The Neutrophils: New Outlook For Old Cells

-THE

NEUTROPHILS New Outlook for Old Cells 2nd Edition

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-THE

NEUTROPHILS New Outlook for Old Cells 2nd Edition

editor

Dmitry 1. Gabrilovich University of South Florida, USA

Imperial College Press

Published by Imperial College Press 57 Shelton Street Covent Garden London WC2H 9HE Distributed by World Scientific Publishing Co. Pte. Ltd. 5 Toh Tuck Link, Singapore 596224 USA ofJice: 27 Warren Street, Suite 401-402, Hackensack, NJ 07601 UK ofice: 57 Shelton Street, Covent Garden, London WC2H 9HE

Library of Congress Cataloging-in-Publication Data The neutrophils : new outlook for old cells I editor, Dmitry I. Gabrilovich. -- 2nd ed. p. cm. Includes bibliographical references and index. ISBN 1-86094-472-8 1. Neutrophis. I. Gabrilovich, Dmitry I. QR 185.8.N47N486 2004 612.1'12-dc22

2004059533

British Library Cataloguing-in-Publication Data A catalogue record for this book is available from the British Library.

Copyright 0 2005 by Imperial College Press

All rights reserved. This book, or parts thereoJ may not be reproduced in any form or by any means, electronic or mechanical, including photocopying, recording or any information storage and retrieval system now known or to be invented, without written permission from the Publisher.

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D EDI CAT10N For the people who made this book possible: Yulia, Sonia, Lev, and Jacob

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Cohntents

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Preface Contributors 1

The Remarkable Neutrophil! Developing a Blueprint for Integrated Cellular Signaling 1. Introduction 1.l. Second messengers 1.2. Integrated cellular signaling 2. The life of the neutrophil 2.1. Bone marrow origin of the neutrophil 2.2. The fate of circulating neutrophils 2.3. Neutrophil stimulators 2.3.1. Do diverse biochemical events underlie stimulation? 2.3.2. Functional maturation 2.4. Neu trophil adherence 2.4.1. Biophysical aspects of adherence 2.4.2. Integrins 2.5. Chemotaxis 2.5.1. Rho family GTPases 2.5.2. Rho-kinase 2.6. Neutrophil priming

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1 2 3 3 4 4 6 6 7 8

10 11 12

12 13 13 15

...

viii

Contents 2.7. The respiratory burst: free radicals in biology 2.7.1. The enzyme involved 2.7.2. Back to Rac 2.8. Neutrophil-endothelial cell communication 3. Integrated cellular signaling 4. Molecular mechanisms of cellular activation 4.1. Protein phosphoylation in cellular activation 4.2. The RaclRho equilibrium, revisited 5. A unifying hypothesis 6. Conclusions References

2

The Neutrophil Respiratory Burst Oxidase 1. Introduction 2. NADPH oxidase components 2.1. Flavocytochrome b 2.2. p47phox 2.3. p67phox 2.4. p40phox 2.5. Rac 2.6. Rap1A 3. Oxidase protein binding interactions 3.1. Flavocytochrorne b-p47Phox interactions 3.2. p40PhoX-p47phox-p67Phox interactions 3.3. Rac interactions 4. Model of NADPH oxidase assembly 5. Oxidant production 5.1. Superoxide anion (0;) 5.2. Hydrogen peroxide (H202) 5.3. Hypochlorous acid (HOC1) 5.4. Hydroxyl radical ( H O ) 5.5. Singlet oxygen (102*) and ozone ( 0 3 ) 5.6. Nitric oxide ( N O )and peroxynitrite (ONOO-) 6. Summary Acknowledgments References

15 16 17 18 19 20

23 26 29 30 31

35 36 37

38 44 48 52 54 55 56

56 59 61 63 66

66 67 68 68 70 70 72 72 73

Contents 3

4

Novel Neutrophil Receptors and Their Signal Transduction 1. Introduction 2. Triggering receptor expressed by myeloid cells (TREM) 2.1. Identification of TREMs 2.2. Characterization of TREMl 2.3. DAP12 and its signal transduction 2.4. TREMl ligand(s) 2.5. Biological function of TREMl 3. Toll-like receptor (TLR) 3.1. Expression of TLRs in neutrophils 3.2. Biologic function of TLRs 3.3. TLR signal transduction pathways 3.3.1. MyD88 dependent pathway 3.3.2. MyD88 independent pathway 4. The Fc receptors (FcR) 4.1. Activation and inhibition of FcR 4.2. FcR-mediated signaling in neutrophils References Mechanisms of Neutrophil Migration 1. Introduction 2. Historical perspective on leukocyte adhesion and emigration (1669-1955) 2.1. The first observations 2.2. Mechanistic insight 3. Molecular adhesive events preceding neutrophil transendothelial migration 4. Integrin regulation of neutrophil transendothelial migration 5. Paracellular neutrophil transendothelial migration 5.1. Endothelial cleft organization 5.2. Tight junctions and preferred transmigration sites 5.3. Adherens junctions 5.4. Gap junctions 5.5. PECAM-1 5.6. CD99

ix

85 86 87 88 88

89 90 90 91 92 92 95 95 95 96 97 98 101

105 106 107

107 108 110 112 114 114 115 120 122 123 125

X

Contents 5.7. JAMS 5.8. J A M - A 5.9. J A M - B and JAM-C 6. Transcytotic neutrophil and transendothelial migration 7. Endothelial permeability responses to neutrophil transendothelial migration 8. Concluding remarks Acknowledgments References

126 127 128 128 132

5

Neutrophils and Apoptosis 1. Introduction 2. Neutrophil apoptosis 3. Regulation of neutrophil apoptosis 3.1. Internal control mechanisms 3.1.1. Caspases 3.1.2. Initiation of apoptosis 3.1.3. Mitochondria and Bcl-2 family proteins 3.2. Regulation of neutrophil apoptosis by external mediators 4. Clearance of apoptotic neutrophils 5. Concluding remarks References

153 154 154 156 156 156 158 160 161 163 164 165

6

Regulation of Neutrophil Functions by Long Chain Fatty Acids 1. Introduction 2. Fatty acids 2.1. De novo synthesis 2.2. Diet 2.3. Phospholipase A2 3. Transport and uptake of fatty acids 4. Metabolism of arachidonic acid and other fatty acids 4.1. General 4.2. Metabolism in neutrophils 4.2.1. Acylation into phospholipids and triglycerides 4.2.2. 5-lipoxygenase

169

140 141 141

170 171

172 172 173 175 176 176 178 178 180

Contents 4.2.3. 12-lipoxygenase 4.2.4. 15-lipoxygenase 4.2.5. Cyclooxygenase 4.2.6. w-oxidation 5. Transcellular metabolism 6. Biological properties of arachidonic acid 6.1. Effects on neutrophil adhesion, cell migration

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181 182 182 183 183 184

184

and chemotaxis 6.2. Activation of the NADPH oxidase 6.3. Stimulation of degranulation 7. Effects of 72-3 fatty acids, eicosapentaenoic and docosahexaenoic acid on neutrophils 8. Regulation of neutrophil functions by metabolites of arachidonic acid 8.1. Products of the lipoxygenase pathway 8.2. Products of the cyclooxygenase pathway 9. Relationship between fatty acid structure and biological function 10. Cytokine induced alteration in neutrophil responses to polyunsaturated fatty acids 11. Neutrophil priming properties of fatty acids 11.1. Alteration of responses to fMLP and P M A 11.2. Antimicrobial activity 11.3. Tissue damage 11.4. Cell surface receptor expression 12. Mechanisms of fatty acid-induced neutrophil activation 12.1. Polyunsaturated fatty acids stimulate neutrophils

independently of lipoxygenase and cyclooxygenase pathways 12.2. Differences in metabolism of long chain and very long chain polyunsaturated fatty acids 12.3. Activation of intracellular signals 12.3.1. Mobilization of intracellular calcium 12.3.2. Heterotrimeric G-proteins 12.3.3. Protein kinase C 12.3.4. Activation o f PLA? bu 20:4n-6 and other fattw acids

185 186 186 187

188 189 190 193 194

195 195 196

197 199

199

200 201 201 203 204 204

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Contents

12.3.5. Activation of the M A P kinases 12.3.6. Activation of sphingomyelinase 12.3.7. Phosphatidylinositol3-kinase 12.3.8. Ion channels 12.3.9. Modulation of the activation status of small GTP

binding proteins 12.4. Evidence for an involvement of PKC, ERK, p38 and PI3K in AA-stimulated superoxide production 12.5. Involvement of ERKlIERK2 and p38 in regulating 5-LOX 13. Modulation of TNFR expression 14. Novel polyunsaturated fatty acids 15. Summary Acknowledgments References 7

Cytokine Production by Neutrophils 1. Introduction 2. General features of cytokine production by human neutrophils 3. Production of specific cytokines by neutrophils 3.1. Chemokines 3.2. Proinflammatoy cytokines 3.3. Anti-inflammatoy cytokines 3.4. Cytokine inhibitors 3.5. Growth factors 4. Cross-talk with others cells 5. Patterns of cytokines production in human neutrophils 5.1. Degranulation 5.2. De novo protein synthesis 5.3. Shedding of membrane-bound cytokine 5.4. Expression of receptor-bound cytokine 5.5. Modulation of PMN-derived cytokine release using various mechanisms 6. Conclusion References

206 207 207 209 209 210 213 213 214 215 218 218 229 229 230

231 231 234 237 239 240 242 243 243 245 246 246 247 248 249

Contents 8

9

Neutrophils in Viral Infections 1. Introduction 2. Inhibition of viruses by neutrophils 2.1. Viral inactivation by oxygen intermediates 2.2. Antibody and complement induced viral inactivation 3. Activation of neutrophils by viruses 3.1. Activation by binding of virus 3.2. Adherence of neutrophils to infected cells 3.3. Activation of oxidative burst activity 3.4. Role of cytokines in neutrophil activation 4. Neutrophil functions inhibited by viruses 5. Neutrophils and influenza A virus 6. Neutrophils and HIV 6.1. Myelodysplastic changes in HIV infection 6.2. HIV infection of neutrophils 6.3. Anti-neutrophil antibodies in HIV infection 6.4. Neutrophil chemotaxis in HIV infection 6.5. Abnormalities in respiratoy burst activity 6.6. Neutrophil cytotoxieity in HIV infection 6.7. Neutrophil defensins inhibit HIV 7. Conclusion References Polymorphonuclear Neutrophils and Cancer: Ambivalent Role in Host Defense Against Tumor 1. Neutrophils are able to promote carcinogenesis 1.1. PMNs may contribute to inflammation associated with tumor development 1.2. PMNs involvement in infection associated carcinogenesis 1.3. Myeloperoxidase and cancer 1.4. Chemokines regulate neutrophil infiltration and activity 1.5. PMNs can promote tumor metastases 2. The role of neutrophils in antitumor reactions 2.1. PMN-mediated tumor destruction 2.2. Cytokine and chemokine-induced PMN anti-tumor activity

xiii 253

253 255 256 257 257 258 259 259 259

260 261 263 2 64 2 64 2 65 2 65 266 267 268

268 269 275

276 2 76

277 2 79 280 281 283 283 286

xiv

Contents 2.3. Neutrophils as effectors of antibody-dependent

290

cell-mediated cytotoxicity against tumor 3. Conclusion

References 10 Use of Colony-Stimulating Factors for Treatment of Neutropenia and Infectious Diseases 1. Characteristics of G-CSF, GM-CSF and its receptors 2. Neutrophil and monocyte development and function 3. Measurement of CSF levels in patients with neutropenia and infectious diseases 4. G-CSF in nonneutropenic animal models of infection

Neonatal sepsis Burn wound injuy Surgical wound infection Bacteremia Intraabdominal infection Pneumonia 5. Clinical studies of the CSFs in infectious diseases 5.1. Neu tropenia 5.2. G-CSF in nonneutropenic patients with pneumonia References 4.1. 4.2. 4.3. 4.4. 4.5. 4.6.

11 Neutrophil Transfusion Therapy in the G-CSF Era 1. Introduction 2. Therapeutic neutrophil transfusions 2.1. Historic experience with therapeutic PMN transfusions

292 294

301 302 303 308

310 311 312 312 313 315 315 318 318 319 322 327 327 329 329

in neutropenic patients 2.2. Modern experience with therapeutic PMN transfusions

331

in the G-CSFera 2.3. Therapeutic PMN transfusionsfor neonatal sepsis

3. Prophylactic neutrophil transfusions 3.1. Historic experience with prophylactic transfusions

337

337 33 7

in neutropenic patients 3.2. Modern experience with prophylactic PMN

transfusions in neutropenic patients

338

Contents

xv 339

4. Methods for PMN collection and transfusion 4.1. Preleukapheresis donor stimulation 4.2. Leukapheresis techniques 4.3. Erythrocyte sedimenting agents 4.4. ?).ansfusion of PMN concentrates

339 340 341 343

Acknowledgments References

344 344

Index

349

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Prefaccce Preface

Five years ago in the preface to the first edition of this book, I wrote about a revived interest in the neutrophil in recent decades. The last five years have witnessed even more remarkable upswing in interest in this cell. Neutrophil has become a test ground for new hypotheses in signal transduction, reactive oxygen production, and mechanisms of cell motility and adhesion. The results of these studies have broad implications for our understanding of the many biological processes. An emerging understanding of the close link between innate and adaptive immunity puts neutrophil into a prominent position in the initiation and regulation of immune responses. It has become increasingly clear that therapeutic manipulation of neutrophils might provide a very important method of treatment of different diseases. In preparing this monograph, the authors tried to pursue two major aims. First, to provide readers with a detailed overview of the recent developments in neutrophil research, as well as to present some topics that are rarely discussed in monographs on neutrophils. Second, to draw the attention of a broad spectrum of researchers from other fields and clinical scientists to this remarkable cell, and to demonstrate how much neutrophil can give in return for exploration by inquisitive minds. This monograph consists of two sections. The first describes basic neutrophil

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Preface

biology, and the second shows how our knowledge of neutrophil biology can be applied in practical medicine. In the first chapter of this monograph, Dr. English introduces a variety of signal transduction pathways that make these cells so important for our understanding of cell biology. He describes why neutrophils may serve as a unique model for investigation of the basic mechanisms of cell activation, since neutrophil as a terminally differentiated cell does not require these pathways for cell division. Dr. English introduces several major players in neutrophil function that are discussed in greater details in other chapters. In the second chapter, Dr. Quinn presents a detailed review of neutrophil respiratory burst oxidase. He provides up-to-date information regarding key structural and functional features of the neutrophil NADPH oxidase and its protein components. In the next chapter, Drs. Chen and Wei describe several novel neutrophil receptors important for its function. They also discuss signal transduction pathways associated with those receptors. Migration to the site of infection or injury is a critical function of neutrophils. In this monograph our readers will find a chapter written by two experts in this field: Drs. Burns and Rumbaut, who discuss recent data pertaining to the mechanisms of neutrophil migration. They provide detailed description of several key receptors involved in cell migration. Special emphasis is on the mechanisms of transendothelial migration of these cells. The fate of neutrophils is discussed by Dr. Rossi and his colleagues in the next chapter. They review recent data on neutrophil apoptosis and its biological significance. Dr. Ferrante and colleagues discuss new data on the effect of fatty acids on neutrophil function. During the last 5 years, this area of investigation has generated a wealth of new interesting and important information with direct clinical implications. The first section concludes with a chapter written by Dr. Collet-Martin and colleagues. They discuss the very interesting and highly important issue of cytokine production by neutrophils. This area was thriving during the last 5 years and new data dramatically expand our understanding of the role of cytokine production by neutrophils in the pathogenesis of many diseases. In the second block of this book we focused on the direct role of neutrophils in different diseases and as a potential tool for therapy. Dr. Roberts has updated his chapter on the role of neutrophils in the

Preface

xix

antiviral response. He includes new information regarding neutrophils involvement in HIV and influenza virus infections. A new chapter in the second edition of the book is focused on the controversial role of neutrophils in cancer. These cells have recently emerged as a major player determining the speed of tumor progression and even the success of certain types of antitumor therapy. The last two chapters discuss the issue of therapeutic utility of neutrophils. Dr. Nelson and colleagues provide new, updated information regarding the use of colony-stimulating factors in the treatment of neutropenia and infectious diseases. Dr. Strauss discusses new data on the benefits and possible pitfalls of neutrophil transfusion therapy. This book is the result of the collective effort of a group of scientists. I am extremely grateful to all contributors to this book, who have kindly found time in the midst of their active research and clinical duties to share with us their knowledge and thoughts. Dmitry Gabrilovich, MD, PhD

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Contributors

Alan Burns Baylor College of Medicine Houston, TX USA Xianghong Chen H. Lee Moffitt Cancer Center University of South Florida Tampa, FL, USA Sylvie Chollet-Martin Service d’H6matologie et d’Immunologie et INSERM u479 Paris, France Maurizio Costabile The Women’s and Children’s Hospital University of Adelaide, Australia

Nadejda V. Cherdyntseva Tomsk University, Tomsk, Russia David C. Dale University of Washington Seattle, WA, USA Dennis English Methodist Research Institute University of Indiana Indianapolis, IN, USA Frederic. Ethuin Service d’H6matologie et d’Immunologie et INSERh4 u479 Paris, France Antonio Ferrante The Women’s and Children’s Hospital University of Adelaide, Australia xx i

xxi i

Contributors

Dmitry Gabrilovich H. Lee Moffitt Cancer Center University of South Florida Tampa, FL, USA Kevin Harvey Methodist Research Institute University of Indiana Indianapolis, IN, USA Charles S . Hii The Women's and Children's Hospital University of Adelaide, Australia Sergei Kusmartsev H. Lee Moffitt Cancer Center University of South Florida Tampa, FL, USA Magdalena Martinez-Losa University of Edinburgh Medical School Edinburgh Scotland, UK Steve Nelson Lousiana State University New Orleans, LA, USA Mark T. Quinn Montana State University MT, USA Lee J. Quinton Lousiana State University New Orleans LA, USA

Robert L. Roberts UCLA School of Medicine Los Angeles, LA, USA Adriano G Rossi University of Edinburgh Medical School Edinburgh Scotland, UK Roland0 Rumbaut Baylor College of Medicine Houston, TX, USA Ronald G . Strauss University of Iowa College of Medicine Iowa City, IA, USA Annemieke Walker University of Edinburgh Medical School Edinburgh Scotland, UK Carol Ward University of Edinburgh Medical School Edinburgh, Scotland, UK Sheng Wei H. Lee Moffitt Cancer Center University of South Florida Tampa, FL, USA

1 -----------The Remarkable Neutrophil! Developing a Blueprint for Integrated Cellular Signaling Denis English"

Neutrophilic leukocytes are dynamic, motile cells that provide the first line of defense against many infecting organisms. In doing so, neutrophils undergo many of the cellular functions that are known to result from activation of signal transduction pathways. As an end stage cell, the neutrophil is not encumbered by machinery necessary for cellular replication, and thereby has provided an attractive model to define pathways involved in signaling events leading to Ca2+mobilization, actin cytoskeletal dynamics, adherence and chemotactic migration and release of toxic oxygen molecules. The realization that virtually all of the neutrophil's signaling pathways converge to coordinate, regulate and orchestrate the cell's effects has resulted in a new appreciation of how cellular responses are integrated by fundamental biochemical processes in a manner designed to optimize cellular responses. *Correspondence to: Denis English, Ph.D. Methodist Research Institute, Experimental Cell Research Program, Methodist Hospital of Indiana, Indianapolis, IN 46201, Rm. 450-E Noyes; phone: 317-962-2663; e-mail: [email protected]. 1

2

The Neutrophils Keywords: neutrophils; polymorphonuclear leukocytes; signal transduction; cell motility; cell activation; second messenger; integrins; Rho family GTPases

1. INTRODUCTION The molecular basis of cellular activation is rapidly being defined. At its current pace, it is likely that most, if not all, of the fundamental aspects of the basis of cellular activation will be identified in the very near future. This prediction is quite surprising in light of the fact that until recently, our understanding of even the most fundamental basis of cellular activation was not known. How hormones exert their myriad effects; how depressants dampen while stimulants inspire; how cells could quickly race toward a defined target, only to stop suddenly, change their appearance and initiate essential functions they were sent to provide; these and other complexities of the biology of the cell are subjects that were not approached experimentally until quite recently. Thus, while inducible cellular functions were well described, their molecular basis was unknown. In fact, it is not at all clear that early investigators appreciated the fact that defined cellular functions were promoted by specific molecular events. As curious as this seems today, it applies to other aspects of cell biology as well as to other areas of research. For example, when we do not know the molecular basis of key aspects of embryonic differentiation and development, we may not even consider one to exist. The molecular basis of reverse differentiation was neither defined nor appreciated because its potential was not revealed until Ian Wilmut cloned Dolly1 Stem cells were not known to be an essential aspect of development until they were identified.*~~ This paradox applies to all aspects of investigation, and it limits in profound ways the scope of our studies. Similarly, one investigator’s realization that a previously undefined and unidentified process must indeed exist is often the foundation of years of un-rewarded investigation by pioneers of the field; the men and women who have carried our understanding of the nature of all living things to the new horizons we now visualize. In science, the relentless thought and work of these individuals have provided the very rationale for our continued quest to understand the basis of life. What powerful force relentlessly drives humans to ponder their own existence is not clear, but the problems that scientists have addressed are

The Remarkable Neutrophil!

3

among the most complex in the universe. Solving them has improved our quality of life and our ability to treat disease.

1 . l . Second Messengers Just over 30 years ago, the identification of cyclic AMP as a receptordependent activator of an intracellular enzyme cascade led to a defining new concept, the concept of a ”second mes~enger”.~ The very concept of intracellular second messengers, relay molecules regulated by extracellular agonists when they engage cellular receptors, dramatically changed our perspective and widened our approach to defining the molecular basis of cellular activation. In fact, this concept provided the very rationale for these initial studies. In this respect, initial studies were carefully designed to determine how membrane receptors regulate levels of intracellular second messengers, and have led to the identification of heterotrimeric proteins that held GTPase activity, now termed G-proteins, as a link between receptor engagement and the activation of specific intracellular target^.^ The pioneering studies of Earl W. Sutherland, Jr. resulted in the award of a Nobel prize in 1971 for his discoveries concerning the mechanisms of the action of hormones. Subsequent studies by Alfred G. Gilman and Martin Robell resulted in a Nobel prize in 1994, for the identification of G-proteins involved in cellular signaling.

1.2. Integrated Cellular Signaling Soon, enzyme cascades activated by second messengers would be termed signal transduction pathways, and their molecular basis would be defined by both those who proposed to alter them to regulate their effects for therapeutic purposes, and by those who desired to define them in order to understand fundamental aspects of homeostasis. The results of studies by these investigators have now converged and led to a very detailed understanding of cell signaling in a very short period of time. We now know that many diverse signaling pathways exist, and that these pathways are highly interrelated in order to operate at maximum efficiency under a variety of conditions to optimally orchestrate cellular function. This chapter will provide the fundamental aspects of the molecular basis of cell activation. In addition, we will explore how many of these

4

The Neutrophils

pathways are integrated in the neutrophil to promote and coordinate this dynamic cells’ response. The neutrophil has been the object of curiosity and investigation by pioneering investigators for centuries. Recent studies of signaling initiated using other cellular models have been greatly clarified by studies using neutrophillic leukocytes. As a result, studies of neutrophil signaling have provided defining aspects of many basic signaling pathways, and this cell’s unique functional attributes have been well exploited to improve our understanding of ”integrated signal transduction.”

2. THE LIFE OF THE NEUTROPHIL 2.1. Bone Marrow Origin of the Neutrophil Neutrophils are born in the bone marrow from committed progenitors, which arise from pluripotential hematopoietic stem cells differentiating in response to specific growth factors (Fig. 1h6 Upon replication, the hematopoietic stem cells of the marrow generate a new stem cell and a committed p r ~ g e n i t o r ,Several ~ . ~ factors that promote stem cell differentiation to myeloid and hence neutrophil development have been identified, but to date, none has been implicated in maintaining rather constant levels of the cell in the circulation throughout the life of a healthy host. Nentrophils delivered to the circulation are mature forms of the initial progeny of myelocytic progenitors, the immature band cells. Unlike mature neutrophils, band cells possess few cytoplasmic granules and lack the segmented nucleus characteristic of the mature neutrophil, which is therefore also commonly referred to as the polyrnorphonuclear neutrophizic granulocyte. While the mature neutrophil generates some protein, the amount of protein the cell manufactures is quite limited. Instead, much of the machinery and molecules needed for neutrophil responses are pre-packaged in the cells’ cytoplasmic granules or plasma membrane, and thereby is available for cellular functions when needed when the cells are initially isolated from blood. After release into the blood, the life of a neutrophil is quite limited.8 Except in disease states, only functionally mature neutrophils are released from the bone marrow; thus few, if any, band cells or other myeloid precursors are found in the circulation of healthy individuals. The basis of

The Remarkable Neutrophil!

5

Fig. 1 The neutrophilic leukocyte. (a) Neutmphils are born in the bone marrow from pluripotential hematopietic stem cells, which manufadure other blood cells as well. The hematopietic stem cell gives rise to myeloid cells, including neutrophils or polymorphonuclear leukocytes. These are derived from earlier stem cell descendants, including the less segmented metamyelocytes,shown here in a stained film of a bone marrow aspirate. (b)However, only mature neutrophils leave the marrow while non-segmented myelocytes are not found in the circulation of healthy individuals. Panel (c) shows a scanning electron micrograph of a neutrophil in the process of ingesting yeast. Killing of microorganisms by neutmphils involves the release of digestive and microbicidal agents into the phagosome during granule fusion with the phagosome. This picture, supplied by Dr.K.B. Pryzwansky,depicts a physiologic portrait of neutrophils as they appear in tissues. In the circulation, neutrophils appear round amidst erythrocytes, platelets and other cells as shown by the illustration in panel (d), where the neutmphil is illustrated in blue, amidst erythrocytes and other blood elements.

this is unclear, but the process ensures that only functionally capable cells are present at the front line. This fact indicates that mature neutrophils exploit such a functional attribute, such as chemotaxis, to leave the bone marrow. Supporting this concept is the fact that blood plasma possesses

6

The Neutrophils

chemoattractive activity for mature neutrophils, but not band cells or other precursors (D English and BR Andersen, unpublished observations). While the levels of circulating neutrophils remain relatively constant in healthy adults, these levels rise quickly during the onset of infection. Many factors have been implicated in the increased number of circulating neutrophils during infection, but again, the basis of this response remains unclear. Certainly, endotoxins released by gram-negative bacteria cause an immediate increase in the levels of circulating neutrophils? but how endotoxemia and other bacterial factors increase levels of circulating neutrophils is not known. Finally, the life of the neutrophil in the circulation is brief, displaying a half-life of approximately 6 hours.* Thus, neutrophil precursors are extremely active, delivering as many as 10l1 cells per day to maintain blood levels of 10000 cells/ ~1 in a healthy adult.

2.2. The Fate of Circulating Neutrophils After the neutrophil leaves the circulation, it does not return. The fact of the cell's brief life span reflects its dynamic and essential role in host defense. The neutrophil reacts quickly and continuously to potential threats, thereby preventing infectious agents from gaining an insurmountable foothold. As the cell ages, its ultimate functional capabilities i n ~ r e a s e , ' ~ ' ~ but the pathways involved ultimately result in either circulatory egress or apoptotic cell death. Thus, optimal function comes at the expense of the cell's brief lifespan. To keep tissues sterile, the neutrophil has no time to multiply or even synthesize protein. Instead, fresh neutrophils continuously replace those worn by age, a process that is both essential and easily perturbed. Thus, neutropenia resulting from chemotherapy or radiation is a limiting factor in cancer treatment, and may quickly result in ovenvhelming infection and death. Active neutrophil progenitors are highly susceptible to mutations, resulting in leukemia and other diseases when neutrophil production from its precursors is uncontrolled.

2.3. Neutrophil Stimulators Neutrophils vigorously respond to factors secreted by bacteria, generated during the host response to bacteria, as well as to factors generated during activation of the coagulation cascade, including bioactive lipids released

The Remarkable Neutrophil!

7

by platelets during clotting. The cell also responds vigorously to hostderived substances, which accumulate at sites of previously damaged tissues, such as crystals of uric acid.15 Uric acid crystals are therefore inflammatory agents that cause tissue damage by recruiting neutrophils from blood and subsequently effecting the release of toxic metabolites from the cells, resulting in severe acute inflammation. Microbial factors that initiate neutrophil responses are either secreted by the invading bacteria or are generated by activation of the "innate" immune system of the host. Many inflammatory stimulants, as well as products of the innate immune system and of the coagulation cascade, activate the host's complement system, resulting in the generation of potent neutrophil agonists, including C5a. Activation of neutrophils by secreted bacterial products is reflected by the cells' response to the synthetic tripeptide, formylated methionyl-luecyl-phenylanine, or FMLP. Physiologically irrelevant agonists, such as phorbol myristic acid, or PMA, which directly activate physiologically relevant signaling pathways, also provoke neutrophil responses. Activation of neutrophil responses also occurs as the cells ingest or phagocytize opsonized particles, such as zymosan, an insoluble extract of the cell wall of yeast. In addition, cytokines such as tumor necrosis factor and interleukin-8,16 as well as many growth factors, including GM-CSF,10*17-18 fine-tune neutrophil signaling pathways, resulting in enhanced or primed responses as well as, in some cases, diminished or anti-inflammatory responses. 2.3.1. Do diverse biochemical events underlie stimulation? The biochemical bases of the cells' response to each of these classes of agonists have been examined in detail and have revealed intricate details of the complex and interrelated signaling mechanisms of the neutrophils' many functions in inflammation. These mechanisms are highly relevant to those employed by other cells, and the multifaceted neutrophil provides a unique model for understanding the interrelationship of a variety of mammalian cell responses. Do responses induced by different agonists result from activation of divergent signaling mechanisms? While early evidence supported this notion, the answer seems to be no. Thus, some stimuli irreversibly initiate the neutrophils' respiratory burst while others do so in a reversible manner,"/l2 and some chemoattractants also

8

The Neutrophils

induce a transient burst of oxidative metabolism while others do n ~ t , ~ no compelling evidence comprises a solid argument that these differences result from activation of any individual function by fundamentally different signaling mechanisms induced by different stimuli. PMA, for example, can induce responses in the absence of extracellular free Ca2+while FMLP cannot, a difference attributable to the unique ability of PMA to induce a key initial response in a Ca2+-freemanner. After the response is induced, it appears to be mediated by the same cellular effectors. Thus, adherence induced by either PMA or FMLP is dependent on extracellular Mg2+,I9and probably results from the same basic biophysical response. NADPH oxidase activation by divergent stimuli may vary in intensity, duration and reversibility, but again, this response presumably has a common biochemical basis.

2.3.2. Functional maturation Circulating neutrophils display limited functions. Upon initial stimulation, the round circulating cells polarize and marginate near sites of challenge by reversible and weak adherence of the cell to the vascular wall (Fig. 2). Key surface receptors needed for subsequent function are mobilized from

Fig. 2 Neutrophil adherence and pseudopod extension. Circulating neutrophils respond to signals generated by infectious invaders and adhere weakly to the endothelial wall. While the nature of the attractive force has not been defined, many adherence molecules are involved, as illustrated in panel (a).After leaving the circulation, the neutrophils migrate to the site of microbial invasion and extend pseudopods, which attach to the invading microorganisms, providing a proactive defense against these invaders (b).

The Remarkable Neutrophil!

9

intracellular storage pools when the cell initially adheres to the endothelium.1020The neutrophil then passes through the formidable vasculature barrier and then to its target by directed chemotaxis. Immobilization there sets the stage for enhanced release of toxic metabolites and ingestion of the microbial predator.21During chemotaxis, adherent foci develop at the cell's leading edge as its elongated tail detaches while focal adhesions dissipate from the extracellularmatrix in a dynamic manner directed by the effects of the stimulus and the extracellular matrix on the moving cell's cytoskeletall plasma membrane dynamics (Fig. 3)."rD This response serves to optimize

Fig. 3 Regional interactions within migrating neutrophils. As neutrophils respond to a gradient of chemoattractant, members of the Rho family of small GTPases re-orient in response to signals from chemoattractant receptors and regulated by the extracellular matrix. At the cell's leading edge, Rac, CDC42 and their effectors, the p-21 activated kinases (PAKs) accumulate. These effectors result in actin reorganization and adherence. Matrix interactions activate integrin signaling, which moves Ca2+ and Rho effectors, particularly Rho-kinase, to the cell's trailing edge. Ca2+ exerts many effects during migration, and the cells cannot migrate in Ca2+-freemedia. Matrix alterations resulting from infection, inflammation and even neutrophil migration itself continuously "change" the environment as the cells move, culminating in arrested migration (and enhanced inflammatory potential), when the cell reaches its target.

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The Neutrophils

chemotaxis by preserving the integrity of the migrating cell, and to limiting damage to tissues of the host. Cytoskeletal alterations resulting from chemotaxis and adherence potentiate the neutrophil’s functional potential when the cell reaches its target.12,14 Tightly adherent at the site, the neutrophil migrates no further, and is instead retained to finish its essential task that it is now optimally equipped to carry out.

2.4. Neutrophil Adherence The first function expressed by a circulating neutrophil anticipating engagement with an invading microbe is enhanced adhesion. Circulating neutrophils are, somewhat obviously, not adherent to the vascular endothelium, which expresses fibronectin. Thus, circulating neutrophils express limited active fibronectin receptors, such as the integrin a5B1. Initial adherence is weak and reversible, but an irreversibly immobilizing adherent response develops as the inflammatory responses progress. Of all of the cells’ responses, perhaps adherence is the least well understood. While mediators and signaling pathways ”involved” in adherence have been well characterized in neutrophils and other cells>4how extracellular signal activation of intracellular pathways actually increases the ability of the outside aspect of the cells’ plasma membrane to adhere to the extracellular matrix is not at all clear, and the reason it is unclear simply defies explanation. Several theories exist. Initial studies postulated the secretion of a specific adherence rnediat~r?~ but recently cytoskeletal and plasma membrane enzymes and adherence molecules activated early during the inflammatory cascade have been implicated as adherence mediators. Indeed, these effect the distribution and activity of cellular adherence molecules, which send clues from the matrix to the cellular interior in a process referred to as ”outside - in” signaling, and they effect the activity of the final arbiters of adherence by activation of ”inside - out” signaling pathways. The agonists involved, the cells’ history and the composition of the extracellular matrix, dictate the degree of cellular adherence. In many cases, the different degrees of adherence have been strongly associated with specific cellular responses initiated by interactions with different matrix components. For example, integrin-dependent linkage of cellular adherence receptors to activation of the enzyme phosphatidylinositol3’-kinase (PIT kinase) has been associated

The Remarkable Neutrophil!

11

with the development of strong adherence at the cells’ leading edge during migration, whereas activation of the enzyme Rho-kinase appears necessary to allow the tail of the migrating leukocytes to detachJ6 Here, inhibition of Rho-kinase inhibits forward movement as the cell simply elongates. 2.4.1. Biophysical aspects of adherence The outer membrane changes that result from activation of these intracellular pathways and lead to attachment or detachment are not clear. Several possibilities exist but none has received much experimental support. The ”Velcro” point of view holds that adherence results from prolonged, high affinity captivating engagement of extracellular adherence receptors by specific molecules in the tissues. However, stimulated neutrophils adhere strongly to both glass and plasti~,’~ which do not possess adherence molecules, pretty much putting a damper on the Velcro concept. In all cases, extracellular Mg2+ is necessary for stimulated adherence, but the cation exerts its influence in unknown ways. Membrane phospholipid changes are probably the basis of enhanced adherence, justifying the postulated involvement of enzymes that alter these phospholipids, since changes in phospholipid components on the cells’ interior parallel changes on the exterior of the membrane bilayer. One candidate is phosphatidic acid, which is generated by the action of phospholipidase D in stimulated n e u t r o p h i l ~Its . ~ functional ~~~~ role is unknown, but it does interact with intracellular messengers and bind critical intracellular sites, which markedly influence cell b e h a v i ~ r . ~ * Generated ~ ~ ~ ” ~ in the plasma membrane, much of the anionic phospholipid would be expected to be deployed to the cells’ exterior in a reversible manner. Extracellular phosphatidic acid disrupts endothelial mono layer^^^ and potentiates neutrophil chemotaxis, tyrosine kinase ’ activation and Ca2+ mobilization. Recent studies have focused on the effects of phosphatidic acid on Raf-1/Ras interactions and other intracellular effects. 36-37 We have found that phosphatidic acid in cells shows an affinity for phosphorylation sites that are preferred substrates of certain tryosine kinases (unpublished), and that tyrosine phosphorylation effects are initiated, or mimicked by, phosphatidic acid (unpublished). In any event, the effects of the phosphatidic acid hydrolyzing enzyme, phosphatidic acid pho~phohydrolase,3~~~~

12

The Neutrophils

are an important determinant of neutrophil function. Our results show an interesting relation between phosphatidic acid affinity and tyrosine phosphorylation in the cells' interior, which may be governed by phosphotyrosine p h o s p h a t a ~ e sThis . ~ ~ work agrees with recent correlations drawn by others and shall perhaps explain the role of PLD in cellular activation.

2.4.2. Integrins Neutrophils possess the adherence integrin p2.Like many cells in suspension, circulating neutrophils possess increased levels of activated protein kinase A (PKA), which limits the effects of products generated during the activation of PI3'-kinase and adherence as well. Injection of chemoattractants such as C5a or FMLP causes the circulating neutrophils to marginate on the endothelial surface in a loose and reversible manner, accounting for the nonpathologic and transient neutropenia associated with kidney dialysis, wherein complement components activated by dialysis tubing are infused into the c i r c ~ l a t i o nHere, . ~ ~ neutrophils accumulate in the pulmonary microvasculature, but the response is not dramatic, and does not cause respiratory distress, as does infusion of potent activators of neutrophil adhesion and the respiratory b ~ r s t . ~ When ' - ~ ~ neutrophils carefully isolated from blood are exposed in suspension to chemoattractants, they exhibit a similar transient and reversible adherent response that mimics their release of oxygen free radicals." This response develops as the cells polarize and weakly aggregate. The response stops quickly (within 1.5-2 min), and the aggregated cells eventually disperse. During this interlude, the cells are refractory to stimulation of the respiratory burst by chemoattractants, but their potential to release toxins upon engagement of other stimuli is enhanced.

2.5. Chemotaxis The initial weak adherence of neutrophils to the vascular wall limits the release of toxic mediators while the circulating neutrophils polarize in anticipation of a chemotactic journey to the site of engagement. This response involves a complex array of individual components, leading to

The Remarkable Neutrophil!

13

organized development and dissolution of actin filaments and focal adhesions along a highly structured cytoskeletal network, as well as contractile processes that operate in a coordinated manner along the length of the cell, a manner directed by integrin signaling. Recently, some of the forces involved in chemotaxis and the dynamics of focal adhesions have been clarified.

2.5.1. Rho family GTPases

Rho family GTPases constitute a family of intracellular messengers that are regulated both by their location and state of activati0n.4~They appear to exert important effects in almost all the functions of the neutrophil, including adherence, oxidative metabolism and migration.45Three Rho family members have received intense study: Rho, Rac and CDC42. During leukocyte migration, Rho appears to exert effects on cellular contraction and detachment, while Rac exerts effects necessary for leading edge adherence and directed migration of polarized cells. CDC42 activates many of the same mediators as does Rac, but its effects appear limited to those involved in cellular morphology and lamillopodia development. Recent studies have demonstrated the localization of Rac at the leading edge of migrating cells where Rho is in fact either inactivated or disintegrated.4648Conversely, at the tailing edge of a migrating leukocyte, activated Rho associates with its effector Rho-kinase to cause detachment and contraction. The effector of Rac that leads to actin development and migration is not clear, but the unique enzyme, Pak-1 appears to play an important role.49The kinase activity of Pak-1 is enhanced over 50-fold when it engages Rac in its GTP ”or activated” form. Its activity is also regulated by products of the activity of PI3’-kinase. The targets of the enzyme that are essential for cell motility are not clear, but myosin, other PI3’-kinase products, the stress kinase p38, and other enzymes known to regulate either actin dynamics or cell contractility have been implicated. 2.5.2. Rho-kinase The target of Rho-kinase that is critical for migration is also unclear, but many candidates have been identified. Under the conditions of leukocyte

14

The Neutrophils

migration, activation of Rho results in the development of a dissipating cortical actin network at the trailing edge of the migrating cell, while Rac signaling mediates the assembly of a fibrous actin network at the cells’ leading edge. During migration, the activities of activated Rho and Rac appear to oppose each other, resulting in continuous detachment and disruption as focal adhesions and actin dissipate at the trailing edge of the cell while they are formed at the leading edge as the cell crawls forward (see Fig. 3). Processes at the neutrophil leading edge are associated with the activation of PI3’-kinase, which generates reactants that regulate the activity of Rho-kinases at the trailing edge. Integrin translocation, Ca2+ mobilization and p38 MAP kinase play an essential role in this dynamic. Thus, focal adhesion kinase (FAK) activated by PI3’-kinase, inhibits the activity of Rho kinases, and Rho kinases sequester another product of Rac activity, protein kinase 2 ( P ~ k - 2 )Chemotaxis .~~ is Ca2+dependent and so is intracellular integrin signalling?’ In cells deprived of Ca2+,the p2integrin moves and remains at the rear of the cell, along with various molecules that initially participated in forming adherence junctions and organized development of filamentous actin at the leading When Ca2+ is not buffered, the integrin recycles to the leading edge via intracellular endosomes. It is likely that while cells move, integrin-dependent changes in local concentrations of free Ca2+play a defining role in chemotaxis by mediating changes in adherence and contractility along the length of the cell. At the leading edge, high levels of free Ca2+may potentiate adherence, actin development, forward contraction and protein tyrosine phosphorylation. Simultaneously, removal of Ca2+ at the trailing edge results in decreased ability of tyrosine kinases to keep Rho effectors in check, and cell contraction mediated by these serine kinases is associated with a loss of adherence, allowing the cell to progress forward. Thus, the role of integrins in altering regional intracellular concentrations of free Ca2+,the role of Rho effectors in adherence and actin dissolution and how the products of PI3’-kinase and other mediators associated with Rac activation and consequent actin assembly and adherence, need to be thoroughly assessed. Several groups now focus on this intriguing problem, since cell migration lies at the very basis of differentiation and development, as well as host defense. The next decade will bring new insights

The Remarkable Neutrophil!

15

that will greatly clarify how a cell moves forward in response to an increasing gradient of chemoattractants.

2.6. Neutrophil Priming The enhanced responsiveness of the neutrophil after the cell leaves the circulation depends neither on adherence nor on Ca2+mobilization but develops in a time- and temperature-dependent manner by a process that involves translocation of a specific subset of cytoplasmic granules to the plasma membrane.I3This process is enhanced by low levels (
2.7. The Respiratory Burst: Free Radicals in Biology Neutrophils have long been known to release hydrogen peroxide, which in association with neutrophil myeloperoxidase and a halide ion, constitutes a

16

The Neutrophils

potent bactericidal system. The basis of hydrogen peroxide generation remained unclear until several investigators identified the neutrophil NADPH oxidase, which generated the reduced oxygen reactant, superoxide ion COT) at the expense of NADPH in stimulated, but not resting, neutrophils. The first indication that neutrophils did, in fact, generate reactive oxygen species came from the classic observation in 1962 of Richard C. who found that neutrophils emitted photons of light, a characteristic of the relaxation of singlet molecular oxygen, upon exposure to bacteria. Subsequently, Joe McCord and colleagues demonstrated that superoxide could be generated in biological systems, and proposed the reaction 0 2 + 0 5 + 2H++H202+ O2,where the derived oxygen was initially thought to exist in the singlet, electrically excited state. While this reaction occurs spontaneously, it is catalyzed by superoxide dismutase (SOD) at rates limited only by diffusion. In the absence of SOD, superoxide, while reactive, persists long enough to inflict damage by initiating free radical reactions chain reactions.54The oxidation of superoxide has since conclusivelybeen demonstrated to be the basis of the neutrophil's "chemiluminescent" response and to constitute the basis of a myeloperoxidaseindependent bactericidal microbial killing ~ystem.5~ While many reactions of superoxide and hydrogen peroxide result in photon generation, the work of Dr. Allen is defining, in that it began the successful search for the free radicals in a mammalian system.

2.7.1. The enzyme involved The fact that superoxide derived from the neutrophil, NADPH oxidase, provides the basis of a physiologically relevant microbicidal system is evident from the etiology of chronic granulomatous disease, which, as a result of defective NADPH oxidase activity, renders patients highly susceptible to infection. The NADPH oxidase is a truly amazing enzyme that is assembled from at least 5 component parts in order to transfer electrons from cytoplasmic NADPH to molecular oxygen.56The superoxide ion is not a potent cytotoxic agent, but its derivates, including the hydroxyl radical are. Initial studies demonstrated that plasma membranes isolated from stimulated neutrophils generated superoxide upon addition of NADPH, whereas membranes from resting cells did not.

The Remarkable Neutrophil!

17

This provided a unique model to define the basis of activation of this enzyme.

2.7.2. Back to Rac After only a few years of studies, several groups determined that the oxidase in the membranes of resting cells could be activated by addition of unseparated cytosol and arachidonic acid to the inactive membrane components, sparking the search for the component responsible. Initially, this was thought to consist of simple protein purification, based on the assumption that a single cytosolic co-factor was responsible. Unraveling activation of the oxidase proved to be much more complex. Ultimately, it was found that 3 or more cytosolic components were involved, including a member of the Rho family of small GTPases, Rac, which prior to this was known to participate in cellular morphological changes, adherence and chemotactic migration. Studies with the neutrophil have recently demonstrated that Rac participates and, in fact, coordinates many neutrophil responses, possibly limiting oxidase activation as it promotes chemotactic migration. The exact role of Rac remains to be defined. Steven Williams, Mary Dinauer and colleagues have clarified this problem using carefully controlled studies with genetically engineered mice, wherein both Rac-1 and Rac-1 expression can be regulated.45Their studies implicate Rac-2, the predominant isoform in neutrophils in both responses. Earlier studies and many recent studies still consider Rac-1, possibly bound to its cytosolic stabilizer, Rho-GDI, in activation of the o x i d a ~ e ?leaving ~ GDIfree Rac available for chemotaxis. GDI, or guanine nucleotide dissociation inhibitor, holds Rac in its GDP or "inactive" form avidly in the cytosol. However, the function of GDI is much more complex, as it targets Rac to specific sites at the cytosol/plasma membrane interface?8 and coordinates the action of Rho phosphorylated at the plasma membrane by PKA and perhaps other kinases in doing There, Rac is displaced from GDI by phosphorylated Rho and enters the membrane at specific targets. How this small molecule can exert such a wide array of activities is not clear. What is clear is that Rac acts in concert with and in opposition to Rho in coordinating neutrophil function and the functions of other cells as well.

18

The Neutrophils

These interactions may prove highly complex, but they define the integrated signaling required to coordinate neutrophil function.

2.8. Neutrophil-endothelial Cell Communication When neutrophil stimulants impinge upon the vascular wall, circulating cells loosely adhere to the vascular endothelium proximal to the area of challenge. The loosely adherent neutrophil then exits the circulation through pores in the permeable endothelium. Processes that lead to vascular permeability have been subjects of intense investigation, since preservation of the endothelial barrier is an essential aspect of homeostasis. Thus, while disruption of the vascular barrier is necessary at specific locations for host defense, if uncontrolled or not restored, barrier breakdown leads to a profound and often fatal leak of fluids from the circulation, which quickly compromises the function of critical organs, including the lung and brain.60t61 Increased vascular permeability during the initial stages of the inflammatory response involves dramatic remodeling of the actin-based cytoskeletal architecture of endothelial cells.@The integrated signaling pathways that lead to a disruption of the vascular barrier and the factors that initiate them in many instances reflect the very same processes that promote neutrophil adherence to and migration through the vascular barrier.62~63 Factors released by stimulated neutrophils as well as metabolites deployed to the neutrophil surface as a consequence of their initial adherence, potentiate vascular permeability and facilitate emigration of the cell through the previously intact endothelial barrier.64-66Not surprisingly, processes evoked in endothelial cells that subsequently restore vascular integrity are similar to those evoked in neutrophils to limit chemotactic migration and thereby trap the cell at sites of challenge after they leave the circulation (D. English and D.N. Brindley, in preparation). Many of the intracellular changes in the endothelium involved in both opening the protective barrier of the vasculature and in closing it as the inflammatory response subsides are similar, if not identical, to the processes that initiate neutrophil adherence to the vasculature, and are triggered by the same stimuli that initiate loose neutrophil adherence to the endothelium.

The Remarkable Neutrophil!

19

The above results again support the fact that neutrophil host defense capabilities increase when the cells leave the circulation. They also support the notion that in the initial phases of the inflammatory response, neutrophil stimuli coordinate neutrophil-endothelial cell communication in a manner that minimizes the pathology associated with enhanced vascular permeability while potentiating subsequent neutrophil functions.

3. INTEGRATED CELLULAR SIGNALING The interrelation of signaling pathways provides convenient and necessary mechanisms to limit certain functions in order to amplify others and conserve metabolic energy. This interrelationship also affords the cell maximal utility in carrying out its role, especially when the cell has multiple roles to carry out. Finally, integrated cell signaling provides a mechanism to limit damage to host tissue by cells that are armed to release toxic metabolites when they arrive at their target. In this respect, our knowledge of the molecular basis of cell activation has growl1 tremendously over the past three decades. While studies of cellular signaling have employed many different cell types, perhaps no cell has been as important in defining individual pathways and in defining how these pathways are interrelated as neutrophilic leukocyte. The utility of the neutrophil in initial studies to define the molecular basis of cellular signaling was based, among other attributes, on the fact that neutrophil functions had been explored and carefully documented for years prior to current investigations of cellular signaling. The neutrophil has played a pivotal role in our present understanding of integrated cellular signaling. The observations described above provide ample evidence that the neutrophil is perhaps the cell of choice for defining the fine details of mechanisms of cellular activation. Thus, the neutrophil has been, and remains, a cell of discovery. Studies using the neutrophil provided the first physiological evidence that receptor-driven phosphoinositide hydrolysis led to mobilization of Ca2+ from intracellular stores, as well as to the development of an agonistdependent system to assess the activation of G-protein receptors in a cellfree system.66The neutrophil has been the defining cell for studies of the

20

The Neutrophils

activation and activities of PI3’-kinase. Indeed, the product of this enzyme, phosphatidylinositol 3,4,5 tris-phosphate, was first observed in the membranes of stimulated n e ~ t r o p h i l sThe . ~ ~neutrophil provided the first cell-free system to examine the biochemical basis of the activation of a multicomponent enzyme system which, as detailed above, exists in an inactive state in membranes recovered from disrupted resting cells but displays abundant activity in membrane fractions of activated cells. The neutrophil was the first mammalian cell used for studies of chemotaxis and the first cell identified that generated oxygen-derived free radicals for a physiologically relevant purpose, providing a new emphasis on the discovery of an enzyme that detoxifies these radicals, superoxide dismutase. Recent studies with the neutrophil have shed new light on the role of Rho in both the initial and final aspects of cellular responses to physiological stimuli; defined how downstream effectors of this GTPase influence cellular activation; implicated CDC42 in morphological changes associated with responses; defined the once elusive role of Rac-2 in cellular metabolic activation; and clarified how integrins relay messages to coordinate cell responses. Assessed together, these studies have defined a new appreciation of the Rho/Rac equilibrium in morphogenesis, adherence, chemotaxis, immobilization, and ultimate cellular functional activation. The neutrophil has attained the leading edge in studies designed to further clarify this equilibrium, as they now define the complex relationship of protein tyrosine kinases and serine/ threonine kinases which are mediated by effectors of both Rho and Rac GTPases. In all these respects, the neutrophil has played a pivotal role in our understanding of cellular signaling, and studies with this cell will certainly add the final chapter to a complete appreciation of the many events involved, and their dynamic interactions.

4. MOLECULAR MECHANISMS OF CELLULAR ACT I VAT I0N After the identification of CAMPas a second messenger leading to activation of enzyme cascades, investigators focused on identifying other second messengers, receptors and agonists that led to the functional

The Remarkable Neutrophil!

21

activation. These studies were initially promoted by the assumption that levels of intracellular free Ca2+ played a key role in many key cellular responses, even though terms such as "signal transduction pathways" had not yet replaced the concept of enzyme cascades as the basis of cellular responses. Although only a short period of time has elapsed since the initial CAMP-dependent signaling pathways and receptors that regulated them were defined, there was a rather prolonged interlude between the discovery of the first second messenger and the initial focus on cellular signaling. Only recently have investigators focused on defining complex pathways wherein signaling mechanisms are orchestrated in a manner that optimizes cellular responses. As we shall see, these responses depend not only on receptors and their targets, but also on the cell's relationship with the extracellular matrix, the cell's history, its biochemical composition and the agonists which impinge upon it. Studies of Ca2+mobilization were initially driven by the concept that increased Ca2+ levels resulted from the influx of extracellular Ca2+. Putney and colleagues implicated phosphatidic acid in Ca2+ delivery from the extracellular environment to the cell's interior.68 The term "calmodulin" was coined to define a certain Ca2+-dependentenzyme that today remains a subject of intense study.69 In addition, signaling effects promoted in neutrophils by metabolites of arachidonic acid led many investigators to dissect the effects of prostaglandins and other arachidonic metabolites in cellular responses, resulting in the award of a Nobel prize to Sune K. Berfstrom, Bengt Samuelsson and John Vane for their discoveries concerning prostanglandins and biologically related substances in 1982. In early 1980, the basis of cellular Ca2+ mobilization remained unclear and captivated the attention of investigators worldwide. Aroused by the pioneering studies of the Hokins in the m i d - 5 0 ~ 7Peter ~ Michell ominously hypothesized in 1983 that membrane inositol phospholipid hydrolysis played a key role in increased cytosolic free Ca2+levels, leading to functional activation in stimulated cell.71The legendary "Michell Hypothesis," published in intricate detail, prompted studies of inositol phospholipid hydrolysis by Burridge and others?2 who demonstrated the existence of inositol containing phospholipids other than phosphatidylinositol (PI). These investigators quickly identified phosphatidylinositol

22

The Neutrophils

3-phosphate (PIP) and phosphatidylinositol3,4-bisphosphate(PIP2) and isolated their products of hydrolysis by the well characterized enzyme, phospholipase C. Along with diacylglycerol, hydrolysis of these lipids by PLC led, expectedly, to inositol phosphate, inositol bisphosphate and inositol trisphosphate (IPI, IP:! and IPS, respectively). Of these water soluble products, IP3 caused an immediate release of Ca2+from storage pools in extracts of broken cells, leading to a search for a cellular receptor linked to PLC activation. Since Ca2+was known to be mobilized in cells treated with agonists of G-protein linked receptors, the putative PLC-linked receptor was initially termed G, and was assessed extensively in several intact cellular systems. The first demonstration of a G-protein-dependent PLC-linked receptor in a cell-free system was made using disrupted neutrophil. Relying on the results of Gilman and colleagues, who had previously demonstrated the agonist-independent activation of G-protein linked receptors with the non-hydrolysable GTP analogue, GTP y-S, Snyderman and associates successfully provoked PIP, hydrolysis in neutrophil plasma membranes by exposing them to GTP y-S ,resulting in the simultaneous generation of IP3 and diacylglycerol in the absence of exogenous Ca2+.73In addition, again based on a model developed by Gilman and colleagues, Snyderman’s group was able to demonstrate a similar response when neutrophil membranes were exposed to physiologically relevant, G-protein receptordependent agonists in the presence of GTP. GTP alone was inactive as the G-protein receptor’s associated GTPase hydrolyzed it quickly, as expected. However, as predicted, GDP-p-S, a non-hydrolysable analogue of GDP was an effective competitive inhibitor of the response effected by agonists in the presence of GTP and by GTP-y-S. Finally, the response in isolated membranes was induced by fluoroaluminates, which Gilman demonstrated were potent activators of inactive, GDP-bound G-proteins, as the metal-halide complex bound guanine avidly, resulting in structural transition to that promoted by GTP. Activation of the enzyme was inhibited by pretreatment of the neutrophils from which the membranes would later be obtained by the toxin of B. pertussis, (commonly referred to as pertussis toxin), which specifically inhibits certain G-proteins by causing their ATP-dependent NADPH ribosylation. These studies conclusively defined a receptor-linked pathway that resulted in CaZf mobilization as a

The Remarkable Neutrophil!

23

result of the hydrolysis of PIP2and subsequent release of IP, in the cellular cytosol. These studies led to an era of incredible productivity that continues today. Shortly after G-protein-receptor linked PLC activation was implicated in cellular activation, other G-protein-linked enzymes were defined. One of the first was mammalian phospholipase D, an enzyme not known to exist prior to 1986. The mammalian homologue of this enzyme, identified years earlier in plants, was first conclusively identified in neutrophillic l e ~ k o c y t e ?as ~ was its activation by chemoattractants, fluoroaluminates, Ca2+and PMA. The linkage of this enzyme to a G-protein coupled receptor was suggested by my work in collaboration with Theodore Gabig,75 as well as subsequent studies of others. Subsequently, a wide array of enzymes, second messengers, adaptor proteins and cellular mediators would be linked to a similarly wide array of G-protein receptor families, including, but not limited to, those that inhibit adenylate cyclase (GJ; those associated with the regulation of members of the Rho and Ras families of small G proteins (for example Gl2II3);those of diverse function, including activation of the phosphoinositide hydrolyzing PLC (Gq); those that activate adenylate cyclase (Gs); those of unknown function (G?); and those that activate phosphatidylinositol 3’-kinase, a unique enzyme that generate previously unidentified inositol-based phospholipids by phosphorylation (for example, the generation of PI 3,4,5 P3 from PI 3,4-PJ. It should be noted that G-protein receptor coupling is rather vague, readily changeable and not at all specific; thus, many Gi receptors have the effect of promoting PI3’ kinase possibly as a result of diminished CAMP affected by an as yet undefined mechanisms, and small GTPases can be regulated in a wide variety of ways as a consequence of receptor engagement. In addition, the receptor’s activation, effectors, and the cellular matrix can markedly alter G-protein receptor coupling and expression.

4.1. Protein Phosphorylation in Cellular Activation While neutrophils played a leading role in the definition of G-protein coupled receptors and their linkage to various signaling pathways, the cell played no role in the identification of another major class of receptor

24

The Neutrophils

systems, the protein tryosine kinase-linked receptors. These receptors exert their effects after they engage agonists by phosphorylating tyrosine residues of intracellular proteins, initially a protein confined to the intracellular portion of the receptor itself. This autophosphorylation often potentiates the receptor’s intracellular tryosine kinase activity, resulting in the tryosine phosphorylation of nearby proteins, an effect that results in their relocation, altered shape and/or functional activity. Initially, relocation was thought to involve the “docking” of these substrates to the autophosphorylated receptor initially activated by the extracellular agonist. Tryosine phosphorylation can exert a variety of effects, including movement specific proteins from the cytosol to the plasma membrane, translocation of membrane or cytosolic proteins to the actin-based cytoskeleton, and relocation of specific proteins to the specific storage sites in the cell. The physical properties that promote these events remain unclear, but probably involve alterations on the hydrophobicity of the substrates. When alterations in tryosine phosphatase alter the structure of the substrate, this alteration usually results in a change in the substrates conformation as well as its activity and its affinity for (and activity of) other molecules in the cellular milieu. Phosphorylation of proteins on serine and threonine amino acids by other protein kinases also alters their location, conformation and function, exerting a marked impact on cellular responses. After the serine/ threonine kinase PKA was identified as a downstream effector of CAMP, studies focused on protein kinase C (PKC), which is activated by anionic phospholipids (such as phosphatidylserine and phosphatidic acid) in the presence of Ca2+,or in a phospholipid and Ca2+-independentmanner by PMA. How these agents activate this ubiquitous kinase remains a mystery, but at the time, PKC was thought to be involved in virtually every cellular response that did not involve PKA. Indeed, along with PKCrelated kinases, including PRKl/2, PKC may also participate in events that depend on, or lead to, activation of PKA. Early studies demonstrated that activated PKC quickly translocated from the cytosol to the plasma membrane, where it is positioned to perpetuating signaling cascades. The ability of diacylglycerol, a product of PLC, to activate the enzyme in the presence of relatively high levels of free Ca2+linked its activity with certain G-protein-linked receptors.

The Remarkable Neutrophil!

25

It is now clear that many isotypes of PKC exist. Specific members of this family of kinases are activated in different ways under various conditions. While protein kinases C and A have a rather wide substrate range, specifically acting serine and threonine kinases, as well as specific tryosine kinases have been identified. These kinases display a high binding affinity for certain substrates in cells, which is regulated by the phosphorylation state of both the substrate and the kinase. However, PKA phosphorylates only serine and threonine residues which are flanked on both their N-and C-terminal aspects by a specific series of relatively hydrophobic amino acids, and antibodies are available that detect with some accuracy these PKA phosphorylated sites. Thus, when intact cells are exposed to ligands that activate intracellular PKA, as well as to pharmaceuticals that activate adenylate cyclase or mimic the effects of CAMP, which activates PKA, the cell’s natural PKA substrates can be identified. Excessive tyrosine phosphorylated proteins were initially identified in transformed cells that displayed defective protein tyrosine phosphatase (PTPase) activity, an observation that led to the award of Nobel prize to J. M. Bishop and Harlold Varmus for their discovery of the cellular origin of retroviral oncogenes in 1989. Shortly thereafter, Edwin G. Krebbs and Edmond Fisher would share the Nobel prize for their discoveries concerning reversible protein phosphorylation as a biological regulatory mechanism. Thus, dysregulated intracellular tyrosine kinases were initially associated with neoplastic transformation. However, tyrosine kinase-linked receptors were soon identified as receptors for various cellular growth factors, and while some cellular growth factors indeed have effects on neutrophils, no tyrosine kinase receptor has, to date, been identified on neutrophils. However, like many other cells, the neutrophil is replete with non-receptor intracellular tyrosine kinases, including members of the Src family of tyrosine kinases, including p6O-Src, Syk, Lyn, Yes, Fyn, as well as other non-receptor tyrosine kinases that are not Src family members. These most notably include focal adhesion kinase (FAK); the related proline enriched tryosine kinase (Pyk-2); the integrinlinked kinase (Ilk); the regulatory subunit of PI3’-kinase, as well as Vav, Rel-1, and Crk. Each of these kinases has been implicated in neutrophil adherence and/or directional migration and the mechanisms they evoke are rapidly being defined. For example, the association of Crk with its

26

The Neutrophils

preferred substrate Cas appears to serve as a molecular switch for the induction of migration, since when tryosine phosphorylated, Cas binds Src and other signaling molecules such as FAK, PTPases and other kinases and appears to localize these molecules to focal adhesions, the site of dynamic actin-based membrane adherence and detachment that is regulated by serine/ threonine kinases and phosphatases which interact with tryosine kinases and phosphatases to direct cellular adherence, polarity and directional migration. Neutrophils also possess several rather non-selective PTPases, which coordinate kinase cascades in complex and ill-defined ways. One of these has been implicated in "cross t a l k between PKA activated signaling and signaling dependent on tryosine phosphorylation, as it possesses a high affinity for PKA phosphorylated proteins. Indeed, several points of interaction between tyrosine and serine/threonine phosphorylation have been identified and may be mediated by serine/threonine kinases activated or translocated by tyrosine phosphorylation or by serine and/or threonine phosphorylation of phosphatases that regulate tyrosine kinases and phosphatases. While the role of these signaling molecules was initially examined in other cell types, recent defining studies linking the activities of these indispensable signaling molecules with those activated by G-protein-linked receptors have been derived from studies with neutrophilic leukocytes. As mentioned above, members of the Rho family of GTPases regulate virtually every aspect of neutrophilic function: adherence, directed migration, cell detachment, and activation of the respiratory burst. Studies with neutrophils have perhaps complicated our once naive yet simplistic appreciation of the diverse role these proteins exert in stimulated cells. However, at the same time, recent studies are clarifying these effects at a level of detail previously unparalleled in studies of cellular signaling. These studies have resulted in evidence supporting a Rho/Rac equilibrium, wherein the two mediators act in contrasting manners in different parts of the polarized cell to coordinate directed migration.

4.2. The Rac/Rho Equilibrium, Revisited Several Rho effectors have been identified, including Rho kinases 1 and 2, protein kinase N and PRC1/2. These proteins are all somewhat

The Remarkable Neutrophil!

27

homologous, have a molecular weight with a range 95-170 kDa, and bind Rho tightly. Like the influence of GDI on Rac, these effectors strongly influence the location of Rho in the cell, especially after the intracellular environment is changed by agonist activation and engagement of integrins. Thus, PKA effectively phosphorylates Rho bound to Rho kinase, and this phosphorylation decreases the affinity of Rho for Rho kinase in aqueous systems.76After PKA phosphorylation, P-Rho quickly detaches from Rho kinase, and is either inactive or binds different effectors with the consequence of inhibition of Rho singaling in aqueous systems, which here present a non-physiological environment. Thus it is quite possible that PKA-dependent phosphorylation of Rho at Ser 188, a process driven by matrix signaling, effects translocation of Rho kinase to the cytosol, where it releases phosphorylated Rho, which avidly displaces Rac from Rho-GDI, initiating membrane targeting of Rac and disrupting normal Rac signaling. In any event, emerging evidence implicates Rho phosphorylation as an initial event in cell activation, an event that leads to Rac signaling, enzyme translocation, and changes in local areas of cellular hydrophobicity. In the plasma membrane, the situation may be complex. Tigyi has recently proposed that Rho phosphorylation promotes its release from Rho-kinase and encumbrance by PKN, a protein kinase C related kinase, which in aqueous systems displays, unlike Rho-kinase, increased affinity for phosphorylated Rho.n Rho-kinase is thought to exist primarily in the cytosol, and its translocation to the plasma membrane may be promoted by Rho-GTP binding. However, the author’s recent studies show the situation to be more complex, with Rho kinase confined to both plasma membrane and cytoskeletal fractions of cells, in excess of levels found in the cytosol, depending on matrix signaling, agonist available and regulatory molecules. Indeed, unlike the parent compound, the GTP-bound form of PKA phosphorylated Rho displays a strikingly enhanced affinity for Rho-GDI in aqueous systems.59 In the membrane, the situation is predictably different, as the affinity of the hydrophobic Rho molecule for its effectors surely changes. This is expected to differ in the membrane of cytoskeleton, as the hydrophobic interactions there changes dramatically. Thus, in aqueous solutions, including both the cytosol and the media used in experiments, phosphorylated Rho shows less affinity for Rho kinase

28

The Neutrophils

Activated Rho kinases, myosin light chain kinase, LIM kinase, PLC related kinases (PKN, PRK-2), M A P kinase 42/44other Rho effectors including mDia, citron, ezrin, CaZ+mobilization, integrin signaling molecules, PLC, PKA. Effects on Fak, sre, pyk-2, and other

Activated: PAK’s, actin binding proteins, inhibitors of actin dissolution, regulators of NADPH oxidase, adherence mediators, inhibitors of integrin signaling, p38 MAP kinase, PI3’ kinase, PDK’s , PKB, cytoskeletal organization, morphological alterations.

+

Rac Signaling

Rhosignaling

+

Oxidative

Inflammation neutrophilia, mobilization, inflammation I

Set Point I

Homeostasis-neutropenia, apoptosis

Circulating Neutrophils Neutrophils

Fig. 4 The Rho/Rac equilibrium. As hypothesized above, cells exist at a certain set point within the Rac/Rho equilibrium. The inflammatory response begins when Rac signaling is activated in neutrophils, resulting in alteration of the activity of PAK’s, P13’ kinase, NADPH oxidase and p38 MAP kinase. These responses lead to increased adherence, directed migration and oxidative activation, processes for which the cell was designed to perform. In the absence of challenge, the neutrophil remains in the circulation unattached. Inflammatory mediators initiate Rac signaling cascades, resulting in adherence, migration, oxidative activation and culminating in inflammation. Resting cells maintain their ”Rho” phenotype as they patrol tissues while circulating in the vasculature. As cells age, Rho signaling results in contraction and apoptosis and the cells are removed from the circulation. Thus, in the responding neutrophil, induction of Rac signaling is of the utmost importance. However, Rho-dependent mechanisms are required to retain homeostasis. Otherwise inflammation runs rampant, resulting in arthritis, hypersensitivity, allergic reactions as well as tissue injury and destruction. Uncontrolled inflammation indeed lies at the root of many diseases. Thus, the possibility of therapeutic approaches designed to limit Rac signaling for inflammatory diseases has received much current attention. However, such therapy may come at a steep price; the function of the neutrophil is vital. To compromise this function leaves patients at risk for infection and dysregulated responses to inflammatory stimuli.

The Remarkable Neutrophil!

29

than unphosphorylated Rho. This result, however, tells us nothing of the affinity of Rho and phosphorylated Rho for Rho kinase in plasma membranes, where the enzyme functions (see Fig. 4).

5. A UNIFYING HYPOTHESIS The information provided above drive home one important fact; Rho family members exert dramatic, key and opposing effects in cellular activation. While many points of crossover exist, activities of Rac are associated with tryosine phosphorylation events related to cellular movement and morphology, while Rho effectors are serine/threonine kinases that maintain cellular homeostasis and effect contraction. As a general rule, an increased activity of Rho effectors is associated with development of cortical actin, cell loosening and detachment, contraction, increased vascular permeability and apoptosis. Conversely, the Rac phenotype consists of a change from a rounded to a spread morphology, tight adherence, increased spreading, loss of cell contacts, disruption of structural integrity and apoptosis. Complex processes like chemotaxis and structural development require finely coordinated responses wherein both phenotypes co-exist. The equation changes when one or the other predominates, and its effectors must be regulated. Thus, chemotaxis may initiate when Rac signaling recruits circulating cells but ceases when Rho signaling stops them. Here, the points of crossover are critical, and our appreciation of them paramount to our appreciation of cellular activation. As an example, as discussed above, the GTP form of phosphorylated Rho complexes avidly to cytosolic Rho-GDI, displacing Rac. There, a specific serine phosphatase may act at the leading edge of the cell to de-phosphorylate Rho and release its entrapped mediator, allowing GDI to once again mediate Rac targeting and activation of downstream effectors. Second, the Rac effector Pak-1 is, in fact, a serine/threonine kinase, which in some manner, critically regulates events that maintain Rac signaling. Another logical point of crossover lies in the activity of PKA, which keeps resting cells dormant, and opposes the integrin-dependent activities of PI3’-kinase. Substrates of PKA include Rho, whose effects on downstream effectors are influenced in many ways by Rho phosphorylation. The affinity of these effectors for Rho and the resultant distribution

30

The Neutrophils

of these effectors are influenced markedly by serine phosphorylation of Rho as well as the tryosine phosphorylation of regulators, such as Fak and Pyk-2. In turn, the activity of both mediators generated by activation of Rac as well as that of Rho effectors is markedly regulated and interrelated. This regulation is evident in the ability of both Src and Fak to dampen the constitutive activity of Rho kinase, and in the ability of Rho kinases to sequester Pyk-2 and possibly other molecules that transmit information when Rac is activated.78 What emerges is a complex interaction wherein the functions of Rac effectors are conveyed by tyrosine kinases to counter those constitutively active by serine/threonine kinases, including protein kinase A. Here, stimulus-dependent activation of Rac promotes function of resting cells, held in abeyance by Rho-dependent signaling. Ultimately, Rho strikes back, and returns the cell to its resting state after its function is carried out in an optimized and orchestrated manner. Integrins facilitate this orchestration, in part by effecting localized redistribution of cytosolic-free Ca2+. Rac is then available to induce final functional activation, including, in the case of the neutrophilic leukocyte, activation of the bactericidal NADPH oxidase. Future research will reveal the important points of crossover between Rac and Rho-mediated signaling pathways, and in doing so, may define the basic differences in the effects of tryosine phosphorylation and serine phosphorylation on cellular responses.

6. CONCLUSIONS The neutrophil remains a cell of discovery. Its diverse functions have been defining in our appreciation of signaling mediated by G-protein receptors, phospholipase C, phospholipase D, PI3’-kinase, and Rho family GTPases. The coordinated functions of the neutrophil provide an attractive model to both explore and define integrated signaling pathways, wherein integration may be highly dependent on alterations of the hydrophobicity of the effectors and their activators. Thus, the fundamental basis of cellular signaling may not be as complex as it seems. One unifying concept is that tryosine phosphoryla tion results in increased hydrophobicity, prompting the translocation of substrates from the cytosol to the cytoskeleton, whereas serine/threonine phosphorylation

The Remarkable Neutrophil!

31

increases hydrophilic affinity of signaling intermediates, resulting in their translocation to the cytosol. In this respect, it is clear that substrates of Rac effectors are indeed hydrophilic while those of Rho are, in general, hydrophobic. Alterations of these characteristics result in a basic and fundamental change in cellular function, and may provide the very basis of the Rho/Rac equilibrium, as defined by studies with the neutrophil.

REFERENCES 1. Dinnyes A, De Sousa P, King T, Wilmut I. Somatic cell nuclear transfer: recent progress and challenges. Cloning Stem Cells 2002; 4331-90.

2. Thomson JA, Itskovitz-Eldor J, Shapiro SS, et al. Science 1998; 282: 1145-1147. 3. Wagers AJ, Weissman IL. Cell 2004; 116:639-648.

4. Krebs EG. The Albert Lasker Medical Awards. 1989; 262:1815-1818. 5. Northup JK, Smigel MD, Gilman AG. J Biol Chem 1982; 257 11416-11423. 6. Moore M. Stem Cells Develop 2004; 13:l-22.

7. Anasassova-Kristeva M. J Hemmat Stem Cell Res 2003; 12:137-154.

8. Mock B, Schauwecker D, English D, et al. J Nucl Med 1988; 29: 1246-1251. 9. Lucht WD, English D, Bernard GR, et al. Am JMed Sci 1987; 294161-168. 10. English D, Broxmeyer HE, Gabig TG, et al. J lmmunoll988; 141:2400-2406. 11. English D, Roloff J, Lukens J. JImmunol1981; 126:1656-1671.

12. English D, Lukens J. J lmmunoll983; 1302450-856. 13. Graves V, Gabig T, McCarthy L, et al. Blood 1992; 80:776-787.

14. English D, Roloff J, Lukens J. Blood 1981; 58:129-134. 15. Weinberger A. Curv Opin Xheumatol 1995; 7:359-363. 16. Siddiqui R, Akard L, Garcia J, et al. Chemotactic migration triggers. Jlmmunol 1999; 162:1077-1083.

17. Mock B, English D. J Lipid Mediators 1990; 2:137-141. 18. Harvey K, Cui Y, Akard L, et al. In: Progress in Clinical and Biological Research (eds. Worthington-White D, Gee A and Gross S). Wiley-Liss & Co, New York, pp. 99-115,1994.

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19. English D, Gabig TG. Blood 1986; 67:1314-1322.

20. Siddiqui R, English D, Garcia JGN. 7 Lab Clin Med 1995; 12518-25. 21. Siddiqui RA, English D, Harvey K, et al. 1L e u b Bid 1995; 58:189-195. 22. Zhelev DV, Alteraifi A. Ann Biomed Engin 2002; 30:356-370,2002. 23. Pioerine LM, Lawson MA, Eddy RJ, et al. Blood 2000; 952471-2481. 24. Shattil SJ, Ginsberg MH. J Clin Invest 1997; 1OO:l-5. 25. Oseas R, Yang HH, Baehner RL, Boxer LA. Blood 1981; 57(5):939-945. 26. Liu L, Schwartz RR, Liu N, Harlen JM. J Immunol2002; 169:2330-2336.

27. English D, Taylor G, Garcia JGN. Blood 1991; 772746-2756.

28. English D. J Lab Clin Med 1992; 120:520-526. 29. Siddiqui RA, English D, Harvey K, et al. J Leuko Bioll995; 58189-195. 30. Siddiqui RA, English D. Cell Signalling 1996; 8:349-354.

31. Siddiqui R, English D. Biochem Biophys Acta 1997; 1349:81-95. 32. English D, Cui Y, Siddiqui RA. Adv Chem Phys Lipids 1996; 80:117-132. 33. English D. Cell Signal 1996; 8:341-347. 34. Siddiqui RA, English D. Biochem Siophys Acta 1999; 1483:l-13. 35. English D, Cui Y, Siddiqui R, et al. J Cell Biochem 1999; 75:105-117. 36. Andersen BT, Rizzo MA, Shome K, Remero G. FEBS Lett 2002; 531:65-68. 37. Gosh S, Strum JC, Sciorra VA, et al. J Biol Chem 1996; 271:8472-8480.

38. English D, Martin M, Siddiqui RA, et al. Biochem J 1997; 324941-950. 39. Taylor GS, Ladd A, Jansen J, et al. Biochem Biophys Acta 1993; 175: 219-224. 40. Cui Y, English D. Cell Signal 1997; 9257-263. 41. Newman JH, Fulkerson WJ, English D, et al. App Phys 1986; 60:1386-1392. 42. Loyd JE, Newman JH, English D, et al. 1App Phys 1983; 54967-276. 43. Rinaldo JE, English D, Levine J, et a!. A m Rev Respir Dis 1988; 137335-345. 44. Wtienne-Mannevlle S, Hall A, Nature 2002; 420:629-637. 45. Gu G, Filippi M-D, Cancelas JA, et al. Science 2003; 302445449,

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46. Parent CB, Blacklock B, Froehlich W, et al. Cell 1992; 95:81-91. 47. Picardino P, Manarini S, Federico L, et al. Biochem j 2004; BJ20040151, (in press). 48. Wang H-R, Zhang Y, Ozdamar B, et al. Science 2003; 302:1775-1779. 49. Bagrodia S, Cerione RA. TIBS 1999; 9:350-355. 50. Rodriguez-Fernandes JL, Sanchez-Martin L, Rey M, et al. J Biol Chem 2001; 276:40518-40527. 51. Leitinger BA, McDowall A, Stanley P, Hogg N. Biochem Biophys ACTA 2000; 1498:91-98. 52. Rodereguez-Fernandez JL, Sanchez-Martin L, de Frutos CA, et al. J Leuka Biol 2002; 71:520-530. 53. Allen RC, Stjernholm RL, Steele RH. Biochem Biophys Res Commun 1972; 47(4):679-684. 54. McCord JM, Wong K, Stokes SH, et al. Acta Physiol Scand 1980; 492:25-30. 55. Horan T, English D, McPherson TA. Clin Immunol Immunopathol 1982; 22~259-269. 56. Babior BM. Curr Opin Immunol2004; 16:42-47. 57. Di-Pon N, Faure J, Molnar G, et al. Biochemistry 2001; 40:10014-10022. 58. del Pozo MA, Alderson NB, Kiosees WB, et al. Science 2004; 303: 839-842. 59. Ellerbroek SMK, Wennerberg K, Burridge K. J Biol Chem 2003; 278: 19023-1 9031. 60. Garcia JGN, Painter, RG, Fenton JW, et al. J Cell Phys 1990; 142186-193. 61. Garcia JGN, Patterson CE, Bahler C, et al. J Cell Phys 1993; 156:541-549. 62. Garcia JGN, Liu F, Verin AD, et al. J Clin Invest 2001; 108:689-701. 63. Liu F, Verin AD, Wang P, et al. A m J Res Cell Mol Biol2001; 24:711-719. 64. Garcia JGN, Verin AD, Herenyiova M, English D. J Appl Phys 1998; 8:1817-1821.

65. Cui Y, English D, Siddiqui RA,Garcia JGN. J Invest Med 1997; 45:388-393. 66. Verghese MW, Smith CD, Snyderman R. Biochem Biophys Res Commun 1985; 127(2):450-457.

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67. Traynor-Kaplan AE, Thompson BL, Harris AL, et al. J Biol Chem 1989; 264(26):15668-15673. 68. Putney JW Jr, Weiss SJ, Van De Walle CM, Haddas RA. Nature 1980; 284(5754):345-347. 69. Sundaram M, Cook HW, Byers DM. Biochem Cell Biol2004; 82(1):191-200. 70. Hokin LE, Hokin MR. J Biol Chem 1958; 233(4):800-804. 71. Michell RH. Biochim Biopkys Acta 1975; 415(1):81-147. 72. Berridge MJ, Dawson RM, Downes CP, et al. Biockem J 1983; 212(2):473-482. 73. Mukaui H. J Biochem 2003; 133:17-27. 74. Billah MM, Eckel S, Mullmann TJ, et al. J Biol Chem 1989; 264(29):17069. 75. English D, Taylor G, Garcia JGN. Blood 1991; 77:2746-2756. 76. Smith A, Brake M, Leitinger B, et al. Cell Sci 2003; 1163123-3133. 77. Nusser N, Gosmanova E, Guo F, et al. Phoshoryltion - medited effector selection of Rho - A: a novel mechanism for moisullating Rho GTPase signaling required for NGF-induced neurite outgrowth. Nature Cell Biol (in press). 78. Okigaki M, Davis C, Falasca M, et al. Proc Nat Acad Sci ( U S A ) 2003; 100:10470-1 0745. 79. Moissoglu K, Gelman IH. J Biol Chem 2003; 278:47946-47959.

2 ----------The Neutrophil Respiratory Burst Oxidase M a r k 1 Quinn"

Neutrophils play an essential role in the body's innate defense against pathogens and are one of the primary mediators of the inflammatory response. To defend the host, neutrophils utilize a wide range of microbicidal products, such as oxidants, microbicidal peptides, and lytic enzymes. The generation of microbicidal oxidants by neutrophils results from the activation of a multi-protein enzyme complex known as the NADPH oxidase, which is responsible for transferring electrons from NADPH to 02,resulting in the formation of a superoxide anion (02'-). During NADPH oxidase assembly and activation, cytosolic oxidase proteins translocate to the phagosome or plasma membrane, where they assemble around a central membrane-bound component known as flavocytochromeb. This process is highly regulated, involving phosphorylation, translocation, and multiple conformational changes. In this chapter, key structural and functional features of the neutrophil NADPH oxidase and its protein components are described.

*Correspondence to: Mark T. Quinn. Veterinary Molecular Biology, Montana State University, Bozeman, h4T 59717; phone: 406-994-5721; fax: 406-994-4303; e-mail: [email protected].

35

36

The Neutrophils Keywords: neutrophils; phagocytes; NADPH oxidase; respiratory burst; reactive oxygen species; inflammation; superoxide anion; oxidants; free radicals; chronic granulomatous disease

1. INTRODUCTION The innate immune response represents a highly conserved strategy used by the host in defense against a wide array of bacterial, fungal, and viral path0gens.l Activation of the innate immune system results in an inflammatory response, which is essential for rapidly controlling infections before they can spread. In addition, it is now clear that cells of the innate immune system contribute to the initiation and subsequent focus of the ensuing adaptive immune response.2 A number of cells contribute to the innate immune response, including phagocytes, NK cells, y6 T cells, and mast cells3Phagocytes are especially critical to the acute inflammatory response, due to their capacity to efficiently engulf and destroy a variety of pathogens. These cells are also known as professional phagocytes and are composed of neutrophils, monocytes, macrophages, and eosinophils. Among this group, neutrophils are the most numerous, are usually the first cell to arrive at the sites of inflammation, and are possibly the most important cellular component of the innate response during an acute infe~tion.~ Neutrophils (also known as polymorphonuclear leukocytes) are normally found circulating in the bloodstream (circulating half-life of -7 hr), migrating through tissues (-2-3 days) and devoting their short lifetime to s~rveillance.~ However, during the acute inflammatory response, the neutrophil lifespan is increased, and large numbers of neutrophils are rapidly recruited to the site(s) of infection, where they function to destroy the invading pathogens. In this capacity, neutrophils serve as one of the body's first line of defense against infection. These cells utilize a wide array of microbicidal weapons to neutralize and kill pathogens, including the release of lytic enzymes and microbicidal peptides from cytoplasmic granules and the production of reactive oxygen species or oxidantsa6 The generation of microbicidal oxidants by neutrophils results from the activation of a multi-protein enzyme complex known as the NADPH oxidase, which is responsible for transferring electrons from NADPH to 02,resulting in the formation of a superoxide anion (02.-h7f8 0 2 ' - is rapidly converted to secondary toxic oxygen species, which can efficiently

The Neutrophil Respiratory Burst Oxidase

37

kill microorganisms and, in combination with the primary and secondary granule contents, constitute one of the primary host defense mechanisms used by n e ~ t r o p h i l sWhile .~ the goal of this process is to destroy the pathogen or offending agent, neutrophil-generated oxidants can also damage host tissues in the inflamed region. Indeed, reactive oxygen species have been reported to be involved in the tissue injury associated with a number of inflammatory diseases.6 The importance of the NADPH oxidase to host immunity is clearly demonstrated by a rare genetic disorder known as chronic granulomatous disease (CGD).'O CGD is characterized by various genetic defects in essential NADPH oxidase components and results in an inactive oxidase. Patients with CGD experience severe, recurrent bacterial and fungal infections and often develop granulomas formed by the fusion of monocytes and macrophages that have phagocytosed bacteria, but are unable to destroy them as a result of a defective NADPH oxidase.1° The classical NADPH oxidase was first described and characterized in neutrophils and other phagocytes, and it was originally thought that this system was restricted only to phagocytes and used solely in host defense. However, studies in the last 10-15 years indicate that similar NADPH oxidase systems are present in a wide variety of non-phagocytic cells, both of leukocyte and non-leukocyte origin.71r12 Although the nature of the nonphagocyte NADPH oxidases is still being defined, it is clear that they are functionally distinct from the phagocyte oxidases, produce much lower levels of 02'-, and appear to play important roles in inter- and intracellular signaling events. It should be noted, however, that structural features of many non-phagocyte oxidase proteins do seem to be similar or identical to those of the phagocyte counterparts. Nevertheless, since this chapter will focus only on the structure and function of the neutrophil NADPH oxidase, the reader is referred to recent reviews for further information on the non-phagocyte NADPH oxidase ~ysterns.l'-'~

2. NADPH OXIDASE COMPONENTS The development of the cell-free assay system for reconstituting NADPH oxidase activity greatly accelerated our understanding of the composition and characteristics of the phagocyte oxidase.14-'6For example, this assay system played a crucial role in demonstrating that both membrane and

38

The Neutrophils

cytosolic components were absolutely required for oxidase activity. Furthermore, cell-free oxidase studies with membranes and cytosol isolated from CGD neutrophils resulted in the identification of key NADPH oxidase proteins. It is now generally accepted that the core oxidase enzyme is composed of four oxidase-specific proteins (p22phox, p47phoX, p67phox,and gp91PhoX) and a GTPase (Rac 1/2). One other oxidasespecific protein (p40phoX) and a second GTPase (RaplA) have also been shown to play roles in regulating oxidase activity; however, their specific roles are still not well understood. Originally, the nomenclature for the various components differed throughout the literature; however, the generally accepted nomenclature for the oxidase-specific components now includes the suffix phox, which refers to phagocyte ~ i d a s e . The ' ~ one exception is gp91phoX, which has also been named N o ~ 2 .Overall, l~ the phox proteins are highly conserved throughout the various species studied to date, confirming the absolute requirement for this system in host defense. Details of each of the individual components are summarized below.

2.1. Flavocytochrome b The first NADPH oxidase component to be identified was a nonmitochondria1 cytochrome b.19 In fact, it was originally thought that the phagocyte NADPH oxidase was composed only of this cytochrome b plus an additional flavoprotein.20,21 At this time, cytochrome b was also known as cytochrome bS5*or b559,due to its or-band absorption maximum of 558-559nmI9 or cytochrome b-245because of its unusually low midpoint reduction potential of -245mV.22 In resting cells, cytochrome b is located primarily in intracellular granules or vesicles, with a small amount present in the plasma membranes (-10%); however, much of the internal cytochrome is mobilized to the membrane during a~tivation?~ A number of pieces of evidence implicated cytochrome b as an essential redox component of the NADPH oxidase; however, the most compelling evidence came from studies showing its spectrum was absent in cells from patients with X-linked CGD.24Subsequently, the gene coding for the defective protein associated with X-linked CGD was identified by a reverse genetics approach and was cloned.25This gene coded for a 54-58 kDa protein containing several potential glycosylation sites; however,

The Neutrophil Respiratory Burst Oxidase

39

it was initially concluded that this X-CGD protein was not cytochrome b but that it could be a protein that interacted with cytochrome b or the f l a v o p r ~ t e i nOne . ~ ~ of the reasons for this conclusion was that the amino acid composition of the predicted X-CGD protein did not resemble that reported for purified cytochrome b.26In addition, the estimated size of ”purified” cytochrome b ranged anywhere from 11-127 kDa, depending on the species and/or purification method. Ultimately, this issue was resolved by Parkos and coworkers, who developed a novel and successful method for purifying neutrophil cytochrome b.27Their studies demonstrated that purified cytochrome b was not a single polypeptide, but was actually a heterodimer of a 91 kDa glycoprotein (known also as the p-chain) and a 22 kDa non-glycosylated protein (known as the a-chain). These proteins are now known universally as gp91PhoXand p22phox,respectively. In addition, Dinauer et dz8 were able to use the antibodies developed in these studies to show that the defective protein in X-linked CGD protein was actually gp91Phox. This finding was confirmed by Teahan et d.F9who sequenced the N terminus of gp91Phoxand found it to be almost identical to that of the X-CGD protein. Further analysis of purified cytochrome b by crosslinking and immunoprecipitation analyses confirmed the heterodimeric nature of the molecule30and eventually led to the cloning and sequencing of p22Phox.31 Sequencing of the cytochrome b subunits provided a major advance in our understanding of this oxidase component; however, a number of aspects of the structure and function of this complex remained unresolved, including the stoichiometry of the subunits. Hydrodynamic analyses of detergent-solubilized cytochrome b were consistent with the complex being either an a, p-type heterodimer or an a, P, P-type heterooligomer,30 and early structural models of cytochrome b were developed around an a, p, p-type complex.32 This issue was resolved by Huang and coworkers,33who used a novel protein sequencing approach to show definitively that cytochrome b was actually a 1:l a, P-type heterodimer of p22phox and gp91PhoX. These findings were subsequently confirmed by Wallach and Sega1,34who utilized a number of biochemical approaches and quantitative Western blotting to establish 1:l stoichiometry. Hydropathy analyses of the predicted p22phoxand gp91p”0xproteins indicated the presence of 2-3 and 4-6 transmembrane regions in these proteins, r e s p e c t i ~ e l y .The ~ ~ ,transmembrane ~~ nature of the cytochrome b

40

The Neutrophils

subunits has also been confirmed by antibody-binding s t ~ d i e s ~and ~”~ by peptide mapping approaches to identify functional domain^.^^-^^ Furthermore, Wallach and Sega143used site-directed mutagenesis to map putative glycosylation sites in gp91phoxand showed that Asnl31,148, and 239 were all glycosylated and, therefore, located on the extracellular surface of the membrane. Based on the information provided from these approaches, tentative topological models have been suggested for gp91Phoxand p22phox(Fig. 1).These models will be used for reference in the remainder of this chapter. Since the primary structure of gp91PhoX showed no homology to known c y t o c h r o m e ~and ~ ~ p22phoxshowed only limited homology to the heme binding region of cytochrome c ~ x i d a s e , ~the * nature of the cytochrome b heme prosthetic groups was not evident. Based on measurement of heme specific activity, Parkos et a1.31 concluded that more than one heme was present in each cytochrome b molecule. Furthermore, their sequence analysis showed that p22phoxcontained only one invariant histidine, resulting in the possibility that the hemes could also be coordinately

Fig. 1 Models of flavocytochrome b showing proposed transmembrane helices and placement of hemes. (A) Shared heme model with one heme coordinated by gp91Phoxhelices 111 and V ( 0 ) and one shared between gp9lPkoX helix V and p22phoX helix 111 ( 0 ) . (B) Non-shared heme model with both hemes coordinated by gp91Phoxhelices 111and V. Proposed sites of glycosylation (Y)and location of other redox components (FAD, NADPH) are indicated.

The Neutrophil Respiratory Burst Oxidase

41

shared between cytochrome b subunits. This idea was supported by subsequent studies showing that the low temperature reduced-minusoxidized absorbance spectrum of the cytochrome b a band was split, indicating the presence of two heme species,44 as well as by electron paramagnetic resonance (EPR) and resonance Raman studies showing that the cytochrome b spectra were compatible with a bihistidinyl, multiheme cytochrome with closely-spaced he me^.^^ We used low-temperature polyacrylamide gel electrophoresis to directly investigate this issue and found that cytochrome b was a bi- or possibly tri-heme molecule with one heme residing completely within gp91PhoX and one apparently shared between gp91PhoX and p22phox.46 This concept was further refined when reevaluation of the averaged heme potential of -245 mV using higher resolution methods showed that cytochrome b contained two non-identical hemes with midpoint redox potentials of -225 and -265 mV.47 Heme incorporation plays an important role in cytochrome b biosynthesis, and Yu et a/.48 found that heme incorporation was important in cytochrome b assembly and stabilization of the heterodimer. Indeed, DeLeo et al.49 showed incorporation of heme by gp65 (the unglycosylated precursor of gp91PhoX) preceded and was required for heterodimer formation. Together, these studies suggest the possibility that the heme could serve as a dimerizing agent, linking gp65 and p22phox.However, there is currently no direct evidence to verify this idea. It is also possible that heme incorporation facilitates conformational changes in the subunits which are required for stable heterodimer formation. Analysis of cytochrome b subunits expressed in transgenic COS7 cell lines, showed that cells expressing gp91PhoX alone exhibited a heme spectrum very similar to neutrophil cytochrome b, with midpoint potentials of -264 and -233mV, while cells expressing p22phoxalone showed no heme spectrum.50 In contrast, co-expression of both gp91PhoX and p22phoX was required to support 0 2 ' production. Thus, these studies suggested that, at least in the COS7 system, gp91PhoX is able to coordinate both hemes in a manner similar to that in native neutrophil cytochrome b. Based on experimental evidence described above, it is well-accepted heterodimer containthat neutrophil cytochrome b is a 1:l gp9lphox:p22PhoX ing two non-identical hemes; however, the exact location of the two hemes is still not completely defined. Two basic models of heme placement are

42

The Neutrophils

currently under consideration. According to the first model, one of the hemes is coordinated within gp91PhoX, while the second heme is coordinately shared between gp9lPhoX and gp9lPhoX5l (Fig. 1A). This model is supported by several additional lines of evidence. For example, the cytochrome b subunits can be separated only under denaturing conditions that result in the loss of the heme spectrum,3° suggesting a role of the heme in bridging the heterodimer. Peptides corresponding to regions adjacent to the invariant histidine of p22PhoX inhibit 9'generation in a cell-free system, suggesting this region is indeed important in cytochrome b function. In addition, missense mutations resulting in a His94 to Arg substitution result in an autosomal form of CGD and absence of cytochrome b.52Significantly, recent studies of Foubert et ul.% showed that a spectrally stable proteolytic product of neutrophil cytochrome b contained fragments of both gp91phoxand p22phox,which is also consistent with the presence of a shared heme. In an alternative model, which is also based on experimental evidence and on modeling similarities between gp91PhoX and yeast iron reductase Frel, Finegold et ul.% proposed that both hemes are stacked within gp91PhoX, between transmembrane helices I11 and V, and coordinated by histidines 101, 115, 209, and 222 (Fig. 1B). Additional support for this model comes from mutagenesis studies showing that replacement of these histidines with leucine or arginine resulted in lost (mutated His 101 or 115) or significantly decreased (mutated His209 or 222) heme spectrum, as well as from data showing that missense mutations in any of these residues results in X-linked CGD.55Furthermore, mutagenesis of the p22PhoXinvariant histidine (His94) showed, in contrast to the CGD mutation described above, that a histidine at this position was not required for flavocytochrome b function, suggesting that heme was not shared between flavocytochromeb subunits in this system.56Given that both models are supported to varying degrees, further studies will be necessary to resolve this issue. As mentioned above, it was originally thought that the NADPH oxidase was composed only of cytochrome b plus an additional flavoprotein; however, the nature of the flavoprotein remained quite elusive. Early on, there was substantial experimental evidence indicating that flavin adenine dinucleotide (FAD) was a cofactor in the oxidase. For example, studies on the detergent-solubilized NADPH oxidase showed that

The Neutrophil Respiratory Burst Oxidase

43

enzyme activity was stabilized or enhanced by the addition of FAD57and was inhibited by FAD analogues58and flavin inhibitor^.^^ The search for the putative flavoprotein led to the identification of candidate proteins with molecular weights of 66 kDa60and 45 kDa;61however, many discrepancies remained, and the ongoing search led to the consideration that the FAD-binding moiety might actually be cytochrome b itself. This issue was eventually resolved by three separate groups who concurrently provided data demonstrating that cytochrome b was indeed a flavocytochrome, based on amino acid sequence homology between gp91Phuxand the ferridoxin-NADP+ reductase (FNR) family of reductases, the absolute requirement for exogenously added FAD to reconstitute oxidase activity partially purified cytochrome b, and the localization of added FAD to gp91phox.62-64 These observations led to the renaming of cytochrome b558to the currently accepted nomenclature of flavocytochromeb558 (a.k.a., flavocytochrome b). Since NADPH is the electron source for the catalysis of O2to 02'-, an NADPH-binding component must be present in or near the enzyme complex. As with the FAD-binding protein, the search for the NADPHbinding moiety also resulted in the identification of several candidates. For example, a number of convincing studies characterized a cytosolic component of -66-67 kDa as the NADPH-binding protein of the oxidase.65,66 Smith et ~ 1 . showed 6 ~ that pre-treatment of cytosol with dialdehyde derivatives of NADPH blocked oxidase activity in a cell-free assay and used affinity labeling to identify a 32 kDa NADPH-binding protein.67 Based on amino acid sequence comparison between gp91phoxand FNR, it was found that regions of gp91phoY were weakly homologous to consensus NADPH-binding sites, suggesting that in addition to binding FAD, flavocytochrome b might also contain an NADPH binding In support of this idea, a rare missense mutation within one of these regions of gp91PhoY(P415H) was identified in a patient with X-linked CGD.68In addition, photoaff inity labeling with azido-NADPH provided direct evidence that gp9lPhoxcontained an NADPH binding site.69Furthermore, Koshkin and Pick showed that relipidated flavocyproviding direct evidence that flavtochrome b alone could generate 02'-, ocytochrome b is able to functionally bind both FAD and NADPH.70 Although it is generally accepted that flavocytochromeb can serve as the

44

The Neutrophils

NADPH-binding component, alternative possibilities are still under consideration, such as p67phox(see below). In summary, flavocytochrome b is a 1:l heterodimer of gp91PhoX and p22Phox;contains two non-identical hemes; binds FAD and NADPH; and appears to contain the entire electron transport apparatus of the NADPH oxidase complex. Based on these observations, it has been concluded that flavocytochromeb is the only catalytic component of the oxidase and acts as a conduit for electron transfer.71Regardless of whether the hemes are shared between subunits or not, the currently accepted pathway involves sequential and stepwise transfer of 2 electrons from NADPH via FAD and two hemes to ultimately reduce 0 2 . On the other hand, flavocytochromeb cannot function independently in the cell and requires several other oxidase proteins for enzymatic activity. Therefore, it can be concluded that these NADPH oxidase proteins, which are described below, must play roles in regulating the ability of flavocytochromeb to efficiently transport electrons.

2.2. p47phox The availability of neutrophils from patients with CGD has been instrumental in research efforts focused on the identification of the NADPH oxidase component proteins and, in conjunction with the cell-free NADPH oxidase assay system, verified that cytosolic proteins were essential for oxidase activity. Early on, Segal et aZ.72 observed that neutrophils from patients with autosomal recessive CGD provided failed to phosphorylate a 44 kDa protein and suggested this protein could be oxidase-related. Through genetic analyses, complementation experiments, and cell-free assays, two groups concurrently identified this protein, which was known previously as neutrophil cytosolic factor 1 (NCFZ) but is now named p47ph0x.73~74 These studies showed that p47Phoxwas required for optimal oxidase activity and was the protein deficient in the most common form of autosomal recessive CGD. Cloning and sequencing of p4rPhoxshowed that it was a protein of 390 amino acids, corresponding to a mass of -42 kDa.75f76 Sequence analysis showed that p47Phoxis a highly basic protein containing a number of potential phosphorylation sites (residues 314-347); tandem Src homology 3 (SH3) domains (residues 163-211 and 227-281); a C-terminal proline-rich domain (residues 360-371 1; and an

The Neutrophil Respiratory Burst Oxidase

45

Fig. 2 Scale model of p47Pbx showing functional domains (see text for details).

N-terminal Phox homology (PX) domain (residues 4-125) that may play a role in phosphoinositide binding (Fig. 2). In resting neutrophil cytosol, p4PX exists in a free form, as well as in an -240 kDa complex, consisting of equimolar (1:l:l)amounts of p47Phox, p67phx, and p4Whx.7,78Following neutrophil activation, the entire complex apparently translocates to and associates with flavocytochrome b.n,78In addition, free p47phoxcan also translocate to the membrane by itself.79 Although the exact role of p47phoxin NADPH oxidase assembly is still unknown, it appears to function in a regulatory role during oxidase activation and deactivation. One possibility suggested recently by Cross and Curnutte is that p4Thxis responsible for electron transfer from FAD to the heme center of flavocytochromeb.80In any case, p4Thxseems to be the first cytosolic component to interact with flavocytochrome b during the assembly processs1B2and its association with flavocytochrome b is a prerequisite for translocation of p6Thxand/or p40phx.81-s3 The assembled complex of flavocytochromeb, p47phx, and p67Phx has been reported to be equimolar,= with an apparent irreversible complex formed between p4Thox and flavocytochrome b.40fi2Not all of the p47Phx present in the cytosol is translocated in the activated cell, however, and only 2-10% of the total protein is detected at the plasma membrane.82In addition, it appears that p4Thx (and p67phox)translocation kinetics closely resemble the kinetics of oxidase activation and that continuous association of these components with the active oxidase complexes is necessary to maintain the respiratory As noted previously, phosphorylation of a protein, putatively p4Thox, was shown to be related to activation of the oxidase. It is now known that

46

The Neutrophils

p47phflxphosphorylation is one of the key intracellular events associated with NADPH oxidase a c t i v a t i ~ n . Six ~ ~ predominant -~~ forms of phosphorylated p47phflxhave been identified in activated neutrophils,ss~yO,yl and it appears that phosphorylation occurs in a step-wise fashion, with four of the most phosphorylated forms of p47phflxassociated with the membrane 30 seconds after activation with PMA and all forms present after 5-15 minutes.88However, in patients with X-linked CGD, two of the most phosphorylated species are not present, indicating that some phosphorylation may occur at the membrane following translocation and/or requires flavocytochrome b i n t e r a c t i ~ n . ~ ~ . ~ ~ A number of kinases have been proposed to participate in p47phox phosphorylation events, including protein kinase C (PKC),y2mitogenactivated protein kinases [p38 MAPK and extracellular signal-regulated kinase (ERK1/2)]y2,y3; p21-activated kinase (PAK)y4;Akty5;casein kinase 2 (CK2)96;and a phosphatidic acid-activated k i n a ~ e One . ~ ~ of the most important kinases is PKC, and it has been shown that p47phoxphosphorylated in vitm by PKC alone can activate the cell-free oxidase system, eliminating the need for amphiphilic activating agentsy8Fontayne et aLYy showed that PKC-dependent phosphorylation occurred at Ser303, 304, 315, 320, 328, 359, 370, and 379; with Ser328 being the most common target. It appears, however, that it is not necessary to phosphorylate all of these serines and that the minimal level of phosphorylation to induce p47phflxunfolding and translocation includes Ser303, 304, and 328.'0° Another key PKC phosphorylation site is located at Ser379, which must be phosphorylated for oxidase activation in vivas5 In addition to these four sites, PKC-mediated phosphorylation at the remaining serines may be utilized for other as yet undefined functions, such as stabilizing the enzyme complex or attenuating oxidase The involvement of various other protein kinases in p47phoxactivation is not as well understood. A number of studies have shown that inhibitors of MAPK/ERK pathways inhibit oxidase activity,101J02 and consensus MAPK substrate sequences are located at Ser345 and 348. Phosphorylation at these residues does not appear to be critical for 02'-generation in phorbol myristate acetate (PMA)-stimulatedcells92,93; however, more recent studies using physiological activators suggest that ERK1/2 (but not p38 MAPK) and PKC actually do cooperate in phosphorylating p47phoxduring oxidase

The Neutrophil Respiratory Burst Oxidase

47

activation. PAKs have also been implicated in p47phoxphosphorylation, and Knaus et ~ 1 found . ~ that ~ neutrophil PAKs could phosphorylate p4ThoX on Ser328, suggesting these kinases may link chemoattractantreceptor stimulation and oxidase activation. Akt is rapidly activated when neutrophils are treated with oxidase activating agents, suggesting Akt may also participate in oxidase activation.lo3Indeed, p47phoxphosphorylation is enhanced in cells expressing membrane-targeted phosphoinositide 3-kinase (PI3-kinase), which constitutively activates AKT?5 and recent studies showed that Akt phosphorylates p47PhoxSer304 and 328.'04J05These results suggest a role for Akt in mediating P13-kinase-dependent phosphorylation of p47PhoX. In contrast to intact neutrophils, production of 02'-in the cell-free system does not require phosphorylation.'06There is, however, a requirement for anionic amphiphilic detergents such as SDS or arachidonic acid.I4-l6It is still not completely clear if amphiphile activation in vitro is mimicking actual physiological events in vim, but it has been suggested that activation with amphiphiles may share mechanistic similarities with activation by pho~phory1ation.l~~ In support of this conclusion, PKC can also activate the cell-free system without added amphiphiles, thus mimicking the phosphorylation-dependent activation process occurring in zlivo.98,108It has also been proposed that phosphorylation causes a conformational change in p47phoxand/or neutralizes the cationic domain of the protein (Fig. 4) in v i m so that it can interact with the membrane,s5,106 and Ago et u1.1°9 found that phosphorylation of only three serines mimicked this conformational change. Similarly, SDS or arachidonic acid may provide a neutralizing negative charge which allows p47Phoxto undergo similar conformational changes in vitro that are likely to occur with phosphorylation in vivo.'l0 In support of this idea, we found that amphiphiles (SDS and arachidonic acid) and PKC phosphorylation both induced similar changes in p47phoxconformation, as determined by changes in tryptophan fluorescence, and that these changes in fluorescence correlated directly with NADPH oxidase activity.ll*Our results, which were subsequently confirmed by Park et u1.,112 provided direct evidence linking conformational changes in p47Phoxto activation of the neutrophil NADPH oxidase. Furthermore, recent studies by Peng et showing that p47Phoxmutants with unmasked SH3 domains are able to fully reconstitute oxidase activity in a cell-free assay lacking arachidonic

48

The Neutrophils

acid, as well as studies by Ago et ~ 1 . showing ~ ~ ' ~ that p47Phoxphosphorylation caused unmasking of phosphoinositide-binding domains, again support a requirement for activation-induced conformational changes. Recently, a novel proline-rich module was identified in p40phoxand p47phox.115 This conserved sequence motif was named the PX domain because of its presence in these phox proteins (Fig. 4). However, it is now known that PX sequences are present in over 150 eukaryotic proteins and serve as phosphoinositide-binding modules.116The p47phoxPX domain is located within an -120-amino acid module encompassing residues 4-125 and has been shown to bind phosphoinositides with apparent preference for phosphatidylinositol 3,4-bisphosphate [ P t d I n ~ ( 3 , 4 ) P ~ ] , ~ ~ ~ although the relative selectivity for various phosphoinositides is not completely r e s ~ l v e d . "The ~ ability to bind phosphoinositides suggests the PX domain plays a role in targeting p47phoxto membranes, and PX domain-mediated targeting of this protein to membranes has been demonstrated.118-120In addition, Karathanassis et ~ 1 . crystallized l ~ ~ the p47phoxPX domain which actually contains two binding pockets, one for PtdIns(3,4)P2and the other for anionic phospholipids, such as phosphatidic acid or phosphatidylserine. They also confirmed previous studies of Hiroaki et u1.,122 who reported that the PX domain was masked by an intramolecular interaction with the C-terminal SH3 domain of p47phox. These findings suggest an additional role for PX modules in proteinprotein binding, and this idea is supported by studies showing that binding of p47ph0xto moesin is mediated by the PX domain, albeit in a phosphoinositide-dependent process.lZ3Furthermore, the identification of a putative phosphatidic acid-binding region is consistent with the ability of phosphatidic acid to activate the 0 x i d a ~ e . I ~ ~

2.3. p67phox Human p67phox,like p47phox,was initially identified as a missing neutrophil cytosolic factor in patients with autosomal recessive CGD.73f74 Originally named NCF2, p67phoxis 526 amino acids long and migrates as an -65-68 kDa protein on SDS-polyacrylamide gels.73~74,125 Sequence analysis showed that p67phoxcontains two SH3 domains (residues 245-295 and 458-517); a proline-rich domain (residues 219-231); and

The Neutrophil Respiratory Burst Oxidase

49

Fig. 3 Scale model of p67Phoxshowing functional domains (see text for details).

four N-terminal tetratricopeptide repeat (TPR) motifs (within residues 6-154)125,126 (Fig. 3). As with p47phox,p67phoxhas been reported to exist in the 240 kDa of the total p67phox cytosolic complex in resting n e ~ t r o p h i l s , and ~ , ~ -10% ~ translocates to the membrane in activated cells.82*wz86 It is apparently the limiting oxidase cofactor in neutrophil cytos01,'~~ and Vergnaud et u1.lZ8 recently verified this notion through complementation studies with neutrophil fractions obtained from p67Phox-deficientCGD neutrophils. There appears to be 2-3 times less p67phoxin neutrophil cytosol compared to p47phox.129J30 Thus, most or all of the p67phoxwould be associated with p47phox.In support of this conclusion, addition of exogenous p67phoxhas been reported to enhance binding of p4Tphoxto the membrane, possibly by binding free p47phox.83 This would be expected if there were always more p47phox available for active complex formation at the membrane. Although less drastic than in p47phox,p67phoxhas been reported to undergo conformational changes following activation in the cell-free assay system,131indicating that molecular interactions reported in the resting cell complex are probably significantly different than those occurring following activation. Most reports agree that in vivo translocation and membrane association of p67phoxis dependent on co-translocation of p47Phoxto the plasma membrane and prior interaction of p4Thoxwith flavocytochrome b.81-84 This conclusion is supported by recent work of Paclet et u1.,132 who used atomic force microscopy to verify that p47phoxpreceded p67phoxand enhanced the affinity of p67Phoxin binding to flavocytochrome b during oxidase activation. It should be noted, however, that in vitro reconstitution

50

The Neutrophils

of NADPH oxidase activity in the absence of p47phoxis possible when relipidated flavocytochrome b and high concentrations of p67phoX and Rac are ~ o m b i n e d , ' ~supporting ~J~ the idea that p67Phoxand Rac play more direct roles in electron transport and suggested a possible direct binding interaction between p67phoxand flavocytochrome b. Subsequent studies showing that chimeric proteins containing truncated p67Phoxfused to Rac were also able to support p47PhoX-independent oxidase activity in vitro135J36 provided further support for this interaction. Ultimately, a direct binding interaction between p67Phoxand flavocytochrome b was demonstrated by Dang et ~ 1 . who ~ ' ~found ~ that binding was enhanced by the presence of Rac, which is consistent with the chimeric proteins studies. The possibility that p67Phoxis the NADPH-binding protein of the oxidase or at least participates in NADPH binding is still an area of debate. Early data suggesting p67phoxmight be the NADPH-binding component were provided by Umei and c o ~ o r k e r s , 6who ~ demonstrated that NADPH dialdehyde specifically labeled a 66 kDa protein in the 9'generating complex of guinea pig neutrophils. In addition, Smith and coworkers66also identified a 66 kDa protein in neutrophils that translocated to the membrane and appeared to contain an NADPH-binding site. Based on these studies and on their subsequent studies identifying the ~ ~ that the dialdehyde-sensitive component, Smith et ~ 1 . ' concluded cytosolic oxidase protein p67phoxwas also an important NADPH-binding protein of the NADPH oxidase and that the oxidase actually contained two NADPH binding sites, one low affinity site in gp91phoxand the other of higher affinity in p67Phox.Furthermore, Dang et u1.*39,140 showed that p67phoxwas able to directly bind NADPH via the TPR domains and catalyze pyridine nucleotide dehydrogenation, further supporting its role in electron transfer. Thus, it is possible that both p67phoxand gp91Phoxmay be involved in NADPH binding, perhaps by creating a shared or cooperative NADPH binding site between the two proteins. This possibility might explain the apparent contradictions between various reports. The interaction between p67phoxand Rac is essential for NADPH oxidase activation and appears to be mediated primarily by binding of Rac to the N-terminal region of p67phox(residues 1-200),'41 although Faris et ~ 1 . found ' ~ ~ that the C-terminus of p67Phoxmay play a role in stabilizing Rac binding. The importance of the N-terminus is demonstrated by the

The Neutrophil Respiratory Burst Oxidase

51

observation that deletion of p67phoxLys58 disrupts the interaction with Rac, resulting in a rare form of autosomal CGD.143Further investigation of the p67PhoxN-terminus resulted in the identification of an array of tandem four TPR motifs (TPR1-4) that serve as a target for Rac bindinglZ6(Fig. 3). In addition, crystallization of the TPR domain bound to Rac further defined the binding site to a domain that is formed by a p hairpin insertion between TPR3 and 4 and by the loops that connect TPRl and 2 and TPR2 and 3, thus representing a novel mode of TPR domain-mediated binding.144,145 Analysis of the p67phoxN-terminus also resulted in the identification of an activation domain encompassing residues 199-210; and Han et ~ 1 . showed l ~ ~ that this domain was required for oxidase activation (Fig. 3). Furthermore, this domain appears to play a role in regulating electron flow from NADPH to flavocytochrome b-associated FAD.147 The role of p67phoxphosphorylation in NADPH oxidase activation has been ambiguous. Previously, it was thought that p67phoxwas phosphorylated during NADPH oxidase activationg1;however, subsequent studies using CGD neutrophils suggested that the phosphorylated 67 kDa protein observed previously in activated neutrophil cytosol was unrelated to p67phox.148 Subsequently, this issue was resolved by El Benna et u1.,149 who used p67phoximmunoprecipitation to clearly show that p67phoxwas phosphorylated in activated neutrophils and that phosphorylation occurred by PKC-dependent and independent pathways. Indeed, the actual phosphorylation site was mapped to Thr233, which is a consensus MAPK substrate sequence,150 and it was found that p67phoxphosphorylation occurred in the cytosol and was independent of any interaction with ' ~ ~ that p67phoxcontained a p47phox.151 Although Ahmed et ~ 1 . reported cryptic PAK phosphorylation site, also located at Thr233, subsequent studies with purified neutrophil PAK showed that p67phoxis actually not a PAK substrate. Recently, Dang et al. 153 showed that p67PhoX is phosphorylated by ERK2 and p38 MAPK in vitro and in intact neutrophils. Phosphorylation occurred at several sites, with the primary ERK2 target localized to the N-terminal fragment and MAPK-mediated phosphorylation primarily in the C-terminus. Interestingly, the C-terminal phosphorylation site(s) appear to be masked in the intact protein and may become accessible only after a conformational change, suggesting an intramolecular interaction involving the TPR domain.

52

The Neutrophils

2.4. p40phox Compared to the other oxidase proteins, relatively little is known about the function of p40phoxin NADPH oxidase function. p4Ophoxwas identified in fractionated resting neutrophil cytosol via its binding to immunoprecipitated p67Ph0x.154 It is 339 amino acids long and migrates as -40 kDa protein on SDS-polyacrylamide gels.'% Sequence analysis of p40Phox revealed a single SH3 domain (residues 175-226)154;an N-terminal PX domain (residues 24-143)l15; and a C-terminal phox and Cdc (PC) motif (residues 283-310)155 (Fig. 4). As with the other cytosolic phox proteins, p40phoxappears to reside within the cytosolic complex.'% p40phoxhas been shown to bind to both p47PhoX and p67Phox;however, it seems to preferentially bind to p67ph0x.156158 Following neutrophil activation, p40phoxhas been reported to cotranslocate to the membrane as part of the 240 kDa complex from the cytosol to the plasma membrane, and it has been proposed to play a role in stabilization of the other components of this complex in the cytosol.'% Furthermore, p40phoX appears to dissociate from the other cytosolic components during the activation p r o c e ~ s .The ~ ~functional ~ , ~ ~ ~ role of p40phox in the oxidase is still not well understood. Tsunawaki et ul.161 found that dissociation of p40phoxfrom the cytosolic complex inhibited the cell-free assay, suggesting that p4OPhoxwas a positive regulator of the oxidase. In contrast, Sathyamoorthy et ~ 1 . reported l ~ ~ that p40phoxinhibited oxidase activity in vitro and in transfected K562 cells, albeit at relatively high p40phoxconcentrations, and proposed that it functioned in downregulating the oxidase by competing with SH3 domain interactions between other oxidase components. More recently, Cross163reported that p40phoxpromotes oxidase activation by increasing the affinity of p47phox for flavocytochrome b by -3-fold. In support of the latter observation, Kuribayashi et ~ 1 . lrecently ~ ~ used a number of approaches to show

Fig. 4 Scale model of p40ph0*showing functional domains (see text for details).

The Neutrophil Respiratory Burst Oxidase

53

p40phoxis probably a positive regulator of the oxidase and enhances translocation of both p47phoxand p67phoxin stimulated cells. In contrast to the p47phoxPX domain (see above), the p4OphoxPX domain preferentially binds phosphatidylinositol 3-phosphate [ P t d I n ~ ( 3 ) P l , ' ~ ~ J ~ ~ suggesting differential targeting of these proteins based on phosphoinositide specificity. Crystallization of the p40phnxPX domain bound to PtdIns(3)Pshowed that it contains a phosphoinositide-binding pocket similar to that in p47Phox;however, the shape and stearic constraints of the binding pocket appear to determine the phosphoinositide specifi~ity.'~~ The physiological role of the p40phoxPX domain is not well understood; however, it is thought that participation in intracellular targeting is likely.116 PtdIns(3)P accumulates in phagosomal membranes,166and Ellson et al.167 suggested that PtdIns(3)P could facilitate oxidase assembly by binding to the p4OphoxPX domain, thereby recruiting p67phox(and possibly other components) to the assembling oxidase via association with p40phox. p40Phoxhas also been reported to bind directly to the neutrophil cytoskeleton,'60 possibly through binding of the PX domain to actin-bindingproteins,'23 suggesting additional modes of recruitment to the oxidase complex. As described above, p40phox was found to bind p67Phx through a unique domain in the C-terminal region of p4Ophox.Further analysis of this interaction using yeast two-hybrid and in vitro binding assays resulted in the identification of a novel protein-protein binding motif (residues 282-309), and because of its presence in p40phoxand Cdc24p, a guanine nucleotide exchange factor in yeast, this motif was designated the PC motif.ls5The PC motif-mediated interaction is not dissociated by anionic amphiphiles in vitro, suggesting that this interaction might be maintained throughout the activation process;15 however, further studies are necessary to verify this conclusion in intact cells. The PC motif target in p67Phoxalso appears to be a novel modular domain (residues 345-427), which is located between the two SH3 domains and is designated the phox and Bem (PB1) domain because of its presence in p67Pbx and Bemlp, a yeast scaffold protein involved in cell polaritylS5 (Fig. 3). PB1 domains exist in a variety of proteins and appear to provide a scaffold for PC motif binding, facilitating protein-protein interactions in a variety of biological processes.168 Like the other cytosolic phox proteins, p40Phoxis phosphorylated dur170mapped the sites of ing NADPH oxidase a~tivation,'~~ and Bouin et al. 170

54

The Neutrophils

phosphorylation to Thr154 and Ser315. Furthermore, it appears that p4oPhoxphosphorylation is catalyzed by PKC.170

2.5. Rac Early on, it was suggested that a cytosolic guanosine triphosphate (GTP)binding factor participated in the NADPH oxidase,171and this factor was concurrently identified by two separate groups as the small GTP-binding protein Rac. 172,173 Rac (Racl and/or Rac2) was found to be required for oxidase activation in cell-free assays, indicating that Rac was responsible for at least part of the GTP sensitivity of the NADPH oxidase and that it was indeed the third required cytosolic cofa~tor.l”J~~ In support of this conclusion, Dorseuil et al.174showed that Rac antisense oligonucleotides caused a dose-dependent inhibition of 02’-production in transformed B lymphocytes, demonstrating a requirement for Rac in the oxidase of intact cells. Subsequently, analysis of Rac-deficient mice showed that neutrophils from these mice had diminished 0 2 ‘ - production; however, the defect could be partially corrected by treating with tumor necrosis factor a (TNFa) prior to stimulation with PMA.175Further studies in Rac2deficient mice showed that the requirement for Rac2 in oxidase activation may be stimulus-specific, and that Rac2 was essential when cells were activated with physiologically-relevant agents, such as fMLF or IgGopsonized ~ a r t i c 1 e s .Since l ~ ~ both Racl and Rac2 are capable of reconstituting oxidase activity in cell-free assays, it was suggested that the ability to achieve partial oxidase activity in neutrophils from Rac2-deficient mice may be due to substitution by Racl. However, Racl cannot substitute completely for Rac2, as shown recently by Glogauer et al.,177 who generated mice with Racl-deficienct neutrophils and showed that oxidase activity is normal in Racl -deficient cells but still markedly diminished in Rac2-deficient cells. These observations support previous studies demonstrating that in the presence of human neutrophil cytosol, RacZ was more active than R a ~ 1 . Furthermore, l~~ nearly all (>96%) of the Rac protein found in human neutrophils is Rac2, indicating that it is the relevant Rac protein in human neutrophils.86In contrast, recent studies indicate that Racl is the predominate isoform in human monocytes and regulates NADPH oxidase activation in these cells.179The physiological importance

The Neutrophil Respiratory Burst Oxidase

55

of Rac2 in the human neutrophil oxidase was substantiated recently when a patient with abnormal neutrophil function was shown to have an inhibitory (dominant-negative) mutation in Rac 2, resulting in decreased oxidase activity and other neutrophil functional defects.*s0J81 In activated neutrophils, Rac translocation corresponds both temporally and quantitatively with p47phoS/p67phos translocation and with oxidase activation.86J82 Furthermore, Rac translocates independently of the other cytosolic oxidase c ~ r n p o n e n t s . ' These ~~,~~ results ~ suggest Rac does not directly mediate phox protein translocation. On the other hand, recent studies suggest Rac activation can induce oxidase assemb1y,ls4possibly through an indirect mechanism involving activation of PAK, thereby leading to p47phox pho~phorylation.~~ Rac has been shown to interact with p67Phos as well as with flavocytochrome b.129Thus, it is likely that Rac can modulate the function of one or more of the proteins of the oxidase. Previous studies showing that components of the oxidase are associated with the cytoskeleton90r91~185 raise the possibility that Rac may play an additional role related to cytoskeletal assembly/association.186

2.6. RaplA The first GTPase to be identified in association with the neutrophil NADPH oxidase was RaplA. RaplA is a member of the Ras superfamily of GTP-binding proteins and was shown to be associated with flavocytochrome b.ls7 The association between flavocytochrome b and RaplA was confirmed using reconstitution procedures in vitro, and RaplA was found to form stoichiometric (1:l)complexes with flavocytochrome b, indicating a direct binding of RaplA to flavocytochromeb.lss Since Rap1A is not required for cell-free oxidase reconstitution,62 the role of RaplA in the oxidase is currently unclear. However, several reports do suggest RaplA may play an important regulatory function in viva For example, Eklund et ul.ls9 found that cytosol immunodepleted of RaplA was unable to support reconstitution of NADPH oxidase activity unless recombinant RaplA was added back. In addition, the subcellular localization of RaplA parallels that of flavocytochrome b, and RaplA translocates with flavocytochrome b in activated neutrophils, supporting a possible functional association of these proteins in the cell.190However, the most

56

The Neutrophils

compelling evidence implicating Rap1A in oxidase function comes from studies showing that transfection of transformed B lymphocytes or differentiated HL-60 cells with dominant inhibitory (17N) mutants of RaplA results in significant inhibition of NADPH oxidase activity in these cell^.'^^,'^* These studies directly support a role for RaplA in the regulation of the oxidase in intact cells. RaplA may also modulate PKC activity, as PKC can be stimulated by R a ~ 1 A . Since l ~ ~ a number of phosphorylation events, including PKC-dependent events, are associated with oxidase activation (see details above), it is plausible that RaplA could modulate phosphorylation of individual oxidase proteins during oxidase activation and/or terminati0~1.l~~ Nevertheless, further studies are necessary to define the exact role of RaplA in this complex system.

3. OXIDASE PROTEIN BINDING INTERACTIONS The realization that multiple proteins were involved in forming the NADPH oxidase complex soon led to studies focused on mapping specific interactions between these components. Importantly, the characterization of protein:protein binding interactions among NADPH oxidase proteins has contributed significantly to our understanding of how the oxidase might assemble in human neutrophils.

3.1. Flavocytochromeb-p47PboxInteractions Early on, a domain at the C-terminus of gp9lPhoxwas found to interact with p4rphox, and peptides mimicking this region inhibited oxidase activity by blocking the association of p47Phoxwith flavocytochrome b.82J94Subsequently, we analyzed the structural basis of this interaction and found that during NADPH oxidase assembly, the C-terminus of gp9lPhoxbinds to p47Phoxin an extended conformation between gp91Phoxresidues 555-564, with immobilization of almost all of the amino acid side chains in this region.195This type of extended conformation is consistent with the biochemical function of this binding site, which contributes to a high affinity, multi-site binding interaction that occurs between p47Phoxand gp91ph0x.40J96 A second p47ph"" binding domain was reported by Leusen et al.,'" who identified a gp91PhoxCGD mutation (D500G),resulting in a non-functional

The Neufrophil Respiratory Burst Oxidase

57

flavocytochromeb, even though the defective flavocytochromeb was present in normal amounts. Cytosolic phox proteins did not translocate in neutrophils from this patient, and a peptide mimicking this region of gp91Phox was found to inhibit oxidase assembly. Thus, these authors concluded that this region was important for binding p47phox.197 Subsequently, another X-linked CGD patient was found with a mutation that resulted in amino acid substitutions in residues 507-509.198 Although this mutation resulted in defective flavocytochrome b, p47phoxand p67Phoxtranslocated normally, and it was suggested that impaired 9’-generation resulted from abnormal electron transfer despite normal oxidase assembly.198Since some of these residues lie within the reported consensus NADPH-binding domain, a potential role of this region in electron transfer is plausible. Thus, although the precise role of this region in oxidase assembly is unknown, it appears to be involved in both NADPH and cytosolic factor binding. As a global approach to identifying biologically-relevant sites of binding interaction between gp91PhoX and p47phox,we utilized random sequence peptide phage display library analysis and mapped two novel p47phoxbinding sites, encompassing gp91PhoX residues 85-93 and 450457, as well as confirmed the C-terminal binding domain in gp91ph0x.40 The domain encompassing gp91PhoX residues 86-93 represents a relatively high-affinity binding site, and peptide mimetics of this domain are potent inhibitors of the oxidase in vitro and in intact neutrophils.lWMore recently, site directed mutagenesis was used to verify that the two arginines in this domain (Arg91 and 92) are essential for flavocytochromeb function, providing further evidence that this region is a relevant p47phoxbinding domain.200In any case, the identification of these sites of binding interaction for cytosolic p47phoxalso provided information about the topology of flavocytochrome b molecule itself, as these regions must be cytosolic for p47Phoxbinding to occur (Fig. 1). In addition to the interaction with gp91PhoX, p47phoxalso binds to p22Phox.Using a peptide mapping approach, Nakanishi et a1?9 reported that a p22-pkox peptide mimicking a proline-rich region encompassing residues 175-194 inhibited 02’generation in the cell-free assay and that the peptide bound to p47PhoX. Subsequently, several groups reported concurrently that p47PhoxSH3 domains interacted with a proline-rich region encompassing residues 149-162 of p22ph0x.201-203 Although the C-terminus

58

The Neutrophils

of p22phoxcontains two proline-rich domains that could be potential targets (residues 149-162 and 175-194), only the first site has been shown to be an SH3 domain target.41In support of this conclusion, an autosomal recessive CGD mutation (P156Q) has been identified within this region of p22Phox, and translocation of p47phoxin activated neutrophils from this patient was undetectable.204Interestingly, this mutant form of flavocytochrome b could be activated with phospholipids in the absence of cytosolic factors, indicating that the P156Q mutation was inhibiting p47p!10xbinding rather than disrupting the structural integrity of p22Ph0x.204 It should be noted, however, that peptide mimetics of the p22Phoxdomains are not as effective at inhibiting 02'-generation when compared to the gp9lphoxpeptides, indicating that the SH3-mediated binding interaction was of lower affinity than the non-SH3 binding Research has also focused on identifying regions in p47Phoxthat participate in binding flavocytochrome b. Nauseef et d 1 O 6 reported that a putative PKC phosphorylation target within the cationic domain of p47phox(residues 323-332) was involved in the assembly of the activated enzyme complex in a phosphorylation-independent manner. In support of this finding, we showed that a larger region of p47phox,encompassing residues 323-342, associated with flavocytochromeb following activation of the oxidase and that peptide mimetics representing this region inhibited 02'-generation in the cell-free oxidase assay and in intact neutrophils, and also inhibited translocation of p47phoxto neutrophil plasma mernbrane2O5 (Fig. 2). Since p47Phoxphosphorylation is essential for oxidase activation, the location of this flavocytochrome b binding site (which is within putative phosphorylation motifs and a highly cationic region of p47phox)is consistent with the hypothesis that charge neutralization of this region by phosphorylation or amphiphiles induces conformational changes, thereby allowing this domain to participate in p47PhoX-flavocytochrome b binding.lll In addition to this domain, the first and/or second SH3 domain of p47phoxis involved in binding to the proline-rich C-terminal domain of p22phoxfollowing a~tivation.~O~-~O~ Overall, p47phox and flavocytochromeb are associated via a high affinity, multi-site binding interaction involving target sites in both gp91Phox and p22PhoX, and this association becomes essentially irreversible once the complex is formed. It is possible that this high affinity interaction may be

The Neutrophil Respiratory Burst Oxidase

59

necessary for p47Phoxto function as a membrane anchor or adaptor for the remaining cytosolic proteins.

3.2. p40phox-p47phox-p6 7phoXI nte ractions

SH3 domains have been shown to play important roles in the intra- and intermolecular association of oxidase protein^.^^.^^^ SH3 domains bind to proline-rich motifs, and it has been proposed that SH3-mediated interactions aid in the stabilization of the cytosolic complex in resting neutrophils and/or assist in the assembly process during oxidase a c t i v a t i ~ n ~ ~ t ~ ~ (Figs. 2 and 3). Indeed, SH3-mediated interactions contribute to the association of all three of the cytosolic phox proteins and may function to align the oxidase proteins during the assembly process and/or facilitate further high affinity, non-SH3 binding interactions. In resting cells, the p22Phox-bindingsurface of the tandem SH3 domains in is thought to be masked by an intramolecular interaction with the cationic domain, which is also the primary target region for activation-induced phosphorylation events207~208 (Fig. 5). As described above, this intramolecular autoinhibitory interaction may also be strengthened by an additional intramolecular association between the N-terminal PX domain and the C-terminal SH3 domain of p47phox.1218122 Importantly, this autoinhibitory interaction, which prevents binding of p47phoxto p22Phox,is during oxidase ctivati~n.~~~ released by phosphorylation of a p47phox h recently proposed an alternative Note, however, that Grizot et al. model, where the role of amphiphiles p47phoxphosphorylation was proposed to disrupt a putative p40Phox-p47PhoX association, rather than release intramolecular autoinhibitory interactions. The p47phoxproline-rich region appears to associate with the C-terminal SH3 domain of p67Phoxin resting cells, although this interaction may not be necessary for in vitro reconstitution of oxidase a ~ t i v i t y .In~ addition, ~~,~~~ de Mendez et uL41 found that a region in the N-terminus of p67Phox(most likely the proline-rich domain) interacted with full-length p47phox(presumably via SH3 binding). The third protein of the cytosolic complex, p40phox, contains one SH3 domain that has also been shown to interact with the C-terminal proline-rich domain of p47phox154~157~158,210(Fig. 4). Thus, this region of p47Phoxseems to bind to both p40phoxand p67phox.

60

The Neutrophils

Fig. 5 Model of NADPH oxidase assembly. Activation/phosphorylation @‘)-induced conformational changes in p47Phoxrelease autoinhibitory interactions to unmask essential binding domains and exposure of PX domains that facilitate membrane targeting and binding of SH3- and non-SH3-mediated binding events. Final interaction of the p67phoXand Rac with flavocytochrome b induces conformational change, resulting in electron flow. See text for a detailed description of the assembly events.

The Neutrophil Respiratory Burst Oxidase

61

SH3 domain-mediated interactions alone likely do not support complete assembly of the active oxidase, and non-SH3 binding interactions between cytosolic phox proteins have also been identified. For example, an undefined p40phoxC-terminal domain was reported to interact with p67ph0x.157J58 Subsequently, this domain was identified as the PC motif155 (Fig. 3). As described above, the PC motif target in p67Phx also appears to be the PB1 modular domain, located between the two SH3 domains.155 Several studies suggest this p40phox-p67Phox interaction may be regulatory in nature to prevent spontaneous oxidase activation162and/or chaperone p67Ph0x.157f214 Interestingly, Rinckel et ~ 1 . 2 ' found ~ that Rac could disrupt the p40PhoX-p67PhoX interaction via p40phoxbinding to the p67phoxN-terminus (possibly the TPR motif), thereby removing the regulatory oversight. We characterized an activa tion-dependent interaction between p47Phox and p67Phox,which was mediated via a common functional domain in p47phoxthat later became occupied by flavocytochrome b.216This mutually exclusive binding domain is located within the cationic region of p47phox,and peptide mimetics of this region blocked the activationdependent association of p47phox with p67phox,as well as the association of p47PhoXwith flavocytochrome b2I6 (Fig. 2). Furthermore, we found that flavocytochrome b and p67phoxboth competed for binding to this p47phox domain, suggesting the binding of p67phoxto this region of p47phoxpresumably occurs prior to the binding of flavocytochromeb.

3.3. Rac Interactions Although Rac translocates normally to the membrane in CGD neutrophils lacking p47phoxor p67Phox,a significantly decreased level of Rac translocation was observed in cells from flavocytochrome b-deficient CGD patients, indicating a necessity for flavocytochromeb (as a potential docking site) for optimal Rac t r a n s l o c a t i ~ nThis . ~ ~ ~conclusion was confirmed by later studies of Diebold and Bokoch?17 who demonstrated a direct physical interaction between Rac2 and flavocytochrome b and showed that this interaction is important in the initial transfer of electrons from NADPH to FAD. The loss of Rac translocation in flavocytochrome b-deficient CGD cells does not exclude the additional possibility that one or more of the

62

The Neutrophils

other cytosolic phox proteins also contribute to this process. Indeed, an interaction between Rac2 and p47phoxwas suggested in studies showing that Rac2 translocation and subsequent association with the membrane were diminished in CGD neutrophils deficient in p47phux.130 The importance of this interaction is unclear; however, as Rac can translocate independently of the other cytosolic factors and can associate with the membrane in the absence of either p47phoxor p67phoX.lz9 In addition, both p47phoxand p67phux can stably associate with the membrane in the absence of R a ~ .Thus, ~ ~a role f ~ for ~ Rac ~ in the regulation of oxidase activity rather than assembly is indicated.219 Rac, like all GTPases, utilizes a common functional mechanism, which is based on the ability of the GTPase to bind and hydrolyze GTP, thus cycling between inactive GDP- and active GTP-bound forms.2z0The N-terminal half of Rac contains two key stretches of sequence involved in GTP binding, and these are designated as the “switch” I region or effector domain (residues 2545) and ”switch” I1 (residues 58-77) region, because these regions have been found to undergo major conformational changes during the GTPase cycle.220Of these regions, it appears that the switch I region plays a key role in oxidase activation, and Rac mutations in the switch I region diminish its ability to activate the oxidase.zzl~zzz Subsequently, it was found that the Rac switch I region binds directly to p67phox and that the target in p67phoxwas the TPR dornain.lz6 Furthermore, crystallization of the Rac-p67Phox TPR complex confirmed a role for the Rac switch I region in stabilizing the interaction with p67phux.144 Rac contains an additional region, the “insert domain” (residues 120-137), which has been reported to be important in oxidase function.2z4The insert domain does not appear to play a role in binding p67phox;rather it has been suggested to bind to another oxidase componenttZz3 which was shown recently to be flavocytochromeb.217 Kreck et uLZz5identified a region near the C-terminus of Racl (residues 178-188) that may also be involved in Rac-mediated assembly of the oxidase, and a peptide mimicking this region of Rac inhibited oxidase activity in cell-free assays. Subsequent analysis of this interaction using peptide scanningzz6and site-directed mutationsz27confirmed the importance of this region, and it was suggested the Rac C-terminal polybasic motif plays an important role in membrane binding. Indeed, Tao et recently 1411223

The Neutrophil Respiratory Burst Oxidase

63

showed that this motif was required for efficient prenylation and for correct membrane localization of Rac.

4. M O D E L OF N A D P H OXIDASE ASSEMBLY As summarized in the previous two sections, the neutrophil NADPH oxidase is composed of multiple proteins that associate with one another through a temporal and spatial array of protein:protein binding interactions, resulting in an active, 02'--generating complex. In resting cells, p40phox,p47phox,and p67phoxexist in a stoichiometric cytosolic complex that is stabilized, in part, by SH3 domain interactions.229 In addition, some free p47Phoxexists, apparently not associated with the complex.79The intramolecular autoinhibitory interactions involving both p47phoxSH3 domains binding to the polybasic domain, as well as the N-terminal PX domain binding to the C-terminal SH3 domain maintain p47phoxin a closed c o n f ~ r m a t i o n ' (Fig. ~ ~ , 5~)~. Additional ~ ~ ~ ~ ~ interactions that seem to be present in the resting cytosolic complex include a highaffinity interaction between the C-terminal proline-rich region of p47phox and the C-terminal SH3 domain of p67phoX202,203,229 and possibly the SH3 domain of p40ph0x,'58,210 although the relative importance of the latter interaction has been debated.229Instead, the most relevant interaction involving p40phoX appears to be a non-SH3-mediated interaction between the p40phoxC-terminal PC motif and the p67phoxPB1 domain.16*Together, these interactions function to stabilize the multi-protein cytosolic complex, as well as any free p47phox,in a resting state. At the same time, flavocytochrome b is also present in an inactive state, which seems to be due to undefined conformational constraints that hinder electron t r a n ~ f e r . ' ~ ~ f ~ ~ ~ As discussed above, multiple phosphorylation events are associated with NADPH oxidase activation. Phosphorylation of p47phox,primarily in the polybasic domain and C-terminus, induces a conformational change that facilitates release of the intramolecular autoinhibitory interaction^^^^^^^^^^ (Fig. 5). Unmasking of the p47phoX autoinhibitory conformation results in exposure of both SH3 domains, the polybasic region, and the PX d ~ m a i n . ' ~ ~ Apparently, "~!~* p47phoxand p67Phoxremain associated throughout these conformational changes via association of the p47Phox proline-rich C-terminus with the p67phox C-terminal SH3 domain.113,202229

64

The Neutrophils

Phosphorylation may also function to neutralize the charge within the exposed polybasic region,lo7 thus enabling the association of this p47phoxdomain with a binding site in p67phox.This would strengthen the interaction of the two proteins during translocation and possibly stabilizing the active, opened conformation of the tandem SH3 domains for binding to flavocytochrome b.216 During oxidase activation, the cytosolic components must translocate to associate with flavocytochromeb; however, it is not clear at what point in this process full activation of the cytosolic proteins is achieved. Phosphorylation occurs in a step-wise fashion, and apparently only the most highly phosphorylated forms of p47phoxassociate with the membrane.88Thus, it is plausible that activation of p47Phoxand the other factors of the cytosolic complex is a cumulative process occurring throughout translocation. Arrival at the membrane would then be coincidental with full exposure of the tandem p47PhoxSH3 domains,207,211 and exposure of the p40phoxand p47PhoxPX d ~ m a i n s . ' ~ ~ J ' ~ J ~ ~ The association of the p47PhoxSH3 domains with p22phoxis a critical step in the assembly process and may facilitate initial docking of p47phox and associated oxidase proteins with flavocytochrome b (Fig. 5). Indeed, p67phoxseems to depend on p47phoxfor binding to the oxidase assembly,s3 although membrane-targeted Rac can serve as a surrogate p67phoxtarget in a modified cell-free assay.232In any case, a correct alignment of p47phox would then insure accurate proximity for binding of the remaining sites of interaction between p47phox/p67Phox and flavocytochrome b, including binding of p47phoxto multiple sites within gp91Phox and binding of the N-terminus of p67Phoxto gp91phox,233 possibly via the activation domain.146It is interesting that p47phoxbinding also appears to be enhanced by the presence of p67phox,s3 suggesting the possibility that the binding of p47phoxchaperones p67phoxto a zone where it can interact with gp91Yhox Additionally, the latter interaction may form a "sandwich" that binding with further anchors p47phox(Fig. 5). During p47phox/p67phDx flavocytochrome b, regulatory input seems to be required to initiate release of p67phoxfrom the cationic domain of p47phox,and this region subsequently binds with high affinity to flavocytochrome b.216The most likely candidate for regulatory input at this stage of oxidase activation appears to be R ~ c .As ~ 'discussed ~ above, Rac translocates independently 38t40

The Neutrophil Respiratory Burst Oxidase

65

of the other cytosolic f a ~ t o r s ' ~and ~ J can ~ ~ bind, via the switch I region, 126,233 126,223 to the p67PhoxN-terminal TPR motifs (Fig. 5). Rac also appears to coassociate with flavocytochrome b,129,217possibly via the Rac insert region.223The relative timing or sequence of the Rac binding interactions in oxidase assembly is unclear; however, Diebold and Bokoch217have recently provided evidence that the Rac:flavocytochrome b interaction may occur prior to the Rac:p67Phoxinteraction and that these events are related to two distinct, Rac-dependent steps in the electron transfer reaction. This idea is supported by earlier kinetic studies showing that oxidase assembly involves an "intermediate state," where electron transfer can proceed from NADPH to FAD (step l), followed by a second step involving transfer from FAD to the heme (step 2).234In these studies, the intermediate state (step 1) was achieved by the binding of p67phoxand Rac to the p47~hox/flav~cytochr~me b complex.234Thus, step 1 may involve direct binding of Rac with flavocytochrome b and involvement of Rac in electron transfer independent of p67phox:l7 or it may involve targeting or modulation of p67phoxby R a ~ . ~ ~ ~ t ~ ~ ~ Events leading from the intermediate state to the fully active oxidase are not well defined; however, step 2 appears to depend on Rac activity, and several models of oxidase regulation by Rac have recently been proposed.219Rac has been shown to disrupt the p40phox-p67phox interaction, and this event is essential for efficient oxidase a c t i v a t i ~ n . Thus, '~~~~~~ although p4Ophoxis required to modulate recruitment of p47phox/p67phox to the membrane via its PB1-PC interactions with p67Phoxand PX domain interactions with the membrane,'@ it subsequently becomes displaced by Rac binding. The binding of Rac and/or release of p40phoxfrom p67Phox could then induce conformational changes in p67Phox,thereby liberating the p47Phoxcationic region for gp91Phox binding216and/or positioning of the p67phoxactivation domain correctly for imminent binding to gp91ph0x.146 Apparently, one or more of these events induces conformational changes in flavocytochrome b, thereby permitting completion of electron transfer In support of this conclusion, from FAD to heme and, ultimately, to 02.234 atomic force microscopy was utilized to demonstrate measurable changes in flavocytochrome b during its transition to the active state.132t236 More recently, Foubert et d Z 3 O reported that amphiphilic agents used in cell-free oxidase activation induced significant conformational changes in

66

The Neutrophils

flavocytochrome b, as determined by monitoring of resonance energy transfer from an external fluorescent probe to the heme, and that these changes in structure could facilitate heme alignment for efficient electron transfer. The nature of the event that directly induces conformational change is still under investigation. In vitro, it has been reported that both p47phoX and p67phox can induce conformational changes in flavocytochrome b, indicating that both can bind;132however, only p67phoX binding induced prod~ction.'~~,'~~~~~ electron flow and 02'-

5. OXIDANT PRODUCTION Neutrophils utilize an extraordinary array of microbicidal mechanisms to destroy and remove infectious agents, and these mechanisms can be generally classified as being oxygen-dependent and oxygeni n d e ~ e n d e n t . Among ~ ~ ~ - ~the ~ ~oxygen-dependent mechanisms, NADPH oxidase activation plays a major and essential role, as demonstrated by the susceptibility of patients with CGD to infectious agents.'O As described below, NADPH oxidase activation results in the generation of 02'-; however, subsequent biochemical events can convert this radical into much more potent microbicidal oxidant species.

5.1. Superoxide Anion (02,-) Activation of the NADPH oxidase catalyzes the univalent reduction of O2 and formation of 02-,240 as described by the following reaction: NADPH

+ 202 -+ NADP' + 202'- + H+

(5.1)

Although a requirement for the oxidase in host defense is well documented, it is apparent that the product of this enzyme ( 0 2 ' - ) is not the primary microbicidal agent. At physiological pH, 02'-itself is relatively unstable, has limited reactivity toward biological molecules, and exhibits minimal antibacterial a ~ t i v i t y .Recently, ~ ~ ~ , ~ Reeves ~~ et ul.243 suggested that 02'accumulation in the phagosome actually functioned to induce K+ influx into the vacuole and corresponding pH stabilization to create

The Neutrophil Respiratory Burst Oxidase

67

optimal conditions for granule protease release and activation. Thus, this novel finding suggests a unique mechanism contributing to neutrophil antimicrobial activity; however, it is clearly not the only mechanism of oxidative killing, as 02'is the precursor to much more potent microbicidal oxidants, as described below.

5.2. Hydrogen Peroxide (H202) At physiologic pH, 02'-is rapidly converted to H202by spontaneous or enzymatic d i s m u t a t i ~ n ~ ~ ~ :

Enzymatic dismutation of 02'-is catalyzed by superoxide dismutases (SOD), which are highly efficient antioxidant enzymes that are abundant in all cells.242The importance of SODSin antioxidant defense is supported by the role of oxidants in aging and disease.245Indeed, additional antioxidant enzymes are present to detoxify H202by dismutation (catalase) or by a multi-step reduction reaction catalyzed by glutathione (GSH)peroxida~e.~~~ or H202,it is surprising Since CGD neutrophils do not generate 02'that they are still able to kill a number of pathogens.l0 One theory proposed to explain this discrepancy is that the microbicidal capacity of CGD neutrophils depends, to some degree, on H202produced by the pathogen itself.247For example, bacterial strains that do not produce H202are .~~~ since many bacteresistant to killing by CGD p h a g o c y t e ~Furthermore, ria express their own catalase and would be able to detoxify HzO2, it was suggested that catalase-producing organisms would be especially resistant to killing by CGD p h a g o c y t e ~Indeed, . ~ ~ ~ catalase-positive organisms cause many of the infections in CGD patients, while catalase-negative organisms rarely infect these individuals.249However, recent studies suggest that this issue is more complicated than previously thought, as certain catalase-deficient organisms have also been shown to be virulent in mouse models of CGD and, therefore must utilize alternative, nonoxidative virulence mechanisms to survive?%

68

The Neutrophils

5.3. Hypochlorous Acid (HOCI) Concurrent with the production of oxidants, neutrophil granules fuse with the membrane, releasing their enzyme contents, including high concentrations of myeloperoxidase (MPO).238MPO utilizes H202to catalyze the oxidation of C1- ions to form hypochlorous acid (HOCl)238: C1-

+ H202 + H++

HOCl + H20

(5.3)

HOCl is a potent oxidant and is cytotoxic to a wide range of pathogens, including bacteria, viruses, and fungi. Indeed, the MPOH202-halidesystem appears to be the most efficient oxygen-dependent microbicidal mechanism in n e ~ t r o p h i l s . ~The ~ ' cytotoxicity of HOCl results from its ability to participate in a variety of oxidation and chlorination reactions.252For example, MPO-generated HOCl oxidizes a-amino acids to a family of reactive aldehydes and tyrosyl radicals that can attack important biological targets. 253,254 HOCl can also oxidize heme groups and iron-sulfur centers.255The main targets for chlorination are primary amines, resulting in the formation of chloramines, as well as pyridine nucleotides, unsaturated lipids, and holes sterol.^^^!^^^ Chloramines seem to play an important role in regulating the inflammatory response and, therefore, could represent a critical product of this p a t h ~ a y . 2 On ~ ~the other hand, MPO deficiencies are relatively common, and individuals with MPO deficiency do not seem to have an increased incidence of infection, except for infection with candid^.^^^ Thus, MPO-deficient neutrophils seem to utilize compensatory MPO-independent, but still oxygen-dependent antimicrobial systems.238

5.4. Hydroxyl Radical (HO') Due to the limited reactivity of 02'-, it has been proposed that it may act through a secondary product (HO) formed by the metal-catalyzed reduction of H202by 02'-, which is commonly known as the Haber-Weiss reaction or 02'--assisted Fenton reaction.259As summarized below, this process requires the presence of redox-active transition metals, such as iron or copper, to catalyze the reaction:

02'-+ Fe3++O2+ Fe2+

(5.4)

The Neutrophil Respiratory Burst Oxidase

+

H202 Fez++H O + OH+ H202 +HO' OH-

02'-

+

+ F$+

+0 2

69 (5.5) (5.6)

According to this reaction, 02'-is required both as a source of H202and as a reducing-agent. It should be noted, however, that 02'-can also reduce HOCl at an appreciable rate, resulting in a second mechanism for HO' generation in neutrophils: 02'-

+ HOCl +H O + 0 2 + C1-

(5.7)

Recent studies suggest that this transition metal ion-independent mechanism of HO' formation represents an important pathway in neutrophils, and HO' generated by this process was shown to oxidatively damage DNA, RNA, cytosolic nucleotides, and proteins.260r261 In addition, Saran et a1.262hypothesized that the highly-reactive HO' might be "stabilized" by reacting with C1- to form an equilibrium with HOC1'- and, if an appropriate target is not reached, could be funneled into chlorine radicals (Cl.), which are also highly reactive:

HO' + C1-

+-+HOC1'- + H" H C1' + H20

(5.8)

HO'is an extremely powerful and highly-reactive oxidant that can attack a wide variety of biological molecules in v i t ~ oBased . ~ ~ ~on its extreme reactivity and on the ability of SOD, catalase, and HO' scavengers to protect against oxidant injury, it has been proposed that HO' may be a major factor contributing to microbicidal activity and inflammatory tissue i n j ~ r y On . ~ the other hand, analysis of PMA-stimulated neutrophils showed that <1%of the 02'-formed was converted to HO', calling into question the biological relevance of neutrophil-generated H0,.261In addition, it has been argued that the cell is maintained in a highly reduced state and contains a number of efficient reducing agents (e.g., GSH, ascorbate, etc.) that can reduce Fe3+ and Cu2+.Thus, it is unlikely that 02.-serves in this role.265Furthermore, intracellular and extracellular iron is highly regulated by iron-binding proteins, such as lactoferrin and transferrin, and iron bound to these proteins does not appear to serve as a catalyst for this reaction.266Currently, the nature of the iron or copper complexes required for metal-catalyzed H O formation in vivo is currently unknown, although one possibility is that they may be

70

The Neutrophils

provided by the cell itself.267Alternatively, the metal ion-independent pathway described in Eq. 5.7 may play an important role in this process.260In any case, the actual role of HO’ in vivo is still unclear and remains to be determined.

5.5. Singlet Oxygen (lo,*) and Ozone (0,) As indicated above, the concurrent generation of high concentrations of oxygen radicals during neutrophil activation provides an ideal environment for the formation of secondary metabolites. One remarkable metabolite formed by HOC1-mediated oxidation of H20z is HOCl + H202 +‘0;+ HC1+ H20

(5.9)

is a highly-reactive and relatively long-lived metabolite that has been implicated as a bactericidal oxidant in the phagosome,268although this conclusion has been debated.255Intracellular production of 1O2 by neutrophils has been demonstrated, and it has been reported that phagocyThe tosing neutrophils convert u p to 20% of O2 consumed to 10;.269 putative mechanism of *O; in bacterial killing is currently unknown, although it has been implicated in DNA damage, lipid peroxidation, and protein oxidation.270An additional mechanism of action was recently suggested by studies of Wentworth et ~ 1 . 1 who 2 ~ ~ reported that antibodies can catalyze the generation of H202and O3 from1O2*via a postulated 1O2 intermediate of dihydrogen trioxide (H203): 1O2* + H20--antibody+H203 +H202 + O3

(5.10)

The highly-reactive and short-lived nature of O3makes it an ideal microbicidal agent, and subsequent studies showed that O3 can be generated during bacterial killing by activated neutrophils and in an inflammatory response in vuivo, thus suggesting its physiological i r n p o r t a n ~ e . ~ ” , ~ ~

5.6. Nitric Oxide (NO) and Peroxynitrite (ONOO-) NO is a free radical that plays essential regulatory roles in a number of physiological processes involving the cardiovascular and neuronal systems; however, NO is also important in innate immunity and has been

The Neutrophil Respiratory Burst Oxidase

71

widely implicated in the inflammatory r e ~ p o n s e .Although ~ ~ ~ , ~ it~ was ~ originally proposed that NO itself was directly cytotoxic, the relatively low reactivity of NO made it difficult to understand how this radical could cause this type of damage.275Indeed, the most recent evidence suggests the toxicity of NO is due to the formation of ONOO-, which is formed by a diffusion-limited reaction between NO and 02.-.275 This is one of the fastest known biochemical reactions and is even fast enough to out-compete SOD for 02'-.276 Furthermore, OONO- exists in equilibrium with its conjugate acid OONOH in physiological buffer and, in the absence of a substrate, decomposes to nitrate (NO3-) via an excited isomer intermediate (ONOOH*)275: NO + 02'+ONOO-

f~OONOH

-+ ONOOH' + NO3-

(5.11)

In the presence of an oxidizable substrate, ONOO- decomposition yields nitrite (NO2->,which is toxic in itself but can also be further converted into toxic metabolites that contribute to the microbicidal effects of ONOO-.277For example, NO2- can be oxidized by H202in a reaction catalyzed by MPO to form a nitrogen dioxide (N02')-likeradical that can nitrate tyrosine and other aromatic c o m p o ~ n d and s ~ promote ~ ~ ~ ~ lipid ~~ per~xidation~~~: N02-

f

H202 + H+ +MPO +N02' + HO-

+ H20

(5.12)

Regardless of the pathways and intermediates involved, significant evidence indicates that ONOO- plays an important role in host defense,280as well as in the pathogenesis of inflammatory disease.275 ONOO- is a potent oxidant that can attack a wide variety of biological tissues, and recent research has implicated ONOO- as one of the damaging agents in a number of pathological inflammatory conditions.275 Although it is clear that significant concentrations of OONO- can be generated in inflammatory TISSUES,275 the actual production of OONO- by human neutrophils in v i m has been a matter of debate. It has been reported that activated human neutrophils can generate ONOO-;2x1282 however, it still remains controversial whether human phagocytes can actually generate significant levels of NO to participate in this process.2x3 Since NO is ubiquitously produced in the vascular system, it has also been suggested that NO derived from endothelial cells can react with

72

The Neutrophils

neutrophil-generated 02'at sites of inflammation, resulting in ONOOformation,284perhaps making the actual source of NO a moot point.

6. SUMMARY The phagocyte NADPH oxidase is an essential component of the human cellular immune response; however, oxidants generated by this system can also contribute to the non-specific tissue damage associated with a variety of inflammatory diseases. Indeed, a number of potent oxidants are generated via reaction of O i - with other radical species or through enzymatic pathways, and it is clear that these metabolites are essential in the inflammatory response. Because of the potential for tissue damage, activation and assembly NADPH oxidase is highly regulated and involves control mechanisms. Activation of the oxidase requires assembly of five proteins with membrane-associated flavocytochrome b, which presumably contains all of the required redox components but cannot on its own catalyze the reaction. By segregating oxidase components into various locations of the cell, neutrophils are able to prevent inappropriate assembly and activation of the oxidase, and thereby, control the onset and duration of the oxidative burst. Only after highly regulated, intricate events involving phosphorylation, translocation, and multiple conformational changes does the oxidase enzyme acquire the ability to generate 0 2 ' - . Thus, understanding the intermolecular interactions occurring among protein components of this system is essential to understanding and/or controlling their function in both microbicidal and inflammatory responses. While the nature of these interactions is becoming increasingly apparent (as summarized in this chapter), further studies in this area will be necessary to define the exact role played by each protein during activation and assembly of the phagocyte NADPH oxidase.

ACKNOWLEDGMENTS I have attempted to cite as many references as possible given the page limitations and apologize if some appropriate references have been inadvertently omitted. This work was supported in part by National Institutes of Health grants AR42426 and HL66575 and the Montana State University Agricultural Experimental Station.

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3

Novel Neutrophil Receptors and Their Signal Transduction Xianghong Chen, Sheng Wei"

Neutrophil-mediated functional responses against infections provide a first line of host defense that occurs rapidly, and is aimed at a wide range of pathogens. Activation of neutrophil function involves the coordinate action of many surface receptors that are either stimulating or inhibiting neutrophil-mediated responses. This chapter will describe the two novel receptors that have been identified recently in neutrophils. (1)TREM, triggering receptor expressed by myeloid cells, is a single Ig domain DNAX adaptor protein 12-associated receptor expressed by cells of the myeloid lineage. TREMs belong to a rapidly expanding family of receptors that include activating and inhibitory isoforms encoded by a gene cluster linked to the MHC. (2) Toll-like receptors (TLRs) function as patternrecognition receptors (PRRS) in mammals and play an essential role in the recognition of microbial components. Stimulation of TLRs by microbial products leads to the induction of antimicrobial genes and inflammatory

*Correspondence to: Sheng Wei. Immunology Program, H. Lee Moffitt Cancer Center & Research Institute, 12902 Magnolia Drive, MRC 4 East, Tampa, Florida 33612, USA; phone: +(1)813-979-3934; e-mail: [email protected].

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responses. In addition, activation of TLRs also induces the development of antigen-specificadaptive immunity. Keywords:TREM; Toll-like receptor; DAPl2; ITAM; ITIM

1. INTRODUCTION Human polymorphonuclear neutrophils (PMNs),which represent 60-70% of circulating leukocytes, are the body's first line of defense in protecting the host against invading microorganisms.l It has now been established that neutrophils serve a complex function in innate immune responses, immune regulation, inflammation, including response to and production of cytokines2 Exposure of neutrophils to cytokines, bacteria, or bacteria1 products activates these cells as part of the innate immune response to clear the pathogen^.^-^ The functional activation of neutrophils results in two significant events: they have enhanced functional activity and prolonged life pan.^,^-'^ Neutrophil activation is a biologic double-edged sword, because inappropriate or excessive neutrophil activation can cause severe tissue damage, contributing to the pathology of a variety of inflammatory diseases? Dysregulation of neutrophil antimicrobial activity has severe clinical consequence^.^^,^^ This is best exemplified in the infection rate observed in cancer patients receiving cytotoxic chemotherapeutic agents with neutropenia or in patients with genetic defects in neutrophil function as seen in chronic granulomatous d i ~ e a s e . ~ , The ' ~ , ' ~ability of neutrophils to combat microbial pathogens is due to a number of specific activities, including: 1)adherence of neutrophils to endothelium; 2) migration or chemotaxis to an inflammatory site; 3) ingestion or phagocytosis into phagosomes; and 4) degranulation and killing.4 These biological events of neutrophils, including adherence, reorganization of microbial pathogens, and response to inflammatory stimuli, migration, phagocytosis, and generation of cytokines are modulated by a large variety of surface receptors. It has been known that many of these receptors are involved in neutrophil activation, including cytokine receptors (such as GM-CSF, IL-8, TNF, etc); immunoglobulin Fc receptors (FcRs); complement receptors (CRs); and multiple receptors for adhesion molecules; as well as receptors for bacteria and microbial products, such as the Toll-like receptors, LPS; and FMLP. Through these receptors, neutrophils can be

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activated and they migrate from the circulation into the local tissue. Expression of these receptors is essential for neutrophil functions such as chemotaxis, phagocytosis, ROS production, and release of cytokines, chemokines, and growth factors that potentiate innate immune and inflammatory responses. In general, these receptors can be divided into four main groups according to their biological function: adherence receptors; chemotactic receptors; phagocytic receptors and cytokine receptors. Most of these receptors have been well illustrated in other chapters or reviews. This chapter will focus on the latest developments and some novel receptors that are involved in neutrophil activation and regulation.

2. TRIGGERING RECEPTOR EXPRESSED BY MYELOID CELLS (TREM) Innate immune responses against infections provide a first line of host defense that occurs rapidly, and is aimed at a wide range of pathogens. Activation of innate responses involves the coordinate action of several cell types, including neutrophils, monocytes, and natural killer cells. Neutrophils are the key players among these cells to control infectious diseases. A hallmark of this immune reaction is its ability to maintain a precarious equilibrium between the extremes of reactivity and quiescence. An important aspect of this ability is encoded in the specificity of the response that can target invading foreign molecules but not self-normal tissue or cells. Equally important is its ability to limit and ultimately terminate a response, inactivating or eliminating the relevant pathways when they are no longer required. This balanced receptor system was originally termed nature killer cells (NK).18-26A key principle of the specificity of NK cells is that they prefer to attack cells that have downregulated classical MHC class I molecule^.'^^^^^^^ To prevent serious cell damage, the activating receptors are usually under the control of MHC class I-specific inhibitory receptors that provide the "off" signal. Since expression of MHC class I molecules is frequently altered as a consequence of tumor transformation or viral i n f e ~ t i o n ? the ~ . ~main ~ function of MHC class I-specific inhibitory receptors is to check the integrity of cells and to avoid damage to normal tissues. This family of MHC class I-specific receptors capable of inhibiting NK cell activation (killer cell inhibitory receptors, KIR) has been described

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both in human and in m o ~ s e ? ~Human * ~ * KIR belongs to the Ig superfamily and its cytoplasmic domains contain two immunoreceptor tyrosinebased inhibition motifs (ITIM).'9,22~32-34 These ITIMs are necessary and sufficient to mediate the inhibitory function of MHC class I NK receptors. Down-regulation of MHC classes I due to viral infection or transformation of cells would reduce inhibition of NK cell positive signaling and may cause initiation of NK activation and target l y ~ i sAlthough .~~ the KIR has not been reported in neutrophils, it has been known that the specificity of neutrophils is determined in part by various stimulatory receptors that function in immune recognition. This was supported by recent progress in identification of activating receptor in neutrophils.

2.1. Identification of TREMs Colonna and colleagues cloned an immunoglobulin superfamily (IgSF)-a new family of receptors, called "triggering receptor expressed by myeloid cells (TREM)family," whose expression appears restricted to various cells of the myeloid lineage?637Currently, at least five members of the TREM family has been identified - TREMl, TREM2, TREM3, TREM4 and TREM5.s39 Among them, TREMl are selectively expressed in neutrophils and a subset of CD14highmonocytes. The stimulation of neutrophoils and monocytes with bacteria (both Gram-positive and Gram-negative) and fungi, or their products, results in significant upregulation of TREMl expression on neut r ~ p h i l s .TREM2 ~ ~ , ~ ~is not constitutively expressed on neutrophils or on monocytes/macrophages. Its expression can be induced, however, on human dendritic cells grown from blood monocytes by culture in GM-CSF and IL4?O TREM3 are expressed on macrophages and also detected at low levels in T cells, but not in NK, B cell, or mast cells. Significantly TREM3 were up-regulated by LPS, but were down-regulated by

2.2. Characterization of TREMl TREMs are encoded as a cluster on mouse chromosome 17 and human chromosome 6. Human TREMl are transmembrane glycoproteins that consist of a single extracellular immunoglobulin-like domain of the V-type, a transmembrane region with a charged lysine residue and a short

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cytoplasmic tail. The TREM receptor shares low sequence homology to each other. The closest TREM relative is NKp44, an activating NK-cell receptor that is encoded by a gene closely linked to the TREM genes. The positively charged lysine residue in the transmembrane regions of TREMs is very critical for activating signal, because it is required for the association of TREMs with an activating adaptor protein, DAP12.%J7J9

2.3. DAP12 and Its Signal Transduction It has been known that TREMs and several other activating NK receptors signal through shared common adaptor molecules, DAP12, which harbors intracytoplasmic immunoreceptor tyrosine-based activation motifs (ITAMs: Y x x L ~ Y x x L / I ) . ~When ~ ~ ~ ' ~these ~ * receptor complexes engage their ligands, the tyrosines in the ITAM are phosphorylated by Src kinase permitting activation of the Syk or ZAP70signal pathway^^^^^' (see Fig. 1).

Fig. 1 TREMl is expressed as a transmembrane receptor complex with the DAP12 adaptor protein. After TREM crosslinking, the DAP12 ITAM tyrosines are phosphorylated, possibly by a protein tyrosine kinase of the Src family, providing a binding site for protein tyrosine kinases of the Syk family. Activated Syk will further recruit or activate downstream signaling molecules, leading to activation of phosphatidylinostol 3-kinase (PDK) and extracellular-signal-regulatedkinase (MAPK/ERK) pathways.

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The ITAM-bearing transmembrane adaptor protein DAP12 was originally described as a component of several NK activating receptor complexes. DAP12 is expressed on the surface of NK cells as a disulfide-bonded homodimer with a very short extracellular domain and a single ITAM in the intracellular region. DAP12 is distinct from other known transmembrane adaptors, and the gene encoding DAN2 is located on human chromosome 19q13.1.24 DAP12 noncovalently associates with at least all three TREMs and other activating receptors in the NK cells, including the CD94/NKG2C receptor for HLA-E, and the NKp44 r e ~ e p t o r s . ~ ~ ~ DAP12 also associates with certain KIRs, which contain a short cytoplasmic domains lacking ITIM sequences; in this case, these KIRs will potentially activate, rather than inhibit, NK lytic activityz1 Phosphorylated DAP12 proteins bind Syk and ZAP-70 protein tyrosine kinases and provide positive signal to trigger cell activation. There is no evidence that activating receptors can use other ITAM-containing transmembrane adaptors.

2.4. TREMl Ligand(s)

No ligands for any of the TREM receptors have been identified. Mouse TREM2A and TREM2B bind to either Gram-positive or Gram-negative bacteria as well as to astrocytoma cell lines?5 Purified anionic bacterial products can inhibit binding of TREM2, suggesting that TREM2 receptors may bind both bacteria and astrocytes via a charge-dependent interaction. Thus, pattern recognition of anionic ligands by TREM2 receptors may extend both to pathogens and to self-Ags. Receptor clustering is thought to occur upon particle binding that in turn generates a phagocytic signal. Several pathways of phagocytic signal transduction have been identified, including the activation of tyrosine kinases and (or) serine/ threonine kinases. Kinase activation leads to phosphorylation of the receptors and other proteins, recruited at the sites of p h a g o ~ y t o s i s . 3 , ~ ~ ~ ~ ~

2.5. Biological Function of TREM1 With the identification of TREMl in neutrophils, an obvious question arises: what are the biological roles of TREMl in neutrophils. It seems clear that they play a critical role in the inflammatory response to

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infection. TREMl is not only expressed in neutrophils, but also in monocytes/macrophages. TREMl is also strongly expressed in infections of human skin and lymph nodes caused by gram-positive and negative bacteria and fungi.37In these lesions, TREMl is expressed in not only neutrophillic infiltrates but also the epithelioid cells of monocytic origin surrounding granulomatous reactions. Conversely, TREMl is poorly expressed or not expressed in granulomatous infections caused by Mycobacterium tuberculosis or in foreign body granulomas. Regardless of the nature of the TREMl ligand, engagement of TREMl on neutrophils and monocytes results in initiation and amplification of inflammatory responses. Ligation of TREMl with a monoclonal antibody stimulates production of the proinflammatory chemokines interleukin (ILk8, monocyte chemoattractant protein (MCP)-l, MCP-3, and macrophage inflammatory protein-la. TREMl triggering also induces secretion of tumor necrosis factor (TNF)-a and IL-la, especially when LPS is used as a costimulus, demonstrating the ability of TREMl to amplify proinflammatory responses induced by TLR.36In addition, LPS and other TLR ligands can upregulate TREMl expression. Thus, TREMl and TLR cooperate in producing an inflammatory burst.48 toryresponse response

3. TOLL-LIKE RECEPTOR (TLR) 3.1. Expression of TLRs in Neutrophils The mammalian TLR is known to consist of ten members and play an effective role in innate immunity. As microbe-recognitionreceptors, TLRs mediate cellular responses to a large array of microbial ligands. TLRs recognize a broad spectrum of ligands, including modified lipids (LPS and bacterial lipoproteins); proteins (flagellin); and nucleic acids (DNA and double-stranded RNA). Different TLR may activate different downstream responses and that these differences may help tailor immune responses to be effective against specific organism^.^^.^^ The Toll-like receptor (TLR) family in mammal comprises a family of transmembrane proteins characterized by multiple copies of leucine-rich repeats in the extracellular domain and IL-1 receptor motif in the

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cytoplasmic domain. Like its counterparts in Dvosophila, TLRs signal through adaptor molecules and could constitute an important and unrecognized component of innate immunity in humans. TLRs characterized so far activate the MyD88/interleukin-l receptor-associated kineas (IRAK) signaling pathway. Because neutrophils are the prototypical innate immune cell and TLRs are the prototypical innate immune receptors, many researchers have started to investigate the role of individual TLRs in neutrophil function?1 Human neutrophils express most of the TLRs so far described: TLRs 1,2,4,5,6,7,8,9, and 10. TLR stimulation on neutrophils can result in the shedding of L-selectin, reduction in chemotaxis; increased phagocytosis; priming of superoxide generation; and the production of a number of cytokines and chemokines. Interestingly, many neutrophil functions can be elicited by purified TLR agonists recognized through specific TLRs, demonstrating that the cellular response to TLR stimulation is more varied than the initiation of proinflammatory gene expression.

3.2. Biologic Function of TLRs Activation of TLRs induces the expression of a variety of host defense genes. These include inflammatory cytokines and chemokines; antimicrobial peptides; costimulatory molecules; MHC molecules; and other effectors necessary to arm the host cell against the invading ~ a t h o g e n . ~ ~ Two -~O important neutrophil functions, chemotaxis and phagocytosis, have been described to be influenced by TLRs: a reduction in chemotaxis to IL-8 and an increase in the phagocytosis of opsonized latex beads upon TLR stimulation in neutrophils.60r61 The reduction of chemotaxis is reminiscent of the defect in neutrophil chemotaxis found in patients with sepsis and suggests this may be due to TLR stimulation of circulating neutrophils. It is tempting to speculate that reduction in neutrophil migration into sites of infection following sepsis-induced TLR stimulation contributes to the complications, including secondary infections, seen in septic patients. A popular hypothesis to explain the existence of multiple TLRs in mammalian genomes is that they allow the innate immune system to identify the class of pathogen encountered in order to subsequently tailor the immune response to best deal with that pathogen.

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TLR2 is the receptor for a variety of microbial ligands, including gram-positive bacteria, peptidoglycan, yeast zymosan, and mycobacterial ara-lipoarabinomannan (araLAM). TLR2 is expressed on the surface of neutrophils as well as on the surface of monocytes. Cell surface expression of TLR2 on neutrophils is modulated by external factors. TLR2 expression can be upregulated by GM-CSF, LPS, and G-CSF, while only minimal effects on monocytes receptor expression are seen.62163 GM-CSF induced increases in expression of TLR2 in 3 of 4 donors in repeated experiments (neutrophils from one individual were refractory to GM-CSF induction of TLR2). One study has demonstrated that TLRl and TLR4 levels are highly variable between donors; for example, TLR4 surface expression ranging from 400 to 3200 molecules per cell, and levels of TLRl ranging from 0 to 5400 molecules per cell. Nevertheless, these experiments are the first to demonstrate that GM-CSF treatment dramatically enhances the functional response of neutrophils to TLR l i g a n d ~ . ~ ~ TLR4 was expressed only weakly by neutrophils. TLR4 is a receptor for gram-negative bacteria, LPS, and some viruses play an essential role in the ability of cells to respond to LPS. This has been demonstrated in both mouse and human cells. A point mutation in the C3H/HeJ TLR4 gene is responsible for the resistance of these mice to LPS.64 TLR4 and TLR2 expressed on neutrophils, like other TLR family members, have a conserved intracellular signaling motif. This signaling motif, which is also found in the intracellular domain of the IL-1 receptor (IL-lR), is responsible for nuclear factor- KB (NF- KB) activation/translocation after TLR or IL-1R receptor engagement and is an essential signaling pathway for IL-1 p and tumor necrosis factor-a (TNF-a)~ e c r e t i o n . ~ ~ GM-CSF primes for enhanced neutrophil responses to microbial ligands in part by increasing the levels of TLR2 expression on the cell surface. Some studies have suggested that the primary neutrophil-stimulating activity of LPS preparations is due to the contaminating TLR2-specific ligand found in commercial LPS preparations. Removal of the TLR2stimulating component by phenol re-extraction significantly diminishes the neutrophil-stimulating activity of the LPS, but does not affect the TLR4-stimulating activity of the LPS and only slightly decreases the monocyte-stimulating activity of the LPS. Thus, monocytes respond strongly to the TLR.l-specific, pure LPS while neutrophils preferentially

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respond to the TLR2-ligand contaminated, partially purified commercial LPS. Nevertheless, TLR4 may play a role in neutrophil responses. Neutrophils do respond to phenol LPS (pure TLR4 ligand) for both IL-8 secretion and superoxide generation. It is interesting to note that although the neutrophil IL-8 secretion response to phenol LPS was enhanced by GM-CSF treatment, the response to commercial LPS showed a greater dose-dependence on GM-CSF treatment than the response to phenol LPS, again suggesting that GM-CSF preferentially enhances TLR2-dependent responses.66 Neutrophil activation in response to the TLR9 agonist nonmethylated CpG-motif-containing DNA (CpG DNA) was undetectable without GMCSF pretreatment. The TLR9-mediated recognition of CpG DNA is essentially nonexistent without pretreatment. Why this exquisite dependence on GM-CSF priming exists only for TLR9-mediated signals is unclear, though it is tempting to speculate that endogenous ligands for TLR9 (CpG DNA) are more prevalent than endogenous ligands for other TLRs, and thus the response must be tightly regulated. A potential mechanism for this GM-CSF induciblity of TLR9 function arises from the subcellular localization of TLR9 compared with the other TLRs - while other TLRs have access to the external environment through their plasma membrane localization; TLR9 is expressed in intracellular vesicles. GM-CSF’s ability to upregulate phagocytosis (and perhaps pinocytosis) would allow TLR9 to recognize internalized hypomethylated CpG DNAs. This suggests that fluid phase uptake of DNA is nonexistent in unstimulated neutrophils and is increased following GM-CSF treatment.67 GM-CSF also increases the magnitude of the response to agonists for TLRs 1, 5, 6, 7, and 8 in human neutrophils. GM-CSF-mediated increase in TLR2 and TLR9 responses is larger than that of the other T L R s . ~ ~ Interestingly, freshly isolated circulating neutrophils express all known TLRs with the exception of TLR3. Although not usually associated with viral infections, neutrophilia is associated with certain respiratory viral infections. TLR3-mediated recognition of double-stranded RNA is a potential mechanism of identifying viruses and viral infections, yet neutrophils do not express this receptor and are unresponsive to doublestranded RNA. It is unclear by which mechanism neutrophils would directly recognize virally infected host cell^.^^!^^

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3.3. TLR Signal Transduction Pathways Upon ligation with their specific binding partners, TLRs induce the expression of a variety of host defense genes. TLRs accomplish this by activating an intracellular signaling pathway conserved from Drosophila to mammals. This signaling pathway is very similar to the one activated by the IL-1R.

3.3.1. MyD88 dependent pathway MyD88 is an adaptor protein, which has a C-terminal TIR domain that mediates its homophilic interaction with the receptor and an N-terminal death domain that engages the death domain of its downstream target IRAK (IL-1 receptor associated kinase). For example, upon ligation with LPS, the TLR receptor recruits MyD88 and its downstream target, IRAK. Then, upon association with MyD88, IRAK, a serine threonine kinase, undergoes autophosphorylation and subsequently dissociated from the receptor complex. Furthermore, phosphorylated IRAK associates with RING-finger containing adapter protein, called tumor necrosis factor (TNF) receptor-activated factor 6 (TRAF6). TRAF6 will be activated by this event and leads to the activation of both NF-KB and MAP kinases. MyD88-deficient mice were generated and found to be completely unresponsive to IL-1, LPS, CpG DNA, lipoproteins, and other immunostimulatory bacterial components. These studies demonstrate that MyD88 is a critical component in the signaling cascades, mediated not only by IL-1R but also TLRs. This signaling pathway plays an essential role in host cells in the response to pathogen-derived immunostimulatory molecule^.^^^^^^^^

3.3.2. MyD88 independent path way

Although all TLRs signal through the conserved signal pathway described above, the additional signal pathways must exist.51This was indicated by the complexity of the TLR-induced cellular responses. An example of the existence of MyD88-independent pathway can be shown by using TLR2 knockout or MyD88 knockout macrophages. Upon stimulation by TLR2 activator, mycoplasmal lipopeptide, the activation of

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NF-KB and MAP kinase are completely abolished in both TLR2 and MyD88 knockout macrophages. However, LPS activation of NF-KBand MAP kinase remains intact in these m a c r o p h a g e ~ . ~These ~ / ~ ~suggest -~~ that there maybe a MyDWindependent signaling pathway that mediates NF-KBand MAP kinase a c t i v a t i ~ nHow . ~ ~ LPS activates NF-KBand MAP kinase in a MyD88-independent manner is not clear. Recently, several components involved in the MyD88-independent signaling pathway have been identified. Subtractive hybridization cloning identified several genes that re-induced in MyD88 knockout macrophages upon activation with LPS, including the novel TIR domain-containing adapter protein (TIRAP) and IFN-y-inducible genes, such as IP-10 (IFN-inducible protein lo), a member of the CXC chemokine family; GARG16 (glucocorticoid attenuated response gene 16); and IRGl (IFN-regulated gene 1). TIRAP contains a small N-terminal region of unknown function and a C-terminal TIR domain that mediates its interaction with TLR4. A dominant negative mutant of TIRAP specifically inhibits TLR4- but not IL-1 or TLR9-induced NF-KB activation, indicating a specificity of TIRAP for the TLR4 pathway. Expression of these genes is TLRCdependent, but MyD88-independent. Therefore, although MyD88 is required for all signaling events downstream of some TLRs, MyD88 is clearly dispensable for some TLR4-induced signals. Thus, MyD88-independent pathways may be involved in IRF-3 activation, as well as induction of type I IFN and IFN inducible geness0

4. THE Fc RECEPTORS (FcR) The FcRs comprise a family of receptors that bind to the Fc portion of immunoglobulin molecules. When binding to FcR, the antibodies indeed provide antigen specificity to a variety of cells, most of which are devoid of antigen recognition structures. FcR recognize not antigens but the Fc portion of the antibodies. Antibody-FcR complexes nevertheless function as membrane receptors for antigen with no predetermined specificity. FcRs exist for every antibody class: FcyR bind IgG; FcaR bind IgA; FcsR bind IgE; FckR bind IgM; and Fc8R bind IgD.76FcRs exist as membrane receptors and as soluble molecules, produced by alternative splicing of FcR transcripts or by proteolysis of membrane receptors. Soluble FcRs

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(sFcRs) retain an affinity for immunoglobulins, and they can display several biological activities. From a functional point of view, these can be divided into two major types: FcR that can trigger cell activation and FcR that ~ a n n o t ? ~ , ~ ~

4.1. Activation and Inhibition of FcR FcRs capable of triggering cell activation possess one or several intracytoplasmic activation motifs, which resemble those of the BCR and TCR signal transduction subunits. These motifs, composed by a twicerepeated YxxL sequence flanking seven variable residues, are now designated immunoreceptor tyrosine-based activation motifs (ITAMs). As described above, these ITAM containing FcRs are referred to as activation receptors and are able to mediate activation signals, which lead to cell activation similar to the signal observed in TREMs-DAP12 signal pathway. FcRs with ITAMs are of two types. FcRs of the first type represent the majority of FcRs; they are multichain receptors composed of a ligandbinding FcRa subunit, associated with one or two signal transduction subunits in the intracytoplasmic domains of which ITAM are located. FcRs of the second type comprise two closely related single-chain IgG receptors, unique to humans, and referred to as FcyRIIA and FcyRIIC. They possess a single ITAM that has 12 residues (instead of 7) between the two YxxL sequences.79 FcRs that do not trigger cell activation have no ITAM. They can also be subdivided into two main categories. FcRs of the first category constitute a family of single-chain IgG receptors, collectively referred to as Fcy NIB, whose intracytoplasmic domain possesses a motif that inhibits cell activation by receptors capable of triggering cell activation. This motif contains a single YxxL sequence that was designated immunoreceptor tyrosine-based inhibition motif (ITIM). FcRs of the second category neither trigger nor inhibit cell activation. They are involved in the transcytosis of immunoglobulins through epithelia. They are the polymeric IgA and IgM receptor (pIgR), and the neonatal FcR for IgG (FcRn). Finally, a human IgG receptor without ITAM, referred to as FcyRIIIB, has no triggering capability by itself but contributes to cell signaling by associating with other FcRs.*O

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4.2. FcR-Mediated Signaling in Neutrophils According to the structure and binding properties of FcyR, three types of receptors have been described. FcyRI (CD64) is a high affinity receptor (70 kDa) for monomeric IgG expressed on monocytes, tissue macrophages and activated neutrophils. FcyRlI (CD32)is a low affinity receptor (40kDa) for monomeric IgG expressed on monocytes, macrophages, neutrophils, B-cells, platelets, epithelial and endothelial cells. The FcyRIII (CD16), also a low affinity receptor for monomeric IgG, is a glycoprotein of 50-70 kDa and is expressed on neutrophils, NK cells, eosinophils and macrophages. The CD16 expressed on neutrophils is glycosylphosphatidyl inositolanchored (CD16B), whereas NK cells and macrophages express a transmembrane form (CD16A).81 FcyRII appears to activate tyrosine kinases and to be phosphorylated on tyrosine during immune complex-mediated cell activation. Fcy RII has been found to associate with specific Src family kinases, including Fgr in neutrophils, and to activate Syk kinase. Ligation of FcyRII leads to the tyrosine phosphorylation of multiple cellular proteins, including phospholipase Cy, Shc, and syk, in addition to FcyRII itself.82 Following stimulation of neutrophils, FcyRIIIb perpetuates inflammation through the production of neutrophil-derived cytokines. In addition to ICs, there exist at least two credible candidates for FcyRIIIbmediated activation of the cells: sFcyRIIIb, which has been shown to bind the P-chain CDllb of the complement receptor (CR) 3 on the surface of neutrophils, and autoantibodies (autoAbs) directed against FcyRIIIb, which have long been reported in autoimmune mice and patients. Both were explored in primary Sjogren’s syndrome (pSS), systemic lupus erythematosus (SLE) and rheumatoid arthritis (RA).83t84 Soluble and insoluble immune complexes containing IgG activate neutrophils by completely separate processes. The major difference resides in the fact that soluble immune complexes are completely unable to stimulate the generation of reactive oxidants or degranulation in unprimed neutrophils. In contrast, insoluble complexes slowly activate a respiratory burst in unprimed neutrophils, but the oxidants that are produced are generated intracellularly. These natural intracellular oxidants are confirmed by the facts that: (a) they cannot be scavenged by the

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extracellular enzymes SOD and catalase; and (b) no oxidants are detected when using the extracellular probes isoluminol or cytochrome; and (c) it is thus likely that insoluble immune complexes are phagocytosed by unprimed neutrophils and hence the reactive oxidants are generated intracellularly, within the phagolysosomes. Similarly, soluble immune complexes did not activate the secretion of myeloperoxidase or lactoferrin from unprimed n e ~ t r o p h i l s . ~ ~ However, when the neutrophils were primed with GM-CSF, dramatic changes in their responsiveness to both insoluble and soluble immune complexes were detected. The most dramatic effect was the ability of soluble immune complexes (and to a lesser effect, insoluble immune complexes) to activate the secretion of reactive oxidants and granule enzymes from primed neutrophils. This activation process was extremely rapid and transient, reaching a peak rate within about two minutes after stimulation, and contrasted with the slower activation of phagocytosis seen with the insoluble complexes. This priming effect was not specific to GMCSF as a number of other proinflammatory cytokines, such as interleukin lp, tumor necrosis factor a,and interferon y could also induce this ability in n e ~ t r o p h i l s . ~ ~ , ~ ~ The roles of FcyRII (CD32) and FcyRIIIb (CD16) in the activation of primed neutrophils by immune complexes were first determined by using blocking assay. Primed neutrophils were preincubated with Fab/F(ab’>, fragments of 3G8 (anti-FcyRIIIb) and IV.3 (anti-FcyRII) before addition of complexes and measurement of luminol chemiluminescence. The production of extracellular oxidants in response to soluble immune complexes was decreased by about 53 (41% of control values ( n = 61, when either FcyRII or FcyRIIIb binding was blocked, with FcyRIIIb blocking having a slightly greater inhibitory effect. However, even when binding to both. receptors was blocked, there was still some detectable chemiluminescence. In contrast, blocking FcyRII had only a slight (24 (5)%,n = 6) inhibitory effect on luminol chemiluminescencestimulated in primed neutrophils by insoluble immune complexes. However, the response was inhibited by 78 (4)%, ( n = 6) when ligation to FcyRIIIb was blocked. Little additional inhibitory effect was seen when binding to both receptors was prevented.68Using an ELISA based on the combination of two monoclonal Abs (mAbs) targeting different epitopes, Youinou and his colleagues have

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reported elevated levels of sFcyRIIIb in pSS, SLE and RA. Chemoattractant stimulation of the cells accelerates their spontaneous apoptosis, and thereby the release of FcyRIIIb, indicating that the excess of sFcyRIIIb originates from apoptotic PMNs within the inflamed tissues.s8 HNA-la/HNA-lb heterozygous neutrophils were incubated with increasing amounts of sFcyRIIIb, and their adherence to endothelial cells was shown to decline in a dose-dependent manner. Similarly, sFcyRIIIb reduced the respiratory burst of neutrophils, while the same amounts of a control protein produced negligible effects. In fact, the diminished superoxide anion production may proceed from the insufficient adherence and the ensuing deficiency of p h a g o c y t o ~ i s . ~ ~ ~ ~ ~ Recently, Zhou’s laboratory has found that direct ligation of FcyRII leads to a respiratory burst, whereas direct ligation of FcyRIIIB does not. Instead, FcyRIIIB cooperates with the neutrophils integrin CR3 (Mac-1, CDllb/CD18) to generate a synergistic respiratory burst. In the synergistic respiratory burst, the two membrane receptors have distinct roles. Ligation of CR3 immobilizes Fcy RII to the adherent plasma membrane by a cytoskeleton-dependent mechanism, and ligation of FcyRIIIB induces appropriate tyrosine kinase activation in the proximity of the immobilized FcyRII. Thus, FcyRII is required, in addition to CR3 and FcyRIIIB, for the synergistic respiratory burst. Immobilization of FcyRII and FcyRIIIB on the adherent neutrophil surface by direct ligation leads to the activation of different Src family kinases. FcyRII is associated with activation and translocation of Fgr to the Triton-insoluble cell fraction; and FcyRIIIB is associated with Hck activation and translocation. The exclusive association of FcyRIIIB with Hck activation is not a property of all GPI-linked proteins in PMN, since immobilization of decay accelerating factor (DAF, CD55) leads primarily to Fgr activation at the adherent membrane. Moreover, DAF cannot substitute for FcyRIIIbB in synergistic activation of the respiratory burst. Ligation of FcyRII and FcyRIIIB activate and translocate distinct Src family members in ne~trophils.~~ Youinou’s study has recently provided direct evidence for a key role of the death promoter Bax as a pro-apoptotic molecule in neutrophils, whereas Bcl-2 is not expressed. In the model, it was found that the percentage and mean fluorescence intensity of Bax-containing neutrophils

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were downregulated by CD16 cross-linking. This is perfectly in line with the report that decreased levels of Bax were normalized by stimulation of the cells with G-CSF and GM-CSF. Caspase-3 has also been shown to be pivotal in spontaneous apoptosis of neutrophils, and indeed they have shown that incubation of neutrophils in the presence of anti-FcgRIIIb F(ab’)* resulted in a reduced caspase-3 activitys8

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12. Pericle F, Liu JH, Diaz JI, et al. Eur J Immunoll994; 24:440444. 13. Torres M, Hall FL, ONeill K. JImmunoll993; 150:1563-1577. 14. Hampton MB, Kettle AJ, Winterbourn CC. Blood 1998; 923007-3017. 15. Glauser MI? Intensive Care Med 2000; 26:S103-110. 16. Kim SK, Demetri GD. Hematol Oncol Clin North Am 1996; 10:377-395.

17. Liu JH, Wei S, Lamy T, Epling-Burnette PK. Blood 2000; 95:3219-3222. 18. Lanier LL. Curr Opin Immunol1997; 9:126-131. 19. Farag SS, Fehniger TA, Ruggeri L, et al. Blood 2002; 100:1935-1947. 20. Yokoyama WM, Plougastel BF. Nut Rev Immunol2003; 3:304-316. 21. Lanier LL. NK cell receptors. Annu Rev Immunol1998; 16:359-393.

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22. Djeu JY, Jiang K, Wei S. A view to a kill: signals triggering cytotoxicity. Clin Cancer Res 2002; 8:636-640. 23. Bakker AB, Wu J, Phillips JH, Lanier LL. Hum lmmunol2000; 61:18-27. 24. Tomasello E, Blery M, Vely E, Vivier E. Semin Immunol2000; 12:139-147. 25. Lanier LL. J E x p Med 2000; 191:1259-1262. 26. Moretta A, Bottino C, Vitale M, et al. Annu Rev Immunol2001; 19:197-223. 27. Karre K. lmmunol Rev 1997; 155:5-9. 28. Soloski MJ. Curr Opin lmmunol2001; 13:154-162. 29. Cosman D, Mullberg J, Sutherland CL, et al. Immunity 2001; 14:123-133. 30. Yokoyama WM. Nut Immunol2000; 1:95-97. 31. Colonna M, Samaridis J. Science 1995; 268:405408.

32. Olcese L, Lang P, Vely F, et al. J lmmunoll996; 156:4531-4534. 33. Campbell KS, Dessing M, Lopez-Botet M, et al. J E x p Med 1996; 184:93-100.

34. Ono M, Okada H, Bolland S, et al. Cell 1997; 90:293-301.

35. Diefenbach A, Raulet DH. Curr Biol 1999; 9:R851-853. 36. Bouchon A, Dietrich J, Colonna M. J Immunol2000; 16449914995. 37. Bouchon A, Facchetti F, Weigand MA, Colonna M. Nature 2001; 410: 1103-1107. 38. Daws MR, Sullam PM, Niemi EC, et al. J Immunol2003; 171:594-599. 39. Chung DH, Seaman WE, Daws MR. Eur J lmmunol2002; 32:59-66. 40. Bouchon A, Hernandez-Munain C, Cella M, Colonna M. J Exp Med 2001; 194:1111-1122. 41. McVicar DW, Taylor LS, Gosselin P, et al. J Biol Chem 1998; 273:32934-32942. 42. Lanier LL, Bakker AB. Immunol Today 2000; 21:611-614. 43. Olcese L, Cambiaggi A, Semenzato G, et al. J lmmunoll997; 158:5083-5086. 44. Moretta A, Sivori S, Vitale M, et al. J E x p Med 1995; 182:875-884. 45. Peiser L, Mukhopadhyay S, Gordon S. Curr Opin lmmunol2002; 14:123-128. 46. Fernandez R, Suchard SJ.J Immunoll998; 160:5154-5162. 47. Gaudry M, Gilbert C, Barabe F, et al. Blood 1995; 86:3567-3574.

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48. Colonna M, Facchetti F. J Infect Dis 2003; 187(Suppl2):S397401. 49. Takeuchi 0, Sat0 S, Horiuchi T, et a / . J lmmunol2002; 169:lO-14. 50. Takeda K, Kaisho T, Akira S. Annu Rev Immunol2003; 21:335-376. 51. Janeway CA Jr, Medzhitov R. Annu Rev lmmunol2002; 20:197-216. 52. Hayashi F, Means TK, Luster AD. Blood 2003; 102:2660-2669.

53. Picard C, Puel A, Bonnet M, et al. Science 2003; 299:2076-2079. 54. Kurt-Jones EA, Mandell L, Whitney C, et a/. Blood 2002; 100:1860-1868.

55. Sabroe I, Jones EC, Usher LR, eta/. JImmunol2002; 168:47014710. 56. Malcolm KC, Worthen GS. J Biol Chem 2003; 278:15693-15701. 57. Ayala A, Chung CS, Lomas JL, et al. Am J Pathol2002; 161:2283-2294.

58. Svanborg C, Frendeus B, Godaly G, et al. J Infect Dis 2001; 183(Suppl 1): S61-65. 59. Marsik C, Mayr F, Cardona F, et al. BYJ Haematol2003; 1213653-656. 60. Fan J, Malik AB. Nut Med 2003; 9:315-321, 61. Solomkin JS, Bass RC, Bjornson HS, et al. Infect Immun 1994; 62:943-947. 62. Zhang G, Ghosh S. J Clin Invest 2001; 107:13-19. 63. Means TK, Golenbock DT, Fenton MJ. Cytokine Growth Factor Rev 2000; 11~219-232. 64. Kurt-Jones EA, Popova L, Kwinn L, et al. Nut Imrnunol2000; 1:398-401. 65. ONeill L. Biochem SOCTrans 2000; 28:557-563. 66. Wollin L, Uhlig S, Nusing R, Wendel A. Am J Respir Crit Care Med 2001; 163:443450. 67. Fossati G, Mazzucchelli I, Gritti D, et al. Int JMol Med 1998; 1:943-951. 68. Fossati G, Bucknall RC, Edwards SW. Ann Rheum Dis 2002; 61:13-19. 69. Jones A, Qui JM, Bataki E, et al. Eur Respir J 2002; 20:651-657. 70. Viuff B, Tjornehoj K, Larsen LE, et al. Am J Pathol2002; 161:2295-2207.

71. Muzio M, Ni J, Feng P, Dixit VM. Science 1997; 278:1612-1615. 72. Medzhitov R, Preston-Hurlburt P, Kopp E, et a / . Mol Cell 1998; 2:253-258. 73. Yamamoto M, Sat0 S, Mori K, et a/. J lmmunol2002; 169:6668-6672.

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74. Takeuchi 0, Hoshino K, Akira S. J Immunol2000; 165:5392-5396. 75. Takeuchi 0, Hoshino K, Kawai T, et al. Immunity 1999; 11:443-451. 76. Hulett MD, Hogarth PM. Adv Immunoll994; 571-127. 77. Fridman WH, Teillaud JL, Bouchard C, et al. J Leukoc Biol 1993; 54:504-512. 78. Delespesse G, Sarfati M, Wu CY, et al. Immunol Rev 1992; 125:77-97. 79. Cambier JC. Immunol Toduy 1995; 16:llO.

80. Daeron M. Annu Rev Immunoll997; 15:203-234. 81. Stein MP, Edberg JC, Kimberly RP,et ul. Clin Invest 2000; 105:369-376. 82. Lamour A, Soubrane C, Ichen M, et al. Eur J Clin Invest 1993; 23:97-101. 83. Hutin P, Lamour A, Pennec YL, et al. Cell-free. Int Arch Allergy Immunol 1994; 103:23-27. 84. Lamour A, Baron D, Soubrane C, et al. J Autoimmun 1995; 8:249-265. 85. Lundqvist H, Follin P, Khalfan L, Dahlgren C. J Leukoc Biol 1996; 59:270-279. 86. Dibbert B, Weber M, Nikolaizik WH, et al. Proc Nut1 Acad Sci USA 1999; 96:13330-13335.

87. Lloyds D, Davies EV, Williams BD, Hallett MB. Br J Rheumutol 1996; 35: 846-852. 88. Youinou P, Renaudineau Y. Biochem SOCTruns 2002; 30819-824.

89. Watson RW, Rotstein OD, Nathens AB, et al. J Immunoll997; 158:945-953. 90. Coxon A, Rieu P, Barkalow FJ, et al. Immunity 1996; 5:653-666. 91. Zhou MJ, Lublin DM, Link DC, Brown EJ. J Biol Chem 1995; 270:13553-13560.

4

Mechanisms of Neutrophil Migration Alan R. Burns,” Roland0 E. Rumbaut

Much is known about the adhesive events that allow flowing neutrophils to interact with the inflamed endothelium, but far less is known about the molecular regulation of neutrophil migration across the endothelium and its effects on vascular permeability. While leukocyte p2 integrins (CD18) typically play a critical role in neutrophil adhesion and emigration, CD18-independent neutrophil emigration can also occur in certain organs (e.g. lung and heart). There is increasing evidence that interendothelial cleft molecules (e.g. connexins, PECAM-I, CD99 and JAMS),play a modulatory role in regulating neutrophil trafficking across the endothelium. As well, it is now clear that neutrophils use both paracellular (migration at endothelial borders) and transcytotic (migration through the endothelial cell body) pathways to move across the endothelium. The molecular regulation of these distinct pathways and the consequences of neutrophil transmigration to vascular permeability are discussed.

*Correspondence to: Alan R. Burns, Ph.D. Section of Cardiovascular Sciences and Leukocyte Biology, Departments of Medicine and Pediatrics, Baylor College of Medicine, Room 5158, One Baylor Plaza, Houston, TX 77030; phone: (713) 798-4371; fax: (713) 790 - 0681.

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The Neutrophils

Keywords: endothelium; leukocytes; inflammation; adhesion molecules; microvascular permeability

1. INTRODUCTION Recruitment of leukocytes (primarily neutrophils) to sites of tissue injury or infection is a hallmark of the acute inflammatory response. Efficient neutrophil extravasation at sites of inflammation requires a coordinated cascade of adhesive and signaling e ~ e n t ~ . Neutrophils ~ J ~ ~ , ~leave ~ ~the , ~ ~ ~ flowing blood stream by first tethering and then rolling on the inflamed endothelium lining the blood vessel lumen. In the systemic circulation, this occurs primarily in post-capillary and collecting venules. Rolling neutrophils that arrest (stop rolling) can become firmly adherent to the endothelial surface. Under favorable conditions, firmly adherent neutrophils will migrate across the endothelium (Fig. 1). Estimates from in nitro models of leukocyte trafficking show the process to be rapid, with transmigration being completed in < 2 min.28201 While there is an extensive database for understanding how adhesion and stimulating molecules (e.g. chemokines and cytokines) and their receptors regulate

Fig. 1 Transmission electron micrograph showing two neutrophils (arrows) migrating out of a venule in a rat mesentery after fMLF superfusion (100nM). Bar = 2 pm.

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neutrophil tethering, rolling, and arrest, much less is known about the transmigration process. Moreover, much remains unknown about the influence of each step in this cascade on the subsequent behavior of leukocytes at sites of acute inflammation. In recent years, it has become apparent that neutrophil transendothelial migration can occur through distinct endothelial sites, either paracellular pathways (between endothelial cells) or transcytotic pathways (through endothelial cells)?2 Neutrophil transendothelial migration may also cause tissue injury, especially if the inflammatory process is activated by non-infectious processes (e.g. trauma, ischemia/reperfusion). Under these conditions, neutrophil activation is often temporally associated with enhanced microvascular permeability.34~110 The sections that follow begin with a historic overview of key scientific studies that lead to our present concept of neutrophil adhesion and transendothelial migration. This historical perspective is a useful tool for enabling us to re-visit the underpinnings of the current emigration paradigm. As well, it provides us with the opportunity to reflect on the scientific acumen possessed by early researchers and once again, acknowledge their outstanding contributions to the field of leukocyte biology. Following these historical reflections, the remaining sections focus on current issues that shape and challenge the way we think about the physical and molecular regulation of neutrophil transendothelial migration and its effects on endothelial permeability.

2. HISTORICAL PERSPECTIVE O N LEUKOCYTE

ADHESION A N D EMIGRATION (1669-1955) 2.1. The First Observations The history associated with the study of leukocyte adhesion and emigration is a distinguished one, with origins that can be traced back to the late 17th century and the discovery of the blood leukocyte by Antoni van Leeuwenhoek in 1669.177The early investigators who studied the phenomenon of adhesion and emigration did so without any knowledge of the existence of adhesion molecules. In fact, it is only within the last 25 years that adhesion molecules have been demonstrated to play a role in neutrophil adhesion and emigration. So who was the first person to

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document and describe leukocyte emigration? Careful inspection of the literature suggests Albrecht von Haller deserves that distinction. In 1756, he documented blood cell adhesion in a frog mesenteric vein as follows: Experiment CXXIV. On four frogs. 22 July. .. . in the small vessels of the venous network, where the globules followed in single file.. . The globules stuck out along the edge of the veins, they gave rise to a half semicircular circumference, and took the shape of a string of beads, because the membrane of the veins is so thin as to become invisible: this same phenomenon does not exist in arteries, where the membranes are thicker?I6[Italicsadded.]

It is well established that, during inflammation, leukocytes preferentially adhere to the veins and not arteries (for a review, see Harlan et ~ 1 . ~ 9 . While von Haller failed to describe the color of these blood globules, his morphological description of blood globules sticking to veins and not arteries is reason enough to think that he was observing leukocyte adhesion. Indeed, one year later in a subsequent report on the movement of blood von Haller appears to have specifically described leukocyte emigration again when he reported that he was:

... struck with the appearance of globules coating the veins like a chaplet of beads and the extravascular appearance of spherical and yellow cells. [From Grant69;italics added.] Red cells appear pale yellow when viewed individually, prompting some to conclude that von Haller’s “yellow cells’’ were probably red blood cells and not leukocytes at all.69However, it is just as likely that von Haller was not describing the extravascular appearance of spherical yellow cells, but rather the extravascular appearance of spherical cells (white cells) and yellow cells (red cells). The amphibian white blood cell is That von Haller did spherical, but the red blood cell is flat and ellip~oid.~ not assign a color to the spherical cells is in keeping with the idea that they have no color. Indeed, years later, Cohnheim would refer to white blood cells as “colorless ~ o r p u s c l e s . ” ~ ~

2.2. Mechanistic Insight By the beginning of the 1 8 4 0 ~the ~ early English investigators began using an experimental approach to show that leukocytes adhere to and emigrate

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from blood vessels in response to inflammatory stimuli (reviewed by Grant).69Of particular note are the studies of Julius Conheim in which he describes for the first time a temporal association between leukocyte emigration and tissue swelling (increased microvascular permeability): Keeping pace with this exodus, emigration, or, as it is also called, extravasation of corpuscular elements there occurs an increased transudation of fluid, in consequence of which the meshes of the mesentery, or the tissues of the tongue, are infiltrated and swell?*

But perhaps Cohnheim's most influential contribution to the study of inflammation was not what he saw but how he interpreted it: Inflammation is the expression and consequence of a molecular alteration in the vessel walls ...it is only and solely the vessel wall which is responsible for the entire series of events ... ?8

This suggestion is remarkable in light of the fact that evidence of a molecular alteration would not be documented for another 100 years. Interestingly, Cohnheim argued so strongly for the role of the endothelium in this process, that he considered leukocyte emigration to be the result of mechanical filtration. He did not believe that the leukocyte actively participated in adhesion or emigration. The type of image Conheim may have seen with his microscope is shown in Fig. 2. In contrast, Elias Metchnikoff believed the entire process of adhesion and emigration could be attributed to the activity of the leukocyte. He believed the accumulation of leukocytes at sites of inflammation was effected by their attraction (sensibility)to a chernotactic substance:

. .. the leukocytes, led by their sensibility and by means of their amoeboid movements, themselves proceed towards the injured spot instead of passively filtering through a ~ e s s e l - w a l l . ' ~ ~ Recognition that the leukocyte might also undergo adhesive changes would go unnoticed until 1955when Allison and colleagues reported that:

... during the course of the inflammatory reaction leukocytes were frequently seen to stick to one another, indicating that the increased adhesiveness characteristic of the inflammatory response is not limited to the endotheli~m.~ [Italics added.]

11 0

The Neutrophils

Fig. 2 Videomicroscopy image of an arteriole (A) and a venule (V)after exteriorization of rat mesentery. Leukocytes (arrows) interact only with the venule. The

black dot on the arteriole is caused by an optical velocimeter. Bar = 10 pm.

The critical observation that the leukocyte also undergoes important adhesive changes that enable it to adhere to, and migrate across, a n inflamed endothelium would have to await the discovery and characterization of leukocyte-endothelial adhesion molecules.

3. MOLECULAR ADHESIVE EVENTS PRECEDING NEUTROPHIL TRANSEN DOTHELIAL MIGRATION

At the sites of inflammation in the systemic circulation, post-capillary and collecting venules are the principal sites for neutrophil adhesion and emigration. Adhesion of neutrophils to the venular endothelium occurs normally in the presence of fluid shear rates ranging from about 150 to 1600 per sec.11,214 All the known members of the selectin family mediate neutrophil rolling on venular endothelium.1,27,48,96,109,111,121,137,138 Selectins are a unique family of adhesion molecules characterized by the juxtaposition of an N-terminal C-type lectin domain, an epidermal

Mechanisms of Neutrophil Migration

11 1

growth factor (EGF) domain and variable numbers of complement regulatory protein-like repeating units.22,95J15J83 The selectin family has three members. L-selectin (CD62L) is expressed on the surface of neutrophils, lymphocytes, monocytes and e o s i n ~ p h i l s .It~ 'localizes ~ to surface projections (ruffles),where it is topographically positioned to facilitate interactions with the inflamed endothelial s ~ r f a c eNeutrophil . ~ ~ ~ ~ activation ~ ~ ~ is associated with rapid (minutes) shedding of L-selectin.Io2P-selectin (CD62P) is found on the surface of activated platelets and endothelial cells. Endothelial P-selectin is stored in specialized cytoplasmic granules known as Weibel-Palade bodies.24f73,74,'39 Stimulation with thrombin, LTC4, histamine calcium ionophore A23187, complement proteins C5b-9 or phorbol esters results in rapid (minutes) translocation to the cell surface. E-selectin (CD62E) is expressed on the surface of activated endothelial cells. A wide range of inflammatory mediators induces E-selectin expression, including IL-1p, TNFa, bacterial endotoxin and substance P.21,22 Selectins are constitutively active when expressed and bind with fast rates of association and dissociation that facilitate rolling in response to the hydrodynamic and normal forces of flowing b l 0 o d . 4 ~ ~ ~The ~~~8'~~ velocity of leukocyte rolling is apparently controlled by at least two factors - the dissociation of selectin/ligand bonds, and level of leukocyte p2 integrin binding to structures on the endothelial surface (e.g. ICAM-1). With the development of mice deficient in one or more selectins, it is now apparent that each of the selectins may mediate leukocyte rolling at different velocities which is also dependent on the dose, type and timing of the inflammatory s t i m ~ l i . " ~ , ' ~ ~ , ' ~ ~ The involvement of p2 integrins is, in contrast to the selectins, activation dependent. For optimum emigration at inflammatory sites, tethered neutrophils apparently must be activated locally.'28The leukocyte integrin family (62 or CD18) exhibits low binding avidity unless activated191!33~127~198 (i.e. undergo conformational changes that increase the affinity of binding182),and then these molecules function to arrest rolling cells. Endothelial ICAM-1 (CD54) is the principal ligand for the p2 integrins, LFA-1 (CDlla/CDlS) and Mac-1 (CDllb/CD18). While many vessels constitutively express ICAM-1, its expression is greatly enhanced following stimulation with inflammatory mediators (e.g. IL-1P, TNFa,

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The Neutrophils

IFNy and endotoxin). VCAM-1 (CD106)is also upregulated by inflammatory cytokine stimulation and serves as a major endothelial ligand for the leukocyte p l integrin, VLA-4 (CD49d/CD29). VLA-4 is readily detected on unactivated murine, but not human, neutrophils. VLA-4 interactions with VCAM-1 can facilitate leukocyte rolling as well as firm a d h e ~ i o n . ~ The transition from rolling to stationary adhesion under conditions of flow is thought to be triggered by chemokines (e.g. IL-8165)that are on the surface of the inflamed endothelial cells.143Activation of p2 integrindependent adhesion by chemokines and chemotactic factors is well documented under static (i.e. absence of hydraulic flow) conditi0ns,9~though the evidence documenting the role for surface bound chemokines in the transition from rolling to stationary adhesion to endothelium under flow conditions is less secure.lgl A variety of ligands for selectins are found on neutrophils,10,*22,146~147~174~195,197,219~224 and binding to these ligands not only accounts for the tethering function of selectins, but may transduce a signal for neutrophil activation.218Cross-linking of L-selectin results in rapid and transient increases in Ca++ flux and the production of reactive oxygen species, IL-8 and TNFa.41,217 p2 integrin adhesive function can also be modulated by cross-linking L - ~ e l e c t i n , 6PSGL-152 ~ J ~ ~ ~ ~and ~ ~the leukocyte receptor(s) for E-se1e~tin.l~~ E-selectin-deficient mice have reduced levels of firmly adherent neutrophils, even though the number of rolling leukocytes in microvessels is not reduced.144

4. INTEGRIN REGULATION OF NEUTROPHIL TRANSENDOTHELIAL MIGRATION Neutrophil transendothelial migration typically depends on at least two events -stimulation of p2 integrins to interact with ICAM-1 or other ligands on the endothelial cell surface, and stimulation of cell locomotion.193 Unstimulated neutrophils are not motile ce11s.192f234 Experimental models of transendothelial migration have been used to establish that concentration gradients of chemotactic factors across endothelial monolayers in vitro or applied to the perivascular space of microvessels in vivo can induce transmigration of n e u t r ~ p h i l s . ~The ~ , 'stimulation ~ ~ ~ ~ ~ ~of motility in vivo is likely to be chemokinetic (random movement) and haptotactic

Mechanisms of Neutrophil Migration

1 13

(contact-mediated). Migration induced by IL-8 can be haptotactic when IL-8 is surface bound to proteoglycans such as heparan ~ u 1 f a t e . I ~ ~ Migration is also directed by adherence to the endothelial cell surface through p2 and possibly p l integrins. Studies in vitro and in vivo using antibodies against CDlla and CDllb demonstrate that both LFA-1 and Mac-1 are involved in t r a n s m i g r a t i ~ n . ~A ~ Jrecently ~ ~ J ~ ~described integrin, a 9 / p l expressed on neutrophils can bind to VCAM-1. Antibodies against a9/ p l can inhibit transendothelial migration under conditions in which VCAM-1 is upregulated on the endothelial cells.211Integrinassociated protein, CD47,a regulatory protein for both leukocytes and endothelium also appears to be involved in tran~migration.~~ Kitayama and colleagues found that antibodies to pl integrins markedly reduced transmigration in vitro, and that transmigration was inhibited by anti-a5 antibody when endothelial cells were grown on the fibronectin but by anti-a6 antibody when monolayers were plated on the laminin.Io4They suggest that the extracellular matrix may induce a transition from the p2 integrins needed for intercellular adhesion to the integrins needed for migration on extracellular matrix as the neutrophil is transmigrating. It is essential to point out that p2 integrins are not an absolute requirement for neutrophil transendothelial migration. In the lung, neutrophils can emigrate into the alveolar airspace via a CD18-independent pathway and the requirement for CD18 is stimulus-spe~ific.~~ Our own studies in mice show that a CDWindependent neutrophil emigration also occurs in the injured heart. While numerous studies have documented a critical role for CD18 in neutrophil infiltration of the postischemic myocardium, we were puzzled by the observation that significant neutrophil emigration persists when CD18 is neutralized or absent.97~132~156~158,208 In our laboratory, using an established mouse model of myocardial ischemia and reperfusion, we found that neutrophil infiltration efficiency was reduced by only 50% in CDWdeficient (null) mice. Neutrophil emigration could be further reduced using an anti-VCAM-1 antibody (given to CD18 null mice 30 minutes prior to ischemia).25That VCAM-1 and CD18 play important roles in neutrophil emigration in the mouse heart is entirely consistent with published studies showing that mouse heart expresses high constitutive levels of vascular VCAM-1 and ICAM-1 (a ligand for CD18).77

1 14

The Neutrophils

To understand more about the molecular regulation of neutrophil transendothelial migration in the heart, we developed an in vitro mouse model of leukocyte trafficking. Using an immunomagnetic cell separation technique, we isolated, characterized and cultured endothelial cells from mouse vena cava and heart.25Culturing endothelium from two different tissue sources allowed us to test the hypothesis that following activation with a common stimulus (endotoxin), CDWindependent neutrophil emigration was a tissue-specific response. Indeed, using freshly isolated peripheral blood neutrophils from wild type or CD18 null mice, we confirmed the tissue specificity of the response by showing that neutrophil migration across endotoxin-activated cultured cardiac endothelium is CDl8-independent while migration across LPS-activated endothelium obtained from inferior vena cava is CD18-dependent. Consistent with in vivo findings, migration of CD18-deficient neutrophils on cardiac endothelial monolayers is blocked by antibodies against 014 integrin or VCAM-I . Collectively, the data support the conclusion that tissue-specific differences in endothelia account, at least partially, for CD18-independent neutrophil transendothelial m i g r a t i ~ n ? ~

5. PARACELLULAR NEUTROPHIL TRANSENDOTHELIAL MIGRATION 5.1. Endothelial Cleft Organization It is widely believed and popularized in review articles that neutrophil migration across the endothelium is primarily paracellular and involves penetration (disruption) of intercellular junctions (zonula occludens or tight junctions; zontlla adherens or adherens junctions). Within the cleft, the neutrophil is thought to interact with endothelial gap junctions and resident adhesion molecules (PECAM-1 (CD31), CD99 and JAMS).The basis for the paracellular migration concept originated with the early electron microscopic observations of Marchesi and Florey which, based on a limited number of serial sections, were interpreted as showing neutrophils (and other leukocytes) passing through interendothelial ~ l e f t s . *This ~ ~ Jconcept ~~ was strengthened by subsequent molecular studies, showing that PECAM-1, CD99 and JAM-A localize to

Mechanisms of Neutrophil Migration

11 5

endothelial clefts and blocking antibodies directed against these molecules impede leukocyte transendothelial migration (see below). In the text that follows, the role of endothelial junctions and cleft-associated adhesion molecules is critically examined.

5.2. Tight Junctionsand Preferred Transmigration Sites The endothelial tight junction (zonulu occludens) is often described as a "belt-like" structure located in the most apical aspect of the intercellular cleft. The junctions are viewed as being "tight" because they prevent macromolecules from moving through the intercellular cleft and limit the lateral diffusion of intrinsic membrane proteins and lipids between the apical and basolateral cell surface domains.65Tight junctions are the first structural barriers a migrating neutrophil encounters as it penetrates the interendothelial cleft (Fig. 3). By thin section transmission electron microscopy and in cross sectional view, tight junctions appear as points of membrane fusion or " k i ~ s e s ' ' . ~Freeze-fracture J~~ micrographs show that the kisses are in fact linear arrays of intramembranous ridges or strands, and depending on fixation conditions, the strands may appear as rows of ~artic1es.l~~ Tight junction strands and particles appear to comprise two distinct transmembrane tetraspan proteins. One of these proteins is occludin and the other is claudin (at least 24 different claudins have been described to date). Trans-homophilic interactions between claudin molecules are critical for tight junction strand formation, whereas trans-homophilic interactions between occludin molecules seem to serve regulatory rather than structural roles in tight junction assembly.64 As a first step towards understanding paracellular neutrophil transendothelial migration, we need to review a few key points concerning endothelial tight junction organization. The first is that tight junctions do not provide a perfect tight seal around the endothelial cell perimeter. Even though the tight junction is belt-like, there are often small (30-60 Angstrom) discontinuities or gaps within the tight junction strands, particularly in venous tight junctions. More importantly, at tricellular corners (the site where three endothelial borders converge) tight junctions are inherently discontinuous. This latter discontinuity arises because of the

11 6

The Neutrophils

Fig. 3 Transmission electron micrographs of HUVEC monolayer showing intercellular junctions within the endothelial cleft. Panel A shows tight junction strands (small arrows) and a gap junction plaque (large arrow). Panel B shows an adherens junction complex (arrows).Bar = 100nm.

inability of the cells to form three-sided junctional contacts. The tricellular comer pore is estimated to have a width of 2 7 0 A n g ~ t r o r n s . ~ ~ ~ Much of what we understand about neutrophil transendothelial migration comes from in vitro studies using freshly isolated peripheral blood neutrophils and looking at their interactions with cultured cytokine-activated human umbilical vein endothelial cell (HUVEC) monolayers (Fig. 4). This brings us to a second key point concerning endothelial tight junctions.

Mechanisms of Neutrophil Migration

1 17

Fig. 4 Neutrophil migration across cytokine-activated HUVEC monolayers. Neutrophils adherent to the monolayer are visible in Panel A (large arrows). Transmigrated neutrophils appear phase dark; trailing tails (uropods) that have not yet penetrated the endothelium remain phase bright (small arrows). Panel B is a cross-sectionalview of a similar monolayer in which neutrophils can be seen above and below the monolayer; the trailing uropod (arrow)on one neutrophil is evident. The diagram shows a surface view of the monolayer with three potential migration sites. Shown are paracellular migration sites at a tricellular corner (A) and bicellular border (B), as well as a transcytotic migration site (C). Bar = 10 pm.

Under conventional culture conditions, tight junctions are poorly developed or absent in HUVEC (and other endothelial cell) monolayers?8 This lack of tight junctions is frequently dismissed as being unimportant to our understanding of leukocyte trafficking, since tight junctions are considered to be loosely organized in post-capillary venules, the principal site of neutrophil emigration in the systemic c i r c ~ l a t i o n .While ' ~ ~ ~ it~ is ~~

1 18

The Neutrophils

true that within the vasculature, venular tight junctions are the least organized (reviewed by Burns3*)and ultrastructural studies suggest 30% of venular tight junctions are open (porous), the size of each pore is only 30-60 h g ~ t r o m s . ' ~These ~,'~~ pores are 200-300 times smaller than the pore (1-2 microns wide) through which a neutrophil penetrates the endothelium. Hence, the so-called "loose" organization of venular tight junctions in vivo is very likely to be a significant physical barrier to neutrophil paracellular transendothelial migration. The fact that in the absence of tight junctions, endothelial intercellular clefts in culture continue to express adherens and gap junction molecules, as well critical adhesion molecules like PECAM-1, JAMS,and CD99 (see below), should not be taken as evidence that these molecules are correctly positioned and functioning normally within the cleft. For example, we know that in HUVEC monolayers lacking tight junctions, occludin (a tight junction regulatory transmembrane protein) is still expressed at endothelial cell borders. In the absence of tight junctions, it seems doubtful that the occludin molecules are functioning normally. Consistent with this notion are immunostaining observations showing that occludin staining patterns along endothelial cell borders are poorly defined (fuzzy) when compared to endothelial cultures expressing organized tight junctionsZ8Whether the topography and function of other cleft molecules is abnormal in endothelial monolayers lacking tight junctions is unknown. However, it only makes sense that good in vitro models of neutrophil trafficking should include endothelial monolayers expressing well formed tight junctions. Lack of tight junction expression in endothelial cell monolayers can be overcome by culturing the cells in medium conditioned by astrocytes. In nojvo, astrocytes secrete an unknown substance(s) that maintains the extensive tight junction network found in brain endothelia. When HUVEC monolayers are cultured in astrocyte-conditionedmedium, they retain the venous organization of umbilical vein tight junctions; they do not form extensive blood brain barrier-like tight junctions.30Using this "improved in nitro endothelial model to study leukocyte trafficking, we found that under static conditions (i.e. absence of hydrodynamic forces) neutrophil migration across cytokine-activated endothelium was exclusively paracellular (i.e. at endothelial borders). Neutrophil migration efficiency was not

Mechanisms of Neutrophil Migration

1 19

compromised by the presence of tight junction networks and, importantly we observed that the majority (77%)of transmigrating neutrophils passed through the endothelium at specialized sites which we termed tricellular corners (i.e. where three endothelial borders converged).28 As mentioned earlier, endothelial tricellular corners are sites of tight junction discontinuity. In their electron microscopic freeze-fracture studies of pulmonary capillaries, Walker and colleagues hypothesized that tricellular corners were ”potential sites for the transient opening and closing of the paracellular pathway ... [and] possible avenues through which white blood cells migrate during inflammatory reactions.” Our own in nitro observations agree with this prediction and show that preferential neutrophil migration at tricellular corners occurs without tight junction protein (occludin, ZO-1 and 2 0 - 2 ) d e g r a d a t i ~ n . ~ ~ Importantly, under hydrodynamic flow conditions that mimic venous shear stress (2 dynes/cm*), 70% of neutrophil migration across cytokine-activated HUVEC monolayers also occurs through tricellular corners. Importantly, the distance a neutrophil moves from the time it arrests (stops rolling) to the time it transmigrates is only 5.5 2 0.70~1.m (under static conditions, the distance is similar 6.8 f 0.9 p,m).66This distance is less than one neutrophil diameter and shows that neutrophils arrest very close to tricellular corners. Mathematical modeling of the process suggested endothelial borders were preferred sites for neutrophil arrest.66 That endothelial borders are sticky sites for leukocyte adhesion is supported by recent atomic force microscopy (AFM) measurements of adhesive interactions between monocytic cells (HL60) and TNFaactivated HUVEC mono layer^.^^^ In this study, a lectin (Concanavalin A) was used to attach an HL60 cell to the AFM cantilever tip and the HL60 cell was then used to probe the endothelial surface. The data show that the force (measured by cantilever deflection) required to detach the HL60 cell from endothelial borders, was twice as high as that needed to detach it from the endothelial cell body. Additional antibody studies suggested HL60 adhesion to endothelial borders is mediated in part by P-selectin, E-selectin, ICAM-1 and VCAM-I, whereas adhesion to the endothelial cell body (i.e. over the nucleus) seems to be VCAM-1-dependent. In our own studies using HUVEC monolayers activated by histamine or

120

The Neutrophils

thrombin, we found that P-selectin was entirely responsible for guiding neutrophils to borders and corners. P-selectin is stored in endothelial Weibel-Palade bodies and rapidly mobilized to the cell surface following stimulation (e.g. histamine or thrombin). Important with regard to the concept of guidance is our finding that P-selectin surface upregulation occurs preferentially along endothelial borders, where it captures neutrophils from the flowing stream and hence by design (intention), targets them for paracellular tran~migration,~~

5.3. Adherens Junctions The adherens junction is another type of "belt-like" structure lying within the endothelial cleft, just beneath the tight junction (Fig. 3). In endothelial cells, VE-cadherin (cadherin-5) is the critical transmembrane molecule that allows the adherens junction to function as a permeability barrier. Antibodies directed at VE-cadherin result in increased endothelial permeability, both in vitro and in '0i'00.68~81,113 In a conceptual model that requires calcium, cis-dimerization between cadherin monomers is thought to be followed by the trans-dimerization of cadherin dimers on the adjacent ce11s.105~160~202 The cytoplasmic tail of cadherins is linked to the cytoskeleton through accessory molecules known as catenins (p- or 7-catenin which in turn is linked to a-catenin).202 While adherens junctions have been implicated in the regulation of endothelial paracellular permeability,26140r81 a point that is often overlooked is the fact that adherens junctions (like tight junctions) are discontinuous at tricellular corners where the margins of three endothelial cells onv verge.^^.^^ Hence, as noted above for tight junctions, preferential neutrophil transmigration at tricellular corner junctional discontinuities would allow them to avoid passing through intact adherens junctions that lie between pairs of adjacent endothelial cells. Initially, it was reported that neutrophil adhesion to cytokineactivated endothelium induced widespread loss (degradation) of VEcadherin and its associated cat en in^.^^ However, subsequent studies established that the apparent loss of adherens junctions was the result of a post-fixation artifact in which neutrophil proteases remain active after fixation (i.e. adherens junction degradation occurred during sample

Mechanisms of Neutrophil Migration

121

p r o c e ~ s i n g ) ? ~Current ~ ' ~ ~ evidence favors the idea that adherens junction degradation is not necessary for leukocyte transmigration. Instead, it appears that adherens junctions undergo a structural reorganization that accommodates the transmigrating leukocyte. The kinetics of VE-cadherin mobility within the cleft during leukocyte transmigration has been studied using real-time microscopy and green fluorescent protein (GFP) coupled VE-cadherin inserted into endothelial cells. Shaw and colleagues showed that endothelial cleft-associated GFP-VE-cadherin moves aside (rather than being degraded) as the leukocyte traverses the cleft and then, within 5 minutes, GFP-VE-cadherin moves back to seal the hole, the so-called "curtain-effect".Iso There is evidence that tight junctions can also slide within the plasma membrane.31 The displacement of tight and adherens junctions may require that the neutrophil signal the endothelium. We know that neutrophil transendothelial migration is associated with an increase in endothelial free calcium, phosphorylation of endothelial myosin regulatory light chains and endothelial isometric tension g e n e r a t i ~ n . ~ ~ ~ ~ ~ , Signaling may be mediated by neutrophil receptor engagement of specific endothelial ligands. Supporting in vitro studies show that antibody ligation of key endothelial ligands (E-selectin, P-selectin and VCAM-1) increases endothelial calcium levels and induces alterations in F-actin dist r i b ~ t i 0 n . Collectively, l~~ the observations hint at an important role for endothelial cytoskeletal changes during leukocyte transmigration. In vitro studies of monocyte migration across human microvascular endothelial monolayers lend support to this concept and show that monocyte transmigration is inhibited when endothelial microfilaments are disrupted by cytochalasin B or latrunculin A.'O' In summary, current evidence suggests neutrophil adhesion to the endothelium generates an outside-in signal triggering endothelial cytoskeletal rearrangements that lead to lateral displacement of adherens junctions and tight junctions. Conceptually, this model of leukocyte transmigration makes sense, particularly since the transmembrane proteins of adherens junctions and tight junctions are known to be intimately tied to the cytoskeleton by cytoplasmic linker proteins (e.g. catenins in the case of VE-cadherin; ZO-1 and 2 0 - 2 in the case of claudins and occludins).59~89~90,179,203,204The mechanism of lateral junction displacement

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The Neutrophils

would also have to be applied to neutrophil migration at tricellular corners, since the width of the resting tricellular corner pore is too small to accommodate a transmigrating neutrophil (see above).

5.4. Gap Junctions Gap junctions are specialized structures for intercellular communication. They are formed from transmembrane proteins known as connexins, and endothelial cells express at least three types of connexin, Cx37, Cx40 and Cx43. Hexameric arrangement of six connexins (Cx) gives rise to a structure known as a hemichannel or connexon. When a connexon from one neighboring cell aligns with that of another, a gap junction channel or pore is formed between the cells. The pore allows for intercellular communication and the passage of macromolecules (up to 900 Da). Multiple pores can exist together (Fig. 3) in the form of a gap junction p l a q ~ e . 5 ~ Unlike tight junctions, gap junction plaques never form belt-like structures around the perimeter of the endothelial cell and hence, they play no physical role in regulating the passage of macromolecules, water or neutrophils across the endothelium. However, they may play signaling role in regulating the passage of neutrophils (and other leukocytes) across the endothelium. Recent studies provide evidence that neutrophils express Cx37, Cx40 and C ~ 4 3 . 9 Based ~ ~ ~ 'on calcein dye coupling experiments, stimulated neutrophil adhesion to HUVEC monolayers results in heterotypic gap junction channel formation and bi-directional dye transfer. Dye coupling between adherent neutrophils and endothelial cells is reduced in the presence of TNFa, but not in the presence of other stimuli (IFNy, endotoxin, thrombin, formyl peptide or phorbol ester). Inhibition of dye coupling by TNFa may be related to its ability to downregulate endothelial gap junctions. Interestingly, when gap junction coupling between neutrophils and endothelial cells is deliberately inhibited with a broad-based gap peptide inhibitor (SRPTEKTVFTV), stimulated neutrophil adhesion to the HUVEC monolayer is unaffected, but the number of transmigrating neutrophils increases (-20%).231 These observations suggest that heterotypic gap junction formation between adherent neutrophils and endothelial cells is regulated by the nature of the inflammatory response, and heterotypic gap junction coupling seems to be a

Mechanisms of Neutrophil Migration

123

negative modulator of neutrophil transmigration. The nature of the inhibitory signal remains to be determined.

5.5. PECAM-1

PECAM-1 is expressed on leukocytes and endothelial cells. It is a 130kDa transmembrane protein belonging to the immunoglobulin (Ig) super family. The extracellular portion is arranged into six globular domains which engage in homophilic interactions and heterophilic interactions with glycosaminoglycans.2,222 The cytoplasmic portion of the molecule appears to have a signaling function, since it supports tyrosine phosphorylation on residues Y663 and Y686 and associates with a number of signaling molecules (SHP-1, SHP-2, SHIP and PLC-y1)78f92,163 That PECAM-1 plays an important role in neu trophil transendothelial migration is suggested by at least four additional key observations: (1)it shows a marked localization to the basolateral membranes of interendothelial clefts14; (2) antiPECAM-1 antibodies or soluble recombinant PECAM-1 block monocyte and neutrophil transendothelial migration150;(3) PECAM-1 ligation is associated with the activation of pl, p2 and p3 integrins19,20~37,44,117f161~209 and (4)antibody epitope mapping studies suggest extracellular domains 1and 2 regulate homophilic interactions, while domain 6 regulates leukocyte migration across the basal lamina.124 Since there is a significant body of evidence supporting a role for PECAM-1 in leukocyte transendothelial migration, it came as a surprise when Duncan and colleagues reported that neutrophil transendothelial migration appeared to be normal in PECAM-1-deficient mice.49The argument has been made that these mice must exhibit compensatory changes in adhesion molecule usage to account for their observed ability to migrate normally across the end0the1ium.l~~ Interestingly, a delay in migration across the basement membrane was noted and the delay was found to depend on the nature of the inflammatory stimulus; it was seen ~~~ mouse studies from in response to IL-1p but not to T N F c x .Additional Sussan Nourshargh's laboratory suggest PECAM-1 homophilic interactions (i.e. neutrophil PECAM-1 binding to endothelial PECAM-1) are necessary for a6/ p l integrin upregulation on neutrophils. Specifically, in response to IL-1 p, PECAM-1-deficient mice and chimeric mice deficient

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The Neutrophils

in either endothelial or leukocyte PECAM-1 (developed by bone marrow transplantation) show reduced neutrophil emigration and reduced ( ~ 6p/l expression. Increased a6/ p l expression on neutrophils facilitates migration across the perivascular basement membrane; when antibody is used to block (w6/pl integrin, neutrophil migration across the perivascular basement membrane is markedly While the preceding studies with PECAM-l-deficient mice clearly demonstrate a role for PECAM-1 in migration across the basal lamina, they were not well suited to study the kinetics of neutrophil migration across the endothelium. As noted earlier, neutrophil transendothelial migration is a rapid process (typically occurring in < 2 minutes) and subtle changes in the ability of the neutrophil to migrate across the endothelium in the absence of PECAM-1 would not have been detected in the studies published to date. While Duncan and colleagues used an in vitro assay to conclude that there was no difference in the net migration of wildtype or PECAM-l-deficient mouse neutrophils across mouse endothelium, interpretation of these data is limited for several reasons. First, the authors used immortalized mouse brain endothelium (bEND3) rather than primary cultures of mouse endothelial cells and these cells did not allow for experimentation with PECAM-l-deficient endothelium. Second, neutrophil transmigration was induced using a very high concentration of IL-8 (500ng/ml) and IL-8 is not a naturally occurring mouse chemokine; MIP-2 and KC are the murine IL-8 orthologs. Third, the mouse neutrophils used in this study were obtained by peritoneal lavage after challenge with thioglycollate. They represent a select population of cells that have already migrated across an endothelium and PECAM-1 expression is downregulated on emigrated neutrophils.210t221 For reasons outlined above, we undertook an investigation to reexamine the impact of PECAM-l-deficiency on neutrophil transendothelial migration. Using our own in vitro model of leukocyte trafficking, we studied the migration behavior of freshly isolated peripheral blood neutrophils across LPS-activated mouse endothelial cells derived from vena cava. Briefly, we investigated two conventional (wildtype neutrophils on wildtype endothelium and PECAM-l-deficient neutrophils on PECAM-1deficient endothelium) and two chimeric (wildtype neutrophils on PECAM-l-deficient endothelium and PECAM-l-deficient neutrophils on

Mechanisms of Neutrophil Migration

125

Table 1 Effect of PECAM-1 Deletion on Neutrophil Adhesion and Transmigration on Cultured Mouse Endothelium Isolated from Vena Cava PECAM-I Expression

LPS (4h)"

Baseline

Endothelium Neutrophil % Adhesion % Migration

+

-

+ -

+

30.3 2 7.7

-

28.8 t 4.7 53.2 2 0.0 29.2 t 1.2

-

+

0 0.6 ? 0.6 0

0

% Adhesion % Migration 65.0 2 10.0

68.2 2 10.3 74.2 t 2.2 79.8 t 7.2

19.4 2 7.8 26.7 2 15.1 0.6 ? 0.6 11.1t 1.7

Data are mean 2 SEM from at least 3 separate experiments, each with 1-5 replicates.

wildtype endothelium) migration situations. In each case, neutrophil transmigration efficiency was normal with one exception. When PECAM1-deficient neutrophils were placed on wildtype endothelium, they showed a marked inability to migrate across endotoxin-activated wildtype endothelium (Table 1). This is the first demonstration, using the PECAM-1 deficiency, that PECAM-1 plays a role in neutrophil transendothelial migration. Several explanations come to mind as to how PECAM-1 is regulating neutrophil transendothelial migration. Conceivably, if PECAM-1 has a signaling role in the neutrophil, a lack of PECAM-1 on the neutrophil surface may result in sub-optimal activation of leukocyte integrins. As well, if PECAM-1 contributes to endothelial barrier function then neutrophils lacking PECAM-1 may not be able to homophilically engage (open) the "PECAM-1 barrier" and neutrophil migration across the endothelium could prove more difficult. Clearly, additional experiments are required to determine exactly how PECAM-1 regulates the migration process.

5.6. CD99 Relatively little is known about CD99 and its ability to regulate leukocyte trafficking. CD99 is a heavily 0-glycosylated 32 kDa type I transmembrane protein originally described as being present in hematopoietic cells. However, a recent study shows that CD99 also resides within the endothelial cleft and it appears to regulate leukocyte transendothelial migration.176 In this study, observations of CD99

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The Neutrophils

transfectants suggest CD99 can engage in homophilic adhesive interactions. Importantly, an antibody directed against CD99 (hec2) blocks monocyte migration across cytokine-treated HUVEC monolayers. Interestingly, the blocking effect is distal to that of PECAM-1 antibody blockade. While anti-PECAM-1 antibodies directed against domain 1 or 2 prevent monocytes from entering the endothelial cleft, monocytes are able to insert a pseudopod into the cleft in the presence of the anti-CD99 antibody, but their progression across the monolayer is inhibited. Whether CD99 regulates neutrophil transmigration is unknown, but neutrophils reportedly (data not shown) express CD99. Clearly, additional studies with neutrophils are warranted given the potential of the molecule to regulate leukocyte transmigration.

5.7. JAMs The junctional adhesion molecules (JAMS)are a small family of Ig molecules implicated in regulating tight junction assembly and leukocyte trafficking. There are three recognized members in the JAM family and the nearly simultaneous discovery and characterization of these molecules in humans and mice has led to difficulties with the nomenclature. For example, human JAM-1 is equivalent to mouse JAM-1; human JAM-2 is equivalent to mouse JAM-3; and human JAM-3 is equivalent to mouse JAM-2. Recently, a new nomenclature scheme was proposed to stabilize the JAM literature. Under the revised nomenclature, human and mouse JAM-1 become JAM-A, human JAM-2 and mouse JAM-3 become JAM-B and human JAM-3 and mouse JAM-2 become JAM-C. The text that follows will use the new nomenclature proposed by M ~ l l e r and ' ~ ~ refer to the JAMs as JAM-A, JAM-B and JAM-C. All three members of the JAM family reportedly localize to endothelial cell borders and JAMs engage in homophilic as well as heterophilic adhesive interaction^.^^^^^,^^^^^^^^ At least for JAM-A, homophilic adhesion is thought to require cis-dimerization and trans-interaction of the NH2terminal domains with JAM-A dimers on the adjacent cell.17,106When JAM-A, JAM-B or JAM-C is transfected into Chinese hamster ovary (CHO) cells or Madin-Darby canine kidney (MDCK) cells, the localization of JAMs to cell-cell borders is associated with decreased paracellular

Mechanisms of Neutrophil Migration

127

permeability; JAM enrichment at borders is not seen at sites where transfectants contact non-tran~fectants.~~~J~~'~J~~~~Z

5.8. JAM-A Immunofluorescence microscopy clearly shows JAM-A staining associated with the endothelial cleft.136J55J81 By confocal microscopy, endothelial JAM-A appears to co-localize with tight junction proteins (AF-6 and cingulin). Immunoprecipitationand glutathione S-transferase(GST)pull-down experiments in epithelial and endothelial cells confirm that JAM-Ainteracts not only with AF-6 and cingulin, but also ZO-1, occludin and PAR-3.1820,91 A model has been proposed in which JAM-A associates with claudin strands through interactions with the PDZ domains of ZO-l.65,91On a functional level, JAM-A appears to regulate tight junction assembly since anti-JAM-A antibodies inhibit transepithelial resistance recovery following T84 monolayer disruption induced by transient calcium depletion.125 Of interest to neutrophil transmigration is the finding that JAM-A is expressed on human neutrophils (and other leukoctye~).*~~ A monoclonal antibody (BV11) against mouse JAM-A inhibits spontaneous and chemokine-induced human monocyte migration across cultured mouse endothelial monolayers as well as across endotoxin-treated mouse endothelial mono layer^.'^^ In v i m , B V l l partially inhibits monocyte migration in a murine model of skin i n f l a m m a t i ~ nand ' ~ ~ it inhibits both monocytes and neutrophils in a murine model of m e n i n g i t i ~However, .~~ BVll did not inhibit leukocyte emigration in a murine model of infectious meningitis, suggesting that a requirement for JAM-A may vary with the nature of the inflammatory stimulus.'18 Interestingly, JAM-Ais also a counter-receptor for the leukocyte p2 integrin CDlla/CD18 (LFA-1). Because LFA-1 binds to the membrane proximal domain 2 region of JAM-A, endothelial JAM-A has the potential to engage in homophilic domain 1 interactions with neutrophil JAM-A as well as heterophilic interactions with neutrophil LFA-1.154 Hence, JAM-Amay serve as a molecular zipper, allowing the migrating neutrophil to maintain close contact with the endothelial membrane lining the cleft, helping to preserve the barrier properties of the endothelium. Additional studies are needed to demonstrate neutrophil JAM-Abinding to endothelialJAM-A.

1 2 8 The Neutrophils

5.9. JAM-B and JAM-C In addition to JAM-B homophilic interactions with JAM-B and JAM-C homophilic interactions with JAM-C, JAM-B and JAM-C can also bind to each other.9 Of potential importance to neutrophil transendothelial migration are the findings that the leukocyte integrins very late antigen-4 (VLA-4, CD49d/CD29) and Mac-1 (CDllb/CD18) can bind JAM-B and JAM-C, respectively. It needs to be mentioned that Mac-1 binding to endothelial JAM-C has not been documented; the observed binding was to platelet JAM-C.’75Also, efficient VLA-4 binding to JAM-B requires previous engagement of JAM-B with JAM-C.43While the p l integrin VLA-4 is expressed on circulating mouse ne~trophils,2~ it is poorly expressed on circulating human neutrophils; its expression on human neutrophils increases after transmigration.108Another important point to consider in the human system is that HUVEC monolayers are frequently used to study neutrophil transmigration in vitro (see above). To date, only JAM-A and JAM-C have been reported to be present in the intercellular clefts of HUVEC m ~ n o l a y e r s l ~ ~ JAM-B ~ ~ / ~has ~ ~not ~ been ’ ~ ~ detected.lZ3 ; Hence, in its current form, the HUVEC assay may be limited to studying neutrophil-endothelial JAM interactions, involving endothelial JAM-A binding to neutrophil JAM-A or neutrophil LFA-1 (see above) and endothelial JAM-C binding to neutrophil Mac-1.

6. TRANSCYTOTIC NEUTROPHIL A N D TRANS EN D O T HELIAL MIG RAT10N In our laboratory, using cultured endothelial monolayers to study neutrophil trafficking, transcytotic migration (i.e. direct penetration of endothelial cell cytoplasm by the neutrophil) is not observed on human endothelium and is observed infrequently ( < l o % ) on dog and mouse endothelium. In vim,silver nitrate perfusion (Fig. 5) can be used to enhance the appearance of endothelial borders and examine leukocyte transmigration sites on the endothelial surface. In rat mesenteric vessels, after mechanical trauma (e.g. exteriorization of the mesentery), leukocytes can be seen emigrating at endothelial tricellular corners (Fig. 5), bicellular borders (Fig. 6 ) and through the body of the endothelial cell

Mechanisms of Neutrophil Migration

129

Fig. 5 Silver perfusion reveals endothelial borders and site of leukocyte transendothelial migration in an inflamed rat mesentery venule. In the top panel, venular endothelial cells (V)are polygonal while arteriolar endothelial cells (A) are spindle-shaped. The leukocyte migrating at a tricellular corner (arrow) is shown at higher magnification in serial optical slices (Panels 1 4 ; 1pm intervals). In Panels 1 and 2, the leukocyte is clearly seen (arrow). Panel 3 shows the migration pore (large arrow) and a portion of the leukocyte lying beneath the endothelium (small arrow) which remains in focus in Panels 4 and 5. The leukocyte does not extend into the focal plane shown in Panel 6. Bar = 20 pm (top panel); 10 pm (lower panels).

(Fig. 6). In other species, and in response to a different stimulus (fMLF), in vivo studies in guinea pi$5 and mouse82suggest neutrophil emigration is primarily, if not exclusively, transcytotic. It is important to recognize that since neutrophil emigration in these studies was observed within 15 minutes of fMLF injection, changes in the expression of endothelial adhesion molecules typically seen after cytokine-activation of

130

The Neutrophils

Fig. 6 Intercellular and transcytotic migration of leukocytes across inflamed rat mesenteric venular endothelium after silver nitrate staining. The luminal portions of three leukocytes are seen in Panel 1 (urruzus);sequential slices show migrating leukocytes (urrmheads) through cell borders (Panels2 and 4) and through the body of an endothelialcell (Panel3). Bar = 20 wm.

endothelium (e.g. upregulation of ICAM-1, VCAM-1 and E-selectin) are unlikely as increased expression of these molecules requires de ~ O V Oprotein synthesis. Changes in adhesion molecule surface topography may be critical for neutrophil guidance to, and paracellular migration at, endothelial borders and corners?O It is also possible that neutrophil agonists like fMLF induce preferential transcytotic migration through the release of neutrophil-derived VEGF. VEGF is a cationic protein of -23 kDa57and present in neutrophil granules.207It is known to induce the formation of transendothelial gaps56 and increase the permeability of microvesselsZ8 and cultured endothel i ~ m .Neutrophil-activation ~ ~ ~ ~ ~ , ~ ~by fMLF results in a dose-dependent release of VEGF (Fig. 7). In a canine study of myocardial injury following reperfusion, VEGF staining is strongly positive in infiltrating neutrophils (Fig. 7)It is tempting to speculate that granule fusion at the site of neutrophil contact with the endothelium results in the controlled and focal release of VEGF and the formation of a transendothelial gap capable of supporting neutrophil transmigration. To find out if the nature of the inflammatory response determines whether a migrating neutrophil utilizes a paracellular route or a transcytotic route, we cultured unstimulated human endothelial monolayers on transwell filters (0.4 micron pore size) and placed lOnM concentrations of fMLF, IL-8, C5a, or LTB, in the lower chamber. Unstimulated neutrophils were placed in the upper chamber and allowed to settle and migrate for

Mechanisms of Neutrophil Migration

13 1

Fig. 7 VEGF expression in neutrophils. The upper panel shows the influence of fMLF stimulation on VEGF release from human neutrophils. The lower panels show a paraffin section of a reperfused injured myocardium in the dog. The panel on the left was stained with a neutrophil-specificantibody. The panel on the right was stained with an anti-VEGF antibody. Note the co-localization of VEGF within the infiltrating neutrophils (arrows). Bar = 10pm.

400s after which the preparation was fixed and silver stained to visualize endothelial borders. Adherent neutrophils were scored as to their location on the endothelial surface (endothelial borders or endothelial cell body); and migrating neutrophils as to the site of endothelial penetration (tricellular, bicellular, or transcytotic). These results were compared with neutrophil adhesion and migration on IL-1 P-activated (10 units/ml, 4 h) HWEC monolayers in the same apparatus. The results show that in all the cases, paracellular migration predominates and the migrating neutrophils show a marked (>64%) preference for tricellular corners. While some transcytotic migration was observed in each case, it was always < 5%. Since our in vitro data suggest the neutrophil's preference for tricellular corners is unaffected by the type (e.g. neutrophil agonist versus

132

The Neutrophils

endothelial stimulant) of inflammatory stimulus, we decided to confirm that neutrophil agonists could indeed elicit transcytotic migration in vivo as reported by other^.^^,^^ Using ddY mice, Hoshi and colleagues reported that neutrophil emigration in the lip, in response to a local injection of fMLF, was preferentially (>80%) transcytotic. Unfortunately, C57BL/6 mice (the mice we use in our laboratory) are relatively insensitive to fMLF and, in our hands, fMLF failed to induce an inflammatory response when injected into the lip. However, another chemoattractant, MIP-2 (a murine ortholog of human IL-8), induced a rapid neutrophil specific inflammatory response in the lip. Using scanning electron microscopy, we examined neutrophil migration sites on the endothelium and found the majority (56%) of migrating neutrophils utilized a transcytotic route (Fig. 5); migration at tricellular corners and bicellular borders was 13% and 31%, respectively. This prompted us to determine whether MIP-2 would elicit neutrophil transcytotic migration behavior in vitro. When MIP-2 (10nM) was placed in the lower well of a transwell chamber, mouse neutrophil migration across mouse endothelium (derived from vena cava) was largely (>90%) paracellular. The reason why MIP-2 elicits preferential transcytotic migration in vim but not in vitvo is unknown and will require further study, However, it needs to be pointed out that transcytotic migration is not always the preferred pathway in vivo. In a separate rabbit lung model of streptococcal pneumonia (a more complex inflammatory setting involving endogenous chemokine and cytokine secretion), we found that 50% of migrating neutrophils cross at tricellular corners and 25% at bicellular borders; the remaining 25% utilize a transcytotic route.32Hence, tissue specific differences and stimulus specific differences may ultimately determine whether neutrophils utilize paracellular or transcytotic migration pathways. Whether the influence of neutrophils on endothelial permeability varies according to the nature of the migration pathway is unknown.

7. ENDOTHELIAL PERMEABILITY RESPONSES T O NEUTROPHIL TRANSENDOTHELIAL MIGRATION Numerous investigators have addressed the question of the influence of neutrophil transmigration on endothelial permeability, and a wide

Mechanisms of Neutrophil Migration

133

variety of discordant results exist, both in vivo and in vitro. Comparison of published studies is complicated in part by a variety of reported measures of ”permeability”; thus, we will present a brief overview of some basic aspects of permeability; a current comprehensive review of microvascular permeability is found in the work by Michel and Curry.141 In general terms, permeability reflects the ease with which substances (water and/or solutes) may be transported across a particular barrier. Transport of water across microvessels (typically capillaries and postcapillary venules) is a function of the hydrostatic and effective oncotic (or colloid-osmotic, a pressure gradient generated by plasma proteins) pressure gradients across microvessels.The magnitude of water movement at given pressure gradients depends on the hydraulic conductivity (L,), which reflects the net volume flux (crn3.s-’) per unit surface area (cm2) per unit pressure (cmH20).L, thus represents a quantitative measure of permeability with regards to water, and may be measured both in cultured endothelial cells and individual microvessels. Abundant experimental evidence demonstrates that L, is not a static parameter, but may be regulated actively by both physiologic and pathologic stimuli. In microvessels in vivo, L, is typically assessed by a technique described initially by Landis114 and modified by Michel et ~ 1 . This ’ ~ ~ technique involves cannulation and perfusion of individual microvessels in situ, and assessment of volume flux at known hydrostatic and oncotic pressures, thus providing a quantitative measure of the ease of volume flux. L, may also be measured across cultured endothelial cell monolayers; a current technique for this purpose was described by Sill et al., and was used in our work for the data shown in Fig. 8. Measurement of L, of cultured endothelial cell monolayers facilitates comparison of their permeability properties with that of single-perfused microvessels. For example, the control values of Lp as illustrated in Fig. 8 in human umbilical vein endothelial cells (HUVEC)were between 5.3 and 6.8 X lop7 cm-1.s-1.cmH20-1, of similar magnitude to control L, values of post-capillary venules of the rat mesentery (1.9 X lop7 cm-’.s-’.cm H20-1:172;2.43 x loT7 cm-1.s-’~cmH20-’:100).However, cultured endothelial monolayers in some published studies have control values of hydraulic conductivity 10- to 20-fold higher than that of microvessels in viv0,131,79,164raising doubts of the validity of those monolayers as

134

The Neutrophils

Aldltioii of PRS or tlwnnlrh

nuombin 111 l l

PBS

I

-+ Q

Addition of mILF 01’ ~ I L t F Pnm

4

I

-o-

rnILF tltILFi

Pnni

R

Fig. 8 Influence of thrombin (positive control) and fMLF-stimulated neutrophil adhesion on HUVEC hydraulic conductivity (L$. In the upper panel, the time course for HUVEC (Lp) during control conditions and in response to thrombin is shown. In the lower panel, the time course for HUVEC (Lp) during the addition of fMLF (10-7M) with or without neutrophils (neutrophil : endothelial cell ratio = 2 : 1) is shown.

representative of the barrier properties of microvessels in viva In excised, isolated organs, the ease of water transport may be quantified by assessment of capillary filtration coefficient (Kf) by a technique first described Kf is the product of L, and the surby Pappenheimer and S0to-Ri~era.l~~ face area available for exchange and thus, at a whole organ level, it represents a quantitative assessment of permeability with regards to volume

Mechanisms of Neutrophil Migration

135

flux. However, since the exchange surface area of an isolated whole organ is unknown, comparison of Kf to L, values measured in single microvessels is limited. Some published studies addressing changes in permeability use various techniques to quantify the rate of edema (or swelling) in a particular vascular bed, such as the rat paw. Although inflammatory increases in permeability (Lp) may certainly induce edema, the rate of edema formation is also a function of other factors independent of permeability, such as blood perfusion, microvascular pressures, interstitial pressures and lymphatic function. Therefore, isolated measures of rates of edema formation are, strictly speaking, not measures of permeability. With regards to transport of solutes, specifically macromolecules, quantitative measures involve assessment of diffusive permeability coefficients (I'd); or the macromolecular flux per unit surface area per concentration gradient in the absence of a pressure difference. In individual microvessels, "apparent" permeability coefficients (P,) are measured with techniques involving microvessel perfusion, such as those described by Huxley et Those coefficients are identified as P, and not Pd, since they are assessed in the presence of volume flux (and conceivably pressure-induced solute flux, or convective flux). By obtaining measures of P, at different hydrostatic pressures, investigators have calculated Pd in single-perfused micro vessel^.^^,'^^ In vitro, Pd may be quantified across cultured endothelial mono layer^,^^'^^^ again allowing comparisons of permeability properties with individual microvessels. Similarly, in whole organs, techniques are available for measuring the product of permeability and surface area (or PS p r o d u ~ t ~ As ~ , is ~ the ~~). case for Kf, comparison of PS values with measures of P, or Pd of individual microvessels or cultured endothelium is limited due to the unknown surface area. Transport of macromolecules across microvessels has been studied by many investigators by means of systemic injection of a specific tracer (typically a labeled macromolecule, such as albumin or similarly sized probe) and monitoring the rate of leakage out of the microvasculature. Some techniques monitor the rate of extravasation of labeled albumin into sites of injection of inflammatory a g o n i s t s , 8 ~and ~~~ others utilize intravital videomicroscopy to monitor the leakage of dye-labeled albumin or dextran upon exposure to the inflammatory

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s t i m ~ l i . " ~Increases ,'~~ in permeability (diffusive permeability coefficients) would enhance the rate of escape of these molecules, though changes in pressure gradients (with changes in pressure-drive macromolecular flux) or changes in lymphatic flow rates may also influence the tissue-to-vessel proportion of the labeled probes. Thus, assessment of macromolecular transport with these techniques (particularly without monitoring microvascular pressure) does not represent a quantitative measure of permeability, but a semi-quantitative assessment of leakage of macromolecules. One technique used frequently in cultured endothelial monolayers to assess permeability is the measurement of transendothelial electrical resistance (TEER) or impedance.28d0,61 Although these techniques represent a quantitative measure of barrier function of endothelial monolayers, comparison to measurements of water or macromolecular permeability of individual microvessels is not feasible, due to limitations in measuring TEER across microvessels. Discordant results on the influence of neutrophil transmigration on permeability have been reported in vivo and in vitro using several of the techniques described above. With regards to the in vivo studies, since the early observations by Cohnheim, numerous investigators have suggested a role for leukocytes in inflammatory changes in permeability.8f23,72,166 In a widely cited study, Wedmore and Williams monitored extravasation of radiolabeled albumin in rabbit skin in response to a variety of agonists such as C5a, fMLF, leukotriene B4 (LTB,), histamine, and bradykinin. Depletion of circulating leukocytes with nitrogen mustard abolished the increases in albumin extravasation induced by the chemotactic stimuli (C5a, FMLF, LTB,), though not those induced by either histamine or bradykinin. Similarly, depletion of neutrophils with anti-neutrophil antibodies has been reported to attenuate PS product to small hydrophilic solutes in a canine model of ischemia-reperfusion.206Leukocyte adhesion via p2 integrins appears necessary for the leukocyte-dependent macromolecular extravasation in response to chemotactic agents, as monoclonal antibodies against 62-integrins attenuate the response^.^,'^^ Similarly, monoclonal antibodies against P2-integrins, as well as those against its endothelial cell ligand ICAM-1, attenuated TNFa-induced increases in Kf in an isolated lung model perfused with neutrophils.lZ6Some

Mechanisms of Neutrophil Migration

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quantitative measures of permeability in single-perfused microvessels also support a direct influence of neutrophil activation on vascular permeability. In excised, single perfused porcine coronary venules, Yuan et reported that incubation of microvessels with C5a-activated neutrophils resulted in significant increases in apparent permeability coefficients to albumin. Further, inhibition of endothelial myosin light chain kinase (MLCK) attenuated the neutrophil-dependent responses, suggesting that MLCK activation mediates the increased permeability associated with neutrophil adhesion. The same group of investigators had reported previously that neutrophils augmented the increases in P, to albumin in response to platelet activating factor.87Similarly, He and colleagues reported that leukocyte adhesion induced by low shear resulted in a nearly 5-fold increase in L, of individual frog mesenteric ~ e n u l e s . ~ ~ Although adhesion to endothelium appears to be necessary for changes in macromolecular leakage, leukocyte adhesion in the absence of changes in macromolecular leakage has been reported in viv0.107~170 Using a cat mesenteric model, Kubes and colleagues described that while both platelet activating factor (PAF) and LTB4 induced similar leukocyte adhesion to venular endothelium, only PAF-induced adhesion was associated with vascular albumin extrava~ation.'~~ Despite similar leukocyte adhesion, leukocyte emigration and superoxide production were noted only in the presence of PAF. They proposed that leukocyte adhesion-dependent functions, such as transmigration and/or oxidant production accounted for the different responses. Zeng et al. have recently reported quantitative permeability measures of single-perfused post-capillary venules of the rat mesentery in the presence of leukocyte adhesion and emigration.232These authors noted that leukocyte adhesion induced by TNFa did not alter single-vessel hydraulic conductivity or apparent permeability coefficients to a-lactalbumin MW 15,000Da. Further, leukocyte emigration induced by fMLF in TNFa-treated rats occurred without altering hydraulic conductivity. These reports in vivo illustrate the complexity of the problem, with some studies demonstrating a clear association between leukocyte activation and inflammatory alterations in microvascular permeability and macromolecular leakage. On the other hand, other in vivo studies demonstrate clearly that leukocyte adhesion and transmigration may occur in the absence of changes in permeability. The reasons for these

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discordant findings as well as the mechanisms responsible for leukocytedependent permeability changes, when present, remain to be clarified. Similar to the case of the in vivo studies, discordant results exist in the published studies addressing the question of the influence of neutrophil adhesion and transmigration on endothelial permeability in vitro. Gautam and colleagues reported that fMLF- or LTB4-activatedneutrophil adhesion to cultured endothelial cells increased endothelial permeability to albumin and decreased TEER.61In that study, neutrophil adhesion (in the absence of transmigration) induced similar changes in permeability as neutrophil transmigration, demonstrating that transmigration was not required for the permeability changes. Further, neutrophil adhesion was associated with increased endothelial cell calcium [Ca2+lof similar magnitude as induced by histamine, an agent well known to increase endothelial permeability. The increases in permeability were dependent on neutrophil adhesion via P2-integrins, as the responses were inhibited by anti-CD18 monoclonal antibody treatment of neutrophils. Additionally, those authors reported that cross-linking of neutrophils with anti-CD18 antibodies resulted in the release of a cationic protein of -25-30 kDa, which increased endothelial [Ca2+]and permeability to albumin, as well as increased leakage of 150kDa dextran across postcapillary venules of the hamster cheek pouch in vivo.62The identity of the neutrophil-derived protein appears to be cationic antimicrobial protein 37 (CAP3763),a neutrophil granule protein which induced endothelial [Ca2+]-dependentcytoskeletal rearrangement, enhanced endothelial permeability and enhanced macromolecular leakage in vivo. Other published studies both support153,168,213 and r e f ~ t ae role ~ ~for ~ neutrophil ~ ~ activation in increasing cultured endothelial cell permeability. Of interest, Rosengren et al. used similar techniques as those described by Gautam et al. and reported that neutrophil-dependent increases in permeability to albumin in response to LTB4 required neutrophil transmigration; adhesion alone did not alter cultured endothelial cell permeability.168Additionally, they performed experiments in vim,applying LTB4to the lumen of microvessels with a micropipette or to the albuminal compartment. Both methods induced significant leukocyte adhesion to post-capillary venules, but only albuminal application of LTB4 (which induced transmigration) resulted in enhanced leakage of

Mechanisms of Neutrophil Migration

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150kDa dextran in hamster cheek pouch in vim. In HUVEC under flow, Su et ~ 1 demonstrated . ~ ~ that ~ neutrophil transmigration, but not rolling nor adhesion, led to increased endothelial [Ca2+]in endothelial cells adjacent to the transmigration site. In uiuo, temporal correlations between changes in endothelial calcium and microvascular L, have been described.76On the other hand, Huang et ~ 1reported . ~ that ~ transmigration of neutrophils induced by either fMLF or LTB4 did not alter either TEER or permeability to albumin of HUVEC. Similarly, data from our laboratory demonstrated that neutrophil transmigration across IL-1 Pstimulated HUVEC did not influence transendothelial electrical impedFigure 9 illustrates data from our laboratory demonstrating that adhesion and/or transmigration of neutrophils across resting HUVEC (which occurs through tricellular corners) did not influence diffusive permeability coefficients to either albumin or 4 kDa dextran. In addition, we assessed the influence of fMLF-stimulated neutrophil adhesion on

fMLF(10”M)

4kd BSA Dextran

+

PMNEC (4:l)

4kd BSA Dextran

I I THROMBIN (1 Wml)

4kd BSA Dextran

Fig. 9 Influence of fMLF-stimulated neutrophil transendothelial migration on HUVEC permeability to bovine serum albumin (BSA) and 4,000 MW dextran, and response to thrombin as a positive control. Data are means -t SEM from 4-6 separate experiments performed in triplicate for each group. *: l’ < 0.05 compared to control value; NS = not significant.

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L,, using the techniques described by Sill et a1.lS4As shown in Fig. 8, fMLF-induced neutrophil adhesion did not influence HUVEC Lp' A limitation in the comparison of the available permeability data in vitvo was alluded to earlier, that some of the published studies are performed on endothelial monolayers with basal permeability more than one order of magnitude higher than of the microvessels in vivu. Alternatively, others fail to determine diffusive permeability coefficients or report only TEER values, thus precluding comparison of the basal permeability properties of the monolayers with that of the microvessels. In summary, numerous studies have shown that adhesion and transmigration of neutrophils is associated with changes in microvascular permeability. In those studies, P2-integrin-dependent adhesion appears necessary for the permeability changes. Neutrophil release of CAP37, oxidants, endothelial changes resulting from transmigration and endothelial MLCK activation, have been proposed as mediators of the neutrophildependent permeability increases. In contrast, numerous other studies demonstrate clearly that transmigration of neutrophils across endothelium (either in v i m or in vitro) can occur without changes in permeability. A satisfactory explanation of these discordant findings remains to be determined.

8. CONCLUDING REMARKS Despite our current understanding that adhesion molecules target neutrophils to sites of inflammation, many questions remain regarding their precise role in neutrophil migration across the endothelium. Moreover, there is controversy over the location of the site or pore through which the neutrophil penetrates the endothelium. In some instances, the neutrophil prefers a paracellular path as it migrates between endothelial cells while at other times a transcytotic route is favored as it penetrates the body of an endothelial cell. The consequences of these two very different migration pathways to microvascular permeability are entirely unknown. The challenge for future studies will be in the identification of the molecular determinants and signaling cascades that dictate whether neutrophil migration is paracellular or transcytotic.

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ACKNOWLEDGMENTS This study was supported by National Institutes of Health Grants AI46773, HL42550, HL070357, and HL64721.

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161. Piali L, Albelda SM, Baldwin HS, et al. Eur Jlmmunol1993; 23:2464-2471. 162. Picker L, Warnock R, Burns A, et al. IPublished erratum appears in Cell 1991 67(6):1267]. Cell 1991; 66921-933. 163. Pumphrey NJ, Taylor V, Freeman S, et al. FEBS Lett 1999; 450:77-83. 164. Qiao R, Siflinger-Birnboim A, Lum H, et al. Am J Physioll993; 265:C439-446. 165. Rainger GE, Fisher AC, Nash GB. Am J Pkysioll997; 272:H114-122. 166. Raud J, Lindbom L. In: lmmunopharmacology of the Microcirculation (ed. Brain SD). Academic Press, London, 1994; pp. 127-170. 167. Rosen H, Gordon S. J Exp Med 1987; 166:1685-1701. 168. Rosengren S, Olofsson AM, von Andrian UH, et al. J Appl Physiol 1991; 713322-1 330. 169. Rot A. Eur J Irnmunoll993; 23:303-306. 170. Rumbaut RE, Harris NR, Sial AJ, et al. Am J Physiol1999; 276:H333-339. 171. Rumbaut RE, Huxley VH. Microvasc Res 2002; 64:21-31. 172. Rumbaut RE, Wang J, Huxley VH. Am J Physiol Heart Circ Pkysiol 2000; 279:H2017-2023. 173. Saito H, Minamiya Y, Kitamura M, et al. Jlmmunol1998; 161:1533-1540. 174. Sako D, Comess KM, Barone KM, et al. Cell 1995; 83:323-331. 175. Santoso S, Sachs UJ, Kroll H, et al. J Exp Med 2002; 196:679491. 176. Schenkel AR, Mamdouh Z, Chen X, et al. Nut lmmunol2002; 3:143-150. 177. Schierbeek A. Antoni Van Leeuwenhoek on the Circulation of Blood. De Graaf, Nieuwkoop, p. 33.

178. Schneeberger EE, Lynch RD. In: Tight Junctions (ed. Cereijido M, Anderson J). CRC Press, Boca Raton, 2001; pp. 19-37. 179. Schnittler HJ. Basic Res Cardioll998; 93:30-39. 180. Shaw SK, Bamba PS, Perkins BN, Luscinskas FW. J lmmunol 2001; 167: 2323-2330. 181. Shaw SK, Perkins BN, Lim YC, et al. Am J Pathol 2001; 159:2281-2291.

182. Shimaoka M, Lu C, Palframan RT, et al. Proc Natl Acad Sci USA 2001; 98:6009-6014. 183. Siegelman M, van dRM, Weissman I. Science 1989; 243:1165-1172.

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184. Sill HW, Chang YS, Artman JR, et al. Am J Physiol 1995; 268:H535-543. 185. Simionescu N, Simionescu M, Palade GE. J Cell Biol 1978; 79:2744. 186. Simionescu N, Simionescu M, Palade GE. Microvasc Res 1978; 15:l-16. 187. Simon DI, Dhen Z, Seifert P, et al. J Clin Invest 2000; 105:293-300. 188. Simon SI, Burns AR, Taylor AD, et al. J Immunoll995; 155:1502-1514.

189. Simon SI, Hu Y, Vestweber D, Smith CW. Immunol2000; 1644348-4358. 190. Smith CW. Microcirculation 2000; 7:385-394. 191. Smith CW, Burns AR, Simon SI. In: Vascular Adhesion Molecules and Inflammation. (ed. Pearson JD). Birkhauser Verlag, Basel, 1999; pp. 37-62. 192. Smith CW, Hollers JC. J Clin Invest 1980; 65:804-812. 193. Smith CW, Marlin SD, Rothlein R, et al. J Clin Invest 1989; 83:2008-2017. 194. Smith MJ, Berg EL, Lawrence MB. Biophys 1999; 77:3371-3383. 195. Snapp KR, Ding H, Atkins K,et al. Blood 1998; 91:154-164. 196. Sperandio M, Forlow SB, Thatte J, et al. J lmmunol2001; 1672268-2274. 197. Spertini 0, Cordey AS, Monai N, et al. J Cell Bioll996; 135:523-531.

198. Springer TA. Cell 1994; 76:301-314. 199. Springer TA. Annu Rev Physiol1995; 573274372. 200. Steeber DA, Engel P, Miller AS, et al. JImmunol1997; 159952-963. 201. Stein B, Khew-Goodall Y, Gamble J, Vadas MA. Transmigration of Leukocytes. In: Endothelium in Clinical Practice: Source and Target of Novel Therapies (ed. Rubanyi GM, Dzau VJ). Marcel Dekker, Inc., New York, 1997; pp. 149-202. 202. Steinberg MS, McNutt PM. Curr Opin Cell Bioll999; 11:554-560. 203. Stuart RO, Nigam SK. Proc Natl Acad Sci USA 1995; 92:6072-6076. 204. Stuart RO, Sun A, Bush KT, Nigam SK. J Biol Chem 1996; 271:13636-13641. 205. Su WH, Chen HI, Huang JP, Jen CJ. Blood 2000; 96:3816-3822. 206. Svendsen JH, Hansen PR, Ali S, et af. Leucocyte depletion attenuates the early increase in myocardial capillary permeability to small hydrophilic solutes following ischaemia and reperfusion. Cardiovasc Res 1993; 27~1288-1294.

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207. Taichman NS, Young S, Cruchley AT, et al. J Leukoc Bioll997; 62:397-400. 208. Tanaka M, Brooks SE, Richard VJ, et al. Circulation 1993; 87:526-535. 209. Tanaka Y, Albelda SM, Horgan KJ, et al. J Exp Med 1992; 176:245-253. 210. Tang Q, Hendricks RL. J Exp Med 1996; 184:1435-1447. 211. Taooka Y, Chen J, Yednock T, Sheppard D. J Cell Bioll999; 145:413420. 212. Thompson RD, Noble KE, Larbi KY, et al. Blood 2001; 97:1854-1860. 213. Tinsley JH, Ustinova EE, Xu W, Yuan SY. Am J Physiol Cell Pkysiol 2002; 283C1745-1751. 214. Von Andrian UH, Hansel1 P, Chambers JD, et al. Am J Physiol 1992; 263:H1034-1044. 215. von Andrian UH, Hasslen SR, Nelson RD, et al. Cell 1995; 82:989-999. 216. von Haller A. In: Deux memoires sur Ie mouvement du sang, et SUY les effefsde la saignee; Fondes SUY des experiences faites SUY des animaux. M-M Bousquet, Paris, 1756; pp. 248-249. 217. Waddell TK, Fialkow L, Chan CK, et al. J Biol Ckem 1994; 269:18485-18491. 218. Waddell TK, Fialkow L, Chan CK, et al. J Biol Chem 1995; 270:15403-15411. 219. Walcheck €3, Moore KL, McEver RP, Kishimoto TK. J CIin Invest 1996; 98:1081-1087. 220. Walker DC, MacKenzie A, Hosford S. Microvascular Res 1994; 48:259-281. 221. Wang SZ, Smith PK, Lovejoy M, et al. Am J Pkysioll998; 275:L983-989. 222. Watt SM, Williamson J, Genevier H, et al. Blood 1993; 82:2649-2663. 223. Wedmore CV, Williams TJ. Nature 1981; 289:646-650. 224. Wilkins PP, Moore KL, McEver RP, Cummings RD. J Biol Ckem 1995; 270:22677-22680. 225. Williams LA, Martin-Padura I, Dejana E, et al. Mol Immunol 1999; 36:1175-1188. 226. Wolf MB. Am J Physioll994; 267H383-399. 227. Woodman RC, Johnston B, Hickey MJ, et a/. J Exp Med 1998; 188:2181-2186. 228. Wu HM, Huang Q, Yuan Y, Granger HJ. Am J Physioll996; 271:H2735-2739. 229. Yadav R, Larbi KY, Young RE, Nourshargh S. Thromb Haemost 2003; 90: 598-606.

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Neutrophils and Apoptosis Annernieke Walker, Carol Ward, Magdalena Martinez-Losa, Adriano G. Ross?+

Neutrophils are key effector cells involved in host defence against invading organisms such as bacteria and fungi. Their over-recruitment, uncontrolled activation and defective removal contribute to the initiation and propagation of many chronic inflammatory conditions. Neutrophil apoptosis is a physiological process that terminates the cells’ functional responsiveness and induces phenotypic changes that render them recognizable by phagocytes (e.g. macrophages). Evidence indicates that neutrophil apoptosis and the subsequent removal of these cells by macrophages occur via mechanisms that do not elicit an inflammatory response and that these processes are fundamental for the successful resolution of inflammation. The molecular mechanisms regulating apoptosis in neutrophils are being elucidated and consequently it is now believed that selective induction of neutrophil is a potential target for therapeutic intervention. Keywords: neutrophil; apoptosis; resolution; macrophage; clearance; caspase

*Correspondence to: Adriano G. Rossi. Centre for Inflammation Research, University of Edinburgh Medical School, Teviot Place, Edinburgh, EH8 9AG, UK; e-mail: [email protected].

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1. INTRODUCTION As highlighted in other chapters, the neutrophil is highly developed for its principal role of fighting invading organisms (e.g. bacteria or fungi). Thus, the neutrophil has evolved a great capacity to generate toxic oxygen metabolites (e.g. 02-,H202, OH-, NO) and an ability to liberate products (e.g. elastase and collagenase) located within different cytoplasmic granules that can destroy or render invading organisms imp0tent.l However, when this normally beneficial response becomes dysregulated, neutrophil-derived toxic products can cause severe tissue damage resulting in the development of chronic inflammatory scenarios. There is now good evidence showing that once invading organisms have been eliminated, neutrophils have to be removed by mechanisms that limit their capacity to cause tissue injury and allow resolution of the inflammatory process to occur. For example, mechanisms exist to reduce or stop neutrophil recruitment and activation, decrease the concentrations of proinflammatory stimuli and augment the generation of mediators with anti-inflammatory potential (e.g. IL-1 receptor antagonist, IL-10, TGF-P). However, it has become apparent that the key mechanisms involved in the successful resolution of inflammation are neutrophil apoptosis and the subsequent removal of these cells by phagocytes.2,3In this chapter, we will focus on these processes and describe the mechanisms controlling this previously under-investigated aspect of neutrophil biology.

2. NEUTROPHIL APOPTOSIS Although researchers have been aware of cell death, especially by necrosis, for many decades, it was only in the early seventies that apoptosis, or programmed cell death, was first formally described as a physiological and highly regulated form of cell death.4,5 Apoptosis has now been shown to be responsible for the physiological death of virtually all cells in every organ and is therefore believed to be important in all aspects of biology. Apoptosis also plays a fundamental role in regulating neutrophilmediated inflammation and inflammatory diseases, as well as in fundamental processes such as embryological morphogenesis and tissue remodeling.

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When cells, including neutrophils, become apoptotic, a chain of highly regulated molecular events occur that result in distinct structural, morphological and biochemical phenotypes. Additionally, apoptotic cells maintain plasma membrane integrity and retain their cytoplasmic granu l e ~ .This ~ , ~is especially important in the neutrophil, since death by necrosis would result in the release of histotoxic intracellular contents that can augment the inflammatory response. Thus, the intact membrane of apoptotic cells renders them still capable of excluding vital dyes such as trypan blue, an important consideration when attempting to evaluate apoptosis experimentally. Apoptotic neutrophils tend to be smaller but more vacuolated as a result of cytoplasmic changes and the typical multilobed nucleus observed in nonapoptotic neutrophils coalesces into one or sometimes several distinct visually disconnected nuclear lobes. The nuclear chromatin condenses into dense, crescent-shaped structures with the nucleolus becoming more prominent. Endogenous endonuclease activation is responsible for internucleosomal cleavage into characteristic DNA fragments of 180-200 base pairs of the chromatin. Like all cells, there are major cell surface changes that occur in neutrophils undergoing apoptosis. For example, the distribution of plasma membrane phospholipid changes dramatically. Phosphatidylserine normally located on the inner leaflet of the plasma membrane of nonapoptotic cells flips onto the external surface of apoptotic cells by the combined efforts of two enzymes (phospholipid scramblase and aminophospholipid translocase).6 Although membrane phosphatidylserine redistribution is a general phenomenon and has been utilized for assessing apoptosis, there are other cell surface changes restricted to particular cell types. The neutrophil, for example, sheds its surface FcyIUII (CD16) when undergoing a p o p t o ~ i s . ~ ~ ~ Importantly, as neutrophils become apoptotic their ability to respond to agonists (e.g. fMLP) is dramatically reduced so that they are no longer capable of undergoing chemotaxis and degranulation and their phagocytic abilities are impaired? Although the biochemical mechanisms responsible for this functional down-regulation remain ill defined, it is thought that loss of molecules important for activation (e.g. specific receptors), phagocytosis (CD16) and adhesion ( L - ~ e l e c t i n )as ~ ,well ~ ~ as changes in secretory pathways are i m p ~ r t a n t . ~It
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The Neutrophils

ability of phagocytes such as macrophages to recognize and phagocytose apoptotic cells from inflammatory sites using mechanisms that do not incite an inflammatory response (see below). Thus human neutrophils in vivo, or when isolated from blood, have a limited lifespan and are destined to die within hours of leaving the bone marrow. In vivo, it has been estimated that neutrophils have a half-life of approximately 6 hours and that neutrophils that leave the vasculature die by apoptosis within 48 hours once their useful inflammatory purposes have been fulfilled." Recently, a novel mechanism for the removal of senescent neutrophils from the circulation has been postulated. It has been demonstrated that aging neutrophils that remain in the circulation upregulate their CXCR4 expression and acquire an ability to migrate towards SDF-la, a process involved in prefer~ tissue culture ential neutrophil homing to the bone marrow in ~ i v o . 'In conditions, isolated human neutrophils undergo constitutive apoptosis with approximately 40-70% of the cells being apoptotic within 20 hours. By 40 hours, virtually the entire population exhibits characteristics of apoptosis. The rate at which these cells die can either be decreased or enhanced by specific treatments and therefore appears to be highly regulated.13J4

3. REGULATION OF NEUTROPHIL APOPTOSIS Since many of the characteristic features of apoptosis are similar in all cell types, there are many evolutionally conserved mechanisms responsible for many of these changes. However, it is apparent that control of the apoptotic process differs from one cell to another and can depend on the types of receptors expressed on the cell. Thus, the control of apoptosis can be highly specific in that neutrophil apoptosis can be regulated without influencing apoptosis of closely related inflammatory cells (e.g. eosinophils)8f13-'6 (Fig. 1).

3.1. Internal Control Mechanisms 3.1 .l.Caspases

Arguably the most important enzymes responsible for the cellular events occurring during apoptosis are the caspase (for cysteinyl wartate-specific p r o t e s ) family of enzymes. Since the initial discovery of caspase 1 (also

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Non-apoptotic neutrophil

Death receptor ligands (e.g., TNF-a GM-CSF, LPS,CSa, LTB,, PAF, IL-8, glucoeorlicoids) Signaling pathways (MAPK, P13K, NF-KB activation, [Ca-I,, [CAMPI,

Apoptotic neutrophil

1 Macrophage ingesting apoptotic neutrophils

Fig 1 Regulation of neutrophil apoptosis and their subsequent removal by macrophages. Once functionally competent non-apoptotic neutrophils have fulfilled their defense against invading organisms, they undergo constitutive apoptosis, a process that can be delayed or enhanced by a number of factors. The apoptotic neutrophil is then rapidly and efficiently ingested by neighboring tissue resident macrophages using specific recognition mechanisms that do not elicit an inflammatory response. Failure of this beneficial physiological process can lead to undesirable enhanced inflammation as apoptotic neutrophils can eventually undergo necrosis with subsequent liberation of their histotoxic contents.

called interleukin-1p-converting enzyme) there have been a total of at least 14 mammalian caspases (caspases 1-14) identified so far. Essentially, one group of caspases (collectively referred to as the caspase-1 subfamily of caspases and consisting of caspases-1, -4, -5,

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Ihe Neutrophils

-11, -12, 13, and -14) are primarily involved in inflammatory processes while the other group referred to as the CED-3 subfamily (consisting of caspases-2, -3, -6, -7, -8, -9, -10) are primarily involved in cell death proce~ses.'~-'~ More recently, caspases of both divisions have been identified in neutrophils, including caspases-1, -3, -4, -6, -7, -8, -9 and -14.20Of the caspases involved in apoptosis, there are two main types: initiator caspases (e.g. caspase-8, -9, -10) and effector caspases (e.g. caspases-3, -6 and -7). The initiator caspases possess long prodomains containing unique motifs, including death effector domains (DED) and CARDS (caspase recruitment domains), allowing association with adapter molecules involved in apoptosis (e.g. DED associates with adapter molecules such as Fas-associated death domain (FADD)).These prodomains also permit oligomerization, culminating in their activation by aut~catalysis.'~ It is these initiator caspases that leads to downstream activation of the effector caspases. The primary function of the effector caspases is the cleavage of key proteins that leads to the characteristic features of apoptotic cells.19 The effector caspases may also be activated by the formation of the "apoptosome," which consists of a complex of cytochrome c, ATP, apoptosis protease-activating factor (Apaf-1) and caspase-9. Apaf-1, like cytochrome c, is released from the inner mitochondria1membrane when an apoptotic signal is received (through the intrinsic or extrinsic pathway). Apoptosome formation allows the activation of caspase-9, which in turn cleaves and activates ~ a s p a s e - 3Although .~~ many caspase substrates have been identified, the number identified in neutrophils is relatively few. The most notable ones identified in neutrophils include PKC-6,22 lamin B, f ~ d r i and n~g ~e l s ~ l i n . ~ ~ 3.1.2. hitiation of apoptosis

Although there are many triggers that can initiate the apoptotic caspasecascade, two principal interrelated pathways exist for the initiation of the apoptotic cascade in all cells. In the neutrophil, apoptotic cell death occurs constitutively, as neutrophils will undergo spontaneous cell death when cultured in vitro, with rates of apoptosis at the time of isolation from the blood being extremely low (usually below 5% of the neutrophil

Neutrophils and Apoptosis

1 59

population being apoptotic), and by approximately 20 hours of culture, the rates of apoptosis can be in excess of 70%.25The precise role of caspases in constitutive apoptosis remains ill-defined, with some groups reporting that constitutive apoptosis proceeds by mechanisms independent of caspase a ~ t i v a t i o n Apoptosis . ~ ~ , ~ ~ of neutrophils can be accelerated or triggered by specific ligation of the so called ”death receptors” (e.g. Fas, TNFRl, Trail-Rl), or through the intrinsic death pathway in response to cellular injury or stress. Although ligation of Fas in neutrophils clearly induces apoptosis, the precise biochemical pathway regulating this death has not been fully defined in neutrophils.28From studies in other cell types, it is believed that Fas ligation results in receptor trimerization and death initiating signaling complex (DISC) formation through interaction of the receptor with its adapter protein FADD.29 Procaspase-8 is then recruited and is activated by autocatalysis. There is good evidence that this process is regulated by a competitive inhibitor of procaspase-8, named c-FLIP that acts by binding to the DISCz9Depending on the cell type, active caspase8 may cleave procaspase-3 directly (type I cells), or cleave the Bcl-2 family member Bid (type I1 cells), to form truncated Bid (tBid) which subsequently acts on the mitochondria to promote the release of cytochrome c.29 The category in which neutrophils fall is unknown but work by Watson and colleagues30 suggested that these cells may be type I1 cells. This group demonstrated that caspase-8 failed to activate procaspase-3 in the presence of the mitochondria1 stabilizing agent bonkrekic acid. The classical pro-inflammatory cytokine TNF-a, as well as promoting cytoskeletal rearrangement and enhancing neutrophil d e g r a d a t i o n responses, has been shown to influence the rate of neutrophil apoptosis. Careful examination of neutrophil apoptosis at early time points (4-8 hours) during culture, in the presence of TNF-a, revealed an enhanced rate of apoptosis, whereas at later time points (18-24 hours), apoptosis was delayed.31It appears that ligation of TNFRl by TNF results in interactions between TNF receptor-associated proteins, such as TRADD (TNF-receptor-associated death domain), FADD (Fas-associated death domain), and caspase 8 and may involve Fas (CD95) and sphingomyelin-ceramide signaling pathways. It has also been demonstrated in

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The Neutrophils

neutrophils that TNF inducing ligand (TRAIL) can also induce apoptosis by interacting with TRAIL receptor^.^^.^^ Although the pathways triggered by ligation of the TRAIL receptors have been partially defined in other cells, they still have to be elucidated in neutrophils.

3.1.3. Mitochondria and Bcl-2 family proteins The central role of cytochrome c in apoptosis is now well established, but due to the scarcity and morphological irregularity of neutrophil mitochondria and the inability of groups to detect this molecule in neut r o p h i l ~ , 2 ~ there ,~~-~ has ~ been some debate concerning the role of cytochrome c in these ~ e l l s .However, ~ ~ , ~ ~we36and more recently Murphy and colleagues20have shown that while cytochrome c still remained illusive at the protein level, cytochrome c is, however, necessary to trigger apoptosis. Further evidence for the role of mitochondria in neutrophils came from work by Altznauer, et al. (2003),37 who showed that Smac/ DIABLO release from mitochondria could be inhibited by blocking calpain activity during Fas-induced and spontaneous apoptosis. Smac/ DIABLO blocks the activity of inhibitor of apoptosis proteins (IAPs) present in the cytoplasm, which in some cases bind directly to caspases, inhibiting their activity3* The Bcl-2 family proteins have a major role in regulating neutrophil apoptosis. Interestingly, however, there appears to be no Bcl-2 in neutrophilsZ8J9despite the presence of other main Bcl-2 family members, such as Bcl-XL, Mcl-1, Al, Bax, Bad and Bid, being identified in these cells8 Bax is arguably the most studied pro-apoptotic Bcl-2 member in mature neutrophils. During apoptosis, Bax translocates from the cytoplasm to the mitochondria and this occurs independently of caspases, since incubation of cells with z-VAD-fmk in the presence of an apoptosisinducing agent does not prevent transl~cation.~~ In the absence of prosurvival signals, neutrophils appear to be primed for apoptosis and this correlates with the Bcl-2 family member profiles. The pro-apoptotic Bcl-2 family members Bax, Bad and Bak are constitutively expressed in neutrophil~,4~,~' whereas the anti-apoptotic members A1 and Mcl-1 are only maintained or increased by inflammatory mediators that delay neutrophil a p o p t ~ s i s . ~ ~ " ~

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3.2. Regulation of Neutrophil Apoptosis by External Mediators Many of the central apoptotic pathways are common to all cells, but discrete differences in their control and implementation allow a window for therapeutically targeting specific cell types. Neutrophils undergo constitutive apoptosis in uituo,but this may be delayed by inflammatory mediators. For example, GM-CSF, G-CSF, LPS, LTB4, PAF, C5a, IL-1p, IL-2, IL-8, TNF-a, IL-15, and IFN-y13J4J6,47,4s can promote neutrophil survival, suggesting that while neutrophils are on balance primed to die, activation of certain critical survival pathways can overcome this "default" state. Thus at inflammatory sites, neutrophils exposed to such mediators would survive longer to aid their defense against invading organisms. Much progress has been made in understanding the signaling mechanisms likely to regulate this process and it appears that these control mechanisms differ somewhat from many other cell types. For example, elevation of intracellular free Ca2+ levels using calcium ionophores (e.g. A23187 and ionomycin) or by mobilization of intracellular Ca2+stores using t h a p ~ i g a r g i n promotes ~t~~ neutrophil longevity, whereas similar treatment in other cells (e.g. eosinophils) induces apopt0sis.4~Similarly, unlike most cell types, increasing intracellular cyclic AMP (CAMP)also delays neutrophil a p o p t o ~ iand s ~ ~seems to be mediated through a mechanism involving altering the balance of pro-survival versus pro-apoptotic proteins and not via protein kinase A (PKA), PI-3 kinase (PI3K), MAPK or mechanisms dependent on t r a n s c r i p t i ~ nThe . ~ ~ control of neutrophil apoptosis is likely to be a reflection of the neutrophil's specialized immune purpose and the environmental milieu in which it must function. For example, a hypoxic environment, found in chronically inflamed sites, delays neutrophil a p o p t o s i ~ , 2 whereas ~ ~ ~ ~hypoxia ~~ induces apoptosis in most other cells types.54Not surprisingly, bacterial lipopolysaccharide (LPS) delays neutrophil a p o p t ~ s i s , ' ~an. ~event ~ that can be enhanced by low levels (<1%)of contaminating monocytes in neutrophil preparation^.^^ Activation of several signal transduction pathways has been identified in LPS-treated neutrophils through the LBP/CD14/MD-2 and Toll-like receptors (TLR4)57triggering MAPK (ERK 1/2 and ~ 3 8 ) : ~ P13K59 and NF-KB signaling.60 Thus the precise involvement of the

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The Neutrophils

signaling pathways appears to depend on the specific survival factor under investigation. Consequently, many signaling pathways have been implicated in the regulation of neutrophil apoptosis, including protein kinase C, MAP kinases, PDK and tyrosine kinases as well as transcriptional pathway^.^^,^^,^^ Furthermore, the likely cross talk between these pathways in neutrophils still remains to be elucidated. Agents used clinically for the treatment of inflammatory disease can also influence neutrophil apoptosis. For example, glucocorticoids, as well as influencing inflammatory cell recruitment and activation, and inflammatory mediator generation can delay neutrophil apoptosis but promote eosinophil and lymphocyte apoptosis.61This effect may explain why steroid therapy is so efficacious in eosinophilic inflammation, such as asthma, but not in chronic obstructive pulmonary disease (COPD), where inflammation appears to be neutrophil driven. Although the mechanisms underlying the divergent effect of glucocorticoids on inflammatory cell apoptosis are currently unknown, it has been suggested that glucocorticoids may differentially influence cytosolic Ca2+concentrations in these cells,62or that the ratios of glucocorticoid receptor isoforms may be different in neutrophils as compared to other cells.63 Other agents which have been shown to induce neutrophil apoptosis, include anti-Fas antibodies/Fas-L, TNF and nitric oxide. It is clear that activation of the Fas pathway promotes neutrophil apoptosis, but whether Fas-L/Fas pathway plays a role in neutrophil apoptosis in vivo remains contr~versial.~~ Nitric oxide (NO), which has powerful vasodilatory effects, also influences neutrophil apoptosis. Recently, we and others have shown that NO donating compounds promote neutrophil apoptosis.65-67Although the mechanisms involved have yet to be fully elucidated, it appears that NO exerts its effects via a cGMP-independent mechanism that involves the simultaneous release of oxygen free radic a l ~Oxidative .~~ dependent mechanisms have also been implicated in neutrophil apoptosis following adhesion molecule-dependent phagocytosis of bacteria or by certain bacterial p r o d ~ c t s , ~and * - ~in ~ apoptosis induced by factors liberated by bacteria71or fungi.14 NF-KBis activated in response to a wide variety of agents in the neutrophil and appears to be a central "switch in determining life or death.60 Perhaps the best example of the role of NF-KBin neutrophil survival is

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during TNF signaling. As mentioned above, TNF enhances neutrophil apoptosis at early time points (-6 h), but at later times (20 h) promotes neutrophil survival.31 This early apoptotic effect was dramatically increased if neutrophils were co-incubated with the fungal metabolite gliotoxin, a reported powerful inhibitor of NF-KB activation.60Subsequently, it was shown that TNF activates NF-KB,limiting early apoptosis and promoting survival of neutrophils at later time points. NF-KBactivation results in the transcription of anti-apoptotic genes necessary if neutrophil longevity is to be prolonged. This is confirmed by incubation of neutrophils with protein synthesis inhibitors in the presence of NF-KBactivating agents, since neutrophil apoptosis still occurs readily. NF-KBtargeting may therefore provide a powerful therapeutic target for the treatment of neutrophil-mediated disease. In line with this, recent studies indicate that NF-KBmay play a role in the apoptotic effects of certain arachidonic acid metabolites. The prostaglandin D2 (PGD2) metabolites AI2PGJ2 and 15dPGJ2 are powerful inducers of apoptosis in neutrophils. AI2PGJ2and 15dPGJ2are PPAR-y agonists, yet in the neutrophil, synthetic PPAR-y and PPAR-a ligands could not replicate the effects nor could they be blocked PPAR-y antagonists. LPS and TNF-a mediated longevity is readily overcome by A12PGJ2and 15dPGJ2,and this is mediated by inhibiting IKBdegradation and therefore NF-KBa~tivation.4~ In the first phase of inflammation, NF-KB activation causes the production of COX-2, an enzyme which controls the formation of PGs, including PGD2 and its metabolites. During the resolution phase, these metabolites can inhibit the activation of NF-KB,production of pro-inflammatory cytokines and ultimately inducing neutrophil apoptosis. Therefore, emerging data suggest that both NF-KBactivation and the formation of PGD2 metabolites may promote the resolution of inflammation by influencing inflammatory cell (including neutrophil) a p o p t ~ s i s . ~ ~ , ~ ' ~ ~

4. CLEARANCE OF APOPTOTIC NEUTROPHILS Once the beneficial function of recruited neutrophils into inflammatory sites has been fulfilled, it is evident that failure of neutrophil removal could lead to tissue damage as these cells would inevitably undergo disintegration and liberate their histotoxic contents. Thus, mechanisms have

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The Neutrophils

evolved to efficiently remove these cells in order to limit potential tissue damage and development of chronic inflammation. A key mechanism for limiting potential neutrophil mediated damage is rapid, efficient and noninflammatory recognition and phagocytosis by macrophages of neutrophils that have undergone apoptosis (Fig. 1).Indeed, it is believed that failure of phagocytes such as the macrophage to scavenge apoptotic cells, may be directly responsible, in part, for the pathogenesis and progression of inflammatory conditions. There is now good evidence in in vitm, experimental inflammatory models and human disease that clearance of apoptotic neutrophils occurs by this process. It was Newman, et al. (1982)” who first reported that aged senescent, but not fresh, isolated human neutrophils were recognized and ingested by inflammatory macrophages. It took several years before it was realized that the process of apoptosis had to have occurred before the neutrophils could be engulfed by macro phage^.^^ Importantly, it was subsequently shown that macrophage ingestion of apoptotic neutrophils does not result in the liberation of histotoxic neutrophil granule contents and generation of macrophage-derived pro-inflammatory mediators.79Indeed, this highly efficient and specific process results in the generation and release of macrophage-derived mediators with potential anti-inflammatory properties (e.g. TGF-a and IL-10).80-82It has become apparent that there exist a number of molecular mechanisms by which phagocytes recognize and ingest apoptotic n e ~ t r o p h i l s . ~ ~ - ~ ~

5. CONCLUDING REMARKS Due to the potential for the development of novel therapeutic targets afforded by specifically inducing neutrophil apoptosis and augmenting macrophage clearance of apoptotic neutrophils, a great deal of effort is being invested in deciphering the precise molecular mechanisms underlying these processes. We believe that the fruits of these efforts will lead to a greater appreciation of the physiological mechanisms involved in the resolution of inflammation and help identify novel therapies aimed at influencing neutrophil apoptosis with the ultimate goal of treating chronic inflammatory diseases where the neutrophil plays a prominent role.

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11. Cannistra SA, Griffin JD. Semin Hematoll988; 25:173-188. 12. Martin C, Burdon PC, Bridger G, et al. Immunity 2003; 19:583-593. 13. Walker A, Ward C, Dransfield I, et al. Curr Drug Targets Infzamm Allergy 2003; 2:339-347.

14. Ward C, Dransfield I, Chilvers ER, et al. Trends Pharmacol Sci 1999; 20:503-509. 15. Walsh GM. Br J Haematol2000; 111:61-67. 16. Simon HU. Immunol Rev 2003; 193:lOl-110. 17. Kumar S. Cell Death Difer 1999; 61060-1066.

18. Leist M, Jaattela M. Nut Rev Mol Cell Biol2001; 2:589-598. 19. Nicholson DW. Cell Death Difer 1999; 6:1028-1042. 20. Murphy BM, ONeill AJ, Adrain C, et al. J Exp Med 2003; 197:625432. 21. Hengartner MO. Nature 2000; 407770-776.

22. Pongracz J, Webb P, Wang K, et al. J Biol Chem 1999; 274:37329-37334. 23. Sanghavi DM, Thelen M, Thornberry NA, et al. FEBS Lett 1998; 422:179-184. 24. Kothakota S, Azuma T, Reinhard C, et al. Science 1997; 278:294-298.

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25. Savill JS, Wyllie AH, Henson JE, et al. J Clin Invest 1989; 83:865-875. 26. Mecklenburgh KI, Walmsley SR, Cowburn AS, et al. Blood 2002; 100: 3008-3016. 27. Harter L, Keel M, Hentze H, et al. J Trauma 2001; 50:982-988. 28. Iwai K, Miyawaki T, Takizawa T, et al. Blood 1994; 84:1201-1208. 29. Tibbetts MD, Zheng L, Lenardo MJ. Nat Immunol2003; 4:404409. 30. Watson RW, ONeill A, Brannigen AE, et al. FEBS Lett 1999; 453:67-71. 31. Murray J, Barbara JA, Dunkley SA, et al. Blood 1997; 90:2772-2783.

32. Daigle I, Simon HU. Swiss Med wkly 2001; 131:231-237. 33. Renshaw SA, Parmar JS, Singleton V, et al. J Immunol2003; 170:1027-1033. 34. Fossati G, Moulding DA, Spiller DG, et al. J Immunol2003; 170:1964-1972.

35. Maianski NA, Mu1 FP, van Buul JD, et al. Blood 2002; 99:672-679. 36. Pryde JG, Walker A, Rossi AG, et al. J Biol Chem 2000; 275:33574-33584.

37. Altznauer F, Conus S, Cavalli A, et al. Biol Chem 2004; 279:5947-5957. 38. Salvesen GS, Duckett CS. Nut Rev Mol Cell Biol2002; 3:401410. 39. Lagasse E, Weissman IL. J Exp Med 1994; 179:1047-1052. 40. Moulding DA, Akgul C, Derouet M, et al. J Leukoc Biol2001; 70:783-792. 41. Cowburn AS, Cadwallader KA, Reed BJ, et al. Blood 2002; 100:2607-2616. 42. Chuang PI, Yee E, Karsan A, et al. Biochem Biophys Res Commun 1998; 249: 361-365. 43. Epling-Burnette PK, Zhong B, Bai F, et al. J Immunol2001; 166:7486-7495. 44. Fulop T Jr, Larbi A, Linteau A, et al. Ann NY Acad Sci 2002; 973:305-308. 45. Moulding DA, Quayle JA, Hart CA, Edwards SW. Blood 1998; 92:2495-2502. 46. Wang JM, Chao JR, Chen W, et al. Mol Cell Bioll999; 19:6195-6206. 47. Ward C, Dransfield I, Murray J, et al. J Immunol2002; 168:6232-6243. 48. Edwards SW, Moulding DA, Derouet M, Moots RJ. Chem Immunol Allergy 2003; 83:204-224. 49. Cousin JM, Haslett C, Rossi AG. Biochem SOCTrans 1997; 25:243S. 50. Rossi AG, Cousin JM, Dransfield I, et al. Biochem Biophys Res Commun 1995; 217892-899.

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51. Martin MC, Dransfield I, Haslett C, Rossi AG. J Biol Chem 2001; 276:45041-45050. 52. Hannah S, Mecklenburgh K, Rahman I, et al. FEBS Lett 1995; 372233-237.

53. Murray J, Walmsley SR, Mecklenburgh KI, et al. Ann NY Acad Sci 2003; 1010:417-425. 54. Riva C, Chauvin C, Pison C, Leverve X. Anticancer Res 1998; 1847294736. 55. Lee A, Whyte MK, Haslett C. J Leukoc Biol 1993; 54:283-288. 56. Sabroe I, Jones EC, Usher LR, et al. JImmunol2002; 168:47014710. 57. Sabroe I, Prince LR, Jones EC, et al. J lmmunol2003; 170:5268-5275. 58. Nolan B, Duffy A, Paquin L, et al. Surgery 1999; 126:406-412. 59. Klein JB, Buridi A, Coxon PY, et al. Cell Signal 2001; 13:335-343. 60. Ward C, Chilvers ER, Lawson MF, et al. J Biol Chem 1999; 274:43094318. 61. Meagher LC, Cousin JM, Seckl JR, Haslett C. J Immunoll996; 156:44224428. 62. Distelhorst CW. Cell Death Differ 2002; 9:6-19. 63. Strickland I, Kisich K, Hauk PJ, et al. J Exp Med 2001; 193:585-593. 64. Ottonello L, Tortolina G, Amelotti M, Dallegri F. J Immunoll999; 162: 36013606, 65. Ward C, Wong TH, Murray J, et a/. Biochem Pkarmncol2000; 59:305-314. 66. Taylor EL, Megson IL, Haslett C, Rossi AG. Biockem Biophys Res Commun 2001; 289: 1229-1 236. 67. Taylor EL, Megson IL, Haslett C, Rossi AG. Cell Death Differ2003; 10:418-430. 68. Coxon A, Rieu P, Barkalow FJ, et al. Immunity 1996; 5653-666, 69. Kagan VE, Borisenko GG, Serinkan BF, et a/. Am J Physiol Lung Cell Mol Pkysiol2003; 285:L1-17. 70. Watson RW. Antioxid Redox Signal 2002; 497-104. 71. Usher LR, Lawson RA, Geary I, et al. J Immunol2002; 168:1861-1868. 72. Lawrence T, Willoughby DA, Gilroy DW. Nat Rev Immunol2002; 2:787-795. 73. Lawrence T, Gilroy DW, Colville-Nash PR, Willoughby DA. Nut Med 2001; 7:1291-1 297. 74. Gilroy DW, Colville-Nash PR. J Mol Med 2000; 78:121-129. 75. Gilroy DW, Colville-Nash PR, McMaster S, et al. FASEB J 2003; 172269-2271.

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76. Colville-Nash PR, Gilroy DW. Drug News Perspect 2000; 13:587-597. 77. Newman SL, Henson JE, Henson I'M. J E x p Med 1982; 156:430-442. 78. Savill JS, Wyllie AH, Henson JE, et al. J Clin Invest 1989; 83:865-875. 79. Meagher LC, Savill JS, Baker A, et al. J Leukoc Bioll992; 52269-273. 80. Fadok VA, Bratton DL, Konowal A, et al. J Clin Invest 1998; 101:89&898. 81. Voll RE, Herrmann M, Roth EA, et al. Nature 1997; 390:350-351. 82. Liu Y, Cousin JM, Hughes J, et al. J Immunoll999; 16236393646. 83. Giles KM, Ross K, Rossi AG, et al. J Imrnunol2001; 167976-986. 84. Savill J, Fadok V. Nature 2000; 407784-788.

85. Savill J, Dransfield I, Gregory C, Haslett C. Nut Rev Immunol2002; 2965-975.

6

Regulation of Neutrophil Functions by Long Chain Fatty Acids Antonio Ferrante," Charles S. Hii, Maurizio Costabile

Long chain fatty acids such as arachidonic acid regulate key neutrophil functional responses, either directly as free fatty acids or through the generation of biologically active metabolites such as leukotrienes. The metabolites of arachidonic can either promote or suppress neutrophil responses. Alteration in specific structural elements on long chain fatty acids modifies their biological properties, giving rise to a network of lipid mediators which regulate the inflammatory reaction. These fatty acids activate several intracellular signaling molecules, including protein kinase c, mitogen activated protein kinases, phospholipase A2 and phosphatidyinositol3 kinase which are relevant to the stimulation of the cellular inflammatory response. Keywords: long chain fatty acids; neutrophils; inflammation intracellular signaling; arachidonic acid; leukotrienes

*Correspondence to: Antonio Ferrante. Department of Immunopathology, The Women's and Children's Hospital, University of Adelaide, 72, King William Road, North Adelaide, SA 5006, Australia; phone: 61 8 81617216; fax: 61 8 81616046; e-mail: [email protected].

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The Neutrophils

1. INTRODUCTION Neutrophils are known to play an important role in protecting against early invasion of tissues by bacteria and other pathogens. Experiments of nature are informative in this manner. Babies born with hereditary forms of neutropenia can die within a few days unless treated with antibiotics. In contrast, a drastic reduction of and dysfunction in T lymphocytes and B lymphocytes do not pose the same level of risk. Even where there are specific defects of the neutrophil functions, e.g. in chronic granulomatous disease, where there is an inability to generate oxygen radicals, life threatening infections occur. There are several key neutrophil functions, which cooperate to contain and digest the invading microorganism. Marginated neutrophils transiently bind to the endothelium at sites close to the infection; they then roll and undergo transendothelial migration under the influence of a chemotactic gradient and accumulate at these sites of infection. Neutrophils then take on the active role of adhering to bacteria, phagocytosing the microorganism and releasing oxygen-derived species and lysosomal enzymes to kill and digest the bacteria. Apart from this well-recognized beneficial effect, this cell is also known to be involved in causing serious damage to tissues when inflammation persists. Besides their role in acute inflammation, recent evidence highlights the neutrophil as playing a significant role in chronic inflammation. This is particularly evident in rheumatoid arthritis, where neutrophils become prominent during the exacerbated episodes of the disease. In addition, they may influence the establishment of a chronic inflammatory response (cell-mediated immunity and resistance to intracellular parasites). Thus, the cell may influence macrophage and T lymphocyte responses, through the release of mediators such as cytokines. Many of these cytokines act through stimulation of the activity of phospholipase A2 and the release of arachidonic acid (AA) (Fig. 1). Recent evidence has shown that polyunsaturated fatty acids influence the process of neutrophil adhesion, chemotaxis, functional cell surface receptor expression, respiratory burst, degranulation and microbial killing. From a mechanistic perspective, mediators may act to not only induce each other’s activity, but act synergistically on neutrophils to maximise responses.

Regulation of Neutrophil Functions by Fatty Acids

171

Immune complexes, Cytokine release Activation complement

- thromboxane Neutrophil activation - neutrophil recruitment - aggregation - adherence - degranulation - oxygen radical production - cytokines Tissue damage

Fig. 1 A schematic representation of the mediator network operating during an infection or autoimmune inflammation. Neutrophils become stimulated by both exogenous and endogenous mediators which include fatty acids (e.g. AA) and their metabolic products. These mediators attract neutrophils to sites of infection or tissue damage and stimulate their antimicrobial and tissue damaging properties. Dotted line shows negative regulation. PLA2:phospolipase A2

2. FATTY ACIDS Fatty acids are characterized by an alkyl chain and a carboxyl group with the basic formula: CH3-(CH2),-COOH.The degree of unsaturation in the molecule is determined by the number of double bonds in the fatty acid

_.--

-

hnrkhnnp Nnrmallv thP are --. in a ris mnfimiratinn and sen*._.*.-^__.-_*I, --.-dniihle hnndq ---.-- -----.-0 - - - - - - - - --.- -r I-_

---I--

1-1

-

arated by a methylene group (-CH2-). The positions of the double bonds _.. . . are numbered from the carboxyl group, with the carboxyl carbon atom as carbon 1. The n-3 polyunsaturated fatty acids have their first double bond between the 3rd and 4th carbon atom, counting from the o or methyl end of the chain; while the n-6 polyunsaturated fatty acids have their first double bond between the 6th and 7th carbon atom. Based on the number of carbon atoms in the fatty acid backbone, the fatty acids are divided

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into short chain (<6 carbon atoms); medium chain (6-12 carbon atoms); long chain (14-22 carbon atoms); and very long chain (>22 carbon atoms) fatty acids. The approved abbreviation of fatty acids involves firstly, "the number of carbon atoms," followed by "the number of double bonds"; for example, 20:4 refers to a 20 carbon fatty acid with 4 double bonds. Fatty acids in the body can be obtained through de novo synthesis in tissues, through the diet or from the hydrolysis of membrane phospholipids.

2.1. De novo Synthesis Human beings can synthesize fatty acids up to 16:O (palmitate)de novu from acetyl coenzyme A by a series of cycles of sequential condensation, reduction, dehydration and reduction. The chain is elongated by two carbon atoms per cycle. 16:O is then elongated to 18:O (stearate) and desaturated to yield 18:ln-9 (oleate). Alternatively, 16:O is desaturated to 16:ln-9 (palmitoleate) and elongated to 18:ln-9. A variety of longer chain fatty acids can be derived from 18:ln-9 by a combination of elongation and desaturation reactions. However, mammalian cells are unable to perform these reactions because they do not express the enzymes, A12 and A15 desaturases to introduce double bonds at carbon atoms beyond (2-9. Consequently,mammalian cells cannot synthesize 18:2n-6 (linoleate) and 18:3n-3 (linolenate). These fatty acids, required by the animal but cannot be synthesized endogenously, are therefore considered as essential fatty acids and are obtained from the diet. The essential fatty acids serve as starting points for the synthesis of longer chain fatty acids, such as 20:4n-6, and the n-3 fatty acids 20:5n-3 (eicosapentaenoic acid, EPA) and 22:6n-3 (docosahexaenoicacid, DHA).

2.2. Diet Dietary fatty acids can be obtained from animal meats, fish, green vegetables, and from oils derived from the above. They mainly occur as triacylglycerols. The 72-3 fatty acids EPA and DHA are abundant in marine oils, and fish-rich diets are another source of these fatty acids. Grain-fed animals are rich in AA.' Diets enriched in specific types of polyunsaturated fatty acids have been of interest because of their potential usefulness in treating a range of human diseases and conditions. In particular, it is well

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173

appreciated that increasing the ratio of n-3 over 72-6 polyunsaturated fatty acids in membrane phospholipids has some beneficial therapeutic effects.' Thus, fatty acid diet manipulations have been used in treating a wide variety of diseases/conditions.

2.3. Phospholipase A2 Stimulation of cells by agonists interacting with specific receptors, such as those for growth factors, thrombin, bradykinin or f-met-leu-phe (fMLP), leads to activation of phospholipases, e.g. phospholipase A2 (PLAJ and phospholipase C/diacylglycerol lipase, which results in the liberation of fatty acids from membrane phospholipids and diacylglycerol, respectively. Several types of PLA2have been described. Besides the well characterized Groups I, 11, I11 and IV, other forms of PLA2 are being recognized and characterizedS2The PLA2, which have been reported to participate in the generation of fatty acids from activated cells, include the calcium-dependent, 85 kDa cytosolic PLA2(cPLA,), and the secretory 14 kDa PLA2 (sPLA2),and a calcium-independent PLA2(iPLA2).2cPLA2, has a preference for AA in the sn-2 position of a phospholipid, while sPLAzand iPLA2do not. sPLA2,secreted from activated cells, and iPLA,, therefore releases AA and other fatty acids from membrane phospholipids. Studies in monocytic cells have suggested that activation of sPLA2 may depend on the transient activation of cPLA,.~n-3 fatty acids, such as 20:5n-3, esterified at the sn-2 position of a phospholipid, are released by c P L A ~Esterified .~ fatty acids can also be liberated from phospholipids/lysophospholipids via the action of phospholipase Al and phospholipase B, the latter possessing both phospholipase A1 and A2 activities. While phospholipase Al and A2 are generally thought to play major roles in phospholipid remodeling, there is some evidence that the activity of phospholipase Al may be regulated by receptor-mediated events: and hence may play a role in ligand-stimulated accumulation of non-esterified fatty acids. Nonesterified fatty acids, which are released by PLA2,have been found to be cell-associated as well as being released into the extracellular space. Consequently, nonesterified fatty acids, which are released from activated cells, can exert paracrine and autocrine effects.

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The Neutrophils

It is well documented that the levels of nonesterified fatty acids are elevated at sites of inflammation. Cell-types, which are likely to contribute to this pool of fatty acids, include monocytes/macrophages, platelets, endothelial cells, chondrocytes, fibroblasts, mast cells, neutrophils and b a ~ t e r i a . ~Although -I~ mature T and B lymphocytes have been reported not to express cPLA2 and that the T cell receptor/CD3 complex is not coupled to cPLA2?O there is some evidence that activated T cells do release 20:4n-6. This is likely to be due to the action of diacylglycerol lipase and/or a CD28-mediated signaling event.21,22 There is also a substantial amount of evidence that some of the above listed cell types also release sPLA2 upon a c t i v a t i ~ nIndeed, . ~ ~ ~the~ ~ ~ ~ plasma of septic shock patients has been found to contain sPLA2 (which is active on monocytic cells), E.coli and synthetic phosphatidy1ethanolamine.l6 Also, sPLA2 isolated from human platelets has been reported to cause the formation of LTB4 in human neut r o p h i l ~It. ~is~ now well recognized that synovial fluid contains high levels of S P L A ~Consequently, .~~ neutrophils which infiltrate into sites of infection or inflammation will be exposed to nonesterified fatty acids in addition to agents such as chemoattractants and bacterial products. The newly recruited cells will in turn release more fatty acids and eicosanoids as they become activated. Table 1 summarizes the range of neutrophil agonists which have been shown to stimulate the activity of either PLA2 and/or release of free 20:4n-6 in neutrophils. Cytokines are a major group of molecules in the network of mediators, which regulate physiological and pathophysiological processes. The cytokines, which have been shown to stimulate the release of 2Ck4n-6, include TNF, GM-CSF and IL-8 (Table 1). Other mediators with this activity include complements (C5a, complement coated zymosen, E . coli); agents such as aggregated IgG or IgG-coated zymosan which act on Fc-yR receptors; and classical neutrophil agonists, fMLP, PMA, Ca2+ionophore (Table 1). Eicosanoids also have the ability to stimulate the release of 20:4n-6 and these include LTB4,5-oxo-ETE and 5-HETE. In addition, many of the above mediators also prime neutrophils for enhanced activation of PLA2 in response to a second ligand.17~1s~29~36~38~41

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175

Table 1 Agonist Induced PLA, ActivationJAA Generation Agonist

Details

C5a

Ca2+independent PLA2 activity stimulated Release of sPLA2

Complement-coated zymosan Complement-coated E.coli

fMLP LXA4, LXB4 Ca2+/PAF

TNF

5-oxo-ETE LTB4 5-HETE GM-CSF AA

Reference

Activation of granuleassociated group I1 PLA~(sPLA~) Release of sPLA2 AA release by cPLA2 Release of AA Measured in Ca2+ depleted human neutrophils AA release LTB, synthesis Minimal AA release cPLA2phosphorylation Some AA release #LA2 phosphorylation Small amount of AA release Increase in cPLA2activity Small increase in cPLA2activity Phosphorylation of cPLA2 Increase in activity of #LA2 Release of AA

3. TRANSPORT A N D UPTAKE OF FATTY ACIDS How fatty acids are taken up by cells remains unclear. It has been proposed that fatty acids firstly become dissociated from albumin and then bind to a fatty acid transporter protein in the plasma membrane. A fatty acid translocase (FAT)with homology to CD36 has also been reported to be involved in the transport of long chain fatty acids?2 There is evidence that fatty acids can also enter the cells by a flip flop me~hanism.4~ These two modes of fatty acid uptake need not be mutually exclusive. Once inside the cell, the fatty

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The Neutrophils

acids are transported to various intracellular sites by the cytosolic fatty acid binding protein (FABP, 14-15 kDa), where they interact with appropriate proteins/structures to evoke cellular response^.^,^^ The precise mechanism by which the fatty acids are taken up by neutrophils is still poorly defined. However, it has been demonstrated that the ability of a fatty acid to partition into the neutrophil plasma membrane is not sufficient to evoke superoxide production?6 Similarly, the observation that saturated fatty acids (lacking biological actions) have a greater ability to partition into the plasma membrane of cytotoxic T lymphocytes than cis unsaturated fatty acids, is inconsistent with biological activity being totally caused by membrane partitioning of a fatty a ~ i d . 4 ~

4. METABOLISM OF ARACHlDONlC ACID A N D OTHER FATTY ACIDS 4.1. General Nonesterified AA is metabolized via a number of pathways, including the lipoxygenase and cyclooxygenase pathways. The products of AA metabolism via the cyclooxygenase pathway are the 2-series prostaglandins and thromboxanes. AA is first converted to PGH2,whichis followed by the formation of PGD2, PGE2,PGF2, and PG12. The types of prostaglandins formed in different tissues vary, depending on the type of prostaglandin synthases being expressed in tissues. For example, PG12 is mainly found in the blood; PGE2 and PGF2, are generated in the kidney and spleen; whereas PGE2, PGF2, and PG12 are synthesized in the heart. The other product of the cyclooxygenase pathway is TXA2, which is produced from PGH, by thromboxane A synthetase. TXA, is mainly synthesized in the lung and platelets. The generation of these eicosanoids is believed to play a major role in the inflammatory reaction in rheumatoid arthritis and psoriasis. The 2-series eicosanoids also increase sensitivity to pain, induce fever, platelet aggregation and thrombosis and act as vasodilators to lower the systemic arterial blood pressure.l The metabolism of AA via the lipoxygenase pathway is catalyzed by three monooxygenase, 5-, 12- and 15-lipoxygenases, which convert AA to either 5-, 12- or 15-monohydroperoxy-eicosatetraenoic acids (HPETE). These HPETE are the precursors of

Regulation of Neutrophil Functions by Fatty Acids

177

5-, 12- or 15-hydroxyeicosatetraenoicacids (HETE). Leukotrienes are another important group of eicosanoids generated by this pathway (Fig. 2). Among the metabolites of AA, LTB4 and 5-HETE stimulate neutrophil chemotaxis, degranulation, respiratory burst, adherence to endothelial cells and the transmigration of neutrophils across vascular barriers,48 (see section on "Regulation of neutrophil functions by metabolites of arachidonic acid"). The products also cause the contraction of smooth muscles in pulmonary airways and the gastrointestinal tract and in this manner promote inflammation and allergic reactions. These proinflammatory products of AA metabolism, along with other peptide inflammatory mediators, therefore form a network which modulates cell responses involved in various physiological response^.^^>^

PLA2

&

4

4

\

(4 series leukotrienes)

(5 series leukotrienes)

Fig. 2 Comparison of products formed from AA (n-6)and the n-3 fatty acids EPA and DHA when metabolized via the lipoxygenase and cyclooxygenase systems. HDoHEs: hydroxydocosahexaenoic acid; HEPE: hydroxyeicosapentaenoicacid.

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The Neutrophils

The n-3 fatty acids, EPA and DHA, can also be metabolized by the lipoxygenase pathway However, the cyclooxygenasepathway preferentially metabolizes EPA (Fig. 2). In the presence of n-3 fatty acids, lower amounts of AA metabolites are formed. The cyclooxygenasepathway gives rise to products with properties different from those of AA-derived metabolites. Metabolism of EPA and DHA by the lipoxygenase and/or cyclooxygenase pathways results in the generation of less active metabolites such as LTB5, TXB3, and 5-hydroxyeicosapentaenoic acid (5-HEPE)in the case of EPA, and a small amount of anti-inflammatory 7-hydroxydocosahexaenoic acid (7-HDoHEs) in the case of DHA in neutrophils and macr~phages.'s',~~ The switching of the metabolic product profile from one which is proinflammatory to one of lower or negligible pro-inflammatory activity by EPA and DHA has been proposed as a mechanism by which n-3 fatty acids exert their anti-inflammatory actions' (Fig.2).

4.2. Metabolism in Neutrophils It has been reported that neutrophils contain 100-2 200pmol/107 cells of AA.53In neutrophils, AA is metabolized mainly via 2 routes: (1)esterification into phospholipids or trigly~erides:~and (2) conversion to various eicosanoids by 5-lipo~ygenase.~~ However, neutrophils have also been reported to metabolize AA via the 12- and 15-lipoxygenases and cyclooxygenase,56albeit at very low levels compared with metabolism via 5-lipoxygenases. For example, stimulation of neutrophils with A23187 caused the formation of 3.51 t 0.22 ng of LTB4 and 0.81 ? 0.08 ng of LTC4/106 cells compared with 0.144 t 0.025ng of TXB2 and 0.15 ? 0.017ng of PGE2 ng/106 cells.57 In neutrophils, LTB4 and related products are metabolized by o-oxidation (see below). The 5-lipoxygenase of neutrophils as in other cell-types can metabolize EPA. Thus, bovine and human neutrophils have been reported to metabolize EPA to 5-hydroxyeicosapentaenoic acid (5-HEPE)57f58 and as discussed above, metabolize DHA to form 7-hydroxydocosahexaenoic acid.

4.2.1. Acylation info phospholipids and triglycerides Fatty acids such as AA, are converted to fatty acyl coenzymeA (FACoA)by fatty acid CoA synthetase. FACoA can then be transported into the inner

Regulation of Neutrophil Functions by Fatty Acids

179

membrane of mitochondria to undergo P-oxidation. Alternatively, they are incorporated into phospholipids, glycosphingolipids, triglycerides, and cholesteryl esters which are involved in membrane biosynthesis, membrane replacement or energy storage. The exchange of intracellular and extracellular fatty acids is a continuous process essential for normal tissue function. Our studies have demonstrated that fatty acids with different chain lengths are handled differently by neutrophils (see "Differences in metabolism of long chain and very long chain polyunsaturated fatty acids"). The incorporation of fatty acids into neutrophil phospholipids is regulated by a variety of ligands. LTBQhas been reported to increase the incorporation of 20:4n-6 into phosphatidylinositol and acyl and alkyl pho~phatidylcholine.~~ Incorporation of 20:4n-6 into phosphatidylinositol was greater than into pho~phatidylcholine.~~ There is some specificity in fatty acid incorporation into neutrophil phospholipids. For example, [3H] 20:4n-6 was found to be incorporated equally into both acyl and alkyl phosphatidylcholine at the sn-2 position.60In resting neutrophils, 20:4n-6 was the only fatty acid that was incorporated into I-0-alkyl-2-lyso-snglycero-3-phosphocholine (lyso-PAF).6118:2n-6 (linoleate), on the other hand, was not found in alkyl phosphatidylcholine.60Labeled saturated fatty acids were incoporated only into acyl phospholipids which contained 18:ln-9 or 18:2n-6 at the sn-2 position.60The specificity in 20:4n-6 incorporation may be lost upon neutrophil activation. Thus, in neutrophil stimulated with A23187, the incorporation of 20:4n-6 into lyso PAF to form alkyl phosphatidylcholine was attenuated by up to 80%.61A possible reason for the loss in specificity was found to be an accummulation of l-O-alk-l'-enyl-2-lyso-sn-glycero-3-phosphoethanolamine which competed with lyso PAE6INeutrophils also incorporate metabolites of 20:4n-6 into their phospholipids. For example, endogenously produced 5-HETE and exogenous 5-HETE are esterified into neutrophil phospholipids and triacylglycerol.62While endogenously generated 5-HETE was esterified equally into phospholipids and triacylglycerol, exogenous 5-HETE was esterified predominantly into triacylglycerol. Similarly, 12-HETE has been found to be esterified into neutrophil phospholipids and trigly~erides.~~ Neutrophils also elongate fatty acids. For example, GLA has been reported to be elongated to dihomo-gamma-LA (DGLA)by n e ~ t r o p h i l s . ~ ~ Other examples of chain elongation in n e ~ t r o p h i l sare ~ ~discussed below.

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The Neutrophils

4.2.2. 5-lipoxygenase In the neutrophils, there is some evidence that 5-lipoxygenase is secreted and the enzyme has been localized to the specific gran~les.6~ Products of the 5-lipoxygenase, which have been detected in neutrophils incubated with AA and A23187, include 5-HETE, 5-diHETE, 5-HPETE, 5-oxo-ETE, LTA4, LTC4, LTD4, LTB4 and LXA4. However, the conversion of AA to LTB4 can be modulated by other fatty acids. For example, linoleic acid (LA) and dihomogammalinoleic acid (DGLA) were found to block LTB4 formation and this was accompanied by the formation of 15-lipoxygenase products from LA (13-hydroxy-octadecadienoicacid; 13-HODE) and DGLA (15-hydroxy-eicosatrienoic acid; 15-HETrE).66Further studies revealed that 15-lipoxygenase products of LA, DGLA and AA directly blocked LTB4 production,66 possibly via inhibition of 5-lipoxygenase. Further studies demonstrated that the inhibitory effect of 15-HETE was dependent on the degree of unsaturation. Thus, analogues with different degrees of unsaturation showed inhibition in the order of 3 double bonds >4 double bonds >2 double bond >O double bonds.6715-HPETE was four-fold more effective than 15-HETE at inhibiting the 5-lipo~ygenase.6~ Diet supplementation with GLA or n-3 fatty acids in healthy volunteers has also been reported to cause a reduction in the ability of their neutrophils to produce LTB468r69i70 The effect of GLA could be due to the formation of DGLA from GLA by chain elongation, subsequently leading to inhibition of LTB4 synthesis by DGLA (see above). However, the effect observed with EPA has been reported to range from minor7* to dramatic70 Incubation of human neutrophils with EPA in vitro has also been found to inhibit LTB4 f~rmation:~an observation which was in agreement with data obtained with neutrophils from volunteers whose diets had been supplemented with n-3 fatty acids. Formation of 5-lipoxygenase products has been reported to be inhibited by pertussis toxin72 and by ibuprofen, an inhibitor of cyclooxygenase. The 5-lipoxygenase was found to be six times less sensitive to ibuprofen compared with cyclooxyg e n a ~ eExtracts .~~ from neutrophils, eosinophils and monocytes have recently been demonstrated to convert 5-HETE to 5-0x0-6, 8, 11, 14eicosatetraenoic acid (5-0x0-ETE) by a highly specific microsomal dehyd r o g e n a ~ e .The 7 ~ ~formation ~~ of 5-0x0-ETE has also been demonstrated in intact neutrophils and m o n ~ c y t e s .In ~ ~unstimulated ,~~ neutrophils, the

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181

level of 5-0x0-ETE is low and most of the 5-HETE is converted to 5-, 20diHETE.77t78Upon stimulation with phorbol 12-myristate 13-acetate (PMA), the ratio of 5-oxo-ETE:5-, 20-diHETE has been found to increase from 0.7 to 1.85. PMA-stimulated neutrophils also produced 5-0x0-ETE from exogenous AA. In these studies, it was found that more 5-0x0-ETE than LTB4 was formed under all conditions. The effect of PMA in the formation of 5-0x0-ETE required the activation of NADPH oxidase but was independent of the formation of ~ u p e r o x i d e Thus, . ~ ~ ~phenazine ~~ methosulphate which converts NADPH to NADP+, but not the generation of superoxide by xanthine/xanthine oxidase, mimicked the actions of PMA on the synthesis of 5-0x0-ETE. Similarly, A23187 stimulated the formation of 5-0x0-ETE and with prolonged incubation with A23187, the amount of 5-0x0-ETE that was formed exceeded that of LTB4.77These studies demonstrate that stimulated neutrophils have the capacity to synthesize a substantial amount of 5-0x0-ETE. The 5-0x0-ETE which accumulates in stimulated neutrophils is metabolized to 5-oxo-20-(OH)-6E, 8Z,11Z,14Z-eicosatetraenoic acids by o - ~ x i d a t i o nHowever, .~~ monocytes have been reported not to form o-oxidized products of ~ - o x o - E T E . ~ ~ When neutrophils were exposed to EPA and PMA or A23187, 5-hydroxy6,8,11,14,17-eicosapentaenoic acid (5-HEPE), 5-0x0-EPE and small amounts of LTB5 and 20-OH-LTB5 were formed,79 demonstrating that EPA, like AA, can be metabolized to form 0x0-derivatives.

4.2.3. 12-lipoxygenase Compared to the 12-lipoxygenase in platelets, the 12-lipoxygenase in neutrophils is relatively inactive. Thus, products of 12-HETE were not formed in stimulated rat neutrophilsq80In fact, it has been suggested that 12lipoxygenase metabolites, if detected in neutrophil preparations, could be produced by contaminating ~latelets.4~ Nevertheless, studies with bovine neutrophils have demonstrated that while intact neutrophils did not metabolize AA via the 12-lipoxygenase,sonicates of bovine neutrophils readily converted AA and LA to their respective 12-lipoxygenase products.81In comparison, incubation of canine neutrophils wth AA has been reported to produce lZHETE, 12,20-diHETE and 12-hydroxyheptadecatrienoic acid.82 With human neutrophils, the production of 12-HETE in the 17000g supernatant of neutrophil homogenates and in intact cells incubated in the

182

The Neutrophils

presence of A23187 have been reported.83fi0In both of these studies, indomethacin was found to be necessary for 12-HETE f o r m a t i ~ n . ~ ~ However, this was not related to the inhibition of the cyclooxygenase since neither acetyl salicylic acid nor ibuprofen, inhibitors of cyclooxygenase, mimicked the action of indomethacin.84

4.2.4. 15-lipoxygenase The 15-lipoxygenasein neutrophils is usually inactive, even in the presence of A23187 and AA. However, it has been reported that 5-, 12- and 15-HETE could stimulate the relak vely inactive enzyme to metabolize AA in the presence of A23187 and nordihydroguaiaretic acid.85The monohydroxy products of LA, 9- and 13-HODEs were found to be less active than the monohydroxy-derivatives of AA at stimulating 15-lipoxygena~e.~~ Similar results were obtained by Fogh et aLS6 In this study, it was found that a number of 5-lipoxygenase inhibitors but not cyclooxygenase inhibitors, diverted the metabolism of AA via 5-lipoxygenase to the 15-lipoxygenasepathway and this was associated with a reduction in LTB4 formation. On the other hand, ibuprofen (9-fold), indomethacin (2-fold) and aspirin (1.5-fold),have been reported to stimulate 15-lipoxygenase in human n e ~ t r o p h i l s The . ~ ~ stimulation of 15-lipoxygenase by ibuprofen was found to occur within 1 min of ibuprofen addition and was reversible.

4.2.5. Cyclooxygenase In many cell types including neutrophils, cyclooxygenase (or prostaglandin endoperoxide synthase) has been localized to the lipid bodiess7 These are inducible cytoplasmic inclusions that develop in cells associated with inflammation. Lipid bodies act as repositories of arachidonyl phospholipids and have been proposed to play a role in the oxidative metabolism of AA to form eicosanoids. As discussed above, stimulated neutrophils produce some PGE2and TXB2?6Produdion of PGE2 was found to be agonist specific. Thus, exposure of human neutrophils to GM-CSF, G-CSF, LPS, urate crystals and fMLP was reported to stimulate PGE2 production.88,s9Cytokine-induced PGE2 production occurs in 2 phases: an early phase (detectable at 20 min) and a late cycloheximide-sensitive phase

Regulation of Neutrophil Functions by Fatty Acids

183

(detected after 4 h). In comparison, neutrophils were found to produce little or no PGE,, TXA, or 6-keto PGFI, in response to M-CSF, IL-1 or IL-3.88,90 The amount of prostanoids produced was dependent on gender. Hence, neutrophils obtained from women have been reported to produce 30% less TXB2 and PGE, than those obtained from meng1Production of PGE2by neutrophils obtained from alcoholics has also been reported to be lower than from neutrophils obtained from non-alc~holics.~~

4.2.6. o-0xidation w-oxidation of LTB4 by LTB4-20-hydrolaseof the cytochrome P450 enzyme family is the major route by which the catabolism of LTB4 in human neutrophils proceeds.93This pathway of LTB4catabolism was found to be exclusive to neutrophils since monocytes, lymphocytes or platelets were not able to produce w-oxidized products of LTB4. This enzyme system adds a hydroxyl moiety to the C-20 (w end) of LTB4 to produce 20-OH-LTB4. Catabolism of exogenous LTB4 is rapid (tl/2of approximately 4 min at 37°C in reaction mixtures containing 1mM LTB4 and 2 X lo7 neutrophils/ml). In addition to 20-OH-LTB4,incubation of neutrophils with AA has widely been reported to result in the production of 20-COOH-LTB4?3,94demonstrating that endogenously-derived LTB4 is also w-oxidized. Neutrophils can also metabolize 5-HETE, 5-0x0-ETE and 12 HETE by w - o ~ i d a t i o n . ~ ~ ~ ~ ~ ~

5. TRANSCELLULAR METABOLISM AA and eicosanoid metabolites released from one cell type can be further metabolized by another ~ e l l - t y p e .For ~ ~ example, in co-incubation experiments, the uptake and further metabolism of [3Hl-12-HETE, produced by prelabelled and activated platelets, to [3Hl 5-,12-diHETE by activated neutrophils has been reported.50However, with unstimulated neutrophils, platelet-derived 12-HETE was converted to 12-,2O-diHETE by the n e ~ t r o p h i l sThese . ~ ~ studies imply that transcellular metabolism facilitates the formation of eicosanoids, which are formed at low levels or not formed by a single cell type alone. In another set of co-incubation experiments, labeled AA which was released from aspirin-pretreated, calcium ionophore-stimulated platelets, had been reported to be taken up by

184

The Neutrophils

activated neutrophils, resulting in the formation of labelled 5- HETE and LTB4.50Cell-cell interaction at the level of eicosanoid metabolism may alter the range and amount of eicosanoids formed at sites of inflammation.

6. BIOLOGICAL PROPERTIES OF ARACHIDONIC ACID 6.1. Effects on Neutrophil Adhesion, Cell Migration and Chemotaxis Human neutrophils treated with arachidonic acid showed increased adhesion to plasma coated surfaces (Table 2). Short term exposure of neutrophils to AA alters the migration properties of the leukocyte.97 At physiologically attainable concentrations, the ability of human neutrophils to migrate in a chemotactic gradient generated with the tripeptide, fMLP and complement (serum activated with yeast particles) was completely inhibited. However, the effect of AA was not specific for the chemotactic response of the cell. Random migration was inhibited concomitantly with the decrease seen in the chemotactic response. This suggests that AA affects the elements involved in cell locomotion. The ability of fMLP to induce chemokinesis was also inhibited by AA. These results suggest that another characteristicof AA is to regulate the accumulation of neutrophils at inflammatory foci. The source of the fatty acid may be the tissues, the bacteria and the infiltrating leukocytes. Table 2 Effects of Arachidonic Acid on Neutrophil Functions Function

Effect

Comment

Adherence Migration

Increased Decreased

Phagocytosis Microbial killing Tissue damage p2integrin expression

Increased Increased Increased Increased

Respiratory burst Degranulation Cytokine synthesis

Induced Induced Suppressed

To plasma coated plastic surfaces Random migration and fMLP/ complement-induced chemotaxis Bacteria/parasites Bacteria/parasites Endothelial cells CR3 (CDllb/CD18) CR4 (CDllc/CD18) Superoxide production Of primary and secondary granules TNF, IL-8

Regulation of Neutrophil Functions by Fatty Acids

185

6.2. Activation of the NADPH Oxidase Neutrophils interacting with various types of soluble agonists and particles undergo an oxygen-dependent respiratory bust, which is associated with the phagocytosis of particles and leads to the release of toxic oxygenderived reactive species (ODRS), such as superoxide, hydrogen peroxide, hydroxyl radical, singlet oxygen and hypochlorous acid. These are responsible for the killing of a range of microorganisms and tumor cells. Perturbation of the neutrophil membrane by either receptor ligation or non-specifically leads to the assembly of NADPH oxidase in the plasma membrane, which catalyzes the reduction of molecular oxygen to superoxide.98-100This oxidase consists of membrane components, cytochrome b558 and FAD, the cytosolic components, p47phox,p67phox,p40PhoXand a small GTP binding protein, rac2.98-100 AA induces the activation of the NADPH oxidase in neutrophils.lo1J02The fatty acid has been shown to be a strong activator of the respiratory burst and the release of ODRS. At optimal agonist concentrations, the response induced by AA was similar to that induced by the phorbol ester, PMA and both of these responses were significantly greater than that induced by fMLP. The characteristics of the response were also examined. fMLP, as previously established, induces a weak to modest respiratory burst which is characterized by a very rapid release of superoxide which peaks within 30 sec and returns to basal levels in the next one to two min. This is quite different to the response induced by PMA, which acts independently of a cell surface receptor and directly activates protein kinase C.lo3At optimal concentrations, the PMA response is characterized by a peak response at 5 2 min and is substantially greater than the fMLP response. The activity of neutrophils stimulated with an optimal concentration of PMA returns to the basal level within 30 minutes. The characteristics of the respiratory burst in response to AA is similar to, but less persistent, than that induced by PMA. AA also stimulates the production of superoxide in reconstituted systems. For this to occur, all the components of the active NADPH oxidase have to be p r e ~ e n t ? ~Compared ,'~~ with intact cells, the concentrations of AA which are needed to evoke these in vitro responses are five to ten times more than those needed to produce the same response in intact neutrophils.

186

The Neutrophils

Interestingly, human monocytes and macrophages treated with polyunsaturated fatty acids showed very poor and often insignificant activation of the NADPH oxidase compared with n e u t r ~ p h i l s . 'However, ~~ pretreating these mononuclear phagocytes with AA, EPA and DHA or the simultaneous addition of fatty acids and either fMLP, PMA or A23187, gave rise to a major respiratory burst response.lo5

6.3. Stimulation of Degranulation Extensive studies on the stimulation of degranulation by AA have been c o n d ~ c t e d .AA ~ ~was ~ ~ found ' ~ ~ to be a complete secretagogue, inducing the release of constituents from both the specific and azurophilic granules, a s shown by the release of vitamin B12 binding protein and P-glucuronidase, respectively. Similarly, endogeously derived AA and other fatty acids have been demonstrated to regulate degranulation and degranulation-dependent receptor expression in intact neutrophils. Hence, neutrophils treated with inhibitors of phospholipase A2108 released less secretory products from both the specific and azurophilic granules in response to A23187. The response to AA in terms of the vitamin B12 binding protein release was greater than that induced by fMLP and PMA.lo7It has been proposed that AA acts by promoting the fusion between granules and plasma membrane.lo9

7. EFFECTS OF n-3 FATTY ACIDS, EICOSAPENTAENOIC AND DOCOSAHEXAENOICACID ON NEUTROPHILS Extensive investigations in our laboratory on the effects of n-3 polyunsaturated fatty acids on neutrophils have yielded some interesting results. Quite unexpectedly and against the perceived anti-inflammatory properties of these fatty acids, n-3 polyunsaturated fatty acids have been shown to activate properties of neutrophils associated with the proinflammatory activity of the cell. This places a different perspective on the concepts held for the last two decades that n-3 fatty acids, e.g. fish oils, can be used to depress the inflammatory reaction in allergic and autoimmune inflammatory diseases.110

Regulation of Neutrophil Functions by Fatty Acids

187

DHA was found to be particularly active compared with EPA in stimulating neutrophil adhesion."' This fatty acid caused a rapid increase in neutrophil adherence which was always greater than that induced by AA.l12 The other polyunsaturated 12-3 fatty acid, EPA, was found to stimulate this property to a lesser extent than AA and DHA.lI2In some cases DHA caused a substantial increase in this response, which was also significantly greater than that induced by AA and EPA. The kinetics of this response induced by AA and the n-3 polyunsaturated fatty acids showed that the response to DHA was greater than that induced by fMLP and PMA.'12 The n-3 polyunsaturated fatty acids were also found to induce marked degranulation of specific and azurophilic granules. It was again evident, on a molar basis, that DHA was much more active than either AA or EPA.107

8. REGULATION OF NEUTROPHIL FUNCTIONS BY METABOLITES OF ARACHIDONIC ACID The metabolism of AA via the lipoxygenase and cylooxygenasepathways generates metabolites which regulate neutrophil functions (Table 3). Some products of the lipoxygenase, such as LTB4, have marked Table 3 Effects of Eicosanoids on Neutrophil Function Lipoxygenase Product

LTB4 LTC4 LTD4 5-HETE 12-HETE 15-HPETE 15-HETE 5-0xo-l5(0H)-ETE 5,15-oxo-diHETE LXA4

Neutrophil Function Chemotaxis

Adhesion

Superoxide Production

+

+ + +

+

+

-

-

-

-

-

+ + -

-

+ + +

-

+

+

Degranulation

+ + -

The + and - sign indicate the presence or absence of activity of the lipoxygenase product.

188

The Neutrophils

pro-inflammatory and neutrophil stimulating activity, but others may show anti-inflammatory activity. In contrast, cylooxygenase products such as PGl and PG2 possess neutrophil-suppressive actions.

8.1. Products of the Lipoxygenase Pathway Although the products generated by the metabolism of AA via the lipoxygenase pathway have been shown to cause activation of neutrophils, recently an inhibitory effect by some of these metabolites has been reported. LTB4 has been of major interest as a neutrophil activator. It has both chemotactic and chemokinetic proper tie^,"^-'^^ stimulates adhesion of neutrophils and release of lysosomal e n z y m e ~ ,and ~ ~ induces ~ ' ~ ~ ~the generation of s u p e r o ~ i d e . l ~ ~ J ~ Thus, this eicosanoid promotes all the steps of the inflammatory reaction with respect to the neutrophil behavior in this response. LTC4 and LTD4 have also been shown to enhance neutrophil adherence properties.11s The hydroxy products, 5-HETE and 12-HETE are chemotactic, although higher concentrations than LTB4 are needed.121,122 However, 15-HETE has little stimulatory effect. The dehydrogenase product of 5-HETE and 5,15-diHETE, namely 5-0x0-ETE and 5-oxo-l5(0H)-ETE, respectively, also stimulate neutrophil c h e m ~ t a x i s . ' ~Degran~lation'~~ ~~'~~ and adherence126are also stimulated by 5-0x0-ETE. Administration of the 15-lipoxygenase product, 15-HETE, has been shown to reduce tissue injury associated with psoriasis vulgaris in humanss6 and carrageenan-induced experimental arthritis.127This is possibly related to the finding that 15-HETE was a potent inhibitor of LTB4induced neutrophil migration and transmigration across endothelium.128 In addition, products of 15-lipoxygenase can also inhibit LTB, formation by inhibiting 5-lipo~ygenase.~~ It also blocked transmigration induced by C5a and fMLP. Interestingly, 15-HETE was significantly more active than either 5-HETE or 12-HETE in inhibiting transmigration. While the 15-HPETE was found not to stimulate any of these neutrophil functions, 15-HPETE caused a marked suppression of cytokine production by neutrophils (unpublished) and macro phage^.'^^ In contrast to the LOX metabolites, the trihydroxytetraene-containing eicosanoids, lipoxins, have counter-regulatory properties on the neutrophils. Despite earlier studies, which showed that the lipoxin A4

Regulation of Neutrophil Functions by Fatty Acids

189

(LXA4)is chemotactic for n e u t r ~ p h i l s ' ~and ~ J ~stimulates ~ the respiratory burst at higher doses130~132 and adherence,l3 there is strong evidence to show that lipoxin A4 (LXA,), its aspirin-triggered 15-epimer (15-epi-LXA4) A4 analogue, have and the stable 15-epi-16-(para-fluoro)-phenoxy-lipoxin potent inhibitory effects on neutrophil chemotactic responses in vitro at nanomolar concentration^.'^'^^ In in v i m studies, 35-epi-16-(para-fluoro)phenoxy-lipoxin A4 has been reported to inhibit neutrophil recruitment in colitis.139 A numanimal models of renal ischemia reperfusion injury138and colitis.139 ber of mechanisms have been proposed to account for this inhibitory action. These include inhibition of inositol trisphosphate generationI4Oand stimulation of the expression in neutrophils of NABl, a transcriptional corepressor identified previously as a glucocorticoid-responsivegene in hamster smooth muscle cells.136The effect of lipoxin A analogue on NABl expression may suggest that lipoxins may have actions that overlap with the anti-inflammatory actions of glucocorticoids. The lipoxins also stimulate the phagocytosis of apoptotic neutrophils by macro phage^.'^' These actions of the lipoxins are consistent with the suggestion that lipoxins promote the resolution of neutrophil-mediated inflammatory responses. The lipoxins are generated within the vascular lumen during plateletleukocyte interactions and at mucosal surfaces via leukocyte-epithelial cell interactions. This process requires the transcellular metabolism of arachidonic acid via the sequential actions of the 15- (in one cell type) and 5- (in the other cell-type) or the 5- and 12-lipoxygenaseenzymatic pathways from two ~ e l l - t y p e s . ' ~The ~ ~dependence '~~ of lipoxin production on the different LOX in different cell-types implies that production of lipoxin lags behind the production of other eicosanoids. Indeed, kinetics studies of the production of lipoxin A4 in clinical and experimental exudates have shown an early coordinated appearance of leukotrienes and prostaglandins which is associated with neutrophil recruitment, followed later by lipoxin biosynthesis which is accompanied by spontaneous resolution of inflammation.

8.2. Products of the Cyclooxygenase Pathway The cyclooxygenase pathway of AA metabolism gives rise to products which modulate neutrophil responses and the inflammatory reaction. Products of the cyclooxygenase pathway contribute to the erythema, pain

190

The Neutrophils

and fever of inflammation. They synergize with other mediators in producing these effects. The effect of the prostaglandins on neutrophil function is, by contrast, largely suppressive. For example, PGE2 inhibits neutrophil aggregation induced by fMLP142and also N L P stimulated chemotaxis by human ne~trophi1s.l~~ PGEI, similarly, has been shown to inhibit the oxidative burst, chemotaxis and phagocytosis by human neutr0phi1s.l~~ In addition, the prostacyclin produced by endothelial cells, PGI, has been shown to inhibit neutrophil adherence.145The mechanism for the anti-inflammatory effects of some products of the cyclooxygenase pathway remains unclear; however, it may be related to their ability to increase intracellular CAMPlevels,146in inhibition of agonist-induced increases in Ca2+,144inhibition of phosphatidylinositol 3 - k i n a ~ e , or '~~ decreased receptor affinity for ligands such as fMLP.147 In contrast to the anti-inflammatory properties of the prostaglandins and prostacyclins, thromboxane A,, which is generated by stimulated n e ~ t r o p h i l senhances , ~ ~ ~ adherence of neutrophils to extracellular matrix (plastic) and thus may play a role in provoking some forms of vascular injury.149Thromboxane A, generation and subsequent selective pulmonary sequestration of neutrophils, is characteristic of several forms of the adult respiratory distress syndrome. Thromboxane B2, the product of thromboxane A2 metabolism, has been reported to be increased in lung following challenge with proinflammatory stimuli such as lipopolysacharide or cigarette smoke, and is accompanied by neutrophil influx into the lung.150~151 Dietary fish oil supplementation reduces thromboxane B2 elicited following LPS ~ha1lenge.l~~ Some evidence suggests that thromboxane-induced neutrophil adhesion to pulmonary microvascular and aortic endothelial cells requires activation of CD18.152

9. RELATIONSHIP BETWEEN FATTY ACID STRUCTURE AND BIOLOGICAL FUNCTION Fatty acids, with different carbon chain length, degrees of unsaturation and position of double bonds, have different physiochemical properties. Accordingly, their uptake, incorporation, interaction with cellular proteins and metabolism may differ dramatically. Extensive studies on this concept by our group have revealed that these impart different types and

Regulation of Neutrophil Functions by Fatty Acids

191

levels of biological activity to neutrophils. The data on this relationship are summarized in Table 4. Studies on neutrophil adhesion showed a relationship between the carbon atom chain length, degree of unsaturation and position of double bonds with the biological activity of the fatty acid.lo6."l The saturated 18:O fatty acid failed to induce any significant increase in adherence. Increased adherence was seen following stimulation with all 18; 20- and 22-carbon polyunsaturated fatty acids. The order of activity was 20:4n-6 > 18:4n-3, 18:3n-6, 18:2n-6> 18:3n-3, 18:ln-9. An examination of the three isomers of 203 (n-6, n-3 and n-9) revealed that they were as effective as 20:4n-6.1°6 Other studies demonstrated that 20:5n-3 and 22:6n-3 were less effective than 20:4n-6 at stimulating adherence, with 20:5n-3 being the least active.'l'

Table 4 The Relationship between Fatty Acid Structural Elements and Biological Effects on Neutrophils Fatty Acid

18:O 18:ln-9 18:2n-6 18:3n-6 18:3n-3 18:4n-3 20:4n-6 20:o 20:5n-3 22:6n-3 24:6n-3 26:6n-3 28:6n-3 30:6n-3 32:4n-6 32:6n-3 34:6n-3

+

Adherence

+

-

+ ++ +++

Respiratory Burst

+ ++

+t

+++ ++++

++

Degranulation Specific

Azurophilic t-

-

t-

+ ++ + t

+++

t-

++ + t

+++

+++ ++++ ++

+ +t ++++

t

+t

++++ -

-

++ +++

Migration Inhibition

++t

++++

++ ++++

f

The and - signs indicate the relative activity between each other for the various neutrophil functional tests. The - sign indicates no activity.

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The Neutrophils

The respiratory burst induced by polyunsaturated fatty acids is also dependent on the structure of the fatty a ~ i d . ~ ~ ~ Poulos , ~ ~ et~ d~l o 2 demonstrated that the ability to stimulate superoxide production by neutrophils was highly dependent on fatty acid carbon chain length. At different concentrations of these fatty acids, it was found that there was a steady decline in activity as the number of carbon atoms of the unsaturated fatty acids increased from 22 -+ 24 +26, having almost no activity once 28 carbon atoms are reached. Further increases to 30 +32 similarly failed to stimulate the respiratory burst102,154 (Table 4). To some extent, this trend was followed in relation to polyunsaturated fatty acid-induced inhibition of random and chemotactic migrati0n.9~18:ln-9 lacked activity, while 18:2n-6 and 18:3n-3 showed partial and significant inhibition of neutrophil random and chemotactic migration. Marked inhibition of these responses were seen with 20:4n-6,20:5n-3 and 22:6n-3. However, the polyunsaturated very long chain fatty acid (32:4n-6)had no activity, illustrating how the carbon chain length of the fatty molecules affects their biological properties. The mono/polyunsaturated fatty acids behaved very similarly with respect to the stimulation of degranulation as with the stimulation of Most evident was their ability to stimulate release from specific granules (release of vitamin BI2 binding protein). However, they also showed activity in inducing release from azurophilic granules (p-glucuronidase), making these complete secretagogues. This was particularly evident with 20:4n-6,22:6n-3 and 18:3n-6. Comparisons between the different structures showed that the ability to induce degranulation of neutrophils was in the order of 22:6n-3 > 20:4n-6 > 20:3n-6 > 20:5n3 > 18:2n-6,18:4n-3,18:ln-9 (Table 4). Evidence has been presented that most of the above activities of fatty acids are dependent on a free carboxyl group. Conversion of the fatty acids 20:4n-6, 20:5n-3 and 20:6n-3 to their methyl esters resulted in complete loss of neutrophil stimulating activity with respect to adherence,"l superoxide production,lo2 degranulation'O6 and migration i n h i b i t i ~ Interestingly, the methyl esters are still capable of partitioning into neutrophil plasma membrane.46This suggests that membrane perturbation is insufficient for biological activity.

Regulation of Neutrophil Functions by Fatty Acids

193

10. CYTOKINE INDUCED ALTERATION IN NEUTROPHIL RESPONSES TO POLYUNSATURATED FATTY ACIDS A variety of mediators is involved in regulating the different phases of the inflammatory reaction. While in many cases we have a comprehensive understanding of the effects of the individual mediators, the ability of these mediators to influence each other's activity remains ill-defined. Cytokines constitute another class of mediators which is generated during inflammation and it is of interest to know whether or not cytokines and polyunsaturated fatty acids act synergistically. This question was recently addressed by Li et ~ 2 . inl which ~ ~ the effects of preexposure of neutrophils to the proinflammatory cytokine, tumor necrosis factor (TNF), on fatty acid-induced superoxide production were examined. Neutrophils pretreated with TNF showed a markedly increased response to a range of fatty acids, such as 18:ln-9, 18:2n-6, 18:3n-3,20:4n-6,20:5n-3 and 22:6n-3, but not the saturated fatty acid 20:O or the hydroperoxy-/ hydroxyderivatives of 20:4n-6. A similar synergistic response was seen with LTB4 and TNF. In contrast and as expected, TNF-treated neutrophils showed no increase in response to PGE,,. In fact, a reduction in the TNF response was observed. These findings illustrate that the combination of two quite different mediators leads to responses which are several fold higher than that achieved with an individual cytokine. Although this network of interaction needs to be studied in more detail, it is evident that a synergistic response is also seen between granulocyte macrophage-colony stimulating factor and polyunsaturated fatty acids.155In addition, a synergistic superoxide response was also seen between polyunsaturated fatty acids and fMLP or PMA.102f156 Besides being evident for superoxide production, this network of interaction is likely to be relevant to other neutrophil responses. Indeed, this is demonstrated by our other finding that TNF and polyunsaturated fatty acids are synergistic with respect to degranulation (Li Y. and Ferrante A., unpublished). Synergistic responses between the lipoxygenase products, LTB4 and 5-oxo-ETE, and TNF have been demonstrated in terms of superoxide prod~ction.~~~~'~~~'~~

194

The Neutrophils

Robinson et al. (34) found that TNF specifically altered the metabolism of phosphatidylinositol, phosphatidic acid, phosphatidylethanolamine and phosphatidylcholine in neutrophils. TNF caused an increase in incorporation of radiolabeled AA into cellular phosphatidylinositol and phosphatidic acid, but the incorporation into phosphatidylcholine and phosphatidylethanolamine was slower. AA was exclusively esterified at the sn-2 position of these phospholipids. There was no change in the labeling pattern of neutral lipids and eicosanoids and the cytokine showed no effect on the distribution of the radiolabel in 1-acyl, 1-akyl and 1-alk-1-enyl subclasses of phosphatidylcholine, phosphatidylethanolamine and triglyceride. TNF did not alter P-oxidation, chain elongation and desaturation of AA. TNF did not activate phospolipases D and C as well as the neutral and acidic sphingomyelinase.

11. NEUTROPHIL P R I M I N G PROPERTIES O F FATTY ACIDS Many studies of microbicidal activity and target cell killing conducted in vitro usually use peripheral blood neutrophils which have not undergone the typical alterations induced by inflammatory mediators. In reality, neutrophils come under the influence of a range of mediators, which will regulate their antimicrobial activity. Over the past decade, evidence has been presented that interactions of neutrophils with microbial, tumor and host tissue targets can be significantly modified by prior exposure of the leukocytes to various mediators. Particular interest has been paid to the role of cytokines in this neutrophil priming response. This priming results in an increase in the neutrophil response to a challenge agonist, observable as an increase in the binding of a ligand, biochemical responses elicited, phagocytosis and in microbial killing and tissue damage.157It has been argued and evidence has been presented that both activated T lymphocytes and macrophages regulate these functions of the neutrophil through the release of ~ y t o k i n e s . l ~ ~ J ~ ~ Some of the most studied cytokines in relation to neutrophil priming for increased antimicrobial activity and tissue damage are TNF, GM-CSF, IFN-7 and lymphotoxin (LT). For example, TNF has been shown to play a critical role in immunity to infection.160Preexposure of neutrophils to TNF

Regulation of Neutrophil Functions by Fatty Acids

195

leads to increased phagocytosis and killing of bacteria and parasites.161J62 Many of these mediators also stimulate the release of 20:4n-6 or alter the activity of PLA2 (Table 1).The released fatty acids may act as second messengers, priming neutrophils for enhanced responses to other mediators.

11.l.Alteration of Responses to fMLP and PMA Our studies have demonstrated that pretreating neutrophils with polyunsaturated fatty acids enhances their capacity to respond to either fMLP or PMA, thereby producing more superoxide than when challenged with a compound alone,102r151(Table 5). The simultaneous addition of a fatty acid and fMLP/PMA also significantly enhances the response to above that observed with one compound alone. On the other hand, a fatty acid per se is unable to stimulate superoxide production in macrophages.lo5The enhancement of superoxide production by macrophages is observed when macrophages are pretreated with a fatty acid or when a fatty acid is added simultaneously with fMLP or PMA. The reasons for the differences in the responses observed between macrophages and neutrophils are unclear. However, it could be related to the inability of fatty acids to stimulate the release of AA in macrophages (see "Activation of intracellular signals").

11.2. Antimicrobial Activity Neutrophils preexposed to polyunsaturated fatty acids show increased killing of intraerythrocytic asexual stages of Plasmodium falciparum.161This Table 5 Modulation of Superoxide Production by PUFAs in Phagocytic Cells EffectdTreatments

FA alone Synergisms With fMLP With PMA Priming for fMLP-induced CL

Neutrophils

Macrophages

AA

EPA

DHA

AA

EPA

DHA

J

J

J

P

P

P

J J

J J

J J

J J

J J

J J

J

J

J

J

J

J

J : active; P:inactive/ poor response; FA: fatty acids; CL: chemiluminescence.

196

The Neutrophils

was seen both with respect to the antibody independent and antibody dependent killing of the parasite by neutrophils. Neutrophils pretreated with polyunsaturated fatty acids showed increased phagocytosis of the parasite and increased production of oxygen radicals. These fatty acids were also able to significantly reduce the parasitemia in murine ma1a1ia.l~~ Extensive investigations show that the fatty acid structure plays a critical role in the ability of the fatty acid to enhance neutrophil parasite killing. Optimal stimulation was seen with polyunsaturated 20-22 carbon fatty acids. The saturated fatty acids 18:O and 20:O had no effect and neither did 18:ln-9 nor 18:2n-6. As the carbon chain length was increased from 22 -+24 4 28, there was a gradual decrease in activity shown by comparing 20:4n-6,24:4n-6 and 28:4n-6. The methyl ester, 15-hydroperoxy and 15-hydroxy derivatives of AA and DHA, showed very little effect, consistent with their inability to stimulate key neutrophil biochemical responses. It was evident from our studies that combined preexposure of neutrophils to TNF and polyunsaturated fatty acids led to a synergistic increase in neutrophil-mediated killing of the parasite.161More recently, we have demonstrated that these polyunsaturated fatty acids, 204n-6 and 22:6n-3, increase the killing of the Staphylococcus uuwus, non-typable Huernophilus influenza and Cundidu ulbicuns by neutrophils (unpublished).

11.3. Tissue Damage In exacerbated inflammation, the nonspecific release of AA may lead to activation of neutrophils and damage to tissue. This is in addition to a cocktail of inflammatory mediators which has been demonstrated to directly kill cells.164We recently addressed this issue with respect to neutrophil-mediated damage of the endothelium.106The finding showed that 20:4n-6 and 226n-3 enhanced the neutrophil-mediated detachment of endothelial cell monolayers. Interestingly, 20:5n-3 was very poor in causing this darnage.lI2Correlating with effects on other neutrophil functions was the relationship between the type of fatty acid structure and ability to augment neutrophil-mediated damage to endothelial cellsIo6(Table 6). Saturated fatty acids, methyl ester forms and hydroperoxy/hydroxy forms of polyunsaturated fatty acids were without effect. There was a slight but insignificant increase in this neutrophil function by 18:ln-9,

Regulation of Neutrophil Functions by Fatty Acids

197

Table 6 Effects of Fatty Acids on Neutrophil-mediated Microbial Killing and Tissue Damage Fatty Acid 18:0 18:ln-9 18:2n-6 18:3n-6 18:3n-3 18:4n-3 20:o 20:3n-6 20:3n-9 20:4n-6 20:5n-3 22:6n-3 22:4n-6 24:4n-6 28:4n-6 32:4n-6

Parasite Killing

++ +++ ++++ +++ +++

Endothelial Cell Damage

++++ + + + ?

+ + ?

+

The number of + signs show the activity relative to each other. The - sign signifies no activity.

18:2n-6, 18:3n-6 and 18:4n-3. For example, 20:4n-6 was 7-8 times more effective than 18:4n-3.1°6 It was also identified that the major mechanism by which polyunsaturated fatty acids prime neutrophils for damage to endothelial cells is through the release of elastase.112The above demonstrates that 20:4n-6 and cytokines share many properties. Thus, both TNF and 20:4n-6 enhance neutrophil microbial killing and phagocytosis, enhance the degranulation and respiratory burst response to fMLP, and inhibit migration of cells in a chemotactic gradient.

11.4. Cell Surface Receptor Expression As described above, polyunsaturated fatty acids (e.g. AA and their metabolic products such as LTB4) alter the antimicrobial and tissue damaging properties of neutrophils. Some of the mechanisms responsible for this priming or enhancement have been partly defined. The basis of the fatty acid-induced enhancement may relate to changes in the surface expression

198

The Neutrophils

of functional receptors on neutrophils. Studies using long chain polyunsaturated fatty acids have shown that, while the saturated fatty acid 20:O had no effect on the expression of p-2 integrin molecules, 20:4n-6,20:5n-3 and 22:6n-3 significantly increased the expression of the complement receptor type 3 (CR3), CDllb/CD18111 (Table 7). The fatty acids also caused a slight but insignificant increase in expression of CR4 (CDllc/CD18) and failed to alter the expression of the leucocyte adhesion functional antigen, LAF-1 (CDlla/CD18). The CDllb/CD18 molecule is a receptor for the C3bi component of complement which is deposited on microorganisms and tissues, promoting neutrophil binding, phagocytosis and damage to these targets. This may explain, at least in part, the increase in bacterial and parasite damage seen with polyunsaturated fatty acid-primed neutrophils (161, unpublished). The CDllb/CD18 molecules are known to also interact with fibrinogen, coagulation factor, bacterial lipopolysaccharideand ICAM-1 in endothelial cells. This may explain the increased adherence properties of neutrophils treated with these fatty acids111,112 as well as their increase in endothelial Table 7 Fatty Acid Induced Changes to Neutrophil p2 Integrins Fatty Acid

Receptor Type CDlla

20:o 20:4n-6 20:5n-3 22:6n-3 LTB4 5-oxo-ETE

5-HETE LTB3 LTBj LTCl LTD4 5-HPETE LTC4

-

CDllb

++++ +++ ++++ +++ ++ + +++ + +

CDllc 5

t

+ -

-

+ + -

Number of + indicates degree of effectiveness. - indicates lack of effect. ? indicates intermediate effect. 4 + indicates very strong activity.

Regulation of Neutrophil Functions by Fatty Acids

199

cell darnage.Io6The increase in CDllb/CDlS expression is likely to be the result of increased degranulation caused by the polyunsaturated fatty acids (see "Stimulation of degranulation"). Ultrastructural and immunofluorescence studies have localized spare CDllb/CD18 to specific and secretory granules and the recruitment of CDllb/CD18 have been tightly correlated with the release of specific granule ~ o n t e n t . ' ~ ~ - ' ~ ~ The effects of products of fatty acid metabolism on surface receptor expression of neutrophils has also been reported and is summarized in Table 7. The lipoxygenase product, LTB4, is a powerful inducer of CR3 expression on neutrophils.168Other eicosanoids with this activity include ~ - O X O - E T which E ' ~ ~ is much more active than 5-HETE and acts via the 5-0x0-ETE receptor.126Although 5-0x0-ETE increases the expression of CDllb, it does not increase the expression of CDlla, CDllc, FcyRII and F C ~ R I I I .LTB3, ' ~ ~ a 5-LOX metabolite from di homo y-linolenic acid (20:3n-6),is also highly active in increasing the expression of CDllb.169J70

12. MECHANISMS OF FATTY ACID-INDUCED NE UT ROPHI L ACT I VAT I ON 12.1. Polyunsaturated Fatty Acids Stimulate Neutrophils Independently of Lipoxygenase and Cyclooxygenase Pathways Because AA gives rise to highly active eicosanoids, these products have been thought to be responsible for the stimulatory properties of AA on neutrophils. However, the effects of 20:4n-6 are unlikely to be due to the metabolism of 20:4n-6. In the first instance, the 20:5n-3, which yields metabolites with lower proinflammatory activity than those derived from 20:4n-6, was just as active as AA in stimulating neutrophil functions. Other evidence has also been provided. When neutrophils were pretreated with either lipoxygenase (caffeic acid or nordihydroguaiaretic acid - NDGA), or cycloxygenase (indomethacin) inhibitors, no effect was observed of AAinduced neutrophil adhesion and respiratory burst.102t'06 Similar observations were made with the migration inhibition properties of polyunsaturated fatty a ~ i d s . 9Pretreatment ~ of neutrophils with either indomethacin or NDGA did not affect the fatty acid-induced

200

The Neutrophils

inhibition of random and chemotactic migration.97Under these conditions, there was near complete inhibition of the cycloxygenase and lipoxygenase pathways. Polyunsaturated fatty acid-induced increase in neutrophil-mediated damage to endothelial cells also occurs independently of the cycloxygenase and lipoxygenase pathways. Under conditions where indomethacin and NDGA inhibited these pathways, the enhancement of neutrophil-mediated endothelial cell damage by AA was not affected.lI2Certain effects of 20:4n-6 in other cell-types are dependent on the formation of metabolites of 20:4n-6. For example, inhibition of gap junctional communication by 20:4n-6 in WB rat liver epithelial cells was prevented by NDGA.171 The effects of 20:4n-6 on some of the intracellular signaling molecules which we have examined are also independent of the metabolism of 20:4n-6 by the lipoxygenases. Thus, we have demonstrated that stimulation of dual phosphorylation of p38 MAP kinase by 20:4n-6 in neutrophils was not inhibited by NDGA.172In comparison, stimulation of the activity of the classlA phosphatidylinositol-3-kinase was inhibited by NDGA.173

12.2. Differences in Metabolism of Long Chain and Very Long Chain Polyunsaturated Fatty Acids Because the activity of the polyunsaturated fatty acids on neutrophils was highly dependent on structure, it was of interest to know whether neutrophils handle the long and very long chain fatty acids differently. A study was undertaken by Robinson et ~ 1 to .compare ~ the incorporation of two tetraenoic very long chain fatty acids, 34:4n-6 and 30:4n-6 with 20:4n-6 into neutral lipids and phospholipids of neutrophil and to examine their conversion into oxygenated derivatives. The findings showed that both 20:4n-6 and 24:4n-6 were readily taken up by human neutrophils. These were esterified into neutral lipids and phospholipids, and elongated by up to four carbon units. However, 30:4n-6 was poorly incorporated and remained essentially in the nonesterified form. Both 24:4n-6 and 30:4n-6 were predominantly esterified into triacyglycerol. Neutrophils poorly @-oxidizedand desaturated the three types of fatty acids. Activation of neutrophils with calcium ionophore, A23187, resulted in the

Regulation of Neutrophil Functions by Fatty Acids

201

generation of different oxygenated products. Metabolism of 20:4n-6 generated mainly 5-HETE and LTB,; 24:4n-6 gave rise to monohydroxylated fatty acids, mainly the 9-hydroxy positional isomer, but not other lipoxygenase and cycloxygenase products. In contrast, 30:4n-6 gave rise to negligible oxygenated fatty acids, suggesting that it is a poor substrate for neutrophil cylooxygenase and lipoxygenase enzymes.

12.3. Activation of lntracellular Signals In order to understand how fatty acids stimulate neutrophil functional responses or alter the cell's response to a second agonist, there is a need to know which intracellular signals are activated by the fatty acids. Although previous studies have found polyunsaturated fatty acids not to stimulate the activity of phospholipase C or D in n e ~ t r o p h i l s , ' ~a recent study, using concentrations less than those used previously, found that 20:4n-6 stimulated the activity of phospholipase D between 0.5-5.0 pM175in a bell shaped manner. Phospholipase D activity had returned to basal level by 5 pM. This would explain why previous studies had failed to detect an effect of 20:4n-6 on phospholipase D activity. Various polyunsaturated fatty acids have been shown to activate a heterogenous group of intracellular signaling molecules (Table 8). These include the heterotrimeric G proteins, the neutral sphingomyelinase, protein kinase C (PKC), the ERK and p38 MAP kinases, and phosphatidylinositol3-kinase (PI3).Fatty acids also stimulate calcium mobilization, the release of rhoGDI from the rac2/rhoGDI complex and modulate ion channel conductance.

12.3.1. Mobilization of intracellular calcium

Ca2+playsa central role in cell physiology. This second messenger regulates diverse functions such as secretion, muscle contraction, metabolism, neuronal excitability, cell proliferation and cell death. The cytosolic Ca2+ concentration is tightly regulated. In the resting cell, Ca2+ is maintained in the nM levels. Upon stimulation, intracellular Ca2+concentrations can Ligand-stimulated increases in the intracellular Ca2+ reach 1 pM.176e177

The Neutrophils

202

Table 8 Intracellular Signals Activated by Polyunsaturated Fatty Acids Fatty acid 18:O 18:ln-9 18:2n-6 18:4n-6 20:o 20:4n-6 20:5n-3 22:4n-6 22:6n-3 24:4n-6 28:4n-6 30:4n-6 32:4n-6

Ca2+ ERKl Mobilization ERK2

p38

SMase

PKC

PLA,

JNK

PI3K

-

-

+ +

+ -

-

+ + + +

+ + +

+

+

+

-

-

-

+ + +

+ + +

+ + +

+

+ +

-

-

-

+ -

The + sign and - sign indicate that the fatty acid is active or nonactive in stimulating the respective function. Smase: sphingomyelinase; ERK: extracellular signal regulated protein kinase; PKC: protein kinase C; PLA2: phospholipase A,; JNK: c-jun N-terminal kinase. PBK: Class 1 A phosphatidylinositol3-kinase.

concentration come mainly from 2 sources: release from intracellular stores, such as the endoplasmic reticulum by inositol trisphosphate or from the sarcoplasmic reticulum by cyclic ADP ribose; and influx via plasma membrane Ca2+channels. Elevated intracellular Ca2+concentrations are then returned to pre-stimulation levels by Ca2+pumps which are located on the plasma membrane and membranes of the endoplasmic and sarcoplasmic reticulum. In neutrophils, stimulation by agonists that bind to the G proteincoupled seven transmembrane-type receptors such as the fMLP receptor, trigger increases in intracellular Ca2+.Polyunsaturated fatty acids have been shown to cause an increase in intracellular Ca2+concentrations in a variety of different cells, including the n e u t r ~ p h i l s . ' ~ Saturated ~ ~ ' ~ ~ ~ fatty '~~ acids failed to mobilize Ca2+.An examination of the Ca2+mobilization properties of polyunsaturated fatty acids with different structural elements was carried out by Hardy et ~ 1 . lThe ~ ~ results showed that 20:4n-6,30:4n-6,22:4n-6 and 18:4n-6 mobilize calcium, whereas 28:4n-6,

Regulation of Neutrophil Functions by Fatty Acids

203

24:4n-6 and 32:4n-6 do not (Table 8). While there is a general trend correlating the degree of Ca2+mobilizationwith ability to stimulate superoxide production, it is evident that discrepancies exist.174The most obvious is that 30:4n-6 is a strong inducer of intracellular calcium mobilization but induces no superoxide response.174It was also interesting that 20:4n-6 releases intracellular Ca2+via a thapsigargin-sensitive pool, while 30:4n-6 mobilizes Ca2+via a thapsigargin-insensitive pool in ne~trophi1s.l~~ 20:4n-6-derived products such as ~ - O X O - E T E 'and ~ ~ , LTB4180 '~~ also trigger calcium transients in neutrophils. LTB4 effects occur via its binding to a receptor and it is believed that 5-0x0-ETE also acts via a specific receptor74 and clearly independently of LTB4 receptors. Other eicosanoids can also stimulate Ca2+mobilization. Thus, 12-HETE and 12HPETE have been shown to stimulate the release of stored Ca2+ in neutrophils.18'

12.3.2. Heterotrimeric C-proteins The heterotrimeric GTP-binding proteins are molecular switches which play crucial roles in transmembrane signaling. Composed of a, p and y submits, the G proteins couple the seven transmembrane type receptors of hormones, growth factors, neurotransmitters and other bioactive molecules, including fMLP and PAF, to their intracellular signaling pathways.182In the resting cell, the ci submit is bound by GDP. Receptor occupancy by a ligand causes a structural change in the receptor which then allows the receptor to interact with a G protein. This permits the exchange of GDP for GTP. The GTP-bound ci submit dissociates from the p'y subunits and activates the signaling molecules, such as adenylate cyclase and phospholipase Cp and phospholipase A2. The Py subunits also activate downstream signaling molecules such as phosphatidylinosito1 3-kinase y.lS3The ci submit also possesses an intrinsic GTPase activity which hydrolyses GTP. This is promoted by the regulator of G protein signaling (RGS). The resultant GDP-bound a subunit then re-associates with the p y subunits, thereby terminating the effector activity. The ability of AA to stimulate GDP/GTP exchange on the heterotrimeric G protein has previously been demonstrated in purified neutrophil membrane preparation^.'^^ There was a positive correlation between the

204

The Neutrophils

ability of fatty acid to increase [35Sl GTPyS binding and to elicit the respiratory burst. The order of effectiveness at causing GTP binding was 20:4n-6 > 18:2n-6 > 18:ln-9. The saturated fatty acids, 14:O and 16:0, were ineffective.

12.3.3. Protein kinase C Protein kinase C (PKC), a family of serine/threonine protein kinases, are classified into three groups: (i) classical PKC (a,PI, PI1 and y), (ii) novel PKC (6, E, 8, q and p); and (iii) atypical PKC (5, L and 1).The classical PKC isozymes are activated by the combination of a phospholipid, calcium and diacylglycerol, and the novel PKC isozymes require phospholipid and diacylglycerol for activation. These forms can be activated in intact cells directly by PMA.lo3The atypical forms require only a phospholipid and are not responsive to I'MA.Io3 Activation of PKC is required for a range of neutrophil activities such as the activation of the NADPH o x i d a ~ e . ' ~ ' ~ ~studies using cell-fkee In~vitro extracts/ purified PKC have shown that many cis-fatty acids, including 18:1n-9,18:2n-6,18:3n-6,20:4n-6,205n-3 and 2 6 n - 3 , stimulate the activity of PKC a,p, y, E and 5 isozymes from rat brain in the presence of very low levels of Ca2+ and/or p h o s p h a t i d y l ~ e r i n e . 'Saturated ~ ~ ~ ~ ~ fatty acids and transfatty acids failed to activate PKC. Hardy et ~ 1 . demonstrated ~ ~ ' that while the very long chain polyunsaturated fatty acids 32:4n-6 and 34:6n-3 activated PKC in vitro, both failed to stimulatea respiratory burst in ne~tr0phils.l~~ The ability of polyunsaturated fatty acids to stimulate PKC in whole cells has been d o c ~ m e n t e d . ' ~This ~~~ is ~summarized ,'~~ in Table 9 for neutrophils and other cell types. In neutrophils, polyunsaturated fatty acids stimulated the translocation of a,PI, PI1 to a particulate fraction. No increase in particulate fraction-associated PKCG or 5 as detected.ln Similarly, polyunsaturated fatty acids also stimulated the translocation of PKCa, PI, pII in macrophageslo5 and of PKCa, 6 and E in WB cells'92(Table 10).

12.3.4. Activation of PLA2 by 20:4n-6 and other fatty acids As shown in Table 1, neutrophils release radiolabeled 204n-6 in response to a variety of external factors. This is due to the activation of PLA2.It has

Regulation of Neutrophil Functions by Fatty Acids

205

Table 9 ActivatiodTranslocation of PKC in vitro and in vivo by Polyunsaturated Fatty Acids Fatty Acid

PKC Activation Cell-free System

Neutrophils

Other Cell Types

+

+ +

+ + +

18:ln-9 18:2n-6 18:3n-6 20:o 20:4n-6 20:5n-3 22:6n-3 32:4n-6 34:6n-3 trans-fatty acids

-

+ + + + -

+: stimulate; -: no effect Table 10 Activation/Translocationof PKC Isozymes by Unsaturated Fatty Acids Activation in System cPKC

Y

+ + + +

E

+

ci

PI PI1

nPKC 6

aPKC

5

+

Translocation in Neutrophils

+ + +

Macrophages

+ + + +

WB Cells

+

+ +

-

cPKC: classical PKC; nPKC; novel PKC; aPKC: atypical PKC: activate; -: no effect

+: stimulate/

been reported that neutrophils express at least three forms of PLA2: sPLA2, cPLA2, iPLA2.24t27t193-195 A number of studies have demonstrated that exogenous 20:4n-6 causes the release of radiolabeled 20:4n-6 from prelabeled n e u t r ~ p h i l s .This ~ ~ . effect ~ ~ has been attributed to the

206

The Neutrophils

formation of LTB4 and the subsequent activation of cPLA2by LTB, binding to its receptor.36However, our results argue against LTB, being a major cause of the fatty acid-stimulated activation of PLA2. Thus, while 20:O was inactive, 18:2n-6, 20:4n-6, 20:5n-3 and 22:6n-3 stimulated the release of 20:4n-6 via both cPLAz and s P L A ~The . ~ ~release of radiolabeled 20:4n-6 and the production of superoxide caused by exogenous 20:4n-6 or 22:6n-3, were blocked by inhibitors of cPLAzor sPLA2. 12.3.5. Activation of the MAP kinases

Mitogen-activated protein (MAP) kinases are proline-directed serine/ threonine kinases which are activated by a wide variety of extracellular signals. Members of the MAP kinases include the extracellular signalregulated kinases (ERK) family (ERKs 1-5, 7, 8); c-jun N terminal kinases (JNK) family; and p38 family. While ERKl and ERK2 are activated by growth factors, serum and some cytokines, JNKl and JNK2, and p38 (also known as stress-activated protein kinases), are activated following the exposure of cells to inflammatory cytokines, bacterial toxins, hyperosmotic stress and UV i r r a d i a t i ~ n . 'The ~ ~ MAP kinases are activated by a cascade of upstream kinases. MAP kinase cascades form crucial links between the receptors at the plasma membrane and the nuclei, since activated MAP kinases have been demonstrated to be present in the nuclei of activated ce11s.197-200 We have previously demonstrated that AA, DHA and EPA stimulated the activity of ERKl and ERK2 in rat liver epithelial WB cells.'92 This effect was dependent on PKC, since PKC depletion resulted in the complete abrogation of AA-induced ERK activation. Our recent studies have also demonstrated that AA also stimulated the activation of ERKl and ERK2 in human neutrophils at concentrations which correlate well with stimulation of superoxide p r o d u ~ t i o n .AA ' ~ ~ and DHA also stimulated the activity of ERK in human macrophages (Huang, Hii and Ferrante, unpublished). AA also stimulated the dual activity of p38 at concentrations which stimulate superoxide p r o d u c t i ~ n . ~ However, ~~,~~~,'~~ AA did not stimulate the activity of JNK in neutrophils although the fatty acid stimulated JNK activity in Jurkat T cells,17zproximal tubular epithelial cellszo1and stromal cells.202Stimulation of JNK activity by AA in

Regulation of Neutrophil Functions by Fatty Acids

207

proximal tubule cells was dependent on the generation of superoxide.201 Given that AA strongly stimulates superoxide production in neutrophils, it is therefore, surprising that AA did not stimulate JNK activity in neutrophils. Stimulation of p38 activity in neutrophils by AA was independent of COX and LOX activities since this effect was not decreased by either NDGA or indometha~in.’~~

12.3.6. Activation of sphingomyelinase Sphingomyelinase (Smase) hydrolyzes membrane sphingomyelin to generate the recently-described second messenger molecule, ~ e r a m i ~ Several different types of sphingomyelinases have been described. These include a neutral, Mg2+-dependentenzyme, localized in the outer leaflet of the plasma membrane; a neutral sphingomyelinase which shows no dependence on divalent cations, resident in the cytosol; and an acidic sphingomyelinase which has no dependence on divalent cations, located in the endosomal/lysosomal compartments of the cell.204Each enzyme appears to act on a distinct pool of sphingomyelin, releasing ceramide. Ceramide causes growth arrest, promotes cell differentiation and induces a p o p t o s i ~20:4n-6 . ~ ~ ~ has been shown to stimulate the hydrolysis of sphingomyelin by the neutral sphingomyelinase in human n e u t r o p h i l ~The .~ activity of the acidic sphingomyelinase was not affected by the fatty acids.205The effect of 20:4n-6 on the activity of the neutral sphingomyelinase was transient, peaking at five min and returning to normal by 10min after exposure. Significant increases in the activity of the enzyme were seen with 2.5 FM of 20:4n-6. Other long chain mono/ polyunsaturated fatty acids also caused the activation of sphingomyelinase in n e ~ t r o p h i l s These . ~ ~ ~ include 18:ln-9, 18:2n-6, 20:5n-3 and 22:6n-3. However, the saturated fatty acids 18:O and 20:0, and the very long chain polyunsaturated fatty acids, 24:4n-6, and 28:4n-6, did not activate the enzyme system.205

1 2.3.7. Phosphatidylinositol3-kinase PI3K is a family of lipid kinases that phosphorylate inositol-containing phospholipids at the D3 position of the inositol ring, resulting in the

208

The Neutrophils

formation of phosphatidylinositol (PtdIns)3 P, PtdIns 3,4 PL, and PtdIns 3, 4, 5 P3.206These kinases are grouped into three classes, I, I1 and 111. Of these, Class Ia and Ib enzymes have been reported in n e u t r o p h i 8 Four different catalytic subunits, pllOa, pllOp, pllOy and pllOF have been found for the Class I enzymes and the pll0 subunit is complexed to an adaptor protein (p85a, p85p and p55-y) derived from three separate geneszo6During activation, the p85 subunit is recruited to tyrosine activation motifs (ITAMs) on the cytoplasmic tail of receptor tyrosine kinases or cytoplasmic tyrosine kinases, resulting in the tyrosine phosphorylation of the p85 subunit.206 Only one Class Ib PI3K, PI3Ky has been identified. PI3Ky is activated solely by G-protein coupled receptors and is composed of a pllOy catalytic subunit and a 101-kDa regulatory subunit.206P13K regulates cell growth, transformation, differentiation, secretory responses, chemotaxis, cell adhesion, apoptosis and cytoskeletal reorganisati0n.2~~ In neutrophils, the activity of PI3K is stimulated by fMLP,208 GMCSF, PAFZ1Oand FcyR ligation. Studies using the pharmacological inhibitors, wortmannin and LY294002, in neutrophils have demonstrated that PI3K is required for Fcy R-mediated responses,211respiratory burst212 and d e g r a n ~ l a t i o n Genetic ~ ~ ~ - ~ evidence ~~ in mice have demonstrated that PI3Ky is responsible for regulating neutrophil chemotaxis.216PI3K also regulates neutrophil-mediated proteoglycan degradation.215 We recently demonstrated that 20:4n-6 stimulated the activity of PI3K.l” This finding has recently been confirmed in another AA-stimulated PI3K was suppressed by NDGA, unlike the activation of p38 by 20:4n-6 (see above). This implies that a LOX metabolite was responsible for the effect. Consistent with this, we demonstrated that 5-HETE also stimulated the activity of PI3K. Activation of PI3K by AA in neutrophils was suppressed by inhibitors which block the activation of the ErbB family of receptors,173suggesting that some of the actions of 20:4n-6 could be mediated by cell surface receptors. Indeed, 20:4n-6 has been reported to stimulate the activation or phosphorylation of the EGF receptor and ErbB4 receptors in the renal proximal tubule epithelial cells and endothelial cells, r e s p e c t i ~ e l y . ’ ~ ~ ~ ~ ~ ~

Regulation of Neutrophil Functions by Fatty Acids

209

12.3.8. lon channels Fatty acids, including AA, are implicated in the direct and indirect modulation of a number of voltage-gated ion channels. For example, in whole cell patch-clamp experiments in rat pulmonary myocytes, external application of AA caused membrane depolarization, acceleration of the rate of rectifier K+ current activation and a marked acceleration of current decay.219The effects were not affected by indomethacin or NDGA, suggesting that AA per se was responsible for these effects. AA also alters the permeability of Na+ channels. Thus, in skeletal muscle, AA can either inhibit or activate Na+ channels, depending on whether it is delivered intracellularly or extracellularly.220The effects of AA on ion channels in neutrophils have also been reported. The human neutrophil NADPH oxidase-associated H+ channel acts as a charge compensator for the electrogenic generation of superoxide and it has been reported that a H+-selective conductance is activated during the respiratory burst in neutrophils.221Although the identity of this H+ channel has not been clearly established, there is some evidence to suggest that the large subunit of the NADPH oxidase cytochrome b558 (gp91PhoX) may act as a H+ channel.221Whole cell patch-clamp studies of neutrophils have demonstrated that externally applied AA amplified a H+-selective conductance.222Thus, AA may also play a role in the respiratory burst by facilitating the dissipation of metabolically generated acid. 12.3.9. Modulation of the activation status of small GTP binding proteins

Fatty acids also alter the function of proteins which regulate the activation status of small GTP binding proteins. For example, AA has been found to inhibit the activity of p21rasGTPase activating protein in ~ i t r 3 This suggests that AA may prolong p21rasfunction. Other in vitro studies have demonstrated that AA also causes the dissociation of rhoGDP Dissociation Inhibitor (rhoGDI) from rhoGDI-rac c0rnplex.2~~ This action can be mimicked by phosphatidic acid and phosphatidylinositol. Given that rac is a component of the neutrophil NADPH oxidase and that only GTP-bound active rac can interact with other components of the NADPH

2 10

The Neutrophils

oxidase and stably translocate to the plasma membrane, it is possible that an important role for intracellular AA is to facilitate rac activation by causing the release of rac from rhoGDI. However, this has yet to be demonstrated in intact neutrophils.

12.4. Evidence for an Involvement of PKC, ERK, p38 and P13K in AA-stimulated Superoxide Production The ability of AA and other polyunsaturated fatty acids to stimulate the translocation of a number of PKC isozymes and to stimulate the activity/phosphorylation of ERK and p38 suggests that these kinases may be involved, at least in part, in mediating the effects of polyunsaturated fatty acids. It has previously been reported that PKC p can directly phosphorylate p47phoxin in nitro assays.225Phosphorylation of p47phoxis currently believed to be a prerequisite for the translocation of p47phoxto the plasma membrane, where it interacts with cytochrome b558. In activated neutrophils and in virally-transformed B lymphoblasts, p47Phoxis phosphorylated on multiple serine residues. Phosphopeptide mapping revealed phosphorylation of serine 303/304, 315, 320, 328, 345/348 and/or 359/370.226-228 In in nitro phosphorylation experiments, it has been found that PKC phosphorylated all of the above serine residues except serine 345/348, while ERK and p38 phosphorylated serine 345/348, with similar r a t e s . 2 2 6 - - 2 2 8 We have demonstrated that the ability of AA to stimulate superoxide production was partially blocked by GFl09203X, PD98059 and SB203580, inhibitors of PKC, MEK and p38, respectively.229Dose-inhibition curves showed that GF109203X, I'D98059 and SB203580 maximally inhibited superoxide production by approximately 80,60 and 55%,respectively. The failure of each of the inhibitors to totally suppress AA-stimulated superoxide production suggests that activation of a number of kinases/ mechanisms are required for the assembly of an active NADPH oxidase and for optimal superoxide production. The effects of a combination of GF109203X, I'D98059 and SB203580, at concentrations close to their IC50 in intact neutrophils, on AA-stimulated superoxide production were therefore determined. Simultaneous addition of these inhibitors suppressed superoxide production in an additive manner. However, total

Regulation of Neutrophil Functions by Fatty Acids

2 11

suppression of AA-stimulated superoxide production was still not observed, even when these inhibitors were used at twice their IC50229 These data suggest that while PKC, ERK and p38 may play some roles in mediating the effects of AA on superoxide production, other mechanisms may also be involved in mediating the actions of polyunsaturated fatty acids on the respiratory burst. For example, rhoGDI has to be released from rac2 and the latter loaded with GTP, before it can translocate to the plasma membrane.227Other signaling molecules, such as phosphatidylinositol 3-kinase (PI3K), could also play a role in mediating the actions of polyunsaturated fatty acids on superoxide prod ~ c t i o nOur . ~ ~recent ~ studies have indeed demonstrated that the ability of AA to stimulate the superoxide production in neutrophils is dependent on 1~13K.173 Another signaling molecule, which may be involved in mediating, at least in part, the effects of AA on the NADPH oxidase, is phospholipase A2. It has been reported that incubation of neutrophils with radiolabeled AA resulted in the release of radiolabeled AA via the activation of PLA2.39Inhibition of cPLA2 with arachidonyltrifluoroketone inhibited fatty acid-stimulated release of radiolabeled AA and superoxide p r o d ~ c t i o nThis . ~ ~ suggests that endogenously generated AA is involved in mediating the actions of exogenously added AA. It also suggests that exogenously added AA and endogenously generated AA may exist as two distinct pools of AA and each pool may regulate different processes in triggering superoxide production. In contrast to neutrophils, monocytes/macrophages do not release radiolabeled AA when exposed to exogenous AA? suggesting that AA does not stimulate the activity of PLA2 in monocytes. This may provide a reason for the inability of polyunsaturated fatty acids per se to trigger a respiratory burst in monocytes/macrophages. On the other hand, other studies have questioned the role of PKC in the action of AA on neutrophil respiratory b u r s t . 2 3 0 Thus, inhibition of PKC by monochloramine, which inhibited the PMA-stimulated respiratory burst, did not affect the AA-stimulated response.230Other observations which are not consistent with an involvement of PKC or other kinases in the action of AA, include the direct stimulation of superoxide production by AA in reconstituted systems in the absence of ATP and Ca2+.104t231 The ability of SDS to mimic the actions of AA on superoxide

21 2

The Neutrophils

production in the cell-free system, has led to the suggestion that AA acts in a detergent-like manner to stimulate superoxide production. However, studies by Corey and R0soff2~~ have excluded a detergent-like action of polyunsaturated fatty acids as a primary mechanism by which fatty acids stimulate superoxide production. It is clear from cell-free studies that higher concentrations of AA are needed to stimulate superoxide production than from intact neutrophils. Thus, in intact neutrophils, AAstimulated superoxide production was easily detectable at 5 JIM or 1ess,lo2 while at least 25 JIM was needed to elicit a detectable response in cell-free systems.231Hence, very high concentrations of AA (82-160 JIM) were used ~ ~ ~the , ~ability ~ ~ of AA to directly stimulate in these in vitro s t ~ d i e s .While superoxide production in cell-free sytems cannot be denied, it is possible that the discrepancy between our results and those of Ogino et uLZ3O in intact neutrophils could be due to the amount of exogenous AA being used. In our studies, we have used AA up to a maximum of 30 JIM (usually 20 JIM), a concentration which is within levels reported to prevail in stimulated c e l l s 2 3 3 and in plasma of human malaria patients.234The response observed at 30 JIM (giving 10-20 fold stimulation above control) was still in the linear part of a dose-response curve. On the other hand, Ogino et ul.230 used 100pM AA to stimulate their neutrophils. At this higher concentration, the rate of AA uptake would be expected to be higher, resulting in more AA being in the intracellular compartment at any given instance than at a lower exogenous AA concentration.Consequently, the higher amount of AA in the intracellular compartment could have created an environment which resembled that of a cell-free system, thereby allowing AA to predominantly and directly stimulate superoxide production without the need of protein kinases. Although we cannot exclude the possibility that AA also interacts with components of the NADPH oxidase in neutrophils which are exposed to low concentrations of exogenous AA, it is unlikely that the stimulatory effect of AA on superoxide production in our studies is mediated entirely via a direct action of the fatty acid on components of the NADPH oxidase. In support of this, our results and those of Abramson et al.'&l demonstrated that AA-stimulated responses could be inhibited by various kinase inhibitors and pertussis toxin. Futhermore, AA-stimulated superoxide production could be inhibited by antagonists of calcium-binding proteins, and inhibitors and substrates of

Regulation of Neutrophil Functions by Fatty Acids

21 3

chymotrypsin-like proteases, thereby arguing against a detergent-like action of AA on the NADPH oxidase in intact n e ~ t r o p h i l s . ~ ~ It is currently not clear how AA and other fatty acids stimulate the activity of cPLA2.One possibility is via PKC, ERK and/or p38, since PKC, ERK and p38 have all been proposed to regulate the activity of cPLA2by p h o ~ p h o r y l a t i o n . On ~ ~ ,the ~~~ other hand, other studies have not found p38 to be responsible for regulating the activity of the c P L A ~This . ~ discrepancy may be due to cell-type differences. Nevertheless, the regulation of cPLA2by AA, which stimulates PKC and the MAP kinase, suggests the existence of a signaling loop involving the enzymes.*73The intracellular signals employed by AA and other fatty acids to stimulate degranulation, adherence and enhance microbicidal activities have not been extensively studied, although the activation of cPLA2 has been suggested to be required for degran~lation.'~~

12.5. Involvement of ERKl/ERKZ and p38 in Regulating 5-LOX Recent studies have demonstrated that the ERKl/ERK2 and p38 signaling modules are involved in regulating the activity of 5-LOX in neutrophils by 20:4n-6. Thus, Werz et ul.237 demonstrated that the ability of 20:4n-6 to cause the formation of 5-LOX products was blocked by inhibitors of the ERK and p38 pathways. These two MAP kinase modules appear to act in concert on 5-LOX activity. ERKl/ERK2 and p38, acting via MAP kinase activated protein kinase2 (MAPKAPK21,were shown to directly phosphorylate 5 LOX on S663 and S271, respectively. Both sites are crucial for the action of 20:4n-6 on 5-LOX acting.237

13. MODULATION OF TNFR EXPRESSION TNF acts via two receptors, TNFRl (55kDa, CD120a) and TNFR2 (75kDa, CD120b),both of which are expressed on ne~trophils.2~~ Both are susceptible to cleavage following cell a c t i v a t i ~ nA. ~ wide ~ ~ range ~ ~ ~ of agonists cause this effect including LPS, fMLP, GM-CSF and opsonized microbial pathogen^?^^-^^ Thus both exogenous and endogenous mediators of inflammation regulate neutrophil TNFR expression. Interestingly, AA caused the up-regulation of TNFR expression on the surface of n e u t r o p h i l ~This .~~~

21 4

The Neutrophils

contrasted with fMLP and LPS which caused a downregulation of TNFR on the same neutrophil population.243No other agonist has been described which increases neutrophil TNFR expression. Both TNFRl and TNFR2 were upregulated by AA. This up regulation was extensive, increasing the receptor number some %fold, and the AA treated neutrophils showed a marked enhancement of TNF-induced superoxide production.243 Perhaps even more intriguing was the finding that the n-3 PUFA, DHA, EPA and LNA not only failed to increase TNFR expression but that these also caused a decrease in expression of both TNFR receptors on n e ~ t r o p h i l sIt. ~is~not ~ clear as to why these fatty acids should behave so differently. This suggests that n-3 PUFA, in contrast to n-6 PUFA (AA), downregulate the inflammatory response by causing a decrease in the neutrophil response to TNF. The mechanism by which AA increased TNFR expression occurred independently of its metabolism via the lipoxygenase and cyclooxygenase pathways.243Evidence has been presented that AA-induced upregulation of these receptors is dependent on the stimulation of intracellular Another key finding in these studies was signals, PKC, ERK and PLA2.243 that AA-pretreated neutrophils showed not a loss in TNFR expression but a substantial increase when challenged with fMLP.243This finding therefore necessitates a modification of the present concept that neutrophils downregulate their TNFR upon activation by inflammatory mediators and thereby provide a source of soluble receptors. The proinflammatory role of AA is therefore also manifested through an increase in the expression of receptors for the proinflammatory cytokine, TNF. Replacing AA with n-3 PUFA, such as DHA and EPA, provides a means of inhibiting inflammation through the ability of these to downregulate TNFR expression.

14. NOVEL POLYUNSATURATED FATTY ACIDS Recently, we have described long chain polyunsaturated fatty acids which contain an oxygen atom in the p position.244One of these P-oxa-21:3n-3has been studied in detai1.245t246 Compared with the natural n-3 PUFA, P-oxa-21:3n-3 was found to retain the immunosuppressive properties but not the neutrophil stimulating properties of the natural fats.245 This further

Regulation of Neutrophil Functions by Fatty Acids

21 5

supports our concept that structural changes to PUFA can lead to dramatic variation in biological properties. Thus, this concept of p-oxa PUFA with selective anti-inflammatory properties compared with the natural 71-3 fatty acids, defines a new strategy in the development of anti-inflammatory agents. The P-oxa-21:3n-3 was also found to be a strong inhibitor of the 5-L0X, several fold more than DHA and EPA.246Consistent with this result was the finding that this novel PUFA caused marked inhibition of both the chronic and acute inflammatory response.245The P-thia PUFA, where, a sulphur atom was placed in the P position, gave rise to similar increases in selectivity for biological activity (unpublished).

15. SUMMARY The inflammatory response to infection and to autoimmune or allergic diseases is characterized by an accumulation of phagocytes at the sites of inflammation. These and other cell-types become activated by microbial products, opsonized particles and proinflammatory mediators. One of the consequences of cell stimulation is the activation of PLA2and the release of AA and other fatty acids. During inflammation and infection, the concentrations of nonesterified fatty acids in vivo have been reported n~~~ that to be in the range that stimulates neutrophils in vitro. E s ~ i e found plasma-free AA levels in human malaria patients were >lo0 p,M and Yasuda et ul.247 reported that the free AA levels in brain were 50 p,M, rising to 500 p,M under ischemic conditions. Activated phagocytic cells per se produced 20-30 p,M AA.248Our studies and those of others have clearly demonstrated that polyunsaturated fatty acids, such as AA stimulate and regulate a number of key functions of the neutrophil, including adhesion, chemotaxis, activation of the respiratory burst and degranulation. These effects are specific, dependent on the activity of signaling molecules and are not due solely to the ability of a fatty acid to the partition into the plasma membrane. Many of the biological properties of AA are retained by the metabolites such as LTB4and 5-0x0-ETE. Fatty acids and metabolites also modify the responses of neutrophils to other endogenous inflammatory mediators at the sites of inflammation. Thus, AA ( 2 3 p,M) acted synergisticallywith fMLP to stimulate superoxide production. It needs to be appreciated that while in vitro

21 6

The Neutrophils

studies on neutrophil functions have used a single fatty acid, in reality at the sites of inflammation, a range of fatty acids and products will be found and hence the collective concentration of these lipids may be quite substantial. Consequently, the behavior of neutrophils is likely to be influenced by AA and other lipid molecules in vivo and it is expected that the findings described in this Chapter deserve major consideration in events of acute and chronic inflammation. Of particular importance is the demonstration that the biological actions of fatty acids on neutrophils can be dramatically altered by specific alterations to the structure of the fatty acids. These include changes to carbon chain length, addition of hydroxy- or hydroperoxy-groups (and their position), degree of unsaturation and masking of the free carboxyl group. This has at least two implications. Firstly, it means that incorporation of different types of fatty acids into membrane phospholipids will result in a change in the composition of the phospholipids and an altered profile of lipid-based second messenger molecules (diacylglycerol, phosphatidic acid and fatty acids) being generated upon cell activation. This is likely to affect the activity of intracellular signaling molecules such as PKC. Secondly, because such alterations in the fatty acid structure yields molecules which exhibit very specific action^,^^,^^^ activity-dictated chemical engineering to produce novel fatty acids244-246 offers potential therapeutic agents for treating a wide range of diseases. The findings outlined in this Chapter are likely to have important implications in our understanding of the inflammatory reaction, during which inflammatory mediators, including lipids, generated from the cells-types such as monocytes, platelets and endothelial cells at the sites of inflammation, results in the activation of PLA2.This results in the liberation of nonesterified fatty acids such as AA from the sn-2 position of membrane phospholipids. While some of the liberated AA are released into the extracellular space, others are cell-associated. AA can interact with and prime neutrophils and monocytes/macrophages for an enhanced respiratory burst. A likely scenario in which fatty acids can regulate the biological functions of neutrophils is proposed (Fig. 3). Although the liberated AA may exist in an immobilized form and may play a direct second messenger role in regulating neutrophil

Regulation of Neutrophil Functions by Fatty Acids

2 17

platelets, endothelial cells, microorganisms,

fMLP, TNF, GM-CSF, IL8 etc \

Lysophospholipids AA

Modulation of

0; production degranulation receptor expression actin reorganization migration inhibition

Fig. 3 Activated neutrophils and other cell types at sites of inflammation/tissue damage release nonesterified fatty acids and metabolites. These exert direct actions on the neutrophils and alter neutrophil responses to bacterial products, cytokines and other proinflammatory agents. The effects of the fatty acids are mediated by a number of intracellular signaling molecules. Abbreviations - PKC: protein kinase C; MAPK: MAP kinases; LOX: lipoxygenase; cPLA2:cytosolic phospholipase A,; sPLA2:secretory phospholipase A,; PUFA: polyunsaturated fatty acid; PI3K: phosphatidylinositol3-kinase; R receptor; FAT fatty acid translocase.

biological responses, some will be metabolized by the lipoxygenases and cyclooxygenases to yield a number of biologically active products. Some of these metabolites such as LTB4 are neutrophil chemoattractants, which

21 8

The Neutrophils

together with the other chemoattractants, including bacterial products and IL8, cause more neutrophils to infiltrate into the inflammatory sites. Once at the sites of inflammation, neutrophils are prevented from leaving by 20:4n-6. As the number of infiltrating cells increases, the levels of fatty acids and metabolites increase. These lipids exert direct effects on the neutrophils and also amplify the responses of the neutrophils to other inflammatory agents such as cytokines. The profile of AA-derived metabolites changes with time as the inflammatory response progresses, switching from the proinflammatory leukotrienes to the counter regulatory lipoxins. Accumulation of lipoxins at the site of inflammation inhibit the influx of additional neutrophils and hence promote the resolution of the inflammatory response. The lipoxins also promote the phagocytosis of apoptotic neutrophils by tissue macrophages, which is also consistent with the resolution of the inflammatory response. Thus, AA and its metabolites participate in the initiation, progression and termination of neutrophil-mediated inflammatory responses.

ACKNOWLEDGMENTS We are indebted to all our colleagues who have contributed towards our work and this has been appropriately referenced. Our work received funding support from the National Health and Medical Research Council of Australia, the Heart Foundation of Australia, the UNDP/World Bank/ WHO special programme for Research and Training in Tropical Medicine and Channel 7 Children’s Research Foundation.

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139. Gewirtz AT, et al. J Immunol2002; 168:5260-5267. 140. Brink C, et al. Pkarmacol Rev 2003; 55:195-227. 141. Mitchell 5, et al. J Am SOCNephrol2002; 13:2497-2507. 142. Wise H. J Leukoc Biol 1996; 60:480-488. 143. Armstrong RA. BY 1Pkarmacoll995; 116:2903-2908. 144. Mikawa K, et al. Prostaglandins Leukot Essent Fatty Acids 1994; 51:287-291. 145. Boxer LA, et al. J Lab Clin Med 1980; 95:672-678. 146. Zurier RB, et al. J Clin lnvest 1974; 5:297-309. 147. Fantone JC, Marasco WA, Elgas LJ, Ward PA. Jlmmunol1983; 130:1495-1497. 148. Goldstein IM, Malmsten CL, Samuelsson B, Weissmann G. Inflammation 1977; 2:309-317. 149. Spagnuolo PJ, Ellner JJ, Hassid A, Dunn MJ. J Clin Invest 1980; 66:406414. 150. Mancuso P, et al. Crit Care Med 1977; 25:1198-1206. 151. Matsumoto K, et al. J Appl Physiol 1996; 81:2338-2364.

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178. Huang JM, Xian H, Bacaner M. Proc Natl Acad Sci USA 1992; 89:6452-6456. 179. Chow SC, Jondal M. 7 Biol Chem 1990; 265:902-907. 180. Naccache PH, Molski TF, Borgeat P, Sha'afi RI. J Cell Physiol 1985; 122: 273-280. 181. Reynaud D, Pace-Asciak CR. Prostaglandins Leukot Essent Fatty Acids 1997; 56:9-12. 182. Cabrera-Vera TM, et al. Endocr Rev 2003; 24765-781. 183. Krugmann S, Cooper MA, Williams DH, et al. Biochem 2002; 362:725-731.

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7 Cytokine Production by Neutrophils Fre‘de‘ric Ethuin, Sylvie Chollet-Martin”

In addition to their phagocytic and killer functions, neutrophils are also a significant source of cytokines, participating in the inflammatory response during infection. A variety of mechanisms are used by the PMN to produce cytokines: release of granular stores of preformed cytokines, de novo synthesis or enzymatic release of membrane-bound form. Keywords: neutrophils; cytokines

1. INTRODUCTION Inflammation is a beneficial host response to foreign microorganisms and involves numerous soluble factors and cell types, including polymorphonuclear neutrophils (PMN) and macrophages. Infiltration and accumulation of PMN within the tissues is a hallmark of the acute inflammatory response. Neutrophils represent a powerful defense system against invading bacteria; they are the first line of defense and play an “Correspondence to: Sylvie Chollet-Martin. Service d’H6matologie et d’Immunologie et INSERM U479, HBpital Bichat, rue Henri Huchard 75018 Paris, France.

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active role in inflammatory response. After challenge by various stimuli, neutrophils have the capacity to release lytic enzymes with potent antimicrobial potential or generate reactive oxygen intermediates, such as superoxide anion. This phenomenon, known as the respiratory burst, occurs at the beginning of the production of potent oxidants, which are essential for bacterial killing and also potentiate inflammatory reactions. In vitro and in vivo, PMN can also produce a variety of proteins upon appropriate stimulation, involved in their effector functions. They can also produce a variety of cytokines, playing an important role in eliciting inflammation, and significantly contributing to the regulation of immune response. This chapter will summarize recent knowledge on the production of cytokines by PMN. For a more extensive report, the reader may consult the last review of M. Cassatella in the previous version of this book. We focus our subject on new cytokines released or new mechanisms involved in the production of cytokines by PMN. The fact that neutrophils can synthesize, store and release a wide array of cytokines necessitates a redefinition of the role of neutrophils in pathophysiology.

2. GENERAL FEATURES OF CYTOKINE PRODUCTION BY HUMAN NEUTROPHILS First, at least in vitro, it is noteworthy that the extent of cytokine production by neutrophils is relatively low, especially when compared with peripheral blood mononuclear cells (PBMC).To investigate whether neutrophils produce a given cytokine (or any protein), it is absolutely mandatory to work with highly purified PMN populations (>99.5%). It is also highly recommended to exclude the possibility of prestimulation of PMN during their isolation procedures, which may be driven, for instance, by contamination of reagents, solutions or labware with trace levels of endotoxin, or by the use of ammonium chloride for erythrocyte lysis. Isolation procedures with Ficoll, dextran sedimentation and hypotonic lysis do not have any stimulatory effects on neutrophils and are recommended. Secondly, a wide range of stimuli capable of inducing cytokine synthesis in PMN has been identified: lipopolysaccharide (LPS); cytokines themselves; phagocytic particles and microorganisms (such as bacteria, fungi and viruses); chemotactic factors (such as formyl-methionyl-leucyl-phenylalanine

Cytokine Production by Neutrophils

23 1

(fMLP); leukotriene €34 (LTM); platelet-activating factor (PAF); the complement component C5a; and neuroimmunomodulatory substances. In general, not only do the magnitude and kinetics of cytokine release vary substantially depending upon the stimulus used, but the pattern of production is thereby influenced to a great extent by the stimulus used. For instance, Suttmann et al. investigated by cDNA microarrays and RT-PCR the capacity of Mycobactevium bovis bacillus Calmette-Guerin (BCG) to stimulate PMN gene expression. Stimulation with BCG alters the expression of various genes for proinflammatory cytokines or chemokines in PMN. An upregulation or de novo synthesis of IL-la, IL-1/3, IL-8, MIP-la, MIP-1P, GROa, TGFP, MCP-1, IL-2Ry, IL-lORa, and IL-6R was detected, whereas genes for IL-9, IL-12aU, IL-15, IL-5Ra, and IL-13Ra were found to be downregulated or switched off.'

3. PRODUCTION O F SPECIFIC CYTOKINES BY NEUTROPH I LS 3.1. Chemokines Chemokines are leukocyte attractants with two major groups based upon the positions of the first two cysteine residues in their primary sequences: the "C-X-C" and the "C-C" subfamilies. RANTES, macrophage inflammatory protein-la (MIP-la), MIP-1P and MIP-1y are members of the CC chemokine subfamily produced by neutrophils when cultured with either LPS or TNFa. They predominantly have monocyte-, eosinophil-, basophiland T lymphocyte-chemotactic properties? By contrast, the C-X-C subfamily predominantly exerts stimulatory and chemotactic activities towards neutrophils.2 Human neutrophils have the capacity to produce a number of CXC chemokines, including interleukin-8 (IL-8/CXCL8), the prototype, growth-regulated oncogene alpha (GROa/CXCLl), IFNy-inducible protein of 10 kDa (IP-lO/CXCLlO), monokine induced by IFNy (MIG/CXCL9), IFN-inducible T cell a chemoattractant (I-TAC), epithelial cell derived and neutrophil-activating properties, 78 amino acids (ENA-78), and cytokine-induced neutrophil chemoattractant (CINC). 1. IL-8 and GROa. IL-8 is one of the potentially most important (and most extensively) studied cytokine produced by neutrophils. PMN are

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the primary targets for IL-8, responding to this mediator by chemotaxis, release of granule enzymes, respiratory burst, upregulation of CRl and CDll /CD18 expression on the surface, and increased adherence to unstimulated endothelial cells.2 In addition, IL-8 has chemotactic activities for T lymphocytes and basophils, though much less effectively than for neutrophils, and is also an angiogenic factor.2Similar to IL-8, GROa acts as a mediator of inflammation, as it has powerful chemotactic and activatory properties on PMN, including degranulation, increased expression of adhesion molecules and in vivo recruitment of neutrophils to sites of inje~tion.~.~ Therefore, generation of GROa by neutrophils may contribute to stimulating the recruitment to, and activation of, further neutrophils at the sites of inflammation, in addition to IL-8. A study by Villard et al.? for instance, has shown that the concentrations of GROa and IL-8 were markedly elevated in BAL of three acute pathologic states: bacterial pneumonia (BPN); adult respiratory distress syndrome (ARDS); and Pneurnocystis carinii pneumonia (PCP). The levels of these two chemokines were higher in the ARDS and BPN groups than in the PCP group; and the levels of GROa were consistently higher than those of IL-8, whereas the BAL levels of both IL-8 and GROa were basically undetectable in 16 subjects of the control groupa5The production of these chemokines by LPS-stimulated neutrophils is negatively modulated by IL-10, The recruitment of neutrophils from the vascular space is an early step in the host innate immune response to bacterial invasion, but seems to be organism specific. Nevertheless, it appears that IL-8 acts directly on neutrophil infiltration, whereas GROa acts indirectly, in part via TNFa production. In vitro, GROa induced TNFa activity in cultured synovial cells, when IL-8 failed to produce TNFa activity from the cells, although equivalent levels of the mRNA expression were induced by IL-8 as compared with GROa. Thus, the functional distinction between IL-8 and GROa may influence the inflammatory responses. Firm adhesion of rolling neutrophils on endothelium after stimuli is dependent on P2 integrins (CD18).At inflammatory sites, LFA-I (CDlla/CD18) appears to be more important than Mac-1 (CDllb/CD18) in neutrophil emigration with different functional contributions. IL-8, GROa, and leukotriene B4 (LTB4) at subnanomolar concentrations induced rapid and optimal rates of LFA-1-dependent adhesion of neutrophils to intercellular adhesion

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molecule (1CAM)-1-coated beads. This LFA-1-dependent adhesion was transient and decayed within 1min after chemoattractant stimulation, whereas Mac-1 adhesion was equally rapid initially but continued to rise for up to 6 min after stimulation. Activation of LFA-1 and Mac-1 by GROW was completely blocked by anti-CXC chemokine receptor R2, but activation of these integrins by IL-8 was most effectively blocked by anti-CXC chemokine receptor R1. Moreover, PMN generation of CXC chemokines as IL-8 and GROa in an autocrine/paracrine mechanism contributes via the suppression of apoptosis to the amplification of the PMN inflammatory response. Suppressing apoptosis, IL-8 and GROa stimulate their own production and PMNs maintain their ability to respond to these chemokines through expression of the CXC receptor^.^ In vitro, CXCRI mediating neutrophil responses to IL-8, remain upregulated after prolonged stimulation. 2. IFNy-inducible protein of 10 kDa (IP-10). IP-10 is produced and released by human neutrophils.6 Despite its structural homology to IL-8, IP-10 is predominantly chemotactic for lymphocytes and NK cells, as opposed to neutrophils. IP-10 is specifically produced in response to IFNy by monocytes, lymphocytes, keratinocytes and endothelial cells. Surprisingly, in neutrophils, IFNy alone had only a modest effect on IP-10 mRNA accumulation. However, stimulation of PMN with IFNy in combination with either TNFa or LPS (but not with Y-IgG or fMLP) resulted in a considerable induction of IP-10 mRNA transcripts, as well as in the extracellular release of the protein. The generation of IP-10 by PMN may significantly contribute to recruiting NK cells, monocytes and activated T lymphocytes to the sites of inflammation.2Although it is still too early to speculate on an eventual in vivo role of neutrophil-derived IP-10, it is noteworthy that preliminary experiments by Cassatella et al. indicate that PMN can also produce MIG, another chemokine homologous to IP-10 and with similar biological properties. 3. Macrophage inflammatory protein-la and p. The ability of stimulated neutrophils to secrete MIP-la and MIP-1P is well documented.7,8MIP-la and MIP-1p act as potent chemotactic/activating factors for monocytes and subpopulations of T lymphocytes, and also activate several effector functions of macrophages and neutrophils. Stimulation of PMN in the presence of both LPS and GM-CSF resulted in

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a synergistic expression of both MIP-la mRNA and protein, compared with LPS alone. Among various stimuli, TNFa exerted a significant effect on MIP-la mRNA expression and secretion. In unpublished experiments, Cassatella et al. could confirm that neutrophils express MIP-la and MIP-1P mRNAs, not only in response to LPS or TNFa, but also to Y-IgG. 4. IL-17 is a cytokine implicated in the regulation of inflammation. It was thought that IL-17 production was restricted to activated T lymphocytes. In a murine model of LPS-induced lung inflammation, IL-17 levels were increased in the bronchoalveolar lavage and the expression of IL-17 mRNA was associated with CD4(+) and CD8(+) cells, but also with neutrophils. IL-17 could then play a physiological role in orchestrating the neutrophil activity in the lungs, following bacterial i n f e c t i ~ n . ~In ,'~ humans, increased levels of IL-17 have been associated with pathological conditions such as rheumatoid arthritis, intraperitoneal abscesses, inflammatory bowel disease, allograft rejection, psoriasis, cancer or multiple sclerosis.ll IL-15 was also shown to be produced by PMN and peripheral blood mononuclear cells (PBMC) in patients with Lyme disease.12 Nevertheless, these results need to be confirmed, with regard to the neutrophil purification methods.

3.2. Proinflammatory Cytokines Besides mononuclear cells, which represent the major source of cytokines in blood, available data indicate that neutrophils produce a wide range of cytokines with the capacity to modulate immune response. In isolated cell preparations, proinflammatory cytokine production by PBMCs is significantly greater compared with PMN. On a per cell basis, PMN produced less than 1.5% of cytokines compared with PBMC. Nevertheless, the capacity of neutrophils to generate proinflammatory cytokines is now firmly established. During infections, neutrophils infiltrate inflammatory sites in large numbers and predominate over other cell types. They could therefore significantly represent a substantial source of cytokines and participate in the cytokine environment. 1. Tumor Necrosis Factor a (TNFa). PMN have the ability to either express TNFa mRNA or secrete the related protein in vitro in response to

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LPS, PMA, fMLP (under specific conditions), IL-lp, IL-2 primed by GMCSF, Candida albicans, E.coli, Staphylococcus aureus, Klebsiella pneumonia, or phagocytosis of Y-IgG. Interestingly, maximal yields of TNFa in neutrophil supernatants in response to Y-IgG, as well as to LPS, were detected after 5-6 h of stimulation, which then declined over time. Thus, if TNFa measurement is carried out after 24 h of PMN stimulation, it is possible to find no TNFa in cell-free supernatants because of proteolytic enzymes (elastase, cathepsin G) which results in TNFa decays with time. TNFa is a potent stimulus of PMN themselves, promoting adherence to endothelial cells and to particles, and leading to increased phagocytosis, respiratory burst activity and degranulation. The ability of PMN to release TNFa in response to so many different stimuli, suggests that granulocytes may exert host defense functions that go beyond the killing of invading microorganisms in septic infections, and may therefore represent a manner whereby neutrophils can activate themselves in an autocrine/paracrine fashion. TNF-related apoptosis-inducing ligand (TRAIL) is a member of the TNF superfamily, inducing apoptosis in tumor and virus-infected cells, but rarely in normal cells. Expression of both TRAIL mRNA and protein and TRAIL receptors has been detected in neutrophils. IFNy upregulates the expression of TRAIL, whereas TNFa is a d o ~ n r e g u l a t o r . These '~ results suggest a role for the TRAIL/TRAIL receptor system in immune surveillance and neutrophil apoptosis. 2. Interleukin-1. Neutrophils induce small amounts of IL-1 (hundreds of picograms as a maximum) or an IL-1-like activity after stimulation with particulate and soluble agents, such as LPS, zymosan or PMA. Moreover, neutrophils stimulated with GM-CSF were observed to express the mRNA and release of both IL-la and IL-1p with regulation at both the transcriptional and post-transcriptional levels. IL-1p mRNA accumulation is also induced in a time- and dose-dependent manner by IL-1p and/or TNFa. Neutrophils produce and release IL-1p not before 5-6 h following stimulation with TNFa. 3. Interleukin-6. The complete lack of IL-6 gene expression in PMN has now been demonstrated. The presence of IL-6 mRNA or protein is considered as a marker for monocyte contamination. There are, however, several published articles showing that PMN expresses IL-6, but

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The Neutrophils

neutrophil preparations were less than 98% pure. Wang and colleague^'^ demonstrated that if neutrophils are prepared with extreme caution, so that monocyte contamination is kept below 0.7%,IL-6 release from PMN is undetectable. 4. Interleukin-12. IL-12 is a heterodimeric cytokine produced mainly by phagocytic cells (monocytes/ macrophages) and antigen-presenting cells (APCs) in response to bacteria, bacterial products, fungi or viruses. IL-12 acts as a proinflammatory and immunomodulatory cytokine on T and NK cells, inducing IFNy production, proliferation and enhanced cytotoxic activity. IL-12 is also a factor of the nonspecific inflammatory response by enhancing IL-8 production by PMN and thus contributing to PMN recruitment at the inflammatory sites.15 Therefore, the ability of neutrophils to produce IL-12 suggests that they may play an active role in the regulatory interactions between innate resistance and adaptive immunity, and, at the same time, favor a Thl-type immune response. In pathological conditions, PMN from patients with systemic lupus erythematosus may have a defect in IL-12 expression, and this defect may be exaggerated in the presence of IFN-y, which normally stimulates IL-12 production. This could account for an increased susceptibility to multiple infections in patients with systemic diseases.16Conversely, during severe sepsis, an upregulation of IL-12 release by PMN can be observed depending on the body compartment (circulating blood or alveolar cells) and the IL-12 isoform (p40or ~ 7 0 1 . ' ~ 5. Interferon (IFN) a. Neutrophils produce IFNa protein as well as mRNA in G-CSF-stimulated cells in a time-dependent manner. By contrast, neither LPS nor fMLP effectively stimulate the expression of IFNa in PMN. Brandt et aI. have shown that PMN accumulate IFNa mRNA in a constitutive manner or upon infection with the Sendai virus.18 Interestingly, the antiviral activity of supernatants recovered from PMN stimulated with Sendai virus was very similar to that detected in PBMC, but much more abundant than those measured from purified T and B cells, emphazing the potentially important role of PMN in host defense against viral infection. 6. Interferon y (IFNy). IFNy is a Thl cytokine mainly produced by T cells, NK cells, and macrophages in response to IL-12. Relatively little

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information is available on PMN secretion of Thl cytokines. It has been suggested that PMN may synthesizeIFNy. PMN have been observed in human end~metrium,'~ as well as in lung and spleen tissue of two different murine models of infection?0,21Yeaman et al. found that isolated PMN released IFNy after IL-12 + TNFa stimulation in vitro, while other investigators failed to detect IFNy after LPS stimulation.22For Hodge-Dufour et al., the early production of IFNy by PMN both acts as a primer in increasing IL-12 production by macrophages and initiates differentiation of TH1 type T lymphocytes for T cell-dependent control of infection.23Personal unpublished data seems to confirm these findings and reveals a new pathway of autocrine and paracrine PMN activation, idenbfying a new role for IFNy, bridging innate and adaptive immune responses via IFNy and IL-12 interactions.

3.3. Anti-Inflammatory Cytokines A particular aspect of inflammatory response is the capacity of a selfregulating network through the production of cytokines and cytokine inhibitors by mononuclear cells and neutrophils. IL-4, IL-10, IL-13 and TGFP play a major role in the regulation of immune responses and are considered to be anti-inflammatory agents mainly due to their actions on monocytes. Nevertheless, these cytokines are also known to participate in the regulation of PMN activities. PMN do not express IL-10 and IL-13.24 1. Interleukin-4. Using intracellular flow cytometry analysis, IL-4 was shown to be produced by PMN. Immunostaining on cytospin preparations of normal granulocytes also confirmed the presence of intracellular IL-4.25Evidence for the presence of functional IL-4 receptors on human neutrophils was demonstrated by Girard et a1?6 IL-4 (and IL-10) suppressed the ex vivo activation state of IFNy- and TNFcx-activated human neutrophils and reduce their phagocytic c a p a c i t i e ~However, .~~ IL-4 induces RNA synthesis in a concentration-dependent manner for cytoskeletal protein actin as well as activates neutrophil cytoskeletal rearrangements participating in neutrophil apoptosis delay.26 Antiinflammatory cytokines can therefore exert powerful regulatory effects on neutrophil functions in an autocrine fashion and IL-4 could be a more potent neutrophil agonist than previously believed.

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UVB irradiation causes modification in the skin at both the cellular subtype and cutaneous cytokine levels. Teunissen et al. have shown that normal human skin exposed to UVB exhibits an infiltration of numerous IL-4 positive cells. These IL-4(+) cells do not express CD3 (T cells), tryptase (mast cells), CD56 (NK cells), nor CD36 (macrophages). By contrast, they coexpress CD15 and CDllb, by a correlation with elastase, indicating that UVB-induced infiltrating IL-4(+) cells are neutrophils.28 Moreover, fluid from irradiated skin, but not from control skin, contained IL-4 protein. In psoriasis patients, upon a single high dose of UVB irradiation, IL-4 expression is enhanced at the level of lesional skin. Piskin et al. have shown that the IL-4 protein detected in the irradiated skin of both healthy controls and these patients, is also associated with infiltrating neutrophils because of coexpression of elastase and CD15, but not CD3.29 Crepaldi et al. have shown that IL-10 enhances the release of IL-1RA from IL-4-stimulated neutrophils, through IL-1RA mRNA stabilization and enhancement of protein de novo synthesis.30The fact that IL-10 strongly upregulates IL-1RA production in IL-4-activated neutrophils reveals a new mechanism in which IL-10 and IL-4 cooperate to negatively modulate the inflammatory responses. 2. Transforming growth factor. Human neutrophils constitutively express TGFPl -P2 mRNA and secrete high levels of the protein in a fully active form.31Interestingly, stimulation of neutrophils with LPS, fMLP or immune complexes for 24 h, results in no difference in the levels of TGFPl protein compared with untreated cells or monocytes. Remarkably, unstimulated PMN secreted approximately five times more TGFP than an equal number of unstimulated monocytes, over a 24 h period in culture. Thus, PMN may represent an important potential source of TGFP which could play an important role in situations such as wound repair, chronic immuno-driven inflammations and immune responses, or in the pathogenesis of fibrotic disease. Neutrophils also store TGFa in cytoplasmic vescicles. TGFa is a polypeptide belonging to the family of EGF-related protein and exerts several effects on target cells, such as mitogenic signaling and promotion of neovascularization. It has also been suggested that TGFa is involved in wound healing and in tumor development. No colocalization of TGFa with components of azurophilic or specific granules or secretory vesicles was observed in

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239

neutrophils, suggesting that TGFa-containing granules differ from the three main kinds of granules or from the rapidly mobilizable pool of secretory vescicles. The findings that neutrophils contain TGFa might help to explain complications caused by chronic inflammation, such as fibrosis and neoplastic transformation.

3.4. Cytokine Inhibitors Endogenous inhibitors of cytokine activity include soluble types I and I1 TNF receptors (sTNF-RI and sTNF-RII) and the IL-1 receptor antagonist (IL-1RA).Anti-inflammatory cytokines IL-4, IL-10, IL-13 and TGFP exert direct anti-inflammatory properties via their ability to repress the production of proinflammatory cytokines. They can also favor the release of cytokine inhibitors such as sTNF-RII and IL-1 receptor antagonist (ILlRA), which are increased by LPS stimulation in purified PMN. IL-4 and TGFP were able to increase the production of IL-IRA. However, only IL-4 was able to further increase IL-1RA production in the presence of LPS. Whereas IL-10 is unable to induce IL-1RA alone, when its production by PMN is induced by TNFa, both IL-10 and IL-4 amplified its release and its presence as a cell-associated form?2 IL-13 and TGF-P do not modulate LPS- nor TNFa-induced IL-1RA production by PMN. Neutrophils take place through IL-1RA mRNA stabilization and enhancement of IL-IRA de novo synthesis. The release of IL-IRA from IL-4-stimulated neutrophils is markedly enhanced in the presence of IL10, but requires 5-6 h, preceded by the activation of Stat3 tyrosine phosphorylation. This latter response to IL-10 was strictly dependent on the The fact that IL-10 strongly upregulates levels of expression of IL-~OILJ%?~ IL-1RA production in IL-4-activated neutrophils, reveals a novel mechanism whereby IL-10 and I L 4 cooperate to negatively modulate the inflammatory responses. Oncostatin M (OSM). OSM is a member of the IL-6 family with the capacity to recruite leukocytes. In a human system in vitro, OSM exhibits a significant effect on leukocyte rolling and adhesion. These doseresponse effects, even at a very low concentration (10ng/ml), are comparable to levels seen with TNFol but are selective for neutrophil recruitment. The molecular mechanism underlying the PMN recruitment

240

The Neutrophils

seemed to be dependent on P-selectin.N Normal human PMN can degranulate and synthesize OSM, and Hurst et al. have shown that OSM synergistically blocked IL-1P-induced IL-8 secretion in combination with the IL-6/sIL-6R ~ o m p l e x ?These ~ . ~ ~results suggest that OSM release from infiltrating neutrophils might contribute to the temporal switch between neutrophil influx and mononuclear cell recruitment seen during acute inflammation.

3.5. Growth Factors

1. Hepatocyte growth factor (HGF). HGF, a growth factor for type I1 pneumocytes, has been shown to be stored in neutrophils, mainly in specific granules. Moreover, in vituo, blood and alveolar neutrophils isolated from patients with acute respiratory failure or pulmonary fibrosis can produce HGF that may take part to the alveolar repair p r o c e ~ s . ~ ~ . ~ ~ Keratinocyte growth factor (KGF) is another growth factor for type I1 pneumocytes and seems to also play a specific role in the process of alveolar repair. Stern et al. have demonstrated that KGF and HGF are present in biologically active concentrations in human pulmonary alveoli in acute respiratory insufficiency and that circulating neutrophils are an important source of HGF.39These results demonstrate a new and beneficial role for neutrophils in terms of factors of repair mechanisms after injury. 2. Vascular endothelial growth factor (VEGF). VEGF, an endothelial cell mitogen, is a growth factor with also potent vascular permeability enhancing and chemoattractant properties for leukocytes. Gaudry’s works have shown that PMA, fMLP, and TNFa triggered a timedependent secretion of VEGF by human neutrophils from degranulation of a preexisting intracellular The subcellular fractionation of human neutrophils showed a granule-specific distribution of the intracellular pool of VEGF in resting neutrophils. Neutrophil-VEGF mRNA and protein expression have also been reported by o t h e r ~ . ~ Ancelin lb~ et al. have recently reported that, among the several VEGF isoforms, VEGF189 (V189) is selectively induced in endometrium during the mid-late phase of the menstrual cycle, together with PMN influx. In transmigration and under-agarose assays, VEGF189 was both chemotactic and chemokinetic for PMN, while VEGF165 was only chemokinetic. All the VEGF isoforms

Cytokine Production by Neutrophils

241

slightly upregulated pl- and P2-integrins and PECAM, but downregulated L-selectin. Even if RT-PCR analysis showed that V165 mRNA was more strongly expressed than V189 mRNA, the major protein isoform secreted after optimal PMN degranulation was V189, located in both azurophilic and specific granules. PMN-derived VEGF can thus modulate PMN migration via an autocrine amplification mechanism.43Therefore, VEGF could participate in the influx of PMN of the acute phase inflammatory response, as well as neovascularization and other endothelial cell alterations. 3. Colony-stimulating factors (CSFs). The colony-stimulating factors are crucial cytokines for hematopoiesis and immune competence of many leukocytes. This group consists of the macrophage-CSF (M-CSF), granulocyte-CSF (G-CSF), granulocyte/macrophage-CSF (GM-CSF), and IL-3. GM-CSF participate in the early recruitment of polymorphonuclear cells and later recruitment of mononuclear cells or dendritic cells, whereas G-CSF plays a role in the proliferation, differentiation, and survival of macrophages and neutrophils. G-CSF also plays an important role in regulating PMN chemokine responsi~eness.~~ Neutrophils do not seem to be high producers of these growth factors, which are efficient inducers of PMN functions. By contrast, the effects of G-CSF are mediated by binding to receptors, expressed on the surface of human neutrophils. Human neutrophil elastase (HNE), stored in the primary granules, is a serine protease, which proteolytically cleaves numerous cytokines and cell surface proteins. HNE resulted in rapid proteolytic cleavage of G-CSF as well as G-CSFR on the surface of cells. This effect is associated with a reduction in cell viability and biologic activity, supporting a role for neutrophil elastase in a negative feedback to granulopoiesis by direct antagonism of G-CSF.45,46 Furthermore, Iwasaki et al. reveal a physiologically secreted human soluble G-CSFR (sG-CSFR)of two different molecular sizes (80 and 85 kDa), correlated with the numbers of neutrophils and which could play an important role in myelopoiesis through their binding to serum G-CSF. Both isoforms of sG-CSFR bind recombinant human G-CSF and a RT-PCR analysis reveals that membrane-anchored G-CSFR and sG-CSFR mRNA is expressed on CDllb + CD15 cells (mature n e ~ t r o p h i l s )The . ~ ~ suppressor of cytokine signaling (SOCS) is the recently cloned cytokine-inducible SH2-containing protein (CIS) and represents a potential modulator of

+

242

The Neutrophils

cytokine signaling. SOCS3 has been shown to be an important negative regulator of cytokines that activate STAT3.48G-CSF are efficient inducers of STAT1 and STAT3 tyrosine phosphorylation in PMN. SOCS3 is a negative regulator of G-CSF signaling in neutrophils and granulopoiesis therefore contributes to n e ~ t r o p e n i a .IL-10 ~ ~ f ~represents ~ also an efficient stimulus of SOCS3/CIS3 mRNA expression in human n e ~ t r o p h i l s . ~ ~ Interestingly, SOCS-3 expression is restricted to macrophages and neutrophils. During sepsis, cytokines and bacterial toxins have the ability to suppress the function of immune cells by upregulating SOCS-3.52

4. CROSS-TALK WITH OTHERS CELLS The cross-talk between different cell populations is an important component of the innate immunity. The following examples emphasize the role of PMN-derived cytokines in this phenomenon. Type I inflammatory cytokines like IFNy are essential for immunity to many pathogens, including intracellular bacteria, parasites and mycobacteria. Dendritic cells (DC) are the key cells in initiating type 1 immunity, but neutrophils are also a source of chemokines and cytokines involved in T helper1 response. For example, T gondii triggered neutrophil production of CC chemokine ligand (CCL)3, CCL4, CCL5, and CCL20, which are chemotactic for immature DC. Moreover, parasite-stimulated PMN induced IL-12p40 and TNFa production or CD40 and CD86 upregulation by DC. Indeed, polymorphonuclear neutrophils exert an important influence on DC activation, as confirmed in vivo by examining splenic DC cytokine production following infection of neutrophil-depleted mice. These animals displayed severely splenic DC IL-12 and TNFa production. In vitvo, supernatants from stimulated neutrophils not only induced chemotaxis of both immature and mature dendritic cells, but also triggered rapid integrin-dependent adhesion of lymphocytes to purified VCAM-1 and ICAM-1, via MIP-3a/CCL20 and MIP-3P/CCL19 production. Neutrophils with the capacity to produce MIG, IP-10 and I-TAC, which are potent chemoattractants for NK cells and Thl lymphocytes, might contribute to the progression, evolution and regulation of the inflammatory response. This regulatory role for neutrophils in DC function during microbial infection suggests that cross-talk between these cell

Cytokine Production by Neutrophils

243

populations is an important component of the innate immune response to infection.53Moreover, neutrophils might orchestrate the recruitment of specialized cell like T lymphocytes and NK cells to the inflamed sites and therefore contribute to the regulation of the immune response. B lymphocyte stimulator (BLyS), a member of the TNF ligand superfamily, is implicated in B cell maturation and survival. Human neutrophils, stimulated by G-CSF or, less efficiently, IFNy, express high levels of BLyS mRNA and release biologically active BLyS in the same magnitude as monocytes or dendritic cells. In activated neutrophils, only soluble BLyS is released, processed intracellularly by a furin-type convertase, and the surface expression of the membrane-bound BLyS is not detected. Scapini et aI. have shown that BLyS serum levels as well as neutrophilassociated BLyS are significantly enhanced after in vivo administration of G-CSF in patients. Neutrophils might represent an important source of BlyS and might play an unsuspected role in the regulation of B cell homeo~tasis.~~ PMN also establish cross-talk with highly specialized tissue. For instance, nociceptin and its receptor, neuropeptide of the opioid family, are present and functional in human neutrophils, and the results identify a novel cross-talk pathway between neural and immune tissues. Freshly isolated PMNs were found to express and secrete nociceptin following degran~lation.~~

5. PATTERNS OF CYTOKINES PRODUCTION IN HUMAN NEUTROPHILS Multiple sources of cytokines are found in PMN: granular pool mobilized following degranulation, plasma membrane pool mobilized upon stimulation, de novo synthesis, and membrane receptor-bound form. Depending on the cytokine, one of these mechanisms is significantly involved, whereas the others are absent or of less importance.

5.1. Degranulation Several cytokines have been shown to be intracellularly stored in the PMN in various granule types. Subcellular fractionation, followed by

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The Neutrophils

western-blotting using specific antibodies, allowed the recent findings, as well as intracellular immunocytochemistry or flow cytometry. For example, degranulation of HGF and VEGF from the specific granules represents the major mechanism of these cytokines released from PMN.43s6 Degranulation is the process by which PMN releases proteins from intracellular vesicles or granules when interacting with surfaces such as endothelium. Degranulation therefore influences PMN functional responses, including adhesion, aggregation, motility and bactericidy. Indeed, endothelial cells stimulated by inflammatory cytokines can express degranulating factors (e.g. IL-8) and adhesion molecules (e.g. Eselectin) for PMN (juxtacrine activation of n e ~ t r o p h i l s )Human .~~ PMN contains primary ("azurophilic") and secondary ("specific") granules as well as gelatinase-containing granules and a population of secretory vesicles. Lactoferrin and myeloperoxidase (MPO) are markers of specific granules, whereas elastase is a marker of the primary granule enzyme. The expression of PMN cell surface degranulation markers involved CD63 and CD66b. Activation of neutrophils results in mobilization of granular contents with secretory vesicles first, followed by gelatinasecontaining, secondary and primary granules. Neutrophil degranulation is an important event in acute inflammatory responses since granules are, for instance, reservoirs for chemotactic factor receptors, as well as integrins, adhesive molecules or l a ~ t o f e r r i n .In ~ ~local , ~ ~and systemic inflammations, neutrophils can also release other toxic products, such as proteins and reactive oxygen species (ROS) used to inactivate foreign microorganisms in the innate immune response. Antimicrobial and potentially cytotoxic substances stored in the neutrophil granules are delivered into the phagosome or to the exterior of the cell following degranulation. However, proteases and hydrolases can also induce vascular and tissue injury when released in an unregulated fashion such as in sepsis or acute respiratory distress syndrome.60Exocytosis allows granular or vesicular fusion with the plasma membrane, resulting in the release of granule/vesicle contents to the cell exterior. This process is primarily the consequence of inflammatory cell activation and mediator elaboration, but could also contribute to tissue damage and remodeling in inflammatory diseases.

Cytokine Production by Neutrophils

245

The intracellular and molecular cascade regulating the mobilization of secretory granules and vesicles uses a convergence of pathways leading to mediator secretion from neutrophils. A role for Src family kinases in a this signaling pathway leading to granule-plasma membrane fusion has been described, as the role of Fgr and Hck involved in the control of adhesion-dependent degranulation in the inflammatory site.61

5.2. De Novo Protein Synthesis Although mature PMN are terminally differentiated cells, numerous studies have made it clear that neutrophils are also capable of de nouo protein synthesis via RNA/protein synthesis capacity with the activation of transcriptional machinery. This gene expression may represent an additional neutrophil function after exposure to stimulus and the transcriptional potential of neutrophils is greater than previously thought. Indeed, numerous genes are expressed principally in neutrophils. Using cDNA microarray containing 240 cytokine genes between the neutrophils and peripheral blood mononuclear cells (PBMCs) obtained from healthy human donors, Koga et al. have shown that 26 genes were expressed in neutrophils at a level ten times higher than those seen in phytohemagglutinin-stimulated PBMCS.~~ IL-8 is one of the potentially most important (and most extensively) studied cytokine produced by neutrophils. Numerous studies showed that PMN not only release substantial amounts of IL-8 into the culture supernatants after LPS stimulation, but also express significant steady-state levels of IL-8 mRNA. Moreover, Cassatella et al. have shown that PMN, stimulated with TNFa, produce IL-8 mRNA in a time- and dose-dependent manner.63The respiratory syncytial virus (RSV) also results in an enhancement of IL-8 mRNA steady-state levels, accompanied by the secretion of IL-8 in a time- and dose-dependent manner, depending on the adherence of viral particles and on a phagocytic event. Involvement of the Fcy-receptors might thus play a role in enhancing the synthesis and/or secretion rate of the de novo-synthesized cytoplasmic IL-8 pool. TGFP, IL-2, IL-12 and IL-13, were all reported to induce IL-8 mRNA expression and secretion by PMN. However, such observations were not confirmed by other authors. It is to be noted that constitutive IL-8 transcripts decrease almost completely

246

The Neutrophils

within a few hours of cell culture in the absence of stimulation. Despite the presence of specific mRNA, secretion of IL-8 by unstimulated cultured human neutrophils is always very low (below lOOpg/ml).

5.3. Shedding of Membrane-bound Cytokine TNFa is first produced as a 26 kDa membrane-associated protein (mTNFa). The expression of the membrane form of TNFa on the PMN surface was shown by Vulcano et al.64 Proteolytic cleavage (shedding) of mTNFa and release of the soluble 17kDa cytokine is due to TNF-alphaconverting enzyme (TACE), which is expressed in resting PMN and can be upregulated during activation (Ref. 65, N. Kermarrec personnal data). Both forms are biologically active, acting during paracrine cell interactions. TACE is also thought to be a potentially important regulator of inflammation by mediating the shedding of several other mediators, such as L-selectin or IL-6R involved in the resolution of inflammation.66 It was shown that neutrophils exposed to a variety of stimuli exhibit a downregulation of both the 55 kDa (TNF-R55) and the 75 kDa (TNF-R75) TNF receptor, by shedding or internalization. The TNF-induced shedding of TNF receptors in neutrophils involved TACE, also named a disintegrin and metalloproteinase (ADAM-17).67This proteolytic cleavage of the extracellular domain of the receptor could generate soluble TNF-binding proteins that prevent excessive bioactivity of the free cytokine and represents a new way of regulating innate immune and inflammatory responses by increasing cytokine receptor shedding.

5.4. Expression of Receptor-bound Cytokine Cytokine receptors can exist in both membrane-bound and soluble forms, binding their ligands with comparable or different affinity. The soluble form is often thought to be an antagonist, by way of competition between the ligand and the membrane counterpart. Nevertheless, some soluble receptors are agonists, mediated by the ligation of the cytokine and its soluble receptor to a second receptor subunit on the target cell (transsignaling). Soluble receptors of the IL-6 family are an example and could play a

Cytokine Production by Neutrophils

247

role in the shift from neutrophil to monocyte recruitment at the inflammatory site. Similarly, cytokine can exist in both membrane-bound and soluble forms. Interleukin-8 is a major chemotactic and activating factor for neutrophils, inducing chemotaxis, degranulation and priming of the respiratory burst. Promoting neutrophil infiltration as well as triggering the release of proteolytic enzymes and reactive oxygen species, IL-8 contributes to the neutrophils-mediated tissue damage during inflammatory diseases. High levels of leukocytes-associated IL-8 were detected in blood samples from patients with sepsis syndrome. Circulating cells may therefore be both a source of IL-8 and a way for a rapid clearance of IL-8 from plasma. Trapping of free cytokines on the cell surface and the internalization of the IL-8 through binding to the chemokine receptor, occur both in vitro and in vivo and represent a component of the innate immune response regulation.68

5.5. Modulation of PMN-derived Cytokine Release Using Various Mechanisms Endogenous or exogenous mediators in vivo and in vitro can modulate cytokine release by PMN using the various mechanisms described above. One single mediator is able to interact simultaneously with degranulation, membrane shedding or mRNA transcription of several cytokines. This capacity of modulating cytokine release is of major importance in local inflammatory sites, where PMNs are present in large numbers. The example of ethanol is summarized below. Ethanol in vivo and in vitvo impairs immune responses in humans. In particular, ethanol inhibits some key functions of neutrophils, like oxidative burst, adhesion molecules expression or chemotaxis. In vitro, Taieb et al. have investigated the impact of ethanol on cytokine production by highly purified PMN. Three cytokines, IL-8, TNFa and HGF, were analyzed. The authors have shown that three different mechanisms of regulation of the cytokines production could be involved. First, ethanol inhibits the production of IL-8 protein and mRNA. Second, TNFa release is decreased via the modulation of TACE expression involved in TNFa

248

The Neutrophils

shedding. Third, degranulation of HGF was also impaired by a clinically relevant ethanol concentration (0.8%), an action that may delay the repair of injured tissue. This disruption of PMN cytokine release by ethanol could contribute to the increased risk of infection in alcoholic patients.65 This figure illustrates these different mechanisms:

6. CONCLUSION Beside monocytes and macrophages, polymorphonuclear neutrophils represent a powerful defense system against invading bacteria and other microorganisms. They are the first line of defense and play an active role in inflammatory response. At the site of infection, polymorphonuclear neutrophils become activated and this activation might set the stage for a subsequent antibacterial immune response. After being challenged by various stimuli, neutrophils have the capacity to release lytic enzymes with potent antimicrobial potential or generate reactive oxygen intermediates, such as superoxide anion. During the past decade, a major role has been evidenced for neutrophils as they can also produce a variety of cytokines upon appropriate stimulation and thus potentiate inflammatory reactions. It is now admitted that neutrophils represent a source of proinflammatory cytokines as well as a source of endogenous cytokine inhibitors. Therefore, infiltrating neutrophils play an important role not only in sustaining the inflammatory response but also in limiting it. Moreover, new functions are proposed for these cells as a regulator of specific immunity or cell proliferation.

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8 Neutrophils in Viral Infections Robert L Roberts*

The interaction between neutrophils and viruses is complex, and the clinical significance is not well established. It is known that neutrophils can inhibit or kill viruses by various mechanisms, including production of oxygen intermediates and phagocytosis in the laboratory. Viruses may activate neutrophils by binding to the surface and by the production of cytokines which occurs in upper respiratory tract infections. Neutrophils may be inhibited viruses such as CMV by direct interaction by suppression of the bone marrow. These interactions between neutrophils and viruses will be discussed in this chapter.

Keywords:neutrophils; viruses; influenza A; HIV; CMV; herpes

I . INTRODUCTION The containment and killing of bacteria are the major functions of neutrophils in host defense. After penetrating the body, factors released by *Correspondence to: Robert L. Roberts, MD, PhD. Professor of Pediatrics, Division of Immunology/Allergy, UCLA School of Medicine, Los Angeles, CA 90095; phone: (310) 825-6777/825-6481; fax: (310) 825-9832.

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The Neutrophils

the bacteria themselves or generated by complement breakdown and by other inflammatory cells attract great numbers of neutrophils to the site of the bacterial invasion. As soon as the bacteria attach to the surface receptors on the neutrophil, they are rapidly sequestered into phagolysomes and then killed by toxins released from the granules and the oxygen radicals produced. The role of neutrophils in viral infections is much less apparent. Although neutrophils can be induced to inactivate viruses in vitro, it is difficult to determine how important this antiviral activity is in vivo. In rhinoviral infections of the upper respiratory tract, or the "common cold," most of the discomfort of the patient appears to be due to the great influx of neutrophils into the nasal secretions and not due to injury to the nasal epithelium by the virus itself. Many other viruses are capable of activating neutrophils in vitro, which may account for the exaggerated inflammatory response in some viral infections if this activation also occurs in vivo. Viruses may also inhibit neutrophil function, and hence, the ability of neutrophils to contain bacterial infections. The influenza A virus may depress many phagocytic activities, making their host more susceptible to bacterial superinfections, these being the most common cause of death in past influenza epidemics. Inhibition of neutrophil function may also play an important role in the present day AIDS epidemics. Although the neutrophil may not be infected by the human immunodeficiency virus (HIV), many defects in neutrophil function have been reported in HIV patients. A large percentage of HIV-infected patients will develop antineutrophil antibodies, which can destroy neutrophils or inhibit their function. Oxidant stress due to low-levels of the antioxidant glutathione, inhibitory HIV proteins and abnormalities in cytokine production may also inhibit neutrophil function. This loss of neutrophil surveillance for bacteria and fungal pathogens makes the patient more susceptible to these microorganisms, and further contributes to their immune deficiency. Impairment in neutrophil function may be even of greater importance in HIV-infected children, who are at a greater risk for more common bacterial infections than adult patients. This may also account in part for the observation that children can become much more ill more quickly than adults even when their CD4 counts are relatively high. In this chapter, the various interactions between neutrophils and viruses will be discussed. We will also examine the role of neutrophils in specific viral infections, including influenza and HIV. By

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understanding these interactions between neutrophils and viruses, we can hopefully devise better therapeutic strategies for the benefit of patients.

2. INHIBITION OF VIRUSES BY NEUTROPHILS Neutrophils are able to inactivate or inhibit viral replication by a number of mechanisms as shown in Table 1. Despite these in vitro studies, most patients with neutropenia or impairment of neutrophil function, as in chronic granulomatous disease (CGD), are not particularly susceptible to severe viral infections. This observation may be due to the fact that other cell populations in these patients, such as monocytes, NK cells, and T lymphocytes, are also able to inhibit viruses more effectively than neutrophils. Normal antibody production is also a strong deterrent to viral infections although neutrophils may play a role in antibody-mediated killing of virally-infected cells. However, the antiviral properties of neutrophils may become of greater significance in patients whose lymphocytes and monocytes are decreased or functionally impaired as occurs in AIDS patients. When neutrophils encounter bacteria, the usual response is phagocytosis which is greatly facilitated by antibody or complement components binding to the bacteria. Viruses may enter neutrophils by phagocytosis Table 1 Inhibition of Viruses by Neutrophils Mechanisms

Viruses

Selected References

Ingestion and inactivation by cationic granular proteins Release of cytokines that attract other cells Release of oxygen intermediates Antibody-dependent cellular cytotoxicity Complement-mediated cytotoxicity Collectin-mediated phagocytosis

Vaccina Herpes

1,2,3,4

Rhinovirus Influenza Herpes Influenza Vaccinia Herpes Varicella Influenza HIV Herpes RSV

5,6

Influenza

15,16

7,8,9

10, 11,12,13 14

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The Neutrophils

but also by other mechanisms, such as fusion with membrane proteins and endocytosis. If entry of the virus activates the neutrophil, then the virus is more likely to be destroyed by the release of granule proteins, such as defensins, and generation of reactive oxygen intermediates. The granule protein defensin will inhibit HIV replication in vitru, and lactoferrin, an iron-binding cationic protein, can inhibit adsorption and penetration of HIV and CMV in cell c u l t ~ r e . ' ~ , ' ~ If entry of the virus does not activate the neutrophil, then the neutrophil may actually act as a reservoir for the virus, allowing it to escape inactivation by other means. Viruses that have been found within neutrophils include vaccinia, herpes simplex, influenzae, CMV, adenovirus, and HIV.I9 In CMV infection, the virus is more readily cultured from neutrophils than from monocytes, although both may contain the virus.20f21 This implies that monocytes may be better at inactivating the virus. Neutrophils may also be a major reservoir for hepatitis B virusF2 Neutrophils produce a number of cytokines that have antiviral effects themselves or that attract other cells with antiviral proper tie^.^^ This would include interleukin (1L)-1 which activates T cells and NK cells, IL-8 that also attracts T cells, interferon-a that activates NK cells, and tumor necrosis factor (TNF)-a that activates T and NK cells. These other cells may then generate more cytokines, such as interferon-gamma, which have also antiviral properties.

2.1. Viral Inactivation by Oxygen Intermediates Oxygen intermediates generated by neutrophils will inactivate viruses in uitro, but its clinical significance has not been proven. Poliovirus and vaccinia virus exposed to the combination of hydrogen peroxide, myeloperoxidase, and halide (which would generate hypophalous acid) ~ ~ been demonstrated that neutrophils from were inactivated in u i t r ~It. has CGD patients are less able to destroy herpes or vaccinia virus as compared with normal control^.^^^^ Our laboratory has also found that neutrophils from CGD patients were unable to inhibit the replication of herpes simplex virus despite stimulation with phorbol myristate acetate which greatly stimulates the inhibition of viral replication by neutrophils from normal controls.26The production of oxygen intermediates also appears to play a role in impairing the infectivity of HIV using a T cell line as targets.27

Neutrophils in Viral Infections 257 T

Although oxygen intermediates, such as superoxide anion, have definite antimicrobial effects, nitric oxide, generated from reactive nitrogen oxide species, may actually enhance viral activity. Nitric oxide may accelerate viral mutations, resulting in more resistant strains. The nitric oxide may also selectively inhibit type 1T cells, leading to increased percentage of type 2 T cells that are less effective in fighting viruses.28

2.2. Antibody and Complement Induced Viral Inactivation Neutrophils are able to lyze virally-infected cells by antibody-dependent cellular cytotoxicity (ADCC), as has been demonstrated using target cells infected with herpes-simplex, varicella-zoster, influenzae, and HIV.29-35ADCC of HIV-infected targets may also be enhanced by cytokines, such as G-CSF and GM-CSF, using neutrophils from normal controls as well as from HIV patients.36The activation and fixation of the complement to virally-infected cells will greatly increase the ability of neutrophils to kill the infected cell. Complement cells will fix to RSV-infected cells in the absence of specific anti bod^^^-^^ and will stimulate lysis by neutrophils. A newly described group of collagenous lectins, named collectins, found in blood and pulmonary fluid, will induce aggregation of influenza A virus (IAV) particles. This aggregation will stimulate binding of IAV to neutrophils as well as prevent inhibition of neutrophil function by IAV.15,16

3. ACTIVATION OF NEUTROPHILS BY VIRUSES Viruses are capable of stimulating various neutrophil functions, as shown in Table 2. The stimulation may occur through direct binding of the virus to the neutrophil, increased adherence of the neutrophil to virallyinfected cells, or release of the cytokines by virally-infected cells. In some viral infections, much of the inflammatory response is due to the activation of neutrophils, as is the case with rhinovirus infections involving the upper respiratory tract. In a study using patients with allergic rhinitis, the patients were inoculated with the rhino virus, and nasal lavage material was collected. The amount of myeloperoxidase (MPO), a component of neutrophils, increased following viral inoculation, indicating neutrophil influx and breakdown.4O

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The Neutrophils Table 2. Activation of Neutrophils by Viruses Neutrophil Function

Virus

Selected References

Adherence to infected cells

Adenovirus Measles Polio RSV Rhinovirus

40

Phagocytosis

Adenovirus Coxsackie CMV Herpes simplex Influenzae Measles Mumps Polio

45

Chemotaxis

Rhinovirus

Oxidative burst

Adenovirus 50,52,53 Hepatitis B Influenzae Japanese encephalitis RSV Sendai

3.1. Activation by Binding of Virus Sendai and myxovirus (strain of IAV) can bind directly to neutrophils in the absence of antibody. Oxidative burst activity will occur within one minute after binding, resulting in the generation of reactive oxygen intermedia t e ~ . 4 ' However, .~~ this oxidative burst activity is "atypical" compared with other stimuli, in that only hydrogen peroxide is released from the cell. The mechanism for the anomaly will be discussed later in the section on IAV. Opsonization of the virus with antibody or complement may enhance binding to neutrophils. This may result in activation of the neutrophil through its Fc or complement receptors which initiate a number of neutrophil activities, including phagocytosis and generation of oxygen intermediate^.^^,^^ In the case of HSV-1, opsonization of viral particles with complement alone will increase binding, but HSV-specific antibody must be present to activate phagocytosis of the v i r ~ s . 4 ~

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259

3.2. Adherence of Neutrophils to Infected Cells

Viral infection of endothelial cells or fibroblasts in vitro will enhance the adherence of neutrophils to these cells, as shown in Table 2. If these neutrophils are activated, injury or death of the infected endothelial cells may O C C U ~ . This ~ ~ ? adherence ~ ~ to infected cells by neutrophils may be enhanced by the presence of virus-specific antibody, as is the case for RSV-infected cells, but is not required for herpes simplex-infected ~ e l l s . ~ ~ ~ Complement will also fix to RSV-infected cells and this will also increase the adherence of neutrophils even in the absence of specific antibody40

3.3. Activation of Oxidative Burst Activity Several in vitro and in vivo studies indicate that oxidative burst activity in neutrophils may be activated directly by viruses or in viral infections. Direct binding of viral particles may initiate an oxidative burst as previously noted, possibly by direct binding to surface glycoproteins on the n e u t r ~ p h i l . ~ Oxidative ~-~I metabolism may also be activated following phagocytosis of viral particles or binding to Fc or complement receptors if the specific viral antibody is present. In clinical studies, it has been observed that neutrophils from the peripheral blood of patients with influenzae or adenoviral infections have elevated resting levels of oxidative metaboli~m.5~2~ This could be due to the direct activation of the neutrophils by virus, although the release of inflammatory cytokines such as interferon-gamma could also increase this activity. Many abnormalities in oxidative burst activity in neutrophils and monocytes from HIV patients have been described, as will be discussed later.

3.4. Role of Cytokines in Neutrophil Activation One mechanism by which viruses may activate neutrophils is by the induction of production of cytokines that activate and increase the number of neutrophils. In a study by Jarjour, et al., subjects with a history of allergic asthma were inoculated with the rhinovirus (strain 16). Infection resulted in significant increases in the absolute circulating blood neutrophil count by day 3 after i n o ~ u l a t i o n Paralleling . ~ ~ ~ ~ ~ these acute changes in circulating neutrophils was an increase in interleukin-8 (IL-8)

260

The Neutrophils

and granulocyte-colony stimulating factor (G-CSF) in nasal secretions. The circulating neutrophil count did correlate with the nasal IL-8 and G-CSF concentrations. Bronchial lavage was also performed, and rhinovirus inoculation also resulted in an increase in bronchial neutrophils at 96 hours. These results suggest that the induction of cytokines, particularly G-CSF, might explain the neutrophil activation in viral infections and may also be responsible for exacerbations of asthma, due to influx of neutrophils in the lungs, in viral infections. Viruses may also play a role in chronic obstructive pulmonary disease by inducing the production of cytokines, such as IL-1B and GM-CSF, that attract neutrophils to the lungs contributing to the inflammatory state.56 Rhinoviruses also induce the production of chemokines that activate n e ~ t r o p h i l s . ~ ~

4. NEUTROPHIL FUNCTIONS INHIBITED

BY VIRUSES There has long been an association between viral infections and neutrophil dysfunction. As previously noted, most patients in the major influenzae epidemics died from bacterial pneumonia, which was likely due to impairment of their neutrophils' capacity to fight bacteria. Table 3 lists some of the neutrophil functions impaired by viruses, with influenzae being listed in every category. Patients with CMV and rubeola infections are also at much greater risk of dying from bacterial infections that is blamed in part on inhibition of neutrophil f u n ~ t i o n . * ~ * ~ ~ " ~ Neutrophil and monocyte functions are also impaired in HIV infection.66 This may be due to the direct effect of the virus, abnormality in cytokine production, formation of antineutrophil antibodies, or depletion of endogenous oxygen scavengers such as glutathione. This inhibition of phagocytic cells further increases the patients' susceptibility to bacterial and fungal infections. Viruses may also cause neutropenia due to impairment of neutrophil maturation in bone marrow. Parvovirus may cause anemia, thrombocytopenia, and neutropenia, which is of particular concern in patients already immunologically compromised by HIV infection or chemotherapy for Other viruses that commonly cause neutropenia in children

Neutrophils in Viral Infections

261

Table 3 Neutrophil Functions Inhibited by Viruses Function

Viruses

Selected References

Chemotaxis

CMV Influenza Rubeola (measles) RSV HIV Herpes simplex Hepatitis B Influenza Mumps CMV Hepatitis B Influenza HIV Para influenza Influenzae HIV CMV

47,48

Phagocytosis

Cytotoxicity, Killing

Oxidative Burst

43,44,58

45,46,47

include RSV, varicella, influenza A and B, measles, mumps, roseola, and r ~ b e l l a . ~The ~ - ~neutropenia l will often develop in the first 48 hours of the illness and may last up to 8 days. The neutropenia may be due to the virus-induced redistribution of the neutrophils from the circulating to the marginating pool, and usually does not put the child at great risk for serious infection. Other viruses such as Epstein-Barr, hepatitis, and HIV may cause a more protracted neutropenia due to infection of the hematopoietic stem cells or formation of immune complexes that bind to neutrophils, causing them to be sequestered in the spleen.","

5. NEUTROPHILS A N D INFLUENZA A V I R U S Many viruses may exert inhibitory effects on neutrophil function as previously noted, but the effect of influenza A virus (IAV) on neutrophils is unique and also of great clinical importance. The clinical importance is due to the thousands of deaths from bacterial pneumonia that occur every year following a bout of flu due to IAV. The interactions between

262

The Neutrophils Table 4 Influenza A Virus (IAV) Effects on Neutrophils

Unopsonized IAV stimulates aberrant oxidative burst on binding to neutrophil Impairs fusion of primary and secondary granules with cell membrane Inhibits lysosome-phagosome fusion Decreases G-protein function in the activation process Interferes with G-protein phosphorylation Inhibits neutrophil chemotaxis Greatly increases binding (>500 times) of neutrophils to IAV-infected epithelium cells Interferes with neutrophil cytoskeletal protein function Stimulates intracellular calcium mobilization Stimulates neutrophil membrane depolarization

virus and neutrophil is unique due to the ability of the virus to activate the cell in an abnormal fashion. These interactions have been examined extensively by Abrainson, Tauber and others, and a listing of some of the interactions is shown in Table 4.6158f74 The IAV has hemagglutinin molecules on its surface that bind to specific residues on glycoproteins on neutrophil membranes, including CD43.75t76 Desialation of the neutrophil by neurominidase treatment, which alters these binding sites, will inhibit IAV binding and activation (or deactivation) of the neutrophil by the virus. Crosslinking of these bound viruses by antibody enhances the ability of the virus to activate the n e ~ t r o p h i l . ~ ~ The exposure to the virus will cause a marked inhibition of phosphorylation of multiple membrane and cytosolic proteins that is part of the activation process that occurs following stimulation with such agents as N-formyl methionyl-leucylphenylalanine (FMLP).77-79 This would suggest that IAV is altering G-protein function in the neutrophil. Binding of IAV to the neutrophil also results in a rise in intracellular calcium that is independent of extracellular calcium, unlike stimulation with FMLP which is dependent on extracellular calcium.6p80 IAV also stimulates the production of hydrogen peroxide from neutrophils, but this occurs without the concorninant release of superoxide anion, as occurs with other activators of oxidative burst activity.81i82 More extensive studies indicate that this stimulation of respiratory burst activity by IAV occurs at an intracellular location rather than at the membrane as would be the normal response.6 Thus, all of the superoxide anion

Neutrophils in Viral infections

263

which is generated first has been converted to hydrogen peroxide by the time it was released from the cell or had been scavenged by the cell components. Stimulation of neutrophils with IAV does not result in extracellular release of granule contents, as would occur with stimulation with agents such as Fh4LPa1 These aberrations in activation that occur with IAV make neutrophil resistant to activation by other stimuli, thus making it much less effective in its normal host defense functions such as chemotaxis and bacterial killing. IAV causes similar defects in nonoxidative neutrophil functions using cells from patients with CGD, suggesting the defects are not dependent on the abnormal release of oxygen intermediates. Inhibition of fusion of primary and secondary granules with the plasma membrane occurs with exposure to the IAV and may account for defects in chemotaxis and other f ~ n c t i o n s . ~ ~ - ~ ~ Some of the abnormalities in neutrophil activation that occur with IAV may be partially prevented by priming the neutrophils with granulocyte, macrophage colony stimulating factor (GM-CSF) and granulocyte colony stimulating factor (G-CSF).86r87 This suggests that cytokines, such as GM-CSF and G-CSF, may be useful in overcoming the inhibition of neutrophil function that occurs in patients infected with IAV, and would have the added benefit of increasing the neutrophil number, thus making patients less susceptible to bacterial infections.

6. NEUTROPHILS AND HIV The devastating effects of HIV infection on the immune system by destruction of CD4 lymphocytes are compounded by the many impairments of neutrophil function that have been reported in this disease. Children infected with HIV are particularly vulnerable to bacterial infections, as their naive immune systems have not developed the repertoire of antibodies needed to combat the more common bacterial infections. The death rates from bacterial infections in AIDS patients are also higher in less developed parts of the world, where antibiotics are readily accessible, as was the case in the pre-antibiotic era when thousands of patients died from bacterial pneumonia during the influenzae epidemics secondary to inhibition of their neutrophils by the influenza virus. The number of

264

The Neutrophils

neutrophils is also decreased in many HIV patients due to antineutrophil antibodies present in many of these patients, myelodysplastic changes in the marrow; and some of the antiretroviral drugs. In this section, we will discuss some of the abnormalities in neutrophil function found in HIV patients, the mechanisms responsible for these abnormalities, and the role of neutrophils in the progression of HIV infection.

6.1. Myelodysplastic Changes in HIV Infection Several reports have indicated that the myeloid precursor cells in the marrow of AIDS patients may be infected with HIV, leading to inhibition of myelopoeisi.~.~*~~ HIV RNA is present in the myeloid precursor cells from bone marrow aspirates of AIDS patients which may alter their diff e r e n t i a t i ~ nDonahue, .~~ et al. reported that bone marrow progenitor cells for monocytes may be infected in vitro with HIV.91Infections of myeloid progenitor cells by HIV was shown more directly by Folks et al., who showed that purified CD34+ cells from human bone marrow could be infected with HIV-1.92 Abnormalities in the myeloid stromal cells have also been reported in the marrow of HIV patients, which may also contribute to the myelodysplastic changes.93

6.2. HIV Infection of Neutrophils Although neutrophils lack the CD4 receptor, which is the usual route of entry of HIV, there is evidence that neutrophils in the peripheral blood of AIDS patients carry the virus. Spear, et al. found that 2 out of 10 patients he studied had neutrophils that contained HIV DNA copies detected by chemiluminescence, and that the number of DNA copies was much less in the neutrophils as compared with the patients’ lymphocyte^.^^ The presence of the HIV DNA in the neutrophils may come about by infection of the myeloid precursor cells as noted above, and the infected neutrophils would likely have a decreased lifespan and abnormal function in the peripheral b l ~ o dThere . ~ ~is ~also ~ evidence ~ that neutrophils may be infected by direct fusion of the HIV envelope with the cell membrane?7s98 In later studies, Gabrilovich, et al. (1993) were able to detect HIV DNA by polymerase chain reaction (PCR) in the neutrophils of 30% of his

Neutrophils in Viral Infections

265

HIV-patient~.~~ Detection of the HIV DNA in neutrophils was more common in symptomatic (47%)than in asymptomatic (18%)patients, and also more common in patients with recurrent bacterial pneumonias and Pneumocystis carinii pneumonia. The detectable HIV DNA was also more common in patients with neutropenia and with low CD4/CD8 ratios.loO They suggested that infection of the neutrophils in these patients led to impairment in neutrophil function.

6.3. Anti-neutrophil Antibodies in HIV Infection Neutropenia occurs in 20% to 40% of AIDS patients and, in addition to faulty myelopoiesis, this neutropenia may be due to autoimmune antibodies to neutrophils or to the deposition of immune complexes on the neutrophil surface.101t102 Although granulocyte-associated immunoglobulins that are not specifically bound may occur in over 20% of asymptomatic HIV-infected subjects,103neutrophil-specific antibodies also occur in many patients.'@ Stroncek, et a1.,'05 studied 100 HIV-infected patients, and found granulocyte antibodies in 66% of the samples by granulocyte immunoflouresence (GIF) and in 21% of the samples by granulocyte agglutination (GA). Further testing showed some of the GIF positive samples were due to the presence of immune complexes on the neutrophils. However, those sera samples positive by GA were due to antibodies directed against neutrophils themselves. The presence of these autoimmune antibodies may contribute to neutropenia in some patients.

6.4. Neutrophil Chemotaxis in HIV Infection

Neutrophil chemotaxis is reported by several investigators to be markedly depressed, although the mechanism underlying this defect is unclear. Defects in adult homosexual males with presumed HIV infection were noted as early as 1984, before the virus itself was identified.Io6This finding was thought to account for the increased number of bacterial infections in these patients and was confirmed in later s t u d i e ~ . ~ ~ ~ * ' Lazzarin, et al., also reported that neutrophil chemotaxis was defective in HIV-infected adult males, regardless of how they acquired the infection.lo9In 1990, Roillides, et al. found neutrophil chemotaxis defective in

266

The Neutrophils

HIV-infected children and was more profound in children with more advanced disease."O They also reported this defect was partially corrected by incubation in GM-CSF. In a longitudinal study of HIV-infected males, neutrophil chemotaxis was 19% inhibited compared with normal controls, and this inhibition increased to 32% after 3 years in follow-up studies in this same group of patients.'ll Phagocytosis of neutrophils and monocytes has also been reported to be depressed in AIDS patients, using Staphylococcus aurem as the target

6.5. Abnormalities in Respiratory Burst Activity The production of ROI by neutrophils is necessary for killing of many bacteria and fungi to which HIV patients are susceptible, but excess production of ROI can damage the host tissue, as occurs in autoimmune disease. Unfortunately, HIV patients are at risk for both infections and autoimmune disease. In an early paper,113nitroblue tetrazolium (NBT) reduction, a measure of ROI production, was increased in patients in the early stages of HIV disease but decreased in patients with advanced AIDS. In 1993, Pitrak1I4 reported that superoxide production was decreased in HIV patients, and that the impairment in superoxide production was more pronounced in patients with lower CD4 cell counts. However, Bandres, et al., using a flow cytometric technique to measure ROI release, found greater ROI production in neutrophils from HIV patients as compared with control^."^ This finding of greater production of ROI with neutrophils from HIV patients was confirmed in 1995, also using a flow cytometric method."6 Laursen, et al., using chemiluminescence to measure ROI production, found lower responses in HIV patients who previously had had Pneurnocytis carinii pneumonia but not in patients with less advanced di~ease."~ Our laboratory reported decreased superoxide production in neutrophils and monocytes from HIV-infected children and adults using the cytochrome C reduction technique in a 2-hour assay. Production of hydrogen peroxide was also decreased in HIV patients in this study that measured production for 90 minutes. Thus, many abnormalities in the respiratory burst activity of neutrophils and monocytes from HIV-infected patients have been reported but the findings are sometimes conflicting. Some investigators report that

Neutrophils in Viral Infections 267 neutrophils from HIV patient are in a more primed state and have a greater respiratory burst activity, while others have found the ability of neutrophils to generate ROI in HIV patients is decreased compared with that of controls. Although some of these differences may be due to differences in technique or the population studied, another explanation might be that neutrophils in these patients do tend to be exposed to more stimuli, such as chronic fungus or parasitic infections, that would place them in a more activated state, requiring less provocation to initiate a respiratory burst. However, HIV patients also have decreased levels of glutathione in their plasma and white cells, which acts as an antioxidant to protect neutrophils from their own ROI. Thus, the respiratory burst may be more easily initiated in HIV patients, but production of ROI cannot be as sustained as long as that of controls, possibly due to the impairment to the neutrophils themselves by their own ROI. This inability of neutrophils to protect themselves may also be reflected by the accelerated rate of apoptosis in neutrophils from HIV patients, as demonstrated by various techniques.'18 The percentage of neutrophils undergoing apoptosis was 2- to 3-fold higher at 3 to 18 hours after isolation in the HIV group. Apoptosis could be greatly reduced in the patients' cells if they were incubated in G-CSF.

6.6. Neutrophil Cytotoxicity in HIV Infection The defects in neutrophil motility and ROI production noted in HIV patients make it not surprising that defects in neutrophil cytotoxicity are also found in HIV patients. Neutrophils from AIDS patients were significantly slower in killing Staphylococcus aureus as compared with normal controls in a study performed at the NIH.l19Roillides, et al., also reported a defect in S. uureus killing using neutrophils from HIV-infected children. He later reported that neutrophils from HIV-infected children were also impaired in their ability to kill Aspergillus fumigatus, and that this defect could be partially corrected with G-CSF.lZ0A defect in killing another fungi, candida, was reported to occur in adult AIDS patients, which was attributed to impairment of nonoxidative killing mechanisms.121 Antibody-dependent cellular cytotoxicity (ADCC)is a mechanism for killing HIV-infected lymphocytes as well as other cells, and defects in

268

The Neutrophils

ADCC have been found in HIV patients. Neutrophils from HIV patients were reported to be defective in ADCC, using herpes infected cells and chicken erythrocytes as target ce11s.122J23 Our laboratory reported that the ADCC of neutrophils from HIV-infected children was defective compared with age-matched controls, using HIV-coated cells as our target.lz4 In a later study, we reported that some of the defects in ADCC in neutrophils from HIV patients could be corrected by N-acetyl-cysteine, which restores the antioxidant glutathione found to be decreased in HIV patients.13 Thus, cytokines such as G-CSF and antioxidants may play a role in improving the defective neutrophil cytotoxicity found in HIVinfected patients.

6.7. Neutrophil Defensins Inhibit HIV The human neutrophil cytoplasmic proteins, the alpha-defensins, were found to inhibit HIV activity. Purified mixtures of alpha-defensins were able to directly inactivate HIV virions as well as to inhibit the replication of HIV in infected CD4 ce11s.125~126 The alpha-defensins'" were found in neutrophils and monocytes but not CD8 cells. Thus, neutrophil defensins do have a number of antiviral properties and this includes inhibition of the HIV virus.

7. CONCLUSION The interaction between neutrophils and viruses is complex and its clinical significance is still not fully understood. Although neutrophils can be shown to inhibit or destroy viruses in vitro,we are not certain how important these antiviral mechanisms are in viva The ability of viruses to inhibit neutrophils, however, is better documented and can be of utmost importance in epidemics of viral disease. Influenza still continues to take its toll, due in large part to its ability to make the patient much more susceptible to life-threatening bacterial infections. The modern epidemic, HIV infection, also results in depression of neutrophil function, which is particularly devastating in children and in underdeveloped countries where antibiotics are much less available. Neutrophils are, however, able to kill HIV-infected cells in the presence of antibodies by ADCC. Neutrophil-mediated ADCC may

Neutrophils in Viral Infections

269

become of greater importance if antiHIV vaccine leads to production of antibodies more efficient in HIV-infected cells.

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89. Rosenberg ZF, Fauci AS. Adv lmmunoll989; 47:377-431. 90. Busch M, Beckstead J, Gantz D, Vyas G. Blood 1986; 68 (Suppl):122A. 91. Donahue RE, et al. Nature 1987; 326:200-203. 92. Folks TM, et al. Science 1988; 242:919-922. 93. Ganser A. Blut 1988; 56:49-53. 94. Spear GT, et al. JInfect Dis 1990; 162:1239-1244. 95. Zon LI, Groopman JE. Sem Hematoll988; 25208-218. 96. Scadden DT, Zon LI, Groopman JE. Blood 1989; 743455-1463, 97. Hoxie JA, Rackowski JL, Haggarty BS, Gaulton GN. J Immunol 1988; 140:786-795. 98. Maddon PJ,et al. Cell 1988; 54:865-874. 99. Gabrilovich DI, et al. J Acquiv Immune Defic Syndr 1993; 6:587-591. 100. Gabrilovich DI, et al. Zhurnal Mikrobiologii, Epidemiologii i lmmunobiologii 1995; 552-55. 101. Minchinton RM, Frazer I. Lancet 1985; 1:936-937. 102. Murphy MF, et al. Lancet 1985; 1:217-218.

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103. Celton JL, et al. Nouu Rev Fr Hernatoll989; 31:187-188. 104. Klaassen RJL, Vlekke ABJ, von dem Borne AEGKr. Br J Haematol 1991; 7 7 403-409. 105. Stroncek DF, et al. J Lab Clin Med 1992; 119:724-731. 106. Valone FH, Payan DG, Abrams DJ, Goetzl EJ. J lnfect Dis 1984; 150267-271. 107. Nielsen H, Kharazmi A, Faber V. Scand J Immunoll986; 24291-296.

108. Martin LS, Spira TJ, Orloff SL, Holman RC. J Leukoc Biol 1988; 44:361-366. 109. Lazzarin A, et al. Clin Exp lmrnunoll986; 65:105-111. 110. Roilides E, et al. J Pediatr 1990; 117:531-540. 111. Flo RW, et al. AIDS 1994; 8:771-777.

112. Pos 0,et al. Clin Exp lmmunol 1992; 88:23-28. 113. Siinnerborg A, Jarstrand C. Scand J lnfect Dis 1986; 18:lOl-103. 114. Pitrak DL, et al. ]Infect Dis 1993; 167:1406-1410. 115. Bandres JC, Musher DM, Rossen RD. Jlnfect Dis 1993; 168:75-83. 116. Elbim C, et al. Blood 1994; 842759-2766. 117. Laursen AL, Rungby J, Andersen PL. J Infect Dis 1995; 172497-505. 118. Pitrak DL, et al. J Clin Invest 1996; 98:2714-2719. 119. Murphy PM, Lane HC, Fauci AS, Gallin JI. J Infect Dis 1988; 158:627-630. 120. Roilides E, et al. Jlnfect Dis 1993; 167905-911. 121. Wenisch C, et al. AIDS 1996; 10:983-987. 122. Shah TP, Sattler FR. J Infect Dis 1987; 155:594-595. 123. Kinne TJ, Gupta S. J Clin Lab Immunoll989; 30:153-156. 124. Szelc CM, Mitcheltree C, Roberts RL, Stiehm ER. ] Infect Dis 1992; 166: 486493. 125. Mackewicz CE, et al. AIDS 2003; 17:F23-32. 126. Zhang L, et al. Science 2002; 298995-1000.

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9 Polymorphonuclear Neutrophils and Cancer: Ambivalent Role in Host Defense Against Tumor Nadejda L: Cherdyntseva, Sergei Kusmartsev, Dmitry 1. Gabrilovich"

Neutrophils are the most abundant cells in the human body. Their ability to take up and destroy microorganisms is well known. The potential role of polymorphonuclear neutrophils (PMN) in antitumor response due to their high cytotoxicity was actively investigated in the recent decades. It turned out that the role of PMN in cancer is very complex and controversial. It appears that these cells contribute both in tumor rejection and tumor promotion. The mechanisms of this dual role are starting to emerge. If we could find ways to manipulate PMN function in cancer, it would open a new exciting opportunity to improve cancer therapy. In this chapter, we will discuss the potential role of PMN in tumor growth and cancer therapy. Keywords: neutrophils; cancer; myeloperoxidase; cytotoxicity; reactive oxygen species *Correspondence to: Dmitry Gabrilovich MD, PhD. H. Lee Moffitt Cancer Center, MRC-2, Room 2067, 12 902 Magnolia Dr. Tampa, FL 33612; e-mail: [email protected]

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The Neutrophils

1. NEUTROPHILS ARE ABLE TO PROMOTE CA RCINOGENESIS 1.l.PMNs May Contribute to Inflammation Associated with Tumor Development It is now well established that tumor growth depends not only on molecular events resulting in malignant transformation but also on the array of different factors produced by tumor-bearing host. Inflammation plays an especially important role in facilitating tumor g r o ~ t h . ' It, ~has ~ ~been ~ suggested that inflammatory cells and cytokines found in tumor tissues contribute to the tumor progression rather than to antitumor response.6 PMNs are the major component of inflammatory reaction. The hypothesis that neutrophils may contribute to carcinogenesiswas based on the early findings that neutrophil's highly reactive oxygen metabolites are involved in bacterial mutagene~is.~.~ Activated PMNs are able to increase the exchanges of sister chromatin in hamster ovary cells, while neutrophils derived from a patient with chronic granulomatosis did not induce similar abnormalities3 These results suggest that neutrophil oxidants may be responsible for the genomic damage. The direct evidence of tumor-induced effect of PMN was obtained in experiments with nude mice inoculated with fibroblasts. Inoculation of fibroblasts, exposed to the activated neutrophils, resulted in the development of tumor.4 Oxygen reactive species (ROS) and arachidonic acid metabolities, specifically prostaglandin E2, are known mediators of macrophage suppressor activ.~ ity. They may directly suppress antitumor NK cell f ~ n c t i o n . ~In, ~addition, reactive oxygen intermediates produced by PMN may also damage nascent fibroblasts, parenchymal cells and endothelial cells and as a result, facilitate tumor spreading. An important mediator of tumor invasion and angiogenesis is gelatinase A (MMP-2), a matrix metalloproteinase that is secreted as an inactive zymogen and activated by proteolytic cleavage.22The activation of zymogen into active MMP-2 requires PMN-derived elastase, cathepsin G, and proteinase-3. Recent studies using human clinical samples have suggested that epithelial cells turnover, is affected by inflammation.10,11 Furthermore, cell proliferation in the sites of chronic inflammation is directly correlated with predisposition to breast, liver, large bowel, urinary bladder, prostate,

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ovary, and skin cancer^.'^-'^ Long-term users of aspirin and nonsteroid antiinflammatory drugs (NSAIDs) have reduced risk of colon, lung, esophagus, and stomach cancer.I5J6These data are consistent with the hypothesis that inflammation directly contributes to tumor development. Chemopreventive action of NSAIDs is the results of their ability to inhibit cyclooxygenases Cox-1 and Cox-2, catalyzing arachidonic acid conversion into prostaglandin, which in turn are the main inducers of inflammatory responses in injured tissue.17 PMNs are the most abundant circulating blood cells. They provide early nonspecific response to infectious agents and injury, and are involved in the inflammatory process. PMNs are known to secrete numerous cytokines and other bioactive molecules that can modulate the recruitment and activity of various inflammatory cells and in turn are able to change their own behavior in response to the different mediators secreted by other cells. This makes PMN highly relevant for our understanding of the mechanisms of tumor development and progression.

1.2. PMNs Involvement in Infection Associated Carcinogenesis Inflammatory reaction is the necessary attribute of host response against infectious agents. Therefore, it was logical to suggest that malignancies might arise from the site of infection and inflammation. There is now evidence pointing to the initiating role of infections in some human malignanc i e ~ . ’Infection ~ , ~ ~ by Helicobucter pylori (H. pylori) is associated with a high risk of gastric cancer and mucosa-associated lymphoid tissue (MALT) lymphoma.13,23,24 H. pylori induce infiltration of gastric mucosa by macrophages, PMNs and lymphocytes. However, this inflammatory response is not sufficient to eliminate the infection agent and results in chronic inflammation. Inflammatory cells generate ROS and nitrogen species. These molecules, in addition to their antimicrobial or antiviral activity, also induce DNA damage in proliferating cells. Interaction of superoxide anion with NO, results in the formation of peroxinitrite, which is a powerful mutagenic agent.25 Peroxinitrite directly modifies protein tyrosine residues and the molecular structure of DNA, resulting in their functional inactivation?6H . pylori is able to convert neutrophil-derived hypochlorous acid through reaction with

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The Neutrophils

self-produced ammonia to cytotoxic NH2C1, thereby increasing gastric have not found the differences in H . pylorimucosa injury. Abe et induced ROS production by neutrophils between patients with gastric cancer and those without. They concluded that the progression from nonmalignant mucosa to cancer might be associated with the time-dependent effect of H . pylori on gastric mucosa through reactive oxygen intermediates produced by neutrophils. Neutrophil-derived enzymes such as protease, neuraminidase, and so on, may also destroy the integrity of mucus or induce the lipid oxidation, resulting in the damage of gastric epithelium. Phospholipases may contribute to mucosal injury by degrading phospholipids and generating the precursor of ulcerogenic compoundsJ8 In some cases, ablation of H . pylori infection correlated with the reversal of inflammation and with the regression of associated tumors.29 Schistosoma haematobium infection is known to be strongly associated with urinary bladder cancer. In experimental studies carried out, it was found that neutrophils activated by inflammatory-associated stimuli could induce chromosomal damage of bladder cell. Furthermore, this injury was detected in chromosome 11, which is commonly altered during bladder carcinogene~is.~~ Neutrophils and mast cells have been reported to potentiate human papilloma virus type 16 induced oncogenesis in transgenic mice predisposed to squamous cell carcinoma.20,21 Although the persistence of viruses is a rather widespread phenomenon in mammals, virus-associated neoplasm occurs relatively rarely in infected individuals. This fact points to the importance of the specific promoters for virus-induced tumor development. Inflammatory mediators probably play a critical role in virus-associated tumor development. In chicken infected with the Rous sarcoma virus, the tumor developed only at the site of the virus injection despite the presence of the virus in the This study has also shown that tissue wound made at a distance from the primary tumor resulted in tumor development at the site of injury. Because tumor was not developed as a result of metastases, the authors consider the wound healing and associated factors as necessary contributors to virus-induced tumor. When inflammation was inhibited with anti-inflammatory drug tumor development was also inhibited. These results suggest that factors associated with inflammation are conducive for tumor development?

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Epithelial turnover in the tissue damaged by ROS during the infection is accompanied by permanent DNA alteration in the proliferatingepithelial cells.20J P53 is known to play a key role in inducing growth arrest or apoptosis after the genotoxic effect of reactive metab~lities.~~ Inhibition of p53 function contributes directly to tumor development because the cells with damaged DNA are able to replicate that result in the accumulated oncogenic mutation^.^^-^^ The activity of p53 can be regulated by migration inhibitory factor (MIF) induced during inflammation.34Chronic attenuation of p53 function by MIF accompanied by massive release of highly reactive oxidants by activated phagocytes, result in DNA damage at the inflammatory sites that might contribute to the tumor d e ~ e l o p m e n t . ~ ~

1.3. Myeloperoxidase and Cancer Myeloperoxidase (MPO) is present primarily in PMNs and to a lesser extent in monocytes. MPO is involved in the production of hypochlorous acid (HCLO), which has a high oxidative potency. MPO is known to be a specific marker of acute myeloid leukemia (AML)36and it is expressed in myeloid precursors and leukemic cells. Besides, MPO has been reported to be involved in other malignant processes such as lung cancer. Reinolds et al?7 demonstrated that the MPO G463A polymorphism was associated with AML. This polymorphism is associated with G/A transition within the promoter region. G allele has been shown to correlate with a 25-fold transcriptional enhancement of the reporter gene through the creation of a strong SP1 transcriptional factor consensus binding site, whereas A allele resulted in a nonactive SP1 binding site. The G-G genotype has been linked with increased MPO expression in leukemic ~ e l l s . 3Higher ~ expression of G allele in leukemic patients may induce DNA damage due to an increased level of MPO-produced ROS.38Importantly, MPO has been shown to be involved in metabolic activation of carcinogenic reactive metabolites: polycyclic aromatic hydrocarbon^^^ and aromatic aminesjOEpidemiological study of the association between lung cancer and MPOG463A polymorphism showed that homozygous allele A correlated with the decreased risk for lung cancer.4143In contrast, a Finish case-control study has demonstrated the association of the A allele with the increased lung cancer risk in older man?4

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The Neutrophils

MPO deficiency seems to be a relatively common disorder affecting 1 out of 2000-4000 i n d i v i d ~ a l s MPO . ~ ~ deficiency is associated with the dysfunction in its processing due to several mutations in MPO gene, which were characterized in recent years.46 According to Hamajima et a low expressing MPO genotype was associated with a reduced risk of H . pylori infection. On the other hand, MPO is responsible for the oxidative inactivation of the alpha-1 protease inhibitors48that may contribute to antitumor host defense, since proteases have been shown to promote carcin~genesis.~~ There is no strong evidence supporting an existing relationship between MPO deficiency and the development of ne0plasm.~~5~

1.4. Chemokines Regulate Neutrophil Infiltration and Activity One of the most important steps in PMN involvement in tumor development and progression is neutrophil migration to the site of inflammation or arising tumor. This process is predominantly regulated by chemokines. The chemokine family includes more than 50 secreted 8-12 kDa proteins that are involved in the chemotaxis of monocytes, lymphocytes, granulocytes (neutrophils, eosinophils, basophils), natural killer cells, dendritic and endothelial cells. Chemokines are able to modulate the expression of adhesion molecules, and the secretion of proteases and other factors, which are essential for the recruitment of effector cells into the inflammatory site. The role of chemokines in tumor biology is unclear because of the conflicting reports pointing to their ability to either inhibit or promote tumor development and the growth and metastasis of established tumor. Chemokines are responsible for the extensive migration of leukocytes to the inflammatory sites during infection, resulting in the promotion of tumor development. The impact of chemokines on tumor growth is controversial. Since tumor-infiltrating leukocytes are able to produce several growth and angiogenic factors, they may enhance tumor growth and spread. Some CXC chemokines that carry a three-amino acid motif, ELR (glutamate-leucine-arginine), which include IL-8, ENA, Gcp2, GROalpha, GRO-beta, have been reported to be chemotactic for endothelial ~ e l l s .Thus, ~ ~ , they ~ ~ may assist angiogenesis during tumor development. Tumor derived IL-8, a major chemotactic factor for neutrophils, has been

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shown to directly promote angiogenesis and tumor metasta~is.5~/~* In contrast, non-ELR CXC chemokines such as PF4, IP-10, MIG have angiostatic properties and inhibit the angiogenic activity of ELR containing ~ h e m o k i n e sChemokines .~~ recruit leukocyte into the tumor site, followed by functional activation, that may also contribute to the host antitumor response. Presently, a large number of data indicate that chemokines may mediate the inhibition of neovascularization, tumor growth, and m e t a s t a s e ~ . ~ *Many , ~ ~ - chemokines ~~ are constitutively expressed in a variety of tumor ~ e l l s .For ~ ~instance, ,~~ various chemokines such as HuMig, MIP-lalpha, beta, IL-8, MCP-1 have been found to be secreted by both primary hepatocellular carcinoma and hepatic metastases of colorectal cancer.58High RANTES expression was detected in breast tumor cells lines (breast adenocarcinoma cells T47D and MCF-7) as well as in histological sections of breast carcinoma.59Granulocyte chemotactic proteins (GCP-2), MCP-1 and MCP-2 have been isolated from osteosarcoma and glioma cells.60,61

1.5. PMNs Can Promote Tumor Metastases Metastases from primary tumors are the prevalent cause of cancer related deaths. Metastasis is a complex process that includes several consequent steps such as tumor cell shedding into the blood stream either by direct invasion into tumor-associated vessels or indirectly via the lymphatic route; and migration through the vascular endothelium into the target organ followed by intensive proliferation there. Despite the evidence of PMN cytotoxic activity against tumor cells, neutrophils can promote primary tumor growth and metastasis under certain circumstance^.^^ According to Starkey et al. neutrophils are able to increase tumor cells attachment to the endothelial monolayer in ~ i t r o The . ~ ~ close interaction of PMN with metastatic tumor cells during extravasation was verified by light and electronic microscope observations.66 PMN derived from tumors have been shown to enhance tumor cell invasiveness and metastatic potential in rat mammary adeno~arcinoma~~ as well as QR-32 murine fibrosar~oma.~~ Thus, depletion of PMN in vim using monoclonal antibody did not inhibit either tumor development or growth of QR-32 tumor cells. In contrast, tumor cell lines established from the mice with

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The Neutrophils

depleted granulocytes, showed a significant decrease in the metastatic incidence as compared with the tumor cell lines obtained from control mice. These results suggest that inflammation is important for tumor cells to acquire metastatic phenotype. To enter into the target organ of metastasis, tumor cells need to interact with the vascular endothelium via adhesion molecules. The repertoire of the surface proteins expressed by vascular endothelium includes granule membrane protein 140 and its ligand67;vascular adhesion molecule (VCAM-1) and its receptor68;and select in^.^^ Besides, tumor cells have been reported to trigger endothelial cells retraction as well as the contactinduced apoptosis resulting in the disruption of endothelial layer integrity followed by facilitation of tumor cell e x t r a v a ~ a t i o n .However, ~~,~~ the exact mechanisms involved in the tumor extravasation are still not clear. Wu et al. have reported data indicating that PMN may assist tumor cells during its transmigration through the vessel e n d o t h e l i ~ mThese .~~ findings show that human PMN incubated with tumor conditioned medium (TCM), prepared from human breast adenocarcinoma cell line MDA-MB-231, strongly facilitate tumor cells migration through normal macro- and microvascular endothelial monolayer in vitro. Although MDA-MB-231 cells have been previously shown to cause endothelial cell apoptosis, they were not able to cross this endothelium barrier without tumor conditioned medium-treated PMN or with aid of non-treated PMN. MDA-MB 231 cells seem to express ICAMl but not p2 integrins (CDlla/CD18, CDllb/CD18) and p3 integrins which are known to be necessary for the process of transendothelial m i g r a t i ~ n .In ~ ~contrast, ,~~ TCM treated PMN have been found to show high expression of CDllb/CD18. PMN treatment with TCM from MDA-MB 231 cells delayed the rates of apoptotic cell death in comparison to the normal neutrophil. These observations may indicate that MDA-MB 231 metastatic breast cancer cell line secretes factors altering PMN phenotype to facilitate tumor cells cross endothelial migration. Because PMN assistance to tumor cell invasion is not mediated by the disruption of endothelium integrity, the authors have considered the hypothesis that p2 integrin ICAM-I interaction between PMN and tumor cells enhances the tumor cell migration through the endothelium in the presence of PMN. The finding that IL-8 increased PMN but not tumor cells migration as well as

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the observation that blocking MAb against adhesion receptors decreased PMN facilitation of tumor cells migration supported this hypothesis. There is evidence for the involvement of elastase in cancer metastaHuman pancreatic and colon cancer cell lines, characterized by different levels of elastase activity, appeared to exhibit different abilities to adhere to TNF-alpha activated human vascular endothelial cells HUVEC. Adhesion of cells with high intracellular elastase activity was more marked in comparison to that of cells with low elastase activity and was inhibited by a potent elastase inhibitor ZD8321. In contrast, adhesion of the tumor cells with low elastase expression was enhanced by neutrophil elastase. In addition, neutrophil elastase has been found to increase the expression of E-selectin (the key molecule in leukocyte-endothelial cells interaction) on HUVEC followed by an elevated concentration of soluble E-selectin in the medium after the PMNs adhesion to HUVEC. Both these effects were inhibited by ZD8321. These findings suggest that one of the biological functions of elastase is to stimulate both E-selectin expression on endothelial cells and adhesion of tumor cells with them, resulting in the facilitation of metastases. Inhibition of elastase activity can be a potent strategy for control of cancer ~netastasis.~~ Thus, the inflammatory components of a developing neoplasm, such as the diverse leukocyte populations with a predominant influx of macrophages and granulocytes, all of which are able to produce reactive species and to secrete a variety of cytotoxic mediators, appear to play a crucial role in epithelial-stromal interaction during neoplastic progression. However, neutrophils also have a high, direct antitumor activity.

2. THE ROLE OF NEUTROPHILS IN ANTITUMOR REACTIONS 2.1. PMN-Mediated Tumor Destruction It is now clear that inflammatory cells are involved in the tumor surveillance. Antitumor cytotoxic effect of PMN in vitro is known to be induced by various stimuli, such as animal or plant lectins, bacterial glucans and other i m m u n ~ r n o d u l a t o r s . ~ Inflammatory ~-~~ PMNs are able to destroy tumor cells effectively due to production of several cytotoxic mediators,

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The Neutrophils

including reactive oxygen and nitrogen intermediates, proteases, membrane-perforating agents and some soluble factors such as IFNs, TNF, and Recently, it has been demonstrated that human PMN conIL-1p.10~18~19~21 stitutively express granzyme B and perforin, the two molecules known as the cytotoxic entity of natural killer cells and of cytotoxic T lymphocytes as well.85In uitro or in vim (inflammatory peritoneal neutrophils) activated PMNs have been shown to lyze tumor cells.80-82There is also a report that unstimulated rat peripheral blood neutrophil were toxic against syngeneic colon cancer cells.83In this study, tumor cell lysis was found to be caused not by reactive oxygen species released by PMNs, but by cytolytic factor, that was spontaneously secreted by neutrophils. This factor has a low molecular weight (less than lOKD), is heat stable and is partially inactivated by chymotrypsin. This points out to its possible granule proteases structure. In contrast, Aeed et al. have shown that nonactivated rat PMNs were not able to lyze both primary and locally recurrent mammary adenocarcin~ma.~~ The study of the mechanisms of the PMA-triggered PMN cytotoxicity against B-lymphoblastoid Daudi cells and erythroleukemic K-562 cells has shown that lyses is promoted by hypochlorous acid (HOC1).86 Only 35% of the generated H202 appeared to be used by PMNs to produce HOC1, while PMNs themselves and target cells consumed the remainder of H202.Neutrophils and tumor cells co-aggregated at an early step of the cytotoxic reaction. PMN-target cell binding was inhibited by mAb J-90, directed against membrane adhesion molecule LFA-1 (leukocyte function-associated antigen-1 or CDlla/CD18). That resulted in the abrogation of tumor cell lysis despite the normal HOCL generation. Calprotectin, one of the most abundant PMN cytosolic proteins, induces apoptotic cell death in various tumor cells.87The induction of cell death of MM46 mouse mammary carcinoma has been markedly inhibited when RNA synthesis inhibitor actinomycin D or the protein synthesis inhibitor cycloheximide, was added to the culture medium 12 h prior to the incubation with the tumor cells. In addition, calprotectin-induced cell death was inhibited by the antioxidant reagents N-acetyl-l-cysteine (NAC) or propyl gallate even 15h after the start of the incubation. Thus, induction of protein synthesis and the generation of oxygen metabolites

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may be essential factors for cell death induction by calprotectin in the early and late phases of reaction, re~pectively.~~ It appears that the mechanisms underlying the PMN cytotoxic effect against tumor cells depend on the origin of the tumor cells and the nature of PMN stimuli. The ability of PMNs to produce ROS in response to stimuli is altered in tumor-bearing animals and in cancer patients. The defect of PMNs to produce ROS was detected in patients with alimentary tract cancer using nitroblue tetrasolium test (NBT).Tumor excision resulted in the improvement of the NBT reaction.88Similarly, PMN ROS production (evaluated by chemiluminescence), in response to recombinant TNF, has been markedly decreased in patients with gastric cancer?l In contrast, breast cancer patients showed a significant increase in the number of blood neutrophils with activated ROS production in comparison to healthy women, and this may be an indication of endogenous stimulation of PMN. However, the ability of PMNs to respond to the stimulus (opsonized zymosan) was significantly de~reased.8~t~O Tumor cell conditioned media can prime PMNs for enhanced release of reactive oxygen intermediate^.^'.^^ Colon adenocarcinoma enhanced superoxide anion release by human PMNs in response to stimulation by opsonized z y m ~ s a n . ~ Urinary ' bladder carcinoma cell line UBC5637 stimulated neutrophil oxidative metabolism directly without the aid of any additional stimuli. This effect of UBC conditioned medium was similar to that of recombinant GM-CSF. The fact that UBC produces GM-CSF suggests that GM-GSF may also be involved in PMN stimula tion.91r93 The ability of Fas ligand (FasL) to impair immune response is well known. Paradoxically, FasL was found to exhibit proinflammatory activity and to recruit and activate n e ~ t r o p h i l s FasL-expressing .~~ tumor induces inflammation associated with the reduction of t~morigenicity.~~ Inoculation of FasL cDNA-transfected hepatoma MH134 cells into normal mice resulted in the tumor eradication after extensive neutrophil influx followed by apoptosis of the tumor cells. In contrast, neutrophil infiltration as well as tumor rejection has not been observed when MH134 cells were inoculated in mice with defect either in the death domain of Fas (DD mutated Fas) or F ~ s L / F ~These s . ~ ~data suggest that PMN apoptosis mediated by FasL-expressing tumor may induce the extensive neutrophil

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The Neutrophils

recruitment resulting in inflammation and in the eradication of tumor. Despite the ability of PMN to destroy tumors, a significant number of PMN-bound tumor targets are able to escape lysis. The resistance of tumor cells to reactive oxygen species can be mediated by antioxidant enzymes (gluthation reductase, SOD, catalase) and other antioxidant fact o r ~The . ~ failure ~ of PMN to kill tumor cells may be caused by catalase inhibition of hydrogen peroxide production in activated n e ~ t r o p h i l sIt .~~ appears that the efficiency of PMN antitumor response in vivo depends on the tumor microenvironment.

2.2. Cytokine and Chemokine-Induced PMN Anti-Tumor Activity Recombinant human granulocyte colony-stimulating factor (rhG-CSF) is currently used for the treatment of cancer patients with neutropenia (see Chapter 11). G-CSF has been reported not only to be a growth factor for the myeloid lineage but to also modulate the function of neutrophils. Recombinant G-CSF enhances phagocytosis, superoxide anion generation, chemiluminescence, microbicidal activity, and ADCC in neutrophils. G-CSF treated PMNs were characterized by increased expression of CD14, CD32, and CD64 molecules. Their binding to available ligands triggers the neutrophil effector functions. However, the significant impairment of CD16 expression, reduction of chemotaxis and in vivo migration into inflammatory sites were attributed to neutrophils after G-CSF administration. These effects may be caused by accelerated bone marrow transit time of myeloid cells.97Indeed, the function of neutrophils in lung cancer patients treated with G-CSF after chemotherapy has been increased or maintained.98Moreover, rhG-CSF administration following chemotherapy significantly stimulated the biosynthesis of human neutrophil peptides localized in the azurophil granules. This finding indicates that rhG-CSF is able to enhance the host defense in compromised patients with n e ~ t r o p e n i aAdministration .~~ of rhG-CSF also augmented the antitumor cytotoxicity of neutrophils.lOO Systemic treatment with other cytokines (TNF-alpha, IL-12, IL-2) may result in effective tumor rejection at least partly mediated by activated neutrophils. Using spontaneously metastasizing mammary adenocarcinoma,

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Cavallo et al. have shown that the key factors mediating IL-12 induced antitumor activity are: (a) indirect inhibition of angiogenesis by secondary cytokines (IFN-gamma and TNF-alpha) and cytokine induced chemokines IP-10 and MIG; (b) injury of tumor vessels by neutrophils; (c) activation of lymphocytes to produce proinflammatory cytokines, antitumor antibodies and cytotoxic T-lymphocytes. In this model PMNs seem to have no inhibitory effect on lung metastases, whereas they were of critical importance for the eradication of primary tumor.lO'The findings that selective depletion of PMN abrogates the antitumor effect of IL-12 as well as the observation of a significantly enhanced number of tumor-infiltratingPMN, 3 h after IL-12 administration, suggest the key role of PMN in IL-12 mediated tumor regression. Massive intratumoral influx of PMN is probably involved in the micro-vessel injury resulting in extensive necrosis of the tumor tissue.lO' TNF-alpha stimulates PMN cytotoxicity against tumor cell of different lines, including erythromyeloid cells K-562, lymphoma Raji, and melanoma M-14. These effects were mediated by hydrogen peroxide, MPO, and cationic prot e i n ~ . ' ~ PMN * ~ ' ~ phenotypic ~ and functional activation has been documented in patients with malignant melanoma and renal cell carcinoma treated with IL-2 infusions.104,10s PMNs appear not only to be responsible for much of the therapeutic efficacy, but also for the systemic toxicity of IL-2 resulting from vascular leak syndrome.10sSystemic cytokine therapy in most cancer patients results in rather poor efficacy due to multiple side effects. Mouse adenocarcinoma TSA, which is apparently nonimmunogenic in BALBc mice, has been shown to become immunogenic after IL-2 gene transfection.106Tumor rejection was associated with dose-dependent IL-2 induced neutrophil infiltration and minor CD8+ T cell influx, while NK cells or CD4+ lympocytes were not affected. This neutrophil related tumor rejection was followed by tumor-specific T-cell mediated immune memory, which required CD4+ lymphocytes.106PMNs have been shown to express IL-2R beta, and gamma chains and IL-2 can directly maintain their viability thereby preventing PMN from being subjected to programmed cell death. Neutrophils activated with IL-2 transduced TSA cells have been reported to have higher tumoricidic activity. This activity was

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abrogated with anti-IL-2 and anti-IL-2R beta Mabs. These data suggest a direct role for IL-2 activated PMNs in tumor cytolysis.*07 Graf et al. demonstrated in the studies using rat T9 glioma cells with the IL-6 transduced gene, that the early antitumor response (10th day) might be mediated by neutrophils followed by glioma-specificT cell activation at the later stages.los Regression of subcutaneously transplanted T9 IL-6 glioma was found to be associated with massive neutrophil infiltration, whereas neutropenic rats did not show tumor regression.108In the case of intracranial injection of IL-6 secreting glioma cells, granulocyte infiltrate was the most visible at day 17 because of delaying neutrophil influx into the immune privileged site. Authors suggested that neutrophils infiltrating IL-6 expressing glioma became activated and initiated tumor destruction via the release of reactive oxygen metabolities, proteolytic enzymes and TNF-a. At the later stage, antigen presenting cells present tumor specific antigens to naive T cells in tumor-draining lymph nodes, followed by the traffic of activated T-lymphocytes into the tumor site and lysis of the remaining tumor cells.lo8Consistent with this hypothesis, Porgador et al. reported that injection of Lewis lung carcinoma secreting IL-6 into nude mice resulted in the decrease of primary tumor formation but not lung metastases, whereas the suppression of both primary tumor and metastasis was observed in normal syngeneic mice.lo9 Murine fibrosarcoma cells genetically altered to produce IL-6 have been reported to reduce metastases in nude and sublethally irradiated mice, suggesting also a non-T-cell mediated antitumor mechanisrn.ll0 In order to understand the mechanisms underlying the antitumor response induced by different interleukin genes transfected into tumor cells, a comparative study was performed using mammary adenocarcinoma cells TSA in BALB/c mice. TSA cells were engineered to release IL-2, IL4, IL-7, IL-10, IFN-a, IFNy and TNF-a. The rejection of TSA-IL-2 and TSA-TNF-a cells was associated with massive neutrophil infiltration; while TSA-IL-4 and TSA-IL-7 cells, with neutrophil influx and poor areas of necrosis; and TSA-IL-10 and TSA-IFN-a cells, with extensive areas of ischemic necrosis and some neutrophil infiltration. IFN-y secreted TSA cells were not rejected but showed delay in growth of tumor, which was characterized by areas of ischemic necrosis in the absence of neutrophils. These findings, in combination with those obtained using the selective

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depletion experiments, demonstrated the importance of various leukocyte populations in rejection of TSA transfected with different interleukins."' To study the effect of chemokines on tumor growth, Chinese hamster ovarian (CHO) cells were transduced with genes for human (hu) IL-8, hu MCP-1, hu-MIP-1-alpha, murine (mu) MCP-1, mu-MIP-1-alpha or muMIP-2. The ability of these cells to form tumors in vivo was evaluated. Expression of huIL-8, MIP-1-a, or muMIP-1-aprevented tumor growth in nude mice despite the absence of the effect on tumor cell growth in nitro. Histological examination of the site of injection displayed infiltration with neutrophil. These findings indicate potent antitumor activity of chemokines released in tumor sites, which may be mediated by recruitment and targeting of PMNs to chemokine secreted tumor cells.112PMN migration from blood into tumor is a complex process that involves several regulatory molecules like selectins, integrins, and interleukins. They assist in the interaction between endothelial cells and PMN, resulting in intratumoral neutrophil recruitment. Neutrophil extravasation is related to induction of ELAM-1 and upregulation of ICAM-1 in the blood vessels, as well as to secondary induction of C X C - c h e m o k i n e ~ . Moreover, ~ ~ ~ J ~ ~ the evidence for macrophage inflammatory protein-2 (MIP-2) production was found in tumor-bearing mice treated with tumor cells, transfected with G-GSF, IL-2, IL-12, and TNF-a genes.l14 MIP-2, also known as cytokineinduced neutrophil chemo-attractant (GRO/KC), is the murine functional homologue of human IL-8. It upregulates PMN integrins as well as ICAM-1 on endothelial cells.l16MIP-2 is produced by tumor-associated macrophages following stimulation with self-secreted or tumor-derived mediators.57 MIP-2 expression is associated with the presence of TNF-a and IL-Ip in tumor microenvironment. It contributesto marked PMN influx and the ability of PMN to release MIP-2 in response to TNF-cY?~ A high level of tumor specific CTL response is necessary for tumor rejection. IFNy, produced by both activated macrophages and Th-1 subset of lymphocytes in a tumor microenvironment, upregulates the expression of endothelial selectins, adhesion molecules ELAM-1, ICAM-1, and chemokine production by tumor-associated macrophages. This results in a marked PMN recruitment into the tumor site. In turn, IFNy-stimulated PMN release chemokines IP-10 and MIG, which are chemoattractants for monocytes and T cells. PMN has been recently shown to activate

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endothelial cells for IL-6 and IL-8 release, independently on ICAM-1 or P2-integrinexpression. This endothelial activation appears to be mediated by microparticle secretion from PMN and was enhanced by inflammatory stimuli, formyl peptide and phorbol ester. These data suggest that membrane microparticles released from stimulated PMNs are inflammatory mediators, which induce activation of endothelial cells followed by cytokine gene induction.l17 Tumor-associated activated PMN also produce neutral proteinase elastase, which can facilitate the infiltration of tumor through the tissue damage.lls These findings point out the crucial role of PMN in antitumor response, involving both specific and nonspecific immune effector cells.

2.3. Neutrophils as Effectors of Antibody-Dependent Cell-Mediated Cytotoxicity Against Tumor Some antibodies can induce tumor cell lysis by activating complementmediated and cell-mediated cytotoxicity (antibody-dependent cellmediated cytotoxicity, ADCC). The effector cells for ADCC are PMNs, monocytes/macrophages, eosinophils and NK cells.119,1*o~121 PMNsmediated ADCC can be triggered through the antibodies binding to neutrophil immunoglobulin receptors FcyRI (CD64), FcyRII (CD32), FcRI (CD89), and F c R I I I . ' ~ ~Mac-1 - ~ ~ ~(C3R, CDIIb/CD18) is essential for PMN Fc-receptor mediated ADCC.128Mac-1 is known to regulate several neutrophil functions, including adhesion, migration, chemotaxis, phagocytosis, activation of respiratory burst enzyme and d e g r a n ~ 1 a t i o n . l ~ ~ Moreover, evidence exists for Mac-1 cooperation with different PMN receptors to provide intracellular ~igna1ing.l~~ The role of Mac-1 in FcRmediated cytotoxicity against tumor targets has been studied using different models, such as Mac-1 deficient mouse PMNs and mouse PMNs transgenic for human FcR. These studies demonstrate the necessity of Mac-1 in FcyR and FcolR-mediated tumor cytotoxicity. The critical role of Mac-1 in ADCC was shown selectively for PMNs because Mac-1 deficient macrophages exhibited normal antibody-dependent cytotoxicity.128 This may reflect different mechanisms by which macrophages and PMNs lyze tumor targets: extracellular PMN cytotoxicity and phagocytosis of tumor cells by m a ~ r o p h a g e s . l ~ ' -Mac-1 ' ~ ~ appeared to be required for

Polymorphonuclear Neutrophils and Cancer

2 91

PMN spreading on Ab-coated targets, but not for Ab-binding with subsequent PMN activation (degranulation and respiratory burst activity).128 Based on the data obtained in in vitro studies, it has been suggested that tumor-specific monoclonal antibody can be used for the recruitment of Fc-receptor bearing cytotoxic cells and thus for facilitation of tumor rejection. However, among the numerous antibodies generated against tumor cells, only a few have demonstrated antitumor e f f i c a ~ y . I ~ ~ - ' ~ ~ Monoclonal antibody 3F8 (anti-GD2 ganglioside mouse Ig G3 mAb) was successfully used for the treatment of cancer patients and experimental tumors in rats.'37 Effective 3F8 mAb-mediated cytotoxicity was caused by human and rat PMN, but was not observed with mice derived PMNs. These differences are likely to be related to the fact that murine PMNs do not express FcyRIIIb, which is necessary for efficient 3 F8 mAb-induced ADCC.138Anti-HLA-DR mAb (Lym-1) plus PMA induced PMN-mediated cytolysis of B-lymphoma cells.139The reactive oxygen species appeared not to be required for the effective cytolysis, because neutrophils from patients with chronic granulomatous diseases were effective despite their inability to demonstrate respiratory burst activity. The FcyRII, Mac-1 but not CD66b molecules were involved in PMA stimulated Lym-1 dependent cytolysis of tumor cells by P M N s . ' ~Monoclonal ~ antibody against cellsurface disialoganglioside (GD2) in neuroblastomas and other tumors has induced neutrophil and mononuclear cell mediated ADCC in vitvo and antitumor response in some neuroblastoma patient^.'^^,^^^ Significant tumor cytotoxicity and tumor growth-inhibiting effect of PMNs, in combination with chimeric anti-GD2 antibody, were found when GD2 was highly expressed on neuroblastoma cells. However, PMNs appeared to promote tumor cell growth when cytotoxicity was not triggered due to the absence of anti-GD2 Abs or sufficient GD2 expre~sion.'~~ These findings suggest the necessity for further optimization of various parameters of treatment like tumor site, antibody concentration, effector cell number and activity, and GD2 expression on tumor cells. Bispecific antibodies (bsAbs) seem to be a promising tool to improve immunotherapy of cancer due to their ability to redirect effector cells towards tumor targets. BsAbs are artificially constructed proteins displaying two different antigen-binding sites that provide the ability to trigger effector cells through a membrane receptor with simultaneous binding to

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The Neutrophils

tumor cells. These interactions result in the target tumor destruction. In early studies investigating bsAbs for cancer treatment, mostly T lymphocytes were used as effector cytotoxic cells. However, T-lymphocyte activation requires costimulatory signals. Moreover, T-cell activation in blood following intravenous bsAbs administration resulted in the severe side effects because of the release of several ~ y t 0 k i n e s . PMNs, l~~ as the most abundant white blood cells, can be better suited as effector cells due to their ability to express high affinity receptor for IgG FcyRI (CD64). CD64 appeared to be an attractive target molecule for Abs based immunotherapy. Neutrophil expression and monocyte upregulation of CD64 are easily achieved in patients by systemic application of G-CSF or I F N - Y Furthermore, the results of clinical trials with CD64 directed immunotherapy in cancer patients have demonstrated excellent tolerability in association with promising antitumor e f f e ~ t ~To. investigate ~ ~ ~ i the ~ ~role ~ of CD64 in bsAbs mediated effect, Heijnen et al. have used human FcyRI transgenic mouse. PMNs isolated from these mice effectively killed human breast cancer cells in the presence of bsAbs against CD64 and '~~ of this antibody in vivo resulted in its HER2/neu in v i t ~ o .Administration binding to PMN, followed by PMN migration to the tumor site. Importantly, these armed PMNs appeared to exhibit strong cytotoxic activity against tumor cells in vitvo without prior s t i m ~ l a t i o n . ' ~ ~ MAb directed against the proto-oncogene product HER2/neu interacts with FcyRI, resulting in enhanced lysis of HER2/neu-expressing tumor cells by G-CSF primed PMN. However, the serum IgG appeared to impair the activation of FcyRI mediated cytotoxicity. In order to avoid this problem, bispecific anti-FcyRI and HER2/neu Ab (BsAb 22 X 520 CD) was constructed, which has been shown to effectively recruit the cytotoxic potential of FcyRI on G-CSF primed PMN regardless of the presence of human serum. PMNs have been armed with these bsmAb during migration into inflammatory sites and are able to specifically lyze HER2/neu expressing tumor cells.'49

3. CONCLUSION The role of neutrophils in the pathophysiology of cancer is still being defined. Neutrophils are thought to be involved in mutagenesis as well as

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carcinogenesis due to their powerful oxidizing agents that may be responsible for the genomic damage. The evidence from animal studies suggests the direct role of activated neutrophils in promoting tumor formation. However, the relevance of these observations to human cancer is not so clear. A significant percentage of human cancers worldwide seem to be associated with bacterial or viral infections. Infection-related chronic inflammatory process, as a result of inadequate host response against invading microorganisms, may promote neoplastic transformation. PMNs as most abundant inflammatory cells, may contribute to accelerated epithelial cell turnover associated with tissue injury. Activated tissue regeneration under the condition of high production of reactive oxygen metabolites by inflammatory cells, may result in DNA damage and genomic alterations. The evidences for damage of p53 function during inflamrnati~n~~ are of critical importance to our understanding of the molecular mechanisms of neoplastic transformation because approximately 50%of all cancers have mutations in p53 gene. PMNs appear to be involved in facilitating the growth and metastasis of established tumor. PMNs along with other leukocytes can contribute to tumor progression by releasing proteases, angiogenic factors and chemokines as well as directly facilitating the tumor cells transendothelial migration.@Understanding the contribution of PMNs in carcinogenesisand tumor growth may lead to the development of new specific approaches to cancer chemopreventionand treatment. On the other hand, it is also clear that the immune system provides protection against a wide variety of pathogens and plays an important role in the host response to tumor cells. PMNs, as one of the key elements of host defense against infection, may be considered as a potent factor in the suppression of tumor growth. New evidence suggests PMN promoting T cell memory response in cancer. Neutrophils were found to be indispensable elements for the eradication of most interleukin gene transfected tumors.20PMN recruitment into the tumor site followed by their activation may result in the direct damage of tumor cells as well as inhibition of angiogenesis and activation of other immune effector cells. PMN mediated ADCC appears in some cases to contribute to immune memo , ~ The . ~ findings ~ ~ of the therapeutic benefit of PMN activation with bispecific Mabs, in combination with G-CSF or IFN-y, may form a foundation for new approaches to cancer treatment.

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The Neutrophils

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10 Use of Colony-Stimulating Factors for Treatment of Neutropenia and Infectious Diseases Lee]. Quinton, David C. Dale, Steve Nelson"

The "colony-stimulating factors" are growth-promoting substances for the hematopoietic cells. This term was introduced by Bradley & Metcalf in 1966, when they demonstrated that specific factors derived from living cells can stimulate hematopoietic precursors to form colonies and clusters of cells in an in nitro culture system.l In a series of critical experiments, they demonstrated that these factors can be detected in serum, urine and other body fluids, using their colony-forming a s ~ a y . They ~ - ~ also demonstrated that endotoxin injections and experimental infections, conditions known to increase blood neutrophil levels, are associated with enhanced production and secretion of these factor^.^,^ Currently, three hematopoietic growth factors are called colony-stimulating factors (CSF).These are granulocyte-CSF (G-CSF), granulocyte macrophage-CSF (GM-CSF) and macrophage-CSF *Correspondence to: Steve Nelson, MD. John H. Seabury, Professor of Medicine, Pulmonary/Critical Care Medicine, Louisiana State University Medical Center, 1901 Perdido Street, Suite 3205, New Orleans, LA 70112 1393; phone: 504-568-4634; fax: 504-568-4295; e-mail: snelsol8lsuhsc.edu 301

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(M-CSF). A fourth factor, originally called multi-CSF, is now usually referred to as Interleukin-3 (IL-3). A number of other interleukins (IL-1 through IL-23), as well as erythropoietin (EPO) and thrombopoietin (TPO), and several other factors, are known regulators of the hematopoietic process. This chapter focuses on G-CSF and GM-CSF, the factors principally influencing neutrophil production and function. Keywords: neutrophil; colony-stimulating factors; therapy

1. CHARACTERISTICS OF G-CSF, GM-CSF AND ITS RECEPTORS G-CSF was first described as a stimulatory factor present in the serum of mice after endotoxin injection^.^,^ Murine and human G-CSF were subsequently identified and purified, and the G-CSF cDNA was subsequently isolated from a bladder carcinoma cell line.8-10Native human G-CSF is an approximately 20-kd glycoprotein containing 174 amino acids, coded for by a gene on chromosome 17q21-q2.11,12Recombinant human G-CSF, which is manufactured in both its nonglycosylated (Filgrastim; Amgen corporation, Thousand Oaks, CA) and glycosylated (Lenograstim; Chugai Pharmaceuticals, Tokyo, Japan) forms, has similar functional and pharmacological effects as the native molecule in vim and in vitro. Murine and human GM-CSF were purified and characterized in 1977, several years before G-CSF.I3Human GM-CSF is a glycoprotein containing 127 amino acids and is coded for by a gene on 5q21-q32, contiguous to the genes for several hematopoietic growth factors.14In contrast to G-CSF, which shows close homology across many species,15 GM-CSF shows greater sequence heterogeneity and is relatively species-specific in its activities. Both GM-CSF and G-CSF are composed of four antiparallel, helical peptide segments connected by amino acid chains, which give the molecules their three-dimensional s t r ~ c t u r e . ' ~ Specificity ~'~ is determined by the CSF's amino acid sequence and the three dimensional structure of its binding domain, as well as the presence and integrity of the cellular receptor. Humans have one class of high affinity receptors for G-CSF, which are composed of two identical molecules, i.e. they are homodirners.ls The G-CSF receptor (G-CSF-R) is a member of the hematopoietin receptor s~perfamily.'~ Although the G-CSF-R contains no intrinsic tyrosine kinase activity, ligation of G-CSF with the G-CSR-R extracellular domain induces the activation of several cytosolic tyrosine kinases. The consequent

Use of Colony-Stimulating Factors 303 signaling cascades involve the Janus protein tyrosine kinase (JAK)family, signal transducers and activators of transcription (STATs), and mitogenactivated protein (MAP) kinases.20 In particular, studies suggest that JAK-1 phosphorylation is critical for downstream activation of STATs 1,3, and 5, which subsequently upregulate the expression of genes necessary for G-CSF-induced granulopoiesis?1t22Distinctive domains of the cytosolic portion of the G-CSF-R, referred to as the membrane-proximal and membrane-distal regions, contain tyrosine residues at positions 704, 729, 744, and 764. All four residues are phosphorylated in response to G-CSF/G-CSF-R signaling in a process required for the proliferation and maturation of myeloid cells bearing the G-CSF receptor.23 By contrast, the CM-CSF receptor is a heterodimer, i.e. composed of two dissimilar transmembrane proteins, an alpha- and a beta chain.l7lz4 The high affinity GM-CSF receptor is found on all types of granulocyte precursors, including eosinophils, as well as blood and marrow monocytes and their precursors. By contrast, G-CSF receptors are present only on cells of the neutrophilic lineage. GM-CSF also activates cells bearing its receptor through the JAK kinases, as well as the Jun kinase pathway to ras-MAP kinase a c t i v a t i ~ nAlthough . ~ ~ ~ ~ ~low affinity G-CSF and GM-CSF receptors have been found on various nonhematopoietic and cancer cells, the functional significance of these receptors is largely unknown.

2. NEUTROPHIL A N D MONOCYTE DEVELOPMENT AND FUNCTION Neutrophils are derived from the common hematopoietic stem cells through the processes of proliferation, differentiation and maturation. Overall, this process normally takes 10 to 14 days, as estimated by in vivo radioisotopic labeling studies. Morphologically, the earliest recognizable neutrophil precursors are the myeloblasts, large cells which have few cytoplasmic granules. Differentiation and maturation involves the condensation of the nuclear chromatin, development of primary and secondary granules, accumulation of adherence proteins and receptors, and a number of other refinements in these cells. A unique feature for cells of the neutrophilic series is the production and storage in the marrow of a large population of relatively mature cells which are normally released from the marrow to the blood in response to infections. Other factors that stimulate the release of

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granulocytes include G-CSF, GM-CSF, glucocorticoids, endotoxin, leukotriene 84, platelet activating factor, C5a, bacterial-derived formylated peptides, and i n t e r l e ~ k i n - 8 . Of ~ ~ these, - ~ ~ G-CSF is particularly effective in the mobilization of both mature neutrophils and neutrophil p r e c u r ~ o r s . Less evident than the capacity of G-CSF to induce cell mobilization is the mechanism by which this phenomenon occurs. The egress of mature and immature granulocytes from the bone marrow involves the disruption of adhesion interactions which normally anchor cells within the bone marrow environment. Among these interactions are those mediated by integrins$ selectins,= and cytokines such as stromal cell-derived factor-1 (SDF-1) and stem cell factor (SCF).34In the currently proposed model of G-CSF-induced mobilization, proteases (elastase, cathepsin G, and matrix metalloproteinases) are released within the bone marrow in response to G-CSF/G-CSF-R signaling. In turn, these proteases cleave intractions between the abovementioned retention molecules and their corresponding l i g a n d ~Proteolytic .~~ degradation of SDF-1 appears to be particularly important during this event, as its levels in bone marrow are significantly reduced in response to exogenous G-CSF treatment?5 The loss of adhesion between the granulocytes and the bone marrow stroma allows granulocytes to respond to migratory stimuli in the peripheral circulation, thus facilitating their exit from the bone marrow. Interestingly, recent data by Semerad et aE. show that G-CSF-induced neutrophil mobilization does not require the expression of the G-CSF-R on bone marrow neutrophils or stromal cells, but instead requires its expression on the surface of other hematopoietic cells within the bone marrow milieu.36To determine this, the authors lethally irradiated wild-type mice, and reconstituted them with various ratios of G-CSFR-deficient and normal hematopoietic cells. Following 7 days of G-CSF treatment, they measured the neutrophil responses in blood and bone marrow and found that G-CSFR deficient neutrophils were mobilized just as well as wild-type neutrophils. However, higher ratios of G-CSFR-deficient hematopoietic cells decreased overall neutrophil mobilization in a dose-dependent manner, irrespective of the presence of G-CSFR on the neutrophils themselves. This suggests that trans-acting factors (i.e. elastase and other proteases) are produced in response to G-CSF signaling, which are then necessary for the effective mobilization of neutrophils into the systemic circulation.

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Neutrophils are formed in the extravascular spaces of the marrow and enter the blood by movement through pores or fenestrae in the marrow vasculature. The short lifespan of neutrophils ( 4 4 hours)37demands a continual release of new cells into the systemic circulation. This is made possible by the maintenance of large stores within the bone marrow, which constitute as many as 99% of the total body neutrophil population under normal conditions.36 Once within the blood, the neutrophils are found in two pools in dynamic equilibrium. About half of the cells flow along with the circulating red blood and are regarded to be in the circulating pool. At least half of the other cells are in the marginal pool. The marginated cells are loosely adherent to vascular endothelial cells throughout the circulation, although the lung, spleen and tissues with capillaries with low blood flow rates may preferentially hold these cells. Adherence proteins, e.g. the selectins which are expressed on the surface of the neutrophils, are thought to regulate the loose and reversible adherence of these cells, which creates the marginal pool. In addition to the adherence proteins, the size and deformability of neutrophils also play a major role in their margination?8 This is particularly important during stress conditions, where the presence of inflammatory mediators lessens the deformability of the circulating neutrophils, thus increasing their sequestration within microvasculature. Firm adherence, mediated by leukocyte integrins, is a necessary and final step before the neutrophils migrate from the blood to the tissues.39 Neutrophils serve as the “first line” cells of the acute inflammatory response in all the body tissues. The rapidity of this response is easily demonstrated by examining the cutaneous inflammatory response by the skin chamber or Rebuck skin window technique. Neutrophils can be measured as migrate to the site of injury and accumulate in large numbers over a few hours. Although bacteremia is a relatively common event in severely neutropenic patients, it is generally a consequence of an inadequate tissue response to contain infection. Ordinarily, bacterial clearance from the blood is a function of the fixed phagocytes lining the vasculature in the spleen, liver, lung, marrow and other tissues. At the inflammatory site, neutrophils engulf bacteria and other foreign debris in a phagocytic vacuole into which microbicidal and proteolytic enzymes are released, resulting in killing and digestion of the invading organism. Many details of this process have been dissected

306 The Neutrophils a

through the recognition of genetic diseases, such as chronic granulomatous disease, and disorders of neutrophil granule proteins such as myeloperoxidase. Most frequently, however, failure of neutrophils to adequately contain a tissue infection is attributable to a deficiency in their number, rather than their function. Because the colony-stimulating factors are potent agents to increase the rate of phagocyte production and distribution, as well as stimulators of phagocyte functions, there are many ways in which they may potentially be used to improve the outcome for infectious diseases. The hematopoietic growth factors play an important role in all stages of neutrophil development and deployment. The earliest precursors, the hematopoietic stem cells, appear to have multiple growth factor receptor^.^^ As the cells mature, the number and function of these receptors evolves, but uniquely, G-CSF and GM-CSF receptors are present both early in the developmental process and on mature neutr~phils.'~J~ In the in vitro colony-forming assay system, G-CSF predominantly stimulates the formation of neutrophilic cells from early precursors, whereas GM-CSF stimulates a more diverse pattern of cell formation, with colonies and clusters of cells of all lineages. Complete maturation of erythroid and megakaryocytic cells, however, requires the addition of other factors, i.e. erythropoietin and thrombopoietin. In vitro incubation of G-CSF and GM-CSF with mature blood neutrophils primes these cells for an enhanced metabolic burst when exposed to a second agonist such as FMLP, opsonized zymosan particles, or other stimuli.41The respiratory burst which follows this response leads to production of microbicidal concentrations of superoxide anion and hydrogen peroxide, thus suggesting that these cytokines may play a direct role in enhancing the killing of bacteria and f ~ n g i . In 4 ~addition, ~~ neutrophils previously exposed to these CSFs have an enhanced antibody dependent, cell-mediated cytotoxicity against certain tumor cells (see Tables 1 and 2). Our best information on the in vivo effects of G-CSF and GM-CSF comes from studies of the administration of these factors to normal human subjects or hematologically normal patients. In this setting, it is known that the proliferation of hematopoietic precursor cells is enhanced by G-CSF and GM-CSF, as reflected in vivo by marrow cell numbers, the proportion of cells in mitosis, and a more rapid transit of cells through

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Table 1 Effects of G-CSF on Phagocytes Precursor cells: stimulation of proliferation and differentiation to neutrophils Effects on mature neutrophils: Enhanced respiratory burst Increased phagocytosis of bacteria and fungi Increased presence of Fcy receptor Increased presence of C3bi receptor (CD-35) Upregulated affinity for the ligand of the LAM-1 receptor Stimulates chemotaxis at low concentrations, decreases at high concentrations Decreases migration in skin chamber assay Enhanced neutrophilic antibody-dependent cell-mediated cytotoxicity (ADCC) against certain tumor cells Delayed apoptosis

Table 2 Effects of GM-CSF on Phagocytes Precursor cells: Stimulation of proliferation and differentiation of precursors to neutrophils, eosinophils and monocyte/macrophages. Effects on mature neutrophil granulocytes: Enhanced respiratory burst Increased phagocytosis of bacteria and fungi Increased presence of C3bi receptor Loss of leukocyte adhesion molecule-1 (LAM-I) Upregulated affinity for the ligand of the LAM-1 receptor Stimulates chemotaxis at low concentrations, decreases at high concentrations Decreases migration on skin chamber assay Enhanced neutrophilic antibody-dependent cell-mediated cytotoxicity (ADCC) against certain tumor cells Effects on mature macrophages/monocytes: Increased in vitro effect against M. avium and M. tuberculosis; Leishmania and Trypanosoma Increased cytokine expression/secretion Enhanced ADDC against tumor cells in vitro Enhanced antitumor response in vitro in combination with endotoxin, interferon gamma and lipopolysaccharides

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The Neutrophils

the marrow to the b l ~ o d .Both ~ ~ G-CSF , ~ ~ and GM-CSF can accelerate neutrophil maturation through the post-mitotic/maturational compartment. For example, administration of G-CSF at a dose of 300 p,g/day will reduce the transit time for neutrophils through this compartment, from approximately six to three days.3O GM-CSF at a dose of 250 pg/kg/day, a dose frequently used clinically, has a somewhat lesser effect in accelerating this transit time.28Autologous studies using radioisotopically labeled neutrophils have shown that this dose of G-CSF will increase neutrophil production rates about si~-fold.~O GM-CSF at 250 p,g/kg/day had a far lesser effect.28The production of monocytes, eosinophils and dendritic cells are also stimulated in v i m and in vitro by GM-CSF; G-CSF has relatively little effect on these cells. Some recent studies suggest that increased antitumor responses and treatment benefits in infectious diseases may accrue from the effects of GM-CSF on monocytes and dendritic cells,46,47 but human studies conclusively demonstrating such benefits are not yet available. Adverse effects are more frequently associated with GM-CSF treatment than with G-CSF therapy (i.e. local skin reactions, malaise and fever). This difference may be due to stimulation of cytokine production (e.g. gamma interferon, tumor necrosis factor) mediated through the monocyte receptors for GM-CSF.

3. MEASUREMENT OF CSF LEVELS 1N PATIENTS WlTH NEUTROPENIA A N D INFECTIOUS DISEASES Under basal conditions in normal subjects circulating, concentrations of G-CSF and GM-CSF are usually undetectable, i.e. less than 50 to 100 picograms per milliliter. The levels of these factors are also quite low in urine and other body fluids and generally only detectable if the fluids are concentrated. For this reason, it has been difficult to identify genetic or clinical conditions attributable to low levels of the CSFs. It has been shown that mice which do not express GM-CSF ("knock-outs") are hematologically normal.48 In contrast, both G-CSF deficiency and the lack of a normal G-CSF receptor lead to chronic n e u t r ~ p e n i a . ~Therefore, ~ . ~ ~ - ~ ~it is clear that G-CSF is required for the maintenance of a normal blood neutrophil count. The mechanism by which constitutively low levels of

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G-CSF regulate this process is yet to be determined. While the absolute number of bone marrow neutrophils and progenitors are decreased in G-CSF-deficient miceF9 recent data by Basu et al. show that the cycling and mobilization of these cells remain relatively intact. In the same study, the authors report a significantly higher rate of apoptosis in early granulocytic lineage cells within the bone marrow. They concluded that the role of G-CSF during steady-state granulopoiesis may be largely due to its survival effects within the bone marrow granulocyte pool. This differs from the physiological response to higher levels of G-CSF, such as those seen during infection or after exogenous G-CSF administration. Under these conditions, G-CSF clearly increases the proliferation, differentiation, and mobilization of granulocytes in a process that is seemingly independent of its effects on a p o p t o ~ i s . ~ ~ Endotoxin infection in normal human subjects and in other species has a profound effect on cytokine production and release, including the CSFs. In man and other species, the G-CSF levels are markedly elevated within the first few hours after endotoxin injection; the time course closely corresponding to that for increased IL-6 and IL-8 after endotoxin, and slightly follows the peaking of levels of tumor necrosis factor.52By contrast, the GM-CSF levels are not significantly elevated in response to endotoxin admini~tration.~~ Commercially available immunoassay systems, as well as bioassay systems, have been used to demonstrate these differences. Patients with neutropenia may have elevation of G-CSF levels, but increases in GM-CSF are rarely ~ b s e r v e d .Understanding ~~,~~ the relationship of neutropenia and CSF levels is confounded by the frequency of fever and inflammation in neutropenic patients, thus making it unclear if changes in the CSFs are directly attributable to the circulating neutrophil level or that they have occurred secondarily in response to exogenous factors (e.g. microbial products or endotoxin) entering the tissues or blood. With the current assay methods, most data suggest that neutropenia must be extreme, i.e. counts less than 0.2 X 109/L to have detectable increased G-CSF levels in patients without overt infections. With naturally occurring infections in both normal and neutropenic subjects, G-CSF levels are increased, although many details of the time course of this response in a clinical setting are yet unknown.55-57 In general, there is a correlation of levels with the severity of sepsis, the highest levels

31 0

The Neutrophils

being detected in patients with bacteremia and septic s h o ~ k .Higher ~~,~~ levels also appear to occur with gram-negative than with gram-positive infection^.^^ By contrast, GM-CSF levels are rarely elevated even with severe infections. As the patients improve, the G-CSF levels gradually return to normal.58,59 Many factors may be involved in determining the pattern of the decline in G-CSF levels over the course of an infection. These include the binding of G-CSF by receptors on immature and mature cells produced in greater number in response to the infection;60reduced production of G-CSF as the infection resolves; and reduced levels of other mediators such as tumor necrosis factor and interleukin-1, which may be modulators of the G-CSF response. It is puzzling that GM-CSF is not detected in the blood with inflammation because it is produced by the same types of cells which produce G-CSF, i.e. fibroblasts and endothelial cells, as well as by T-lymphocytes. Currently, it is believed that it is produced locally, i.e. in the bone marrow or at sites of inflammation, and acts locally to modulate hematopoiesis and the inflammatory response at the tissue level.

4. G-CSF IN NONNEUTROPENIC ANIMAL MODELS OF INFECTION Contrary to the widespread belief that infectious diseases are no longer a serious threat to life in any but the developing countries, mortality from infections in the United States has, in fact, increased in recent years.61 Between 1980 and 1992, mortality attributable to infectious disease rose from 41 to 65 deaths per 100000 population in the United States - a 58% increase. Deaths resulting from respiratory tract infections increased from 25 to 30 per 100000, a 20% increase, while deaths from septicemia increased by 83%,from a mortality rate of 4.2 to 7.7 per 100000. These statistics clearly underscore the need for adjuvant therapies in the treatment of severe infections, such as sepsis. One promising strategy for upregulating the host defense system of the infected patient focuses on the use of G-CSF; this cytokine has been studied much more intensively than GM-CSF because of its clearer role in regulating the neutrophil response. The efficacy of G-CSF, either alone or in combination with antibiotic therapy, has been studied in a variety of

Use of Colony-Stimulating Factors 3 1 1 Table 3 Effects of G-CSFin Nonneutropenic Animal Infection Models

Increased production of neutrophils during infection Increased neutrophil delivery into the site of the infection Increased bactericidal activity of neutrophils Additive to synergisticeffects of G-CSF with antibiotic therapy Reduction in the burden of the infection and mortality

nonneutropenic animal infectious disease models (Table 3). These include neonatal sepsis, burn wound injury, surgical wound infection, bacteremia, intraabdominal sepsis and pneumonia.

4.1. Neonatal Sepsis Neonatal sepsis due to group B streptococci remains a significant cause of morbidity and mortality. Developmental immaturity in neonatal phagocytic defenses is a predisposing factor.62Depletion of the marrow neutrophil storage pool with profound neutropenia typically precedes death. Studies suggest that the observed deficit in neutrophil supply may be due to an inadequate endogenous G-CSF response.63In vitro stimulation of blood monocytes from preterm neonates produces less G-CSF than do monocytes recovered from term neonates or adults.@As a result, during an infection, circulating levels of G-CSF may not rise appropriately in order to ensure a steady supply of neutrophils. These observations suggest that exogenous G-CSF may be of benefit in the treatment of group B streptococcal infection in this patient population. In one study, neonatal rats were infected subcutaneously with group B streptococci and then treated with one of the following regimens: no antibiotics or G-CSF (control); G-CSF given once at the time of infection; ampicillin and gentamicin starting 24 hours after infection; or both G-CSF and antibiotics by these same dosing schedules.65At 72 hours, the survival rate for the animals receiving both G-CSF and antibiotics was 91% versus 4% for the control group; 9% for the animals treated with G-CSF alone; and 28% for the animals treated with antibiotics only. When G-CSF was administered prophylactically prior to the bacterial challenge, a similar synergistic effect on survival was seen for G-CSF in addition to antibiotics,

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The Neutrophils

compared with antibiotics alone. Current data gathered from randomized controlled clinical trials show that CSF treatment for neonatal sepsis is well-tolerated, and significantly decreases the mortality in septic infants who are also neutropenic.66 However, insufficient evidence presently exists in humans supporting the regular use of either G-CSF or GM-CSF in the treatment of established infections, or as a prophylaxis to prevent infection.

4.2. Burn Wound Injury Infection continues to be a major problem following burn injury. Multiple immune defects have been demonstrated after thermal injury, including inhibition of both the production and function of n e ~ t r o p h i l sTo . ~ study ~ the effects of G-CSF after thermal injury, mice were burned and their wounds inoculated with Pseudomonas aeruginosa.68 The animals were randomized to receive either G-CSF or placebo starting at the time of injury and bacterial seeding, then twice daily thereafter. The mice receiving G-CSF showed an enhanced myelopoietic response, as assessed by significant increases in the absolute neutrophil count, bone marrow cellularity, and the number of myelopoietic progenitor cells. The addition of antibiotic therapy to G-CSF significantly improved survival compared with burn infected control mice or the animals that received either G-CSF or antibiotic therapy alone.

4.3. Surgical Wound Infection In an experimental model of surgical wound infection, P. aeruginosa was inoculated into the thigh muscle of mice.69G-CSF was administered immediately after infection and for 2 days thereafter. Greater than 90% of the control animals died, while only 50% of animals treated with G-CSF succumbed to their infection. G-CSF greatly enhanced the influx of neutrophils into the infected tissue site, which resulted in a significant reduction in the number of viable bacteria compared with control animals. In this study, the authors also examined the relationship between the number of circulating neutrophils and the efficacy of antibiotic therapy in this intramuscular infection model. Mice were treated with

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3 13

cyclophosphamide to make them granulocytopenic or with G-CSF to induce neutrophilia prior to the onset of infection. The therapeutic effect of the aminoglycoside netilmicin, was not significantly affected by the number of neutrophils in the blood. In contrast, the therapeutic effect of ceftazidime, a €3-lactamantibiotic, was significantly affected by the number of circulating neutrophils at the time of infection. Thus, selection of a particular antibiotic may also be an important consideration when utilizing G-CSF as an adjunct therapy. Because some antibiotics are known to concentrate within neutrophils, McKenna et al. hypothesized that G-CSF might increase the antibiotic uptake into these cells as a way of enhancing their functionyo This effect would potentially result in "targeting" antibiotic delivery to an infected site and be particularly useful in parts of the body where antibiotic concentrations are typically lower compared with serum concentrations, such as the lung. Ciprofloxacin is a quinolone antibiotic which is known to concentrate within neutrophils three to four times greater than the extracellular concentration. McKenna et al. isolated human neutrophils and incubated them with G-CSF for 1 hour. Ciprofloxacin was then added and the cells were incubated for an additional hour. G-CSF increased the intracellular-to-extracellular concentration of ciprofloxacin approximately lO-f~ld.~O Interestingly, in a recently published trial of G-CSF in patients with multilobar pneumonia, a subgroup receiving both G-CSF and ciprofloxacin had an improved outcome.71 In a recent clinical trial, the potential prophylactic effects of G-CSF were explored in patients with esophageal cancer undergoing esophagectomy. In this phase-I1 trial, 19 patients received subcutaneous G-CSF 2 days prior to, and up to 7 days following, surgery. Compared with historical controls, neutrophil phagocytosis and oxidative burst were enhanced. Furthermore, the incidence of infection and mortality was lower in this patient population, perhaps identifying a role for G-CSF in the prevention of postoperative infection.n

4.4. Bacteremia In a study by Haberstroh and colleagues, 15 intravenously catheterized pigs were given a constant infusion of live P. amginma, reaching a final blood

31 4 The Neutrophils concentration of approximately lo3 colony-forming units (CFU)/ml, not unlike the concentration observed in baderemic patients.” Seven of the animals received G-CSF 30 minutes before the start of the bacterial infusion, and eight received placebo. Two animals in the placebo group died, whereas all of the animals treated with G-CSF survived. The blood endotoxin levels in control animals increased steadily during the first 24 to 36 hours, and then gradually declined. In the G-CSF treated animals, the peak endotoxin levels were approximately 50% lower compared with the peak values in control animals. A similar pattern was observed with the levels of TNF in the circulation. Thus, the use of G-CSF in this model system resulted in lower systemic cytokine levels and improved survival. As noted previously, it is important to appreciate that there are critical differences between hematopoietic growth factors. Whereas G-CSF is specific in stimulating the proliferation, differentiation, and functional activities of neutrophils, GM-CSF also exerts profound effects on cells of macrophage lineage. These differences can have dramatic effects in the infected host. Havill et al. pretreated mice with either GM-CSF or G-CSF prior to an intravenous endotoxin challenge, and monitored mortality over the following 72 hours.74Prior treatment with GM-CSF converted a nonlethal endotoxin challenge to one with 50% mortality. For these experiments, a dose of GM-CSF was selected which did not increase circulating neutrophil levels. G-CSF, which increased the absolute neutrophil count by approximately 80%, caused no enhancement of endotoxin induced lethality. Therefore, this effect on mortality was presumably due to the effect of GM-CSF on the mononuclear phagocyte cell population, priming them for enhanced release of proinflammatory cytokines. In this study, there was a 20-fold increase in the serum TNF levels in the animals receiving GM-CSF prior to the endotoxin challenge, whereas G-CSF had no effect on the TNF response. Similar to these observations, Tiegs et al. reported that GM-CSF enhanced endotoxin-induced organ injury and mortality in mice.75 Furthermore, administration of a neutralizing anti-GM-CSF monoclonal antibody prior to the endotoxin challenge significantly improved survival. These observations lend support to the hypothesis that the macrophage, as opposed to the neutrophil, may be the primary effector cell type mediating, in large part, the lethal consequences of sepsis.

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In a murine model of systemic E . coli infection, Noursadeghi et al. showed that elevated serum levels of G-CSF produced during an acute phase response to casein are protective. Mice pretreated subcutaneously with casein had accelerated bacterial clearance and enhanced neutrophil function. This protection was abolished in the presence of a neutralizing G-CSF antibody. Pretreatment with recombinant G-CSF resulted in the same protection, supporting a potential prophylactic effect of G-CSF treatment in the prevention of bacterial infection?6

4.5. lntraabdominal Infection Lundblad et al. utilized a model of cecal ligation and puncture to simulate intraabdominal sepsis.77These investigators focused on the burden of bacterial infection and blood levels of cytokines and endotoxin, as well as evidence of neutrophil-mediated tissue injury. None of the organs examined in those animals that were treated with G-CSF showed histopathological evidence of neutrophil-mediated injury. Furthermore, as in the previous study, blood levels of bacteria, endotoxin, and TNF were consistently lower in the G-CSF-treated animals compared with placebo-treated animals. In contrast to these observations, Toda et al. reported that in their model of cecal ligation and puncture, administration of GM-CSF failed to improve survival and appeared to cause the animals to succumb more rapidly to their infection.78In a model of intraabdominal sepsis utilizing agar pellets implanted with live E. coli, Zhang et al. showed that G-CSF increased the number of neutrophils responding to the infection within the peritoneum by approximately 3-fold and increased survival from 38% to 78%.79Furthermore, the bactericidal activity of these neutrophils recovered from the peritoneal cavity was significantly enhanced compared with vehicle-treated animals. Studies in a model of cecal ligation and puncture have also shown that G-CSF increases the phagocytic function of both circulating and peritoneal neutrophils80

4.6. Pneumonia Examination of the lung may be particularly useful in defining the role of tht. CSFs and other cytokines in thc. host response to infection. The cellular

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The Neutrophils

population of the uninfected lung is almost exclusively composed of alveolar macrophages and these cells can be readily obtained by bronchoscopy. Alveolar macrophages recovered by bronchoalveolar lavage from patients with pneumonia spontaneously release G-CSF, whereas alveolar macrophages from healthy controls produce G-CSF only after endotoxin stimulation.81This G-CSF response in patients with pneumonia most likely serves at least two purposes. It would act locally, along with other cytokines and inflammatory mediators, within the lung to increase the functional activity of neutrophils entering the infected lung. It would also function systematically to stimulate the bone marrow to ensure an ongoing supply of additional effector cells needed to eradicate the infection. In contrast to certain other cytokines, such as TNF, IL-I, and IL-8, G-CSF is not compartmentalized within the lung.82In studies of patients with unilateral pneumonia, these other cytokines have been shown to remain localized within the lung and remain undetectable in the serum of these pneumonia patient^.^^,^^ By contrast, increased lung levels of G-CSF in bronchoalveolar lavage fluid following intratracheal E. coli precede similar increases in the systemic circulation. In the same study, G-CSF gene expression was localized to alveolar macrophages and airway epithelial cells following the intrapulmonary challenge, with no change detected in selected extrapulmonary tissues.82Thus, lung-derived G-CSF becomes decompartmentalized, and enters the intravascular space. The appearance of G-CSF in the systemic circulation is associated with increased numbers of myeloid progenitors in the bone marrow, blood, and spleen 48 hours after intratracheal E. coli (Nelson et al., unpublished data). Similar results are seen following intratracheal administration of recombinant G-CSF, which subsequently induces systemic granulopoiesis and increases the number of circulating n e ~ t r o p h i l s(Nelson ~~ et al., unpublished data). Taken together, these results endorse a functional role for the selective decompartmentalization of G-CSF witnessed during an intrapulmonary infection. One important determinant of the specific patient populations that might benefit from CSF therapy is how host factors or underlying illness may affect the endogenous cytokine responses to infection. Alcohol is known to significantly increase patient susceptibility to a variety of infections, particularly bacterial phneumonia.86--88 Although this relationship is

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3 17

widely appreciated, the basic mechanisms remain unclear. Numerous in vitro and in vivo studies have reported ethanol-induced defects in neutrophil function, including adherence, mobilization, and d e l i ~ e r y . ~ ~ - ~ l Furthermore, alcohol-abusing patients frequently fail to initiate a leukocytosis in response to their infection, which markedly increases their likelihood of succumbing to the infection.92These observations suggest that the ability of the host to generate an appropriate neutrophil response is both essential for survival and may be impaired by alcohol abuse. Nelson et al. investigated the effects of G-CSF in ethanol-treated rats with experimentally induced pneumonia.93Rats were pretreated with G-CSF or placebo for 2 days, and then intraperitoneal alcohol or saline was administered, followed by an intratracheal challenge with Klebsiella pneumoniae. At 4 hours after the intratracheal challenge, G-CSF augmented the recruitment of neutrophils into the lungs of control animals and significantly attenuated the adverse effects of ethanol on neutrophil delivery into the infected lung. G-CSF also enhanced the bactericidal activity of the lung in both the control and ethanol-treated rats. All of the 12 intoxicated control rats with pneumonia died within 72 hours of infection, whereas only 1 of 12 rats treated with G-CSF died. Subsequently, these investigators showed that alcohol suppresses the normal serum G-CSF response to a bacterial infection in vivo and that G-CSF can attenuate the adverse effects of alcohol on several vital neutrophil functions in vitro, including the expression of adhesion molecules and p h a g o c y t ~ s i s . ~ ~ - ~ ~ Splenectomy is a known risk factor for increased morbidity and mortality resulting from pneumococcal pneumonia.97 In a murine model, G-CSF administered from 24 hours before challenge to 3 days after challenge improved survival among splenectomized animals exposed to an aerosol challenge with Streptococcus pneumoniae. The survival rate among splenectomized G-CSF-treated mice was 70% compared with 20% in the splenectomized control animals.98 Smith et al. studied the effect of G-CSF in a rabbit model of gramnegative pneumonia and sepsis.99Rabbits were inoculated transtracheally with Pasteurella multocida and treated 24 hours later with penicillin G and G-CSF or placebo once daily for up to 5 days. All the rabbits underwent careful histologic examination at the time of death or when sacrificed on day 6. In these animals, sepsis-induced leukopenia was a predictor of significantly

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The Neutrophils

improved survival with G-CSF therapy (57% compared with 39% in controls). Interestingly, the majority of this survival benefit occurred within the first 24 hours of treatment with G-CSF, which was prior to the onset of G-CSF-induced neutrophilia. Histologic examination of these animals did not demonstrate evidence of organ toxicity related to G-CSF therapy. Several studies have shown that pulmonary host defense is suppressed in the presence of extrapulmonary infection.100-102 Attalah et al. recently showed that bacterial peritonitis inhibits pulmonary neutrophil recruitment following intratracheal endotoxin or Pseudornonas aer~ginosa.'~~ In the same study, G-CSF treatment prior to the intrapulmonary challenge restored pulmonary neutrophil recruitment, and enhanced the clearance of pulmonary infection.

5. CLINICAL STUDIES OFTHE CSFS IN INFECTIOUS DISEASES 5.1. Neutropenia The CSFs have had a major impact upon the treatment of neutropenic patients. There are many types and causes for neutropenia, e.g. congenital and acquired neutropenias; acute and chronic neutropenias; and neutropenias occurring with and without other defects in host defenses. Mechanistically these disorders can also be described as abnormalities of the production, maturation or distribution of these cells.lo4Severe neutropenia, i.e. blood neutrophil counts less than 0.5 X lo9cells/L, generally results in infections if it lasts more than a few days. Cancer chemotherapy and marrow ablation for bone marrow transplantation are common causes of this type of neutropenia. Randomized controlled trials have established that the CSFs are effective at ameliorating neutropenia in this setting by hastening marrow recovery and shortening the duration of severe neutropenia. Recently, guidelines for the use of CSFs in these settings have been p u b l i ~ h e d . ~ ~ ~ , ' ~ ~ Patients with congenital neutropenia, cyclic neutropenia and idiopathic neutropenia, sometimes referred to as "severe chronic neutropenia," have a defect in neutrophil production which leads to a life-long risk of recurrent infection^.'^^ They frequently have mouth ulcers, gingivitis,

Use of Colony-Stimulating Factors 3 19 sinusitis and cervical lymphadenopathy. Life-threatening infections, e.g. pneumonia, neutropenic colitis, deep tissue abscesses and bacteremia can also occur, especially in the most severely affected patients. The precise cellular or genetic causes for most disorders are not yet known. At present, they are not attributable to recognized defects in the production of the CSFs or in the structure or function of the CSF receptors. Characteristically, bone marrow examination in these patients shows relatively normal numbers of other hematopoietic cells, but a deficiency in cells of the neutrophil lineage. Generally, there are some early precursors, but a deficiency in the number of the more mature cells. In cyclic neutropenia, the severity of this defect in the marrow varies in a regular oscillatory fashion.lo8 Clinical trials of the CSFs for the treatment of severe chronic neutropenia were begun in 1987. A randomized controlled trial clearly established the effectiveness of G-CSF for these conditions, with more than 90% of patients responding to an increase in their blood neutrophil counts to normal levels, with a concomitant decrease in the occurrence of fever and infection^.'^^ Detailed clinical studies have shown that patients with cyclic neutropenia and idiopathic neutropenia respond to relatively low doses of G-CSF, i.e. 1 to 3 pg/kg/day, administered subcutaneously on a daily or alternate day basis. Patients with congenital neutropenia generally have lower counts, a more severe marrow defect, and require higher doses of G-CSF."O There are now several hundred patients who have been treated generally with daily or alternate day G-CSF for more than five years, with few long-term adverse effects. One group of patients, patients with congenital neutropenia, was known to be at risk of conversion to acute myelogenous leukemia before the availability of the CSFs."l Since the availability of the CSFs, this occurrence has been better documented, but it is unclear if treatment affects this evolution. GM-CSF is much less effective than G-CSF for these patients.

5.2. C-CSF in Nonneutropenic Patients with Pneumonia Four trials have recently been completed studying the> effect of G-CSF in nonneutropenic patients with pneumonia. The first I rial was a phase I study of 30 nonntwtroptmic patients hospitalizt~lwith communityacquired piiei*monici(( 'AP) ''? A11 t h e pz tien+; I . W E ~ Cd~ iirtx,i t r ( m o t i Q ;

320

The Neutrophils

antibiotics in addition to G-CSF (75-300 K g ) subcutaneously daily for a maximum of 10 days. Overall, the median change in the absolute neutrophil count from baseline was approximately 200% and the peak was achieved by day 4 of G-CSF administration. Aside from mild bone pain, no adverse pulmonary or systemic side effects occurred that were attributable to G-CSF. A phase 111, double-blind, placebo-controlled trial of recombinant human G-CSF for the treatment of hospitalized patients with CAP has recently been c ~ n c l u d e d . "This ~ was a multicenter trial involving 756 patients enrolled in 71 centers in the United States, Canada, and Australia. Participants in this study were randomized to receive 300 kg/day G-CSF (376 patients) or placebo (380 patients) in addition to conventional antibiotic therapy. Treatment duration was u p to 10 days and the length of the study observation period was 28 days or until death. The primary objectives of this study were to determine the safety and effect of G-CSF on TRM. TRM (time to resolution of morbidity) was defined as an index of several clinical variables which are useful in determining if a patient with pneumonia is benefiting from therapy.l14 In this study, in order to reach TRM, a patient had to have either an improved or a stable chest radiograph; resolve their tachypnea; become afebrile; and improve or normalize their oxygenation. Mortality was low (6%) in this study and the length of stay was only 7 days. Both variables were unaffected by G-CSF treatment. Similarly, TRM was 4 days in each treatment group. In the intent-to-treat analysis, G-CSF did increase the blood neutrophils 3-fold; significantly accelerated radiological resolution of pneumonia; and reduced serious complications (i.e. ARDS and disseminated intravascular coagulation (DIC)). Post hoc analyses showed that these benefits were more pronounced in patients with multilobar (>2 lobes) pneumonia. In this study, there were 261 patients with multilobar pneumonia (G-CSF, n = 138; placebo, n = 123) and 28% of these patients were admitted to an ICU at study entry. G-CSF administration was safe and welltolerated in this study. G-CSF has also been studied in the treatment of patients with pneumonia and severe sepsis.115Eighteen patients were randomized in a 2:l ratio to G-CSF (300 Fg/day intravenously) or placebo for a maximum of

Use of Colony-Stimulating Factors

32 1

5 days in addition to standard therapy. Inclusion criteria included a chest radiograph compatible with pneumonia, a respiratory pathogen on gram stain or culture, fever, tachycardia, tachypnea or need for mechanical ventilation, and either hypotension despite volume resuscitation requiring vasopressors or, in the absence of shock, two end organ dysfunctions (metabolic acidosis, ARDS, acute renal failure, DIC). Three of the 12 G-CSF-treated patients and 4 of 6 placebo-treated patients died. Septic shock resolved in 9 of 10 G-CSF-treated patients and none of the 4 placebo-treated patients. ARDS resolved in 2 of 5 G-CSF-treated patients and 1 of 4 placebo-treated patients. G-CSF was well-tolerated in these septic patients. Based on the favorable trends seen in these studies, additional trials have been performed in patients with multilobar pneumonia and in patients with severe pneumonia with sepsis. In one trial, 480 patients with multilobar community-acquired pneumonia were randomized to receive 300 kg/day G-CSF (237 patients) or placebo (243 patients).71 Treatment was combined with standard therapy, and was continued for 10 days, or until the white blood cell achieved levels of 75.0 X lo9 cells/L. G-CSF treatment was well-tolerated and increased the WBC counts. In addition, there was a trend toward decreased mortality in patients receiving G-CSF, although this effect did not reach statistical significance. In another more recent multicenter clinical trial, 701 patients with bacterial pneumonia were randomized to receive 300 kg/day G-CSF (348 patients) or placebo (353 patients).'16 G-CSF treatment was continued for 5 days or until the white blood cells achieved levels of 75.0 X lo9 cells/L or above. Although G-CSF treatment was safely-tolerated while significantly increasing white blood cell counts, it had no significant effect on mortality or any other major endpoint of the study. However, patients receiving G-CSF in combination with a quinolone antibiotic showed a noticeable trend toward decreased mortality compared with patients treated with placebo and quinolone (29% vs. 40%). While this trial failed to show a significant effect of G-CSF therapy on the outcome of patients with pneumonia and severe sepsis, more controlled studies in the future may better account for the heterogeneous nature of this patient population and their underlying conditions.

322

The Neutrophils

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3. Metcalf D, Stanley ER. Aust 1E x p Biol Med Sci 1969; 47:453466. 4. Metcalf D. Immunol Cell B i d 1987; 65 (Pt 1):35-43. 5. Cheers C, Haigh AM, Kelso A, et a/. Infect Immun 1988; 56:247-251. 6. Burgess AW, Metcalf D. Int J Cancer 1980; 26:647-654. 7. Nicola NA, Metcalf D, Johnson GR, Burgess AW. Blood 1979; 54:614427.

8. Nicola NA, Metcalf D, Matsumoto M, Johnson GR. 9017-9023.

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9. Souza LM, Boone TC, Gabrilove J, et al. Science 1986; 232:61-65. 10. Welte K, Platzer E, Lu L, et al. Proc Natl Acad Sci U S A 1985; 82:1526-1530.

11. Simmers RN, Webber LM, Shannon MF, e t a / . Blood 1987; 70:330-332. 12. Welte K, Gabrilove J, Bronchud MH, et al. Blood 1996; 88:1907-1929. 13. Wong GG, Witek JS, Temple PA, et al. Science 1985; 228:810-815. 14. Huebner K, Isobe M, Croce CM, Golde DW, Kaufman SE, Gasson JC. Science 1985; 230~1282-1285. 15. Han SW, Ramesh N, Osborne WR. Gene 1996; 175:lOl-104. 16. Hill CP, Osslund TD, Eisenberg D. Proc Natl Acad Sci U S A 1993; 90:5167-5171. 17. Nicola NA, Smith A, Robb L, et al. Ciba Found Symp 1997; 204:19-27. 18. Avalos BR, Gasson JC, Hedvat C, et al. Blood 1990; 75:851-857. 19. Ward AC, van Aesch YM, Gits J, et al. 1 Exp Med 1999; 190:497-507. 20. Tidow N, Welte K. Curr Opzn Hematoll997; 4:171-175.

21. Shimoda K, Feng J, Murakami H, et al. Blood 1997; 90:597404. 22. Shimozaki K, Nakajima K, Hirano T, Nagata S. 25184-251 89.

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23. Basu S, Dunn A , W,ii.ti A. Int 1Mol Med 2002; 10:3-10. 24. Di1’e1~1011: t i ~ > ~ i j(~ , F’ord lt CFr c’t a\. I,

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25. Gomez-Cambronero J, Veatch C. Life Sci 1996; 592099-2111. 26. Watanabe S, Itoh T, Arai K. J Allergy Clin Immunoll996; 98:S183-S191.

27. Dale DC, Fauci AS, Guerry D IN, Wolff SM. J Clin Invest 1975; 56:808-813. 28. Dale DC, Liles WC, Llewellyn C, Price TH. Am J Hematoll998; 57:7-15.

29. Jagels MA, Hugli TE. JImrnunoll992; 148:1119-1128. 30. Price TH, Chatta GS, Dale DC. Blood 1996; 88:335-340. 31. Wang J, Mukaida N, Zhang Y, et al. J L e u b c Bioll997; 62:503-509. 32. Papayannopoulou T, Nakamoto B. Proc Nut1 Acad Sci USA 1993; 90: 9374-9378. 33. Da WM, Zhang M, Zhang BL, et al. Zkongguo Ski Yan Xue Ye Xue Za Zki 2002; 10:240-242. 34. Lapidot T, Petit I. Exp Hematol2002; 30:973-981. 35. Petit I, Szyper-Kravitz M, Nagler A, et al. Nut Immunol2002; 3:687-694. 36. Semerad CL, Liu F, Gregory AD, et al. Immunity 2002; 17:413423. 37. Bicknell S, van Eeden S, Hayashi S, et al. Am J Respir Cell Mol Biol 1994; 10:16-23. 38. Doerschuk CM. Microcirculation2001; 8:71-88. 39. Babior BM, Golde DW. Hematology, 5th ed. (eds. Williams WJ, et al.). McGraw-Hill, New York, 1995; pp. 773-779. 40. Ogawa M. Blood 1993; 81:2844-2853. 41. Sullivan GW, Carper HT, Mandell GL. Blood 1993; 81:1863-1870. 42. Dale DC, Liles WC, Summer WR, Nelson S. J Infect Dis 1995; 172:1061-1075. 43. Harmenberg J, Hoglund M, Hellstrom-Lindberg E. Eur J Haematol Suppll994; 55:1-28. 44. Turzanski J, Crouch SP, Fletcher J, Hunter A. Br J Haematol1997; 96:46-54. 45. Sullivan GW, Gelrud AK, Carper HT, Mandell GL. Proc Assoc Am Physicians 1996; 108:455-466. 46. Jones TC. Med Oncoll996; 13941-147. 47. Tarr PE. Med Oncoll996; 13:133-140. 48. Stanley E, Lieschke GJ, Grail D, et al. Proc Natl Acad Sci U S A 1994; 91: 5592-5596.

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49. Lieschke GJ, Grail D, Hodgson G, et al. Blood 1994; 84:1737-1746. 50. Mitsui T, Watanabe S, Taniguchi Y, et al. Blood 2003; 101:2990-2995. 51. Semerad CL, Poursine-Laurent J, Liu F, Link DC. Immunity 1999; 11:153-161. 52. Kuhns DB, Alvord WG, Gallin JI. J Infect Dis 1995; 171:145-152. 53. Kojima S, Matsuyama T, Kodera Y, et al. Blood 1996; 87:1303-1308. 54. Mempel K, Pietsch T, Menzel T, et al. Blood 1991; 771919-1922. 55. Cebon J, Layton JE, Maher D, Morstyn G. Br J Haematoll994; 86:265-274. 56. Kragsbjerg P, Jones I, Vikerfors T, Holmberg H. Thorax 1995; 50:1253-1257. 57. Pauksen K, Elfman L, Ulfgren AK, Venge P. Br J Haematoll994; 88:256-260. 58. Kawakami M, Tsutsumi H, Kumakawa T, et al. Blood 1990; 76:1962-1964. 59. Waring PM, Presneill J, Maher DW, et a[. Clin Exp Immunol1995; 102:501-506. 60. Layton JE, Hockman H, Sheridan WP, Morstyn G. Blood 1989; 74:1303-1307. 61. Pinner RW, Teutsch SM, Simonsen L, et al. IAMA 1996; 275:189-193. 62. Cairo MS. Neonatal neutrophil host defense. Am J Dis Child 1989; 143:4046. 63. Liechty KW, Schibler KR, Ohls RK, et al. Biol Neonate 1993; 64:331-340. 64. Schibler KR, Liechty KW, White WL, Christensen RD. Blood 1993; 82: 2478-2484. 65. Cairo MS, Mauss D, Kommareddy S, et al. Pediatr Res 1990; 27612-616. 66. Carr R, Modi N, Dore C. Cochrane Database Syst Rev 2003; CD003066. 67. Mooney DP, Gamelli RL, OReilly M, Hebert JC. Arch Surg 1988; 123: 1353-1 357. 69. Yasuda H, Ajiki Y, Shimozato T, et al. Infect lmmun 1990; 58:2502-2509. 70. McKenna PH, Nelson S, Andresen J. Am J Respir Crit Care Med 1996; 153s: A535. 71. Nelson S, Heyder AM, Stone J, et al. J Infect Dis 2000; 182970-973. 72. Schafer H, Hubel K, Bohlen H, et al. Ann Hematol2000; 79:143-151. 73. Haberstroh J, Breuer H, Lucke I, et al. Shock 1995; 4216-224. 74. Havill AM, Anderson JW, Karoch JW, Nelson S. Am Rev Respir Dis 1991; 143S3236. 75. Tiegs G, Barsig J, Matiba B, et al. J Clin Invest 1994; 932616-2622.

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76. Noursadeghi M, Bickerstaff MC, Herbert J, et al. J Immunol2002; 169:913-919. 77. Lundblad R, Nesland JM, Giercksky KE. Crit Care Med 1996; 24820-826. 78. Toda H, Murata A, Oka Y, et al. Blood 1994; 83:2893-2898. 79. Dunne JR, Dunkin BJ, Nelson S, White JC. J Surg Res 1996; 61:348-354. 80. Zhang P, Bagby GJ, Stoltz DA, et al. Crit Care Med 1998; 26:315-321. 81. Tazi A, Nioche S, Chastre J, et al. Am J Respir Cell Mol Biol 1991; 4:140-147.

82. Quinton LJ, Nelson S, Boe DM, et al. J Infect Dis 2002; 185:1476-1482. 83. Boutten A, Dehoux MS, Seta N, et al. A m J Respir Crit Care Med 1996; 153~336-342. 84. Dehoux MS, Boutten A, Ostinelli J, et al. A m J Respir Crit Care Med 1994; 150:710-716. 85. Nelson S, Bagby G, Andreson J, et al. Respir Dis 1991; 143S3710. 86. Capps JA, Coleman GH. JAMA 1923; 80750. 87. Kolb D, Gunderson EK. Drug Alcohol Depend 1982; 9:181-189. 88. Osler W. The Principles and Practice $Medicine. Appleton, New York, 1905, 89. Brayton RG, Stokes PE, Schwartz MS, Louria DB. N Engl J Med 1970; 2821123-128. 90. Gluckman SJ, MacGregor RR. Blood 1978; 52:551-559. 91. Hallengren B, Forsgren A. Actu Med Scand 1978; 204:43-48. 92. Limson BM, Romansky MJ, Shea JG. Antibiot Annu 1955; 3:786-793. 93. Nelson S, Summer W, Bagby G, et al. J Infect Dis 1991; 164:901-906. 94. Nelson S, Bagby G, Mason C, Summer W. A m 115S:A14.

J Respir Crit Care Med 1995;

95. Zhang P, Nelson S, Summer WR, Spitzer JA. Alcohol Clin Exp Res 1997; 21: 773-778. 96. Bagby GJ, Zhang P, Stoltz DA, Nelson S. Alcohol Clin Exp Res 1998; 22: 1740-1 745, 97. Gopal V, Bisno AL. Arch lntern Med 1977; 137:1526-1530. 98. Hebert JC, OReilly M, Gamelli RL. Arch Surg 1990; 125:1075-1078. 99. Smith WS, Sumnicht GE, Sharpe RW, et al. Blood 1995; 86:1301-1309.

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100. Frevert CW, Warner AE, Kobzik L. A m J Respir Cell Mol Biol 1994; 11:716-723. 101. Nelson S, Summer WR, Bagby GJ. In: The Physiological and Pathological Effects of Cytokines (eds. Dinarello CA, Kluger MJ, Powanda MC, Oppenheim JJ). Witey-Liss, Inc., New York, 1990; pp. 141-146. 102. White JC, Nelson S, Winkelstein JA, et al. J Infect Dis 1986; 153:202-208. 103. Attalah HL, Azoulay E, Yang K, et al. Crit Care Med 2002; 30:2107-2114. 104. Dale DC. Neutropenia. In: Hematology, 5th ed. (eds. Williams WJ, et al.). McGraw-Hill, New York, 1995; pp. 815-824. 105. American Society of Clinical Oncology. J Clin Oncol 1994; 12:2471-2508. 106. Boogaerts M, Cavalli F, Cortes-Funes H, et al. Ann Oncoll995; 6237-244. 107. Welte K, Dale D. Ann Hematoll996; 72:158-165.

108. Dale DC, Hammond WP. Blood Rev 1988; 2178-185. 109. Dale DC, Bonilla MA, Davis MW, et al. Blood 1993; 81:2496-2502. 110. Dale DC. Stem Cells 1995; 13:94-100.

111. Bonilla MA, Dale D, Zeidler C, et al. Br JHaematol1994; 88:723-730. 112. Deboisblanc BP, Mason CM, Andresen J, et al. Respir Med 1997; 91:387-394. 113. Nelson S, Belknap SM, Carlson RW, et al. CAP Study Group. J Infect Dis 1998; 178:1075-1080. 114. Daifuku R, Movahhed H, Fotheringham N, et al. Respir Med 1996; 90: 587-592. 115. Wunderink R, Leeper K Jr, Schein R, et al. Chest 2001; 119:523-529. 116. Root RK, Lodato RF, Patrick W, et al. Crit Care Med 2003; 31:367-373.

11 Neutrophil Transfusion Therapy in the G-CSF Era Ronald G. Strauss"

Following donor stimulation with G-CSF and corticosteroids, up to 8 X 10'O neutrophils can be collected for transfusion. Several case reports and studies of patient groups without randomized concurrent control subjects have suggested success in treating infections in neutropenic patients with these high doses of neutrophils. However, success has not been consistent -particularly for invasive fungal infections - and properly designed clinical trials must be done to establish whether or not efficacy truly exists. Keywords: neutrophil transfusions; granulocyte transfusions; granulocytecolony stimulating factor

1. INTRODUCTION Current blood bank technology - including donor stimulation with granulocyte colony stimulating factor (G-CSF) and corticosteroids - permits *Correspondenceto: Ronald G. Straws, MD. Department of Pathology, C250 GH, University of Iowa College of Medicine, Iowa City, IA 52242-1009; phone: 319-3560387; fax: 319-356-0331; e-mail: [email protected].

327

328

The Neutrophils

the collection of large numbers of neutrophils (PMNs), or granulocytes, as a standard blood component (Granulocytes,Pheresis) to treat patients with neutropenia or PMN dysfunction who have developed severe infections. This chapter analyzes the use of PMN transfusions - called granulocyte transfusions by convention - as an adjunct to antimicrobial drugs in either the treatment, or the possible prevention, of certain types of infections. Serious and repeated infections with bacteria, yeast and fungus are well documented to be a consequence of severe neutropenia (<0.1 X 109/L blood PMNs) or PMN dysfunction (e.g. chronic granulomatous disease).'a Even with modern antimicrobial drugs, the mortality of systemic infections with progressive tissue involvement, that occur in patients with severe neutropenia due to a markedly diminished myelopoiesis, approaches loo%, unless severe neutropenia is reversed fairly quickly. Previous attempts to prevent infections in severely neutropenic patients using prophylactic PMN transfusions have achieved only modest success (i.e. the rates of certain infections were reduced, but PMN transfusions failed to prevent all infections), and the transfusions were toxic and expensive. Similarly, the use of therapeutic PMN transfusions to treat infections in neutropenic patients has diminished strikingly over the past several years despite many reports that have demonstrated their efficacy in certain experimental and clinical setting^.^ This lack of enthusiasm for PMN transfusions can be explained, at least in part, by an overall diminished need for PMN replacement in many patient groups owing to: 1) the availability of more effective antimicrobial drugs to prevent and treat infections in neutropenic patients; 2) the development of recombinant hematopoietic cytokines or growth factors; and 3) the widespread use of peripheral blood progenitor cell (PBPC) transfusions, rather than bone marrow, to hasten marrow recovery and to shorten the duration of severe neutropenia following transplantation using either myeloablative or nonmyeloablative techniques. Recombinant cytokines such as G-CSF and granulocytelmacrophage colony stimulating factor (GM-CSF) are glycoprotein growth factors that enhance the production, differentiation and function of myeloid cell^.^,^ Most importantly, G-CSF has revolutionized the collection of either PMNs for granulocyte transfusion therapy or of hematopoietic PBPC and stem cells for transplantation? Both G-CSF and GM-CSF have been given to patients following chemotherapy to accelerate marrow recovery, to reduce the rate of developing infections, and to diminish the need for prolonged

Neutrophil Transfusion Therapy in the G-CSF Era

329

ho~pitalization.~ Despite this success in diminishing the development of neutropenic infections, the role of G-CSF and GM-CSF as treatment for already established infections is not as firmly documented. Thus, it seems reasonable to consider additional therapies, such as modern therapeutic PMN transfusions, when severe bacterial, yeast or fungal infections progress - despite therapy with antibiotics and recombinant cytokines. However, the role of PMN transfusions remains controversial. For many physicians, the preference to use multiple antimicrobial drugs plus cytokines to treat infected neutropenic patients and the corresponding reluctance to prescribe PMN transfusions have been reinforced by knowledge that, historically, PMN transfusions provided limited benefit because they provided woefully inadequate numbers of PMNs. However, modern PMN transfusion therapy in the G-CSF era is markedly different because very large numbers of PMNs can be collected from normal donors stimulated with G-CSF and corticosteroids using modern leukapheresistechniques. This fact, along with the historical success of PMN transfusion in treating bacterial infections, even when PMNs were given in relatively low doses? justifies a critical reassessment of the possible rules for PMN transfusions.

2. THERAPEUTIC NEUTROPHIL TRANSFUSIONS 2.1. Historic Experience with Therapeutic PMN Transfusions in Neutropenic Patients In the first edition of this book, 34 papers that reported the use of PMN transfusions to treat infections in severely neutropenic patients (<5 x i09/L blood PMNs) were reviewed? Because the earlier papers are quite dated and reported results using PMNs collected from donors who were either unstimulated or stimulated with corticosteroids only, they will not be included in this updated chapter - with the exception of the seven controlled trial^.^-'^ In these seven reports, most of which evaluated small numbers of patients, the response of infected neutropenic patients to treatment with PMN transfusions plus antimicrobial drugs (transfusion group) was compared with that of comparable patients, evaluated concurrently, who were given antimicrobial drugs alone (control group). Importantly, even in these historic trials, some measure of success was achieved in five of the seven controlled studies transfusing relatively modest doses of PMNs (Table 1).In three of the seven studies,lG12a significant overall benefit for

330

The Neutrophils Table 1 Seven Historic Controlled Studies of Therapeutic PMN Transfusions in Neutropenic Patients Transfusion Group

Ref. No.

Patients Survival (%)

7 8 9

12 17 39

82 78 46

10

13

75

11 12 13

17 17 48

76 59 63

Control Group Dose x 1010 5.9 (F) 0.4 (C) 2.0 (F) 0.6 (C) 1.7 (F) 0.4 (C) 2.2 (F) 2.7 (C) 0.5 (C)

HLA-WBC* Patients Survival Success

(%I NO-NO No-Yes No-Yes

19 22 37

62 80 30

Partial No Partial

No-Yes

14

36

Yes

No-Yes Yes-Yes NO-NO

19 13 47

26 15 72

Yes Yes No

* Donor-recipient compatibility was enhanced by HLA matching or white blood cell crossmatch. F = filtration leukapheresis; C = centrifugation leukapheresis.

PMN transfusions was found, and in two additional ~tudies,7~ certain subgroups of patients were found to benefit significantly (i.e. partial success). Although therapeutic PMN transfusions were successful to some degree in five of the seven historic controlled studies, success was not uniform, as evidenced by two negative report^.^.^^ One explanation for the inconsistent success of the controlled studies is evident when the adequacy of PMN transfusion support is analyzed critically (Table 1).Patients in the three trials reporting overall success10-12received relatively high doses of PMNs (i.e. "high" for the historic period of time) collected from donors who were selected with the use of techniques intended to improve donor-recipient leukocyte compatibility. In contrast, the four trials with either partial or no success provided, what is now known to be, inadequate transfusion support. Doses of PMNs were extremely low (0.4-0.5 X 1O'O per transfusion). In addition to problems with inadequate PMN dose, investigators in two studies7,13made no provisions for the possibility of leukocyte incompatibility and selected donors solely on the basis of erythrocyte compatibility. Data from the seven historic controlled PMN transfusion therapy trials7-13 were analyzed formally by metaana1y~is.l~ The impressions were

Neutrophil Transfusion Therapy in the G-CSF Era

33 1

that marked variations of both the dose of PMNs transfused and the survival rate of the control subjects were primarily responsible for differing success rates of these early studies. In clinical settings in which the survival rate of nontransfused control subjects was low, the study subjects receiving adequate doses of PMNs benefited from PMN transf~sions.'~ The authors concluded that neutropenic patients with infections that carry a high mortality rate should be considered for therapeutic PMN transfusions, provided PMNs are given in adequate dosages.

2.2. Modern Experience with Therapeutic PMN Transfusions in the G-CSF ERA Fungal, yeast and mold infections continue to occur frequently in patients with either severe neutropenia or PMN dysfunction, and they pose a major challenge to the efficacy of therapeutic PMN transfusions. Recipients of hematopoietic progenitor cell transplants - particularly marrow transplant patients - often become severely neutropenic, exhibit PMN dysfunction, and manifest defective cellular and humoral immunity for months following transplantation. Altered immunity is particularly profound when the marrow is T-lymphocyte depleted to diminish graftvs-host disease. Hence, all types of infection pose a threat, with fungal and yeast infections being the major problem^.'^^'^ In a series of 1186 marrow transplant patients, 10% developed a noncandidal fungal infection, with '~ the marrow graft is only 17% of infected patients s ~ r v i v i n g . When depleted of T lymphocytes, the rate of infection is increased by two- to seven-fold above that occurring with standard marrow transplantation.I6 To date, data are insufficient to determine the proper role of PMN transfusions in treating fungal and yeast infections. Historically, some report^'^-^^ have supported the efficacy of therapeutic PMN transfusions in fungal and yeast infections, whereas others have disagreed.23 These studies all involved PMN transfusions with relatively low PMN doses. Now that stimulation of donors with G-CSF permits collection of PMN concentrates containing 4 to 10 x 1O1O PMNs per each leukapheresis procedure, a possible role for modern "high dose" PMN transfusions has been suggested as treatment for patients with these difficult infections.

332

The Neutrophils

At this time, no randomized clinical trials of therapeutic PMN transfusions collected following G-CSF donor stimulation have been reported to establish either the efficacy or the potential toxicity of modern ”high dose” PMN transfusion therapy. However, several case reports (Table 2) and small inadequately controlled studies (Table 3) have been publi~hed~ with ~ ” varying ~ findings. To summarize the case reports, Clark et al.24and Catalan0 et al.25 each reported single patients with aplastic anemia undergoing progenitor cell transplantation with fungus infections that responded favorably to strikingly different doses of PMNs. Similarly, Ozsahin et a1.26 and Bielorai et ~ 2 1 each . ~ ~ reported single patients with chronic granulomatous disease and fungal infections that responded favorably to PMN transfusions during the transplantation period. Also, Bielorai et a1.28 reported a single patient with acute leukemia and sepsis with progressive, antibioticresistant bacteria whose infection cleared slowly with PMN transfusions. Table 2 Case Reports of Modern PMN Transfusions using PMNs Collected from G-CSF Stimulated Donors Ref. No.

PMNs X loloper each GTX

Stimulation

Leukapheresis

Outcomes

24

5.3*

G-CSF 4-10 pg/kg

Dextran 1OL processed

One patient with fungus recovered

25

1.9

G-CSF 300 pg/kg

Not described

One patient with fungus recovered.

26

3.1

G-CSF 5w/kg

Hetastarch 5-7Lprocessed

One patient with fungus recovered.

27

7.0*

G-CSF 5Pg/k

Not described

One patient with fungus recovered.

28

4.8-6.8

Not described

Not described

One patient with vancomycinresistant Entercococcus recovered.

* Assumptions made as PMN dose expressed X 1O1O unclear in these reports. Dose calculated that would be given to a 70 kg recipient for Clark et al. and Bielorai et al.

Neutrophil Transfusion Therapy in the G-CSF Era

333

Table 3 Inadequately Controlled Studies of Modern PMN Transfusions using PMNs Collected from G-CSF Stimulated Donors Ref. PMNs Stimulation No. X loloper each GTX

Leukapheresis Outcomes

29

4.1

G-CSF 5 yg/kg

Pentastarch 7L processed

30

5.9*

G-CSF 10 y.g/kg

Dextran 100% (3 of 3) success 10L processed with bacterial infection. 0% (0 of 5) success with progressive fungus. 67% (2 of 3) success with stable fungus.

31

3.5*

G-CSF 5 yg/kg-orPrednisolone

82% (14 of 17) success Hetastarch 6.4 L processed with bacterial infection. 54% (7 of 13) success with fungal infection.

32

8.2

G-CSF 600 Fg-plusDexamethasone 8mg

100% (4 of 4) sucess with Hetastarch 10L processed bacterial infection. 0% (0 of 8) success with invasive fungus. 57% (4 of 7) success with yeast infection.

33

8.1*

Hetastarch or G-CSF 600 Pg -plusDexamethasone 8 mg Pentastarch G-CSF 600 Wg 10 L processed G-CSF 600 Pg

Bacterial infection: 55% success unrelated donor 75% success family donor Yeast infection: 70% success unrelated donor 40% success family donor Fungus / mold infection: 15%success unrelated donor 25% success family donor

G-CSF 5 yg/kg -and/orDexamethasone 3 mg/m2

40% (10 of 25) success with multiple-organism infections.

5.4' 4.6*

34

5.1-10.6

Pentastarch 6-10L processed

60% (9 of 15) success with fungus (11patients) and yeast (4 patients)

* Assumptions made as PMN dose expressed X 101Ounclearin these reports. Dose calculated that would be given to a 70kg recipient for Peters et ul. PMN dose calculated using values for range of leukocytes collected, percentage of collected cells classified as being myeloid and volume of units collected for Grigg et al. Hubel et al. used three groups of donors (unrelated given G-CSF plus dexamethasone, unrelated given G-CSF only and family members given G-CSF only).

334

The Neutrophils

Obviously, in these single case reports of very complicated patients, it is impossible to firmly ascribe the good outcome to the PMN transfusions. The inadequately controlled studies of groups of patients (Table 3) are summarized in the following paragraphs: Hester et ~ 1 transfused . ~ ~ 15 patients with hematologic malignancies and infections. PMNs were collected from donors stimulated only with G-CSF and selected without regard for leukocyte compatibility. Although PMN transfusions were successful in most patients, it was not possible to distinguish between responses of fungus and yeast infections. Grigg et ~ 1 transfused . ~ ~ 11 patients. Eight patients had hematologic malignancies and progressive infections - five of the eight undergoing progenitor cell transplantation and three receiving chemotherapy. Three additional patients who were undergoing progenitor cell transplantation had stable fungus infections. PMNs were collected from donors stimulated only with G-CSF and selected without regard for leukocyte compatibility. Success was excellent for bacterial and stable fungus infections, but was quite poor for progressive fungus infections with organ dysfunction - a pattern seen by others. Peters et ~ 1 transfused . ~ ~ 30 patients with hematologic disorders - 18 undergoing progenitor cell transplantation. PMNs were collected from donors stimulated with either G-CSF or prednisolone and selected without regard for leukocyte compatibility. The exact PMN dose transfused is uncertain because values from 0.9 X 1O1O to 14.4 x 1O1O per each transfusion can be calculated from the data reported, and it was impossible to distinguish the success of PMN transfusions collected from G-CSF from that of prednisolone stimulated donors. However, the outcome of bacterial infections appeared to be superior to that of fungus infections. Price et ~ 1 transfused . ~ ~ 19 patients with hematologic malignancies, 16 of whom had received progenitor cell transplants and three who were in the pretransplant period. PMNs were collected from donors stimulated with both G-CSF and dexamethasone.Although the donors were selected without regard for leukocyte compatibility, the recipients were documented not to exhibit evidence of leukocyte alloimmunization at

Neutrophil Transfusion Therapy in the G-CSFEra

335

study entry, Bacterial infections responded well while yeast modestly. However, despite very high PMN doses, the result for invasive fungus infections was dismal - findings similar to Grigg et aL30 Hiibel et aZ.33 expanded the study of Price et aZ.32 to a total of 74 patients experiencing progenitor cell transplants. Although not a randomized trial, comparisons were made with selected control patients (N= 74) who did not receive PMN transfusions. PMNs were collected either from unrelated donors given either G-CSF and dexamethasone or G-CSF only, or from family members given G-CSF only. Hetastarch was used for single leukapheresis procedures, and pentastarch was used when donors experienced repeated leukapheresis. The results in patients given PMN transfusions is shown by Table 4; comparative success in nontransfused control patients was approximately 90% for bacterial, 40% for yeast and 30% for fungal/mold infections - values similar to those of PMN transfusion recipients, with nontransfused controls actually having better success than PMN recipients for bacterial infections ( p = 0.04). As a potential adverse effect, allogeneic transplant recipients given PMN transfusions from unrelated donors experienced significantly more ( p = 0.04) grade IV graft-vs-host disease compared with controls not given PMN transfusions. However, relatively few patients were assessed for this endpoint, and firm conclusions are not warranted. Nonetheless, the lack of clear benefit and the possibility of toxicity are of concern. Lee et a1.34 transfused 25 patients with hematologic malignancies, many of whom were infected with multiple organisms. Thus, the outcome of infections with specific types of individual organisms could not be determined. PMNs were collected from donors stimulated with G-CSF alone (66%of donors); G-CSF plus dexamethasone (25% of donors); or dexamethasone alone (8% of donors). Of the patients with sepsis, 50% (2 of 4) responded favorably, and 38% (8 of 21) of patients with progressive localized infections responded favorably. Grigull et a1.35 transfused four infected neutropenic children with PMN transfusions collected from G-CSF stimulated donors. Although there seemed to be improvement of the infections, two of the four patients died fairly early, and reported clinical details were too incomplete to permit inclusion in Table 4.

336

The Neutrophils

Table 4 Twelve Controlled Studies of Prophylactic PMN Transfusions in Neutropenic Patients Ref. No

42 43 44 45 46 47 48 49 50 51 52 53

Dose X 1O1O

2.1 1.2 1.5-2.2 0.7 1.6 NR 0.07 1.2 0.9 1.5 2.6 1.2

Frequency

Daily Daily Daily Daily Daily Daily NR Alternate Days Daily Alternate Days Twice Weekly Daily

Matching

Success

HLA

WBC

No No Yes No Yes Yes Yes No No No Yes No

Yes Yes Yes No No No No No No No Yes No

Yes Yes Yes Partial Partial Partial Partial No No No No No

WBC = white blood cell; NR = not reported.

No firm conclusions can be drawn from these somewhat anecdotal reports of modern therapeutic PMN transfusions for several reasons: 1) satisfactory concurrent control subjects were not included (i.e. randomly assigned to receive either no PMN transfusions at all or "historic" PMN transfusions collected from donors stimulated only with corticosteroids); 2) the number of patients reported, generally, was quite small; and 3 ) PMN collection methods varied with a broad range of PMN doses transfused. However, based on these preliminary findings, bacterial infections appeared to respond well to modern PMN transfusions; relatively mild fungus and yeast infections responded only modestly well; while serious fungus and mold infections with tissue damage often resisted even the large doses of PMNs transfused with modern PMN transfus i o n ~ . ~Thus, ~ , ~ the ~ , ~precise ~ role of modern therapeutic PMN transfusions, collected from donors stimulated with G-CSF plus corticosteroids, awaits definition by randomized clinical trials. Clearly, it is not warranted to conclude that "high dose" therapeutic PMN transfusions are efficacious and safe - particularly, for patients with invasive nonbacterial infections.

Neutrophil TransfusionTherapy in the G-CSF Era

337

2.3. Therapeutic PMN Transfusions for Neonatal Sepsis Newborn infants (neonates) are susceptible to severe bacterial and viral infections - particularly, preterm infants born after prolonged premature rupture of the membranes. PMNs isolated from the blood of neonates exhibit both quantitative and qualitative abnormalities that may contribute to the increased incidence, morbidity, and mortality of bacterial infections. Thus, a rationale exists for combining PMN transfusions with antibiotics to treat septic neonates - subjects in whom both neutropenia and PMN dysfunction have been demonstrated - and neonatal PMN transfusions were discussed at length in the previous edition of this book.6 Six controlled trials3641assessed the role of PMN transfusions in treating neonatal infections. Although four of the six36--39 found a significant benefit for PMN transfusions, the controlled studies, when assessed by metaanalysis, were insufficiently homogeneous to permit clear recommendations regarding efficacy.14The dosage of PMNs transfused was identified as the primary reason for disagreements between studies. When leukapheresis PMN concentrates were transfused, they were beneficial. When buffy coats obtained from whole blood units were transfused, they were not beneficial. Thus, the role of therapeutic PMN transfusions for neonatal infections is unclear at present, and, because they are transfused only rarely at this time, they will not be discussed further.

3. PROPHYLACTIC NEUTROPHILTRANSFUSIONS 3.1. Historic Experience with ProphylacticTransfusions in Neutropenic Patients Existing reports of 12 controlled trials (Table 4)indicate that prophylactic PMN transfusions were historically of marginal value.42--53 Overall, the benefits seemed few, while the risks and expenses were substantial. However, some measure of success was found in seven of 12 studies!z48 The remaining five studies failed to show a benefit for prophylactic PMN tran~fusions.4~-~~ In none of these five negative studies were large numbers of PMNs obtained from matched donors and transfused daily. Thus, in a situation analogous to that of the negative therapeutic PMN transfusion

338

The Neutrophils

trials, the failure of prophylactic PMN transfusions might be explained by inadequate transfusion therapy. When data from eight of the controlled prophylactic PMN transfusion trials were analyzed by formal metaanaly~is?~ many of the impressions of the clinical analysis were confirmed - specifically, that the variability in the dosage of PMNs transfused, inconsistent attempts to provide leukocyte-compatible PMN transfusions, and the varying duration of severe neutropenia were primarily responsible for the differing success rates of the reported studies. It was recommended that, in the design of future trials of prophylactic PMN transfusions, provision should be made to transfuse high doses of compatible PMNs.

3.2. Modern Experience with Prophylactic PMN Transfusions in Neutropenic Patients Clearly data are insufficient for prophylactic PMN transfusions to be recommended at this time as a standard or routine therapy for severely neutropenic patients. However, consideration should be given to renewed investigation of prophylactic PMN transfusions, particularly in hematopoietic progenitor cell transplant patients. Progressive infections with yeast and fungus often occur in transplant recipients because these patients are severely neutropenic for 1-3 weeks; exhibit PMN dysfunction for several weeks; and manifest defective cellular and humoral immunity for months following transplantation - particularly, when T-lymphocytes in the marrow are depleted to diminish graft-vs-host disease. In one study of such patient^?^ 10% of 1186 marrow transplant patients developed a noncandidal fungal infection, with only 17%of infected patients surviving. Hematopoietic progenitor cell transplantation is undergoing marked technological changes with both autologous and allogeneic marrow transplantation being supplanted by transfusion of PBPC and stem cells collected following cytokine (usually G-CSF) donor stimulation. This technique is being increasingly used because it is convenient, economical and, most importantly because it leads to relatively rapid engraftment, with recovery to a blood PMN count of at least 0.5 X 109/L within 7-14 days after transfusion of PBPCs. In some patients, the period of severe neutropenia (< 0.5 x 109/L) lasts only a few days, suggesting that prophylactic PMN transfusions, given in high-dose (i.e. from G-CSF stimulated donors),

Neutrophil Transfusion Therapy in the G-CSF Era

339

might eliminate severe neutropenia via post-transfusion elevation and fairly prolonged circulation of patient blood PMN counts. Preliminary studies have been reported to establish the feasibility and to develop methods for such an approach.56 However, the efficacy of prophylactic PMN transfusions must be investigated by properly designed randomized trials.

4. METHODS FOR PMN COLLECTION

AND TRANSFUSION 4.1. Preleukapheresis Donor Stimulation A major limitation of PMN transfusions has been the inability to consistently transfuse adequate numbers of perfectly functioning PMNs. In any attempt to transfuse satisfactory PMN concentrates, PMNs must be collected by automated leukapheresis with the use of an erythrocyte sedimenting agent such as hydroxyethyl starch (HES).57,58 In addition, donors must be stimulated with a drug such as adrenal corticosteroid (e.g. dexamethasone or prednisone) and/or G-CSF a few hours before collection to increase the donor blood PMN count - with the use of G-CSF highly desired. Under the stress of a severe bacterial infection, the marrow in an otherwise healthy adult will produce between lo1* and lo1* PMNs in 24 hours. PMN concentrates collected from healthy donors who are not stimulated with corticosteroids or G-CSF will contain between lo9 and ZO'O PMNs per each leukapheresis procedure - about 1% of a healthy marrow's daily output. Obviously, donor stimulation is mandatory to achieve even a hope of a reasonable PMN dose for each transfusion. Donor stimulation with properly timed corticosteroids ( 24 hours before leukapheresis) will permit the collection of 1 to 2.5 X 1O1O PMNs per each leukapheresis procedure.58Stimulation with G-CSF alone or in combination with corticosteroids will produce PMN concentrates with quite variable PMN yields, depending on the G-CSF dose and schedule of administration. Yields of 4 to 8 X 1O1O PMNs per each leukapheresis procedure are achieved regularly with some investigators reporting yields as great as 10 to 14 X 10'O PMNs per each leukapheresis procedure.59 Experience with G-CSF in normal donors is still somewhat limited, leading to concerns about potential toxicity and questions about the correct means to obtain informed consent from donors (i.e. need for institutional

340

The Neutrophils

review board oversight versus simple consent for leukapheresis PMN donation). Thus, the proper role of G-CSF, with or without concomitant corticosteroid, as a PMN donor stimulator is still being defined. Donors have received varying doses of G-CSF by a variety of routes and schedules of administration. When given subcutaneously as a single dose of 300 pg or as 5 pg/kg, G-CSF seems to have acceptable toxicity - usually minor musculoskeletal pain that can be relieved by acetaminophen or i b u p r ~ p h e nAlthough .~~ the long-term effects of recombinant cytokines on bone marrow function in humans will not be completely clarified until after years of observations, it is widely believed that G-CSF will exert no long-term hematopoietic effects when administered briefly to healthy individuals. Regardless, G-CSF is not approved by the Food and Drug Administration for stimulation of normal PMN donors, and written consent of some kind should be obtained. Currently, an optimal donor stimulation is achieved by giving 8 mg of dexamethasone orally and 300 to 480pg of G-CSF subcutaneously approximately 12 hours before leukapheresis begins.59Because flexibility of timing is often important for scheduling PMN donation, a timing "window" of 8 to 16 hours between dexamethasone and G-CSF administration and leukapheresis will permit convenient PMN collection. Of note, a recent study of a relatively small number of repeat PMN donors suggested that adrenal corticosteroids might cause posterior subcapsular cataracts after multiple PMN donations.60Although this report must be confirmed by a study involving larger numbers of PMN donors, it seems logical to either include this cautionary information in donor consent forms or to stimulate PMN donors using only G-CSF until the issue is resolved - accepting the fact that PMN yields will be decreased by about 25% or so without the addition of de~amethasone.~~

4.2. LeukapheresisTechniques An optimal PMN transfusion therapy depends on the consistent preparation of satisfactory PMN concentrates that contain large numbers of functional PMNs. By regulation, each transfusion must deliver at least 1 X 1O'O PMNs to an adult patient.61 As noted in the preceding discussion of G-CSF donor stimulation, a more acceptable modern dose for treating

Neutrophil Transfusion Therapy in the G-CSF Era

341

adults is 4 to 8 X 1O1O PMNs per each transfusion. Although leukapheresis techniques may vary among blood centers, there is general agreement that donors must be stimulated with corticosteroids and/or G-CSF; that an erythrocyte sedimenting agent must be used throughout the entire leukapheresis procedure; and that relatively large volumes of donor blood (e.g. 10 liters) need to be processed using a continuous-flow blood separator. Because it is very difficult to process adequate volumes of donor blood with discontinuous-flow techniques, this type of blood separator is seldom, if ever, used for PMN collections.

4.3. Erythrocyte Sedimenting Agents It has been known for decades that an erythrocyte sedimenting agent such as HES, dextran, or modified fluid gelatin is required during centrifugation leukapheresis for optimal granulocyte collection.62Many cytapheresis centers prefer HES (either hetastarch or pentastarch) over the other agents because it has proven to be relatively safe. However, concern has been raised over the prolonged persistence of hetastarch in the bloodstream after leukapheresis - particularly, following serial collections at intervals of only a few days.63,64 Thus, efforts were made to replace hetastarch with pentastarch - an HES solution that has been reported to enhance PMN yields during leukapheresis, yet is promptly eliminated from the bloodstream and has fewer effects on c o a g u l a t i ~ nHowever, .~~~~~ as will be discussed later, the efficacy of pentastarch has been questioned, and it is now preferred to use hetastarch whenever possible. HES is a complex polysaccharide consisting of glucose units connected primarily by a-1:4 glycosidic linkages, with a-1:6 linkages serving as branch points. Hydroxyethyl groups are attached by ether linkages to carbon atoms C2, C3, and/or C6. Solutions of HES contain mixtures of HES molecules that vary in size rather than being of uniform size and weight. The molecular weights of HES molecules in both hetastarch and pentastarch overlap considerably,but hetastarch solutions contain larger molecules with more sites of hydroxyethylation than does pentastarch. It is important to note that the hydroxyethylation ratios among available carbon atoms (C2, C3, and C6) vary with different solutions and can greatly influence the properties of the seemingly identical HES preparations. Thus,

342

The Neutrophils

two starch solutions with identical molecular weights and hydroxyethyl molar substitution values can exert markedly different biological effects when infused, simply because they have different C2C6 hydroxyethylation ratio values.67In other words, the number of hydroxyethyl groups may be the same within the HES molecule, but the biological properties will differ because of the arrangement of hydroxyethyl groups per available carbons. Thus, for accurate comparison of different HES solutions, all biochemical properties must be known, as these are a "family" of products with almost unlimited heterogeneity. Unfortunately, the C2:C6 hydroxylation ratio is often not reported in HES studies, leading to difficulties in comparison and interpretation of results. The optimal type of HES for PMN collection is controversial. In an uncontrolled multicenter trial, pentastarch appeared to be an efficacious and safe erythrocyte-sedimenting agent for use during centrifugation leukapheresi~.~~ However, the efficacy of pentastarch for PMN collection was challenged. In two s t ~ d i e s ,pentastarch ~ ~ , ~ ~ was thought to exert effects on donor erythrocyte sedimentation rates to a lesser extent than hetastarch and, consequently, pentastarch was predicted by a PMN collection efficiency equation to be less effective in enhancing PMN yields. This prediction was later supported by a controlled clinical tria170 in which steroid-stimulated donors underwent paired PMN collections separated by 2 weeks to 7 months - in which they received 500mL of either 10% pentastarch or 6% hetastarch. Approximately 7 L of donor blood were processed at a 1:13 starch:donor blood ratio. In 92% of the donors, hetastarch procedures were more efficient. The PMN yield (mean ? SD) was 2.3 2 0.7 X 1O1O with hetastarch versus 1.4 2 0.076 X 1O1O with pentastarch. It is unclear why pentastarch performed so poorly in the more recent s t u d i e ~ , ~ ~compared -~O with performance results in the initial multicenter Until this issue is resolved, it is prudent for each center preparing PMN concentrates to perform continuing quality assessment of its leukapheresis program. The average PMN yield obtained by processing 10 L of donor blood following corticosteroid only stimulation, and using some type of HES at a 1:13 starch:donor blood ratio, should be between 1.5 and 2.5 X 1O1O; after G-CSF plus corticosteroid stimulation, the PMN yield should be consistently between 4.0 and 8.0 X 1O'O. Hetastarch seems most

Neutrophil Transfusion Therapy in the G-CSF Era

343

likely to achieve these goals consistently, but it seems reasonable, for donors experiencing repeat leukapheresis at brief intervals, to use pentastarch because of its more rapid elimination from the bloodstream and its lesser effect on c ~ a g u l a t i o nIf. ~ ~ ~ ~yields ~ PMN are not satisfactory with pentastarch, consideration should be given to switching to hetastarch, even during serial donations.

4.4. Transfusion of PMN Concentrates PMN concentrates should be transfused as soon as possible after collection - preferably within 6 hours - since PMN functions begin to deteriorate rapidly. Because some delay between collection and transfusion is inevitable, PMN concentrates are usually stored briefly at 22"C, with little or no agitation. However, since aberrations of nearly all functions become evident within 24-72 hours and PMNs are collected using an "open" system, PMN concentrates stored for more than 24 hours should be transfused only in study settings - although G-CSF diminishes PMN apoptosis and, eventually, the permitted length of storage for PMN concentrates might be lengthened. Each institution must assess local needs for PMN transfusions. If, despite optimal antimicrobial and other supportive therapy, neutropenic patients suffer significant morbidity or mortality from infections, PMN transfusions should be considered. Once PMN transfusions have been prescribed, they must be given effectively: 2 4 X 1O1O PMNs per dose with G-CSF and corticosteroid donor stimulation; 2 2 X 1O1O PMNs per dose with corticosteroid only stimulation (less desirable); and never less than 1X 1Olo PMNs per dose. Transfusions are continued daily until the infection has resolved or until blood PMNs have risen above 0.5 X 109/L, independent of PMN transfusions (i.e. sustained without need for continued transfusions) - a decision often difficult because of post-transfusion increases in blood PMN counts with modern PMN transfusions. Thus, in practice, it is often practical to continue daily PMN transfusions until the blood PMN count is sustained at > 1.5 X 109/L for a few days, then to stop transfusions for a day or so, and to observe the blood PMN count. If it falls to < 0.5 x 109/L, PMN transfusions are resumed, unless the infection has resolved.

344

The Neutrophils

On the basis of studies of the circulation and migration of indium-llllabeled PMN171,72it seems logical that patients with evidence of alloimmunization (platelet refractoriness or the presence of leukocyte antibodies) should receive PMN transfusions collected from donors selected to be leukocyte compatible by HLA-matching and/or leukocyte crossmatching. Dutcher et ~ 1 . ~found ' that transfused, labeled PMNs failed to reach sites of infection in 78% of alloimmunized patients, while localization of PMNs was normal in nonalloimmunized patients. McCullough et al." reported similar findings and also documented reduced recovery and circulating half-life in some alloimmunized patients. However, it has not been clearly shown that attempts to improve donor-recipient leukocyte antigen compatibility do, in fact, increase the success of PMN transfusions in alloimmunized patients particularly, when modern "high-dose" PMN concentrates collected from G-CSF stimulated donors are transfused. Thus, a requirement cannot be mandated for leukocyte-matched PMN transfusions.

ACKNOWLEDGMENTS Supported by National Institutes of health, Transfusion Hemostasis Clinical Trials Grant 1 UO1 HL 72028-01.

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5. Anderlini P, Przepiorka D, Champlin R, Korbling M. Blood 1996; 88: 2819-2825. 6. Strauss RG. In: The Neutrophils: New Outlook for the Old Cells (ed. Gabrilovich D). Imperial College Press, London, 1999; pp. 345-362. 7. Alavi JB, Root RK, Djerassi I, et al. N Engl J Med 1977; 296:706-711.

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10. Herzig RH, Herzig GP, Graw RG Jr, et al. N Engl J Med 1977; 296:701-705. 11. Higby DJ, Yates JW, Henderson ES, Holland JF. N Engl J Med 1975; 292: 761-766. 12. Vogler WR, Winton EF. Am J Med 1977; 63:548-555. 13. Winston DJ, Ho WG, Gale RP. Ann Intern Med 1982; 97:509-515. 14. Vamvakas EC, Pineda AA. J Clin Apheresis 1996; 11:l-9. 15. Morrison VA, Haake RJ, Weisdorf DJ. Am J Med 1994; 96:497-503. 16. Pirsch JD, Maki DG. A n n Intern Med 1986; 104:619-632. 17. Clarke K, Szer J, Shelton M, et al. Bone Marrow Transplant 1995; 16:723-726.

18. Spielberger RT, Falleroni MJ, Coene AM, Larson RA. Clin Infect Dis 1993; 16:528-530. 19. Swerdlow B, Deresinski S. Am JMed 1984; 76:162-165. 20. Chow HS, Sarpel SC, Epstein RB. BZood 1980; 55:546-551. 21. Chow HS, Sarpel SC, Epstein RB. Blood 1982; 59:328-333. 22. Strauss RG. Hematol Oncol Clin North Am 1994; 8:1159-1166. 23. Bhatia S, McCullough JJ, Perry EH, et al. Transfusion 1994; 34226-231. 24. Clarke K, Szer J, Shelton M, et al. Bone Marrow Transplant 1995; 16:723-726. 25. Catalan0 L, Fontant R, Scarpato N, et al. Haematologica 1997; 8271-72. 26. Ozsahin H, von Planta M, Muller I, et al. Blood 1998; 922719-2724. 27. Bielorai B, Toren A, Wolach B, et al. Bone Marrow Transplant 2000; 26: 1025-1028. 28. Bielorai B, Neumann Y, Avigad I, et al. Med Pediatr Oncol2000; 34:221-223. 29. Hester JP, Dignani MC, Anaissie EJ, et al. J Clin Apheresis 1995; 10:188-193. 30. Grigg A, Vecchi L, Bardy P, et al. Aust N Z J Med 1996; 262313-818. 31. Peters C, Minkov M, Matthes-Martin S, et al. Br J Haematol 1999; 106:689-696. 32. Price TH, Bowden RA, Boeckh M, et al. Blood 2000; 95:3302-3309.

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38. Cairo MS, Rucker R, Bennets GA, et al. Pediatrics 1984; 742387-892. 39. Cairo MS, Worcester C, Rucker R, et al. ] Pediatr 1987; 110:935-941. 40. Baley JE, Stork EK, Warkentin PI, Shurin SB. Pediatrics 1987; 80:712-720. 41. Wheeler JG, Chauvenet AR, Johnson CA, et al. Pediatrics 1987; 79:422-425. 42. Mannoni P, Rodet M, Vernant JP, et al. Blood Transfus Immunokaematol 1979; 22~503-508. 43. Gomez-Villagran JL, Torres-Gomez A, Gomez-Garcia P, et al. Cancer 1984; 541734-738. 44. Clift RA, Sanders JE, Thomas ED, et al. N Engl J M e d 1978; 298:1052-1056. 45. Strauss RG, Connett JE, Gale RP, et al. N Engl] Med 1981; 305597-603. 46. Hester JP, McCredie KB, Freireich EJ. In: Care of the Child with Cancer. American Cancer Society, Atlanta, 1979; pp. 93-97. 47. Buckner CD, Clift RA, Thomas ED, et al. Infection 1983; 11:243-247. 48. Curtis JE, Hasselback R, Bergsagel DE. Can Med Assoc ] 1977; 117341-346. 49. Schiffer CA, Aisner J, Daly PA, et al. Blood 1979; 54:766-770. 50. Sutton DMC, Shumak KH, Baker MA. Plasma Tker Transfus Techno1 1982; 3:45-52. 51. Ford JM, Cullen MH, Roberts MM, et al. Transfusion 1982; 22:311-315. 52. Cooper MR. Personal communication update of earlier study. In: Collection and Transfusion (eds. Cooper MR, Heise E, Richards F, et al.). Academic Press, San Diego, 1981; pp. 436439. 53. Winston DJ, Ho WG, Young LS, Gale RP. Am ]Med 1982; 68:893-900. 54. Vamvakas EC, Pineda AA. J Clin Apheresis 1997; 12:74-81. 55. Pirsch JD, Maki DG. Ann Intern Med 1986; 104:619-624. 56. Adkins D, Spitzer G, Johnson M, et al. Transfusion 1997; 37737-748.

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57. Strauss RG, Rohert PA, Randels MJ, Winegarden D. Granulocyte collection. J Clin Apheresis 1991; 6:241-245.

58. Strauss RG, Hester JP, Vogler WR, et al. Transfusion 1986; 26258-262. 59. Liles WC, Rodger E, Dale DC. Transfusion 2000; 40:642-644. 60. Ghodsi Z , Strauss RG. Cataracts in neutrophil donors stimulated with adrenal corticosteroids. Transfusion 2001; 41:1464-1468. 61. Fridey JL, Chair. Standards for blood banks and transfusion services. 21st ed. Bethesda MD: American Association of Blood Banks, 2002:36. 62. Mishler JM, Hadlock DC, Fortuny IE, et al. Blood 1974; 44:571-581. 63. Ring J, Sharkoff D, Richter W. Vox Sang 1980; 39:181-185. 64. Maguire LC, Strauss RG, Koepe JA, et al. Transfusion 1981; 21:347-353. 65. Treib J, Baron J-F, Grauer MT, Strauss RG. Intensive CareMed 1999; 25:258-268. 66. Strauss RG, Pennell BJ, Stump DC. Transfusion 2002; 4227-36. 67. Treib J, Haass A, Pindur G, et al. Thromb Haemost 1995; 74:1452-1456. 68. Lee JH, Cullis H, Leitman SF, Klein HG. J Clin Apheresis 1995; 10:19&202. 69. Lee JH, Klein HG. Tvansfusion 1995; 35:384-388.

70. Lee JH, Leitman SF, Klein HG. Blood 1995; 86:4662-4666. 71. Dutcher JP, Schiffer CA,Johnston GS, et al. Blood 1983; 62354-358. 72. McCullough J, Weiblen BJ, Clay ME, et al. Blood 1981; 58:164-169.

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Index

a 4 integrin 114 ci6/Pl integrin 123 p l integrin 123 P2 integrin 123,198 p3 integrin 123 P-oxa PUFA 215 o-oxidation 183 5-HETE 179 5-oxo-ETE 193 actin cytoskeletal dynamics 1 adherence 10 adherens junction 114 adhesion 106 adhesion (L-selectin) 155 adhesion molecule 107,317 AF-6 127 Akt 46 alcohol 316,317 antibiotic 311-313,320 antibody-dependent cellular cytotoxicity (ADCC) 267 anti-neutrophil antibodies 265

antioxidant 67 “apparent” permeability coefficients (PJ 135 apoptosis 153,309 arachidonic acid 47,170 ARDS 320,321 astrocytes 118 atomic force microscopy (AFM) 119 bacteremia 311,313 bacterial-derived formylated peptides 304 bactericidal oxidant 70 Bcl-2 160 blood 316 bone marrow 304,305,309,310,316, 318,319 bradykinin 136 burn wound injury 311,312 C5a 136,161,304 Ca2+ 161,201 Ca2+mobilization 11

349

350 lndex cadherin-5 120 cancer 275,277-279,281,284,286, 291-293 capillary filtration coefficient (Kf) 134 Cas 26 casein kinase 2 (CK2) 46 caspase 156 catalase 67 catenin 120 cathepsin G 304 cationic antimicrobial protein 37 (CAP371 138 CDlla/CD18 111 CDllb/CD18 100,111 CD18 105 CD47 113 CD49d/CD29 128 CD54 111 CD62E 111 CD62L 111 CD62P 111 CD99 105 CD106 112 chemoattractant 7 chemokine 280,287,293 chemotactic migration 1 chemotaxis 155,265 chemotherapy 318 chloramine 68 chronic granulomatous disease (CGD) 37,306 chronic inflammatory condition 153 chronic obstructive pulmonary disease (COPD) 162 cingulin 127 ciprofloxacin 313 claudin 115 connexin 122 connexon 122

Crk 25 cross talk 26 cx37 122 Cx40 122 cx43 122 cyclic AMP 3,161 cyclooxygenase 176 cytochalasin B 121 cytochrome b 38 cytochrome bSs838 cytochrome b-245 38 cytochrome c 159 cytokine 86,229-231,234,237,239, 241-243,247’248,253,316 cytoskeletal network 13 cytoskeleton 55 cytotoxicity 267,275,284,286,290 DAP12 89 defensin 268 degranulation 186 dendritic 308 dextran 139 differentiation 303,309,314 diffusive permeability coefficients (Pd) 135 docosahexaenoic acid 172 edema 135 eicosanoid 174 eicosapentaenoic acid 172 elastase 304 electron transfer 65 electron transport 44 emigration 107 endothelial calcium 139 endothelial cell 310 endothelial cell calcium 138 endothelial permeability 132

Index endothelium 105,124 endotoxin 6,304,309,314-316 eosinophil 36,156,303,308 erythroid 306 erythropoietin (EPO) 302,306 E-selectin 111 ethanol 317 extracellular signal-regulated kinase (ERK1/2)] 46 F-actin 121 Fas 158 fatty acid CoA synthetase 178 Fc receptors (FcR) 96 FcyRIII (CD16) 155 Fenton reaction 68 fibroblasts 310 filgrastim 302 flavin 43 flavin adenine dinucleotide (FAD) 42 flavocytochrome 43 flavocytochromeb 35 flavoprotein 38,42 fMLF 129,136 fMLP 155 focal adhesion kinase 25 freeze-fracture 115 gap junctions 122 G-CSF antibody 315 G-CSF receptor (G-CSF-R) 302-304, 308 G-CSF therapy 318 G-CSF/G-CSF-R 303 G-CSF-deficient mice 309 glucocorticoid 162,304 glutathione 268 glutathione (GSH)-peroxidase 67 GM-CSF (“knock-outs”) 308

351

GM-CSF receptor (GM-CSF-R) 303 gp91PhoX38 G-protein 3 G-protein receptor 19 graft-vs-host disease 331,335,338 granule 37 granulocyte 303,309 granulocyte colony stimulating factor (G-CSF) 161,267,301-304,306, 308-321,327 granulocyte/macrophage colony stimulating factor (GM-CSF) 99,161,266,301-304,306, 308-310,312,314,315,319 granulopoiesis 309,316 guanosine triphosphate (GTP) 54 Haber-Weiss reaction 68 hamster cheek pouch 139 hematopoiesis 310 hematopoietic growth factor 306 hematopoietic stem ceII 306 hematopoietin 302 heme 41 heterotrimeric GTP-binding proteins 203 histamine 119,136 HIV patients 266 host defense 37 HUVEC 117 hydraulic conductivity 133 hydrogen peroxide 15,67 hydroxyl radical 68 hypochlorous acid 68 ICAM-1 111 IFN-y 161 IL-1 316 IL-lp 161

352

Index

IL-2 161 IL-6 309 IL-8 124,161,309,316 IL-15 161 impedance 136 increased vascular permeability 18 infection 309-311,313,315-319,328, 329,331-338,343 inflammation 36,106,170,309,310 inflammatory disease 37 inflammatory response 35 innate 35 innate immune response 36,87 innate immune system 36 insert domain 62 integrated cellular signaling 3 integrin-linked kinase 25 integrins 282,289,304,305 interleukin-1 310 interleukin-3 (IL-3) 302 interleukin-8 7,304 intraabdominal infection 315 intraabdominal sepsis 311 intracellular signals 201 ion channels 209 JAK 303 JAM 105 JAM-1 126 JAM-2 126 JAM-3 126 JAM-A 126 JAM-B 126 JAM-C 126 Janus protein tyrosine kinase (JAK) 303 KC 124 killer cell inhibitory receptor, KIR 87 kinase 46

L, 133 latrunculin A 121 lenograstim 302 leukapheresis 329-333,335,337, 339-343 leukemia 319 leukocyte 37 leukopenia 317 leukotriene B4 (LTB,) 136,161,188,304 LFA-1 111 lipid peroxidation 70 lipopolysaccharide (LPS) 161 lipoxin A4 189 lipoxygenase 176 liver 305 L-selectin 111 lung 132,305 Mac-1 111 macrophage 36,153,164,314,316 marrow 303,305,306,308,319 matrix metalloproteinases 304 maturation 303 M-CSF 302 megakaryocytic cells 306 MHC class I-specific inhibitory receptors 87 Michell Hypothesis 21 microbial factor 7 microbicidal 35 MIP-2 124,132 mitochondria 160 mitogen-activated protein (MAP) kinase 46,161,206,303 mobilization 304,309 moesin 48 monocyte 36,121,303,308,311 morphogenesis 20 mortality 310

Index multi-CSF 302 MyD88 95 MyD88/interleukin-l 92 myeloblast 303 myeloid progenitor 316 myeloperoxidase 68,275,279,306 myosin light chain kinase (MLCK) 137 n-3 polyunsaturated fatty acid 171 n-6 polyunsaturated fatty acid 173 NADPH 43 NADPH oxidase 16,35,185 NCF2 48 necrosis 154 neonatal sepsis 311,312 neutropenia 308,309,311,318,319, 328,331,337-339 neutropenic 305,309,318 neutrophil activation 86 neutrophil cytosolic factor 1 (NCFI) 44 neutrophil 35,36,153,170,229-231, 234,237,240-242,245,246,248, 253,275,276,278,280,281,284, 286,290,292,303-306, 308-320,328 neutrophil function 184 neutrophilia 313,318 neutrophilic leukocyte 1 NF-KB 161,162 nitric oxide (NO) 70,162 non-phagocyte oxidase 37 occludin 115,127 oxidants 35 oxidase 46 oxygen metabolites 154 ozone 70

353

p21-activated kinase (PAK) 46 p22phDx38 p38MAPK 46 p40phoX38 p47phox38 p67phox38 PAF 161 PAR-3 127 PDZ domains 127 PECAM-1 105 peroxynitrite 70 phagocyte 36 phagocyte oxidase 37 phagocytosis 155,253,255,317 phagosome 35,66 phosphatidic acid 11 phosphatidic acid-activated kinase 46

phosphatidylinositol3,4-bisphosphate (PtdIns(3,4)P2)48 phosphatidylinositol3,4,5-trisphosphate 20 phosphatidylinositol3-kinase 207 phosphatidylinositol 3’-kinase 10

phosphatidylinositol3-phosphate (PtdIns(3)P) 53 phosphatidylserine 155 phosphoinositide 3-kinase (PI3-kinase) 47 phosphoinositide 45 phospholipase A2 173 phosphorylation 35 Phox and Bem (PB1) domain 53 Phox and Cdc (PC) motif 52 Phox homology (PX) domain 45 PI-3 kinase (PDK) 161 PKA 24 PLA2 204 plasma membrane 10 Plasmodium falciparum 195

354

index

platelet activating factor (PAF) 137, 304 pneumonia 311,315-317,319-321 polymorphonuclear leukocytes 36 potent bactericidal system 16 progenitor 309 programmed cell death 154 proliferation 303,306,309,314 proline-rich domain 44 prostaglandin 163 protein kinase 2 14 protein kinase A (PKA) 161 protein kinase C (PKC) 24,46,204 P-selectin 111 PTPase 26 Racl 54 Rac2 54 RaplA 38 reactive oxygen species 36,284, 286,291 Rel-1 25 respiratory tract infections 310 Rho effectors 14 Rho family GTPases 13 Rho/Rac equilibrium 20,31 Rho-GDI 27 Rho-kinase 11 rolling 110 scanning electron microscopy 132 SDF-1 304 second messenger 3 selectin 110,304,305 sepsis 317,320,321,332,335,337 septicemia 310 shear 110 signal transducers and activators of transcription (STATs) 303

signal transduction 89 signal transduction pathways 1 singlet oxygen 70 small GTP binding proteins 209 sphingomyelinase 207 spleen 305,316 splenectomy 317 Src 26 Src homology 3 (SH3) 44 stem cell factor (SCF) 304 streptococcal pneumonia 132 stromal cell-derived factor-1 (SDF-1) 304 superoxide anion 35 superoxide dismutases 67 superoxide ion (0,) 16 superoxide production 266 surgical wound infection 311,312 Syk kinase 98 tetratricopeptide repeat (TPR) 49 therapy 313,316,320,321 thrombin 119 thrombopoietin (TPO) 302,306 tight junction 114 T-lymphocyte 310 TNF-cx 161 TNFRl 159 Toll-like receptor 85 Trail-R1 159 transcytotic migration 131 transendothelial electrical resistance (TEER) 136 transmigration 107 transmission electron microscopy 115 TREMl ligand 90 tricellular corner 116 triggering receptor expressed by myeloid cell (TREM) family 85

Index tumor metastase 281 tumor necrosis factor (TNF) 7,193, 309,310,314-316 tyrosine kinase 14,98

very late antigen-4 128 virus 253,278 VLA-4 128 Weibel-Palade bodies 111

Vav 25 VCAM-1 112 VE-cadherin 120 VEGF 130

ZO-1 127

zonula adherens 114 zonula occludens 114

355