The Neutrophil: An Emerging Regulator of Inflammatory and Immune Response (Chemical Immunology)

The Neutrophil

Chemical Immunology and Allergy Vol. 83

Series Editors

Johannes Ring Munich Luciano Adorini Milan Claudia Berek Berlin

The Neutrophil An Emerging Regulator of Inflammatory and Immune Response

Volume Editor

Marco A. Cassatella

Verona

32 figures, 5 in color and 12 tables, 2003

Basel · Freiburg · Paris · London · New York · Bangalore · Bangkok · Singapore · Tokyo · Sydney

Chemical Immunology and Allergy Formerly published as ‘Progress in Allergy’ (Founded 1939), continued 1990–2002 as ‘Chemical Immunology’ Edited by Paul Kallos 1939–1988, Byron H. Waksman 1962–2002

Marco A. Cassatella Professor, Department of Pathology, Section of General Pathology, Verona

Bibliographic Indices. This publication is listed in bibliographic services, including Current Contents® and Index Medicus. Drug Dosage. The authors and the publisher have exerted every effort to ensure that drug selection and dosage set forth in this text are in accord with current recommendations and practice at the time of publication. However, in view of ongoing research, changes in government regulations, and the constant flow of information relating to drug therapy and drug reactions, the reader is urged to check the package insert for each drug for any change in indications and dosage and for added warnings and precautions. This is particularly important when the recommended agent is a new and/or infrequently employed drug. All rights reserved. No part of this publication may be translated into other languages, reproduced or utilized in any form or by any means electronic or mechanical, including photocopying, recording, microcopying, or by any information storage and retrieval system, without permission in writing from the publisher. © Copyright 2003 by S. Karger AG, P.O. Box, CH–4009 Basel (Switzerland) www.karger.com Printed in Switzerland on acid-free paper by Reinhardt Druck, Basel ISSN 1660–2242 ISBN 3–8055–7552–1

Contents

XI Preface

1 Transcription Factor Activation in Human Neutrophils A. Cloutier, P.P. McDonald, Sherbrooke, Qué. 2 The STAT Family of Transcription Factors 3

Activation of STAT Proteins in Neutrophils: The Early Studies and the Proteolysis Issue

4

Activation of STAT Proteins by Colony-Stimulating Factors in Human Neutrophils

6

Activation of STAT Proteins by IFN␥ in Human Neutrophils

7

Activation of STAT Proteins by IL-10 in Human Neutrophils

8

Activation of STAT Proteins by Other Agents in Human Neutrophils

8

Concluding Remarks and Future Directions

9 The NF-␬B Family of Transcription Factors 10

Expression and Activation of NF-␬B Pathway Components in Human Neutrophils

12

Regulation of NF-␬B Activity by Reactive Oxygen Derivatives in Neutrophils

13

Potential Role of NF-␬B in Neutrophil Apoptosis

14

Concluding Remarks and Future Directions

15 Other Transcription Factors Potentially Involved in Neutrophil Activation 15

The Ets Family of Transcription Factors

15

The C/EBP Family of Transcription Factors

16

The AP-1 Family of Transcription Factors

17 Conclusion 17 References 24 Phenotypic and Functional Changes of Cytokine-Activated Neutrophils C. Galligan, T. Yoshimura, Frederick, Md. 25 28 29 30 31 33 35 38 38

Chemokine Receptors in PMN Mobilization and Activation Cytokine Involvement in PMN Emigration PMN Priming PMN Granule Release The Role of PMN in Adaptive Immunity Expression of a DC Phenotype by Cytokine-Activated PMN Gene Expression in Cytokine-Activated PMNs Conclusions References

45 Expression of MHC Class II Antigen and Coreceptor Molecules in Polymorphonuclear Neutrophils G.M. Hänsch, C. Wagner, Heidelberg 46 47 51 51 53 60 61

Induction of MHC Class II Synthesis and Surface of PMN in Culture Expression of MHC Class II Antigens in vivo Upregulation of Costimulatory Molecules on PMN in vitro and in vivo Expression of CD83 on PMN Functional Consequences of MHC Class II and Coreceptor Expression Conclusion References

64 Phenotypic and Functional Change of Neutrophils Activated by Cytokines Utilizing the Common Cytokine Receptor Gamma Chain D. Girard, Pointe-Claire, Qué. 64 66 66 66 66 67 68 70 71

The Polymorphonuclear Neutrophil Cells The IL-2 Family Cytokines or ‘␥c Users’ Generalities Pleiotropy and Redundancy Signaling: The Jak-STAT Pathway The Jak-STAT Pathway in Neutrophils Interleukin-2 Interleukin-4 Interleukin-7

Contents

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72 73 74 74 75 76

Interleukin-9 Interleukin-15 Interleukin-21 Production of ␥c User Cytokines by Neutrophils Summary and Future Perspectives References

81 The Evolving Role of the Neutrophil in Chemokine Networks S.S. Cheng, S.L. Kunkel, Ann Arbor, Mich. 82 82 84 85 86 86 87 88 90 90

Chemokines and Chemokine Receptor Involvement in Neutrophil Recruitment CXCR1 and CXCR2 CCR1 CCR2, CCR3, and CCR6 Other Receptors Regulation of Chemokine Receptor Expression Chemokine Networking during Acute Inflammation Neutrophil-Derived Chemokines Contribute to Inflammation Conclusions References

95 Neutrophil Production of IL-12 and Other Cytokines during Microbial Infection E.Y. Denkers, L. Del Rio, S. Bennouna, Ithaca, N.Y. 98 98 100 102 103 104 107 108 109 109

Microorganisms Inducing Neutrophil Cytokine Production Triggering Pathways Involved in PMN Release of IL-12 and Other Cytokines Cytokine Modulation of Neutrophil IL-12 Production Preformed versus Newly Synthesized Cytokines Neutrophil Subsets Influence of PMN on T Cell Subset Selection during Infection Neutrophils as Triggers of Immunity Conclusions and Future Directions Acknowledgments References

115 Novel Pathways and Endogenous Mediators in Anti-Inflammation and Resolution C.N. Serhan, B. Levy, Boston, Mass. 117 First Things First: ‘The Battle Within’ 117 Cell-Cell Interactions in Chemical Mediator Production 118 ASA-Triggered Mediators

Contents

VII

120 15-LO-Initiated Pathway 123 Temporal and Spacial Considerations in LX Formation and the Role of Exudate Prostanoids 124 Leukotriene A4-Dependent LX Biosynthetic Route 126 LX Biosynthesis in Selectin-Deficient Mice: Diminished Cell-Cell Interactions 127 Evolution and LXs: Other Species That Produce LX 128 ATL Circuit 129 Unexpected Benefits with ASA 129 ASA-Dependent Generation of 15-Epi-LXA4 in Experimental Exudates and ASA-Induced Asthma Patients 130 Metabolically Stable Analogs LXA4 and ATL; New Leads to Resolution? 134 LXs and ATL Associated with Human Diseases and Disease Models 135 Experimental Models of Disease: ATL and LX 135 Lung Disease 136 Enteritis 136 Nephritis 136 Conclusions 138 Acknowledgments 138 References 146 Regulation of Vascular Permeability by Neutrophils in Acute Inflammation L. Lindbom, Stockholm 147 147 149 150 151 152 152 154 155 159 159 160 160

Neutrophil Recruitment Neutrophil Interactions with Vascular Endothelium Integrin-Mediated Firm Adhesion Neutrophil Diapedesis Structural Basis of Alteration in Endothelial Barrier Function Neutrophil-Induced Increase in Vascular Permeability Endothelial Responses to Neutrophil Activation Oxidant-Mediated Endothelial Barrier Dysfunction Role of Neutrophil Cationic Proteins in Regulation of Endothelial Barrier Function Role of Endothelial Gap Formation in Leukocyte Transendothelial Migration Concluding Remarks Acknowledgments References

167 Neutrophils and Angiogenesis: Potential Initiators of the Angiogenic Cascade R. Benelli, A. Albini, D. Noonan, Genoa 168 Neutrophils and Angiogenesis 168 Chemokines and Angiogenesis

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169 170 170 170 171 172 172 173 173 174 175 175 178 178

Soluble Mediators Released by Neutrophils Vascular Endothelial Growth Factor IL-8 Hepatocyte Growth Factor Platelet Activating Factor Neutrophils and the Endometrium Neutrophils as Antiangiogenesis Effectors Free Radicals Angiostatin Angiostatic Cytokines Anti-Inflammatory Agents To Induce or Inhibit Angiogenesis: What Makes the Difference? Acknowledgments References

182 Neutrophils in the Antitumoral Immune Response E. Di Carlo, Chieti; G. Forni, Orbassano/Turin; P. Musiani, Chieti 182 The New ‘Immunological Identity’ of Neutrophils 182 Neutrophil-Endothelial Interactions 183 Neutrophil-Centred Cytokine Network 184 The Neutrophil Dilemma: Is It ‘Tumor-Stimulatory’ or ‘Tumor-Inhibitory’? 186 Preclinical Studies in Animal Models of Tumor Cure and Prevention: Evidence of Neutrophil Anticancer Potential 186 Cytokine Gene-Transfected Tumors 189 Chemokine Gene-Transfected Tumors 189 TNF Ligand-Transfected Tumors 190 The Role of Neutrophils in Antitumor Immune Memory Rejection 191 Targeting Immunoprevention: The New Frontier in Anticancer Strategies 193 Neutrophils and Anticancer Therapy in Humans 193 News from Clinical Trials 195 Patients Eligible for Neutrophil-Stimulating Immunotherapy 196 Future Developments 196 Improvement of Ab-Based Immunotherapy 197 Vaccination ⫹ Cytokines for Tumor Prevention 197 Acknowledgment 197 References 204 Regulation of Neutrophil Apoptosis S.W. Edwards, D.A. Moulding, M. Derouet, R.J. Moots, Liverpool 204 205 207 209

Overview of Neutrophil Apoptosis Morphological and Molecular Features of Apoptotic Neutrophils Extracellular Factors That Regulate Neutrophil Apoptosis Molecular Control of Apoptosis

Contents

IX

209 Death Receptors and Caspases 210 Mitochondria and Cytochrome c 210 The Bcl-2 Family of Proteins 212 Regulation of Neutrophil Apoptosis and Survival 213 Bcl-2 Family Expression in Human Neutrophils 213 Properties of MCL-1 217 Properties of A1 (Bfl-1) 217 Mechanisms Leading to Neutrophil Death 218 Roles of A1 and Mcl-1 in Control of Neutrophil Apoptosis 219 Acknowledgments 219 References 225 Author Index 226 Subject Index

Contents

X

Preface

Contrary to what is traditionally thought, the neutrophil is a remarkably versatile cell. Indeed, while the ability of neutrophils to transcribe many genes is no longer a matter of debate, the research conducted in recent years has brought forward exciting discoveries that have greatly broadened our knowledge on the functional role of this cell type and uncovered novel links involving neutrophils in unsuspected physiopathologic processes. For instance, evidence on their capacity to change the phenotype under specific circumstances, or on their active involvement in the resolution of inflammation (other than in its regulation), or also on their unquestionable regulatory role in angiogenesis and tumor fate, or else on their response to, and release of, a wide variety of cytokines and chemotactic molecules, has made it clear that the obsolete concept of the neutrophil as a ‘terminally differentiated, short-lived, cell devoid of transcriptional activities’ found in most biomedical textbooks is certainly an old-style assumption which underestimates the multiple and amazing functional capacities of this cell type. Time is now ripe for pathologists, cell biologists, physicians, and, why not, the same immunologists, to definitively change such an outdated view and start thinking that neutrophils, in the context of inflammatory and immune responses, can no longer be regarded as cells that only release preformed mediators and kill pathogens. With these thoughts in my mind, I found it appropriate to edit a volume in which the progress recently made in the ‘neutrophil’ field would be described, emphasized and critically discussed as well. The present book is an attempt to offer an up-to-date coverage of what are in my view the most significant advances that appeared in the scientific

XI

literature in the last decade regarding some of the novel aspects of the biology of neutrophils. To this end, leading scientist(s) of the respective fields were asked to give their contributions and all did so with admirable results. Obviously, what is presented herein cannot be exhaustive. Since there already exist several books and a series of excellent reviews focusing on more classical aspects of neutrophil physiology [for example, 1], which I encourage you to read anyway, I decided not to include an initial chapter describing the more general features and properties of neutrophils. In brief, the opening article deals with a new emerging field of research, that is the transcriptional control of neutrophil gene expression. Cloutier and McDonald summarize the progress made in elucidating some of the regulated transcriptional processes in the context of inducible gene expression in human neutrophils. To date, only a handful of reports have addressed the issue of transcription factor expression and activation in neutrophils and thus our current understanding of this important facet of neutrophil biology is still very incomplete. In the following article, Galligan and Yoshimura describe the effects of proinflammatory cytokines on neutrophils. In particular, the authors make an update on the latest findings in this rapidly expanding area of research, highlighting, among other aspects, the novel concept that cytokine-exposed neutrophils during inflammatory responses may acquire new properties and thus exist at various stages of activation and differentiation. The notion that neutrophils, under appropriate circumstances, might play a role in local antigen presentation and T cell proliferation has been outlined in the third article by Hänsch and Wagner. These authors recapitulate all the observations and the current experiments which demonstrate that neutrophils, activated under specific conditions either in vitro or in vivo, can express characteristic features of dendritic cells, including MHC class II antigens, CD83 and costimulatory receptors such as CD80 and CD86. In the fourth article, Girard describes what is known about the responsiveness of neutrophils to cytokines utilizing the common cytokine receptor gamma chain (␥c) such as IL-2, IL-4, IL-7, IL-9, IL-15 and IL-21, which were thought to activate mainly B, T, or NK cells. According to the results published to date, IL-4 and IL-15 seem to represent the more potent agonists for neutrophils. Further studies are, however, necessary to better elucidate the in vivo functional significance of the interactions between neutrophils and ␥c agonists. The important issue concerning neutrophil participation in chemokine networking has been addressed by Cheng and Kunkel. Specifically, these authors outline the more recent advances on the range of chemokines and chemokine receptors involved in neutrophil recruitment during acute inflammation. Furthermore, they update the reader on the ability of neutrophils to produce chemokines [2], another recently discovered function of neutrophils that reinforces the notion

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XII

that these cells may greatly influence not only early cell trafficking and activation during the inflammatory response but also the ultimate progression or resolution of inflammation. Denkers et al. discuss, in the subsequent chapter, the importance of neutrophils as an in vivo source of IL-12 and other cytokines during infectious diseases. The data obtained by using Toxoplasma gondii and its antigens or Candida albicans infection and other animal models have provided compelling evidence that neutrophil-derived cytokines during specific infectious diseases may play a critical role in determining the outcome of the infections. In addition, convincing results have made it clear that neutrophils may also modulate T cell effector function and may exert immunoregulatory effects on T cell subset selection, particularly with regard to Th1 generation. By speculating on possible models explaining how this might occur, Denkers et al. suggest that neutrophils are in a position to play a master role in orchestrating the innate immune response, and influencing, in turn, the development of acquired immunity. Serhan and Levy, in the context of the mechanisms carried out to control the inflammatory processes, cover the latest results regarding the effects of the novel class of endogenous anti-inflammatory lipid mediators that include lipoxin, 15-epi-lipoxin and related molecules. These products are generated to dampen the host response, to prevent neutrophil-mediated tissue injury and to orchestrate resolution. Lipoxins and related analogs represent useful tools to evaluate the potential of pharmacological manipulation of the inflammatory process as a means to develop new and selective anti-inflammatory therapies with reduced unwanted toxic side effects. I am sure we will hear more and more about these lipids in the future. Three more original articles then follow. The first one, written by Lindbom, reports the most recent advances on the molecular mechanisms by which activated neutrophils can cause increased vascular permeability during acute inflammation. The author discusses the mediators and molecular interactions that are potentially involved in mediating neutrophil-induced alteration in vascular permeability. The second one, written by Benelli et al., deals with a novel and in a certain sense unpredictable function of neutrophils, i.e. their ability to directly regulate the angiogenic process. On taking into account the information currently available, these researchers speculate on the environmental circumstances, occurring for instance during inflammation, which could render the neutrophil either a potential inducer of new vascularization, or, alternatively, a repressor of it. Finally, in the third article, Di Carlo et al. illustrate the role of neutrophils in tumor biology. They outline that tumor-associated neutrophils appear to greatly influence either in a positive or in a negative manner the dynamics that lead to tumor development. As in the case of angiogenesis, it is emerging that neutrophils can operate in two ways: they can behave very

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actively in immunosurveillance, but they may also sometimes favor malignant growth and progression via the release of growth/angiogenic factors and matrix-degrading enzymes. The authors highlight the real possibilities of transferring experimental results to a clinical setting, as they speculate that the neutrophil anticancer potential may prove to be an effective way to fight cancer. The last article, compiled by Edwards et al., covers in detail what is known about the molecular processes that regulate neutrophil cell death and survival. It is well known that neutrophils undergo spontaneous apoptosis within 24 h after leaving the bone marrow and that proinflammatory cytokines can delay this apoptosis. Edwards et al. report on the most recent information on the morphological and molecular features of apoptotic neutrophils, on the extracellular factors that regulate neutrophil apoptosis and on the molecular control of neutrophil apoptosis. Understanding completely all these mechanisms may certainly help to develop better therapeutic strategies for intervention in those inflammatory disorders in which neutrophils contribute to disease pathology. In this book, I have deliberately avoided including a chapter covering the topic regarding the capacity of neutrophils to produce cytokines. Exhaustive reviews are already available [3–5]. However, this does not mean that this area of research failed to advance. Actually, there have been a number of remarkable insights. First of all, I would like to emphasize that the in vivo studies are rapidly increasing in number, and, more importantly, they are reproducing the in vitro findings. Interestingly, these in vivo studies are elucidating the pathophysiologic meaning of neutrophil-derived cytokines, thus confirming that this function constitutes an important aspect of neutrophil biology. Furthermore, recent exciting observations have greatly extended the list of cytokines that, under specific experimental conditions, are produced by neutrophils in vitro. Let me mention here the reports demonstrating, for instance, that neutrophil could express and produce (1) macrophage inflammatory protein-3␣ (MIP-3␣/ CCL20) and MIP-3␤/CCL21 [6], two chemokines that have been suggested to play a fundamental role in dendritic cells trafficking to mucosal surfaces and lymphoid organs, (2) hepatocyte growth factor (HGF) [7, 8], a cytokine that plays key roles in angiogenesis and in the attenuation of disease progression as an intrinsic repair factor, (3) endothelin-1 [9], a peptide with potent vasoconstrictive capabilities, inotropic and mitogenic actions, and a relevant role in the maintenance of vascular tone and blood pressure in healthy subjects, and (4) B lymphocyte stimulator (BLyS) [10], a novel member of the TNF ligand superfamily that is fundamental for B cell maturation and survival. The possible implications of all these observations need to be explored by in vivo studies, but it is clear that they might have an important biological significance in health and disease.

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I am confident that the overviews provided in this volume are comprehensive and I hope that they will stimulate more research to contribute towards the advancement in the field of neutrophils. I am fully convinced that there are still many unsolved issues related to the properties of neutrophils and their contribution to tissue homeostasis. Let me thank all the colleagues who have so generously contributed to this volume. They agreed with great enthusiasm to participate to this project, making the task to assemble the whole book very easy. It has been a pleasure for me to interact with them. Finally, I also wish to thank the staff at Karger for their patience and valuable assistance in bringing this volume to light. Marco A. Cassatella

References 1 2 3 4 5 6

7 8

9 10

Witko-Sarsat V, Rieu P, Descamps-Latscha B, Lesavre P, Halbwachs-Mecarelli L: Neutrophils: Molecules, functions and pathophysiological aspects. Lab Invest 2000;80:617–653. Scapini P, Lapinet-Vera JA, Gasperini S, Calzetti F, Bazzoni F, Cassatella MA: The neutrophil as a cellular source of chemokines. Immunol Rev 2000;177:195–203. Cassatella MA: Cytokines Produced by Polymorphonuclear Neutrophils: Molecular and Biological Aspects. Berlin, Landes Co., 1996, pp 1–199. Cassatella MA: Neutrophil-derived proteins: Selling cytokines by the pound. Adv Immunol 1999; 73:369–509. Matsukawa A, Yoshinaga M: Neutrophils as a source of cytokines in inflammation. Histol Histopathol 1999;14:511–516. Scapini P, Laudanna C, Pinardi C, Allavena P, Mantovani A, Sozzani S, Cassatella MA: Neutrophils produce biologically active macrophage inflammatory protein-3␣(MIP-3␣)/CCL20 and MIP-3␤/CCL19. Eur J Immunol 2001;31:1981–1988. McCourt M, Wang JH, Sookhai S, Redmond HP: Activated human neutrophils release hepatocyte growth factor/scatter factor. Eur J Surg Oncol 2001;27:396–403. Grenier A, Chollet-Martin S, Crestani B, Delarche C, El Benna J, Boutten A, Andrieu V, Durand G, Gougerot-Pocidalo MA, Aubier M, Dehoux M: Presence of a mobilizable intracellular pool of hepatocyte growth factor in human polymorphonuclear neutrophils. Blood 2002;99:2997–3004. Cambiaggi C, Mencarelli M, Muscettola M, Grasso G: Gene expression of endothelin-1 (ET-1) and release of mature peptide by activated human neutrophils. Cytokine 2001;14:230–233. Scapini P, Nardelli B, Nadali G, Calzetti F, Pizzolo G, Montecucco C, Cassatella MA: G-CSF-stimulated neutrophils are a prominent source of functional BlyS. J Exp Med, in press.

Marco A. Cassatella, Department of Pathology, Section of General Pathology, Strada le Grazie 4, I-37134 Verona (Italy)

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Cassatella MA (ed): The Neutrophil. Chem Immunol Allergy. Basel, Karger, 2003, vol 83, pp 1–23

Transcription Factor Activation in Human Neutrophils Alexandre Cloutier, Patrick Pierre McDonald Pulmonary Division, Faculty of Medicine, Université de Sherbrooke, Sherbrooke, Québec, Canada

Neutrophils are by far the most abundant leukocyte population, representing about 60% of all circulating leukocytes. They are terminally differentiated cells in which the apoptotic program is constitutive, and are best known for their role as professional phagocytes. Neutrophils are typically the first leukocytes to migrate into inflammatory sites, where they accumulate in large numbers, and unleash a variety of cellular responses against microorganisms and foreign particles. Foremost amongst these responses is the phagocytosis of non-self targets and a microbicidal response involving the generation of oxygenderived reactive intermediates and the release of various lytic enzymes [1]. Activated neutrophils also synthesize lipid mediators such as leukotriene B4 (a neutrophil chemoattractant) and platelet-activating factor (a vasoactive lipid), which facilitate the extravasation and recruitment of additional granulocytes into inflamed tissues. As a result, neutrophils have been traditionally regarded as an important first line of defense against pathogens and other immunogenic material. In the last decade or so, another key facet of neutrophil biology has emerged, that is, the ability to express a sizeable number of genes whose products lie at the core of inflammatory and immune responses. These include growth factors, cell surface receptors, adhesion molecules, cytokines such as TNF␣, IL-1␤, IL-12, and TGF␤, and a wide array of chemokines such as IL-8, Gro␣, Mip-1␣/␤, Mip-3␣/␤, IP-10, MIG, I-TAC, and others [reviewed in 2]. The production of inflammatory cytokines and chemokines by neutrophils is

PPMcD is a Scholar of the Canadian Institutes for Health Research.

typically preceded by (and largely dependent upon) an accumulation of the corresponding mRNA transcripts [reviewed in 2]. In the particular case of IL-1␤, IL-8 and Mip-1␣, inducible gene expression was further shown by us and others to reflect an increased transcriptional activity [3–5]. Collectively, these studies have considerably furthered our appreciation of neutrophil function, and have prompted a growing number of investigators to begin elucidating the regulation of inducible transcriptional processes in these cells. The object of this review is to provide an overview of the current state of knowledge regarding the issue of transcription factor activation in the context of inducible gene expression in human neutrophils.

The STAT Family of Transcription Factors

The STATs (for signal transducers and activators of transcription) are cytoplasmic proteins that represent signaling endpoints for various cytokines, growth factors and hormones [6–8]. Not surprisingly, the inducible expression of a number of inflammatory genes is under the partial or full control of the STAT proteins. Seven STAT genes have been identified, termed STAT1, STAT2, STAT3, STAT4, STAT5A, STAT5B, and STAT6 [6, 9]. Additionally, various isoforms exist for each STAT family member, due to alternative splicing or posttranslational proteolytic processing (as in the case of STAT3 and STAT5). Upon cell stimulation, the STATs are rapidly recruited to SH2 domains within the cytoplasmic tail of cell surface receptors, and become tyrosine-phosphorylated by Janus kinases (JAKs) that are bound to cytokine receptors, or by an intrinsic receptor kinase activity in the case of growth factor receptors [10]. Tyrosinephosphorylated STAT proteins can then dimerize into various DNA-binding configurations; STAT1, STAT3, and STAT5A/5B can all form homo- or heterodimers, whereas STAT4 and STAT6 can only homodimerize [11]. By comparison, STAT2 does not homodimerize, but rather forms a complex with STAT1 and p48/IRF-9 [12–15]. STAT protein dimers are swiftly translocated to the nucleus, where they can bind specific DNA sequences in the promoter region of target genes; such binding occurs with varying affinities, depending on each dimer’s composition. For instance, the STAT1-STAT2-p48 complex recognizes an ISRE motif (for interferon-stimulated response element), whereas many other STAT dimers bind to variants of the GAS motif (for gammainterferon-activated sequences) [8]. Finally, many STAT proteins can become phosphorylated on serine residues (in addition to tyrosine residues); this is thought to contribute to STAT-driven gene transactivation [7, 16]. Signaling through the JAK/STAT cascade is under negative regulation by at least three distinct mechanisms. First, several cytosolic and nuclear protein

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tyrosine phosphatases were shown to inhibit JAK activity and STAT activity [17–24]. Another class of negative regulators includes the cytokine-inducible SH2 protein (CIS), and the SOCS proteins (for suppressors of cytokine signaling). The genes encoding CIS, SOCS-1, SOCS-2, and SOCS-3 are rapidly induced through the JAK/STAT pathway in response to cytokines, hormones, or growth factors, and the matching proteins inhibit the JAK/STAT in a negative feedback loop, thereby terminating signaling [25–28]. A third type of negative regulation involves the PIAS proteins (for protein inhibitors of activated STATs), which can directly interact with STAT dimers and interfere with their ability to bind DNA [29–31]. Activation of STAT Proteins in Neutrophils: The Early Studies and the Proteolysis Issue The first studies to address whether STAT proteins can be activated in neutrophils were published 5 months apart in 1995–1996. In one of them, Tweardy et al. [32] investigated the effect of several neutrophil agonists on this response. Whole-cell extracts from neutrophils disrupted by freeze-thaw cycles were analyzed in an electrophoretic mobility shift assay (EMSA) using a human serum-inducible element (hSIE/m67) oligonucleotide probe. A specific complex was induced in cells treated with G-CSF, but could not be supershifted using antibodies raised against individual STAT proteins. The authors concluded that granulocytes express a novel STAT-like protein, which they called STAT-G [32]. At about the same time, Bovolenta et al. [33] also reported that neutrophil extracts contain a G-CSF-inducible complex that can bind to a hSIE/m67 probe or to a 39-bp oligonucleotide spanning the ␥-interferon response region (GRR) of the Fc␥RI/CD64 promoter. In contrast with the previous study, however, they were able to identify STAT3 within the inducible complex, along with STAT1 as a minor participant. Bovolenta et al. [33] further showed that G-CSF treatment promotes the tyrosine phosphorylation of STAT3 in neutrophils, a finding which would be independently confirmed at a later date [34]. They concluded that G-CSF is a potent STAT3 activator in neutrophils. The discrepancy between the conclusions reached in these two early studies was only the first of many conflicting results which in the years to come would abound in studies of STAT activation in neutrophils, and which all share a common root. Bovolenta et al. [33] correctly attributed the difference to the procedures used to prepare neutrophil extracts. On the one hand, nitrogen cavitation (which they used) was already known as a cell disruption procedure that is gentle enough to preserve the integrity of neutrophil organelles such as nuclei and cytoplasmic granules [35]. On the other hand, repeated freeze-thaw cycles had often been used to break open the protease-rich neutrophil granules [35, 36]. Thus, should the STATs be vulnerable to the action of neutrophil

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proteases, then the cell disruption procedure could greatly affect the ensuing results. And this is precisely what subsequent studies would establish. In a follow-up to their original article, the Tweardy laboratory reported that the G-CSF-inducible complex could be partially supershifted using different anti STAT3 antibodies than the one used in their prior study, and a STAT3␣ band was now detectable by immunoblot in the same nuclear extracts [37]. A weaker 72-kD species was also detected, whose abundance was dramatically increased if the cells were disrupted in the absence of the potent serine protease inhibitor, DFP (as in their previous study). Together, these results strongly suggested that the STAT3 form(s) present in various extracts from freeze-thaw disrupted neutrophils was partially proteolyzed. In a paper entirely dedicated to the issue of transcription factor degradation by neutrophil proteases which was published at the same time, we demonstrated that classical cell disruption procedures (i.e. detergent lysis, successive freeze-thaw cycles) indeed result in the partial degradation of all STAT proteins examined (i.e. STAT1, STAT3, STAT5), even in the presence of numerous protease inhibitors [38]. Expectedly, degraded STAT proteins or complexes were found to migrate faster, both on SDS-PAGE and in native gels, and sometimes eluded detection altogether when the antibodies used happened to recognize a proteolyzed epitope. On the contrary, the integrity of STAT proteins was consistently preserved if neutrophils were instead disrupted by nitrogen cavitation [38], so that the various STAT proteins remained fully reactive with all antibodies tested. Collectively, these studies greatly emphasize the necessity to shield transcription factors from the action of endogenous proteases, especially when working with a cell type known to be particularly replete with proteolytic enzymes. Activation of STAT Proteins by Colony-Stimulating Factors in Human Neutrophils Aside from documenting the problematic issue of STAT proteolysis in neutrophil studies, the reports presented in the previous section have shown that G-CSF, a critical regulator of granulopoiesis and of neutrophil maturation, can induce the phosphorylation and nuclear recruitment of STAT3 and of STAT1 in human neutrophils. Although a direct link between this phenomenon and the induction of target genes has yet to be demonstrated in neutrophils, there exist at least two scenarios which would fit the bill. The inducible expression of the high-affinity IgG receptor, CD64, is known to be largely STAT-dependent in other leukocytes, and CD64 mRNA rapidly accumulates following the G-CSF-mediated STAT activation in neutrophils [33]. Similarly, both SOCS-1 and SOCS-3 are rapidly induced by G-CSF in neutrophils [39], and SOCS protein expression is known to be a STAT-driven process [40].

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GM-CSF is another growth factor which exerts numerous actions in mature neutrophils, including the priming of many neutrophil responses, the induction of several inflammatory molecules, and the inhibition of constitutive apoptosis. Investigation of the signaling pathways underlying these actions of GM-CSF led to the realization that much like G-CSF, GM-CSF also has the ability to activate STAT proteins in neutrophils. In this regard, Brizzi et al. [41] initially reported that in response to GM-CSF, both STAT1 and STAT3, as well as the upstream kinase, JAK2, rapidly become tyrosine phosphorylated. Accordingly, GM-CSF promoted the formation of an hSIE-binding complex in EMSA that could be supershifted using antibodies to either STAT1 or STAT3 [41]. Noteworthy is that the GM-CSF-induced complex migrated faster than an authentic STAT1/STAT3 heterodimer [41]; given that neutrophil extracts were prepared following detergent lysis in the presence of few protease inhibitors, this observation indicates that STAT proteins were partially proteolyzed, as we would later establish [38]. The ability of GM-CSF to activate JAK2 was confirmed by Al-Shami et al. [42], who further showed that both STAT5B and STAT3 become tyrosine-phosphorylated under these conditions. Accordingly, DNA affinity precipitation of neutrophil extracts using a GAS motif led to the detection of STAT5B by immunoblot, indicating that tyrosine phosphorylated STAT5 can bind a specific DNA sequence [42]. By comparison, STAT1 was constitutively phosphorylated in unstimulated cells (an unusual occurrence), which is perhaps why no increase in STAT1 phosphorylation was noted in response to GM-CSF. We independently confirmed that GM-CSF induces the nuclear mobilization and DNA binding activity of STAT5 in neutrophils [38]. A similar effect of GM-CSF was noted in the case of STAT3 and (to a lesser extent) STAT1 ([38] and our unpubl. data). More recent publications further confirmed the ability of GM-CSF to induce JAK2 and STAT3 tyrosine phosphorylation, and to promote the DNA binding of complexes containing STAT1, STAT3, and STAT5 [34, 43, 44]. One of these studies also confirmed our earlier demonstration that detergent lysis of neutrophils in the presence of few protease inhibitors results in a truncated STAT5 protein that no longer reacts with C-terminal antibodies [38, 43]. Some novel findings were also reported. For instance, Kuroki and O’Flaherty [34] showed that in response to GM-CSF, STAT3 becomes phosphorylated not only on tyrosine residues, but also on serine residues. In another recent study, Epling-Burnette et al. [44] provided evidence for a link between the effect of GM-CSF on neutrophil survival and its ability to activate the JAK/STAT pathway. They reported that pretreating the cells with AG-490 (a selective inhibitor of JAK2 and JAK3) interfered with the ability of GM-CSF to induce STAT3 tyrosine phosphorylation, and attenuated its antiapoptotic effect.

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Collectively, the above studies have established that in neutrophils, GM-CSF can activate several STAT proteins, as well as the upstream kinase, JAK2. Several lines of evidence also indicate that STAT5 probably represents the predominant target of GM-CSF action in neutrophils, with STAT3 a close second. This said, the actual impact of STAT activation on GM-CSF-elicited gene expression remains to be convincingly demonstrated in neutrophils. Some potential candidates include anti-apoptotic proteins, as indicated above [44]. In this regard, GM-CSF is known to stimulate the mRNA and protein expression of Mcl-1 [44–46], and it was recently reported that a JAK inhibitor (AG-490) could partially prevent this response [44]. Other STAT-dependent genes that are induced by GM-CSF in neutrophils are CIS and SOCS-3, as recently reported by Cassatella et al. [47]. Activation of STAT Proteins by IFNg in Human Neutrophils The pleiotropic cytokine, IFN␥, is a well-known STAT activator in many cell types. In neutrophils, IFN␥ directly induces several genes, and modulates the expression of numerous others; such is the case, notably, of many inflammatory cytokine/chemokine genes [reviewed in 2]. A common characteristic of all these genes is that their induction is known to depend, at least partially, on STAT activation – as demonstrated in other cellular models. The first study to investigate whether IFN␥ can activate STAT proteins in neutrophils reported that nuclear extracts from IFN␥-treated cells contained an inducible complex that migrated much faster in EMSA than authentic STAT1 homodimers from IFN␥stimulated HepG2 cells, and that failed to react with a C-terminal anti-STAT1 antibody [32]. These characteristics raised the possibility that STAT1 might have been degraded; this possibility was considerably strengthened when it later turned out that in the same study, G-CSF treatment of neutrophils led to the detection of partially degraded STAT3-containing complexes, as detailed in a previous section of this chapter. When we reinvestigated the matter, we therefore ensured that neutrophil extracts were prepared under conditions that avoid the damage caused by endogenous proteases (i.e. nitrogen cavitation in the presence of an elaborate antiprotease cocktail). In EMSA analyses, IFN␥ was found to rapidly promote the binding of a doublet to the gamma-interferon response region (GRR) of the Fc␥RI promoter, and the inducible complex was indistinguishable from that induced by IFN␥ in autologous monocytes [48]. In extracts from both cell types, the doublet could be supershifted by various antibodies raised against STAT1 (but not against other STAT family members), and could also be displaced by antiphosphotyrosine antibodies [48]. These results clearly demonstrated that IFN␥ can activate STAT1 in neutrophils. In another study published the same year, we confirmed that the difference between our data and that of the earlier study reflected the different cell disruption procedures employed (nitrogen

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cavitation in our case, as opposed to detergent lysis in the presence of few protease inhibitors). Indeed, we showed that classical procedures (detergent lysis, sonication, freeze-thaw) invariably resulted in the detection of a truncated STAT1 band by immunoblot, and in faster-migrating native complexes which no longer reacted with some anti-STAT1 antibodies [38]. Subsequent studies independently confirmed that IFN␥ has indeed the ability to activate STAT1 in neutrophils [44], and that STAT1 becomes tyrosine phosphorylated in response to IFN␥ in neutrophils [47]. The impact of IFN␥-mediated STAT1 activation on inducible gene expression remains to be formally demonstrated in neutrophils. Nevertheless, it is to be expected that the rapid accumulation of Fc␥RI/CD64 mRNA following neutrophil exposure to IFN␥ is due in large part to STAT1 activation by the latter, based on observations made in other inflammatory cells. Similarly, both CIS1 and SOCS-3 are rapidly induced by IFN␥ in neutrophils [47], and the IFN␥-elicited induction of CIS/SOCS proteins has been shown to be STAT-dependent in other cell types [49, 50]. Finally, the induction of several inflammatory cytokines and chemokines (including IL-12, IP-10, MIG, I-TAC, and others) in neutrophils requires IFN␥ as a costimulus [2], and on the basis of studies conducted in other leukocytes, a role for IFN-activated STAT1 (and perhaps STAT2 as well) can be envisaged. Activation of STAT Proteins by IL-10 in Human Neutrophils IL-10 is an important anti-inflammatory cytokine which downregulates the inducible expression of many proinflammatory cytokines in neutrophils [51–56]. Up to very recently, however, there was no indication that IL-10 could exert direct effects toward neutrophils (as opposed to modulating ongoing responses). Because IL-10 is known to induce Fc␥RI expression in monocytes via its ability to activate STAT proteins [57–59], we investigated whether IL-10 might exert a similar action towards neutrophils. Disappointingly, IL-10 failed to induce either STAT activation or Fc␥RI gene expression [48]; the latter (negative) finding was also reported by another group [60]. This led us to propose that in neutrophils, IL-10 receptors were either insufficiently expressed or somehow dysfunctional with respect to STAT activation [48]. The issue was recently revisited by the group of M.A. Cassatella [61], who showed that whereas circulating neutrophils abundantly express IL-10R2 on their surface, their IL-10RI surface expression is very low. More importantly, neutrophils could be made to express substantial levels of surface IL-10R1 after a 4-hour culture, and this effect was further enhanced in the presence of LPS [61]. Under these conditions, neutrophils now responded to IL-10 in terms of STAT1 and STAT3 tyrosine phosphorylation and DNA binding in EMSA [61]. Thus, IL-10 can be an effective STAT inducer in human neutrophils, provided that both chains of its receptor are expressed.

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Because IL-10 is known to induce very few genes in neutrophils, the consequences of its ability to activate STAT proteins are not easy to fathom. This said, the ability of IL-10 to induce SOCS-3 gene and protein expression was greatly enhanced in neutrophils expressing the IL-10RI, relative to freshly isolated cells, in which the STATs are not activatable by IL-10 [47, 61]. Thus, it seems reasonable to suggest that the IL-10-elicited induction of SOCS-3 expression might represent, at least in part, a STAT-driven process – as observed in other cell types [62]. Activation of STAT Proteins by Other Agents in Human Neutrophils Various other stimuli have been shown to activate the STATs in neutrophils. Amongst them, prolactin was shown to promote the tyrosine phosphorylation of STAT1, and to induce the formation of a specific GAS-binding complex in EMSA [63]. The nature of the complex remains uncertain, as supershift assays were inconclusive; a probable reason for this outcome is that neutrophil extracts were prepared using a detergent-based kit [63]. Nevertheless, prolactin rapidly induced the expression of SOCS-2 and IRF-1 in neutrophils [63], consistent with a potential role for STAT activation in gene expression. Similarly, pituitary growth hormone was shown to induce the tyrosine phosphorylation of both JAK2 and STAT3 in neutrophils [64]. Finally, more classical neutrophil agonists such as formylated peptides (fMLP), C5a, and phorbol esters were shown to induce the phosphorylation of STAT3 on serine, but not on tyrosine residues [34]. The biological significance of STAT3 serine phosphorylation by these stimuli, however, remains poorly understood. Concluding Remarks and Future Directions A recurring theme in many of the studies addressing STAT activation in neutrophils has been the detection of truncated or unidentifiable STAT proteins and native complexes; this has repeatedly led to the conclusion that new isoforms must be expressed in neutrophils. In all cases, however, neutrophil disruption was carried out using procedures that cause the solubilization of intracellular granule contents, something which can have dire consequences, given that all STAT proteins can be cleaved by neutrophil proteases ([38] and our unpubl. data). Thus, the question of whether novel STAT isoforms are expressed in neutrophils remains far from certain. Future studies therefore ought to take advantage of protocols which make it possible to circumvent the adverse effects of protease release, so that unequivocal conclusions can be drawn. Beyond these (essentially technical) considerations, further studies are needed to extend our understanding of STAT activation in neutrophils. First, the activation of STAT family members other than STAT1/STAT3/STAT5 needs to be further investigated, especially since all STAT proteins are expressed in these

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cells (our unpubl. data). In this regard, indirect evidence for STAT4 activation in neutrophils was recently reported [65]. Similarly, a more thorough characterization of the conditions leading to STAT serine phosphorylation could help better understand the potential function of this response in primary cells such as neutrophils. Other promising research avenues include the activation of JAK/Tyk by neutrophil agonists, and the negative regulation of the JAK/STAT pathway in neutrophils (by SOCS and PIAS proteins, as well as by phosphatases), which has only begun to be investigated. Finally, it is likely that STAT activation is involved in the expression of several neutrophil genes. Examples include surface receptors such as CD64, whose gene is induced by several STAT activators [33, 47, 48]; the antiapoptotic protein, Mcl-1, which is inducible by GM-CSF [44–46], and the CIS/SOCS family members, which are induced by most STAT-activating stimuli in neutrophils [39, 47, 61]. However, the actual impact of STAT activation on inducible gene expression still awaits a formal demonstration in neutrophils.

The NF-␬B Family of Transcription Factors

The ubiquitously expressed NF-␬B transcription factor is widely recognized as a central regulator of inflammatory and immune processes. This transcription factor exists as a dimer whose constituent proteins belong to the NF-␬B/Rel family. All of these proteins have the ability to bind DNA through a conserved motif, the Rel homology domain, which is also required for homoand heterodimerization, resulting in multiple variants of NF-␬B [66]. Individual members comprise the p65/RelA, c-Rel, and RelB proteins, as well as the products of the Nfkb1 and p52 Nfkb2 genes, whose mature forms (p50 and p52) are derived from larger precursors. Latent NF-␬B dimers are complexed to cytoplasmic inhibitor proteins, collectively termed I␬B proteins. Upon cell stimulation, multiple intracellular signals rapidly converge on a multimeric I␬B kinase complex (IKK), which phosphorylates I␬B on N-terminal serine residues [67, 68]. This is soon followed by I␬B ubiquitination, a process which leads to its proteolytic degradation by the proteasome [69]. This in turn unmasks the nuclear localization signal of NF-␬B constituent proteins, which can then enter the nucleus and induce the transcription of target genes. Amongst the latter, the gene encoding I␬B-␣ is very rapidly expressed [70, 71], and a portion of the newly synthesized inhibitor protein accumulates in the nucleus where it can dissociate NF-␬B dimers from DNA, and retarget them to the cytoplasm [72]. Other examples of ␬B-dependent genes are the ones encoding cytokines such as TNF␣, IL-1␣/␤, IL-1ra, IL-12; chemokines such as IL-8, Gro␣, Mip-1␤, and IP-10, or surface receptors such as ICAM [73, 74]. Because

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all of these genes are rapidly expressed by neutrophils in response to stimuli such as LPS or TNF␣ [2], which are known NF-␬B activators [73], it was only a matter of time before researchers would start investigating the ability of neutrophils to activate NF-␬B. Expression and Activation of NF-kB Pathway Components in Human Neutrophils In the first study to address this issue, we extensively characterized the expression and activation of various components of the NF-␬B system in human neutrophils [75]. We found that neutrophils express NF␬B1/p50, p65/RelA and c-Rel, whereas RelB and NF␬B2/p52 (or its precursor, p100) were undetectable. In contrast to most other cell types, in which the bulk of NF-␬B/Rel proteins is cytoplasmic, unstimulated neutrophils featured equivalent amounts of the NF-␬B/Rel proteins in the cytoplasm and nucleus [75] – an observation that was independently confirmed at a later date [76]. We also showed that the three NF-␬B/Rel proteins present in neutrophils form various heterodimers (i.e. p50/RelA, p50/c-Rel or p65/c-Rel) that are all physically associated with I␬B-␣ in resting cells [75]. Together, these observations clearly indicated that the basic requirements for a functional NF-␬B cascade were fulfilled in human neutrophils. Indeed, stimulation of these cells with LPS, TNF␣, or IL-1␤ led to the rapid loss of I␬B-␣ and concomitant nuclear accumulation of NF-␬B/Rel proteins, resulting in a transient induction of NF-␬B DNA binding activity [75]. By supershift analysis, the major inducible complex was shown to contain p50, RelA, and (to a lesser extent) c-Rel. Within 20–30 min of stimulation, an accumulation of I␬B-␣ mRNA transcripts was detectable, and the I␬B-␣ protein was re-expressed shortly thereafter. This correlated with the termination of nuclear NF-␬B DNA binding activity [75]. Other neutrophil agonists were also found to activate NF-␬B in these cells – in particular, chemoattractants such as platelet-activating factor, the formylated tripeptide, fMLP, and leukotriene B4. However, they proved to be weaker NF-␬B activators than LPS or TNF␣, and they also elicited a more delayed response [75]. Because all the aforementioned stimuli can induce the expression of I␬B-␣ and of many other ␬B-dependent genes in neutrophils (in particular, those encoding inflammatory cytokines and chemokines), and in view of their ability to activate NF-␬B in these cells, we proposed that NF-␬B activation must underlie their action towards human neutrophil gene expression. Conversely, neutrophil modulators that do not directly induce inflammatory cytokine expression (such as GM-CSF, G-CSF, IFN␣, IFN␥, IL-8, and IL-10) did not activate NF-␬B in these cells [75]. In a study published at about the same time, Browning et al. [77] reported that fMLP activates NF-␬B in human monocytes, but not in neutrophils, even

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after stimulation with LPS or TNF. Because p50 and RelA were barely detected in neutrophil extracts, these authors attributed the lack of NF-␬B activation to insufficient amounts of p50 and RelA [77]. However, it must be pointed out that neutrophil extracts were prepared in the presence of a single protease inhibitor (PMSF), which was used at a low concentration, and in a paper that appeared the following year, we showed that NF-␬B/Rel proteins suffer extensive proteolytic degradation under these conditions [38]. This outcome turned out to be a general consequence of neutrophil disruption by conventional procedures, whereas disruption of neutrophils by nitrogen cavitation consistently resulted in the recovery of intact NF-␬B proteins and DNA-binding complexes, which were invariously recognized by antibodies directed at various parts of the proteins [38, 75]. Thus, the apparent discrepancy between our initial study and that of Browning et al. stemmed from the different cell disruption procedures used, and the consequences thereof on neutrophil NF-␬B components. A number of subsequent studies, by us and others, confirmed the ability of neutrophils to activate NF-␬B under various conditions. In particular, the activation NF-␬B by bacterial LPS or TNF has been abundantly corroborated [76, 78–92]. In one of those studies, Sugita et al. [81] made the interesting observation that NF-␬B activation by LPS varies significantly depending on the origin of the LPS used, and (to a lesser extent) on the presence or absence of serum. In addition to the aforementioned stimuli, several other agents were described as NF-␬B activators in neutrophils. For instance, we reported that IL-15 (but not IL-2) induces NF-␬B activation and IL-8 production in these cells, albeit to a lesser extent than LPS [79]. In another study, we showed that the phagocytosis of yeast particles also leads to NF-␬B activation, an effect which was enhanced by opsonization of the yeast particles with IgG [78]. In agreement with these findings, Wakshull et al. [87] later reported the activation of NF-␬B in neutrophils ingesting PGG-glucan, a complex sugar derived from the cell wall of the yeast Saccharomyces cerevisiae. The ability of neutrophils to activate NF-␬B in response to phagocytic activity was also observed during the uptake of opsonized Staphylococcus aureus [82]. Interestingly, the authors of that study also reported that disrupting neutrophils by detergent lysis resulted in an NF-␬B DNA-binding activity that migrated faster than genuine p50/RelA dimers, and that could not be supershifted. Mixing these neutrophil extracts with T cell extracts also led to the degradation of NF-␬B complexes present in the latter. By contrast, preparing neutrophil extracts in the presence of the natural serine protease inhibitor, ␣1-antitrypsin, yielded NF-␬B complexes that had an almost normal mobility in EMSA, and that were partially reactive with an anti-RelA antibody [82]. These observations confirm our data showing that failure to counter the action of endogenous neutrophil proteases results in the degradation of NF-␬B constituents [38].

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Finally, two recent studies by Vancurova et al. [91] added new insights to our current understanding of the NF-␬B system in neutrophils. In one instance, they found that TNF induces NF-␬B more potently in neutrophils from newborn individuals, relative to neutrophils from adult donors; this correlates well with the elevated production of IL-8 and IL-1␤ reported in neonatal neutrophils [93, 94]. These authors also showed that dexamethasone inhibits NF-␬B induction by TNF in both newborn and adult neutrophils [91]; we observed a similar effect of dexamethasone in neutrophils from adult donors (our unpubl. data). In a later study, the same group demonstrated that I␬B-␣ is distributed in approximately equal amounts between the cytoplasm and the nucleus of neutrophils [76] – in stark contrast to most other cell types. Moreover, TNF stimulation led to the degradation of I␬B-␣ in both cellular compartments, in addition to its known ability to promote the nuclear recruitment of NF-␬B/Rel proteins [76]. This effect of TNF could be partially blocked by MG-132, a proteasome inhibitor that is known to inhibit NF-␬B [95], as well as by the PKC␦ inhibitor, rottlerin [76]. Regulation of NF-kB Activity by Reactive Oxygen Derivatives in Neutrophils Endogenous reactive oxygen intermediates (ROI) have been implicated in the regulation of NF-␬B activation in several cellular models [reviewed in 96, 97]. Because neutrophils probably produce more ROI than any other cell type, they must be particularly well protected from the adverse effects of ROI, and indeed, they have been shown to resist high concentrations of exogenous hydrogen peroxide [98]. Thus, transcription factor activation might not be substantially affected by endogenous ROI in neutrophils. In a recent study, Carballo et al. [86] examined the effect of H2O2 on calcineurin activity and NF-␬B activation in neutrophils. Following pretreatment of the cells with a catalase inhibitor, AMT (3-amino-1,2,4-triazole), exogenous H2O2 inhibited both processes. Though the authors proposed that H2O2 exerts its effect on NF-␬B through its ability to inhibit calcineurin activity, their data shows that calcineurin was unaffected by H2O2 if catalase was not deliberately inhibited [86]. This would imply that under these conditions, H2O2 does not affect NF-␬B either. Unfortunately, this was not addressed experimentally. In this regard, however, Vollebregt et al. [82] reported that the activation of NF-␬B observed in phagocytosing neutrophils was unchanged in the presence of various oxidant scavengers (such as exogenous catalase, superoxide dismutase, or methionine), and that accordingly, exogenous H2O2 did not activate NF-␬B. We also observed that exogenous H2O2 fails to induce NF-␬B in neutrophils (our unpubl. data). While these results definitely support the notion that endogenous oxidants have little impact on NF-␬B in neutrophils, a recent study reached the opposite

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conclusion. Following the stimulation of whole blood, Zouki et al. [90] separated total leukocytes and used a FACS procedure to detect individual transcription factor subunits in the resulting nuclei. The authors gated on singlet or doublet nuclei (which purportedly represent PBMC and neutreuphil nuclei, respectively) to discriminate between the two leukocyte populations. Stimulation of whole blood with TNF␣ and IL-1␤ resulted in the nuclear accumulation of RelA in both neutrophils and PBMC [90]. Pretreatment of whole blood with pyrrolidine dithiocarbamate, an antioxidant that can function as an NF-␬B inhibitor [99], suppressed this cytokine-induced nuclear mobilization of RelA [90]. However, some aspects of the FACS procedure preclude unambiguous conclusions. First, although it is quite likely that the two nuclei populations do represent neutrophils and PBMC, it must be hoped that they are free from cross-contamination. For instance, any aggregation of PBMC nuclei would place them within the (widely distributed) ‘doublet’ population that contains neutrophil nuclei. This would be very problematic, given that the ability of PBMC to activate NF-␬B dwarfs that of neutrophils [75]. Similarly, a correlation between the data obtained using this FACS procedure, on the one hand, and either immunoblot detection of nuclear Rel proteins or EMSA analysis of specific NF-␬B binding, on the other hand, has yet to be demonstrated in neutrophils. Another major difficulty in interpretation arises from the fact that stimulations and inhibitor pretreatments were carried out in whole blood, from which leukocytes are later isolated. Thus, it cannot be excluded that the modulation of a given neutrophil function might result from factors being released by other cells during stimulation in whole blood. Indeed, Niwa et al. [92] reported that the TNF-induced NF-␬B activation observed in isolated neutrophils was only slightly inhibited following pyrrolidine dithiocarbamate pretreatment. In conclusion, while exogenous ROI such as H2O2 do not seem to affect NF-␬B activation in neutrophils, some uncertainty subsists concerning whether endogenous ROI can affect the ability of neutrophils to activate NF-␬B. Clearly, further work is required to definitively settle the issue. Potential Role of NF-kB in Neutrophil Apoptosis A singular characteristic of neutrophils is that they undergo spontaneous apoptosis. Whereas this process and its modulation by various agents have been extensively studied in neutrophils [as recently reviewed in 100 and in this book], the molecular events involved remain poorly understood. Given its central role in the induction of various antiapoptotic proteins, the NF-␬B pathway could represent an important mechanism whereby neutrophil apoptosis is regulated, as described in many other cellular models [reviewed in 101]. This possibility was first explored by Ward et al. [83], who showed that various NF-␬B inhibitors increased the constitutive apoptosis of neutrophils.

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One such inhibitor, gliotoxin (a fungal metabolite), was found to synergistically increase the proapoptotic effect of TNF observed at early time points, and to suppress the survival effect of LPS [83]. Because both LPS and TNF are potent NF-␬B activators [75], these results indicated that gliotoxin might interfere with the protective effect of NF-␬B on apoptosis. Indeed, preincubation of neutrophils with gliotoxin was found to inhibit NF-␬B activation by either LPS or TNF [83]. The authors concluded that NF-␬B plays a crucial role in regulating neutrophil apoptosis. Similar conclusions were reached by other investigators. For instance, Niwa et al. [92] reported that although TNF did not affect neutrophil apoptosis at early time points (i.e. within 2 h of incubation), a proapoptotic effect of TNF was observed once the cells were rendered unable to activate NF-␬B. Similarly, Nolan et al. [84] reported that the inhibition of neutrophil apoptosis observed after a 12- to 18-hour incubation with TNF␣ or LPS was no longer observed in cells pretreated with the proteasome inhibitor, PSI-I, which was also found to inhibit the nuclear translocation of RelA in neutrophils. By contrast, Dunican et al. [102] reported that the inhibition of neutrophil apoptosis observed following an overnight incubation with TNF␣ was only slightly affected by prior treatment of the cells with SN50, a specific inhibitor of NF-␬B. This said, no evidence was presented by the authors to confirm that SN50 actually prevented NF-␬B activation by TNF in their hands. Finally, Ward et al. [85] recently examined the effect of prostaglandin D2 and of its metabolites on neutrophil apoptosis. Amongst the latter, both ⌬12PGJ2 and 15dPGJ2 were found to be proapoptotic in neutrophils, and to suppress both the LPS-elicited inhibition of neutrophil apoptosis and the LPS-induced degradation of I␬B-␣. Collectively, these various studies indicate that NF-␬B is likely to play a protective role in the context of neutrophil apoptosis. Concluding Remarks and Future Directions Whereas NF-␬B activation (and the related cellular events) has been relatively well characterized in neutrophils stimulated by various agonists, many questions subsist. For instance, a role for ROI in this process remains unclear. Another potentially important issue is whether NF-␬B constituent proteins become phosphorylated in response to neutrophil stimulation. A more general aspect of neutrophil activation that requires further investigation is the determination of the signaling steps upstream of I␬B degradation and/or NF-␬B subunit phosphorylation. Finally, the collective evidence gathered to date makes it very likely that NF-␬B activation plays an important role in inducible gene expression in neutrophils. However, the actual impact of NF-␬B on neutrophil gene expression still awaits a formal demonstration in neutrophils. Conversely, there is good evidence that NF-␬B plays a protective role in the context of spontaneous as well as modulated neutrophil apoptosis, but the

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targets of this NF-␬B action have not been identified yet. Thus, there is little doubt that the continuing study of NF-␬B activation in neutrophils should bring about significant new advances in the next few years.

Other Transcription Factors Potentially Involved in Neutrophil Activation

In addition to the STAT and NF-␬B families, other transcription factors have been reported to be expressed in neutrophils, and others still are likely to be present. These will be briefly covered in this section. The Ets Family of Transcription Factors Transcription factors of the Ets family share a DNA-binding domain spanning about 85 amino acids, termed the Ets domain, and bind to variants of a purine-rich DNA sequence. Among these factors, PU.1 is expressed specifically in hematopoietic lineages, and is critically involved in neutrophil differentiation, as demonstrated in a knockout mouse model [103]. High levels of PU.1 mRNA have been shown to be expressed in mature human neutrophils [104], and constitutive binding of PU.1 to its cognate sequence within the CD11b or gp91phox promoters has been shown in EMSA using neutrophil nuclear extracts [104, 105]. Although the resulting complex was specific, it migrated much faster than PU.1 complexes from monocyte extracts [104, 105], and only reacted with certain antibodies [104, 105]. Thus, the PU.1 recovered from the neutrophil extracts appears to be partially degraded. We indeed demonstrated that various transcription factors become degraded when neutrophils are disrupted by detergent lysis or other conventional means [38], as in the above studies [104, 105]. In summary, PU.1 is expressed in mature neutrophils, and constitutively binds DNA; whether this binding can be further induced or whether it can be modulated at all, remains uncertain at this stage. As a result a role for PU.1 in inducible gene expression by mature neutrophils remains speculative. The C/EBP Family of Transcription Factors The CCAAT/enhancer-binding proteins (C/EBP) are transactivators known for their involvement in the regulation of acute phase and inflammatory protein expression [106]. The family comprises several isoforms, which can homo- and heterodimerize through their basic leucine zipper region [107], or associate with other transcription factors, including NF-␬B [108, 109]. Activation of the C/EBP proteins involves their phosphorylation in a negative regulatory domain [110, 111]. In a similar manner to PU.1, some C/EBP family

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members have been described as essential for granulopoiesis and terminal neutrophil differentiation [112]. Additionally, C/EBP proteins are known to participate in the induction of several inflammatory mediators expressed in mature human neutrophils, notably cytokines and chemokines. In murine granulocyte-committed progenitor cells, C/EBP␣, ␤, and ␦ exist as nuclear proteins [113, 114]; nuclear C/EBP␣ has also been detected in human promyeloid HL-60 cells [114]. In addition, C/EBP␧ mRNA is strongly induced during the in vitro granulocytic differentiation of human primary CD34⫹ cells or of HL-60 cells [115]. While these findings suggest that human neutrophils must express at least some C/EBP proteins, the matter has not been thoroughly addressed to date. One group recently reported that in nuclear extracts from resting or activated neutrophils, the C/EBP␣ and C/EBP␤ proteins were undetectable, and that no binding to a C/EBP probe occurred in EMSA [80, 116]. However, these extracts were prepared following detergent lysis of neutrophils in the absence of elastase class protease inhibitors, a protocol which we have shown to entail the proteolysis of many transcription factors in neutrophils [38]. Thus, further studies are required to determine if C/EBP transcription factors are expressed and functional in neutrophils. The AP-1 Family of Transcription Factors The ubiquitously expressed AP-1 transcription factor typically consists of combinations of Jun and Fos family proteins, which bind as dimers to a common sequence. Dimerization occurs via a basic leucine zipper domain within the Jun/Fos proteins, which also makes it possible for them to associate with members of other transcription factor families [117, 118]. Upon cell stimulation, c-Jun and JunD are phosphorylated by JNK (for Jun N-terminal kinase), and c-Fos is phosphorylated by FRK (for Fyn-related kinase); these modifications have been shown to greatly enhance the transactivation potential of AP-1 complexes [119–122]. Several inflammatory mediators generated by neutrophils are known to be either AP-1-driven, or to require AP-1 for full promoter activity. These include TNF, IL-1, IL-1ra, IL-8, and ICAM [123–127]. Despite this, the question of whether AP-1 is functional in neutrophils has remained unanswered thus far. It has been reported that the genes encoding c-Fos, c-Jun, JunB, and JunD are constitutively expressed in neutrophils [128–130], and that they can be rapidly upregulated in response to various agonists [130–134]. Whether the corresponding proteins are expressed at all, however, remains to be demonstrated. In one study, Page et al. [80] observed a moderate increase in AP-1 binding in EMSA when neutrophils were stimulated for 60 min with LPS. Because no supershifts were performed, and because the inducible AP-1 complex was partially displaced by a nonspecific competitor, it is difficult to determine whether the DNA-binding activity was made up of

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AP-1 dimers or of other constituents. Clearly, this is another area that requires further investigation.

Conclusion

Advances made in the last decade have forced a reassessment of the traditional view according to which the role of neutrophils is limited to that of professional phagocytes, by clearly documenting the ability of neutrophils to express genes encoding numerous inflammatory mediators that are known to profoundly influence the course of inflammatory reactions. As a result, the study of how neutrophil gene expression is regulated has gained considerable momentum in recent years. Despite recent progress, however, several important questions remain. Perhaps the most critical issue is that of the actual impact of transcription factor activation on inducible gene expression in neutrophils. Other challenges include the identification of transcriptional regulators (other than NF-␬B and the STATs) that can be activated in neutrophils, the characterization of the upstream signaling pathways, and the elucidation of how transcriptional activation is negatively regulated in neutrophils. Together, such investigations will inevitably lead to a better understanding of transcriptional regulation in neutrophils – a goal whose importance is perhaps best illustrated by the fact that it could help identify new targets for pharmacological intervention. This in turn could be particularly relevant to numerous chronic inflammatory disorders in which neutrophils predominate over other inflammatory cells, and therefore represent a likely source for various inflammatory mediators that are found in high levels in these conditions.

References 1 2 3

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Patrick P. McDonald, PhD, Centre de recherche clinique, 3001, 12e avenue Nord, pièce 4849, Sherbrooke, Qué. J1H 5N4 (Canada) Tel. ⫹1 819 346 1110, ext. 14849, Fax ⫹1 819 564 5377, E-Mail [email protected]

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Cassatella MA (ed): The Neutrophil. Chem Immunol Allergy. Basel, Karger, 2003, vol 83, pp 24–44

Phenotypic and Functional Changes of Cytokine-Activated Neutrophils Carole Galligan, Teizo Yoshimura Laboratory of Molecular Immunoregulation, Center for Cancer Research, National Cancer Institute at Frederick, Frederick, Md., USA

Polymorphonuclear leukocytes (PMN) are the most abundant leukocyte population in the general circulation and comprise two thirds of the circulating leukocytes. PMN arise in the bone marrow (BM) from CD34
stem cells under the influence of regulatory cytokines [1]. The exact mechanism involved in PMN release from BM is not known; however, cytokines are involved in the process since in vivo administration of G-CSF, GM-CSF, IL-3, stem cell factor, IL-1 or IL-8 results in significant mobilization of PMN and hematopoietic progenitor cells (HPC) [1]. Clinically, cytokine treatment is employed in the collection of stem cells for BM transplantation [2]. Therefore, cytokines are involved in PMN maturation and the subsequent release into the bloodstream. Approximately 10 million PMN are produced and released into the bloodstream every minute in the average adult human, but the number of circulating cells remains constant [3]. This is because circulating PMN have a relatively short half-life (6–10 h), after which time they undergo apoptosis and are removed from the circulation. The phagocytic Kupffer cells of the liver play a major role in PMN removal [4, 5], and BM [5] can also function in this capacity. Therefore, the circulating pool of PMN is constantly replenished with mature PMN. Tissue injury induces a rapid mobilization of PMN out of the general circulation and into the tissues. The life span of the tissue-infiltrating PMN is considerably prolonged. Twenty-four hours after the onset of the inflammatory reaction, a large number of PMN are still present at the inflammatory site despite the cessation of PMN influx after 1–4 h [6]. Stimulation with GM-CSF, G-CSF, IFN-, IL-1, IL-2, IL-4, IL-6 and IL-15 can rescue PMN from preprogrammed apoptosis [7]. The specific genes required for PMN apoptosis

(or survival) are not known. The expression of PMN surface CD16 (FcRIIIb) has been correlated with PMN survival and function, and GM-CSF maintains CD16 surface expression [8]. PMN apoptosis also follows the downregulation of many surface markers [9]. Therefore, cytokines can circumvent the normal cycle of PMN apoptosis and removal and prolong their survival. In vivo, tissue-infiltrating PMN never exist in a cytokine-free environment; these cells are constantly surrounded by cytokines and require them for both maturation and survival. So far, the majority of functional studies have been performed on peripheral blood PMN that have previously been exposed to cytokines in the course of development. These PMN are traditionally referred to as resting PMN. PMN phenotype(s) and function(s) are dependent on the compartment the cells were isolated from, the age of the cells and the previous exposure to cytokine(s) stimulation. This is likely one of the major reasons that varying results have been observed. PMN are rapidly recruited to sites of inflammation and are vital participants in preprogrammed ‘innate’ host defenses. Cytokines can influence all aspects of PMN activation, including recruitment, release of cytokines, oxidative products, prostaglandins, and enzymes, thus contributing to innate immunity. In addition, they also help initiate adaptive immune responses as will be discussed. This review will attempt to summarize the effects of cytokine activation of PMN from a physiological and functional perspective.

Chemokine Receptors in PMN Mobilization and Activation

The chemokines are a family of cytokines with low molecular weights that predominately regulate the trafficking of leukocytes. There are currently 40 known chemokines and 16 chemokine receptors, with the majority of the chemokines binding to more than one receptor [10, 11]. The chemokine family can be subdivided into four groups based on the location of the first two cysteines: C, C-C, C-X-C and C-X3-C. Five C-X-C chemokine receptors and 11 C-C chemokine receptors have been identified to date [11]. Interaction of chemokines with their receptors on PMN not only induces directional migration but also activates PMN adhesion, degranulation and surface marker expression. Circulating human PMN express both CXCR1 and CXCR2, the receptors for IL-8/CCL8 and GCP-2/CXCL6 and IL-8/CCL8, GRO ,,/CXCL1–3, NAP-2/CXCL7, GCP-2/CXCL2 and ENA-78/CXCL5, respectively. Circulating PMN infiltrate into injured tissues after encountering these CXCR1 or CXCR2 ligands containing an ELR motif. In mice there is only one receptor for the ELR
CXC chemokines (CXCR2), and mice lacking the CXCR2 receptor do not mobilize PMN in response to peritoneal challenge with inflammatory

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Table 1. Cytokine regulation of chemokine receptor expression on PMN Chemokine receptors

Cytokine regulation

Ref. no.

CXCR1

↑ G-CSF ↓ TNF-

14 15

CXCR2

↑ G-CSF ↓ TNF- ↓ TNF-

14 15 17

CXCR4

↑ G-CSF in BM ↓ IFN- ↓ GM-CSF ↓ G-CSF

20 22 26 26

CCR1

↑ IFN- ↑ GM-CSF

22 28

CCR2

↑ in chronic inflammatory sites in vivo

30

CCR6

↑ TNF- ↑ IFN-

CX3CR1

↑ in infiltrating inflammatory leukocytes

106 106 35

stimulants [12]. CXCR1 and CXCR2 levels can be modulated by cytokine exposure (table 1). LPS [13, 14] and TNF- [15, 16] can downregulate both receptors, whereas another report states that TNF- selectively downregulates CXCR2 on human PMN in vitro [17]. In vitro exposure to G-CSF has been found to elevate levels of both CXCR1 and CXCR2 [14]. Therefore, cytokine exposure can alter PMN mobilization and activation in response to subsequent chemokine stimulation. Chemokine receptors also play a role in PMN release from the BM since CXCR4-deficient mice retained fewer PMN precursors in the BM [18]. Studies of G-CSF receptor knockout mice, partially reconstituted with normal stem cells, mobilized HPC in response to G-CSF, indicating a role for some functional G-CSF receptors in this process [19]. The mechanism is thought to be through G-CSF-stimulated elastase release by PMN, which in turn degrades the chemokine SDF-1/CXCL12, the ligand for CXCR4. It was therefore postulated that mobilization of HPC from the BM represents an escape from the effects of SDF-1/CXCL12 [20]. Further evidence suggests that mature PMN are involved in HPC release, since mice with antibody-induced neutropenia failed to mobilize HPC in response to IL-8/CXCL8 during the neutropenic stage, but mobilized HPC in response to IL-8/CXCL8 during the PMN

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recovery phase [21]. Therefore, functional PMN can secrete enzymes required for CXCR4-mediated HPC release from the BM. Although HPC express CXCR4, the expression of CXCR4 by circulating PMN is controversial. Several in vitro studies have shown that circulating PMN express CXCR4 [22], and migrate or flux calcium in response to SDF-1/ CXCL12 [23, 24], whereas another study did not observe these effects [25]. IFN- was reported to inhibit CXCR4 mRNA accumulation in some donors [22]. It was recently shown that the expression of CXCR4 was not detected in freshly isolated PMN, but it became apparent during in vitro incubation [26]. This spontaneous CXCR4 expression was suppressed most potently by IFN-, and also by IFN-, G-CSF and GM-CSF [26]. Cytokine-induced inhibition of CXCR4 expression in vitro contradicts the in vivo data that tissue-infiltrating PMN showed increased CXCR4 levels [27]. Since CXCR4 levels are modulated in PMN and many cytokines downregulate CXCR4, it has been suggested that PMN may use SDF-1/CXCL12 for baseline trafficking under normal, but not under inflammatory conditions. A definitive proof of this hypothesis awaits experiments in a PMN-specific conditional CXCR4 knockout animal, since systemic CXCR4 knockout is embryonically lethal and specific PMN defects cannot be assessed. In tissue-infiltrating, activated PMN, the expression of CXCR1 and CXCR2 is downregulated [27], but the expression of several CC chemokine receptors is upregulated. In vitro studies have shown that IFN- [22] or GM-CSF [28] upregulates CCR1 expression on human PMN. Since CCR1 expression is essential for PMN-mediated host defense in a murine system in vivo [29], induction of CCR1 appears to have a biological consequence. CCR2 expression was observed on rodent PMN isolated from chronic inflammatory sites in vivo [30] and these cells exhibited a chemotactic response to the agonist MCP-1/CCL2. CCR2 knockout mice showed impaired recruitment of PMN into lungs after intratracheal administration of CCL2 and LPS [31], but the recruitment of PMN in response to intraperitoneal thioglycollate was not a affected [32]. However, CCR2 expression has not been shown on PMN in vitro, suggesting that CCR2 expression by PMN may only occur during specific chronic inflammatory conditions. CCR3 expression has been reported to be induced on PMN by IFN-; however, previous reports of CCR3 expression on PMN are considered to be the result of eosinophil contamination [33]. PMN express low levels of CX3CR1 [34], and infiltrating inflammatory leukocytes, including PMN, displayed elevated levels of CX3CR1 in a rat glomerulonephritis model [35]. PMN also express the orphan chemokine receptor CCRL-2 in response to TNF- stimulation but the functional significance of this receptor is not known [Galligan and Yoshimura, unpubl. observations]. Thus, PMN express many different chemokine receptors either constitutively or

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in response to cytokines. Additional studies are necessary to elucidate the role of individual chemokine receptors on PMN recruitment/activation in inflammatory responses.

Cytokine Involvement in PMN Emigration

In order for PMN to be effective in countering invasive infectious agents, they must leave the general circulation and transmigrate through the endothelial cell (EC) layer and extracellular matrix (ECM) to arrive at the inflammatory site. Leukocyte transmigration is a multistep process involving cell rolling, activation, firm adhesion and transmigration [36–38]. The first step in leukocyte emigration is selectin-mediated cell rolling on the EC layer. This is followed by  integrin-mediated arrest of leukocytes on EC and subsequent transmigration through the EC barrier into the tissues. PMN rolling is mediated by both PMN- and EC-expressed selectins and carbohydrates [36]. L-selectin (CD62L), a transmembrane glycoprotein, contributes to physiological leukocyte rolling [39] and is rapidly shed from PMN cell surface upon activation [40]. Cytokine stimulation alters selectin expression on both PMN and EC. In vitro treatment of PMN with cytokines or proinflammatory mediators such as TNF-, IL-1, C5a, LTB4, phorbol ester or LPS induces L-selectin shedding [40]. Calmodulin, an intracellular calcium regulatory protein, also induced L-selectin shedding mediated by a cell surface metalloproteinase [41]. Cytokine-activated PMN presumably use a similar mechanism to induce L-selectin shedding. Elevated levels of serum L-selectin were also observed in vivo after G-CSF administration [42]. PMN rolling also involves the interaction of P-selectin expressed on activated endothelium and its counter-receptor P-selectin glycoprotein ligand-1 (PSGL-1) on PMN. P-selectin binding to PMN was lost under conditions that caused the release of proteinases, such as cathepsin G and elastase. fMLP, IL-8, or C5a induced integrinmediated stationary adhesion of PMN that were already rolling on a P-selectin-expressing platelet monolayer, and adherent PMN rapidly released elastase [43]. Elastase release was also observed from EC-adherent PMN, following stimulation with TNF- or IL-1 [44]. Both elastase and cathepsin G have a negative feedback effect and induce a loss of PMN PSGL-1 and inhibit P-selectin binding [45]. G-CSF also downregulates PSGL-1 expression on PMN [46]. Thus, cytokine stimulation is involved in PMN rolling and may also be involved in the transition from rolling to a stationary state. The net effect of these stimulants is to promote PMN transmigration into tissues. Integrins are normally present in an inactive state and stimulation is required to rapidly alter the configuration that allows adhesion. PMN primarily

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use 2 integrins for a strong, stationary adhesion to the EC surface. 2 integrins are known to be activated to become adhesive by outside-in signaling, and this change is mediated by chemokines [47]. Activated PMN, but not freshly isolated PMN, bind a specific antibody against cell surface CD18, and this capacity has been attributed to a conformational change in the activated integrin molecule [48]. Stimulation of PMN with a combination of fMLP, PF-4 and GM-CSF induces CD11b (CR 3) cleavage by a currently undefined serine protease [49], suggesting that cytokine stimulation of PMN may also be important in detaching PMN from the endothelium. CD11b has also been shown to be the major integrin involved in PMN migration through the epithelial cell layer [50]. PMN 1 integrins are induced on the cell surface in association with transendothelial migration, and cross-linking of 2 integrins has been reported to trigger this event [51]. Once through the EC layer, the PMN must traverse the ECM, and additional adhesion molecules, such as 1 integrins as well as collagen, fibronectin and laminin receptors, are likely involved in the process. PMN express the 1 integrin, very late antigen 9 (VLA-9), and cell surface expression of VLA-9 was upregulated following fMLP stimulation [52]. VLA-9 was one of the adhesion molecules involved in PMN migration through fibroblast layers in vitro, and may be involved in PMN migration through the ECM. Taken together, cytokine stimulation regulates various aspects of PMN transmigration to sites of inflammation.

PMN Priming

PMN have been shown to undergo ‘priming’ when exposed to cytokines. In general, exposure to a priming agent results in the potentiation of subsequent downstream events. TNF- has long been known to prime PMN and induces PMN to secrete currently undefined products that inhibit phagocytosis, oxidative metabolism and migration of freshly isolated PMN [53]. On the other hand, TNF--treated PMN show an enhanced oxidative burst upon subsequent treatment with fMLP, C5a or opsonized zymosan, compared to unprimed cells [54]. GM-CSF, G-CSF, IL-15, LTB4 and PAF prime PMN for an enhanced oxidative burst in response to fMLP [54, 55]. IL-8 also primed PMN for enhanced fMLP-stimulated O 2 release in combination with suboptimal concentrations of TNF-, GM-CSF or G-CSF [56]. Therefore, the cytokine environment can alter the responsiveness of PMN to subsequent stimulation. Although the precise mechanisms involved in cytokine modulation of PMN function are not known, some of the priming effects have been attributed to receptor upregulation following PMN degranulation [57].

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Inducible secreted mediators Secretory vesicles CR1 CD11b uPA receptor/CD87 CD16/FcRIII fMLP receptor CD14 CD10 CD13 CD45 C1q receptor

PAF C5a LTB4 TNF- G-CSF GM-CSF G-CSF IL-1 GM-CSF IL-8 TNF-

GPI-80

Specific granules CD11b CD15 uPA receptor fMLP receptor Fibronectin receptor Laminin receptor Thrombospondin receptor TNF receptor CD66 CD67 Vitronectin receptor TNF-

Azurophil granules CD63 CD68

TNF-* fMLP* C5a* IL-8*

Gelatinase granules CD11b fMLP receptor uPA receptor

Fig. 1. PMN granules are released following cytokine stimulation. *  Requires cell adhesion in addition to cytokine stimulation.

PMN Granule Release

PMN possess four distinct granules that contain enzymes, receptors, adhesion molecules and proteins with antimicrobial activity [57]. Cytokines can induce expression of receptors and adhesion molecules through degranulation and thus alter the phenotype and functional characteristics of PMN (summarized in fig. 1, table 2). There is a distinct order to the release of PMN cytoplasmic granules. Secretory vesicles are readily mobilized, followed by gelatinase and specific granules and finally by azurophil granules. Secretory vesicles are released during the process of PMN rolling on activated endothelium, and induction for their release is mediated either through selectins [58] or by cytokines secreted by activated endothelium [44]. In vivo, stimulation with GM-CSF [8] or G-CSF [59] was found to rapidly mobilize secretory vesicles. In vitro, TNF-, G-CSF, GM-CSF, IL-1, PAF, C5a and LTB4 upregulated secretory vesicle markers on PMN surfaces [54, 60–64]. G-CSF administration in vivo resulted in specific granule release as measured by increased surface expression of CD15/sialyl Lewisx [65] and CD32/FcRII [59]. PMN-specific granules contain IL-10 receptors and the expression of cell surface IL-10 receptors was found to be upregulated after LPS, TNF- or GM-CSF stimulation [66]. IL-10 receptors may serve to downregulate the proinflammatory activities of the PMN. Specific granules also contain fibronectin, laminin and vitronectin receptors, and increased

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Table 2. Cytokine induction of PMN granule release Cytokine

Granule marker

Ref. no.

GM-CSF

CD16 CD35 CD11b/CD18 CR1 fMLP receptor IL-10 receptor

8 59, 60 54 54 54 66

G-CSF

CD35 CD15

60 65

TNF-

CD35 CD87 IL-10 receptor lactoferrin gelatinase

60 63 66 68 68

IL-1

CD35

60

PAF

CD35 CD11b/CD18

64 62

LTB4

CD35

60

C5a

CD10 CD13 CD16 CD45

61 61 61 61

IL-8

CD11b/CD18 CR1 fMLP receptor

54 54 54

expression after degranulation may alter the adhesive characteristics of the PMN and allow enhanced transmigration through the ECM [57]. TNF- has also been reported to release azurophil granules from whole blood ex vivo [67] and from fibronectin-adherent PMN concurrently stimulated with fMLP and cytochalasin B [68]. fMLP, C5a and IL-8 were able to induce azurophil granule release by PMN adherent to platelet monolayers [43]. Therefore, cytokine stimulation induces PMN granule release, altering the phenotype and function of the PMN.

The Role of PMN in Adaptive Immunity

In addition to their role in innate immunity, in vivo studies suggest that PMN can contribute to the development of adaptive immunity as evidenced by

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models of delayed-type hypersensitivity (DTH) reactions. PMN depletion with an anti-PMN antibody resulted in a reduction in the subsequent monocyte/ macrophage infiltration and consequently diminished DTH reactions in both mice [69] and rats [70]. Pretreatment of animals with an anti-IL-8 antibody resulted in decreased PMN recruitment and tissue edema in a DTH reaction in rabbits [71]. These observations suggest that PMN may be the source of ‘signals’ that mobilize delayed mononuclear cell infiltration. A number of PMN-derived signals may participate in this process. For example, MCP-1/CCL2, a CC chemokine that has potent monocyte chemotactic activity in vitro and in vivo [72], was produced by infiltrating PMN and shown to be functionally important for the development of DTH reactions by recruiting monocytes and T cells [73]. Recently, the central role of MCP-1 in the development of DTH was demonstrated in MCP-1-deficient mice. Although the number of infiltrating PMN was similar in MCP-1 knockout and wild-type animals following antigen stimulation in sensitized mice, mononuclear cell recruitment was significantly reduced in MCP-1-deficient mice [74]. These results suggest that MCP-1 is expressed in vivo by infiltrating PMN and the expression of MCP-1 appears to be required for delayed cellular immune responses. Human PMN produce very low levels of MCP-1 after overnight incubation in a medium containing fetal calf serum [75]. This low level of spontaneous expression could not account for the in vivo observations that MCP-1 was produced by PMN in chronic inflammation, including DTH [73, 76, 77]. However, PMN activation with supernatants from phytohemagglutinin (PHA)-stimulated human peripheral blood mononuclear cells (PHA-sup) resulted in high levels of MCP-1 expression [78]. Unlike the early expression of MIP-1 or IL-8 by TNF--stimulated PMN (2–4 h), PHA-sup-induced MCP-1 expression was delayed (table 3). It required 16 h of stimulation and both early protein synthesis and tyrosine phosphorylation were involved in the process [78]. TNF- played a major role in stimulating PMN. In addition to TNF-, an unidentified 60-kD factor was also involved in the activity of the PHA-sup [79]. The 60-kD factor(s) altered the responsiveness of PMN to TNF- since TNF- stimulation of PMN that were primed with the 60-kD factor resulted in MCP-1 expression after only 4 h of stimulation. PMN activation by TNF- was mediated through TNFR-p55 but the expression levels of the receptor were not altered following overnight incubation with the 60-kD factor. Therefore, it was hypothesized that this unknown factor may alter the intracellular signaling pathway and allow MCP-1 production. Thus, PMN primed by a PBMC product can maximize PMN responsiveness to TNF-. PMN also express CCL20/MIP-3 and CCL19/MIP-3 [80]. Substantial amounts of both CCL20 and CCL19 proteins were released from PMN when

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Table 3. Time course of delayed chemokine protein expression in human PMN Chemokine

Stimulus

0–1 h

4h

21–24 h

42–48 h

MCP-1 [78] MIP-3 [80]

PHA-sup



–









TNF- LPS

 

–















MIP-3 [80]

TNF- LPS IL-10
LPS

 – –

ND ND ND

– – –











ND  Not determined.

stimulated with either TNF- or LPS (table 3). CCL20 is chemotactic for memory T cells [81] and B cells [82], whereas CCL19 attracts B cells, T cells [83, 84] and activated NK cells [85]. In addition, CCL20 and CCL19 are involved in dendritic cell (DC) trafficking to mucosal surfaces and lymphoid organs [86]. CCR6 is a unique receptor for CCL20 [87], also known as liver and activationregulated chemokine (LARC) [88] or exodus [89], and for  defensins [90]. It is expressed on immature DC [91], IL-2-stimulated T cells [88], memory T cells and B cells [92]. Immature DC express high levels of CCR6. Following cytokine stimulation, they downregulate CCR6, but upregulate CCR7 [93]. In PMN, CCL20 is expressed before CCL19. Therefore, the early expression of CCL20 by PMN may regulate immature DC trafficking through CCR6, and later expression of CCL19 may regulate mature DC trafficking. The differential expression of these two chemokines suggests a role for activated PMN in controlling immature and mature DC trafficking [80], promoting their capacity to activate T cells.

Expression of a DC Phenotype by Cytokine-Activated PMN

PMN have been found at many inflammatory and autoimmune disease sites. Since they are phagocytic cells, it has been proposed that they may have the capacity to develop into antigen-presenting cells (APC) and trigger an adaptive immune response [94]. The expression of major histocompatibility complex (MHC) and costimulatory receptors has been attributed exclusively to APC, which traditionally do not include PMN. However, recent evidence suggests that PMN can express both MHC class I and II as well as costimulatory molecules under certain circumstances [95–98].

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GM-CSF

HLA-DR
CD40 CD83
/ CD86

GM-CSF
TNF-
IFN- Circulating PMN HLA-DR  CD40  CD83  CD86 

TNF-
IFN- (PHA-sup)

Allogenic MLR
/ MCP-1
CCR6

HLA-DR
Allogenic MLR CD40
MCP-1


CD83
CCR6
CD86

HLA-DR
/ Allogenic MLR CD40
/ MCP-1


CD83
CCR6
CD86 

Fig. 2. Phenotypic and functional changes in PMN after cytokine activation suggesting that inflammatory PMN are heterogeneous.

GM-CSF is a well-known survival factor for PMN [99, 100]. In contrast, TNF- is one of the most potent activators of PMN and induces the expression of many genes [101]. IFN- is known to potentiate cytokine production by PMN activated by other cytokines [102]. Stimulation of PMN with different combinations of these three cytokines resulted in several distinct PMN phenotypes (fig. 2) [94]. GM-CSF-activated PMN expressed MHC class II and presented superantigens to T cells [103], but did not express CD40, CD83 or CCR6. In contrast, PMN activated with TNF- and IFN- expressed CD83 but not CD40 or HLA-DR. The lack of MHC class II suggests that these PMN do not present antigens, including superantigens. PMN activated by a combination of all three cytokines expressed MHC class II, CD40 and CD83, and are likely to present superantigens. None of the stimuli used induced CD86, an important costimulatory molecule expressed on DC. This may be the reason why PMN were not able to activate a significant level of allogeneic MLR. Further activation of the cells with CD40 ligand may result in the expression of CD86, since CD40 has been shown to induce CD86 expression in DC [104]. Freshly isolated PMN have been shown to express MHC class I and process bacteria, via an undefined alternative processing pathway, and present antigen to T cells [95]. PMN isolated from patients with Wegener’s granulomatosis expressed MHC class II, CD80 and CD86 [105]. It was reported that PMN acquired this phenotype when cultured with T cells or in the presence of T-cell-derived cytokines. In vitro stimulation of PMN with IFN- or GM-CSF alone or in combination resulted in an upregulation of MHC class II, CD80 and

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CD86 on PMN cell surface [96, 97]; however, expression levels were donordependent [103]. These cytokine-stimulated PMN were functionally able to induce proliferation of a tetanus-specific T cell line [97]. PMN isolated from sites of chronic inflammation in vivo and LPS- or IFN--stimulated PMN in vitro expressed B7-1 molecules [98]; however, the functional capacity of this costimulatory molecule has not been evaluated. Cytokine-stimulated PMN were shown to express increased CCR6 [106]. As described above, expression of CCR6 appears to be involved in the trafficking of immature DC [107]. Thus, we hypothesize that PMN-expressing CCR6 might be in the process of acquiring features characteristic of DC. Our hypothesis is supported by other findings that cytokine-activated PMN could express MHC class II and present antigens, and highly purified, lactoferrin-positive, immediate precursors of end-stage PMN could be reverted in their functional maturation program and driven to acquire the characteristics of DC [108]. TNF- stimulation induced high levels of CCR6 mRNA expression in PMN, whereas IFN- induced low levels and the two cytokines together exhibited considerable synergy [106]. Priming was not necessary to induce CCR6 expression. Approximately 160 binding sites for CCR6 ligand (CCL20) were detected [91] with an equilibrium dissociation constant of 1.6 nM on TNF- and IFN--activated PMN. While the Kd of CCL20 binding to PMN was in the range previously reported for CCL20 binding to CCR6-transfected cells [88, 91, 109, 106], the number of CCR6 binding sites on PMN was considerably lower compared to that on immature DC, which expressed approximately 42,000 binding sites per cell [109]. Nevertheless, cytokine-induced CCR6 on PMN is functional as shown by chemotactic responses of these cells to the ligand CCL20 [106]. However, CCR7, normally expressed on mature DC [107], could not be detected on PMN even after 4 days of incubation with TNF- or IFN-, indicating that these PMN do not parallel the maturation program of DC [106]. Additional studies are required to fully understand the role of PMN in adaptive immunity; however, in vitro experiments suggest that PMN, under appropriate circumstances, can play a role in antigen presentation and T cell proliferation.

Gene Expression in Cytokine-Activated PMNs

Originally it was thought that PMN were terminally differentiated and incapable of synthesizing new proteins; however, in the last 20 years, it has been revealed that PMN can synthesize and secrete a plethora of proteins. Gene expression in peripheral blood PMN has been the focus of several recent studies (table 4). In a 3-directed cDNA library prepared from unstimulated human

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Table 4. Representative genes identified from several independent PMN cDNA arrays showing genes that are upregulated or downregulated Normal PMN [110]

Upregulated genes 2-Microglobulin MHC I HLA-Cw HLA-E heavy chain fMLP receptor C5a receptor IL-8 receptor

4-hour TNF [78]

4-hour LPS [121, 122]

Murine 4-hour LPS [123]

24-hour PHAstimulated PBMC supernatant [78]

IL-8 MIP-1 MIP-1 A1

annexin III leukocyte elastase factor phosphostathmin protein phosphatase I

IL-8 MIP-1 MIP-1 MCP-1

p21Waf1/Cip1 DDR1

-catalytic subunit protein tyrosine kinase 9 nonmuscle myosin heavy chain moesin

KC TNF serum amyloid A prostaglandin synthetase A20 glycoprotein ZP3 L-CCR

Pre-B cell colonyenhancing factor IL-8

A1 p21Waf1/Cip1 DDR1

MIP-2

Downregulated genes myeloid cell nuclear differentiation antigen

myeloid cell nuclear differentiation antigen

peripheral blood granulocytes [110], 1,142 clones were sequenced and 748 independent sequences were identified. Approximately 20% of the genes consisted of nuclear proteins such as DNA-binding proteins, secretory proteins such as cytokines, and membrane proteins such as MHC proteins and receptors, indicating that PMN can maintain their gene expression without further activation. A cDNA array analysis was performed with PMN stimulated for 4 h with TNF- or stimulated overnight with PHA-sup [78]. The expression of chemokine genes, such as IL-8, MIP-1 and MIP-1, was upregulated in both TNF-- and PHA-sup-treated PMN. The expression of MCP-1 was only upregulated in PHA-sup-activated PMN. This shows a distinct difference in PMN responsiveness to different cytokine stimulation. The expression of antiapoptosis protein A1, cyclin-dependent kinase inhibitor p21Waf1/Cip1 and receptor tyrosine kinase discoidin domain receptor (DDR1) was upregulated with either treatment. Upregulation of A1 was shown to have an important role in PMN survival [111], thus indicating that cytokine-stimulated PMN can prolong their survival. Induction of p21Waf1/Cip1 was shown to positively correlate with growth

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arrest associated with monocyte-macrophage differentiation [112], and a similar event occurs during PMN activation. DDR1 was cloned originally from a placenta cDNA library; later it was found to be expressed in highly metastatic tumor cells and was suggested to be involved in tumor progression [113]. The ECM component, collagen, was identified as the ligand of DDR1 [114, 115]. Peripheral blood PMN do not express DDR1, but the expression is upregulated after in vitro culture [116]. In vitro experiments have shown that DDR1a may be involved in leukocyte migration through ECM [116]. Thus, PMN may use DDR1 to interact with collagen of the ECM during inflammation. An additional cDNA microarray study of approximately 7,000 genes was analyzed with PHA-sup-activated PMN [Yoshimura et al., unpubl. observations]. The expression of about 400 genes was upregulated by at least 2-fold. These genes included indole 2,3-dioxygenase (IDO), TNF-inducible protein TSG-6, and MCP-1. Expression of several ESTs was also upregulated in these cells. Many of the cDNAs were previously cloned from other cell types. IDO was cloned originally from IFN--activated fibroblasts and converts tryptophan and other indole derivatives to kinurenin and contributes to the inhibition of intracellular pathogens, such as Toxoplasma gondii and Chlamydia psittaci [117]. PMN are important in the resistance of T. gondii [118] and C. psittaci [119]. TSG-6 was cloned from TNF--activated fibroblasts and forms a stable complex with components of the plasma protein inter--inhibitor. These two proteins synergize to inhibit plasmin, the major fibrinolytic enzyme in the clotting cascade. Plasmin also has the ability to activate matrix metalloproteinases, which are responsible for most of the ECM degradation associated with inflammation. This implies that TSG-6 may be involved in a negative feedback control of the inflammatory response [120]. Another human cDNA array study was performed on LPS-stimulated PMN (4 h) and revealed 134 genes that were upregulated [121, 122]. These included chemokines, cytokines, signaling molecules and transcriptional regulators. Proteomic analysis revealed upregulation of the proinflammatory molecules: annexin III, leukocyte elastase inhibitor, and the signaling molecules: phosphostathmin, protein phosphatase I and -catalytic subunit, as well as structural proteins such as protein tyrosine kinase 9-like, nonmuscle myosin heavy chain and moesin [121]. A cDNA array analysis of murine PMN stimulated with LPS was performed. In this study, PMN were isolated from the BM and stimulated for 1 h in vitro with LPS and compared to PMN isolated from the lung of endotoxinstimulated animals in vivo [123]. In vivo, 423 genes were upregulated and 401 genes were downregulated; however, only 8 genes were upregulated after in vitro LPS activation and none were downregulated. The genes that were found to be upregulated both in vivo and in vitro were the chemokine KC, TNF, serum amyloid A, prostaglandin synthetase, A20, glycoprotein ZP3, a putative

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-chemokine receptor and macrophage inflammatory protein-2. The marked difference in the number of genes upregulated after in vivo or in vitro stimulation is likely the result of a complex cytokine network triggered by LPS in vivo. Taken together, these studies reinforce the idea that resting circulating PMN are not terminally differentiated cells with a limited capacity to transcribe genes, but rather can be activated by cytokines to prolong their survival and enhance their gene expression capacity. PMN have the capacity to express many genes, and different stimuli result in different patterns of gene expression.

Conclusions

PMN are multifunctional cells that play a role in both innate immune responses as well as adaptive immune responses. Although circulating PMN are short-lived, once they infiltrate into tissues in response to injury or foreign invaders, their life span is prolonged and they can be activated to perform multiple functions associated with innate and adaptive immune responses. Cytokine stimulation of PMN results in many functional and phenotypic changes in PMN that enhance their functional responses and allow the cell to respond to environmental factors. Such cells in turn also influence the environment. Many different phenotypic forms of PMN have been generated in vitro, suggesting that during inflammatory or immune responses PMN may exist at various stages of activation and differentiation. Cytokine regulation of PMN function is currently an expanding area of research and we have only just begun to scratch the surface. Future studies will, hopefully, determine the full range of receptors and cytokines that can be induced in cytokine-activated PMN, and their functional significance in inflammatory and/or immune diseases.

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Dr. Teizo Yoshimura, Laboratory of Molecular Immunoregulation, National Cancer Institute at Frederick, Bld 559, Rm. 9, Frederick, MD 21702-1201 (USA) Tel.
1 301 846 5518, Fax
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Cassatella MA (ed): The Neutrophil. Chem Immunol Allergy. Basel, Karger, 2003, vol 83, pp 45–63

Expression of MHC Class II Antigen and Coreceptor Molecules in Polymorphonuclear Neutrophils G. Maria Hänsch, Christof Wagner Institut für Immunologie, Universität Heidelberg, Heidelberg, Germany

Polymorphonuclear neutrophils (PMN) have outgrown their image as short-lived terminally differentiated cells and it is now generally accepted that in addition to their role as ‘first-line defense’ PMN may also acquire regulatory functions, particularly by secreting cytokines. An important advance in unravelling the proinflammatory and regulatory role of PMN derived from the observation that the life span of PMN is extended when the cells are appropriately stimulated [1, 2]. While PMN in the peripheral blood undergo spontaneous apoptosis within 6 h, their life span is extended under pathological conditions, for example in patients with trauma, burns, or infectious diseases [3–6]. To some extent, the pathophysiological in vivo conditions can be reproduced by culturing PMN of healthy donors with a variety of proinflammatory cytokines or other stimulators [examples can be found in 1, 2, 7–11]. Escape from apoptosis, as outlined in more detail by Edwards et al. [12], is dependent on protein synthesis and is associated with numerous changes, among others de novo protein synthesis, for example of cytokines, of proteolytic enzymes, chemokine and other surface receptors [examples in 6, 13–20]. This review focuses on a very special aspect of PMN activation, namely the differentiation to cells with dendritic-like phenotype and function. As will be described in detail below, PMN can be activated to de novo synthesize and express MHC class II antigens and the costimulatory receptors CD80 and CD86, all required for antigen presentation to T lymphocytes with no other function known so far. The notion that PMN can differentiate to dendritic-like cells is further supported by the finding that CD83, a surface antigen thought to be specific for dendritic cells, is also de novo synthesized, and expressed on the cell surface upon stimulation.

Differentiation to cells with dendritic-like properties links PMN to the specific immune response and represents a further connection between innate and adaptive immunity.

Induction of MHC Class II Synthesis and Surface of PMN in Culture

Constitutive expression of MHC class II antigens is restricted to professional antigen-presenting cells, such as dendritic cells, B cells or cells of the monocyte/macrophage lineage. There is, however, ample evidence that MHC class II antigen expression can be induced in eosinophils [21–24] and also in tissue cells. Particularly well analyzed are keratinocytes [25], thymic epithelial cells [26], tubular epithelial cells [27], endothelial cells [28], or synovial cell fibroblasts [29]. PMN of healthy individuals do not express MHC class II antigens. Induction of MHC class II antigens in PMN precursor cells [30], but also in mature PMN was described in response to either interferon-
(IFN-
), granulocyte/macrophage colony-stimulating factor (GM-CSF) or interleukin-3 (IL-3) [31, 32]. In our own study [33], we found that MHC class II-specific mRNA was measurable within 8–24 h after stimulation of PMN with IFN-
and/or GM-CSF and that surface expression increased for up to 48 h (an example is shown in fig. 1a, b). At that time, the majority of PMN were still viable, as measured by the expression of CD16, binding of annexin V or staining of DNA with propidium iodide. An interesting observation, already made by Gosselin et al. [31], was that the surface expression of MHC class II on PMN was donor-dependent: PMN of some individuals acquired more MHC class II than those of others. Basically, we could confirm this finding. In the last years, we have analyzed MHC class II expression in more than 30 healthy donors upon stimulation with either IFN-
, GM-CSF or a combination thereof. In all, MHC class II expression could be induced, but a wide variation was seen. Expression ranged from 5.5% MHC class II-positive PMN to 35% (median 21%; mean 23% when measured 48 h after adding 100 U/ml IFN-
per 106 PMN). From analysis of the data obtained by cytofluorometry, but also by confocal laser scan microscopy it appeared that only a portion of PMN was expressing MHC class II antigens following stimulation. Whether this reflects transient expression or a true subpopulation cannot be decided at this stage (fig. 1c). It is important to note that de novo synthesis of MHC class II antigens is not invariably associated with escape from apoptosis and PMN activation. As reported in the literature [31, 32] and also in our hands, GM-CSF and IFN-


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alone or in combination induced MHC class II synthesis, whereas other cytokines, known to activate other PMN functions or to delay constitutive apoptosis, did not or only to a very minor degree do so. Among those tested were IL-6, IL-8, IL-10, NAP-2 or TNF. Moreover, also the classic PMN activators such as C5a, fMLP or LPS failed to induce MHC class II synthesis. A profound stimulatory signal was derived from T cells: when highly purified PMN were cocultivated with T cells in dishes coated with cross-linked anti-CD3, a massive upregulation of MHC class II antigen on PMN was seen (table 1). Antibodies either to the IFN-
receptor or to IL-8 were not or only in part inhibitory, suggesting involvement of other T cell-derived factors or engagement of surface receptors. Irrespectively, however, of the nature of the T-cell-derived signalling molecules, these data indicate interactions between T cells and PMN, resulting in PMN activation and – as will be described below – in consequence also in the activation of T cells. When PMN were activated in whole blood, either with heparin or sodium citrate as anticoagulant, both, IFN-
or GM-CSF induced MHC class II expression 2- to 3-fold higher than that seen with isolated cells (data of five donors are shown in fig. 1d). Moreover, also cytokines unable to stimulate isolated PMN rather efficiently caused upregulation of MHC class II when added to whole blood cultures. This was seen for TNF or IL-2, and to a lesser degree for LPS (table 2). There are multiple explanations, including more suitable culture conditions, activating cytokines provided by cells other than PMN, costimulatory serum factors or cell-cell interactions. Moreover, the stress put on cells during the purification procedure should not be underestimated as a possible factor modifying the response to activating signals. Regardless of the reason for the enhanced response of PMN in whole blood, the data might be relevant with regard to the in vivo situation, because induction of MHC class II antigens on PMN in vivo might be more proficient than anticipated from the in vitro studies with purified PMN. Indeed, therapeutic application of IFN-
or GM-CSF, e.g. for mobilization of stem cells, leads to the appearance in the peripheral blood of MHC class II-positive PMN [33–36]. With these ideas in mind we set out to look for MHC class II expression on PMN under in vivo conditions.

Expression of MHC Class II Antigens in vivo

Acute and chronic inflammatory diseases are invariably associated with the activation of leukocytes, including the PMN. Because of the easy accessibility, MHC class II expression was tested on PMN derived from the peripheral

Expression of MHC Class II Antigen and Coreceptor Molecules

47

HLA DP-DQ-DR PE

100 101 102 103 104

HLA DP-DQ-DR PE

100 101 102 103 104

100 101 102 103 104

CD 67 FITC

HLA DP-DQ-DR PE

Unstimulated PMN

PMNIFN-
GM-CSF

2

3 Counts

1

Freshly isolated PMN

d

0 20 40 60 80 100 120

HLA DP-DQ-DR PE 3

48h

100 101 102 103 104 CD 67 FITC

c 2

56.74%

100 101 102 103 104 CD 67 FITC

b 1

100 101 102 103 104

100 101 102 103 104

CD 67 FITC 100 101 102 103 104

Mouse lgG2a PE Mouse lgG2a PE

100 101 102 103 104

Isotype controls

100

101

102 103 HLA DR

104

20

40

60

80 100 120

PMN stimulated 48h

D1 D2 D3 D4 D5

0

45 40 35 30 25 20 15 10 5 0

Counts

MHC class-positive PMN (%)

24h

Mouse lgG1 FITC

100 101 102 103 104 Mouse lgG1 FITC

1

27.25%

100 101 102 103 104

100 101 102 103 104

a

Unstimulated PMN

Isolated PMN

Hänsch/Wagner

PMN in whole blood

100

101 102 103 HLADP-DQ-DR PE

48

104

Table 1. Induction of MHC class II antigens of CD80 and CD86 during coculture of PMN with T cells and staphylococcus enterotoxin E (SEE) PMN cultivated with

AIM/2.5% NHS T cells T cells activated by anti-CD3 T cells  SEE SEE IFN-
 GM-CSF

MHC class II

CD80

day 1

day 2

day 1

2.5 5.6 7

3.5 24 19

0 0 3

9 2.5 6

23 4 12

4 0 3

CD86 day 2

day 1

0 6.9 9.5

1.5 8 9

2 19 27

15 2.5 9

35 6 19

11 3 7

day 2

% Positive cells as measured by cytofluorometry; isotype controls were 1% positive cells; data representative for one of three similar experiments. NHS  Normal human serum.

blood, being well aware that this most probably is not the primary site of action of any leukocyte. We included patients with various chronic inflammatory diseases, such as primary vasculitis, systemic lupus erythematosus (SLE) or rheumatoid arthritis, but also patients with acute bacterial infections. Pilot studies revealed that only in Wegener’s granulomatosis, a primary vasculitis, was MHC class II expression on PMN found in some patients. This finding prompted a large prospective study, including up to now 85 patients with the diagnosis of Wegener’s granulomatosis. Wegener’s granulomatosis is a necrotizing vasculitis of small vessels of unknown etiology. It is usually treated with immunosuppressive drugs resulting

Fig. 1. Induction of MHC class II antigens on PMN in culture. PMN were cultivated with AIM-V, 2.5% heat-inactivated autologous serum with or without IFN-
(100 U/ml) plus GM-CSF (50 U/ml). a Surface expression of MHC class II was measured after 24 and 48 h. An antibody to CD66b (former CD67) was used to identify the PMN population; isotype controls are on the left; the middle panel shows PMN without cytokines. b RT-PCR products generated using MHC class II-specific primers and PMN-derived RNA are shown. PMN had been cultured for 24 h with AIM-V/NHS (1), IFN-
(100 U/ml) (2), and GM-CSF (50 U/ml) (3). The left panel shows the respective RT-PCR products for -actin. c By confocal laser microscopy and cytofluorometry it was seen that only a subpopulation of PMN acquired MHC class II antigens. d Induction of MHC class II antigens on PMN in whole blood. Whole blood (heparinized peripheral blood of healthy donors) was cultivated with IFN-
(100 U/1 ml blood) for 48 h; then upregulation of MHC class II on PMN was measured by cytofluorometry and compared to that obtained with PMN isolated from the same donor. D1–D5 designate five individual donors.

Expression of MHC Class II Antigen and Coreceptor Molecules

49

Table 2. Induction of MHC class II antigens on PMN by various means Means of activation (48 h)

Isolated PMN %

Whole blood %

IFN-
 GM-CSF (100 U/ml each) TNF (2 ng/ml) IL-2 (30 U/ml) LPS (0.1 g/ml)

25  19 (n  5) 2.5  2 (n  6) 5.5  4 (n  4) 6.5  5 (n  3)

49  24 (n  5) 24  17 (n  3) 45  7 (n  3) 14  11 (n  5)

PMN had been isolated by centrifugation through PolymorphPrep and positive selection by anti-CD15 beads (MAGS) and were suspended in AIM-V containing 2.5% autologous heat-inactivated serum. In all experiments MHC class II expression was measured by cytofluorometry; the values were corrected for isotype controls; only viable cells were considered in experiments where 90% of PMN had survived. Data represent mean  SD of independent experiments using cells of different donors.

in some patients in a quite stable ‘inactive’ disease, sometimes even with complete remission, whereas in other patients relapses occur, requiring again aggressive immunosuppression. In our study we had three groups of patients, those with active disease before receiving immunosuppressive therapy, others with inactive disease and immunosuppressive therapy, mostly low-dose steroids, and a third group with inactive disease and without immunosuppression. MHC class II-positive PMN were only found in the patients with active disease, but not in patients under immunosuppressive therapy or patients with inactive disease [37, 38] (fig. 2a). In fact, when patients with active disease were receiving immunosuppressive drugs, the MHC class II expression declined within a day or two (data summarized in fig. 2b). These data are consistent with the fact that PMN of patients receiving low-dose corticoids cannot be induced to upregulate MHC class II in vitro in response to IFN-
or GM-CSF [38] and that steroids prevent the upregulation of MHC class II antigens on PMN of healthy donors (fig. 2c). So far, we have only found MHC class II-positive PMN in patients with active Wegener’s granulomatosis, and in 3 patients with active Churg-Strauss vasculitis. Whether this indicates a certain disease specificity cannot be decided at this stage. A more reasonable explanation could be that all the patients with SLE or rheumatoid arthritis had received some type of immunosuppressive therapy, which would prevent upregulation of MHC class II. In patients with acute bacterial infections massive upregulation of activation-associated surface receptors was seen, including the high-affinity Fc receptor CD64 and the LPS receptor CD14, but in none of the patients was MHC class II found on PMN from the peripheral blood (fig. 2d). The patients

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varied widely with regard to the infectious species, localization of the infection, the ensuing disease and the antibiotic regimen as well. Therefore, it is unlikely that we had selected a special ‘nonresponder’ group. More localized inflammatory reactions and also different cytokines might rather account for the different activation pattern of PMN.

Upregulation of Costimulatory Molecules on PMN in vitro and in vivo

The only known function of MHC class II antigens is the presentation to T cells of antigens. To fully activate T cells, costimulatory signals are required, which are delivered by the interaction of receptors expressed on the antigenpresenting cells with their counterreceptors on T cells. Multiple pairs of costimulatory receptors have been described, which in part act synergistically. Of those costimulatory molecules, ICAM-1 (CD54) and leukocyte-functional antigen-3 (LFA-3; CD58) are constitutively expressed on PMN [39]. Others, like CD80 and CD86, both being ligands for CD28 on T cells, are not expressed on naive PMN, but are de novo synthesized in response to IFN-
, GM-CSF or a combination thereof [33, 40]. Highly purified PMN acquired only very small amounts of CD80 (average 5%) or CD86 (average 12%). Following coculture with T cells, or when cultivated in whole blood up to 20% PMN became positive for CD80 and 40% for CD86 (fig. 3a). Expression of CD80 and of CD86 was also found in vivo on PMN of patients with Wegener’s granulomatosis, when the disease was active. Consistent with the in vitro data, the CD86 expression was more pronounced than the expression of CD80, and again similar to MHC class II, the expression of CD80 and CD86 decreased rapidly under immunosuppressive therapy (fig. 3b).

Expression of CD83 on PMN

CD83, a member of the Ig family with a binding capacity for sialic acid, is widely used as a marker for dendritic cells [41]. Its expression can also be induced in monocytes [42] and, as was shown more recently, also in PMN [39, 43]. Upon culture with IFN-
a dendritic-like PMN bearing CD83, CD80, CD86 and MHC class II antigens was seen [39]. Of note was that TNF, though a weak inducer of MHC class II antigens, induced synthesis of CD83 to a higher extent than IFN-
did [44]. In vivo, expression of CD83 was seen on PMN of patients with bacterial infections (fig. 4) but not on PMN of patients with Wegener’s granulomatosis

Expression of MHC Class II Antigen and Coreceptor Molecules

51

a

b

MHC class II-positive PMN (%)

MHC class II-positive PMN (%)

25

*

20

15

Immunosuppressive therapy

10

Active disease

60

Patients with Wegener’s granulomatosis

30

Immunosuppressive therapy

50

40

30

20

10

Healthy donors

5

0 1

d

0 Active disease

Inactive disease

2

3

4

5

Patients with bacterial infections CD64

100

Healthy donors Patients

c

Native PMN  IFN-
48h

Positive PMN (%)

80

CD14 60

40

20

Steroidtreated PMN  IFN-
48 h Anti-DR

MHC II

0

Fig. 2. Induction of MHC class II expression in vivo. a MHC class II expression on PMN was measured by cytofluorometry on PMN (identified by CD66b) of patients with Wegener’s granulomatosis. PMN of patients with active disease (n  25) were positive for MHC class II, but not PMN of patients with inactive disease with (n  46) or without (n  14) immunosuppressive therapy or PMN of healthy donors (n  40). The group of patients with active disease was different from all others (largest p  0.0016 when t test

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[44], though the latter were expressing MHC class II antigens. One interpretation would be that the two antigens are regulated independently of each other, as the in vitro data imply as well. Also possible, however, are different kinetics of appearance or of downregulation from the surface. As pointed out above, the peripheral blood is certainly not the best source for obtaining activated PMN; thus giving an interpretation of these data is somewhat premature.

Functional Consequences of MHC Class II and Coreceptor Expression

So far there is evidence that PMN differentiate to cells with a dendritic-like phenotype. With the expression of MHC class II and the costimulatory molecules CD80 and particularly CD86, but also ICAM-1 and LFA-1 they possess the prerequisites of antigen-presenting cells. Whether or not PMN can indeed efficiently activate T cells in an MHC class II-restricted and MHC class II-dependent manner is still under investigation. Two approaches were taken to test for the functional capacity of differentiated PMN: staphylococcus enterotoxin (SE), a so-called superantigen, was used and alternatively, peptide antigens, requiring to some extent processing and presentation to autologous antigen-specific T cells. SE as superantigen binds to a variety of MHC class II allotypes outside the specific peptide binding groove. The MHC class II-superantigen complex then attaches to the T cell receptors, but again not to the antigen-specific site. Rather, binding to constant framework sequences is observed [for review, see 45]. The advantages of using superantigens instead of peptides are obvious: (1) there is no need for antigen processing; (2) there is only some selectivity with regard to the DR allotype, and (3) no antigen-specific T cells are needed and stimulation is possible with heterologous cells. Since the majority of T cells respond to a given superantigen, a very robust proliferation is obtained, making this experimental approach highly reproducible and easy to handle. When PMN induced to express MHC class II were cocultivated with T cell and SE, a well-studied superantigen, proliferation of T cells was seen [33, 46].

was used) (*). b As shown for 5 patients with active disease (1–5), MHC class II expression on PMN declined under immunosuppressive therapy, usually within 1–2 days. c PMN were cultivated with IFN-
with (gray peak) or without dexamethasone (black line; 1  10–7 M); then MHC class II surface expression was measured. d PMN of patients with bacterial infections (gray boxes; n  16) are activated as seen by upregulation of CD14 and CD64, but they are negative for MHC class II. The open boxes contain data from healthy donors (n  20).

Expression of MHC Class II Antigen and Coreceptor Molecules

53

a R1

PMN

R1 CD80

R3

CD66b

R2

R4

CD86

T cells R2

CD3

b

30

Patients with Wegener’s granulomatosis

50

Patients with Wegener’s granulomatosis

25

20

15

Immuno- Healthy suppressive donors therapy

10

CD86-positive PMN (%)

CD80-positive PMN (%)

40 Immunosuppressive therapy

30

20

Healthy donors

10

5

0

0 Active disease

Inactive disease

Active disease

Inactive disease

3

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Proliferation could be induced in peripheral T cells, but also in established T cell lines and could be inhibited by antibodies to MHC class II antigens or to CD86 (data summarized in fig. 5). There is still some disagreement with regard to the requirement of CD80/CD86 for superantigen presentation. Under some experimental conditions, there is apparently no need for engagement of CD28, the counterreceptor for CD80/CD86 on T cells. Our data, however, also indicate dependency of T cell proliferation on CD86-CD28 interaction. About twice as much PMN than monocytes were required to induce similar T cell proliferation. Considering, however, that only 20–40% of PMN expressed MHC class II, the superantigen-presenting capacity of PMN is in about the range of monocytes, at least under experimental conditions. A rather intriguing finding was that MHC class II expression, as well as expression of CD86, increased during the coculture of PMN with the T cells, suggesting that T-cell-derived signals were activating the PMN. This interpretation is in line with the experiment cited above (fig. 3), showing that coculture of PMN with autologous T cells activated by cross-linked anti-CD3 resulted in a massive upregulation of MHC class II on PMN within 24–48 h (table 1). It is also of note that in the absence of an exogenous stimulus, such as cross-linked anti-CD3, cocultivating isolated T cells with PMN resulted in mutual activation of both of the cells: in PMN an increase of IL-8 and induction of IFN-
was seen, preceding upregulation of MHC class II, in T cells upregulation of IL-2 followed an increase of IFN-
and IL-8 (a representative experiment is shown in fig. 6). How the mutual activation in the absence of SE occurs is still under investigation; first data point to engagement of surface receptors resulting in low level activation of the cells. Indeed, some proliferation of T cells was also noted. Presentation of superantigens to T cells is not only a pragmatic approach to explore functional activity of MHC class II antigens, but might also be relevant for the handling of superantigens in vivo. There is increasing evidence that bacterial or viral superantigens participate in the development of pathological conditions, which are also associated with PMN infiltration, making it worthwhile to analyze further superantigen presentation by PMN [47–49]. Fig. 3. a Induction of CD80 and CD86 on PMN of healthy donors. Isolated PMN were cocultivated with autologous T cells for 48 h, then cells were analyzed by cytofluorometry. By forward-side scatter analysis the two cell populations could be identified, R1 being PMN (CD66b-positive, gray peak) and R2 being T cells (CD3-positive, gray peak). The lines represent the isotype controls. The PMN were also positive for CD80 and CD86. b CD80 and CD86 were measured on PMN of patients with Wegener’s granulomatosis. On PMN of patients with active disease (n  11) CD80 and CD86 expression was seen, but not on PMN of patients with inactive disease with (n  21) or without (n  7) immunosuppressive therapy. The group of patients with active disease was different from all others (largest p  0.04 when t-test was used).

Expression of MHC Class II Antigen and Coreceptor Molecules

55

CD83-positive PMN (%)

Counts 10 20 30 40 50 60 70 80 90

80

60

40

p0.02 20

0

0

100

101

a

102

103

104

Healthy donors

CD83 PE

Patients

b

Fig. 4. Expression of CD83 in vivo. By cytofluorometry, expression of CD83 on PMN of patients with bacterial infections was seen. a As an example cells of a patient with a local staphyolococcus infection are shown (thick line  anti-CD83; thin line  isotype contol). b Data of 16 patients and 20 healthy donors are summarized (the boxes contain 50% of the values; the small squares represent the highest and the lowest values, respectively).

12,000

8,000

6,000

4,000

2,000

10,000 8,000 6,000 4,000

0 T cell clone (D894)PMNMHC II 200pg SEE

2pg SEE

No SEE

b

80,000 60,000

* 40,000

*

20,000

2,000

0

a

100,000 T cell proliferation (cpm)

T cell proliferation (cpm)

T cell proliferation (cpm)

10,000

0 IFN-
- No treated PMN PMN

Untreated No PMN PMN

c

PMNMHC II IgG anti- anti T cells MHC II CD86  SEE

Fig. 5. Induction of proliferation of the PHA-propagated T cell clone D894 by PMN and staphylococcus enterotoxin E (SEE). a, b T cell proliferation, measured as uptake of 3H thymidine, in dependency of SEE concentration and IFN-
-pretreated PMN. c T cell proliferation induced by IFN-
-pretreated PMN and SEE could be inhibited by antibodies to MHC class II antigens or to CD86. *  Proliferation significantly different from IgG (largest p  0.001).

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PMN

Coculture

SSC-Height

PMN IL-8

102

T cells T cells

103 FSC-Height

IFN-


Fig. 6. Coculture of T cells with PMN causes mutual activation. Highly purified PMN were cultivated with autologous T cells for 24 h. Then cytokine poduction (IL-8 and IFN-
) was measured by cytofluorometry of saponin-treated cells. The gray peaks represent data of cultivated cells, the black lines the cells before culture. The isotype controls are not shown for clarity; they, however, were negative.

50,000

T cells (line FRI-T)

PMN T cells TT 16,000

8,000 6,000 4,000

PTDC Brefeldin B

0

Wortmannin

2,000 PTDC

TT

PMN (aut)TT PMN (het)TT

PMN (aut) PMN (het)

*

10,000

Brefeldin B

*

12,000

Wortmannin

*

Proliferation (cpm)

*

IgG Anti-CD86 Anti-MHC II Anti-ICAM-1 Anti-CD18

Proliferation (cpm)

20,000

10,000

a

Monocytes

Pulsed with TT for 3h

30,000

0

PMN

14,000

40,000

b

Fig. 7. Induction of proliferation of a TT-specific T cell clone (FRI-T) by PMN and TT. a Either autologous (aut) or heterologous (het) PMN were used, pretreated with IFN-
/ GM-CSF for 24 h. Proliferation could be induced by autologous PMN only, and could be inhibited by antibodies to either CD86, MHC class II, ICAM-1 or CD18. b When PMN were pulsed with TT for 3 h, proliferation of T cells was also induced. When Nf -B translocation (PTDC  ammonium pyrrolidonedithiocarbamate) or exocytosis (brefeldin B) was added to PMN and TT inhibitors of PI kinase, proliferation was inhibited. *  Proliferation different from IgG (largest p  0.002).

Expression of MHC Class II Antigen and Coreceptor Molecules

57

Lavage

103

103

Peripheral blood

Side scatter

PMN 102

Side scatter 102

PMN

101

T cells

101

T cells

102 Forward scatter

103

102 103 Forward scatter

PMN

100

b

0

0

20

20

40

Counts 40 60

Counts 60 80

80

100

100 120

a

101

102

103

104

102

103

104

HLA DR-DQ-DP PE

200

80

160

Counts 40 60

120 80

20 0

0 100

101

100

HLA DR-DQ-DP PE

40

Counts

100

101

c

102 CD83 PE

103

104

100

101

102

103

104

CD83 PE

Fig. 8. Expression of MHC class II and CD83 on locally activated PMN. From a patient undergoing surgery because of an acute S. aureus-induced bursitis, cells were intraoperatively taken as lavage from the infectious site and from the peripheral blood. a The cell

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With regard to the ability of PMN to present peptide antigen, there is some discrepancy between the data in the literature [46, 50] and our own observations [33, 38]. While we have no conclusive explanation, one possibility appears to be the more complex situation when analyzing the presentation of peptide antigens. In contrast to superantigens, the presentation of peptides to T cells requires processing of protein antigens by the antigen-presenting cells, followed by attachment of a processed peptide to the MHC class II molecules and transfer of the MHC class II-peptide complex to the cell surface. Since the peptide binding groove formed by the two chains of the MHC class II antigens varies with the HLA-DR allotype, there is some selectivity with regard to the peptide being presented. Hence, depending on the HLA-DR allotype, different peptides of the same protein will be presented. Within the T cell repertoire, only a limited number of T cells will recognize the MHC class II-peptide complex, and, provided that costimulatory molecules are present, will proliferate. Therefore, to study antigen presentation by PMN, but also by professional antigen-presenting cells, peripheral T cells are not particularly helpful, since the proliferation rate is low. To circumvent this problem, we established tetanus toxoid (TT)-specific T cell lines from a healthy, recently vaccinated donor using her B cells and monocytes as antigen-presenting cells. After repeated cycles of restimulation, cells with a high proliferative response to TT and low spontaneous growth were selected and then cocultivated with autologous PMN and TT. Before culture, both PMN and T cells were highly purified using magnetic bead selection to avoid contamination with professional antigen-presenting cells. After coculture for 3–5 days, proliferation of T cells was seen, which could be inhibited by antibodies to MHC class II or to CD86. To some extent, also antibodies to ICAM-1 and to CD18 were inhibitory. Using the obligatory controls, such as absence of antigen, heterologous versus homologous PMN or no PMN at all, the interpretation of these experiments is that PMN are also able to present peptide antigens to T cell (fig. 7). In some experiments, PMN were pulsed with the antigen for 3–6 h, and then cocultivated with the T cells. When Wortmannin, a phosphatidylinositol 3-kinase inhibitor, was present during antigen uptake by PMN, the T cell proliferation was inhibited, indicating that TT needed to be processed (fig. 7). Proliferation could also be inhibited when the PMN had been pretreated with brefeldin B or cytochalasin D, supporting the notion that TT had to be processed, distribution, as judged by forward-side scatter analysis, was remarkably alike. b, c PMN taken from the inflamed site, however, were positive for MHC class II and to some degree for CD83 (thick lines, the thin lines represent the isotype controls) while PMN of the peripheral blood were not positive or only to a minor degree.

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attached to MHC class II and recycled to the cell surface. That processed peptides can be presented to T cells by PMN and induce proliferation is in agreement with previous reports by others [51] and in line with the observation that also other nonprofessional antigen-presenting cells, particularly eosinophils [21–24] or endothelial cells [28], are able to present peptide antigens in an MHC class II-restricted manner. There is no question that PMN are able to degrade proteins; whether they also have the accessory molecules for loading the peptides to the MHC class II molecules is still under investigation. First data also indicate the presence of HLA-DM; the analysis of its function, however, awaits further investigation. The in vivo relevance of MHC class II expression on PMN is still a matter of speculation. One might argue that only around 10% of the PMN in the peripheral blood acquire MHC class II antigens (as seen in patients with active Wegener’s granulomatosis) and that this is too small a number to be of relevance. It has, however, to be considered that PMN are the most abundant cells, and that 10% represents a large number of cells, exceeding that of circulating dendritic cells and even of monocytes. Moreover, the peripheral blood from where the PMN were derived is certainly not the site of interaction either with antigen or with T cells. Where PMN and T cells come across each other is not yet known; one possibility is at infectious sites or inflammatory areas where both, PMN and T cells, are present in a cytokine-rich environment. First data analyzing the cellular infiltrates of patients with a Staphylococcus aureusmediated bursitis revealed the presence of both PMN and T cells, the PMN being highly positive for MHC class II and CD83. The data suggest a local activation, because cells of the peripheral blood, measured at the same time, contained much less activated PMN (fig. 8); these data may be taken as a first indication for PMN-T cell interaction in vivo.

Conclusion

Mature PMN can still further differentiate: by de novo protein synthesis, cytokines are produced, the surface receptor pattern changes and PMN acquire new functions. Differentiation, however, is not uniform: depending on the microenvironment, particularly on cytokines, but also on surrounding cells, PMN do acquire various phenotypes. Here we presented evidence that PMN when cultured in the presence of T cells or T cell-derived cytokines differentiate to cells with a dendritic-like phenotype and function. Moreover, we presented evidence that dendritic-like PMN are also generated in vivo under pathological conditions, e.g. during acute inflammation.

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Whether antigen presentation by PMN and the activation of the specific T cell response is of great physiological or pathophysiological relevance remains to be clarified; it is, however, an intriguing possibility, considering the large number of PMN.

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(letter). Blood 1996;87:3068. Hänsch GM, Radsak M, Wagner C, Reis B, Koch A, Breitbart A, Andrassy K: Expression of major histocompatibility class II antigens on polymorphonuclear neutrophils in patients with Wegener’s granulomatosis. Kidney Int 1999;55:1811–1818.

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Iking-Konert C, Vogt S, Radsak M, Wagner C, Hänsch GM, Andrassy K: Polymorphonuclear neutrophils in Wegener’s granulomatosis acquire characteristics of antigen-presenting cells. Kidney Int 2001;60:2247–2262. Iking-Konert C, Csekö C, Wagner C, Stegmaier S, Andrassy K, Hänsch GM: Transdifferentiation of polymorphonuclear neutrophils: Acquisition of CD83 and other functional characteristics of dendritic cells. J Mol Med 2001;79:464–474. Windhagen A, Maniak S, Gebert A, Ferger I, Wurster U, Heidenreich F: Human polymorphonuclear neutrophils express a B7-1-like molecule. J Leukoc Biol 1999;66:945–952. Zhou LJ, Schwarting R, Smith HM, Tedder T: A novel cell surface molecule expressed by human interdigitating reticulum cells, Langerhans cells and activated lymphocytes is a new member of the Ig superfamily. J Immunol 1992;149:735–742. Zhou LJ, Tedder TF: CD14 blood monocytes can differentiate into functionally mature CD83 dendritic cells. Proc Natl Acad Sci USA 1996;93:2588–2592. Yamashiro S, Wang JM, Gong WH, Yan D, Kamohora H, Yoshimura T: Expression of CCR6 and CD83 by cytokine-activated human neutrophils. Blood 2000;96:3958–3963. Iking-Konert C, Wagner C, Denefleh B, Hug F, Schneider M, Andrassy K, Hänsch GM: Up-regulation of the dendritic cell marker CD83 on polymorphonuclear neutrophils (PMN): Divergent expression in acute bacterial infection and chronic inflammatory disease. Clin Exp Immunol 2002;130:501–508. Fleischer B, Schrezenmeier H: T-cell stimulation by microbial ‘superantigens’. Immunol Res 1990;10:349–355. Fanger NA, Liu C, Guyre PM, Wardwell K, O’Neil J, Guo TL, Christian TP, Mudzinski SP, Gosselin EJ: Activation of human T cells by major histocompatibility complex class II expressing neutrophils: Proliferation in the presence of superantigen, but not tetanus toxoid. Blood 1997;89: 4128–4135. Skov L, Baadsgaard O: Bacterial superantigens and inflammatory skin disease. Clin Exp Dermatol 2000;25:57–61. Yoh K, Kobayashi M, Yanaguchi N, Hirayama K, Ishizu T, Kikuchi S, Iwabuchi S, Muro K, Nagase S, Aoyagi K, Kondoh M, Takemura K, Yamagata K, Koyama A: Cytokines and T-cell responses in superantigen-related glomerulonephritis following methicillin-resistant Staphylococcus aureus infections. Nephrol Dial Transplant 2000;15:1170–1174. Llewelyn M, Cohen J: Superantigens: Microbial agents that corrupt immunity. Lancet Infect Dis 2002;2:156–162. Prior C, Townsend PJ, Hughs DA, Haslam PL: Induction of lymphocyte proliferation by antigenpulsed human neutrophils. Clin Exp Immunol 1992;87:485–492. Reali E, Guerrini R, Moretti S, Spisani S, Lanza F, Tomatis R, Traniello S, Gavioli R: Polymorphonuclear neutrophils pulsed with synthetic peptides efficiently activate memory cytotoxic T lymphocytes. J Leukoc Biol 1996;60:207–213.

G.M. Hänsch, Institut für Immunologie der Universität Heidelberg, Im Neuenheimer Feld 305, D–69120 Heidelberg (Germany) Tel. 49 6221 564071, Fax 49 6221 565536, E-Mail [email protected]

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Cassatella MA (ed): The Neutrophil. Chem Immunol Allergy. Basel, Karger, 2003, vol 83, pp 64–80

Phenotypic and Functional Change of Neutrophils Activated by Cytokines Utilizing the Common Cytokine Receptor Gamma Chain Denis Girard INRS-Institut Armand-Frappier, Université du Québec, Pointe-Claire, Québec, Canada

In the past few years, the phenomenon of cytokine redundancy has been, and is still, the object of several investigations. Collectively, these latter demonstrate that the utilization of a common receptor subunit by distinct cytokines partly explains the overlapping biological activities shared by different cytokines. At another level, identification of several common intracellular proteins involved in cell signaling following cytokine binding to its receptor also explains such a redundancy phenomenon. Among the different cytokines known to utilize a common receptor subunit, interleukin-2 (IL-2), IL-4, IL-7, IL-9, IL-15 and more recently IL-21, all share the common cytokine receptor ␥ chain (␥c). The discovery that neutrophils express ␥c at their surface is recent. The bulk of this chapter will focus specifically on the role of the different ␥c users on neutrophil cell physiology.

The Polymorphonuclear Neutrophil Cells

Polymorphonuclear (PMN) neutrophils are important cells of the immune system involved in host defense. Except for erythrocytes and platelets, PMNs are the most abundant cell type in circulation and have a limited life span (halflife of ⬃12 h in circulation). This may explain why the number of PMN cells remains relatively stable in healthy individuals [1–4]. Cell turnover must therefore be under strict control for preventing diseases and this is probably why PMNs spontaneously undergo apoptosis without addition of special reagents in

the milieu [1–4]. One can imagine that if the basal apoptotic rate of PMNs is suppressed or delayed by an agent this could be deleterious for an organism during an inflammatory state, since PMNs will perpetuate tissue damage by releasing their toxic products. The concept that resolution of PMN-mediated inflammation could be achieved in part through induction of neutrophil apoptosis has gained increasing attention in the past few years [4–8]. Therefore, elimination of apoptotic PMNs during an inflammatory state represents an interesting avenue of research for the development of future potential therapeutic strategies. Thus, studying and discovering molecules that modulate the PMN apoptotic rate is of great importance in biology and medicine. This is probably why many investigators are interested in discovering molecules involved in the regulation as well as in elucidating the mechanism involved in neutrophil apoptosis. PMNs, typically known to be involved in innate immunity, are the first cell type to arrive at inflamed sites. Neutrophils can adhere to endothelial cells, transmigrate and crawl toward a corresponding chemotatic gradient (chemotaxis). PMNs are phagocytes that can eliminate invading pathogens via oxygendependent and oxygen-independent mechanisms. The former refers to the respiratory burst leading to the generation of reactive oxygen species (O⫺ 2, H2O2, HOCl) whereas the second refers to the capacity of these cells to degranulate and release their potent toxic degradative products. PMNs were previously characterized as terminally differentiated cells with little protein synthesis activity. However, it is now becoming increasingly clear that these cells are more capable of protein synthesis than previously thought. It is generally accepted that PMNs play roles in host responses that extend well beyond their capacities to function as phagocytes and cell-releasing cytotoxic compounds. Various cytokines are among proteins that are produced by PMNs [8]. Because of this, these cells have the potential to perform active functions in both afferent and efferent limbs of the immune response [9]. PMNs are known to be an important source of products implicated in tissue damage and inflammation such as various granule enzymes, reactive oxygen metabolites, leukotriene B4, platelet-activating factor, and various cytokines (IL-1␣, IL-8, IL-12, TNF-␣, TGF-␤, GRO-␣), to name a few [10]. The importance of PMNs in inflammation is further reinforced by the observation that various PMN priming and activating agents such as IL-1␤, IL-8, GM-CSF, TGF-␤, C5a, C9, and more recently, IL-15, are present in the synovial fluids from rheumatic patients [11–13]. The same arsenal and response used by neutrophils for host defense could also be very deleterious for an organism when deregulation occurs. This is known as the neutrophil paradox where the defending cell becomes an enemy. Because of this, and knowing their role during inflammation, it is very important

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to carefully understand the mode of action of neutrophil agonists as well as to identify new ones. The IL-2 Family Cytokines or ‘␥c Users’

Generalities IL-2, IL-4, IL-7, IL-9, IL-15, and IL-21 are all members of the hematopoietin cytokine family. Each member is believed to have the structure of a four ␣-helical bundle type I cytokine [14]. The IL-2 family cytokines are known to mediate their biological activities via cytokine receptor class I constituents such as IL-2/15R␤, IL-4R␣, IL-7R␣, IL-9R␣, IL-21R␣, and the common ␥c chain that is shared by all receptors to these cytokines. Collectively, they constitute the IL-2 family cytokines. For simplicity, the terminology ␥c users will be used throughout this review for designing IL-2, IL-4, IL-7, IL-9, IL-15 and IL-21. Of note, two additional chains, IL-2R␣ and IL-15R␣ that are not members of the type I cytokine receptor, are known to be receptor subunits for IL-2 and IL-15, respectively. Thus, in addition to the ␥c receptor subunit [15,16], the IL-2R is known to be composed of two other chains, IL-2R␣ and IL-2/15R␤ [17] whereas the IL-15R contains its specific ␣ chain designated IL-15R␣ [18]. In addition to ␥c, receptors to IL-4, IL-7, IL-9 and IL-21 possess one chain conferring the specificity that is IL-4R␣ [19], IL-7R␣ [20], IL-9R␣ [21], or IL-21R␣ [22], respectively. Of note, two types of IL-4R exist: the one that is composed of the IL-4R␣ and the ␥c chains is designated the IL-4R type I whereas the IL-4R type II is composed of IL-4R␣ and IL-13R␣ [23, 24]. Pleiotropy and Redundancy The terms cytokine pleiotropy and cytokine redundancy are used to describe the ability of a single cytokine to exert multiple actions and the ability of multiple cytokines to exert similar actions, respectively [25]. Of note, the IL-2 family of cytokines uses both of these phenomena. In particular, IL-2 and IL-15 possess redundant biological activities. The cytokine redundancy phenomenon is largely explained by the fact that several cytokines utilize not only common receptor subunits, but also common intracellular cell signaling molecules. An example of this phenomenon includes the IL-2R and the IL-15R that share the ␥c and the IL-2/15R␤ subunits (originally named IL-2R␤) that, upon activation, will recruit Jak-1/3 and STAT3/5 for cell signaling [26]. Signaling: The Jak-STAT Pathway It is well established that cytokines mediate their biological activities via binding to a specific cell surface receptor [27]. In addition, it is well characterized

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that cytokine binding induces tyrosine phosphorylation of an increasing number of intracellular proteins [27, 28]. Recent studies have identified some of these proteins as members of the Janus kinase (Jak) family of cytoplasmic proteins tyrosine kinases. Members of the Jak family are Jak-1, Jak-2, Jak-3, and Tyk-2 and those of the STAT family are STAT1–6. Jak-3 is known to be associated with ␥c. The simplified general picture is that following ligation of a cytokine to its specific receptor, activated Jaks, that are associated with receptor components, phosphorylate other cytoplasmic proteins belonging to the family of transcription factors called the signal transducers and activator of transcription (STATs). The Jak kinases phosphorylate critical tyrosines on the receptors, which serve as docking sites for STATs that are associated with it via their SH2 domains. Activated STATs dissociate from the receptor and dimerize in the cytosol and translocate to the nucleus where they stimulate the transcription of a particular gene. These events represent the Jak-STAT pathway that is shared by all members of the hematopoietin cytokine receptor family including the ␥c users. The description of the detailed cell signaling events involved in response to IL-2, IL-4, IL-7, IL-9, IL-15, and IL-21 is well beyond the scope of the present chapter. Excellent reviews on this subject have been published [15, 26, 29, to cite a few]. This being said, I will simply summarize the utilization of the Jak-STAT pathway, which is the most important for the present purpose. However, it is important to keep in mind that other cell signaling pathways may be activated following ␥c user receptor binding. Among the four Jak kinases, Jak-1, Jak-2, Jak-3, and Tyk-2, the ␥c users all signal via Jak-1 and Jak-3 [30, 31]. This could be explained by the ability of Jak-1 to associate with the chain conferring the specificity for each receptor, namely IL-2R␤ [32, 33], IL-4R␣ [34], IL-7R␣ [35], and IL-9R␣ [36], and probably IL-21R␣ [37], whereas Jak-3 associates primarily with ␥c [33, 35]. The importance of the ␥c was demonstrated by the discovery that mutations in ␥c cause X-linked severe combined immunodeficiency (SCID) [30, 38], also named the ‘bubble boy’ disease. In this disease, both cellular and humoral immunity are defective. In fact, T and NK cells do not develop and even if B cells are present, they are nonfunctional [30, 38]. Interestingly, mutations in Jak-3 were found to cause an autosomal recessive form of SCID [39] and the essential role of Jak-3 in lymphoid development was established [40]. This clearly demonstrated the important role of the Jak-STAT signaling pathway. The Jak-STAT Pathway in Neutrophils The pituitary hormone GH was found to trigger the tyrosine phosphorylation of Jak-2 and STAT3 in PMNs [41]. Prolactin was found to enhance Jak-2 phosphorylation in peripheral blood mononuclear cells but not in granulocytes whereas it was able to induce phosphorylation of STAT1 in neutrophils [42].

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Caldenhoven et al. [43] demonstrate that, in addition to STAT1␣, STAT3 was also activated by IFN-␥ in human PMNs. GM-CSF was reported to activate Jak-2, STAT1, STAT3, and STAT5b but not Jak-1, Jak-3, or Tyk-2, STAT2, STAT4, or STAT6 [28, 44, 45]. In other studies, STAT5 was found to be involved in myeloid cell proliferation and neutrophil differentiation and Jak-2 was activated in neutrophils from patients with severe congenital neutropenia [46, 47]. Collectively, the above observations indicate that human neutrophils express all Jaks and STATs identified [28, 41–47]. Despite this, there is presently no reported study in the literature dealing with the utilization of the Jak-STAT pathway in ␥c user-induced neutrophils. We have recently demonstrated that IL-15 induces the phosphorylation of Jak-2, p38 mitogen-activated protein kinase and extracellular signal-regulated kinase 1 and 2 [48]. The description of the numerous biological activities mediated by the ␥c users on their numerous targets cannot be fully covered in this review. For the present purpose, I will detail the role of each of these cytokines in neutrophil cell physiology.

Interleukin-2

Among the different ␥c users, IL-2 has gained more attention concerning the activation of neutrophils. However, the role of IL-2 in neutrophil cell physiology is still obscure and many observations remain to be confirmed by different laboratories. Although we have previously found that this cytokine does not modulate human neutrophil apoptosis [49], another team has found that this cytokine can delay this biological process [50]. This discrepancy may be explained by the different experimental conditions used. We have incubated neutrophils at 10 ⫻ 106 cells/ml in the presence of 10% autologous serum versus 2 ⫻ 106 cells/ml in the presence of 5% commercially available human AB serum. One important variable is the cell density used, since as reported by others [51], IL-6 suppression of neutrophil apoptosis was found to be neutrophil concentrationdependent. The influence of cell density for evaluation of the modulation of neutrophil apoptosis by IL-2 remains to be assessed. More recently, another team reported that IL-2 does not modulate human neutrophil apoptosis [52], confirming our results. Curiously, they also incubated cells at 2 ⫻ 106 cells/ml, but in the presence of 10% fetal calf serum suggesting the possibility that both the concentration and source of serum are also important variables. In our hands, we have observed that IL-2 was not able to induce neutrophil phagocytosis of opsonized sheep red blood cells in vitro [49]. MacDonald et al. [53] found that neutrophils synthesize and release IL-8 in response to IL-15, but not to IL-2. Moreover, a nuclear factor-kappaB (NF-␬B) DNA-binding activity

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Table 1. Activation of neutrophil functions by the cytokines utilizing the common cytokine receptor ␥c Cytokine

O⫺2

Cell Cell shape spreading change

␾

RNA synthesis

Protein synthesis

Apoptosis

Ref. no.

IL-2 IL-4 IL-7 IL-9 IL-15

no yes no no variable

no yes no no yes

no yes no no yes

no yes no no yes

weak yes no no yes

weak yes ND ND yes

variable delay no effect no effect delay

IL-21

no

ND

no

no

ND

ND

no effect

49, 50, 52 63, 64 81 57 49, 52, 57, 89, Unpubl. Unpubl. observations

ND ⫽ Not done; Unpubl. ⫽ Pelletier et al., unpubl. observations.

was enhanced in nuclear extracts of IL-15-treated neutrophils, which could be supershifted by antibodies to p50 or RelA, but this was not observed in nuclear extracts of IL-2-induced neutrophils. In one study, in vivo administration of IL-2 in humans suffering from advanced cancers was found to suppress FcR expression (CD16) in neutrophils and chemotaxis [54]. In this case, one can imagine that an indirect effect of IL-2 during in vivo treatment may lead to activation of cells other than neutrophils that could synthesize soluble factors that would inhibit neutrophil responses. In contrast to other ␥c users (table 1), we never observed a direct effect of IL-2 on the neutrophil functions tested in vitro including: O⫺ 2 production, cell spreading, cell shape, phagocytosis, RNA synthesis, apoptosis, and IL-2R component expression [49]. However, this does not rule out the possibility that IL-2 can activate these cells. In fact, we have previously published that IL-2 can induce GM-CSF-induced de novo protein synthesis in a concentration-dependent manner [55]. This indicates that although IL-2 alone cannot induce some neutrophil functions, it can act on primed cells. Interestingly, it was recently demonstrated that IL-2 has the ability to induce protein tyrosine kinases in human neutrophils [56]. In addition, IL-2 was found to induce the association of IL-2R␤, lyn, and MAP kinase ERK-1 in these cells [56]. However, the functional end point of such tyrosine kinase activation remains unclear, since the authors did not correlate this with any neutrophil function. What is clear, however, is that human neutrophils express IL-2R␤ and ␥c on their cell surface, but not IL-2R␣ [55–61]. We have explained the lack of direct effect of IL-2 on human neutrophil functions by the fact that these cells do not express the full high affinity IL-2R (␣␤␥), and that although IL-2 can bind to human neutrophil cell surface with an intermediate affinity, this is not sufficient to modulate the tested functions. One can imagine that a ‘cosignal’ is

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lacking, probably similar to the one occurring during in vivo IL-2 treatment or during coincubation of neutrophils with GM-CSF and IL-2 [55]. IL-2 was also found to induce IL-8 and TNF-␣ in human neutrophils [59, 61], but this remains to be confirmed by others. As indicated by the authors, it may be possible that IL-8 gene expression is indirectly induced by the fact that cells are preactivated by adherence [61]. This is in agreement with our observation that neutrophils did not directly respond to IL-2, but did so when activated by GM-CSF [55]. In summary, I am more comfortable with the statement that IL-2 is a weaker neutrophil agonist when compared with other ␥c users such as IL-4 [62] and IL-15 [49], as discussed in subsequent sections.

Interleukin-4

One of the first studies, which reported that IL-4 could activate PMNs, was that of Boey et al. [63]. They found that IL-4 enhanced neutrophil-mediated killing of opsonized bacteria, but could not itself induce a respiratory burst. However, IL-4 was found to increase, in a concentration-dependent manner, the respiratory burst mediated by the peptide formyl-methionyl-leucyl-phenylalanine (fMLP). This was not a generalized phenomenon, since IL-4 did not potentiate this response when mediated by phorbol myristate acetate, calcium ionophore A23187, or zymosan-treated serum. The same study also reported that IL-4 enhanced the ability of neutrophils to phagocytose opsonized sheep erythrocytes. IL-4 was found to antagonize the biological action of IL-1 by inducing the expression and release of IL-1 type II receptor, the decoy target for IL-1 [64]. Bober et al. [65] found that IL-4 induces neutrophilic maturation of the promyelocyte HL-60 cells. In addition, IL-4 was capable of sustaining the neutrophil maturation of HL-60 cells that had been pretreated for 24 h with DMSO. In this study, IL-4 induced phagocytic responses of freshly isolated human neutrophils. The response was specific, since treatment with anti-human IL-4 abolished phagocytic stimulation. It was also reported that IL-4 treatment stimulated migration of resting neutrophils toward zymosan-activated serum and human IL-5. Consistent with its anti-inflammatory properties, IL-4 was found to induce interleukin-1 receptor antagonist (IL-1Ra) in neutrophils [66–68]. We found that human neutrophils express the IL-4R␣(CDw124) component on their surface and that IL-4 induces RNA and protein synthesis [62]. Based on observations of the induction of morphological cell shape changes and spreading onto glass, it also activates cytoskeletal rearrangements [62]. In addition, we found that IL-4 can delay neutrophil apoptosis and induce IL-8 production. In one study, Wertheim et al. [69] demonstrated that IL-4 could inhibit IL-8 production, but this was

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observed in LPS-induced neutrophils. Others have observed that IL-4 inhibits LPS-induced prostaglandin E2 (PGE2) as well as LPS-induced cyclooxygenase-2 (COX-2) protein expression in neutrophils [70]. Later, the same authors investigated the molecular mechanism of LPS-induced COX-2 expression in human PMNs and found that both ERK and P38 (MAPK) were involved in this response as well as in LPS-induced PGE2 production [71]. IL-4 was found to inhibit this by down regulating the activation of p38 (MAPK) [71]. Zaitsu et al. [72] have studied the effect of IL-4 on A23187-stimulated synthesis of the potent chemotactic factor leukotriene B4 in human neutrophils. They found that IL-4 enhanced this response and increased mRNA expression and protein synthesis of leukotriene A4 hydrolase. More recently, it was reported that a single exposure of normal human skin to ultraviolet B radiation induced an infiltration of numerous IL-4⫹ cells that were identified as neutrophils [73], based on the observations that they coexpressed CD15 and CD11b, showed a clear association with elastase, and had a multilobed nucleus. In one other study, the expression of CD23 (the low affinity receptor for IgE) on human neutrophils isolated from rheumatoid arthritis patients was monitored by immunofluorescence [74]. Results showed a strong association between the ability of neutrophils to express CD23 and rheumatoid arthritis, and that such expression may be regulated by GM-CSF, IFN-␥ and IL-4. The above observations clearly indicate that IL-4 is an important neutrophil activator in vitro. Recently, one study was conducted in order to assess the capacity of IL-4 and IL-10 to block neutrophil activation in an ex vivo human model system, and to confirm their effect on neutrophil function in an animal model of arthritis [75]. In the rat adjuvant arthritis model, treatment with systemic murine IL-4 (mIL-4; and mIL-10) was found to be effective against even the most severely diseased [75]. IL-4 (and IL-10) was effective in lowering the absolute neutrophil cell number recovered and the neutrophil activation state in the joint synovia. Both cytokines reduced the phagocytic activation of human neutrophils in response to proinflammatory cytokines. Collectively, the results demonstrate that IL-4 (and IL-10) can exert powerful regulatory effects on neutrophil function that translate into a therapeutic response in a disease model of arthritis. The authors concluded that treatment with IL-4 (or IL-10) alone or in combination might therefore be very useful in the management of patients with rheumatoid arthritis [75].

Interleukin-7

There is little data in the literature concerning the activation of mature neutrophils by IL-7. Prior our interest in this area of research, recombinant

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human IL-7 was reported to mobilize murine myeloid progenitor cells from the bone marrow to the spleen, blood, and liver [76–77] and to increase the number of immature murine granulocytes in the spleen in vivo [78]. Although it had previously been documented that myeloid cells express IL-7R, these studies were performed in murine models [79] and data on humans were unavailable. Intriguingly, in contrast to murine models, it was reported that freshly isolated human granulocytes failed to bind biotinylated IL-7, suggesting that functional IL-7R was absent or too weakly expressed on these cells [80]. Because we and others [55, 58] have found that mature human neutrophils constitutively express the ␥c chain, which is known to be a constituent of the IL-7R component [20], we have examined the ability of IL-7 to modulate a whole array of neutrophil responses and studied the expression of the IL-7R␣(CDw127) component. We found that IL-7 was unable to induce a respiratory burst, phagocytosis, cytoskeletal reorganization, and RNA synthesis in these cells [81]. In addition, IL-7 was unable to modulate neutrophil apoptosis. The absence of activation was explained by the fact that mature human neutrophils did not express the IL-7R␣ chain on their cell surface, as assessed by flow cytometry [81]. From this study, we concluded that expression of ␥c on human neutrophils is insufficient in itself to modulate neutrophil responses with respect to the studied functions. Therefore, it cannot be proposed, based on the above observations, that mature neutrophils are targets of IL-7.

Interleukin-9

The IL-9R, like the IL-7R, is composed of the ␥c subunit and one additional chain, the IL-9R␣. Although the IL-9 receptor ␣ chain alone is sufficient to confer high-affinity binding, both chains are needed for signal transduction. We have demonstrated that mature human neutrophils do not express the IL-9R␣ chain on their cell surface and this is correlated with the inability of IL-9 to induce directly (or in priming experiments) O⫺ 2 production, cell spreading, cell shape changes, phagocytosis, RNA synthesis and with the inability to modulate apoptosis [57]. More recently, another team reported the expression of a functional IL-9R (which include the IL-9R␣ chain) on the surface of human neutrophils from asthmatic donors [82]. Curiously, they found that the IL-9R␣ transcript was infrequently detected in some neutrophil preparations from healthy donors, whereas it was detected in 100% of preparations from asthmatic patients. Knowing that eosinophils are the most predominant contaminating cells present in neutrophil preparations, it is tempting to speculate that the presence of IL-9R␣ transcripts in neutrophil preparations from healthy subjects originated from contaminating eosinophils, a cell type that is increased in

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asthmatic patients [83]. Although they observed that neutrophils expressed the IL-9R␣ chain on their surface as assessed by flow cytometry, cells were not preincubated, in contrast to our study, with an excess of human serum before incubating them with the specific anti-IL-9R␣ antibody. Therefore, some unspecific binding may have occurred in their assay. The only biological assay in response to IL-9 performed by these authors was the measurement of IL-8 production. Again, the role of contaminating eosinophils cannot be ruled out, knowing that the latter can produce high amounts of IL-8 [84, 85]. Because of the discrepancy between these two studies, it will be important to reevaluate the role of IL-9 on neutrophils.

Interleukin-15

IL-15 is a recently discovered cytokine [86] known to share many biological actions with IL-2 on T cells, NK cells, and B cells. Both IL-2 and IL-15 are ⬃14- to15-kD members of the four alpha-helical bundle family of cytokines sharing basically T cell growth activity. Their overlapping actions are not surprising since their specific receptors were found to share two common components, ␥c and IL-2R␤ [86]. The third subunit for each receptor is however distinct and is referred to IL-2R␣ and IL-15R␣ [18, 86]. We have previously documented that IL-15 is a neutrophil agonist [49]. This cytokine induces RNA synthesis, de novo protein synthesis, phagocytosis, and delays apoptosis. This was correlated with the fact that human PMNs express the recently identified IL-15R␣ subunit [53, 57]. More recently, IL-15, unlike IL-2, was found to induce the production of the potent neutrophil chemoattractant IL-8 and activation of NF-␬B [53]. In addition, it has been reported that IL-15 could not inhibit the ability of the plant lectin Viscum album agglutinin-I (VAA-I) to induce neutrophil apoptosis, and this was correlated with an inhibition of de novo protein synthesis induced by VAA-I [86]. IL-15 was found to enhance in vitro neutrophil functional responses in patients with human immunodeficiency virus (HIV) infection; namely, chemotaxis and fungicidal activity were increased by IL-15 [88]. In another study, incubation with IL-15 was found to enhance secretion of IL-8 by neutrophils and the amount secreted was increased by costimulation with heat-inactivated Candida albicans [89]. The authors also reported that IL-15 could prime the neutrophil respiratory burst in response to fMLP, but was not sufficient to trigger the production of superoxide in cells exposed to C. albicans. IL-15 also increased the ability of neutrophil to phagocytose heat-killed C. albicans organisms in a dose-dependent manner without opsonization by antibodies or complementderived products. In a recent study, IL-15 was found to induce the simultaneous

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secretion of IL-1␤ and its natural inhibitors IL-1R␣ and sIL-1RII by human neutrophils isolated from normal and tumor-bearing hosts [90]. IL-15 also induced IL-␤ and IL-1R␣ secretion by neutrophils from healthy controls, but not by neutrophils from cancer patients. However, a priming effect of IL-15 was noted on IL-1␤ production by LPS-stimulated cells in oral cavity cancer [90]. We have recently not only confirmed that human PMNs secreted IL-1R␣ as measured by ELISA, but also found that IL-15 induces de novo synthesis of IL-1R␣ [unpubl. data]. Collectively, these observations clearly indicate that neutrophils are important targets of the proinflammatory cytokine IL-15.

Interleukin-21

To date, no data are available in the literature concerning the role of IL-21 on neutrophil cell physiology. Our preliminary results indicate that this cytokine does not modulate human neutrophil phagocytosis and apoptosis [Pelletier et al., unpubl. data]. Other experiments need to be performed to clearly establish whether or not IL-21 is a neutrophil agonist. Whether or not these cells express the IL-21R␣ chain remains to be determined. Production of ␥c User Cytokines by Neutrophils

When appropriately activated, neutrophils are able to secrete several cytokines and chemokines including IL-1R␣, IL-8, IL-12, macrophage inflammatory protein (MIP)-1␣, or IFN-␥ [8, 11, 53, 90, 91]. Despite this, their ability to produce ␥c user cytokines has not been well documented. In fact, only one study reported that human neutrophils produce IL-4 [92]. This was demonstrated by intracellular flow cytometry, immunostaining performed on cytospin preparations, and by a sensitive ELISA. This correlates well with the previously mentioned study, which reported that a single exposure of normal human skin to ultraviolet B radiation induced an infiltration of numerous IL-4⫹ cells identified as neutrophils [73]. However, we failed to detect IL-15 in the culture of neutrophils isolated from healthy donors following a 24-hour in vitro treatment in the presence of classical neutrophil agonists such as fMLP, GM-CSF or TNF-␣ (unpubl. observations). To date, there is no other study reported in the literature in this matter. Of note, the secretion of IL-15 is known to be under tight control [93, 94] and to date although mRNA transcripts are widely expressed, its detection at the protein level is not common. Macrophages appear to be the principal source of IL-15 protein [95].

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IL

-1

5R

IL-4R

IL-21R ?

IL-21R␣

IL-

9R

␣

? IL-

9R

2R

IL-

␥c

IL-2/IL-15R␤

IL-4R␣

IL-15R␤

Fig. 1. Cell surface expression of the ␥c user receptor components on human neutrophils.

Summary and Future Perspectives

Taken together, the preceding observations indicate clearly that among the ␥c users, IL-4 and IL-15 are potent human neutrophil activators (table 1). When studying cell surface expression of all known ␥c user receptor components on PMNs, it was found that these cells express IL-2R␤, IL-4R␣, ␥c, and IL-15R␣, but not IL-2R␣, IL-7R␣, nor IL-9R␣ (fig. 1). A correlation between the expression of all IL-4 and IL-15 receptor components and the restricted ability of IL-4 and IL-15 to directly activate PMNs is evident. PMNs do not respond to IL-2, IL-7, or IL-9 and do not express IL-2R␣, IL-7R␣, and IL-9R␣ [49, 53, 55, 57, 62, 63]. The studies described in this review clearly indicate that human PMNs can respond to cytokines that were initially thought to activate mainly B, T, or NK cells. Future research, particularly the elucidation of signaling events in IL-4- and IL-15-induced neutrophils, will be of great interest. Both of these cytokines play important roles during inflammation and are potent neutrophil agonists. Studies in this area are in progress in my laboratory. We have recently demonstrated that IL-15 induces the phosphorylation of Jak-2, p38 mitogen-activated protein kinase and extracellular signal-regulated kinase 1 and 2 [48]. We have recently performed experiments dealing with the role of each ␥c user on the recruitment of neutrophils in vivo. Using the murine air pouch model, our data indicate that IL-2, IL-4 and IL-15 attract neutrophils [unpubl. data]. Another important aspect

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would be to study the role of ␥c users in preactivated neutrophils. As previously mentioned, de novo protein synthesis is increased by IL-2 when cells are incubated in the presence of GM-CSF [55]. I believe that interesting results and observations in this area of research will soon emanate in the literature and this will be of help for researchers working in inflammation or related fields.

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Denis Girard, PhD, INRS-Institut Armand-Frappier, 245 boul Hymus, Pointe-Claire (PQ), Canada, H9R 1G6 (Canada) Tel./Fax ⫹1 514 630 8847/8850, E-Mail [email protected]

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The Evolving Role of the Neutrophil in Chemokine Networks Sara S. Cheng a, Steven L. Kunkel b a Graduate Program in Cellular and Molecular Biology and bDepartment of Pathology, University of Michigan Medical School, Ann Arbor, Mich., USA

Polymorphonuclear phagocytes, commonly referred to as neutrophils, are short-lived, phagocytic leukocytes that circulate the blood in large numbers. Their main function is in antibacterial or antifungal immune responses, where they comprise a major component of host defense [1, 2]. Neutrophil phagocytosis and production of toxic substances work together to effectively eradicate infectious pathogens. However, the very functional characteristics of neutrophils that make them efficient antipathogen effectors also carries the potential for extensive host tissue damage when acute inflammatory responses are overly large or do not resolve themselves appropriately. Since the acute inflammatory response is both protective yet potentially dangerous to the host, a better understanding of neutrophil recruitment and the mechanisms regulating neutrophil influx may aid in the development of therapeutic agents against infectious disease as well as pathoinflammatory disorders. The innate immune system relies on a series of cytokine cascades to achieve a rapid and massive neutrophil influx when needed. The first step of this cascade consists of the activation of sentinel cells, usually tissue macrophages or mast cells, by bacterial or fungal products. Macrophages then immediately release potent early response cytokines, such as IL-1␤ and TNF-␣, that act in a paracrine manner on nearby stromal cells and in an autocrine manner on the macrophages themselves, causing the release of massive amounts of proinflammatory chemokines. The local production of appropriate chemokines ultimately leads to the transendothelial migration of neutrophils from the peripheral blood. While a robust neutrophilic response is adaptive in the setting of infection it poses potential harm to the host, since overly robust production of neutrophilderived substances can have pathological consequences. For example, cytokineinduced neutrophil infiltration is thought to be one cause of vascular leak and

edema associated with acute inflammation [3]. Neutrophil degranulation releases many oxidants and proteolytic enzymes [2, 4, 5], which are meant to target the offending pathogen. However, release of these powerful substances can also lead to destruction of normal cells and dissolution of connective tissue at the site of inflammation [6–8]. Such neutrophil-dependent tissue damage underlies the loss of organ function associated with many pathoinflammatory disorders, including glomerulonephritis, ischemia-reperfusion injury of the heart, and acute respiratory distress syndrome. In addition to their involvement in acute inflammation, periodic neutrophilic infiltration is also typical of certain chronic inflammatory conditions such as rheumatoid arthritis [9], psoriasis [10], inflammatory bowel disease [11], and asthma [12], and may contribute to the progression of these diseases. Chemokines are a large family of small secreted peptides that are known for their essential roles in leukocyte trafficking and activation [13]. These peptides exert their biological effects via binding to seven transmembrane G proteincoupled receptors that can activate a diverse array of signaling pathways within cells. Each chemokine can be categorized according to the number and spacing of conserved N-terminal cysteines into one of four subclasses, CC, CXC, C, or CX3C. The chemokine subclasses have limited specificity for different leukocyte subsets. The CXC chemokine subfamily can be further subdivided based on the presence or absence of a three amino-acid sequence consisting of glutamic acid-leucine-arginine, termed an ‘ELR’ motif. In ELR-positive CXC chemokines, the ELR motif precedes the first cysteine in the primary amino acid sequence of the peptide, while in ELR-negative CXC chemokines, this motif is absent. Interestingly, the ELR motif confers a distinct functionality to these peptides with regard to their activity on neutrophils in that ELR⫹ CXC chemokines are well-known for their potent neutrophil chemotactic properties [14]. In addition, other chemokines have recently been implicated in neutrophil recruitment as well. This review will address the range of chemokines and receptors involved in neutrophil recruitment during acute inflammation, exploring in particular new interactions that have been discovered over the past several years. In addition, emerging data is presented concerning neutrophil participation in chemokine networking, which demonstrates that neutrophils are dynamic cells that can influence the subsequent nature of the inflammatory response.

Chemokines and Chemokine Receptor Involvement in Neutrophil Recruitment

CXCR1 and CXCR2 Several members of the CXC family of chemokines are well-characterized, potent neutrophil chemoattractants (table 1). CXCL8, the most well-characterized

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Table 1. Chemokine receptor involvement in neutrophil recruitment Subfamily

Chemokine receptor

Major ligands

Involved in neutrophil recruitment?

CXC

CXCR1

CXCL8 (IL-8) CXCL6 (GCP-2) CXCL1, 2, 3 (GRO␣␤␥) CXCL5 (ENA-78) CXCL6 (GCP-2) CXCL7 (NAP2) CXCL10 (IP-10) CXCL9 (Mig) CXCL11 (I-TAC) CXCL12 (SDF-1␣) CXCL13 (BLC)

Yes [83]

CCL3 (MIP-1␣) CCL9 (MIP-1␥) CCL7 (MCP-3) CCL5 (RANTES) CCL23 (MPIF-1) CCL2, 8, 7, 13, 12 (MCP-1, -2, -3, -4, -5) CCL11, 24, 26 (eotaxin-1, -2, -3) CCL7 (MCP-3) CCL13 (MCP-4) CCL5 (RANTES) CCL20 (MIP-3␣) Multiple ligands

Yes (murine) [30]

CXCR2

CXCR3

CXCR4 CXCR5 CC

CCR1

CCR2 CCR3

CCR6 CCR4, 5, 7, 8, 9, 10

Yes [83]

No

No No

Yes [46] No

Yes [49] No

C

XCR1

XCL1 (lymphotactin-␣) XCL2 (lymphotactin-␤)

No

CX3C

CXC3CR1

CX3CL1 (fractalkine)

No [53]

New nomenclature as proposed by Zlotnik and Yoshie [84] is shown, with older nomenclature indicated in parentheses.

neutrophil-specific human chemokine, can be produced by almost every nucleated cell in the body under the appropriate stimulatory conditions [15, 16]. Inducible human CXCL8 is thought to be a key effector of neutrophil recruitment and activation in acute inflammation [17]. CXCL8 production is under both positive and negative regulation from factors such as cytokines [16, 18], adhesion [16], bacterial products [19], and oxidative stress [20].

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Unstimulated human peripheral blood neutrophils are known to express relatively high levels of the CXCL8 receptors, CXCR1 and CXCR2 [21]. Upon stimulation with the corresponding ligands, these cells respond with robust chemotaxis [22], as well as increased adhesion [22, 23], superoxide generation [24], release of proteolytic enzymes [22], and enhanced bactericidal capacity [25]. CXCL8 binds to both CXCR1 and CXCR2, but signaling occurs via different structural components and with different kinetics [21, 26]. GCP-2 is also thought to bind and activate both CXCR1 and CXCR2 [27]. Other neutrophil chemoattractants such as CXCL1, CXCL2, CXCL3, CXCL5, and CXCL7 bind and act exclusively through CXCR2 [28]. While murine models are used extensively in the study of inflammation, it seems that the human and murine chemokine systems differ slightly in structure. While several murine ELR⫹ CXC chemokines have been identified, none of them are structurally homologous to human CXCL8. In the murine genome, MIP2, KC, and LIX are generally considered functional homologues of human CXCL8 and CXCL1 [26]. In the mouse, MIP2 and KC are thought to act via binding of murine CXCR2; no murine homologue of the CXCR1 receptor has yet been identified [29, 30]. In addition to the ELR⫹ chemokines mentioned, a new ELR⫺ CXC chemokine that specifically recruits neutrophils, called lungkine, has also been identified [31]. Both CXCR1 and CXCR2 are major players in neutrophil recruitment in a wide range of inflammatory situations, within the context of host defense as well as pathoinflammatory diseases [32]. CXC-chemokine-mediated neutrophil recruitment and activation is the main component of host defense against a large number of invasive microorganisms including microorganisms such as Klebsiella [33], Escherichia [34–36], Nocardia [37], and Toxoplasma [35, 38]. Mice that are lacking CXCR2 are highly susceptible to infection with these pathogens due to their lack of neutrophil-mediated killing. Considerable effort has been focused on CXCR1 and CXCR2 as pharmaceutical targets for therapies for acute inflammatory conditions. Blockade of these receptors with various antibodies or chemical antagonists have proven to be effective in reducing acute inflammation [36, 39, 40]. While such studies are promising, the existence of these receptors on other cell types, such as endothelial cells and macrophages [41, 42], necessitates careful study to rule out possible side effects of these antagonistic reagents. CCR1 CCR1 was identified on both human and murine neutrophils several years ago, but it was unclear if this receptor was truly mediating neutrophil chemotaxis. Recently, several papers have emerged demonstrating that activated human neutrophils can upregulate this receptor [43, 44]. Two different proinflammatory

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cytokines, IFN-␥ and GM-CSF, have been shown to upregulate CCR1 and confer responsivity to CCR1 ligands in assays of intracellular calcium release and chemotaxis [43, 44]. CCR1 expression on activated neutrophils seems to be important in vivo, since CCR1 knockout mice have increased mortality when infected with Aspergillus fumigatus [30], a fungal pathogen controlled mainly by neutrophils. While CCR1 is necessary for neutrophil recruitment in this model of fungal infection, it is not necessary for neutrophil recruitment in helminthmediated keratitis [45], suggesting stimulus- or organ-specific usage of this chemokine receptor by the neutrophil. CCR2, CCR3, and CCR6 CCL2, the main ligand for CCR2, has generally been regarded as a mononuclear chemoattractant. Its prolonged expression during inflammatory processes is thought to explain the sustained influx of mononuclear cells, which express the CCR2 receptor, during chronic inflammation. However, recent evidence shows that in the context of chronic inflammation, intravascular neutrophils upregulate CCR2 [46]. In a model of adjuvant-induced chronic vasculitis, neutrophils were shown to have novel expression of CCR2 and to exhibit transendothelial migration into mesentery preparations injected with CCL2. In addition, CCL2 was able to induce chemotaxis in vitro of neutrophils isolated from adjuvant-immunized rats but not normal rats [46]. This provides a mechanism for the continued recruitment of neutrophils after the acute expression of CXC chemokines has resolved. CCR3, while generally considered an eosinophil and Th2 lymphocyte chemokine receptor, is also upregulated on IFN-␥-stimulated neutrophils. The main CCR3 ligand, CCL11, can stimulate chemotaxis in vitro [44], suggesting that in certain inflammatory environments, eotaxin may chemoattract neutrophils. However, recent studies suggest that the biological role of CCL11 involves a negative effect on neutrophil influx. In a model of complementinduced acute lung injury, neutralization of CCL11 resulted in a decrease in neutrophil recruitment into the lung [47] . Furthermore, CCL11 was shown to decrease the production of neutrophil-specific CXC chemokines by peritoneal macrophages in this model. In another study, CCL11 decreased the production of CXC chemokines from human endothelial cells [48], suggesting that this chemokine may affect neutrophil trafficking indirectly by suppressing neutrophil-specific chemokine production from stromal cells. It is quite possible that direct effects of CCL11 on neutrophils via the CCR3 receptor may also result in decreased tissue sequestration of neutrophils, by heterologous desensitization of CXC chemokine-induced migration, or by inducing a hyperadhesive phenotype in these cells. Such possibilities remain to be explored experimentally.

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CCR6 is another CC-type chemokine receptor that can be expressed by activated neutrophils. Neutrophils activated by TNF-␣ or conditioned medium from PHA-stimulated macrophages express increased amounts of surface CCR6, and display chemotactic migration in response to the CCR6 ligand CCL20 [49]. CCR6 is a receptor implicated in the trafficking of dendritic cells [50]. Since activated neutrophils also express CD83 [49], a marker for mature dendritic cells [51], it is possible that activated neutrophils can acquire phenotypic characteristics similar to dendritic cells during the course of their maturation. Other Receptors Neutrophils express several other chemokine receptors including HCCR-1, CXCR3, CXCR4, CXCR5, and CX3CR1 [52, 53]. However, the function of these chemokine receptors on neutrophils has not yet been elucidated. In addition, several chemokines that are active on neutrophils bind to receptors that are not yet identified. A recent report demonstrates the importance of the ELR⫺ CXC chemokine CXCL10 and the CC chemokine CCL7, in neutrophil recruitment to the lung, although the receptors involved have not been identified [54]. CXCL4 is a CXC chemokine that induces adhesion and exocytosis of neutrophils independently of the known chemokine receptors for this ligand [55]. This receptor and perhaps others remain to be identified.

Regulation of Chemokine Receptor Expression

Neutrophil expression of CXCR1 and CXCR2 is highly regulated by environmental factors. CXCR1 and CXCR2 can be downregulated by bacterial products [56], proinflammatory cytokines [57], and CD45 activation [58], while G-CSF can upregulate these two receptors [57]. Regulation of chemokine receptor expression may be a mechanism for resolution of acute inflammatory processes. TNF-␣ and LPS, while amplifying the inflammatory process, also result in downregulation of CXCR1 and CXCR2 expression [56]. This downregulation occurs via a metalloproteinase-dependent cleavage of surface receptors, and renders neutrophils less responsive to CXCL8 in terms of oxidant production and chemotaxis [59]. Local downregulation of chemokine receptors on elicited neutrophils is probably an important mechanism for resolution of the acute inflammatory response. However, when intravascular neutrophils are prematurely activated, such as occurs during bacterial sepsis, the resulting downregulation of CXC chemokine receptors may render these cells unable to exit the vascular space

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and lead to a defect in neutrophil-dependent innate immune responses. In fact, decreased neutrophil recruitment to peripheral tissues, such as the lung, is a commonly observed phenomenon in septic patients and leads to an increase in pulmonary infections [60]. The mechanisms behind this decreased migratory ability are thought to include downregulation of CXC receptors on intravascular neutrophils, as well as abolishment of the chemokine gradient between the tissues and the vascular space. However, it is unclear if this is the full story. Some groups report that abolishment of the MIP-2 gradient does not abolish CXCL8-stimulated neutrophil recruitment to the periphery [61, 62], suggesting that other factors may directly affect CXC-dependent neutrophil migration. The documentation of CC receptors on neutrophils is a fairly recent finding, so much remains to be elucidated about the relationship between the constitutively expressed CXC receptors and the activation-induced CC receptors on these cells. The functional relationships and relative signaling strengths between these receptors are not known. Since neutrophil activation can both downregulate CXC receptors and upregulate CC receptors, it seems likely that this dichotomy has important functional consequences during the course of an inflammatory process. When CXC-mediated neutrophil responses seem to be downregulated, such as during the course of sepsis, it is possible that CC chemokines could be functional substitutes. Such a switch in responsiveness might be therapeutically exploited by drugs targeting neutrophil hyporesponsiveness or hyperactivation.

Chemokine Networking during Acute Inflammation

A multitude of different neutrophil-active chemokines are produced during the course of an acute inflammatory process in vivo. The multiplicity of chemokines acting on neutrophils provides some functional redundancy to the system. For example, murine models have demonstrated that selective neutralization of a single CXC chemokine, such as MIP2 or KC, cause low or modest changes in the host response to infection with either Pseudomonas aeruginosa [63], Legionella, [64], or Nocardia asteroides [37], while CXCR2 blockade in these models causes striking defects in neutrophil recruitment and bacterial clearance. Therefore, it seems that the large number of CXCR2 ligands present in the mouse provide a level of functional redundancy that ensures a prompt and robust neutrophil response. In addition to their functionally redundant roles in neutrophil recruitment, the effects of the CXCR2 ligands KC and MIP2 on murine neutrophils may differ depending on their compartmental localization. Thioglycollate or glycogen-induced peritonitis leads to large local increases in MIP2, but large

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Table 2. Regulation of neutrophil-derived chemokines Neutrophil-derived chemokine

Chemotactic for

Stimulants for release

Suppressors of release

CXCL8

Neutrophils

IL-10

CCL3 CCL4 CCL2 CXCL10 Defensins-1, -2, -3

Monocytes Monocytes Monocytes T lymphocytes T lymphocytes, dendritic cells

TNF-␣, IL-1b, LPS, GM-CSF LPS LPS TNF-␣

IL-10, IL-13 IL-10

CXCL8

systemic increases in KC [65]. Because of this, MIP2 might be more relevant to the recruitment of neutrophils to the peritoneum, while the systemic increases in KC may desensitize CXCR2-mediated responses on intravascular neutrophils. This suggests that the host may be able to titrate the level of neutrophilic inflammation by controlling the ratio of systemic and local chemokine expression, resulting in a well-controlled system. In another example, in a model of cecal ligation and puncture-induced bacterial sepsis, significant increases in MIP2 were only seen in the lung, while increases in KC were only detected in the peritoneum [66]. Furthermore, MIP2 was found to be important in neutrophil recruitment, while KC was not tied to neutrophil recruitment, but rather exerted direct effects on hepatocyte function. Thus, compartmental specificity of CXC chemokine expression can lead to distinctly different functional profiles for each chemokine.

Neutrophil-Derived Chemokines Contribute to Inflammation

Neutrophils were once thought to be terminally differentiated, ‘mindless’ soldiers that are programmed strictly to phagocytose and kill infectious pathogens. However, in addition to their role as early-response phagocytic cells, neutrophils are also a source of many different cytokines and other mediators. Since neutrophils are often the first leukocyte type to infiltrate an inflammatory sight in large numbers, they have the potential to influence the course of the ongoing inflammatory process by secreting various substances, including chemokines (table 2). Neutrophils are an important integration point for various environmental signals, which can positively or negatively regulate neutrophil chemokine

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production. These cells are robust producers of CXCL8, a fact which results in rapid amplification of the acute inflammatory response by two different mechanisms: (1) recruitment of more neutrophils, and (2) prevention of apoptosis in recruited neutrophils [67, 68]. Many environmental factors stimulate neutrophil CXCL8 production. GM-CSF, itself an antiapoptotic agent, causes the release of CXCL8 [69]. Proinflammatory cytokines such as IL-1 and TNF-␣, as well as bacterial LPS [70], stimulate the production of CXCL8 [70]. During the course of a typical inflammatory response, the early infiltrating neutrophils are often eventually replaced with a mononuclear infiltrate. This shift in the type of leukocytes found at the inflammatory site usually signifies either the resolution of the lesion or a progression to chronic inflammation. A mechanism for this temporal shift may be provided by neutrophils present in the acute infiltrate. Neutrophils stimulated by the CXC chemokine CXCL8 release sIL-6R. Studies have shown that sIL-6 is an important modulator of leukocyte recruitment, since IL-6 knockout mice have increased MIP2 production and neutrophil recruitment upon inflammatory challenge [71]. Increased neutrophil recruitment in these mice may be mediated by direct effects on chemokine production. When sIL-6R is added together with IL-6 to peritoneal mesothelial cells, these factors suppress the cytokine-induced production of CXC chemokines (CXCL8 and CXCL1-␣) from peritoneal mesothelial cells, while concomitantly directly increasing the production of a CC chemokine (CCL2) by peritoneal mesothelial cells and endothelium [72, 73]. Thus, the local action of CXC chemokines on elicited neutrophils may lead to a suppression of more CXC chemokine production and the eventual release of a CC chemokine, making these cells important players in the cytokine cascade. In addition, other mononuclear cell-specific chemokines such as CCL3 [74], CCL4–1␤ [74] and CCL2 [75] are released by neutrophils when stimulated by LPS, proinflammatory cytokines, and meningococcal vesicles [76]. Secretion of these chemokines by neutrophils at the inflammatory site is thought to lead to the eventual influx of mononuclear cells. Neutrophil participation in cytokine networks may constitute a link between innate and acquired immunity. CXCL8-induced degranulation of neutrophils has been shown to cause the release of defensins, novel antibacterial peptides that disrupt the cytoplasmic membrane of microorganisms [77]. Defensins also act as chemoattractants for immature dendritic cells and T lymphocytes in vitro [78, 79], and have been shown to recruit T cells via binding to a chemokine receptor, CCR6 [77]. Furthermore, early neutrophil recruitment has been shown to be a prerequisite for later T cell influx in vivo, suggesting that neutrophil-derived T cell chemoattractants are indeed important in recruiting lymphocytes to inflammatory sights [78, 80]. Therefore, neutrophil-derived CXCL8 may act in an autocrine/paracrine loop to stimulate the release of

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neutrophil defensins, which may facilitate the acquired immune response via recruitment of dendritic and T cells. The mechanism of action of cytokines and drugs that modulate acute inflammation likely involves effecting neutrophil-derived chemokines. IL-13, IL-10, and IL-4, cytokines known to have potent immunomodulatory properties, exert some of their biological effects at the level of neutrophil activation by downregulating the release of neutrophil-derived chemokines [81, 82]. In addition, anti-inflammatory compounds, such as corticosteroids, suppress CXCL8 production in neutrophils [70], supporting the idea that neutrophil-derived chemokines play an important role in the development of inflammation.

Conclusions

Due to their large numbers early in the acute inflammatory response, neutrophils are ideally situated to influence the subsequent nature of the inflammatory process. While early studies suggested that neutrophils were fairly simple phagocytes that responded solely to CXC chemokines, recent findings have demonstrated that these initial characterizations were incomplete. Rather than being terminally differentiated cells, neutrophils are capable of synthesizing new proteins and acquiring new functionalities depending on the local environment. Some of these newly expressed proteins include chemokines and chemokine receptors, which may play important roles in the ultimate progression or resolution of inflammation. Of particular interest is the emerging data concerning the expression of CC chemokine receptors on neutrophils, and the apparent dichotomy between CXC- and CC-type chemokine receptors on these cells. It seems certain that continuing investigations into novel chemokine receptor expression on neutrophils will yield findings of therapeutic interest to a variety of inflammatory processes.

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Lee WL, Downey GP: Neutrophil activation and acute lung injury. Curr Opin Crit Care 2001;7:1. Heinzelmann M, Mercer-Jones MA, Passmore JC: Neutrophils and renal failure. Am J Kidney Dis 1999;34:384. Fujie K, Shinguh Y, Inamura N, Yasumitsu R, Okamoto M, Okuhara M: Release of neutrophil elastase and its role in tissue injury in acute inflammation: Effect of the elastase inhibitor, FR134043. Eur J Pharmacol 1999;374:117. Peichl P, Ceska M, Effenberger F, Haberhauer G, Broell H, Lindley IJ: Presence of NAP-1/IL-8 in synovial fluids indicates a possible pathogenic role in rheumatoid arthritis. Scand J Immunol 1991;34:333. Terui T, Ozawa M, Tagami: Role of neutrophils in induction of acute inflammation in T-cellmediated immune dermatosis, psoriasis: A neutrophil-associated inflammation-boosting loop. Exp Dermatol 2000;9:1. Kucharzik T, Walsh SV, Chen J, Parkos CA, Nusrat A: Neutrophil transmigration in inflammatory bowel disease is associated with differential expression of epithelial intercellular junction proteins. Am J Pathol 2001;159:2001. Linden A: Role of interleukin-17 and the neutrophil in asthma. Int Arch Allergy Immunol 2001; 126:179. Baggiolini M, Dewald B, Moser B: Human chemokines: An update. Annu Rev Immunol 1997; 15:675. Strieter RM, Polverini PJ, Arenberg DA, Walz A, Opdenakker G, Van Damme J, Kunkel SL: Role of C-X-C chemokines as regulators of angiogenesis in lung cancer. J Leukoc Biol 1995;57:752. Kunkel SL, Strieter RM, Chensue SW, Basha M, Standiford T, Ham J, Remick DG: Tumor necrosis factor-alpha, interleukin-8 and chemotactic cytokines. Prog Clin Biol Res 1990;349:433. Standiford TJ, Strieter RM, Kasahara K, Kunkel SL: Disparate regulation of interleukin 8 gene expression from blood monocytes, endothelial cells, and fibroblasts by interleukin 4. Biochem Biophys Res Commun 1990;171:531. Harada A, Sekido N, Akahoshi T, Wada T, Mukaida N, Matsushima K: Essential involvement of interleukin-8 (IL-8) in acute inflammation. J Leukoc Biol 1994;56:559. Lugering N, Kucharzik T, Gockel H, Sorg C, Stoll R, Domschke W: Human intestinal epithelial cells down-regulate IL-8 expression in human intestinal microvascular endothelial cells; role of transforming growth factor-beta 1 (TGF-beta1). Clin Exp Immunol 1998;114:377. Strieter RM, Chensue SW, Basha MA, Standiford TJ, Lynch JP, Baggiolini M, Kunkel SL: Human alveolar macrophage gene expression of interleukin-8 by tumor necrosis factor-alpha, lipopolysaccharide, and interleukin-1 beta. Am J Respir Cell Mol Biol 1990;2:321. Verhasselt V, Goldman M, Willems F: Oxidative stress up-regulates IL-8 and TNF-alpha synthesis by human dendritic cells. Eur J Immunol 1998;28:3886. Feniger-Barish R, Ran M, Zaslaver A, Ben-Baruch A: Differential modes of regulation of cxc chemokine-induced internalization and recycling of human CXCR1 and CXCR2. Cytokine 1999; 11:996. Huber AR, Kunkel SL, Todd RF 3rd, Weiss SJ: Regulation of transendothelial neutrophil migration by endogenous interleukin-8. Science 1991;254:99. DiVietro JA, Smith MJ, Smith BR, Petruzzelli L, Larson RS, Lawrence MB: Immobilized IL-8 triggers progressive activation of neutrophils rolling in vitro on P-selectin and intercellular adhesion molecule-1. J Immunol 2001;167:4017. Van Dervort AL, Lam C, Culpepper S, Tuschil AF, Wesley RA, Danner RL: Interleukin-8 priming of human neutrophils is not associated with persistently altered calcium fluxes but is additive with lipopolysaccharide. J Leukoc Biol 1998;64:511. Djeu JY, Matsushima K, Oppenheim JJ, Shiotsuki K, Blanchard DK: Functional activation of human neutrophils by recombinant monocyte-derived neutrophil chemotactic factor/IL-8. J Immunol 1990;144:2205. Richardson RM, Pridgen BC, Haribabu B, Ali H, Snyderman R: Differential cross-regulation of the human chemokine receptors CXCR1 and CXCR2. Evidence for time-dependent signal generation. J Biol Chem 1998;273:23830. Wolf M, Delgado MB, Jones SA, Dewald B, Clark-Lewis I, Baggiolini M: Granulocyte chemotactic protein 2 acts via both IL-8 receptors, CXCR1 and CXCR2. Eur J Immunol 1998;28:164.

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28

29

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31 32 33 34

35

36

37

38 39

40 41

42

43 44

45

46

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Wuyts A, Proost P, Lenaerts JP, Ben-Baruch A, Van Damme J, Wang JM: Differential usage of the CXC chemokine receptors 1 and 2 by interleukin-8, granulocyte chemotactic protein-2 and epithelial-cell-derived neutrophil attractant-78. Eur J Biochem 1998;255:67. Gao JL, Kuhns DB, Tiffany HL, McDermott D, Li X, Francke U, Murphy PM: Structure and functional expression of the human macrophage inflammatory protein 1 alpha/RANTES receptor. J Exp Med 1993;177:1421. Gao JL, Wynn TA, Chang Y, Lee EJ, Broxmeyer HE, Cooper S, Tiffany HL, Westphal H, Kwon-Chung J, Murphy PM: Impaired host defense, hematopoiesis, granulomatous inflammation and type 1-type 2 cytokine balance in mice lacking CC chemokine receptor 1. J Exp Med 1997; 185:1959. Rossi DL, Hurst SD, Xu Y, Wang W, Menon S, Coffman RL, Zlotnik A: Lungkine, a novel CXC chemokine, specifically expressed by lung bronchoepithelial cells. J Immunol 1999;162:5490. Matsukawa A, Hogaboam CM, Lukacs NW, Kunkel SL: Chemokines and innate immunity. Rev Immunogenet 2000;2:339. Standiford TJ, Kunkel SL, Strieter RM: Role of chemokines in antibacterial host defense. Methods Enzymol 1997;288:220. Godaly G, Bergsten G, Hang L, Fischer H, Frendeus B, Lundstedt AC, Samuelsson M, Samuelsson P, Svanborg C: Neutrophil recruitment, chemokine receptors, and resistance to mucosal infection. J Leukoc Biol 2001;69:899. Del Rio L, Bennouna S, Salinas J, Denkers EY: CXCR2 deficiency confers impaired neutrophil recruitment and increased susceptibility during Toxoplasma gondii infection. J Immunol 2001; 167:6503. Olszyna DP, Florquin S, Sewnath M, Branger J, Speelman P, van Deventer SJ, Strieter RM, van der Poll T: CXC chemokine receptor 2 contributes to host defense in murine urinary tract infection. J Infect Dis 2001;184:301. Moore TA, Newstead MW, Strieter RM, Mehrad B, Beaman BL, Standiford TJ: Bacterial clearance and survival are dependent on CXC chemokine receptor-2 ligands in a murine model of pulmonary Nocardia asteroides infection. J Immunol 2000;164:908. Bliss SK, Gavrilescu LC, Alcaraz A, Denkers EY: Neutrophil depletion during Toxoplasma gondii infection leads to impaired immunity and lethal systemic pathology. Infect Immun 2001;69:4898. Auten RL, Richardson RM, White JR, Mason SN, Vozzelli MA, Whorton MH: Nonpeptide CXCR2 antagonist prevents neutrophil accumulation in hyperoxia-exposed newborn rats. J Pharmacol Exp Ther 2001;299:90. McColl SR, Clark-Lewis I: Inhibition of murine neutrophil recruitment in vivo by CXC chemokine receptor antagonists. J Immunol 1999;163:2829. Addison CL, Daniel TO, Burdick MD, Liu H, Ehlert JE, Xue YY, Buechi L, Walz A, Richmond A, Strieter RM: The CXC chemokine receptor 2, CXCR2, is the putative receptor for ELR⫹ CXC chemokine-induced angiogenic activity. J Immunol 2000;165:5269. Bonecchi R, Facchetti F, Dusi S, Luini W, Lissandrini D, Simmelink M, Locati M, Bernasconi S, Allavena P, Brandt E, Rossi F, Mantovani A, Sozzani S: Induction of functional IL-8 receptors by IL-4 and IL-13 in human monocytes. J Immunol 2000;164:3862. Cheng SS, Lai JJ, Lukacs NW, Kunkel SL: Granulocyte-macrophage colony stimulating factor up-regulates CCR1 in human neutrophils. J Immunol 2001;166:1178. Bonecchi R, Polentarutti N, Luini W, Borsatti A, Bernasconi S, Locati M, Power C, Proudfoot A, Wells TN, Mackay C, Mantovani A, Sozzani S: Up-regulation of CCR1 and CCR3 and induction of chemotaxis to CC chemokines by IFN-gamma in human neutrophils. J Immunol 1999;162:474. Hall LR, Diaconu E, Patel R, Pearlman E: CXC chemokine receptor 2 but not C-C chemokine receptor 1 expression is essential for neutrophil recruitment to the cornea in helminth-mediated keratitis (river blindness). J Immunol 2001;166:4035. Johnston B, Burns AR, Suematsu M, Issekutz TB, Woodman RC, Kubes P: Chronic inflammation upregulates chemokine receptors and induces neutrophil migration to monocyte chemoattractant protein-1. J Clin Invest 1999;103:1269. Guo RF, Lentsch AB, Warner RL, Huber-Lang M, Sarma JV, Hlaing T, Shi MM, Lukacs NW, Ward PA: Regulatory effects of eotaxin on acute lung inflammatory injury. J Immunol 2001; 166:5208.

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48 49 50

51 52 53

54

55 56

57

58

59

60 61 62

63

64

65

66

67

Cheng SS, Lukacs NW, Kunkel SL: Eotaxin/CCL11 suppresses IL-8/CXCL8 secretion from human dermal microvascular endothelial cells. J Immunol 2002;168:2887. Yamashiro S, Wang JM, Yang D, Gong WH, Kamohara H, Yoshimura T: Expression of CCR6 and CD83 by cytokine-activated human neutrophils. Blood 2000;96:3958. Cook DN, Prosser DM, Forster R, Zhang J, Kuklin NA, Abbondanzo SJ, Niu XD, Chen SC, Manfra DJ, Wiekowski MT, Sullivan LM, Smith SR, Greenberg HB, Narula SK, Lipp M, Lira SA: CCR6 mediates dendritic cell localization, lymphocyte homeostasis, and immune responses in mucosal tissue. Immunity 2000;12:495. Zhou LJ, Tedder TF: Human blood dendritic cells selectively express CD83, a member of the immunoglobulin superfamily. J Immunol 1995;154:3821. Patel L, Charlton SJ, Chambers JK, Macphee CH: Expression and functional analysis of chemokine receptors in human peripheral blood leukocyte populations. Cytokine 2001;14:27. Combadiere C, Salzwedel K, Smith ED, Tiffany HL, Berger EA, Murphy PM: Identification of CX3CR1. A chemotactic receptor for the human CX3C chemokine fractalkine and a fusion coreceptor for HIV-1. J Biol Chem 1998;273:23799. Michalec L, Choudhury BK, Postlethwait E, Wild JS, Alam R, Lett-Brown M, Sur S: CCL7 and CXCL10 orchestrate oxidative stress-induced neutrophilic lung inflammation. J Immunol 2002; 168:846. Zucker MB, Katz IR: Platelet factor 4: Production, structure, and physiologic and immunologic action. Proc Soc Exp Biol Med 1991;198:693. Khandaker MH, Xu L, Rahimpour R, Mitchell G, DeVries ME, Pickering JG, Singhal SK, Feldman RD, Kelvin DJ: CXCR1 and CXCR2 are rapidly down-modulated by bacterial endotoxin through a unique agonist-independent, tyrosine kinase-dependent mechanism. J Immunol 1998; 161:1930. Asagoe K, Yamamoto K, Takahashi A, Suzuki K, Maeda A, Nohgawa M, Harakawa N, Takano K, Mukaida N, Matsushima K, Okuma M, Sasada M: Down-regulation of CXCR2 expression on human polymorphonuclear leukocytes by TNF-alpha. J Immunol 1998;160:4518. Mitchell GB, Khandaker MH, Rahimpour R, Xu L, Lazarovits AI, Pickering JG, Suria H, Madrenas J, Pomerantz DK, Feldman RD, Kelvin DJ: CD45 modulation of CXCR1 and CXCR2 in human polymorphonuclear leukocytes. Eur J Immunol 1999;29:1467. Khandaker MH, Mitchell G, Xu L, Andrews JD, Singh R, Leung H, Madrenas J, Ferguson SS, Feldman RD, Kelvin DJ: Metalloproteinases are involved in lipopolysaccharide- and tumor necrosis factor-alpha-mediated regulation of CXCR1 and CXCR2 chemokine receptor expression. Blood 1999;93:2173. Wagner JG, Roth RA: Neutrophil migration during endotoxemia. J Leukoc Biol 1999;66:10. Schleiffenbaum B, Fehr J, Odermatt B, Sperb R: Inhibition of leukocyte emigration induced during the systemic inflammatory reaction in vivo is not due to IL-8. J Immunol 1998;161:3631. Remick DG, Green LB, Newcomb DE, Garg SJ, Bolgos GL, Call DR: CXC chemokine redundancy ensures local neutrophil recruitment during acute inflammation. Am J Pathol 2001; 159:1149. Tsai WC, Strieter RM, Mehrad B, Newstead MW, Zeng X, Standiford TJ: CXC chemokine receptor CXCR2 is essential for protective innate host response in murine Pseudomonas aeruginosa pneumonia. Infect Immun 2000;68:4289. Tateda K, Moore TA, Newstead MW, Tsai WC, Zeng X, Deng JC, Chen G, Reddy R, Yamaguchi K, Standiford TJ: Chemokine-dependent neutrophil recruitment in a murine model of Legionella pneumonia: Potential role of neutrophils as immunoregulatory cells. Infect Immun 2001;69:2017. Call DR, Nemzek JA, Ebong SJ, Bolgos GR, Newcomb DE, Wollenberg GK, Remick DG: Differential local and systemic regulation of the murine chemokines KC and MIP2. Shock 2001; 15:278. Mercer-Jones MA, Shrotri MS, Peyton JC, Remick DG, Cheadle WG: Neutrophil sequestration in liver and lung is differentially regulated by C-X-C chemokines during experimental peritonitis. Inflammation 1999;23:305. Dunican A, Grutkoski P, Leuenroth S, Ayala A, Simms HH: Neutrophils regulate their own apoptosis via preservation of CXC receptors. J Surg Res 2000;90:32.

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81 82

83 84

Dunican AL, Leuenroth SJ, Grutkoski P, Ayala A, Simms HH: TNFalpha-induced suppression of PMN apoptosis is mediated through interleukin-8 production. Shock 2000;14:284. McCain RW, Dessypris EN, Christman JW: Granulocyte/macrophage colony-stimulating factor stimulates human polymorphonuclear leukocytes to produce interleukin-8 in vitro. Am J Respir Cell Mol Biol 1993;8:28. Wertheim WA, Kunkel SL, Standiford TJ, Burdick MD, Becker FS, Wilke CA, Gilbert AR, Strieter RM: Regulation of neutrophil-derived IL-8: The role of prostaglandin E2, dexamethasone, and IL-4. J Immunol 1993;151:2166. Xing Z, Gauldie J, Cox G, Baumann H, Jordana M, Lei XF, Achong MK: IL-6 is an antiinflammatory cytokine required for controlling local or systemic acute inflammatory responses. J Clin Invest 1998;101:311. Marin V, Montero-Julian FA, Gres S, Boulay V, Bongrand P, Farnarier C, Kaplanski G: The IL-6soluble IL-6Ralpha autocrine loop of endothelial activation as an intermediate between acute and chronic inflammation: An experimental model involving thrombin. J Immunol 2001;167:3435. Hurst SM, Wilkinson TS, McLoughlin RM, Jones S, Horiuchi S, Yamamoto N, Rose-John S, Fuller GM, Topley N, Jones SA: Il-6 and its soluble receptor orchestrate a temporal switch in the pattern of leukocyte recruitment seen during acute inflammation. Immunity 2001;14:705. Kasama T, Strieter RM, Standiford TJ, Burdick MD, Kunkel SL: Expression and regulation of human neutrophil-derived macrophage inflammatory protein 1 alpha. J Exp Med 1993;178:63. Yamashiro S, Kamohara H, Wang JM, Yang D, Gong WH, Yoshimura T: Phenotypic and functional change of cytokine-activated neutrophils: Inflammatory neutrophils are heterogeneous and enhance adaptive immune responses. J Leukoc Biol 2001;69:698. Lapinet JA, Scapini P, Calzetti F, Perez O, Cassatella MA: Gene expression and production of tumor necrosis factor alpha, interleukin-1beta (IL-1beta), IL-8, macrophage inflammatory protein 1alpha (MIP-1alpha), MIP-1beta, and gamma interferon-inducible protein 10 by human neutrophils stimulated with group B meningococcal outer membrane vesicles. Infect Immun 2000;68:6917. Yang D, Chertov O, Bykovskaia SN, Chen Q, Buffo MJ, Shogan J, Anderson M, Schroder JM, Wang JM, Howard OM, Oppenheim JJ: Beta-defensins: Linking innate and adaptive immunity through dendritic and T cell CCR6. Science 1999;286:525. Chertov O, Michiel DF, Xu L, Wang JM, Tani K, Murphy WJ, Longo DL, Taub DD, Oppenheim JJ: Identification of defensin-1, defensin-2, and CAP37/azurocidin as T-cell chemoattractant proteins released from interleukin-8-stimulated neutrophils. J Biol Chem 1996;271:2935. Yang D, Chen Q, Chertov O, Oppenheim JJ: Human neutrophil defensins selectively chemoattract naive T and immature dendritic cells. J Leukoc Biol 2000;68:9. Taub DD, Anver M, Oppenheim JJ, Longo DL, Murphy WJ: T lymphocyte recruitment by interleukin-8 (IL-8). IL-8-induced degranulation of neutrophils releases potent chemoattractants for human T lymphocytes both in vitro and in vivo. J Clin Invest 1996;97:1931. Kasama T, Strieter RM, Lukacs NW, Burdick MD, Kunkel SL: Regulation of neutrophil-derived chemokine expression by IL-10. J Immunol 1994;152:3559. Ohta TM, Kasama T, Hanyuuda M, Hatano Y, Kobayashi K, Negishi M, Ide H, Adachi M: Interleukin-13 down-regulates the expression of neutrophil-derived macrophage inflammatory protein-1 alpha. Inflamm Res 1998;47:361. Murphy PM: Neutrophil receptors for interleukin-8 and related CXC chemokines. Semin Hematol 1997;34:311. Zlotnik A, Yoshie O: Chemokines: A new classification system and their role in immunity. Immunity 2000;12:121.

Dr. Steven L. Kunkel, Department of Pathology, University of Michigan Medical School, 1301 Catherine Street, Ann Arbor, MI 48109-0602 (USA) Tel. ⫹1 734 936 1020, Fax ⫹1 734 764 2397, E-Mail [email protected]

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Cassatella MA (ed): The Neutrophil. Chem Immunol Allergy. Basel, Karger, 2003, vol 83, pp 95–114

Neutrophil Production of IL-12 and Other Cytokines during Microbial Infection Eric Y. Denkers, Laura Del Rio, Soumaya Bennouna Department of Microbiology and Immunology, College of Veterinary Medicine, Cornell University, Ithaca, N.Y., USA

The concept that neutrophils are an important source of cytokines such as IL-12 is a relatively new idea that is gaining increasing acceptance as more studies are reported. Polymorphonuclear leukocytes (PMN) are exquisitely tuned to respond to microbial challenge, and it is well-known that these cells accumulate rapidly (often within minutes or hours) and in large numbers at sites of infection. Neutrophils generally produce lower amounts of cytokine on a per cell basis than do macrophages or dendritic cells (DC) [1]. Nevertheless, PMN are by far the most common leukocyte type. The fact that they often vastly outnumber other cells at sites of infection argues very strongly that neutrophils are a physiologically important source of cytokines that play a critical role in determining the outcome of infectious disease. A number of recent studies, described in this chapter, provide compelling evidence that this is the case, particularly with regard to IL-12. The cytokine IL-12 itself is a 70-kD heterodimer composed of constitutive 35-kD and inducible 40-kD subunits and is produced by macrophages, DC and neutrophils [2]. The primary function of IL-12 is to promote IFN-
responses by driving differentiation of naïve T lymphocytes into Th1 cells, as well as by promoting NK cell production of this cytokine. IL-12 also plays an important role in CD8 T lymphocyte differentiation into cytolytic T cell effectors that can also be an important IFN-
source [2]. Whether IL-12 functions in Th1 differentiation to instruct cell fate through chromatin remodeling, or whether the cytokine selects T lymphocytes whose fate has been prespecified by master regulatory molecules such as T-bet is an area of current debate [3, 4]. Whatever the case, IL-12 is regarded as the central cytokine which bridges innate and acquired

Table 1. Microbes and microbial products inducing neutrophil IL-12

Organism

Ref. no.

Fungi C. albicans

80, 81

Protozoa T. gondii P. berghei ANKA T. cruzi

42, 43, 62 88 73

Bacteria LPS M. avium L. pneumophila

10, 12 84 82

Viruses Herpes simplex virus

99, 100

HIV

101

immunity. To the extent that neutrophils produce IL-12 during infection, these cells may also be regarded as crucially important in linking innate and acquired immunity. PMN are often described as short-lived cells that undergo apoptosis within 24 h of leaving the bone marrow [5]. This is thought to be a protective mechanism minimizing host tissue damage caused by release of toxic neutrophil mediators, particularly in the absence of ongoing infection. Nevertheless, TNF- and other proinflammatory cytokines can increase neutrophil life span [6–8]. Inasmuch as neutrophils themselves produce cytokines such as TNF- in response to microbial pathogens (see below), this pathway may function as an autocrine or paracrine loop, allowing extended PMN cytokine secretion and microbicidal effector function during infectious disease [9]. Neutrophils have been reported capable of secreting a wide spectrum of cytokines and chemokines [for thorough reviews, see 10, 11]. Production of IL-12 by human PMN was reported soon after discovery of this cytokine [12]. Many studies demonstrate production of cytokines (mostly proinflammatory) and chemokines (in particular, IL-8) by neutrophils in response to microbial stimulation. The number of reports directly demonstrating PMN IL-12 production during infection is rather limited (table 1), but in most of these cases there is evidence that neutrophil production of this cytokine has profound functional consequences. Work in our laboratory focuses on the ability of the protozoan Toxoplasma gondii and its antigens to stimulate neutrophil IL-12 production (fig. 1, 2). The list of microbes inducing neutrophil IL-12 synthesis is likely to grow as the capacity of PMN to produce this crucial immune-initiating

Denkers/Del Rio/Bennouna

96

600

IL-12 (p40)

TNF-

400

Cytokine (pg/ml)

500 300 400 200

300 200

100 100 0

0 Med

TZ

STAg

Med

TZ

STAg

Fig. 1. Production of IL-12 p40 and TNF- by mouse neutrophils during culture with T. gondii. Neutrophils were purified from the bone marrow of C57BL/6 mice using a Percoll gradient, then cultured with live tachyzoites (TZ; 1:2 ratio of parasites to cells) and soluble tachyzoite lysate antigen (STAg; 25 g/ml). After 18 h of culture, supernatants were collected for cytokine ELISA. Med  Medium.

b

a

Fig. 2. IL-12-positive neutrophils during T. gondii infection. Mouse peritoneal cells were obtained 6 h after i.p. injection of RH strain tachyzoites. Cells were subsequently fixed, permeabilized, stained with anti-IL-12 p40 antibody (shown in green), and nuclei were counterstained with propidium iodide (shown in red). a Arrows show tachyzoites within macrophages. These cells display little or no evidence of intracellular IL-12, in contrast to surrounding neutrophils. b Higher magnification of granulocytes showing punctate cytosolic staining for IL-12. The bar is equivalent to 5 m. This figure is reprinted with permission from Bliss et al. [62], copyright 2000, The American Association of Immunologists, Inc.

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97

cytokine becomes more fully appreciated. In this review, we discuss several emerging themes that collectively underscore the importance of neutrophils as a source of cytokines such as IL-12 during infectious disease.

Microorganisms Inducing Neutrophil Cytokine Production

As shown in table 1, although the number of infections in which PMN IL-12 production has been shown is relatively limited, the pathogens involved are diverse. This suggests that neutrophil IL-12 production may be a common feature of many microbial infections. In addition to the organisms in table 1, neutrophil depletion studies in mice reveal that the cells are involved in protective immunity to many additional microbial pathogens. For example, severity of infections with Aspergillus fumigatus, Strongyloides ratti, Yersinia enterocolitica, Chlamydia spp., Salmonella typhimurium and Listeria monocytogenes are increased in the absence of neutrophils [13–20]. PMN have been shown to be capable of releasing a wide spectrum of cytokines and chemokines in response to in vitro microbial stimuli [1, 10]. Thus, while neutrophils can act as microbicidal effectors through phagocytosis and release of toxic molecules, it seems likely that in many infections production of cytokines such as IL-12 will prove to be involved in the protective effects of PMN.

Triggering Pathways Involved in PMN Release of IL-12 and Other Cytokines

Intracellular signaling pathways leading to IL-12 and other cytokine production in PMN are not yet well-defined. Nevertheless, the Toll-like receptors (TLR) are likely to play an important role in recognizing pathogen-associated molecules, and this may involve activation of NFB family members and mitogen-activated protein kinase (MAPK) pathways that are present in PMN [21–24]. Ten TLR have so far been described in humans and mice [25]. TLR together mediate recognition of a diverse array of microbial products such as peptidoglycans, lipoproteins, bacterial flagellin, bacterial DNA and protozoal glycophosphoinositols. Neutrophils express TLR4 and CD14, which are involved in recognition of LPS [26, 27]. PMN have also been shown to express most or all other TLR, albeit at varying levels with respect to each other and to other cells such as macrophages [28]. Neutrophils in culture display spontaneous TLR2, but not TLR4, upregulation over time, and the early TLR2 increase can be prevented by the presence of LPS [27]. Thus, regulation of TLR during microbial infection is likely to be complex.

Denkers/Del Rio/Bennouna

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Gene knockout mice which lack the TLR adapter molecule MyD88 display defective T. gondii-induced neutrophil IL-12 production during infection [29]. DC and macrophage IL-12 production in response to T. gondii is also severely curtailed in the absence of MyD88. These results implicate TLR in Toxoplasma-induced IL-12 production by PMN, as well as other cell types. The identity of TLR involved in T. gondii recognition is not known, but neither TLR2 nor TLR4 are required for resistance to infection, unlike MyD88 or IL-12 itself [29]. While parasite-triggered neutrophil and DC IL-12 release is greatly reduced in the absence of MyD88, small but significant amounts of the cytokine are nonetheless produced. For the case of DC, this appears to be mediated by a novel pathway involving signaling by the parasite through chemokine receptor CCR5 [30]. Possibly, PMN can also be triggered to release IL-12 through this receptor. The NFB protein c-Rel is required for the ability of LPS to stimulate IL-12 production in macrophages [31]. It is therefore interesting that c-Relindependent IL-12 production occurs during T. gondii infection, and in particular that neutrophil expression of this cytokine is nondefective in c-Rel/ mice [32]. Combined with the above result, this suggests that MyD88-dependent signaling during Toxoplasma infection triggers a c-Rel-independent pathway leading to IL-12 production in neutrophils and other innate immune cells. Several studies have shown that PMN production of cytokines during microbial stimulation requires activation of MAPK. Using specific MAPK inhibitors, neutrophil IL-8 production in response to LPS, Mycoplasma fermentans membrane lipoproteins, type III group B streptococci, as well as TNF- and GM-CSF was found to be dependent upon p38 MAPK [22–24, 33]. The ERK1/2 and p38 MAPK are also involved in LPS and M. fermentans membrane lipoprotein-stimulated IL-8 production, as determined by inhibitor studies with PD98059 and SB203580 [22]. In addition to the role of TLR described above, PMN can be triggered to release cytokines during phagocytosis of opsonized microbial antigens. Neutrophils phagocytosing various bacterial microbes, including Salmonella typhimurium, Pseudomonas aeruginosa and Staphylococcus aureus, are induced to release high amounts of IL-8 and MIP-1 [34]. Both Fc
RIIa (CD32) and Fc
RIIIb (CD16) are expressed by PMN, and the high affinity receptor Fc
RI (CD64) is inducible with IFN-
[35]. The complement receptors CR1 (CD35) and CR3 (CD11b/CD18) may also mediate phagocytosis of pathogens [36]. Another way that the complement system can be involved in triggering PMN cytokine production is illustrated by studies on Cryptococcus neoformans. Capsular material from this fungal pathogen induces neutrophil IL-8 secretion by an indirect mechanism involving activation of C3a and C5a. The latter two complement components appear to be the direct mediators of IL-8 release in PMN [37].

Neutrophil Cytokine Production during Microbial Infection

99

Neutrophils express high and low affinity receptors for N-formylmethionyl-leucyl-phenylalanine (fMLP), and triggering with this formylated peptide has been reported to induce TNF- production in human PMN [38]. Nevertheless, G-protein-coupled receptors such as those that recognize fMLP have also been implicated in downregulating both TNF- and IL-12 production [39, 40]. Therefore, the importance of fMLP receptors in triggering neutrophil release of IL-12 and other cytokines is unclear, and indeed they may have a greater role in modulating proinflammatory cytokine responses by PMN and other cell types. TNF- alone stimulates transcription of IL-8 and MIP-1 genes, resulting in high level secretion of these chemokines in culture [34, 41]. Studies with the p38 MAPK inhibitor SB20358 suggest that TNF- as well as GM-CSFstimulated IL-8 production is dependent upon the activity of this MAPK [33]. In our laboratory, we found that T. gondii antigen stimulates PMN TNF- production, and also that recombinant TNF- added to human PMN is a potent inducer of MIP-1 and MIP-1 gene transcription [42]. In addition, transcription of the chemokines driven by T. gondii antigen is partially ablated in the presence of neutralizing anti-TNF- antibody. While TNF- alone is capable of inducing neutrophil production of the chemokines IL-8, MIP-1 and MIP-1 [34, 41, 42], it does not appear to be involved in eliciting IL-12. This is because addition of TNF- to human PMN fails to induce secretion of IL-12, and thioglycollate-elicited neutrophils from TNFRI knockout mice are capable of IL-12 production in response to protozoan stimulation [12, 43]. Related to these observations, IL-12, itself, possesses the capability of increasing PMN IL-8 production and release [44]. The cytokines IL-15 and IL-2, that have in common many biological activities and whose receptors are partially shared [45], stimulate PMN production of IL-8 [46–48]. A role for IL-18 in neutrophil activation, including production of IL-8, IL-1, and TNF-, is suggested by recent studies [49]. Together, these results suggest autoregulatory loops in which PMN are initially induced to release cytokines such as TNF- and IL-12. The cytokines, in turn, through binding to their receptors would induce synthesis of chemokines such as MIP-1/MIP-1 and IL-8. Thus, it seems likely that microbial infection will result in production of several key cytokines which can induce cytokine and chemokine secretion by PMN.

Cytokine Modulation of Neutrophil IL-12 Production

Regulation of IL-12 production has been most thoroughly examined with respect to triggering by LPS. Endotoxin stimulation of human PMN induces

Denkers/Del Rio/Bennouna

100

mRNA accumulation and secretion of p40 protein, but little biologically active IL-12 p70 is produced without addition of IFN-
. Induction of IL-12 p40 mRNA is distinct from that of TNF-, in that the former is not apparent before 20 h, whereas maximal levels of TNF- are achieved after only 1.5 h of culture. In our studies, T. gondii induces relatively high amounts of IL-12 relative to TNF-, but for LPS the reverse pattern of cytokine production occurs [42, 43]. These results indicate nonidentical control of these cytokines in PMN. While IFN-
promotes IL-12 release, its effects on other cytokines and chemokines produced by neutrophils are complex. IFN-
inhibits early induction of chemokines such MIP-1, MIP-1, and IL-8 [50–52]. For the case of MIP-1, this has been shown to occur through transcriptional inhibition [51]. Nevertheless, during extended cultures, IFN-
augments production of these same chemokines [52, 53]. Production of TNF- and IL-1 at all time points is enhanced by IFN-
, and this may underlie the enhancing effects of IFN-
when cytokine production is analyzed during longer-term culture periods [53]. Although IL-4 is most frequently associated with Th1 responses, this cytokine has been implicated in early Th1 response induction during Leishmania major infection [54]. IL-4 also appears required for development of protective Th1 responses during infection with the fungal pathogen Candida albicans, at least in part through effects on PMN [55]. In vivo expression of IL-12 p40 in macrophages and neutrophils can be blocked by administration of anti-IL-4 antibody in this system, and in IL-4–/– mice PMN do not upregulate IL-12 p40 unless given recombinant IL-4. In vitro priming with IL-4 greatly increases IL-12 release from neutrophils, and this correlates with upregulation of IL-4 receptor expression [55]. Production of IL-6 is also promoted by IL-4 priming in both PMN and macrophages. During toxoplasmosis, IL-4 displays either protective or exacerbatory roles, depending on the phase of infection, and the cytokine has been implicated in sustaining CD4 T lymphocyte IFN-
production [56, 57]. It is possible that these phenomena also stem from effects on neutrophils. IL-10, an important downregulator of inflammation, decreases levels of IL-12 mRNA and protein secretion by neutrophils [12], and has similar effects on secretion of chemokines such as MIP-1, MIP-1, and IL-8 [58]. The inhibitory effect of IL-10 requires de novo protein synthesis and is therefore only apparent after several hours of in vitro culture [58]. Full PMN responsiveness to IL-10 is acquired only after exposure to LPS, and correlates with upregulation of the IL-10R1 component of the IL-10 receptor complex [59]. Subsequent Stat3 tyrosine phosphorylation leads to inhibition of proinflammatory cytokines by still poorly defined mechanisms.

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Preformed versus Newly Synthesized Cytokines

A number of reports indicate that neutrophils express cytokines as preformed stores without the need for prior microbial stimulation. In particular, IL-12, IL-6, and MIP-2 appear to be present in mouse neutrophils in the absence of infection [60–62], and IL-4 has been reported in PMN from normal human donors [63]. This has important implications for the relative role of PMN as a significant source of cytokines during the early immune response to microbial infection. For the case of T. gondii, we found that intraperitoneal tachyzoite injection induces a rapid influx of IL-12-positive PMN (fig. 2). However, although neutrophils are only a minor population in control animals injected with endotoxinfree PBS, approximately 75% of these PMN are IL-12-positive [62]. A similar result was found in noninjected animals, ruling out the possibility that injection alone provides a stimulus for PMN IL-12 induction. Furthermore, approximately 40% of the neutrophils in peripheral blood from noninfected mice are IL-12-positive, arguing against exit from the peripheral blood as a stimulus for IL-12 synthesis [62]. The MIP-2 chemokine, which neutrophils also express as preformed stores, is important because it is one of the major mediators that mobilize PMN during infection [64–66]. Expression of MIP-2 mRNA in normal mice appears largely confined to neutrophils in the bone marrow, while both bone marrow and splenic neutrophils express MIP-2 protein [60]. However, after Yersinia enterocolitica infection there is a large increase in splenic PMN with MIP-2 mRNA and protein. These results suggest that MIP-2 gene expression occurs during PMN maturation to generate cells with preformed MIP-2. Nevertheless, during infection neutrophils retaining MIP-2 mRNA expression are recruited to the spleen. In addition, TNF- was found to upregulate MIP-2 expression [60]. These results suggest that mature PMN contain preformed MIP-2, but that in addition expression of this chemokine can be induced or maintained by mediators such as TNF-, or by microbial antigen itself. Neutrophil IL-12 is likely regulated in a similar manner, because in addition to preformed cytokine, we have found that thioglycollate-elicited PMN upregulate IL-12 p40 mRNA during in vitro stimulation with T. gondii antigen [43, 62]. Together, these results support the concept that PMN arrive at the site of infection or inflammation equipped to immediately release certain cytokines and chemokines. Sustained production dependent upon gene induction and protein synthesis can also occur, and this may be promoted by neutrophil survival cytokines such as TNF- and GM-CSF. Neutrophils possess at least four distinct types of granules (azurophil, specific, gelatinase, and secretory) which contain unique sets of polypeptides,

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and whose formation is coordinated with cell maturation [67–69]. Expression of IL-12 is associated with Ly6Ghigh cells, suggesting that acquisition of this cytokine is a late event in neutrophil maturation [62, 70]. By morphological examination, IL-6 and MIP-2 expression is also associated with mature PMN [60, 61]. The results suggest that cytokines such as IL-12, IL-6 and MIP-2 may be stored within secretory or possibly gelatinase granules of the neutrophil, whose contents would be released when appropriately stimulated. However, immunoelectron microscopy will be required to determine the precise location of preformed cytokines in PMN.

Neutrophil Subsets

Most studies on PMN cytokine production suggest these cells as a source of proinflammatory mediators such as IL-12 [10]. Recent studies also indicate that PMN can produce IFN-
[71, 72]. Neutrophils may also produce Th2 cytokines such as IL-10 and IL-4 [63, 73, 74], and in some PMN studies (described below) neutrophils are implicated in promoting Th2 responses. This raises the question of whether there exist discrete neutrophil subsets that can direct Th1 or Th2 responses based upon distinct cytokine-production profiles. There is clear variation in neutrophil surface markers such as Gr-1 and F4/80, suggestive of discrete PMN populations [62, 65]. It is not clear whether this reflects distinct PMN lineages, or – perhaps more likely – different neutrophil activation states. Regardless, it is possible that the phenotypic variability is an indication of underlying functional heterogeneity, and this may include cytokine-secreting potential. Other antigen-presenting cells (APC), most notably DC, can be divided into subpopulations that display potential to direct either Th1 or Th2 responses, but it is not yet clear how this occurs. It has been suggested that Th1- and Th2inducing DC exist as separate developmental lineages [75, 76]. Other studies provide evidence that DC activation status, determined by pathogen-derived signals or the cytokine microenvironment, determines whether DC of a given lineage induce Th1 or Th2 differentiation [77–79]. These issues are currently a matter of debate, and they may also apply to neutrophils. Studies on Candida infection support the concept that neutrophils can be induced to release either pro- or anti-inflammatory cytokines, depending upon antigenic stimulus. Thus, mouse PMN cultured with a nonhealer C. albicans strain produce IL-10, while an attenuated yeast strain induces IL-12 [74, 80]. In similar fashion, PMN isolated from mice genetically resistant to Trypanosoma cruzi were reported to produce IL-12 p40 and IFN-
, in contrast to cells from susceptible animals [73]. As described above, a subset of PMN from noninfected

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mice express preformed stores of IL-12, raising the question of whether the remaining IL-12-negative neutrophils express other cytokines. Additional studies suggest that IL-10 and IL-4 may also be present in neutrophils in the absence of infection, although in our own work we do not detect PMN positive for these particular cytokines [62, 63, 74]. Existence of distinct cytokinesecreting neutrophil subsets, and how they might be generated, remain areas to be explored.

Influence of PMN on T Cell Subset Selection during Infection

Neutrophils produce IL-12 during microbial infection, but is this physiologically relevant? Studies on several bacterial, fungal and protozoan infections suggest dysregulated T cell responses in the absence of PMN, consistent with a loss of an IL-12 source. Some of the most compelling evidence for the ability of PMN to influence immunity through cytokine production comes from the pioneering studies of Romani et al. [81] working in a C. albicans model system. Depletion of PMN in mice infected with a healing Candida strain results in increased susceptibility and emergence of IL-4 CD4 T lymphocytes that are otherwise undetectable [80]. When PMN-depleted animals are reconstituted with recombinant IL-12 emergence of Th2 cells is prevented, in vitro CD4 IFN-
production is increased, and resistance is restored [74]. Parallel findings have been reported in neutrophil-depleted mice undergoing infection with Legionella pneumophila, in which IL-12-positive PMN have been identified [82]. Thus, the normally strong Th1 response in the lungs of infected animals, characterized by high amounts of IL-12 and IFN-
, is substantially decreased in neutropenic mice [82]. This is accompanied by corresponding increases in IL-4 levels and susceptibility. Although IL-12 therapy has no effect on resistance in neutrophil-depleted mice undergoing L. pneumophila infection, IFN-
administration reverses susceptibility of the animals. Neutrophils also appear to play a protective nonphagocytic role in systemic Mycobacterium tuberculosis infection. Neutropenic mice display higher numbers of bacteria in the liver, and this is associated with lower levels of IFN-
and inducible nitric oxide synthase mRNA [83]. Of relevance to these observations, splenic neutrophils from mice infected with the related microorganism Mycobacterium avium release IL-12, as well as TNF- and IL-1, when cultured with M. avium in vitro [84]. PMN display immunoregulatory properties during T. cruzi infection in a manner that is mouse-strain-dependent. Peritoneal exudate PMN from resistant

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BALB/c animals display IL-12 p40 mRNA, in apparent contrast to PMN from the genetically susceptible C57BL/6 mouse strain [73]. Neutrophil depletion of BALB/c mice exacerbates infection. This is accompanied by decreased Th1 cytokines (IL-12 p40, IFN-
, TNF-) and increases in the IL-10 response in spleens of infected animals, suggesting a Th1 to Th2 switch [73]. In contrast, during T. cruzi infection of susceptible C57BL/6 mice, neutrophil depletion induces resistance to disease and enhances Th1 cytokine production. Neutrophils have also been implicated in induction of the CD4 Th2 response in L. major-infected BALB/c mice [85]. Thus, the early burst of IL-4 that instructs Th2 differentiation in this model is prevented by neutrophil depletion, and draining lymph node T cells remain IL-12-responsive, unlike cells from nondepleted mice. A slightly different effect occurs in PMN-depleted mice undergoing T. gondii and Plasmodium berghei infections. In these cases, while Th1 responses are decreased, there is no corresponding increase in Th2 cytokines. We found that during acute Toxoplasma infection in neutrophil-depleted mice, both Th1 (IFN-
, TNF-, IL-12 p40) and Th2-associated cytokines (IL-10) are lower (fig. 3), resulting in lethal systemic pathology and increased parasite numbers [86]. In these studies, we also found that neutrophil depletion leads to decreases in splenic populations of CD4 and CD8 T lymphocytes, as well as NK1.1 cells during infection, although the basis for this has yet to be investigated. Neutrophil depletion prevents development of cerebral malaria caused by P. berghei ANKA and decreases sequestration of monocytes and microhemorrhage in the brain [87]. This is associated with downregulated levels of IFN-
, TNF-, IL-2, and IL-12 p40 in the brain, with no corresponding Th2 cytokine increases. Peritoneal exudate neutrophils, obtained after intraperitoneal injection of P. berghei ANKA-parasitized red blood cells, contain IL-12 p40 mRNA, but it is not clear whether this represents parasite-induced or preformed IL-12 message [88]. Likewise, the PMN were found also to express mRNA for IL-18, IL-10, IFN-
and TNF-, and the Th1 chemoattractants MIG, MIP-1, and IP-10. In studies on T. gondii, M. tuberculosis, and C. albicans, depletion of neutrophils has an effect only when treatment is performed during early infection [80, 83, 86]. Neutrophil depletion initiated 2 or more days after infection does not generally exacerbate the course of disease, suggesting that neutrophils exert their protective effects during initiation of the immune response. Interestingly, when mice are immunized with a low virulence Candida strain, then neutrophil-depleted immediately prior to challenge, protective immunity is abrogated. This suggests that PMN may also exert effects on T cell effector function [80].

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Fig. 3. Defective cytokine production from cultured splenocytes of neutrophildepleted mice. C57BL/6 mice were administered either anti-Gr-1 (Ly6G) antibody or a control rat IgG on days 2, 0, 2, and 4, and i.p. infected with ME49 strain T. gondii cysts on day 0. On days 2, 4, 6, and 8 postinfection, mice were euthanized, and spleens collected for culture. Splenocytes were incubated in medium for 3 days, then cell-free supernatants were harvested for cytokine level determination by ELISA. 䊉  Mice administered control antibody; 䊊  neutrophil-depleted mice. This figure is adapted from data in Bliss et al. [86] and is published with permission from the American Society of Microbiology.

PMN depletion late during overwhelming C. albicans infection results in an increase in resistance [80], possibly because a fungal septic shock response mediated by dysregulated PMN cytokine release is avoided [80]. We found a similar ameliorating effect of neutrophil depletion using a cytokine shock model in which mice are sensitized to endotoxin with D-galactosamine (D-gal). Thus, injection of T. gondii-soluble lysate into D-gal-sensitized animals causes rapid death mediated by IL-12, TNF-, and IL-
. However, PMN depletion rescues mice from the lethal cytokine shock response induced by administration of T. gondii antigen to D-gal-sensitized mice [89]. The above studies provide convincing data that neutrophils exert immunoregulatory effects on T cell subset selection, particularly with regard to Th1 generation. Nevertheless, some caution must be exercised in interpreting results in which the RB6-8C5 monoclonal antibody, which recognizes Ly6G [90], is used to deplete PMN. This is because the antibody also removes a subset of CD8 T cells, and the Ly6G molecule has been reported to occur on the murine equivalent of plasmacytoid DC that produce IFN- during viral infection [20, 91]. There is clearly a need to develop alternative neutrophildepleting antibodies to completely evaluate the in vivo immunoregulatory effects of PMN on developing T cell responses.

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Neutrophils as Triggers of Immunity

Substantial evidence suggests that neutrophils may influence the developing T cell response during infection, but it is not yet clear how or where this might occur. One straightforward possibility is that PMN in secondary lymphoid organs could play a role in T cell differentiation by acting as cytokine-producing APC. Neutrophils have been reported capable of expressing MHC class II antigens as well as costimulatory molecules such as CD28 [92, 93]. Insofar as they also produce IL-12, neutrophils possess the potential capability of delivering the three obligatory signals required to generate polarized T cell subsets during infection [78]. It is also possible that PMN IL-12 release in the lymphoid microenvironment could polarize T cells that are activated by cognate interaction with APC such as DC or macrophages. Arguing against these models are observations that neutrophils comprise only a minor population in lymphoid organs, and that infection is not usually associated with major PMN increases in draining lymphoid tissues. Alternative hypothetical models, which are not necessarily mutually exclusive, incorporate the well-known ability of neutrophils to rapidly localize to sites of infection during microbial pathogenesis (fig. 4). This is mediated by PMN CXCR2 that would recognize CXCR2 ligands (e.g., IL-8, MIP-2) present as chemokine gradients in infected tissues [64–66, 94]. In turn (fig. 4a), neutrophils can be triggered to produce chemokines such as MIP-3, MIP-1 and MIP-1 that are chemoattractants of immature DC [95, 96]. Subsequent PMN-driven DC recruitment to centers of infection would thereby promote contact between microbial antigens and APC (fig. 4a). Pathogen-triggered PMN cytokine and chemokine release, as well as microbial antigen itself, could promote DC maturation and optimal activation [97]. In addition to TNF-, IL-12 may be involved in this process [98]. Alternatively, neutrophils that phagocytose microbial antigen could themselves be engulfed by DC, providing a source of antigen and activation cues for the latter. The DC, primed by neutrophils and armed with antigen, might then travel to lymphoid tissues where they would promote T cell activation and subset selection (fig. 4a). Alternatively (fig. 4b), neutrophils recruited to a focus of infection might initially release IL-12 in response to microbial pathogen. Then, as newly generated antigen-specific T cell effectors are recruited from the secondary lymph nodes to the infection site, neutrophil IL-12 might play a role in stabilizing the Th1 phenotype. Antigen presentation to T cells would subsequently result in IFN-
release at the site of infection. In these ways, neutrophils, through release of cytokines and chemokines, might play a master role in orchestrating the innate immune response that in turn influences development of acquired immunity.

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a

Blood

Site of infection Microbial pathogen

CXCR2-dependent PMN recruitment MIP-1, MIP-1 Recruitment and IL-12, MIP-3 activation of DC; TNF- migration to 2º lymphoid organs

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CXCR2-dependent PMN recruitment IFN-
IL-12 IL-12

Phenotype stabilization of newly arrived Th1 cells; IFN-
release in response to APC Ag

IL-12

IFN-


IFN-


Fig. 4. Instructive role of neutrophils in peripheral tissues during the early immune response. a Production of CXCR2-binding chemokines in infected tissues would result in rapid neutrophil recruitment. In response to microbial pathogen PMN would release cytokines such as IL-12 and TNF-, and chemokines such as MIP-1, MIP-1, and MIP-3, at least in some cases from preformed stores. Chemokine release could promote recruitment of immature DC, maximizing their exposure to microbial antigens. PMN cytokines and chemokines, acting in addition to, or in combination with, microbial antigens could activate DC. Activated DC, armed with peptide antigens, would migrate to the draining lymph nodes to initiate T cell responses. b Neutrophils, recruited to the site of infection, would release IL-12 in response to microbial antigen. Antigen-specific activated T cells would soon begin to arrive at the focus of infection, and Th1 phenotypic stabilization might be promoted by PMN-derived IL-12. These T cells would release effector cytokines such as IFN-
in response to peptide antigen presented by tissue APC.

Conclusions and Future Directions

The simple fact that neutrophils are a source of cytokines and chemokines argues, albeit not particularly elegantly, that these cells play a role in cytokine immunoregulation during infection. The data described in this chapter provide compelling supportive evidence that this is the case. Nevertheless, a challenge

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for the future is to determine precisely how this occurs, and to define the molecular control points in neutrophils that lead to protection or pathology during microbial infection. Such an understanding can be expected to lead to new strategies of intervention to improve the outcome of infectious disease in the clinical setting. Acknowledgments We thank Dr. George Yap (Brown University) for critical review of this manuscript, and members of the Denkers laboratory for insightful discussions. We apologize to scientists whose work was not cited due to space limitations. LDR is supported by a Fulbright Fellowship from the Council for International Exchange of Scholars, and our work on neutrophils is supported by NIH grant AI47888.

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in response to pulmonary infection with Norcardia asteroides. J Leukoc Biol 2002;72:373–381. Yeaman GR, Collins JE, Currie JK, Guyre PM, Wira CR, Fanger MW: IFN-gamma is produced by polymorphonuclear neutrophils in human uterine endometrium and by cultured peripheral blood polymorphonuclear neutrophils. J Immunol 1998;160:5145–5153. Chen L, Watanabe T, Watanabe H, Sendo F: Neutrophil depletion exacerbates experimental Chagas’ disease in BALB/c, but protects C57BL/6 mice through modulating the Th1/Th2 dichotomy in different directions. Eur J Immunol 2001;31:265–275. Romani L, Mencacci A, Cenci E, Spaccapelo R, Del Sero G, Nicoletti I, Trinchieri G, Bistoni F, Puccetti P: Neutrophil production of IL-12 and IL-10 in candidiasis and efficacy of IL-12 therapy in neutropenic mice. J Immunol 1997;158:5349–5356.

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Maldonado-Lopez R, De Smedt T, Michel P, Godfroid J, Pajak B, Heirman C, Thielemans K, Leo O, Urbain J, Moser M: CD8alpha and CD8alpha– subclasses of dendritic cells direct the development of distinct T helper cells in vivo. J Exp Med 1999;189:587–597. Pulendran B, Smith JL, Caspary G, Brasel K, Pettit D, Maraskovsky E, Maliszewski CR: Distinct dendritic cell subsets differentially regulate the class of immune response in vivo. Proc Natl Acad Sci USA 1999;96:1036–1041. MacDonald AS, Straw AD, Bauman B, Pearce EJ: CD8– dendritic cell activation status plays an integral role in influencing Th2 response development. J Immunol 2001;167:1982–1988. Kalinski P, Hilkens CMU, Wierenga EA, Kapsenberg ML: T-cell priming by type-1 and type-2 polarized dendritic cells: The concept of a third signal. Immunol Today 1999;20:561–567. Maldonado-Lopez R, Moser M: Dendritic cell subsets and the regulation of Th1/Th2 responses. Semin Immunol 2001;13:275–282. Romani L, Mencacci A, Cenci E, Del Sero G, Bistoni F, Puccetti P: An immunoregulatory role for neutrophils in CD4 T helper subset selection in mice with candidiasis. J Immunol 1997;158: 2356–2364. Romani L, Bistoni F, Puccetti P: Initiation of T-helper cell immunity to Candida albicans by IL-12: The role of neutrophils. Chem Immunol 1997;68:110–135. Tateda K, Moore TA, Deng JC, Newstead MW, Zeng X, Matsukawa A, Swanson MS, Yamaguchi K, Standiford TJ: Early recruitment of neutrophils determines subsequent T1/T2 host responses in a murine model of Legionella pneumophila pneumonia. J Immunol 2001;166: 3355–3361. Pedrosa J, Saunders BM, Appelberg R, Orme IM, Silva MT, Cooper AM: Neutrophils play a protective nonphagocytic role in systemic Mycobacterium tuberculosis infection of mice. Infect Immun 2000;68:577–583. Petrovsky M, Bermudez LE: Neutrophils from Mycobacterium avium-infected mice produce TNF-alpha, IL-12, and IL-1 beta and have a putative role in early host response. Clin Immunol 1999;91:354–358. Tacchini-Cottier F, Zweifel C, Belkaid Y, Mukankundiye C, Vasei M, Launois P, Milon G, Louis J: An immunomodulatory function for neutrophils during the induction of a CD4 Th2 response in BALB/c mice infected with Leishmania major. J Immunol 2000;165:2628–2636. Bliss SK, Gavrilescu LC, Alcaraz A, Denkers EY: Neutrophil depletion during Toxoplasma gondii infection leads to impaired immunity and lethal systemic pathology. Infect Immun 2001;69: 4898–4905. Chen L, Zhang ZH, Sendo F: Neutrophils play a critical role in the pathogenesis of experimental cerebral malaria. Clin Exp Immunol 2000;120:125–133. Chen L, Sendo F: Cytokine and chemokine mRNA expression in neutrophils from CBA/NSlc mice infected with Plasmodium berghei ANKA that induces experimental cerebral malaria. Parasitol Int 2001;50:139–143. Marshall AJ, Denkers EY: Toxoplasma gondii triggers granulocyte-dependent, cytokine-mediated lethal shock in D-galactosamine sensitized mice. Infect Immun 1998;66:1325–1333. Fleming TJ, Fleming ML, Malek TR: Selective expression of Ly-6G on myeloid lineage cells in mouse bone marrow. RB6-8C5 mAb to granulocyte-differentiation antigen (Gr-1) detects members of the Ly-6 family. J Immunol 1993;151:2399–2408. Nakano H, Yanagita M, Gunn MD: CD11c()B220()Gr-1() cells in mouse lymph nodes and spleen display characteristics of plasmacytoid dendritic cells. J Exp Med 2001;194: 1171–1178. Venuprasad K, Banerjee PP, Chattopadhyay S, Sharma S, Pal S, Parab PB, Mitra D, Saha B: Human neutrophil-expressed CD28 interacts with macrophage B7 to induce phosphatidylinositol 3-kinase-dependent IFN-
secretion and restriction of Leishmania growth. J Immunol 2002;169: 920–928. Gosselin EJ, Wardwell K, Rigby WF, Guyre PM: Induction of MHC class II on human polymorphonuclear neutrophils by granulocyte/macrophage colony-stimulating factor, IFN-gamma, and IL-3. J Immunol 1993;151:1482–1490. Frendeus B, Godaly G, Hang L, Karpman D, Svanborg C: Interleukin-8 receptor deficiency confers susceptibility to acute pyelonephritis. J Infect Dis 2001;183:S56-S60.

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95 Scapini P, Laudanna C, Pinardi C, Allavena P, Mantovani A, Sozzani S, Cassatella MA: Neutrophils produce biologically active macrophage inflammatory protein-3 (MIP-3)/CCL20 and MIP-3/CCL19. Eur J Immunol 2001;31:1981–1988. 96 Vecchi A, Massimiliano L, Ramponi S, Luini W, Bernasconi S, Bonecchi R, Allavena P, Parmentier M, Mantovani A, Sozzani S: Differential responsiveness to constitutive vs. inducible chemokines of immature and mature mouse dendritic cells. J Leukoc Biol 1999;66:489–494. 97 Bennouna S, Denkers EY: Neutrophils orchestrate early recruitment and activation of dendritic cells during microbial infection, in preparation. 98 Grohmann U, Belladonna ML, Bianchi R, Orabona C, Ayroldi E, Fioretti MC, Puccetti P: IL-12 acts directly on DC to promote nuclear localization of NF-B and primes DC for IL-12 production. Immunity 1998;9:315–323. 99 Daheshia M, Kanangat S, Rouse BT: Production of key molecules by ocular neutrophils early after herpetic infection of the cornea. Exp Eye Res 1998;67:619–624. 100 Kanangat S, Thomas J, Gangappa S, Babu JS, Rouse BT: Herpes simplex virus type 1-mediated up-regulation of IL-12 (p40) mRNA expression. Implications in immunopathogenesis and protection. J Immunol 1996;156:1110–1116. 101 Vecchiarelli A, Monari B, Palazzetti B, Bistoni F, Casadevall A: Dysregulation in IL-12 secretion by neutrophils from HIV-infected patients. Clin Exp Immunol 2000;121:311–319.

Dr. E.Y. Denkers, Department of Microbiology and Immunology, College of Veterinary Medicine, Cornell University, Ithaca, NY 14853–6401 (USA) Tel. 1 607 253 4022, Fax 1 607 253 3384, E-Mail [email protected]

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Cassatella MA (ed): The Neutrophil. Chem Immunol Allergy. Basel, Karger, 2003, vol 83, pp 115–145

Novel Pathways and Endogenous Mediators in Anti-Inflammation and Resolution Charles N. Serhan, Bruce Levy Center for Experimental Therapeutics and Reperfusion Injury, Department of Anesthesiology, Perioperative and Pain Medicine, Brigham and Women’s Hospital, Harvard Medical School, Boston, Mass., USA

Many endogenous chemical mediators are known to orchestrate the host response controlling the inflammatory response which involves the recognition of self and nonself by leukocytes [1, 2]. These chemical mediators or ‘signals’ regulate the traffic of leukocytes and the cardinal signs of inflammation. The classic eicosanoids such as prostaglandins and leukotrienes play key roles and exert a wide range of actions in responses of interest in host defense and inflammation (fig. 1) [3]. The scope and range of chemical mediators identified has, in recent years, expanded considerably [2] to include novel lipid mediators [4, 5], new cytokines and chemokines, gases such as nitric oxide and carbon monoxide [reviewed in 6, 7], and reactive oxygen species as well as new roles for nucleotides as mediators such as adenosine [8]. Among the most recent to be uncovered in this diverse class of compounds is inosine monophosphate, which downregulates neutrophil (PMN) trafficking [9] (see http://serhan.bwh.harvard.edu). When generated in elevated levels as in human disease, many of these small molecules or chemical signals are thought to be ‘proinflammatory’, generated to promote and amplify inflammation. Uncontrolled, these compounds in excess are thought to lead to chronic inflammation (fig. 1). Recent results from the author’s laboratory [7, 10, 11] and now from other laboratories indicate that endogenous lipid-derived mediators are generated to dampen the host response and orchestrate resolution [2, 12, 13]. The lipoxins (LX) were the first and defining members to be identified and recognized as endogenous anti-inflammatory lipid mediators of resolution [14] (fig. 1). In this regard, these compounds can function as ‘braking signals’ for PMN and

Resolution of inflammation Host defense Chemical mediators

PMN infiltration

Acute inflammation Chemical mediators Amplification

Chemical mediators ‘New roles and uncharted terrain’

Chronic inflammation Prostaglandins Leukotrienes

Resolution

Time

Lipoxins • ‘Stop’ PMN • Stimulate monocyte • Macrophage clearance

Fig. 1. Chemical mediators in inflammation resolution: counterregulatory signals.

eosinophils during local inflammation to direct specific subtypes of leukocyte traffic to a proresolution or homeostatic state [10]. It is of particular interest that aspirin (ASA), a widely used NSAID with many beneficial properties [15] in addition to its well-appreciated action to inhibit prostaglandins [16], also triggers the endogenous generation of 15-epimeric LX via acetylation of cyclooxygenase-2 (COX-2) at sites of inflammation in vivo [17] (see below) that carry both anti-inflammatory and antiproliferative actions [11, 18, 19]. This pathway is a previously unappreciated and completely novel mechanism of drug action for a therapeutic agent that has intriguing implications for targeted structure-based drug design as well as the targeted use of ASA itself. But more importantly, results from these investigations and those reviewed below help to further illustrate the importance of endogenous generation of lipid mediators with anti-inflammatory proresolving properties. A traditional approach to developing anti-inflammatory drugs, as in other human conditions amenable to pharmacologic interventions, is the use of inhibitors and/or receptor antagonists directed at the ‘proinflammatory’ mediators. This approach namely the focus on making synthetic inhibitors of enzymes and receptor level antagonists has and continues to enjoy considerable clinical and commercial successes [2, 20], but are not without significant unwanted side effects [21–23]. Therefore, the emergence of endogenous mechanisms involved in counterregulation of responses by local chemical signals, mediators and pathways that may lead to correction of local tissue injury that can occur as unwanted ‘bystander’ events via host defense and acute inflammation not only opens new avenues to begin to chart this relatively unappreciated system in human immunobiology [24, 25], but also provides an opportunity to explore

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new therapeutic approaches based on these new endogenous pathways. This approach holds promise for the design of new resolution-targeted drugs that could reduce the possibilities for unwanted toxic side effects [26, 27]. First Things First:‘The Battle Within’

PMN are within the first cellular line of defense, and by their ability to chemotax and phagocytize microbes they can protect the host from infection. These cells can also give rise to PMN-dependent vascular endothelial cell injury and contribute to increased vascular permeability, edema, and further release of chemoattractants [1]. Leukotriene B4 (LTB4) is among the more potent PMN chemoattractants [3] and can participate in tissue injury via recruiting more PMN in pathophysiologic scenarios [28]. LXs are trihydroxytetraene-containing members of the family of eicosanoids that are, among other in vivo sites, rapidly produced within vascular lumen via platelet-leukocyte interactions [29, 30]. Transcellular biosynthesis during cell-cell interactions [18, 19, 29], which occur in vivo, are events that can now be visualized [31] by processes that are activated during the temporal events of multicellular responses such as inflammation, atherosclerosis, and thrombosis [for a recent review, see 25, also 26]. Cell-cell interactions that occur during these events can evoke transcellular biosynthetic routes that lead to amplification signals such as with leukotrienes [32], prostaglandins [33] or to braking signals that can also involve novel compounds that have yet to be uncovered. Thus, these LX branches illustrated in figures 1 and 2 can involve cell-cell interactions that appear to be highly redundant to produce ‘endogenous stop signals’ and/or ‘antiinflammatory signals’ that are proresolution. On the other side of this equation, the pathways that generate leukotrienes, for example, evoke cellular events that are considered to advance leukocyte recruitment, inflammation, and the inappropriate liberation of proinflammatory agents and mediators [2, 26, 34, 35]. Cell-Cell Interactions in Chemical Mediator Production

Platelet-leukocyte interactions and/or platelet-leukocyte microaggregates [31] promote the formation of LX by transcellular conversion of the leukocyte (fig. 2) 5-lipoxygenase (5-LO) epoxide product LTA4. Once thought to be only an intracellular intermediate in leukotriene production, it is now clear that LTA4 released by activated leukocytes is available for enzymatic conversion by neighboring cell types [8, 36]. When platelets are adherent, the human platelet 12-LO converts LTA4 to LXA4 and LXB4 [for a review of mechanistic details with recombinant 12-LO, see 25]. Human platelets that do not produce LX on

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Membranes

Stimuli

LX biosynthesis

Arachidonic acid

cPLA2

15-LO

Eosinophils, monocytes, epithelial (T)

COOH Agonist PI, PIP2

S

Primed storage ‘release’

OOH

OOH COOH

PMN O (O)H COOH S

LXA4 hydrolase HO

O

HO (O)

LXB4 hydrolase

OH

OH RS

COOH

COOH

S

S

R S

HO

HO

LXA4

OH

LXB4

Fig. 2. LX biosynthesis: 15-LO-initiated pathway.

their own become a major source of LXs, given the abundance of platelets in vivo and their highly active 12-LO. This appears to be a major route for LX generation, particularly when the platelet COX-1 is inhibited via a NSAID or the like. 15-LO-initiated LX production is most clearly illustrated by airway epithelial cells, monocytes or eosinophils, which upregulate their 15-LO when exposed to cytokines such as IL-4 or IL-13 [26, 37, 38]. When these cell types are activated, they generate and release 15S-HETE, which is rapidly taken up and converted by PMN to LXs via the action of their 5-LO. This event not only leads to LX biosynthesis, but also ‘turns off’ leukotriene formation. 5-LO conversion of 15R-HETE also results in inhibition of leukotriene biosynthesis [19].

ASA-Triggered Mediators

15R-HETE is a major product of arachidonic acid (see fig. 3) in several cell types when COX-2 is upregulated after acetylation by ASA [18, 19].

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‘Stops’ PMN

Aspirin

HO

S516 COX-2

OH

R

C CH3

OH

Leukocytes COOH

COOH

R O

R 5-LO

OH 15R-HETE

COOH

15-Epi-LXA4

OH OH

Inhibition X

COOH

S RR

Aspirin HO

COX-1 and 2 O COOH O OH

OH

15-Epi-LXB4 Blocks cell proliferation

Synthases Prostanoids The classic eicosanoids

Fig. 3. ASA blocks prostaglandin generation and triggers 15-epi-LX biosynthesis via acetylation of COX-2.

Evidence was sought for an alternate basis for ASA’s anti-inflammatory therapeutic actions because many beneficial new actions have been documented in recent clinical studies. Also inflammation is now recognized as the basis for many human diseases including heart and lung disease [39]. These new potential therapeutic indications for ASA include decreasing incidence of lung, colon, and breast cancer [reviewed in 40], and prevention of cardiovascular diseases [41]. Inhibition of cyclooxygenase and biosynthesis of prostaglandins can account for many of ASA’s known therapeutic properties [34]; however, its ability to regulate PMN-mediated inflammation or cell proliferation remains of interest [42]. Unexpectedly, a new mechanism for ASA’s anti-inflammatory impact in vivo was uncovered that involves COX-2-bearing cells such as vascular endothelial cells or epithelial cells and their coactivation with PMN. In these experiments or in vivo models, inflammatory stimuli (i.e., TNF-
, LPS, etc.) induce COX-2 to generate 15R-HETE when ASA is administered [18]. This hydroxy-containing intermediate carries a carbon-15 alcohol in the R configuration that is rapidly converted by activated PMN to 15 epimeric LXs, or LXs that carry their 15 position alcohol in the R configuration [25] rather than

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O

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15S in the native lipoxygenase-derived LX, results from LO:LO interaction (see fig. 2, 3). The native 15S-containing LXs regulate human PMN responses that are relevant to inflammation and reperfusion injury. These responses include: inhibition of (1) FMLP and LTB4-induced chemotaxis [43], (2) adhesion and transmigration with endothelial cells [44], (3) cytokine formation such as IL-1 [45], and (4) transmigration through epithelial cells [46] as well as (5) enhanced antimicrobial actions of epithelial cells [47]. These actions of LXA4 and ASA-triggered lipoxins (ATL), their endogenous epimers (15-epi-LX), were first uncovered in experiments with isolated cell types in vitro [24] and now also demonstrated in several murine models of acute inflammation and second organ reperfusion injury (see table 1 and the original reports of Takano et al. [48, 49]; Hachicha et al. [45]; Clish et al. [50]; Bandeira-Melo et al. [51]; Chiang et al. [17, 52]). Some general points can now be made as follows from these results. First, PMN infiltration in vivo to lung, skin, and sites of wound healing is dramatically and potently inhibited by both intravenous and topical application of stable analogs of either LXA4 or 15-epi-LXA4 (ATL); second, 15-epi-LXA4 and LXA4 analogs inhibit IL-1, TNF-
and IL-8 expression while stimulating IL-4 release in vivo [45, 53] and interact in a stereoselective fashion with a common receptor on human and murine leukocytes; third, bioactive ATL and LXA4 analogs compete with [3H]-LXA4 binding to LXA4 receptors (ALX) [48]; fourth, LX regulate angiogenesis [11]; fifth, LX stimulate endogenous antimicrobial activities [47]. These actions of LX and ATL analogs are likely to be mediated by these specific LX receptors present in rodent (mouse and rat) [74] and human cells [54]. LXB4 is a positional isomer of LXA4, carrying alcohol groups at carbon 5S, 14R, and 15S positions, instead of at the carbon-5S, 6R, and 15S positions present in LXA4 (fig. 2). ASA-triggered LXB4 carries a 15R alcohol, hence 15-epi-LXB4 (see fig. 3). Although LXA4 and LXB4 can show similar activities in some biological systems [44], in many others they each show distinct actions (Tamaoki et al. [55]; reviewed in Serhan [25]). For example, 15-epi-LXB4 is a more potent inhibitor of cell proliferation than LXA4 or 15-epi-LXA4 [25] (fig. 3).

15-LO-Initiated Pathway

The first LX biosynthetic route reported in 1984 rationalized the generation of LXs by routes involving insertion of molecular oxygen into the carbon (C) 15 position of arachidonic acid, predominantly in the S configuration (see fig. 2). This pathway implicated the involvement of the human 15-LO in the generation of bioactive molecules [56], and was of interest not only because

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Table 1. Actions of LXs, ATL stable analogs and novel omega n–3 PUFA-derived resolvins in inflammation resolution ATLM

Response/action

References

LXA4, ATL, and stable analogs

Interact with specific receptors on leukocytes (PMN, monocytes, eosinophils), endothelial cells, epithelial cells and fibroblasts and regulate their function

Fiore et al. [130], Chiang et al. [106], Gronert et al. [54], Kang et al. [131], Levy et al. [26]

LX, ATL, and stable analogs

Inhibit PMN-mediated inflammation in skin, lung and kidney in murine models Transgenic ALX receptor gives dampened response to challenge

Takano et al. [49], Chiang et al. [106], Clish et al. [50], Badr et al. [67], Godson et al. [124], Levy et al. [132]

ATL, LX stable analogs

Protect from PMN-mediated lung injury in second organ reperfusion injury

Chiang et al. [52]

LXA4, ATL, and stable analogs

Enhance macrophage phagocytosis of apoptotic leukocytes

Godson et al. [124]

LXA4, ATL, and their stable analogs

Redirect chemokine, cytokine expression and gene regulation (IL-4↑, IL-1↓, IL-8↓) Block TNF-
actions Induce corepressor expression and protective genes

Hachicha et al. [45], Sodin-Semrl et al. [123], Gewirtz et al. [53], Qiu et al. [27]

15-Epi-LXB4, LXA4 and ATL stable analogs

Inhibit cell proliferation

Clària et al. [19], Fierro et al. [11]

LXA4 and ATL analogs

Enhance clearance and accelerate resolution of pulmonary edema Reduce COX-2 traffic in pain responses Antiangiogenic properties

Bandeira-Melo et al. [51], Levy et al. [26], Serhan et al. [133], Fierro et al. [11]

Resolvins: 18R series, 15R series EPA-derived mediators and 17R series docosanoids

Block TNF-
-stimulated PMN recruitment in vivo Block human PMN transmigration

Serhan et al. [4, 5]

For further details, see text and Serhan et al. [4, 5]. ATL  Aspirin-triggered lipoxin, 15RLXA4; ATLM  aspirin-triggered lipid mediators.

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of the new trihydroxytetraene structures uncovered, but also because the role of the human 15-LO was not known. The field was seemingly complicated for some because of dense and disparate observations in the literature filled with many observations not directly connected to either human physiology or pathology [for review, see 57]. It is now clear that there are several human as well as mammalian 15-LO isotypes that are temporal and spacial in their functional role(s) [26]. Also, many 15-LO-related genes that are related at the nucleotide level have been cloned from the plant kingdom in recent years with unknown functions in their tissue of origin. In human tissues, 15-LO is found to be abundant in human eosinophils, alveolar macrophages, monocytes, and epithelial cells; its functional expression is controlled by cytokines and regulated primarily by IL-4 and IL-13 [37, 38, 58, 59]. These two cytokines are implicated as negative regulators of the inflammatory response or anti-inflammatory cytokines [60]. Results from studies on the actions of LX implicate LXA4 and LXB4 as potential anti-inflammatory or protective compounds, findings that appear to be substantiated with the use of LX analogs in in vivo animal models (table 1). The oxygenation of arachidonic acid at the C15 position generates 15-HpETE. The 15S-hydroperoxy form and/or 15S-HETE, the reduced alcohol form, can each serve as a substrate for 5-LO in leukocytes. These transformations can occur within the cell type of origin or via transcellular biosynthetic routes in humans. The initial product of the 5-LO’s action on 15-HpETE is a 5S-hydroperoxy, 15S-hydro(peroxy)-DiH(p)ETE, which is converted to a 5,6-epoxytetraene. The 5-LO is also regulated by cytokines such as GM-CSF and IL-3 [61–63], and 5-LO is highly expressed in human PMN and monocytes. Once formed, the 5(6)-epoxytetraene is rapidly converted by hydrolases to either 5S, 6R, 15S-trihydroxy-7,9,13-trans-11-ciseicosatetraenoic acid, trivial name LXA4, and/or, via LXB4 hydrolase, to 5S,14R,15S-trihydroxy-6,10,12-trans-8-cis-eicosatetraenoic acid, termed LXB4 (fig. 2). LXA4 and B4 are each vasoactive, primarily vasodilatory in most isolated organs and in vivo models tested [64, 65], and they both regulate leukocyte functions [24, 66, 67], perhaps serving as downregulatory molecules. They required micromolar levels, however, to give vasoactions and thus, since only nano- to subnanomolar amounts are needed for leukocyte actions, their main role appears to be that of regulating leukocyte responses (see table 1). Concomitant with the biosynthesis of LXs by the 15-LO-initiated route, leukotriene biosynthesis is blocked at the 5-LO level [10, 19], resulting in an inverse relationship between leukotriene and LX biosynthesis. Thus, when LXs are generated by PMN from conversion of extra-PMN 15-HETE carrying its alcohol in either the R or S configuration (fig. 2), leukotriene formation is dramatically reduced, while LXs and 15-epi-LX are formed and released (see below).

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Results indicate that primed leukocytes from individuals with inflammatory disorders such as asthma that are exposed to various cytokines and other inflammatory stimuli in vivo generate LXs entirely from endogenous sources of arachidonic acid from a single cell type. Also, the finding that primed cells [68–71] as well as cells from peripheral blood of individuals with various diseases can generate LXs from endogenous sources of arachidonic acid raises important questions with respect to our current understanding of the generation of eicosanoids in inflammatory diseases. Namely, what is the temporal relationship between each of the classes of eicosanoid during the progression of an inflammatory response from acute to chronic status? Does diet affect the temporal relationships between classes? Information along these lines will help in understanding the biological impact of each class of compound since in experimental models they regulate key responses during multicellular events. This is particularly evident in view of findings with transgenic rabbits that overexpress 15-LO in a macrophage-specific fashion [72]. When the 15-LO-overexpressing rabbits were fed an atherogenic diet, the expression of 15-LO was unexpectedly found to have a protective impact on the development of atherosclerosis; that is, rabbits fed an atherogenic diet that did not overexpress 15-LO developed atherogenic plaques, whereas those rabbits overexpressing the 15-LO showed a markedly reduced potential for plaque formation [72]. It is likely that this impact of 15-LO overexpression in these studies can be causally related to the biosynthesis and local anti-inflammatory mediators. Indeed, detailed analyses of these transgenic rabbits overexpressing 15-LO showed a reduced inflammatory phenotype and enhanced LX products [134].

Temporal and Spacial Considerations in LX Formation and the Role of Exudate Prostanoids

Another recognized source of LX biosynthesis involves a new form of ‘priming’ that involves the esterification of 15-HETE (fig. 2) in inositolcontaining phospholipids within the membranes of human PMN [73]. Cells rapidly esterify 15-HETE into their inositol-containing lipids, which, upon subsequent agonist stimulation, release 15-HETE from this source, which is further transformed. The deacylated mono-HETEs are released and transformed, in this case, to LX, or perhaps to other eicosanoids that have yet to be discovered. This pathway suggests that precursors of LX biosynthesis can be stored within membranes of inflammatory cells and then released by stimuli activating PLA2 [73]. This form of ‘membrane priming’ to generate bioactive lipid mediators has implications in the second messengers generated, such as 15-hydroxy-PIP2 and diglyceride, which contain 15-HETE that may alter their intracellular signaling

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Lipid mediator class switch

O COOH

HO

OH COOH

HO OH

Cyclooxygenases Stimuli

PGE2

OH

LXA4

P CREB CRE

15-LO 5-LO

Fig. 4. Eicosanoid class switching during inflammation. Background (left) histology shows leukocyte infiltration and the resolution of the tissue without leukocytes (right). Temporal relationships are discussed in the text. H/E courtesy of Dr. Birgitta Schmidt, Children’s Hospital/Harvard Medical School.

activities. As is the case with 15S-HETE, 15R-HETE is also rapidly esterified into membrane phospholipids [19] and could probably serve as a reservoir for 15-epi-LX formation. In this context, LX appear in experimental models of inflammation after both prostanoids and leukotrienes as exudates begin to resolve [26] (fig. 4). Moreover, initially prostaglandins PGE2 and PGD2 produced early in the time course of expected inflammation induce both transcription and translational production of 15-LO, which lags in inflammation to set up the resolution circuit or biosynthetic apparatus (fig. 5), namely expression of the lipoxygenases required to produce LX that appear later in the time course of expected acute inflammation (fig. 4, 5) [26].

Leukotriene A4-Dependent LX Biosynthetic Route

In humans, the other route recognized for LX biosynthesis occurs between cell types of the vasculature. These intravascular sources of lipoxygenase products sharply contrast interstitial or mucosal origins. This pathway is best studied

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PGE2 increases 15-LO RNA Interactions with a CRE PGE and PGD 6

GGACGTC

7

CRE 6259-65

cAMP 1

2 34 5 6

7

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9 10 11 12 13 14

IL-4 RE TATA Human neutrophils

Fig. 5. Regulation of 15-LO.

with the interaction of human PMN with platelets. The key insertion of molecular oxygen in this biosynthetic scheme involves 5-LO within human PMN and the 12-LO, which is present in high amounts in human platelets [29, 68, 75, 76]. Platelet-leukocyte interactions in the biosynthesis of LXs and other eicosanoids serve as a model of cell-cell interactions in other tissues and cell types because of the ease with which we are able to obtain sufficient numbers of PMN and platelets to carry out experiments in vitro. Unprimed or apparently naive PMN from peripheral blood of healthy volunteers apparently do not generate appreciable quantities of LXs on their own [29, 68], and greater than 50% of their LTA4 formed from the 5-LO is released to the extracellular milieu, presumably for transcellular biosynthetic cell-cell communications [36]. Activated platelets adhere to PMN in whole blood and they can ‘pick up’ LTA4 released by leukocytes and convert it using the 12-LO to a carbonium cation intermediate in a reaction mechanism that is similar to the 15-LO [29, 77]. In this pathway, the platelet 12-LO abstracts hydrogen at carbon 13 and inserts molecular oxygen into the C15 position of LTA4 and converts it to a cation intermediate that opens either to LXB4 when attacked by water at the C14 position or LXA4 when attacked by water at the C6 position [78–80]. The formation of both LXB4 and LXA4 is unique to the 12-LO; 15-LO does convert LTA4 to the 5(6)epoxytetraene and then nonenzymatically opens to LXA4 and its isomers without a high yield generation of LXB4. This new activity of 12-LO, for which a functional role of the enzyme in platelets had remained elusive, indicates that the 12-LO serves as a LX synthase in human platelets [29]. Observations with isolated intact platelets from human peripheral blood were confirmed with recombinant 12-LO. 12-LO converts LTA4 to both LXA4 and LXB4 [80].

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The enzyme undergoes suicide inactivation with LTA4 as substrate for LXB4 but continues to produce LXA4. This catalytic regulatory mechanism has implications for the separate biological actions of LXA4 and LXB4 when generated within the vasculature, which is the likely place for platelets and leukocytes to adhere and interact [31, 81]. Thus, cell-cell interactions as exemplified here with platelets-leukocytes can result in the formation of new molecules by transcellular communication that downregulate cell function (see below).

LX Biosynthesis in Selectin-Deficient Mice: Diminished Cell-Cell Interactions

This type of cellular LX circuit was demonstrated in an animal model of glomerular nephritis where platelet-leukocyte interactions lead to the generation of LXs in vivo [82]. Antibodies against P-selectin, a mediator of plateletleukocyte adhesion, block transcellular LX biosynthesis both in vitro and during glomerular nephritis in vivo [83]. PMN infiltration was unexpectedly more pronounced during acute nephrotoxic serum nephritis in P-selectin ‘knockout’ mice than in wild-type mice because it was anticipated that infiltration of PMN in the P-selectin-deficient mouse would be reduced, not enhanced as observed. Consistent with this notion, P-selectin-deficient mice show a reduced efficiency of transcellular LX generation [82]. LXA4 levels are restored and the difference in PMN infiltration is eliminated when the P-selectin-deficient mice are infused with wild-type platelets. These observations raise the possibility that plateletPMN adherence and transcellular biosynthesis are important inflammatory events that regulate PMN recruitment via initiating the formation of lipid mediators that suppress proinflammatory responses. LX generation by platelets during platelet-PMN microaggregates [31, 81] within the vascular lumen may thus have an important impact in regulating the entry of recruiting and infiltrating PMN. Transcellular biosynthetic circuits and networks as illustrated by the model of platelet-PMN interactions in particular can yield both LXs and leukotrienes. Platelets can generate both pro- and anti-inflammatory molecules. If LXs act as ‘braking signals’ for the recruitment of leukocytes, it is likely that molecular switches are thrown that direct eicosanoid product profiles from a proinflammatory to an ‘anti-inflammatory phenotype’. In this regard, cytokines, as well as the local redox potential of the inflammatory microenvironment, play important roles in the generation of local levels of LXs, particularly within platelets. Drugs and other agents that can reduce the intracellular platelet level of glutathione such as nitroprusside enhance LX generation by platelets and block the formation of cysteinyl-containing LT [68].

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GM-CSF and IL-3 regulate expression of 5-LO and IL-4 and IL-13 specifically regulate the gene expression of 15-LO [37, 38, 58, 61, 62]. To date, specific cytokine regulators of 12-LO expression in human megakaryocytes have not been observed [78]. Functional integration of these pathways and their regulation by cytokines requires further studies but clearly provides cells and tissues with diverse routes to generate LX. This redundant biosynthetic capacity to produce LXs in human tissues is orchestrated by cell-cell interactions of diverse cell types and is likely to reflect the basic functional requirements for these compounds in regulating essential tissue responses. The current appreciation of cell-cell interactions, namely, that PMNplatelet microaggregates do indeed form in vivo [31, 81] and that key eicosanoid biosynthetic enzymes are regulated by specific cytokines [37, 58], provides new concepts for appreciating the complex signaling networks involved in LX formation [32] and actions. GM-CSF enhances LX generation during receptormediated PMN and platelet interactions [68], and TH2 cells can produce the 15-LO, regulating cytokines IL-4 and IL-13, which are in the microenvironment [37, 38, 60]. Thus, an in vivo microenvironment, particularly in a specific disease state, is highly likely to contain specific cytokines and lipid mediators such as PGD2 or PGE2 [26] that regulate transcription and translation of the 15-LO type 1 (fig. 4, 5) that can in turn enhance the production of LXs and possibly other novel bioactive lipid mediators active within the local milieu. LX can be produced by several additional routes by this type of transcellular event. For example, bidirectional interactions can involve PMN-released LTA4 [84] that is converted by the 15-LO abundant in epithelial cells [85], particularly in tracheal epithelial cells, to generate LXs via a LTA4-dependent mechanism. Hence, LTA4 is also pivotal in LX biosynthesis, as it is a substrate for the 15-LO and can be converted to LX [29, 85]. A less prominent biosynthetic route to their formation involves 5,6-dihydroxyeicosanoids, which are also substrates for conversion to LX production [86]. These reactions can include both 15-LO conversion or 12-LO conversion of 5,6-dihydroxyeicosanoid substrates to LX [86], which are enhanced by cell-cell adhesion interactions [32].

Evolution and LXs: Other Species That Produce LX

LX are evolutionarily conserved molecules in that they are generated in large quantities (i.e. ⬃microgram amounts/incubation) by a variety of fish tissues (i.e., trout, catfish) including leukocytes and brain [87]. The levels generated by isolated fish cells are ⬃10–100 times greater than the amount generated by human cells in coincubation, suggesting that they possess important

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roles in fish. Their role in fish brain is not known, but LX inhibit proliferation of fish lymphocytes [87]. Phylogenetic analyses have shown that LX are also generated by frog leukocytes and thrombocytes [88]. In trout, LXA4 stimulates chemotaxis of trout macrophages [87], a response that is shared by human monocytes [89], but not by human PMN [48]. Produced in human marrow, LX are implicated in regulating myelopoiesis [90]. Thus, it appears that LX have a primitive physiological function that has evolved and changed in humans (see table 1) but has held the LX structure conserved (see fig. 2).

ATL Circuit

A third major group of novel tetraene-containing eicosanoids was recently uncovered that are products of an unanticipated origin (see fig. 3) [18, 19]. In this transcellular biosynthetic scheme, COX-2 (also known as PGHS-2), expressed in either endothelial cells or epithelial cells after exposure to proinflammatory cytokines such as IL-1, LPS, or TNF, switches its catalytic activity in the presence of ASA, generating 15R-HETE instead of prostaglandin intermediates. In this setting, ASA inhibits prostaglandin biosynthesis by both COX-1 and COX-2 [reviewed in 91]. PGHS-2, when acetylated in endothelial or epithelial cells, is not enzymatically inactive. Instead, this isozyme converts endogenous arachidonic acid to 15R-HETE, which is released and transformed via transcellular routes to form 15-epi-LXs by leukocytes in close proximity. The activated and, in most instances, adherent leukocytes in this scenario possess 5-LO and transform 15R-HETE to a 5(6)-epoxytetraene within leukocytes, which carries its C15 position alcohol in the R configuration (see fig. 3). This proposed common intermediate leads to the formation of both 15-epi-LXA4 and 15-epi-LXB4, which each carry the R configuration at C15. 15-epi-LXA4 proves to be more potent than native LXA4 in inhibiting PMN adhesion [18, 24], and 15-epi-LXB4 inhibits cell proliferation [19]. The R configuration increases their bioactivity in both molecules. Recombinant PGHS-2, when acetylated by ASA, switches its substrate binding to an altered conformer that favors the generation of 15R-HETE [92]. The well-known in vivo inhibition of PGH2 by ASA is readily demonstrable with recombinant enzyme with the appearance of lipoxygenase activity or 15R-generating activity within minutes which reduces sharply with time. The interest in this pathway is that PGHS-2 is present in abundance in inflammatory reactions and in disease states including colon cancer [40, 91], and is in place when individuals take ASA for its therapeutic benefit. Hence, not only is COX-2 likely to be in place in vivo when ASA is taken, it is more than likely that inflammatory cell types are adhering and in position in these scenarios.

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Unexpected Benefits with ASA Most biomedical scientists are accustomed to thinking of ASA as simply another inhibitor of eicosanoid biosynthesis. The more recent results clearly establish that ASA can trigger the biosynthesis of novel compounds, namely, the ATL, which can serve as potential endogenous anti-inflammatory signals or mediators of some of ASA’s newly recognized beneficial actions. These relatively recently recognized beneficial actions of ASA include prevention of myocardial infarction [41, 93] and protection from colorectal adenoma, as well as other forms of cancer [40, 94]. The mechanism of ASA’s beneficial actions is likely to include the biosynthesis of 15-epi-LX and related compounds and could represent a novel mechanism for the first but now ancient synthetic drug for which a wealth of toxicology data is available worldwide. Because of ASA’s widespread use and lack of a ‘unifying mechanism’ to explain its many newly appreciated actions, it is important to gain a more complete understanding of its cellular impact and potential site(s) of its additional therapeutic value.

ASA-Dependent Generation of 15-Epi-LXA4 in Experimental Exudates and ASA-Induced Asthma Patients

It was important to determine whether 15-epi-LXA4 could be detected in animal experimental models or in patient-derived materials; to this end experiments were first carried out with a mouse peritonitis model [17]. In this model, COX-2 protein levels were upregulated by intraperitoneal injection of LPS and peritonitis was initiated by intraperitoneal injection of casein. Upregulation of COX-2 was demonstrated not only by enzyme activity but also by Western blot analysis. In these experiments, the immunoreactive bands were observed at ⬃70 kD in peritoneal lavage samples from LPS-treated mice. At 4 h after leukocyte infiltration was initiated, approximately 25  106 cells were obtained from peritoneal lavage of each mouse. The exudate leukocyte populations were ⬃73% PMN and 10% monocytes (and/or macrophages), respectively, as determined by H/E staining and enumeration by light microscopy. ASA was administrated by intraperitoneal injection, to test whether ASA treatment of the mice results in the generation of 15-epi-LXA4 during an inflammatory event (see protocol timeline [17]). The collected peritoneal exudates from each mouse were incubated in the presence or absence of the agonist divalent cation ionophore A23187 without addition of exogenous substrates, and samples from individual mice were analyzed separately using a newly developed specific ELISA method and LC-MS-MS system [17]. When one or two doses of ASA were administered, the values for 15-epiLXA4 were ⬃1.5 and 1.8 ng/5 ml peritoneal lavage per mouse [17]. Without

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administration of ASA, approximately 0.5 ng of 15-epi-LXA4/5 ml lavage was associated with peritoneal exudates from each mouse, suggesting that additional routes may be operative in vivo to produce 15-epi-LX in an ASA-independent fashion. Naive animals (without any treatment) gave very low levels (0.2 ng/ 5 ml peritoneal lavage per mouse) of 15-epi-LXA4. The physiological relevance of these values obtained in the absence of experimental challenge is currently not clear, but might reflect at present unappreciated P450-dependent routes of 15R-HETE production [95]. Since LPS upregulates COX-2 [40] and can induce PMN recruitment, the possibility was tested that animals treated with LPS could give rise to 15-epi-LXA4 in the absence of casein. The results obtained showed a low level of 15-epi-LXA4 generation in these LPS-treated animals in the presence or absence of ASA (0.25 and 0.30 ng/5 ml peritoneal lavage per mouse, respectively), again suggesting that LPS alone is not sufficient to elicit PMN infiltration into peritoneal lavage. Casein-induced PMN infiltration and ASA are required in this scenario to generate statistically significant levels of 15-epi-LXA4. These results demonstrated that ASA administration in murine peritonitis gives inflammatory exudates that generate 15-epi-LXA4 in appreciable levels from endogenous substrate within these inflammatory cells, thus establishing a biosynthetic circuit for ATL/15-epi-LX generation in vivo. These techniques and methods were used to evaluate ATL and LXA4 formation in ASA-tolerant and ASA-intolerant asthmatics and their relation to LTC4. Of interest, the ASA-tolerant subjects generated both LX and ATL, but the ASA-intolerant patients proved to have a diminished capacity to generate ATL and LX upon ASA challenge [96]. The lower levels of these potentially protective mediators could contribute to the pathobiology of this chronic disorder in that the disease state is not only characterized by the overproduction of proinflammatory mediators but the loss or reduction in LX and ATL that may keep inflammation in check and ‘self-limited’. Also, a reduction and alteration in LX generation were found in patients with chronic liver disease [97] and chronic myelogenous leukemia [90, 98–101]. These diseases contrast with recent findings in localized juvenile periodontitis, namely that LXA production is upregulated [102], following atherosclerotic plaque rupture [103], and with nasal polyps [85]. Together, these findings indicate that alterations in LX levels may be linked to the pathophysiology of several human diseases.

Metabolically Stable Analogs LXA4 and ATL; New Leads to Resolution?

Since LT biosynthetic enzyme inhibitors and LT receptor antagonists are of limited use and only in certain clinical settings [2, 20], it was clear that other

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LX and ATL analogs resist conversion Recombinant enzyme in all

NADH formation (Abs)

0.03

LX conversion

LX and ATL stable analog criteria Delay inactivation Increase potency 500 MW

0.02

0.01

Analog 0.00 0

50

100

150

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s

Fig. 6. LX analog design: rapid enzymatic screen. 䊏  LXA4; 䊉  LXB4; 䉭  LX analog.

new approaches were needed. In this regard, LX stable analogs were prepared for in vivo studies that were designed as potential mimetics of the inhibitory actions noted for LXA4 [24] and for LXB4 [104] in vitro. Both LXA4 and ATL stable analogs were strategically synthesized and screened using a recombinant dehydrogenase screen assay (fig. 6) [24] as a relatively inexpensive and rapid screen to design suitable analogs that might function in vivo by resisting rapid inactivation (see fig. 7). Several of these analogs were scaled up via total organic synthesis to examine in more detail and were tested for their ability to inhibit PMN infiltration and changes in vascular permeability in vivo in several murine models (table 1). 15(R/S)-methyl-LXA4, having a methyl group at C15 position (racemate 15R/S), is an analog of both the ASA-triggered 15-epi-LXA4 and native LXA4, and 16-phenoxy-LXA4, which has a phenoxy group at C16 position, is an analog of native LXA4 that prevents enzymatic inactivation with recombinant 15-dehydrogenase in vitro [24; see 105] (table 1 and references within). These LX and ATL analogs that proved to be bioactive also act via ALX [48] and receptor chimeras [106]. These LX stable analogs (for example, see fig. 7) are topically active and when applied to mouse ears, they inhibit both PMN infiltration and vascular permeability changes in a concentrationdependent fashion [48, 49]. For example, at 130 nmol per ear, the degree of inhibition of PMN infiltration was more than 90% for both analogs, with apparent IC50 noted at ⬃13–26 nmol per ear range for each analog. In the same concentration range, these two LXA4 stable analogs also inhibited the vascular

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Metabolic inactivation and select analogs HO

OH

HO

OH

COOH

COOH

HO

OH

CH3

15(R/S)-methyl-LXA4 Inactivation HO

OH

HO

OH COOCH3

COOH

O O 15-Oxo-LXA4

F

OH 15-Epi-16-(p-fluoro)-phenoxy-LXA4

Fig. 7. LX metabolic inactivation: biotemplates and screening. LX are rapidly inactivated via specific enzymes. The endogenous structures were used as templates to synthesize analogs.

permeability, namely extravasation of Evans blue. At 130 nmol per ear, the inhibition of vascular permeability change was 98% for 15(R/S)-methyl-LXA4, and 85% for 16-phenoxy-LXA4, respectively, and their impact was noted visually. The inhibition of vascular permeability changes paralleled the ‘stopping or blocking’ of PMN infiltration with both the ATL and LX analogs. Selected LXA4 analogs were also directly compared to the actions of both native LXA4 and the LTB4 receptor antagonist U-75302. In addition, the impact of LXB4 analogs that resist enzymatic inactivation were evaluated [104]. When applied topically at 26 nmol per ear, the stable analogs were 3–4 times more potent than native LXA4. Of the LX stable analogs tested in this model, 15(R/S)-methyl-LXA4 was the most potent (70% inhibition). These blocking or inhibitory actions of LX and ATL on PMN infiltration and vascular permeability changes were significantly greater than topically applied native LXA4 (p  0.05), indicating that these analogs also increase bioavailability as topical agents because LXA4 and these analogs are within a similar potency range in in vitro assays of leukocyte responses [24, 104]. A 16-p-fluoro derivative of 16-phenoxy-LXA4 was prepared for these experiments to assess whether fluorination of the phenoxy ring could enhance potency. Results indicate that 16-(p-fluoro)-phenoxy-LXA4 was also potent and retained the activity at levels comparable to 16-phenoxy-LXA4. Both of the two LXB4 analogs inhibited PMN infiltration and vascular permeability. The S enantiomer, 5(S)-methyl-LXB4,

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Dorsal air pouch PMN in exudate

Dermis PMN

Day 6 Muscle layer

2 1

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a te

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TNF-
ATL analog i.v.

Ex ud

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Cells106

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Innermost lining

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Leukocyte trafficking

TNF-
ATL analog i.v.

Fig. 8. Murine dorsal air pouch. Illustration of the exudate formation and blockage of PMN infiltration in the air pouch by ATL and LX analogs. *  p  0.05 by Student’s t-test.

was significantly more potent than 5(R)-methyl-LXB4, indicating a preferred stereoselectivity for inhibition. The rank order of inhibitory potency was 15(R/S)-methyl-LXA4  16-p-fluoro-phenoxy-LXA4 ⬃ 5(S)-methyl-LXB4  16-phenoxy-LXA4  5(R)-methyl-LXA4 for both PMN infiltration and vascular permeability changes. These also proved to be potent inhibitors of PMN infiltration in the dorsal air pouch (fig. 8) with potency 100 times that of ASA [50] and redirecting cytokine formation and action [45]. A small panel of known potent inflammatory mediators was examined to test the specificity and/or generality of the actions of LX analogs using murine ear skin inflammation as an endpoint. PGE2 dramatically augmented the LTB4induced PMN infiltration and vascular permeability change, although the effects of this prostaglandin by itself were minimal. PMA, a tumor promoter and topical irritant that bypasses cell surface receptors, caused concentrationdependent changes in both PMN infiltration and vascular permeability, and 100 ng PMA per ear was chosen for further evaluation. Several potent agents such as FMLP, C5a, IL-8, platelet-activating factor (PAF), or LTD4 did not give significant changes in these parameters at amounts applied as high as 1–25 g compared to LTB4, suggesting that they are not topically active, perhaps because

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they do not gain access in intact skin tissues to locations that would establish a chemotactic gradient [49]. In vitro, LX and ATL analogs block PAF, FMLP and IL-8 actions on human PMN [53, 107] and in whole blood [108]. The impact of 15-epi-LXA4 was also evaluated using a stable analog, 15-epi-16-(p-fluoro)-phenoxy-LXA4, designed to enhance intravenous stability by slowing metabolic inactivation [50]. This LX analog not only inhibited LTB4 but also blocked PGE2 plus LT-enhanced inflammation. Of interest, it also inhibited PMA-induced PMN influx with little impact at 24 h on PMA-induced vascular leakage. Thus, LXA4 stable analogs clearly inhibited native mediators as topically active agents and partially blocked PMA-evoked actions that were restricted to inhibition of PMN influx [49]. The results with PMA-induced inflammation and vascular damage also indicate that a major component of the observed vascular leakage with PMA is not mediated by PMN-dependent mechanisms.

LXs and ATL Associated with Human Diseases and Disease Models

The native or endogenous LXs are generated in human organs and are associated with a variety of inflammatory events, with the first in vivo demonstration in bronchial lavage [109]. LXA4 and LXB4 are formed in nasal polyps [85] and, of interest, LXA4 is generated in nasal lavage from ASA-sensitive asthmatics [110] and in experimental nephritis [83]. Along these lines, Chavis et al. [69] proposed that LX are useful biomarkers of asthma, and Thomas et al. [71] proposed that LXs are biomarkers of long-term clinical improvement in arthritic patients. The list of diseases and tissues in table 2 is not exhaustive, and is likely to represent examples of in vivo scenarios where cell-cell interaction is accelerated and the generation of LXs is detected because of the abundance of cytokines and cell-cell interactions in these settings. Rupture of the atherosclerotic plaque leads to rapid generation of LXA4 in the intracoronary artery [103]. As stated above, LX are also generated by normal human bone marrow [90, 98]. During chronic myelocytic leukemia, platelets lose 12-LO. The enzymatic loss of 12-LO coincides with blast crisis, that is when platelet peripheral blood numbers are abnormal and do not function normally [90]. In this phase of the disease the platelets from these patients also lose their ability to generate LX because of this loss in the 12-LO platelet pathway. In view of the literature amassed to date and actions elucidated for LX, this early finding in chronic myeloid leukemia patients may be important and possibly represents a gene defect causally related to the blast crisis observed in chronic myelocytic leukemia.

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Table 2. LXs and ATL in tissues and diseases Asthma ASA-sensitive asthmatics Angioplasty-induced plaque rupture Angiogenesis: neovascularization Bone marrow generation

Glomerulonephritis Periodontitis Rheumatoid arthritis Inflammatory bowel disease

See text for details.

Experimental Models of Disease: ATL and LX Lung Disease Both LX and 15-epi-LX are generated during respiratory inflammation. LXA4 (0.4–2.8 ng/ml) has been detected in bronchoalveolar lavage and pleural fluid from individuals with inflammatory lung disease [26, 109]. In an experimental model of asthma, LX formation at peak airway inflammation is similar to LTB4 yet one to two log orders less than cysteinyl LTs and PGE2 [111]. An LX stable analog, designed using the 15-epi-LXA4 structure as a template, markedly inhibits (with as little as 1 g; ⬃0.05 mg/kg) murine allergen-driven airway hyperresponsiveness and inflammation. Targeted expression of human ALX to murine leukocytes in transgenic mice also dramatically inhibits allergen sensitization, eosinophil trafficking and airway inflammation. Airway reactivity is not similarly reduced in hALX-tg animals, raising the possibility that the LX stable analog may inhibit airway hyperresponsiveness at distinct sites of action in the airway (e.g., CysLT1 receptors). With an ED50 of less than 0.05 mg/kg in this murine model of asthma, the LX analog compares favorably with both the CysLT1 receptor antagonist montelukast (0.03 mg/kg in rats) and the synthetic glucocorticoid dexamethasone (0.5–3 mg/kg in mice) [112–114]. Together, these findings indicate that endogenous LX formation during acute inflammation is temporally dissociated from LT and PG and that ALX activation by endogenous ligands or an LX stable analog evokes potent ‘stop’ signals for airway responses. Of note, diminished formation of these counterregulatory mediators has been identified in severe forms of human illness. For example, LX and 15-epi-LX biosynthesis is defective in stimulated whole blood from ASA-intolerant asthmatic individuals [96] and PMN from patients with severe, steroid-dependent asthma [115]. Decreased production of these counterregulatory substances may predispose the host to more severe, inflammatory responses.

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Enteritis These chemical ‘braking’ signals for inflammation might also be operative in intestinal inflammation. Nanomolar concentrations of the ALX agonists LXA4, 15-epi-LXA4 and select structural analogs inhibit TNF-
-stimulated PMN adherence to colonic epithelia and block TNF-
-initiated release of IL-8, MCP-1 and RANTES, disruption of mucosal architecture and colonocyte apoptosis in colonic mucosa ex vivo [116]. In a murine model of inflammatory colitis, oral 15-epi-LXA4 analog (10 g/day) prevents dextran sodium sulfateinduced weight loss, hematochezia and mortality [117]. In this regard, LXA4 and 15-epi-LXA4 appear to activate a ‘molecular shield’ for mucosal protection against gram-negative bacteria, such as Salmonella typhimurium, by increasing the mucosal expression of bactericidal/permeability-increasing protein [47] and preventing NFkappaB activation [117]. Together, these findings suggest new therapeutic approaches for the treatment of enteritides. Nephritis LXs also display potent counterregulatory actions on PMN renal tissue accumulation. In an experimental model of glomerulonephritis, ex vivo exposure to LXA4 downregulates PMN recruitment to glomeruli [83]. This LXprotective effect is blocked during the heterologous phase of nephrotoxic serum nephritis in P-selectin-deficient mice [82], emphasizing the importance of cellcell interactions in transcellular LX biosynthesis in vivo. Moreover, increased renal expression of 15-LO in rat kidney both increases endogenous LX formation and protects the transgenic animals from immune complex glomerulonephritis [118]. In addition to inflammatory diseases of the glomeruli, a 15-epi-LXA4 analog is also protective in experimental ischemic acute renal failure, displaying potent inhibition of functional, morphological and inflammatory responses to cross-clamping of the renal pedicles [119]. In aggregate, interventional trials of LX, 15-epi-LX and select analogs in animal models of renal inflammation or injury have consistently demonstrated potent therapeutic effects, providing a rationale for further exploration of their efficacy in renal diseases. Of interest, new methods have recently been described for LXA4 extraction from urine, which should enable further investigation of LX and 15-epi-LX generation and their metabolism in renal tissues and other human disease [120].

Conclusions

Several new methods were developed including both ELISA and liquid chromatography-tandem mass spectrometry (LC/MS/MS)-based lipidomics for

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the ASA-triggered 15-epi-LXA4 and new lipid mediators [5] that proved highly sensitive and stereoselective compared with its natural epimer LXA4, at the level required to selectively interact with 15-epi-LXA4 [17]. Utilizing this ELISA, it was possible to obtain evidence to establish that 15-epi-LXA4 generation proceeds via transcellular biosynthesis during heterotypic leukocyteleukocyte interactions [17] and the first evidence for ASA-dependent 15-epi-LXA4 generation by inflammatory exudate cells from murine models and humans with ASA-sensitive asthma. Also, stable LX and ATL analogs (see table 1) were designed and found to be potent blockers of both PMN infiltration and PMN-mediated vascular permeability changes. These compounds were specifically designed to resist rapid inactivation and proved to resist the rapid conversion by cells and/or isolated recombinant enzymes that were each used as a rapid screening procedure [24, 104] to more swiftly focus the efforts for rational design. Regulated gene expression and assembly of the LX biosynthetic circuits within the micromilieu in vivo require upregulation of key enzymes by cytokines such as IL-4 and IL-13 (fig. 5) that also control the expression of one of the receptors for LXA4 denoted as ALX [121]. This was also recently found with acute production of PGE and PGD2 levels [26]. Hence, both the temporal and spacial components in LX formation and actions are key determinants in their bioimpact. LX and ATL each appear to be the first recognized members of a new class of mediators, namely, endogenous mediators of anti-inflammation or proresolution. Along these lines, PGE2 can and may display anti-inflammation in certain settings [122], but in most cases in vivo this prostanoid dramatically enhances inflammation evoked by leukotrienes or other agents [49]. This likely reflects the numerous receptor isoforms as well as their differential coupling mechanisms with PGE2 that clearly play a very diverse role in human physiology. The results obtained with ATL and LX analogs (see table 1 and references within) display potent and stereoselective actions within the sub- to nanomolar range, substantiating the actions of endogenous LX and ATL in several in vivo models. These pathways (fig. 2, 3) are likely to be important in vivo in human host defense [47]. They join the many other local mediators that govern local inflammation in vivo such as select cytokines (IL-10, IL-4, IL-13), proteins of interest in resolution [12]. In this context, LX and ATL receptor activation not only inhibits proinflammatory events such as IL-6 gene expression [123] but stimulates IL-4 generation in vivo [45] and enhances the nonphlogistic phagocytosis of apoptotic PMN by macrophages [124], a key event in clearance and resolution. Because the integrated response of the host is essential to health and disease, it is important to achieve a more complete understanding of the molecular and cellular events governing the formation and actions of endogenous mediators of resolution that appear to control both the magnitude and duration

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of inflammation. Given the present body of evidence with LX and ATL, it is not surprising that others have recently found a protective action for COX-2 in cardiovascular disease [125], as current treatments may not be optimal for certain indications [126] and new evidence for COX-3 [127, 128]. Characterizing useful experimental systems with clinically relevant endpoints that are predictive of human disease will also require a multidisciplinary approach as well as a shift in our current notions [6] regarding the role of lipid-derived chemical mediators in inflammation resolution and the relative contribution of these likely highly redundant circuits in host defense [see 26, 129]. Acknowledgments Many thanks to Mary Halm Small for expert assistance in the preparation of this manuscript. This work was supported in part by grant No. R01-GM38765 (CNS) and K08-HL03788 (BDL) from the National Institutes of Health.

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Majno G, Joris I: Cells, Tissues and Disease: Principles of General Pathology. Cambridge, Blackwell Science, 1996, p 974. Gallin JI, Snyderman R, Fearon DT, Haynes BF, Nathan C: Inflammation: Basic Principles and Clinical Correlates. Philadelphia, Lippincott Williams & Wilkins, 1999, p 1360. Samuelsson B, Dahlén SE, Lindgren JÅ, Rouzer CA, Serhan CN: Leukotrienes and lipoxins: Structures, biosynthesis, and biological effects. Science 1987;237:1171–1176. Serhan CN, Clish CB, Brannon J, Colgan SP, Chiang N, Gronert K: Novel functional sets of lipid-derived mediators with antiinflammatory actions generated from omega-3 fatty acids via cyclooxygenase 2-nonsteroidal antiinflammatory drugs and transcellular processing. J Exp Med 2000;192:1197–1204. Serhan CN, Hong S, Gronert K, Colgan SP, Devchand PR, Mirick G, Moussignac R-L: Resolvins: A family of bioactive products of omega-3 fatty acid transformation circuits initiated by aspirin treatment that counter pro-inflammation signals. J Exp Med 2002;196:1025–1037. Serhan CN, Ward PA: Molecular and Cellular Basis of Inflammation. Totawa, Humana Press, 1999. Serhan CN, Haeggström JZ, Leslie CC: Lipid mediator networks in cell signaling: Update and impact of cytokines. FASEB J 1996;10:1147–1158. Krump E, Picard S, Mancini J, Borgeat P: Suppression of leukotriene B4 biosynthesis by endogenous adenosine in ligand-activated human neutrophils. J Exp Med 1997;186:1401–1406. Qiu F-H, Wada K, Stahl GL, Serhan CN: IMP and AMP deaminase in reperfusion injury downregulates neutrophil recruitment. Proc Natl Acad Sci USA 2000;97:4267–4272. Serhan CN: Lipoxin biosynthesis and its impact in inflammatory and vascular events. Biochim Biophys Acta 1994;1212:1–25. Fierro IM, Kutok JL, Serhan CN: Novel lipid mediator regulators of endothelial cell proliferation and migration: Aspirin-triggered-15R-lipoxin A4 and lipoxin A4. J Pharmacol Exp Ther 2002;300: 385–392. de Waal Malefyt R: Role of interleukin-10, interleukin-4, and interleukin-13 in resolving inflammatory responses; in Gallin JI, Snyderman R, Fearon DT, Haynes BF, Nathan C (eds): Inflammation: Basic Principles and Clinical Correlates. Philadephia, Lippincott Williams & Wilkins, 1999, pp 837–849.

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Diamond P, McGinty A, Sugrue D, Brady HR, Godson C: Regulation of leukocyte trafficking by lipoxins. Clin Chem Lab Med 1999;37:293–297. Maddox JF, Serhan CN: Lipoxin A4 and B4 are potent stimuli for human monocyte migration and adhesion: Selective inactivation by dehydrogenation and reduction. J Exp Med 1996;183: 137–146. Marcus AJ: Aspirin as prophylaxis against colorectal cancer. N Engl J Med 1995;333: 656–658. Vane JR: Adventures and excursions in bioassay: The stepping stones to prostacyclin; in Les Prix Nobel: Nobel Prizes, Presentations, Biographies and Lectures. Stockholm, Almqvist & Wiksell, 1982, pp 181–206. Chiang N, Takano T, Clish CB, Petasis NA, Tai H-H, Serhan CN: Aspirin-triggered 15-epi-lipoxin A4 (ATL) generation by human leukocytes and murine peritonitis exudates: Development of a specific 15-epi-LXA4 ELISA. J Pharmacol Exp Ther 1998;287:779–790. Clària J, Serhan CN: Aspirin triggers previously undescribed bioactive eicosanoids by human endothelial cell-leukocyte interactions. Proc Natl Acad Sci USA 1995;92:9475–9479. Clària J, Lee MH, Serhan CN: Aspirin-triggered lipoxins (15-epi-LX) are generated by the human lung adenocarcinoma cell line (A549)-neutrophil interactions and are potent inhibitors of cell proliferation. Mol Med 1996;2:583–596. Showell HJ, Cooper K: Inhibitors and antagonists of cyclooxygenase, 5-lipoxygenase, and platelet activating factor; in Gallin JI, Snyderman R (eds): Inflammation: Basic Principles and Clinical Correlates. Philadelphia, Lippincott Williams & Wilkins, 1999, pp 1177–1193. Malaviya R, Abraham SN: Role of mast cell leukotrienes in neutrophil recruitment and bacterial clearance in infectious peritonitis. J Leukoc Biol 2000;67:841–846. Swan SK, Rudy DW, Lasseter KC, Ryan CF, Buechel KL, Lambrecht LJ, Pinto MB, Dilzer SC, Obrda O, Sundblad KJ, Gumbs CP, Ebel D, Quan H, Larson PJ, Schwartz JI, Musliner T, Gertz BJ, Brater DC, Yao SL: Effect of cyclooxygenase-2 inhibition on renal function in elderly persons receiving a low-salt diet: A randomized, controlled trial. Ann Intern Med 2000; 133:1–9. Cannon GW, Caldwell JR, Holt P, McLean B, Seidenberg B, Bolognese J, Ehrich E, Mukhopadhyay S, Daniels B: Rofecoxib, a specific inhibitor of cyclooxygenase 2, with clinical efficacy comparable with that of diclofenac sodium: Results of a one-year, randomized, clinical trial in patients with osteoarthritis of the knee and hip. Rofecoxib Phase III Protocol 035 Study Group. Arthritis Rheum 2000;43:978–987. Serhan CN, Maddox JF, Petasis NA, Akritopoulou-Zanze I, Papayianni A, Brady HR, Colgan SP, Madara JL: Design of lipoxin A4 stable analogs that block transmigration and adhesion of human neutrophils. Biochemistry 1995;34:14609–14615. Serhan CN: Lipoxins and novel aspirin-triggered 15-epi-lipoxins (ATL): A jungle of cell-cell interactions or a therapeutic opportunity? Prostaglandins 1997;53:107–137. Levy BD, Clish CB, Schmidt B, Gronert K, Serhan CN: Lipid mediator class switching during acute inflammation: Signals in resolution. Nat Immunol 2001;2:612–619. Qiu F-H, Devchand PR, Wada K, Serhan CN: Aspirin-triggered lipoxin A4 and lipoxin A4 up-regulate transcriptional corepressor NAB1 in human neutrophils. FASEB J 2001;15:2736–2738 (available at www.fasebj.org). Varani J, Ward PA: Mechanism of neutrophil-dependent and neutrophil-independent endothelial cell injury. Biol Signals 1994;3:1–14. Serhan CN, Sheppard KA: Lipoxin formation during human neutrophil-platelet interactions. Evidence for the transformation of leukotriene A4 by platelet 12-lipoxygenase in vitro. J Clin Invest 1990;85:772–780. Edenius C, Stenke L, Lindgren JA: On the mechanism of transcellular lipoxin formation in human platelets and granulocytes. Eur J Biochem 1991;199:401–409. Lehr H-A, Frei B, Arfors K-E: Vitamin C prevents cigarette smoke-induced leukocyte aggregation and adhesion to endothelium in vivo. Proc Natl Acad Sci USA 1994;91:7688–7692. Brady HR, Serhan CN: Adhesion promotes transcellular leukotriene biosynthesis during neutrophil-glomerular endothelial cell interactions: Inhibition by antibodies against CD18 and L-selectin. Biochem Biophys Res Commun 1992;186:1307–1314.

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Herschman HR: Recent progress in the cellular and molecular biology of prostaglandin synthesis. Trends Cardiovasc Med 1998;8:145–150. Samuelsson B: From studies of biochemical mechanisms to novel biological mediators: Prostaglandin endoperoxides, thromboxanes and leukotrienes; in Les Prix Nobel: Nobel Prizes, Presentations, Biographies and Lectures. Stockholm, Almqvist & Wiksell, 1982, pp 153–174. Serhan CN, Prescott SM: The scent of a phagocyte: Advances on leukotriene B4 receptors. J Exp Med 2000;192:F5–F8. Fiore S, Serhan CN: Phospholipid bilayers enhance the stability of leukotriene A4 and epoxytetraenes: Stabilization of eicosanoids by liposomes. Biochem Biophys Res Commun 1989;159: 477–481. Nassar GM, Morrow JD, Roberts LJ 2nd, Lakkis FG, Badr KF: Induction of 15-lipoxygenase by interleukin-13 in human blood monocytes. J Biol Chem 1994;269:27631–27634. Levy BD, Romano M, Chapman HA, Reilly JJ, Drazen J, Serhan CN: Human alveolar macrophages have 15-lipoxygenase and generate 15(S)-hydroxy-5,8,11-cis-13-trans-eicosatetraenoic acid and lipoxins. J Clin Invest 1993;92:1572–1579. Libby P: Atherosclerosis: The new view. Sci Am 2002;286:46–55. Levy GN: Prostaglandin H synthases, nonsteroidal anti-inflammatory drugs, and colon cancer. FASEB J 1997;11:234–247. Savage MP, Goldberg S, Bove AA, Deutsch E, Vetrovec G, Macdonald RG, Bass T, Margolis JR, Whitworth HB, Taussig A, Hirshfeld JW, Cowley M, Hill JA, Marks RG, Fischman DL, Handberg E, Herrmann H, Pepine CJ: Effect of thromboxane A2 blockade on clinical outcome and restenosis after successful coronary angioplasty. Circulation 1995;92:3194–3200. Sneader W: The discovery of aspirin: A reappraisal. BMJ 2000;321:1591–1594. Lee TH, Horton CE, Kyan-Aung U, Haskard D, Crea AE, Spur BW: Lipoxin A4 and lipoxin B4 inhibit chemotactic responses of human neutrophils stimulated by leukotriene B4 and N-formyl-L-methionyl-L-leucyl-L-phenylalanine. Clin Sci 1989;77:195–203. Papayianni A, Serhan CN, Brady HR: Lipoxin A4 and B4 inhibit leukotriene-stimulated interactions of human neutrophils and endothelial cells. J Immunol 1996;156:2264–2272. Hachicha M, Pouliot M, Petasis NA, Serhan CN: Lipoxin (LX)A4 and aspirin-triggered 15-epiLXA4 inhibit tumor necrosis factor 1
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Gronert K, Martinsson-Niskanen T, Ravasi S, Chiang N, Serhan CN: Selectivity of recombinant human leukotriene D4, leukotriene B4, and lipoxin A4 receptors with aspirin-triggered 15-epiLXA4 and regulation of vascular and inflammatory responses. Am J Pathol 2001;158:3–9. Tamaoki J, Tagaya E, Yamawaki I, Konno K: Lipoxin A4 inhibits cholinergic neurotransmission through nitric oxide generation in the rabbit trachea. Eur J Pharmacol 1995;287: 233–238. Serhan CN, Hamberg M, Samuelsson B: Lipoxins: Novel series of biologically active compounds formed from arachidonic acid in human leukocytes. Proc Natl Acad Sci USA 1984;81:5335–5339. Ford-Hutchinson AW: Arachidonate 15-lipoxygenase; characteristics and potential biological significance. Eicosanoids 1991;4:65–74. Sigal E, Conrad DJ: Human 15-lipoxygenase: A potential effector molecule for interleukin-4. Adv Prostaglandin Thromboxane Leukot Res 1994;22:309–316. Katoh T, Lakkis FG, Makita N, Badr KF: Co-regulated expression of glomerular 12/15-lipoxygenase and interleukin-4 mRNA in rat nephrotoxic nephritis. Kidney Int 1994;46:341–349. Anderson GP, Coyle AJ: TH2 and ‘TH2-like’ cells in allergy and asthma: Pharmacological perspectives. Trends Pharmacol Sci 1994;15:324. Pouliot M, McDonald PP, Borgeat P, McColl SR: Granulocyte/macrophage colony-stimulating factor stimulates the expression of the 5-lipoxygenase-activating protein (FLAP) in human neutrophils. J Exp Med 1994;179:1225–1232. Ring WL, Riddick CA, Baker JR, Munafo DA, Bigby TD: Lymphocytes stimulate expression of 5-lipoxygenase and its activating protein in monocytes in vitro via granulocyte-macrophage colony stimulating factor and interleukin-3. J Clin Invest 1996;97:1293–1301. L’Heureux GP, Bourgoin S, Jean N, McColl SR, Naccache PH: Diverging signal transduction pathways activated by interleukin-8 and related chemokines in human neutrophils: Interleukin-8, but not NAP-2 or GROA
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96 Sanak M, Levy BD, Clish CB, Chiang N, Gronert K, Mastalerz L, Serhan CN, Szczeklik A: Aspirin-tolerant asthmatics generate more lipoxins than aspirin-intolerant asthmatics. Eur Respir J 2000;16:44–49. 97 Clària J, Titos E, Jiménez W, Ros J, Ginès P, Arroyo V, Rivera F, Rodés J: Altered biosynthesis of leukotrienes and lipoxins and host defense disorders in patients with cirrhosis and ascites. Gastroenterology 1998;115:147–156. 98 Stenke L, Näsman-Glaser B, Edenius C, Samuelsson J, Palmblad J, Lindgren JÅ: Lipoxygenase products in myeloproliferative disorders: Increased leukotriene C4 and decreased lipoxin formation in chronic myeloid leukemia. Adv Prostaglandin Thromboxane Leukot Res 1990;21B: 883–886. 99 Stenke L, Edenius C, Samuelsson J, Lindgren JA: Deficient lipoxin synthesis: A novel platelet dysfunction in myeloproliferative disorders with special reference to blastic crisis of chronic myelogenous leukemia. Blood 1991;78:2989–2995. 100 Stenke L, Nasman-Glaser B, Edenius C, Samuelsson J, Palmblad J, Lindgren JA: Lipoxygenase products in myeloproliferative disorders: Increased leukotriene C4 and decreased lipoxin formation in chronic myeloid leukemia. Adv Prostaglandin Thromboxane Leukot Res 1991;21B:883–886. 101 Stenke L, Mansour M, Edenius C, Reizenstein P, Lindgren JA: Formation and proliferative effects of lipoxins in human bone marrow. Biochem Biophys Res Commun 1991;180:255–261. 102 Pouliot M, Clish CB, Petasis NA, Van Dyke TE, Serhan CN: Lipoxin A4 analogues inhibit leukocyte recruitment to Porphyromonas gingivalis: A role for cyclooxygenase-2 and lipoxins in periodontal disease. Biochemistry 2000;39:4761–4768. 103 Brezinski DA, Nesto RW, Serhan CN: Angioplasty triggers intracoronary leukotrienes and lipoxin A4. Impact of aspirin therapy. Circulation 1992;86:56–63. 104 Maddox JF, Colgan SP, Clish CB, Petasis NA, Fokin VV, Serhan CN: Lipoxin B4 regulates human monocyte/neutrophil adherence and motility: Design of stable lipoxin B4 analogs with increased biologic activity. FASEB J 1998;12:487–494. 105 Clish CB, Levy BD, Chiang N, Tai H-H, Serhan CN: Oxidoreductases in lipoxin A4 metabolic inactivation. J Biol Chem 2000;275:25372–25380. 106 Chiang N, Fierro IM, Gronert K, Serhan CN: Activation of lipoxin A4 receptors by aspirintriggered lipoxins and select peptides evokes ligand-specific responses in inflammation. J Exp Med 2000;191:1197–1207. 107 Gewirtz AT, Fokin VV, Petasis NA, Serhan CN, Madara JL: LXA4, aspirin-triggered 15-epi-LXA4, and their analogs selectively downregulate PMN azurophilic degranulation. Am J Physiol 1999; 276:C988–C994. 108 Filep JG, Zouki C, Petasis NA, Hachicha M, Serhan CN: Anti-inflammatory actions of lipoxin A4 stable analogs are demonstrable in human whole blood: Modulation of leukocyte adhesion molecules and inhibition of neutrophil-endothelial interactions. Blood 1999;94:4132–4142. 109 Lee TH, Crea AE, Gant V, Spur BW, Marron BE, Nicolaou KC, Reardon E, Brezinski M, Serhan CN: Identification of lipoxin A4 and its relationship to the sulfidopeptide leukotrienes C4, D4, and E4 in the bronchoalveolar lavage fluids obtained from patients with selected pulmonary diseases. Am Rev Respir Dis 1990;141:1453–1458. 110 Levy BD, Bertram S, Tai HH, Israel E, Fischer A, Drazen JM, Serhan CN: Agonist-induced lipoxin A4 generation: Detection by a novel lipoxin A4-ELISA. Lipids 1993;28:1047–1053. 111 Levy BD, De Sanctis GT, Devchand PR, Kim E, Ackerman K, Schmidt BA, Szczeklik W, Drazen JM, Serhan CN: Multi-pronged inhibition of airway hyper-responsiveness and inflammation by lipoxin A(4). Nat Med 2002;8:1018–1023. 112 De Bie JJ, Hessel EM, Van Ark I, Van Esch B, Hofman G, Nijkamp FP, Van Oosterhout AJ: Effect of dexamethasone and endogenous corticosterone on airway hyperresponsiveness and eosinophilia in the mouse. Br J Pharmacol 1996;119:1484–1490. 113 Jones TR, Labelle M, Belley M, Champion E, Charette L, Evans J, Ford-Hutchinson AW, Gauthier JY, Lord A, Masson P: Pharmacology of montelukast sodium (Singulair), a potent and selective leukotriene D4 receptor antagonist. Can J Physiol Pharmacol 1995;73:191–201. 114 Trifilieff A, El-Hashim A, Bertrand C: Time course of inflammatory and remodeling events in a murine model of asthma: Effect of steroid treatment. Am J Physiol Lung Cell Mol Physiol 2000; 279:L1120-L1128.

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115 Bonnans C, Vachier I, Chavis C, Godard P, Bousquet J, Chanez P: Lipoxins are potential endogenous antiinflammatory mediators in asthma. Am J Respir Crit Care Med 2002;165: 1531–1535. 116 Goh J, Baird AW, O’Keane C, Watson RW, Cottell D, Bernasconi G, Petasis NA, Godson C, Brady HR, MacMathuna P: Lipoxin A4 and aspirin-triggered 15-epi-lipoxin A4 antagonize TNFalpha-stimulated neutrophil-enterocyte interactions in vitro and attenuate TNF-alpha-induced chemokine release and colonocyte apoptosis in human intestinal mucosa ex vivo. J Immunol 2001;167:2772–2780. 117 Gewirtz AT, Collier-Hyams LS, Young AN, Kucharzik T, Guilford WJ, Parkinson JF, Williams IR, Neish AS, Madara JL: Lipoxin A4 analogs attenuate induction of intestinal epithelial proinflammatory gene expression and reduce the severity of dextran sodium sulfate-induced colitis. J Immunol 2002;168:5260–5267. 118 Munger KA, Montero A, Fukunaga M, Uda S, Yura T, Imai E, Kaneda Y, Valdivielso JM, Badr KF: Transfection of rat kidney with human 15-lipoxygenase suppresses inflammation and preserves function in experimental glomerulonephritis. Proc Natl Acad Sci USA 1999;96: 13375–13380. 119 Leonard MO, Hannan K, Burne MJ, Lappin DW, Doran P, Coleman P, Stenson C, Taylor CT, Daniels F, Godson C, Petasis NA, Rabb H, Brady HR: 15-Epi-16-(para-fluorophenoxy)-lipoxin A4-methyl ester, a synthetic analogue of 15-epi-lipoxin A4, is protective in experimental ischemic acute renal failure. J Am Soc Nephrol 2002;13:1657–1662. 120 Romano M, Luciotti G, Gangemi S, Marinucci F, Prontera C, D’Urbano E, Davi G: Urinary excretion of lipoxin A4 and related compounds: Development of new extraction techniques for lipoxins. Lab Invest 2002;82:1253–1254. 121 Gronert K, Gewirtz A, Madara JL, Serhan CN: Identification of a human enterocyte lipoxin A4 receptor that is regulated by IL-13 and IFN- and inhibits TNF-
-induced IL-8 release. J Exp Med 1998;187:1285–1294. 122 Dahlén B: Leukotrienes as Mediators of Asthma Induced by Aspirin and Allergen. Stockholm, Department of Thoracic Medicine, Karolinska Institutet, 1993, p 68. 123 Sodin-Semrl S, Taddeo B, Tseng D, Varga J, Fiore S: Lipoxin A4 inhibits IL-1 beta-induced IL-6, IL-8, and matrix metalloproteinase-3 production in human synovial fibroblasts and enhances synthesis of tissue inhibitors of metalloproteinases. J Immunol 2000;164: 2660–2666. 124 Godson C, Mitchell S, Harvey K, Petasis NA, Hogg N, Brady HR: Cutting edge: Lipoxins rapidly stimulate nonphlogistic phagocytosis of apoptotic neutrophils by monocyte-derived macrophages. J Immunol 2000;164:1663–1667. 125 Shinmura K, Tang X-L, Wang Y, Xuan Y-T, Liu S-Q, Takano H, Bhatnagar A, Bolli R: Cyclooxygenase-2 mediates the cardioprotective effects of the late phase of ischemic preconditioning in conscious rabbits. Proc Natl Acad Sci USA 2000;97:10197–10202. 126 Marcus AJ, Broekman MJ, Pinsky DJ: COX inhibitors and thromboregulation. N Engl J Med 2002;347:1025–1026. 127 Chandrasekharan NV, Dai H, Roos KLT, Evanson NK, Tomsik J, Elton TS, Simmons DL: COX-3, a cyclooxygenase-1 variant inhibited by acetaminophen and other analgesic/antipyretic drugs: Cloning, structure, and expression. Proc Natl Acad Sci USA 2002;99:13926–13931 (www.pnas.org/ cgi/doi/10.1073/pnas.162468699). 128 Warner TD, Mitchell JA: Cyclooxygenase-3 (COX-3): Filling in the gaps toward a COX continuum? Proc Natl Acad Sci USA 2002;99:13371–13373 www.pnas.org/cgi/doi/10.1073/pnas. 222543099. 129 Aliberti J, Hieny S, Reis e Sousa C, Serhan CN, Sher A: Lipoxin-mediated inhibition of IL-12 production by DCs: A mechanism for regulation of microbial immunity. Nat Immunol 2002; 3:76–82. 130 Fiore S, Maddox JF, Perez HD, Serhan CN: Identification of a human cDNA encoding a functional high affinity lipoxin A4 receptor. J Exp Med 1994;180:253–260. 131 Kang Y, Taddeo B, Varai G, Varga J, Fiore S: Mutations of serine 235–237 and tyrosine 302 residues in the human lipoxin A4 receptor intracellular domains result in sustained signaling. Biochemistry 2000;39:13551–13557.

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132 Levy BD, De Sanctis GT, Devchand PR, Kim E, Ackerman K, Schmidt BA, Szczeklik W, Drazen JM, Serhan CN: Multi-pronged inhibition of airway hyper-responsiveness and inflammation by lipoxin A4. Nat Med 2002;8:1018–1023. 133 Serhan CN, Fierro IM, Chiang N, Pouliot M: Nociceptin stimulates neutrophil chemotaxis and recruitment: Inhibition by aspirin-triggered 15-epi-lipoxin A4. J Immunol 2001;166:3650–3654. 134 Serhan CN, Jain A, Marleau S, Clish C, Colgan SP, Stahl GL, Chan L, Petasis NA, Van Dyke TE: Reduced inflammation and tissue damage in transgenic rabbits overexpressing 15-lipoxygenase and endogenous anti-inflammatory lipid mediators, in preparation.

Prof. Charles N. Serhan, Director Center for Experimental Therapeutics and Reperfusion Injury, Brigham and Women’s Hospital, Thorn Building for Medical Research, 7th Floor, 75 Francis Street, Boston, MA 02115 (USA) Tel. 1 617 732 8822, Fax 1 617 582 6141, E-Mail [email protected]

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Regulation of Vascular Permeability by Neutrophils in Acute Inflammation Lennart Lindbom Department of Physiology and Pharmacology, Karolinska Institutet, Stockholm, Sweden

The acute inflammatory reaction constitutes the first line of defense against noxious stimuli and represents the initial phase in the healing process following tissue injury. Changes at the microcirculatory level, namely increased local blood flow, enhanced microvessel permeability, and recruitment of circulating leukocytes, are characteristics of the inflammatory response and give rise to the clinical signs of inflammation – redness, heat, swelling and pain. The physiological fluid exchange and solute transport between blood and tissue is strictly controlled by the selective barrier capacity of the endothelium [1]. In normal conditions, the permeability to macromolecules is restricted whereas water and small solutes pass freely across the microvascular barrier under homeostatic control by the hydrostatic and colloid osmotic pressures in intra- and extravascular compartments. In inflammation, the endothelial barrier is compromised and becomes more permissive for the exchange of large molecules, leading to efflux of plasma proteins from the vasculature and subsequent edema formation. It has been known for a long time that a number of inflammatory mediators, e.g. histamine, bradykinin and the cysteinyl leukotrienes, through direct interaction with their receptors on the endothelial cell (EC), have the capacity to induce such permeability changes in the postcapillary venules of the microvasculature. However, it has become increasingly clear that the loss of the integrity of the endothelial barrier may also occur in conjunction with leukocyte trafficking across the vessel wall. Several in vivo studies have shown that extravasation of neutrophil granulocytes is associated with an increase in vascular permeability [2–6]. Moreover, it is evident from these observations that the capacity of various chemoattractants to induce hyperpermeability depends entirely on the presence of neutrophils, or more exactly, an intact

adhesive function of these cells. However, the detailed mechanisms responsible for neutrophil-induced alteration in vascular permeability have remained obscure. Opening of the endothelial barrier to allow extravasation of leukocytes and plasma components constitutes a physiological response of vital importance in host defense. Yet, neutrophil-induced derangement of the barrier function is also considered to contribute to vascular dysfunction in a variety of acute and chronic inflammatory disorders [7]. Consequently, a better understanding of the signaling events involved in the neutrophil-EC cross talk may provide interesting targets for therapeutic intervention in inflammatory disease.

Neutrophil Recruitment

Recruitment of circulating leukocytes is a key event in inflammatory reactions. Upon tissue insult, polymorphonuclear leukocytes (PMN), predominantly the neutrophilic granulocytes, are the first white blood cells to leave the microvasculature and enter the site of infection or injury. These cells respond to chemical signals generated in the inflamed tissue and are chemotactically activated to penetrate the vessel wall and invade the extravascular space. The number of factors known to stimulate leukocyte extravasation has grown in recent years with the characterization of the extensive family of chemokines [8, 9]. Like the classical chemoattractants, such as complement factor 5a, leukotriene B4 (LTB4) or platelet-activating factor, chemokines act on G-proteincoupled receptors and stimulate directional movement through complex intracellular signaling machineries [10, 11]. Once having reached the extravascular space, the primary function of PMN is to phagocytose and destroy invading microorganisms and foreign material as well as tissue debris. The capacity of PMN to execute their functions relies on the release of proteolytic and bactericidal granule proteins and on the generation of reactive oxygen species, all of which are biologically active compounds that may cause damage not only to invading pathogens but also to host tissue.

Neutrophil Interactions with Vascular Endothelium

Neutrophilic granulocytes constitute the vast majority of the PMN population in the blood, accounting for 50–60% of the total circulating leukocytes in man. Following release from the bone marrow, they circulate in the bloodstream in a nonactivated state, having a half-life of approximately 4–10 h, after which they are cleared from the circulation. At sites of infection or injury the neutrophils become activated and adhere to the endothelial lining of the vessel

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a

b Fig. 1. a In vivo micrograph illustrating the sequential steps in the leukocyte extravasation process: rolling along the endothelial lining, firm adhesion to and transmigration through the vessel wall, and further migration in extravascular tissue. See text for a more detailed description of the different steps. b In vivo micrograph showing neutrophil-induced leakage of plasma from small venules in the hamster cheek pouch. Intravenously injected FITC-dextran (molecular weight: 150,000) serves as a plasma tracer.

wall. Intravascular adhesion and subsequent extravasation comprise a coordinated series of interactions between leukocytes and EC precisely regulated by the activity of distinct cell surface adhesion molecules [12]. This response, like the plasma fluid and protein efflux accompanying an inflammatory insult, is normally confined to the postcapillary venular region of the microvasculature (fig. 1). Initially, free-flowing leukocytes are captured by the endothelium and bind with intermittent low-affinity interactions that give rise to rolling of leukocytes along the endothelial lining. These adhesive events, characterized by rapid on and off rates, are mediated primarily by the selectin family of adhesion molecules [13, 14]. All three selectins, L-selectin (CD62L) on leukocytes and P-selectin (CD62P) and E-selectin (CD62E) on EC, bind to carbohydrate moieties presented on the surface of the reciprocal cell [15]. These molecules have different expression patterns. Whereas L-selectin is constitutively expressed on leukocytes, activation of the endothelium is required for surface expression of the endothelial selectins. Stimulation by certain proinflammatory mediators, such as histamine and thrombin, results within minutes in translocation of preformed P-selectin from cytoplasmic stores (Weibel-Palade bodies) to the cell surface. E-selectin, on the other hand, is upregulated more slowly in response to cytokine activation. Accordingly, leukocyte rolling seen early after tissue injury is primarily P-selectin-dependent, whereas rolling in later phases is increasingly dependent on E-selectin [16, 17]. Regardless of the molecular nature of the rolling interaction, the speed of the moving leukocytes is effectively slowed down by this mechanism enabling them to detect signals from the vicinity and to initiate firm adhesion to the endothelium [18].

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Integrin-Mediated Firm Adhesion The subsequent step in the extravasation process comprises arrest and firm attachment to the vessel wall. Neutrophil firm adhesion is mediated primarily by the ␤2 integrin complex (CD11/CD18). Integrins are a large family of transmembrane receptors that connect the cell cytoskeleton with the extracellular environment [19]. Integrin receptors are composed of two noncovalently linked subunits, a larger ␣-chain and a smaller ␤-chain. The ␣- and ␤-subunits associate with each other in different combinations to form a receptor with high specific affinity for distinct ligands. Integrin receptors can be found on almost all cell types and mediate physical binding to cells and matrix components. The ␤2 integrins are the most abundant integrin receptors found on leukocytes. They share the common ␤2 subunit (CD18) which combines with either of four different ␣-chains into ␣L␤2 (CD11a/CD18, LFA-1), ␣M␤2 (CD11b/CD18, Mac-1), ␣X␤2 (CD11c/CD18, p150,95), and ␣D␤2 (CD11d/CD18) [20]. The expression of the ␤2 integrins is restricted to leukocytes and the receptor distribution differs among the various leukocyte subsets. In neutrophils, CD11a/ CD18 and CD11b/CD18 are the most important for firm adhesion to endothelium. In resting leukocytes, integrins are maintained in a conformationally low affinity state. Exposure of neutrophils to activating stimuli, such as chemokines and other chemoattractant molecules, induces rapid conversion of CD11b/ CD18 from a low to a high ligand affinity state, and, at a slower rate, increases surface expression through translocation of preformed receptor complexes from intracellular storage pools [21]. This scenario takes place in inflamed tissue when the rolling leukocyte encounters an activating stimulus, e.g. a chemokine, presented on the endothelial surface, which via intracellular signaling pathways induces instantaneous activation of the ␤2 integrins and recognition of endothelial counterreceptors, resulting in firm attachment to the vessel wall. Ligands for the ␤2 integrins on EC include adhesion molecules belonging to the immunoglobulin gene superfamily, i.e. intercellular adhesion molecule 1 and 2 (ICAM-1 and -2) [12, 20]. Both ICAM-1 and ICAM-2 are constitutively expressed on EC; however, the level of expression of ICAM-1 can be largely increased by transcriptional regulation triggered by cytokines (e.g. IL-1 and TNF-␣) and bacterial lipopolysaccharide, substantially enhancing neutrophil binding. On the other hand, interaction with the ICAMs appears in many situations not sufficient to explain the rapid binding of neutrophils to vascular endothelium in response to chemoattractant stimulation, which may suggest additional physiological ligand structures for ␤2 integrins on the endothelium [22]. The significance of ␤2 integrins for neutrophil emigration was early indicated by the leukocyte adhesion deficiency type I syndrome, which is characterized by recurrent infections and poor wound healing as a result of impaired

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phagocyte recruitment to inflammatory loci due to absent or deficient ␤2 integrin expression [23]. That ␤2 integrins are absolutely required for neutrophil adhesion to vascular endothelium in vivo has been clearly illustrated by intravital microscopy observations demonstrating that a function-blocking antibody against the common ␤2 chain (CD18) completely inhibits neutrophil adhesion (and subsequent extravasation) stimulated by various chemoattractants [5]. The importance of ␤2 integrins for the leukocyte response in a wide variety of inflammatory conditions has later been confirmed in a large number of studies [12]. Integrins connect via the cytoplasmic domain to intracellular cytoskeletal and signaling molecules, thus forming linkages between the cytoskeleton and the cell environment. Besides being involved in neutrophil attachment, the ␤2 integrins transmit signals from the surroundings that initiate and sustain vital PMN responses [24, 25]. Ligand binding triggers lateral receptor aggregation and clustering in the plasma membrane, which increases substrate-binding avidity and signaling capacity. The concerted actions of soluble mediators and integrin engagement result in cytoskeletal rearrangement and coordinate downstream activities such as motility, degranulation, and oxidant production. Adhesion greatly potentiates the effect of soluble mediators on activation of respiratory burst and release of neutrophil granule contents as compared to stimulation of PMN in suspension [26, 27]. This potentiating effect is missing in the presence of function-blocking antibodies to CD11/CD18 and is impaired in neutrophils from leukocyte adhesion deficiency type I syndrome patients [28, 29]. The intracellular signaling reactions initiated by integrin ligation and which link adhesion to downstream functional responses in PMN are complex and not known in detail but supposed to involve tyrosine kinases of the FAK and the Src families [24, 25]. Neutrophil Diapedesis Unlike the well-characterized intravascular adhesive interactions between leukocytes and EC, the mechanisms controlling the subsequent passage through the vessel wall are not well defined. Although it is generally believed that the majority of leukocytes leave the microvasculature by squeezing through the junctions between adjacent EC, evidence has also been presented for a transcellular route [30]. Neutrophils appear to migrate preferentially at endothelial tricellular intersection points where tight junctions are discontinuous [31]. Distinct molecular interactions have been suggested to guide the cell through the junction. Two separate sets of homophilic interactions, involving PECAM-1 and CD99, respectively, are supposed to act in series in this process [32]. In vitro, combined blockade of PECAM-1 and CD99 essentially abolishes diapedesis of monocytes (effect on neutrophils unknown) [33]. On the other hand, results from PECAM-1-deficient mice indicate only a moderate and

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stimulus-specific role for this molecule in regulating leukocyte transmigration [34, 35]. Antibody-blocking experiments in vivo have suggested a functional role in transendothelial migration also for the tight junction protein JAM-1 [36], possibly via interaction with the leukocyte integrin LFA-1 (CD11a/CD18) [37]. However, as for PECAM-1 involvement, conflicting data are available as to the importance of JAM-1 in controlling cell movement across the endothelium [38]. Moreover, the integrity of the EC junction may in itself determine the leukocyte’s ability to migrate through the paracellular cleft. Several reports have suggested that disorganization of junction-associated proteins and dissociation of cell contacts will facilitate transmigration [39, 40].

Structural Basis of Alteration in Endothelial Barrier Function

The major barrier to macromolecular efflux from blood to tissue is the EC layer itself. However, the glycocalyx meshwork of glycoproteins, sialoconjugates and proteoglycans on the luminal surface of the endothelium and the basolateral basement membrane may have additional restrictive influences on protein transport. In inflammation, the microvascular permeability to macromolecules is dramatically enhanced. These changes are typically confined to postcapillary venules. Even though exchange of macromolecules may occur via transcellular routes, the increased fluid and protein flux in inflammation most likely takes place through openings between the EC [1]. Early observations by Majno and Palade [41] revealed the formation of intercellular gaps in postcapillary venular endothelium in response to edematogenic mediators. This morphological adjustment has later been confirmed in a number of studies [42, 43], and has been shown to be associated with the structural rearrangement of the EC cytoskeleton [44–47]. It was originally proposed that gap formation is due to active contraction of the EC [48], which is supported by findings that the impaired barrier function is subsequent to activation of contractile elements within the cell. Several reports have shown the involvement of Ca2⫹-dependent phosphorylation of myosin light chains and activation of the actomyosin complex in enhanced EC permeability [44, 46, 47, 49]. However, the mechanism of active contraction has been disputed and gaps have instead been suggested to arise primarily because of disintegration of the lateral cell-cell adhesive contacts which may be accompanied by passive EC retraction due to inherent tensile forces [50–52]. In either case, for interendothelial gaps to form, EC contraction/retraction has to coincide with the uncoupling of junctional cell contacts. Several junctional complexes connecting neighboring EC can be distinguished, two of which are the tight junctions and the adherens junctions. These structures are formed

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by transmembrane proteins which, through their extracellular domains, promote homotypic adhesion between cells and interact through their cytoplasmic regions with cytoskeletal proteins and actin filaments [53, 54]. Tight junctions represent the most apical junction structure and contain the transmembrane proteins occludin, claudins and the recently identified junction adhesion molecule JAM-1 [55]. Compared to epithelia, this junctional complex forms a less restrictive barrier for solute exchange in the vascular endothelium, particularly in postcapillary venules where tight junctions appear to be less well developed [56]. Instead, the more basolaterally positioned adherens junction has been suggested as a major structure regulating macromolecular permeability in the microvasculature. The transmembrane proteins characteristic of adherens junctions belong to the cadherin superfamily. EC specifically express VE-cadherin that via the intracellular catenins interacts with the actin cytoskeleton [57]. Phosphorylation of adherens junction proteins and disruption of their adhesive properties have been implicated in the control of vascular permeability and the impaired barrier function in response to edema-promoting agents [58–60]. The functional roles of junctional complexes in regulating paracellular permeability in normal and pathological conditions have in recent years been the subject for intense research and work in the field has been comprehensively reviewed elsewhere [52, 53, 61–63]. Regardless of the mechanistic origin for openings in the endothelial barrier in inflammation (cell contraction or junctional protein disintegration or a combination of both), these gaps will permit increased solute and fluid transport across the vessel wall. The fluid movement is determined by convective forces and a rise in intravascular hydrostatic pressure will enhance fluid efflux considerably. Vasodilating agents, which themselves are devoid of permeabilityincreasing activity, thus act synergistically with gap-inducing mediators and largely potentiate plasma leakage from the vasculature [64]. The contribution of this mechanism to different forms of clinical inflammatory edema is substantial because of a plethora of mediators in these situations having a profound vasodilatory capacity as reflected by the commonly observed redness and heat of the inflamed tissue.

Neutrophil-Induced Increase in Vascular Permeability

Endothelial Responses to Neutrophil Activation Leukocyte recruitment and plasma fluid efflux represent physiological changes that are critical in the normal immune response to invading and noxious stimuli. Therefore, mechanisms that allow transvascular passage of the blood constituents while at the same time preventing irreversible loss of barrier

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function are required. Excessive leukocyte activation, on the other hand, may cause persistent damage to the endothelium and contribute to vascular dysfunction in various forms of inflammatory diseases. Studies carried out two decades ago clearly demonstrated that emigration of neutrophils, stimulated by chemotactic mediators, is accompanied by leakage of plasma from the vasculature and that these cells are in a position to trigger permeability changes separate from those evoked by direct-acting edematogenic mediators [2–4]. A fundamental role has been attributed to the leukocytic ␤2 integrins in this process. Numerous reports have shown that the capacity of activated neutrophils to cause plasma leakage critically depends on an intact adhesive function of the CD11/CD18 complex. Blockage of the physical interaction between leukocytes and EC with antibodies results in more or less complete abrogation of the evoked permeability change [5, 65–68]. It was originally believed that plasma might escape in the wake of the emigrating leukocyte. However, more recent data suggest that the neutrophil may not need to penetrate the endothelial lining to induce openings in the barrier, but rather, adhesion per se is sufficient to provoke these alterations [69–71]. This is supported by the promptness with which this response occurs upon chemoattractant stimulation. Thus, early cross talk between the activated neutrophil and the EC is important for the induction of the change in permeability. Docking of neutrophils to the luminal surface of the endothelium triggers a rapid and transient increase in EC cytoplasmic free Ca2⫹ [69, 70, 72]. This Ca2⫹ response, which precedes an increase in monolayer permeability, is dependent on ␤2 integrin-mediated adhesion but does not require transmigration. Interestingly, similar ␤2 integrin-dependent signaling has been demonstrated for NK cell-induced mobilization of Ca2⫹ in EC, a response that was shown to require a precedent Ca2⫹ flux in the leukocytes [73]. In some disagreement with these reports are the findings indicating that initial adhesion may be insufficient to trigger a Ca2⫹ response, but rather, Ca2⫹ mobilization occurs in EC that are immediately adjacent to the leukocyte during subsequent transmigration [74]. The observation that clamping of EC cytosolic Ca2⫹ with cell permeant calcium chelators prevents a neutrophil-induced permeability increase signifies a compulsory role for Ca2⫹-dependent regulatory processes in the evoked changes [69, 75]. This may involve phosphorylation of EC myosin light chains and actomyosin reorganization leading to conformational changes of the EC cytoskeleton and loosening of inter-EC contacts. Rearrangement of actin filaments within the cell body is a typical morphological pattern associated with enhanced endothelial permeability and interference with the formation or stability of the filamentous network will influence barrier function [61]. Activation of myosin light chain kinase (MLCK) and actin-myosin contraction is a Ca2⫹/calmodulin-dependent event and adherent neutrophils have been

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shown to stimulate such enzyme activity [76, 77]. Pharmacological inhibition of MLCK also largely prevents the increase in permeability induced by activated neutrophils [78]. Taken together, these reports suggest that integrindependent neutrophil adhesion to the endothelial lining triggers a rise in EC cytosolic free Ca2⫹, MLCK activation and rearrangement of the actin cytoskeleton. This chain of events supports a model whereby tension generated by the contractile elements induces adjacent cells to separate, which then causes the formation of interendothelial gaps. Besides active EC contraction other mechanisms likely contribute to enhanced permeability following PMN adhesion. Phosphorylation of junctionassociated proteins and downregulation of adhesive contacts between neighboring EC represent yet another mechanism by which activated leukocytes may trigger EC hyperpermeability [79, 80]. Neutrophil binding to the EC surface has been shown to cause disorganization of the VE-cadherin junctional complex, which is accompanied by an increase in EC permeability [66, 81]. However, whether or not this response is due to a physiological leukocyte-toendothelium signaling event is at present unclear [82]. Moreover, the fact that an antibody blockade of the VE-cadherin function results in increased permeability to both solutes and leukocytes [39, 40, 58] does not necessarily mean that disruption of VE-cadherin-adhesive properties is a regulatory mechanism utilized by leukocytes to open up the endothelial barrier. Nonetheless, since junction proteins directly associate with cytoskeletal actin filaments, it is well conceivable that contractile and junctional mechanisms act synergistically to cause openings in the endothelial barrier in response to PMN activation. As modeled for neutrophil transepithelial migration, these events may also occur in a sequential manner; initial MLCK-dependent cell contraction followed by junctional remodeling [83]. Oxidant-Mediated Endothelial Barrier Dysfunction Knowing that neutrophil adhesion to the endothelial lining initiates a series of active events in adjacent EC, eventually leading to increased paracellular permeability, the question arises of how signal transfer is accomplished in the cellular cross talk between leukocytes and endothelium. Neutrophils are capable of generating highly reactive oxygen species and carry in their granules a multitude of preformed bioactive and lysosomal agents, all of which have the capacity to cause injury to adjacent cells and matrix structures. As such, these compounds may contribute to endothelial barrier dysfunction in inflammation [7, 84]. However, it is reasonable to consider that these neutrophil products are intended primarily for microbial killing and destruction of foreign particles, and teleologically, they should not be released until the neutrophil has encountered the stimulus in the extravascular space. Further, from a physiological

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regulatory point of view it would seem disadvantageous that the neutrophil should make use of potentially harmful mechanisms to open up the endothelial barrier. Nonetheless, during situations of oxidant stress abundant reactive oxygen material is formed (e.g. superoxide anions and hydrogen peroxide) and during the last two decades a multitude of data have emerged showing that these agents may cause clear-cut alterations in endothelial barrier function [63], not only through inducing plasmalemma lipid peroxidation and cell disintegration but possibly also via activation of signaling pathways leading to an elevation of intracellular free Ca2⫹ [85], MLCK activation [86], and reorganization of junctional proteins [87, 88]. Oxidants that could signal these events may originate not only from activated leukocytes but may be generated by the EC themselves upon neutrophil adhesion [89]. However, the involvement of reactive oxygen species in neutrophil-evoked permeability increase is controversial and a large number of studies have failed to show any participation of such molecules, but rather, that this type of leakage occurs via oxygen-radical independent mechanisms [65, 67, 90]. It is worth noting that oxidants may stimulate endothelial production of proinflammatory mediators as well as increased expression of cell adhesion molecules that in turn will promote leukocyte adhesion [91–93]. Thus, oxidants may via such mechanisms indirectly contribute to neutrophil-dependent endothelial responses and enhanced vascular permeability. Role of Neutrophil Cationic Proteins in Regulation of Endothelial Barrier Function The obligatory role of ␤2 integrins in PMN-induced alteration in endothelial barrier capacity suggests that signaling via these receptors is involved in the evoked EC responses. Integrin-mediated ligation of ICAM-1 may directly initiate signaling events into EC and trigger reorganization of endothelial actin filaments [89, 94, 95]. On preactivated endothelium, neutrophil engagement also of other EC adhesion molecules has been shown to cause a rise in intracellular free calcium and cytoskeletal rearrangement that is CD18-independent [96]. However, ␤2 integrin-mediated adhesion may lead to EC activation not primarily because of direct interaction with endothelial counterreceptors but rather via indirect mechanisms. Adherence-dependent engagement of ␤2 integrins and transmembrane signaling is known to cause PMN activation [25, 28] and to initiate secretory events as well as respiratory burst [26, 97, 98]. In fact, outside-in signaling by ␤2 integrins, simulated through antibody cross-linking of CD11b/CD18, triggers secretion of PMN-derived factors that provoke increased permeability of EC monolayers independent of direct engagement of EC counterreceptors [75]. Further, cell-free supernatant obtained after CD18 cross-linking in suspended PMN induces EC responses indistinguishable from

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that induced by direct PMN activation and adhesion to EC. This indicates that stable soluble factors may account for at least part of the permeability-increasing activity of adherent PMN. Such factors may be preformed and originate from intracellular storage organelles. Indeed, neutrophils are equipped with distinct granule compartments, containing an array of bioactive glycoproteins, which become exocytosed at various stages of cell activation [99]. On the basis of their protein content these granules have been classified in azurophilic (primary), specific (secondary), and gelatinase (tertiary) granules, and so-called secretory vesicles. Upon neutrophil activation, the secretory vesicles are considered to be the most readily mobilized compartment followed by the gelatinase and specific granules, and lastly the azurophil granules. The function of these storage organelles is not only to provide enzymes for hydrolytic substrate degradation, but also to secrete their contents for destruction of microorganisms and to regulate various physiological and pathological processes, including inflammation. For this purpose they carry antimicrobial and cytotoxic substances, neutral proteinases, acid hydrolases, and a pool of cytoplasmic membrane receptors [99]. Over the last two decades, there has been a certain focus on PMN-derived granule proteins as potential mediators of leukocyte-driven plasma leakage, in particular on that stored in the azurophil granule. This granule compartment contains, among several other proteins, the serprocidin family of cationic glycoproteins. Three members of this family, cathepsin G, elastase, and proteinase 3, are serine proteases, while the fourth one, azurocidin, also known as CAP37 and heparin binding protein (HBP), is a proteolytically inactive homologue [99, 100]. Elastase, cathepsin G, and HBP/CAP37 have all been shown to stimulate increased permeability of EC monolayers [101–103]. However, diverging views exist as to the mechanisms by which these cationic proteins exert their effect on monolayer integrity. With regard to elastase, it is suggested that the protein acts primarily through enzymatic action [102], possibly via proteolysis of EC cadherins [104]. Interestingly, it has recently been demonstrated that activated PMN retain exocytosed elastase on the cell surface and that this membrane-bound elastase pool is localized to the leading front of the migrating cell [105]. Such a mechanism may serve to protect the protease from being degraded by protease inhibitors in plasma, and to target enzymatic activity to the particular endothelial junction being traversed. This may contribute to focal disruption of the VE-cadherin complex, which was recently shown to be restricted to areas in contact with the migrating cell [106]. On the other hand, involvement of neutrophil elastase in the prompt increase in vascular permeability upon neutrophil adhesion is disputed by observations indicating that EC dysfunction due to elastase release seems to occur only after a fairly long incubation of EC with PMN (possibly because of slow mobilization of the azurophil

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granules) and requires prestimulation of leukocytes or EC with cytokines/ lipopolysaccharides [107, 108]. At variance with the notion that neutrophil cationic proteins alter endothelial barrier function due to proteolytic activity, data are at hand indicating a nonenzymatic effect of these molecules on vessel wall permeability, presumably via charge interactions [101, 103, 109–111]. Using an intravital microscopy model, it was shown that the polyanion dextran sulfate, but not specific enzyme inhibitors, is effective in neutralizing plasma leakage induced by exogenous elastase or polylysine [111]. More interestingly, similar results are obtained when instead the chemoattractant LTB4 is used as a stimulus [110, 112]. In the latter case, activated leukocytes are responsible for the plasma leakage because LTB4 itself has no direct permeability-increasing activity on the endothelium [4]. The capacity of polyanions to antagonize neutrophildependent permeability responses in vivo has been confirmed in more recent studies [113]. However, in direct contrast to the aforementioned studies, opposite findings with regard to the effect of enzyme inhibitors and polyanions, respectively, have been obtained in a model for neutrophil-evoked permeability changes in cultured EC in vitro [102]. The reason for these contrasting results is unknown, but may reflect distinct characteristics of EC in culture as compared to endothelium in vivo. Recent data from our own laboratory indicate that the proteolytically inactive serprocidin family member HBP/CAP37 has a central signaling role in the cell-cell cross talk between PMN and EC. We have demonstrated that antibody-induced cross-linking of ␤2 integrins, mimicking adhesiondependent receptor engagement, triggers the release of neutrophil-borne cationic proteins that provoke a rise in cytosolic free Ca2⫹ in adjacent EC, rearrangement of actin filaments, and increased EC paracellular permeability [75]. These responses, which are identical to those achieved by chemoattractant stimulation of PMN, depend on the presence of HBP/CAP37 in the secreted material [103]. Apparently, since HBP lacks enzymatic activity and is able to stimulate active responses in EC, a nonlytic mechanism underlying its permeability-increasing activity is suggested. Interestingly enough, even though other cationic proteins can be detected in the PMN-derived secretion, no evidence was found for the involvement of elastase and/or cathepsin G in the evoked EC responses. In this regard, two characteristics of HBP may serve to explain a unique contribution of this protein in the PMN-induced increase in EC permeability. First, compared to the other serprocidins, HBP carries a large number of positively charged amino acid residues concentrated to one side of the protein, creating a strong dipole moment in the molecule that is likely important for its activity. It is possible that the basic patch interacts with negatively charged proteoglycans on the EC surface by which EC conformational

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Chemoattractant

PMN granule

Cationic protein

Fig. 2. Proposed mechanism for neutrophil-induced alteration in EC barrier function. At sites of inflammation, neutrophils become activated by a chemoattractant, which stimulates adhesion to the endothelial lining through activation of ␤2 integrins. Engagement of the ␤2 integrins (e.g. ICAM-1 binding) initiates intracellular signaling events that trigger degranulation and release of cationic proteins, one of which corresponds to the inactive serine protease homologue HBP/CAP37. HBP, and possibly other granule proteins, induces structural rearrangements in adjacent EC, likely through interacting with proteoglycans on the EC surface, which then leads to the formation of interendothelial gaps and plasma fluid efflux.

changes are induced through as yet unidentified mechanisms. The strong affinity of HBP and other neutrophil cationic proteins for binding negatively charged molecules likely explains the neutralizing capacity of polyanions observed in many model systems with regard to neutrophil-induced permeability changes. Second, the notion that the azurophil granule pool is only slowly mobilized upon cell activation does not seem compatible with the idea that members of the serprocidin family are indeed responsible for triggering the rapid changes in EC permeability following PMN adhesion. However, HBP/CAP37 is also stored in a more readily mobilized compartment located close to the plasma membrane and is rapidly released upon cell activation, supporting a role for this protein already in the very initial phase of neutrophil activation [114]. Thus, the neutrophil-derived cationic protein HBP/CAP37 fulfils the criteria of a signaling link between activated PMN and EC, which in a paracrine manner regulates EC barrier function in conjunction with neutrophil trafficking in inflammation (fig. 2). Yet, the mechanisms by which neutrophil cationic proteins may activate signaling pathways in EC and stimulate reorganization of cytoskeletal and junctional complexes remain obscure.

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Role of Endothelial Gap Formation in Leukocyte Transendothelial Migration

While there is no doubt that activated neutrophils trigger openings in the endothelial barrier that allows plasma to escape from the vasculature, it is not clear whether such openings also serve the function to facilitate passage of the migrating cell. Findings demonstrating that inhibition of the VE-cadherin function results in coincident increases in endothelial permeability and neutrophil transmigration speak in favor of a causal relationship between EC layer integrity and the capacity for leukocytes to penetrate the barrier [40, 58]. Further, inhibition of MLCK and actin-myosin contraction in EC reduces neutrophil transendothelial migration [77, 115]. The notion of an active role for the endothelium in neutrophil diapedesis is supported by earlier findings demonstrating that clamping of EC intracellular free Ca2⫹ prevents not only the increase in EC monolayer permeability triggered by adherent neutrophils, but also the transmigration of these cells [69]. At variance with these observations are findings indicating that neutrophils might traverse the endothelium seemingly without the need for an increase in EC permeability. Burns et al. [31, 116] have presented evidence that the barrier capacity of EC monolayers is preserved and that tight junctions remain intact during neutrophil transmigration. The authors linked these findings to the notion that neutrophils migrate preferentially at endothelial tricellular corners where junctional complexes are discontinuous. Whether or not activated neutrophils trigger an increase in endothelial permeability also seems to depend on the number of leukocytes that are in contact with the EC. Studies have shown, for a low PMN:EC ratio, that neutrophils transmigrate without causing any measurable change in EC permeability, whereas in the case of a large number of PMN, transmigration is accompanied by a permeability increase [69, 117]. In a more physiological in vivo setting further evidence has been provided that neutrophil transmigration might occur independent of changes in permeability. While neutrophil-dependent LTB4induced macromolecular leakage was largely prevented in animals treated with dextran sulfate, neutrophils extravasated in equal numbers as compared to animals receiving no such treatment [112]. The question as to what extent these events can be dissociated and whether neutrophils might escape from the vasculature without creating interendothelial gaps needs further evaluation.

Concluding Remarks

Results derived from in vivo models clearly illustrate that neutrophil trafficking in acute inflammation is accompanied by an increase in vascular

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permeability. Neutrophil-induced adjustment of endothelial barrier capacity represents an important regulatory mechanism in immune surveillance allowing blood components to leave the vasculature and enter into extravascular tissue. However, loss of barrier integrity due to neutrophil activation may also contribute to vascular dysfunction in inflammatory disease. The precise mechanisms with which neutrophils exert their effects on vascular permeability are not entirely clear. Available data suggest that neutrophil adhesion to the endothelial lining induces phosphorylation of cytoskeletal and junctional proteins causing remodeling of the actin cytoskeleton and EC junctions. The distinct signaling pathways triggering these events and their respective contribution to EC hyperpermeability have yet to be elucidated in more detail. Further, the molecular interactions and mediators involved in the signal transfer between the activated PMN and the adjacent EC are ill-defined, and data in the literature are in many respects inconsistent. This may depend on different model systems used, stimulus strength and temporal resolution, and calls for a need to better define the experimental conditions that prevail in each specific case. To this end, it will be important to verify and confirm the functional relevance in vivo of conclusions reached in various models in vitro. Although much information has been gained in recent years about the leukocyte-endothelial cross talk in inflammation, an incomplete understanding of the signaling mechanisms involved in leukocyte-induced alteration in vascular permeability warrants further research. A deeper insight into these mechanisms may contribute to better therapeutic strategies for intervention in inflammatory disease conditions. Acknowledgments The authors’ work is supported by the Swedish Medical Research Council, the Swedish Foundation for Health Care Sciences and Allergy Research, the Swedish Heart-Lung Foundation, Inga Britt and Arne Lundbergs Foundation, and Karolinska Institutet.

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Lennart Lindbom, Department of Physiology and Pharmacology, Karolinska Institutet, SE–171 77 Stockholm (Sweden) Tel. ⫹46 8 7287207, Fax ⫹46 8 332047, E-Mail [email protected]

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Cassatella MA (ed): The Neutrophil. Chem Immunol Allergy. Basel, Karger, 2003, vol 83, pp 167–181

Neutrophils and Angiogenesis: Potential Initiators of the Angiogenic Cascade Roberto Benelli, Adriana Albini, Douglas Noonan Istituto Nazionale per la Ricera sul Cancro, Genoa, Italy

Mature neutrophils are able to regulate several aspects of the immune response; their influence extends beyond innate immunity, as they can modify lymphocyte maturation and behavior. As a ‘ready-to-use’ cell, the neutrophil stores in its granules several soluble mediators that can be rapidly secreted in both physiological and pathological conditions. They are the first cell types to respond to most inflammatory agents. When activated as an effector cell, the granulocyte is also able to synthesize an array of factors and to rapidly produce oxygen and hypochlorite radicals that can destroy pathogens. These same radicals can be the cause of tissue damage and in some cases are responsible for severe pathologies linked to abnormal neutrophil activity. As a leading cell in inflammation, the neutrophil initiates a cascade that results in a full immune response (see other chapters in this volume). One endpoint of inflammation is tissue reconstruction, including vascularization. Angiogenesis is a well-known endpoint in chronic inflammatory diseases such as arthritis [1], and inflammatory cells appear to play a crucial role during tumor progression and in particular the angiogenic switch from hyperplasia to carcinoma [2]. Among the soluble mediators and radicals produced by neutrophils there are several potential regulators of the angiogenic process. Neutrophils appear to also have the potential for playing a direct role in the angiogenic process, although the direct link between angiogenesis and neutrophils has received little attention to date. Recent studies have suggested that these cells can play a primary role in the angiogenic response and may play a much more important part in several pathologies related to angiogenesis than previously thought; here we review the evidence supporting this hypothesis.

Neutrophils and Angiogenesis

Chemokines and Angiogenesis One of the early indications that inflammatory cells may participate in angiogenesis was the observation that the CXC chemokine IL-8 (CXCL8) induced angiogenesis in in vivo models [3, 4]. The subsequent observation that other CXC chemokines instead inhibited angiogenesis gave rise to the hypothesis that an ELR motif, characteristic of CXCL8, was associated with a proangiogenic phenotype while the absence of this motif indicated an antiangiogenic activity [5]. The role of the ELR sequence in relation to angiogenic activity was supported by mutagenesis studies which demonstrated that alteration of the ELR motif was associated with a loss of angiogenic potential [6]. With time, it became clear that the ELR motif was indicative of chemokines that bind to and activate the chemokine receptor CXCR2 [7]. Another series of non-ELR chemokines with potent antiangiogenic activity were ligands for the CXCR3 receptor, while the angiogenic activity for other CXC chemokines varies: some appear to have proangiogenic activity (e.g. CXCL12, a ligand for CXCR4) and others antiangiogenic activity (e.g. CXCL4) [8–10]. The CXCR2 ligands are all chemokines that are potently active on neutrophils. Surprisingly, in the corneal micropocket assay for angiogenesis little inflammatory response to CXCR2 ligands was observed [6], although there was a significant angiogenic response. This lack of inflammatory response to these potent neutrophil chemoattractants may be due to the immune privilege of the anterior chamber of the eye [11, 12]. The consistent angiogenic response obtained suggested that these ligands could directly interact with endothelial cells. Clearly some endothelial cells can express the CXCR2 receptor and will migrate in response to these ligands in vitro [13]. Studies on the angiogenic activity of CXCL8-derived ‘muteins’ of the ELR sequence further supported the hypothesis of direct interactions with endothelial cells. ELR mutant proteins did not induce angiogenesis in rodent models in vivo or endothelial cell chemotaxis in vitro, but retained chemotactic activity for human neutrophils [6]. The retention of neutrophil chemotactic activity in these mutant proteins with low affinity for CXCR2 suggested that they have a high affinity for other chemokine receptors expressed on human neutrophils, such as CXCR1, although this was not directly addressed. Whether CXCR2 ligands induce angiogenesis directly through CXCR2 on endothelial cells or indirectly through neutrophils and other leukocyte mediators has recently become controversial. It is clear that neutrophils play an important role in regulating angiogenesis in the endometrium, as discussed in detail below. While microvascular endothelial cells have been shown to express CXCR2 [13], other endothelial cells clearly neither express this receptor nor

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respond to ligands for CXCR2 [14, 15]. We recently confirmed the angiogenic activity of CXCR2 ligands in the Matrigel plug angiogenesis assay in vivo [16]. However, in this model neutrophil depletion of the animals was found to completely abrogate the angiogenic response to these chemokines, implicating a key role for neutrophils in the angiogenic process induced by CXCR2 ligands [16]. Finally, generic inflammatory stimuli such as bacterial lipopolysaccharides (LPS) also induced angiogensis in vivo, suggesting that the inflammatory stimulus leads to an angiogenic cascade [16], as would be expected from clinical observations in chronic inflammation [1]. Blocking of CXCR2 has been shown to inhibit angiogenesis and fibrosis in a lung inflammation model [13], but not to alter neutrophil infiltration, again suggesting that other neutrophil chemotactic factors actively recruited neutrophils but did not induce angiogenesis in these models. The effects of neutrophil depletion were not reported. The biological responses induced by the closely related receptors CXCR1 and CXCR2 are clearly quite different [17]. For example, activation of CXCR2 on neutrophils causes chemotaxis, whereas binding to CXCR1 also triggers the respiratory burst [18]. Taken together these data may suggest the following: (1) The biological response elicited in neutrophils in the absence of CXCR2 may be profoundly different from that induced by CXCR2 activation, where the latter is associated with induction of an angiogenic cascade by these cells. This could well be the result of changes in the balance between the pro- and antiangiogenic activities of neutrophils. (2) The cellular targets of CXCR2 ligands may vary among different anatomical sites, depending upon the immune privilege status of the site and the CXCR2 expression status of the endothelial cells in the specific compartment. These intriguing data clearly indicate that additional studies are required to fully investigate the mechanisms of action of the CXCR2 chemokines in angiogenesis-associated human pathophysiology.

Soluble Mediators Released by Neutrophils

Neutrophils contain two main types of granules known as primary and secondary granules. Primary (azurophilic) granules are true lysosomes containing collagenases, elastase, cathepsin E and myeloperoxidase, and mediate pathogen degradation and often tissue damage. Secondary (specific) granules are large stores for soluble mediators and do not contain acid proteases. Many stimuli (i.e. fMLP and TNF-␣) cause only a release of the contents of the secondary granule into the extracellular environment. These neutrophil products direct the initial phases of the inflammatory response and modulate tissue reconstitution. The molecules with known angiogenic activity released by neutrophils include vascular endothelial growth factor (VEGF), IL-8, hepatocyte growth

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factor (HGF) and platelet activating factor (PAF). IL-8 and PAF (which are neosynthesized) can in turn act on neutrophils, self-propagating PMN recruitment. Vascular Endothelial Growth Factor VEGF is known as the principal growth/survival factor for the endothelial cell. Gaudry et al. [19] were the first to describe VEGF storage and secretion by human neutrophils. Increasing amounts of VEGF are released by activated neutrophils from 15 min to 2 h after challenge with PMA. The VEGF titer obtained from 107 neutrophils reached 700 pg/ml after 2 h. The quantification of the intracellular pool of VEGF showed similar amounts of protein. These data showed that neutrophils would be the first source of VEGF during any inflammatory event, releasing this factor in the early stages of inflammation. The physiopathological consequences of this activity are still underestimated; it should also be noted that VEGF has a significant vascular permeability activity, and this most likely contributes to edema and facilitates leukocyte recruitment in inflammation. IL-8 This chemokine is one of the most potent chemoattractants for neutrophils; however, it is also secreted by these cells in response to many stimuli, apparently inducing a self-propagating accumulation at the inflammatory site. IL-8 is present in neutrophils as a ‘ready to use’ protein in secondary granules, and it can also be rapidly expressed in any activating situation. IL-8 has the ability to modulate neutrophil responses; its higher affinity binding to CXCR2 induces chemotaxis while binding to CXCR1 triggers the respiratory burst [18]. When wound healing experiments were performed on CXCR2 knockout mice [20], decreased recruitment of neutrophils and keratinocytes was observed, and neovascularization was retarded. This is consistent with our demonstration that chemokine ligands for CXCR2 induce a strong angiogenic response with conspicuous neutrophil infiltration in normal mice, while mice made neutropenic failed completely to induce any vascular recruitment [16], supporting the observation that angiogenic activity can be mediated by neutrophils. Hepatocyte Growth Factor HGF is a pleiotropic cytokine with strong angiogenic properties. TNF-␣, LPS or fMLP stimulation induce a significant increase of HGF release in neutrophils (755, 484 and 565 pg/ml, respectively) compared to controls (118 pg/ml) [21]. The analysis of total HGF release following cell lysis indicated that HGF is released from preconstituted intracellular stores, as already shown for VEGF. Interestingly, unlike most sources where HGF is released as

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a propeptide that requires proteolytic cleavage for activation, the HGF stored in and released from neutrophils is already in an active conformation [21]. No influence of HGF on PMN physiology was reported; however, in addition to VEGF, proinflammatory mediators also stimulated HGF release from PMN intracellular stores [21]. As this is the first report concerning neutrophils and HGF, further studies are needed to evaluate the part of this mediator in neutrophil-sustained angiogenesis. Platelet Activating Factor PAF is a phospholipid produced by platelets, leukocytes and vascular endothelial cells with a powerful agonist activity on specific G-protein-coupled receptors. Its role as an early amplifier of the inflammatory signal is quite unspecific as neutrophils and vascular cells are both sources and targets of PAF. Due to its lipidic origin, PAF is not stored in neutrophil granules but it is neosynthesized following most types of stimulation (bacteria, chemokines, cytokines, C5) [22]. The primary role of PAF appears to be the enhancement of neutrophil-endothelium interaction, promoting the extravasation of these leukocytes [23]. Different concentrations of PAF can elicit distinct patterns of neutrophil activation as shown in a bovine model by Swain et al. [24]. Swain et al. demonstrated that lower concentrations of PAF promote neutrophil sensitivity and interaction by selective degranulation (secondary/specific granules), upregulation of adhesion molecules (Mac-l and L-selectin), and increased actin polymerization. In contrast, higher PAF concentrations can promote more direct bactericidal responses, including the release of reactive oxygen species (ROS) and primary granule enzymes. PAF is also an angiogenic molecule [25] that can directly recruit vascular cells [26], an activity mediated by nitric oxide (NO) production [27]. PAF also appears to be able to induce the transcription of several angiogenic mediators through the activation of NF␬B [28, 29]. Among the NF␬B-dependent mediators investigated, only anti-VEGF antibodies or soluble VEGF receptors yielded a significant inhibition of the angiogenic effect of PAF, while antibodies against TNF-␣, IL-1␣ or bFGF were ineffective [29], indicating that VEGF is the most likely mediator of the angiogenic activity of PAF. In addition, activated neutrophils can also release potent chemoattractants for monocytes and macrophages such as the chemokine MCP-1 [30]. Macrophages can in turn release angiogenic factors such as VEGF [31–33], which can further maintain and propagate inflammatory angiogenesis. All these soluble factors contribute to the global angiogenic stimulus that neutrophils can exert in activating conditions; as these phagocytes are heavily recruited in any inflammatory site, their potential contribution to the initial phases of the angiogenesis process should no longer be ignored.

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Neutrophils and the Endometrium

Extensive studies on the tissue remodeling process in the endometrium have supported a role for neutrophils in angiogenesis. In 1994 Kelly et al. [34] examined the ability of endometrium explants and chorion cells in culture to synthesize and release IL-8. In the endometrium, the stage of the menstrual cycle did not affect IL-8 production, which was constant, and only high-dose progesterone reduced IL-8 levels. These observations suggested that the endometrium could be a target for neutrophil recruitment. Six years later Mueller et al. [35] identified the leukocyte populations infiltrating the endometrium and the source of endometrial VEGF. Immunohistochemical analysis of VEGF-positive cells in 31 bioptic samples of normal endometrium showed that, in addition to epithelial and stromal endometrial cells, the predominant cells that stained for VEGF were neutrophil granulocytes. Neutrophils were more abundant in the secretory phase of the menstrual cycle, making the authors postulate that neutrophil granulocytes infiltrating the human endometrium could regulate cyclical endometrial vascular proliferation by VEGF production. The more recent studies on endometrial angiogenesis by Gargett and coworkers [36, 37] related the expression of focal VEGF associated with microvessels to endothelial cell proliferation. The analyses were extended to the three layers of human endometrium at various stages of the menstrual cycle. Immunohistochemical analysis of 18 healthy samples showed that the percentage of proliferating vessels was higher in proliferative as compared to secretory endometrium, but only in the basalis layer. The percentage of VEGFstained endothelium was significantly higher in the proliferative than in the secretory endometrium. The most intensely VEGF-stained microvessels were observed in the subepithelial capillary plexus, followed by the functionalis and then the basalis. The detection of VEGF corresponded to areas of proliferating vessels for the three layers of tissue. VEGF associated with microvessels was found in marginating and adherent neutrophils, suggesting that these leukocytes could be the source of intravascular VEGF for the vasculature of endometrium.

Neutrophils as Antiangiogenesis Effectors

Neutrophils also show several activities that could potentially inhibit angiogenesis, through either direct activities of soluble mediators or downstream production via proteolysis.

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Free Radicals Under strong activating stimuli, neutrophils engage in respiratory burst activity that results in free radical production and secretion. In inflammation, the concomitant presence of ROS and a reduction of free oxygen can cause endothelial cell apoptosis [38]. Endothelial cells actively produce and use NO and ROS (especially H2O2) as intracellular mediators of signal transduction and, extracellularly, for communication with vascular smooth muscle cells [39–42]. In this way we could assume that NO and ROS could act as proangiogenic factors at low concentrations (normally produced by the endothelial cell itself), while they could cause endothelial cell toxicity and death at high concentrations (when released by neutrophils during acute inflammation). NO is a known downstream mediator of VEGF receptor activation [43]. ROS are able to react with NO forming highly toxic radicals [44]; this block of NO-mediated signals could impair VEGF activity. The simultaneous rise in free radicals and decrease in VEGF would clearly be detrimental for endothelial cells and enhance their apoptosis. These observations suggest that while the partial activation of neutrophils would act as a proangiogenic determinant, the occurrence of the respiratory burst could rapidly invert this angiogenic balance and even kill the endothelium (fig. 1). This brings us back to the dissimilar activities of proangiogenic chemokines activating CXCR2 and the potentially antiangiogenic activity of neutrophil recruitment in the absence of CXCR2, favoring full neutrophil activation and tissue damage. In the perspective of neutrophil-targeted therapies, only the occurrence of an acute inflammation would be active against endothelial and, probably, tumor cells, while submaximal neutrophil recruitment would lead to angiogenesis and tumor growth (fig. 1). Angiostatin This peptide fragment of plasminogen was originally described as a tumorderived antiangiogenic agent [45]. Angiostatin (AST) has been reported to target the endothelial cell, blocking the angiogenic process and causing the apoptosis of activated vascular cells [46–48]. Recently Scapini et al. [49] have demonstrated that PMN elastase, released from primary neutrophil granules, can degrade plasminogen to the kringle 1–3 form fragment of AST. This neutrophil-derived AST inhibited endothelial cell proliferation in vitro and the vascularization of VEGF-enriched Matrigel plugs in vivo, displaying a full antiangiogenic potential. This study indicates that the activation of a neutrophilmediated response can lead to the indirect production of specific antiangiogenic molecules. As AST generation is linked to elastase release, stored in primary granules, only degranulating neutrophils would be a source of this inhibitor.

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Plasminogen VEGF HGF IL-12 (mouse)

Elastase Angiostatin

Stored in secondary granules

Stored in primary granules

Angiogenic activities

Antiangiogenic activities Neosynthesis IP-10, IL-8, PAF IL-12, MIG, radicals

Fig. 1. The opposing activities of neutrophils in angiogenesis. Fully activated neutrophils can suppress angiogenesis (left side) through neutrophil elastase that converts plasminogen to angiostatin and synthesis of angiostatic cytokines and oxygen radicals. In contrast, partially (i.e. CXCR2) activated neutrophils (right side) release angiogenic growth factors that can initiate an angiogenic cascade.

We have recently shown that neutrophils themselves are a target of AST [16]. AST inhibited the recruitment of PMN stimulated by CXCR2-binding chemokines and fMLP in vitro. When the same stimuli were used to induce an angiogenic response in the Matrigel plug model in vivo, a strong infiltration of neutrophils, leading endothelial cell invasion and vascularization, was observed. The addition of AST to the gels caused an almost complete inhibition of PMN infiltration and blocked subsequent angiogenesis. As AST is produced by and is active on neutrophils, it could represent a physiological feedback signal regulating the inflammatory response. The observations of the involvement of AST in neutrophil-regulated angiogenesis and inflammation could impact the development of new therapeutic strategies. Angiostatic Cytokines Although neutrophils are a source of soluble angiogenic factors, as discussed above, they also store or produce cytokines that can inhibit angiogenesis. Neutrophil secondary granules contain IL-12 [50, 51], an immunemodulatory cytokine able to exert antiangiogenic effects possibly mediated

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through the induction of secondary soluble effectors (i.e. IFN-␥ and the CXCR3 ligand IP-10). In addition, the neutrophil, when exposed to inflammatory stimuli, can directly synthesize the chemokines IP-10 and MIG [52], both CXCR3 chemokine ligands with potent antiangiogenic potential [53]. Anti-Inflammatory Agents Recent studies have indicated that anti-inflammatory agents also inhibit angiogenesis. This is particularly highlighted by studies on nonsteroidal antiinflammatory drugs demonstrating that COX-2 inhibitors are also strong inhibitors of angiogenesis [54–56], an activity that may be involved in the chemopreventive properties of this class of molecules as well [54–56]. Flavonoids from green tea, a beverage linked both to cancer prevention [57–59] and inhibition of angiogenesis [60] and inflammation [61–64], may also target neutrophils. A recent study has demonstrated that green tea flavonoids are extremely potent inhibitors of neutrophil elastase [62]; these molecules may also inhibit other aspects of neutrophil function. In inflammation, the neutrophil is the first effector cell to intervene. However, their presence is transitory, these cells rapidly undergo apoptosis, and in the later phases of inflammation the recruitment of these cells is restrained. The generation of inhibitory molecules such as AST, and diffusion of high-level IL-8 release leading to a loss of chemotactic gradients and receptor downregulation are possible mechanisms suppressing neutrophil infiltration. The role of the neutrophil in chronically inflamed tissue, such as in arthritis and tumors, is less well studied. Investigation of the presence of neutrophils in these tissues using specific markers would help in understanding the potential long-term contributions of these cells to inflammatory angiogenesis. To Induce or Inhibit Angiogenesis: What Makes the Difference?

The review of studies regarding the involvement of neutrophils in angiogenesis strongly indicates that they may have a role in angiogenesis, but also generates several legitimate questions. As both pro- and antiangiogenic factors can be induced by the same inflammatory or microbial stimuli, what makes the difference as to which activity predominates? Do CXCR2 ligands target the endothelial cell directly, or is their angiogenic potential neutrophil-mediated? Is the neutrophil an inducer or a repressor of endothelial cell recruitment/ activation and under which circumstances? Only extended studies will answer these questions, but we can speculate now that the picture will be composite and controversial. The abundant literature on macrophages and angiogenesis could be a clear example of the complex evolution of this field.

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The involvement of CXCR2 ligands in angiogenesis is still controversial regarding the direct or indirect ability, by these molecules, to recruit the endothelium and cause angiogenesis. Several reports maintain the ‘direct’ hypothesis indicating that the ELR motif present in most alpha chemokines would confer the ability to directly activate CXCR2 on microcapillary cells [13]. Alteration of the ELR sequence in CXCL8 was accompanied by the lack of endothelial response in rodent models while migration of human neutrophils was not affected [6]. Human neutrophils also express CXCR1, a receptor for IL-8, however there appears to be no CXCR1 homologue in murine systems; it would be of interest to investigate whether murine neutrophils could respond to ELR mutants of IL-8 and the biological activity of these mutants in in vivo murine models. Direct recruitment of endothelial cells does not necessarily imply that vascularization will follow. Once activated and extravasated, endothelial cells become VEGF-dependent, and an absence of this growth factor leads to the apoptotic death of activated endothelial cells. Neutrophils are rapidly migrating cells that are the first to enter any tissue under an acute inflammatory stimulus. These phagocytes would reach the target tissue before endothelial cells in response to a stimulus effective on both cell types. It is possible that these cells could help prepare a suitable environment for subsequent endothelial cell invasion, releasing angiogenic factors such as VEGF and HGF (fig. 1). Our studies on the angiogenic activity of HIV Tat have suggested that neutrophils may indeed play this role [65]. HIV Tat actively recruits endothelial cells [66], monocytes [67, 68] and neutrophils [69]. We had previously observed using Tat in the Matrigel sponge model that neutrophils frequently directed the formation of clefts in the Matrigel, followed by macrophages, fibroblasts and finally endothelial cells which then lined these clefts to form vessels [70]. It appeared that neutrophils not only condition the environment, but physically open a passage for endothelial cells towards a particular site in what could be considered a model of ‘acute angiogenesis’, with the introduction of an avascular tissue containing a pluripotent angiogenic factor. The neutrophil may well be a unique regulator of the initial phases of inflammatory angiogenesis, exerting a pro- or antiangiogenic switch that the endothelium then executes. Our murine model of chemokine-induced angiogenesis, where only CXCR2 is present, clearly shows that the absence of neutrophils blocks subsequent endothelial recruitment in response to CXCR2 agonists [16]. However, neutrophils, depending on the type and intensity of the stimulus, could also produce an environment unfavorable to vascularization, producing several inhibitors including AST (through elastase) and radicals (fig. 1). Neutrophils need to act in large numbers, the concentration of these phagocytes has been shown to be crucial for an effective immune reaction against pathogens [71].

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Neutrophil activation by a wide range of stimuli immediately results in the expression and release of IL-8, inducing the recruitment of other phagocytes needed to reach a ‘critical mass’ necessary for efficient defense. During acute inflammation in the presence of pathogens, neutrophils are immediately committed to a killer activity based on the release of stored mediators that also leads rapidly to the death of the neutrophils themselves; the expression of additional molecules in time is restricted. This is associated with the release of IL-8, with triggering of CXCR1, a coinducer of degranulation. We hypothesize that elastase and radicals would act as main neutrophil angiogenesis regulators at the inflammatory site, along with VEGF, HGF and PAF. VEGF would mainly improve vessel permeability, helping the extravasation of new phagocytes; HGF would stimulate epithelial cells to scatter through the entire area and ‘envelope’ the damaged tissues, and PAF to enhance leukocyte extravasation and blood clotting. The involvement of neutrophils and endothelial cells would be restricted with time by neutrophil elastase generated AST, while the release of active radical species could reduce endothelial cell survival. The resulting picture is typical of acute inflammation: edema and purulence, massive leukocyte infiltration and poor neovascularization. A similar scenario is seen in acute forms of arthritis, where strong inflammation and tissue destruction without angiogenesis is observed [1]. On the other hand, the presence of neutrophils in chronic inflammation (i.e. psoriasis, rheumatoid arthritis) and in tumors (i.e. head and neck cancers) could favor angiogenesis. These leukocytes would be recruited by suboptimal partially activating chemokine or cytokine signals. Lower concentrations of TNF-␣ and IL-1 accompanied by a local sustained expression of CXCR2 agonists and a lack of pathogen stimuli would induce a chronic recruitment of neutrophils. Here neutrophils would be involved in a sustained release of secondary granule contents without involvement of primary granules or radical production. The result would be a secretory and neosynthetic behavior where the continuous turnover of infiltrating neutrophils would act chronically as a cell factory for soluble mediators. The extracellular environment would be enriched with a continuous supply of VEGF, HGF and IL-8, thereby resulting in an evident angiogenic response. Gillitzer and Goebeler [17] have shown that neutrophils are the source of IL-8 at inflammatory sites in wound healing, while endothelial cells preferentially express Gro-␣. This observation is in line with the consequential steps of neutrophil recruitment: a first ‘call’ by endothelialderived Gro-␣ active on the CXCR2 receptor mediates chemotaxis, and successive neosynthesis of IL-8 by neutrophils which causes recruitment at lower doses (active on CXCR2) and activation and degranulation of secondary granules at higher doses (by CXCR1 triggering). This would cause the presence of a chronic neutrophil-infiltrate acting, as observed for the endometrium, as a source of angiogenic factors.

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These hypotheses merit further investigation in several pathologies, in particular cancer, where often only well-established tumors are studied. In cancer, the role of neutrophils could be linked to the initial steps of progression, suggesting that it would be best to utilize animal models with spontaneous tumor occurrence to investigate these phenomena. Some intriguing and countercurrent hypotheses could additionally be extrapolated from clinical practice, as most high-dose anticancer therapies also cause severe neutropenia requiring GM-CSF or G-CSF treatment; one could ask whether these drugs only inhibit tumor growth directly or whether they could reduce tumor angiogenesis by neutrophil depletion as well. It is clear that the presence of neutrophils may be the primary checkpoint for angiogenesis in a wide array of different pathologies, from rheumatoid arthritis and psoriasis to tumor progression, as well as in physiological processes such as endometrial angiogenesis. In summary, the role of neutrophils as both promoters and inhibitors of the angiogenic process should not be overlooked. This potential ‘yin-yang’, double faced role of inflammation and inflammatory cells, particularly regarding their role in cancer, is now receiving increasing attention [72, 73, 74]. Acknowledgments We wish to acknowledge Prof. Marco Cassatella for helpful comments. This work is supported by the Fondazione San Paolo, CNR Progetto Giovani, AIRC and ISS Progetto di Cooperazione Italia-USA.

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Douglas Noonan, Istituto Nazionale per la Ricera sul Cancro, largo Rosanna Benzi 10, I–16132 Genova (Italy) Tel. ⫹39 010 5737361, Fax ⫹39 010 5737231, E-Mail [email protected]

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Neutrophils in the Antitumoral Immune Response E. Di Carloa, G. Forni b,c, P. Musiani a a

Department of Oncology and Neurosciences, ‘G. d’Annunzio’ University, Chieti, bDepartment of Clinical and Biological Sciences, University of Turin, Orbassano, and cCenter for Experimental Research and Medical Studies (CERMS), Ospedale San Giovanni Battista, Torino, Italy

The New ‘Immunological Identity’ of Neutrophils

The immunological functions of neutrophils are currently the subject of a new wave of enthusiastic research. Until a few years ago, in fact, neutrophils were simply regarded as innate immunity effectors. It is now clear that they are immune-regulatory cells involved in a continuous cross talk with other leukocyte subsets and endothelial cells (ECs). Neutrophil-Endothelial Interactions Vascular ECs are the gatekeepers of the tissues as seen from the bloodstream and regulate neutrophil trafficking at the site of inflamed tissues [1]. Extravasation of neutrophils is a multistep process mediated by adhesion molecules, multifunctional inflammatory cytokines and chemotactic factors [2]. Adhesion molecules known as selectins (P-, E-, and L-selectins also known as GMP140, ELAM-1, and LAM-1, respectively) tether free-flowing neutrophils to the endothelium of the postcapillary venules and mediate a transient interaction that subsequently becomes a firm adhesion via the recognition of ␤2 integrin Mac-1 (CD11b/CD18) – and/or leukocyte function-associated antigen-1 (LFA-1, CD11a/CD18) – intercellular adhesion molecule-1 (ICAM-1) [3]. Inflammatory cytokines, namely interleukin-1 (IL-1), tumor necrosis factor (TNF), granulocyte-macrophage colony-stimulating factor (GM-CSF) and chemokines, particularly those of the CXC family [growth-related oncogene (GROs), epithelial neutrophil-activating-78 (ENA-78), granulocyte chemotactic

protein-2 (GCP-2), neutrophil-activating peptide-2 (NAP-2) and IL-8], regulate the adhesion kinetics by chemoattracting neutrophils and inducing adhesion molecule expression [1, 4, 5]. Tight adhesion of neutrophils to ECs triggers their double activation, followed by the release of cytokines and mediators which recruit other leukocyte subpopulations to become part of the complex neutrophil-endothelial cross talk [6, 7]. The release of monocyte chemotactic protein-1 (MCP-1/CCL2) [8], GROs/CXCL1-2-3 [9] and interferon (IFN)inducible protein 10 (IP-10/CXCL10) [10] by activated ECs, in fact, leads to the recruitment of monocytes, neutrophils, NK and dendritic cells (DCs), or T lymphocytes [5]. Neutrophil-Centered Cytokine Network Their adhesion to ECs enables neutrophils to be fully responsive to and release several factors and mediators of cellular communications [6, 7]. Microenvironmental signals stimulate their production of a variety of cytokines, such as proinflammatory IL-1␣/␤, TNF-␣, IFN-␣/␤ GM-CSF, IL-6, IL-12 and anti-inflammatory IL-1 receptor antagonist (RA), and transforming growth factor-␤ (TGF-␤) [11]. These then act on ECs, neutrophils, macrophages, DCs and T and B cells to regulate nonspecific and specific immune responses. The direction of a specific response may be influenced by neutrophil release of MCP-1, macrophage inflammatory proteins-1␣ and ␤ (MIP-1␣ and ␤) (CCL3 and 4) and MIP-3␣ and ␤ (CCL20 and 19) which recruit monocytes/ macrophages, immature (MIP-1␣/␤ and MIP-3␣) and mature (MIP-3␤) DCs and T cells to initiate the response and skew it towards a Th1 type profile [12, 13]. The importance of neutrophils in the development of delayed-type hypersensitivity (typical Th1-type reaction), in fact, depends on their production of MCP-1 leading to monocyte and lymphocyte infiltration [14]. Alternatively neutrophils may release CXC chemokines. These amplify their own recruitment (GROs/CXCL1-2-3 and IL-8) or promote T and NK cell recruitment (monokine induced by IFN-␥, MIG/CXCL9; IP-10/CXCL10; IFN-inducible T cell ␣ chemoattractant, I-TAC/CXCL11) and Th2 immune responses [12]. Defensins, small cysteine-rich cationic proteins, and cathepsin G, a neutral serine proteinase (both stored in the azurophil granules of neutrophils), are now seen as important immune regulatory tools [15, 16]. In addition to their direct antimicrobial activity [15] defensins, whose functional overlap with chemokines has recently been confirmed, enhance phagocytosis, promote neutrophil recruitment, boost the production of proinflammatory cytokines and regulate complement activation, thus upregulating innate host inflammatory defenses [15]. They also contribute to adaptive immune responses by selectively attracting immature DCs, CD8⫹ T cells,

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CD45RA⫹ and CD45RO⫹ memory T cells, and enhance antigen-specific humoral responses [15, 17]. The newly discovered ability of ␤ defensins 2 and 3 to bind the chemokine receptor CCR6 (similarly to MIP-3␣) on immature DCs [18] has recently been exploited to carry weakly immunogenic tumor antigens to their receptor on professional antigen-presenting cells (APCs) [18]. The resulting humoral, protective, and therapeutic antitumor immunity demonstrates the significance of the ability of the defensins to link innate and adaptive immunity. Cathepsin G is a potent chemoattractant for monocytes, macrophages and T cells, and has been implicated as a neutrophil-derived signal that induces mononuclear cell and T-cell-dependent immune responses [17]. Both cathepsin G and ␣ defensins stimulate IFN-␥ and IL-4 cytokine production by immune spleen cells by enhancing either the Th1 or the Th2 limb of the immune response [17]. In this scenario, neutrophils modulate the balance between humoral and cell-mediated immunity and are engaged in a complex cross talk with immune and endothelial cells that bridge innate resistance and adaptive immunity. The intriguing finding of MHC class II molecule expression by activated neutrophils has extended the range of their potential immunological functions [19]. Selected cytokines, such as TNF-␣ and IFN-␥, induce neutrophil expression of CD83 [20], a typical marker of differentiated or activated human DCs that regulate the development of cellular immunity by interacting with its ligand on resting monocytes and a subset of activated CD8⫹ T cells [21]. Under the same stimuli they express functional CCR6 (the receptor of MIP-3␣/CCL20) and by adding GM-CSF they also express CD40, a receptor for contactdependent T cell help [20]. These findings mean that under appropriate conditions neutrophils differentiate to acquire a new phenotype and functions through which they can more actively promote the conversion of acute to chronic inflammation and to adaptive immunity [20]. Discovery of these new and unsuspected neutrophil functions has led to speculation about their role in the antitumoral response.

The Neutrophil Dilemma: Is It ‘Tumor-Stimulatory’ or ‘Tumor-Inhibitory’?

Tumors consist on the whole of tumor, endothelial, stromal and infiltrating cells, especially innate immunity cells [22]. Granulocytes are an uncommon component of both human and animal tumors. In animal models, they may sometimes favor malignant growth and progression [23, 24]. Nevertheless, recent studies suggest that they are active in immunosurveillance against

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several tumors [25, 26]. These contrasting findings are probably expressions of the interplay between the kind and amount of cytokines and chemotactic factors naturally released by tumor cells [27, 28] or tumor-associated ECs and reactive cells, and the degree of recruitment and activation of the intermingled granulocytes. Thus, depending on the cytokine milieu in which they act, tumor-associated neutrophils may became a source of growth factors for tumor cells, angiogenic factors for ECs and matrix-degrading enzymes promoting angiogenesis and tumor progression. In a mouse model of squamous cell carcinoma (SCC) pathogenesis, the lack of matrix metalloproteinase MMP-9 production by reactive cells, particularly neutrophils, reduced keratinocyte hyperproliferation at all neoplastic stages and decreased the incidence of invasive tumors, suggesting its role in tumor progression [29]. In addition to their direct genotoxic and carcinogenic potential exerted by the release of reactive oxygen species (ROS) [30], that also possess immunosuppressive effects [31], neutrophils can feed tumor cells releasing growth factors such as epidermal growth factor, TGF-␤ and platelet-derived growth factor and/or ECs by releasing well-known angiogenic factors, such as basic fibroblast growth factor (bFGF), vascular endothelial growth factor (VEGF), platelet-activating factor, TNF-␣ hepatocyte growth factor and the ELR (glutamic acid-leucine-arginine) ⫹ CXC chemokines IL-8 and GROs [11, 12]. GRO-␣/KC/CXCL-1 expression by murine SCC promotes tumor growth and metastasis in association with neutrophil infiltration and vessel proliferation by a host CXCR-2-dependent mechanism [32]. In humans, SCC may develop in the skin, upper airway and digestive tract, lung and cervix, often in association with neoplastic epithelial cell production of GRO-␣ and IL-8, which recruit neutrophils and also promote neoangiogenesis and hence tumor growth [33, 34]. However, IL-8 expression by IL-8 genetransfected Chinese hamster ovary cells decreases their tumorigenicity when injected subcutaneously (s.c.) into nude mice [35]. The early and highly cytotoxic neutrophil infiltrate generated when immunocompetent mice are injected s.c. with IL-8 gene-transfected fibrosarcoma cells also inhibits tumor growth [36]. Thus, the intratumoral level of IL-8 or other neutrophil chemoattractants, and the functional state of the recruited neutrophils, may differentially affect tumor behavior. This will also depend on the tumor progression stage at which they operate. The balance between neutrophil-mediated tumor promotion and inhibition is frequently shifted towards tumor growth as the stimuli prevailing in a ‘naive’ tumor microenvironment switch on the ‘nursing’ function of neutrophils [24].

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Immunostimulating interventions (described later) may elicit the antitumor potential of neutrophils by triggering their production of cytotoxic mediators, angiostatic factors or chemotactic and activating molecules for other leukocyte subsets (described above). The cytotoxic mediators of tumor and EC killing include IL-1␤, TNF-␣ and IFNs [37–39], defensins [highly toxic against several types of tumor cells, 40], proteases [such as elastase and cathepsin G, particularly injurious to ECs, 41] and ROS, such as nitric oxide, hydrogen peroxide (H2O2) and hypochlorous acid (HOCl) [42]. Oxidants injure tumor cells by acting synergistically with protease and/or by inactivating plasma antiproteases to allow proteases to operate. After their LFA-1- or Mac-1-dependent recognition of the target tumor or EC surface, activated neutrophils release HOCl, resulting in tumor cell lysis, microvessel injury and matrix degradation [43]. On the other hand, neutrophils act ‘antiangiogenically’ by releasing IP-10 and MIG (upon TNF-␣ and IFN-␥) or IFN-␣ [upon granulocyte colonystimulating factor (G-CSF)] [12]. In addition, it has recently been observed that they generate a bioactive angiostatin-like fragment inhibiting bFGF plus VEGF-induced EC proliferation in response to inflammatory stimuli [44]. Other neutrophil-mediated tumor cell killing mechanisms involve crosslinking of the Fc receptor (R) on the neutrophil surface with immunoglobulin (Ig) Fc fragment [45]. This interaction triggers several antitumor activities, including superoxide generation, cytokine and enzyme release, azurophil granule exocytosis, phagocytosis and antibody (Ab)-dependent cellular cytotoxicity (ADCC) [46]. After neutrophil-target cell contact, in which the Mac-1-ICAM-1 interaction has a fundamental role [47], subcellular tumor cell cytolysis mechanisms, e.g. oscillatory release of oxygen metabolites such as enzymes, drive multihit pericellular proteolysis (membrane rupture events) culminating in a dramatic tumor cell collapse [48, 49]. Preclinical Studies in Animal Models of Tumor Cure and Prevention: Evidence of Neutrophil Anticancer Potential

Early manipulation of the tumor microenvironment by means of selective immunological maneuvers engages circulating neutrophils as prompt, first-line innate immunity effectors that do not have to ‘ask for tumor antigens’ to be able to destroy cancer cells. Cytokine Gene-Transfected Tumors Immunomodulatory cytokines have been used to stimulate nonspecific immune reactions against tumors. Over the last 10 years, cytokine gene transfer

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a

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Fig. 1. Immunohistochemical aspects of TS/A-IL-12 cell rejection area 3 days after s.c. injection of transfected tumor cells in (1 ⫻ 105) BALB/c mice. Recruitment of numerous neutrophils (acetone-fixed cryostat section tested with antigranulocytes, RB6-8C5 mAb) (a) and marked production of MIP-2 (acetone-fixed cryostat section tested with anti-MIP-2 Ab) by infiltrating reactive cells (b). Original magnification. ⫻400.

strategies in animal models have been employed to dramatically increase intratumoral cytokine availability, avoid the side effects of systemic administration and lead to intratumoral recruitment and activation of neutrophils [50, 51]. A wide range of immunoregulatory molecules sustainedly released by engineered tumor cells, namely IL-1, IL-2, IL-4, IL-7, IL-12, IFN-␣/␤, G-CSF and TNF-␣, quickly built up a massive local reaction characterized by numerous granulocytes, which results in the rejection of engineered tumor cells and the establishment of a significant immunity against the wild-type parental tumor [50–53]. A much slower kinetics of tumor rejection follows the local release of IL-10 by gene-transfected mouse mammary carcinoma cells (TS/A-IL-10) [54]. Initial tumor growth, during which the anti-inflammatory activity of IL-10 inhibits reactive cells and their proinflammatory cytokine production, is followed by ELAM-1 expression [55], possibly induced directly by IL-10 in peripheral tumor microvessels [56]. The resulting neutrophil influx and interaction with ECs play a key role in leading to a delayed, but efficacious antitumor reaction with complete tumor eradication. Endothelial activation and expression of ELAM-1 and ICAM-1 are also features of the immunological rejection of tumor cells engineered to release G-CSF, IL-2, IL-4, TNF-␣ and IFN-␣/␤ and IL-12 [53, 57–59]. Adhesion molecules are induced or upregulated by the cytokine primarily released and/or by the downstream secondary mediators, such as CXC chemokines [60]. MIP-2, the murine functional homologs of human IL-8, upregulates the binding affinity of integrins on granulocytes and of their counterreceptor, ICAM-1, on ECs [5, 60]. A strong expression of MIP-2 is usually found in granulocytes, macrophages and ECs of an IL-1 plus TNF-␣-rich tumor microenvironment, as in IL-12 gene-transfected tumors (fig. 1) [59, 61]. Implantation in nude mice of

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a

b

c

d Fig. 2. Histopathological features of TS/A-IL-2 and TS/A-IL-10 cell rejection areas 3 days (a, c) and 8 days (b, d) after s.c. injection of transfected tumor cells (1 ⫻ 105) in BALB/c mice. TS/A-IL-2 cells elicited a massive neutrophil influx (a) which was followed by colliquative necrosis of TS/A-IL-2 tumor (c). TS/A-IL-10 cells elicited a later recruitment of neutrophils that surrounded the blood vessels and frequently destroyed their walls (b), resulting in areas of ischemic-hemorrhagic necrosis (d). Original magnification. ⫻400.

IL-12 and IL-15 gene-cotransfected, MHC class I-negative, human small cell lung cancer N592 was followed by prompt, complete rejection in association with a rapid influx of granulocytes and a marked production of MIP-2 and nitric oxide [59]. IL-12 may activate neutrophils by increasing reactive oxygen metabolite production [62]. Neutrophils are also targeted by IL-15, which promotes their degranulation and release of IL-8 [63], thus regulating granulocyte trafficking. Surprisingly, the combined antitumor effect of IL-12 and IL-15 was also evident in IFN-␥-deficient mice in which rejection of cotransfected cells, driven by granulocyte and CD8⫹ cells, was always accompanied by substantial MIP-2, IL-1␤, TNF-␣ and GM-CSF production [61]. Basically, the histopathological features of the neutrophil-mediated immunological rejection which follows inoculation of cytokine gene-transfected tumors are of two kinds. Colliquative necrosis, as in the case of TS/A cells engineered to release IL-2 (fig. 2a, c), IL-4, TNF-␣ may be observed when neutrophil cytotoxicity is mainly directed against tumor cells, while a predominantly ischemic and/or hemorrhagic necrosis is evidenced when the main

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neutrophil target is the vascular endothelium as in the case of TS/A cells engineered to release IL-10 (fig. 2b, d), IFN-␣ or IFN-␤ [50–55, 57–59]. Interestingly, it has recently been observed that in a murine model of prostate cancer the inhibition of tumor growth by IL-12 gene therapy was associated with a morphological pattern of localized sheets of neoplastic apoptotic cells in close association with necrosis containing neutrophils [64]. Depletion of neutrophils resulted in the loss of this pattern of apoptosis and reduced growth suppression. This indicates that rather than direct EC or tumor cell destruction neutrophils may indirectly support an apoptotic pathway [64]. Chemokine Gene-Transfected Tumors Chemokine genes have been transduced in a variety of experimental tumors. Gene transfection of either CC chemokines, which attract monocytes, DCs and lymphocytes [65, 66], or CXC chemokines, which preferentially attract neutrophils (some of them B and T cells), has been used to evoke antitumor reactions [67, 68]. Neutrophils were actively involved in these reactions via different pathways. Neutrophils intratumorally recruited by highly expressed endothelial adhesion molecules along with APCs and lymphocytes play a crucial role in the rapid rejection of TS/A cells engineered to release liver-expressed chemokine (LEC) [65]. LEC/HCC-4/CCL16 is a CC chemokine which attracts human monocytes and DCs, but not neutrophils [69], whose recruitment and activation may be orchestrated by the locally induced secondary proinflammatory cytokine and chemokine cascade [65]. Injection of immunocompetent or nude mice with P815 mastocytoma cells transfected with the gene of the CC chemokine MCP-3/CCL7 resulted in peritumoral accumulation of DCs and intratumoral recruitment of neutrophils [66]. The expression of CCR1 and CCR3 (MCP-3 receptors) on neutrophils may be upregulated by IFN-␥ induced in T and/or NK cells so that they can directly respond to MCP-3 [70] or, alternatively, recruited by secondary downstream mediators. Removal of neutrophils caused an evident delay of MCP-3-elicited tumor rejection, suggesting their cooperative role in tumor inhibition. The marked and rapid granulocyte influx and tumor necrosis observed in nude mice challenged with human tumor cells engineered to release IL-8 or human MIP-1␣/CCL3, and murine MIP-1␣ [67] demonstrates that both CXC (IL-8) and CC (MIP-1␣) chemokines regulate granulocyte traffic and functions, and provides further evidence that granulocytes suppress tumor growth by different ways. TNF Ligand-Transfected Tumors CD95 (APO/Fas) ligand (CD95L) is a member of the TNF family predominantly expressed by activated T and NK cells, but also by tumors of

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diverse cellular origin [71]. CD95L trimerizes surface CD95 expressed by target cells that subsequently undergo apoptosis [71]. Elimination of tumor-reactive T cells by cytotoxic CD95L⫹ tumors may be an active way of circumventing rejection by the host and has been called ‘tumor counterattack’ [72]. However, mice injected with syngeneic CD95L-expressing tumor cells did not develop tumors and displayed a strong neutrophil-mediated response against the grafted cells [73, 74]. Moreover, it was shown that in vivo depletion of neutrophils with anti-GR1⫹ allowed subsequent growth of CD95L⫹ tumors. An elegant experiment performed to establish whether tumors use CD95L to downregulate an antitumor immune response utilized perforin knockout (PKO; which excludes perforin as mediator of tumor destruction) anti-Kb TCR (which enable specific allogeneic recognition between tumor cells and T cells) transgenic mice injected with CD95L⫹ tumor cells bearing tumor antigen Kb and caspase inhibitor crmA [75]. Despite the impairment of four major T cell killing systems (perforin, CD95L, TNF, TRAIL), CD95L⫹ tumors were eliminated in TCR transgenic PKO mice. The massive presence of neutrophils at the tumor site suggests they are the cause of tumor rejection. CD95L does not directly exert chemotaxis on neutrophils. Secondary mediators, such as IL-8 inducible by CD95L in endothelial cells (ECs) [76], or released by macrophages ingesting apoptotic cells [77], alternatively, the apoptosis of neutrophils with CD95⫹ tumors may trigger the extensive recruitment of neutrophils [74]. Recruited neutrophils kill CD95L⫹ tumor cells by mechanisms that still remain to be investigated. OX40L is a member of the TNF ligand superfamily and is expressed on activated professional APCs, including DCs [78]. Its receptor, OX40, is expressed primarily on activated CD4⫹ T lymphocytes [79]. Effective OX40L-OX40 interaction requires intact CD28 and CD40 signals [79]. OX40L may provide a sustained T cell boost. C26 colon carcinoma cells transfected with OX40L or GM-CSF only were not rejected by the syngeneic host even if a strong neutrophil influx was observed in both cases. Cotransfection with costimulatory molecule and cytokine gene led to tumor cell rejection in association with very impressive and amplified neutrophil recruitment and activation [Gri et al., unpubl. data]. Tumor rejection was achieved through the cooperation of neutrophils, CD8⫹, CD4⫹ T cells and APC-mediated DC40-CD40L cosignalling. Thus, the synergistic activity of OX40L and GM-CSF resulted in a coordinated and useful elicitation of nonspecific and specific immune cells. In this context, neutrophils were engaged in the cross talk between activated APC and CD4⫹ T cells to sustain, along with CD8⫹ T cells, the effector phase of the antitumor response. The Role of Neutrophils in Antitumor Immune Memory Rejection As revealed by immunohistochemical and ultrastructural analysis and confirmed by selective depletion experiments, neutrophils are of crucial importance

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in a variety of cytokine- or chemokine-induced tumor rejections, often in cooperation with CD8⫹ T lymphocytes [50, 51]. Furthermore, establishment of a specific immune memory against the parent tumor usually follows rejection. Neutrophils have not yet proved to be of great importance in the elaboration of a significant memory. In IFN-␥ gene-transfected tumors the granulocyte infiltrate was almost absent and rejection occurred in only 25% of mice [51]. The immune memory, however, always provided complete protection against the parental tumor. Recent discoveries suggest that involvement of neutrophils in the ‘building phase’ of antitumor immune memory should be reconsidered based on (1) their ‘APC potential’ [19, 20] and (2) the ability of IFN-␥, which is important in the elaboration of the antitumor immune memory, to promote granulocyte differentiation toward an APC phenotype and function [20]. Neutrophils are certainly one of the effector arms involved in destruction of the second inoculum after the establishment of an antitumor immune memory [54, 65, 80]. The cytokines released by engineered tumor cells play a leading role in skewing the memory reaction toward Th1 or Th2 [50]. However, secondary rejection is not simply the work of T cells since the selective removal of granulocytes impairs or even abolishes rejection after establishment of either a Th1- or a Th2-deflected immune memory [50]. It may well be that the cytolytic activity of PMN is guided by factors secreted by T cells. Targeting Immunoprevention: The New Frontier in Anticancer Strategies Neutrophil involvement in the antitumor effect of IL-12 was also observed when it was used to prevent mammary cancer spontaneously arising in Her-2/neu transgenic mice (BALB-neuT H-2d) and that summarizes the multistep process of human mammary cancer [81]. Lifetime and time/dose-limited IL-12 schedules, respectively, hamper mammary carcinogenesis [81] and cure preneoplastic (atypical hyperplasia) and early neoplastic (in situ carcinoma) lesions [82]. This is mainly due to activation of nonspecific immune mechanisms leading to angiogenesis inhibition and vascular damage [82–84] (fig. 3). The numerous neutrophils surrounding the preneoplastic lesions (fig. 3b) in close proximity to injured microvessels (fig. 3d) are thought to cooperate in the significant delay of mammary carcinogenesis by IL-12. Since IL-12 administration alone does not effectively decrease the incidence of cancer-related deaths [81], it was associated with vaccination with allogeneic (H-2q) tumor cells expressing the Her-2/neu oncogene product (p185) [85]. This reduced the tumor incidence by 90% and the lifetime of the treated mice was more than doubled. Their mammary glands displayed a few hyperplastic foci heavily infiltrated by granulocytes, macrophages and CD8⫹ cells. The high titers of specific anti-HER-2/neu Abs and the ineffectiveness of the treatment in

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e Fig. 3. BALB-neuT mice receiving four ‘shorter and lower’ IL-12 courses consisting of 100 ng/week for 4 weeks, starting at 7 weeks (when full-blown atypical mammary hyperplasia was already evident) followed by a 3-week rest. At 14 weeks of age mice still displayed hyperplastic foci in the form of nests of p185neu ⫹ neoplastic epithelial cells (formalin-fixed, paraffin-embedded section tested with anti-Neu, C-18 Ab) (a). These foci, however, were heavily infiltrated by reactive leukocytes that were almost entirely GR1⫹ cells (granulocytes) (b) adherent to ELAM-1-expressing microvessel endothelium (acetone-fixed cryostat section tested with anti-E selectin mAb) (c). Endothelial walls (acetone-fixed cryostat section-tested with anti-CD31 mAb) showed evident signs of severe damage (arrows) (d). When the experiment ended in the 32nd week of age, the healed animals (about 50%), instead of multiple invasive lobular carcinomas, as observed in control mice, showed a few foci of atypical hyperplasia surrounded by a dense fibrotic stroma (revealed as blue-green by trichrome staining) as the result of ischemic-hemorrhagic necrosis (e). Original magnification. a, c, d ⫻630. b, e ⫻400.

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B cell-deficient mice, along with a peculiar brisk infiltration of neutrophils in close contact with the neoplastic mammary epithelial cells, strongly suggest that ADCC [48] is involved. Recently, electroporated DNA p185 vaccine has been demonstrated to cure in situ carcinoma of HER-2/neu transgenic mice by means of anti-p185 Abs and INF␥-releasing T cells. Here, too, neutrophils, which along with professional APCs and CD8⫹ lymphocytes massively overcome the basal membrane to achieve tight contact with p185-expressing neoplastic cells, were deeply involved in tumor regression [Quaglino et al., unpubl. data].

Neutrophils and Anticancer Therapy in Humans

Systemic cytokine therapy has been tested in a variety of human tumors. However, the toxic side effects of the high dose schedules required to obtain antitumor effects in advanced cancers have frequently impeded its clinical application [86–88]. During infusion therapy with well-known neutrophil-activating cytokines such as IL-2 and TNF-␣, the disease response was closely associated with a respiratory burst of neutrophils, their production of oxidants, such as HOCl and upregulation of their ␤2 surface integrins [43, 88, 89], such as Mac-1, which regulate neutrophil adhesion, migration, phagocytosis, degranulation and neutrophiltumor cell interactions [47]. Paradoxically, neutrophils themselves may be responsible for vascular leak toxicity and adverse cytolytic effects produced by high cytokine doses [89, 90]. One of the latest cytokines tested in cancer patients on the strength of its powerful antitumor activity in different experimental systems is IL-12 [64, 83–85]. Its ability to cure well-established tumors in murine models mainly rested on the cooperative action of CD8⫹ cells and granulocytes, which markedly filled peri- and intratumoral blood vessels [82, 84]. Unfortunately, the use of IL-12 in humans is complicated by dose-limiting toxicity in the form of neutropenia and other hematological disorders [86]. At present, systemic administration of neutrophil-stimulating cytokines is mostly restricted to G-CSF used as adjuvant management accompanying more conventional therapy [91], which prevents dose-limiting neutropenia, restores neutrophil functions and makes cancer chemotherapy safe and tolerable. News from Clinical Trials Cytokine-based immunotherapy and chemotherapy have been much improved. Even so, the prognosis of advanced tumors is still poor. Tumordirected Abs, particularly bispecific (Bs) Abs (containing one tumor-directed specificity and one directed toward a cytotoxic trigger molecule on leukocytes),

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increase the specificity and efficacy of both immunological and more conventional oncological treatments [92]. The antitumor activity of Abs is mediated either directly, e.g. by blocking growth factor receptors or induction of apoptosis, or indirectly by redirecting effector cells toward tumor cells, thus exploiting the mechanism of ADCC [92]. As T and NK cell-directed BsAb approaches are hindered by difficulties in mobilizing and activating T and NK effector cells, attention is now being focused on BsAbs that target myeloid effectors. Neutrophils are attractive effector cells for Ab-directed immunotherapy [93]. They are the most populous effector cell subset in the body and display fast recruitment and very rapid cytotoxicity in vivo [92]. Two classes of IgG receptors (Fc␥RIIa, CD32 and Fc␥RIIIb, CD16) and one class of IgA receptors (Fc␣RI, CD89) have been identified on human PMNs, whereas the Fc␥RI (CD64) expression is inducible on PMN by stimulation with G-CSF, GM-CSF and IFN-␥ [94]. Fc␣RI is the most effective FcR for PMN-mediated tumor cell killing and IgA1 antitumor Abs are most efficient in recruiting peripheral blood PMNs, whereas they are less effectively recruited by IgG Abs [95], though these are most frequently used in clinical practice. Trastuzumab (herceptin) is the first humanized monoclonal IgG Ab to be approved for therapeutic use and has improved the survival of women with HER-2/neu (erbB-2)-positive metastatic breast cancer [96]. Its mechanism of action in vivo is still unclear, though it would seem to antagonize the constitutive growth-signalling properties of the HER-2/neu system, reverse resistance to cytokines and enlist immune cells to attack and kill the tumor target [97]. Along with other effector cells, granulocytes are heavily involved in herceptin-dependent cellular cytotoxicity, which was further enhanced in G-CSF- or GM-CSF-treated patients [98]. New-generation Abs against various solid tumors and hematological malignancies are being investigated in phase I and II trials. Against solid tumors, Abs anti-HER-2/neu or epidermal growth factor receptor are among the best examples that PMNs may contribute to Ab efficacy [99–101]. CD16-directed BsAb, 2B1, targeting HER-2/neu and Fc␥RIII, has been tested in a phase I trial in different cancers overexpressing p185. Excitingly, half the treated patients developed autologous Abs reacting with both intra- and extracellular domains of HER-2/neu. This reflects induction of active antitumor immunity [92] and indicates that a ‘vaccine effect’ is induced in vivo upon Fc␥R-directed targeting of carcinomas. It remains to be seen whether Fc␥RIIIexpressing neutrophils can function as APCs rather than effector cells in this context. BsAb MDX-H210 (CD64 X HER-2/neu) has been tested alone and in conjunction with granulocyte-activating cytokines, namely G-CSF, IFN-␥ and

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GM-CSF, in phase I/II metastatic breast, ovary, lung, colon, renal cell carcinoma and hormone refractory prostate cancer trials [92, 102]. Combinations with these cytokines, which significantly increase the recruitment and activation of neutrophils and upregulate their surface expression of Fc␥RI, have resulted in higher amounts of tumor cell killing and excellent clinical responses (reduction of tumor lesions or stabilization). s.c. administration of IL-2 has also been shown to trigger Fc␥RI expression on human peripheral blood neutrophils and hamper disease progression in renal cell carcinoma and low-grade nonHodgkin lymphoma patients [103]. In hematological malignancies, tumor cells are relatively accessible to Abs and effector cells. Malignant B cells display high susceptibility to neutrophilmediated ADCC. Rituximab, a chimeric anti-CD20 monoclonal Ab generated by fusing the variable region of murine Ab to the gamma and kappa constant region of human Ab, enhances interaction with immune effectors involved in Ab-induced tumor cell lysis and is widely used to treat low-grade, non-Hodgkin’s B cell lymphoma. Its high cytotoxicity is increased by G-CSFstimulated neutrophils [104]. A new anti-HLA-DR Ab, Lym-1, is highly effective against malignant B cells and its efficacy significantly improves when neutrophil’s patients are stimulated with GM-CSF [105]. Patients Eligible for Neutrophil-Stimulating Immunotherapy The advantage of nonspecific antitumor management, such as exploitation of the antitumor potential of neutrophils, is that it can be directly applied to a broad range of individuals, irrespective of the type of their tumor-associated antigens. Its real curative potential, however, is confined to patients with a low tumor load or minimal residual disease. Thus, individuals with preneoplastic or early neoplastic lesions and those whose primary tumor mass has been removed (minimal residual disease) may be envisaged as potential candidates. Selection of candidates must take account of the significant fraction of patients whose tumors lack MHC class I [106, 107] or are so poorly differentiated and immunogenic that tumor-specific immunotherapy is doomed to failure. Under appropriate immunoregulatory stimuli, such as those obtained by combining the effects of IL-12 and IL-15 [59], granulocytes may lead to tumor destruction and cooperate in antitumor immune memory reactions to prevent relapses. An aging immune system displays functional dysregulation and a gradual decline in responsiveness to antigens and tumors. Immunosenescence reduces a granulocyte’s ‘appetite for killing’ and increases neutrophil apoptosis as a consequence of altered oxidative metabolism [108, 109]. Immunological manoeuvers may restore neutrophil functions against infections and cancer and also constitute a noninvasive ‘soft’ approach, free from significant side effects

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and thus particularly suitable for elderly patients with poor chemotherapy tolerance. Neutrophils are immunologically and functionally defective in HIVinfected patients and this may increase their susceptibility to infections and malignancy. Decreased chemotaxis and killing activity, altered respiratory burst response and accelerated apoptosis have been reported [110]. Furthermore, neutrophils from these patients spontaneously produce increased amounts of ROS. These are implicated in the pathophysiology of HIV infection [111] and because of their genotoxicity should also be implicated in the genesis of cancer-related HIV. Neutrophils targeting immunomodulatory molecules may be useful as adjuvants to conventional therapy in this setting. IL-15 selectively acts on neutrophils and completely reverses their dysfunctions in active antiretroviral therapy-treated patients with long-term HIV suppression, including those with treatment failure [112]. Future Developments Improvement of Ab-Based Immunotherapy As PMNs substantially contribute to Ab efficacy in vivo, the selection of novel target antigens capable of recruiting them as effector cells for ADCC is of great significance. It has recently been observed that the HER-2/neu intracellular domain contributes substantially to effective Ab-mediated tumor cell killing by PMNs [101]. In hematological malignancy, for example, Abs against HLA class II or related epitopes, such as Lym-1 or Lym-2 [105], were highly effective in recruiting PMNs as effector cells, whereas CD19 Abs were ineffective. Differences in the ability of target antigens to initiate signal transduction or interact with the target cell cytoskeleton, or even the ability to form immunological synapses may explain this difference. Coadministration of cytokines might augment neutrophil-mediated ADCC [98–100] via mechanisms ranging from upregulation of FcR and ␤2 integrin expression on neutrophils to stimulation of their cellular functions, namely phagocytosis, degranulation and production of oxygen radicals. Exciting results are emerging from the use of humanized Ab-cytokine (particularly GM-CSF) fusion protein against neuroblastoma cells. This construct, in fact, significantly enhances PMN-driven ADCC by increasing expression and activation of Mac-1, adhesion and spreading onto tumor cells, and azurophil granule exocytosis [113]. Tumor cell killing by granulocytes was most potently triggered by Fc␣RIdirected bispecific constructs [92]. Recent studies indicate that simultaneous engagement of Fc␣RI and Fc␥RI results in enhanced PMN-mediated tumor cell lysis [114], suggesting that new Ab cocktails, e.g. combinations of IgG and IgA Abs, may improve the efficacy of current clinical protocols.

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Vaccination ⫹ Cytokines for Tumor Prevention One of the most ambitious goals of anticancer research is tumor prevention [115]. In models employing animal genetically predestined to develop tumors, costimulation of nonspecific and tumor-specific immune mechanisms by combining vaccination with allogeneic tumor cells and systemic cytokines (such as IL-12) secures the most effective prevention [85]. The finding that granulocytes operate as an active component of nonspecific immunity and cooperate to ‘specifically’ kill target cells (by ADCC and perhaps by other mechanisms) at the time of tumor onset certainly opens new possibilities to exploit their potential to prevent rather than cure cancer. Translation of experimental results to the clinical setting, however, requires further efforts on the part of both scientists and clinicians. The former must: (1) elaborate DNA constructs for the preparation of vaccines and thus avoid the ethical and biological objections to the use of allogeneic tumor cells in humans, and (2) select the most effective immunomodulatory cytokine to recruit and activate circulating granulocytes. The planning of clinical trials implies: (1) selection in the light of genetic screening or exposure to high carcinogen doses of individuals with a high risk of developing cancer, and (2) the setting of the time at which to start the treatment and, obviously, the most effective and best-tolerated schedule readily acceptable by ‘not yet patients’.

Acknowledgment This work was supported by grants from the Italian Association for Cancer Research (AIRC), MURST 40%, and European Cancer Immunome Programme (EUCIP). We thank Mrs. C. Colangelo for editorial help and Prof. John Iliffe for careful review of the manuscript.

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103 Sconocchia G, Cococcetta NY, Campagnano L, Amadori S, Iorio B, Boffo V, Ferdinandi V, Del Principe I, Adorno D, Casciani CU: Subcutaneous administration of interleukin-2 triggers Fcgamma receptor I expression on human peripheral blood neutrophils in solid and hematologic malignancies. J Immunother 2001;24:374–383. 104 van der Kolk LE, de Haas M, Grillo-Lopez AJ, Baars JW, van Oers MH: Analysis of CD20dependent cellular cytotoxicity by G-CSF-stimulated neutrophils. Leukemia 2002;16:693–699. 105 Ottonello L, Epstein AL, Mancini M, Tortolina G, Dapino P, Dallegri F: Chimaeric Lym-1 monoclonal antibody-mediated cytolysis by neutrophils from G-CSF-treated patients: Stimulation by GM-CSF and role of Fc gamma-receptors. Br J Cancer 2001;85:463–469. 106 Doyle A, Martin WJ, Funa K, Gazdar A, Carney D, Martin SE, Linnoila I, Cuttitta F, Mulshine J, Bunn P: Markedly decreased expression of class I histocompatibility antigens, protein, and mRNA in human small-cell lung cancer. J Exp Med 1985;161:1135–1151. 107 Seliger B, Ritz U, Abele R, Bock M, Tampe R, Sutter G, Drexler I, Huber C, Ferrone S: Immune escape of melanoma: First evidence of structural alterations in two distinct components of the MHC class I antigen processing pathway. Cancer Res 2001;61:8647–8650. 108 Wenisch C, Patruta S, Daxbock F, Krause R, Horl W: Effect of age on human neutrophil function. J Leukoc Biol 2000;67:40–45. 109 Butcher S, Chahel H, Lord JM: Review article: Ageing and the neutrophil: No appetite for killing? Immunology 2000;100:411–416. 110 Pitrak DL, Tsai HC, Mullane KM, Sutton SH, Stevens P: Accelerated neutrophil apoptosis in the acquired immunodeficiency syndrome. J Clin Invest 1996;98:2714–2719. 111 Elbim C, Pillet S, Prevost MH, Preira A, Girard PM, Rogine N, Hakim J, Israel N, GougerotPocidalo MA: The role of phagocytes in HIV-related oxidative stress. J Clin Virol 2001;20: 99–109. 112 d’Ettorre G, Forcina G, Lichtner M, Mengoni F, D’Agostino C, Massetti AP, Mastroianni CM, Vullo V: Interleukin-15 in HIV infection: Immunological and virological interactions in antiretroviral-naive and -treated patients. AIDS 2002;16:181–188. 113 Metelitsa LS, Gillies SD, Super M, Shimada H, Reynolds CP, Seeger RC: Antidisialoganglioside/ granulocyte macrophage-colony-stimulating factor fusion protein facilitates neutrophil antibodydependent cellular cytotoxicity and depends on FcgammaRII (CD32) and Mac-1 (CD11b/CD18) for enhanced effector cell adhesion and azurophil granule exocytosis. Blood 2002;99:4166–4173. 114 van Egmond M, van Spriel AB, Vermeulen H, Huls G, van Garderen E, van de Winkel JG: Enhancement of polymorphonuclear cell-mediated tumor cell killing on simultaneous engagement of fcgammaRI (CD64) and fcalphaRI (CD89). Cancer Res 2001;61:4055–4060. 115 Quaglino E, Rovero S, Cavallo F, Musiani P, Amici A, Nicoletti G, Nanni P, Forni G: Immunological prevention of spontaneous tumors: A new prospect? Immunol Lett 2002;80:75–79.

Emma Di Carlo, ‘G. d’Annunzio’ University, Anatomia Patologica, Ospedale Clinicizzato ‘SS. Annunziata’, Via dei Vestini, I–66013 Chieti (Italy) Tel. ⫹39 0871 357395, Fax ⫹39 0871 540079, E-Mail [email protected]

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Regulation of Neutrophil Apoptosis Steven W. Edwardsa, Dale A. Mouldinga, Mathieu Deroueta, Robert J. Mootsb a

School of Biological Sciences and bDepartment of Medicine, University of Liverpool, Liverpool, UK

Overview of Neutrophil Apoptosis

Of all the cells of the immune system, the neutrophil has the shortest half-life, estimated to be between 6 and 18 h. This short half-life arises from the fact that bloodstream neutrophils constitutively undergo apoptosis [1–4]. The development of neutrophils from blast cells within the bone marrow requires a period of approximately 15 days to reach maturation [5, 6], and yet astonishingly, once these mature cells are released into the circulation, they will only survive for a few hours. The reasons for this are unclear, but may be related to the potential toxicity of neutrophils and hence their capacity to inflict tissue damage [7], which can be potentially life threatening if they are inappropriately activated. A process by which these cells naturally turnover, without the need to generate a positive death signal, may help protect against the possibility of large numbers of activated cells persisting in the circulation for long periods of time. However, the consequence of this short half-life of circulating neutrophils is that the bone marrow must release up to 5 ⫻ 1010 neutrophils on a daily basis in order to provide the threshold levels required for effective protection against infections [5]. Synthesis and release of such vast numbers of neutrophils on a daily basis to maintain adequate levels for protection can be impossible for some immunosuppressed patients (e.g. cancer patients undergoing chemotherapy or radiotherapy). In these circumstances such patients can be severely impaired in their ability to fight infections [5, 8]. In contrast to the short half-life of circulating cells, neutrophils recruited into tissues have a much longer survival time because they become rescued from this constitutive apoptotic pathway. Precisely how long they can survive

in tissues is not known with certainty and much will depend upon their local environment and the balance between pro- and anti-apoptotic factors present within this environment. In vitro, neutrophils can be kept alive for several days by exposure to agents such as cytokines [1, 4, 9, 10], and it is not unreasonable to assume that similar survival times can be encountered when exposed to similar agents within tissues. Such factors slow down but do not prevent apoptosis and neutrophils will eventually become apoptotic and subsequently cleared from the site by tissue macrophages or other phagocytic cells. Apoptotic neutrophils are incapable of mounting their normal cellular responses such as chemotaxis, degranulation, adherence, phagocytosis and activation of the respiratory burst [10, 11]. Also, the expression of many cell surface receptors is markedly decreased (e.g. by receptor shedding or internalisation), the nuclear lobes contract and the chromatin begins to fragment [12, 13]. Other cell surface changes occur (see later) that aid recognition of apoptotic neutrophils by phagocytic cells [14]. However, the plasma membrane remains intact and acts as a permeability barrier, at least during the early stages of apoptosis, such that cellular components (e.g. proteases) are retained within the apoptotic neutrophil. If the cells were to die by necrosis and the plasma membrane ruptured, then these cytoplasmic components would be released into the tissue spaces. In view of the large numbers of neutrophils that can be present within inflammatory foci, cytoplasmic contents released from necrotic neutrophils would be likely to overwhelm local anti-proteinase and anti-oxidant activity and tissue damage would result [7]. Apoptosis of neutrophils followed by their phagocytosis by macrophages or fibroblasts provides a mechanism for the safe removal of neutrophils from the site of inflammation thereby minimising the risk of bystander tissue damage [14, 15]. This safe removal of effete neutrophils is essential for the resolution of inflammation. Not surprisingly then, perturbation of the normal apoptotic pathway of tissue neutrophils can enhance their survival times and thereby extend their capacity to secrete their toxic products (e.g. reactive oxidants and proteinases), which may then contribute to the tissue damage that is often associated with inflammatory conditions [7]. Understanding the molecular processes that regulate neutrophil cell death and survival could therefore lead to benefits in the treatment of inflammatory disorders in which neutrophils contribute to disease pathology.

Morphological and Molecular Features of Apoptotic Neutrophils

Bloodstream neutrophils possess a characteristic multilobed nucleus and a granular cytoplasm. A distinctive feature of these cells is the expression of the

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Non-apoptotic Apoptotic

Multilobed nucleus Intact chromatin CD16-positive Annexin V-negative Functional

Condensed nucleus Degraded chromatin CD16-negative Annexin V-positive Non-functional

Fig. 1. Morphological and functional changes in neutrophils during apoptosis. The properties of non-apoptotic and apoptotic neutrophils are shown. For details, see text.

receptor, Fc␥RIIIb (CD16), which is present on the cell surface at a density of approximately 100–200,000 copies per cell [16–18]. This receptor is linked to the plasma membrane via an easily cleaved glycosyl-phosphatidylinositol anchor [19–21] and is constantly shed from the cell surface, but continuously replenished by mobilisation of preformed stores, present on the membranes of secretory vesicles to the plasma membrane [22]. The receptor is also actively synthesised by neutrophils [23] and hence the processes of biosynthesis and mobilisation of stores together ensure that surface levels are maintained to replenish receptor lost by shedding. This shedding occurs constitutively, but is enhanced (via the activity of a metalloproteinase) when the neutrophils are activated [24]. Apoptotic neutrophils have distinct morphological properties (fig. 1). The cells are round and compact, but the most striking feature is the appearance of a condensed, rounded nucleus, as the nuclear lobes contract. Apoptotic neutrophils cannot respond to activating signals and they lose expression of CD16 and hence become CD16– [12, 13, 18]. Changes in chromatin structure also occur, and in common with other apoptotic cells, the chromatin fragments into a distinctive ‘ladder’ of nucleosome length fragments [11, 25]. These changes in CD16 expression and chromatin structure form the basis for a sensitive flowcytometric assay to simultaneously measure both of these events [18]. Apoptotic neutrophils are also reported to have a decreased expression of other receptors (e.g. CD31, CD50, CD66) and such decreased expression may serve to prevent receptor:ligand interactions [12], and hence decrease the ability to recognise and respond to activation signals in the environment.

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Other changes occur on the surface of apoptotic neutrophils, notably the appearance of phosphatidylserine residues on the cell surface [26]. Normally these residues are present on the inner leaflet of the plasma membrane, but flip to the outer leaflet upon apoptosis. This phosphatidylserine exposure aids in the recognition of apoptotic neutrophils by macrophages or fibroblasts. Fibroblasts that phagocytose apoptotic neutrophils utilise both ␣3␤3 integrins and lectinlike receptors. These appear to recognise carbohydrate structures on the surface of the apoptotic cells. The phagocyte adhesion molecule CD36 co-operates with ␣3␤ to bind thrombospondin (that is secreted by phagocytes) that forms a molecular bridge between phagocytic receptors and structures on the apoptotic neutrophil [14, 26, 27]. The exposure of phosphatidylserine on the cell surface forms the basis of another sensitive assay to detect apoptosis in neutrophils, utilising flow cytometry or confocal microscopy [28]. This is based on the fact that phosphatidylserine binds annexin V in the presence of Ca2⫹. If the annexin V is fluorescently labelled (e.g. by FITC or Cy3), then apoptotic neutrophils become fluorescently labelled.

Extracellular Factors That Regulate Neutrophil Apoptosis

A variety of pro-inflammatory cytokines and other agents have been shown to readily delay neutrophil apoptosis. In vitro such agents include IL-1␤, IL-2, IL-4, IL-15, INF-␥, G-CSF, GM-CSF and LPS [29–35]. Agents such as sodium butyrate can also delay neutrophil apoptosis [11, 36], as can glucocorticoids [37]. The effects of glucocorticoids on neutrophil apoptosis contrast its effects on eosinophils which show accelerated apoptosis in response to this agent [38]. The reported effects of TNF␣ are conflicting, and its effects very much depend upon the concentrations used and the time of exposure [39]. TNF␣ rapidly induces apoptosis in a subpopulation of blood neutrophils, but delays apoptosis in the surviving cells [40]. These dual and contradictory effects may be related to the ability of TNF␣ to stimulate either a cell death pathway (e.g. involving caspase activation) or a cell survival pathway (e.g. involving activation of the transcription factor, NF-␬B) in different subpopulations of neutrophils (see later). Neutrophils are also susceptible to Fas-mediated apoptosis [41]. They express significant levels of Fas and some reports indicate that they can or cannot also express Fas ligand [42, 43]. Co-expression of Fas and Fas ligand may indicate an autocrine method for constitutive apoptosis. Triggering of cell adhesion has also been shown to stimulate intracellular signals in neutrophils that result in enhanced survival [44]. Related to this phenomenon, endothelial transmigration can delay apoptosis, presumably by a mechanism that also involves stimulation of adhesion receptors [45]. These

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phenomena are likely to be of extreme importance in extending the survival of neutrophils as they migrate from the bloodstream into infected tissues. Thus, when neutrophils are recruited into an inflamed or infected tissue, delayed apoptosis can extend their life span and hence their capacity to carry out their cytotoxic functions. In infections, this is beneficial in that delayed apoptosis can ensure effective killing of the pathogens. However, in inflammatory diseases, extended neutrophil survival may mean that the cells have an increased capacity to inflict tissue damage via secretion of their cytotoxic arsenal, before they are cleared by macrophages and the inflammation is resolved. In contrast to observations in other cell types, hypoxia (decreased oxygen tensions) can delay neutrophil apoptosis [46, 47]. Similarly, anti-oxidants such as catalase (but not superoxide dismutase) added to neutrophil cultures in vitro can delay apoptosis. There are also reports that neutrophils from CGD neutrophils (that have defective ability to generate reactive oxidants via an impaired NADPH oxidase) have delayed apoptosis in vitro [48]. Thus, a scenario may be envisaged in which local oxygen tensions may play an important role in neutrophil survival: low tensions may promote survival, but reactive oxidants generated via the respiratory burst following the reduction of O2 to O⫺2 , may be pro-apoptotic. Thus, neutrophil survival will be finely balanced by the local conditions of oxygenation. These considerations are extremely important because infected or inflamed tissues are invariably relatively low in oxygen tensions, compared to oxygen tensions found in the circulation. The O2 tension in arterial blood is in the range of 11–13% O2 and can be as low as 2% O2 in tissues. Indeed, it has been determined that a concentration gradient of 2–5% O2 exists in cells that are only 400 ␮m from a capillary and that rapidly metabolising cells (e.g. tumour cells) may function at 0.2% O2, only 200 ␮m from a capillary [49]. Thus the neutrophil will be exposed to a great range of O2 tensions, moving from a relatively O2-rich environment in the circulation, to hypoxia or near anoxia within tissues. Such changes in O2 tension will therefore have profound effects on their function and their survival kinetics. The levels of reactive oxidants that they can generate at these inflamed sites will also be important in determining their fate and residence time within the tissue. Induction of apoptosis has been shown to result in depletion of cellular levels of the anti-oxidant glutathione, not via its oxidation, but rather by its extrusion from the cell [reviewed in 50]. Conversely, increasing cellular levels of glutathione (by incubation with the glutathione precursor, N-acetylcysteine) was shown to inhibit Fas-mediated apoptosis, but had no effect on spontaneous neutrophil apoptosis [51]. It may be that inhibition of the glutathione pump (e.g. by methionine or cystathionine) may be required to delay spontaneous neutrophil apoptosis following enhancement of cellular glutathione levels [reviewed in 50].

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Molecular Control of Apoptosis

Apoptosis and survival of most cells and tissues are controlled by a number of key sets of proteins and processes: (1) death receptors, which are responsible for binding ligands that activate the caspases to promote apoptosis, (2) cytochrome c release from mitochondria, and (3) the Bcl-2 family of proteins. Death Receptors and Caspases The death receptors are responsible for binding extrinsic factors (such as Fas and TNF␣) that activate apoptotic pathways in cells. For example, binding of the Fas ligand to the Fas receptor results in trimerisation of the receptor and the recruitment of FADD protein (Fas-activated death domain). This process itself results in the binding, dimerisation and subsequent activation of caspase 8 [52–55]. Caspase 8 is one member of the caspase family, all of which are normally inactive in non-apoptotic cells [56, 57]. 14 family members have so far been identified and all are cysteine proteins that possess a common pentapeptide sequence (-QACRG-) which constitutes the active site. All caspases cleave their targets at aspartic acid residues. In non-apoptotic cells they exist as inactive monomeric precursors, containing a pro-domain together with large and small monomeric subunits. Upon proteolytic cleavage to remove the prodomain, monomers dimerise to assemble into active dimers with protease activity. Curiously, caspases can be activated by other caspases. Thus, some caspases (e.g. caspases 8 and 9) are termed regulatory caspases whilst others (e.g. caspases 1 and 3) are termed effector caspases and it is these latter enzymes that are directly responsible for carrying out the apoptotic processes leading to cell death [58, 59] in neutrophils. Thus, a caspase cascade is triggered upon initiation of apoptosis. This cascade may serve two purposes. First, it may function as a signal amplification mechanism, whereby a low level of extracellular signal is amplified to generate high levels of intracellular death signals. Second, the cascade may guard against inappropriate activation of the effector proteases, their activation relying on a complex series of signals and intermediates generated via a complex activation pathway. Activation of caspase 8 by FADD or TRADD (TNFR1-associated death domain) initiates a series of events in cells that culminates in apoptosis. Activated caspase 8 can then activate other caspases that ultimately lead to disablement of cellular functions and the acquisition of the apoptotic phenotype. Targets for activated effector caspases include actin (disruption of which regulates cell shrinkage and blebbing) and cleavage of poly(ADP ribose) polymerase thus preventing repair of fragmented DNA [25]. Apart from direct breakdown and cleavage of structural components, activated caspases can also cleave regulatory enzymes and alter their function, e.g. focal adhesion kinase

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and MAP kinases. They may also amplify an apoptotic signal by the inactivation of apoptosis inhibitors, e.g. IAP (inhibitor of apoptosis) or anti-apoptotic Bcl-2 family proteins. Mitochondria and Cytochrome c In some instances (perhaps in cells containing low levels of caspase 8), activation of procaspase 8 is an insufficient trigger to fully initiate the caspase cascade. In these circumstances, the activated caspase 8 must interact with the intrinsic cellular machinery required to initiate and then activate the death programme. The release of cytochrome c from mitochondria is now recognised to be a key event in the initiation of apoptosis. Activated caspase 8 can also catalyse the cleavage and subsequent activation of Bid, a Bcl-2 family member (see later) that is a mitochondrial cytochrome c-releasing factor. Released cytochrome c can then interact with Apaf-1 (requiring dATP), resulting in its dimerisation [60–62]. This promotes the dimerisation and activation of caspase 9, thereby forming a complex termed the apoptosome. This in turn activates the effector caspases 3, 6 and 7, thereby triggering the death programme. However, experiments showing that apoptosis can occur in cells incubated with effective capsase inhibitors indicate that pathways of apoptosis can also occur in the absence of the activity of these enzymes [63, 64]. A key event in preventing apoptosis is thus the retention of cytochrome c within mitochondria. The permeability transition pore complex is formed between the inner and outer mitochondrial membranes and is reported to control protein release from the intermembrane space. The permeability transition pore complex comprises the adenine nucleotide transporter, the voltage-dependent anion channel and possibly other proteins such as the benzodiazepine receptor and cyclophilin D [65]. Thus, cells possess specialised systems and processes for retaining cytochrome c within mitochondria to ensure survival, as well as systems that can rapidly mobilise this molecule when the apoptotic pathway is triggered. The Bcl-2 Family of Proteins This family of proteins plays a central role in the early life/death decisions of cells. The first family member discovered was Bcl-2 itself (identified as being overexpressed in B cell lymphoma) but since its discovery many other family members have been identified on the basis of the presence of conserved sequence motifs or BH domains (Bcl-2 homology domains) within their structures [66, 67]. Curiously, members belonging to this family can have antiapoptotic effects (e.g. Bcl-2, Bcl-XL) or pro-apoptotic effects (e.g. Bax, Bak, Bid, Bak) [68]. The number and types of BH domains present within these family members dictate their properties and function in the apoptotic process. Some family members possess 4 domains, whereas others contain only one.

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The hallmark feature of the Bcl-2 proteins is their ability to form heteroor homodimers [69–71]. The BH domains are the key to this property and form ␣-helical structures that serve as protein:protein interaction motifs. For example, the BH4 domain (found in most, but not all anti-apoptotic family members) may be responsible for molecular interactions with calcineurin and Raf-1 [72, 73]. The importance of the BH4 domain is emphasised by the fact that in cells transfected with a mutant or truncated form of Bcl-2 (an antiapoptotic protein) apoptosis is accelerated [74, 75]. There is also some evidence that this BH4 domain may be involved in control of the voltage-dependent anion channel of mitochondria [76]. Some pro-apoptotic proteins (e.g. Bik, Bid and Bad) only possess the BH3 domain. Consequently, this has led to the idea that this domain is the ‘minimal’ death domain required for the activity of some pro-apoptotic proteins. Insights into the functions of Bcl-2 family proteins have come from studies of the BH domains of Bcl-XL and its interactions with the pro-apoptotic protein, Bak. The BH1, 2 and 3 domains of Bcl-XL form a hydrophobic pocket, into which the BH3 domain of Bak (and perhaps that of other pro-apoptotic proteins) may fit, thereby preventing it from activating the downstream events (e.g. caspase activation) that trigger the death pathway [77]. However, molecular modelling indicates that in some pro-apoptotic proteins, the BH3 domain is normally not exposed on the periphery of the protein, but rather is buried within the core of the molecule. Such proteins may therefore require some form of activation step (e.g. phosphorylation or proteolytic cleavage) to expose the BH3 domain, thereby activating the molecule. An example of regulation of Bcl-2 family protein function by posttranslational modification is seen with Bad [78–80]. In the presence of an anti-apoptotic signal Bad is phosphorylated by PKA and PKB (Akt) and is then sequestered by 14-3-3 protein within the cytoplasm [81–83]. Dephosphorylation of Bad (perhaps by calcineurin) results in release of Bad from 14-3-3 protein and relocalisation with mitochondria, where it interacts with anti-apoptotic Bcl-2. It then exposes its BH3 domain and inserts into the hydrophobic cleft of Bcl-2 resulting in cytochrome c release. A major function of the Bcl-2 family members is to regulate cytochrome c release via their interaction with the outer mitochondrial membrane. As described above, cytochrome c release is an important first step in initiating apoptotic events in cells because of its ability to interact with and thereby activate Apaf-1. However, mitochondria can also release a number of other proteins during apoptosis, such as AIF, certain procaspases, catabolic enzymes, adenylate kinase 2 and SMAC/Diablo. The role of these proteins in the apoptotic process is not known with certainty. Anti-apoptotic proteins of the Bcl-2 family possess membrane anchoring domains at their carboxy terminus that target the

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molecules to mitochondria, but also to the ER and nuclear envelope [84–88]. Whilst anti-apoptotic proteins are generally localised to these membranes, proapoptotic proteins are often located within the cytoplasm of non-apoptotic cells. However, these pro-apoptotic proteins may translocate to the mitochondria where they exert their apoptotic effects [89–93]. For example, as described above Bad may translocate from the cytoplasm to the mitochondria following its dephosphorylation and dissociation from 14-3-3 protein, and cleavage of Bid to generate a short C-terminal fragment (tBid) aids in the oligomerisation and insertion of Bax into the mitochondrial membrane [94]. Bim is normally localised within the microtubules via association with LC8 dynein light chain and then translocates to the mitochondria in response to apoptotic stimuli. Once translocated to the mitochondria, pro-apoptotic proteins with BH1–3 domains (e.g. Bax) may insert into the membrane to destabilise it such that cytochrome c release is promoted, whereas those with only a BH3 domain may interact with anti-apoptotic proteins (e.g. Bcl-2, Bcl-XL) to induce conformational changes that again result in release of cytochrome c. The Bcl-2 family proteins thus play fundamental roles in mitochondrial protein permeability, either by acting as pore formers or by regulating the activity of intrinsic channels.

Regulation of Neutrophil Apoptosis and Survival

Two important questions are raised by the facts that (1) bloodstream neutrophils have a very short half-life, constitutively undergoing apoptosis and (2) pro-inflammatory cytokines can delay this apoptosis. First, what processes are responsible for the rapid onset of apoptosis in blood neutrophils? Second, what pathways are triggered by cytokines that result in neutrophil survival? The first clues to understanding these phenomena came from the observations that inhibitors of transcription (e.g. actinomycin D) and translation (cycloheximide) further accelerated the rate of apoptosis of blood neutrophils [11, 95, 96]. These observations indicate that survival of these cells requires the de novo biosynthesis of a protein(s) with a high turnover rate. The second important clue to understanding the processes regulating death and survival came from the observation that agents that delay neutrophil apoptosis almost always increase gene expression. Cytokines such as GM-CSF (which is a potent delayer of neutrophil apoptosis) induce a variety of effects of neutrophils, including the induction of tyrosine phosphorylation of proteins, a process intimately involved with the ‘priming’ response of this cytokine. However, GMCSF-mediated rescue of neutrophils from apoptosis could also be blocked (at least in part) by cycloheximide [97]. This would suggest that cytokine-mediated

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rescue from apoptosis also requires de novo biosynthesis, perhaps of the same protein(s) required for survival of bloodstream neutrophils. Bcl-2 Family Expression in Human Neutrophils Despite some early reports to the contrary [98, 99] it is now generally accepted that blood neutrophils do not express the ‘classical’ anti-apoptotic proteins Bcl-2 and Bcl-XL [1, 31, 100]. Importantly, these proteins are also undetectable in neutrophils exposed in vitro to agents (e.g. cytokines) that delay apoptosis. This indicates that the survival mechanisms stimulated by cytokines do not involve the induction of these survival proteins. Curiously, mRNA for Bcl-XL is detectable in neutrophils (in RNase protection assays) and its levels are increased upon GM-CSF exposure [100], but the protein is not detected. Possible explanations for this discrepancy between mRNA expression and protein expression may be that either (1) translation of Bcl-XL mRNA does not occur in these circumstances perhaps requiring a second, as yet unidentified signal or (2) the protein translated by this message is not recognised by the commonly used antibodies. Human neutrophils express a range of pro-apoptotic proteins, including Bax, Bad, Bik, Bak and Bid [1, 98, 100–102]. These proteins are expressed at fairly high levels and they have a relatively long half-life. In most reports, their cellular levels in neutrophils remain fairly constant when the cells age naturally or when their apoptosis is either experimentally delayed or accelerated. There is one report that indicates a decrease in Bax levels during exposure of neutrophils to GM-CSF [103] but this finding has not been repeated in other experiments [100]. It is possible that cytokine treatment may alter the function rather than the absolute levels of death proteins so that they become non-functional during delayed apoptosis. An example of this phenomenon is Bad, which is phosphorylated by Akt/PKB, thereby downregulating its function [81, 104, 105]. If neutrophils do express neither Bcl-2 nor Bcl-XL, then how is their survival regulated? The answer lies in the fact that they express Mcl-1 [31, 100], an anti-apoptotic protein of the Bcl-2 family that has some unusual properties compared to other members of the family. These properties of Mcl-1 make it ideally suited to its function in the survival of neutrophils, cells which must respond very quickly to either pro- or anti-inflammatory signals in their environment so that their role in infections and inflammation is tightly controlled. Properties of MCL-1 MCL-1 was originally cloned as an ‘early induction’ gene during differentiation of the myeloid cell line, ML-1 [106]. Sequence analysis revealed that the protein encoded by the cDNA was 37.3 kD, much larger than Bcl-2 (21 kD) or Bcl-XL (29 kD). The size of the protein detected in Western blots is 40/42 kD.

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Sequence analysis identified three putative BH domains (BH1–3), but BH4 is absent from Mcl-1. Almost all other anti-apoptotic proteins possess this BH4 domain, the only exceptions being Mcl-1 and A1 (see later). As this domain is thought to be required for molecular interactions with other proteins, an absence of BH4 from Mcl-1 and A1 suggests that these anti-apoptotic proteins interact with a different set of proteins as Bcl-2 and Bcl-XL. PEST sequences and Arg:Arg domains are also present in Mcl-1 and these motifs are often present in proteins that are subject to rapid turnover [107]. Indeed, the half-life of Mcl-1 in cells has been estimated as between 1 and 5 h, depending upon the environment in which the cells are exposed [100, 108, 109]. However, deletion of the PEST region did not significantly alter the half-life of a GFP:Mcl-1 fusion protein expressed in U-937 cells [110]. Perhaps features other than this PEST region also regulate Mcl-1 turnover. Whatever the mechanisms involved, Mcl-1 has the fastest turnover rate of the anti-apoptotic Bcl-2 family members and hence the shortest half-life. Sequence analysis also identified a putative membrane anchor domain at the carboxy terminus, and this has been confirmed experimentally. Deletion of this domain results in the loss of mitochondrial localisation of a GFP:Mcl-1 fusion protein in U-937 cells, resulting in diffuse, cytoplasmic distribution [110]. Recently, an alternatively spliced variant of Mcl-1 has been identified (Mcl-1S) that arises from exon skipping. This short form of Mcl-1 possesses only a BH3 domain and has pro-apoptotic functions when transfected into cell lines [111, 112]. MCL-1 has been shown to be expressed in a variety of cells and tissues. Its overexpression in transfection studies results in enhanced cell survival, confirming its role as an anti-apoptotic protein [113–115]. In many cell types, expression of Mcl-1 is transient, often occurring at certain stages of development or differentiation [101, 116–118]. Indeed, in mice, an MCL-1 gene knockout is lethal, confirming its key role in development [119]. It is often expressed independently of Bcl-2, and may provide for transient protection against apoptosis at certain times in differentiation/development. For example, its expression may be triggered before that of Bcl-2 during differentiation of B cells [120], and it is transiently expressed during differentiation of U-937 cells [28]. Such an expression of Mcl-1 independently of Bcl-2 and Bcl-XL indicates that it must play a distinct role in cell survival. It may be that Mcl-1 functions at different subcellular sites or perhaps interacts with different death proteins. There is emerging evidence to show that enhanced Mcl-1 expression can confer a malignant phenotype on cells [121]. For example, overexpression of Mcl-1 is thought to be responsible for the impaired apoptosis and resistance to chemotherapy of malignant myeloma cells [122]. Specific antisense disruption of Mcl-1 expression in differentiating U-937 cells is sufficient to induce apoptosis in the absence of changes in the expression of any other pro- or anti-apoptotic Bcl-2 proteins

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Treatment GM-CSF

CF ⫺

Inverse ⫹

⫺

⫹

AS ⫺

⫹

Mcl-1 40/42 kD

Bax 21 kD

Bak 24 kD

Caspase 3 17/19 kD Actin 45 kD

Fig. 2. Depletion of Mcl-1 protein levels by antisense treatment. Neutrophils were reversibly permeabilised with streptolysin O and incubated with 20 ␮M carboxyfluorescein (CF), inverse antisense (Inverse) or antisense sequence 1 (AS) of chimeric oligodeoxynucleotides. Suspensions were incubated in either the absence (⫺) or presence (⫹) of GM-CSF. Western blots were analysed on aliquots of 106 cells, 4 h after permeabilisation. Actin (Ponceau S-stained blots) is shown to indicate equivalence of loading. Representative blot of 3 independent experiments. Antisense sequences and structures are given by Moulding et al. [28].

expressed in these cells [28]. All of this evidence points to Mcl-1 having a specialised role in control of apoptosis particularly at certain times in their differentiation/developmental programme. Bloodstream neutrophils express both mRNA and protein for Mcl-1 [31, 47, 100, 123]. Agents that accelerate neutrophil apoptosis (e.g. cycloheximide) decrease Mcl-1 levels, whereas agents that delay neutrophil apoptosis (e.g. GMCSF, IL-1␤, TNF␣) all increase or sustain Mcl-1 levels. Indeed, there is a very close correlation between Mcl-1 levels and neutrophil survival: low levels of Mcl-1 are associated with apoptosis whereas high levels of Mcl-1 are associated with survival. This would imply a central role for Mcl-1 as the controlling factor for neutrophil survival. However, more compelling evidence for the importance of Mcl-1 in regulating survival comes from experiments in which Mcl-1 is specifically depleted by antisense oligodeoxynucleotides. Antisense treatment results in a marked depletion of Mcl-1 in both control and GM-CSF-treated cells (fig. 2), and in parallel with this specific depletion, neutrophil apoptosis is accelerated (fig. 3).

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0

* Apoptosis (%)

60

0.8

Mcl-1 protein

0.4 80

1.2

*

40

1.6

20

0 CF

Inv AS

AS

Fig. 3. Antisense disruption of Mcl-1 accelerates neutrophil apoptosis: relationship between Mcl-1 levels and apoptosis. Apoptosis was assessed from cytospins prepared 4 h after streptolysin O permeabilisation. Results shown are means (⫾SD) from the 3 independent experiments shown in figure 2. Significant differences from carboxyfluorescein (CF) and inverse antisense (Inv AS) controls are indicated by *p ⱕ 0.05. An inverted graph of Mcl-1 protein expression at 4 h from these 3 experiments is shown to demonstrate the relationship between Mcl-1 expression and levels of apoptosis. Neutrophils were incubated in the presence of 50 U/ml GM-CSF.

Mcl-1 mRNA is cytokine-regulated in neutrophils, thus providing a mechanism for the rapid increase in cellular levels of this protective protein [98]. Thus, the protein has a naturally high turnover rate, and is rapidly induced via transcription and translation. When transcription ceases, the naturally high turnover rate of the protein will result in a rapid decline in cellular levels below the critical threshold value necessary for protection against apoptosis. These molecular properties of Mcl-1 therefore make it an ideal molecule to function as the key regulator of neutrophil survival. The following model is thus proposed to explain how it can play a pivotal role in the fate of these acutely regulated cells. Blood neutrophils express Mcl-1 protein, which is sufficient to ensure the survival of these cells by counteracting the effects of pro-apoptotic proteins. In the absence of continuous expression of this protein (via transcription and translation), the natural turnover of this protein and short half-life will result in a rapid decrease in its cellular levels. When these levels fall below a critical threshold, its protective effects are lost. The activity of the pro-apoptotic proteins then

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predominates and the cells undergo apoptosis. When the cells receive inflammatory signals (e.g. inflammatory cytokines, integrin engagement, transendothelial migration), then Mcl-1 transcription is activated and cellular levels are increased or sustained. This de novo biosynthesis of Mcl-1 will ensure cell survival until the effects of the signal on Mcl-1 transcription are lost. Again, the naturally high turnover rate of the protein will result in a decrease in the cellular levels to below the critical threshold and apoptosis will occur. The apoptotic neutrophils will then be phagocytosed by macrophages or fibroblasts. Changes in Mcl-1 stability (increased or decreased stability) may also play a role in the rate of Mcl-1 turnover and hence its ability to protect against cell death. Antisense disruption of Mcl-1 protein levels in human neutrophils can decrease the levels of this survival factor in control or GM-CSF-untreated cells (fig. 2). These decreases in protein levels occur in the absence of changes in the levels of other members of the Bcl-2 family. As Mcl-1 levels are decreased by antisense treatment, there is a corresponding increase in the cellular levels of the active form of caspase 3 (fig. 2). There is also a marked correlation between cellular levels of Mcl-1 protein and apoptosis (fig. 3). As the levels of this protein are decreased by antisense treatment, so the rate of apoptosis of the cells increases. Properties of A1 (Bfl-1) The only other anti-apoptotic gene product implicated in neutrophil survival to date is A1 (Bfl-1). Human A1 (Bfl-1) was originally identified from sequence analysis of a cDNA clone isolated from human liver 22 weeks after gestation whose predicted amino acid sequence showed 72% homology to murine A1 [124, 125]. It is expressed in a variety of tissues, including some tumours and its molecular mass predicted from cDNA analysis is 20.1 kD. Like Mcl-1 it lacks a BH4 domain, again suggesting that it interacts with a different set of proteins as Bcl-2 and Bcl-XL. Knockout mice heterozygous for A1 deficiency have increased rates of spontaneous apoptosis compared to controls [126]. However, studies in human cells are frustrated by the fact that no antibody exists that reliably detects human A1. Thus, studies on this molecule in human neutrophils are largely restricted to the mRNA level. Indeed, A1 transcripts are abundant in human neutrophils and are cytokine-regulated [100, 127]. A1 mRNA also has a very short half-life (comparable to that of Mcl-1) [100]. These data indicate that A1 may function alongside Mcl-1 in human neutrophils to control cell function, but a more definitive role for A1 awaits reliable studies at the protein level. Mechanisms Leading to Neutrophil Death As described above, decreased levels of Mcl-1 (due to limited transcription and turnover) are a trigger for progression into apoptosis. Thus, loss of this

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protective protein leads to activation of the cellular apoptosis machinery that disables cellular functions and results in the acquisition of the apoptotic phenotype. The events that trigger this progression into apoptosis following Mcl-1 depletion, involving caspase activation, are similar to those described above in other cell types. Roles of A1 and Mcl-1 in Control of Neutrophil Apoptosis Whilst neutrophils do not express the archetype survival proteins Bcl-2 and Bcl-XL, it is curious to ask why they should express two different family members, with apparently similar roles in protection against apoptosis? A simple answer to this question may be that this merely represents redundancy: it is beneficial to depend upon the activities of two separate gene products, rather than only one. This redundancy may ensure survival if one of the genes were defective and an active protein were not expressed. Neutrophil death and survival need to be rapidly regulated in order for the cell to respond quickly and appropriately to signals in its local environment to control its function. This rapid regulation of survival could be achieved by posttranslational modification of proteins, but this does not appear to be the case. Instead, survival appears to be regulated by the balance between the rapid induction (by transcription and translation) and rapid turnover of survival factors, resulting in increased survival and apoptosis, respectively. The specialised properties of Mcl-1 and A1 may fit the requirements to fulfil roles as acute regulators of death and survival of neutrophils. However, it is intriguing to speculate that Mcl-1 and A1 may play specialised, but overlapping roles in neutrophil survival. Mcl-1 has been reported to be localised mainly to mitochondria [109, 110], whereas the subcellular location of A1 is reported to be cytoplasmic [128, 129]. Indeed, sequence analysis implies that A1 lacks a defined transmembrane anchor at its carboxy terminus. If these proteins are localised to different subcellular compartments, then they could both function to protect against apoptosis, but via different pathways, perhaps by interacting with different death proteins. It is also likely that A1 and Mcl-1 expression are regulated by different signalling pathways leading to transcription. A1 expression is clearly dependent upon activation of the transcription factor, NF-␬B [130]. However, there is no evidence to implicate this transcription factor in the control of Mcl-1 expression. Indeed, mutagenesis of a putative NF-␬B site in the promoter of the MCL-1 gene had no effect on transcription of a luciferase reporter gene [131]. Numerous signalling pathways have been implicated in regulation of neutrophil survival and cell death, as well as those stimulated when neutrophil apoptosis is delayed by cytokines. These pathways are not fully delineated and are reviewed by Akgul et al. [1].

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Acknowledgments We thank the Wellcome Trust, UK, the Arthritis Research Campaign and Mersey Region Kidney Fund for financial support.

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113 Reynolds JE, Yang T, Qian L, Jenkinson JD, Zhou P, Eastman A, Craig RW: Mcl-1, a member of the Bcl-2 family, delays apoptosis induced by c-Myc overexpression in Chinese hamster ovary cells. Cancer Res 1994;54:6348–6352. 114 Reynolds JE, Li J, Craig RW, Eastman A: bcl-2 and mcl-1 expression in Chinese hamster ovary cells inhibits intracellular acidification and apoptosis induced by staurosporine. Exp Cell Res 1996;225:430–436. 115 Zhou P, Qian L, Bieszczad CK, Noelle R, Binder M, Levy NB, Craig RW: Mcl-1 in transgenic mice promotes survival in a spectrum of hematopoietic cell types and immortalization in the myeloid lineage. Blood 1998;92:3226–3239. 116 Zhou P, Qian L, Kozopas KM, Craig RW: Mcl-1, a Bcl-2 family member, delays the death of hematopoietic cells under a variety of apoptosis-inducing conditions. Blood 1997;89:630–643. 117 Kaufmann SH, Karp JE, Svingen PA, Krajewski S, Burke PJ, Gore SD, Reed JC: Elevated expression of the apoptotic regulator Mcl-1 at the time of leukemic relapse. Blood 1998;91:991–1000. 118 Yang T, Buchan HL, Townsend J, Craig RW: Mcl-1, a member of the Bcl-2 family, is induced rapidly in response to signals for cell differentiation or death, but not to signals for cell proliferation. J Cell Physiol 1996;166:523–536. 119 Rinkenberger JL, Horning S, Klocke B, Roth K, Korsmeyer SJ: Mcl-1 deficiency results in peri-implantation embryonic lethality. Genes Dev 2000;14:23–27. 120 Lømo J, Smeland EB, Krajewski S, Reed JC, Blomhoff HK: Expression of the Bcl-2 homologue Mcl-1 correlates with survival of peripheral blood B lymphocytes. Cancer Res 1996;56:40–43. 121 Okaro AC, Deery AR, Hutchins RR, Davidson BR: The expression of antiapoptotic proteins Bcl-2, Bcl-X(L), and Mcl-1 in benign, dysplastic, and malignant biliary epithelium. J Clin Pathol 2001;54:927–932. 122 Zhang B, Gojo I, Fenton RG: Myeloid cell factor-1 is a critical survival factor for multiple myeloma. Blood 2002;99:1885–1893. 123 Leuenroth SJ, Grutkoski PS, Ayala A, Simms HH: The loss of Mcl-1 expression in human polymorphonuclear leukocytes promotes apoptosis. J Leukoc Biol 2000;68:158–166. 124 Choi SS, Park I-C, Yun JW, Sung YC, Hong S-I, Shin H-S: A novel Bcl-2 related gene, Bfl-1, is overexpressed in stomach cancer and preferentially expressed in bone marrow. Oncogene 1995; 11:1693–1698. 125 Choi SS, Park SH, Kim UJ, Shin HS: Bfl-1, a Bcl-2-related gene, is the human homolog of the murine A1, and maps to chromosome 15q24.3. Mamm Genome 1997;8:781–782. 126 Hamasaki A, Sendo F, Nakayama K, Ishida N, Negishi I, Nakayama KI, Hatakeyama S: Accelerated neutrophil apoptosis in mice lacking A1-a, a subtype of the bcl-2-related A1 gene. J Exp Med 1998;188:1985–1992. 127 Chuang PI, Yee E, Karsan A, Winn RK, Harlan JM: A1 is a constitutive and inducible Bcl-2 homologue in mature human neutrophils. Biochem Biophys Res Commun 1998;249:361–365. 128 Orlofsky A, Somogyi RD, Weiss LM, Prystowsky MB: The murine antiapoptotic protein A1 is induced in inflammatory macrophages and constitutively expressed in neutrophils. J Immunol 1999;163:412–419. 129 D’Sa-Eipper C, Subramanian T, Chinnadurai G: bfl-1, a bcl-2 homologue, suppresses p53-induced apoptosis and exhibits potent cooperative transforming activity. Cancer Res 1996;56:3879–3882. 130 Zong WX, Edelstein LC, Chen C, Bash J, Gelinas C: The prosurvival Bcl-2 homolog Bfl-1/A1 is a direct transcriptional target of NF-␬B that blocks TNFalpha-induced apoptosis. Genes Dev 1999;13:382–387. 131 Akgul C, Turner PC, White MRH, Edwards SW: Functional analysis of the human Mcl-1 gene. Cell Mol Life Sci 2000;57:684–691.

Professor S.W. Edwards, School of Biological Sciences, Life Sciences Building, University of Liverpool, Liverpool L69 7ZB (UK) Tel. ⫹44 151 794 4363, Fax ⫹44 151 794 4349, E-Mail [email protected]

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Author Index

Albini, A. 167

Forni, G. 182

Benelli, R. 167 Bennouna, S. 95

Galligan, C. 24 Girard, D. 64

Noonan, D. 167

Cheng, S.S. 81 Cloutier, A. 1

Hänsch, G.M. 45

Serhan, C.N. 115

Kunkel, S.L. 81

Wagner, C. 45

Levy, B. 115 Lindbom, L. 146

Yoshimura, T. 24

Del Rio, L. 95 Denkers, E.Y. 95 Derouet, M. 204 Di Carlo, E. 182 Edwards, S.W. 204

Moulding, D.A. 204 Musiani, P. 182

McDonald, P.P. 1 Moots, R.J. 204

225

Subject Index

A1, apoptosis regulation 217, 218 Adaptive immunity, polymorphonuclear neutrophil role 31–33 Angiogenesis cancer 178 endometrium remodeling 172 HIV Tat induction mechanism 176 inflammatory disease 167 neutrophil modulators angiostatin 173, 174 anti-inflammatory agents 175 CXCR2 ligands 168, 169, 175, 176 cytokine inhibitors 174, 175 hepatocyte growth factor 170, 171, 176, 177 induction vs inhibition 175–178 interleukin-8 168, 170, 177 platelet activating factor 171 reactive oxygen species 173 vascular endothelial growth factor 170, 172, 176, 177 Angiostatin, neutrophil modulation of angiogenesis 173, 174 AP-1 dimerization 16 neutrophil synthesis 16, 17 target genes 16 Apoptosis, neutrophils CD16 expression 206 cytokine regulation 207, 212, 213 glutathione loss 208 hypoxia effects 208

life span of neutrophils 24, 45, 64, 204 molecular regulation A1 217, 218 Bcl-2 210–213 caspases 209, 210 cytochrome c loss from mitochondria 210 death receptors 209 Mcl-1 213–218 morphology 205–207 nuclear factor-␬B role 13, 14 phosphatidylserine translocation 207 survival factors 24, 25, 204, 205, 207, 208 Aspirin, promotion of lipoxin synthesis 116, 118–120, 128–130 Asthma, lipoxin role 129, 130, 135 Bcl-2, apoptosis regulation 210–213 Bone marrow, polymorphonuclear neutrophil release 24, 26 Cancer angiogenesis 178, 185 antitumor therapy 193 gene transfer studies chemokines 189 cytokines 186–189 stimulating immunotherapy bispecific antibodies 193–195 patient selection 195, 196 principles 193, 194 prospects 196

226

tumor necrosis factor ligands 189, 190 interleukin-12 therapy 191, 193 neutrophils antitumor activity overview 186 feeding 185 immune memory rejection role 190, 191 tumor components 184, 185 vaccination plus cytokines for prevention 197 CAP37, see Heparin binding protein Caspases, apoptosis regulation 209, 210 Cathepsin G functions 184 vascular permeability modulation 156 CCL19, polymorphonuclear neutrophil adaptive immunity role 32, 33 CCL20, polymorphonuclear neutrophil adaptive immunity role 32, 33 CD16, expression on apoptotic cells 206 CD83, polymorphonuclear neutrophil expression 51, 53 C/EBP activation 15, 16 isoforms 15, 16 neutrophil synthesis 16 Chemokines, see also specific chemokines adaptive immunity role 32, 33 angiogenesis regulation CXCR2 ligands 168, 169, 175, 176 interleukin-8 168, 170, 177 classification 25, 82 expression in cytokine-activated polymorphonuclear neutrophils 36 inflammation role of neutrophil cytokines 88–90 networking during acute inflammation 87, 88 neutrophil recruitment 82–86 receptor expression on polymorphonuclear neutrophils bone marrow release role 26, 27 CCR1 84, 85 CCR2 85 CCR3 85 CCR6 35, 86 CXCR1 25–27, 82–84, 169

Subject Index

CXCR2 25–27, 82–84, 168, 169 CXCR4 27 cytokine regulation 26 miscellaneous receptors 86 regulation of expression 86, 87 tissue-infiltrating cell expression 27, 28 regulation of neutrophil expression 88 tumor transfection studies 189 Circulating levels, polymorphonuclear neutrophils 24 Complement, cytokine release regulation in neutrophils 99 Cytochrome c, apoptosis regulation 210 Cytokine receptor ␥ chain, polymorphonuclear neutrophil expression 64, 75, 76 Defensins, functions 183, 184 Degranulation, see Granule release, polymorphonuclear neutrophils Dendritic cell polymorphonuclear neutrophil phenotype and cytokine activation 33–35, 45, 184 subpopulations 103 DNA microarray, cytokine-activated polymorphonuclear neutrophil expression studies 35–38 Elastase, vascular permeability modulation 156 Emigration, cytokines in polymorphonuclear neutrophil emigration 28, 29 Endometrium, remodeling 172 Endothelium, see Vascular permeability Enteritis, lipoxin 136 Ets, neutrophil synthesis 15 Extracellular matrix, polymorphonuclear neutrophil transversal 29 Fas ligand, tumor transfection studies 189, 190 N-Formyl-methionyl-leucyl-phenylalanine, interleukin-12 production role 99

227

Glutathione, loss in apoptosis 208 Granule release, polymorphonuclear neutrophils cytokine regulation 30, 31 granule types 169 tissue damage 81, 82 Granulocyte colony-stimulating factor cancer treatment 193 granule release role 30, 31 STAT activation 3, 4 Granulocyte-macrophage colony-stimulating factor granule release role 30, 31 polymorphonuclear neutrophil phenotype effects 34 STAT activation 4, 5 Growth hormone, STAT activation 8 Heparin binding protein, vascular permeability modulation 156–158 Hepatocyte growth factor, neutrophil modulation of angiogenesis 170, 171, 176, 177 15-HETE, esterification in lipoxin biosynthesis priming 123, 124 Human immunodeficiency virus neutrophil defects 196 Tat, angiogenesis induction mechanism 176 Innate immunity, polymorphonuclear neutrophil triggering 107 Integrins neutrophil-endothelium interactions 148, 149, 153, 155 polymorphonuclear neutrophil emigration role 28, 29 structures 149 Interferon-␥ interleukin-12 production regulation 101 polymorphonuclear neutrophil phenotype effects 34 STAT activation 5, 6 Interleukin-1 granule release role 30, 31 neutrophil synthesis 1, 2 Interleukin-2 Jak-STAT signaling 66–68

Subject Index

pleiotropy 66 polymorphonuclear neutrophil activation 68–70 redundancy 66 structure 66 Interleukin-4 interleukin-12 production regulation 101 Jak-STAT signaling 66–68 pleiotropy 66 polymorphonuclear neutrophil activation 70, 71 production 74 redundancy 66 structure 66 Interleukin-6, preformed stores in neutrophils 102, 103 Interleukin-7 Jak-STAT signaling 66–68 pleiotropy 66 polymorphonuclear neutrophil activation 71, 72 redundancy 66 structure 66 Interleukin-8 angiogenesis role 168, 170, 177 cytokine stimulation of production 100 granule release role 30, 31 neutrophil synthesis 1, 2, 99, 100 tumor angiogenesis 185 Interleukin-9 Jak-STAT signaling 66–68 pleiotropy 66 polymorphonuclear neutrophil activation 72, 73 redundancy 66 structure 66 Interleukin-10 interleukin-12 production regulation 101, 103, 104 STAT activation 7, 8 tumor transfection studies 187 Interleukin-12 antitumor therapy 191, 193 functions 95, 96 polymorphonuclear neutrophil production

228

cytokine modulation 100, 101 functions 104–107 inducing microorganisms 96, 98 overview 96 preformed stores 102–104 prospects for study 108, 109 triggering pathways 98–100 structure 95 tumor transfection studies 187–189 Interleukin-15 Jak-STAT signaling 66–68 pleiotropy 66 polymorphonuclear neutrophil activation 73–75 production 74 redundancy 66 structure 66 tumor transfection studies 187, 188 Interleukin-21 Jak-STAT signaling 66–68 pleiotropy 66 polymorphonuclear neutrophil functions 74 redundancy 66 structure 66 JAM-1, neutrophil diapedesis role 151, 152 Janus kinase signaling through STATs 2, 3, 67, 68 types 67 Leukotrienes antagonist comparison studies with lipoxins 132 leukotriene A4-dependent biosynthesis of lipoxins 124–126 neutrophil recruitment 147 Life span, polymorphonuclear neutrophils 24, 45, 64, 204 Lipopolysaccharide, polymorphonuclear neutrophil gene expression response 37, 38 Lipoxins biosynthesis aspirin promotion of synthesis 116, 118–120, 128, 129

Subject Index

15-HETE esterification in priming 123, 124 leukotriene A4-dependent biosynthesis 124–126 15-lipoxygenase-initiated pathway 117, 118, 120, 122, 123 regulation 137 selectin-deficient mouse studies 126, 127 transcellular biosynthesis 117, 118, 126, 127 disease association asthma 135 enteritis 136 nephritis 136 overview 134, 135 15-epi-LXA4 induction by aspirin in animal models and asthma 129, 130 stable analog 134 inflammation resolution 115, 116 species distribution 127, 128 therapeutic use leukotriene antagonist comparison studies 132 neutrophil infiltration inhibition 131–133 prospects 137, 138 rational 116, 117, 130, 131 stable analog generation 131 vascular damage prevention 131–134 types and functions 121 Major histocompatibility complex, polymorphonuclear neutrophil expression class II molecules active disease studies 47, 49–51 donor-dependence of surface expression 46 functional consequences 53, 55, 59, 60, 61 induction by cytokines 46 specificity of cytokine induction 46, 47 costimulatory molecule expression 51, 53, 55 overview 33–35, 45

229

Mcl-1 antisense disruption 214, 215, 217 half-life 214 neutrophil expression and apoptosis role 215–218 regulation of expression 218 structure 213, 214 tissue distribution 213 MCP-1, polymorphonuclear neutrophil adaptive immunity role 32 MIP-1␣, neutrophil synthesis 1, 2 MIP-2, preformed stores in neutrophils 102, 103 Mitogen-activated protein kinase, interleukin-12 production role 99 Nephritis, lipoxin 136 Nuclear factor-␬B activation in neutrophils 10–12 apoptosis role 13, 14 DNA binding 9 gene targets 9, 10 inhibitor 9 interleukin-12 production role 99 prospects for study 14, 15 reactive oxygen intermediate modulation 12, 13 structure 9 OX40L, tumor transfection studies 190 PECAM-1, neutrophil diapedesis role 150, 151 Phosphatidylserine, translocation in apoptosis 207 Platelet activating factor, neutrophil modulation of angiogenesis 171 Priming, cytokines in polymorphonuclear neutrophil priming 29, 65 Prolactin, STAT activation 8 PU.1, neutrophil synthesis 15 Reactive oxygen species neutrophil modulation of angiogenesis 173 nuclear factor-␬B modulation 12, 13 Rolling, polymorphonuclear neutrophils 28

Subject Index

Selectins deficient mouse studies of lipoxin synthesis 126, 127 neutrophil-endothelium interactions 148, 182, 183 L-selectin, polymorphonuclear neutrophil emigration role 28 STATs activation in neutrophils early studies 3, 4 granulocyte colony-stimulating factor 3, 4 granulocyte-macrophage colony-stimulating factor 4, 5 growth hormone 8 interferon-␥ 5, 6 interleukin-10 7, 8 prolactin 8 prospects for study 8, 9 dimers 2 isoforms 2 Jak signaling 2, 67, 68 types 2 T cell antitumor immune memory rejection 190, 191 delayed-type hypersensitivity 183 recruitment 183 subset selection by polymorphonuclear neutrophils during infection 104–106 Tat, angiogenesis induction mechanism 176 Toll-like receptor, neutrophil expression 98, 99 Tumor, see Cancer Tumor necrosis factor-␣ polymorphonuclear neutrophils gene expression response 36, 37 granule release role 30, 31 phenotype effects 34, 35 polymorphonuclear neutrophil priming 29 tumor transfection studies of ligands 189, 190 Vascular endothelial growth factor, neutrophil modulation of angiogenesis 170, 172, 176, 177

230

Vascular permeability endothelial barrier structure 151, 152 inflammatory reaction and mediators 146 lipoxin effects 131–134 neutrophils diapedesis 150, 151 endothelium interactions adhesive contact downregulation 154 cationic proteins in regulation of barrier function 155–158 endothelial cell calcium flux and contraction 153, 154

Subject Index

extravasation 147, 148 integrins 148, 149, 153, 155 junction protein phosphorylation 154 oxidant-mediated barrier dysfunction 154, 155 prospects for study 159, 160 selectins 148, 182, 183 gap formation in transendothelial migration 159 recruitment 147, 183 Wegener granulomatosis, polymorphonuclear neutrophil MHC class II expression 49, 50

231