Mast Cells in Allergic Diseases (Chemical Immunology)

Mast Cells in Allergic Diseases Chemical Immunology and Allergy Vol. 87 Series Editors Johannes Ring Munich Luciano...

Author: H. Saito | Y. Okayama

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Mast Cells in Allergic Diseases

Chemical Immunology and Allergy Vol. 87

Series Editors

Johannes Ring Munich Luciano Adorini Milan Claudia Berek Berlin Kurt Blaser Davos Monique Capron Lille Judah A. Denburg Hamilton Stephen T. Holgate Southampton Gianni Marone Napoli Hirohisa Saito Tokyo

Mast Cells in Allergic Diseases

Volume Editors

Hirohisa Saito Tokyo Yoshimichi Okayama Yokohama

38 figures, 10 in color, and 8 tables, 2005

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 Kalos 1939–1988, Byron H. Waksman 1962–2000

Hirohisa Saito Department of Allergy and Immunology National Research Institute for Child Health and Development Setagaya, Tokyo Japan

Yoshimichi Okayama Laboratory for Allergy Transcriptome RIKEN Research Center for Allergy and Immunology Yokohama City, Kanagawa Japan

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 2005 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 3–8055–7948–9

Contents

XIII Preface H. Saito, Tokyo

1 Regulation of Mast Cell Development M. Shiohara, K. Koike, Matsumoto 1 1 2 3 5 6 7 9 9 9 10 11 13 13 14 16

Abstract Phenotypic Characteristics of Mast Cells Human Mast Cells Are Derived from Multipotential Hematopoietic Progenitors Human Mast Cell Development on Stimulation with SCF Alone in Serum-Deprived Culture SCF-Dependent Human Mast Cell Development Is Regulated by Various Factors Thrombopoietin Interleukin-9 Interleukin-16 Nerve Growth Factor Interleukin-3 Interleukin-4, Interleukin-6 Retinoids Interferon Property of Cultured Mast Cells Conclusions and Future Directions References

V

22 Regulation of Mast Cell Activation through Fc␧RI S. Yamasaki, T. Saito, Yokohama 22 Abstract 22 Structure and Function of FcR on Mast Cells 23 Function of FcR␥ in Mast Cell Activation 23 Hypersensitivity Mediated through Fc␥R on Mast Cells 24 FcR␥-ITAM-Dependent and Independent Mast Cell Response through Fc␧RI 25 Regulation of Mast Cell Survival vs. Degranulation through Fc␧RI 29 Acknowledgments 29 References 32 Role of Oxidants in Mast Cell Activation Y. Suzuki, T. Yoshimaru, T. Inoue, O. Niide, C. Ra, Tokyo 32 Abstract 32 ROS and Regulation of the Cellular Redox Balance 33 Generation of ROS in Non-Phagocytic Cells through the NOX/DUOX Family 35 Role of ROS in Mast Cell Activation 35 Fc␧RI Signaling in Mast Cells 36 Generation of ROS in Mast Cells and Basophils 37 Signal Transduction Pathway for ROS Generation 37 Role of Oxidants in Fc␧RI Signaling and Allergy 39 Conclusions 40 Acknowledgements 40 References 43 Roles of Adaptor Molecules in Mast Cell Activation C. Tkaczyk, S. Iwaki, D.D. Metcalfe, A.M. Gilfillan, Bethesda, Md. 43 43 44 45 46 46 47 47 48 48 49 49 51 52

Abstract Adaptor Molecules in Mast Cells Constitutive Protein-Protein Interactions Inducible Protein-Protein and Protein-Lipid Interactions SH2 Domains and PTB Domains PH Domains Receptors and Receptor Subunits as Adaptor Molecules Fc␧RI and Fc␥RI: ␤ and ␥ Subunits Fc␥RIIb and gp49b Kit Transmembrane Adaptor Molecules LAT (Linker for Activation of T Cells) NTAL (Non-T Cell Activation Linker) Cbp/PAG (Csk-Binding Protein/Phosphoprotein Associated with GEMs)

Contents

VI

52 Cytosolic Adaptor Molecules 53 SLP-76 (Src Homology 2 Domain-Containing Leukocyte Protein of 76 kDa) 53 Vav 54 Grb2, Gads and Gab1 55 3BP2 55 Cbl and Dok 56 Conclusions 56 References 59 Eicosanoid Mediators of Mast Cells: Receptors, Regulation of Synthesis, and Pathobiologic Implications J.A. Boyce, Boston, Mass. 59 Abstract 59 Introduction 60 MC-Associated Eicosanoids 60 PGD2 60 Biosynthesis 62 Actions of PGD2 in Humans: Direct Challenges 62 Receptors for PGD2 and Receptor-Mediated Functions in vitro 62 Effects on Leukocytes 63 Effects on Smooth Muscle 63 Functions of PGD2 and Its Receptors in vivo: Mouse Models 63 Allergen-Induced Pulmonary Inflammation 64 Cysteinyl Leukotrienes 64 Biosynthesis 65 Actions in Humans: Direct Challenges 65 Receptors for cysLTs and Receptor-Mediated Functions in vitro 66 Effects on Smooth Muscle and Endothelial Cells 66 Effects on Leukocytes 67 Functions of cysLTs and Their Receptors in vivo: Pharmacologic Studies in Humans 67 Asthma 68 Functions of cysLTs and their Receptors in vivo: Disease Models in Mice 68 Microvascular Responses 68 Allergen-Induced Pulmonary Inflammation 69 Dendritic Cell Maturation and Migration 69 Pulmonary Fibrosis 69 Regulation of Eicosanoid Synthesis by MCs 69 Heterogeneity of Eicosanoid Generation by Tissue MC Subsets 70 Regulation of Eicosanoid Pathways in vitro by Exogenous Cytokines 70 PGHS/PGDS Pathway 72 5-LO/LTC4S Pathway 72 Summary 73 References

Contents

VII

80 Role of Mast Cell Proteases in Tissue Remodeling H. Saito, Tokyo 80 80 80 81 82 83 83

Abstract Introduction Effect of Tryptase on Tissue Cell Proliferation and Remodeling Effect of Tryptase on Airway Smooth Muscle Cells and Epithelium Chymase and Other Proteases Related to Tissue Remodeling Conclusion References

85 Mast Cell Mediators in Airway Remodeling C.K. Oh, Torrance, Calif. 85 85 86 89 90 92 92 94 95

Abstract Introduction Mast Cells and Their Mediators in Asthma Identification and Characterization of PAI-1 in Mast Cells Mechanisms of PAI-1 Action in ECM Accumulation Regulation of PAI-1 Expression Effect of PAI-1 in the Development of Airway Remodeling Conclusion References

101 Mast Cell-Derived Cytokine Expression Induced via Fc Receptors and Toll-Like Receptors Y. Okayama, Yokohama 101 101 102 103 104 108 108

Abstract Introduction Fc␧RI-Mediated Cytokine Expression by MCs TLR-Mediated Cytokine Expression by MCs Fc␥R-Mediated Cytokine Expression by MCs Conclusions References

111 Mast Cells in Allergic Airway Disease and Chronic Rhinosinusitis R. Pawankar, Tokyo 111 111 112 112 114 115 115 116

Abstract Introduction Phenotypes and Distribution of Mast Cells in Allergic Rhinitis Nasal Mast Cells as a Source of Multifunctional Cytokines Mast Cells-IgE-Fc␧RI Cascade in Allergic Airway Disease Nasal Mast Cells, Adhesion Molecules and Extracellular Matrix Proteins Nasal Mast Cell – Structural Cell Interactions Mast Cell Migration and Regulation of Mast Cell Phenotypes in Allergic Nasal Epithelium

Contents

VIII

116 116 117 118 118 120 120 120 121 123 124

Versatile Roles of Mast Cells in IgE-Mediated Allergy Immediate Phase Response Late Phase Response Chronic Allergic Inflammation Highlighting the Roles of Mast Cells in Asthma Implications of Mast Cells in Rhinitis and Asthma Future Potential Therapy Targeting Mast Cell-IgE-Networking: A Global Approach for Rhinitis and Asthma Humanized Monoclonal Antibodies against IgE Mast Cells in Chronic Rhinosinusitis Conclusion References

130 Chemokine Receptor Expression by Mast Cells M. Juremalm, Uppsala; G. Nilsson, Stockholm/Uppsala 130 130 131 133 135 135 136 137 137 137 138 138 138 139 139 139 140 141 141 142 142

Abstract Introduction Chemokines and Their Receptors Chemokine Receptors Complex Ligand-Receptor Chemokine Network Expression of Chemokine Receptors by Mast Cells CCR1 and CCR4 CCR2 CCR3 CCR5 CX3CR1 CXCR1 and CXCR2 CXCR3 CXCR4 Functional Aspects of Chemokine Receptors on Mast Cells Migration Degranulation and Cytokine Secretion Viral Infection Conclusions Acknowledgment References

145 Mast Cell ␤2-Adrenoceptors L.J. Kay, P.T. Peachell, Sheffield 145 145 146 148 150 151

Abstract Introduction Mast Cell Stabilisation Tolerance Pharmacogenetics Conclusion

Contents

IX

151 Acknowledgement 152 References 154 Potential Role of Stem Cell Factor in the Asthma Control by Glucocorticoids C.A. Da Silva, N. Frossard, Illkirch 154 154 155 156 157 159 159

Abstract Introduction Glucocorticoids and Asthma Glucocorticoids and Mast Cells Glucocorticoids and SCF Conclusions References

163 Mast Cell Ion Channels P. Bradding, Leicester 163 164 164 164 168 171 173 175 175 176

Abstract Mast Cells in Asthma and Allergy Critical Role of Ion Channels in Mast Cell Activation Mast Cell K⫹ Channels Mast Cell Cl⫺ Channels Mast Cell Ca2⫹ Channels Na⫹ Channels P2X Receptors Hypothetical Mast Cell Electrical ‘Excitation’ Cycle References

179 Using Mast Cell Knock-In Mice to Analyze the Roles of Mast Cells in Allergic Responses in vivo M. Tsai, M.A. Grimbaldeston, M. Yu, S.-Y. Tam, S.J. Galli, Stanford, Calif. 179 Abstract 181 ‘Mast Cell Knock-In Mice’ as a Model for Studying Mast Cell Functions in vivo 184 Mast Cells in Reactions of Immediate Hypersensitivity 184 IgE-Dependent Local or Systemic Reactions 186 Anaphylaxis or Local Immediate Hypersensitivity Reactions in Actively Immunized Mice 188 Mast Cells in Allergic Inflammation in the Airways: Mouse Models of Asthma 189 Mast Cells in Allergic Inflammation in the Skin: Delayed Hypersensitivity, Contact Hypersensitivity (CHS) and Atopic Dermatitis 189 Update on the Controversy Regarding the Roles of Mast Cells in CHS and Delayed Hypersensitivity 190 Potential Roles of Mast Cells in Certain CHS Responses 191 Potential Roles of Mast Cells in Other Models of Allergic Inflammation in the Skin

Contents

X

192 Conclusions 193 References 198 Mast Cell-Specific Genes – New Drug Targets/Pathogenesis H. Saito, Tokyo 198 Abstract 198 Introduction 199 Mast Cells vs. Other Cell Types 203 Activated vs. Resting Mast Cells 204 Glucocorticoid Sensitivity of Mast Cell Transcripts 205 Mast Cell Subset-Specific Genes 207 Human Mast Cells vs. Mouse Mast Cells 209 Computational Modeling of Human Mast Cells in silico 209 References 213 Author Index 214 Subject Index

Preface

XI

Preface

Mast cells were named after the Greek word ‘mastos’, which means breast, in 1878 by Paul Ehrlich because he believed that the intracellular granules contained nutrients. The discovery of IgE and its association with mast cell histamine release provided the initial understanding of the role of mast cells in asthma and acute allergic reactions. Thus, the cross-linking of high-affinity IgE receptors on mast cells was regarded as the essential mechanism involved in allergic diseases and was the major target for the therapeutic development of drugs. After the 1980s, allergic inflammation characterized by eosinophil recruitment into tissues was found to be essential in asthma pathology. Then, inhaled corticosteroids, which prevent eosinophilic inflammation but not mast cell degranulation, were widely recognized as the first-line therapy for asthma. The role of mast cells was thus considered relevant only for the early asthmatic response to allergen challenge, but less involved in asthmatic reactions found in patients having chronic symptoms. Recently, the difference between asthma and eosinophilic bronchitis lacking airway hyperresponsiveness and airflow obstruction was found to be infiltration of airway smooth muscle by mast cells [1]. Mast cells produce a variety of lipid mediators, chemokines, cytokines, and enzymes that may interact with airway smooth muscle cells and cause hyperreactivity to constrictive stimuli and proliferation. More recently, Oguma et al. [2] have described an exemplary investigation of the prostaglandin D2 (PGD2) receptor gene (PTGDR) as a candidate for a role in the susceptibility to asthma in young adults. PGD2 is almost exclusively produced by activated mast cells but not other cell types and can evoke airway

XIII

hypersensitivity and the chemotaxis of T cells, eosinophils and basophils through interaction with two different receptors: one is prostanoid DP receptor (translated from PTGDR) and the other is chemoattractant receptor-homologous molecule expressed on Th2 cells (CRTH2). Cytokines generated by both resident and freshly recruited cells are responsible for the initiation and coordination of many local processes, including allergic inflammation and tissue remodeling. In IgE-dependent allergic inflammation it would be logical to expect that the mast cell would generate a spectrum of cytokines directed at initiating and maintaining allergic inflammation. Indeed, human mast cells were found to generate multiple cytokines and chemokines, which activate a variety of cell types including T cells and eosinophils. Mast cells are now recognized as tissue-dwelling effector cells that play multiple roles not only in immediate-type allergic reaction but also in innate immunity, inflammation, angiogenesis, and tissue remodeling. In this book, we have focused on the roles of mast cells in allergic diseases and discuss the future direction of discovering drugs. Another implication of this book is to understand mast cells at the system level. System biology is a research category to understand biology at the system level by examining the structure and dynamics of cellular and organismal functions, rather than the characteristics of isolated parts of a cell or organism [3]. Understanding the properties of systems may have an impact on the future of medicine. The most feasible application of systems biology research is to create a detailed model of cell regulation, focused on particular signal-transduction cascades. It is expected to provide system-level insights into mechanism-based drug discoveries. Such models may help to identify feedback mechanisms that offset the effects of drugs and predict systemic side effects. Hirohisa Saito, Tokyo

References 1 2 3

Brightling CE, Bradding P, Symon FA, Holgate ST, Wardlaw AJ, Pavord ID: Mast-cell infiltration of airway smooth muscle in asthma. N Engl J Med 2002;346:1699–1705. Oguma T, Palmer LJ, Birben E, Sonna LA, Asano K, Lilly CM: Role of prostanoid DP receptor variants in susceptibility to asthma. N Engl J Med 2004;351:1752–1763. Kitano H: Systems biology: a brief overview. Science 2002;295:1662–1664.

Preface

XIV

Saito H, Okayama Y (eds): Mast Cells in Allergic Diseases. Chem Immunol Allergy. Basel, Karger, 2005, vol 87, pp 1–21

Regulation of Mast Cell Development Masaaki Shiohara, Kenichi Koike Department of Pediatrics, Shinshu University School of Medicine, Matsumoto, Japan

Abstract Human mast cells originated from multipotential hematopoietic progenitors in bone marrow (BM). A large proportion of these progenitors were CD34⫹CD38⫺c-kit⫹ cells and CD34⫹CD38⫹c-kit⫹ cells. Cloning of stem cell factor (SCF) contributed to the development of culture systems of human mast cells from different sources including BM mononuclear cells, CD34⫹ cord blood (CB) cells, and fetal liver cells. SCF could support mast cell development from CD34⫹ CB cells in the serum-deprived culture system. The cumulative mast cell number reached 1010-fold the input quantity at 50 weeks of SCF-containing serumdeprived culture. CB-derived mast cells expressed tryptase, chymase, and histamine similar to mast cells residing in tissues. The surface expression of Fc␧RI, however, was very low on fetal liver- or CB-derived mast cells. Factors that had effects on SCF-dependent human mast cell development were divided into four types: (1) factors that stimulated both proliferation and maturation: SCF; (2) factors that stimulated only proliferation: thrombopoietin, interleukin (IL)-9; (3) factors that inhibited proliferation, but stimulated maturation: IL-4, IL-6, and (4) factors that inhibited proliferation and maturation: retinoids. Copyright © 2005 S. Karger AG, Basel

Phenotypic Characteristics of Mast Cells

Human mast cells were first discovered by Ehrlich [1] in 1877 as cells with metachromatic granules in their cytoplasm. Mast cells are 6–12 ␮m in diameter, and distributed throughout the body in tissues including skin, lungs, nasal mucosa, and gut. In contrast to other lineages of hematopoietic cells, mast cells are rarely found in peripheral blood [2]. Mast cells play an important role as primary effector cells in allergic disorders such as bronchial asthma, atopic dermatitis, and allergic rhinitis [2, 3]. In rodents, mast cells have been classified into at least two phenotypically distinct subpopulations, i.e., mucosal mast cells (MMC) and connective tissue mast cells (CTMC) [4–7]. MMC and CTMC originate from bone marrow (BM) hematopoietic progenitors, and change their phenotypes depending on conditions

around the cells [8–11]. In humans, two types of mast cells have been identified on the basis of protease expression: one phenotype is positive only for tryptase (designated as MCT), and the other is positive for both tryptase and chymase (designated as MCTC). MCTC also express cathepsin G and carboxypeptidase A. MCT are predominantly found in intestinal mucosa and alveolus, whereas MCTC are found in skin and intestinal submucosa [12, 13]. Li et al. [14] reported the presence of mast cells that express chymase, but not tryptase, in intestinal submucosa and salivary glands.

Human Mast Cells Are Derived from Multipotential Hematopoietic Progenitors

In the murine system, it has been elucidated that mast cells originate from hematopoietic stem cells in vivo [15] or multipotential hematopoietic progenitors in vitro [16]. Mast cell precursors depart from BM and migrate into connective or mucous tissues, where they differentiate into the mature form [2]. Kirshenbaum et al. [17] demonstrated that human mast cells are derived from CD34⫹ BM cells in vitro. Födinger et al. [18] showed that donor mast cells are detectable 198 days after allogeneic BM transplantation. These observations suggest that human mast cells are also generated by hematopoietic progenitors. To assess the differentiative potential of human mast cell-producing colony-forming cells, we performed a two-step culture assay [19]. Individual CD34⫹ BM cells were initially incubated with stem cell factor (SCF) plus thrombopoietin (TPO). When the number of cells in each well reached more than 10, the cells were divided into two aliquots. One half of the sample was replated in a well containing SCF⫹TPO for the expression of neutrophil and mast cell lineages. The other half was recultured in a well containing granulocyte colony-stimulating factor, SCF, TPO, flt3 ligand (FL), granulocytemacrophage colony-stimulating factor (GM-CSF), interleukin (IL)-3 and erythropoietin for the expression of neutrophil, macrophage, megakaryocyte, and erythroid lineages. Mast cells are derived from multilineage colony-forming cells which have the potential to differentiate into neutrophil/mast cell lineages, neutrophil/macrophage/mast cell lineages, or neutrophil/macrophage/mast cell/ erythroid lineages. A large proportion of these multipotential progenitors were CD34⫹CD38⫺c-kit⫹ cells and CD34⫹CD38⫹c-kit⫹ cells. These results indicate the clonal origin of human mast cells from primitive BM progenitors. Additionally, they support the existence of common precursors for granulocytes and mast cells as described by Kitamura et al. [15]. However, the developmental pathway common to mast cell and lymphoid lineages remains unclear. Taken together with the results from other studies using a clone-sorting method,

Shiohara/Koike

2

Table 1. Serum-deprived culture of human mast cells A. Content of culture medium CD34⫹ cells or cultured mast cells 1% bovine serum albumin 300 ␮g/ml fully iron-saturated human transferrin 16 ␮g/ml soybean lecithin 9.6 ␮g/ml cholesterol 10-100 ng/ml stem cell factor Alpha-medium in 24-well culture dish B. Culture conditions 5% CO2, 5% O2, and 90% N2 in 100% humidity

human mast cell progenitors express CD13, CD 33, CD38, and CD34, c-kit, but rarely possess HLA-DR [20, 21]. Human Mast Cell Development on Stimulation with SCF Alone in Serum-Deprived Culture

The SCF-c-kit receptor signal transduction pathway is essential for the development of murine mast cells, since both W and Sl mice, which have mutations in the locus of the c-kit receptor and SCF, respectively, are deficient in mast cells [22, 23]. In humans, SCF is also a pivotal growth factor that promotes the development of mast cells. Initially, the culture of cord blood mononuclear cells (CB MNCs) on a layer of 3T3 fibroblasts derived from Swiss albino mouse skin yielded mast cells expressing both tryptase and chymase [24]. Subsequently, SCF was identified as a main growth-promoting factor secreted from fibroblasts [25]. After SCF was cloned in 1990 by several groups [26–28], studies of mechanisms for mast cell proliferation and differentiation progressed rapidly. Simultaneously, culture systems of human mast cells from different sources including BM MNCs, CD34⫹ CB cells, and fetal liver cells, were developed [15, 16, 29, 30]. However, the purity of the mast cells remained at 40–85%. When a serum-deprived culture system (table 1) was used, SCF alone supported a progressive, steady increase of mast cell production until 50 weeks from CD34⫹ CB cells [31]. The cumulative cell number reached 1010-fold the input quantity at 50 weeks of culture. According to immunocytochemical staining, almost all of the 4-week cultured cells reacted with anti-tryptase monoclonal antibody (mAb), but were negative for chymase. After 36 weeks, a vast majority of the cells were positive for both tryptase and chymase (fig. 1). Our single CD34⫹ cell culture showed that SCF acts as a proliferative rather than survival factor in mast cell development from CB hematopoietic progenitors [19].

Mast Cell Development

3

a

b

c Fig. 1. Cytological characteristics of cultured human mast cells grown with SCF. Cytochemical and immunologic staining of 40-week cultured cells grown with 10 ng/ml of SCF from CD34⫹ CB cells were performed on cytocentrifuged samples. Staining of cells with May-Grünwald-Giemsa (a), with a mAb for tryptase (b), and with a mAb for chymase (c). Orig. magn. ⫻1,000. Reprinted with permission from the American Society of Hematology.

Shiohara/Koike

4

Additionally, the cultured mast cells grown with 100 ng/ml of SCF were larger and contained more intracellular histamine than those grown with 10 ng/ml of SCF [31]. Thus, SCF acts as a stimulator of both the growth and maturation of human mast cells. Furthermore, a serum-deprived culture system would be appropriate for obtaining large quantities of highly enriched human mast cells by stimulation with SCF alone. Association of mast cells with cardiomyopathy: Patella et al. [32] reported that histamine and tryptase content and mast cell density were higher in heart tissue of patients with idiopathic dilated and ischemic cardiomyopathy compared to control hearts. Moreover, cardiac mast cells in these patients store SCF in the secretory granules, indicating that SCF might represent an autocrine factor sustaining mast cell hyperplasia in heart tissue in these patients. Mast cell chymase is involved in angiotensin II forming systems, in which angiotensin-converting enzyme plays major roles [33]. When tissue macrophages phagocytize cholesterol in hypercholesterolemia, the cells are activated and secrete various types of cytokines. Mast cells migrate to local sites in response to these chemotaxic factors and release chymase by degranulation, which converts angiotensin I to angiotensin II [34]. Since angiotensin II induces the contraction of blood vessels and proliferation/hypertrophy of vascular smooth muscle cells, this substance plays an important role as a causative agent in atherosclerosis and cardiomegaly. STI571-induced apoptosis: STI571 is known to be clinically active in patients with chronic myeloid leukemia and other Philadelphia chromosomepositive leukemias [35–38], since it binds to the ATP-binding site of the target kinase and prevents the transfer of phosphate from ATP to the tyrosine residues of various substrates. From the evidence that STI571 is also a competitive inhibitor of c-kit [38, 39], we tested the possibility of using the compound for the clinical treatment for allergic disorders. STI571 at concentrations of 10⫺6 M or higher almost completely abolished the SCF-dependent generation of progeny from CB-derived cultured mast cells through an inhibition of the tyrosine phosphorylation of c-kit [40]. The compound also suppresses the early phase of mast cell development. As presented in figure 2, the extinction of mast cell growth induced by STI571 may be due largely to apoptosis according to flow cytometric analysis and gel electrophoresis.

SCF-Dependent Human Mast Cell Development Is Regulated by Various Factors

Recent studies have elucidated that many factors modulate human mast cell development. Some molecules stimulate, while others inhibit, the proliferation and maturation of mast cells.

Mast Cell Development

5

1,000

1

2 1

Day 0

800 600

2

3

86% 1. G0 /G1 2. S⫹ G2/M 13%

400 200 0 0 1,000

50 1

100

150

200

250

2

Day 4

800 600

1. G0 /G1 44% 2. S ⫹ G2/M 5%

400 200 0

a

0

50

100

150

200

250

b

Fig. 2. STI571 induces apoptosis of cultured mast cells grown with SCF. Ten-week cultured mast cells were replated into 20 ng/ml of SCF alone or in combination with 10⫺6 M STI571 for 4 days. a The incidence of viable cells (cells in G0/G1 phase and cells in S⫹G2/M phase) relative to total cells was evaluated by flow cytometry. The arrow indicates the significant appearance of cells with less than 2N. b DNA was extracted from the cultured mast cells, and gel electrophoresis was performed. Lane 1, DNA molecular-weight marker, 100 bp DNA ladder; lane 2, SCF alone; lane 3, SCF⫹STI571.

Thrombopoietin TPO was cloned as a potent stimulator of megakaryocytopoiesis [41–44]. Moreover, TPO was demonstrated to support the growth of hematopoietic progenitors, especially multipotential progenitors, in combination with other cytokines including SCF, IL-3, and FL [45, 46]. We found that SCF alone generated only a small number of tryptase-positive cells from normal CD34⫹ BM cells, but the addition of TPO to the cultures containing SCF resulted in the production of tryptase-positive cells from 3 weeks until at least 15 weeks (fig. 3) [19]. Some of the cells reacted with an anti-chymase mAb as well. The cultured mast cells grown with SCF⫹TPO express c-kit, but not c-mpl. Additionally, 15-week-old cultured mast cells grown in SCF⫹TPO maintained their absolute number on stimulation with SCF for 3 weeks, but not in the presence of TPO. Moreover, the addition of TPO to the cultures containing SCF did not influence the total viable cell number. Thus, TPO may stimulate an early stage of mast cell development in concert with SCF, and the subsequent growth appears to be supported by SCF alone. Single cell cultures indicated that the CD34⫹CD38⫺ c-kit⫹ cells and CD34⫹CD38⫹c-kit⫹ cells are responsible for the SCF⫹TPOdependent mast cell production.

Shiohara/Koike

6

100

15 75 10

%

Number of viable cells per well (⫻104)

Peroxidase-positive cells Tryptase-positive cells

SCF⫹ TPO SCF TPO No growth factors

20

5

25 0

0 0

a

50

3

6

9

Weeks in culture

12

15

0

b

2

3

6

9

12

15

Weeks in culture

Fig. 3. Combination of SCF and TPO stimulates mast cell production by CD34⫹ bone marrow (BM) cells in serum-deprived liquid culture. a CD34⫹ BM cells (1 ⫻ 104 per well) were plated in serum-deprived liquid culture medium supplemented with 10 ng/ml of SCF and/or 10 ng/ml of TPO. The viable cells were enumerated serially. b Time course of the relative frequency of tryptase-positive cells and peroxidase-positive cells grown with SCF⫹TPO.

Interleukin-9 IL-9 is a cytokine produced by Th2 cells, and acts on various types of cells involved in the allergic immune response [47]. In our serum-deprived liquid cultures, IL-9 apparently enhanced mast cell production on stimulation with SCF from CD34⫹ CB cells (an approximately 8-fold increase of the SCF value at 4 weeks) [48]. In methylcellulose cultures of CD34⫹ CB cells, IL-9 increased both the number and size of mast cell colonies grown with SCF. Furthermore, SCF⫹IL-9 caused an expansion of the progenitors that were capable of becoming mast cell colony-forming cells in short-term liquid cultures of CD34⫹ CB cells, the level being markedly greater than the value obtained with SCF alone. However, IL-9 neither augmented the SCF-dependent generation of progeny nor supported the survival of 6-week cultured mast cells. The frequency of tryptase-positive cells and chymase-positive cells in the cultures grown with SCF⫹IL-9 increased in parallel with the values in the cells grown with SCF alone. Additionally, there was no difference in the intracellular histamine level of the cultured mast cells between the two groups. Taken together, IL-9 may potentiate SCF-dependent human mast cell generation at the progenitor level, but does not appear to influence differentiation into the mast cell lineage. We then elucidated whether the target cells of SCF⫹IL-9 stimulation were identical to those of SCF⫹TPO stimulation (table 2). In the cultures containing CD34⫹CD38⫹ cells, the number of mast cell colonies supported by SCF⫹IL-9 was equivalent to that supported by SCF⫹TPO. When SCF, IL-9 and TPO were

Mast Cell Development

7

Table 2. Mast cell colony growth by CD34⫹CD38⫺ and CD34⫹CD38⫹cord blood (CB) cells stimulated with SCF, IL-9, or TPO, alone or in combination Clonal cell cultures

SCF SCF⫹IL-9 SCF⫹TPO SCF⫹IL-9⫹TPO

CD34⫹CD38⫺ cells

CD34⫹CD38⫹ cells

0 1.7 ⫾ 1.5 16.0 ⫾ 4.0* 24.2 ⫾ 4.0*

11.8 ⫾ 4.9 107.5 ⫾ 18.7* 112.0 ⫾ 13.1* 145.0 ⫾ 12.8*

] ]

† †

]

†

]

†

]

†

CD34⫹CD38⫺ and CD34⫹CD38⫹ CB cells sorted by flow cytometry were plated at 150 cells and 750 cells, respectively, in dishes each containing 10 ng/ml SCF, 100 U/ml IL-9, or 10 ng/ml TPO, alone or in combination. The data represent the mean colony number ⫾ SD from quadruplicate dishes. *Significantly different from SCF alone. †Significant difference among the two- or three-factor combinations.

added together, the mast cell colony number was significantly larger than with the two-factor combinations. In contrast, the proliferative response of CD34⫹CD38⫺ cells to SCF⫹IL-9 was markedly lower than the value obtained with SCF⫹TPO. RT-PCR analysis of IL-9 receptor mRNA supported the differential effect of IL-9 on the two subsets of CD34⫹ cells. These results suggest that SCF⫹IL-9 stimulates mainly the growth of a mature subset of mast cell progenitors, whereas the target cells of SCF⫹TPO belong to a primitive subset as well as a mature subset of the progenitors. In addition, the mature mast cell progenitors that respond to SCF⫹IL-9 may not always be identical to those that respond to SCF⫹TPO. Another difference between the two treatments is progeny type: a great majority of cultured cells grown with SCF⫹IL-9 were positive for tryptase after 4 weeks of culture, whereas SCF⫹TPO yielded significant numbers of myeloid cells as well as mast cells. Thus, IL-9 may act as a potentiator selective for the mast cell lineage in the presence of SCF. The addition of IL-9 increased numbers of mast cell colonies grown with SCF from CD34⫹ peripheral blood (PB) cells in children with or without asthma [48] (fig. 4). It is of interest that mast cell progenitors of asthmatic patients responded to SCF⫹IL-9 to a greater extent than those of normal controls. A significant difference was also found with SCF stimulation [49]. Thus, mast cell progenitors of asthmatic patients may possess superior potential in terms of responsiveness to mast cell growth-promoting cytokines than those of nonallergic subjects. Studies of IL-9-transgenic and -deficient mice clearly showed that IL-9 contributes to airway hyper-responsiveness, mucus hypersecretion and

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* * *

*

Number of mast cell colonies

15

10

5

SCF SCF ⫹ IL-9

SCF SCF ⫹IL-9

Control

Asthma

Fig. 4. Comparison of the ability to form mast cell colonies of CD34⫹ peripheral blood (PB) cells from children with or without asthma. CD34⫹ PB cells from asthmatic patients or normal controls were cultured at 500 cells in dishes each containing 100 ng/ml of SCF with or without 100 U/ml of IL-9. After 4 weeks, aggregates were scored as colonies if the constituent cells numbered 30 or more. The results expressed are the means for 6 asthmatic patients and 5 normal controls. *Statistical significance.

mast cell infiltration [50–53]. Taken together, the results suggest that IL-9 is a candidate gene for asthma. Interleukin-16 IL-16 is a multifunctional cytokine and a ligand for CD4 [54]. When buffy coat cells of PB of asthma patients were cultured for 21 days with SCF and IL-16, tryptase-positive/chymase-positive cell numbers increased 20-fold compared to those with SCF alone [55]. Nerve Growth Factor In the murine system, nerve growth factor (NGF) stimulates the proliferation and differentiation of BM-derived mast cells in the presence of IL-3 [56]. NGF is also associated with phenotypic changes of MMC and CTMC [56]. In humans, NGF inhibited mast cell apoptosis synergistically with SCF through TrkA on the cell surface, which is a high affinity receptor of NGF [57]. NGF is also reported to increase chymase content in mast cells [58]. Interleukin-3 IL-3 has been shown to stimulate the proliferation of BM-derived mast cells and survival of CTMC in murine system [59, 60]. Initially, Durand et al. [61] reported that a cocktail of SCF and IL-3 is necessary for human mast cell

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differentiation from CD34⫹ CB cells. On the other hand, in our serum-deprived culture, IL-3 had the ability to neither produce human mast cells, nor stimulate SCF-dependent mast cell production [31]. Valent et al. [30] reported similar results to ours. Taken together, IL-3 does not have the ability to induce mast cell differentiation, but functions as a growth factor of basophils and eosinophils in humans. These results provide an example of a species-specific growth factor requirement in mast cells. Interleukin-4, Interleukin-6 IL-4 has been demonstrated to diminish the number of mast cells that develop in response to SCF [62, 63]. In addition, IL-4 has various biological effects on human cultured mast cells including the up-regulation of the expression of functional high-affinity IgE receptor (Fc␧RI), intercellular adhesion molecule-1 and lymphocyte function-associated antigen-1 [64–67]. Toru et al. [68] reported that IL-4 promotes the morphologic maturation of human cultured mast cells in accordance with the increase in chymase expression. Nakahata et al. [69] demonstrated that IL-6 was a requisite for the growth of sufficient numbers of almost 100% pure mast cells from CB cells in a serumcontaining liquid culture medium supplemented with SCF. On the other hand, in our serum-deprived liquid culture, the addition of IL-6 to cultures containing mast cells resulted in a substantial reduction in the number of progeny grown with SCF [31], as presented in figure 5. This IL-6-mediated inhibition of mast cell growth may be due in part to suppression at the precursor level, according to the results of a clonal cell culture assay. Moreover, a flow cytometric analysis showed that the cultured mast cells grown in the presence of SCF⫹IL-6 have decreased c-kit expression. Therefore, one possible explanation is that the IL-6mediated reduction of c-kit expression results in the growth retardation of mast cells. The exposure of cultured mast cells to SCF⫹IL-6 also caused substantial increases in cell size, the frequency of chymase-positive cells and the intracellular histamine level compared with the values obtained with SCF alone (fig. 5). The flow cytometric analysis revealed low but significant levels of expression of IL-6 receptor (IL-6R) and gp130 on the cultured mast cells grown with SCF. The addition of either anti-IL-6R Ab or anti-gp130 Ab abrogates the biological functions of IL-6. Thus, IL-6 appears to modulate SCF-dependent human mast cell development directly via an IL-6R-gp130 system. Both IL-4 and IL-6 decrease the numbers of cultured mast cells but also increase intracellular histamine levels on stimulation with SCF (fig. 5) [31]. Comparative assays of the effects of IL-6 with those of IL-4 on SCF-dependent mast cell development revealed that significantly higher concentrations of histamine in cell lysates are obtained in the cultures with SCF⫹IL-6. Thus, IL-6 may be a more potent stimulator of the intracellular histamine content of mast

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Tryptase-positve chymase-positve cells Tryptase-positve chymase-negative cells SCF SCF ⫹ IL-4 SCF ⫹ IL-6 SCF⫹ IL-4 ⫹ IL-6

* *

*

†† †

*

* 0

1 2 3 Number of cells per well (⫻105)

†† †† ††

*

††

4

0

10

20

30

40

Intracellular histamine level (pg/cell)

Fig. 5. Comparison of effects of IL-6 with those of IL-4 on human mast cell development supported by SCF. Ten-week cultured mast cells (2 ⫻ 104) grown with CD34⫹ CB cells were incubated in culture wells containing 100 ng/ml of SCF, 50 ng/ml of IL-6, or 20 ng/ml of IL-4, alone or in combination. After 2 weeks, the viable cells were enumerated, and then processed for immunocytochemical staining with an anti-tryptase or anti-chymase mAb. At the same time, histamine amounts in cell lysates were analyzed. Significantly different from SCF alone (*p ⬍ 0.0001). Significantly different among the two- or three-factor combinations (†p ⬍ 0.0005, ††p ⬍ 0.0001).

cells supported by SCF than is IL-4. When SCF, IL-4 and IL-6 are used together, both the inhibition of cell growth and the elevation of intracellular histamine levels are further amplified compared with the values obtained with the two-factor combinations. Taken together, the results suggest that the two cytokines use different regulatory mechanisms. Retinoids Retinoids are derivatives of vitamin A, and have various effects on many cell types including hematopoietic stem cells [70–73]. Notably, alltrans-retinoic acid (ATRA) is an indispensable agent in induction therapy for acute promyelocytic leukemia, and used widely in the clinic [74, 75]. Retinoids exert their effects through nuclear receptors, RAR/RXR and/or RXR/RXR [76–78]. In our serum-deprived culture, both ATRA and 9-cis-RA remarkably inhibited SCF-dependent mast cell production (fig. 6) [79]. These effects are not due to the induction of apoptosis, but to cell cycle arrest. These inhibitory effects of RA on mast cell proliferation are more prominent on tryptase-positive cells than immature CD34⫹ cells. Interestingly, RA decreases the intracellular histamine content of mast cells (table 3). Both RARs and RXRs consist of three subsets of receptors (␣, ␤, and ␥), which have distinct patterns of distribution and functions in human tissues [77, 78]. RT-PCR analysis of nuclear retinoic acid receptors and experiments using synthetic retinoids showed that effects of RA on proliferation and intracellular levels of histamine of mast cells are exerted through RAR␣. These results suggest that RA is an inhibitor

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Number of cells in well (⫻104)

16

12

*

8 ** 4 **

**

0 0

10⫺11 10⫺10 10⫺9 10⫺8 10⫺7 ATRA (M)

Fig. 6. Dose response to all-transretinoic acid (ATRA) of mast cell growth supported by SCF. 1 ⫻ 104 10-week cultured mast cells were incubated in wells containing SCF at 100 ng/ml with ATRA at 10⫺11 to 10⫺7 M. After 2 weeks, the viable cells were enumerated. Significantly different from SCF alone (*p ⬍ 0.0005, **p ⬍ 0.0001).

Table 3. Effects of retinoids on properties of cultured mast cells supported by SCF Stimuli

Mast cell size, ␮m

Histamine content pg/cell

SCF SCF⫹ATRA SCF⫹9-cis-RA SCF⫹Ro13-7410 SCF⫹Ro25-7386 SCF⫹IL-4 SCF⫹IL-6

21.5 ⫾ 3.6 13.3 ⫾ 3.3* 13.0 ⫾ 3.2* 13.7 ⫾ 3.6* 20.1 ⫾ 3.7 25.0 ⫾ 3.3* 29.7 ⫾ 5.3*

3.00 ⫾ 0.47 1.44 ⫾ 0.18† 1.41 ⫾ 0.10† 1.47 ⫾ 0.24† 2.63 ⫾ 0.77 6.24 ⫾ 0.96* 15.84 ⫾ 2.17*

1 ⫻ 104 10-week cultured mast cells were incubated in culture wells containing SCF, retinoids, IL-6, or IL-4, alone or in combination. The diameter of tryptase-positive cells was measured by calculating the average of two perpendicular diameters on glass slides. At the same time, intracellular histamine concentrations were determined by radioimmunoassay. SCF, 100 ng/ml; ATRA, 9-cis-RA, Ro13-7410 (selective RAR agonist), Ro25-7386 (selective RXR agonist), 10⫺7 M; IL-4, 20 ng/ml; IL-6, 50 ng/ml. Significantly different from SCF alone (*p ⬍ 0.0001, †p ⬍ 0.005)

of both the proliferation and differentiation of mast cells, and a possible antiallergic drug. Roles of retinoids in serum-containing culture: As described above, there are some differences in the proliferation and maturation of mast cells between fetal bovine serum (FBS)-containing and serum-deprived cultures. Accordingly, we attempted to identify circulating factor(s) affecting the development of

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human mast cells. Similar to FBS, human serum suppressed the growth of tryptase-positive cells from CD34⫹ CB cells or 20-week cultured mast cells under stimulation with SCF in a dose-dependent manner [80]. It is demonstrated that IL-4, IL-6, GM-CSF, retinoic acid and transforming growth factor (TGF)-␤1 suppress the proliferation of human mast cells or the HMC-1 mast cell line [63, 79, 81–83]. Among them, retinol and TGF-␤1 are present at high levels in human serum: mean concentrations are approximately 2 ⫻ 10⫺6 M and 40 ng/ml, respectively. An RAR␣ antagonist, but not neutralizing anti-TGF-␤1, counteracted the serum-induced suppression of human mast cell proliferation. Thus, our results suggest that retinol and its derivatives act as a circulating regulator for human mast cell growth. RAR␣ antagonist may be a useful tool to obtain higher numbers of mast cells in FBS-containing cultures. Interferon Interferon-␥ (IFN␥) has been shown to inhibit murine mast cell proliferation [84]. In humans, CD34⫹ BM cell-derived mast cells cultured for 7 days with 100 ng/ml SCF and 10 ng/ml IL-3 then recultured with SCF and 1,000 U/ml IFN␥1b significantly decreased in number and incidence of granular metachromasia, compared to the culture with SCF alone [85]. Similar results were obtained when CD34⫹ BM cells were cultured from day 0 with SCF and IFN␥-1b. In contrast, Yanagida et al. [86] reported that IFN␥ promotes the survival of human cultured mast cells. CB-derived mast cells cultured with SCF and IL-6 died after withdrawal of these cytokines because of apoptosis. Addition of IFN␥ to the culture suppressed apoptosis and prolonged their survival in a dose-dependent manner. Neutralizing Abs to IFN␥ or to the IFN␥ receptor canceled the survival-promoting effects of IFN␥, indicating that IFN␥ exerts these effects via a specific receptor.

Property of Cultured Mast Cells

CB-derived cultured mast cells generated with SCF alone or in combination with other factor(s) in vitro exhibit many similarities to mast cells residing in tissues, i.e., expression of tryptase, chymase, and histamine. The surface expression of Fc␧RI, however, which is maintained at a high level on mast cells in tissues, is very low on fetal liver- or CB-derived mast cells, indicating that SCF with or without IL-3 or IL-6 is not enough to express a high level of Fc␧RI [61, 64, 86]. Consistent with these observations, IgE-dependent mediator release was low in CB-derived cultured mast cells [64]. Another possible explanation is ontogenetic immaturity, based on comparative analyses of the maturation properties of cultured mast cells grown from CD34⫹ CB cells and CD34⫹ PB cells on stimulation with SCF alone [87, 88]. Despite an apparent positivity for c-kit and

Mast Cell Development

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100

* % histamine release

80

60

40

20

0 CB

PB

Fig. 7. Histamine release induced by Fc␧RI cross-linking from cord blood (CB)derived and peripheral blood (PB)-derived cultured mast cells. Fifteen-week-old mast cells were replated into serum-containing culture medium supplemented with SCF (10 ng/ml), IL-4 (20 ng/ml), and IL-6 (50 ng/ml). After 1 week, the cells were treated with anti-human IgE after sensitization with IgE. The results are expressed as % histamine release. *Significant difference between the two groups at p ⬍ 0.05.

tryptase, there was a significant difference in the tryptase and chymase enzymatic activities of 15-week cultured mast cells between the two groups. Additionally, PB-derived cultured mast cells contain more histamine than CBderived mast cells. Comparing the difference in expression of the Fc␧RI␣ chain, PB-derived mast cells, but not CB-derived mast cells, substantially released histamine in response to the cross-linkage of Fc␧RI with IgE and anti-IgE (fig. 7). Kanbe et al. [89] reported the development of mast cells from human tissues other than hematopoietic organs. Human skin-derived mast cells can proliferate in serum-deprived medium supplemented with SCF. These cells proliferated 160-fold after 4 weeks of culture compared to the number at the start, and were positive for both tryptase and chymase. Additionally, skin-derived cultured mast cells expressed Fc␧RI␣ on the cell surface at high levels, and showed degranulation through Fc␧RI. Conclusions and Future Directions

The proliferation and differentiation of human mast cells are controlled by a variety of physiologically active molecules. Based on published results,

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Proliferation

Maturation

1. SCF

2. TPO, IL-9 ( IL-16)

3. IL-4, IL-6

4. Retinoids (IFN␥)

Fig. 8. Effects of various factors on mast cell proliferation and maturation. Stimulatory and inhibitory effects of each factor, respectively; ⫽ no effect.

,

⫽

most of these factors can be divided into at least four types, as presented in figure 8: (1) factors that stimulate both the proliferation and maturation of human mast cells: SCF; (2) factors that stimulate only the proliferation of human mast cells: TPO, IL-9, (IL-16); (3) factors that inhibit the proliferation, but stimulate the maturation of human mast cells: IL-4, IL-6, and (4) factors that inhibit both the proliferation and maturation of human mast cells: retinoids (IFN␥). The roles of mast cells in innate and acquired immune responses have become clearer. Notably, regarding the kinetics and function of mast cells in allergic diseases, an increase in the number of circulating mast cell colony-forming cells was reported in this allergic disorder [48, 49]. These results suggest quantitative and developmental aberrations as well as functional abnormalities of mast cell in asthma. Further advances will enable us to apply order-made therapies to individuals with allergic diseases. Mast cells have been shown to be closely related with the onset and progression of various human diseases such as atherosclerosis, cardiomegaly, hypertension [33, 90, 91], multiple sclerosis [92], and sudden infantile death syndrome [93, 94]. A better understanding of the development and function of mast cells in vivo in addition to studies in vitro is necessary to analyze the pathophysiology of these mast cell-related diseases. Recently, the reconstitution of human hematopoiesis in genetically immunodeficient mice was reported. Nakahata et al. [95] reported that NOD/SCID (non-obese diabetic/severe combined immunodeficient)/␥cnull mice showed the engraftment of human hematopoietic cells and multilineage mature cells including mast cells. In the murine system, highly enriched mast cells, which

Mast Cell Development

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are derived from wild-type or genetically altered mouse embryonic stem cells, can be generated using the in vitro differentiation system [96]. Furthermore, these genetically manipulated mast cells can be transplanted into native mast cell-deficient mice. These in vivo models will facilitate the use of biochemical and molecular techniques for the analysis of the development and function of mast cells in various conditions.

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Smeland EB, Rusten L, Jacobsen SE, Skrede B, Blomhoff R, Wang MY, Funderud S, Kvalheim G, Blomhoff HK: All-trans-retinoic acid directly inhibits granulocyte colony-stimulating factorinduced proliferation of CD34⫹ human hematopoietic progenitor cells. Blood 1994;84: 2940–2945. Rusten LS, Dybedal I, Blomhoff HK, Blomhoff R, Smeland EB, Jacobsen SE: The RAR-RXR as well as RXR-RXR pathway is involved in signaling growth inhibition of human CD34⫹ erythroid progenitor cells. Blood 1996;87:1728–1736. Huang ME, Ye YC, Chen SR, Chai JR, Lu JX, Zhoa L, Gu LJ, Wang ZY: Use of all-trans-retinoic acid in the treatment of acute promyelocytic leukemia. Blood 1988;72:567–572. Chomienne C, Ballarini P, Balitrand N, Amar M, Bernard JF, Boivin P, Daniel MT, Berger R, Castaigne S, Degos L: Retinoic acid therapy for promyelocytic leukaemia. Lancet 1989;ii: 746–747. Pfahl M: Vertebrate receptors: molecular biology, dimerization and response elements. Semin Cell Biol 1994;5:95–103. Mangelsdorf P, Evans RM: The RXR heterodimers and orphan receptors. Cell 1995;83:841–850. Chambon P: A decade of molecular biology of retinoic acid receptors. FASEB J 1996;10:940–954. Kinoshita T, Koike K, Mwamtemi HH, Ito S, Ishida S, Nakazawa Y, Kurokawa Y, Sakashita K, Higuchi T, Takeuchi K, Sawai N, Shiohara M, Kamijo T, Kawa S, Yamashita T, Komiyama A: Retinoic acid is a negative regulator for the differentiation of cord blood-derived human mast cell progenitors. Blood 2000;95:2821–2828. Ishida S, Kinoshita T, Sugawara N, Yamashita T, Koike K: Serum inhibitors for human mast cell growth: possible role of retinol. Allergy 2003;58:1044–1052. Saito H, Ebisawa M, Tachimoto H, Shichijo M, Fukagawa K, Matsumoto K, Iikura Y, Awaji T, Tsujimoto G, Yanagida M, Uzumaki H, Takahashi G, Tsuji K, Nakahata T: Selective growth of human mast cells induced by steel factor, IL-6, and prostaglandin E2 from cord blood mononuclear cells. J Immunol 1996;157:343–350. Du Z, Li Y, Xia H, Irani AM, Schwartz LB: Recombinant human granulocyte-macrophage colonystimulating factor (CSF), but not recombinant human granulocyte CSF, down-regulates the recombinant human stem cell factor-dependent differentiation of human fetal liver-derived mast cells. J Immunol 1997;159:838–845. Xia HZ, Du Z, Craig S, Klisch G, Noben-Trauth N, Kochan JP, Huff TH, Irani AM, Schwartz LB: Effect of recombinant human IL-4 on tryptase, chymase, and Fc␧ receptor type I expression in recombinant human stem cell factor-dependent fetal liver-derived human mast cells. J Immunol 1997;159:2911–2921. Takagi M, Koike K, Nakahata T: Antiproliferative effect of IFN-␥ on proliferation of mouse connective tissue-type mast cells. J Immunol 1990;145:1880–1884. Kirshenbaum AS, Worobec AS, Davis TA, Goff JP, Semere T, Metcalfe DD: Inhibition of human mast cell growth and differentiation by interferon␥-␥1b. Exp Hematol 1998;26:245–251. Yanagida M, Fukamachi H, Takei M, Hagiwara T, Uzumaki H, Tokiwa T, Saito H, Iikura Y, Nakahata T: Interferon-␥ promotes the survival and Fc␧RI-mediated histamine release in cultured human mast cells. Immunology 1996;89:547–552. Nilsson G, Forsberg K, Bodger MP, Ashman LK, Zsebo KM, Ishizaka T, Irani AM, Schwartz LB: Phenotypic characterization of stem cell factor-dependent human foetal liver-derived mast cells. Immunology 1993;79:325–330. Kikuchi T, Ishida S, Kinoshita T, Sakuma S, Sugawara N, Yamashita T, Koike K: IL-6 enhances IgE-dependent histamine release from human peripheral blood-derived cultured mast cells. Cytokine 2002;20:200–209. Kanbe N, Kanbe M, Kochan JP, Schwartz LB: Human skin-derived mast cells can proliferate while retaining their characteristic functional and protease phenotypes. Blood 2001;97:2045–2052. Hamada H, Terai M, Kimura H, Hirano K, Oana S, Niimi H: Increased expression of mast cell chymase in the lungs of patients with congenital heart disease associated with early pulmonary vascular disease. Am J Respir Crit Care Med 1999;160:1303–1308. Hara M, Matsumori A, Ono K, Kido H, Hwang MW, Miyamoto T, Iwasaki A, Okada M, Nakatani K, Sasayama S: Mast cell cause apoptosis of cardiomyocytes and proliferation of other intramyocardial cells in vitro. Circulation 1999;100:1443–1449.

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Secor VH, Secor WE, Gutekunst CA, Brown MA: Mast cells are essential for early onset and severe disease in a murine model of multiple sclerosis. J Exp Med 2000;191:813–822. Platt MS, Yunginger JW, Sekula-Perlman A, Irani AM, Smialek J, Mirchandani HG, Schwartz LB: Involvement of mast cells in sudden infant death syndrome. J Allergy Clin Immunol 1994;94: 250–256. Holgate ST, Walters C, Walls AF, Lawrence S, Shell DJ, Variend S, Fleming PJ, Berry PJ, Gilbert RE, Robinson C: The anaphylaxis hypothesis of sudden infant death syndrome: Mast cell degranulation in cot death revealed concentrations of tryptase in serum. Clin Exp Allergy 1994;24: 1115–1122. Nakahata T, Toru H: Cytokines regulate development of human mast cells from hematopoietic progenitors. Int J Hematol 2002;75:350–356. Tsai M, Tam SY, Wedemeyer J, Galli SJ: Mast cells derived from embryonic stem cells: A model system for studying the effects of genetic manipulations on mast cell development, phenotype, and function in vitro and in vivo. Int J Hematol 2002;75:345–349.

Kenichi Koike, MD, PhD, Prof. Med. Department of Pediatrics, Shinshu University School of Medicine 3-1-1, Asahi, Matsumoto 390–8621 (Japan) Tel. ⫹81 0263 372642, Fax ⫹81 0263 373089 E-Mail [email protected]

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Saito H, Okayama Y (eds): Mast Cells in Allergic Diseases. Chem Immunol Allergy. Basel, Karger, 2005, vol 87, pp 22–31

Regulation of Mast Cell Activation through Fc␧RI Sho Yamasaki, Takashi Saito Laboratory for Cell Signaling, RIKEN Research Center for Allergy and Immunology, Yokohama, Kanagawa, Japan

Abstract The cross-linking of Fc␧RI on mast cells by IgE and antigen (Ag) initiates activation cascades that lead to allergic responses. Fc␧RI is composed of an ␣ and a ␤ monomer, and a ␥ homodimer, and the ␤ and ␥ chains possess immunoreceptor tyrosine-based activation motifs (ITAMs). Through the phosphorylation of ITAMs, activation signals are transmitted intracellularly. Mast cells are also activated through Fc␥R-FcR␥ by an immune complex, which often results in hypersensitivity. We defined FcR␥-signal-dependent and -independent mast cell responses by analyzing FcR␥⫺/⫺ mice reconstituted with mutant FcR␥-ITAM. Most of the Fc␧RI-mediated activations by IgE(⫹Ag), such as induction of degranulation, arachidonic acid metabolism, cytokine production and systemic anaphylaxis, are dependent on signaling through FcR␥-ITAM. On the other hand, IgE without Ag induces the upregulation of surface Fc␧RI expression and mast cell survival. The former is independent of and the latter is dependent on FcR␥-ITAM. As a molecular mechanism for the generation of diverse responses through Fc␧RI, we found that the quantity and the duration of the FcR␥ signal determine the degranulation and the survival of mast cells, respectively. Furthermore, such a sustained FcR␥-signal-induced survival is mediated by autocrine cytokine production. In this review, the in vivo function of FcR␥ and the signal regulation for the distinct responses of mast cells through Fc␧RI are discussed. Copyright © 2005 S. Karger AG, Basel

Structure and Function of FcR on Mast Cells

Antigen (Ag)-specific recognition and responses in the immune system are mediated by T-cell receptor (TCR), B-cell receptor (BCR) and Fc receptor (FcR). Whereas TCR and BCR exhibit their diversity for recognizing a variety of Ags through rearrangement of their variable gene segments, FcR mediates Ag-specific recognition through binding with Ag-specific antibody.

The Ag-recognition signal through these receptors is transmitted into intracellular signaling molecules through signaling subunits of the receptor complex that contains the immunoreceptor tyrosine-based activation motif (ITAM) within its cytoplasmic region. The activation signals through FcR are also mediated through the ITAM-bearing ␥ chain of FcR (FcR␥), similar to the Ig␣ and ␤ chains of BCR and the CD3 chain of TCR. Furthermore, similar to BCR or TCR, the cross-linking of FcR by Ab plus multivalent Ag or aggregated Abs induces the sequential activation of Src- and Syk-family protein tyrosine kinases, which is essential for the downstream cascades of cell activation [1]. The precise molecular mechanism of FcR signaling has been extensively reviewed [2–5]. The high-affinity IgE receptor, Fc␧RI, is predominantly expressed on mast cells and basophils. Fc␧RI exists as a tetramer composed of an ␣ and a ␤ monomer, and a ␥ homodimer, on these cells in rodent. The ␤ and ␥ chains each contain an ITAM within their cytoplasmic domains. Fc␧RI can bind to IgE in the absence of Ag with high-affinity (affinity constant ⫽ 109–1010 M⫺1) [6] compared to Fc␥R for IgG binding (affinity constant ⫽ 108 M⫺1) [7]. This step has been considered to be a form of passive ‘sensitization’ prior to subsequent Ag cross-linking. However, several studies have demonstrated that IgE alone can actively promote the upregulation of cell surface Fc␧RI expression, cytokine production, and mast cell survival [8–10]. Mast cells also express FcR for IgG, which is a trimer composed of Fc␥RIII (CD16) and an FcR␥ dimer. Several reports have shown that mast cell function is also regulated through Fc␥R by IgG, mainly through the immune complex (IC) [4, 11]. The triggering of mast cells either through Fc␧RI by IgE or through Fc␥RIII by IgG induces the phosphorylation of FcR␥ which, in turn, initiates further activation signals.

Function of FcR␥ in Mast Cell Activation

Hypersensitivity Mediated through FcgR on Mast Cells That Fc␥R on macrophages and NK cells mediates Ab-mediated cytotoxicity and inflammation has been analyzed extensively [4, 11]. In the analysis of the induction mechanism of the Arthus reaction, an IC-induced hypersensitivity, Sylvestre and Ravetch [12] first demonstrated that Fc␥RIII is responsible for the induction of the Arthus reaction, and mast cells are, surprisingly, responsible for the induction of the Arthus response. On the other hand, in the analyses of skin vasculitis induced by a monoclonal Ab (mAb) spontaneously derived from the autoimmune-prone mice, MRL-lpr/lpr, which has the characteristics of a cryoglobulin and a rheumatoid factor against IgG2a [13], we also found that Fc␥R on mast cells is responsible for the induction of skin vasculitis [14].

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The administration of this mAb (IgG3) induced skin vasculitis in wildtype (WT) mice but not in FcR␥-KO mice. The failure of the induction of skin vasculitis in the FcR␥-KO mice was not due to the lack of formation of IC precipitate as the IC between IgG3 and self IgG2a was similarly precipitated in the WT and KO mice. The in vivo transfer of various FcR⫹ cells into the mAb-treated FcR␥-KO mice that had IC precipitation in skin blood vessels clearly identified that mast cells and not macrophages are responsible for inducing skin vasculitis. Moreover, as the FcR␥-KO mice lacked the surface expression of both Fc␧RI and Fc␥RIII, we identified Fc␥RIII to be the responsible receptor and TNF␣ to be the major mediator for the induction of IC-induced skin vasculitis by means of the transfer experiments. These results demonstrate that the activation of mast cells through Fc␥R induces hypersensitivity via the production of inflammatory mediators. Furthermore, these observations may imply that not only are mast cells passively activated by soluble Ab and Ag, they are also actively surveying ICs in vivo, probably in order to clear them. The in vivo type I hypersensitivity reaction mediated through Fc␥R has been demonstrated in IgE-deficient mice [15]. FcRg-ITAM-Dependent and -Independent Mast Cell Response through Fc␧RI In vitro analyses have demonstrated that the FcR␥ chain is critical for mediating most of the activations by IgE⫹Ag, whereas the Fc␧RI␤ chain plays a role in augmenting mast cell activation. We analyzed the function of FcR␥ITAM-mediated signals in vivo by creating transgenic (Tg) mice expressing fulllength and mutant FcR␥ (either cytoplasmic deletion or ITAM mutation) with the FcR␥-KO background. Analysis of bone-marrow-derived mast cells (BMMCs) from these Tg mice revealed the differential requirement of FcR␥ITAM for various mast cell functions. The surface expression of Fc␧RI depends on the presence of FcR␥ but is independent of FcR␥-ITAM. Mast cell activations, such as the induction of degranulation, arachidonic acid metabolism, and cytokine production, are all dependent on signaling through FcR␥-ITAM. In other words, Fc␧RI␤-ITAM is not sufficient for mast cell activation because FcR␥-ITAM mutation completely abrogated the activation of these pathways. Furthermore, in vivo anaphylaxis, which resulted from this activation upon the administration of IgE plus Ag, is induced in only mast cells with full-length FcR␥. The stimulation of mast cells with IgE alone (IgE(⫺Ag)), which has been previously referred to as passive sensitization, induces the upregulation of surface Fc␧RI expression, cytokine production and mast cell survival [9, 10]. In contrast, the stimulation with IgE⫹Ag induces degranulation but not mast cell survival. Although these distinct responses are mediated through the same receptor, Fc␧RI, as BMMC from Fc␧RI␣-KO and FcR␥-KO mice did not show any responses upon

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Degranulation Cytokine production PG metabolism Systemic anaphylaxis Survival Cytokine production

⫹ ⫹ ⫹ ⫹ ⫹ ⫹

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Fig. 1. FcR␥-ITAM-dependent and -independent mast cell responses induced by IgE. Transgenic mice expressing full-length (WT) and mutant (cytoplasmic deletion, ⌬CT, and ITAM mutation, YF) FcR␥ were crossed with FcR␥-KO (⫺/⫺) mice. BMMC from these mice were stimulated with IgE in the presence or absence of Ag (DNP-OVA). Differential requirement of FcR␥-ITAM was clearly observed. Only cell surface expression and IgE-induced upregulation of Fc␧RI are independent of FcR␥ signaling, whereas others, including IgE(⫺Ag)-induced survival, are dependent on FcR␥-ITAM signaling.

stimulation with IgE(⫺Ag), the molecular mechanism for the divergence remains unknown. An analysis of BMMC from FcR␥-ITAM mutant Tg mice demonstrated that the IgE(⫺Ag)-induced upregulation of surface Fc␧RI expression is independent of FcR␥-ITAM. On the other hand, the IgE(⫺Ag)-induced mast cell survival and cytokine production were found to be dependent on FcR␥-ITAM [16]. Thus, our analysis has clarified FcR␥-ITAM-dependent and -independent mast cell responses upon stimulation through Fc␧RI, as summarized in figure 1 [16]. Furthermore, the distinct responses of degranulation vs. mast cell survival upon stimulation with either IgE(⫹Ag) or IgE(⫺Ag) are mediated through the single signal motif, FcR␥-ITAM, within the same receptor, Fc␧RI, in the same cellular context.

Regulation of Mast Cell Survival vs. Degranulation through Fc␧RI

How does the same motif of FcR␥ within the Fc␧RI complex mediate different responses such as degranulation vs. mast cell survival upon stimulation

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with IgE in the presence or absence of Ag? One can postulate that IgE⫹Ag induces extensive cross-linking of the receptors, thereby resulting in the increased phosphorylation of FcR␥-ITAM and further downstream signals. To determine whether the cellular output is regulated by the quantity of the FcR␥ signal, we expressed CD8/FcR␥ chimera (CD8/␥) in BMMC from FcR␥⫺/⫺ mice and manipulated the strength of the FcR␥ signal by anti-CD8 cross-linking [17]. The cross-linking of CD8/␥ was sufficient for the induction of mast cell survival and degranulation. Mast cell survival was induced by stimulation that was weaker than that needed for degranulation in terms of anti-CD8 concentration and the valency of the chimera. CD8/␥ is expressed as a dimer with a disulfide bond between the extracellular domains of CD8. When the critical cysteine residues were mutated to prevent dimerization, degranulation but not mast cell survival was markedly impaired upon cross-linking with the same concentration of anti-CD8. A recent report has also indicated that weak to moderate stimulation favors mast cell survival, whereas a strong signal is required for degranulation [18]. However, sustained Erk activation seems to regulate mast cell survival even when the activation signal is sufficiently strong to elicit degranulation. In other cell types, the activation of Erk is known to induce cell survival [19, 20], and in our system, both high and low concentrations of anti-CD8 induced Erk activation, which is well correlated with mast cell survival induction. The generation of sustained Erk activation by active MEK induced BMMC survival. These results suggest that the duration and the quantity of the FcR␥ signal determine mast cell survival and degranulation, respectively (fig. 2). How can IgE trigger signals in the absence of Ag? We initially hypothesized that IgE binding may induce the association with lipid raft, as observed in the initial step of IgE(⫹Ag) stimulation [21]. However, raft-targeted FcR␥ did not induce mast cell survival at all, suggesting that cross-linking of the receptor may be still required for the survival induction even within lipid raft [17]. It is known that the IgE(⫺Ag)-induced effect is largely dependent on the individual IgE clone, which also supports that the cross-linking of Fc␧RI is caused by the aggregation between IgE itself and/or the assembly with unknown molecules [22]. This seems to be mediated by the V region of IgE, at least in the case of anti-hapten (DNP) IgE, because monomeric hapten blocks several IgE(⫺Ag)-induced effects [18]. Some IgE clones such as SPE-7 can induce degranulation as well as mast cell survival in the absence of Ag. Although the quantity and the duration of stimulation appear to be mutually exclusive because strong cross-linking is accompanied by the rapid internalization of the receptor, it is possible to suppose an intermediate situation that induces both degranulation and mast cell survival. Indeed, SPE-7 has been reported to induce the substantial internalization of Fc␧RI in the absence of Ag, suggesting that the signal is sufficiently strong to evoke degranulation. These individualities

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Sustained

Sustained

Fig. 2. Quantity and duration of FcR␥ signal determine mast cell responses. Application of strong and transient input signal, such as IgE(⫹Ag) or anti-CD8⫹secondary Ab, on CD8/␥ induced only degranulation. On the other hand, weak and sustained signal delivered by IgE(⫺Ag) or a low concentration of anti-CD8 induced only mast cell survival. These two parameters, quantity and duration, are mutually exclusive in a normal situation, because strong cross-linking is accompanied by the rapid receptor internalization. Strong and sustained signal triggered by IgE⫹immobilized Ag or a high concentration of anti-CD8 may induce both degranulation and mast cell survival simultaneously.

may be explained by applying the general stimulation scheme in figure 2 to each IgE clone (fig. 3). One IgE clone, anti-dansyl (clone 27–74), induced neither mast cell survival nor degranulation [unpubl. observation], possibly due to its weak cross-linking ability (fig. 3, left panel). On the other hand, another IgE that induced moderate cross-linking transduced a sustained signal and induced mast cell survival (fig. 3, middle panel). One possible explanation for the strong cross-linking ability of SPE-7 is the existence of two distinct conformations that enable IgE to bind to two distinct Ags [23]. Further precise molecular characterization of polyclonal IgE in allergic patients will promote our understanding of the physiological significance of the Ag-independent response of IgE and its heterogeneity on the progression of allergic disease. Although the duration of the FcR␥ signal and the sustained Erk activation are critical to the IgE-induced mast cell survival, the downstream mechanism is still controversial [8]. Kalesnikoff et al. [24] have suggested that the IgE(⫺Ag)mediated survival is mediated, at least in part, by a soluble factor. We found that IgE(⫺Ag) induces rapid and abundant IL-3 production compared to that by IgE(⫹Ag) stimulation. Further, the IgE-induced survival was severely impaired by anti-IL-3 and in IL-3⫺/⫺ BMMC, indicating that IL-3 is the major factor responsible for the IgE-induced survival [25]. Accordingly, the IgE(⫺Ag)induced upregulation of Bcl-xL was abolished in IL-3⫺/⫺ BMMC.

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Stimulation Cross-linking capacity IgE clone

IgE (⫺Ag) Weak 27–74

Medium H1-DNP-␧-206

IgE (⫹Ag) Strong SPE-7

Quantity

Degranulation

Survival

Stronger Any Degranulation

Survival

Duration

Duration

Duration

Receptor internalization:

⫺

⫺

⫹

⫹⫹⫹

Output:

⫺

Survival

Degranulation survival

Degranulation

Fig. 3. Model for various effects of individual IgE clones. One IgE clone, anti-dansyl (27–74), induces neither mast cell survival nor degranulation, possibly due to its weak crosslinking ability (left). Another IgE that induces moderate cross-linking can transduce a sustained signal and induce mast cell survival (middle). Certain IgE clones, such as anti-DNP IgE (SPE-7), possess self-aggregating activity and are sufficiently strong to induce degranulation. However, because the internalization rate is lower than the Ag cross-linking rate, the signal duration becomes sufficiently long to induce mast cell survival simultaneously.

The IgE-induced mast cell survival has not been clearly verified in vivo [8]. The presence of a normal number of mucosal mast cells in Fc␧RI␣⫺/⫺ mice and FcR␥⫺/⫺ mice [unpubl. observation] indicates that this mechanism may not be essential for the normal generation and homeostasis of mast cells [26]. However, Kitaura et al. [22] have reported that the implantation of IgE-producing hybridoma cells in the mouse peritoneal cavity has led to the slightly increased survival of mucosal mast cells. Furthermore, recent work has revealed that IgE(⫺Ag) plays a significant role in immune responses in vivo, possibly through chemokine/ cytokine production from mast cells [27]. In addition, as helminth or nematode infection is known to induce mastocytosis [28], the response to parasite infection may reflect a more physiological function of the IgE-mediated mast cell survival. As parasite infection also promotes a rapid increase in the expression of non-specific IgE in addition to that of parasite-specific IgE [29], such IgE may induce mast cell survival in the absence of Ag in specific tissues. Although the role of IgE in parasite-induced mastocytosis is controversial [30–32], splenic mastocytosis induced by the primary infection of Trichinella spiralis is greatly impaired in IgE⫺/⫺ mice [33], whereas mucosal mastocytosis is impaired in IL-3⫺/⫺ mice during Stronglyoides venezuelensis infection [34]. Together, these observations imply that the IgE-induced autocrine production of IL-3 from mast

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Fc␧Rl

IgE ⫹ Ag Ag

Strong cross-link YY YY IgE

Strong & transient

Transient Erk Ca2⫹

YY Y

IgE Y YY YY Weak cross-link

Weak & sustained

Sustained Erk BcI-xL

Degranulation

IL-3

Survival

Fig. 4. Distinct signaling mechanism and cellular outputs through IgE(⫹Ag) vs. IgE(⫺Ag). IgE(⫹Ag) induces the strong cross-linking of Fc␧RI, which is accompanied by receptor internalization. Thus, the signal becomes strong and transient. The strong signal determines degranulation (left panel). On the other hand, IgE(⫺Ag) can induce the weak cross-linking of Fc␧RI. As the weak cross-linking induced by IgE(⫺Ag) does not induce receptor internalization, the receptor signal becomes weak and sustained. The sustained Erk activation is critical for IL-3 transcription and autocrine production, which lead to the upregulation of Bcl-xL and survival (right).

cells may support mastocytosis induced by parasite infection, although its importance may differ depending on the type of parasite. In conclusion, ligand differences may be interpreted by Fc␧RI as quantitative and/or durational differences, and may be converted into qualitatively distinct output through respective signaling pathways (fig. 4). Acknowledgments We would like to thank many collaborators who kindly provided reagents, mice and valuable information, and T. Kawakami for valuable discussion. We also thank all members of our laboratory, particularly D. Sakurai, S.Y. Park, M. Kohno and E. Ishikawa, for helpful discussion and support; and H. Yamaguchi for secretarial assistance. This work was supported by grants from the Ministry of Education, Culture, Sports, Science and Technology, Japan.

References 1 2

Daeron M: Fc receptor biology. Annu Rev Immunol 1997;15:203–234. Kinet JP: The high-affinity IgE receptor (Fc␧RI): From physiology to pathology. Annu Rev Immunol 1999;17:931–972.

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3 4 5 6 7 8 9 10 11 12 13

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19 20 21 22 23 24 25 26 27 28 29

Turner H, Kinet JP: Signalling through the high-affinity IgE receptor Fc␧RI. Nature 1999;402: B24–B30. Ravetch JV, Bolland S: IgG Fc receptors. Annu Rev Immunol 2001;19:275–290. Siraganian RP: Mast cell signal transduction from the high-affinity IgE receptor. Curr Opin Immunol 2003;15:639–646. Kulczycki A Jr, Metzger H: The interaction of IgE with rat basophilic leukemia cells. II. Quantitative aspects of the binding reaction. J Exp Med 1974;140:1676–1695. Unkeless JC, Eisen HN: Binding of monomeric immunoglobulins to Fc receptors of mouse macrophages. J Exp Med 1975;142:1520–1533. Kawakami T, Galli SJ: Regulation of mast-cell and basophil function and survival by IgE. Nat Rev Immunol 2002;2:773–786. Asai K, Kitaura J, Kawakami Y: Regulation of mast cell survival by IgE. Immunity 2001;14:791–800. Kalesnikoff J, Huber M, Lam V: Monomeric IgE stimulates signaling pathways in mast cells that lead to cytokine production and cell survival. Immunity 2001;14:801–811. Hogarth PM: Fc receptors are major mediators of antibody based inflammation in autoimmunity. Curr Opin Immunol 2002;14:798–802. Sylvestre DL, Ravetch JV: Fc receptors initiate the Arthus reaction: Redefining the inflammatory cascade. Science 1994;265:1095–1098. Reininger L, Berney T, Shibata T: Cryoglobulinemia induced by a murine IgG3 rheumatoid factor: Skin vasculitis and glomerulonephritis arise from distinct pathogenic mechanisms. Proc Natl Acad Sci USA 1990;87:10038–10042. Watanabe N, Akikusa B, Park SY: Mast cells induce autoantibody-mediated vasculitis syndrome through tumor necrosis factor production upon triggering Fc␥ receptors. Blood 1999;94:3855–3863. Oettgen HC, Martin TR, Wynshaw-Boris A: Active anaphylaxis in IgE-deficient mice. Nature 1994;370:367–370. Sakurai D, Yamasaki S, Arase K: Fc␧RI␥-ITAM is differentially required for mast cell function in vivo. J Immunol 2004;172:2374–2381. Yamasaki S, Ishikawa E, Kohno M: The quantity and duration of FcR␥ signals determine mast cell degranulation and survival. Blood 2004;103:3093–3101. Kitaura J, Xiao W, Maeda-Yamamoto M: Early divergence of Fc␧ receptor I signals for receptor up-regulation and internalization from degranulation, cytokine production, and survival. J Immunol 2004;173:4317–4323. Marshall CJ: Specificity of receptor tyrosine kinase signaling: Transient versus sustained extracellular signal-regulated kinase activation. Cell 1995;80:179–185. Werlen G, Hausmann B, Naeher D: Signaling life and death in the thymus: Timing is everything. Science 2003;299:1859–1863. Holowka D, Baird B: Fc(␧)RI as a paradigm for a lipid raft-dependent receptor in hematopoietic cells. Semin Immunol 2001;13:99–105. Kitaura J, Song J, Tsai M: Evidence that IgE molecules mediate a spectrum of effects on mast cell survival and activation via aggregation of the Fc␧RI. Proc Natl Acad Sci USA 2003;100:12911–12916. James LC, Roversi P, Tawfik DS: Antibody multispecificity mediated by conformational diversity. Science 2003;299:1362–1367. Kalesnikoff J, Lam V, Krystal G: SHIP represses mast cell activation and reveals that IgE alone triggers signaling pathways which enhance normal mast cell survival. Mol Immunol 2002;38:1201. Kohno M, Yamasaki S, Tybulewicz VL: Rapid and large amount of autocrine IL-3 production is responsible for mast cell survival by IgE in the absence of antigen. Blood 2005;105:2059–2065. Dombrowicz D, Flamand V, Brigman KK: Abolition of anaphylaxis by targeted disruption of the high affinity immunoglobulin E receptor ␣-chain gene. Cell 1993;75:969–976. Bryce PJ, Miller ML, Miyajima I: Immune sensitization in the skin is enhanced by antigenindependent effects of IgE. Immunity 2004;20:381–392. Nawa Y, Ishikawa N, Tsuchiya K: Selective effector mechanisms for the expulsion of intestinal helminths. Parasite Immunol 1994;16:333–338. Ehigiator HN, Stadnyk AW, Lee TD: Extract of Nippostrongylus brasiliensis stimulates polyclonal type-2 immunoglobulin response by inducing de novo class switch. Infect Immun 2000;68: 4913–4922.

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Perrudet-Badoux A, Binaghi RA, Boussac-Aron Y: Trichinella spiralis infection in mice. Mechanism of the resistance in animals genetically selected for high and low antibody production. Immunology 1978;35:519–522. Watanabe N, Katakura K, Kobayashi A: Protective immunity and eosinophilia in IgE-deficient SJA/9 mice infected with Nippostrongylus brasiliensis and Trichinella spiralis. Proc Natl Acad Sci USA 1988;85:4460–4462. Onah DN, Uchiyama F, Nagakui Y: Mucosal defense against gastrointestinal nematodes: Responses of mucosal mast cells and mouse mast cell protease-1 during primary Strongyloides venezuelensis infection in FcR␥-knockout mice. Infect Immun 2000;68:4968–4971. Gurish MF, Bryce PJ, Tao H: IgE enhances parasite clearance and regulates mast cell responses in mice infected with Trichinella spiralis. J Immunol 2004;172:1139–1145. Lantz CS, Boesiger J, Song CH: Role for interleukin-3 in mast-cell and basophil development and in immunity to parasites. Nature 1998;392:90–93.

Takashi Saito Laboratory for Cell Signaling RIKEN Research Center for Allergy and Immunology 1-7-22 Suehiro-cho, Tsurumi-ku Yokohama, Kanagawa 230–0045 (Japan) Tel. ⫹81 45 503 7038, Fax ⫹81 45 503 7036, E-Mail [email protected]

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Saito H, Okayama Y (eds): Mast Cells in Allergic Diseases. Chem Immunol Allergy. Basel, Karger, 2005, vol 87, pp 32–42

Role of Oxidants in Mast Cell Activation Yoshihiro Suzuki, Tetsuro Yoshimaru, Toshio Inoue, Osamu Niide, Chisei Ra Division of Molecular Cell Immunology and Allergology, Nihon University Graduate School of Medical Sciences, Tokyo, Japan

Abstract Reactive oxygen species (ROS), such as superoxide, hydrogen peroxide (H2O2), and hydroxyl radical, have for a long time been considered as accidental by-products of respiratory energy production in mitochondria and as being useless and rather deleterious to biological systems. Contrary to such a classical view, accumulating evidence indicates that upon stimulation of divergent receptor systems, ROS are intentionally produced and even required for appropriate signal transduction and biological responses. Work by our group and that of others have shown that stimulation of mast cells through the high-affinity IgE receptor (Fc␧RI) induces the production of ROS such as superoxide and H2O2 possibly by the phagocyte NADPH oxidase homologue and that these endogenously produced oxidants have important functions in regulation of various mast cell responses, including degranulation, leukotriene secretion, and cytokine production. Subsequent studies have defined particular biochemical pathways that can be targeted by ROS and/or cellular redox balance. More recent research reveals that ROS may also play an important role in mast cell activation by divergent allergy-relevant environmental substances, for instance heavy metals and polycyclic aromatic hydrocarbons. This review summarizes current knowledge on the role of endogenous oxidants in mast cell activation. Copyright © 2005 S. Karger AG, Basel

ROS and Regulation of the Cellular Redox Balance

It is well known that respiratory energy production in mitochondria has a considerable advantage in generating energy compared to glycolysis. As the terminal electron acceptor for oxidative phosphorylation, molecular oxygen (O2) plays an essential role in various processes. This type of oxygen use however results in the production of a variety of reactive oxygen species (ROS) such as

superoxide (O2⫺), hydrogen peroxide (H2O2) and hydroxyl radical (⭈OH) as accidental by-products [1]. ROS readily react with cellular macromolecules, either damaging them directly, or setting in motion a chain reaction in which free radical is passed from one molecule to another, resulting in extensive damage of cellular structures such as the membrane. In addition to the respiratory energy production, various intra- and extracellular systems are involved in the production of ROS (collectively referred to as the prooxidant system). For instance, ultraviolet and ionizing radiation like ␥-rays are capable of producing ROS such as H2O2 and ⭈OH. Phagocytes including neutrophils and macrophages produce large amounts of ROS, O2⫺ and H2O2, via activation of the phagocyte NADPH oxidase in response to microbial infection and soluble stimuli. The catalytic moiety of the phagocyte NADPH oxidase is the membrane-associated flavocytochrome gp91phox and activation of the gp91phox system occurs through assembly of the cytosolic regulatory proteins, p40phox, p47phox, and p67phox, and small GTP binding protein Rac [for a review, see 2]. This respiratory burst however may force other types of cells to become exposed to considerable levels of ROS. Thus, ROS have been considered as being useless and rather deleterious to biological systems. To overcome these ultimately toxic oxygen species, early forms of life needed to develop simultaneously an effective defensive system that could cope with unwanted ROS [3]. This system, called the antioxidant system, contains molecules capable of either scavenging and/or detoxifying ROS, blocking free radical chain reactions, or removing transition metals which can serve as a ready source of free electrons. Consequently, an aerobic existence is accompanied by a persistent state of oxidative siege, where the survival of a given cell is determined by the balance between ROS and antioxidants [4] (fig. 1). It is not surprising therefore that oxidative stress (a term which refers to the imbalance between the occurrence of oxidants and antioxidants) causes cell dysfunction and death.

Generation of ROS in Non-Phagocytic Cells through the NOX/DUOX Family

The concept of oxidative stress defines a dynamic situation in which the balance between the occurrence of oxidants and antioxidants in biological systems can indirectly influence cellular functions and phenotypes through an effect on other cellular components, particularly redox-sensitive functional groups and proteins. However, the term ‘stress’ often carries a rather negative nuance. In terms of a positive role for ‘oxidative stress’, it is proposed more accurately to use a term such as ‘oxidant-mediated regulation’. In relation to

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H2O2

O⫺ 2 H2O2 HOCI

NADPH oxidase Phagocytic cells

Ligand TCR BCR PDGFR TNFR

⫺ NOX/DUOX O2

Activation

Radiation (UV, ␥-rays)

O2⫺ Non-phagocytic cells ROS Nucleus

Mitochondria

NF-␬B, AP-1 Antioxidants (SOD, catalase, GSH, etc.)

Gene expression

Fig. 1. ROS and regulation of the cellular redox balance. Cells maintain suitable redox equilibrium by balancing the levels of oxidants and antioxidants such as superoxide dismutase (SOD), catalase, and glutathione (GSH). Activation through divergent receptors on the cell surface including T-cell receptor (TCR), B-cell antigen receptor (BCR), platelet-derived growth factor receptor (PDGFR), and tumor necrosis factor-␣ receptor (TNFR) may evoke the generation of ROS by increasing mitochondrial respiration and/or by activating ROS-generating system such as NOX/DUOX family to produce O2⫺. Phagocytes including neutrophils produce large amounts of ROS, including O2⫺, H2O2, and HOCl in response to microbial infections and soluble stimuli, which may force other types of cells to become exposed to considerable levels of ROS. Increases in the intracellular levels of ROS may regulate gene expression through the modulation of redox-sensitive transcription factors such as NF-␬B and AP-1.

this concept, accumulating evidence indicates that ROS act as signal intermediates in intracellular signaling to activation of mitogen-activated protein kinase (MAPK) family members, gene expression, and/or cell proliferation [5–9]. In the last decade it has been revealed that non-phagocytic cells like epithelial cells, smooth muscle cells, and endothelial cells also generate O2⫺. Our group and others have demonstrated that upon cell activation lymphocytes generate ROS, primarily O2⫺, and that ROS and/or an altered redox balance may regulate various cell functions including adhesion, proliferation, and apoptosis [6, 10–15]. Although production of ROS was frequently attributed to mitochondrial respiration, in some cases, use of inhibitors suggested that the actual source might be a flavoprotein that is similar to gp91phox. Subsequent studies revealed the occurrence of a new family of homologues of gp91phox, the

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NADPH oxidase (NOX)/dual oxidase (DUOX) family in non-phagocytic cells. The NOX/DUOX family now includes NOX1 (initially referred to as Mox1, NOH-1 [9, 16]), which is predominantly expressed in colon, NOX3 (gp91–3 [17]) cloned from fetal kidney, NOX4 (Renex [18]) found in kidney cortex, NOX5 found predominantly in testis, spleen, and lymph nodes. The NOX family members have almost the same length as gp91phox (⬃560–580 amino acids) [19] (gp91phox is also referred to as NOX2). The DUOX family members are longer (⬃1,550 amino acids) because of their N-terminal extensions consisting of two EF hands (presumed binding) motifs, an additional transmembrane helix, and a peroxidase homology domain. The DUOX family includes DUOX1 (initially termed Thox1) and DUOX2 (Thox2) [20, 21]. The NOX enzymes have been proposed to function in generating ROS as mediators of signal transduction relating to growth, angiogenesis, and apoptosis [for a review, see 22].

Role of ROS in Mast Cell Activation

Fc␧RI Signaling in Mast Cells Mast cells play a critical role in allergic reactions. Mast cells express the high-affinity IgE receptor (Fc␧RI) on the cell surface and aggregation of Fc␧RI by IgE-antigen complexes initiates a cascade of intracellular signaling events that lead to degranulation, inflammatory mediator release, and cytokine production, contributing to allergic and inflammatory reactions [23, 24]. Fc␧RI is a tetramer of ␣-, ␤-, and ␥-chain homodimers [25], of which the ␣-chain binds IgE, while the ␤- and ␥-chain mediate intracellular signaling through the receptor. Like divergent receptors found in lymphocytes, Fc␧RI lacks intrinsic enzyme activity but the ␤- and ␥-chains contain the immunoreceptor tyrosinebased activation motif (ITAM), which is critical for cell activation through these receptors [26, 27]. The ␤-chain ITAM is recognized as an important site of interaction with Lyn for signal transduction. It is now clear that the ␤-chain acts as a signal amplifier in mast cells and is important for augmenting allergic reactions [28–31]. In humans, it has been demonstrated that the ␥-chain can act as an autonomous signaling molecule, whereas the ␤-chain can amplify early activation signals through the ␥-chain by more than 5-fold [28]. The ITAM consensus sequence contains tyrosine residues, which are phosphoreceptor sites for receptor-associated tyrosine kinases and once phosphorylated, serve as a docking site for cytoplasmic proteins that contain the Src-homology-2 (SH2) domain. It is believed that aggregation of Fc␧RI causes tyrosine phosphorylation of the ␤-chain ITAM by activated Lyn, and subsequent phosphorylation of Syk and the ␥-chain ITAM, which in turn leads to downstream signals including Ca2⫹ mobilization and activation of protein kinase C and MAPK family

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members [27, 32]. The signal transduction pathway in mast cells has been extensively reviewed elsewhere and will not be further addressed here. Generation of ROS in Mast Cells and Basophils Earlier studies demonstrated that rat peritoneal mast cells generated a substantial level of ROS. Both pharmacological agents (mercury and gold salts, compound 48/80, Ca2⫹ ionophores) and physiologically relevant stimuli (antigen, nerve growth factor, substance-P) stimulated the generation of intracellular ROS, although the nature of the oxidant generated was not defined [33, 34]. However, some investigators claimed that the production of ROS observed in rat peritoneal mast cells was attributed to contaminating macrophages rather than to mast cells by themselves [35]. Thus, there are conflicting data regarding whether mast cells can actually generate ROS. We have succeeded in detecting the generation of ROS in RBL-2H3 cells (rat mast cell line) and bone marrowderived cultured mast cells [36–38]. The production of ROS is sensitive to diphenyleneiodonium, which inhibits the phagocyte NADPH oxidase and other flavoproteins, suggesting that a homologue of NADPH oxidase, possibly a NOX/DUOX family member, is the actual source of oxidants. Our previous works indicated that mast cells can produce O2⫺ and H2O2 in response to the elevation of cytosolic Ca2⫹ concentration ([Ca2⫹]i). Because NOX5 and presumably DUOX1/DUOX2 can be activated in response to the rise in [Ca2⫹]i, these enzymes might be responsible for the generation of ROS in mast cells. We detected the generation of ROS especially O2⫺ in human leukocytes, predominantly basophils, which were stimulated through Fc␧RI. Similar generation of ROS was observed when leukocytes from patients sensitized with mite allergens were challenged with the relevant allergen but not irrelevant allergen [37]. This indicates that the oxidative burst at least of basophils is allergenspecific, suggesting its relevance to allergic reactions. The fact that divergent environmental substances relevant to allergy can induce and/or enhance the generation of ROS may support the view. Mercuric chloride is well known to cause type I and IV allergic reactions. At relatively high concentrations the compound can induce the generation of ROS in rat peritoneal mast cells and at lower concentrations it augments antigen-induced ROS generation [33]. We recently revealed that heavy metals silver and gold, both of which induce severe allergic inflammation in humans, can directly activate mast cells through a ROS-dependent signaling pathway [39]. Polycyclic aromatic hydrocarbons (PAHs) are major components of diesel exhaust particles found in respirable particles of air pollutants and these consumption products of fossil fuel exacerbate allergic inflammation. PAHs such as benzo(a)pyrene and benzo(a)pyrene quinones were found to enhance antigen-induced ROS production and mediator release in human basophils [40].

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Signal Transduction Pathway for ROS Generation Studies employing Lyn- or Syk-deficient mice have demonstrated a critical role of both tyrosine kinases in Fc␧RI-mediated mast cell activation [41, 42]. The Tec family kinases are another class of tyrosine kinases that are implicated in mast cell activation. Btk, Itk, and Tec are expressed in primary cultured mast cells and both Btk and Itk are also activated upon Fc␧RI engagement, suggesting their functional roles in mast cell activation [43]. To gain further insights into roles of these signaling components in the generation of ROS, we tested the effects of divergent pharmacological inhibitors of them on the generation of ROS. As a result, Src family kinase(s), Syk kinase, and the phosphoinositide3-kinase (PI-3K) may be necessary for the generation of ROS [38]. Lyn is a major Src family kinase in mast cells and binds to Fc␧RI␤ loosely even in resting cells and is activated immediately upon Fc␧RI engagement to bind to Fc␧RI␤ tightly and transduce downstream signals [44]. It is established that the ␤-chain ITAM is an important site of interaction with Lyn for signal transduction [27, 32], suggesting that the ␤-chain ITAM is also important for the induction of ROS production. To test this hypothesis, we generated BMMCs, in which all tyrosine residues of the ␤-chain ITAM are replaced by phenylalanine and compared the generation of ROS in BMMCs expressing the wild-type and the mutated type of the ␤-chain. The results revealed that the association between Lyn and Fc␧RI␤ via the ␤-chain ITAM is necessary for the PI-3K-dependent generation of ROS [unpubl. observations]. The Btk-selective inhibitor LFM A-13 indicated that Btk may also be involved in the generation of ROS in mast cells. Activation of PI-3K results in the production of phosphatidylinositol-3,4, 5-triphosphate (PIP3). The interaction of the pleckstrin homology (PH) domain with PIP3 then leads to targeting of Tec family kinases to specific membrane microdomains (lipid rafts), which is a critical step for their activation upon antigen receptor stimulation. For full activation of Tec family kinases, a second step, phosphorylation of a tyrosine residue within the activation loop of the kinase domain by Src family kinases, may also be required [for a review, see 45]. Because Src family kinases and PI-3K are required for the generation of ROS, and destruction of lipid rafts abolishes the generation of ROS [unpubl. observations], the translocation of Btk to lipid rafts and phosphorylation by Src family kinases might be a critical step in the signal transduction pathway for the generation of ROS. The putative signal transduction pathway is illustrated in figure 2.

Role of Oxidants in Fc␧RI Signaling and Allergy

Because the generation of ROS in mast cells is sensitive to diphenyleneiodonium, the reagent is useful to investigate especially the role of O2⫺ in mast cell

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Fc␧RI aggregation Fc␧RI␤ chain-Lyn binding (tight) Lyn activation Syk activation

PI-3K activation

NOX/DOUX activation?

PIP3 production Btk activation

ROS generation

LAT phosphorylation

Regulation?

Formation of Ca2+ signalsomes

Ca2+ mobilization

Fig. 2. Model for generation and roles of ROS in mast cells. Cross-linking of Fc␧RI on mast cells or basophils results in aggregation of the receptor and initiates a cascade of intracellular events inducing the rise in cytosolic Ca2⫹ concentration, which is required for degranulation, LT secretion, and cytokine production. Upon Fc␧RI aggregation, the association between Lyn and Fc␧RI␤ via the ␤-chain ITAM may become tight enough to transduce downstream signals such as recruitment and activation of Syk and PI-3K. Activation of PI-3K results in the production of PIP3, which in turn may lead to the recruitment of Tec family kinase (BtK) to specific membrane microdomains (lipid rafts) and activation of Btk. These events may collectively or separately lead to activation of the NOX/DUOX family, evoking the generation of multiple species of oxidants. Some of these oxidants may be required for the formation and/or maintaining of macromolecular complex (Ca2⫹ signalsomes), thereby regulating Ca2⫹ mobilization.

responses. Blockade of the generation of ROS could prevent Fc␧RI-mediated degranulation and LT secretion in BMMCs [38]. Basically the same results were obtained with RBL-2H3 mast cells and human leukocytes, predominantly basophils. Furthermore, blocking O2⫺ generation also suppressed mediator secretion in allergen-challenged cells from patients with atopic dermatitis [37]. These observations imply that O2⫺ plays a common facilitating role in mediator release under pathophysiological conditions. The SOD mimetic MnTBaP possesses both superoxide dismutase and peroxidase activity in vitro and inhibits T-cell receptor-mediated ROS generation [14] and is expected to abolish Fc␧RI-mediated ROS generation. MnTBaP also suppressed Fc␧RImediated mediator release at a comparable dose. The glutathione peroxidase mimetic ebselen was employed to examine the selective role of H2O2 in redox regulation of mast cell responses. Interestingly, the effects of ebselen were

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different from those of MnTBaP. In particular, ebselen had no significant inhibitory effect on degranulation, although it abolished cytokine production. Collectively, these observations suggest selective roles of O2⫺ and H2O2 in the regulation of distinct mast cell responses. The next question is how ROS regulate chemical mediator release (degranulation) in mast cells. The observations that blockade of ROS generation and scavenging ROS reduce divergent responses suggest the common targets of ROS in the signal transduction pathways of mast cells. As described above, one of the earliest steps upon activation of mast cells is increased tyrosine phosphorylation of several proteins. Therefore, we examined whether blocking ROS generation affected the tyrosine phosphorylation of cellular proteins. In the initial studies, some proteins including the focal adhesion kinase (FAK) pp125FAK, a molecule critical for intracellular signaling to degranulation, were identified as likely targets of ROS [36]. Subsequent studies demonstrated that these molecules lie in the downstream of the activation of Ca2⫹ influx. In addition, blockade of ROS generation as well as scavenging ROS also impaired Ca2⫹ mobilization [38]. Thus, endogenously produced ROS and exogenously added ROS commonly affect Ca2⫹ mobilization. These observations support the view that Ca2⫹ mobilization is a primary target of ROS and/or altered redox status. Recent work indicated that Fc␧RI stimulation leads to the formation of macromolecular complex analogous to Ca2⫹ signalsomes of T and B cells and contains some of the same components, including Syk, the linker for activation of T cells (LAT), Btk, Itk, SLP-76, Vav1, and PLC␥1. Assembly of this LATorientated complex is tyrosine phosphorylation-dependent and requires Syk and Lyn, and is essential for full activation of Ca2⫹ mobilization [32, 46]. In this respect it should be noticed that tyrosine phosphorylation of PLC␥-1/2 and LAT is abolished by inhibiting ROS generation or by scavenging ROS [38], which suggests the possibility that ROS are required for the formation of and/or maintaining the Ca2⫹ signalsomes (fig. 2).

Conclusions

Generation of ROS is a common signal transduction event in a variety of functional responses in mast cells. ROS play a critical role in the regulation of mast cell signaling including Ca2⫹ mobilization, although further studies are necessary for revealing the molecular mechanisms of ROS generation and of the regulation of different cellular responses. More recent research has indicated that environmental substances that induce and/or exacerbate allergic inflammation such as heavy metals and diesel exhaust particles also stimulate and/or enhance Fc␧RI-mediated ROS generation and oxidant-regulated mast

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cell activation. Thus, better understanding and specific targeting of the generation of ROS in mast cells might offer a promising new approach for the treatment of allergic disorders.

Acknowledgements The authors wish to thank Satoshi Nunomura (Division of Molecular Cell Immunology and Allergology, Nihon University Graduate School of Medical Sciences) for his collaboration and help. The authors thank National Institute of Health Sciences (Japan Collection of Research Biosources) for providing RBL-2H3 (cell No. JCRB0023). The authors thank the Ministry of Science, Sport, and Education of Japan and Nihon University for financial support.

References 1 2 3 4 5

6

7 8 9 10 11

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Halliwell B, Gutterridge JM: Role of free radicals and catalytic metal ions in human disease: An overview. Methods Enzymol 1990;186:1–85. Babior BM: The respiratory burst oxidase. Adv Enzymol 1992;65:49–88. McCord JM, Fridovitch I: Superoxide dismutase. An enzyme function for erythrocuprein (hemocuprein). J Biol Chem 1969;244:6049–6055. Davies KJ: Oxidative stress: The paradox of aerobic life. Biochem Soc Symp 1995;61:1–31. Ushio-Fukai M, Alexander RW, Akers M, Griendling KK: p38 mitogen-activated protein kinase is a critical component of the redox-sensitive signaling pathways activated by angiotensin II. Role in vascular smooth muscle cell hypertrophy. J Biol Chem 1998;273:15022–15029. Lee JR, Koretzky GA: Production of reactive oxygen intermediates following CD40 ligation correlates with c-Jun N-terminal kinase activation and IL-6 secretion in murine B lymphocytes. Eur J Immunol 1998;28:4188–4197. Sundaresan M, Yu ZX, Ferrans VJ, Irani K, Finkel: Requirement for generation of H2O2 for platelet-derived growth factor signal transduction. Science 1995;270:296–299. Burdon RH: Superoxide and hydrogen peroxide in relation to mammalian cell proliferation. Free Radic Biol Med 1995;18:775–794. Suh YA, Arnold RS, Lassegue B, Shi J, Xu X, Sorescu D, Chung AB, Griendling KK, Lambeth JD: Cell transformation by the superoxide-generating oxidase Mox1. Nature 1999;401:79–82. Suzuki Y, Ono Y: Serum factors and the cellular redox status regulate cellular responsiveness to MHC class II-triggered homotypic B cell adhesion. Biochem Biophys Res Commun 1998;249:31–37. Suzuki Y, Ono Y: Involvement of reactive oxygen species produced via NADPH oxidase in tyrosine phosphorylation in human B- and T-lineage lymphoid cells. Biochem Biophys Res Commun 1999;255:262–267. Suzuki Y, Ono Y, Hirabayashi Y: Rapid and specific reactive oxygen species generation via NADPH oxidase activation during Fas-mediated apoptosis. FEBS Lett 1998;425:209–212. Tatla S, Woodhead V, Foreman JC, Chain BM: The role of reactive oxygen species in triggering proliferation and IL-2 secretion in T cells. Free Radic Biol Med 1999;26:14–24. Devadas S, Zaritskaya L, Rhee SG, Oberley L, Williams MS: Discrete generation of superoxide and hydrogen peroxide by T-cell receptor stimulation: Selective regulation of mitogen-activated protein kinase activation and fas ligand expression. J Exp Med 2002;195:59–70. Hamano T, Iwasaki T, Ogata A, Hashimoto N, Kakishita E: The molecular mechanism in activationinduced cell death of an Ag-reactive B cell clone. Clin Exp Immunol 2002;128:436–443. Banfi B, Maturana A, Jaconi S, Arnaudeau S, Laforge T, Sinha B, Ligeti E, Demaurex N, Krause KH: A mammalian H⫹ channel generated through alternative splicing of the NADPH oxidase homolog NOH-1. Science 2000;287:138–142.

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Kikuchi H, Hikage M, Miyashita H, Fukumoto M: NADPH oxidase subunit, gp91phox homologue, preferentially expressed in human colon epithelial cells. Gene 2000;254:237–243. Geiszt M, Kopp JB, Varnai P, Leto TL: Identification of renox, an NAD(P)H oxidase in kidney. Proc Natl Acad Sci USA 2000;97:8010–8014. Lambeth JD, Cheng G, Arnold RS, Edens WA: Novel homologs of gp91phox. Trends Biochem Sci 2000;25:459–461. Dupuy C, Ohayon R, Valent A, Noel-Hudson MS, Deme D, Virion A: Purification of a novel flavoprotein involved in the thyroid NADPH oxidase. Cloning of the porcine and human cdnas. J Biol Chem 1999;274:37265–37269. Dupuy C, Pomerance M, Ohayon R, Noel-Hudson MS, Deme D, Chaaraoui M, Francon J, Virion A: Thyroid oxidase (THOX2) gene expression in the rat thyroid cell line FRTL-5. Biochem Biophys Res Commun 2000;277:287–292. Lambeth JD: NOX enzymes and the biology of reactive oxygen. Nat Rev Immunol 2004;4:181–189. Turner H, Kinet JP: Signaling through the high-affinity IgE receptor Fc␧RI. Nature 1999;402: B24–B30. Kinet JP: The high-affinity IgE receptor (Fc␧RI): From physiology to pathology. Annu Rev Immunol 1999;17:931–972. Ra C, Jouvin MH, Kinet JP: Complete structure of the mouse mast cell receptor for IgE (Fc␧RI) and surface expression of chimeric receptors (rat-mouse-human) on transfected cells. J Biol Chem 1989;264:15323–15327. Cambier JC: Antigen and Fc receptor signaling. The awesome power of the immunoreceptor tyrosine-based activation motif (ITAM). J Immunol 1995;155:3281–3285. Nadler MJ, Matthews SA, Turner H, Kinet JP: Signal transduction by the high-affinity immunoglobulin E receptor Fc␧RI: Coupling form to function. Adv Immunol 2000;76:325–355. Lin S, Cicala C, Scharenberg AM, Kinet JP: The Fc(␧)RI␤ subunit functions as an amplifier of Fc(␧)RI␥-mediated cell activation signals. Cell 1996;85:985–995. Dombrowicz D, Lin S, Flamand V, Brini AT, Koller BH, Kinet JP: Allergy-associated FcR␤ is a molecular amplifier of IgE- and IgG-mediated in vivo responses. Immunity 1998;8:517–529. Hiraoka S, Furumoto Y, Koseki H, Takagaki Y, Taniguchi M, Okumura K, Ra C: Fc receptor ␤ subunit is required for full activation of mast cells through Fc receptor engagement. Int Immunol 1999;11:199–207. Donnadieu E, Cookson WO, Jouvin MH, Kinet JP: Allergy-associated polymorphisms of the Fc␧RI ␤ subunit do not impact its two amplification functions. J Immunol 2000;165: 3917–3922. Rivera J: Molecular adapters in Fc(␧)RI signaling and the allergic response. Curr Opin Immunol 2002;14:688. Wolfreys K, Oliveira DB: Alterations in intracellular reactive oxygen species generation and redox potential modulate mast cell function. Eur J Immunol 1997;27:297–306. Brooks AC, Whelan CJ, Purcell WM: Reactive oxygen species generation and histamine release by activated mast cells: Modulation by nitric oxide synthase inhibition. Br J Pharmacol 1999;128: 585–590. Swindle EJ, Hunt JA, Coleman JW: A comparison of reactive oxygen species generation by rat peritoneal macrophages and mast cells using the highly sensitive real-time chemiluminescent probe pholasin: Inhibition of antigen-induced mast cell degranulation by macrophage-derived hydrogen peroxide. J Immunol 2002;169:5866–5873. Matsui T, Suzuki Y, Yamashita K, Yoshimaru T, Suzuki-Karasaki M, Hayakawa S, Yamaki M, Shimizu K: Diphenyleneiodonium prevents reactive oxygen species generation, tyrosine phosphorylation, and histamine release in RBL-2H3 mast cells. Biochem Biophys Res Commun 2000;276: 742–748. Yoshimaru T, Suzuki Y, Matsui T, Yamashita K, Ochiai T, Yamaki M, Shimizu K: Blockade of superoxide generation prevents high-affinity immunoglobulin E receptor-mediated release of allergic mediators by rat mast cell line and human basophils. Clin Exp Allergy 2002;32:612–618. Suzuki Y, Yoshimaru T, Matsui T, Inoue T, Niide O, Nunomura S, Ra C: Fc␧RI signaling of mast cells activates intracellular production of hydrogen peroxide: Role in the regulation of calcium signals. J Immunol 2003;171:6119–6127.

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Suzuki Y, Yoshimaru T, Matsui T, Ra C: Silver activates calcium signals in rat basophilic leukemia2H3 mast cells by a mechanism that differs from the Fc␧RI-activated response. J Immunol 2002;169:3954–3962. Kepley CL, Lauer FT, Oliver JM, Burchiel SW: Environmental polycyclic aromatic hydrocarbons, benzo(a)pyrene (BaP) and BaP-quinones, enhance IgE-mediated histamine release and IL-4 production in human basophils. Clin Immunol 2003;107:10–19. Costello PS, Turner M, Walters AE, Cunningham CN, Bauer PH, Downward J, Tybulewicz VL: Critical role for the tyrosine kinase Syk in signalling through the high affinity IgE receptor of mast cells. Oncogene 1996;13:2595–2605. Nishizumi H, Yamamoto T: Impaired tyrosine phosphorylation and Ca2⫹ mobilization, but not degranulation, in lyn-deficient bone marrow-derived mast cells. J Immunol 1997;158:2350–2355. Kawakami Y, Yao L, Miura T, Tsukada S, Witte ON, Kawakami T: Tyrosine phosphorylation and activation of Bruton tyrosine kinase upon Fc epsilon RI cross-linking. Mol Cell Biol 1994;14: 5108–5113. Scharenberg AM, Kinet JP: Initial events in Fc␧RI signal transduction. J Allergy Clin Immunol 1994;94:1142–1146. Miller AT, Berg LJ: New insights into the regulation and functions of Tec family tyrosine kinases in the immune system. Curr Opin Immunol 2002;14:331–340. Rivera J, Cordero JR, Furumoto Y, Luciano-Montalvo C, Gonzalez-Espinosa C, Kovarova M, Odom S, Parravicini V: Macromolecular protein signaling complexes and mast cell responses: A view of the organization of IgE-dependent mast cell signaling. Mol Immunol 2002;38: 1253–1258.

Yoshihiro Suzuki, PhD Division of Molecular Cell Immunology and Allergology Nihon University Graduate School of Medical Sciences 30-1 Oyaguchikami-cho Itabashi-ku, Tokyo 173–8610 (Japan) Tel. ⫹81 3 3972 8111, Fax ⫹81 3 3972 8227, E-Mail [email protected]

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Roles of Adaptor Molecules in Mast Cell Activation Christine Tkaczyk, Shoko Iwaki, Dean D. Metcalfe, Alasdair M. Gilfillan Laboratory of Allergic Diseases, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, Md., USA

Abstract The release of pro-inflammatory mediators from mast cells generally occurs following antigen-dependent aggregation of the high-affinity receptors for IgE (Fc␧RI) expressed on the cell surface. Under the appropriate conditions, however, other receptors including the high-affinity receptor for IgG (Fc␥RI), Kit, the C3a complement component receptor, and adenosine receptors, can also induce or potentiate mast cell activation. In contrast, receptors such as the Fc␥RIIb low-affinity IgG receptor, and gp49b, when co-ligated with Fc␧RI, down-regulate mast cell activation. The driving force by which the Fc␧RI, the Fc␥RI, Kit, and potentially other receptors, lead to mast cell degranulation, arachidonic acid metabolism and cytokine gene expression, is a series of tyrosine kinase-mediated protein phosphorylation events which result in recruitment and subsequent activation of signaling enzymes. Similar processes are required by gp49b and Fc␥RIIb for the down-regulation of mast cell activation. The cellular localization and sequence of these events, the subsequent amplification and diversification of the signaling cascade, and potentially, the termination of these events, are regulated by an important group of signaling proteins termed adaptor molecules. In this chapter, we discuss the structure and properties of these molecules and how these proteins regulate the cellular processes associated with receptor-mediated mast cell activation. Copyright © 2005 S. Karger AG, Basel

Adaptor Molecules in Mast Cells

The term adaptor molecule describes a diverse group of proteins whose primary function is to act as linkers between signaling proteins regulating cell activation. These molecules can be thought of as components of a molecular scaffold or framework which tethers and compartmentalizes crucial signaling enzymes in the receptor complex. Mast cell adaptor molecules can be grouped into three distinct categories (fig. 1): (i) receptors such as Kit, and receptor

i. Receptors and receptor subunits Fc␧RI LAT

␣

␥

Kit ␤

ii. Transmembrane adaptors

NTAL

Iyn

iii. Cytosolic adaptors

Signaling enzymes

Kit

Cytosolic adaptors Kinase Kinase Cytosolic adaptors MAPK

Syk Cytosolic adaptors MAPK PI-3 Kinase

Degranulation

Cytokines

PLC␥1

PI-3 Kinase

Mast cell growth differentiation survival

Arachidonic acid metabolites

Fig. 1. Role of adaptor molecules in transduction of signals following antigen-mediated aggregation of the Fc␧RI and SCF-mediated activation of Kit. The details of the interactions mediated by these adaptor molecules are discussed in the text.

subunits such as the ␤ and ␥ of the Fc␧RI, which contain docking sites for associating molecules on their cytosolic tails; (ii) transmembrane adaptor molecules such as PAG, LAT and NTAL which have no ligand-binding sites on their extracellular domain, but which have multiple docking sites for associating signaling molecules on their cytosolic tails, and (iii) cytosolic adaptor molecules such as Gab1, Gads, Grb2, Shc, SLP-76, Sos, and Vav which bind associating signaling molecules via a number of different motifs. Critical to the function of adaptor molecules, therefore, is the ability to interact with other signaling proteins in either a constitutive or inducible manner.

Constitutive Protein-Protein Interactions

Although a number of binding domains which regulate constitutive protein-protein interactions have been described [1], in mast cells, these interactions are generally a consequence of Src homology 3 (SH3) domains being present on one or both of the interacting molecules. SH3 domains are highly

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Recognition sequence/ Binding domain Example

-PxxP-/SH3 domain Sos/Grb2

-RxxK-/SH3 domain SLP-76/Gads

SH3 SH3

RxxK SH3

PxxP SH3

Constitutive protein-protein interactions

SH3 domain/SH3 domain Grb2/Vav

Y

Recognition sequence/ Binding domain Example

pY/SH2 domain Fc␧RI␥ⲐSyk

PTB

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pY/PTP domain NGF receptor/Shc

PH

Y

SH2 SH2

Inducible protein-protein/lipid interactions

PIP3 /PH domain Membrane/PLC␥2

Fig. 2. Protein-protein interactions involved in the binding of adaptor molecules to associating signaling molecules.

conserved sequences of approximately 60 amino acids which interact with associating signaling molecules by three alternative mechanisms (fig. 2): (i) binding of an SH3 domain present in one molecule, to a specific proline-rich motif (core consensus sequence: -PxxP-) in an associating signaling molecule, for example, the binding of the SH3 domain of the cytosolic adaptor molecule, Grb2, to the proline-rich sequence of the associating adaptor molecule/GTP exchanger, Sos [1]; (ii) binding of an SH3 domain present in one molecule to the consensus sequence -RxxK- present in an associating molecule, for example, the binding of the SH3 domain of the cytosolic adaptor molecule, Gads, to the -RxxK- motif of SLP-76 [2], and (iii) the dimerization of an SH3 domain of one molecule with the SH3 domain present in another signaling molecule, for example, the binding of the SH3 domain of Grb2 to the SH3 domain of the associating adaptor molecule/GTP exchanger, vav [3] (fig. 2).

Inducible Protein-Protein and Protein-Lipid Interactions

Although a number of inducible protein-protein interactions have been described [1, 4], such interactions, important for the control of mast cell activation

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by adaptor molecules, are dependent on the presence of Src homology 2 (SH2) domains, phosphotyrosine-binding (PTB) domains and/or pleckstrin homology (PH) domains on one or more of the interacting signaling molecules.

SH2 Domains and PTB Domains SH2 domains are conserved regions of approximately 100–120 amino acids which bind specific tyrosine-containing sequences following phosphorylation of these residues by activated tyrosine kinases. Specificity of these interactions is determined by the residues ⫹1 to ⫹4, C-terminus to the phosphorylated tyrosine residues. Thus, for example, the tandem -YxxL- motifs present in the Fc␧RI␥ chain when phosphorylated by Src kinases, will bind the dual SH2 domains of Syk with high affinity [5], and the multiple -YxN- motifs present in the transmembrane adaptor molecules, LAT and NTAL, when phosphorylated by either Src kinases or Zap70 tyrosine kinase family members, will bind the SH2 domain of Grb2 [6]. Protein-protein interactions involving phosphotyrosine-SH2 domain interactions can be reversed by the activation of tyrosine phosphatases such as SHP1 and SHP2. By this means, signaling pathways involving these interactions can be finely regulated. PTB domains are spans of approximately 100–170 residues found in a number of cytosolic adaptor molecules such as Shc and Dok-2 but not in receptor subunits or transmembrane adaptor molecules [7]. Whereas SH2 domains can only bind sequences containing phosphorylated tyrosine residues, PTB domains recognize sequences which contain either phosphorylated tyrosines or non-phosphorylated tyrosines [4], therefore, these interactions are both dependent and independent of the action of tyrosine kinases. Unlike SH2phosphotyrosine interactions, the sequence determinants for PTB domaintyrosine/ phosphotyrosine interactions are immediately N-terminal to the tyrosine residues [4, 7].

PH Domains PH domains are spans of approximately 120 residues which are found in a number of signaling proteins such as PLC␥1 and the Src-related tyrosine kinase, Btk, but not in receptor subunits or transmembrane adaptor molecules. These domains interact with membrane-associated phosphoinositides following the phosphorylation of these lipids by the action of the lipid kinase, phosphoinositide 3-kinase (PI3-kinase). These interactions are reversible following dephosphorylation of the phosphoinositides by the lipid phosphatases SHIP and PTEN [8]. Thus, as with SH2 domains, responses requiring interactions of PH domains with membrane lipids can be finely regulated.

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Fc␧RI ␣

a

Fc␥RI

␤

␥

ITAM’s

Fc␧RI␥ ITAM

DGY YTGL

Fc␧RI␤ ITAM

DRV

YEEL

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Fc␥RIII ␣

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Fc␥RIIb ITIM

b

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ITIM

␥

ITAM's

S T R NQ E T

YETL

NI Y S A T

YSTL Y x x L/I

ITYSSL I/V/L/S xY x x L/V

Fig. 3. Structure of Fc␧RI and Fc␥RI receptors expressed on mast cells (a) and the sequences of the ITAMs and ITIMs present in these receptors (b). [From Tkaczyk et al. [44], with permission of the publisher.]

Receptors and Receptor Subunits as Adaptor Molecules

Fc␧RI and FcgRI: b and g Subunits The Fc␧RI comprises a single ␣ subunit, a single ␤ subunit and a homodimeric ␥ subunit (fig. 3). The extracellular domain of the Fc␧RI␣ subunit is responsible for the high-affinity binding of IgE, whereas the ␤ and ␥ chains are responsible for recruiting signaling molecules which initiate the downstream cellular activation cascade. As such, these subunits can be considered as the earliest adaptor molecules involved in mast cell activation via the Fc␧RI. The Fc␧RI␥ but not the Fc␧RI␤ chain is also associated with the Fc␥RI␣ [9] (fig. 3). This may explain the fact that both similarities and differences in signaling and mediator release following both Fc␧RI and Fc␥RI aggregation have been observed in human mast cells [10]. Contained within the cytoplasmic domains of the ␤ and ␥ chains of Fc␧RI are phosphotyrosine-containing SH2 domain-binding sequences termed immunoglobulin receptor tyrosine activation motifs (ITAMs) [11]. These sequences comprise a span of approximately 24 amino acids containing tandem YxxL/I sequences which are targets for Src family member tyrosine kinases

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(fig. 3) [8]. In the case of the Fc␧RI, and likely the Fc␥RI in mast cells, the major Src kinase responsible for this action is Lyn. The phosphorylated ␤ chain ITAM, thus, provides a docking site for the single SH2 domain contained in Lyn [8], and the tyrosine phosphatases SHP1 and SHP2 [8]. In contrast, phosphorylation of the Fc␧RI␥ chain results in recruitment of the tyrosine kinase Syk via binding of its dual SH2 domains [5]. Activation of Syk following Lyn-induced phosphorylation and auto-/trans-phosphorylation in turn results in multiple other binding sites for signaling molecules being generated by Syk-induced phosphorylation of downstream adaptor molecules (fig. 1). FcgRIIb and gp49b Co-ligation of Fc␥RIIb and/or gp49b with Fc␧RI results in down-regulation of the secretory responses elicited by the Fc␧RI. Unlike the Fc␧RI, these receptors are single chains, and the inhibitory responses mediated by the receptors are dependent on binding of signaling molecules to specific tyrosine-containing motifs termed immunoglobulin receptor inhibitory motifs (ITIM) present in the cytosolic tails of these molecules (fig. 3). Co-ligation of Fc␥RIb and/or gp49b with the Fc␧RI, results in the ITIMs becoming tyrosine phosphorylated by the action of Src family tyrosine kinases thereby providing docking sites for the SH2 domains of the tyrosine phosphatases SHP1 and SHP2 on GP49b, and the phosphoinositide phosphatase SHIP, on Fc␥RIIb. Thus, these molecules respectively terminate tyrosine kinase-mediated and PI3-kinase-mediated signaling pathways. Kit The growth factor receptor, Kit, is not only required for mast cell growth, differentiation, and survival, but is also required for optimal degranulation and cytokine production in response to Fc␧RI aggregation [12, 13]. Kit contains multiple tyrosines within the cytosolic domain that become docking sites for SH2 and PTB domains of associating signaling molecules following tyrosine phosphorylation [14]. Unlike the Fc␧RI, Fc␥RI, Fc␥RIIb and GP49b receptors, Kit possesses inherent catalytic activity, therefore can undergo auto- and transphosphorylation following stem cell factor (SCF)-induced dimerization [14]. However, the Src kinases Lyn and Fyn may also be required for the interaction of some of these signaling molecules with the receptor and subsequent downstream signaling [14]. The binding sites/consensus motifs for the SH2 domains for a number of associating signaling molecules have been mapped on human Kit, e.g. pY569 binds SHP2, pY570 binds SHP1 and both of these residues appear to bind Src kinase family members and Shc; pY721 binds PI3-kinase, and pY936 binds PLC␥1. An activating mutation (V560 to G560) associated with stromal cell

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carcinoma in the intestine appears to prevent binding of the SH2 domain of SHP1 to its docking site thereby preventing SHP1-dependent down-regulation of Kit signaling [14].

Transmembrane Adaptor Molecules

There is emerging evidence that signaling events associated with the Fc␧RI and other receptors take place in discrete regions within the cell membrane which are rich in cholesterol, glycolipids, and specific glycolipidanchored proteins. These regions are termed glycolipid-enriched microdomains (GEM fractions), or more commonly, lipid rafts. Signaling proteins become resident in these microdomains as a consequence of palmitoylation of a specific sequence in the juxtamembrane regions of these proteins. One of the major groups of lipid raft-associated signaling proteins involved in mast cell activation are the transmembrane adaptor molecules. These molecules are essential for the recruitment of other adaptor molecules and signaling enzymes into the receptor-signaling complex. A number of transmembrane adaptor molecules have been described in hematopoietic cells and the readers are referred to several excellent reviews on this subject [6, 15, 16]. In this chapter, however, we describe the three major transmembrane adaptor molecules that regulate mast cell function, namely LAT, NTAL and Cbp/PAG (fig. 4). These molecules have a common characteristic structure comprising a short extracellular domain, a transmembrane domain and an extended cytoplasmic domain, which contains the palmitoylation site and multiple tyrosine residues. When these residues are phosphorylated by members of the Src and Zap-70 family of tyrosine kinases, SH2 domain-containing cytosolic adaptors and signaling enzymes are recruited as described below. Cbp/PAG is an inhibitory regulator of mast cell activation whereas LAT and NTAL are likely involved in the positive regulation of mast cell signaling, although these molecules have also been suggested to negatively regulate degranulation under specific conditions. LAT (Linker for Activation of T Cells) LAT is a molecule of 36–38 kDa, originally discovered in T cells and subsequently found expressed in NK cells, platelets, and mast cells [6]. It has been demonstrated in both rodent and human mast cells, that Fc␧RI aggregation induces LAT tyrosine phosphoryiation via Lyn and/or Syk activation. There are ten potential target tyrosines for these kinases in the cytosolic tail of LAT and, following phosphorylation, these become docking sites for cytosolic adaptor

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Transmembrane adaptor molecules NTAL PAG

N-terminus

PxxP

PxxP

LAT

C-terminus

Cbl-b

SH2

Dok1

PH

SH2

Gab2

PH

PxxP

Gads

SH3

SH2

PxxP SH3

Grb2

SH3

SH2

SH3

SHC

SH2

SLP76

PxxP PxxP

PTB PxxP

SH2

Sos Vav

PxxP PH

: Palmitoylation site

: Ring finger domain

: Phosphotyrosine

: Leucine zipper domain

SH3

SH2

SH3

: GTP exchanger

Fig. 4. Structure of transmembrane adaptor molecules and cytosolic adaptor molecules expressed in mast cells.

molecules including Grb2 and SLP-76 [6], and signaling enzymes including PLC␥1, PLC␥2, and PI3-kinase [6]. In mouse BMMCs, pY136 has been mapped to the binding of PLC␥, and the -YxN- motifs containing tyrosines 175, 195 and 235, when phosphorylated, bind Grb2. LAT appears to play a major role in degranulation and cytokine production in mast cells. In this respect, mast cells derived from the bone marrow of LAT⫺/⫺ mice exhibit a profound defect in the ability to degranulate and generate cytokines including IL-2, IL-4, IL-6 and TNF-␣ in response to Fc␧RI aggregation [17]. In addition, siRNA targeted towards LAT in human mast cells resulted in an inhibition of antigen-dependent degranulation [12]. The reduced response in BMMCs was associated with similar defects in the phosphorylation of SLP-76, PLC␥1 and PLC␥2 phosphorylation, MAP kinase activation and calcium mobilization in the absence of defective phosphorylation of the Fc␧RI␤ and ␥ chains and Syk [17]. Deletional analyses in BMMCs have revealed that the four distal tyrosines are required for the ability of LAT to regulate calcium mobilization and degranulation, particularly Y136 which binds PLC␥1 [18]. Taken together, these data suggest that LAT may be a primary transmembrane adaptor molecule responsible for the regulating the PLC␥1mediated component of calcium mobilization and degranulation.

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NTAL (Non-T Cell Activation Linker) NTAL is a 30 kDa transmembrane adaptor molecule, structurally and functionally related to LAT. However, unlike LAT which is found in T cells but not B cells, NTAL is expressed in B cells but not resting T cells [19]. NTAL is also expressed in mast cells, monocytes and NK cells. Why mast cells should express both the major transmembrane adaptor molecules found in B and T cells is unclear, however as discussed below, this may provide a mechanism by which primary pathways regulated by one transmembrane adaptor molecule may be amplified or down-regulated by another. In both human and mouse mast cells, NTAL becomes rapidly and heavily tyrosine phosphorylated following Fc␧RI aggregation [12, 19]. In addition, SCF-mediated Kit activation in these cell types also induces tyrosine phosphorylation of NTAL and this response synergizes with that induced by Fc␧RI aggregation. In contrast to the Fc␧RI-induced response, however, Kit-mediated NTAL phosphorylation was observed in the absence of detectible LAT phosphorylation [12]. This suggests that Fc␧RI and Kit induce NTAL and/or LAT phosphorylation by differential signaling pathways. The observation that Fc␧RI-mediated, but not Kit-mediated NTAL phosphorylation, was ablated in mast cells derived from the bone marrow of Lyn⫺/⫺ mice [12] implies that Fc␧RI-mediated NTAL phosphorylation may be mediated by Lyn and/or Syk, whereas Kit may directly phosphorylate NTAL independently of Lyn and/or Syk. A role for NTAL in human mast cell function has been suggested by studies demonstrating that reduced NTAL and/or LAT expression, following incubation of the cells with NTAL and/or LAT targeted siRNA [12], results in defective degranulation in response to antigen and a reduction in the capacity of SCF to potentiate this response. The precise role(s) that NTAL plays in mast cell function, however, remains unclear. Studies conducted in transgenic mice expressing NTAL in LAT-deficient T cells suggest that NTAL may function as LAT but without the capacity to bind PLC␥1. As with LAT, however, NTAL possesses multiple other potential tyrosine phosphorylation sites in the cytosolic tail which may bind to SH2 domains of associating signaling molecules. In B cells, tyrosine phosphorylated-NTAL binds Grb2, Sos, Cbl and SLP-65, [19, 20] and binding of Grb2 to NTAL has been mapped to the -YxN- motifs containing tyrosines 136, 193 and 233 which are critical for NTAL function. These data suggest that there may be a degree in overlap of functions of LAT and NTAL in activated mast cells. This conclusion has been further supported by the recent studies conducted in NTAL⫺/⫺ BMMCs demonstrating enhanced degranulation in these cells which was associated with a marked elevation in LAT phosphorylation following antigen challenge when compared to wild-type responses [21]. In contrast, the residual degranulation observed in LAT⫺/⫺

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BMMCs was virtually abolished in LAT⫺/⫺/NTAL⫺/⫺ double knockout BMMCs. Furthermore, our studies in human mast cells demonstrated that the concurrent addition of siRNAs targeted towards NTAL and LAT was more effective at blocking degranulation than when added separately [Tkaczyk and Gilfillan, unpubl. observations]. Taken together, these data suggest that the phosphorylation of both molecules is independently required for mediator release to take place or, alternatively, LAT may regulate the principle pathway leading to mediator release from mast cells and NTAL may control a pathway which amplifies these responses; a role which has been previously suggested for the ␤ chain of the Fc␧RI [22]. It is possible that at lower levels of Fc␧RI aggregation, phosphorylated NTAL may recruit molecules such as Grb2 and perhaps other molecules which would potentiate degranulation, whereas at higher levels of aggregation, NTAL may recruit molecules such as Cbl which would down-regulate degranulation. This may be one possible explanation for the bell-shaped dose-response curves observed with increasing concentrations of antigen and would also explain the apparently conflicting results from the NTAL⫺/⫺ BMMC studies compared to the NTAL⫺/⫺/LAT⫺/⫺ BMMC and human mast cell siRNA studies described above. Cbp/PAG (Csk-Binding Protein/Phosphoprotein Associated with GEMs) Cbp/PAG is a transmembrane adaptor molecule of 80–90 kDa, which is a negative regulator of cell signaling [6]. This molecule was initially discovered in developing tissues including brain, lung, thymus, spleen and testis, however, it is also expressed in T cells and mast cells [23–25]. In resting RBL 2H3 cells, Cbp/PAG is constitutively associated with the negative regulator of Src kinases, Csk. Cbp/PAG becomes tyrosine phosphorylated upon Fc␧RI aggregation and this results in increased binding to the SH2 domain of Csk. Tyrosine 314 is critical for this association and for the localization of Csk to plasma membrane. Overexpression of Cbp/PAG in RBL-2H3 cells results in a dramatic reduction in degranulation and calcium mobilization following antigen stimulation, however, these changes are not seen following PMA and Ca2⫹ ionophore A23187 administration [24]. These data support the conclusion that Cbp/PAG negatively regulates Fc␧RI-mediated mast cell activation by down-regulation of Src kinases such as Lyn and/or Fyn following recruitment of Csk.

Cytosolic Adaptor Molecules

In contrast to the transmembrane adaptor molecules whose ability to interact with associating molecules is solely dependent on the tyrosine residues in their cytosolic tail being phosphorylated, cytosolic adaptor molecules possess

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both docking sites and binding motifs which allows interaction with multiple associating molecules (fig. 4). By this means, these proteins link transmembrane adaptor molecules, other cytosolic adaptor molecules, and/or signaling enzymes to form macromolecular receptor-signaling complexes which regulate downstream-signaling processes. Fc␧RI aggregation induces the formation of at least two such macromolecular complexes: one organized around LAT and the cytosolic adaptor molecules SLP-76, Gads, Vav, and Grb2-Sos-Shc, and one organized around Gab2, Fyn, and PI3-kinase [26, 27]. The transmembrane adaptor molecule binding these latter molecules has yet to be determined, however NTAL is a potential candidate for this role. The first complex controls PLC␥1 phosphorylation leading to enhanced calcium mobilization and degranulation, and the MAP kinase pathway which regulates arachidonic acid metabolism and gene transcription. The second complex likely regulates the PI3-kinase pathway which also contributes to calcium flux, degranulation and cytokine production. Thus the ability of the individual cytosolic adaptor molecules to influence mast cell function is highly dependent on the specific molecules they bind upon cell activation. SLP-76 (Src Homology 2 Domain-Containing Leukocyte Protein of 76 kDa) SLP-76, which is primarily expressed in T lymphocytes and myeloid cells [28], contains multiple N-terminal tyrosine residues, a proline-rich region and a C-terminal SH2 domain. Upon Fc␧RI aggregation, SLP-76 becomes associated with LAT through Gads [29]. Thus, mast cells derived from SLP-76-deficient mice present the same phenotype as do LAT-deficient mast cells. In this respect, these cells display similar defects in degranulation, calcium mobilization, PLC␥1 phosphorylation, and IL-6 secretion after Fc␧RI aggregation. Furthermore, SLP-76-deficient mice are resistant to passive cutaneous anaphylaxis [29]. Studies using BMMC derived from SLP-76-deficient mice reconstituted with different SLP-76 mutants, demonstrated that the N-terminal tyrosine residues are involved in Vav binding and the proline region is necessary for association with Gads and PLC␥1 [30]. These two regions are therefore essential for mast cell function. However, the SH2 domain of SLP-76 which binds ADAP (Adhesion and Degranulation Promoting Adapter Protein) is not essential, but is only required for optimal mast cell function [30]. Vav Vav is a guanine nucleotide exchange factor which also contains multiple binding domains including two SH3 domains, a single SH2 domain, and a single PH domain. Upon Fc␧RI aggregation, Vav becomes tyrosine phosphorylated thereby recruiting and inducing the Rac/Cdc42/Rho pathway, which

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results in the activation of Jun N-terminal kinase (JNK) [31] and induction of specific cytokine genes including IL-2, IL-6, and IFN-␥ [32]. BMMC derived from Vav-deficient mice, thus, are defective in their ability to generate these cytokines. In contrast, they do not show major defects in degranulation and the production of several other cytokine despite displaying defects in PLC␥1 and PLC␥2 phosphorylation and calcium flux [33]. The fact that degranulation is not affected despite the decrease in calcium flux in Vav⫺/⫺ BMMC suggests that there is a component of degranulation independent of PLC␥1. This conclusion is supported by our findings in human mast cells where we observed that antigen-induced degranulation is regulated by an early PLC␥1-dependent, PI3kinase-independent component, and a delayed PI3-kinase-dependent component [34]. Grb2, Gads and Gab1 The Grb2 family members Grb2 (growth factor receptor bound 2), and Gads (Grb2-like adaptor downstream of Shc), which are expressed in mast cells, present the common structure of SH3 domains on the N- and C-termini separated by a single SH2 domain. Grb2 is a molecule of 25 kDa, which has been shown to be a key player in the initiation of MAP kinase pathway leading to cytokine synthesis and release after mast cell activation [8]. Grb2 is constitutively bound to the Ras guanine exchange factor, Sos, through binding to its SH3 domain [35]. The SH2 domain recognizes the consensus sequence -YxNwhen phosphorylated. By this means, upon Fc␧RI aggregation, the Grb2-Sos complex is recruited to the receptor-signaling complex by binding to these sequences in both LAT and NTAL [6]. In addition, upon Fc␧RI aggregation, the SH2 domain of Grb2 binds another cytosolic adaptor molecule, Shc, following its phosphorylation by Syk [36]. This Shc-Grb2-Sos thus activates the RasRaf-MAP kinase pathway. Gads is a protein of 40 kDa, which is similar in structure to Grb2, except that it contains a proline-rich region and glutamine residues between its C-terminal SH2 and SH3 domains [37]. This molecule is expressed in T cells and mast cells and has been demonstrated to act as a linker between SLP-76 and LAT [1], and appears to be integral to the regulation of the PLC␥1-dependent regulation of calcium flux and degranulation. Gab2 (Grb2 associating binder 2) is a molecule of 98 kDa, containing a proline-rich SH3-binding domain, an N-terminal PH domain, and multiple tyrosine residues, which when phosphorylated, provide binding sites for Grb2, Shc, the tyrosine phosphatase SHP2, and the p85 subunit of PI3-kinase. BMMCs derived from Gab2-deficient mice present defective PI3-kinase-dependent signaling following Fc␧RI aggregation [26] in the absence of similar defects in LAT phosphorylation or activation of MAP kinase. In addition, in vivo passive

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cutaneous anaphylaxis reactions were reduced in these mice compared to the reactions in wild-type mice. 3BP2 One final cytosolic adaptor molecule that apparently plays a role in the positive regulation of Fc␧RI-mediated mast cell activation is 3BP2 [38]. This molecule contains a proline-rich SH3 domain-binding sequence, a PH domain, an SH2 domain, and a -YxN- Grb2-binding site. A role for this molecule in mast cell activation has been proposed based on its rapid tyrosine phosphorylation following Fc␧RI aggregation in RBL 2H3 cells, its ability to bind LAT and Syk, and from studies demonstrating that overexpression of the 3BP2-SH2 domain in RBL 2H3 cells resulted in inhibition of Fc␧RI-mediated PLC␥ phosphorylation, calcium mobilization and degranulation [38]. Further studies involving 3BP2 knockout mice or siRNA knock-down approaches, however, will be required to determine the exact role of this molecule in mast cell activation. Cbl and Dok Cbl and Dok are two separate classes of cytosolic adaptor molecules, however, they share the common property of negatively regulating mast cell activation. Two forms of Cbl are expressed in mast cells, Cbl-b and c-Cbl. These molecules possess an SH2 domain, a proline-rich SH3-binding domain, and multiple tyrosine residues, which are substrates for tyrosine kinases. They are members of the family of ubiquitin ligases and it has been shown that c-Cbl catalyzes the ubiquitination of Fc␧RI and Syk, through binding to Syk following Fc␧RI aggregation [39]. Studies in c-Cbl knockout mice however suggest that this does not interfere with the ability of Fc␧RI to induce degranulation [40]. In contrast, Cbl-b⫺/⫺ BMMCs exhibited an increase in degranulation and calcium flux which was associated with increased phosphorylation of Syk and PLC␥, suggesting that Cbl-b but not c-Cbl is involved in the negative regulation of Fc␧RI-mediated degranulation. This conclusion was supported by studies conducted in RBL 2H3 cells which demonstrated that overexpression of Cbl-b in lipid rafts resulted in inhibition of phosphorylation of Fc␧RI, Syk, PLC␥, Erk, and I␬␬, resulting in reduced calcium influx, degranulation, and cytokine release [41]. The molecules binding to Cbl in mast cells have not yet been determined however, it has been reported in T cells that Cbl-b can bind PI3-kinase, Vav and Grb-2, therefore, it is possible that such interactions also occur in mast cells. As with Cbl, Dok proteins negatively regulate mast cell activation. These molecules possess an N-terminus PH domain, a PTB domain, seven prolinerich SH3 domain-docking sites and multiple tyrosine residues which are targets for phosphorylation by tyrosine kinases including Bcr-Abl, Lyn and Tec. In mast cells, Dok-1 is phosphorylated in response to both Fc␧RI aggregation and

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Kit activation. Following subsequent phosphorylation, Dok-1 and Dok-2 recruit Nck, CrkL and CAS. In RBL 2H3 cells, Dok-1 was found to be constitutively associated with the Fc␧RI, and overexpression of Dok-1 resulted in a marked reduction in Fc␧RI-mediated activation of the Ras-Raf-MAP kinase pathway and TNF-␣ production, suggesting that Dok-1 may negatively regulate both degranulation and cytokine production. The ability of Fc␥RIIb to inhibit Fc␧RImediated signaling has also been suggested to involve Dok-1 by forming a complex with SHIP and Grb2 and/or thereby inhibiting calcium mobilization and ERK1/2 phosphorylation [42, 43].

Conclusions

As discussed in this chapter, it is evident that adaptor molecules play an essential role in the organization of the signaling pathways utilized for both positive and negative regulation of receptor-mediated mast cell activation. The complexity of the macromolecular interactions involving these adaptor molecules is becoming more apparent as the roles of these signaling components in mast cell activation are explored. This degree of complexity however is apparently required to finely regulate the processes required for the generation and/or release of the diverse groups of mediators following mast cell action. As novel adaptor molecules likely remain to be identified, the role of these molecules in mast cell activation will continue to be an area of intense research.

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Alasdair M. Gilfillan Laboratory of Allergic Diseases, National Institute of Allergy and Infectious Diseases National Institutes of Health, Bldg 10, Rm 11C206 10 Center Drive MSC 1881, Bethesda, MD 20892-1881 (USA) Tel. ⫹1 301 4968757, Fax ⫹1 301 4808384, E-Mail [email protected]

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Saito H, Okayama Y (eds): Mast Cells in Allergic Diseases. Chem Immunol Allergy. Basel, Karger, 2005, vol 87, pp 59–79

Eicosanoid Mediators of Mast Cells: Receptors, Regulation of Synthesis, and Pathobiologic Implications Joshua A. Boyce Division of Rheumatology, Immunology and Allergy, Brigham and Women’s Hospital, Boston, Mass., USA

Abstract When activated by diverse stimuli, mast cells mobilize arachidonic acid through cytosolic phospholipase A2, and rapidly generate both prostaglandin D2 and leukotriene C4, the parent molecule of the cysteinyl LTs. While initially recognized for their bronchoconstricting and vasoactive properties, these two eicosanoids are now known to serve diverse and pivotal functions in effector cell trafficking, antigen presentation, immune cell activation, matrix deposition, and fibrosis. This chapter reviews the biological functions for each eicosanoid and their respective receptor classes, discusses the mechanisms by which the generation of eicosanoids by mast cells is regulated, and considers the potential pathobiologic and therapeutic ramifications in host defense, inflammation, and allergic diseases. Copyright © 2005 S. Karger AG, Basel

Introduction

The constitutive residence of mast cells (MCs) at mucosal, submucosal, and perivascular locations suggests that they are strategically situated to initiate immune and inflammatory responses. Indeed, MCs are essential in animal models of innate antibacterial immunity [1] and in the effector arm of the adaptive immune response to helminthic worm infections [2], and initiate allergic responses in humans [3]. Along with other mediators (preformed amines and proteases, induced cytokines and chemokines), activated MCs generate and release newly formed eicosanoids (leukotriene [LT]C4, prostaglandin [PG]D2) in abundance. These eicosanoids participate in the regulation of vascular permeability, cause smooth muscle constriction, alter patterns of antigen presentation, recruit

effector cells, activate stromal cells, and induce fibrosis. This chapter focuses on the biological functions cysteinyl LTs (cysLTs) and PGD2, the receptors involved, the regulation of the synthesis of these mediators by MCs, and the pathobiologic implications of these regulatory processes.

MC-Associated Eicosanoids

On cross-linkage of their high-affinity Fc receptors for immunoglobulin (Ig)E (Fc␧RI) or other stimuli that elicit sustained elevations of intracellular calcium, MCs generate two dominant eicosanoids; LTC4, the parent of the cysLTs, and PGD2 [4–7]. Activated MCs also secrete smaller amounts of LTB4, a powerful chemoattractant for neutrophils and effector lymphocytes [8]. Each eicosanoid is produced de novo from arachidonic acid, a 20-carbon fatty acid that is liberated from cell membrane phospholipids by an 85-kDa cytosolic phospholipase A2 (cPLA2) [9] (fig. 1), which requires activation by intracellular calcium flux and phosphorylation by mitogen-activated protein kinases (MAPKs) [10]. Fc␧RI-dependent cysLT generation by RBL-2H3 cells, a transformed rat MC line, required phosphorylation of protein kinase C-␦ by the small GTPase Rac1, resulting in MAPK phosphorylation and cPLA2 activation [11]. Activated cPLA2 translocates to intracellular membranes, where it provides arachidonic acid for the subsequent synthesis of eicosanoids. MCs from mice lacking cPLA2 are unable to generate any eicosanoids, indicating that cPLA2 is indispensable for this function [12]. The downstream metabolic pathways necessary for the generation of cysLTs and PGD2, the major end-products of MC activation, are outlined below, and their bioactivities are discussed.

PGD2 Biosynthesis Arachidonic acid is converted to the intermediate PGH2, the precursor of all bioactive PGs, by prostaglandin H synthase (also known as cyclooxygenase or COX). MCs express both constitutive PGHS-1 and inducible PGHS-2 [13] (fig. 1). Both of these enzymes provide PGH2 for conversion to PGD2 by a terminal hematopoietic PGD2 synthase (H-PGDS), the enzyme responsible for PGD2 generation by MCs [14]. A second PGDS, termed lipocalin (L)-PGDS, shares little sequence homology with H-PGDS and is responsible for PGD2 generation in the central nervous system [15]. L-PGDS is not expressed by MCs. Following its synthesis by MCs, PGD2 is released extracellularly by

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Cellular membrane cPLA2

SCF

Arachidonic acid SCF IL-10, IL-1␤, TLR ligands

PGHS-1

FLAP 5-LO

PGHS-2 PGD2

SCF RasGRP4 MITF

LTA4

9␣, 11␤-PGF2

PGD2

IL-4

LTC4s

PGJ2

5-HETE

LTA4H

LTB4

6-trans-LTB4

LTC4

Receptors: CysLT1 CysLT2

LTD4

Receptors: CysLT1 CysLT2

⌬GT ⌬12-PGJ2

IL-3, IL-5

5-HPETE

PGDS

Receptors: DP1 DP2

IL-3, IL-5

DiP 15-deoxy- ⌬

12,14-PGJ2

Receptor: PPAR␥

LTE4

Receptors: CysLT1 CysLT2

Fig. 1. Eicosanoid-synthesizing pathways of MCs and likely regulatory control points. cPLA2 liberates arachidonic acid from the cell membrane for subsequent conversion to cysLTs and PGD2 through the 5-LO/LTC4S pathway (white) or the PGHS/PGDS pathway (gray). Receptors responsible for mediating the effects of each end-product are indicated. Exogenous and endogenous regulatory factors for each step are delineated by the clear arrows.

a process involving a PG transporter protein [16], where it exerts diverse receptor-mediated effects in allergic and immune responses, as well as the regulation of sleep. PGD2 is metabolized non-enzymatically to 15-deoxy-⌬12,14PGJ2 (15-dPGJ2), a ligand for peroxisome proliferator-activated receptor (PPAR)-␥ [17], and to 9␣,11␤-PGF2 [18], the latter of which is excreted in urine, and is used in clinical studies as an index of MC activation.

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Actions of PGD2 in Humans: Direct Challenges Inhalation challenge of atopic human subjects with specific allergen sharply increases the levels of PGD2 detected in bronchoalveolar lavage (BAL) fluid [19], as well as the levels of 9␣,11␤-PGF2 in both serum and urine [18]. These events are thought to reflect the activation of local MCs. Even without allergen challenge, BAL fluids obtained from subjects with asthma contain higher levels of PGD2 than do BAL fluids obtained from non-asthmatic atopic subjects, suggesting that ongoing PGD2 production by MCs is characteristic of asthma [20]. When administered by inhalation to subjects with asthma, exogenous PGD2 induces bronchconstriction with roughly 10-fold greater potency than does histamine [21]. Inhalation of PGD2 also potentiates airway hyperresponsiveness (AHR) to subsequent challenges with either histamine or methacholine [22]. PGD2 also induces wheal-and-flare responses in the skin of normal human subjects when injected subcutaneously [23], and is also implicated in the episodic vasodilation observed in patients with systemic mastocytosis [24]. Taken together, these observations suggest that that MC-derived PGD2 may contribute to smooth muscle contraction, vascular leak, and edema that are typical features of asthma exacerbations and allergic responses, and may also potentiate responses to other physiologically relevant bronchoconstrictors. Receptors for PGD2 and Receptor-Mediated Functions in vitro Two PGD2-specific G protein-coupled receptors (GPCRs) have been cloned and characterized, respectively termed the DP1 [25, 26] and DP2 receptors (also known as chemoattractant receptor-homologous molecule expressed on Th2 cells [CRTH2]) [27]. Human DP1 receptor mRNA is expressed strongly in retina and small intestine [26], while mouse DP1 receptor mRNA is highly expressed in ileum, followed by lung, stomach, and uterus [25]. Human DP2 receptor mRNA is expressed in almost all tissues, with relatively high expression in various regions of brain, heart, stomach, adrenal gland, liver, small intestine, thymus, and placenta [28]. Both DP receptors bind PGD2 with high affinity (low nM range). While DP1 receptors use pertussis toxin (PTX)-resistant Gs proteins for their signaling and induce elevations in intracellular levels of cyclic adenosine monophosphate (cAMP) [26], DP2 receptors signal using PTX-sensitive Gi proteins and decrease cAMP levels in response to PGD2 [28]. The deduced amino acid sequence of the DP1 receptor is similar to other prostanoid receptors, whereas the DP2 receptor displays higher identity to chemokine receptors. Effects on Leukocytes Both DP1 and DP2 receptors are expressed on a number of leukocyte subsets. Consistent with their structural and signaling differences, DP1 and DP2 mediate disparate biological effects on these cells. PGD2 acts as a potent chemoattractant

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for eosinophils, basophils, and Th2 lymphocytes through DP2 receptors in vitro [27]. Stimulation of human basophils with a DP2 receptor-selective agonist, 13, 14-dihydro-15-keto (DK)-PGD2, also potentiates their exocytosis in response to Fc␧RI cross-linkage [29]. In contrast, a DP1 receptor-selective agonist, BW245C, inhibits Fc␧RI-dependent exocytosis by basophils. DK-PGD2 induces human eosinophils to degranulate [30], whereas BW245C prolongs eosinophil survival in vitro but does not induce degranulation. Human monocytes and monocyte-derived dendritic cells (DCs) respond chemotactically to PGD2 or DK-PGD2, but not to BW245C [31]. Conversely, PGD2 and BW245C, but not DK-PGD2, inhibit the migration of DCs in response to the chemokines CCL5 and CCL19, which are instrumental to the recruitment of DCs to regional lymph nodes in vivo. Moreover, stimulation of DCs with PGD2, 15-dPGJ2, or BW245C inhibited their generation of the Th1-promoting cytokine IL-12 and the Th1-active chemokine CXCL10 in response to either dust mite antigen or LPS. When naive T cells were co-cultured with antigen-pulsed DCs, their secretion of IL-4 by was enhanced when the DCs had been stimulated with either PGD2 or BW245C during their stimulation with antigen [31]. Thus, PGD2 may use both DP1- and DP2-mediated mechanisms to control DC migration and maturation, with a profile that may favor the induction of a Th2 response. Such functions are supported by data derived from animal models (see below). Effects on Smooth Muscle Earlier studies implicated the Gq-coupled thromboxane A2 (TXA2) receptor (known as the TP receptor) in mediating PGD2-dependent bronchconstriction in vivo, based on studies in humans using an orally-active TP receptor antagonist, BAY u3405 (ramatroban) [32]. Consistent with this hypothesis, a PGD2 metabolite, 8-epi-prostaglandin F2␣, binds the TP receptor in vitro [33]. Interestingly, a stable metabolite of TXA2, 11-dehydro-TXB2, is a full agonist for recombinant DP2 receptors [34]. Thus, PGD2 and TXA2, two products of PGH2 metabolism, may be functionally promiscuous due to reciprocal crossreactivity of their metabolites on each other’s receptors in the airways. Since ramatroban has subsequently proven to block DP2 receptors [35], the identity of the receptor(s) responsible for mediating PGD2-induced bronchoconstriction remains undefined [36]. To date, no specific antagonists of DP receptors have been made available for therapeutic use in humans. Functions of PGD2 and Its Receptors in vivo: Mouse Models Allergen-Induced Pulmonary Inflammation Several lines of evidence from mouse studies support key roles for PGD2 in allergen-induced pulmonary inflammation in vivo. Allergen-sensitized transgenic

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mice that ectopically express L-PGDS in the lung generate higher levels of PGD2 than do sensitized wild-type control mice when they are challenged by allergen inhalation [37]. The challenged transgenic mice also exhibited higher bronchial eosinophil counts, higher lung levels of T-helper class 2 (Th2)type cytokines IL-4 and IL-5, and lower levels of the Th1 cytokine interferon (IFN)-␥, when compared with wild-type controls. In another study, the administration of a single dose of inhaled PGD2 to allergen-sensitized wild-type mice dramatically potentiated the expression of Th2 cytokines in the lung in response to subsequent inhalation of low-dose antigen [38]. This effect was associated with strongly induced expression of macrophage-derived chemokine, a selective chemoattractant for Th2 cells, by the lung in response to PGD2. Thus, PGD2 favors Th2 responses in the lung under experimental conditions where its levels are high. Mice lacking DP1 receptors exhibited markedly diminished pulmonary inflammation, lung Th2 cytokine expression, and AHR in a mouse model of allergen-induced lung inflammation [39], despite developing elevations of allergen-specific IgE that were comparable to those observed in the sensitized wild-type control animals. In a mouse model of in vivo antigen presentation, the intratracheal administration of BW245C, but not DK-PGD2, blocked the migration antigen-loaded DCs to regional lymph nodes [40]. This observation is consistent with the aforementioned capacity of DP1 receptormediated stimulation of DCs to block subsequent chemokine-mediated migration responses in vitro [31]. Together, these studies suggest that the prominent functions of PGD2 in experimentally-induced allergic pulmonary inflammation likely reflect both direct and indirect effects on effector cell chemotaxis and activation, antigen presentation, and Th2 cell stimulation. It is noteworthy that the epithelially-derived anti-inflammatory protein, uteroglobulin, homeostatically inhibits Th2-polarized pulmonary inflammation by interfering with DP receptordependent signaling events [41]. The therapeutic potential of drugs that specifically target PGD2 synthesis or DP receptors in allergic diseases thus seems high. Cysteinyl Leukotrienes Biosynthesis LT production by MCs requires the reversible translocation of 5-LO from the nucleoplasm or cytosol to the perinuclear region [42]. 5-LO acts in concert with 5-LO-activating protein (FLAP) to convert arachidonic acid sequentially to the unstable intermediates 5-hydroperoxyeicosatetraenoic acid (5-HPETE) and then to LTA4 [43]. LTA4 is either converted by LTA4 hydrolase [44] to LTB4 (the dominant event in neutrophils) or is conjugated to reduced glutathione by LTC4S [45], an integral perinuclear membrane protein with homology to FLAP,

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to form LTC4, the first committed molecule in cysLT synthesis. LTC4 is released by a distinct, energy-dependent export mechanism [46] involving multidrug resistance protein (MRP)-1 [47]. LTC4 is converted sequentially to LTD4 by extracellular ␥-glutamyl transferase [48] or by a more specific ␥-glutamyl leukotrienase [49], and then to LTE4 by a dipeptidase [50]. LTE4, the least bioactive of the cysLTs, is also the most stable in extracellular fluids and is excreted in the urine without additional modification [51]. Actions in Humans: Direct Challenges As is the case for PGD2, BAL fluids obtained from allergen-challenged subjects with asthma contain increased quantities of cysLTs compared with those obtained from atopic subjects without asthma [52]. Urinary levels of LTE4 increase during spontaneous exacerbations of asthma, and decrease during attack-free intervals [51]. CysLTs exert powerful contractile effects on isolated human bronchi [53] and pulmonary vascular smooth muscle [54] in vitro, as well as isolated guinea pig ileum [55]. Subsequent direct challenges performed on human subjects validated these smooth muscle-based activities in vivo. In one such study, inhaled LTE4 induced bronchoconstriction in asthmatic subjects at concentrations ⬃3 log-fold lower than did histamine [56]. In another study, LTE4 inhalation induced the recruitment of eosinophils, as well as smaller numbers of neutrophils, to the bronchial mucosa of human subjects with asthma [57]. Intradermal challenge of normal human subjects with nanomolar amounts of LTC4, LTD4, and LTE4 induced sustained wheal-and-flare responses [23]. These early laboratory and clinical observations implicated the cysLTs in the pathophysiology of bronchconstriction and vascular leak, and suggested the possible involvement of cysLTs in the smooth muscle dysfunction and inflammation of asthma. These disease-related functions were later confirmed by studies in human subjects using biosynthetic inhibitors and receptor antagonists (see below). Receptors for cysLTs and Receptor-Mediated Functions in vitro As is the case for PGD2, cysLTs bind and activate two specific GPCRs, respectively termed CysLT1 and CysLT2 receptors. Both receptor classes have been cloned and characterized in human [58, 59] and mouse [60, 61]. These two receptors are only loosely homologous to one another (38% amino acid sequence identity for the human receptors), and exhibit different respective profiles of ligand affinity. CysLT1 receptors preferentially bind LTD4 (Kd ⬃1 nM ) over LTC4 (Kd ⬃10 nM), whereas CysLT2 receptors bind LTC4 and LTD4 with equal affinity (Kd ⬃10 nM). Both CysLT1 and CysLT2 receptors bind LTE4 with comparatively low (Kd ⬃100 nM) affinity. In humans, CysLT1 receptors are richly expressed by bronchial smooth muscle cells [58], and peripheral blood leukocytes [62]. CysLT1 protein localizes to neutrophils,

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eosinophils, macrophages and MCs in nasal biopsies from patients with chronic rhinitis [63]. CysLT2 receptors show an overlapping tissue distribution with that of CysLT1 receptors [59], but are far more abundant than are CysLT1 receptors on certain endothelial cell populations [64]. Moreover, the tissue distribution of CysLT2 receptor mRNA, but not that of CysLT1 receptor mRNA, includes the brain, adrenal glands, and cardiac Purkinje cells [59, 61]. The differences between CysLT1 and CysLT2 receptors in structure, ligand binding, and distribution of expression correlate with divergent biologic functions both in vitro and in vivo as noted below. Effects on Smooth Muscle and Endothelial Cells In addition to their contractile effects, cysLTs also induce proliferation of smooth muscle in vitro. Stimulation of cultured human bronchial smooth muscle cells potently amplified their proliferation in response to exogenous mitogens such as epidermal growth factor (EGF) [65], IL-13, or transforming growth factor-␤ [66]. In one study, LTD4-mediated proliferation was blocked by pranlukast, a selective competitive antagonist for CysLT1 receptors, but curiously was resistant to blockade by zafirlukast, another CysLT1 receptor antagonist [65]. These studies did not reveal a residual CysLT2-dependent effect. While CysLT1 is dominant for cysLT-dependent effects on airway smooth muscle, CysLT2 receptors are dominant for cysLT-dependent calcium signaling in human umbilical vein endothelial cells [64], even though these cells do express CysLT1 receptors. In vivo studies using selective receptor blockade and receptor-null mouse strains (see below) further support distinct functions of CysLT1 and CysLT2 receptors in smooth muscle and microvascular responses, respectively. Effects on Leukocytes Several immune cell types (macrophages, eosinophils, MCs) express both CysLT1 and CysLT2 receptors [62, 63, 67–71] suggesting the potential for these two receptors to regulate certain inflammatory cellular functions in a complementary manner. CD34⫹ progenitor cells from human peripheral blood exhibit calcium flux, actin polymerization, and migration in response to stimulation with cysLTs in vitro, each of which are blocked by pretreatment with a CysLT1 receptor-selective antagonist, MK571. In contrast, LTD4-mediated calcium fluxes in mixed populations of mature human granulocytes are resistant to inhibition by MK571, suggesting that CysLT2 receptors are acquired during later stages of granulocyte development [72]. Human MCs (hMCs) derived in vitro from umbilical cord blood respond to exogenous cysLTs with a strong calcium flux [69] and phosphorylation of extracellular signal-regulated kinase (ERK) [70], both of which were blocked completely by MK571. Priming of these hMCs with recombinant IL-4 markedly enhanced their sensitivity to

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cysLT-mediated calcium fluxes [69], especially that mediated by LTC4, and modestly enhanced surface expression of CysLT2 receptors without altering CysLT1 receptor expression [71]. Surprisingly, the nucleotide uridine diphosphate (UDP) induced a calcium flux in IL-4-primed hMCs that was also sensitive to blockade with MK571, and that cross-desensitized with LTC4, indicating a potential IL-4-inducible alteration in receptor specificity. LTC4, LTD4, and UDP all induced the generation of IL-5, TNF-␣, macrophage inflammatory protein (MIP)-1␤, and IL-8 by IL-4-primed hMCs, but not by their unprimed replicates [71]. While cysLT-and UDP-induced production of IL-5, TNF-␣, and MIP-1␤ was substantially attenuated by MK571, the production of IL-8 in response to these same ligands was MK571-resistant [70]. Both MK571 and a FLAP inhibitor, MK886, decreased IL-5 and TNF-␣ production by primed hMCs stimulated by Fc␧RI cross-linkage, implying autocrine signaling by cysLTs in MCs [71]. An ‘intracrine’ role for LTC4 was implicated in the secretion of preformed IL-4 by human peripheral blood eosinophils stimulated by chemokines or IL-16 [73]. The latter effect occurs independently of CysLT1 and CysLT2, and involves an uncharacterized intracellular receptor. Thus, cysLTs, acting through the complementary yet distinct functions of CysLT1 and CysLT2 receptors and possibly through a third intracellular receptor, can amplify or induce cytokine generation by effector cells, which could explain some of their effects in acute and chronic inflammatory responses (see below).

Functions of cysLTs and Their Receptors in vivo: Pharmacologic Studies in Humans Asthma Orally-active inhibitors of 5-LO [74] and of the CysLT1 receptor [75] both improved indices of lung function and asthma control compared with placebo in human subjects with asthma. Treatment of human asthmatic subjects with CysLT1 receptor antagonists improved their FEV1, while decreasing sputum eosinophil counts [76]. Pretreatment of allergic asthmatic human subjects with Pranlukast attenuates the decrease in airflow during both early and late asthmatic responses induced by allergen challenge [77]. Orally administered CysLT1 receptor antagonists also attenuate bronchoconstriction in response to experimental challenges with exercise [78], cold air [79], and inhalation of adenosine [80]. Intravenously administered montelukast, another CysLT1 receptor-selective antagonist, substantially increased measures of airflow compared with placebo in a group of patients presenting to the emergency room with acute asthma who also received standard treatment with bronchodilators and glucocorticoids [81]. These observations confirm the involvement of cysLTs in the development of airflow obstruction in

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both experimentally-induced and naturally occurring asthma in humans, and with a prominent role for the CysLT1 receptor in exacerbations. Functions of cysLTs and their Receptors in vivo: Disease Models in Mice Microvascular Responses Mice with a targeted deletion of LTC4S are unable to generate cysLTs in most tissues and organs, and completely lack MC-derived cysLTs [82]. While cysLTs do not cause bronchoconstriction in mice [83, 84], both LTC4Sdeficient mice and CysLT1 receptor-deficient mice display markedly less tissue swelling in response to a localized MC-dependent cutaneous allergic response (passive cutaneous anaphylaxis (PCA)) than do wild-type controls [82, 85]. Surprisingly, mice lacking CysLT2 receptors behave similarly to those lacking LTC4S or CysLT1 receptors in the PCA model [86], but unlike the other two knockout strains exhibit no decrement in plasma exudation in a zymosaninduced model of peritonitis. These observations confirm that the regional differences in CysLT1 and CysLT2 receptor expression correspond to distinct functions for each. Specifically, the two receptors cooperate in the microvasculature of the skin, but not that of the peritoneum, to induce permeability changes. Allergen-Induced Pulmonary Inflammation While the LTC4S, CysLT1, and CysLT2 knockout strains have not been studied in the context of experimentally-induced allergic pulmonary inflammation to date, mice lacking 5-LO showed attenuated eosinophilic bronchial inflammation, AHR, and allergen-specific IgE compared with wild-type controls when subjected to allergen sensitization and challenge [87, 88]. A similar phenotype was recognized in mice lacking cPLA2, which cannot generate either LTs or PGs [89]; IgE levels were not reported in this study. These studies are consistent with prominent functions for 5-LO pathway products in the induction and/or effector phases of allergic responses. In another model of ovalbumin-induced allergic pulmonary inflammation in mice, where the period of allergen challenge was extended to 76 days to elicit changes of airway remodeling, the administration of the selective CysLT1 receptor antagonist montelukast during the challenge phase sharply decreased lung expression of Th2 cytokines and bronchial eosinophil numbers, while inhibiting smooth muscle hypertrophy and collagen deposition [90]. These findings are thus consistent with the mitogenic effect of cysLTs on bronchial smooth muscle suggested by in vitro studies [65, 66].

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Dendritic Cell Maturation and Migration The decrement in allergen-specific IgE identified in 5-LO knockout mice [87, 88] suggested the potential involvement of cysLTs in proximal events in the allergic immune response. Mice lacking MRP1 cannot secrete cysLTs, and show defective migration of DCs in a model of cutaneous hypersensitivity [91]. Mouse myeloid DCs exposed to exogenous cysLTs during ex vivo pulsing with dust mite antigen show augmented production of IL-10 and attenuated IL-12 generation compared with antigen pulsing alone. Conversely, treatment of these DCs with CysLT1 receptor-selective antagonists during antigen pulsing attenuates IL-10 generation and augments IL-12 production [92]. Blockade of CysLT1 receptors on myeloid DCs during stimulation with dust mite allergen in vitro substantially diminished their ability to support an eosinophil-dominated inflammatory response to inhaled allergen following their adoptive transfer into the tracheas of naive recipient mice. Thus as is the case for PGD2, cysLTs participate in the regulation of dendritic cell maturation responses, with consequent downstream effects on antigen-mediated T-cell responses, supporting a potential role for MC-derived eicosanoids in regulating proximal events in mucosal immunity. Pulmonary Fibrosis Previous studies demonstrating that mice lacking cPLA2 [93] and 5-LO [94] were each protected from pulmonary injury induced by challenge with bleomycin led to studies examining the potential role of cysLTs in this process. Wild-type C57/BL6 mice exhibited marked neutrophilic inflammation in response to intratracheal administration of bleomycin, followed by fibrosis. Mice lacking LTC4S and CysLT2 receptors each exhibit substantial protection from bleomycininduced inflammation and fibrosis, whereas CysLT1 receptor-deficient mice were not protected [86]. Thus the pro-fibrotic effects of the cysLTs are entirely CysLT2 receptor-dependent in this model. The involvement of cysLTs as mediators of fibrosis could relate to the long-held suspected role of MCs as mediators of fibrotic diseases.

Regulation of Eicosanoid Synthesis by MCs

Heterogeneity of Eicosanoid Generation by Tissue MC Subsets In both rodents and humans, there is strong evidence that MCs in different tissue compartments vary widely in their capacity to produce eicosanoids. The effector phase of the immune response to intestinal helminths in rodents is characterized by a marked hyperplasia of MCs in the involved intestinal mucosa [95].

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These ‘mucosal’ MCs are necessary for elimination of several worm species, and in contrast to the MCs residing constitutively in adjacent connective tissues (‘connective tissue MCs’), require normal T cells for their development in vivo [96]. A parallel exists in humans, as MC numbers in the intestinal mucosa are markedly depleted in tissue samples from patients with T-cell immunodeficiencies, even though adjacent connective tissues contain normal numbers of MCs [97]. Moreover, MC hyperplasia in the involved mucosal epithelium is also a feature of asthma, rhinitis, conjunctivitis, and allergic esophagitis in humans [98–101]. The selective mucosal epithelial hyperplasia of MCs in allergic or parasitic diseases likely reflects the costimulatory effects of Th2-derived cytokines, several of which (IL-3, IL-4, IL-5, IL-6, IL-9, IL10) synergize in vitro with the obligate MC growth factor, stem cell factor (SCF), to enhance proliferation and/or cytoprotection of MCs in mouse [102–104], human [105–107], or both species. MCs isolated from the enzymatically dispersed intestinal mucosa from rats that had been infected experimentally with the nematode Nippostrongylus brasiliensis generated abundant quantities of PGD2, cysLTs, and LTB4 when challenged ex vivo with Fc␧RI cross-linkage [108]. In contrast, the MCs harvested from the peritoneal cavities of the same rats (a location where MC development is not affected by T cells) generated only PGD2 when activated. MCs obtained from human tissue specimens also display marked regional variability in LT generation. MCs from normal human skin generate small amounts of cysLTs (3.5 ng/106 cells) following Fc␧RI cross-linkage in vitro [4], while human uterine, lung, and intestinal MCs generate between 6- and 10-fold more cysLTs than do skin hMCs [5–7, 109, 110]. By contrast, all human MC subsets produce PGD2 ranging from ⬃20 ng/106 cells (for intestinal MCs) to as much as 80 ng/106 cells (for uterine MCs). Thus both human and rodent MCs exhibit heterogeneity of eicosanoid production, with relatively wide (10-fold) variations in cysLT production among MC subpopulations, compared with abundant and somewhat less variable amounts of PGD2 generation. These observations suggest that the PGHS/PGDS pathway may be developmentally regulated and, at least in part, constitutively expressed. In contrast, the prominence of 5-LO/LTC4S pathway activity in MCs from mucosal locations (particularly in mucosal inflammation) implies that this pathway may be inducible by T-cell-derived cytokines and other elements of the inflammatory microenvironment.

Regulation of Eicosanoid Pathways in vitro by Exogenous Cytokines PGHS/PGDS Pathway While hMCs absolutely require SCF for their derivation in vitro, an immature population of mouse bone marrow-derived MCs (mBMMCs) can be developed

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in vitro independently of SCF using medium supplemented with recombinant IL-3. These IL-3-derived mBMMCs express cPLA2, constitutive PGHS-1, and PGDS, and generate small amounts of PGD2 when activated by Fc␧RI crosslinkage ex vivo. Treatment of mBMMCs with recombinant SCF upregulated their expression of cPLA2, PGHS-1, and hematopoietic PGDS and primed them for augmented PGD2 generation in response to Fc␧RI cross-linkage [111]. In another study, stimulation of IL-3-derived mBMMCs with a triad of SCF, IL-10, and IL-1␤ induced their expression of PGHS-2, and directly elicited PGD2 synthesis in a delayed, PGHS-2-dependent manner [112]. These in vitro studies imply that PGD2 generation by MCs can be regulated by exogenous factors that control levels of expression of the terminal synthase, PGDS, as well as upstream enzymes, including the constitutive and inducible forms of PGHS. IL-1␤ acts through receptors that induce NF-␬B activation and share signal transduction mechanisms with toll-like receptors (TLRs) [113]. Since TLRs are also expressed by MCs [114] and induce PGHS-2 expression in other cell types [115], it is tempting to speculate that PGHS-2 and PGD2 biosynthetic capability may be upregulated in MCs during their innate responses to microbial pathogens (fig. 1). MCs express a highly lineage-restricted guanine nucleotide exchange factor, RasGRP4 [116]. The immature human MC line, HMC-1, expresses a nonfunctional splice variant of RasGRP4, and does not express PGDS or generate substantial amounts of PGD2 when activated by calcium ionophore. Transfection of HMC-1 cells with wild-type human RasGRP4 induces their expression of PGDS and permits their ionophore-mediated generation of PGD2. Conversely, inhibition of RasGRP4 expression using small interfering RNA in a rat peritoneal MC line down-regulates PGDS expression [116]. The micropthalmia transcription factor (MITF) is a basic-helix-loop-helix-leucine-zipper protein highly restricted in its expression to MCs and melanocytes, and regulates the expression of several MC-specific proteases. Recently, MITF was shown to be important for transactivation of the H-PGDS gene. Moreover, mBMMCs derived from MITF-deficient mice failed to express H-PGDS, and did not generate PGD2 when activated [117]. It is thus likely that the obligate constitutive interaction between SCF and its receptor, Kit, which is required for normal MC development in vivo [118], initiates downstream lineage-restricted effectors such as RasGRP4 and MITF that are responsible for the transcriptional and/or post-transcriptional control of PGDS expression. This constitutive control system may explain why all rodent and human tissue MC subsets studied to date produce PGD2 in abundance. Moreover, MCs likely have the capacity to augment their production of PGD2 by upregulating proximal enzymes such as PLA2 and/or PGHS-2 to in circumstances of inflammation (fig. 1).

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5-LO/LTC4S Pathway T-cell-derived cytokines that mediate MC development at inflamed mucosal surfaces also control 5-LO/LTC4S pathway activity in both mouse and human MCs in vitro. When mBMMCs are developed in vitro with a combination of SCF and IL-10, they proliferate markedly in response to IL-3 [119]. This proliferative response is associated with sharply upregulated expression of 5-LO, FLAP, and LTC4S, and a marked increase in Fc␧RI-mediated cysLT generation compared with control mBMMCs maintained under the original conditions of SCF and IL-10. Cord blood-derived hMCs derived in the cytokine triad of SCF, IL-6, and IL-10 generate few cysLTs when activated by Fc␧RI cross-linkage, but produce nanogram quantities of PGD2 [120], a profile reminiscent of that reported for MCs from normal human skin [4]. Priming of these hMCs with IL-4 for 5 days resulted in a 27-fold increase in Fc␧RI-dependent cysLT production, with only a 2-fold increase in PGD2 generation. This priming event was associated with profound and rapid (within hours) IL-4-dependent induction of LTC4S mRNA and protein expression by hMCs, increasing the activity of the terminal enzyme necessary for the conversion of LTA4 to LTC4. Priming of cultured hMCs with either IL-3 or IL-5 also modestly increased their biosynthetic capacity for cysLT, without any quantitative changes in cPLA2, FLAP, 5-LO, or LTC4S expression. This priming event relates to a nuclear import of cytosolic 5-LO stores. Priming with IL-4 in the presence of either IL-3 or IL-5 augments Fc␧RI-mediated cysLT generation to ⬃30 ng/106 hMCs. The synergy between the Th2 cytokines for amplifying the Fc␧RI-mediated cysLT-generating capacity of MCs is due to their actions at non-redundant distal (IL-4) and proximal (IL-3, IL-5) control points of the 5-LO/LTC4S pathway (fig. 1), indicating a process of coordinate regulation of the pathway that may occur during allergic mucosal inflammation. This process could provide enhanced cysLT synthesis to mediate smooth muscle constriction, vascular leak, and inflammatory cell recruitment and activation in Th2-dominated allergic inflammation. If sustained, enhanced 5-LO/LTC4S pathway activity could lead to matrix overgrowth and smooth muscle hyperplasia, as is observed in the histopathology of chronic asthma, nasal polyposis, and other clinical circumstances where both Th2 cytokines and increased cysLT generation occur concomitantly.

Summary

The ability of MCs to synthesize and secrete both PGD2 and LTC4 in substantial quantities is unique among hematopoietic cells, and likely evolved as effector properties that facilitate MC functions in host defense, homeostasis, and tissue repair. Although some functions of these two eicosanoid classes

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appear redundant or complementary in asthma and allergic inflammation, the widely heterogeneous profiles of eicosanoid production by the MCs from different microenvironments, and the differential loci of regulation for each pathway, also implies separate and distinct functions for each product. The identification of receptors for both PGD2 and cysLTs, the definition of functions for each receptor, and the development of receptor knockout mice have each advanced the biology of eicosanoids substantially. CysLT1 receptor antagonists and 5-LO inhibitors have validated clinical use. The development of CysLT2 antagonists and orally-active antagonists of DP receptors await therapeutic exploration.

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Joshua A. Boyce, MD, Assoc. Prof. Med. Harvard Medical School, Co-Director, Research Section of Inflammation and Allergic Diseases Division of Rheumatology, Immunology and Allergy, Brigham and Women’s Hospital Smith Research Building, Rm 626, 1 Jimmy Fund Way, Boston, MA–02115 (USA) Tel. ⫹1 617 5251261, Fax ⫹1 617 5251260, E-Mail [email protected]

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Saito H, Okayama Y (eds): Mast Cells in Allergic Diseases. Chem Immunol Allergy. Basel, Karger, 2005, vol 87, pp 80–84

Role of Mast Cell Proteases in Tissue Remodeling Hirohisa Saito Department of Allergy and Immunology, National Research Institute for Child Health and Development, Setagaya, Tokyo, Japan

Abstract Mast cells specifically localize within the bronchial smooth muscle bundles in patients with asthma but not in normal individuals. Tryptase and other proteases such as chymase are abundant in mast cell granules and have recently been reported to be involved in the proliferation of airway smooth muscle cells. In this chapter, therefore, the role of proteases other than fibrolytic activity is reviewed. Copyright © 2005 S. Karger AG, Basel

Introduction

Mast cells, but not T cells or eosinophils, localize within the bronchial smooth muscle bundles in patients with asthma but not in normal individuals or those with eosinophilic bronchitis. Smooth muscle mast cell density correlates significantly with indices of bronchial hyperresponsiveness, and is likely to be an important factor determining the asthmatic phenotype [1]. Tryptase and other proteases such as chymase are abundant in mast cell granules and have recently been reported to be involved in the proliferation of airway smooth muscle cells. In this chapter, therefore, the role of proteases other than fibrolytic activity is reviewed.

Effect of Tryptase on Tissue Cell Proliferation and Remodeling

On the basis of their amino acid sequences, tryptase is just a serine proteinase related to trypsin expressed and stored in mast cells rather than the

pancreas. On the basis of their biochemical and biological features, however, tryptases show little family likeness to trypsin and most other trypsin-like proteases. The intriguing discrepancies have been explained by the crystal structure of the tryptase tetramer [2]. For the specific effect of tryptase, heparin binding is reported to be necessary [3]. Proteolytically activated receptors (PARs) represent a subset of seven transmembrane G-protein-coupled receptors that mediate cell activation events by receptor cleavage at distinct scissile bonds located within receptor amino termini. PAR-1 to PAR-4 genes can be identified within a PAR gene cluster spanning approximately 100 kilobases at 5 q13. [4]. Mast cell tryptase was initially found to regulate PAR-2 expressed by rat colonic myocytes [5]. Tryptase and other trypsin-like enzymes can interact with PAR-2, thus this action differs from its protease activity. As a mitogenic factor for tissue cells, tryptase was reported in 1991 to promote the proliferation of fibroblasts in vitro [6]. Later, various tryptase activities were found to be mediated via PAR-2 such as the activation of colonic myocytes [7] and aortic smooth muscle cells [8]. At least regarding the fibroblast activation, tryptase is considered to be one of the active mediators among all mast cell-derived mediators and cytokines [9].

Effect of Tryptase on Airway Smooth Muscle Cells and Epithelium

Contribution of mast cell tryptase to hyperplasia of airway smooth muscle cells was reported in 1995 using cultured dog tracheal smooth muscle cells [10]. These authors found that tryptase is a potent mitogen for airway smooth muscle cells and that tryptase-induced mitogenesis is not a nonspecific effect of all serine proteinases, because thrombin, another proteinase with mitogenicity for fibroblasts, did not stimulate increases in cell counts. This observation probably suggests that this may be via activation of PAR-2 that had been found later. Then, PAR-2 was found to be present in human bronchial smooth muscle. Activation via PAR-2 results in constriction of human airway smooth muscle [11]. Tryptase-induced mitogenesis in airway smooth muscle cells requires activation of ERK1 and 2 that these responses depend partially, but not completely, upon tryptase’s properties as a protease [12]. In addition to PAR-2-mediated activation, therefore, tryptase also acts as a protease for the proliferation of dog tracheal smooth muscle cells. However, the same authors reported that tryptase’s mitogenic effects in human airway smooth muscle cells are via nonproteolytic actions [13]. These authors found that irreversibly abolishing tryptase’s catalytic activity does not alter its effects on increases in DNA synthesis, and concluded that ␤-tryptase is a potent mitogenic serine protease

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in cultured human airway smooth muscle cells and its growth stimulatory effects in these cells occur predominantly via nonproteolytic actions. Thus, for the mitogenesis of human airway smooth muscle cells, PAR-2 is the major signal pathway of the effect of tryptase. Compared to normal human airway epithelium, PAR-2 expression is upregulated among the asthmatic airway epithelium. The upregulated expression is not influenced by inhaled corticosteroids [14]. Regarding different activities between tryptase and other trypsinlike enzymes, Compton et al. [15] have examined these effects on the relaxation of rat aorta. Removal of heparin from tryptase tetramer or surface sialic acid from the target cells completely abolished the effect of tryptase but not other trypsin-like enzymes. On the other hand, both tryptase-induced and other trypsin-like enzyme-induced relaxation was inhibited by either removal of the endothelium or pretreatment of the tissue with NG-nitro-L-arginine methyl ester (L-NAME), suggesting an endothelium-derived nitric oxide mechanism. Mast cell-derived tryptase acts on human airway smooth muscles and epithelium, and thus plays a role in tissue remodeling. However, its mechanisms may be dependent on both tryptase-unique protease activity and PAR-2 that are expressed by the target cells.

Chymase and Other Proteases Related to Tissue Remodeling

Mast cell chymase is abundantly expressed by human skin mast cells but is scantly expressed by human lung mast cells. Contrary to the effect of tryptase which both skin and lung mast cells abundantly express, chymase inhibits the proliferation of human airway smooth muscle cells [16]. However, chymase degrades the smooth muscle cell pericellular matrix, suggesting that the local release of mast cell chymase may have profound effects on airway smooth muscle cell function and airway remodeling. The clinical effect of chymase has been reported regarding atopic dermatitis more frequently than regarding asthma such as increase in chymase expression among patients with atopic dermatitis [17]. Recently, Iwanaga et al. [18] have found that polymorphism of the mast cell chymase gene (CMA1) promoter region is associated with serum total immunoglobulin E levels in adult atopic dermatitis but is not associated with asthma. Chymase is also known to abundantly exist in mast cell granules present in the human heart. Regarding this issue, chymase is reported to participate in acute inflammation and the remodeling process of viral myocarditis [19]. Other abundant mast cell-derived proteases such as carboxypeptidases and matrix metalloproteinases are reported to work on the fibrolytic process and are reviewed in another chapter.

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Conclusion

Mast cells seem to play a crucial role in airway remodeling by releasing tryptase on smooth muscle and epithelium, and may play a role in skin tissue remodeling by releasing chymase in an IgE-dependent manner in allergic diseases.

References 1 2 3

4 5

6 7

8

9 10

11

12

13

14

15

16

Brightling CE, Bradding P, Symon FA, Holgate ST, Wardlaw AJ, Pavord ID: Mast-cell infiltration of airway smooth muscle in asthma. N Engl J Med 2002;346:1699–1705. Sommerhoff CP: Mast cell tryptases and airway remodeling. Am J Respir Crit Care Med 2001; 164:S52–S58. Humphries DE, Wong GW, Friend DS, Gurish MF, Qiu WT, Huang C, Sharpe AH, Stevens RL: Heparin is essential for the storage of specific granule proteases in mast cells. Nature 1999;400:769–772. Hoogerwerf WA, Hellmich HL, Micci MA, Winston JH, Zou L, Pasricha PJ: Molecular cloning of the rat proteinase-activated receptor 4 (PAR-4). BMC Mol Biol 2002;3:2. Corvera CU, Dery O, McConalogue K, Bohm SK, Khitin LM, Caughey GH, Payan DG, Bunnett NW: Mast cell tryptase regulates rat colonic myocytes through proteinase-activated receptor 2. J Clin Invest 1997;100:1383–1393. Ruoss SJ, Hartmann T, Caughey GH: Mast cell tryptase is a mitogen for cultured fibroblasts. J Clin Invest 1991;88:493–499. Corvera CU, Dery O, McConalogue K, Bohm SK, Khitin LM, Caughey GH, Payan DG, Bunnett NW: Mast cell tryptase regulates rat colonic myocytes through proteinase-activated receptor 2. J Clin Invest 1997;100:1383–1393. Molino M, Raghunath PN, Kuo A, Ahuja M, Hoxie JA, Brass LF, Barnathan ES: Differential expression of functional protease-activated receptor-2 (PAR-2) in human vascular smooth muscle cells. Arterioscler Thromb Vasc Biol 1998;18:825–832. Gailit J, Marchese MJ, Kew RR, Gruber BL: The differentiation and function of myofibroblasts is regulated by mast cell mediators. J Invest Dermatol 2001;117:1113–1119. Brown JK, Tyler CL, Jones CA, Ruoss SJ, Hartmann T, Caughey GH: Tryptase, the dominant secretory granular protein in human mast cells, is a potent mitogen for cultured dog tracheal smooth muscle cells. Am J Respir Cell Mol Biol 1995;13:227–236. Schmidlin F, Amadesi S, Vidil R, Trevisani M, Martinet N, Caughey G, Tognetto M, Cavallesco G, Mapp C, Geppetti P, Bunnett NW: Expression and function of proteinase-activated receptor-2 in human bronchial smooth muscle. Am J Respir Crit Care Med 2001;164:1276–1281. Brown JK, Jones CA, Rooney LA, Caughey GH: Mast cell tryptase activates extracellular-regulated kinases (p44/p42) in airway smooth-muscle cells: Importance of proteolytic events, time course, and role in mediating mitogenesis. Am J Respir Cell Mol Biol 2001;24:146–154. Brown JK, Jones CA, Rooney LA, Caughey GH, Hall IP: Tryptase’s potent mitogenic effects in human airway smooth muscle cells are via nonproteolytic actions. Am J Physiol 2002;282: L197–L206. Knight DA, Lim S, Scaffidi AK, Roche N, Chung KF, Stewart GA, Thompson PJ: Proteaseactivated receptors in human airways: Upregulation of PAR-2 in respiratory epithelium from patients with asthma. J Allergy Clin Immunol 2001;108:797–803. Compton SJ, McGuire JJ, Saifeddine M, Hollenberg MD: Restricted ability of human mast cell tryptase to activate proteinase-activated receptor-2 in rat aorta. Can J Physiol Pharmacol 2002;80: 987–892. Lazaar AL, Plotnick MI, Kucich U, Crichton I, Lotfi S, Das SK, Kane S, Rosenbloom J, Panettieri RA Jr, Schechter NM, Pure E: Mast cell chymase modifies cell-matrix interactions and inhibits

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mitogen-induced proliferation of human airway smooth muscle cells. J Immunol 2002;169: 1014–1020. Badertscher K, Bronnimann M, Karlen S, Braathen LR, Yawalkar N: Mast cell chymase is increased in chronic atopic dermatitis but not in psoriasis. Arch Dermatol Res 2005;296:503–506. Iwanaga T, McEuen A, Walls AF, Clough JB, Keith TP, Rorke S, Barton SJ, Holgate ST, Holloway JW: Polymorphism of the mast cell chymase gene (CMA1) promoter region: Lack of association with asthma but association with serum total immunoglobulin E levels in adult atopic dermatitis. Clin Exp Allergy 2004;34:1037–1042. Kitaura-Inenaga K, Hara M, Higuchi K, Yamamoto K, Yamaki A, Ono K, Nakano A, Kinoshita M, Sasayama S, Matsumori A: Gene expression of cardiac mast cell chymase and tryptase in a murine model of heart failure caused by viral myocarditis. Circ J 2003;67:881–884.

Hirohisa Saito, MD, PhD Department of Allergy and Immunology National Research Institute for Child Health and Development 2–10–1, Okura, Setagaya, Tokyo 157–8535 (Japan) Tel. ⫹81 3 5494 7027, Fax ⫹81 3 5494 7028, E-Mail [email protected]

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Mast Cell Mediators in Airway Remodeling Chad K. Oh Division of Allergy and Immunology, Department of Pediatrics, UCLA School of Medicine, Harbor-UCLA Medical Center, Torrance, Calif., USA

Abstract Increasing evidence indicates that airway remodeling plays an important part in asthma pathogenesis. However, mechanisms underlying airway remodeling are not yet fully elucidated. Plasminogen activator inhibitor (PAI)-1 is the main inhibitor of the fibrinolytic system and is known to play an essential role in tissue remodeling. Recent evidence indicates that chronic asthma may lead to tissue remodeling such as subepithelial fibrosis and extracellular matrix (ECM) deposition in the airways. Recently, the mast cell (MC), which plays a major role in asthma, is found as a novel source of PAI-1 and a large number of MCs expressing PAI-1 are infiltrated in the airways of patients with severe asthma. Furthermore, PAI-1-deficient mice show reduced ECM deposition in the airways of a murine model of chronic asthma. In a human study, the 4G allele frequency was significantly higher in the asthmatic patients than in the control group. In view of the findings that the 4G allele is associated with elevated plasma PAI-1 level, elevated PAI-1 level in the lung may contribute to the development of airway remodeling. In summary, MCs may play an important role in the pathogenesis of asthma in part by producing PAI-1. Further studies evaluating the mechanisms of PAI-1 action may provide a new paradigm in airway remodeling and lead to the development of a novel therapeutic target. Copyright © 2005 S. Karger AG, Basel

Introduction

Hallmarks of airway remodeling include thickening of the reticular basement membrane, mucus gland hypertrophy, angiogenesis, deposition of extracellular matrix (ECM) components, and increased smooth muscle mass [1]. Although structural changes may result in reduced airflow, the role of remodeling in determining asthma severity is controversial [2–5]. The relationship between inflammation and remodeling in asthma is not fully established. Although remodeling may be a consequence of repeated injury and persistent inflammation,

recent evidence suggests that airway inflammation and remodeling can be parallel or interdependent processes [2, 5–7]. Recent studies demonstrated the inefficacy of anti-inflammatory therapy in some patients with asthma, suggesting that an unregulated pathologic tissue remodeling process occurs in spite of adequate anti-inflammatory therapy [8, 9]. Tissue remodeling usually involves two distinct processes: physiologic remodeling or regeneration, which is the replacement of injured tissue by parenchymal cells of the same type, and pathologic remodeling, which is the replacement by ECM. Pathologic remodeling eventually leads to altered restitution of airway structure such as subepithelial fibrosis and increase in smooth muscle and mucus gland mass [5]. Plasminogen activator inhibitor (PAI-1) is the key inhibitor of the fibrinolytic system, which comprises an inactive proenzyme, plasminogen, that can be converted to the active enzyme, plasmin. Plasmin degrades fibrin into soluble fibrin degradation products. Two physiologic plasminogen activators (PAs) have been identified: the tissue-type PA (tPA) and the urokinase-type PA (uPA). tPAmediated plasminogen activation has a main role in the dissolution of fibrin in the circulation. On the other hand, uPA binds to a specific cellular receptor (uPAR), resulting in enhanced activation of cell-bound plasminogen. The main function of uPA is known to be in the induction of pericellular proteolysis via the degradation of matrix components or via activation of latent proteinases or growth factors. Inhibition of the fibrinolytic system may occur either at the level of the PA, by specific PAIs such as PAI-1, PAI-2, and PAI-3, or at the level of plasmin, mainly by ␣2-antiplasmin [10]. The fibrinolytic system and its interaction with the matrix metalloproteinase (MMP) system are schematically represented in figure 1. The PAIs belong to the serine protease inhibitor (serpin) family. PAI-1, a 50-kD glycoprotein, is the main PAI secreted in vivo and is a potent fast-acting and irreversible inhibitor of tPA and uPA. It forms stoichiometric complexes with active PAs, which are subsequently endocytosed and degraded. The role of PAI-1 in asthma is unknown, although PAI-1 may play an important role in other tissue repair processes such as pulmonary fibrosis and renal fibrosis [11, 12]. We and others recently demonstrated that PAI-1 may play an important role in asthma [13–15]. The implicated mechanisms of PAI-1 action in these studies involve inhibiting fibrinolysis and the MMP system which are crucial in the tissue remodeling process [13]. In this review, we will focus on the role of mast cells and their mediators including PAI-1 in the pathogenesis of asthma.

Mast Cells and Their Mediators in Asthma

Airway inflammation is a cardinal feature of asthma. Mast cells, eosinophils and T cells are the three major cell types implicated in airway inflammation [16].

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Activation/conversion Inhibition Pro-MMP tPA PAI-1 Plasminogen

Positive feedback

Plasmin

uPA

␣2-Antiplasmin

MMP TIMP

Fibrin degradation

ECM degradation

Fig. 1. The fibrinolytic system and its interaction with the MMP system. Plasmin degrades fibrin and can also convert latent MMPs into the active forms, which degrade ECM.

T cells are activated in patients with asthma [17, 18] and orchestrate the bronchial inflammatory response through the release of multifunctional cytokines [19–21]. Eosinophils are abundant in asthma and their numbers correlate with the degree of airway hyperresponsiveness (AHR) [22, 23]. MC accumulation in the airways and increased levels of specific MC-derived mediators such as tryptase in bronchoalveolar lavage fluid (BALF) of patients with asthma indicate the role of MCs in the pathogenesis of this disease. MC-deficient or MC-reconstituted mice have been used to demonstrate the critical role of MCs in the pathophysiology of asthma. One such model is a mouse with a mutation of the W locus (W/Wv) that encodes for the c-kit receptor [24]. These mice have virtually no tissue MCs due to their inability to respond to stem cell factor which is essential in MC growth and differentiation from the hematopoietic progenitor cells. William et al. [25] demonstrated that MCs are essential in recruitment of eosinophils into BALF and lung tissues and in the development of AHR in MC-reconstituted mice after allergen stimulation. In contrast, Masuda et al. [26] reported that epithelial remodeling and AHR caused by repeated allergen challenge are independent of MCs, although MCs play a role in the development of allergen-induced subepithelial fibrosis despite airway inflammation. MCs initiate immediate reactions such as acute bronchoconstriction through IgE-mediated release of preformed and newly formed mediators from granules within the cell. Many of these mediators have direct spasmogenic activity on the smooth muscle of the airways [27]. Histamine is one of the most abundant preformed mediators in MCs and is released upon exocytosis of the

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granule [28]. Histamine causes bronchoconstriction, increased mucus secretion, vasodilation, and increased vascular permeability of the airways. MCs also produce considerable amounts of newly formed eicosanoid mediators, the cysteinyl leukotrienes (LTs) and prostanoids, as well as platelet-activating factor (PAF) [29, 30]. The levels of histamine, PGD2 and its metabolite PGD9␣, and 11␤-PGF2 are elevated in the BALF of sensitized individuals after allergen challenge [31, 32]. PGD2 causes bronchoconstriction, vasodilation, and increased vascular permeability. PGD2 also directly activates eosinophils, causes neutrophil chemotaxis, and inhibits platelet aggregation [33]. LTC4 is converted to LTD4 which is a potent mediator of asthma [34, 35]. Levels of LTs are increased in the BALF of patients with asthma. The level of LTC4 is also increased in bronchial biopsies of patients with aspirin-sensitive asthma [36]. When agents that either inhibit the synthesis of LTs or block the LT receptor were administered to patients with asthma, they produced a rapid improvement in pulmonary function in some of these patients, demonstrating that LTs play an important role in asthma [37]. PAF is a potent chemoattractant of other inflammatory cells such as eosinophils, neutrophils, monocytes, and macrophages. A combination of PAF and IL-5 may have synergistic effect in eosinophil chemotaxis [38]. PAF acetylhydrolase degrades PAF and deficiency of this enzyme may be associated with severe asthma [39]. On the other hand, PAF antagonists improve AHR in some asthmatics [40]. Taken together, these findings suggest that MC-derived mediators play an important role in AHR and airway inflammation in patients with asthma. MCs produce a variety of cytokines [41–44]. The range of cytokines originated by MCs is similar to that produced by T-helper 2 (TH2) cells which play a central role in atopic asthma [19]. MC-derived TNF-␣ in mice has an important role in neutrophil recruitment and a critical protective role in a murine endotoxic shock model [45, 46]. In murine models, TNF-␣ upregulates the expression of E-selectin and intracellular adhesion molecule-1 on endothelial cells that may facilitate the trafficking of both eosinophils and neutrophils to the inflammatory site [47]. Bradding et al. [48] showed that MC-associated TNF-␣ was significantly increased in asthmatics in immunohistochemical analysis of endobronchial biopsy specimens. However, no TNF-␣ immunoreactivity was present in either T cells or eosinophils. This shows that MCs are a major source of TNF-␣ in bronchial asthma. MCs produce chemoattactants, including the C-X-C chemokine IL-8, a potent neutrophil chemoattractant [49]. Stimulated human MC lines express mRNA for IL-1, IL-3, and platelet-derived growth factor [50]. A lymphocyte-specific chemokine, lymphotactin, is released from activated MCs [51], suggesting that MCs contribute to the recruitment of lymphocytes to areas of allergic inflammation. Furthermore, MCs produce C-C chemokines, I-309 and TCA3, respectively, after Fc␧RI cross-linking

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or cell-to-cell contact with T lymphocytes [52–54]. Murine MCs also produce IL-1, IL-2, IL-3, and nerve growth factor and contain transcripts for IL-10, IL-12, RANTES (regulated upon activation, normal T cell expressed and secreted), and the macrophage inflammatory protein (MIP) family of chemokines. Cytokines and growth factors that are relevant to the proliferation and activation of fibroblasts, cells implicated in the structural changes in the asthmatic airway, have also been identified in MCs. Murine MC-derived TGF-␤1, as well as TNF-␣, induce a transient and marked increase of type I collagen mRNA in dermal fibroblasts after IgE-dependent activation [55]. Basic fibroblast growth factor, which promotes fibroblast differentiation and angiogenesis, is found in the majority of MCs from normal skin and lung and in tissue samples characterized by fibrosis, hyperplasia, and neovascularization. Although MCs generate a series of cytokines and chemokines, the relative contributions of MC-produced cytokines in the airways of asthmatics in comparison with those produced by eosinophils or T lymphocytes remain to be determined. MCs are an important source of chymases and tryptases degrade a variety of extracellular peptides and proteins, including vasoactive intestinal peptide, a bronchodilating neuropeptide [56, 57]. Tryptases inactivate procoagulant proteins, prevent the deposition of fibrin, and activate uPA. Tryptases also activate MMPs which are crucial in the tissue remodeling process [58], stimulate the growth and differentiation of fibroblasts [59], and induce airway smooth muscle cell hyperplasia [60]. Furthermore, tryptases promote the influx of circulating inflammatory cells into inflamed tissues [58]. The mitogenic activity of tryptases on fibroblasts and smooth muscle cells could promote the subepithelial deposition of collagen types III and V, as well as increasing airway wall thickness [61]. In addition to the chymase and tryptases, MCs are an important source of MMPs such as MMP-2 and MMP-9 and their specific inhibitor, tissue inhibitor of metalloproteinase (TIMP)-1 [62–64].

Identification and Characterization of PAI-1 in Mast Cells

To determine which genes play a key role in asthma, we and others have identified and characterized some of differentially expressed MC-derived genes by using cDNA microarray or subtraction library [65–69]. We screened 7,075 genes to identify those that were upregulated in stimulated MCs. Human mast cell line (HMC)-1 cells were exposed to a combination of phorbol myristate acetate and calcium ionophore (A23187) to achieve maximal stimulation [70]. Among the inducible genes that were identified, PAI-1 mRNA was induced at the highest level followed by uPA receptor mRNA (table 1). Other PA genes such as tPA and uPA were not significantly induced. We next examined

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Table 1. Differential expression of genes participating in MMP and PA systems in HMC-1 cells1 Gene name

Differential expression

Plasminogen activator inhibitor, type 1 Plasminogen activator, urokinase receptor Metalloproteinase domain 17 Tissue inhibitor of metalloproteinase 1 Plasminogen activator, tissue-type (t-PA) Plasminogen activator, urokinase-type (u-PA)

⫺34.2 ⫺4.2 ⫺3.2 ⫺2.7 ⫺1.2 ⫺1.1

Plasminogen activator inhibitor, type II

⫹1.8

1

Differential expression; absolute numbers correlate to the degree of differential expression between resting and activated cells. ‘⫺’ or ‘⫹’ represents upregulation or downregulation of the gene after activation, respectively.

MC-derived PAI-1’s ability to neutralize tPA activity by measuring net tPA activity [70]. The tPA activity in the supernatants of unstimulated HMC-1 cells was very high. The activity was almost completely absent in the supernatants of HMC-1 cells after stimulation. Restored tPA activity by neutralizing PAI-1 show that this reduction in tPA activity was due to inhibition by PAI-1. The total effect of PAI-1 secretion in the fibrinolytic system of human MCs was determined by performing a clot lysis assay [70]. Supernatants from unstimulated HMC-1 cells induced clot lysis. No clot dissolution was seen with supernatants from stimulated HMC-1 cells. When the cells were pretreated with neutralizing antibody against PAI-1 before stimulation and the supernatants were added to a synthetic fibrin meshwork, the clot lysis effect was fully recovered in the supernatants from the MCs pretreated with the neutralizing antibody, suggesting MC-derived PAI-1 completely suppresses tPA activity and converts a fibrinolytic environment to a fibrinogenic environment.

Mechanisms of PAI-1 Action in ECM Accumulation

It is well established that PAI-1 inhibits fibrinolysis and MMP activation. PAI-1 inhibits fibrinolysis by blocking the conversion of plasminogen to plasmin. PAI-1 also plays a role in the control of MMP activation. The MMP system is comprised of the MMPs and their inhibitors (TIMPs) that contain several conserved motifs and a zinc binding site. The MMP family contains at least 28 known members, which are grouped according to their substrate specificity

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[71–73]. The collagenases (MMP-1, -8, -13) degrade fibrillar forms of interstitial collagen. The gelatinases (MMP-2 & -9) are specific for denatured collagens and collagen-IV of the basement membrane. Stromelysins (MMP-3, -10, -11) cleave non-collagen components such as fibronectin, laminin, and vitronectin. Metalloelastase (MMP-12) cleaves elastin and membrane type (MT)-MMP (MMP-14) cleaves various collagens and non-collagen components. The MMPs are secreted in the extracellular space in catalytically latent forms because of the binding of the active site zinc atom to an unpaired cysteine of the propeptide domain. Disruption of the cysteine-zinc bond by conformational change or by limited proteolysis leads to the opening of the switch. Then the autocatalytic cleavage of the propeptide yields the active enzyme [74]. The activation of MMPs may also occur through the cleavage by MT-MMPs. The activation of MMP-2 at the cell surface is due to MT1-MMP. Active MMP-2 is then released into the extracellular space, but may also remain at the cell surface, where it has been shown to bind to the integrin ␣v ␤3 [75]. In vitro, plasmin directly activates proMMP-1, -3, -9, -10 and -13 [76–79, 81], whereas proMMP-2 is indirectly activated by plasmin [80]. Several active MMPs are also able to activate other proMMPs, indicating positive feedback mechanisms. For instance, MMP-3 can activate proMMP-9 [82], and MMP-3 & -10 can superactivate procollagenase, generating collagenase with higher specific activity [77, 79]. In addition to its role in the fibrinolysis and MMP pathways, PAI-1 promotes cell migration. uPA-deficient mice show defect in recruitment of T cells and macrophages and succumb to bacterial (Cryptococcus neoformans) infection [83]. They are also deficient in supporting the growth and malignant development of chemically-induced melanomas [84]. Furthermore, uPARdeficient mice exhibit a reduced ability to recruit neutrophils to the peritoneum upon inflammatory stimuli [85]. Although PAI-1 generally inhibits cell adhesion and migration by blocking the action of uPA, endothelial cell recruitment to tumor sites is totally abolished in PAI-1-deficient mice [86]. The proteolytic and non-proteolytic effects of uPA are interconnected through PAI-1. PAI-1 binds to uPA not only in solution, but also when uPA is receptor-bound and, therefore inhibits cell surface plasminogen activation, plasmin formation, and proteolytic stimulation of cell migration [87, 88]. PAI-1 is located in the ECM in a vitronectin (VN)-bound form, and VN binding also influences its structure preventing its conversion into a latent form. The aminoterminal region of VN (the somatomedin domain) contains the PAI-1 binding site close to the RGD sequence mediating binding to integrins [89]. Binding of VN to PAI-1 and integrins is mutually exclusive. Cell adhesion onto VN is inhibited by PAI-1 in a process that does not require its active site. Through this mechanism, PAI-1 inhibits the migration of cells on VN substrate [90, 91].

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Regulation of PAI-1 Expression

The PAI-1 gene contains at its 5⬘ regulatory end several known consensus cis regulatory elements, which bind trans activating factors such as Sp1, activated protein-1 (AP-1), nuclear factor-␬B (NF-␬B), Smad3 and Smad4, and others [92–96]. The PAI-1 gene transcription is activated by inflammatory cytokines, especially IL-1␤ [97], TNF-␣ [98], and TGF-␤ [99], and non-specific protein kinase C activators such as phorbol myristate acetate [70, 100]. Inhibition of PAI-1 has been less extensively studied, but suppression has been reported with interferon-␥, nitric oxide, natriuretic factors, and lipid-lowering drugs [101–103]. The plasma level of circulating PAI-1 has been shown to be genetically controlled, and a polymorphism in the 5⬘ gene promoter has been described. Two alleles, 4G and 5G, at position -674 in the promoter region, are encountered, and the plasma level of PAI-1 has been shown to be higher in patients with the 4G/4G genotype than in those with the 5G/5G genotype, while the 4G/5G genotype has intermediate values [94]. The 4G/4G genotype is reported to be associated with an increased risk of myocardial infarction in adult male patients. The molecular mechanisms involved in the increased synthesis of PAI-1 by the 4G allele as compared with the 5G allele appears to be related to the binding of NF-␬B to the cis regulatory region, which is partially inhibited by a regulatory protein, binding to the 5G sequence but not, or to a lesser extent, to the 4G sequence. Under IL-1 stimulation of the cells, the PAI-1 gene transcription rate is higher with the 4G allele than with the 5G allele [104].

Effect of PAI-1 in the Development of Airway Remodeling

Fibrosis is due to the abnormal accumulation of ECM in basement membranes and interstitial tissues [105]. The abnormal ECM in fibrosis is made of an excess of normal components of ECM such as fibronectin, laminin, proteoglycans, and collagen type IV, but also of an accumulation of proteins that is not found in the normal ECM such as collagen type I and III [106]. These latter proteins characterize the scarring process and are usually irreversibly deposited in the fibrotic tissues. The lung parenchymal cells themselves may undergo a fibroblastic trans differentiation and overproduce the ECM components. Proliferation of fibroblasts and myofibroblasts within the lung are also involved in the fibrogenic process. On the other hand, the ECM can be degraded, and it is likely that the fibrogenic process may also result from a deficit in ECM degradation. However, the relationship between ECM degradation and fibrogenesis is more complex than initially suspected, since abnormal ECM accumulation is often

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preceded or combined with an increased expression of ECM-degrading enzymes [107]. This increased proteolytic activity is presumably required for degradation of the normal ECM by infiltrating inflammatory and fibroblastic cells and its replacement by abnormal ECM. The fibrinolytic and MMP systems are two main systems involved in degrading ECM in the lung [108]. Damaged alveoli during inflammatory lung diseases can be repaired by replacement of injured alveolar cells, restoration of damaged ECM, and clearance of plasma proteins that have leaked into the alveolar space. Plasmin plays an important role in this repair process by being involved in cell migration, modulation of inflammatory activity, and breakdown of fibrin and other ECM. This latter function of plasmin may be important for limiting scar formation by dissolving the provisional matrix on which fibroblasts invade and secrete interstitial collagens. The normal alveolar space has net fibrinolytic activity due to the presence of uPA [109, 110]. However, during many acute and chronic inflammatory lung disorders, fibrin accumulates in lung tissue [111]. The fibrinolytic activity is decreased in BALF from patients with the adult respiratory distress syndrome [109, 110], idiopathic pulmonary fibrosis [112], sarcoidosis [112, 113], and bronchopulmonary dysplasia [114]. All of the above diseases have been associated with the development of pulmonary fibrosis. PAI-1 is the major inhibitor of PAs not only in plasma [10], but also in the alveolar space [110]. Elevated levels of PAI-1 have been observed in BAL specimens obtained from patients with adult respiratory distress syndrome and have been shown to reduce the fibrinolytic capacity of the fluid [109, 110]. A similar pattern of depressed fibrinolysis can be seen in a variety of animal models of lung injury [115]. Bleomycin-induced lung injury is an established murine model of human pulmonary fibrosis. PAI-1-deficient mice were resistant to pulmonary fibrosis after bleomycin-induced lung injury, presumably due to accelerated fibrinolysis [11]. On the other hand, PAI-1 transgenic mice suffered a severe lung injury and ECM deposition after bleomycin challenge. Furthermore, the level of PAI-1 gene expression strongly correlates with the amount of collagen deposition in lung tissues, suggesting that the balance of fibrinolytic activity within the lung is an important determinant of the pulmonary response to inflammatory injury. We demonstrated that PAI-1 promotes ECM deposition in the airways of a murine model of chronic asthma [116]. When the mice were challenged with ovalbumin (OVA) for 4 weeks, PAI-1 production was increased by 4-fold in lung tissue and by 5-fold in BALF of wild-type (WT) mice. Both PAI-1deficient and WT mice showed similarly increased numbers of peribronchial eosinophils (20-fold) and goblet cells (4-fold) and OVA-specific IgE levels (7-fold) after OVA challenge compared with saline challenge. When hydroxyproline assay was performed, the levels of collagen deposition were 2-fold less in lung tissue from PAI-1-deficient mice than WT mice after OVA challenge

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Table 2. Transmission disequilibrium test for the 4G and 5G alleles in asthma and atopy

Asthma 4G 5G Atopy 4G 5G

Transmitted

Not transmitted

␹2

p value

44 23

23 44

6.6

0.0139

28 20

20 28

1.3

0.3123

[116]. We also determined whether PAI-1 promotes collagen deposition in the airways of OVA-challenged mice by inhibiting the activity of MMP-9 by measuring MMP-9 activity in lung homogenates and BALF of PAI-1-deficient and WT mice. After OVA challenge, MMP-9 activity was approximately 3-fold higher in lung tissue and BALF from PAI-1-deficient mice than WT mice. This suggests that PAI-1 may promote ECM deposition by inhibiting MMP-9 activity. We also demonstrated that PAI-1 promotes irreversible fibrin deposition by comparing the amounts of water-insoluble fibrin in PAI-1-deficient and WT mice [116]. The amounts of total lung water-insoluble fibrin were minimal in PAI-1-deficient and WT mice after saline challenge. The amounts of waterinsoluble fibrin were 4-fold less in lung tissue from PAI-1-deficient mice than in WT mice after OVA challenge. Taken together, these data suggest that PAI-1 may promote ECM deposition by inhibiting fibrinolysis and MMP-9 activity. With regards to human asthma, we recently reported increased expression of PAI-1 in lung MCs from fatal asthmatics by double immunofluorescence colocalization [70]. We then demonstrated that the 4G allele is preferentially transmitted to asthmatic children (table 2). Later, Buckova et al. [15] also demonstrated that the 4G allele is associated with asthma. These data suggest that elevated PAI-1 levels in the lung may be associated with the development of asthma.

Conclusion

MCs are a major source of PAI-1 and a large number of MCs expressing PAI-1 are infiltrated in the airways of patients with fatal asthma. Furthermore, PAI-1-deficient mice show reduced ECM deposition in the airways of a murine model of chronic asthma probably by enhancing MMP-9 activity and fibrinolysis. Genotyping studies suggested that elevated PAI-1 levels in the lung may contribute to the development of asthma. In summary, MCs may play an important

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role in the development of airway remodeling in part by producing PAI-1 in the asthmatic airway. Further studies evaluating the mechanism by which PAI-1 promotes ECM deposition in the asthmatic airway may provide a new paradigm in airway remodeling and novel therapeutic targets.

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Chad K. Oh, MD UCLA School of Medicine Harbor-UCLA Medical Center, Bldg N-25 1000 W. Carson St., Torrance, CA–90509 (USA) Tel. ⫹1 310 2224162, Fax ⫹1 310 3202271, E-Mail [email protected]

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Mast Cell-Derived Cytokine Expression Induced via Fc Receptors and Toll-Like Receptors Yoshimichi Okayama Laboratory for Allergy Transcriptome, RIKEN Research Center for Allergy and Immunology, Yokohama City, Kanagawa, Japan

Abstract It is now well established that human mast cells (MCs) are a source of several multifunctional cytokines. Preformed immunoreactive tumor necrosis factor-␣ (TNF-␣) has been observed within human skin, and pulmonary MC granules are released after IgE-dependent activation. Recent studies in animal models indicate that mouse MCs may play a protective role in host defense against bacteria through production of TNF-␣, mainly as a result of Tolllike receptor 4 (TLR4)- or CD48-mediated activation. Moreover, several recent observations in animal models have indicated that MCs may also play a pivotal role in coordinating the early phases of autoimmune diseases through production of TNF-␣, particularly as a result of Fc␥RIII-mediated activation by autoantibodies. The questions now concern how MCs modulate immune responses and what cytokines MCs release through activation of each receptor. Since we recently identified functional TLR4 and Fc␥RI on human MCs, in this study we used high-density oligonucleotide probe arrays (GeneChip) to compare each of the receptormediated gene expression profiles with the Fc␧RI-mediated gene expression profile. The results indicated that human MCs might modulate the immune system in a receptor-specific manner by releasing cytokines in quantitatively and qualitatively different ways. Copyright © 2005 S. Karger AG, Basel

Introduction

Mast cells (MCs) are believed to play a pivotal role in allergic inflammation, host defense, and coordination of the early phases of certain autoimmune diseases. MCs are localized at the host-environment interface, such as in perivascular areas and mucosae, where they encounter both antigens and invading pathogens. Tissue MCs contain tumor necrosis factor-␣ (TNF-␣) in their

granules and release it by triggered exocytosis [1–3]. The rapid release of TNF-␣ is noteworthy because of the pleiotropic proinflammatory effects of this cytokine and because its release by tissue MCs is more rapid than by other cell systems [4, 5], which require induction of synthesis after cell activation. Since TNF-␣ is released by MCs via Fc␧RI, Fc␥RI, and Toll-like receptor 4 (TLR4), the rapid release of TNF-␣ by MCs may be important not only to innate immunity but to initiation of adaptive immunity in infection, allergy, and autoimmune diseases [6–8]. However, it is unclear how MCs modulate immune responses through each receptor and what cytokines and chemokines are released by MCs in response to stimulation of each receptor. In this review we have attempted to clarify the function of MCs in infection, autoimmune diseases, and allergic diseases by comparing the cytokine profiles expressed by activation of MCs through TLR4, Fc␥RI, and Fc␧RI.

Fc␧RI-Mediated Cytokine Expression by MCs

Fc␧RI is expressed on MCs and is believed to be responsible for allergendependent allergic responses [9]. Immediate hypersensitivity is an immune reaction that is initiated by antigen binding to IgE pre-attached to MCs and leads to the secretion of inflammatory mediators. Following aggregation of Fc␧RI, MCs release biogenic amines and proteases, including tryptase and chymase, and they also synthesize prostaglandin D2 (PGD2) and leukotriene C4 (LTC4). These mediators cause bronchoconstriction, mucus secretion, and edema formation [9]. The role of MCs in potentiating the late allergic response has in part been attributed to MC-dependent secretion of proinflammatory cytokines and chemokines [9], and the inflammatory infiltrates of the latephase reaction are rich in eosinophils, basophils, and TH2 lymphocytes. To identify a specific Fc␧RI-mediated gene expression profile, we compared Fc␧RI-mediated gene expression profiles with the TLR4-mediated gene expression profile by means of high-density oligonucleotide probe arrays (GeneChip) [10]. The results showed both a shared core response and LPS- or antigen-specific programs of gene expression in the MCs. The shared core response genes included NF-␬B-related genes and those encoding TH2 cytokines, such as IL-5 and IL-13 [10]. Analysis of genes whose expression is specifically induced via Fc␧RI showed that the genes included those encoding growth factors such as IL-3, M-CSF, amphiregulin, epiregulin and inhibin ␤, and chemokines, such as CCL7 (MCP-3) and CCL11 (eotaxin). The results indicate that MCs may not only recruit eosinophils, basophils, and TH2 lymphocytes to inflammation sites but also maintain the survival of these cells, resulting in the amplification of allergic inflammation.

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TLR-Mediated Cytokine Expression by MCs

A number of studies have confirmed that MCs play a critical role in host immune defense against gram-negative bacteria through the release of TNF-␣. As shown in two different models of acute bacterial infection, MC-deficient W/Wv mice are less efficient in clearing pathogenic bacteria in cecal-ligation- and puncture (CLP)-induced peritonitis or from the lungs of mice intranasally challenged with Klebsiella pneumoniae [11, 12]. CD48 has been demonstrated to be the MC membrane receptor for Escherichia coli expressing the fimbrial adhesion molecule FimH [13], and the interaction between CD48 and bacterial FimH results in MC degranulation and concomitant bacterial uptake via lipid rafts [13]. Murine MCs have been reported to express TLR2 and TLR4 [14, 15]. Peptidoglycan from Staphylococcus aureus stimulates murine bone-marrowderived MCs (mBMMCs) to produce TNF-␣, IL-4, IL-5, IL-6, and IL-13, but not IL-1␤, in a TLR2-dependent manner, and TLR2-dependent MC stimulation results in MC degranulation and Ca2⫹ mobilization [14]. mBMMCs and murine MC line MC/9 express TLR4 mRNA [14–16], and when activated by E. coliderived LPS, murine MCs produce TNF-␣, IL-1␤, IL-6, and IL-13, but not IL-4 or IL-5 [14, 15]. On the other hand, Masuda et al. [16] reported that LPS induces production of TH2-associated cytokines, such as IL-5, IL-10, and IL-13 by mBMMCs. Inhibition of c-Jun N-terminal kinase (JNK) activation significantly suppresses both IL-10 and IL-13 expression at both the mRNA and protein level. Although inhibition of p38 did not down-regulate the mRNA induction, it moderately decreased production of all three cytokines in response to LPS, suggesting that the LPS-mediated production of these three cytokines is distinctly regulated by mitogen-activated protein kinases [16]. A study in an MC-dependent model of acute sepsis revealed higher mortality among TLR4mutated BMMC-reconstituted W/Wv mice, and that TLR4 deficiency in BMMCs in mice results in significantly higher mortality because of defective neutrophil recruitment and the production of proinflammatory cytokines in the peritoneal cavity [14]. These findings in murine models suggest that MCs play an important role in the expression of innate immunity through TLR4. In humans, cord-blood-derived MCs (CBMCs) express mRNA for TLR1, TLR2, and TLR6, but not TLR4 [17]. The putative TLR2/TLR6 activators bacterial peptidoglycan and yeast zymosan are potent inducers of GM-CSF and IL-1␤, and also induce substantial short-term cysteinyl leukotriene generation [17]. By contrast, the putative TLR2/TLR1 activator, a synthetic triacylated lipopeptide, induces short-term degranulation, but does not induce cysteinyl leukotriene production [17]. Varadaradjalou et al. [18], on the other hand, have reported that both LPS from E. coli and peptidoglycan induce

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a significant release of not only TNF-␣ by CBMCs, but histamine, IL-5, IL-10, and IL-13. We investigated expression of TLR4 on human adult peripheralblood-derived cultured MCs and discovered functional expression of TLR4 that is up-regulated by IFN-␥ exposure [10]. To systematically explore how human MCs modulate the immune system in response to pathogens and antigens, we used GeneChip to compare the LPS-induced gene expression profiles in MCs with the Fc␧RI-mediated profiles. In addition to expressing NF-␬B-related genes, MCs also expressed an antiviral-response gene program in response to IFN-␥, and LPS sustained its expression. Interestingly, LPS induced IL-12 mRNA expression in human MCs in the presence of IFN-␥. Comparison with the LPS-stimulated gene expression profile in peripheral blood mononuclear cells revealed that the LPS-stimulated MCs specifically induce a TH2 cytokine and chemokines against TH2 cells and eosinophils. The cytokine profiles induced through TLR4 and Fc␧RI are summarized in table 1.

Fc␥R-Mediated Cytokine Expression by MCs

Numerous studies have reported a correlation between the number and/or distribution of MCs and development of autoimmune diseases, such as multiple sclerosis and rheumatoid arthritis. For example, gene expression profiling of multiple sclerosis brain lesions detected a high proportion of transcripts of proteases and other inflammatory mediators derived from MCs [19, 20]. Experimental allergic encephalomyelitis (EAE) is an experimental model of multiple sclerosis, a chronic inflammatory disorder of the central nervous system characterized by a breach of the blood-brain barrier, mononuclear cell infiltration of white matter, and eventual demyelinization. Brown and colleagues [21] showed that mice lacking MCs (W/Wv mice) develop EAE later and less severely than control wild mice in response to injection of different myelin components, such as myelin oligodendrocyte glycoprotein. Complementation of W/Wv mice with immature MCs derived in vitro restores typical EAE susceptibility. MC function appears to be dependent on MC expression of Fc␥R, especially, Fc␥RIII [21], and this has been found to be true in animal models of rheumatoid arthritis [22] and bullous pemphigoid [23]. Immune complexes may aggregate Fc␥R, causing the release of TNF-␣, which recruits neutrophils [6, 7]. Because of the increasing body of evidence from animal models that MCs are recruited into allergic reactions by non-IgE-dependent mechanisms and the fact that Fc␥RIII has been shown to induce murine MC degranulation and anaphylaxis

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Table 1. Cytokine/chemokine profiles expressed in human MCs following aggregation of Fc␧RI and activation via TLR4 Resting

Fc␧RI

TLR4⫹IFN-␥

Cytokines G-CSF M-CSF GM-CSF IFN-␣ IFN-␤ IFN-␥ IL-1␣ IL-1␤ IL-2 IL-3 IL-4 IL-5 IL-6 IL-7 IL-10 IL-12 IL-13 IL-15 IL-16 IL-17 IL-18 IL-22 TNF-␣

⫺ ⫺ ⫺ ⫺ ⫺ ⫺ ⫺ ⫹ ⫺ ⫺ ⫺ ⫺ ⫺ ⫺ ⫹ ⫺ ⫺ ⫺ ⫹ ⫺ ⫹ ⫺ ⫺

⫹ ⫹⫹ ⫹⫹ ⫺ ⫺ ⫺ ⫺ ⫹⫹ ⫺ ⫹ ⫺ ⫹⫹ ⫹⫹ ⫺ ⫹ ⫺ ⫹ ⫺ ⫹ ⫺ ⫹ ⫹ ⫹⫹

⫹ ⫺ ⫹⫹ ⫺ ⫺ ⫺ ⫺ ⫹⫹⫹ ⫺ ⫺ ⫺ ⫹⫹ ⫹⫹ ⫺ ⫹⫹ ⫹ ⫹ ⫹ ⫹ ⫺ ⫹ ⫺ ⫹⫹⫹

Chemokines CCL1 I-309 CCL2 MCP-1 CCL3 MIP-1␣ CCL4 MIP-1␤ CCL5 RANTES CCL7 MCP-3 CCL8 MCP-2 CCL11 Eotaxin CCL15 MIP-1␥ CXCL1 GRO␣ CXCL2 GRO␤ CXCL3 GRO␥ CXCL4 PF4

⫺ ⫹ ⫹ ⫹ ⫺ ⫹ ⫹ ⫺ ⫺ ⫹ ⫺ ⫺ ⫺

⫹⫹ ⫹⫹ ⫹⫹ ⫹⫹ ⫺ ⫹⫹ ⫹ ⫹⫹ ⫹ ⫹⫹ ⫹⫹ ⫹⫹ ⫺

⫹⫹ ⫹ ⫹⫹ ⫹⫹ ⫹⫹ ⫹ ⫹⫹ ⫺ ⫺ ⫹⫹ ⫹⫹ ⫹⫹ ⫺

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Table 1 (continued)

CXCL5 CXCL8

ENA78 IL-8

Resting

Fc␧RI

TLR4⫹IFN-␥

⫺ ⫹

⫹⫹ ⫹⫹

⫺ ⫹⫹

For aggregation of Fc␧RI, human MCs were precultured with human myeloma IgE (1 ␮g/ml) for 24 h, and then activated with anti-IgE (3 ␮g/ml) for 6 h. For TLR4-mediated MC activation, human MCs were precultured with IFN-␥ for 24 h, and then activated with LPS (100 ng/ml) for 6 h. Total RNA was extracted and used for GeneChip analysis. The expression levels were simplified and expressed as ⫺, ⫹, ⫹⫹, and ⫹⫹⫹, where ⫺ ⫽ no mRNA is detectable; ⫹ ⫽ mRNA is detectable, and ⫹⫹⫹ ⫽ higher expression levels than ⫹⫹.

[24, 25], we hypothesized that human MCs also express Fc␥ receptors, and that their expression is affected by specific factors produced in the microenvironment. We therefore first used RT-PCR to confirm that resting human MCs contain mRNA for FcgRIa1, FcgRIb2, FcgRIIA, FcgRIIb1, FcgRIIb2, and FcgRIII [26, 27], and then investigated whether proinflammatory cytokines affect the expression of Fcg receptors on human MCs. The results showed that IFN-␥ upregulates the expression of FcgRIa1 and FcgRIb2 [26]. Fc␥RIb2, however, appears not to be present on the surface membrane and not to bind to either monomeric or complexed IgG [28]. Thus, according to the results of RT-PCR, human MCs express the high-affinity IgG receptor, which is the gene product of FcgRIA, and FcgRI mRNA expression is maximal between 4 and 8 h, when the increase is ⬃10-fold over the baseline level. This was confirmed by flow cytometry, which showed that IFN-␥ exposure increased Fc␥RI expression on human MCs from ⬃2 to 44%. The intensity of surface expression of Fc␥RI was maximal at 24 h, and it appeared to plateau from 24 through 48 h. Expression of Fc␧RI, Fc␥RII, and Fc␥RIII was unaffected. Expression of Fc␥RII and Fc␥RIII in human MCs was ⬃45 and ⬃0.5%, respectively [26]. Although murine MCs express a functional Fc␥RIII, Fc␥RIII protein expression is minimal and unaltered by permeabilization [27]. Thus, human MCs express the high-affinity IgG receptor, Fc␥RI, and a low-affinity IgG receptor, Fc␥RII, on their surface, and expression of Fc␥RI is up-regulated by IFN-␥. Aggregation of Fc␥RI on human MCs resulted in histamine release, PGD2 and LTC4 generation, and production of a variety of cytokines. Proinflammatory cytokines, such as TNF-␣ and IL-1␤, are more up-regulated in MCs following aggregation of Fc␥RI than following aggregation of Fc␧RI [29]. We compared TNF-␣ production by MCs following aggregation of Fc␧RI and Fc␥RI, and activation through TLR4. As shown in figure 1, maximal

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1,400

TNF-␣ (pg/106 MCs)

1,200 1,000 800 600 400 200 0 0

2 Time (h)

6

Fig. 1. TNF-␣ production by human MCs following aggregation of Fc␧RI and Fc␥RI and activation via TLR4. For aggregation of Fc␧RI, human MCs were precultured with human myeloma IgE (1 ␮g/ml) in the presence of 25 ng/ml of IFN-␥ for 24 h, and then activated with anti-IgE (3 ␮g/ml) for 6 h (䉱). For aggregation of Fc␥RI, MCs were precultured with human IgG1 (1 ␮g/ml) in the presence of IFN-␥, and then activated with anti-IgG1 (3 ␮g/ml) for 6 h (䊏). For TLR4-mediated MC activation, MCs were precultured with IFN-␥, and then activated with LPS (100 ng/ml) for 6 h (䊉). Control cells were preincubated either with IgE (⌬), IgG1 (䊐) or medium alone (䊊) in the presence of IFN-␥, and then incubated with medium alone for 6 h. TNF-␣ in the cell supernatants was measured by ELISA.

TNF-␣ production in response to aggregation of Fc␥RI and activation via TLR4 was about 2- and 6-fold that induced by aggregation of Fc␧RI, respectively. Activation of IFN-␥-treated human MCs sensitized with 1 ␮g/ml aggregated IgG1 also resulted in 15–30% degranulation (␤-hexosaminidase release). No degranulation was observed when heat-generated aggregates of IgG2, IgG3, or IgG4 were used [30]. Activation with aggregated IgG1 led to PGD2 and LTC4 generation as well as enhanced IL-3, IL-13, GM-CSF, and TNF-␣ production [30]. Next, we investigated Fc␥RI-dependent activation of specific signal transduction molecules and assessed the relative involvement of these events in human MC degranulation and TNF-␣ production following Fc␥RI and Fc␧RI aggregation. Fc␥RI aggregation resulted in the phosphorylation of src kinases and p72syk, and subsequent tyrosine phosphorylation of multiple substrates [31]. Inhibitor studies revealed that these responses are required for degranulation and TNF-␣ synthesis. Both Fc␥RI and Fc␧RI aggregation also activated the MAP kinases ERK1/2, JNK and p38, and MAP kinase activation was necessary for TNF-␣ synthesis, but not degranulation in response to stimulation of both receptors. Thus, the signaling events in human

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MCs following aggregation of Fc␥RI were generally similar to those observed following Fc␧RI aggregation. The one exception was that, although phosphatidylinositol-3-kinase was activated after both Fc␧RI and Fc␥RI aggregation, only Fc␥RI appeared to require this molecule for degranulation [31]. Since both the Fc␧RI ␣-chain and the Fc␥RI ␣-chain require FcR␥ (common ␥-chains) for signal transduction [31], the Fc␧RI and Fc␥RI-induced gene expression profiles, especially the cytokine profiles, appeared to be similar, but, as described above, the amounts of some of the cytokines that were released were different.

Conclusions

MCs are major effector cells in allergic inflammation and release mainly TH2-type cytokines following aggregation of Fc␧RI. The phenotype of MCs may change with the microenvironments; for example, IFN-␥ induces expression of a variety of receptors, such as Fc␥RI and TLR4, on their surface. Stimulation of MCs induces expression of IL-12 through TLR4. However, IL-12 mRNA is not up-regulated by aggregation of Fc␧RI. In addition, proinflammatory cytokines, such as TNF-␣ and IL-1␤, are more up-regulated in MCs following the aggregation of Fc␥RI than following aggregation of Fc␧RI. Thus, human MCs exhibit tailored receptor-specific immune responses, suggesting that human MCs may play an important role in innate and adaptive immunity.

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Yoshimichi Okayama, MD, PhD Laboratory for Allergy Transcriptome RIKEN Research Center for Allergy and Immunology 1-7-22 Suehiro-cho, Tsurumi-ku, Yokohama 230–0045 (Japan) Tel. ⫹81 45 5037034, Fax ⫹81 45 5037033, E-Mail [email protected]

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Mast Cells in Allergic Airway Disease and Chronic Rhinosinusitis Ruby Pawankar Department of Otolaryngology, Nippon Medical School, Tokyo, Japan

Abstract Conventional knowledge tells us that mast cells are only important in the acute IgEmediated reactions as seen in anaphylaxis, asthma and rhinitis. Yet, in recent years, much evidence has accumulated on the versatile role of mast cells in allergic inflammation. Here, we describe the novel and potential roles of mast cells in the late phase allergic reaction as well as in chronic allergic inflammation. Mast cells in patients with allergic rhinitis and asthma produce Th2 type cytokines, induce IgE synthesis in B cells and can autoactivate itself via the mast cell-IgE-Fc␧RI cascade. In addition, mast cells upregulate the production of a variety of cytokines/chemokines in epithelial cells and fibroblasts and induce the recruitment of basophils, T cells and eosinophils into sites of allergic inflammation as well as their own intraepithelial accumulation. Furthermore, mast cells express MMPs and interact with extracellular matrix proteins and ASM and may play a role in nasal and bronchial hyperresponsiveness as well as tissue remodelling. In chronic rhinosinusitis with nasal polyps, the potential role of mast cells not only in orchestrating eosinophilic inflammation but also in the genesis and perpetuation of nasal polyp formation via Fc␧RI and TLR mediated activation is also of growing interest. Copyright © 2005 S. Karger AG, Basel

Introduction

Mast cells have long been considered to primarily serve as important effector cells for acute IgE-associated allergic reactions (like anaphylaxis or the immediate phase allergic reaction in allergic rhinitis and asthma). However, this represents too narrow a perception of the function of these cells. Mast cells are tissue-resident cells and an important source of a variety of inflammatory mediators, cytokines and chemokines. Thus they not only orchestrate various aspects of the IgE-associated immune responses including the late phase allergic response and chronic allergic inflammation through the release of these mediators, but also via cell-cell interaction by which they regulate the function of other cells.

The nasal mucosa is the first barrier of the entire respiratory tract that encounters various pathogens like viruses or bacteria. Mast cells are strategically located at the interface between environmental and mucosal surfaces and recent evidence suggests that these cells play a critical role in innate immunity to bacterial infection and that they can be activated by viral proteins. In this review, we will discuss the novel and potential roles of mast cells in allergic airway diseases and chronic rhinosinusitis.

Phenotypes and Distribution of Mast Cells in Allergic Rhinitis

Human mast cells originate from CD34⫹ hematopoietic progenitors and undergo maturation in the tissues under specific factors like stem cell factor (SCF) present within the microenvironment. Phenotypically distinct subsets of mast cells have been accepted in rodents, based on their distinct staining characteristics, Tcell dependency, and functions, namely connective tissue mast cells, and mucosal mast cells [1–5]. In humans too, two types of mast cells have been recognized based on the neutral proteases they express, tryptase- and chymase-positive mast cells (MCTC) which contain tryptase together with chymase, cathepsin-G like protease, and mast cell carboxypeptidase, whereas tryptase-positive type mast cells (MCT) contain tryptase, but lack the other neutral proteases present in MCTC [6]. In humans and many other mammalian species, the numbers of mast cells in normal tissues exhibit considerable variation according to the anatomic site. Moreover, the numbers of mast cells vary in association with the underlying inflammatory or immunologic condition [7–10]. In atopic diseases like allergic rhinitis and asthma, mast cells are known to accumulate within the epithelial compartment of the target organ. In fact, there is a selective increase of MCT cells in the epithelial compartment of the nasal mucosa of allergic rhinitics [11, 12]. Also after allergen provocation, the increase in intraepithelial mast cells occurs even as early as 30 min after allergen challenge [13]. Moreover, the number of mast cells in the epithelial compartment and the levels of histamine release from these cells in the epithelium correlates with the symptom score [14]. By contrast, in normal subjects and non-allergic rhinitis there are few mast cells in the epithelial compartment of the nasal mucosa [12].

Nasal Mast Cells as a Source of Multifunctional Cytokines

Allergic inflammation is characterized by many cytokine-dependent processes including the induction of IgE synthesis (IL-4, IL-13); eosinophil

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recruitment, development and survival [IL-3, IL-5, granulocyte-macrophage colony-stimulating factor (GM-CSF), IL-16, certain C-C chemokines like RANTES and eotaxin]; recruitment of monocytes and T cells (IL-16, certain C-C chemokines like RANTES), and basophil recruitment (TNF-␣, IL-4) or enhanced mediator production (IL-3, IL-4, the C-C chemokine, macrophage inflammatory protein [MIP]-1␣) [15, 16]. Moreover, cytokines promote the allergic inflammation by enhancing the recruitment of leukocytes through the upregulation of adhesion molecules like P-selectin, E-selectin, vascular cell adhesion molecule-1 (VCAM-1) on vascular endothelial cells [15–17]. Finally, cytokines can also critically influence the development and perpetuation of chronic allergic inflammation. Mast cells represent a potential source of many cytokines that might influence allergic inflammation, and the synthesis and release of these products can be induced via IgE-dependent mechanisms [18–23]. When mouse mast cells are activated via the Fc␧RI, they express increased levels of mRNA for a range of cytokines (IL-1␣, IL-3, IL-4, IL-5, IL-6, and GM-CSF and MIP-1␣, MIP1␤, and several other C-C chemokines) and also secrete substances with corresponding activities [18]. In humans too, recent studies have clearly shown that mast cells are a potential source of several cytokines, including IL-4, IL-5, IL-6, IL-7, IL-8, IL-10, IL-13, TNF-␣, and basic fibroblast growth factor [19]. Bradding et al. [22] first reported the expression of IL-4, IL-5, and IL-6 in bronchial mast cells of atopic asthmatics. It is now well known that bronchial as well as nasal mast cells (NMC) in patients with asthma and allergic rhinitis are an important source of Th2 type cytokines IL-4, IL-5, IL-6, and IL-13 [20–23]. Moreover, NMC from patients with perennial allergic rhinitis (PAR) to house dust mite not only express the protein but also release significant amounts of IL-4, IL-5, IL-6, and IL-13 when stimulated by specific mite antigen [20, 21]. Whereas T cells are an important source of IL-4 and IL-13, the levels of IL-4 and IL-13 released by mite antigen-activated NMC is significantly greater than that of mite antigen-activated T cells [24]. In addition to Th2 cytokines, NMC express several other cytokines and chemokines like GM-CSF, TNF-␣, IL-7, IL-8, and TGF-␤ which regulate the growth and activation of a variety of cells [12, 25]. While mast cell-derived cytokines are capable of upregulating allergic inflammation, at certain points in the natural history of these complex processes, cytokines derived from mast cells (TGF-␤) may also contribute to the downregulation of the response. However, it is also of interest that there is a kind of heterogeneity in the cytokine profile between subsets of mast cells in that tryptase-positive MCT mast cells mainly express IL-5, IL-6 and IL-7 whereas tryptase-positive, chymase-positive MCTC mast cells preferentially express IL-4 [26]. IL-13 is produced by both subsets [23]. In this context, we refer to the study on isolated

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skin mast cells (which are nearly all MCTC) that were found to express high levels of IL-4 immunoreactivity, but almost no immunoreactivity for IL-5 or IL-6 [27]. Such differences in the distribution of cytokine expression between subsets of mast cells even within the same target organ suggest the possibility of distinct roles for subsets of mast cells in various aspects of allergic inflammation.

Mast Cells-IgE-Fc␧RI Cascade in Allergic Airway Disease

Ishizaka and Tomioka first described the presence of IgE receptors on mast cells and that these cells could be activated to degranulate upon cross-linking of the Fc␧RI-bound IgE with bivalent or multivalent antigen. Therefore, the expression of the high-affinity IgE receptor (Fc␧RI) in mast cells and basophils is critical to the development of allergic diseases. The Fc␧RI is a tetrameric structure comprising of an ␣-subunit, a ␤-subunit and two disulfide linked ␥-subunits [28–30]. The extracellular portion of the Fc␧RI␣ chain contains the entire IgE-binding site [31, 32], and the ␤-subunit is considered to be largely within the cell membrane, spanning it 4 times so that both the amino- and carboxy-termini are within the cytoplasm [33]. The ␤- and ␥-subunits are known to be involved in signal transduction [34–36]. Studies in Fc␧RI␣ chain-deficient mice have demonstrated the inability of these mice to exert allergen-induced anaphylaxis even with normal number of mast cells [37]. Several lines of evidence indicate that mast cells or basophils must display the high-affinity IgE receptors on their surface to be able to have significant IgE-antigen-specific effector function. NMC in PAR patients exhibit increased expression of the Fc␧RI, and IL-4 as well as IgE can upregulate the Fc␧RI expression in mast cells [20, 38, 39]. This enhanced expression of the Fc␧RI in NMC was associated with an increase in IgE bindability as well as increased mediator release [20, 21, 24]. Furthermore, the Fc␧RI expression in NMC correlated well with the levels of serum IgE [20]. These findings taken in concert with the observations of Pastorello et al. [40] who demonstrated a strong positive correlation between the levels of serum IgE and clinical symptoms, in symptomatic patients with allergic rhinoconjunctivitis, suggest a very important role for NMC in regulating chronic allergic inflammation. Moreover, mast cells can induce IgE synthesis in B cells and several studies have demonstrated that local IgE synthesis occurs at sites of allergic inflammation [24, 41, 42]. Putting together all these observations, one can perceive a very important role for the mast cell in perpetuating allergic inflammation via the mast cell-IgE-Fc␧RI cascade [21].

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Nasal Mast Cells, Adhesion Molecules and Extracellular Matrix Proteins

In addition to cytokines, a number of cell surface molecules are involved in the recruitment of inflammatory cells into specific sites of inflammation [43]. Lymphocytes and mast cells in the tissues are surrounded by other cells like fibroblasts and epithelial cells as well as extracellular matrix (ECM) proteins (e.g., collagen, fibronectin and laminin) [43, 44]. Therefore, the interaction of these inflammatory cells with structural cells like fibroblasts, epithelial cells and ECM may be important in the perpetuation of chronic allergic inflammation. In PAR patients, NMC express higher levels of VLA-4 and VLA-5 and IgE-mediated activation of NMC induced the release of greater levels of IL-4, IL-13 and TNF-␣ when the NMC were cultured on fibronectin-coated culture plate [45]. Thus, mast cell-ECM interactions may contribute to the enhancement of mast cell activation especially when the levels of IgE and Ag in the microenvironment are rather low. This in fact may explain at least in part the phenomenon of nasal and bronchial hyperresponsiveness. VCAM-1 and ICAM-1 are the counterreceptors or ligands for ␤1 and ␤2 integrins, respectively. A striking feature in allergic inflammation is the selective accumulation of activated eosinophils and basophils, without increased numbers of neutrophils. In this context, one may refer to the studies of Lee et al. [46] who demonstrated that ICAM-1 expression in the nasal mucosa of allergic rhinitics was not upregulated after stimulation with antigen but on the other hand, VCAM-1 was upregulated at 24 h post-allergen challenge. Since VLA-4 is not expressed by neutrophils, the selective upregulation of VCAM-1 in the nasal mucosa may result in the selective accumulation of activated eosinophils, basophils and lymphocytes, without increase in numbers of neutrophils. It is known that IL-4 and IL-13 can upregulate VCAM-1 expression in endothelial cells. This indicates that mast cells and lymphocytes may contribute to the recruitment of eosinophils and basophils through the IL-4/IL-13-induced upregulation of VCAM-1 expression in the nasal mucosa.

Nasal Mast Cell – Structural Cell Interactions

Mast cell activation may directly or indirectly promote the release of cytokines from other resident cells in the respiratory tract, such as macrophages, epithelial cells, vascular endothelial cells, fibroblasts, and nerves. Cytokines released in these responses then contribute to the vascular and epithelial changes and to the angiogenesis that are so prominent. In fact, mast cell mediators like histamine and tryptase upregulate the production of RANTES and

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GM-CSF from nasal epithelial cells (NEC), and IL-4 and IL-13 in synergy with TNF-␣ upregulates eotaxin and TARC production in NEC and nasal fibroblasts [47, 48]. Moreover, mast cells themselves express vascular endothelial growth factor (VEGF), and IL-4 from mast cells can upregulate the VEGF expression in endothelial cells [49]. Mast Cell Migration and Regulation of Mast Cell Phenotypes in Allergic Nasal Epithelium

Structural cells like epithelial cells (EC) and fibroblasts (FB) are an important source of not only SCF but also several chemokines. While SCF is a wellknown chemotactic factor for mast cells, the low levels of SCF in the epithelial compartment as compared to the high levels in the deep lamina propria goes against the possibility that SCF induces the intraepithelial accumulation of mast cells. Interestingly, the levels of RANTES (but not eotaxin and SCF) was significantly greater in the nasal epithelium than in the lamina propria of PAR patients and RANTES was a more potent chemotactic factor for cultured human mast cells as compared to SCF or eotaxin [50]. Furthermore, a proportion of mast cells in the nasal mucosa of allergic rhinitics expressed both CCR3 and CCR5 (both ligands for RANTES), and histamine as well as tryptase upregulated the production of RANTES from NEC. Therefore, one could consider that mast cells do not only orchestrate the recruitment or migration of other immune cells like eosinophils and basophils, but also regulate their own migration and accumulation into the allergic epithelial compartment. While SCF may not be so important for the intraepithelial accumulation of mast cells at sites of allergic inflammation, its role in regulating the phenotype of mast cells cannot be underplayed. It is clear now that high levels of SCF favor the development and survival of MCTC and low levels of SCF induce a decrease in the chymase expression and reduction in the number of MCTC, virtually not affecting the tryptase expression. Therefore, the low level of SCF in the allergic nasal epithelium as compared to that in the deep lamina propria may explain at least in part why there is a selective accumulation of MCT in the allergic nasal epithelium. By contrast, high numbers of MCTC are distributed in the deep lamina propria where the levels of SCF are about 10 times higher [51, 52]. Versatile Roles of Mast Cells in IgE-Mediated Allergy

Immediate Phase Response It is conventionally believed that mast cells when activated via the high-affinity IgE receptor react by undergoing several morphological changes

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Early phase reaction Sneezing

Ag

Fc␧RI IL-4

IL-4/ IL-13

lgE class switch IgE

IgE

B CD40 IL-4

VLA-4 VLA-5 IL-4/ IL-13

Mast cell

Nasal obstruction

Chronic allergic inflammation

Rhinorrhea

Histamine Leukotriene Prostaglandin

Late phase reaction IL-4/ IL-13/ TNF-␣ CD3⫹ Eosinophil Basophil VCAM-1

Fig. 1. Versatile roles of mast cells in the early phase reaction, late phase allergic reaction as well as in chronic allergic inflammation [modified from R. Pawankar, Clin Exp Allergy, 2000].

including swelling of the cytoplasmic granules and subsequent solubilization of its granule contents. Histamine, tryptase, PGD2 and LTC4 are among the mast cell products that can be detected immediately after exposure to allergens. Histamine induces vasodilation, increased vascular permeability and increased glandular secretion in the ipsilateral as well as contralateral sides through neural reflexes. Prostaglandins like PGD2 also cause edema by vasodilation, and increased vascular permeability. Histamine acts on the sensory nerve endings of the trigeminal nerve to cause sneezing, histamine and leukotrienes act on the mucous glands to cause rhinorrhea and histamine, leukotrienes and prostaglandins act on the blood vessels to cause increased vascular congestion and nasal obstruction (fig. 1). Late Phase Response The late phase allergic reaction occurs as a result of the infiltration of a variety of inflammatory cells like eosinophils, basophils and T cells and the subsequent release of a number of soluble products like prostaglandins, leukotrienes, platelet-activating factor, ECP, MBP and so on. Tissue eosinophilia is an important aspect of the late phase allergic reaction. Mast cells can orchestrate the late phase reaction by inducing the infiltration of eosinophils not only through the upregulation of VCAM-1 (TNF-␣, IL-4, IL-13) on endothelial cells but also through release of eosinophil-chemotactic factors like platelet-activating factor and leukotriene B4. In addition, mast cells can enhance eosinophil survival through the release of granulocyte-macrophage colony-stimulating factor and IL-5. Interestingly, strong correlations were reported to exist between the numbers of IL-4-, IL-5- and TNF-␣-positive mast cells and the number of tissue

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eosinophils in atopic asthma and allergic rhinitis [22, 53]. These recruited eosinophils (also basophils and T cells) then promote the further progression of the inflammatory response by providing additional sources of certain cytokines (that can also be produced by mast cells stimulated by ongoing exposure to allergen), as well as new sources of cytokines and other mediators that may not be produced by mast cells. In fact, time kinetics of cytokine secretion from purified lung or NMC from PAR patients have shown that upon IgE-mediated stimulation induces the secretion of IL-13, TNF-␣ and IL-4 as early as from 2 to 6 h and peaked at 24–48 h [54]. Again, TNF-␣ is constitutively expressed in mast cells and can be released within 2 h of IgE-mediated stimulation. Klein et al. [55] have shown that activation of mast cell products in fragments of human skin in vitro, resulted in the upregulation of E-selectin expression in adjacent vascular endothelial cells and this was attributed to the release of TNF-␣. Moreover, interaction of mast cells with ECM further enhances the IgE-mediated cytokine release from mast cells [45]. Mast cells can further contribute to the eosinophil/T-cell infiltration in the late phase allergic reaction through the histamine-induced upregulation of RANTES, and GM-CSF and the IL-4-, IL-13-, TNF-␣-induced upregulation of eotaxin and TARC from NEC [47, 48]. Taken together, these studies strongly suggest that the mast cell is a key effector cell in the late phase reaction (fig. 1). Chronic Allergic Inflammation Most recent studies also suggest that the mast cell has the potential to regulate allergic inflammation by inducing IgE synthesis in B cells. Under allergic inflammatory conditions, ‘primed’ mast cells express high levels of the highaffinity receptor for IgE and the ligand for the surface antigen CD40, involved in T/B-cell interactions leading to immunoglobulin production, as well as Th2-type cytokines, IL-4 and IL-13 [20]. Mast cells also have the potential to function as antigen-presenting cells with the ability to shift T cells into Th2 subtypes [20, 56, 57]. These findings suggest that mast cells can modulate the allergic response by acting directly on B cells and inducing IgE synthesis. Furthermore, the locally synthesized IgE itself can upregulate the Fc␧RI expression in mast cells. The augmented Fc␧RI in mast cells can bind increased number of IgE-Ag complexes which in turn can enhance the sensitivity of mast cells to allergen resulting in the enhancement of the production of immunomodulatory cytokines and chemical mediators, forming an important positive feedback amplification loop involving the IgE-IgE-receptor-mast cell cascade [21] (fig. 1). Highlighting the Roles of Mast Cells in Asthma

As in allergic rhinitis, mast cells are primary effector cells in asthma and play crucial roles in the early phase response, late phase response as well as

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chronic allergic inflammation [58–61] Mast cell numbers are increased in the bronchial epithelium of atopic asthmatics [62]. Besides the epithelial compartment, mast cells are widely distributed in specific compartments of the asthmatic airways (i.e. the bronchoalveolar space [63], beneath the basement membrane, in close proximity to blood vessels, near submucosal glands and in the ASM bundles [64]. In fact a close correlation between mast cell numbers in the bronchial smooth muscle of asthmatics and the degree of airway hyperresponsiveness to methacholine is well established [64]. Mast cells in asthmatics release a variety of mediators (histamine, cysteinyl leukotriene C4 (LTC4), PGD2, tryptase and chymase), as well as cytokines and chemokines which induce and sustain chronic inflammation in asthma by regulating the recruitment and function of T cells, macrophages, ASM and EC. As mentioned earlier in this chapter, increasing evidence suggests that histamine can regulate the activity and cytokine/chemokine release from other cells. Histamine induces lysosomal enzyme release and IL-6 and TNF-␣ production from human lung macrophages [65] and CysLTs exert a variety of responses on bronchial smooth muscle, human lung macrophages, mast cells and peripheral blood leukocytes via the CysLTR1 and CysLTR2 [66–69]. Besides the above-mentioned mediators, mast cells in asthmatic airways express a wide spectrum of cytokines (e.g. IL-4, SCF, IL-5, IL-6, IL-8, IL-13, IL-16, transforming growth factor-␤ (TGF-␤), IL-25, TNF-␣ and granulocyte macrophage-colony stimulating factor (GM-CSF)) [70–72]. IL-16 is chemotactic for CD4⫹ T cells and IL-25 can induce IL-4 and IL-13 gene expression, and this may contribute to mast cell-ASM interactions as well as in amplifying allergic inflammation via the mast cell-IgE-Fc␧RI cascade [73]. IL-3, IL-5, and GM-CSF from mast cells can enhance histamine release and IL-4 synthesis in basophils, whereas IL-4 enhances the production of proinflammatory mediators (PGD2 and LTC4) and various cytokines by mast cells [74, 75]. Thus, Th2 (IL-4, IL-5, IL-3) cytokines can induce changes in the biosynthetic pathways of mast cells at sites of allergic inflammation. SCF, a principal growth, differentiating and chemotactic factor for human mast cells, is expressed and released by lung mast cells [76]. Several mast cell mediators like histamine, tryptase, LTC4, TNF-␣ and TGF-␤1 also exhibit fibrogenic activity [77]. Mast cells that are treated with IL-4 express Toll-like receptor 4 (TLR4), which when activated by lipopolysaccharide (LPS) induces the release of Th2 cytokines [78]. Activation of mast cells via TLR2 and TLR3 selectively induce the release of leukotrienes, GM-CSF and IL-1␤ [78–80]. Human cultured mast cells also express TLR1, TLR2, TLR4, TLR5, TLR6, TLR7, and TLR9 [80]. These findings highlight several novel mechanisms whereby endogenous, bacterial and viral proteins can activate human mast cells, thus providing the means by which viruses and bacteria can induce asthma exacerbations.

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Structural alterations of the airways, defined as ‘tissue remodelling’, account for a progressive and irreversible loss of lung function in chronic asthma. The positive correlation between the thickness of the tenascin and laminin layers and mast-cell density in asthmatics suggests that mast cells may have a role in tissue remodeling [81]. Mast cells, which are closely associated with blood vessels and are increased at angiogenic sites, can contribute to various aspects of angiogenesis [82]. Mast cells synthesize and release various pro-angiogenic factors (histamine, tryptase, TGF-␤, IL-8 and VEGF) and the production of VEGF by mast cells is increased by PGE2 and other cAMPelevating agents [83–85].

Implications of Mast Cells in Rhinitis and Asthma

The unique microlocalization of mast cells in specific nasal and lung tissue compartments, the ability of these cells to migrate to the epithelial compartment where they are easily activated by allergens or pathogens, the powerful effector repertoire, the recognition of their different microbial-related activating ligands and their plasticity in response to various signals, suggest that mast cells have a central role in allergic airway diseases like rhinitis and asthma. However, mast cells at different stages of maturation might have different, or even, in some cases, protective roles in the appearance of the asthmatic phenotype. The extent to which each effector cell contributes toward each phase of allergic inflammation and tissue repair remains to be fully elucidated.

Future Potential Therapy Targeting Mast Cell-IgE-Networking: A Global Approach for Rhinitis and Asthma

Humanized Monoclonal Antibodies against IgE A monoclonal antibody raised against the C␧3 domain of IgE molecule is MAE11 and its humanized form used in clinical trials is the Rhu-MAb-E25 (E25) [86]. This region of the IgE molecule binds to the IgE receptor (Fc␧RI). Complexing free IgE with the mAb (MAE11) prior to its linking with the Fc␧RI prevents its binding to the IgE receptor. Moreover, MAE11 only binds to free IgE and does not bind to receptor bound IgE, and is therefore non-anaphylactic. Since Fc␧RI expression on mast cells/basophils is regulated by IgE and upregulated in allergic patients [38, 87], treatment with the anti-IgE MAb not only decreases free IgE levels to 1% of pretreatment levels but also downregulates Fc␧RI expression [88]. A study using E25 mAb in ragweed pollen-induced rhinitis showed only a small effect probably because the dose infused was insuf-

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ficient [89] and only patients with a dose of 300 mg/4 weeks E25 had a reduction of H1-antihistamine use of over 60% greater than placebo whilst symptoms were reduced by over 20%. E25 was administered to over 300 patients for periods up to 1 year and no serious adverse event was reported. In another study of 251 adult subjects with a history of SAR and a positive skin test response to birch pollen, 300 mg of rhumAb-E25 or placebo were given 2 or 3 times during the season, depending on baseline IgE levels [90]. These researchers showed that E25 prevented the seasonal increase in nasal symptoms, with a reduced intake of rescue medication as well as improved quality of life in patients with seasonal allergic rhinitis and was well tolerated. In a study of a total of 221 subjects, Kuehr et al. [90] showed that in patients with SAR to birch pollen combination therapy of SIT and the humanized anti-IgE mAb reduced symptom load over the two pollen seasons by 48% (p ⬍ 0.001) as compared to SIT alone. Thus, anti-IgE therapy can be considered to be useful for the treatment of allergic rhinitis, particularly for polysensitized patients.

Mast Cells in Chronic Rhinosinusitis

Chronic rhinosinusitis (CRS) is a multifactorial chronic inflammatory disease of the upper airway occurring with or without nasal polyps (NP) that are characterized histologically by the infiltration of inflammatory cells, predominantly eosinophils and/or neutrophils [91, 92]. Mast cells are key inflammatory cells that are known to play crucial roles in not only IgE-mediated diseases but also in non-IgE mediated inflammatory diseases. NP are characterized by massive tissue edema, resulting from a leakage of plasma through widened endothelial junctions in the blood vessels. The typical histological characteristics include edematous fluid with sparse fibrous cells, and few mucous glands with no innervation, squamous metaplasia of the surface epithelium, proliferation of stromal and epithelial elements and a thickening of the basement membrane. Other characteristics of NP include the existence of different types of epithelium from respiratory pseudostratified to transitional epithelium and a lowered density of goblet cells. The cellular components comprise a variety of cells including eosinophils, mast cells, lymphocytes, neutrophils and plasma cells. In a majority of NP, eosinophils comprise more than 60% of the cell population, except in cystic fibrosis. However, this eosinophilic inflammation is unrelated to atopy and therefore is also seen in polyps from non-atopic patients. While there is an increase in activated T cells (CD45RO⫹), mast cells and plasma cells are also significantly increased as compared to the normal nasal mucosa [93–95]. As in allergic rhinitis an increased expression of a variety of cytokines like IL-5, IL-6, IL-8, IL-13, TNF-␣, TGF-␤ [95–101], adhesion molecules like ICAM-1,

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VCAM-1, E-selectin and P-selectin and chemokines (e.g. RANTES, eotaxin) is well documented in CRS with NP [102]. By contrast to mast cells in allergic rhinitis which selectively express Th2-type cytokines as opposed to those in infective rhinitis, mast cells in NP from both atopic or non-atopic patients express Th2-type cytokines IL-5, IL-6 and IL-13 [103, 104]. Besides cytokines and chemokines, the levels of tryptase, histamine and ECP in polyp tissue and the nasal lavages from patients with NP are significantly higher as compared to those without NP [105, 106]. Indeed, the good correlation between the levels of histamine/tryptase and ECP suggest a potential role for mast cells in orchestrating eosinophil infiltration [105, 106]. There is also evidence for remodeling in NP with increase in basement membrane thickening and tissue degradation. MMPs are known to play a role in cell migration, edema and ECM degradation. Our recent studies have shown an increase in MMP-9 in NP with relatively low levels of TIMP-1 and -2 [107]. Moreover, the levels of MMP-9 were in good correlation with the levels of ECP and tryptase [107]. Furthermore, mast cells themselves expressed MMP-9 and mast cell tryptase and chymase could upregulate the production of MMP-9 from NP EC, suggesting an important role for mast cells in not only inducing eosinophil infiltration but also in the ECM degradation in NP. While allergenand IgE-activated mast cells could autoactivate themselves via the IgE-IgEreceptor-mast cell cascade, recently we demonstrated that mast cells activated by LPS (bacterial product) produced increased levels of IL-13 and TNF-␣ which could further promote the infiltration of T lymphocytes and eosinophils via increased production of TARC and RANTES [R. Pawankar unpubl. data]. Again, an increase in the levels of TGF-␤ in NP can contribute to the stromal fibrosis seen in NP. Also, TGF-␤ can upregulate eosinophilic inflammation by enhancing the IL-4- and LPS-induced production of eotaxin and VEGF in NP FB and TGF-␤ and the expression of VEGF [108, 109] important for angiogenesis and edema. Taken together, mast cells may contribute to NP growth and the remodeling process, and the latter may be a sequel of chronic inflammation (fig. 2). Various bacterial and viral products can activate Fc␧RI through diverse mechanisms. Bacterial (protein A and L from Staphylococcus aureus and Peptostreptococcus magnus, respectively) and viral (gp120 from HIV-1) Ig superallergens activate Fc␧RI⫹ cells by interacting with IgE to release proinflammatory mediators and cytokines in vitro and, in some settings, in vivo [110, 111]. Bachert et al. [112] have shown the presence of specific IgE to staphylococcal enterotoxins A and B in NP and described that the levels of IgE correlated with the eosinophilic infiltration. These researchers demonstrated multiclonal IgE, including specific IgE to SEA and SEB in 50% of bilateral eosinophilic NPs. Toll-like receptors (TLRs), a family of pattern recognition

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Aspirin intolerance

Nasal epithelium

GM-CSF RANTES Eotaxin TARC Eosinophil T cells

MBP ECP ICAM-1

IL-1 TNF-␣

Macrophage IL-4, IL-13, TNF-␣ FC␧RI / Histamine TLR Tryptase/chymase TGF-␤ Neutrophil

Fibroblasts

PGs 5 HETES

Increased collagen/ fibronectin MMP-9

VCAM-1 Albumin

Albumin

Virus, Bacteria, Allergens

IgE

Superantigens Immune complex

Histamine, LTs PGs, Kinins IL-5, IL-6, IL-13, TNF-␣

Cellular and Interstitial Edema Growth and persistence of nasal polyps

Fig. 2. Potential role of mast cells in nasal polyps [modified from R. Pawankar, Curr Opin Immunol, 2002].

receptors that are crucial for cellular responses to a variety of microbial agents, have been identified on mouse BMMCs (TLR2, TLR4 and TLR6) and on human umbilical cord blood-derived mast cells (TLR1, TLR2 and TLR6) [113]. Malaviya et al. [114] showed that mast cells have a pivotal role in innate host immune responses to Gram-negative bacteria through the release of TNF-␣. In another study, LPS and PGN induced a significant release of not only TNF-␣, but also IL-5, IL-10 and IL-13 by human mast cells, and this was mediated through interactions with TLR4 or with TLR2, respectively. Also, activation via TLR induces increased release of leukotrienes suggesting such a mechanism probably playing a role in the genesis of NP in patients with aspirin sensitivity. We recently demonstrated that NP mast cells express TLR2 and TLR4 [Pawankar R et al., unpubl. observations]. Thus, activation of mast cells via the TLR may be one of the mechanisms by which mast cells contribute to the immune and inflammatory events in CRS with NP which has a multifactorial etiology.

Conclusion

The paradigm of mast cell activation in allergic rhinitis, asthma or CRS is evolving from a reactionary system in which granule contents are released and

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generated following stimulation via the Fc␧RI, or to a proactive system in which the mast cell discriminates between a variety of stimuli and chooses its response accordingly. Therefore, targeting the mast cell-IgE-Fc␧RI cascade or manipulation of TLR signaling may be promising targets as a therapeutic option in allergic airway diseases and CRS.

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103 Pawankar R: Nasal polyposis: An update. Curr Opin Allergy Clin Immunol 2003;3:1–6. 104 Hamilos DL, Leung DY, Wood R, Cunningham L, Bean DK, Yasruel Z, Schotman E, Hamid Q: Evidence for distinct cytokine expression in allergic versus non-allergic chronic sinusitis. J Allergy Clin Immunol 1995;96:537–544. 105 Pawankar R: Mast cells in rhinitis; in Watanabe T, Timmerman H, Yanai K (eds): Histamine Research in the New Millennium. Amsterdam, Elsevier Science, 2001, pp 369–374. 106 Di Lorenzo G, Drago A, Esposito Pellitteri M, Candore G, Colombo A, Gervasi F, Pacor ML, Purello D’Ambrosio F, Caruso C: Measurement of inflammatory mediators of mast cells and eosinophils in native nasal lavage fluid in nasal polyposis. Int Arch Allergy Immunol 2001;125: 164–175. 107 Pawankar R, Watanabe S, Nonaka M, Ozu C, Aida M, Yagi T: Differential expression of matrix metalloproteinase-2 and -9 in the allergic nasal mucosa and nasal polyps. J Allergy Clin Immunol 2004;113:S332. 108 Nonaka M, Pawankar R, Fukumoto A, Yagi T: Synergistic Induction of eotaxin in fibroblasts by IL-4 and LPS: Modulation by TGF-␤. J Allergy Clin Immunol 2002;109:S38. 109 Nonaka M, Pawankar R, Fukumoto A, Ogihara N, Sakanushi A, Yagi T: Induction of eotaxin production by interleukin-4, interleukin-13 and lipopolysaccharide by nasal fibroblasts. Clin Exp Allergy 2004;34:804–811. 110 Genovese A, Bouvet JP, Florio G, Lamparter-Schummert B, Bjorck L, Marone G: Bacterial immunoglobulin superantigen proteins A and L activate human heart mast cells by interacting with Immunoglobulin E. Infect Immun 2000;68:5517–5524. 111 Genovese A, Borgia G, Bjorck L, Petraroli A, de Paulis A, Piazza M, Marone G: Immunoglobulin superantigen protein L induces IL-4 and IL-13 secretion from human Fc␧RI⫹ cells through interaction with the ␬ light chains of IgE. J Immunol 2003;170:1854–1861. 112 Bachert C, Gevaert P, Holtappels G, Johansson SG, van Cauwenberge P: Total and specific IgE in nasal polyps is related to local eosinophilic inflammation. J Allergy Clin Immunol 2001;107: 607–614. 113 Marshall JS, McCurdy JD, Olynych T: Toll-like receptor-mediated activation of mast cells: Implications for allergic disease? Int Arch Allergy Immunol 2003;132:87–97. 114 Malaviya R, Georges A: Regulation of mast cell-mediated innate immunity during early response to bacterial infection. Clin Rev Allergy Immunol 2002;22:189–204.

Prof. Ruby Pawankar, MD, PhD Department of Otolaryngology Nippon Medical School, 1-1-5, Sendagi Bunkyo-ku, Tokyo 113–8603 (Japan) Tel./Fax ⫹81 3 5802 8177, E-Mail Pawankar_Ruby/[email protected]

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Saito H, Okayama Y (eds): Mast Cells in Allergic Diseases. Chem Immunol Allergy. Basel, Karger, 2005, vol 87, pp 130–144

Chemokine Receptor Expression by Mast Cells Mikael Juremalma, Gunnar Nilssona,b a

Department of Genetics and Pathology, Uppsala University, Uppsala, and Department of Medicine, Karolinska Institutet, Stockholm, Sweden

b

Abstract There is a growing interest in the role of chemokines and their receptors in the determination of mast cell tissue localization and how chemokines regulate mast cell function. At least nine chemokine receptors (CXCR1, CXCR2, CXCR3, CXCR4, CX3CR1, CCR1, CCR3, CCR4 and CCR5) have been described to be expressed by human mast cells of different origins. Seven chemokines (CXCL1, CXCL5, CXCL8, CXCL14, CX3CL1, CCL5 and CCL11) have been shown to act on some of these receptors and to induce mast cell migration. Mast cells have a unique expression pattern of CCR3, CXCR1 and CXCR2. These receptors are mainly expressed intracellularly on cytoplasmic membranes. Upon an allergic activation, CCR3 expression is increased on the cell surface and the cell becomes vulnerable for CCL11 treatment. Chemokines do not induce mast cell degranulation but CXCL14 causes secretion of de novo synthesized CXCL8. Because of the expression of CCR3, CCR5 and CXCR4 on mast cell progenitors, these cells are susceptible to HIV infection and mast cells might therefore be a persistent HIV reservoir in AIDS. In this review, we summarize the knowledge about chemokine receptor expression and function on mast cells. Copyright © 2005 S. Karger AG, Basel

Introduction

Mast cells originate from CD34-positive hematopoietic stem cells in the bone marrow and circulate in the blood as progenitors lacking granules and functions characteristic for mature mast cells. The committed progenitors enter into diverse peripheral tissues where they complete their final maturation. Mature mast cells are ubiquitously distributed throughout the body, especially in the perivascular spaces and connective tissues of the skin, respiratory and gastrointestinal tracts. Although the number of mast cells in tissues normally is

relatively constant, the number can rapidly increase as a response to tissue injury, infection, etc. Both the transit of mast cell precursors to various tissues and the migration of mast cells within a tissue are regulated by a number of different chemoattractants of which chemokines and their receptors have emerged as important components.

Chemokines and Their Receptors

Chemokines are a group of small (8–14 kDa), mostly basic heparin proteins whose main function is the regulation of cell trafficking of various types of leukocytes. Chemokines are distinguished from other cytokines by being the only members of the cytokine family that act on the superfamily of seven transmembrane, G-protein-coupled receptors (GPCRs). Approximately 50 different human chemokines have been identified to date. Most of the chemokines have been discovered during recent years due to the development of EST (expressed sequence tag) databases, bioinformatics and completing of the human genome [1]. The sequence identity between chemokines is relatively low. In spite of that, their three-dimensional structure shows a remarkable homology due to the same monomeric fold. This fold, consisting of three ␤-strands, a carboxy-terminal helix and a flexible amino-terminal region, is conferred by a four conserved cysteine motif that forms two characteristic disulfide bridges. Four classes of chemokines have been defined based on the arrangement of the conserved cysteine (C) residues in the NH2-terminal domain of the mature proteins: the CXC, CC, C, and the CX3C (fig. 1). The CXC and CC chemokines are the major chemokine families, since only two C chemokines and one CX3CL1 chemokine have been described. CX3CL1 is unique among the chemokines since it has a multimodular structure consisting of a chemokine domain fused to a mucin-like stalk plus a transmembrane domain, which anchors the molecule to the plasma membrane, and a cytoplasmic domain. Consistent with this, it functions as an adhesion molecule by binding directly to its receptor CX3CR1. The CXC family can further be subclassified into those that contain the sequence glutamic acidleucine-arginine (ELR motif) near the N-terminal to the first cysteine and those that do not. There has been some confusion regarding the nomenclature of the chemokine system. The chemokines were originally named according to their function or after the cells that produced them. Subsequently, the simultaneous identification of chemokine sequences by many laboratories resulted in several names. In order to eliminate any confusion, a new systemic nomenclature has been adopted where the chemokines are named after to which receptor subtype they bind and in which order they were discovered [1] (table 1).

Chemokine Receptors

131

Family

Structure

CXC

CXC

CC

CC

C

C ␤1

␤2

␤1

␤2

␤3 C

␤2

␤3 C

␤2

␤3

C

C

␤1 C

C XXX C

CX3C

␤1

Chemokine

␤3 C

C

Mucin stalk

Cytoplasmic domain

Fig. 1. Structural features of the chemokine family.

Table 1. Chemokines and their corresponding receptors Systemic name

Common namesa

Chemokine receptor(s)

CXC chemokines CXCL1 CXCL2 CXCL3 CXCL4 CXCL5 CXCL6 CXCL7 CXCL8 CXCL9 CXCL10 CXCL11 CXCL12 CXCL13 CXCL14 CXCL15 CXCL16

GRO␣ GRO␤ GRO␥ PF4 ENA-78 GCP-2 NAP-2 IL-8 Mig IP-10 I-TAC SDF-1 ␣/␤ BCA-1 BRAK Unknowna

CXCR1, CXCR2 CXCR2 CXCR2 CXCR3b CXCR2 CXCR1, CXCR2 CXCR2 CXCR1, CXCR2 CXCR3a,b CXCR3a,b CXCR3a,b CXCR4 CXCR5 Unknown Unknown CXCR6

C chemokines XCL1 XCL2

Lymphotactin/SCM-1␣ SCM-1␤

XCR1 XCR1

CX3C chemokine CX3CL1

Fractalkine

CX3CR1

Juremalm/Nilsson

132

Table 1 (continued) Systemic name

Common namesa

Chemokine receptor(s)

CC chemokines CCL1 CCL2 CCL3 CCL3L1 CCL4 CCL5 CCL6 CCL7 CCL8 CCL9/10 CCL11 CCL12 CCL13 CCL14 CCL15 CCL16 CCL17 CCL18 CCL19 CCL20 CCL21 CCL22 CCL23 CCL24 CCL25 CCL26 CCL27 CCL28

I-309 MCP-1 MIP-1␣/LD78␣ LD78␤ MIP-1␤ RANTES Unknown MCP-3 MCP-2 Unknown Eotaxin Unknown MCP-4 HCC-1 HCC-2 HCC-4 TARC PARC MIP-3␤/ELC MIP-3␣/LARC 6Ckine/SLC MDC MPIF-1 Eotaxin-2 TECK Eotaxin-3 CTACK MEC

CCR8 CCR2 CCR1, CCR5 CCR1, CCR5 CCR5 CCR1, CCR3, CCR5 Unknown CCR1, CCR2, CCR3 CCR3, CCR5 CCR1 CCR3 CCR2 CCR2, CCR3 CCR1, CCR5 CCR1, CCR3 CCR1, CCR2 CCR4 Unknown CCR7 CCR6 CCR7 CCR4 CCR1 CCR3 CCR9 CCR3 CCR10 CCR3, CCR10

a

The common names given in this table are based on human chemokines. Unknown chemokine denotes that no counterpart has been found in humans.

Chemokine Receptors Chemokines mediate their function by binding to seven transmembranespanning GPCRs. By definition, CC chemokines are only able to bind CC receptors and the CXC receptors bind only the CXC chemokines. Six CXC receptors (CXCR), these being CXCR1–CXCR6, and ten CC receptors (CCR), CCR1–CCR10, have been described. In addition, one receptor for C chemokines

Chemokine Receptors

133

N C

C C

Extracellular C Transmembrane segment Intracellular

DRYLAIVHA C

Fig. 2. General structure of chemokine receptors.

(XCR1) and one for the CX3C chemokine (CX3CR1) have been reported [2] (table 1). Furthermore, two other chemokine-binding proteins have been shown with capacity to bind chemokines but not to cause any functional responses in cells upon chemokine binding, called Duffy and D6, and subsequently excluded from the nomenclature system. To date, there also exist more than 30 distinct virally encoded chemokine and chemokine receptor mimics in the herpes virus, poxvirus and retrovirus families. The sequences of chemokine receptors have 25–80% aa identity, indicating a common ancestor and there are several features frequently found among chemokine receptors (fig. 2). These include a length of 340–370 aa; an acidic flexible N-terminal segment; the sequence DRYLAIVHA, or a variation of it, in the second intracellular loop; a short basic third intracellular loop, and a cysteine in each of the four extracellular domains. The flexible N-terminus is believed to be important in receptor activation, because modification of this region has been shown to affect activity. Mutagenesis has indicated that the ligand-binding site of chemokine receptors is highly complex, being composed of multiple non-contiguous domains and at least two distinct binding subsites – one for docking and the other for triggering. Chemokine binding to GPCRs activates a complex network of intracellular signalling pathways. In the classical view of GPCR, chemokine receptor signalling is mediated by pertussis toxin-sensitive heterotrimeric G␣i␤␥ protein activation and subsequently dissociation into the G␤␥ complex and the GTP-bound G␣i subunit. The G␣i subunit and G␤␥ complex may then couple to downstream effectors such as ion channels, and diverse enzymes and generation of a variety of second messenger systems, such as calcium, cAMP, and phospholipids, as well as a concerted interplay of kinase cascades downstream of small GTPases. Most chemokines share the ability to activate G-protein-sensitive phospholipase C isoforms, resulting in inositol 3,4,5-trisphosphate generation and elevation of intracellular calcium. However, the functional requirement for calcium is questionable as chemotaxis can be detected in situations where calcium mobilization

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cannot be detected, suggesting that other biochemical events are probably more important. Several chemokines have been shown to activate alternative signalling pathways, such as Janus kinase (JAK) family members and specific associated STAT (signal transducer and activators of transcription) members. Similar to the cytokine receptor family, activation of the JAK-STAT pathway have been described to also involve homo- and heterodimerization of some chemokine receptors, and that signalling through the JAK-STAT pathway is independent of G-protein signalling [3]. Complex Ligand-Receptor Chemokine Network Although some chemokine receptors appear to have a single specific chemokine ligand, e.g. CXCR4, that specifically binds only CXCL12, it is more common that a single chemokine is able to act as a ligand for multiple receptors and that one receptor can bind multiple chemokines (table 1). Chemokine receptors are expressed on different types of leukocytes. Some are restricted to specific cell types, whereas others are more widely expressed. To make it more complex, distinct receptor subtypes specific for the same chemokine and for the same function can be co-expressed on the same cell. Further, distinct chemokines acting at separate receptors co-expressed on the same cell can induce the same cellular response, and the same receptor can sort signals from different ligands to distinct signalling pathways. Furthermore, it has become evident that the interplay of the receptors and ligands in physiological conditions is even more complicated by the presence of agonist and antagonist activities. The CXCR3 agonists CXCL9, CXCL10 and CXCL11, involved in responses driven by Th1 cells, can act as antagonists of CCR3, which is postulated to play a role in Th2-driven responses. Additionally, the CCR3 agonist CCL11 is a weak agonist for CCR2 and CCR5, whereas the CCR1/CCR2/CCR3 agonist CCL7 is an antagonist for CCR5. Evidently, cross-reactivity seems to be a fundamental property of the chemokine system. Further characterization of the binding potentials of all known chemokines is expected to reveal additional examples of cross-reactivity. The complexity of the chemokine system might seem exaggerated, but may be a way to regulate cellular responses and functions in different cell types and create a robust system of overlapping ligands and receptors that protect the host.

Expression of Chemokine Receptors by Mast Cells

To date, five CCRs, CX3CR and four CXCRs have been found to be expressed as mRNA and/or as proteins by mast cells. Although a considerable number of chemokine receptors are expressed on mast cells, only a rather limited

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number of chemokines seem to act on these receptors. One example is CCR1, which is a receptor for CCL3, CCL5, CCL7, CCL14, CCL15, CCL16 and CCL23, where only CCL5 seems to act on this receptor expressed by mast cells. Thus, the functional properties of individual receptors may vary depending on the cellular distribution of the receptor. The research concerning chemokine receptors expressed by mast cells has been performed on a number of different mast cell lines, primary mast cells, and mast cells derived in vitro using different protocols for differentiation. The sources have been rodents as well as humans. Furthermore, some studies have been analyzing chemokine receptors expressed on mast cells in situ, using different tissues. It is important to keep in mind that chemokine receptor expression differs between species and between different tissues. Furthermore, cytokines present (or missing) in the medium used for mast cell differentiation in vitro also have an effect on the expression. In addition, a certain chemokine receptor can be expressed on a small portion of the mast cells in a given tissue, e.g. only 14% of the mast cells in the lung express CCR3 [4]. Taken together, this has led to several discrepancies in the literature on chemokine receptors expressed by mast cells.

CCR1 and CCR4 The first chemokine shown to induce mast cell migration was CCL5/ RANTES [5, 6]. CCL5 binds to several receptors, i.e. CCR1, CCR3, CCR4 and CCR5. Of these receptors all, but CCR5, have been demonstrated on tissue mast cells in situ. CCR1 and CCR4 are expressed on airway mast cells in allergic asthma [K. Amin and G. Nilsson, unpubl. observation], and CCR1 also on mast cells in the lamina propria in oral lichen planus [7]. CCR3 is expressed mainly on MTTC-type mast cells, resembling connective tissue type, found in the skin, gut and intestinal submucosa [4]. Of the ligands binding to CCR1 and CCR4, only CCL5 induces a migratory response mediated by both CCR1 and CCR4 [8]. CCL17 and CCL22, agonists for CCR4, causes calcium flux in in vitro developed human mast cells without stimulating migration or degranulation [M. Juremalm and G. Nilsson, unpubl. observation]. Thus, the physiological role of CCL17 and CCL22 for mast cell functions remains to be elucidated. Subcutaneous injection of CCL5 into the rat paw causes an accumulation of mast cells in the sole, suggesting a direct effect of CCL5 on chemokine receptors expressed on mast cells [9]. In contrast, intradermal injection of CCL5 into human skin results in infiltration of eosinophils and T lymphocytes, but not mast cells [10]. This is in accordance with lack of CCR1 and CCR4 expression on human dermal mast cells [I. Harvima and G. Nilsson, unpubl. observation].

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CCR2 CCR2 is a receptor for CCL2, CCL7, CCL13 and CCL16. None of these chemokines have been shown to have an effect on human mast cells, but CCL2 induces migration in bone marrow-derived murine mast cells [11]. Although expression of CCR2 transcripts in bone marrow-derived mast cells has been described [12], expression of CCR2 on human mast cells is equivocal. CCR3 CCR3 was first described as a specific eosinophilic receptor, regulating infiltration of eosinophils after interacting with CCL11/eotaxin. However, CCR3 is expressed as well on basophils, Th2 lymphocytes and mast cells. The expression of CCR3 by mast cells is unique in that the majority of CCR3 is expressed intracellularly on granules and not to any high degree on the cell surface [13]. Upon IgE receptor aggregation, CCR3 is translocated from the intracellular compartment and expressed on the cell surface. It has been suggested that this unique expression pattern of CCR3 facilitates the emigration of mast cells from the intraepithelial compartment of the lung rather than recruitment of them in an allergic inflammation. Using an in vitro system, culturing cord blood mononuclear cells in the presence of SCF, IL-6 and IL-10, it was demonstrated that 4-week-old progenitor mast cells express CXCR2, CXCR4, CCR3 and CCR5, but after 10 weeks only CCR3 remained expressed [14]. This suggests that chemokine receptors are differentially expressed during mast cell differentiation. The expression of CCR3 by tissue mast cells appears to be organ-specific, expressed mainly by MCTC in gut, lung and skin tissue [4]. Thus, CCR3-positive mast cells are more abundant in skin dermis and intestinal submucosa than in intestinal mucosa and lung interstitium. A role for CCR3 expressed on mast cells in vivo comes from studies using CCR3⫺/⫺ mice. One study showed an increase in the number of tracheal intraepithelial mast cells, especially after allergen challenge [15]. In contrast, such an increase was not observed in jejunal intraepithelial mast cells after helminth infection [16]. Furthermore, no change in dermal mast cell numbers was observed in an allergic skin model [17]. Thus, the role of CCR3 in regulating mast cell migration into inflamed tissues is much more complex compared to eosinophils, where CCR3 is a requirement for recruitment of the cell [15]. CCR5 CCR5 is expressed on mast cell progenitors circulating in the blood, or developed in vitro [14, 18, 19]. The human mast cell line LAD-1 also expresses CCR5 [20]. However, a physiological role for CCR5 expressed on mast cells is not clear. CCR5 binds CCL3, CCL3L1, CCL4, CCL5, CCL8 and

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CCL14, but none of these have been shown to have a clear direct effect on human mast cells. CX3CR1 The ligand to CX3CR1, CX3CL1, is unique because it is synthesized as a large type I membrane protein with an N-terminal glycosylated membrane proximal domain (fig. 1). This form of CX3CL1 functions as an adhesion molecule for CX3CR-expressing cells. Soluble, chemotactically active CX3CL1 can be released by proteolytic cleavage. CX3CL1 is expressed on several cell types including neurons, renal glomeruli, dendritic cells and melanocytes, some of which mast cells are closely associated with, suggesting a direct interaction between CX3CL1 and mast cells. CX3CR is expressed on both cultured murine bone marrow derived mast cells and human dermal foreskin mast cells [21]. CX3CL1 induces a migratory response in these cells, without inducing degranulation. CXCR1 and CXCR2 Expression of CXCL8 (IL-8) is a common feature for many inflammations, both acute and chronic. Several of these inflammations, such as rheumatoid arthritis, psoriasis, etc., are also associated with mast cell hyperplasia. CXCL8 acts on both CXCR1 and CXCR2, where CXCR1 only binds CXCL8, whereas CXCR2 is more promiscuous binding to seven chemokines (table 1). CXCR1 and CXCR2 are expressed on human mast cells, both in vitro developed and skin mast cells [22, 23]. CXCR1 and CXCR2 are expressed on the cell surface but also on cytoplasmic membranes including granules [22]. Whether mast cell degranulation results in a translocation of the receptors to the cell surface, as in the case for CCR3, thereby increasing the level of surface expression and susceptibility to agonist stimulation, has not been investigated. Expression of CXCL8 is a common feature of many inflammations, both acute and chronic. Several of these inflammations, e.g. psoriasis, rheumatoid arthritis and others, are associated with mast cell hyperplasia suggesting a specific recruitment of mast cells to the inflamed tissue. The role of mast cell expressed CXCR2 in these diseases remains however to be described. CXCR3 Most of the studies on chemokine receptor expression on mast cells have been performed on in vitro differentiated mast cells, which may not reflect the true or complete picture of chemokine receptor expression on mast cells. It is well known that receptor expression fluctuates during differentiation and during pathophysiological settings where exposure to external stimuli affects the expression of receptors. This means that expression of chemokine receptors on mast

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cells differ in, for example, TH1- and TH2-polarized inflammations. One example is the expression of CXCR3 on synovial mast cells from patients with rheumatoid arthritis [24], an expression which has not been detected on in vitro developed mast cells. A hallmark of rheumatoid arthritis is infiltration of leukocytes into synovial tissue. Two of the chemokines highly expressed in rheumatoid arthritis are the IFN-␥-inducible CXCL9 and CXCL10, both ligands for CXCR3. Interestingly, the predominant CXCR3-expressing cells in rheumatoid arthritis have been shown to be mast cells [24]. Thus, CXCR3 expression might play a role in the recruitment and function of mast cells in the synovial tissue of patients with rheumatoid arthritis. CXCR4 CXCR4 is an important receptor for the transit of cells from the bone marrow into the circulation and for the recruitment of progenitors to certain tissues. Human mast cell progenitor cells developed in vitro express CCR3, CCR5, CXCR2 and CXCR4 [14]. Of these receptors, CXCR4, is likely to be of importance for mast cell progenitors and their recruitment and transmigration over the endothelial cell layer [25]. CXCR4 is co-expressed with Kit, the receptor for stem cell factor, on a small portion of cells circulating in the blood, resembling mast cell progenitors [26]. Culturing these cells in the presence of SCF and IL-6 results in the development of mast cells [26].

Functional Aspects of Chemokine Receptors on Mast Cells

By definition, chemokines are chemotactic cytokines regulating the migration of especially hematopoietic cells. However, the binding of chemokines to their receptors can induce a number of different cellular activities depending on the target cell. Besides migration, chemokines promote adhesion by regulating the expression of integrins, and stimulate release of inflammatory mediators from leukocytes. Chemokines also regulate angiogenesis and hematopoiesis. Besides these actions, some of the chemokine receptors are co-receptors for HIV infection. Thus, chemokine receptors are multifunctional in that they can provide signals regulating several different cellular events. Migration Chemokine receptors expressed by mast cell progenitors and mature tissue mast cells are most likely involved in directing the progenitors from the circulation into the tissue where the mature. Mature mast cells express a somewhat different set of chemokine receptors compared to the progenitors, and this differs also between different mast cell phenotypes (fig. 3). Mast cells at sites of

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CCR3 CXCR2 CXCR4

Circulating mast cell progenitor

Endothelium

Tissue mast cells

MCT CCR1 CCR4

MCTC CCR3 CXCR1 CXCR2 CXCR3

Fig. 3. A model for chemokine receptor expression during mast cell differentiation. Changes in chemokine receptor expression by mast cells of different phenotypes are indicated.

inflammations might exhibit enhanced levels of chemokine receptors, either because of cytokines expressed in the microenvironment or dependent on activation of the cells [27]. These mast cells might therefore have an enhanced susceptibility to chemokine stimulation. However, these differences still need to be elucidated. The diversity of chemokine receptors expressed on different mast cells most likely reflects the specific recruitment of mast cells to particular tissues. Human mast cells, either the HMC-1 cell line, in vitro developed mast cells, or mast cells purified from tissues, have been shown to migrate in vitro in response to CXCL1, CXCL5, CXCL8, CXCL14, CX3CL1, CCL5 and CCL11 [4–6, 8, 14, 21–23, 25, 26]. Thus, a rather limited number of chemokines, especially of the CCL family, have so far been demonstrated to be chemoattractants for human mast cells. Degranulation and Cytokine Secretion Several of the previously described non-IgE-dependent histamine-releasing factors acting on basophils were found to be chemokines. CCL2 is one of the most potent secretagogues for basophils, but also CCL3, CCL5, CCL7 and CCL8 are potent factors causing basophilic degranulation [28]. In contrast to basophils, chemokines seem not to be secretagogues for mast cells causing degranulation or eicosanoid mediator release [11, 29, 30]. Although not causing degranulation on its own, CCL3 binding to CCR1 expressed on rat RBL-2H3 cells synergistically enhance IgE receptor-mediated degranulation [31]. Thus, chemokines like CCL3, and possible other chemokines, have the potential to affect mast cell degranulation in an allergic setting.

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It is becoming more and more evident that mast cells have the capacity to secrete cytokines without undergoing exocytosis. This is most likely of importance for the pathophysiological contribution of mast cells in chronic inflammations that are not associated with release of granule mediators such as histamine. One such example of degranulation-independent release of cytokines is CXCL12/ SDF-1␣ that induces secretion of de novo synthesized IL-8 from the human mast cell line HMC-1 [25]. Interestingly, CXCL12 does not induce secretion of TNF-␣, IL-1␤, IL-6, GM-CSF, IFN-␥, or CCL5. This suggests that mast cells stimulated by CXCL12 in vivo may specifically release IL-8 that acts as an important chemoattractant for neutrophils. Mast cell-dependent neutrophil recruitment has been shown to be critical in a number of events, such as bacterial infection, immune complex-mediated injury, and IgE-mediated responses in the skin and gastric mucosa. CCL11 provides yet another type of effect on mast cell secretion. As already mentioned, mast cells express the CCL11 receptor, CCR3, mainly intracellularly in their secretory granules. Upon IgE receptor activation the expression of CCR3 on the surface of activated mast cells increases, and the cells become vulnerable for CCL11 treatment. A few hours after IgE receptor activation, treatment with CCL11 causes an increase in Fc␧RI-dependent secretion of IL-13 [13]. It is important to note that CCL11 neither causes any degranulation on its own, nor acts synergistically on Fc␧RI-dependent release. It is only when mast cells have degranulated and intracellular CCR3 is mobilized to the cell surface that CCL11 potentiates release of IL-13. Viral Infection One of the most exciting developments in chemokine receptor-associated pathogenesis comes from the discovery that some chemokine receptors function as co-receptors together with CD4 for HIV infections. Both in vitro developed mast cell progenitors and a metachromatic mast cell/basophilic-like cell in peripheral blood express the co-receptors CCR3, CCR5 and CXCR4, and are susceptible to an M-tropic strain of HIV-1 [18, 19]. Furthermore, HIV-1 Tat protein is a potent chemoattractant for human mast cells via interaction with CCR3 [32]. Considering the recruitment of committed mast cell progenitors to multiple tissues, the long life span of mast cells and their susceptibility to HIV-1 infection, suggests that mast cells can be a widespread and persistent HIV reservoir in AIDS.

Conclusions

Although there has been recent progress in our understanding of chemokine receptors expressed on mast cells, our knowledge about their role for mast cell

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migration and function in vivo is still very limited. The vast majority of the information comes from cell lines or mast cells derived in vitro. We know that there is considerable mast cell heterogeneity at the biochemical and functional levels in vivo and that this heterogeneity is also reflected on the level of chemokine receptor expression (fig. 3). Thus, characterization of chemokine receptor expression by mast cells located in different tissues, healthy or from different diseases, is highly desired. Furthermore, the precise role that each of the chemokines has on chemokine receptors expressed on tissue mast cells and how they cooperate with one another remains largely unknown. The picture of chemokine expression by tissue mast cells is far from complete why future advances in chemokine receptor expression by mast cells are certain to follow. This will also be of importance for the evaluation of new drugs developed against chemokines or chemokine receptors and how these will target mast cells in mast cell-mediated inflammations.

Acknowledgment Gunnar Nilsson is supported by The Swedish Research Council – Medicine and The Swedish Cancer Foundation.

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Dr. Gunnar Nilsson Clinical Immunology and Allergy Unit Department of Medicine, Karolinska Institutet SE–171 76 Stockholm (Sweden) Tel. ⫹46 8 517 70205, Fax ⫹46 8 335724, E-Mail [email protected]

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Mast Cell ␤2-Adrenoceptors Linda J. Kay, Peter T. Peachell Molecular Pharmacology, University of Sheffield, The Royal Hallamshire Hospital, Sheffield, UK

Abstract The human lung mast cell is a crucial effector cell in the mediation of asthma. Activation of mast cells by allergens, and other insults, leads to the elaboration of a wide variety of autacoids that cause bronchoconstriction and promote inflammation. Of the drugs that are used to treat asthma, only bronchodilator ␤2-adrenoceptor agonists are effective at inhibiting the elaboration of mediators from mast cells. Both short- and long-acting ␤2-adrenoceptor agonists are effective inhibitors of mast cells although there are differences in the degree of inhibitory activity attained with a given agonist. Human lung mast cells express a homogeneous population of ␤2-adrenoceptors. However, the density of ␤2-adrenoceptors differs from preparation to preparation and this may influence the extent to which agonists stabilise mast cell activity. Tolerance to the mast cell-stabilising activity of ␤2-adrenoceptor agonists can be readily demonstrated. As a generalisation, agonists that are more effective inhibitors of mediator release also induce greater levels of tolerance although weaker agonists induce greater levels of tolerance than might be expected. However, the extent of tolerance does not correlate with the degree of ␤2-adrenoceptor loss. The inhibitory activity of agonists and the extent of tolerance observed may be influenced by genetic polymorphisms in the gene for the ␤2-adrenoceptor. Copyright © 2005 S. Karger AG, Basel

Introduction

The mast cell has long been recognised as central in the mediation of allergic disorders. As a corollary, the human lung mast cell has a prominent role in the mediation of asthma, especially asthma that has an allergic basis. The IgEdependent activation of mast cells by allergens leads to the generation of a wide variety of autacoids that can cause bronchoconstriction and promote inflammation. It follows that stabilising mast cell activity would be expected to be of benefit in the treatment of asthma. Of the drugs that are currently used to treat

asthma, only bronchodilators appear to be able to attenuate mast cell activity to an appreciable degree. This property of bronchodilators is likely to be of additional therapeutic benefit over and above the primary action of these drugs which is to relax airway smooth muscle. Over the past few years, we have been investigating, in some detail, the effects of bronchodilator ␤2-adrenoceptor agonists on human lung mast cell function. Apart from the obvious relevance that such studies might have from the therapeutic perspective, the lung mast cell has proved to be a very useful primary human cell system in which to study ␤2-adrenoceptors. The aim of this review will be to discuss the role and regulation of ␤2-adrenoceptors in human lung mast cells. As such, the review will focus on (a) the inhibitory effects of ␤2-adrenoceptor agonists on mast cells, (b) tolerance to the mast cell-stabilising properties of ␤2-adrenoceptor agonists and (c) the influence that genetic polymorphisms of the ␤2-adrenoceptor may have on the response of mast cells to agonists.

Mast Cell Stabilisation

In 1937, Schild [1] demonstrated that adrenaline inhibited the antigendriven release of histamine from guinea pig lung. Later studies on primate lung tissue demonstrated that catecholamines were effective inhibitors of histamine release from lung fragments challenged with antigen [2, 3]. As mast cells constitute the tissue source of histamine, these data suggested that the effects of catecholamines were mast cell-directed. Since the pioneering work of Schild, a host of studies has appeared demonstrating that bronchodilators are effective inhibitors of the IgE-dependent release of histamine from mast cells. However, it should be stressed that these agents are also very effective inhibitors of the stimulated generation of the eicosanoids, prostaglandin D2 (PGD2) and cysteinyl-leukotrienes [4]. Indeed, ␤-adrenoceptor agonists are both more potent and efficacious inhibitors of eicosanoid generation than histamine release, which in the clinical context, could be a particularly important property of these drugs since cysteinylleukotrienes are especially potent bronchoconstrictors. Pharmacological characterisation of the adrenoceptor mediating the inhibitory effects of catecholamines on mast cells has been provided both by functional approaches and by radioligand binding. The first comprehensive study attempting to characterise the mast cell adrenoceptor was performed on human lung fragments [5]. The effects of a range of agonists, selective for different adrenoceptor subtypes, on IgE-dependent mediator release from lung fragments were determined. Based on the rank order of potency of the agonists,

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the data suggested that the agonists mediated their effects on mast cells by engaging ␤2-adrenoceptors. These findings, based on functional assays, are supported by our own recent radioligand-binding studies in membranes derived from highly purified mast cells [6]. Displacement of the radioligand, iodinated cyanopindolol, from membranes by the antagonists ICI118551 (␤2-selective) and CGP20712A (␤1-selective) is consistent with the expression of a homogeneous population of ␤2-adrenoceptors by mast cells. Although clinically useful short- (salbutamol, terbutaline) and long-acting (salmeterol, formoterol) ␤2-adrenoceptor agonists are effective stabilisers of mast cells, the extent to which these agents attenuate mast cell activity is highly variable among preparations. For example, salbutamol, in some mast cell preparations, is as efficacious as the full agonist isoprenaline (non-selective ␤-adrenoceptor agonist) whereas, in others, it acts as a partial agonist as the maximal inhibitory response it attains is lower than that of isoprenaline [7]. Similarly, variable inhibitory activities in mast cells can be seen with alternative ␤2-adrenoceptor agonists such as terbutaline and salmeterol [7, 8]. Formoterol, on the other hand, acts as a full agonist in all preparations [8]. These findings probably have some clinical bearing and may explain the variable benefit that ␤2-adrenoceptor agonists provide among asthmatics. The underlying mechanism for the very variable inhibitory responses is probably related to variable receptor reserves among mast cell preparations, higher receptor reserve equating with a better inhibitory response in mast cells to a ␤-adrenoceptor agonist [9]. Indeed, measures of receptor density in membranes generated from purified mast cells indicate that there can be as much as ten-fold differences in the amounts of ␤2-adrenoceptor expressed by different mast cell preparations [6]. Differences in receptor density are highly likely to influence the extent of inhibitory activity displayed by agonists [10]. The mechanism by which ␤2-adrenoceptor agonists inhibit mast cell responses is not known. However, the stabilisation of mast cells by these drugs is probably mediated by cyclic AMP. The ␤2-adrenoceptor is G-protein linked to adenylate cyclase and activation of the receptor is known to cause intracellular elevations in cyclic AMP. Exposure of mast cells to ␤2-adrenoceptor agonists causes increases in cyclic AMP and there is a good correlation between the extent to which a given ␤-adrenoceptor agonist elevates mast cell cyclic AMP and the degree to which the agonist inhibits mast cell responses [11]. These findings imply a role for cyclic AMP and, by association, cyclic AMP-dependent protein kinase (PKA) as the intracellular machinery mediating the inhibitory effects of ␤-adrenoceptor agonists. Although the targets of PKA are not known, by extrapolation with the situation for the human basophil [12], it is possible that agents that elevate cyclic AMP may, at least in part, interfere with the mobilisation of intracellular calcium which occurs following mast cell activation.

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Tolerance

Although bronchodilators are the most commonly prescribed medication for the treatment of asthma, one factor that could disadvantage their continued use is the development of tolerance. In clinical practice, it is difficult to assess the extent to which tolerance is an issue but under controlled study conditions, the development of tolerance can be demonstrated suggesting that it should be considered as a potential drawback to the use of bronchodilators. Additionally, it has been demonstrated, both in vivo and in vitro, that tolerance to the mast cell-stabilising properties of bronchodilators occurs more readily than tolerance to bronchodilation [13–16]. This could create a situation in which the release of spasmogenic and pro-inflammatory mediators from mast cells could occur unchecked, the deleterious effects of which could be masked by the smooth muscle relaxant effects that bronchodilators would continue to provide. At the molecular level, tolerance most probably reflects ␤2-adrenoceptor desensitisation. Desensitisation is a multi-step process that involves, by stages, uncoupling, sequestration and down-regulation of the receptor [17]. Phospho rylations of the receptor, mediated by both PKA and G-protein receptor kinase (GRK), are believed to be instrumental in mediating receptor desensitisation [18]. We have investigated ␤2-adrenoceptor desensitisation in mast cells primarily in a functional context. Thus, we can readily demonstrate that long-term incubation (24 h) of mast cells with a ␤-adrenoceptor agonist impairs the subsequent ability of a ␤-adrenoceptor agonist to inhibit the IgE-mediated release of histamine [11, 19]. The functional desensitisation appears to be ␤-adrenoceptormediated since co-incubation of an agonist with the ␤-adrenoceptor antagonist, propranolol, during the desensitisation incubation protects against desensitisation [19]. Moreover, the desensitisation is ‘homologous’ as overnight treatment with a ␤-adrenoceptor agonist has little effect on the ability of either PGE2 (an alternative receptor-mediated activator of adenylate cyclase) or forskolin (a direct activator of adenylate cyclase) to inhibit IgE-mediated histamine release from mast cells demonstrating that the desensitising treatment acts specifically to attenuate ␤2-adrenoceptor-mediated effects [7, 8]. Although, in general terms, functional desensitisation is observed following long-term treatment with both short- and long-acting agonists, it should be noted that pre-treatment of mast cells with either salmeterol or formoterol promotes substantially greater levels of functional desensitisation than either salbutamol or terbutaline [7, 8]. The reason for this difference in desensitising capability is not immediately apparent but it is possible that the lipophilic nature of the long-acting agonists, relative to the short-acting agonists, may contribute to the enhanced degree of functional desensitisation.

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1.0

Isoprenaline Fenoterol

Intrinsic activity

0.8

Formoterol Terbutaline

Salbutamol Clenbuterol

0.6 0.4 Salmeterol 0.2

Dobutamine

0 Agonist

Fig. 1. Mast cells were incubated for 10 min with or without a given ␤-adrenoceptor agonist (10⫺10⫺10⫺5 m) before challenge with an optimal releasing concentration of antihuman IgE for 25 min. After this incubation, the cells were centrifuged and histamine content in the supernatants was assayed. Maximal (Emax) inhibitory responses were determined and intrinsic activities of the agonists evaluated relative to the full agonist, isoprenaline. Hence, intrinsic activity was determined by: (Emax for an agonist ⫼ Emax for isoprenaline). Values are means and are based on 8–23 experiments [adapted from 11].

More recently, we have investigated whether agonist efficacy influences desensitisation [11]. Prevailing opinion, based on studies with transfected cell systems, suggests that drugs with higher efficacy are liable to induce greater levels of receptor desensitisation [20]. This reasoning is based on an assumption that the conformational state induced following activation of the receptor by an agonist also serves as a substrate for kinases that phosphorylate the receptor and, thereby, target the receptor for the desensitisation pathway [18]. We have examined this concept in human lung mast cells and, using intrinsic activity as an indirect measure of a drug’s efficacy, investigated whether the intrinsic activity of a range of ␤2-adrenoceptor agonists influences the extent of functional desensitisation (see figure 1 for a discussion on intrinsic activity and an indication of the agonists that were used in these experiments). Mast cells were treated (24 h) with concentrations of agonists calculated to occupy a high proportion (⬃88%) of the available receptors, then washed extensively and then the ability of isoprenaline to inhibit IgE-mediated histamine release determined. The studies indicated that there was a statistically significant correlation between the extent of desensitisation observed and the intrinsic activity of an agonist supporting the concept that drug efficacy influences desensitisation [11]. However, this relationship breaks down at lower receptor occupancies as the degree of functional desensitisation observed with partial agonists, such as

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salbutamol and salmeterol for example, was higher than expected. These findings could suggest that, in the clinical context, bronchodilators may cause greater levels of tolerance than hitherto appreciated at concentrations that are, therapeutically, relatively ineffective. Moreover, these data suggest that the mechanisms involved in mediating desensitisation at high and lower receptor occupancies differ and the suggestion from these experiments also exists that different agonists induce desensitisation by different mechanisms. These issues are, currently, being explored. We have also investigated the effects of desensitising treatments on ␤2-adrenoceptor expression in mast cells [7, 8, 11]. Surprisingly, treatments with agonists that can lead to very substantial levels of functional desensitisation are accompanied by modest changes in receptor density that are not significant statistically [11]. This finding is in stark contrast to similar studies in cells transfected with the ␤2-adrenoceptor in which treatments with full and partial agonists can lead to substantial levels of receptor down-regulation [21]. That little down-regulation of ␤2-adrenoceptors is observed following desensitising treatments in mast cells could suggest that uncoupling of the receptor and/or sequestration of the receptor, rather than receptor degradation, are more prominent processes contributing to the desensitisation of mast cell ␤2-adrenoceptors.

Pharmacogenetics

Recent studies have demonstrated that the ␤2-adrenoceptor is polymorphic [22]. At least 11 polymorphic positions have been identified both within the promoter region and the coding block of the gene for the ␤2-adrenoceptor. Moreover, studies by others have shown that these polymorphisms may influence the function and expression of the ␤2-adrenoceptor [22]. These polymorphisms could, therefore, influence the degree of therapeutic benefit bronchodilators afford in asthma [23]. We have investigated the influence that polymorphisms may have on the inhibitory activity of ␤-adrenoceptor agonists in mast cells. Our data show that, of all the polymorphic positions studied, position 164 (thr to ile), which is found in the fourth trans-membrane domain, may influence agonist activity [24]. The potency of isoprenaline, as an inhibitor of IgE-mediated histamine release, was lower in mast cell preparations expressing the less frequent allele (ile). These data are in broad agreement with studies in transfected cell systems showing that agonists are less effective activators of adenylyl cyclase in preparations expressing ile164 as opposed to those expressing thr164 [22]. The suggestion has been made that ␤2-adrenoceptors expressing ile164 may couple less efficiently to G-protein.

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We have also investigated whether polymorphisms influence the functional desensitisation of ␤2-adrenoceptor-mediated responses in mast cells. Reports have shown that two polymorphic positions, 16 and 27, found within the extracellular N-terminus may affect ␤2-adrenoceptor desensitisation in transfected cell systems [22]. Of these two positions, we have established that position 27 influences desensitisation in mast cells. Mast cell preparations expressing ␤2-adrenoceptors with glu27 were resistant to desensitising treatments when compared with preparations expressing gln27 [25]. Overall, these data suggest an influence of polymorphisms in the context of how effectively bronchodilators might stabilise mast cell responses in vivo. However, whether determining genotype can be accurately predictive of how well an individual either responds to a bronchodilator or demonstrates susceptibility to tolerance is questionable given the large variability in responses observed even within a given genotype in our studies [24]. Indeed, the suggestion has been made that trying to correlate responses with a single polymorphism may not be as rigorous an approach as trying to correlate responses with multiple polymorphisms across the ␤2-adrenoceptor gene (haplotype). It remains to be seen whether associations between haplotype and ␤2-adrenoceptor function in mast cells offer any advantage over correlations between single polymorphisms and ␤2-adrenoceptor function.

Conclusion

The ␤2-adrenoceptor has been the most extensively studied G-proteincoupled receptor. This large body of assimilating literature on the ␤2-adrenoceptor has provided invaluable information on the mechanisms involved in G-proteincoupled receptor activation and regulation. However, the majority of these studies has been performed in transfected cells where the manipulated expression of high densities of ␤2-adrenoceptors, in an unusual environment, may not necessarily reflect physiological reality. Moreover, transfected cell systems, despite their obvious value, could not hope to represent the full gamut of biological variability. As such, the human lung mast cell may continue to serve as a very useful primary human cell system to complement studies of the ␤2-adrenoceptor in transfected cell systems.

Acknowledgement Work in the authors’ laboratory has been supported by grants from Asthma, UK.

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References 1 2

3 4

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Schild H: Histamine release in anaphylactic shock of isolated lungs of guinea pigs. Quart J Exp Physiol 1937;26:165–179. Orange RP, Austen WG, Austen KF: Immunological release of histamine and slow-reacting substance of anaphylaxis from human lung. I. Modulation by agents influencing cellular levels of cyclic 3⬘,5⬘-adenosine monophosphate. J Exp Med 1971;134:136–148. Assem ESK, Schild HO: Beta-adrenergic receptors concerned with the anaphylactic mechanism. Int Arch Allergy Appl Immunol 1969;45:62–69. Undem BJ, Peachell PT, Lichtenstein LM: Isoproterenol-induced inhibition of immunoglobulin E-mediated release of histamine and arachidonic acid metabolites from the human lung mast cell. J Pharmacol Exp Ther 1988;247:209–217. Butchers PR, Skidmore IF, Vardey CJ, Wheeldon A: Characterisation of the receptor mediating the anti-anaphylactic effects of ␤-adrenoceptor agonists in human lung tissue in vitro. Br J Pharmacol 1980;17:663–667. Chong LK, Chess-Williams R, Peachell PT: Pharmacological characterisation of the ␤-adrenoceptor expressed by human lung mast cells. Eur J Pharmacol 2002;437:1–7. Chong LK, Suvarna K, Chess-Williams R, Peachell PT: Desensitization of ␤2-adrenoceptor-mediated responses by short-acting ␤2-adrenoceptor agonists in human lung mast cells. Br J Pharmacol 2003;138:512–520. Scola AM, Chong LK, Suvarna SK, Chess-Williams R, Peachell PT: Desensitisation of mast cell ␤2-adrenoceptor-mediated responses by salmeterol and formoterol. Br J Pharmacol 2004;141: 163–171. Drury DEJ, Chong LK, Ghahramani P, Peachell PT: Influence of receptor reserve on ␤-adrenoceptor-mediated responses in human lung mast cells. Br J Pharmacol 1998;124:711–718. MacEwan DJ, Kim GD, Milligan G: Analysis of the role of receptor number in defining the intrinsic activity and potency of partial agonists in neuroblastoma ⫻ glioma hybrid NG108-15 cells transfected to express differing levels of the human ␤2-adrenoceptor. Mol Pharmacol 1995;48: 316–325. Scola AM, Chong LK, Chess-Williams R, Peachell PT: Influence of agonist intrinsic activity on the desensitisation of ␤2-adrenoceptor-mediated responses in mast cells. Br J Pharmacol 2004;143:71–80. Botana LM, MacGlashan DW: Differential effects of cAMP-elevating drugs on stimulus-induced calcium changes in human basophils. J Leuk Biol 1994;55:798–804. Cockcroft DW, McParland CP, Britto SA, Swystun VA, Rutherford BC: Regular inhaled salbutamol and airway responsiveness to allergen. Lancet 1993;342:833–837. O’Connor BJ, Aikman SL, Barnes PJ: Tolerance to the nonbronchodilator effects of inhaled ␤2-agonists in asthma. N Engl J Med 1992;327:1204–1208. Van der Heijden PJCM, Van Amsterdam JGC, Zaagsma J: Desensitization of smooth muscle and mast cell ␤-adrenoceptors in the airways of the guinea pig. Eur J Respir Dis 1984;65(suppl 135): 128–134. Chong LK, Peachell PT: Beta-adrenoceptor reserve in human lung: A comparison between airway smooth muscle and mast cells. Eur J Pharmacol 1999;28:378:115–122. Ferguson SSG: Evolving concepts in G protein-coupled receptor endocytosis: The role in receptor desensitization and signaling. Pharmacol Rev 2001;53:1–24. Kohout TA, Lefkowitz RJ: Regulation of G protein-coupled receptor kinases and arrestins during receptor desensitization. Mol Pharmacol 2003;63:9–18. Chong LK, Morice AH, Yeo WW, Schleimer RP, Peachell PT: Functional desensitization of ␤ agonist responses in human lung mast cells. Am J Respir Cell Mol Biol 1995;13:540–546. Clark RB, Knoll BJ, Barber R: Partial agonists and G protein-coupled receptor desensitization. Trends Pharmacol Sci 1999;20:279–286. Williams BR, Barber R, Clark RB: Kinetic analysis of agonist-induced down-regulation of the ␤2-adrenergic receptor in BEAS-2B cells reveals high- and low-affinity components. Mol Pharmacol 2000;58:421–430.

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Kirstein SI, Insel PA: Autonomic nervous system pharmacogenomics: A progress report. Pharmacol Rev 2004;56:31–52. Taylor DR, Kennedy MA: Beta-adrenergic receptor polymorphisms and drug responses in asthma. Pharmacogenomics 2002;3:173–184. Kay LJ, Chong LK, Rostami-Hodjegan A, Peachell PT: Influence of the thr164ile polymorphism in the ␤2-adrenoceptor on the effects of ␤-adrenoceptor agonists on human lung mast cells. Int Immunopharmacol 2003;3:91–95. Chong LK, Chowdry J, Ghahramani P, Peachell PT: Influence of genetic polymorphisms in the ␤2-adrenoceptor on desensitization in human lung mast cells. Pharmacogenetics 2000;10: 153–162.

Peter T. Peachell Molecular Pharmacology, University of Sheffield The Royal Hallamshire Hospital (Floor M) Glossop Road, Sheffield S10 2JF (UK) Tel. ⫹44 114 2712063, Fax ⫹44 114 2261348, E-Mail [email protected]

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Saito H, Okayama Y (eds): Mast Cells in Allergic Diseases. Chem Immunol Allergy. Basel, Karger, 2005, vol 87, pp 154–162

Potential Role of Stem Cell Factor in the Asthma Control by Glucocorticoids Carla Alexandra Da Silva, Nelly Frossard EA 3771, ‘Inflammation and Environment in Asthma’, Faculté de Pharmacie, Illkirch, France

Abstract Asthma is an allergic disease characterized by inflammation that includes an increase in the number and activation of mast cells in the airways. Glucocorticoids, on the other hand, diminish inflammation as well as the number of mast cells in this disease. Stem cell factor (SCF) is a major growth factor for human mast cells, inducing chemotaxis as well as survival of the mast cells. SCF induces proliferation and differentiation of immature mast cells from CD34⫹ progenitors. It also potentiates the IgE-dependent activation of mast cells. Furthermore, SCF expression is reduced in the airways of asthmatic patients treated with glucocorticoids. Thus, SCF could be involved in mast cell-associated diseases such as asthma. We here review the main effects of glucocorticoids in asthma and on mast cells, with a special interest on SCF, as a potential therapeutic target in asthma. Copyright © 2005 S. Karger AG, Basel

Introduction

Inhaled glucocorticoids have revolutionized asthma treatment and are currently the first-line disease-modifying drug for this condition [for review, see Allen et al., 2003]. Glucocorticoids have a major anti-inflammatory effect in the airways of persons with asthma. They reduce the number of inflammatory cells, such as eosinophils or mast cells [for review, see Barnes et al., 1998] and at the same time reduce activation of these cells by inhibiting the release of mediators by T lymphocytes and macrophages for instance [for review, see Barnes et al., 1998]. Glucocorticoids inhibit production of numerous proteins – cytokines, chemokines, and growth factors – involved in the recruitment and

survival of these inflammatory cells and thus cause their numbers to reduce in the bronchi of subjects with asthma.

Glucocorticoids and Asthma

Glucocorticoids exert an important effect on the structural cells of the bronchi. In vitro they inhibit the proliferation of bronchial smooth muscle cells [Stewart et al., 1995] and fibroblasts [Durant et al., 1986] and inhibit collagen production by fibroblasts [Perez et al., 1992]. They may therefore reduce both smooth muscle hyperplasia and subepithelial fibrosis of the bronchi in subjects with asthma. After 6 weeks [Olivieri et al., 1997], 4 months [Trigg et al., 1994], or 6 months [Hoshino et al., 1998] of glucocorticoid treatment, the basement membrane in the bronchi of these subjects is significantly thinner. The effect of glucocorticoids on bronchial smooth muscle hyperplasia has not been described, however. Inhaled glucocorticoid treatment reduces bronchial hyperresponsiveness, which is one of the most disabling characteristics of asthma [for reviews, see Allen et al., 2003; Barnes et al., 1998]. Chronic glucocorticoid treatment reduces non-specific bronchial hyperresponsiveness to histamine [De Baets et al., 1990; Trigg et al., 1994], cholinergic agonists [Djukanovic et al., 1992; Olivieri et al., 1997], and also specific allergens [De Baets et al., 1990; Gauvreau et al., 1996]. The mechanisms for this glucocorticoid-induced improvement in bronchial hyperreactivity are not well known. The resolution of bronchial inflammation by these drugs is probably partly responsible. Bronchial smooth muscles may also play an important role in this reduction, however [for review, see Hirst and Lee, 1998]. Glucocorticoids inhibit expression of the receptors involved in bronchial smooth muscle contraction. For example, in vitro, they inhibit the interleukin (IL)-1␤-induced overexpression of bradykinin B2 receptors [Schmidlin et al., 1998], and in vivo they induce the transcription of the ␤2-adrenergic receptor, which is involved in relaxation [Baraniuk et al., 1997; Mak et al., 1995]. Although glucocorticoids are currently the most effective anti-inflammatory treatment for asthma, some patients with asthma do not respond to them, and a few are completely resistant [Barnes et al., 1995]. This phenomenon of resistance may depend on several molecular mechanisms: increased synthesis or activation or both of pro-inflammatory transcription factors such as activated protein (AP)-1 and nuclear factor (NF)-␬B, reduced expression of the glucocorticoid receptor (GR), or altered GR affinity for its ligands [Adcock, 1996]. Another possibility is the overexpression of GR␤, an isoform with little affinity for glucocorticoids and, especially, that induces no effect [Bamberger et al., 1996]. Recent data also indicate that the mitogen-activated protein (MAP)

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kinases may play a role in the development of resistance to glucocorticoids in asthma. That is, p38 activation by IL-2 and IL-4 induces phosphorylation of GR at the serine residues and thereby reduces the receptor’s affinity for glucocorticoids [Irusen et al., 2002]. Moreover, the monocytes and T lymphocytes of glucocorticoid-resistant subjects, compared with glucocorticoid-sensitive subjects, express more of the c-fos subunit of AP-1 [Lane et al., 1998]. These results are consistent with the higher phosphorylation rates of c-Jun and of Janus kinase (JNK), which lead to AP-1 activation. After 9 days of daily treatment with 40 mg of prednisolone, immunohistochemical analysis of skin biopsy specimens from a tuberculin-induced model of dermal inflammation in humans showed that phosphorylation of both JNK and c-Jun was suppressed in subjects with glucocorticoid-sensitive asthma but not in glucocorticoid-resistant subjects [Sousa et al., 1999]. These results suggest that glucocorticoid resistance may be due in part to the inability of the glucocorticoids to inhibit JNK activation.

Glucocorticoids and Mast Cells

In vitro glucocorticoids have no effect (as measured by histamine release) on mast cell degranulation in the airways activated by an IgE-dependent pathway; this is equally true for isolated mast cells, mast cells from isolated human bronchi, and mast cells from human lung fragments [Cohan et al., 1989; Schleimer et al., 1983]. On the other hand, glucocorticoids inhibit the stem cell factor (SCF)-induced degranulation of rat mast cells [Taylor et al., 1996a]. The effect of glucocorticoids on the release of mediators derived from arachidonic acid is controversial. Some studies report that they do not affect mast cell degranulation [Cohan et al., 1989; Schleimer et al., 1983], while others find they inhibit it [Heiman and Crews, 1984; Rider et al., 1996]. Moreover, glucocorticoids inhibit the production of the IL-3, IL-4, IL-5, IL-8 and TNF-␣ cytokines by human and murine mast cells in vitro [Eklund et al., 1997; Lippert et al., 1996; Sewell et al., 1998] and in vivo [Bradding et al., 1995; Wershil et al., 1995]. One of the principal mechanisms by which glucocorticoids reduce inflammation and bronchial hyperreactivity in asthma may depend on diminution in the number of mast cells. Glucocorticoid treatment, even of short duration, substantially reduces – by approximately 62% – the number of mast cells infiltrated into the bronchi of subjects with asthma [Bentley et al., 1996; Djukanovic et al., 1992, 1997; Jeffery et al., 1992; Laitinen et al., 1992; Olivieri et al., 1997; Trigg et al., 1994]. Nonetheless, glucocorticoids do not seem to affect all bronchial mast cells of all phenotypes in the same way. They reduce the number of tryptase-positive mast cells (MCT) but not of tryptase-chymase-positive mast cells (MCTC) in the bronchial epithelium [Bentley et al., 1996]. The mechanisms

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of this diminution are not known. In vitro, glucocorticoids inhibit the development of bone marrow-derived mast cells [Eklund et al., 1997; Irani et al., 1995]. Nonetheless, glucocorticoids appear to only inhibit directly mast cells freshly differentiated by SCF administration. Adding glucocorticoids later, that is, 3 weeks after mast cell differentiation, does not inhibit the proliferation of mature mast cells [Irani et al., 1995]. Glucocorticoids may affect the number of mast cells in the tissues by inhibiting production of the growth factors necessary for the mast cells’ development and survival. They do not induce apoptosis of isolated mast cells in vitro [Finotto et al., 1997; Tchekneva and Serafin, 1994], while in vivo in mice, they reduce the number of cutaneous mast cells by inhibiting SCF production [Finotto et al., 1997].

Glucocorticoids and SCF

The Kit ligand, or SCF [Huang et al., 1990; Martin et al., 1990; Zsebo et al., 1990] acts as an important growth factor for human and murine mast cells [for reviews, see Broudy, 1997; Galli et al., 1994; Kassel et al., 2001]. It induces proliferation and differentiation of immature CD34⫹ progenitors in the bone marrow and peripheral blood in vitro, chemotaxis and adhesion of the mast cell to the extracellular matrix, and inhibition of their apoptosis. SCF by itself induces mast cell hyperplasia in vivo after subcutaneous injection in humans [Costa et al., 1996; Dvorak et al., 1998]. It increases the antigen-induced degranulation of human pulmonary mast cells, and itself induces mast cell degranulation both in vitro and in vivo [for review, see Kassel et al., 2001]. Glucocorticoids have been shown to affect SCF – both its expression and its activity. In vivo in mice, glucocorticoids reduce the number of cutaneous mast cells by increasing their apoptosis, as shown by DNA fragmentation assessed with the TUNEL method (terminal deoxynucleotidyl transferase mediated dUTP nick end labeling) [Finotto et al., 1997]. This reduction is linked to the inhibition of SCF production by structural cells. When exogenous SCF is administered at the moment of glucocorticoid treatment, the number of mast cells does not decrease. This strongly suggests that glucocorticoids reduce the number of mast cells by inhibiting SCF production [Finotto et al., 1997]. In humans, glucocorticoid treatment of patients with nasal sinus polyposis reduces the number of structural and epithelial cells and fibroblasts expressing SCF [Kim et al., 1997]. How glucocorticoids affect SCF overproduction in the airways of asthma patients is not known. Some observations offer evidence to support the hypothesis that glucocorticoids reduce the number of mast cells in the bronchi by inhibiting SCF production. This is the case in recent studies showing that mast cell number as well as SCF mRNA and protein expression

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are reduced within the airways of asthmatic patients treated with glucocorticoids [Da Silva et al., submitted]. In vitro, SCF promotes the differentiation of mast cells that express tryptase [Kirshenbaum et al., 1992] but not the development of those expressing chymase [Kirshenbaum et al., 1992]. If SCF plays a role in human bronchi, in particular by permitting MCT development, glucocorticoid inhibition of SCF production would affect the number of MCT mainly and the number of MCTC to a lesser extent. As we now know, that is the case: glucocorticoids diminish the number of MCT in the bronchial epithelium of persons with asthma [Bentley et al., 1996]. In vitro, glucocorticoids inhibit SCF production in cultures of fibroblasts derived from nasal-sinus polyps [Kim et al., 1997] and of cutaneous fibroblasts [Finotto et al., 1997]. Nonetheless, in other cell types, glucocorticoids are reported to have opposite effects, increasing SCF expression in bone marrow stromal cells [Linenberger et al., 1995; Thalmeier et al., 1996] but without affecting this expression in mesenchymal cells at all [Haynesworth et al., 1996]. These differences may be interpreted as reflecting the variable effects of glucocorticoids depending on the cell type used. Nonetheless, the differences in experimental conditions, and especially in the time of cell stimulation by glucocorticoids, may be responsible for these different effects. A 4-hour glucocorticoid treatment of fibroblasts derived from nasal sinus polyps inhibited SCF production [Kim et al., 1997], while in the bone marrow-derived fibroblasts, a treatment of 24–48 h stimulated SCF production [Linenberger et al., 1995; Thalmeier et al., 1996]. Other in vitro works showed biphasic regulation of constitutive SCF expression as a function of time [Kassel et al., 1998]. That is, glucocorticoids inhibit expression of SCF mRNA by human pulmonary fibroblasts at 2.5 h but increase this expression at 24 h [Kassel et al., 1998]. More recently, Da Silva et al. [2003] showed that glucocorticoids inhibit SCF mRNA and protein expression induced in inflammatory conditions, mimicked in vitro by the pro-inflammatory cytokine IL-1␤, in human lung fibroblasts in culture. This inhibition occurs from 1 h after treatment with glucocorticoids [Da Silva et al., 2003]. The structure of the SCF gene, and especially its promoter region, provides material for hypotheses about how glucocorticoids may affect SCF transcription. The promoter region of the SCF gene contains a sequence partly homologous to the glucocorticoid-responsive element [Taylor et al., 1996b]. It also contains responsive elements for transcription factors that may be inhibited by GRs like NF-␬B, AP-1 and the cAMP-responsive element binding protein [Da Silva et al., 2003; Taylor et al., 1996b]. Transfection experiments using the SCF promoter region show that deletion of the glucocorticoid-responsive elementlike element abolishes the inhibition by glucocorticoids of the IL-1␤-enhanced SCF expression in human lung fibroblasts [Da Silva et al., 2003], showing that

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in vitro, glucocorticoids can directly affect the SCF expression in inflammatory conditions in a transcriptional level.

Conclusions

Glucocorticoids control the asthma disease by reducing the number and activation of infiltrated mast cells within the airways. The expression of the major mast cell growth factor, SCF, is reduced in the airways of asthmatic patients treated by glucocorticoids, and in vitro studies support the direct regulation of SCF by glucocorticoids in inflammatory conditions. SCF may have some role in the inflammatory changes observed in mast cell-associated diseases like asthma, and constitutes a potential new therapeutic target in the control of the mast cell number and activation in the asthmatic disease.

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Carla A. Da Silva EA 3771, ‘Inflammation and Environment in Asthma’ Faculté de Pharmacie, BP 24 FR–67401 Illkirch Cedex (France) Tel. ⫹33 390 244196, Fax ⫹33 390 244309, E-Mail [email protected]

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Saito H, Okayama Y (eds): Mast Cells in Allergic Diseases. Chem Immunol Allergy. Basel, Karger, 2005, vol 87, pp 163–178

Mast Cell Ion Channels Peter Bradding Department of Infection, Immunity and Inflammation, University of Leicester Medical School, Glenfield Hospital, Leicester, UK

Abstract IgE-dependent activation of human mast cells is a central feature of allergy. However, alternative non-IgE-mediated signals derived from the complex inflammatory milieu are also important in driving chronic mast cell activation in many tissues. Irrespective of the point from where pathological secretion is sustained, all mechanisms will change the activity of the final ‘effector’ ion channels involved in normal stimulus-secretion coupling. IgE-dependent activation of human mast cells is characterised by an influx of extracellular Ca2⫹, which is essential for subsequent release of both preformed (granule-derived) mediators and newly generated autacoids and cytokines. In addition, flow of ions such as K⫹ and Cl⫺ are likely to play an important role in mast cell activation through their effect on cell membrane potential and thus Ca2⫹ influx. Emerging evidence suggests that mast cells express a number of channels carrying K⫹, Cl⫺ and Ca2⫹. With current techniques it will be possible to identify the molecular origin of these channels and define precisely their role in mast cell function. Mast cell ion channels offer a novel target for the attenuation of allergic disease. Copyright © 2005 S. Karger AG, Basel

Mast Cells in Asthma and Allergy

Mast cells are central to the pathophysiology of allergic disease through their immunomediator secretory activity in response to IgE-dependent activation. This is manifest in a variety of guises, from acute life-threatening episodes of anaphylaxis due to food allergy, the strictly seasonal symptoms due to grass pollen-induced summer rhinoconjunctivitis (hay fever), to the chronic ongoing mediator release evident in bronchial asthma. When IgE bound to the highaffinity IgE receptor (Fc␧RI) is cross-linked by allergen, complex signalling pathways are engaged culminating in the secretion of preformed granulederived mediators, newly generated products of arachidonic acid metabolism, and newly synthesised cytokines.

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Critical Role of Ion Channels in Mast Cell Activation

IgE-dependent activation of both human and rodent mast cells is characterised by an influx of extracellular Ca2⫹, which is essential for subsequent release of both preformed (granule-derived) mediators and newly generated autacoids and cytokines [1]. However, excitable cells (e.g. cardiac tissue, neurons) and non-excitable cells (e.g. leukocytes) express K⫹ and Cl⫺ channels which are likely to play an important role in cell activation responses through their effect on membrane potential. Thus the negative cell membrane potential generated by open K⫹ channels for example has been shown to enhance Ca2⫹ influx [2, 3]. This occurs because the store-operated Ca2⫹ channels (SOCC) that carry Ca2⫹ into cells conduct larger currents at negative membrane potentials [4]. This principal is highlighted in T cells where specific inhibition of the voltage-dependent K⫹ channel Kv1.3 by the scorpion toxin margatoxin inhibits T cell proliferation, IL-2 secretion and delayed-type hypersensitivity responses [5]. Mast Cell Kⴙ Channels

At rest, rat basophilic leukaemia (RBL) cells and rodent bone marrowderived mast cells express exhibit a strongly inwardly-rectifying K⫹ conductance carried by the inwardly-rectifying K⫹ channel Kir2.1 (fig. 1) [6]. This sets the cell membrane potential close to the equilibrium potential for K⫹ at around ⫺80 mV. When RBL cells are activated by antigen, they undergo a phase of depolarisation, in part due to Ca2⫹ influx and possibly due to Kir closure, and then partial repolarisation thought to be due to K⫹ efflux [7, 8]. This repolarisation is attenuated by charybdotoxin, a blocker of the voltage-gated K⫹ channel Kv1.3, the large conductance calcium-activated K⫹ channel bKCa, and the intermediate conductance K⫹ channel iKCa1 [8]. However, repolarisation is not inhibited by specific blockers of Kv1.3 or bKCA, suggesting that charybdotoxin

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is exerting its effect via the iKCa1 channel. In parallel with its effects on repolarisation, charybdotoxin also attenuates antigen-induced 86Rb⫹ efflux (an indicator of K⫹ channel opening), and suppresses degranulation by about 40% after maximal antigenic stimulation [8]. This supports a role for iKCa1 in promoting mediator release from RBL cells but this current has never been demonstrated in these cells electrophysiologically using the patch clamp technique. A further outwardly-rectifying K⫹ conductance has been identified in rodent mucosal mast cells but not rat peritoneal mast cells. It does not appear after antigenic stimulation and is not activated by Ca2⫹, but is activated by pertussis toxin-sensitive G protein-coupled receptors [9]. It is activated by adenosine, and so it has been proposed that this is the mechanism by which adenosine potentiates antigen-induced secretion in these cells [10]. The regulation of cell membrane potential in human lung mast cells (HLMC), human peripheral blood-derived mast cells (HPBMC) and human bone marrow-derived mast cells (HBMMC) is clearly distinct from that in RBL cells. The majority of these human mast cells are electrically silent at rest, with resting membrane potential measured electrophysiologically around 0 mV (fig. 2a). Although we have demonstrated the presence of mRNA for Kir2.0 family members in human mast cells [11, 12], we have never recorded a Kir current in several hundred cells [3, 13, 14]. Following Fc␧RI cross-linking by anti-IgE, human mast cells open rapidly a K⫹ conductance with electrophysiological features suggesting it is carried by iKCa1 (fig. 2b–f) [3, 14]. Thus there is an acute transition in the cell membrane potential from around 0 mV to about –45 mV (fig. 2a). Human mast cells express iKCa1 mRNA, and the same current is opened by the iKCa1 opener 1-ethyl-2-benzimidazolinone (1-EBIO) [3]. Charybdotoxin attenuates HLMC secretion by about 30% after maximal activation with anti-IgE, but the effect is quite variable [14]. However, we have shown recently that 1-EBIO enhances Ca2⫹ influx in HLMC following submaximal IgE-dependent activation, and that this is accompanied by a dose-dependent increase in histamine release (fig. 3) [3]. This is consistent with the observation that blockade of iKCa1 in T cells attenuates antigen-induced Ca2⫹ influx, cytokine secretion and cell proliferation. The opening of this channel can therefore be considered as a means of increasing the ‘gain’ of an immunological stimulus. iKCa1 expression increases in T cells following mitogenic stimulation. Interestingly, we have observed an increase in functional expression of iKCa1 in HLMC maintained in culture with SCF, IL-6 and IL-10 [3]. This is consistent with the observation that HLMC maintained in culture with SCF are far more ‘reactive’ than when freshly isolated, and in many respects appear more representative of the mast cells present in asthmatic BAL [3, 15]. It will be interesting to assess whether asthmatic mast cells demonstrate increased iKCA1

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expression, contributing to their increased basal and immunological secretory state. Targeting iKCA1 in allergy may prove to be particularly attractive since there is the potential to attenuate pathological IgE-dependent secretion, but leave other innate responses [reviewed in 16] which do not require acute Ca2⫹ influx such as those mediated via Toll-like receptors intact. a linear current develops which demonstrates rectification at positive potentials. Note the negative shift in reversal potential to ⫺70 mV (arrow) indicating that the current is likely to be carried predominantly by K⫹. d Raw current from the same cell as in c after anti-IgE demonstrating appearance of current immediately following voltage steps and no decay during a 100-ms pulse. The voltage protocol is shown inset. e A further cell after anti-IgE recorded in 5 and 140 mM external K⫹. Inward current increases, outward current decreases and reversal potential shifts from ⫺50 to 0 mV (arrows) on switching from 5 to 140 mM external K⫹, confirming the presence of a K⫹ current. Note also the inward rectification in 140 mM K⫹. f Another cell after antiIgE demonstrating the typical whole cell current. Following removal of extracellular Ca2⫹ there is a marked and fully reversible decrease in the whole cell current with a shift in reversal potential from ⫺55 to 0 mV (arrows) indicating that the K⫹ component is carried by a Ca2⫹-activated K⫹ channel. A smaller outwardly-rectifying Ca2⫹-insensitive current remains; the reversal potential of 0 mV suggests it may be carried by Cl⫺ or non-selective cations. Reproduced from Duffy et al. [14], with permission of The American Association of Immunologists, Inc., © 2001.

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There is marked functional and phenotypic heterogeneity between human mast cells both within the same organ and in different tissue sites [17]. Interestingly, using DNA microarrays, human skin mast cells express mRNA for the ␣ subunit of the large conductance calcium-activated K⫹ channel bKCa and its ␤4 subunit [12]. The ␣ subunit was not expressed by cord blood-derived mast cells or HLMC, and we have never seen a bKCa current in HLMC, HPBMC or HBMMC. If bKCa currents are present in skin mast cells, this differential channel expression could well contribute to the heterogeneity observed between mast cell phenotypes. Mast Cell Clⴚ Channels

A moderately outwardly-rectifying Cl⫺ conductance has been described in RBL, rodent bone marrow-derived and rat peritoneal mast cells both at rest and following antigenic stimulation. The molecular identity of this channel is unclear but Kulka et al. [18] recently demonstrated by RT-PCR that rat cultured mast cells express mRNA for the voltage-dependent Cl⫺ channels ClC-2, -3, -4, -5 and -7, although rat peritoneal mast cells expressed only ClC-7. These channels each have distinct electrophysiological signatures, and the one most closely resembling the rodent mast cell Cl⫺ conductance is ClC-3. This channel is widely expressed across cell types, and appears to play an important role in the regulation of cell volume [19]. Such a role during mast cell activation is plausible due the changes in cell volume initiated following degranulation. It has also been suggested that the rodent mast cell Cl⫺ conductance promotes Ca2⫹ influx and degranulation. However, interpreting the role of Cl⫺ currents during cell activation is fraught with difficulties. This is firstly because intracellular Cl⫺ concentration varies widely between cells. Physiological extracellular Cl⫺ concentration is about 100 mM, so if intracellular Cl⫺ concentration is in the region of 30 mM as has been estimated for RPMC [20], Cl⫺ currents will contribute to membrane polarisation since reversal potential for Cl⫺ at these concentrations is about ⫺40 mV, and this will theoretically promote Ca2⫹ influx. In support of this, putative blockers of rodent mast cell Cl⫺ channels such as 5-nitro-2-(3-phenylpropylamino)benzoic acid (NPPB) and 4,4⬘ -diisothiocyanato-2,2⬘ -disulphonic acid (DIDS) attenuate histamine secretion from rodent mast cells, but only in the ␮M range [20, 21]. However, NPPB also inhibits Ca2⫹ influx through SOCC [21], and although DIDS attenuates secretion and blocks Cl⫺ channels, it does not inhibit antigen-induced 36Cl⫺ uptake in RPMC which occurs rapidly, while the appearance of Cl⫺ channel activity is delayed [20]. DIDS also binds secretagogues and secretory products, and it does not inhibit the nerve growth factor/lysophosphatidylserine-induced

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serotonin release from rat peritoneal mast cells, a process which is dependent on Ca2⫹ influx [22]. Furthermore, although sodium cromoglycate is a potent blocker of the IgE-dependent Cl⫺ conductance in RBL cells [23], it is only a weak antagonist of secretion from HLMC. Conversely, if intracellular Cl⫺ concentration is similar to extracellular Cl⫺ concentration, then Cl⫺ channel opening will depolarise the cell and antagonise Ca2⫹ influx. This may indeed be the case, since in HMC-1 we have estimated that the intracellular Cl⫺ concentration is about 66 mM from the extrapolated reversal potential of the single channel conductance of a resting Cl⫺ current (see below) [13]. With this scenario, one could hypothesise that since the appearance of the Cl⫺ current is delayed following IgE-dependent activation, it actually represents a negative feedback pathway to provide a brake to the secretory response. Firm molecular identification of the channels present and selective inactivation, for example with small interfering RNA is needed. The cystic fibrosis transmembrane conductance regulator (CFTR) has also been identified in rodent mast cells with RT-PCR and immunohistology. Since cAMP potentiated Cl⫺ flux, and the CFTR blocker diphenylamine-2-carboxylate inhibits secretion, it is suggested that CFTR may also contribute to the activation of these cells [24]. We have identified several Cl⫺ currents in human mast cells both at rest and following cell activation. The most striking current is a Cl⫺ current which demonstrates extreme outward rectification from about ⫹50 mV [13, 14, 25]. This is present in all cells of the human mast cell line HMC-1, and about 10% of HLMC (fig. 4). The electrophysiological properties of this channel most closely resemble those of heterologously expressed ClC-4 and ClC-5 and both HMC-1 and HLMC express mRNA for ClC-5 but not ClC-4 [13, and HLMC RT-PCR, unpubl. data]. However there are some subtle differences in the electrophysiology of the HMC-1 current and heterologously expressed ClC-5, for example the anion permeability of the HMC-1 channel is nitrate⬎iodide⬎chloride while that for cloned ClC-4 and ClC-5 is nitrate⬎chloride⬎iodide. This HMC-1 ClC-5-like current was resistant to blockade by a number of Cl⫺ channel blockers including DIDS, niflumic acid, and chlorotoxin, but was blocked by tamoxifen. Interestingly, in the same dose range that tamoxifen modulated channel activity, it inhibited the proliferation of both HMC-1 and HLMC in long-term culture [25] (fig. 5). The concentrations required were higher than those required to modulate oestrogen receptor function, but are still achieved in vivo with clinically relevant dosing. This raises the possibility that tamoxifen may be useful for the treatment of mast cell proliferative disease for which no useful treatment currently exists. HLMC also exhibit a moderately outwardly-rectifying Cl⫺ conductance in some cells at rest, and open a similar current following IgE-dependent

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Fig. 4. Representative examples of whole cell electrophysiological recording of a ClC-5-like current in the human mast cell line HMC-1. a Typical whole cell raw membrane current recorded from an HMC-1 cell (left graph). The cell was clamped at a holding potential of ⫺20 mV and 100-ms voltage commands were applied from ⫺140 to ⫹140 mV in 10-mV steps (shown on the right). b Current-voltage relationship from the same cell as in a. Note the extreme outward rectification. c Current-voltage relationship demonstrating the reduction of the outwardly-rectifying current and positive shift in reversal potential after substituting extracellular Cl⫺ with methanesulphonate ions. d Current-voltage relationship showing the relative permeability of iodide and nitrate ions compared to chloride ions. e Current-voltage relationship showing that raising the extracellular K⫹ concentration from 5 to 140 mM has no effect on the outwardly-rectifying current. Note however that a small inward current is revealed by the higher concentration.

activation [14]. The macroscopic features of this current are similar to that in rodent mast cells and heterologously expressed ClC-3. HMC-1 and HLMC also express ClC-3 mRNA [12, 13], but whether ClC-3 carries this current awaits knockdown experiments. This current appears relatively slowly after IgEdependent activation, taking about 10 min to peak. It seems unlikely therefore

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that it contributes to the rapid release of histamine, but could contribute to the release of arachidonic acid metabolites. Alternatively, as suggested above, it could provide a break to the secretory response. Using DNA microarrays, mRNA expression for ClC-3 was also present in skin and cord blood mast cells, and ClC-7 mRNA was expressed by HLMC and cord blood mast cells but not skin mast cells [12]. The ClC voltage-dependent channels are believed to have predominantly intracellular roles regulating ionic composition of intracellular organelles [26]. However, there is also another family of intracellular Cl⫺ channels the CLIC family, which are also located in the membranes of intracellular structures [26]. Their role in cell physiology remains uncertain, but there was clear expression of CLIC-4 in all types of mast cells tested on the DNA microarray and CLIC-2 in skin mast cells alone. Lastly HLMC and HPBMC express a distinct Ca2⫹-activated Cl⫺ conductance which is activated by ionophore or by dialysing the cell with a high (e.g. 500 nM) concentration of Ca2⫹ in the patch clamp pipette [14]. Interestingly, this current is not activated by Fc␧RI cross-linking. The reason for this, its role in mast cell physiology and its molecular identify remain unknown. None of the known cloned Ca2⫹-activated Cl⫺ channels were expressed in our DNA microarray study.

Mast Cell Ca2ⴙ Channels

While K⫹ and Cl⫺ channels may modulate the secretory response, the critical requirement for the release of preformed and newly generated mediators

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from HLMC is the influx of Ca2⫹ from the extracellular fluid [27]. The channels carrying these currents and the signalling pathways that activate them are therefore of great interest as novel targets for the development of anti-secretory drugs. In rodent mast cells there is evidence of Ca2⫹ influx through both nonselective cation channels and SOCC [28]. The SOCCs activated in several leukocytes including rodent mast cells following depletion of intracellular stores are also known as calcium release-activated Ca2⫹ (CRAC) channels. Recent data has shown a close association between Ca2⫹ influx through CRAC in RBL cells and both degranulation and arachidonic acid metabolism resulting in the secretion of granule mediators and LTC4 respectively [29]. Possible candidates for both CRAC and the non-selective cation channels are the transient receptor potential (TRP) family of channels, some of which are reported to be controlled by the filling state of intracellular stores [30]. TRP channels are classified by their homology, rather than function or selectivity. Channels from three main TRP protein subfamilies have been implicated in Ca2⫹ signalling, including members of the canonical/TRPC subfamily, melastatin/TRPM subfamily and vanilloid/TRPV subfamily. TRPV6 (Cat1, EcaC2) in particular expresses many features of ICRAC, i.e. high Ca2⫹ selectivity, steep inward rectification, blockade by low (10 ␮M) concentrations of La3⫹, loss of selectivity in the absence of divalent cations, and single channel conductance to Na⫹ in divalent-free conditions of ⬃60 pS [31]. However, there are some important differences between Cat1 and ICRAC suggesting that they are different channels [32]. For example, Cat1 displays a mode of voltage-dependent gating that is fully absent in CRAC and originates from the voltage-dependent binding/unbinding of Mg2⫹ inside the channel pore. Several TRP channels from the TRPC and TRPV subfamilies are poorly selective for cations, and so could contribute to the rodent mast cell non-selective Ca2⫹ influx pathway. Using high-density oligonucleotide probe arrays, we have recently described the mRNA expression profile for TRP channel genes in human mast cells [12]. We identified the expression of TRPC1 in skin mast cells, but not lung or cord blood-derived mast cells (CBMC), TRPV2 in all three types of cells, and TRPM2 in HLMC and CBMC but not skin mast cells. These differences between mast cell phenotypes could again contribute to their functional heterogeneity. While the oligonucleotide array technique is highly specific, it is not so sensitive, so it is likely that these mast cell populations express further TRP family members. Furthermore, several TRP channels of interest such as TRPV6 were not represented on the chips used, so could not be assessed. TRPV2 has also been identified more recently in RBL cells and mouse bone marrow-derived mast cells [33]. It is expressed in the plasma membrane and since the cells exhibit a rise in cytosolic free Ca2⫹ and degranulate at ⬎50⬚C, which is dependent on the presence of extracellular Ca2⫹, it appears to

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be biologically active. While such temperature activation may be relevant to skin responses to thermal stimuli, it seems unlikely that this would be a physiological mechanism for activation in the lungs. This suggests other as-yet unidentified factors may be involved in its gating. Interestingly, the cytosolic Ca2⫹ signal in RBL cells following Fc␧RI aggregation is markedly different from that seen in HLMC. In RBL cells there are wide transient oscillations in the concentration of cytosolic free Ca2⫹ [Ca2⫹]i while in HLMC there is a sustained rise in [Ca2⫹]i (fig. 6) [3, 34, 35]. This may be a consequence of the different K⫹ and Cl⫺ channels identified in the two cell types, and similar differences may well exist for the Ca2⫹ influx pathways. Naⴙ Channels

Na⫹ channels are gated by either membrane voltage (Nav channels) or second messengers such as the epithelial Na⫹ channels [36]. The role of Nav channels in excitable conducting tissue such as nerves and cardiac tissue is clear. As the cell membrane depolarises, a critical threshold is reached which opens the channel resulting in a rapid influx of sodium and further membrane depolarisation. These channels are made up of ␣ subunits which carry the Na⫹ current, and accessory ␤ subunits which modify channel gating and serve as adhesion molecules mediating both homophilic cell adhesion and adhesion to extracellular matrix proteins [37, 38]. Such channels have rarely been reported in nonexcitable cells such as leukocytes [39]. In our DNA microarray study, we identified mRNA for the ␣ subunits Nav1.8 and the recently cloned SCN12A in all three types of mast cell studied, together with the ␤1.1 subunit. We have not seen voltage-dependent Na⫹ currents in human mast cells using the patch clamp technique but it remains a possibility that a small subset of human mast cells could express such channels. Interestingly, T lymphocytes express a voltagedependent and amiloride-sensitive Na⫹ channel which cannot be recorded electrophysiologically under baseline conditions, but appears within 30 min of co-incubation of the cells with peptide-primed antigen-presenting cells [40]. The ␤1.1 subunit could also play a role in mast cell adhesion, and since mast cells are intimately related to peripheral nerves geographically within tissues, might provide a mechanism for their co-localisation. Parekh [41] has described the presence of a novel GTP-dependent nonvoltage-activated Na⫹ channel in RBL cells. This was not present in cultured rodent mast cells, and it has been suggested therefore that perhaps this relates to the malignant phenotype of the RBL cell line, either directly contributing to neoplastic transformation or as a result of it.

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Fig. 6. Typical changes in intracellular free Ca2⫹ [Ca2⫹]i following optimal IgE-dependent activation in (a) six individual RBL cells activated with 1 ␮g/ml antigen (DNP-BGG) and (b) two individual HLMC activated with 1:1,000 goat anti-human IgE. Secretagogues were added at the time indicated by the arrows. Note the marked oscillations in the RBL cells compared to the relatively smooth plateau in the HLMC. RBL cell data courtesy of Clare Fewtrell and Paul Millard.

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P2X Receptors

The P2X family of purinoceptors consists of 7 members and these are ATPgated ion channels, which carry cations non-selectively. On our DNA microarrays human mast cells from all sources expressed P2X1 and P2X4 but only blood-derived mast cells activated with anti-IgE expressed P2X7 [12]. This latter observation is in keeping with a previous study by Schulman et al. [42] in which P2X7 was not expressed on HLMC but was expressed by the HMC-1 cell line. Schulman et al. also showed that ATP enhances IgE-dependent histamine release from HLMC but that this appears to be mediated solely via P2Y receptors. However, P2X receptors are readily desensitised by ATP, and often need to be ‘re-sensitised’ in vitro by first destroying extracellular ATP with apyrase [43]. Further experiments along these lines will help determine whether these P2X1 and P2X4 receptors are expressed in a functional form by human mast cells.

Hypothetical Mast Cell Electrical ‘Excitation’ Cycle

Since mast cells recover and regranulate following IgE-dependent activation, we have proposed a hypothetical cycle of electrical activity analogous to that in excitable tissue such as ventricular myocardium, where a co-ordinated cell activation response requires an orderly sequence of ion channel gating and regulation of membrane potential [44]. However, rather than occurring over milliseconds, in mast cells this electrical cycle would evolve over hours or even days (fig. 7). However, rapid millisecond side-cycles may operate to afford specific rapidly alternating channel activity where tight control of ion flow is required, e.g. between Ca2⫹-activated K⫹ or Cl⫺ channels with SOCC (fig. 7, grey arrowheads). In summary, there are important differences in the ion channels expressed by rodent and human mast cells. The marked difference in the intracellular Ca2⫹ response following Fc␧RI aggregation between RBL cells and HLMC may be an important consequence of this. This profound difference between rodent and human mast cells may explain in part the significant phenotypic differences between species. While rodent models may be particularly convenient for studying channel physiology, caution is required before extrapolating any observations to mast cells in human disease. Targeting the Ca2⫹ influx pathway in human mast cells, either directly, or indirectly via modulation of K⫹ channels, offers the prospect of inhibiting allergen-induced disease but leaving other Ca2⫹-independent mast cell functions such as their response to bacterial infection via Toll-like receptors intact.

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IgE-dependent activation Non lgE-dependent activation (proteases, SCF) ‘Resting’

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Fig. 7. Simplified hypothetical mast cell electrical excitation cycle based on currents in human mast cells. In disease, the cells may become hyperreactive due to the side-cycle indicated by the grey arrowheads. The delayed Cl⫺ current might attenuate this process by producing membrane depolarisation. Reproduced from Bradding and Conley [44], with permission of Blackwell Publishing Ltd., © 2002.

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Caulfield JP, Lewis RA, Hein A, Austen KF: Secretion in dissociated human pulmonary mast cells. Evidence for solubilization of granule contents before discharge. J Cell Biol 1980;85:299–312. Fanger CM, Rauer H, Neben AL, Miller MJ, Rauer H, Wulff H, Rosa JC, Ganellin CR, Chandy KG, Cahalan MD: Calcium-activated potassium channels sustain calcium signaling in T lymphocytes. Selective blockers and manipulated channel expression levels. J Biol Chem 2001;276:12249–12256. Duffy SM, Berger P, Cruse G, Yang W, Bolton SJ, Bradding P: The K⫹ channel iKCA1 potentiates Ca2⫹ influx and degranulation in human lung mast cells. J Allergy Clin Immunol 2004;114:66–72. Hoth M, Penner R: Depletion of intracellular calcium stores activates a calcium current in mast cells. Nature 1992;355:353–356. Koo GC, Blake JT, Talento A, Nguyen M, Lin S, Sirotina, Shah K, Mulvany K, Hora DJ, Cunningham P, Wunderler DL, McManus, OB, Slaughter R, Bugianesi R, Felix J, Garcia M, Williamson J, Kaczorowski G, Sigal NH, Springer MS, Feeney W: Blockade of the voltage-gated potassium channel Kv1.3 inhibits immune responses in vivo. J Immunol 1997;158:5120–5128. Wischmeyer E, Lentes KU, Karschin A: Physiological and molecular characterization of an IRKtype inward rectifier K⫹ channel in a tumour mast cell line. Pflügers Arch 1995;429:809–819. Labrecque GF, Holowka D, Baird B: Characterization of increased K⫹ permeability associated with the stimulation of receptors for immunoglobulin E on rat basophilic leukemia cells. J Biol Chem 1991;266:14912–14917.

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Narenjkar J, Marsh SJ, Assem ES: Inhibition of the antigen-induced activation of RBL-2H3 cells by charybdotoxin and cetiedil. Eur J Pharmacol 2004;483:95–106. McCloskey MA, Cahalan MD: G protein control of potassium channel activity in a mast cell line. J Gen Physiol 1990;95:205–227. Qian YX, McCloskey MA: Activation of mast cell K⫹ channels through multiple G protein-linked receptors. Proc Natl Acad Sci USA 1993;90:7844–7848. Dewson GD, Conley EC, Bradding P: Multigene family isoform profiling from blood cell lineages. BMC Genomics 2002;3:22–22. Bradding P, Okayama Y, Kambe N, Saito H: Ion channel gene expression in human lung, skin, and cord blood-derived mast cells. J Leuk Biol 2003;73:614–620. Duffy SM, Leyland ML, Conley EC, Bradding P: Voltage-dependent and calcium-activated ion channels in the human mast cell line HMC-1. J Leuk Biol 2001;70:233–240. Duffy SM, Lawley WJ, Conley EC, Bradding P: Resting and activation-dependent ion channels in human mast cells. J Immunol 2001;167:4261–4270. Flint KC, Leung KB, Hudspith BN, Brostoff J, Pearce FL, Johnson NM: Bronchoalveolar mast cells in extrinsic asthma: A mechanism for the initiation of antigen specific bronchoconstriction. Br Med J 1985;291:923–926. Bradding P, Holgate ST: Immunopathology and human mast cell cytokines. Crit Rev Oncol Haematol 1999;31:119–133. Church MK, Okayama Y, Bradding P: Functional mast cell heterogeneity; in Busse, WW, Holgate ST (eds): Asthma and Rhinitis. Boston, Blackwell Scientific, 1994, pp 209–220. Kulka M, Schwingshackl A, Befus AD: Mast cells express chloride channels of the ClC family. Inflamm Res 2002;51:451–456. Duan D, Winter C, Cowley S, Hume JR, Horowitz B: Molecular identification of a volume-regulated chloride channel. Nature 1997;390:417–421. Friis UG, Johansen T, Hayes NA, Foreman JC: IgE-receptor activated chloride uptake in relation to histamine secretion from rat mast cells. Br J Pharmacol 1994;111:1179–1183. Reinsprecht M, Rohn MH, Spadinger RJ, Pecht I, Schindler H, Romanin C: Blockade of capacitive Ca2⫹ influx by Cl⫺ channel blockers inhibits secretion from rat mucosal-type mast cells. Mol Pharmacol 1995;47:1014–1020. Roloff T, Wordehoff N, Ziegler A, Seebeck J: Evidence against the functional involvement of outwardly rectifying Cl⫺ channels in agonist-induced mast cell exocytosis. Eur J Pharmacol 2001;431:1–9. Romanin C, Reinsprecht M, Pecht I, Schindler H: Immunologically activated chloride channels involved in degranulation of rat mucosal mast cells. EMBO J 1991;10:3603–3608. Kulka M, Gilchrist M, Duszyk M, Befus AD: Expression and functional characterization of CFTR in mast cells. J Leuk Biol 2002;71:54–64. Duffy SM, Lawley WJ, Kaur D, Yang W, Bradding P: Inhibition of human mast cell proliferation and survival by tamoxifen in association with ion channel modulation. J Allergy Clin Immunol 2003;112:970–977. Jentsch TJ, Stein V, Weinreich F, Zdebik AA: Molecular structure and physiological function of chloride channels. Physiol Rev 2002;82:503–568. Church MK, Pao GJ, Holgate ST: Characterization of histamine secretion from mechanically dispersed human lung mast cells: Effects of anti-IgE, calcium ionophore A23187, compound 48/80, and basic polypeptides. J Immunol 1982;129:2116–2121. Fasolato C, Hoth M, Matthews G, Penner R: Ca2⫹ and Mn2⫹ influx through receptor-mediated activation of nonspecific cation channels in mast cells. Proc Natl Acad Sci USA 1993;90: 3068–3072. Artalejo AR, Ellory JC, Parekh AB: Ca2⫹-dependent capacitance increases in rat basophilic leukemia cells following activation of store-operated Ca2⫹ entry and dialysis with high-Ca2⫹containing intracellular solution. Pflügers Arch 1998;436:934–939. Clapham DE, Runnels LW, Strubing C: The TRP ion channel family. Nat Rev Neurosci 2001;2: 387–396. Yue L, Peng JB, Hediger MA, Clapham DE: CaT1 manifests the pore properties of the calciumrelease-activated calcium channel. Nature 2001;410:705–709.

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Voets T, Prenen J, Fleig A, Vennekens R, Watanabe H, Hoenderop JGJ, Bindels RJM, Droogmans G, Penner R, Nilius B: CaT1 and the calcium-release activated calcium channel manifest distinct pore properties. J Biol Chem 2001;276:47767–47770. Stokes AJ, Shimoda LM, Koblan-Huberson M, Adra CN, Turner H: A TRPV2-PKA signaling module for transduction of physical stimuli in mast cells. J Exp Med 2004;200:137–147. Kim TD, Eddlestone GT, Mahmoud SF, Kuchtey J, Fewtrell C: Correlating Ca2⫹ responses and secretion in individual RBL-2H3 mucosal mast cells. J Biol Chem 1997;272:31225–31229. MacGlashan D Jr: Single-cell analysis of Ca2⫹ changes in human lung mast cells: Graded vs. all-ornothing elevations after IgE-mediated stimulation. J Cell Biol 1989;109:123–134. Goldin AL: Resurgence of sodium channel research. Annu Rev Physiol 2001;63:871–894. Malhotra JD, Kazen-Gillespie K, Hortsch M, Isom LL: Sodium channel ␤ subunits mediate homophilic cell adhesion and recruit ankyrin to points of cell-cell contact. J Biol Chem 2000;275: 11383–11388. Srinivasan J, Schachner M, Catterall WA: Interaction of voltage-gated sodium channels with the extracellular matrix molecules tenascin-C and tenascin-R. Proc Natl Acad Sci USA 1998;95: 15753–15757. DeCoursey TE, Chandy KG, Gupta S, Cahalan MD: Voltage-dependent ion channels in T-lymphocytes. J Neuroimmunol 1985;10:71–95. Lai ZF, Chen YZ, Nishimura Y, Nishi K: An amiloride-sensitive and voltage-dependent Na⫹ channel in an HLA-DR-restricted human T cell clone. J Immunol 2000;165:83–90. Parekh AB: Nonhydrolyzable analogues of GTP activate a new Na⫹ current in a rat mast cell line. J Biol Chem 1996;271:23161–23168. Schulman ES, Glaum MC, Post T, Wang Y, Raible DG, Mohanty J, Butterfield JH, Pelleg A: ATP Modulates anti-IgE-induced release of histamine from human lung mast cells. Am J Respir Cell Mol Biol 1999;20:530–537. Buell G, Michel AD, Lewis C, Collo G, Humphrey PP, Surprenant A: P2X1 receptor activation in HL60 cells. Blood 1996;87:2659–2664. Bradding P, Conley EC: Human mast cell ion channels. Clin Exp Allergy 2002;32:979–983.

Dr. Peter Bradding Department of Respiratory Medicine, Glenfield Hospital Groby Road, Leicester LE3 9QP (UK) Tel. ⫹44 116 2563998, Fax ⫹44 116 2502787 E-Mail [email protected]

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Saito H, Okayama Y (eds): Mast Cells in Allergic Diseases. Chem Immunol Allergy. Basel, Karger, 2005, vol 87, pp 179–197

Using Mast Cell Knock-In Mice to Analyze the Roles of Mast Cells in Allergic Responses in vivo Mindy Tsaia, Michele A. Grimbaldestona, Mang Yua, See-Ying Tama, Stephen J. Gallia,b Departments of aPathology and bMicrobiology and Immunology, Stanford University School of Medicine, Stanford, Calif., USA

Abstract It is well established that mast cells are important effector cells mediating the acute phase of IgE-associated allergic disorders, but their roles in late phase reactions and chronic allergic inflammation are not well defined. Here we describe an experimental approach for analyzing mast cell functions in vivo by comparing the biological responses in wild-type mice, genetically mast cell-deficient mice, and ‘mast cell knock-in mice’ (mast cell-deficient mice selectively repaired of their mast cell deficiency). Studies using ‘mast cell knock-in mice’ have indicated that mast cells can contribute importantly to IgE-associated late phase reactions and to chronic allergic inflammation. Moreover, ‘mast cell knock-in mice’ containing adoptive-transferred mast cell populations with defined alterations in the expression of specific mast cell products can be used to characterize the mechanisms by which mast cells contribute to the expression of the response of interest. Copyright © 2005 S. Karger AG, Basel

Allergic disorders are caused by aberrant immune responses to common but otherwise innocuous environmental antigens called allergens [1, 2]. The hallmark of allergy is the development, in genetically susceptible individuals, of allergen-specific, Th2 cytokine-dependent IgE responses [1, 2]. Tissue mast cells and basophils, a type of circulating granulocytes, are the primary effector cells responsible for the acute phase of IgE-associated allergic disorders. These two cell types each express high-affinity IgE receptors (Fc␧RI) on their surface

and exhibit partially overlapping function in IgE-associated reactions; however, it has become clear that mast cells and basophils represent two distinct cell lineages [3, 4]. Of these two cell types, mast cells have been the more extensively studied for their involvement in IgE-associated allergic disorders and for their roles in certain protective immune responses to parasites [1, 3, 5, 6]. In IgE-dependent responses in mast cells (and basophils), the ‘cross-linking’ of Fc␧RI-bound IgE molecules with multivalent antigen induces the aggregation of Fc␧RI, resulting in the activation of multiple cascades of downstream signaling events [1–3, 5–9]. Such Fc␧RI-dependent mast cell activation leads to the release of histamine and other preformed mediators stored in the cells’ cytoplasmic granules, the de novo synthesis of proinflammatory lipid mediators, such as prostaglandins and leukotrienes, and the synthesis and secretion of many growth factors, cytokines and chemokines [1–3, 5–10]. Although there is general agreement that Fc␧RI-dependent mast cell activation is important for key manifestations of the acute phases of allergic responses in human subjects, the roles of mast cells in late phase reactions and chronic allergic inflammation are not as well understood. However, several lines of evidence, especially that derived from mouse studies, indicate that mast cells can contribute as effector cells to multiple aspects of both late phase reactions and chronic allergic inflammation and that mast cells may also exhibit immunoregulatory functions in allergic disorders [1, 3, 5, 8–10]. For example, many mast cell-derived effector molecules, such as histamine [11], LTB4 [12], and PGD2 [12], can exhibit immunomodulatory functions in allergic inflammation, e.g., by influencing the recruitment, phenotype and/or functional responses of dendritic cells (DCs), T cells or B cells [3, 10–12]. Furthermore, mast cells can produce an array of cytokines, chemokines and growth factors that can enhance the recruitment, trafficking and function of other inflammatory cells and/or have direct effects on the cells that normally reside in the tissues at sites of allergic reactions, such as epithelial cells, smooth muscle cells, and fibroblasts [1, 3, 5, 7–10]. More recently, the identification and characterization of co-stimulatory molecules expressed by mast cells has indicated that mast cells can interact directly with other immune cells in the regulation of the sensitization and/or effector phases of allergic reactions [3, 7, 9, 10, 13]. Although these findings have supported the hypothesis that mast cells can have important roles in allergic responses beyond functioning as effector cells during the acute phase of reactions to allergens, it has been quite challenging to prove that mast cells can indeed significantly contribute to late phase reactions and chronic allergic inflammation. Among the issues that have complicated our efforts in achieving a clear picture of the roles of mast cells in these settings are the following: (1) IgE-associated immune responses usually involve complex

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and redundant effector and immunoregulatory mechanisms. For example, many activities and products detected in mast cells can also be expressed by other inflammatory cells that participate in the same response [8, 9]. (2) Mast cell activation frequently results in the release of a variety of mediators, chemokines and cytokines with diverse and, sometimes, opposing activities [11]. (3) Distinct mast cell populations can exhibit functional plasticity and phenotypic heterogeneity, adding another level of complexity to the analysis of mast cell functions in vivo [5, 6, 8, 9, 14]. (4) Depending on the details of the protocols used to elicit particular experimental models of immune responses in mice, mast cells have been shown to be either essential or dispensable in the expression of such responses; these include models of disorders associated with allergic inflammation, such as asthma [8, 15], as well as models of other immune responses, such as contact hypersensitivity [16]. (5) There are differences in certain aspects of allergic immune responses in mice and humans [8, 17]; accordingly, caution should be exercised in extrapolating findings obtained in mouse models of allergic disorders to those in humans. In this review, we will describe an experimental approach for analyzing many proposed mast cell functions in vivo by comparing the expression of biological responses in wild-type mice, genetically mast cell-deficient mice, and ‘mast cell knock-in mice’, i.e. mast cell-deficient mice that have been selectively repaired of their mast cell deficiency. We will also review findings from studies that have used such ‘mast cell knock-in mice’ to identify and characterize some of the diverse contributions of mast cells to the expression of specific examples of allergic disorders, including active or passive anaphylaxis and asthma.

‘Mast Cell Knock-In Mice’ as a Model for Studying Mast Cell Functions in vivo

Normal mast cell development and survival, and therefore mast cell functions, are disrupted in c-kit/W mutant animals that either lack expression of the c-kit receptor or express c-kit with significantly reduced tyrosine kinase activity. Several mast cell-deficient rodents, such as KitW/KitW-v, KitW-41/KitW-41 and KitW-f/KitW-f mice, as well as KitW-s/KitW-s rats, have been used to study mast cell function in vivo [14, 18, 19], but the most commonly used animal model for such studies is the WBB6F1-KitW/KitW-v mouse. Adult KitW/KitW-v mice ordinarily have ⬍1% of the wild-type levels of skin mast cells, and contain no detectable mast cells in the peritoneal cavity, respiratory system, gastrointestinal tract or other sites [20]. However, these mice also exhibit other abnormalities due to the lack of normal c-kit function, including macrocytic anemia,

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sterility, and a virtual lack of skin melanocytes and interstitial cells of Cajal [14, 18, 19]. Although the lack of tissue mast cells in KitW-sh/KitW-sh mice was documented some time ago [21], these mice only recently have been investigated as another model for studies of mast cell functions in vivo [22, 23]. Unlike other c-kit/W mutations that affect the c-kit coding region and that result in abnormalities affecting multiple cell lineages, the ‘W-sash’ (Wsh) mutation, an inversion mutation in the transcriptional regulatory elements upstream of the c-kit transcription start site on mouse chromosome 5 [24], has been reported primarily to impair the development of mast cells and melanocytes [25, 26]. And like WBB6F1-KitW/KitW-v mice, C57BL/6-KitW-sh/KitW-sh mice are profoundly mast cell-deficient [21–23, 25, 26] and can be reconstituted with mast cells with normal c-kit function by adoptive transfer of such cells into the peritoneal cavity [22, 23] or via the intradermal or intravenous routes [23]. However, compared to KitW/KitW-v mice, KitW-sh/KitW-sh mice exhibit fewer phenotypic abnormalities; for example, KitW-sh/KitW-sh mice are neither anemic nor sterile. As a result, KitW-sh/KitW-sh mice may offer some significant advantages over KitW/KitW-v mice as a model for analyzing certain mast cell functions in vivo. For example, the fertility of the KitW-sh/KitW-sh mice is especially useful when one wishes to produce mast cell-deficient mice that also have other defined genetic abnormalities; in the case of the sterile KitW/KitW-v mice, more complex and expensive breeding strategies must be employed for this purpose [27]. The lack of mast cells in genetically mast cell-deficient c-kit mutant mice can be selectively repaired (i.e., mast cell reconstitution can be achieved without replacing the recipient’s other hematopoietic cells with those of donor origin) by adoptive transfer of genetically compatible, in vitro derived mast cells (fig. 1) [28]. Such mast cells can be derived from the bone marrow cells of either the congenic wild-type mice or various transgenic or knockout mice (fig. 1). Bone-marrow-derived cultured mast cells (BMCMCs) can be administered by intravenous, intraperitoneal or intradermal injection [14, 18, 28] or by direct injection into the anterior wall of the stomach [29], thus producing so-called ‘mast cell knock-in mice’ (fig. 1). Notably, depending on the anatomical site in which they reside, such adoptively transferred BMCMCs can gradually acquire multiple phenotypic characteristics of the corresponding native mast cell populations found in the congenic wild-type mice [28, 30]. In certain anatomical sites, such as the skin and peritoneal cavity, the locally injected BMCMCs acquire both a tissue distribution and numbers, as well as phenotypic characteristics, that closely resemble those of the corresponding mast cell populations in the wild-type mice [14, 18, 28, 30]. However, in other sites, such as the lung, the distribution and/or

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Bone marrow- or ES cell-derived cultured mast cells (BMCMCs or ESCMCs)

Wild-type mouse or mouse with specific gene mutation(s) In vitro differentiation Wild-type ES cells or ES cells with specific gene mutation(s)

i.d. i.p. i.v.

Mast cell-deficient mouse

Mast cell knock-in mouse

Wild-type mouse

Experimental studies (e.g. of allergic inflammation)

Fig. 1. ‘Mast cell knock-in mouse’ model for analyses of mast cell function in allergic responses. Wild-type mast cells, or mast cells with specific genetic alterations, are generated from hematopoietic stem cells, such as those in bone marrow or fetal liver, of wild-type mice or of mice with specific genetic alterations of interest. Alternatively, using in vitro differentiation, embryonic stem (ES) cell-derived cultured mast cells can be generated from wild-type or genetically-altered ES cells. These mast cells can then be transplanted into mast cell-deficient c-kit mutant mice, such as WBB6F1-KitW/KitW-v or C57BL/6-KitW-sh/KitW-sh mice, to produce ‘mast cell knock-in mice’. The in vitro derived mast cells can be injected via different routes (intravenous, intraperitoneal or intradermal) for selective local (i.d., i.p.) or systemic (i.v.) reconstitution of various mast cell populations. Mast cell function(s) in allergic or other biological responses can then be analyzed by comparing the responses in wild-type, c-kit mutant mast cell-deficient and selectively mast cell-reconstituted (i.e. ‘mast cell knock-in’) mice. The contribution(s) of specific mast cell products (surface structures, signaling molecules, secreted products, etc.) can be analyzed by comparing expression of the responses of interest in mast cell knock-in mice reconstituted with either wild-type mast cells or mast cells derived from mice or ES cells that lack, or express genetically-altered forms of, such products.

numbers of the adoptively transferred mast cells may differ from those of the corresponding native populations of mast cells in the wild-type mice [15, 31]. Therefore, in any studies employing ‘mast cell knock-in mice’, it is important to quantify the numbers of mast cells in the recipient animals and to assess their tissue distribution, in comparison to the corresponding features of the native mast cell populations in the congenic wild-type mice. In some types of experiments, it may also be important to evaluate certain aspects of the phenotype of the reconstituted mast cells as well. Because variable amounts of time are required for the appearance of morphologically identifiable mast cells in different anatomical sites after the adoptive transfer of bone marrow cells [20, 32] or BMCMCs [33], one must choose carefully the time interval between the

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initial injection of mast cells into the mast cell-deficient mice and the subsequent use of the mast cell-reconstituted mice for experimental analysis. The full expression of certain features of mast cell-dependent responses may be observed in normal mice even when relatively low levels of mast cell activation have been achieved [34]. Thus, the reconstitution of wild-type levels of mast cells may not be necessary in order for ‘mast cell knock-in mice’ to express features of mast cell-dependent responses that mimic, in magnitude, those in the corresponding wild-type mice [e.g., see 15, 31]. However, in other settings, aspects of biological responses elicited at tissue sites in ‘mast cell knock-in mice’ that contain smaller populations of mast cells than in the corresponding tissues in wild-type animals also may be smaller in magnitude than those in the wild-type animals. In addition to BMCMCs, mast cells can be generated in vitro from other sources of hematopoietic cells [35] or from embryonic stem (ES) cells [19, 36, 37] for transfer into mast cell-deficient mice. ES cell-derived mast cells (ESCMCs) that are generated from genetically-altered ES cells can be used to analyze the function of specific mast cell products in vivo, after the transfer of such ESCMCs into the skin [36] or peritoneal cavity [37] of genetically mast celldeficient mice (fig. 1).

Mast Cells in Reactions of Immediate Hypersensitivity

IgE-Dependent Local or Systemic Reactions Allergen- and IgE-dependent mast cell mediator release initiates multiple inflammatory cascades that can mediate most of the pathology associated with type I or ‘immediate’ hypersensitivity responses, including anaphylaxis [2, 5, 38–41]. However, at least in the mouse, there is now abundant evidence that anaphylaxis also can occur by mechanisms that are fully independent of IgE [42] or mast cells [39, 43–47]. Accordingly, it is useful to distinguish between the roles of mast cells in ‘purely’ IgE-dependent responses (such as those elicited by antigen challenge in mice which have been passively sensitized with antigen-specific IgE), and the role of mast cells in active anaphylaxis and other ‘immediate hypersensitivity’ reactions, which are elicited in mice that have been actively sensitized with antigen. The roles of mast cells in IgE-dependent responses have been studied by transferring antigen-specific IgE or injecting anti-IgE antibodies into wild-type mice, genetically mast cell-deficient mice and, in some cases, mast cell knockin mice. These studies have shown that mast cells contribute significantly to all aspects of nearly every IgE-dependent immediate hypersensitivity reaction analyzed so far, including local responses (e.g., ‘passive cutaneous anaphylaxis’

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reactions) and systemic anaphylactic responses. Mast cells are required for the cardiopulmonary changes [38–40], hypothermia [41], and death [38–40] associated with IgE-dependent systemic responses. IgE-mediated activation of mast cells can also enhance airway responsiveness to cholinergic stimulation [31]. Notably, even though mast cells are thought to be the most critical effector cells in IgE-dependent systemic anaphylaxis, mast cell hyperplasia induced by the chronic treatment of wild-type mice with stem cell factor, the c-kit ligand and the major regulator of mast cell survival and development [5, 6, 18], does not enhance the severity of IgE-induced systemic anaphylaxis [48]. This can be attributed to the phenotypic and functional changes that are induced in mast cells by such treatment. Whatever the explanation for the findings in that study, they illustrate that the intensity of IgE- and mast cell-dependent biological responses does not necessarily correlate solely with the numbers of mast cells in the affected tissues. Other studies using ‘mast cell knock-in mice’ have revealed the essential roles of mast cells in several acute and late phase features of IgE-dependent responses, which were elicited locally after the passive transfer of the antibody to the skin [49, 50] or the gastrointestinal tract [29, 51]. In the skin, these mast cell-dependent tissue changes include acute tissue swelling [49, 50], local extravasation of fibrinogen and deposition of fibrin [49], the recruitment of leukocytes during the ‘late phase’ of the response [29, 50], and the enhancement of type 1 collagen mRNA levels in fibroblasts at the site of the reaction [52]. Such studies have also provided evidence that the recruitment of circulating inflammatory cells, including neutrophils and monocytes, to sites of acute IgE- and mast cell-dependent responses in the skin or stomach is promoted by mast cell-derived TNF [50, 51]. The central role of Fc␧RI in mediating the ‘classic’ IgE- and mast celldependent anaphylaxis pathway has been demonstrated using mice deficient in the expression of Fc␧RI [40, 41]. Miyajima et al. [40] showed that Fc␧RI ␣-chain ⫺/⫺ mice do not develop significant mast cell degranulation or cardiopulmonary changes, nor do these mice exhibit significant mortality, during attempts to elicit IgE-dependent passive systemic anaphylaxis. Nevertheless, in vivo studies conducted in mice deficient in Fc␥RIIB or Fc␥RIII receptor expression suggest that IgE-dependent passive systemic anaphylaxis can be modulated by the expression of Fc receptors for IgG. Fc␥RIIB⫺/⫺ and Fc␥RIII⫺/⫺ mice can exhibit augmented and attenuated IgE-dependent systemic anaphylaxis responses, respectively [53]. These findings indicate that at least some IgE-mediated responses represent the summation of effects resulting from high-affinity binding of IgE to Fc␧RI as well as low affinity interactions of IgE with Fc␥RIII and Fc␥RII receptors expressed on mast cells.

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Anaphylaxis or Local Immediate Hypersensitivity Reactions in Actively Immunized Mice Reactions of active anaphylaxis can be induced locally or systemically by the administration of certain protein antigens or haptens to mice that have been previously sensitized with the same agents. In mice, the acquired immune responses that are elicited by active antigen sensitization and associated with antigen-specific IgE are usually also associated with antigen-specific IgG1 [40, 41]. The IgG1-dependent component of reactions to antigen challenge in such actively immunized mice probably involves the participation of many cell types, including macrophages [41], granulocytes [46], and platelets [54], as well as mast cells [55]. For example, in an active model of systemic anaphylaxis induced by goat anti-mouse IgD antibody (goat IgG), it has been shown that macrophages, rather than mast cells, contribute importantly to the expression of IgG1-dependent responses [41]. Platelet activating-factor appears to be an important mediator for this mast cell-independent anaphylaxis pathway [41]. Based on passive transfer studies and other lines of evidence, the elicitation of IgG1-dependent systemic anaphylaxis in mice generally requires much higher doses of antigen than does the elicitation of IgE-dependent systemic reactions [40, 41, 56]. Another active anaphylaxis pathway, that apparently involves IgE but not mast cells, also has been reported. In a mouse model of active fatal anaphylaxis induced by penicillin V (Pen V), Pen V induced an IgE-associated biphasic response that was correlated with early and late phase production of platelet activating-factor [57]. Studies in KitW/KitW-v mice indicated that mast cells were not required for the expression of either the immediate or late phase responses induced by Pen V [57]. While evidence was presented to indicate that the responses to Pen V were dependent on IgE but not IgG1, it would be of interest to attempt to elicit Pen V-induced active anaphylaxis in IgE⫺/⫺ mice, as such study would provide further evidence that this is an entirely IgE-dependent model system. Although the cells responsible for Pen V-induced anaphylaxis have not been identified, basophils represent one attractive candidate, as mast cells and basophils are the only cells that express high-affinity IgE receptors in the mouse. Antigen- and IgE-dependent activation of NK cells, induced via the binding of IgE to Fc␥RIII receptors, has also recently been demonstrated [58]. This finding suggests the possible involvement of NK cells, in addition to mast cells and basophils, in some IgE-dependent immune responses. A platelet-mediated example of immediate hypersensitivity has recently been reported by Cara et al. [54]. In this model system, ovalbumin (OVA)induced immediate hypersensitivity reactions were found to be mast celldependent when they were elicited in the skin. However, the reactions could be

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elicited in the skeletal muscle in the absence of mast cells and inhibited by an anti-platelet antibody. Even though fatal active anaphylaxis can be elicited in mice that lack signaling via Fc␧RI (due to the absence of the IgE binding ␣ chain of that receptor), such mice do not develop either the marked tachycardia, or the rapid and significant changes in pulmonary conductance and compliance, which are associated with active anaphylaxis in the corresponding wild-type mice [40]. Thus, even though this model of OVA-induced active anaphylaxis can be elicited in the absence of the IgE/Fc␧RI/mast cell pathway, the clinical features of the responses differ significantly from those of the reactions that are elicited in mice in which the IgE/Fc␧RI/mast cell pathway is intact. In summary, studies in wild-type mice, mast cell-deficient mice, and mice deficient in either the ␣ chain of the Fc␧RI or the ␥ chain common to Fc␧RI and Fc␥RIII, indicate that both IgE and IgG1 antibodies can contribute to active systemic anaphylaxis in the mouse, with the IgE-dependent component being largely mast cell-dependent. The IgG1-dependent component, on the other hand, probably involves the participation of many cell types, such as macrophages [41], granulocytes [46], platelets [54], as well as mast cells, which can also be activated for antigen-specific mediator release by IgG1- and Fc␥RIII-dependent pathways [55]. Mast cells thus are able to contribute to immediate hypersensitivity, in the setting of active systemic anaphylaxis, by both IgE/Fc␧RI- and IgG1/Fc␥RIII-dependent mechanisms. Mast cell-deficient mice have also been used to investigate the roles of mast cells in local expressions of intestine anaphylaxis induced by active immunization. For example, the role of mast cells in promoting the enhanced secretion of ions (primarily Cl⫺) by the small intestine during active intestinal hypersensitivity was investigated using mast cell-deficient mice that had been repaired of their mast cell deficiency non-selectively by the transfer of wild-type bone marrow cells. This work indicated that mast cells can contribute significantly to this response, in part, by influencing the function of intestinal nerves [59]. Finally, an immunological-specific pathway that can produce acute mast cell-dependent inflammation independently of IgE or IgG1 has recently been reported. Redegeld et al. [60] have shown that that hapten-specific immunoglobulin light chains can induce skin mast cell degranulation and immediate hypersensitivity-like responses in wild-type mice but not in mast cell-deficient mice. Although the receptors for Ig-free light chains have yet to be identified, these studies point to yet another mast cell-dependent pathway that can contribute to immediate hypersensitivity responses. The extent to which such a mechanism might contribute to antigen-specific immune responses in humans remains to be determined.

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Mast Cells in Allergic Inflammation in the Airways: Mouse Models of Asthma

The importance of mast cells (as opposed to other potential effector cells) in the chronic inflammation and other long-term tissue alterations observed in asthma and other IgE-associated disorders has been debated for some time [8, 17]. Although only a few studies have investigated models of asthma using ‘mast cell knock-in mice’, such analyses have shown that one key role of mast cells in allergic asthma is to amplify the expression of multiple features of the pathology that can be elicited in actively immunized animals. For example, studies in mouse models of asthma that either omitted artificial adjuvants at the time of antigen sensitization [15, 61] or employed relatively low doses of antigen for sensitization or challenge [62, 63] have revealed that mast cells can directly or indirectly enhance the magnitude of multiple features of the asthmatic responses, including airway hyperreactivity to cholinergic stimulation [15, 62, 63], infiltration of eosinophils [15, 63] and other leukocytes into the airways and/or bronchoalveolar lavage fluid [61] and the numbers of proliferating cells in the airway epithelium [15]. We have recently established a chronic mouse model of asthma that exhibits multiple features shared with human asthma, including airway hyperreactivity to cholinergic stimulation, chronic inflammation of the airways, increased numbers of airway goblet cells, increased lung collagen deposition, and increased numbers of mast cells in the airways, including some within the epithelium [61]. Moreover, we have shown that optimal expression of these features of such chronic asthma model is mast cell-dependent [61]. On the other hand, the direct contribution of mast cells to various features of allergic asthma can be masked (or redundant) in other models of asthma, including very commonly used models that employ artificial adjuvants and relatively high doses of antigen for sensitization and challenge [15, 64–67]. In our opinion, a reasonable generalization from studies in various models of asthma in mice is that the importance of mast cells in the expression of allergic inflammation and other features of asthma (and probably other IgE-associated responses) in mice is greatest when relatively weak stimulants/inducers are used to induce the response. We also propose that such models may have particular relevance to the human setting, in which the sensitization of the subject and the elicitation of reactions in patients with atopic (or allergic) asthma can occur with low doses of antigen [68]. The contribution of mast cells to ‘non-atopic asthma’ has been analyzed in a model of IgE-independent airway responsiveness induced by epicutaneous sensitization with dinitrofluorobenzene followed by intra-airway challenge with dinitrofluorobenzene sulfonic acid. These studies have shown that mast cells

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are required for the changes in vascular permeability, mucus production and bronchoconstriction induced by dinitrofluorobenzene sulfonic acid challenge in the acute and late phases, as well as for the tracheal hyperreactivity detected 24 or 48 h after challenge [69]. Although the mechanisms responsible for mast cell activation in this model remain to be fully defined, mast cell-derived TNF has been implicated in the priming of the sensory nerve endings to enhance the tracheal hyperpermeability reactions [70]. In addition to IgE and antigen or other immunological mechanisms, other stimuli can activate airway mast cells, both in mouse models of asthma and in other settings. For example, adenosine, acting at least in part via adenosine A3 receptors on mast cells, can augment airway inflammation and airway responsiveness [71, 72]. Adenosine-stimulated human mast cells have recently been shown to induce IgE synthesis in B lymphocytes, providing further support for the hypothesis that mast cells can promote allergic inflammation by enhancing the development or magnitude of adaptive immune responses associated with IgE production [73]. Activation of complement, with subsequent activation of mast cells, may also contribute to the pathology in certain models of asthma in mice [74] and possibly in humans [74].

Mast Cells in Allergic Inflammation in the Skin: Delayed Hypersensitivity, Contact Hypersensitivity (CHS) and Atopic Dermatitis

Update on the Controversy Regarding the Roles of Mast Cells in CHS and Delayed Hypersensitivity Experimental models of CHS to chemical haptens have been widely used to investigate mechanisms of immune sensitization and the subsequent antigenspecific T activation [75]. While such reactions are classified as ‘type IV’ hypersensitivity responses in the Gell and Coombs classification [76], several groups have employed mast cell-deficient mice to investigate the possibility that mast cells can contribute to the expression of these reactions. However, these studies have produced disparate findings: some reports strongly implicated mast cells as important contributors to the responses [16, 77, 78], whereas others detected no involvement of mast cells in such reactions [43, 79–82]. A detailed review of the protocols employed in these studies revealed that a number of variables might have influenced whether or not mast cells contributed importantly to the CHS responses analyzed. These include the chemical structure (and perhaps chemical stability) of the hapten tested, the concentrations of hapten used (in both the sensitization and elicitation phases), the choice

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of vehicle for administering the hapten in the sensitization and challenge phases of the model, and the genotype of the mast cell-deficient mice employed. Moreover, the variability in the experimental results obtained in these studies of mast cell functions in CHS is quite reminiscent of the findings obtained when the role of mast cells was assessed in different mouse models of asthma (as discussed above). In these and probably many other settings, whether mast cells have a very important or entirely redundant role in a particular immune response may reflect the specific features of the protocol employed. Again, it seems reasonable to propose that the role of the mast cell in such complex biological responses is likely to be most evident when the ability of mast cells to amplify aspects of the response (that otherwise would only weakly be expressed) is most critical in achieving a high level of local expression of the reaction.

Potential Roles of Mast Cells in Certain CHS Responses Recent evidence suggests that, when mast cells do play a role in the expression of CHS or delayed-type hypersensitivity reactions, mast cells can contribute to these reactions through several distinct mechanisms. For example, studies using mast cell knock-in mice indicate that mast cell-derived TNF and MIP-2 can contribute significantly to the recruitment of neutrophils in one model of CHS [78]. In another study that employed BMCMC-reconstituted KitW-f/KitW-f mice, Villa et al. [83] reported that mast cells can contribute to the tissue swelling associated with a delayed-type hypersensitivity response to a subcutaneous challenge with OVA. In the same study, it was also shown that mast cell-reconstituted KitW-f/KitW-f mice developed higher titers of antigenspecific IgE and IgG1 than did KitW-f/KitW-f mice [83]. However, there was no difference in the production of antigen-specific IgE between KitW-f/KitW-f and wild-type mice [83]. Thus, such a finding is consistent with the possibility that mast cells can augment the production of antigen-specific antibodies in vivo, at least in KitW-f/KitW-f mice [83]. An additional mechanism by which mast cells can contribute to the expression of CHS was recently reported by Bryce et al. [16]. This group demonstrated that IgE antibodies are required for optimal immune sensitization in a model of oxazolone (Ox)-induced CHS. In this model of CHS to Ox, both IgE⫺/⫺ or KitW/KitW-v mice exhibited significant impairment in the expression of the tissue swelling elicited by hapten sensitization and challenge. Both types of mutant mice also exhibited a reduction in the numbers of DCs that emigrated from the epidermis in response to Ox in the sensitization phase of the CHS reactions. Normal levels of CHS to Ox (as well as normal levels of egress of DCs from the epidermis upon initial hapten challenge) were observed in IgE⫺/⫺

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mice that had received passive transfer of IgE, even though the IgE transferred did not have specificity for the hapten tested (Ox). The ‘antigen non-specific’ effect of IgE in promoting CHS in this system required signaling via the FcR common ␥ chain. Moreover, IgE was required for optimal cutaneous expression, 1 h after initial challenge of the skin with Ox, of mRNA for a mast cell-associated protease (mouse mast cell protease-6), as well as for several cytokines (IL-1␤, IL-6 and TNF) and a chemokine (MCP-1) known to have effects on DC migration, maturation and function. Accordingly, Bryce et al. [16] speculated that physiological levels of IgE are required for optimal immune sensitization in certain models of CHS. In such models, IgE can mediate antigen-independent, but Fc␧RI-dependent, priming effects on dermal mast cells that render these cells better able to produce cytokines and chemokines, which can in turn enhance DC migration, maturation and/or function. Potential Roles of Mast Cells in Other Models of Allergic Inflammation in the Skin Increased numbers of mast cells and/or morphological evidence of mast cell activation have been detected in a variety of conditions associated with chronic inflammation in the skin [84–87]. However, the mechanisms which regulate mast cell numbers in these settings, or by which mast cells might contribute to these responses, are not fully understood. Alenius et al. [88] examined the involvement of mast cells in a mouse model of allergic dermatitis which was elicited by repeated epicutaneous sensitization with OVA. In this model, the extent of inflammatory cell infiltration observed in the OVA-sensitized skin in mast cell-deficient mice was similar to that observed in the congenic wild-type mice. However, the levels of total serum IgE and IFN-␥ mRNA expression in the sensitized skin were significantly increased in KitW/KitW-v mice compared to those in the wild-type mice. Although ‘mast cell knock-in mice’ were not tested, these findings are consistent with the possibility that mast cells can contribute to chronic allergic inflammation via effects on IFN-␥ production and the regulation of serum IgE levels. Using functional genomics approaches to study mast cell function, our group recently identified Rab guanine nucleotide exchange factor 1 (RabGEF1) as a novel negative regulator of Fc␧RI-dependent mast cell activation [89]. RabGEF1 binds to Ras and negatively regulates the Ras/Raf/ERK signaling cascade. Mast cells derived from Rabgef1⫺/⫺ mice exhibit enhanced degranulation and increased release of cytokines and lipid mediators in response to IgE and antigen stimulation. Moreover, mice lacking RabGEF1 develop severe skin inflammation and exhibit increased numbers of mast cells at the sites of such skin lesions, with some mast cells showing histological evidence of degranulation.

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These mice also develop significantly elevated levels of serum IgE and histamine [89]. Since the development of skin lesions and the increases in mast cell numbers appear to progress simultaneously in the skin of Rabgef1⫺/⫺ mice, it is possible that mast cells contribute to the cutaneous inflammation in these mice. While the mechanism(s) that contribute to the mast cell activation detected in the skin of Rabgef1⫺/⫺ mice remain to be elucidated, a number of factors may be involved. These include the high levels of IgE in the blood of Rabgef1⫺/⫺ mice (since IgE itself can induce mast cell activation in the absence of known antigen [3, 6]) and the high basal levels of Ras activity that have been detected in mast cells derived from Rabgef1⫺/⫺ mice [89]. Further analysis of the skin lesions in Rabgef1⫺/⫺ mice may help to reveal some of the mechanisms that link mast cell activation to the expression of specific features of skin pathology associated with chronic allergic inflammation.

Conclusions

The development of the ‘mast cell knock-in mouse’ model has greatly facilitated the direct analysis, in vivo, of specific hypotheses regarding the roles of mast cells in allergic responses, as well as in many other biological responses. Such studies strongly support the notion that mast cells are critically involved in, if not essential for, the expression of many aspects of the allergic responses that involve Th2 cells and IgE. In the immediate phase of the IgE-dependent reactions, such as those observed in animals which have been passively sensitized with IgE, mast cells appear to be essential for all aspects of virtually every response so-far analyzed, with the possible exception of anaphylactic reactions to certain penicillin-related antigens. Studies in ‘mast cell knock-in mice’ also indicate that mast cells can contribute importantly to certain aspects of IgE-associated ‘late phase reactions’ and to the chronic tissue remodeling associated with at least some models of chronic asthma. In these settings, mast cells appear to be able to amplify significantly the magnitude of the reactions, some of which nonetheless can still occur, albeit at reduced levels, in the absence of this cell type. One of the attractive aspects of the ‘mast cell knock-in mouse’ model is that it can permit a fine dissection of the pathogenesis of specific allergic responses into components that require mast cells, that do not require mast cells but that can be significantly influenced by this cell type, or that can be expressed normally in the complete absence of the mast cell. And, in those instances in which mast cells are essential for, or significantly contribute to the response, one may employ ‘designer’ mast cell knock-in mice, containing adoptivelytransferred mast cell populations with defined alterations in the expression of

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specific surface structures, signaling components or secreted products, to characterize in detail the mechanisms by which mast cells contribute to the expression of the response of interest (fig. 1). Despite the importance of the mast cell in many aspects of allergic responses, there is no doubt that effector cells other than mast cells, especially T cells, eosinophils, and effector cells that can interact directly or indirectly with IgE (or, in the mouse, with IgG1), including basophils, may also have important roles in Th2- and IgE-associated responses, especially those that develop over long periods of time.

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Arase N, Arase H, Hirano S, Yokosuka T, Sakurai D, Saito T: IgE-mediated activation of NK cells through Fc␥RIII. J Immunol 2003;170:3054–3058. Perdue MH, Masson S, Wershil BK, Galli SJ: Role of mast cells in ion transport abnormalities associated with intestinal anaphylaxis. Correction of the diminished secretory response in genetically mast cell-deficient W/Wv mice by bone marrow transplantation. J Clin Invest 1991;87: 687–693. Redegeld FA, van der Heijden MW, Kool M, Heijdra BM, Garssen J, Kraneveld AD, Van Loveren H, Roholl P, Saito T, Verbeek JS, Claassens J, Koster AS, Nijkamp FP: Immunoglobulin-free light chains elicit immediate hypersensitivity-like responses. Nat Med 2002;8:694–701. Yu M, Tsai M, Tam SY, Galli SJ: Mast cells contribute to multiple features of a model of chronic asthma in mice. J Allergy Clin Immunol 2005;115:S188. Abstract no. 749. Kobayashi T, Miura T, Haba T, Sato M, Serizawa I, Nagai H, Ishizaka K: An essential role of mast cells in the development of airway hyperresponsiveness in a murine asthma model. J Immunol 2000;164:3855–3861. Kung TT, Stelts D, Zurcher JA, Jones H, Umland SP, Kreutner W, Egan RW, Chapman RW: Mast cells modulate allergic pulmonary eosinophilia in mice. Am J Respir Cell Mol Biol 1995;12: 404–409. Nogami M, Suko M, Okudaira H, Miyamoto T, Shiga J, Ito M, Kasuya S: Experimental pulmonary eosinophilia in mice by Ascaris suum extract. Am Rev Respir Dis 1990;141:1289–1295. Okudaira H, Nogami M, Matsuzaki G, Dohi M, Suko M, Kasuya S, Takatsu K: T-cell-dependent accumulation of eosinophils in the lung and its inhibition by monoclonal anti-interleukin-5. Int Arch Allergy Appl Immunol 1991;94:171–173. Brusselle GG, Kips JC, Tavernier JH, van der Heyden JG, Cuvelier CA, Pauwels RA, Bluethmann H: Attenuation of allergic airway inflammation in IL-4-deficient mice. Clin Exp Allergy 1994;24:73–80. Takeda K, Hamelmann E, Joetham A, Shultz LD, Larsen GL, Irvin CG, Gelfand EW: Development of eosinophilic airway inflammation and airway hyperresponsiveness in mast celldeficient mice. J Exp Med 1997;186:449–454. Liu LY, Swenson CA, Kelly EA, Kita H, Jarjour NN, Busse WW: Comparison of the effects of repetitive low-dose and single-dose antigen challenge on airway inflammation. J Allergy Clin Immunol 2003;111:818–825. Kraneveld AD, van der Kleij HP, Kool M, van Houwelingen AH, Weitenberg AC, Redegeld FA, Nijkamp FP: Key role for mast cells in nonatopic asthma. J Immunol 2002;169:2044–2053. Van Houwelingen AH, Kool M, de Jager SC, Redegeld FA, van Heuven-Nolsen D, Kraneveld AD, Nijkamp FP: Mast cell-derived TNF-␣ primes sensory nerve endings in a pulmonary hypersensitivity reaction. J Immunol 2002;168:5297–5302. Tilley SL, Tsai M, Williams CM, Wang ZS, Erikson CJ, Galli SJ, Koller BH: Identification of A3 receptor- and mast cell-dependent and -independent components of adenosine-mediated airway responsiveness in mice. J Immunol 2003;171:331–337. Zhong H, Shlykov SG, Molina JG, Sanborn BM, Jacobson MA, Tilley SL, Blackburn MR: Activation of murine lung mast cells by the adenosine A3 receptor. J Immunol 2003;171:338–345. Ryzhov S, Goldstein AE, Matafonov A, Zeng D, Biaggioni I, Feoktistov I: Adenosine-activated mast cells induce IgE synthesis by B lymphocytes: An A2B-mediated process involving Th2 cytokines IL-4 and IL-13 with implications for asthma. J Immunol 2004;172:7726–7733. Gerard NP, Gerard C: Complement in allergy and asthma. Curr Opin Immunol 2002;14:705–708. Grabbe S, Schwarz T: Immunoregulatory mechanisms involved in elicitation of allergic contact hypersensitivity. Immunol Today 1998;19:37–44. Cooms RPA, Gell PGH: The Classification of Allergic Reactions Underlying Disease. Oxford, Blackwell Scientific, 1963, pp 317–337. Askenase PW, Van Loveren H, Kraeuter-Kops S, Ron Y, Meade R, Theoharides TC, Nordlund JJ, Scovern H, Gerhson MD, Ptak W: Defective elicitation of delayed-type hypersensitivity in W/Wv and Sl/Sld mast cell-deficient mice. J Immunol 1983;131:2687–2694. Biedermann T, Kneilling M, Mailhammer R, Maier K, Sander CA, Kollias G, Kunkel SL, Hultner L, Rocken M: Mast cells control neutrophil recruitment during T cell-mediated delayed-type hypersensitivity reactions through tumor necrosis factor and macrophage inflammatory protein 2. J Exp Med 2000;192:1441–1452.

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Mindy Tsai, DMSc Department of Pathology, Stanford University School of Medicine 269 Campus Drive, CCSR#3255 Stanford, CA 94305 (USA) Tel. ⫹1 650 7360071, Fax ⫹1 650 7360073, E-Mail [email protected]

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Saito H, Okayama Y (eds): Mast Cells in Allergic Diseases. Chem Immunol Allergy. Basel, Karger, 2005, vol 87, pp 198–212

Mast Cell-Specific Genes – New Drug Targets/Pathogenesis Hirohisa Saito Department of Allergy and Immunology, National Research Institute for Child Health and Development, Setagaya, Tokyo, Japan

Abstract It has become possible to see all the expressed genes present in a cell (transcriptome) at once using microarray. We have applied microarray technology in various studies involving allergic diseases. Although we and others have discovered various novel molecules crucially involved in the pathogenesis of the disease, transcriptome assay is now expected as a tool for understanding the whole molecule balancing, i.e., system biology. Here I introduce examples of our trials for understanding the whole functional roles of mast cells as has been published in the web database for transcriptomes expressed by several mast cell types and various cell types. In the near future, we will be able to construct human mast cell models in silico (in a computer) by analyzing integrative information regarding the genome, transcriptome and proteome of mast cells, and will be able to test our hypotheses without having to perform in vitro tests. Copyright © 2005 S. Karger AG, Basel

Introduction

By complete reading of the human genome sequence, we have obtained tools for understanding everything about the transcriptome and the proteome, the whole functional elements in a cell. It is expected that we will be able to predict the results more precisely than before which may be obtained from basic researches or clinical trials for new treatment by utilizing such a comprehensive assay [1, 2]. Transcriptome analysis may be categorized in two ways depending on the approach: One is the aim to find an important molecule in which function or structure has not been fully described, and the other is the comprehension of the whole system controlling a cell and the simulation of human disease models in silico, i.e., in a computer. As such, the 21st century is considered to

be the era of ‘system biology’ [3], aiming at the comprehension of total function of a cell, an organ, or the human body using bioinformatics and functional genomics. One will be able to use computational modelling to analyze integrative biological functions. There are several different types of approaches to predict the total cellular function as a consequence of interaction of all molecules present in a cell. Most of the approaches, however, simulate the final cellular function based on the description in the literature but not on the whole genomic information. This is because most genomic techniques such as proteomics are still premature and impractical, except transcriptome analysis using microarray technology. This cell type-specific transcriptome database is also applied for understanding the system biology. Here, I introduce the mast cell-specific transcriptome database and discuss it in relation to the literature.

Mast Cells vs. Other Cell Types

In transcriptome research for finding novel key role molecules, many investigators have already reported such findings related to allergic inflammation mainly using mixed peripheral blood or tissue cells as samples [2]. However, these mixed cell populations sometimes make interpretation of the results difficult. Up-regulation of transcriptional levels in a crude tissue is often due to the increase in recruitment of a certain inflammatory cell type, and does not mean the increase in the transcriptional level in a single cell. On the other hand, mRNA is unstable so that complicated procedures for purification of a certain cell type should be avoided. Thus, computational identification of celltype specificity of a certain key role molecule found in crude tissues may be preferable. For this purpose, we constructed the cell type-specific transcriptome database (http://www.nch.go.jp/imal/GeneChip/JACI.htm) (table 1). To understand the total functional system in the human body, however, we have to know the whole system in an organ. In order to do so, we have to examine the whole systemic functions in a cell. At the end of the last century, we obtained a revolutionary tool, i.e., microarray, for understanding the transcriptome. Using the above database, it became possible to understand the total function of various types of a single cell at least at the transcriptome level. Comprehension of cell type-specific genes using comparative transcriptome assays is the first step for constructing the human body in the future. Mast cells are known to play versatile roles such as in evoking allergic reaction and in controlling physiological reactions such as blood pressure [4], and innate immunity. However, functions of a substantial number of genes or molecules remain undiscovered. Novel genes that are highly expressed by mast

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Table 1. Mast cell-, basophil- and eosinophil-selective transcripts for ion channels and receptors Transcript (GenBank Accession No.)

Cell-type selectivity

Ion channels Ca2⫹ channel type A1 D (BE550599)

Basophils, eosinophils

Ion channels Histamine H4 receptor (AF312230.1) Prostaglandin E receptor type 3a2 (X83858.1) C3a receptor (U62027.1) Chemokine receptor CCR3 (NM_001837.1) CRTH2* (NM_004778.1) EMR-1* (NM_001974.1) Adenosine A3 receptor (NM_000677.2) P2Y2 purinergic receptor (NM_002564.1) GPR105 purinergic receptor (NM_014879.1)

Basophils Basophils Basophils, eosinophils Basophils, eosinophils Basophils, eosinophils Basophils, eosinophils Eosinophils Eosinophils Eosinophils

Other receptors Fc␧RI ␣ (BC005912.1) HTm4 (L35848.1) IL-3 receptor ␣ (NM_002183.1) CD244 NK cell receptor† (NM_016382.1) Fibroblast growth factor receptor 2† (NM_022969.1) IL-5 receptor ␣ (M75914.1) Siglec 8 (NM_014442.1) CD117 c-KIT (NM_000222.1)† Siglec 6 (D86358.1) Fc␧RI (NM_000139.1) Low density lipoprotein receptor† (NM_000527.2) TRK* receptor (NM_002529.2)

Basophils Basophils Basophils Basophils, eosinophils Basophils, eosinophils Basophils, eosinophils Eosinophils Mast cell Mast cell Mast cell, basophils Mast cell, basophils Mast cell, basophils

Cell type-selective transcripts were chosen from approximately 20,000 transcripts (GeneChip U133A) based on the following criteria; (1) the average mRNA expression level of each gene (obtained from three independent experiments) in a certain cell type must be 3-fold or greater than the maximal level in other cell types, and (2) must be significantly (p ⬍ 0.01) greater than that in other cell types. (3) The expression level provided with ‘absence’ call by GeneChip Software should be observed only once or not at all in the three independent experiments. (4) For the transcripts preferentially expressed for the two different cell types, the average normalized expression levels in the two cell types should be within 3-fold of each other. *CRTH2 ⫽ Chemoattractant receptor-homologous molecule expressed on Th2 cells; EMR ⫽ epidermal growth factor-like module-containing mucin-like receptor; TRK ⫽ tropomyosin-related kinase neurotrophin. † Transcripts that are highly expressed by more than five different normal organs (http:// www.lsbm.org/index_e.html).

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RBC Plate CD4 CD8 CD19 CD56 CD14 Neu Eos Baso Mast

Fig. 1. Clustering analysis of transcriptomes expressed by mast cells and ten different types of blood cells. Approximately 22,000 kinds of mRNA transcripts were examined in RBC, platelets (Plate), CD4⫹ cells, CD8⫹ cells, CD19⫹ cells, CD56⫹ cells, CD14⫹ cells, neutrophils (Neu), eosinophils (Eos), basophils (Baso) and mast cells (Mast). Blue lines represent transcripts with lower expression and red lines represent those with higher expression. As shown here, a highly mast cell-expressing transcript is not always mast cell-specific.

cells may be easily found by examining the mast cell transcriptome database. By comparing to transcriptomes expressed by other cell types, however, one may find the mast cell-expressing genes are not mast cell-specific (fig. 1). Thus, elucidating the whole information related to the mast cell type-specific functions compared to the other cell types is important especially for pharmaceutical development, because one may get side effects on physiologically important organs by using drugs targeting various organs. The safety of any candidate drug must be evaluated by comparing its efficacy on these granulocytes with its toxicity to physiologically important organs. Eosinophils and basophils as well as mast cells are crucially involved in allergic reactions and inflammation [5]. On the other hand, neutrophils kill bacteria and sometimes induce systemic vasculitis or multiple organ damage under certain conditions [6, 7]. Mast cells, basophil- and/or eosinophil-specific genes could be potential therapeutic targets for allergic diseases because these granulocytes play a crucial role in allergic inflammation [8]. Activation of these cells is generally characterized by an influx of extracellular calcium (Ca2⫹), which is essential for subsequent release of granule-derived mediators, newly generated

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lipid mediators and cytokines [9]. Flow of other ions plays an important role during granulocyte responses because they regulate cell membrane potential and thus influence Ca2⫹ influx [10]. Treatment of mast cells and basophils with pertussis toxin inactivates the Gi-type of G-proteins and abolishes degranulation but not the influx of Ca2⫹ induced by non-immunological ligands such as thrombin and N-formylpeptide [11]. Thus, the granulocyte degranulation pathway is sometimes Ca2⫹-independent and is G-protein-dependent. Indeed, the thrombin-activated receptors and formylpeptide receptors are classified as G-protein-coupled receptors (GPR), having a seven-transmembrane region [12]. As such, ion channels and GPR both play essential roles in degranulation as well as other cellular functions important for granulocytes, and are thought to be good targets for drug development [13]. Receptor genes and ion channel genes are found only in 5 and 1.3% of all genes present in the human genome, respectively [14]. However, receptors and ion channels are respectively found in 45 and 5% of the molecular targets of all known drugs [13, 15, 16]. Beside their physiological importance, receptors including GPR and ion channels are considered to be marketable, and targeting these molecules should be efficient for pharmaceutical development. In other words, we can concentrate on approximately 2,000 genes as practical drug targets, and can ignore approximately 20,000 other genes present in the human genome [1]. As such, the cell type-selective transcriptome expression of seven types of leukocytes (basophils, eosinophils, neutrophils, CD4⫹, CD8⫹, CD14⫹ and CD19⫹ cells), platelets, fibroblasts and mast cells were examined using high-density oligonucleotide probe arrays (GeneChip, Affymetrix, Santa Clara, Calif., USA). Then the expression of granulocyte-selective genes for ion channels, GPR and other receptors were determined. It is estimated that approximately 50 genes are selectively expressed by mast cells, eosinophils, and basophils among all genes present in the human genome [17]. The representative druggable genes, GPCRs that were selectively expressed by mast cells, basophils and/or eosinophils using Affymetrix U133A GeneChip (approximately 20,000 known genes can be detected) were as follows; HRH4 (histamine H4 receptor), PTGER3 (prostaglandin E receptor type 3a2), ADORA3 (adenosine A3 receptor), P2RY2 (purinergic receptor), GPR44 (chemoattractant receptor-homologous molecule expressed on Th2 cells; CRTH2), EMR1 (egf-like module containing, mucinlike, hormone receptor-like sequence 1), CCR3 (chemokine receptor 3), and C3AR1 (C3a receptor). As such, analysis of cell type-selective druggable genes from database searches is expected to minimize unexpected adverse effects and the efforts required for drug discovery (fig. 2). When a new drug is first marketed, findings regarding its efficacy and safety are commonly based on the experience of several thousand people who have been treated in controlled clinical trials. Despite extensive tests, rare adverse events can easily escape

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~2,000 genes in the genome

Filtering through genomic information

~2,000 marketable genes

Filtering through celll type-specific information

~50 anti allergic druggable genes

Fig. 2. Filtering of ‘druggable’ genes through genomic information. Only approximately 22,000 genes were found to be contained in our genome. Through marketable (⫽ easy-to-obtain specific antagonist or agonist) merits, 2,000 genes may be filtered as drug targets. Using a cell type-specific transcriptome database, they may be further subclassified to be antiallergic drug targets. The estimated number of the antiallergic (mast cell-, basophil- and eosinophil-specific) drug targets was approximately only 50.

detection. Thus, these studies are also expected to minimize unexpected adverse effects related to the drugs.

Activated vs. Resting Mast Cells

Even before microarray techniques were developed, mast cells were known to produce multiple cytokines through transcription of these genes for inducing allergic inflammation, i.e. tumor necrosis factor (TNF)-␣ [18], granulocytemacrophage colony-stimulating factor (GM-CSF) [19], I-309/TCA-3 (CCL1, i.e., CC-chemokine ligand 1) [20], monocyte chemoattractant protein (MCP)-1 (CCL2) [21], macrophage inflammatory protein (MIP)-1␣ (CCL3) [20, 22], MIP-1␤ (CCL4) [20], and interleukin (IL)-3 [23], IL-4 [24], IL-5 [25], IL-6 [20, 26], IL-8 [20, 22], IL-9 [26], IL-10 [27], IL-13 [28, 29], and IL-16 [30] following IgE-dependent activation. A transcriptome assay for human mast cells seems to be of particular interest as to whether they can produce other unidentified cytokines. So far, two databases are available regarding human mast cell transcriptome after IgE-mediated activation (http://genome-www5.stanford. edu/cgi-bin/publication/viewPublication.pl?pub_no ⫽ 157, and http://www. nch.go.jp/imal/Mast/Blood_Dec1_2002sup.xls) [31, 32]. According to their microarray data, the results were not very unexpected regarding the types of inflammation-related cytokines and chemokines they produced. Most of these genes expressed by human mast cells after aggregation of Fc␧RI were identical to the previous reports using non-array methods [18–30]. It was confirmed that Th1 cytokine- and CXC-chemokine-derived Th1 cells are not really expressed

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by activated human mast cells. Interesting findings among these microarray data may be up-regulation of some genes related to tissue remodeling or immune regulation such as IL-11 [31] and 4-1BB [32]. Mast cells express Toll-like receptors (TLR) which recognize various microbial components [33], and can be activated via challenge with these components. Mouse cultured mast cells express TLR2, 4 and 6 and produce a variety of cytokines including IL-13 in response to these TLR ligands such as LPS [34, 35] and release preformed mediators in response to peptide glycan, a TLR2 ligand [36]. Human cord blood-derived cultured mast cells express TLR2 and 6 and produce a variety of cytokines such as GM-CSF [37, 38]. On the other hand, adult peripheral blood progenitor-derived cultured mast cells do not significantly respond to TLR stimulation. However, after preincubation with IFN-␥, the expression of TLR4 is up-regulated and LPS can induce a variety of transcripts in the peripheral blood-derived cultured mast cells. The transcriptome in LPS-stimulated and INF-␥-primed human mast cells is somewhat different from that in anti-IgE-stimulated human mast cells. These LPS-stimulated mast cells produce more TNF-␣ and RANTES (CXCL5), and produce less IL-5 [39]. Although many investigators have recently reported the positive effect of various TLR ligands on mast cell activation, mast cell biologists should be cautious about the tricks in TLR experiments which are known among researchers investigating innate immunity. TLR9 is almost exclusively expressed by plasmacytoid dendritic cells and B cells [40]. Even ⬍0.1% of contamination with these cell types may thus produce a false-positive result in CpG motifchallenged experiments. The possibility of contamination with other TLRexpressing cell types should also be excluded from the result obtained by almost all mast cell populations. LPS is also known to be contaminated everywhere even in a TLR3 ligand poly(I:C) [41], so that appropriate pure reagents should be carefully employed. According to the cell type-specific database (http:// www.nch.go.jp/imal/GeneChip/JACI.htm), mast cells express low levels of TLR2, 4 and 6. The other TLRs are not detectable. On the other hand, monocytes and neutrophils abundantly express almost all TLRs. We should carefully consider the biological significance of the presence of these TLRs on mast cells.

Glucocorticoid Sensitivity of Mast Cell Transcripts

It has been reported that ⬎20 cytokines and chemokines are expressed by different populations of activated mast cells [18–39]. Thus, the finding that activated mast cells express yet another cytokine, by itself, is not a significant finding. Because glucocorticoids inhibit the expression of numerous harmful

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cytokines and additional factors in mast cells [42] and other cell types, glucocorticoids are used as the first-line therapy for patients with asthma and other inflammatory disorders. Despite the clinical improvement, glucocorticoidtreated asthmatic patients continue to produce large amounts of sputum. This finding has led to the speculation that a glucocorticoid-unresponsive factor might be present in abundance in the lungs of asthmatic patients that helps induce airway epithelial cells to increase their production of mucin. Okumura et al. [43] used a GeneChip approach to identify those transcripts that are preferentially induced in activated human mast cells, and reported that the level of the transcript that encodes the cytokine amphiregulin (AREG) is markedly increased in mast cells that have been activated via Fc␧RI. They also reported that amphiregulin is the only one glucocorticoid-unresponsive cytokine, and several receptor transcripts such as OX40L are also glucocorticoid-unresponsive [43]. AREG is a member of the epidermal growth factor (EGF)/transforming growth factor-␣ family of cytokines that binds to the EGF receptor and promotes the growth of normal epithelial cells. Many dermatologists are interested in AREG because the increased expression of this cytokine in keratinocytes is believed to contribute to the development of psoriasis [44]. Up-regulation of amphiregulin protein expression was observed in mast cells of asthmatic lung specimens, but not those in normal control subjects, and that significantly correlated with the extent of goblet cell hyperplasia in the mucosa of bronchial asthmatics [43]. After exposure to antigens, human mast cells are considered to induce sputum production via release of amphiregulin. Other than AREG, only a few cytokine transcripts exist.

Mast Cell Subset-Specific Genes

For a long time we were not able to obtain a substantial amount of human mast cells necessary for genome-wide comprehensive analyses such as transcriptome. The methods of generating a substantial number of human mast cells from umbilical cord blood [45] and from adult peripheral blood [46] have recently been established. More recently, since the development of microarray technology, it has become possible to examine the levels of whole transcripts present in a cell using approximately 105 cells. Thus, it is now possible to examine small cell populations such as tissue-derived mast cells or blood basophils in comparison with cultured human mast cells. The expression levels of ⬎10,000 transcripts in cultured human mast cells derived from umbilical cord blood progenitors and from adult peripheral blood progenitors have been investigated [47]. It was found that the transcript for Fc␧ receptor ␣-chain is selectively down-regulated in cord blood-derived human

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mast cells compared to adult peripheral blood progenitor-derived mast cells. This down-regulation may be controlled by epigenetic mechanisms present in hemopoietic progenitors since the expression level of Fc␧ receptor ␣-chain was down-regulated even under the same culture conditions. Indeed, neonatal cord and adult peripheral blood hemopoietic progenitors can produce cell types having somewhat different profiles [48, 49]. Kashiwakura et al. [50] have recently reported the transcriptome analysis of human tonsillar mast cells, which locate in the interfollicular areas and may interact with T cells. A large fraction of the gene expression profiles of cultured tonsillar mast cells were comparable to other mast cells including cultured lung mast cells. However, MIP-1␣ (CCL3) and MIP-1␤ (CCL4) were expressed in resting cultured tonsillar mast cells but not in resting cultured lung mast cells. TNF-␣ expression was also up-regulated in tonsillar mast cells following Fc␧RI aggregation. It has been reported that CCL3 and CCL4 have the ability to recruit T cells into lymph nodes and mast cells are one of the major sources of CCL4 [51], and that both TNF-␣ concentration and the recruitment of circulating T cells were increased within draining lymph nodes following peripheral mast cell activation [52]. T cells thus may be recruited by tonsillar mast cells. In agreement with the results of chymase expression of tissue mast cells [53], cultured tonsillar mast cells also showed higher intensity of the expression than cultured lung mast cells. It is of particular interest that the tissue-specific nature of human mast cells persists after a long period of culture. Kambe et al. [54] have reported a similar observation regarding human skin-derived cultured mast cells. Reactivity toward substance P of the human skin-derived cultured mast cells was not lost even after extensive proliferation of the mast cells under the standard serum-free culture system supplemented with stem cell factor. Bradding et al. [55] have reported that human skin-derived cultured mast cells express somewhat different types of ion channel profiles in comparison with lung and cord blood-derived mast cells, although the transcriptome profiles of these mast cells were mostly identical. These three reports indicate that cultured mast cells retain some of their characteristics derived from the original tissues, even after proliferation at 15fold increased number of mast cells under the standard culture conditions. Although the microenvironment can change the characterization of mast cells [56], some effects of the microenvironment may last long after removal of the environmental factors. These findings may suggest that mast cell progenitors committed to a certain mast cell subtype might be selectively recruited to the tissue. Alternatively, the tissue-specific characters of mast cells may be determined by epigenetic alteration occurring in their progenitors. The mast cell progenitors may be epigenetically influenced by environmental factors during their stay in the tissue. Environmental conditions sometimes modify the

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composition of the genomic structure or nucleic acid molecules such as CpG methylation, and this modification sometimes lasts for several generations [57].

Human Mast Cells vs. Mouse Mast Cells

Animal disease models have been used as surrogates for humans and have been informative. The use of mouse models for diseases related to allergy and immunology has increased dramatically because of the rapidly developing technologies and immunological tools to block specific pathways or to selectively knockout genes that are important for processes that contribute to the pathogenesis of the disease. By using these technologies, responsible genes have been found for several types of severe combined immunodeficiency and common variable immunodeficiency. Controversy does exist, however, as to the relevance of these models of allergic diseases such as asthma [58, 59]. Clinical trials sometimes fail due to the fact that the results obtained in animal studies cannot be reproduced in man. For instance, anti-IL-5 (eosinophil growth factor) antibody completely blocked the airway hypersensitivity related to eosinophil inflammation in experimental animal models of asthma [60]. However, the therapeutic application of humanized anti-IL-5 antibody did not improve the bronchial hypersensitivity of asthmatics in spite of a marked decrease in eosinophil number [61]. One of the reasons why allergic disease models sometimes fail to reproduce the human diseases is the complexity of human allergic diseases. Total loss of the gene function usually induces severe symptoms but not mild symptoms as seen in multifactorial diseases such as allergic diseases. Until now, we have not succeeded in reproducing even the simplest mouse model for a hygiene hypothesis as to whether low levels but not high levels of endotoxin in an environment induces allergic diseases. Rodent mast cells are common experimental tools but are somewhat different from their human counterparts in their responses to certain cytokines and antiallergic drugs [62, 63]. Thus, in 2002, we examined the genome-wide gene expression in cultured human and mouse mast cells (triggering IgE-mediated allergic reaction) to find molecules similarly regulated and expressed by the two mast cell types using a preliminary comparative interspecies database [32]. After completion of human and mouse genome sequencing, information regarding many more human and mouse orthologous genes became available at the genome-wide level, which facilitates interspecies comparisons. Thus, we constructed and published on-line an interspecies comparison database (http:// bio.mki.co.jp/en/results/_notes/comparativeDB_index.html), which opens human and mouse mast cell transcriptomes regarding orthologous genes. Genes in which expression levels were markedly up-regulated both in human and mouse mast

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PYCS, CEACAM1 PHLDA1, CCL5 DTP, OLR1 GADD45B, IL5 INHBA, RGC32 CSF1, TNF, RRAD SERPINE1, RIL MMP19, CCL4 RIL, IL3, CCL1

Fig. 3. Identification of orthologous genes that similarly regulated in human and mouse mast cells by hierarchical clustering analysis. Although clustering analysis tends to enhance the slight differences, orthologous genes containing several cytokines and chemokines, i.e., CCL5 (RANTES), IL5, CSF1 (M-CSF), TNF, CCL4 (MIP-1␤), IL3 and CCL1 (I-309) were found in the same cluster. CCL3 (MIP-1␣) and CSF2 (GM-CSF) were also found to be similarly regulated in both human and mouse mast cells after IgE receptor aggregation. PGN2 (major basic protein) and AREG (amphiregulin) were representatively found to be human mast cell-unique up-regulated genes. See our website displaying interspecies comparison database (http:// bio.mki.co.jp/en/results/comparativeDB/comparativeDB_index.html).

cells after stimulation via high-affinity IgE receptor (Fc␧RI) were examined using hierarchical clustering analysis (fig. 3). The clustering analysis tends to enhance the slightly different patterns. However, the following genes including several cytokine/chemokine genes were found in the same cluster. They were PYCS, CEACAM1, PHLDA1, CCL5, DTR, OLR1, GADD45B, IL-5, INHBA, RGC32, CSF1, TNF, RRAD, SERPINE1 (plasminogen activator inhibitor-1), RIL, MMP19, CCL4, IL3, and CCL1 (I-309), indicating that Fc␧RI-mediated induction of these genes is highly conserved between human and mouse. Among these orthologous genes, we found that PRG2 (major basic protein) mRNA levels were abundantly expressed by human mast cells but not by mouse mast cells. Studies on the function of molecules highly expressed only in mouse cells have to be carefully interpreted with regard to their potential function in humans. Interspecies comparison studies of whole genome expression should be useful for interpretation of experimental data from animal models of human pathogenesis. To simulate the human body in computational model, we still have to use many animal models. In the near future it will be possible to select the information obtained from animal models just where the orthologous genes are similarly functioning. The mouse disease models should not be used any more where the key orthologous genes are differently working in human and mouse.

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Computational Modeling of Human Mast Cells in silico

In the near future, not only will we be able to construct human mast cell models in silico by analyzing integrative information regarding the genome, transcriptome and proteome of mast cells, but we will also be able to test our hypotheses without performing any in vitro tests. To translate human mast cell models into reality, many issues should be resolved. Especially proteomics for understanding functional modification of proteins should be more advanced. Compared to transcriptome assays, the present techniques for analyzing proteome are neither really comprehensive nor high-throughput and are laborintensive. However, we do not need to wait until we fully comprehend the functional roles of proteome in mast cells. Bioinformatics combined with a transcriptome database may compensate for the lack of information regarding proteome, although even now bioinformatics alone without genomic information can mimic the behavior of a cell [64]. We now need to know what types of mRNA transcripts are reliable for predicting their functional roles as proteins in silico.

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36 37

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40 41

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45

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47

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50 51

52

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55 56

57 58 59 60

61

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Kashiwakura JI, Yokoi H, Saito H, Okayama Y: T cell proliferation by direct cross-talk between OX40 ligand on human mast cells and OX40 on human T cells. J Immunol 2004;173:5247–5257. Tedla N, Wang HW, McNeil HP, Di Girolamo N, Hampartzoumian T, Wakefield D, Lloyd A: Regulation of T lymphocyte trafficking into lymph nodes during an immune response by the chemokines macrophage inflammatory protein (MIP)-1␣ and MIP-1␤. J Immunol 1998;161: 5663–5672. McLachlan JB, Hart JP, Pizzo SV, Shelburne CP, Staats HF, Gunn MD, Abraham SN: Mast cellderived tumor necrosis factor induces hypertrophy of draining lymph nodes during infection. Nat Immunol 2003;4:1199–1205. Irani AM, Bradford TR, Kepley CL, Schechter NM, Schwartz LB: Detection of MCT and MCTC types of human mast cells by immunohistochemistry using new monoclonal anti-tryptase and anti-chymase antibodies. J Histochem Cytochem 1989;37:1509–1515. Kambe N, Kambe M, Kochan JP, Schwartz LB: Human skin-derived mast cells can proliferate while retaining their characteristic functional and protease phenotypes. Blood 2001;97: 2045–2052. Bradding P, Okayama Y, Kambe N, Saito H: Ion channel gene expression in human lung, skin and cord blood-derived mast cells. J Leukoc Biol 2003;73:614–620. Jippo T, Lee YM, Ge Y, Kim DK, Okabe M, Kitamura Y: Tissue-dependent alteration of protease expression phenotype in murine peritoneal mast cells that were genetically labeled with green fluorescent protein. Am J Pathol 2001;158:1695–1701. Borish L, Steinke JW: Beyond transcription factor. Allergy Clin Immunol Int 2004;16:2–27. Persson CGA: Mice are not a good model of human airway disease. Am J Respir Crit Care Med 2002;166:6–7. Gelfand EW: Mice are a good model of human airway disease. Am J Respir Crit Care Med 2002;166:5–6. Hamelmann E, Oshiba A, Loader J, Larsen GL, Gleich G, Lee J, Gelfand EW: Anti-interleukin-5 antibody prevents airway hyperresponsiveness in a murine model of airway sensitization. Am J Respir Crit Care Med 1997;155:819–825. Leckie MJ, ten Brinke A, Khan J, Diamant Z, O’Connor BJ, Walls CM, Mathur AK, Cowley HC, Chung KF, Djukanovic R, Hansel TT, Holgate ST, Sterk PJ, Barnes PJ: Effects of an interleukin-5 blocking monoclonal antibody on eosinophils, airway hyper-responsiveness, and the late asthmatic response. Lancet 2000;356:2144–2148. Shichijo M, Inagaki N, Nakai N, Kimata M, Nakahata T, Serizawa I, Iikura Y, Saito H, Nagai H: The effects of anti-asthma drugs on mediator release from cultured human mast cells. Clin Exp Allergy 1998;28:1228–1236. Okayama Y, Church MK: Comparison of the modulatory effect of ketotifen, sodium cromoglycate, procaterol and salbutamol in human skin, lung and tonsil mast cells. Int Arch Allergy Immunol 1992;97:216–225. Tomita M: Whole-cell simulation: A grand challenge of the 21st century. Trends Biotechnol 2001;19:205–210.

Hirohisa Saito, MD, PhD Department of Allergy and Immunology National Research Institute for Child Health and Development 2-10-1, Okura, Setagaya, Tokyo 157-8535 (Japan) Tel. ⫹81 3 5494 7027, Fax ⫹81 3 5494 7028, E-Mail [email protected]

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Author Index

Boyce, J.A. 59 Bradding, P. 163 Da Silva, C.A. 154

Kay, L.J. 145 Koike, K. 1 Metcalfe, D.D. 43

Frossard, N. 154 Galli, S.J. 179 Gilfillan, A.M. 43 Grimbaldeston, M.A. 179 Inoue, T. 32 Iwaki, S. 43 Juremalm, M. 130

Niide, O. 32 Nilsson, G. 130 Oh, C.K. 85 Okayama, Y. 101 Pawankar, R. 111 Peachell, P.T. 145

Ra, C. 32 Saito, H. 80, 198 Saito, T. 22 Shiohara, M. 1 Suzuki, Y. 32 Tam, S.-Y. 179 Tkaczyk, C. 43 Tsai, M. 179 Yamasaki, S. 22 Yoshimaru, T. 32 Yu, M. 179

213

Subject Index

Activation, mast cells adaptor molecules, see Adaptor molecules difficulty of study 180, 181 Fc␧RI role FcR␥-ITAM-dependent and -independent mast cell response 24, 25, 35, 36 hypersensitivity mediation 23, 24 ion channels 164–175 reactive oxygen species allergy role 37–39 Fc␧RI signaling and generation 35–37 transcriptome analysis 203, 204 Adaptor molecules 3BP2 55 Brb2 54 Cbl 55, 56 classification 43, 44 constitutive protein–protein interactions 44, 45 cytosolic adaptor molecules 52–56 Dok 55, 56 Fc␧RI subunits 47 Fc␥RI subunits 47, 48 Fc␥RIIb 48 Gab2 54, 55 Gads 54 gp49b 48 inducible protein–protein and proteinlipid interactions overview 45

PH domains 46 PTB domains 46 SH2 domains 46 Kit 48, 49 LAT 49, 50 NTAL 51, 52 SLP-76 53 transmembrane adaptor molecules 49–52 Vav 53, 54 ␤2-Adrenoceptor, mast cells agonists bronchodilation 146 stabilization of mast cells 146, 147 tolerance induction 148–150 polymorphisms and pharmacogenetics 150, 151 Airway remodeling mast cell plasmin activator inhibitor-1 extracellular matrix accumulation mechanisms 90, 91 gene expression characterization 89, 90 regulation of expression 92 role 92–95 pathogenic remodeling 86 physiologic remodeling 86 structural changes 85 Allergic airway disease chronic allergic inflammation 118 immediate-phase response 116, 117 immunoglobulin E humanized monoclonal antibodies for treatment 120, 121

214

late-phase response 117, 118 mast cell migration and phenotype regulation in nasal epithelium 116 mast cell phenotype and distribution in allergic rhinitis 112, 116 nasal mast cells adhesion molecules and extracellular matrix proteins 115 IgE-Fc␧RI cascade 114 production of cytokines 112–114 structural cell interactions 115, 116 Allergy leukotriene response 65, 68 prostaglandin D2 response 62–64 reactive oxygen species role in mast cell activation 37–39 Apoptosis, STI571 induction 5, 6 Asthma ␤2-adrenoceptor agonist therapy, see ␤2-Adrenoceptor, mast cells glucocorticoid therapy airway remodeling, see Airway remodeling anti-inflammatory effect 154, 155 bronchial hyperresponsiveness reduction 155 mast cell number reduction 156, 157, 159 resistance 155, 156 stem cell factor suppression 157–159 immunoglobulin E humanized monoclonal antibodies for treatment 120, 121 leukotriene response 67, 68 mast cell mediators 86–89, 119, 120 Atopic dermatitis, mast cell role 191, 192 Basophil Fc⑀RI signaling and reactive oxygen species generation 35–37 transcriptome analysis 200–202 3BP2, mast cell activation role 55 Brb2, mast cell activation role 54 Calcium channels, mast cell expression and function 171–173 Cardiomyopathy, mast cell role 5

Subject Index

Cbl, mast cell activation role 55, 56 CCR1, mast cell expression 136 CCR2, mast cell expression 137 CCR3, mast cell expression 137 CCR4, mast cell expression 136 CCR5, mast cell expression 137, 138 CD34⫹ bone marrow cells, mast cell precursors 2, 3 Chemokines classification 131 complexity of ligand-receptor network 135 mast cell functions degranulation and cytokine secretion 140, 141 migration 139, 140 prospects for study 141, 142 viral infection 141 nomenclature of types and receptors 131–134 receptors homology 134 mast cell receptors CCR1 136 CCR2 137 CCR3 137 CCR4 136 CCR5 137, 138 CXCR1 138 CXCR2 138 CXCR3 138, 139 CXCR4 139 CX3CR1 138 overview 135, 136 signaling 134, 135 structure 131 Chloride channels, mast cell expression and function 168–171 Chronic rhinosinusitis, mast cell role 121–123 Connective tissue mast cell (CTMC), phenotype 1, 2 Contact hypersensitivity, mast cell role 189–191 Corticosteroids, see Glucocorticoids Cultured mast cells Fc␧RI expression 13, 14

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Cultured mast cells (continued) protease expression 13, 14 stem cell factor cofactors in mast cell development 5–13 differentiation and proliferation role in culture 3, 5 Toll-like receptor expression 119 transcriptome analysis from different tissues 205–207 CXCR1, mast cell expression 138 CXCR2, mast cell expression 138 CXCR3, mast cell expression 138, 139 CXCR4, mast cell expression 139 CX3CR1, mast cell expression 138 Cyclic AMP (cAMP), mast cell stabilization 147 Cytokines, see also specific cytokines mast cell expression mediation by receptors Fc␧RI 102, 105, 106 Fc␥RI 104, 106–108 Toll-like receptors 103–106 nasal mast cell production in allergy 112–114 Delayed hypersensitivity, mast cell role 189, 190 Dendritic cell (DC), leukotriene response 69 Dok, mast cell activation role 55, 56 Eicosanoids, see specific eicosanoids Eosinophil, transcriptome analysis 200–202 Fc␧RI cultured mast cell expression 13, 14 eicosanoid signaling, see specific eicosanoids functional overview 22, 23 mast cell activation FcR␥-ITAM-dependent and -independent mast cell response 24, 25 hypersensitivity mediation 23, 24, 180 mast cell cytokine expression mediation 102, 105, 106

Subject Index

mast cell survival versus degranulation regulation 25–29 signal transduction 35–37 structure 22, 23 subunits as adaptor molecules 47 Fc␥RI mast cell cytokine expression mediation 104, 106–108 subunits as adaptor molecules 47, 48 Fc␥RIIb, adaptor molecule 48 Formoterol, mast cell stabilization 147 Gab2, mast cell activation role 54, 55 Gads, mast cell activation role 54 Glucocorticoids anti-inflammatory effect 154, 155 bronchial hyperresponsiveness reduction 155 mast cell number reduction 156, 157, 159 resistance in asthma 155, 156 stem cell factor suppression 157–159 transcript sensitivity studies in mast cells 204, 205 gp49b, mast cell activation role 48 Histamine, bronchodilator effects on mast cell release 146 Immunoglobulin E (IgE) humanized monoclonal antibodies for allergic airway disease treatment 120, 121 receptor, see Fc␧RI Interferon-␥ (IFN-␥), mast cell development role 13, 15 Interleukin-3 (IL-3), mast cell development role 9, 10 Interleukin-4 (IL-4), mast cell development role 10, 11, 15 Interleukin-6 (IL-6), mast cell development role 10, 11, 15 Interleukin-9 (IL-9), mast cell development role 7–9, 15 Interleukin-16 (IL-16), mast cell development role 9, 15

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Ion channels, mast cells calcium channels 171–173 chloride channels 168–171 electrical excitation cycle 175, 176 potassium channels 164, 165, 167, 168 P2X receptors 175 sodium channels 173 Kit ligand, see Stem cell factor mast cell activation role 48, 49 transgenic mice, see Mast cell knock-in mice Knock-in mice, see Mast cell knock-in mice LAT, mast cell activation role 49, 50 Leukotrienes allergic response 65 biosynthesis cytokine regulation 72 heterogeneity of eicosanoid generation by tissue mast cell subsets 69, 70 overview 64, 65 bronchodilator effects on mast cell release 146 endothelial cell effects 66 leukocyte effects 66, 67 pharmacologic studies human asthma 67, 68 mouse models allergen-induced pulmonary inflammation 68 dendritic cell maturation and migration 69 microvascular responses 68 pulmonary fibrosis 69 receptors 65, 66 smooth muscle cell effects 66 therapeutic targeting 72, 73 Mast cell knock-in mice asthma model studies 188, 189 immediate hypersensitivity reaction studies anaphylaxis or local immediate hypersensitivity reactions in actively immunized mice 186, 187

Subject Index

immunoglobulin-E-dependent local or systemic reactions 184, 185 Kit knockout mice for mast cell deficiency rescue by cultured mast cells 182 types 181, 182 mast cell grafts generation 182, 184 phenotypes and characterization 182–184 prospects for study 192, 193 skin allergy studies atopic dermatitis 191, 192 contact hypersensitivity 189–191 delayed hypersensitivity 189, 190 Mitogen-activated protein kinase (MAPK), glucocorticoid resistance role in asthma 155, 156 Mucosal mast cell (MMC), phenotype 1, 2 NADPH oxidase/dual oxidase, reactive oxygen species generation in non-phagocytic cells 33–35 Nasal mast cell (NMC) adhesion molecules and extracellular matrix proteins 115 cytokine production 112–114 IgE-Fc␧RI cascade in allergic airway disease 114 structural cell interactions 115, 116 Nasal polyps, mast cell role 121–123 Nerve growth factor (NGF), mast cell development role 9 NOD/SCID mouse, mast cell grafting 15, 16 NTAL, mast cell activation role 51, 52 Oxidative stress, see Reactive oxygen species Penicillin V, murine anaphylaxis model 186 PH domains, inducible protein–protein interactions 46 Plasmin activator inhibitor (PAI-1) airway remodeling role 92–95 extracellular matrix accumulation mechanisms 90, 91 functional overview 86

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Plasmin activator inhibitor (PAI-1) (continued) mast cell expression gene expression characterization 89, 90 regulation of expression 92 Potassium channels, mast cell expression and function 164, 165, 167, 168 Prostaglandin D2 (PGD2) allergen-induced pulmonary inflammation studies in mice 63, 64 allergic response 62 biosynthesis cytokine regulation 70, 71 heterogeneity of eicosanoid generation by tissue mast cell subsets 69, 70 overview 60, 61 bronchodilator effects on mast cell release 146 leukocyte effects 62, 63 receptors 62 smooth muscle effects 63 therapeutic targeting 72, 73 PTB domains, inducible protein–protein interactions 46 P2X receptors, mast cell expression and function 175 Reactive oxygen species (ROS) mast cell activation allergy role 37–39 Fc␧RI signaling and oxidant generation 35–37 NADPH oxidase/dual oxidase generation in non-phagocytic cells 33–35 Retinoids, mast cell development role 11–13, 15 Salbutamol, mast cell stabilization 147 Salmeterol, mast cell stabilization 147 SH2 domains, inducible protein–protein interactions 46 SH3 domains, constitutive protein–protein interactions 44, 45 SLP-76, mast cell activation role 53 Sodium channels, mast cell expression and function 173

Subject Index

Stem cell factor (SCF) cardiomyopathy role 5 cofactors in mast cell development 5–13 mast cell differentiation and proliferation in culture 3, 5 STI571-induced apoptosis role 5, 6 suppression by glucocorticoids in asthma 157–159 STI571, apoptosis induction in cultured mast cells 5, 6 T cells leukotriene effects 66, 67 prostaglandin D2 effects 62, 63 Th2 response in allergy 179 Terbutaline, mast cell stabilization 147 Thrombopoietin (TPO), mast cell development role 6, 15 Tolerance, induction by ␤2-adrenoceptor agonists 148–150 Toll-like receptors (TLRs) activated mast cell expression 204 chronic rhinosinusitis mast cell expression 123 cultured mast cell expression 119 mast cell cytokine expression mediation 103–106 Transcriptome, mast cells activated versus resting cells 203, 204 approaches for analysis 198, 199 comparison with other immune cells 199–203 computational modeling of human cells 209 cultured cells from different tissues 205–207 drug discovery opportunities 202, 203 glucocorticoid sensitivity studies 204, 205 human versus mouse cells 207, 208 Transgenic mice, see Mast cell knock-in mice Vav, mast cell activation role 53, 54

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