following Surgical Procedures Bilateral adrenalectomies carried out on patients suffering from Cushing’s syndrome are the most frequent surgical cause of adrenal insufficiency. L. Primary AD Due to Genetic Disorders Rare disorders (such as hypoplasia of the adrenal gland, adrenal hemorrhage due to trauma at birth, or maternal Cushing’s disease) may be responsible for the onset of congenital adrenal insufficiency [204,209,218]. The genetic disorders associated with hypoadrenalism (Table 1) are discussed below. 1. Adrenoleukodystrophy Adrenoleukodystrophy (ALD), also known as brown Schilder’s disease, is a hereditary X-linked disorder that affects about 1/20,000 males [219]. The disease is associated with elevated levels of circulating very long-chain fatty acids (VLCFA), which are the markers of this disease [220]. A progressive accumulation of VLCFA at the level of the target organs leads to damage in either the central or the peripheral nervous system or in the adrenal cortex. This syndrome is caused by mutations in a gene located in the terminal segment of the Xq28 chromosome [221]. In male population adrenal failure can manifest itself at any age, but it is often present before the age of 15. The most common neurological presentation (45%) is a rapidly progressive cerebral ALD in childhood, which manifests itself before the age of 10 and leads to severe disability and death within a few years. The slowly progressive adult type (35%), also known as adrenomyeloneuropathy, presents most commonly between the ages of 20 and 40 and affects the spinal cord. A small proportion of ALD patients can manifest adrenal failure without the involvement of the nervous system. ALD is the third most frequent etiological cause of an AD unassociated with either autoimmunity or tuberculosis in males. Therefore, a diagnosis of ALD should consider regarding all males as having primary adrenal failure if the adrenal cortex autoantibodies are negative [222]. Up to 20% of heterozygous females also develop a late-onset, mild form of neurological disorder resembling adrenomyeloneuropathy [219]. Cases of overt adrenal failure in these females are rare, but in many cases a subclinical glucocorticoid deficiency following ACTH-test stimulation can occur [223]. The pathology of the adrenals in these cases reveals that the zona glomerulosa is normal, but that the zona fasciculata contains groups of degenerated cortical cells, which, with the progression of the disease, can affect the whole adrenal cortex in the absence of any type of lymphocytic infiltration [36,219]. As

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the disease progresses, the adrenal glands undergo a gradual process of atrophy, so that they may show up as either slightly or markedly atrophic at NMR [220]. NMR of the brain often reveals symmetrical demyelinations in the parieto-occipital region. 2. Adrenal Hypoplasia Congenita Adrenal hypoplasia congenita (AHC) is an X-linked, recessive disorder affecting 1 per 12,500 births, which reveals primary adrenal insufficiency due to a failure in the development of the layers of the adrenal cortex [219]. In addition to hypoadrenalism, these patients can also have delayed puberties resulting from hypogonadotrophic hypogonadism. The adrenal glands are small in these cases [36]. The locus for AHC has been mapped to the Xp21 chromosome. The AHC gene has been defined as a DAX-1 (a dosage-sensitive sex reversal–AHC critical region) and can be identified on the X chromosome. More than 30 different types of DAX-1 gene mutations have already been described [219]. 3. Adrenal Hypoplasia as Part of a Contiguous Gene Deletion Syndrome Another form of adrenal insufficiency is characterized by certain typical clinical manifestations: faces with hyperthelorism and strabism and drooping mouths, muscular dystrophy, short stature, anorchia, or cryptorchidism. This an X-linked disease associated with a glycerol kinase deficiency, and the genetic locus has been mapped to the Xp21 chromosome [224]. 4. Kerns-Sayre Syndrome The Kerns-Sayre syndrome is a mitochondrial cytopathy caused by deletions in the mitochondrial DNA, which, in addition to causing adrenal insufficiency, is characterized by certain neurological manifestations and endocrine deficiencies [219]. 5. Corticotropin Resistance Syndromes Corticotropin (ACTH) resistance syndrome is another genetic disease associated with adrenal insufficiency consisting of the following disorders: familial glucocorticoid deficiency and triple A syndrome [219,225,226]. Familial glucocorticoid deficiency is a rare, autosomal, recessive disorder characterized by a failure to thrive, hypoglycemia, pigmentation, and recurrent infections. Mutations in the ACTH receptor gene have been detected in about 40% of patients with this disorder [219]. The disease results in an absence of the zonae fasciculata and reticularis, inducing both high levels of ACTH and low levels of cortisol, without any impairment in mineralocorticoid production. The triple A syndrome is characterized by adrenocortical failure, achalasia, and alacrimia, and is an autosomal, recessive disorder associated with mutations in a gene on the 12q13 chromosome. The incidence of this syndrome is 1 in 20,000 [219]. 6. Congenital Adrenal Hyperplasia Congenital adrenal hyperplasia (CAH) consists of a group of autosomal, recessive disorders and is due to a defect in one of the enzymes involved in the steroidogenesis process [36]. The most common of these disorders is 21-hydroxylase deficiency, which is present in about 90% of cases of CAH. The gene that encodes 21-hydroxylase is located in the class III HLA on chromosome 6p21 [219]. The occurrence of 21-hydroxylase deficiency produces a variety of defects in both cortisol and aldosterone synthesis, because this

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enzyme catalyzes the conversion, in the glucocorticoid pathway, of 17-hydroxyprogesterone to 11-deoxycortisol and, in the aldosterone pathway, of progesterone to deoxycorticosterone. These defects induce a chronic increase in the production of ACTH. The hyperproduction of ACTH induces an overproduction of steroid precursors—in particular, of 17␣hydroxyprogesterone. This chronic adrenal stimulation can lead to adrenal hyperplasia and to the hyperplasia of congenital adrenal tissue in the testis. NMR can reveal glands that manifest from a normal to a diffusely hyperplastic state (Fig. 5i). There are three different forms of 21-hydroxylase deficiency: classical salt wasting, milder classical simple virilizing, and nonclassical or late-onset. Among other enzyme defects, only 3␤-hydroxysteroid dehydrogenase deficiency is associated with adrenocortical failure [219]. Lipoid CAH is the most severe form of CAH, characterized by an impaired synthesis of all the adrenal and gonadal steroid hormones, and is caused by mutations in the steroidogenic acute regulatory protein (StAR) gene, which is located on the 8p11 chromosome. 7. Smith-Lemli-Opitz Syndrome The Smith-Lemli-Opitz syndrome results from mutations in the sterol-␦-7 reductase gene, which catalizes the final step in cholesterol biosynthesis process and leads to the occurrence of primary adrenal insufficiency. The incidence of this disorder is 1 in 20,000–30,000. The syndrome can present together with mental retardation, microcephaly, congenital cardiac abnormalities, syndactyly, polydactyly, and the incomplete development of the male genitalia in young boys [224]. IV. SECONDARY AND TERTIARY ADRENAL INSUFFICIENCY The prevalence of secondary adrenal insufficiency is quite rare. The causes are listed in Table 18. V. CLINICAL PRESENTATION OF ADRENOCORTICAL INSUFFICIENCY The clinical manifestations of AD depend on the type of adrenocortical insufficiency (primary or secondary), on the modality of the onset (acute or chronic), and on the stage

Table 18 Causes of Secondary Adrenocortical Insufficiency Pituitary tumors (primary or metastatic) Craniopharyngioma Pituitary surgery Pituitary traumas Pituitary radiation Pituitary autoimmunity (lymphocytic hypophysitis) Empty sella syndrome Sheehan’s syndrome (postpartum pituitary necrosis) Pituitary infiltrative processes (sarcoidosis, histiocytosis X, TBC), amyloidosis Hypothalamic tumors

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Table 19 Symptoms of Adrenocortical Insufficiency Manifestations Weakness Anorexia Gastrointestinal Salt craving Myalgias Neuropsychiatric

Incidence (%) 100 100 50–70 10–20 10–20 10–15

of life at onset (congenital or acquired). Furthermore, the clinical manifestations of AD are often superimposed on, or associated with, other disorders. A. Chronic Primary Adrenocortical Insufficiency The symptoms of primary adrenocortical insufficiency are listed in Table 19. The general symptoms include weakness, fatigue, anorexia, myalgias and arthralgias, gastrointestinal symptoms including abdominal cramps, nausea, vomiting, and diarrhea [204]. These patients sometimes feel an urgent need to eat salt. Depression and psychosis may also be present in most of these patients [204]. The signs of AD are listed in Table 20. The weight loss is due to anorexia, deyhydratation, and vomiting. The blood pressure is low, and postural hypotension is present. The most specific sign of primary AD is hyperpigmentation of the skin, the nails, and the mucosal surfaces; this symptom is present only in patients with chronic primary AD. Based on the study of the natural history of autoimmune AD, we have observed that the skin hyperpigmentation usually tends to appear many months after the increase in ACTH levels (see Table 8). High levels of ACTH in the plasma stimulate the melanocytes to synthesize melanin. This hyperpigmentation of the skin is most evident in the normally hyperpigmented areas (e.g., the areolae or the genitalia), in the areas exposed to the light (e.g., the face), or in the areas that are prone to traumas (e.g., the elbows); also, the areas exposed to traumas in the mucosae become pigmented. This type of hyperpigmentation is also evident in the palmar creases. Any scars already in existence before the onset of the adrenal failure remain unpigmented, whereas those recently acquired become pigmented. B. Secondary and Tertiary Adrenocortical Insufficiencies Most of the clinical symptoms of secondary adrenocortical insufficiency are similar to those of primary AD, but the signs differ because the hypotension in the secondary form

Table 20 Signs of Adrenocortical Insufficiency Manifestations Weight loss Orthostatic hypotension (most evident in primary) Hyperpigmentation (present only in primary)

Incidence (%) 100 100

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is less pronounced, because the mineralcorticoids are in general not greatly impaired, and hyperpigmentation is absent because the levels of ACTH are either normal or low [14,201]. C. Acute Adrenocortical Insufficiency (Adrenal Crisis) An adrenal crisis can present as a shock. In general, it is refractary to therapy if glucocorticoids are not introduced as part of the therapy. The crisis can arise either in a patient with undiagnosed AD, in a patient taking a low pharmacological dose who is submitted to stress (e.g., surgical, infectious, pregnancy, dyarrhea), or in a patient who has WaterhouseFriderichsen syndrome [204].

VI. LABORATORY DIAGNOSIS The main data concerning this subject are summarized in Table 21. Hyponatremia, hypochloremia, and hyperkalemia, together with a reduction of plasmatic osmolality, are the characteristics of adrenal insufficiency. Both hypercalcemia and an hypercreatininemia can also be present. The hemochrome reveals the presence of eosinophilia in conjunction with both lymphocytosis and anemia; increased levels of thyrotropin can also be present [204]. The morning level of ACTH is increased and that of cortisol is reduced in cases of primary adrenal failure, whereas cortisol levels are reduced, together with either a normal or low level of ACTH, in the secondary forms of adrenal insufficiency. Aldosterone plasma levels are low and plasma renin activity is increased (in primary adrenocortical insufficiency). Testosterone levels are reduced among female patients. In patients without any clinical signs of adrenocartical insufficiency but where ACAs/21-OHAbs are present, an ACTH test using either high or low doses of synthetic ACTH is carried out in order to reveal a subclinical adrenal dysfunction [14,70,72,73]. Patients with clinical AD should be examined for evidence of ACAs and/or 21OHAbs. If they prove to be negative and are males, an evaluation of VLCFA levels should also be performed. If VLCFA are absent, tests for antiphospholipid antibodies and antinuclear antibodies, for amyloidosis, for HIV, for cytomegalovirus, for the presence of antibodies to fungi, and for hemochromatosis should be carried out on these patients.

Table 21 Laboratory Findings in Patients with AD Laboratory findings

Frequency (%)

Hyponatremia Hyperkalemia Hypochloremia Hypercalcemia Hyperazotemia Increased TSH levels Anemia Eosinophilia Lymphocytosis

100 50–70 60–80 10–15 50–60 30 40–50 10–15 10–15

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VII. GENETIC STUDY Genetic study has already been discussed in the relevant sections of this chapter concerning the various forms of primary adrenocortical insufficiency (see above). VIII. IMAGING In patients with primary AD, the adrenal glands should be examined using CT or NMR. In cases of secondary AD, the pituitary should be studied by means of NMR. These techniques can provide important information about the etiology of AD. The characteristic adrenal patterns of the various forms of primary AD have discussed above, and Fig. 5 shows images of adrenals in patients with primary clinical and preclinical AD. IX. THERAPY In cases of adrenal crisis, therapy consists of saline infusions, with i.v. injections of glucocorticoids (hydrocortisone, dexamethazone) every 6–12 hours depending on the half-life of the drug, until the patient’s hydroelectrolytes are balanced. When the patient’s condition has improved and the symptoms have been stabilized, an oral dose of 20–30 mg/day of hydrocortisone or 25–50 mg/day of cortisone acetate (divided into two or three doses) can be given. It is important to use the smallest dose that relieves the patient’s symptoms. Patients with primary adrenal insufficiency also require 50–100 ␮g/day of fludrocortisone given in one oral dose. The problems connected with overreplacement therapy include the appearance of symptoms and signs of an iatrogenic Cushing’s syndrome. Patients should increase the levels of the oral therapy in case of stress events (e.g., infections, surgical procedures, dental care, dehydratation), but parenteral glucocorticoid therapy should follow where vomiting occurs. A double-blind study of replacement therapy for adrenal androgens (dehydroepiandrosterone), involved 24 women with adrenal insufficiency. After the patients had taken 50 mg of dehydroepiandrosterone orally each morning for 4 months, this drug significantly improved some aspects of their sexuality [227]. X. ETIOLOGICAL DIAGNOSIS OF AD The presence of ACAs and/or 21-OHAbs, mainly at high titers, has become the gold standard for the diagnosis of autoimmune AD. Imaging of the adrenal glands can confirm the diagnosis of autoimmune AD, revealing the presence of normal/atrophic glands. Following diagnosis, further investigation should be carried out in order to identify in patients with autoimmune AD the presence or absence of other clinical autoimmune diseases (e.g., chronic thyroiditis, Graves’ disease, type 1 diabetes mellitus, amenorrhea, chronic hypoparathyroidism, vitiligo, alopecia, pernicious anemia, atrophic gastritis, autoimmune hepa䉳 Figure 7 Etiological flowchart of adrenocortical insufficiency. (From Ref. 15.) PA ⳱ antiphospholipid antibodies; VLCFA ⳱ very long–chain fatty acids; ACA ⳱ adrenal cortex autoantibodies; 21-OH Abs ⳱ 21-hydroxylase autoantibodies; ANA ⳱ antinuclear antibodies; APS ⳱ autoimmune polyendocrine syndrome.

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titis, primary biliary cirrhosis) or other correlated diseases (e.g., chronic candidiasis, malabsorption, keratoconjunctivitis, ectodermal dystrophy, asplenia, cholelithiasis, cancer). In addition, all patients with autoimmune AD should be tested for evidence of the main organ- and non–organ-specific autoantibodies. This approach makes it possible to identify whether a patient with autoimmune AD is affected by either a clinical or subclinical or potential APS-1, APS-2, APS-4 or an isolated AD. Finally, a genetic study should be carried out on these patients in order to identify the class II HLA genes, as regards APS2, APS-4 and isolated AD, and the AIRE gene mutations, with regards to APS-1. A search for either clinical or latent AD and other autoimmune diseases should be carried out among the first-degree relatives of patients with AD. In the cases that prove to be negative for ACAs/21-OHAbs, a CT or NMR scan of the adrenal glands should be performed in order to obtain a differential diagnosis. These techniques have greatly improved the diagnosis of nonautoimmune primary AD. A test for VLCFA (principally in male patients) is needed in patients with either normal or atrophic adrenals. If this test is positive, a diagnosis of adrenoleukodystrophy is usually made. In this case, the neurological manifestations typical of this disease should be looked for in both the patients and their relatives, and an NMR of the brain should be performed. In the presence of a marked enlargement of the adrenal glands, either with or without calcifications, one of the following conditions needs to be considered: tuberculosis, other types of granulomatosis, amyloidosis, and either primary or metastatic adrenal cancer. A detailed personal history should be taken for all such cases, and the markers of neoplasias, the serological markers of infections and a Mantoux intradermoreaction, should be investigated. In addition, an NMR of the abdomen and the chest should be performed. If necessary, a CT-guided fine needle biopsy can be performed. If amyloidosis is suspected, a biopsy of the rectal mucosa should then be performed. If the adrenal enlargement is due to an adrenal hemorrhage or to an infarction, tests for SLE or other collagen diseases, antiphospholipid syndrome and other coagulation disorders, and information about the use of drugs that modify blood coagulation will be also necessary. If a small/normal, hyperintense adrenal gland is present, hemochromatosis can be ruled out and determinations regarding both sideremia and transferrinemia should be carried out and the necessary genetic studies set up. Furthermore, the presence of clinical manifestations of the disease in other organs must be investigated. With reference to cases of either congenital or young patients with primary AD, the other clinical manifestations present in these rare conditions should be investigated. Based on this information, specific genetic studies may be useful to reach a diagnosis. A flowchart for the diagnosis of primary AD is presented in Fig. 7. XI. CONCLUDING REMARKS In recent years considerable advances have been made in both the diagnosis of and prognosis for AD. With regard to cases of autoimmune AD, the discovery of the main adrenal and gonadal autoantigens has permitted the development of more sensitive assays for the measurement of adrenal cortex and gonadal autoantibodies. These tests allow for better identification of those patients with autoimmune AD, together with premature autoimmune ovarian failure. Furthermore, these tests allow for the screening of subjects at risk of developing autoimmune AD (i.e., first-degree relatives, children with chronic candidiasis or chronic hypoparathyroidism, or patients with other autoimmune diseases). Cases of potential or subclinical AD can be better individuated, predicted, and prevented by means

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of this approach. Furthermore, the use of CT or NMR provides a more detailed evaluation of the adrenal glands in the cases of primary adrenal insufficiency. The identification of AIRE gene mutations among different populations allows for the identification in hot spot areas of the disease the health carriers among unaffected family members of APS-1 patients or in the general population. Furthermore, the study of the mutated proteins of the AIRE gene will allow for a better understanding of the mechanisms of self tolerance and autoimmunity. Finally, the recent identification of various genes that induce adrenocortical failure has great implications regarding both clinical and prenatal diagnoses. ACKNOWLEDGMENTS I would like to thank all those who have cooperated with me over the years regarding the study of the clinical, genetic, immunological, morphological, and endocrine aspects of AD. The author is also grateful to Mrs. Helen Maguire for her assistance in reviewing this manuscript. This work was partially funded by a grant from MURST (Ministero dell’Universita` e della Ricerca Scientifica e Tecnologica) 2001–2003 Roma, Italia. Sulla Eziopatogenesi del Morbo di Addison. REFERENCES 1. Eustachi B, Fallopio G. Tabulae Anatomicae Opuscola Anatomica. Venezia 1563. 2. Addison T. On the constitutional and local effects of disease of the suprarenal capsules. In a collection of the published writings of the late Thomas Addison, M.D., physician to Guy’s Hospital 1855. London: New Sydenham Society, 1868. 3. Brown-Sequard CE. Recherches experimentales sur la physiologie e la pathologie des capsules surrenales. Acad Sci Paris 1856; 43:422–425. 4. Trousseau A. Bronze Addison’s disease. Arch Gen Med 1856; 8:478–485. 5. Cushing HW. The Pituitary Body and its Disorders. Philadelphia: JB Lippincott, 1912. 6. Harris GW. Neural control of pituitary gland. Physiol Rev 1948; 28:139–179. 7. Hartman FA, MacArthur CJ, Hartman WE. Substances which prolongs life of adrenalectomized cats. Proc Soc Exp Biol Med 1927; 25:69–70. 8. Stewart GN, Rogoff JM. The influence of extracts of adrenal cortex on the survival period of adrenalectomized dogs and cats. Am J Physiol 1929; 91:254–264. 9. Swingle WW, Pfiffner JJ. Preparation of an active extract of suprarenal cortex. Anat Rec 1929; 44:225. 10. Rowntree LG, Greene CH, Swingle WW. The treatment of patients with Addison’s disease with the ‘‘cortical hormone’’ of Swingle and Pfiffner. Science 1930; 72:482–483. 11. DeFremery P, Laqueur E, Reichstein T, spanhoff RW, Uyldert JE. Corticosterone, a cristallized compound with the biological activity of the adrenocortical hormone. Nature 1937; 139: 26. 12. Steiger M, Reichstein T. Desoxycorticosterone (21-oxyprogesterone aust-3-oxy-atio cholensa¨ure). Helv Chem Acta 1937; 20:1164–1179. 13. Simpson SL. The use of synthetic desoxycorticosterone acetate in Addison’s disease. Lancet 1938; 2:557–558. 14. Oelkers W. Adrenal insufficiency. N Engl J Med 1996; 335:1206–1212. 15. Betterle C, Dal Pra C, Mantero F, Zanchetta R. Autoimmune adrenal insufficiency and autoimmune polyendocrine syndromes: autoantibodies, autoantigens, and their applicability in diagnosis and disease prediction. Endocrine Rev 2002; 23:327–364. 16. Eason RJ, Croxon MS, Perry MC, Somerfield SD. Addison’s disease, adrenal autoantibodies and computerized adrenal tomography. NZ Med J 1982; 95:569–573.

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25 Premature Ovarian Failure ANNEMIEKE HOEK Academic Hospital Groningen, Groningen, The Netherlands HEMMO A. DREXHAGE Erasmus Medical Center, Rotterdam, The Netherlands

I. INTRODUCTION One of the important functions of the immune system is the discrimination between self and nonself, or perhaps better between danger and nondanger. Currently, such discrimination is thought to be made through a series of complicated and multistep interactions between various cells and components of the immune system. Immune cells sometimes erroneously establish an immune reaction towards self during conditions of apparent nondanger. If such immune reactions are so aberrantly and vigorously self-directed, they may inflict pathological damage on tissues. So-called autoimmune diseases are the consequence. Autoimmune diseases can be divided into two main categories: organ-specific and systemic. In organ-specific autoimmune diseases, the immune attack is confined to one organ or organ system, while in systemic autoimmune diseases the damage is widespread and often the consequence of immune complex formation. In the majority of organ-specific autoimmune diseases, target tissues are of endocrine character; hence this category of autoimmune disease is often referred to as endocrine autoimmune disease. There is evidence that some cases of premature ovarian failure (POF) are endocrine autoimmune diseases due to a faulty recognition of self in the ovary by the immune system. POF is a syndrome characterized by menopause before the age of 40 years. The patients suffer from anovulation, leading to hypoestrogenism and infertility. The etiology of the syndrome is heterogeneous. Karyotypic abnormalities, such as numerical and structural defects of the X chromosome, genetic defects, prenatal mumps infection, and metabolic disorders, have all been described as inducing POF due to oocyte destruction or depletion of ovarian follicles. Also, chemotherapy, especially multidrug regimes and radia537

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tion, are well known causes of iatrogenically induced POF. An autoimmune etiology of POF is established in cases of oophoritis and concomitant Addison’s disease. These cases of POF can be placed in the group of endocrine autoimmune diseases like autoimmune thyroiditis, insulin-dependent diabetes mellitus (IDDM), and Addison’s disease. There remains, however, a group of POF cases without a known etiology, and these are referred to as idiopathic. The purpose of this chapter is to further evaluate the autoimmune phenomena in idiopathic POF cases and to describe the evidence for the autoimmune pathogenesis in POF cases with concomitant Addison’s disease. II. DEFINITION AND CLINICAL PRESENTATION OF POF Women in western countries experience menopause at an average age of 50 years [1–3]. The cumulative risk of natural menopause is based on age-specific incidence rates. The annual incidence rates of natural menopause per 100,000 person-years were 10 for ages 15–29 and 76 for ages 30–39. In the age group 40–44 the incidence of natural menopause increases sharply to 881 per 100,000 person-years. The age that separates premature from normal menopause is by definition 40 years and thus arbitrary. POF was defined by de Moraes-Ruehsen and Jones in 1967 [4] as an unphysiological cessation of menses before the age of 40 years and after puberty (secondary amenorrhea). The incidence of POF in a population under the age of 40 years is estimated to be 0.9% [5]. Women with POF have a hypergonadotropic-hypoestrogenic hormone profile. Patients with POF are mainly troubled by infertility due to the cessation of ovarian function. They have a typical menstrual history of normal age at menarche [6,7] followed by regular periods. The disease thereafter presents either with oligomenorrhea or abrupt amenorrhea such as is seen after pill-stop [8–10]. A family history of POF is incidentally obtained [10–13]. Fifty percent of patients with POF experience vasomotor symptoms, such as hot flushes and sweating boosts [10,14,15], due to the hypoestrogenic status. Other troubling symptoms are atrophy of the vagina and the urological tract, leading to vaginitis, dyspareunia, and cystitis. The diagnosis of POF rests upon the clinical picture and the demonstration of elevated gonadotropin levels. The level of follicle-stimulating hormone (FSH) is higher than that of luteinizing hormone (LH) [16]. Serum levels of FSH greater than 40 IU/L are the hallmark of the diagnosis. POF presents itself not as an all-or-none phenomenon, and the precise timing of onset is often impossible. The disease may have a fluctuating course, with high gonadotropin levels that return to normal some time later, and a later regain of ovulatory functions and even pregnancy [17–22]. Nelson et al. [23] examined 65 POF patients by weekly estradiol sampling and sonography. In 50% of the cases follicular activity could be demonstrated, and 16% of cases regained ovulatory function. Follicle biopsies were carried out in 6 patients, and these showed luteinized Graafian follicles. A systematic review evaluating spontaneous pregnancies and pregnancies after therapeutic interventions revealed a 5–10% chance to conceive following diagnosis [24]. III. THE VARIOUS ETIOLOGIES OF POF POF is a heterogeneous disorder with a multicausal pathogenesis, and chromosomal [25–32], genetic [11–13,33–40], enzymatic [41,42], iatrogenic [43–48], infectious [49,50], or autoimmune (AIRE gene) [51,143,144] aberrations may all form the basis for the disappearance of ovarian follicles (see Table 1). These aberrations may hit the ovary

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Table 1 Etiology of POF Ref. Iatrogenic causes

Infections Metabolic causes Chromosomal causes

Genetic causes

Autoimmune causes

Radiation Chemotherapy Surgery Mumps Galactosemia XO-Turner syndrome mosaicism XXX female Deletions Xq13–Xq26 FMR1: premutation fragile X syndrome BEPS1: blepharophimosis epicantus inversus syndrome ATM: ataxia telangiectasia Mutations in the FSH-receptor gene Associated with mutated AIRE gene on chromosome 21: APECED Associated with adrenal autoimmunity

43–48

49,50 41,42 25–32

11–13, 33–40

51, 143, 144

Idiopathic Source: Adapted from Ref. 24.

at any stage of life, including the prepubertal, pubertal, or reproductive life span [52]. Despite the extensive list of known etiologies of POF (Table 1), such etiologies are rare, and the majority of cases are idiopathic. Kinch et al. [53] the first to identify two histopathological types of POF: the afollicular form, where there is a total depletion of ovarian follicles/oocytes and hence a permanent loss of ovarian function, and the follicular form. Although this histological classification suggests a sharp division between the follicular and afollicular forms, there is evidence that some cases of POF originally of the follicular type may progress to an afollicular stage. This is particularly the case in blepharophimosis [35–37], galactosaemia [41,42], and in the animal models of autoimmune oophoritis (see later). In the follicular form, follicular structures are still preserved, and hence a possibility of either spontaneous or induced return of ovarian function exists. The follicular form can be subdivided into oophoritis (inflammation of follicles), ovaries with a few follicles present, and ovaries in which numerous primordial follicles are present—the so-called resistant ovary syndrome (ROS) [54]. Some ROS cases are presently considered as due to FSH receptor mutations, but such mutations are rare and mainly found in the Finish population [40]. Hence we are left with a considerable number of cases of POF that are idiopathic, and ovarian autoimmunity is a possible cause of both the afollicular and oophoritis forms of such idiopathic cases. IV. LESSONS FROM ANIMAL MODELS TO CONSIDER SOME FORMS OF IDIOPATHIC POF AS ENDOCRINE AUTOIMMUNE DISEASES Before giving the pros and cons for considering POF an autoimmune disease, it must be noted that similar failures of endocrine organs are nowadays classified as due to autoim-

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mune destruction. These endocrine failures include autoimmune hypothyroidism (thyroid failure), type 1 diabetes (failure of the islets of Langerhans), and most forms of idiopathic Addison’s disease (failure of the adrenal cortex). There are ethical and technical restrictions to studying the etiology and pathogenesis of such autoimmune diseases in humans. Reliance on animal models is in part a recognition of the primacy of patient safety—primum non nocere—first do no harm [55]. In humans the endocrine organ–specific autoimmune diseases often have sub- or nonclinical prodromal phases, which are difficult to study since signs and symptoms are virtually absent. In animal models these studies can be done. Unlike humans, animals with endocrine organ–specific autoimmune diseases can be bred to study and manipulate inheritance. They can be biopsied and autopsied. Their genome can be altered. Therapies to prevent or reverse the disease can readily be tested. Over the past 50 years a plethora of animal models of various endocrine organ–specific autoimmune diseases have been developed. These animal models have greatly contributed to the knowledge concerning the etiology and the pathogenesis of endocrine organ–specific autoimmune diseases and their possible prevention and treatment. A word of caution is, however, necessary when trying to extrapolate data obtained in these animal models to the human situation. The animal models clearly show a caricature of the more complex human disorder. The animal disease is often studied in specifically inbred animals to generate homogeneous and extreme forms of the autoimmune diseases. In this way the disease will not only differ from the human disorder but also between various animal models. Hence, general conclusions drawn from studies in one of the animal models should always be verified in other animal models and in patients. Recent studies in the animal models, patients, and their relatives have culminated in the awareness that endocrine organ–specific autoimmune diseases must be regarded as polygenic diseases, in which the penetrance of a combination of genes is strongly influenced by environmental factors (Fig. 1). First, multiple genes determine part of the aberrant immune response towards self. The most important genes are those in the MHC region [56–58]. However other genes are also involved, including those with a role in the regulation of the immune response in general, e.g., the CTLA-4 gene [59], genes determining aberrancies in the target gland eliciting the abnormal self response [60,61], genes playing a role in the sensitivity of the target gland to the autoimmune attack [62], and genes controlling T-cell development and differentiation [63,64]. However, genetic polymorphisms or mutations are clearly not always explaining the etiology. Monozygotic twin studies, for example, have shown a concordance rate ranging from 80% for thyroid autoantibody positivity [65], 30–40% for type 1 diabetes [66], to a meager 20% for Graves’ disease [67]. This demonstrates the important role of environmental eliciting factors in the development of these diseases. Efforts to understand more precisely the etiopathogenesis of endocrine organ–specific autoimmune diseases in humans have involved animal models of the diseases that develop spontaneously, or are induced by either environmental perturbations or by genetic manipulations (transgenes and knockouts). A. Spontaneous Animal Models of Endocrine Autoimmune Diseases 1. The BB-DP Rat The BB-DP rat is primarily a model for autoimmune diabetes [55], and inbred BB-DP rats develop spontaneously a T-cell–dependent, ketosis-prone diabetes that is clinically very similar to type 1 diabetes in humans. BB-DP rats are special in that they have a

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Figure 1 The combined effects of several genes and environmental factors must act together to bring about a disturbed mutual interaction between various subtypes of immune cells and of these cells with target cells to elicit an endocrine organ–specific autoimmune disease.

profound T-cell lymphopenia. The lymphopenia is a recessive trait, and the animals are lymphopenic from birth due to a mutation in one of the immune-associated nucleotide (Ian) genes, i.e., the Ian-5 gene (idd1) on rat chromosome 4 [64]. The diabetes develops in most DP lines at around the age of 8–12 weeks, and there is no sex difference. There also exist sublines of the BB-DP rats that lack the Ian-5 mutation in idd1 and are not lymphopenic and do not develop diabetes. These lines are referred to as diabetes resistant, or BB-DR. The peripheral lymphopenia of the BB-DP rat is primarily due to a lack of RT6Ⳮ T cells. RT6 is a marker for regulatory T cells. Transfers of RT6Ⳮ T cells from BB-DR rats to BB-DP rats prevent disease [68]. Although diabetes-resistant BB-DR rats are sufficient in RT6Ⳮ T cells, they are still prone to diabetes: infection with Kilham rat virus (KRV) is a known inducer of autoimmune diabetes in these rats [69]. The virus does not infect islet cells, but the macrophages of the animal. This perturbs the immune system of the BB-DR rats, resulting in changes in the balance from Th2 to Th1 mechanisms [70]. Also, treatment with poly I:C induces diabetes in these rats. BB-DP rats also suffer from a form of focal lymphocytic infiltration that under normal conditions does not lead to hypothyroidism [71]. Yet thyroid failure becomes apparent after hemi-thyroidectomy of the animals. Aggravation of focal infiltrations can also be observed when the animals are fed a high-iodine diet [72,73]. The thyroiditis is genetically linked to the MHC class II RT1u rather than to the Ian-5 gene mutation [74]. Oophoritis, to our knowledge, does not occur in BB-DP rats.

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2. The NOD Mouse The NOD mouse is predominantly studied for its diabetes and sialo-adenitis, [75]. The NOD mouse has been extensively studied since the 1980s. NOD mice develop at an early age (from 5 weeks of age onwards) an initially non-destructive peri-insular infiltration of dendritic cells, accessory macrophages, T cells, and B cells that persists for several weeks before it develops into a destructive form of insulitis (from 12 weeks of age onwards). Mild diabetes follows. Typically female mice are more severely affected. There are many genetic loci (over 15) on different chromosomes that associate with diabetes and/or insulitis and/or sialoadenitis in the NOD mouse. The most important diabetic loci (idd1 loci) in the mouse are linked to the MHC complex; NOD mice express an unique I-A locus, i.e., I-Ag7 (histidine as residue number 56 and serine as residue 57, homologous to ‘‘diabetogenic’’ HLA-DQ ␤ nonaspartic acid 57 containing alleles in the human), but lack expression of I-Ea (homologous to DR ␣ in humans) [56,57]. Idd1 is not related to sialoadenitis development; here Idd5 and Idd3 are thought to play a prime role, but data are inconsistent [76,77]. There exists a subline of NOD mice that, under normal dietary conditions, exhibits a prevalence of around 5% thyroiditis, but when kept on a continuously high-iodine diet spontaneously develops autoimmune thyroiditis in virtually all animals [78]. This subline is characterized by an alternative MHC haplotype, namely the I-Ak allele instead of the I-Ag7 on the NOD background; the mice are called NOD-H2h4 mice. In the majority of regular NOD strains there is only occasionally an association of diabetes with thyroid infiltrations. In general, the incidence of thyroiditis is very low in the regular NOD mouse [79]. But again, certain dietary iodine regimens (leading to thyrocyte necrosis) have a triggering effect on thyroiditis development [79]. This shows the importance of a local factor (high antigen release, necrosis) in combination with a dysregulated immune system (NOD mouse background) in the development of at least this endocrine organ–specific autoimmune disease [79,80]. To our knowledge there are no reports on the presence of oophoritis in the NOD mouse. 3. Obese Strain Chickens One of the oldest models of endocrine organ-specific autoimmune disease is the obese strain (OS) chicken, which suffers from a form of lymphocytic thyroiditis with a rapid onset of hypothyroidism, which resembles severe Hashimoto’s disease of humans in many clinical, histopathological, serological, and endocrinological aspects [81]. The first genetic theory of endocrine organ-specific autoimmunity as a polygenic trait was proposed by Cole, based on breeding studies with this bird [82]. Approximately three genes encode a susceptibility of the thyroid to attack by the immune system (one of them recessive), and the remaining one or two genes encode a hyperreactivity of the immune system [62]. Iodine levels in food are an important environmental factor in the development of the thyroiditis in the OS chicken, and the severity of the disease can be manipulated using iodine [83]. Application of antioxidants delays the onset of the disease, illustrating the importance of oxidative reactions in the toxicity of iodine [83]. The role of the stress system in the development of the disease in chickens is illustrated by an altered immunoendocrine communication via the HPA axis in this strain of birds [84]. The OS chicken shows a hyporesponsiveness to glucocorticoids, in particular

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to inhibitory factors released by this stress hormone in immune cells [84]. Moreover, low levels of the central opioid peptide -endorphin have been shown in the hypothalamus of OS chickens before onset of the disease, i.e., in the embryonic stage. A further decrease in this brain peptide was observed at the first signs of thyroid mononuclear infiltration [85]. Similar HPA axis–regulating disturbances have been shown in another animal model of organ-specific autoimmune disease; for example, the Lewis rat is sensitive to experimental allergic encephalomyelitis (EAE) elicited by immunizations with myelin antigens [86]. There are to our knowledge no reports on the presence of oophoritis in the OS chicken. B. Experimentally Induced Animal Models of Endocrine Autoimmune Disease 1. Excessive Exposure to Autoantigen Classical models for the induction of organ-specific autoimmune disease are models that make use of immunization with autoantigen in an adjuvant, e.g., injections of thyroglobulin (Tg), H/K ATPase [87], or myelin basic protein (MBP) [88] in Freund’s complete adjuvant (FCA) leading to experimental allergic thyroiditis (EAT), experimental allergic gastritis (EAG), or experimental allergic encephalomyelitis (EAE), respectively. In general, diseases are transient in these models, depending on the animal used for sensitization. Obviously these models cannot be used to study the very early phases of spontaneous autoantigen presentation in wild-type occurring endocrine organ–specific autoimmune diseases. The models have, however, been proven useful in the study of effector mechanisms playing a role in autoimmune diseases as well as some preventive and therapeutic interventions. Immunization with Crude Ovarian Antigens. Experimental autoimmune oophoritis can be induced in animals, such as the rat and the Balb/c mouse, using immunization with bovine or rat ovarian extracts in complete Freund’s adjuvant [89–91]. The immunization establishes an autoimmune oophoritis as early as day 14 after immunization, with infiltration of the ovaries by immune cells. The autoimmune nature of the oophoritis is underlined by a positive delayed-type hypersensitivity (DTH) reaction towards the injected ovarian antigens by day 14, illustrating the existence of a cell-mediated immune reaction to the ovarian antigens. A similar experimental allergic autoimmune oophoritis could also be induced by passive transfer of peripheral blood lymphocytes, spleen cells, and T- and Bcell–enriched cell suspensions from ovarian antigen–immunized rats to naive recipients, indicating that T cells and B cells are important in the pathogenesis of the disease [91]. Antiovarian antibodies in the serum of the affected animals are not detectable earlier than day 28 [89], and the reproductive capacity of the rats, measured by the litter size, could be correlated to the titer of the antiovarian antibodies. Moreover, passive immunization of rats with rabbit anti-rat ovarian serum gave a temporary dose-dependent reduction in litter size [92], showing a clear role of antibodies in the pathogenesis of the disorder. The antibodies produced in this experimental oophoritis animal model are thought to either interfere with zona pellucida antigens inhibiting fertilization and/or to disturb ovulation [93]. Histological examination of the ovarian tissue at day 14 after immunization showed characteristic perivenous accumulations of lymphocytes and macrophages as well as plasma cells [89–90]. The infiltrate was found beneath the tunica albuginea and in the interfollicular tissue, as well as in the granulosa layer of follicles. Occasionally cell infil-

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trates were found in the external theca. The large secondary follicles and corpora lutea seemed unaffected, in contrast to the primordial and small secondary follicles; while the number of follicles and corpora lutea decreased, the number of atretic follicles increased. It is evident comparing the histology of this experimental oophoritis rat model to the known cases of human autoimmune oophoritis that there are major differences. In human autoimmune oophoritis (see later), the main targets are the steroid-producing cells of the theca of maturing follicles and the corpus luteum and not the interfollicular space, the secondary and primordial follicles such as seen in this animal model. This implies that this model may have only limited value in the study of human autoimmune oophoritis and POF. Immunization with Zona Pellucida Antigens. Immunization of New Zealand white rabbits with heterologous zona pellucida (ZP) antigens shows an induction of ovarian failure due to follicle depletion. Immunization experiments with porcine ZP in rabbits showed development of ZP antibodies in the immunized animals. It was demonstrated that rabbits actively immunized with ZP proteins ceased to ovulate in response to hCG administration [93]. Immunization of rabbits induced a marked reduction in follicles and an atretic appearance of primary follicles. Growing follicles disappeared completely by 30 weeks postimmunization. The reduction in the number of normal follicles was accompanied by a striking increase in the number of oocyte-free cell clusters. An oophoritis such as seen in immunization experiments with crude ovarian extracts was not detected [94]. The alteration in ovarian function and histology in rabbits could be correlated with the presence of serum antibodies to ZP glycoproteins. These studies and the histological pictures indicate, first, that the antibodies to ZP alter ovarian function and histology by interfering with cells during the stage of follicle differentiation at which ZP proteins are being synthesized [94] and, second, that the model might be of relevance in the study of human POF in the absence of adrenal autoimmunity (see later). It has been hypothesized that premature depletion of ovarian follicles might represent the human counterpart of this animal model. However, Starup and Pedersen showed in ovaries of ROS patients a normal ultrastructural appearance of the early follicles [95], and no oocyte-empty follicle remnants, such as described by Skinner et al. in the rabbit model [94]. On the other hand, Scully et al. reported on two cases of ROS in whom hyalinization of preantral follicles was described [96,97]. The proteins of the zona pellucida are conserved among mammals [98]. ZP3 is a major ZP glycoprotein that functions as a sperm receptor [99], and mouse and human ZP3 proteins are 67% identical. A 15-amino-acid peptide of ZP3 was shown to induce oophoritis in (C57BL/6xA/J) F1 (B6AF1) mice after immunization in Freund’s complete or incomplete adjuvant. ZP3-specific T-cell responses and antibodies directed to ZP3 were detectable in these ZP3 immunized animals [98]. In an adoptive transfer experiment to naive mice, ZP3-specific CD4Ⳮ T cells were sufficient for induction of the oophoritis without observable antibody production to the zona pellucida. The ZP3-specific CD4Ⳮ T cells mainly produced interleukin (IL)-2, interferon (IFN)-␥, and tumor necrosis factor (TNF), but not IL-4, indicating that the disease-specific T cells belonged to the TH1 subset of CD4Ⳮ T cells. Tung [100] subsequently very elegantly showed that a transfer of T cells only directed to a small T-cell epitope of ZP3 (15-amino-acid peptide) differed from the adoptive transfer of T cells to whole ZP3, and that this transfer could result in a full-blown autoimmune oophoritis, with serum antibodies to native ZP3 and preferential binding of the

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antibody to the zona pellucida in vivo (apart from a T-cell response to the self-peptide and histomorphologically confirmed oophoritis). Crucial in the experiments was the presence of the ovaries during the antigen-specific CD4Ⳮ T-cell transfer [100]. The phenomenon shows that B cells autoreactive to ovarian antigens can be generated after T-cell transfer and that these cells can be activated by ZP3-specific CD4Ⳮ T cells to produce antibodies that are directed to and bind ZP3 in vivo. It is thought that the ovarian antigen required for antibody production in this model is provided by the normal ovaries, since the zona pellucida antigens may be generated through the physiological process of follicular atresia (epitope spreading). Another mechanism by which the ovarian autoantibodies can be induced in experimental animals is by idiotype mimicry of autoantigens in the absence of the antigen itself. Tung et al. [101] investigated whether a nonovarian peptide could be recognized by ZP3specific T cells. The author detected, by searching the protein sequence library, nonovarian peptides sharing sufficient residues with ZP3. Interestingly, the ␦-chain of the murine acetylcholine receptor and the ZP3 peptide had certain homology. The ZP3 peptide derivate and the ␦-chain of the acetylcholine receptor both elicited severe oophoritis, and they also stimulated the ZP3-specific T-cell clone to proliferate. Through the mechanism of Tcell peptide mimicry, using a ␦-chain of the murine acetylcholine receptor, autoimmune oophoritis could be elecited by clonal activation of ZP3 specific pathogenic T cells [101]. Hence, T-cell epitope mimicry as autoimmune disease mechanism was detected in the murine model, and this mimicry may explain the clinical association between POF and myasthenia gravis. However, in humans there does not exist a homology between ZP3 and the acetylcholine receptor [102]. Still, it remains remarkable that there is a marked coincidence between POF and myasthenia gravis. The notion that in human oophoritis a perineural infiltration of the ovarian hilus nerves is seen might also indicate a shared pathogenic mechanism between ovarian and neuronal diseases. 2. Thymectomy Models Neonatal thymectomy in Balb/C mice at day 3 after birth results in oophoritis, as well as in other organ-specific autoimmune manifestations, such as thyroiditis, gastritis, and parotitis [103–105]. The inflammations are characterized by the presence of T-cell infiltrates in the affected organs and the development of organ-specific antibodies in the serum. There is a strict temporal relationship between the development of the autoimmune syndrome and the day of thymectomy, which occurs between the second and fifth days after birth [105,106]. The recent interest in CD4ⳭCD25Ⳮ T cells as a specific subpopulation of thymusderived regulatory or suppressor T cells has a historical association with the day 3 mouse thymectomy model [105,107]. Day 3 neonatal thymectomy-induced autoimmune disease is due to a lack of CD4ⳭCD25Ⳮ T-cell migration into the periphery, since these regulatory cells typically migrate out of the thymus in this early period and since injection of purified CD4ⳭCD25Ⳮ T cells into neonatally thymectomized mice prevents the development of the various autoimmune syndromes, including oophoritis. CD4ⳭCD25Ⳮ T cells develop in the thymus via a distinct pathway of thymic selection requiring the expression of endogenous TCR ␣ chains on the cells for selection since CD4ⳭCD25Ⳮ T cells are absent in TCR transgenic mice on a RAG-deficient background. A feature of CD4ⳭCD25Ⳮ T cells is that the cells themselves are anergic to mitogenic stimuli, but are in addition capable of suppressing the proliferation of CD4ⳭCD25ⳮ T cells when cultured together. Such suppression can be abrogated by the addition of IL-2 or stimulation with anti-CD28

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antibodies. The mechanisms of suppression by CD4ⳭCD25Ⳮ T cells are not clarified yet, but are presently the subject of intensive research [108,109]. The histopathological events of oophoritis in thymectomized mice occur in an orderly manner. Initially the oophoritis is evident as a patchy or diffuse infiltration of lymphocytes; later developing follicles are clearly affected, and monocytes, macrophages, neutrophils, and plasma cells are found between and within ovarian follicles. The onset of puberty markedly potentiates the oophoritis, indicating that a change in antigen profile due to the gonadotropin stimulation is important. The oophoritis is most severe between 4 and 14 weeks after thymectomy. This is accompanied by loss of ova and collapse of ovarian follicles. Autoantibodies are detected in the circulation by week 4, with a peak between weeks 7 and 9. Autoantibodies are directed towards oocytes, zona pellucida, and in lower titers to steroid-producing cells such as the granulosa cells, the theca cells, and the luteal cells. The inflammation subsides after 14 weeks, and the ovary becomes atrophic [103–105]. IgG-producing plasma cells are found, but not frequently. The overall picture of the oophoritis is one of a cell-mediated autoimmune reaction. The histological and serological manifestations of murine autoimmune oophoritis are hence comparable to the histological and serological picture of human autoimmune oophoritis in association with Addison’s disease. It is, however, remarkable that the adrenal glands are unaffected in neonatally thymectomized mice, even in the presence of antibodies to steroid-producing cells. However, immunomodulation using cylosporin A after birth does affect the adrenals [110]. As the inflammation of the ovaries subsides, serum antioocyte and antizona antibodies sometimes decrease to undetectable levels at day 150–360 when oocytes have completely disappeared from the atrophic ovary [103–105]. An absence of serum autoantibodies therefore does not exclude an autoimmune etiology of the ovarian disease. This finding may be of relevance in patients with adrenalitis and/or amenorrhea; detection of steroid cell antibodies may not always be expected unless they are looked for in an early stage of the disease. With regard to the genetics of this model, certain strains of mice are susceptible, such as BALB/c and A/J mice, whereas other strains (C57bl/6J, DBA/2 mice) are resistant. Since susceptibility and resistance are not associated with the MHC haplotype (H2) of the mice, these antigen-presenting molecules are apparently of minor importance in this model. Using susceptible and resistant mouse strains and backcrosses of these strains in combination with a microsatellite approach, a locus has been found on chromosome 16, controlling the abrogation of the tolerance to ovarian autoantigens due to neonatal thymectomy on day 3 [111]. This so-called Aod1 locus was associated with the presence of oophoritis in mice. Interestingly, the markers on chromosome 16 failed to exhibit a significant linkage to the concomitant ovarian atrophy in this mouse oophoritis model. Rather, this atrophy exhibited an association with markers on mouse chromosome 3. Two regions on the distal arm of chromosome 4 (Gasa1 and Gasa2) might be involved in the gastric autoimmunity of the mice [87]. 3. Transgenic and Knockout Animal Models The spectacular progress made in transgenic and knockout technology has provided powerful new tools for the investigation of the fundamental aspects of endocrine organ–specific autoimmune diseases. There has been great interest in creating transgenic and knockout models, especially in the field of NOD mouse diabetes. It must be noted, however, that the transgenic and knockout models are often quite artificial and far away from the etiopathogenesis of wild-type endocrine organ–specific diseases. They are hence only suitable for studying certain aspects of the endocrine autoimmune diseases.

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A relevant model for POF is the AIRE gene knockout model. Recently such knockout mice have become aivailable, and the mice show characteristics of APS type 1 [51]. In these mice the immune system seems to have developed normally, but reacts abnormally when challenged with immunization with the experimental antigen hen egg lysozyme (HEL) in complete Freund’s adjuvants. The peripheral T cells of the AIRE knockout mice then show a three- to fivefold increased proliferation. The mice also fail to reproduce. Female mice show lymphocytic infiltrates in the margin of the atrophied ovary, and some of the mice lacked follicles, suggesting an autoimmune-induced atrophy. Moreover, 75% of the animals showed autoantibodies against liver, testis, pancreas, and adrenal gland, such as is also seen in humans with APS type 1. It has been suggested that when an excessive environmental influence triggers an immune response in these mice, the animals start to produce an exaggerated response, spreading to various endocrine autoantigens and leading to the autoimmune attack towards the various glands. V. ANIMAL MODELS REVEAL A GENERAL ‘‘BLUEPRINT’’ FOR THE ETIOPATHOGENESIS OF ENDOCRINE AUTOIMMUNE DISEASES Studies in virtually all of the above-listed animal models have shown that the pathogenesis of an autoimmune failure of a gland is generally a multistep process, requiring several genetic and environmental abnormalities (or variants) to converge before full-blown disease develops. Hence, endocrine organ–specific autoimmune diseases are the outcome of an unfortunate combination of various genetic traits and environmental circumstances (Fig. 1) that by themselves need not be harmful, and may even be advantageous. With regard to the local initiation of endocrine autoimmunity, all the spontaneous animal models of endocrine autoimmune disease (the BB-DP rat, the NOD mouse and the OS chicken) [112,113], the thymectomy mouse model [87], and the transgenic models [114] have shown that the process starts with a local increase in the numbers of dendritic cells (DC) and macrophages (M␾) in the glands to be affected. DCs are MHC class II positive and antigen-presenting cells (APC) par excellence—essential not only for the initiation of immune responses (the stimulation of naive T cells [112,115]), but also for tolerance induction (the deletion of autoreactive T cells and the generation of regulator T cells). M␾ have various functions, ranging from the production of factors for wound healing and remodeling of bone [116] via the phagocytosis and degradation of unwanted material to the regulation of immune responses. The early accumulation of DC and M␾ is indispensable for the development of endocrine organ–specific autoimmune diseases, since prevention of their accumulation in the pancreas of NOD mice [117,118], in the pancreas of BB-DP rats [119] or the brain of Lewis rats [88] results in prevention of diabetes and EAE development. Splenic lymphocytes from macrophage-depleted NOD mice fail to transfer diabetes to recipients [120]. Early attraction signals for the DC and M␾ to come to the endocrine glands can be of environmental origin and inflammatory in nature. An early nonspecific necrosis of glandular cells caused by (1) toxins, e.g., iodine in the thyroid [121,122] and streptozotocin in the islets [56], (2) bacterial infections, e.g., Heliobacter pylori in the stomach [87], and (3) viral infections, e.g. EMC-D virus in the islets, with the concomitant release of self antigens and pro-inflammatory and chemotactic factors for DC and M␾ have all been described as eliciting factors for endocrine organ–specific autoimmunity. However, it is remarkable that in the spontaneous animal models of endocrine autoimmunity, DC and M␾ accumulate in the glands in the absence of any obvious inflammatory condition of

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the gland [123,124]. In an attempt to explain this discrepancy, we have collected evidence that DC and M␾ may accumulate in endocrine glands not only to exert a function in inflammation and removal of cell debris, but also to regulate the growth and function of the glandular tissue. It is important to note that DC and M␾ are normal constituents of the thyroid, the islets, and other glandular structures and that the cells have been proven to regulate the growth and function of glandular cells in vitro [125–127] predominantly via IL-1 and IL-6 signals. The recognition that such cells constitute a recruitable cellular force that, on the one hand, is capable of regulating tissue homeostasis but, on the other hand, is capable of initiating immune responses has implications for our understanding of the induction phase of endocrine organ–specific autoimmune diseases. Minor inborn errors in metabolism and minor aberrances in the structure, growth, and function of tissues may therefore necessitate an influx and local activation of DC and M␾ to regulate tissue homeostasis. This noninfectious influx and activation might serve as a first step on the way to endocrine organ–specific autoimmunity. There is an argument for such a view: very early (and even fetal) abnormalities in the growth potential and hormone production of thyrocytes, salivary gland cells, and islet cells have been reported in the OS chicken, the BB-DP rat, and the NOD mouse. The destiny of DC and M␾ accumulated in tissues is to enter the lymphatics [112,113] and to travel to the draining lymph nodes while transporting antigens to these nodes. In these nodes the cells are able to elicit an immune response, and indeed in the above-described animal models of endocrine autoimmune disease, the early glandular accumulation of DC and M␾ is followed by an expansion of autoreactive CD8Ⳮ and CD4Ⳮ T cells and the production of autoantibodies in the reacting draining lymph nodes (Fig. 2). In this context it is worthy noting that it is presently believed that DC and M␾ induce tolerance towards collected autoantigens when they are under steady-state, i.e., noninflammatory, conditions. If inflammatory conditions prevail in the tissues, the DC switch to a maturation program and become DC1 (Fig. 2) and are able to initiate aggressive immune responses. Apparently DC and M␾ are not in steady-state conditions in the early influxes in endocrine autoimmunity. Evidence has recently been collected that aberrant cross-talk between abnormally differentiated DC and abnormally functioning T cells in the draining lymph nodes of the animal models of spontaneous endocrine autoimmune disease is a prerequisite to initiate the excessive permanent destructive immune response towards endocrine self-antigens characteristic of endocrine autoimmune diseases. Various mechanisms exist for maintaining T-cell tolerance in normally draining lymph nodes (so-called peripheral tolerance mechanisms), such as: 1. The induction of anergy in potential autoreactive T cells via the presentation of antigens in the context of MHC class II molecules, but in the absence of costimulatory molecules 2. Via the downregulatory activity of regulatory T cells 3. Via so-called activation-induced t-cell death/apoptosis (AITCD) of potential autoreactive T cells The above animal models are practically all characterized by one or more defects in one or more of these T-cell tolerance mechanisms. Many of the models show a defect in regulatory T cells. The mouse thymectomy model is known for its specific lack of CD4ⳭCD25Ⳮ T regulator cells [87]. The BB-DP rat also lacks a particular subpopulation

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Figure 2 Immunopathological events that take place during the development of endocrine organ–specific autoimmune diseases. Initiation phase: In a first afferent phase APC accumulate in the gland. The APC influx can be induced by nonspecific inflammatory stimuli, e.g., the necrosis of target cells by toxins or viruses. The accumulated and activated APC take up relevant autoantigens and leave the tissue to travel to the draining lymph nodes. In the draining lymph nodes the APC seek contact with T cells and B cells. Apparently an aberrant immune response results in these conditions of endocrine autoimmunity. Under normal conditions the APC would have reinforced tolerance. The text of this chapter lists the various abnormalities found in animal models underlying this aberrant regulation of the immune response. Generated cytotoxic T cells attack the glandular target cells and more antigens are released. B cells produce autoantibodies, which are in the majority of endocrine organ–specific autoimmune diseases just markers of the process (except in Graves’ disease). Expansion and destruction phase: In later phases the autoimmune reaction is perpetuated and expanded. The generated autoreactive T cells and B cells gain access to the target glands and often form focal accumulations. Such focal accumulations are mostly harmless for the target cells. When a switch takes place from the APC to Th1-stimulating cells (DC1), a more aggressive type of inflammation is induced. In such inflammations IFN-␥ activates scavenger macrophages to kill off the target cells. E ⳱ endocrine cell; B ⳱ B cell; DC ⳱ dendritic cell; M␾ ⳱ macrophage; Th1 ⳱ T-helper 1 cell; abs ⳱ antibodies; NO ⳱ nitric oxide.

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of T cells, the so-called RT6Ⳮ T cells, which clearly forms a regulator (suppressor) population [68]. The OS strain of chickens has defects in its regulatory T-cell system [62]. The NOD mouse has defects in T-cell apoptosis, leading to a decreased ability of the cells to undergo AITCD, a major mechanism in both central and peripheral tolerance induction [130]. Moreover, NOD mice are characterized by abnormal thymic architecture [131,132]. Interestingly, the spontaneous animal models of endocrine autoimmune disease additionally show aberrations in the differentiation of DC and M␾. Studies in the BB-DP rat and NOD mouse have established that in both models aberrations exist in the maturation process of these myeloid cells from their precursors [112]. There is a shift in balance between M␾ and DC development in favor of the M␾, leading to an enhanced maturation of scavenger M␾ in the animals. In addition, the macrophages show an enhanced migration and an enhanced production of pro-inflammatory factors [133]. This hyperreactivity of scavenger M␾ likely contributes to the enhanced cytotoxic potential of the cells for target glandular cells in endocrine organ–specific autoimmunity. At the same time there is a maturation defect in the DC compartment of the animals. This defect leads to a low expression of MHC class II and CD80 and CD86 molecules on the generated DC of these animals, resulting in a low capacity of the APC to stimulate T cells, particularly via the MHC class II TCR and CD80-CD28 route [134–137]. Precisely such triggering via the latter route is essential to prevent the development of diabetes in the BB-DP rat and NOD mouse model [129] in vivo treatment of BB-DP rats with the stimulatory ␣-CD28 Mabs JJ319 and JJ316 completely prevents the development of both insulitis and diabetes, whereas treatment with a blocking ␣-CD80 Mab accelerates diabetes development. Knocking out CD80-CD28 interaction in the NOD mouse model has also proven to result in an acceleration of the disease [138]. Another piece of evidence that fully active and mature DC are required for optimal tolerance induction in the BB-DP rat is the observation that the immature DC of the BB-DP rat cannot sufficiently expand the RT6Ⳮ T-cell population, which represents the suppressor cell population in the rat system [136]. In sum, the aberrant function of T cells and the aberrant differentiation and maturation of DC and M␾ in the animal models of endocrine autoimmune disease likely contribute to the poor tolerogenic capability of the animals and the heightened and permanent aggressiveness to endocrine tissues. Armed with this knowledge on the development and the appearance of endocrine autoimmune disease in animal models, we will consider some forms of POF as endocrine autoimmune diseases.

VI. POF IN ASSOCIATION WITH ADRENAL AUTOIMMUNITY One of the first signs that autoimmunity could be responsible for a failure of ovarian function came from the observation that ovarian failure could precede the onset of Addison’s disease by 8–14 years [139]. Addison’s disease is an uncommon disorder (40–110 per million) caused by a deficiency of adrenocortical hormones. The prevalence is highest in the fourth decade of life, and there is a marked female preponderance (2.5:1). The nature of idiopathic Addison’s disease in the majority of patients in developed countries is now regarded as autoimmune [140], in contrast to the nature of the disease in developing countries, which is still mainly due to tuberculosis [141]. Autoimmune Addison’s disease seldom develops in isolation, and several other endocrine glands and organs are generally affected [142], leading to an autoimmune polyglandular syndrome (APS) (see Chapter 24). Two main forms of APS can be clinically discerned in which oophoritis occurs.

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APS type 1 mainly affects children and is characterized by the association of mucocutaneous candidiasis, hypoparathyroidism, and Addison’s disease. Ovarian failure is often part of the syndrome. The basic defect of APS type 1 is a mutation in the autoimmune regulator gene (AIRE gene) on chromosome 21Q22.3 [143,144]. APS type 2 is characterized by adrenal failure in association with hypothyroidism. The latter mainly occurs in the fourth decade of life and has a female preponderance. Within families several generations can be affected, and the mode of inheritance is compatible with an autosomal dominant trait with incomplete penetrance. The chromosomal defect has not yet been identified. In this syndrome only 25% of women have an amenorrhea and 10% have a classical POF [145,146]. With regard to POF in general, the literature indicates that 2–10% is associated with Addison’s disease and/or adrenal autoimmunity [147]. A. Antibodies in Patients with Adrenal Autoimmunity and/or Addison’s Disease Two varieties of adrenal antibodies have been recognized in the sera of patients with Addison’s disease using indirect immunofluorescence (IIF) and cryostat sections of human or monkey adrenal glands. One variety demonstrated reactivity with the three layers of the adrenal cortex (ACA) only [148,149], whereas the other variety also reacted with cytoplasmic antigens of other steroid-producing cells present in the ovary, testis, and placenta [148,150,151]. This latter subvariety of adrenal cytoplasmic antibodies was called steroid-cell antibodies (StCA), and its reactivity could be absorbed by adrenal homogenates, thus confirming the cross-reactivity with adrenal cytoplasmic antibodies [152]. There is an absolute association between the presence of StCA and that of ACA, the former only being detectable when the latter is also present. StCA are of the IgG type and bind within the ovary to the hilar cells, the cells of a developing follicle, such as theca and granulosa cells, and to the corpus luteum cells [148,151]. Female patients with a primary amenorrhea and Addison’s disease almost all have a detectable serum titer of StCA; of patients with a secondary amenorrhea and Addison’s disease, 60% show these antibodies (Table 2). In the absence of clinically overt gonadal failure, StCA have been described in about 15–20% of patients with clinical or latent Addison’s disease [145]. In the follow-up of StCA-positive addisonian patients, about 40% of females developed ovarian failure in a period of 10–15 years, whereas in males the StCA did not herald gonadal failure (however, numbers of studied patients were small). Heterogeneity exists between APS type 1 and 2 in relation to StCA (Table 1): 60–80% of patients with hypoparathyroidism and Addison’s disease (APS type 1) and 25–40% of patients with APS type 2 show these antibodies. In APS type 1 without Addison’s disease, 10% of patients show StCA. The high prevalence of StCA in patients with APS type 1 probably explains the common association with gonadal failure seen in this group, and the appearance of the StCA in a female patient with APS type 1 without adrenocortical or ovarian failure signals a high risk of their development [149,153,154]. The sensitivities/specificities/predictive values in females with APS type 1 who initially had normal adrenocortical and ovarian function were 1.0/0.56/0.50 for StCA in predicting ovarian failure and 0.86/0.83/0.86 for StCA in predicting adrenocortical failure [154]. The mere presence of an autoantibody in the serum of a patient is certainly not evidence for the pathogenic significance of the antibody; the autoantibody may also be the consequence of cellular destruction, such as is seen after the destruction of cardiac

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Table 2 StCA Prevalence in Disease and Controls Ovarian failure Unselected infertility/amenorrhea With autoimmune thyroid disease or type 1 diabetes With Addison’s disease primary and secondary amenorrhea Addison’s disease (without ovarian failure) Isolated cases With hypoparathyroidism/candidiasis (type I APGS) With autoimmune thyroid disease (type II APGS) Type I APGS, mucocutaneous candidiasis, and hypoparathyroidism without Addison disease Autoimmune thyroid disease or type 1 diabetes Healthy controls

⬍1% 5–10% 100% 60% 10–25% 60–100% 25–40% 10% ⬍1% ⬍1%

Source: Adapted from Refs. 145, 152, and 154.

muscle cells in myocardial infarction giving rise to anti–heart cell antibodies. It has, however, been shown that sera of patients with APS type 1 and Addison’s disease positive for ACA and StCA are cytotoxic for cultured granulosa cells in the presence of complement, when high titers of these antibodies were demonstrated in 9 out of 23 cases [155]. Complement-dependent cytotoxicity of StCA might indeed be one of the mechanisms leading to destruction of steroid-producing cells in vivo and thus to ovarian failure. Considerable progress has been made with regard to the identification of the target antigens of ACA and possibly of StCA [156]. It has been found that the adrenal cytochrome p450 enzyme 21-hydroxylase (21–04) is the major autoantigen recognized by autoantibodies present in patients with Addison’s disease [157,158], either in the form of isolated adrenal failure or associated with hypothyroidism (APS type 2). In APS type 1 it is thought that autoantibodies are directed to other members of the cytochrome p450 enzyme family, namely to the p450 side-chain cleavage enzyme (p450 scc) and to 17-␣-hydroxylase (17-␣-OH) [158–162]. However, there is some confusion on this subject, and not all investigators could confirm the presence of these autoantibodies in APS type 1 (negative results p450 scc [163]; 17-␣-OH [163,164]). Of the steroidogenic p450 enzymes, 21-OH is adrenal-specific, 17-␣-OH is expressed in both adrenals and gonads, whereas p450 scc is present in adrenal, gonads, and placenta. Targets of the StCA are thus 17-␣-OH and the p450 scc enzyme. Apart from autoantibodies, another strong argument for considering StCA-positive ovarian failure as an autoimmune disease is the histology of the ovarian lesions. B. Histology of Ovaries in Patients with POF in Combination with Adrenal Autoimmunity and/or Addison’s Disease Table 3 [165–175] gives an overview of the reported histology of POF, including the reported cases of histologically confirmed oophoritis. All StCA-positive cases had lymphocytic oophoritis, and of all lymphocytic oophoritis cases reported, 78% had StCA. The macroscopic appearance of ovaries with lymphocytic oophoritis was either cystic (50% of the cases), with smaller and larger cysts, or normal.

1 5 1 1 1 1 1 1 1 1 1 1 1991 1 1 Without adrenal/steroid cell antibodies 1982 1 1 1987 1 1990 1 1 1

Cases ⫹ ⫹ ⫹ ⫹ ⫹ ⫹ ⫹ ⫹ ⫹ ⫹ ⫹ ⫹ ⫹ ⫹ ⫺ n.a. ⫺ n.a. n.a. ⫺

⫺ n.a. ⫺ ⫺ n.a. ⫺

Adrenal

⫹ ⫹ ⫹ ⫺ ⫹ ⫹ ⫺ ⫹ ⫹ ⫺ ⫺ ⫹ ⫹ ⫹

Ovary

Antibodies

Granulomatous oophoritis n.a. thyroid abs⫹ n.a. n.a. ⫺

Testis Addison’s disease 5 X Thyroid abs⫹, gastric abs⫹ ⫺ Addison’s disease 3 years later Thyroid abs⫹ Thyroid abs⫹ Testis abs⫹ Testis abs⫹, gastric abs⫹ Thyroid abs⫹, Hashimoto’s disease Gastric abs⫹, ANA⫹ ⫺ ⫺ Thyroid abs⫹

Other antibodies/diseases

Histology of Ovaries in Relation to Adrenal/Steroid Cell Antibody Profile of POF Patients

With adrenal antibodies 1968 1980 1981 1984 1986 1987 1987 1988 1989 1990

Year

Table 3

⫹ ⫹ ⫹ ⫹ ⫹ ⫹

⫹ ⫹ ⫹ ⫹ ⫹ ⫹ ⫹ ⫹ ⫹ ⫹ ⫹ ⫹ ⫹ ⫹

Oophoritis

175 172

147

173

150 165 166 167 168 169 97 170 171 172

Ref.

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Concerning the pattern of microscopic infiltration, there is a marked similarity in the reported cases of lymphocytic oophoritis in the different compartments of the ovary. In most cases the primordial follicles are unaffected, as is the cortex of the ovary. It is the developing follicle that is predominantly infiltrated by mononuclear inflammatory cells. There is a clear pattern of increasing density with the infiltration with more mature follicles. Preantral follicles are only surrounded by small rims of lymphocytes and plasma cells, whereas larger follicles have a progressively denser infiltrate, usually in the external and internal theca. The granulosa layer is usually spared in this process until luteinization of the degenerating follicle occurs. Cysts were luteinized with a marked infiltration in the cyst wall and destruction of the lining cells. Atretic follicles and, when present, corpora lutea or corpora albicantia were infiltrated as well. Mild infiltration might be seen in the medulla and hilar region of the ovaries. There is a perivascular and, surprisingly, a perineural infiltration in the hilus of the ovary [172]. This pattern of infiltration confirms that indeed steroid-producing cells are major targets of autoimmune attack. Immunohistochemical analysis of lymphocytic oophoritis reveals that the inflammatory cells are mainly formed by T lymphocytes (CD4Ⳮ and CD8Ⳮ), with a few B cells, together with large numbers of plasma cells. Macrophages and natural killer (NK) cells can also be found. The plasma cells mainly secrete IgG, but also IgA or IgM [169,167], which probably indicates the local production of ovarian autoantibodies. That T cells are important in the ovarian destructive autoimmune reaction is mainly supported by data generated in the animal models of autoimmune lymphocytic oophoritis. The involvement of T cells in human oophoritis is suggested by a case report on a patient with autoimmune thyroiditis, adrenalitis, and POF in whom migration-inhibiting factor (MIF) production towards ovarian as well as testicular antigens was found [176]. The MIF test is an oldfashioned, yet sensitive antigen-specific test for the production of a cytokine, MIF, by peripheral blood T lymphocytes when cultured in the presence of specific antigens. In conclusion, shared antibodies and antigens, histology, and neonatal thymectomy and experimental allergic animal models point in the direction of a clear autoimmune origin of POF in combination with Addison’s disease and/or adrenal autoimmunity.

VII. OVARIAN AUTOIMMUNITY IN PATIENTS WITH IDIOPATHIC POF A. Histology of Ovaries in Idiopathic POF Patients The histological picture of ovaries of POF patients without adrenal autoimmune disease is summarized in Table 4 [177–189]. Approximately 60% of such cases of POF lack ovarian follicles, and in these cases fibrotic ovaries are found. In 40% of the cases ovarian follicles are detectable, varying in amount from few to numerous. About 10% of such follicular cases have numerous follicles, and these latter are probably ROS cases. It is important to note that cases of lymphocytic oophoritis are rarely found in POF patients in the absence of adrenal autoimmunity/Addison’s disease (Table 3.). Muechler et al. showed the presence of immunoglobulins in such ovaries using direct immunofluorescence: in 50% of cases they found vascular wall staining (IgA, IgM or IgG), and in 30% the stroma and the follicular cells were positive for immunoglobulins [189]. Remarkable again in the histology of these cases was the absence of lymphocytic oophoritis. Hypothetically, autoantibodies to the ovary may have been present in the ovary without reaching detectable levels in the serum or inducing a local inflammation. It must also be noted that Muechler et al.’s data have not been confirmed by others, and in fact the histology of POF

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Table 4 Histology of Ovaries of POF Patients Without Adrenal/Steroid Cell Antibodies Year

Cases

No. of follicles

Few follicles

Numerous follicles

Ref.

1965 1970 1970 1972 1973 1977 1978 1979 1982 1982 1984 1985 1987 1990 1991

9 7 7 10 15 3 8 8 9 19 43 14 10 12 17

6 5 4 8 7 2 3 2 5 14 27 9 10 5 12 119 (55%)

1 2 3 2 6 1 3 4 4 0 16 2 0 7 2 53 (25%)

2 0 0 0 2 0 2 2 n.a. 3 n.a. 5 0 n.a. 3 19 (9%)

53 177 178 179 180 181 182 183 184 174 185 186 87 188 189

Total

in the absence of adrenal autoimmunity/Addison’s disease does not support an immune pathogenesis of the disease. This also applies to the atrophy found in the majority of cases; this phenomenon may represent the end stage of an autoimmune process directed against ovarian structures (such is seen in animal models), but it may also represent a final depletion of oocytes due to genetic or environmental factors. B. Autoantibodies in Patients with Idiopathic POF POF has been described in association with several autoimmune disorders other than adrenal autoimmunity, e.g., myasthenia gravis, Graves’ disease, type 1 diabetes, alopecia, pernicious anemia, hypohysitis, Sjo¨gren’s disease, primary biliary cirrhosis, and systemic lupus erythematosus (SLE). Thyroid autoimmunity is the most prevalent (up to 14%) associated endocrine autoimmune abnormality reported in POF patients without an adrenal autoimmune involvement, followed by the presence of parietal cell antibodies (up to 2%), type 1 diabetes (up to 4%), and myasthenia gravis or positivity for acetylcholine receptor antibodies (up to 2%) [147,190]. However, the general prevalence of positivity for thyroid antibodies and gastric parietal cell antibodies is—if at all—only slightly greater than that found in normal populations. It is, however, remarkable that type 1 diabetes and myasthenia gravis, both relatively uncommon autoimmune diseases (⬍⬍1%), are relatively frequently found in POF patients (2–4%). Whether this high frequency is due to publication bias or to a shared underlying immune-dysregulating factor remains to be established. SLE, antinuclear antibodies, and rheumatoid factors have also been reported with a higher than normal frequency in POF patients. A relationship of POF to SLE is further strengthened by the finding by Moncayo-Naveda et al. of the presence of antiovarian antibodies in 84% of female SLE patients [191].

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C. Ovarian Autoantibodies in Patients with Idiopathic POF Strong support for the autoimmune character of isolated POF would be the presence in patients’ serum of antibodies to ovarian structures. The major conclusion drawn from several investigations using IIF on gonadal tissue (animal or human) is that patients are negative for StC-Abs [152–154,192–196]. However, positive results regarding antiovarian antibodies have been found using assay methods other than IIF. Using immunoperoxidase staining, Cameron et al. [197] detected antiovarian antibodies in the serum of 4 out of 10 patients with incipient ovarian failure [still regular menses, but elevated serum FSH levels; an assumed early stage of POF: incipient ovarian failure (IOF)]. Damewood et al. found antiovarian antibodies in POF cases that did not react with the steroid-producing cells, but did react with the granulosa cells and the oocytes of primary follicles in 9 out of 14 POF cases [198]. Coulam and Ryan, using a radioimmunoassay with radioactive-labeled crude human ovarian extracts, found that the sera of 14 out of 15 POF patients bound stronger to these ovarian antigens in comparison to control sera [199]. In a later study [200] a much smaller proportion, namely, 30 out of 110 (27%) POF patients, were positive in this assay system. Luborsky et al. [201], using an ELISA with human oocyte and ovarian homogenates, found 70% positivity in the POF patient group. The autoantibodies were directed to the crude ovarian homogenates and/or towards the oocyte homogenates. Moncayo-Naveda et al. [191], using an ELISA with corpus luteum extracts of bovine origin, found that antiovarian antibodies could be detected in the sera of patients with primary and secondary infertility, endometriosis, and Addison’s disease. They also found positivity in the sera of women with other autoimmune disorders, such as in 84% of SLE patients who did not suffer from ovarian failure. Moreover, it was also shown that IVF could lead to seroconversion of antiovarian antibodies, a result of either the use of high dosages of gonadotropins or the multiple ovarian traumas during the IVF procedure [202]. Wheatcroft et al. [203] showed that antiovarian antibodies could be secondary to ovarian cell destruction. They first established, using an ELISA with two separate human ovarian antigen preparations, that 24 and 60% of POF sera showed activity. However, frequent crossreactivity with fallopian tube preparations occurred, and patients with Turner syndrome and iatrogenic causes of ovarian failure such as chemotherapy and radiation were also positive [203]. Yet another specific set of ovarian antibodies playing a possible role in POF might be the antibodies to the zona pellucida. The zona pellucida is the acellular matrix surrounding developing and ovulated oocytes and is also detectable in atretic follicles. Autoantibodies to zona pellucida have been described as a cause of infertility in women. In women with unexplained infertility, these antibodies were seen in 5.6% of cases, whereas positivity was seen in only 1.7% of normal controls [204]. Zona pellucida antibodies were thought to interfere with sperm-oocyte interaction, thus inducing infertility. Animal models have, however, demonstrated that the zona pellucida antibodies interfere with follicular development, and the presence of these antibodies in experimental animals leads to follicular depletion and amenorrhea. It has thus become gradually clear from studies on antiovarian antibodies that the presence and clinical activity of POF do not correlate with the presence of these antibodies in serum. Moreover, the results indicate that although ovarian antibodies are common in POF, their pathogenic role remains questionable. They might well be the consequence rather than the cause of the disease.

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D. Receptor Autoantibodies in Patients with Idiopathic POF Conflicting results have also been obtained in investigations into so-called receptor antibodies. Receptor antibodies are directed to membrane receptors for hormones, and these antibodies can mimic the action of the hormone if they have a similar specificity and affinity for the receptor. Stimulating antibodies to the TSH receptor are the cause of hyperthyroidism and goiter formation in patients with Graves’ disease [205]. On the other hand, receptor antibodies may also block the action of the corresponding hormone if they lack a stimulatory action but still bind to the receptor. Blocking receptor antibodies have been described as causal for myasthenia gravis (blocking antibodies to the acetylcholine receptor), some forms of insulin-resistant diabetes (blocking antibodies to the insulin receptor), and primary hypothyroidism (blocking antibodies to the TSH receptor [206]). It is thus a small step to the idea that receptors such as LH and FSH receptors might become targets for blocking antibodies, and such hypothetical antibodies would be a cause of ovarian failure. Austin was the first to develop an assay system to test the hypothetical presence of blocking LH receptor antibodies in patients with POF [207]. When the sera of POF patients were incubated with radiolabeled hCG and LH receptor preparations of rat or human corpora lutea, no difference in binding was observed when compared with controls. Tang and Faiman [208] used bovine testicular membranes to detect serum factors in POF patients interacting with the gonadotropin receptors. In only one POF patient—with rheumatoid arthritis, vitiligo, and Hashimoto’s thyroiditis—was interference with the FSH receptor detected. Later, Chiauzzi et al. were the first to find clear evidence for immunoglobulins blocking the FSH receptor in two patients with POF and myasthenia gravis. The authors used an FSH-binding assay with rat testicular membranes [209]. Our group [210] reported on the presence of antibodies able to block the growth of granulosa cells in 21 of 26 patients with POF. Protein A–sepharose purified IgG fractions obtained from the serum of these patients interfered with FSH-induced granulosa cell growth in rat ovaries measured by DNA synthesis. Four of the 26 patients with these granulosa cell growth–blocking antibodies had Cy-Ad antibodies; one also had StCA. Moncayo et al. found evidence of antibodies in patients treated for sterility (note: no POF patients) that recognize the hCG receptor of human or bovine origin in an ELISA [211]. A proportion of these antibodies bound to the unoccupied LH/hCG receptor, while others reacted only to the complex formed by the hormone plus the receptor. Wheatcroft, et al. could not reproduce these results [203]. The group did find LH- blocking activity in IgG preparations from 2 of 10 women with POF using an assay of LH-stimulated testosterone production by rat Leydig cells [203]. We could not detect these antibodies in our patient group [210]. Recent data using cloned human LH and FSH receptors indicate that human gonadotropin receptors are highly selective for their human ligands [212,213], and this selectivity may also apply for the receptor antibodies. Therefore, Anasti et al. used recombinant human gonadotropin receptors to detect a putative presence of immunoglobulins directed against the gonadotropins or their receptors in sera of patients with POF [214]. They were unable to demonstrate the presence of blocking antibodies to LH or FSH receptors in any of the 38 POF patients studied. In conclusion, the data on receptor antibodies in POF are not conclusive; antibodies to the LH and FSH receptors may exist, but their precise role and prevalence require further studies. Support for POF being related to the above-described other autoimmune disorders has also been given in an immunogenetic study by Walfish, et al. who reported an associa-

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tion of POF with HLA-DR3 and HLA-B35, with a relative risk of 4.3 and 2.99, respectively, for the presence of POF [215]. That study involved 22 POF patients without autoimmune adrenal pathology. Others were, however, unable to confirm this association in later studies on larger groups of POF patients, of whom only a small number had associated Addison’s disease [216,217]. E. Cellular Immune Abnormalities in Patients with Idiopathic POF Recent literature [218] in the field of thyroid autoimmunity and type 1 diabetes indicates that immune cells, such as CD4Ⳮ Th1 lymphocytes, macrophages, and CD8Ⳮ T-cytotoxic cells, are more important in the destruction of endocrine cells in endocrine autoimmunity than autoantibodies. So what is the evidence of such immune cell involvement in idiopathic POF? Though the data on the numbers of CD3Ⳮ, CD4Ⳮ, and CD8Ⳮ T cells vary among the reported studies [187,195,119–223], a consistent pattern of an increased number of activated T cells (as defined by MHC class IIⳭ or IL-2RⳭ) is evident in the majority of the studies [187,221–223]. Similar increased numbers of activated peripheral blood T cells have been described in other autoimmune endocrinopathies, such as recent-onset Graves’ disease [224], type 1 diabetes [225], and Addison’s disease [226]. A word of caution is, however, needed, since we recently observed that postmenopausal women may also show raised numbers of activated peripheral T cells [223]. Estrogen substitution lowered the number of activated peripheral T cells in women with POF, although not to completely normal levels. Ho et al. also demonstrated the importance of estrogen status for the number of peripheral blood lymphocyte subsets [227]. We therefore consider the hypergonadotropic hypoestrogenic hormone status present in POF patients and postmenopausal women as partly responsible for the raised numbers of activated blood T cells. Another more direct indication of the involvement of the T-cell system in the pathogenesis of POF is given in the experiments of Pekonen et al., who detected in several cases of POF a positive MIF test towards gonadal antigens [195]. The cumulative data on T cells in the literature might thus be some support for a T-cellular autoimmune response towards gonadal antigens in POF, but again the question of consequence or cause must be addressed. With regard to peripheral B-cell numbers, two of three reports [219,220,223] detected an increase in the number of peripheral blood B cells [220,223]. Ho et al. were able to correlate the raised numbers of peripheral blood B cells to the presence of various autoantibodies [220]. We were unable to confirm this correlation [223]. A similar increase in the number of peripheral B cells has been observed in other autoimmune endocrinopathies. It is therefore not unreasonable to interpret the raised numbers of peripheral blood B cells as a sign of activation of the humoral immune system crucial for autoantibody production because estrogen substitution in POF women did not lower the raised number of peripheral B cells [223]. With regard to the number and activity of peripheral NK cells in POF, two reports have been published. We showed a decrease in the number of peripheral CD56Ⳮ/CD16Ⳮ/ CD3⬃ NK cells [223]. Pekonen et al. showed a decreased activity (lysis of K562 cells) of normal numbers of peripheral blood NK cells in 30% of POF women [195]. A lowered activity of NK cells has also been described in patients with Graves’ disease [228], and it was hypothesized that these lowered numbers of NK cells or the lowered activity of the cells might ‘‘disinhibit’’ B cells and hence stimulate autoantibody production. On the

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other hand, it has been hypothesized that a decreased activity of the peripheral blood NK cells indicates a susceptability to viral infection increasing the chance for a viral oophoritis. However, there is hardly any clinical or histopathological evidence for viral infection in POF. Van Kasteren et al. [229] studied lymphocyte subsets in women with elevated early follicular FSH and infertility (incipient ovarian failure: IOF) and compared data with those of POF patients and healthy controls. Changes in lymphocyte subsets appeared to be linear from controls via IOF to POF patients, showing an increase in B cells and decreases in CD8Ⳮ T cells and NK cells. An interesting new avenue is the study of the number and functions of monocytes and monocyte-derived APC, e.g., DC, in endocrine autoimmune disease. Abnormal functions of monocytes and monocyte-derived APC (abnormal chemotaxis, abnormal interaction with T cells) have been found by our group and others in type 1 diabetes [230] and Graves’ disease [231]. Studies have also been extended to POF, and similar disturbances were found that were not correctable by estrogen substitution. The abnormalities in the function of peripheral monocytes, monocyte-derived APC, T cells, and B cells in patients with POF seem to be part of a more complex cell-mediated immune abnormality, including defects in the delayed-type hypersensitivity (DTH) reactivity to Candida antigen [232] and MIF production of peripheral T cells towards this commensal antigen [219]. Although we do not understand the clinical significance of these general defects and abnormalities in cell-mediated immunity in POF patients (patients did not show recurrent infections), they might be related to an immunodysregulation leading to this form of autoimmunity. It must be noted in this respect that patients with chronic mucocutaneous candidiasis (part of the APS type I) and patients with recurrent vaginal candidiasis (who do show DTH abnormalities to candida) also show a raised incidence of autoantibodies towards ovarian antigens [233]. Whether the APS type 1 syndrome (where there is a connection between candidiasis and oophoritis) represents the extreme of a spectrum of disorders combining T-cell deficiencies with ovarian autoimmunity requires further investigation. VIII. CONCLUSION POF in association with Addison’s disease/adrenal autoimmunity (2–10% of cases) is an endocrine autoimmune disorder. This view is supported by the presence of autoantibodies to steroid-producing cells in the patients, the characterization of shared autoantigens between adrenal and ovarian steroid-producing cells, and the histological picture of the ovaries of such cases (lymphoplasmacellular infiltrate particularly around steroid-producing cells). The existence of an animal model for the autoimmune syndrome of adrenalitis/ oophoritis (the neonatal thymectomy mouse model) lends additional support to this view. There is some (debatable) evidence that certain cases of idiopathic POF may belong to the group of endocrine autoimmune diseases. The positive, albeit circumstantial evidence consists of the fact that these cases of POF show similar cellular immune abnormalities as other endocrine autoimmune diseases such as type 1 diabetes, Graves’ disease, and Addison’s disease. These cellular immune abnormalities include abnormalities in the numbers and/or function of peripheral monocytes, monocyte-derived APC, and subsets of T cells and B cells. Another point of positive evidence might be the more than normal association of POF with type 1 diabetes and myasthenia gravis. The data on antiovarian antibodies and antireceptor antibodies are not conclusive, since these antibodies—although found by the majority of authors—might be the consequence than the cause of the disease.

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Another point of concern is that the histology of POF in the absence of Addison’s disease/ adrenal autoimmunity rarely shows oophoritis (⬍3%).

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169. Sedmak DD, Hart WR, Tubbs RR. Autoimmune oophoritis: a histopathological study involved ovaries with immunologic characterisation of the mononuclear cell infiltrate. Int J Gynecol Pathol 1987; 6:73–81. 170. Wolfe CDA, Stirling RW. Premature menopause associated with autoimmune oophoritis. Case report. Br J Obstet Gynecol 1988; 95:630–632. 171. Biscotti CV, Hart WR, Lucas JG. Cystic ovarian enlargement resulting from autoimmune oophoritis. Obstet Gynecol 1989; 74:492–495. 172. Bannatyne P, Russell P, Shearman RP. Autoimmune oophoritis: a clinicopathologic assessment of 12 cases. Int J Gynaecol Pathol 1990; 9:191–207. 173. Lonsdale RN, Roberts PF, Trowell JE. Autoimmune oophoritis associated with polycystic ovaries. Histopathology 1991; 19:77–81. 174. Russell P, Bannatyne P, Shearman RP, Fraser IS, Corbett P. Premature hypergonadotropic ovarian failure: clinicopathological study of 19 cases. Int J Gynecol Pathol 1982; 1:185–201. 175. Friedman CI, Gurgen-Varol F, Lucas J, Neff J. Persistent progesterone production associated with autoimmune oophoritis. J Reprod Med 1987; 32:193–196. 176. Edmonds M, Lamki L, Killinger DW, Volpe R. Autoimmune thyroiditis, adrenalitis and oophoritis. Am J Med 1973; 54:782–787. 177. Emperaire JC, Audebert A, Greenblatt RB. Premature ovarian failure. Am J Obstet Gynecol 1970; 108:445–449. 178. Zarate A, Karchmer S, Gomez E, Castelazo-Ayala L. Premature menopause. Am J Obstet Gynecol 1970; 106:110–114. 179. Sharf M, Isreali I, Graff G. The value of ovarian biopsy in the diagnosis and treatment of amenorrhoea related sterility. Obstet Gynecol 1972; 39:89–94. 180. Starup J, Sele V. Premature ovarian failure. Acta Obstet Gynecol Scand 1973; 52:259–268. 181. Falk RJ. Euestrogenic ovarian failure. Fertil Steril 1977; 28:502–503. 182. Duignan NM. Sex hormone levels and gonadotrophin release in premature ovarian failure. Br J Obstet Gynaecol 1978; 85:862–867. 183. Board JA, Redwine AO, Moncure CW, Frable WJ, Taylor JR. Identification of differing etiologies of clinically diagnosed premature menopause. Am J Obstet Gynecol 1979; 134: 936–944. 184. Rebar RW, Erickson GF, Yen SSC. Idiopathic premature ovarian failure: clinical and endocrine characteristics. Fertil Steril 1982; 37:35–41. 185. Menon V, Logan Edwards R, Butt WR, Bluck M, Lynch SS. Review of 59 patients with hypergonadotrophic amenorrhoea. Br J Obstet Gynaecol 1984; 91:63–66. 186. Aiman J, Smentek C. Premature ovarian failure. Obstet Gynecol 1985; 66:9–14. 187. Miyaka T, Sato Y, Takeuchi S. Implications of circulating autoantibodies and peripheral blood lymphocytes for the genesis of premature ovarian failure. J Reprod Immunol 1987; 12:163–171. 188. Rebar WR, Connolly HV. Clinical features of young women with hypergonadotropic amenorrhea. Fertil Steril 1990; 53:804–810. 189. Muechler EK, Huang K, Schenk E. Autoimmunity in premature ovarian failure. Int J Fertil 1991; 36:99–103. 190. Hoek A, Schoemaker J, Drexhage HA. Premature ovarian failure and ovarian autoimmunity. Endocrine Rev 1997; 18:107–133. 191. Moncayo-Naveda HE, Moncayo R, Benz R, Wolf A, Lauritzen C. Organ specific antibodies against ovary in patients with system lupus erythematosus. Am J Obstet Gynecol 1989; 160: 1227–1229. 192. de Moraes-Ruehsen M, Blizzard RM, Garcia-Bunuel R, Jones GS. Autoimmunity and ovarian failure. Am J Obstet Gynecol 1972; 112:693–703. 193. Friedman S, McCormick JN, Fudenberg HH, Goldfien A. Ovarian antibodies in disorders of ovarian function. Clin Immunol Immunopathol 1972; 1:94–103.

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26 Myasthenia Gravis ¨ BEL, and HANS KONRAD MU ¨ LLERALEXANDER MARX, PHILIPP STRO HERMELINK University of Wu¨rzburg, Wu¨rzburg, Germany

I. INTRODUCTION Myasthenia gravis (MG) is a rare neurological disorder characterized by severe, sometimes life-threatening muscle weakness. MG is a prototypical model for antibody-mediated autoimmune diseases, since the effector pathway (i.e., autoantibodies binding to a known target antigen) has been confirmed by many clinical and experimental settings. The target antigen in MG, the acetylcholine receptor (AChR), and the effects of autoantibody binding have been well characterized. A point that merits particular consideration is the observation that MG is almost invariably associated with pathological alterations of the thymus. There is now very good evidence that MG is not a single disease, but rather a common symptom shared by a variety of pathogenetically different diseases. Recent scientific work has helped to characterize a new subgroup of patients with ‘‘seronegative’’ MG (autoantibodies against a muscle-specific receptor tyrosine kinase, MuSK) as opposed to classical ‘‘seropositive’’ MG (autoantibodies against the AChR). MG occurs in two age groups with significant clinical and epidemiological differences. In up to 70% of patients, MG presents between the age of 10 and 40 years (early-onset MG) and is usually associated with significant lymphofollicular hyperplasia of the thymus (thymitis). In elderly patients (lateonset MG), MG may be associated with either thymic epithelial tumors (thymomas) or thymic atrophy. This review will focus on current pathogenetic concepts in the different MG-associated entities. For a review of the Lambert-Eaton myasthenic syndrome, which will not be covered here, see Refs. 1–4. II. THE PATHOPHYSIOLOGY OF MYASTHENIA GRAVIS Transmission at the neuromuscular junction depends on release of acteylcholine (ACh) from the motoneuron and its interaction with AChRs on the postsynaptic membrane. 571

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Binding of ACh to the AChR leads to the transient opening of ion channels, resulting in an increased endplate potential. If the resulting electrical potential exceeds a threshold amplitude, it generates an action potential that spreads along the muscle fiber and triggers the release of Ca2Ⳮ with subsequent muscle contraction. Muscle action is the net effect of hundreds to thousands of single muscle fibers stimulated by the neuromuscular transmission. Under normal conditions, the endplate potential is well beyond the potential required for muscle fiber contraction [5]. Since the numbers of AChRs at the neuromuscular endplate in patients with MG are decreased, the resulting endplate potential is not sufficient to evoke an action potential and thus neuromuscular transmission fails in many synapses, leading to clinically manifest muscle weakness [6]. In general, the degree of reduction of AChRs correlates with the clinical severity of MG, although clinically strong muscles may have reduced AChR numbers [7]. Compensatory mechanisms at the neuromuscular endplate of individual muscles, so-called safety factors, may account at least in part for this phenomenon [8]. III. CLINICAL FEATURES For a detailed review of the clinical features, see Ref. 9. MG is a rare disease with a prevalence of 12–18/100,000 population [10]. MG patients are at increased risk to develop other autoimmune diseases, such as Graves’ disease, Hashimoto’s thyroiditis, or rheumatoid arthritis [11,12]. In a majority of patients, ocular muscle weakness, usually presenting as ptosis and/ or diplopia [13], is the initial symptom. In the following months, 85% of patients develop generalized myasthenic symptoms involving facial and bulbar muscles as well as limb and respiratory musculature. Especially involvement of the respiratory muscles may be life-threatening (myasthenic crisis) and may require assisted ventilation. For clinical purposes, the severity of MG is graded using the modified Ossermann classification system [14]. In only about 15% of patients does MG remain confined to the ocular region (purely ocular MG) [15]. The underlying causes for the preferential weakness of extraocular muscles (EOM) are largely enigmatic. EOM may have a low safety factor, or the AChR in EOM may have antigenic properties different from other muscles [16], including the occurrence of fetal AChRs at the neuromuscular junction, as detected in rat EOM [17] or higher content of the adult-type ε subunit of the AChR [18]. About 40% of patients with pure ocular MG have no detectable AChR autoantibodies at all in conventional detection assays [19]. In light of all current evidence, the possibility that non-AChR epitopes specific for EOM may be responsible for the preferential involvement of this region cannot be ruled out. IV. DIAGNOSIS Clinical history and physical examination are of great importance in the diagnosis of MG. However, a definitive diagnosis requires further tests, including effect of ACh-esterase inhibitors (Tensilon test), electromyography, and determination of anti-AChR antibodies in serum. By inhibition of the enzyme ACh-esterase, which degrades ACh released into the synaptic gap, intravenous application of edrophonium (Tensilon) leads to at least a partial, temporary improvement of strength in an objectively weak muscle (positive Tensi-

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lon test). However, false-negative tests are not infrequent in MG patients, and repeated testing is recommended in cases where MG is considered clinically. False positives may occur in a number of motor neuron diseases, Lambert-Eaton syndrome, or even healthy control persons [20]. Single-fiber electromyography (SFEMG) is the most sensitive test in the detection of neuromuscular transmission abnormalities [21]. In myasthenic muscles, repetitive muscle fiber stimulation may lead to a decrease in evoked action potential. Reduced response of the compound muscle action potential is detectable in up to 90% of MG patients and may be particularly helpful in difficult diagnostic situations, but the specificity of the test is limited [22]. The gold standard for diagnosis of MG is measurement of AChR autoantibodies, since positive detection of these antibodies is virtually specific for MG. However, AChR antibodies are detectable in only about 85% of patients. In some cases the antibody titers are only marginally elevated. The majority of antibody-negative (seronegative) MG patients can be expected to harbor antibodies to a muscle-specific receptor tyrosine kinase (MuSK) (see below) [23]. However, established tests for use in clinical routine are not yet available.

V. GENETIC BACKGROUND IN THE PATHOGENESIS OF MG Genetic factors are believed to play an important role in the susceptibility to MG, since there is a considerable concordance rate of monozygotic twins and an increased risk of MG in relatives of patients with MG [24]. Moreover, MG patients and their relatives are at an increased risk to develop other autoimmune diseases such as Hashimoto’s thyroiditis and lupus erythematosus, suggesting that genetic defects of immune regulation might contribute to the predisposition to MG [25]. MG, like other autoimmune disorders, is probably a multigenic diesase [26,27]. Association with particular major histocompatibility complex (MHC) loci has attracted particular interest, but the correlation of MG with MHC haplotypes is weak and varies among different ethnic populations [28–30]. Data obtained from animals with EAMG suggest that properties of the molecular structure of the MHC class II contribute to the predisposition to MG [31]. While MG in young Caucasian women shows moderate association with HLA isotypes B8, DR3, and DQw2 [28,29], the predisposing isotypes in thymoma are DR2 and A24 and DR2/B7 in atrophic thymus. Surprisingly for a disease assumed to depend on MHC class II–restricted T-cell help, the strongest association is with the class I molecule B8 [15]. Whether this may hint at an immunoregulatory role of CD8Ⳮ T cells is not clear. Alternatively, other genes close to class I locus with potential interest for the pathogenesis of MG such as tumor necrosis factor (TNF), heat-shock proteins, and TAP transporters have been discussed [32–34].

VI. THE ACETYLCHOLINE RECEPTOR The AChR is a heteromeric 250 kDa transmembrane glycoprotein made up of five subunits arranged around a central channel [35–37]. The different subunits are highly conserved and share considerable interspecies homology [38]. There are two isoforms—fetal and adult—which are developmentally regulated (Fig. 1). Both isoforms contain two ␣ subunits and one ␤ and one ␦ subunit. The two isoforms differ by a ␥ subunit in fetal AChR instead

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Figure 1 (a) Architecture of the postsynaptic membrane. ACh molecules pass through the central vestibule of the AChR channel before entering tunnels to their binding sites. Cations leave or enter the channel on the cytoplasmic side of the membrane through narrow openings in the channel wall. These narrow openings are framed by negatively charged residues excluding anions from the vicinity of the transmembrane pore. Rapsyn is thought to be attached to the innermost end of the AChR. (b) Diagram of AChR␣-subunit depicting the four hydrophobic transmembrane domains (M1–M4), the ACh-binding site (ACh), the ‘‘very immunogenic cytoplasmic epitope-␣’’ (VICE-␣), and the ‘‘main immunogenic region’’ (MIR). (c) Diagram of fetal and adult AChR subtypes and the location of the MIR. The broken lines in (b) and (c) indicate that the MIR is not a single epitope. Instead, for different anti-MIR antibodies, different nearby or remote residues contribute to the whole MIR in addition to the critical ␣67–76 core epitope. (Figure reprinted from Ref. 184).

of the adult ε subunit. Each subunit has an extracellular domain and two transmembrane domains [9]. A high proportion of polyclonal antibodies from MG patients has been shown to bind to a site of the extracellular domain of the AChR␣ subunit called the main immunogenic region (MIR) [39]. The MIR contains several epitopes, since antibodies of different specificities can bind to it [40,41]. High-resolution microscopic techniques have shown that the MIR is located at an exposed position of the AChR␣ subunit that allows autoantibodies to cross-link neighboring AChRs, thus facilitating accelerated receptor internalization and degradation [42]. Not all AChR antibodies, however, bind to the MIR. Each of the AChR subunits contains antibody-binding sites, and a small subset of patients has been identified whose antibodies bind only to adult form, i.e., to the ε subunit of the AChR [43]. In experimental animals, injection of denatured AChR results in the production of antibodies to cytoplasmic sites of the AChR on various subunits [44–47]. The main binding site on the ␣ subunit corresponding to sequence ␣373–380 has been termed ‘‘very immuno-

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genic cytoplasmic epitope alpha’’ (VICE-␣) [44]. The other major epitope recognized after immunization is the VICE-␤ epitope, located at ␤354–359, one of the phosphorylation sites of the AChR [45]. Indeed, a small subgroup of MG patients has been shown to produce low titers of anti-VICE-␣ antibodies [48].

VII. AUTOANTIBODIES IN NONTHYMOMA MG In the majority of patients, MG is caused by polyclonal serum autoantibodies of various idiotypes directed against the muscle form of the nicotinic AChR [49–51]. Autoantibody levels can be accurately measured, for example, by immunoprecipitation of 125I-␣BuTx–labeled AChRs extracted from human muscle [50,52]. Although alterations of the autantibody titer in an individual patient correlate well with changes in clinical status [53], the serum autoantibody concentration per se does not correspond well with the clinical severity of the disease [54]. However, the causative role of autoantibodies to the AChR in the pathogenesis of MG has been firmly established by the following experiments: (1) clinical disease including the characteristic morphological changes at the neuromuscular junctions can be passively transferred to experimental animals by injection of patients’s IgG antibodies into mice [55–57]; (2) injection of purified AChR into experimental animals causes a very similar disease termed experimental autoimmune myasthenia gravis (EAMG) [58,59]; (3) plasma exchange, which removes circulating autoantibodies, leads to a dramatic transient improvement in muscle function in the great majority of patients [60]. There are three basic mechanisms by which antibodies in MG have been shown to impair neuromuscular transmission. First, antibodies can mediate direct damage of the postsynaptic membrane by complement activation [61]. Second, cross-linking with the AChR can lead to accelerated receptor internalization and degradation [62,63]. Third, autoantibodies can block binding of ACh released into the synaptic cleft to the receptor [64]. The poor correlation between autoantibody levels and clinical disease severity has been explained by different properties of the autoantibodies in different patients (e.g., the ability to bind and activate complement) as well as different safety factors. The majority of autoantibodies (60–70%) directed against the AChRa subunit recognize a sequence including the MIR. From in vitro AChR protection assays using monoclonal antibody fragments directed against the different subunits [65,66], it has been concluded that the weak correlation between antibody level and clinical symptoms may be in part related to different titers of anti-␣-subunit (anti-MIR) antibodies [67]. In support of these data titers of autoantibodies with anti-AChR␣-subunit specificity have been reported to correlate better with clinical status than total anti-AChR antibody titers [9]. About 10–20% of patients with otherwise typical MG do not have increased AChR autoantibody serum levels, hence the designation seronegative myasthenia gravis. Although it is not clear at the moment whether seronegative MG is a single entity, passive transfer experiments have shown that seronegative MG is also an antibody-mediated disease [33,68,69]. Experiments with IgGs of seronegative patients pointed to a musclespecific antigen other than AChR. In more than 70% of cases, IgG from seronegative patients bind to the muscle-specific receptor tyrosine kinase (MuSK). MuSK is involved in agrin-induced clustering of AChRs during synapse formation and is also expressed at mature neuromuscular junctions [23,70]

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VIII. CELLULAR IMMUNITY IN NONTHYMOMA MG: ROLE OF T CELLS The production of AChR autoantibodies depends on MHC class II restricted Th1 and Th2 cells [71–75]. In studies with AChR peptide libraries, T cells of most patients with generalized MG recognize all AChR subunits, including the fetal-type ␥ subunit [76,77]. The anti-␥-subunit T-cell response has been reported to correlate with ocular manifestations [77–79]. Moreover, CD4Ⳮ T cells of patients with ocular MG recognized preferentially peptides of the ␥ and virtually never of the ε subunit [77]. This is very surprising considering the observation that, on the B-cell level, the ε subunit appears to be a preferential target of pure ocular myasthenics [52]. In contrast to the high anti-␥-subunit T-cell responses, T-cell reactivities to ␣-, ␤-, and ␦-subunit peptides are variable and generally low in pure ocular MG, and CD4Ⳮ responses are generally much lower than those observed for generalized MG patients [77]. The heterogeneity of the T-cell AChR epitope specificities both in individuals and among different patients has been interpreted to indicate that there may be no major immunodominant T-cell epitope in humans [77,80]. The relevance of investigations based on T-cell stimulation with small peptides, however, has been questioned [81,82], since it is generally assumed that processed native AChR stimulates the T cells that direct autoantibody synthesis in vivo [24]. Importantly, recent data on T-cell clones recognizing native human AChR demonstrate that the relevant peptide can be generated by AChR processing in vivo. Furthermore, the restricted epitope specificity of these clones [83,84], as opposed to the broad peptide reactivity of T cells reported by Conti-Fine et al. [77], suggests that there only few T cells are capable of recognizing epitopes naturally processed from the AChR protein. Therefore, these data have been interpreted by the Oxford group to indicate the existence of an immunodominant T-cell epitope on the AChR ε subunit in humans with a new candidate susceptibility allele, DR52a [85]. Clearly, many more clones need to be studied before definite conclusions can be drawn. Whether autoreactive T-cell repertoires in the various myasthenia subtypes are different is controversial [77,81,86–89]. Most importantly, AChR-reactive T cells occur both in the majority of nonmyasthenic controls [87–90] and in a wide range of aninial species [91]. These T cells, belonging to the normal T-cell repertoire, are not anergized but are naive [81], suggesting that MG does not result from a lack of AChR-specific T-cell tolerance. However, future studies will have to exclude the existence of MG-specific autoaggressive T cells (i.e., of T cells absent from the normal repertoire) in terms of antigen specificity, cytokine profile, or ability to provide B-cell help. IX. ROLE OF THE THYMUS IN MYASTHENIA GRAVIS MG is almost invariably (90%) associated with morphological changes of the thymus, thus making a pathogenetic role of the thymus very likely. A. The Thymus in Early-Onset Seropositive MG In the vast majority of early-onset MG, the thymus shows lymphofollicular hyperplasia (thymitis). In thymitis, the perivascular spaces are extended by B cells forming folicles and germinal centers. By disruption of basal membranes, medulla and perivascular spaces fuse [92,93]. Both T and B cells isolated from thymuses of myasthenic patients are more responsive to AChR than cells derived from the peripheral blood [87]. Both normal and

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myasthenic thymuses contain a peculiar, noninnervated muscle cell of unknown function that express AchRs, so-called myoid cells [94–96]. Myoid cells are located in close contact with immunocompetent cells and may play an important role in central tolerization against muscle antigens. In contrast, breaking of tolerance, e.g., through attack of myoid cells by interdigitating dedritic cells [96,97] might lead to an self-sustaining immune response against AChRs. Another arguement supporting an etiological role of the thymus in the pathogensis of MG is the fact that up to 85% of patients experience clinical improvement after thymectomy [98–100]; 35% even achieve drug-free remission [101]. B. The Thymus in Seronegative Myasthenia Gravis The histological picture in seronegative MG is somewhat different from changes in seropositive cases in that lymphofollicular hyperplasia is usually mild. The main histological change results from an increase of mature T cells in perivascular spaces [34]. Whether these changes also lead to a breakdown of the interface between perivascular spaces and thymic medulla has so far not been investigated. C. The Thymus in Nonthymoma Late-Onset MG So-called thymic atrophy in myasthenia gravis is encountered in 10–20% of MG patients [102]. Because of distinct epidemiological and genetic findings (onset after age 40, male predilection, association with HLA B7 and DR2) and a short course of disease, thymus atrophy is not considered an end stage after thymitis. Morphologically, except from a slight increase in medullary B cells and interdigitating reticulum cells [92], the thymuses in these patients are equivalent to those in age-matched controls. In particular, the number of myoid cells per thymic tissue area follows the same age-related decline [96]. X. TREATMENT Current treatment options for MG include thymectomy, medications with cholinesterase inhibitors, corticosteroids, immunosuppressive agents (azathioprine, cyclosporine, methotrexate, cyclophosphamide) [103,104] and immunmodulatory techniques such as plasmapheresis or intravenous immunoglobulins [104]. With these treatments, myasthenic symptoms can be controlled in the vast majority of patients [15]. The benefit of thymectomy in patients with seronegative MG or thymus atrophy awaits further statistical support [83,105–108]. After surgery, MG in thymoma patients may take an unpredictable course, but usually also shows gradual improvement (own observation). However, even complete surgical removal of thymoma plus residual thymus is often not followed by a decline of autoantibody titers [109]. XI. PATHOGENETIC CONCEPTS IN MG A. Etiology While many steps in the pathogenesis of MG have been clarified (see below), the disease trigger has remained enigmatic except for D-penicillamine–induced MG [110]. Infections have long been thought to be such triggers, but how they could break T-cell tolerance remains controversial. It has been suggested [111] that superantigens expressed by bacteria or viruses might unspecifically stimulate antigen-presenting cells and unprimed AChR-

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specific T cells that occur in the normal human T-cell repertoire. Autoimmunity in this situation may be elicited only in the context of a suitable genetic background, as suggested for multiple sclerosis. More popular has been the view that microbial antigens crossreacting with self antigens may trigger autoreactivity [9,112]. However, experimental evidence for such molecular mimicry is still lacking [9]. Cross-reactivity could happen at both the B- and T-cell levels. Molecular mimicry on the B-cell level involving epitopes of the AChR or other autoantigens relevant in MG has been reported previously [81,113–117], but whether these epitopes are important in vivo has not been elucidated [24,34,113]. In particular, it has not been dernonstrated that B cells can elicit antigenspecific autoreactive T-cell activation by shared B- plus T-cell epitopes, although B cells in mice can contribute to the diversification of immune responses [118]. There is some experimental evidence that molecular mimicry on the T-cell level could play a role in initiating autoreactivity even if only one T-cell epitope is involved [71]. This situation is now described as ‘‘determinant spreading,’’ as described below [85,119]. In the context of the human disease (MG), endogenous AChR could be released from peripheral skeletal muscle or thymic myoid cells as a consequence of either an inflammatory response in the vicinity of MG endplates [120,121] or the abnormal attack of interdigitating dendritic cells on myoid cells in thymitis [96,97]. The mechanisms of autoantigen release and autopresentation, however, are unknown [24,122]. Once initiated, the process may be selfsustaining due to the constant release of endogenous autoantigen. Detecting the (triggering) events among the secondary effects will obviously be the challenge of future experiments and epidemiological investigations addressing the etiology of MG. B. Determinant Spreading One of the pivotal mechanisms of autoimmunization [85,119], the concept of determinant spreading, comes from experimental autoimmune myasthenia gravis (EAMG), where this phenomenon has been observed in a number of species [123]. Determinant spreading refers to the broadening of an immune response against many epitopes of one or more antigen(s), while the triggering immunogen has only a limited number of epitopes. When the immunogen is part of the target antigen, the process is called intramolecular determinant spreading. When the immune reaction spreads to unrelated target antigens (e.g., from an immunizing virus to an autoantigen with shared epitopes), the process is referred to as intermolecular determinant spreading. In EAMG, there is evidence for both intra- and intramolecular determinant spreading. When rabbits are immunized with peptides covering the human sequence ␣138–199, most of the animals initially produce non-myasthenogenic antibodies against the human peptide. However, later and after boosting with the human peptides, many animals start producing antibodies to native rabbit AChR epitopes and develop EAMG [85]. Of note, the myasthenogenic autoantibodies react more strongly with rabbit than with human AChR, and a majority is directed against the rabbit MIR while they do not cross-react with the triggering ␣138–199. The anti-peptide antibodies do not cross-react with the native rabbit AChR, and the antipeptide antibody levels are not correlated with the anti-native AChR autoantibody levels. From these findings, it was concluded that immunization with the human sequence ␣138–199 led to autoimmunization to self (rabbit) AChR [85]. The mechanisms of determinant spreading probably involve T-cell epitopes that are part of the peptide used for immunization (e.g., the sequence ␣146–162 within the ␣138–199 peptide). T cells activated by an MHC-presented xenogeneic AChR sequence

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may subsequently recognize the same or a homologous sequence processed from the autologous AChR. The source of the autologous AChR for determinant spreading in EAMG is not clear so far. Autologous AChRs are probably not derived from the myoid cells of the thymus (see below), given the observation that immunization of rats with Torpedo AChR does not induce lymphofollicular thymic hyperplasia (thymitis), which is characteristic of early-onset myasthenia in humans [124]. More likely, anti-Torpedo-peptide antibodies may induce an inital release of autologous AChR from the neuromuscular junction after complement-mediated lysis. After onset of an intramuscular inflammation, cytotoxic T cells may help to further aggravate muscular damage and thus in AChR release, since inflammation inside the muscle (as it occurs in EAMG and MG) can induce MHC class I (and II) [125] on muscle cells, which then become targets for cytotoxic T cells [122]. In spite of many unresolved questions, the finding that immunization with short linear peptides can induce a complex antibody response against conformation-dependent epitopes of the native AChR clearly raises the possibility that cross-reacting T-cell epitopes may be involved in the pathogenesis of MG [85,126–128]. C. Pathogenetic Concepts in Lymphofollicullar Hyperplasia (Thymitis) Some authors considered lymphofollicular thymitis a secondary phenomenon following the sensitization of T cells in the periphery, recirculation to the thymus, and restimulation there [34,129]. However, we and others favor a primary intrathyrnic pathogenesis of MG, as suggested by Wekerle almost 20 years ago [130]. According to this hypothesis, AChR on thymic myoid cells are primarily involved in the triggering of MG in lymphofollicular thymitis. Three findings support this notion: (1) A substantial percentage of autoantibodies in thymitis-associated MG specifically recognize the fetal type of AChR (131); (2) fetaltype AChR (i.e., AChR with a ␥ instead of an ε subunit) are expressed on thymic myoid cells, but not on extrathymic muscle [94,131] except, probably, for multiple-innervated ocular muscles [132]; (3) extrathymic immunization with the AChR can induce EAMG in animals, but does not elicit lymphofollicular thymitis [124]. In support of this concept, Kirchner et al. [96] reported abnormal clusters of myoid cells and antigen-presenting dendritic cells in lymphofollicular hyperplasia (LFH). Since myoid cells remain negative for MHC class II in MG and therefore are unable to present antigen to T cells [94,96], it is thought that the abnormal clustering enables dendritic cells to more efficiently take up AChR released from myoid cells. Processing of engulfed AChR in dendritic cells might result in a quantitatively improved presentation of AChR peptides to potentially AChRspecific T cells that have been found in increased numbers in thymuses with LFH [87,88]. Finally, the thymus with lymphofollicular thymitis is known to be the single most important organ where anti-AChR autoantibodies are produced both in absolute terms and on a per plasma cell basis [133]. Once produced, the autoantibodies may react not only with peripheral muscle AChR but also with AChR on thymic myoid cells. Whether such an antibodymediated or cytotoxic mechanism is the basis of the increased apoptosis of thymic myoid cells in MG [97] has yet to be investigated. As elegantly shown by the transplantation of thymitis specimens into SCID mice (resulting in the prolonged production of human antiAChR autoantibodies in these immunodeficient mice), such thymuses contain all the necessary constituents of a complete and self-sustaining immune reaction [134–136]. The mechanisms eliciting lymphofollicular thymitis are not known.

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D. Pathogenesis of MG in Thymic Atrophy There have been few experimental data on the pathogenesis of MG associated with thymic atrophy that occurs in the late-onset type of myasthenia gravis. Data on the heterogeneous occurrence of autoantibodies to striational antigens [137], particularly titin [138], also suggest a heterogeneity among late-onset MG patients. Pathogenetic models have not been suggested so far. Given that thymoma-associated MG and late-onset MG associated with thymic atrophy share similarities with respect to striational autoantibodies and the usefulness of thymus surgery [137–139], it is tempting to speculate that atrophic thymuses, in conjunction with genetic and environmental susceptibility factors, might contribute to the pathogenesis of MG by similar mechanisms as thymomas (see below). Specifically, we speculate that in late-onset MG the thymus might still contribute new T cells to the peripheral T-cell repertoire. By this principally physiological mechanism [140,141], the newly generated but autoimmunity-prone T cells may gradually replace the tolerant ‘‘historic’’ T cells from the preatrophic era [126]. XII. THYMOMA-ASSOCIATED (PARANEOPLASTIC) MG Thymomas are tumors of the thymic epithelium with the capacity to mature and export thymocytes. There is a large body of evidence showing that the pathogenesis of thymomaassociated MG differs from LFH-associated MG [34,85,86,142]. A broad spectrum of paraneoplastic autoimmune diseases occur either in isolation or associated with MG in thymoma, such as systemic lupus erythematosus, thyroiditis, or pure red cell aplasia. In some of these paraneoplastic syndromes the autoantigens and autoantibodies and their pathogenic relevance have been characterized. Some authors found no increase of nonmuscle autoimmune diseases in MG-associated thymoma patients [143,144]. Concurrent autoimmunity against four apparently unrelated types of autoantigens is highly characteristic of paraneoplastic MG. These autoantigens are (1) the AChR [145], (2) striational muscle antigens, including titin [146], (3) neuronal antigens [147], and (4) cytokines (IL-12, IFN-␣) [148]. Antibodies to IL-12 and IFN-␣ might be involved in the pathogenesis of paraneoplastic MG and are sensitive markers to detect thymoma recurrences [148]. Autoimmunity to the ryanodine receptor is also highly characteristic but less frequent [115,149]. Titers of antiryanodine receptor antibodies significantly correlate with clinical MG severity [115,150], while antititin autoantibodies as markers for disease severity are not unequivocally established [150,151]. A common theme shared by MG-associated thymomas is the occurrence of mRNA coding for the autoantigens mentioned [152–154]. Except for the cytokines, however, there appears to be an apparent lack of the respective proteins [127,149,155–157], although it is not excluded that translation into very small amounts of autoantigenic protein occurs, with potential implications for autoimmunization [158–161]. A major morphological difference between thymoma and TFH is the presence of AChR-expressing myoid cells in LFH but their absence in thymomas. A striking functional difference is the general absence of autoantibody production inside thymomas [162]. XIII. CELLULAR IMMUNITY IN PARANEOPLASTIC MG A. The Role of T Cells The pathogenesis of paraneoplastic MG is probably heterogeneous considering diverse morphological and functional findings [142]. However, MG-associated thymomas share common features:

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1. All MG-associated thymomas share morphological and functional features with the normal thymus (type A, AB, or B1-3 thymomas according to the WHO classification). In particular, they provide signals for the homing of immature hematopoietic precursors and promote their differentiation to apparently mature T cells [163,164]. It is not yet clear whether a few type A thymomas might be an exception to this rule, since they generate at most very few T cells. 2. MG-associated thymomas are enriched for autoreactive T cells with specificity for the AChR␣ subunit and the ε subunit [77,90,164–166]. There is strong evidence that some are generated by intratumorous, non-tolerogenic thymopoiesis [165,166]. These T cells are restricted to the minority HLA isotypes DP14 and DR52a, which are infrequent in MG patients without thymoma [165]. Furthermore, intratumorous and blood T cells exhibit unusual autoantigen specificities [166] 3. All MG-associated thymomas export naive mature T cells [167–169]. Very recently we found that MG-positive and MG-negative thymomas differ with respect to their capacity to complete maturation and export of the CD4 T-cell lineage, i.e., to generate CD4ⳭCD45RAⳭ naı¨ve T-cells [169]. 4. Almost all MG-associated thymomas exhibit reduced expression of MHC class II molecules on neoplastic epithelial cells [34,170]. In parallel, thymopoiesis in thymomas is quantitatively less efficient [163,170]. In addition, reduced MHC class II levels might be one reason why thymopiesis in thymomas is not tolerogenic, i.e., qualitatively abnormal [171]. B. Diverse Features of MG-Associated Thymomas Apart from shared features among MG-associated thymomas, there are also features that are clearly diverse. One such feature is the abnormal (hyper)expression of proteins that are unrelated to the autoantigens but express AChR-, titin-, or ryanodine receptor–like epitopes in neoplastic epithelial cells [127,149,157,166]. These antigens occur in a subset of thymomas only. While the role of such cross-reacting proteins as specific immunogens has been seriously questioned [166,172], it appears likely that abnormally expressed proteins might disturb the normal pool of endogenous peptides for presentation by MHC class II proteins on thymoma epithelial cells [170]. Since quality and quantity of thymic endogenous epithelial cell peptides have a major impact on T-cell selection and tolerance induction [173–176], altered expression of endogeneous proteins in thymoma might be an indirect mechanism resulting in non-tolerogenic intratumorous T-cell development [142]. Loss of heterozygocity (LOH) for the MHC locus in neoplastic epithelium appears to be particularly frequent among MG-associated thymomas, although more cases need to be evaluated. This LOH may result in a MHC chimerism between the hemizygous thymoma epithelium (presumed to perform T-cell selection) and the heterozygous intratumorous dendritic cells and the peripheral immune system. MHC chimerism might be one among many mechanisms of nontolerogenic T-cell selection in a subset of thymomas [177–179]. XIV. PATHOGENETIC MODEL OF PARANEOPLASTIC MYASTHENIA GRAVIS A. Etiology The mechanisms that activate the autoimmunity-prone T-cell repertoire, i.e., the etiologies triggering the MG-provoking autoimmune cascade, have not been defined. We have ob-

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served paraneoplastic MG following unspecific infectious or traumatic triggers, but in most cases no major event can be identified [142]. Whether CD8Ⳮ T cells (122) and genetic susceptibility [180,181] are of etiological relevance has yet to be proven. B. Pathogenesis Taking current evidence into account, the following model can be envisaged for paraneoplastic MG: the vast majority (90–95%) of MG-associated thymomas contribute to autoimmunization by nontolerogenic thymopoiesis and export of naive but potentially autoreactive mature T cells to the periphery [167–169]. Export of naive T cells might gradually replace the normally tolerant, thymus-derived T-cell repertoire by a chimeric autoimmunity-prone T-cell repertoire derived from both the thymoma and the thymus. To become pathogenetically relevant, nonactivated but potentially autoantigen-reactive T cells must become activated (either in the residual thymus or in the periphery) in order to provide help for autoantibody-producing B-cells outside the thymoma [182]. At this stage of pathogenesis a role of a ‘‘CTLA-4 low’’ phenotype [183] and of anti-IL-12 or antiIFN-␣ autoantibodies [148] can be hypothesized facilitating the CD4 T-cell–dependent production of anti-AChR autoantibodies. Generation of the autoimmune T-cell repertoire inside the thymoma may depend on quantitatively and/or qualitatively altered expression of MHC molecules on the tumor epithelium, changed selecting peptide repertoires, and inefficient tolerization (negative selection) of the resulting mature naı¨ve thymocytes. However, this model does not explain why nontolerogenic thymopoiesis in type AB and B thymomas results in only a narrow spectrum of autoantibodies and an even narrower spectrum of autoimmune diseases, with MG outnumbering cytopenias or central nervous system alterations by far [142,165]. It is tempting to speculate that the lack of myoid cells in thymomas might play a role in this respect. ACKNOWLEDGMENT Alexander Marx and Philipp Stro¨bel are supported by Grant QLRT-2000-01918 of the European Community. REFERENCES 1. Lang B, Newsom-Davis J. Immunopathology of the Lambert-Eaton myasthenic syndrome. Springer Semin Immunopathol 1995; 17(1):3–15. 2. Lang B, Waterman S, Pinto A, Jones D, Moss F, Boot J, Brust P, Williams M, Stauderman K, Harpold M, Motomura M, Moll JW, Vincent A, Newsom-Davis J. The role of autoantibodies in Lambert-Eaton myasthenic syndrome. Ann NY Acad Sci 1998; 841:596–605. 3. Sherer Y, Shoenfeld Y. A malignancy work-up in patients with cancer-associated (paraneoplastic) autoimmune diseases: pemphigus and myasthenic syndromes as cases in point (review). Oncol Rep 1999; 6(3):665–668. 4. Dalmau J, Gultekin HS, Posner JB. Paraneoplastic neurologic syndromes: pathogenesis and physiopathology. Brain Pathol 1999; 9(2):275–284. 5. Waud DR. A review of pharmacological approaches to the acetylcholine receptors at the neuromuscular junction. Ann NY Acad Sci 1971; 183:147–157. 6. Stalberg E, Trontelj JV, Schwartz MS. Single-muscle-fiber recording of the jitter phenomenon in patients with myasthenia gravis and in members of their families. Ann NY Acad Sci 1976; 274:189–202.

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27 Rheumatoid Arthritis MAURIZIO CUTOLO University of Genoa, Genoa, Italy RAINER STRAUB University Hospital Regensburg, Regensburg, Germany

I. INTRODUCTION The involvement of the hypothalamic-pituitary-adrenal (HPA) and the hypothalamic-pituitary-gonadal (HPG) axis seems crucial in the development and maintenance of inflammatory and autoimmune conditions such as rheumatoid arthritis (RA) [1]. In female patients the immune response seems to depend more on the HPA axis, whereas male patients seems to depend more on the HPG axis. In particular, hypoandrogenism may play a pathogenetic role in male RA patients and in other autoimmune conditions (i.e., systemic lupus erythematosus), since androgens are considered natural immunosuppressors [2]. Conversely, estrogens seem to exert immunoenhancing activities, at least on the humoral response. A range of physical/psychosocial stressors are also implicated in the activation of the HPA axis and the related altered HPG function in RA [3,4]. Finally, increased levels of neuropeptides detected in RA synovial fluids suggest that the peripheral nervous system might contribute to the generation of the synovial inflammation (i.e., neurogenic inflammation) [2–8]. A major unknown in the pathogenesis of RA is why immunomediated inflammation begins and develops within joints. A central role in understanding RA pathogenesis lies in the comprehension of arthrotopism of antigens and inflammatory cells for joints and in learning what specific receptors, mediators, and chemotactic gradients are active in focusing the immune-mediated inflammation within the synovial tissue. Undoubtedly, the synovial tissue in RA can be regarded as the target tissue, in which the sexual dimorphism in immune response to relevant trigger antigens is present and involves mainly synovial macrophages as well as fibroblasts and lymphocytes [9–12]. In 593

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contrast to the cell-centered models of RA pathogenesis, in which proliferation of antigenspecific T cells determines various manifestations of RA synovitis, such as B-cell stimulation, leukocyte infiltration, and cytokine synthesis, a key role for synovial macrophages and fibroblasts is presently recognized in which the inflammatory changes in the RA synovium are originated and maintained by paracrine and autocrine mechanisms [13–16]. Therefore, an intricate balance with bidirectional interactions between soluble mediators released by the neuroendocrine system (i.e., steroid hormones and neurotransmitters) and products of activated cells of the immune/inflammatory system (i.e., cytokines from macrophages) maintains the homeostasis in the presence of immune/inflammatory synovitis [17,18].

II. THE HPA IN RHEUMATOID ARTHRITIS The inflammatory cytokines (i.e., IL-6, IL-1, TNF-␣) as soluble products of the activated immune system stimulate the production of corticotropin-releasing hormone (CRH) in the hypothalamus. CRH release leads to pituitary production of adrenocorticotropic hormone (ACTH), followed by glucocorticoid secretion by adrenal cortex and indirect perturbations of gonadal function [19,20] (Fig. 1). It is now recognized that young females affected by recent stressful conditions (interpersonal stressors, surgical or infectious events) activating the HPA with associated low plasma adrenal androgens [i.e., dehydroepiandrosterone sulfate (DHEAS)] and recent use of contraceptive pills are the best candidates for the onset of autoimmune disorders, including RA [21–23]. The stress system has multiple levels and is comprised of neuroendocrinological (i.e., HPA axis), psychological, and environmental components [24]. The physiological balance between stress and the immune system may be disrupted as a consequence of various pathological insults, including sustained exposure to stress, abnormal immune reactions to infections, or both [25]. Recently, intact ACTH secretion but impaired cortisol response in patients with active RA has been described, and this observation was consistent with a relative adrenal glucocorticoid insufficiency, the latter already suggested 40 years earlier [26,27]. Increased HPA axis function is a normal response to the stress of inflammation and might be mediated by central and peripheral actions of circulating cytokines. In addition to IL-1 and TNF␣, IL-6 appears to be a major factor mediating interactions between the activated immune system and both the anterior pituitary cells and the adrenal steroidogenesis [28]. However, recent studies in RA patients have shown that overall activity of the HPA axis remains inappropriately normal and is apparently insufficient to inhibit ongoing inflammation, at least in early untreated arthritic patients [29]. More recently, another study showed a significantly altered secretion of adrenal androgens in non–glucocorticoid-treated premenopausal RA patients [23]. Baseline concentrations of dehydroepiandrosterone (DHEA) and its sulfate metabolite DHEAS were found to be significantly lower in chronic RA than in normal subjects. In addition, and during low-dose ACTH testing, DHEA production was found to be significantly lower in chronic RA patients than in controls [23]. Low levels of plasma DHEA and DHEAS were found in the same study to be significantly correlated with early morning low cortisol concentrations and high basal levels of IL-6 in RA patients [23]. Early morning IL-6 peak values were found to be higher in RA patients than in controls and significantly correlated to morning CRP levels and Ritchie’s index [30].

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Figure 1 Schematic representation of the hypothalamic-pituitary-adrenal (HPA), hypothalamicpituitary-gonadal (HPG), and sympathetic nervous systems. The (neuroendocrine) hormonal products of the different systems act at the level of the synovial tissue.

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The observation of reduced DHEA production, combined with normal cortisol production during oCRH and ACTH testing, further supports the concept of the presence of an adrenal hypofunction in active RA patients [31] (Fig. 1). IL-6 had a strong effect on steroid release and may be one of the factors controlling the long-term adrenal response to stress. This cytokine is able to act synergistically with ACTH on adrenal cells to stimulate the release of corticosterone [32,33]. The reduced cortisol and adrenal androgen secretion observed during testing in RA patients not treated with glucocoticoids should be clearly regarded as a ‘‘relative adrenal insufficiency’’ in the setting of a sustained inflammatory process, as shown by high IL-6 levels [34]. In a very recent investigation on salivary cortisol levels in patients with recent-onset RA, afternoon concentrations in patients with high disease activity did not drop, as did the cortisol levels in healthy controls and RA patients with low disease activity [35]. This indicates that activation of the HPA axis is possible but insufficient.

III. GONADAL HORMONE INTERACTIONS WITH SYNOVIAL CELLS Generally, the macrophage-derived inflammatory cytokines (i.e., IL-6, IL-1, TNF-␣), as soluble products of the synovial arthritis induce indirect perturbations of the gonadal function [20]. Significantly lower androgen concentrations (i.e., testosterone, androstenedione, and DHEAS) are detected in the serum as well as in the synovial fluid of male and female RA patients [36,37]. Low serum androgens seem to characterize other immunomediated rheumatic diseases, such as systhemic lupus erythematosus and systemic sclerosis [38,39]. We investigated the capacity of cultured synovial macrophages to metabolize androgens and found that these cells were able to metabolize testosterone into the bioactive metabolite dihydrotestosterone (DHT). Therefore, macrophages contain the key enzymes of steroidogenesis, in particular 5␣-reductase. Furthermore, we analyzed interleukin-1␤ (IL-1␤) production by primary cultures of synovial RA macrophages following exposure to physiological concentrations of testosterone (10ⳮ8 M) and observed a significant decrease in IL-1␤ levels in conditioned media after 24 hours (p ⬍ 0.05) [40] (Fig. 2). Interestingly, the effects of estrogens on IL-1␤ synthesis by macrophages seems to be dose-dependent, and a negative relationship has been found between IL-1 mRNA levels and estrogen concentrations in human peripheral monocytes (precursors of tissue macrophages) and pelvic macrophages [41]. Thus, low estrogen levels (10ⳮ8 M) stimulate both IL-1 mRNA levels and IL-1 protein synthesis, whereas higher levels (pharmacological, 10ⳮ6/10ⳮ5 M) are inhibitory, suggesting a biphasic response. Similar results were obtained when TNF-␣ mRNA levels were evaluated on cultured RA synovial macrophages treated with estrogens (F. Di Giovine, personal communication). We concluded that gonadal steroids may act directly on human macrophages and may interfere with some of their functions via receptor-dependent mechanisms. Therefore, the combined presence of low immunosuppressive androgens and high immunoenhancing estrogens in the synovial fluid might encourage immunomediated synovitis in RA [42] (Fig. 2). However, if low androgen levels in RA synovial fluid can be explained by HPA inhibitory effects on HPG, how can one explain the high estrogen levels found in both female and male synovial fluids [43,44]?

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Figure 2 Effects of androgen and estrogens on the cells of the synovial tissue immune system involved in the inflammatory response in rheumatoid arthritis (left). The effects of the inflammatory cytokines (IL-1, IL-6, TNF-␣) at the level of aromatase are reported. Locally produced inflammatory cytokines are able to markedly stimulate aromatase activity (P450 Arom) in the synovial tissue with resultant increased conversion of androgens (testosterone and androstenedione) to estrogens (estrone and estradiol, respectively) (right). In addition, IL-6 has been found to mediate an increase in reductive 17␤-hydroxysteroid dehydrogenase (17␤-HSD) activity that converts estrone to the biologically more active 17␤-estradiol. These effects might explain low androgens and high estrogens in synovial RA fluids, as well as their effects on synovial cells.

The appropriate explanation might originate from studies showing that the inflammatory cytokines (i.e., IL-6, IL-1, TNF-␣) are able to markedly stimulate aromatase activity in peripheral tissues [45–47]. In particular, in tissues rich in macrophages a significant correlation was found between aromatase activity and IL-6 production [47]. The aromatase-enzyme complex is involved in the peripheral conversion of androgens (testosterone and androstenedione) to estrogens (estrone and estradiol, respectively). Therefore, its increased activity induced by locally produced inflammatory cytokines (i.e., TNF-␣, IL-1, IL-6) might explain low androgen and high estrogen levels in synovial RA fluids as well as their effects on synovial cells [48] (Fig. 2). In addition, IL-6 has been found to mediate an increase in reductive 17␤-hydroxysteroid dehydrogenase (17␤-HSD) activity that converts estrone to the biologically more

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active 17␤-estradiol [49]. Studies on the influence of sex hormones upon synoviocyte apoptosis and proto-oncogene expression offer further perspectives on the pathogenesis and therapy of synovitis in RA [50]. Generally, estrogens seem to stimulate cell proliferation and to inhibit apoptosis, whereas androgens seem to induce apoptosis. Consequently, low levels of androgens, as frequently observed in sera and synovial fluids of RA patients, may reduce synoviocyte apoptosis and enhance synovial tissue proliferation, as also observed for ovarian granulosa cells [51]. Therefore, activation-induced macrophage apoptosis may serve to reduce the destructive potential of inflammatory macrophages [52]. Recent studies showed that testosterone therapy dramatically suppresses lymphocyte infiltration in, and significantly improves the functional activity of, lacrimal glands in the MRL/lpr female mouse model of Sjo¨gren’s syndrome [53]. A subsequent study showed that androgen treatment influences the expression of proto-oncogenes, with decreased bcl-2 and c-myb mRNA levels, as well as apoptotic factors in salivary and lacrimal tissues of the same model of Sjo¨gren’s syndrome [54]. We induced an increased apoptosis in primary cultures of RA synovial macrophages by treatment with physiological concentrations (10ⳮ8 M) of testosterone [55]. The proapoptotic effect of testosterone was significantly increased (p ⬍ 0.001) by concomitant treatment with methotrexate (50 ␮g/mL). If sex hormones well as cytotoxic agents modulate synovial macrophage apoptosis, such an approach might be important for the control of RA [56,57].

IV. NEUROTRANSMITTER INTERACTIONS WITH IMMUNE SYSTEM CELLS In addition to paracrine and systemic gonadal hormone influence on synovial cells, it is crucial to discuss paracrine neurotransmitter effects because the synovial tissue is richly innervated [58]. At least three different types of nerve fiber enter the synovial tissue by various routes: 1. The sympathetic nervous system accompanies blood vessels and the small nerve fibers end in the surrounding of these vessels [59]. These efferent nerve fibers store neurotransmitters such as norepinephrine, neuropeptide Y, endogenous opioids (i.e., methionine enkephalin), and adenosine triphosphate (ATP) in the peripheral nerve endings [60]. ATP is converted to adenosine in the extracellular space by ecto-5′-nucleotidase, which is located at the surface of macrophages and other cells (CD73) in the vicinity of the nerve terminal [61]. 2. The primary sensory afferent nerve fibers with the two neurotransmitters substance P and calcitonin gene-related peptide (CGRP) also innervate the synovial membrane [62]. 3. Vasoactive intestinal peptide belongs to the nonadrenergic noncholinergic type of peripheral nerve fiber, which are also present in this tissue [63]. The effects of these neurotransmitters on immune cells are not uniform. Generally, it seems that neurotransmitters of the sympathetic nervous system (norepinephrine, adenosine, methionine-enkephalin) are anti-inflammatory, whereas the main neurotransmitter of the sensory nervous system — substance P — is pro-inflammatory in all aspects. On the other hand, vasoactive intestinal peptide is more anti-inflammatory) (Fig. 1).

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At the moment it is widely accepted that substance P, a neurotransmitter of the sensory afferents, is pro-inflammatory. For example, substance P stimulates IL-1, IL-2, IL-8, TNF, NF-␬B, and superoxide anion production from various types of cells [60–68]. Local administration of substance P antagonists or dorsal rhizotomy markedly reduces the severity of inflammation in animal models [69–71]. Furthermore, substance P is chemotactic for human monocytes, and it sensitizes articular afferent nerve fibers in normal and inflamed knee joints, which leads to increased mechanosensitivity, pain, and continuous increased substance P release into the lumen of the joint [72,73]. Aside from the proinflammatory mechanisms influenced by substance P, the effects of substance P also serve to continuously sense painful stimuli in the periphery (Fig. 1). With respect to the sympathetic nervous system and its neurotransmitters, the situation is not as uniform as with substance P. Since norepinephrine (NE) or adenosine, which are colocalized in vesicles of the sympathetic nerve terminal, are ligands of different receptor subtypes with opposing intracellular signal transduction pathways, completely different effects may arise depending on the local concentration [74]. At high concentrations of about 10ⳮ6 –10ⳮ4 M (in the synaptic cleft), NE acts on ␣- and ␤-adrenergic receptors and adenosine on A1, A2, and A3 adenosine receptors, respectively (Fig. 1). However, at low concentrations (ⱕ10ⳮ7 M) effects are mediated mainly via ␣-receptors or A1 adenosine receptors, respectively. Stimulation of the ␤-adrenoceptor and the A2receptor leads to an intracellular increase of cyclic AMP and, thus, marked downregulation of arthritogenic TNF, IL-12, or interferon-␥ [75–78]. In contrast, stimulation via the ␣2adrenoceptor (cAMP decrease) even stimulates TNF secretion [79]. Furthermore, ligation of ␤-adrenoceptors and A2 receptors has been shown to generally induce anti-inflammatory mechanisms in animal models of inflammation [80,81]. Moreover, the anti-inflammatory mechanism of low-dose methotrexate is accomplished by extracellular adenosine, which binds at A2 receptors [82]. Hence, early reports on the modulation of arthritis by the sympathetic nervous system demonstrate pro-inflammatory effects, while more recent studies show anti-inflammatory effects of the sympathetic nervous system [83–85]. The differential effects of the sympathetic nervous system are dependent on either ␣2- or ␤adrenergic stimulation, respectively [86]. Furthermore, opioids from the sympathetic nerve terminals were demonstrated to have anti-inflammatory properties, and intra-articular injection of morphine (␮-opioid receptor agonist) has anti-inflammatory effects [87–89]. These peptides exert a peripheral analgesic effect at sensory articular afferents or a central analgesia on the spinal level [90,91]. Taken together, increased sympathetic nervous system activity is accompanied by release of high amounts of norepinephrine, adenosine, and opioids, which induces an anti-inflammatory effect. Recent studies suggest that the density of pro-inflammatory sensory nerve fibers remains high in the inflamed tissue, whereas the density of anti-inflammatory sympathetic nerve fibers decreases [92]. This may lead to a shift towards a more pro-inflammatory situation, as demonstrated in Crohn’s disease and during wound healing [93,94]. This shift may be a relevant factor in the chronicity of the disease. Since norepinephrine (via ␤adrenoceptors) and cortisol help each other to maintain the respective signal transduction pathway, a decrease in their relative concentrations in the synovial tissue may synergistically lead to a pro-inflammatory state [95,96]. Furthermore, catecholamines induce androgen secretion in endocrine cells via ␤-adrenergic receptors and might increase the antiinflammatory effects through androgen, which may also be a relevant mechanism in local synovial cells [97,98]. Thus, loss of norepinephrine and other sympathetic neurotransmit-

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ters due to retraction of sympathetic nerve fibers will be accompanied by a pro-inflammatory state. V. CONCLUSIONS Neuroendocrine mechanisms represent modulating factors for the immune response and inflammatory reactions. The involvement of the neuroendocrineimmune system in the pathophysiology of rheumatoid arthritis is now more clear and suggests a common pathway for other autoimmune rheumatic diseases [88,100]. However, further studies to address the effects of neurotransmitters together with gonadal hormones in target cells of the synovial tissue are in progress and need great attention [101–105]. REFERENCES 1. Bijlsma JWJ, Cutolo M, Masi AT, Chikanza I. Neuroendocrine immune basis of the rheumatic diseases. Immunol Today 1999; 20:298–301. 2. Masi AT, Bijlsma JWJ, Chikanza IC, Pitzalis C, Cutolo M. Neuroendocrine, immunological, and microvascular systems interactions in rheumatoid arthritis; physiopathogenetic and therapeutic perspectives. Sem Arthritis Rheum 1999; 29:65–81. 3. Cutolo M, Prete C, Walker J. Is stress a factor in the pathogenesis in autoimmune rheumatic diseases. Clin Exp Rheum 1999; 17:515–518. 4. Walker JG, Littlejohn G, McMurray NE, Cutolo M. Stress system activation in rheumatoid arthritis: a multilevel approach. Rheumatol 1999; 38:1050–1057. 5. Cutolo M, Castagnetta L. Immunomodulatory mechanisms mediated by sex hormones in rheumatoid arthritis. Ann NY Acad Sci 1996; 784:534–541. 6. Masi AT, da Silva JAP, Cutolo M. Perturbations of the hypothalamic pituitary gonadal axis in rheumatoid arthritis. In Neuroendocrine Immune Mechanisms of Rheumatic Diseases Chikanza IC, Ed: Ballie`re’s Clinical Rheumatology, Oxford 1996:295–331. 7. Cutolo M. Do sex hormones modulate synovial macrophages in rheumatoid arthritis. Ann Rheum Dis 1997; 56:281–284. 8. Matucci-Cerinic M, Konttinen Y, Generini S, Cutolo M. Neuropeptides and steroid hormones in arthritis. Curr Op Rheum 1998; 10:220–235. 9. Cutolo M, Sulli A, Barone A, Seriolo B, Accardo S. Macrophages, synovial tissue and rheumatoid arthritis. Clin Exp Rheumatol 1993; 11:331–339. 10. Zvaifler NJ. Macrophages and the synovial lining. Scand J Rheumatol 1995; 24(suppl 101): 67–75. 11. Cutolo M, Accardo S, Villaggio B, Barone A, Sulli A, Coviello DA, Carabbio C, Felli L, Miceli D, Farruggio R, Carruba G, Castagnetta L. Androgen and estrogen receptors are present in primary cultures of human synovial macrophages. J Clin Endocrinol Metab 1996; 81: 820–827. 12. Burmester GR, Stuhlmuller B, Keyszer G, Kinne RW. Mononuclear phagocytes and rheumatoid synovitis. Arthritis Rheum 1997; 40:5–18. 13. Van Lent PELM, Holthuynsen AEM, Van der Berseelaar L, Van Rooijen N, Van de Putte LBA, Van der Berg WWWB. Role of macrophage-like synovial lining cells in localization and expression of experimental arthritis. Scand J Rheumatol 1995; 24(suppl 101):83–89. 14. Mulherim D, Fitzgerald O, Bredniham B. Synovial tissue macrophage populations and articular damage in rheumatoid arthritis. Arthritis Rheum 1996; 39:115–124. 15. Tak PP, Smeets TJM, Daha MR. Analysis of the synovial cell infiltrate in early rheumatoid synovial tissue in relation to local disease activity. Arthtitis Rheum 1997; 40:217–225. 16. Cutolo M. Macrophages as effectors of the immunoendocrinologic interactions in autoimmune rheumatic diseases. Ann NY Acad Sci 1999; 876:32–43.

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80. Malfait AM, Malik AS, Marinova-Mutafchieva L, Butler DM, Maini RN, Feldmann M. The beta2-adrenergic agonist salbutamol is a potent suppressor of established collagen-induced arthritis: mechanisms of action. J Immunol 1999; 162:6278–6283. 81. Cronstein BN, Naime D, Firestein G. The antiinflammatory effects of an adenosine kinase inhibitor are mediated by adenosine. Arthritis Rheum 1995; 38:1040–1045. 82. Cronstein BN. Molecular therapeutics. Methotrexate and its mechanism of action [see comments]. Arthritis Rheum 1996; 39:1951–1960. 83. Levine JD, Moskowitz MA, Basbaum AI. The contribution of neurogenic inflammation in experimental arthritis. J Immunol 1985; 135:843–847. 84. Basbaum AI, Levine JD. The contribution of the nervous system to inflammation and inflammatory disease. Can J Physiol Pharmacol 1991; 69:647–651. 85. Lorton D, Bellinger D, Duclos M, Felten SY, Felten DL. Application of 6-hydroxydopamine into the fatpads surrounding the draining lymph nodes exacerbates adjuvant-induced arthritis. J Neuroimmunol 1996; 64:103–113. 86. Coderre TJ, Chan AK, Helms C, Basbaum AI, Levine JD. Increasing sympathetic nerve terminal-dependent plasma extravasation correlates with decreased arthritic joint injury in rats. Neuroscience 1991; 40:185–189. 87. Walker JS, Howlett CR, Nayanar V. Anti-inflammatory effects of kappa-opioids in adjuvant arthritis. Life Sci 1995; 57:371–378. 88. Binder W, Walker JS. Effect of the peripherally selective kappa-opioid agonist, asimadoline, on adjuvant arthritis. Br J Pharmacol 1998; 124:647–654. 89. Stein A, Yassouridis A, Szopko C, Helmke K, Stein C. Intraarticular morphine versus dexamethasone in chronic arthritis. Pain 1999; 83:525–532. 90. Russell NJ, Schaible HG, Schmidt RF. Opiates inhibit the discharges of fine afferent units from inflamed knee joint of the cat. Neurosci Lett 1987; 76:107–112. 91. Lombard MC, Besson JM. Electrophysiological evidence for a tonic activity of the spinal cord intrinsic opioid systems in a chronic pain model. Brain Res 1989; 477:48–56. 92. Miller LE, Wessinghage D, Ju¨sten H-P, Scho¨lmerich J, Straub RH. Lack of sympathetic nerve fibers in human synovial membrane in long-standing rheumatoid arthritis (RA) as compared to osteoarthritis (OA). Arthritis Rheum 1999; 42:S247. 93. Geboes K, Collins S. Structural abnormalities of the nervous system in Crohn’s disease and ulcerative colitis. Neurogastroenterol Motil 1998; 10:189–202. 94. Reynolds ML, Fitzgerald M. Long-term sensory hyperinnervation following neonatal skin wounds. J Comp Neurol 1995; 358:487–498. 95. DiBattista JA, Martel-Pelletier J, Cloutier JM, Pelletier JP. Modulation of glucocorticoid receptor expression in human articular chondrocytes by cAMP and prostaglandins. J Rheumatol Suppl 1991; 27:102–105. 96. Cotecchia S, De Blasi A. Glucocorticoids increase beta-adrenoceptors on human intact lymphocytes in vitro. Life Sci 1984; 35:2359–2364. 97. Anakwe OO, Moger WH. Beta 2-adrenergic stimulation of androgen production by cultured mouse testicular interstitial cells. Life Sci 1984; 35:2041–2047. 98. Hernandez ER, Jimenez JL, Payne DW, Adashi EY. Adrenergic regulation of ovarian androgen biosynthesis is mediated via beta 2-adrenergic theca-interstitial cell recognition sites. Endocrinology 1988; 122:1592–1602. 99. Cutolo M. Sex hormone adjuvating therapy in rheumatoid arthritis. Rheum Dis Clin North Am 2000; 26:881–895. 100. Straub RH, Gluck T, Cutolo M, Georgi J, Helmke K, Scholmerich J, Vaith P, Lang B. The adrenal steroid status in relation to inflammatory cytokines IL-6 and TNF in polymyalgia rheumatica. Rheumatology 2000; 39:624–631. 101. Cutolo M, Villaggio B, Sulli A, Seriolo B, Giusti M. CYP17 gene polymorphisms and androgen levels in postmenopausal patients with rheumatoid arthritis. Clin Exp Rheumatol 2000; 18: 420–421.

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102. Bijlsma JWJ, Straub RH, Masi AT, Lahita RG, Cutolo M. Neuroendocrine Immune Mechanisms in Rheumatic Diseases. Trends Immunol 2002; 23:59–61. 103. Cutolo M, Seriolo B, Villaggio B, Pizzorni C, Craviotto C, Sulli A. Androgens and estrogens modulate the immune and inflammatory responses in rheumatoid arthritis. Ann NY Acad Sci 2002; 966:131–142. 104. Cutolo M, Straub RH, Masi AT, Bijlsma JWJ, Lahita R, Bradlow HL. Altered neuroendocrine immune (NEI) networks in rheumatology. Ann NY Acad Sci 2002; 966:xiii. 105. Whitacre CC. Sex differences in autoimmune disease. Nature Immunol 2001; 2:777–780.

28 Aging and Neuroimmunoendocrinology RAINER STRAUB University Hospital Regensburg, Regensburg, Germany MAURIZIO CUTOLO University of Genoa, Genoa, Italy

I. EVOLUTIONARY ASPECTS OF AGING Two questions concerning evolutionary aspects of the aging process should be asked: 1. Do specific genes evolutionarily conserved in the genome shape the aging process? 2. Why must we consider the nervous system, the endocrine system, and the immune system when we talk about the aging process? With respect to the first question, we can easily respond with ‘‘no’’: The reproductive period of a Homo erectus, who lived 1 million years ago, normally lasted from 12 to 25 years of age. Fossil remains suggest a human life expectancy of approximately 25 years for humans in this period [1]. Thus, natural selection has limited opportunity to exert a direct influence over the process of aging because the advantageous or disadvantageous genes are not conserved after the reproductive period. As recently pointed out [2], this situation allows a wide range of alleles with late deleterious effects to accumulate over generations with little or no check. Under these conditions we might expect considerable heterogeneity in the distribution of such alleles among individuals within the population. Another theory suggested that pleiotropic genes with good effects early in life—e.g., fertility with elevated levels of estrogens in females—would be favored by selection even if these genes had bad effects at later ages [2]. In conclusion, no advantageous or disadvantageous genes are specifically conserved for successful aging. 607

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With respect to the second question, we first consider a single cell such as a bacterium. Here, the process of aging will obviously depend on the cell’s capacity to cope with different conditions such as oxidative damage, nonenzymatic shaping of molecules, mitochondrial mutations, defects in cell cycle control, mitotic dysregulation, genome instability, telomere shortening, and other chromosomal pathologies. Research on aging often focuses on the single-cell level. However, during evolution single cells started to arrange networks of distinct cell types. Paracrine factors such as cytokines or hormones became important as transmitters. In this new context, an aging cell influenced a remote cell in the same tissue, and the remote cell perceived this information in order to respond with its own set of mediators. In such a dynamic situation, aging of one single cell in the tissue can lead to age-related processes throughout the tissue. The situation became more complex when specialized tissue appeared, leading to the development of organs. Organs moved away from each other to build independent regions in a body. As these distant organs developed, complicated transport systems became necessary in order to exchange information: vessels for blood or lymph (humoral pathway) and nerves (neuronal pathway). In inflamed tissue, local cells produce mediators that can appear in the circulation or stimulate sensory nerves in order to announce the local problem to remote organs such as the brain. The brain responds by activating the hypothalamic-pituitary-adrenal (HPA) axis or the hypothalamus–autonomic nervous system axis [3–6]. In a very similar way, age-related changes of one organ due to age-dependent changes of a group of single cells within the organ may be announced to distant sites in the body. Thus, multiple interactions of distant organs appear during aging and may contribute to the overall aging process in a nonlinear dynamic way. No advantageous or disadvantageous genes are evolutionarily conserved in a specific way to allow perfect healthy aging and to increase life span in a single group of cells, organs, or the entire body. This is also true for evolutionarily conserved factors that mediate the cross-talk between cells and organs. Thus, when one supersystem ages, another supersystem is probably also aging. II. AGING OF THE NERVOUS SYSTEM In rats and mice, sympathetic innervation is reduced in many organs [7–12]. Loss of innervation during aging depends on tissue type, species, strain, and microenvironment (reviewed in Ref. 8). In addition, ␤-adrenoceptors on different cells increase with age [8,13–16]. However, there may be subsensitivity to ␤-adrenergic agonists with age, probably due to an altered signaling cascade (reviewed in Refs 8 and 17). With respect to autonomic nerve function, it has been demonstrated that heart rate variability [18] and pupillary oscillations [19], are significantly reduced in the elderly. During the process of aging, the interaction of these autonomic nervous pathways is altered, which leads to attenuation of autonomic reflex amplitudes. Other autonomic responses become hyperresponsive, such as the vascular tone due to an augmented vasoconstrictor (␣adrenergic) and a reduced vasomotor (␤-adrenergic) responsiveness [17]. This increased sympathetic response with age is accompanied by elevated levels of norepinephrine in the circulation [20–22] and increased sympathetic nerve activity [23–25]. We confirmed that plasma norepinephrine levels were significantly increased in healthy aged people [26]. In a further analysis of these data, we demonstrated that plasma levels of norepinephrine were elevated in relation to serum concentrations of cortisol [26]. In conclusion, there is

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an overall shift to norepinephrine in relation to cortisol and, further, in relation to other adrenal hormones. Sympathetic innervation of the tissue at many sites decreases, whereas norepinephrine plasma concentration and ␤-adrenoceptor density increases during aging (Table 1). This pattern may be consistent with a compensatory activation of remaining noradrenergic neurons and alterations of signaling through adrenoceptors with a concomitant increase in plasma norepinephrine in relation to serum cortisol and other adrenal hormones.

Table 1 Typical Age-Related Changes in the Nervous, Immune, and Endocrine Systems Increase Nervous system Muscle sympathetic nerve activity (human) Sympathetic response to stimuli (human) Plasma norepinephrine (human) Vascular tone Axonal degeneration in atria (rat) Mesenteric substance P innervation (rats) ␤-Adrenoceptors on different cells (human, rodents) Immune system (all human) Repertoire degeneracy of B lymphocytes Autoantibody production Memory type of T cells (oligoclonal) T-helper lymphocyte type II response Serum IL-6 Serum TNF Ratio of serum TNF/serum soluble TNF receptor 1

Endocrine system (all human, serum, or plasma) Nadir concentration of cortisol Follicle-stimulating hormone Luteinizing hormone in female Parathyroid hormone Adrenocorticotropic hormone

Decrease Cardiovascular autonomic function (human) Pupillary autonomic function (human) Sudomotor function (human) Innervation of the lacrimal glands (rats) Innervation of lymph nodes (rats, mice) Innervation of the spleen (rats) Innervation of the kidneys (rats) Innervation of the trachea (rats) Immunoglobulin hypermutation Affinity maturation of B lymphocytes HLA class II expression B7–2 expression on B lymphocytes CD28 expression of T cells IL-2 secretion Respiratory burst from neutrophils Phagocytosis of neutrophils Dendritic cell traffic Natural killer cell function Number of naive T lymphocytes Serum soluble vascular cell adhesion molecule-1 Acrophase concentration of cortisol in female Androstenedione Growth hormone Dehydroepiandrosterone and its sulfate Progesterone Testosterone Aldosterone Melatonin Calcitonin Vitamin D

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III. AGING OF THE IMMUNE SYSTEM Important changes of the immune system in the aged are the following [27–29]: there is a loss of phagocytic capacity and decreased generation of radicals (respiratory burst), an increase in ‘‘early’’ cytokines (IL-1␤, IL-6, TNF), and a reduction in dendritic cell trafficking. With respect to the adaptive immune system, a decrease in T-cell and B-cell proliferation, repertoire degeneration of B cells, decreased numbers of naive cells, increase in memory cell type, and a shift from T-helper 1 to T-helper 2 responses were noted (Table 1). Furthermore, expression of important surface molecules for intercellular interaction, such as B7-2, CD28, and HLA class II, is reduced in the elderly. In our studies, we found an increase in the serum concentration of soluble IL-2 receptor, IL-6, and the ratio of TNF/soluble TNF receptor 1, which indicate the presence of an activated immune system [26,30]. These changes were paralleled by a decrease in serum soluble vascular cell adhesion molecule-1 (sVCAM-1) [26]. Interestingly, a combination of immune status indices can predict survival [28,31,32]. In conclusion, it is obvious that the innate and the adaptive immune systems deteriorate during the aging process and that changes in immune system responsiveness are important predictors of survival.

IV. AGING OF THE ENDOCRINE SYSTEM This topic has been extensively reviewed (Table 1) [33–35]. In summary, adrenosenescence is accompanied by low serum levels of androstenedione (ASD), dehydroepiandrosterone (DHEA), DHEA sulfate (DHEAS), progesterone, and aldosterone. Gonadosenescence is demonstrated by low serum levels of 17␤-estradiol (female) and testosterone (male). Decreased serum levels of growth hormone and insulin-like growth factor are typical for somatosenescence, and ‘‘calcium-senescence’’ is demonstrated by low serum levels of calcitonin and vitamin D but elevated levels of parathyroid hormone. We were recently able to demonstrate that serum cortisol levels remained elevated in relation to serum concentrations of other adrenal hormones such as ASD, DHEA, DHEAS, and progesterone in healthy subjects [36]. This is probably an important compensatory mechanism of the adrenal glands in order to maintain synthesis of cortisol in the aging adrenal cortex at the expense of other adrenal steroid hormones. In conclusion, during aging there is an enormous decrease in peripheral hormones and an increase in some pituitary hormones (not growth hormone) (Table 1). However, cortisol decrease is smaller in relation to the decline of other peripheral hormones. This may result in a preponderance of cortisol effects in relation to other hormones of the adrenal glands.

V. AGING OF ONE SUPERSYSTEM INVOLVES ANOTHER In earlier years, we and others demonstrated that serum concentration of IL-6 significantly increases with age [30]. This phenomenon has also been found in tissue [37], which may be a consequence of decreased serum testosterone, estrogens, and DHEA [30,38,39]. Daynes et al. demonstrated in mice that administration of DHEAS, which is converted into the active DHEA by a sulfatase, inhibits spontaneous IL-6 secretion in aging mice [40]. In human subjects, serum levels of IL-6 are inversely correlated to serum levels of DHEA [30], and DHEA inhibits IL-6 secretion from human and mouse mononuclear

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cells [30,41]. This indicates that aging of one supersystem may induce typical age-related changes in another supersystem (Fig. 1). In another investigation of healthy subjects [42], we found significantly lower serum levels of cortisol in relation to plasma adrenocorticotropic hormone (ACTH) with age only in women [42]. This particular ratio as the dependent variable was explained by the independent variables 17␤-estradiol and IL-6 serum levels in a multiple linear regression model [42]. Serum levels of TNF did not influence the model. This may indicate that increasing serum levels of IL-6 together with decreasing serum concentrations of 17␤estradiol are responsible for the decrease in serum cortisol in relation to plasma ACTH in women. This is another example of the interaction of two global systems during the aging process (Fig. 1). In a third example in healthy human subjects, we were recently able to demonstrate that the age-related decrease in serum sVCAM-1 was closely related to an age-dependent decrease in serum levels of progesterone [43]. Furthermore, progesterone facilitated the surface expression of VCAM-1 on human umbilical vein endothelial cells. This indicates that the age-related loss of progesterone is probably responsible for the loss of surface VCAM-1 and sVCAM-1 (Fig. 1). Such an age-related loss of surface expression of VCAM1 would lead to a deterioration in immune surveillance due to a decrease in patrolling leukocytes [43]. A fourth example helps to understand the general shift from T-helper type 1 to Thelper type 2 immune responses during aging. There is now much evidence that cortisol

Figure 1 Four examples of cross-influence of the supersystems during the aging process. Abbreviations: ↓, decreased serum/plasma level or cell surface expression; ↑, increased serum/plasma level; ACTH, adrenocorticotropic hormone; DHEA, dehydroepiandrosterone; IL-6, interleukin-6; VCAM1, vascular cell adhesion molecule-1.

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and norepinephrine (via ␤-adrenoceptors) induce a shift into the direction of a T-helper 2–mediated immune responses [44–59]. Furthermore, cortisol and norepinephrine pathways (␤-adrenergic) help each other in different cell types [60–63]. We mentioned above that blood levels of norepinephrine increase relative to cortisol and other adrenal steroids, which probably supports an overall shift to T helper type 2 immune responses (Fig. 1). This shift is observed in elderly people. It can lead to autoimmune phenomena [64], accumulation of intracellular microbes, tumor growth (due to a missing T-helper type 1 immune response with cytotoxic immune reactions), and acceleration of atherosclerosis (due to a T-helper type 2 immune response–mediated increase in LDL oxidation by activated human monocytes) [65,66]. Furthermore, this would support the generation of autoantibodies by nonspecific activation of the humoral immune pathway. Thus, a general increase in norepinephrine and cortisol in relation to other hormones may lead to an unfavorable preponderance of T-helper type 2 immune reactions, which may contribute to typical age-related diseases. These examples clearly demonstrate that aging of one supersystem influences aging of another supersystem (Fig. 1). In previous chapters it was demonstrated that the process of aging changes the interplay of the nervous, immune, and endocrine systems. This process takes place in a longitudinal way from birth to death of an individual in form of a ‘‘vicious spiral’’ of aging [26]. One testable hypothesis is that the influence of the supersystems on each other may lead to a slowly progressing aging process of these global systems. Such a mechanism would be an addition to many known cellular aging factors. The image of a vicious spiral indicates that new and better statistical and computational techniques are necessary to unravel the nonlinear complexity of the aging process. VI. CHRONIC INFLAMMATORY DISEASES IN THE AGING PROCESS Premature atherosclerosis has been recognized as a major determinant of morbidity and mortality in patients with systemic lupus erythematosus, rheumatoid arthritis, and probably also inflammatory bowel disease [67–70]. Furthermore, premature osteoporosis is a typical feature of chronic inflammatory diseases even in a situation without prior corticosteroid treatment due to strong inflammation [71–77]. Atherosclerosis and osteoporosis are only two typical age-related problems that occur early in the course of inflammatory diseases. An inflammation-dependent decrease in sex steroids and an increase in circulating serum levels of proinflammatory cytokines such as TNF and IL-6 may be significant causative factors for these disease-related problems. It has been repeatedly demonstrated that serum levels of adrenal and gonadal androgens are low in patients with chronic inflammatory diseases such as rheumatoid arthritis [78–80], systemic lupus erythematosus [81,82], progressive systemic sclerosis [83], polymyalgia rheumatica [84,85], pemphigus and psoriasis [86], and inflammatory bowel diseases [87]. The most prominent decrease is observed with respect to the serum levels of DHEA and DHEAS. This decline of DHEAS is very similar to the decline during the aging process. Since DHEA is converted to testosterone and 17␤-estradiol in peripheral cells [88], loss of DHEA may be deleterious for the maintenance of the peripheral sex hormone pool, for example, in bone. Lack of DHEA may be an important factor in increased serum levels of IL-6 [30,40], which are elevated in these chronic diseases. Interestingly, substitution therapy with DHEA improved systemic lupus erythematosus in two double-blind, placebo-controlled studies [89,90]. In an open study on patients with ulcerative colitis and Crohn’s disease, we were able to detect a beneficial effect of DHEA [91].

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The positive effect of DHEA was observed with respect to disease activity as well as bone mineral density [92,93]. In two recent reviews the authors outlined a possible beneficial effect of a parallel therapy with DHEA plus corticosteroids due to its anti-inflammatory and antiglucocorticoid effects [94,95]. We recently observed a preponderance of the sympathetic nervous system relative to the cortisol-producing system in patients with inflammatory bowel disease [96]. Such a shift is also observed during aging, when plasma levels of norepinephrine are higher in relation to cortisol. This may lead to an unfavorable shift into the direction of T-helper lymphocyte type 2 responses. In addition, it seems reasonable that endocrinoimmunosenescence might predispose to inflammatory conditions in the elderly, which may lead to diseases such as polymyalgia rheumatica (PMR) [97]. PMR patients demonstrate an altered HPA axis function at disease onset, prior to corticosteroid treatment, and during the course of the disease, which is indicative of a reduced responsiveness of the HPA axis [85,98]. This aspect reinforces some similarities between PMR and elderly onset of rheumatoid arthritis. Several factors might be involved in adrenal hypofunction, such as concomitant physiological decline of adrenal steroidogenesis during aging, chronic or acute stress system activation, chronic infections (e.g., chlamydia pneumonia or herpesviruses), altered adrenal hormonal pathways (interindividual and genetic differences), and age-related changes of gonadal hormone biosynthesis. The abrupt onset of PMR, with symptoms reminiscent of steroid withdrawal syndrome (i.e., myalgia, malaise, fever, pain, depression, sleepiness, anorexia) or adrenal insufficiency, as well as their dramatic and rapid disappearance following corticosteroid administration, may well represent strong clinical evidence for PMR as an HPA axis–driven disease. VII. CONCLUSIONS The supersystems are closely interrelated, and, thus, aging of one supersystem necessarily involves aging of another supersystem. Better understanding of the physiological interplay of the immune, endocrine, and nervous systems during aging may help to define therapeutic targets and strategies to modulate parts of the vicious spiral of aging, for example, with a well-balanced and thoroughly tested combination of hormone-replacement therapies. This may lead to new therapeutic principles which could improve the health of aging people. Longitudinal studies similar to the Framingham study are necessary in order to detect key factors in the aging process. Among others, parameters of the three supersystems must be included in these longitudinal surveys. REFERENCES 1. Baur M, Ziegler G. Die Odyssee des Menschen—Es begann in Afrika. Mu¨nchen: Econ Ullstein List Verlag, 2001. 2. Kirkwood TB, Austad SN. Why do we age. Nature 2000; 408:233–238. 3. Chrousos GP. The hypothalamic-pituitary-adrenal axis and immune-mediated inflammation. N Engl J Med 1995; 332:1351–1362. 4. Besedovsky HO, Del Rey A. Immune-neuro-endocrine interactions. Endocr Rev 1996; 17: 64–102. 5. Straub RH, Westermann J, Scho¨lmerich J, Falk W. Dialogue between CNS and immune system in lymphoid organs. Immunol Today 1998; 19:409–413.

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48. Nusslein HG, Trag T, Winter M, Dietz A, Kalden JR. The role of T cells and the effect of hydrocortisone on interleukin-4- induced IgE synthesis by non-T cells. Clin Exp Immunol 1992; 90:286–292. 49. van der Poll T, Barber AE, Coyle SM, Lowry SF. Hypercortisolemia increases plasma interleukin-10 concentrations during human endotoxemia—a clinical research center study. J Clin Endocrinol Metab 1996; 81:3604–3606. 50. Norbiato G, Bevilacqua M, Vago T, Taddei A, Clerici A. Glucocorticoids and the immune function in the human immunodeficiency virus infection: a study in hypercortisolemic and cortisol-resistant patients. J Clin Endocrinol Metab 1997; 82:3260–3263. 51. Vieira PL, Kalinski P, Wierenga EA, Kapsenberg ML, de Jong EC. Glucocorticoids inhibit bioactive IL-12p70 production by in vitro-generated human dendritic cells without affecting their T cell stimulatory potential. J Immunol 1998; 161:5245–5251. 52. Visser J, Boxel-Dezaire A, Methorst D, Brunt T, de Kloet ER, Nagelkerken L. Differential regulation of interleukin-10 (IL-10) and IL-12 by glucocorticoids in vitro. Blood 1998; 91: 4255–4264. 53. Dandona P, Aljada A, Garg R, Mohanty P. Increase in plasma interleukin-10 following hydrocortisone injection. J Clin Endocrinol Metab 1999; 84:1141–1144. 54. Verhoef CM, van Roon JA, Vianen ME, Lafeber FP, Bijlsma JW. The immune suppressive effect of dexamethasone in rheumatoid arthritis is accompanied by upregulation of interleukin 10 and by differential changes in interferon gamma and interleukin 4 production. Ann Rheum Dis 1999; 58:49–54. 55. Feldman RD, Hunninghake GW, McArdle WL. Beta-adrenergic-receptor-mediated suppression of interleukin 2 receptors in human lymphocytes. J Immunol 1987; 139:3355–3359. 56. Sanders VM, Baker RA, Ramer-Quinn DS, Kasprowicz DJ, Fuchs BA, Street NE. Differential expression of the beta2-adrenergic receptor by Th1 and Th2 clones: implications for cytokine production and B cell help. J Immunol 1997; 158:4200–4210. 57. Elenkov IJ, Papanicolaou DA, Wilder RL, Chrousos GP. Modulatory effects of glucocorticoids and catecholamines on human interleukin-12 and interleukin-10 production: clinical implications. Proc Assoc Am Phys 1996; 108:374–381. 58. Szabo C, Hasko G, Zingarelli B, Nemeth ZH, Salzman AL, Kvetan V, Pastores SM, Vizi ES. Isoproterenol regulates tumour necrosis factor, interleukin-10, interleukin-6 and nitric oxide production and protects against the development of vascular hyporeactivity in endotoxaemia. Immunology 1997; 90:95–100. 59. Paul-Eugene N, Kolb JP, Damais C, Abadie A, Mencia-Huerta JM, Braquet P, Bousquet J, Dugas B. Beta 2-adrenoceptor agonists regulate the IL-4-induced phenotypical changes and IgE-dependent functions in normal human monocytes. J Leukoc Biol 1994; 55:313–320. 60. Oikarinen J, Hamalainen L, Oikarinen A. Modulation of glucocorticoid receptor activity by cyclic nucleotides and its implications on the regulation of human skin fibroblast growth and protein synthesis. Biochim Biophys Acta 1984; 799:158–165. 61. Gruol DJ, Campbell NF, Bourgeois S. Cyclic AMP-dependent protein kinase promotes glucocorticoid receptor function. J Biol Chem 1986; 261:4909–4914. 62. DiBattista JA, Martel-Pelletier J, Cloutier JM, Pelletier JP. Modulation of glucocorticoid receptor expression in human articular chondrocytes by cAMP and prostaglandins. J Rheumatol Suppl 1991; 27:102–5:102–105. 63. Straub RH, Gu¨nzler C, Miller LE, Cutolo M, Scho¨lmerich J, Schill S. Anti-inflammatory cooperativity of corticosteroids and norepinephrine in rheumatoid arthritis synovial tissue in vivo and in vitro. FASEB J 2002; 16:993–1000. 64. Wilder RL. Hormones, pregnancy, and autoimmune diseases. Ann NY Acad Sci 1998; 840: 45–50:45–50. 65. Fong LG, Albert TS, Hom SE. Inhibition of the macrophage-induced oxidation of low density lipoprotein by interferon-gamma. J Lipid Res 1994; 35:893–904.

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66. Folcik VA, Aamir R, Cathcart MK. Cytokine modulation of LDL oxidation by activated human monocytes. Arterioscler Thromb Vasc Biol 1997; 17:1954–1961. 67. Urowitz M, Gladman D, Bruce I. Atherosclerosis and systemic lupus erythematosus. Curr Rheumatol Rep 2000; 2:19–23. 68. Manzi S, Wasko MC. Inflammation-mediated rheumatic diseases and atherosclerosis. Ann Rheum Dis 2000; 59:321–325. 69. Levy PJ, Tabares AH, Olin JW. Lower extremity arterial occlusions in young patients with Crohn’s colitis and premature atherosclerosis: report of six cases. Am J Gastroenterol 1997; 92:494–497. 70. Geissler A, Andus T, Roth M, Kullmann F, Caesar I, Held P, Gross V, Feuerbach S, Scho¨lmerich J. Focal white-matter lesions in brain of patients with inflammatory bowel disease. Lancet 1995; 345:897–898. 71. Henderson NK, Sambrook PN. Relationship between osteoporosis and arthritis and effect of corticosteroids and other drugs on bone. Curr Opin Rheumatol 1996; 8:365–369. 72. Cortet B, Guyot MH, Solau E, Pigny P, Dumoulin F, Flipo RM, Marchandise X, Delcambre B. Factors influencing bone loss in rheumatoid arthritis: a longitudinal study. Clin Exp Rheumatol 2000; 18:683–690. 73. Gough AK, Lilley J, Eyre S, Holder RL, Emery P. Generalised bone loss in patients with early rheumatoid arthritis. Lancet 1994; 344:23–27. 74. Teichmann J, Lange U, Stracke H, Federlin K, Bretzel RG. Bone metabolism and bone mineral density of systemic lupus erythematosus at the time of diagnosis. Rheumatol Int 1999; 18:137–140. 75. Trapani S, Civinini R, Ermini M, Paci E, Falcini F. Osteoporosis in juvenile systemic lupus erythematosus: a longitudinal study on the effect of steroids on bone mineral density. Rheumatol Int 1998; 18:45–49. 76. El Maghraoui A, Borderie D, Cherruau B, Edouard R, Dougados M, Roux C. Osteoporosis, body composition, and bone turnover in ankylosing spondylitis. J Rheumatol 1999; 26: 2205–2209. 77. Will R, Palmer R, Bhalla AK, Ring F, Calin A. Osteoporosis in early ankylosing spondylitis: a primary pathological event. Lancet 1989; 2:1483–1485. 78. Masi AT, Josipovic DB, Jefferson WE. Low adrenal androgenic-anabolic steroids in women with rheumatoid arthritis (RA): gas-liquid chromatographic studies of RA patients and matched normal control women indicating decreased 11-deoxy-17-ketosteroid excretion. Semin Arthritis Rheum 1984; 14:1–23. 79. Cutolo M, Balleari E, Giusti M, Monachesi M, Accardo S. Sex hormone status of male patients with rheumatoid arthritis: evidence of low serum concentrations of testosterone at baseline and after human chorionic gonadotropin stimulation. Arthritis Rheum 1988; 31: 1314–1317. 80. Sambrook PN, Eisman JA, Champion GD, Pocock NA. Sex hormone status and osteoporosis in postmenopausal women with rheumatoid arthritis. Arthritis Rheum 1988; 31:973–978. 81. Lahita RG, Bradlow HL, Ginzler E, Pang S, New M. Low plasma androgens in women with systemic lupus erythematosus. Arthritis Rheum 1987; 30:241–248. 82. Straub RH, Zeuner M, Antoniou E, Scho¨lmerich J, Lang B. Dehydroepiandrosterone sulfate is positively correlated with soluble interleukin 2 receptor and soluble intercellular adhesion molecule in systemic lupus erythematosus. J Rheumatol 1996; 23:856–861. 83. Straub RH, Zeuner M, Lock G, Scho¨lmerich J, Lang B. High prolactin and low dehydroepiandrosterone sulphate serum levels in patients with severe systemic sclerosis. Br J Rheumatol 1997; 36:426–432. 84. Nilsson E, de la TB, Hedman M, Goobar J, Thorner A. Blood dehydroepiandrosterone sulphate (DHEAS) levels in polymyalgia rheumatica/giant cell arteritis and primary fibromyalgia. Clin Exp Rheumatol 1994; 12:415–417.

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29 Quality of Life in Asthma and Rhinitis JEAN BOUSQUET and PASCAL DEMOLY Montpellier University, Montpellier, France PHILIPPE J. BOUSQUET Montpellier University, Nimes, France

I. INTRODUCTION Asthma is a chronic disease that can place considerable restrictions on the physical, emotional, and social aspects of patients’ lives and may have an impact of their careers. The importance of emotional factors and restriction of social life may be greater when symptoms are not adequately controlled, but the underlying disease itself may cause distress, especially when its evolution is unpredictable, as with asthma [1]. Medical care and health practitioners can increase these difficulties. Many asthmatics do not completely appreciate the impact of the disease on their social life and claim they lead ‘‘normal’’ lives, when their concept of normality may be based on adjustments and restrictions they have already incorporated into their lifestyles or alternatively because they mask their restriction, wanting to ‘‘live like others.’’ Most patients suffering from asthma cannot be cured, and their management should be directed to a reduction in symptoms and an improvement in lifestyle measured in terms of health-related quality of life (HRQL). It is now recognized that allergic rhinitis comprises more than the classical symptoms of sneezing, rhinorrhea, and nasal obstruction. In the last decade an increasing effort has been made to understand the socioeconomic burden of rhinitis in terms of effects on HRQL. It has been acknowledged in several consensus reports that allergic rhinitis is associated with impairments in patients’ day-to-day functioning at home, at work, and in school [2–4]. With the introduction of a questionnaire designed to measure rhinitis-associated impairments of HRQL [5] it became clear that patients may be bothered by sleep disorders, emotional problems, and impairment in activities and social functioning. In general terms patients with allergic rhinitis are impaired in physical and mental functioning, including vitality and the perception of general health [6]. 619

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II. METHODS FOR MEASURING QUALITY OF LIFE A. Generic Questionnaires Generic questionnaires measure physical, mental, and psychosocial functions in all health conditions irrespective of the underlying disease and can be used in the general population. These include the Sickness Impact Profile [7], the Nottingham Health Profile, and the Medical Outcomes Survey Short Form 36 (SF 36), which has been used to characterize patients with asthma or rhinitis [6,8] and to evaluate the effects of treatment on HRQL [9]. The advantage of generic instruments is that the burden of illness across different disorders and patient populations can be compared. The disadvantage, however, is that the instruments miss depth and may not be responsive enough to detect changes in general health states in spite of important changes in disease-related problems [10]. Health-related quality of life has become an essential part of health-outcome measurement in chronic disorders. However, only recently have health professionals focused on HRQL assessment in children and adolescents [11]. The recently published Child Health Questionnaire (CHQ-PF50) is a useful generic instrument to comprehensively assess quality of life, in particular when comparing young people with different chronic disorders [12]. B. Disease-Specific Questionnaires Specific instruments have been designed by asking patients what kind of problems they experience related to their disease. Both frequency and importance of impairments find expression in the questionnaires. These instruments have the advantage that they describe more accurately the disease-associated problems of the patients. Moreover, they seem to be more responsive to changes in HRQL than generic instruments. 1. Rhinitis Quality-of-Life Questionnaires Specific instruments for rhinitis patients from different age groups have been developed. The Rhinoconjunctivitis Quality-of-Life Questionnaire (RQLQ) [5] and the Rhinitis Quality-of-Life Questionnaire [13] have been tested in adult patients with seasonal allergic rhinitis and perennial allergic rhinitis, respectively. The 28-item Rhinoconjunctivitis Quality-of-Life Questionnaire (RQLQ) has strong measurement properties, but for large clinical trials, surveys, and practice monitoring, where efficiency is important, a shorter questionnaire was developed [14]. The Adolescent RQLQ questionnaire was developed to address patients in the age range of 12–17 years [15]. This questionnaire is a slightly modified version of the adult version, as problems in doing (school) work and the problem of feeling generally unwell appeared to be more important in adolescents than in adults. The RQLQ has been adapted to many different cultures and languages [16,17]. A Pediatric RQLQ has been developed for children aged 6–12 years [18]. This questionnaire differs from others in that children are less bothered by emotional problems and rhinitis interferes less with their day-to-day life. The RQLQ has been used in several trials focused on the effect of nasal glucocorticosteroids [18–22], H1 antihistamines [23], and the combination of glucocorticosteroids and H1 antihistamines [24] on rhinitis-related QOL. 2. Asthma Quality-of-Life Questionnaires Several specific instruments for asthma patients from different age groups have also been developed, several of which can be used in adult asthmatics (e.g., Asthma Quality-of-Life

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Questionnaire) [25–28]. The St. George’s Respiratory Questionnaire [29] appears to be a valid measure of HRQL in asthma [30]. A new tool was developed for monitoring of asthma outcomes: the ITG Asthma Short Form [31]. The reliability and validity of the Asthma Quality-of-Life Questionnaire (AQLQ) have been evaluated [32]. These questionnaires have been translated into several languages and can be used in many countries [33]. Electronic questionnaires are also available [34]. Several asthma-specific HRQL instruments have been designed for use in children and adolescents [11,35–37]. However, it is not clear which source we should use to measure HRQL in children with asthma—the children themselves or their parents [38]. The Pediatric Asthma Quality-of-Life Questionnaire [39] has shown responsiveness to change over time, but it lacks age specificity with regard to psychosocial issues and comprehensiveness of quality-of-life assessment. Another interesting questionnaire is the Adolescent Asthma Quality of Life Questionnaire (AAQOL) [40]. The Pediatric Asthma Caregiver’s Quality-of-Life Questionnaire (PACQLQ) measures the impact of child asthma symptoms on family activity (CGAct) and parental anxiety (CGEmot) [41]. In contrast, the Childhood Asthma Questionnaire [42] provides three different versions for different target ages. However, its generic section is not reflective of the respondent’s health status. Other asthma-specific instruments have major conceptual deficiencies when used as a single measure for quality-of-life assessment [43,44]. In the absence of a single ideal instrument, the use of batteries of HRQL instruments is therefore recommended, and further research is required to identify the impact that age and developmental status have on quality-of-life assessment. In clinical trials, specific questionnaires have been widely used, and differences between an intervention and placebo or between interventions have been reported. As to whether statistically significant difference is clinically important [45], several studies have attempted to answer this question [26,46,47]. The standard error of measurement may also be used to identify important changes on the AQLQ [48]. III. QUALITY OF LIFE IN RHINITIS Using a generic questionnaire (SF-36) [49], HRQL was found to be significantly impaired in patients with moderate to severe perennial allergic rhinitis compared with normal subjects [6]. Using the same questionnaire, HRQL was impaired, but to a lesser extent in patients suffering from seasonal allergic rhinitis [50], suggesting that prolonged allergen exposure impaired HRQL more than seasonal exposure. A new instrument examining satisfaction in 32 aspects of daily life, the Satisfaction Profile (SAT-P), was used in seasonal allergic rhinitis and found to correlate with SF-36 data [51]. Although HRQL questionnaires are of great interest to assess the overall effect of a disease on HRQL in a group of individuals, these questionnaires do not appear to be of sufficient sensitivity to be used in individual patients. The functional impairment in patients with moderate to severe perennial rhinitis [6] is comparable with the limitations perceived by asthmatic patients with a moderate to severe disease [52], and the extent to which asthma and rhinitis comorbidities are associated in HRQL has been elucidated [53]. Answers to the SF-36 questionnaire from 850 subjects recruited in two French centers participating in the European Community Respiratory Health Survey, a population-based study of young adults, were analyzed. Both asthma and allergic rhinitis were associated with an impairment in HRQL. However, 78% of asthmatics also had allergic rhinitis. Subjects with allergic rhinitis but not asthma

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(n ⳱ 240) were more likely than subjects with neither asthma nor rhinitis (n ⳱ 349) to report problems with social activities, difficulties with daily activities as a result of emotional problems, and poorer mental well-being. Patients with both asthma and allergic rhinitis (n ⳱ 76) experienced more physical limitations than patients with allergic rhinitis alone, but no difference was found between these two groups for concepts related to social/ mental health. As asthma was not found to further impair the HRQL in subjects with allergic rhinitis in the areas of mental disability and well-being, and as subjects with asthma often also suffer from allergic rhinitis, further studies on quality of life in asthma should ensure that the impairment in quality of life attributed to asthma does not result from concomitant allergic rhinitis. Sinusitis is a common feature of rhinitis, and the SF-36 and a sinusitis-specific HRQL measure, the Chronic Sinusitis Survey (CSS), have shown that sinus surgery may improve the quality of life of sinusitis patients [54–56]. Recognizing that rhinosinusitis is a disabling disease, other specific instruments such as the Rhinosinusitis Disability Index (RDI) [57] and the 31-item Rhinosinusitis Outcome Measure (RSOM-31) [58] have been introduced. Individuals with chronic rhinosinusitis and allergic rhinitis have the greatest level of disability as assessed by the RDI, while those with aspirin triad are least affected [59]. Rhinosinusitis also impairs HRQL in children, as shown in a study that used the Child Health Questionnaire [60]. This questionnaire measures in parallel both the childs and parents’ perceptions of health by means of separate parent proxy report (Child Health Questionnaire–Parent Form 50) and child self-report (Child Health Questionnaire–Child Form 87) questionnaires concerning physical and psychosocial functioning. The impact of recurrent ENT infections on children’s social life during the first 4 years of life is not easily captured. Indirect information can be obtained by use of a specific questionnaire that measures parental quality of life [61]. Rhinitis-related HRQL appears to be moderately correlated to the more classical outcome variables used in clinical trials, such as daily symptom scores [51] and nasal hyperreactivity [62]. These observations are in line with the results of studies comparing disease-specific HRQL in asthmatics with asthma symptoms, peak flow, and bronchial hyperresponsiveness [25,63]. It has been posited that the classical outcome variables may only partially characterize the disease of the patient. From that point of view it has been suggested to measure HRQL along with the conventional clinical indices [64]. When using HRQL outcomes in clinical trials, the question arises whether a change in HRQL is clinically important. It has been shown that in HRQL instruments that use a 7-point scale, the minimal important difference of quality-of-life score per item is very close to 0.5 [65]. Using the SF-36 it was shown that oral H1 antihistamines were able to reduce HRQL scores in perennial and seasonal rhinitis [9,66,67]. Generally, the effect on HRQL runs parallel with the effect on conventional medical outcome measures. However, in some studies differences can be found. In the study evaluating the combined effect of glucocorticosteroids and H1 antihistamines, no differences were seen between patients treated with H1 antihistamine and glucocorticosteroids versus glucocorticosteroids alone in terms of HRQL, whereas for some patient-rated symptoms the combination was found superior [24]. This might indicate that patients perceive differences in efficacy not captured by conventional symptom scores. Alternatively, as the RQLQ was not designed to measure the short-term variations in disease status that appear in SAR, it may not demonstrate the rapid improvement of HRQL under antihistamine treatment [68]. Patients with chronic conditions may adapt themselves to their disease. As the perception of patients is clearly important in the management of disease and patient

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compliance, measurement of this dimension by HRQL questionnaires in clinical trials may be justified. Finally, HRQL indices may be predictive of placebo and medication response to treatment for allergic rhinitis [69]. The daily use of either intranasal corticosteroids or histamine(1) receptor antagonists has proved to be efficacious in the treatment of seasonal allergic rhinitis. Most patients, however, use these medications as needed. Two studies compared the effectiveness of asneeded use of histamine(1) receptor antagonists with that of intranasal corticosteroids in the treatment of seasonal allergic rhinitis and found that intranasal fluticasone is more effective in terms of symptom reduction and HRQL than loratadine [70]. Other rhinitis treatments were shown to improve HRQL. These treatments include leukotriene receptor antagonists [71], allergen immunotherapy in pollinosis [72], and omalizumab, an anti-IgE monoclonal antibody [73,74]. In the future, with more data available, it is possible that HRQL measurements may represent the primary outcome measure for clinical trials. IV. QUALITY OF LIFE IN ASTHMA Symptoms of disease reported by patients reflect the effects of the disease process within the individual and the person’s physical and mental ability to tolerate or otherwise cope with the limitations on their functioning. Generic HRQL scales may be used to detect the importance of social life impairment, and all nine SF-36 category scores were highly significantly correlated with the severity of asthma [52]. The HRQL of patients with epilepsy was compared with that of patients with angina pectoris, rheumatoid arthritis, asthma, and chronic obstructive pulmonary disease using SF-36 [75]. The results indicated that the HRQL of a representative sample of patients with epilepsy is good when compared with other chronic disorders, although it is reduced in several dimensions compared with a general reference population. Patients with rheumatoid arthritis and COPD scored lowest on the physical function scales, while rheumatoid arthritis patients reported the most pain. Clinical predictors of HRQL depend on asthma severity [76]. The AQLQ is a useful measure for low-income asthmatics [77]. It has similar psychometric properties during acute hospitalization and subsequently in an outpatient setting [78]. A study showed that the failure of prehospital management to prevent hospitalization in most cases stems from a failure to implement currently recommended actions or treatments for exacerbations and can be assessed using HRQL [79]. An asssociation between HRQL and consultation for respiratory symptoms was found in the DIMCA program [80]. The experiences of young people at home and at school were reported [81]. Asthma restricted their lives at school and recreationally, but they were actively involved with their condition and its management. The study revealed that while prescribed medicines in the form of inhalers were used as the primary means of coping with asthma episodes, the young people were concerned about being dependent on such medicines, in line with more general ambivalence in late modern cultures about the long-term use of prescribed medicines. It also demonstrated how social relations in particular contexts help to determine the extent to which asthma episodes can be managed. In the Childhood Asthma Management Program (CAMP), it was examined whether the burden of childhood asthma would compromise psychological adaptation and if the degree of compromise increases with disease severity [82]. Mild to moderate asthma was found to have modest effects on the daily life but not the psychological health of this group of children. Variation in the psychological characteristics of these children was, as is the case for most children, tracea-

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ble to the overall psychological adaptation of their families. The child health questionnaire (CHQ) was tested in a sample of moderate- and low-income inner-city children with asthma ranging in age from 5 to 12 years [12]. Inner-city children had a lower score than those living elsewhere [83]. The Paediatric Asthma Caregiver’s Quality of Life Questionnaire (PACQLQ) measures the impact of a child’s asthma symptoms on family activity and parental anxiety. It was found to be sensitive to group measures of change in symptoms over 3 months among preschool children. The relationship between asthma severity, family functioning, and the HRQL of children with asthma was examined in one study [84]. There was a significant relationship between the mental health of children with asthma and family functioning, but no significant relationship between their physical health and family functioning. These findings suggest that the domains comprising the HRQL of children with asthma are related to both disease and nondisease factors. A better understanding of these relationships will facilitate the development of new interventions to help children with asthma. Some studies of asthma severity, atopic status, allergen exposure, and quality of life in elderly persons showed an impairment of HRQL in elderly asthmatics [85]. Most clinical trials in asthma have focused on outcomes that are primarily of importance to the clinician, such as symptom medication scores and the measurement of the pulmonary function. Fewer have assessed whether patients feel better and can function better in day-to-day activities. One of the aims of treatment of asthma is to improve patient well-being, which incorporates the concept of patients’ perceptions of how they feel and their ability to function in their everyday life (HRQL). The weak association between conventional measures of clinical status in asthma and asthma-specific quality of life means that quality of life must be measured directly [86]. Health status (or HRQL) measurement is an established method for assessing the overall efficacy of treatments for asthma [87]. Although it is important to use HRQL in clinical trials, there are several drawbacks that should be avoided [88]. Both generic and disease-specific HRQL scales are valid measures of quality of life, but disease-specific scales are likely to be more capable of detecting smaller changes in the health status of patients with bronchial asthma and hence may be chosen as the instrument in a clinical trial [89]. When the Sickness Impact Profile was compared with the AQLQ and the Living-with-Asthma Questionnaire (LWAQ), only disease-specific questionnaires were found to be responsive to treatment [90]. Health-related quality of life was found to be improved by many therapeutic interventions, including inhaled corticosteroids [91–95], long-acting 2-agonists [63,96], leukotriene receptor antagonists [97–100], and anti-IgE monoclonal antibodies [101,102]. The use of antiallergenic mattress covers results in significant reductions in Der p 1 concentrations in carpet-free bedrooms. However, in patients with moderate to severe asthma, airways hyperresponsiveness, clinical parameters, and HRQL were not affected by this allergen avoidance measure [103]. Education is important in the management of asthma, and self-management reduces incidents caused by asthma and improves quality of life [104–110].

REFERENCES 1. Goeman DP, Aroni RA, Stewart K, Sawyer SM, Thien FC, Abramson MJ. Patients’ views of the burden of asthma: a qualitative study. Med J Aust 2002; 177(6):295–299.

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30 Atopic Dermatitis ¨ HM and THOMAS LUGER MARKUS BO University of Mu¨nster, Mu¨nster, Germany

I. INTRODUCTION Atopic dermatitis (AD) is a common chronic pruritic inflammatory skin disorder occurring with increasing frequency in children and adults in western countries. Patients with AD have dry skin (xerosis), eczema, lichenification, and pruritus that is often etremely difficult to treat. The etiology of AD is currently believed to include an immunodysfunction and a skin barrier defect. Immunologically, a Th1/Th2 imbalance appears to result in an allergenspecific hypersecretion of IgE and an increased release of interleukin (IL)-4 and IL-5 but reduced secretion of IL-2 and interferon-␥ (IFN-␥) (reviewed in Ref. 1). However, as the German synonymon for AD, ‘‘neurodermitis,’’ suggests, neurobiological factors have long been proposed to play an important role in AD. In many anecdotal and investigative reports psychological problems, such as psychic lability, chronic anxiety, mood deviations, or insufficient coping with stress, were delineated as key features of AD [2]. Moreover, as early as 1968, when Szentivanyi formulated his ‘‘␤-adrenergic theory of the constitutional basis of atopy,’’ neuromediators or neurotransmitters have been implicated in the pathogenesis of atopy [3]. During the last two decades, our knowledge of the complex interrelations between neuropeptides, the immune system, and the skin has increased dramatically. The skin as the largest organ of the human body not only is a target organ for many neuropeptides, but is also capable of expressing many neuropeptides by itself [4]. One characteristic example is the skin proopiomelanocortin (POMC) system that represents an apparent analogy to the hypothalamic-pituitary-adrenal (HPA) axis induced by stress [5]. As the biological actions of many neuroptides become increasingly transparent, investigation on the neurobiological basis of AD is currently an intense area of research for many laboratories. 631

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In this chapter we will systematically review selected neuropeptides and neurotransmitters in patients with AD with emphasis on their potential role in regulating immune and inflammatory responses. To facilitate comprehensive reading of the chapter, we have briefly outlined the biochemistry, physiology, and principal immunoregulatory activities of each neuropeptide/neuromediator before addressing its role in AD. For detailed information concerning the biology and immunology of each neuromediator, the reader is referred to special review articles and citations herein. It was also our intention to address open questions and to suggest novel or interesting future directions in the field of neuropeptides/ neuromediators and AD. II. THE CORTICOTROPIN/PROOPIOMELANOCORTIN SYSTEM The POMC system is the central component of the HPA axis, which integrates stress and largely controls hormonal homeostasis of the human body. POMC was first characterized as a pituitary-derived prohormone with a molecular weight of approximately 31 kDa. It is the precursor of adrenocorticotropin (ACTH), melanocyte-stimulating hormones (␣-, ␤-, ␥-MSH), and the endogenous opioid ␤-endorphin (␤-ED) (reviewed in Ref. 5). Generation of these bioactive peptides is a complex process and involves the enzymatic action of specific prohormone convertases that belong to the subtilisin-kexin family. In contrast to ACTH and ␤-ED, ␣-MSH requires posttranslational modification, i.e., N-terminal acetylation and C-terminal amidation, in order to obtain bioactivity. In the pituitary gland, transcription of the POMC gene and processing of the translated POMC precursor into the biocative POMC peptides is under the control of corticotropin-releasing hormone (CRH), which regulates POMC gene activity, production and release of ACTH, MSH peptides, and ␤-ED. CRH, ACTH, the MSH peptides as well as ␤-ED elicit their biological actions by means of binding to specific cell membrane receptors that all belong to the superfamily of G-protein–coupled receptors with seven transmembrane domains: CRH binds to the CRH receptor family (CRH-1R and CRH-R2), ACTH and the MSH bind to the so-called melanocortin receptors (MC-R), while ␤-ED binds to the ␮-opioid receptor (MOR) and, to a lesser extent, to the ␦-opioid receptor. It is established that the skin is a target organ as well as a source for CRH, melanocortins, and ␤-ED (reviewed in Refs. 4–6). CRH and CRH-R1 gene expression have been detected in rodent and human skin at the RNA and protein level. In contrast to the MOR, which seems to have a limited distribution within the skin, MC-R, especially MC-1R, are ubiquitously expressed in the majority of resident skin cells. In vitro studies have shown that epidermal melanocytes and keratinocytes, Langerhans cells, microvascular dermal endothelial cells, and sebocytes express the MC-1R (reviewed in Refs. 5 and 6). Moreover, POMC gene expression as well as immunoreactive amounts of ACTH, MSH peptides, and ␤-ED have been detected in the majority of the above cell types as well as in human skin in situ or ex vivo. The fact that inflammatory stimuli such as ultraviolet light and pro-inflammatory cytokines induce CRH expression, POMC synthesis, and release of POMC-derived peptides in many cell types of the skin has led to the concept of a cutaneous CRH/POMC system in analogy to the bona fide HPA axis. It is noteworthy to mention that both CRH and ␣-MSH exert a wide range of immunomodulatory activities on various cell of the immune system as well as on resident cells of the skin (reviewed in Refs. 5–7). Pro-inflammatory and anti-inflammatory properties have been reported for CRH (reviewed in Ref. 5). Intradermal injection of CRH in mice and rats resulted in mast cell degranulation, which could be blocked by a CRH-R1 antago-

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nist. On the other hand, CRH has been shown to act as a potent vasoconstrictive and antiinflammatory agent when applied topically or injected subcutaneously in various animal models. In the majority of in vivo and in vitro studies addressing the immunomodulatory activity of ␣-MSH, this neuropeptide had anti-inflammatory properties (reviewed in Refs. 5–7). ␣-MSH suppressed the sensitization and elicitation phase of the cutaneous immune response to contact allergens and induced hapten-specific tolerance. In another mouse model of experimentally induced vasculitis, ␣-MSH suppressed endothelial cell damage induced by lipopolysaccharide and reduced expression of the intercellular adhesion molecule-1, vascular adhesion molecule-1, and E-selectin [8]. In vitro, ␣-MSH upregulated expression and release of IL-10 by human monocytes while expression of IL-1, IL-6, and TNF-␣ were downregulated. ␣-MSH also downregulated the expression of MHC class I molecules and of CD86 and CD40 on monocytes and dendritic cells. Interestingly, ␣MSH, by modulating the function of antigen-presenting cells, also regulates IgE synthesis in B lymphocytes stimulated with CD40 and IL-4. In a variety of target cells ␣-MSH inhibits activation of NF-␬B, a transcription factor induced by various inflammatory stimuli (reviewed in Refs. 5–7). Interestingly, this inhibitory action of ␣-MSH can be significantly blocked by ␤2-adrenergic blockade [9]. Contradictory effects have been reported for ␣-MSH with regard to its effect on mast cells (reviewed in Ref. 6). In murine mast cells ␣-MSH in vitro inhibited antigen-stimulated histamine release, enhanced IL-3–dependent proliferation, and downregulated expression of IL-1␤, TNF-␣, and lymphotactin, while in unstimulated human skin mast cells ␣-MSH increased histamine release. In light of the above activities orchestrated by CRH, ␣-MSH, and ACTH, which also trigger the release of glucocorticoids by the adrenals, it is conceivable that any imbalance of the HPA axis may lead to an alteration in the net outcome of inflammatory and immune responses, including those of the skin. This concept is clearly supported by studies on Lewis (LEW/N) rats with a genetically blunted HPA axis [10]. Accordingly, these animals are highly susceptible to pro-inflammatory stimuli, including streptococcal cell walls. Further studies have shown that a blunted HPA axis also predisposes to the development of other inflammatory disorders, as demonstrated in animal models for lupus erythematosus and autoimmune thyroiditis (reviewed in Ref. 11). With regard to the HPA axis in patients with AD, work in the late 1980s suggested some deviations in the circadian serum cortisol profile [12]. Further studies addressing the integrity of the HPA axis and its response to exogenous stimuli have recently provided evidence for a blunted response of cortisol and ACTH in patients with AD [13]. Rupprecht and coworkers studied 24-hour cortisol secretion and the cortisol, ACTH, and ␤-ED responses to CRH in such individuals versus healthy control subjects matched for sex and age. The net response to CRH was significantly attenuated for both cortisol and ACTH in patients with AD, while 24-hour secretion of cortisol and the ␤-ED response did not differ between normal controls and patients with AD. To assess the relevance of these data with a physical stimulus, the same investigative team performed exhausting incremental graded bicycle exercise and measured adrenal and pituitary hormones in 14 patients with AD as well as in normal control subjects. Patients with AD displayed a significantly lower increase in peripheral blood level of norepinephrine as compared to the less affected patient group but no substantial difference in the net responses of ACTH and cortisol when compared to healthy individuals [14]. In another study on 15 children with AD and control subjects, the stress response during performance of the so-called Trier Social Stress Test for Children was evaluated by salivary cortisol measurements and continuous measurement of the heart rate via a wireless transmission device [15]. As expected, there

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was an increase in the heart rate response during performance of the test, but no differences in the resting heart rate or heart rate reactivity between children with AD and healthy controls were found. However, children with AD exhibited a blunted cortisol response, which was related to neither a different subjective stress rating nor an atopy-specific personality profile. Matsuda et al. investigated adrenal function in patients with severe AD to clarify if adrenocortical suppression could be due to overused topical corticosteroids [16]. They performed ACTH tests and measured basal serum cortisol in two patient groups, one that had not used topical corticosteroids for at least 3 months and one that had topical corticosteroids on a daily basis prior to the 3-week hospitalization. In accordance with the above finding of an altered HPA axis in patients with AD, both groups showed an reduced cortisol serum level and a suppressed response to ACTH. With regard to ␤-ED, it was furthermore reported that patients with AD had significantly increased basal serum levels as compared to normal control subjects and that the increased ␤-ED levels correlated with some clinical parameters of the disease [17,18]. It was speculated that the increased ␤-ED could be derived from inflammatory cells in lesional skin or from activation of the HPA axis by psychoneural factors in the mechanism of chronic stress. Collectively, these findings clearly indicate abnormalities in the secretion pattern of selected POMC peptides into the systemic circulation and support the concept of an altered HPA axis in patients with AD. However, whether the disclosed abnormalities are specific for AD awaits further investigation. Many chronic inflammatory and neoplastic skin disorders including psoriasis, alopecia areata, keloids, squamous cell carcinoma, and melanoma are associated with increased in situ expression of POMC peptides (reviewed in Refs. 5 and 6), which may ‘‘spill over’’ into systemic circulation. Increased serum levels of ␤endorphin have also been detected in psoriasis and systemic sclerosis [18,19], suggesting that increased POMC expression and release of POMC-derived peptides is a reactive phenomenon. Whether the increased serum level of ␤-ED is related to the blunted response of cortisol and ACTH in patients with AD is unclear. It is possible that increased peripheral production of POMC-derived peptides such as ␤-ED contributes to the hyporesponsiveness of the HPA axis in atopic individuals, possibly by downregulating or desensitizing receptors for POMC peptides. Since ACTH binds not only to MC-2R (the bona fide ACTH receptor) but also to MC-1R with similar affinity as ␣-MSH, it may be interesting to investigate if immunocompetent cells or resident skin cells from patients with AD display differences in MC-1R expression or have abnormal responses towards ␣-MSH. III. CATECHOLAMINES Evaluation of the adrenergic response and studies on catecholamines are retrospectively some of the earliest research activities establishing a link between the nervous system and AD. The biosynthesis of the catecholamines from L-phenylalanine is a multistep process and involves the action of phenylalanine hydroxylase, tyrosine hydroxylase, L-aromatic amino acid decarboxylase, dopamin-␤-hydroxylase, and phenylethylethanolamine-Nmethyltransferase (PNMT). Both phenylalanine hydroxylase and tyrosine hydroxylase require the essential cofactor (6R)L-erythro-5,6,7,8-tetrahydrobiopterin (6BH4). The biological action of catecholamines is mediated by adrenoreceptors, which have been cloned and which are classified into various subtypes. These receptors are also expressed on epidermal keratinocytes [20] as well as on several cells of the immune system, including lymphocytes [21] and Langerhans cells [22]. In the latter cell type both epinephrine and norepinephrine inhibited antigen presentation, as recently shown. Local injection of epinephrine inhibited

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the contact hypersensitivity reaction to epicutaneously applied haptens, and this effect could also be elicited by injection of epinephrine at a distant site, suggesting a systemic effect [23]. In the skin, catecholamine release does not seem to be restricted to the sympathetic nervous system. The human epidermis has the capacity for total synthesis and degradation of catecholamines, including the pathway for de novo synthesis and recycling of 6BH4 [23,24]. As the skin releases and degrades catecholamines, which act on immunocompetent and accessory cells, an imbalance of this homeostasis may alter the integrity of the skin immune system. The fact that patients with AD have distinct clinical abnormalities such as white dermographism, delayed blanching, acetylcholine-stimulated bronchial hyperreactivity, increased histamine, and abnormal sweating and pilomotor smooth muscle responses suggested in the late 1960s an imbalance of the ␤-adrenergic system towards an ␣1-response [3]. The majority of subsequent studies focused on the adrenergic response of peripheral blood polymorphonuclear cells or lymphocytes from patients with AD. The detected abnormalities are complex, sometimes controversial, and include deviations on the receptor and postreceptor level. In general, cells from patients with AD displayed a weaker increase in intracellular cAMP after adrenergic agonist stimulation than cells from control subjects [25–27]. Some investigators found decreased numbers of ␤-adrenoreceptors [28], while others did not [29,30]. It was reported that atopic peripheral blood leukocytes display a reduced affinity of ␤-adrenergic binding sites [28,29], an increased ratio of ␣- to ␤adrenoreceoptors [31], as well as an elevated phosphodiesterase activity [28]. In addition to peripheral blood leukocytes, an altered response of cultured epidermis to ␤-adrenergic stimulation has been reported [32]. Earlier studies also disclosed a high activity of catecholo-methyl transferase in lesional skin of patients with AD [33]. Schallreuter et al. detected significantly higher levels of total 6BH4 in uninvolved epidermis of patients. Cell extracts from suction blister roofs of nonlesional skin furthermore revealed only half of the normal activity of PNMT as compared to normal subjects. On the other hand, the norepinephrinedegrading enzyme monoamine oxidase A was significantly upregulated [34]. However, the concept of ␤-adrenoreceptor weakness was challenged since the decreased cAMP response of leukocytes from patients with AD was not limited to adrenergic agonist stimulation but was also detectable upon treatment with histamine or prostaglandin E1 [35]. Since histamine, isoproterenol, or prostaglandin are capable of desensitizing peripheral mononuclear leukocytes to subsequent stimulatory concentrations of any of the agonists, it was speculated that heterologous desensitization could be another explanation for the reduced cAMP synthesis in AD [36]. It is possible that chronic exposure to increased systemic or locoregional levels of catecholamines may likewise contribute to the impaired ␤-adrenergic response in AD (homologous desensitisation). Indeed, increased basal plasma levels of norepinephrine have been demonstrated in 41 adults with severe AD [37,38]. However, other investigators examining the adrenomedullary function of patients with AD by measuring catecholamine levels and cAMP in the resting state as well as in response to various stimuli including histamine infusion were unable to detect any significant differences as compared to normal subjects. In addition, plasma clearance of exogenously administered epinephrine was similar between patients with AD and normal volunteers [39]. Whether these changes in catecholamine synthesis and degradation are related to increased noradrenergic innervation, as observed in the skin of patients with AD, is unknown. Collectively, these puzzling findings indicate that several alterations within the catecholamine/␤-adrenergic homeostasis do exist in patients with AD. In recent years the subject of catecholamines and adrenoreceptors in patients with AD has been somewhat

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dormant. Since the pathogenetic mechanism of the phenomenon, which led to the hypothesis of Szentivanyi, remains unclear, further studies are needed. In light of the recently discovered immunomodulatory activities of catecholamines on dendritic cells and other immunocompetent cells, future studies addressing these effects in cells of patients with AD may be of special interest.

IV. MELATONIN Melatonin (MLT, N-acetyl-5-methoxytryptamine), a methoxy derivative of serotonin, was orginally characterized as a pineal gland–derived hormone. The biosynthesis of MLT starts from tryptophan and involves several enzymes including tryptophan hydroxylase, 5-hydroxytryptophan decarboxylase, N-acetyltransferase, and N-hydroxyindole-O-methyltransferase (HIOMT). The latter enzyme is the most restrictive enzyme in terms of organ distribution. Due to its amphiphilic character, MLT penetrates into every tissue and crosses the blood-brain barrier and the placenta. MLT easily enters the cytoplasm of its target cells but has also been detected in other subcellular compartments including the nucleus and the mitochondria. MLT mediates its biological action by binding to high-affinity membrane and nuclear MLT receptors. Other actions, for example, the property of MLT to act as a scavenger for the hydroxyl radical (䡠OH), may be mediated by a nonreceptor mechanism. The pineal gland is the major source of circulating MLT, and peripheral blood levels become uniformely low after surgical removal of the pineal gland. However, several lines of evidence show that MLT is also synthesized in nonpineal tissues. First, MLT can be detected in bacteria and invertebrates that lack a pineal gland; second, MLT has been found in several nonpineal tissues including the eye, gut, ovary, and bone marrow. Although some tissues and body fluids may have the ability to accumulate or concentrate MLT derived from the circulation, several investigative teams have confirmed the expression and biological activity of enzymes involved in MLT biosynthesis. Recently, Slominski et al. detected the presence of HIOMT as well as MLT in human skin and in various cutaneous cell types, respectively [40]. Although MLT was initially characterized as a regulator of melanin dispersion in amphibian melanocytes, no such effect has been confirmed in human melanocytes until now. In the mammalian system, most attraction of MLT has come from its fluctating peripheral blood level generated by the suprachiasmatic nuclei and entrained by the light/ dark cycle (reviewed in Ref. 41). Accordingly, MLT plasma levels peak at 3:00–4:00 a.m. and become undetectable during the daytime. The pattern of rhythmic oscillation between the MLT plasma level and that of cortisol is therefore inversely correalated. In addition to its role as an effector of the chronobiotic clock, it is known that MLT can alter certain immune functions in vitro and in vivo (reviewed in Refs. 42–44). However, the exact role of MLT in immune function remains controversial, and many contradictory studies have been published. Specific binding sites for MLT have been reported in immunocompetent cells such as in circulating lymphocytes, splenocytes, and thymocytes of rodents as well as in human T-helper cells. In immunocompromised mice, treatment with MLT enhanced antibody production and restored impaired T-helper cell functions. MLT also increased antigen presentation by splenic macrophages, enhanced MHC class II molecule expression, and upregulated production of IL-1 and TNF-␣. On the other hand, MLT suppressed the synthesis of IFN-␥ and TNF-␣ in peripheral blood mononuclear cells of

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some blood donors. In analogy to the immunomodulatory activity of ␣-MSH, MLT reduced NF-␬B DNA binding in the rat spleen. Whether MLT plays a decisive role in the pathogenesis of AD is unclear. Two studies have addressed the question as to whether alterations in MLT secretion occur in patients with AD. In one study the authors measured MLT serum levels every 2 hours starting at 8:00 a.m. in 18 patients with severe AD [45]. In 6 patients with low melatonin levels, the circadian rhythm was found to be abolished, and in 8 patients a decreased nucturnal increase in MLT was observed as compared to normal volunteers. Only 4 patients had a normal pattern of MLT secretion. There was no relationship between the pattern of MLT secretion, clinical severity of AD, patient’s history, or duration of the disease. From these data the authors concluded that patients with AD have a dysfunction of pineal MLT secretion. In a recent study by Rupprecht et al., plasma levels of MLT were analyzed in 13 patients with AD in relation to disease activity [46]. The circadian pattern of MLT secretion was maintained in all patients with AD as compared to 10 normal individuals. However, the total amount of MLT secreted over 24 hours was lower and the increment between the diurnal and nocturnal MLT values was significantly smaller in atopics than in normal controls. Subsequent measurements 10 days after hospitalization as well as under remission 1 year after initial presentation revealed increased total MLT plasma levels and a tendency towards a normalized circadian secretion pattern in the patients with AD. Since sympathetic fibers transmit light-induced stimuli to the pineal gland, it has been hypothesized that the altered MLT secretion may be linked to the ␤-adrenoreceptor weakness of atopic individuals. However, the secretory activity of the pineal gland is regulated by many other central pinealopetal projections, some of which contain neuropetides also implicated in the pathogenesis of AD. Alterations in MLT secretion occur in many other unrelated disorders including psoriasis, anorexia nervosa, diabetes, and various psychiatric diseases [41–44]. Therefore, it remains to be determined if an altered secretion pattern, a differential receptor expression on immunocompetent cells, or abnormalities in cutaneous synthesis/degradation of MLT plays a pathogenetic role in AD.

V. SUBSTANCE P As a mediator of neurogenic inflammation [47], substance P (SP) has gained particular attention in the field of neurobiological research on patients with AD. SP is a member of the tachykinin family of neuropetides that also include neurokinin A, neurokinin B, neuropeptide K, and neuropeptide-␥ (reviewed in Ref. 48). All members are biochemically characterized by the conseverd minimal C-terminal amino acid sequence FXGLM-NH2. Production and release of SP occurs mainly in the central nervous system. In the human skin SP immunoreactivity has been found in dermal sensory nerves, often associated with blood vessels, mast cells, or hair follicles. In addition, SP immunoreactive nerve fibers have been identified in the epidermis. Interestingly, expression of SP and other tachykinins are induced by pro-inflammatory such as IL-1 and lipopolysaccharide. The biological effect of SP are mediated by the so-called neurokinin receptors (NK-R), which belong to the superfamily of G-protein–coupled receptors and which are closely related at the structural level. The three cloned NK-Rs bind SP as well as neurokinins A and B with differential affinity. They are expressed in the brain but also in a number of peripheral tissues, including the gut, lung, immune system, and skin. In the latter, functional NK-R have

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been detected in keratinoyctes, fibroblasts, endothelial cells, Langerhans cells, and mast cells (reviewed in Ref. 48). Functionally, SP can be considered as the prototype of a pro-inflammatory neuropeptide (reviewed in Ref. 48). SP released from sensory neurons after noxious stimuli induces a triple response consisting of erythema, edema, and itching. This phenomenon is mediated via release of histamine by mast cells as well as via a direct effect of SP on endothelial cells. In endothelial cells as well as in epidermal keratinoyctes, SP upregulates the expression of pro-inflammatory cytokines including IL-1 and IL-8 and enhanced their in vitro proliferation. When injected into human skin, SP induces neutrophilic and eosinophilic infiltration, luminal translocation of P-selectin, and enhanced expression of E-selectin, the latter two adhesion molecules crucially involved in leukocyte recruitment. The important role of SP as a pro-inflammatory neuropeptide has been confirmed in animal models. Accordingly, NK-1R knockout mice are protected from inflammatory reactions mediated by SP and related tachykinins. Interestingly, it was recently shown that therapeutic UVA irradiation reduces the expression of NK-1R on dermal blood vessels and epidermal keratinocytes in patients with AD [49], supporting a pro-inflammatory role of SP-mediated reactions in this disease. With regard to SP and leukocytes, it has been demonstrated that peripheral blood mononuclear cells from patients with AD express binding sites for SP [50]. SP increased the release of both IFN-␥ and IL-4 at physiological concentrations, and this effect was different in patients with AD and normal subjects [51]. In another study SP also had a significant percentage-enhancing effect on these cytokokines and the ratios of IFN-␥:IL-4 production were markedly elevated in the SP-treated peripheral blood mononuclear cells from patients with AD [52]. The wheal-and-flare response to intradermal injection SP and related neuropeptides has been evaluated in patients with AD. Coulson and Holden did not detect any significant difference in the flare areas between controls and atopics. The wheal volumes after injection with SP and histamine were larger in patients with AD, and this was explained by an exaggerated wheal reaction to histamine in atopics [53]. Others have reported a statistically significant reduction in both the wheal and flare responses to SP, whereas the itch response was not different in atopics and control subjects [54]. This is in accordance with a blunted histamine and SP response in dogs with AD [55]. Several studies have addressed the question if the number of nerve fibers immunoreactive for SP and other neuropeptides are increased in patients with AD. Of note, many of the pro-inflammatory neuropeptides including SP, calcitonin gene–related peptide (CGRP), and vasointestinal active peptide (VIP) are often colocalized in the same fiber [56]. The studies addressing the amount of these neuropeptides revealed partially contradictory findings. Early studies revealed increased levels of SP and VIP in suction blister fluids from patients with AD [57]. In a later study using whole homogenates from lesional skin of patients with AD, this could not be confirmed for SP [58,59]. Immunohistochemical studies revealed perivascular expression of SP in lesional skin in most patients with AD, while no such staining was detectable in normal skin [60]. The same authors failed to detect any changes in the expression of other neuropeptides, including CGRP, a finding confirmed by another group [61]. In contrast, Tobin et al. detected an increased number of immunoreactive sensory nerve fibers for both SP and CGRP in lesional skin of patients with AD [62]. In summary, it appears that cutaneous expression of sensory neuropeptides with proinflammatory activities, especially SP, is increased in patients with AD. Whether the

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altered in vivo response of atopic skin to SP is related to quantitative changes in cutaneous expression or increased systemic circulation of SP and other neuropeptides is unknown. With regard to the partially controversial findings on neuropeptide expression in eczematous skin, it is likely that methodological differences, e.g., use of frozen tissue sections versus paraffine-embedded material, explain some of the above discrepancies. On the other hand, quantification of immunoreactive structures remains problematic. Since the density and fine structure of peripheral nerves in the skin of patients with AD depend on the acuity and morphology of the cutaneous eczematous lesions [63,64], this factor has to be considered in previous as well as in future studies. Further studies addressing the expression, synthesis, and degradation of SP and related sensory neuropeptides including their receptors are needed. VI. NEUROTROPHINS Two members of the neurotrophin (NT) family of neuropeptides, NT-4 and nerve growth factor (NGF), have recently been implicated as a pathogenetic link between the nervous system and the immune system in patients with AD. The receptors for NGF and NT-4 include the 75 kDa NT receptor (p75NTR) as well as members of the trk family, which form heterocomplexes with each other to modulate NT actions. Both p75NTR and the members of the trk family are expressed in human skin and have been detected in epidermal keratinoyctes, melanocytes, dermal fibroblasts, mast cells, and immunocompetent cells (reviewed in Ref. 4). In addition to playing crucial roles as regulators of survival, development, proliferation, differentiation, and function of neurons of the central and peripheral nervous system, NTs have a variety of immunomodulatory properties (reviewed in Ref. 65), including regulation of lymphocyte proliferation, IL-2 receptor expression, and immunoglobulin synthesis. On mast cells, NGF has been shown to act as a secretagogue. Of note, NTs are also synthesized by various cells of the immune system, including lymphocytes, macrophages, mast cells, and eosinophils as well as by resident cells of the skin such as keratinocytes, Merkel cells, and dermal fibroblasts (reviewed in Ref. 4). Toyoda et al. determined the levels of NGF and SP in the plasma of 52 patients with AD as compared to 35 nonatopic individuals [66]. Patients with AD had significantly elevated plasma levels of both neuropeptides. There was a positive correlation between the plasma levels of NGF and SP. In addition, there was a significant correlation between the plasma levels of NGF and SP and the disease activity evaluated by three different scoring systems. These data are suggestive for a systemic involvement of both NGF and SP. The source for the elevated plasma NGF in patients, however, remains speculative. Since expression of NGF in the epidermis is increased in psoriasis as well as in several other inflammatory dermatoses [66,67], it is likely that the elevated NGF in the plasma of patients with AD is derived from the skin. Although it is conceivable that increased systemic NGF levels influence the skin immune system, the exact role of NGF remains to be determined. Like other neuropeptides, plasma levels of NGF are elevated in a variety of diseases, including lupus erythematosus and systemic sclerosis (reviewed in Ref. 65). In another study, expression of NT-3 and NT-4 was investigated in human epidermal keratinocytes in vitro and in situ [68]. Cultured human keratinocytes expressed NT-4, while dermal fibroblasts expressed NT-3. In vitro stimulation with IFN-␥, a marker cytokine for AD, upregulated NT-4 expression in keratinoyctes. Immunohistochochemical studies on normal human skin revealed that NT-4 expression is expressed in epidermal keratinocytes and NT-3 in the dermal compartment. In prurigo lesions of AD, very intense epidermal

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immunostaining for NT-4 was detected. Since NT-4 promotes survival and outgrowth of neurons, it is possible that the increased number of sensoric nerve fibers observed in lesional skin of patients with AD is due to increased epidermal expression of NT-4 or other NT. VII. VASOACTIVE INTESTINAL PEPTIDE AND PITUITARY ADENYLATE CYCLASE ACTIVATING POLYPEPTIDE Both vasocactive intestinal peptide (VIP) and pituitary adenylate cyclase activating polypeptide (PACAP) are members of the glucagon/secretin family and bind with different affinity to the same group of receptors, recently identified as VPAC receptors. Three different VIP/PACAP receptors have been cloned, which were defined as PVR1 (⳱ PACAPR-1), VPAC1R (⳱ VIP1R), and VPAC2R (⳱ VIP2R) (reviewed in Refs. 69–71). VPAC1R and VPAC2R have been detected in human skin as well as in immunocompetent cells [72,73]. VIP and PACAP have been identified as mediators of inflammation. Accordingly, they cause vasodilatation and stimulate histamine release from mast cells [74–77]. Moreover, they both exert a variety of immunomodulating effects as they downregulate the production of pro-inflammatory cytokines but upregulate suppressor factor production by macrophages. In addition, VIP and PACAP turned out to downregulate the expression costimulatory molecules (CD80, CD86) on activated macrophages [78]. VIP and PACAP also inhibit the LPS-induced macrophage production of CXC as well as CC chemokines [79]. Moreover, VIP was also found to significantly induce the migration of immature DC but to arrest that of mature D [80]. Furthermore, PACAP inhibits the LPS-stimulated production of TNF-␣, and both VIP as well as PACAP downregulate Fas ligand expression and thus suppress antigen-induced apoptosis of mature T lymphocytes [81,82]. In vivo, using a mouse model of collagen-induced arthritis, PACAP was shown to have a beneficial effect via inhibiting the release of pro-inflammatory cytokines [83]. A possibly important role of VIP and VPAC2R in allergic reactions was recently proposed. Accordingly, application of VIP into human skin prior to challenge was found to inhibit the elicitation of allergic contact dermatitis [84]. Moreover, VPAC2R transgenic mice showed significant elevations of serum IgE and IgG1 as well as eosinophils, whereas their DTH was downregulated. In contrast, in VPAC2R-deficient mice an enhanced DTH but a diminished immediate-type hypersensitivity was observed [85,86]. Thus, VIP appears to cause an immuno-deviation from Th1 to Th2 and may thereby promote type I allergic responses. In the skin, immunoreactivity for VIP is predominantly observed in the dermis associated with blood vessels, sweat or apocrine glands, hair follicles, Merkel cells, or mast cells [48,74,87]. PACAP is present in cutaneous autonomic and capsaicin-sensitive sensory nerve fibers [88]. In human skin, PACAP can be detected in the dermis, around hair follicles, and close to sweat glands [89,90]. Furthermore, PACAP-positive fibers were found in lymphoid tissues and lymphocytes [69]. The possible role of VIP in Th2-driven diseases such as atopic dermatitis is supported by the finding of an increased number of VIP-positive nerve fibers in atopic skin [59,60]. In addition, increased levels of VIP were detected in suction blister fluid derived from lesional skin of patients with atopic dermatitis [57]. VIII. CONCLUSION AND OUTLOOK Evidence has been accumulated that patients with AD have abnormal neuropeptide function, i.e., abnormal secretion, blunted inductivity, altered cutaneous expression, synthesis,

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and degradation as well as altered agonist responses. Despite these findings, it is apparent that we are only beginning to understand the role of even a single neuropetide in AD. Many of the studies have addressed peripheral blood levels of neuropeptides, resulting in novel findings but warranting deeper functional investigation. Others have focused on expression of distinct neuropeptides in lesional skin of patients with AD. Except for some early work on ␤-adrenoreceptor function, nothing has been learned about expression and function of CRH, ACTH, and ␣-MSH receptors, which mediate the multiple activities of the central players of the HPA axis. Is the in vitro or in vivo response of immunocompetent cells from patients with AD to these hormones as altered as the blunted response of the HPA axis suggests? One problem in research on neuropeptides and AD is the lack of an appropriate animal model for AD that would allow more functional studies on the above neuropeptides. Accordingly, in all of the presented studies on neuropeptides and patients with AD it is impossible to distinguish between cause and effect. An alternative strategy may be the use of neuropeptide knockout mice or animals with a signaling-deficient neuropeptide receptor in order to assess the potential relevance of a given neuropeptide on immune and inflammatory functions. Despite these future challenges in basic research on neuropeptides and AD, it can be anticiapted that in the future selected neuropeptide agonists or their antagonists will become available as novel anti-inflammatory therapeutic agents for patients with AD.

REFERENCES 1. Boguniewicz M, Leung DY. Pathophysiologic mechanisms in atopic dermatitis. Semin Cutan Med Surg 2001; 20:217–225. 2. Ginsburg IH, Prystowsky JH, Kornfeld DS, Wolland H. Role of emotional factors in adults with atopic dermatitis. Int J Dermatol 1993; 32:656–660. 3. Szentivanyi A. The beta adrenergic theory of the atopic abnormality in bronchial asthma. J Allergy 1968; 42:203. 4. Slominski A, Wortsman J. Neuroendocrinology of the skin. Endocr Rev 2000; 21:457–487. 5. Slominski A, Wortsman J, Luger T, Paus R, Solomon S. Corticotropin releasing hormone and proopiomelanocortin involvement in the cutaneous response to stress. Physiol Rev 2000; 80: 979–1020. 6. Bo¨hm M, Luger TA. The role of melanocortins in skin homeostasis. Horm Res 2000; 54: 287–293. 7. Luger TA, Brzoska T, Scholzen TE, Kalden DH, Sunderkotter C, Armstrong C, Ansel J. The role of alpha-MSH as a modulator of cutaneous inflammation. Ann NY Acad Sci 2000; 917: 232–238. 8. Scholzen TE, Sunderkotter C, Kalden DH, Brzoska T, Fastrich M, Fisbeck T, Armstrong CA, Ansel JC, Luger TA. Alpha-melanocyte stimulating hormone prevents lipopolysaccharideinduced vasculitis by down-regulating endothelial cell adhesion molecule expression. Endocrinology 2003; 144:360–370. 9. Ichiyama T, Sakai T, Catania A, Barsh GS, Furukawa S, Lipton JM. Inhibition of peripheral NF-␬B activation by central action of ␣-melanocyte-stimulating hormone. J Neuroimmunol 1999; 99:211–221. 10. Sternberg EM, Hill JM, Chrousos GP, Kamilaris T, Listwak SJ, Gold PW, Wilder RL. Inflammatory mediator-induced hypothalamic-pituitary-adrenal axis activation is defective in streptococcal cell wall arthritis-susceptible Lewis rats. Proc Natl Acad Sci USA 1989; 86:2374–2378. 11. Wick G, Hu Y, Schwarz S, Kroemer G. Immunoendocrine communication via the hypothalamo-pituitary-adrenal axis in autoimmune diseases. Endocr Rev 1993; 14:539–563.

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12. Heubeck B, Schonberger A, Hornstein OP. Are shifts in circadian cortisol rhythm an endocrine symptom of atopic eczema. Hautarzt 1988; 39:12–17. 13. Rupprecht M, Hornstein OP, Schluter D, Schafers HJ, Koch HU, Beck G, Rupprecht R. Cortisol, corticotropin, and beta-endorphin responses to corticotropin-releasing hormone in patients with atopic eczema. Psychoneuroendocrinology 1995; 20:543–551. 14. Rupprecht M, Salzer B, Raum B, Hornstein OP, Koch HU, Riederer P, Sofic E, Rupprecht R. Physical stress-induced secretion of adrenal and pituitary hormones in patients with atopic eczema compared with normal controls. Exp Clin Endocrinol Diabetes 1997; 105:39–45. 15. Buske-Kirschbaum A, Jobst S, Hellhammer DH. Altered reactivity of the hypothalamus-pituitary-adrenal axis in patients with atopic dermatitis: pathologic factor or symptom. Ann NY Acad Sci 1998; 840:747–754. 16. Matsuda K, Katsunuma T, Iikura Y, Kato H, Saito H, Akasawa A. Adrenocortical function in patients with severe atopic dermatitis. Ann Allergy Asthma Immunol 2000; 85:35–39. 17. Glinski W, Brodecka H, Glinska-Ferenz M, Kowalski D. Increased concentration of betaendorphin in the sera of patients with severe atopic dermatitis. Acta Derm Venereol 1995; 75: 9–11. 18. Glinski W, Brodecka H, Glinska-Ferenz M, Kowalski D. Increased concentration of betaendorphin in sera of patients with psoriasis and other inflammatory dermatoses. Br J Dermatol 1994; 131:260–264. 19. Glinski W, Brodecka H, Glinska-Ferenz M, Kowalski D. Neuropeptides in psoriasis: possible role of beta-endorphin in the pathomechanism of the disease. Int J Dermatol 1994; 33:356–360. 20. Gazith J, Cavey MT, Cavey D, Shroot B, Reichert U. Characterization of the beta-adrenergic receptors of cultured human epidermal keratinocytes. Biochem Pharmacol 1983; 32: 3397–3403. 21. Watanabe Y, Lai RT, Yoshida H. The beta-adrenoreceptor in human lymphocytes. Clin Exp Pharmacol Physiol 1981; 8:273–276. 22. Seiffert K, Hosoi J, Torii H, Ozawa H, Ding W, Campton K, Wagner JA, Granstein RD. Catecholamines inhibit the antigen-presenting capability of epidermal Langerhans cells. J Immunol 2002; 168:6128–6135. 23. Schallreuter KU, Wood JM, Lemke R, LePoole C, Das P, Westerhof W, Pittelkow MR, Thody AJ. Production of catecholamines in the human epidermis. Biochem Biophys Res Commun 1992; 189:72–78. 24. Schallreuter KU, Lemke KR, Pittelkow MR, Wood JM, Korner C, Malik R. Catecholamines in human keratinocyte differentiation. J Invest Dermatol 1995; 104:953–957. 25. Busse WW, Lee TP. Decreased adrenergic responses in lymphocytes and granulocytes in atopic eczema. J Allergy Clin Immunol 1976; 58:586–596. 26. Reed CE, Busse WW, Lee TP. Adrenergic mechanism and the adenyl cyclase system in atopic dermatitis. J Invest Dermatiol 1976; 67:333. 27. Parker CW, Kennedy S, Eisen AZ. Leukocyte and lymphocyte cyclic AMP responses in atopic eczema. J Invest Dermatol 1977; 68:302–306. 28. Cooper KD, Chan SC, Hanifin JM. Lymphocyte and monocyte localization of altered adrenergic receptors, cAMP responses, and cAMP phosphodiesterase in atopic dermatitis. A possible mechanism for abnormal radiosensitive helper T cells in atopic dermatitis. Acta Derm Venereol Suppl 1985; 114:41–47. 29. Pochet R, Delaspess G, Demaubuege J. Characterization of beta-adreoreceptors on intact circulating lymphocytes from patienst with atopic dermatitis. Acta Derm Venereol Suppl 1980; 92: 26–29. 30. Galant SP, Underwood S, Allred S, Hanifin JM. Beta adrenergic receptor binding on polymorphonuclear leukocytes in atopic dermatitis. J Invest Dermatol 1979; 72:330–332. 31. Szentivanyi-A. JM, Heim O, Schultze P, Szentivanyi-J P. Adrenoreceptor binding studies with 3-H-(dihydroalprenolol) and 3-H-(dihydroergocryptine) on membrnaes of lymphocytes from patienst with atiopic disease. Acta Derm Venereol 1980; 92:19.

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32. Carr RH, Busse WW, Reed CE. Failure of catecholamines to inhibit epidermal mitosis in vitro. J Allergy Clin Immunol 1973; 51:255–262. 33. Bamshad J. Catechol-O-methyl transferase in skin of patients with atopic dermatitis. J Invest Dermatol 1969; 52:100–102. 34. Schallreuter KU, Pittelkow MR, Swanson NN, Beazley WD, Korner C, Ehrke C, Buttner G. Altered catecholamine synthesis and degradation in the epidermis of patients with atopic eczema. Arch Dermatol Res 1997; 289:663–666. 35. Archer CB, Hanson JM, Morley J, MacDonald DM. Adrenoceptor function in atopic dermatitis: in vitro and in vivo observations. Acta Derm Venereol Suppl 1985; 114:93–97. 36. Safko MJ, Chan SC, Cooper KD, Hanifin JM. Heterologous desensitization of leukocytes: a possible mechanism of beta adrenergic blockade in atopic dermatitis. J Allergy Clin Immunol 1981; 68:218–225. 37. Ionescu G, Kiehl R. Plasma catecholamine levels in severe atopic eczema. Allergy 1988; 43: 614–616. 38. Ionescu G, Kiehl R. Abnormal plasma catecholamine levels in severe atopic eczema (AE). Allergy 1990; 45:477–478. 39. Archer CB, Dalton N, Turner C, Macdonald DM. Investigation of adrenomedullary function in atopic dermatitis. Br J Dermatol 1987; 116:793–800. 40. Slominski A, Pisarchik A, Semak I, Sweatman T, Wortsman J, Szczesniewski A, Slugocki G, McNulty J, Kauser S, Tobin DJ, Jing C, Johansson O. Serotoninergic and melatoninergic systems are fully expressed in human skin. FASEB J 2002; 16:896–898. 41. Geoffriau M, Brun J, Chazot G, Claustrat B. The physiology and pharmacology of melatonin in humans. Horm Res 1998; 49:136–141. 42. Liebmann PM, Wolfler A, Felsner P, Hofer D, Schauenstein K. Melatonin and the immune system. Int Arch Allergy Immunol 1997; 112:203–211. 43. Nelson RJ, Drazen DL. Melatonin mediates seasonal changes in immune function. Ann NY Acad Sci 2000; 917:404–415. 44. Reiter RJ, Calvo JR, Karbownik M, Qi W, Tan DX. Melatonin and its relation to the immune system and inflammation. Ann NY Acad Sci 2000; 917:376–386. 45. Schwarz W, Birau N, Hornstein OP, Heubeck B, Schonberger A, Meyer C, Gottschalk J. Alterations of melatonin secretion in atopic eczema. Acta Derm Venereol 1988; 68:224–229. 46. Rupprecht M, Berg P, Koch HU, Hornstein OP. Twenty-four hour melatonin concentrations in patients with active atopic eczema and after recovery. Eur J Dermatol 1995:80–83. 47. Richardson JD, Vasko MR. Cellular mechanisms of neurogenic inflammation. J Pharmacol Exp Ther 2002; 302:839–845. 48. Scholzen T, Armstrong CA, Bunnett NW, Luger TA, Olerud JE, Ansel JC. Neuropeptides in the skin: interactions between the neuroendocrine and the skin immune systems. Exp Dermatol 1998; 7:81–96. 49. Staniek V, Liebich C, Vocks E, Odia SG, Doutremepuich JD, Ring J, Claudy A, Schmitt D, Misery L. Modulation of cutaneous SP receptors in atopic dermatitis after UVA irradiation. Acta Derm Venereol 1998; 78:92–94. 50. Ostlere LS, Gordon DJ, Ayliffe MJ, Rustin MH, Pereira RS, Holden CA. Substance P binding to peripheral blood mononuclear leukocytes in atopic dermatitis. Acta Derm Venereol 1997; 77:260–263. 51. Gordon DJ, Ostlere LS, Holden CA. Neuropeptide modulation of Th1 and Th2 cytokines in peripheral blood mononuclear leucocytes in atopic dermatitis and non-atopic controls. Br J Dermatol 1997; 137:921–927. 52. Kang H, Byun DG, Kim JW. Effects of substance P and vasoactive intestinal peptide on interferon-gamma and interleukin-4 production in severe atopic dermatitis. Ann Allergy Asthma Immunol 2000; 85:227–232. 53. Coulson IH, Holden CA. Cutaneous reactions to substance P and histamine in atopic dermatitis. Br J Dermatol 1990; 122:343–349.

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77. Williams TJ. Vasoactive intestinal polypeptide is more potent than prostaglandin E2 as a vasodilator and oedema potentiator in rabbit skin. Br J Pharmacol 1982; 77:505–509. 78. Ganea D, Delgado M. Neuropeptides as modulators of macrophage functions. Regulation of cytokine production and antigen presentation by VIP and PACAP. Arch Immunol Ther Exp (Warsz) 2001; 49:101–110. 79. Delgado M, Ganea D. Inhibition of endotoxin-induced macrophage chemokine production by vasoactive intestinal peptide and pituitary adenylate cyclase-activating polypeptide in vitro and in vivo. J Immunol 2001; 167:966–975. 80. Dunzendorfer S, Kaser A, Meierhofer C, Tilg H, Wiedermann CJ. Cutting edge: peripheral neuropeptides attract immature and arrest mature blood-derived dendritic cells. J Immunol 2001; 166:2167–2172. 81. Delgado M, Munoz-Elias EJ, Kan Y, Gozes I, Fridkin M, Brenneman DE, Gomariz RP, Ganea D. Vasoactive intestinal peptide and pituitary adenylate cyclase-activating polypeptide inhibit tumor necrosis factor alpha transcriptional activation by regulating nuclear factor-␬B and cAMP response element-binding protein/c-Jun. J Biol Chem 1998; 273:31427–31436. 82. Delgado M, Ganea D. Vasoactive intestinal peptide and pituitary adenylate cyclase-activating polypeptide inhibit antigen-induced apoptosis of mature T lymphocytes by inhibiting Fas ligand expression. J Immunol 2000; 164:1200–1210. 83. Abad C, Martinez C, Leceta J, Gomariz RP, Delgado M. Pituitary adenylate cyclase-activating polypeptide inhibits collagen-induced arthritis: an experimental immunomodulatory therapy. J Immunol 2001; 167:3182–3189. 84. Lundeberg L, Mutt V, Nordlind K. Inhibitory effect of vasoactive intestinal peptide on the challenge phase of allergic contact dermatitis in humans. Acta Derm Venereol 1999; 79: 178–182. 85. Voice JK, Dorsam G, Lee H, Kong Y, Goetzl EJ. Allergic diathesis in transgenic mice with constitutive T cell expression of inducible vasoactive intestinal peptide receptor. FASEB J 2001; 15:2489–2496. 86. Goetzl EJ, Voice JK, Shen S, Dorsam G, Kong Y, West KM, Morrison CF, Harmar AJ. Enhanced delayed-type hypersensitivity and diminished immediate-type hypersensitivity in mice lacking the inducible VPAC(2) receptor for vasoactive intestinal peptide. Proc Natl Acad Sci USA 2001; 98:13854–13859. 87. Harvima IT, Viinamaki H, Naukkarinen A, Paukkonen K, Neittaanmaki H, Harvima RJ, Horsmanheimo M. Association of cutaneous mast cells and sensory nerves with psychic stress in psoriasis. Psychother Psychosom 1993; 60:168–176. 88. Moller K, Zhang YZ, Hakanson R, Luts A, Sjolund B, Uddman R, Sundler F. Pituitary adenylate cyclase activating peptide is a sensory neuropeptide: immunocytochemical and immunochemical evidence. Neuroscience 1993; 57:725–732. 89. Steinhoff M, McGregor GP, Radleff-Schlimme A, Steinhoff A, Jarry H, Schmidt WE. Identification of pituitary adenylate cyclase activating polypeptide (PACAP) and PACAP type 1 receptor in human skin: expression of PACAP-38 is increased in patients with psoriasis. Regul Pept 1999; 80:49–55. 90. Odum L, Petersen LJ, Skov P, Ebskov LB. Pituitary adenylate cyclase activating polypeptide (PACAP) is localized in human dermal neurons and causes histamine release from skin mast cells. Inflamm Res 1998; 47:488–492.

31 Neuroendocrine Control of Th1 and Th2 Responses: Clinical Implications ILIA ELENKOV National Institute of Mental Health, National Institutes of Health, Bethesda, Maryland, U.S.A.

I. INTRODUCTION The T-helper (Th)1/Th2 balance is critically skewed, one way or the other, in several common human diseases, such as acute and chronic infections, autoimmunity, atopy/ allergy, and tumor growth [1,2]. These diseases frequently develop and progress in settings of hyperactivity or hypoactivity of specific neuroendocrine pathways [3–8]. Evidence accumulated over the last decade also suggests that various neuroendocrine mediators are involved in the control of the Th1/Th2 balance. Thus, the question arises: Are the changes in the Th1/Th2 balance and the activity of neuroendocrine pathways seen in the course of some common human diseases causally linked, or are these associations an epiphenomenon? This chapter addresses the view that several hormones and neurotransmitters through modulation of Th1/Th2 balance influence the onset and the progress of infectious, autoimmune, and atopic/allergic diseases or tumor growth. A complete discussion of these neuroendocrine regulatory effects is beyond the scope of this chapter. Nevertheless, the available data on the regulatory actions of cortisol, epinephrine, norepinephrine (NE), estrogen, progesterone, 1,25-dihydroxyvitamin D3, histamine, and adenosine on Th1/Th2 balance are sufficient to illustrate the relevant concepts. Clearly these hypotheses require further investigation, but the answers should provide critical insights into mechanisms underlying a variety of common human diseases. II. THE TH1/TH2 PARADIGM: ROLE OF TYPE 1 AND TYPE 2 CYTOKINES The immune system is classified into innate (or nonspecific, natural) and adaptive (or specific, acquired) immunity. Innate immunity provides a rapid, nonspecific host response 647

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against different bacteria, viruses, or tumors that precedes the adaptive immunity. Moreover, the innate immunity also has an important role in determining the nature of downstream adaptive immune responses. Thus, immune responses are regulated by antigenpresenting cells (APCs)—monocytes/macrophages and dendritic cells—and by natural killer (NK) cells, which are components of innate immunity, and by the recently described T-helper (Th) lymphocyte subclasses Th1 and Th2, which are components of adaptive (acquired) immunity. Th1 cells primarily secrete interferon (IFN)-␥, interleukin (IL)-2, and tumor necrosis factor (TNF)-␤, which promote cellular immunity, whereas Th2 cells secrete a different set of cytokines, primarily IL-4, IL-10, and IL-13, which promote humoral immunity [1,2,9] (Fig. 1). Naive CD4Ⳮ (antigen-inexperienced) Th0 cells are clearly bipotential and serve as precursors of Th1 and Th2 cells. Among the factors currently known to influence the differentiation of these cells towards Th1 or Th2, cytokines produced by cells of the innate immune system are the most important. Thus, IL-12, produced by activated monocytes/macrophages or other APCs, is a major inducer of Th1 differentiation and hence cellular immunity. This cytokine acts in concert with NK-derived IFN-␥ to further promote Th1 responses [9]. APC-derived IL-12 and TNF-␣, in concert with NK cell– and Th1-derived IFN-␥, stimulate the functional activity of T-cytotoxic cells (Tc), NK cells, and activated macrophages, which are the major components of cellular immunity. The type 1 cytokines IL-12, TNF-␣, and IFN-␥ also stimulate the synthesis of nitric oxide (NO) and other inflammatory mediators that drive chronic delayed type inflammatory responses. Because of these crucial and synergistic roles in inflammation IL-12, TNF-␣ and IFN-␥ are considered the major pro-inflammatory cytokines [1,2,9]. Th1 and Th2 responses are mutually inhibitory. Thus, IL-12 and IFN-␥ inhibit Th2 cells activities, while IL-4 and IL-10 inhibit Th1 responses. IL-4 and IL-10 promote humoral immunity by stimulating the growth and activation of mast cells and eosinophils, the differentiation of B cells into antibody-secreting B cells, and B-cell immunoglobulin switching to IgE. Importantly, these cytokines also inhibit macrophage activation, T-cell proliferation, and the production of pro-inflammatory cytokines [1,2,9]. Therefore, the Th2 (type 2) cytokines IL-4 and IL-10 are the major anti-inflammatory cytokines. III. SYSTEMIC EFFECTS OF STRESS HORMONES—GLUCOCORTICOIDS AND CATECHOLAMINES Previous studies have shown that glucocorticoids suppress the production of TNF-␣, IFN␥, and IL-2 in vitro and in vivo in animals and humans [10,11]. Recent evidence indicates that glucocorticoids also act through their classic cytoplasmic/nuclear receptors on APCs to suppress the production of the main inducer of Th1 responses IL-12 in vitro and ex vivo [12,13]. Since IL-12 is extremely potent in enhancing IFN-␥ and inhibiting IL-4 synthesis by T cells, the inhibition of IL-12 production by APCs may represent a major mechanism by which glucocorticoids affect the Th1/Th2 balance. Thus, glucocorticoidtreated monocytes/macrophages produce significantly less IL-12, leading to their decreased capacity to induce IFN-␥ production by antigen-primed CD4Ⳮ T cells. This is also associated with an increased production of IL-4 by T cells, probably resulting from disinhibition from the suppressive effects of IL-12 on Th2 activity [14]. Furthermore, glucocorticoids potently downregulate the expression of IL-12 receptors on T and NK cells. This explains why human peripheral blood mononuclear cells (PBMCs) stimulated with immobilized anti-CD3 lose their ability to produce IFN-␥ in the presence of glucocorticoids [15]. Thus, although glucocorticoids may have a direct suppressive effect on Th1 cells, the overall

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Figure 1 Role of APCs, Th1 and Th2 cells, and type 1/pro-inflammatory and type 2/anti-inflammatory cytokines in the regulation of cellular and humoral immunity. Cellular immunity provides protection against intracellular bacteria, protozoa, fungi, and several viruses, while humoral immunity provides protection against multicellular parasites, extracellular bacteria, some viruses, soluble toxins, and allergens (see text). Solid lines represent stimulation, while dashed lines inhibition. Ag, antigen; APC, antigen-presenting cell; NK, natural killer cell; T, T cell; B, B cell; Th, T-helper cell, Tc, T-cytotoxic cell; Eo, eosinophil; IL, interleukin, TNF, tumor necrosis factor; IFN, interferon. (From Ref. 6.)

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inhibition of IFN-␥ production by these cells appears to result mainly from the inhibition of IL-12 production by APCs and from the loss of IL-12 responsiveness of NK and Th1 cells. It is particularly noteworthy that glucocorticoids have no effect on the production of the potent anti-inflammatory cytokine IL-10 by monocytes [12,16]; yet, lymphocytederived IL-10 production appears to be upregulated by glucocorticoids (Table 1). Thus, rat CD4Ⳮ T cells pretreated with dexamethasone exhibit increased levels of mRNA for IL-10 [17]. Similarly, during experimental endotoxemia or cardiopulmonary bypass, or in multiple sclerosis patients having an acute relapse, treatment with glucocorticoids is associated with increased plasma IL-10 secretion [16,18,19]. This could be the result of a direct stimulatory effect of glucocorticoids on T-cell IL-10 production and/or a block on the restraining inputs of IL-12 and IFN-␥ on monocyte/lymphocyte IL-10 production. In contrast to catecholamines (CAs), glucocorticoids have a direct effect on Th2 cells by up-regulating their IL-4, IL-10, and IL-13 production [13,17]. Systemically, CAs suppress type 1/pro-inflammatory cytokine production, Th1 activities, and cellular immunity, but boost type 2/anti-inflammatory cytokine production, Th2, and humoral responses (Table 1). Both NE and epinephrine through stimulation of ␤2-adrenergic receptors (ARs) potently inhibit the production by monocytes and dendritic cells of the main inducer of Th1 responses, IL-12 [12,20,21]. Epinephrine appears to be a strong inhibitor of IL-12 production, exhibiting an EC50 of 10ⳮ9 M [12]. Since IL-12 is extremely potent in enhancing IFN-␥ and inhibiting IL-4 synthesis by Th1 and Th2 cells, respectively, the inhibition of IL-12 production may represent one of the major mechanisms by which CAs affect the Th1/Th2 balance. Thus, in conjunction with their ability to suppress IL-12 production, ␤2-AR agonists inhibit the development of Th1-type

Table 1 Effectsa of Hormones and Neurotransmitters on Type 1 and Type 2 Cytokine Production Type 1 Hormone Cortisol 1,25(OH)2 Vitamin D3 Estrogenb Progesterone Norepinephrine Epinephrine Histamine Adenosine a

Type 2

IL-12

TNF-␣

IFN-␥

IL-4

IL-10(Mo)

IL-10(Ly)

IL-13

↓ ↓

↓ ↓

↓ ↓

↑ —

— —

↑ ↑

↑ ?

— — ↓ ↓ ↓ ↓

↓ ↓ ↓ ↓ ↓ ↓

? ? ↓ ↓ ↓ ↓

— ↑ ↑c ↑c ↑c ?

— — ↑ ↑ ↑ ↑

↑ — ↑c ↑c ? ?

? ? ? ? ? ?

Most of these effects are systemic ones. Note that the effects of hormones and neurotransmitters in some local responses might be different (see text). b Estrogens might also have indirect effects in vivo by enhancing the activity of the stress system (see text). IL, interleukin; TNF, tumor necrosis factor; IFN, interferon, Mo, monocyte-derived; Ly, lymphocyte-derived;—, no effect. c Th2 cells do not express ␤2-adrenergic and possibly H2-histamine receptors, thus catecholamines and histamine might upregulate IL-4, IL-10 (Ly), and IL-13 indirectly, through disinhibition, i.e., by removing the inhibitory restraints of IL-12 and IFN-␥ on Th2 cells.

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cells, while promoting Th2-cell differentiation [20]. NE, epinephrine, and ␤-AR agonists inhibit the production of TNF-␣ by monocytes, microglial cells, and astrocytes [22–24]. CAs also suppress the production of IL-1, an effect that is mostly indirect via inhibition of TNF-␣ and potentiation of IL-10 production [25,26]. While suppressing type 1 cytokine production, CAs appear to upregulate the production of type 2 cytokines by APCs. Thus, the production of IL-10, one of the most potent anti-inflammatory cytokines, induced by LPS in human monocytes or mouse peritoneal macrophages, is potentiated by NE and epinephrine, an effect that is ␤2 AR mediated and cAMP-PKA dependent [12,27]. Similarly, the production of IL-6, a cytokine that exerts both pro- and anti-inflammatory effects but possesses mostly Th2-type activities (previously known as BCDF, B-cell differentiation factor), is also upregulated by CAs [28,29]. It appears that ␤2-ARs are expressed on Th1 cells, but not on Th2 cells [30]. This may provide an additional mechanistic basis for the differential effect of CAs on Th1/Th2 functions. In both murine and human systems, ␤2-AR agonists inhibit IFN-␥ production by Th1 cells, but do not affect IL-4 production by Th2 cells [30,31]. Furthermore, cAMP levels increase in Th1 cells following terbutaline exposure, but not in Th2 cells [30]. In vivo, increasing sympathetic outflow and endogenous production of CAs in mice by selective ␣2-AR antagonists or application of exogenous CAs or ␤-AR agonists results in inhibition of LPS-induced TNF-␣ and IL-12 production [32,33]. CAs also appear to exert tonic inhibition on the production of pro-inflammatory cytokines in vivo. Thus, application of propranolol, a ␤-AR antagonist that blocks their inhibitory effect on cytokine-producing cells, results in substantial increases of LPS-induced secretion of TNF-␣ and IL-12 in mice [32,33]. In IL-10–deficient C57BL/6 IL-10 (ⳮ/ⳮ) mice, plasma levels of IL-12 are about 70-fold higher than in their counterparts, suggesting tonic inhibitory effect of IL-10 on IL-12 production. Injection of isoproterenol, while augmenting the IL-10 response in C57BL/6 IL-10 (Ⳮ/Ⳮ) mice, inhibits IL-12 production in both C57BL/6 IL-10 (Ⳮ/Ⳮ) and C57BL/6 IL-10 (ⳮ/ⳮ) mice [33]. Thus, the inhibition of IL-12 production appears to be independent of the increased release of IL-10. In humans, the administration of the ␤2-AR agonist salbutamol results in inhibition of IL-12 production ex vivo [20], while acute brain trauma that is followed by massive release of CAs triggers secretion of substantial amounts of systemic IL-10 [34]. Salbutamol also induces an increase of the ex vivo release of IL-4, IL-6, and IL-10 [35]—these effects are most likely indirect, due to the removal of the inhibition by type 1 cytokines on Th2 cells (see Fig. 1, Table 1, and text above). Thus, systemically, both glucocorticoids and CAs, through inhibition and stimulation of type 1 and type 2 cytokine secretion, respectively, cause selective suppression of cellular immunity and a shift towards Th2-mediated humoral immunity, rather than generalized immunosuppression. This is further substantiated by studies showing that stress hormones inhibit effector function of cellular immunity components, i.e., the activity of NK, Tc, and activated macrophages. For example CAs are potent inhibitors of NK-cell activity, both directly, acting on ␤2-ARs expressed on these cells, or indirectly, through suppression of the production of IL-12 and INF-␥, which are essential for NK-cell activity [12,36]. It appears that NK cells are most sensitive to the suppressive effect of stress; indeed, NKcell activity has been used as an index of stress-induced immunosuppression in many studies [37,38]. The stress hormone–induced Th2 shift may have both beneficial and detrimental consequences (see clinical implications below). Although interest in the Th2 response was initially directed at its protective role in helminthic infections and its pathogenic role in

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allergy, this response may have important regulatory functions in countering the tissuedamaging effects of macrophages and Th1 cells [2]. Thus, an excessive immune response, through activation of the stress system, may trigger a mechanism that inhibits Th1 but potentiates Th2 responses. This important feedback mechanism may protect the organism from ‘‘overshooting’’ with type 1/pro-inflammatory cytokines and other products of activated macrophages with tissue-damaging potential. IV. HISTAMINE Histamine is one of the major mediators of acute inflammation and allergic reactions. These actions are largely mediated by activation of H1 histamine receptors. Recent evidence indicates, however, that histamine may also have important immunoregulatory functions via H2 receptors expressed on immune cells [39]. Thus, histamine inhibits TNF-␣, but potentiates IL-6 production [40,41]. Since TNF-␣ is primarily a type 1 cytokine and IL6 promotes B-cell differentiation, these data suggest that histamine may promote humoral immunity. Recently, we and others have shown that histamine inhibits the secretion of human IL-12 but stimulates the production of IL-10 by human monocytes and dendritic cells [42,43], opening the possibility that histamine could favor Th2-dominated immunity. Cimetidine, a H2 receptor antagonist, blocked the effects of histamine on IL-12 and IL10 production, while dimaprit, a H2 receptor agonist, mimicked dose-dependently the effect of histamine. Monocytes are the main IL-12– and IL-10–producing cells in LPSstimulated human peripheral blood, and human monocytes express H2 receptors [42,44]. Because Ro 20–1724, a phosphodiesterase inhibitor, potentiated the effect of histamine on both IL-12 and IL-10 secretion, it appears that H2 receptors on monocytes appear to mediate the effect of histamine via an increase of intracellular cAMP. In addition, histamine via H2 receptors inhibits IFN-␥ production by Th1-like cells, but has no effect on IL-4 production from Th2 clones [45]. Thus, histamine, similarly to CAs, appears to drive a Th2 shift at the level of both APCs and Th1 cells (Table 1). Through this mechanism, allergen/antigen-IgE–induced release of histamine might participate in a positive feedback loop that promotes and sustains a shift to IgE production. V. LOCAL VERSUS SYSTEMIC EFFECTS The above general conclusion about the effects of stress hormones on Th1/Th2 balance may not pertain to certain conditions or local responses in specific compartments of the body. Thus, the synthesis of transforming growth factor (TGF)-␤, another type 2 cytokine with potent anti-inflammatory activities, is differentially regulated by glucocorticoids: it is enhanced in human T cells but suppressed in glial cells [46]. In addition, NE, via stimulation of ␣2-ARs can augment LPS-stimulated production of TNF-␣ from mouse peritoneal macrophages [47], while hemorrhage, a condition associated with elevations of systemic CA concentrations, increases the expression of TNF-␣ and IL-1 by lung mononuclear cells via stimulation of ␣-ARs [48]. Because the response to ␤-AR agonist stimulation wanes during maturation of human monocytes into macrophages [49], it is possible that in certain compartments of the body, the ␣-AR–mediated effect of CAs becomes transiently dominant. Interestingly, in vitro, long-term incubation with low concentrations of the synthetic glucocorticoid dexamethasone can indeed activate alveolar macrophages, leading to increased LPS-induced IL-1␤ production [50]. Exposure of rats to mild inescapable electrical

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footshock stress also results in increased IL-1␤ and TNF-␣ production by alveolar macrophages [51]. The upregulation of pro-inflammatory cytokine production in in vivo conditions appears to be dependent of intact sympathetic innervation and ␤-ARs. However, the effect is most likely indirect, since in vitro a direct modulatory effect of CAs on LPSinduced IL-1␤ by alveolar macrophages was not demonstrated. It can be envisaged that the stress-induced changes in alveolar macrophage activity result from ␤-AR–mediated alveolar type II epithelial cell activation, leading to release of surfactant and/or other factors [51]. Through the above mechanisms, CAs may actually boost local cellular immune responses in a transitory fashion. This is further substantiated by the finding that CAs potentiate the production of IL-8 by monocytes and epithelial cells of the lung [52,53], thus probably promoting recruitment of polymorphonuclear leukocytes in local inflammation. Again, this effect appears to be an indirect one. Recent evidence indicates that epinephrine promotes IL-8 production by human leukocytes via an effect on platelets. Thus, IL-8 levels in samples containing platelets and stimulated with LPS and epinephrine were significantly higher than control samples containing no platelets and activated platelets are able to induce endothelial secretion of IL-8 [54,55]. Anatomically, a close spatial relationship between sympathetic and peptidergic nerve fibers on one hand, and macrophages and mast cells on the other, is frequently observed (see Ref. 38). Neuro-macrophage and neuro-mast cell connections are not restricted to the preformed lymphoid organs and tissues, but are also regularly encountered in virtually all somatic and visceral tissues. Substance P (SP) and peripheral corticotrophin-releasing hormone (CRH) (see also below), which are released from sensory peptidergic neurons, are two of the most potent mast cell secretagogues [56–59]. Furthermore, recent evidence indicates that SP upregulates both TNF-␣ and IL-12 production by human and murine monocytes and macrophages [60–62]. This adds further complexity to the local effects of stress hormones in conjunction with other neurotransmiters and/or mediators [38,63]. Thus, in summary, while stress hormones suppress Th1 responses and pro-inflammatory cytokine secretion and boost Th2 responses systemically, they may affect certain local responses differently. Further studies are needed to address this question (see Fig. 2 and text below). VI. CRH–MAST CELL–HISTAMINE AXIS Apart from its central effects, CRH is also secreted peripherally at inflammatory sites (peripheral or immune CRH) and influences the immune system directly, through local modulatory actions [64]. Immunoreactive CRH is identified locally in experimental carrageenan-induced subcutaneous aseptic inflammation, streptococcal cell wall– and adjuvantinduced arthritis, and retinol-binding protein (RBP)–induced uveitis, and in human tissues from patients with various autoimmune/inflammatory diseases, including rheumatoid arthritis, autoimmune thyroid disease, and ulcerative colitis. The demonstration of CRH-like immunoreactivity in the dorsal horn of the spinal cord, dorsal root ganglia, and sympathetic ganglia support the hypothesis that most of the immune CRH in early inflammation is of peripheral nerve rather than immune cell origin (see Ref. 65). Peripheral CRH has proinflammatory, vascular permeability–enhancing and vasodilatory actions. Systemic administration of specific CRH antiserum reduces the inflammatory exudate volume and cell number in carrageenan-induced inflammation and RBP-induced uveitis and inhibits stress-induced intracranial mast cell degranulation [58,64]. In addition, CRH administra-

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Figure 2 Simplified scheme of the effects of locally released NE, SP, and CRH on macrophage activity. Note that the anti-inflammatory effect of CAs exerted through stimulation of ␤2-ARs depends on the presence and expression of ␣-ARs on macrophages and mast cells and other local mediators or factors (see text for details). Solid lines represent stimulation, while dashed lines inhibition. Ag, antigen; CRH, corticotropin-releasing hormone (peripheral); IL, interleukin; NE, norepinephrine; TNF, tumor necrosis factor; SP, substance P. (From Ref. 38.)

tion to humans or nonhuman primates causes major peripheral vasodilation manifested as flushing and increased blood flow and hypotension [66]. An intradermal CRH injection induces a marked increase of vascular permeability and mast cell degranulation [59]. Importantly, this effect is mediated through CRH type 1 receptors and is stronger than the effect of an equimolar concentration of C48/80, a potent mast cell secretagogue [59]. Therefore, it appears that the mast cell is a major target of immune CRH. This concept has an anatomical prerequisite: in blood vessels, periarterial sympathetic plexuses are closely associated with mast cells lining the perivascular regions, and plexuses of nerve fibers (noradrenergic and peptidergic) within lymphoid parenchyma are also closely associated with clusters of mast cells. Interestingly, recent evidence suggests that urocortin, a newly discovered member of the CRH family, which binds to the same receptors as CRH, is produced by human lymphocytes and Jurkat T lymphoma cells [67,68]. High expression of urocortin immunoreactivity was recently demonstrated in the synovial lining cell layer, subsynovial stromal cells, blood vessels endothelial cells, and mononuclear inflammatory cells from joints of rhematoid arthritis (RA) patients. In addition, urocortin also stimulated IL-1␤ and IL-6

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secretion by PBMC in vitro [69]. Thus, this peptide may also participate in the peripheral CRH receptor-mediated inflammatory response. Histamine, a major product of mast cell degranulation, is a well-recognized mediator of acute inflammation and allergic reactions. These actions are mainly mediated by activation of H1 histamine receptors and include vasodilation, increased permeability of the vessel wall, edema, and, in the lungs, bronchoconstriction. Thus, it is conceivable that CRH activates mast cells via a CRH receptor type 1–dependent mechanism leading to release of histamine and other contents of the mast cell granules that subsequently cause vasodilatation, increased vascular permeability and other manifestations of inflammation. Thus, the activation of CRH–mast cell–histamine axis through stimulation of H1 receptors may induce acute inflammation and allergic reactions, while through activation of H2 receptors it may induce suppression of Th1 responses and a Th2 shift (see Fig. 2 and text above).

VII. ESTROGEN AND PROGESTERONE Estrogens alter the cytokine profile of T cells towards Th2 phenotype by upregulating the production of IL-10 and inhibiting TNF-␣ secretion (Table 1). In the presence of high doses of estrone (E1), estradiol (E2), and estriol (E3), the majority of the antigen-specific T-cell clones show enhancement of antigen- and anti-CD3–stimulated human IL-10 production [70–72]. These observations are relevant to the finding that E2 may polyclonally increase the production of IgG, including IgG anti-dsDNA, in systemic lupus erythematosus (SLE) patients’ PBMC by enhancing B-cell activity and by promoting IL-10 production—evidence that supports the involvement of E2 in the pathogenesis of SLE [73]. E2 and E3 decreases LPS-induced TNF-␣ production by splenic macrophages and T cells by inhibition of the transcription factor NF-␬ B [71,72,74]. Some studies suggest that E2 has biphasic effects on secretion of TNF-␣, with enhancement occurring at low doses of E2 and inhibition at high concentrations [71,74,75]. Interestingly, E2 does not affect the production of IL-12 and IL-10 by murine splenic macrophages and human monocytes [74,76]. Progesterone is a steroid hormone that typically increases during pregnancy and is essential for the maintenance of pregnancy. Progesterone also favors Th2 development mainly through induction of IL-4 and IL-5 and through inhibition of TNF-␣ production. Progesterone decreases steady-state levels of TNF-␣ mRNA in LPS-activated mouse macrophages. In addition, the production of intracellular and secreted TNF-␣ is also decreased by progesterone [77]. Importantly, progesterone, at concentrations comparable to those present at the materno-fetal interface, induces the development of Ag-specific CD4Ⳮ T-cell lines and clones that show enhanced ability to produce IL-4 and IL-5 without affecting the secretion of IL-10 [70,78] Moreover, progesterone also induces the expression of IL-4 mRNA and production in established human Th1 clones [78] (Table 1). Estrogens and progesterone are most likely to drive a substantial Th2 shift only at concentrations associated with pregnancy (up to 35, 000 pg/mL) [70]. Thus, at these doses and higher, in the above-mentioned studies IL-4 and IL-10 secretion are stimulated and TNF-␣ inhibited. Since steroid hormones accumulate at higher concentrations in target or source tissues than in the peripheral circulation, a localized Th2-like environment with potential for an impact on immunity is more likely to occur at concentrations achieved during pregnancy (see also pregnancy and autoimmune diseases, below).

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However, it should be pointed out that most of the actions of ovarian hormones, and specifically of estrogens, might be indirect rather than direct on cytokine-producing cells. Estrogen enhances the activity of the stress system, i.e., it enhances cortisol and catecholamine production [79]. The secretion of the hypothalamic CRH is essential for activation of the peripheral arms of the stress system, i.e., the hypothalamic-pituitaryadrenal (HPA) axis and systemic sympathoadrenal system [80]. The CRH gene contains a functional estrogen-responsive element [81], which may explain higher levels of CRH in the CRH neurons of female rats and the higher HPA axis responsiveness of women [82]. In fact, pregnant women and women receiving high-dose estrogen treatment have elevated plasma cortisol levels compared to control individuals [83]. Estradiol also decreases corticosteroid receptor levels in the hypothalamus, the anterior pituitary, and the hippocampus, resulting in decreased corticosteroid feedback and increased stress system activation [79]. Conversely, estrogen deficiency, such as observed in the postpartum period and at menopause, is expected to result in a hyporesponsive HPA axis and suboptimal cortisol and catecholamine production. In addition, estrogens are potent inhibitors of the extraneuronal uptake of norepinephrine (uptake-2) [84]. Through this mechanism, estrogens, via an increase of local levels of catecholamines, are probably able to amplify the effects of catecholamines on pro-/anti-inflammatory cytokine balance (see Fig. 3). In other words, interactions between the stress system and estrogens have the potential to significantly modulate the production and actions of cortisol and catecholamines. Thus, it appears likely that the combined direct and indirect effects of estrogens on TNF␣, IL-12 production relative to IL-10 production are possibly very large. The interaction between estrogens and stress system hormones probably contributes to the dramatic changes in autoimmune disease in the context of pregnancy (see below) and changing the reproductive status. Declining estrogen levels may facilitate the development of cellmediated autoimmune diseases such as RA, whereas high estrogen levels may promote autoimmune diseases associated with humoral immunity such as SLE (see text above). Progesterone appears to magnify these effects. Interestingly, an estrogen deficiency has also been linked to induction of bone loss by enhancing T-cell production of TNF-␣ [85]. The differentiation of cells of the monocyte lineage into mature osteoclasts is specifically induced by the TNF-related factor RANKL (receptor activator of NF-␬B ligand). T cells from ovariectomized mice produce increased amounts of TNF, which augments RANK-Induced osteoclastogenesis [86]. This evidence indicates that the enhanced T-cell production of TNF resulting from increased bone marrow T-cell number might represent a key mechanism by which estrogen deficiency induces bone loss in vivo [86,87]. VIII. 1, 25-DIHYDROXYVITAMIN D3 The steroid hormone 1,25-dihydroxyvitamin D3 (calcitriol) is one of the key regulators of plasma calcium/phosphate and adequate mineralization of bone matrix. Calcitriol exerts its effect via specific vitamin D receptor (VDR), a member of the superfamily of steroid/ thyroid/vitamin A hormone receptors. Studies in recent years have provided ample evidence that 1,25-dihydroxyvitamin D3 plays a role in immunoregulation. High-affinity VDRs are expressed constitutively in monocytes and are induced in lymphocytes following activation by mitogens. At a cellular and molecular level, 1,25-dihydroxyvitamin D3 preferentially targets Th1 activity by inhibiting the secretion of both IFN-␥ and IL-2 and by suppressing the production of the pro-Th1 cytokine IL-12 by APCs [88]. The hormone

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Figure 3 A proposed simplified model of the role of different hormones in regulation of innate and T-helper (Th) 1 and Th2 cytokine profiles during pregnancy. Hypothalamic CRH stimulates the secretion of pituitary ACTH, which in turn triggers the secretion of cortisol from the adrenal cortex. During human pregnancy, the placenta is the major source of circulating CRH. The placenta also secretes IL-10, which may stimulate humoral and suppress cellular immunity. The sympathetic system innervates all peripheral tissues, including blood vessels and lymphoid organs. Upon activation, the sympathetic nerve terminals in these organs release NE locally and into the blood stream. Cortisol, NE, 1,25-dihydroxyvitamin D3, estradiol, and progesterone have multiple and divergent effects upon the immune system. *Cortisol does not affect the production of IL-10 by monocytes/ macrophages (see text). Note that cortisol and estradiol upregulate IL-10 production by Th2 lymphocytes. In addition, estradiol stimulates the activity of the CRH neurons and increases local NE concentrations by blocking its uptake. Thus, in vivo, estradiol might amplify the effects of cortisol and NE. The net result of these complex hormonal effects is suppression of IL-12 and TNF-␣ production by monocytes, whereas peripheral lymphocytes secrete less IFN-␥ and IL-2 but more IL-4 and IL-10, particularly in the third trimester (see text). This hormonally induced Th2 shift may suppress Th1-related diseases such as RA and MS during pregnancy, while the rebound of IL-12 and TNF-␣ production and Th1 responses in the postpartum may facilitate the flares or the onset of these diseases. Please note that several other factors besides hormones (e.g., antibodies, soluble cytokine receptors, etc.) most likely also involved in the modulation of Th1/Th2 balance during pregnancy and postpartum are not discussed here. ACTH, adrenocorticotropic hormone; CRH, corticotropin-releasing hormone; IL, interleukin; LC, locus ceruleus; NE, norepinephrine; PVN, paraventricular nucleus; Th, T-helper lymphocyte; TNF, tumor necrosis factor. (From Ref. 76.)

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inhibits IL-12 production by activated macrophages and dendritic cells by downregulation of NF-␬B activation and binding to the p40-␬B sequence [89]. Interestingly, 1,25-dihydroxyvitamin D3 has little or no effect on IL-4 production but enhances IL-10 secretion by dendritic cells and IL-10 and IL-5 by PBMC [90–92]. In addition, 1,25-dihydroxyvitamin D3 does not suppress, at least acutely, the production of IL-10 by human monocytes from whole blood cultures ex vivo (A.A. Link, G.P. Chrousos, I.J. Elenkov, unpublished observations). Thus, similarly to glucocorticoids, 1,25-dihydroxyvitamin D3 may upregulate lymphocyte-derived IL-10, but does not affect the production of IL-10 by monocytes. The above-mentioned suggests that 1,25-dihydroxyvitamin D3 may selectively inhibit Th1 functions and favor Th2 responses (see Table 1). Therefore, the development of less hypercalcemic analogs of 1,25-dihydroxyvitamin D3 might open a new therapeutic area in autoimmunity and organ transplantation. In fact, it has recently been shown that administration of such analogs by inhibiting IL-12 and Th1 development prevents or ameliorates chronic-relapsing experimental allergic encephalomyelitis (EAE) and autoimmune diabetes in mice [93,94]. In addition, the clinical improvement in psoriasis after application of calcipotriene, a synthetic analog of 1,25-dihydroxyvitamin, has been linked to the reduction of IL-8 and the increase of IL-10 production induced by this drug [95].

IX. ADENOSINE Inflammation, ischemia, and tissue injury represent pathological states in which intracellular ATP metabolism is accelerated, resulting in an enhanced release from metabolically active cells of the endogenous purine nucleoside adenosine. Adenosine exerts potent antiinflammatory and immunosuppressive effects mediated mainly by A2 receptors: diminished leukocyte accumulation, inhibition of C2 production, and reduction of the superoxide anion generation [96–98]. We have recently shown that the adenosine analogs NECA and CGS-21680 dosedependently inhibit the production of IL-12 induced by LPS in whole blood and isolated human monocytes cultures [99]. Because A2a receptors are characterized by their highaffinity binding of the agonist CGS-21680, this pharmacological profile of the response implies involvement of A2a receptors. Moreover, the inhibitory effect of CGS-21680 is potentiated by a phosphodiesterase inhibitor and prevented by an inhibitor of the type I and II PKA. Thus, ligand activation of A2a receptors trough stimulation of cAMP/PKA pathway appears to mediate the inhibition of IL-12 production by human monocytes [99]. This is consistent with previous studies showing that adenosine analogs inhibit, also via stimulation of A2 receptors, the secretion of the pro-inflammatory cytokine TNF-␣ by human monocytes [100]. Adenosine analogs potentiate, however, the production of IL-10 in vitro by human monocytes, in human whole blood ex vivo, and in vivo in endotoxemic mice [99,101,102]. This indicates that adenosine expresses a Th1/Th2 modulatory profile similar to CAs and histamine (Table 1). Thus, conditions related to increased local concentrations of adenosine, through inhibition of IL-12 and TNF-␣, and potentiation of IL-10 production from monocytes may simultaneously mediate an inhibition of Th1 responses and a shift towards Th2 dominance.

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X. CLINICAL IMPLICATIONS A. Intracellular Infections A major factor governing the outcome of infectious diseases is the selection of Th1- versus Th2-predominant adaptive responses during and after the initial invasion of the host by the pathogen. Stress-induced Th2 shift may, therefore, have a profound effect on the susceptibility of the host to infections and/or may influence the course of infections, particularly intracellular ones, the defense against which is primarily through cellular immunity mechanisms (Table 2). Cellular immunity, particularly IL-12– and IL-12–dependent IFN-␥ secretion in humans, seems essential in the control of mycobacterial infections [103]. In the 1950s, Holmes (see Ref. 3) reported that individuals who had experienced stressful life events were more likely to develop tuberculosis and less likely to recover from it. Although it is still a matter of some speculation, stress hormone–induced inhibition of IL-12 and IFN-␥ production and the consequent suppression of cellular immunity might explain the pathophysiological mechanisms of these observations [12]. Helicobacter pylori infection is the most common cause of chronic gastritis, which in some cases progresses to peptic ulcer disease. The role of stress in promoting peptic ulcers has been recognized for many years [104]. Thus, increased systemic stress hormone levels, in concert with an increased local concentration of histamine, induced by inflammatory or stress-related mediators, may skew the local responses towards Th2 and thus might allow the onset or progression of a Helicobacter pylori infection. HIV-positive patients have IL-12 deficiency, while disease progression has been correlated with a Th2 shift. The innervation (primarily sympathetic/noradrenergic) of lymphoid tissue may be particularly relevant to HIV infection, since lymphoid organs represent the primary site of HIV pathogenesis. In fact, as recently shown, NE, the major sympathetic neurotransmitter released locally in lymphoid organs [105,106], is able to directly accelerate HIV-1 replication by up to 11-fold in acutely infected human PBMCs [107]. The effect of NE on viral replication is transduced via the ␤-AR–adenylyl cyclase–cAMP–PKA signaling cascade [107]. In addition, it has been found that the induction of intracellular cAMP by a synthetic, immunosuppressive, retroviral envelope peptide causes a shift in the cytokine balance and leads to suppression of cell-mediated immunity by inhibiting IL-12 and stimulating IL-10 production [108]. Progression of HIV infection is also characterized by increased cortisol secretion in both the early and late stages of the disease. Increased glucocorticoid production, triggered by the chronic infection, was recently proposed to contribute to HIV progression [109]. In addition, the HIV-1 accessory protein, Vpr, acts as a potent coactivator of the host glucocorticoid receptor, rendering lymphoid cells hyperresponsive to glucocorticoids [110]. Thus, on one hand, stress hormones suppress cellular immunity and directly accelerate HIV replication, while, on the other hand, retroviruses may suppress cell-mediated immunity using the same pathways by which stress hormones, including CAs and glucocorticoids, alter the Th1/Th2 balance. In a recent study, an association was demonstrated between stress and the susceptibility to common cold among 394 persons who had been intentionally exposed to five different upper respiratory viruses. Psychological stress was found to be associated in a dosedependent manner with an increased risk of acute infectious respiratory illness, and this risk was attributed to increased rates of infection rather than to an increased frequency of

Excessive Th2 responses

Th1 protects

Allergy (Atopy)

Tumors

The role of stress in autoimmunity and allergy is more complex, see text for details. Th, T helper; IL, interleukin; TNF, tumor necrosis factor; IFN, interferon; RA, rheumatoid arthritis; MS, multiple sclerosis; ATD, autoimmune thyroid disease; SLE, systemic lupus erythematosus.

a

Th2 shift, deficit of IL-12 and TNF-␣, overproduction of IL-10 Th2 shift, deficit of IL-12, overproduction of IL-4, IL-10

Excessive Th2 response

Suppressed cellular immunity, deficit of IL-12, TNF-␣, overproduction of IL-10

Th1 shift, overproduction of IL-12, TNF-␣, IFN-␥; deficit of IL-10

Increased levels of stress hormones, histamine, and adenosine may contribute to suppression of cellular immunity resulting in infectious complications Hypoactive stress system may facilitate/sustain the Th1 shift and flares of these autoimmune diseases Stress (Th2 shift) may induce/facilitate flares of SLEa Stress hormone (and histamine)–induced Th2 shift may induce/facilitate/sustain allergic reactionsa Stress hormone-, histamine-, and adenosine-induced Th2 shift may contribute to increased susceptibility to or progression of certain tumors

Suppressed cellular immunity and IL-12 and IFN-␥ production, overproduction of IL-10, Th2 shift

Th2 protects?

Excessive Th1 response

Stress-induced Th2 shift may contribute to increased susceptibility to or progression of these infections

Role of hormones

Suppressed cellular immunity, deficit of IL-12, and IFN-␥, Th2 shift with progression of infection

Pathogenic response

Th1 protects

Host response

Autoimmunity RA, MS, ATD, type 1 diabetes mellitus SLE

Infections Mycobacterium tuberculosis Helicobacterium pylori HIV Common cold viruses Major injury

Condition

Putative Pathophysiological Roles of Hormone-Induced Alterations of Th1/Th2 Balance in Certain Infections, Infectious Complications After Major Injury, Autoimmune/Inflammatory, Allergic, or Neoplastic Diseases

Table 2

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symptoms after infection [111]. Thus, stress hormones, through their selective inhibition of cellular immunity, might play important roles in the increased risk of an individual to acute respiratory infections caused by common cold viruses. B. Major Injury Major injury (serious traumatic injury and major burns) or major surgical procedures often lead to severe immunosuppression, which contributes to infectious complications and, in some cases, to sepsis, the most common cause of late death after trauma. A strong stimulation of the sympathetic nervous system (SNS) and the HPA axis correlates with the severity of both cerebral and extracerebral injury and an unfavorable prognosis (see Ref. 34). In patients with traumatic major injury, and in animal models of burn injury, the suppressed cellular immunity is associated with diminished production of IFN-␥ and IL-12 and increased production of IL-10, i.e., a Th2 shift [112]. A recent study indicated that systemic release of IL-10 triggered by SNS activation might be a key mechanism of immunosuppression after injury. Thus, high levels of systemic IL-10 documented in patients with ‘‘sympathetic storm’’ due to acute accidental or iatrogenic brain trauma were associated with high incidence of infection [34]. Additionally, in a rat model, the increase of IL-10 was prevented by ␤-AR blockade [34]. During ischemia or hypoxia, local adenosine concentrations increase to the micromolar range [113]. Thus, a massive release of adenosine during major injury may mediate, through inhibition of IL-12 and TNF-␣, and potentiation of IL-10 production, part of the substantial immunosuppression that occurs in these patients. Therefore, stress hor- mone, histamine, and adenosine secretion triggered by major injury via an induction of a Th2 shift may contribute to the severe immunosuppression observed in these conditions (Table 2). C. Autoimmunity Several autoimmune diseases are characterized by common alterations of the Th1 versus Th2 and IL-12/TNF-␣ versus IL-10 balance. In RA, multiple sclerosis (MS), type 1 diabetes mellitus, autoimmune thyroid disease (ATD), and Crohn’s disease (CD), the balance is skewed towards Th1 and an excess of IL-12 and TNF-␣ production, whereas Th2 activity and the production of IL-10 are deficient. This appears to be a critical factor that determines the proliferation and differentiation of Th1-related autoreactive cellular immune responses in these disorders [114]. On the other hand, SLE is associated with a Th2 shift and an excessive production of IL-10, while IL-12 and TNF-␣ production appear to be deficient. Taking into consideration the Th2-driving effects of stress hormones systemically, one could postulate that a hypoactive stress system may facilitate or sustain the Th1 shift in MS or RA, and stress system hyperactivity may intensify the Th2 shift and induce or facilitate flares of SLE (Table 2). Animal studies and certain clinical observations support this hypothesis. 1. Stress System Activity in RA and MS Recent studies suggest that suboptimal production of cortisol is involved in the onset and/ or progression of RA [63,115,116]. Most patients with RA have relatively ‘‘inappropriately normal’’ plasma cortisol levels in the setting of severe, chronic inflammation, characterized by increased production of TNF-␣, IL-1, and IL-6. Since these cytokines are powerful stimulants to the HPA axis and cortisol production, we would have expected significantly elevated plasma cortisol levels in RA patients. The available data suggest that the HPA

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axis response is blunted in these patients. Whether this abnormality is primary or secondary has not been established [116]. Several lines of evidence indicate that the sympathetic-immune interface is defective in MS and its experimental model, experimental allergic encephalomyelitis (EAE). Thus, sympathetic skin responses are decreased and lymphocyte ␤-ARs are increased in progressive MS [117]. The density of ␤-ARs on CD8Ⳮ T cells are increased two- to threefold compared with age-matched controls [118,119]. Similarly, in the preclinical stage of EAE the NE content in spleen is reduced, accompanied by an increase of splenocyte ␤-ARs density [120]. A defective or hypoactive SNS is most likely to be a ‘‘causative’’ factor for the upregulation of ␤-ARs observed in MS [121]. Furthermore, isoproterenol and terbutaline, ␤-AR and ␤2-AR agonists, respectively, were reported to suppress chronic/ relapsing EAE in Lewis rats [122,123]. The latter observation might have resulted from the above-discussed effects of CAs and ␤-AR agonists on the production of type 1 cytokines. Interestingly, dimaprit, an H2 histamine receptor agonist, is also able to reduce the clinical severity and pathology of EAE in both C57BL/6- and iNOS-deficient mice [124]. These data provide a rationale for exploring H2 receptor activation for therapeutic value in MS (see histamine above). Recent data suggests a ‘‘protective’’ role of SNS in RA or its experimental models in animals. Thus, in arthritis-prone Lewis rats sympathectomy with 6-OHDA enhanced the severity of adjuvant-induced arthritis [125,126]. In this animal model of arthritis, selective sympathetic denervation of the reactive secondary lymphoid organs, the popliteal and inguinal lymph nodes, was achieved with local injection into the fat pads surrounding these lymph nodes [125,126]. This denervation resulted in earlier onset and enhanced severity of inflammation and bone erosions compared with nondenervated rats. The ‘‘protective’’ role of SNS is further substantiated by a recent study demonstrating that the ␤2AR agonist salbutamol is a potent suppressor of established collagen-induced arthritis in mice [127]. This drug had a profound protective effect as assessed by clinical score, paw thickness, and joint histology. Additionally, in in vitro experiments salbutamol reduced IL-12 and TNF-␣ release by peritoneal macrophages and blocked mast cell degranulation in joint tissues. Recent studies in humans also suggest a defective SNS in RA. In patients with RA diminished autonomic responses were observed after cognitive discrimination and the Stroop color–word interference tests [128]. Patients with long-term RA have a highly significant reduction of sympathetic nerve fibers in synovial tissues, which was dependent on the degree of inflammation [129]. Thus, the reduction of sympathetic nerve fibers in the chronic disease may lead to uncoupling of the local inflammation from the antiinflammatory input of SNS. Interestingly, in RA synovial tissues it appears there is preponderance of about 10:1 for primary sensory, substance P–positive fibers as compared with sympathetic fibers [129]. Since substance P is powerful pro-inflammatory agent, via release of histamine and TNF-␣ and IL-12, such preponderance may lead to an unfavorable proinflammatory state, supporting the disease process of RA. In addition, a recent study also indicates that there is a decrease in G-protein–coupled receptor kinase (GRK) activity in lymphocytes of RA patients, particularly the GRK2 and GRK6 subtypes [130]. The GRKs are responsible for the rapid loss of receptor responsiveness despite continuous presence of the agonist, a process known as homologous desensitization. The decrease in GRK2 activity in RA appears to be mediated by cytokines such as IL-6 and IFN-␥. Local proinflammatory cytokines or a hypoactive SNS may, therefore, mediate the changes in coupling of ␤-ARs to G-proteins observed in RA patients.

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2. Pregnancy, Postpartum and Autoimmune Disease Activity Some autoimmune diseases like RA and MS often remit during pregnancy, particularly the third trimester, but have an exacerbation or their initial onset during the postpartum period [115,1–133. The risk of developing new-onset RA during pregnancy, compared to nonpregnancy, is decreased by about 70%. In contrast, the risk of developing RA is markedly increased in the postpartum period, particularly the first 3 months (odds ratio of 5.6 overall and 10.8 after first pregnancy). In women with multiple sclerosis, the rate of relapses declines during pregnancy, especially in the third trimester, increases during the first 3 months of the postpartum, and then returns to the prepregnancy rate [133]. In apparent contrast, autoimmune diseases that present with symptoms associated predominantly with antibody-mediated damage, such as SLE and specifically immune complex–mediated glomerulonephritis, tend to develop or flare during pregnancy [131,2–136]. A decrease in the production of IL-2 and IFN-␥ by antigen- and mitogen-stimulated peripheral blood mononuclear cells, accompanied by an increase in the production of IL4 and IL-10, is observed in normal pregnancy. The lowest quantities of IL-2 and IFN-␥ and the highest quantities of IL-4 and IL-10 are present in the third trimester of pregnancy [137]. Placental tissues from mothers at term express high levels of IL-10 [138], while IL-10 is present in the amniotic fluid of the majority of pregnancies, with higher concentrations found at term compared with the second trimester [139]. We have recently found that during the third trimester of pregnancy, ex vivo monocytic IL-12 production was about threefold and TNF-␣ production approximately 40% lower than postpartum values [76]. These studies suggest that type 1/pro-inflammatory cytokine production and cellular immunity are suppressed, and there is a Th2 shift during normal pregnancy, particularly the third trimester (see Fig. 3). The third trimester of pregnancy and the early postpartum is also known to be associated with abrupt changes of several hormones. Thus, during the third trimester of pregnancy, urinary cortisol and NE excretion and serum levels of 1,25-dihydroxyvitamin D3 are about two- to threefold higher than postpartum values [76]. This is accompanied by the well-known marked elevations of estradiol and progesterone serum concentrations. The data reviewed here are consistent with the view that the increased levels of cortisol, NE, 1,25-dihydroxyvitamin D3, estrogens, and progesterone in the third trimester of pregnancy might orchestrate the improvement in autoimmune diseases, such as RA and MS via suppression of type 1/pro-inflammatory (IL-12, IFN-␥, and TNF-␣) and potentiation of type 2/anti-inflammatory (IL-4 and IL-10) cytokine production. Conversely, this particular type of hormonal control of pro-/anti-inflammatory cytokine balance might contribute to the flaring up of SLE observed during pregnancy. Postpartum, the hormonal state abruptly shifts. The deficit in hormones that inhibit Th1-type cytokines and cell-mediated immunity might permit autoimmune diseases such as RA and MS to first develop or established disease to flare up [76,115,140]. D. Allergy/Atopy Allergic reactions of type 1 hypersensitivity (atopy), such as asthma, eczema, hay fever, urticaria, and food allergy, are characterized by dominant Th2 responses, overproduction of histamine, and a shift to IgE production. The effects of stress on atopic reactions are complex, at multiple levels, and can be in either direction. Stress hormones acting at the level of APCs and lymphocytes may induce a Th2 shift and, thus, facilitate or sustain atopic reactions, but this can be antagonized by their effects on the mast cell. Glucocorticoids and

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CAs (through ␤2-ARs) suppress the release of histamine by mast cells, thus abolishing its pro-inflammatory, allergic, and bronchoconstrictor effects. Thus, reduced levels of epinephrine and cortisol in the very early morning could contribute to nocturnal wheezing and have been linked to high circulating histamine levels in asthmatics [141]. This might also explain the beneficial effect of glucocorticoids and ␤2-agonists in asthma. Infusion of high doses of epinephrine, however, causes a rise in circulating histamine levels that may be due to an ␣-adrenergic–mediated increase in mediator release (see Ref. 141). Thus, severe acute stress associated with high epinephrine concentrations and/or high local secretion of CRH could lead to mast cell degranulation. As a result, a substantial amount of histamine could be released, which consequently would not antagonize, but rather amplify, the Th2 shift through H2 receptors, while in parallel, by acting on H1 receptors it could initiate a new episode or exacerbate a chronic allergic condition. Glucocorticoids alone or in combination with ␤2-AR agonists are broadly used in the treatment of atopic reactions, and particularly asthma. In vivo, ex vivo, and in vitro exposure to glucocorticoids and ␤2-agonists result in a reduction of IL-12 production, which persists at least several days [12,14,20]. Thus, glucocorticoid and/or ␤2-AR agonist therapy is likely to reduce the capacity of APC to produce IL-12, to suppress greatly the synthesis of type 2 cytokine in activated, but not resting T cells, and to abolish eosinophilia [14]. If, however, resting, (cytokine-uncommitted) T cells are subsequently activated by APCs preexposed to glucocorticoids and/or ␤2-AR agonists, enhanced IL-4 production could be induced, with limited IFN-␥ synthesis [14]. Thus, although in the short term the effect of glucocorticoids and ␤2-AR agonists might be beneficial, their long-term effects might be to sustain the increased vulnerability of the patient to the allergic condition. This is substantiated by the observations that both glucocorticoids and ␤2-AR agonists potentiate IgE production in vitro and in vivo [142,143]. E. Tumor Growth Low levels of IL-12 have been associated with tumor growth, as opposed to tumor regression observed with administration of IL-12 delivered in situ or systemically (see Ref. 144). On the other hand, local overproduction of IL-10 and TGF-␤, by inhibiting the production of IL-12 and TNF-␣, and the cytotoxicity of NK and Tc cells, seems to play an inappropriate immunosuppressive role, as seen for example in melanoma [145]. Stress can increase the susceptibility to tumors, tumor growth, and metastases. In animals, ␤-AR stimulation suppresses NK-cell activity and compromises resistance to tumor metastases [146]; stress decreases the potential of spleen cells to turn into antitumor Tc against syngeneic B16 melanoma, and it significantly suppresses the ability of tumorspecific CD4Ⳮ cells to produce IFN-␥ and IL-2 [147]. High concentrations of histamine have been measured within colorectal and breast cancer tissues and treatment with cimetidine, an H2 histamine antagonist, correlates with increased survival in patients with colorectal cancer [42,148]. Our results suggest that by preventing the effect of histamine on IL-12 and IL-10 production, cimetidine may contribute to a restoration of Th1 and local cellular immune responses. The above-described adenosine-induced inhibition of IL-12 production may be relevant to the immunosuppression observed in solid tumors, such as adenocarcinomas of the lung and colon, where hypoxic conditions cause accumulation of high concentrations of extracellular adenosine that may contribute to inhibition of cellular immunity [149,150]. These data suggest that stress hormone–, histamine-, and/or adenosine-induced inhibition

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of IL-12 and potentiation of IL-10, TGF-␤ production, and subsequent suppression of cellular immunity may contribute to the increased growth of certain tumors.

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32 HIV Infection and the Central Nervous System MARCUS KAUL and STUART A. LIPTON The Burnham Institute, La Jolla, California, U.S.A.

I. INTRODUCTION Human immunodeficiency virus-1 (HIV-1), in addition to destroying the immune system, can also induce neurological disease that culminates in frank dementia. The worldwide development of HIV-related disease is alarming, with more than 36 million existing infections and about 20 million deaths [1]. HIV infection with acquired immunodeficiency syndrome (AIDS) opportunistic infections may affect the central nervous system (CNS), but the virus itself can also induce a number of neurological syndromes [2]. Neuropathological conditions directly triggered by HIV-1 include peripheral neuropathies, vacuolar myelopathy, and a syndrome of cognitive and motor dysfunction that has been designated HIV-associated dementia (HAD) [2]. A mild form of HAD is called minor cognitive motor disorder (MCMD) [2,3]. This chapter reviews recent developments in HIV-1–associated disease of the CNS, in particular HAD and MCMD. The mechanisms of HAD and MCMD remain poorly understood, but the discovery in the brain of cellular binding sites for HIV-1, the chemokine receptors, promises new insights. Interestingly, HIV-1 in the brain productively infects only macrophages and microglia, but injury and apoptotic death occur in neurons. Neurotoxins from macrophages, microglia, and astrocytes constitute the predominant pathway to neuronal injury, though direct effects of viral proteins might contribute. The released neurotoxic factors excessively stimulate neurons, thus leading to excitotoxicity with subsequent breakdown in neurons of vital cellular functions in a manner shared with other neurodegenerative diseases. Advances in understanding the molecular mechanisms of the disease-defining events provide hope for improved therapeutic intervention. 673

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Highly active antiretroviral therapy (HAART) has resulted in at least a temporary decrease in the incidence of HAD [4,5]. However, HAART does not appear to provide complete protection from or reversal of HAD [6,7], and the prevalence of dementia seems to be increasing as people live longer with AIDS [8,9]. Currently, no specific treatment for HAD exists, mainly because of an incomplete understanding of how HIV infection triggers neuronal injury and apoptosis. The major pathway for HIV entry into the CNS is thought to be via infected monocytes. The predominant pathogenesis of HAD is believed to involve activation of monocytic cells (macrophages and microglia) and their subsequent release of toxins that lead to neuronal and astrocytic dysfunction. Macrophages and microglia can be activated by HIV infection itself, by interaction with viral proteins, or by immune stimulation due to concurrent infection or other factors [10]. Although neurons themselves are not productively infected by HIV-1, it is possible that the direct effects of viral proteins on neurons may also contribute to neurodegeneration. The extracellular and intracellular signaling pathways leading to macrophage or microglial activation as well as those induced in neurons and astrocytes are potential therapeutic targets for the prevention or treatment of HAD. II. EPIDEMIOLOGY OF HAD IN THE ERA OF HAART In the early 1990s the prevalence of HAD was estimated to be as high as 20–30% in those individuals with advanced HIV disease and low CD4 cell counts [11]. Numerous experts believe that HAD is currently the most common cause of dementia worldwide among people aged 40 or less, and it is a significant independent risk factor for death due to AIDS [12]. With the advent of HAART, the incidence of HAD has decreased to as low as 10.5% [13], but in recent years the incidence of HAD as an AIDS-defining illness has actually increased [6]. The proportion of new cases of HAD displaying a CD4 cell count greater than 200 per ␮L is also increasing [13]. Moreover, a less fulminant form of neurological dysfunction, termed minor cognitive/motor disorder (MCMD), may be more prevalent than frank dementia in the HAART era and remains a significant independent risk factor for AIDS mortality [12]. These findings suggest that HAART does not provide complete protection from the development of HAD. Unfortunately, HIV protease inhibitors and several of the nucleoside analogues penetrate poorly into the CNS [14], perhaps allowing early CNS infection to evolve independently over time in a protected brain reservoir. Thus, despite the finding that improved systemic control of viral replication is associated with a decrease in the incidence of HAD, the question of whether this effect will be long lasting has been hotly debated [7,15]. In fact, distinct viral drug-resistance patterns in the plasma and cerebrospinal fluid (CSF) compartments have recently been reported [16]. Thus, it is possible that the proportion of HIV-infected individuals who develop disability secondary to HAD will increase while improvements in control of peripheral viral replication and the treatment of opportunistic infections continue to extend survival times, resulting in an increase in the overall prevalence of MCMD and HAD. A better understanding of the pathogenesis of HAD, including viral and host factors, is needed in order to identify therapeutic targets for the prevention and treatment of this neurodegenerative disease. III. ROUTE OF HIV ENTRY INTO THE BRAIN AND INITIATION OF HAD HIV arrives in the CNS early in the course of infection, and the virus primarily resides in microglia and macrophages. However, infection of these cells may not suffice to initiate

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neurodegeneration [15]. It has been proposed that factors associated with advanced HIV infection in the periphery, thus outside the CNS, are important triggers for events leading to dementia. One such factor could be an elevated number of circulating monocytes that express CD16 and CD69. These activated cells tend to adhere to the normal endothelium of the brain microvasculature, transmigrate, and might then trigger a number of deleterious processes [15]. The blood-brain barrier (BBB) plays a crucial role in HIV infection of the CNS [15,17]. Microglial and astrocytic chemokines, cell migration/chemotaxis-inducing cytokines, such as monocyte chemoattractant protein (MCP)-1, appear to regulate migration of peripheral blood mononuclear cells through the BBB [17]. In fact, a mutant MCP-1 allele that causes increased infiltration of mononuclear phagocytes into tissues has recently been implicated in an increased risk of HAD [18]. Histological studies in specimens from HIV-1–infected humans and simian immunodeficiency virus (SIV)–infected rhesus macaques show that lymphocytes and monocytes migrate into the brain [19,20]. However, the pathophysiological relevance of CNS-invading lymphocytes in HAD is not clearly established [20]. Cellular migration also involves adhesion molecules, and increased expression of vascular cell adhesion molecule-1 (VCAM-1) has been implicated in mononuclear cell migration into the brain during HIV and SIV infection [21]. It has also been suggested that the inflammatory cytokine tumor necrosis factor-alpha (TNF-␣) opens a paracellular route for HIV-1 across the BBB [22]. A vicious cycle of immune dysregulation and BBB dysfunction might be required to achieve sufficient entry of infected or activated immune cells into the brain to cause neuronal injury. Concerning the virus, variations of the envelope protein gp120 might also influence the timing and extent of events, allowing viral entry into the CNS and leading to neuronal injury [23]. Interestingly, alterations in the BBB have been observed in transgenic mice expressing the HIV envelope protein gp120 in a form that circulates in plasma [24], suggesting that circulating virus or envelope proteins may cause BBB dysfunction during the viremic phase of primary infection. HIV infection within the central nervous system can be followed by measurement of viral RNA in the CSF. Several groups have reported a positive correlation between CSF viral load and the observed degree of cognitive dysfunction in patients with HAD or MCMD [25–27]. CSF viral load also appears to correlate with viral load in brain measured by quantitative PCR [26,28], and the highest concentrations of virus are observed in those subcortical structures most frequently affected in patients with HAD [28]. IV. NEUROPATHOLOGY OF HIV INFECTION AND PATHOGENESIS OF HAD The neuropathology associated with HIV infection in the brain, termed HIV encephalitis, is characterized by widespread reactive astrocytosis, myelin pallor, microglial nodules, activated resident microglia, multinucleated giant cells, and infiltration predominantly by monocytoid cells, including blood-derived macrophages. However, numbers of HIVinfected cells, multinucleated giant cells or viral antigens in CNS tissue do not correlate well with measures of cognitive function [29,30]. The pathological features most closely associated with the clinical signs of HAD include increased numbers of microglia [29], elevated TNF-␣ mRNA in microglia and astrocytes [31], evidence of excitotoxins [32,33], decreased synaptic and dendritic density [30,34], and selective neuronal loss [35,36]. Several groups have demonstrated that HAD is associated with evidence of neuronal apoptosis

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[37–39], but this finding is not clearly associated with viral burden [37] or a history of dementia [40]. The topographic distribution of neuronal apoptosis is correlated with evidence of structural atrophy and closely associated with markers of microglial activation, especially within subcortical deep gray structures [40], which may show a predilection for atrophy in HAD. The neuropathology observed in HAD coupled with extensive research on both in vitro and animal models of HIV-induced neurodegeneration have led to a fairly complicated model for the pathogenesis of HAD. As with other emerging models of neurodegenerative disease, it is likely that a construct similar to the multi-hit model of oncogenesis will be the most effective way to understand all of the factors involved in the pathogenesis of HAD. Figure 1 schematizes potential intercellular interactions that can lead to neuronal degeneration in the setting of HIV infection. Macrophages and microglia can be infected by HIV-1, but they can also be activated by factors released from infected cells, including cytokines and shed viral proteins such as gp120. Variations of the HIV-1 envelope protein gp120, in particular in its V1, V2, and V3 loop sequences, have been implicated in modulating the activation of macrophages and microglia [23]. Factors released by activated microglia affect all cell types in the CNS, resulting in upregulation of cytokines, chemokines, and endothelial adhesion molecules [3,10,15]. Some of these molecules may contribute to neuronal damage and apoptosis via direct or indirect routes. Additionally, activated microglia release excitatory amino acids (EAAs) and related substances, including, quinolinate, cysteine, and the amine NTox [41–45]. EAAs released by infected or activated microglia can induce neuronal apoptosis through a process known as excitotoxicity, which engenders excessive Ca2Ⳮ influx and free radical (nitric oxide and superoxide anion) formation by overstimulation of glutamate receptors [46]. Some HIV proteins, such as gp120 and Tat, have also been reported to be directly neurotoxic, although high concentrations of viral protein may be needed or neurons may have to be cultured in isolation to see these direct effects [47,48]. It is important to note that, even in the absence of extensive viral invasion of the CNS, toxic viral proteins and factors released from microglia and glutamate set free by astrocytes may act synergistically to promote neurodegeneration.

V. CHEMOKINE RECEPTORS IN HAD Entry of HIV-1 into target cells such as T cells or macrophages/microglia occurs usually after binding of the viral envelope protein gp120 to chemokine receptors in conjunction with CD4. Generally, T cells are infected via the ␣-chemokine receptor CXCR4 and/or the ␤-chemokine receptor CCR5. In contrast, monocytes, macrophages, and microglia are primarily infected via CCR5 or CCR3, but CXCR4 may also be involved [49–51]. Chemokine receptors can also be present on neurons and astrocytes in the brain [52,53], although these cells are not thought to harbor productive infection. In vitro studies suggest that specific chemokine receptors mediate HIV-associated neuronal damage while others may serve a protective role [47,54]. Three chemokine receptors (with their respective ligands in parentheses) are of particular interest in HAD: CXCR4 (SDF-1␣/␤/␥), CCR5 (RANTES or MIP-1␣/␤), and CX3CR1 (fractalkine). CXCR4 is expressed on neurons, microglia, astrocytes, and endothelia in the brain [55–57]. SDF-1 is produced by astrocytes, macrophages, neurons, and Schwann cells [58–60], and an increase in SDF-1 mRNA has been detected in HIV encephalitis [53].

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Figure 1 A current model of neuronal injury induced by HIV-1 infection: Immune-activated and HIV-infected brain macrophages (M⌽)/microglia release potentially neurotoxic substances. These substances include quinolinic acid and other excitatory amino acids such as glutamate and L-cysteine, arachidonic acid, PAF, NTox, free radicals, TNF-␣, and probably others. These factors from macrophages and also possibly from reactive astrocytes contribute to neuronal injury, dendritic and synaptic damage, and apoptosis as well as to astrocytosis. A major pathway of entry of HIV-1 into monocytoid cells is via gp120 binding, and therefore it is not surprising that gp120 (or a fragment thereof) is capable of activating uninfected macrophages to release similar factors to those secreted in response to frank HIV infection. Macrophages bear CCR5 and CXCR4 chemokine receptors on their surface in addition to CD4, and gp120 binds via these receptors. Some populations of neurons and astrocytes have been reported to also bear CXCR4 and CCR5 receptors on their surface, raising the possibility of direct interaction with gp120. Macrophages/microglia and astrocytes have mutual feedback loops (bidirectional arrow). Cytokines participate in this cellular network in several ways. For example, HIV infection or gp120 stimulation of macrophages enhances their production of TNF-␣ and IL1␤ (arrow). The TNF-␣ and IL-1␤ produced by macrophages stimulate astrocytosis. Arachidonate released from macrophages impairs astrocyte clearing of the neurotransmitter glutamate and thus contributes to excitotoxicity. In conjunction with cytokines, the ␣-chemokine SDF-1 stimulates reactive astrocytes to release glutamate in addition to the free radical nitric oxide (NO•), which in turn may react with superoxide to form the neurotoxic molecule peroxynitrite. NO might also activate extracellular matrix metalloproteinases (MMPs), which can then proteolytically affect neurons, and also cleave membrane-anchored fractalkine [77,93]. Neuronal injury is primarily mediated by overactivation of NMDARs with a resultant excessive increase in intracellular Ca2Ⳮ levels. This in turn leads to overactivation of a variety of potentially harmful signaling systems, the formation of free radicals, and release of additional neurotransmitter glutamate. Glutamate subsequently overstimulates NMDARs on neighboring neurons, resulting in further injury. This final common pathway of neurotoxic action can be blocked by NMDAR antagonists. For certain neurons, depending on their exact repertoire of ionic channels, this form of damage can also be ameliorated to some degree by calcium channel antagonists or non-NMDAR antagonists. Additionally, agonists of ␤-chemokine receptors, which are present in the CNS on neurons, astrocytes, and microglia, can confer partial protection against neuronal apoptosis induced by HIV/gp120 or NMDA. (Modified from Ref. 3.)

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Chemokine receptors are seven membrane-spanning, G-protein–coupled receptors, and as such trigger intracellular signaling events. For example, SDF-1␣ can modulate synaptic transmission in the rat cerebellum by increasing the intracellular Ca2Ⳮ concentration [61]. In isolated hippocampal neurons, SDF-1␣ causes Ca2Ⳮ- and cAMP-dependent CREB transcription factor activation [47]. SDF-1␣ also increases ERK-1 and -2 kinase activity in neurons, similar to gp120 from HIV-1IIIB, a CXCR4 (or X4)–preferring strain of the virus [62]. SDF-1␣/␤, HIV-1 virions, and supernatants of HIV-infected monocytederived macrophages reportedly signal by a Gi-dependent decrease in cAMP and an increase in IP3 and [Ca2Ⳮ]i; these signals correlate with enhanced synaptic transmission, activation of caspase-3, and neuronal apoptosis [56]. In cerebrocortical neurons and neuronal cell lines, picomolar concentrations of X4 preferring or dualtropic gp120 (as well as intact virus) can induce neurotoxicity via CXCR4 receptors [51,54,63]. This apoptotic death appears to be mediated predominantly via the release of microglial toxins rather than by direct neuronal damage in mixed neuronal/glial cultures that mimic the cellular composition of the intact brain [54]. However, nanomolar concentrations of SDF-1␣/␤ interacting with CXCR4 can induce apoptotic death of cerebrocortical neurons in the absence of microglial signaling, suggesting a possible direct interaction with neurons while interaction with astrocytes can also occur [54]. Interestingly, inhibition of p38 mitogen-activated protein kinase (MAPK) prevents the neurotoxicity of both gp120 and SDF-1 in these mixed cultures (Fig. 2) [54]. In contrast to these findings, somewhat higher concentrations of SDF-1␣ have been reported to provide neuroprotection from X4 preferring gp120-induced damage of isolated hippocampal neurons [47]. Clearly, the results obtained on isolated neurons may be different from those observed in mixed

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neuronal/glial cultures that more closely resemble the repertoire of cells found in the brain, as discussed further below. The ␤-chemokine receptor CCR5 is expressed by neurons, microglia, and astrocytes [52], and its role in HAD appears to be distinct from the effect of the ␣-chemokine receptor CXCR4. While individuals lacking functional CCR5 are at reduced risk for HIV infection, in vitro studies have shown that activation of this chemokine receptor by RANTES or 䉳 Figure 2 Cellular signaling in HAD—pathways engaged in neuronal injury and apoptosis. (Middle panel) Neuronal signaling in HAD: Overstimulation of the NMDAR is triggered by neurotoxins released from HIV-infected or immune-stimulated macrophages/microglia and by impaired clearance of glutamate that under normal conditions would have been taken up by astrocytes. Consequently, excessive Ca2Ⳮ influx into neurons triggers activation of p38 MAPK, mitochondrial Ca2Ⳮ overload and cytochrome c (Cyt c) release, free radical generation [nitric oxide (NO•) and reactive oxygen species (ROS)], caspase activation, and ultimately apoptosis. NMDARs are physically tethered to neuronal nitric oxide synthase (nNOS), facilitating its activation. NO passing out of the cell may activate MMPs and trigger an extracellular proteolytic pathway to neuronal injury. Inside the cell, the Bcl2 family members Bad, Bax, and Bid promote apoptosis mediated by glutamate, ROS, and TNF-␣, respectively. Bcl2 prevents apoptosis, apparently by attenuating cytochrome c release and ROS production. Activation of the p38 MAPK pathway by a Ca2Ⳮ-mediated mechanism and by oxidative stress may lead to phosphorylation/activation of transcription factors involved in apoptosis. Stimulation of the ␣-chemokine receptor CXCR4 can also induce several pathways in neurons, including activation of p38 MAPK, which leads to apoptosis. In contrast, activation of the ␤chemokine receptor CCR5 initiates an as yet uncharacterized neuroprotective pathway that interferes with toxicity triggered by HIV/gp120 or excessive stimulation of NMDARs. The chemokine fractalkine (Fkn) is released from neurons subsequent to excitotoxic injury and may represent feedback signaling onto nonneuronal cells. (Left panel) Microglial/macrophage signaling in HAD: HIV/gp120 (gp) interacts with chemokine receptors CXCR4 or CCR5 in conjunction with CD4 to stimulate or infect (if the entire virus is present) microglia and macrophages. Natural ligands of CXCR4 (i.e., the ␣-chemokine SDF-1) and CCR5 (i.e., the ␤-chemokines MIP-1␤ and RANTES) interfere with HIV/gp120 binding and signaling. However, only the ␤-chemokines can prevent the neurotoxic effect of activated microglia and macrophages. Neurons release the ␦-chemokine fractalkine (Fkn), which activates microglia. Hence, Fkn may mediate communication between neurons and glia. The HIV envelope protein gp120 triggers a signaling pathway that involves p38 MAPK, a pivotal factor in immune stimulation of macrophages, which in turn activates the transcription factor MEF2C. HIV/gp120 induces the release of neurotoxic substances, including excitatory amino acids, arachidonic acid, and related molecules such as PAF, which engenders neuronal glutamate release; gp120 also induces release of inflammatory cytokines, such as TNF-␣. Inflammatory cytokines can activate adjacent microglia/macrophages and astrocytes, and thus indirectly contribute to brain injury. (Right panel) Astrocyte signaling in HAD: Astrocytes express the HIV coreceptors CXCR4 and CCR5 in addition to other chemokine receptors, but lack CD4. Therefore, astrocytic reactivity may be influenced by natural ligands of chemokine receptors. Via these chemokine receptors, astrocytes may possibly be stimulated by a CD4-independent effect of HIV/gp120. Astrocytes are activated by inflammatory cytokines, including TNF-␣, IL-1␤, and IFN-␥. Exposure to arachidonic acid released from macrophages/microglia and cytokine activation results in impaired glutamate uptake, increased glutamate release, and induction of iNOS, leading to release of potentially neurotoxic nitric oxide. TNF-␣, released from macrophages/microglia, and SDF-1 stimulate astrocytes to release glutamate. TNF-␣ also promotes expression of the astrocytic fractalkine (Fkn) receptor, CX3CR1. Stimulation of CX3CR1 on astrocytes induces release of a soluble factor that triggers microglial proliferation. (Modified from Ref. 128.)

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MIP-1 ␤ protects neurons from gp120-induced apoptosis [47,54]. Additionally, HIV-infected patients with higher CSF concentrations of ␤-chemokines MIP-1␣/␤ and RANTES perform better on neuropsychological measures than those with low or undetectable levels of ␤-chemokines [64]. These findings suggest that once HIV infection is established, distinct ␤-chemokines may actually be protective against progression to HAD. Individuals with the CCR5⌬32 allele express less than normal or no CCR5 protein depending on heterozygosity or homozygosity, respectively. Interestingly, these human beings are highly, but not completely, protected against infection with HIV-1 [65–68]. In fact, some homozygous CCR5-deficient AIDS patients display a rapidly progressing form of HIV disease [69,70]. Other ␤-chemokines, such as MCP-1, are elevated in the CSF of patients with HAD and may correlate with CSF viral load [71,72]. A mutant allele of MCP-1 has been linked to increased protein expression and monocyte infiltration into tissues, as well as accelerated disease progression to AIDS and elevated risk of dementia [18]. CX3CR1 is a chemokine receptor found on microglia and possibly neurons and has been reported to interact with HIV-1 in vitro [73]. Fractalkine, a chemokine expressed in both a membrane-bound and soluble form by neurons, binds to CX3CR1, possibly mediating neuronal-microglial interaction [17]. Fractalkine and CX3CR1 have been found to be upregulated in pediatric patients with HIV encephalitis and reportedly mediate both macrophage recruitment and neuroprotection from gp120 or Tat, at least in vitro [74,75]. These two actions would seemingly exert opposing effects on HAD. In rat microglia, fractalkine signaling increases intracellular Ca2Ⳮ, activates Akt, and triggers chemotaxis [76]. A potential role of fractalkine in HAD is suggested by its cleavage from the neuronal cell surface in response to excitotoxic injury (see below) [77].

VI. MACROPHAGES AND MICROGLIA IN HAD Macrophages and microglia play a crucial role in HAD because they are the only resident cells that can be productively infected with HIV-1 in the CNS [3,10], although a nonproductive or latent infection of astrocytes has been observed in pediatric patients [78]. HIV-1–infected macrophages and possibly lymphocytes migrate into the brain and constitute the major route of viral entry into the CNS (reviewed in Ref. 15). We and others have shown that HIV-infected or immune-stimulated macrophages/microglia produce neurotoxins, and macrophages/microglia are required for HIV-1 or gp120-induced neurotoxicity [41,42,79]. These macrophages/microglia damage neurons by releasing excitotoxic substances that produce excessive activation of glutamate receptors, primarily of the Nmethyl-D-aspartate subtype (NMDAR). Additionally, indirect neurotoxicity is probably mediated by macrophage- and microglial-derived inflammatory cytokines, such as IL-1␤ and TNF-␣, arachidonate and its metabolites including platelet-activating factor (PAF), chemokines, matrix metalloproteinases (MMPs), and viral proteins [3,10,80,81]. Chemokine and cytokine signaling in microglia promote p38 MAP kinase activity, which in turn phosphorylates/activates the transcription factor MEF2C (Fig. 2). Pharmacological inhibition of p38 MAPK prevents microglial induction of TNF-␣ and inducible nitric oxide synthase (iNOS) gene expression in response to inflammatory stimuli [82]. The p38 signal transduction pathway appears to be involved in mediating both microglial activation and neuronal injury [3,54,83], suggesting that modulation of this kinase may be an important therapeutic target for the prevention of HAD (see below).

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VII. ASTROCYTES IN HAD Astrocytosis (proliferation of activated astrocytes) as well as occasional astrocyte apoptosis occur in association with HAD and are also observed in the gp120 transgenic mouse [39,84,85]. We have found an increase in the number of astrocytes labeled with antibodies against activated caspase-3 in postmortem cerebrocortical tissue from human patients with HAD, suggesting ongoing injury to astrocytes in the setting of HIV [3]. Astrocytes may contribute to the production or maintenance of excitotoxins like glutamate in several ways (Fig. 2). For example, the normal reuptake of glutamate by astrocytes is impaired and release of astrocytic glutamate is induced by several factors derived from activated macrophages/microglia, including SDF-1, TNF-␣, and arachidonic acid [86–89]. Stimulation of metabotropic (G-protein linked) glutamate receptors (mGluRs) on astrocytes may lead to increased [Ca2Ⳮ]i and further release of glutamate [90]. In addition to contributing to dysregulation of EAA homeostasis, astrocytes may play an important role in relaying or amplifying neurotoxic signals that emanate from activated or HIV-infected microglia. First, the HIV protein Tat can induce astrocytic expression of the ␤-chemokine MCP-1 [91]. Secreted MCP-1 is a chemoattractant factor for monocytes/microglia and may recruit these cells into an environment, where they subsequently release cytokines and EAAs. In fact, increased MCP-1 secretion has been linked to the promotion of HAD [18]. Second, cytokines and viral proteins promote the induction of iNOS within astrocytes [92]. The nitric oxide (NO) thus released may then react with superoxide anion (O2ⳮ) to form neurotoxic peroxynitrite (ONOOⳮ), similar to the reaction that can occur within neurons after excessive NMDAR stimulation. In addition, NO may also activate MMPs, which are secreted by stimulated macrophages/microglia as a consequence of HIV infection in the brain [80,81]. Indeed, we have recently found that nitrosylation can activate MMP-9 and convert the enzyme into a neurotoxin [93].

VIII. MECHANISMS OF NEURONAL INJURY AND APOPTOSIS IN HAD How HIV infection results in neuronal injury continues to remain a controversial topic. While there is general agreement that HIV does not infect neurons, the primary cause of neuronal injury remains in question. There is evidence to support multiple theories for neuronal injury by various viral proteins, including Tat, Nef, Vpr, and the Env proteins gp120 and gp41. These findings have led to at least two different theories on how HIV results in neuronal injury in the brain. The theories can be described as the ‘‘direct injury’’ hypothesis and the ‘‘indirect’’ or ‘‘bystander effect’’ hypothesis. These two theories are in no way mutually exclusive, and currently available data support a role for both theories, although an indirect form of neurotoxicity appears to predominate. The theory that HIV proteins can directly injure neurons without requiring the intermediary function of nonneuronal cells (microglia and/or astrocytes) is supported by experiments showing that viral envelope proteins are toxic in serum free primary neuronal culture [47] or in neuroblastoma cell lines [94]. In these experimental paradigms, the impact of neurotoxic cytokines and EAAs secreted from nonneuronal cells is minimized because serum-free neuronal cultures contain few if any nonneuronal cells and neuroblastoma lines do not contain cells of other phenotypes. The HIV coat protein gp120 interacts with several members of the chemokine receptor family (see above), and the direct form of HIVinduced neuronal injury may be mediated by chemokine receptor signaling. Indeed, experi-

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ments aimed at blocking chemokine receptor signaling can in some cases prevent HIV/ gp120-induced neuronal apoptosis [56,75]. Moreover, some chemokines, such as SDF1, appear to be directly neurotoxic via stimulation of ␣-chemokine receptors [54,63]. Additionally, nanomolar concentrations of gp120 have been reported to interact with the glycine-binding site of the NMDAR [95], suggesting another mechanism by which HIV/ gp120 may have a direct effect on neuronal cell death. However, these concentrations are quite high compared to the picomolar amounts of gp120 that can mediate indirect neuronal injury via microglial stimulation (see below). HIV/Tat can be taken up into PC-12 cells by a receptor-mediated mechanism [48] and may also have a direct effect on neurons by potentiating the response to excitotoxic stimuli [96]. Experiments using cultured hippocampal neurons revealed that HIV/Vpr may be directly neurotoxic through formation of a cation-permeable channel [97]. However, the meaning of all of these in vitro findings must be interpreted in the context of the limitations of the experimental paradigm and concentration of HIV proteins employed. Most of the experimental results described above were obtained in the absence of nonneuronal cells and therefore a predominantly indirect effect would not be detected. In addition, the concentrations of HIV proteins employed were frequently well above the picomolar or lower range thought to be present in brain or CSF from patients with HAD. Apoptotic neurons do not co-localize with infected microglia in HAD patients [98], supporting the hypothesis the HIV infection causes neurodegeneration through the release of soluble factors. Therefore, the propensity for cell-cell interactions mandates that disease pathogenesis in vitro be approached in a ‘‘mixed’’ neuronal/glial primary culture system that recapitulates the type and proportion of cells normally found in the intact brain (Fig. 1). Systems designed to study the effect of soluble factors released from microglia have included mixed cerebrocortical cultures from human fetal brain directly infected with HIV [98], severe combined immunodeficiency (SCID) mice inoculated with HIV-infected human monocytes [99], and mixed rodent cerebrocortical cultures exposed to picomolar concentrations of the envelope protein HIV/gp120 [54,100,101]. Using such in vitro models, we and others have found evidence for a predominantly indirect neurotoxic effect that occurs due to the response of nonneuronal cells to HIV infection or shed HIV proteins, as described previously. Much of the data supporting the theory of indirect neuronal injury stems from experiments designed to examine the toxicity of HIV envelope proteins or supernatants of infected macrophages [41,79,102]. Picomolar concentrations of HIV/gp120 induce injury and apoptosis in primary rodent and human neurons [56,79,98,100]. In our hands, the predominant mode of HIV/gp120 neurotoxicity to cerebrocortical neurons requires the presence of macrophages/microglia [10,54]. HIV1–infected or gp120-stimulated mononuclear phagocytes release neurotoxins that stimulate the NMDAR, as described earlier. NMDAR antagonists can ameliorate neuronal cell death in vitro due to HIV-infected macrophages or purified recombinant gp120 [103,104], and in vivo in gp120 transgenic mice [105]. Excessive stimulation of the NMDAR induces several detrimental intracellular signals that contribute to neuronal cell death by apoptosis or necrosis, depending on the intensity of the initial insult (Fig. 2) [46]. If the initial excitotoxic insult is fulminant, the cells die early from loss of ionic homeostasis, resulting in acute swelling and lysis (necrosis). If the insult is more mild, neurons enter a delayed death pathway known as apoptosis [46]. Neuronal apoptosis after excitotoxic insult involves Ca2Ⳮ overload, p38 MAP kinase activation, release of cytochrome c from mitochondria, activation of caspases, free radical formation, lipid peroxidation, and chromatin condensation [106–108]. The scaffolding

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protein PSD-95 (postsynaptic density-95) links the principal subunit of the NMDAR (NR1) with neuronal nitric oxide synthase (nNOS), a Ca2Ⳮ-activated enzyme, and thus brings nNOS into close proximity to Ca2Ⳮ via the NMDAR-operated ion channel [109]. Excessive intracellular Ca2Ⳮ overstimulates nNOS and protein kinase cascades with consequent generation of deleterious levels of free radicals, including reactive oxygen species (ROS) and nitric oxide (NO) [110]. NO can react with ROS to form cytotoxic peroxynitrite (ONOOⳮ) [110]. However, in alternative redox states, NO can activate p21ras [111] and inhibit caspases [112] via S-nitrosylation (transfer of the NO group to critical cysteine thiols), thereby attenuating apoptosis in cerebrocortical neurons. In addition to the intracellular effects of NO, we have recently identified an extracellular proteolytic pathway to neuronal injury that is mediated by nitrosylation and subsequent activation of MMP-9 [93]. Proteolytically active MMP-9 induces neuronal death presumably by disrupting the cellular mechanism(s) that allow essential attachment to the extracellular matrix and neighboring neurons. Furthermore, we have found that neurons exposed to HIV/gp120 and grown in mixed cerebrocortical cultures containing astrocytes and microglia demonstrate release of mitochondrial cytochrome c, caspase activation, chromatin condensation, and apoptosis, which is blocked by inhibition of the p38 MAP kinase [54,113]. In addition to chemokines and EAAs, HIV-infected or gp120-activated microglia also release inflammatory cytokines, including TNF-␣ and IL-1␤. Among other actions, both of these cytokines stimulate release of L-cysteine from macrophages, and pharmacological blockade of IL-1␤ or antibody neutralization of TNF-␣ prevents this release [45]. Under physiological or pathophysiological conditions, L-cysteine can stimulate NMDARs and lead to neuronal apoptosis [45]. TNF-␣ is capable of stimulating apoptosis in human neurons [114], but an indirect route of injury cannot be excluded. Expression of TNF-␣ and its receptor are elevated in brain from patients with HAD [31]. Experiments aimed at addressing the question of interactions between neurotoxins associated with HAD have revealed that TNF-␣ and HIV/Tat synergize to promote neuronal death, and this effect is prevented by antioxidants [115]. It remains possible that TNF-␣ can activate caspases within neurons via TNF-␣ receptor-1 (TNFR1), since TNFR1 is found on at least some neurons, and it can trigger caspase-8 activation. Indeed, we have found that antibody neutralization of TNF-␣ or inhibition of caspase-8 prevents the neurotoxicity of HIV/ gp120 in cultured cerebrocortical neurons [113], and caspase-8 activity can directly or indirectly activate caspase-3, leading to apoptosis. These findings suggest that inflammatory cytokines, including TNF-␣ and IL-1␤, may have important synergistic roles in HIVassociated neuropathology [3]. Transgenic (tg) mice expressing HIV-1/gp120 in their CNS manifest neuropathological features that are similar to the findings in brains of AIDS patients, including reactive astrocytosis, increased number and activation of microglia, reduction of synapto-dendritic complexity, loss of large pyramidal neurons [84], and induction of MMP-2 [116]. In addition, these gp120 tg mice display significant behavioral deficits, such as extended escape latency, and reduced swimming velocity and spatial retention [117]. In gp120 tg mice, neuronal damage is ameliorated by the NMDAR antagonist memantine [105]. Memantine-treated gp120 tg and non-tg control mice retain a density of presynaptic terminals and dendrites that is similar to untreated non-tg/wild-type controls but significantly higher than in untreated gp120 tg animals [105]. This finding confirms the hypothesis that the HIV-1 surface glycoprotein is sufficient to initiate excitotoxic neuronal injury and

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death. It also shows that an antagonist of NMDAR overstimulation can ameliorate HIVassociated neuronal damage in vivo. IX. STRATEGIES FOR THERAPY OR PREVENTION OF HAD Presently, an effective pharmacotherapy for HAD is not available. Previous approaches to cope with HAD reflect the challenging complexity inherent in the treatment of patients with AIDS (reviewed in Refs 118 and 119). Previous and current therapeutic approaches include various antiretroviral compounds, alone or in combination: (1) reverse transcriptase inhibitors, including Zidovudine, Didanosine, Zalcitabine, Stavudine, and Lamivudine, and (2) protease inhibitors, such as Saquinavir, Ritonavir, and Indinavir. Of these, only Zidovudine has been shown to cross the blood-brain barrier to some extent, and Zidovudine has a beneficial effect on HAD, but the effect is not long-lasting. The other antiretroviral drugs may not penetrate the brain sufficiently to eradicate the virus in the CNS. Thus an adjunctive treatment besides antiretroviral drugs is needed. Based on the evolving pathogenesis of HAD described above, several potential therapeutic strategies to attenuate neuronal damage are worth exploring. Among others, agents warranting consideration include NMDAR blockers, chemokines, chemokine and cytokine receptor antagonists, p38 MAPK inhibitors, caspase inhibitors, and antioxidants (free radical scavengers or other inhibitors of excessive NO or ROS). NMDAR antagonists have been shown to attenuate neuronal damage due to either HIV-infected macrophages or HIV/gp120, both in vitro and in vivo. The open-channel blocker memantine prevents excessive NMDAR activity while sparing physiological function [103,120]. Also, unlike other NMDAR antagonists tested in clinical trials to date, memantine has proven both safe and effective in a number of phase III clinical trials for Alzheimer’s disease and vascular dementia. The results of a large, multicenter NIHsponsored clinical trial using this agent in patients with HAD are expected soon, and improved second-generation drugs are currently under development. Previous small clinical trials of a voltage-activated calcium channel blocker, nimodipine, and a PAF inhibitor suggested some therapeutic benefit but were not conclusive [121,122]. An additional clinical trial using the antioxidant drug selegiline is aimed at combating the effects of excitotoxicity by minimizing the impact of free radicals [123]. Chemokine receptors allow HIV-1 to enter cells and as such are major potential therapeutic targets in the fight against AIDS [50,124]. Antagonists of CXCR4 and CCR5 inhibit HIV-1 entry and are being assessed in clinical trials [50,124]. However, the benefit of inhibitors of chemokine receptors for HIV-associated neurological complications, although likely, remains to be shown [15]. Interestingly, certain chemokines have been shown to protect neurons, even though the virus does not productively infect neurons. In particular, ␤-chemokines and fractalkine prevent gp120-induced neuronal apoptosis in vitro [54,75,125], and, similarly, some ␤-chemokines can ameliorate NMDAR-mediated neurotoxicity [125]. Additionally, HIV-infected patients with higher CSF concentrations of MIP-1␣/␤ and RANTES performed better on neuropsychological measures then those with low or undetectable levels [64]. These findings support the hypothesis that selected ␤-chemokines may represent a potential treatment modality for HAD. Neuronal apoptosis appears to be one of the hallmarks of neurodegenerative diseases, including HAD [37]. Since caspases carry out the apoptotic program, caspase inhibitors may be helpful in preventing detrimental neuronal loss [126]. As detailed above, caspases have been implicated in HIV-related neuronal damage. However, caspase inhibitors are

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not currently available in a form deliverable to the CNS or targeted to degenerating neurons. With further advances in the caspase field, such drugs may be developed. Care must be exerted to avoid inhibitors that promote oncogenic processes or interrupt physiological circuits. Finally, p38 MAPK inhibitors have been shown to abrogate neuronal apoptosis due to excitotoxicity, HIV/gp120 exposure, or ␣-chemokine (SDF-1) toxicity [54,127]. The pharmaceutical industry is currently developing p38 inhibitors for a variety of inflammatory-and stress-related conditions, such as arthritis, and this may expedite trials for CNS indications such as HAD. One important lesson from HAD is that the synergy between the excitatory and inflammatory pathways to neuronal injury and death may, at least in part, be common to other CNS disorders including stroke, spinal cord injury, and Alzheimer’s disease. As such, the development of new therapeutic approaches for HAD is likely to have substantial impact on several other important neurodegenerative diseases. ACKNOWLEDGMENTS We sincerely apologize to our colleagues whose works we could not cite owing to space and reference limitations. M. K. is supported by the American Foundation for AIDS Research. S. A. L. is supported by the National Institutes of Health. S. A. L. is or has been a consultant to Allergan, Alcon, and Neurobiologicial Technologies, Inc. in the field of neuroprotective agents. REFERENCES 1. Piot P, Bartos M, Ghys PD, Walker N, Schwartlander B. The global impact of HIV/AIDS. Nature 2001; 410(6831):968–973. 2. McArthur JC. Neurologic manifestations of AIDS. Medicine (Baltimore) 1987; 66(6): 407–437. 3. Kaul M, Garden GA, Lipton SA. Pathways to neuronal injury and apoptosis in HIV-associated dementia. Nature 2001; 410(6831):988–994. 4. Dore GJ, Hoy JF, Mallal SA, Li Y, Mijch AM, French MA, Cooper DA, Kaldor JM. Trends in incidence of AIDS illnesses in Australia from 1983 to 1994: the Australian AIDS cohort. J AIDS Hum Retrovirol 1997; 16(1):39–43. 5. Ferrando S, van Gorp W, McElhiney M, Goggin K, Sewell M, Rabkin J. Highly active antiretroviral treatment in HIV infection: benefits for neuropsychological function. AIDS 1998; 12(8):F65–70. 6. Dore GJ, Correll PK, Li Y, Kaldor JM, Cooper DA, Brew BJ. Changes to AIDS dementia complex in the era of highly active antiretroviral therapy. AIDS 1999; 13(10):1249–1253. 7. Major EO, Rausch D, Marra C, Clifford D. HIV-associated dementia. Science 2000; 288(5465):440–442. 8. Clifford DB. Human immunodeficiency virus-associated dementia. Arch Neurol 2000; 57(3): 321–324. 9. Lipton SA. Treating AIDS dementia [letter; comment]. Science 1997; 276(5319):1629–1630. 10. Lipton SA, Gendelman HE. Seminars in medicine of the Beth Israel Hospital, Boston. Dementia associated with the acquired immunodeficiency syndrome. N Engl J Med 1995; 332(14): 934–940. 11. McArthur JC, Hoover DR, Bacellar H, Miller EN, Cohen BA, Becker JT, Graham NM, McArthur JH, Selnes OA, Jacobson LP, et al. Dementia in AIDS patients: incidence and risk factors. Multicenter AIDS Cohort Study. Neurology 1993; 43(11):2245–2252.

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Kaul and Lipton or nitric oxide/superoxide in cortical cell cultures. Proc Natl Acad Sci USA 1995; 92(16): 7162–7166. Meucci O, Fatatis A, Simen AA, Bushell TJ, Gray PW, Miller RJ. Chemokines regulate hippocampal neuronal signaling and gp120 neurotoxicity. Proc Natl Acad Sci USA 1998; 95(24):14500–14505. Liu Y, Jones M, Hingtgen CM, Bu G, Laribee N, Tanzi RE, Moir RD, Nath A, He JJ. Uptake of HIV-1 Tat protein mediated by low-density lipoprotein receptor-related protein disrupts the neuronal metabolic balance of the receptor ligands. Nat Med 2000; 6(12):1380–1387. He J, Chen Y, Farzan M, Choe H, Ohagen A, Gartner S, Busciglio J, Yang X, Hofmann W, Newman W, Mackay CR, Sodroski J, Gabuzda D. CCR3 and CCR5 are co-receptors for HIV-1 infection of microglia. Nature 1997; 385(6617):645–649. Michael NL, Moore JP. HIV-1 entry inhibitors: evading the issue [news] [see comments]. Nat Med 1999; 5(7):740–742. Ohagen A, Ghosh S, He J, Huang K, Chen Y, Yuan M, Osathanondh R, Gartner S, Shi B, Shaw G, Gabuzda D. Apoptosis induced by infection of primary brain cultures with diverse human immunodeficiency virus type 1 isolates: evidence for a role of the envelope. J Virol 1999; 73(2):897–906. Rottman JB, Ganley KP, Williams K, Wu L, Mackay CR, Ringler DJ. Cellular localization of the chemokine receptor CCR5. Correlation to cellular targets of HIV-1 infection. Am J Pathol 1997; 151(5):1341–1351. Zhang L, He T, Talal A, Wang G, Frankel SS, Ho DD. In vivo distribution of the human immunodeficiency virus/simian immunodeficiency virus coreceptors: CXCR4, CCR3, and CCR5. J Virol 1998; 72(6):5035–5045. Kaul M, Lipton SA. Chemokines and activated macrophages in gp120-induced neuronal apoptosis. Proc Natl Acad Sci USA 1999; 96:8212–8216. Sanders VJ, Pittman CA, White MG, Wang G, Wiley CA, Achim CL. Chemokines and receptors in HIV encephalitis. AIDS 1998; 12(9):1021–1026. Zheng J, Thylin MR, Ghorpade A, Xiong H, Persidsky Y, Cotter R, Niemann D, Che M, Zeng YC, Gelbard HA, Shepard RB, Swartz JM, Gendelman HE. Intracellular CXCR4 signaling, neuronal apoptosis and neuropathogenic mechanisms of HIV-1-associated dementia. J Neuroimmunol 1999; 98(2):185–200. McManus CM, Weidenheim K, Woodman SE, Nunez J, Hesselgesser J, Nath A, Berman JW. Chemokine and chemokine-receptor expression in human glial elements: induction by the HIV protein, Tat, and chemokine autoregulation. Am J Pathol 2000; 156(4):1441–1453. McGrath KE, Koniski AD, Maltby KM, McGann JK, Palis J. Embryonic expression and function of the chemokine SDF-1 and its receptor, CXCR4. Dev Biol 1999; 213(2):442–456. Gleichmann M, Gillen C, Czardybon M, Bosse F, Greiner-Petter R, Auer J, Muller HW. Cloning and characterization of SDF-1gamma, a novel SDF-1 chemokine transcript with developmentally regulated expression in the nervous system. Eur J Neurosci 2000; 12(6): 1857–1866. Stumm RK, Rummel J, Junker V, Culmsee C, Pfeiffer M, Krieglstein J, Hollt V, Schulz S. A dual role for the SDF-1/CXCR4 chemokine receptor system in adult brain: isoform-selective regulation of SDF-1 expression modulates CXCR4-dependent neuronal plasticity and cerebral leukocyte recruitment after focal ischemia. J Neurosci 2002; 22(14):5865–5878. Limatola C, Giovannelli A, Maggi L, Ragozzino D, Castellani L, Ciotti MT, Vacca F, Mercanti D, Santoni A, Eusebi F. SDF-1alpha-mediated modulation of synaptic transmission in rat cerebellum. Eur J Neurosci 2000; 12(7):2497–2504. Lazarini F, Casanova P, Tham TN, De Clercq E, Arenzana-Seisdedos F, Baleux F, DuboisDalcq M. Differential signalling of the chemokine receptor CXCR4 by stromal cell-derived factor 1 and the HIV glycoprotein in rat neurons and astrocytes. Eur J Neurosci 2000; 12(1): 117–125.

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33 Opioid Receptors and HIV Infection BURT M. SHARP University of Tennessee Health Science Center, Memphis, Tennessee, U.S.A

I. INTRODUCTION For more than two decades, the direct immunomodulatory effects of opiate alkyloids and opioid peptides have been recognized. For example, both ␤-endorphin and opioid peptide agonists selective for the delta subtype of opioid receptor (DOR) are known to modulate mitogen and T-cell receptor (TCR)–induced thymic and splenic T-cell proliferation and cytokine production [1–3]. Thus, selective DOR agonists, such as [D-Ala2-D-Leu5]-enkephalin (DADLE) and deltorphin, have been shown to attenuate the anti-CD3-ε–induced proliferation of highly purified murine splenic CD4Ⳮ and CD8Ⳮ T cells [2]. In similar experiments, higher concentrations (10ⳮ8 –10ⳮ6 M) of DADLE and deltorphin partially suppressed interleukin (IL)-2 production. Moreover, ␤-endorphin has been shown to amplify concanavalin-stimulated calcium mobilization by splenic T cells [4]. Although these studies provide pharmacological evidence for the presence of DORs on T cells, understanding the mechanism(s) underlying these and other functional effects has been hampered by the lack of direct evidence for the expression of DORs by immune cells. Recent studies have resolved this dilemma by demonstrating the expression of opioid receptor mRNAs and receptor proteins on lymphocytes and other cells involved in host defense and immunity. Interest in the immune effects of opiates and endogenous opioids has been engendered by the search for a deeper understanding of the pathogenesis of human acquired immunodeficiency syndrome (AIDS). This disorder is especially prevalent in drug-abusing populations that take drugs intravenously (iv). Although the use of HIV-contaminated needles may account for some of the increased prevalence of AIDS in drug-abusing populations, a wealth of research has suggested that iv drugs per se may modify the immune response to HIV. Indeed, one of the earliest studies in vitro showed that morphine dramatically increased HIV p24 antigen expression by co-cultures of normal and virally infected 693

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human peripheral blood mononuclear cells [5]. This review will focus on advances in our understanding of the expression of lymphoid opioid receptors and their effects on intracellular signaling and the expression of HIV-1. II. CLASSICAL AND ATYPICAL OPIOID RECEPTORS ON IMMUNE CELLS In a 1979 report, Wybran et al. made seminal observations regarding the direct effects of opiates and opioids on immune cells [6]. They showed that morphine and methionine enkephalin had direct, yet opposite, effects on the rosetting of human T cells to sheep red blood cells. Based on our present understanding, these effects indicate that opiates affect the CD2 receptor expressed by T cells. From these disparate functional effects of morphine and methionine enkephalin, Wybran drew the inference that different subtypes of opioid receptors are expressed by T cells. These receptors show preferential interactions with and confer different, and sometimes opposite, cellular responses to specific opiates and opioids. A. Identification of Classical Opioid Receptor Transcripts in the Immune System Opioid receptor transcripts for the ␮, ␦, and ␬ subtypes have been detected on immune cells using primarily reverse transcription with polymerase chain reaction (RT-PCR). First, DOR mRNA was identified in simian peripheral blood mononuclear cells [7]. Thereafter, human T-, B-, and monocyte cell lines and murine lymphocytic cell lines were shown to express DOR transcripts [8]. Our group [9] reported a 98% identity between the sequence of the PCR transcript amplified from 90% pure murine lymph node and splenic T cells (Balb/c) and the murine DOR mRNA originally reported in 1992 [10]. Freshly obtained murine splenocytes expressed very low levels of DOR mRNA that increased after splenocytes were cultured in the absence of mitogens [9,11]. Using RNA extracts from fresh Tcell–enriched populations (CD1) that were 85% positive for Thy-1, we subsequently observed approximately 1 DOR transcript per T cell by quantitative competitive RT-PCR amplification [12]. Thus, the very low basal expression of murine DOR transcripts in mature T cells may be common across many strains. Several laboratories have identified transcripts for ␮- and ␬-opioid receptors (MOR, KOR). MOR was detected in rat peritoneal macrophages and in human and simian peripheral blood mononuclear cells [13,14]; monocytes, granulocytes, and CD4Ⳮ T cells were MOR positive in humans [14] KOR transcripts were detected in human and monkey peripheral blood lymphocytes and in immature thymic CD4ⳮ CD8ⳮ T cells [15,16]. B. Regulation of Classical Opioid Receptor Transcript Expression Of the three opioid receptor subtypes, the regulation of DOR transcript expression has been studied most extensively. In the absence of mitogens, we found that cell culture increased DOR transcript expression by 10- and 20-fold after 24 and 48 hours, respectively [12]. In addition, DOR transcript expression increased linearly with increasing cell density when splenocytes were in culture for 48 hours [11]. To determine whether this induction was dependent on a soluble factor, supernatant transfer experiments were performed. Cells were cultured at relatively low density (1.2 ⳯ 106 cells/cm2) along with supernatants obtained from cells that had been cultured at either low or high density (3.0 ⳯ 106 cells/

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cm2). The expression of DOR mRNA was unaffected by supernatants from cells cultured at high density [11]. Therefore, cell-cell interactions, rather than a soluble factor, appear to mediate the enhanced expression of DOR transcripts by splenocytes cultured in the absence of mitogens. Two laboratories have reported that T-cell activation is associated with the enhanced expression of DOR transcripts. Stimulation with concanavalin A, a T-cell mitogen [17], or cross-linking the T-cell receptor (TCR) with anti-CD3-ε [12] increased DOR mRNA in murine CD4Ⳮ T cells. Using quantitative competitive PCR, our laboratory reported that anti-CD3-ε doubled the number of DOR transcripts per T cell in comparison to the effect of culture alone [12]. DOR copy number increased from 10 per T cell (culture alone) to 22 at 24 hours and from 20 to 42 at 48 hours. In experiments with actinomycin D, the effect of anti-CD3-ε on DOR transcription was determined indirectly. Actinomycin D did not affect the rate of transcript degradation (apparent half-life of 6 h) in antiCD3-ε–stimulated T cells, suggesting that stability was unaffected [12]. Hence, increased transcription appears to account for the anti-CD3-ε–induced expression of DOR mRNA. Anti-CD3-ε activates T cells through the early mobilization of intracellular calcium and stimulation of protein kinase C (PKC). Therefore, we studied the effects of these intracellular mediators on DOR transcript expression. Phorbol myristate acetate (PMA) in the presence or absence of ionomycin affected DOR transcript expression in a manner opposite to that observed with anti-CD3-ε. PMA alone or in combination with ionomycin inhibited the induction of T-cell DOR mRNA observed when splenocytes were cultured without mitogens [12]. This suppression was evident in T cells separated from splenocytes that had been cultured for 24 and 48 hours. In addition, PMA inhibited the anti-CD3ε–induced expression of DOR transcripts. The mouse DOR gene promoter region contains two sequences similar to the consensus TRE (phorbol ester–responsive element) and TRE-like elements. Although the function of these TRE elements is unknown, studies have shown that both of these elements can mediate activation [18,19], as well as inhibition [20] of gene expression, depending on the cellular context. Phorbol esters have been shown to inhibit gene transcription in a wide variety of cell types. For example, in HL-60 human myeloid leukocytes, PMA inhibited transcription of the P2U-purinergic nucleotide receptor, a 7-transmembrane G-protein–coupled receptor [21]. Since the activation of PKC by PMA inhibits DOR gene expression, but anti-CD3-ε (an activator of PKC) has the opposite effect, it is possible that other TCR-dependent intracellular effectors deliver a dominant positive signal(s), which enhances DOR expression. The activation of PKC by PMA may shift this balance, suggesting that different isoforms of PKC may be activated by PMA and/or the duration and magnitude of PKC activation by PMA may be significantly different from antiCD3-ε. Recent studies on the regulation of MOR and KOR transcript levels have shown that both are affected by morphine. Using competitive RT-PCR, increased MOR mRNA was detected in morphine-treated human lymphocytic cells [22]. In addition, treatment with morphine intensified a single band detected by Western blotting with an anti-MOR antibody at approximately 50 kDa. Morphine exerted similar effects on monkey lymphocytes [22]. Taking a similar approach with the CEM ⳯ 174 human lymphocytic cell line, morphine 10 ␮M was shown to enhance the expression of KOR mRNA within 24 hours of treatment [23]. The KOR-selective agonist U50,488H also induced KOR mRNA, and both were blocked by opioid receptor antagonists [23,24]. Thus, opiate alkyloids such

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as morphine may paradoxically upregulate the expression of lymphoid MOR and KOR transcripts through as yet undescribed mechanisms. C. Identification and Regulation of Classical Opioid Receptors (KOR and DOR) Using Indirect Fluorescence and Immunofluorescence Labeling KOR has been detected on murine thymocytes by fluorescence flow cytometry using a high-affinity agonist conjugated to fluorescein (FITC-AA) and amplified with biotinylated anti-fluorescein IgG and extravidin-R-phycoerythrin [25]. More than 50% of double-positive thymocytes (CD4Ⳮ/CD8Ⳮ) were KOR positive [26], and most of the thymocytes expressing KOR were double positive. In contrast, murine splenocytes expressed KOR on less that 25% of freshly obtained CD4Ⳮ T cells and 16% of B lymphocytes [27]. These findings suggest that T-cell maturation is associated with a decline in KOR expression. Recently, mitogenic stimulation in vitro was reported to increase KOR expression by splenic T cells; this was especially evident on the CD8Ⳮ subset [28]. Recent studies from our laboratory have identified DOR on splenocytes by immunofluorescence [29]. Balb/c V␤8.1 mice received a single injection of the superantigen staphylococcal enterotoxin B (SEB), which activates the TCR, and spleens were obtained at various time intervals thereafter. SEB enhanced both DOR mRNA and immunofluorescence, detected by epifluorescence microscopy with digital image analysis. Approximately 50% of the total T-cell population expressed DOR immunofluorescence within 15 hours of SEB treatment, compared to less than 10% of control T cells. DOR expression was elevated for 24 hours, then gradually declined toward control levels by 72 hours. Relative DOR immunofluorescence per cell increased approximately twofold in a subpopulation equivalent to 26.8 Ⳳ 8.6% of all T cells. In this subpopulation, fluorescence intensity was more than 2 standard deviations above the mean level in the control group. These studies indicate that DOR is expressed in vivo by T cells through a TCR-dependent mechanism. Using flow cytometry, we have shown that phytohemagglutinin (PHA) stimulated the expression of DOR immunofluorescence by human peripheral blood T cells [30]. By 48 hours 50% of the CD4Ⳮ and CD8Ⳮ T cells expressed DOR, and more than 90% of DOR was on these T cells. The cell surface markers CD45RA and CD45RO were used to assess whether DOR is present on naı¨ve or memory T cells, respectively [30]. PHA stimulated the expression of DOR in similar fractions of both CD45RA- and CD45ROpositive T cells. In additional studies (unpublished observations), we have shown that antiCD3-ε induced the expression of DOR immunofluorescence by CD4Ⳮ and CD8Ⳮ T cells obtained after splenocyte culture (Fig. 1). D. Identification and Characterization of Atypical Receptors for Opiates and Opioid Peptides on Immune Cells by Radioligand Binding Among the atypical sites, 3H-morphine binding to resting thymocytes has been reported with a relatively low affinity for morphine (Kd 艐 100 nM)[31]. Similar sites on human peripheral blood macrophages showed low-affinity, naloxone-insensitive binding for morphine, designated ␮3[32]. Lastly, our group characterized high-affinity, naloxone- and morphine-insensitive binding of ␤-endorphin on both murine splenocytes and U937 cells, a human promonocytic cell line [33,34]. ␤-Endorphin binding was saturable and sensitive

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Figure 1 Immunofluorescence detection of DOR on CD4Ⳮ and CD8Ⳮ human peripheral blood T cells. After 48 or 96 hours in culture with PHA 5 (␮g/mL) or vehicle (saline), PBMC were labeled with rabbit anti-DOR and mouse anti-h-CD4 or anti-h-CD8; normal rabbit sera (NRS) and CyChrome mouse IgG1, ␬ (Isotype), were used as controls. Anti-DOR was detected with a fluorescein avidin-biotin-antirabbit Ab complex, whereas anti-CD4 or -CD8 was detected with Cy-Chrome (directly conjugated). Cytofluorometric analyses were performed using an EPICS XL flow cytometer (Coulter) equipped with an argon laser, filtered for excitation at 488 nm and emission at 526 and 682 nm. (a) Representative distributions of cells positive for DOR and CD4 or CD8 immunofluorescence in the PBMC from 1 donor cultured with PHA or saline for 48 hours. The value within each quadrant is the percentage of the total cells analyzed in the experiment detected within that quadrant. In unstimulated PBMC, the fraction of cells positive for DOR/CD4 (right upper quadrant) was greater in those labeled with primary antisera than NRS/Isotype. PHA increased the fraction of cells expressing both DOR/CD4 and DOR/CD8. (b) Quantitative analysis of the fraction of PBMC positive for the indicated immunfluorescent labels in blood obtained from four donors (values are mean Ⳳ SEM). Fluorescence detected in the presence of NRS/Isotype was subtracted from the immunofluorescence signal emitted by the primary Ab. Compared to unstimulated cultures (saline), PHA 5 (␮g/ mL) significantly increased both the fraction of DORⳭ cells in PBMC and the fractions of double positive cells (i.e., DORⳭCD4Ⳮ) in both T-cell subsets at 48 hours. The effects of PHA were sustained at 96 hours in both T-cell subsets. Analysis of differences was performed with ANOVA (F ⳱ 52.5, p⬍0.0001 for DORⳭ; F ⳱ 51.1, p⬍0.0001 for DORⳭCD4Ⳮ; F ⳱ 27.1, p⬍0.0001 for DORⳭCD8Ⳮ). Group comparisons of the following treatments utilized Scheffe’s test: saline and PHA at the same time interval (*p⬍0.001), PHA at 48 hours and 96 hours (p ⬍ 0.05). (From Ref. 30.)

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Figure 1 Continued.

to cations, GTP-␥S, and incubation with PMA [33–35]. In contrast to neuronal opioid receptors, this ␤-endorphin–binding site required the C-terminus of ␤-endorphin (␤-endorphin28–31), and N-acetyl-␤-endorphin was virtually equipotent to ␤-endorphin. Thus, both morphine and ␤-endorphin can bind to lymphoid cells through naloxone-insensitive sites that are distinctly different and appear to modulate immune functions related to cellular proliferation [31,36,37]. E. Opioid Receptor–Mediated Intracellular Signaling in the Immune System In neuronal tissues, adenylyl cyclase is inhibited by all three opioid receptor subtypes. To avoid the problem of immune cell heterogeneity, cell lines often have been utilized to investigate these issues in lymphoid tissue. In the R1.1 thymoma cell line, the KOR inhibited basal and forskolin-stimulated cAMP production in a pertussis toxin–sensitive manner, consistent with coupling to Gi proteins [38]. Using a human Jurket-derived Tcell line that stably expresses DOR (DOR-Ju.1), we also found that DADLE (IC50 10ⳮ11 M) inhibited forskolin-stimulated cAMP production in a pertussis toxin–sensitive manner [39]. Finally, in studies with human peripheral blood lymphocytes, a biphasic effect of methionine enkephalin on intracellular cAMP concentrations was reported: low concentrations (e.g., 10ⳮ12 M) elevated cAMP within 15 minute, whereas nM concentrations reduced cAMP levels by 2 hours [40]. The effect of antagonists on these cAMP responses was not determined in normal lymphocytes, and, therefore, the opioid receptor specificity is unknown.

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Several laboratories have studied opioid receptor–induced phosphorylation of the mitogen–activated protein kinases (MAPKs) ERKs 1 and 2 in lymphoid cell lines. Two groups reported that the DORs expressed by DOR-Ju1.1 cells rapidly stimulate phosphorylation of ERKs 1 and 2 in a ras-independent manner [41,42]. The protein tyrosine kinase inhibitor herbimycin A reduced the DADLE-induced phosphorylation of ERKs 1 and 2 by 70% [41]. Furthermore, in CEM ⳯ 174 lymphocytic cells, morphine stimulated the expression of proteins involved in the MAPK cascade and increased both the expression and phosphorylation of ERK 1 and 2 [43]. Thus, in lymphoid cell lines, DOR and MOR are coupled through as yet undefined pathways, which activate ERK 1 and 2. In lymphoid cell lines, the effects of DORs on the phosphorylation of ERKs 1 and 2 differ from those reported in normal mature T cells. Recently, DADLE was shown to inhibit anti-CD3-ε–induced phosphorylation of ERKs 1 and 2 in murine splenic T cells [29]. DOR expression was induced in vivo with staphylococcal enterotoxin B, spleens were harvested, and then splenocytes were treated with DADLE prior to a 5-minute stimulation with anti-CD3-ε. In a concentration-dependent manner, DADLE (nM) reduced antiCD3-ε-stimulated ERK phosphorylation by as much as 50%, whereas DADLE alone was ineffective. Moreover, the kinetics of anti-CD3-ε–induced ERK phosphorylation were unaffected by DADLE [29]. Thus, DORs appear to inhibit anti-CD3-ε–induced ERK phosphorylation, rather than accelerating ERK dephosphorylation. These inhibitory actions of DORs all require preincubation with DOR agonists prior to TCR cross-linking. Recent investigations in our laboratory (unpublished) have focused on the effects of DADLE on the phosphorylation of activating transcription factor-2 (ATF-2), a member of the ATF/CREB family. It is known that stimulation by ultraviolet radiation or proinflammatory cytokines rapidly activates c-Jun N-terminal kinase (JNK) and induces the phosphorylation of ATF-2, which is necessary for its transcriptional activity [44,45]. ATF2 has been implicated in cytokine gene expression, such as tumor necrosis factor (TNF)␣, following engagement of lymphocyte antigen receptors [46]. Moreover, during the late stages of T- and B-lymphocyte activation, ATF-2 binding to DNA probes containing cAMP-response element (CRE) consensus sequences is markedly enhanced [47]. To induce DOR expression in our studies, murine splenocytes were stimulated with anti-CD3-ε in vitro and then rested. Thereafter, Western blotting was used to demonstrate that DADLE dose-dependently induced phosphorylation of ATF-2 (p-ATF-2). This effect was rapid and abolished by naltrindole, a DOR-specific antagonist. Anti-JNK immunoprecipitates also were prepared from DADLE-treated splenocytes. Western immunoblots of these immunoprecipitates, developed with anti-p-ATF-2, demonstrated that DADLE induced the phosphorylation of two p-ATF-2 bands, and the one at 71 kDa was most affected. Thus, in DADLE-stimulated splenocytes, JNK proteins are specifically associated with the most highly phosphorylated bands of p-ATF-2 (e.g., 71 kDa). These findings strongly suggest that JNK pathways are involved in mediating the DOR-dependent phosphorylation of ATF-2. Taken together with previous observations, these studies suggest that lymphocyte DORs can affect MAPK activation indirectly by modulating TCR-dependent stimulation (e.g., ERK1,2) and directly by TCR-independent stimulation (e.g., JNK). III. OPIOID RECEPTORS AFFECT HIV EXPRESSION IN PERIPHERAL BLOOD MONONUCLEAR CELLS All three opioid receptor subtypes are known to modify the expression of HIV in humans peripheral blood mononuclear cells (PBMC) infected with various HIV-1 isolates in vitro.

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Reminiscent of the original observations of Wybran, opposite effects on HIV expression have been reported, depending on the opioid receptor subtype. Whether HIV expression is amplified or suppressed, these effects uniformly require pretreatment of cell cultures with opiates or opioid agonists prior to the introduction of virus, suggesting that these ligands may alter the expression and/or function of key signaling intermediates or membrane receptors involved in early processes mediating viral entry. In addition to these intriguing observations made in vitro, a few studies indicate that MOR agonists modify the expression of lentiviruses (e.g., simian immunodeficiency virus and feline immunodeficiency virus) used to model in vivo HIV infections in other species. A. Effects of ␮-, ␦-, and ␬-Opioid Receptor Agonists on HIV Expression In Vitro Morphine was the first opioid receptor ligand shown to modify HIV expression [5]. Using a coculture assay consisting of phytohemagglutinin (PHA)-activated normal PBMC and cells infected with a viral isolate (HIV-1AT) from one patient, morphine pretreatment significantly enhanced the growth of HIV as reflected by the accumulation of p24 antigen in the medium. Interestingly, an inverted U–shaped dose response to morphine was reported, with maximal stimulation at 10ⳮ12 M. The potentiation by morphine was stereospecific and was blocked by naloxone and ␤-funaltrexamine, strongly suggesting the involvement of ␮-opioid receptors. Recently, morphine was shown to induce CCR5, a chemokine co-receptor for HIV, which also is used by simian immunodeficiency virus (e.g., SIVmac239) to enter mononuclear cells. In CEM ⳯ 174 human lymphocytic cells, enhanced CCR5 transcript and protein expression were detected within 12–24 hours of stimulation in the presence of 10 nM to 10 ␮M of morphine [48]. In addition, pretreatment with morphine increased indices of viral expression, such as syncytium formation and reverse transcriptase activity, in cells infected with SIVmac239. Similar observations were made after CEM ⳯ 174 cells were treated with methadone [49]. These findings provide a potential mechanism whereby morphine may promote HIV expression by increasing the number of CCR5 co-receptors available to facilitate viral uptake into immune cells. However, the effects of morphine on CXCR4, the principal chemokine co-receptor for T-tropic strains of HIV, remain to be determined (Fig. 2). DOR agonists were the first opiates/opioids reported to suppress HIV expression. Using DOR-transfected Jurkat cells (DOR-Ju.1), we observed that pretreatment with the DOR-specific peptide deltorphin or the DOR-specific alkaloid-like agonist benzamide—4-[[2,5-dimethyl-4-(2-propenyl)-1-piperazinyl](3-methoxyphenyl)methyl]N [2S[(S*),2a,5b]]-(9Cl) (SNC-80)—dose-dependently inhibited the production of p24 antigen after infection with HIV-1AT [50]. An inverted U–shaped dose response was evident, with maximal inhibition at 10ⳮ11 M SNC-80 or 10ⳮ15 –10ⳮ13 M deltorphin. By 10ⳮ7 M, no inhibition was evident with these ligands, and the DOR-specific antagonist naltrindole abolished these effects. The physiological significance of the foregoing observations was further evaluated by investigating the effects of pretreating normal human peripheral blood CD4Ⳮ T cells with SNC-80 prior to infection with two different strains of HIV [30]. Pretreatment for 24 hours, but not 1 hour, was effective at inhibiting p24 production by cells infected with the HIV T-tropic strain HIV-1AT or the monocytotropic strain SF162. Again, inverted U–shaped dose-response profiles were found, with maximal inhibition at 10ⳮ10 M SNC-

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Figure 2 Effect of SNC-80 on p24 Ag expression by human peripheral blood CD4Ⳮ T cells infected with (top) HIV-1 MN or (bottom) SF162. Purified activated CD4Ⳮ lymphocytes were incubated with the designated concentrations of SNC-80 for 1 hour prior to infection with the MN or SF162 strains of HIV-1 at a multiplicity of infection of 0.02. After 2 hours of incubation with HIV-1 at 37⬚, CD4Ⳮ lymphocytes were cultured for 3 days with PHA and SNC-80. Four subjects were studied, and the data are expressed as the mean % inhibition of p24 Ag expression (in the vehicle control groups, p24 Ag levels were 6683 Ⳳ 1537 pg/mL and 16,174 Ⳳ 460 pg/mL for the MN and SF162 strains, respectively) for the group. SNC-80 significantly suppressed p24 Ag expression by both strains of HIV-1. Statistical analyses were made by ANOVA (for HIV-1 MN and SF162, respectively: F ⳱ 37.1, p ⬍ 0.0001; F ⳱ 14.1, p⬍0.0001) and between-group comparisons of SNC-80 concentrations were performed with Fisher’s least significant difference (**p⬍0.0001 for SNC-80 10ⳮ10 M vs. 10ⳮ6 or 10ⳮ14 M; *p⬍0.01 for SNC-80 10ⳮ8 M vs. 10ⳮ6 M or 10ⳮ12 M vs. 10ⳮ14 M). (From Ref. 30.)

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80 against both HIV strains. Naltrindole 10ⳮ10 M uniformly abolished the effects of 10ⳮ10 M SNC-80. As previously discussed [30], the requirement for preincubation with DOR agonists and the suppressive effects of these agents on two strains of HIV suggest that early cellular events necessary for viral uptake or replication may be involved. These studies also suggest that adjunctive immunotherapy with DOR agonists may be beneficial in conjunction with antiviral therapy. KOR agonists have also been shown to suppress HIV expression in PBMC. Preincubation with the KOR-specific agonist U50,488 at 10ⳮ7 M maximally inhibited HIV p24 antigen production (approximately 60%) by activated CD4Ⳮ T cells [51]. The KORspecific antagonist nor-binaltorphimine (nor-BNI) blocked this. Using immunofluorescence, 34% of the CD4Ⳮ T cells were positive for KOR. In a subsequent study, these authors reported that U50,488 at 10ⳮ10 M maximally suppressed expression of the chemokine co-receptor CXCR4 on CD4Ⳮ T cells by approximately 45% [52]. In addition, U50,488 inhibited HIV-1 IIIB envelope glycoprotein–mediated membrane fusion with CD4Ⳮ T cells, and an inverted U–shaped dose-response profile was observed. Thus, KOR-dependent suppression of HIV expression is associated with reduced viral entry and expression of CXCR4 by CD4Ⳮ T cells. B. Effects of Morphine on Lentivirus Expression In Vivo There have been two published reports with sufficient subjects to provide insight into the effects of morphine on lentivirus expression in vivo. In a pilot study with six rhesus monkeys compared to historical controls, morphine 3 mg/kg was injected at consecutive 6-hour intervals for 2 years [53]. This dose is sufficient to avoid obvious signs of withdrawal in the interval between injections. The six subjects, who were successfully infected with SIVsmm9, were all surviving at 2 years in the absence of signs of wasting. This differs from the expected 2-year survival rate of approximately 50%. A well-controlled investigation using this regimen is currently ongoing (personal communication). In the second report, the weekend opiate abuser who is not dependent or addicted was modeled in the cat using feline immunodeficiency virus (FIV)[54]. Sixteen animals received vehicle, FIV, morphine, or the combination; morphine was delivered as a single injection per day for 2 consecutive days per week. The morphine regimen delayed FIVdependent progression of disease as indicated by delayed onset of lymph node enlargement, delayed or absent deficits in evoked auditory potentials and a trend toward diminished viral load. Taken together, these studies suggest that the schedule of morphine administration may be a critical determinant of the cellular and systemic response to pathogenic lentiviruses. Thus, when administered to avoid both dependency and multiple episodes of subsequent withdrawal, morphine may improve survival by unknown mechanisms.

IV. SUMMARY Multiple laboratories have reported clear evidence for the regulated expression of opioid receptors by cells involved in host defense and immunity. At both the protein and mRNA levels, there is especially good evidence for DOR and KOR on immune cells in several species. In the case of KOR, expression is greater in compartments (e.g., thymus) containing immature cells. Both DOR and KOR expression are enhanced in activated cells, and

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T cells demonstrate increased DOR expression as a function of increasing cell density, independent of TCR activation. Although DOR agonists can stimulate ERK phosphorylation in lymphoid cell lines, the opposite occurs in normal T cells. These agonists alone do not affect ERK phosphorylation; however, following preincubation, they inhibit antiCD3-ε–induced ERK phosphorylation. In addition, DOR agonists stimulated AFT-2 phosphorylation, apparently by activating JNK. These results suggest that lymphocyte DORs can affect MAPK activation indirectly by modulating TCR-dependent stimulation (e.g., ERK1,2) and directly by TCR-independent stimulation (e.g., JNK). In contrast to the stimulative effect of activated MORs, DORs and KORs suppress HIV expression in vivo in CD4Ⳮ T cells. The suppression by KORs is associated with the downregulation of chemokine CXCR4 expression and viral entry. Based on these observations, opioid receptors appear to be part of an immunomodulatory system that can respond to both opioid neuroand immunopeptides secreted locally by innervating neurons and immune cells, respectively. The existence of this system may offer novel targets for immunotherapy, such as opioid adjunctive treatment of AIDS in combination with antiviral therapy. REFERENCES 1. Linner KM, Quist HE, Sharp BM. Met-enkephalin-containing peptides encoded by proenkephalin A mRNA expressed in activated murine thymocytes inhibit thymocyte proliferation. J Immunol 1995; 154:5049–5060. 2. Shahabi NA, Sharp BM. Anti-proliferative effects of delta opioids on highly purified CD4Ⳮ and CD8Ⳮ murine T-cells. J Pharmacol Exp Ther 1995; 273:1105–1113. 3. Gilmore W, Weiner LP. Beta-endorphin enhances interleukin-2 (IL-2) production in murine lymphocytes. J Neuroimmunol 1988; 18:125–138. 4. Shahabi NA, Heagy W, Sharp BM. ␤-Endorphin enhances concanavalin-stimulated calcium mobilization by murine splenic T cells. Endocrinology 1996; 137:3386–3393. 5. Peterson PK, Sharp BM, Gekker G, Portoghese PS, Sannerud K, Balfour HH. Morphine promotes the growth of HIV-1 in human peripheral blood mononuclear cell cocultures. AIDS 1990; 4:869–873. 6. Wybran J, Appelboom T, Famaey J-P, Govaerts A. Suggestive evidence for receptors for morphine and methionine-enkephalin on normal human blood T-lymphocytes. J Immunol 1979; 123:1068–1070. 7. Chuang LF, Chuang TK, Killam KF, Chuang AJ, Kung HF, Yu L, Chuang RY. Delta opioid receptor gene expression in lymphocytes. Biochem Biophys Res Commun 1994; 202: 1291–1299. 8. Gaveriaux C, Peluso J, Simonin F, Laforet J, Kieffer B. Identification of kappa- and deltaopioid receptor transcripts in immune cells. FEBS Lett 1995; 369:272–276. 9. Sharp BM, Shahabi NA, McKean D, Li MD, McAllen K. Detection of basal levels and induction of delta opioid receptor mRNA in murine splenocytes. J Neuroimmunol 1997; 78:198–202. 10. Evans CJ, Keith DE, Morrison H, Magendzo K, Edwards RH. Cloning of a delta opioid receptor by functional expression. Science 1992; 258:1952–1955. 11. Sharp BM, Li MD, Matta SG, McAllen K, Shahabi NA. Expression of delta opioid receptors and transcripts by splenic T cells. In: Conti A , Maestroni GJM , McCann SM, Eds. Neuroimmunomodulation Perspectives at the New Millennium. Vol. 917. New York: Annals of the New York Academy of Sciences, 2000:764–770. 12. Li MD, McAllen K, Sharp BM. Regulation of delta opioid receptor expression by anti-CD3ε, PMA, and ionomycin in murine splenocytes and T cells. J Leukocyte Biol 1999; 65:707–714. 13. Sedqi M, Roy S, Ramakrishnan S, Elde R, Loh HH. Complementary DNA cloning of a ␮opioid receptor from rat peritoneal macrophages. Biochem Biophys Res Commun 1995; 209: 563–574.

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34 Mechanisms of Cytokine-Induced Sickness Behavior ROBERT DANTZER, JAN-PIETER KONSMAN, and PATRICIA PARNET INRA-INSERM U394, Bordeaux, France

I. INTRODUCTION The subjective feelings of sickness, in the form of malaise, lassitude, fatigue, numbness, coldness, muscle and joint aches, and reduced appetite, are well known to everyone who has experienced an episode of viral or bacterial infection. Due to their commonality, these symptoms are frequently ignored by physicians. They are considered as an uncomfortable, but rather banal, component of the pathogen-induced debilitation process that affects a sick individual. This view has turned out, however, to be incorrect. The psychological and behavioral components of sickness represent, together with the fever response and the associated neuroendocrine changes, a highly organized strategy of the organism to fight infection [1]. This strategy, referred to as ‘‘sickness behavior,’’ is triggered by the proinflammatory cytokines that are produced by activated cells of the innate immune system in contact with specific pathogen-associated molecular patterns (PAMPs). These cytokines include mainly interleukin-1 (IL-1␣ and IL-1␤), IL-6, and tumor necrosis factor (TNF)-␣. The mechanisms that mediate the behavioral effects of peripherally released cytokines on the brain have been elucidated over the last decade. IL-1 and other cytokines act on the brain via two communication pathways: (1) a neural route represented by the primary afferent neurons that innervate the body site where the infectious process takes place, and (2) a humoral pathway that involves the production of IL-1 by phagocytic cells in the circumventricular organs (CVOs) and choroid plexus in response to circulating PAMPs or cytokines, followed by its diffusion to brain target areas [2]. The objective of this chapter is to present the current knowledge about the way this communication system 707

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is organized and regulated and the implications of these advances for our understanding of brain physiology and pathology. II. PERIPHERAL PROINFLAMMATORY CYTOKINES AND CYTOKINE EXPRESSION IN THE BRAIN A. Origin of Peripheral Cytokines Infectious microorganisms that invade the body encounter a first line of defense represented by tissue macrophages and liver Kupffer cells. These phagocytic cells express pattern recognition receptors in the form of Toll-like receptors (TLR). TLRs are defined by the presence of a conserved cytoplasmic signaling domain, the Toll/IL-1 receptor homology domain, that signals via the nuclear transcription factor NF-␬B [3]. Peritoneal macrophages and Kupffer cells express TLR4s that recognize lipopolysaccharide (LPS), a component of the cell wall of gram-negative bacteria [4,5]. Gram-positive bacteria are recognized by TLR2Rs that are present on the same phagocytic cells. Activation of TLR4 by LPS results in the production of the proinflammatory cytokine IL-1 [6,7]. IL-1 is then able to induce its own synthesis and the synthesis of other cytokines potentiating its effect (TNF-␣ and IL-6) or antagonizing it (the so-called anti-inflammatory cytokines such as the specific antagonist of IL-1 receptors, IL-1Ra, and IL-10). Although proinflammatory cytokines are often thought of as hormones, they are to be distinguished from other intercellular communication signals. To be released by its cellular sources, a particular cytokine needs in general de novo synthesis. Once released, a typical cytokine is biologically active at nano- to picomolar concentrations and often act in a paracrine or autocrine manner on various target cells to exert a wide range of actions (pleiotropy). B. Peripheral Cytokines Induce Sickness Peripheral administration of IL-1 and/or TNF-␣ mimics all nonspecific symptoms of sickness, including fever, activation of the hypothalamic-pituitary-adrenal (HPA) axis, reduction of food intake and other behavioral activities, and withdrawal from the physical and social environment [8]. Conversely, administration of cytokine antagonists abrogates the physiological and behavioral effects of the cytokine inducer lipopolysaccharide. These findings indicate that proinflammatory cytokines mediate the clinical signs of the host response to infection. The physiological and behavioral changes that are characteristic of sickness appear to be mediated in the central nervous system (CNS). Fever, for instance, represents a regulated rise in body temperature due to increased thermogenesis and decreased thermal loss in response to an elevated setpoint for the regulation of body temperature. Given that body temperature setpoint is controlled by temperature-sensitive neurons in the preoptic hypothalamus, this indicates that IL-1 acts in the CNS to induce fever. In the same manner, glucocorticoid hormone release is controlled by adrenocorticotropin hormone (ACTH) secretion from the pituitary, which, in turn, is stimulated by corticotropin-releasing hormone (CRH) from the hypothalamus. The fact that prevention of CRH release or action inhibits the activation of the HPA axis in response to peripheral administration of IL-1 indicates that IL-1 also acts in the CNS to stimulate glucocorticoid release [9]. The proposed action of IL-1 in the CNS raises the question as to how peripheral IL-1 signals the brain. Like other proinflammatoy cytokines, IL-1 is a relatively large hydrophilic peptide that cannot cross the blood-brain barrier passively. In addition, IL-1 and other cytokines are considered as short-range communication molecules that act predomi-

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nantly in an autocrine or paracrine manner rather than in an hormonal manner. For this reason, several models of immune to brain communication have been proposed for the action of these cytokines on the nervous system, from the induction of prostaglandins in those brain areas that are devoid of a functional blood-brain barrier to the existence of specific transporters [10]. C. Neural Transmission of the Cytokine Message The hypothesis that cytokines act indirectly on the CNS by activating afferent nerves was based on the recognition that two of the cardinal signs of inflammation, calor (heat) and dolor (pain), are of sensory nature, which implies that inflammatory mediators released at the site of injury or infection are able to signal the brain. When cytokines or LS are injected into the abdominal cavity, they induce inflammation of the peritoneal wall. One of the major routes of visceral sensibility is represented by the afferent branches of the vagus nerves. These branches contain in their perineural sheath macrophages and dendritic cells that produce IL-1 in response to an intraperitoneal injection of LPS [11]. Sensory neurons of the vagus nerves express receptors to IL-1, and circulating IL-1 stimulates vagal sensory activity [12]. The role of the vagus nerves in the transmission of information from the periphery to the brain has been confirmed by vagotomy experiments in which the vagus nerves are sectioned under the diaphragm. Using this approach, vagal afferents have been shown to mediate the induction of sickness behavior as well as the neural activation of the brain stem, hypothalamus, and limbic structures in response to peripherally administered LPS and IL-1 [2,13,14]. The decreased response of vagotomized animals to proinflammatory cytokines is not due to an inability to mount a peripheral cytokine response, since vagotomy does not alter plasma levels of cytokines nor the ability of peritoneal macrophages to produce cytokines [15]. Furthermore, vagotomized animals are still able to develop a fullblown episode of sickness in response to IL-1 injected by other routes than intraperitoneally, including the subcutaneous, intravenous, and intracerebroventricular routes [16,17]. The importance of the neural pathway in the transmission of the immune message from the periphery to the brain is not the same for all components of sickness behavior. In particular, vagal afferents are less important for the cytokine-induced fever and activation of the hypothalamic-pituitary adrenal axis than for cytokine-induced sickness behavior since vagotomized rats did not develop the behavioral alterations characteristic of sickness while they were still able to mount a fever [14]. These findings indicate that other pathways of communication function in parallel with the neural pathway [2]. D. Humoral Transmission of the Cytokine Message A slower pathway of transmission from the immune system to the brain is represented by the production of molecular intermediates at the level of the blood-brain interface in response to circulating cytokines or PAMPs. Prostaglandins of the E2 series represent the main mediators of cytokine-induced fever and activation of the hypothalamic-pituitary adrenal (HPA) axis, since pretreatment with specific inhibitors of the prostaglandin synthesizing enzyme cyclooxygenase 2 (COX-2) attenuates these responses [18,19]. The synthesis of PGE2 is dependent on the induction of COX-2 and the enzyme prostaglandin E synthase, both of which are expressed in endothelial cells of cerebral blood vessels and possibly perivascular macrophages after intravenous IL-1␤ administration [20]. These small lipophilic compounds diffuse into the brain parenchyma and act on neuronal EP3

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or EP4 receptors in the brainstem and hypothalamic neural structures that are involved in the control of the HPA axis activity and the regulation of body temperature. These brain areas include the catecholaminergic brainstem nuclei, the paraventricular nucleus of the hypothalamus (PVN), and the ventromedial preoptic area (VMPO) (Fig. 1) [20–22]. The reduction in social behavior and the anorexia that develop in response to peripheral LPS and IL-1 are mediated by brain IL-1 since these responses are attenuated by intracerebroventricular administration of the IL-1 receptor antagonist [2,14,23]. In response to peripheral LPS, IL-1␤ is synthesized by macrophage-like cells in the circumventricular organs and choroid plexus where the blood-brain barrier is deficient [24]. This is certainly due to the action of circulating LPS on TLR-4 receptors that are present on the

Figure 1 Prostaglandin-dependent and -independent mechanisms in IL-1 actions in the brain. Note that in both cases the molecular intermediates represented, respectively, by PGE2 and IL-1 can activate their target structures directly or indirectly via distant neuronal pathways projecting to the target brain areas. The neuroendocrine activation by circulating IL-1 is mediated by prostaglandin activation of the PVN neurons that contain CRH and of noradrenergic A1 neurons that project to the neuroendocrine PVN, whereas the fever response to circulating IL-1 is mediated by PGE2dependent activation of the VMPO nucleus, which in turn activates descending projections of the PVN. Behavioral depression is mediated by IL-1 expressed by macrophage-like cells in the circumventricular organs and choroid plexus that activates limbic structures directly or indirectly via a neuronal pathway originating from the area postrema. AP: area postrema; BLA: basolateral amygdala; CeA: central amygdala; COX-2: cyclooxygenase-2: CP: choroid plexus; CVO: circumventricular organ; EP: prostaglandin receptors; MPO: medial preoptic area of the hypothalamus; PGE: porstaglandins of the E series; PVN: paraventricular nucleus of the hypothalamus.

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same cells. Another possibility is that IL-1 is produced in response to circulating cytokines. IL-1 can act on neuronal IL-1 receptors in the area postrema, the circumventricular organ of the brain stem [25]. This results in the activation of a neuronal pathway projecting to the parabrachial nucleus and from there to the central amygdala (CeA) and the bed nucleus of the stria terminalis (BNST). IL-1␤ may also propagate by volume transmission from the choroid plexus into the surrounding brain parenchyma to reach relatively distant structures such as the baso-lateral amygdala (BLA) that contains neurons expressing IL-1 receptors [26]. These two pathways, of which the respective importance remains to be elucidated, would be responsible for the behaviorally depressing effects of IL-1. In the same manner, diffusion of IL-1␤ from the median eminence to the arcuate nucleus would be responsible for cytokine-induced anorexia (Fig. 1). The fast neural pathway and the slow humoral pathway are likely to converge in a manner that is still unknown to promote the brain expression of IL-1 since electrical stimulation of the vagus nerve induces the expression of brain IL-1 [27], and vagotomy abrogates the induction of expression of brain IL-1 in response to intraperitoneal LPS and IL-1 [28,29]. The exact nature of the neuromediators responsible for the behavioral effects of IL-1 is still unknown. The effects of proinflammatory cytokines on brain neurotransmitters are grossly similar to those of other stressors, but this similarity breaks off at the regional level [30]. More detailed neuroanatomical studies are still required to identify the neurotransmitter content of those neuronal structures that are activated directly or indirectly by cytokines, so that the role of the putative neurotransmitter mediator can be assessed by micropharmacology intervention techniques.

III. MOLECULAR BASIS OF SICKNESS BEHAVIOR A. Role of IL-1 The availability of species-specific recombinant cytokines makes possible an assessment of the range of physiological and behavioral actions of the proinflammatory cytokines produced during an infectious episode using pharmacology experiments in which the cytokine under investigation is administered alone or in combination with other cytokines to healthy animals. IL-1 is an important cytokine for the induction of sickness behavior. Peripheral and central administration of IL-1␤ alone induces all the central components of the acute phase reaction, including fever, HPA axis activation, and behavioral depression [31]. In contrast, IL-6 has pyrogenic and corticotropic activities but no behavioral activity [32]. These findings do not imply that IL-1␤ is the sole cytokine that mediates sickness behavior. In accordance with the concept of a cytokine network, a given cytokine never acts alone, but in the context of other cytokines that potentiate or oppose its activity. IL-6, for instance, potentiates the behaviorally depressing effects of IL-1␤ [32]. Such complementary interactions between proinflammatory cytokines can be more easily addressed when one cytokine is missing from the cytokine network because the gene for this cytokine or its receptor has been deleted by the technique of homologous recombination. IL-6 knockout mice, for example, are less sensitive to the behavioral effects of LPS or IL-1␤ injected peripherally or centrally [33]. In the same manner, IL-1 type I receptor knockout mice are still responsive to the behaviorally depressing effects of LPS, whereas they no longer respond to peripheral or central IL-1 [34]. In these mice, the blockade of another proinflammatory cytokine, TNF-␣, by a fragment of its soluble receptor injected

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centrally abrogates the behaviorally depressing effects of LPS, whereas it has no effect in wild-type mice. As already mentioned, IL-1 appears to be the predominant mediator of sickness behavior in the brain since blockade of its action by central administration of the interleukin-1 receptor antagonist attenuates cytokine-induced sickness behavior measured by either depression of social exploration [35] or reduction of food intake [23,35]. The involvement of brain IL-1 in the depressing effects of LPS on food intake has been confirmed by an experiment using mice that are deficient in the IL-1␤–converting enzyme (ICE). This enzyme is also known as caspase-1, and it processes inactive pro-IL-1␤ into mature IL-1␤. ICE knockout mice are less sensitive to the depressing effects of LPS on food intake when LPS is injected into the lateral ventricle of the brain, whereas they do not differ from controls in their response to intraperitoneal LPS [36]. B. Receptor Mechanisms of the Effect of IL-1 in the Brain 1. IL-1 Receptors The effects of IL-1 on its cellular targets are mediated by several receptor subtypes featuring an extracellular domain with three immunoglobulin-like domains, a single transmembrane domain, and an intracellular domain that is more or less elaborated depending on the type of IL-1 receptor. The type I IL-1 receptor (IL-1RI) that mediates all of the known biological effects of IL-1 has a relatively long intracellular domain that is articulated with adapter proteins. The type II IL-1 receptor (IL-1RII) is a negative regulator of the IL-1 system and functions as a decoy receptor. Its intracellular domain is very short and has no signaling function. The additional IL-1 receptor accessory protein (IL-1 RacP) is necessary for IL-1 signal transduction since binding of IL-1 to the type I IL-1 receptor leads to the formation of a heterodimeric complex with this accessory protein, whereas binding of IL-1ra to the type I IL-1 receptor prevents the formation of this complex. This makes it possible to understand why IL-1RacP knockout mice are resistant to the effect of IL-1 both at the periphery and in the brain [37]. 2. Are Brain IL-1 Receptors the Same as Those Identified in Other Tissues? All biochemical techniques used so far to characterize IL-1Rs on brain cells have shown a striking similarity between brain IL-1 receptors and those found on peripheral immune and nonimmune cells. Most members of the IL-1Rs family have been cloned from transformed lines of blood cells. The descriptive work on the type and localization of IL-1 receptors present in the brain is based on autoradiographic detection of radioiodinated ligands such as IL-1␣, IL-1␤, or IL-1ra, polymerase chain reaction detection of a small fragment of the cDNA encoding IL-1Rs, and immunohistochemical detection with antibodies raised against epitopes of IL-1Rs from peripheral blood cells. Since these techniques do not allow one to conclusively determine whether brain IL-1Rs and peripheral IL-1Rs are the same, it can be assumed that the difficulty of cloning new brain-specific IL-1Rs indicates an equivalence between peripheral and brain receptors. In agreement with this assumption, detection of IL-1Rs in the brain using in situ hybridization or Rnase protection assay with full-length cDNA or long riboprobes [25] indicates that the same IL-1RI mRNA is present in peripheral and central nervous tissue. Moreover, autoradiography studies on knockout mice for IL-1RI have confirmed that this receptor is responsible for all IL1–binding sites in the mouse brain [34]. Yet this last result was unexpected in view of

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the presence of both IL-1RI and IL-1RII in the mouse brain. The most likely explanation for the lack of IL-1RII binding in the brain of IL-1RI knockout mice is the small amount of IL-1RII and the relatively low sensitivity of in vitro binding techniques. In agreement with this interpretation, immunohistochemistry studies have revealed the expression of IL-1RII but not IL-RI in the mouse hypothalamus [38], despite the fact that this expression was undetected with the use of binding techniques [39]. These results emphasize the need for multiple methods to study brain cytokine receptors in order to confirm their presence or absence in this organ. 3. Localization of Brain IL-1 Receptors In the rat, the very first study carried out to localize IL-1–binding sites made use of quantitative autoradiography to show that IL-1 receptors were widely spread across the brain, with the highest level in the granular layer of the dentate gyrus, the granule cell layer of the cerebellum, the hypothalamus, and the pyramidal cell layer of the hippocampus. These findings were interpreted as indicating a neuronal localization [40]. Seven years later, Ericsson et al. [25] used in situ hybridization with a 1.35 kb cDNA probe to determine the distribution of IL-1RI in rat brain. Their results can be compared to results from similar but less extensive studies [41,42]. IL-1RI mRNA expression was localized in nonneuronal cells in structures at the interface between the brain parenchyma and its fluid environments, such as the choroid plexus and the endothelial cells of the brain vasculature. Neuronal expression appeared mostly in the hippocampus and was also detected in a few cell groups of the basolateral nucleus of the amygdala and the basomedial nuclei of the hypothalamus. It was not possible to determine the exact nature of the few labeled cells observed in the arcuate nucleus and the area postrema. Compared with IL-1RI, much less is known about the expression of IL-1R2 mRNA in the rat brain, IL-1R2 mRNA appears to be undetectable in the normal adult brain but is induced in the dentate gyrus, the hippocampus, and the basolateral amygdaloid nucleus in response to a systemic injection of kainic acid. IL-1R2 mRNA can also be observed 24 hours later in neurons of the medial and median preoptic area, dorsomedial and paraventricular hypothalamic nuclei, and various thalamic nuclei [43]. In contrast with the relatively localized expression of IL-1RI and IL-1R2 mRNA, rat IL-1RacP mRNA detected by RNase protection assay is quite ubiquitous and expressed in large quantities in many brain regions (hypothalamus, cortex, hippocampus, and cerebellum) [44]. The presence of IL-1RacP in brain areas that are devoid of IL-1RI calls into question the exact role of this accessory protein in the rat brain. In the mouse, the first evidence for the presence of IL-1 receptors in the brain was obtained by demonstration of the expression of IL-1RI and IL-1R2 mRNA [45,46] as well as radioactive IL-1␣–binding affinity [47,48]. Later on, immunohistochemistry made it possible to confirm the protein expression and neuronal localization of the two subtypes of IL-1 receptors [49]. IL-1R1 and IL-1R2 were found to be expressed on neuronal soma in the granular cell layer of the dentate gyrus and the CA1-CA4 pyramidal cells fields of Ammon’s horn of the hippocampus. Similarly, both IL-1R isoforms are expressed on ependymal epithelial cells, choroid plexus epithelial cells, and Purkinje cells of the cerebellum. Interestingly, IL-R2 but not IL-R1 was detected on neuronal soma and proximal cell processes in the hypothalamic paraventricular grey matter. No clear evidence of immunolabeling on vascular endothelial cells and meninges was found. However, choroid plexus epithelial cells and ventricular ependidymal epithelial cells expressed easily detectable IL1R1 and IL-1R2, which is in accordance with their role in the entrance of IL-1 into the

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brain. Only limited immunoreactivity for IL-1R was detected on astroglial cells of normal adult mouse brain, although in mice previously infected with moloney murine leukemia, an abundant expression of IL-1R1 was observed on reactive astrocytes. IL-1RacP localization is similar in the mouse and rat brain [50,51]. Surprisingly, 125 I–IL-1 binding studies in IL-1RacP KO mice did not reveal a lower affinity of the binding sites, indicating that IL-1R1 and IL-1RacP do not necessarily form in this structure the heterodimeric complex that is normally required for high-affinity binding. Again, these findings argue for a not yet defined role of IL-1RacP in the rodent brain. 4. Functionality of Brain IL-1R Administration of IL-1␤ or IL-1␣ into the lateral ventricle of the brain or directly into the brain parenchyma induces the typical signs of sickness behavior. These effects are mediated by brain IL-1 receptors since they are abrogated by local administration of IL-1Ra into the brain [8]. In order to determine which subtype of IL-1 receptor mediates the behavioral effects of IL-1, passive immunization experiments, antisense technology, and mouse knockout strategies have been used. Blockade of IL-1RI with a specific neutralizing antibody totally abrogated the behavioral effects of centrally and peripherally injected IL-1␤ in mice [52]. Blockade of IL-1RII potentiated the suppressing effect of IL-1␤ on food intake [53]. Blockade of brain IL-1RI by antisense oligonucleotides abrogated the anorexic but not the adipsic effects of i.c.v. IL-1␤. IL-1RI–deficient mice were found to be no longer responsive to the behaviorally depressing effects of IL-1 injected at the periphery or directly into the brain. This was not due to an inability to mount a sickness response, since they were still responsive to LPS [34]. In the same manner, IL-1RacP knockout mice no longer responded to IL-1 injected into the lateral ventricle of the brain [37]. The signaling pathways that mediate the behavioral effects of IL-1 have not yet been elucidated. Since I␬-B␣ is strongly expressed in the CVOs and choroid plexus in conditions of peripheral immune stimulation [54], the effects of IL-1 at this level are likely to be dependent on the transcription factor NF-␬B. In accordance with this hypothesis, central administration of a NF-␬B inhibitor peptide was found to block the somnogenic and pyrogenic effects of peripheral IL-1␤ in rabbits [55]. The same is not necessarily true in the brain parenchyma, since activation of mitogen-activated protein (MAP) kinase pathways appears to be predominantly involved in the neural effects of IL-1 investigated so far. Those include IL-1␤–induced inhibition of long-term potentiation in perforant path–granule cell synapses [56] and IL-1␤–induced elevation in intracellular free calcium levels in rat cortical synaptosomes [57].

IV. ENDOGENOUS ANTI-INFLAMMATORY MOLECULES Cytokine-induced sickness behavior is normally a fully reversible phenomenon. This implies that the expression and action of cytokines in the brain are tightly regulated. The molecular mechanisms involved in this regulation have been elucidated (Fig. 2). In addition to their potent effects at the periphery, glucocorticoids potently down regulate the expression and action of cytokines in the brain [58]. However, many other molecules share a similar activity, including anti-inflammatory cytokines such as IL-10 [59] and peptides such as insulin-like growth factor-I [60], vasopressin [61,62], and ␣-melanocortin.

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Figure 2 Regulatory influences on the expression and action of proinflammatory cytokines in the brain. Solid arrows represent positive effects, whereas dotted arrows represent inhibitory influences.

V. PATHOPHYSIOLOGICAL IMPLICATIONS Based on the previous evidence, sickness behavior appears to be nothing other than the outward expression of a reversible episode of cytokine expression and action in the brain in response to peripheral immune stimulation. However, sickness behavior is more than that. Sickness behavior has motivational properties in the sense that it reorganizes the organism’s perception and action in a way that depends on the environmental constraints to deal as efficiently as possible with infection [8,63]. Much evidence points to fever and anorexia as effective disease-coping strategies. During parasitic infections, for instance, anorexia appears to promote an effective immune response in the host and allows it to become more selective in its diet, so as to select foods that either minimize the risk of infection or are the source of anti-infectious compounds [64]. In terms of mechanisms, this implies that cytokines, in addition to their direct effects on gastrointestinal motility and secretion [65], act in the hypothalamus to uncouple metabolic demands from the regulation of food intake. The exact manner in which this is achieved is still unknown, although recent evidence implicates the melanocortin system in this action [66]. Whatever the case, the resulting effect can become disastrous when anorexia and elevated energy expenditure last for too long or occur out of context, as is the case in HIV-associated wasting [67,68] and in cancer cachexia [69]. Because of their potent effects on mood and behavior, cytokines have also been proposed to play a causative role in the pathophysiology of depression [70]. Although many pieces are still missing from the puzzle, there is compelling evidence for an intersection between cytokine-induced sickness behavior and depression and even some leads for possible intermediate mechanisms, such as the induction of an enzyme known as indoleamine-2,3-dioxygenase that degrades tryptophan along the kynurenine pathway and therefore depletes brain serotonin.

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VI. CONCLUSION Sufficient evidence is now available to accept the concept that the brain recognizes cytokines as molecular signals of sickness. The action of both peripheral and central IL-1 induces sickness behavior and activation of limbic structures, albeit with different kinetics. Clarifying the way the brain processes information generated by the innate immune system has been accompanied by a progressive elucidation of the cellular and molecular components of the intricate system that mediates cytokine-induced sickness behavior. However, we are still far from understanding the whole. Among the hundreds of genes that proinflammatory cytokines can induce in their cellular targets, only a handful has been functionally examined. In addition, a dynamic view of the cellular interactions that occur at the brain sites of cytokine production and action is still lacking, together with a clarification of the mechanisms that favor the transition toward pathology.

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35 Immunotherapy of Neuroendocrine Tumors MATTHIAS SCHOTT, JOCHEN SEISSLER, and WERNER A. SCHERBAUM Heinrich-Heine University of Duesseldorf, Duesseldorf, Germany

I. INTRODUCTION Many malignant endocrine tumors are rare diseases with poor prognosis. Differentiated tumors are characterized by the expression and secretion of hormones of the origin tissue (corticotropin, calcitonin, parathyroid hormone, insulin, glucagon, or gut peptides) or the expression of neuroendocrine markers including chromogranin A, neuron-specific enolase, and synaptophysin [1–3]. The therapy of choice for these malignancies is radical surgical revision of the tumor masses. Despite progress in radiation and chemotherapy regimes, many metastatic forms remain incurable by conventional therapies. Developments in immunology within the last two decades have revealed increasing information about the molecular basis of tumor-host interactions. The convergence of information resulting from basic studies in cellular immunology, along with increasing sophistication in biotechnology, which has made biological reagents available in pharmacological amounts, has opened novel possibilities for the development of effective immunotherapies for patients with different kind of cancers, including neuroendocrine malignancies. This chapter will discuss new developments in cellular immunotherapies, e.g., dendritic cell–based protocols, gene therapy, as well as the introduction of cytokines and antibodies for the treatment of neuroendocrine malignancies. II. IMMUNOTHERAPY WITH DENDRITIC CELLS A. Dendritic Cells and Immunity Antigen-presenting cells (APCs) play a major role in the initiation of B- and T-lymphocyte responses. Among the APCs, dendritic cells (DCs) have been identified as the most impor721

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tant cell type that integrates signals from peripherial tissues and immune cells resulting in coordinated immune response of the innate and the adaptive immune systems. The activation of T lymphocytes depends on the binding of the T-cell receptor to its specific antigen presented by molecules of the major histocompatibility complex (MHC) (human leukocyte antigen, or HLA) and the binding of co-stimulatory molecules including CD28B7 or CD40-ligand-CD40 interactions. Both signals are exclusively provided by stimulated APCs [4–6]. HLA class I molecules that present endogeneous antigens are the prereqisite for the stimulation of CD8Ⳮ cytotoxic T lymphocytes (CTLs), whereas HLA class II present exogeneous antigens and activates CD4Ⳮ helper T (Th) cells. DCs are unique in that they possess the capability to present exogeneous antigens not only on class II but also on class I molecules. This explains that DCs can induce a strong Th-cell response and a CTL immune response as observed in virus infections (Fig. 1) [7–10]. Myeloid DCs including Langerhans cells and monocyte-derived DCs are the primary candidates as adjuvants for immunotherapies because they are highly efficient inducers of T-cell immunity depending on the cell maturation and activation [11]. In the immature state DCs have a high capacity to sample antigens in peripheral organs and present it in low levels without co-stimulatory factors. This results in T-cell inactivation and the

Figure 1 Interaction between dendritic cells and lymphocytes. Dendritic cells (DCs) express antigens on MHC class I and class II molecules to CD8-positive cytotoxic lymphocytes (CTLs) and CD4-positive T-helper (Th) lymphocytes. Activation of lymphocytes is dependent on the presence of costimulators such as CD40, CD80, and CD86 and the release of cytokines, especially IL-12. Interaction between DCs and Th cells further activates both cell types and increases their ability to stimulate CTLs, which then are licensed to kill tumor cells by a granzyme-dependent mechanism. In addition, DCs are able to stimulate natural killer (NK) cells that mediate lysis of tumor cells with downregulated MHC class I molecules.

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maintainence of peripheral tolerance. In the presence of proinflammatory signal, e.g., lipopolysaccharides or TNF-␣, DCs strongly upregulate the expression of HLA and costimulatory molecules [11,12], migrate to local lymph nodes or the spleen, provide antigens and stimulatory signals to naive and memory T cells, and secrete large amounts of stimulatory cytokines (IL-12, IL-6, TNF-␣) supporting the development of Th1 cells and CTLs [13–17]. Therefore, DCs may represent an optimal adjuvant for the induction of a strong CTL reaction required for antiviral or antitumor therapies. B. Dendritic Cell Vaccination In patients with established cancers, the immune system fails to recognize and destroy tumor cells. It is thought that most tumor cells are poorly antigenic by the downregulation of MHC class I molecules [18], the lack of expression of novel tumor antigens, and/or the active immunosuppression of effector cells of the innate and aquired immune system [19]. Some tumors have been shown to produce IL-10 or TGF-␤, both of which are involved in the downregulation of costimulatory molecules and the inhibition of DC maturation leading to a decreased T-cell stimulatory capacity [20–22]. Several in vitro studies have shown that vaccination with IL-10–pretreated DC prime Th2 cells decrease the activation of CTLs or even participate in the induction of immunosuppressive regulatory T cells or tumor antigen–specific T-cell anergy [23–26]. It is important to note that the in vivo anergy of T cells and the decreased immunostimulatory capacity of DCs can be recovered by in vitro generation and activation of DCs [27]. This has been shown using DCs generated from CD34Ⳮ bone marrow cells, leukapheresis, and peripheral blood monocytes [28–32]. It is possible to isolate large numbers of DCs by culturing in media supplemented with GM-CSF and IL-4, which can be activated by treatment with various stimuli such as TNF-␣ (Fig. 2), monocyte-conditioned medium, or immunostimmulatory DNA (CpG motifs) [31–34]. Several studies also demonstrate that DCs can be loaded with antigens by in vitro incubation with soluble antigen in the form of whole proteins, which need to be processed in the cell for the presentation on MHC molecules, or synthetic peptides which can directly bind to MHC class I molecules. Alternatively, antigens can be delivered to DCs using whole tumor lysates, undefined acideluted peptides from autologous tumors, tumor cell–derived mRNA, transfection with antigen-specific mRNA or cDNA, fusion of DCs with tumor cells or DCs transduced with retroviral and adenoviral vectors coding for tumor antigens. In animals these strategies were successfully used to induce protective and therapeutic antitumor response against various tumor types [35–42]. In humans it was also shown that antigen-loaded DCs possess a high immunogenic capacity. Dhodapkar and coworkers described a specific Th1 cell and CTL response after a single injection of tetanus toxoid or influenza matrix peptide-pulsed mature DCs in healthy volunteers [43]. However, in cancer patients tolerance or T-cell anergy may need to be overcome in order to elicit an efficient response. It may be necessary to generate fully matured, strongly activated DCs with high-level expression of MHC and costimulatory molecules as well as secretion of bioactive IL-12 to overcome the in vivo T-cell anergy and elicit a strong Th1 and CTL response [17,44]. In addition, the number of DCs and the antigen dose needs to be established for each antigen in order to avoid induction of anergy or tolerance by a low or a too high dose as well as high frequency of immunization [15]. The most important point in DC vaccination may be the choice of tumor antigen. In humans there are only few tumors such as papilloma-associated tumors, myeloma, or

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Figure 2 Dendritic cells. Microscopic picture of human, monocyte-derived mature dendritic cells after in vitro culturing with granulocyte-macrophage colony-stimulating factor, interleukin-4, and tumor necrosis factor-␣.

melanoma where tumor-specific antigens or shared antigens are identified [45]. To overcome this dilemma, several studies used antigens that are expressed only in cells from which the tumor has been derived (tumor-associated antigens) or whole tumor preparations. The use of one tumor protein or peptide may induce a specific immune response against a defined antigen. Whole tumor cell mRNA, tumor lysate, or cell fusion offer the possibility to widen the immune response against several tumor-associated or mutated antigens, which may reduce the risk that the tumor cells can escape the immune attack, but bears the risk of inducing a harmful autoimmune response against self antigens. Therefore, an optimal DC vaccination protocol needs to be established for each tumor type. Several clinical trials have been performed to assess the benefit and the side effects of this novel therapy. C. DC Immunotherapy in Nonendocrine Tumors During the past few years, clinical trials have documented the generation of antitumor immunity and clinical responses in hematopoietic tumor sarcoma and carcinoma. Most experience has been gained in patients with metastasized melanoma. Intranodal injection of DCs pulsed with a mixture of HLA-A2–restricted MART-1, gp100 and tyrosinase peptides or MAGE-1 and MAGE-3 peptides binding to HLA-A1 and/or tumor lysate

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results in a cellular immune response in 11 of 16 patients as assessed by delayed-type hypersensitivity (DTH) reactivity together with 2 complete and 2 partial responses of metastases [46]. Similar results were reported by Thurner and coworkers following a DC administration pulsed with a single (MAGE-3A1) peptide [47]. Other studies demonstrated a clinical response in only 2 of 14 and 3 of 16 patients using DCs pulsed with a pool of HLA class I restricted MAGE, Melan-A, Mart-1, tyrosinase, or gp100 peptides [48,49]. Immunological reactivities were seen in some patients as assessed by the measurement of melanoma peptide–specific DTH reactions, significant expansion of antigen-specific CTLs, and antigen-specific IFN-␥ production, respectively. Interestingly, Banchereau and coworkers showed that the clinical success correlates with the number of antigen-specific reactivities [50]. Because of the availability of several cell-specific antigens such as prostate-specific antigen (PSA) and prostate-specific membrane antigen (PSMA), prostate cancer represents a promising model for the use of cell-associated antigens as targets for DC immunization therapies. Tjoa and coworkers reported on partial tumor regression in 9 of 33 patients suffering from advanced disease stage after intravenous infusion of PSMA peptide pulsed DCs [51]. Other studies have confirmed the initial results of a clinical response in about one third of treated cases [52,53]. Interestingly, the latter study demonstates that immunization with larger dosages of DCs with a reduced number of infusions has the same efficacy as low doses applied more frequently, as has been done in previous studies [53]. An alternative approach to treat prostate cancer may represent the use of RNA for transfection of DCs. One study demonstrated the induction of polyclonal prostate cancer–specific CTLs in all patients treated with metastatic prostate cancer following stimulation with PSA mRNA transfected DCs and a transient clearance of circulating tumor cells in the peripherial blood [54]. Studies in patients with B-cell lymphoma reported on specific cellular responses and a complete remission in one patient after vaccination with DCs pulsed with idiotype protein (Id) [55]. Another report showed objective clinical responses in 4 of 18 patients with residual B-cell lymphoma after treatment with Id pulsed DCs [56]. In addition, alternative strategies for the loading of DCs with antigens have been analyzed in preclinical trials. Heiser and coworkers described the in vitro induction of tumor-specific CTLs against renal cancer cells after vaccination with DC transfected with tumor mRNA [57]. Other in vitro studies suggest that the fusion of autologous and allogenic DCs with tumor cells will induce the generation of specific CTLs which mediate the lysis of autologous human ovarian and breast cancer cells [58,59]. The side effects observed in all clinical trials were minor, including erythema, indurations and pain at the site of injection, low-grade fever, and bouts of sweating and chills. This indicates low toxicity of DC-based immunotherapies. Some melanoma studies reported on localized vitiligo and the induction of TSH-receptor antibodies or antinuclear antibodies. Vaccination-associated severe autoimmune reactions have not been observed thus far. D. Dendritic Cell Vaccination Studies in Endocrine Malignancies The majority of metastasized endocrine carcinomas are incurable by conventional radioand chemotherapy and, therefore, have a fatal outcome. New strategies for the treatment of these cancers are needed. The major problem for an immunotherapy of endocrine tumors is the lack of specific tumor antigens. This dilemma may be overcome by the use of tumor cell preparations (proteins or mRNA) or antigens specifically expressed in tumor cells

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(specific enzymes involved in the synthesis of hormones or polypeptide hormones). Both approaches were tested in pilot studies in patients with advanced disease stages with the aim to minimize the growth of metastatic lesions or to control the hormone activity. One patient suffered from a parathyroid hormone (PTH) carcinoma, which caused hypercalcemic symptoms including severe bone pain, profound weight loss, and extreme muscle weakness. A second patient had a metastasized neuroendocrine pancreas carcinoma (NEC) which was strongly positive for chromogranin A (ChA). After several rounds of vaccinations with tumor lysate (TL) pulsed DCs, both patients showed a dose-dependent proliferation of PBMCs towards yet unidentified antigen(s) within the tumor lysate (TL) and a positive DTH reactivity, suggesting the induction of TL-specific T lymphocytes. Immunohistochemical analysis of a skin biopsy in the patient with the NEC revealed an infiltration with CD4Ⳮ and CD8Ⳮ T lymphocytes, providing indirect evidence for the induction of tumor-specific cytotoxic immunity [60,61]. In this patient the therapy was accompanied by a steady decrease of the tumor marker ChA and a slight tumor regression. In the second patient serum PTH and calcium levels steadily increased. Therefore, the immunization protocol was changed and a synthetic PTH peptide was used for DC pulsing in combination with the T-helper antigen KLH [62]. Although the tumor masses did not decrease significantly, serum PTH declined following vaccination, suggesting a partial destruction of tumor cells. During follow-up, the patient still needed repeated bisphosphonate therapies, and she eventually died of pneumonia. Despite the devastating outcome, this case demonstrated for the first time the ability to induce cytotoxic immunity in an endocrine carcinoma using a polypeptide hormone as antigen [63]. These results prompted us to design a protocol for DC vaccination of patients with metastasized medullary thyroid carcinoma (MTC). MTC, arising from the parafollicular, calcitonin-producing C cells, represents an aggressive, usually slowly growing tumor occurring in both sporadic and familial forms such as multiple endocrine neoplasia type 2 (MEN 2) [64]. The tumor cells express and secrete the polypeptide hormone calcitonin as well as carcinoembryonic antigen (CEA), both of which are well-established tumor markers to monitor metastatic MTC. Seven patients were included in the study and immunized by subcutaneous injections of 2–5 ⳯ 105 DCs loaded either with CEA-peptide and calcitonin (n ⳱ 6) or calcitonin only (n ⳱ 1) [65]. After DC vaccination all patients developed a DTH reaction, which was found by immunohistochemistry to be mediated by the infiltration with CD4-positive and CD8-positive T lymphocytes. In addition, three patients developed a significant T-cell proliferation of peripheral blood lymphocytes in response to calcitonin and CEA. Posttreatment lymphocytes responded with high-level IFN-␥ secretion in some patients after stimulation with calcitonin or CEA, whereas IL-4 production was only slightly increased [66]. These data indicate the induction of a Th1dominated cellular immune response against calcitonin and CEA in the majority of patients. Clinical follow-up revealed that 3 of the 7 treated patients developed a clinical response with a decrease in the serum levels of calcitonin and CEA, and 3 patients had a stable disease. One patient who failed to develop a T-cell response to either calcitonin or CEA developed further tumor progression. Among the responders one subject rejected all radiologically detectable liver metastases and developed an almost complete regression of pulmonary metastases during the treatment period of 14 months (Fig. 3) [65]. During further follow-up, however, this patient again showed tumor progression with the appearance of novel metastates, indicating an only temporary success of the DC vaccination. It may be speculated that the tumor cells might have downregulated the tumor-associated antigens calcitonin and/or CEA or MHC class I molecules in order to escape the induced

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Figure 3 Clinical response to dendritic cell vaccination. CT scan of a patient with medullary thyroid carcinoma before (left) and 13 months after (right) initiation of dendritic cell vaccination shows complete rejection of liver metastases (top) and a near complete regression of pulmonary metastases (bottom). Arrows, sites of lesions before vaccination. (From Ref. 65.)

immune attack. This hypothesis needs to be investigated in future studies. Recently, another study reported on DC vaccination in 3 MTC patients using TL pulsed DCs. In all patients significant cytotoxic acitivity of T cells was observed against autologous tumor cells [67]. Although clinical responses were not described in the latter study, the data of both trials suggest that DC vaccination is sufficient to induce a cellular antitumor response and may provide benefit in some patients with advanced endocrine carcinomas. It is obvious that the immunization procedures must be optimized and standardized in future studies to improve the induction of a specific CTL response and successfully eliminate tumor cells. Clinical trials should also include patients with minimal disease, in whom it may be more feasible to obtain a clinically effective antitumor immunity. The current available data raise hope that within the next few years it may be possible to transfer DC-based vaccination of endocrine malignancies from an experimental approach to clinical practise. III. CYTOKINES FOR THE TREATMENT OF NEUROENDOCRINE TUMORS The use of cytokines may represent another possible approach to treat neuroendocrine carcinomas. Within the last decade different cytokines have been tested, of which interferon (IFN)-␣ and interleukin (IL)-2 might be the most important in this context.

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IFN-␣ has antiproliferative activity against neuroendocrine tumors through mechanisms different from and sometimes complementary to somatostatin analogs (as octreotide), which are also used as antiproliferative agents in neuroendocrine carcinomas. In fact, IFN-␣ increases the non–MHC-restricted cytotoxicity against tumor cells. It also increases the secretion of immunoglobulins at low doses by plasma cells and exerts a direct cytotoxic effect on tumor cells. The molecular mechanisms of the direct antitumor effect of IFN-␣ are still not completely clear. It has been suggested that IFN-␣ may deprive tumor cells of essential metabolites such as glucose or tryptophane. Moreover, IFN-␣ activates a series of transcriptional factors that elicit the arrest of DNA synthesis [68–71]. Different authors have used IFN-␣ for the treatment of neuroendocrine carcinomas, e.g., medullary thyroid carcinoma (MTC) or carcinoids. Bajetta et al. reported on the remarkable activity of IFN-␣-2a in the treatment of MTC and carcinoids, with an improvement in symptoms in about two third of treated patients as well as some partial and one complete response [72]. These results were further improved by a combination with somatostatin analogs, e.g., octreotide and slow-release lanreotide [73,74]. Using the same substances Frank and coworkers reported only prolonged survival in the responder group of patients with carcinoids, but no diminished mortality [75]. Another promising cytokine for the treatment of neuroendocrine tumors is IL-2. In vitro and in vivo studies have proposed the use of IL-2 in the treatment of MTC. Tumors caused by implanted MTC cells in animals can be inhibited in their growth and even eradicated by local secretion of IL-2. This cytokine leads to the development of antitumoral immunity by stimulating the proliferation of cytotoxic and helper T cells, causing the activation of natural killer cells and enhancing their cytolytic function as lymphokineactivated killer cells [76–79]. Conversely, severe side effects due to systemic administration of high doses of IL-2 have limited its clinical use in the treatment of cancer. In summary, these data demonstrate the ability of IL-2 to elicit specific antitumor immune response, which may in the future lead to a IL-2–based vaccine for the treatment of MTC. Such vaccine strategies are at present strictly experimental, but they open new options and thus deserve further investigation.

IV. GENE THERAPY A completely different way to induce antitumoral immunity involves gene therapy consisting of retroviral, adenoviral, RNA, or plasmid DNA transfection of eukaryotic cells. Up to now there are only a few reports on gene therapy for the treatment of neuroendocrine carcinomas. A principal advantage of DNA vaccines with immunostimulatory properties is that they can be easily produced and purified in large quantities. Once in cell nuclei, the plasmids persist as circular nonreplicating episomes; they are not integrated into the host’s genome, resulting in long-term expression of the encoded proteins by the host’s cells [80,81]. The major advantage of DNA vaccination is that the in vivo synthesized protein can enter both the MHC class I and class II processing pathways to favor effective antigenspecific cellular and humoral immunity. This approach has been shown to elicit in vivo immune responses against a broad range of infectious agents [82] and against certain tumor antigens, including B-cell lymphoma idiotype [83], an epitope derived from mutated p53 [84], free hCG -subunit [85], prostate-specific antigen [86], and melanoma-associated antigens such as gp100 [87] and gp75 [88].

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A German research group applied this method to a murine MTC model [89]. To prove the basic principle, the authors investigated whether DNA immunization based on gun-mediated gene transfer is able to induce cellular and humoral response against the human calcitonin (hCT) precurser preprocalcitonin (PPCT). This precursor was used instead of hCT because this larger molecule has a greater probability of containing suitable epitopes than mature hCT. Previous reports have also shown that the co-delivery of vectors encoding cytokines such as IL-2, IL-12, IFN-␥, or (GM-CSF) can direct the nature of the resulting immune response and augment the efficacy of DNA vaccines. Therefore, the authors used GM-CSF and IFN-␥ to enhance the immune response [90–92]. GM-CSF appears to be potent in augmenting cellular and humoral immune responses [93–95], possibly due to the differentiation of primitive hematopoietic precursors into dendritic cells [96], activating antigen-presenting cells [97], or increasing the expression of MHC class II molecules on antigen-presenting cells [98], thus enhancing their antigen-presenting ability. The authors were able to demonstrate PPCT-specific proliferative cellular and antibody responses to antigen-DNA vaccine delivered in the presence of the GM-CSF gene. Moreover, the group of animals immunized with hPPCT and GM-CSF expression plasmids not only developed higher levels of anti-hPPCT antibodies, but also showed an earlier time point of seroconversions compared with the group immunized with hPPCT DNA alone. In contrast to the results obtained with GM-CSF, co-delivery of IFN-␥ expression plasmid resulted in a decreased magnitude of antibody response against hPPCT compared with immunization with hPPCT expression plasmid alone. These findings confirmed previous reports on the regulation of the antibody response by IFN-␥ [92]. One explanation for this observation could be that IFN-␥ is a cytokine that tends to enhance Th1-like [99] and suppress Th2-like responses, leading to a decreased B-cell response. To overcome the aforementioned problem of systemic IL-2 administration, some investigators transfected IL-2–complementary DNA into tumor cells. The results have shown that IL-2–secreting tumor cells lose their tumorigenicity and induce an efficient immune response after implantation into syngeneic animals [76,77]. Retroviruses and adenoviral vectors may represent an alternative approach to the use of xenogeneic cells for local interleukin delivery. In small MTCs, intratumoral injection of replication-defective adenoviral vectors containing murine IL-2 complementary DNA induces tumor regression in two thirds of animals. The treatment of large tumors led to the stabilization of tumor size in ⬃70% of cases, but the procedure was not able to completely abolish them [78,79]. In a similar approach Yamazaki and coworkers also used a recombinant replication-defective adenoviral vector, which, however, expressed murine IL-12, driven by the CALC-I promotor [100]. The authors reported tumor regression in over 60% of animals within 3 weeks of immunization. A completely different model for achieving cell specificity for gene therapy was demonstrated by Minemura and coworkers, who showed rendered sensitivity to ganciclovir of human and rat tumor C cells following adenoviral transfection with herpes simplex virus thymidine kinase [101]. These results had, however, not yet been confirmed in in vivo studies. V. ANTIBODY-BASED IMMUNOTHERAPY Different trials have reported on encouraging in vivo results in patients with nonendocrine malignancies following treatment with monoclonal antibodies (mAb). Most convincing are data from patients with non-Hodgkin’s lymphoma receiving anti-CD20 mAbs who experienced better overall survival rates compared to patients treated with only conven-

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tional (chemo-)therapy [102]. The antitumor effector function of these unconjugated mAbs in cancer therapy is complex. Different mechanisms such as antibody-dependent cellular cytotoxicity and complement-mediated cytolysis as well as induction of apoptosis and cell cycle arrest have been observed [103,104]. In neuroendocrine carcinomas, only limited data are available. Zhang and DeGroot reported on the in vitro antitumor effect of calcitonin-specific mAbs on a rat MTC cell line shown by a decreased [3H]thymidine incorporation [105]. The antitumor effect was manifested by apoptosis and cell cycle arrest. This approach has not been tested in vivo in humans yet. A predominantly nonimmunological way to induce tumor cell destruction may involve the use of monoclonal antibodies labeled with radionucleotides. Pioneer work has been done using 131I-labeled anti-CEA (carcinoembryonic antigen) mAb for the treatment of MTC [106,107]. Behr and coworkers reported significant tumor regression and cure in a mouse model after combined radioimmunochemotherapy with 131I-labeled anti-CEA mAb [107]. Juweid and coworkers reported a median 55% reduction of tumor markers in 7 of 15 patients and a dramatic tumor mass regression in another patient followed by a radionucleotide-labeled anti-CEA mAb treatment [106]. The most important side effect of this treatment was myelosuppression in some patients. Up to now no additional data are available to determine whether this or a comparable mAb-based approach could be used in clinical routine for the treatment of patients with advanced MTC or other neuroendocrine carcinomas. A totally different way to induce a Th2-predominant immune response resulting in antigen-specific antibody production might be the use of Freund’s complete adjuvant, a water–in–mineral oil emulsion containing heat-killed Mycobacterium species. Usually this adjuvant is considered unacceptable for routine human vaccination bacause of granuloma and abscess formation at immunization sites [108]. It has, however, been used for tumor immunotherapy without serious problems in patients with lung cancer [109,110]. Abscess formation may be minimized by the use of small volume and intracutaneous injections. Bradwell and Harvey used this approach to treat one patient with a metastasized parathyroid carcinoma [62]. Within 4 weeks after initial vaccination with parathyroid hormone peptide together with Freund’s adjuvant, the patient developed high titers of specific antibodies together with a dramatic calcium decrease over 6 months of therapy. Even though this patient died during follow-up, this approach could be used to control excess hormone production in several types of neuroendocrine tumors.

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36 Glucocorticoid Resistance in Inflammatory Diseases DENIS FRANCHIMONT Erasme University Hospital, Brussels, Belgium GEORGE CHROUSOS National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, Maryland, U.S.A.

I. INTRODUCTION Despite the broad use of glucocorticoids in inflammatory diseases (Table 1), a major question remains unanswered: why some patients with inflammatory diseases respond well to glucocorticoids while others are refractory or need chronic glucocorticoid therapy to maintain their disease in remission. Indeed, some patients are steroid-sensitive (50–75%), while others are steroid-dependent (30–35%) or resistant (10–15%). Most steroid-dependent or steroid-resistant patients develop a cushingoid appearance or metabolic dysfunction, which suggests that their poor glucocorticoid sensitivity is limited to the immune system rather than involving nonimmune tissues. Recent studies have revealed that both the process of inflammation or the disease itself and the genetic and constitutional background of the patient may influence his or her variable response to glucocorticoids [1–4]. II. MECHANISMS OF CELL- AND TISSUE-SPECIFIC GLUCOCORTICOID RESISTANCE The clinical response to glucocorticoids can sometimes be correlated with in vitro leukocyte sensitivity to glucocorticoids. Several mechanisms of tissue glucocorticoid resistance have been described (Fig. 1). Thus, cytokines can decrease glucocorticoid sensitivity of 737

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Table 1 Glucocorticoid Therapy in Inflammatory/Autoimmune, Allergic, and Infectious Diseases Inflammatory diseases Rheumatoid arthritis Connective Diseases Lupus, Sjögren’s syndrome, scleroderma, and dermatomyositis Inflammatory bowel diseases Autoimmune hepatitis Multiple sclerosis Glomerulonephritis and nephritis Autoimmune thrombopenia Vasculitis Wegener’s granulomatosis Sarcoidosis

Allergic diseases

Infectious diseases

Allergic rhinitis Atopic dermatitis Eczema

Septic shock Acute respiratory distress syndrome

Asthma Anaphylactic shock Churg-Strauss syndrome Hypereosinophilia

Meningitis

tissues by interfering with the glucocorticoid signaling pathway. Glucocorticoid sensitivity seems to be modulated by cytokines in various in vitro studies. Some cytokines, such as interleukin (IL)-1␤, IL-6, tumor necrosis factor (TNF)-␣, interferon (IFN)-␥, IL-2, IL-4, and IL-13, modulate glucocorticoid receptor number and binding affinity with parallel changes of glucocorticoid sensitivity [5–11]. While glucocorticoid receptor (GR)␣ is the classic receptor that binds to glucocorticoids and transduces their biological activities, GR␤ does not bind to glucocorticoids and exerts weak dominant negative activity on GRmediated genomic actions [12]. Cytokines such as IL-2 and IL-4 have been shown to enhance GR␤ expression, contributing to a decreased glucocorticoid response following exposure to these cytokines [13–15]. Very interestingly, neutrophils demonstrate higher constitutive GR␤ expression than peripheral blood mononuclear cells (PBMCs), and this expression is further enhanced after IL-8 exposure [16]. This may explain the relative resistance of neutrophils to glucocorticoid-induced cell death and their enhanced survival in the presence of glucocorticoids during inflammation. Also, certain cytokine-stimulated transcription factors may interact in the nucleus with ligand-activated GR␣ and prevent ligand-activated GR␣ from binding to glucocorticoid responsive elements (GREs) or from exerting its transcriptional actions. Excessive nuclear factor (NF)-␬B, activator protein (AP)-1, and signal transducers and activators of transcription (STAT) expression and activity in an inflammatory site might inhibit glucocorticoid activity, contributing to a decrease in glucocorticoid sensitivity [17–19]. NF-␬B is a key regulator of the transcriptional activity of many cytokines. NF-␬B can be viewed as an intracellular amplification mechanism that exacerbates/sustains chronic inflammatory processes. Indeed, cytokines activate NF-␬B, leading to a positive feed-forward cycle at an inflammatory site [3,20]. Enhanced activation and expression of P65 inside the nucleus inhibits the ability of glucocorticoids to transactivate their GRE complementary DNA sequence [17,21]. Type II cytokines exert their cellular effects through the Jak/Stat signaling pathways. The transcription factor STAT5 of the type II cytokine receptors, such as IL-2 R␣ and IL-7 R␣, directly interacts with the glucocorticoid receptor so that the STAT5/GR␣ complex strongly suppresses the response of a GRE-containing promoter to glucocorticoids [19]. Cytokines

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may also directly target the glucocorticoid receptor through posttranslational mechanisms. Phosphorylation and dephosphorylation of the receptor may participate in the ligand activation/inactivation, recycling, and turnover of the receptor. Mitogen- activated phosphokinase (MAPK) family members, extracellular regulated kinases (ERK), and c-Jun Nterminal kinase (JNK) and p38 MAPK (p38) phosphorylate GR and its transcriptional activity [22,23]. This could represent an early repression effect of mitogenic and proinflammatory signals on the expression of GR-dependent genes. These examples help explain the ‘‘acquired’’ cytokine-induced glucocorticoid resistance during inflammation. Ligand-activated glucocorticoid receptors regulate the transcription of responsive genes by forming complexes with the recently discovered coregulators (i.e., coactivators or corepressors) of transcription and several chromatin modulators. These molecules not only alter chromatin structure but also enhance or inhibit the transduction of the trancriptional signal of the ligand-activated GR␣ to the general transcription complex, which includes RNA polymerase II (RNPII) and its ancillary factors [24,25]. The combinatorial interactions of coactivators and corepressors with the nuclear receptors may explain the cell and tissue selectivity of nuclear receptor actions [26]. Their relative levels may vary from tissue to tissue and can be stoichiometrically limiting between nuclear receptors and transcription factors in a particular tissue. A change in this flexible mix could lead to hypersensitivity and/or resistance to steroid hormones with hormonal and/or tissue predilection. It is fascinating that these coregulators not only enhance or repress nuclear receptor transcription but could undergo posttranslational modifications and integrate multiple signals from different signaling pathways such as the MAP kinase pathway.

Figure 1 Mechanisms of glucocorticoid resistance. GR ⳱ glucocorticoid receptor; GRE⳱glucocorticoid-responsive element; 11␤HSD⳱hydroxysteroid dehydrogenase; TF⳱transcription factor; STAT⳱signal transducer and activator of transcription; CREB⳱cAMP-responsive element-binding protein, P65 ⳱ nuclear factor-␤B subunit.

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Finally, the expression and activity of 11␤-hydroxysteroid dehydrogenase, a key enzyme in the regulation of intracellular glucocorticoid active metabolite concentration, may be modulated by cytokines, enhancing or decreasing glucocorticoid sensitivity [27]. Recently there has been a growing interest in the multidrug resistance (MDR)-1 gene product P-glycoprotein 170, which is a membrane-based drug efflux pump that transports MDR substrates, such as glucocorticoids, out of the cells, thereby lowering their intracellular active concentration. While independent of disease activity, P-glycoprotein 170 may play an important role in the response of inflammatory bowel disease and rheumatoid arthritis patients to glucocorticoid therapy [28,29]. III. GLUCOCORTICOID RESISTANCE IN INFLAMMATORY, ALLERGIC, AND AUTOIMMUNE DISEASES Glucocorticoid resistance in inflammatory diseases does not appear to be related to primary genetically determined glucocorticoid resistance even though a few mutations and polymorphisms have been described in familial glucocorticoid resistance and in the normal population [4,30–33]. Differential glucocorticoid-dependent transcriptional activation and repression have been observed among human glucocorticoid receptor variants associated with general glucocorticoid resistance [34]. The dexamethasone-induced repression of transcription from elements in the promoter of the intercellular adhesion molecule-1 via NF-␬B seemed in fact more efficient for the D641V variant GR than for the wild-type GR; however, the patients with this mutation did not have clinically significant immunosuppression. Five glucocorticoid receptor cDNA polymorphisms have been reported in the normal population, which were at variance with the original sequence reported by Hollenberg et al. [35,36]. In a population of 216 healthy subjects, a reduced response in a short dexamethasone suppression test as a marker of relative glucocorticoid insensitivity was observed in 20 otherwise healthy persons. However, this relative glucocorticoid insensitivity was not associated with any of these polymorphisms [37]. Nevertheless, this selected sample of the general population could represent the lowest section of a normal distibution of glucocorticoid sensitivity, defined by differences in the GR signal transduction system unrelated to GR polymorphisms. Finally, alterations of postreceptor mechanisms in the glucocorticoid signaling pathway and transcription machinery may account for resistance in some patients with familial glucocorticoid resistance [38]. An appropriate definition of dependence and resistance is necessary to tackle this major inflammatory disease issue with relevant clinical research protocols. A glucocorticoid-dependent patient responds normally to glucocorticoids but relapses once glucocorticoids are completely withdrawn and/or at dose tapering. A glucocorticoid-resistant patient does not respond to glucocorticoids. These two concepts are oftenly misused and are sometimes confusing. A. Asthma Glucocorticoid resistance in asthma is often associated with a decreased inhibition of peripheral blood mononuclear cell proliferation and cytokine secretion by glucocorticoids. In these patients, glucocorticoid resistance appears related to alterations of T-cell glucocorticoid receptor density and binding affinity and/or to changes in postreceptor mechanisms, while glucocorticoid receptor density and binding affinity in mononuclear cell subpopulation remain unaffected [39–41]. Hyperactivation of T lymphocytes with overexpression of

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IL-2R and HLA-DR reported by many groups in glucocorticoid-resistant patients suggests inflammation-induced glucocorticoid resistance [39,40]. Two different populations of steroid-resistant (SR) asthma patients have been described: Type 1, defined by decreased GR binding affinity, and Type 2, defined by decreased GR number per cell [15]. Type 1 SR asthma was shown to be reversible and secondary to inflammation, namely IL-2 and IL-4 dependent, while Type 2 SR asthma appeared to be genetically determined [15,41]. Resistance to glucocorticoid therapy could be due to a primary defect in the GR signal transduction pathway. However, no consistent GR polymorphisms have been reported to date in the hGR cDNA from glucocorticoid-sensititive and -resistant asthma patients [42–44]. The ability of the GR to bind to GREs seems impaired in glucocorticoid-resistant asthma patients because of a reduced number of receptors available for binding to DNA [45]. Indeed, increased levels of AP-1 DNA binding with increased basal and PMAstimulated protein levels of c-fos were recognized in glucocorticoid-resistant compared to glucocorticoid-sensititive asthma patients. Pretreatment of PBMCs from glucocorticoidresistant asthma patients with c-fos antisense oligonucleotides enhanced GR-DNA binding activity, suggesting that overexpression of c-fos was responsible for the decreased GRDNA binding observed in these patients [42,46]. Finally, recent studies have suggested that this decreased binding to DNA could be related to a markedly increased hGR␤/hGR␣ ratio such as observed in peripheral blood and bronchial lavage cells from these patients. These hGR␤/hGR␣ ratio changes appear reversible and cytokine inducible [13,14,42, 46,47]. B. Rheumatoid Arthritis The 50% decrease of GR number/cell reported in patients with rheumatoid arthritis did not appear to influence the in vitro glucocorticoid sensitivity of PBMCs from these patients [13,14,42,46–49]. However, leukocyte MDR-1 expression might affect glucocorticoid requirements for maintenance of remission in patients with systemic lupus erythematosus and influence the disease outcome in patients with rhumatoid arthritis [28]. C. Inflammatory Bowel Disease There is a lack of evidence of any change of glucocorticoid receptor number and binding affinity in the mucosa and/or in peripheral mononuclear cells from patients with inflammatory bowel disease [50]. Moreover, in contrast to glucocorticoid resistance, dependence in Crohn’s disease did not appear to be related to altered glucocorticoid sensitivity but rather to an excessive, although sensitive, ongoing inflammatory process [51]. A third of the patients with severe ulcerative colitis are resistant despite sustained intravenous glucocorticoid treatment. This resistance observed in ulcerative colitis is correlated with poor glucocorticoid sensitivity of proliferating peripheral blood lymphocytes [52,53]. This poor glucocorticoid response can be explained by a higher detection rate of hGR␤ expression in these glucocorticoid-resistant patients [54]. HGR␤ seemed to change over time in these patients, and its expression appeared reversible. HGR␤ could represent a possible predictive molecular marker of glucocorticoid resistance. Independent of disease activity, highly constitutive MDR (P-glycoprotein 170) expression appears to be associated with poor medical response to glucocorticoids in both Crohn’s disease and ulcerative colitis patients [29].

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D. Septic Shock and Acute Respiratory Distress Syndrome Loss of ability to downregulate proinflammatory cytokine production is an early event in the pathophysiological course of lethal acute respiratory distress syndrome (ARDS) [55,56]. Endogenous glucocorticoids do not always seem effective in suppressing lifethreatening systemic inflammation, even though the degree of cortisolemia frequently correlates with severity of illness and mortality rate [57,58]. Failure to suppress inflammation could be due to inadequacy of, and/or tissue resistance to, the levels and duration of endogenous cortisol elevations [59]. Recent randomized studies have shown that prolonged exogenous glucocorticoid administration—at doses that are clearly pharmacological for normal persons—compensate adequately for the inability of target organs to respond to glucocorticoids, restore glucocorticoid anti-inflammatory action, and, therefore, normalize glucocorticoid sensitivity [60]. Improvers had declining inflammatory cytokine levels over time, and cellular findings included a progressive rise in all aspects of GR␣-mediated activity and a concomitant reduction in NF-␬B–mediated activity (regulated inflammatory response). By contrast, nonimprovers had persistent and exaggerated elevation in plasma inflammatory cytokine levels over time, and cellular findings included only a modest increase in GR␣-mediated activity and a progressive escalation in NF-␬B activation over time (dysregulated inflammatory response) [60]. Indeed, the systemic inflammation-induced glucocorticoid resistance observed in patients with ARDS or septic shock returned to normal with prolonged GC treatment at moderate doses [59,61,62].

IV. PHARMACOGENETICS AND GENOMICS OF GLUCOCORTICOID THERAPY Physicians face major dilemmas on a day-to-day basis. When treating a patient with glucocorticoids, they cannot predict whether or not this patient will be suffering from a glucocorticoid-dependent or glucocorticoid-resistant inflammatory disease rather than a glucocorticoid-sensitive disease. First, they must be aware of the concomitant factors that may interfere with the diagnosis of glucocorticoid dependence and resistance, because glucocorticoid-sensitive disease may be mistaken for a glucocorticoid-dependent disease. These concomitant factors are disease-related complications, such as infections, taking certain drugs, environmental factors, inadequate doses or excessive catabolism of glucocorticoids, inefficient administration routes, and compliance of the patient. Second, physicians may consider the disease type, behavior, and severity when using glucocorticoids to treat patients. These relevant parameters are important because of the long-term adverse effects of glucocorticoids. As far as the disease type, there is a remarkable difference in the prevalence of steroid dependence and resistance even in similar inflammatory diseases (e.g., Crohn’s disease and ulcerative colitis). Also, a clinical response to glucocorticoids is to be expected from a specific disease behavior or clinical pattern [63,64]. Today, predicting glucocorticoid response would be possible on the basis of analysis of host genetic factors. There are approximately 1.4 million single nucleotide polymorphisms (SNPs) across the human genome, 60,000 of which may account for the variability observed in response to treatment with glucocorticoids. There are two complementary methods to look for SNPs associated with drug responsiveness: screening the SNP map of each patient based on SNP-linkage disequilibrium and analyzing candidate SNPs of selected genes known to be involved in a drug or disease pathway. For example, several groups have observed an increased prevalence of two 308-single-base-pair polymorphisms

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of the TNF-␣ promoters (TNF1 and TNF2) among glucocorticoid-sensitive and glucocorticoid-dependent patients suffering from autoimmune hepatitis or Crohn’s disease [65,66]. However, analyzing candidate SNPs may provide biased associations giving that several SNPs from a single gene interact with each other to produce a drug’s response phenotype. Therefore, combinations of SNPs (or haplotypes) will provide a much more predictable drug response. In the near future, pharmacogenetics research will give physicians access to the genetic and constitutional factors that influence treatment response. Although clinical and genetic information will be available before treating the patient with glucocorticoids, clinical evaluation of disease response to treatment is vague and not always accurate. Therefore, the biological profile and dynamics of the disease will offer a precise picture of the disease course and response. Microarray technologies and proteomics offer an opportunity to analyze global gene and protein expression in a timely manner. For example, the effect of glucocorticoids on the gene expression profile of peripheral blood mononuclear cells from healthy donors with DNA chip microarray has been determined [67]. This immediate picture of global gene regulation allows profiling of the glucocorticoid-mediated pharmacological effects. Also, specific molecular markers can be targeted for quick analysis of disease response given the magnitude or pattern of their regulation. Many potential molecular markers, such as the scavenger receptor CD163, have been identified and could be used for clinical application to predict response to glucocorticoids. Again, pharmacogenomics and proteomics may represent an invaluable tool in the decision and assessment of treatment response.

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Index

Acetylcholine immune role, 265, 266, 275, 278 and mast cells, 370 and myasthenia gravis, 571–572, 573–581 and ovarian failure, 555 Acquired immune deficiency syndrome (AIDS), 172, 177, 395, 702. See also Human immunodeficiency virus Acquired immunity, 7–8 Acromegaly, 84, 175 ACTH. See Adrenocorticotrophic hormone Actinomycin D, 695 Activating transcription factor–2 (ATF–2), 699 Activator protein (AP)–1, 56, 738 Acute phase proteins (APP), 23 Acute phase response (APR), 23 Adaptive immune response aging effect, 610 cell types, 4 description, 18–19 and GCs, 56–58, 67–72. See also Glucocorticoids and HLA complex, 421 and immunoglobulins, 5

[Adaptive immune response] and MHC, 9–10, 18 and neuroendocrine system, 23–24, 38, 90–91 and noradrenaline, 382 and sickness behavior, 707–715 and stress, 36–38 summary, 381–382 Th1 vs. Th2 cells, 658–664. See also Th1 cells; Th2 cells ADCC. See Antibody-dependent cellmediated cytotoxicity Addison’s disease calcifications, 506, 514, 515, 517 and diabetes insipidus, 447, 448 and gender, 500, 511, 513, 516, 517 high risk patients, 499 isohormonal therapy, 453–454 organ-specific autoantibodies, 452, 509 and ovarian failure, 538, 546, 550–551 primary, 491–519 and cellular immunity, 500–502 chronic vs. acute, 519–521 clinical entities, 513 clinical presentation, 519–521 747

748 [Addison’s disease] diagnosis, 521–523 etiology, 493 genetics, 517–519, 524 histopathology, 494–495 and humoral immunity, 495–497 hypogonadism, 500 isolated, 511–513 pathogenesis, 513, 517–519 polyglandular syndromes, 503–511, 513, 523–524 prevalence, 492–494 stages, 497–500 therapy, 523 secondary, 515–519, 520 and sodium, 521 and stress, 497 and syphilis, 515 and testosterone, 521 and thyroid-replacement therapy, 510 Adenosine, 658, 661, 664 Adenosine monophosphate. See Cyclic AMP Adenosine triphosphate (ATP), 598 Adenoviruses, 729 Adenylate cyclase, 24, 109–111 Adenylyl cyclase, 194, 659, 698 Adhesion. See also Intercellular adhesion molecule 1; Vascular adhesion molecule 1 dopamine, 270–273, 275 and glucocorticoids, 399 and glutamate, 278 and GnRH, 284 LE-CAMS, 13 and natriuretic peptide, 312–314 and oxytocin, 343 selectins, 13, 312–313 somatostatin, 199, 279 substance P, 282 and thymocytes, 326–327 and VIP, 294 Adhesion proteins, 13, 16 Adipose tissue, 59, 85. See also Leptin Adrenal cytoplasmic antibodies, 551 Adrenal glands in Addison’s disease, 494–497, 514–517. See also Addison’s disease and aging, 608, 613 bilateral massive hemorrhage (BMAE), 515

Index [Adrenal glands] congenital hyperplasia (CAH), 518–519 cytokine receptors, 90 and EAE, 400 genetic disorders, 517–519 and IL–1, 90 and leptin, 93–94 and mast cells, 366 and ovarian failure, 546 and rheumatoid arthritis, 594–596 and TNF-␣, 90 tumors, 515. See also Hypothalamicpituitary-adrenal axis Adrenal hypoplasia congenita (AHC), 517–518 Adrenaline. See Epinephrine Adrenergic blockade and dendritic cells, 383–383 and IL–10, 661 and mast cells, 364 and MSH, 633 Adrenergic nervous system and antigen presentation, 382–386 and atopic dermatitis, 635 ␤-agonists, 382, 651, 664 cytokine production, 386–390 and HIV, 659 ␤-Adrenergic receptors (␤2AR) and allergic reactions, 663–664 and atopic dermatitis, 635, 637 ␤AR–adenylyl cyclase–cAMP-PKA cascade, 659 and B cells, 276 and epinephrine, 266–267 and glucocorticoids, 399–400 and IgE, 664 and IL–1␤, 267 and multiple sclerosis, 661–662 and norepinephrine, 276 and pro-inflammatory cytokines, 653 and rheumatoid arthritis, 599, 662 and Th1 vs. Th2, 270, 650–651, 652 and tumors, 664 Adrenocortical insufficiency. See Addison’s disease Adrenocorticotrophic hormone (ACTH) and adaptive immune response, 23–24 Addison’s disease test, 502, 521 and aging women, 611 and atopic dermatitis, 632–634

Index [Adrenocorticotrophic hormone (ACTH)] and AVP, 441 and cytokines, 85, 88 and fever, 708 and glucocorticoids, 73 and inflammation, 53–54 and leptin, 92, 93, 94 as POMC, 25, 36 receptor-blocking antibodies, 500 resistance syndrome, 518 and rheumatoid arthritis, 594 and signaling pathways, 32 and vagus nerve, 88 Adrenoleukodystrophy, 517 Affinity maturation, 129 Age. See also Children and Addison’s disease, 444, 499, 504, 511, 513, 516, 517 and APS–2, 509 diabetes insipidus, 442, 444 diabetes mellitus, type 1, 424, 425, 427 and GH/IGF-I axis, 173, 174, 180 and T-cells, 558 of thymus, 324 Aging and adaptive immune response, 610 chronic inflammatory diseases, 612–613 evolutionary aspects, 607–608 and leukocytes, 611 of nervous system, 608–609 and norepinephrine, 608–609 and TNF, 610 Ag purified protein derivative (PPD), 74 Akt, 34 Aldosterone, 521 Allergen immunotherapy, 623 Allergic encephalomyelitis, experimental adrenalectomy effect, 400 animal model, 401–402, 543 and 1,25-dihydroxyvitamin D3, 151–152, 658 and glucocorticoids, 68–69 and stress, 661–662 Allergic reactions. See also Delayed-type hypersensitivity; Histamine; Rhinitis and CRH, 55, 655 and epinephrine, 634–635 and glucocorticoids, 58, 68–69, 72, 663–664 and IgE, 19, 623

749 [Allergic reactions] intestinal anaphylaxis, 370 and leukocytes, 3, 4 and mast cells, 365–366, 370, 655, 663–664 and stress, 55, 663–664 and substance P, 366 and Th2 response, 227, 663–664 and tryptase, 362 and VIP, 295–296, 640 Alopecia areata, 506, 509 Alzheimer’s disease, 369, 684 Amino acids, 264 Aminoglutethimide, 516 Amyloidosis, 516 Analgesia, 53, 55, 599 Androstenedione, 596, 597, 610 Anemia. See Pernicious anemia Angiogenesis, 200 Animal models acute arthritis, 90 for atopic dermatitis, 632–633, 641 autoimmunity pathogenesis, 547–550 BB-DP rat, 540–541, 550 Hashimoto thyroiditis, 68, 542–543 for HIV, 172, 682 of inflammatory disease, 401–402 Lewis rat, 543, 662 for lupus, 68 for multiple sclerosis, 68 NOD mouse, 542, 546, 550 obese-strain chickens, 542–543, 550 for opiate abusers, 702 for ovarian failure, 543–550, 554 for stress, 542–543 transgenic and knockout, 546–547 Antalarmin, 55, 84 Antibodies. See also Autoantibodies; Humoral immune response; Immunoglobulins ACTH-receptor-blocking, 500 adrenal cortex, 551 adrenal cytoplasmic, 551 antinuclear, 555 antityrosine hydroxylase, 506 AVP, 444–447, 451–453 to dsDNA, 130 to extractable nuclear antigens, 506 lupus, 130 monoclonal, 729–730

750 [Antibodies] parietal cell, 506 steroid producing, 500, 506, 551–552, 559 Antibody-dependent cell-mediated cytotoxicity (ADCC) immunoglobulin role, 5 NK cell role, 4 Antidiuretic hormone (ADH). See Arginine vasopressin Antigen binding sites, 11 Antigenic competition, 67 Antigen presentation, and glucocorticoids, 56–57, 70 Antigen presenting cells (APC) and vitamin D3, 656–658 Antigen-presenting cells (APC) adrenergic regulation, 382–386 in allergic reactions, 663–664 and catecholamines, 650 and glucocorticoids, 648 and histamine, 652 and MHC proteins, 11, 19 monocyte-derived, 559 thymic vs. peripheral, 343 thyroid cells as, 469 and tumor immunity, 391 types. 4. See also Dendritic cells; Macrophages; Monocytes Langerhans cells, 382–386, 390, 634 and VIP/PACAP, 292, 296–297 and vitamin D3, 146–148 Antigen presenting proteins, 1, 9–11 Antigens in antitumor vaccine, 723–724, 725–726 autoand APS–1 and APS–2, 552 diabetes, 422–424, 433 thyroid, 462–463, 467–468 vs. self, 340–349 in cancer immunotherapy, 725 cluster differentiation (CD), 339, 342 cytotoxic T-lymphocyte antigen–4, 510–511 extractable nuclear (ENA), 506 HIV p24, 693–694, 700 and HLA complex, 421 melanoma, 728 and myasthenia gravis, 571, 580 p24, 693–694, 700 with repeating haptens, 3 self-, 340–342, 390 tumor-associated, 390–391

Index Antigen specificity and B-cell receptor, 2 devlopment of, 15 and glutamate, 266 Antigen specific memory cells, 297 Antigen-specific therapy, for thyroid disorders, 476 Antihistamines, 622, 652, 664 Antiinflammatories, 250. See also Pituitary adenylate cyclase activating polypeptide; Vasoactive intestinal peptide CRH, 633 and glucocorticoids, 399 IGF–2, 348 listing, 714 melanocyte-stimulating hormone, 633 and natriuretic peptides, 308–311 neurotransmitters, 598 norepinephrine and cortisol, 599 and pregnancy, 663 Antinuclear antibodies, 555 Anti-phospholipid syndrome, 516 Antipsychotic drugs, 221 Anxiety, 24, 390 AP–1, 397 Apoptosis of dendritic cells, 151 and diabetes, 424 and dopamine, 276 estrogen effect, 598 Fas-mediated, 174, 176, 424, 464, 640 glucocorticoid-induced, 219–220, 324–325, 738 and IGF-I, 165, 179 inhibition of, 26, 31, 34, 70 of lymphocytes, 19, 219–220. See also under B lymphocytes; T lymphocytes T-helper cells, 297 of macrophages, 598 of neurons, 395, 675–678, 681–684 of neutrophils, 175, 179 noradrenaline effect, 276 and prolactin, 215, 219, 324 TCR-induced, 74 testosterone effect, 598 of thymocytes, 325 thyroid cells, 463–464, 469 and tolerance, 19 and VIP, 324–325 VIP/PACAP, 297–298, 640 and vitamin D3, 151

Index Appetite loss, 710, 715 APS–1. See Autoimmune polyglandular syndromes APS–2. See Autoimmune polyglandular syndromes APS–4, 511 Arachidonic acid, 677, 679 Arginine vasopressin (AVP) antibodies, 444–447, 451–453 and CRH, 53 and HPA axis, 52 secretion, 439–442 Aromatase, 597 Arthritis. See also Rheumatoid arthritis and genes, 402 and HPA axis, 90 and tryptase inhibitors, 362 and vitamin D3, 151 Asplenia, 506 Asthma and CRH, 55 and glucocorticoid receptor, 404, 740–741 and leukotrienes, 624 and mast cells, 55, 365–366 quality of life, 619, 620–621, 623–624 and Th2 response, 663 and tryptase inhibitors, 362 Astrocytes, 680 Activating transcription factor–2 (ATF–2), 699 Atherosclerosis, 309–311, 315, 612 Atopic dermatitis animal models, 632–633, 641 and catecholamines, 634–636 corticotropin/proopiomelanocortin, 632–634 melatonin, 636–637 and monocytes, 633, 635 and neurons, 638, 639–640 neurotrophins, 639 and norepinephrine, 633, 635 and selectins, 633 and stress, 631, 633–634 and substance P, 637–639 symptoms, 631, 635 and VIP, 638, 640 Adenosine triphosphate (ATP), 598 Atrial natriuretic peptide (ANP) and adhesion, 311–314 immune system links, 307–308

751 [Atrial natriuretic peptide (ANP)] and inflammation, 306, 309–311 and phosphorylation, 310 and pituitary gland, 23 receptors, 306, 309, 312 Atropine, 364 Autoantibodies and Addison’s disease, 452, 495–499, 509, 517, 523, 551, 553–554 in APS–1 and APS–2, 509, 551–551 glutamic decarboxylase (GAD), 420, 452, 506 liver-kidney, 506 and myasthenia gravis, 571, 573, 575, 580, 582 nonpathogenic causes, 551–552 and ovarian failure, 545, 554, 555–558 to parathyroid gland, 504 parietal cell, 506, 555 receptor, 557–558, 571, 573 steroid-producing (StCAs), 500, 506, 551–552, 559 and thymomas, 580 thyroid, 463, 467, 470, 557 tryptophan hydroxylase, 506 Autoimmune disorders. See also Autoimmune polyglandular syndromes; Comorbidity animal models, 539–550 pathogenesis blueprint, 547–550 and apoptosis, 19 and B–1 cells, 17 comorbidity, 448 criteria, 427, 444, 494 and estrogen, 130–132, 472–473 and Fas/FasL pathway, 19 gastrointestinal, 506 and gender, 426, 444, 504, 551. See also Addison’s disease and glucocorticoids, 399–404 and heat shock proteins, 433, 573 organ-specific, 447–448, 452, 465, 547–550 hepatitis, 506, 508, 509, 743 versus systemic, 537 and prolactin, 222–225 and somatostatin, 200 and TH3 cells, 19 and Th2 response, 661 and thymus, 345 and VIP/PACAP, 248–255 and vitamin D3, 148–151

752 Autoimmune lymphoproliferative syndrome (ALPS), 19 Autoimmune polyglandular syndromes (APS) APS–1, 503–509, 513, 523–524 animal model, 547 APS–2, 509–511, 513, 523–524 APS–4, 511 and ovarian failure, 551 Autoimmunity. See also Self-tolerance and B-cells, 15, 129, 134–135, 137 and dendritic cells, 469, 474, 547–548, 550, 559 endocrine-based, 547–550 genetics, 343, 462, 471–472, 475 and HLA complex, 469. See also Human leukocyte antigen complex and inflammation, 548 and interleukins, 661 and macrophages, 469, 470, 547–548, 550 and MHC, 473, 475, 547, 550 and T-cells, 340–342 and testosterone, 131, 472–473 and T-helper cells, 548, 661 of thyroid. See Thyroid, autoimmune and TNF-␣, 661 Autonomic nervous system, 608–609. See also Sympathetic nervous system Avidity model, 342 AVP. See Arginine vasopressin Azidothymidine (AZT), 172, 219 B7.1. See CD80 B7.2. See CD86 Bacterial infections and Addison’s disease, 513–515 and autoimmunity, 547 and complement proteins, 9, 16 endotoxin tolerance, 94–95 gram-negative, 708 and IL–12, 6 intracellular, 18 and leukocytes, 3 and myasthenia gravis, 577 mycobacteria, 6, 659 and thyroid, 473 Bad, 34 Barbiturates, 516 Basal ganglia, 506 Basophils and glucocorticoids, 58

Index [Basophils] and IgE, 5 leukemic, 368 and priming, 362 vasoactive amines, 4 BB-DP rat, 540–541, 550 B-cell lymphoma, 725, 728 B-cell receptors (BCRs) and immunoglobulins, 5, 128 role, 2–3, 11–12, 19, 128 Bcl–2 and autoreactive B-cells, 129, 135, 137 and glucocorticoids, 75 Bcl-xL, 215 Benzamide, 700 Biogenic amines, 264 Biomarkers for APS–1 related diseases, 507 diabetes, type 1, 423–424 for neuroendocrine tumors, 726 pharmacogenetics, 743 for prostate cancer, 725 Blastomycosis, 515 Blood-brain barrier and cytokines, 23, 86–87, 395, 708 and HIV dementia, 675, 684 and MHC, 11 and T- and B-cells, 264 Blood flow, 371 Blood pressure, 305–315 Blood vessels and Addison’s disease, 506 and aging, 608 constriction, 113–114, 633 and CRH, 653–654 and IL–1, 87 and mast cells, 364, 654 and natriuretic peptide, 55, 309–315 permeability, 55, 309–315, 343, 364 and substance P, 343 B lymphocytes. See also Apoptosis in adaptive immune response, 19 and Addison’s disease, 495, 513 aging effect, 610 anti-DNA, 131, 136 apoptosis, 129, 138, 173, 176, 276 autoreactivity, 15, 129, 134–135, 137 ␤2AR, 266, 276

Index [B lymphocytes] and CpG, 3 and cytokines, 6, 16 development, 14, 15, 128–130 and diabetes insipidus, 445, 446 differentiation, 2, 128–129, 173, 216, 648 and estrogen, 129–135, 137, 138 follicular, 129, 130, 136–137, 138 genetics, 222 and glucocorticoids, 58, 70 and growth hormone, 165–167, 177 homing ability, 16 and IFN-␥, 729 and IGF–1, 171, 172–173, 179 and IL–4 and IL–10, 648 and immunoglobulins, 5, 11–12, 17, 19 marginal zone, 129, 130, 135, 137–138 memory vs. naı¨ve, 3, 19, 128–129 and MSH, 633 and myasthenia gravis, 576, 578 and neurotransmitters, 195, 266 and opioid receptors, 696, 699 and ovarian failure, 552, 558–559 and prolactin, 27, 28, 31, 135–137, 138, 210, 216, 222 role, 2–3, 292 and tamoxifen, 134–135, 137 and T-cells, 3, 129, 136–137 and thyroid autoimmunity, 469–470 transitional types, 128–130 unusual subsets, 17 Bone loss, 612, 656 Bone marrow and cell development, 14–15 grafts/transplants, 174, 180, 419 and growth hormone, 174 and IGF-I, 171, 174 and neurotransmitters, 265 and prolactin, 210, 226 Bone marrow grafting, 174, 180 Bone marrow stem cells, 200 Bone marrow transplants, 419 Brain. See also Blood-brain barrier; Central nervous system communication with, 707 HIV entry route, 674–675 IL–1 receptors, 712–714 and mast cells, 369 and prostaglandin, 709 and testes, 118–121

753 Brain-derived nerve growth factor (BDNF), 369 Bromocriptine, 136–137, 219 Burns, 659–661 Bystander suppression, 433 C3a, 16 C4a, 16 C5a, 16 Calcifications, 506, 514, 515, 517 Calcitonin, 726, 729 Calcitonin-gene related peptide (CGRP) and arthritis, 250 and atopic dermatitis, 638 and IL–10 and IFN-␥, 270 and mast cells, 361, 371 receptor subtypes, 275 and rheumatoid arthritis, 598 and substance P, 371 Calcitriol, 656–658 Calcium and acetylcholine, 278 and Addison’s disease, 506, 514, 515, 517, 521 and aging, 610 and APS–1, 507 and HIV dementia, 676, 678, 679, 682–683 and macrophages, 146 and MAPK, 33 and mast cells, 362, 368 and myasthenia gravis, 572 in parathyroid carcinoma, 726 release of, 24 and sickness behavior, 714 and T-cell activation, 695 and thymocytes, 324 and vitamin D3, 149, 151 Cancer. See also Leukemia; Lymphoma; Tumors and Addison’s disease, 515 of lung, 276, 342, 730 melanoma, 664, 723–724, 728 neuroendocrine tumors, 725–729, 730 of pancreas, 726 of parathyroid, 726 and Th1 response, 664 Cancer cells and dopamine, 276 and GnRH, 284 and MHC class I, 723

754 [Cancer cells] and somatostatin, 279 tumor antigens, 725 Cancer vaccines, 391, 723–724 Candidiasis and ovarian failure, 559 and polyglandular syndromes, 503–504, 506, 507, 513 Capsaicin, 361, 364 Carbohydrate recognition receptors (LECAMs), 13 Carcinoembryonic antigen (CEA), 726 Carcinoid tumors, 728 Carcinomas, with APS–1, 506 Cardiovascular hormones. See Atrial natriuretic peptide ␤-Casein, 213 Caspases, 683, 684–685 Catecholamines and allergic reactions, 663 and atopic dermatitis, 634–636 and cytokines, 86 and estrogen, 656 and IFN-␥, 651 and IL–4, 651 and IL–8, 653 local effects, 653 and monocytes, 653 and rheumatoid arthritis, 599 systemic effects, 650–652 and testes, 114–116 Cathepsin G, 172 C3 convertase, 7–9 CCR2, 469 CCR3, 676 CCR4, 6 CCR5 and autoimmune thyroid disorder, 469 and HIV, 676, 679, 680, 684, 700 and homing, 6 CCR7, 16 CCR8, 6 CD, 9–13, 339, 342 CD1, 17 CD3, 11–12, 433 anti-CD3, 695, 699 CD4Ⳮ cells, 2, 3. See also T-helper cells CD5Ⳮ cells, 105 CD8Ⳮ T cells. See Cytotoxic T cells CD11b, 16 CD11c, 16

Index CD16, 675 CD20, 729–730 CD22, 138 CD25, 342 CD28, 12 CD40 and dendritic cells, 381, 389–390 and MSH, 633 and prolactin, 137 CD44, 171 CD45, 19 CD56, 17 CD69, 675 CD80 and autoimmunity, 550 and dendritic cells, 12 and MHC, 56 and monocytes, 56 and Toll signaling, 17 and VIP/PACAP, 292, 640 CD86 and autoimmunity, 550 and dendritic cells, 12, 56 and MHC, 56 and MSH, 633 and Toll signaling, 17 and VIP/PACAP, 292, 640 CD94, 17 CD132, 71 CD163, 57, 743 CD94-NKG2, 17 Celiac disease, 429, 506, 510 Cell-associated proteins, 9–13 Cell cycle, 71, 179 Cell-mediated immunity and Addison’s disease, 500–502 aging effect, 610 catecholamine effect, 650–652 cell types, 1–4 description, 18–19 GH and IGF-I effects, 177 glucocorticoid effect, 648–650, 651 IL–12 role, 648 and myasthenia gravis, 576, 578, 580–581 and neuroendocrine system, 25–28 and neurotransmitters, 264–275 and NGF, 365 and noradrenaline, 382 and ovarian failure, 558–559 and prolactin, 216–217

Index [Cell-mediated immunity] and Th2 shift, 659 and thyroid, 462–464. See also Th1 cells Cell-mediated toxicity antibody-dependent cytotoxic, 4, 5 Central diabetes insipidus. See Diabetes insipidus Central nervous system (CNS). See also Blood-brain barrier; Brain and HIV, 673–685 immune defenses, 4 immune-neuroendocrine interaction, 36, 393–395 inflammation, 11, 366–367, 405 and mast cells, 362, 366–367, 369 and MHC, 11 and neurotransmitters, 264 pain control areas, 52 and sickness behavior, 708 VIP/PACAP role, 297 C-fibers, 361, 367 CGRP. See Calcitonin-gene related peptide Chemoattraction and glucocorticoids, 399 MCP–1, 311–312 and NGF, 365 and serotonin, 276–277 and VIP, 294 Chemokine receptors and HIV dementia, 678, 681–682, 684 profiles, 6 Chemokines and HIV dementia, 676–680, 681–682, 684 and natriuretic peptides, 311–315 structure and role, 6 and thymocyte migration, 321 and thymus, 322, 338 and thyroid autoimmunity, 469, 470 and VIP/PACAP, 640 Chemotherapy, 180 Children asthma, 619, 620–621, 623–624 atopic dermatitis, 633–634 Crohn’s disease, 177 rheumatoid arthritis, 177, 181 rhinitis, 619–620, 621–623 Chloride ion, 367 Cholecystokinin, 506

755 Cholesterol, 93, 111 Cholinergic blockers, 364, 367 Chromatin, 682, 739 Chromogranin A, 726 Chronic lymphocytic leukemia, 404 Cimetidine, 652, 664 Circadian rhythm, 53, 400, 633, 636–637 CLIP peptide, 475 Clostridium difficile, 370 Cluster differentiation (CD) antigens, 339, 342 CNS. See Central nervous system Coccidioidomycosis, 515 Cyclooxygenase–2 (COX–2), 309, 361, 709 Cognitive disorders, 673, 674 Colitis, 54, 404, 612, 741 Colony-stimulating factors (CSFs), 15, 71. See also Granulocyte-macrophage colony stimulating factor Common cold, 659 Comorbidity. See also Autoimmune polyglandular syndromes with Addison’s disease, 503–511, 515, 523–524, 538, 546, 550–551 asthma and allergic rhinitis, 621 with diabetes insipidus, 448–449 with ovarian failure, 538, 546, 550–551, 555 Complement proteins activation, 7–8, 19, 23 and glucocorticoids, 70, 71 Complement receptors, 16 Compound 48/80, 369 Congenital adrenal hyperplasia (CAH), 518–519 Conjunctivitis, 506 Connective tissue–type mast cells (CTMC), 359 Contiguous gene deletion syndrome, 518 Contraceptive pills, 594 Co-receptors, 12 Corticosteroid-binding globulin (CBG), 68 Corticosterone, 398. See also Glucocorticoids Corticotropin-releasing factor (CRF), 116, 441 Corticotropin, 53 Corticotropin/proopiomelanocortin, 632–634

756 Corticotropin-releasing hormone (CRH) and adaptive immune response, 23–24 and allergy, 55, 655 and atopic dermatitis, 632, 633 and cytokines, 85–87 and estrogen, 656 and fever, 708 genetics, and receptors, 632 and glucocorticoids, 68–69 and histamine, 653–655 and IL–6, 55 and IL–1␤, 86 and inflammation, 54–56, 84, 632, 653 and leukocytes, 55 and macrophages, 55 and mast cells, 55, 366–367, 369, 632, 653–655 and rheumatoid arthritis, 54, 594 role, 23, 51–53 Corticotropin resistance syndrome, 518 Cortisol and aging, 610–611 and atopic dermatitis, 633–634 and AVP, 441 and estrogen, 656 medication effects, 516 and norepinephrine, 599 and rheumatoid arthritis, 594, 599, 661 and septic shock, 742 and T-helper cells, 612 Cortisol-binding globulin, 404 Cortisone, 366 Cortistatin–17, 197 Co-stimulatory proteins, 12–13 Courting behavior, 371 Coxsackie virus, 346–347, 428 CpG, 3, 17 CREB, 246, 678, 699 CRF. See Corticotropin-releasing factor CRFR2, 114 CRH. See Corticotropin-releasing hormone Crohn’s disease and cortisol-binding globulin, 404 DHEA therapy, 612 drug responsiveness, 743 and GC receptors, 741 and IGF–1, 177 and nerve fibers, 599 and Th2 response, 661 and VIP/PACAP, 250–255 Cryptococcosis, 515 Cushing’s syndrome, 84

Index CXC, 640 CXC12, 321 CXCL12, 321 CXCR3, 6, 469 CX3CR1, 676, 679, 680 CXCR4, 676, 679, 684, 700–702 CXCR5, 16 Cyclic AMP and atopic dermatitis, 635 ␤AR–adenylyl cyclase–cAMP-PKA cascade, 659 and HIV dementia, 678 and opioid receptors, 698 and prolactin, 210 and rheumatoid arthritis, 599 and Th1 cells, 651 and thyroid, 462 Cyclic AMP-response element (CRE), 699 cyclic AMP-response element binding protein (CREB), 246, 678, 699 Cyclophilin B, 214 Cyclosporin, 151–152, 432, 546 CYP2A6, 506 CYPIA2, 506 Cysteine, 6, 677, 683 Cystic disease fluid, 15, 213 Cystic fibrosis, 506 Cystitis, 363, 367 Cytochrome c, 683 Cytochrome P450, 506, 552 Cytokine-induced neutrophil chemoattractant (CINC), 399 Cytokine receptors class I and class II, 28 Cytokines and ACTH, 85, 88 aging effect, 610 and antitumor immunotherapy, 727–728, 729 and B cells, 6, 16 and blood-brain barrier, 395 and CRH, 85–87 and dendritic cells, 386–390 and dopamine, 276 and glucocorticoids, 69–70, 76, 89, 399, 714, 738, 740 gp130-dependent, 53 and growth hormone, 53 as hormones, 395 and HPG axis, 108–111

Index [Cytokines] and hypothalamus, 85–88 and leptin, 85 and mast cells, 359 and neuroendocrine system, 53–54, 325–330, 707–715 neuropeptide-induced, 280 and NK cells, 4 and noradrenaline, 386–390 and oxytocin, 325–326 and pituitary gland, 88–90 and pituitary hormones, 23, 24 pro- and anti-inflammatory, 52, 58. See also Pro-inflammatory cytokines production of, 18–19 release of, 16, 17, 23 role, 2, 15 and sickness behavior, 708–715 signaling, 136, 709 and somatostatin, 279 sources and functions, 7 and stress, 53–54, 648–652 subgroups, 6 and T-cells, 2, 6, 18, 279, 294–296 TH1, 6, 18, 19 TH2, 6, 19, 58 and thymus epithelium, 322 and thyroid autoimmunity, 464–467, 476 transport system, 87 Cytomegalovirus, 67–68, 400, 515 Cytotoxic T-cells activation, 389–390, 510–511 in adaptive immune response, 18 and Addison’s disease, 510–511, 513 and autoimmunity, 548 development, 15–16, 339 and diabetes, type 1, 424 and growth hormone, 177 and Hashimoto thyroiditis, 463 and HLA complex, 722 and IFN-␥, 18 and MHC proteins, 9, 10–11, 12, 15–16 and neuroendocrine tumor, 726 and neurotransmitters dopamine, 269–270 serotonin, 277 somatostatin, 198–199 VIP/PACAP, 296–297 opioid receptors, 696 and ovarian failure, 554

757 [Cytotoxic T-cells] and parathyroid gland, 504 and prolactin, 215 prostate cancer-specific, 725 role, 2, 296–297 and thyroiditis, 464, 470 and vitamin D3, 154 Cytotoxic T-lymphocyte antigen–4 (CTLA–4), 510–511, 513, 582 DADLE ([D-Ala2-D-Leu5]-enkephalin), 693, 698–699 DC. See Dendritic cells Degranulation of eosinophils, 3–4 mast cells, 363–366, 367, 370, 654, 664 Dehydroepiandrosterone (DHEA) and aging, 610, 612 and rheumatoid arthritis, 594, 596 Delayed-type hypersensitivity and diabetes prevention, 433 and glucocorticoids, 76, 398 and ovarian failure, 543, 559 and Th1/Th2 cells, 72 and VIP, 295–296, 640 Deltorphin, 700 Dementia, HIV-associated epidemiology, 675–676 pathogenesis, 676–684 therapy, 684–685 Dendritic cells. See also Antigen-presenting cells (APC) and aging, 610 antigen-loading, 725 apoptosis, 151 and autoimmunity, 547–548, 550, 559 thyroid, 469, 474 differentiation, 148, 548, 550 follicular, 14 as immunotherapy, 721–727 for cancer, 723–727 and LPS, 723 and lymphoid system, 13–15 and MHC–2, 4 and MSH, 633 (nor)epinephrine, 382–384, 386–390 and norepinephrine, 382–384, 386–390 pattern recognition, 17 and prolactin, 137, 216 regulation, 382–386 role, 4, 12, 18, 292, 381–382, 390

758 [Dendritic cells] and self-tolerance, 390 and stress, 390 and tamoxifen, 135 and T-helper cells, 389, 390 and VIP/PACAP, 292 and vitamin D3, 151, 656–658 Depression, 390, 715 Dermatitis. See Atopic dermatitis Desmopressin, 454 Determinant spreading, 578–579 Dexamethasone and glucocorticoid resistance, 740 and IL–2, 399 and IL–12, 398 and IL–1␤, 652 and prolactin, 210 Diabetes classification, 418, 419–420 and prolactin, 223 and vitamin D3, 658 Diabetes insipidus autoimmune involvement, 345, 443–446 stages, 450–453 AVP role, 439–441, 444–446 comorbidity, 448–449 diagnosis, 442–443, 446, 451–453 etiology, 441–443 and IL–2, 445 and IL–1␤, 441 and neurons, 347 and ovarian failure, 448 and pituitary gland, 442, 443, 445, 452, 454 primary vs. secondary, 442, 446 therapy, 453–454 Diabetes mellitus, type 1 with Addison’s disease, 506, 510 animal models, 540–542, 546, 550 autoimmunity evidence, 418–419, 422–423, 452 ␤-cell destruction mechanism, 345–349, 424 cellular markers, 423–424 Coxsackie virus, 428 epidemiology, 424–427 etiology, 427–430 genetics, 421–422, 540–541 and glucocorticoids, 399 immune changes, 422–424

Index latent (LADA), 420, 447, 449 and MHC–2, 347 and nicotinamide, 432 and ovarian failure, 538, 555 in polyglandular syndromes, 506, 509 prediction, 419, 423, 425 prevalence, 417, 426–427 prevention, 430–434, 453, 550 and Th1 cells, 223, 424, 430, 433 and Th2 cells, 223, 661 therapy, 425, 510 and thymus, 345–349 vaccines, 347–349, 433 and vitamin D3, 149–150 , 151, 154–155 Differentiation of B-cells, 2, 128–129, 173, 216, 648 CD molecules, 10 of complement proteins, 7–9 dendritic cells, 148, 548, 550 of Ig expression, 5 of immune system cells, 14–16 macrophages, 58, 227, 550 mast cells, 359 into memory cells, 297–298 of MHC polymorphisms, 10 monocytes, 4, 656 NK cells, 14–15 of PRRs, 17 resting T-cells, 4 of T-cells, 18, 72–75, 291–295, 339–342 T-helper cells. See T-helper cells, subtypes thymocytes, 319–325, 343 VIP/PACAP effect, 291–295 DiGeorge syndrome, 338–339 1,25-Dihydroxyvitamin D3. See Vitamin D3 Dimaprit, 662 Diplopia, 572 DNA vaccines, 728–729 Dopamine and adhesion, 270–273, 275 and IL–2, 269–270 and lung cancer, 276 and PRL, 23, 24, 35, 36–38 receptor subtypes, 274 and T cells, 266, 269–273, 275–276

Index Dopaminergic agents, 221 Dynorphins, 27

Eczema, 55, 390. See also Atopic dermatitis Eicosanoids, 361 Elastase, 172 Encephalitis, HIV, 675–676 Encephalomyelitis. See Allergic encephalomyelitis, experimental Endocrine autoimmunity, 547–550 Endorphins and atopic dermatitis, 632–634 binding site, 696–698 immune role, 27–28 and ovarian failure, 543 Endothelial-leukocyte adhesion molecule (ELAM–1), 399 Endothelium, 309–313 Endotoxins and Addison’s disease, 516 and LHRH-LH, 108, 111 protection from, 248–249 tolerance to, 94–95 Enkephalins, 27, 598–599 Environment assessment criteria, 427 and autoimmunity start, 547 and dendritic cells, 390 hygiene hypothesis, 430 and mast cells, 371 and thyroid autoimmunity, 472–475 Eosinophils and glucocorticoids, 58, 70 identification, 3 and IL–4 and IL–10, 648 and substance P, 638 and Th2 response, 664 and VIP/PACAP, 295 Eotaxin, 399 Epinephrine and contact hypersensitivity, 634–635 and dendritic cells, 384 and IL–8, 653 and IL–10, 651 and IL–12, 650 and T-cells, 267 Th1 response, 650

759 Epithelia, 5 thymic epithelial cells, 321–324, 326–228 Erythema, 638 E-selectin. See Selectins Esophagus, 506 17␤-Estradiol, 597–598, 610, 656 Estrogen and autoimmune disorders, 130–132 of thyroid, 472–473 and B-cells, 129–135, 137, 138 and IL–1, 596 and IL–12, 656 and lupus, 130–135, 137–138, 655, 656 and rheumatoid arthritis, 128, 596, 598, 656 and T-cells, 325 and Th1/Th2 cells, 653, 655, 656 Estrogen receptors and prolactin, 213 selective modulators (SERMs), 134–135 and thymocytes, 330 Estrogen-responsive elements, 132–135 Ethnicity and diabetes, 426 and polyglandular syndromes, 503, 508 and thyroid autimmunity, 471 Etomidate, 516 Evolution, 10, 607–608 Excitory amino acids, 676, 677 Excitotoxicity, 676, 682, 683, 684 Exercise, 400, 402–403 Extracellular regulated kinases (ERKs), 678, 699, 739 Extractable nuclear antigens (ENA), 506 Eyes Graves ophthalmopathy, 200, 499 keratoconjunctivitis, 506 ptosis, 572 uveitis, 653 Fas/FasL pathway apoptosis, 174, 176, 424, 464, 640 and autoimmune disorders, 19 and diabetes, 424 and growth hormone, 174, 176 and Hashimoto thyroiditis, 463–464 and IFN-␥, 464 role, 18 and VIP, 26–27 VIP/PACAP, 296–297, 640

760 Fatty acids, very long chain (VLCFA), 517, 521, 524 Fc receptors, 5, 11, 19, 433 Fertility female. See Ovarian failure male, 116 Fetal overgrowth syndrome, 164–165 Fetal thymic organ cultures (FTOC), 74, 174, 345 Fever, 23, 708–709 Fibronectin and dopamine, 270–273, 275–276 and somatostatin, 199, 279 substance P effect, 282 FK506, 152 Follicle-stimulating hormone (FSH) and IL–6, 24 and ovarian failure, 538, 539, 556, 557, 559 Follicle-stimulating hormone-releasing hormone (FSH-RH), 23 Follicular dendritic cells (FDCs), 14 Fractalkine, 676, 677, 679, 680 Free radicals. See also Nitric oxide and CRH, 55 and HIV dementia, 677, 679, 682, 684 lipid peroxidation, 682 and thyroid, 474, 542 Fyn, 34, 35

Gab fragment, 11 Gammaglobulin, 506 Gamma globulin, 506 Ganciclovir, 729 Gastric cancer, 510 Gastritis and AVP antibodies, 447, 448 and Helicobacter pylori, 659 and polyglandular syndromes, 506, 509 Gastrointestinal tract amyloid deposits, 516 autoimmune disorders, 506 and mast cells, 369–371 GC. See Glucocorticoids Gender and aging, 611 and autoimmune disorders, 426, 444

Index [Gender] Addison’s disease, 500, 511, 513, 516, 517 and polyglandular syndrome, 504, 551 and CRH, 656 and myasthenia gravis, 545, 555, 573 and polyglandular syndrome, 504 and rheumatoid arthritis, 593, 594, 596, 598 and T-cells, 558 General adaptation syndrome (GAS), 66 Gene therapy, 476, 742 Genetics and Addison’s disease, 517–519, 524 of antigen-presenting proteins, 9–11 of APS–1, 508–509, 513, 523, 524, 551 animal model, 547 of APS–2, 510–511, 513, 523 and arthritis, 402 for B-cells, 222 ␤-casein, 213 contiguous gene deletion syndrome, 518 CREB transcription factor, 678, 699 CRH receptors, 632 cytokine signaling suppressors, 136 and diabetes type 1, 421–422, 540–541 and DiGeorge syndrome, 338–339 and drug responsiveness, 742–743 estrogen-responsive elements, 132–135 glucocorticoids, 397–405, 740–743 receptor mutations, 403–404 resistance, 738–740 and Graves’ disease, 422 for growth hormone, 165–169, 179 and HIV, 680, 699 HLA complex, 421–422, 426 for IFN-␥, 227 for IGF-I, 169–170, 171, 179 for IGF-II, 164–165 and insulin, 344–347 for leptin, 84, 94 for LHRH, 108 and MHC, 10, 728 of myasthenia gravis, 573 and opioid receptors, 694–696 POMC, 32, 53, 632 and prolactin, 30, 210–212, 215, 217–221, 223 RAG, 339–340 and self-tolerance, 343 for somatostatin receptors, 194–197 survival gene, 30

Index [Genetics] for T-cell receptors, 319, 339 and thyroid autoimmunity, 462, 471–472, 475 Germinal centers, 129 GH/IGF-I axis. See Growth hormone/insulinlike growth factor–1 (GH/IGF–1) axis Ghrelin, 164, 169 Glandular swelling, 14 Glia, 23, 292, 395. See also Microglial cells Glomerulonephritis, 663 Glucocorticoid receptors cofactors, 404–405 description, 396–398 and GC resistance, 738, 740, 742 mutations, 403–404 and resistance, 739–742 Glucocorticoids (GCs). See also Hypothalamic-pituitary-adrenal axis acute vs. chronic administration, 398 and adaptive immune response and accute phase proteins, 23 cell-mediated, 648–650, 651 description, 56–58, 393–395 humoral, 69, 76 immunosuppression, 23, 36, 58–59, 67–70, 395 inflammation, 19, 56–58, 90–91, 399–404 regulatory role, 90–91 stimulation, 70–72 and adrenoleukodystrophy, 517 and allergic reactions, 58, 68–69, 72, 663–664 animal model, 542–543 and apoptosis, 219–220, 324–325, 738 and B-cells, 58, 70 and cytokines, 69–70, 76, 89, 399, 714, 738, 740 dose dependence, 399 and endotoxin tolerance, 94–95 excess/deficiency, 399, 402–404 familial, 518 and fever, 708 genetics, 397–405, 740–743 and HIV, 404–405 and HPA axis, 68–69 and IgE, 664 and immunosuppression, 23, 36, 58, 67–70, 395

761 [Glucocorticoids (GCs)] and inflammation, 19, 56–58, 90–91, 395, 399–404 and interferons IFN-␣, 68, 70–71, 72 IFN-␥, 51, 58, 59, 738 IL–1, 58, 70–71, 652, 738 IL–2, 51, 58, 70–71, 738 IL–4, 58, 72, 648, 738 IL–5, 69, 70, 72 IL–6, 59, 70–71 IL–7, 70, 326 IL–8, 69, 71, 738 IL–12, 59, 72, 398, 404, 648–650, 664 IL–13, 58, 650, 738 and interleukins and Jak/STAT pathway, 738 and leptin, 90, 93 and leukemia, 404 and LIF, 71 and leukocytes, 56, 740 local effects, 652–653 and LPS, 76, 94, 652 and lupus, 68, 399, 403–404, 740 and lymph nodes, 51 and macrophages, 23, 58, 648, 652 and MAPK, 403, 739 and mast cells, 58 and MHC, 56, 58, 70 and MIF, 89 and MIPs, 69 and monocytes, 57–58, 72, 648 and NF-␬B, 56, 397, 738, 740, 742 and PBMC, 648, 738, 740, 743 and PBMCs, 648, 738, 740, 743 preparative effect, 76 and RANTES, 69, 399 regulator, 53 research history, 65–68 resistance, 403–405, 737–743 vs. dependence, 740, 742 and rheumatoid arthritis, 66, 403, 599, 741 and rhinitis, 622 and selectins, 399 and self-tolerance, 343 and stress, 30, 38, 648–653 synergies, 70–72 systemic effects, 648–650 and tachykinins, 343 and T-helper cells apoptosis, 174 Th1/Th2, 19, 72, 399, 648–650

762 [Glucocorticoids (GCs)] therapeutic use, 56, 66, 69, 72, 76, 742–743 and thymocytes, 74–75, 323, 324, 343 and thymus, 51, 72–73, 325 and TNF-␣, 58, 71, 738 transport, 405 Glutamate, 266, 277–278 Glutamate receptors and HIV dementia, 676, 677, 680, 682–683, 682–684 subtypes, 274–275, 278 Glutamic decarboxylase (GAD) autoantibodies, 420, 452, 506 GM-CSF. See Granulocyte-macrophage colony stimulating factor GnRH (Gonadotropin-releasing hormone), 282–285 Gonadotropins, 26 Gonads, 506, 509. See also Ovarian failure; Testes Gonatotropin-releasing hormones (GnRH), 282–285 gp41, 681 gp120, 675, 676, 678, 681, 683 gp130, 53 G-protein–coupled receptor kinase (GRK), 662 G-protein–coupled receptors (GPCRs), 26–27, 632, 637, 678 Grafts, 148–154 bone marrow, 174, 180, 419 of human T-cells, in mice, 326 Graft-vessel disease (GVD), 201 Granulocyte colony-stimulating factor (GCSF), 200 Granulocyte-macrophage colony stimulating factor (GM-CSF). See also Proinflammatory cytokines and antitumor vaccine, 723, 729 and glucocorticoids, 69, 70–71 and growth hormone, 172–174 and prolactin, 218 role, 6 and signaling, 31 Granulocytes complement receptors, 16 and growth hormone, 172 and prolactin, 213, 216 and somatostatin, 194 Granulomatous disease, 16

Index Graves’ disease and Addison’s disease, 509 dendritic cells, 469 genetics, 422, 471–472 lymphoid cell infiltrates, 470 and multiple sclerosis, 465 and NK cells, 558 pathogenesis, 463, 465–466, 472–475 potential phase, 447 and pregnancy, 473 and prolactin, 224 and receptor autoantibodies, 557 therapy, 475–476 and thymus, 346 Graves ophthalmopathy, 200, 499 Growth hormone (GH) and aging, 610 and AIDS, 172, 177 and B-cells, 165–167, 177 and cytokines, 53 and Fas/FasL, 174, 176 and granulocytes, 172 and HIV, 180 and humoral response, 176 and IL–2, 169 and IL–8, 175 immune system expression, 165–169 and innate immunity, 174 and interleukins, 24, 169, 175, 325 and leukocytes, 167–169 and LPS, 169 macrophages, 175 and MHC–2, 173 and monocytes, 167, 175 and PHA, 167, 169 and prolactin, 24, 27, 221 release of, 163–164 replacement therapy, 176 and signaling pathways, 31 somatomedin hypothesis, 164 synergies, 173, 177 therapeutic use, 180–181 and thymocytes, 173–174, 180, 323, 324–325 and thymulin, 326 Growth hormone/insulin-like growth factor–1 (GH/IGF–1) axis circuitry, 328 and PBMC, 167–171 and pituitary hormones, 175 and T-cells, 177 therapeutic potential, 180–181 and thymocytes, 173–174, 180

Index Growth hormone receptors, 28, 31 Growth hormone-releasing hormone (GHRH). See also Somatostatin and hypothalamus, 164 and IL–1␣, 24 and leukocytes, 169 and pituitary hormones, 23 and thymus, 324 Hashimoto thyroiditis animal model, 542–543 and B-cells, 469–470 and CRH, 54 and diabetes insipidus, 447, 448, 449 and Fas/FasL pathway, 463–464 genetics, 462, 471–472, 475 and glucocorticoids, 68 and HPA axis, 402 and IFN-␥, 466 and IL–1␤, 464 lymphoid cell infiltrates, 470 and myasthenia gravis, 573 pathogenesis, 463–465, 466–467, 472–475 and perforin/granzymes, 463 and prolactin, 224 therapy, 475–476 and Th2 response, 661 thyroglobulin, 468 Hassall’s corpuscles, 173 Healing and mast cells, 359 and neurons, 599 and stress, 402–403 and Th1/Th2, 659–661 Heat shock proteins, 310, 433, 573 Heavy chain switching, 19 Helicobacter pylori, 659 Helper T-cells. See T-helper cells Hemochromatosis, 516 Hemorrhage, 220 Hepatitis, autoimmune, 506, 508, 509, 743 Herpes simplex virus, 729 Heyman nephritis, 151 Highly active antiretroviral therapy (HAART), 674 Hippocampus, 656, 678, 682 Histamine and atopic dermatitis, 638 and cancers, 664 and CRH, 653–655 and glucocorticoids, 58

763 [Histamine] and IFN-␥, 652 and IL–12, 652 and mast cells, 4, 19, 366, 368, 369, 653–655 and MSH, 633 and NGF, 365 and serotonin, 363, 370 and Th2 response, 652, 664 and traumatic injury, 661 and tryptase, 362 and vagal nerve, 364 Histiocytosis X, 446 Histoplasmosis, 515 HLA complex. See Human leukocyte antigen (HLA) complex Hot flushes, 538 HPA axis. See Hypothalamic-pituitaryadrenal axis Human chorionic gonadotropin (hCG), 112–114 Human immunodeficiency virus–1 (HIV) and Addison’s disease, 515 and dementia, 673–685 and MAPK, 678, 680, 682, 685 MIP role, 676, 679, 684 and monocytes, 674, 675 and RANTES, 676, 680, 684 TNF-␣, 675, 677, 679, 680, 683 and genetics, 680, 699 and glucocorticoids, 404–405 gp120 envelope protein, 675, 676, 678, 681, 683 and growth hormone, 180 and IFN-␥, 679 and IL–12, 659 and metalloproteinases, 677, 680 and neurons, 675–678, 681–685 and norepinephrine, 659 and opioid receptors, 699–702 p24 antigen, 693–694, 700 and PBMC, 675, 693–694, 699–702 and sickness behavior, 715 and Th2 response, 659 Human leukocyte antigen (HLA) complex and APS–2/Addison disease, 510, 513 and diabetes insipidus, 446 and diabetes mellitus, type 1, 421, 426 genetics, 421–422, 426 and immunotherapy, 722 and myasthenia gravis, 573 and ovarian failure, 558 and thyroid autoimmunity, 469

764 Humoral immune response. See also Th2 cells and Addison’s disease, 495–497 and B-cells, 5, 11–12, 17, 19 description, 5–9, 18, 19 GH and IGF-I effects, 176 glucocorticoid effect, 58 heavy chain switching, 19 IL–4 and IL–10 roles, 648 and pituitary hormones, 23 and sickness behavior, 707–715 and thyroid autoimmunity, 464–467 Hydrocortisone, 398. See also glucocorticoids 17 ␣-Hydroxylase, 552 21-Hydroxylase, 495–497, 552 11␤-Hydroxysteroid dehydrogenase, 404, 740 17␤-Hydroxysteroid dehydrogenase, 597–598 Hygiene hypothesis, 430 Hypergammaglobulinemia, 506 Hypergonadism, 506, 509 Hyperkalemia, 521 Hypocalcemia, 597–508 Hypochloremia, 521 Hypogonadism, 506, 509, 513, 518 Hypoparathyroidism, 503–504, 507 Hypophysitis, 506, 509 Hypothalamic-pituitary-adrenal axis (HPA axis) and adaptive immune response, 19, 23, 36, 90–91 and aging, 608, 613 and atopic dermatitis, 632–634 and autoimmune disorders, 400–404 brain site, 720 and cytokines, 85–90 description, 52–53 disruption causes, 402–405 and estrogen, 656 and glucocorticoids, 68–69 and IFN-␥, 53 and IL–6, 52, 53 imbalances, 395 and inflammation, 36, 52, 53, 90, 366, 400–404 and leptin, 85, 91–94 and LIF, 89–90 measurement, 402 POMC, 632 regulation of, 393–394

Index [Hypothalamic-pituitary-adrenal axis (HPA axis)] and rheumatoid arthritis, 593, 594–596, 661 and sickness behavior, 708, 709 and stress, 36, 366–367 and thymulin, 24–25 and TNF-␣, 52, 53 Hypothalamic-pituitary-gonadal (HPG) axis and cytokines, 108–111 and neural hypothalamic testicular path, 116–118 and rheumatoid arthritis, 593 Hypothalamic-pituitary-thyroid (HPT) axis, 53 Hypothalamic-testicular pathway and cytokines, 111–116 main components, 120–121 structure-function, 118–120 Hypothalamus aging effect, 608 and cytokines, 85–88 and diabetes insipidus, 347, 442, 446, 453 and estradiol, 656 and prolactin, 209 Hypoxia, 661, 664 ICA, 452 IFN-␥. See Interferon-␥ IGF–1. See Insulin-like growth factor 1 Ii CLIP peptide, 475 Immunity acquired, 7–8 adaptive, 18–19 antitumor, 225–226 cell-mediated, 1–4, 18 humoral, 5–9, 18, 176. See also Humoral immune response innate, 7, 8, 16–17 and stress, 36 Immunodeficiency. See also Human immunodeficiency virus–1 AIDS, 172, 177, 702 SCID, 172, 682 Immunoglobulin A (IgA) and Addison’s disease, 506 and epithelia, 5 and ovarian failure, 554 and prolactin, 216 and somatostatin, 199

Index Immunoglobulin D (IgD) role, 5 Immunoglobulin E (IgE) and allergic rhinitis, 19, 623 and asthma, 624 and ␤2-adrenergic agonists, 664 and B-cells, 648 and glucocorticoids, 58, 70, 72, 664 and histamine, 652 and mast cells, 5, 19 and somatostatin, 199 and tryptase, 362–363 Immunoglobulin G (IgG) and ADCC, 4 and estrogen, 655 and IGF-I, 176 and leukocytes, 4, 175 and LH, 557 and ovarian failure, 554, 557 and placenta, 5 subsets, 19 and thyroiditis, 464 Immunoglobulin M (IgM) and B-cell subset, 17 and diabetes, 429 and ovarian failure, 554 role, 5, 19 Immunoglobulins and ADCC, 5 adhesion proteins, 13 antigen binding site, 11 and B cells, 2–3, 11 KIRs, 17 and leukocytes, 9 structure, 5 transport, 19 Immunosuppressants and APS–1, 508 for myasthenia gravis, 577 and vitamin D3, 151–154 Immunosuppression and bromocriptine-induced, 136 and glucocorticoids, 23, 36, 58, 67–70, 395 and malnutrition, 85 and prolactin, 218–221 and traumatic injury, 659–661 and tumors, 664, 723 Immunotherapy. See also Vaccines with allergens, 623 antitumoral, 724–730 with cytokines, 727–728

765 [Immunotherapy] with dendritic cells, 721–727 gene therapy-induced, 728–729 for lymphoma, 725, 728, 729–730 and MHC, 728 monoclonal antibodies, 729–730 Immunotyrosine activation motifs (ITAMs), 11 Indoleamine–2,3-dioxygenase, 715 Infections. See Bacterial infections; Parasitic infections; Viral infections Infertility, in women, 556 Inflammation. See also Pro-inflammatory cytokines and ACTH, 53–54 and aging, 612–613 and autoimmunity, 548 of blood vessels, 506 and CNS, 11, 366–367 and CRH, 54–56, 84, 632, 653 glucocorticoid resistance, 737–743 and glucocorticoids, 19, 56–58, 90–91, 395, 399–404 animal models, 401–402 and HPA axis, 36, 52, 53, 90, 366, 400–404 and IGF-I, 171, 179 and IL–6, 53 in innate immune response, 16 and leukocytes, 3–4, 311–312 and macrophages, 4 and mast cells, 359, 362–364, 366 in vitro studies, 368 mediators, 608 and MIF, 89 and natriuretic peptide, 308–311, 315 and neurotransmitters, 598 and NF-␬B, 56, 738 NF-␬B role, 738 and NGF, 365 and sensory neurons, 358 and serotonin, 276–277, 277 and sickness behavior, 709 and somatostatin, 84, 197–200 VIP/PACAP role, 245–248, 297 Inflammatory bowel disease, 741 Inflammatory diseases and aging, 612–613 and CNS, 405 and GC resistance, 740–743 and HPA axis, 90

766 Inflammatory mediators, 16, 399. See also Nitric oxide; Prostaglandins Injury, 659–661 Innate immunity and complement proteins, 7–8 description, 16–18 GH and IGF-I effects, 174–176 and noradrenaline, 382 self/non-self, 56 Inositol phosphate, 24 Insulin dosage reduction, 510 and genetics, 344–347 and multiple sclerosis, 347 in newly-diagnosed patients, 425 and prolactin, 210 as secondary intervention, 431–432 Starling curve production, 420 Insulin-like growth factor 1 (IGF–1). See also Growth hormone/insulin-like growth factor–1 and aging, 610 and apoptosis, 165, 179 and B-cells, 171, 172–173, 179 and cell-mediated immunity, 177 and Crohn’s disease, 177 and genetics, 169–170, 171, 179 and granulocytes/PBMC, 172–174 and humoral immunity, 176–177, 714 and IFN-␥, 347–349 and IL–1␤, 171, 178 and IL–7, 173 and IL–8, 179 and immune proteins, 171–172 and immune receptors, 169–170 and inflammation, 171, 179 and innate immunity, 174–176 and leukocytes, 179–180 and macrophages, 171–172 and MHC–2, 173 and monocytes, 171, 179–180 and phosphorylation, 165 system description, 164–165 and thymocytes, 324, 326, 328, 330 and TNF-␣, 171, 178 Insulin-like growth factor 2 (IGF–2), 164–165, 344–349 Insulin-related genes, 344–347 Integrins and dopamine, 275–276 role of, 13 and somatostatin, 279 and substance P, 282

Index Intercellular adhesion molecule 1 (ICAM–1) and glucocorticoids, 399, 740 and macrophages, 215 and MSH, 633 and natriuretic peptide, 312–313 and NF-␬b, 312–313 and prolactin, 215, 224 role, 13 Interdigitating cells, 14, 18 Interferon-␣ (IFN-␣), 68, 70–72 and diabetes, 428–429 and myasthenia gravis, 580, 582 and neuroendocrine tumors, 728 and thyroid gland, 474–475 Interferon-␤ (IFN-␤), 154 Interferon-␥ (IFN-␥). See also Proinflammatory cytokines and antitumor immunotherapy, 726, 729 and atopic dermatitis, 639 and B-cells, 729 and catecholamines, 651 and CGRP, 270 and cytotoxic T-cells, 18 and Fas/FasL pathway, 464 and fever, 23 genetics, 227 and glucocorticoids, 51, 58, 59, 738 and Hashimoto thyroiditis, 466 and histamine, 652 and HIV dementia, 679 and HPA axis, 53 and IGFs, 347–349 and IL–12, 648 and infections, 6, 18 and LPS, 23 and melatonin, 636 and norepinephrine, 390 and pituitary gland, 36 and pregnancy, 663 and prolactin, 24, 36, 217, 224, 227 and somatostatin, 198 and substance P, 281, 638 and T-helper cells, 18, 648, 659 and TNF-␣, 175 and VIP/PACAP, 295 and vitamin D3, 146–148, 151 Interferon regulatory factor (IRF), 30, 36, 136 Interleukin-1 (IL–1). See also Proinflammatory cytokines and adrenal glands, 90 and blood vessels, 87

Index [Interleukin-1 (IL–1).] brain receptors, 712–714 and estrogen, 596 and fever, 23 and glucocorticoids, 58, 70–71 and IL–6, 22, 23 and melatonin, 636 and MSH, 633 and neuroendocrine system communication role, 22–24 and CRH, 55 HPA axis, 52, 53, 66 and prolactin, 220 receptors (IL–1R), 57, 708, 712–714. See also Toll-like receptors and sickness behavior, 708–714 and substance P, 631 and vagus nerve, 87–88 and VIP, 266 and viruses, 66 and vitamin D3, 146 Interleukin–1␣(IL–1␣), 24, 325 Interleukin–1␤(IL–1␤) and aging, 610 and ␤-adrenergic receptors, 267 and ␤2AR, 267 and CRH, 86 and diabetes insipidus, 441 and estrogen, 596 and glucocorticoids, 59, 652, 738 and growth hormone, 177, 325 and Hashimoto thyroiditis, 464 and HIV dementia, 679, 683 and IGF-I, 171, 178 and leptin, 84 and MSH, 633 and prolactin, 325 and rheumatoid arthritis, 596 and sickness behavior, 709–712 and somatostatin, 199 and stress, 653 and testes, 111–116, 121 and TNF-␣, 171 Interleukin-2 (IL–2) and Akt, 34 and antitumor immunotherapy, 728, 729 and CRH, 55 and diabetes insipidus, 445 and endorphins, 27 and glucocorticoids, 51, 58, 70–71, 738 and growth hormone, 169

767 [Interleukin-2 (IL–2)] and IGF-I, 179 and LPS, 23 and nitric oxide, 24 and pregnancy, 663 and prolactin, 29, 227 and serotonin, 277 somatostatin, 198, 274, 279–281, 324 and stress, 53 substance P, 274, 281–282 and substance P, 274 and tamoxifen, 134 and T-cells, 6, 18, 26, 269–270 and thyroid, 473 and TRH, 26 and VIP, 274 and VIP/PACAP, 292, 294–295 and vitamin D3, 147 See also Pro-inflammatory cytokines Interleukin-2R (IL–2R), 71, 227, 738 Interleukin-3 (IL–3), 34, 69, 70 Interleukin-4 (IL–4). See also Antiinflammatories and antitumor immunotherapy, 726 and B-cells, 648 and catecholamines, 651 and glucocorticoids, 58, 72, 648, 738 and histamine, 652 and MSH, 633 and pregnancy, 663 and progesteroine, 655 and prolactin, 29–30, 210, 223 role, 18, 19, 648 and substance P, 270, 638 and thyroid, 466 and VIP, 253, 295 and VIP/PACAP, 295 and vitamin D3, 147, 151 Interleukin-5 (IL–5) and glucocorticoids, 69, 70, 72 and progesteroine, 655 and VIP, 295 and vitamin D3, 656–658 Interleukin-6 (IL–6). See also Proinflammatory cytokines and adrenal glands, 90 and aging, 610, 612 and catecholamines, 651 and CRH, 55 and diabetes insipidus, 441 and fever, 23 and FSH, 24

768 [Interleukin-6 (IL–6).] and glucocorticoids, 59, 70–71, 738 and growth hormone, 177–178, 325 and histamine, 652 HPA axis, 52, 53 and IL–1, 22, 23 and inflammation, 53 and Jak/STAT pathway, 53 and leptin, 84 and LHRH, 108 and LPS, 109–111 and mast cells, 363 and MSH, 633 neuroendocrine effects, 24 and NGF, 363 and oxytocin, 326 and prolactin, 36, 220, 325 release, 23 and rheumatoid arthritis, 594, 597–598 and sickness behavior, 708, 711 and somatostatin, 199, 201 and VIP, 24 and VIP/PACAP, 23, 246, 249, 266 Interleukin-6R (IL–6R). See also Proinflammatory cytokines and glucocorticoids, 59, 70–71 Interleukin-7 (IL–7) and glucocorticoids, 70, 326 and IGF-I, 173 and thymocytes, 321, 339 Interleukin-7R(IL–7R) and glucocorticoids, 71 Interleukin-8 (IL–8) and catecholamines, 653 and epinephrine, 653 and glucocorticoids, 69, 71, 738 and growth hormone, 175 and IGF-I, 179 and leukocytes, 653 and neutrophils, 738 and platelets, 653 and somatostatin, 199, 201 and vitamin D3, 658 Interleukin-10 (IL–10). See also Antiinflammatories and adenosine, 658 and adrenergic blockade, 661 and autoimmune disorders, 661 and B-cells, 648 and CGRP, 270

Index [Interleukin-10 (IL–10).] and epinephrine, 651 and estrogen, 655 and glucocorticoids, 58–59, 399, 404, 650 and histamine, 652 and HIV, 659 and IGF–2, 348 and injury, 661 and MHC, 19 and MSH, 633 and neuroendocrine system, 23 and norepinephrine, 386–390, 651 and pregnancy, 663 and progesterone, 655 and prolactin, 223, 227 role, 18, 648, 714 and sickness behavior, 708 and Th2, 723 and thyroid, 466, 467, 476 and tumors, 664, 723 and VIP, 253 and vitamin D3, 656–658 Interleukin-11 (IL–11) Interleukin–11R (IL–11R), 71 Interleukin-12 (IL–12). See also Proinflammatory cytokines and adenosine, 658 and antitumor immunotherapy, 729 and autoimmune disorders, 661 ␤-adrenergic agonists, 382, 651, 664 and cell-mediated immunity, 18, 648 and dexamethasone, 398 and epinephrine, 650 and estrogen, 656 and glucocorticoids, 59, 72, 398, 404, 648–650, 664 and histamine, 652 and HIV, 659 and IFN-␥, 648 infection type, 6 and myasthenia gravis, 580, 582 and norepinephrine, 386–390 and pregnancy, 663 and prolactin, 216 and T-helper cells, 18, 648, 649 and tumors, 664 and vitamin D3, 146, 148, 656–658 Interleukin-12R (IL–12R), 72 Interleukin-13 (IL–13), 58, 650, 738 Interleukin-16 (IL–16), 277

Index Intestines lymphangiectasia, 506 and mast cells, 367, 369–371 and MDR proteins, 405, 741 permeability, 367 In vitro fertilization (IVF), 556 Iodine, 473–474, 542 IRF (Interferon regulatory factor), 30, 36 Iron, 516 Irritable bowel disease, 151 Ischemia, 661 Isohormonal therapy, 453 Jak/STAT pathway description, 31–32 and dexamethasone, 399 and glucocorticoids, 738 and gp130-dependent cytokines, 53 and prolactin, 36–38, 213–214 and VIP/PACAP, 246 c-Jun N-terminal kinase (JNK), 26, 33, 699, 739 Keratoconjunctivitis, 506 Kerns–Sayre syndrome, 518 Ketoconazole, 516 Kidneys, 151, 181 cancer cells, 725 Killer cell inhibitory receptors (KIRs), 17 LAK cells, 217 Langerhans cells, 382–386, 390, 634, 722 Lanreotide, 199, 201, 728 L-DOPA, 276 Lectins, 94 Leishmania, 6 Lentivirus, 702 Leprosy, 6 Leptin, 84–85, 91–94 Leukemia and glucocorticoids, 404 and glutamate, 278 and mast cells, 368 and prolactin, 225–226 Leukemia inhibitory factor (LIF). See also Selectins and ACTH, 24, 89–90 and glucocorticoids, 71 and HPA axis, 53–54 and Jak/STAT pathway, 53 and oxytocin, 325–326 and signaling pathways, 32, 36 and stress, 53

769 Leukocyte endothelial cell cell-adhesion molecules (LE-CAMs), 13 Leukocytes. See also Polymorphonuclear leukocytes and aging, 611 and allergic reactions, 3, 4 associated proteins, 9–13 and chemokines, 6 and CRH, 55 and glucocorticoids, 56, 740 and growth hormone, 167–169 and IGF-I, 179–180 and IL–8, 653 and inflammation, 3–4, 311–312 in innate response, 16 natriuretic peptide effect, 311–315 and opioid receptors, 695 and prolactin receptors, 213, 214–215 and somatostatin, 27, 200 and substance P, 638 Leukotriene C, 58 Leukotriene receptors, 623, 624 Lewis rat, 543, 662 Leydig cells, 109–113, 116–118, 121 LHRH. See Luteinizing hormone-releasing hormone LIF. See Leukemia inhibitory factor Lipids, 17, 682 Lipopolysaccharides (LPS) and dendritic cells, 723 and glucocorticoids, 76, 94, 652 and growth hormone, 169 and IFN-␥, 23 and leptin, 84–85 and luteinizing hormone, 108 and natriuretic peptides, 308–309 receptors for, 57 role, 17, 23 and sickness behavior, 708, 710, 711 and substance P, 637 and testes, 109–111 and vagus nerve, 87–88 and VIP/PACAP, 247 and vitamin D3, 146 Listeria monocytogenes, 382 Liver and acute phase proteins, 23 autoimmune hepatitis, 506, 508, 509, 743 and growth hormone, 177–179

770 [Liver] and IGF–1, 165 and natriuretic peptide, 314 Liver-kidney autoantibodies, 506 L-NAME, 86 Local effects, 652–653 LPS. See Lipopolysaccharides L-selectin. See Selectins Lung, 653 Lung cancer, 276, 342, 730 Lupus. See Systemic lupus erythematosus Luteinizing hormone (LH) and hCG, 117–118 and LPS, 108 and ovarian failure, 538, 557 and thymus, 24–25 Luteinizing hormone-releasing hormone (LHRH), 23, 24–25, 108 Lymphangiectasia, 506 Lymph nodes and autoimmunity, 548 and glucocorticoids, 51 and immune system, 14, 18 immunotherapy injection, 724 innervation, 659 and neurotransmitters, 265 Lymphocyte progenitor, 14–15 Lymphocytes, 281. See also B lymphocytes; T lymphocytes Lymphofolicular hyperplasia, 579 Lymphoid system in adaptive immune response, 18 and chemokines, 6 and macrophages, 13–14, 15 and mast cells, 15 and neurotransmitters, 265 stress effect, 51 Lymphokine-activated killer (LAK) cells, 17, 217 Lymphoma and GnRH, 284 immunotherapy, 725, 728, 729–730 non-Hodgkin’s, 729–730 and prolactin, 225 and somatostatin, 200 Lymphoma cells, 215 Lymphotactin, 633 Lysis by cells, 2, 4, 5 by MAC, 8

Index [Lysis] and prolactin, 225 Lysosomal storage disease, 516

MAC (Membrane attack complex), 8 Macrophage colony stimulating factor (M-CSF), 70 Macrophage inflammatory proteins MIP–1␣, 69, 224, 676, 679, 684 Macrophage migration inhibitory factor (MIF), 88–89 Macrophages. See also Antigen-presenting cells (APC) apoptosis, 598 and autoimmunity, 469, 470, 547–548, 550 and calcium, 146 and CRH, 55 and cytokines, 6 differentiation, 58, 227, 550 and glucocorticoids, 23, 58, 648, 652 and growth hormone, 175 and HIV dementia, 680, 683 and IGF-I, 171–172 and lymphoid system, 13–14, 15 and melatonin, 636 and natriuretic peptides, 308–309 and nerve fibers, 653 and neurons, 653 and neurotransmitters, 598 and opioid receptors, 694, 696 and ovarian failure, 554 pattern recognition, 17 and progesterone, 655 and prolactin, 216, 219, 220 role, 4, 16, 245, 292 and self-tolerance, 342 and sex hormones, 596, 598 and somatostatin, 197, 199 stress, 653 and testosterone, 596 and VIP/PACAP, 245, 246–248, 292, 640 Macropinocytosis, 4 Major histocompatibility complexes (MHCs) in adaptive immune response, 9–10, 18, 19 class I (MHC–1), 339, 342–343 and antitumor immunotherapy, 726–727 and cancer cells, 723 and KIRs, 17

Index [Major histocompatibility complexes (MHCs)] and MSH, 633 and myasthenia gravis, 579 vs. class II, 10–11 class II (MHC–2) and autoimmunity, 473, 475, 547, 550 and CD80 and CD86, 56 and dendritic cells, 4 and diabetes, 347 GH and IGF-I effect, 173 and glucocorticoids, 56, 58, 70 and melatonin, 636 and myasthenia gravis, 573, 579 and self-tolerance, 339, 343 and substance P, 282 and genes, 10, 728 and immunotherapy, 728 in innate immune response, 17 in NOD mouse, 542 and ovarian failure, 546 peptide complex, 4 structure-function, 9–10 and T-cells, 9, 10–11, 12 T-cell receptors, 12, 15–16 and thymomas, 581 Malabsorption, 506. See also Celiac disease Malnutrition, 85 Mannin-binding lectin (MBL), 8 MAPK. See Mitogen-activated protein kinases Mast cells activation, 363–364 and allergic reactions, 365–366, 370, 655, 663–664 and asthma, 55, 365–366 and blood vessels, 364, 654 and calcium, 362, 368 and CNS, 362, 366–367, 369 connective-tissue type, 359 and CRH, 55, 366–367, 369, 632, 653–655 deficiency, 370, 371 degranulation, 363–366, 367, 370, 654, 664 differentiation, 359 gastrointestinal tract, 369–371 and glucocorticoids, 58 and histamine, 4, 19, 366, 368, 369, 653–655 and IgE, 5, 19

771 [Mast cells] and inflammation, 359, 362–364, 366, 368 and interleukins, 363, 648 and leukemia, 368 location, 358–359, 369–371 and lymphoid system, 15 and migraine, 369 and MSH, 633 mucosal (MMC), 359 and multiple sclerosis, 369 and nervous system, 363–364, 367–371, 653 and NGF, 363, 365–366, 369, 639 and pituitary gland, 369 and priming, 362–363 progenitors, 359 role, 4, 19, 359–363, 366–371 and serotonin, 364, 368 and somatostatin, 27 and stress, 366–367 and substance P, 359–361, 363, 364, 369, 370, 638, 653 and TNF-␣, 361, 363 tryptase, 362, 369 types, 359 MCP–1. See Monocyte chemoattractant protein–1 MDR proteins. See Multidrug-resistance (MDR) proteins Median eminence, 87, 395 Melanocortin, 25–26, 714, 715 Melanocortin receptors (MCRs), 25–26, 632 Melanocyte-stimulating hormone (MSH), 632–633 Melanoma, 664, 723–724, 728 Melatonin, 636–637 Memantine, 683 Membrane attack complex (MAC), 8 Memory cells antigen-specific, 297 B lymphocytes, 3, 19, 128–129 T lymphocytes, 12, 19, 297–298 and thyroiditis, 461 Menstrual disorders, 538 Metabolic disorders, 516 Metabolism, 715 Metaclopramide, 324 Metalloproteinases and HIV, 677, 680 MMP–3, 224 MMP–9, 683 and prolactin, 224 and somatostatin, 201 and VIP/PACAP, 250

772 Methionine enkephalin, 598–599 Methotrexate, 599 Metyrapone, 516 Microglial cells, 4, 674–675, 676, 677, 680 Microsomal antigen. See Thyroid peroxidase MIF (Macrophage migration inhibitory factor), 88–89 MIF (Migration-inhibiting factor), 554, 558, 559 Migraine headaches, 55–56, 369 Migration of Langerhans cells, 382–386 of mast cell progenitors, 359 of thymocytes, 327–328 Migration-inhibiting factor (MIF), 554, 558, 559 Milk proteins, 429 Mimicry, 578 MIP. See Macrophage inflammatory proteins Mitochondria, 518, 683 Mitogen-activated protein kinases (MAPK) and glucocorticoids, 403, 739 and HIV dementia, 678, 680, 682, 685 JNK family, 33 and natriuretic peptide, 310–311 and opioid receptors, 699 p38 MAPK, 680, 682, 685, 739 p38 MAPK/NF-␬B pathway, 23 and prolactin, 213, 215, 228 role, 32–33 and sickness behavior, 714 and somatostatin, 194 MMP. See Metalloproteinases Mofetil, 154 Molecular mimicry, 578 Molecules, receptor, 11 Monoamine oxidase, 635 Monoclonal antibodies, 729–730 Monocyte chemoattractant protein–1 (MCP–1) and glucocorticoids, 399 and HIV dementia, 675, 680 and leukocytes, 311–312 Monocytes and adenosine, 658 and atopic dermatitis, 633, 635 and catecholamines, 653 differentiation, 4, 656 and glucocorticoids, 57–58, 72, 648

Index [Monocytes] and growth hormone, 167, 175 and HIV dementia, 674, 675 and IGF-I, 171, 179–180 and ovarian failure, 559 role, 4, 16 and somatostatin, 199 and substance P, 599 and vitamin D3, 146–147 Morphine, 693–696, 699, 700, 702 ␣-MSH, 25–26 MSH (Melanocyte-stimulating hormone), 632–633 Mucosal mast cells (MMC), 359 Multi-drug resistance (MDR) proteins, 405, 740. See also P-glycoprotein 170 Multiple endocrine neoplasia type 2 (MEN 2), 726 Multiple sclerosis animal model, 68 and glucocorticoids, 309, 650 and Graves’ disease, 465 and insulin-derived peptides, 347 and mast cells, 369 and neurotransmitters, 264 and pregnancy, 128, 663 and prolactin, 224 and stress, 661–662 and Th1/Th2 cells, 465, 661 Muscle-specific tyrosine kinase receptor (MuSK), 573, 575 Myasthenia gravis and B cells, 576, 578 clinical features, 572 determinant spreading, 578–579 diagnosis, 572–573 etiology, 577–580, 581–582 experimental autoimmune (EAMG), 578–579 genetics, 573 and heat shock proteins, 573 and IL–12, 580, 582 and lupus, 573 and MHCs, 573, 579 and ovarian failure, 545, 555, 557 paraneoplastic, 580–582 pathophysiology, 571–572 seronegative, 575, 577 target antigen, 571 and T-cells, 576, 578, 580–581

Index [Myasthenia gravis] therapy, 577 and thymoma, 575–576, 577 and thymus, 576–577, 580 and tumors, 580 Mycobacteria, 6, 659 Mycophenolate mofetil, 154 Nail dystrophy, 506 NaⳭ ion, 277 Naı¨ve B-cells, 3, 128 Naı¨ve T-cells, 12, 18 T-helper cells, 72, 228 Nasal polyposis, 404 Natriuretic peptides. See Atrial natriuretic peptide (ANP) Natural killer (NK) cells adhesion proteins, 13 differentiation, 14–15 and Graves’ disease, 29–30, 217, 225, 227 and growth hormone, 175 KIRs, 17 and ovarian failure, 554, 558–559 and POMC peptides, 25 and prolactin, 29–30, 217, 225, 227 role, 4, 18 and thyroid, 470 Natural killer (NK) receptors, 17, 400 Nematodes, 370 Nephritis, 151 Nerve growth factor (NGF) and atopic dermatitis, 639 and lupus, 639 and mast cells, 363, 365–366, 369, 639 Neuroendocrine system aging effect, 608–612 and cell-mediated immunity, 25–28 and cytokines, 53–54, 325–330 class I cytokine receptors, 28 growth hormone effects, 31 and IL–1, 22–24, 52, 53, 55, 66 and immune system, 22–25, 36–38, 51–53, 90–91, 265, 366, 395 immune cell hormone receptors, 25–28 and prolactin, 29–30 signaling, 22–25, 31–35 stress effect, 36–38, 51–53, 265, 366 and thymus, 329–330 and TNF-␣, 58, 71, 738 tumor localization, 200

773 Neuroendocrine tumors, 725–729, 730 Neurohypophysial receptors, 342–343 Neurokinin A, 343, 360, 637 Neurokinin receptors, 637–638 Neuronal pathways, 87–88 hypothalamic-testicular, 116–121 Neurons apoptosis, 675–678, 681–684 and atopic dermatitis, 638, 639–640 and diabetes insipidus, 347 and HIV, 675–678, 681–685 and HPA axis, 52 and macrophages, 653 and mast cells, 363–364, 368–371 and TNF-␣, 361, 363, 683 Neuropeptides. See also Tachykinins; Vasoactive intestinal peptide and atopic dermatitis, 639, 640–641 as neurotransmitters, 254 neurotrophins, 639 T cell stimulation, 270, 280 and thymocytes, 321–325 and thymus epithelium, 322, 323 Neuropeptide Y, 91–92, 275 Neurotensin, 342 Neurotoxins, 674 Neurotransmitters. See also Acetylcholine; Calcitonin gene-related peptide; Dopamine; Glutamate; Gonatotropinreleasing hormones; Growth hormone-releasing hormone; Neuropeptides; Serotonin; Somatostatin; Substance P; Vasoactive intestinal peptide and B-cells, 195, 266 categories, 264 description, 263 and inflammation, 598 and macrophages, 598 and multiple sclerosis, 264 and rheumatoid arthritis, 598–600 and T-cells, 264–275 outcome factors, 268–275 specific neurotransmitters, 266, 275–285 and T-helper cells. See T-helper cells and thymus, 322 Neutrophils GH and IGF-I effects, 175, 179 and glucocorticoids, 57–58, 738

774 [Neutrophils] and IL–8, 738 and mast cells, 361 and POMC peptides, 25 role, 3, 16 and substance P, 638 NGF. See Nerve growth factor Nicotinamide, 432 Niemann–Pick disease, 516 Nitric oxide (NO) and glucocorticoids, 399 and HIV neuron damage, 677, 679 and HP axis, 86 and IL–2, 24 and natriuretic peptides, 309 and prolactin, 36 role, 395 and Th1 response, 648 Nitric oxide synthase (NOS) and HIV dementia, 679, 680, 683 and IL–2, 24 and IRF, 215 and natriuretic peptides, 309 Nitrogen, reactive, 19 NK receptors, 17, 400 NKT cells, 17–18 N-methyl-D-aspartate (NMDA) antagonists, 684 receptors, 680, 682–684 NOD mouse, 542, 546, 550 Non-Hodgkin’s lymphoma, 729–730 Noradrenaline. See Norepinephrine Norepinephrine and aging, 608–609 and atopic dermatitis, 633, 635 and cortisol, 599 and CRH, 52, 53 and dendritic cells, 382–384, 386–390 and HIV, 659 and IFN-␥, 390 and interleukins, 386–390, 651 and LH, 118 receptor subtypes, 274 and rheumatoid arthritis, 598–600 and T-cells, 266, 270, 276 and Th1 cells, 266, 389, 550, 650 and Th2 cells, 266, 390, 612 NT–3, 639 NT–4, 639 Nuclear factor AT (NF-AT), 152, 297

Index Nuclear factor IL–6 (NF IL–6), 70 Nuclear factor-␬B (NF-␬B) and adhesion, 312–313 and glucocorticoids, 56, 397, 738, 740, 742 and ICAM–1, 312–313 and inflammation, 56, 738 and melatonin, 636 and MSH, 633 and natriuretic peptide, 313–314 and prolactin, 213–214 role, 738 and sickness behavior, 708, 714 and VIP/PACAP, 246, 297 and Vitamin D3, 656–658

Obese strain chicken, 542–543, 550 Obesity and growth hormone, 180 and 11␤-hydroxysteroid dehydrogenase, 404 and IL–6, 59 and leptin, 91, 94 Octreotide, 198, 199, 200–201, 728 OKT3, 433 Omalizumab, 623 Oncostatin M, 70, 71 Opioid receptors ␦ (DOR) and corticotropin/proopiomelanocortin, 632 and ERK1,2 phosphorylation, 699 immunofluorescence detection, 697 and T-cells, 703 T-cell receptors, 693 as therapy, 702 transcript expression, 694–696 and HIV, 699–702 intracellular signaling, 698–699 ␬ (KOR), 694, 695, 696, 702, 703 ␮ (MOR), 632, 694, 695, 699, 703 on splenocytes, 696 Opioids, endogenous, 599. See also Endorphins Oral contraceptives, 594 Oral mucosa, 506, 507 Organum vasculosum of the laminae terminalis (OVLT), 87, 395 Osteoporosis, 612, 656

Index Ovarian failure, premature (POF) and Addison’s disease, 538, 546, 550–554 (a)follicular, 539, 554 animal models, 543–550, 554 and B-cells, 552, 558–559 and diabetes insipidus, 448 and diabetes mellitus, type 1, 538, 555 diagnosis, 538 etiology, 537–540, 557 and FSH, 538, 539, 556, 557, 559 histology, 552–555 idiopathic, 554–559 incipient, 556, 559 and lupus, 555 and macrophages, 554 and MHC, 546 and monocytes, 559 and myasthenia gravis, 545, 555, 557 and NK cells, 554, 558–559 therapy, 510 Oxygen. See also Free radicals lack of, 661 reactive species, 3, 683 Oxytocin and IL–6, 325 and LIF, 325 and phosphorylation, 343 and pituitary hormones, 23 and self-tolerance, 342–343 PACAP. See Pituitary adenylate cyclase activating polypeptide Pain, 53, 55, 599, 709 Pain control areas, 52 Pancreatic carcinoma, 726 Pancreatic insufficiency, 506 Pancreatic islet ␤ cells, 345–347 Paracoccidioidomycosis, 515 Parasitic infections and diabetes, 430 and leukocytes, 3 and mast cells, 370 and sickness behavior, 715 Th2 response, 295, 651 Parathyroid carcinoma, 726 Parathyroid hormone, 730 deficiency, 503–504 Parathyroid hormone–related protein, 27 Paraventricular nucleus (PVN), 86–87 Parietal cell antibodies, 506 Patch clamp electrophysiology, 261

775 Pathogen-associated molecular patterns (PAMPs), 17, 707, 709 Pattern recognition receptors (PRRs), 16–17, 18, 56 Peptide/MHC complexes, 4 Peptidergic pathway, 360–361 Perforin/granzymes, 18, 463 Peripheral blood lymphocytes, 281 Peripheral blood mononuclear cells (PBMC) and GH/IGF-I axis, 167–171 and glucocorticoids, 648, 738, 740, 743 and HIV, 675, 693–694, 699–702 and melatonin, 636–637 opioid receptors, 694, 699–702 and prolactin, 215, 217 and signaling, 136 and somatostatin, 194, 198 and substance P, 638 and urocortin, 654–655 and vitamin D3, 656–658 Peripheral blood polymorphonuclear cells (PBPC), 635 Peripheral immune tolerance, 19 Permeability of blood vessels, 55, 309–315, 343, 364 of intestinal wall, 367 Pernicious anemia, 506, 509, 510 Peyer’s patches, 195 P450C21, 93 P450 c11 ␹-hydroxylase, 73 p59FYN, 34–35 P-glycoprotein 170, 740, 741. See also Multi-drug resistance (MDR) proteins PHA. See Phytohemagglutin Phagocytes, 3, 16, 610, 707 Phagocytosis and aging, 610 and cell types, 3, 4, 14, 359 in innate response, 16, 175 Phenytoin, 516 Phorbol mristate acetate (PMA), 695 Phosphatidylinositol–3-kinase (PI3K), 34 Phosphodiesterase, 635 Phosphorylation of CREB, 246 of GC receptor, 739 and HIV dementia, 680 and IGFs, 165

776 [Phosphorylation] and natriuretic peptide, 310 and opioid receptors, 699, 703 and oxytocin, 343 and prolactin, 214–215 and signaling pathways, 11–12, 32–35, 136 Phylogeny, 358 Phytohemagglutin (PHA), 167, 169, 265 Pigmentation, 497 PI3K, 34 Pineal gland, 637 Pituitary adenylate cyclase activating peptide (PACAP), 23, 26, 242, 640. See also Vasoactive intestinal peptide Pituitary adenylate cyclase activating peptide (PACAP) receptors, 243–244, 291, 296 Pituitary gland and cytokines, 88–90 and diabetes insipidus, 442, 443, 445, 452, 454 and dopamine, 36 and growth hormone, 168–169 and IFN-␥, 36 and leptin, 94 and mast cells, 369 and POMC, 632 Pituitary hormones and aging, 610 GH and IGF-I effects, 175 IFN-␥ effect, 36 regulation, 23 Plasmatic osmolality, 521 Platelet-activating factor (PAF), 680 Platelet-derived growth factor (PDGF), 171 Platelets, 27, 653 Pollinosis, 623 Polyendocrinopathy, 503–509 Polyglandular syndromes. See Autoimmune polyglandular syndromes Polymorphonuclear leukocytes and catecholamines, 653 description, 3–4, 16. See also Leukocytes and somatostatin, 27 Polymyalgia rheumatica, 613 POMC. See Proopiomelanocortin Potassium, 521 Pregnancy and Addison’s disease, 497

Index [Pregnancy] and B-cells, 222 and diabetes prediction, 424–425 and glomerulonephritis, 663 and glucocorticoids, 66, 399 and Graves’ disease, 473 and IFN-␥, 663 and IgG, 5 and interleukins, 663 and lupus, 127, 663 and multiple sclerosis, 128, 663 and PRL, 24, 222–225 and rheumatoid arthritis, 128, 223, 662–663 and Th2 response, 655 and thymus, 324 and thyroid autoimmunity, 473 Pregnane X receptor (PXR), 405 Premature ovarian failure. See Ovarian failure Prenatal infection, 424 Preprocalcitonin (PPCT), 729 Priming, 362–363 PRL. See Prolactin PRL-releasing peptide (PrRP), 33 Progesterone, 655, 656 Progesterone receptors, 213 Pro-inflammatory cytokines and aging, 612 and aromatase, 597 and autoimmunity, 550 and ␤2ARs, 653 and catecholamines, 651 and dendritic cells, 723 and GH/IGF-I axis, 177–178, 179, 181 and glucocorticoids, 399 and HIV, 683 and hypothalamic-testicular pathway, 111–116 and LPS, 108 and NGF, 365 and pregnancy, 663 and substance P, 638 synergies, 648 synergy, 648 and testes, 109–111 and VIP/PACAP, 246, 248–249 Prolactinomas, 222 Prolactin (PRL) and adaptive immune response, 23, 24

Index [Prolactin (PRL)] and autoimmune disorders, 222–225 and B-cells, 27, 28, 31, 135–137, 138, 210, 216, 222 deficiency effects, 218–219, 221 and dendritic cells, 137, 216 and dopamine, 23, 24, 35, 36–38 and estrogen receptors, 213 excess, 219–220, 221–222, 224 genetics, 30, 210–212, 215, 217–221, 223 and Graves’ disease, 224 and growth hormone, 24, 27, 221 and Hashimoto thyroiditis, 224 hematopoesis, 225, 226 and IFN-␥, 24, 36, 217, 224, 227 and immune system cells, 24, 216–217 immunoregulatory effects, 29–30, 217–222, 228–229 and interleukins IL–1, 220 IL–2, 227 IL–4, 29–30, 210, 223 IL–6, 36, 220, 325 IL–10, 223, 227 IL–12, 216 and Jak/STAT pathway, 36–38 and leukemia, 225–226 and lupus, 136–137, 138, 222–223 and lymphocytes, 27 and lysis, 225 and macrophages, 216, 219, 220 and malignancies, 225–226 and MAPK, 213, 215, 228 multiple sclerosis, 224 and neuroendocrine system, 29–30 and NF-␬B, 213–214 and NK cells, 29–30, 217, 225, 227 and PBMCs, 215, 217 and phosphorylation, 214–215 and rheumatoid arthritis, 223–224 and signaling, 29–35, 136, 213–215, 220–221, 226–229 sources, 208–210 and stress, 24, 30 and T-cells, 27, 29–31, 34, 210, 213, 217, 227 and Th1 cells, 223, 227 and thymocytes, 323, 330 and TNF-␣, 210, 213, 220 Prolactin receptors (PRLR), 136 expression sites, 17, 212–213, 217

777 [Prolactin receptors (PRLR)] and leukemic cells, 225 and leukocytes, 213, 214–215 and signaling, 31, 34, 136 signal transduction, 213–215 structure, 28, 210–212 Proopiomelanocortin (POMC) and atopic dermatitis, 632–634 and CRH, 52–53 derived peptides, 25, 32, 36, 634 and LIF, 89 and pituitary gland, 632 Prostaglandin E2 (PGE2) and fever, 709 and NGF, 363 and TNF-␣, 361 Prostaglandins, 86, 87 and glucocorticoids, 399 role, 395 and testes, 112 Prostaglandins (PGE2), 171 Prostate cancer, 725 Protease-activated receptors (PAR), 362 Protease inhibitors, 684 Protein kinase A (PKA), 26–27 ␤AR–adenylyl cyclase–cAMP-PKA cascade, 659 Protein kinase C (PKC), 35, 695 Proteins. See also Complement proteins activator protein (AP)–1, 56, 738 acute phase proteins, 23 Ag purified protein derivative (PPD), 74 antigen presenting, 1, 9–11 CDs, 9–13 cell-associated, 9–13 co-stimulatory, 12–13 CREB-binding, 246 excitory amino acids, 676, 677 gp120, 675, 676, 678, 681, 683 heat shock, 310, 433, 573 HIV–1 accessory, 404, 681 gp120, 675, 676, 678, 681, 683 macrophage-inflammatory, 224, 676, 679, 684 MDR, 405, 740, 741 in milk, 429 monocyte chemoattractant (MCP–1), 311–312, 399, 675, 680 multi-drug resistance (MDR), 405, 740 parathyroid hormone–related, 27 retinol-binding, 653

778 [Proteins] steroidic acute regulatory (StAR), 93, 111, 116, 121 transporters, 404–405 vasoactive, 4 PRRs. See Pattern recognition receptors Pruritis, 638. See Atopic dermatitis PSD–95 protein, 683 Pseudorabies virus (PRV), 118–119 Psoriasis, 390, 634 and melatonin, 637 and NGF, 639 and vitamin D3, 658 Psychotropic drugs, 226 p38 MAPK. See Mitogen-activated protein kinases (MAPK) Ptosis, 572 p24 antigen, 693–694, 700 Puberty, 118, 518 Quality of life, 623–624 questionnaires, 619–621 Radioactivity, 474 Radionucleotides, 730 RAG genes, 339–340 Raloxifen, 134 RANTES and glucocorticoids, 69, 399 and HIV dementia, 676, 680, 684 Rapamycin, 151–153 Ras, 34, 699 Ras/Raf/MAPK pathways, 32–33 Reactive nitrogen species, 17 Reactive oxygen species, 3, 683. See also Free radicals Rearrangement excision circles (TRECs), 339–340 Receptor autoantibodies, 557–558 Receptor molecules, 11 Regulatory mechanisms, 18–19 Regulatory T-cells (TREG), 342 Renal cancer cells, 725 Renal failure, 181 Resistant ovary syndrome, 539, 544 Respiratory distress syndrome, 742 Reticulin, 506 Retinoic acid, 210 Retinol-binding protein, 653 Retroviruses, 659, 673–685, 729 Reverse transcriptase inhibitors, 684 Rheumatic diseases, 506

Index Rheumatoid arthritis in children, 177, 181 and cortisol, 594, 661 and CRH, 54, 594 and estrogen, 128, 596, 598, 656 and glucocorticoids, 66, 399, 403, 741 and gonadal hromones, 596–598 and HPA axis, 593, 594–596, 661 and IL–1␤, 596 and IL–6, 594, 597–598 and MDR proteins, 405 and MIP, 224 and neurotransmitters, 598–600 and norepinephrine, 598–600 pathogenesis, 593–594 and pregnancy, 128, 223, 662–663 and prolactin, 223–224 and somatostatin, 197, 200–202 and stress, 594, 661–662 and stress response, 594, 661–662 and substance P, 598–599, 662 and testosterone, 596, 597–598 and Th2 response, 661 and urocortin, 654–655 and VIP, 598 and VIP/PACAP, 249–250 Rheumatoid factors, 555 Rhinitis and leukotrienes, 623 medication, 622–623 and quality of life, 619–620, 621–623 and tryptase inhibitors, 362 Rhodopsin, 28 Ribavirin, 475 Rickets, 154–155 Rifampin, 516 RNA polymerase II (RNPH), 739 Ro 20–1724, 652 Rsk, 35 RU 486, 67, 91, 400 Ryanodine, 580, 581

Salbutamol, 662 Salmonella typhimurum, 6, 400 Sarcoidosis, 201, 516 Scavenger receptors, 56–57 Schilder’s disease, 517 Schistosomiasis, 295

Index Schmidt’s syndrome (APS–2), 509–511, 513, 523–524 SCID (Severe combined immunodeficiency syndrome), 172, 682 Scleroderma, 223 Sclerosis, systemic, 633 SDF–1, 676, 678, 682 Selectins and adhesion, 13, 312–313 and atopic dermatitis, 633 and glucocorticoids, 399 and MSH, 633 and natriuretic peptide, 312 and substance P, 638 Selective estrogen receptor modulators (SERMs), 134–135 Selegiline, 684 Self-antigens, 340–349 Self recognition, 17, 342 Self-tolerance, 19, 339–349, 390, 548–549. See also Autoimmunity Self-vaccination, 347–349 Sensory neurons and inflammation, 358 and mast cells, 367 and rheumatoid arthritis, 599, 662 and substance P, 638 and TNF-␣, 361, 363 Sepsis. See also Tissue damage after injury, 659–661 and HG/IGF-I axis, 177 and leptin, 85, 94 Septic shock and Addison’s disease, 516 and glucocorticoids, 400, 742 and LIF, 53–54 and natriuretic peptide, 315 toxic shock syndrome, 248–249 and VIP/PACAP, 248–249 Serotonin depression mechanism, 715 and histamine, 363, 370 and IL–16, 277 and inflammation, 276–277 and mast cells, 364, 368 T-cell receptors, 266, 275 Sertoli cells, 111 Severe combined immunodeficiency syndrome (SCID), 172, 682 SH2-containing phosphatase (SHP2), 34–35 Sheehan’s syndrome, 221 Shock, 25. See also Septic shock

779 Sialo-adenitis, 542 Sickness behavior mechanisms, 707–714 uses, 714–715 Signaling and B-cell receptors, 11–12 to brain, 709 by chemokine receptors, 678, 681–682 by cytokines, 709 dendritic and T-cells, 548, 550 to dendritic cells, 381 GC receptors, 74–75, 740 of growth hormone, 177 immune system, 11–12, 18, 31–38 leptin, 84–85, 91 mast cell–nerve communication, 367–371 neuroendocrine system, 22–25, 31–38 and neurotransmitters, 35, 36–38, 268 opioid receptor-mediated, 698–699 prolactin role, 29–35, 213–215, 220–221, 226–229 somatostatin role, 194, 279 T-cell receptors, 74–75 by Toll-like receptors, 17 and VIP, 26 Signaling pathways. See also Fas/FasL pathway; Jak/STAT pathway ␤AR–adenylyl cyclase–cAMP-PKA, 569 brain and testes, 118–121 glucocorticoid, 599, 737–740 for HIV dementia, 679, 680 IL–3, 34 Jak-STAT, 31–32, 399. See also Jak/ STAT pathway JNK, 26, 33, 699 and neuroendocrine system, 31–38 p59FYN, 34–35 and phosphatidylinositol–3-kinase/Akt, 34 and phosphorylation, 11–12, 32–35 p38 MAPK/NF-␬B, 23 Ras/Raf/MAPK, 32–33 SH2-containing phosphatase, 34–35 for sickness behavior, 708–711, 714 thymic, 34–35 vav, 34–35 ZAP–70, 34–35 Signal transducers and activators of transcription (STATs), 56, 738 Stat5, 216, 220, 226 –228. See also Jak/ STAT pathway

780 Sinusitis, 622. See also Rhinitis Sjo¨gren’s syndrome, 506, 598 Skin abscesses, 16 atopic dermatitis, 632–640 catecholamine release, 634–636 corticotropin/proopiomelanocortin, 632–634 eczema, 55, 390 erythema, 638 and hemochromatosis, 516 pigmentation, 497 and VIP/PACAP, 640 vitiligo, 448, 506, 509 Smith–Lemli–Opitz syndrome, 519 Social withdrawal, 710 SOCS–3, 227–228 Sodium, 277, 305–315, 521. See also Atrial natriuretic peptide Sodium iodide symporter (NIS), 462, 466, 473 Somatomedin hypothesis, 164 Somatostatin analogs, 193, 199, 200–201 and antitumor immunotherapy, 728 and growth hormone, 164 and IFN-␥, 198 immune system sources, 196–198 and inflammation, 84, 197–200 and interleukins IL–2, 198, 274, 279–281, 324 IL–8 and IL–6, 199, 201 and leukocytes, 27, 200 and lymphoma, 200 and macrophages, 197, 199 and MAPK, 194 and mast cells, 27 and monocytes, 199 and PBMCs, 194, 198 in platelets, 27 and rheumatoid arthritis, 197, 200–202 signaling role, 194, 279 and spleen, 195, 197, 200 and substance P, 199 and T-cells, 195, 197, 198–199, 278–281 therapeutic use, 200–202 and thymocytes, 324, 325 Somatostatin receptors, 194–196, 198–201, 275 SOX9, 506 SOX10, 506

Index Spleen and APS–1, 506 and B cells, 14, 128–129 and melatonin, 636–637 and neurotransmitters, 265 and somatostatin, 195, 197, 200 and stress, 664 Splenocytes, 696 Stat5, 216, 220, 226 –228 Stem cell factor (SCF), 359, 362–363 Stem cells, 200 Steroidic acute regulatory protein (StAR), 93, 111, 116, 121 Steroid-producing cell autoantibodies (StCAs), 500, 506, 551–552, 559 Stomach, 506, 510 Streptococcal cell-wall (SCW) polysaccharide, 66–67, 90, 400 Stress. See also Catecholamines; Glucocorticoids acute vs. chronic, 398, 402 and Addison’s disease, 497 and allergic reactions, 55, 663–664 animal model, 542–543 and atopic dermatitis, 631, 633–634 and catecholamines, 650–652 and common cold, 659 and cytokines, 53–54, 648–652 and dendritic cells, 390 and estrogen, 656 and glucocorticoids, 30, 38, 648–653 and HPA axis, 52–54 and interleukins, 53, 653 and leptin, 91–94 and LIF, 53 local effects, 652–653 and lupus, 661 and macrophages, 653 and mast cells, 366–367 and multiple sclerosis, 661–662 neuroendocrine-immune interaction, 36–38, 51–53, 265, 366 and neurotransmitters, 265 and PRL, 24, 30 research history, 66 and rheumatoid arthritis, 594, 661–662 and Th2 response, 648–652, 659–664 and TNF-␣, 653 and tumors, 664 Stroop color–word test, 662

Index Substance P and allergic reaction, 366 and atopic dermatitis, 637–639 and CGRP, 371 and glucocorticoids, 400 and HPA axis, 52 and IFN-␥, 281, 638 and IL–1, 631 and IL–2, 274, 281–282 and IL–4, 270, 638 and leukocytes, 638 and LPS, 637 and mast cells, 359–361, 363, 364, 369, 370, 638, 653 and MHC–2, 282 and monocytes, 599 and neutrophils, 638 and NGF, 639 and PBMCs, 638 and peripheral blood lymphocytes, 281 priming with, 363 receptor subtypes, 275 and rheumatoid arthritis, 598–599, 662 role, 281–282, 395 and selectins, 638 and somatostatin, 199 and Th1/Th2, 270, 281 and thymus, 343 Suramin, 516 Surfactants, 653 Sympathetic nervous system aging effect, 608–609 and HIV, 659 and mast cells and macrophages, 653 and multiple sclerosis, 661–662 and rheumatoid arthritis, 598–600, 661–662 and Th1/Th2, 390 Synergies with AVP, 441 cortisol and norepinephrine, 599 with glucocorticoids, 70–72 with growth hormone, 173, 177 in pro-inflammatory cytokines, 648 Tat/TNF-␣, 683 with vitamin D3, 151–154 Syphilis, 515 Systemic lupus erythematosus and apoptosis, 19 and atherosclerosis, 612 and AVP antibody, 447

781 [Systemic lupus erythematosus] and complement proteins, 9 DHEA therapy, 612 and estrogen, 130–135, 137–138, 655, 656 and glucocorticoids, 68, 399, 403–404, 740 and MDR proteins, 405 and myasthenia gravis, 573 and NGF, 639 and ovarian failure, 555 and pregnancy, 127, 663 and prolactin, 136–137, 138, 222–223 and stress, 661 and Th2 response, 661 and vitamin D3, 151 Systemic sclerosis, 634 Tachykinins. See also Neurokinin A; Substance P and mast cells, 358, 360, 367 receptors, 281 and self-tolerance, 343 subtypes, 281 Tamoxifen, 134–135, 137, 138 Tat, 676, 682, 683 TATA-box binding protein, 246 T cell–independent (TI) responses, 3 T-cell receptors (TCRs) costimulation, 56 and diabetes, 433 genes, 319, 339 and glucocorticoids, 74–75 and MHC, 12, 15–16 for neurotransmitters, 266 and opioid receptors, 693, 699 rearrangement excision circles (TRECs), 339–340 and serotonin, 266, 275 and signaling, 11–12, 56, 74–75 TEC. See Thymic epithelial cells Tec signaling pathway, 34–35 Teeth, 506 Testes. See also Gonads and brain, 118–121 and catecholamines, 114–116 and hypothalamus, 116–120 and IL–1␤, 111–116 and LPS, 109–111 Testosterone and Addison’s disease, 521

782 [Testosterone] and aging, 610 and autoimmunity, 131 of thyroid, 472–473 and hypothalamic-testes path, 109–121 and macrophages, 596 and rheumatoid arthritis, 596, 597–598 Th0 cells, 279, 648 Th1 cells. See also Cell-mediated immunity; T-helper cells, subtypes and adenosine, 658 aging effect, 610, 611–612 and antitumor immunotherapy, 726, 729 and autoimmunity, 661 ␤-adrenergic agonists, 382, 651 and cancer, 664 and catecholamines, 651 and CRH–mast cells–histamine, 653–655 and delayed-type hypersensitivity, 72 and diabetes, 424, 430, 433 and epinephrine, 650 and estrogen, 653, 655, 656 and glucocorticoids, 19, 72, 399, 648–650 and histamine, 652 and HIV, 659 and hygiene hypothesis, 430 and interleukins, 18, 648 and multiple sclerosis, 465 and neuropeptides, 270, 280 and norepinephrine, 266, 389, 550, 650 and progesterone, 655 and prolactin, 223, 227 response description, 18 and substance P, 270 and thyroid autoimmunity, 465–467, 475 and TNF-␣, 648 and VIP/PACAP, 247–248, 249, 253, 292–295 and Vitamin D3, 151, 656–658 Th2 cells. See also Humoral immune response; T-helper cells, subtypes and adenosine, 658 aging effect, 610, 611–612 and allergic reactions, 227, 663–664 and asthma, 663 and atopic dermatitis, 640 and autoimmune diseases, 661 and catecholamines, 651 and CRH–mast cells–histamine, 653–655 and diabetes, type 1, 661 and estrogen, 653, 655, 656

Index [Th2 cells] and glucocorticoids, 19, 72, 399, 648–650 and histamine, 652 and hygiene hypothesis, 430 and IFN-␥, 18 and IL–12, 648, 659 IL–10–pretreated DC prime, 723 and lupus, 661 and monoclonal antibodies, 730 and multiple sclerosis, 465, 661 and mycobacterial infections, 659 and neuropeptides, 270 and norepinephrine, 266, 390 and parasitic infections, 295, 651 and progesterone, 655 and prolactin, 223, 227 response description, 18 and rheumatoid arthritis, 661 and stress, 648–652, 659–664 and substance P, 281 and Th1 cells, 18 and thyroid autoimmunity, 465–467, 661 and traumatic injury, 661 and tuberculosis, 659 and VIP/PACAP, 247–248, 249, 292–294, 297–298 and Vitamin D3, 151, 656–658 Th3 cells, 19 TH1 cytokines, 6, 18, 19. See also Granulocyte-macrophage colony stimulating factor; Interferon-␥; Tumor necrosis factor (TNF) TH2 cytokines, 6, 19, 58. See also Interleukin–4; Interleukin–10 T-helper cells. See also Th2 cells and ACTH, 25 in adaptive immune response, 18 and antigen-presenting proteins, 9 apoptosis, 297 and APS–2, 513 and autoimmunity, 548 and autoreactive B-cells, 138 and cortisol, 612 and cytokines, 6, 18, 294–296 cytotoxicity in, 296 and dendritic cells, 722 development, 15–16, 339 and diabetes insipidus, 445 and estrogen, 653, 655, 656 function, 2, 3, 294–296, 389–390

Index [T-helper cells] and glucocorticoids, 19, 70, 72, 174, 648–650 and growth hormone, 180 and HIV, 700–702 and IFN-␥, 18, 648, 659 and IGF-I, 173 and IL–12, 18 and melatonin, 636 and MHC proteins, 11, 12 and myasthenia gravis, 573 and neuroendocrine tumor, 726 and neurotransmitters ␤-adrenergic receptors, 266, 270, 651 dopamine, 269–270 neuropeptide stimulation, 280 and serotonin, 277 somatostatin, 196, 279–281 VIP/PACAP, 247–248, 249, 253, 292 and norepinephrine, 612, 650 opioid receptors, 694, 695, 696, 702–703 and ovarian failure, 544–545, 554 and prolactin, 213, 216, 227 subtypes. See also Th1 cells; Th2 cells clinical implications, 658–664 and CRH–mast cells–histamine, 653–655 and dendritic cells, 389, 390 differentiation, 72, 227–228, 247–248, 291–295, 390, 399, 648 and hygiene hypothesis, 430 local vs. systemic effects, 648–652, 652–653 Th0, 279, 648 Th3, 19 and thyroiditis, 461, 469–470 tumor-specific, 664 and vitamin D3, 147, 148, 149–154 Thymic epithelial cells (TEC) and extracellular matrix, 326–328 and glucocorticoids, 73 receptor expression, 321–324 and self-recognition, 339–346 and signaling, 34–35 Thymic nurse cells (TNC), 321, 339–342 Thymitis, 579 Thymocytes differentiation, 319–325, 343 and estrogen receptors, 330 GH and IGF-I effects, 173–174, 180, 323–325

783 [Thymocytes] and glucocorticoids, 74–75, 323, 324, 343 and IL–7, 321, 339 migration, 321, 327–328 opioid receptors, 696 and prolactin, 210 receptor expression, 321–325 and somatostatin, 324, 325 and VIP, 290 Thymomas, 580–582 Thymosin, 24–25, 321 Thymulin, 24–25, 326 Thymus atrophy, 580 and chemokines, 322, 338 developmehntal biology, 338–339 and diabetes type 1, 345–349 and glucocorticoids, 51, 72–73, 325 and hormone release, 24–25 and immune system, 13, 15 and insulin-related genes, 344–347 MHCs, 13–14 microenvironment, 320–321 and myasthenia gravis, 576–577, 580 and natriuretic peptides, 308 neuroendocrine control, 329–330 and neurotransmitters, 265 and somatostatin, 195 and self-reactive T-cells, 340–342 and substance P, 343 thymectomy models, 545–546 and thyroid hormones, 473 Thyroglobulin (Tg), 462, 464, 468, 473–474, 475 Thyroid, autoimmune. See also Graves’ disease; Hashimoto thyroiditis and estrogen/androgens, 472 genetics, 462, 471–472, 475 and iodine, 473 and pregnancy, 473 and T-cells, 467–471, 473 and T-helper cells, 461, 469–470 Th1/Th2, 465–467, 475, 661 vaccine, 476 Yersinia enterocolitica, 473 Thyroid carcinoma, medullary, 726–729 Thyroiditis. See also Hashimoto thyroiditis and Addison’s disease, 509 animal models, 541–542 and HPA axis, 402

784 [Thyroiditis] and IL–10, 466, 467, 476 and MHC–2, 473, 475 and ovarian failure, 538 and TEC, 346 Thyroid peroxidase (TPO), 462, 464, 466, 468, 470, 473–474 Thyroid-replacement therapy, 510 Thyroid-stimulating antibodies (TSAB), 463, 467, 470 Thyroid stimulating hormone (TSH), 168 Thyrotropin receptor (TSHR), 462, 466, 467–468, 473 Thyrotropin-releasing hormone (TRH), 26, 33, 224 Thyrotropin (TSH), 26, 462, 466 agonists, 463, 467 Thyroxine (T4), 462 Tissue damage, 53–54, 651–652 Tissue removal, 56–57 Tissue repair, 371 Titin, 580, 581 TLR4, 708, 710 T lymphocytes. See also Apoptosis activation, 198, 695, 699 in adaptive immune response, 4, 18–19 and Addison’s disease, 495, 501 anergy, 723 and antigen-presenting proteins, 9 apoptosis and dopamine, 276 and glucocorticoids, 36, 70 and growth hormone, 174 and IGF–1, 179 and ovarian failure, 550 and VIP/PACAP, 640 and vitamin D3, 151 autoreactivity, 148 and B-cells, 3, 129, 136–137 CD4Ⳮ. See T-helper cells CD8Ⳮ. See Cytotoxic T cells co-stimulatory proteins, 12 and cytokines, 6, 16, 18–19 development, 13–16, 72–75, 173, 319–321, 339–342 differentiation, 18, 72–75, 291–295, 339–342 and estrogen, 325 function, 1–2, 13, 294–297 and glucocorticoids, 58–59, 71, 72–75

Index [T lymphocytes] and growth hormone, 31, 166–167, 173 helper vs. cytotoxic, 2, 12. See also Cytotoxic T-cells; T-helper cells homing ability, 16 and IGF-I, 173, 174, 179 and IL–2, 6, 18, 26, 269–270 and leptin, 85 memory vs. naı¨ve, 12, 19, 297–298, 461 and MHC, 9, 10–11, 12, 15–16 mitogenic response acetylcholine, 265, 278 and growth hormone, 167, 169 and IGF–1, 171 and prolactin, 29–30 somatostatin effect, 279 and myasthenia gravis, 576, 578, 580–581 and neurotransmitters acetylcholine, 265, 278 dopamine, 270–273, 275–276 glutamate, 277–278 GnRH, 284 outcome factors, 268–275 serotonin, 276–277 somatostatin, 195, 197, 198–199, 278–281 specific neurotransmitters, 266–267 substance P, 281–282 VIP/PACAP, 291–298 and norepinephrine, 266, 270, 276 opioid receptors, 693, 695, 699, 703 and ovarian failure, 544–545, 554, 558–559 in postmenopausal women, 558 and PRL, 27, 29–31, 34, 210, 213, 217, 227 proliferation inhibitors, 71 regulatory T-cells (TREG), 19, 342 and rheumatoid arthritis, 250 RT6Ⳮ, 550 self-reactive, 340–342 and self-tolerance, 548–549 and tamoxifen, 134, 135 and thyroid autoimmunity, 467–471, 473 unusual subsets, 17 and vitamin D3, 146, 148, 152, 154 TNF-␣. See Tumor necrosis factor

Index Tolerance and antigen-presenting cells, 548 and apoptosis, 19 of autoreactive B-cells, 135, 138 and dendritic cells, 550 and diabetes, type 1, 433 to endotoxins, 94–95 of grafts, 148–151 of self, 19, 339–349, 390, 548–549 Toll-like receptors (TLRs) activation, 17 and glucocorticoids, 57 and lipopolysaccharides, 57 and sickness behavior, 708 Toxic shock syndrome, 248–249 Tr1, 19 Transforming growth factor ␣ (TGF-␣), 70 Transforming growth factor ␤ (TGF-␤) and adaptive immune response, 19 and glucocorticoids, 70 and prolactin, 225 and Th1/Th2 cells, 652 and tumors, 664, 723 Transplants, bone marrow, 174, 180, 419 Transport proteins, 404–405 Trauma, 659–661. See also Stress TRH. See Thyrotropin-releasing hormone Triiodothyronine (T3), 323, 326, 462 Triple A syndrome, 518 Tryptase, 362, 369 Tryptophan hydroxylase, 506 Tuberculosis, 200, 513–515, 659 Tumor-associated antigens, 390–391 Tumor necrosis factor-␣ (TNF-␣). See also Pro-inflammatory cytokines and adenosine, 658 and adrenal glands, 90 and aging, 610 and autoimmune disorders, 661, 699 and bone loss, 656 and catecholamines, 651 and dendritic cells, 723 and diabetes insipidus, 440–441 and eicosanoids, 361 and estrogen, 596, 655, 656 and Fas/FasL pathway, 464 and fever, 23 and glucocorticoids, 58, 71, 738 and growth hormone, 175, 177 and histamine, 652

785 [Tumor necrosis factor-␣ (TNF-␣)] and HIV dementia, 675, 677, 679, 680, 683 and HPA axis, 52, 53 and IFN-␥, 175 and IGF–1, 171, 178 and IL–1␤, 171 and leptin, 84 and LHRH, 108 and LPS, 23, 94, 109–111 and mast cells, 361, 363 and melatonin, 636 and MSH, 633 and natriuretic peptide, 309–315 neuroendocrine effects, 24, 53 and neurons, 361, 363, 683 and NGF, 363 and pregnancy, 663 and progesterone, 655 and prolactin, 210, 213, 220 and sickness behavior, 708, 711–712 and somatostatin, 199 and stress, 653 and Tat, 683 and Th1 response, 648 and traumatic injury, 661 and VIP/PACAP, 249, 266 PACAP alone, 640 and vitamin D3, 146 Tumor necrosis factor-␣ (TNF␣), 68 Tumor necrosis factor promoters, 743 Tumors of adrenal glands, 515 carcinoid, 728 and immunity against, 4, 225, 390–391 immunotherapy with cytokines, 727–728 with dendritic cells, 724–727 gene therapy-induced, 728–729 monoclonal antibodies, 729–730 and interleukins, 664, 723 localization of, 200 multiple endocrine, type 2 (MEN 2), 726 and myasthenia gravis, 580 neuroendocrine, 725–729, 730 and stress, 664 vaccines, 390–391, 723–724, 725–727 Turner’s syndrome, 175, 556 Tympanic membrane, 506 Tyrosine hydroxylase (TH), 35

786 Tyrosine kinases Jak family, 31–32 muscle-specific receptor (MuSK), 573, 575 and opioid receptors, 699 U50,488, 702 Ulcerative colitis, 54, 404, 612, 741 Ultraviolet radiation (UVA), 638 Urocortin, 114, 654–655 Uterus, 217 Uveitis, 653 Vaccines administration route, 390 for cancer, 390–391, 723–724, 725–727 for diabetes, type 1, 347–349, 433 DNA, 728–729 for thyroid disorders, 476 Vagus nerve and capsaicin, 364 and cytokines as hormones, 395 and IL–1, 87–88 and LPS, 87–88 and mast cells, 367, 370 and sickness behavior, 709, 711 Vascular cell adhesion molecule 1 (VCAM–1) and aging, 610, 611 and glucocorticoids, 399 and HIV dementia, 675 and MSH, 633 and NF-␬B, 312 role, 13, 15 Vasoactive amines, 4 Vasoactive intestinal peptide (VIP) and allergic reactions, 295–296, 640 and atopic dermatitis, 638, 640 early research, 242 and Fas, 26–27 and IL–1, 266 and IL–2, 274 and IL–4, 253, 295 and IL–5, 295 and IL–6, 23, 24 and IL–10, 253 and PACAP and atopic dermatitis, 640 autoimmune disorders, 248–255 and Fas, 296–297, 640

Index [Vasoactive intestinal peptide (VIP)] and IFN-␥, 295 and IL–6, 246, 249, 266 and LPS, 247 macrophages, 245–248, 292, 640 NF-␬B, 246, 297 and rheumatoid arthritis, 249–250 and T-cells, 26–27, 291–298 and Th1/Th2, 247–249, 253, 292–295, 297–298 and TNF-␣, 249, 266 receptors, 243–244, 253, 291, 296 and rheumatoid arthritis, 598 signaling role, 26 and thymocytes, 290 Vasoconstriction, 113–114, 633 Vasodilation and aging, 608 by CRH, 55, 653–654 in innate immune response, 16 and mast cells, 367 and polymorphonuclear leukocytes, 4 and somatostatin, 200 Vasopressin, 23, 114, 714 Vav, 34–35 Vectors, 729 Very long chain fatty acids (VLCFA), 517, 521, 524 Viral infections. See also Human immunodeficiency virus and aging, 613 and autoimmunity, 547 and cellular immunity, 4 cytomegalovirus, 67–68, 400, 515 and diabetes, 346, 424, 427–429 and glucocorticoids, 67–68, 400–401 and IFN-␥, 6, 18 and IL–1, 66 and myasthenia gravis, 577 during pregnancy, 424 pseudorabies, 118–119 retroviruses, 659, 673–685, 729 Virion-associated protein (Vpr), 404 Vitamin D3 biological profile, 145–146 deficiency, 154–155 and dendritic cells, 151, 656–658 epidemiology, 154–155 and immune system cells, 146–147 Th1/Th2, 151, 656–658 immunomodulation paths, 147–149

Index [Vitamin D3] and interferons IFN-␤, 154 IFN-␥, 146–148, 151 and interleukins, 146, 147, 151, 656–658 and irritable bowel syndrome, 151 and lupus, 151 and monocytes, 146–147 and NF-␬B, 656–658 and PBMC, 656–658 and PBMCs, 656–658 and pregnancy, 663 synergies, 151–154 synthetic analogs, 149 therapeutic use, 149–151 Vitamin D receptor, 656–658 Vitiligo, 448 and polyglandular syndromes, 506, 509

787 VLA–4, 15 Vpr, 682 Waterhouse–Friderichsen syndrome, 516 Wegener’s granulomatosis, 201 Wheal-and-flare response, 638 Wolman disease, 516 Women. See also Ovarian failure, premature; Pregnancy menstrual disorders, 538 postmenopausal, 558 Xenobiotics, 226 Yersinia enterocolitica, 473 ZAP–70, 34–35 Zidovudine, 684

About the Editors

VINCENT GEENEN, M.D., Ph.D., is Research Director for the National Fund of Scientific Research of Belgium (FNRS) at the Liege Center of Immunology, Professor of Developmental Biology at the University of Liege, and Clinical Head in Endocrinology at Liege University Hospital, B-4000, Liege-Sart Tilman, Belgium. He has given more than 80 invited lectures and authored or coauthored more than 140 papers and book chapters. Dr. Geenen holds memberships in the Endocrine Society, the American Association of Immunologists, the European Neuroendocrine Association, the American Association of Diabetes, and the International Federation of Neuroendocrinology, among many organizations. The recipient of several research awards, associate editor of NeuroImmunoModulation, and review editor for the International Diabetes Monitor, he received the M.D. (1982) and Ph.D. (1987) degrees from the University of Liege, Liege-Sart Tilman, Belgium. GEORGE CHROUSOS, M.D., is Chief of the Pediatric and Reproductive Endocrinology Branch of the National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, Maryland, as well as Professor of Pediatrics at Athens University Medical School, Athens, Greece. He has authored or coauthored over 500 original research articles to date and is among the most highly cited physician-scientists in the world. Dr. Chrousos has received many national and international awards for his contributions to medicine. A Fellow of the American Academy of Pediatrics and the American College of Physicians and member of numerous other professional organizations, Dr. Chrousos received the M.D. (1975) and Sc.D. (1977) degrees from Athens University, Greece.