Current Topics in Membranes and Transport, Volume 35: Mechanisms of Leukocyte Activation

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Current Topics in Membranes and Transport -~

VOLUME 35

Mechanisms of Leukocyte Activation

This Page Intentionally Left Blank

Current Topics in Membranes and Transport Edited by Arnost Kleinzeller D~~partment of Physiology University of Pennsylvania School of Medicine Philadelphia, Pennsylvania

VOLUME 35

Mechanisms of Leukocyte Activation Guest Editors Sergio Grinstein

Ori D. Rotstein

Divi.\ion of Cell Biology The Hospitul t o r Sick Children Toronto, Ontario, Cunudu

Depurtment of Surge? Univer.\rty oj Toronto Toronto, Onfurio. Canada

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ACADEMIC PRESS, INC Harcourt Brace Jovanovich, Publishers

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1

Contents

Contributors, xi Preface, xv Yale Membrane Transport Processes Volumes, xvii Chapter 1. Genomic Organization and Polymorphism of the T Cell Receptor AGNES CHAN AND TAK W. MAK

I . Introduction, 1 11. Genomic Organization of the T Cell Receptor Genes, 2 111. Conclusion, 12 References, I3

Chapter 2. Chemoattractant Receptors as Regulators of Phagocytic Cell Function MARILYN C. PIKE

I. Physiology of Phagocytic Cell Function Mediated by Chemoattractants, 19 11. Chemoattractant Receptor Quantification, 21 111. Characterization of Biologically Relevant Chemoattractant Receptors, 23 References. 36

Chapter 3. Involvement of GTP-Binding Proteins in T- and 8-Lymphocyte Activation Signaling JOHN G. MONROE 1. Introduction, 45 i i . Involvement of G Proteins during Lymphocyte Activation, 50 HI. Future Perspectives, 57 References, 59

vi

CONTENTS

Chapter 4. Signal Transduction by GTP Binding Proteins during Leukocyte Activation: Phagocytic Cells GARY M. BOKOCH

I. Introduction. 65 GTP Binding Rcgdatory Proteins. 66 GTP Binding Proteins as Mediators of Neutrophil Activation, 74 The G I P Binding Protein Cornpositton of the Neurrophil. X I Mechanisms for Regulation of Signal Transduction in thc Ncutrophil, 87 Concluzions. 91 References, 92

11. 111. IV. V. V1.

Chapter 5. Monovalent Ion Transport and Membrane Potential Changes during Leukocyte Activation: Lymphocytes BRUCE SELIGMANN

I Introduction, 104 11 Mcmbianc Potential Change$, 105 111 pH Change\, 108 IV NnlK-ATPace of I yniphocytec, I10 V Anion Channels, 1 I2 VI Cation Channel$, I17

V11 Conclusion, 118 References, 12 I

Chapter 6. Monovalent Ion Transport and Membrane Potential Changes during Activation in Phagocytic Leukocytes ELAINE K . GAILIN AND I.ESI.IE C MCKINNEY I. 11. Ill. IV. V.

Introduction, 127 Ionic Basis of' the Resling Mcrnbranc Potential, 128 Ionic Channels, Pumps, and Carriers, 133 Rolc of Membrane Potential and Ionic Conductancca in Phagocytc Function, 140 Summary, 147 Relerences. 148

Chapter 7. Cytosolic Calcium Changes during T- and B-Lymphocyte Activation: Biological Consequences and Significance ERWIN W. GELFAND

I . Introduction. 153 11. Measurement of Cellular Ca? + Content and [CaL I,, 154 111 Cytosolic Calcium Changes in Activated T 1.ymphocytes. 155 IV. Cytosolic Calcium Changes in Activated B Lymphocytes, 165 V. Summary. 170 References. 171 +

vii

CONTENTS

Chapter 8. Cytoplasmic Calcium in Phagocyte Activation FRANCESCO D1 VIRGILIO, OLLE STENDAHL, DIDIER PITTET, P. DANIEL LEW, AND TULLIO POZZAN 1. 11. 111. IV. V. V1.

Introduction. 180 Measuring and Manipulation (CaZ+], in Intact Phagocytes, 180 Basic [Ca2+], Homeostatic Mechanisms in Phagocytes. 184 The Rise and Fall of [CaZ+1,. Changes in [Ca2' 1, and Phagocyte Activation, 187 The Role of Ca2+ as a Second Messenger Is Questioned in Phagocytic Cells, 193 Conclusions: Ca2+ May Not Be All, After All, 197 References, 198

Chapter 9. Role of Intracellular pH in Lymphocyte Activation THOMAS H. STANTON AND KAROL BOMSZTYK

I. Introduction, 207 Intracellular pH Changes in Nonlyrnphoid Cclls, 208 Intracellular pH Changes in T Lymphocytes, 210 N a + / H + Antiport Activity and pH, in B Cell Differentiation, 216 Conclusion, 222 References. 223

11. 111. IV. V.

Chapter 10. Regulation and Functional Significance of Cytoplasmic pH in Phagocytic Leukocytes CAROL J. SWALLOW, SERGIO GRINSTEIN, AND OR1 D. ROTSTEIN

I . Mechanisms of Cytosolic pH Regulation in Phagocytic Leukocytes, 227 11. Role of Cytoplasmic pH in Phagocytic Cell Activation and Modulation of Function, 234 111. pH, Changes during Phagocytic Cell Differentiation: Regulation and Significance. 239 References, 243

Chapter 11. Phosphoinositide Metabolism in Lymphocyte Activation ROBIN HESKETH, J. C. METCALFE, S. R. PENNINCTON, AND LOUISE R. HOWE I . Introduction, 250 11. T Cclls, 253 111. T Cell Proliferation, 257

IV. T Cell Responses in Cellular Immunity, 279 V. B Cells, 282 VI. Summary, 287 References, 287

viii

CONTENTS

Chapter 12. Phosphoinositide Metabolism during Phagocytic Cell Activation ALEXIS E. TRAYNOR-KAPLAN

I. Introduction, 303 II. Phosphatidic Acid Metabolism, 305 Ill. Role of Enzynics in Phosphoinositide Metabolism in Neutrophil Activation, 310 IV. Conclusions, 324 References. 325

Chapter 13. The Role of Arachidonic Acid Metabolltes in Lymphocyte Activation and Function MARK L. JORDAN 1. Introduction, 333 11. Effects of Arachidonic Acid Metaholitcs on Lyrnphocytc Activation and Function. 335

111. Lymphocyte Synthesis of Arachidonic Acid Metabolites, 340 IV. The Effects of Lipoxygenase Inhibitors on Lymphocyte Activation and Function, 342 V. Conclusions, 345 References, 345

Chapter 14. Mechanisms Regulating the Production of Arachidonate Metabolites in Mononuclear Phagocytes RONALD J. UHING, MAITHEW S. COWLEN, AND DOLPH 0. ADAMS

I . Introduction, 340 11. Involvement of Eicosanoid Production in Host-Defense Mechanisms, 350

111. Biochcniical Mechanisms Involved in Eicosanoid Production. 353 IV. Potential Transductioiial Mechanisins Involved in the Stimulation of Macrophage Eicosanoid Production, 358 V. Platelet Activating Factor as an Autocrine Component of Eicosanoid Production, 362 V1. The Relationship of Eicosanoid Production to Macrophage Development, 364 VII. Summary, 367 Kcfcrences. 367

Chapter 15. Role of Cyclic Nucleotides in Lymphocyte Activation VOLKHARD KAEVER AND KLAUS RESCH 1. Introduction. 375 I I . Cyclic Nucleotides as Potential Activation Signals, 377 Ill. Modulatory Ktfccts of Cyclic Nucleotides in Lymphocyte Activation, 385 IV. Interrelation of Cyclic Nucleotides with Other Signal Transduction Pathways: Possible Mechanisms of Cross-Talk, 389 V. Conclusions, 390 References. 39 I

ix

CONTENTS

Chapter 16. Alterations in Cyclic Nucleotides and the Activation of Neutrophils JOAN REIBMAN, KATHLEEN HAINES, AND GERALD WEISSMANN 1. Introduction, 399 11. Receptor-Mediated Activation of Adenylate Cyclase in Neutrophils, 400

Ill. IV. V. VI. VII. VIII.

Cyclic AMP in Activated Neutrophils, 406 Effect of Elevated CAMP o n Neutrophil Responses, 408 Effect of CAMP on lntracellular Signals, 412 Altered Cyclic Nucleotide Responses in Disease, 416 Cyclic GMP in Neutrophils, 417 Conclusion, 419 References, 4 I9

Chapter 17. induction of Protein Phosphorylation during Leukocyte Activation WILLIAM I,. FARRAR, DOUGLAS K . FERRIS, DENNIS F. MICHIEL, AND DIANA LlNNEKlN

I . Introduction, 425 Protein Serine/Threonine Kinases, 426 Protein Tyrosine Kmases, 430 Lymphoid Cells and Phosphorylation, 432 Myeloid Cell Activation, 442 Summary and Perspectives, 455 References, 4.58

11. 111. IV. V. V1.

Chapter 18. The Role of Phosphorylation in Phagocyte Activation ALFRED 1. TAUBER, ANAND B . KARNAD, AND IRENE GINIS

I. Introduction, 469 11. Neutrophil Activation by PK-C, 471

Ill. Phosphorylation Studies, 477 IV. PK-C Activation of NADPH-Oxidase, 483 V. Conclusion, 48.5 References, 486

Chapter 19. Activation of Lymphocytes by Lymphokines GORDON B . MILLS 1. Introduction, 496 11. Lymphokines Are Nonspecific in Activity yet Function within a Specific Immune System, 496 Ill. Pleiomorphic Function of Lymphokines. 497 IV. Protein Structure, 498 V. Gene Structure, 498 VI. Lymphokine Receptors, 500

CONTENTS

X

VII. Lymphokines as Initiation and Progression Factors, 50.5 VIII. Cell Activation, 506 IX. Trdnsmembranc Signaling by Lymphokines, SOX X . Cross-Talk hetween Antigen and Lymphokine Receptors, 52 I XI. Suiiuiiary, 523 References, 524

Chapter 20. Role of Cytokines in Leukocyte Activation: Phagocytic Cells MICHAEL A. WEST 1. Introduction, 538 11. Phagocyte Activation. 538

111. IV. V. VI.

Major Cytokines. 541 Role of Cytokines in Neutrophil Activalion, 545 Rolc of Cytokines in Macrophagc-Monocytc Activation, 549 Summary: Cytokine Efl'ects on Activation of Phagocytic Cells, 561 Rcfcrcnccs. 561

Chapter 21. Protooncogene Expression following Lymphocyte Activation ROGER M. PERLMUTTER AND STEVEN F. ZIECLER

I. Introduction, 571 Altcrations in Oncogene Expression in Stimulated T Lymphocytes, 573 i\lterdtiOnS in Protooncogcnc Expression in Stimulated B Lymphocytes, 578 Lyrnphocytc Activation Modulates Expression of the Ick Gene. 579 Future Directions: The Underlying Complexity of Lymphocyte Activation, 58 I Referenccs. S X 2

11. 111. IV. V.

Chapter 22. Early Gene Expression in the Activation of Mononuclear Phagocytes DOLPH O ADAMS. b I bWAKl P JOHNSON. AND KONALD J UHlNC I Introduction, 5x7 11 Eddy Genes, 589 111 Molcculdr Mcclianisins ot Maciophagc Acrivatioii, 5'90 Iv Eddy Genes in Mdcrophdgc Actlvdtlon. 5 % V Concluzions and Future Directions, 597 Refercnccs. 598

Index, 603

Contributors

Numbers in parenthcaes indicate the pages on which thc authors' contributions begin

Dolph 0. Adams, Departments of Microbiology, Immunology, and Pathology, Laboratory of Cell and Molecular Biology of Leukocytes, Duke University Medical Center, Durham, North Carolina 27710 (349, 587) Gary M. Bokoch, Department of Immunology, Research Institute of Scripps Clinic, La Jolla, California 92037 (65) Karol Bomsztyk, Division of Nephrology, Department of Medicine, University of Washington, Seattle, Washington 98 195 (207) Agnes Chan, Departments of Medical Biophysics and Immunology, The Ontario Cancer Institute, University of Toronto, Toronto, Ontario, Canada M4X 1K9 ( 1 ) Matthew S. Cowlen, Department of Pathology, Laboratory of Cell and Molecular Biology of Leukocytes, Duke University Medical Center, Durham, North Carolina 277 10 (349) Francesco Di Virgilio, C.N.R. Center for the Study of the Physiology of Mitochondria, and Institute of General Pathology, 1-35131 Pddova, ltaly (179) William L. Farrar, Laboratory of Molecular Immunoregulation, Cytokine Mechanisms Section, National Cancer Institute-Frederick Cancer Research Facility, Frederick, Maryland 21701 (425) Douglas K. Ferris, Program Resources IRC., Frederick Cancer Research Facility, Frederick, Maryland 21701 (425)

Elaine K. Gallin, Department of Physiology, Armed Forces Radiobiology Research Institute, Bethesda, Maryland 208 14 ( I 27) xi

xi i

CONTRIBUTORS

Erwin W. Gelfand, Division of Basic Sciences, and The Raymond and Beverly Sacklcr Foundation Laboratory, Department of Pediatrics, National Jewish Center for Immunology and Respiratory Medicine, Denver, Colorado 80206 (153) Irene Ginis, Departments of Medicine and Pathology, Boston University School of Medicine, Boston, Massachusetts, 021 18 (469) Sergio Grinstein, Division of Cell Biology, The Hospital for Sick Children, Toronto, Ontario, Canada (227) Kathleen Haines, Departments of Medicine and Pediatrics, New York University Medical Center, New York, New York 10016 (399) Robin Hesketh, Department of Biochemistry, University of Cambridge, Cambridge CB2 IQW, England (249) Louise R. Howe, Institute of Cancer Research, Chester Beatty Laboratories, London SW3 6JI3, England (249) Stewart P. Johnson, Department of Pathology, Laboratory of Cell and Molecular Biology of Leukocytes, Duke University Medical Center, Durham, North Carolina 27710 (587) Mark L. Jordan, Division of Urology, School of Medicine, Univcrsity of Pittsburgh, Pittsburgh, Pennsylvania 15213 (333) Volkhard Kaever, Division of Molecular Pharmacology, Department of Pharmacology and Toxicology, Medical School 1Iannover, D-3000 Hannover 61, Federal Republic of Germany (375) Anand B. Karnad, Department of Hematology/Oncology, East Tennessee State University, Johnson City, Tennessee 376 14 (469) P. Daniel Lew, Division of Infectious Diseases, University of Geneva, CH-121 1 Geneva, Switzerland (179) Diana Linnekin, Laboratory of Molecular Immunorcgulation, Cytokine Mechanisms Section, National Cancer Institute-Frederick Cancer Research Facility, Frederick, Maryland 21701 (425) Tak W. Mak, Departments of Medical Biophysics and Immunology, The Ontario Cancer Institute, University of Toronto, Toronto, Ontario, Canada M4X IK9 (1)

Leslie C. McKinney, Department of Physiology, Armed Forces Radiobiology Research Institute, Bethesda, Maryland 208 14 (127)

CONTRIBUTORS

xiii

J. C. Metcalfe, Department of Biochemistry, University of Cambridge, Cambridge CB2 IQW, England (249) Dennis F. Michiel, Laboratory of Molecular Immunoregulation, Cytokine Mechanisms Section, National Cancer Institute-Frederick Cancer Research Facility, Frederick, Maryland 2 1701 (425) Gordon B. Mills, Oncology Research, Toronto General Hospital, Toronto, Ontario M5G 2C4, Canada (495) John G. Monroe, Department of Pathology and Laboratory Medicine, University of Pennsylvania School of Medicine, Philadelphia. Pennsylvania 19 104 (45) S. R. Pennington, Department of Human Anatomy and Cell Biology, University of Liverpool, Liverpool L69 3BX, England (249) Roger M. Perlmutter, Howard Hughes Medical Institute, and Departments of Immunology, Medicine, and Biochemistry, University of Washington, Seattle, Washington 98195 (571) Marilyn C. Pike, Arthritis Unit, Harvard Medical School, Massachusetts General Hospital, Boston, Massachusetts 021 14 (19) Didier Pittet, Division of Infectious Diseases, University of Geneva, CH- 12 I 1 Geneva, Switzerland (1 79) Tullio Pozzan, Institute of General Pathology, University of Ferrara, Ferrdra, Italy ( I 79) Joan Reibman, Departments of Medicine and Pulmonary Medicine, New York University Medical Center, New York, New York 10016 (399) Klaus Resch, Division of Molecular Pharmacology, Department of Pharmacology and Toxicology, Medical School Hannover, D-3000 Hannover 6 I , Federal Republic of Germany (375) Ori D. Rotstein, Department of Surgery, Toronto General Hospital, University of Toronto, Toronto, Ontario, Canada (227) Bruce Seligmann, InflammationiOsteoarthritis, Enzymology Research, CibaGeigy Pharmaceuticals Corporation, Summit, New Jersey 07901 (103) Thomas H. Stanton, Division of Nephrology, Department of Medicine, University of Washington, Seattle, Washington 98 195 (207) Olle Stendahl, Department of Medical Microbiology, University of Linkoping, S-581 85, Linkoping, Sweden (179)

XIV

CONTRIBUTORS

Carol J . Swallow, Departnicnt of Surgcry, Toronto General Hospital, University of Toronto, Toronto, Ontario, Canada (227) Alfred I. Tauber, Departments of Medicine and Pathology, Boston Univcrsity School of Medicinc, Boston, Massachusetts 02 I I8 (469) Alexis E. Traynor-Kaplan,' Department of Immunology, Research Institute of Scripps Clinic, La Jolla, California 92037 (303) Ronald J. Uhing, Dcpartmcnt of Pathology, Laboratory of Cell and Molecular Biology of Leukocytes, Duke University Medical Ccntcr, Durham, North Carolina 27710 (349, 587) Gerald Weissmann, Dcpartmcnt of Medicine, Division of Kheumatology, New York University Medical Center, New York, New York 10016 (399) Michael A. West, Department of Surgery, Hennepin County Medical Center, Minneapolis, Minncsota 55415 (537) Steven F. Ziegler,' Howard Hughes Medical Institute, and Departments of Immunology, Medicine, and Biochemistry, University of Washington, Seattle, Washington 98195 (571)

'Present address: Department of Mcdicine, UCSD Medical Center, University nf California. San Diego, San rhego, Calilurnia 92103. 2PPrcscnt address: Immunex Corporation, Scattle, WA 98101.

Over the past decade, our knowledge of the contribution of leukocytes to the maintenance of normal health and to the pathogenesis of disease has blossomed. One area of investigation which has received particular attention is the basic mechanisms underlying leukocyte activation. This volume reviews the processes of cell activation in lymphocytes and phagocytic cells. The contributors are actively engaged in basic research related to their topics, and have attempted to provide a “state-of-the-art” review of the field. We hope the reader finds this book both timely and topical.

SERGIO GRINSTEIN OR1 D. ROTSTEIN

xv

This Page Intentionally Left Blank

Yale Membrane Transport Processes Volumes

Emile L. Boulpaep (ed.). (1980). “Cellular Mechanisms of Renal Tubular Ion Transport”: Volume 13 of Current Topics in Membranes and Transport (F. Bronner and A. Kleinzeller, eds.). Academic Press, New York. William H. Miller (ed.). (1981). “Molecular Mechanisms of Photoreceptor Transduction”: Volume 15 of Current Topics in Membranes and Transport (F. Bronner and A. Kleinzeller, eds.). Academic Press, New York. Clifford L. Slayman (ed.). (1982). “Electrogenic Ion Pumps”: Volume 16 of Currenl Topics in Membranes and Transport (A. Kleinzeller and F. Bronner, eds.). Academic Press, New York. Joseph F. Hoffman and Bliss Forbush 111 (eds.). (1983). “Structure, Mechanism, and Function of the Na/K Pump”: Volume 19 of Current Topics in Membranes and Trunsport (F. Bronner and A. Kleinzeller, eds.). Academic Press, New York . James B. Wade and Simon A. Lewis (eds.). (1984). “Molecular Approaches to Epithelial Transport”: Volume 20 of Current Topics in Membranes and Transport (A. Kleinzeller and F. Bronner, eds.). Academic Press, New York. Edward A. Adelberg and Carolyn W. Slayman (eds.). (1985). “Genes and Membranes: Transport Proteins and Receptors”: Volume 23 of Current Topics in Membranes and Transport (F. Bronner and A. Kleinzeller, eds.). Academic Press, Orlando. Peter S. Aronson and Walter F. Boron (eds.). (1986). ‘“a+-H+ Exchange, Intracellular pH, and Cell Function”: Volume 26 of Current Topics in Membrunes and Transport (A. Kleinzeller and F. Bronner, eds.). Academic Press, Orlando. Gerhard Giebisch (ed. ). (1987). “Potassium Transport: Physiology and Pathophysiology ”: Volume 28 of Current Topics in Membranes and Transport (F. Bronner and A. Kleinzeller, eds.). Academic Press, Orlando. xvii

xviii

YALE MEMBRANE TRANSPORT PROCESSES VOLUMES

William S . Agnew, Toni Claudio. and Frederick J . Sigworth (eds.). (1988). “Molecular Biology of lonic Channels”: Volume 33 of Ciirrmt Topics in Membrcinrs cind Trunsport (J. F. Hoffman and G . Giebisch, eds.). Academic Press, San Diego. Stanley G. Schultz (ed.). (1989). “Cellular and Molecular Biology of Sodium Transport”: Volume 34 of Current Topics in Mcmbrunes and Trunsport (J. F. llol‘lrnan and G. Giebisch, cds.). Academic Press, San Diego. Toni Claudio (ed.) ( 1990). “Protein--Membrane Interactions”: Volume 36 of Current Topics in Membranes and Transport ( J . F. Hoffman and G. Giebisch, eds.). Academic Press, San Diego.

CLRRENT TOPICS IN MEMBRANES AND TRANSPORT, VOLUME 35

Chapter 7

Genomic Organization and Polymorphism of the T Cell Receptor AGNES CHAN AND TAK W. MAK Departments of Medical Biophysics and lmmunology The Ontario Cancer Institute University of Toronto Toronto, Ontario, Canadu M4X l K 9

1.

Introduction Genomic Organization of thc T Cell Receptor Genes A. The a Chain Genes B . The f3 Chain Genes C. The y Chain Genes D. The 8 Chain Genes 111. Conclusion References 11.

1.

INTRODUCTION

Two major cell types, the T and B lymphocytes, are responsible for the regulation of the immune system. The B lymphocytes recognize antigens by means of surface receptors known as the immunoglobulin molecules. These molecules are encoded by noncontiguous genes composed of variable (V), diversity (D), joining (J), and constant (C) segments. The availability of large numbers of V, D, and .I gene segments, frequent imprecise joining of the rearranged segments, and somatic mutation during B cell replication provide diversity to these immunoglobulin molecules (for review, see Tonegawa, 1987). Like immunoglobulins, the germline T cell receptor (TcR) genes are also composed of noncontiguous V, D (in p and 6 only), J, and C gene segments (Takihara et ul., 1988a,b; Toyonaga and Mak, 1987; Yanagi et al., 1984, 1985; Sim et al., 1984; Hedrick et al., 1984; Chien et al., 1984). During T cell 1

Copyright 0 19W hy Academic Press, Inc All nghls of reproduction in any form reserved

2

AGNES CHAN AND TAK W. MAK

ontogeny, thcsc V, D, and J (or VJ) gene segments rearrange to form a unique TcR gene encoding a functional variable domain, which is joined to the C region scqucnces by RNA splicing following transcription. This process of rcarrangement is similar to that observed in thc inimunoglobulin loci during B cell development. The usage of different V, D, and J segments to produce functional ct or p chains is presumably random, proceeding by positivc or negative selection of effective T cells. On thc othcr hand, depending on the developmental stages of anatomical locations, there is a preferential usage of the variable and constant y and 6 chain genes (Ito et al., 1989; Groh et al.. 1989; Asarnow et c i l . , 1988; 1988; Loh c/ ul., 1988; Goodman and Takihara et al., 1989a; Korman el d., Lefrancois, 1988; fleilig and Tonegawa, 1986). Likc immunoglobulins, the TcK are diversified by somatic recombination events, hut unlikc immunoglobulins, these receptor proteins have not been shown to undergo further diversification by somatic mutation. The protein structure of the two TcR heterodimers and their possible role in antigen recognition have been reviewed extensively (see Brenner t’t ul., 1988; Wilson et al., 1988; Caccia e / a / . , 1988a,b; Davis and Bjorkman, 1988; Toyonaga and Mak, 1987; Kroncnberg et al., 1986) and will not be discussed hcrc. Instead, we will summarize findings on the genomic organization of thc TcR genes. Polymorphisms of thc TcR ctf3 variable gene regions also will be discussed.

II. GENOMIC ORGANIZATION OF THE T CELL RECEPTOR GENES A. The a Chain Genes The complete a chain locus is locatcd on chromosome 14 in both man (Caccia et al., 198.5) and mouse (Dembic et al., 1985). The variable domain of the ct chain is encoded by only two gene segments (V and J ) (Yanagi et al., 198.5). The V gene segments are composed of two exons separated by an intron of 90 to 400 base pairs (bp). The first exon codes for the signal peptide, and the second exon codes for thc last five residues of the signal peptide and for the first 100 amino acids of the variable domain of thc a chain (Yoshikai et al., 198.5, 1987). Analysis of human V, genes from cDNA library derived from the peripheral lymphocytes suggested that there are about 40-50 V,, gcnc scgments in the ct chain locus (Yoshikai et al. , 1987). However, since the usage of these dinerent V, gene segments is not random, the preceding statistical estimatcs arc probably lower than the actual availablc number of V, gene segments. Based on nucleotide sequences and Southern blot hybridizations, these V, gene segments can be grouped into 22 subfamilies, half of which contain a single member on the

3

1. GENOMIC ORGANIZATION AND POLYMORPHISM OF TcR

classification basis of greater than 75% homology at the nucleotide level (Wilson et al., 1988; Klein et al., 1987). The number of variable region region gene segments in mouse is estimated to be about 100 (Arden et al., 1985; Chou et al., 1986). Based on Southern blot hybridization analysis, these V, gene segments can be grouped into 13-20 subfamilies. All of these subfamilies have multiple members, ranging from two to ten (Arden et al., 1985). The genomic organization of the human V,, gene segments was determined using pulsed field gel electrophoresis (Griesser el ul., 1988). Using 16 V, probes derived from cDNAs encoding the different subfamilies, the locations of these genes are mapped with respect to each other. The five rare restriction enzymes used are MIuI, Sun, SfiI, Clul and Xhol. Half of the V, gene subfamilies are found on a 230 kb SfiI fragment. The relative positions of each of these V,, gene segments are shown in Fig. 1. It is worth noting that at least two pairs of members of the V, subfamilies, V,13/V, I I and V,,4/V,,5, can be found together at different locations of the V, locus as far as 600 kb apart. This observation supports the view that gene duplication was a common mode of increasing the V, gene diversity during evolution. In mouse, deletion mapping and pulsed field gel electrophoresis have shown that members of the different V, subfamilies are interspersed across the V, locus with the V region occupying more than 100 kb (Wilson et al., 1988). The J, gene segments, located centromeric to the V, region, are dispersed

Mlu

CENTROMER

1

,

I

1

,

1

1

1

TELOMER

3,11.14

Va Families

1.2.7.2.8.2

6.14.16

'2.9

Mlu I

Sol I Cla I

Sfl Xho I

HIOOkb

FIG. I . Marco restriction map of the human TcR, chain locus. (After Griesser rt u / . , 1988.) Fragments obtained with each enzyme are illuatratcd to scale below the subregions with which hybridization has been demonstrated (arrows).

4

AGNES CHAN AND TAK W. MAK

over an area of at least 50 kb in man and 70 kb in mouse (Hayday et id., 198Sa; Winoto et af., 1985; Yoshikai et 01.. 1985). Each of the J, genes are separated on average by I kb (Hayday et u l . , 1985a), with the first J,, gene located about 4 kb upstream to the single constant region (C,) (Hayday et al., 1985a; Winoto et a l . , 1985). Unlike the V,, J, gene segment usage appears to be random. From the distribution of J, gene usage in ci chain cDNA clones, statistical calculation indicates that there may be more than 100 J, gene segments in the human ci chain locus (Barth et a / . , 1985; Toyonaga and Mak, 1987), although only 46 J,, gene segments have been identified by nucleotide sequence analysis so far (Yoshikai et a / . , 1985, 1986; Klein rt d.,1987). The J, gene segments are very highly variable at the nucleotide level, with an average nucleotide identity ranging !rom 40 to 60% (Klein et al., 1987). Hence, the J, gene segments have not been grouped into subfamilies. In the murine IY chain locus, 32 J, gene segments have been sequenced (Arden et d.,1985). Of those sequenced, most are encoded by distinct gene segments, hence the repertoire of J,, gene segments is much greater than 30. D-region coding sequences have not been identified in the a chain. However, examination of the sequences of cDNA clones reveals the presence of additional nucleotidcs at the junctional region not encoded in the gcrmline genome (Klein et u l . , 1987). These nuclcotides are thought to be the resujt of N-region diversification (Alt ~t ul., 1984; Klein ('1 d . , 1987). One constant region gene is found in the OL chain locus. The gene segment is composed of four exons. The first exon encodes the extracellular constant domain, and the second exon encodes the extracellular domain hingelike region. The third exon encodes the transmembrane and the cytoplasmic regions, while the fourth exon encodes only the 3' untranslated region (Hayday el rrl.. I985a). Kecombination signals, similar to those of the immunoglobulins, are also present in both human and mouse (Y chain loci. They are found flanking the ci chain segments, with long spacers 3' to the V, gcnes and shott ones 5' to the J,, (11.. gene segments (Hayday r t d . , 1985a; Winoto ct a / . , 1985; Yoshikai 1985). Restriction fragment length polymorphism (RFLP) of the human V,, gene scgments was defined by digesting genomic DNA from unrelated individuals with different restriction enzymes. Using 12 different V, probes, polymorphisms were associated with half of the subfamilies studied (Chan et al., 1989a,b). Most of the probe-enzyme combinations reveal one predominant hybridization pattern with only a few individuals displaying a second pattern, indicating that the degree of polymorphism of the human ci chain locus is limited (Table 1).RFI,P is also observed around thc C, gene region (Robinson and Kindt, 1987; Ball e t a / ., 1987; So et d., 1987). In the murine system, extcnsive polymorphisms were found in the V,, and J, gene segments i n most strains (Chou i't al., 1986; Arden ct ul., 1985). KFLP is c9t

1. GENOMIC ORGANIZATION AND

5

POLYMORPHISM OF TcR

TABLE I POLYMORPHISMS I N HUMAN V, GENE SEGMENT SUBFAMILIES~~,~ Subfamily

v, 1 v2 V,"3 v-5 V,"6 v,7 V-8 V,IO V,,I v,, I 2 v-13 V',16

'

EcoRI

BamHI

Hind111

ND

-

-

+

-

-

-

ND

+

~

-

ND -

-

-

-

-

+ +

-

-

-

-

-

-

+ -

-

-

-

-

-

+

-

-

Fnim Chan et a / . ( I 98Ya). t , Probeienzyme combinations that detected polymorphism. -, probeienzyme combinatlons that failed to detect polymorphism; ND, not determined. r'

1'

observed when genomic DNAs from inbred strains were digested with restriction enzymes and hybridized with different V, probes (Singer et al. , 1988; Arden et al., 1985). Detailed analysis of one V, subfamily showed the presence of immunoglobulin-like hypervariable regions within the coding sequences. The variability of these regions is mainly due to nucleotide substitutions (Chou et al., 1986). Interestingly, comparisons of patterns obtained from RFLP analysis with various autoimmune mice strains showed that they share a common pattern (Singer et al. , 1988), and this pattern is quite different from that of the wild-type strains.

B. The

p Chain Genes

The TcR, chain locus is located on chromosome 7 in the human (Caccia et al., 1984; Isobe et ul., 1985) and on chromosome 6 in the mouse (Caccia et al., 1984). The variable gene segments are composed of two exons. The first exon codes for the signal peptide, which is about 50 nucleotides long, and the second exon codes for the last five residues of the signal peptide and for the majority of the variable domain (Siu et a l . , 1984). Both cysteines involved in the intradomain disulfide-linkage are encoded by the V, gene segments. Nucleotide sequences and Southern blot hybridizations have been used to estimate that there are at least 20 V p chain gene segments in the mouse (Barth et ul., 1985; Behlke et ul., 1985) and more than 60 V, gene segments in the human (Concannon et

AGNES CHAN AND TAK W. MAK

6

d., 1987; Kimura et ul., 1986). Based on DNA sequence analysis and Southern blot hybridization studies, the rnurinc and human V, gene segments can be grouped into 17 and 20 subfamilies, respectively. Thirteen of the human V, subfamilies are found to consist of a single member each. In the mouse, there are 17 single-member families (Wilson et al.. 1988). By using pulse field electrophoresis, chromosomal walking, and cosinid and phage cloning, the locations of both the human and murinc V, gene segments have been determined (Fig. 2 ) (Wilson et ul., 1988; Lee et al., 1987; Lai et al., 1987, 1988; Lindsten et al., 1987; Chou el al., 1986). In human, the V, gene segments and the two D,-J,-C, clusters are found on a SfiI fragment spanning approximately 600 kb. All 40 V, gene segments identified are mapped to the 5' end of the two D,-J,-C, clusters (Lai et ul., 1988). In the mouse, 20 of the 22 known Vg gene segments are located on a 300 kb DNA fragment. All of the V, gene segments, except V,14, are located upstream of the C, regions. A functional murine T cell clone has been found to have the V,14 gene segment located 10 kb 3' to C,2 in a reverse transcriptional orientation (Malissen et u l . , 1986). Two three-member V, subfamilies, V,5/8, are found to be together at different locations in the murine V, locus (Wilson et al., 1988). Downstream to the V, genes are the diversity (D,) and joining (J,) gene segments. The cluster of D, gene segments, consisting of approximately 600 nucleotides, is 5' to the cluster of J, region sequences, which is 2 to 5 kb 5' to t 1984; Siu et ul,, 1984; Toyonaga er ( I / . , the constant (C,) regions (Clark c ~ ul., 1985). Two constant region genes are found in the p chain locus for both human and mouse. The coding sequences of the two Cp genes, C,1 and Cp2, are highly homologous with only six amino acid differences in humans and four amino acid differences in mice (l'oyonaga ct al., 1985; Caccia 6'1 Ul., I988a). The C, gene i s divided into four exons, with the first two exons encoding most of the extracellular constant domain. The third cxon encodes the majority of the transmembrane protein region, while the fourth exon encodes the cytoplasmic coding sequences and the 3' untranslatcd region (Caccia et ill., 1988a).

FIG. 2. Physical map of the TcR, chain locus in (A) human and in ( B ) mouse. (Aftcr Lai er d., d .19x7; ~ Lce er a / . . 1987; Malisscn er d..1986.)

1087, 1988; Wilson ri o l . , 1988; Chou et

Hurimntal arrows abovc each gcne segment indicate its transcriptional oricntation. Pscudo-VI, genes are marked with an asterisk.

7

1. GENOMIC ORGANIZATION AND POLYMORPHISM OF TcR

Recombinational signals, similar to those of the immunoglobulins, are also found flanking the V,, D,, and J, genes (Clark et al., 1984; Malissen et al., 1984; Kavaler et al., 1984; Siu et a / . , 1984). Polymorphisms of the human p chain genes have been documented by several laboratories (Concannon et al., 1987; Robinson and Kindt, 1985; Berliner et al., 1985). RFLP are found to associated with 12 of the 14 V, subfamilies studied. For most V, enzyme/probe combinations, one predominant hybridization pattern is observed with only a few individuals displaying a second pattern (Concannon et al., 1987) (Table 11). RFLP analysis of 20 different inbred strains of mice with a variety of V, probes also showed variation in restriction fragment length (Wilson et al., 1988; Behlke et al., 1986). Among those strains that showed variations are NZW, SJL, C57BR, C57L, and SWR. The TcRp locus of NZW mice was found to have undergone a deletion resulting in removal of an 88 kb DNA segment. This segment contains C,1, D,2, and the entire J,2 cluster (Kotzin et al., 1985). The other four strains have deleted half of the known V, gene segments (Behlke et ul., 1986).

C. The y Chain Genes The TcR, chain locus is located in chromosome 13 in the mouse (Kranz et al., 1985) and on the short arm of chromosome 7 in humans (Murre ef al., 1985). The organization of the murine y gene locus is quite distinct from either the a or p chains (Fig. 3). Analysis of the genomic organization of the y chain genes in TABLE 11 POLYMORPHISMS I N HUMANV, GENESEGMENT SUBFAMILIES~,~'

+ + + -

+ + -

From Concannon el a / . (1987). + , Prohelzn/ymc comhinations that detect polymorphism; probelenryme combinations that failed t o detect polymorphism. 0

~,

8

AGNES CHAN AND TAK W. MAK

A

-

I 1.1

#m - Mm+JI

Jy C y l

VY

1.2 1.3 1.4 1.5 1.5 1.6 1.7 1.E

JIJI9

A

2

3 B

I

4

Jy

Cy2

- r - - ~ n n l.l,I.Z,l.3

CYS

2.1,2.3

JI

-65kb -Okb--335kb+

8

t l G . .\ . Schematic diagram of the gcnomic (A) human and ( B ) murinc y chain locus. (Aftcr Brenncr rt a / . . 1988.) (A) Nomenclature oT the linman y chain locus is according to Qucrtermous et trl. (1987) and Strauss ef al. (1987). There are four !,ubfamilies of V, gene scgments. The largcst subfamily is V,I which consists of ninc closely related nicmbcrs; however, four of them are pscudtrgencs as indicated with ii 9 ( R ) Nomcnclaturc of the murinc y chain locus is according to Garman rf ul. (1986). Parentheses surrounding V,I .3-J3-CY3 indicate that thc orientation of this cluster relative to other clusters is unknown.

BALBic and B10 rnicc reveals threc cross-hybridizing constant regions (C,l, C,2, C,3) and a unique C,4 constant region (Born et a/., 1986; Hayday ct d., 1985b; lwamoto rt ul., 1986; Saito c't al., 1984). Each C, segment is composed of three exons. Thc first exon encodes the extracellular constant domain, and the second exon encodes the connector peptidc, while the third exon encodes thc transmembrane cytoplasmic sequences (Hayday et (11.. 1985b; Garman et ul., 1986). Although the C,I , C,2, and C,3 genc segments are highly homologous, there arc important difkrences betwcen them. In BALB/c mice, C,1, has one potential N-glycosylation site, while C,2 has none, but both code for functional proteins, and C,3 is a psuedogene. Each of the C, genes, except C,1, is associated with its own V and J gene segments. C,I has been found to associate with four V segments and one J segment (Garman rt u / , , 1986; Heilig and Tonegawa, 1986; Trauneckcr et u l . , 1986; Pelkonen c:t ul.. 1987). Similar to the a and f3 chain gcnes, the murinc V, gene segmcnts are also composed of two exons. The first exon codes for the signal peptide, and the sccond exon codes for the majority of the variable domain of the y chain (Hayday et d . , 1985b). Using the nomenclature of Garman et ui. (19861, five V, subfamilics have been defined. Four of the subfamilies contain only one member each, with the exception of V,1 which consists of three members. Members within the V,I subfamily displayed up to 88% homology at the amino acid level (Hayday el a/., 198%). The usage of V, gcnes in the adult thymic population is nonrandom, with most cells expressing the product of a single rearranged gene, V,ZJ,lC,I (Pardoll et ul.,1987). On the other hand, V,3 is preferentially used in fetal thymocytes (Ito et al., 1989; lieilig

1. GENOMIC ORGANIZATION AND POLYMORPHISM OF TcR

9

and Tonegawa, 1986). There are four J regions in the murine y locus, each associated with its own C region. At the amino acid level, J,I and J,2 are identical, whereas J,4 differs from them at 50% of the residues (Hayday et al., 1985b: Iwamoto et al., 1986). The genomic organization of the human y genes is similar to that of murine and human p chain genes. The y locus spans roughly 160 kb (Strauss et ul., 1987), consisting of at least 14 V segments, 5 J segments, and 2 C segments (LeFranc and Rabbits, 1985; Murre et al., 1985; Quertermous et al., 1987; LeFranc et al., 1986a; Dialynas et al., 1986; Forster et al., 1987; Huck and LeFranc, 1987; Huck et al., 1988) (Fig. 3). The V, gene segments can be divided into four subfamilies (LeFranc et al., 1986~;Yoshikai et ul., 1987). Most of the subfamilies consist of one member each. The largest subfamily is V,I which consists of nine closely related members; however, only five are able to encode functional peptides (Strauss et al., 1987; Forster et al., 1987). Comparisons among V sequences reveal 76-9 1 % homology at the amino acid level among members of this subfamily (LeFranc et al., 1986c; Brenner et al., 1988). Homology between subfamilies, however, is only about 20-40% (LeFranc et al., 1986a; Forster et a[., 1987; Huck et d., 1988). Like the TcR, chain, the V, segment is encoded in two exons, a hydrophobic leader segment and a variable region. At least five joining (J) segments have been identified in the human y locus (LeFranc et al., 1986a,b; Quertermous et al., 1986; Huck and LeFranc, 1987; Tighe et al., 1988). These segments can be divided into two groups, with J,1.1, J,1.2, and J,I .3 located upstream of C,1, and J,2.1 and J,2.3 located upstream of C,2. J,1.3 and J,2.3 are identical to each other at the amino acid level, differing by only a single nucleotide, whereas J,I . I and J,2.1 have roughly 70% homology with each other at the amino acid level. J,1.2 does not have a counterpart that associates with C,2; J,l.l, 1.2, and 1.3 share about 50% homology with each other at the amino acid level (Brenner et al., 1988). Two constant regions are found in the human y chain locus. Both C, genes have a structure similar to that of the C , and C, genes, with an extracellular constant domain followed by transmembrane and cytoplasmic portions. The human C,I gene has three exons (LeFranc et al., 1986~).The first exon codes for the immunoglobulin-like domain, the second exon codes for the connector peptide that includes a cysteine residue, and the third exon codes for the transmembrane and intracytoplasmic portions of the polypeptide. Both exons I and 111 are highly conserved between C,I and C,2, but exon I1 shows considerable differences. The most striking difference is that the C,2 gene has multiple copies of the second exon (Littman et d., 1987; Krangel et al., 1987; LeFranc et al., 1986a), but none of these code for the cysteine residue which is thought to be important for interchain disulfide linkage (Krangel et al., 1987). The human TcR, chain locus is found to be polymorphic (Li et ul., 1988; Forster et al.. 1987; LeFranc et ul., 1986b). RFLP has been observed in the

10

AGNES CHAN AND TAK W. MAK

region flanking the V,2 segment (LeFranc et a / . , 1986a) as well as in the C,2 region (Li P G ul.. 1988). D. The 6 Chain Genes The 6 chain genes are embedded in the a chain locus, with the diversity (D,), joining (J,), and constant (C,) regions in between the V, and J, gene segments (Fig. 4). This unusual location results in the deletion of the 6 locus upon rearrangement of V, and J, segments. In humans, there is only one C, region (Takihara et ul., 1988a; lsobe et ul., 1988), which consists of four exons, whose organization is very similar to that of the C, exons. This similarity in genomic organization suggests that C, and C, may have arisen from a gene duplication event. The first exon of the C, gene encodes most of the extracellular constant domain. The second exon encodes a hingelike region, and the third exon encodes the entire transmembrane and intracytoplasniic segment, whereas the fourth exon contains the 3' untranslated sequences (Takihara et al., 1988a,b). Three joining segments, J,1, J,2, and J,3, are found 12, 5.7, and 3.4 kb upstream of the first exon of C, (Takihara et al.. 1988a; Hata et al., 1989). Like the f3 chain locus, functional diversity (D) segments are present in the 6 locus. There are at least 23 D, regions (Loh et al., 1988; Takihara et al., 1988a). D,I and D,2, which can be productively translated in all three reading frames, are found 1 and 9.6 kb upstream of J,I (Takihara et a / . , 1988a,b). The three J, and the two to three D,

A

D8I

082 J8I

J62

J63 EXONl 2 3

4

V63

70kb from C a

B

FIG. 4. Gcrioriiic organization of the (A) human arid ( B ) rriurinc TcKp, chain locua. (A) The schematic diagram ofthe genomic human S chain locus is adapted from Takihara ct a/. (1989b). V, D, J, and C region gene seginents are shown and transcriptional orientations are indicated with open arrows. Boxes indicate the coding exons of different Vp,El8, J6. and C K ,rcspcctively. (B) Organization of the murine 8 locus is adapted from Chien et a/. (1987h) and Brenner e t a / . (1988, Fig. I I).

1. GENOMIC ORGANIZATION AND POLYMORPHISM OF TcR

11

gene segments are confined within a 40 kb region upstream of the human C, locus. There appears to be only a very limited number of V, gene segments, and they are exclusively associated with J, and C, gene sequences, although V, and V, gene segments are not segregated from each other and, in fact, are found to be interspersed within the locus (Satyanarayana et al., 1988; Hata et a / . , 1989). Six different V, subfamilies have been characterized so far (Takihara et al., 1989a,b; Hata eta/., 1989). The usage of these V,s is not random, with most of the cells using V,I and V,2 subfamilies (Takihara et al., 1989b; Loh et al., 1987). The majority of these subfamilies seems to consist of a single member each. V,1 is found 8.5 kb downstream of the V,13. I gene segment, and both V segments are in the same transcriptional orientation. Another V, gene segment, V,17.1, is located between V,1 and the D,, J,, C, region (Satyanarayana et al., 1988). V,3, on the other hand, is about 2-3 kb 3' of C, in an inverted transcriptional orientation to the D,, J,, C, region (Takihara et al., 198Ya; Hata et al., 1989). Analysis of a TcR, clone, KT041, showed that V,3 is linked to D,1, D,2, J,3, and C, and that this message encodes a potentially functional TcR, chain (Takihara et al., 1989b), suggesting that chromosomal inversion is also an important rearrangement mechanism in the TcR, chain locus. The V,s display very low levels of amino acid sequence identity with each other (Hata et al., 1989). Sequence comparisons of V,s to V,s also indicate that there is no significant homology between these V gene segments, except for the V,4 and V,6.1 subfamilies (Takihara er al., 1989a; Yoshikai ef al., 1986). These V gene segments are very similar to each other with only 11 nucleotide differences between them. The human TcR, locus conserves a 12/23 bp spacer paradigm in which J, possesses a 12 bp and V, a 23 bp spacer, while the D, segments have a 12 bp-D,-23 bp spacer motif. In the murine system, the single copy C, gene is located 75 kb upstream of the C, gene (Chien et a / . , 1987a). There are at least two J, segments, J,1 and J,2, which lie 5' to the C, region. In addition, two D, elements, D,1 and D,2, are found 5' of the J segments (Chien et al., 1987a,b). The murine C, region, like those of the other TcR chain constant genes, is composed of four exons encoding an extracellular constant domain, a hingelike region, a transmembrane domain, and an intracytoplasmic segment. The two joining segments, J,1 and J,2, are found 13 and 5.6 kb upstream of the C, segment. J,l is highly homologous in consensus sequences to other TcR J regions, but J,2 shares very little similarity with J,1 or with any of the published J segments (Chien et al., 1987b). Six V, region gene sequences have been reported so far (Chien et al., 1987a,b; Elliott et al., 1988; Korman et al., 1988). Of these, two are quite similar to the V,,7 genes, whereas the others are only distinctly related in sequence to other known V gene sequences. V,5 is the most commonly used V, gene in adult thymic y/6 cells (Elliott et a / . , 1988; Korman et al., 1988; Ito et al., 1989), whereas V,I is preferentially expressed in early fetal thymocytes. The V,5 gene is located 2.5

12

AGNES CHAN AND TAK W. MAK

kb 3' of the constant (C,) region (Iwashima ct id.,1988; Korman et ul., 1989); thus it is in an inverted orientation to the J,-C, region. V,I, on the other hand, is located at least 40 kb 5' of the D,2 gene segment (Iwashima et al., 1988). IIeptamer and nonamer elements separated by 12 bp are found flanking the 5' ends of both the D and J segments, whereas these elements are separated by 23 bp at the 3' ends of the D segments. This organization allows the incorporation of more than one D segment into the rearranged TcR, genes. In fact, clones reflecting V-D,2-J81 and V-D,1-D,2-J8l joining events have been documented (Chien et al., 1987a,b). RFLP studies involving the J,2 region of the human 6 chain have been reported by Chuchana et d.(1989).

111.

CONCLUSION

The genes encoding the two forms of TcK, aP and y6, have now been cloned and largely characterized in both the human and murine systems. It has been established that they are distinct from the immunoglobulin genes, although their basic genomic organization and mechanisms of recombination are similar to those of the I3 cell antigen-recognition genes. The actual mechanism by which the TcR recognizes antigens and MIIC encoded molecules is still not known, but preliminary data have suggested that there is a strict conservation of an amino acid positioned in the p chain that is specific for antigen recognition (Fink et NI., 1986; Hedrick et ai., 1988; Winoto ct al., 1986). Also, a model has been proposed to explain the interaction between the TcR a p chains and antigens bound to MHC molecules (Davis and Bjorkman, 1988). Like the TcR a p chains, the y6 chains may also recognize antigen on the surface of other cells, although this has so far been reported only in one instance. The proliferative and cytotoxic responses expressed by this y6 cell line are toward an allogcnic target cell (Matis el d., 1987). Thus, further experiments are needed to determine whether y6 cells can also recognize foreign antigens in association with self-MHC encoded molecules. Recently, y6 hybridomas have been found to recognize conventional antigens, such as those expressed by Mycobucterium tuberculosis (O'Brien el al., 1989). The same hybridomas could be stimulated by using purified protein derivative (PPD) from the bacterium. The antigenic component of this PPD preparation is homologous to eukaryotic heat shock protein. RFLP studies, especially in the V, and V, regions, supply information which can be used to assess any role the TcR polymorphisms may have in the susceptibility to autoimmune diseases. Preliminary population studies have yielded both positive and negative results in looking for associations of TcR, and TcK, KFLPs with human autoimmune diseases such as systemic lupus erythematosus +

1. GENOMIC ORGANIZATION AND POLYMORPHISM OF TcR

13

(Dunckley et ul., 1988), insulin-dependent diabetes mellitus (Hoover and Capra, 19871, Graves disease (Oksenberg et al., 1989; Weetman et al., 1987), and rheumatoid arthritis (Sakkas et al., 1987). Further studies using families and a larger population sample may help clarify the discrepancy. REFERENCES Alt, F. W., Yancopoulos. G . D., Blackwell, T. K., Wood, C., Thomas, E . , Bos, M., Coffman, R . , Roscnberg, N., Tonegawa, S., and Baltimore, D. (1984). Ordered rearrangement of immunoglobulin heavy chain variable region segments. EMBO J . 3, 1209-1219. Arden, B . , Klotz, J. L., Siu, G . , and Hood, L. (1985). Diversity and structure of genes of the a family of mouse T cell antigen receptor. Nature (London) 317, 783-787. Asamow, D. M . , Kuziel, W. A . , Bonyhadi. M . , Tigelaar, R. E . , Tucker, P. W., and Allison, J. P. (1988). Limited diversity of y8 antigen receptor genes of Thy 1 dendritic epidermal cells. Cell 55, 837-847. Ball, E. J . , Dombrauxky, L., Hoover. M., Capra, J. D., and Strastny, P. (1987). Restriction fragment length polymorphisni of the human T cell receptor alpha gene. lmmunogenetics 26, 48-55. Barth, R., Kim, B., Lan, N . , Hunkapiller, T., Sobieck, N . , Winoto, A., Gershenfeld, H . , Okada. C . , Hamburg. D . , Weissman, I . , and Hood, L. (1985). The murine T cell receptor employs a limited repertoire of expressed VB gene segments. Nurrrre (London) 316, 5 17-523. Behlkc, M. A., Spinella, D. G., Chou, H. S., Sha, W., Hart, D. L., and Loh, D. Y. (1985). T cell receptor p chain expression:dependence on relatively few variable region genes. Science 229, 566-570. Behlke, M. A., Chou, H. S . , Huppi, K.. and Loh, D. Y. (1986). Murine T cell receptor mutants with deletions of @-chainvariable region genes. Immunology 83, 767-771. Berliner, N . , Duby, A. D., Morton, C. C.. Leder, P.. and Seidman, J. G. (1985). Detection of a frequent restriction fragment length polymorphism in the human T cell antigen receptor beta chain locus. J . Clin. Invest. 76, 1283-1285. Born, W., Rathbun, G., Tucker, P., Marrack, P., and Kappler, J. (1986). Synchronized rearrangement of T cell y and p chain genes in fetal thymocyte development. Science 214, 479-482. Brenner, M. B . , Strominger, J. L., and Krangel, M. (1988). The y6 T cell receptor. Immunology 43, 133- 192. Caccia, N., Kronenberg, M . , Saxe, D., Haars, R . , Bruns, G., Goverman, J., Malissen, M . , Willard, H., Simon, M., Hood, L., and Mak, T. W. (1984). The T cell receptor p chain genes are located on chromosome 6 in mice and chromosome 7 in humans. Cell 37, 1091-1099. Caccia, N., BNnS, G . A. P., Kirsch, I.R . , Hollis, G. R., Bertness, V., and Mak, T. W. (1985). T cell receptor cy chain genes are located on chromosome 14 at 14ql1-14q12 in humans. J . Exp. Med. 161, 1255- 1260. Caccia, N., Toyonaga, B., Kimura, N., and Mak, T. W. (1988a). The a and @ chains of the T cell. I n ”The T Cell Receptor” (T. W. Mak, ed.), pp. 9-51, Plenum, New York. Caccia, N., Takihara. Y., and Mak, T. W. (l988b). The y-8 heterodimer a second T cell receptor‘! In “The T Cell Receptor” (T. W. Mak, ed.). Plenum, New York. Chan, A., Du, R. P., Reis, M., Mcskc, L. M., Sheehy, M . , Baillie, E . , and Mdk, T. W. (1989a). Human T cell receptor V m gene polymorphism. Exp. Clin. Immunogenet. (in press). Chan, A., Du, R.-P., Reis. M . , Baillie, E.. Sheehy, M., and Mak, T. W. (1989b). Polymorphism of human T cell receptor alpha chain variable genes: identification of a highly polymorphism V gene probe. lnr. J . Immunoi. (in press). Chien, Y . , Becker, D., Lindsten, T., Okamurd, M . , Cohen, D., and Davis, M. (1984). A third type of murine T cell receptor gene. Nurure (London) 312, 31-35. Chien, Y. S., Iwashima, M., Kaplan, K. B., Elliott, J. F., and Davis, M. M. (1987a). A new T ccll +

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receptor gene located within thc alpha k u s and expressed early in T cell differentiation.Nutrtre (London) 321, 677-682. Chien. Y. S., Iwashia, M., Wettstein. D. A., Kaplan, K . B., Elliott, J. F., Born, W., and Davis, M. M. (l987b). T cell receptor S gene rcanangements in early thymocytes. Narure (London) 330, 722-727 Chou. H., Anderson, S . , Louie, M., Godambe, S., Pozzi, M., Behlkc, M . . Huppi, K., and Imh, D. (1987). Tandem linkagc and unusual RNA splicing of thc T cell receptor p chain variable rcgion genes. Proc. Null. Arad. Sci. U.S.A. 84, 1992. Chou, H. S . , Behlke, M. A . , Godambe, S . A . . Russell, J. H., Brooks, C. G., and Loh, D. Y. (1986). T cell receptor genes i n an alloreactivc CTI. clone: implications for rearrangement and germline diversity of variable gene scgnients. EMBO J . 5 , 2149-2155. Clark. S. P., Yoshikai, Y., Siu, G . , Tayler, S., Hood, L., and Mak, T. W. (1984). ldcntification of a divcrsity segment of the human T cell receptor beta chain, and comparison to the analogous murinc element. Nature (Lundon) 311, 387-389. Concannon, P., Gatti, R . A , . and Hood, L. E. (1987). Human T cell receptor V p gene polymorphism. J . Exp. M e d . 165. 1130-1140. Chuchana, P., Souz, Z . , Ghanem, N., Brackly, F., LeFranc, G., and LeFranc, M.-P. (10x9). Two Kpnl restriction fragment alleles of the human T cell rcceptor delta (TRD) joining segment J2. Nucl. Acid Res. 17, 1275. Davis, M. M., and Bjorkinan, P. J. (1Y88). T cell antigen receptor genes and T cell recognition. Nature (London) 334, 395-402 Bannworth, W., Taylor, B. A., and Steinmetz, M. (1985). The gene encoding the T cell Dembic, Z., receptor a cham maps cloac to the Np-l locus on mouse chromosome 14. Nuture (London) 314, 271-273. Dialynas, D. P., Murre, C., Quertermous, T., Boss. J. M., Leiden, J. M., Seidman, J. G . , and Strominger, J. L. ( 1986). Cloning and sequence analysis of complementary DNA encoding an aberrantly rearranged human T cell y chains. Proc. Nuti. Acad. Sci. U . S . A . 83, 2619-2623. Dunckley, H.. Gatenby. P. A , , and Serjeantson, S. W. (1988). T cell rcceptor and HLA class I1 RFLK in system lupus erythematosus. Irnmunogendics 27, 392-395. Elliott, J. F., Rock, E. P., Patter, P. A., Davis, M. M.. and Chien, Y. M. (1988). The adult T cell 6 chain is diverse and distinct from thc fetal thymocytes. Nolure (Lundon) 331, 627-631. Fink, P. J., Matis, L. A . , Sorger, S. B., and Hcdrick. S. M. (1986). The structure of the T cell receptor for antigen is correlated with T cell specificity. Year Immitnol. 3, 60. I-orster, A,, Huck, S . , Ghanem, N . , I.eFranc, M. P., and Rabbits, T. H. (1987). New subgroups in the human T cell rearranging V, gene locus. EMBO J . 6, 1945-1950. Garman, R. D., Doherty, P. J . , and Raulet, D. H. (1986). Diversity rearrangement, and exprcssion of murine T cell gamma genes. Cell 45, 733-742. Goodman, T., and Lefrancois. L. (1988). Exprcssion of the y-6 T cell receptor on intestinal CD8+ intraepithelial lymphocytes. Nulure ( h n d o n ) 333, 855-858. Griesser, H., Champagne, E . , Tkachuk, D.. Takihara, Y., Lalande, M., Baillie, E., Minden, M., and Mak, T. W. (1988). The human T cell receptor y-6 locus: a physical map of the variable, joining and constant region genes. Eur. J . Immunol. 18, 641-644. Groh, V., Porcclli, S . , Fabbi, M., I.anier, L. L., Picker, L. J., Anderson, T., wdrnkc, R . A , , Bhan, A. K . , Stromingcr, J. L., and Brenner, M. B. (1989). I-luman lymphocytes bearing T cell receptor y i 6 are phenotypically diverse and evenly distributed thoroughout the lymphoid system J . Exp. Med. 169, 1277-1294. Hata, S., Clabhy, M., Devlin, P., Spits, H., de Vries, J. E., and Krangel, MI. S . (1989). Diversity and organization of human T cell receptor 6 variable gene segments. .I. Exp. Med. 169, 41-57. Hayday, A . C., Diamond. D. J., Tmigawa, G . , Heilig, J. S . , Folsom, V., Saito, H., and Tonegawa, S. (1985a). Unusual organization and diversity of T cell reccptor a chain genes. Nature (London) 316, 828-832.

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Hayday, A. C., Saito, H., Gillies, S. D., Kranz, D. M., Tanigawa, G . , Eisen, H. M., and Tonegawa, S. (1985b). Structure, organization, and somatic rearrangement of T cell gamma genes. Cell 40, 259-269. Hedrick, S. M., Nielsen, E. A., Kavaler, J., Cohen, D. I., and Davis, M. M. (1984). Sequence relationships between putative T cell receptor polypeptides and immunoglobulins. Nature (London) 308, 153-158. Hedrick, S. M., Engel, I., McElligott, D. L., Fink, P. J., Hsu, M.-L., Hansburg, D., and Matis, L. A. (1988). Selection of amino acid sequences in the beta chain of the T cell antigen receptor. Science 239, 1541. Heilig, J. S . , and Tonegawa, S . (1986). Diversity of murine gamma genes and expression in fetal and adult T lymphocytes. Nature (London) 322, 836-840. Hoover, M. L., and Capra, J. D. (1987). HLA and T cell receptor genes in insulin-dependent diabetes mellitus. Diabetes Metab. Rev. 3, 835-863. Huck, S . , and LeFranc, M. P. (1987). Rearrangements to the JPI, JP and JP2 segments in the human T cell rearranging gamma gene (TcRr) locus. FEBS Lett. 224, 291-296. Huck, S . , Darivach, P., and Lefranc, M. P. (1988). Variable region genes in the human T cell rearranging gamma (TRG) locus: V-J junction and homology with the mouse genes. EMBO J . 7, 71 9-726. lsobe, M., Russo, G . , Haluska, F. G . , and Croce, C. M. (1988). Cloning of the gene encoding the S subunit of the human T cell receptor reveals its physical organization within the a-subunit locus and its involvement in chromosome translocations in T cell mallgnancy. Proc. Natl. Acad. Sci. U.S.A. 85, 3933-3937. Ito, K., Bonneville, M., Takagaki, Y . , Nakanishi, N., Kanagawa, O., Krecko, E., and Tonegawa, S . (1989). Different y6 T cell receptors are expressed on thymocytes at different stages of devclopment. Proc. Natl. Acad. Sci. U.S.A. 86, 631-635. Iwamoto, A , , Rupp, F., Ohashi, P. S . , Walker, C. L., Pircher, H., Joho, R., Hengartner, H . , and Mak, T. W. (1986). T cell specific y genes in C57B1110 mice. J. Exp. Med. 163, 1203-1212. Iwashima, M., Green, Davis, M. M., and Chien, Y. H. (1988). Variable region (V,) gene segment most frequently utilized in adult thymocytes is 3’ of the constant (C,) region. Proc. Natl. Acad. Sci. U.S.A. 85, 8161-8165. Kavaler, J . , Davis, M. M., and Chien, Y. J. (1984). Localization of a T cell receptor diversity region element. Nurirre (London) 310, 42 1-423. Kimura, M., Toyonaga, B., Yoshikai. Y., Triebel, F., Debre, P . , Minden, M., and Mak, T. W. (1986). Sequences and diversity of human T cell receptor p chain variable region genes. J . Exp. Med. 164, 739-750. Klein, M., Concannon, P., Everett, M., Kim, L., Hunkapiller, T., and Hood, L. (1987). Diversity and structure of human T cell receptor a chain variable region genes. Proc. Natl. Acad. Sci. U.S.A. 84, 6884-6888. Korman, A. J., Marusic-Galesic, S . , Spencer, D., Kruisbeek, A. M., and Raulet, D. H. (1988). Predominant variable region gene rearrange by y/S T cell receptor-bearing cells in the adult thymus. J. Exp. Med. 168, 1021-1040. Korman, A. J., Maruyama, J., and Raulet, D. H. (1989). Rearrangement by inversion of a T cell receptor 6 variable region gene located 3’ of the 6 constant region gene. Proc. Nut/. Acad. Sci. U.S.A. 86, 267-271. Kotzin, B. L., Ban, V. L., and Palmer, E. (1 985). A large deletion within the T cell receptor beta chain gene complex in New Zealand white mice. Science 229, 167-171. Krangel, M. S., Band, H., Hata, S., Mclean, J., and Brenner, M. B. (1987). Structurally divergent human T cell receptor y proteins encoded by distant C, genes. Science 237, 64-67. Kranz, D. M., Saito, H., Disteche, C. M., Swissheim, K., Pravtcheva, D., Ruddle, F. H., Eisen, H. N . , and Tonegawa, S. (1985). Chromosomal locations of the murine T cell receptor a chain gene and the T cell y gene. Science 227, 941-945.

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Kronenberg, M., Siu, G . , Hood, L. E., and Shastin, N. (1986). The molecular genetics of the T cell antigen receptor and T cell antigen recognition. Annic. Rev. Inimunol. 4, 529-591. I a i , E., Barth. R., and Hood, L. (1987). Genomic organization of the mouse T cell rcccptor p gene family. Proc. Nail. Acad. S r i . U.S.A. 84, 3846. Lai. E., Concannon, P., and Hood, L. (1988). Conserved organization of the human and murine T cell receptor p gene families. Narure (Loncfun)311, 543-546. 1.c~.N . . Linsten. T.. and Davis, M. (1987). Chromosomal organization of the murine T cell rcccptor p chain locus. J . Cell Bioc-hem. 110, 227. LeFranc, M. P.. and Rabbits, T. H. (1985). Two tandemly organized human genes encoding the 'I ccll y constant region sequences show multiple rearrangement s in diffwcnt T cell types. Nuricre (London) 316, 464-466. LcFraric, M. P., Furster, A , , Baer, R., Stinson, M. A , , and Rabbits, T. H. (1986a). Diversity and rearrangement of the human T cell rearranging y genes: Nine germline variahle genes belonging to two subgroups. Cell 45, 237-246. LeFranc, M. P., Forster, A . , and Rabbits, T. H. (1986b). Genetic polymorphism and exon changes of the conbtant rcgionh of the human T cell rearranging gene y. Pruc. Nurl. Acad. Sci. U.S.A. 83, 9.596-9600. LeFranc, M.-P., Forster, A , , and Rabbits, T. H. (1986~).Rearrangement of two distinct T cell y chain variable region genes in human D N A . Nururr (London) 319, 420. Li, Y., Szabo. P., and Posnett, D. N . (1988). Molecular genotypes of the human T cell receptor y chain. J . Immuriol. 140, 1300- 1303. L-indsten, T.. Fowlkes. B. J , Sandson, L. E., Davia, M. M., and Chien, Y. (1987). Transient rearrangements of the T cell antigen receptor a locus in early thymocytc. J. Exp. Mud. 166, 761-765. Littman, D. R . , Newton, M., Crommie, D., Ang. S . I.., Siedman, T. G . , Gettncr, S. M., and Weiss, A . (1987). Charactenzation of an expressed CD3 associated Ti y chain reveals C r domain polymorphism. Nutrrre (London) 326, 85-88. Imh, E. Y., Lanier, L. L., 'hrck, C W., Littman, D. R., Davis, M. M., Chien, Y. H., and Weiss. A . (1987). Identification and sequence of a fourth human T cell antigen receptor chain. Nurure (London) 330, 569-572. ].oh. E. Y.. Cwirla, S., Serafini, A . T., Phillips, J. H., and Lanier, L. L. (1988). Human T-cell receptor 6 chain: Genomic organization. diversity, and expression in populations of cclls. Proc. Nutl. Acocl. Sci. U . S . A . 85, 9714-9718. Maliasen, M., Mirard, K., Mjolsness. S . , Kronbenberg. M.. Goverman, J., Hunkapillcr, T., Prystowsky. M. R . . Fitch, F., Yoshikai, Y . . Mak, T. W., and Hood, L. E. (1984). Mouse T-cell antigen receptor: Structure and organization of constant and joining gene segments encoding thc p polypeptide. Cell 37, I 101. Malissen, M., McCoy, C., Blanc, D., Trucy, J . , Devaux, C., Schmitt-Verhulst. A . , Fitch, F., Hood, L . , and Malissen, R. (1986). Direct evidence for chromosomal inversion during T cell receptor p chain rearrangements. Nuture (London) 319, 28-33. Matis, L. A,, Cron. R., and Bluestone, J. A . (1987). Major histocompatihility complex-linked specificity of y8 receptor bearing T lymphocytes. Nuture (London) 330, 262-263. M u m , C., Waldman, R. A,, Morton. C. C.. Bongiovanir, K. G . , Waldmann, T. A . , Shows, T. B., and Seidman, J. G. (1985). Human y chain genes are rearranged in lcukcmic T cells and map to the short arm of chrornosome 7. Nuture (London) 316, 547-552. O'Brien, R. L., IIapp, M. P.. Dallas, A . , Palmer, E . , Kuho. R., and Born, W. K. (1989). Stiinulation of a nia.jor subset of lymphocytes expressing T cell receptor y8 by an antigen derived from Mycobucterium tuberculosis. Cell 57, 667-674. Oksenberg. J. R., Sherritt, M., Gegovich, A. B., Erlich, H. A.. Bernard, C. C., Cavalli-Sforta, L. L., and Steinman, L. (1989). T cell receptor V, and C, alleles associated with multiplc sclcrosis and myasthenia gravis. Proc. Nutl. Acud. Sci. U . S . A . 86, 988-992.

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Pardoll, D. M., Kruisbeek, A. M.. Fowlkes. B. J . , Coligan, J. E., and Schwartz, R. H. (1987). The unfolding story of T cell receptor y. FASEB J. I , 103-109. Pelkonen, J., Trannecker, A., and Karjalainer, K . (1987). A new mouse TcR V, gene that shows remarkable evolutionary conservation. EMBU J , 6 , 1941- 1944. Quertermous, T., Strauss, W., Murre. C., Dialynas, D. P., Strominger, J. L., and Seidman, J. G. (1986). Human T-cell y genes contain N segments and have marked junctional variability. Nature (London) 322, 184-187. Quertermous, T., Strauss, W. M., van Dongen, J. J. M., and Seidman, J. G. (1987). Human T cell y chain joining regions and T cell development. J. Immunol. 138, 2687-2690. Robinson, M. A.. and Kindt. T. J. (1985). Segregation of polymorphic T-cell receptor genes in human families. Proc. Nut/. Acad. Sci. U . S . A . 82, 3804-3808. Robinson, M. A., and Kindt, T. J. (1987). Genetic recombination within the human T-cell receptor a-chain gene complex. Proc. Natl. Acad. Sci. U . S . A . 84, 9089-9093. Saito, H . , Kranz, D. M., Takagaki. Y., Hayday, A. C., Eisen, H. N., and Tonegawa, S. ( 1984). Complete primary structure of a heterodimeric T-cell receptor deduced from cDNA sequences. Nature (London) 309, 757-763. Sakkas, L. I . , Demaine, A. G . , Walsh, K. J., and Panayi, G. S. (1987). Restriction fragment length polymorphism for the T cell receptor a and chain genes in rehematoid arthritis. Arthritis Rheum. 30, 232-233. Satyanarayana, K., Hata, S., Devlin, P., Roncarolo, M. G., De Vries, J. E., Spits, H., Strominger. J. L., and Krangel, M. S. (1988). Genomic organization of the human T-cell antigen-receptor a/6 locus. Proc. Nut/. Acad. Sci. U.S.A. 85, 8166-8170. Sim, G . , Yague, J., Nelson, J . , Marrack, P., Palmer, E., Augustin, A., and Kappler, J. (1984). Primary structure of human T cell receptor a chain. Nature (London) 312, 771-775. Singer, P. A., McEvilly, R. J.. Balderas, R. S . , Dixon, F. J . , and Theofilopoulos, A. N. (1988).Tcell receptor a-chain variable-region haplotypes of normal and autoimmune laboratory mouse strains. Proc Nut/. Acad. Sci. U . S . A . 85, 7729-7733. Siu, G., Kronenberg, M., Straus, E., Haars, R . , Mak, T. W., and Hood, L. (1984). The structure, rearrangement and expression of Dp gene segments of the murine T cell antigen receptor. Nature (London) 311, 344-350. So, D., Joh, S., Bailey, C . , and Owen, M. J. (1987). A new polymorphic marker of the T-cell antigen receptor a chain genes in man. Immunogenetics 25, 141-144. Strauss, W., Quertermmous, T., and Seidman, J. G. (1987). Measuring the human T cell receptor ychain locus. Science 237, 1217-1219. Takihara, Y., Tkachuk. D.. Michalopoulos, E., Champagne. E . , Reimann, J . , Minden, M., and Mak, T. W. (1988a). Sequence and organization of the diversity, joining. and constant region genes of the human T-cell &chain locus. Proc. Natl. Acad. Sri. U . S . A . 85, 6097-6101. Takihara, Y., Champagne, E., Griesser, H., Kimura, N . , Tkachuk, D., Reimann, J., Okada, A , , Ah, F. W., Chess. L., Minden, M., and Mak, T. W. (198Xb). Sequence and organization of the human T-cell 6 chain gene. Eur. J. Immunol. 18, 283-287. Takihara, Y., Reimann, J., Michaiopoulos, E.. Ciccone. E., Moretta, L., and Mak, T. W. (1989a). Diversity and structure of human T cell receptor S chain genes in peripheral blood yiS-bearing T lymphocytes. J . Exp. Med. 169, 393-405. Takihara, Y., Champagne, E . , Ciccone. E., Moretta, L., Minden, M., and Mak, T. W. (1989b). Organization and orientation of a human T cell receptor 8 chain V gene segment that suggcsts an inversion mechanism is utilized in its rearrangement. Eur. J. Immunol. (in press). Tighe, L., Forster. A., Clark, D., Boylston, A., Lavenir. I . , and Rabbitts, T. H. (1987). Unusual forms of T cell y mRNA in a human T cell leukemia cell 1ine:implications for the y gene expression. Eur. J . Immunol. 17, 1729-1736. Tonegawa, S. (1987). Somatic generation of immune diversity. Biosci. Rep. 8, 3-26.

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Toyonaga, B . , and Mak, T. W. (1987). Genes of‘the T cell antigen receptor in normal and rnaIignant T cells. Annu. Rev. Immunol. 5 , 585-620. Toyonaga, B., Yoshikai, Y., Vadasz, V., Chin, S., and Mak, T. W. (1985). Organization and scquences of the diversity, joining and constant region gencs of the human T cell receptor p chain. Proc. Nurl. Acad. Sci. U . S . A . 82, 8624-8628. Traunecker, A , , Oliveri, F.,Allen, N., and Karjahinen, K. (1986). Normal T cell development is 5, 1589-1602. possible without “functional” a chain genes. EMBO .I. Weetman, A. P., So, A. K., Koe, C.. Walport, M. J., and Foroni, L. (1987). T cell receptor a chain V rcgion polymorphism linked to primary autoinimune hypothyroidism but not Grave’s disease. Hum. Immunol. 20, 167- 173. Wilson, R . K . , Lai, E.,Concannon, P., Barth, R. K., and Hood, L. E. (1988). Structure, organization and polymorphism of murine and human T-cell receptor a and p chain gene families. Immunol. Rev. 1, 149-171. Winoto, A , , M.jolsners, S . , and Hood, L. (1985). Genomic organizntion of the genes encoding the mouse T cell receptor a chain. Nature (London)316, 832. Winoto, A , , Urban, J . , Lan, N., Covemian, J . , Hood, L., and Hamburg, D. (1986). Predominant use of the V, gene segment in mouse T cell receptors for cytochrorne C. Nature (London) 324, 679. Yanagi, Y., Yoshikai, Y., Legett, K . , Clark. S.,Aleksander, I., and Mak, T. W. (1984). A human T cell specific cDNA clone encodes a protein having extensive homology to immunoglobulin chains. Nuture (London) 308, 145. Yanagi, Y., Chan, A , . Chien, B., Minden, M., and Mak, T. W. (1985). Analysis of cDNA clones specific for hurhdn T cells and a and p chains of the T cell receptor heterodimer from a human T ccll lines. Proc. Natl. Acud. Sci. U . S . A . 82, 3430. Yoshikai, Y . , Clark, S . , Taylor, S . , Sohn, V., Wilson, R . , Minden, M . , and Mak, T. W. (1985). Organization and sequences of the variable, joining and constant region genes of the human T cell receptor a chain. Nature (London) 316, 837. Yoshikai, Y., Kimura, N., Toyonaga, B.. and Mak, T. W. (1986). Sequences and repertoire of human T cell receptor a chain variable region genes in mature T lymphocytes. J . Exp. Mrd. 164, 90103.

Yoshikai, Y., Toyanaga. B.. Koga, Y., Kimura, N., Griesser, H., and Mak, T. W. (1987). Repettoire of the human T cell garnma genes:high frequency of nonfunctional transcripts in thymus and rnaturc T cells. Bur. 1.Immunol. 17, 119-126.

CURRENT TOPICS IN MEMBRANtS A N D TRANSPORT. VOLUME. 35

Chapter 2 Chemoattractant Receptors as Regulators of Phagocytic Cell Function MARILYN C. PIKE Arthritis Unit Harvard Medicai School Massachusetts General Hospital Boston, Massachusetts 021 14 I. Physiology of Phagocytic Ccll Function Mediated by Chemoattractants A. The Biologically Relevant Chemoattractants 9. Chemoattractants as Cellular Activators 11. Chemoattractant Receptor Quantification A. Methodology B. Analysis of Chemoattractant Direct Binding Data 111. Characterization of Biologically Relevant Chemoattractant Receptors A. N-Formylated Oligopeptide Chemoattractant Receptors B. Chemoattractant Receptors for LTB4 C. Chemoattractant Receptors for C5a D. Interleukin I (IL-I) Receptors References

1. PHYSIOLOGY OF PHAGOCYTIC CELL FUNCTION MEDIATED BY CHEMOATTRACTANTS A. The Biologically Relevant Chemoattractants The localization of phagocytes to sites of intrusion by foreign materials was first described by Metchnikoff more than a century ago (Metchnikoff, 1891). Despite this, it was not until 1962 that in vitro methods were developed to quantitate this important function of inflammatory cells. Boyden (1962) is the first individual credited with developing an in vitro device for measuring the directed migration of inflammatory cells. Using what came to be known as the Boyden chamber, this investigator showed that incubation of serum with immune 19

Copynght 0 1990 by Academic Press, Inc All nghta of reproductinn in any form reserved

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complexes led to the formation of chemotactic activity. Subsequent studies showed that this activity was derived from activation of the complement cascade and was specifically identified by Snyderman and his colleagues as C5a, a cleavage product of the fifth component of complement (Shin et ul., 1968; Snyderman ('t a / . , 1968). C5a possesses not only chemodttractant activity but also potent anaphylatoxic activity. The molecule has been purified and sequenced and is now known to exert its biological activity through specific receptors on the cell surface of inflammatory cells (Chenoweth and Hugli, 1978; Hugli and Mullcr-Eberhard, 1978). Other biologically relevant chemoattractants are those derived from bacterial culture supernatants that were subsequently identified as N-formylated oligopcptides. Chemotactic activity associated with bacterial proteins was noted as early as 1967 (Keller and Sorkin, 1967; Ward et a!., 1968; lernpel et al., 1970); however the nature of these agents was not identified until 1975, at which time Schiffmann and co-workers (1975) made the important discovery that N-terminal formylated di- and tripeptides were chemotactic for neutrophils and macrophages. This finding was the single most important contribution to the subsequent identification of high affinity chcnioattractant reccptors on phagocytic cells. N-formylated peptides have been isolated from bacterial culture filtrates (Marasco et al., 1983) and are thought to represent a primitive recognition system of eukaryotic cells for bacteria. These blocked peptides have also bcen isolated from eukaryotic mitochondria1 preparations (Carp, 1982) and may be important for the accumulation of phagocytic cells at sites of necrotic tissues. Another biologically relevant class of chemoattractants is those derived from lipid metabolism in phagocytic cells. In 1975, Turner ri al. first noted that oxidized components of polyenoic fatty acids, such as arachidonic acid, are chemotactic for neutrophils. The most active lipid derived chemoattractant resulting from lipid oxidation has been identified as leukotriene B, (LTB,) whose chemical nature is S(S), 12(R)-dihydroxy-6,8-truns-10,14-cis-eicosatetraenoic acid (5,12-diHETE) (Ford-Hutchinson rt d., 1980; Goetzl and Pickett, 1980). This substance mediates the chemotactic response of leukocytes via a specific cell surface receptor distinct from those which bind C5a or the formylated oligopeptides (Goldman and Goetzl, 1982; Kreisle and Parker, 1983). Another biologically relevant chemoattractant is platelet factor 4 (PF4), a heparin-binding protein released from platelet a-granules (Deuel et ul., 198 1). It has not been determined whether this substance binds to specific leukocyte cell surface receptors. Another potential biologically important mediator of leukocyte chemotaxis and cellular activation is interleukin 1 (IL-1). Early in v i m studies using partially purified IL-1 showed that it was cheniotactic and induced activation of oxidative metabolism in neutrophils (Luger et a!., 1983; R. J. Smith et ( i l . , 1985). In addition, injection of purified I L 1 causes accumulation of neutrophils in vivo at r .

2. CHEMOATTRACTANT REGULATION OF PHAGOCYTES

21

the injection site (Beck et al.. 1983; Granstein et al., 1986). Later studies employing recombinant IL- 1 molecules show conflicting effects on polymorphonuclear leukocyte (PMN) function (Georgilis et al., 1987; Yoshimura et ul., 1987). The discovery of specific IL-1 binding sites on murine peritoneal and human peripheral blood PMN (Parker et al., 1989) suggests that it does in fact play a direct role in the regulation of neutrophil inflammatory functions. Other biologically relevant chemotactic factors have been described, including a lymphocyte derived chemotactic factor (LDCF) (Altman et al., 1975) and a crystal induced chemotactic factor (CCF) produced by PMN undergoing phagocytosis of urate crystals (Spilberg and Mehta, 1979; Spilberg et al., 1976, 1977). LDCF is 12,500 daltons and is distinct from C5a, but the difficulty in purifying this material from culture supernatants of purified lymphocytes has precluded the identification of its specific membrane receptors. Specific receptors for CCF have been described on the surface of human PMN (Spilberg and Mehta, 1979).

B. Chemoattractants as Cellular Activators It has become apparent that chemoattractants mediate not only the directed migration of leukocytes but also induce a series of coordinated biochemical events, including ion fluxes (Gallin and Rosenthal, 1974; Gallin and Gallin, 1977), cytoskeletal rearrangements associated with morphological polarization (Hoffstein et al., 1977; Stossel et al., 1977), changes in lipid metabolism (Pike et al., 1979; Hirata et al., 1979; C. D. Smith et al., 1985; Pike and DeMeester, 1988), activation of protein kinases (Pike et al., 1986; Nishihira et al., 1986), production of superoxide anion, and release of lysosomal enzymes (Goldstein et al., 1973; KlebanoR and Clark, 1978). Chemoattractant-mediated biological and biochemical responses can be divided into two major categories: (1) those functions and biochemical processes that are triggered by low concentrations of chemoattractants such as cellular migration, and (2) functions such as activation of the oxidative burst associated with 10- to 100-fold higher concentrations of chemotactic factors. This chapter will review the physiological and biochemical parameters of the most thoroughly described chemoattractant receptors whose physiology may provide a molecular basis for these complex functions of inflammatory cells.

II. CHEMOATTRACTANT RECEPTOR QUANTIFICATION

A. Methodology Advances have been made in the study of leukocyte chemoattractant receptors mainly because of the availability of large numbers of single cell suspensions of

22

MARILYN C. PIKE

leukocytes from either peripheral blood or peritoneal exudates as well as the availability of stable, high-affinity, highly purified, radioactive ligands. The criteria needed to establish that an observed binding site is in fact a receptor that mediates a particular biological response are as follows: (1) the radioligand must be chemically pure and physiologically active; (2) there should be a finite number of binding sites on a given cell; (3) the binding of the ligand should be saturable; (4) the concentration range over which the ligand occupies the receptors should be comparable to the concentration range over which it elicits a biological response; ( 5 ) the specificity and stereospecificity of agonists and antagonists for producing their biological activities should exactly parallel their interaction with specific receptor sites; and (6) the kinetics of binding of agonists and antagonists should reflect the kinetics of the chemotactic and secretory responses (Williams et ul. 1977; Williams and Lefkowitz, 1978). The most important consideration for accurately measuring chemoattractant receptors by direct binding studies is adequate separation and quantification of bound vs. free ligand under equilibrium conditions (Boeynaems and Dumont, 1975). Two separation methods have been employed to measure chemoattractant receptors: ( I ) filtration of ligand plus cell or membrane preparations through glass fiber filters using a vacuum filtration manifold (Williams and Letkowitz, 1978; Pike and Snyderman, 1988); or (2) centrifugation of ligand and receptor mixtures through an inert substance such as silicone oil, Ficoll-Hypaquc, or a mixture of n-butyl phthalate and dionyl phthalate (7:2,v/v) (Goldman and Goetzl, 1982; Kreisle and Parker, 1983). Real time analysis of the binding of fluoresceinated chernoattractants to intact cells can also be performed using a technique which monitors the quenching of free chemoattractant by an antibody to fluorescein (Sklar et al.. 1982; Sklar and Finney, 1982). This technique is particularly useful for studying the real time association of chemoattractant receptor occupancy with the kinetics of degranulation and superoxide production.

B. Analysis of Chemoattractant Direct Binding Data Classic Scatchard techniques or computer programs which utilize least-squares curve fitting analysis can be used to analyze direct binding data. Important assumption5 used in these techniques are that binding is at equilibrium and that there is no significant destruction of the ligand. The two most widely used computer programs employed for analysis of receptor binding data are SCTFIT (DeLean et al., 1982) and LIGAND (Munson and Kodbard, 1980), which have been adapted for use with personal computing systems. Rigorous statistical estimates of multiple receptor classes can be obtained with thcsc programs provided an appropriate sampling of data points is employed.

2. CHEMOATTRACTANT REGULATION OF PHAGOCYTES

23

111. CHARACTERIZATION OF BIOLOGICALLY RELEVANT CHEMOATTRACTANT RECEPTORS A. N-Formylated Oligopeptide Chemoattractant Receptors 1. INITIALDESCRIPTION A N D BIOCHEMICAL CHARACTERIZATION

Early studies by Schiffmann et al. ( 1 975) and Showell et al. (1 976) examining the strict structural specificity of a series of synthetic N-formylated peptides for producing chemotactic and secretory responses in rabbit peritoneal PMNs suggested that these agents may exert their effects through specific cell surface receptors. In 1977, Williams et al. (1977) and Aswanikumar et al. (1977) independently described the direct binding of synthetic, tritiated N-formylated peptides to human peripheral blood PMNs and rabbit peritoneal exudate PMNs, respectively. The ligand, N-formylmethionylleu~yl[~H]phenylalanine(FML[~H]P) was used by Williams et al. (1977) to define the presence of these receptors on human PMNs. These receptors have an average equilibrium disassociation constant (K,) of 10- 14 nM with 35,000-60,000 sites/cell. Initial studies performed at 37°C showed that binding was rapid with a t , l 2 of 2.5-3.0 minutes and was reversible by chemical dilution using 1000-fold excess of unlabeled peptide. Subsequent studies have shown that this peptide is extensively degraded by longer incubations at 37°C (Yuli and Snyderman, 1986), necessitating performance of binding assays at 24°C or 0°C. A series of formylated chemotactic peptides was used to study the stereospecificity of the binding site by demonstrating that there was an excellent correlation ( r = 0.99) between the concentrations of peptides that produced half-maximal inhibition of binding of FML[”H]P and half-maximal chemotactic responses for PMNs (Williams et at., 1977). These findings were the first to demonstrate the presence of a specific receptor for a chemoattractant on human PMNs. Subsequent studies by the same laboratory showed that this ligand could identify specific receptors for N-formylated peptides on guinea pig peritoneal macrophages (Snyderman and Fudman, 1980). Aswanikumar et al. (1977) developed a similar but more stable agonist, N formylnorleucylle~cyl[~H]phenylalanine (FNL[3H]P), to describe oligopeptide chemoattractant receptors on the surface of rabbit peritoneal PMNs. These cells possess more receptors/cell (100,000) which have a higher average affinity (K, = 1.5 nM) than do human peripheral blood PMNs. The differences in peripheral blood PMN and exudate PMN receptor number and affinity have subsequently been explained by translocation of an intracellular, preformed receptor pool localized in specific granules to the cell surface upon exposure of PMNs to low concentrations of chemoattractants and other cellular activators encountered during migration from the blood vessels to tissue inflammatory sites (Zimmerli et al., 1986).

24

MARILYN C.PIKE

Another agonist which has been useful for the characterization of N-formylated oligopeptide chemoattractant receptors is fNle-Leu-Phe-Nle-Tyr-Lys, which was developed for its ability to be iodinatcd at the Tyr residue and fluoresceinatcd via the Lys residue (Niedcl et al., 1979a,b). Using this peptide, Niedel rt al. (1979a,b) estimated that there were 100,000 receptors/human PMN that had an average affinity of 1 nM. 'Fable I summarizes thc characteristics of N-formylated oligopeptide receptors described on various cell types using different ligands. For a more complete description of initial studies describing oligopeptide chcmoattractant receptors, see Pike and Snyderman (1984). Several laboratories have attempted to biochemically characterize the oligopeptide chemoattrdctant receptor employing ligand cross-linking studies. Using a variety of cross-linking methods with the ligand tNle-Leu-Phe-Nle-['2sI]Tyr-Lys, Niedel et al. (1980) and Dolmatch and Niedel (1983) identified a polypeptide on SDS-PAGE with an apparent molecular wcight of 55.000-70,000. These investigations have shown that papain trcatment of intact neutrophils results in the production of a 35,000 dalton receptor fragment that had biological activity identical to that of the intact receptor. Subsequent studies by Schmitt et al. (1983) using a covalent affinity labeled Mle-Leu-Phc-Nle-Tyr-Lys confirmed a molecular weight of 50,000-60,000 for the receptor. In these studies, however, two major distinct entities of M , = 50,000 with an isoelectric point of 6.0 and Mr = 60,000 with an isoelectric point of 6.5 were identified. The receptor has been localized to predominantly plasma TABLE I N-FORMYLATED OLIGOPTPTIDT CHEMOATTRACTANT RECEPTORS O N VARIOUS IXUKOLYTES" C H A R A C T t K I S I I C S OF

Agonist used

FMLI3H]P Human PMN Guinea pig macrophages Equine PMN Rat PMN FNL[ ?HIP Rabbit PMN PNLPN[ I25I1TL Human PMN Human monocyks F[ 75S]MI.P

Human irionocytes a

Average K , (nM)

Receptordccll ( X I 0 3)

10.0-22.3

15-60

11.0

10

0.52 9.9

1.5 1.o

0.6 4.1 100

I20

1 .I-2.7

10-18

30.2

84

Adapted from Pikc and Snyderman (1988).

2. CHEMOATTRACTANT REGULATION OF PHAGOCYTES

25

membrane but is also translocated to a Golgi-rich cell fraction (Painter et al., 1982). A major problem encountered in solubilizing the oligopeptide chemoattractant receptor is its inactivation by detergents such as Nonidet P-40 and Triton X-100. Marasco et al. (1985) have succeeded in solubilizing a receptor preparation from rabbit neutrophil membranes in 3 4(3-cholamidopropyl)dimethylammonio]-1propanesulfonate (CHAPS) and digitonin and from intact cells with CHAPS alone. This solubilized receptor maintained its ability to bind the iodinated hexapeptide and to cross-link a photoactivated derivative of the same ligand. Unfortunately, further attempts at purifying this receptor to homogeneity have been unsuccessful due to its lability in detergent solutions. Following the initial characterization of the formylated peptide receptor on intact leukocytes, studies performed in cellular membrane fractions indicated that the receptor existed in at least two affinity states, whose interconvertibility was regulated by guanine nucleotides. Koo et al. (1982) found that the receptor existed in high affinity (Kd = 0.53 k 0.01 M )and low affinity (Kd = 24.4 1.21 nM) states, with approximately 25% of the receptors being in the high affinity state. This was confirmed by Snyderman et al. (1983) using guinea pig peritoneal exudate macrophages. Seligmann et al. (1 982) also demonstrated nonlinear Scatchard analysis of FML['HIP binding to human PMN membranes consistent with heterogeneity of binding sites.

*

2. REGULATION OF N-formylated Oligopeptide Chemoattractant Receptors

a. Guanine Nucleofide Regulatory Proteins. Several studies have shown that pertussis toxin, which catalyzes the ADP-ribosylation and inactivation of specific guanine nucleotide regulatory proteins (Katada and Ui, 1982; Okajima and Ui, 1984), inhibits several chemoattractant-mediated functional and biochemical responses in leukocytes (Okajima e f al., 1985; Verghese et al., 1985; Volpi et al., 1985). Superoxide anion generation, release of lysozyme, calcium uptake as measured by increases in quin2 fluorescence, chemotaxis, arachidonic acid release, and the production of inositol phosphates in response to multiple chemoattractants are inhibited by pertussis toxin (Brandt et al., 1985; Okajima et al., 1985; Goldman et a l . , 1985a; Lad et ul., 1985; Verghese et al., 1985). Studies by Koo et al. (1983) and Snyderman et al. (1983) have shown that nonhydrolyzable analogues of GTP, such as p[NH]ppG, cause a reversible decrease in the percentage of high affinity chemoattractant receptors labeled by FML[3H]P in human PMN and guinea pig macrophage membranes. The hydrolysis of polyphosphoinositides in human PMN membranes by the chemoattractant FMLP has also been shown to require guanine nucleotides at

26

MARILYN C. PIKE

low concentrations of calcium (C. D. Smith et al., 1985). Taken together with the receptor data, it has been postulated that chemoattractant receptor mediated transmembrane signaling requires interaction of the occupied receptor with a G protein, followed by GTP hydrolysis and interaction of the G protein with a phospholipase C (PLC) (Verghese and Snydernian, 1983; C. D. Smith et al., 1985; Volpi et ul., 1985; Snyderman et al., 1986). This leads to activation of the PLC and hydrolysis of phosphoinositides, with generation of inositol phosphates. These substances cause increases in intracellular calcium concentrations (Berridge, 1984; Majerus et al., 1986). The nature of the specific C protein that interacts with the oligopeptide chemoattractant receptor is under active investigation. Polakis et al. (1985) have shown that the oligopeptide chemoattractant receptor from HL60 cells co-purifics with a GTP binding protein that is distinct from Gi, the pertussis toxin-sensitive G protein that is involved in regulation of adenylate cyclase. The G protcin associated with the chemoattractant receptor has an cu-subunit of s40 kDa (Polakis et ul., 1985; Gierschik et al.. 1986; Verghese et al., 1986) that is immunologically distinct from G"-CYand G,-a, which are associated with adenylate cyclase regulation (Lad el ul., 1977; Burgisser et al., 1982). Thus it appears that a unique G protein, termed G,, represents the species necessary to couple the oligopeptide chemoattractant receptor to PLC.

b. Altared Expression of Chemoattractanc Receptors Producwl by Secretion of Secondary Grunules. An important mechanism regulating thc expression of forniylated peptide receptors in human PMN has been provided by Gallin and coworkers ( 1978). These investigators found that treatment of human neutrophils with agents that cause limited secretion of secondary granules such as phorbol esters increase the number of receptors labeled by FML[3H]P in the plasma membrane (Flctcher and Gallin, 1980). These receptors have a lower average affinity than those originally expressed on the plasma membrane. Neutrophil subcellular fractions enriched for specific granule markers have been shown to bind FML[3H]P (Fletcher and Gallin, 1983). Gardner et al. (1986) have shown that the newly expressed receptors on the plasma membrane of neutrophils treated with PMA have the same apparent molccular weight (55,O00-75,000) as thc receptor on the surface of unstiniulated cells. In addition, the newly expressed rcceptors are expressed in two isoelectric forms (isoelectric points = 5.8 and 6.2) which are similar to those found in the plasma nicmbrane of unstimulated cells. The expression of the receptors following limited secondary granule secretion is not dependent upon new protein synthesis (Fletcher et al., 1982). Thus, neutrophil specific granules contain a pool of preformed formyl peptide receptors, which, upon expression in plasma membrane, allow the neutrophil to respond to ever-increasing Concentrations of chemoattractants at inflammatory sites. This hypothesis is supported by the descriptions of patients with recurrent infections

2. CHEMOATTRACTANT REGULATION OF PHAGOCYTES

27

who have been found to have a specific granule deficiency in their neutrophils (Boxer et al., 1982; Gallin et al., 1982). These defects are accompanied by a diminished capacity of the patient’s cells to express new FML[3H]P binding sites following treatment with agents that cause secretion of specific granules (Gallin et al., 1982). c. Tumor Necrosis Factor and Granulocyte-Monocyte Colony Stimulating Factor (GM-CSF) as Regulators o j Chemoattractant Receptor Expression. Several laboratories have demonstrated that GM-CSF regulates the affinity and functional activity of neutrophil formylated peptide receptors. GMCSF modulates the function of mature peripheral blood neutrophils by inhibiting chemokinesis and priming neutrophils for enhanced oxidative metabolism in response to FMLP (Gasson et al., 1984; Weisbart et al., 1985). Weisbart et al. (1986) have shown that brief (5-15 min) exposure of human neutrophils to physiological concentrations of biosynthetic human GM-CSF enhances chemotaxis in response to FMLP. This is accompanied by a three-fold increase in “high-affinity” receptors labeled by FML[3H]P. More prolonged treatment of cells with GM-CSF (1-2 hr) caused a reduction in the average affinity of the receptors from Kd = 29 nM to Kd = 99 nM for FMLr3H]P (Weisbart et al., 1986). The shift to expression of lower affinity receptors is accompanied by the disappearance of the enhancement of chemotaxis and the appearance of an enhanced oxidative response. These investigators did not determine whether the increased receptors were derived from preformed intracellular stores. Atkinson et al. (1988a) have confirmed that recombinant human GM-CSF results in enhanced superoxide production by human neutrophils in response to FMLP which was accompanied by a decrease in the chemotactic responsiveness of the cells. Unlike Weisbart et al. (1986), these investigators noted these GMCSF effects on PMN functional activity as early as 5 min following exposure to GM-CSF. Alterations in oxidative metabolism were accompanied by a change in the binding of FML[3H]P (Atkinson et al., 1988a). Unstimulated neutrophils expressed both high (Kd = 4 nM, 2000 sitedcell) and low affinity (Kd = 220 nM, 40,000 sites/cell) formylated peptide receptors. Incubation with GM-CSF caused no change in the total number of binding sites but converted all expressed receptors to one intermediate affinity of Kd = 30 nM. These findings differ significantly from those reported by Weisbart et al., which may be related to differences in calcium and magnesium concentrations used during the FML[3H]P binding assays (Atkinson et al., 1988a). Further studies by English et al. (1988) have demonstrated that priming of the oxidative response by GM-CSF in human neutrophils is not reversible by washing the cells prior to stimulation by FMLP and that extracellular calcium is not required for functional enhancement. In addition, no alterations in 32P-labeledphospholipids were produced by GM-CSF in neutrophils. These authors concluded that GM-CSF released at sites of infec-

28

MARILYN C.PIKE

tion or inflammation in vivo may accelerate the functional maturation of circulating PMNs which eventually localize at inflammatory sites (English ef a / ., 1988). Another biologically active molecule produced as a result of ongoing inflammation is tumor necrosis factor CY (TNF-a) (Beutler and Cerarni, 1986). Recombinant human TNF-a has also been shown to enhance superoxide anion production by human neutrophils treated with FMLP and to cause a concomitant decrease in neutrophil chemotaxis (Atkinson et a l ., 1988b). TNF-a produced cffects on FMLL-IHJPbinding similar to those observed by the same investigators for GM-CSF (Atkinson et ul., 1988a). These workers noted a linearization of Scatchard plots performed in the presence of TNF-a which showed no change in the total number of binding sites but showed one class of sites with an affinity of K , = 40 nM. This contrasted with untreated cells which exhibited two classes of receptors with aifinities of K,, = 2 nM and K,, = 180 nM. It is unclear how the changes in chemoattractant receptor affinity would account for the increase in the oxidative burst observed in the presence of TNF-a. d . Regulation of Oligopeptide Chemouttructant-Receptor Expression by the. Extrucellulur Matrix. The directed migration of PMNs in vivo requires attachment of the cells to the basement membrane and subsequent diapedesis through the tissue along an increasing gradient of chemoattractant (Gallin and Quie, 1978; Snyderrnan and Goetzl, 1982). A component of all basement membranes, laminin is a high molecular weight (900,000) matrix protein (Timple and Heilwig, 1979; Von der Mark and Kuhl, 1985) which has been shown to augment the chernotaxis of neutrophils in response to chemoattractants and, by itself, stimulates the motility of peritoneal exudate PMNs (Terranova et al., 1986). Receptors for laminin exist in small numbers on unstimulated PMNs, and low concentrations of cellular activators, such as chemoattractants and phorbol esters, induce the expression of additional laminin receptors on the cell surface (Yoon el al., 1987a). These cryptic laminin receptors are stored within secondary granules in 1987a). Studies in this laboratory were undertaken to deterPMNs (Yoon et d., mine whether laminin altered the oxidative burst of human PMNs (Pike et af., 1989). Preliminary observations by Yoon et al. (1987b) indicate that supcroxide production was increased by incubation of human PMNs with laminin. Other studies indicated that concentrations of laminin ranging from 5 to 100 Fg/ml increased lysozyme release and superoxide anion production in response to FMLP by as much as 69% (Pike et uf., 1989). These results could be explained by changes in cell surface oligopeptide ehemoattractant receptor expression in that incubation of normal PMNs with concentrations of laminin ranging from 5 to 75 p,g/ml increased the binding of I9 nM FML[’H]P by 3 5 4 0 % (Fig. 1) (Pike et al., 1989). This corresponded to as much as a 2.1 -fold increase in the number of chemoattractant receptors/cell from 35,965 to 75,110 in the presence of 50

2. CHEMOATTRACTANT REGULATION OF PHAGOCYTES

29

Larninin (Fg/rnl) FIG. 1 . Effect of laminin on the binding of FML17H]Pto normal human PMNs-dose response. PMNs (8 X 106iml) were preincubated with incubation buffer alone or containing the indicated concentration of laminin in solution for 15 min at 24°C before the addition of 19 nM FML[3H]P 2 10 )LM unlabeled FMLP. Specific binding was calculated following an additional incubation for 60 min i of normal FML[3H]P binding = (E/C)100, where E is the specific binding of 19 nM at 24°C. 9 FML[”H]P in the presence of the indicated concentration of laminin, and C is the specific binding of FML[3H]P in the presence of buffer alone. (From Pike cr ul., 1989.)

Fg/ml laminin (Fig. 2A and 2B). In addition, the Kd was increased in the presence of laminin from 10.6 nM to 29.5 nM, indicating a lower average affinity of the newly expressed receptors. The effects of laminin were reversed by washing the cells prior to addition of FML[3H]P and were not observed when organelle depleted cytoplasts were used in the receptor binding assay. These results suggested that laminin, in the presence of chemoattractant, induced the expression of new chemoattractant receptors from an intracellular source. To confirm this, we examined the effects of laminin on chemoattractant receptor expression in PMNs from a patient with a specific granule deficiency (Pike et al., 1989). The laminin-induced increase in FMLP[3H]P receptors expressed on patient PMNs was only l 1-2 l % of that seen in normal PMNs. These findings have led to the formulation of the following hypothesis. When circulating in the blood stream, human PMNs may be unable to respond to laminin contained in the basement membranes of vessel walls because they lack sufficient receptors for this glycoprotein on their cell surface. When the cell is exposed to low levels of a chemoattractant substance emanating from inflamed tissue, more laminin receptors are exposed on the cell surface, allowing binding of the cell to laminin molecules within the blood vessel wall. The binding of laminin then results in expression of additional chemoattractant receptors, which are released from intracellular granules. These new receptors, which possess a lower affinity, are capable of responding to increasing concentrations of the chemoattractant as the PMNs approach the inflammatory focus. Occupation of these receptors then

A ,035

.03C

,020

,015

,010

,005

0

fMet-Le~-[~H]Phe Bound (nM) B a

a a/---

'04/ f

/

f Met-Leu-['H]

I ' 0

Phe (nM)

2. CHEMOATTRACTANT REGULATION OF PHAGOCYTES

31

leads to further release of IysosomaI enzymes and superoxide anions to destroy the invading substance (Pike et a/., 1989). This proposed model of the interaction of the attachment protein, laminin, with human PMNs may provide a molecular amplification mechanism for the egress of leukocytes from the circulation and eventual localization at inflammatory sites.

B. Chemoattractant Receptors for LTB, 1. INITIAL DESCRIPTION A N D BIOCHEMICAL CHARACTERIZATION LTB, evokes a maximal chemotactic stimulus in human neutrophils at concentrations of 20-100 nM (Goetzl and Pickett, 1981). Other products of the lipoxygenase pathway, such as 5-HETE, 1 I-HETE, and 12-HETE, are also chemotactic for human neutrophils but are less potent than LTB, (Lewis et al., 1981). This substance, while being a potent chemotactic agent, is a rather poor activator of the oxidative burst and produces less than a third of the maximal secretion of P-glucuronidase and lysozyme as that produced by other chemotactic stimuli (Serhan et al., 1982; Rollins et at., 1983). The binding of ['HILTB, to intact human neutrophils was described independently by two different laboratories (Goldman and Goetzl, 1982; Kreisle and Parker, 1983). In both cases, the ligand was isolated from [3H]arachidonatelabeled, ionophore-stimulated human neutrophils. Kreisler and Parker ( 1983) demonstrated nonreversible binding of ['HJLTB, to intact neutrophils at 4°C. An estimate of the binding constant (200 nM) in these studies was made by calculating the 50% inhibitory concentration of [ 14C]LTB, added simultaneously with [3H]LTB,. The number of receptors per cell was calculated to be 390,000. Goldman and Goetzl(l982) similarly showed specific binding of [3H]LTB, to human neurrophils wing a similar methodology to Kreisler and Parker except that binding studies were performed at 0°C. The number of sites per cell estimated by Scatchard analysis was 26,000-40,000, and the average Kd ranged between 11 and 14 nM.Structural analogues of LTB,, including 5-HETE and 5(5), 12(5)-dihydroxyeicosa-6,8,I0-trans- 14-cis-tetraenoic acid, competitively

FIG. 2. Effect of laminin on the binding of FML[.'H]P to normal human PMNs-binding isotherm and scatchard analysis. (A) Normal human PMNs (8 X I0"iml) were preincubated with 50 Fgiml laminin for 15 min at 24°C prior to the addition of concentrations of FML("HJP ranging from 0.5 to 28 nM in the presence and absence of 10 I.M unlabeled FMLP. Cells were incubated an additional 60 min at 2 4 T , and specific binding was measured. Closed circles, 50 Fgirnl laminin; open circles, buffer alone. (€3) Scatchard analysis of data presented in A. The Kd and total number of receptors per cell were calculated from the x intercept and slope of the lines obtained from linear regression analysis. Receptorsicell in the presence of buffer alone, 35,965; receptors/cell in the presence of laminin, 75,110. The Kdsfor buffer and laminin treated cells were 10.6nM and 29.5 nM, respectively. (From Pike et ul., 1989.)

32

MARILYN C.PIKE

inhibited the binding of [3HJLTB,. C5a and FMLP did not affect the binding of LTB,, indicating that these agents bind to distinct receptors (Goldman and Goetzl, 1982). These same investigators subsequently reexamined the nature of the LTB, receptor on human neutrophils. Two classes of LTB, receptors were defined on intact PMNs-a high affinity subset which comprised approximately 4400 sitesicell with a K,, of 0.4 nM and a low affinity, 270,000 sitdcell subset with a Kd of 61 nM (Goldman and Goetzl, 1984). A5 with the formyl peptide receptor, it wa5 postulated that the high affinity receptors mediate chemotactic functions of the cells, while the low affinity mediate lysosomal enzyme release and generation of superoxide. The majority of human neutrophil LTB, receptors are localized in the plasma membrane (Goldman et al., 1987; Goetzl et a/., 1988). Unlike formyl peptide receptors, intracellular granules contained minor amounts of [WJLTB, binding capacity. The human PMN LTB, receptor has been solubilized in CHAPS. The solubilized receptors exhibited only high affinity binding of [3H]LTB, and had a specificity identical to the high affinity binding site identified on the intact neutrophil (Goetzl er al., 1988). Covalent affinity cross-linking of the LTB, receptor in PMN plasma membrane enriched fractions was achieved using the analogue [3H]N-(3-aminopropy1)leukotriene B, amide (['H]LTB,-APA) (Goldman et ul., 1985b). This agent bound to a 60,000 dalton protein resolved by SDS-PAGE, with one mean K , of 2.3 nM.

2 . MODULATION OF LTB, RECEPTORSBY G ~ J A N I N E NUCLEOTIDE REGULATORY PROTEINS Similar to the formyl peptide chemoattractant receptor, the binding of ['HILTB, to human PMN membranes is inhibited by GTP and nonhydrolysable GTP analogues (Sherman et al., 1988). The inhibition of L3H]LTB, binding was shown to be reflective of a reversible conversion of approximately 29% of the high affinity LTB, receptors to binding sites manifesting the characteristics of the low affinity receptors on intact PMNs. The association of G proteins with LTB, receptors in human PMNs was further demonstrated by the finding that physiological concentrations of LTB, increased the binding of the nonhydrolysable GTP derivative ['HJGMP-PNP to leukocyte plasma membranes (Sherman et ul., 1988). In addition, pertussis toxin incubation of intact human PMNs results in preferential expression of low affinity LTB, receptors (Goldman er a / ., 1985b). Goetzl el ui. (1988) have proposed a model for the interaction of LTB, receptors, G proteins, and guanine nucleotides that is similar to that described for the formylated peptide chemoattractant receptor.

2. CHEMOATTRACTANT REGULATION OF PHAGOCMES

33

3. REGULATION OF LTB, RECEPTOR EXPRESSION The differential expression of LTB, receptors of high versus low affinity has been shown to be regulated via several mechanisms. Incubation of human PMNs with concentrations of LTB, up to 10 nM has been shown to progressively reduce the expression of high affinity receptors with no changes noted in the expression of the low affinity species (Goldman and Goetzl, 1984). The loss of high affinity receptors under these conditions correlated with a complete loss of LTB,-specific chemotactic responsiveness and a partial loss of the migratory response to C5a. Degranulation responsiveness to both LTB, and C5a was maintained under these conditions, confirming the hypothesis that preferential expression of low affinity receptors enhances transduction of this cellular response, while the high affinity receptors mediate chemotaxis (Goldman and Goetzl, 1984). LTB, receptor expression was also found to be regulated by lipopolysaccharide administration (Goldman et al., 1986). It is well known that bacterial infection and sepsis result in altered PMN function. Goldman et al. (1986) showed, in an animal model for gram-negative sepsis, that intravenous injection of endotoxin into rabbits caused decreased chemotactic responsiveness of the peripheral blood PMNs to LTB, and C5a but not to FMLP used as chemoattractants. The decreased chemotactic responsiveness was correlated with 68% fewer high affinity sites for LTB, and a 51% decrease in [12s1]C5abinding. The decreased ['2sI]C5a binding was accompanied by a small decrease in the apparent Kd for C5a. Interestingly, intravenous endotoxin injection caused an eight-fold increase in FMLP binding to rabbit neutrophils, which was not associated with changes in chemotactic or degranulation responses (Goldman er al., 1986). Decreased expression of C5a and LTB, receptors may contribute to the neutrophil dysfunction that accompanies sepsis. Several investigators have demonstrated that protein kinase C (PKC) activation may regulate the expression of LTB, receptors. Phorbol ester induced activation of PKC in rabbit neutrophils completely blocked LTB, and FMLP induced degranulation and changes in cytosolic calcium (Naccache et al., 1985). O'Flaherty et ul. (1986) showed that PKC activation leads to a reduction in the number of LTB, high affinity receptors. This finding was confirmed by Goldman (1987) in human neutrophils using oleoylacetylglycerol (OAG) as an activator of PKC . It remains to be determined how PKC activation alters LTB, receptor expression and LTB, elicited functional responses in PMNs. In other receptor systems, such as the epidermal growth factor (EGF) receptor, PKC has been shown to decrease receptor expression transiently, and this is thought to be due to phosphorylation of the EGF receptor itself (Cochet et a l ., 1984; Davis and Czech, 1984). Alternatively, PKC-mediated phosphorylation of the inhibitory G protein which regulates adenylate cyclase has been shown to suppress its func-

34

MARILYN C. PIKE

tion (Katada et a / ., 1985). It has been postulated that PKC may either phosphotylate an LTB, receptor-associated G protein or the receptor itself (Goldman, 1987). Another point of action by PKC may be at the level of receptor-mediated PLC activation since it has been demonstrated in other systems that phorbol esters inhibit this enzyme and decrease inositol triphosphate formation (Orellana et d., 1987). In addition, PKC activation has been shown to lead to increased inositol triphosphate phosphomonoesterase activity, which may further attenuate calcium-mediated responses in PMNs (Connolly et ul., 1986). C. Chemoattractant Receptors for C5a 1.

INITIAL

DESCRIPTION A N D BIOCHEMICAL CHARACTERIZATION

Human CSa is a glycoprotein whose amino acid content comprises a molecular weight of 8400 and whose carbohydrate portion accounts for 3000 (Fernandez and Hugli, 1976). The molecule is chemotactic for human PMNs at eoncentrations as low as 1.0 nM (Femandez ef a l . , 1978). The binding of purified lZ51lahcled CSa to human leukocytes was first described by Chenoweth and Hugli (15178). The binding of [lZSII]C5ato intact human PMNs was found to be saturable but not reversible at 24°C. Half-maximal binding of ['251]C5a occurred at a concentration of 3-7 nM, and it was estimated that there were 100,000-300,000 sitedcell. Structural specificity studies showed that native C5a was the most potent a molecule from which inhibitor of binding of [ lZ51]C5a,followed by C5adesargr the COOH-terminal arginine was removed by carboxypeptidase enzymes (Chenoweth and llugli, 1980). C5adesnrgis approximately ten-fold Less active than native C5a. A derivative of CSa that is not chemotactic, C5a( 1-69) also bound to the receptor at a lower affinity, indicating its potential usefulness as a competitive antagonist for CSa. A synthetic pentapeptide that mimics the COOH-tcrniinal linear sequence of C5a. L-niethionyl-r.-glutaminyl-L-leucyl-L-ar~inine, lacks both biological activity and the ability to interact with the C5a binding site (Chenoweth and Hugli, 1980). The CSa receptor has been photoaffinity labeled with the probe p-azidobenzoyl-2-mercapto-1-ethylamide-CSa (Johnson and Chenoweth, 1985). This derivative of C5a maintained its ability to specifically bind to both neutrophil and U937 cells. The apparent molecular weight of the molecule labeled by covalent attachment of this compound in both neutrophil and U937 cell plasma membranes was 52,000, which is similar to the size of the other functionally active chenioattractant receptors. 2. REGUI.ATIONOF LEUKOCYTE C5a RECEPTORS Several studies have shown that the C5a receptor is regulated quite differently lrom the forniyl peptide receptor. Phospholipase C treatment of human PMNs

2. CHEMOATTRACTANT REGULATION OF PHAGOCYTES

35

results in decreased C5a receptor expression but in an increase in formylated oligopeptide receptors (Nelson et al., 1982). Alternatively, sulfinpyrazone and phenylbutazone inhibit both the biological response and binding of FMLP to leukocytes but does not change CSa-mediated responses (Dahinden and Fehr, 1980). The tissue response of CSa receptors is also regulated differently when compared to formyl peptide receptors. Rabbit peritoneal exudate PMNs contain an increased number of FMLP receptors as compared to peripheral blood leukocytes while biological responses of the two cells to CSa d o not differ (Tsung et al., 1980). In addition, human synovial fluid monocytes show less binding of CSa than do peripheral blood monocytes, while no difference is seen in FMLP binding (Ohura et a / . , 1985). As mentioned in Section B3, decreased binding of CSa to peripheral blood PMN in rabbits is noted following intravenous injection of endotoxin, while enhanced binding of FMLP is noted under the same experimental conditions (Goldman et al., 1986). Studies performed by Bender er ul. (1987) examined the effects of leukocyte cellular activation on CSa receptor expression. Using fluoresceinated chemotactic factors, including C5a, f-Met-Leu-Phe-Lys, and casein, receptor expression was monitored following cellular activation with PMA, CSa, or FMLP. As noted by other laboratories, low concentrations of PMA (0.5 to SO ng/ml) increased the binding of the fluoresceinated formylated peptide and of casein (Bender et al., 1987). Similar treatments decreased CSa binding, and complete loss of C5a binding was noted at concentrations of PMA greater than 5 ng/ml. The addition of catalase, superoxide dismutase, or protease inhibitors did not prevent the decreased binding of CSa, nor was the inhibition of binding due to degradation of the ligand. The inhibition of C5a binding produced by PMA treatment was correlated with a loss of functional responsiveness to this chemoattractant (Bender et al., 1987). The differential regulation of chemoattractant receptors by cellular activation may be important for sequential functioning of leukocytes during the inflammatory response.

D. lnterleukin 1 (11-1) Receptors Early studies using IL-1 purified from cellular supernatants showed that it stimulated the degranulation, oxidative metabolism, and chemotaxis of neutrophils (R. J. Smith et ul., 1985; Luger et al., 1983). Subsequent, careful studies using recombinant l L - l a and IL- I(3 showed inconsistent effects of these molecules on PMN function (Georgilis et ul., 1987; Yoshimura et al., 1987). A number of in vivo studies using both purified human IL-1 and recombinant I L l a have demonstrated that intraperitoneal injection of these substances results in the accumulation of Ph4Ns (Beck et al., 1986; Granstein et al., 1986; Parker et a / ., 1989). Although these studies cannot distinguish between direct vs. indirect

36

MARILYN C. PIKE

eifects of 1L-l on PMN function, they suggest that 11-1 may, at a minimum, modulate the chemotaxis and subsequent activation of these cells. Studies by Parker ci cil. (1989) using recombinant human 12sl-laheled 1L-1 showed that both human peripheral blood PMNs and murine exudate PMNs contain specific, high aginity receptors for this molecule on their surfaces. Murinc peripheral exudate PMNs contained approximately I700 receptors/PMN with an apparent average K , of 0.1 nM. Both unlabeled, human rIL-la and r l L I p inhibited the binding of the radioligand, and human PMNs were capable of internalizing ('2sI]1L-1 at 37°C. These receptor studies were not accompanied by a correlation with effects on PMN functional responsiveness other than the demonstration of increased PMN accumulation in vivo upon injection of ILla into the peritoneal cavities of mice (Parker et d., 1989). These findings for the first time demonstrate specific, high affinity binding sites for rIL- la and r l L 1 p on the surface of PMNs. 1L-1 may not, in fact, produce direct cffects on YMN functional responses but may result in modulation of responscs to other known chemoattractants or cellular activators. Indeed, it has been demonstrated that IL- 1 trunsmodulates receptors for EGF on fibroblasts by rapidly, but transiently, decreasing the affinity of the EGF receptor. Furthcr studies of the interaction with II, 1 and chemoattractant receptor expression should elucidate whether this important cytokine modulates PMN inflammatory responses. REFERENCES Altnian, 1.. C.. Chassey, B., and Macklcr. B . F. (1975). Physicochemical characterization ofchemotactic lymphokines produced by human T and R lymphocytes. J. Irnmunol. 110, 18-21. Aswanikumar, S., Corcoran. B., Schiffman. E . , Day, A. R . , Freer, R. J . , Showell, H. J.. and Pert, C. R (1977). Demonstration of a receptor on rabbit neutrophils for cheniotactic peptides. Biochern. Biophys. Ker. Commun. 74, 810-817. Atkinson, Y H., Lopez, A . F., Marasco, W. A , , I.ucas, c'. M . , Wong, Ci. G . , Burnb, C . P., and Vadas, M. A. ( I98Xa). Recombinant human granulocyte-macrophage colony-stimulating factor (rH-Gm-CSF) re~ulatesf-Met-Leu-Phereceptors on human neutrophils. Irnmunolu~y64, 5 I9s2s. Atkinson, Y. H.. Marasco, W. A . . I.oper, A. F., and Vadas. M. A. (1988b). Recombinant human tumclr necrosis factor-n: Regulation of N~formylmcthionyIleucylphenylalariine rcceptor affinity and function on human neutrophils. J . Clin. Invest. 81, 759-765. Beck. G . , Habicht, ti. S . , Benach, J . L., and Miller, F. (1986). Interleukin I : A conimon endogenous mediator of inflammation and the local Schwartzman reaction. J. Imniunol. 136, 30253030

Heck. G., Habicht, Ci. S . , Bcnach, J. L., and Millcr, F. (1986). Interleukin-1: A common endogcnous niediatoi- of inflammation and the local Shwartzman reaction. J. Ifnniunol. 136, 3025. Bender, J . G., Van Epps, D. E., and Chenoweth, D. E. (1987). Independent regulation of human neutrophil chemotactic receptors after activation. J. Immunol. 139, 3028~3033. Berridge, M. J. ( 1984). Inositol trisphosphate and diacylglyccrol as second messcngcrs. Bioclrem. J . 220, 345-360. Beutler, R . . and Cerami, A . (1986). Cachcctin and tumor necrosis factor as two sides of the same biological coin. Nature (London) 320, 584-588.

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Boeynaems, J. M., and Dumont, J. E. (1975). Quantitative analysis of the binding of ligands to their receptors. J . Cyclic Nucleotide Res. 1, 123-142. Boxer, L. A., Coatcs, T. D., Haak, R. A., Wolach, J. B., Hoffstein, S., and Baehner, R. L. (1982). Lactoferrin deficiency associated with altered granulocyte function. N. Engl. J. Med. 307, 404410. Boyden, S . (1962). The chemotactic effects of mixtures of antibody and antigen on polymorphonuclear leukocytes. J. Exp. Med. 115, 453-466. Brdndt, S. J., Dougherty, R. W., Lapetina. J. E . , and Niedel, J. E. (1985). Pertussis toxin inhibits chemotactic peptide-stimulation of inositol phosphates and lysosomal enzyme secretion in human leukemic (HL-60) cells. Proc. Narl. Acad. Sci. U.S.A. 82, 3277-3280. Burgisser, G., DeLean, A . , and Lefkowitz, R. J. (1982). Reciprocal modulation of agonist and antagonist binding to muscarinic cholinergic receptor by guanine nucleotides. Proc. Natl. Acad. Sci. U.S.A. 79, 1732-1736. Carp, H. ( 1982). Mitochondria1 N-formylmethionyl protcins as chemoattractants for neutrophils. J. Exp. Med. 155, 264-275. Chenoweth, D. E., and Hugli, T. E. (1978). Demonstration of a specific C5a receptor on intact polymorphonuclear leukocytes. Proc. Natl. Acad. Sci. U.S.A. 75, 3943-3947. Chenoweth, D. E., and Hugli, T. E. (1980). Human C5a and C5a analogs as probes of the neutrophil C5a receptor. Mol. Immunol. 17, 151-161. Cochet, C. G . , Gill, N . , Meisenhelder, J., Cooper, J. A., and Hunter, T. (1984). C-kinase phosphorylates the epidermal growth factor receptor and reduces its epidermal growth factor stimulated tyrosine protein kinase activity. J . Biol. Chem. 259, 2553-2560. Connolly, T. M., Lawing, W. J., Jr., and Majerus, P. W. (1986). Protein kinase C phosphorylates human platelet inositol trisphosphate 5’-phosphomonoesterase increasing the phosphatase activity. Cell 46, 951-958. Dahinden, C., and Fehr, J. (1980). Receptor-induced inhibition of chemotactic factor-induced neutrophil hyperactivity by pyrazolon derivatives. Definition of a chemotactic peptide antagonist. J. Clin. Invest. 66, 884-891. Davis, R. J., and Czech, M. P. (1984). Tumor promoting phorbol diesters mediate phosphorylation of the epidermal growth factor receptor. J . B i d . Chem. 259, 8545-8558. DeLean, A., Hancock, A. A , , and Lefkowitz, R. J. (1982). Validation and statistical analysis of computer modeling method for quantitative analysis of radioligand binding data for mixtures of pharmacological receptor subtypes. Mol. Pharmacol. 21, 5- 16. Deuel, T. F., Senior, R. M . , Chang, D.. Griffin, G. L., Heinrikson, R. L., andKaiser, E. T. (1981). Platelet factor 4 is chemotactic for neutrophils and monocytes. Proc. Natl. Acad. Sci. U.S.A. 78, 4584-4587. Dolmatch, B., and Niedel, J. (1983). Formyl peptide chemotactic receptor: evidence for an active proteolytic fragment. J . B i d . Chem. 258, 7570-7577. English, D., Broxmeyer, H. E., Gabig, T. G . , Akard, L. P., Williams, D. E., and Hoffman, R. (1988). Temporal adaptation of neutrophil oxidative responsiveness to n-formyl-methionylleucyl-phenylalanine. J . Immunol. 141, 2400-2406. Fernandez, H. N., and Hugli, T. E. (1976). Partial characterization of human C5a anaphylatoxin. 1. Chemical description of the carbohydrate and polypeptide portions of human C5a. J . Immunol. 117, 1688-1694. Fernandez, H. N., Henson, P. M., Otani, A., and Hugli, T. E. (1978). Chemotdctic response to human C3a and C5a anaphylatoxins: 1. Evaluation of C3a and C5a leukotaxis in vitro and under stimulated in vivo conditions. J . Immunol. 120, 109-1 15. Fletcher, M. P., and Gallin, J. I. (1980). Degranulating stimuli increase the availability of receptors on human neutrophils for the chemoattractant met-Leu-Phe. J . Immunol. 124, 1585-1588.

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Fletcher, M. P., and Gallin. J . I . (1983). Human neutrophils contain an intracellular pool of putative receptors for the chemoattractanl N-forrnylmethionylleucylphenylalanine.Blood 62, 792-799. Fletcher, M. P., Seligmann, B. E., and Gallin, J . 1. (19x2). Correlation of‘ human neutrophil secretion, chemoattractant receptor mohilization and enhanced functional capacity. J . Immrtnol. 128, 941-948. Ford-Hutchinson, A. W., Bray, M. A , , Doig, M. V., Shipley, M. E.. and Smith, M. J. H. (1980). Lcukoti-iene K , a potent chemokinetic and aggregating substance released from polymorphonuclear leukocytes. Ntr/ure (London) 286, 264 265. Gallin, E. K . , and Gallin, J. I. (1977). Interaction of chemotactic factors with human macrophages: Induction of transinenibrane potential changes. J . Cell Biol. 75, 277-289. Gallin, J. I . , and Quie, P. G. (1978). “Leukocyte Chemotaxis: Methods, Physiology, and Clinical Implications.” Raven, New York. Gallin, J. I.. and Rosenthal, A. S. (1974). The regulatory role of divalent cations in human granulocytc chemotaxis: Evidence for an association between calcium exchanges and rnicrotubulc assembly. J . Cell B i d . 62, 594-609. Gallin, 1. I., Wright. D. G., and Schiffmann, E. (l97X). Role of secretory cvents in modulating hunian neutrophil chcmotaxis. J . Clin. Invrst. 62, 1364- 1374. Gallin. J 1.. Fletcher, M. P., Seligmann, H. E . , Hotfstein, S., Cehrs, K., and Motincssa, N. (1982). Human neutrophil-specific granule deficiency: A niodcl to assess the role of neutrophil-specific granules in the evolution of the inflammatory response. Blood 59, 1317-1329. Gardner, J. P., Mclnick. D A . , Malech. H. L. (1986). Characterization of the formyl peptidc cheniotactic receptor appearing at the phagocytic cell surface after exposure to phorholmyristate acetate. J . Immunol. 136, 1400- 1406. Gasson. J. C., Weisbart, R. H., Kauhnan. S. E., Clark, S. C . , Hewick, R. M., Wong, G. G . , and Colde, D. W. ( 1984). Purified human granulocyte-macrophage colony-stimulating Iactor: direct action on neutrophils. Scirnc 226, 1339- 1342. Gcorgilis. K . , Shaefer. C.. Dinarello, C. A , . Henderson, W. R., and Gallin, J . 1. (19x7). Human recombinant interleukin- 1 beta has no effect on intraccllular calcium or other functional rcsponses of neutrophils. J . Immimol. 138, 3403-3407. Cierschik, P., hlloon, J.. Milligan. G . , Pines. M., Gallin, J. I . , and Spiegel, A . (1986). Immunochcmical evidence for a novel pertussis toxin substrate in human neutrophils. J . Biul. Chem. 261, 8058-8062. Goctzl, E. J., and Pickett, W. C. (1980). The human PMN leukocyte cheniotactic activity of complex hydroxy-eicosatetrdcnoic acids (HETES). J . Immunol. 125, 1789- 179 I . Goctzl, E. J . , and Pickett, W. C. (1981). Novel structural determinants of the human neutrophil tivity of leukotriene B. J . E,rp. Mrtl. 153, 482-487. Goct71. E. J., Sherman. J. W., Ratnoti’. W. U.. Harvey, J. P., Eriksson. E., Seaman, W. E., Baud. L., and Keo. C. H. ( 1988). Receptor-specific mechanisms for the response of human leukocytes to leukotrienes. Ann. N.Y. Acad. Sci. 524, 345-355. Goldman, D. W. (1987). Activation of protein kinase C (PKC) decreases leukotriene B4 (LTB,) receptor expression on human neutrophils. Fed. Pruc.. Fed. Am. SOC. Exp. Biol. 46, 606 (A), Goldman. D. W., and Coetzl, E. J . (1982). Specific binding of leukotricnc B4 to receptors on human polyniorphonuclear leukocytes. J . Immrtnol. 129, 1600- 1604. Goldman, D. W., and Goetzl, E. J. (1984). Heterogeneity of human polymorphonuclear leukocyte receptors for leukotriene B4. ldentirication of a subset of high affinity receptors that transduce the chemotactic response. J . fhp. Mrd. 159, 1027-1041. Goldman. D W., Chang, F. H., Cifford, H. K . , Goetzl, E. J., and Bourne, H. R. (IYXSa). Pertussis toxin inhibition of cheniotactic factor-induced calcium mobilization and function in human polymorphonuclear leukocytes. J . Exp. Med. 162, 145-156.

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Goldman, D. W., Gifford, L. A., Young, R. N., and Goetzl, E. J. (1985b). Affinity labeling of human neutrophil (N) receptors for leukotriene B4.Fed. Pror., Fed. Am. Soc. Exp. Biol. 44,781 (A). Goldman, D. W., Enkel, H., Gifford, L. A , , Chenoweth, D. E., and Rosenbaum, J. T. (1986). Lipopolysaccharide modulates receptors for leukotriene B4.C5a, and formyl-methionyl-leucylphenylalanine on rabbit polymorphonuclcar leukocytes. J. Imrnunol. 137, 1971- 1976. Goldman, D. W., Gifford, L. A , , Marotti. T., Koo, C. H., and Goetzl, E. J. (1987). Molecular and cellular properties of human polymorphonuclear leukocyte receptors for leukotriene B4.Fed. Pror., Fed. Am. Soc. Exp. Biol. 46, 200-203. Goldstein, I . , Hoffstein, S . . Gallin, J., and Weissmann, G. (1973). Mechanisms of lysosomal enzyme release from human leukocytes: Microtubule assembly and membrane fusion induced by a component of complement. Proc. Narl. Acad. Sci. U . S . A . 70, 2916-2920. Granstein, R. D., Margolis, R., Mizel, S . B., and Sander, D. N . (1986). In vivo inflammatory activity of epidermal cell derived thymocyte activating factor and recombinant interleukin- I in the mouse. J. Clin. Invest. 77, 1026-1027. Hirata, F., Corcoran, B. A , , Venkatasubramanian, K., Schiffrnann, E., and Axelrod, J. (1979). Chemoattractants stimulate degradation of methylated phospholipids and release of arachidonic acid in rabbit leukocytes. Proc. Nafl. Acad. Sci. U . S . A . 76, 2640-2643. Hoffstein, S . , Goldstein, I. M., and Weissmann, G . (1977). Role of microtubule assembly in lysosomal enzyme secretion from human polyniorphonuclear leukocytes. J. C e / l Biol. 73, 242256. Hugli, T. E., and Muller-Eberhard, H. J. (1978). Anaphylatoxins. Adv. Immunol. 26, 1-53. Johnson, R. J., and Chenoweth. D. E. (1985). Labeling the granulocyte C5a receptor with a unique photorcactive probe. J . Biol. Chem. 260, 7161-7164. Katada, T., and Ui, M. (1982). Direct modification of the membrane adenylate cyclase system by islet-activating protein due to ADP-ribosylation of a membrane protein. Proc. Nail. Acad. Sci. U . S . A . 79, 3129-3133. Katada, T., Gilman, A. G . , Wantanabe, Y., Bauer, S . , and Jakobs, K. H. (1985). Protein kinase C phosphorylates the inhibitory guanine-nucleotide binding regulatory component and apparently suppresses its function in hormonal inhibition of adenylate cyclase. Eur. J. Biochem. 151, 431437. Keller, H. U . , and Sorkin, E. (1967). Studies on chemotaxis V. On the chemotactic effect of bacteria. Inr. Arch. Allergy Appl. Immunol. 31, 505-517. Klebanoff, S. J., and Clark, R. A. (1978). “The Neutrophil: Function and Clinical Disorders.” North-Holland Publ., New York. Koo, C., Lefkowitz, R. J.. and Snyderman, R . (1982). The oligopeptide chemotactic factor receptor on human polymorphonuclear leukocyte membranes exists in two affinity states. Biorhem. Biophys. Res. Commun. 106, 442-449. Koo, C . , Lefkowitz, R. J., and Snyderman, R. (1983). Guanine nucleotides modulate the binding affinity of the oligopeptide chemoattractant receptor on human polyniorphonuclear leukocytes. J. Clin.Invest. 72, 748-753. Kreisler, R. A., and Parker, C. W. (1983). Specific binding of leukotriene B4 to a receptor on human polymorphonuclear leukocytes. J. Exp. Med. 157, 628-641. Lad. P. hl., Welton, A . F., and Rodbell, M. (1977). Evidence for distinct guanine nucleotide sites in the regulation of the glucagon receptor and of adenylate cyclase activity. J. Biol. Chem. 252, 5942-5946. Lad. P. M., Olson, C. V.. Grewal, I. S . , and Scott, S. J. (1985). A pertussis toxin-sensitive GTPbinding protein in the human neutrophil regulates multiple receptors, calcium mobilization, and lectin-induced lapping. Proc. Natl. Acad. Sci. U . S . A . 82, 8643-8647.

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Lewis, R. A.. Goetzl, E. J . , Drazen, J. M., Sotter, N . A , , Austen, K. F., and Corey, E. J. (1981). Functional characterization of synthetic leukotriene B and its stereochemical isomers. J. Exp. Med. 154, 1243-1248. Luger, T. A., Charon, J. A., Colot. M . , Micksche, M . , and Oppenheini, J . J . (1983). Chemotactic properties of partially purified epidermal cell-derived thymocyte activating factor (ETAF) for po~ymorphonuc~edr and mononuclear cells. J. Immunol. 131, 8 16-820. Majerus, P. W., Connolly, T. M., Deckmyn, H . , Ross, T. S . , Brass, T. E . , lshii, H . , Bansal, V. S., and Wilson, D. B . (1986). The metabolism of phosphoinositide-derived messenger molecules. Science 234, 1519- 1526. Marasco, L. A , , Krutzsch, H., Shaisl, H. J., Feltner, D. E., Nairn, R., Becker. E. L., and Ward, P. as the maA. (1983). Purification and identification of formyl-methionyl-leucyl-phenylalanine jor peptidc neutrophil chemotactic factor produced by E. culi. Svmp. Inr. Congr. Immunol. Sci., S/h. Kvoro. Marasco, W. A . , Becker, K . M., Feltncr, D. E., Brown, C. S . , Ward, P. A,, and Nairn, R. (1985). Covalent affinity labeling, detergent solubilization, and fluid-phase characterization of the rabbit neutrophil fonnyl peptide chemotaxis receptor. Biochemisrry 24, 2227-2236. Metchnikoff, E. (1891). Lecture on phagocytosis and immunity. Br. Med. J. I, 213-217. Munson, P. J.. and Rodhard, L). (1980). LICAND: A versatile computerized approach for characterization of ligand-binding systems. Anal. Biochem. 107, 200-229. Naccachc, P. H., Molski, T. F. P., Borgeat, P., White, J. R.. and Sha’afi, R. 1. (1985). Phorbol esters inhibit the f-Met-Leu-Phe and leukotriene B4-stimulated calcium mobilization and enzymc secretion in rabbit neutrophils. J. B i d . Chem. 260, 2125-2131 Nelson, R. D., Piegcl. V. D., and Chcnoweth, D. W. (1982). Human neutrophil peptide receptors; mobilization mediated by phospholipase C. Am. J. Purhol. 107, 202-2 I 1 . Niedel, J. E., Kahane, 1.. and Cuatrccasas, P. (1979a). Receptor-mediated internalization of fluorescent chcniotactic peptide by human neutrophils. Science 205, 1412- 1414. Niedel, J. E., Kahanc, I . , and Cuatrccasas, P. (l979b). Receptor-mediated uptake and degradation of 12sl chcinotactic peptide by human neutrophils. J. B i d Chem. 254, 10700-10706. Niedel, J., Davis, J.. and Cuatrecasas, P. (1980). Covalent affinity labeling of the formyl peptide cheniotactic receptor. J. Biol. Chem. 255, 7063-7066. Nishihira, J., McPhail, L. C., and O’Flaherty, J. T. (1986). Stimulus-dependent mobilization of protein kinasc C. Biochem. Eiophvs. Res. Commun. 134, 587-594. O’Flaherty, J. T., Redrnan, J. F., and Jacobson, D. P. (1986). Protein kinase C rcgulates leukotriene B4 receptors in human neutrophils. FEBS Len. 206, 279-282. Ohura, K . , Katona, I . , Chenoweth. D., Wahl, L., and Wahl, S . (1985). Chemoattractant receptors on peripheral blood (PB) monocytes and receptor modulation in inflammation. Fed. Proc., Fed. Am. Soc. E.rp. B i d . 44, 1268. (Abstr.) Okajima, P., and Ui, M . (1984). ADP-ribosylation of the specific membrane protein by isletactivating protein, pertussis toxin associated with inhibition of a chemotactic peptide-induced arachidonic release in neutrophils. A possible role of toxin substrate in Ca2+ mobilizing hiosignalling. J. B i d . Chem. 259, 13863- 13871. Okajima, F., Katada, T., and Ui. M. ( I Y X S ) . Coupling of the guanine nucleotide regulatory protein to chemotactic peptidc in neutrophil membrane, and its uncoupling by islet-activating protein, pertussis toxin. A possible role of toxin substrate in Ca2 -mobilizing receptor-mediated signal transduction. J. Biol. Chem. 260, 6761-6768. Orellana, S . , Solski, P. A,, and Brown. J. H. (1987). Guanosine 5’-0-(thiotriphosphate)-dcpendent inositol trisphosphate formation in membrdncs is inhibited by phorbol ester and protein kinase C. J. R i d . Chem. 262, 1638-1643. Painter, R. C., Schmitt, M., Jesaitis, A. J.. Sklar, L. A , , Aeissnar, K . , and Cochrane, C. G . (1Y82). +

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Photoaffinity labeling of the N-formyl peptide receptor on human polymorphonuclear leukocytes. J . Cell. Biochem. 20, 203-214. Parker, K. P., Benjamin, W. R., Waffica, K. L., and Kilian, P. L. (1989). Presence of I L I receptors on human and munnc neutrophils: Relevance to IL-1 mediated effects in inflammation. J. Immunol. 142, 537-542. Pike, M. C., and DeMeester, C. A. (1988). Inhibition of phosphoinositide metabolism in human polymorphonuclear leukocytes by S-adenosylhomocystene. J . B i d . Chem. 263, 3592-3599. Pike, M . C., and Snyderman, R. (1984). Leukocyte chemoattractant receptors. In “The Receptors” (P. M. Conn, ed.), Vol. I , pp. 223-259. Academic Press, New York. Pike, M. C., and Snyderman, R. (1988). Leukocyte chemoattractant receptors. Merhods Enzymol. 162, 236-245. Pike, M. C . , Kredich, N. M., and Snyderman, R. (1979). Phospholipid methylation in macrophages is inhibited by chemotactic factors. froc. Nafl. Acad. Sci. U.S.A. 76, 2922-2926. Pike, M. C . , Jakoi, L., McPhail, L. C.. and Snyderman, R. (1986). Chemoattractant-mediated stimulation of the respiratory burst in human polymorphonuclear leukocytes may require appearance of protein kinase activity in the cells particulate fraction. Blood 67, 909-913. Pikc, M. C . , Wicha, M. S . . Yoon, P.. Mayo, L., and Boxer, L. A. (1989). Laminin promotes the oxidative burst in human neutrophils via increased chemoattractant receptor expression. J . Immunol. 142, 2004-201 I Polakia. P. G . , Lching, R . J., and Snyderman, R. (l98X). The formyl peptide chemoattractant receptor copurifies with a GTP-binding protein containing a distinct 40 kDa pertussis toxin substrate. J . Biol. Chem. 263, 4969-4916. Rollins, T. E . , Zanolari, B., Springer. M. S . , Guindon, Y., Zamboni, R., Lau, C.-K., and Rokach, J . (1983). Synthetic leukotriene B4 is a potent chemotaxin but a weak secretagogue for human PMN. Prosruglandins 25, 28 1-289. Schiffmann, E., Corcoran, B . A , , and Wahl, S . M. (1975). N-formylmethionyl peptides as chemoattractants for leukocytes. froc. Nu?/. Acad. Sci. U.S.A. 72, 1059-1062. Schmittt, M., Painter, R. G., Jesaitis, A. J., Preissner, K., Sklar, L. A,, and Cochrdne, C. G. (1983). Photoaffinity labeling of the N-formyl peptide receptor binding site of intact human polymorphonuclear leukocytes. J . B i d . Chem. 258, 649-654. Seligmann, B . E., Fletcher. M. P., and Gallin, J. I . (1982). Adaptation of human neutrophil responsiveness to the chemoattractant N-formylmethionylphenylalanine.J . Biol. Chem. 257, 62806286. Serhan, C. N., Radin, A , , Smolen, J. E., Korchak, H . , Samuelsson, B., and Weissmann, G. (1982). Leukotriene B4 is a complete secretagogue in human neutrophils: A kinetic analysis. Biochem. Biophys. Res. Commun. 107, 1006-1012. Sherman, J. W., Goetzl. E. J., and Koo, C. H . (1988). Selective modulation by guanine nucleotides of the high atfinity subset of plasma membrane receptors for leukotriene B, on human polymorphonuclear leukocytes. J . Immunol. 140, 3900-3904. Shin, H . S . , Snyderman. R., Friedman, E., Mellors, A., and Mayer, M. D. (1968). Chemotactic and anaphylatoxic fragment, cleaved from the fifth component of guinea pig complement. Science 162, 361-363. Showell, H . J., Freer, R. J . , Zigmond, S . H . , Schiffmann, E., Aswanikumar, S . , Corcoran, B. A., and Becker, E. L. (1976). The structure-activity relations of synthetic peptides as chemotactic factors and inducers of lysosomal enzyme secretion for neutrophils. J . Exp. Med. 143, 11541169. Sklar. L. A , , and Finney, D. A. (1982). Analysis of ligand-receptor interactions with the fluorescence activated cell sorter. Cyfomefry3, 161-165. Sklar, L. A.. Jesaitis, A. J . , Painter, R. G . , and Cochrdne, C. G . (1982). Ligandireceptor intemaliza-

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tion: A spectroscopic analysis and a comparison of ligand binding, cellular response, and internalization by human ncutrophils. J . C’e/l. Biochem. 20, 192-202. Smith, C. D . . [ m e , B. C., Kusaka, I . , Verghese, M. W., and Snyderman. R. (1985). Chcmoattractant receptor induced hydrolysis of phosphatidylinositoI~4,5 bisphosphate in human polyniorphonuclear leukocyte membranes: requirement of a guanine nucleotide regulatory protein. J. B i d . Chem. 260, 5875-5878. Smith, K. J . . Spezlate. S . C..and Bowman. B. J. (1985). Properties of interleukin-1 as a complete secretagoguc for human neutrophils. Riochem. Biophys. Res. Commun. 130, 1233- 1240. Snydcrman, R., and Fudman, E. J. (1980). Demonstration of a chemotactic factor receptor on macrophapcs. J . Immunol. 124, 2754-2757. Snyderman, R., and Goetzl, E. L. (1982). Molecular and cellular mechanisms of leukocyte chernotaxis. Scisnce 213, 830-837. Snyderman, R., Gewurz, H., and Mergenhagen, S. E. (1968). Interactions of the complement system with endotoxic lipopolysaccharide. Generation of a factor chemotactic for polyrnorphonuclear leukocytes. J. Exp. Mrd. 128, 259-275. Snydcrnian, R . , Pike, M . C., Edge, S., and Lane, B. (1983). A chemoattractant rcccptor on macrophagcs exists in two atYinity statcs regulated hy guanine nucleotides. J . Cell B i d . 98, 444-448. Snyderman, R . , Smith, C. D., and Verghese, M. W. (1986). Model for leukocyte regulation by chemoattractant receptors: Roles of a guanine nucleotide regulatory protein and polyphosphoinositidc metabolism. J . Leukor:yrr R i d 40, 785-800. Spilherg, I.. and Mehta, J. (1979). Denionstration of a specific neutrophil receptor for a cell dcrivcd chcniotactic factor. J. C h . Invesr. 63, 8.5 -88. Spilherg, I . . Gallacher, A., Mehta, J., and Mandcll, B. (1976). Urate crystal induced chemotactic factor, isolation and partial characterization. J . Clin. It7vest. 58, 815-8 19. Spilherg, I . , Rosenbcrg, D., and Mandcll, B. (1977). Induction of arthritis hy purified cell-derived chcmotactic factor: Role of chernotaxis and vascular permeability. J . Clin. Invest. 59,582-585. Stossel, T. P. ( 1977). Contractile proteins in phagocytosis: An example of cell surface-to-cytoplasm communication. Fed. Proc., Fed. Am. Soc. Exp. 36, 2181-2184. Tempel, T. R . , Snyderman, R.. Jordan, H. V., and Mergenhagen. S. E. (1970). Factors from saliva and oral bacteria, chemotactic for polyrnorphonuclear leukocytes: Their possiblc rolc in gingival inflainmation. J . Periodunto/. 41, 7 I . Terranova, V. P., DiFlorio, R., Hujanen, E. S.,Lyall. R. M.. Liotta, I.. A , , Thorgcirsson, U . . Sicgal, G . P , and Schiffmann, E. (1986). Laminin promotea rabbit neutrophil motility and attachment. J . Clin. Invest. 77, 1180-1 186. Tinipl, K.. and Heilwig. R . (1979). Laminin: A glycoprotein from basement membrane Chem. 254, 9933-9937. Tsung, P., Showell. H. J., and Becker, E. L. (1980). Surl‘ace rnembranc enzyme, chemotactic peptide binding activities and chemotactic responsiveness of rabbit peripheral and peritoneal neutrophils. I,iflurnmutiun 4, 271-280. Turner, S . R.. Camphell, 1. A , . and Lynn, W. S. (1975). Polymorphonuclcar leukocytc chemotaxis towards oxidized lipid components of cell membranes. J . Exp. Med. 141, 1437-1441. Verghesc, M . W , and Snyderman, R. (1983). Hormonal regulation of adenylate cyclase in macrophage membranes is regulated by guaninc nucleotides. J . Immunol. 180, 869-873. Vcrghese, M . W., Sinith. C. D., and Snyderman, R. (1985). Potenlial role for a guanine nucleolide regulatory protein in chcmoattractant receptor-mediated polyph~sphoin~sitidemetabolism, Ca?+ inohilirration and receptor response in leukocytes. Eiuchem. fiiophys. Xes. Cornmuri. 127, 450-457. Verghese, M., Uhing, R. J., and Snyderman, K. (1986). A pertussis/choleratoxin-sensitiveN protein

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may mediate chemoattractant receptor signal transduction. Biochern. Biophys. Res. Commun. 138, 887-894. Volpi, M., Naccache, P. H . , Molski, T. F. P., Shefcyk, J., Huang, C. K., Marsh, M. L., Munoz, J . , Becker, E. L., and Sha’afi, R. I. (1985). Pertussis toxin inhibits the formyl-methionyl-leucylphenylalanine but not the phorbol ester stimulated changes in ion fluxes protein phosphorylation and phospholipid metabolism in rabbit neutrophils, role of the “G-proteins” in excitation response coupling. Proc. Narl. Acad. Sci. U.S.A. 82, 2708-2712. Von der Mark, K., and Kuhl, U. (1985). Laminin and its receptor. Biochim. Biophys. Acta 823, 147160. Ward, P. A , , Lepow, I. H., and Newman, L. J. (1968). Bacterial factors chemotactic for polymorphonuclear leukocytes. Am. J. Pathol. 52, 725-736. Weisbart, R. H., Golde, D. W., Clark, S. C., Wong, G . G . , and Gasson, J. C. (1985). Human granulocyte-mdcrophage colony-stimulating factor is a neutrophil activator. Nature (London) 314, 361-363. Weisbart, R. H., Golde, D. W., and Gasson, J. C. (1986). Biosynthetic human GM-CSF modulates Immunol. . 137, 3584-3587. the number and affinity of neutrophil ,f-Met-Leu-Phe receptors. .I Williams, L. T., and Lefkowitz, R. J. (1978). “Receptor Binding Studies in Adrenergic Pharmacology.” Raven, New York. Williams, L. T., Snyderman, R., Pike, M. C., and Lefiowitz, R . J. (1977). Specific receptor sites for chemotactic peptides on human polymorphonuclear leukocytes. Proc. Nutl. Acad. Sci. U.S.A. 74, 1204-1208. Yoon, P. S . , Boxer, L. A . , Mayo, L. A,, Yang, A. Y., and Wicha, M. S . (1987a). Human neutrophil laminin receptors: Activation-dependent receptor expression. J . Immunol. 138, 259-265. Yoon, P. S . , Wicha, M. S., and Boxer, L. A. (1987b). Potentiation of chemoattractant-stimulated superoxide production in human granulocytes by laminin. Clin. Res. 35, 63344. Yoshimura, T., Matsushima, K . , Oppenheim, J. T., and Leonard, E. J. (1987). Neutrophil chemotactic factor produced by lipopolysaccharide (LPS)-stimulated human blood mononuclear leukocytes. Partial characterization and separation from interleukin-1 (IL1). J . Immunol. 139, 78793. Yuli, I . , and Snyderman, R. (1986). Extensive hydrolysis of N-formyl-L-methionyl-L-leucyl-~-[3H]phenylalanineby human polymorphonuclear leukocytes. J. B i d . Chem. 261, 49024908. Zimmerli, W., Seligmann, B., and Gallin, J. I. (1986). Exudation primes human and guinea pig neutrophils for subsequent responsiveness to the chemotactic peptide N-formylmethionyleucylphen ylalanine and increases complement component C3hi receptor expression. J . C/in. Invest. 17, 925-933.

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CUKKENT TOPICS IN MEMBRANES AND TRANSPORT. VOLUME 35

Chapter 3

Involvement of GTP-Binding Proteins in T- and B-Lymphocyte Activation Signaling JOHN G . MONROE Department of Pathology and Laboratory Medicine University of Pennsylvania School of Medicine Philadelphia, Pennsylvania 19104

I. Introduction Involvement of G Protcins during Lymphocyte Activation A . T-Lymphocyte Activation B. B-Lymphocyte Activation Ill. Future Perspectives References 11.

Activation signaling in T and B lymphocytes is a complex process in which, in its most simplistic form, primary activation signals generated through receptors for antigen are potentiated, or in some cases modified, by secondary signals generated through receptors for lymphokines. In this discussion 1 will present current experimental evidence supporting the involvement of G proteins in primary and secondary receptor-mediated signaling during T- and B-lymphocyte activation. Before presenting this material, however, I will introduce some general background information regarding G protein studies from other systems which will provide a critical framework from which to discuss studies specific to lymphoid systems.

1.

INTRODUCTION

Cells interact with one another and with their environment via plasma membrane associated receptors. Interactions of these receptors with signal molecules 45

Copyrighi 0 1990 by Academic P r c s Inc All rights of rcpruduilion in any lorm rcserved

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JOHN G.MONROE

secreted by other cells (i.e., neurotransmitters, hormones, or growth factors) or environmental stimuli (i.e., antigens) initiate a cascade of biochemical events that serve to transduce the cxtracellular signal across the plasma membrane. Once inside the cell, this signal is then translated by the cell into an appropriate response (i.e., cell division, diffcrcntiation, or secretion). Cell surface reccptors differ greatly with regard to their mechanisms of signal transduction. For example, ligand binding to the insulin or epidermal growth factor receptor leads to direct modification of the kinase activity associated with the extensive cytoplasmic domain of these receptors (Carpenter, 1984; Czech, 1985), thereby allowing signal transduction by tyrosine-specific phosphorylation of intracellular substrates. Ligand binding to the acetylcholine receptor causes direct conformational changes within the multispanning regions of this receptor (Lester, 1977; Raftery et al., 1980). The consequence of these changes is opening of a specific ion channel, allowing influx of cations into the cell. Many receptors without intrinsic enzymatic or ion channel activity operate via receptor-associated transducing proteins. The most commonly used and well studied of these signal transduction molecules are the guanine nucleotide binding proteins or G proteins. Originally identified and characterized for the P-adrenergic receptor, G protcins have been shown to be involved in signal transduction for a large number of plasma membrane associated receptors (reviewed in Gilman, 1987; Stryer and Bourne, 1986). G proteins are believed to be primarily associated with the membranes, a conclusion based upon the requirement for detergent soluhilization during their isolation and purification. However, there is some evidence that they can, in some instances, behave as soluble proteins (Sternweis, 1986; Rodbell, 1985). These molecules are a heterotrimeric complex composed of a,p, and y subunits. In the inactive state, the guanine nucleotide binding site associated with the a subunit is occupicd by GDP (Gilman, 1987). Receptor coupled activation of the G protein is associated with exchange of GTP for GDP, and subsequent deactivation is associated with hydrolysis of bound GTP (Gilman, 1987). Agents which can occupy the nucleotide binding site and mimic GTP but which cannot be hydrolyzed (i.e., GTPyS or fluoride in the presence of aluminum) result in enhanced G protein activity (Gilman, 1987; Bokoch et ul., 1983; Stein et al., 1985). On the other hand, agents which effectively compete for binding of GTP (i.e., GDPPS) uncouple the G protein from receptor-mediated signaling. The a subunits of G proteins are the largest of the three components (39-52 kDa) and have been the most extensively studied. Alpha subunits are characterized based upon sequencc homologies and sensitivity to bacterial toxins from Vibrio cholera (CT) or Borcietellu pertussis (PT). Both toxins result in the covalent modification of the a subunit by catalyzing ADP-ribosylation in the presence of NAD to specific sites of susceptible G proteins (Gilman, 1987). Covalent modification by CT is associated with inhibition of receptor-coupled

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47

GTPase activity (Cassel and Selinger, 1977; Abood er al., 1982) and results in elevation of CAMP levels in the P-adrenergic receptor system (Gilman, 1984). PT catalyzed modification appears to block interactions between susceptible G proteins and their associated receptors (Gilman, 1987). Given these effects, one would predict that CT inactivation would potentiate the receptor-G protein signal whereas PT inactivation would be associated with uncoupling of receptormediated cellular effects. Susceptibility to toxin catalyzed ADP-ribosylation allows assignment of G proteins into four general groups: those susceptible to CT only, to PT only, to both toxins, or to neither toxin. Originally, the terms G, and G, were used to indicate G proteins in the adenylate cyclase system and were distinguished by their specific susceptibility to CT and PT, respectively (Gill and Meren, 1978; Cassel and Pfeuffer, 1978; Katada and Ui, 1982a,b). Functionally, these two groups have been expanded to include any G protein with the characteristic toxin susceptibility regardless of receptor or effector enzyme association. Thus, CT substrates are referred to as G,-like and PT substrates as G,-like G proteins. Sequence comparisons between a subunits of various G proteins have shown significant class variation with at least four variants of a, and three of a,known to exist (Robishaw er al., 1986a,b; Nakada et al., 1986; Itoh et al., 1986; Graziano et al., 1987; Bray er al., 1986; Jones and Reed, 1987). The most conserved regions between subtypes and families is within the guanine nucleotide binding region (Neer and Clapham, 1988), which is not unexpected given the conserved function of this site for all G proteins. Regions of variability between a subtypes do not appear to be randomly distributed. It is therefore tempting to speculate that these regions are important in receptor or effector interactions. Like the a subunit, subclasses of P and y subunits exist (Sugimoto et al., 1985; Fong et al., 1987; Gao et al., 1987; Roff et al., 1985; Gierschik et al., 1985; Sternweis and Robishaw, 1984). However, because of the difficulty in isolating and purifying to homogeneity individual p and y subunits, progress towards the characterization of these subunits has lagged behind that for the a subunits. Interestingly, it has been observed that in some instances Py subunit complexes are interchangeable with different a subunits (Katada et al., 1984; Northup et al., 1983), but whether this interchange affects the specificity of the receptor or effector association has not been established. Speculation as to the function of the p and y subunits has ranged from conferring receptor specificity and binding (Cerione et a l . , 1985) to anchorage to the plasma membrane (Sternweis, 1986). In addition, some evidence exists to suggest that py complexes may be capable of regulating the effector enzyme activity (Katada er al., 1984; Jelsema and Axelrod, 1987; Kim et al., 1989). The mechanism by which G proteins interact with receptors is still unclear. The controversy surrounds the question of whether the a subunit alone confers

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JOHN G.MONROE

specificity by mediating the interaction with the receptor or whether f3y subunits are also involved. The observation that pertussis toxin catalyzed ADP-ribosylation of G,,, G,,,. and G,, inhibits the ability of receptor to activate the G protein (Gilmdn, 1987)provides indirect evidence for this association. Further support of this idea is the finding that the unc mutant of the S49 lymphoma (Haga et ul., 1977), in which an arginine to proline substitution at position 372 of the a subunit mimics the effect of ADP-ribosylation, exhibits uncoupling of receptormediated G, activation (Sullivan et c i l . , 1987) but is capable of activating adenylate cyclase in response to cholera toxin, AIF,-, and nonhydrolyzable GYP analogs. For each of these studies, it is unclear whether covalent modification or mutation of the ci subunit prevents its direct interaction with the receptor or affects its association with Py which then affects the ability of G protein to interact with the receptor. Our own studies in the B-lymphocyte antigen receptor system have shown co-precipitation of [35Slmethionine labeled receptor following immunoprecipitation using anti-a subunit specific antibodies (A. Yellen, D. Manning, and J. G. Monroe, unpublished observations). However, we cannot as yet formally rule out the possibility that f3y subunits are not involved in the association of a to this receptor. More direct evidence for a subunit association with receptor conies from studies that show that purified (Y subunit can interact with isolated receptor. In the f3-adrenergic receptor system, Cerione et d.( 1985) observed ligand induced GDP-GTP exchange by the a subunit in the presence of purified receptor. However, the observed levels of exchange were suboptimal and could be augmented by the presence of Py, suggesting that some receptor interaction mediated through f3y occurs. Consistent with this notion is the finding that the f3-y subunits of transducin (G,) are required for the binding of this G protein to rhodopsin (Fung, 1983). Furthermore, studies by Kelleher and Johnson (1988) show that both the and cy subunits of transducin can block light-dependent phosphorylation of rhodopsin by rhodopsin kinase. That this phosphorylation has been shown to be inhibited by the binding of transducin to rhodopsin argues that f3y as well as cy may be involved in the G protein recognition of receptors. The general mechanism by which G protein coupling of receptors to second messenger-generating systems is accomplished has been inferred from studies in the P-adrenergic receptor-adenylate cyclase system. While thoroughly reviewed elsewhere (Gilman, 1987; Stryer and Bourne, 1986), a few critical concepts need to be mentioned before discussing more specific examples of G protein involvement in lymphocyte activation. The signal transducing G proteins are activated when GDP bound to the a subunit in its inactive form is exchanged for GTP (see Fig. 1). In the absence of receptor signaling this exchange occurs very slowly. However, receptor activation increases the rate of this exchange. The mechanism by which ligand binding

49

3. GTP-BINDING PROTEINS IN LYMPHOCYTE ACTIVATION

GTP

1

2

3

4

GDP

+ P,

FIG. 1 . General mechanism of action of G proteins in receptor signaling. Ligand (L) interaction with receptor (1) leads to conformational changes in the cytoplasmic domain of the receptor which allows G protein to interact with the receptor (2). Subsequent to receptor interaction, exchange of GTP for bound GDP occurs by the a subunit. Following GTP-GDP exchange, the a subunit disassociates from its Py subunits and receptor and then associates with and activates the effector molecule (E). The intrinsic GTPase activity of the a subunit converts the bound GTP to GDP allowing its reassociation with Py (4)and thereby inactivating the G protein.

to the receptor changes the exchange rate of the ci subunit is unknown. Possibly conformational changes imposed by ligand binding might allow a receptor-(; protein interaction, thereby altering the affinity of the ci subunit for GTP. Following GDP-GTP exchange, the ci subunit is believed to dissociate from the Py complex (Gilman, 1987), although this has only been demonstrated for detergent-solublized G proteins. The a subunit-GTP complex is then able to modify the target effector (i.e., adenylate cyclase or phosphodiesterase) to initiate second messenger generation. As mentioned previously, there is evidence that the by complex may also be able to regulate effector activity in some systems (Kim et al., 1989). Signal transduction by the G protein is terminated when the intrinsic GTPase activity of the ci subunit hydrolyzes the GTP to GDP causing the ci subunit to reassociate with the Py complex and revert to the inactive state. An inability to hydrolyze GTP as in the case of nonhydrolyzabie GTP analogs such as GTPyS or in the case of the p21 product of the ras oncogene (Gibbs et ul., 1984; Satoh et a / . , 1987) results in constitutive G protein activity. Based on the generalized scheme for G protein mediated receptor-effector coupling depicted in Fig. 1, several experimentally addressable predictions can be made. If a G protein is involved in transducing signals of a particular receptor: (1) receptor activation will be associated with an increase in the rate of GTP binding and elevated GTPase activity; (2) direct activation of the G protein will

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JOHN G.MONROE

mimic the effect of receptor activation on the specific associated effector system; and, (3) inhibition of the G protein will block receptor-effector coupling. The extent to which these predictions have been evaluated for receptor-driven lymphocyte activation will now be addressed.

II. INVOLVEMENT OF G PROTEINS DURING LYMPHOCYTE ACTIVATION Despite much active research in the G protein field over the past few years, the study of G protein involvement for lymphocyte-associated receptors has only recently begun. The reasons for the late involvement of lymphocyte biologists in this area is due in part to the relatively recent identification and characterization of specific receptors involved in lymphocyte activation and the second messenger systems used in transducing their activation signals. Because the majority of studies to date have focused on G protein involvement during primary activation signaling of resting lymphocytes, 1 will in this discussion concentrate on the T- and B-lymphocyte receptors for antigen. However, studies of G protein involvement in secondary signaling, through lymphokine receptors, will be discussed where appropriate.

A. T-Lymphocyte Activation 1. T-LYMPHOCYTE ANTIGENRECEPTOR The T-lymphocyte receptor for antigen (TCR) is composed of two disulfidelinked glycoproteins. These proteins, called 01 and p, bear constant region (structural) and variable region (ligand binding) domains (Allison and Lanier, 1987; Toyonaga and Mak, 1987; Chapter 1, this volume). The a p heterodimer recognizcs and binds processed antigen in association with products or the MHC (Yague et al., 1985; Dembic et al., 1986). The cytoplasmic domains of the TCR polypeptide chains are extremely short (Hedrick et ul., 1984) and devoid of any known enzymatic activity, making it initially difficult to understand the mechanism by which this receptor transduces primary activation signals following antigen-MHC binding to the T lymphocyte. It is now appreciated that this is accomplished via a complex of membrane-associated molecules called the CD3 complex (Clevers et al., 1988). In contrast to the 01p chains, the polypeptidcs coinprising the CD3 complex possess much longer intracellular domains which may then interact with intracellular transducer molecules or directly be involved in enzyme-catalyzed reactions (Clevers et a l . , 1988). The TCR/CD3 complex belongs to a large family of receptors whose activation signals are transduced at least partially via phospholipase C (PLC)catalyzed

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51

hydrolysis of inositol phospholipids (Imboden et al., 1987; Weiss et a / . , 1986, 1988; Chapter 6, this volume). Given the analogy to other receptor systems involving coupling to PLC and subsequent inositol phospholipid hydrolysis (PI hydrolysis), one would predict coupling to be mediated via G proteins (Smith et al., 1986; Cockcroft and Gomperts, 1985; Aub et al., 1987; Paris and Pouyssegur, 1987; Brass ef u l . , 1986). In human peripheral blood T lymphocytes, direct activation of G protein by AIF, - or GTPyS results in generation of inositol phosphates, breakdown products of PLC-catalyzed inositol phospholipids (Mire-Sluis et al., 1987). While these studies show that PLC can be coupled to G protein in T lymphocytes, they do not address whether there is G protein involvement in the coupling of the TCR/CD3 complex to PLC-catalyzed PI hydrolysis. Using cholera toxin to covalently modify the presumed G protein intermediate between the TCR/CD3 complex and PI-specific PLC, Imboden et al. (1986) showed that CT pretreatment of the T-lymphocyte tumor line Jurkat abrogates the PI hydrolysis response following signaling through this receptor. This effect of CT is unlikely due to uncoupling of the TCR/CD3 complex from PLC. In contrast to PT-catalyzed ADP-ribosylation of CY subunits which uncouples G protein from receptor and thus results in receptor-response uncoupling, CT-catalyzed modification leads to specific inhibition of the GTPase activity associated with the CY subunit (Gilman, 1987). The consequence of this latter effect would actually be potentiation of the coupled response as the modified G protein would no longer be subject to deactivation subsequent to GTP hydrolysis. This is in fact what is observed in the P-adrenergic-adenylate cyclase system (Gilman, 1984). Therefore, the inhibitory effects observed by Jmboden et a!. (1986) with respect to PI hydrolysis are most likely explained by CT effects on other substrates or by CT-mediated (through G,) upregulation of adenylate cyclase leading to an increase in cAMP levels. Although Imboden et al. have argued against cAMP involvement in this phenomenon, this latter interpretation is consistent with the studies of Lerner et al. (1988). These investigators also showed that CT blocks anti-TCR/CD3 stimulated PI hydrolysis and Ca2+ increases. However, they found that this inhibition correlated with increased levels of cAMP and that pharmacological elevation of cAMP also inhibited the PI response following stimulation through the Tlymphocyte antigen receptor. There have been other reports concluding that CT-catalyzed ADP-ribosylation of receptor-linked G proteins can uncouple the receptor from subsequent biochemical responses (Xuan et al., 1987; Lo and Hughes 1987). However, in all cases it cannot be completely ruled out that the observed effect is not due to CTinduced activation of G,. While Lo and Hughes (1987) showed that forskolin did not mimic the effect of CT, they did not verify that they were indeed elevating cAMP levels by this treatment. The most compelling evidence for involvement of G proteins in the transduc-

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JOHN G. MONROE

tion of TCR/CD3 coniplcx generated signals are those involving ligand-induced changes in ornithine decarboxylase (ODC) activity. One of the earliest metabolic events following anti-CD3 or PHA stimulation of T lymphocytes is elevation of ODC activity, occurring within minutes following stimulation (Mustelin et al., 1986; Mustelin, 1987). Depletion of guanine nucleotides inhibits ODC activation in response to anti-CD3 antibodies (Mustelin, 1987), and this effect is reversed by guanine or GTP. Furthermore, exogenous GTPyS can induce ODC in the absence of TCR/CD3 triggering, further suggesting that a GTP-binding protein couples TCR/CD3 to the ODC response. Similarly, using a cloned CTL line, Schrezenmeier et (11. (1988a,b) showed that TCRKD3-triggcrcd granule release of CTL-spccific serine esterases could be inhibited by exogenous GDPPS, while the response could be triggered by GTPyS. GDPpS specifically blocks G proteins by occupying the GTP-binding site of the cy subunit, preventing GDP-GTP exchange (Stryer and Bourne, 1986). In summary, it is clear that G proteins mediate coupling between the TCR/CD3 complex and downstream physiological responses and functional events. It is not so clear as to whether the PI response thought critical to TCRICD3 signal transduction is coupled via G protein. By analogy to other receptor systems where signals are transdueed all or in part by PLC-catalyzed inositol phospholipid hydrolysis (Smith et ul., 1986; Cockcroft and Gomperts, 1985; Aub et al., 1987; Paris and Pouyssegur, 1987; Brass et a l . , 1986), it is very likely that the Tlymphocyte antigen receptor is coupled to a guanine nucleotide binding protein. While available experimental evidence is consistent with G protein involvement in signal transduction through the TCRICD3 complex on T lynlphocytes, this evidence is indirect and inconipletc. Published studies are few in number and, as discussed, often conflicting. Appropriate mechanistic studies, such as those outlined in Section I, are critical to a definitive description of signal transduction through this receptor. More complete studies of this type have been performed for the T-lymphocyte interleukin 2 receptor. 2.

~ N T E R L E U K I N2

RECEPTOR (IL-2R)

Primary activation signals generated through the TCK/CD3 complcx arc by themselves insutficient to initiate and sustain proliferation by T lymphocytes. For proliferation, the T lymphocyte (specifically TI,I and Tc..,L) must receive lymphokinc-mcdiatcd secondary stimulation through the IL-2R (Robb, 1984). Studies by Evans et (11. (1987) argue for a role for G proteins in the signal transduction following interaction of IL-2 with its receptor. Using isolated plasma membranes from an 1L-2 dependent, T-lymphocyte cell line, they observed increased GTPase activity following IL-2 binding. The G protein involved belongs to the G, family as 1L-2 stimulated GTPase activity was inhibited by PT. In addition, the investigators observed an incrcase in the rate of labeled GTP bind-

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53

ing to membranes stimulated with IL-2. Binding was inhibited by unlabeled GDP and GTPyS but not ADP or ATP. ADP-ribosylation studies showed the PT substrate to be a 41 kDa protein.

B. 6-Lymphocyte Activation B Lymphocytes recognize antigen via a surface form of immunoglobulin. -Like the T-lymphocyte aP heterodimer, surface immunoglobulin (sIg) possesses a very abbreviated putative cytoplasmic domain. Both sIgM and sIgD, the two receptors found on primary mature B lymphocytes (Goding, 1978; Monroe et al., 1983), have cytoplasmic domains which are comprised of only three amino acids (Tucker et al., 1982). Thus, the B-lymphocyte antigen receptor is unlikely to possess any intrinsic enzymatic activity to mediate ligand-generated signal transduction. Like the T-lymphocyte receptor for antigen, slg signaling results in generation of the second messengers inositol trisphosphate and diacylglycerol resultant from PLC-catalyzed PI hydrolysis. Association of G proteins with slg has been inferred from studies by two laboratories. Using the murine B cell lymphoma WEHI-23 I , Gold et al. (1987) have shown stimulation of PI hydrolysis by exogenous GTPyS. Importantly, GTPyS augments the effect of ligand-induced sIg cross-linking with respect to PI hydrolysis, suggesting that the G protein involved is associated both with sIg and PLC. Similar findings using nontransformed murine splenic B lymphocytes were reported by Harnett and Klaus (1988). Further evidence for a G protein involvement in coupling slg to PI hydrolysis was provided by the observation that the inhibitor GDPPS could block ligand-induced PI hydrolysis through slg (Gold et ul., 1987; Harnett and Klaus, 1988). These two studies provide indirect evidence for G protein association with slg and are consistent with a role for this molecule in coupling sIg to PI hydrolysis. More direct evidence comes from our own studies (Monroe and Haldar, 1989). As discussed previously, if a G protein is coupled to a particular receptor system, a prediction is that ligand binding to that receptor will cause an increase in the rate of GTP-GDP exchange by the membrane-associated G protein. Using isolated plasma membranes from WEHI-23 I or nontransformed murine splenic B lymphocytes, we have observed an increase in the rate of GTPyS binding following sIg cross-linking by anti-receptor antibodies (anti-Ig) (Fig. 2). This increase was specific for guanine nucleotides as it could be inhibited by unlabeled GDP but not ADP (Table I). Furthermore, we have observed an increase in the level of GTPase activity associated with these plasma membranes following stimulation by ligand (Table 11). Characterization of the slg associated G protein has been somewhat confusing. Gold et at. (1987) have concluded that the sIg-linked G protein in WEHI-23 1 is insensitive to both PT and CT. Similarly, Harnett and Klaus (1988) have reported that the G protein associated with this receptor in untransformed murine splenic

54

JOHN G . MONROE

1

Splenic B cells

4.0

2.0

t

WEHI-231

1.5

3.0

2.0

0.5

n

0.0

.n

U

5

0

IU

10

Time (minutes) FIG. 2 Stimulation of specific ["SIGTPyS binding to membranes from untransformed murine splenic B lyniphocytes and WEHI-231. Isolated nicnibrancs (25 pg) froni unstimulatcd WEHI-231 or nwrine splenic I3 lymphocytes were suspended in binding buffer (10 mM Tris-HCl. pH 7.8; I0 mM MgCI,; 1 nlM EDTA; 0.2% BSA; 0.5 i r l M ascorbic acid; 2 nlM adcnyl-5-iniidodiphosphatc). Mcnibranes were stimulated at 37°C with anti-l* antibodies (@-a)or buffer (0-O), then placed on ice. Binding reactions wcrc initiated by addition of 25 nM [3s]GTPyS and 10 J L anti-p ~ antibodies. ~ 1ice-cold Reactions were continued for thc indicated times and then stopped by addition of 4 5 0 ~ of stopping buffcr (2SpM unlabeled CTPyS; 10 mM Tris-HCI. pH 7.4; 100 IMNaCI; 0.1% Lubrol). Free-labeled nucleotidcs wcrc separated by filtration thi-ough 0.45 phf nitroccllulosc IiiciIibrdnCS and washed with 25 nil of stopping buffer without GTPyS. (After Monroe and Haldar, 1989.)

TABLk I N L J C L ~i i )Ot IS i w i t i c I I Y ot I H E ANTI-JL STIMULATED INCREASE IN Gl'P-BlNDlNG l3Y Pt A 9 M A MTMRRANFS t K O M M U K ~BNLYMPHOCYTtS" ~ Stimulus None Anti-pL" Anti-l* Anti-p

Competitor'

GTPyS bound (pmolimg membrane protein)

~

0.55 t 0.10

-

1.34 I 0 . 1 6

ADP GDP

1.31 0.13 0.23 2 0.13

*

After Monroe and Haldar (19x9). Binding reactions on isolated plasma mcmbrdnes from the miurine I3 lymphoma were performed as described for Fig. 2. Reactions were termi(1

6

nated at 10 min. "

Compctitors were prescnl at 2.5 p M Present at I U ~ g i i n l .

3. GTP-BINDING PROTEINS IN LYMPHOCME ACTIVATION

55

TABLE I1 EFFECTOF A N T I - pSTIMULATION ON CTPASE ACTIVITY BY ISOLATED PLASMA MEMBRANES FROM MURINE B LYMPHOCYTES~ Stimulus None Anti-p

P, release (pmollmgimin)

5.1 C 0.2 7.2 ? 0.1

‘1 CTPase activity was determined by measuring the release of TO,’-from [y-z2P]GTP. The reaction mixture contained 10 mM Tris-HCI, pH 7.4, 100 nm [y-?ZP]GTP, 0.1 mM ATP, 3 mM creatinr phosphate, 75 U/ml creatine phosphokinase, and 10 kgiml of gost anti-mouse pchain antibody in stimulated reaction mixes (total volume 50 111). The reaction was initiated by addition of 25 p g of isolated membrane protein from unstimulated WEHI-231 B-lymphoma cells. The reaction was stopped al’ter 5 min at 37°C by addition of SO0 &I of 5% (wiv) Norit A in potassium phosphate, pH 7.0. After centrifugation, 10 pI of the supernatant was counted for 3*P0,3 , and P, released was calculated.

B lymphocytes to be PT insensitive. Our own studies (Monroe and Haldar, 1989) conflict with these results. We have observed inhibition of both ligand-stimulated GTP binding (Table 111) as well as PLC-catalyzed inositol phospholipid hydrolysis (Table IV) following PT pretreatment. The reason for the discrepancy between our results and those of Gold et ul. and Harnett and Klaus is unclear. Our studies employed isolated plasma membranes from WEHI-23 1 while the studies of Gold et al. utilized intact cells. When we repeated our studies using intact cells, we have generally been unable to observe reproducible inhibition of PI hydrolysis following sIg signaling in PT pretreated cells. We believe that the inability to demonstrate PT sensitivity in the intact cell system may reflect an inability to modify 100% of the G protein. In the isolated membrane system, ADP-ribosylation may be significantly more efficient. Supporting this conclusion are studies of Pobiner et al. (1985) where it was shown that a nonlinear relationship exists between the degree of PT catalyzed ADP-ribosylation and the extent of attenuation of the effector response. Incomplete ribosylation of even less than 20% of the G proteins still resulted in complete receptor-mediated responses. In addition, it has been argued that much longer incubation times are required for PT modification of intact cells (Katada and Ui, 1980; Pobiner et d . , 1985), suggesting that effects on intact cells may be relatively inefficient. However, it should be noted that PT treatment of intact WEHI-23 l cells was effective in completely inhibiting the function of the G protein operative in LPS-mediated

56

JOHN G. MONROE

TABLE 111 TOXINPRETREATMENT ABROGATES THE ANTI-p STIMULATED INCREASE I N GTP BINDINGBY ISOLATED MEMBRANES FROM M L I R I NBELYMPHOCYTEV

PERTUSSIS

Pertussis toxin

Stimulus

pretreatment"

None

-

Anti+ Anti-p.

-

None

-t

GTPyS b o u n d ( p m o l i m g m e m b r a n e protein) 0.60 2 0.03 1.29 0.11 0.62 ? 0.02 0.58 5 0.10

*

+

'4 Binding reactions were performed (in iwlaled rncnihrancs lrom the inurine 8-lytnphonia WEHI-231 exactly as described for Pig. L Binding reaction5 were terminated at 5 min. Pertussis toxin catalyzed ADP-ribmylation was accoinpliahcd as follows: membranes (I mgiinl) lrom unstimulated WEHI-231 B-lymphoma cella were pretreaied ( I S rnin al 30°C) with or withoul 7.5 pg/ml olactivated pertussis toxin in 150 niM potassium phosphate. p H 7 5, I m M NAD i , 0.5 tnM ATP, I0 iiiM thytnidinr, and SO (IM G I P . After Monroe and lialdar (1989).

''

TABLE 1V HYDROLYSIS RESULTING FROM ANTI+ STIMULATION OF I s o L A r m MEMBRANES I S INHIBITED B Y PERTUSSIS TOXINPRETREATMENT"

INOSITOI. Ptl(JSPHOL1PID

Stimuluq

Pertussis toxin pretreatmentb

None Anti-p. Anti-p

None

-

+ +

[ 3H]Inositol phosphate production (cpni)

65 5 5 386 + I 1 66 ? 8 25 2 5

fl Relative inotitol phospholipid hydrolysis wad determined by ineasuremen1 of relative production oi watcr-soluble labeled inositol phosphates. WEHI-231 cells were labeled with [ 'H]myo-inositol as described previously (Monroe rl u l . , 1989). I5dated inembranes from labeled WEHI-23 I wcrc pretreated with pertussis toxin a5 described for Table 111. Pretreated or untreated membranes (75 p g protein) were suspended in Hepea huffcred saline ciinlaining 5% feral hrivine semtn and 10 rnM LiCl and incubated for 45 min at 37°C with or without 10 kgiml of goat anti-mouse p antihody. Reactions were terminated, and levels ot inowol phosphates producctl wcrc tlolcriiiined exactly as described previously (Monroe r / a / . . 19x9). After Mmiroe and Haldar (19x9).

3. GTP-BINDING PROTEINS IN LYMPHOCYTE ACTIVATION

57

signaling (Jakeway and DeFranco, 1986). Therefore, additional studies are indicated in order to resolvc this area of dispute. It should also be noted here that Klaus et ul. ( 1 987) have shown inhibition of ligand-induced DNA synthesis following pretreatment of murine splenic B lymphocytes with CT. More proximal activation events associated with sIg signaling, such as increased class 11 antigen expression (Klaus et ul., 1987) and PI hydrolysis (Gold et a / ., 1987), were not shown to be inhibited by CT treatment. Agents which facilitate a rise in CAMP were shown (Klaus et ul., 1987) to only partially inhibit ligand-stimulated DNA synthesis allowing the possibility that some of the observed inhibitory effects were due to CT-mediated inactivation of a sIg associated G protein. However, this was considered the less likely explanation by the authors. Again, as discussed previously, the predicted effect of CTinduced modification should be a potentiation of the effect rather than inhibition. Although inhibition of G protein coupled cellular effects has been reported (lmboden et al., 1986; Xuan et af., 1987; Lo and Hughes, 1987), in all cases the observed effects cannot be conclusively disassociated from CT-mediated activation of G,.

111.

FUTURE PERSPECTIVES

What is most clearly apparent from the above discussion is that much is still unresolved with regard to G protein involvement in receptor-driven lymphocyte activation. Lack of understanding occurs in part because at one level there is incomplete characterization of the G protein involved. While evidence is consistent with G protein involvement in coupling the TCR/CD3 to specific T cell responses, very little has been accomplished with respect to the biochemical characterization of this signal transducing molecular complex. Furthermore, the biochemical pathways to which these G proteins are coupled are unknown. Coupling of this receptor system to the PLC-catalyzed hydrolysis of inositol phospholipids is inferred from data that are neither direct nor definitive. Studies, such as those described for the 1L-2 receptor, are indicated in order to establish directly coupling of a G protein to the TCRICD3 complex as well as to show definitively G protein involvement as a signaling intermediary molecule between this receptor and PLC-catalyzed PI hydrolysis. Studies in the B-lymphocyte system have been more complete. Data discussed previously clearly establish G protein coupling to PLC-mediated PI hydrolysis in these cells. However, G protein association with surface immunoglobulin as well as its involvement in coupling receptor to PI hydrolysis are not as definitive. Our studies may begin to fill in some of these gaps. GTP binding studies and ligandinduced changes in the rate of GTPase activity provide strong evidence that one

58

JOHN G. MONROE

or more G proteins are associated with sIg. Furthermore, our ability to show inhibition of slg-mediated PI hydrolysis following PT pretreatment is supportive of thosc studies of Gold ei al. (1987) and Harnett and Klaus ( 1 988) who showed uncoupling of slg and PI hydrolysis in permeabilized B lymphocytes in the presence of the G protein inhibitor GDPPS. Several issues remain to be resolved in the B-lymphocyte system. For the most part, these arc central unresolvcd issues in all reccptor systems in which G protein involvement has been addressed. The first issue centers around the number of G proteins associated with this receptor. Whether multiple G proteins are involved in coupling sIg to the same or different second messenger systems is not known. Evidence exists to suggest that signaling pathways distinct from PI hydrolysis are operative in slg signaling (Mond et al., 1987). Whether coupling of these pathways occurs through a G protein and, if so, if this linkage depends upon a distinct G protein can at this point only be speculated upon. Similarly, there is evidence (Ashkenazi et al., 1989) that shows that distint G proteins can regulate the level of PLC-catalyzed PI hydrolysis through different muscarinic acetylcholine receptor subtypes in the same cell. Given prior studies where G proteins from different receptor systems have been shown to interact with a particular receptor, one could speculate that sIg may, at least under certain physiological conditions, be associated with more than one G protein species involved in PLC coupling. Perhaps this might explain the differences in PT sensitivity in our system and that of Gold er (11. (1987). This last point retlects another issue in which very little is known in the sIg or any other receptor system. Within any given cell there arc undoubtedly multiple G proteins coupling various receptors to specific biochemical pathways and differential changes in cellular physiology. The mechanism by which the specificity of the G protein for a particular receptor/sccond messenger system is achieved is a central question to the understanding of receptor-mediated signaling but nonetheless remains unresolved. Discussed in Section 1, the molecular basis for G protein interactions with receptor and effector enzyme systems, and therefore specificity, has not been delineated. Whether it is this type of mechanism which confers specificity to the receptor-(; protein-effector system or whether G proteins are inherently of limited specificity and appropriate associations are insured through cellular compartmentalization remains to be evaluated. How G proteins interact with their receptors within the plasma membrane is particularly problematic for slg. Although G proteins appear to be intimately associated with the cytoplasmic surface of the plasma membrane, evidence argues against the notion that they are transmembrane proteins or are even partially embedded in the membrane (Chabre, 1987). Thus, it appears that G proteins interact with receptors via the protruding cytoplasmic domains (O’Dowd et ul., 1988). However, in contrast to the P-adrenergic receptor and the T cell receptor through the associated CD3 complex, sIgM or slgD do not possess significant

3. GTP-BINDING PROTEINS IN LYMPHOCYTE ACTIVATION

59

cytoplasmic domains. Therefore, the mechanism by which they interact with G proteins is unclear. Whether the G protein has access to the transmembrane domain of these receptors or whether these data argue that intermediary proteins analogous to the CD3 complex must exist between slg and G protein are important areas for future research. REFERENCES Abood, M. E., Hurley, J. B.. Pappone, M.-C., Boumc, H. R., and Stryer, L. (1982). Functional homology between signal-coupling proteins. J . B i d . Chem. 257, 10540- 10543. Allison, J. P., and Lanier, L. L. (1987). The structure, function, and serology of the T cell antigen receptor complex. Anu. Rev. Immunol. 5, 503-540. Ashkenazi, A,, Peralta, E. G . , Winslow, J. W., Ramachandran, J., and Capon, D. J. (1989). Functionally distinct G proteins selectively couple different receptor to PI hydrolysis in the same cell. Cell 56, 487-493. Aub, D. L., Gosse. M. E., and Cote. T. E. (1987). Regulation of thyrotropin-releasing hormone receptor binding and phospholipase C activation by a single GTP-binding protein. J . B i d . Chem. 262, 9521-9528. Bokoch, G. M., Katada, T., Northup, J. K . , Hewlett, E. L., and Gilmdn, A. G. (1983). Identification of the predominate substrate for ADP-ribosylation by islet activating protein. J . B i d . Chem. 258, 2072-2075. Brass, L. F., Laposata, M., Banga, H. S., and Rittenhouse, S. E. (1986). Regulation of the phosphoinositide hydrolysis pathway in thrombin stimulated platelets by a pertussis toxinsensitive guanine nucleotide-binding protein. J . Biol. Chem. 261, 16838- 16847. Bray, P., Carter, A., Simmons, C., Guo, V.. Puckett, C . , Kamholtz, J . , Spiegel, A., and Nirenberg, M. ( 1986). Human cDNA clones for four species of G,,, signal transduction protein. Proc. Nutl. A m d . Sci. U.S.A. 83, 8893-8897. Carpenter, G . (1984). Properties of the receptor for epidermal growth factor. Cell 37, 357-358. Cassel, D., and Pfeuffer, T. (1978). Mechanism of cholera toxin action: Covalent modification of the guanyl nucleotide-binding protein of the adenylate cyclase system. Proc. Natl. Acad. Sci. U.S.A. 75, 2669-2673. Cassel, D.. and Selinger, 2 . V. 1. (1977). Mechanism of adenylate cyclase activation by cholera toxin: Inhibition of GTP hydrolysis at thc regulatory site. Proc. Narl. Acad. Sci. U . S . A . 74, 3308-331 1 . Cerionc, R. A , , Staniszcwski, C., Benovic. J. L . , LeRowitz, R. J., Caron, M. G . , Giershik, P., Somers, R., Spiegel, A. M., Codina, J.. and Birnbaumer, L. (1985). Specificity of the functional interactions of the (3-adrenergic receptor and rhodopsin with guanine nucleotide regulatory proteins reconstituted in phospholipid vesicles. J . Biol. Chem. 260, 1493- 1500. Chabre, M. (1987). The G protein connection: is it in the membrane or the cytoplasm? Trends Biochem. Sci. 6, 213-215. Clevers, H., Alarcon, B., Wileman, T., and Terhorst, C. (1988). The T cell receptorlCD3 complex: A dynaniic protcin enscmblc. Annu. Rev. Immunol. 6, 629-662. Cockcroft, S., and Gomperts, B. D. (1985). Role of guanine nucleotide binding protein in the activation of polyphosphoinositidc phosphodicstcrasc. Nuture (London) 314, 534-536. Czech, M. P. (1985). The nature and regulation of the insulin receptor: Structure and function. Annu. RPV. Physiol. 47, 357-381. Dembic, Z . , Haas. W., Weiss. S., McCubrcy. J . , Kiefer, H., vonBoehmer, H., and Steinmetz, M. (1986). Transfer of specificity by murine (Y and p T cell receptor genes. Nuture (London) 320, 232.

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JOHN G. MONROE

Evans, S. W.. Beckner, S. K., and Farrar. W. L. (1987). Stimulation of specific GTP binding and hydrolysis activities in lyniphocyte membrane by intcrleukin-2. Nufirrc. (London) 325. 166- 168. Fung, H K. W.. Aniatrada, T. T., Birrcn, B . W.. and Simon, M. F (IW7). Distinct l'oriiis of the p subunit of GTP-binding regulatory proteins identified by molecular cloning. Pror. Nut/. Acod. Sci. U . S . A . M,3792-3796. Ring. B . K . K. (1983) Characterization of transducin from bovine retinal rod outer segments. 1. Separation and reconstitution of the subunits. J . B i d . L'hrm. 258. 10495-10502. Gao. B . , Gilnian, A. G . , and Robishaw, J. I). (1987). A second fiiriii of the p subunit of signaltransducing G proteins. Proc. Not/. Acud. Sci. U . S . A . 84, 6122-6125. Gibbs, J. B., Sigal, S., Poe, M., and Scolnick. E. M. (1984). Intrinsic GTPase activity distinguishes normal and oncogcnic I'US p21 molecules. f ro ( ,.Nutl. Arad. Sci. U.S.A. 81, 5704-5708. Gierschik, P., Codinn, J., Sirnmons, C.. Bimbaumer, L . , arid Spicgcl, A. (1985). Antisera against a guanine nucleotide binding protein froiii retina cross-react with thc fi subunit of the adenylyl cyclasc-associated guanine nucleotide binding proteins N, and N,. Pror. Nurl. A w d . Si,i. U.S.A. 82, 727-731, Gill, D. M.. and Meren, R . (1978). AIWribosylation uf membrane proteins catalyzed by cholera toxin: Basis of the activation of adcnylatc cyclase. Froc.. NLI//.Ac.crcl Sci. 1l.S.A. 75, 30503054. Gilman, A. G. (19x4). G proteins and dual control of adenylate cyclase. C P / /36, 577-579. Gilnian. A . G. (1987). G proteins: Transducers of receptor-generated signals. Annu. Rev. Binchern. 56, 615-649. Goding, J. (1978). Allotypcs of IgM and IgD rcccptors in the muusc: A probc for lymphocytc ditferentiation. Conremp. Top. Immumhiol. 8. 203-243. Gold, M. R., Jakeway. J. P., and DeFrancu, A. L. (1987). Involvement of a guanine nucleotidebinding component in membrane IgM-stimulated phosphoinositide breakdown. J . Irnmunol. 139, 3604 -3613 Graziano. M . P., Casey, 1'. J.. and Gilman, A. G . (1987). Expression of cDNAs for G proteins in Escherichia coli. J . Biol. Chem. 262,11375- I 138 I . Haga, T.. Ross. E. M., Anderson. H. J . , and Gilnlari. A. G. (1977). Adenylate cyclase pcrinanently uncoupled from hormone receptors in a novel variant of 549 mouse lymphoma cells. Pruc. N d . A i u d . Sri. U.S.A. 74, 2016-2020 Haldat, and Monroe, J. G . . (1989). Submitted for publication. Harnetr. M. M., and Klaus. G . C . B. (1988). G protein coupling ul' antigen receptor-stimulated pulyphosphoinositide hydrolysis in B cells. J. irnmunol. 148, 3135-3 139. Hedrick, S. M.. Nielwn. E. A , , Kavalcl-. J., Cohen, D. I., and t h i s . M . M. (1984). Sequcncc relationships between piitativc 'I-cell receptor polypeptides and immunoglobulins. Nurrrrc ( I m dc>n) 308, 153-158. linboden, J. R . . Shoback, D. M.. Pattison, G . , and Stobo, J. D. (19R6). Chulcra toxin inhibits the Tcell antigen receptor-mediated increases in inositol trisphosphate and cytoplasmic frcc calcium. P m r . Nut/. Actrd. Sci. U . S . A . 83, 5673-5077. Imbodcn, J . , Wcyand. C., and Gurunzy. J. ( 1987). Antigen rccognirion by a human T cell clone lcads to increases in inositol trisphosphate. J . /mmurto/. 138, 1322- 1324. Ituh, H . , Komsa, T., Nagata, S., Nakamura, S . , Katada, T., IJi. M . , Iwai, S.. Ohisuka, E., Kawasaki, H . , Suzucki. K., and Kaziro. Y. (1986). Molecular cloning and sequence detcmiinat i o n of cDNAs for a subunits of the guanine nuclcotide-binding proteins G , , G,, and G, from rat brain. f r o c Nutl. Acad. Sci. U . S . A . 83, 3776-3780. Jakcway. J. P., and DcFranco. A . 1.. (1986). Pertussis toxin inhibition of B ccll and macrophage responses to bacterial lipopolysaccharide. Science 234, 743-746. Jclsema, C. L., and Axclrod, J. (1987). Stimulation of pbospholipase A2 activity in bovine rod outcr segmcnts hy the beta ga111ma subunits of transducin and its inhibition by thc alpha subunit. froc.. Narl. Actid. Sci. U . S . A . M,3623-3627

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Jones, D. T., and Reed, R. R . (1987). Molecular cloning of five GTP-binding protein cDNA species from rat olfactory neuroepithelium. J. Biol. Chem. 262, 14241- 14249. Katada, T.. and Ui, M. (1980). Slow interaction of islct activating protein with pancreatic islets during primary culturc to cause reversal of a-sdrenergic inhibition of insulin secretion. J. B i d . Chem. 255, 9580-9588. Katada, T., and Ui, M. (1982a). Direct modification of the membrane adenylate cyclase system by islet-activating protein due to ADP-ribosylation of a membrane protein. Proc. Nut/. Acad. Sci. U . S . A . 79, 3129-3133. Katada, T., and Ui, M . (1982b). ADP ribosylation of the specific membrane protein of C6 cells by islet-activating protein associated with modification of adenylate cyclase activity. J. B i d . Chern. 257, 7210-7216. Katada, T., Bokoch, G. M . , Northup, J. K . , Ui, M., and Gilman, A. G. (1984). The inhibitory guanine nucleotide-binding regulatory component of adenylate cyclase. J . Biol. Chern. 259, 3568-3577. Kellehler, D. J., and Johnson, G. L. (1988). Transducin inhibition of the light-dependent rhodopsin phsophorylation: Evidence for Py subunit interaction with rhodopsin. Mol. Phurmacol. 34, 452-460. Kim, D., Lewis, D. L., Graziadei. L., Neer, E. J., Bar-Sagi, D . , and Clapham, D. E. (1989). Gprotein py-subunits activate the cardiac inuscarinic K + -channel via phospholipase A,. Nature (London) 337, 557-560. Klaus, G. G . B., Vondy, K., and Holman, M. (1987). Selective effects of cholera toxin on the activation of mouse B cells by different polyclonal activators. Fur. J. Immunol. 17, 1787-1792. Lerner, A., Jacobson, B., and Miller, R. A. (1988). Cyclic AMP concentrations modulate both calcium flux and hydrolysis of phosphatidylinositol phosphates in mouse T lymphocytes. J. Immunol. 140, 936-940. Lester, H. A. (1977). The response to acetylcholine. Sci. Am. 236, 106-1 18. Lo, W. W. Y., and Hughes, J. (1987). A novel cholera toxin-sensitive G-protein (G,) regulating receptor-mediated phosphoinositide signaling in human pituitary clonal cells. FEES Lett. 220, 327-33 1. Mire-Sluis, A. R., Hoffbrand, A. V., and Wickremasinghc, R. G . (1987). Evidence that guaninenucleotide binding regulatory proteins couple cell-surface receptors to the breakdown of inositol-containing lipids during T-lymphocyte mitogenesis. Biochem. Biophys. Rrs. Commun. 148, 1223-1231. Mond. J. J., Feuerstein, N., Finkelman, F. D., Huang, F., Huang, K.-P., and Dennis, G. (1987). Blymphocyte activation mediated by anti-immunoglobulin antibody in the absence of protein kinase C. Proc. Nut/. Acad. Sci. U.S.A. 84, 8588-8592. Monroe, J. G . , and Haldar, S . (1989). Involvement o f a specific guanine nucleotide binding protein in receptor immunoglobulin stimulated inositol phospholipid hydrolysis. Biochirn. Biuphys. Acta (in press). Monroe, J. G., Havran, W. L., and Cambier, J. C. (1983). B lymphocyte activation: entry into cell cycle is accompanied by decreased expression of IgD but not IgM. Eur. J. Immunul. 13, 208213. Monroe, J. G., Seyfert, V. I,.. Owen, C. S.. and Sykes, N . (1989).Isolation and characterization o f a B-lymphocyte mutant with altered signal transduction through its antigen receptor. J. Exp. Med. 169 (in press). Mustelin, T. (1987). GTP dependence of the transduction of mitogenic signals through the T3 complex in T lymphocytes indicates the involvement of the G-protein. FEES Lett. 213, 199203. Mustelin, T., Poso, H., Iivanainen, A., and Andersson, L. C. (1986). myo-inositol reverses Li+induced inhibition of phosphoinositide turnover and ornithine decarboxylase induction during carly lymphocyte activation. Eur. J. Irnmunol. 16, 859-861.

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Nakada, T., Tanabc, T.. Takahashi, H., Noda. M., Haga, K., Haga, T.. Ichiyama, A., Kangawa, K.. Hiranaga, M.. Matsuo, H . , and Numa, S. (1986). Primary structure of the a-subunit of bovine adenylatc cyclase-inhibiting G-protein deduced from the cDNA sequence. FEBS Lett. 197, 305-310. Neer, E. J . , and Clapham, D. E. (1988). Roles of G protein subunits in transmembrane signalling. Nature (London) 333, 129- 134. Northup, 1. K . , Stemweis, P. C., and Gilman, A. G. (1983). The subunits of thc stimulatory regulatory component of adenylate cyclase. J. Biol. Chem. 258, 11361- 11368. O'Dowd, B. F., Hnatowich, M., Regan, J. W.. Leader, W. M., Caron, M. G . , and Lcfkowitz, R. J. ( 1988). Site-directed mutagenesis of the cytoplasmic domains of the human P2-adrenergic rcccptor. .I. Biol. Chem. 263, 15985- 15992. Paris, S., and Pouyssegur, J. (1987). Further evidence for a phospholipase C-coupled G protein in hamster fibroblasts. J. B i d . Chem. 262, 1970-1976. Pohiner, B. F., Hewlett. E. I.., and Garrison, J. C. (1985). Role of N, in coupling angiotcnsin rcceptors to inhibition of adcnylate cyclase in hcpatocytes. J . Biol. C'hem. 260, 16200- 162OY. Raftery, M. A , , Hnnkapiller, M. W., Strader, C. D., and Hood, L. E. (1980). Acetylcholine receptor: Complex of homologous subunits. Science 208, 1454 -~ 1457. Robb, R. J. (1984). Interleukin 2: The molecule and its function. Immunol. Ti& 5 , 203-209. Robishaw, J. D., Russell, D. W.. Harris, B. A , , Smigel, M. D.. and Gilman, A. G . (1986a). Deduced primary structure of the LY subunit of the GTP-binding stimulatory protein of adenylate cyclase. Pruc. Nut/. Acad. Sci. U . S . A . 83, 1251-1255. Robishaw, J. D., Smigel, M. D., and Gilman, A. G . (1986b). Molecular basis for two forms of the G protein that stimulates adenylate cyclase. J. Biol. Chem. 261, 9587-9590. Rodbell, M . (1985). Programmable messengers: A new theory of hormone action. Trends Biochem. P i . 10, 461-464. Roff, D. J . , Applebury, M. L.. and Stemwies, P. C. (1985). Relationships within the family of GTPbinding proteins isolated froin bovine central nervous system. J. Biol. Chem. 260, 1624216249. Satoh, T., Nakamura, S., and Kaziru, Y. (1987). Induction of ncurite formation in PC12 cells by microinjection of proto-oncogenic H a m s protein preincubated with guanosine-S'-O-(3-tlliotriphuspatc). Mol. Cell. Biul. 7 , 4553-4556. Schrezenmeier, H., Ahnert-Hilger, C . , and Flcischer, B. (1988a). A T cell receptor-associated GTPbinding protein triggers T cell receptor-mediated granule exocytosis in cytotoxic T lyrnphocytcs. J . Immund. 141, 3785-3790. Schrezenmeier, H . . Ahncrt-Hilgcr, G.. and Fleiseher, B . (1988b). Inactivation of a T cell receptorassociated GTP-binding protein by antibody-induced modulation of the T cell receptoriCD3 complex. J. E.rp. M e d . 168, 817-822. Smith, C. D.. Cox. C. C., and Snyderman, R. (1986). Receptor-coupled activation of phosphninositide-specific phospholipase C by an N protein. Science 232, 97- 100. Stein, P. J . . Halliday, K. R . , and Rasenick, M. M. (1985). Photoreceptor CiTP binding protein mediates lluoride activation of phosphodiesterase. J . B i d . Chem. 260, 908 1-9084. Sternwcis, P. C. (1986). The purified a subunit of G,, and G, from bovine brain require py for association wilh phospholipid vesicles. J. R i d . Chem. 261, 63 1-637. Sternwcis, P. C., and Robishaw, 1. D. (1984). Isolation of two proteins with high affinity for guaninc nucleotides from membranes 01 bovine brain. J. Biul. Chem. 259, 13806- 13813. Stryer, L . . and Bourne, H. R. (1986). G proteins: A family of signal transducers. Annu. Rev. Cell. B i d . 2, 391-419. Sugimoto. K . , Nukada. T.. Tanabe, T., Takahashi, H., Noda, M., Minamino, N., Kanagawa, K . . Matsuo, H., Hiroae, T.. Inayama, S., and Numa, S . (1985). Primary structure of the P-subunit of bovinc transducin deduced from the cDNA sequence. FEBS Lerf. 191, 235-240.

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Sullivan, K. A,, Miller, R. T., Masters, S . B., Beiderman, B., Heideman, W., and Bourne, H. R . (1987). Identification of receptor contact site involved in receptor-(; protein coupling. Nature (London) 330, 758-760. Toyonaga, B . , and Mak, T. (1987). Genes of the T cell antigen receptor in normal and malignant T cells. Annu. Rev. Immunol. 5 , 585-620. Tucker, P. W., Cheng, H.-L., Richards. J. E., Fitzmaurice, L., Muchinski, J. F., and Blattner, F. R. (1982). Genetic aspects of IgD expression: 111. Functional implications of the sequence and organization of the C.. gene. Ann. N . Y . Acad. Sci. 399, 26-38. Weiss, A , , Imbodcn, J . , Hardy, K., Manger, B., Terhorst, C . , and Stobo, J. (1986). The role of the T3/antigen receptor complex in T cell activation. Annu. Rev. Immunol. 4, 593-619. Weiss, E. R., Kelleher, D. J., Woon, C. W., Soparkar, S . , Osawa, S., Heasley, L. E., and Johnson, G . L. (1988). Receptor activation of G proteins. FASEB J . 2 , 2841-2848. Xuan, Y.-T., Su, Y.-F., Chang. K.-J., and Watkins, W. D. (1987). Apertussisicholera toxin sensitive G-protein may mediate vasopressin-induced insoitol phosphate formation in smooth muscle cell. Biochem. Bioph.ys. Res. Commun. 146, 898-906. Yague, J., White, J., Coleclough, C., Kappler, J., Palmer, E., and Marrack, P. (1985). The T cell receptor: The CY and chains define idiotype, and antigen and MHC specificity. Cell 42, 81.

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C U R R h N T TOPICS IN M t M B R A N E S A N D TRANSPORT. VOLUME 35

Chapter 4 Signal Transduction by GTP Binding Proteins during Leukocyte Activation: Phagocytic Cells GARY M . BOKOCH Department of fmmunofogy Research Institute of Scripps Clinic La Jollu, Culdorniu 92037

I 11

111

IV.

V.

VI .

Introduction G T P Binding Regulatory Proteins A . The Receptor-Coupled (Oligomeric) G T P Binding Proteins B. The Low Molecular Weight G T P Binding Proteins G T P Binding Proteins as Mediators of Neutrophil Activation A. Evidence for the Interaction of Chemoattractant Receptors with a Pertussis Toxin-Sensitive G T P Binding Protein B. Nonpcrtussis Toxin Substrate GTP Binding Proteins: Evidence for Roles in Neutrophil Secretion and NADPH Oxidase Activation C . N-Formyl Peptide Receptor Affinity States: Evidence That These States Can Be Defined as Particular Forms of L-R-G The G T P Binding Protein Composition of the Neutrophil A. Oligomeric (Receptor-Coupled) G Proteins of the Neutrophil B. Low Molecular Weight G Proteins of the Neutrophil Mechanisms for Regulation of Signal Transduction in the Neutrophil A. CAMP-Dependent Inhibitory Pathways B. Protein Kinase C Inhibitory Pathway C. Regulation by Translocation~PhysicaiSegregation Conclusions References

1.

INTRODUCTION

Polymorphonuclear leukocytes and macrophages play major roles in the body’s defense against bacterial infection, as evidenced by the often life-threatening infections acquired by patients with neutropenia, leukemia, or congenital diseases 65

Vopyright (c) IYW hy Acadcmic P r c s . Inc. All rights 01 reproduction i n any lorm reserved

66

GARY M. BOKOCH

affecting leukocyte structure and function. In order to perform this function, leukocytes migrate from the circulation to sites of tissue damage or inflammation under the influence of chcmoattractant factors produced by various humoral or cellular immunologic processes at these sites. Well-defined chemoattractants include the N-formyl peptides, which are by-products of bacterial protein synthesis-secretion; CSa, a polypeptide cleavage product of complement; leukotrienc R,, an ardchidonic acid rnetabolitc; and platelet activating factor, a bioactive alkyl phospholipid. Upon reaching the site of infection or inflammation, leukocytes phagocytize and destroy microorganisms and damaged tissue with an array of microbicidal oxidants, proteolytic enzymes, and antimicrobial peptides. Under certain circumstances, the excessive or inappropriate release of thcse highly destructive agents can result in undesirable tissue damage. Therefore, in addition to their normal role in the inflammatory response, leukocytes can also be considered the primary cellular mediators of pathologic inflammation. The process of leukocyte activation involves a variety of cellular responses, including cell shape changes, aggregation, phagocytosis, granule enzyme secretion, and stimulation of the respiratory burst which generates superoxide anion (0, ). All of these processes, as well as the initial chemotactic responses, are dependent o n a series of distinct events: (1) the binding of chemoattractant ligands by specific cell surface receptors, (2) the transduction of the binding event signal into an intracellular signal for cell activation, and ( 3 ) the stimulation (or inhibition) of the appropriate biochemical pathways that lead to cell functions. Since leukocytes are callcd upon to react to many chemoattractant stimuli with multiple (potential) functional responses under a wide range of physiologic circumstances, the leukocyte must be capable of rapid, but highly coordinated and regulated, responses. It is clear that part of this capability is a result of the transduction, coordination, and regulation of chemoattractant receptor signals by GTP binding regulatory proteins. A goal of this article will be to describe current knowledge of GTP binding protein composition and function in the neutrophil, placing this information in the context of what is understood about GTP binding protein-mediated signal transduction in more well-defined systcms. We will also attempt to point out areas in which more biochemical and mechanistic detail is required to validate current hypotheses about GTP binding protein function in the neutrophil.

II. GTP BINDING REGULATORY PROTEINS The GTP binding proteins’ comprise a growing superfamily of proteins that utilize the binding and hydrolysis of GTP to mediate interactions between protein ’For the purposcs of this article. we will refer to all GTP binding protcins in general as “G proteins.” although it should be noted that sonic authors referred to herein utilize the term G proteins solcly for that class of heterotrimeric, receptor-coupled CTP binding protcins.

67

4. GTP BINDING PROTEINS IN PHAGOCMIC CELLS

macromolecules. This GTP binding protein-catalyzed interaction can result in a signal being propagated and amplified (as in the case of the “classical” receptorcoupled GTP binding proteins), a process being spatially directed (as by the elongation factors involved in protein synthesis), or in other events requiring reversible interaction of macromolecules. The basic catalytic cycle is outlined in Fig. 1, and can be divided into four stages: 1. Basal state: In its basal or inactive state, the GTP binding protein (G) exists in the form of the GDP bound complex. 2 . Nucleotide exchange reaction: The release of GDP from the nucleotide binding site is catalyzed by a “receptor” or guanine nucleotide exchange factor (R), resulting in the subsequent binding to the unoccupied site of GTP. This GGTP complex is the activated form of the GTP binding protein. 3 . Activation of efector: The active G-GTP complex has a high affinity for, and interacts with, the effector (E). This interaction converts the effector to an active conformation. 4. Termination: The interaction of G with E is terminated by an intrinsic GTPase activity in G which converts G-GTP to G-GDP. The inactive GDP bound form has a lower affinity for E, resulting in the release and deactivation of E.

This GTP binding protein activation cycle thus utilizes the hydrolysis of the high-energy phosphodiester bond of GTP to switch the GTP binding protein from an active to an inactive conformation. While the basic mechanism is similar for all types of GTP binding proteins, the regulation and operational details of the cycle will be modified depending upon the kinetic parameters of each step, the subunit composition of the GTP binding protein involved, the stoichiometries of the interacting macromolecules, and so on. Thus the behavior of a GTP binding protein in a particular cell system can appear substantially different from other GTP binding proteins in another cell system. Stimulus

I

R -R*

1

GTP

G -GDP

GDP

G-GL E

n

X FIG. I .

GTP binding protein activation-deactivation cycle.

Y

68

GARY M. BOKOCH

A. The Receptor-Coupled (Oligomeric) GTP Binding Proteins 1. STRUCT~JRE AND

FUNCTION

A family of GTP binding proteins that couple cell surface receptors to thcir intracellular effector systems has been described (Table I). The receptor-coupled C proteins arc characteristically heterotrimcrs made up of distinct alpha, beta, and gamma subunits (Fig. 2). An overview of the receptor-coupled G proteins will be presented here, but the interested reader is referred to several excellent reviews (Gilman, 1987; Lochrie and Simon, 1988; Neer and Claphani, 1988; Stryer iind Bournc, 1986) for details. The alpha subunits of the known receptor-linked G proteins range in size from 39,000 to 52,000 MW. These alpha subunits contain the binding site for guanine nucleotides and the GTP hydrolytic activity of the G protein. The alpha subunit also contains sites which can be ADP-ribosylated in an NAD-dependent manner by toxins produced by Vibrio chiderue (cholera toxin) or Bordetcllu pcv-tussis (pertussis toxin). This toxin-catalyzed covalent modification of the G protein alpha subunit can either stimulate the functional activity of the G protein (i.e., as does cholera toxin to G,) or rcsult in inhibition of the G protein function (i.e., as does pertussis toxin to GI). These bacterial toxins have thus proven extremely useful in identifying systems in which the receptor-linked G proteins are inTABLE 1 CHARACTERISTICS OF PURIFIED AND/OR CI.ONED

Alpha subunit designation

M, 52

G,

44.5

G, G,

?

Gi

3

I

Go

Toxin scnsitivity

G

PROTEINS"

Receptor

Effector

CI' CT

P-adrenergic, PGE,, glucagon, many others

Adenylate cyclase (stimulates) Ca2+ Channcls (heart)

F T

Adcriylatc cyclase (inhibits) Phospholipase C ? K + Channels (heart) Ca2+ Channels (neuronal

40.4 40.5 40.5

PT

Muscarinic, a,-adrenergic, N-furmyl pcptide, inany others

31,

PT

Muscarinic, opiate, others'?

PT,CT?

cclls) ctw

%',

40 40.4

G,

40.9

PT'CT PT,CT?

-

Rhodopsin

cGMP Phosphodieskrase

?

'? Phospholipase C

Subscript designalit)n\. s , stimulatory with regard to iidcnylatc cyclase; I , inhibitory with regard ((1 ililcnylatc c y c h e ; o, "other"; 1 , transducin from rodh (r) or cones (c): 2 , arbitrary designation 0 1 original repon (Fong el a / . . 1988) CT indicates cholera toxin; IT indicates pertushis toxin. M, indicates molecular weight dctcrmined from amino acid composition ol' cloncd protein. (1

69

4. GTP BINDING PROTEINS IN PHAGOCYTIC CELLS

THE G PROTEIN SUPER FAMILY Family I

Family fl

The ReceptorXoupled (Oligomeric) G Proteins (MW = 39,000 - 52,000)

The Low Molecular Weight G Proteins (MW = 19,000 - 28,000)

0 ' I

a

"

1. R+G-+E

1. R ? E - G A P ?

2.

2. Ras, rho, ral, rab, rap, smg-p25, Gpiacenta 7

3.

aPy Subunit Structure

3. Single Known Subunit 4. Activation-Subunit

Dissociation

4. Activation-

1

5. Substrates for cholera andlor pertussis toxin

5. Substrates for botulinum toxin

FIG. 2. Characteristics of the members of the G protein superfamily.

volved, as was the case for the neutrophil (see Section 111,A). The alpha subunits of G, and at least one form of G, have recently been shown to be posttranslationally myristoylated (Buss et a / . , 1987). The molecular cloning of the G protein alpha subunits has revealed extensive sequence homologies not only between the various classes of receptor-coupled G protein but also between the former and the low molecular weight (rus-related) GTP binding proteins and the elongation factors. These areas of highly conserved primary structure are most pronounced in the guanine nucleotide binding portions of the G proteins, and such information has been utilized to develop structural models of the G protein alpha subunits (Masters et ul., 1986) by placing the primary structure information within the framework of three-dimensional structures obtained by X-ray crystallographic analysis of EF-Tu and rus (DeVos et ul., 1988; Jurnak, 1985). Most G protein alpha subunits are associated with two forms of beta subunits, with molecular weights of 35,000 and 36,000. Transducin, however, is only associated with a 36,000 MW form of beta subunit. The beta subunits exist as a tight, but noncovalent, complex with the gamma subunit(s). There appear to be at least three forms of G protein gamma subunits. It has been suggested that some of the specificity of interaction of a G protein with particular receptors or effectors may be regulated by distinct oligomeric combinations of the alpha-betagamma subunits. The beta-gamma subunit complex is hydrophobic, and in light of the relative hydrophilicity of the alpha subunits, it has been hypothesized that the beta-

70

GARY M. BOKOCH

gamma complex serves as a membrane anchor for the alpha subunits. There is evidence that the association of alpha subunits with receptors requires the presence of beta-gamma (Florio and Sternweis, 1985; Fung, 1983). Whether this reflects a specific requirement of these subunits for the receptor interaction or is merely a result of the ability of beta-gamma to allow alpha subunits to associate with the phospholipid membrane is not clear. In terms of G protein function, the beta-gamma subunit complex has largely been considered to bind, and thereby inactivate, the active alpha subunit. This relatively passive role of beta-gamma complex has been called into question by data that indicate the ability of these subunits to stimulate ion channels (Logothetis et ul., 1988) or phospholipase A, (Jelsema and Axelrod, 1987; Kim ef uf., 1989) as well as to directly inhibit the catalytic unit of adenylate cyclase (Katada rt ul., 1986a). In the yeast Saccharumyces cerevisiur, genetic evidence directly indicates transduction of mating factor receptor signals by yeast G protein beta-gamma subunits. It has been demonstrated that deletion (or mutation) of G alpha subunits in yeast causes a constitutively active cell state (Jahng ef a / . , 1988), while disruption of gcnes encoding either beta or gamma subunits will prevent cell activation by yeast mating factors (Whiteway et u l . , 1989). These findings also suggest a mechanism for a single G protein to activate more than one effector. Overall, there is extensive evidence that the G proteins listed in Table 1 directly interact with receptors and couple them to various effector systems (see reviews in Gilman, 1987; Lochrie and Simon, 1988; Neer and Clapham, 1988; Stryer and Bourne, 1986). The most conclusive demonstrations of this have come from reconstitution studies utilizing purified receptor, G protein, and effector components. Such studies have been carried out in particular with the p- and aadrenergic receptors (Ixfkowitz and Caron, 1987), muscarinic receptors (Florio and Sternweis, 1985; Kurose p t u l . , 1986; Haga et al., 1986), and rhodopsin (Kuhn, 1986). Mechanistic information derived from reconstitution, as well as other in vitru studies, has been utilized to develop the models of G protein activation described in the following sections.

2. DISSOCIATION MODEI.OF RECEPTOR-COUPLED G PROTEIN ACTIVATION The dissociation model of G protein activation (Fung, 1983; Gilman, 1987; Sniigcl et al., 1984a) proposes that the binding of GTP to the G protein (stimulated by receptor, see Fig. 3) is accompanied by the dissociation of the betagamma subunit complex from the GTP-bound alpha subunit. The basis for this model comes from the observation of this phenomenon in detergent solution. All oligomeric G proteins dissociate when activated by nonhydrolyzable guanine nucleotide analogs or AIF,-. That such a mechanism is operative in the intact cell membrane is suggested by a number of experimental observations. In partic-

4. GTP BINDING PROTEINS IN PHAGOCYTIC CELLS

71

t

A

indicates the lour FIG. 3. Activation-deactivation cycle of the receptor-coupled G proteins. affinity form of receptor, while indicates the high affinity form of receptor.

fl

ular, the ability of exogenous Gi alpha subunit to reverse the inhibition of adenylate cyclase by hormones in membranes can be most simply interpreted in terms of the complexation by the added alpha subunits of released beta-gamma subunits which act to inhibit the adenylate cyclase (Katada er al., 1984a,b). Similarly, the ability of exogenous beta-gamma subunits to interfere with the stirnulatory effects of G protein alpha subunits on their effectors suggests such a mechanism is operative. Direct evidence for hormone receptor-mediated G protein subunit dissociation in native membranes has recently been presented for G, (Iyengar et al., 1988; Ransnas and Insel, 1988). The occurrence of G protein subunit dissociation within the membrane is significant for several aspects of signal transduction. First, since the receptorlinked G proteins all share functionally similar beta-gamma subunits, the receptor-induced release of these subunits would potentially allow the activation of two effector systems simultaneously or, alternatively, the inhibition of a second messenger system antagonistic to the activated system. A second possibility is that the dissociation of the G protein alpha subunit from the hydrophobic betagamma complex might allow release of the activated alpha subunits from the plasma membrane to the cytosol. Such release could allow the regulation of nonplasma membrane associated enzymes or events by membrane receptor or could provide a means to regulate the system by changing the stoichiometry of membrane receptor-G protein components. 3. THE RECEPTOR-GPROTEIN REGULATORY CYCLE The activation of G protein by receptor (Gilman, 1987; Lefkowitz and Caron, 1987; Smigel et al., 1984b) involves the basic guanine nucleotide binding cycle

72

GARY M. BOKOCH

previously described, superimposed with a cycle of G protein subunit dissociation, as depicted in Fig. 3. The rate limiting step for activation of the oligomeric G proteins is the release of GDP from the nucleotide binding site, which allows GTP to enter this site and form activated G alpha. This nucleotide exchange step is catalyzed by the hormone receptor, which in its hormone-liganded form is able to interact sufficicntly with G-GDP to drive this reaction. The beta-gamma subunit complex appears to be required for the interaction and exchange reaction to occur. Amplification of the hormone signal can occur due to the catalytic action of hormone-receptor, leading to activation of multiple G proteins within the lifetime of the activated receptor, or by the stimulation of multiple effector units by Ga-GTP. Thermodynamic considcrations dictate that the interaction of receptor with G protein leading to guanine nucleotide exchange has reciprocal effects upon the rclative affinity of receptor for hormone. Thus, one obscrves that the receptor exists in two affinity states: (1) a low affinity state in which the receptor is in a free form unassociated with G protein. This form can represent the receptor itself or the equivalent form that results when receptor is in the presence of G-GTP or GDP (i.c., not associated); and ( 2 ) a high atfinity state that represcnts the hormone-receptor-(; protein complex that occurs in the absence of guanine nucleotide bound to G . Reconstitution studies in both membrane and in phospholipid vesicle systems utilizing purified components have demonstrated the ability of G protein to produce the high affinity form of receptor. Thus the demonstration of guanine nucleotide-sensitive interconversion of a receptor bctween high and low affinity forms for hormone is presumptive evidence for the intcraction of that receptor with a G protein. 6. The Low Molecular Weight GTP Binding Proteins The rus proteins are a family of GTP binding proteins with molecular weights of -2 1,000 that exhibit considerable homology at the mechanistic and sequence level with the rcceptor-coupled GTP binding proteins (Barbacid, 1987). The protein products of the rus protooncogene family are involved in normal cell differentiation and proliferation. Mutation or abnormal expression of these proteins can result in malignant transformation, and 20-40% of human tumors are associated with the rus oncogene (depending upon tissue type). The rus proteins appear to exist as monomeric “alpha” subunits which can bind and hydrolyze GTP (Fig. 2). They are largely membrane associated and require the attachment of a palmitic acid group to the carboxy-terminal region of the protein in order to effectively insert into the membrane (Barbacid, 1987; Sefton and Buss, 1987). The importance of the GTP binding and hydrolytic capabilities of rus for activity of the protein has been demonstrated by mutational analysis, in which modifica-

4. GTP BINDING PROTEINS IN PHAGOCYTIC CELLS

73

tion of discrete regions of the protein involved in GTP binding or hydrolysis results in activation (or inhibition) of the transforming potential of rus. Because of these, as well as other homologies between rus and the oligomeric G proteins, it is widely perceived that ras proteins interact with specific receptor (i.e., guanine nucleotide exchange factors) and effector systems. The ras proteins have been reported to serve as substrates for the tyrosine kinase activity of several types of growth factor receptors (Barbacid, 1987; Kamata and Feramisco, 1984) and are likely to serve as normal cellular transducers of growth factor receptor signals. The exact cellular signals transduced by rus are as yet unknown, although there are numerous, albeit inconclusive, reports of effects upon phospholipases C and A, (Barbacid, 1987). Recently, a putative effector of the rus proteins (termed GAP, for GTPase activating protein) has been identified and cloned (Trahey and McCormick, 1987; Gibbs et ul., 1988; Vogel et al., 1988). A region of the ras primary structure (aa 35-45) has been indicated by mutagenic analysis to be involved with the effector function of ras (Barbacid, 1987). The interaction of GAP with ras has been shown to correlate well with those forms of the protein that maintain the integrity of this effector domain (Adari et al., 1988). This suggests that GAP could be the ras effector protein, although it is also reasonable to interpret this data in terms of amino acids 35-45 being a GAP domain required for ras function and not the “effector” domain per se. It is intriguing that sequence analysis of cloned GAP indicates significant homologies with the adenylate cyclase catalytic unit, phospholipase C, and tyrosine kinases (Vogel et al., 1988). Over the past several years, a large number of ras-related proteins have been identified by molecular cloning techniques utilizing probes derived from the previously identified N-, K-, or H-rus proteins. These proteins exhibit significant sequence homologies with rus, particularly in the sites involved with guanine nucleotide binding and hydrolysis. Such proteins include R-ras (Lowe et ul., 1987), rho (Madaule and Axel, 1985), rul (Chardin and Tavitian, 1986), rub (Touchot et ul., 1987), and rap (Pizon et al., 1988). Additionally, several low molecular weight proteins that exhibit sequence homology with ras have been identified in yeast, including ARF (Sewell and Kahn, 1988), SEC4 (Salminen and Novick, 1987), and YPTl (Schmitt et al., 1986). The latter two proteins have been demonstrated to participate in constitutive secretion in yeast, with YPTl involved in endoplasmic reticulum to Golgi transport, and SEC4 in Golgi to plasma membrane trafficking. The ability of a number of low molecular weight GTP binding proteins to bind [cx - ~ ~P I G Tafter P SDS-polyacrylamide gel electrophoresis-transfer to nitrocellulose (a property not exhibited by receptor-associated oligomeric G proteins) has made it increasingly clear that the low molecular weight G proteins exist in many cell types (Bhullar and Haslam, 1987; Bokoch and Parkos, 1988). Some of

74

GARY M. BOKOCH

these low molecular weight proteins are being purified from mammalian tissues and characterized. Many of these purified proteins have been found to be the products of the ras-related genes previously identified by cloning methodologies. Thus, rho has been purified from bovine adrenals (Narumiya et al., 1988) and brain (Yamamoto et at., 1988), rup from bovine brain (Kawata ef ul., 1988) and human neutrophils (Bokoch e t al., 1988b), and c-Ki-ras from bovine brain (Yamashita e t al., 1988). Other, potentially distinct low molecular weight G proteins that have been purified include Gp ( p designating placenta) (Evans et a / . , 1986), ARF from rabbit liver (Kahn and Gilman, 1984), and smg25 from bovine brain (Kikuchi et ul., I988b). The cellular roles of these proteins have yet to be elucidated, although they have been implicated as participating in processes such as protein trafficking, cell growth and differentiation, Ca2 ' mobilization, microtubule function and assembly, and phospholipasc activation. The botulinum toxins are potent neurotoxins produced by strains of Clostridium /~otu/inum.A cornponcnt of botulinum toxin, termed the C3 ADPribosyltransferase, can catalyze the NAD-dependent ADP-ribosylation of 20,000-24.000 MW GTP binding proteins (Rosener et ul., 1987). Identified substrates for this toxin include rho (Kikuchi crt a / . , 1988a; Quilliam et ul., 1989; Narumiya et a / ., 1988) and neutrophil GZZK(Bokoch et a / . , 1988b). Botulinum C3 toxin can produce effects on cell secretion (Mege et a / ., 1988; Banga rt ul., 1988) and differentiation (Rubin et al., 1988) that appear to be mediated through the low molecular weight G protein substrates of this toxin. Stoichiometric ADPribosylation of rho by botulinum toxin does not inhibit that protein's ability to bind or hydrolyze guanine nucleotides (Kikuchi ef al., 1988a; Quilliam rt ul,, 1989). Thc toxin may exert effects upon the interactions of substrate proteins with a GAP-like effector.

111. GTP BINDING PROTEINS AS MEDIATORS OF NEUTROPHIL ACTIVATION A. Evidence for the Interaction of Chemoattractant Receptors with a Pertussis Toxin-Sensitive GTP Binding Protein There is substantial evidence to indicate that neutrophil (and macrophage) chemoattractant receptors interact with, and have their signals transduced via, GTP binding proteins (reviewed in Ornann et al., 1987; Snyderman e l ul., 1986; Sklar, 1986). While the majority of this evidence comes from studies on the N formyl peptide receptor, many of the conclusions are likely to also apply to C5a, leukotriene B,, platelet activating factor, and Fc receptors.

4. GTP BINDING PROTEINS IN PHAGOCYTIC CELLS

75

1 . GUANINE NUCLEOTIDE REGULATtON OF N-FORMYL

PEPTIDERECEPTOR AFFINITY The existence of two forms of the N-formyl peptide receptor, varying in their relative affinity for binding N-formyl peptide ligands and interconvertible by guanine nucleotides, has been demonstrated both in equilibrium binding studies (Koo et ul., 1983; Snyderman et al., 1984) and in kinetic analyses (Sklar et al., 1987). ADP-ribosylation of the neutrophil G protein substrate by pertussis toxin abolishes the formation of the high affinity form of the receptor. Since these observations are consistent with the predictions of the receptor-G protein interaction cycle described in section II,A, this suggests the coupling of a chemoattractant receptor to a pertussis toxin-sensitive GTP binding protein. We will describe in more detail the applicability of such a model to the neutrophil Nformyl peptide receptor in Section III,C. Direct evidence that the guanine nucleotide sensitive, high affinity state of the N-formyl peptide receptor results from an interaction with a GTP binding protein comes from reconsitution studies in both membrane and phospholipid vesicle systems. In these studies, endogenous neutrophil G proteins were either inactivated by pertussis toxin (Kikuchi et a/., 1986) or separated from the detergentsolubilized N-formyl peptide receptor by wheat germ agglutinin chromatography (Williamson et ul., 1988), and both conditions resulted in the loss of high affinity N-formyl peptide binding. Addition of a mixture of purified Go-G, proteins obtained from brain restored high affinity N-formyl peptide binding. While not indicative of the identity of the actual G protein responsible for the formation of the high affinity state of the N-formyl peptide receptor in vivo, the ability to reconstitute the high affinity form of the receptor with purified (albeit mixed) G proteins argues that this represents a r e c e p t o r 4 protein complex.

2. ACTIVATION OF G PROTEIN FUNCTION(S) ny N-FORMYL PEPTIDES The demonstration that the N-formyl peptides can directly stimulate the biochemical functions associated with G proteins indicates that the N-formyl peptide receptor interacts with a G protein(s) in a fashion consistent with the receptor-G protein interaction cycle. Thus, N-formyl peptides have been shown to stimulate both guanine nucleotide binding and GTP hydrolysis activities of neutrophil membranes in a pertussis toxin-sensitive manner (Hyslop et ul., 1984; Lad et al., 1985; Okajima et al., 1985). The ability of a series of N-formyl peptides to stimulate GTP hydrolysis has been correlated with their known efficacy for stimulation of granule enzyme secretion from neutrophils (Becker et al., 1987).

76 3 . PERTUSSIS TOXIN INHIBITION BY THE N-FORMYI. PEPTIDES

GARY M. BOKOCH OF NEU'I'ROPHIL

ACTIVATION

The ability of pertussis toxin to functionally uncouple G proteins from their associated receptors inhibits cellular responses resulting from this interaction. This property has been utilized to demonstrate inhibition of thc activation of neutrophil functions by the N-formyl peptide receptor in pertussis toxin-treated cells. Chemotaxis, shape change, aggregation, granule enzyme secretion, and superoxide production have all been shown to be sensitive to pertussis toxin treatment, as has Ca2' mobilization (Molski et al., 1984) and phospholipase C and phospholipase A, activation by N-formyl peptides (Omann et al., 1987; Snydcrman ef al., 1986; Sklar, 1986). Bokoch and Gilman (1984) and Okajima and Ui (1984) demonstrated that the effect of pertussis toxin to inhibit neutrophil activation can he dircctly correlated with the toxin-catalyzed ADP-ribosylation of a 40,000 MW membrane substrate. The ability of both pertussis toxin and cholera toxin to label a 40,000 MW membrane protein in ncutrophils has been shown to be modulated when labcling is performed in the presence of N-forrnyl peptide ligands. The presence of ligand inhibits the ability of pertu toxin to ADP-ribosylatc its 40,000 MW substrate in neutrophil membranes (Matsumoto rt a/., 1987). Although not rigorously demonstrated, this may indicate G protein subunit dissociation induced by ligand-occupied N-formyl peptide receptor. The presence of ligand also enhances the ability of cholera toxin to ADP-ribosylate a 40,000 MW substrate in neutrophil membranes (Verghese et al., 1986; Gierschik and Jakobs, 1987), which may be the same protein that serves as the pertussis t o x i n substrate. This effect is likely due to the N-formyl peptide receptor-catalyzed release of guanine nucleotide from G protein, since cholera toxin labeling of the 40,000 MW protein is known to occur only in the absence of guanine nucleotides. Both phenomena can be intcrpreted as indicative of direct interactions between the N-formyl peptide receptor and a G protein transduction partner. 4. G PROTEIN COUPI.ING OF T H E N-FORMYtJ PEPTIDE RECEPTOR TO A SPECIFIC EFFECTOR SYSTEM: PHOSPlIOLIPASP c

The involvement of a GTP binding protein(s) in mediating the coupling of chemoattractant receptor to the enzyme phospholipase C has been indicated by several studies utilizing broken cell systems. The ability of nonhydrolyzable guanine nucleotide analogs to stimulate the formation of inositol polyphosphatcs in pcrmcabilizcd neutrophils (Cockcroft and Gomperts, 1985; Stutchfield and Cockcroft, 1988) and mast cells (Cockcroft et al., 1987) dcnionstrated that known G protein activating agents were capable of stimulating a membrane-

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associated phospholipase C. Analogous stimulatory effects have been obtained with the G protein activator, NaF. Snyderman and colleagues (1986; Smith et ul., 1985, 1986) utilized a neutrophil membrane system to demonstrate that the coupling of the N-formyl peptide receptor to phospholipase C required the presence of GTP. The ability of the N-formyl peptides to stimulate polyphosphatidylinositol breakdown in the presence of GTP was inhibited by pretreatment of the neutrophils with pertussis toxin. A mechanism by which the G protein may reduce the Ca2 requirement for phospholipase C activation to physiological levels has been proposed by this group. Such data are consistent with studies of receptor-mediated phospholipase C activation in a large number of systems (Berridge, 1987). While the evidence presented, when taken in total, provides a strong case for the coupling of the N-formyl peptide receptor to cell activation through a membrane-associated phospholipase C, several points should be emphasized. At the level of the receptor, there is little evidence for the physical association of the receptor with a particular G protein. Polakis et ul. (1988) have shown that a detergent-solublized N-formyl peptide receptor comigrates with a 40,000 MW pertussis toxin substrate through several chromatographic steps and that ligand binding to this receptor retains sensitivity to guanine nucleotides. Jesaitis et ul. (1988b) have been able to identify forms of the receptor that differ in apparent hydrodynamic size and show that they are interconvertible by guanine nucleotides. The ability to form a physical complex of receptor with purified G protein has not been demonstrated. Similarly, at the level of the putative G protein-phospholipase C interaction, there has been no physical association of a neutrophil G protein with a phospholipase C demonstrated. Nor has it been shown that exogenous G protein (liganded or unliganded) can directly stimulate phospholipase C activity in membranes or other phospholipase preparations. The availability of purified neutrophil G proteins (see Section IV) and the development of appropriate reconstitution systems should allow such definitive studies to be conducted in the near future. +

B. Nonpertussis Toxin Substrate GTP Binding Proteins: Evidence for Roles in Neutrophil Secretion and NADPH Oxidase Activation The evidence for a pertussis toxin-sensitive GTP binding protein being involved in the transduction of N-formyl peptide receptor signals to phospholipase C does not rule out the possibility that additional GTP binding proteins might participate in receptor signaling or in regulating processes initiated by receptor. Evidence for the participation of such proteins in both granule enzyme secretion and NADPH oxidase activation has been obtained.

GARY M. BOKOCH

I . SECRETION Both pernieabilizcd neutrophil and mast cell systems have been utilized to demonstrate the ability of nonhydrolyzable guanine nucleotide analogs to either stimulate secretion in the absence of CaZ+ or to enhance secretion at low CaZ+ concentrations (Barrowman et (ti., 1986; Howell el al., 1987; Cockcroft ct d., 1987; Stutchfield and Cockcroft, 1988; Smolen and Stoehr, 1986). The inactive guanine nuclcotide GDPpS can antagonize the secretion induced by active GTP analogs as well as the secretion caused by increases in cytosolic Ca2' . The stimulatory effects of guanine nucleotides occur in the apparent absence of phospholipase C activation and can be distinguished from the generation of IP, or diacylglycerol. Similar stimulatory effects of guanine nucleotides on mast cell and neutrophil secretion have been demonstrated after introduction to the cell cytosol by patch pipette (Fernandez et d., 1987; Lindau and Nusse, 1987, 1989). The inability of pertussis toxin pretreatment of cells to inhibit secretion induced by GTPyS has been argued to indicate the G protein involved is a nonpertussis toxin substrate. This argument per se has little value, since the effects of GTPyS upon G protein activity are generally resistant to pertussis toxin-catalyzed ADPribosylation (Aktories et ul., 1983; Katada et al., 1986a; Smith et a/., 1987). However the inability of pertussis toxin to change the kinetics of secretion by such GTP analogs (Lindau and Nusse, 1987) or to inhibit Ca*+-induced secretion (Barrowman ct d . , 1986) might suggest such a conclusion is still valid. The putative GTP binding protein involved in neutrophil and mast cell secretion may act to stimulate fusion of granule membranes with the plasma membrane (Gomperts, 1986). Overall, the data obtained in ncutrophils and mast cells is consistent with a large number of studies implicating GTP binding proteins in exoeytotic secretion from a wide variety of mammalian cells (Gomperts, 1986; Burgoyne, 1987). The existence of GTP binding proteins (YPTl and SEC4) required for constitutive 1986; Salsccretory processes has been demonstrated in yeast (Schmitt et d., minen and Novick, 1987). Both YPTl and SEC4 exhibit significant degrees of sequence homology with the r m (p21) proteins, and mammalian homologues of YPTl have been identified (Touchot et 01.. 1987; Segev et ul., 1988). It has been reported that the microinjection of the oncogenic forms (activated forms) of rus proteins into mast cells can stimulate the degrdnuhtion of these cells (Bar-Sagi and Gomperts, 1988). The participation of such a rus or ras-related protein in the normal leukocyte secretory process is suggested. 2. NADPH OXIDASE ACTIVATION Data analogous to that obtained for neutrophil secretion has been obtained to implicate a GTP binding component in activation-assembly of the NADPH

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oxidase system. Thus, nonhydrolyzable guanine nucleotide analogs and NaF enhance the formation of superoxide anion in reconstituted oxidase systems consisting of membrane, cytosol, and arachidonic acid or SDS (Gabig et al., 1987; Siefert et al., 1986; Siefert and Schultz, 1987; Della Bianca et al., 1988; Ligeti et al., 1988). This effect, as well as the ‘‘basal’’ oxidase activity of the system, is antagonized by GDP or GDPpS. Stimulation of NADPH oxidase activity by NaF can occur in the apparent absence of phospholipase C activity (Della Bianca et al., 1988). Membranes from pertussis toxin or cholera toxin treated cells are capable of generating superoxide at normal rates in cell free systems (Gabig et ul., 1987), although the lack of effect of pertussis toxin treatment upon GTPyS-induced oxidase activation is again of dubious significance. Attempts to localize the GTP-activatable component argue that it is cytosolic and upon activation either becomes associated with the membrane NADPH oxidase complex or allows additional cytosolic proteins to associate with the complex (Doussiere et al., 1988). Although cytosolic components apparently able to restore NADPH oxidase activity to autosomal recessive forms of chronic granulomatous disease have been reported to bind to GTP-agarose matrices, the significance of this is doubtful, as the complexes could be eluted from these columns with adenine as well as guanine nucleotides (Volpp et al., 1988). The recent report that rap 1 protein associates with the cytochrome b of neutrophils (Quinn et al., 1989) suggests this protein could represent the GTP binding protein involved in the stimulation of NADPH oxidase activity by guanine nucleotides.

C. N-Formyl Peptide Receptor Affinity States: Evidence That These States Can Be Defined as Particular Forms of L-R-G While the N-formyl peptide receptor has been described as a typical G proteincoupled receptor, with characteristics predicted by the ternary complex model first developed for the adrenergic receptors (DeLean et al., 1980), it is worthwhile presenting some of the evidence that indicates this model accurately describes the multiple receptor affinity states observed in the intact neutrophil or broken cell preparations. Equilibrium binding studies using radiolabeled N-formyl peptides to examine receptor affinity states in neutrophil or macrophage membranes (Mackin et al., 1982; Seligmann et ul., 1982; Koo et al., 1983; Snyderman et al., 1984) demonstrated both high and low affinity forms of the receptor. Addition of guanine nucleotides to the system resulted in loss of some of the high affinity sites and an increase in the proportion of low affinity sites, with no overall change in receptor number. Subsequent studies in a permeabilized neutrophil system (Sklar et al.,

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I %7), using analytical techniques capable of kinetic analysis of receptor affinity, demonstrated that the majority ( 2 90%) of the formyl peptide receptors of the cell could form a high affinity state capable of being converted to a low affinity state by the addition of guanine nucleotides. The evidence that the high affinity state in these studies represents the complex of LK-G and that the low affinity state is L-R is as follows: ( 1 ) The high affinity form is converted to the low affinity form by guanine nucleotides. (2) A similar effect is produced by ADPribosylation of neutrophil G protein by pertussis toxin or by N-ethylmaleimide, both of which are conditions that result in the uncoupling of K from G . (3) Reformation of the high affinity state occurs when ADP-ribosylated membranes are reconstituted with a mixture of G proteins (Kikuchi et ul., 1986; Williamson et al.. 1988). Sklar and associates (1984, 1989a,b; Sklar, 1986) have attempted to apply this paradigm to the receptor forms observed in intact neutrophils. They can demonstrate the formation of three states of the formyl pcptide receptor on the intact neutrophil, and these can be assigned to various forms of L, R, and G (or X) as follows. 1. Low ufinity receptor (LR). (a) This form occurs transicntly after cell activation and is characterized by a dissociation t , / * for ligand of 10-20 seconds, similar to the low affinity form observed in permcabilized cells or membranes. (b) It can be stabilized in cells exhaustively ADP-ribosylated by pertussis toxin and depleted of nuclcotidc tri- and diphosphates (energy dcpleted). In the presence of nucleotide triphosphates, the LR form is rapidly ( f , / 2 = -10 SCC) converted to a form, LRX. 2. High clffiniry receptor (LRG). (a) This form is associated with cell activation, as evidenced by the ability of agents (i.e., pertussis toxin) able to prevent its formation to shut off activation, and is transient in intact cells with normal high levels of guanine nucleotide di- and triphosphates. (b) Formation of a pertussis toxin-sensitive, high affinity form not normally seen in intact cells that is likely to be LKG can be observed in cells energy depleted and lacking nucleotide diand triphosphates. 3. High ufiniv receptor Z LRG (LRX). (a) ‘This form occurs subsequent to cell activation and is not associated with signal transduction. (b) The existence of this high affinity form in the intact, energy (re, guanine nucleotide) replete cell would preclude its identity as LKG. (c) Formation is insensitive to pertussis toxin. (d) Formation is dependent upon the availability of ATP.

-

The LRX form of the receptor may be analogous to phosphorylated and desensitized forms of the P-adrenergic receptor or rhodopsin. “X” could also be an “arrestin-like” protein able to bind and prevent interaction of the receptor with G protein, as occurs in the aforementioned systems (Wilden et al., 1986). LRX at some point may be associated with the neutrophil cytoskeleton and

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therefore may be the high affinity, cytoskeletal-associated form reported by Jesaitis et al. (1984, 1985). Loss of guanine nucleotide-sensitive, high affinity N formyl peptide binding in permeabilized cells in the presence of Ca’+ (Sklar et a/., 1987) may relate to the formation of LRX. The formation of a receptor with the characteristics of LRX has also been described in neutrophil membranes (Snyderman and Pike, 1984). Thus, at the levels of analysis currently available, the multiple affinity states observed for the N-formyl peptide (and likely other) chemoattractant receptor meet the operational criteria for “typical” G protein-coupled receptors and can be assigned as various L-R-G(X) forms on this basis. The concept that distinct isoforms of the N-formyl peptide receptor with different relative affinities exist and are associated with distinct aspects of neutrophil activation no longer appears tenable.

IV. THE GTP BINDING PROTEIN COMPOSITION OF THE NEUTROPHIL A. Oligomeric (Receptor-Coupled) G Proteins ofthe Neutrophil Neutrophils have been reported to contain at least three pertussis toxin substrates. One of these, with an apparent molecular weight of 43,000, appears to be a minor component of neutrophil membranes, and its identity and significance are unknown (Iyengar et a f . , 1987). The other two pertussis toxin substrates, with alpha subunits of 40,000 and 41,000 MW are present in substantially greater quantities in neutrophils (based upon comparative labeling with pertussis toxin and 132P]NAD)and are likely to represent and GI.3,as indicated by the evidence presented in the following section. Neutrophils do not contain any detectable Go or transducin-like GTP binding proteins nor do they contain detectable G,., (Gierschik et a / . , 1986, 1987; Bokoch et a/., 1987; Goldsmith et a / . , 1987; Oinuma et a/., 1987). 1 . G” (GI-?) a. Purification and Characterization. The major neutrophil pertussis toxin substrate, with an alpha subunit of 40,000 MW has been purified and characterized by a number of laboratories (Gierschik et a/., 1987; Oinuma et a / ., 1987; Dickey et af., 1987; Uhing et a / . , 1987). While this protein has also been referred to as G,, indicating association with the chernoattractant receptor, or G,, indicating coupling to phospholipase C, we prefer the term G,, indicating its identity as the major neutrophil toxin substrate without premature assumption of its function. It is likely that G,, is actually a Gi.*.

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G, has been purified both in the absence (Oinuma rt d., 1987; Dickey et al., 1987; Uhing et a / . , 1987) and in the presence (Gierschik et al., 1987) of the activating ligand AIF,-. In the absence of AIF, , G, purifies as a complex of alpha (40,000 MW) and beta-gamma subunits, exhibiting thc typical oligomeric structure of the receptor-coupled G proteins. Purified G, is associated with both 35,000 and 36,000 MW forms of beta subunit. Functional interaction of the 0, alpha subunit with beta-gamma subunits has bccn dctnonstrated by the ability of ncutrophil beta-gamma subunits to support pertussis toxin-catalyzed ADPribosylation of thc resolved G, alpha subunit (Oinuma et al., 1987) as well as to enhance binding of GTPyS to the isolated alpha subunit (Gierschik ef al., 1987). G,-derived beta-gamma subunits are interchangeable with beta-gamma subunits derived from bovine brain G proteins in such assays as ADP-ribosylation by pertussis toxin and inhibition of adenylate cyclase activity (Oinuma et ul., 1987) and appear indistinguishable by immunological criteria (Gierschik et a/., 1987; Oinuma et ul., 1987). lmmunoblot analyses with a variety of sera from several laboratories have shown that GI, does not cross-react with transducin- or Gospecific antisera. GI, does react with Gi-?specific antisera but not with antisera specific for Gi-l (Gierschik rt cil., 1987; Oinuma et al., 1987; Goldsmith et a / . , 1987; Murphy et d . , 1987). Protcolytic mapping of the G, alpha subunit with chymotrypsin, trypsin, and Stciphyloc.oc.c.i~suurtus V-8 protease demonstrated that it was distinct from the 41,000 MW pertussis toxin substrates of brain, neutrophils, and rabbit liver (Oinuma et (I/. , 1987; Uhing et al., 1987; Bokoch et ul., 198th). which are likely to represent Gi., and Gi-3 (Murphy ct al., 1987; Mumby el ( J I . , 1987). Gi.? has been cloned from a diEerentiated U937 library (Didsbury e f a l . , 1987), and the cxpression of G,.2 message in differcntiated HL60 cells (Murphy et a / ., 1987) has been demonstrated. These data strongly indicate that GI, is a form of G,-,, and this conclusion is tentatively confirmed by amino acid sequence analysis of unique peptide fragments dcrivcd from human neutrophil G,, alpha (T. Amatruda and G. M . Bokoch, unpublished observations). While a 40,000 MW protein in human neutrophils suggested to be G, has been shown to be a cholera toxin substrate in the absence of guanine nucleotides (Verghese et d . , 1986; Gicrschik and Jakobs, 1987), purified G, has not been demonstrated to serve as a substrate for this toxin. Comparison of the levels of the 40,000 MW (G,) versus the 41,000 MW neutrophil pertussis toxin substrates by protein staining, imniunoblotting, and two-dimensional gel electrophoresis indicate that G, represents -80-90% of the total toxin substrate in mature neutrophil membranes (Bokoch et c i l . , 1988a), as opposed to a more equal distribution of the two forms in undifferentiated HL60 cells (Uhing pi ul., 1987). Analysis by quantitativc Western blotting of total Gi alpha subunit in highly purified human neutrophil membranes indicates membranc levels of -750 pmol/mg menibrane protein (Bokoch et ul., 1988a). Little characterization of the guanine nucleotide binding properties of G, has

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been done, although such studies have been performed with Gi.2 derived from brain (Katada et a / ., 1987) and lung (Morishita et al., 1988). The ability of G, to interact with N-formyl peptide receptors is suggested by chromatographic comigration of the two proteins through a partial purification procedure (Polakis et al., 1988), but no direct evidence of specific G, coupling to chemoattractant receptors has been reported. Purified G, has likewise not been shown to associate with or modulate the activity of neutrophil (or other) phospholipase C or phospholipase A,. The identity of G, with G, or G, is thus still undetermined. b. Subcellulur Locdization ofG,. Subcellular fractions of human neutrophils resolved on density gradients have been analyzed for the presence of neutrophil G proteins by [3sSS]GTPySbinding, pertussis toxin labeling, and immunoblotting with subunit specific antisera (Bokoch et al., 1988a; Rotrosen et al., 1988). Bokoch and associates observed the presence of pertussis toxin substrate activity in both plasma membranes and cytosolic fractions but did not detect such activity in fractions associated with either specific granule or azurophil granule markers. The cytosolic toxin substrate appears to be the alpha subunit of G,, as evidenced by identical proteolytic digestion patterns of the cytosolic substrate and purified membrane-derived G, alpha. This cytosolic form of G, likely represents the free or uncomplexed form of G , alpha subunit, since ( I ) there is no beta subunit detectable in cytosol with beta-specific antibody blots; (2) the pertussis toxincatalyzed labeling of the cytosolic alpha subunit is markedly enhanced by the addition of exogenous beta-gamma subunits, consistent with the known inability of free G protein alpha subunits to serve as effective pertussis toxin substrates (Neer et al., 1984; Katada et al., 1986b); and (3) the hydrodynamic parameters of the cytosolic toxin substrate are indicative of an M , = 42,300. Total cytoplasmic levels of G, alpha observed in this study, while not quantitated by immunologic methods, were estimated from pertussis toxin labeling to be approximately one-third to one-half of those in the plasma membrane pool. Rotrosen et al. (1988) also reported plasma membrane and cytosolic forms of G, alpha subunit but additionally observed pertussis toxin substrates associated with specific granule markers. Their immunologic analysis indicated that this granule pool was enriched in the 41,000 MW (Gie3)alpha subunit. Apparent translocation of this specific granule associated pool to the plasma membrane was observed in cells stimulated to degranulate with Met-Leu-Phe. The distribution of the total Gi between various fractions was estimated in this study to be -60% plasma membrane, -35% specific granule-enriched fraction, and -5% cytoplasmic. The lack of detectable Gi in specific granule associated fractions reported by Bokoch et al. (1988a) and the presence of such a pool in this study do not seem attributable to contamination of granule fractions with plasma membranes. In light of the existence of a granule fraction in neutrophils that is often associated with specific granule markers and that can be mobilized by various

a4

GARY M. BOKOCH

neutrophil “priming” agents (Fletcher and Gallin, 1980), the discrepancy may be a result of the relative degrees of priming of the cells utilizcd in the two studies.

Purification of pertussis toxin substrates from an undifferentiated llL60 cell line revealed the presence of nearly equal amounts of both 41,000 and 40,000 toxin substrates (Uhing et a l . , i 987). The 4 I ,000 MW alpha subunit differs in its chymotryptic digestion pattern from the 40,000 MW alpha subunit (G,,). Since this 4 1,000 MW alpha subunit does not substantially cross-react with G,-, or Gi specific antibodies (Goldsmith et u l . , 1987), it is likely to be Gi.3 (although the possibility that it represents an undescribed form of Gi is not ruled out). Gi.rrhas been cloned from a differentiated human HL60 cell library and has been shown to be expressed in HL60 cells (Didsbury and Snyderman, 1987; Murphy et a l . , 1987).

3. G, The existence of G, in neutrophils can be inferred from thc ability of various hormones (P-adrcnergic agonists, PGE,, histamine, etc.) and G, activators (guanine nucleotides, NaF, cholera toxin) to stimulatc adcnylate cyclase activity in intact cclls as well as membranes (Zurier et ul., 1973; Kivkin rt d.,1975; Verghese et ul., 1985; Bokoch, 1987; Sha’afi and Molski, 1988). Cholera toxin labels the 45,000 MW alpha subunit of G, in neutrophil membranes (Verghese el al., 1986; Gierschik and Jakobs, 1987), and this subunit is also detectable with specific G, antisera (G. M. Bokoch and L. A . Ransnas, unpublished observations). Studies of neutrophil adenylate cyclasc rcveal certain as yet unexplained characteristics of this system, including relatively low adenylate cyclase activity, particularly in response to stimulatory hornioncs, and the inability of exogenously added beta-gamma subunits to inhibit adenylate cyclase activity (Bokoch, 1987). These biochemical data may relate to the unusual stoichiometries of G protein subunits in neutrophil membranes (see Section IV,C), as well as to as yet unidentified propertics of the relevant adenylate cyclase system components. An understanding of the neutrophil G,-activated adenylate cyclasc system assumes importance in light of the inhibition of ncutrophil activation produced by hormones able to stimulate CAMP formation in these cells (see Section V,A).

B. Low Molecular Weight G Proteins of the Neutrophil The low molecular weight C proteins exist as monomers with molecular weights characteristically ranging from 19,000 to 28,000. The existence of at

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least four distinct low molecular weight GTP binding proteins in human neutrophils can be demonstrated. With the realization that many of the low molecular weight proteins comigrate on SDS-polyacrylamide gels, it is likely that this number will increase as these proteins are resolved and their corresponding cDNAs cloned and identified. Two proteins of estimated molecular weight 24,000 (G24K)and 26,000 (G,,,) have been described in human neutrophil membranes that retain [ C~-~*P]GTP binding capability after SDS-polyacrylamide gel electrophoresis-electroblotting (Bokoch and Parkos, 1988). Binding is specific for guanine versus adenine nucleotides and is insensitive to treatment with 10 mM N-ethylmaleimide. A third protein of apparent molecular weight of 22,000 (G,,,) does not have this capability but does bind [”SIGTPyS in its native form in an N-ethylmaleimidesensitive manner. None of these proteins appear to be proteolytic breakdown products of the larger known G proteins, as they do not cross-react with antisera specific for disparate regions of G, primary structure nor with antibody AS69, a common peptide G protein antibody (Mumby et a / ., 1986). Additionally, these proteins do not serve as either pertussis or cholera toxin substrates (Bokoch and Parkos, 1988; Bokoch et al., 1988b). G22Kcross-reacts with a monoclonal antibody to a conserved portion of the N-, H-, or K-rus proteins known to be involved in guanine nucleotide binding. It is immunochemically distinct from ras, however, because it does not react with monoclonal antibody Y 13-259, which recognizes N-, H-, and K-rus. Aminoterminal sequence determination indicated differences between G,,, and the known ras proteins (Bokoch ef a f . , 1988b). We have subsequently obtained a full-length clone from a differentiated HL60 library using this sequence information (L. A . Quilliam, C. J. Der, and G. M. Bokoch, unpublished observations) and have identified the clone as identical to the recently described ras-related protein, rap I (Pizon et ul., 1988). It is interesting to note that the clone obtained has a threonine substituted for the glutamine usually present at amino acid position 61 in the ras proteins. This substitution is known to result in reduced GTPase activity and an enhancement of the transforming capability of normal rus (Der et al., 1986). The rap 1 protein in neutrophils may therefore exist in an “activated” state. The guanine nucleotide binding and GTP hydrolytic properties of G,,, are currently under study in our laboratory (Bokoch e t a / ., 1988~).While the function of rap 1 in the neutrophil is unknown, it has been found to copurify with the cytochrome b component of the NADPH oxidase (Quinn et a/., 1989). Neutrophils contain a -22,000 MW membrane botulinum toxin substrate that comigrates with purified G,,, and is immunoprecipitated by monoclonal antibody 142-24EOS (Bokoch et al., 1988b). Isolated G,,, does not serve as an efficient botulinum toxin C, substrate but, in the presence of a cytosolic protein of unknown identity, can be ADP-ribosylated to an extent of 0.6 mol ADPribose/mol of protein (Quilliam ef al., 1989). Since the amino acid sequence

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obtained from G,,, indicated the presence of the rup 1 protein, it is possible that rap 1 is the endogenous neutrophil botulinum toxin substrate, and this possibility is currently under investigation. However, the likelihood that G,,, as isolated may contain multiple rcrs-related proteins docs not allow this conclusion to be definitively made as yet. A botulinum toxin substrate in bovine brain and adrenal 1989; cells has been identified as rho (Kikuchi et nl., 1988a; Quilliam et d., Narumiya et al., 1988). Botulinurn toxin-catalyzed ADP-ribosylation of rho differs from the neutrophil substrate in that a cytosolic factor does not appear to be required. This difference, as well as the inability of neutrophil G,, to crossreact with anti-rho polyclonal antibodies (Quilliam et ul., 1989), suggests the neutrophil substrate may not be rho per se. Recently, a 22,000 MW protein with 58% amino acid sequence homology with human rho has been cloned from the HL60 library (Didsbury et al., 1989). This protein, termed ruc, can serve as a botulinum toxin C, substrate when expressed in COS cells. rac may represent the novel botulinum toxin substrate activity found in G,,,. The potential use of botulinum toxin C, ADP-ribosyltransferase as a tool to identify cell functions regulated by the low molecular weight G protein substrate in neutrophils is evident. An additional low molecular weight G protein, ARF (ADP-ribosylation factor), can be shown to be present in neutrophils using ARF-specific antibodies (K. A . Kahn and G . M. Bokoch, unpublished observations). AKF is a 19,000 MW GTP binding protein known to be required as a cofactor for the ADP-ribosylation Stirnulatory Signals N-fPep

0

Inhibitory Signals

0

PK-C

Calmodulin

1

1

I PK-A 1

FIG. 4. Stirnulatory and inhibitory G prolein-rncdiated pathways in the neutrophil. N-Pep, N forinyl peptides; PL-C, phusphulipasc C; DAG, diacylglyccrul; AC, adcnylate cyclase; PK-C or A, protein kinase C or A; G,,, the major ncutrophil pertussis toxin substratc; G,, the stirnulatory regulatory G protein of adenylate cyclase; ISO, isoproterenol. This figure was contributed by Heinz Muellcr and Larry A. Sklar.

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of G, by cholera toxin (Kahn and Gilman, 1986),although it is likely ARF may have additional cellular functions as well. More recently, a clone for ral protein in a differentiated HL60 library has been identified (Weber et al., 1989). The expression of this protein in mature neutrophils or its possible identity with G,,, or G26,Khas not yet been determined (Fig. 4).

V. MECHANISMS FOR REGULATION OF SIGNAL TRANSDUCTION IN THE NEUTROPHIL A. CAMP-Dependent Inhibitory Pathways The situation which exists in the neutrophil with regard to activation of cell function clearly emphasizes the misleading nomenclature currently in use for the GTP binding proteins. In the past, G proteins have been classified according to their role in the adenylate cyclase system. Neutrophil activation by chemoattractant receptors involves a G protein of the class referred to as “Gi,” even though in the neutrophil the activation of this protein leads to stirnulatory effects on cell function. Conversely, hormones that stimulate adenylate cyclase via “GS,” in neutrophils cause a marked inhibirion of neutrophil activation through chemoattractant receptors. These respective classes of GTP binding proteins thus deserve opposite designations with regard to the process of neutrophil activation. The activation of neutrophil responses, such as enzyme release and superoxide formation, by chemoattractants can result in tissue damage during the directed migration of these cells to sites of inflammation. Mechanisms to regulate these activities may provide the appropriate balance between stimulatory and inhibitory events in the neutrophil such that tissue damage is minimized. Physiologic regulators of the CAMP-inhibitory pathway may be released by neuronal, endothelial, immune, or other cells in response to encounters with activated neutrophils. Increases in cellular cAMP levels induced by hormonal mechanisms (i.e., via PGE,, P-adrenergic hormones, histamine, and adenosine), or by forskolin, cholera toxin, and the membrane permeable cAMP analog, dibutyrylCAMP, antagonize neutrophil functions. These include granule enzyme release and superoxide generation (Zurier et a / . , 1973; Ignarro et al., 1974; Smolen et al., 1980; Simchowitz et al., 1980;Fantone et al., 1984; Tecoma et al., 1986), as well as others (Zurier et al., 1973; Rivkin er a l . , 1975; Hill et al., 1975). The Nformyl peptides themselves produce transient elevations of neutrophil cAMP levels (Jackowski and Sha’afi, 1979; Simchowitz et al., 1980; Smolen et al.. 1980). This does not occur through the direct coupling of N-formyl peptide receptor to G, (Verghese et al., 1985; Bokoch, 1987) but may reflect the elevation of cellular Ca2 stimulated by these chemoattractants (Verghese et al., 1985). It has been suggested that this transient increase in cAMP may serve to +

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terminate responses to the N-formyl peptides. Synergistic increases in cAMP can be produced by formyl peptides and P-adrenergic hormones or cholera toxin (‘Tecoma et ul., 1986; Bokoch, 1987; Cronstein et al., 1988). The mechanisms by which CAMP might regulate neutrophil activation have been investigated in a number of studies. Catecholamines inhibit oxidant production stimulated by N-formyl peptides in a manner clearly consistent with catecholamine binding to a P,-adrenergic receptor (Mueller et al., 1988). Blockade of P-adrenergic receptors with an irreversible antagonist indicated full inhibitory etfects can occur with as many as 50-60% of the receptors blocked (Mueller er a l . , 1988). These data indicate that marked amplification of this inhibitory signal must occur in order for the output of fewer than 10’ P-adrenergic receptors to inhibit the stirnulatory output of 5-10 x lo4 N-formyl peptide receptors. lsoproterenol did not inhibit the binding of N-formyl peptide ligands to rcccptor, indicating blockade of cell activation is subsequent to the ligand binding stcp 1988; Mueller and Sklar, 1989). Stimulation of oxidant produc(Mueller (Jr d., tion by phorbol myristate acetate (PMA), the diacylglycerol analog OAG, calcium ionophore A23 187, or NaF was not attenuated by isoproterenol, forskolin, or dibutyryl-CAMP, indicating the oxidant generating system per sc is not inhibited and that the inhibitory step is proximal to protein kinase C activation or elevation of intracellular Ca2+ (Mueller et u l . , 1988; Mueller and Sklar, 1989). However, Cronstein et al. (1985) reported inhibition of these same stimuli by adenosine andogs. The ability of N-formyl peptides to stimulate GTPase activity in cell sonicates was disrupted by isoproterenol, suggesting that the receptor-(; protein coupling may be directly modified by the action of isoproterenol (Mueller and Sklar, 1989). Interestingly, the effect of isoproterenol to inhibit the chemoattractant-stimulated GTPase activity was lost in a membrane system, suggesting that soluble elements (e.g., CAMP-dependent protein kinase) may play a role in modulating the inhibitory effects of isoproterenol and CAMP. Cronstein et al. (1985, 1988) have reported data that they interpret as indicating cAMP is not involved in mediating the effects of adenosine to inhibit neutrophil activation. They cite ( I ) the inability of adenosine to increase cellular cAMP levels in the absence of phosphodiesterase inhibitors or N-forniyl peptides, ( 2 ) the inability of adenosine analogs to inhibit secretion, even though this is inhibited by other CAMP elevating stimuli, and (3) the inability of phosphodiesterase inhibitors to potentiate the inhibitory effects of adenosine or its analogs on neutrophil function. Mueller and Sklar ( 1 988) examined the ability of various protein kinase inhibitors (the isoquinoline sulfonamides) to block the effects of cAMP elevating agents on neutrophil activation. The protein kinase antagonists H-7, H-8, and H-9 were able to effectively block isoproterenol, histamine, adenosine, PGE, forskolin, and dibutyryl-CAMP-mediated inhibition of superoxide anion generation. The direct action of these antagonists upon the putative neutrophil CAMP-

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dependent protein kinase was not examined. While the data discussed previously would suggest either the chemoattractant receptor or its associated G protein transduction partner as likely targets of the CAMP-dependent protein kinase, this has not yet been demonstrated. Direct phosphorylation of agonist-occupied padrenergic receptors has been shown to inhibit their ability to couple effectively to G, (Sibley et al., 1987), and an analogous situation exists for the rhodopsintransducin system (Kuhn and Dreyer, 1973). The occurrence of similar mechanisms in termination of neutrophil responses can only be suggested indirectly by analogy at this point.

B. Protein Kinase C Inhibitory Pathway The ability of activators of protein kinase C to inhibit the receptor-stimulated breakdown of polyphosphatidylinositols into inositol phosphates and diacylglycerol has been reported in a number of cell systems, including the neutrophil. This effect is presumably the result of a protein kinase-mediated phosphorylation reaction (Sha’afi et al., 1986). Smith et al. (1987) and Kikuchi et (11. ( I 987) have attempted to localize the point in the chemoattractant signal transduction pathway at which protein kinase C may act. In intact cells, treatment with phorbol myristate acetate (PMA) inhibited chemoattractant-induced inositol triphosphate generation. Membrane preparations from PMA pretreated cells also exhibited a reduced ability to hydrolyze phosphatidylinositol 4,5-diphosphate when stimulated by either fMLP and GTP, GTPyS, or GTP itself at low Ca2+ concentrations, but not when stimulated by high concentrations of C a 2 + . Binding of fMet peptides to the PMA treated membranes was not altered, nor was the stimulation by formyl peptides of the rate of GTPyS binding to the membranes. Both studies suggest that activation of protein kinase C can disrupt coupling of G protein to phospholipase C. Whether the G protein or the phospholipase C, or some third protein, is the (putative) substrate for protein kinase C-catalyzed phosphorylation leading to uncoupling of this interaction has not been determined. Gi in human platelets can be phosphorylated by a protein kinase Cmediated mechanism (Katada ct nl., 1985), as can the catalytic unit of adenylate cyclase (Yoshimasa et a l . , 1987). C. Regulation by Translocation-Physical Segregation

The existence of cytosolic forms of G, alpha subunits suggests the potential for regulation of signal transduction by physical dissociation-association of G, alpha subunits from-to the plasma membrane (Bokoch et al., 1988a). The release of G , alpha subunits stimulated by chemoattractants could allow the transduction of receptor signals to intracellular sites-enzymes not associated directly with the plasma membrane. Another possibility is that cytoplasmic G, alpha can

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serve as a source of transduction units able to be recruited to the membrane upon chemoattractant ligand binding, a situation somewhat analogous to the translocation of protein kinase C to the plasma membrane. The factors that can regulate G,, distribution in the neutrophil are as yet undetermined. It is known that G protein beta-gamma subunits are relatively hydrophobic when compared to the isolated G alpha subunits, and thcsc subunits can promote the association of alpha subunits with phospholipid membranes or receptors (Sternweis, 1986; Fung, 1983; Florio and Sternweis, 1985). Quantitation of the levels of G, alpha versus G beta-gamma subunits in neutrophil membranes by immunoblotting (Bokoch rt al., 1988a) indicates G alpha is present at levels -3- to 4-fold in excess of G beta-gamma subunits (30 7.5 pg/mg membrane versus 8.0 k 1.6 pg/mg membrane). This absence of stoichiometric beta-gamma subunits could account for the presence of the G, alpha subunits in the cytosol of fractionated cells. Whether alpha subunit release occurs under physiologically relevant activating conditions is unknown. If this distribution is regulated solely by the association of G, alpha subunits with betagamma subunits, it is expected that G protein activation and consequent subunit dissociation would result in alpha subunit release. The observation that GTPyS, a known G protein activator which causes subunit dissociation, will stimulate Gi alpha subunit release from membranes has been reported (Milligan et al., 1988). The ability of formyl peptides to inhibit the labeling of G, alpha by pertussis toxin has been suggested to be due to the dissociation of G, subunits under the 1987). It has not influence of the ligand occupied receptor (Matsumoto rt d., been shown that chemoattractant receptor activation results in GI, dissociation from the membrane. The continued association of GI, alpha subunits with the plasma membrane in the apparent absence of stoichiometric beta-gamma subunits suggests additional factors play a role in regulating G protein distribution. The observation that Gi and G, alpha subunits can be covalently modified by myristic acid is likely to be relevant, as the covalent attachment of lipid to proteins is known to stabilize their interaction with the membrane bilayer (Sefton and Buss, 1987). Whether G, is fatty acylated in the neutrophil has not yet been determined. It is of interest though to note that Adcrem et al. (1986) have reported the stimulus-dependent incorporation of [‘Hlmyristic acid into specific macrophage proteins, with changes in myristoylation occurring over periods of minutes. A major stimulus for myristoylation in the macrophage was bacterial lipopolysaccharide (LPS). This agent has bccn shown to “prime” cells such as the neutrophil and macrophages for enhanced responsiveness to chemotactic factors. The roles of myristoylation and G protein translocation in such priming phenomena have not been determined but could result in enhanced signal transduction by recruitment via acylation of G protein transduction units from a cytoplasmic pool. The cellular effects of the colony stimulatory factors (CSFs) have been reported to be sen-

*

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sitive to pertussis toxin (He et al., 1988; Imamura and Kufe, 1988), and tumor necrosis factor has been reported to stimulate the activity of a pertussis toxinsensitive GTPase in HL60 cell membranes, as well as to increase the rate of specific GTPyS binding to these membranes (Imamura et al., 1988). The possible role of G protein mobilization and modification as a component of the priming process warrants further investigation. Jesaitis et ul. (1988a) and Painter et al. (1987) have provided data that indicate the ligand-induced segregation of N-formyl peptide receptors into a membrane domain enriched in cytoskeletal markers and possibly depleted in G protein. The receptor in this down-regulated form exists in a high affinity state which is insensitive to guanine nucleotides and which appears to be functionally uncoupled from its G protein transduction partner. The formation of this receptor state was inhibited by dihydrocytochalasin B and appeared to be similar to a form of the formyl peptide receptor previously shown to co-isolate with neutrophil cytoskeleton (Jesaitis et ul., 1985). The regulation of receptor4 protein interactions by physical uncoupling and segretion appears analogous to the process of homologous desensitization observed for the P-adrenergic receptor-G, system (Harden, 1983). Similar regulatory mechanisms may thus be operative in the neutrophil chemoattractant receptor-(; protein transduction system.

VI.

CONCLUSIONS

The evidence that has been discussed in the preceding pages strongly suggests that G,, as the major neutrophil pertussis toxin substrate, is the likely means by which chemoattractant receptors couple to cell activation in the neutrophil. As a novel form of “Gi” (Gi.J, G, may serve to link chemoattractant receptors rather specifically to phospholipase C. What is necessary at this point, however, is the convincing biochemical demonstration of these expectations. In a similar fashion, the discovery of the low molecular weight G proteins in the neutrophil is of extreme interest in terms of the cellular transduction process. The existence of these proteins makes them logical candidates for the mediators of guanine nucleotide effects on granule exocytosis or oxidative burst activity. Additional roles might involve protein translocation, phospholipase regulation, and cell differentiation and priming events. The possibility that these proteins may interact with chemoattractant receptors directly is exciting and needs to be rigorously examined. Clearly a total understanding of the signal transduction process in the neutrophil will require definition of the functional roles of these low molecular weight GTP binding proteins. The continued analogies between the neutrophil chemoattractant receptor-(; protein system and the P-adrenergic receptor-adenylate cyclase system tempts us to extend this analogy once more. We are at a stage in the analysis of signal

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transduction in the leukocyte that the adenylatc cyclase system was at in the early 1980s. Thc outlines and components of the transduction system have been identificd, hut the detailed analysis of these components at the biochemical and structural level has just begun. The success in purifying and cloning the G protein component(s1 of this pathway, as well as the development of elegant methods of analysis of the macromolecular assemblies involved, indicate that such details will be forthcoming. While the continued use of the analogies between more well-dcfincd rcccptorG protein systems and that of the neutrophil provides a useful guide for experimental design, one should not overlook the novel aspects of thc ncutrophil signal transduction system. The existence of unique macromolecular components and their existence in unique stoichiomctrics within the plasma membrane, the existence of cytoplasmic forms of G,, the demonstration of such leukocyte-specific phenomena as “priming,” and the potential of novel leukocyte regulatory mechanisms governing chemoattractant receptor-(; protein-effector interactions all indicate that a full understanding of neutrophil signal transduction will require more than just the transfer of principles from transduction systems in other cells. Relating the biochemistry to thc actual cell physiology of the neutrophil will require the development of ingenious experimental approaches to dissect the architecturc of the transduction pathways in silu, as well as in the test tube. ACKNOWLEDGMENTS

I apologize in advance to any colleagues whose work may have been inappropriately excluded from the references. The format of this article made extensive referencing ditficult, and it wa5 necessary to list rcferences with the view that they would be used to access other appropriate articles. I would like to thank Dr. Larry A. Sklar. Dr. Heinz Mueller, Dr. Ronald C. Weingarten, and Dr. Lawrcncc A. Quilliam fur their suggcstions and coiiiiiicnts during the preparation of this chapter. Additional thanks to Monica Hartlett and Janet N. Bokoch for editorial assistance. REFERENCES Adari, H., Lowy, D. R.. Williamsen, B. M.. Der, C. J., and McCormick, P. (1988). Cuanosine triphosphatase activating protein (GAP) interacts with the p21 rux cfector binding domain. Science 240, 518-.521. Aderem, A . A,, Keum, M. M., Pure, E..and Cohn, 2. A. (1986). Bacterial Iipopolysaccharides, phorbol niyristatc acetate, and zymosan induce the inyristoylation of specific macrophage proteins. Proc. Nurl. A m d . Sci. U.S.A. 83, 5817-5821. Aktories, K . . Schultz. G . , and Jakohs. K H . (1987). Adenylatc cyclase inhibition and C;TPase stimulation by somatostatin in S49 lymphoma cyc-variants are prevented by islet-activating protein. FERS f.rif. 158, 169-173. Banga. H . S . , Gupta, S. K., and Feinstcin, M. B. (1988). Botulinum toxin D ADP-ribosylates a 22-24 KDa memhrane protein in platelet%and HI.-60 cells that is distinct from p21 r u r Biochern. Biophvs. Res. Cummirn. 155, 263-269. Barbacid, M. (1987). rus Genes. Artnu. Rei. Biochern. 56, 779-828. Harrowinan. M. M.. Cockcroft, S.,and Gonipcrts, B. D. (1986). Twu roles for guanine nucleotides in the stimulus-secretion sequence of neutrophils. Narurr (Imidon) 319, 504-507.

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Bar-Sagi, D., and Gomperts, B. D. (1988). Stimulation of exocytotic degranulation by microinjection of the ras oncogene protein into rat mast cells. Oncogene 3, 463-469. Becker, E. L., Yanaho, Y.. and Kermode, J. C. (1987). Nature and functioning of the pertussis toxinsensitive G protein of neutrophils. Biomed. Phurmacother. 41,289-297. Berridge, M. J. (1987). Inositol trisphosphate and diacylglycerol: Two interacting second messengers. Annu. Rev. Biochem. 56, 169-193. Bhullar, R. P., and Haslam. R . J. (1987). Detection of23-27 kDA GTP-binding proteins in platelets and other cells. Biochem. J. 245, 617-620. Bokoch, G . M. (1987). The presence of free G protein Pi? subunits in human neutrophils results in suppression of adenylate cyclase activity. J . B i d . Chem. 262, 589-594. Bokoch, G . M., and Gilman, A. G. (1984). Inhibition of receptor mediated release of arachidonic acid by pertussis toxin. Cell 39, 301-303. Bokoch, G. M., and Parkos, C. A. (1988). Identification of novel GTP binding proteins in the human neutrophil. FEBS Left. 227, 66-70. Bokoch, G . M., Sklar, L. A,, and Smolen, J . E. (1987). Guanine nucleotide regulatory proteins as transducers of receptor-stimulated neutrophil activation. Inf. J. Tissue Reacf. 9, 285-294. Bokoch, G. M., Bickford, K . , and Bohl, B. P. (1988a). Subcellular localization and quantitation of the major neutrophil pertussis toxin substrate. Gn. J . Cell B i d . 106, 1927-1936. Bokoch, G. M.. Parkos, C. A., and Mumby, S . M. (1988b). Purification and characterization of the 22,000-dalton GTP-binding protein substrate for ADP-ribosylation by botulinum toxin, G22k. J . B i d . Chem. 263, 16744-16749. , Bokoch, G . M.. Quilliam, L. A., and Liu, B. M. (1988~).Biochemical properties of G 2 2 ~ the neutrophil botulinum toxin G protein substrate. J. Cell B i d 107, 705a. Burgoyne, R . D. (1987). Control of exocytosis. Nature (London) 328, 112-113. Buss, J. E., Mumby, S . M.. Casey, P. J., Gilman, A. G . , and Sefton, B. M. (1987). Myristoylated a subunits of guanine nucleotide-binding regulatory proteins. Proc. Nafl. Acad. Sci. U.S.A. 84, 7493-7497. Chardin, P.. and Tavitian, A. (1986). The rul gene: a new ras related gene isolated by the use of a synthetic probe. EMBO J . 5 , 2703-2705. Cockcroft, S . , and Gomperts, B. D. (1985). Role of guanine nucleotide binding protein in the activation of polyphosphoinositide phosphodiesterase. Nature (London) 314, 534-536. Cockcroft, S . , Morell, T. W., and Gomperts, G. D. (1987). The G proteins act in series to control stimulus-secretion coupling in mast cells: Use of neomycin to distinguish between G proteins controlling polyphosphoinositide phosphodiesterase and exocytosis. J . Cell B i d . 105, 27452750. Cronstein, B. N . , Kramer, S . B., Rosenstein, E. D.. Weissmann, G . , and Hirschhom, R. (1985). Adenosine modulates the generation of superoxide anion by stimulated human neutrophils via interaction with a specific cell surface receptor. Ann. N . Y . Acad. Sci. 451, 291-301. Cronstein, B. N., Kramer, S . B., Rosenstein, E. D., Korchak, H. M . , Weissmann, G . , and Hirschhorn, R. (1988). Occupancy of adenosine receptors raises cyclic AMP alone and in synergy with occupancy of chemoattractant receptors and inhibits membrane depolarization. Biochem. J. 252, 709-7 15. DeLean, A,, Stadel, J. M., and Lefkowitz, R. J. (1980). A ternary complex model explains the agonist-specific binding properties of the adenylate cyclase-coupled beta-adrenergic receptor. J. B i d . Chem. 255, 7108-71 17. Della Bianca, V., Grzeskuwiak, M., Dusi, S . , and Rossi, F. (1988). Fluoride can activate the stimulation of phosphoinositide turnover and protein respiratory burst independently of Ca kinase C translocation in primed human neutrophils. Biochern. Biophys. Res. Commun. 150, 955-964. Der, C. J., Finkel, T., and Cooper, G . M. (1986). Biological and biochemical properties of human ras-H genes mutated at Codon 61. Cell 44, 167-176. +

+

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DeVos, A. M.. Tong, L., Milburn, M. V., Matias. P. M., Jananrik, J . , Noguchi, S., Nishimura, S . . Miura, K., Ohtsuka, E.. and Kim, S. H. (1988). Three-dimensional structure of an oncogene protcin: Catalytic domain of human c-H-rar p21. Scienre 239, 888-893. Dickey. B. F., Pyun, H. Y.. Willianison, K . C., and Navarro, J. (1987). Idcntification and purification of a novel G protein from neutrophils. FEBS Lett. 219, 28Y-292. Didshury, J. K . , and Snyderman, R. ( I 987). Molecular cloning of a new G protein. Evidence for two GialPh,-likeprotein families. FEBS Len. 219, 259-263. Didshury, J. K., Ho, Y.-S., and Snyderman, R. (1987). Human G, protein alpha-subunit: Deduction of amino acid structure trom a clvncd cDNA. FEBS Lett. 211, 160 164. Ilidsbury, J., Weher, R. F., Bokoch, G . M., Evans, T., and Snyderrnan, R. (1989). Rnc, a novcl rusrelated family of protcins that are hotulinum toxin substrates. J . B i d . Chrm. 264, 1637816382.

Doussierc, J., Pilloud, M.-C., and Vignais, P. V. (1988). Activation of bovine neutrophil oxidase in a cell f'rec system. GTP-dependent formation of a complex bctween a cytosolic factor and a membrane protein. Biochem. Biophys. Rrs. Cornmun. 152, 993- 1001. Evans, T., Brown. M. L., Fraser, E. D., and Northup, J. K. (1986). Purification of the major GTPbinding proteins from human placental rnemhranes. J. B i d . Chem. 261, 7052-70.59. Rntone, J. C., Marasco, W. A., Elgas, 1,. J . , and Ward, P. A. (1984). Stimulus specificity of prostaglandin inhihition of rabbit polynioiphonuclear leukocyte lysosomal enzyme release and supcroxide anion production. Am. J. Porhd. 115, 9- 16. Fernandez, J. M., Lindau, M., and Ecksiein. F. (1987). lntracellular stimulation of mast cells with guaninc nucleotides mimics antigenic stimulation. FEBS Lert. 216, 89-93. Fletcher, M. P., and Gallin. J. I.(1980). Degranulating stimuli increase the availability of receptors on human neutrophils for the chemoattractant fMet-Leu-Phe. J. Immunol. 124, 1.5X5-- 1588. Florio. V. A., and Sternweis, P. C. (198.5). Reconstitution of resolved muscarinic cholinergic receptors with purified GTP binding proteins. J. B i d . Chem. 260, 3477-3483. Fond. [I. K. W., Yoshimoto, K . K., Eversole-Circ, P., and Simon, M. 1. (1988). Identification of a GTP-hinding protein alpha subunit that lacks an apparent ADP-ribosylation site for pertussis toxin. Pror. Natl. Acud. Sci. U . S . A . 85, 3066-3070. Fung, B. K.-K. (1983). Characterization of transducin from bovine retinal rod outer segments. 1. Separation and reconstitution of the subunits. J. R i d . Chem. 258, 10495- 10502. Gabig. T. G., English, D., Akard, L. P., and Schell, M. S. (1987). Regulation of neutrophil NADPH oxidase activation in a cell-frce system by guanine nucleotides and fluroide. J . D i d . ('hem. 262, 1685-1690.

Gibbs, I. B., Schaber, M. D., Allard, W. J., Sigal, I. S . , and Scolnick, E. M. (1988). Purifications of r m GTPase activating protein from bovine brain. Proc. Nail. Acod. Sci. U.S.A. 85, 5026-5030. Gierschik, P., and Jakohs. K. H . (1987). Rcceptor-mediated ADP-ribosylation of a phospholipase Cstimulating G Protein. FEBS L e / / . 224, 219-223. Cierschik, P., Falloon. J.. Milligan, G . , Pines, M . , Gallin, .I. I . , and Spiegel, A. (1986). Immunochemical evidence for a novel pertussis toxin substrate in human neutrophils. J . B i d . Chem. 261, 8058-8062. Gierschik, P.. Sidiropoulous, D., Spiegel, A., and Jakobs, K. H. (1987). Purification and immunochemical characterization of the major pertussis toxin-sensitive guanine nucleotide binding protein of bovine neutrophil membranes. Eur. J . Biuchem. 165, 185- 194. Gilman. A. G . ( 1987). G Proteins: Tranducers of rcceptor-generated signals. Annu. Rev. Biochem. 56, 615-649. Goldsmith, P., Gierschik, P., Milligan, G., Unson, C. G . , Vinitsky, R.. Malech, H. L., and Spiegel, A . M. (1987). Antibodies directed against synthetic peptides distinguish bctwcen GTP-binding proteins in neutrophil and brain. J. B i d Chem. 262, 14683-34688. Gomperts. B. D. (1986). Calcium shares the limelight in stimulus-secretion coupling. Trends B i d . Sci. 11. 290-292.

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Haga, K., Haga, T., and Ichiyama, A . (1986). Reconstitution of the muscarinic acetylcholine receptor. J . Biol. Chem. 261, 10133-10140. Harden, T. K. ( 1983). Agonist-induced dcscnsitization of the beta-adrenergic reccptor-linked adenylate cyclase. Pharmacol. Rev. 35, 5-32. He, Y., Hewlett, E. L., Tcmeles, D., and Quesenberry, P. (1988). Inhibition of interleukin 3 and colony-stimulating factor 1 -stimulated marrow cell proliferation by pertussis toxin. Blood 71, 1 187- 1 195. Hill, H. R . , Estensen, R. D., Quie, P. G., Hogan, N. A., and Goldberg, N. D. (1975). Modulation of human neutrophil chemotactic responses by cyclic-3‘,5’-guanosine monophosphate and cyclic-3’,5‘-adenosine monophosphate. Merab. Clin. Exp. 24, 447-456. Howell, T. W., Cockcroft, S.. and Gomperts, B. D. (1987). Essential synergy between Ca+ + and guanine nucleotides in exocytotic secretion from penneabilized rat mast cells. J . Cell Biol. 105, 191- 197. Hyslop, P. A., Oades, 2 . G., Jesaitis, A. J., Painter, R. G., Cochrane, C. G., and Sklar, L. A . ( 1984). Evidence for N-formyl peptide chcmotactic peptide-stimulated GTPase activity in human neutrophil honiogenates. FEES Len. 166, 165- 169. Ignarro, L. J.. Lint, T. F.. and George, W. J. (1974). Hormonal control of lysosomal enzyme release from human neutrophils. J . Exp. Med. 139, 1395-1414. Imamura, K., and Kufe, D. (1988). Colony-stimulating factor I-induced Na+ influx into human monocytes involves activation of a pertussis toxin-sensitive GTP-binding protein. J . B i d . Chem. 263, 14093- 14098. Imamura, K., Sherman, M. L., Spriggs, D., and Kufe, D. (1988). Effect of tumor necrosis factor on GTP binding and GTPase activity in HL-60 and L929 cells. J. Biol. Chem. 263, 10247-10253. Iyengar, R., Rich, K. A., Herberg, J. T., Grenet. D.,Mumby, S., and Codina, J. ( I 987). Identification of a new GTP-binding protein (a Mr = 43,000 substrate for pertussis toxin). J . Biol. Chem. 262, 9239-9245. lyengar, R . , Rich, K. A., Herberg, J. T., Premont, R. T., and Codina, J. (1988). Glucagon receptormediated activation of Gs is accompanied by subunit dissociation. J . B i d . Chem. 263, 1534815353. Jackowski, S., and Sha’afi, R. 1. (1979). Response of adenosine cyclic 3’,5’-monophosphate level in rabbit neutrophils to the chemotactic peptide formyl-methionyl-leucyl-phenylalanine.Mol. Pharmarol. 16, 473-481. Jahng, K.-Y., Ferguson, J., and Reed, S. 1. (1988).Mutations in a gene encoding the alpha subunit of a Saccharomyces cerevisiae G protein indicate a role in mating pheromone signaling. Mol. Cell. Biol., 8, 2484-2493. Jelsema, C. L., and Axelrod, J. (1987). Stimulation of phospholipase A2 activity in bovine rod outcr segments by the p/y subunits of trdnsducin and its inhibition by the a subunit. Proc. Narl. Acad. Sci. U.S.A. 84, 3623-3627. Jesaitis, A. J., Naemura, J. R . , Sklar, L. A , , Cochrane, C. G., and Painter, R. G. (1984). Rapid modulation of N-formyl chemotactic peptide receptors on the surface of human granulocytes. Formation of high-affinity ligand-receptor complexes in transient association with cytoskeleton. J . Cell Biol.98, 1378- 1387. Jesaitis, A. J., Tolley, J. O., Painter, R. G., Sklar, L. A,, and Cochrane, C. G. (1985). Membranecytoskeleton interactions and the regulation of chemotactic peptide-induced activation of human granulocytes: the eflects of dihydrocytochalasin B. J. Cell. Biochem. 27,241-253. Jcsaitis, A. J., Bokoch, G . M., Tolley, J. O., and Allen, R. A. (1988a). Lateral segregation of neutrophil chemotactic receptors into actin- and fodrin-rich plasma membrane microdomains depleted in guanyl nucleotide regulatory proteins. J . Cell B i d . 107, 921-928. Jesaitis, A . J., Tolley, J. 0..Bokoch, G. M., and Allen, R. A. (1988b). Regulation of the interaction of chemoattractant receptor and G-proteins in the plasma membrdne of human neutrophils. J . Cell Biol. 107, 56A.

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CURRENT TOPICS IN MEMHRANES AND TRANSPORT. VOLUME 35

Chapter 5

Monovalent Ion Transport and Membrane Potential Changes during Leukocyte Activation: Lymphocytes BRUCE SELIGMA" InfZbmmutionlOsteourthritis,Enzymofogy Research Ciba-Geigy Pharmaceuticals Corporation Summit,New Jersey 07901

I. 11.

111.

IV,

V.

VI.

VII.

Introduction Membrane Potential Changes A . Limitations of the Methodology B. Effect of Stimuli C. Effect Altered Membrane Potential Has on Activation D. Calcium and Membrane Potential E. Summary pH Changes A. Effect of Stimuli B. SUmmdry NaiK-ATPase of Lymphocytes A. General Comments B . Effect of Stimuli C. Effect Inhibition of NaiK-ATPase Has on Mitogenesis D. Summary Anion Channels A. HC03iCl Antiport B . Voltage-Second Messenger Dependent Anion Channels C. Summary Cation Channels A. General Comments B. Voltage-Dependent Potassium Channels C. Voltage-Dependent Sodium Channels D. Summary Conclusion References

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Copyright 0 IYw hy Academic Prcih. Inc. All rights of reproduction In any form reserved.

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INTRODUCTION

The activation of lymphocytes involves complex changes in ion fluxes (permeability) across the plasma and intraccllular membranes, alterations in the intraccllular concentrations of ions, and changes in the binding of ions to membrane components and intracellular proteins (Grinstein and Dixon, 1989). Thcsc changes may altcr the ion gradients across membranes; regulate intracellular and organelle pH, osmotic equilibrium, and membrane potential; and drive ion solute transport and cncrgy mctabolisni. The complexity of ion transport and binding maintains cellular homeostasis and permits large changes in ion flux and concentration to occur without neccssarily gcncrating a change in membrane potential and pH or, alternatively, without imposing a large demand on energy metabolism, It is the complexity of ion transport that enables the cell to utilize the selective ionic permeability of lipid membranes to advantage. Basic to this system are the ion pumps, which couple energy metabolism of ATP to the net accumulative transport of ions across membranes. If a stitnulus induces changes in thc transport of specific ions across the plasma membrane due to the opening or closing of receptor or second messenger operated ion channels, there is a tendency of ion pump systems to countcr these altcrations in permeability, since the pump systerns are sensitive to any changes in the ionic composition of the cell and its environment. In addition, voltage-dependent ion transport systems come into play to either counter or amplify ionic processes that produce a small initiating change in membrane potcntial. Jon gated channels open (or close) to increase (or decrease) the transport of select ions in response to changes in the concentration of a different ion. Ion exchange antiport mechanisms operate to passively exchange one ion for another in response to the initial stimulus-induced perturbation of the ionic equilibrium. As will be discussed, this complcx array of cellular ionic machinery with which cells may be equipped is found in lymphocytes. Work can be accomplished by, or for, the cell through manipulation of membrane permeability to ions or through changes in the extracellular or intracellular ionic milieu. When dealing with the cellular activation of lymphocytes one must consider several general possibilities: (1) ionic changes induce a change in membrane potential or pH, which is the effector event; ( 2 ) ionic changes alter the free and hence bound concentration of an eft'ector ion necessary as a cofactur or facilitator in a binding or enzymatic process; (3) ionic fluxcs indirectly drive activation through processes such as (co)transport of an effector solute; (4) ionic changes induce ligand-receptor internalization; and ( 5 ) ionic changes regulate and alter the metabolic state of the cell. However, before these concepts can be addressed, the ionic mechanisms must be defined and linkcd to mitogcncsis in general and then to specific points along the mitogenesis pathway. The data will be discussed in this regard.

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There is one aspect that will not be considered in this review because of insufficient data-the subcellular compartmentalization of ionic changes. Obviously this is quite important in the metabolism of cells and is the function of mitochondria and sarcoplasmic reticulum. It is equally probable that subcellular compartmentalization occurs within the cytoplasm and underlying select areas of the plasma membrane, but only with the advent of fluorescent probe microscopic image analysis and electron probe image analysis have these aspects of ion regulation begun to be addressed. The focus of these studies has largely been on divalent cations for which the methods were initially developed and which are dealt with in another chapter. One should anticipate that the number of publications concerning this area of research will be increasing exponentially.

II. MEMBRANE POTENTIAL CHANGES

A. Limitations of the Methodology Changes in ionic permeability or gradients can result in changes in membrane potential. The data regarding membrane potential changes in lymphocytes are controversial, in part because indirect charged lipophilic probes have been used to assess this property. These studies can be divided into two broad categories, those based on positively charged probes (cyanine dyes and TPMP) and those based on negatively charged probes (oxonol dyes). Both types of charged probes are accumulated by mitochondria, which have a negative membrane potential relative to the cytosol. Because of this, the concentration of a positive probe will be greater in the mitochondrial compartment than in the cytosol, while the concentration of a negatively charged probe will be lower in the mitochondrial compartment than in the cytosol. If the mitochondrial compartment size is large relative to the total intracellular volume of the cell, or if a significant change in the size of the mitochondrial compartment occurs due to a change in the number of mitochondria, or if a change in the mitochondrial metabolic state alters the mitochondrial potential, the effect will be particularly reflected in changes in the distribution (fluorescence) of a positively charged probe, such as the cyanine dyes. To interpret such data it is necessary to separate the mitochondrial contributions from the total changes in probe distribution-fluorescence (Felber and Brand, 1983; Wilson and Chused, 1985; Wilson et al., 1985; Bramhall et ul., 1976; Rink et a/., 1980; Deutsch et at., 1979; Johnson et a/., 198I; Lark et al., 1975). Mitochondria1 poisons have been employed to eliminate this component, but in a metabolically active cell such as lymphocytes, where inhibition of mitochondrial activity might cause activation of early pathway components (due in part to raised intracellular calcium concentration) while inhibiting penultimate

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mitogenic events, this approach leaveb serious questions when it conies to interpretation of the results (Felber and Brand, 1983; Johnson rt ut., 1981; Laris et ut., 1975; Brand and Felber, 1984). Furthermore, the cyanine dyes themselves are toxic at high concentrations, particularly to B lymphocytes (Wilson et a/., 1985; Rink rt al., 1980).

B. Effect of Stimuli Despite these problems, a great deal of' work has bccn carried out with these probes, and the results have proved quite useful. Studies with TPMP, cyanine dyes, and oxonol dyes all indicate that the resting membrane potential of B and T lymphocytes ranges from -60 to -70 mV (Wilson and Chused, 1985; Wilson et af.,1985; Rink et al., 1980; Dcutsch et al., 1979). While several reports indicate that the calcium ionophore and concanavalin A (ConA) induce changes in mcnibrane potential (Bramhall et ul.. 1976; Shapiro, 1981; Monroe and Cambier, 1983), results using low (nontoxic) concentrations of cyaninc dyes or oxonol dyes do not confirm these findings. Instead the data indicate that low-dose calcium ionophore A23 187, causing mitogenesis, induces hyperpolarization of T lymphocytes (Wilson and Chused, 1985; Wilson et at., 1985).

C. Effect Altered Membrane Potential Has on Activation An alternative approach to define the role of membrane potential is to subject lymphocytes to conditions causing depolarization or hyperpolarization and observe the effect this has on activation. Progressive substitution of potassium for sodium in the extracellular media depolarizes T lymphocytes, though these cells resist any change in membrane potential until the extracellular potassium conccntration is quite high. This resistance to depolarization suggests voltage-sensitive channels are progressively activated to restore membrane potential when it is perturbed (Wilson and Chused, 1985; Gelfand et al., 1984). Furthermore, concurrent with the depolarization of membrane potential, proliferation of phytohemagglutinin (PHA) stimulated cells is inhibited. Gramicidin and nystatin also depolarize membrane potential and inhibit PHA induced proliferation, while depolarization in high potassium media has no effect on IL-2 stimulated proliferation (Gelfand et al., 1984). Thus the absence of proliferation of PHA stimulated cells is attributable to inhibition of 1L-2 synthesis. IL-2 receptor expression by PHA stimulated cells depolarized in high potassium is normal, as is the activation process once exogenous IL-2 is supplied. This effect of depolarization (on proliferation via effects on IL2 synthesis-secretion) may involvc effects on calcium entry into the cells and hence be indirect since ionomycin overcomes the inhibitory effect of high potassium. In similar studies, depolarization had no effect on antibody capping on B cells (Montecucco et ul., 1980).Hyperpolarization of the membrane potential of lymphocytes by treatment

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with valinomycin also has an inhibitory effect on proliferation (Daniele and Holian, 1976). However, these results must be interpreted with caution, since it is quite possible that valinomycin reaches the mitochondria and exerts its effects on the cell at this point.

D. Calcium and Membrane Potential More detailed studies of the relationship between calcium and membrane potential using ionomycin to stimulate calcium entry reveal that ionomycin induces T lymphocytes to hyperpolarize though a quinine-sensitive mechanism. The hyperpolarization and the ionomycin induced rise in intracellular calcium were both prevented by raising the extracellular potassium concentration, inhibiting the rise in intracellular calcium, or by addition of quinine (Wilson and Chused, 1985; Ishida and Chused, 1988; Tatham and Delves, 1984). A sodiumdependent depolarization occurs under these conditions where the hyperpolarization is blocked. In view of evidence presented subsequently on characterization of voltage-dependent potassium channels, these data suggest that this hyperpolarization is mediated through an outward conducting potassium channel sensitive to intracellular calcium. The data obtained from B lymphocytes are not as consistent. Studies from one group indicate that B cells depolarize when treated with ionomycin (Wilson and Chused, 1985). However, a different group of investigators report that anti-IgM and ionomycin both stimulate a hyperpolarization that is dependent on intracellular calcium such that in the absence of calcium a sodium-dependent depolarization is observed as in T cells (Tatham and Delves, 1984). A possible explanation for the contradictory results is the report that B cells are resistant to ionomycin induced calcium rise (Ishida and Chused, 1988). This resistance is apparently due to these cells exhibiting a large stimulated increase in calcium ATPase pump activity, which drives calcium out of the cell, countering the influx caused by ionomycin until the ionomycin reaches a concentration that swamps the pump effects. Thus the concentration of ionomycin used in studies becomes a critical point.

E. Summary There is little evidence that any substantial change in membrane potential occurs in response to stimulation of lymphocytes, but instead it appears that such changes are inhibitory. These cells have extensive ionic machinery which affords resistance to depolarizing changes over physiologic ranges of potassium concentration. Depolarization (such as suspension of cells in high potassium) prevents intracellular calcium from increasing and inhibits IL-2 secretion, preventing mitogenesis by a mechanism that can be bypassed by the addition of exogenous IL-2 or by raising intracellular calcium.

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111.

pH CHANGES

A. Effect of Stimuli Without any p l l regulatory mechanism the intracellular pH of lymphocytcs would be expcctcd to be approximately one unit bclow the extracellular level due to the high (-60 to -70 mV) membrane potential of these cells (Gelfand eta!., 1987). This would be cytotoxic to the cells. Instead, the pH gradient of lymphocytes is cstiniated to be only 0.1-0.5 pH units more acidic than the external pH when measured in media at various pHs between 6.6 and 7.6 (Dcutsch rt ui., 1979). These measurements may be affected by pH gradients across the nuclear membrane, which constitutes 70-80% of the total lymphocyte volume, but nonethe-less indicate that lymphocytes regulate their intracellular pH to maintain it at a nontoxic value. Activation of T lymphocytes with IL-2 causes an increase in intracellular pH by mechanisms that are sensitive to amiloride and require extracellular sodium, indicative of Na/H-antiport exchange (Mills et ul., 1985a). Amiloride itself inhibits proliferation, but analogs that etfectively inhibit the pH change do not inhibit proliferation. Thus the amiloride effect is apparently not mediated through inhibition of the Na/H-antiport exchange system, and the pH change is not required for proliferation. Similar results are found using PHA as the mitogen for T lymphocytes and the same amiloride analogs (Mills et a / . , 1986). An effect of phorbol esters on pH has also been reported (Grinstein et ul., 1985a). Phorbol esters induce an alkalinization in the pH of T lymphocytes of approximately 0.5 pH units through an amiloride sensitive mechanism dependent upon extracellular sodium which implicates a Na/H-antiport system. Based on a study using a radiolabcled analog of amiloride there arc approximately 8000 antiports in the membrane of lymphocytes (Dixon et a/., 1987). The activity of Na/H exchange is sufficient to permit measurement of a net dccrease in proton content and a net incrcase in sodium content of thc cclls. Amiloride sensitive cell swelling is associated with this process, attributed to the direct effects of the Na/H exchange as well as to increased HCO,/CI exchange. The intracellular HCO, concentration would tend to increase due to alkalinization and the diffusion of CO, into the cell. This in turn would exchange for chloride, leading to a net osmotic gain of chloridc. Evidence fur this mechanism is based on the dependence of amiloride inhibitable swelling on HCO, - and chloride containing media (Grinstein ef d.,1985a). In addition, membrane potential, measured using an oxonol dyc, becomes hyperpolarized in a ouabain and amiloride sensitive manner, consistent with the expectation that the rise in intracellular sodium concentration would stimulate Na/K-ATPase pump activity, hyperpolarizing the cells. Similar observations of coupling bctween NaiCl antiport, HCO,/CI exchange, and NaiK-ATPase pump activity and osmotic regulation of swelling have ~

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been made following acid loading or equilibration of cells in hypotonic media to induce osmotic shock upon resuspension in normal media (Grinstein et al., 1984a,b). A slight hyperpolarization (detected using a cyanine dye) is detected following recovery from acid loading, but in the case of the osmotic shock experiments, no change in membrane potential is observed. Swelling was also not affected by ouabain, suggesting the NaiK-ATPase activation was a secondary event. Concanavalin A also stimulates a sodium-dependent alkalinization of intracellular pH, as do lipopolysaccharide (LPS) and PHA (Hesketh et al., 1985; Mills et ul., 1986). Antibodies interacting with the T3T receptor of the leukemic T cell line HPB-ALL stimulate a calcium-dependent alkalinization (Roscoff and Cantley, 1985; Gerson et al., 1982). However, recent studies with human peripheral blood T cells demonstrated that the calcium ionophore ionomycin, PHA, ConA, and WGA caused an acidification of pH that was directly dependent on the raised intracellular calcium concentration and due to the generation of protons (Gelfand et ul., 1988). This acidification was partly counteracted by the NaiH-antiport induced alkalinization. The synergistic interaction of phorbol esters with these agents and other mediators that do not directly induce mitogenesis (anti-CD3 antibodies OKT3 and UCHT-I , etc.) to enhance proliferation was accompanied by an increase in intracellular calcium concentration and acidification of pH (Cheung et al., 1988). These results are consistent with the observation that stimulation of early activation events (c-fos mRNA transcription) in PHA stimulated rat thymic lymphocytes is not inhibited by amiloride analogs, nigericin, or low sodium and is instead associated with acidification rather then alkalinization (Grinstein et al., 1988a). Another report indicates no change in pH upon stimulation with lectins (Rogers et al., 1983). Differentiation is induced by several of the agents listed above, namely LPS and phorbol esters. Phorbol esters and LPS enhance Na/H exchange leading to alkalinization and inducing differentiation in a pre-B cell line (Roscoff et al., 1984). 1L-1 also stimulates alkalinization in B cells (Calalb et al., 1987). Amiloride blocks the surface expression of IgM which is induced by phorbol esters, LPS, and IL-I (Roscoff and Cantley, 1983; Roscoff et al., 1984; Stanton et al., 1986). In addition, surface IgM is expressed when the intracellular sodium concentration of cells is raised, as occurs following treatment with monensin or ouabain alone or in combination.

B. Summary These studies, taken together, suggest that there are two pathways regulating pH, one of which is dependent on the net rise in calcium concentration due to uptake into the cell and which induces acidification. The other depends on the NaiH antiport and is not dependent on intracellular calcium. A stimulus causing

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only calcium rise would induce acidification. A stimulus inducing both a calcium rise and activation of protein kinase C would simultaneously induce both alkalinization and acidification, with the net result being dependent upon the time course and summed magnitude of the two opposing processes. A stimulus causing thc generation of diacylglycerol alone without raising intracellular calcium would be expected to cause alkalinization of intracellular pH, swelling, and hyperpolarization of the resting membrane potential due to the stimulation of the NaiH-antiport system. The proposed mechanism for this is through phosphorylation of the Na/H exchanger or regulatory subunit, causing a change in the pH sensitivity of the system and hence inducing the exchanger to establish a gradient of different magnitude. Support for this was obtained using inhibitors of protein kinase C and demonstrating that the antiport system is not stimulated in cells depleted of either ATP or protein kinase C (Grinstein et al., 1985a,b, 1986). The role of pH regulation appears foremost to be restricted to maintaining homeostasis at a nontoxic pH. It would seem that the pH changes reflect this maintenance feature of the system rather than acting as an activation mechanism for proliferation. It remains to be detcrmined if differentiation is mediated or modulated by intracellular pH changes, a study that could be accomplished using the same amiloride analogs as used in the proliferation studies cited previously. Possible cffccts on specific pH-sensitive enzymatic activities have not been pursued because the role of pH appears more relegated to maintaining homeostasis than in stimulation of mitogenesis.

IV. Na/K-ATPase OF LYMPHOCYTES

A. General Comments It is clear from the data presented previously that lymphocytes have a complex array of ionic mechanisms to keep membrane potential at close to unstimulated levels even though large changes in specific ionic flux and in ionic gradients may occur that do regulate lymphocyte function. The Na/K-ATPase “ion pump” utilizes metabolic energy in the form of ATP to transport, or “pump,” sodium out of the cell and potassium into the cell, maintaining a gradient of each ion with the intracellular concentration of potassium high compared to the extracellular concentration (roughly 120 mM : 5 mM) and the intracellular sodium concentration low rclativc to the extracellular concentration (roughly 20 mM : 120 mM). Pump activity is dependent on, and stimulated by, the extracellular potassium concentration and intracellular sodium concentration. Since the Na/K-ATPase is an energized system, it is possible to generate a net charge displacement across thc plasma niembrane, an electrogenic potential. This results from the unequal exchange of several potassium ions for each sodium ion but is offset by the endogenous potassium ion “leak” out of the cell (down its concentration gradient),

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which occurs by passive permeability mechanisms. In unpublished studies we conducted investigating 42K transport in lymphocytes, greater than 95% of total potassium influx into B lymphocytes is inhibited by ouabain, while influx into T lymphocytes is much less sensitive. This observation indicates that B lymphocytes have unusual potassium permeability-exchange properties, namely, all potassium permeability in unstimulated B cells is via the Na/K-ATPase rather than through passive exchange mechanisms. T lymphocytes have a typical arrangement between the contribution of the Na/K-ATPase and total potassium permeability. 6. Effect of Stimuli The Na/K-ATPase activity increases when T and B lymphocytes are stimulated to proliferate by a variety of agents (Quastel and Kaplan, 1970; Averdunk and Lauf, 1975; Segel and Lichtman, 1976; Kaplan, 1978; Segel et al., 1979; Deutsch and Price, 1982; Negendank and Collier, 1976). The increase in activity is attributable to a rapid two- to threefold increase in the rate of pump activity. This is detectable within 3 min of stimulation with ConA, lasts for 3 hr, and is followed by an actual increase in the number of Na/K-ATPase pump sites in the membrane seen at 5 hr following ConA stimulation (Prasad et al., 1987) and as early as 20 min following stimulation with PHA (Kaplan, 1978). In these studies, the measurement of pump activity was based on calculation of ouabain-inhibitable potassium, rubidium, or sodium permeability, while the measurement of pump sites was determined by measuring ouabain binding. Ouabain binds competitively to the extracellular potassium binding site of the Na/K-ATPase. While generally pump activity is sensitive to the intracellular and extracellular concentrations of sodium and potassium, the mitogen stimulated increase in pump activity is dependent on an amiloride sensitive increase in intracellular sodium concentration (from 20 mM to 50 mM) and requires sufficient extracellular sodium to provide a gradient for its entry into the cell. Consistent with the association of increased Na/K-ATPase activity with proliferation, a decrease in Na/K-ATPase activity is associated with the process of terminal differentiation and cessation of proliferation (Kaplan, 1979; Kaplan and Owens, 1981).

C. Effect Inhibition of NaiK-ATPase Has on Mitogenesis The obligatory correlation of this increased Na/K-ATPase activity with proliferation is based on several observations. First, proliferation does not occur if cells are maintained in low sodium media or conditions that inhibit the amiloride sensitive sodium influx and activation of the NA/K-ATPase. Second, synthesis and blast formation are inhibited by ouabain in a manner that can be reversed by simply washing the inhibitor out or overcome by the addition of excess potassium (Quastel and Kaplan, 1968).

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D. Summary The proposed mechanism for the regulation of proliferation by the Na/KATPase is that within the first 3 min of mitogen stimulation there is induction of an amiloride sensitive influx of sodium down its chemical gradient into the cell (Segel et al., 1979; Prasad et d . ,1987; Owens and Kaplan, 1980; Rothenberg et d . ,1983; Grinstein e t a / . , 198Sb; Felber and Brand, 1983; Deutsch et al., 1981, 1984; Schuldiner and Rozengurt, 1982; Mills et al., 1985b). The resultant increase in intracellular sodium concentration (from 20 mM to SO mM) in turn activates the Na/K-ATPase already in the membrane within this same 3 min, which restores the sodium and potassium gradients. Over time, additional pump sites appear in the membrane to sustain the increased pump activity. The kinetics of these events vary some from report to report and appear much slower in murinc systems (Prasad et d.,1987; Owens and Kaplan, 1980), but as a generality, the mechanism appears conserved across lymphocyte T and B cell types and species. The mechanism by which the amiloride sensitive sodium influx is stimulated rcmains speculative. Protein kinase C can stimulate this exchange system (Grinstein et ul., 1985b), but it is not clear that stimulation of protcin kinasc C is necessary for lymphocyte proliferation (Rothenberg et ul., 1983). Demonstration that the amiloride sensitive sodium influx is the sole stimulus of increased pump activity has not been feasible because inhibitor studies, while useful in short term expcrimcnts to define ionic mechanisms, are not reliable when investigating the long term (48 hr) proliferative function of cells because of the toxicity and lack of specificity of the inhibitors, amiloride and the ionophore monensin.

V.

ANION CHANNELS

A. HCO,/CI Antiport The HCOJCI antiportcr or exchanger is a ubiquitous anion transport mechanism. In the lymphocyte, the transport of chloride is linked to the Na/H antipurter, specifically as a result of the HCO, transmembrane distribution being dictated by the pH gradient through the intracellular proton concentration (Mason ct al., 1989; Grinstein et al., 1988b). The effect of this anion exchanger is to causc a slight acidification of pH, as well as add to the intracellular buffering capacity of the cell. ~

B. Voltage-Second Messenger Dependent Anion Channels A large conductance voltage-dependent anion channel has been described in B lymphocytes that is nonselective (transporting C1- as well as aspartate) (Mc-

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Cann et al., 1989). This channel is insensitive to intracellular calcium, is found in clusters, and displays multiple conductance states active at depolarized potentials and inactive at the normal resting potential. This latter characteristic distinguishes this large anion conductance from the chloride conductances that operate and may be directly involved in maintenance of the resting potential of cells (Bosma, 1986; Blatz and Magleby, 1985). The regulatory factors (ligands, ions) for this channel await definition; however, the existing data indicate this channel would activate upon membrane depolarization and hence possibly have a role in the activation process. Channels can be operated by ligand binding to extracellular membrane receptors (receptor operated channels) or through the binding of second messengers. Such an anion channel, generating an outward C1 current, has been described in both Jurkat human T lymphocytes and normal Epstein-Barr virus transformed B lymphoblasts. This channel is observed upon imposing a depolarizing potential and depends on the presence of CAMP and calcium but is not calcium gated (Chen er al., 1989). This means that for the channels to open in the depolarized state calcium is required, but removal of calcium does not cause the channels to close. In whole cell experiments, CAMP is required, and in excised patches, the channels open if the catalytic subunit of the CAMP-dependent kinase is added in the presence of ATP. This latter evidence demonstrates that the channel itself, or a regulatory subunit, requires phosphorylation for the channels to open. This channel is defective in cystic fibrosis, having lost the dependence on CAMPdependent kinase. Instead the transformed cells from these patients exhibit only voltage-dependent chloride channels.

C. Summary The anion channels in lymphocyte activation have not been sufficiently studied. The HCO,/CI antiporter is of particular interest. While the observation has been made that there is a second messenger operated anion channel, its role in mitogenesis is questionable sincc this channel is abnormal in cells from patients with cystic fibrosis, for which there is no clear link to a defect in mitogenesis.

VI.

CATION CHANNELS

A. General Comments Cation channels can be divided into the cation exchange systems, such as the ATP-dependent Nd/K pump and Nai H antiport discussed above, voltage-dependent cation channels, and receptor or second messenger activated cation channels. Calcium channels are discussed elsewhere and thus will not be reviewed in this chapter. Instead this section will be confined to sodium and potassium channels.

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6. Voltage-Dependent Potassium Channels

I . CHARACTERISTICS One predominant type of potassium channel has been described in human cells and investigated extensively in T lymphocytes using patch clamp methods (Cahalan et ul., 1987; Mills et d., 1985b; Bregestovski et al., 1986; Chandy et d., 1984; DeCoursey et a/., 1985a.b; Lee et al., 1985; Deutsch el al., 1986a,b; Matteson and Deutsch, 1984; Chandy et d., 1985). Voltagc-dcpcndcnt potassium channels have also bccn described in B lymphocytes (McCann rt a/., 1987; MacDougall el ul., 1988; Choquet et al., 1987). The T lymphocyte channel is voltagc dcpcndent, opening as the membrane potential is depolarized to generate an outward current of potassium ions leaving thc cell with a halfmaximal conductance being observed at a potential of approximately -40 mV, and is sensitive to the extracellular potassium concentration. The effect of channel opening would tend to reverse the depolarization, causing the cell membrane potential to repolarize. The channels exhibit delayed rectification, meaning that if thc depolarization state is maintained the channels inactivate and close. The channels are not opened by calcium, and in fact, the data suggest that as intracellular calcium concentrations are raised to millimolar levels the numbcr of potassium channels that open upon dcpolarization progressively decrease, though the conductance per open channel is not affected (Bregestovski et al., 1986; Chandy et a / . , 1984; DeCoursey et al., 1985b). The T lymphocyte potassium channel is blocked by the classical organic ion potassium channel blockers tetraethylammonium (TEA) and 4-aminopyridine, as well as by quinine and cetiedil, which typically block calcium activatcd potassium channcls. The calcium channel antagonists diltiazem, nifedipine, verapamil, and polyvalent cations (La3 , Zn2 , Ni2 , Co2 , Mn2 ) also block this channel. In addition, the calmodulin antagonists trifluroperazine and chlorpromazine block the T lymphocyte potassium channcl. The mechanism of channcl inhibition of these agents is varied, but typically the effect is not a result of preventing the channels from opening, but instead the agents accelerate the rate of closing or make the channels refractory so that once each closes it is ditficult to open the channel again. The inhibitors also shift the membrane potential threshold for opening to more positive values, so that a larger depolarization is necessary to open the channels in the first place. It was reported that increasing thc concentration of extracellular calcium or Ba2 caused the potassium channels to inactivate more rapidly because these ions entcrcd thc open channels and were “trapped,” preventing the transport of potassium (Grissmer and Cahalan. 1989a). Raising the extracellular potassium concentration or adding rubidium slowed calcium and Ba’ induccd inactivation. consistent with the proposcd mechanism that these divalent cations entcr the same channel space as potassium. +

+

+

+

+

+

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The EL-4 cell line exhibits an additional type of potassium channel that opens when the membrane potential hyperpolarizes and is sensitive to the intracellular potassium concentration (Cahalan et al., 1985). This channel also exhibits delayed rectification. Murine T cells exhibit a channel that has all the properties of the human cell outward rectifying potassium channel, classified as “n” type (DeCoursey et al., 1985b, 1987a,b; Cahalan et al., 1985). This n channel opens to produce a halfmaximal current at approximately -40 mV and is blocked by TEA (1Cs0 8-16 mM) and polyvalent cations (Co2 and La3 as well as the other inhibitors of the human potassium channel. This channel is also blocked completely by 5 nM charybdotoxin (Lewis and Cahalan, 1988). The channels inactivate upon prolonged depolarization and exhibit accumulated inactivation upon repeated depolarizing pulses leading to a progressive reduction in current and ultimately permanent closure. TEA binds sufficiently rapidly that it is possible to show it not only reduces single channel conductance, but it is able to bind to open channels and prevent their inactivation (Grissmer and Cahalan, 1989b). However, not all murine lymphocytes exhibit this channel. A different type of potassium channel has been identified on some cells (though both have been found on the same cell), classified as an “1” potassium channel. This 1 channel differs in that it is activated to produce a half-maximal conductance at a more positive membrane potential, approximately - 10 mV, does not exhibit accumulated inactivation upon repeated depolarizing pulses, has very high sensitivity to block by TEA (50-100 pM IC5,,), and is not blocked by the polyvalent cations Co2+ and La3 , though these ions do cause the point at which the channels open to give a half-maximal conductance to shift to approximately 30 mV more positive. A third type of potassium channel has been characterized and classified as n‘ (Lewis and Cahalan, 1988). Like the n type channel, n’ potassium channels are blocked completely by 5 nM charybdotoxin but are even less sensitive to TEA (IC50 -100 mM) and, like 1 channels, do not exhibit accumulated inactivation upon repeated depolarization pulses. A second messenger CAMP-regulated, voltage-dependent outward rectifying potassium channel has been demonstrated in murine B cells following stimulation with LPS to induce blast formation, as well as in pre-B cell lines (Choquet et al., 1987). The channel opens upon depolarization of the cell membrane potential with a half-maximal current generated at approximately -20 mV. Similar to the potassium channels of T cells, this channel is sensitive to extracellular potassium (opening at more positive potentials in high potassium media), blocked by Co2+, but not activated by calcium. Instead, it is inactivated by high intracellular calcium concentrations. Regulation of conductance by cAMP distinguishes this channel from those discussed previously. Raising intracellular cAMP levels effectively decreases peak channel conductance, not as a result of gross inactivation or shift in voltage dependence but as a result of accelerating +

+

+

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both the rate of activation and inactivation so the net effect is that channels remain open for a more transient period during each depolarizing pulse. The adenylate cyclase activator forskolin plus the phosphodiesterase inhibitor theophyllin produce reversible inhibition of the outward potassium currents, associatcd with an increase in the rate of potassium channel inactivation. The adenylate cyclase inhibitor adenosine blocks the inhibitory effect of forskolin. 2. KOLE IN MITOGENESIS Stimulation of resting '1' lymphocytes with ConA, allogeneic cells, or phorbol esters causes an immediate increase in the number of outward rectifying potassium channels on human cells. A similar delayed increase in the number of n type channels occurs on rnurine cells stimulated with ConA (Chandy rt u!., 1984; DeCoursey et al., 1985a,b; Deutsch ct a / ., 1986b). However the overall increase on the murine cells is more striking because the resting cells express only about 10 channels/cell, with activation inducing an increase to 400 channels/cell within 24 hr. while the resting human cells express approximately 100 channels/cell, a number which increases only about 70% following stimulation. Cell volume also increases, but the murine cells still exhibit about a ten-fold increase in channel density. Furthermore, the murine cells exhibiting the largest number of channels represent the cells that are most rapidly proliferating (DcCoursey ei NI., 1987a,b). Kapidly dividing immature murine T cells from the thymus also exhibit (40-fold) greater numbers of potassium channels than mature resting T lymphocytes (Cahalan et ul., 1987). An increased number of potassium channels is induced following IL-2 stimulated proliferation of a murine cell line (L2), and the number of channels returns to normal upon removal of I L 2 (Lee rt d., 1986). This accompanies the return of the cells to their resting quiescent state. PHA and ConA also change the voltage dependence of the channels so that they open more readily upon dcpolarization by shifting the point of half-maximal conductance approximately 10 mV more negative. However, unlike ConA, PHA causes a decrease in the number of potassium channels on human cells, suggesting the number may not be as important as other aspects of the potassium channel characteristics (Lee et al., 1985). The outward rectifying potassium channel of human cells and the n type potassium channel found on murine cells appear to be essential for mitogenesis based on inhibitor studies (Chandy et a l . , 1984; DeCoursey et al., 1985b, 1987a, 1987b; Mills et al., 1985b). Proliferation of human T lymphocytes stimulated with PHA, ConA, phorbvl esters, OKT-3, or allogeneic cells is inhibited by TEA, 4-aminopyridine, quinine, diltiazem, and verapamil at the same pharmacologic concentrations at which the potassium channel is blocked. The TEA analog tetramethlyammonium, which does not block potassium channels, is without effect, demonstrating the specificity of this inhibition. Killing of allogeneic target cells by cytotoxic T cells is also inhibited by the potassium channel blockers, as is killing by NK cells (Cahalan et al.,

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1987; Schlichter e t a / . , 1986). Most, but not all, stimulated protein synthesis is inhibited by TEA and 4-aminopyridine. This includes IL-2 production but not increased expression of the 1L-2 receptor. The inhibition by TEA is seen at high concentrations, which block the n and n' type murine channels, but not at low concentrations, which completely block the 1 type channel, indicating that the 1 channels are not required for mitogenesis. Inhibition of proliferation by TEA and 4-aminopyndine requires that they be added during the first 10 hr following the mitogenic stimulus, and the effect is reversible. Furthermore, these blockers are without effect on a cell line (CCRF-HSB-2) that lacks the potassium channel (Mills et al., 1985b). The inhibition of proliferation and cytotoxicity is reversed in all cases by washing the cells free of inhibitor, as is blockade of the potassium channel, demonstrating that the effects on proliferation are not due to toxicity (a concern considering the long incubation times necessary for effect). In addition to the observation that the proliferation of a mutant cell line lacking potassium channels is not inhibited by potassium channel blockers, the expression of the 1 type channel by murine cells provides additional evidence that normal potassium channel activity is required for mitogenesis. This channel is particularly associated with diseased MRL mice which carry the lpr (lymphoproliferation) gene mutation and diseased C3H lpr and gld deficient mice (DeCoursey er al., 1987a,b; Cahalan er a f . , 1987; Chandy er al., 1986; Grissmer er a / ., 1988). These diseased mice exhibit a lupus-like autoimmune syndrome. During the first few months before spontaneously developing lymphoproliferation of functionally abnormal T cells, the T lymphocytes of these mice exhibit the normal distribution of small numbers of either n or 1 type channels. However, associated with the onset of disease and functionally abnormal T cells, the phenotype changes to expression of large numbers (20-fold more) of 1 type channels on those cells that exhibit the abnormal phenotype and function. It is not certain whether this association is causative or merely a closely associated epiphenomenon.

C. Voltage-Dependent Sodium Channels Voltage-dependent inward current sodium channels have been identified in mouse T lymphocytes but appear restricted to cells exhibiting predominantly n type potassium channels. They have been found only infrequently in human T lymphocytes (DeCoursey et al., I987a). These channels open with a half-maximal conductance at approximately -48 mV and are reversibly blocked by tetrodotoxin, tetramethylammonium, and Cs . Similar to the potassium channel, ConA induces a tenfold increase on the sodium channel number over a 24 hr period. However, distinctly different from the case of the potassium channels, blockade of sodium channels with tetrodotoxin had no effect on ConA stimulated proliferation. Murine NK clonal cells also exhibit this channel in high numbers, but again +

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BRUCE SELIGMANN

tetrodotoxin is not effective in inhibiting the killing function of these cells (Chan1986). dy et d.,

D. Summary The data from T lymphocyte studies indicate that the outwardly rectifying potassium channel is required for mitogenesis. There are insufficient data to assess the mitogenic requirement for the inwardly rectifying potassium channel of T cells, the CAMP-dependent potassium channel of T cells, the potassium channel of B cells, and sodium channels. There remain several unresolved questions concerning the fundamental mechanistic role of the T cell potassium channel in mitogenesis. First, though the evidence is quite extensive that this channel is required by normal cells, a cell line deficient in the potassium channel undergoes apparently normal mitogenesis. Thus this cell line has either developed an alternative mechanism to overcome its potassium channel defect or the potassium channel mediated step in activation has been bypassed. The answer to these questions may prove to be quite informative in defining the mechanistic role of the potassium channel in mitogenesis. The association of type 1 channels with the abnormally functioning T cells found in mouse models of autoimmune disease suggests the possibility that this channel may have a causativc role in the abnormal function of these cells. Whether there is a cause and effect role of the 1 type channels is under active investigation by the group that identified this channel.

VII.

CONCLUSION

The purpose of this review is not to collect all the data that have been published [as done in an excellent review by Grinstein and Dixon (1989)] but instead to focus on the conclusions that can be reached from the existing reports and indicate where additional experimentation is necessary. At the outset several questions were posed that should be addressed before concluding that this area of investigation is complete; namely, when dealing with the correlation of ionic events with the cellular activation of lymphocytes, one must address the following general possibilities: ( 1 ) ionic changes induce a change in membrane potential or pH which is the effector event; (2) ionic changes alter the free and hence bound concentration of an effector ion necessary as a cofactor or facilitator in a binding or enzymatic process; (3) ionic fluxes indirectly drive activation through processes such as (co)transport of an effector solute; (4) ionic changes induce ligand-receptor internalization; and (5) ionic changes regulate and alter the metabolic state of the cell. It is clear that there is still insufficient information to

5. ION TRANSPORT IN LYMPHOCYTE ACTIVATION

119

address all these questions. What is clear is that lymphocytes possess an extensive array of ionic mechanisms that are altered upon activation and that serve to maintain a framework of ionic homeostasis at or near the state of the resting cell (Fig. 1). Activating ligands and second messenger agents, such as protein kinase C, change the ionic state of the cell, and in maintaining homeostasis, the fluxes of many ions are altered and there is a great deal of ionic and metabolic activity generated that provides numerous points for modulating the activation of these cells. Causing the lymphocyte to deviate from this homeostasis either through the use of inhibitors or through the nonphysiologic manipulation of the intracellular or extracellular ionic milieu inhibits mitogenesis. Thus, lymphocytes are resistant to any actual depolarization of their resting membrane potential, but once depolarized by raising the extracellular concentration of potassium sufficiently proliferation is inhibited. It is postulated that this occurs through secondary effects on intracellular calcium that result in inhibition of IL-2 synthesis. Similarly, inhibition of the Na/H-antiport system, which maintains pH, modulates longterm cell competency, though not early activation events. The Na/K-ATPase fits into this scheme of conserved cellular homeostasis, with inhibitors preventing proliferation. However there is little evidence to suggest that an actual alteration in membrane potential, pH, or the Na/K-ATPase is a signaling mechanism for mitogenesis, and it is thus fairly safe to conclude that these general ionic mechanisms do not have a direct role in the activation pathway. Thus option 1, that membrane potential or pH changes are the effector events, is likely not to be the case. Option 5 , that ionic changes modulate the metabolic state of the cell, therefore, ionic homeostasis is required to maintain the metabolic state of the cell, is clearly one aspect that does apply. Numerous ion channels have been described using patch clamp methods, but while satisfying with respect to the amount of detail obtained about their regulation, again conclusions regarding the role of these specific ionic channels in mitogenesis cannot be reached at this time. The best evidence for a requirement in mitogenesis comes from the studies on the voltage-dependent, outward rectifying potassium channel where specific channel-blocking organic and inorganic ions inhibit mitogenesis of normal lymphocytes through effects at the level of IL-2 synthesis. Likewise the association of, and switch to, a different type of potassium channel (1 type) by diseased cells from murine models of autoimmune disease suggest there may be a correlation between potassium channel function and lymphocyte dysfunction. However, these observations need to be directly correlated with the point in activation which is dependent upon the potassium channel function. Figure 1 summarizes the ion conductances that have been identified in lymphocytes. No attempt is made to assign signficance to, or define any changes which occur upon, activation. At that time the remaining options listed previously ( 2 , 3 , and 4) can begin to be addressed. Finally, though there is an indication that calcium-dependent potassium chan-

120

A-

GI

t/

K

n n’l K

CAMP

-70 FIG. 1 .

mV

Diagram of the ion conductance mechanisms that have heen identified in lymphocytes.

( I ) NaiK-ATPase; (2) calcium ATPase; (3) magnesium ATPdse; (4)NaiH antiport; ( 5 ) HCOdCI antipon; (6) receptor-second messenger operated calcium channel; (7) voltage-gated. nonselective anion channel; ( 8 ) voltage-gated sodium channel; (9) voltage-gated, CAMP-dependent calcium activated anion channel; ( 10) calcium-dependent potassium channel; ( I 1) volldge-gated, CAMP-dependent, calcium-sensitive potassium channel; ( 1 2) voltage-gated potassium channel that opens at more negative (hyperpolarized)potentials; (13),( 14). and (15) voltage-gated potassium channels that open at more positive (depolari~ed)potentials. The magnitude of the resting membrane potential is indicated as well as the relative iuri cuncentratiuns (largcr font size, grcatcr concentration).

nels may cxist in lymphocytes, and have a significant role in activation, this has not been confirmed by patch clamp studies. ydking the approach that the indirect studies implicating a calcium-dependent potassium channel are correct, then the studies reported with cyanine and oxonol dyes in T and B lymphocytes point to one very interesting fact; namely, significant changes in the ionic mechanisms of lymphocytes can be induced by the manner in which the cells are held, particularly with regard to calcium and temperature. Thus it appears worthwhile to address carefully the manner in which lymphocytes are handled prior to and during study, as well as to thc ionic and cofactor content of the pipette. On the other hand, it is also quite likely that the indirect inhibitor studies are misleading due to the inhibitors having a different selectivity in lymphocytes than in other cells, as is, for instance, the case with the lymphocyte voltage-dependent potassium channel which is blocked by calcium channel blockers. In this regard,

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121

patch clamp studies directed toward investigating anion channels may prove particularly revealing. In general, the studies that are underway to define ligand and second messenger operated channels should provide greater insight into the role lymphocyte ionic mechanisms have in activation. Extending the indirect fluorescent probe studies in this direction would also be quite useful, particularly with the application of high resolution quantitative fluorescence image analysis now possible using confocal microscopy. Thus the question has been answered in the affirmative that the ionic homeostasis of lymphocytes is perturbed by the process of mitogenesis, and these cells respond with a wide array of transport mechanisms to maintain this homeostasis and the metabolic state of the cell. Questions remain as to whether specific ionic conductances, channels, or binding to effector proteins are necessary components of the direct activation pathway, and if so, how do they act as effectors. REFERENCES Averdunk. R., and Lauf, P. K. (1975). Effects of mitogens on sodium-potassium transport, 3Houbain binding, and adenosine triphosphate activity in lymphocytes. Exp. Cell Res. 93, 33 I342. Blatz, A. L., and Magleby, K. L. (1985). Single chloride-selective channels active at resting meinbrane potentials in cultured rat skeletal muscle. Eiophys. J . 47, 119-123. Bosma, M. M. (1986). Chloride channels in neoplastic B lymphocytes. Eiophvs. J. 49, 413. Bramhall, J. S . , Morgan, J . I . , Penis, A. D., and Britten, A. Z. (1976). The use of a fluorescent probe to monitor alterations in trans-membrane potential in single cell suspensions. Eiochem. Eiophys. Res. Commun. 72, 654-662. Brand, M. D., and Felber, S . M. (1984). Membrane potential of mitochondria in intact lymphocytes during early mitogenic stimulation. Eiochem. J . 217, 453-459. Bregestovski, P., Redlkozubov, A., and Alexeev, A. (1986). Elevation of intracellular calcium reduces voltage-dependent potassium conductance in human T cells. Nature (London) 319,776778. Cahalan, M. D., Chandy, K . G . , DeCoursey, T. E., and Gupta, S. (1985). Avoltage-gated potassium channel in human T-lymphocytes. J. Physiol. (London) 358, 197-237. Cahalan, M. D., Chandy, K . G . , DeCoursey, T. E., Gupta, S . , Lewis, R. S . , and Sutro, J. B. (1987). Ion channels in T-lymphocytes. Adv. Exp. Med. B i d . 213, 85-101. Calalb, M. B., Stanton, T. H.. Smith, L., Cragoe, E. J., and Bomsztyk, K. (1987). Recombinant human interleukin- I-stimulated Na+ / H + exchange is not required for differentiation in pre-B lymphocyte cell line. J. Eiol. Chem.262, 3680-3684. Chandy, K . G., DeCoursey. T. E., Cahalan, M. D.. McLaughlin, C. and Gupta, S. (1984). Voltagcgated K channels are required for T-lymphocyte activation. J. Exp. Mud. 160, 369-385. Chandy, K . G., DeCoursey, T. E., Cahalan, M. D., and Gupta, S. (1985). Electroimmunology: the physiologic role of ion channels in the immune system. J . Immunol. 135, 787-791. Chandy, K . G., DeCourbey, T. E., Fischbach, M., Talal, N . , Cahalan, M. D., and Gupta, S . (1986). Altered K + channel expression in abnormal T-lymphocytes from mice with the lpr gene mutation. Science 233, 1197-1200. Chen, J. H . , Schulman, H.. and Gardncr. P. (1989). A CAMP-regulated chloride channel in lymphocytes that is affected in cystic fibrosis. Science 243, 657-660.

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Cheung, R. K . , Grinstein, S . , and Gelfand, E. W. (1988). Mitogenic and non-mitogenic ligands trigger a calcium-dependent cytowlic acidification in human T lyniphocytes. J. Immunol.141, 1648-164 I.

Choqiiet, D., Sarthou, P., Primi, D., Cezcnave, P. A., and Korn, H. (1987). Cyclic AMP-modulatd potassium channels in murinc B cells and their precursors. Science 235, 121 1-1214. Daniele. R. P., and Holian, S. K. (1976). A potassium ionophore (valinomycin) inhibits lymphocyte proliferation by its efiects on the cell membrane, fror. Narl. Acad. Sci. U.S.A. 73. 3599-3602. lIcCoursey, T. E., Chandy, K. G . , Gupta, S . , and Cahalan, M . D.(l985a). Voltage-dependent inn channels in T-lymphocytes. J . Neuruinrmunol. 10, 71 -95. DeCoursey, T. E., Chandy, K . G., Gupta, S . , and Cahalan, M. D. (1985b). Voltage-gated K + channels in human T-lymphocytes: a role in mitogenesis'! Nature (London) 307, 465-468. DeCoursey, T. E., Chandy, K. G . , Gupta, S.,and Cahalan, M . D. (1987a). Mitogen induction of ion channels in murinc T-lymphocytes. J . Gen. Physiol. 89, 405-420. DeCourxy, T. E . . Chandy, K. G . , Gupta, S . , and Cahalan, M. D. (1987b). Two types of potassium channels in murine T-lymphocytes. J. Gen. Physiol. SY, 379-404. Deutbch, C. J . , and Price, M. (1982). Role of extracellular Na and K in lymphocyte activation. J . CC'ell BiOl. 113, 73-79. Dcutsch, C. J., Holian, A , , Holian, S . K . , Daniclc, R . P., and Wilson, D. F. (1979). Transmernbrane electrical and pH gradients across human erythrocytcs and human peripheral lymphocytes. J . Cull. Physiol. 99, 79-94. Deutsch, C . , Price, M. A., and Johansson, C. (1981). A sodium requirement for mitogen-induced proliferation in human pcripheral blood lymphocytes. Exp. Cell Re.\. 136, 359- 369. Deutsch, C. J . . Taylor, J. S . , and Price, M. A. (1984). pH homeostasis in human lymphocytes: modulation by ions and mitogen. J . Cell Riol. 98, 885-894. Deutsch, C. J . , Krause, D., and Lee, S. C. (1986a). Voltage-gated potassium conductance in Tlymphocytes stimulated with phorbol ester. J . Physiol. (London) 372, 405-423. Deutxh, C., Patterson, J . , Price. M . , Lee, S., and Prystowsky, M. (1986b). Volume regulation in cloned T-lyiiiphocytes. Oioph.ys. 1. 49, 162. Dixon. S. J., Cohen, S . , Cragoe, B. J., Jr., and Grinstein, S . (1987). Estimation of the number and turnover rate of Na' / H + exchangers in lymphocytes. J . Riol. Chem. BZ62, 3626-3632. Felber, S. M . , and Brand, M. D. (1983). Concanavalin A causes an increase in sodium pernicability and intraccllular sodium content of pig lymphueytcs. Biorhern. J. 210, 893-897. Gclfand, E. W., Mills, G. B., Cheung, R. K . . and Grinstein, S. (1984). Role of membrane potential in the regulation of lectin-induced calcium uptake. J . Cell Physiol. 122, 533-539. Gelfand, E. W., Mills, G . B., Cheung, R. K . , Lee, J. W., and Grinstein, S. ( I 987). Transnicmbrane ion lluxcs during activation of human T-lymphocytes: role of C a t t' N a + / H + exchange and phospholipid turnover. Immunol. Rev. 95, 59-87. Gelfand, E. W., Cheung, R. K . , and Grinstein, S. (1988). Calcium-dependent intracellular acidification doininatcs the pH response to mitogen in huiiian T cells. J. Immunol. 140, 246-252. Gerson, D. F., Kiefer, H . , and Eufe. W. (1982). lntracellular pH of mitogen-stimulated lymphocytes. Science 216, 1009-1010. Grinstein, S . , and Dixon, S. J. (1989). Ion transport, membrane potential, and cytoplasmic pH in lymphocytes: changes during activation. Phvsiul. Rev. 69, 4 17-48 1. Grinstein, S., Cohcn, S., and Rothstein, A. (1984a). Activation of Na-' / H + exchange in lymphocytes by osniotically induced volume changes and by cytoplasmic acidification. J. G m . Ph.ysiol. 82, 619-638. Grinstein, S., Cohen, S . , and Rothstein, A. (1984b). Cytoplasmic pH regulation in thymic lymphocytes by an amiloride-sensitive N a + / H + antiport. J . Gen. Physiol. 83, 341-369. Grinstin, S., Cohen, S . , Goetz, J. D., Rothstein, A., and Gelfand, E. W. (198%). Characterization

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of the activation of Na+ / H + exchange in lymphocytes by phorbol esters: change in cytoplasmic pH dependence of the antiport. Prof. Nurl. Acud. Sri. U.S.A. 82, 1429-1433. Grinstein, S . , Cohen, S . , Goetz, J. D., and Rothstein, A. (1985b). Osmotic and phorbol esterinduced activation of Na+ /H exchange: possible role of protein phosphorylation in volume regulation. J . Cell B i d . 101, 269-276. Grinstein, S., Mack, E., and Mills, G. B. (1986). Osmotic activation of the N a + / H + antiport in protein kinase C-depleted lymphocytes. Biochem. Biophys. Res. Commun. 134, 8- 13. Grinstein, S., Garcia, S . J . , and Mason, S. J. (1988a). Differential role of cation and anion exchange in lymphocyte pH regulation. Cibo Found. Symp. No. 139, 70-86. Grinstein, S . , Smith, J. D., Onizuka, R . , Cheung, R. K., Gelfand, E. W., and Benedict, S. (l988b). Activation of N a + / H + exchange and the expression of cellular proto-oncogenes in mitogenand phorbol ester-treated lymphocytes. J . Biol. Chem. 263, 8658-8665. Grissmer, S . , and Cahalan, M. D. (1989a). TEA prevents inactivation while blocking open K + channels in human T lymphocytes. J . Gen. Phusiol. 93, 609-630. Grissmer, S . , and Cahalan, M. D. (1989b). Divalent ion trapping inside potassium channels of human T lymphocytes. Biophys. J. 55, 203-206. Grissmer, S., Cahalan, M. D., and Chandy, K. G. (1988). Abundant expression of type 1 K + channels: A marker for lymphoproliferative diseases? J. Immunol. 141, 1137-1 142. Hesketh, T. R . , Moore, J . P.. Morris, J . D. H . , Taylor, M. V., Rogers, J . , Smith, G . A , , and Metcalfe, J. C. (1985). A common sequence of calcium and pH signals in the mitogenic stimulation of eukaryotic cells. Nature (London) 313, 481-484. Ishida, Y., and Chused, T. M. (1988). Heterogeneity of lymphocyte calcium metabolism is caused by T cell-specific calcium sensitive potassium channel and sensitivity of the calcium ATPase pump to membrane potential. J . Exp. Me&. 168, 839-852. Johnson, L. V.. Walsh, M. L., Bockus, B. J.. and Chen. L. B. (1981). Monitoring of relative mitochondria1 membrane potential in living cells by fluorescence microscopy. J . Cell B i d . 88, 526-535. Kaplan, J. G . (1978). Membrane cation transport and the control of proliferation of mammalian cells. Annu. Rev. Physiol. 40, 19-41. Kaplan, J. G. (1979). Activation of cation transport during lymphocyte stimulation: the molecular theology of spinning metabolic wheels. Trends Biochem. Sci. 4, N147-149. Kaplan, J. G . , and Owens, T. (1981). The cation pump as a switch mechanism controlling proliferation and differentiation in lymphocytes. Biosci. Rep. 2, 577-58 I . Laria, P. C . , Bahr, D. P., and Chaffee, R. R . J. (1975). Membrane potentials in mitochondria1 preparations as measured by means of a cyanine dye. Biochim. Biophys. Acto 376, 415-425. Lee, S. C., Krause, D., and Deutsch, C. (1985). Increased voltage-gated K + conductance in Tlymphocytes stimulated with phorbol ester. Biophys. J . 47, 147a. Lee, S . C., Sabath, D. E., Deutsch, C . , and Prystowsky, M. B. (1986). Increased voltage-gated potassium conductance during interleukin 2-stimulated proliferation of a mouse helper T-lymphocyte clone. J . Cell Biol. 102, 1200-1208. Lewis, R. S . , and Cahalan, M. D. (1988). Subset-specific expression of potassium channels in developing murine T lymphocytes. Science 239, 77 1-774. McCann, F. V., Keller, T. M., and Noelle, R. J. (1987). Ion channels in murine B-lymphocytes. J. Gen. Physiol. 90, 29. McCann, F. V., McCarthy, D. C . , Keller. T. M., and Noelle, R. J. (1989). Characterizationofa large conductance non-sclectivc anion channel in B lymphocytes. Gel/. Signall. 1, 3 1-44. MacDougall, S. L., Grinstein, S . , and Gelfand, E. W. (1988). Activation of CaZ+-dependent K + channels in human B lymphocytes by anti-immunoglobulin. J . Clin. Invest. 81, 449-454. Mason, M. J., Smith, J. D., Garcia-Soto, J. J., and Grinstein, S . (1989). Internal pH-sensitive site +

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couples CI -HCO,- exchange to Na+-H+ antiport in lymphocytes. Am. J . Phvsiol. 256, C428-C433. Mattcson, D. R . , and Deutsch. C. (1984). K channels in T lymphocytes: a patch clamp study using monoclonal antibody adhcsion. Nuture (London) 307, 468-47 I . Mills, G.B., Cheurig. K . K . , Grinstein, S., and Gelfand, E. W. (1985a). Increase in cytosolic free calcium concentration is an intracellular messenger for the production of intcrlcukin 2 hut not for the expression of the interleukin 2 receptor. J . Immunol. 134, 1640-1643. Mills, G . B . , Cragoe, E. J . , Jr., Gelfand, E. W., and Grinstein, S . (1985b). lnterleukin 2 induces a rapid increase in intracellular pH through activation ofa Na+ /H antiport. J. B i d C‘hrrn. 260, 12500 - 12507. Mills, G . B . . Chcung, R . K., Cragoe. E . J., Jr., Grinstein, S . , and Gelfand, E. W. (1986). Activation of the NaC / H antiport is not required for the lectin-induced proliferation of human T-lymphocyles. J . Immunol. 136, 1150-1 154. Monroe. J. G . , and Camhier, J. D. ( 1983). B cell activation. 1. Antl-itnmunoglobulin-iu[iuccd receptor cross linking results in a decrease in plasma membrane potential of murinc B lymphocytes. J . E.rp. Mrrl. 157, 2073-2086. Montecucco, C.. Rink, T. 1.. Pozzan. T., and Mctcalfe, J. C. (1980). Triggcrmg of lymphocyte capping appears not to require changes in potential o r ion fluxes across the plasma membrane. Biolhim. Biuphvs. Acru 595, 65-70. Negendank, W. G . . and Collier, C. R. (1976). Ion contents of human lymphocytes: the effects of concanavalin A and ouhain. Exp. Cell Rrs. 101, 31-40. Owens, T., and Kaplan, J. G. ( 1 ‘980). Increased cationic fluxes in stimulated lymphocytes of the mouse: response of enriched R- and T-cell subpopulations to B- and T-cell mitogens. Cun. J . Biuchrm. 58, 83 1-839. Prasad. K. V. S . , Severini, A , , and Kaplan, J. G. (1987). Sodium ion influx in proliferating lymphocytes: an early component of the mitogenic signal. Arch. Biochcm. Biuphy. 252, 5 1552s. Quastel. M R., and Kaplan, J . G. (1968).Inhibition by ouhain of human lymphocyte transformation induced by phytohemagglutin in v i / r n Nature (London) 219, 198-200. Quaatcl. M. R., and Kaplan, J. G . (1970). Early stimulation of potassium uptake in lymphocytes treated with PHA. Exp. Cell Rrs. 63, 230-~233. Rink, ‘r. J., Montecucco, C.. Hesketh. T. R., and Tsein, R. Y. (1980). Lymphocyte membrane potential assessed with fluoresccnt prohes. Biochim. Biophys. Actu 595, 15-30. Kogcrs, J., Hesketh, T. K , Smith, G. A,, and Metcalfe. J. C. (1983). lntracellular pH of stimulated thyinocytes measured with a new fluorescent indicator. J . Biol. Chern. 258, 5994-5997. Rovmff, P. M., and Cantley, L. C. (1983). Increasing the intracellular Na concentration induces differentiation in a pre-B lymphocyte cell line. Proc. Narl. Acud. Sci. U.S.A. 80, 7547-7550. Roscoff. P. M.. and Cantley, L. C. (1985). Stimulation of the T.3-T cell receptor associated Ca + influx enhances the activity of the Na+ / H + exchanger in a leukemic T cell line. J . Biol. U I ~ I T I . 260, 140.53- 14059. Koscoff, P. M.. Stein. L., and Cantley, L. C. (1984). Phorbol esters induce differentiation in a pre-€3 lyrriphocyte cell line by enhancing Na’ / I f + exchange. J. R i d . Chrm. 259, 70.56-7060. Kothenherg, P.,Glascr, I>. , Schlesingcr, P..and Cassel, D. (1983). Activation of Na+ /H exchange by epidermal growth factor elevates intracellular pH in A431 cells. J . B i d . Clzrm. 258, 1264412653. Schlichter, L.. Sidell, N., and Hagiwara, S. (1986). Potassium channels mediate killing by human natural killer cells. froc. Nut/. Arud. Sci. U.S.A. 83, 451-455. Schuldincr, S., and Ruzengurt, E. (1982). Na+/H+ antiport in Swiss 3T3 cells: mitogenic stitnulation leads to cytoplasmic alkalinization. froc. Nut/. Acad. Sci. U.S.A. 79, 7778- 7782. +

+

+

+

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Segel, G. B., and Lichtman, M. A. (1976). Potassium transport in human blood lymphocytea treated with phytohemagglutinin. J. Clin. Invest. 58, 1358- 1369. Segel, G. B., Simon, W., and Lichtman, M. (1979). Regulation of sodium and potassium transport in phytohemagglutinin-stimulated human blood lymphocytes. J . Clin. Invest. 64, 834-841. Shapiro, H. M. (1981). Flow cytometric probes of early events in cell activation. Cytometry 1, 301312. Stanton, T. H . , Maynard, M . , and Bomsztyk, K. (1986). EEect of interleukin-1 on intracellular concentration of sodium, calcium, and potassium in 70213 cells. J. B i d . Chem. 261, 56995701. Tatham, E. R.. and Delves, J. (1984). Flow cytometric detection of membrane potential changes in murine lymphocytes induced by concanavalin A . Biochem. J . 221, 137-146. Wilson, H. A . , and Chuaed, T. M. (1985). Lymphocytes membrane potential and Cai- sensitive potassium channels described by oxonol dye fluorescence measurements. J . Cell Physiol. 125, 72-8 I . Wilson, H. A , , Seligmann, B. E., and Chused, T. M. (1985). Voltage sensitive cyanine dye fluorescence signals in lymphocytes: plasma membrane and mitochondria1 components. J . Cell Physiol. 125, 61-71, +

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CURRENT TOPICS IN MEMBRANES A N D TRANSPORT. VOLUME 15

Chapter 6 Monovalent Ion Transport and Membrane Potential Changes during Activation in Phagocytic Leukocytes ELAINE K . GALLIN AND LESLIE C . MCKINNEY Department o j Physiology Armed Forces Radiobiology Research Institute Bethesda, Maryland 20814

Introduction Ionic Basis of the Resting Membrane Potential A . Intracellular Ion Concentrations of Macrophages and Neutrophils B. Resting Mcmbranc Potential 111. Ionic Channels, Pumps, and Carriers A . Terminology B. Ionic Channels in Macrophages C. Ionic Channels in Neutrophils D. Pumps and Carriers in Macrophages and Neutrophils IV. Role of Membrane Potential and Ionic Conductances in Phagocyte Function A . Membrane Potential B. Ionic Conductances V. Summary References I.

11.

1.

INTRODUCTION

Phagocytic leukocytes (macrophages, neutrophils, and eosinophils), like all other cells, regulate the permeability of their membranes to various ions in order to establish ionic gradients, maintain a resting membrane potential, and control the ionic composition of the intracellular milieu. Much progress has been made in characterizing the various channels, pumps, and carriers that regulate ion transport across neutrophil and macrophage membranes. Since little data on ionic 127

Copyright 0 1990 by Acadcniic Press, InL All right? of rcproduction in any form re\eNcd

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ELAINE K. GALLIN AND LESLIE C. MCKINNEY

transport in eosinophils exist, this chapter will focus only on the macrophage and thc neutrophil. Studies on the ionic basis of the resting membrane potential, thc properties of several well-characterized ionic conductances and ion transporters of the macrophage and neutrophil, and the role of membrane potential and ionic conductances or transport mechanisins in phagocyte function will be reviewed.

II. IONIC BASIS OF THE RESTING MEMBRANE POTENTIAL A. lntracellular Ion Concentrations of Macrophages and Neutrophils The resting membrane potential (resting Vm) of a cell is determined by the concentration of ions inside and outside the cell and the permeability of the membrane to those ions. Values for the intra- and extracellular concentrations of the physiologically important ions are given in Table I. With these values the equilibrium potential (Eio,,)for a given ion can be calculated using the Nernst equation: Eicrn= RT/zF In [ion],,/[ionJi, where R is the gas constant, T is temperature, z is valence, and F is Faraday's constant. The resting membrane potential of the cell will go toward the equilibrium potential of the ion with the TABLE I NEUTROPHILSA N D MACHOPHAGES

IONIC CONCTN I HATION GRADIENTS I N

Intracdlular concentration Extraccllular concentration

Ion

Na' K+ CI Ca2' H'

(IIIM)

136- 145 4.5 100-106 4.3-5.3

pH, 7.35-7.45

(mM) Human neutrophil

25" 120" 80/' 100 nM' pH, 7.25''

Human macrophage

?4? 128' 10Y 4 0 nM' pH. 7.1"

Siinchowitr er (if. (1982). Siinchowitz and De Weer (1986). Lew er ol (19x4); vcin 'l'schamer et oi (19Xb). 4 Siiiichowitz and R o w (1985), Grinstein and Furiiyii ( I O X b ) (' Human peripheral blood rnonocyce\ (Itice L'I d,, 1987h) Alveolar rnacrophagcs (Stickle er a / . . 1984). 8 L. C.McKinney (unpublished observations). h Sung rr a / (1985). ' Meliiied rrol. (1981). YUllng P I d.(1984). 1 McKinney and Moran (1989). '1

b 1

J

Murk 1774 cells 10"

162", 1531 44 ' 87 nM'

pH, 7.5.'

6. ION TRANSPORT IN PHAGOCYTE ACTIVATION

129

largest permeability. Changes in the external or internal concentration of a permeant ion, or the permeability of the membrane to a given ion, will change the resting membrane potential. It is experimentally difficult to change one of these parameters without secondarily affecting another. For example, even simple procedures, such as modifying the ionic composition of the bathing solution or storing cells below 37”C, can lead to altered intracellular ion concentrations. Transient exposure of cells to ammonium chloride, which occurs during some isolation procedures, induces pH transients that can also disrupt cell function (Pfefferkorn, 1984).

B. Resting Membrane Potential Resting membrane potential can be measured directly, by impaling cells with microelectrodes, or indirectly, using radiolabeled lipophilic cations or fluorescent dyes that partition themselves across the membrane according to membrane voltage. Two different types of microelectrodes (intracellular and patch electrodes) have been used to measure Vm. lntracellular microelectrodes have narrow tip diameters and high resistance and are used to impale cells directly. Although stable Vms as low as -75 mV have been recorded in some macrophages using intracellular microelectrodes (Gallin and Livengood, 198I), impaling small cells, such as phagocytes, often results in membrane damage and induction of leak current. To correct for the effects of microelectrode damage, Ince er al. (1983, 1986) estimate the membrane potential of macrophages from the rapid transients recorded immediately after microelectrode penetration. Since the advent of the patch clamp technique (Hamill et al., 1981), intracellular microelectrodes have been used less frequently. Patch electrodes have wider tip diameters (and therefore a lower resistance) and are used to suction a tiny patch of membrane into the electrode, where a mechanically stable, electrically tight seal (the so-called “gigaohm seal,” on the order of 10’ Q) is formed between the cell membrane and electrode glass (see Fig. I for illustration). Application of additional suction causes the small patch of membrane to rupture without disrupting the seal, allowing electrical contact between the patch pipette solution and the cell interior. Membrane potential measurements are made immediately after rupture of the membrane before diffusion of the pipette contents into the cell is completed. 1. MACROPHAGES

Human Macrophagrs. Resting membrane potentials in macrophages derived from human peripheral blood monocytes have been measured with both intracellular and patch electrodes. Using intracellular electrodes, values of -36 (Ince et al., 1987b) and -42 mV (McCann ef al., 1983) were obtained from (1.

130

ELAINE K. GALLIN AND LESLIE C. MCKINNEY

Cell Attached Patch

-

Excised

inside-Out Patch

Patch

Fic,. I . Schematic rcprcscntation 01 four diRcrcnt patch clamp recording configurations

human monocytes maintained in culture for at least 5 days. Measurements with patch electrodes have yielded consistently more negative values. Gallin and McKinney ( 1988a) reported that human peripheral blood monocytes cultured for I I- 15 days had a resting Vm of -5 I mV. Approximately one-third of these cells expressed an inwardly rectifying K conductance (which is described later in this chapter); these cells had an average resting Vm of -56 mV, slightly more ncgative than that found in cells that did not express an inwardly rectifying conductance (-42 mV). The resting membrane potential of human alveolar macrophages cultured under adherent conditions for 1 day following isolation had a resting Vm of -56 niV; interestingly, these cells were considerably more depolarizcd (resting Vm = - 15 mV) when freshly isolated (Nelson rt d., 1985). Values for the resting Vm for human macrophages are substantially more positive than the equilibrium potential for K for these cells (EK is approximately -81 mV; see Table I), indicating that resting Vm is not established simply by K permeability ( P K ) . Ion substitution experiments demonstrated that human macrophages were depolarized either by raising external K or reducing external chloride, while varying external “a] had no effect on resting Vm (Ince et al., 1987b). In this study, the relationship between membrane potential and external K and C1 concentration was best described assuming that cells were permeable to both ions, with a P, : P,, ratio of 4.3. I). Mitrine M u c w p h a p ’ s . Murine macrophages have a considerably more negative resting Vm than human macrophages. Early studies with niicroelectrodes showed a biphasic distribution of membrane potentials; the average resting Vms of these two groups were -28 mV and approximately -75 mV (Callin and Livengood, I98 1). The group of cells that had a more negative

131

6 . ION TRANSPORT IN PHAGOCYTE ACTIVATION

resting Vm exhibited markedly nonlinear I-V relationships and exhibited two stable states of resting Vm. Similar behavior has been reported in basophilic leukemia cells (Lindau and Fernandez, 1986). The group of macrophages with smaller resting Vm (-28 mV) had I-V relationships with little, if any, rectification and probably represented cells damaged by microelectrode penetration, since subsequent patch clamp studies by two different groups of investigators demonstrated that mouse peritoneal macrophages maintained for at least 24 hr in culture have resting Vm values of -80 to -90 mV (Ypey and Clapham, 1984) and -70 to -80 mV (Randriamampita and Trautmann, 1987). The closeness of these values to E , suggests that murine macrophages have little resting permeability to ions other than potassium. In the murine macrophage-like cell line 5774 (Snyderman et al., 1977), resting membrane potentials have been measured using several different techniques, and the influence of adherence on resting Vm has been systematically studied. Three groups have measured the resting potential of nonadherent 5774 cells, each using different methods. Sung et al. (1985) reported a value of -14 mV, using the lipophilic cation tetraphenylphosphonium (TPP ); McCaig and Berlin ( 1983) reported - 35 mV using triphenylmethylphosphonium (TPMP ); and Ehrenberg et al. ( 1 988), using rhodamine-6G, reported a value of -26 mV. Within 6 to 8 hr after adherence, 5774 cells acquire a more negative resting Vm (Sung et al., 1985; McKinney and Gallin, 1989). Values for adherent cells, determined by patch electrode measurements (Gallin and Sheehy, 1985; McKinney and Gallin, 1988; Randriamampita and Trautmann, 1987), radioisotope distribution (Sung et a / ., 1985), and fluorescent dyes (Ehrenberg et al., 1988) are similar, and cluster around -70 mV. The closeness of these values to E , and the observation that Vm is well predicted by the Nernst equation for K (Gallin and Sheehy, 1985) indicate that adherent 5774 cells have little permeability to ions other than potassium. The predominant ion channel present in adherent 5774 cells is an inwardly rectifying K channel; a block of this channel with 1 mM barium depolarizes 5774 cells by approximately 30 mV. Recent studies indicate that the increase in resting Vm that occurs during the first 6-8 hr following adherence is due to an increase in the density of inwardly rectifying K channels in the membrane (McKinney and Gallin, 1989). +

+

c. Contribution ojNa / K Pump to Vm. The electrogenic Na /K pump contributes to the resting Vm of both human and murine macrophages. Gallin and Livengood (1983), recording from mouse spleen macrophages, reported that the Na + / K + pump contributed -7 mV to the resting potential in the steady state. Under nonsteady state conditions, that is, after cells had been sodium-loaded in the cold and were then rewarmed, the pump hyperpolanzed the macrophage by as much as 30 mV. Ince et a/.(1987b) demonstrated that the Na+.lK+ pump contributed - 1 1 mV to the resting Vm of human peripheral blood monocytes. +

+

+

+

132

ELAINE K. GALLIN AND LESLIE C. MCKINNEY

Thesc values arc in accord with calculations showing that the maximal possible contribution that the Na / K pump can make to the resting Vtn of a cell under steady state conditions (assuming a coupling ratio of 3 K + /2 Na ’ ) is - 10 mV (Thomas, 1972). +

d . O.scil1ution.s in Vm. lntracellular microelectrode studies have shown that macrophages (human or murine) can exhibit oscillations in membrane potential from a resting level of -30 or -40 niV to potentials near E , (approximately -80 mV; Gallin et al., 1975; Dos Reis and Oliveira-Castro, 1977; Gallin and Gallin, 1977). These oscillations were ascribed to the activation of a calciumdependent K conductance, since they involved an incrcase in conductance, reversed near B,, were blocked by EGTA, and were induced by either ionomycin (Gallin et al., 1975: Dos Reis and Oliveira-Castro, 1977) or the intracellular injection of calcium (Persechini et ul., 1981). Although the oscillations were not blocked by tetraethylarnmonium chloride (TEA-CI, 50 d), they could be blockcd by quinine ( I .5 mM) and barium (20 mM) (Araujo et (11.. 1986). Incc et al. (1984) concluded that these oscillations were not physiological but were induced by a leak of external calcium into the cell following impalement by microelectrodes. However, recent experiments with the patch clamp technique, where electrode-induced leak current was negligible, confinned the presence of spontaneous oscillations (see section “Bursting Inward K Channel”; Callin, 1989). In addition, Kruskal and Maxfcld (1987) have demonstratcd that spontaneous oscillations in [Cali can occur in macrophages after adherence. Thus, spontaneous oscillations in membrane potential may occur undcr physiological conditions. 2. NEUTROPHILS No direct electrophysiological measurements have been made of thc resting V m of neutrophils. However, measurements with the indirect probes TPMP(Seligmann and Gallin, 1983), and TPP+ (Mottola and Romeo, 1982), and with the fluorescent dye 3,3’-dipropylthiadicarbocyanine[diS-C,(S)] (Simchowitz et d.,1982; Simchowitz and De Weer, 1986) have yielded resting Vm values for suspended neutrophils of -54, -67, and -53 mV, respectively. It is not known whether adherent neutrophils have similar resting membrane potentials. From measurements of resting V m and ion fluxes, Simchowitz et al. (1982) calculatcd the relative permeability of the membrane to K , Na , and C1+ to be 10: 1 : 1 . At normal [K],, (4.5 mM), the deviation of the resting V m from E , ( - 8 5 mV) can be accounted for by the small permeability to sodium. Above 10 mM IK],, Vm follows the equilibrium potcntial for potassium. The Na+ / K + pump contributes -9 mV to the resting potential in the steady state. Under nonsteady state conditions, when cells are sodium-loaded and allowed to recover, the pump hyperpolarized the cells by as much as 30 mV. +

+

6. ION TRANSPORT IN PHAGOCYTE ACTIVATION

133

111. IONIC CHANNELS, PUMPS, AND CARRIERS

A. Terminology Ion channels are integral membrane proteins through which ions passively flow down their electrochemical gradients at rates exceeding lo6 ionslsec. Ion channels are characterized by their conductance (ease with which ions flow through channels), ionic selectivity, pharmacology, gating properties (factors which control channel opening and closing), and kinetics (rates of opening and closing). Conductance is defined as the inverse of resistance, is expressed in Siemens (S), and from Ohm’s law, is equal to the current divided by the potential across the channel. Channel gating can be controlled by chemical ligands, voltage, or other factors, such as intracellular calcium. Information about the ionic selectivity of a channel can be obtained by measuring the reversal potential of the current through the channel. The reversal potential (the potential at which the current through a channel reverses direction) is determined by the equilibrium potential ( E ) for the permeant ion. When the membrane potential is equal to the equilibrium potential for a given ion, no current will flow. When the membrane potential is above or below the equilibrium potential for an ion, current will flow out of or into the cell, depending on the driving force for that ion. Ion channels can be permeant to more than one type of ion. Some channels allow current to flow more easily in one direction than in the other direction, a property called rectification. Besides ion channels, the other important family of proteins involved in ion transport are energy-dependent pumps and carriers. These proteins d o not mediate the passive flow of ions down their electrochemical gradients but transport ions against an electrochemical gradient in an energy-dependent manner. The term “ion pump” is applied to those transporters that require the hydrolysis of ATP to translocate ions, and “ion carrier” refers to transporters that do not hydrolyze ATP but derive energy from existing ionic gradients. 6. Ionic Channels in Macrophages Electrophysiological studies at the whole-cell or single-channel level have demonstrated that macrophages exhibit voltage-gated and calcium-gated ionic currents. Four different K currents and an anion current have been identified and are described in detail later. A nonselective cation channel that is activated following the binding and cross-linking of the receptor for IgGy2b/y, also has been described (Young et a l . , 1983b,c) and will be discussed in the section on the role of ion channels in signal transduction. In addition to these channels, several other channels of different conductances have been reported in patch clamp studies (McCann et al., 1987; Ince ef al., 1987a, 1988), but since these have not been well characterized, they will not be discussed in this review.

134

ELAINE K. GALLIN AND LESLIE C. MCKINNEY

1 . VOLTAGE-DEPENDENT INWARDLY RECTIFYING CONDUCTANCF (K,)

An inwardly rectifying K current (Fig. 2A) that activates at voltages negative to -50 mV was first described in intracelluiar microelectrode studies of mouse spleen and thioglycolate-induced macrophages that had been cultured for several weeks (Gallin and Livengood, 1981; Gallin, 1981) and has since been characterized in cultured human macrophages (Callin and McKinney, 1988a), the

A

-10

-160

1

1'

500 pA

VH = -80 m V

d 1 1 0 msec

B

1 0 . 5 nA

V H = -33 m V

C

0

.

I

VH = - 6 0 rnV FIG.2. Whole-cell current records from patch clamp recordings uf 3774 cells (A and C) and a human mdcrophage (B). Cells were stepped to the indicated potentials. (A) Data from McKinney and Gallin ( I Y X X ) . (B) Data from Callin and McKinncy (1988a). (C) Data from Gallin and McKinncy ( I9XXb).

6. ION TRANSPORT IN PHAGOCYTE ACTIVATION

135

murine macrophage-like cell line 5774.1 (Gallin and Sheehy, 1985), and in mouse peritoneal macrophages (Randriarnampita and Trautmann, 1987). It is similar to the inwardly rectifying K current characterized in several other cell types, including invertebrate egg cells (Hagiwara et al., 1976), frog skeletal muscle (Leech and Stanfield, 1981), heart muscle (Giles and Shibata, 198% and rat basophilic leukemia cells (Lindau and Fernandez, 1986). It has a steep voltage dependence and shows time-dependent inactivation for voltage steps negative to - 120 mV. A unique property of this current is that its activation depends not only on membrane voltage but also on [K], (Gallin and Sheehy, 1985). It is blocked by external barium in a voltage-dependent manner (McKinney and Gallin, 1988). Single-channel currents whose properties corresponded to the macroscopic inwardly rectifying K current measured in whole cells have been described in both 5774.1 cells (McKinney and Gallin, 1988) and human peripheral blood derived macrophages (Gallin and McKinney, 1988a). The single-channel conductance in cell-attached patches with 145 mM [K], in the patch electrode was 29 pS, and the extrapolated reversal potential was near E,. No outward current was noted at potentials above E,, indicating that rectification occurs at the singlechannel level. Averaged single-channel currents showed time-dependent inactivation below -120 mV and were blocked by barium. Both whole-cell and single-channel conductances were proportional to the square root of [K],. The inwardly rectifying K conductance plays a role in maintaining the membrane potential near E,. As discussed in the previous section on membrane potential, macrophages that exhibited this conductance had more negative resting membrane potentials than cells that did not have this conductance (Gallin and McKinney, 1988a), and barium, which completely abolished this current, depolarized cells by 20 mV or more (Gallin and Sheehy, 1985). A brief report by Moody-Corbett and Brehm ( 1987) indicated that in rat thymus-derived macrophages the inwardly rectifying current was reduced by acetylcholine and muscarine. Further studies need to be done to determine whether modulation of the Ki conductance by these agents is functionally relevant. The dependence of the K i conductance on [K], is likely to be important, since the macrophage is present at sites of dying tissue, and therefore, is exposed to elevated [K],. In addition to depolarizing the macrophage (because E , will become less negative), increases in [K],, will amplify the K, conductance, increasing inward rectification, thereby making the I-V relationship more nonlinear. Thus, the macrophage will remain sensitive to depolarizing stimuli, even though it is depolarized by high [K],. 2. LARGECAI.CIUM AND VOLTAGE-ACTIVATED K CONDUCTANCE (KLCa) Single-channel studies in human macrophages have demonstrated a largeconductance K channel (240 pS in symmetrical K ; 110 pS in 150 mM "a],,/

136

ELAINE K. GALLIN AND LESLIE C. MCKINNEY

5 nM [K],,) activated by both voltage and [Cali (Gallin, 1984; McCann e l u l . , 1987). Whole-cell currents corresponding to the activity of thesc channels havc also bccn characterized and are shown in Fig. 2B (Gallin and McKinney, 1988a).

The channel is blocked by charybdotoxin, a preteinaceous component of scorpion toxin known to block Ca-activated K channels in other cells (Miller et NI., 1985), and TEA-CI ( I S m M ) (Gallin and McKinney, 1988a). Increasing [Cali from to l o p 7 M greatly increascd the probability of channel opening. However, in thc macrophage, as in skeletal muscle cclls, large increases in intracellular Ca2 (to greater than 10 M ) are required to activate channels at negative membrane potentials (Gallin and McKinney, I988a). Studies using Caindicator dyes have reported [Cali increases following physiological stimulation that are too low (in the range of 0.2 to 1 pM; Conrad and Rink, 1986; Kruskal and Maxfield, 1987) to activate Kldc, channels at negativc membrane potentials. Therefore, either the intracellular calcium sensitivity in situ is different from that of the excised patch, or it is unlikely that these channels are open very often at rest or during stimulation. A third possibility is that these channels function in intracellular compartments where [Cal, levels may be high. Reshly isolated human peripheral blood monocytcs do not exhibit Ca-activated K + channels, but macrophages cultured for 2 to 30 days do (Gallin and McKinney, 1988a). In these studies, expression was maximal after 7 days in culture, when 90% of patchcs from human macrophages contained the channel. Since freshly isolated peripheral blood monocytes are phagocytic and chemotactic, the presence of KLCnchannels in the plasma membrane must not be required for those functions. +

3. CALCIUM-ACTIVATED INWARDLYRECTIFYING K CONDUCTANCE (Kit,,) In addition to the voltage-dependent inwardly rectifying K channel (K,) previously described, a calcium-activated inwardly rectifying K channel ( KicJ has been identified in human macrophages (Gallin, 1989). Exposing macrophages to either ionomycin or platelet-activating factor, two substances known to transiently increase intracellular calcium, induced bursting channel activity in cellattached patches. Single-channel conductances (with 150 mM KCI in the pipette) for inward and outward currents were 37 pS and approximately 10 pS, respectively. Channel activation was not voltage dependent. Ion substitution experiments indicated that the channel was permeable to K and impermeable to either sodium or chloride. I he Klca channcl was differentiated from the Ki channel on the basis of its calcium sensitivity, conductance (37 vs. 29 pS for inward currents), its kinetics (bursting vs. nonbursting), its lack of voltagc dependence, and its differing sensitivity to block by external barium. Three millimolar barium, a concentration .

7

6. ION TRANSPORT IN PHAGOCYTE ACTIVATION

137

that completely blocked the voltage-dependent K, channel (McKinney and Gallin, 1988), did not significantly block the Kica channel at rest and produced only a partial block when the patch was hyperpolarized (Gallin, 1989). Similar inwardly rectifying calcium-activated K channels have been described in lymphocytes (Mahaut-Smith and Schlicter, 1989), red blood cells (Grygorckyk and Schwartz, 1983), and HeLa cells (SauvC et al., 1986, 1987). Randriamampita and Trautmann ( 1 987) recorded whole-cell currents in mouse peritoneal macrophages and 5774 cells (under conditions of high [Ca],), which they concluded were due to activation of a voltage insensitive calcium-activated K conductance. However, these whole-cell currents did not exhibit rectification. It is likely that Kica channels, rather than KL.Cachannels, are responsible for the oscillatory hyperpolarizations in membrane potential described in macrophages since ( I ) the Kip, channel is active at the resting Vm following exposure to ionomycin, while the KLCa channel is not; (2) the bursting pattern of the Kica channel is oscillatory; and (3) activity of the Kica channel is associated with oscillatory changes in membrane potential (Gallin, 1989). In macrophages, the channel activity induced by ionomycin is often associated with a shift in DC current level, indicating that the cell membrane hyperpolarizes. This observation is consistent with early microelectrode studies that demonstrated that the calcium ionophore A23 187 induced membrane hyperpolarizations in macrophages (Gallin et al., 1975). 4. INACTIVATING OUTWARD K CONDUCTANCE (KJ An inactivating (transient) outward K conductance (Fig. 2C) has been described at the whole-cell current level in resident mouse peritoneal macrophages (Ypey and Clapham, 1984), cultured human monocytes (Nelson et a/., 1986), and two macrophage cell lines, 5774.1 (Gallin and Sheehy, 1985; Randriamampita and Trautmann, 1987) and P388D1 (Sheridan and Bayer, 1986). This conductance activates at potentials positive to -50 mV, inactivates over a time course of seconds, and is blocked by 4-aminopyridine (Ypey and Clapham, 1984; Gallin and Sheehy, 1985). Similar outward currents have been described in detail in T lymphocytes (Cahalan et al., 1985). Ypey and Clapham (1984) characterized a 16 pS channel whose properties can account for the behavior of the outward current. Outward K current appears to be variably expressed with time in culture, but no consistent pattern of expression across different cell types has yet been observed. Ypey and Clapham ( 1 984), using resident mouse peritoneal macrophages, reported that this conductance was absent during the first day after isolation but was present in 96% of cells cultured for 1-4 days. Randriamampita and Trautmann (1987) also recorded outward currents in mouse peritoneal macrophages cultured for 1-2 days but found that the currents decreased after 5-6

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ELAINE K. GALLIN AND LESLIE C . MCKINNEY

days in culture. This finding is consistent with earlier studies in which outward K current was not found in long-term (2-6 weeks) cultured mouse peritoneal (Gallin and Livengood, 1981) and spleen macrophages (Gallin, 1981). In 5774 cells, inactivating outward current was sometimes noted 1-8 hr after adherence but was rarely present in long-term adherent cultures (Gallin and Sheehy, 198.5). Recently, Jow and Nelson (1989) demonstrated that trcating peripheral blood monocytes for 24 hr with bacterial lipopolysaccharide (LPS) increased the percentage of cells expressing outward K current from near 0% to 30%. In mouse peritoneal macrophages, mechanical stimulation caused by perfusing the bathing medium increased the outward K current (Randrianiampita and Trautrnann, 1987). 5 . PHYSIOLOGICAL ROLE OF K CONDUCTANCES

The role of K conductances in phagocyte function is poorly understood. Ilowever, it is clear that K conductances serve some of the same functions in phagocytes that they do other cells. For example, inwardly rectifying K i channels maintain membrane potential near E, in the niacrophage as well as in other cells. The outward inactivating K current rnay be important for restoring membrane potential to negative values after the cell is depolarized, and Ca-activated conductances may have a similar role after transient increases in [Cali. In lymphocytes, potassium channels are important in volume regulation (Lee el al., 1988). Modulation of K permeability might influence intracellular K levels, which could influence synthetic processes in the cell (Shinohara and Piatigorsky, 1977; Villereal and Cook, 1987; Lau et c d . , 1988) as well as receptor-mediated endocytosis (Larkin ef crl., 1983). The contractile machinery of the macrophage rnay be influenced by changes in [Kli, since it contains an actin-modulating protein, acumentin, whose activity is modified by changes in [KI, (100-200 nlM) (Southwick rt a / . , 1982). A few studies have investigated the effect of pharmacologic blockers of specific K channels on phagocyte functions. In 5774 cells, barium (2 mM), which blocks the K, channel, does not block chemotaxis in response to endotoxin activated mouse serum, release of hydrogen peroxide following stimulation with PMA, or phagocytosis of opsonized red blood cells (E. K. Gallin, unpublished observations). Therefore, it is unlikely that the Ki conductance plays a crucial role during these events. In human rnacrophages, TEA, which blocks K,,c., channels, did not inhibit chernotaxis toward fMLP (E. K . Gallin, unpublished observations). This observation, together with the finding that freshly isolated human peripheral blood rnonocytes do not exhibit KI-c;, channels (even though they are capable of carrying out phagocytosis and chemotaxis), indicates that the KLCo channels (at least those on the cell membrane) are not required for these functions. Further studies examining the effects of blockers of the other two types of K channels may help to clarify the role of K conductances in phagocyte function.

139

6. ION TRANSPORT IN PHAGOCYTE ACTIVATION

6. CHLORIDE CONDUCTANCE Schwarze and Kolb (1984) described an ion channel present in mouse peritoneal macrophages that had a very large conductance (340 pS), had several subconductance states, and was poorly selective for chloride (that is, the selectivity ratio, P,, : P,,, was only 5 : 1). It was seen in cell-attached patches only after treatment with the calcium ionophore A23 187; in quiescent patches, excision of the membrane activated the channel. It was reported that these channels could be elicited by perfusing cells with zymosan during cell-attached patch recordings (Kolb and Ubl, 1987). They were activated by both depolarizing and hyperpolarizing voltage steps, exhibited voltage-dependent inactivation, and behaved as if they were controlled by two independent voltage-sensitive gates. Similar models have been used to describe the behavior of voltage-dependent gap junctions, and it was suggested that the chloride channel may play a role in intercellular communication (Schwarze and Kolb, 1984). However, Randriamampita and Trautmann ( 1 987) have demonstrated that octanol, a blocker of gap junction channels, does not block these channels, indicating that they are not related to gap junction channels.

C. Ionic Channels in Neutrophils Only one electrophysiological study has been performed on neutrophils. This study, which used patch-clamp techniques to examine ionic channels during stimulation with the chemotactic peptide N-formylmethionylleucylphenylalanine (fMLP; von Tscharner et al., 1986) demonstrated that addition of fMLP to the bath during cell-attached patch recordings induced two different calcium-activated, cation-nonselective channels. The presence of fMLP in the patch pipette did not increase the probability of channel opening, indicating that the channels activated by fMLP were not directly coupled to the fMLP receptor. These channels appeared to be calcium activated since depleting [Cali, by loading cells with fura-2, prevented fMLP-induced channel activation. Treating cells with saponin, to increase /Cali, activated channels in absence of fMLP. Two types of singlechannel currents, having conductances of 18-25 and 4-6 pS, were identified. Ion substitution experiments indicated that these channels were equally permeable to K, Na, and Ca. (A later section in this review will cover other studies that have characterized the ionic events that occur during fMLP stimulation.)

D. Pumps and Carriers in Macrophages and Neutrophils As has been previously discussed, both the macrophage and neutrophil have a ouabain-sensitive Na+ /K pump that is electrogenic and contributes to the resting potential of these cells. Properties of the Na /K pump have been best +

+

+

140

ELAINE K. GALLIN AND LESLIE C. MCKINNEY

characterized in the neutrophil (Sirnchowitz et al.. 1982), where it has been shown that 9.5% of Na emux and 63% of K influx are mediated by the pump. Although chloride transport has not been well characterized in the macrophage, it has been extensively characterized in the neutrophil (Simchowitz and De Weer, 1986; Simchowitz vt d.,1986). Unlike some cells in which intracellular chloride passively follows membrane potential, the neutrophil actively maintains [Clli at a value fourfold higher than equilibrium. There are three main pathways for chloride transport in the neutrophil: active uptake, passive diffusion, and CI- /CI self-exchange. The bulk (70%) of chloride influx and efflux is due to CI-ICI- self-exchange via a carrier that may mediate CI-/HCOq exchange under other conditions. This exchanger is somewhat different from the anion exchanger in red blood cells in that it is insensitive to disulfonic stilbenes and othcr classical inhibitors of anion exchange in the red blood cells (Simchowitz and DeWeer, 1986). Active uptake of chloride accounts for 20% of total chloride influx and is mediated by a carrier that can be inhibited by ru-cyano-4hyclroxycinnamate (CHC), ethacrynate, and furosemide. About 8% of chloride influx and 30% of chloride eftlux is via passive ditt'usion. Characterizations of Na+ /H exchange (Simchowitz, 1985a,b; Grinstein and Furuya, 1986), Na+ /Ca2+ exchange (Simchowitz and Cragoe, 1988), and the calcium ATPase (Klempner, 1985) havc also been carried out for the neutrophil, but since these transporters havc to do with the regulation of intracellular pH and calcium, which arc discussed in other chapters, they will not be discussed here. +

+

+

IV. ROLE OF MEMBRANE POTENTIAL AND IONIC CONDUCTANCES IN PHAGOCYTE FUNCTION A. Membrane Potential While it is clear that various substances that activate phagocytes produce changes in membrane potential, these changes are not necessarily required for signal transduction. This conclusion is based on studies in which the membranc potential of phagocytes was depolarized to approximately 0 mV by incubating cells in either high ( I 20- 1 50 mM) K medium or gramicidin. If either a negative membrane potential or voltage-dependent conductances are required for a particular phagocyte function, then these treatments should affect that function. It must be noted that these studies should be interpreted with caution, since exposing cells to solutions of altered ionic composition can affect parameters other than membrane potential. For example, high K solutions are usually made by substituting K for Na, and decreases in extracellular Na can lead to alterations of intracellular pH (Nasmith and Grinstein, 1986) and changes in the number of surface receptors in phagocytes (Roberts et a l . , 1984).

6. ION TRANSPORT IN PHAGOCYTE ACTIVATION

141

Neither phagocytosis nor chemotaxis is inhibited by depolarizing the phagocyte with high K medium. Neutrophils depolarized by high K can still migrate in response to fMLP (Showell and Becker, 1976; Mottola and Romeo, 1982). Roberts et a / . (1984) demonstrated that the number of neutrophils migrating in high K-low Na medium is increased, but this increase is due to the reduction in extracellular Na and not to the increase in K. In these studies, decreasing extracellular Na also stimulated the preferential secretion of secondary granules and increased the mobilization of fMLP receptors. Pfefferkorn (1984) reported that 5774 cells ingest the opsonized protozoan parasite Trypanosoma gondii normally in 120 mM K medium. Phagocytosis of unopsonized zymosan by murine peritoneal macrophages also occurs normally in high K medium. However, in these cells high K medium prevents the subsequent induction of phospholipase activity that normally occurs after the ingestion of zymosan, although it does not block the activation of phospholipase by A23187 (Aderem et at., 1984). The authors concluded that high K blocked the signal that coupled the ingestion of opsonized zymosoan particles to activation of the phospholipase. In summary, experiments indicate that, even though high K medium may affect events that occur subsequent to migration or phagocytosis, neither of these two functions require that the phagocyte have a negative membrane potential. Data on the relationship between membrane potential and the oxidative burst are less consistent. This variability appears to relate in part to the different stimuli used to elicit the oxidative burst and in part to effects of sodium removal. Kitagawa and Johnston (1985) have shown that murine macrophages that are depolarized by either high K or gramicidin release the same quantity of superoxide in response to stimulation by phorbol myristate acetate (PMA) as control macrophages and that depolarization, itself, does not induce the release of superoxide. Similar results were noted when fh4LP-induced superoxide release was measured in guinea pig alveolar macrophages depolarized by incubation in I 10 mM Ki35 mM Na medium. In these cells, further increasing (K],, to 142 mM (and decreasing "a), to 4 mM) decreased superoxide production by 25%, but this decrease was due to the decrease in "a],, rather than the increase in [K], (Holian and Daniele, 1982). In neutrophils, Korchak and Weissmann (1980) demonstrated that replacing extracellular Na with either K or choline decreased superoxide generation in response to concanavalin A or immune complexes, and Simchowitz ( 1 9 8 5 ~ )showed that superoxide production decreased by 70% in neutrophils incubated in medium containing N-methyl-D-glucamine instead of Na. In contrast to these findings, Luscinskas et al. (1988) noted an increase in immune complex-induced superoxide production in human neutrophils following exposure to 120 mM K/O mM Na medium. However, when neutrophils were incubated in 120 mM choline/O mM Na medium, a similar increase in superoxide was noted, indicating that the increased release of superoxide in high K medium was not due simply to membrane depolarization.

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ELAINE K. GALLIN AND LESLIE C. MCKINNEY

B. Ionic Conductances 1. CHANGES

DURING

MATURATION OR AFTER “ACTIVATION”

Electrophysiological studies in both human macrophages and 5774 cells have examined the effects of maturation and adherence on the expression of ionic conductances. Expression of the large conductance Ca-activated K channel in human peripheral blood monocytes increased during the first 7 days in culture, a time period during which monocytes mature into macrophages; less than 5% of cell-attached patches obtained from cells 24 hr after plating exhibited this channel, while greater than 80% of the patches obtained 5 days in culture did (Gallin and McKinney, 1988a). Channel expression was a function of time in culture, and not adherence, since cells grown for the same amount of time under nunadherent conditions also expressed KLCa channels. In contrast, in 5774 cells, adherence appears to induce an increase in the density of voltage-dependent inwardly rectifying channels, which increases approximately twofold during the first 6-8 hr following adherence (McKinney and Gallin, 1988). The increase in Ki channel density can account for the observed hyperpolarization (from approximately -20 mV to -70 mV) that also occurs in 3774 cells over the same time course (Sung et a / ., 1985; McKinney and Gallin, 1989). Two studies have examined the etfect of activating stimuli on the expression of ionic currents. As was previously mentioned, Jow and Nelson (1989) showed that treating cultured human peripheral blood derived macrophages for 24 hr with LPS increased the percentage of cells expressing the inactivating outward K current from near 0% to approximately 30%. In 5774. I cells, however, the same treatment did not increase the K,, or the Ki conductance (McKinney and Gallin, 1988). Similar results were noted after 3774 cells were exposed to ionizing radiation, a treatment that induces more activated cells (Gallin et al.. 1985). 2. SIGNALTRANSDUCIION A wide variety of techniques have been used to examine the role of ionic conductances in signal transduction. Measurements have been made of changes in membrane potential, ion fluxes, intracellular Ca, and pH in intact cells, vesicles, and reconstituted receptor preparations. This chapter will briefly review only those studies that have examined the ionic events following ligand binding to the two best studied phagocyte receptors, those for fMLP and the Fc region of immunoglobulin. (1. j M L P . The early events that follow thc binding of fMLP to the membrane include changes in membrane potential and ion transport. Studies with fluorescent dyes, such as di-0-C,(3) and di-S-C,(S), have shown that fMLP induces an initial depolarization followed by repolarization that is completed within 8- 10

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6. ION TRANSPORT IN PHAGOCYTE ACTIVATION

min (Seligmann and Gallin, 1983; Tatham et al., 1980; Lazzari et al., 1986). The fMLP-induced depolarization requires a stimulus concentration of at least 10- * M ,while lower concentrations induce either no change (Di Virgilio et al., 1987) 1986). or a slight hyperpolarization (Lazzari et d., The ionic basis of the fMLP-induced membrane potential changes has not been adequately resolved. Varying external Na from I to 122 mM did not change the amplitude of the membrane depolarization, indicating that the depolarization does not involve a Na conductance (Seligmann et ul., 1980). Von Tscharner et ul. (1986) have suggested that the transient depolarization is caused by an influx of cations through the calcium-dependent, cation-nonselective ion channels induced by fMLP. It is plausible that calcium provides a signal leading to depolarization, since it has been demonstrated that an increase in [Ca], precedes the depolarization (Lazzari et u l . , 1986). However, Di Virgilio et al. (1987) have shown that, even in Ca-depleted cells where no increase in intracellular calcium occurs, fMLP can induce a depolarizing response, although it is diminished. Simultaneous measurements of membrane potential and intracellular calcium changes in single cells will be necessary to resolve the temporal relationship between changes in [Cali and Vm. The repolarization phase of the WLP-induced membrane potential changes may be dependent on external calcium (Tatham et a!., 1980). If so, it is reasonable to speculate that a calcium-dependent K channel may be called into play for repolarization to occur. This possibility is supported by two findings. First, the membrane repolarization is diminished when the external K concentration is increased (Seligmann et a)., 1980), and second, in alveolar macrophages a component of the K efflux stimulated by the dipeptide fMP is blocked by quinine, a well-known inhibitor of calcium-activated K channels (Holian and Daniele, 1982). Thus, while there is a general agreement on the time course and direction of membrane potential changes following fMLP stimulation, the changes in membrane permeability underlying these events need to be better defined. They may involve the stimulation of electrogenic ion transporters. Ion flux studies have demonstrated that the Naf / K + pump is stimulated following fMLP application, which would lead to membrane hyperpolarization (Naccache et al., 1977). The effect of fMLP on the Na+/K +-ATPase may be direct (Becker et al., 1978) or may be secondary to other intracellular ion changes, such as increased sodium (Simchowitz, 1985a). Simchowitz and Cragoe (1988) have characterized an electrogenic Na+ /Ca2 exchanger in neutrophils that transports one Ca ion into the cell in exchange for three Na ions. This exchanger is activated by WLP, which could account for a portion of the observed increase in intracellular calcium. Finally, fMLP directly stimulates Na /H exchange (Simchowitz, 1985a,b). Although this transport system is not electrogenic, the changes in intracellular Na or pH, which are substantial, could affect other ion transporters +

+

+

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ELAINE K. GALLIN AND LESLIE C. MCKINNEY

and conductances. It should be noted, however, that the changes in intracellular pH induced by fMLP may bc blunted in vivo in the presence of physiological bufffers. The purpose of thc fMLP-induced membrane potential changes arc unclear, since cells can migrate (Showell and Becker, 1976) and produce an oxidative burst (Holian and Danicle, 1982) when depolarized by high K. However, the observation that fMLP fails to induce membranc potential changes in neutrophils from patients with chronic granulomatous disease, a condition in which phagocytes are incapable of producing an oxidative burst (Seligmann and Gallin, 1980). supports the view that the membrane depolarization (or evcnts leading to i t ) is linked to the oxidative burst, even if the depolarization is not required for the activation of the oxidasc. A close association between membrane depolarization and the oxidative burst was also demonstrated in a study that measured tMLP and PMA-induccd membrane potential changes and supcroxidc release in HL60 cells at varying stages of differentiation (Kitagawa ct ( I / . , 1984). Similarly, Di Virgilio et a / . (1987) demonstrated that the dose-response relationship for the IMLP-induced depolarization in neutrophils was identical to the dose-responsc relationship for fMLP-induced activation of the NADPH oxidase. This study also showed that the increase in intracellular calcium that occurs during fMLP stimulation is reduced when neutrophils are depolarized and enhanced when they are hyperpolarizcd during fMLP stimulation. Therefore, fMLP-induced depolarixation may serve to limit the influx of calcium into the cell following stimulation.

6. Fc Receptor. Phagocytes can ingest particles by nonreceptor mediated nicchanisnis or through receptors for the Fc domain of immunoglobulin G and the C3b fragment of coniplemcnt (Silvcrstcin e6 a / ., 1977). The ionic events that underlie different kinds of phagocytosis may differ. For example, in ncutrophils, C3b-mediated phagocytosis is calcium-independent, while Fc-mediated phagocytosis is calcium-dependent (Lew et ul., 1985). In this scction we will review only the studies on thc ionic events occurring during Fc-mediated phagocytosis. A series of studies done by Young et a/. ( 1 983a-c) have demonstrated that ligand binding to the Fc receptor (FcR) is associated with the activation of ionic channels. The first of these indicated that the binding and cross-linking of the y2blyl FcR by IgG or inimunc coniplexes depolarized 5774.1 cells by 7 mV (Young al., 1983a). This study, done by monitoring membranc potential indirectly with TPP, showed that dcpolarization required a multivalent ligand and was dependent on external sodium. In a related study, in which purified y2blyl FcKs were inserted into lipid vesicles, ligand binding to FcR-containing proteoliposomes increased their cation permeability (Young ef a/., 1983b). Finally, Young et a/. ( 1 9 8 3 ~demonstrated ) that the addition of ligand to bilayers containing the FcR induced cation-selective ion channels. The channels had a conductance of 60 pS in symmetrical I M KCI and dccreased in activity within several minutcs of adding ligand.

6. ION TRANSPORT IN PHAGOCYTE ACTIVATION

145

If the FcR-ligand complex is an ionic channel, then changes in membrane potential or ionic conductances should be evident during electrophysiological recordings from cells that are ingesting IgG-coated particles or aggregated IgG (algG). Several studies have been done that directly monitored channel activity in intact macrophages or macrophage membranes before and after the addition of IgG. Nelson et a / . (1985) recorded whole-cell currents as well as single-channels in human alveolar macrophages exposed to aIgG. The application of aIgG to cells during whole-cell recordings produced an inward current that diminished with successive applications of aIgG, indicating that the response desensitized. In cell-attached patches, channel activity was noted only when the electrode contained aIgG and not when aIgG was applied to the bath. The channels had a unitary conductance of 350 pS in symmetrical 140 mM NaCl Hanks solution. Changing the permeant cation from Na to K did not affect the reversal potential, indicating that if the channel was a cation channel, it was nonselective. Unfortunately, similar responses have not been noted in patch clamp studies of 5774 cells exposed to ligands that bind to and cross-link the Fc receptor (D. J. Nelson, personal communication). In addition, the difference between the value of conductance obtained in this study (350 pS in physiological saline) and that obtained by Young et al. ( 1 9 8 3 ~on ) the isolated FcR (60 pS in symmetrical 1 M KCI) is puzzling and needs to be investigated further. There is evidence that the Fc receptor-ligand complex, in addition to acting as a ionic channel itself, may indirectly activate ionic channels through a second messenger (Lipton, 1986; Ince et af., 1988). In one study, IgG,, was added to the bath during a cell-attached recording from P388D1 cells (Lipton, 1986). Following the addition of IgG2,,, multiple current amplitudes were evident, representing either several different types of channels or a single-channel type with different subconductance states; the smallest channels had conductances of 3545 pS and were cation selective. Channel activity could be maintained following excision of the patch, and activity was modulated by changes in [Ca],. Therefore, it was suggested that these channels were activated by [Ca], increases that occur following binding and cross-linking of the Fc receptor. (The role of calcium in phagocytosis is discussed in another chapter.) Using a similar experimental protocol in patch clamp recordings from cultured human macrophages, Ince et af. (1988) demonstrated transient changes in background current along with the activation of several types of channels with conductances ranging from 26 to 163 pS. Some of these channels reversed near E,, but the ionic selectivity of these channels was not investigated. In contrast to the previous studies that reported ionic channel activation during Fc-mediated phagocytosis, exposure of murine macrophages to aIgG or the monoclonal antibody 2.462 during whole-cell patch clamp recordings did not induce membrane currents (Randriamampita and Trautmann, 1987). In addition, the resting Vm values obtained from whole-cell recordings immediately after the macrophages had ingested opsonized red blood cells were identical to those

-1 >

,

min

FIG.3. Current clamp recordings obtained with a 2 M KCI filled microelectrode (resistance > 60 m n ) before and during ingestion of opsonized red blood cells by a thioglycollate-induced mouse peritoneal macrophage grown in tissue culture for about 2 weeks. Photomicrographs were obtained at the indicated time during the voltage tracing. Small current pulses shown in the bottom tracing were injected through the recording electrode to monitor changes in the ionic conductance of the cell. During the time period in which the four photomicrographs were taken, the macrophage ingested at least two red blood cells. and its resting membrane potential (-33 mV) and conductance remained relatively constant.

6. ION TRANSPORT IN PHAGOCYTE ACTIVATION

147

obtained prior to phagocytosis. Thus, Randriamampita and Trautmann (1987) concluded Fc-mediated phagocytosis can occur under conditions where no detectable conductances are activated. Early studies carried out in our laboratory using intracellular microelectrodes produced similar findings. That is, intracellular recordings from mouse peritoneal macrophages before and during ingestion of opsonized red blood cells indicated that phagocytosis could occur without any changes in membrane potential or in input resistance (E. K . Gallin, unpublished observations). The data from one of these recordings are shown in Fig. 3 . The discrepancy between the findings shown in Fig. 3 (as well as those of Randriamampita and Trautman, 1987) and those presented in previous paragraphs indicate that the ionic events occurring during Fc-mediated phagocytosis are not fully understood.

V.

SUMMARY

This chapter has reviewed the ionic basis for the resting membrane potential in the neutrophil and the macrophage; properties of the different types of ionic channels, pumps, and carriers that are present in these cells; and the possible role of membrane potential and ionic conductances in phagocyte function. A great deal of progress has been made in this area. The resting membrane potentials of both neutrophils and macrophages are known, and a number of the ion channels and carriers in the phagocyte membrane have been characterized. In the macrophage, several K channels that are voltage or calcium sensitive have been described, as well as a large conductance chloride channel and cation channel activated by ligand binding to the IgGy2b/y,receptor. In the neutrophil, only a nonselective cation channel has been described, which appears following exposure to fMLP. Voltage-dependent calcium and sodium channels have not been found in either neutrophils or macrophages. It is possible that calcium channels similar to those described in human T lymphocytes (Kuno and Gardner, 1987) also exist in phagocytes but that they have not been detected to date because of their very small single-channel conductance and activation by second messengers, rather than by voltage. Although we know that ionic conductances and membrane potential can change during maturation or activation, the functional role(s) of the different ionic conductances and transporters that have been described in the neutrophil and the macrophage is still unclear. The two stimuli that have been the most thoroughly studied in terms of their effects on ionic transport are fMLP and IgG. Both of these stimuli activate ionic channels, increase ionic fluxes, change membrane potential, and increase intracellular calcium. However, it is not yet known how these events are coupled to the functional responses of the phagocyte to fMLP or immunoglobulins.

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ELAINE K. GALLIN AND LESLIE C. MGKINNEY ACKNOWLEDGMENTS

The authors thank Dr. Louis Simchowitz for critically reviewing several sections of the manuscript. This work was supported by the Armed Forces Radiobiology Research Institute, Defense Nuclear Agency. undcr work unit 00020. Views presented in this paper are those of the authors; no endorsement by the Defense Nuclear Agency has heen given or should be inferred. REFERENCES

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potassium, and calcium across rabbit polymorphonuclear leukocyte membranes. J. Cell Biol. 73, 428-444.

Nasmith, P., and Grinstein, S. (1986). Impairment of Na+ / H + exchange underlines inhibitory effects of Na+-free media on leukocyte function. FEES Lett. 202, 79-85. Nelson, D. J., Jacobs, E. R., Tang, J. M., Zeller, J. M., and Bone, R. C. (1985). Immunoglobulin Ginduced single ionic channels in human alveolar macrophage membranes. J. Clin. Inveut. 76, 500-507. Nelson, D. J . , Rufer, L., Nakayama, T., and Zeller, J. M. (1986). Phorbol ester block of voltagedependent K current in monocyte-derived macrophages. Biophys. J. 49, 164a. Persechini, P. M., Araujo, E . G., and Oliveira-Castro, G. M. (1981). Electrophysiology of phagocytic membranes; induction of slow membrane hyperpolarizations in macrphages and macrophage polykaryons by intracellular calcium injection. J. Membr. Biol. 61, 81-90. Pfefferkorn, L. (1984). Transmembrane signalling: an ion-flux-independent model for signal transduction by complexed Fc receptor. J . Cell Biol. 99, 2231-2240. Randriamarnpita, C., and Trautmann, A. (1987). Ionic channels in murine macrophages. J. Cell Biol. 105, 761-769. Roberts, R., Mounessa, N., and Gallin, J. I. (1984). Increasing extracellular potassium causes calcium dependent shape changes and facilitates concanavalin A capping in human neutrophils. J. Immunol. 132, 2000-2006. Suave, R., Simoneau, C . , Monette, R., and Roy, G. (1986). Single channel analysis of the potassium permeability in HeLa cancer cells: Evidence for a calcium-activated potassium channel of small unitary conductance. J. Membr. Biol. 92, 269-282. Suave, R., Simoneau, C., Parent, L., Monette, R., and Roy, G. (1987). Oscillatory activation of calcium-dependent potassium channels in HeLa cells induced by histamine HI receptor stimulation: A single channel study. J. Membr. B i d . 96, 199-208. Schwarze, W., and Kolb, H. A. (1984). Voltage dependent kinetics of an anionic channel of large unit conductance in macrophages and myotube membranes. ffluegers Arch. 402, 281-291. Seligmann, B. E., and Gallin, J. I. (1980). Use of lipophilic probes of membrane potential to assess human neutrophil activation. J. Clin. Invest. 66, 493-503. Seligmann, B. E., and Gallin, J. I. (1983). Comparison of indirect probes of membrane potential utilized in studies of human neutrophils. J. Cell. Physiol. 115, 105-115. Seligmann, B. E., Gallin, E. K . , Martin, D. L., Shain, W., and Gallin, J. I. (1980). Interaction of chemotactic factors with human polymorphonuclear leukocytes: Studies using a membrane potential-sensitive cyanine dye. J. Membr. Biol. 52, 257-272. Sheridan, R. E., and Bayer, B. M. (1986). Ionic membrane currents induced in macrophages during cytolysis. Fed. Proc., Fed. Am. Soc. Exp. Biol. 45, 1009a. Shinohara, T., and Piatigorsky, J. (1977). Regulation of protein synthesis, intracellular electrolytes and cataract formation in vitro. Nature (London) 270, 406-41 1. Showell, H., and Becker, E. (1976). The effect of external K + and Na+ on the chemotaxis of rabbit peritoneal neutrophils. J. Immunol. 116, 99-104. Silverstein, S., Steinman, R., and Cohn, Z. (1977). Endocytosis. Annu. Rev. Biochem. 46, 665722. Simchowitz, L. (1985a). Chemotactic factor-induced activation of Na+ / H + exchange in human neutrophils. I. Sodium fluxes. J. Biol. Chem. 260, 13237-13247. Simchowitz, L. (1985b). Chemotactic factor-induced activation of Na+ /H + exchange in human neutrophils. 11. Intracellular pH changes. J. Biol. Chem. 260, 13248-13255. Simchowitz, L. (1985~).lntracellular pH modulates the generation of superoxide radicals by human neutrophils. J. Clin. Invesr. 76, 1079- 1089. Simchowitz, L., and Cragoe, E. J., Jr. (1988). Na+/Ca+ exchange in human neutrophils. Am. J . Physiol. 254, C150-Cl64.

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Simchowitz, L., and Dc Weer, P. (1986). Chloride movements in human neutrophils. J. Gen. Phy.yia/. 88, 167-194. Simchowitz, L., and Row, A . (1985). Regulation of intracellular pH in neutrophils, J. Gen. Physiol. 85, 443 -470. Simchowitz, L . , Spilberg, I . , and De Weer, P. (1982). Sodium and potassium fluxcs and membrane potential of human neutrophils. J . Gerz. Physiol. 79, 453-479. Simchowin. L . , Ratzlaff, R., and De Weer, P. (1986). Anion/anion exchange in human neutrophils. J . t i u n . Plzysiol 88, 195-217 Snyderman, R . , Pike, M . C., Fischer, D. G . , and Korcn, H. S. (1977). Biologic and biochemical activitiea of continuous macrophage cell lincs P388DI and 5774. I,J . Immitnol. 119, 20602066. Southwick, F., Tatsumi, N., and Stosscl, T. (1982). Acumentin. an actin-modulating protein of rabbit pulmonary macrophages. Biochemistry 21, 632 1-6326. Stickle. I). F., Daniele, K. P. and Holian, A . (1984). Cytosolic calcium, calcium fluxes, and regulation of alveolar macrophage superoxide anion production. J . Cell. Physiol. 121, 458 466.

Sung, S.-S. J . , Young, J. D.-E., Origlio. A . M., Helple, J . M., Kahack, H. R . , and Silverstein, S. C. (1985). Extracellular ATP perturbs transmembrane ion fluxes, elevates cytgsolic [Caz+1, and inhibits phagocytosis in niuuse macrophages. J . Riol. Chern. 260, 13442.- 13449. Tatham, P. E. R., Delves, P. J . , Shen, L., and Roitt, I. M. (1980). Chemotactic factor-induced membrane potential changes in rabbit ncutrophils monitored by the fluorescent dye 3,3-dipropylthiadicarbocyanineiodide. Biochim. Biophys. Acra 602, 285-298. Thomas, R. C. (1972). Electrogenic sodium pump in nerve and muscle cells. Physiol. Rev. 52, 563594. Villercal, M. L., and Cook, J. S. (1987). Regulation of active amino acid transport by growth rddted changes in membrane potential in human macrophages. J . B i d . Chem. 253, 8257-8262. von Tschamer, V., Prod’hom. B., Raggiolini. M.. and Reuter. H . (1986). Ion channels in human neutrophils arc activatcd hy a rise in the free cytosolic calcium concentration. Nature (Londori) 324, 369-372. Young, J. D., Unkcless. J. C., Kaback, H. R . , and Cohn, Z. A. (1983a). Macrophage membrane potential changes associated with y2blyl Fc receptor-ligand binding. Proc. Nufl. Acad. Sci. U.S.A. 80, 1357-1361. Young, J. I)., Unkeless, J. C . , Kaback. H . R., and Cohn. Z. A . (1983h). Mouse macrophage Fc reccptor for IgG y2blyl in artificial and plasma membrane vesicle functions as a liganddependent ionophore. Proc. Nut/. Acad. Sci. U.S.A. 80, 1636- 1640. Young, J. D., Unkeless. J. C., Young, T. M., Mauro, A . , and Cohn, Z. A. (1983~).Role for niouse macrophage IgG Fc receptor as ligand-dependent ion channel. Nature (London)306, 186- 189. Young, J. D., KO. S. S . , and Cohn, Z. A . (1984). The increase in intracellular free calcium associated with IgGy2biyl Fc receptor-ligand interactions: Role in phagocytosis. Proc. N u l . Acad. S c i . U.S.A. 81. 5430-5434. Ypey, D. L., and Clapham, D. E. (1984). Development of a delayed outward-rectifying K + conductance in cultured mouse peritoneal macrophages Proc. Nu//.Acad. Sci. U . S . A . 81, 3083-3087.

CURRENT TOPICS IN MEMBRANES AND TRANSPORT. VOLUME 35

Chapter 7 Cytosolic Calcium Changes during T- and B-Lymphocyte Activation: Biological Consequences and Significance ERWIN W. GELFAND Division of Busic Sciences and The Raymond and Beverly Sackler Foundation Laboratory Department of Pediutrics National Jewish Center ,for Immunology und Respiratory Medicine Denver, Colorudo 80206 I . Introduction 11. Measurement of Cellular Ca2+ Content and [Ca2+], 111. Cytosolic Calcium Changes in Activated T Lymphocytes A. Receptor-Activated Calcium Uptake and Internal Store Release B. Calcium-Dependcnt Changes in Cytosolic pH C. Calcium-Dependent Hyperpolarization D. Biological Significance of Changes in Cytosolic Calcium in Ligand-Activated T Cells IV. Cytosolic Calcium Changes in Activated R Lymphocytes A . Receptor-Activated Calcium Uptake and Internal Store Release B. Ligand-Activated Changes in Cytosolic pH C. Calcium-Dependent Hyperpolarization D. Calcium-Dependent Depolarization E. Biological Significance of Changes in Cytosolic Calcium in Ligand-Activated B Cells V. Summary References

1.

INTRODUCTION

Increases in the concentration of cytoplasmic free calcium ([Caz+Ii) appear to play a major role in the activation and initiation of lymphocyte responses to antigen or cross-linking antibodies directed against surface receptors. Receptors on both T and B cells are generally linked to a polyphosphoinositide-specific 153

('npynght 0 1990 by AcademiL Prssr InL All nghis nl repruduclion in any form rocrvrd

154

ERWIN W. GELFAND

phosphodiesterdse, such that receptor-ligand binding triggers inositol phospholipid hydrolysis, Ca2 mobilization, and activation of protein kinase C. These changes precede the entry of cells into the G, phase of the cell cycle, where they acquire responsiveness to additional signals and progress through the cell cycle. The individual steps in the triggering of T cell proliferation have been reasonably well characterized. Some of these events may lead to or require different ion transport properties. T cell proliferation involves an autocrine-paracrine system that utilizes a lyrnphocytotrophic hormone, interleukin 2 (1L-2), and a highaffinity receptor complex, the I L 2 receptor (IL-2R). Activation of T cells results in the expression of IL-2R and the synthesis and secretion of IL2. Optimal production of IL-2 requires the presence of antigen presenting cells (APC) or macrophages. Progression of the cells through G , into S phase follows I L 2 binding to the high-affinity l L 2 R complex, generally after 10-12 hr (Smith, 1988). In contrast to T lymphocytes, characterization of the individual steps in B cell activation has been less complete. Cross-linking of surface immunoglobulin (Ig), la, or addition of antigen leads to B cell activation. Progression through the cell cycle or differentiation also requires the interaction of a series of interleukins (e.g., IL4, 1L-6) with newly expressed surface receptors. In both T and B cells, activation of the cells and triggering of different ion transport mechanisms have been associated with specific changes in gene expression that accompany the entry of cells into the cell cycle. Among the earliest events studied have been the increased expression of the protooncogenes cfos and c-myc, which have been implicated in the control of cell proliferation. Induction of the c-fos gene is followed by the accumulation of c-myc mRNA. Since maximal c-jos mRNA is obscrved 30-60 min after ligand binding and the accumulation of c-myc mRNA reaches its maximum 1-2 hr after stimulation, it has bcen easier to establish links between ion transport and gene expression than with cell proliferation, assessed 2-3 days later. +

II. MEASUREMENT OF CELLULAR Ca2+ CONTENT AND [Ca2+], Early studies advocating a role for Ca2 in lymphocyte mitogenesis relied on measurements of total exchangeable Ca2 using 4sCa2 labeling. Flux studies with 45CaZ+indicated the existence of Caz+ entry pathways that were stimulated following lectin binding (Whitney and Sutherland, 1972; Milner, 1979; Allwood et al., 197 1 ; Freedman et al., 1975). Major advances in this area were achieved with the introduction of dyes or probes with known Ca2+ affinities and specialized instruments that optimize the detection of changes in fluorescence emission (Grynkiewicz et a l . , 1985; Paradiso et al., 1987). +

+

+

155

7. CALCIUM CHANGES DURING LYMPHOCYTE ACTIVATION

The use of fluorimetry to measure [Ca2+Ii offers distinct advantages over Ca2 measurements using radioisotopes. Fluorimetry is the least biologically disruptive technique available and permits faster tracking of [Ca2 Ii changes. Much of the work carried out in lymphocytes has used flow cytometry or spectrofluorimetric equipment, the latter measuring changes in average intracellular fluorescence intensity over time. The most accurate fluorescent probes have distinctive dual-excitation or -emission characteristics, permitting ratioing of the fluorescence signals in the free or Ca2 -bound state. The introduction of digital image processing equipment provides, at a singlecell level, the ability to localize and quantitate biochemical events within the cell, to distinguish the heterogenous behavior of cells, and to monitor changes in cation concentrations associated with cell-cell interactions. In many cell types examined in this way, including lymphocytes, ligand binding results in repetitive [Ca2 Ii spikes (Jacob ef al., 1988). The spiking frequency appears dependent on the concentration of ligand or extent of receptor occupation, so that at the highest concentrations of ligand, the spikes may fuse, resulting in a maintained elevation of [Ca2+Ii(Wilson et ul., 1987). +

+

+

+

111. CYTOSOLIC CALCIUM CHANGES IN ACTIVATED T LYMPHOCYTES A. Receptor-Activated Calcium Uptake and Internal Store Release More than a decade ago, lectin binding to human or murine lymphocytes was shown to increase significantly exchangeable Ca2 and to increase 45Ca2 uptake. In addition, lectin-induced proliferation of T cells was shown to be dependent on the presence of extracellular Ca2 (Whitney and Sutherland, 1972; Milner, 1979) and could be inhibited by Ca2+-channel blockers; T cell proliferation could be stimulated by the calcium ionophore A23 187 (Maino et al., 1974; Luckasen et al., 1974). Confirmation that lectin binding did increase [Ca2 Ii followed the introduction of the fluorigenic probe quin2. In both murine or human T cells loaded with quin2, addition of lectins, such as phytohemagglutinin (PHA), concanavalin A (ConA), wheat germ agglutinin (WGA), anti-CD3, anti-CD2, anti-T-cell receptor complex, or anti-Thy-1 antibodies, as well as specific antigen, resulted in a rapid increase in [Ca2+Ii,roughly two to fivefold over basal levels (Tsien et a l . , 1982; Gelfand et al., 1984; Roifman et a l . , 1986; Hesketh et al., 1983; A. Weiss et af., 1984; lmboden and Stobo, 1985; O’Flynn et al., 1984; Nisbet-Brown et al., 1985; M. J. Weiss et al., 1984; Kroczek et al., 1986). In quin2 loaded resting T cells, for the most part, these changes in [Ca2+Iiwere monophasic (Tsien et af., 1982; Gelfand et a l . , 1984). In contrast, in quin2 loaded Jurkat cells, addition of ligand resulted in a biphasic curve (A. Weiss et al., 1984). +

+

+

+

156

ERWIN W. GELFAND

Because of the elevated Ca2 -buffering capacity of quin2, better resolution of the changes in [Ca2+Ii has been achieved with the newer probes indo-1 and 1988a; O'Flynn et al., 1985). Activation of T cells loaded fura-2 (Gelfand et d., with these dyes is associated with a biphasic [Ca2+ li response (Fig. 1 A): an initial peak, which lasts for about 1 min and a lower, but sustained, platcau phasc. +

1 . CIIARACTEKIZATION 01; 'IME ICa2 '

Ii RESPONSE

It appears that in lymphocytes, as in other ccll types, thc initial peak is independent of extracellular Ca2+ and results from the release of Ca2+ from internal stores. 'These stores are primarily in the endoplasmic reticulum and arc released in response to receptor activation of phosphatidylinositol hydrolysis and liberation of inositol 1,4,5-trisphosphate (InsP,) (Imboden and Stobo, 1985). On the other hand, the sustained plateau is dependent on extracellular Ca2 and is inhibited by Ca2 -channel blockers, implying that CaZ uptake is primarily responsible for thc plateau phase (Gelfand et al., 1988a). Further dissociation of the two phases of the [Ca2+1, response has been achievcd by loading the cells with high concentrations of quin2 (Ives and Daniel, 1987) or the compound BAPTA [ I ,2-bis-(2-aminophcnoxy)ethane-N,N,N',N'tetraacetic acid] (Gelfand et a l . , 1988a). The latter is a high-affinity C a 2 + chelator that docs not interfere with fluorescent determinations using indo-1. In cells loaded with BAPTA plus indo- I , addition of ligand leads to a monophasic curve (Fig. 1B) similar to that seen in quin2 loaded cells. This indicates that BAPTA can cornplctely buffer the finite pool of CaZ+ released from internal stores. The sustained plateau is retained in BAPTA-loaded cells as the large uptake of Ca2 across the plasma membrane overcomes the chelating capacity of the drug. We have shown that the sustained clevation of [Ca2+1, is due to unidirectional +

+

+

+

b

a

PHA

C

PHA

PHA

U

2 min

'

FIG. I . Dissociation of the components of the [Ca' 1, response. Addition of PHA (I0 Fgiml) to indu-l loaded T cclls results in a biphasic response (a). In BAIYTA-loaded cells (b), the initial transient response is no longer detected. When cells arc depolari7~dby increasing extracellular K +, only the initial transient response is observed (c).

157

7. CALCIUM CHANGES DURING LYMPHOCYTE ACTIVATION

Ca2+ influx and not to any reduction in Ca2+ efflux. In these studies we distinguished cation influx and efflux using Mn2 , a cation that permeates Ca2+ selective channels in other cell types (Hallam, 1985). In contrast to Ca2+, which leads to an increase in indo-1 fluorescence, the binding of Mn2 to indo-1 quenches fluorescence. The rate of indo-1 fluorescence quenching can be used to monitor Mn2* uptake, an indirect measure of Ca2+ uptake. As shown in Fig, 2A, when cells suspended in Ca2 -free medium are exposed to Mn2 , fluorescence intensity declines at a constant rate. Sudden chelation of external Mn2+ with EGTA stops the progression of quenching without a reversal or increase in fluorescence. This indicates that Mn2 efflux from the cells is minimal and that the rate of quenching is a valid measure of unidirectional divalent cation uptake. Ligand-activated fluxes were monitored in the same way. As shown in Fig. 2B, addition of Mn2+ established a constant rate of decline in fluorescence. Subsequent addition of PHA to peripheral blood T cells is followed by a transient increase in fluorescence intensity, presumably from mobilization of Ca2 from internal stores. Following this initial increase in indo-1 fluorescence, the rate of indo- 1 fluorescence quenching increased significantly, consistent with an increased rate of divalent cation uptake. Addition to EGTA arrested the progression +

+

+

+

+

+

a Mn2+

1

U

2 min

b Mn2+

'----FIG. 2. Mn2+ uptake assessed by the rate of indo-1 fluorescence quenching. T cells, loaded with indo-I, were suspended in Ca*+-free medium to which 0.25 mM Mn2+ was added. In (a), EGTA (2 mM) was added. Where indicated in (h), PHA (10 pglml) was added, followed by EGTA. In leiu of calibration (in the prescncc of Mn* ) the relative fluorescence change is indicated (AF/F). +

158

ERWIN W. GELFAND

of quenching without inducing any recovery of fluorescence, indicating minimal, if any, change in divalent cation efflux as a consequence of ligand binding. 2. ROLEOF ANTIGEN-PRESENTING CELLS IN TRIGGERING THE [CaZ 1, RESPONSE +

Antigen presenting cells (APC) play a critical role in the induction of specific T cells to secrete I L 2 and proliferate in response to antigen. In the absence of accessory cells, many ligands directly trigger increases in [Ca2+li,despite the fact that they do not induce I L 2 secretion or proliferation in the absence of APC. Certain anti-CD3 antibodies provide examples of this type of ligand. Other ligands do require APC for triggering increases in [Ca2+Ii, and as demonstrated for suboptimal concentrations of PHA (Mills et al., 1985a) or Staphylococcus uureus protein A (Lederman el al., 1984), the APC-dependent increases in [Ca2+],, IL-2 secretion, or cell proliferation are not MHC restricted. The responses of antigen-specific T cell clones are MHC restricted. Following addition of' APC and antigen to these T cells, the changes in [Ca2+], are slow to develop unless the APC have been pre-incubated with antigen prior to contact with the T cells (Nisbet-Brown et al., 1985). If MHC-compatible APC are allowed to process antigen and are then fixed, incubation with specific T cells can trigger a [Ca2+ll response as well as I L 2 R expression but not IL-2 secretion or cell proliferation (Nisbet-Brown et d.,1987). Addition of non-fixed, non-MHC compatible APC can now trigger 1L2 secretion and cell proliferation (Table I), suggesting that some APC-dependent signaling, especially for I L 2 production, is not MHC-restricted and may not involve the T3-Ti complex.

3. HETEROGENEITY AMONG T CELLSA N D

THE

[Ca2+IiRESPONSE

Triggering of the [Ca2+1, response in T cells can occur via one of several distinct pathways. As discussed previously, triggering can be achieved through

RESPONSES OF

TABLE 1 ANTIGEN-SPECIFIC T CE1.I. CI.ONES Proliferation

Addition Clone

+0

+ (APC/Ag)autol

+ (APC/Ag)allo + (APC/Ag)autol-fixed

+ (APC/Ag)autol-fixed + APC allo + (APC/Ag)autol-fixed + APC allo-fixed

[Ca'+], ~

+++ +++ +++ +++ ~

IL-ZR

-1L-2

'IL-2

-

-

-

ti+

++

+++

-

-

-

+ ++ +

-

+++ ++ +++

+

-

7. CALCIUM CHANGES DURING LYMPHOCYTE ACTIVATION

159

the binding of antigen in the context of MHC products, A second pathway is through the T3-Ti complex with anti-CD3 (Imboden and Stobo, 1985), antiTCR (O'Flynn et al., 19851, or anti-clonotypic antibodies (Imboden and Stobo, 1985). Both pathways require the surface expression of the T3 receptor complex. T3-negative variants are also unresponsive to PHA (Ohashi et al., 1985). Thus Ca2+ mobilization appears regulated in some way by the TCR complex. A further extension of this potential regulation is the correlation between the expression of the CD3-S-q subunits and phosphoinositide hydrolysis following stimulation through the TCR complex (Mercep et al., 1988). A third pathway of T cell activation is through the CD2 molecule. Activation of CD3- T cells can be achieved through CD2 (Moretta et al., 1987). Within populations of peripheral blood lymphocytes the [Ca2+Ii response to mitogenic stimuli may be heterogenous. This heterogeneity may be related to the immunophenotype. Rabinovitch et al. (1986) reported that the [Ca2+Iiresponse to anti-CD3 antibody or PHA is greater among CD4' cells than CD8+ cells. Further heterogeneity of the [Ca2+],response may be observed between immature and mature T cells (Finkel et al., 1987). Using anti-T-cell receptor antibodies as ligand, immature murine thymocytes showed a much reduced Ca2 influx compared to mature cells, which could reflect a lower Ca2+ channel frequency in immature T cells or less efficient activation-opening of existing channels. +

OF [Ca2f)iCHANCES DURlNC ACTIVATION 4. REGULATION

There are essentially two basic mechanisms by which [Ca2+], can be increased following ligand binding: releasing Ca2 from intracellular storage sites and transporting Ca2 from the extracellular space across the plasma membrane. A third possibility, the decrease in Ca2 efflux in activated cells, does not appear to play a major role. By analogy with other tissues, receptor-ligand interaction of T cells results in the hydrolysis of phosphatidylinositol bisphosphate (PtdInsP,), liberating InsP,. Inositol trisphosphate (and conceivably its phosphorylated derivative, inositol 1,3,4,5-tetrakisphosphate)is presumed to mediate the release of intracellular stores and the transient [CaZ Ii response in T cells as well. Activation of murine thymocytes with ConA (Taylor et al., 1984) or Jurkat cells with anti-CD3 or anticlonotypic antibodies (Imboden and Stobo, 1985) is associated with increases in InsP, production. In Jurkat cells, stimulation of the antigen receptor also is associated with an increase in activity of the InsP,-kinase, leading to substantial increases in the tetrakisphosphate (Imboden and Pattison, 1987). The mechanism(s) underlying the sustained elevation of [Ca2'1, in lymphocytes is less well understood. The most important structures controlling Ca2 uptake are proteins in the lipid bilayer of the plasma membrane that function as +

+

+

+

+

160

ERWIN W. GELFAND

“channels” or “pores” through which Ca2 moves. The molecular configuration of these structures determines their selectivity for the ions that can traverse the channels as well as the membrane potential-dependent “gating” of these channels. In contrast to excitable cells, such as nerve or muscle, which express voltage-gated Ca2+ channels, T and B lymphocytes do not express such channels and differ from excitable cells in many other ways. In the absence of voltgeactivated channels, depolarization of the cells does not result in increases in ICa2+Ii. To the contrary, depolarization of the cells using ion substitution, nystatin, or gramicidin results in a marked inhibition of receptor-activated increases in Ca2+ uptake (Gelfand et ul., 1984; Oettgen et al., 1985), in contrast, release from internal stores is unaffected in depolarized cells (Fig. 1C). These effects of membrane potential on ligand-stimulated increases in [CaLi li suggest that they are mediated through a conductive pathway and that maintenance of a normal resting membrane potential is necessary for receptor-activated Ca2 uptake and ultimately cell prolifcration. The effects of membrane potential on Ca2’ influx are presumably mediated by changes in the Ca2 electrochemical gradient, with depolarization decreasing the force driving Ca2 inward, or possibly by voltage-dependent inactivation of a ligand-gated channel. However, Ca2 permeable channels opened following receptor activation have been difiicult to detect in T cells. Kuno et al. have detected PHA-induced increases in Ca2 current in cloned human T cells (Kuno et ul., 1986; Kuno and Gardner, 1987). The opening of these channels was independent of voltage, and PHA increased Ca2 current primarily by increasing the frequency of channel openings without affecting mean opcn time or single channel conductance. Physiological concentrations of InsP, could activate these channels, thus coupling InsP, production to both Ca2+ influx and Ca2 release from internal stores. Regulation of Ca2+ influx and release from stores may also be expressed at levels other than the state of membrane potential. In murine T cells, activation of protein kinase C by phorbol esters or exogenously added diacylglycerol leads to a decrease in [Ca2 li implying some form of feedback inhibition (Tsien el al., 1082; Rogers rt at., 1983). Protein kinase C activation may directly or indirectly inhibit phospholipase C, resulting in decreased InsP, production and reduced Ca2+ release from stores and Ca2+ influx (Nishizuka, 1988). Addition of TPA may also lead to an increase in Ca2+ efflux in some cells by activating the Ca2+ pump (Schimmel and Hallarn, 1980). Surprisingly, addition of TPA does not affect ligand-induced increases of [Ca2 1, in human T cells (Gelfand et al., 1985). In several cell types there is a relationship between InsP, and specific guanine nucleotidc (GrP)-mediated effects on [Ca2 Ii. The mechanism is unclear, especially since there may be two separate systems or InsP, production may bc regulated by a GTP regulatory process (Gill et al.. 1986). Signaling through the +

~+

+

+

+

+

+

+

+

+

161

7. CALCIUM CHANGES DURING LYMPHOCYTE ACTIVATION

T cell antigen receptor on Jurkat cells is blocked by cholera toxin, but it does not appear to be through a G protein-mediated phenomenon (Imboden et a / ., 1986). B. Calcium-Dependent Changes in Cytosolic pH An amiloride-sensitivc, electroneutral Na+ /H antiport that plays a major role in both cell volume regulation and cytosolic pH (pH,) regulation has been detected in most mammalian cells including lymphocytes (Mahnensmith and Aronson, 1985; Cala, 1985; Thomas, 1984). Various growth factors and mitogens activate this exchanger, resulting in cytosolic alkalinization. In mitogen-stimulated lymphocytes, the primary route for Na+ entry is the antiport. Phorbol esters activate the antiport, presumably through activation of protein kinase C (Grinstein et al., 1985). Mitogenic lectins can activate the antiport independently of protein kinase C (Grinstein et a/., 1986). In rodent and human T cells and T cell lines, mitogens and anti-CD3 antibody activate the antiport as detected by increases in Na+ uptake, cytosolic Na+ concentration, and pH, (Hesketh et al., 1985; Grinstein et a / . , 1987; Rosoff and Cantley, 1985a). When added to IL-2R-expressing cells, IL-2 also activates the Na+ / H + antiport, resulting in rapid and sustained increases in pHi (Mills et al., 1985b). Although activation of murine T cells with mitogenic lectins or human T cell lines with anti-CD3 antibody usually results in stimulation of the antiport and increased pHi, this is not always the case (Ives and Daniel, 1987). We were surprised to find that in resting human T cells, addition of ligands such as PHA, ConA, WGA, and anti-CD3 antibody resulted in a decrease in pH, (Gelfand et al., 1988b; Cheung et al., 1988). This decrease in pH, was accentuated in Na+ free medium or in the presence of amiloride analogs. It thus appears that although the Na-+/ H + antiport is concomitantly stimulated, in activated human T cells the acidification response predominates. Using mitogenic and non-mitogenic ligands (Fig. 3) as well as ionomycin (Gelfand et a/., 1988b), we established that the acidification response was [Ca2+],-dependent and was only observed with Ca2 -mobilizing ligands (Cheung et al., 1988). Further, only ligand-induced uptake of extracellular Ca2+ triggered the acidification response; release of internal stores alone was insufficient. This Ca2+-dependence for the net decrease in pH, of activated human T cells is not paralleled by an analogous requirement for the alkalinization response of activated murine T cells or human T cell lines. In these situations, ligandinduced changes in [ Ca2 1, did not appear to be required for stimulation of the antiport (Hesketh et a l . , 1985; Grinstein et al., 1987), although Ca2+ influx enhanced the activity of the exchanger in an activated human T cell line (Rosoff and Cantley, 1985a). The biological significance of activation of the Na /H antiport remains +

+

+

+

+

162

ERWIN W. GELFAND a

b

PHA

7.0

d

C

'

ConA

ConA

f

4

--350 -170

NaCl

[Ca2'], 1 --__, nM -

4;:;

PHi

'7.1

f

WGA

U

2 min FIG. 3. Demonstration of the Ca2 + requirement for changes in pH, induced by mitogenic (PHA, ConA) and nan-mitogenic (WGA) lectins. [Ca2 1, was monitored following indo-1 loading of the cells, and for pHi determinations, cells were loaded with BCECF and suspended in NMG+ (Na frrc) mediun~.NaCl (80 mM) was added to show that the N a + / H + antiport was active. +

+

questionable, at least in the early period following cell activation. Inhibition of the antiport using amiloride analogs did not affect T cell proliferation (Mills et ul., 1986) or 112-dependent cell proliferation (Mills el al., 1985b), at least in HC0,--containing medium. In HC0,- -free medium we found that the early expression of c7fbs, induced by lectins or phorbol ester, was also unaffected by inhibition of the antiport (Grinstein et ul., 1988).

C. Calcium-Dependent Hyperpolarization The membrane potential of unstimulated lymphocytes ranges from - 50 to -70 mV, representing primarily the diffusion potential of K + (Deutsch et nf.,

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7. CALCIUM CHANGES DURING LYMPHOCYTE ACTIVATION

1979; Rink et al., 1980) and to a lesser extent of Na+ or other ions (Wilson and Chused, 1985). Variable changes in membrane potential have been observed in porcine (Felber and Brand, 1983), murine (Kiefer et al., 1980; Felber and Brand, 1983), and human T cells (Deutsch et al., 1979; Segel et ul., 1979), following ligand binding and monitoring membrane potential using cationic radioactive probes and cyanine dyes. Introduction of oxonol dyes provides a more reliable measurement of membrane potential (Tsien et al., 1982; Tatham and Delves, 1984; Tatham et al., 1986). Using these dyes, Tsien et al. (1982) first noted that lectin-induced increases in [Ca2+], in murine T cells were associated with a hyperpolarization. This was presumed to be secondary to Ca2+-activated K + conductance, since it was dependent on the external K and Ca2 concentration and was insensitive to ouabain. Further support for the existence of Ca2 -activated K channels in T cells is derived from the demonstration of Ca2 -dependent hyperpolarization in the presence of low concentrations of Ca2+ ionophores (Tsien et al., 1982; Gukovskaya and Zinchenko, 1985; Grinstein and Cohen, 1987), the relationship between mitogen-induced membrane potential changes and [Ca2'1, in human T cells (Wilson and Chused, 1985; Tatham and Delves, 1984; Tatham et a l . , 1986), and the measurement of 86Rb fluxes (Segel et al., 1979; Grinstein et al., 1983). Attempts to detect these channels by patch clamping have been unsuccessful (Fukushima et al., 1984; Cahalan et al., 1985). We have demonstrated Ca2 activated K channels in human T cells (unpublished). Addition of Ca2 -mobilizing ligands results in a small (3-5 mV) degree of hyperpolarization. Addition of charybdotoxin, a derivative of scorpion venom that is a selective inhibitor of Ca2 -activated K channels (Gimenez-Gallego et al., 1988), results in a reversal of the potential (i.e., depolarization) following ligand binding without affecting the increases in [Ca2+],. At least three types of voltage-gated K + channels have been described in T cells, and their distribution may vary among different T cell subsets (DeCoursey et al., 1987; Chandy et al., 1986; Lewis and Cahalan, 1988). It is possible that mitogen-induced hyperpolarization of T cells is due to the activation of these voltage-gated K + channels (DeCoursey et al., 1984), although this is controversial (Matteson and Deutsch, 1984; Schlichter et al., 1986). Despite a common sensitivity to some drugs, these K channels differ significantly from the Ca2 activated K channels described previously. Chandy et al. (1984) proposed that ligand-induced Ca2 entry may be through the opened voltage-gated K + channels. However, this does not appear to be so since (1) K channel blockers have minimal effect on lectin-induced increases in [Ca2+Il, (2) membrane depolarization failed to increase [Ca2+I, although K channels would be expected to open, and (3) the inhibitory effects of the K + channel blockers 4-aminopyridine or tetraethylammonium on T cell proliferation appeared to be nonspecific (Gelfand et af., 1986a). +

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D. Biological Significance of Changes in Cytosolic Calcium in Ligand-Activated T Cells 1 . CaZ+-DEPENDENTA N D Ca2+-INDEPENDENT

T CELLRESPONSES The different stages that T cells traverse betwecn activation and cell proliferation have different requirements for changes in [CaZ J i . Depletion of extracellular CaZ+ or inhibition of Ca’ uptake with CaZ -channel blockers results in inhibition of cell proliferation and 1L-2 synthesis-secretion but does not affect normal expression of functional 1L-2R (Gelfand et al., 1986b). Since these manipulations leave the activated release from internal stores intact (Gelfand et al., 1988a), the critical changes for IL-2 synthesis appear to be due to CaZ+ uptake, with release from internal stores alone not being sufficient or essential. In contrast, the induction of IL-2R appears to be Ca2 ’ -independent, since elimination of any detectable changes in [Ca*+I i (for example in BAPTA-loaded cells suspended in CaZ -fe e medium) does not affect 1L-2R expression in ligandactivated cells (Gelfand et 01.. 1986b). ?‘here are additional lines of evidence that support the pivotal role of Ca2+ uptake in the regulation of T cell proliferation. Depolarization of the plasma membrane just prior to receptor-ligand activation by Ca’ -mobilizing ligands results in a marked reduction of Ca2+ uptake but with normal increases in [Ca2+Iifrom internal store release. In these cells, whereas 1L-2R expression is normal and the cells proliferate in response to exogenous 1L-2, there is marked inhibition of 1L-2 synthesis-secretion (Gelfand et al., 1987a). Increasing cytosolic CaZ+ with the ionophore ionomycin to the levels seen with ligands in normally polarized cells restores IL-2 synthesis-secretion and T cell proliferation. Our data also confirm that this membrane-potential sensitive step is unidirectional Ca’+ uptake and not an increase in Cazi efflux. In addition to the Ca2 -dependent events triggered by most CaZ -mobilizing ligands, these agents also trigger a Ca2 -independent event(s). For example, treatment of T cells with the phorbol ester 12-0-tetradecanoylphorbol-13-acetate (TPA) bypasses the need for Ca2i uptake (Gelfand et a l ., 1985). In TPA-treated cells, receptor-ligand interaction, even in the absence of demonstrable increases in [Ca2+l i , results in IL-2 synthesis and T cell proliferation. The nature of these alternative Ca2 -independent signaling pathways remains to be defined and may involve the activation of protein kinase C. It is also interesting to note that, unlike the effects of TPA on activated murine T cells, pretreatment of human T cells with TPA does not affect ligand-induced changes in [Ca2 Ii (Gelfand et d., 1985). The importance of changes in [CaZ+J i for T cell activation and I L 2 production has also been challenged (Sussman er al., 1988). In an antigen-specific murine T cell hybridoma, IL-2 secretion was normal following cell activation, +

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7. CALCIUM CHANGES DURING LYMPHOCYTE ACTIVATION

although there were no detectable changes in either InsP, production or lCa2 + I i . These data underscore the potential importance of alternative signaling pathways in certain cell systems. Following IL-2 binding to its receptor, a number of biochemical changes can be demonstrated. Among these is stimulation of the Na /H antiport. We (Mills et al., 1 9 8 5 ~ and ) others (Legrue, 1987; Abraham et al., 1987) have, however, failed to demonstrate any early changes in [Ca2+Ii or a requirement for such changes in IL2-dependent proliferation. +

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2 . CYCLOSPORIN-MEDIATED EFFECTSON LIGAND-INDUCED IN [Ca2+], CHANGES

Several of the cyclosporins are potent immunosuppressive agents which initially appeared to act primarily by preventing 1L-2 gene transcription and synthesis-secretion (Kronke et ul., 1984; Granelli-Piperno et al., 1984). It is now clear that these drugs must act at several different levels. We have shown that the immunosuppressive cyclosporins, but not a non-immunosuppressive analog, can inhibit PHA-induced Ca2 uptake and result in membrane depolarization (Gelfand et al., 1987b). The degree of depolarization and the kinetics of the depolarizing effect are not sufficient to totally explain the marked reduction in PHA-induced changes in (Ca2+], seen after a 30 min pre-incubation period but may contribute to the effects of the drugs on [Ca2+ I t uptake. Cyclosporin A has also been shown to block the CD3-TCR-stimulated Ca2 influx-dependent activation of the N a + / H + exchanger in a human T cell line (Rosoff and Terres, 1986). +

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IV. CYTOSOLIC CALCIUM CHANGES IN ACTIVATED B LYMPHOCYTES

A. Receptor-Activated Calcium Uptake and Internal Store Release 1. CHARACTERIZATION OF THE ICa2 +Ii RESPONSE

In parallel to observations in T cells, cross-linking of the B-lymphocyte antigen receptor initiates the degradation of PtdlnsP, with the resultant formation of two intracellular messengers, diacylglycerol and InsP, (Ransom et al., 1986; Bjisterbosch et al., 1986). The similarities between T and B lymphocytes extend to the findings that [Ca2+ I, changes can be induced by anti-Ig antibodies as well as specific antigen in antigen-specific B cell clones (Partain et al., 1986). Furthermore, as in T cells, receptor-ligand activation of indo-l loaded B cells is usually associated with a biphasic increase in [Ca2+li(Bjisterbosch et al., 1986;

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Roifman et al., 1987; MacDougall et a / ., 198th). The initial peak is independent of extracellular Ca2 + ,cannot be inhibited by Ca2 +-channel blockers, and is associated with increases in InsP, production. The sustained rise in [Ca2 li is dependent on extracellular Ca2 and can be inhibitcd by Ca2 -channel blockers. Further dissociationpf the [Ca2 + I i response can also be achieved by loading the cells with BAPTA. Addition of anti-lgD to human B cells induced the greatest changes in [Ca2 Ii; anti-IgM induced intermediate changes; anti-IgG, the lowest changes. The changes induced by anti-IgM and anti-IgD were not additive. Surprisingly, antiIgG induced the greatest increases in InsP, production (Roifman et al., 1987). The phorbol ester TPA may (Ledbetter et al., 1988) or may not (Roifman et al., 1987) affect anti-Ig-induced increases in [Ca2+liof human B cells. Binding of an anti-CD19 antibody to human B cells consistently decreased the [Ca2+J i increases induced by anti-IgM (Ledbetter er al., 1988; Pezzutto et al., 1987). In contrast, on their own, anti-CD19 antibodies stimulated increases in [Ca2+ I i (Pczzutto t’t ul., 1987). In murine cells, ligand binding to mlgM or mIgD for as little as 3 min renders the cell unable to respond to subsequent ligand binding to the reciprocal receptor (Cambier et al., 1988). This “desensitization” is long-lived, does not reflect modulation of mlg, and appears not to be mediated solely by activation of protein kinase C. Lipopolysaccharide (LPS) rapidly stimulates an increase in PtdlnsP, turnover and InsP, production in the pre-B-lymphocyte line, 702/3 (Rosoff and Cantley, 1985b). There is also rapid elevation of [Ca2+liwhich is independent of cxtracellular Ca2 ,suggesting release from internal stores. The cytochalasins A, B, D, and E also induce a rapid and sustained elevation of intracellular Ca2 which derives largely from the influx of extracellular Ca2 , although a small, transient elevation in [Ca2+ I i is independent of extracellular Ca2 (Baeker et al., 1987). Pretreatment of murine B cells with a phorbol ester results in inhibition of the B cell [Ca2+Ii response to anti-Ig (Wilson et al., 1987), LPS (Rosoff and Cantley, 1985b), and the cytochalasins (Baeker et al., 1987). As described for T cells, we distinguished Ca2+ influx and efflux using Mn2+ and the rate of fluorescence quenching. The sustained anti-Ig induced changes in [Ca2+Iiwere shown to be due to increased unidirectional Ca2+ influx (Fig. 4). +

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2. REGULATIONOF [Ca2+IiCHANGES

DURING

ACTIVATION

The mechanisms resulting in and regulating the sustained elevations in [Ca2+], in ligand-activated B cells are not well defined. As described for T cells, the sustained increases are dependent on external Ca2 and can be inhibited by Ca2 -channel blockers, implying the presence of a Ca’ channel for uptake (MacDougall et al., 1988a,b). Although Fukushima and Hagiwara (1983, 1985) have characterized a voltage-gated CaZ conductance in murine myeloma cell +

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B

Mn*+

-1

U

U

'FIG.4. Demonstration of unidirectional Ca2+ influx in ligand-activated human B cells. Uptake was monitored by the rate of indo-l fluorescence quenching. B cells loaded with indo-1 and BAPTA were suspended in Ca2+-free medium to which 0.75 mM Mn2+ was added. Where indicated, antiIgM or EGTA (2 mM) was added.

lines, the putative anti-Ig stimulated Ca2+ channel in human B cells is quite different. In fact, depolarization of human B cells did not increase resting [Ca2+Ii levels and reduced anti-Ig sustained increases in [Ca2 Ii (MacDougall et al., 1988a). Release from internal stores is unaffected by membrane depolarization. The human B cell Ca2+ channel, similar to that described in T cells, is ligand-gated and not voltage-gated. Release of Ca2 from internal stores and entry across the plasma membrane also behave differently towards artificially imposed changes in [Ca2+Ii. In B cells pre-pulsed with ionomycin to elevate baseline levels of [Ca2+Ii,addition of anti-IgM could still trigger the transient increase due to internal store release, but the sustained phase was progressively reduced as basal levels of [Ca2+Ii were gradually increased (Gelfand rt LIE., 1989). The increases in [Ca2 Ji in murine B cells, which are dependent on mIg crosslinking, can be inhibited by elevation of intracellular cyclic AMP levels and by activation of protein kinase C (Coggeshall and Cambier, 1985; Wilson er al., 1987). Further, activation of phospholipase C by mIgM appears to involve a guanine nucleotide-dependent step that is insensitive to both pertussis toxin and cholera toxin (Gold et nl., 1987). Dexamethasone also markedly inhibits anti-Ig +

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ERWIN W. GELFAND

stimulated PtdlnsP, hydrolysis and increases in [Ca2 ji, but the mechanisms remain to be defined (Dennis et al., 1987).

B. Ligand-Activated Changes in Cytosolic pH There is ample evidence in murine B cell lines for the presence of an amiloride-scnsitive, electroneutral Na+ /HI exchanger. Addition of LPS (Rosoff and Cantley, 1983), phorbol esters (Rosoff et d., 1984), and IL-I (Stanton et I.. 1986) results in murine B cell cytosolic alkalinization, and in the 702/7 B cell line, B cell differentiation (Rosoff and Cantley, 1983).Other than the responsc to 11- 1 (Calalb et a l . , 1987), alkalinization was amiloridc-sensitive and required extracellular Na ’ . Acid-loading experiments have demonstrated the prcsence of the Na+/El+ exchange system in human B cell lines (Grinstein et d . , 1984). This exchange system and its importance in LPS-induced proliferation of normal human B lymphocytes has becn demonstrated (Gaidano et a!., 1989). Both LPS and TPA were shown to activate the exchanger in resting, normal human B cells (Gaidano ct d., 1989). Surprisingly, there was a significant lag of 10-15 min, and this was very different from the results with TPA in human T cells (Gelfand et ul., 1988b). The regulation of pHi is clearly different in human B cells since iononiycin does not alter pH, in these cells (E. W. Gclfand, unpublished observations). +

C. Calcium-Dependent Hyperpolarization If Ca2 uptake is through a channel, then the opening of Ca2 channels by cross-linking of mIg should be associated with an increase in membrane conductance. Although there are many similarities in ion transport between activated T and B lymphocytes, initial studies of murine B cells suggested that ligandinduced activation resulted in membrane depolarization, not hyperpolarization (Monroe and Cambier, 1983). Wilson and Chused (1985) found that T and not B cells display a Ca2 -induced hyperpolarization. Consistent with these findings, Ransom ef al. (1986) found that B cell mitogens known to elcvatc [Ca2 Ii or A23187 also induced a marked depolarization. These studies may have been compromised by the methods used to monitor membrane potential changes. Data from our laboratory differ significantly (MacDougall et al., 1988a,b). We found that treatment of human tonsillar B cells with anti-lg resulted in a significant (5- 10 mV) hyperpolarization. Several lines of evidence suggested that this hyperpolarization was due to Ca2 -sensitive K conductance. The potential change was still present when Na+ was substituted for by N-methylglucamine (NMG + ) but not whcn K was substituted for Na+ . Second, a similar potcntial change was demonstrated when [Ca2+Iiwas clevated by ionomycin, and third, the shape of the hyperpolarization mirrored the changes in [CaZ+li.Eliminating +

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7. CALCIUM CHANGES DURING LYMPHOCYTE ACTIVATION

the sustained phase of the [Ca2+li response abolished the prolonged hyperpolarization. These data identify, for the first time, the presence of Ca2 -sensitive K + channels in human B cells. +

D. Calcium-Dependent Depolarization These increases in K conductance and the resultant hyperpolarizing effect could have masked the putative Ca2+ current that would accompany Ca2+ uptake through conductive channels. To further define the mechanism of Ca2 entry into ligand-activated B cells, we maximized our ability to detect Ca2+ currents by increasing the amount of Ca2 entering the cells at the same time as delaying the activation of Ca2 -dependent K conductance (MacDougall et ul., 1988b). Using BAPTA-loaded cells suspended in Ca2+ -free medium, addition of anti-IgM resulted in a marked depolarization (Fig. 5A). The depolarization was further enhanced when Ca2 was added to cells initially activated in Ca2 -free medium (Fig. 5B) (MacDougall et af., 1988b). The initial depolarization was dependent on extracellular N d + (it was not observed in NMG -containing medium), which permeates through ligand-gated Ca2+ channels when the cells are activated in the absence of extracellular Ca2 (Fukushima and Hagiwara, 1985). The second phase of depolarization noted on addition of Ca2+ is compatible with entry of Ca2 through anti-lg gated Ca2 channels. These receptor-gated Ca2 channels in human B cells share some properties with the voltage-gated Ca2+ channels of excitable cells in that they are Ca2+ selective under physiological conditions yet become permeable to Na+ in the absence of external C a 2 + . They do differ from voltage-gated channels in that ligand-gated channels cannot be +

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FIG. 5 . Effects of removal and addition of extracellular Ca2+ on anti-IgM induced changes in membrane potential. B lymphocytes, loaded with 10 p % 4BAPTA, were equilibrated with bis-oxonol in Ca2+-free solutions containing (A) NaCl or (B) NMG-CI. Where indicated, anti-IgM or Ca2+ (2 mM) was added.

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ERWIN W. GELFAND

activated by depolarization and are inhibited by calcium-channel blockers only at higher (micromolar) concentrations (MacDougall er ul., 1988b).

E. Biological Significance of Changes in Cytosolic Calcium in Ligand-Activated B Cells The possible role of ligand-induced changes in ICa2 1, for B cell differentiation, maturation, and proliferation is less clear than results in T cells. This is especially so since the phorbol esters are often required as co-mitogens in the proliferative response to anti-lg (Roifmann et ul., 1987). Capping of surface Ig in response to anti-Ig appears to be Ca2+-independent (Pozzan et al., 1982). In contrast, anti-Ig or calcium ionophore induction of Ia on murine B cells appears to be Ca2 -dependent and dependent on the production of InsP, (Ransom and Chambier, 1986). The failure of anti-Ig alone to simulate B cell proliferation despite the changes in [CaZ+1, suggests that additional signals are required. Until these signals are defined, it is uncertain whether changes in [Ca2+Ii are as essential to B cell proliferation as they appear to be for T cell proliferation. +

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V. SUMMARY Stimuli of very diverse nature cause increases in [Ca2+Ii in both T and B lymphocytes, and these increases in turn presumably give rise to a wide spectrum of cellular effects. Control mechanisms for [Ca2 Ii are located both in the plasma membrane and within the cells. Changes in [Ca2+li in both types of lymphocytes arise from a combination of internal store release and Ca2 uptake across the plasma membrane. Alteration in Ca2 efilux does not appear to play a role in the net increases in [Ca2 ' Ii. In both T and B cells, release from internal stores is easily dissociated from Ca2+ uptake. Unlike excitable cells, Ca2 is not transported through voltage-gated channels in lymphocytes. Instead, in both T and B cells, ligand-gated Caz+ -selective and conductive channels appear to be opcrative with membrane dcpolarization inhibiting ligand-induced entry of Ca2 . Changes in [Ca2 li appear to be essential for some responses, contributory to others, and irrelevant to others. The connection of changes in [Ca2+1,, especially due to Ca2 ' uptake, and receptor-activated phosphatidylinositol hydrolysis is not entirely clear in many situations. Although great strides have been made in understanding the potential role of [Ca2 li in stimulus-receptor coupling of cells, future studies are rcquired to distinguish those responses in which elevation of [CaZ' J i may be merely the consequence. of ligand-binding from those responses in which the elevation of [Ca2+Ii is prerequisite for cell activation. +

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Supported in part by NlH grant #A1 26490. Dr. Gelfand is a Scholar of the Raymond and Beverly Sackler Foundation. REFERENCES Abraham, R . T., Ho, S . N., Bama, T. J., and McKean, D. J. (1987). Transmembrane signaling during interleukin 1-dependent T-cell activation. J . Eiol. Chem. 262, 2719-2728. Allwood, G . , Asherson, G. L., Davey, M. J., and Goodford, P. H. (1971). The early uptake of radioactive calcium by human lymphocytes treated with phytohemagglutinin. Immunohgv 21, 509- 5 16. Baeker, T. R., Simons, E. R . , and Rothstein, T. L. (1987). Cytochalasin induces an increase in cytoaolic free calcium in murine B lymphocytes. J . Immunol. 138, 2691-2697. Bjisterbosch, M. K . , Rigley, K. P., and Klaus, C. G. B. (1986). Cross-linking of surface immunoglobulin on B lymphocytes induces both intracellular Ca* + release and Ca2+ influx: analysis with indo-l . Biochem. Biophys. Res. Commun. 137, 500-506. Cahalan, M. D., Chandy, K. G., DeCoursey, T. E., and Gupta, S. (1985). A voltage-gated potassium channel in human T lymphocytes. J . Physiol. (London) 358, 197-237. Cala, P. M. (1985). Volume regulation by Amphiuma red blood cells: Strategies for identifying alkali nietal/H+ transport. Fed. Proc.. Fed. Am. Soc. Exp. B i d . 44,2500-2507. Calalb, M . B., Stanton, T. H., Smith, L.. Cragoe. E. J., and Bomsztyk, K. (1987). Recombinant human interleukin-I-stimulated Na+ iH exchange is not required for differentiation in pre-8 lymphocyte cell line, 70213. J . Biol. Chem. 262, 3680-3684. Cambier, J., Chen, Z. Z . , Pasternak, J . , Ransom, J . , Sandoval, V., and Pickles, H. (1988). Ligandinduced desensitization of B-cell murine immunoglobulin-mediated Ca2 mobilization and protein kinase C translocation. Proc. Nail. Acad. Sci. U . S . A . 85, 6493-6497. Chandy, K. G., DeCoursey, T. E., Cahalan, M. D., McLaughlin, C., and Gupta, S. (1984). Voltagegated potassium channcls are required for human T lyiiiphocyte activation. J. Exp. Med. 160, 369-385. Chandy, K . G . , DeCoursey, T. E., Fischbach, M . , Tala], N., Cahalan, M. D., andGupta, S. (1986). Altered K + channel expression in abnormal T lymphocytes from mice with one Ipr gene mutation. Science 233, 1197-1200. Cheung, R. K., Grinstein, S . , and Gelfand, E. W. (1988). Mitogenic and non-mitogenic ligands trigger a calcium-dependent cytosolic acidification in human T lymphocytes. J . Immunol. 141, 1648- 1651. Coggeshall, K. M., and Cambier, J. C. (1985). B-cell activation: VI. Studies of modulators of phospholipid mctabolism suggest an essential role for diacylglycerol in transmembrane signalling by mIg. J . Immunol. 134, 101-106. DeCoursey, T. E., Chandy, K. G . , Gupta, S . , and Cahalan, M. D. (1984). Voltage-gated K + channels in human T lymphocytes. A role in mitogenesis. Nature (London) 307, 465-471. DeCoursey, T. E., Chandy, K. G., Gupta, S . , and Cahalan, M. D. (1987). Mitogen induction of ion channels in murine T lymphocytes. J . Gen. Phvsiol. 89, 405-420. Dennis, G., June C. H., Mizuguchi, I., O'Hara, J., Witherspoon, K., Finkelman, F. D., McMillan, V., and Mond, J. J. (1987). Glucocorticoids suppress calcium mobilization and phospholipid hydrolysis in anti-lg antibody-stimulated B cells. J . Immunol. 139, 2516-2523. Deutsch, C. J . , Holian, A , , Holian, S. K . , Daniele, R. P., and Wilson, D. F. (1979). Transmembrane electrical and pH gradients across human erythrocytes and human peripheral lymphocytes. J . C d . Phvsiol. 99, 79-94. +

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Felbcr, S M., and Brand, M. D. (1983). Early plasma-membrane-potential changes during stimulation of lymphocytes by cuncanavalin A . Biochem. J . 83, 885-891. Finkcl. T. H., McDuEic. M . , Kappler. J. W., Marrack. P., and Cambier, J. C. (1987). Both immature and mature T cells mobilize Ca2+ in response to antigen receptor crosslinking. Nuture (Londofl) 330, 179- I8 1 . Freedman, M. H., Rafl', M. C , and Gompcrts. H. (1975). Induction of incrcased calcium uptake i n mouse T lyniphocytes by concanavalin A and its modulation by cyclic nuclcotides. Nuntrc. (London) 255, 378-382. Rkushima. Y., and Hagiwara, S . (1983). Volbagc-gated Ca2+ channels in mouse myeloma cells. Proc. Nnrl. Arud. Sci. U . S . A . S O , 2240-2242. Rkushima, Y., and Hagiwara, S. ( 1985). Currents carried by monovalent cationa through calcium channels in mouLe neoplastic B lymphocytes. J. Physiol. (London) 358, 255-284. Fukushinia, Y , , Hagiwara, S . , and Henkart, M . (1984). Potassium current in clonal cytotoxic T lymphocytes from the mouse. J. Physiol. (London) 351, 645-656. Gaidano, G . , Cihigo. D., Schena, M., Bergui, I-.. Treves, S . . Turrini, F., Cappio, F. C., and Bosia, A . ( 1989). Na /H exchange activation mediales the lipopolysaccharide-induccdproliferation of human R lymphocytes and is impaired in malignant B-chronic lymphocytic leukemia lymphocytes. J. Immunol. 142, 913-918. Gelfand, E. W., Cheung, R . K., and Grinskin. S . (1984). Role of inembrane potential in the regulation of lectin-induced calcium uptake. J . Cell. Phvsiol. 121, 533-539. Gell'and, E. W., Cheung. R . K., Mills, G . B . . and Grinstein. S.(1985). Mitogens trigger a calciumindependent signal for proliferation in phorbol ester treated lymphocytes. Nu!ure (London) 315, 4 19-420. Cielfand, E. W., Cheung, R. K . , and Grinstein, S. (1986a). Mitogen-induccd changes in Ca2+ permeability are not mediated by voltage-gatcd K channelb. J. H i d . Ckem. 261, I 1520+

+

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11523. Gelfand, E. W., Chcung, R. K., Grinstein, S . , and Mills,

G.B. (1986b). Characterization of the role

for calcium influx in mitogen-induced triggering of human T cells. Identification of calciumdependent and calcium-independent signals. Eur. J. Irnmunol. 16, 907-9 12.

Gelfand, E. W., Cheung, R . K . , Mills, G . H.,andGrinstein, S. (1987a). Rolc of membrane potential in the response of human T lymphocyka to phytohemagglutinin. J . Imrnrtnul. 138, 527-531. Gclfand, E. W., Chcung, R . K . , and Mills, G. B. (1987b). The cyclosporins inhibit lymphocyte activation at more than one site. J. Immunol. 138, 1 115-1 120. Gclfand, E. W., Cheung. R. K., Mills, G . B . , and Grinstein, S . (1988a). Uptake of extracellular CaZ and not recruilrricnt from internal stores is essential for T-lymphocyte proliferation. h r . J. Immunol. 18, 917-922. Gelfand, E. W., Chcung, K. K., and Grinstein, S . (198813). Calcium-dependent intracellular acidification dominates the pH response to mitogen in human T cells. J. Immunul. 140, 246-252. Gelfand, E. W., MacDougall, €3. K., and Grinstein, S . (1989). Independent regulation of Ca2+ entry and release from internal stores inactivated B cells. J. E.xp Med. 170, 315-320. Gill, D. L., Ueda, T., Chueh, S. H . , and Noel, M. W. (1486). Ca2+ release from endoplasinic reticulum is mediated by a guanine nucleotide regulatory mechanism. Nuturu (London) 320, 461-464. Gimencz-Gallego, G.,Navia, M. A , . Reubcn. I . P., Katz, G , M., Kaczorowski, G . J . , and Garcia. M. Z. (1988). Purification, sequence, and rnodcl structure of charyhdotoxin, a potent selective inhibitor of calcium-activated potassium channels. Pro<*.Natl. Actrd. Sri. U.S.A. 85, 33293333. Gold. M. R . , Jakway, J. P.. and DeFranco, A. L. (1987). Involvement of a guanine nuclcotidebinding component in membrane IgM-stimulated phosphoinositide breakdown. J. Immitnol. 139. 3604-3613. +

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receptor cross-linking results in a decrease in the plasma membrane potential of murine B lymphocytes. J. Exp. Med. 157, 2073-2081. Moretta, A,, Poggi, A , , Olive, D., Bottino, C., Fortis, C., Pataleo, G., and Moretta, L. (1987). Selection and characterization of T-cell variants lacking molecules involved in T-cell activation (T3 T-cell receptor, T44, and T11): Analysis of the functional relationship among different pathways of activation. Proc. Nail. Acad. Sci. U.S.A. 84, 1654-1658. Nisbet-Brown, E., Cheung, R. K., and Gelfand, E. W. (1985). Antigen-dependent increase in cytosolic free calcium in specific human T lymphocyte clones. Nature (London) 316, 545-547. Nisbet-Brown, E. R., Lee, J . W. W., Cheung, R. K., and Gelfand, E. W. (1987). Antigen-specific and nonspecific mitogenic signals in the activation of human T cell clones. J. Immunol. 138, 3713-3719. Nishizuka, Y. (1988). The molecular heterogeneity of protein kinase C and its implications for cellular regulation. Mature (London) 334, 661-665. Oettgen, H. C., Terhorst, C., Cantley, L. C., and Rosoff, P. M. (1985). Stimulation of the T3-T cell receptor complex induces a membrane-potential-sensitive calcium influx. Cell 40, 583-590. O’Flynn, K . , Linch, D. C., and Tatham, P. E. R. (1984). The effect of mitogenic lectins and monoclonal antibodies on intracellular free calcium concentration in human T lymphocytes. Biochem. J. 219, 601-606. O’Flynn, K . , Zanders, E. D . , Lamb, J. R., Beverley, P. C. L., Wallace, D. I., Tatham, P. E. R., Tax, W. J. M.. and Linch, D. C. (1985). Investigation of early T-cell activation: analysis of the effect of specific antigen, interleukin 2 and monoclonal antibodies on intracellular free calcium concentration. Eur. J. Immunol. IS, 7-1 I . Ohashi, P. S . , Mak, T. W., Van den Elden, P., Yanagi, Y., Yoshikai, Y., Calman, A. F., Terhorst, C . , Stobo, J. D., and Weiss, A. (1985). Reconstitution of an active surface T3iT-cell antigen receptor by DNA transfer. Mature (London) 316, 606-609. Paradiso, H. M., Tsien, R. Y., and Machen, T. E. (1987). Digital image processing of intracellular pH in gastric oxyntic and chief cells. Nature (London) 325, 447-450. Partain, K . , Jensen, K . , and Aldo-Benson, M. (1986). Inositol phospholipid and intracellular calcium metabolism in B lymphocytes stimulated with antigen. Biochem. Biophys. Res. Commun. 140, 1079-1085. Pezzutto, A , , Dorken, B., Rabinovitch, P. S . , Ledbetter, J. A,, Moldenhauer, G., and Clark, E. A. (1987). CD19 monoclonal antibody HD37 inhibits anti-immunoglobulin-induced B cell activaI Immunol. . 138, 2793-2799. tion and proliferation. . Pozzan, T., Arslan, P . , Tsien, R . Y., and Rink, T. J. (1982). Anti-immunoglobulin, cytoplasmic free calcium and capping in B lymphocytes. J . Cell Biol. 94, 335-340. Rabinovitch, P. S . , June, C. H., Grossmann, A,, and Ledbetter, J. A. (1986). Heterogeneity among T cells in intracellular free calcium responses after mitogen stimulation with PHA or anti-CD3. Simultaneous use of indo-1 and immunofluorescence with flow cytometry. J . Immunol. 137, 952-961. Ransom, J. T., and Cambier, J. C. (1986). B cell activation. V11. Independent and synergistic effects of mobilized calcium and diacylglycerol on membrane potential and I-A expression. J. Imrnunol. 136, 66-72. Ransom, I. T., Hams, L. K . , and Cambier, J. C. (1986). Anti-Ig induces release of inositol I ,4,5trisphosphate which mediates mobilization of intracellular Ca2+ stores in B lymphocytes. J. Irnmunol. 137, 708-714. Rink, T. J., Montecucco, C., Hesketh, T. R., and Tsien. R. Y. (1980). Lymphocyte membrane potential assessed with fluorescent probes. Biochim. Biophys. Acta 595, 15-30. Rogers, J.. Hesketh, T. R., Smith, G. A,, and Metcalfe, J. C. (1983). Intracellular pH of stimulated thymocytes measured with a new fluorescent indicator. J. B i d . Chem. 258, 5994-5997. Roifman, C. M., Mills, G. B., Cheung, R. K., and Gelfand, E. W. (1986). Mitogenic response of

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human thyrnocytes: ldentification of functional Ca2 +-dependent and independent signals. Cfin. Ex/). Immuiiol. 66, 139- 149. Roifman, C. M . , Mills, G . B . , Stewart, D., Cheung, R. K . . Grinstein, S . , and Gelfand, E. W.

(1987). Response of human B cells to different anti-immunoglobulin isotypes: absence of a correlation between early activation events and ccll proliferation. Eur. J . Irnmunol. 17, 17371742. Ruaoff, P M . , and Cantlcy, L. C, (1983). Incrcasing the intracellular Na+ concentration induces differentiation in a pre-B lymphocyte cell line. Proc. N u / / . Acad. Sci. U.S.A. 80, 7547-7550. Rosoff. P. M., and Cantlcy, L. C. (1985a). Stimulation of the T3-T cell receptor associated Caa+ intlux enhances the activity ofthe Na 1H exchanger in a leukemic human T cell line. J. Riol. C.’hettl. 260, 14053- 140S9. RoioE, P M . and Cantley, L. C. ( I 9XSb). Lipopolysaccharidc and phorbol esters induce differentiation hut have opposite effects on phosphatidylinositol turnover and CaZ+ mobilization in 70213 prc-B lymphocytes. J . B i d . Chem. 260, 9209-9215. Rosoff, P. M . , and Terrcs, G. (1986). Cyclosporine A inhibits Ca2+-dependent stimulation of the Na+ / H + antiport in human T cells. J. Cell B i d . 103, 457-463. Rosoff, P. M., Stein, L . F . , and Cantley. L . C. (1984). Phorbol esters induce dikrentiation rn a preB lymphocyte ccll line by enhancing N a + / H + exchange. J . B i d . Chem. 259, 7056-7060. Schimmel, S . D.. and Hallam, T. (1980). Rapid alteration in CaZ+ content and fluxcs in phorbol-12inyristate 13-acetate treated myohlasts. Biochem. Biophys. Res. Commun. 92, 624-630. Schlichter, L . . Sidell. N . , and Hagiwara, S. (1986). K + channels are expressed early in human Tcell developnicnt. Proc. Not/. Acud. Si,i. U.S.A. 83, 5625-5629. Segel, G. B . , Gordon, B. R., Licbtnian, M . A , , Hollander, M . M., and Klempcrer, M . R. ( 1979). Exodus of 42K and XhRb from rat thymic and human blood lymphocytes exposed to phytohemagglutinin. I. CeN. Physiol. 1 0 , 109-1 18. Smith, K . A . ( 1 9 X X ) . Interleukin-2: Inccption. impact, implications. Science 240, 1169-1 176. Stanton, T. H., Maynard, M . , and Bomsztyk, K . (1986). Effect of interleukin-l on intracellular concentration of sodium, calcium, and potassium in 702/3 cells. J . Biol. Chem. 261, 5699+

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CmRENT TOPICS IN MEMBRANES A N D TRANSPORT, VOLUME 35

Chapter 8

Cytoplasmic Calcium in Phagocyte Activation FRANCESCO DI VIRGILIO,* OLLE STENDAHL, f DIDIER PITTET,$ P. DANIEL LEW,,$ AND TULLIO POZZANj *C.N.R. Center for the Study of the Physiology of Mitochondria and Institute of General Pathology 1-35131 Padovu, Italy TDepartment of Medical Microbiology University of Linkoping S-581 85, Linkoping, Sweden $Division of Infectious Diseases University of Genevu CH-121I Geneva, Switzerland §Institute of General Pathology University of Ferrara Ferrara, Italy

I. 11.

Introduction Measuring and Manipulating [Ca2+], in Intact Phagocytes A. Indicators of [Ca2+li B . Methods for Trapping Ca2 Indicators into the Cytoplasm 111. Basic [Ca2+ 1, Homeostatic Mechanisms in Phagocytes A. Systems for Ca2+ Extrusion across the Plasma Membrane B . Intracellular Ca2 Sequestration: The Calciosome IV. The Rise and Fall of [Ca2+],.Changes in [Ca2+Ii and Phagocyte Activation A. Studies in Cell Populations B. Studies in Single Cells V. The Role of Ca2+ as a Second Messenger Is Questioned in Phagocytic Cells A. Ca2+ Dependency of Metabolic Responses in Phagocytes B. Ca2+ Dependency of Motility in Phagocytes VI. Conclusions: Ca2+ May Not Be All, After All References +

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Copyright 0 1990 by Academic Press. Inc. All right, uf rcpruduction in any lum reserved

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1.

INTRODUCTION

Changes in the cytosolic free calcium concentration ([Ca2 ],)l have evolved as a key intracellular messenger system to couple external stimuli to a variety of complcx cellular responses (Campbell, 1983). In phagocytic cells (i.e., neutrophils and macrophages) changes in [Ca2 Ii follow stimulation of several receptors and are thought to be involvcd in the rcgulation of adherencc, chemotaxis, phagocytosis, degranulation, and production of toxic oxygen metabolites (Treves and Pozzan, 1988; Di Virgilio er d., 1989b). The introduction of new, noninvasive techniques to monitor [CaZ+li,not only in cell populations but also in singlc cells, has extended greatly our knowledge of the mechanisms underlying [CaZ li homeostasis in resting and activated cells (Tsien et al., 1982; Campbell and Hallet, 1983; Grynkiewicz rt al., 1985). However, the role of changes in ICa2 +Ii in many cellular functions, in particular during the multiple and complex processes of oriented locomotion and phagocytosis, is still controversial. Growing evidence clearly argues against the simplistic hypothesis that an increase in [Ca2+I i is a necessary and sufficient stimulus for triggering phagocyte activation (Pozzan er ul., 1983; Di Virgilio and Gomperts, 1983; Lew ut ul.. 1984; Di Virgilio o r ul., 1984; Treves and Pozzan, 1988). It is now clear that different synergistic or antagonistic intracellular messengers are contemporarily or sequentially generated by receptor ligation, a rise in [Ca2+li being only one of them (Berridge, 1984, 1987; Bemdge and lrvine, 1984). The crucial qucstion to be answered in phagocyte biology is whether [Ca2+lichanges are necessary for, or merely associated with, cellular responses. Several reviews dealing with [Ca2 Ii homeostasis in phagocytic cells have appeared (Treves and Pozzan, 1988; Snyderman and Uhing, 1988; Di Virgilio c ~ t (I/. , l989b). The reader is referred to them and to the original articles cited for a comprehensive discussion of the status of the art in this field. In this contribution, we have aimed to summarize briefly what we consider the most significant progresses. +

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II. MEASURING AND MANIPULATING [Ca2+Ii IN INTACT PHAGOCYTES Unlike a few years ago, when a serious problem was a lack of suitable techniques for measuring [Ca2 li, today two major alternatives are available to +

'Abbreviations: [Caz I,. cytosolic free calcium concentration; PMA, phurbol 12-myristatc I Racetate; DMSO, diiuethyl sulfoxide; ConA, concanavalin A; CAMP, cyclic AMP; EGTA, ethyleneglycol-bis(~-aminocthylether)-N,N,N',N'-tctraacetic acid; EDTA, ethylcnediaminetetraacetic acid; Ins I .4,5P3, inositol I ,4,5-trisphosphate; Ptdlns, phosphatidylinositol; PtdIns4P, phosphatidylinositol 4-phosphate; PtdIns4,5P2, phosphatidylinositol 4,5-hisphosphate; ER, endoplasmic reticulum; SR, sarcoplasniic rcticulum; TKH, thyrotrupin-releasiny hormone. +

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Ca2+ researchers: photoproteins and carboxylate dyes. A wider range of possibilities is offered for loading the Ca2+ probes into the cell cytoplasm. A. Indicators of [Ca2+], Ca2 -sensitive photoproteins are a family of coelenterate proteins endowed with the unique property of undergoing decomposition with emission of photons on interaction with calcium ions (Blinks et ul., 1978). The amount of photons emitted is a function of [Ca2+].The most commonly used Ca2+-sensitive photoproteins are aequorin and obelin , extracted from the jellyfish Aequorea forskoleu and from the hydroid Obelia geniculutu, respectively (Thomas, 1982). The main advantages of photoproteins are the negligible extra Ca2 buffering capacity provided and the high sensitivity of the technique. The major disadvantages so far have been the painstaking loading procedure and the troublesome calculations needed for accurate quantitative determination of [Ca2+IIi(Blinks et al., 1978; Thomas, 1982). quin2 is the prototype of the Ca2+ indicators of the tetraipentacarboxylate family (Tsien et al., 1982) and had been extensively used, particularly in neutrophils, until 1986. Since late 1985, fura-2 and indo-1 (Grynkiewicz et al., 1985) have become commercially available, and these two new dyes are slowly replacing quin2 in most applications. Fura-2 and indo- 1 have several advantages over quin2. The higher fluorescence and quantum yield, the lower affinity for Ca2 , and the larger shift of the excitation spectrum on Ca2 binding make the use of the second generation dyes ideally suited for measuring [Ca2+Ii in cell suspensions and in single cells (Cobbold and Rink, 1987; Malgaroli et al., 1987). Tsien and co-workers introduced two new Ca2+ indicators, fluo-3 and rhod-2 (Minta et ul., 1989), whose main advantage is an absorbance peak in the 500-nm region, a property that abolishes the potential toxic effects of UV irradiation. A communication reporting the application of fluo-3 to the measurement of [Ca2 Ii in red blood cells has already appeared (Hinds et al., 1989), and we anticipate that these dyes will also encounter wide general acceptance. The extraordinary success of the tetraipentacarboxylate Ca2+ indicators over the past years is essentially due to the ease of intracellular trapping, the straightforward calibration of the signal in terms of [Ca2+Ji,and the simple and inexpensive equipment needed for measurement (Capponi et al., 1986; Rink and Pozzan, 1985; Milani et al., 1989). Moreover, fura-2 and indo- I are more suitable than photoproteins for quantitative image analysis of [Ca2+Iiand, unique among [Ca2+Ii indicators, can also be used for fluorescence-activated cell sorting. Tetraipentacarboxylate dyes, on the other hand, present a number of drawbacks, notably significant Ca2 buffering capacity (higher with quin2), extracytoplasmic trapping, leakage from intact cells, and shifts in the fluorescence spectrum dependent on the viscosity of the intracellular environment (Tsien et ul., 1982; Malgaroli et ul., +

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1987; Meldolesi et u l . , 1987). It is worth mentioning that the Ca2+ buffering capacity of quin2, often considered the major disadvantage of this probe, can be exploited under certain experimental conditions and for selected purposes in the investigation of cellular responses (Di Virgilio et ul., 1984; Lew et ul., 1984, 1986).

B. Methods for Trapping Ca2+ Indicators into the Cytoplasm Photoproteins are high molecular weight polypeptides and therefore impermeant across the plasma membrane. Historically, the traditional technique for photoprotein trapping has been microinjection. Although this technique has developed with a great degree of sophistication, it is still rather laborious, and at the best, a few hundred cells can be injected in one day. Thus direct comparison of biochemical data with [Ca2+Iimeasurements in the very same cells is usually not possible. Numerous alternative approaches have developed to bypass the microinjection step. Among them scrape-loading (McNeil et al., 1984; Steinberg e f ul., 1987b), reversible permeabilization of the plasma membrane (Steinberg et ul., 1987a; Di Virgilio et ul., 1988b), and fusion with photoprotein-loaded erythrocyte ghosts (Hallet and Campbell, 1982) are the most popular. A literature search in this field, however, reveals that very few laboratories have employed these techniques for loading Ca2+ probes into phagocytes, indicating that they are still far from being routinely applicable without special skill. A very elegant alternative has been suggested by Campbell and co-workers (Campbell er a l . , 1988). These authors succeeded in incorporating mRNA purified from Obeliu genirulutu into human neutrophils by using uptake of mRNA-loaded immunoliposomes. Translation of the mRNA within the neutrophils led to the synthesis of apo-obelin which could be regenerated to its active form by the addition of the prosthetic group coelenterazine to the neutrophil suspension. Obelin thus generated appeared to be cytosolic since challenge of the neutrophils with agonists known to increase [Ca2+ I i elicited the expected increase in obelin chemiluminescence (Campbell et d., 1988). The techniques mentioned previously, with the exception of mRNA loading, are in principle also applicable to cytoplasmic trapping of the new fluorescent Ca2+ probes synthesized by Tsien and colleagues. The most commonly used method for incorporating these dyes, however, is incubation of the cells with the hydrophobic, intracellularly cleavable, acetoxymethylester derivative of the dye (Tsien et ul., 1982; Rink and Pozzan, 1985; Capponi et al., 1986; Tsien and Pozzan, 1989). As an alternative, in murine macrophages and macrophage tumor cell lines, such as 5774 cells, fluorescent Ca2+ indicators can be loaded by reversible permeabilization of the plasma membrane with extracellular ATP (Steinberg et ul., 1987a). This nucleotide, in its tetra-anionic form ( A T P . ) opens in the plasma membrane large pores permeable to solutes of MW of up to

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830 (Cockcroft and Gomperts, 1979; Steinberg et al., 1987a). Rapid resealing of these lesions can be achieved simply by increasing the concentration of divalent cations of the medium (i.e., by chelating ATP-). Reversible, ATP'--dependent permeabilization has not been used extensively in studies of [Ca2+Iihomeostasis mainly for two reasons: (1) not all phagocytic cells so far tested can be permeabilized according to this procedure; for example, while murine macrophages are easily permeabilized by ATP- , human neutrophils are not; and (2) it is more expensive than ester loading and some cell types are not robust enough to survive after resealing (F. Di Virgilio, unpublished observations). A relevant advantage of A T P - -dependent permeabilization is that molecules other than Ca2 indicators (e.g., other impermeant dyes, hydrophilic drugs, nucleotides, and small peptides) can be trapped at the same time (Gomperts, 1983; Steinberg et al. , 1987a; Di Virgilio ef al., 1988b). Figure 1, for example, shows an application of reversible A T P - permeabilization of the plasma membrane of mouse macrophages to the study of phagocytosis. Plated macrophages were first permeabilized with 5 mh4 ATP in the presence of EGTA and of the fluorescent anionic dye Lucifer Yellow then resealed and presented with IgG-coated red blood cells. As shown by the fluorescent illustration (Fig. 1b), permeabilization allowed free access of Lucifer Yellow (and EGTA) to the cytoplasm, with the exception of the erthrocyte-containing phagosomes which appear as black dots. The implication of this experiment for the understanding of the mechanism of particles engulfment will be discussed later on (see Section IV). +

a

b

FIG. 1. Macrophages loaded with Lucifer Yellow and EGTA by reversible ATP4--penneabilization ingest IgG-coated red blood cells. Mouse macrophages were permeabilized with 5 mM ATP, loaded with Lucifer Yellow and EGTA, and presented with IgG-coated erythrocytes as described in Steinberg er al. (1987a) and Di Virgilio E I ul. (1988b). (a) Phase; (b) fluorescence.

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Dye loading as the acetoxymethylcster derivative still remains the technique of choice in most cases. Several reviews concerned with the most important aspects of this approach have appeared. (Capponi et ul.. 1986; Rink and Pozzan, 1985; Tsien and Pozzan, IY8Y; Milani rt d., 1989; Di Virgilio e t a l . , 1989b). Suffice it to say that, among phagocytes, neutrophils are one of the cell types in which ester loading is more efficient and extracytosolic trapping and leakage are nearly negligible. Macrophages and related cell lines, on the other hand, require the addition of inhibitors of organic anion carriers throughout loading and experimentation to avoid sequestration and leakage of the Ca2+ indicators (Di Virgilio et al., 1988a,c, 1989a). “Leaky cells” have become a popular modcl for the investigation of [Ca2+ li homeostasis. The plasma membrane of most cells can be made permeable selectively, without impairing the permeability of intracellular membranes, by treatment with detergents specific for cholesterol, such as saponin or digitonin (Brooks and Treml, 1983; Dunn and Holz, 1983). Alternatively, cells can be exposed to brief pulses of high voltage discharge (Baker and Knight, 1978). This latter procedure punches small holes in the plasma membrane that admit molecules of a molecular weight of up to a few thousand but, as a significant advantage over detergents, does not allow leakage of most cytoplasmic proteins. Finally, cells can be exposed to toxins, such as streptolysin-0 or staphylococcal u toxin, that make nonselective holes (6 = 3 nm) in the plasma membrane. A basic assumption in leaky cells is that by virtue of the large lesions generated in the plasma membrane, ICa2+Ii is the same both extracellularly and intracellularly. In fact, this is not always the case, as shown by Biden et al. (1986) in electrically permeabilized GH, cells. Leaky cells are also advantageous with respect to classical experiments with subcellular fractions in that organelles can be studied in situ, and yet they are accessible to pharmacological agents. Therefore, studies in leaky cells complement nicely those in intact cells. New electrophysiological approaches have also been applied to phagocytes (Gallin and Sheehy, 1988). The patch clamp technique has been used to investigate agonist-induced [Ca’ + I i rises in neutrophils (von Tscharner et al., I986a) and ion changes accompanying phagocytosis in macrophages (Randriamampita and Trautmann, 1987; lnce et al., 1988; Gallin and Sheehy, 1988).

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BASIC [Ca2+Ii HOMEOSTATIC MECHANISMS IN PHAGOCYTES

A. Systems for Ca2+ Extrusion across the Plasma Membrane In resting neutrophils and macrophages, [Ca2+Iiis about 100-150 nM (i.e., four orders of magnitude lower than in the extracellular milieu). Interestingly,

8. CYTOPLASMIC CALCIUM IN PHAGOCYTE ACTIVATION

185

ICa2+], of the highly undifferentiated HL60 leukemia cells or of the macrophage-like tumor cell line 5774 is indistinguishable from that of their normal counterparts (Lew et al., 1986; Di Virgilio et al., 1988a,b). Furthermore, treatment of HL60 cells with differentiating agents, such as DMSO or PMA, does not alter [Ca2+1, acutely (within 10 min of addition) or after days in culture, at a time when this leukemic cell line has acquired a differentiated, neutrophil-like phenotype (T. Pozzan, unpublished observations). In macrophages, as well as in all other eukaryotic cells, low [Ca2+Ii is the result of a complex equilibrium among influx from the external environment, intracellular buffering, organellar sequestration, and efflux across the plasma membrane. It is worth pointing out that the long-term control of [Ca2+], is governed by the equilibrium between influx and efflux across the plasma membrane and that intracellular organelles can only play a role as short-term regulators. While in many excitable cells the Na+/Ca2+ antiport is one of the main systems for Ca2+ extrusion (Baker, 1972), no conclusive evidence exists for a significant role for this exchange mechanism in phagocytes. Rather, various groups have reported the presence of a Ca2 -ATPase in plasma membraneenriched fractions prepared from neutrophils and macrophages (Lew and Stossel, 1980; Lagast et al., 1984; Volpi et al., 1983). The Ca2+-ATPase of neutrophil plasma membrane has a high affinity for Ca2+ ( K , between 0.2 and 0.4 pA4) and for ATP (K,,, between 0.2 and 0.36 pA4), is inhibited, unlike that of intracellular organelles, by micromolar concentrations of vanadate (Lagast et al., 1984) and its V,,,, can be doubled by activation of protein kinase C (Lagast et al., 1984). Whether this latter effect depends on direct phosphorylation of the enzyme or is the end result of a chain of protein kinase C-activated reactions has not been established yet. +

B. lntracellular Ca2+ Sequestration: The Calciosome Ca2 buffering power of the soluble cytoplasm (i.e., that independent of transport processes) has been studied in detail by von Tschamer et al., ( 1 986b). During the past few years much attention has been devoted to the Ca2+ buffering capacity due to intracellular organelles. Phagocytes have become the experimental model of choice for these investigations (Prentki et al., 1984; Volpe et al., 1988). Leaky neutrophils can reduce ambient [Ca2+] to 200 nM, a level very close to that measured in intact cells. Uptake by at least two types of vesicular structures contribute to the setting of [Ca2+]: mitochondria and a microsomal store dependent on ATP for Ca2 accumulation (Prentki et al., 1984). Given the small number of mitochondria present in neutrophils, this result was rather unexpected. However, mitochondria appear to play a role only when [Ca2+] is above 600-700 nM (Prentki et al., 1984) as expected on the basis of the low +

+

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ET AL.

affinity for Ca2 of the mitochondria1 uniport (Carafoli, 1987). The ATP-dependent microsomal Ca2 store is endowed with two properties relevant to the regulation of [Ca2 Ii homeostasis under physiological conditions: (1) it can pump Ca2 ’ at physiological [Ca2 ’ 1;; and ( 2 ) it is the target of Insl,4,5P,, the intracellular messenger thought to be responsible for agonist-induced mobilization of intracellular Ca2+ (Berridge, 1984, 1987; Berridge and Irvine, 1984). In early studies this microsomal store was suggested to be identifiable with either endoplasmic reticulum (ER) or a specialized portion of it, for example, rough EK (Streb et al., 1983, 1984; Prentki rt ul., 1984). Recent experiments from our laboratories suggest that a new organelle named “calciosome” by our group, may be responsible for most of the high affinity intracellular Ca2+ pumping activity and Ins 1,4,SP,-dependent Ca2 release (Volpe et ul., 1988; Hashimoto rr d . , 1988; Pozzan et al., 1988; Krause et ul., 1989). The identification of calciosomes as unique subcellular entities is based on both subcellular fractionation and on the immunolocalization in the same organelle of a calsequestrin-like Ca’ -binding protein and of a Ca2 -ATPase, two bona fide markers of a Ca2 store (Volpe et d . , 1988). By Pereoll-density centrifugation and free-tlow electrophoresis of homogenates of HL60 cells, a light density fraction containing 7080% of total Insl,4,SP3-sensitive, Ca2 accumulating activity was obtained (Volpc et al., 1988). This fraction was selectively enriched with a 60 kDa Ca2 binding protein that shares a number of properties with the skeletal muscle calsequestrin (CS), including immunological reactivity (Volpe et al., 1988). Immunofluorescence with anti-CS antibodies (Abs) demonstrated in both HL60 cells and neutrophils that this antigen, in resting cells, is distributed homogeneously throughout the cytoplasm, while the nucleus is patently negative (0. Stendahl, unpublished observations). In HL60 cells immunoelectronmicroscopy with gold-tagged anti-CS Abs revealed the presence of numerous CS-positive vesicles of an approxiniate diameter of 50-250 nm, clearly distinct from other morphologically identifiable structures (Volpe et ul., 1988). Similar results were also obtained in cells from liver and pancreas (Hashimoto et ul., 1988). By double labeling with anti-CS and anti-ATPase Abs, it was also shown that, in these tissues, the two antigens colocalize in about 50% of the cases. Only a minority ( 5 % ) of the organelles were labeled with CS only (Hashimoto et al., 1988). In no cell type have bona fide ER elements been detected by either Abs. Conversely, anti-cytochrome p-450 Abs (i.e., Abs specific for a typical ER marker) did not stain CS or Ca2 -ATPase-positive vesicles (Volpe et al., 1988; Hashimoto et al., 1988). On the basis of these observations we have proposed that calciosomes are the functional equivalent of sarcoplasmic reticulum (SR) in nonmuscle cells. This conclusion is further supported by the observation that the Ca2 -ATPase of microsomal stores in nonmuscle cells is both structurally (similar molecular weight and inimunoreactivity) and functionally (low sensitivity to vanadate) correlated to that of SR. Evidence suggesting that calciosomes are the +

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physiological target of Ins 1,4,5P, is only indirect so far. The most important clue remains the finding that in subcellular fractions from HL60 cells, Ca2 -pumping activity copurifies with 1nsl,4,5P3 binding, Ins 1,4,5P,-dependent Ca2 release, and a CS-like protein (Volpe et al., 1988; D. Pittet, and P. D. Lew, unpublished observations). Identification of the calciosomes as the physiological Ins 1,4,5P, target could allow the study of a crucial problem in phagocyte physiology (i.e., whether local [Ca2 li gradients are generated because of the redistribution of Ca2 -releasing organelles or as a consequence of the local synthesis of second messengers). +

+

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IV. THE RISE AND FALL OF [Ca2+],. CHANGES IN [Ca2+], AND PHAGOCYTE ACTIVATION A. Studies in Cell Populations With the important exception of tumor promoters, all soluble stimuli of phagocyte responses (chemoattractants, Abs, arachidonic acid metabolities, extracellular nucleotides, Ca2+ ionophores, etc.) cause a rise in [Ca2+Ii(Di Virgilio and Gomperts, 1983; Pozzan et al., 1983; Treves et al., 1987; Treves and Pozzan, 1988; Greenberg et al., 1988; Di Virgilio et al., 1989b). Increases in [Ca2+J i have a dual origin; mobilization from intracellular stores and influx from the extracellular medium. The relative importance of the two sources depends on the cell type, the concentration and nature of the agonist, and the incubation conditions. The [Ca2+liresponse when particulate stimuli are used is more complex. Latex particles were ingested by both neutrophil suspensions and neutrophil monolayers without detectable increases in [Ca2 Ii whether obelin or quin2 were used as (Ca2+Iiindicators (Campbell and Hallet, 1983), but ingestion of IgG or C3b/bi-coated yeasts was accompanied in quin2 loaded human neutrophils by a long lasting increase in [Ca2+Ii that preceded or occurred simultaneously to particle engulfment (Lew et ul., 1985; Sawyer et al., 1985). In inflammatory mouse peritoneal macrophages plated on glass coverslips and loaded with either aequorin, fura-2, or quin2, on the other hand, phagocytosis of IgG-coated erythrocytes did not increase [Ca2+Ii(McNeii et a l . , 1986; Di Virgilio et al., 1988b), in striking contrast with observations by the same group and by other investigators in the macrophage cell line 5774 (Young et al., 1984; Di Virgilio et al., 1988b). Challenge of 5774 or inflammatory macrophage suspensions with IgG-coated erythrocytes always resulted in a rise in [Ca2+li (Di Virgilio et al., 1988b). The reason for this variability between cell suspensions and cell monolayers is not completely understood and may depend on the heterogeneity, asyncronicity, and subcellular localization of the [Ca2 Ii response to particulate agonists. +

+

I aa 1 . Ca'

FAANCESCO DI VIRGIL10 ET AL.

'

M O B I L ~ Z ArlON FROM I N T R A C E L L U L A R S T O R E S

To date there is a general consensus of opinion on the basic mechanism of Gal+ mobilization in eukaryotic cells and in phagocytes as well. The role of In5 1,4,5P, generated from Ptdlns4,5P2 hydrolysis is undisputed and is bascd on an impressive amount of data. To mention only the most relevant evidence: ( 1 ) lnsl,4,SP, is generated rapidly on ligation of Ca2 -mobilizing receptors (Di Virgilio e r al., 1985; Lew c't a l . , 1986); ( 2 ) the threshold concentration of agonist for lnsl,4,SP3 formation and Ca2 mobilization is very similar (Lew et a/., 1086); (3) submicromolar Ins I ,4,5P, concentrations can induce release of Ca2 from menibrane-bound stores in permeabilized phagocytes as well as in micro1984; Spat rt al., 1986; Volpe el d., 1988); (4) soma1 fractions (Prentki et d., high affinity lnsl,4,5P3 receptors are found in pernicabilized neutrophils (Spat ct c d . , 1986); and ( 5 ) inhibition of lnsl,4,5P3 formation, for example, by pretreatment with pertussis toxin, results in inhibition of CaZ+ mobilization (Bockoch and Gilman, 1984; Molski et d., 1984; Krausc et NI., 1985). Two main issues remain to be solved: ( 1) the nature of the lns1,4,5P3-sensitive intracellular CaL store; and (2) whether lnsl,4,5P3 formation is rapid enough to account for the tast kinetics of Ca' mobilization. With respect to the former point, there is good evidence, as described in detail previously, indicating that the Ins 1,4,5P,sensitive Calf store in phagocytes may be a novel organelle, the calciosorne. With respect to the latter point, it should be noted that in human neutrophils stimulated by high Met-Leu-Phe concentrations, the onset of [Ca2 lirise due to CU' release from intracellular stores occurs within 0.2-0.3 sec from the addition of the agonist (Wymann et ul., 1987). The earliest changes in Ins1 .4,5P,3 concentration have been detected after 1 sec of stimulation of GH,C, cells with TRH (Xashjian et ( I / . , 1987). We are not aware of any measurement of Insl,4,5P, performcd in a subsecond time scale in phagocytes. +

+

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2. C a 2 + INFLUX The rncchanism of receptor-regulated Ca2 influx is more controversial. The evidence in favor of a Ca2 influx in phagocytes can be summarized as follows: ( I ) uptake of 45Ca is stimulated by surface-acting agonists (Naccache rt al., 1977); (2) the kinetics of the ICa2+li increase induced by receptor ligation is biphasic, the second, slower and prolonged phase being observed only in thc presence of extracellular Ca2 (Andersson el u l . , 1987); (3) receptor-directed agonists increase severalfold the influx of Mn2+ (Andersson rt a/.,1987; Merrit et ul., 1989; Pittet et u I . , 1989); and (4) impermeable divalent and trivalent cations, such as La-< , Co2+, and Ni2+ , completely inhibit Mn2+ influx and the second delayed phase of [Ca2 li rise (Andersson rt al., 1987; Merrit et d., 1989). The mechanism underlying Ca2 influx is still unclear. Several hypoth+

+

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esis, none of which has yet been conclusively proved, have been proposed in the past. Weissmann and co-workers (Sheran et al., 1982) suggested that leukotriene B, (LTB,) and phosphatidic acid may function as Ca2 ionophores. The former possibility seems to be excluded on the basis of two observations: (1) LTB, is coupled via a specific membrane receptor to PtdIns4,5P2 hydrolysis and therefore to Ins1,4,5P3 generation; and (2) exogenous LTB, is one of the weakest stimulators of Ca2 influx in neutrophils (Lew et al., 1987; Merrit et al., 1989). The latter possibility has been formally excluded by Holmes and Yoss (1983). Furthermore, we have observed that [ 3H]phosphatidic acid formation in 13H]glycerol labeled neutrophils is nearly completely suppressed by compound Ro5922, an inhibitor of diacylglycerol kinase, without impairement of Met-Leu-Pheinduced Ca2 influx (S. Treves and T. Pozzan, unpublished observations). However, Weissmann's group has shown that, in artificial liposomes, phosphatidic acid has a weak Ca2+ ionophoric activity (Blau and Weissmann, 1988), thus providing new data in favor of the original model. The most popular hypothesis for receptor-regulated Ca2 influx in phagocytes and in other eukaryotic cells is that of a second messenger operated channel (SMOC). Ca2+ itself is obviously a candidate for such a role. Von Tscharner et al., (1986a) have shown, using inside-out patches of neutrophil plasma membrane, that [Ca2+Ii itself can regulate the opening probability of a nonspecific cation channel. This observation is consistent with the report by Pittet et al. (1989), showing that extracellular Ca2 potentiates met-Leu-Phe-induced influx of Mn2+ in DMSO-differentiated HL60 cells. Nasmith and Grinstein (1987a) have challenged the role of [Ca2+Ii in the modulation of Ca2 influx on the basis of the observation that, in the presence of Met-Leu-Phe, there is a relevant Ca2+ influx even when [Ca2+ I i is at resting level. This finding has been confirmed by Merrit et al. (1989) who showed an agonist-dependent, Ni2+inhibitable influx of Mn2+ at or below resting [Ca2+Ii. An alternative and currently fashionable model for the regulation of Ca2 SMOCs maintains that lnsl,4,5P3 and/or Insl ,3,4,5P, might act as the intracellular modulators of these channels. The results of Pittet et al. ( 1 989) argue in favor of a primary role of both a [Ca2 li elevation and Insl ,3,4,5P, production, while the kinetics of Insl,4,5P3 formation seem incompatible, at least in differentiated HL60 cells, with a regulation of Ca2+-SMOCs by this compound. The issue of the modulation of agonist-induced Ca2+ influx appears as confused in phagocytes as in all other cell types, and no firm conclusion can be drawn at this time. +

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3 . FEEDBACKMECHANISMS

a. Protein Kinase C . Great attention has been devoted over the past few years to the feedback control of Ca2+ release from intracellular stores and Ca2+

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influx. It has become increasingly clear that the ubiquitous, Ca2+-activated enzyme protein kinase C (Kikkawa and Nishizuka, 1986) plays a pivotal role in the feedback regulation of agonist-induced [Ca2+li increases (Vicentini et a/., 1985; Naccache et ul., 1985). Protein kinase C is activated by tumor promoters, compounds largely used in the investigation of responses of phagocytic cells long before the identification of protein kinase C as their target. PMA, the most commonly used tumor promoter, stimulates several phagocyte responses (secretion, NADPH oxidase activation, and phagocytosis via the complement receptor) without increasing lCa2+Ii. In fact, it inhibits the increases in [Ca2+Iiinduced by other stimuli (Lagast et al., 1984; Naccache et al., 1985). Under extreme nonphysiological conditions (absence of Na from the extracellular medium), phorbol esters can induce a small rise in [Ca2~+li (Nasmith and Grinstein, 1987b). Fallman et u l . , (1989) have shown that PMA can also cause a slow increase in lnsl,4,5P3 levels in neutrophils, but the mechanism underlying this cffect remains obscure. Feedback regulation by protein kinase C is exerted at difierent levels and by a number of mechanisms: ( 1 ) protein kinase C stimulation increases the activity of the plasma membrane Ca2+-ATPase (Lagast et u / . , 1984); (2) by causing depolarization of plasma membrane potcntial , protein kinase C stimulation reduces the driving force for Ca2 entry (Di Virgilio et ul., 1987); ( 3 ) depolarization of plasma membrane potential in neutrophils also reduces Ins 1,4,5P, generation and therefore mobilization of Ca2 from intracellular stores (Di Virgilio et al., 1987; D. Pittet and D. P. Lew, unpublished observations); and (4) by acting either on the G protein coupling surface receptors to phospholipase C or directly on the phospholipase C itself, protein kinase C-dependent phosphorylation(s) reduces the generation of lnsl,4,5P3. +

+

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b. Cyclic AMP. Another important feedback mechanism for the regulation of [Ca2 li in phagocytes involves changes in the intracellular concentration of cyclic AMP (CAMP). Stimuli that increase intracellular CAMPconcentration are potent inhibitors of metabolic activation of phagocytes (Weissmann et a/., I97 I ; Smolen et ul., 1980; Simchowitz et al., 1980). The intracellular pathway that mediates this inhibition is not known, but there is evidence that it may involve a decreased agonist-dependcnt generation of Insl,4,5P, and DAG, and consequently smaller increases in [Ca2 li and a reduced activation of protein kinase C (De Togni t.t ul., 1984; Della Bianca et a / . , 1986a). Although an inhibition of the phosphoinositidase by CAMP was proposed some years ago (RittenhouseSimmons, 1979; Watson ct d.,l9X4), it is not known whether this effect is exerted directly or rather is mediated via an interaction with a regulatory unit (e.g., a G protein). An additional site of inhibition by CAMP is at the level of resynthesis of PtdIns, PtdIns4P, and Ptdlns4,5P2 (Della Bianca et ul., 1986a). A reduced availability of the precursor phospholipids in the plasma membrane +

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would further decrease stimulated Ins I ,4,5P, and DAG generation and therefore cell activation. Although attempts have been made to unravel the interrelationships between the CAMP and the Ca2 t- pathways, the molecular mechanism of the interaction between these two fundamental second messengers in phagocytic cells still remains obscure in spite of the large body of observations accumulated during the last 20 years.

B. Studies in Single Cells The availability of the new carboxylic dyes has allowed an old dream of morphologists to come true: perform on the same single cells microscopic observations and biochemical measurements. Although the technique is far from straightforward and the set-up still fairly expensive, this approach has already produced valuable and often unexpected information on [Ca2 Ii homeostasis in resting and stimulated phagocytes. Sawyer ef al. f 1985) first documented localized [Ca2+Ii increases in a quin2 loaded neutrophil ingesting a zymosan particle coated with IgG. The same authors also described a [Ca2+Ii increase localized at the leading edge of a locomoting neutrophil. More recently, several laboratories reported observations in a simpler system: phagocytes adherent to a glass coverslip coated with different ligands (Kruskal et al., 1986; Kruskal and Mayfield, 1987; Jaconi et al., 1988). Both neutrophils and inflammatory peritoneal macrophages undergo a transient increase in [Ca2+ I i immediately before spreading on a substrate (Fig. 2). The surprising observation is that many phagocytes (up to 40% of plated cells) exhibited, after spreading, a spiking behavior (Fig. 3). [Ca2+Iioscillated with a variable period of up to an average concentration of about 400 nM for up to 30 min (Kruskal and Mayfield, 1987). It is not surprising that the oscillatory behavior had escaped the attention of investigators given that the oscillations seemed to occur asyncronously in individual cells and therefore could be seen only with single cell analysis, not to mention that adherent cells are quite different from cells in suspension both morphologically and functionally, and it could well be that “swimming” neutrophils do not undergo oscillations in [Ca2 Ii. In neither macrophages nor neutrophils did the oscillations in [Ca2+Iishow an absolute requirement for external C a 2 + , although in the latter cells removal of’ Ca2+ caused a slow fading of the [Ca2+Ii spikes (Kruskal and Mayfield, 1987; Jaconi et ul., 1988). Ligation of the Fc receptor was not required for either the spike in [Ca2+Iipreceding spreading nor for the subsequent oscillations (Kruskal and Mayfield, 1987). In neutrophils that were not “oscillating” and exhibited a stable [Ca2 Ii base line, low concentrations of Met-Leu-Phe ( I O - ~lo- lo-” M) elicited sustained oscillations in [Ca2+J i (Jaconi et al., 1988). Higher concentrations of the chemotactic peptide (10V6 M ) induced a single ICa2+ 1, transient followed by a stable line (Jaconi et a l . , 1988). The relevance to cell physiology of these oscillations is not obvious, and any +

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FRANCESCO DI VlRGlLlO ET AL.

550

450

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Time (sec) FIG. 2. (a) Time course of ICa2+ 1, i n a spreading macrophapc in the presence of extracellular Ca”. Inset: detail of [Car+], from 210-410 scc. (b) [Ca2+], in a spreading macrophage in thc ahscncc of extracellular Ca: . The arrow indicates when spreading wab first detected. [Reprinted with perrriission from Kruskal and Maxfield (1987). J . Cell B i d 105, 2685--2693.] +

simple relationship to cell motility has yet to be detected. We feel, however, that an “oscillating [ Ca’ +Ii’’ describes the intracellular situation much more realistically than docs a steady level of [Ca2 J i . It has to be expected that a cell. whether it is moving on a substrate or not, is never really idle. On the contrary, pseudopods are continually extending from the cell body, “sensing” the environment around, and sending messages through the cytoplasm. Of course this does not mean that “resting” [Ca2+], is an empty abstraction and that the normal +

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300

A

7

A

A A A

A A

A

A

A A A

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5: c)

=! ’.2

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lime (min) FIG. 3 . Spontaneous [Ca’+ 1, oscillations in a single neutrophil adhered to a fibronectin-coated coverslip. [Reprinted with permission from Jaconi et ul. (19x8). J. Biol. Chem. 263, 10557- 10560.1

intracellular condition in an unstimulated cell is that of an oscillating [Ca2+ji. A cell adhering and spreading onto a substrate is something very remote from a “resting” cell, and it is quite possible that oscillations in 1Ca2+Iiare due to the engagement of adhesion receptors. Only future experiments correlating oscillations in [Ca2+ I i to functional responses will clarify this intriguing issue.

V. THE ROLE OF Ca2+ AS A SECOND MESSENGER IS QUESTIONED IN PHAGOCYTIC CELLS A. Ca2+ Dependency of Metabolic Responses in Phagocytes The response to the occupancy of surface receptors in phagocytes is one of the most complex biological phenomena observed in single cells. Depending on the type of receptor and the experimental conditions, phagocytes adhere to matrix proteins or to other cells; change shape and undergo oriented locomotion; engulf and digest microbes, particles, and debris; secrete granular content either in the medium or into the phagosome; synthesize and secrete a number of arachidonic acid metabolites; and, last but not least, produce large amounts of microbicidal oxygen radicals (Hallet, 1989). In each of these processes a role for [Ca2+Iihas

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been proposed and general consensus holds that a rise in (Ca2+Ii is a necessary step in the chain of events leading to many of the mentioned responses (Campbell, 1983). It is also accepted, however, that some stimuli, alone or in combination with other agents, can bypass the requirement for an increase in [Ca2+1,. Most of the current research in this field is aimed at the identification of the [Ca2 +Ii-sensitive step in the transduction chain, but at this time, very little progress has been made in this direction, and the reader is referred to reviews on this subject (Rossi, 1986; Hallet, 1989). There appear to be documented conditions, with both membrane receptor-directed and membrane-permeable agonists, in which activation of the NADPH oxidase and secretion of granules from neutrophils can be clearly dissociated from any measurable increase in ICa2 li (Pozzan et al., 1983; Di Virgilio et al., 1984; Grzeskowiak et ul., 1986; Rossi, 1986; Rossi et al., 1986, 1989). A rise in [Ca2+Iiseems to be neither necessary nor sufficient for the triggering of these responses, although it is conceivable that under physiological conditions it synergizes with other receptor-generated mediators at some step of the activation chain (Pozzan et al., 1983; Di Virgilio et al., 1984; Rossi et al., 1989). A role for [Ca2+Ii has been proposed in the mechanism of “priming,” a process in which products released from mononuclear cells (but also pharmacological agents) augment cytotoxic activity of phagocytes (Koenderman et ( I / . , 1989). However, this is also disputable, since several priming agents do not cause any increase in ICa2+li (Della Bianca et al., 1986b; Koenderman et al., 1989). It is more likely that several intraccllular signals are generated by a single receptor among which a rise in [Ca2 J i is only one and maybe not even the most important. A given metabolic response may require only one of these intracellular changes; others may need several acting in concert (Dewald ct al., 1988). An example of this hypothesis is the suppression of arachidonic acid release from [Ca2+Ii-depleted, phagocytosing neutrophils, while generation of 0,- is unaffected (Rossi et at., 1989). +

+

B. Ca2+ Dependency of Motility in Phagocytes I . THEACTINNETWORK It is also difficult to rationalize the involvement of lCa2+li in cell motility. One of the most striking features of phagocytes is their capacity to emit pseudopodia, ingest extracellular particles, and orient and migrate in a chemotactic gradient (Hallet, 1989; Silverstein et al., 1977, 1989). Several of the proteins that make up the contractile apparatus of striated muscle are also present in the cytoplasm of phagocytes (Southwick and Stossel, 1983; Stossel, 1988) and arc concentrated in the subcortical area of the cell and in the extending lamellipodia and pseudopodia (Valerius et a!., 198 I ; Stendahl et ul., 1980). During stimulation of phagocytes with chemotactic peptides or with aggregated IgG, transient polymerization of actin can be detected (Sheterline and Hopkins, 1981; Sklar et

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at., 1985; Greenberg er af., 1989). Further support for the role of the actin filaments in motility is the long-standing observation that inhibition of actin polymerization with cytochalasins blocks phagocytosis. Muscle contraction is a well-known Ca2 +-dependent process. Some of the proteins extracted from phagocytes (e.g., gelsolin) are regulated in vitro by physiological Ca2+ concentrations (Southwick and Stossel, 1983; Stossel et al., 1985). In the presence of Ca2 , gelsolin binds to and severs the actin filaments and prevents further polymerization of actin monomers. Removal of gelsolin allows rapid repolymerization of actin (Stossel et ul., 1985). Therefore the conclusion that motility in phagocytes is Ca2 -dependent and Ca2 -regulated seems to be almost inescapable. +

+

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2. THE PHAGOCYTIC RESPONSE Over the past few years, however, the conclusion that [Ca2+Ii controls motility in neutrophils and macrophages has been challenged by several observations. First, Campbell and Hallet (1983) showed that ingestion of latex particles occurred in neutrophils without detectable changes in [Ca2+Iiand was not prevented by loading EGTA into the cytoplasm. Our group then showed that phagocytosis by human neutrophils of C3b/bi-coated oil particles was unaffected by chelation of [Ca2 li (Lew el al., 1985). Later Di Virgilio et al. (1988b) provided evidence that Fc receptor-mediated phagocytosis was [Ca2 Ii independent in macrophages loaded with either quin2 in the absence of extracellular Ca2+ or with EGTA by A T P - permeabilization, as shown in Fig. 1 ([Ca2+Iiclamped at 1-10 nM). Finally Rossi et ul. (1989) also demonstrated that neutrophils could ingest ConA coated yeast cells in a completely [Ca2+Ii-independent manner. The intriguing aspect of the phagocytic response is that ingestion via both the complement and the Fc receptors is accompanied by an increase in [Ca2+li whether examined in cell populations (Young et al., 1984; Lew et al., 1985; Di Virgilio et ul., 1988b) or in single phagocytosing neutrophils (Sawyer et al., 1985; Stendahl et al., 1988), yet this Ca2 signal is not necessary for phagocytosis. Soluble stimuli, such as aggregated IgG and a monoclonal Ab (2.4g2) against the Fc receptor for IgG,, also raised [Ca2+Ii,indicating that the Ca2+ signal is not merely coincidental to ingestion but depends on ligation of the specific receptors engaged in phagocytosis (Lew et al., 1985; Di Virgilio et al., 1988b). To render the picture more complicated, there are well documented instances in which ligation of the Fc receptor and ingestion of IgG-coated particles by macrophage monolayers did not trigger any change in [Ca2 I, (McNeil et al., 1986; Di Virgilio et a / . , 1988b). Results obtained with intracellular Ca2+ indicators are in agreement with observations by Randriamampita and Trautmann ( I 987) performed with the patch clamp technique. These authors investigated putative changes in conductance triggered by ligation of Fc receptors in adherent murine macrophages and were not able to detect any. On the other hand, Ince et +

+

+

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al. (1988), did demonstrate changes in ionic channel currents during phagocyto-

sis by human macrophages. It is obvious from the reported conflicting results that the issue of the association of phagocytosis with intracellular changes in (cat)ion concentration needs to be clarified further. While discrepancies between human and mouse cells might be simply due to species differences, the reason for the lack of a Ca2 response in adherent macrophages as opposed to macrophage suspensions is unknown. Given that mouse and human cells carry three different types of Fc receptors, one reason for this could be that different Fc receptors are involved in sending the Ca2+ signal and in mediating phagocytosis. In other words, i t may be hypothesized that in adherent macrophages there is a preferential ligation of only the receptor competent for phagocytosis, while in macrophage suspensions and in neutrophils all Fc receptors, also those coupled to [CaZ J i elevations, are bound and activated. Difficult as it is to be tested, this hypothesis does not seem to be true because particle ingestion and a [Ca2+Iirise are caused by both the murine receptors FcI and Fcll (Silverstein ef al., 1989). More likely is the possibility that adherence and spreading somehow change the physiology of the phagocyte. While they do not impair phagocytosis (in fact very often enhance it), they turn off the Ca2+ signal associated with Fc receptor ligation. In light of the fact that in neutrophil suspensions [in contrast to macrophage or neutrophil monolayers (0.Stendahl, unpublished observations)] ingestion of particles via the Fc receptor is inhibited by chelation of [Ca2+ J i (Lew et al., 1985), it could be suggested that spreading itself "primes" or activates the phagocyte and makes ingestion independent of [Ca2+Ii. As discussed previously, adhesion and spreading induce dramatic changes in phagocyte [Ca2+ I i levels and these [Ca2 li oscillations may function to prime the phagocyte machinery. Phagocytosis was also remarkably resistant to pertussis toxin, another classical inhibitor of the InsP,-Ca*+ pathway in phagocytes (Lad el al., 1986; M. Fallman and 0. Stendahl, unpublished observations). Experiments from one of our laboratories suggest that diacylglycerol (DAG), and therelore protein kinase C, may be involved in phagocytosis by human neutrophils (Anderson et al., 1988; Stendahl et al., 1989; M. Fallman and 0. Stendhal, unpublished observations). The interesting observation was that the main source of DAG during ingestion of C3b/bicoated particles was not Ptd1ns4,SP2, since formation of DAG can also be measured in conditions under which phospholipase C is inhibited and Insl,4,5P, formation completely suppressed (Stendahl et al., 1989). PtdIns4,SP2 hydrolysis is not necessary for actin assembly either (Bengtsson et al., 1988). The identity of this additional source of DAG during engulfment is probably phosphatidylcholine, since it has also been observed that this phospholipid can undergo agonistactivated breakdown with generation of DAG (Besteman et ul., 1986; Fallman et cil., 1989).These results from Stendahl's group cast some doubts on the physiological relevance of the Ptdlns4,SP2-based mechanism for regulating actin assembly proposed by Stosscl (1988). +

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3. PHAGOCYTE LOCOMOTION The picture is not any more coherent in the case of chemotaxis. Some evidence indicates that exposure of phagocytes to chemoattractant causes heterogeneity in the distribution of [Ca2+], (Sawyer et al., 1985; Brundage and Fay, 1989). Zigmond and her co-workers, however, demonstrated that lowering [Ca2 1, with ionophore and EGTA did not affect neutrophil chemotaxis (Zigmond et al., 1988). Furthermore, quin2 loaded neutrophils, while showing decreased random migration, were still capable of increasing their location when subjected to a chemotactic field. Surprisingly, whereas Sawyer et al. (1985) detected an increase in [Ca2+ ], localized at the leading edge of a neutrophil moving toward an IgG-coated particle, Marks and Maxfield (1989) observed no correlation between the direction of the movement and the localization of the [Ca2 Ii increases in a neutrophil migrating toward a complement-coated particle. Rather, according to these authors, [Ca2 J i seemed to oscillate non-homogeneously throughout the cytoplasm. +

+

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4. ACTINASSEMBLY During the complex motile responses of phagocytosis and of oriented locomotion, neutrophils undergo repeated cycles of assembly and disassembly of the actin filament scaffolding. Current wisdom has maintained for many years that this rearrangement is controlled by changes in [Ca2+Ii (Stossel et al., 1985). Evidence presented so far clearly suggests that phagocytosis proceeds unimpeded in the virtual absence of [Ca2+Iiand chemotaxis may require changes in [Ca2+Ii that are not quite localized as one would expect. But how is actin assembly’itself affected by [Ca2+Ii manipulations in intact cells? The issue is being investigated currently in several laboratories, and the published evidence also hints at the basic [Ca2+], independence of actin polymerization whether induced by PMA or chemotactic peptides in neutrophils or by aggregated IgG in macrophages (Sklar et ul., 1985; Sheterline et ul., 1986; Carson et ul., 1986; Sha’afi et a l ., 1986; Zigmond et al., 1988; Greenberg et al., 1989). The kinetics of depolymerization may be somewhat slower in the absence of [Ca2+Iithan in its presence, although the issue is not yet completely clarified.

VI.

CONCLUSIONS: Ca2+ MAY NOT BE ALL, AFTER ALL

“The calcium ion has an unusual importance in biological phenomena, and the literature concerning its effects extremely voluminous,” wrote Lewis Victor Heilbrun in 1937, as cited by Anthony K. Campbell in a review article (Camp-

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bell, 1986). More than 50 years later, there are no doubts as to the second part of Heilbrunn’s statement [a literature search reveals that over 4100 papers dealing with Ca2 ’ were published in 1988 (U.S. National Library of Medicine, 1989)l. On the other hand, it has become clear over the years that Ca2+, although important, “shares the limelight” (Gompcrts, 1986) as an intracellular mediator of biological responses with a number of equally important other molecules (CAMP, InsP,, DAG). It has also become clear that the mere association of a ccllular response to a change in [CaZ+],does not imply a causal relationship between the two events. The challenge for future research in phagocyte physiology will be the discrimination of those responses that are [Ca2+],-activated and [Ca2+Ii-dependent from those simply associated with a change in [Ca2+Ii, the latter being due to the ligation of multifunctional receptors generating multiple signals and reactions. With the help of the new Ca2 chelators, which allow the manipulation of [CaZ+liin intact cells, answers to these questions are already becoming available. Yet we know nothing of the “missing messenger(s)” that mediates phagocyte responses in those instances in which no generation of known intracellular signals has been documented. +

ACKNOWLEDGMENTS The authors wish to thank Drs. S . C. Silverstein, T. H. Steinberg, and S . Greenberg who allowed reproduction of unpublished experiments performed with their help and counseling. This work was supported by grants from MPI (40% and 60%), from CNR (Special Project Oncology no. 88.00639.44 and Biotechnology and Bioinstrumentation). and from AIRC to F.Di V. and T.P.; by grants from the Swedish Cancer Association. Swedish MRC (no. 6956,5968) to 0 , s . . and by Fonds National de la Rcchcrche Scieniifique Suissr (no. 3.829.O.XX) 10 D.P.I-. REFERENCES Andersson. T., Dahlgren, C., Pozzan. T., and Lew, D. P. (1987). Characterization ofwet-Leu-Phe receptor-mediated Ca2 influx across thc plasma membrane of human neutrophils. Mol. Plmrm a d . 30, 437-443. Andersson, T., Fallnian, M., Lew, D. P., and Stendahl, 0. (1988). Does protein kinase C control receptor-mediated phagocytosis in human neutrophils? FEBS Lert. 239, 37 1-375. Mol. B i d . Baker, P. F. (1972). Transport and metabolism of calcium ions in nerve. Prog. Bioph~v.~. 24, 177-223. Baker, P. F., and Knight, D. E. (1978). Calcium-dependent exocytosis in bovine adrenal medullary cells with leaky plasma membranes. Narure (Londun) 276, 620-622. Bcngtsson, T., Rundquist, I.. Stendahl, O., Wymann. M., and Andersson, T. (1988). Increased breakdown of phosphatidylinositol 4.5-bibphosphate is not an initiating factor lor actin assemhly in human neutrophils. J . B i d . Chem. 263, 17385- 17389. Berridge, M. J . ( 1 984). Inositoltnsphosphdte and diacylglycerol as second messengers. Biochem. J . 220, 345-360. Berridge, M. J. ( 1987). Inositol trisphosphate and diacylglycerol: two interacting second messengers. Antiu. Rev. Biochem. 56, lS9-193. Berridgc, M . J . , and Irvinr, R. K. (1984). inositoltrisphosphate, a novcl sccond messenger in cellular signal transduction. Narure (London)312, 315-321.

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Besterman, J. M., Durino, V., and Cuatrecasas, P. (1986). Rapid formation of diacylglycerol from phosphatidylcholine: A pathway for generation of a second messenger. Proc. Narl. Acad. Sci. U.S.A. 83, 6785-6789. Biden, T. J . , Wollheim, C. B., and Schlegel, W. (1986). Inositol I ,4,5-trisphosphate and intracellular Ca*+ homeostasis in clonal pituitary cells (GH3). J . B i d . Chem. 261, 7223-7229. Blau, L., and Weissmann, G. (1988). Transmembrane calcium movements mediated by ionomycin and phosphatidic acid in liposomes with fura-2 entrapped. Eiochernisrry 27, 5661-5666. Blinks, J. R . , Mattingly, P. H., Jewell, B. R., Van Leeuwen, M., Harrer, G. C . , and Allen, D. G . (1978). Practical aspects of the use of aequorin as a calcium indicator: assay, preparation, microinjection and interpretation of signals. Merhods Enzymol. 57, 292-328. Bockoch, G. M., and Gilman, A. G. (1984). Inhibition of receptor-mediated release of arachidonic acid by pertussis toxin. Cell 39, 301-308. Brooks, J. C., and Treml, S. (19x3). Catecholamine secretion by chemically-skinned cultured chromaffin cells. J , Neuruchem. 40, 468-473. Brundage, R. A., and Fay, F. S . (1989). The role of Ca2+ in the polarization and chemotaxis of newt eosinophils. J . Cell B i d . 107, 7a. (Abstr.) Campbell, A. K . (1983). “lntracellular C a 2 + . Its Universal Role as a Regulator.” Wiley, New York. Campbell, A. K. (1986). Lewis Victor Heilbrunn. Pioneer of calcium as an intracellular regulator. Cell Calcium 7, 287-296. Campbell, A. K., and Hallet, M. B. (1983). Measurement of intracellular calcium ions and oxygen radicals in polymorpho-nuclear leukocyte-erythrocyte ghost hybrids. J. Physiol. (London) 338, 537-550. Campbell, A. K . , Patel, A. K., Razavi, Z. S . , and McCapra, F. (1988). Formation of the Ca2+activated photoprotein obelin from apo-obelin and mRNA inside human neutrophils. Biochem. J . 252, 143-149. Capponi, A , , Lew, D. P., Schlegel, W., and Pozzan, T. (1986). Use of intracellular calcium and membranc potential fluorescent indicators in neuroendocrine cells. Methods Enzymol. 124, 1 16- 135. Carafoli, E. (1987). lntracellular calcium homeostasis. Annu. Rev. Biochem. 56, 395-433. Carson, M., Weber, A , , and Zigmond, S. H. 11986). An actin-nucleating activity in polymorphonuclear leukocytes. J . Cell Eiol. 103, 2707-2714. Cobbold, P. H., and Rink, T. J . (1987). Fluorescence and bioluminescence measurement of cytoplasmic free calcium. Biochem. J . 248, 313-328. Cockcroft, S . , and Gomperts, B. D. (1979). ATP induces nucleotide permeability in rat mast cells. Nature (London) 279, 541 -542. Della Bianca, V., De Togni, P., Grzeskowiak. M., Vicentini, L. M., and Di Virgilio, F. (1986a). Cyclic AMP inhibition of phosphoinositide turnover in human neutrophils. Biuchim. Eiuphys. Actu 886, 441-447. Della Bianca, V., Grzeskowiak, M., Cassatella, M., Zeni, L., and Rossi, F. (1986b). Phorbol 12, myristate 13, acetate potentiates the respiratory burst while inhibits phosphoinositide hydrolysis in human neutrophils. Bioand calcium mobilization by formyl-methionyl-leucyl-phenylalanine chem. Biophys. Res. Cumrnun. 135, 556-565. De Togni, P., Cabrini, G., and Di Virgilio, F. (1984). Cyclic AMP inhibition offlet-Leu-Phedependent metabolic responses in human neutrophils in not due to its effects on cytosolic C a 2 + , Biochem. 1. 224, 629-635. Dcwald, B., Thelen, M.. and Baggiolini, M. (1988). Two transduction processes are necessary for neutrophil activiation by receptor agonists. J . Eiol. Chem. 263, 16179- 16184 Di Virgilio, F., and Gomperts. B. D. (1983). Cytosol Mg2+ modulates Ca2f ionophore induced secretion from rabbit neutrophils. FEES L e / / . 163, 315-3 18. Di Virgilio, F., Lew, D. P., and Pozzan, T. (1984). Protein kinase C activation of physiological

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Hashimoto, S . , Bruno, B., Lew, D. P., Pozzan, T., Volpe, P., and Meldolesi, J. (1988). Immunocytochemistry of calciosonies in liver and pancreas. J. Cell B i d . 107, 2523-253 I . Hinds, T. R . , Lindner, A., and Vincenzi, F. F. (1989). Estimation of intracellular free calcium in red blood cells. J . CeN B i d . 107, 7%. (Abstr.) Holmes, R. P., and Yoss, N. L. (1983). Failure of phosphatidic acid to translocate Ca2+ across phosphatidylcholine membranes. Nature (London) 305, 637-638. Ince, C., Coremans, J. M. C. C., Ypey, D. L., Leijh, P. C., Verveen, A. A,, and Van Furth, R . (1988). Phagocytosis by human macrophages is accompanied by changes in ionic channel currents. J . Cell Biol. 106, 1873-1878. Jaconi, M. E. E., Rivest, R. W., Schelegel, W., Wollheim, C. B., Pittet, D., and Lew. D. P. (1988). Spontaneous and chemoattractant-induced oscillations of cytosolic free Caz+ in single adherent human neutrophils. J. B i d . Chem. 263, 10557-10560. Kikkawa, U., and Nishizuka, Y. (1986). The role of protein kinase C in transmembrane signalling. Annu. Rev. Cell B i d . 2, 149-178. Koenderman, L., Yazdanbakhsh, M., Roos, D., and Verhoeven, A. J. (1989). Dual mechanism in priming of the chemoattractant-induced respiratory burst in human granulocytes. J. Immunol. 142, 623-628. Krause, K . H., Schlegel, W., Wollheim, C. B., Andersson, T., Waldvogel, F. A,, and Lew. D. P. (1985). Chemotactic peptide activation of human neutrophils and HL60 cells. J. Clin. Invest. 76, 1348-1354. Krause, K. H., Pittet, D., Volpe, P., Pozzan, T., Meldolesi, J., and Lew, D. P. (1989). Calciosome, a sarcoplasmic reticulum-like organelle involved in intracellular Caz+ -handling by non-muscle cells: studies in human neutrophils and HL60 cells. Cell Calcium 10, 351-361. Kruskal, B. A., and Maxfield, F. R. (1987). Cytosolic free calcium increases before and oscillates during frustrated phagocytosis in macrophages. L Cell B i d . 105, 2685-2693. Kruakal, B . A . , Shak, S . , and Maxfield. F. R. (1986). Spreading of human neutrophils is immediately preceded by an increase in cytoplasmic free calcium. Proc. Nut/. Acad. Sci. U.S.A. 83, 29192923. Lad, P. M . , Olson, C. V., and Grewal. I. S . (19%). A step sensitive to pertussis toxin and phorbol esters in neutrophils regulates chemotaxis and capping but not phagocytosis. FEES Lett. 200, 91-96. Lagasl, H., Pozzan, T., Waldvogel, F. A.. and Lew, D. P. (1984). PMA stimulates the ATPdependent calcium transport by the plasma membrane of neutrophils. J . Clin. Invest. 73, 878883. Lew, P. D., and Stossel, T. P. (1980). Calcium transport by macrophage plasma membrane. J. B i d . Chem. 255, 5841-5846. Lew, P. D., Wollheim, F. A., Waldvogel, F. A., and Pozzan, T. (1984). Modulation ofcytosolic-free calcium transients by changes in intracellular-buffering capacity: correlation with exocytosis and 02- production in human neutrophils. J . Cell B i d . 99, 1212-1220. Lew, D. P., Andersson, T., Di Virgilio, F.. Pozzan, T., and Stendahl, P. (1985). Ca*+-dependent and Ca2+-independent phagocytosis in human neutrophils. Nature (London) 315, 509-51 I . Lew, P. D., Monod, A., Krause, K . H., Waldvogel, F. A., Biden, T. F., and Schlegel, W. (1986). The role of cytosolic calcium in the generation of inositol I ,4,5-trisphosphate and inositol 1,3,4trisphosphate in HL60 cells: differential effects of chemotactic peptide receptor stimulation at distinct Ca2+ levels. J. B i d . Chem. 261, 13121-13127. Lew, D. P., Monod, A., wdldvogel, F. A . , and Pozzan, T. (1987). Role of cytosolic free calcium and phospholipase C in leukotriene B4 stimulated secretion in human neutrophils: comparison with the chemotactic peptide M L P . Eur. J. Biochem. 162, 161-168. McNeil, P. L., Murphy, R. F., Lanni, F., and Taylor, D. L. (1984). A method for incorporating macromolecules into adherent cells. J. Cell Biol. 98, 1556-1564.

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Rossi, F., Grzeskowiak, M . , and Della Bianca, V. (1986). Double stimulation with FMLP and ConA restores the activation of the respiratory burst but not of the phosphoinositide turnover in Ca2+ depleted human neutrophils. A further example of dissociation between stimulation of the NADPH oxidase and phosphoinositide turnover. Biochem. Biophys. Res. Commun. 140, 1-1 1. Rossi, F.. Della Bianca, V., Grzeskowiak, M., and Bazzoni, F. (1989). Studies on molecular regulation of phagocytosis in neutrophils. Con A-mediated ingestion and associated respiratory burst independent of phosphoinositide turnover, rise in [Ca2+I, and arachidonic acid release. J. Intmimol. 142, 1652- 1660. Sha’afi, R. I . , Shefcyk, J . , Yassin, R.. Molski, T. F. P., Volpi, M., Naccache, H . , White, J. R., Fein, M. B., and Becker, E. L. (1986). Is a rise in intracellular concentration of free calcium necessary or sufficient for stimulated cytoskeletal associated actin? J. Cell B i d . 102, 14591463. Sawyer, D. W., Sullivan, J. A , , and Mandell, G . A. (1985). lntracellular free calcium localization in neutrophils during phagocytosis. Science 230, 663-666. Sheran, C. N . , Fridovich, J . , Goetzl, E. 1.. Dunham, P. B . , and Weissmann, G. (1982). Leukotriene B4 and phosphatidic acid are Ca2+ ionophores. J. B i d . Chem. 257, 4746-4752. Sheterline, P., and Hopkins, C. R. (1981). Transmembrane linkage between surface glycoproteins and components of the cytoplasm in neutrophil leukocytes. 1.Cell B i d . 90, 743-754. Sheterline, P., Rickard, J. E., Boothroyd, B., and Richards, R. C. (1986). Phorbol ester induces rapid actin assembly in neutrophil leukocytes independently of changes in [Ca* +Iiand pH,. J . Muscle Res. Cell Moril. 7 , 405-412. Silverstein, S. C . , Steinman, R. M., and Cohn, Z . A. (1977). Endocytosis. Annu. Rev. Biochem. 46, 669-722. Silverstein, S. C . , Greenberg, S . . Di Virgilio, F., and Steinberg, T. H. (1989). Phagocytosis. In “Fundamental Immunology” (W. Paul, ed.) pp. 703-720. Raven, New York. Simchowitz, L., Fischbein. L. C.. Spilberg, I . , and Atkinson, J. P. (1980). Induction of a transient elevation in intracellular levels of adenosinc-3’,5’-cyclic monophosphate by chemotactic factors: an early event in human neutrophil activation. J. Immunol. 124, 1482-1491. Sklar, L. A., Omman, F. M . , and Painter, R . G . (1985). Relationship of actin polymerization and depolymerization to light scattering in human neutrophils: dependence on receptor occupancy and intracellular C a z + . 1.Cell B i d . 101, 1161-1166. Sniolen, J. E., Korchak, H. M.. and Weissmann, G. (1980). Increased levels of cyclic adenosine-3’,5’-monophosphate in human polymorphonuclear leukocytes after surface stimulation. J . Clin. Invest. 65, 1077-1085. Snyderman, R., and Uhing, R. J. (1988). Phagocytic cells: Stimulus-response coupling mechanisms. In “Inflammation: Basic Principles and Clinical Correlates” (J. I. Gallin, I. M. Goldstein, and R. Snydcnnan, eds.), pp. 309-323. Raven, New York. Southwick, F. S . , and Stossel, T. P. (1983). Contractile proteins in leukocyte function. Sem. Haemarol. 20, 305-321. Spat, A , , Bradford, P. G., McKinney, J . S., Rubin, R. P., and Putney, J. W., Jr. (1986). A saturable receptor for 32P-inositol 1,4,5-trisphosphate in hepatocytcs and neutrophils. Nature (London) 319, 514-516. Steinberg, T. H., Newman, A. S . , Swanson, J. A., and Silverstein, S . C. (1987a). ATP- permeabilizes the plasma membrane of mouse macrophages to fluorescent dyes. J . B i d . Chem. 262, 8884-8888. Steinberg, T. H., Newman, A. S . , Swanson, J. A., and Silverstein, S. C. (1987h). Macrophages possess probenecid-inhihitable organic anion transporters that remove fluorescent dyes from the cytoplasmic matrix. J . Cell B i d 105, 2695-2702. Stendahl, O., Hartwig. J. H., Brotschi, E. A , , and Stossel. T. P. (1980). Distributionof actin-binding protein and myosin in macrophages during spreading and phagocytosis. J. Cell Biol. 84, 215224.

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Stcndahl, O., Krausc, K. H . , Volpe, P., Gil, J., Rilke, L). Meldolesi, J., and Lew. D. P. (1988). Calciosomes, the inositol I ,4,5-trisphosphate sensitive Ca2 store, are redistributed in human neutrophils during phagocytosis and degranulation. Clin. Res. 36, 472a. ( Ahstr.) Stossel. T. P. (1988). The mechanical responses of white blood cells. In “Inflammation: Basic Principles and Clinical Correlates” (J. 1. Gallin. I. M. Goldstein, and R. Snyderman. eds.) pp. 325-342. Raven, New York. Stossel, T. P., Chaponnier, C., Ezzel, R. M., Hartwig, J. H . , Janmey, P. A,, Kwiatowski, D. J . , Lind, S. E., Smith. D. R., Southwick, F. S . , Yin, H. L.,and Zanes, K S.(1985). Non-muscle actin-binding proteins. Annu. Rev. Cell B i d . I , 353-402. Streb. H . , Irvinc, R. F . . Benidge, M . J., and Schulz, 1. (1983). Release of C a 2 + from a non mitochondria1 intracellular store in pancreatic acinar cells by inositol 1.4.5-trisphosphate. Niitrrre (Lontlorr) 306, 67-69. Streb, H.. Rayerdoffrr, E.. Haase, W., Irvinc, K . P., and Schulz, 1. (1984). Etfect of inositol 1,4,5trisphosphare on isolated subcellular fractions of rat pancreas. J . Memhr. Biol. 81, 241 253. ydshjian, A . H., Heslop. J. P., and Berridge, M. J. (1987). Subsecond and sccond changes in inositol polyphosphates in GH4CI cells induced by thyrotropin-releasing hormone. Biochem. J. 243, 305-308. Thomas, M. V. (1982). “Techniques in Calcium Research.” Academic Press, New York. Trcves, S., and Pozzan, T. (1988). Stimulus-activation coupling in neutrophils: an overview. In “Clinical Immunology. Vol. 1: Human Inflammatory Diseases” (C. Maronc, L. M. Lichtenstein. M. Condorelli. and A. S . Fauci. eds.), pp. 263-271. B. C. Decker, Toronto. Trevca, S., Di Virgilio. F.. Vaselli, G . M., and Pozzan, T. (1987). Etrect of cytochalasins on cytosolic-free calcium concentration and phosphoinositide inetaholism in leukocytes. Exp. Cell KPS. 168, 285-298. Tsien, R . , and PoZzdn, T. (1989). Measurement of cytosolic free Ca2+ with q u i d . Merhod.9 Enzyrnol. 172, 230-262. Tsien, R. Y.. Pozzan, T., and Rink, T. J. (19x2). Calcium homeostasis in intact lymphocytes: cytoplasmic free calcium monitored by a new, intracellularly trapped fluorescent indicator. J. Cell B i d . 94, 325-334. U . S . National Library of Medicine. Medline on Silver Platter. (1989). Silver Platter Int., Boston, Massachusetts. Valerius, N . H . , Stendahl, 0..Hartwig, J. H., and Stossel, T. P. (1981). Distribution of actin-binding protein and myosin in PMN during locomotion and phagocytosis. Cell 24, 195-200. Vicentini, L. M., Di Virgilio, F.. Ambrosini, A . , Pozzan, T., and Meldolesi, J. (1985). ’ h i o r promoter phorbol 12-myristatc, 13-acetate inhibits phosphoinositide hydrolysis and cytosolic Ca? risc induced by the activation of niuscarinic receptors in PC12 cells. Biochem. Biophvs. Re.$. Commun. 127, 310-317. Volpe, P., Krause, K . H . , Hashimoto, S..Zormto, F., Pozzan. T., Meldolesi, J . , and Lew, D. P. (1988). Calciosome, a cytoplasmic organelle: the inositul 1,4,5-trisphosphate-sensitiveCaL’ store of non-muscle cells. Pro[.. Natl. Arad. Sci. U.S.A. 85, 1091-1095. Volpi, M., Naccache, P. H . , and Sha’afi, R. 1. (1983). Calcium transport in inside-out membrane vesicles prepared from rabbit ncutrophils. J. Biol. Ckem. 258, 4153-4158. von Tscharner. V., Proud’hom, R . , Baggiolinl. M., and Reutcr, H. (1986a). Ion channels in human neutrophils activated by a rise in free cytosolic calcium concentration. Nuture (London) 324, 369-372. von Tschamcr, V., Deranlau, D. A . , and Baggiulini, M. (1986b). Calcium fluxes and calcium buffering in human neutrophils. J . Biol. Chem. 261, 10163-10168. Watson, S . P.,McConnel, R. ‘I_. and Lapetina, E. (1984). The rapid formation of inositvl phosphates in human platelets by thrombin is inhibited by prostacyclin. J . B i d . Chern. 259, 13199-13203. Weissniann. G . , Zurier, R. B . , Spieler, P. J . , and Coldstein, 1. M. (1971). Mechanism of lysosomal +

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enzyme release from leukocytes exposed to immune complexes and other particles. J. Exp. Med. 134, 1495-1505. Wymann, M. P., von Tschamer, V., Deranleau, D. A , , and Baggiolini, M. (1987). The onset of the respiratory burst in human neutrophils. Real time studies of H20, formation reveal a rapid agonist-induced transduction process. J. B i d . Chem. 262, 12048-12053. Young, J. D.-E., KO, S . S . , and Cohn, Z. A . (1984). The increase in intracellular free Ca2+ associated with JgG 2b/ I Fc receptor-ligand interaction: role in phagocytosis. Pruc. Nut/. Acud. Sri. U.S.A. 81, 5430-5434. Zigmond, S. H., Slonczewski, J. L., and Carson, M. (1988). Polymorphonuclear leukocyte locomotion is insensitive to lowered cytoplasmic Ca2+ levels. Cell Motil. Cyroskel. 9, 184-1239,

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CURRENT TOPICS IN MEMBRANES AND TRANSPORT, VOLUME 35

Chapter 9

Role of Intracellular pH in Lymphocyte Activation THOMAS H . STANTON AND KAROL BOMSZTYK Division of Nephrology Department of Mdicine University of Washington Seattle, Washingcon 98195

I. Introduction 11. Intracellular pH Changes in Nonlymphoid Cells A. Regulation of pH, by Na+ /H Antiport B . pH, Changes in Sea Urchin Eggs C. pH, Changes in Fibroblasts 111. Intracellular pH Changes in T Lymphocytes A. Effects of Mitogen Stimulation on pH, of T Lymphocytes B. Role of pH, in Controlling Mitogen- or Phorbol Ester-Stimulated Oncogene Expression C. Role of [Ca2+]i in Mitogen-Stimulated pH, Changes D. Effects of Antibody-Induced Activation of T Lymphocytes on pH, E. Role of pH, in T Lymphocytes Activated by Growth Factors IV. N a + / H + Antiport Activity and pH, in B Cell Differentiation V. Conclusion References +

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Biochemical events associated with lymphocyte activation are initiated on binding of ligand or lectin to the cell surface, leading to a cascade of signals transmitted from the membrane to the cell interior. Activation of certain pathways results in entry into the cell cycle, while others prepare the cell for increased metabolic activity or maintain homeostasis. Since most intracellular enzymatic reactions have a marked pH dependence, lymphocytes must maintain their intracellular pH (pH,) within certain limits even in the face of varying external pH. Therefore, an active regulatory mechanism must be present to 207 Copynght 0 1990 by Academic Press. lnc All rights o l reproduction In any fonri reserved

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extrude H + produced by metabolic activity and to oppose the approximately 60m V negative membrane potential which acts to drive H into the cell (Boron, 1984; Moolenar, 1986; Roos and Boron, 1981). Changes in pHi may also act as second messengers, transmitting the signal provided by receptor binding of growth factors or cytokines (Pouyssegur et al., 1982; Moolenar et ul., 1984; Koch and Leffer, 1979). For example, decreased or increased pHi may lead to increased activation of previously quiescent enzyme systems, transcription of mRNA, activation of DNA binding proteins, or release of stored biologically active material. +

II. INTRACELLULAR pH CHANGES IN NONLYMPHOID CELLS A. Regulation of pH, by Na+/H+ Antiport The best-studied mechanism for controlling pHi is the Na /H antiport, an electroneutral exchanger which couples transport of Na+ and H ' across the membrane in a 1 : I exchange (Aronson, 1985; Boron, 1983; Roos and Boron, 1 9 8 1 ) . T h e N a t / H + antiporttransportsNa+, H + , a n d L i + b u t n o t K + , K b + , or divalent cations. In lymphocytes, for example, there are approximately 8000 high affinity sites per cell that, when activated, can exchange Na+ for H + at the rate of 2000 cycleslsec (Dixon et al., 1987). The antiport is normally activated on increased production of intracellular H + from metabolic activity and is a primary mechanism for extruding metabolically generated H . It has been proposed that thc antiport has a modifier site that binds H , leading to an allosteric change which in turn leads to increased binding of H to the transport site (Paris and Pouyssegur, 1983). The N a + / H + antiport is thus the primary mechanism opposing intracellular acidosis (Aronson et al., 1982). The diuretic drug amiloride [3,S-diamino-6-chloro-N-/(diaminomcthylene)pyrazinecarboxamide] is a potent and specific inhibitor of the Na+ /H antiport (Paris and Pouyssegur, 1983). It acts competitively at the Na' binding site, blocking transport of Na+ . It also appears to lower noncompetitively the affinity o f thc antiport for H + binding. Aniiloride has an IC,,, of approximately 3-4 kM. However, several analogs in which the 5-amino group is substituted with alkyl or alkenyl groups yield compounds that inhibit Na+ / H exchange at approximately 100-fold lower concentration (L'Allemain et a / ., 1984a). Amiloride analogs such as dimethylamiloride, ethylpropylamiloride, ethylpropenylamiloride, and others have proved useful in studying N a + / H + exchange becausc they can be used at concentrations that block the Na ' / H antiporter but do not block protein synthesis, protein kinase c , or protein phosphorylation. These are all side effects that can occur at concentrations of aniiloride required to inhibit Na / H exchange (Leffert et ril., 1982: Lubin and Cahn, 1981). +

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An early elevation of intracellular [Ca2 ] ([Ca2 1,) has been postulated to trigger Na+ / H + activity through activation of Ca2+ requiring enzymes (Owen and Villereal, 1982; Villereal et al., 1985). The rise in [Ca2 li could be derived either from a release of Ca2+ from internal stores or an influx of extracellular Ca2-+.Huang and colleagues (1987) found that in smooth muscle cells, chelation of intracellular Ca2 by the Ca’+ binding agent quin2 significantly blunted thrombin-induced Na+ /H exchange activation. However, increased [Ca2 J i alone was not sufficient to induce Na+ / H + activation. Moolenar and colleagues ( 1984) found that phorbol ester ( 12-U-tetradecanoylphobolI3-acetate) increased N a + / H + exchange and pH, without any change in [Ca2+Ii, suggesting that activation of protein kinase C , and not increased [Ca2+],, is required to activate fibroblast Na+ / H + exchange as did (Siffert and Akkerman, 1988). Similarly, Rozengurt and colleagues (Lopez-Rivas and Rozengurt, 1983; Vara and Rozengurt, 1985) found that epidermal growth factor and insulin increase pH, of mouse 3T3 fibroblasts without increased [Ca2 + I i . +

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6. pH, Changes in Sea Urchin Eggs Early demonstrations of the importance of intracellular pH for cell activation were the reports from Mazia and Ruby (Mazia and Ruby, 1974) on sea urchin eggs. They found that exposure of unfertilized eggs to elevated pH even for short periods of time led to DNA synthesis and the bioelectric changes that normally signify fertilization. It appeared that a pHi rise was the important event since only weak bases, such as NH,OH, which as NH, can penetrate the membrane to the intracellular space, caused activation (Winkler and Grainger, 1978). In addition, it was found that normal fertilization also leads to a rise in pHi, which is caused by an exchange of extracellular Na+ into the cell for intracellular H + to the outside resulting in an elevation of pH, (Johnson el al., 1976).

C. pH, Changes in Fibroblasts Growth of mammalian cells in culture is also profoundly affected by pH,. Mouse 3T3 fibroblasts growing in sparse cultures can be rendered quiescent by removing serum (Zetterburg and Engstrom, 198 I). Such serum-starved cells can be induced to proliferate again by brief exposure to an alkaline medium. The growth stimulatory effect is directly proportional to the pH of the medium to which the cells are exposed and not reversed by subsequent exposure of the cells to acid media, suggesting that an increase in pH, is sufficient to trigger the cells to grow. This suggestion was supported by a series of reports from L’Allemain, Pouyssegur, and colleagues that strongly implicate activation of the plasma membrane Na+ / H antiport and a rise in pHi as critical to growth factor-induced fibroblast +

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proliferation. First, they found that the degree of inhibition of growth factorinduced proliferation by amiloride, or its structural analogs, was directly correlated with the aff'inity of binding to the antiporter and the degree of inhibition of N a + / H + exchange (L'Allemain et al., 1984a). Second, a tight correlation of dose responsiveness was found between the ability of the growth factor thrombin to induce pHi rise and DNA synthesis (L'Alleniain et d., 1984b). Lastly, mutant cell lines lacking a Na+ /H antiport could not be induced by growth factors to either increase pH, or to proliferate at neutral or acidic pH (Pouyssegur et al., 1984). These experiments were carried o u t in HCO, free media, which renders the Na / H + antiport the main mechanism for pH, regulation. However, when the experiments were carried out in the presence of HC0,- (activating HCO,- /Clexchange which can augment or replace the N a + / H + antiport), dimethylamiloride did not block DNA synthesis (Besterman et al., 1984). Evidence thus exists that control of cytoplasmic pH may not only be important in maintenance of intracellular metabolic homeostasis, but in addition, cytoplasmic alkalinization appears to play a role in activation of sea urchin eggs and quiescent fibroblasts. In fibroblasts, and probably in sea urchin eggs, this is due to activation of the plasma membrane Na+ / H antiport. +

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111. INTRACELLULAR pH CHANGES IN T LYMPHOCYTES

A. Effects of Mitogen Stimulation on pH, of T Lymphocytes A role for pH, in activation of lymphocytes similar to that seen in sea urchin eggs and fibroblasts has been difficult to establish. Treatment of T lymphocytes with mitogens that lead to cellular activation and proliferation has resulted in both increases and decreases in pHi. These contrasting results appear to correlate with the source of cells. Mitogenic stimulation of normal resting mouse or rat T lymphocytes results in increased pHi, while similar treatment of normal resting human T lymphocytes results in intracellular acidification. Early reports by Gerson and colleagues (1982; Gerson and Kiefer, 1983) showed Na /H exchanger-dependent cytoplasmic alkalinization in ConA-activated mouse spleen cells within 6 hr of stimulation and again after approximately 20 hr. At 20 hr, the rise in pH, was correlated with increased DNA synthesis, suggesting that increased Na+ / H antiport activity and pHi may play a role in the cell activation process. However, it was not demonstrated that the pHi rise is required for activation. Rink et al. ( 1982) found no early increase in pH, of pig lymphocytes or mouse thymocytes on stimulation with mitogenic doses of ConA. However, when fresh+

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ly isolated mouse thymocytes were rendered quiescent by culture for 16 hr, Hesketh et al. (1985) were able to show that ConA induced a 0.1-unit increase in pH,. The rise was first noticeable 1-2 min after addition of ConA. The rise was preceded by, but not dependent on, a rise in [CaZ+Ii.Phorbol esters also induced an early pHi rise but by an independent mechanism, since sequential treatment of the cells with ConA followed by phorbol ester, or vice versa, leads to pHi increases after each addition. Similar results were obtained in rat thymic lymphocytes by Grinstein and colleagues (1987) who found that ConA induced an early (within 2 min) rise in pH, of approximately 0.2 pH units. The rise was inhibited by Na -free media or by the presence of ethylpropylamiloride, indicating a role for N a + / H + exchange. Similar to mouse thymocytes, the pH, rise was associated with, but not dependent on, a rise in [Ca2+li.The ConA-induced pH, rise was also independent of protein kinase C activity, since neither potent antagonists nor depletion of protein kinase C activity inhibited the pHi rise. In contrast to the mouse, thymocytes from the rat did not need a 16-hr incubation to induce quiescence before lectin-induced alkalinization could be demonstrated. The previously discussed experiments clearly demonstrated that increased N a + / H + exchange and pHi are associated with T lymphocyte activation but d o not address whether it is a requirement. Mills et al. (1986) further explored this by incubating human peripheral blood lymphocytes (PBL) with the mitogen phytohemagglutinin (PHA) in the presence of dimethylamiloride, ethylpropylamiloride, or ethylpropenylamiloride. The presence of these inhibitors at concentrations that are expected to block Na / H exchange had no effect on PHA-induced proliferation or expression of cell surface IL-2 receptors. These results show that increased Na /H exchange is not required for cellular activation. However, since HC0,- was included in the incubation media to maintain cell viability, it is possible that increased HCO, - /C1- exchange could have elevated pH, independently of Na+ / H antiport activity and led to intracellular alkalinization. Contrasting results between rodent and human lymphocytes were found when the pH, of human PBL, responding to lectin stimulation, was actually measured. Yamaguchi et al. (1986) investigated whether pHi was actually elevated in either T cell-enriched or B cell-enriched human PBL on stimulation with PHA, pokeweed mitogen (PWM), or allogeneic cells. It was found that neither mitogenic concentrations of PHA or PWM nor allogeneic cells induced a detectable rise in pHi either within the first 10 min or between 2 and 6 hr in either the T cell or B cell population. In addition, a concentration of dimethylamiloride, which was shown to block N a + / H + exchange, blocked neither DNA synthesis in the T cell population nor Ig production by the B cell population. HC0,- was again included in the incubation media, leaving open the possibility that increased HCO, /C1- exchange could have elevated pHi. Additional contrasting data between rodent and human lymphocytes were +

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found by Gelfand et ul. (1988) who showed that PHA actually stiniulated a decrease in pH, of human PBL. The acidification was magnified in low Na’ media and in the presence of ethylpropylamiloride, indicating participation of the Naf / H + antiport to offset the accumulation of intracellular H C . Acidification was dependent on an increase in [Ca2+],, most of which was a result of an influx from outside. Acidification was blocked when outside [Ca2+] was lowered and proceeded when outside [ Ca2 J was returned to normal. The source of H is probably increased intracellular metabolic activity. In addition to PHA, similar acidification and [Ca*+], increases were seen in response to ConA, WGA, and UCHI’-1, an anti-CD3 (cell surface molecules associated with the T cell receptor for antigen) antibody (Cheung et a l . , 1988). Interestingly, all mitogens tested induced acidification, but only PHA and ConA induced PBLs to proliferate. WGA and UCHT-1, while inducing acidification and [Ca2+Ii rise, did not induce proliferation. There is thus no correlation bctween pH, and proliferation. Similar results were obtained by Fischer ct ul. (1988). When human PBL were stimulated with mitogenic concentrations of PHA or ConA, proliferation was induced and a rise in [Ca*+], was seen. Examination of the data suggests acidification after exposure to ConA or PHA occurred, though not as dramatic as in other data (Gelfand et al., 1988; Cheung et ul., 1988). In HCO,--free media, mitogen induced proliferation was not blocked by methylisobutylamiloride, indicating that Naf / H exchange was not required for proliferation. Unless other pH, regulating mechanisms arc active, Fischer’s results in HCO, -free media suggest that not only is Na+ / H exchange not required but an increase in pH, is also not required. The mitogen stimulation results suggest a difference in the pH, response between rodent and human lymphocytes. lntracellular pli changes, either up or down, result from ligand binding and may retlect changes in metabolic H + generation or in the activation of H ’ transport mechanisms. Initial acidification or alkalinization appears not to be required for early lymphocyte activation or DNA synthesis (Gelfand et al.. 1987). However, it is likely to play a role in the regulation of pH,, which is essential for a variety of cellular processes. The contrast between mitogen-stimulated alkalinization seen in rodent T lymphocytes vs. the acidification seen in human T lymphocytes may reflect a more rapid attempt to reestablish homeostasis by rodent cells in the face of increased metabolic activity. +

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B. Role of pH, in Controlling Mitogen- or Phorbol Ester-Stimulated Oncogene Expression Newly transcribed mRNA and the resultant translated protein signify early events leading to functional activity of the cell or to proliferation. Thus, this

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transcription can be used as a measure of the lymphocyte activation initiated by binding of ligand or lectin to the cell surface. Another advantage of using mRNA transcription as a measure of lymphocyte activation is that some new mRNA transcripts are detectable within 30 min to 1 hr. One such early event is the increased expression of mRNA for the protooncogene c+s (Heldin and Westermark, 1984). Expression occurs within 30 min after stimulation with growth factors, mitogen, or phorbol esters. c-fos encodes a protein located in the nucleus that, in combination with another oncogene c-jun, binds to chromatin and may act to alter chromosome structure, allowing new transcription of certain genes. c-fos may thus be an important regulator of cell activation (Marx, 1987). Because c-fos can be measured within 30 min, control of its expression can be tested under conditions where pH, and Na+ can be continuously monitored or controlled. This is an advantage because some experiments investigating the role of pH, in cell activation were performed under conditions where increased pH, might have occurred unknowingly. For instance, experiments demonstrating that amiloride analogs block increases in Na+ / H + activity and pH, were performed in HCO, --free media to prevent the involvement of HCO,-/CI - exchange. However, in order to maintain cell viability, HC0,- was added in experiments to evaluate the role of N a + / H + exchange or pH, on cell proliferation. Therefore, it is possible that increases in pH, were occurring under the latter conditions via HC0,-/Cl- exchange. Grinstein et a/. (1988) found that PHA-stimulated human T lymphocytes to express cfos mRNA in 30 min. pH, was monitored during the time course of the experiment. In normal medium, PHA did not alter pH,, but in Na+-free or ethylpropylamiloride-containingmedium, the pH, actually decreased. These results are similar to those of Gelfand et al. (1988) and suggest that N a + / H + exchange has been activated but is balanced by metabolic H + generation. In contrast to its varying effects on pHi, PHA stimulated a nearly equal rise in c-fos mRNA expression in normal, Na -free, and ethylpropylamiloride-containing media. Furthermore, when pH, was increased by setting it equal to pH,, by the K + / H + exchanging ionophore nigericin, PHA did not alter pH,, and the relative increase in expression of c-fos mRNA was equal to normal cells. Thus, it appears that increasing neither Na / H exchange nor pHi is required or sufficient for PHA-stimulated c-fos mRNA expression. Grinstein et al. (1988) also found similar results with rat thymocytes. In these cells, phorbol ester plus the Na+ ionophore ionomycin enhanced Na /H exchange and induced c-fos mRNA expression. However, when Na /H exchange was blocked by Na -free media or ethylpropylamiloride, the expression of c-fos mRNA induced by phorbol ester plus ionomycin was not significantly reduced from the level obtained by the same treatment of cells in normal media. These results again point out that +

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increased Na+ /H exchange or pHi is neither required nor sufficient to induce c:fos mRNA expression. +

C. Role of [Ca2+], in Mitogen-Stimulated pH, Changes Changes in [Ca2t]i have been proposed as a mechanism to stimulate activation of the Na / H + antiport. This suggestion is based on results of experiments on fibroblasts. For instance, Villereal and colleagues (Owen and Villareal, 1982; Villereal et a / . , 1985) found that Ca2+ ionophores would activate Na / H + exchange and that blockage of the Ca2 requiring enzyme calmodulin blocked Na /H exchange activation. Mitogen stimulation of lymphocytes leads to either intracellular alkalinization or acidification. In mouse or rat lymphocytes where mitogen-induced alkalinization occurs, mobilization of Ca2+ is not required to achieve an increased pHi. Hesketh found that lowering the [Ca2+Jof the media to a point where the increased [Ca2 Ii was no longer observed had no effect on ConA-induced increase in pH, (Hesketh et ul., 1985). Similar results were reported in rat thyrnocytes by Grinstein et al. (1987). In thymocytes, changes in [Ca2+lIiwere buffered by loading the cells with a high concentration of the Ca2 binding agent quin2. Under these conditions, or in Ca2+-free media, the ConA-induced increase in pH, still occurs. Furthermore, the Ca2+ ionophore ionomycin could not induce a pHi rise, while it did induce a dramatic spike in [Ca2+Ii. These results suggest that activation of the Na+/H+antiport by mitogen in lymphocytes is independent of Ca2 . In contrast, the acidification seen in human PBL was clearly dependent on an increase in [Ca2 Ii.Gelfand et al. (1988) found that an influx of Ca2 from the outside, which could be eliminated in Ca2 -free media containing EGTA, blocked PHA-induced acidification. However, blockage of Ca2 release from internal stores by RAPTA, in the presence of extracellular Ca2+, only slightly inhibited PHA-induced acidification, suggesting a requirement for an influx of extracellular Ca2 . Other mitogens that induce increased [Ca2+Iicorrelate with those that cause intracellular acidification. In summary, Ca2 is not required for mitogen to activate Na+ /Ht- exchange in lymphocytes but appears to play a role in mitogen-stimulated acidification. +

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D. Effects of Antibody-Induced Activation of T Lymphocytes on pH, Binding to specific cell surface antigens has been found to lead to cellular activation. Antibodies reactive to CD3 (part of the antigen-specific T cell receptor complex), especially in the presence of monocytes, have been shown to induce T

215

9. ROLE OF INTRACELLULAR pH IN LYMPHOCYTE ACTIVATION

cell proliferation, expression of 1L-2 receptors, and secretion of IL-2 (Weiss and Imboden, 1987). Additionally, the cell surface receptor for sheep erythrocytes found on all T cells and thymocytes and termed CD2 can serve as an activation site (Howard et ul., 1981). Stimulation with various combinations of anti-CD2 antibody leads to T cell proliferation (Meur et al., 1984; Yang et al., 1986). Fischer et trl. (1988) observed that stimulation with a combination of anti-CD2 antibodies, VIT13 plus 9.6 or VIT13 plus T1 l,, leads to a rise in pHi and in proliferation. However, in the presence of methylisobutylaniiloride, anti-CD2 induced proliferation was not blocked. Indeed proliferation was enhanced. These experiments were carried out in HCO, - -free media so that a pHi rise potentially mediated by HCO, - /C1- exchange could not circumvent the dimethylamiloride-blocked pHi rise. Stimulation of human lymphocytes with anti-CD3 antibodies recognizing various T3-Ti epitopes leads to more variable results. Treatment with the anti-CD3 antibody UCHT- 1 (Cheung et u l . , 1988) leads to acidification and a concomitant increase in [Ca2+Ii similar to results seen with PHA, ConA, or WGA. In contrast, treatment with OKT3, another anti-CD3 antibody, affected neither pH, nor [Ca2+Ii.Neither of these antibodies, in the absence of monocytes, leads to significant proliferation. Another set of anti-CD3 antibodies, VIT-3 and VIT-3b, was heterogeneous in its effect on pHi (Fischer et al., 1988). In seven out of 30 experiments, pH, was raised in T cell preparations depleted of monocytes. Potentially, the heterogeneity of response could be due to varying contamination by monocytes. It is not clear if the lack or heterogeneity of T cell responses to antiCD3 could be affected by the inclusion of sufficient monocytes to act as accessory cells, facilitating T cell activation. In contrast to its effects on normal resting T cells, anti-CD3 appears to have a more profound effect on the human leukemic T cell line HPB-All (Rosoff and Cantley, 1985). Three different anti-CD3 antibodies, OKT3, WT-3 1, and T40/25, recognize different epitopes of the T3-Ti receptor. Treatment of HPBALL, in HC0,--free media, with any of these antibodies induced a 0.15-unit pH, rise within 1-2 min of being added to culture. The rise in pH, was blocked in Na+ -free media or by addition of dimethylamiloride, indicating that activation of the Na+ /H antiport was responsible for the pH, rise. In addition, activation of the Na /H antiport was dependent on an influx of Ca2 from the outside. However, in this study, the authors did not examine the role of pHi changes on cell function. One event involving I L 2 that may depend on increased Na+/H+ exchange and pHi is the secretion of I L 2 by the human T cell line Jurkat (Rosoff and Terres, 1986). Jurkat is induced to secrete lL-2 by simultaneous stimulation with anti-CD3 antibody and phorbol ester. Dimethylamiloride at a concentration that should block Na /H exchange activity blocked I L 2 secretion, suggesting that there may be a role for increased pHi in mediating IL-2 secretion. +

+

+

+

+

+

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THOMAS H. STANTON AND KAROL BOMSZTYK

E. Role of pH, in T Lymphocytes Activated by Growth Factors

In contrast to the mitogen-induced intracellular acidification of human PBL, T cell growth Factors elicit alkalinization. The cell surface receptor for T cell growth factor IL-2 is composed of two polypeptides of 55 and 75 kDa in size (Green and Leonard, 1986; Sharon et u l . , 1986). When combined on the cell surface, these polypeptides form a high affinity receptor for l L 2 (Teshigawara el ul., 1987). IL-2 binding to its receptor is a required and sufficient event to induce the intracellular process leading to cell proliferation. Binding of 1L-2 to its receptor leads to intracellular alkalinization in HC0,- -free media due to activation of Na+ / H exchange (Mills er al., 1985). However, approximately 1000fold more IL-2 is required to induce alkalinization than to induce proliferation. Furthermore, the proliferative response to IL-2 has reached a plateau at an 1L-2 concentration that does not induce any alkalinization above baseline (Mills and May, 1987). In addition, IL-2 driven proliferation proceeds in HC0,- -frce media whcn Na /H exchange has been blocked by addition of dimethylamiloride or ethylpropylarniloride. Thus, similar to the action of mitogen or antibody on rodent T lymphocytes, these results suggest that neither activation of Na+ / I I + exchange nor a rise in pH, is required for IL-2-driven proliferation. +

+

+

IV. Na+/H+ ANTIPORT ACTIVITY AND pH, IN B CELL DIFFERENTIATION We have utilized the murine pre-B like cell line 70Z/3 to investigate carly events leading to lymphocyte activation. 70Z/3 was derived from a nitrosoureaderived tumor as described previously (Paige et al., 1978). This cell line does not express easily detectable levels of IgM on its cell surface. However, it does express IgM heavy chains but not light chains intracellularly. One of the K light chain gene allcles is rearranged, and the lack of cell surface IgM expression is due to very low level expression of K mKNA (Maki et of.,1980). We and others have shown that IL-1, IFN-y, and LPS can induce 70Z/3 to synthesize K mRNA, which can be detected in approximately 4-6 hr (Calalb er al., 1987; Perry and Kelly, 1979; Smith et ul., 198; Weeks and Sibley, 1988), and cell surface IgM (Stanton and Maynard, 1984; Giri el ul., 1984). When K mRNA levels are elevated by 6 hr, IgM becomes detectable on the surface of 70-90% of the cells by 24 hr. In this sense 70Z/3 can be said to have undergone “differentiation.” Expobure of 70213 to 1L-l (Calalb et al., 1987), IFN-y, (Smith el ul., 1988), or LPS (Rosoff and Cantley, 1983a), leads to activation of the Na+ /H + antiport, resulting in increased intracellular Nat concentration (“a+ 1,) and pH,. Treatment with IL-I resulted in increased “a+], (Stanton et al., 1986) and increased

217

9. ROLE OF INTRACELLULAR pH IN LYMPHOCYTE ACTIVATION

pHi (Calalb et al., 1987) first noted at 1 hr and peaking at 4 hr (Fig. 1). The IL- 1 induced increase in "a+], and pHi was abolished by pretreatment with the amiloride analog methylisobutylamiloride (Fig. 1). These experiments were performed in the presence of HCO, - . Since methylisobutylamiloride has been shown not to affect Na+-HCO,- cotransport (Alpern, 1985; Grass1 and Aronson, 1986), it is likely that increased N a + / H + exchange is responsible for the increased [Na+ji and pHi. Methylisobutylamiloride had no effect on the ability of IL-1 to induce cell surface IgM expression or to increase K light chain mRNA expression (Fig. 2 ) (Calalb et a/., 1987). [Na 1, and pH, were measured at the same time point that K mRNA was measured to insure that an unidentified pH, rise did not influence differentiation. At this time point, IL- 1 plus methylisobutylamiloride treated cells had no increased [Na+li or pHi. However, K mRNA expression in IL-1 plus methylisobutylamiloride-treatedcells was as great as in cells treated with IL- 1 +

r IL- lo(+MIA

r IL-IOC

0.075

-

-

*

pHl = 7.05 0.04 T

- _ - _ - pH, _ _= 7.06 - - t- 0.02 ----0.025

--_-

I

6.0

I

I

I

L

,

1

1

2

4

6

I

-

-2.0i

1

1

I

2

4

6

Tirndhovsl

Tlme(hours1

FIG. I . Time course of rIL-I-induced changes in pH, and "a+ l i in the presence or abscnce of methylisobutylamiloride (MIA) in 70213 cells. Aliquots of 1 X IOh cells were incubated with or without 20 unitsiml rlL-l u and assayed for pH, and "a+ 1, at each time point in the presence or absence of 5 X M methylisobutylamiloride. Data arc presented as the difference between rIL-l a-treated cells and a paired control done at the same time. The broken line represents the average baseline values for the control. Each data point is the mcan difference t S . E . of an average of nine cxpcriments done in triplicate. Asterisk (*) indicates at least p i0.05 statistical significance determined by paircd Student's f Lest (Calalb ef al., 1987).

218

THOMAS H. STANTON AND KAROL BOMSZTYK

MIA r IL-lo(+MIA r IL-lH

Untreated FIG. 2. Analysis by cytoplasmic dot hybridiLation of K light chain iriRNA levels in rlI,-l induced 70213 cells with or without methylisobutylamiloride. Cytoplasm was prepared and used at a concentration of 0 . 5 X 106 cellsidot after 5 hr incubation in RPMl 1640 media with or without 20 unitsiml 0 1 rIL-I OL and with or without 5 X 10 - h M methylisobutylamiloride (Calalh PI d., 1987).

alone (Fig. 2). This demonstrates that increased [Na + I i or pH, is not required for IL- 1 -induced 702/3 differentiation. We had initially found that amiloride itself inhibited IL-1 induced cell surface IgM expression (Stanton el ul., 1986). This was likely due to the higher concentration of amiloride over methylisobutylamiloride required to inhibit Na / H exchange, perhaps resulting in inhibition of protein synthesis. IFN-y treatment of 702/3 resulted in effects similar to those seen after 1L-1 1988). [Na ' 1, and pH, increased after 30 niin and treatment (Smith et d,, persisted for 4 hr. At 4 hr the pHi rise and Na+ rise were completely inhibited by methylisobutylamiloride and are thus likely due to Nai / H + antiport activity. At 30 min the pH, rise was only 30% inhibited by methylisobutylamiloride. This pH, rise was not due to Na+ /H exchange, since at the same time point, [Na l i did not rise. The methylisobutylamiloride-insensitivepH, rise was also seen in Na+ -free, HCO, - -free, or Ca2 -free media and in media that contained inhibitors of Cl-/OH- exchange. It is thus unlikely that the N a + / H + exchangeindependent pH, rise is due to Ca2 / H + exchange, C1- /OH- exchange, or Na+-HC0,- cotransport. We have not yet identified the Na+/H antiportindependent mechanism. As in the case of IL1,methylisobutylamiloride had no effect on the ability of IFN-y to induce expression of cell surface IgM or K light chain mRNA (Smith ei al., 1988). Therefore, it appears that N d + / H + exchange is not required for IFN-y-induced activation of 702/3. It is possible that the methylisobutyl+

+

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9. ROLE

OF INTRACELLULAR pH IN LYMPHOCYTE ACTIVATION

219

amiloride-insensitive pH, rise is important for IFN-y-induced K mRNA expression; however, it is not by itself sufficient, since IL-I-induced pH, rises could be prevented at all time points and 70213 were still induced to express K mRNA. It has been shown in 702/3 and other systems that phorbol ester can stimulate N a + / H + exchange (Rosoff et a/., 1984; Grinstein e t a / . , 1985, 1986a,b). Phorbol ester is known to be a specific, high affinity activator of protein kinase C by substituting for diacylglycerol, the product of membrane metabolism that normally activates protein kinase C (Kikkawa et al., 1983). We extended this observation by investigating the role of protein kinase C in inducing Na+/H+ after 1L-1 or IFN-y treatment (Ostrowski et a/., 1988). We found that indeed IL-I and IFN-y cause a translocation of protein kinase C activity from the cytosol to the membrane. The translocation peaked at approximately 30 min for both IL I and IFN-y and returned to starting levels by 60-120 min. To analyze the requirement for translocation of protein kinase C in activation of N a + / H + exchange, we employed the protein kinase C inhibitor H-7 [ 1 -(5-isoquinolinesulfonyl)-2-methylpiperazine]. A 100 pM concentration of H-7 inhibited protein kinase C activity over 80% but had no effect on either IFN-y or IL-l-induced activation of N a + / H + exchange (Fig. 3). Furthermore, depletion of protein kinase C activity by incubating 70Z/3 with phorbol ester for 72 hr had no effect on the ability of IL-I or IFN-y to induce enhanced Na+ / H + exchange. Thus, as is the case with thymocytes (Grinstein et al., 1987) and smooth muscle cells (Huang et ul., 1987), our results suggest that Na /H exchange can be enhanced via a protein kinase C independent pathway. Rosoff and Cantley investigated effects of the lectin LPS and phorbol esters on 70Z/3 (Rosoff and Cantley, 1983a; Rosoff et al., 1984). They found that both agents increased [Na+li and pHi and that these effects were blocked by methylisobutylamiloride or amiloride. It was thus concluded that activation of the N a + / H + antiport was responsible for the increased [Na+Ii and pHi. Amiloride also blocked LPS- and phorbol ester-induced expression of cell surface IgM, suggesting a requirement for increased pHi or [Na+Ii to induce differentiation of 70Z/3 with these agents. At the concentration they used, we also found amiloride blocked I L I induced 70213 differentiation (Stanton et al., 1986). However, lower concentrations of methylisobutylamiloride still blocked increases in "a+], and pH, but did not block differentiation, suggesting that N a + / H + exchange is not required for expression of K mRNA (Calalb et al., 1987). It is possible, however, that IL-1 and IFN-y induce differentiation of 70Z/3 in a manner different from LPS or phorbol ester and that the latter agents do require increased Na+ / H exchange activity to induce differentiation. The conclusion by Rosoff and colleagues that activation of Na+ / H + exchange is required for differentiation of 70Z/3 was strengthened by the finding that the pharmacological agent monensin, a Na+ ionophore, leads to increased "a+ 1, and pH, as well as 70Z/3 IgM expression (Rosoff and Cantley, 1983a). Monensin +

+

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220

THOMAS

H.STANTON AND KAROL BOMSZTYK

0.12

I 0

pn 1

2

-

7.wfo.u2 4

3

5

Tim (hwn)

pH -6.90f0.02

i

I

-0.04

0

1

1

I

I

3 Time (hours)

4

2

5

FIG.3 . Time course of rIL-1 induced changes i n pH, in 702/3 treated with or without H-7. Aliquots of 1 X 10" cells were assayed for pH, at the indicated time points. Data are prescnted as the difference in pH hetween rIL-1 (Y (20 units/nil) treated cells and paired control at the same time point. Broken line repreacnts the averagc baseline for cells not treated with rlL-I. Each data point represents a mean difieience 2 S.E. ot at least five cxperinienla pcI tiinc point donc in triplicate. Astcrisks indicate at leaat p <: 0.01 (*) and p < 0.05 (**). Statistical significance was determined by paired Student's r rest. (A) N o H-7; (B) plus 100 pM ti-7 (Ostrowski ~t ul., 1988).

could induce IgM expression in the presence of a concentration of amiloride that blocked the LPS- or phorbol ester-induced activity of the N a + / H + antiport. Since monensin raises [Na ' 3, and pH,, circumventing the amiloride blockade of the Na+ / H + antiport, it was concluded that increased [Na ' 1, or pH, was sufficient for inducing cell surface IgM cxpression.

9. ROLE OF INTRACELLULAR pH IN LYMPHOCYTE ACTIVATION

221

These results contrast with our results with IL-I and IFN-y that show that Na+ /H exchange is not required for 70Z/3 differentiation. We further investigated the ability of monensin and other weak bases to activate 70213. When 70Z/3 cells were treated with either monensin, methylamine, or NH,Cl, there was a rapid (<30 sec), transient rise in pH, of approximately 0.1 units. By 3-4 min, pH, was beginning to return to baseline. As has been noted previously (Rosoff and Cantley, 1983a), monensin also induced a rise in [Na+Ii. In contrast, methylamine and NH,CI led to a fall in [Na +Ii over the same time. Each of these agents induces expression of cell surface IgM after 24 hr of exposure (Fig. 4). That this was true IgM expression was confirmed by immunoprecipitation and SDS polyacrylamide gel analysis of radioiodinated cell surface IgM heavy and light chains from monensin. methylamine, NH,CI, LPS, and IFN-y treated cells but not from control untreated cells. In the case of LPS and IFN-y, the surface expressed 1gM was the result of newly synthesized heavy and light chains as seen by their incorporation of [35S]methionine. In contrast, monensin, methylamine, and NH,CI did not lead to increased incorporation of [35S]methionine into IgM heavy or K light chains. Furthermore, Western blots showed that the level of K chain was increased with LPS and IFN-y but not with the weak bases or monensin. Therefore, the increased surface IgM expression was not the result of decreased degradation of the intracellular pool of K chain. LPS- and IFN-y-induced expression of IgM is also dependent on increasing the level of K light chain mRNA. Monensin, methylamine, and NH,CI did not lead to any increase in expression of K mRNA (Fig. 5). Since these agents neither +

100

NH4Cl

I

'-

Monensln

Z 70

FlTC FLUORESCENCE FIG. 4. Cell surface IgM expression on 70213 cells after stimulation with weak bases, monensin, or IFN-y. 70213 at 0.5 X IOh cells1ml were cxposed for 24 hr to 25 mM methylamine, 12.5 mM NH4CI, I p.g/ml monensin. or 50 unitsiml IFN-y. Cells were stained with FITC-labeled goat antimouse p. chain. Immunofluorescence was measured using flow cytomctry with 5000 cells counted per experiment. FI'TC fluorescence is expressed on a three-decade log scale. Dead cells were electronically gated out by their right angle and forward scatter.

222

THOMAS H. STANTON AND KAROL BOMSTTYK

Control LPS IFN-Y IL-I

Methylam ne Monensin NH,CI Rti.5 . Analysis by cytoplasmic dot hybridization of K light chain mRNA levels in 702/3 treated with weak bascs, rnonensin, LPS, IFN-y, or IL-I. Cclls were treated for 4 hr with 10 pg/ml LPS,50 unitsirnl IFN-y, 20 unitsirnl rIL-I, 25 mM rnethylamine, I kg/ml monensin, or 12.5 mM NH,CI. Cytoplasmic RNA was prepared and used at a concentration of 0.5 X loGcellsidot.

increase K mRNA nor induce synthesis of new heavy or light chains, it is possible that they cause cell surface expression of IgM by mobilizing a preexisting pool of IgM to the surface. Indeed, we have noted morphological changes of 70213 after methylamine treatment that appear to show a migration of IgM-covered intracellular vesicles to the cell surface.

V.

CONCLUSION

Lymphocyte activation has been shown to cause pH, to either increase or decrease. The time course for altering pH, is generally rapid, within 1-10 min, especially in T lymphocytes, but may take 30-60 min in B lymphocytes stimulated with cytokines. Increased pHi most often results from increased activity of

9. ROLE OF INTRACELLULAR pH IN LYMPHOCYTE ACTIVATION

223

the Na / H antiport. Increasing the activity of the antiport is not dependent on changes in [Ca2+Ji nor is it dependent on protein kinase C activity, though protein kinase C can activate the Na+ / H + antiport in some settings. Decreased pH, occurs in mitogen- or antibody-stimulated human T lymphocytes. Decreased pH, appears to require an influx of extracellular C a 2 + . Under conditions of normal extracellular pH, changes in pH, are neither required nor sufficient for either lymphocyte proliferation or differentiation. Instead, the changes in pHi that accompany lymphocyte activation may serve to modulate lymphocyte response to ligand or may be important to maintain intracellular homeostasis in the face of increased metabolism. +

+

ACKNOWLEDGMENT Supported by grant DCB-87022.5 from thc National Science Foundation and a grant from the NW Kidney Foundation. KB is an Established Investigator of the American Heart Association. REFERENCES Alpern, R. ( I 985). Mechanism of basolateral membrane H /OH - /HCO - transport in the rat proximal tubule. J . Gen. Physiol. 86, 613-636. Aronson, P. ( I 985). Kinetic properties of the plasma membrane Na+ /H exchanger. Annu. Rev. Physiol. 47, 545-560. Aronson. P., Nee, J . , and Sufim, M. (1982). Modifier role of internal H + in activating the N a + / H + exchanger in renal microvillus membrane vesicles. Nature (London) 299, 161- 163. Besterman, J., Tyrey, S.,Cragoe, E., and Cuatracasas, P. (1984). Inhibition of epidermal growth factor induced mitogenisis by amiloride and an analog: Evidence against a requirement for N a + / H + exchanger. Proc. Natl. Acad. Sci. U.S.A. 81, 6762-6766. Boron, W. (1983). Transport of H + and ionic weak bases and acids. J . Membr. B i d . 72, 1-16. Boron, W. (1984). The basic connection. Nature (London) 312, 112. Calalb, M. B., Stanton, T. H., Smith, L., Cragoe, E. J . , Jr., and Bomsztyk, K. (1987). Recombinant human interleukin I-stimulated Na+/H+ exchange is not required for differentiation in pre-B lymphocyte cell line, 70213. J . B i d . Chem. 262, 3680-3684. Cheung, R. K., Grinstein, S.,and Gelfand, E. W. (1988). Mitogenic and non-mitogenic ligands trigger a calcium-dependent cytosolic acidification in human T lymphocytes. J . Immunol. 141, 1648- I65 I . Dixon, S . J., Cohen, S., Cragoc, E. J . , Jr., and Grinstein, S. (1987). Estimation of the number and turnover rate of N a + / H + exchangers in lymphocytes. J . B i d . Chem. 262, 3626-3632. Fischer, G . F., Holter, W., Majdic, O., Cragoe, E. J., Jr.. and Knapp, W. (1988). T cell stimulation via CD2 molecules is regularly accompanied by an increase in cytoplasmic pH. J . Immunol. 141, 404-409. Gelfand, E. W., Mills, G. B . , Cheung, R. K., Lee, J. W. W., and Grinstein, S. (1987). Transmembrane ion fluxes during activation of human T lymphocytes: role of CaZ+, N a + / H + exchange and phospholipid turnover. Immunol. Rev. 95, 59-87. Gelfand, E., Cheung, R . , and Grinstein, S. (1988). Calcium dependent intracellular acidification dominates the pH response to mitogen in human T cells. J. Immunol. 140, 246-252. Gerson, D., and Kiefer, H. (1983). Intracellular pH and the cell cycle of mitogen stimulated murine lymphocytes. J. Gen. Physiol. 114, 132-141. Gerson, D., Kiefer, H., and Eufe, W. (1982). Intracellular pH of mitogen stimulated lymphocytes. Science 216, 1009. +

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Ciiri, I., Kincade, P., and Mizel, S . (1984). lnterlcukin 1 mediated induction of k-light chain synthesis and surface immunoglobulin expression on pre-B cell. J fmmunol. 132, 223-228. Grassl, S . , and Aronson. P. (1986). Na + /HC03- co-transport in badatera] nienibranc vesicles isolated from rabbit renal cortex. J . B i d . Chem. 201, 8778-8783. Green, W., and Ixonard, W. (1986). The intcrleukin 2 receptor. Annu. Rev, Irnmunol. 4, 69. Grinstein, S . , Cohen, S . , Goetz, J., Rothstein, A,, and Gelfand. E. (1985). Characterization of activation of Na+ / H + exchange in lymphocytes by phorbol esters. Proc. Narl. Acad. Sci. U . S . A . 82, 1429. Grinstein, S., Goetz-Smith, J. D., Stcwart, D., Beresford. B. J., and Meliors, A. (1986a). Protein phosphorylation during activation of N a C /H exchange by phorbol esters and by osmotic shrinking. J . B i d . Chem. 261, 8009-8016. Grinatein, S . , Mack, E., and Mills, G . B. (198hh). Osmotic activation of the N a + / H + antiport in protein kinase C-depletcd lymphocytes. Biochern. Biiphys. Res. Commun. 134, 8- 13. Grinstein, S., Smith, J. D., Rowatt, C., and Dixon, S . J. (1987). Mechanism of activation of lymphocyte N a + / H + exchange by concanavalin A . J . R i d . Chcm. 262, 15277-15284 Grinstein, S . , Smith, J . , Onizuka, R . , Cheung, R . , and Gclfand, E. (1988). Activation o f N a + / H + exchange and the expression of cellular proto-oncogenes in mitogcn and phorbol ester treated lymphocytes. J. B i d . Chem. 263, 8658-8665. Heldin, C., and Westermark, B. (1984). Growth factors: mechanism of action and relation to oncogenes. Cell 37, 9-20. Hcskcth, T. R . , Moore, J. P., Morris, J. D. H., Taylor, M. V., Rogers, J., Smith, G. A , , and Metcalfe, J . C. (1985). A common sequcnce of calcium and pH signals in the mitogenic stimulation of eukaryotic cells. Narure (London) 313, 481. Howard, F., Ledhetter, J., Wong, J., Biebcr, G.. Stinson, E., and Herrenberg, L. (1981). A human T lymphocyte differentiation marker defined by monoclorial antibodies that block E-rosette formation. J . Immunol. 126, 2117. Huang, C., Cognan, M. G., Cragoe, E. J . , Jr., and Ives, H. E. (1987). Thrombin activation of the N a + / H + exchanger in vascular smooth muscle cells. J. B i d . Chem. 262, 14134-14140. Johnson, J.. Epel, D., and Paul, M. (1976). Intraccllular pH and activation of sea urchin eggs after fertilization. Nuture (London) 262, 661 -664. Kikkawa, U . , Takai, Y., Tdnaka, Y., Miyake, R . , and Nishizuka, Y. (1983). Protein kinase C possible receptor protein of tumor promoting phorbol esters. J. Biol. Chem. 258, 11442-1 1445. Koch. K . , and Leffer, H. (1979). Increased sodium ion influx is necessary to initiate rat hepatocyte proliferation. Cell 18, 157- 163. L'Allemain. G . , Franchi, A , , Cragoe, E. J., Jr., and Pouysscgur, J. (1984a). Blockade of the Na + / H antiport abolishing growth factor-induced DNA synthesis in fibroblasts. 1. B i d . Chem. 259, 4313-4319. L'Allemain, G., Paris, S . , and Pouysscgur. J. (1984b). Growth factor action and intracellular pH regulation in fibroblasts. J. A i d . Chem. 259, 5809-5815. LcfYert, H., Koch, H., Fchimann, M . , Heiser. W., Lad, P., and Skelly, H. (1982). Amiloride blocks cell free protein synthesis at levels attained inside cultured rat hepatocytes. Biochem. Biophys. Res. Commun. 108, 738-745. Lopez-Rivas, A., and Rozengurt, E. (1983). Serum rapidly mobilizes calcium from an intracellular pool in quiescent fibroblastic cells. Biochem. Biophys. Res. Commun. 114, 240-247. Lubin, M., andCahn, F. (1981).Amiloride, protein synthesis, and activation ofquiescent (GO) cells. J. Cell B i d . 91, 6. Maki, R., Kearny, J., Paige, C., and Tonegawa, S. (1980). Immunoglobulin gene rearrangements in immature B cells. Science 209, 1366-1371. Marx, J. (1987). Thefis gene as "master switch". Science 237, 854-856. +

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Mazia, D., and Ruby, A. (1974). DNA synthesis turned on in unfertilized sea urchin eggs by treatment with NH,OH. Exp. CellRes. 85, 167-172. Meur, S . , Hussey, L., Fabbi, M., Fox, D., Acuto, O., Fitzgerald, K . , Hodgdon, J . , Protentis, J . , Schlossman. S . , and Reinherz, E. (1984). An alternative pathway ofT-cell activation: a functional role for the 50 kd TI I sheep erythrocyte receptor protein. Cell 36, 897-905. Mills. 0. B., and May, C. (1987). Binding of interleukin 2 to its 75-kDa intermediate affinity receptor is sufficient to a activate Na + /H exchange. J. Immunol. 139, 4083-4087. Mills, G. B., Cragoe, E. J., Jr., Gelfand, E. W., and Grinstein, S. (1985). Interleukin 2 induces arapid increase in intracellular pH through activation of a Na + /H + antiport. J. B i d . Chem. 260, 1250012507. Mills, G. B., Cheung, R. K . , Cragoe, E. J., Jr., Grinstein, S . , and Gelfand, E. W. (1986). Activation of the Na+ /H antiport is not required for lectin-induced proliferation of human T lymphocytes. J. Immund. 136, 1150-1 156. Moolenaar, W. H. (1986). Effects of growth factors on intracellular pH regulation. Annu. Rev. Physiol. 48, 363-376. Moolenaar, W. H., Tertoolen, L. G . J., and de Laat, S . W. (1984). Phorbol ester and diacylglycerol mimic growth factors in raising cytoplasmic pH. Nature (London)42, 371-374. Ostrowski, J., Jeier, K . E., Stanton, T. H., Smith, L. L.. and Bomsztyk, K. (1988). lnterferon(gamma) and interleukin- 1 alpha induce transient translocation of protein kinase C activity to membranes in a B lymphoid cell line. J . B i d . Chem. 263, 13786-13790. Owen, N., and Villereal, M. (1982). Effects of intracellular calcium antagonist TMB-8 on serum stimulated Na+ influx in human fibroblasts. Biochem. Biophys. Res. Commun. 109, 762-768. Paige, C., Kincade, P., and Ralph, P. (1978). Murine B cell leukemia line with inducible surface immunoglobulin expression. J. lmmunol. 121, 64 1-647. Paris, S . , and Pouyssegur, J. (1983). Biochemical characterization of the amiloride-sensitive Na+ /H antiport in Chinese hamster lung fibroblasts. J . B i d . Chem. 258, 3503-3508. Perry, R., and Kelly, D. (1979). Immunoglobulin messenger RNA in murine cell lines that have characteristics of immature B lymphocytes. Cell 18, 1333- 1342. Pouyssegur, J., Chambard, J., Franchi, A., Paris, S . , and Van Ohberghen-Schilling, E. (1982). Growth factor activation of an amiloride sensitive Na /H exchange system in quiescent fibroblasts. Pro<. Nod. Aead. Sri. U.S.A. 79,3935-3939. Pouyssegur, J., Sardet, C . , Franchi, A , , L'Allemain, G., and Paris, S . (1984). A specific mutation abolishing Na+ / H + antiport activity in hamster fibroblasts precludes growth at neutral and acidic pH. Proc. Natl. Acad. Sci. U.S.A. 81, 4833-4847. Rink, R. J., Tsien, R. Y.,and Pozzan, T. (1982). Cytoplasmic pH and free Mg2+ in lymphocytes. J . Cell B i d . 95, 189-196. Roos, A , , and Boron, W. (1981). lntracellular pH. Phvsiol. Rev. 61, 296-434. Rosoff, P. M., and Cantley, L. C. (1983a). Increasing the intracellular Na+ concentration induces differentiation in a pre-B lymphocyte cell line. Proc. Natl. Acad. Sci. U.S.A. 80, 7547-7550. Rosoff, P. M., and Cantley, L. C. (1983b). Lipopolysaccharide and phorbol esters induce differentiation but have opposite effects on phosphatidylinositol turnover and Caz+ mobilization in 70213 pre-B lymphocytes. J. B i d . Chem. 260, 9209-9215. Rosofi, P. M . , and Cantley, L. C. (1985). Stimulation of the T3-T cell receptor-associated Ca*+ influx enhances the activity of the Na + /H exchanger in a leukemic human T cell line. J. B i d . Chem. 260, 14053-14059. Rosoff, P. M., and Terres. G. (1986). Cyclosporine A inhibits Ca2+-dependent stimulation of N a + / H + antiport in human T cells. J. Cell B i d . 103, 457-463. Rosoff, P. M., Stein, L. F.. and Cantley, L. C. (1984). Phorbol esters induce differentiation in a PreB-lymphocyte cell line by enhancing N a + / H + exchange. J. B i d . Chem. 259, 7056-7060. +

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Sharon, M., Klausner. R . , Cullen, B., Chizzonite, R., and Leonard, L. (1986). Novel interleukin 2 receptor subunit detected by crosslinking under high affinity conditions. Science 234, 859. Siffert, W., and Akerman, J. W. N. (1988). Protein kinase C enhances Ca2+ mobilization in human platelets by activating N a + / H + exchange. J . Biol. Chem. 263, 4223-4229. Smith, L. L., Stanton, T. H., Calalh, M. B., and Bomsztyk, K. (1988). Recombinant m u r k interferon-(gainma)-induced differentiation of pre-B lymphocytes is associated with Na+ /H exchange-dependent and -independent cytoplasmic alkalinization. J . B i d . Chem. 263, 73597363. Stanton, T.. and Maynard, M. (1984). Cytokine effects on a B ccll line. Immunol. Left. 7, 301-307. Stanton, T. H.. Maynard, M., and Bomsztyk, K. (1986). Effect of interleukin-I on intraccllular concentration of sodium, calcium, and potassium in 70213 cells. J. Biol. Chem. 261, 56995701. Teshigawara, K., Wang, H., Kato, K., and Smith, K. (1978). Interleukin 2 high atfinity receptor expression requires two distinct binding proteinc. J . Exp. Mrd. 165, 223. Vara, F., and Rozengurt. E. (1985). Stimulation of Na' / H + antiport activity by epidermal growth factor and insulin occurs with out activation of protein kinase C. Biochrm. Biophys. Res. Commun. 130, 646-653. Villereal, M . , Owen, N . , Vincenti, L., Mix-Muldoon, L., and Jamieson, G . (1985). Mechanism for growth factor induced increase in Na+ / H +exchange and rise in C a + activity in cultured human fibroblasts. Cuncer Rrs. 3, 417-424. Weeks, R..and Sibley, C. (1988).Inducible expression of transfcctcd kappa light chain by LPS and IFN-(gamma) in the murine B lymphoma 702/3. J. Immunol. 140, 1312-1320. Weiss, A , , and Imboden, 1. B. (1987). Human T lymphocyte activation. Adv. Immunol. 41, 1-31. Winkler, M . , and Grainger, J. (1978). Mechanism of' action of NH&I and other weak hases in thc activation of sea urchin eggs. Nuture (London) 273, 536-538. ~dlndgUchi,D. T., Sakai, R., Bahn, L . , Cragoe, E. J . , Jr., and Jordan, S . C. (1986). Amiloride inhibition of DNA synthesis and immunoglobulin production by activated human peripheral blood niononuclear cells is independent of sodiumihydrogen antiport. J . Immunol. 137, 13001304. Yang, S . . Chouaib, S., and duPont, B. (1968). A common pathway for T lymphocyte activation involving both the CD3-Ti complex and CD2 sheep erythrocyte receptor determinants. J . Immunol. 137, 1097. Zctterberg, A , , and Engstrom, W. (1981). Mitogenic effect of alkaline pH on quiescent, serumstarved cells. Proc. Natl. Acad. Sci. U . S . A . 78, 433444338, +

CURRENT TOI'ICS IN MEMBRANES AND TRANSPORT, VOLUME 15

Chapter 10 Regulation and Functional Significance of Cytoplasmic pH in Phagocytic Leukocytes CAROL J. SWMLOW,* SERGIO GRINSTEIN, f AND ORI D . ROTSTEIN" *Department of Surgery Toronto Generul Hospitul University of Toronto Toronto, Onturio, Canudu TDivision of Cell Biology The Hospitul for Sick Children Toronto, Ontario, Canada

Mechanisms of Cytosolic pH Regulation in Phagocytic Leukocytes A. N a + / H + Exchange B. CI-/HC03- Exchange C. Na+-Dependent C1- /HC03- Exchange D. ATP-Dependent H + Extrusion 11. Role of Cytoplasmic pH in Phagocytic Cell Activation and Modulation of Function A. Migration B . Microbicidal Activity C. Implications o f Altered pH, on Phagocyte Function in Vivo D. The Physiological Role of Proton Extrusion in Phagocytic Cells 111. pH, Changes during Phagocytic Cell Diffcrentiation: Regulation and Significance A. Granulocytic Differentiation B. Monocytic Differentiation C. Macrophage Activation References

I.

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MECHANISMS OF CYTOSOLIC pH REGULATION IN PHAGOCYTIC LEUKOCYTES

Like other mammalian cells, phagocytic leukocytes regulate their cytoplasmic pH (pH,) within very narrow boundaries. This is accomplished by the parallel 227 Copyright Q 19Sil by ALademic Press lnc All nghts of repruduction In dny tom rcirrved

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operation of a multiplicity of systems that transport acid equivalents into or out of the cells, usually in the form of protons or bicarbonate. The requirement for the simultaneous existence of seemingly redundant pathways is not understood, but it stresses the importance of pH, homeostasis for normal leukocyte function. This section describes individually the nature and properties of the pHi regulatory systems reported in phagoeytic cells (see Table I).

A. Na+/H+ Exchange The most widely studied mechanism of pHi regulation in leukocytes is an electroneutral exchanger (antiport) of Na for H + . This antiport has been detected in rabbit (Molski et al., 1980) and human (Grinstein and Furuya, 198th; Simchowitz and Cragoe, 1987) neutrophils, in peritoneal macrophages from rats (Rotstein et al., 1987a) and mice (Swallow e t a l . , 1988), in human blood monocytes (Grinstein et al., 1984), and in transformed myeloid cell lines, including the nionocytic U937 cells (Ladoux et al., 1987b) and HL60 cells differentiated along both the monocytic and granulocytic pathways (Ladoux et d., 1987a; Restrepo et a / . , 1988). While only a few of these systems have been studied in detail, the properties described seem to be universal and very similar to those of N a + / f I + antiports in other cell types (see Decker and Dietcr, 1988; Grinstein et d.,1989, for reviews). For simplicity, the information available, obtained mostly from human neutrophils, is generalized below. +

TABLE 1 MECHANISMS OF pH, REGULATION I N PHAGOCYTIC CELLS Cell typc

Na' / H + antiport

Rabbit neutrophils Human neutrophils

HL60 cell\ U937 cells

Molski et a / . (1980) Grinstein and Furuya (1986b); Simchowitz and Crdgoe (1987) Rotstein et ul. (1987a) Swallow ef a!. (1988) Ladoux et al. (1987b) Ladoux et ( I / . (1987a); Restrepo et (I/. (1988) Simchowitz and Roos (1985); Simchowitr and De Wccr ( I 986) Rcstrepo et ul. (1988) Ladoux er a/. ( 1987b)

Munne macrophages

Swallow rr d.(1988)

Rat macrophages Murine macrophages U937 cells HL60 cells CI IHCO,

exchdnge

Nn+-dependent C1- IHCO, exchdnge ATP-dependent H + ex tru\lon

References

Humdn neutrophila

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The antiport has been studied by measuring the influx of radioactive Na+ or the changes in intracellular or extracellular pH that accompany Na /H exchange. Comparison of the rates of intra- and extracellular pH change indicates that acid equivalents, likely H , are translocated outward across the plasma membrane. The net rate of acid extrusion is comparable to the rate of Na+ uptake, indicating that the stoichiometry is approximately one-to-one. Accordingly, the exchange process is electroneutral and insensitive to changes in transmembrane electrical potential. Inhibitors of other cation transport systems, such as ouabain and furosemide, have little effect on Na+ / H + exchange in leukocytes. In contrast, the antiport is very susceptible to inhibition by micromolar concentrations of amiloride and related pyrazine derivatives (Simchowitz and Cragoe, 1986). In particular, analogs where the 5-amino group of amiloride was replaced by short alkyl groups were much more potent (up to 100-fold) than amiloride itself. In all cases, the inhibition is very fast and readily reversible, and at least for amiloride, inhibition is fully competitive with extracellular Na+ , suggesting that the drug binds to the externally facing transport site (Grinstein and Furuya, 1986b). As in other cells, the antiport of leukocytes is selective for Na+ and Li+ , while K , Rb , and Cs , as well as organic cations, are not transported. Whereas the affinity for Li+ is higher than that for N a + , the latter ion, which is the physiological substrate, is translocated more effectively. Extracellular H can also occupy the transport site and effectively compete with N a + , so that decreasing the extracellular pH results in inhibition of forward (Na JH i ) exchange. Complete suppression of exchange was observed at pH, < 6.3. Inhibition of forward exchange was also obtained when the intracellular Na+ concentration was increased, possibly due to competition with H t i for the internal transport site (Grinstein and Furuya, 1986b). Contrary to the effects of extracellular pH, an intracellular acidification greatly accelerated the rate of Na+,/H + i exchange. A linear relationship was found between the rate of exchange and pHi in the 6.5 to 7.2 range. At pHi 2 7.2, the antiport of otherwise untreated cells is virtually quiescent, but a marked activation is noted as the cytoplasm is rendered acidic. This behavior is consistent with a role of the exchanger in pH, homeostasis, defending the cells against their spontaneous tendency to become acidic due to metabolic acid production and electorphoretic H (equivalent) influx. The pronounced sensitivity of the antiport to pH, has been attributed to the existence of an allosteric “modifier” site that controls the rate of countertransport. When protonated, the internally oriented allosteric site activates exchange, thereby protecting the cytosol against excessive acidification. At higher pHi, deprotonation of the modifier curtails the activity of the antiport, preventing alkalinization of the cytosol beyond the physiological level. Though in resting cells the antiport is practically quiescent at normal pHi, +

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activation can be obtained when the cells are stimulated by any one of a variety of agents. In cells suspended in nominally bicarbonate-free media, the activation is manifested as an elevation of the intracellular pH (Molski et al., 1980; Weisman et al., 1987; Simchowitz, 1985a). Several lines of evidence indicate that this alkalosis is due to stimulation of Na+ / H exchange: ( I ) the pH, change is accompanied by a marked increase in the rate of Na+ uptake; (2) the alkalinization is eliminated by omission of extracellular Na+ , and (3) both the pH, change and the acceleration of Na uptake are prevented by amiloride and its analogs (Molski r f ul., 1980; Simchowitz, 1985a; Wright et al., 1986; Weisman er d.,1987). In fact, in the presence of amiloride or in the absence of Nai , stimulation induces a decrease of pH,, which has been attributed to metabolic acid production (Grinstein et al., 1985). Stimulation of Na /H exchange has been reported for both neutrophils and macrophages. The list of agents that stimulate cation exchange includes the chemotactic peptide formylmethionylleucylphenylalanine (fMLP), the phorbol ester 12-0-tetradecanoylphorbol-13-acetate (TPA), leukotriene B,, and the divalent cation ionophore A23187 (Molski et d.,1980; Grinstein and Furuya, 1984; Sumimoto et al., 1988; Volpi or al., 1985). Sha’afi and colleagues have investigated the mechanism of activation of the antiport in some detail. Their studies suggest that surface receptors convey the activating message to the cell interior through a GTP-binding (G) protein that is susceptible to inhibition by pertussis toxin (Volpi et a / . , 1985). The G protein then activates phospholipase C, which releases diacylglycerol from phosphoinositides, thereby stimulating protein kinase C. The kinase presumably phosphorylates the antiport itself or an ancillary protein, inducing activation of Na+ / H + exchange. Support for this model comes from several findings. First, the activation of the antiport can be mimicked by exogenously added phospholipase C (Weisman et u / . , 1987) and by phorbol esters, which directly stimulate C kinase (Molski et a / ., 1986). Second, elevation of cytosolic [Ca2 1 by means of ionophores, which can activate the endogenous phospholipase C, is associated with activation of the antiport (Satoh ef ul., 1985). Third, inhibitors of protein kinase C, such as H-7 and polymyxin, prevent the stimulation of Na+ / H + exchange induced by fMLP (Molski et al., 1986). At the kinetic level, stimulation of the antiport by leukocyte activators is manifested as an alkaline shift in the pH, dependence of N a + / H + exchange. This shift apparently reflects an altered behavior of the “modifier,” inasmuch as this allosteric site largely determines the pH, sensitivity of the exchanger. According to this model, the set point of the modifier is adjusted upward by ~ 0 . 2 5 pH units, which is the magnitude of the alkalosis recorded in cells stimulated with fMLP or other activators. It is conceivable that this change in pH, sensitivity is brought about by phosphorylation of the antiport itself or of a vicinal protein. +

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6. CI- /HCO,-

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Exchange

An anion exchange pathway has been described in the plasma membranes of phagocytic cells (Simchowitz and Roos, 1985; Simchowitz et al., 1986; Simchowitz and De Weer, 1986; Restrepo et al., 1988). The exchange process is independent of the presence of cations and translocates anions across the membrane with a 1 : 1 stoichiometry. As a result, transport is electroneutral and potential insensitive. The physiological substrates appear to be CI - and HCO, - , the predominant inorganic anions. The kinetic properties of the anion exchanger of neutrophils, measured as C1- I T L - exchange, have been studied in detail by Simchowitz et al. (1986). These authors found that, as expected for carriermediated transport, anion exchange in neutorphils displays trans-stimulation, competition with other anions, and saturability. An apparent K , of 5-22 mM was determined for CI- uptake, depending on the substitute anion used. Unlike the anion exchanger of most other cells, that of neutrophils was found to be relatively insensitive to disulfonic stilbene derivatives. Instead, exchange was effectively inhibited by a-cyano-4-hydroxycinnamate. In contrast to the mature human neutrophils, the promyelocytic HL60 cells, which can differentiate along the granulocytic line, were susceptible to inhibition by 4,4'-diisothiocyanostilbene-2,2'-disulfonate (DIDS) and by pentachlorophenol (Restrepo et al., 1988). Like the Na+ / H + antiport, the anion exchanger is believed to play a central role in the regulation of pHi in phagocytic cells. The extrusion of intracellular HC0,- (a base equivalent) in exchange for external CI- tends to lower the cytosolic pH. Several findings suggest that this reaction is important for the recovery of physiological pH, following an intracellular alkalosis. Alkali-loaded neutrophils recover their normal pHi within minutes. The recovery process is enhanced by addition of HC0,- and is inhibited by omission of extracellular CI- or by the presence of a-cyano-4-hydroxycinnamate (Simchowitz and Roos, 1985). These findings are consistent with activation of (C1 -),/(HCO, -)iexchange upon intracellular alkalinization, possibly due to the accumulation of intracellular HCO, in the cytoplasm. In HL60 cells, the anion exchanger is believed to play the opposite role, namely to promote the net uptake of HC0,- in exchange for cellular C1- , thereby elevating pHi. Restrepo et al. (1988) found that addition of DIDS or pentachlorophenol to these cells resulted in a significant drop in pHi. This acidification can be accounted for by inhibition of anion transport, assuming that (CI -),/(HCO, -)<,exchange is active constitutively, contributing to the maintenance of pHi above the level predicted by electrochemical equilibrium. This assumption would require the existence of an independent mechanism whereby C1- is actively accumulated inside the cells. The existence of such a process has not been confirmed in HL60 cells, where Na+-K+-2Cl- co-transport, a likely -

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ET AL.

candidate, has been ruled out (Restrepo et al., 1988). However, a 2-deoxyglucose-sensitive and presumably active component of Cl - uptake was described in neutrophils (Simchowitz and De Weer, 1986). It remains to be established whether the rate of C1- uptake through this pathway is compatible with the rates of DIDS-sensitive H equivalent extrusion recorded in HL60 cells. +

C. Na+-Dependent CI- /HCO,-

Exchange

In several cell types, all or part of the exchange of intracellular CI- for external HCO, is dependent on the availability of extracellular Na+ . Such behavior is due to the existence of a distinct transporter, which mediates the uptake of Na ' and HCO,- in exchange for C1- and possibly H ' . In the process, two acid equivalents are cxtruded by the cells, driven mainly by the inward Na+ gradient. A similar system has been described in a phagocytic line, the prornonocytic U937 cells (Ladoux et af.,1987b). In these cells, the Na+-dependent anion exchanger can be detected as a Na - and bicarbonate-dependent recovery from an acid load. This protocol, together with isotopic measurements of Na and CI- fluxcs were used by Ladoux et al. (1987b) to characterize the system. Briefly, these authors found that whereas Na'. supported the exchange of (HC0,-),, for (CI- ) i , Li+ and K + were ineffective. As expected, the exchange process was precluded by omission of extracellular HC0,3- and by depletion of intracellular CI- . Transport was also inhibited by DIDS, with a of 10 pM. Though the Na+-dependent anion exchanger shares the sensitivity to stilbenes with the Na -independent exchanger described above, the two transporters appear to be separate molecular entities, as indicated by pharmacological studies (Madshus and Olsnes, 1987). Indeed, in U937 cells only the Na -dependent exchanger was detected, whereas no evidence was found for cation-independent CI - /HCO, - exchange. Na ' -dependent anion exchange in U937 leukocytes and in other cells is stimulated by intracellular acidification. This property, together with the net extrusion of two acid equivalents effected during each transport cycle, suggests that this exchanger is involved in the defense of pH, against acid loading as well as in the maintenance of resting pH,, which tends to acidify spontaneously due to metabolic acid production and electrophoretic accumulation of H cquivalents. ~

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D. ATP-Dependent H -c Extrusion Like other cells, rnurine macrophages recover from an imposed acid load within minutes. The recovery of pH, is accompanied by an increased rate of extracellular acidification, indicative of transmembrane flux of acid equivalents.

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10. CYTOPLASMIC pH IN PHAGOCYTIC LEUKOCYTES

Unexpectedly, a sizable component of this pHi regulatory process was found to persist in the absence of extracellular Na+ (ruling out Na+ / H exchange and Na+ -dependent anion exchange) and in the nominal absence of HC0,- (making C1- /HCO, exchange an unlikely candidate) (Swallow et al., 1988). That these transport systems are not responsible for the Na+ - and HC0,--independent recovery was confirmed using amiloride and disulfonic stilbenes, which failed to inhibit the pH, change. Instead, H + extrusion was found to be blocked by N ethylmaleimide, N,N’-dicyclohexylcarbodiimide,p-chloromercuribenzene sulfonate, and 7-chloro-4-nitrobenz-2-oxa- 1,3-diazole (NBD-CI). These reagents are known inhibitors of H +-transporting ATPases. That an active H pump is involved was also suggested by the ATP dependence of the pH, recovery of macrophages (Swallow et al., 1988). The putative H pump is probably different from the mitochondrial F,F, ATPase and from the gastric E,E, ATPase in that it was not inhibited by oligomycin or by vanadate, respectively. Instead the pharmacological profile of the macrophage plasma membrane “pump” resembles that of vacuolar or microsomal H + pumps. In particular, preliminary results indicate that recovery from an acid load is blocked by micromolar concentrations of bafilomycin A , , a macrolide antibiotic that at low concentrations selectively blocks vacuolar but not mitochondrial or E,E, type ATPases (Bowman et al., 1988). The detailed mechanism of ATP-dependent H extrusion in macrophages has not yet been elucidated. However, recovery from an acid load is accompanied by hyperpolarization of the plasma membrane (C. J. Swallow et al., unpublished observations), suggesting an electrogenic mechanism. The charge neutralizing ions that must accompany the substantive H + movement observed during pH, recovery have not been identified. They are unlikely to be cations entering the cell, since recovery from an acid load persists in isotonic sucrose lightly buffered with Hepes-Tris. Instead, cellular anions may accompany the exit of H + equivalents. In accordance with this interpretation, partial inhibition of H extrusion was recorded in cells depleted of C1- . Conceivably, nitrate or organic anions could also serve as counterions, facilitating H + extrusion. With the exception of some acid secreting epithelia, such as the renal collecting duct, plasma membrane H pumps have not been described in animal cells other than macrophages and osteoclasts. The latter cells utilize the pumps to create an acidic compartment wherein bone is resorbed. In macrophages, the role of the putative pumps has not been definitely established. They may contribute to the acidification of the phagosomal compartment. In addition, they may play a role in the regulation of intracellular pH. This specialized mechanism of pH, regulation would be advantageous to macrophages in the acidic milieu of tumors and abscesses, where the concentration of HC0,- is low and N a + / H + exchange is at least partially inhibited. +

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11.

ROLE OF CYTOPLASMIC pH IN PHAGOCYTIC CELL ACTIVATION AND MODULATION OF FUNCTION

Engulfment and digestion of invading microorganisms in one of the primary functions of the phagocytic cell population (i.e., neutrophils and macrophages). In order to achicve this end, these cells must migrate from the bloodstream to the site of the foreign pathogens, where they subsequently ingest and kill the infecting bacteria. As noted in the previous section, the pH response of neutrophils and macrophages to most stimuli in vitm consists of an initial acidification phase followed by an alkalinirdtion phase. In the in vivo setting, these cell types must occasionally function within a markedly acidic microenvironment (Bryant et d., 1980). Thus, an understanding of how changes in pH, contribute to cell activation and modulation of function may provide important insight into cell function and dysfunction within an inflammatory focus. The major aims of this section are ( 1 ) to review the potential contribution of alterations in cytoplasmic pH to cell activation, (2) to summarize the role of cytoplasmic pH in modulating cell function, and (3) to consider potential functional roles of proton extrusion by phagocytic cells. Because relatively little functional information regarding macrophages is presently available, the discussion will focus mainly on neutrophils. In broad terms, neutrophil function can be divided into two major areas: (1) migration and (2) microbicidal activity.

A. Migration Activation of the actin-based cytoskeletal network is considered to be responsible for changes in cell shape, membrane ruffling, and cell migration following exposure to chcmotactic peptides (White et at., 1983; Fechheimer and Zigmond, 1983). Activation of this network, as measured by actin polymerization or changes in cell shape (Sklar et al., 1985), has been used as an early indication of the cell response to these stimuli. Using this endpoint, several lines of evidence suggest that the initial acidification phase associated with exposure to chemoattractants, such as fMLP and leukotriene B,, plays a role in initiating cell migration (Naccache, 1987; Yuli and Oplatka, 1987; Naccache ef al., 1989). These include: ( I ) cell acidification induced by agonists is accompanied by actin polymerization, (2) prevention of agonist-induced acidification with protonophores obviates actin polymerization (Yuli and Oplatka, 1987), and (3) the time course and dose responses for agonist-induced acidification and actin polymerization are similar (Faucher and Naccache, 1987). The link between cytoplasmic acidification and activation of the cytoskeletal network is further strengthened by studies in which acidification was induced by propionic acid (reviewed in Naccache, 1987). Over a range of concentrations from 0.5 to 30 mM, propionic acid is able to initiate actin polymerization. In addition, at concentrations as low as 5 mM,

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propionic acid causes a light scatter response (a measure of cell shape change) that approximates that of a chemoattractant dose of fMLP ( 10 M) (Faucher and Naccache, 1987). Cytoplasmic acidification in response to both treatments is comparable. The mechanism(s) underlying the ability of chemoattractants and weak acids to induce actin polymerization is not completely defined. Pertussis toxin blocks actin polymerization in response to propionic acid without inhibiting acidification, indicating that the effect of the acidification is, at least in part, mediated through G proteins (Molski and Sha’afi, 1987). Weak acids may initiate activation of actin polymerization via mobilization of the second messenger calcium. In this regard, a comparable elevation in cytoplasmic calcium occurs in response to both fMLP- and propionic acid-induced acidification (Naccache et al., 1988). However, previous studies have demonstrated that a rise in cytoplasmic calcium is neither necessary nor sufficient for actin polymerization (Sha’afi et al., I986), thereby making this mechanism unlikely. Alkalinization mediated by Na+ /H antiport activity in response to cytoplasmic acidification also appears to play no role in initiating the light scatter response. Activation of antiport occurs temporally after this response, and in addition, the shape change is not prevented by the specific Na+ /H antiport inhibitor, amiloride, or by removing Na+ from the medium (Faucher and Naccache, 1987). The effect of cytoplasmic acidification does not appear to be mediated through the release of neutrophil-derived cell activators, a phenomenon that occurs in response to acid treatment of neutrophils (Zigmond and Hargrove, 1981). The propionic acid-induced scatter response is not affected by treatment of the cells with an inhibitor of leukotriene synthesis or an antagonist of platelet activating factor (Faucher and Naccache, 1987). This suggests that the effect is not mediated by either of these two chemoattractants. In addition, treatment of cells with 2 mM HCl, which reduced extracellular pH by 0.3 units, had no effect on light scattering. This is in contrast to the effect of propionic acid, which had no effect on extracellular pH. Indeed, it appears that absolute cytoplasmic acidification is not required for the light scatter response. Treatment of cells alkalinized with ammonium chloride with fMLP or propionic acid induces a light scatter response without lowering pH below resting levels (Naccache et al., 1989). This suggests that a change in pH rather than cytoplasmic acidification per se is the responsible initiating event. Having summarized the association between cytoplasmic acidification in response to chemotactic peptides and the initiation of actin polymerization, a causal relationship between the two and their contribution to subsequent cell migration remains to be defined. The finding that neutrophils from patients with chronic granulomatous disease exhibit normal migration, despite their failure to acidify following stimulation with an agonist (Grinstein et al., 1986), makes it unlikely that acidification alone is the crucial signal for migration. Furthermore, ~

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studies by Naccache and colleagues ( 1989) demonstrated that prevention of agonist-induced acidification using a cytoplasmic calcium chelator did not ablate the cytoskcletal rearrangement occurring in response to cell stimulation. These results differ from those reports by Yuli and Oplatka (1987). As noted, these authors demonstrated that protonophorcs prevented the light scatter in eutrophils initiated by exposure to f'h4LP and propionic acid. Presumably, the protonophores did so by abolishing the initial acidification phase. However, huge doses ( 10 M) were required to completely prevent the light scatter response, suggesting the possibility of a nonspecific effect. At doses commonly used for dissipation of proton gradients (3.3-7.5 KM), there was only a minimal effect on the light scatter. In addition, these authors did not measure cytoplasmic pII in cells treated with protonophores. Indeed, one might expect that, due to the negative membrane potential of neutrophils, protonophores might themselves induce an initial acidification. Regardless of whether acidification signals actin polymerization, it is clear that thc absolute level of cytoplasmic pH is able to modulate the chemotactic response of neutrophils to various stimuli. Sirnchowitz and Cragoe (1986) demonstrated a linear correlation betwccn net migration (the difference between directed and random motility) and pH, over an intracellular pH range of 6.8-8.6. In general, cytoplasmic acidification relative to resting pHi (pH 7.25) impaired migration, whether acidification was achieved through inhibition of the Na+ / H antiport or through acid loading of the cell. These results are corroborated by other studies in which pH, was not measured directly but was effectively reduced by maneuvers such as reducing extracellular pH or substituting extracellular Na with other cations (Showell and Becker, 1976; Rabinovitch et a l . , 1980; Kotstein et a l . , 1988). At elevated cytoplasmic pH, Simchowitz and Cragoe ( 1986) found that net migration was stimulated. Up to pH 7.7, increased net migration was the result of a progressive increase in directed migration with no change in random motility. At cxtracellular pH levels greater than 7.7, both directed migration and random migration decreased progressively. The fact that the latter decreased more than the former resulted in an ovcrall incrcasc in nct migration. Thcsc authors postulatcd a major role for pH, in the regulation of actin polymerization as a mechanism for its modulatory effect on cell migration. In this regard, cytoplasmic alkalinization by exposure to ammonium chloride causes a sustained actin polymerization in neutrophils (Faucher and Naccache, 1987). Further studies arc required to clarify the mechanism by which this effect enhances cell migration in response to chemoattractants. ~

+

B. Microbicidal Activity 1 . R t S P I R A T O R Y BURST

Toxic oxygen radicals generated following stimulation of the respiratory burst in both neutrophils and macrophages are an integral part of these cells' micro-

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bicidal armamentarium. Although the time course of initiation of the respiratory burst corresponds well with the early acidification phase following cell stimulation, there is presently no evidence that acidification serves as a signal for the respiratory burst. Rather, it has been suggested that the acidification is a consequence of the generation of metabolic acids by the respiratory burst (Grinstein and Furuya, 1986a), although later studies have disputed this (Naccache et al., 1989). Cytoplasmic pH appears to modulate agonist-induced generation of oxygen radicals. Reduction of pH, impairs activation of the respiratory burst in response to both fh4LP (Simchowitz, 1985b) and TPA (Nasmith and Grinstein, 1986). The most obvious explanation for this effect is that an acidic intracellular milieu impairs the NADPH oxidase system, which displays a pH optimum in the 6.8 to 7.9 range in vitro (Tauber and Goetzl, 1979). Clearly, several other enzyme systems involved in this cascade may also be involved in this effect. By contrast, an alternative explanation must be cited to account for the stimulation of superoxide production at pH, levels > 7.22, as reported by Simchowitz (1985b). While the mechanism underlying this enhancement was not specifically examined, it did not appear to be due to increased receptor-ligand interaction at elevated pH.

2. LYSOSOMAL DEGRANULATION Oxygen-independent bacterial killing, in particular via lysosomal degranulation, is the other major microbicidal system present in phagocytic cells. Recent studies have demonstrated that cytoplasmic acidification in macrophages causes the rapid movement of lysosomes to the periphery of the cell (Heuser, 1989). This movement occurs along the distribution of the microtubules and is prevented if microtubules are disrupted. The significance of this lysosomal trafficking is not clear, but a role in mobilizing lysosomes to the cell surface in preparation for exocytosis or for fusion with phagosomes may be possible. Further studies are required to examine these considerations.

C. Implications of Altered pH, on Phagocyte Function in Vivo Both in vitro and in vivo studies (Bryant et al., 1980; Silver et al., 1988) have documented markedly reduced pH levels within the local microenvironment of phagocytic cells. For example, in clinical abscesses, pH levels as low as 5.7 have been reported. Such low extracellular pH levels are capable of reducing cytoplasmic pH to levels at which both cell migration and microbicidal activity are markedly reduced if the normal pH regulatory mechanisms are not functioning. Although this concept has not been validated in vivo, in vitro evidence suggests that the acidic milieu present within clinical abscesses might render phagocytic

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cells incapable of maintaining their pH, within the physiological range (Grinstein and Furuya, 1986b; Swallow et al., 1988). Taken together, this suggests a potential role for impaircd pH, regulation in causing phagocytic cell dysfunction in vilw.

Within abscess containing anaerobic bacteria, the short chain fatty acid byproducts of bacterial metabo~isni(Gorbach rr at., 1976) may further exaggerate pH, reduction, thereby resulting in further inhibition. Support for this hypothesis is derived from studies in which the short chain fatty acid succinate, a byproduct of anaerobic bacterial metabolism, was examined for its effect on neutrophil pH, and oxygen consumption in response to TPA at several different extracelluar pH levels (Rotstein era!., 1987b). At extracellular pH 6.5 or less, the addition of 30 mM succinate to the incubation medium significantly reduced neutrophil pH, when compared to succinate-free medium. Presumably, the weak acid operated as a protonophore, shuttling protons across the plasma membrane (Fig. 1). The augmentation of cytoplasmic acidification effected by succinate at low pH accounted for the virtually complete abolition of the respiratory burst, as measured by oxygen consumption. These studies have been morc closely related to the in vivo setting by the investigation of crude Bacteroirlcs jkagiiis filtrates (Rotstein et ( I / . , 1989). This bacterial species, which is the most frequently recovered anaerobe from intra-abdominal infection, has been shown to secrete a factor(s) during its growth in virru that impairs both neutorphil migration (Rotstein rt t i / . ,

Extracellular space Low pH

Succlnate 2 H* +

-l

+H2Succlnate

Cytopl asml c space Neutral pH

J,

reduces pH

SuccinatC+ 2 H+

F K . I . Schematic diagram of nicchanism by which siiccinate reduces cytoplasniic pH in neutrophila. At reduced extracellular pH, undissociated succinic acid traverses the plasma membranc. Within the cytoplasmic compartment succinic acid dissociates, thereby releasing protons in the intracellular milieu.

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1987a) and killing functions. Subsequent studies revealed that the combined effect of several short chain organic acids generated by this bacterial species after it had reached the stationary phase of growth (as would occur in abscesses) was entirely responsible for the inhibition. Furthermore, the inhibitory effect was due to the ability of these metabolic acids to reduce neutrophil pHi. These studies suggest that the local microenvironment of an infection may interact with bacterial products to impair local host defense mechanisms, thereby encouraging persistence of the infection. Recent studies have documented similar inhibition of human peritoneal macrophage function following their incubation in commercial peritoneal dialysis solution containing 35 mM lactate and with a pH of 5.3 (Topley et al., 1988).

D. The Physiological Role of Proton Extrusion in Phagocytic Cells Cellular mechanisms responsible for movement of protons from the cytoplasmic compartment to the extracellular space have generally been considered to serve primarily as regulators of cytoplasmic pH within the physological range. The recent description of an ATP-dependent proton extrusion pump in peritoneal macrophages, which is constitutive to the plasma membrane, suggests other possible roles (C. J. Swallow et al., unpublished observations; Swallow et al., 1988). Active proton extrusion has been shown to reduce the pH of the pericellular microenvironment surrounding activated macrophages to levels as low as 4.2 (Silver et al., 1988). Such low pH levels are bacteriostatic and also favour the lytic activity of acid proteases released into the microenvironment by macrophage degranulation. Thus, the macrophage proton extrusion pump may represent a functional activity of the cell aimed at optimizing the local milieu for bactericidal and possibly tumoricidal activity. In this regard, osteoclasts, which arise from the same granulocyte-macrophage progenitor cell as do macrophages (Mundy and Roodman, 1987), have a proton ATPase extrusion pump. This pump serves to acidify the local milieu between the cell and its bony substrate and contributes to bone resorption (Baron et a / ., 1985). Further studies examining the potential functional role of the proton pump in macrophages may provide information as to its importance in vivo.

111. pH, CHANGES DURING PHAGOCYTIC CELL DIFFERENTIATION: REGULATION AND SIGNIFICANCE Changes in pH, and its regulation have been reported to accompany functional activation in several cell types (see Grinstein et al., 1989; Busa, 1986, for reviews). Since in many cases, treatment with activating or differentiating agents

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was associated with stimulation of Na / H + exchange, it was postulated that a Na /H ' antiport-mediated cytoplasmic alkalinization might act as a trigger for cellular activation or differentiation (Grinstein et al., 1989; Busa, 1986; Rosoff and Cantley, 1983; Rosoff ct u / ., 1984). This section will examine the evidence suggesting that alterations in pH, and its regulation play a role in the differentiation of phagocytic leukocytes. This question has been approached mainly by studying immature leukemic cell lines induced to differentiate in either the granulocytic or monocytic direction. +

A. Granulocytic Differentiation Several groups have used the human promyelocytic cell line HL60 to study pH, and its regulation over the course of differentiation into granulocyte-like cells. Ladoux et al. ( 1 9 8 7 4 reported a progressive increase in resting pH, from 7.04 to 7.37, measured in HCO, -free medium at pH,, 7.4, over the course of 5 days of treatment with retinoic acid, an agent that induces granulocytic differentiation. Alterations in pH, consistently preceded functional maturation by 1-2 days. Exposure to another differentiating agent, dimethyl sulfoxide (DMSO), for 5 days had a similar effect on pH,, while 5 days of treatment with a nondifferentiating analog of retinoic acid, etretinate, had no effect on pH,. Based on these correlations, the authors speculated that changes in pH, might be causally related to differentiation in these cells. Further evidence suggested that the retinoic acidinduced increase in resting pH, was attributable to an increase in N a + / H + antiport activity: Blocking Na /H exchange with ethylisopropylamiloride (EIPA) reduced pH,, measured in 11CO,--frec medium, to -6.89 in both undifferentiated and differentiated cells. In addition, the rates of EIPA-sensitive acid extrusion and 22Na uptake were 1.7 and 2.05 times greater, respectively, in differentiated than in undifferentiated cells during recovery from an imposed acid load. Finally, rates of pH, recovery and 22Na uptake following acid loading were equivalent in undifferentiated and differentiated cells analyzed in HCO, - -containing Na+ medium in the presence of EIPA. Taken together, these data suggested that the activity of the Na -dependent HCO, - IC1- exchanger remained constant throughout differentiation, while the activity of the Na ' / H + exchanger increased approximately twofold. Costa-Casnellie et al. (1987, 1988) compared the kinetic properties of the Na / H exchanger in immature and DMSO-differentiated HL60 cells. Na / H ' exchange was activated by either Li -loading or by cytoplasmic acidification and measured as the dimethylaniiloride (DMA)-sensitive 22Na uptake and acid extrusion; these experiments were done in HCO, -free media. The K,,, for Na+ of the antiporter was consistently greater in immature than mature cells, at all pH, levels tested. This indicated that the "a+ lo required to drive forward Na+ / H + exchange at a given rate was lower in differentiated than in undifferentiatcd cells. The authors noted that this alteration in the properties of the Na / H exchanger +

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preceded functional differentiation by several days and suggested that the modified exchanger might be required for expression of the mature granulocytic phenotype. However, at pHi levels within the physiological range, the absolute values of K,llNa+ in both cell types were well below the expected physiological [Na+IO (K,,Na+ at pH, 7.25 was 15 mM in immature and 10 mM in mature cells). It follows that the external site of the Na+ /H antiporter would normally be fully saturated with Na+ in both undifferentiated and differentiated cells. The demonstrated alteration in Kn,Nd+ with differentiation is therefore unlikely to be associated with any alteration in Na+ /H exchange activity under physiological conditions, which raises doubts as to its potential role in facilitating the differentiation process. Restrepo et ul. (1987, 1988) addressed the question of whether alterations in pHi and its regulation would still be observed if immature and mature cells were studied in HCO, .- -containing solutions. They showed that activation of the Na+ / H exchanger via protein kinase C was different in undifferentiated and DMSO-differentiated HL60 cells: pHi, measured in HCO, -containing medium, increased in a DMA-sensitive fashion upon TPA treatment of differentiated but not of undifferentiated cells (Restrepo et al., 1987). This is consistent with an alteration in the modulation of Na / H exchange during differentiation. However, this group subsequently found (Restrepo et al., 1988) that resting pHi, measured in 20 mM HCO, medium at pH, 7.4, was similar in undifferentiated, DMSO-differentiated, and retinoic acid-differentiated HL60 cells (7.00, 6.98, and 7.02, respectively). Furthermore, the effects of DMA, dihydro-DIDS, and a combination of the two inhibitors, on resting pHi were the same in undifferentiated and DMSO-treated cells, suggesting that the contributions of the N a + / H + and Na -dependent HCO, - / C 1 ~exchangers to maintaining resting pHi were similar in both cell types. These authors went on to emphasize that inhibition of N a + / H + exchange with DMA resulted in an insignificant decrease in pH, in cells tested in 20 mM HC0,- medium (-0.01 and -0.05 in undifferentiated and DMSO-differentiated cells, respectively). It was therefore concluded that any contribution made by Na / H exchange to maintaining resting pHi was of little importance under physiological conditions, in both undifferentiated and DMSO-differentiated HL60 cells. Although there might be an alteration in the kinetic properties of the Na+ / H exchanger with differentiation, this would have little effect on resting pHi in physiological, HCO, containing media. This is consistent with the failure of Restrepo el al. (1988) to demonstrate any alteration in pH, upon granulocytic differentiation. In summary, there is some evidence for an enhancement of Na+ / H antiport activity with granulocytic differentiation in HL60 cells. It now appears, however, that the increase in resting pHi originally reported to accompany differentiation was an artifact of measuring pHi in HCO, free solutions. Whatever the physiological importance of the enhanced NaC / H exchange activity may be, if any, it is apparently not related to an associated alteration in resting pH,. +

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CAROL J. SWALLOW ET AL.

6. Monocytic Differentiation Investigation of pHi and its regulation during monocytic differentiation has been less extensive, but the results obtained thus far parallel those for granulocytic differentiation. HL60 cells treated with butyrate, 1,25dihydroxyvitamin D, recombinant y human interferon, or TPA differentiate into monocyte-macrophage-like cells. Ladoux et al. (1988a) found that, in HC0,- -free solution, treatment with each of these agents was associated with an increase in pHi from 7.00 in undifferentiated cells to 7.16, 7.20, 7.13, and 7.23, respectively, in differentiated cells. Similarly, retinoic acid-induced monocytic differentiation of U937 leukemic cells was accompanied by an increase in pIIi from 7.03 to 7.23 (Ladoux et a [ . , I988a,b). When the differentiated cells were treated with EIPA in HC0,--free solution, resting pHi was reduced to -6.95. To compare Na+ / H + antiport activity in undifferentiated and differentiated U937 cells, rates of EIPAsensitive '?Na uptake and acid extrusion were measured during recovery from acid loading; these were increased 2.3- and 2.1-fold, respectively, in differentiated compared to undifferentiated cells. Based on these results, the authors concluded that the increase in pHi that followed treatment with differentiating agents was due to an increase in N a + / H exchange activity. Significantly, Ladoux ei UI. ( 1988a) found that blocking the Na+ /H ' antiport by incubation in EIPA did not prevent the appearance of the differentiated monocytic phenotype. Thus, the increase in N a + / H exchange activity that accompanied monocytic differentiation was likely a consequence of the differentiation process, rather than a trigger for it. While Ladoux et ul. (1988b) did not report resting pH, levels for U937 cells in HCO, - -containing medium, they did present evidence that both undifferentiated and retinoic acid-differentiated cells could recover from acid loading via Na dependent HCO, IC1- exchange. Rates of EIPA-insensitive and HC0,- -stimulated "Na uptake and acid extrusion were equivalent in undifferentiated and differentiated cells, indicating that differentiation was not associated with an enhancement of Na+ -dependent HCO, /CI- exchange activity. If resting pII, in physiological, HCO, - -containing medium is set by the Na -dependent HC0,- /C1 exchanger in these cells, the increase in Na+ / H exchange activity that apparently accompanies monocytic differentiation is unlikely to be associated with any alteration in resting pH, levels under physiological conditions. +

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C. Macrophage Activation We have investigated the potential role of pH, in macrophage activation. The transition from the resident to the activated state is characterized by the acquisition of specialized functional capacities, including the ability to undergo an

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enhanced respiratory burst in response to various stimuli. Because the activity of the NADPH oxidase is markedly pH sensitive, we hypothesized that a difference in pH, or its regulation might exist between resident and activated macrophages. Such a difference could potentially account for the enhanced NADPH oxidase activity characteristic of activated cells. When examined in HCO, - -free medium, neither resting pH, nor Na+ /H exchange activity differed significantly between lipopolysaccharide-elicited and resident murine peritoneal macrophages. This suggests that any alteration in Na+ / H + antiport activity that occurs during monocytic differentiation, if present, is complete prior to the mature resident macrophage stage and that NADPH oxidase activity becomes enhanced during cellular activation through some mechanism other than an alteration in PHi. Cytoplasmic alkalinization due to stimulation of Na /H exchange has been documented in many cell types in response to a wide variety of growth factors (see Grinstein el al., 1989, for review). The fact that these studies were carried out in HCO,--free solutions did not prevent speculation that a N a + / H + antiport-mediated increase in pHi might serve as an essential signal in the initiation of growth or differentiation. More recently, studies conducted in physiological HCO, - -containing media have revealed both the dominance of the Na -dependent HCO,-- /C1- exchanger in determining resting pH, and the complexity of the pH, response to mitogenic stimulation (Bierman et al., 1988; Ganz er al., 1989; Thomas, 1989; Szwergold er a / . , 1989). Similarly, it seems likely that resting pHi in both immature and mature HL60 and U937 cells is set chiefly by the Na -dependent HCO, - /Cl exchanger, rather than by the Na+ / H antiport. While there is some evidence for an alteration in the kinetic properties of the Na+ /H exchanger during phagocytic leukocyte differentiation, Na+ -dependent HCO,-/Cl- exchange activity does not appear to change. It is therefore not surprising that resting pHi measured in HCO, -containing medium is the same before and after differentiation. Measurement of pHi in HCO;--free solutions led to the erroneous conclusion that a cytoplasmic alkalinization accompanied, and was necessary for, phagocytic cell differentiation. It is now apparent that the enhanced Na /H antiport activity observed in differentiated cells is not a cause, but rather a consequence, of the differentiation process. +

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ACKNOWLEDGMENTS Original work in the authors' laboratories was supported by the Medical Research Council of Canada and the National Cancer Institute (Canada). C. J. S. is the Merck-Frost Surgical Infectious Disease Fellow and a Medical Research Council of Canada Fellow. S . G . is a Medical Research Council Scientist. REFERENCES Baron, R., Neff, L.. Louvard, D . , and Courtoy, P. J . (1985). Cell-mediated extracellular acidification and bone resorption: Evidence for a low pH in resorbing lacunae and localization of a 100 kD lysosornal membrane protein at the osteoclast ruffled border. J . Cell B i d 101, 2210-2222.

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Rierman, A. J.. Cragoe. E. J., Jr., de Laat. S. W., and Moolenaar, W. H. (19x8). Ricarhonatc determines cytoplasmic pH and suppresses mitogen-induced alkaliniration in fibroblastic cells. .I. Biol. Chem. 263, 15253-152.56. Bowman, E. J.. Siebers, A , , and Altendorf. K . (1988). Bafiloniycins: A class of inhibitors of membranc ATPases from microorganisms, animal cells, and plant cells. Proc,. Nut/. Acad. Sci. U.S.A. 85, 7972-7976. Bryant. R . E . , Rashad, A. L . . Mazza, J. A,. and Hammond, U . (1980). Beta-lactamasc activity in himan pus. J . Inject. Dis. 142. 94-601. Busa. W. B . (1986). The proton as an integrating effector in metabolic activation. Curr. Top. Mernhr. Trtrtwp. 26, 291-305. Costa-Casncllie, M. R., Segel. G . B.. Cragoe. E. J . . Jr., and Lichtnman. M. (1987). Characterization of the Na / H + exchanger during maturation of HL-60 cells induced hy dimethyl sulfoxicie. J . Biol. Chcrr~.262, 9093 -9097. Costa-Casnellic, M. R . , Scgcl, G . B.. and Lichtman, M. (1988). The N a + / H + exchanger in immature and iiia~uregranulocytic HL-60 cells: Property changes induced by intracellular acidification and cell maturation. J . B i d . Chrm. 263, I I8SI-11855. Decker, K.. and Dieter, P. (1988). The ~timiilu~-activatcd Na+/ H i exchange in macrophagcs, neutrophils and platelets. In “pH Homeo ,is’’ (D. Haussinger. ed.), pp. 79-96. Academic Press, San Diego, CA. Faucher, N . , and Naccache. P. H. (1987). Relationship between pH, sodium. and shape changes in clicii~otactic~factnr-stimulated human neutrophils. J. Cell. Physiol. 132, 483-491. Fechhcimer, H., and Zigmond, S. H. ( 1983). Changes in cytuskcletal protein of pulyniorphonuclear leukocytes induced hy chemotactic peptidcs. Cell Motil. 3, 349-361. Ganz. M . B . , Boyarsky. G . , Sterzel, R. B., and Boron, W. F. (1989).Arginine vasopressin enhances pH, regulation in the presence of HCOi by stimulating three acid-base transport systcrns. Nrrrurt (London) 337, 648-65 I . (iorbach, S. L., Mayhcw, J. W., Bartlett, J. G . , Thadepalli. H.. andonderdonk, A. B. (1976). Rapid diagnosis of anaerobic infections by direct gas-liquid chromatography of clinical specimens. J . C‘li)i, Invesr. 57, 478-484. Grinstein, S . , and Furuya, W. ( 1984). Amiloride-sensitive Nd + / H exchange in human neutrophils: mechanism of activation by chemotactic tactor5. Biochem. Biophvs. Res. Commun. 122, 7.55762. Grinstein, S., and Furuya, W. (1986a). Cytoplasmic pH regulation in phorbol ester-activated human neutrophils. A m . J. P/iysinl. 251, C55SC65. Grinstein. S.,and Furuya, W. (1986b). Characterization of the amiloride-sensitive Na+/H+ antiport of human neutrophils. Am. J. Physiol. 250, C283-C291, Grinstein, S . . Goetz, J. D . . Furuya, W., Rothstein. A , , and Gelfand. E. W. (1984). Amiloridesensitive Na+ /H+ exchange in platelets and leukocytes: detection by electronic cell sizing. A m . J . Physiol. 247, C293-C298 Grinstein. S . , Elder, B., and Rruya, W. (198.5). Phorbol ester-induced changes ofcytoplasmic pH i n neutrophils: role of cxocytosis in Na+ /H ’ exchange, Am. J. Physiol. 248, C379-C3Xh. Grinstein. S . . Fumya, W., and Biggar, W. D. (1986). Cytoplasmic pH regulation in noriiial and abnormal neutrophils. Role of superoxide generation and Na+/ H + exchange. J. Biol. C h r m . 261, 512-514. Grinstein, S., Rotin. D.. and Mason, M. (1989). N a + / H + exchange and growth factor-induced cytosolic pH changes. Role in cellular proliferation. Biuchim. ,Yiophy.s. Actu 988, 73-97, Heuser, J. 11989). Changes i n lyaowme shape and distribution correlated with changes in cytoplasmic pH. J . Cell B i d . 108, 855-864. I.adoux, A., Cragoe, E. J., Jr., Geny, B., Abita, J. P., Rclin, C. (1987a) Differentiation of human prornyelocytic HIhO cell> by retinoic acid is ompanied by an increase in the intracellular pli: The role of the Nai / H exchange system. J . Eiol. Chent. 262, X I 1-816.

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Ladoux, A , , Krawice, I . , Cragoe, E. J., Jr., Abita, J. P., and Frelin, C. (1987b). Propertics of the Na+ -dependent CI -- /HCOi exchange system in U937 human leukemic cells. Eur. J . Biochern. 170, 43-49. Ladoux, A., Damais, C., Krawicc, I.. Abita. J. P., and Frelin, C . (l988a). An increase in intracellular pH is a general response ofpromonocytic cells to differcntiating agents. FEBS Lett. 234, 353-356. Ladoux, A., Miglierina, R . . Krawice. I . , Cragoc, E. J . , Jr., Abita, J. P., and Frelin, C. (1988b). Single-cell analysis of the intracellular pH and its regulation during the monocytic differentiation of U937 human leukemic cells. Eur. J . Bzochem. 175, 455-460. Madshus, 1. H., and Olsnes, S. ( 1987). Selective inhibition of aodium-linked and sodium-independent bicarbonateichloride antiport in Vero cells. J . B i d . Chem. 262, 7486-7491. Molski, T. F. P., and Sha’afi, R. I . (1987). lntracellular acidification, guanine nucleotide binding proteins and cytoskeletal actin. Cell Motil. 8, 1-6. Molski, T. F. P., Naccachc. P. H . , Volpi, M.. Wolpert, L. M . , and Sha‘afi, R. 1. (1980). Specific modulation of the intracellular pH of rabbit neutrophils by chemotactic factors. Biochem. Biophys. Res. Commun. 94, 508-514. Molski, T. F. P.. Ford, C.. Weisinan, S. J., and Sha’afi, R. I. (1986). Cell alkalinization is not necessary and increased sodium intlux is not sufficient for stimulated supcroxide production. FEBS Lett. 203, 267-272. Mundy, C. R., and Roodman, G. D. (1987). Osteoclast ontogeny and function. In “Bone and Mineral Research” (W. A. Peck, ed.), Vol. 5, p. 209. Elsevier, Amsterdam. Naccache, P. H. (1987). Signals for actin polymerization in neutrophila. Biomed. Pharmacorher. 41, 297-304. Naccache, P. H., Caon, A. C . . and McColl, S. R. (1988). Propionic acid-induced calcium mobilization in human neutrophils. J . Cell. Physiol. 136, I18- 124. Naccache, P. H.. Therrien, S., C a m , A. C . , Liao, N.. Gilbert, C., and McColl, S. R. (1989). Chemotactic-induced cytoplasmic pH changes in cytoskeletal reorganization in human neutrophils. Relationship to the stimulated calciuin tansients and oxidative burst. J . Immunol. 142, 2438-2444. Nasmith, P. E., and Grinstcin, S. (1986). lmpairment of N a + / H + exchange underlies the inhibitory eftect of Na+-free media on leukocyte function. FEBS Letr. 202, 79-85. Rabinovich. M., DeStefano. M. J., and Dzienzanowski, M . A (1980). Neutrophil migration under agarose: Stimulation by lowered medium pH and osmolarity. J . Reticuloendothel. Soc. 27, 1 R9200. Restrepo, D., Kozody, D. J.. and Knauf, P. A. (1987). Changes in N a + i H + exchange regulation upon granulocytic differentiation of HL-60 cells. A m . J . Phvsiol. 253, C6 l9-C624. Restrepo. D., Kozody, D. J.. Spinelli, L. J.. and Knauf, P. A. (1988). pH homeostasis in promyclocytic leukemic HL60 cells. J . Gen. Physiol. 92, 489-507. Rosoff, P. M . , and Cantley, L. C. (1983). Increasing the intracellular Na+ concentration induces differentiation in a pre-B lymphocyte cell line. Proc. Narl. Acad. Sci. U.S.A. 80, 7547-7550. Rosoff, P. M., Stein, L. F.. and Cantley, L. C . (1984). Phorbol esters induce differentiation in pre-Blymphcoyte cell line by enhancing N a + / H + exchangc. J. B i d . Chem. 259, 7056-7060. Rotstein, 0. D., Houston, K.. and Grinstein, S. (1987a). Control of cytoplasmic pH by N a + i H + exchange in rat peritoneal macrophages activated with phorbol ester. FEES Left. 215, 223-227. Rotstein, 0. D., Nasmith, P. E.. and Grinstein, S. (1987b). The Bacreroidrs by-product, succinic acid, inhibits neutrophil respiratory burst by reducing intracellular pH. Infect. Imrnun. 55, 864870. . Rotstein, 0. D., Wells, C. L . , Pruett. T. L.. Sorenson, J . J., and Simmons, R. L. ( 1 9 8 7 ~ )Succinic acid production by Bacreroides fiagilis: a potential virulence factor. Arch. Surg. 122, 93-98. Rotstein, 0. D., Fiegel. V. D.. Simmons. R. L., and Knighton, D. L. (1988). The deleterious effect of reduced pH and hypoxia on neutrophil migration in virro. J . Surg. Res. 45, 298-303. ~

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Rotbtein, 0. D., Vittorini, T., Kao, J., McBurney, M. I . , NdSmith, P. E., and Grinstein. S . (1989). A soluble Bacteruides by-product impairs phagocytic killing of Escherichia coli by neutrophils. Inject. Itnmuti. 57, 745-753. Satoh, M., Nanri, H . . Takeshige, K , and Minakami, S . (1985). Pertussis toxin inhibits intracellular pH changes in human neutrophils stimulated by N-formyl-methionyl-leucyl-phcnylalanine. Biochum. Biophys. Res. Cotntnun. 131, 64-69. Sha’afi, R. I . , Shefcyk, J . , Yassin, R., Molski, T. F. P., Volpi, M., Naccache, P. H., White, J. R., Feinstein, M. B.. and Becker. E. L. (1986). Is a rise in intracellular concentration of free calcium necessary or sufficicnt for stimulated cytoskcletal-assaciatcd actin’?J . Cell Biol. 102, 1459- 1463, Showcll, H. J . , and Becker, E. L. (1976).The ctfects of external H + and Na+ on the chemotaxis of rabbit peritoneal neutrophils. J . Irntnunol. 116, 99- 105. Silver, 1. A., Murrils. R. J., and Etherington, D. J. (1988). Microelectrode studies on the acid microenvironment beneath adherent macrophages and osteoclasts. Exp. Cell Res. 175, 266276. Simchowitz, L. ( 1985a). Intracellular pH modulates the generation of superoxide radicals by human neutrophils. J . Clin.Invest. 76, 1079- 1089. Simchowitz, L. (1985b). Chemotactic factor-induced activation of Na /H + exchange in human neutrophils. J . B i d . C‘hrm. 260, 13248- 13255. Simchowitz, L.,and Cragoe, E. J . , Jr. (1986). Inhibition of chemotactic Pactor-activated Na /H exchange in human neutrophils by analogues of amiloride: structure-activity rclationships in the amiloride series. Mol. Pharmucol. 30, I 12- 120. Simchowitz. L., and Cragoe. E. J., Jr. (1987). Intracellular acidification-induced alkali metal cation/H+ exchange in human neutrophils. J . Grn. Phystol. YO, 737-762. Simchowitz. L.. and De Weer, P. (1986). Chloride movements in human neutrophils: diffusion, exchange, and active transport. J . Get?. Physiol. 88, 167-194. Simchowitz, L., and Roos, A . (1985). Regulation of intracellular pH in human neutrophils. J. G m . Physid. 85, 441-470. Simchowitz, L., RatzlaK, R., and Dc Weer, P. (1986). Anionianion exchange in human ncutorphils. J. Gen. Phvsiul. 88, 195-217. Sklar, L. A., Omann, G . M., and Painter, R. G. (1985). Relationship of actin polymerization and depolymerization to light scattering in human neutrophils: Dependence on receptor occupancy and intracellular C a 2 + . J . Cell B i d . 101, 1161-1166. Surnimoto, H . , Satoh, M., Takeshige, K.,Cragoe, E. J . , Jr., and Minakami. S . (1988). Cytoplasmic pH change induced by leukotriene B, in human neutrophils. Biochim. Biopiiys. A r m 970, 3138. Swallow, C . J., Grinstein, S., and Rotstein, 0.D (1988). Cytoplasmic pH regulation in inacrophages by an ATP-dependcnt and N.N’-dicyclohexylcarbodiimidc-bensitivemechanism. J . B i d . Chem. 263, 19558-19563. Szwergold, B. S., Brown, T. R., and Freed, J. J . (1989). Bicarbonate abolishes intracellular alkalinization in mitogen-stimulated 3T3 cells. J . Cell. Physiol. 138, 227-235. and Coet7.l. E. J. (1979). Structural and catalytic properties of the solubilized supcroxTduber, A . I., ide-generating activity of human polymorphonuclear leukocytes. Solubilization. stabilization in solution and partial characterization. Biochernistp 18, 5576-5584. Thomas, R. C. (1989). Bicarbonate and pH, response. Nature (London) 337, 601. Topley, N., Alobaidi, H. M. M., Davies. M., Coles, G . A., Williams, J. D., and Lloyd, D. (1988). The ctfect of dialysatc on peritoneal phagocyte oxidative metabolism. Kidne~vItit.34, 404-41 1. Volpi, M., Naccache, P. H . , Molski, T. F. P., Shefcyk, J., Huang, C.-K., Marsh, M. L., Munoz, J . . Becker, E. L . , and Sha’afi, R. I . (1985). Pertussis toxin inhibits,fmet-leu-phe but not phorbol +

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ester-stimulated changes in rabbit neutrophils: Role of C proteins in excitation response coupling. Proc. Nut/. A u d . Sci. U.S.A. 82, 2707-2112. Weisman, S. J., Punzo, A . , Ford, C., and Sha’afi, R . I. (1987). lntracellular pH changes during neutrophil activation: Na+/H antiport. J . Lcdocvte B i d . 41, 25-32. White, J. R . , Naccache, P. H . , and Sha’afi, R. 1. (1983). Stimulation by chemotactic factor of actin association with the cytoskeleton in rabbit neutrophils: effects of calcium and cytochalasin B . J . B i d . Chern. 258, 14041-14047. Wright, J., Schwartz, J. H., Olson, R., Kosowsky. 1. M.,and Tauher, A . I. (1986). Proton secretion by the sodiumihydrogen ion antiporter in the human neutrophil. J . Clin. Invest. 77, 782-788, Yuli, I . , and Oplatka, A. (1987). Cytosolic acidification as an early transductory signal of human neutrophil chemotaxis. Scienc.e 235, 340-342. Zigmond, S . , and Hargrove, R. L. (1981). Orientation of PMN in a pH gradient. Acid-induced release of a chemotactic factor. J. Irnmund 126, 478-481. +

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CURRENT TOPICS IN M € M B R A N t S A N D TRANSPORI. VOLUME 35

Chapter I I

Phosphoinositide Metabolism in Lymphocyte Activation ROBIN HESKETH, J . C. METCALFE, S . R. PENNINGTON, * AND LOUISE R. H O W E f Department of Biochemistry University ?f Cambridge Cambridge CB2 IQW, England *Department of Human Anutomy and Cell Biology University of Liverpool Liverpool L69 3BX, Eirglund flnstitute of Cancer Research Chester Beatty Laboratories London SW3 6JB, England 1. Introduction 11. T Cells A. The T Cell Receptor B. Other T Cell Surface Antigcna 111. T Cell Proliferation A . Mitogenic T Cell Activation by Antibodies and Lectins B. Rcplenishment of the Phosphatidylinositol 4.5-Bisphosphate Pool C . How Do Lymphocyte Surface Receptors Cause Phosphatidylinositol 4,5-Bisphosphate Hydrolysis'? D. Inositol 1,4,S-Trisphosphate Release and Metabolism E. Correlation of Inositol Phosphates Accumulation with [Ca2+Ii Responses F. Protooncogene Activation and the Phosphatidylinositol Cycle G . Does Phosphatidylinositol 4,5-Bisphosphate Hydrolysis Activate Protein Kinase C'? H. Protein Kinase C Translocation I . The Effect of CAMP on T Cell Function J. Negative Signals in T Cell Mitogenesls IV. T Cell Responses in Cellular Immunity A . Natural Killer Cells B. T Cell-Mediated Cytotoxicity V. B Cells VI. Sumniary References

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1.

INTRODUCTION

When K. H. Michell wrote what was to become a classical review on phosphatidylinositol metabolism (Michell, 1975), only 8 of the 401 references he cited referred to lymphocytes. These emanated from just five research groups and were composed mainly of the pioneering studies of Fisher and Mueller (1968, 1971). In the 14 years since the publication of that review, however, lyniphocytic cells have become a major focus for studies of how metabolites of phosphatidylinositol regulate cellular processes. The intense interest in them derives both from the central role they play as the key effector cells of the immune response and from their convenience as model systems for the study of cell growth control. Lymphocytes are divided into two major classes. B lymphocytes (B cells) are derived from the bone marrow without passing through the thymus, and when stimulated by antigen, they differentiate into plasma cells, synthesizing and secreting antibody. T cells are lymphocytes that mature in the thymus into two major subclasses defined by the expression of the cell surface molecules CD4 and CD8. The major roles of T lymphocytes are the recognition of antigen and, in cooperation with B cells, the generation of humordl immunity against thymusdependent antigens. CD4+ T cells include the effector cells responsible for delayed hypersensitivity, for promoting the development of cytotoxic effector cells and active suppressor cells, and for helping B cells to differentiate (Th cells). CD8 T cells comprise cytotoxic effector cells and active suppressor cells ('Is) that can inhibit delayed hypersensitivity reactions and B cell ininiunoglobulin synthesis. T cells normally rccognise a foreign antigen on the surface of a cell only when it is associated with self-antigens of the major histocompatibility complex (MHC). Cytotoxic T cells usually respond to foreign antigen in association with Class 1 MHC antigens and Th lymphocytes to antigen associated with Class 11 (la) antigens. The heterogeneity of preparations of primary lymphocytes has for many years indicated the desirability of using cloned, homogeneous cell lines for studying cither the immunological responses of lymphocytes or the niolecular biology of lymphocyte growth activation. The methods for cloning mitogen-stimulated T +

'It should bc noted that the initial designations of human T cell receptors as TI. T2. ctc., have now been superceded by a revised nomenclature based on cluster designations (CD). At least 78 human leukocyte cell surface markers have thus far been descrihed by thcir cluster designations, the rcccptors of grcatcst relevance in the present context being CD2 (TI I), CD3 (T3), CD4 (T4), CD8 (T8), CD25, which is the interleukin 2 receptor recognized by anti-Tac antibody, CD28 (T44), and CD45 (T200) (Sell. 1987). T9 and Ti are not included in cluster designations. The CD designations have been generalty used throughout this rcvicw to apply both to the human surface antigens and to the homologous murine proteins. W. Knapp er a / . have prepared a summary table of antlgen classification agreed to at the Fourth International Workshop on Leukocyte Differentiation Antigens, 1989, published in Inimuno/ogy T d u j , August 1989.

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cells (Rozenszajn et ul., 1975; Benn-Sasson et al., 1975) have led to the development of long-term cultures of antigen-specific and alloreactive murine and human T lymphocytes (Sredni et ul., 1981). Some of the data relating to PtdIns metabolism that have now accumulated from studies using such clones will be discussed in this review, in addition to data from primary cell preparations. The stimulation of a vast range of cell types by specific agonists is now known to cause hydrolysis of the phospholipid phosphatidylinositol 4,5-bisphosphate (PtdlnsP,) in the plasma membrane with the consequent intracellular release of inositol 1,4,5-trisphosphate [Ins( 1,4,S)P,] and diacylglycerol (DAG), a phenomenon often loosely referred to as “stimulation of phosphatidylinositol turnover.” PtdInsP, is a phosphorylated derivative of phosphatidylinositol (PtdIns), which is itself a minor component of the plasma membrane, comprising approximately 10% of the total lipids. The pools of phosphorylated derivatives of PtdIns, phosphatidylinositol 4-phosphate (PtdInsP), and PtdlnsP, account for only 5% of the total polyphosphoinositides and are maintained by the actions of specific kinases and phosphatases of high enzymatic activity that have been characterized in several types of cells (O’Shea et u l . , 1986; Whitman et d . , 1987). It is generally considered that the functional significance of stimulated PtdlnsP, hydrolysis derives from the actions of the two breakdown products, the Ins( 1,4,5)P, generated causing the release of calcium from intracellular stores, thereby elevating the free cytosolic concentration of Ca2 ([Ca2+Ii), and the other product, DAG, activating protein kinase C. It is now clear that lymphocytes follow this general pattern in that stimulation of each of the types of effector cells described previously by appropriately presented specific agonists has been shown to cause PtdInsP, hydrolysis. There are several quantitatively minor phosphoinositides and one inositol phosphate derivative that have not yet been isolated from lymphocytes: phosphatidylinositol 3-phosphate has been detected in fibroblasts and astrocytoma cells (Whitman et ul., 1988; Stephens et a / . , 1989), inositol 1,2-cyclic 4,5-trisphosphate (Tarver et a/., 1987) and phosphatidylinositol trisphosphate (PtdlnsP,; Traynor-Kaplan et (I/. , 1988) in platelets, and PtdInsP, and phosphatidylinositol 3,6bisphosphate in human vascular smooth muscle cells (Auger et ul., 1989). It seems possible that PtdlnsP, may be generated by the action of a membrane-bound, receptor-regulated PtdInsP,-3-kinase that has been detected in some tissues (Irvine et ul., 1988) but has not yet been reported in lymphocytes. With these exceptions, all of the known derivatives and metabolites of PtdIns have been detected in lymphocytes, although for many the concentrations are too low to permit reliable quantitation. It seems probable, therefore, that lymphocytic cells possess all the enzymes necessary to generate the entire family of PtdIns derivatives and metabolites (Fig. I). It may be noted, however, that although all lymphocytes appear capable of initiating PtdInsP, hydrolysis and do so when activated by a wide variety of agents, this response is not caused by all mitogens that stimulate proliferation. For example, lipopolysaccharide is a potent mitogen for B lymphocytes but does not cause detectable +

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ROBIN HESKETH ET AL. I n s ( 1 ,3.4,5)P4

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FIG. I .

L

f

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lnsP6

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ns ( 1 , 3 , 4 , 6 )p4+

L lns(3.4)%

t

Ins ( 1 . 3 , 4 , 5 , 6 )P5

t In s ( 3 , 4 . 5 , 6 )p4

Pathways of phosphatidylinositol 4.5-bisphosphate metabolism

accumulation of inositol phosphates (see Section V). Furthermore, a number of lymphokines that can play crucial roles in proliferation have been shown to be without etfect on Ptdlns metabolism. These include interleukin I (IL- I), which has pleiotropic effects including the capacity to substitute for the tumor promoter 12-0-tetradecanoylphorbol13-acetate (PMA) as a co-mitogen with antigen or monoclonal antibody (mAb) against the T cell antigen receptor (TCR) but does not stimulate PtdlnsP, hydrolysis in the human lymphoma cell line Jurkat (Kosoff et d.,1988), interleukin 3, which regulates the proliferation of pluripotent stem cells (Whetton et a / . . 1986), and although more controversially, interleukin 2 (IL-2), which is essential for the proliferation of almost all types of T cells (see Section 111,A). It is therefore evident that, although the “second messengers” Ins( 1,4,5)P, and DAG may be important in the activation of lymphocytic cells by many agonists, not all co-mitogens generate this response. In this review the term “activation” applied to any of the specific examples of lymphocyte effector function mentioned above will refer to the initiation of the chain of events leading to DNA synthesis as a consequence of signal transduction across the plasma membrane. The acute responses stimulated will, of course, differ between subclasses of cells. In lymphocytes, and in other eukaryotic cells, there is a wide variety of ligands that interact with specific receptors on cells to cause mitosis. but it is evident that there are only a few signal transduction mechanisms operating across the plasma membrane. The primary mitogenic signals from specific receptors that have bcen identified are: (1) activation of PtdlnsPz hydrolysis, (2) activation of CAMP-dependent kinases via G,, and (3) activation of tyrosine kinases intrinsic to the mitogen receptor structure. The coupling of these primary signals to effector systems depends on cell phenotype, as clearly demonstrated by responses to the activation of CAMP-dependent kinases. This is a primary mitogenic signal in some cells (Swiss 3T3 fibroblasts, Schwann cells, ete.) but a potent inhibitor of mitogenic stimulation in lymphocytes for most species that have been examined (Novogrodsky et al., 1983; Farrar rt u / . , 1987). Furthermore, although many growth factor receptors possess endogenous tyrosine kinase activity the enhancement of which appears to be essen-

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11. PHOSPHOINOSITIDE METABOLISM IN LYMPHOCYTES

tial for mitogenesis [e.g., the epidermal growth factor receptor on fibroblasts (Honegger et al., 1988)], no such domain has thus far been demonstrated in any lymphocyte receptor (although see Section III,G for a more detailed discussion of this point). It is, of course, far from certain that all of the primary mitogenic signals that are generated by mitogen receptors have been identified. In particular, the diversity of G proteins that have been cloned with as yet unidentified functions leaves open the possibility that there are other members of this set of signaling mechanisms to be discovered. However, the detection of PtdInsP, hydrolysis as the earliest response to stimulus-activation coupling in almost all types of lymphocytes has made the mechanism by which receptors interact with the Ptdlns system and the second messenger roles of the hydrolysis products a major focus of interest.

II. T CELLS

A. The T Cell Receptor After resisting attempts at identification for many years, a detailed picture of the antigen receptor (TCR) on T lymphocytes is now emerging. The best characterized component is Ti, a disulfide-linked a-P heterodimer of M , 80-90 kDa, the subunits of which show strong amino acid sequence homology (Williams and Barclay, 1988). The Ti glycoprotein complex appears to carry the entire antigenic specificity of T cells (Dembic et al., 1986). Even in the T cell clone A10, which produces two functional a-chain genes, a unique .-P receptor appears to be responsible for T cell dual rccognition, that is, for mediating both self-MHC restricted recognition and allo-MHC recognition (Malissen et al., 1988). A minor subset of CD3 cells express the alternative y/6 form of Ti, the function of which is unknown (Brenner et a / . , 1986; Lanier and Weiss, 1986; Hochstenbach ez al., 1988). Although the Ti heterodimer defines both MHC restriction and the specific recognition of antigen, it has emerged that Ti is expressed on the surface of normal T cells in noncovalent association with at least five other polypeptides (Borst et a / . , 1983; Kanellopoulos et al., 1983; Meuer et a / . , 1983a; Baniyash et al., 1988a; Clevers et al., 1988). These are now collectively known as the CD3 complex and comprise CD3-y, -6, -E, -5 and -q chains (Samelson et al., 1985a; Oettgen et al., 1986; Baniyash et al., 1988a) (Fig. 2). The polypeptides of the CD3 complexes in human and mouse cells are closely homologous. On human cells these are glycoproteins (gp) of 25 kDa (CD3-y) and 20 kDa (CD3-6) and a non-glycosylated protein of 20 kDa (CD3-E); the murine homologues are gp21 (CD3-y), gp26 (CD3-6), and p25 (CD3-E). The major additional polypeptide associated with CD3 is the 16 kDa chain, which exists principally as a disulfide-linked homodimer, but in approximately 20% of the receptors, is found in a disulfide linkage with an additional polypeptide, q, +

<

<

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ROBIN HESKETH ET AL.

26 25

45 45

21

a-P

Y 5

E

16 22 16 16

q-r

r-5

Rc,. 2. l’hc rnurine T cell aotigcn receptor.

of 22 kDa (Brenner rt d., 1985; Allison and Lanier, 1985; Samelson et ul., 198Sb; Baniyash rt a / . , 1988a). There is some evidence from niurine T cell hybridoma mutants defective in the synthesis of 5 chain that the availability of this molecule may regulate the expression of the TCR at the cell surface (Sussman er al.. 1988). In addition to thc Ti-CD3 complex, there are other cell surface receptors that, on binding their specific ligand, may stimulate the activation of T cells. Those defined on human cells are CD2 (Meuer et al., 1984a), CD4 (murine L3T4)(Carre1 et al., 1988), CD28, and Thy- I , and in mice Thy- 1 (Gunter et al., 1986) and several products of the Ly-6 locus (Yeh et al., 1987), including the T cell activating protein (TAP). Thy-1 and the Ly-6 gene products are mcmbers of the growing class of membrane proteins known to be anchored by attachment to glycosyl-phosphatidylinositol(gly-PtdIns; Low, 1987). This group also includes ornithine decarboxylase, one of the most rapidly activated enzymes following the stimulation of growth in many types of cclls (Scott er al., 1985). Some lymphocyte surface molecules can bc expressed in either a normal, transmembrane spanning form or attached vici gly-PtdIns. Examples of these include the lymphocyte function associated antigen 3 (LFA-3), which occurs on antigen-prescnting cells (Seed, 1987; Dustin et d., 1987), and probably Thy-1 and TAP (Ishihara et c r l . , 1987). The reason for the evolution of gly-Ptdlns protcin anchors for lymphocyte proteins is unknown. In other cells insulin has been shown to stimulate the hydrolysis of gly-Ptdlns, and the InsP-gly released mimics the effects of insulin on a variety of enzymes (Mato et al., 1987). In a T cell-enriched human spleen cell population, mitogenic stimulation has been shown to initiate the

11. PHOSPHOlNOStTlDE METABOLISM IN LYMPHOCYTES

255

synthesis of a set of gly-Ptdlns molecules concurrently with the induction of expression of insulin receptors (Gaulton et a / ., 1988). These molecules were hydrolyzed in response to insulin binding or by the addition of exogenous PtdInsspecific phospholipase C. There is, however, no evidence that activation of Thy- 1 by mAb causes cleavage of the protein from the gly-PtdIns anchor (Peyron et u l . , 1988). In other studies, the mitogenic response to monoclonal antibodies against the Qa-2 antigen, a Class 1 murine MHC product that is normally attached to PtdIns (Waneck et ul., 1988). was abrogated when the receptor was genetically engineered to contain a transmembrane protein anchor. These observations suggest, therefore. that lymphocytes may be capable of releasing InsPgly intracellularly to function as a second messenger in a manner analogous to that proposed for other cells, although it remains to be shown that this process is involved in proliferation. Stimulation of the TCR can induce lymphocyte proliferation, and it has been known for many years that a variety of polyclonal plant lectins are effective mitogens for T cells (Nowell, 1960). These include concanavalin A (ConA) and phytohemagglutinin (PHA), which bind to the carbohydrate moieties of cell surface proteins, including the TCR complex (Chilson et al., 1984). The capacity of the other receptors defined above (CD2, CD4, CD28, Thy-I, or TAP) to activate the mitogenic pathway has been revealed by the use of monoclonal antibodies (mAb). Although not stressed in some reports, it seems clear that, in addition to stimulation of at least one of these receptors, T cell proliferation normally requires the participation of macrophages (Tax et al., 1983; Moretta et c r l . , 1985), which synthesize and secrete IL- I and, in vivo, play a crucial role in antigen presentation. Accessory cells appear to cross-link the surface receptors of lymphocytes vicr the interaction of their Fc receptors with the Fc region of the mAb bound to the lymphocyte receptor (Tax et al., 1983, 1984; Landegren et ul., 1984; Smith et u /., 1986; Wakasugi et a/., 1985; Ceuppens et al., 1985; Benichou el d . , 1987). The in v i m immobilization of mAb may present a crosslinking matrix to the T cell that more closely resembles the in vivo situation. In general, it appears that T cell lines, normally established by mitogenic stimulation, d o not require accessory cells for proliferation, presumably because most clones express IL-2 receptors. Thus T cell clones may be activated by crosslinking mAbs with anti-Ig or by prior immobilization of anti-Ti or antLCD3 on beads (Meuer rt al.. 1983b, 1984b). There is one report of a similar result for human peripheral lymphocytes (Geppert and Lipsky, 1987), but in general, primary lymphocytes and also the Jurkat cell line are not stimulated by immobilized antibody alone (Manger et ul., 1985; Williams et al., 1985; Ledbetter et a/., 1986; Weiss et u l . , 1986) and require an additional mitogenic signal. For all lymphocyte phenotypes, including primary cells, this additional signal can be delivered by PMA (van Lier et d., 1988), which can fully substitute for accessory cells.

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An alternative approach to studying the role of the Ti-CD3 complex has been to derive CD3 - clones from human peripheral blood lymphocytes (Pantaleo et a/., 1988). In this method, cells were labeled with fluorescent monoclonal antibodies, separated by fluorescence activated cell sorting, and CD3 cells were cloned in the prcsence of autologous feeder cells. Two distinct CD3- cell phenotypes were selected, expressing either CD2 and CD7 or only CD7. Neithe,rcell type expressed CD4, CD8, or CD28 antigens but both were cytolytically active against a broad range of tumor target cells. In cither CD3YCD2+CD7+ or CD3-CD2 CD7 cells, PHA caused increases in lCa2 ' li and accumulation of InsP,, InsP2, and lnsP that were similar to those observed in cells expressing the Ti-CD3 complex. In the CD2 subset, anti-CD2 mAbs also induced both responses, and when the CD2 receptor was completely down-regulated by prolonged (24 hr) exposure to saturating concentrations of antibody, the responsivencss to PHA was unaffected. The principal conclusion from these data is that activation of these cell clones does not require the presence of thc Ti-CD3 receptor complex. Furthermore, it is evident that CD2 does not exert a regulatory role equivalent to that of Ti-CD3 in mature T cells and that neither Ti-CD3 nor CD2 is the functional receptor for PIIA in thcse cells. Thus in these clones, neithcr the increases in ICaL li and inositol phosphatcs accumulation stimulated by mAbs or by PHA nor the cytolytic activity of the cells required the presence of a functional Ti-CD3 complex. The stimulation of proliferation, shown elsewhere to be dependent on phosphorylation of this complex (see Section II,G), was not assayed. ~

+

+

+

B. Other T Cell Surface Antigens Thcre is a further category of lymphocyte cell surface molecules that arc themselves incapable of activating proliferation but that appear to play an auxiliary role in antigen recognition and may regulate subsequent cellular responses. These include CD8 (murine Lyt-2/3), CD16, CD45, LFA-I, and ILI. For an cxtensive summary of lymphocyte surface proteins, the reader is referrcd to Horejsi and B a d (1988). On the basis of the observations that monoclonal antibodies to the CD4 or CD8 glycoproteins inhibit the T cell response to antigen presentation, CD4 and CD8 are thought to stabilize the interaction between the T cell, antigen, and thc antigen-presenting cell (Sanniento et al., 1980; Marrack et d.,1983). The simplest interpretation of these results is that these surface glycoproteins function merely to locate accessory cells on the surface of T cells to facilitate activation through the Ti-CD3 complex. It is becoming evident, however, that CD4, CD8, and other regulatory surface receptors can themselves transduce either positive or negative signals across thc plasma membrane (see Section III,A, K). In addition to these receptors, a variety of glycoproteins appear on the surface

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257

of T cells in a specific sequence following activation. These include the receptors for 1L-2, transferrin, and insulin, the antigens 4F2 and EA-I, and the activation inducer molecule (AIM, Cebrian el al., 1988), all of which are detectable before DNA synthesis. Antigens that appear after S phase include HLA-DR, CB. I , Ta-I, TLiSAI, T10, and VLAl. Until recently it was considered that all thymocytes were capable of expressing the IL-2 receptor and that for both primary cells and clones autocrine stimulation by IL-2 was an essential step in the proliferative process. It should be noted, however, that 1L-2-independent pathways have now been demonstrated; for example, some T cell clones secrete B cell stimulatory factor (interleukin 41, which is used by these cells as an autocrine growth factor, rather than IL-2 (Lichtman et ul., 1987). The IL-2-dependent pathway remains, however, the best understood autocrine mechanism for the clonal expansion of T lymphocytes. It appears that the activation of the IL-2 gene may be regulated by the production of the nuclear factor of activated T cells (NFAT-I); this protein seems to be expressed only in activated T cells, being detectable some 10-25 min before IL-2 transcription (Shaw et al., 1988). The normal mechanism by which IL-2 secretion and IL-2 receptor expression is initiated in T cells appears to be through the action of 1L-1 secreted by macrophages. Thus, in the absence of macrophages (or exogenously added IL-I), neither ConA, PHA, nor anti-CD3 mAbs are mitogenic for human T cells (Williams et a/., 1985; Palacios, 1985; Schcurich et at., 1985; Kaibuchi et ul., 1985). In murine cells, the effect of macrophages in stimulating IL-2 receptor production following activation of the TCR was also caused by PMA which down-regulated the TCR complex (Ando et al., 1985). In human cells the action of macrophages or PMA was required for IL-2 receptor expression in C D 4 + cells, whereas activation of the TCR alone was sufficient in CD8+ cells. The induction of detectable amounts of 1L-2 receptors on the surface of quiescent T cells requires approximately 8 hr from the time of mitogenic stimulation, and their appearance is frequently used as an indication that T cells have been activated. Using this assay, Klausner rt a / . (1987) have concluded that phosphorylation of the TCR is an absolute requirement for activation by antibodies directed against surface molecules.

111. T CELL PROLIFERATION A. Mitogenic T Cell Activation by Antibodies and Lectins It is now considered that, in many cell systems activated by agonists causing the hydrolysis of PtdInsP,, the cooperative action of the two products, Ins( 1,4,5)P, elevating [ Ca2 1, and DAGactivatingprotein kinase C, is ultimatelyresponsiblefor the responses of the cell. One of the most compelling pieces of experimental +

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evidence supporting this hypothesis for lymphocyte activation is the potent comitogcnic action of the Ca2 ionophore A23 I87 and the tumor promoter PMA on mouse T cells (Maino et al., 1974; Truneh et czl., 1985). In the hypothesis, these “opportunistic” mitogens bypass the physiological pathway acting through PtdInsP, breakdown by increasing [Ca2 1, and activating protein kinase C directly as theirrespective primary responses, which are assumed to beobligatory secondary responses for mitogenic stimulation by monoclonal antibodies or lectins. In all T cell lines or primary T lymphocytes thus far examined, including the sub-population of cells bearing the yS receptor (Bismuth et al., 1988), the interaction of mitogenic lectins, mitogenic anti-CD3 antibodies, or specific antigen with the Ti-CD3 complex caused hydrolysis of PtdInsP,, accumulation of inositol phosphates, and an increase in [ C G + Ii (Hesketh et al., 1983b; Moore et a / . , 1984; Taylor et al., 1984; M. J. Weiss C’Z ul., 1984; lmboden and Stobo, 1985; O’Flynn et al., 1985; Shapiro et ul., 1985; Sasaki et d., 1987; Taylor et al., 1988). The principal evidence associating the Ti-CD3 complex with Ptdlns(4,5)P, hydrolysis and the elevation of [Ca2+li has come from studies of a mutant of a human T cell leukemia line (Jurkat) that is unable to synthesize mKNA for the Ti @ chain, has a rcduced level of ct chain transcription, and expresses no detectable Ti-CD3 on its surface (Saito et ul., 1987). After transfection with Ti @ chain cDNA, the cells express a fully functional Ti-CD3 receptor and the responses of the wild-type cells to PHA or anti-CD3 antibody, namely an increase in ICa*+ l i , Ptdlns(4,S)P2 hydrolysis, and synthesis of IL-2, arc restored (Alcover et u l . , 1988). Furthermore, T cell hybridoma mutants expressing decreased amounts of CD3-5-q did not accumulate inositol phosphates when stimulated either by antigen or mAb against CD3-E (Mercep et a/. , 1988). This suggested that the presence of CD3-a, p, y, 6, E, and 5 chains was insufficient to couple activation of the TCR to Ptdlns, hydrolysis, although it does not exclude the possibility that other signals generated by the TCR may be transduced without the participation of CD3-q. Studies using monoclonal antibodies directed against different epitopes of CD2 glycoproteins or against the Ti-CD3 complex showed that any of these antibodies caused rapid hydrolysis of both PtdInsP and PtdInsP, in a cloned cell line (JA3) derived by mutagenesis from IL-2-producing Jurkat T-ALL cells (Pantaleo e t a / . 1987). The concentrations of the polyphosphoinositides declined by approxirnately 50% within 1 Inin and recovered to initial lcvels after a further 2 min. In this study, the stimulated release of DAG was also measured by prelabeling the cells with I3HH]arachidonicacid; transient increases in 1,2-DAG of nearly twofold occurred during the first 5 min of activation by each of the antibodies, but within 15 min, the concentration in unstimulated cells had been restored. There was no increase in 1,3-DAG. A number of IL-2-dependent T cell clones have been used to study the early +

+

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events in T cell activation. Thus in the clone HA1.7, the mitogenic mAb UCHTl , which binds to CD3, caused rapid hydrolysis of PtdInsP, and an increase in [Ca2+ J i (Linch sr a l . , 1987). These responses were shown to correlate with enhanced phosphorylation of PtdInsP to PtdInsP, (Cockcroft et al., 1987), which, it was claimed, occurred more rapidly than the activation of phospholipase C and thus constituted the primary event. For some inexplicable reason, however, this work did not include the resolution of isomers of InsP,, and the data presented are not strong enough to substantiate such a radical claim. An interesting discovery has been the Ipr mutation in T cells (Scholz el al., 1988). lpr cells express CD3 and have normal levels of protein kinase C activity and [Ca2+ I i but do not show agonist-induced PtdlnsP, breakdown. Thus the homozygous mutation causes loss of activation by antigen or mitogens, but the cells proliferate in response to PMA and A23 187 as d o their normal counterparts. Thus it appears that lpr cells have a defect in the signaling capacity of the TiCD3 complex or in the PtdInsP, phospholipase or in some coupling entity. The concentration of InsPs in the resting mutant cells appeared greatly to exceed that in the wild-type cells, however, which may have masked the extent of PtdlnsP, breakdown in the lpr cells. Nevertheless, these cells provide a promising system for study. As mentioned earlier, there is a general requirement for IL-2 stimulation as an intermediate step in the normal mitogenic pathway of T lymphocytes. However, whether the binding of IL-2 activates PtdInsP, hydrolysis remains controversial. Bonvini er al. (1987) found that the murine cytotoxic 1L-2-dependent T cell lines CT6 and CTB6 rapidly accumulated InsP, InsP2, and InsP, (the latter maximal after 1 min) following addition of IL-2 and that there was subsequent enhancement of synthesis of polyphosphoinositides, although very high concentrations of IL-2 were required to produce these responses. Mills er al. (1986), however, showed that in human or mouse IL-2-sensitive T cells, IL-2 did not cause the accumulation of Ins( 1,4,5)P,, Ins( 1 ,4)P2, or InsP, nor did it affect the metabolism of PtdIns, PtdlnsSP, or Ptdlns(4,5)P, as measured by [32P]orthophosphate incorporation; nevertheless, 1L-2 was mitogenic, and PHA or ConA stimulated polyphophoinositide metabolism and the accumulation of inositol phosphates. A similar lack of effect of IL-2 on polyphosphoinositide metabolism was observed in cloned murine cytotoxic T cells (Kozumbo et al., 1987) and in a murine helper T cell clone (Howe, 1989), while the accumulation of inositol phosphates and increased synthesis of PtdIns were observed in response to ConA or specific antigen. In the cytotoxic T cells the stimulation of polyphosphoinositide metabolism was rapid (<2.5 min) and sustained for 6 to 8 hr, a period that correlated with the synthesis and secretion of macrophage-activating factor (MAF). These authors therefore concluded that lymphokine production by these cytotoxic cells was associated with activated PtdIns metabolism.

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6. Replenishment of the Phosphatidylinositol 4,s-Bisphosphate Pool Coincident with the release of large amounts of inositol phosphates from PtdlnsP, in T cells stimulated by ConA, PtdInsP, was shown to be resynthesised, thereby replcnishing the precursor of Ins( 1,4,5)P, (Taylor ct cil., 1984). In these cells either ConA, A23187, or PMA stimulated both synthesis of Ptdlns, detected as enhanced [’H]inositol incorporation after 30-60 min (Moore et al., 1982), and a net increase in PtdIns phosphorylation to PtdlnsP and PtdlnsP,, which was maximal within 10 min (Taylor ~t (11.. 1984). Similarly, in mouse spleen cells, either ConA or PMA activated the PtdIns and PtdInsP kinases (Boon et ul., 1985), although stimulated synthesis of PtdIns was not observed. In principle, the replenishment of the PtdInsP, pool following mitogenic stimulation would enable continued release of Ins( I ,4,5)P, from PtdtnsP, to be maintained. In fact, there is no evidence that detectable concentrations of Ins( 1,4,5)P, are present after the initial transient has subsided, and it is therefore clear that more complex mechanisms determine the Ins( 1,4,5)P, level (see Section 111,D). The enzymes responsible for maintaining the pool of PtdlnsP, are being characterized. For example, the phosphatidylinositol kinase in rnurine T lymphocyte membranes is a Mg’+-dependent, 60 kDa, integral membrane protein that is inhibited by PtdlnsP or PtdlnsP, but not by other phospholipids (O’Shea et al., 1986). As discussed earlier, proliferation of T cells requires the presence of macrophages, and an interesting study by Grier and Mastro (1988) investigated the effect of accessory cell depletion on Ptdlns metabolism in bovine lymph node lymphocytes. In normal preparations of these cells, ConA stimulated the rapid hydrolysis and resynthesis of PtdInsP, and the accumulation of inositol phosphates in a manner similar to that described for other types of lymphocytes. When macrophages were removed, DNA synthesis in response to ConA was reduced by 85%, the accumulation of InsPs was reduced by 40%, and there was no detectable resynthesis of PtdlnsP,. 11- 1 or PMA, together with ConA, restored DNA synthesis in the absence of accessory cells but there was no stimulation of Ptdlns metabolism, consistent with the results of Abraham et al. (1987). Thus in the absence of macrophages, substantial inhibition of the normal PtdIns responses occurred together with almost total inhibition of the proliferative response. When macrophage-depleted cells were treated with concentrations of ConA that were supra-optimal for mitogenesis, however, InsPs accumulated to the levcls measured in thc presence of macrophages. This suggested that high concentrations of thc polyvalent ligand enhanced the cross-linking of surface receptors, thereby promoting responses normally generated by the antigen-presenting cells, and this appears to be consistent with the proliferative responses

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stimulated by immobilized mAbs in the absence of macrophages (Williams et al., 1985; Geppert and Lipsky, 1987; see also Section 11,A).

C. How Do Lymphocyte Surface Receptors Cause Phosphatidylinositol 4,5Bisphosphate Hydrolysis? The failure to detect any endogenous enzymatic activity (for example, tyrosine protein kinase) in any of the mitogenic T cell receptors suggests that PtdInsP, breakdown may represent the primary mitogenic signal generated by these receptors or at least be a component of the overall primary signal. An attractive model for the activation of PtdInsP, hydrolysis is that stimulatory receptors are coupled to the PtdInsP,-specific phospholipase C via guanine nucleotide-binding regulatory proteins (G proteins). A wide range of signal transduction processes are now known to be mediated by G proteins (Neer and Clapham, 1988), and there is evidence that this category includes the hydrolysis of Ptdlns(4,5)P, by phospholipase C (Cockroft and Gomperts, 1985; Haslam and Davidson, 1984; Litosch et d., 1985; Jackowski et a / . , 1986; Ashkenazi et al., 1989). Suggestive evidence that such coupling can occur in T cells has come from studies of plasma membranes and transient premeabilization experiments. When lysed murine thymocyte membranes were pre-labeled with ['H]inositol, the accumulation of inositol phosphates was maximally stimulated by GTPyS (10 p,M) but was not stimulated by GTP or ATPyS (Zilberman et ul., 1987) (Fig. 3). Enhanced accumulation of inositol phosphates has also been observed following treatment of Jurkat cell membranes with GTPyS or NaF and AICI, (Sasaki and Hasegawa-Sasaki, 1987). The activity of ornithine decarboxylase (ODC), which is elevated 4- to 5-fold within 10 min of mitogenic stimulation, has been assayed in cells that had been transiently permeabilized by the addition of ATP in the absence of Ca2+ and Mg2+ to permit the uptake of GTPyS or Ins(1,4,5)P3. Either of these agents. acting intracellularly, caused an increase in the activity of ODC equivalent to that following the extracellular binding of ConA (Mustelin et a / . , 1986). Neomycin inhibited the response, presumably by binding to polyphosphoinositides thereby preventing their hydrolysis, although this drug has other cellular targets. ODC was not activated by DAG, suggesting that this enzyme was not regulated by protein kinase C. In a separate study, GTPyS was shown to release Ca2 + from permeabilized murine thymocytes (Ueda et al., 1988). Taken together, these results suggest that a G protein can couple mitogenic receptors to a PtdlnsP, phospholipase C in T cells and that the rapid activation of ODC is independent of protein kinase C. A phosphoinositide-specific phospholipase C has been cloned and sequenced (see Section III,F), and two major isozymal forms have been isolated from porcine lymphocytes (Carter and Smith, 1987). The enzymes from pig mesenteric lymphatic tissue had a pH optimum for

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X

8 -

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+ c)

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0 10 0 10 0 10 0 10

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0.1

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FIG. 3 . Releasc of InsP, + InsP, from lyscd ihymocytei by CTPyS. Cells were diluted into lysis medium with or without GTPyS at 37°C. After 2 min TrisiKCl buffer containing thc indicated concentrations of Ca'+ and Mg'+ was added. The reactions were ztopped after 40 min. (Ahcr Zilberman ri ti!., 1987.1

Ptdlns hydrolysis of 5.5 and were Ca2+-dependent. Nevertheless, the observations that early responses to antibodies to the Ti-CD3 complex were not affected by pertussis toxin (Ledbetter el NI., 1987a) suggests that the G proteins involved in coupling activated receptors to these enzymes are not the classical G, or G, variety that couple hormonal receptors to adenylate cyclase (Gilman, 1987). A study of murine thyniocytes revealed that two GTPyS-binding proteins of M,. 25 kDa and 40 kDa were associated with ConA receptors (Wang PZ d.. 1989). 'The 25 kDa protein appears to be distinct from the CD3-E (25 kDa) subunit, which, on the basis of sequence analysis, lacks a GTP-binding domain (Clevers rt ul., 1988). The 40 kDa protein was identified as Gi, while the smaller protein appeared to enhance the activity of phosphoinositide-specific phospholipase C. ConA was shown to inhibit partially the increase in CAMPcaused by prostaglandin El or isoproterenol, so these results appear to indicate that ConA receptors on T cells can interact with at least two species of G proteins. The possible mechanisms by which activation of the TCR or other lymphocyte receptors could cause PtdlnsP, breakdown may be summarized as follows. The second messengers generated may be solely products of PtdlnsP, hydrolysis or may include other directly or indirectly activated signals.

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I . The simplest mechanism is direct activation by the receptor of PtdlnsP, hydrolysis: in addition to effects on lCaZ li of Ins( I .4,5)P, and, possibly, InsP, and the potential of DAG and [ C a 2 + l ito modulate the activity of protein kinase C, the indirect products of this reaction may have functions as yet unknown. 2. The receptor may have multiple, direct targets: these would include the stimulation of PtdInsP, hydrolysis and may also include protein tyrosine kinases and as yet unknown targets. 3. Activation via the same coupling (G?) protein to both a phosphodiesterase and to a tyrosinc kinase or to other, unknown mechanism(s) of second messenger generation. 4. Activation via different coupling proteins of PtdInsP, hydrolysis, protein tyrosine kinase(s) or to other, unknown mechanisms of second messenger generation. +

D. lnositol 1,4,5-Trisphosphate Release and Metabolism Although it has been known for many years that the mitogenic activation of lymphocytes stimulated Ptdlns metabolism (Fisher and Mueller, 1968, I97 t ; Maino et al., 1975; Allan and Michell 1977), it is only within the last 5 years that measurements of the intracellular release of the resolved isomer Ins( I ,4,5)P, have been made in thesc cells (Moore et ul., 1984; Taylor et al., 1984). The first report of the accumulation of inositol 1,3,4,5-tetrakisphosphate[Ins( 1 ,3,4,5)P,] in lymphocytes was by Stewart et al. (1986) using Jurkat cells stimulated with a monoclonal antibody to the TCR. We have carried out a detailed study of the patterns of inositol phosphates production in normal mouse thymocytes stimulated with ConA (Zilberman et u l . , 1987). The principal results, shown in Fig. 4, represent typical patterns of changes in the concentrations of inositol phosphates in lymphocytes activated by cross-linking ligands. The maximum concentration of Ins( 1,4,5)P, was reached approximately 1 min after addition of ConA when the increases in the amounts of InsP, and the two isomers of InsP,[Ins( 1,4,5)P, and inositol 1,3,4-trisphosphate [Ins( I ,3,4)P,J), were in the order of InsP, > Ins( I ,4,5)P, > Ins( 1,3,4)P,. The Ins( 1,4,5)P, maximum coincided closely with the peak in the[Ca2+ I, transient (see Section 111,E;see also Hesketh et al., 1985). There was a transient maximum in the amount of InsP, after 2 min followed by a further slow accumulation. The accumulation of Ins( 1,3,4)P, was monotonic, reaching 50% of the amount of InsP, after 20 min. From experiments measuring the concentrations of the inositol phosphates within 10 sec of mitogen addition, it was concluded that there was no very rapid increase in Ins( I ,4,5)P, preceding the increase in InsP,. This was consistent with the delay of 10-15 sec in the [Ca2+Iiresponse to ConA in these cells (see Section 111,E; see also Hesketh et ul., 1983a, 1985).

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Fici. 4 Production o f inoiitol phosphates in mouse thymocytcs in response to ConA. [7H]inusitollabclcd cells were treated with ConA (10 pg/ml). The inositol phosphates were assayed by high pressurc liquid chromatography, and the amounts are expressed relative (%) to the total d.p.rn. in [3H]PtdIns in cell aliquots. A and B show time courses for 20 rnin and 60 sec after ConA addition. (After Zilbemian a / ., 1987 ) (31

The transient accumulation of Ins( 1,4,S)P, could be due to the termination of PtdlnsP, breakdown within a few minutes of ConA addition or to a substantial increase in the rate at which Ins( 1,4,5)P, is converted into other products (InsP, and/or InsP,). The removal of ConA from the cell surface after 5 min with amethyl mannoside caused a rapid decrease in the InsP, + InsP, pool at an initial rate comparable with the initial rate of production of inositol phosphates in response to ConA (Fig. 5). If it is assumed that all of the InsP, and InsP, is derived from the initial release of Ins( 1,4,5)P, from PtdlnsP,, these data show that, rather than there being a transient burst of PtdlnsP, hydrolysis, Ins( 1,4,S)P, must be produced at an approximately constant rate while the ConA receptors remain activated. It may be noted that, although thymocytes contain at least 5-fold more InsP, than the combined amounts of InsP, and InsP,, there is no significant change (< 10%) in the concentration of InsP, on mitogenic stimulation (Fig. 4A). It seems unlikely, therefore, that InsP, acts as a signal in response to ConA in these cells, and its concentration was also unaffected by activation of the TCK in Jurkat cells (Stewart et al., 1987). It is of interest to note the effect of the microfilament-inhibiting drug cytochalasin D. Although not itself affecting Ptdlns metabolism, cytochalasin D caused a slight increase in the accumulation of InsP, InsP,, and lnsP, in response to ConA (Treves et ui., 1987b). Cytochalasin D has previously been shown to amplify the mitogenic response of T lymphocytes (Pozzan et ul., 1981). In murine B cells,

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r

CON A

0

d-MM

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PIC;.5. Reversal of inositol phosphate rcsponscs to ConA. Cells labeled with ['Hlinositol were treated with I Fgiml ConA (closed symbols). Open symbols indicate the pattern of inositol phosphates production when a-methyl mannoside (SO M )was added 5 min after ConA. (After Zilberman el a / . . 1987.)

cytochalasin has been reported to stimulate phosphoinositide metabolism, increasing the amounts of InsPs (including InsP,) over 30 min (van Haelst and Rothstein, 1988). These studies of activated lymphocytes have proceeded in parallel with analyses of the roles of polyphosphoinositide metabolites in a variety of other cell systems. Thus Irvine et al. (1985) first showed that when receptor-mediated PtdInsP, hydrolysis was activated in the rat parotid gland, two isomers of InsP, were produced. There was a transient increase in Ins( 1,4,5)P,, which is the initial product from PtdInsP, breakdown, and a slower, persistent increase in Ins( 1,3,4)P, similar to that observed in murine T cells (Fig. 4). Accumulation of Ins( 1,3,4,5)P, concomitant with that of Ins( 1,3,4)P, has been observed in carbachol-stimulated cerebral cortical slices (Batty er d . , 1985), and small amounts of InsP, and InsP, have been found in stimulated GH, pituitary cells (Heslop et al., 1985) and in mammalian brain (Vallejo et a l . , 1987). When ['H]Ins( 1,4,5)P, was incubated with rat brain homogenates, Ins( 1,4,5)P, was converted sequentially into Ins( 1,3,4,5)P, and Ins( 1,3,4)P, (Irvine et ul., 1986). These results indicate, therefore, that the pattern of PtdIns metabolism stimulated by many ligands that activate lymphocytes is broadly similar to that established in many other types of cells.

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E. Correlation of lnositol Phosphates Accumulation with [Ca2+], Responses The development of the fluorescent indicators quin2 (Tsien et ul., 1982) and fura-2 (Grynkiewicz et ul., 1985) has permitted the direct measurement of [Ca*+Ii in intact cells, which in unstimulated eukaryotic cells is normally approximately 100 nM (Hesketh t ~u/ / . , I983b). The increase in lCa2 ’ Ii occurring in activated T cells is composed of a transient component commonly reaching 500 nM, which is independent of extracellular C a 2 + , and a sustained elevation of approximately 100 nM, which requires influx of Ca2 across the plasma membrane (A. Weiss er al., 1984; Metcalfe et al., 1985; Oettgen rt a / . , 1985). MAbs to either Ti-CD3 or CD2 increased [Ca2 ’ Iiin unstimulated human lymphocytes or T cell clones from 100 nM to approximately 300 nM (Alcover et al., 1986; Goldsmith and Weiss, 1988). The [Ca2 liresponse to anti-CD3 included a large transient component, complete within 10 min when a stable concentration of 200 nM was reached. The response to anti-CD2 was slower, reaching a new constant value of 300 nM after 10 min with no significant transient component, although subsequent studies (Pantaleo e t a / ., 1987) have shown that some release of Ca2 ’ from intracellular stores does occur in response to antiLCD2 antibody. Single cell studies carried out by flow cytometry combined with immunofluorescence have essentially confirmed these observations but indicated that differential responses occurred between subsets of cells (Kabinovitch rt al., 1986). Thus, in peripheral blood lymphocytes, the lCa2 + I i increase in response to PHA or mAb against CD3 was greater in CD4 than in CD8 cells, and a proportion of the cells showed no change in [Ca’+ li in response to PHA. In Jurkat cells, however, there was a (Ca2 litransient in essentially all cells in response to mAb against the CD3 complex. The similarity between the kinetics of the transient increases in [Ca2+ Ii and in Ins( I ,4,5)P, is consistent with the hypothesis that this isomer is responsible for releasing Ca2 from the endoplasniic reticulum, thereby accounting for the transient component of the [Ca2 li response, which is essentially independent of lCa2 J, (Irvine, 1989). Compelling evidence that one was a Consequence of the other only appeared with the demonstration that, in common with other cell systems (Streb et a/., 1983), the addition of InsP, to permeabilized lymphocytes caused a lCa2 Iitransient similar to those generated by mitogens (Imboden and Stobo, 1985). This work used Jurkat cells and mixed isomers of InsP,, but a similar response to Ins( 1,4,5)P, has been obtained in human cells (Eberl and Schnell, 1987) and in rat thymocytes permeabilized with digitonin (Gukovskaya rt (I/., 1986); in the latter study, the Ca2 released was shown to be from nonmitochondria] stores. There is convincing evidence that to cause lymphocyte proliferation a crosslinking ligand must remain bound to its plasma membrane receptor until the cells +

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are committed to S phase (i.e., for up to 20 hr). Removal of the mitogen before this point reverses the sustained increase in [ C a 2 + l iand causes the cells to fall back into the quiescent state (Hesketh et ul., 1983a). The inositol phosphates responses following removal of ConA from the cell surface with a-methyl mannoside (Fig. 5 ) are consistent with the reversal of the persistent component of the [Ca2+Ii response, in that they iniply that there is continuous production of Ins( 1,4,5)P, at an approximately constant rate as long as the receptors remain activated by ConA. Similar results have also been reported in Jurkat cells by Imboden and Weiss (1987), who also showed that when a-methyl mannoside was used to displace ConA from the TCR, the intracellular Ca2+ pool was refilled by influx of extracellular Ca2’ . The reduction of the extracellular Ca2+ concentration to 100 nM had very little effect on the release on Ins( 1,4,5)P,, although the persistent component of the [Ca2 liresponse was abolished (Zilberman et a / ., 1987). The rapid increase in Ins( 1,4,5)P, was, however, sustained for at least 5 min, in contrast to the transient appearance of this isomer in normal medium. There was also a very large decrease in the amount of InsP,, suggesting that the kinase responsible for the conversion of Ins( 1,4,5)P, to InsP, was Ca2 -dependent. Supporting evidence for this conclusion came from experiments with lysed T cells that indicated that the rate of this conversion increased substantially between 0.1 FM and 1 p M free Ca2 (Zilberman et id., 1987). Of the large number of inositol phosphate metabolites produced in lymphocytes (Fig. I), lns(1,4,S)P, remains the only one for which a role in generating a subsequent response has been established. It has, however, been proposed that Ins( 1 ,3,4,5)P,, acting together with Ins( 1,4,5)P,, is responsible for sustained elevations in [Ca2 li in agonist-stimulated cells caused by extracellular Ca2 entry (Irvine and Moor, 1986). A more refined version of this model (Irvine, 1989) proposed that InsP, acts to release Ca2+ from a pool distinct from the endoplasmic reticulum, which may be the “calciosomes” detected by Volpe et al. (1988). This pool, maintained by influx of Ca2+ across the plasma membrane, would therefore be linked to the endoplasmic reticulum pool by InsP, via a mechanism that may be GTP-dependent, as suggested by the data of Mullaney et a / . (1987). One function of the calciosome pool would be to maintain a constant, net intracellular Ca2 concentration that would otherwise decline as some of the Ca’+ released by 1ns(1,4,5)P3 was pumped out by the plasma membrane Ca2+-ATPase. It is also possible that the coordinated regulation of ICa2 Iiby Ins( 1,4,S)P, and InsP, may generate the periodic oscillations in [Ca2+ li that have been observed in many types of cells (Cobbold et u l . , 1989), but it should be noted that oscillations in the concentration of any inositol phosphates have not yet been detected in any cell. Some direct evidence that InsP, can cause Ca2 intlux has come from voltage clamping experiments on single acinar cells (Morris et al., 1987). These experi+

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ments indicated that a sustained increase in the Ca2+-activated K current could not be produced by either Ins(l,4,5)P3 or InsP, acting alone, but the two inositol phosphates togethcr were able to generate a persistent increase in current that was dependent on external Ca2 . In activated thymocytes, the rapid accumulation of InsP,, which is maintained at high levels in the cells (Fig. 4), does have the characteristics to act as a sustained intracellular signal in response to ConA by, for example, opening Ca2+ channels in the plasma membrane to cause the persistent rise in [Ca2+ I i . Such channels have been detected by the patch clamp technique in T cells stimulated by PHA (Kuno et ul., 1986). The evidence from the use of this technique in Jurkat T cells, howevcr, is that Ins( I ,4,5)P3 alone, produced after stimulation viu Ti-CD3 or CD2 (Gardner ef al., 1989), can activate single channel conductance and that its target may be the mitogensensitive, voltage-independent Ca2 channel activated by the TCR (Kuno and Gardner, 1987). Consistent with this suggestion is the observation that when the ligand-TCR complex was disrupted, the decline in [Ca2+j i preceded that in InsP,, thus indicating that InsP, alone was insufficient to maintain the increase in [Ca2+li(Imboden and Weiss, 1987). In contrast to these findings, Hill et (11. (1988) have shown that, in electropermeabilizcd neoplastic rat liver epithelial cells, Ins( I,3,4,5)P, lowcred lCa2+]i from the sustained elevated level caused by the inositol trisphosphate isomer lns(2,4,5)P,, which is not metabolized. This sequestration of Ca2 was caused by Ins( 1 ,3,4,S)P4acting alone, its presumed receptor being distinct from that for Ins( I,4,5)P,. An InsP, receptor has not yet been detected in lymphocytes, but a high affinity binding site ( K , 4 nM) has been demonstrated in bovine adrenal cortical microsomes (Enyedi and Williams, 1988). In a detailed study of the effect of monoclonal antibodies on [CaZi Ii Ledbetter et al. (1987b, 1988a) showed that substantial (between 50 and 400%) increases in [Ca2 Ii were caused when antibodies bound to a number of T cell differentiation antigens were cross-linked by a second antibody. In human T cells the antibodies were directed against CD2, CD4, CD5, CD6, CD7, CD8, Tp44, and CDw18 and in mouse cells against Thy-I .2, Lyl, Ly9, ThB, and Lyt-2. None of these antibodies alone caused proliferation, even after cross-linking, although cross-linking of CD2 or Tp44 induced the cxpression of IL-2 receptors. The increase in lCa2 li caused by each cross-linked antibody was completely inhihited by pertussis toxin, a result that suggests the involvcment of a G protein in the signaling mechanism but is difficult to reconcile with the observation that pertussis toxin is itself mitogenic in many types of T cells (Morse et al., 1977; Rosoff rt ul., 1987; Strnad and Carchman, 1987). Anti-L3T4 (CD4) antibody did not affect [Ca2+ I t , in agreement with our observations on a T cell clone (see Section II1,K). In apparent conflict with the data of Ledbetter et ul. (1977b), Carrel et al. (1988) showed that human T cells proliferated in response to a mAb (B66.6) +

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against CD4, which caused a rapid [Ca2+Iitransient, and Alcover et al. (1988) demonstrated IL-2-dependent proliferation following activation of the CD2 receptor, which caused PtdInsP, breakdown and an increase in [Ca2+Ii. MAbs against the CD2 receptor have also been shown to be mitogenic for peripheral lymphocytes, but not for thymocytes, even though 1L-2 receptor expression was induced in the latter. However, CD2 could stimulate IL-2-dependent proliferation of thymocytes in the presence of anti-CD28 (Yang et ul., 1988; Pierres et ul., 1988). This observation has been confirmed by van Lier et al. (1988), but although they showed that anti-CD2 together with anti-CD28 stimulated proliferation, they were unable to detect any increase in [Ca2+li.The contradictions between these data and the results of Ledbetter et al. (1987b) may result from the different epitopes recognized by mAbs directed against the same receptor. Thus Meuer et ul. (1984a) demonstrated the existence of three distinct epitopes on CD2, and proliferation occurred following simultaneous activation of two of these. This work was extended by He et al. (1988) who confirmed that antibody to a single CD2 epitope was not mitogenic but showed that two different antiCD2 mAbs, for example, OX54 and 0x55, did stimulate proliferation. A third antibody, also to CD2 (0X34), blocked the mitogenicity of the combination of OX54 and 0x55. The same authors have transfected Jurkat cells with rat CD2 cDNA and shown that deletion of either 110 or 76 of the 116 residues in the cytoplasmic domain of CD2 virtually abolished the [Ca2 Ii response caused by rat anti-CD2 monoclonal antibodies. The significance of the cytoplasmic regions of CD2 that appear to activate the ionic response remain to be resolved, but it seems probable that the presence of a cross-linking antibody, as used in the experiments of Ledbetter et al. (1987b), or a second antibody against a different epitope results in a conformationai change in the receptor that promotes signal transduction. +

F. Protooncogene Activation and the Phosphatidylinositol Cycle The stimulation of mitogenesis in T lymphocytes is correlated with the coordinated transcriptional activation of over 60 genes during the first 6 hr following addition of mitogen (Zipfel et ul., 1989). This large number of induced genes includes the protooncogenes C ~ ~ Oand S c - m y . the products of which are considered to play an essential role in regulating cell growth and which are presumed to be released from the normal controling mechanisms when cells undergo transformation. c-fus transcription is detectable within 5 min of the addition of mitogens to T cells, and this renders attractive the suggestion that the activation of gene expression is coupled to the earliest known primary response in these cells, namely the hydrolysis of PtdlnsP, and the subsequent ionic responses. This has been

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investigated in detail in T cells where it is evident that c-jos and c-myc transcription can be activated either by cross-linking ligands (ConA), by A23187, or by PMA (Moore et al., 1985).Thus there arc both lCa2 I,-dependent and [Ca2 Iiindependent activation mechanisms. For activation of the TCR by ConA, expression of both c $ x and c-myc appears to be largely independent of either the (Ca?-t 1, responses caused by cross-linking the receptors or of protein kinase C (Moore P I l i l . , 1985). Furthermore, for eukaryotic cells in general, no evidence has yet accrued linking early ionic responses caused by PtdlnsP, breakdown to the activation of protooncogene expression. Paradoxically, howcver, there is indirect evidence that protooncogene products may modulate PtdlnsP metabolism. This has mainly come from studies of fibroblasts that show patterns of growth factor-activated PtdinsP, breakdown, ionic responses, and rapid activation of c-fos and c-myc expression that are closely similar to those of leukocytes (Hesketh C t d., 1988). The initial observation was that in chick embryo fibroblasts transformed by the avian sarcoma virus UR2, the transforming protein of which is the tyrosine kinase p6XV-"", immunoprecipitates of p6Xv-r~'.y also possessed Ptdlns kinase activity (Macara el a / . , 1984). PtdInsPz inhibited the PtdIns kinase activity and autophosphorylation of p68, and [ 32P]labeled cells showed enhanced incorporation into PtdlnsP and PtdInsP, and accumulation of Ins( 1,4)P, and Ins( 1,4,5)P,. Similar results were obtained when pp6OV-"'"'was immunoprecipitated from RSVtransfected cells (Sugimoto e l a / . , 1984), and in SV40-transformed NIH 3T3 fibroblasts, the Ptdlns-PtdlnsP kinase activity that imniunoprecipited with the middle T antigen-pp6@-""' complex was induced in parallel with middle T antigen (Kaplan e t a / ., 1986).The original interpretation of these results was that the PtdIns kinase activity was borne by the transforming protein. This has proved incorrect. Whitman et ul. (1987) have now shown that the type I PtdIns kinase of fibroblasts is specifically associated with the middle T antigen-~p60'-~~" complex or with p~60"-"'~' and can be separated therefrom (see also Fukui and Hanafusa, 19x9). Type I PtdIns kinase also co-precipitated with the platelet-derived growth factor (PDGF) factor (which both stimulates PtdlnsP, hydrolysis and possesses intrinsic tyrosine kinase activity), and the amount of activity in anti-phosphotyrosine immunoprecipitates increased 50-fold after PDGF stimulation. Courtneidge and Heber (1987) have confirmed that the complex of middle T antigen and pp60"."" that can be immunoprecipitated from pol yoma transformed cells includes a PtdInsP kinase, but they made the interesting additional observation that this kinase itself undergoes tyrosine phosphorylation. Similar results have been obtained in 3T3 fibroblasts transformed by polyoma middle T antigen, v+n.s, or v-xis (Kaplan et ul., 1987). The significance of the formation of such complexes in activated and transformed cells remains obscure. It should be noted that the type I kinase phosphorylates the inositol ring in the 3 position and is therefore not directly involved +

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in the production of PtdlnsP,. Nevertheless, the observation that one class of polyoma mutants defective in transformation was also low in PtdIns kinase activity but had normal tyrosine kinase activity underlines its possible importance (Kaplan et a / ., 1986). Clearly it provides a possible mechanism by which protooncogene and transforming oncogene products could interact with the mediators of normal growth control. A striking discovery has come from studies of the avian sarcoma virus CTlO, the transforming protein of which is p47RaR-crk (Mayer et a / . , 1988). The expression of this protein in infected cells elevates the level of tyrosine phosphorylation, and several regions of the protein show strong sequence homology with domains in the noncatalytic region of other cytoplasmic oncogenes that are also tyrosine kinases (e.g., c-src). The simultaneous discovery of three similar homologous regions in a cloned PtdIns-specific phospholipase C (Mayer el a / . , 1988; Stahl et al., 1988), albeit transposed with respect to c-src, suggests that several tyrosine kinases share a common regulatory mechanism with this phospholipase. These regions may compete for the same regulatory factors, so that, for example, the lipase could deplete negative factors thus increasing tyrosine phosphorylation (Mayer ef at., 1988). No evidence for interactions between the products of protooncogenes and the enzymes of PtdIns metabolism in lymphocytes has yet been reported, but the close similarity between the known, early mitogenic responses in these cells and those in fibroblasts suggests that they could occur in lymphocytes. G. Does Phosphatidylinositol 4,5-Bisphosphate Hydrolysis Activate Protein Kinase C? It is evident from the foregoing that T cell mitogens generally increase Ca2 I through the action of Ins(1,4,S)P3. It is also clear that the direct activation of protein kinase C by PMA can constitute an effective co-mitogenic signal in T cells. In intact lymphocytes, however, it is far from established that the other product of PtdInsP, breakdown, DAG, causes the concomitant activation of protein kinase C. Indeed, the concentration of DAG in resting T cells is high, and it is not clear how the DAG released from PtdInsP, could constitute a sensitive signaling mechanism. We have argued elsewhere (Metcalfe et al., 1985) that, although protein kinase C is a Ca2 -dependent enzyme, the ConA-induced increase in [Ca*+ Ii in thymocytes is insufficient to cause its activation and furthermore that the DAG released from PtdInsP, does not activate protein kinase C by more than 5 % of the maximal increase that can be caused by PMA. The evidence for this conclusion is (1) neither polyclonal mitogens (ConA) nor mAb against cause detectable redistribution of enzymatic activity within 30 min, (2) cross-linking ligands did not cause the phosphorylation of an 80 kDa protein that appears to be the T cell homologue of the specific substrate for the enzyme in fibroblasts (Hesketh ei ul., +

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1989) nor was this protein phosphorylated in response to A23187, and (3) macrophage-depleted T cells require both PMA and a cross-linking mitogen to stimulate maximal DNA synthesis (Kaibuchi et d., 19x5). In apparent conflict with these deductions, however, are the observations that, in a niouse T cell clone, down-regulation of protein kinase C by chronic treatincnt with phorbol esters resulted in the loss of the proliferative response to mitogens or to anti-TCR antibody (Valge et ul., 1988). Nevertheless, these cells still proliferated in response to IL-2. Similar results were obtained with a mouse cytotoxic T cell clone (Redondo et ui., 1988). These data were interpreted as indicating that protein kinase C is required for the expression of IL-2 receptor in response to activation of the TCR but not in response to IL-2. These results indicate the existence of at least two pathways for proliferation in T cells, one of which is independent of protein kinase C. The report of Chauhan and Brockerhoff (1988), although not yet substantiated in lymphocytes, has implications for the role of protein kinase C in signaling pathways; they found that in rat brain the enzyme was activated 50 times more efficiently by PtdIns(4,5)P2 than by DAG and, furthermore, that the effect was specific for that phosphoplipid , neither Ptdlns or PtdInsP causing significant activation. The phosphorylation of components of the TiLCD3 complex caused by the direct activation of protein kinase C and by antigenic activation of the TCR has been extensively studied by Klausner and his colleagues. In addition to the rapid stimulation of Ptdlns(4,5)P2 hydrolysis and the modulation of [Ca2 Ii, activation of the TCR complex also causes serine phosphorylation of the y subunit of CD3 and weaker phosphorylation of CD3-6. This was interpreted to be a consequence of protein kinase C activity because a similar phosphorylation pattern occurred on treatment o f T cells with PMA (Samelson et ul., 19x6, 1987; Patel et u / . , 1987; Klausner et ul., 1987). Either anti-CD3 antibody or PMA promoted the down-regulation of the complex (Meuer et al., 1983b; Cantrell et ul., 1985; Samelson et al.. 1985a). A very striking finding has been that antigenically stimulated murine T cells undergo an additional phosphorylation reaction on CD3-5, but in this case, the protein is the target of a tyrosine kinase (Baniyash et ( I / . , 3988b; Veillette et a l . , 1989). Tyrosine phosphorylation of CD3-5 was not affected by the depletion of cellular protein kinase C (Valge et d., 1988). CD3-< is present in much smaller amounts than are the other components of the TCR (Minami et a / ., 1987), and only approximately 10% of CD3-5 molecules underwent phosphorylation. We have observed that antibodies to CD3 or Ti stimulate significant increases in the total phosphotyrosine content of the cells (Hesketh et a/. , 1989); therefore. although both human and murine forms of CD3-[ contain six cytoplasmic tyrosine residues (Weissman e t a / . , 1988), it seems probable that there are additional other cellular targets for tyrosine kinases activated in T cells.

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It is now established that growth factors and mitogens that cause proliferation of many types of mammalian cells rapidly increase the endogenous tyrosine kinas, activity of their specific receptors (e.g., the fibroblast receptors for plateletderived growth factor, insulin, and epidermal growth factor). As was pointed out earlier (Section III,C), no tyrosine kinase activity or protein kinase amino acid signature has so far been found within any T cell receptor. Nevertheless, the detection of a phosphotyrosine protein “loitering with intent” in the activated Ti-CD3 complex and the elevated levels of phosphorylated tyrosine in these cells suggests strongly that this receptor does indeed stimulate a tyrosine kinase and thus belongs within the family of receptors bearing endogeneous tyrosine kinase activity, and we suspect that tyrosine kinase activation may be a fundamental step in the normal mitogenic pathway in lymphocytes. In this context, it may be noted that in murine cells, CD4 and CD8 surface antigens appear to be functionally associated with ~ 5 6 / ( .the ~ , lymphocyte-specific tyrosine kinase (Veillette el ul., 1988). In this work, CD4 and CD8 co-immunoprecipitated with p56/1‘~. and the results suggested the existence of a mechanism analogous to that activated by fibroblast growth factors in which cross-linking of the CD4-CD8 surface antigens might activate the associated tyrosine kinase. This model is supported by the observation that anti-CD4 antibody caused rapid tyrosine phosphorylation of the TCR 5 subunit that was equivalent to that observed in response to antigen (Veillette r t a l . , 1989). Despite the similarity between the patterns of serine phosphorylation of CD3-y and CD3-E following activation by PMA or via the TCR, the evidence that ligands activating the TCR promote the activity of protein kinase C remains unconvincing. For example, a recent study on human T lymphocytes has shown that the lectin wheat germ agglutinin (WGA) was as effective as anti-T3 Ab or PHA in increasing [Ca2 ’ J i and each caused accumulation of inositol phosphates (Clevers et al.. 1986). Nevertheless, WGA alone was not mitogenic but was a potent co-mitogen when added with PMA. If it is assumed that the only target of PMA is protein kinase C, thesc observations also suggest that when the TCR is cross-linked by WGA, the DAG released from PtdInsP, does not cause significant activation of protein kinase C. Further data consistent with this conclusion have been provided by Rister and Sage (1986) who obtained conditions for mouse T cells under which ConA gave PtdInsP, breakdown but not significant IL-2 expression or DNA synthesis. Co-addition of PMA activated both late responses. These authors concluded that the activation of protein kinase C by ConA-induced PtdlnsP, hydrolysis either did not occur or was quantitatively insufficient to promote the later responses. They also pointed out that the [Ca2+ I i increase could cause protein kinase C to bind to the plasma membrane (see Section III,H), but if this event is synonymous with the activation of the enzyme, then it too is inadequate.

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H. Protein Kinase C Translocation It is established in a number of cell types that the hydrolysis of PtdlnsP, arising from the activation of receptors is correlated with an alteration in the subcellular distribution of protein kinase C. In T cells, anti-CD3 antibody caused the translocation of 60% of the cytosolic activity of protein kinase C to the plasma membrane (Nel ef ul., 1987). Sarnelson ot ul. (I98Sb) showed that, although antigenic activation of thc TCK caused scrine phosphorylation of CD3-8, both CD3-6 and CD3-E were phosphorylatcd in response to PMA. These results indicate that even if protein kinase C was being stimulated via the TCR, the responsc was not quantitatively the same as that causcd by PMA. In fact, we have shown that activation of the niurine TCR by the polyclonal mitogen ConA caused no detectablc redistribution of protein kinase C during the first 30 min after stimulation (Hesketh e/ ul., 1989). Furthermore, the mAb G7, which recognises Thy- 1 (Gunter et u l . , 19841, did not modulate the activity or distribution of the cnzyme; G7 requires the co-addition of IL-2 or PMA to stimulate proliferation. PMA, however, caused essentially complete transfer of protein kinase C activity from the cytosol to the plasma membrane within 30 min, with the disappearance of any detectable activity occurring during the subsequent 6 hr. The observations contrast with those of Grove and Mastro ( I 987); using bovine lymph node cells, they found that PMA or ConA had very similar effects on protcin kinase C activity and distribution, with eithcr agent causing a 3- to 4-fold increasc in activity in the particulatc fraction within 5 min of addition and no significant dccrease in total activity within 30 min. The apparent inconsistencies between different assays of protein kinase C distribution may derive from artifacts introduced during the fractionation procedure. In addition, however, the observations of Manger et ul. (1987) on translocation of the enzyme in Jurkat cells stimulated with mAb against CD3 are of interest. These authors found substantial and sustained transfer of enzymatic activity to the membrane fraction when the antibody was immobilized but a much smaller, transient effect when solublc antibody was uscd. Howcver, regardless of the manner of antibody presentation, PMA was required to obtain maximal DNA synthesis, suggesting that cven the major redistribution of enzymatic activity caused by immobilized antibody represented a relatively insignificant activation in terms of the requirement for prolifcration. In B cells, anti-lg caused rapid transfer ( 2 rnin) of 60% of the total protein kinase C activity from thc cytosol to a membrane fraction with a subsequent, slow recovery of activity in the cytosol during the following 15 min (Chen et al., 1986; Cambier et ul., 1987). In a later report. Mond et ul. (1987) showed that in cells deplcted of protein kinasc C , anti-lg no longer caused mitogenesis but still induced the expression of MHC Class I1 molecules on the cell surface. The ribonucleoside 8-rncrcaptoguanosine rcstored B cell proliferation in protein

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kinase C-depleted cells, as assayed by the increased synthesis of a nuclear protein (p40, PI 5 ) whose appearance correlated with mitogenesis. Stimulation of B cells with antibodies against Ia antigcns, however, caused a different pattern of protein kinase C redistribution (Cambier et al., 1987). A similar rapid loss of cytosolic enzyme activity occurred, but translocation was almost exclusively to the nucleus. Anti-la antibodies increased the concentration of cAMP in B cells, and it was also shown that CAMP analogues also mediated translocation of protein kinase C to the nucleus. In view of the fact that protein kinase C is a protein that contains zinc-fingers, it seems probable that the enzyme regulates the expression of genes associated with differentiation, the response stimulated by elevation of CAMP.

1. The Effect of cAMP on T Cell Function Although, as mentioned in the introduction, cAMP is a powerful activator of many cellular responses, including growth and differentiation, it has been firmly established that it is a potent inhibitor of the early steps in the activation of lymphocyte proliferation (Novogrodsky et a / ., 1983; Farrar et a / ., 1987), including those following stimulation by IL-2. Nevertheless, it has been known for a long time that lymphocytes possess receptors for prostaglandins (PGE, and PGE,), although the elevation of cAMP by PGE,, PGE,, or dibutyryl cAMP inhibited lymphocyte-mediated cytolytic activity (Henney et al., 1972). Thc effects of increased cAMP concentration are presumed to occur through the action of CAMP-dependent protein kinases, and lymphocytes have been shown to contain both types (I and 11) of these kinases (Kemp et a / . , 1975). Grove and Mastro (1987) have shown that ConA activates the type I1 kinase in bovine lymph node cells but has little effect on the activity of type I. We have shown that specific patterns of protein phosphorylation were induced in human or murine T cells by forskolin (which activates adenylate cyclase directly) or by analogues of CAMP, but these patterns were not identical with that obtained in response to ConA (Hesketh et al., 1989). Furthermore, there is no change in the concentration of cAMP in T cells during the first 30 min after stimulation by ConA (Moore et al., 1983). In studies of the effect of cAMP on PtdIns metabolism in intact cells, both the Ins( 1,4,5)P, response to ConA and the transient component of the [Ca2+ I i signal in mouse T cells were substantially inhibited by 8-bromo-CAMP (Taylor et d., 1984; Hesketh et al., i985). A similar observation was made by Kaibuchi et al. (1982), who showed that, although in hepatocytes the stimulation of PtdIns metabolism was not inhibited by CAMP, in human peripheral T cells, the incorporation of j2P into Ptdlns was inhibited in the presence of PGE,, which increased the concentration of cAMP by 10-fold. This suggested that the PtdIns kinases were independent of the CAMP-dependent kinase in hepatocytes but not

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in lymphocytes. The stimulation of Ptdlns metabolism by antigen or by anti-TCR antibodies was also blocked by increasing cellular concentrations of cAMP (O’Shea et d.,1987). This general pattern of results was confirmed and extended in a more recent study of murine T cells by Lerner et ul. (1988), who showed that cholera toxin, forskolin, PGE,, or isoproterenol reduced the increase in inositol phosphates and [Ca’ li normally caused by ConA. The twofold increase in cAMP caused by these agents reduced the responses to ConA by 50%. Lerner et al. pointed out that cAMP could exert its effects by (1) reducing the activity of the PtdlnsP,-specific phospholipase C, ( 2 ) reducing the synthesis or availability of phospholipase C substrates, (3) altering the specificity of a phospholipase C substrate(s), or (4) activating an lnsP phosphatase. The observation that 8-bromo-CAMP slightly increased PtdlnsP, synthesis and inhibited its breakdown by ConA (Taylor et a / . , 1984) suggests that cAMP does not diminish the resynthesis of phospholipase C substrates. It may be noted that experiments with isolated plasma membranes indicated that a CAMP-dependent protein kinase can stimulate the formation of polyphosphoinositides (Sarkadi rt u l . , 19x3). These experiments measured the phosphorylation of membrane lipids in the presence of \Y-~,P\ATPand 32P was incorporated exclusively into phosphatidic acid, PtdInsP, and PtdInsP,. Thc relative importance of the other possible targets of CAMP-dependent protein kinases remains to be resolved. These observations do, however, clearly establish that two of the major transmembrane signaling systems in lymphocytes, those involving adenylate cyclase and PtdInsP, hydrolysis, interact with one another in a regulatory network sometimes referred to as “cross-talk.” There is some inconsistency between the above observations on murine T cells and some work on the human T lymphoma cell line Jurkat (Imboden et d., 1986). In these cells it was found that, although cholera toxin inhibited T cell antigen receptor-mediated increases in InsP, and [Ca, Ii, its effects were not mimicked by raising the concentration of cAMP using forskolin or 8-bromoCAMP. These authors suggested that cholera toxin was acting viu a G protein distinct from the G,, that regulates the transduction of signals to adenylate cyclase. Nevertheless, other studies on Jurkat cells indicate that the normal, cholera toxin-sensitive G,, coupling mechanism operates in these cells (Mary et ui., 1987). It has also been reported that aluminium fluoride, which can directly activate Gh,, causes breakdown of Ptdlns, PtdlnsP, and PtdlnsP,, together with the accumulation of InsPs (O’Shea et u l . , 1987). It was concluded, howcver, that the effects of AlF4 ~,which also included phosphorylation of CD3-y and C D h , were not due to increased concentrations of cAMP and the mechanism remains obscure. The complexity of CAMP-mediated effects is illustrated by the fact that, despite its inhibition of proliferation, cAMP appears to promote some of the early responses associated with the activation of lymphocytes. Thus in the murine +

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cytoxic cell clone CT6, an increase in cAMP activated transcription of c-jos and c-myh mRNA (Farrar et al., 1987) and could substitute for IL-l in inducing expression of IL-2 receptors (Shirakawa er a / . , 1988). The synthesis of 1L-2 itself, however, was inhibited by PGE, or PGE,. IL-2 alone stimulated cTfos, c-myc, and c-myb transcription (Beckner and Farrar, 198.5), and the activation of c-myc was antagonised by 8-bromo-CAMP (Farrar el al., 1987). It is known that the c-fos gene upstream regulatory region includes a CAMP-dependent element (Sassone-Corsi et al., I988), and it is evident that there are multiple targets for cAMP among the genes activated during the early stages of the proliferative response. In CT6 cells, the reduction in adenylate cyclase activity caused by IL-2 was accompanied by translocation of protein kinase C to the plasma membrane (Beckner and Farrar, 198.5). The redistribution of protein kinase C has been shown to correlate with a reduction in the rate of PtdIns metabolism in a number of types of cells, including a T cell hybridoma stimulated by anti-Thy-I or antiTCR (Klausner et ul., 1987). In the latter work, antibody to either the TCR Echain or Thy-l activated serine and tyrosine kinases. Both antibodies caused PtdInsP, breakdown, which was inhibited by CAMP, a result consistent with the inhibition by cAMP of the [Ca2+Iiresponse in S49 T lymphoma cells (Taylor et a/., 1988). The decrease in PtdlnsP, hydrolysis correlated directly with a decline in the extent of phosphorylation of 0 3 - y . Prolonged depletion of protein kinase C by treatment with PMA caused the loss of phosphorylation of CD3-y by antigen, anti+, or anti-Thy-I antibody. The effects on CD3-< were complex; all the ligands activated tyrosine kinase but only antigen-stimulated tyrosine phosphorylation was blocked by CAMP. O’Shea et al. (1987) suggested that the most likely mechanism for the inhibition of PtdlnsP, breakdown by cAMP was that the receptor became uncoupled from the G protein activating phospholipase C , that is, the phosphorylated G protein is unable to couple. Therefore, for cells activated via the TCR, the protein that couples the receptor to a tyrosine kinase is a target for a CAMP-dependent kinase. In general, cAMP appears to exert similar inhibitory effects in B cells, blocking c-myc transcription and protein synthesis as well as blocking DNA synthesis. In the human lymphoblastoid cell line LA350, for example, incorporation of [32P]orthophosphateinto phosphatidic acid and PtdIns and the accumulation of inositol phosphates occurred within 1.5 min of the addition of anti-p antibody in common with the responses of normal B cells (Shearer et al., 1988). IgM secretion was subsequently increased in these cells, but all these responses were blocked by the presence of either dibutyryl cAMP or theophylline. There are conflicting data obtained, for example, from spleen cells from athymic nude mice (Wiener et af., 1987), in which anti-Ig caused a 2-fold transient increase in cAMP that was maximal in 2 min. This response was inhibited by 60% in the presence of PMA, but when forskolin was used to activate adenylate cyclase

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directly, the further addition of PMA doubled the increase in CAMP, and anti-lg also enhanced the effect of forskolin.

J. Negative Signals in T Cell Mitogenesis In further studies on the effect of cross-linking receptors, Ledbetter ei crl. (1988b) showed that the leukocyte common antigen CD45 can act as a positive or a negative regulator of signals generated by different receptors. CD4S is of particular interest because it is expressed on both T cells and B cells. When CD45 was cross-linked to CD3, CD2, or CD28 on T cells or to CD19 or Bgp95b on B cells, the increase in [ C a 2 + l inormally following the binding of specific antibody to these receptors was completely abolished. Conversely, however, anti-CD4 antibody caused a large, transient [Ca2+Ii response when CD4 was cross-linked to CD45 that was not obtained when unassociated CD4 was activated. Proliferation stimulated by antibodies to CD3, CD19, or Bgp95 was also substantially inhibited when CD45 was cross-linked to these receptors. It has now been shown that CD45 is a tyrosine phosphatase (Tonks et al., 1988). This was shown by purification and biochemical analysis of the protein, together with the determination of sequence homology with the major low molecular weight tyrosine phosphatase from human placenta. These discoveries emphasize the probable importance of tyrosine phosphorylation in T cell activation and suggest that the signals delivered by CD4S may be due to alteration of the phosphorylation state of tyrosyl residues in membrane receptors when they are closely juxtaposed to CD45. There is evidence from studies on cytotoxic T cells that activation of the CD3 complex can generate inhibitory effects (Leo et a/., 1987). This report indicated that mAb to CD3-e inhibited antigen-specific cell lysis by cloned, rnurine cytotoxic cells but stimulated non-specific lysis and proliferation. In this example, however, rather than representing the generation of a negative signal, the inhibitory effect may simply indicate steric interference by the mAb with the normal cell-cell interaction. In the T cell clone 4 (Tomonari, 19851, a variety of ligands have been identified that either activate or block mitogenic stimulation. IL2 and antibodies to Ti and Thy-1 stimulated DNA synthesis, but activation by anti-Thy-l or anti-Ti antibodies was blocked by each other or by IL-2 or by antibodies to the L3T4 and LFA-1 antigens. Stimulation by IL-2 was blocked by anti-Thy-1 or anti-Ti antibodies, but this block was reversed by antibodies to L3T4 or to LFA-1. Consistent with this complex pattern is the finding that concentrations of anti-Thy- I antibody high enough to cause the release of detectable amounts of IL-2 from clone 4 cells caused auto-inhibition of the mitogenic response. Some, but not all, of these interactions have been reported for other T cell clones, hybridomas, or primary cultures. In T cell hybridoma studies, antiLL3T4 antibody has been

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reported to have no effect on the accumulation of inositol phosphates and the increase in [Ca2-c 1, caused by anti-Thy-1 antibody (Rosoff et al., 1987). Our own observations are, however, contradictory in that we find that occupation of the L3T4 receptor completely inhibits the primary signal generated by activation of the Thy-1 receptor (Howe, 1989). It may be noted that agents that blocked the Ptdlns(4,5)P2 response to anti-Thy- 1 antibody (e.g., anti-L3T4 antibody) substantially inhibited the accumulation of both czfos and c-myc mRNA; other agents that blocked the mitogenic response, but not PtdInsP, hydrolysis (e.g., anti-LFA-l antibody or IL2), did not affect activation of these genes. In view of the mounting evidence that the activation of these protooncogenes is necessary but not sufficient for the stimulation of cell proliferation (see Section Ill,G), it will be of interest to extend the analysis of their expression to all of the agents that inhibit mitogenic stimulation.

IV. T CELL RESPONSES IN CELLULAR IMMUNITY It is now established that there are a number of distinct lymphocyte subtypes, normally present in mammals, that possess cytotoxic capability. For example, some natural killer (NK) cells and cytotoxic T cells recognise and lyse their target cells directly, whereas in antibody-dependent, cell-mediated cytotoxicity, IgG usually binds to the target cells and the effector cell then binds to the Fc portion of IgG.

A. Natural Killer Cells Natural killer cells are large granular lymphocytes that are spontaneously and nonspecifically cytolytic for some tumor cell lines, virus-infected cells, and some normal cells. It is established that the action of NK cells does not require the T cell antigen reccptor heterodimer (Ti), and there is evidence that the Ly5 differentiation antigen functions as the NK receptor. Purified NK cells will grow in vitro in the presence of interleukin 2, although clones vary in their cytotoxic specificity. The activation of some receptors on N K cells by mAbs can directly stimulate PtdInsP, hydrolysis in a manner analogous to that occurring in other T cells. For example, CD16 ligands, which stimulate the synthesis of interferon y or tumor necrosis factor, elevate [Ca2t]i and release InsP,s and InsP, (Cassatella et ul., 1989). It may be noted that the production of cytokines in response to CD 16 was [Ca2 ],,-dependent, whereas the IL-2-stimulated synthesis was independent of (Ca2 J., The mechanism of cytotoxicity of NK cells has largely been studied using cloned cells in v i m , an example of such a clone being the rat leukemic cell line +

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(RNK-16) that lyses the NK targct cell line YAC-1 but does not interact with tumor cells that are resistant to NK cell lysis in vivo (Reynolds et a / . , 1984). RNK-16 cells do not express a functional flsubunit for the ’Ti heterodimer and thus resemble NK cells. Interaction of RNK-16 cells with the YAC-1 target cells causes the accumulation of InsP, InsP,, and InsP, in the RNK-16 cells (Seaman et a / ., 1987). The accumulation of InsP, was maximal after 2 min and declined thereafter, returning to the levels in cells incubated without targct tumor cclls after 15 min. The isomers of InsP, were not separated in this study. InsP, accumulation correlated with lysis [i.e., not generated with NK-resistant tumor cell lines EL-4 and K562 (erythroleukernia)]. The monoclonal antibody OX34 blocked the increase in InsP, and target cell lysis, although OX-34 did not itself stirnulate PtdInsP, hydrolysis unless it was cross-linked by F(ab’), fragments. Although cross-linked OX34 caused a transient increase in [Ca2+liin RNK-16 cells, it was not shown in this study that such an increase occurred during lysis of targct cells. Indcpendent studies of cytotoxic T cells, however, have shown that many such cells undergo a transient increase in [Ca2+li when they contact their target, and that in the latter there is a slower but much greater increase in [Ca2 1, that is sustained (Poenie et ul., 1987). These data were obtained by continuous imaging of cells loaded with the fluoresccnt indicator fura-2 (Grynkiewicz e/ cil., 1985) and, although the initial event leading to the [Ca2 Ii transient in cytotoxic T cells is the perturbation of the T cell receptor when it interacts with the target cell, it is evident from the imaging studies that the maximum elevation of [Ca’ 1, occurs in the intracellular region that is furthest from the point of contact with the target. This indicates the existence of a mechanism by which calcium channels in the plasma membrane of cytolytic cells may be opened, and it seems plausible that a similar mechanism may operate in N K cells, albeit activated by a different membrane receptor. More recently, NK cell-enriched lymphocytes from human peripheral blood have been shown to accumulate InsP,, InsP,, and lnsP when they interact with susceptible target cells (K562 and Jurkat) but not with resistant targets (LarsEBV transformed in virro; Chow et ul., 1988; Steele and Brahmi, 1988). Thc interaction was dependent on extracellular Ca2+ , and the extent of cell lysis was enhanced by phorbol esters or the synthetic diacylglycerol derivative I-oleoyl-2acetyl-sn-glycerol (OAG) and strongly inhibited by sphingosine. As the latter has been shown to inhibit protein kinase C (Hannun et al., 1986), these results werc interpreted as indicating a key role for this enzyme in cytotoxicity, consistent with earlier reports (Russell, 1984; Orosz et al., 1983; Orosz and Roopenian, 1985). The observation that quercetin or dibutyryl CAMP and theophylline blocked bath lytic activity and the stimulation of Ptdlns(4,S)P2 hydrolysis in NK cells (Steele and Brahmi, 1988) suggests that the activity of adenylate cyclase may regulate PtdIns(4,S)P2 metabolism. This is consistent with previous observations in B cells (Coggeshall and Carnbier, 1984) and T cells in which the +

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agonist-stimulated incorporation of 32P into Ptdlns was 50% inhibited by PGE, or dibutyryl CAMP (Kaibuchi et a / . , 1982).

B. T Cell-Mediated Cytotoxicity It seems probable that the exocytosis of proteins called perforins, which insert into the membranes of target cells and depolarize the membrane potential, represents the mechanism of action of cytotoxic T cells. The perforins are functionally related to C9, the terminal, lytic component of complement (Young et a/., 1986). In view of the comprehensive evidence correlating the hydrolysis of PtdIns(4,5)P2 and an increase in [Ca2+J i with agonist-induced secretion in many types of cells (Beaven et al., 1987), it is perhaps unsurprising that recent studies of cloned cytotoxic T cells have shown that the interaction with specific target cells stimulates both these responses in the T cells (Treves rt a / ., I987a). When the stimulus was provided by appropriately sensitized antigen-presenting cells, [Ca2 li in the target T cells increased 5-fold, although the response was much slower than when the surface receptors are directly cross-linked, taking 10-20 min to develop (Shapiro etal., 1985). The source of Ca2 was intracellular, although it may noted that, using fura-2, the maximum increase detected was only approximately threefold (to 300 nM) compared with maxima of > 1 KM commonly observed in mast cells. A further parallel with secretory cells was found in that PMA, which can stimulate secretion in the absence of a detectable increase in [Ca2+ J i (Rink er al., 1983), rendered this T cell clone cytotoxic for unrelated target cells (Russell, I987a). A23 187 also stimulated 1986; Schrezenmeier et a[., 1985; Treves et d., nonspecific lysis (Treves et al., 1987a). Although the evidence relating to cytotoxic T cells is still relatively sparse, it appears that, in general, their activation by interaction with target cells causes a pattern of stimulated PtdlnsP, hydrolysis and [Ca2 li elevation that is presently indistinguishable from that occurring during the activation of other types of lymphocytes. It appears also that exocytosis from these cells is regulated by the synergistic interaction of protein kinase C and [Ca2+ ],-dependent reactions, as it is in the acute secretory rcsponses of mast cells and platelets. In marked contrast to this picture, however, is the recent report by O’Rourke and Mescher (1988) that, although murine cytotoxic T cells were equally effective at lysing target cells in the presence or absence of extracellular Ca2 , under Ca2 -free conditions, there was no stimulation of PtdInsP, hydrolysis. Furthermore, the mitogen ConA was as effective as antigen in stimulating PtdIns metabolism in the cytotoxic T cell clones in normal medium (0.42 mM C a 2 + ) , but the ConA responses were also completely inhibited by the presence of 4 rnM EGTA, although the free Ca2+ concentration under these conditions was not reported. The Ca2+ dependence of PtdlnsP, breakdown described by O’Rourke and Mescher contradicts the results +

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of Treves et a / . (1987a) discussed previously and, furthermore, challenges the generally held view that agonist-stimulated PtdInsP, hydrolysis is [Ca2 1 , independent. Similar experiments on normal murine thymocytcs have shown that lowering [Ca2+ I,, to 100 nM (approximately ICa2+Ii in unstimulated cells) caused a large reduction in the amount of InsP, to approximately the same level as Ins( 1,4,S)P,, and the amount of Ins( I ,4,5)P, was sustained at an elevated level for at least 5 min, in contrast to the transient appearance of the isomer in normal niedium (Zilberman et al., 1987). The aggregate amount of InsP, and InsP, isomers was decreased by approximately 30% at S min in low Ca2 medium, and it was concluded that the external Ca2 concentration might exert a significant effect on receptor coupling to Ins( 1,4,S)P, fomiation. Nevertheless, it was pointed out that if the total increase in inositol phosphates, including InsP, and InsP, is ultimately derived from the Ins( 1,4,S), released, then the effect of low external Ca2 in Ins( 1,4,5)P, release must be less than 1 S%, since the amount of lnsP accumulated was substantially greater than that of any other inositol phosphate and was little affected by the removal of extracellular Ca2+. It is impossible to rationalize the clone data of O’Kourke and Mescher with these findings in primary T cells or with the data of Treves er a / . (1987a), although it is possible that thc clonal phenotypes possess a Ptdlns coupling system that is abnormally sensitive to extracellular Ca2 . It seems evident that these cells do generate an alternative signal(s) from the activated T cell receptor the nature of which is as yet unknown. In view of the emerging evidence from other work that such signal(s) exist, these cell clones may prove to be a valuable system fur further study. +

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V. B CELLS The principal function of B cells is to synthesize and secrete specific antibody in response to antigen, thereby mounting the humoral component of the immune response. The control of this activation process is complex and involves macrophages and complement. as well as hclper T (Th) cells and antigen binding to Ig receptors on the surface of B cells. The essential feature is that an individual clone of B cells is stimulated to proliferate, differentiating into plasma cells, which are short-lived but secrete massive amounts of antibody. The study of the biochemical signaling pathways in €3 cells has principally focused on those activated by specific receptors for antigen (slg) or for the Fc region of IgG (FcR) and by the T cell-derived lymphokines, B cell stimulatory factor [BSFl or interleukin 4 (IL-4)], and B cell growth factor I1 (BCGF 11 or 1L-5). In addition to these, direct (cognate) Th cell interaction with antigen and class I1 MHC determinants on the B cell surface occurs. Most of the B cell growth factors, for example I L 4 and the T cell mitogen ConA, are not capable of fully activating B cells but drive the cells into a pre-activated state in which they express MHC class I1

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antigens (la) on their surface and can respond to anti-lg addition by rapidly synthesising DNA. Antigen-specific activation by Th cells causes a significant proportion of B cells to express the B cell specific activation antigen BLAST-2 (CD23) approximately 16 hr later. The first report that the activation of receptors on B cells could modulate PtdIns metabolism showed that anti-IgM antibody caused increased 32P incorporation into PtdIns (Maino et a / . , 1975). Direct evidence that PtdInsP, hydrolysis was occurring with the release of [3HJlnsP3 and I3H]1,2-DAG was provided by Bijsterbosch et a / . (1985); Mizuguchi et a/. (1986a) found that anti-IgD or antiIgG antibodies also caused PtdIns(4,5)P2 hydrolysis. The use of fluorescent Ca2 indicators showed that the kinetics of early changes in [Ca2 Ii coincided approximately with the accumulation of inositol phosphates, as found in other cell types (Pozzan eta/., 1982; LaBaer et al., 1986). Confirmation that Ins( 1,4,5)P, releases intracellular Ca2 in B lymphocytes, as it does in other cells, was obtained when Ins( 1,4,5)P3 was shown to be the first product to accumulate after stimulation with antibody to surface immunoglobulin (anti-Ig) and that, when added to permeabilized cells, it released Ca2+ from the endoplasmic reticulum (Ransom et al., 1986). Ins( 1,4,5)P, appears to be rapidly converted to Ins( I ,3,4,5)P,, as in other cells (Fahey and DeFranco, 1987). These and many other observations have led to a picture of receptor coupling to PtdlnsP, hydrolysis broadly similar to that in T cells; a more detailed discussion of the earlier developments in B lymphocyte activation may be found in reviews by DeFranco (1987) and Klaus et ul. (1987). In common with the responses to some T cell receptors, cross-linking surface immunoglobulin (slg) on B lymphocytes induces both release of Ca2+ from intracellular stores and Ca2+ influx across the plasma membrane. Using the fluorescent indicator indo-1 , Bijsterbosch et a/. (1986) showed that anti-Ig transiently raised [Ca2+Iifrom 100 nM to 1 p.M, due to internal release, after which [Ca2+Ii declined to a new, stable value of 350 nM. The sustained level was [Ca’+],,-dependent. Using flow cytometry to study single cells, Chused et al. (1987) showed that anti-b antibody generated a [Ca2+Ii response in a larger proportion of B cells than anti-p. antibody and that the maximum transient concentration was also greater (2.6 p M compared to 1.7 FM). Work from the same laboratory (Wilson rt ul., 1987) also showed that when the Ig receptor was cross-linked to the Fcy receptor, the [Ca2 li transient declined more rapidly, in parallel with the concentration of InsP, (Wilson et a / . , 1987). These authors showed that, although agents that blocked Ca2 channels in muscle or nerve also blocked B cell activation, those channels were not responsible for the sustained increase in [Ca2 1;. The results of Bijsterbosch et a/. (1986) suggest that inositol phosphates, as well as causing release of intracellular C a 2 + , may cause C a 2 + influx into B cells, as may occur in T cells. It is not clear, however, why the cross-linking experiment of Wilson et al. generated a different [Ca2+ I i response. The sustained, but not the transient, component of the [Ca2+Iiresponse in B +

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cells can also be generated by ConA, and the response was reversed by the addition of a-methyl mannoside (Bijsterbosch et al., 1986). In this article the authors were unable to detect any accumulation of InsP, following the addition of ConA, although in a subsequent paper they described the accumulation as “minimal” (Bijsterbosch and Klaus, 1986). This suggested that different mechanisms operate in B cells and T cells in that the sustained increase in [Ca2-+li caused by ConA is similar but almost independent of PtdlnsP, hydrolysis in the former cells. In fact, the data were not really definitive enough to conclude the existence of different mechanisms responding to ConA in the two cell types; InsP, was assayed 30 sec and 1 hr after ConA addition and, although there was no clear increase within the first period, there was a 3-fold stimulation after 1 hr. As the rise in [Ca2+li did not reach a maximum until 5-10 min after ConA addition (Klaus et ( J I . , 1987), it seems probable that the mechanism is PtdlnsP,dependent. In B cells, therefore, it appears that reagents that cross-link slg cause a PtdInsP,-dependent increase in (Ca2 I,. Thus F(ab’), fragments of rabbit antimouse Ig rapidly stimulated both these responses (Bijsterbosch et al., 1985) while monomeric Fab anti-Ig did not. A more detailed study using monoclonal anti-p and anti-6 antibodies suggested that there was a correlation between their mitogenicity and the amount of inositol phosphates released (Klaus et nl., 1987). Similar studies using a human lymphoblastoid B cell line also indicated that anti-F caused the rapid accumulation of InsP, InsP,, and InsP,, as well as inducing secretion of IgM (Shearer et u l . , 1988). In the latter, dibutyryl CAMPor theophylline blocked the stimulation of PtdIns metabolism, an effect similar to that observed in T cells (see Section 111,J). In spite of these similarities, it is evident that B cells can be activated by pathways independent of inositol phosphates. Thus, although anti-lg is established as causing an increase in [Ca2+Ii and the accumulation of [3H]InsP, and [‘HI I,2-diacylglycerol (Bijsterbosch et a l . , 1985), neither lipopolysaccharide (LPS) nor PMA caused any perturbation of PtdIns metabolism. LPS is a strong mitogen for B cells (Greaves and Janossy, 1972), and Bijsterbosch et al. found that, in common with anti-lg, LPS or PMA each induced RNA synthesis after 6 hr. Stimulation of PtdInsP, breakdown and increase in [Ca2+li in response to’, anti-lg, but not LPS, was also found by Partain et al. (1986). In apparent conflict with the findings were the responses of a pre-B cell line, 70213, which does not express surCice IgM (Rosoff and Cantley, 1985). In these cells either LPS or PMA induced differentiation to IgM positive cells by enhancing Na /H cxchange, but LPS also increased [Ca’ Iiand the rate of hydrolysis of PtdlnsP?, responses that were antagonised by PMA. It is not clear why these cells differ in their response from mature B cells. The question of which ligands activate RNA synthesis in B cells is, however, controversial. In human B cells Walker et al. ( 1986) found that, although anti-Ig stimulated InsP, formation, neither anti-Ig, +

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A23187, nor PMA caused RNA synthesis. Nevertheless, each of these agents induced CD23, p45, and BK. 19.9 surface antigens to appear between 6 and 24 hr. Ca2 ionophores (A23 187 or ionomycin) do not cause RNA synthesis in B cells, but DNA synthesis was accelerated following prior treatment with ionophores when the cells were subsequently challenged with Ig (Klaus et al., 1985). Murine B cells stimulated with anti-Ig rapidly accumulated InsPs with a concurrent elevation of [CaZi Ii,depolarization of the plasma membrane, and loss of protein kinase C from the cytosol (Justement et al., 1986). IL-4, however, stimulated none of these responses, even though either 1L-4 or anti-lg caused enhanced expression of Ia. IL-4 appears to prime B cells so that on subsequent exposure to anti-lg or LPS they rapidly enter S phase. IL-4 also increased the phosphorylation of a 44 kDA membrane protein that was unaffected by anti-Ig. In a less extensive study, Mizuguchi et al. (1986b) also showed that IL-4 did not modulate PtdInsP, metabolism or [Ca2 +I,. They also observed that although the combination of A23187 and PMA caused an increase in [Ca2+Ii and protein phosphorylation, IL-4 was still required for proliferation. This result provides further evidence that the plasma membrane receptors deliver an additional mitogenic signal to those provided by the hydrolysis of PtdInsP,. Further evidence for the existence of different activation mechanisms for B cells is provided by the responses to dextran sulfate and LPS (Morelec et ul., 1988). Dextran sulfate induced the synthesis of polyphosphoinositides [PtdlnsP and PtdIns(4,5)P2] but did not promote the accumulation of InsP,; LPS had no effect on PtdIns metabolism and, in fact, inhibited the response to dextran sulfate. Thus the mechanisms of action of each of these agents are different from each other and also from that of anti-Ig. It may be noted that the synthesis of IL-1 by the macrophage line P388D, and the relief by LPS of the inhibition of growth of the B lymphoma cell line WEHI-231 by anti-IgM were blocked by pertussis toxin (Jakway and DeFranco, 1986). IgM stimulated the accumulation of inositol phosphates, including Ins( I ,3,4,5)P,, in WEHI-231 cells, and this response was not susceptible to the actions of either pertussis or cholera toxin (Gold et a [ . , 1987), suggesting that it is not G protein coupled. Nevertheless, when these cells were permeabilized with saponin, there was a rapid accumulation of InsP, and InsP, following the addition of GTPyS. Although LPS, which is mitogenic for B cells, can activate protein kinase C, PMA alone is not mitogenic; thus, LPS must generate a signal(s) in addition to the activation of protein kinase C, and it seems probable that this is transduced via a G protein(s), although its nature is as yet unknown. The most illuminating experiments concerning the coupling of receptors to PtdInsP, hydrolysis in B cells have been the work of Harnett and Klaus (1988) using mouse cells permeabilized with streptolysin 0. They were able to show that GTPyS increased the accumulation of InsP, and InsP, and that this response was further enhanced by the addition of either polyclonal anti-Ig antibody or +

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monoclonal anti-p. or anti-S antibody. These effects were not antagonized by pertussis toxin, in contrast to the corresponding result of Ledbetter et ul. (1987a) for several T cell agonists, although there was some inhibition (-30%) by cholera toxin. Cholera toxin, however, strongly inhibited the proliferative response to F(ab’)2 anti-lg without reducing that caused by LPS. The concentration of cholera toxin required to produce these effects did not apparently elevate CAMP significantly in B cells. It is, of course, possible that the eKect of cholera toxin in B cells is mediated through G proteins other than the classical G , that regulates adenylate cyclase. This explanation, together with the results of the experiments with GTP analogues, suggests that in B lymphocytes, as in other cells, specific G proteins couple antigen receptors to the polyphosphoinositidespecific phospholipase C. Flow cytometric analysis of B cells loaded with the f-lourescent indicator indo- 1 have confirmed that a significant proportion of the [ Ca2+ Ii response to the cross-linking of surface Ig was due to release from intracellular stores (Ransom et a / ., 1988). These studies also detected an increase in the permeability of the plasma membrane and the resulting Ca2+ influx contributed the remainder of the change in [Ca2+ I,. The modulation of membrane permeability to CaZ+ was attributed to a breakdown product of PtdInsP, or a metabolite thereof on the basis of its inhibition by neomycin. Studies of the antigen-specific interaction between cloned allo-specific human Th cells and class II MHC determinants on allogeneic B cells showed that high levels of expression of the activation antigen BLAST-2 occurred with no detectable change in [Ca2+Ii or inositol lipid metabolism. (Chartash et al., 1988). When the cells were stimulated viu their sIg receptors by anti-IgM antibody, F(ab’),, or Staphylococcus aztreus protein A. however, there was increased incorporation of [32P]orthophosphateinto phosphatidic acid and PtdIns, accumulation of inositol phosphates, and an increase in [Ca2+ I i , but BLAST-2 expression was not induced. The kinetics of the induction of BLAST-2 expression are not dissimilar to those of the I L 2 receptor in T cells, but none of the known second messengers appears to be involved in the initiation of the response. Finally, having regard to the earlier discussion of possible interactions between protooncogene products and the enzymes of PtdIns metabolism, it may be noted that, although studies of protooncogene expression in activated B cells are less comprehensive than for T cells, it is established that anti-IgM antibodies or LPS stimulates transcription of c-myc (Smeland et a / . , 1985; Kelly et al., 1983). Hapten-conjugated soluble antigen also activated c-myc expression (Snow et al., 198h), although this was insufficient to cause proliferation of the cells even in the presence of IL-5, consistent with the evidence from T lymphocytes and other cells that expression of c-myc might be necessary for proliferation but that additional intracellular signals are also required. Evidence that overexpression of c-myc could be associated with transformation was provided by Adams et ul. (1985) who transfected murine germ cells with the c-myc gene juxtaposed to an

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lg heavy chain enhancer; the resulting transgenic mice developed tumors of B lineage cells “which appeared to be monoclonal in origin.”

VI.

SUMMARY

The activation by specific ligands of essentially all classes of lymphocytes can cause the hydrolysis of PtdlnsP, with the transient accumulation of Ins( 1,4,5)P, and the elevation of [Ca2+Ii in a manner closely analogous to that occurring during the activation of many other types of cells. In both T cells and B cells there is a considerable amount of data indicating that G proteins couple activated receptors to a PtdInsP,-specific phospholipase C, although at present the evidence is indirect. At least one other PtdIns metabolite, Ins( l ,3,4,5)P, may play a role in sustaining [Ca2 Ii at the elevated levels commonly observed after stimulation. There is suggestive, but not definitive, evidence that these very rapid responses may be necessary for the stimulation of differentiation and proliferation. It is also clear that lymphocytes possess receptors that, under certain conditions, are capable of delivering negative signals to the cell by inhibiting PtdInsP, hydrolysis and the [Ca2+ I , response. It appears that the other product of PtdlnsP, hydrolysis, diacylglycerol, may stimulate the activity of protein kinase C in intact lymphocytes of some species and subclasses and cause rapid intracellular redistribution of protein kinase C activity (e.g., anti-Ig on 13 cells), but this pathway is not generally activated (e.g., in murine T cells). There is increasing evidence that the stimulation of some lymphocyte receptors activates tyrosine protein kinases in the plasma membrane. This may be a crucial step in lymphocyte activation, but there is no evidence at present to indicate whether it is regulated by products of PtdIns metabolism. Although an increase in the concentration of CAMP inhibits some early steps in lymphocyte activation, its detailed mechanism remains elusive. It is clear, however, that CAMPcan inhibit the ligand-induced hydrolysis of PtdInsP,, and it is possible that the normal coupling between activated receptors and the PtdInsP,-specific phospholipase C , mediated by a G protein, is modulated by CAMP-dependent protein kinase phosphorylation of the coupling protein. There is as yet no evidence in lymphocytes indicating an interaction between protooncogene or oncogene products and enzymes regulating Ptdlns metabolism, as occurs in fibroblasts. Nevertheless, the fact that the activation of B or T cells promotes a pattern of protooncogene expression closely resembling that observed in other cells, including fibroblasts, suggests that such interactions may also take place in lymphocytes.

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REFERENCES Abraham, R . T., Ho, S. N . , Bama. T. J . , and McKean, D. J. (1987). Transmembrane signaling during interleukin 1 -dependent T cell activation: interactions of signal I - and signal 2-type mediators with the phusphoinusitide-dependent signal transduction mechanism. J . B i d . Chem. 262, 2119-2128.

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Adams, J. M., Harris, A. W., Pinkert, C. A , , Corcoran, L. M.. Alexander, W. S., Cory, S . , Palmiter, R . D., and Brinster, R. L. (1985). The c-mvc oncogene driven by immunoglobulin enhancers induces lymphoid malignancy in transgenic mice. Nature (London) 318, 533-538. Alcovcr, A , , Wciss, M. J., Daley, J. F., and Reinherz. E. I.. (1986). The T11 glyeoprotein is functionally linked to a calciuni channel i n precursor and mature T-lineage cells. Proc. Nut/. Ai.cld. Si.;. U . S . A . 83, 2614-2618. Alcover, A , , Alherini, C.. Acuta. O., Clayton. L. K.. Transy, C . , Spagnoli, G . C., Moingeon, P., Lopez. P.. and Reinherz, E. L. (1988). Interdependence of CD3-Ti and CD2 activation pathways in human T lymphocytes. E M R O J. 7, 1973-1977. Allan, D., and Michell. K. H. (1977). A comparison of the effects of phytohaeiuagglutinin and 01 calcium ionophore A23 I87 o n the metabolism of glycerolipids in small lymphocytes. Biochem. .I. 164, 389-397. Allison, J. P., and Lanier, L. L. (19851. Identification of antigen receptor-associated structures on murine T cells. Nancre (London) 314, 107-109. Ando. I . . Hariri. G . . Wallace, D.. and Beverley, P. (1985). Tumor promoter phorbol esters induce unresponsiveness t o antigen and expression of interleukin 2 receptor on T cells. Eur. J. Irnrnuno/. 15, 196-199. Ashkcnazi, A , , Peralta, E. G . . Winslow, J. W., Rarnachandran. J . , and Capon, D. J. (1989). Functionally dictincr G proteins sclcctivcly couple different receptors to PI hydrolysis in the sanic cell. Cell 56, 487-493. Auger, K . R., Serunian. I.. A . . Soltoff. S . P . . Libby, P., and Cantley, L. C. (1989). PDGF-dependent tyrosinc phosphorylation stimulates production of novel polyphosphoinoaitides in intact cells. Cell 57, 167-175. Raniyash. M.. (iarcia-Moraka, P., Bonifacino, J. S . , Samelson, L. E., and Klausner, R. D. (1988a). Disulfide linkage of the 5 and chains of the T cell receptor. J . Biol. Chem. 263, 9874-9878. Baniyash. M., Garcia-Morales, P., Luong, E . , Samelson, L. E., and Klausner, R. D. (1988b). T h e T cell antigen receptor 5 chain is tyrosine phosphorylated upon activation. .I. B i d . Chem. 263, 18225-18230. Ratty. I. R . , Nahorski, S . R., and Irvine, R. I;. (1985). Rapid formation of inositol 1,3,4,5tetrakisphosphate following muncarinic receptor sthubation of rat cerebral cortical slices. Biochetn. J. 232, 211-215. Beaven, M. A., Cuthrie. D. F.. Moore, J. P.. Smith, G . A , , Hesketh, T. R . , and Metcalfc, J. C. (1987). Synergistic bigriala in the mechanism of antigen-induced cxocytosis in 2H3 cells: evidence for an unidentified signal required for histamine release. J. Cell B i d . 105, 1129-1 136. Beckner, S . K . , and Farrar, W. L. (1985). Interleukin 2 modulation of adcnylatc cyclase. J. Oiol. Chem. 261, 3043-3047. Benichou. G.. Kanellopoulos, J. M., Wallon, C . , Boue, F.. and Delfraissy, I.-F. (1987). Interferon-y restores T lymphocyte proliferation of nonrcsponders to IgG I antiLCD3 via the induction of Fc,, receptors on monocytes. Eur. J. Immunol. 17, 1175-1 181, Benn-Sasson. S . Z., Paul, W. E., Shevach, E. M., and Green, I. (1975). 111 v i m selection and extended culture of antigen-specific T lymphocytes. J. Immunol. 115, 1723-1730. Bijsterhosch, M. K . , and Klaus, G. G . (1986). Concanavalin A induces Ca2+ mobilization, but only minimal inositol phospholipid breakdown in mouse B cells. J . Immunol. 137, 1294- 1299. Bijsterbosch, M . K . , Meade, C. J . . Turner, G. A.. and Klaus, G. G . (1985). B lymphocyte receptors and polyphosplioinositide degradation. Cell 41, 999- 1006. Bijsterboach, M. K . , Rigley, K . P.. and Klaus, G . G . (1986). Cross-linking of surface immunoglobulin on B lymphocytes induces both intracellular Ca2+ rclcasc and C i 2 influx: analysis with indo-l , Biochm. Biophys. Res. Commun. 137, 500-506; erratum, Rrochem. Riophvs. Res. Cornmum 138, 503 ( 1986). Bismuth, G.. Faure. F.. Thcodorou, I . , Debre, P., and Hercend, T. (1988). Triggering of the

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Ych, E. 'K H . , Reiser. H . , Daley, J . , and Rock, K. L. (1987). Stimulation o f T cells vio the TAP molecule. a membcr in a family of activating protcins encoded in the Ly-6 locus. J . fmrnrrnol. 138, 91-97. Young, J. D.-E.. Cohn, Z . A , , and Podack, E. R . (19x6). 'The ninth component of complenient and the purc-forming protein ( pcrforin I ) from cytotoxic T cells: structural, immunological and functional similarities. Science 233, 184- 190. Zilherman, Y.. Howe, L. R . . Moore, J. P., Hesketh. T. R . , and Metcalle, J. C . (1987). Calcium regulates inositol 1 ,?,4,5-tetrakisphosphatcproduction in lyscd thyinocytcs and in intact cells stimulated with concanavalin A . EMBO J . 6, 957-62. Zipfcl. P. F., Irving, S . G . , Kelly, K . , and Sicbcnlist, U. (1989). Complexity of thc primary response to mitogenic activation of human T cclls. Mol. C e l l . Biol. 9, 1041-1048.

CURRENT TOPICS IN MEMBRANES AND TRANSPORT. VOLUME 35

Chapter 12 Phosphoinositide Metabolism during Phagocytic Cell Activation ALEXIS E. TRAYNOR-KAPLAN’ Department of Immunology Research Institute of Scripps Clinic La Jolla, California 92037

Introduction Phosphatidic Acid Metabolism A. Phosphatidic Acid: Metabolite or Mediator’? B. Diglyceride Kinase: Its Role in Signal Transduction C. Contribution of Phospholipase D Activity to Phosphatidic Acid Production D. Lysophosphatide AcyltrdIISferaSe III. Role of Enzymes in Phosphoinositide Metabolism in Neutrophil Activation A. Phosphoinositide Synthesis: Background B , Phosphatidylinositol Kinases: Possible Links to Receptor Activation C. Polyphosphoinositide Phosphatases D. Phospholipase C: Comparison of Its Response to Different Stimuli E. Phospholipase A2: Its Contribution to Phosphoinositide Hydrolysis IV. Conclusions Referenccs I. 11.

1.

INTRODUCTION

For years after Hokin and Hokin (1953) first demonstrated the selective incorporation of 32P into phosphatidylinositol (Ptdlns) and phosphatidic acid (PtdH) in pancreatic slices and its accelerated metabolism during enzyme secretion, papers documenting the “PI effect” in a variety of cell types appeared. Initially, the PI effect was thought to be linked to secretory events and was measured as an increase in the amount of radioactive label incorporated into PtdIns or one of its derivatives, which included phosphatidylinositol 4-phosphate (PtdInsP), phosI Present address: Department of Medicine, UCSD Medical Center, University of California, San Diego, San Diego, California 92103.

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Copyright 0 1990 by Academic Pres.;. Inc All right? of rcpruduchon in any form reserved.

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E.TRAYNOR-KAPLAN

phatidylinositol 4,5-bisphosphate (PtdInsP,), or phosphatidic acid (PtdH). Jones and co-workers ( 1979) confirmed an earlier proposal of Hokin and Hokin (1964) by identifying a phospholipase C (PLC) activity that hydrolyzed phosphoinositides, producing inositol phosphates and diacylglycerol (DAG). Ptdlns, not PtdInsP,, was thought to be the predominant substrate at that time. The history of these developments has been expertly reviewed by Michell (1986). Through a separate line of investigation in Japan, DAG was found to stimulate the calciumactivated protein kinase C (PKC) (Takai et al., 1979), leading to the hypothesis that one of the primary elements in phosphoinositide metabolism important in signal transduction was DAG (Nishizuka, 1983). When phospholipase C-mediated PtdInsP, hydrolysis was linked to receptor activation (Kirk et al., 1981; Mitchell and Kirk, 1981) and the resultant production of inositol 1,4,S-trisphosphate [Ins( I ,4,5)P,J linked to calcium elevation (see review in Berridge and Irvinc, 1984), a large number of studies documenting increases in InsP, and DAG in a variety of cell types were spawned. Functional consequences of these metabolic pathways are considered in this volume in chapters concerning Ca2 regulation (see Chapter 10) and protein phosphorylation (see Chapter 18). By comparison to the attention given since 1983 to PLC activity, relatively little work has been done on the other enzymes in the pathway of PtdIns metabolism. However, evidence for another signal pathway has emerged. In human neutrophils, chemotactic peptide stimulated the increase in a previously unidentified phosphoinositide, PtdInsP,, putatively PtdIns(3,4,5)P3 (Traynor-Kaplan et (I/. , 1988), which is probably the result of the activity of a novel PtdIns kinase. A tyrosine kinase-linked Ptdlns-3-kinase has been identified in immunoprecipitates of middle T/pp60c-,src'from middle T-transformed NIH 3T3 fibroblasts and in antiphosphotyrosine immunoprecipitations from platelet derived growth factor (PDGF)-stimulated BALB/C-3T3 fibroblasts which catalyzes the formation of the previously unidentified Ptdins(3)P (Whitman ef a/., 1988). Although PtdIns kinase activity had been previously linked to the PDGF receptor (Whitman et al., 1985, 1987; Kaplan et u l . , 1986, 1987), a more recent study has demonstrated the link between PDGF receptor stimulation and the novel PtdIns-3-kinase activity in intact cells (Auger ct id., 1989). In neutrophils, a combination of recent developments suggests calcium and diacylglycerol fluctuations may be inadequate to account for many responses (see Section IV). Therefore, in this chapter, we explore the possibility that other enzymes in phosphoinositide metabolism, in addition to PLC, may be linked to receptor activation in phagocytes, particularly neutrophils. Phosphatidic acid (PtdH), an intermediary that serves both as a precursor and a product of phosphoinositide metabolism, will be discussed first. Evidence will be presented suggesting that PtdH is a mediator in ncutrophil signal transduction. Relative contributions of different enzymatic pathways for the production of PtdH will be assessed for +

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their role in signal transduction. Then we will discuss the enzymes directly involved in phosphoinositide synthesis and breakdown.

II. PHOSPHATIDIC ACID METABOLISM A. Phosphatidic Acid: Metabolite or Mediator? PtdH is a central intermediary in phospholipid synthesis, serving as both a precursor and a product in the “PtdIns cycle” (see Figs. 1 and 2). Agents which stimulate phospholipase C hydrolysis of phosphoinositides invariably result in synthesis of PtdH. Therefore, activities of enzymes in this pathway have an impact on Ptdlns metabolism. Pathways for the synthesis of PtdH are depicted in Fig. 1. Three enzymes participate directly in the formation of PtdH: (1) DAG kinase, ( 2 ) phospholipase D (PLD), and (3) lysophosphatidic acid acyltransferase. Depending on the label used, the radioactivity in PtdH will reflect different metabolic pathways. For instance, [‘*P]ATP will label PtdH formed via the diglyceride kinase path, whereas radiolabeled fatty acids will become incorporated into PtdH through monoacylglycerol or lysolipid acyltransferase. Radiolabeled glycerol will also become incorporated into PtdH through these pathways. The radiolabel in PtdH formed through PLD activity will depend on the precursor phospholipid and will be discussed in greater detail in Section I1,C. The formation of PtdH is of interest since recent evidence suggests it could participate in superoxide production. PtdH concentrations of 10- 100 ~J.M stimulated NADPH-dependent superoxide production in a plasma membrane fraction from pig neutrophils (Bellavite et al., 1988). The kinetics of the formation of Glucose

I

DHAP AcylDHAP

GP

\L

.Y

PA Kinase

DAG

FIG. I . Synthesis of phoaphatidic acid (PA). Three different routes for the synthesis of phosphatidic acid are depicted: ( I ) de novo synthesis by way of dihydroxyacetone phosphate (DHAP) or glycerol phosphate (GP) followed by acylation of lysophosphatidic acid (lysoPA) by a lysophosphatide acyltransferase to phosphatidic acid: ,(2)synthesis from diacylglycerol (DAG) via DAG kinase; and ( 3 ) phospholipase D (PLD) hydrolysia of phospholipid head groups.

306

ALEXIS E. TRAYNOR-KAPLAN

PC, PE, etc. LysoPA

o/"

DAG

PA

CDP-DG

lo

PI

FIG.2 . Metabolism of phosphatidic acid. Phosphatidic acid (PA) can be hydrolyzcd to DAG through the action of phoaphatidate phosphohydrolase ( 1 ). It can be converted to phosphatidylinositol (PI) through the cytidine diphosphate-diglyceride (CDP-DAO) inositol phosphatidyltransferase (3) and phosphdtidylinosilol aynthase (4)path or it can by hydrolyzed by phospholipase A? (PLA2) (2) to lysophosphatidic acid (IysoPA).

PtdH make it a plausible candidate for mediating superoxide formation in neutrophils. Although [ '2P]PtdH rises slowly in neutrophils stimulated with chemotactic agents, the mass of PtdH accumulates quickly, reaching a peak at 30 sec (Cockcroft, 1984). The time course is consistent with PtdH being a precursor for superoxide production. Through comparison of neutrophil responses to formyl peptide, phorbol esters, and concanavalin A, Korchak and co-workers (1988b) concluded that although DAG elevation in conjunction with calcium may be necessary, they are not adequate to elicit superoxide generation. Their data suggested PtdH may also contribute to oxidant production.

B. Diglyceride Kinase: Its Role in Signal Transduction DAG kinase, a Mg2 -ATP dependent phosphokinase, performs two roles potentially important for signal transduction. ( 1 ) It phosphorylates diacylglycerol in an ATP dependent fashion, resulting in the formation of PtdH which may itself be a physiological mediator (see Section 11,A). (2) By lowering levels of the DAG it may provide a mechanism whereby the cell regulates activation of protein kinase C. Its activity becomes elevated following stimulation and does not appear to be regulated by intracellular calcium. +

1 . OBSERVATIONS OF DAG KINASE ACTIVITY I N CELLFRACTIONS

Call and Rupert (1973) attempted to identify DAG kinase activity in human neutrophils but failed, probably as a result of inadequate inactivation of endoge-

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nous proteases. However, in later studies, evidence was obtained for DAG kinase activity in the plasma membrane in formyl peptide-stimulated rabbit neutrophils (Bennett et al., 1982). Cockcroft and co-workers (1984) later demonstrated DAG kinase activity in a light plasma membrane fraction which also contained PUS activity. One mechanism for regulating DAG kinase activity could be compartmentalization, thereby limiting substrate availability. Recently, translocation of DAG kinase activity from the cytosol to the membrane was reported in formyl peptide- or phorbol ester-stimulated neutrophils (Ishitoya et al., 1987). The V,,, was elevated in the translocated enzyme although the K,, was not altered. Therefore, translocation of DAG kinase could be a common mechanism by which formyl peptide and phorbol esters stimulated PtdH synthesis in neutrophils. 2. ELEVATION OF DAG KINASE ACTIVITY DURING NEUTROPHIL ACTIVATION Large increases in the incorporation of 32P0, into PtdH have been measured in neutrophils, macrophages, and HL60 cells in response to a variety of activating agents (Cockcroft et al., 1980; Volpi et al., 1983; Dougherty et al., 1984; Cockcroft, 1984). Although PtdH can be formed by the action of PLD (see Section II,C), this cannot account for the increases in 32P incorporation into PtdH. Therefore, the increase in PtdH measured with 32Pis probably secondary to PLC activity which increased substrate availability to DAG kinase. Exogenous DAG is rapidly converted to PtdH when added to HL60 cells (May et al., 1986). This, taken together with the finding of Bellavite and co-workers (1988) that PtdH can stimulate NADPH-dependent superoxide production, suggests an alternate pathway to PKC activation by which DAG could induce superoxide formation. This is consistent with the delay in superoxide production induced by DAG relative to that induced by formyl peptide. Recently an inhibitor of DAG kinase, R59 022, has become commercially available and been utilized in analysis of the role of DAG and PtdH in neutrophil activation. Unfortunately, it appears to also stimulate PLC or PLD, causing elevation of both DAG and PtdH (Mahadevappa, 1988). 3. MECHANISM OF INCREASE I N DAG KINASE ACTIVITYIN STIMULATED NEUTROPHILS It has yet to be determined whether the activity of DAG kinase is stimulated directly through receptor activation or is only limited by substrate availability. When digitonin-permeabilized neutrophils were treated with PLC (Bacillus cereus), a 100-fold increase in radioactivity in PtdH was observed (A. E. TraynorKaplan and L. A. Sklar, unpublished observations), suggesting that in permeabilized cells substrate availability is rate limiting for DAG kinase. Substrate

308

ALEXIS E. TRAYNOR-KAPLAN

availability could be regulated by enzyme translocation. The increase in PtdH in neutrophils stimulated with the chemotactic peptide varies with the individual donor but appears to remain proportional to the number of receptors expressed on the cell surface (A. E. Traynor-Kaplan and L. A. Sklar, unpublished observations). Although many investigators have reported the pertussis toxin sensitivity of the PLC-mediated hydrolysis of PtdInsP, (see Section lIl,D,2), very few have mentioned the relationship between the increase in (32P]PtdH formation and G proteins. However, a dependence of formation of ["PIPtdH on G proteins is expected if the PtdH synthesis is limited only by DAG availability which is regulated by PLC activity. In fact, we observe that pertussis toxin pretreatment inhibits the formation of ["PJPtdH (Traynor-Kaplan et a / ., 1989). Some evidence suggests that the increase in DAG kinase activity in activated neutrophils is not secondary to calcium elevation. Calcium is not necessary for the formyl peptide-stimulated increase in [32P]PtdHin neutrophils where the rise in intracellular calcium is buffered with the nonfluorescent chelator MAPTAM (Korchak et al., 1988a). Interestingly, these investigators observed a potentiated elevation of [32P]PtdH in calcium-buffered cells stimulated with chemoattractants. In summary, DAG kinase activity appears to increase following neutrophil activation. Preliminary evidence suggests this activity is independent of the rise in intracellular calcium (Korchak et al., 1988a). One mechanism for this increase could be a receptor-stimulated translocation of the enzyme from cytosol to plasma membrane (Ishitoya et d., 1987) which may increase substrate availability. In turn, the product of this enzyme activity, PtdH, may play a role in superoxide production. Increases in PtdH parallel cell activation when neutrophils are stimulated by a variety of agents. (Cockcroft, 1984; Volpi et al., 1983; Korchak et a / ., 1988a). Finally, PtdH stimulants NADPH-dependent superoxide production in pig neutrophil membranes (Bellavite ct al., 1988).

C. Contribution of Phospholipase D Activity to Phosphatidic Acid Production PLD activity also results in the formation of PtdH. Recent findings suggest PLD activity in neutrophils may also be important in signal transduction. As mentioned previously (see Section II,B), DAG kinase activity is elevated in activated neutrophils, contributing to a portion of the mass of PtdH generated. However DAG kinase activity cannot account for all the PtdH generated, suggesting PLD may also be contributing. In contrast to DAG activity, PLD-mediated production of PtdH is poorly measured with the 32Plabel. Mass measurements or labeling with radioactive glycerol or fatty acids are more effective means of detecting PLD-

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mediated increases in PtdH. Discrepancies between the mass of PtdH generated and the specific activity of [ 32P]PtdH from stimulated neutrophils led Cockcroft (1984) to postulate PLD activity as a mode for receptor-mediated decreases in PtdIns levels. Later work suggested that this PLD activity was limited to PtdIns and that the hydrolysis of polyphosphoinositides was the result of PLC activity (Cockcroft et al., 1985). In a later summary of this research Cockcroft (1986) abandoned this idea, noting (1) the loss in PtdIns was much less than the gain in PtdH, (2) discrepancies in the fatty acid compositions of the newly synthesized PtdH and PtdIns, and (3) the lack of evidence at that time for a PtdIns-specific PLD. However these arguments may need to be reexamined in light of a PtdInsspecific phospholipase D recently identified in the postnuclear fraction of human neutrophils (Balsinde er al., 1988). The activity of this enzyme increased following treatment of the neutrophils with A23187, suggesting that this enzyme may be stimulated during cell activation, possibly as a consequence of calcium elevation. Although calcium is not necessary for the formyl peptide-stimulated increase in [32P]PtdH in neutrophils when the rise in intracellular calcium is buffered (Korchak et al., I988b), this does not indicate whether PLD-mediated elevation of PtdH is secondary to calcium elevation. This needs to be tested using mass measurements of PtdH in calcium-buffered cells. Cockcroft (1984) compared the mass of formyl peptide-stimulated PtdH produced in cells depleted of calcium by incubation for 90 min with 100 pM EGTA in the absence of added calcium to that produced in cells in which the calcium had been restored. The mass of PtdH production in the calcium-depleted cells was not significantly different from unstimulated cells, suggesting that PLD-mediated formation of PtdH is dependent on calcium elevation. This is in direct contrast to [32P]PtdH formation described earlier, which is apparently independent of calcium elevation. It becomes important to ascertain what mass [32P]PtdH (PtdH formed through diglyceride kinase) contributes to the total PtdH mass in order to resolve these separate observations. Another pathway by which the mass of PtdH could be increasing is through IysoPtdH acyltransferase. The relative contribution to the elevation in PtdH in stimulated cells from this pathway needs to be evaluated before we can assess the importance of PLD activity in elevating the mass of PtdH. Other labeling paradigms similar to those described below have been employed to measure PLD activity. One technique has utilized the unique property of PLD to catalyze transphosphatidylation in the presence of an alcohol. When the alcohol is ethanol, this results in the formation of phosphatidylethanol Stimulation of a PLD-mediated hydrolysis of PtdCholine by chemotactic peptide was obtained in HL60 cells labeled with alkyl-[32P]lysoPtdCho. Alkyl-[32PllysoPtdCho becomes incorporated in cells into alkyl-[32P]PtdCholeaving ATP unlabeled. PLD activity cleaves the choline leaving alkyl-[”P]PtdH. Indeed, when these labeled cells were stimulated with chemotactic peptide, there

310

ALEXIS E. TRAYNOR-KAPLAN

ensued a rapid increase in alk~l-[~*P]PtdH (Pai e t a / . , 1988a,b). When ethanol was present, alkyl-[ 32P]phosphatidylethanol was formed in addition to alk ~ l - [ ~ ~ P ] P t dproviding H, further evidence for PLD activation. In summary, increases in the mass of PtdH and the identification of a receptormediated increase in PLD activity indicate that PLD is contributing to the production of PtdH in activated neutrophils. However, it is unlikely that the substrate for the PLD is exclusively phosphoinositides. Since the receptor-stimulated increase in the mass of PtdH produced is blocked by chelating calcium, PLD activity in neutrophils may be dependent on the elevation of intracellular calcium. This should be determined in the future.

D. Lysophosphatide Acyltransferase Lysolipids can be converted to phospholipids by the action of lysophosphatide acyltransferase. Increases in lysolipids and free arachidonic acid could result from an inhibition of this enzyme. Investigators have found that the diacylglycer01s dioctanoylglycerol and oleylacetylglycerol inhibit lysophosphatide acyltranferase in macrophages (Goppelt-struebe et a l . , 1987). Inhibition of lysophosphatide acyltransfersase was also observed in plasma membranes from T lymphocytes under conditions unfavorable for PKC activation. The authors postulated diacylglycerols inhibited this enzyme activity through membrane perturbation.

111. ROLE OF ENZYMES IN PHOSPHOINOSITIDE METABOLISM IN NEUTROPHIL ACTIVATION

A. Phosphoinositide Synthesis: Background The phosphoinositides, along with phosphatidylglycerol (PG) and diphosphatidylglycerol (DPG) can be differentiated from the bulk of phospholipids by their route of formation. While most phospholipids, including PtdCho and PtdEth, are formed from DAG in a three-step process, Ptdlns, PG, and DPG are synthesized from PtdH via cytidine diphosphate (CDP) diglyceride. PtdH is converted to CDP-diglyceride (more sensibly phosphatidyl-CMP or CMP-phosphatidate) by a PtdH : cytidyltransferase. Inositol is substituted for CMP by the action of PtdIns synthase (CDP-DG : inositol phosphatidyltrdnsferase). The phosphate on the PtdH is preserved through this step and remains with Ptdlns. The evidence for this pathway has been reviewed (Agranoff and Bleasdale, 1978; Michell, 1986). From the time Ptdlns synthase was first identified in 1958 (Agranoff et a / ., 1958) until recently, it was thought to exist exclusively in the endoplasmic reticulum. Since DAG kinase and Ptdlns kinases are localized primarily in the plasma

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31 1

membrane and PtdH : cytidyltransferase and PtdIns synthase are in the endoplasmic reticulum, transfer of phospholipids between membrane compartments is thought to be an important part of phosphoinositide synthesis. Transfer proteins bind phospholipids and transfer them between membranes. Specific transfer proteins have specificity for different phospholipids. PtdIns-specific transfer proteins have been described in other cells (Demel et d.,1985; Van Paridon et d., 1987), but the potential role for regulation of phospholipid metabolism through transfer proteins has not been explored. Experiments have identified a saltextractable Ptdlns synthase localized in the plasma membranes of rat pituitary tumor cells (Cubitt and Gershengorn, 1989). If this is in fact a general phenomenon, then the importance of transfer proteins for phosphoinositide synthesis will have to be reevaluated.

B. Phosphatidylinositol Kinases: Possible Links to Receptor Activation Polyphosphoinositides are formed from PtdIns through the actions of kinases that phosphorylate specific hydroxyls on the inositol ring. In resting neutrophils that have been incubated with 32Pfor 1-2 hours, relatively little [32P]PtdInshas been found (Traynor-Kaplan et ul., 1988). Nevertheless, close to 80% of all the label incorporated into phospholipids can be found in PtdIns(4)P and Ptdlns(4,5)P2, indicating these kinases are very active even in unstimulated neutrophils. The rapid recovery of labeling in PtdInsP, following stimulated PLC hydrolysis in intact cells indicates that these enzymes continue to be active subsequent to stimulation. Two separate kinases, phosphorylating PtdIns and PtdIns(4)P, respectively, were recently identified in a human neutrophil plasma membrane fraction (Pike and Arndt, 1988). Both enzymes utilized ATP preferentially to GTP, were Mg2 dependent, and were inhibited by millimolar concentrations of Ca2 .They were distinguished by their detergent solubility. PtdIns kinase was readily extracted in detergent, whereas PtdlnsP kinase was not. In a separate investigation, Pike and DeMeester ( 1988) found that S-adenosylhomocysteine is a competitive inhibitor of PtdIns kinase activity in human neutrophil plasma membrane fractions. This was consistent with their observations of decreased [32P]PtdInsPand inositol phosphate production in intact neutrophils subjected to conditions leading to the rise of intracellular levels of S-adenosylhomocysteine. This finding could be related to the previous observation of an inhibition of monocyte chemotaxis under conditions that resulted in elevated concentrations of intracellular S-adenosylhomocysteine (Pike et al., 1979). Most PtdIns kinases have considerable substrate specificity and will only phosphorylate at one site. Until recently, two classes of enzymes were thought to be largely responsible for the synthesis of polyphosphoinositides: PtdIns-4kinase, phosphorylating PtdIns on the 4 position of the inositol ring, and +

+

312

ALEXIS E. TRAYNOR-KAPLAN

Ptdlns(4)P-5-kinase, which recognizes PtdIns(4)P as a substrate and adds a phosphate onto the 5 position of the inositol ring. However, a phospholipid kinase activity (PtdIns-3-kinase) that copurifies with pp60v-,srt'and with the polyoma virus middle T/pp6OC-""' complex has been identified (Whitman et al., 1988) (Fig. 3). The PtdIns-3-kinase phosphorylates phosphoinositides on the 3 position of the inositol ring, generating PtdIns(3)P from Ptdlns. Studies (Auger ef al., 1989) indicate that it will also phosphorylate PtdIns(4)P and PtdIns(4,5)P2, generating PtdIns(3,4)P2 and PtdIns(3,4,5)P3, but not the soluble inositol phosphates. Therefore, it is distinct from the cytosolic inositol kinases. The PtdIns-3-kinase is a substrate for middle T/pp60C-src tyrosine kinase and is phosphorylated on both the tyrosine and serine residues. The PtdJns-3-kinase activity becomes associated with the PDGF receptor in vascular smooth muscle cells within 15 scc of addition of PDGF to cells, which coincidcs with tyrosine phosphorylation of an 85,000 dalton protein (Auger et u/., 1989).'This coincides with thc appearance of PtdJns(3)P and Ptdlns(3,4)P2 in these cells. A transient elevation of a previously unidentified phospholipid, PtdInsP, (putatively Ptd1ns(3,4,5)P3) (Traynor-Kaplan et al., 1988), in chemotactic peptide-stimulated neutrophils has been described. This, in conjunction with the subsequent observation of an activation-linked elevation in PtdIns(3,4)P2 (Traynor-Kaplan er al., 1989),suggests the PtdIns-3-kinase may also be present in

P1(3,4,5)P, FIG, 3. Putative pathways in the formation of novel polyphosphoinositides. Phosphatidylinositol (PI) can bc convcrtcd to phobphdtidylinositol4-phosphate [P1(4)P]by the action of phosphatidylinositol-4-kinase ( I ) which has been identified i n neutrophils (Pike and DeMcester, 1988). This is the commonly known pathway which contributes to the majority of PIP formed in cells. Alternatively PI can be converted to phosphatidylinositol 3-phosphate[P1(3)P] by a phosphatidylinositol-3-kinase( 2 ) , like the one recently identificd by Whitman et a / . (1988). Phosphatidylinositol-3-kinascwill also phosphorylate Pl(4)P and phosphatidylinositol 4.5-bisphosphatc [Pl(4.S)P2J to phosphatidylinositol 3.4-bisphosphate [P1(3,4)P2] and phosphatidylinositol 3,4,5-trisphosphate [P1(3,4,5)P3], rcspcctivcly. Although P1(3.4,)P2and PIP7 [putatively P1(3,4,5)P3] have been idcntified in neutrophils, the phosphatidylinobitol-3-kinasehas yet to be isolated. Finally, PI(4)P is converted to P1(4,5)P2 by a phosphatidylinositolphosphate-5-kinase( 3 ) .This enzyme may also convert P1(3.4)Pzto P1(3,4,5)P7, hut this has yet to bc demonstrated.

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neutrophils and interact in a G protein-dependent fashion with the chemotactic peptide receptor. Its activity does not appear to be dependent on an elevation in intracellular calcium. The phospholipid kinase in neutrophils may also be a substrate for tyrosine kinase. Guanine nucleotide-stimulated tyrosine phosphorylation has been identified concomitant with superoxide production in electroporated neutrophils (Nasmith et al., 1989). The polypeptide phosphorylation pattern was distinct from that observed with DAG, indicating the activation of other protein kinases in additional to PKC. Tyrosine kinase activity associated with A231 87 and retinoic acid stimulation has also been identified in HL60 cells (Chapekar et al., 1987).

C. Polyphosphoinositide Phosphatases Phosphomonoesterases that specifically remove phosphates from the 4 or 5 position of polyphosphoinositides have been identified in erythrocytes (Palmer and Mack, 1985). These enzymes exhibit considerable substrate specificity in that they only hydrolyze the phosphate from the 4 position or the 5 position but not both. The EDTA-sensitive phosphomonoesterase that removes the 5 phosphate from PtdIns(4,5)P2 (Palmer, 1981) is somewhat less effective in removing the 5 phosphate from lysoPtdIns(4,5)P2 and again less effective in cleaving the 5 phosphate from Ins( I ,4,S)P,. Therefore, it is unlike the cytosolic 5-phosphomonoesterase, which hydrolyzes the 5 phosphate from Ins( 1,4,S)P, and Ins(1,3,4,5)P4 (Downes et al., 1982). There is minimal hydrolysis of the 4 phosphate from any inositol compound using this enzyme preparation. A separate enzyme, which was not inhibited by EDTA, cleaved the phosphate from the 4 position of the lipid but not the inositol phosphate. A Mg2+-dependent conversion of PtdInsP, to PtdInsP in neutrophil plasma membranes suggested the presence of a phosphomonoesterase in neutrophils (Cockcroft et al., 1984).

D. Phospholipase C: Comparison of Its Response to Different Stimuli Phospholipase C has been identified in bacteria as well as mammalian cells. While bacterial PLCs have wide ranging substrate specificity, most mammalian PLC is specific for phosphoinositides (Waite, 1987). PLC capable of hydrolyzing PtdInsP, was first identified in brain tissue (Thompson and Dawson, 1964) and has since been found in the cytoplasm and plasma membranes of many cells (see review in Downes and Michell, 1985). 1. PLC ISOLATIONJ N NEUTROPHILS

A PLC activity was identified in a light plasma membrane fraction from human and rabbit neutrophils that were stimulated with 0.3-0.5 mM Ca2+ and

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hydrolyzed polyphosphoinositides but not PtdIns, PtdCho, or PtdH (Cockcroft et al., 1984). More recently, a PtdIns-specific PLC has been identified in the cytosolic fraction from sucrose gradients of rat neutrophil sonicates (Mackin and Stevens, 1988). PtdlnsP, will also act as a substrate for this protein with a K, of 109 pM vs. 240 pkl for Ptdlns. This P K activity has a pH optimum between 5 . 5 and 6.0 and is dependent on calcium up to 100 pM with half-maximal activity being reached around 10 nM. Although it is similar to a platelet PLC (Chau and Tai, 1982) in its substrate specificity and cytosolic location, it is unaffected by many inhibitors that attenuate platelet P E activity.

2 . LINKAGE OF PLC TO FORMYL PEPTIDE RECEPTOR. G PROTEINS A N D CALCIUM ELEVATION IN NEUTROPHILS Since the earlicr work in this area has been thoroughly reviewed (Cockcroft, 1986), this summary will be brief and focus on more recent developments. Hydrolysis of PtdInsP, by PLC in formyl peptide-stimulated human and rabbit neutrophils has been inferred from the increases in I3H] InsP, and DAG measured in concert with a transient decrease in radioactivity in PtdlnsP, (Dougherty et al., 1984; Bradford and Rubin, 1985; Cockcroft ef al., 1985). Considerable data from neutrophils and HL60 cells indicate this event is regulated through a guanine nucleotide regulatory (G) protein (Verghese et al., 1985; Cockcroft and Gomperts, 1985; Brandt et a/. , 1985; Smith et u l . , 1986). The lines of investigation elucidating the role of G proteins in formyl peptide-stimulated phosphoinositide hydrolysis have been reviewed by Snyderman and Verghese (1 987), and Bokoch (Chapter 4, this volume). Further investigation indicated the formyl peptide-activated G protein lowered the calcium requirement for the PLC hydrolysis to physiological, unstimulated concentrations (Smith et ul., 1986). The Ins( 1,4,5)P, released during PLC hydrolysis of PtdInsP, is believed to cause release of calcium from intracellular stores, elevating the intracellular calcium concentrations in a variety of cells (Berridge and Irvine, 1984). In permeabilized human neutrophils, Prentki and co-workers ( 1984) identified a non-mitochondria1 calcium sequestering pool that released calcium in the presence of 2.5 pkl Ins( 1,4,5)P,. This is well within the range of Ins( 1 ,4,5)P, concentrations occurring within the first few seconds of stimulation in neutrophils, and the time course of Ins( 1,4,5)P, production is rapid enough to be a precursor of calcium elevation (Dougherty et al., 1984; Bradford and Rubin, 1985; Cockcroft et u l . , 1985; Di Virgilio et ul., 1985; Krause ef al., 1985; Lew et al.. 1986; Dillon et a / . , 1987a). More evidence has been obtained recently that InsP, is operating through a receptor-mediated mechanism. A saturable receptor for Ins( 1,4,5)P, in rabbit neutrophils and hepatocytes was described by Spaet and co-workers (1986). In macrophages, three proteins were identified as binding sites for isotope-labeled arylazide derivatives of InsP, (lshimatsu et u / . , 1988).

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These findings suggest that PLC hydrolysis of PtdlnsP, precedes and contributes to calcium elevation in neutrophils. Studies with intracellular calcium buffers confirm this conclusion. Although PLC activity has a calcium requirement, it does not appear that it is above basal concentrations of intracellular calcium. Lower than physiological levels of calcium (less than 30 nM) inhibited Ins( I ,4,5)P, production in HL60 cells, whereas Ins( 1,4,5)P, production was normal when calcium levels were maintained at normal resting values with the intracellular calcium chelator-indicator quin2 (Lew et ul., 1986). Further support for this conclusion came when the elevation of intracellular calcium was buffered with nonfluorescent MAPTAM, permitting an independent measurement of the calcium concentrations with fura. In these experiments, phosphoinositide hydrolysis proceeds normally following formyl peptide stimulation (Korchak et al., 1988a), although superoxide generation is prevented. 3. OTHERINOSITOL PHOSPHATES APPEARING DURING NEUTROPHII. ACTIVATION It is difficult to measure inositol phosphate production from [3H]myo-inositollabeled neutrophils because of their limited viability once isolated from the blood which limits the amount of time the cells can be incubated with [3H]myoinositol. Most studies in neutrophils of inositol phosphate production have utilized short incubation periods that definitely do not permit labeling to isotopic equilibrium. One way of overcoming this problem is by using HL60 cells. Dillon et al. (1987a) dealt with this difficulty by developing culture conditions where neutrophils could be incubated with [3H]myo-inositol for 24 hr. Until 1984, all InsP, was thought to exist as one isomer Ins( 1,4,5)P3. At that time Irvine identified a second isomer of InsP,, Ins( 1,3,4)P, (Irvine et al., 1984, 1985). This was followed by the discovery of Ins(1,3,4,5)P4 (Irvine and Moor, 1986). Ins(1,4,5)P3, Ins( 1,3,4)P,, and Ins( 1 ,3,4,5)P, have all been identified following stimulation of rabbit and human neutrophils and HL60 cells (Dougherty et al., 1984; Bradford and Rubin, 1985; Krause et al., 1985; Lew et al., 1986; Dillon et al., 1987a). All of these molecules have been reported to increase with stimulation but with different time courses and different calcium dependencies. Formyl peptide-induced increases in Ins( 1,4,5)P3 and Ins( I ,3,4,5)P4 were detected at 5 sec, both peaking between 15 and 30 sec and returning to baseline after several minutes. The relative quantity of InsP, production was approximately one-fifth that of Ins(1,4,5)P3. In addition to Ins(l,4)P2, another isomer of InsP, has also been identified in human neutrophils, lns(3,4)P2 (Dillon et ul., 1987b). Both Ins( 1,3,4)P, and lns(3,4)P2 increase with formyl peptide stimulation, although the time courses of these increases were dclayed relative to Ins( 1,4,5)P, and Ins( 1,4)P2. When the rise in intracellular calcium was blocked by pretreatment

316

ALEXIS E. TRAYNOR-KAPLAN

with ionomycin and EGTA, Ins( 1,3,4)P, and lns(3,4)P2 production was markedly reduced, whereas the production of the other inositol phosphates was unaffected. lhis is consistent with another report where calcium chelation with quin2 prevented the formyl peptide-induced increase in calcium and thereby inhibited production of Ins( 1,3,4)P, but not Ins( 1,4,S)P, in HL60 cells (Lew et ul., 1986). Results from the same study indicated calcium may be sufficient to elicit a rise in Ins( 1,3,4)P,. Ionomycin stimulated a calcium-dependent increase in Ins( 1,3,4)P, but inhibited formyl peptide-stimulated Ins( 1,4,S)P,. Therefore, Ins( I ,3,4)P, production appears to be at least partially secondary to calcium elevation, whereas Ins( I ,4,S)P, production can be inhibited at high calcium concentrations. 4. ROUTEOF FORMATION OF INOSlTOL PHOSPHATES lnasitol phosphates in neutrophil cytoplasm can arise from the action of PLC on phosphoinositides or from the activity of cytosolic enzymes such as inositol kinases and phosphatases present in the cytoplasm. Only Ins( 1,4,S)P, is thought to arise from PLC hydrolysis of phosphoinositides. The other inositol phosphates are thought to be the result of the activity of a variety of cytosolic kinases and phosphatases, some of which have been reported to exist in neutrophils. In studies with lysates of human neutrophils, Dillon and co-workers (1987b) identified an ATP-dependent 3-kinase which converted Ins( I ,4,S)P, to Ins( 1,3,4,S)P, as well as phosphomonoesterases which hydrolyzed the 5 , 1, and 4 phosphates successively. Originally, when Ins( 1,4,5)P, and Ins( 1 ,3,4,S)P4 were identified, potential phospholipid precursors were not known (Irvine et al., 1985). However, phospholipid precursors for InsP, and Ins( l ,3,4)P, have been identified in human neutrophils (Traynor-Kaplan et ul., 1988, 1989). Therefore, a portion of lnsP, and Ins( 1,3,4)P, appearing in stimulated neutrophhs could be arising from PLC hydrolysis of PtdlnsP, and PtdTns(3,4)P2. Theoretically, inositol phosphates could also be gencrated as a result of PLD hydrolysis of polyphosphoinositides. These inositol phosphates would not have a phosphate on the I position. Of the inositol phosphates detected in neutrophils, only lns(3,4)P2 and some of the inositol monophosphates could arise from PLD activity. However, since both lns(3,4)P2 and PLD activity (see Section II,D) appear to be calcium dependcnt, this is a possibility. Are Other lnositol Phosphates Signals.? Ins( 1 ,3,4,S)P4 has been proposed to play a role in calcium elevation (Irvine et a / . , 1985). In lacrimal acinar cells, both InsP, and InsP, have been shown to act synergistically in stimulating a calcium-dependcnt K channel (Morris et al. , 1987). More recently, evidence has been shown indicating that InsP, may be involved in the resequestration of calcium (Hill et al., 1988). ['Hlinositol 1,3,4,S-tetrakisphosphatebinding sites were observed in the crude membranc fraction from HL60 cells (Bradford and +

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317

Irvine, 1988). Although Ins( 1,3,4)P, and the isomers of InsP, do not stimulate calcium flux, other as yet unidentified signal functions have not been precluded.

5. COMPARISON OF DIFFERENT STIMULANTS ON PLC ACTIVATION Evidence is accumulating that receptors for other ligands may also be linked to PLC activity through a G protein. Feltner et al. (1986) have found that C5a and LTB, are linked to a plasma membrane-bound, pertussis toxin-sensitive GTPase from rabbit neutrophils. Omann et a1. (1987) demonstrated that LTB, stimulates phosphoinositide hydrolysis in neutrophils to a lesser extent than formyl peptide in contrast to early studies where no phosphoinositide hydrolysis was measured in response to LTB, (Volpi et a / ., 1985). More recently, LTB,-stimulated increases have been compared to formyl peptide-stimulated increases in Ins( 1,4,5)P,, Ins( I ,3,4)P,, and Ins( 1 ,3,4,5)P, (Dillon etal., I987b). The rise in all species was comparable for the two stimulators and the peak levels were almost as high in LTB,-stimulated cells. However, levels of all three inositol phosphates fell more rapidly in cells stimulated with LTB,. LTB,-induced calcium elevation and actin polymerization is pertussis toxin sensitive (Omann et al., 1986). The response of neutrophils to substance P has been characterized (Serra et al., 1988). These investigators observed superoxide formation, exocytosis, elevation of cytosolic calcium, and inositol phosphate production following stimulation with substance P. However, these responses appear to be a function of the COOH-terminal fragment which has sequence homology with the formyl peptide. Therefore, it is unclear if substance P is acting through a site distinct from the formyl peptide receptor. ATP has been found to stimulate inositol phosphate production and calcium elevation in HL60 cells in a partially pertussis toxin-sensitive manner through an extracellular P,-purinergic receptor (Dubyak et al., 1988). Interestingly, although concanavalin A causes an elevation of intracellular calcium and superoxide production it does not appear to stimulate PLC hydrolysis of PtdInsP,. Neither PtdInsP, hydrolysis nor InsP, elevation was detectable following concanavalin A stimulation (Korchak et al., 1988b), although PtdIns hydrolysis and an increase in radiolabeled DAG and (32P]PtdHwere observed. Levels of PtdH and DAG were about half of that obtained with formyl peptide stimulation. 6. PHOSPHOLIPASE C A N D PROTEIN KINASE ACTIVITY

a. Cyclic AMP-Dependent Protein Kinuse: Receptor-Mediated Inhibition of PLC in Neutrophils. There is a group of agents that inhibits some aspects of formyl peptide-stimulated neutrophil activation, including phosphoinositide hydrolysis and superoxide production (Takenawa et al., 1986; Tecoma e f al., 1986). Most of these agents interact with a pathway operating through receptors

318

ALEXIS E. TRAYNOR-KAPLAN

linked to adenylatc cyclase, causing increases in cyclic AMP and stimulating cyclic AMP-dependent protein kinase. These agents include prostaglandin E,, padrenergic agonists, such as isoprotcrenol, and adenosine receptor agonists. Agents that cause an elevation of intracellular cyclic AMP, such as forskolin and dibutyryl cyclic AMP, will also attenuate neutrophil responses to formyl peptide stimulation. There is now evidence that this inhibition of PLC activity is at the level of the G protein since isoproterenol inhibits GTPase in broken cell preparations (Mueller et al., 1988; Mueller and Sklar, 1989). h. Protein Kinase C and PLC. In contrast to cell activators such as formyl peptide and LTB,, which stimulate cclls via a membrane receptor, phorbol esters stimulate cell activation at a point distal to the cell surface r e c e p t o r 4 protein complex. An intracellular binding site for phorbol esters has been identified as the phospholipid-dependent protein kinase C (Castagna et al., 1982; Nishizuka, 1983), which has been demonstrated to be stimulated by phorbol esters. Phosphoinositide hydrolysis does not appear to mediate phorbol- 12-myristate- 13acetate (PMA) stimulated cell activation, although protein kinase C activators do affect phosphoinositide metabolism. Although both phorbol esters and formyl peptide stimulate an increase in [32P]PtdH, formyl peptide stimulates PtdInsP, hydrolysis, whereas phorbol esters do not. Despite this absence of phosphoinositide-specificPLC activity, phorbol esters stimulate incrcases in diacylglycerols and alkylacylglycerols in human neutrophils (Rider and Neidel, 1987), suggesting other phospholipids may be hydrolyzed by PLC in these experiments. There are two-way interactions between phosphoinositide metabolism and protein kinase activity. ( I ) PLC hydrolysis of phosphoinositidcs leads to the production of diacylglycerols which are stimulators of protein kinase C, which is present in large quantities in neutrophils (see Chapter 18). (2) Stimulation of protein kinase C can, in turn, modulate phosphoinositide metabolism. Decreases in formyl peptide-stimulated inositol phosphate production have been observed in neutrophils and HL60 cells pretreated with 2-200 nM phorbol ester (Kato et d., 1986; Kikuchi et al., 1986; Della Bianca et al., 1986; Smith et al., 1987; Tyagi et al., 1988). Kato and co-workers (1986) found PtdInsP, hydrolysis still occurred following formyl peptide in guinea pig neutrophils pretreated with 100 nM PMA for 3 min, although inositol phosphate production was reduced. PtdInsP, levels remained depressed for 3 min, whereas they had recovered in neutrophils stimulated with formyl peptide alone. The authors interpreted their findings as resulting from PMA inhibition of PtdIns kinase activity, although PMA did not inhibit PtdIns or PtdInsP kinase in neutrophil plasma membranes. Most of the hydrolysis of PtdInsP, was abolished in GTPyS- but not C a 2 + stimulated membranes from neutrophils pretreated with PMA( 16- 160 nM), suggesting that PMA is causing dissociation of PLC from the G protein and not

12. PHOSPHOlNOSlTlDE METABOLISM IN PHAGOCYTES

31 9

inhibiting PLC activity directly (Smith et ul., 1987). It is unclear, however, if this can account for the inhibition of InsP, production in neutrophils preincubated with low priming concentrations of PMA (3 nM) (Della Bianca et al., 1986). Similarly, Tyagi et al. (1988) found that DAG is elevated following formyl peptide stimulation of human neutrophils primed with 2 nM PMA while inositol phosphate production is depressed. Oddly, DAG, but not phorbol esters, stimulated inositol phosphate production and calcium elevation, suggesting DAG could be influencing phosphoinositide metabolism through a non-PKC-dependent mechanism (Restrepo et a / ., 1989). Another protein kinase other than PKC has been identified in rabbit neutrophils activated with chemoattractants (Huang and Laramee, 1988). Decreases in radiolabel in PtdInsP, could result in less radiolabeled InsP, released following stimulation in PMA treated cells. There is some evidence suggesting that PKC activation lowers ATP levels in HL60 cells, which could reduce levels of polyphophoinositides. Dawson et al. (1987) observed a 40% reduction in ATP levels in HL60 cells stimulated with DAG. Although PI kinase activity in vitro is maximal at 1 mM ATP, decreases in phophoinositides are reported in phagocytic cells stimulated with PKC activators. Therefore, it is necessary to measure phosphoinositide hydrolysis as well as inositol phosphate production when assessing the effects of PKC activators on PLC activity. 7. HYDROLYSIS OF PtdInS-ANCHORED PROTEINS WITH PLC

There is a class of proteins which has been found to be linked through PtdIns to the plasma membrane in a variety of cells from protozoans to mammals (see review in Low, 1987). PLC hydrolyzes the inositol linkage, releasing the protein from the membrane. Some of these proteins are receptors. In neutrophils, one of the receptors for the constant region of immunoglobulin G (FcRIII) has been found to be linked to PtdIns. Formyl peptide stimulation causes release of FcRIII, probably through PLC-mediated hydrolysis of the PI anchor (Huizinga et a / . , 1988).

E. Phospholipase A,: Its Contribution to Phosphoinositide Hydrolysis 1 . GENERAL CONSIDERATIONS

PLA, has two functions in phagocytes: ( I ) hydrolysis of bacterial and other exogenous phospholipids, and (2) signal transduction through the release of arachidonic acid and its metabolites and possibly lysolipids (see Chapter 22). It may be difficult to distinguish between PLA, activity which participates in signal transduction from PLA, activity associated with phagocytic and lysosomal func-

320

ALEXIS E. TRAYNOR-KAPLAN

tions. Initial studies of PLA, activity in ncutrophils which focused on hydrolysis of bacterial phospholipids have been reviewed (Elsbach, 1980; Waite, 1987). Because of its proximity to reccptors, a receptor-linked PLA, activity in the plasma membrane is more likely to participate in the early phases of signal transduction than a granule-associated PLA, preparation which is not linked to receptor activation and may function to hydrolyze bacterial phospholipids (Traynor and Autii, 1981). PLA, activity has been measured in plasma membrane (Franson et a / . , 1977; Victor er a / . , 1981) and azurophilic and specific granule fractions (Franson et d . , 1974; Smith and Waite, 1986) of neutrophils. PLA, activity is associatcd with cell activation. There is evidence that it may be linked through a G protein to the formyl peptide receptor in rabbit neutrophils. Bokoch and Gilman ( I 984) were able to block arachidonic acid production in guinea pig neutrophils by pretreating the cells with pertussis toxin. There is evidence that pertussis toxin-sensitivc release of arachidonic acid in rabbit neutrophils stimulated with platelet activating factor is independent of a rise in intracellular calcium, suggesting that PLA, stimulation is directly linked to physiological receptors (Tao et al., 1989). However, this is in contrast to the findings of G. M. Bokoch and L. A. Sklar (personal communication) who observed that arachidonic acid release could be preventcd by buffering intracellular calcium with quin2. Increases in activity of PLA, have been observed in some plasma membrane fractions from rabbit peritoneal ncutrophils activated by formyl peptide (Bormann et a l . , 1984) but not others (Matsumoto et d . , 1988). PLA, activity in neutrophils has been inferred from the increases in arachidonic acid and arachidonic acid metabolites released during phagocytosis (Franson and Waite, 1978; Waitc et al., 1979; Walsh et a / ., 1981) as well as during activation by A23187 and phorbol esters (Volpi e t a / . , 1985). Formyl peptide stimulation of arachidonic acid release was observed in rabbit neutrophils by Bokoch and Reed (1981). In one study, arachidonate release was not observed in chemotactic peptide-stimulated human neutrophils, although externally provided arachidonate was converted to 5-lipoxygenasc products (Clancy er al., 1983). These investigators may have missed arachidonate release, however, by failing to trap it with BSA.

2 . ARE PHOSPtlOINOSITIDES SUBSTRATES FOR PLA, I N S T I M U L A T E D PHAGOCYTES'? A variety of PLA, preparations have been described in phagocytic cells, which as a group will hydrolyze most phospholipids. However, from the point of view of signal transduction, it is more interesting to know what the endogenous substrates are, how closely they are linked to receptor activation, and the time courses of their hydrolysis following physiological stimulation. There is considcrable evidence that arachidonyl Ptdlns is a substratc for PLA, in neutrophils and

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macrophages. Phosphoinositides have proportionally more arachidonic acid, usually in the 2 position, than other phospholipids. One approach to studying this question has been to label cells with radiolabeled arachidonic acid and observe its subsequent fate on stimulation. Stimulation of rabbit peritoneal neutrophils with forrnyl peptide enhanced incorporation of arachidonic acid into PtdIns in a dosedependent manner (Rubin et d . , 1981). Twice as much ['Hlarachidonyl PtdIns was degraded than PtdCho in human neutrophils stimulated with opsonized zymosan or A23187 (Walsh ef ul., 1981). Platelet activating factor also stimulated the incorporation of radiolabeled fatty acids into PtdEth, PtdCho, and PtdIns of guinea pig granulocytes, probably as a result of reacylation of lysolipids (Tou, 198 I). In another study, 80% of the arachidonic acid generated in response to zymosan was reported to come from PtdIns (Garcia Gil et al., 1982). They also observed an increase in the production of arachidonyl DAG, indicating arachidonyl containing phospholipids were also substrates for PLC. This indicates that a decrease in levels of an arachidonyl containing phospholipid without a quantitative analysis of the resultant lysolipids would not necessarily indicate hydrolysis with PLA,. Therefore, the greater decrease in ['Hlarachidonyl PtdlnsP and PtdlnsP, vs. [RH]glycero-PtdInsPand -PtdInsP, observed by Yano and co-workers ( 1983) in forrnyl peptide-stimulated rabbit neutrophils could be explained by either increased PLA, activity or preferential hydrolysis by PLC of arachidonyl containing phosphoinositides. Cockcroft and Allen (1984) compared the fatty acid compositions of Ptdlns, PtdH, and DAG in unstimulated human neutrophils to cells stimulated with formyl peptide for 10 sec and 2 min vs. unstimulated cells. They observed a decrease in arachidonic acid in PtdIns with concomitant increases in stearic and oleic acids. However, relative levels of arachidonic acid were unchanged in PtdH and DAG following 10 and 120 sec of stimulation. The overall fatty acid compositions of DAG and PtdH were similar to each other and different from the fatty acid composition of PtdIns, indicating that only a portion of the pools of DAG and PtdH participated in the PtdIns cycle. Wiederhold et al. (1988) reported a preferential incorporation of [.
322

ALEXIS E. TRAYNOR-KAPLAN

by Meade et u1. (1986), but from their double-label experiments determined that the primary source of the arachidonic acid was PtdCho. In contrast, formyl peptide was ineffective in eliciting arachidonic acid release without the presence of cytochalasin B. Phospholipid deacylation has been measured in human neutrophil subfractions following stimulation of prelabeled cells with A23187 (Smith and Waite, 1986). Loss of [3H]arachidonic acid from Ptdlns-PtdSer and, to a lesser extent, from PtdCho was observed in all fractions. The specific granules were subject to the greatest loss from the Ptdlns-PtdSer fraction, whereas the azurophil granules had the greatest proportional loss of radioactivity. Although still substantial, the least amount of deacylation was in the denser plasma membrane fraction. However, the denser plasma membrane fraction had the least loss from [I4C]stearic acidlabeled phospholipids. Since stearic acid is usually in the sn-1 position and arachidonate in the sn-2 position, this indicates that the denser plasma membrane fraction was devoid of PLA, activity. PLA, activity, in the absence of PLA, activity, is niore likely to be associated with signal transduction. In contrast, both activities are likely to be present during bacterial lysis. Shibata et ul. (1988) obtained a PLA, from macrophage plasma membrane which had a 10-fold greater hydrolysis of [ ''C]arachidonic acid from PtdIns as from PtdCho or PtdEth, whereas Alonso ef a]. (1986) identified a cytosolic phospholipase in human neutrophils that favors arachidonyl PtdCho as substrate. Therefore, phosphoinositides are substrates for PLA,, and their hydrolysis appears to increase during phagocytosis or A23 187 stimulation in macrophages, rabbit neutrophils, and human neutrophils. Most studies report a significant portion of the rise in arachidonic acid production following receptor stimulation to be from PLA, hydrolysis of Ptdlns. However, since there are substantial amounts of PtdCho hydrolyzed, it is difficult to assess from the radiolabel studies the relative masses of PtdCho and Ptdlns hydrolyzed. Furthermore, the relative contributions of different pathways may vary in cells from distinct species stimulated with different stimuli. To add to the complexity, in a physiological setting in which neutrophils are phagocytosing bacteria, much of the arachidonic acid generated could be arising from hydrolysis of bacterial phospholipids (Traynor and Autii, 1981).

3. LYSOLIPID PRODUCTION

IN

PHAGOCYTES

If phosphoinositides are indeed the primary substrate for PLA, in neutrophils, lysophosphoinositides should also be formed. Lysophosphoinositides are particularly amphipathic, partitioning equally between aqueous and organic phases. As a result, most methods for extracting phospholipids, which are derived from a Folch procedure using chloroform, methanol, and HCI, incompletely extract lysolipids as well as lysophosphoinositides into the organic phase, leaving up to 50% in the

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aqueous phase (Bjerve et a / ., 1974). Further complications can arise from prolonged exposure to HCI, frequently present in extraction mixtures, which hydrolyze phospholipids generating lysolipids. Lack of ability to measure increases in lysophospholipids (Walsh e t a / ., 1983; Meade ef ul., 1986) has been attributed to the efficient activity of an acyl-CoA : lysophospholipid acyltransferase which has been identified in rabbit neutrophils (Elsbach et al., 1972). Using a butanol extraction procedure which recovered 75% of the exogenously added, radiolabeled lysolipids Meade and co-workers (1986) investigated the subsequent metabolism of lysolipids in rabbit neutrophils. They showed that lysoPtdCho and IysoPtdH were reacylated and that this was accelerated in the presence of A23 187. In contrast, over 50% of the exogenous lysoPtd['H]Ins was further deacylated to glycerophosphoinositol with only 4% being reacylated into Ptdlns. However, since only the acyl chains of IysoPtdCho and IysoPtdH were radiolabeled, further deacylation would not have been detected. Takenawa and co-workers (1983) measured a gradual elevation in lysoPtdIns, IysoPtdCho, IysoPtdEth, and IysoPtdH in guinea pig neutrophils stimulated with A23187 over a 10 min period. Formyl peptide stimulated a comparable rise in IysoPtdEth, slightly less in lysoPtdCho, and only after 10 min in IysoPtdIns, with no significant change in IysoPtdH. In macrophages, zymosan stimulated a decrease in PtdIns and concomitant increase in IysoPtdIns and glycerophosphoinositol, suggesting that it was hydrolyzed by both a PLA, and a PLA, (Emilsson and Sundler, 1984). An increase in IysoPtdlns in a macrophage cell line was also observed following stimulation with lipid A precursors (Zoeller et ul., 1987). Thus, considerable evidence points to the appearance of IysoPtdIns with most routes of activation. 4. ROLE OF PKC

IN

PLA, ACTIVATION

Translocation of PLA, activity from cytosol to membranes has been observed in macrophages stimulated with 1-oleyl 2-acetyl glycerol (Schonhardt and Ferber, 1987). Phorbol esters cause release of arachidonic acid from macrophages, possibly from a unique phospholipid lyso(bis)PtdH which is extraordinarily elevated in macrophages and is concentrated in lysosomes (Cochran et ul., 1985). PMA stimulated the incorporation of radiolabeled fatty acids into PtdEth, PtdCho, and PtdIns of guinea pig granulocytes, possibly through reacylation of lysolipids (Tou, 1981). This is in contrast to the inhibition of acyltransferase by DAGs in macrophages (see Section 11,E). Phorbol esters had different effects on lipid metabolism in human vs. rabbit neutrophils. They enhance incorporation of [14C]arachidonic acid into PtdIns but not other phospholipids in rabbit neutrophils. In contrast, PtdSer, PtdCho, PtdEth, PtdH, and PtdIns become labeled in human neutrophils (Kramer ct d.,1984). PKC activation, however, may not be the mode of action of phorbol esters in stimulating PLA, in neutrophils. Although phorbol esters potentiated the A23 187-induced arachidonic acid re-

324

ALEXIS E. TRAYNOR-KAPLAN

lease in rabbit (Volpi et ul., 1985) and human neutrophils (Galbraith, 1988), the calcium-dependent release in human neutrophils stimulated with either DAG or phorbol ester was not affected by PKC inhibitors. Therefore, phorbol esterinduced arachidonic acid release may not be through a PKC-dependent mechanism. Similarly, formyl peptide-stimulated increases in arachidonic acid may not be through a PKC-dependent mechanism. H-7, a PKC inhibitor, did not alter the receptor-mediated release of arachidonic acid in HL60 cells (Billah and Siege], 1987). On the other hand, another product of PLA, activation, lysolipids, may modulate PKC activity (Hannun and Bell, 1987; Oishi et ul., 1988).

IV. CONCLUSIONS Earlier studies pointed to calcium as the primary intracellular mediator of formyl peptide stimulation in neutrophils until Lew and co-workers found that calcium changes alone could not mediate neutrophil activation. With the discovery of DAG stimulation of PKC (Nishizuka, 1984), many investigators pursued the possibility that it was the other signal component. Since PLC hydrolysis led to the production of both putative mediators, it became a new focus of signal transduction research. However, superoxide production independent of calcium elevation or PKC activation has been observed by Grinstein and Furuya (1988) in lormyl peptide-stimulated electropermeabilized ncutrophils. This further confirmed the findings of Rossi and co-workers who had succeeded in dissociating phosphoinositide hydrolysis, calcium elevation, and superoxide production (Grzeskowiak et al., 1986; Rossi et al., 1986; Della Bianca et al., 1988). Korchak and co-workers (1988b), through comparing calcium, DAG, and other components of phosphoinositidc metabolism following stimulation with different agents, also concluded that DAG and calcium elevation alone was not sufficicnt to stimulate superoxide and postulated PtdH may be another mediator. Nasmith and co-workers (1989) suggested an independent pathway for eliciting superoxide production through tyrosine phosphorylation by a previously unidentified tyrosine kinase. This finding could be related to the appearance of novel phosphoinositides reported to occur in activated neutrophils (TraynorKaplan et al., 1989). The receptor-mediated elevation of levels of PtdInsP, [putatively Ptdlns(3,4,5)P3] and Ptdlns(3,4,)P, indicates a PtdIns-3-kinase is stimulated in activated neutrophils. A Ptdlns-3-kinase recently identified in immunoprccipitates from pp60v-~srcand smooth muscle cells is associated with tyrosine kinase activity (Whitnian et al., 1988; Auger et al., 1989). Clearly, signal transduction pathways in neutrophil activation are more complex than once thought. PL,C is only one of many enzyme activities in the phosphoinositide pathway that appears to be linked to receptor activation. However, it may be helpful to differentiate between enzymes whose activity is in-

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creased as a result of PLC activity and those whose activation is independent of PLC. For instance, enzymes which require elevated intracellular calcium could be activated as a result of PLC-mediated rise in InsP,. It will be important to ascertain definitively which of the enzyme activities are secondary to an elevation in intracellular calcium. PLD and PLA, may fall into this category. On the other hand, DAG kinase activity may increase as a result of increased substrate availability resulting from PLC action. It has yet to be determined if PtdIns-3kinase stimulation can be dissociated from PLC activity. ACKNOWLEDGMENTS

I would like to express my gratitude to Drs. Gary M. Bokoch, Larry A. Sklar, Gerry R. Boss, and Kiersten Dharmsathaphorn and to Barbara Thompson for their constructive comments and proofreading. The author’s research has been supported by NIH grant number A1 17354. REFERENCES Agranoff, B . W., and Bleasdale, J. E. (1978). The acetylcholine phospholipid effect: What has it told us? What is it trying to tell us? In “Cyclitols and Phosphoinositides” (W. W. Wells and F. Eisenberg, Jr., eds.), pp. 105-120. Academic Press, New York. Agranoff, B. W., Bradley, R. M., and Brady, R. 0. (1958). The enzymatic synthesis of inositol phosphatide. J . B i d . Chem. 233, 1077-1083. Alonso, F., Henson, P. M., and Leslie, C. C. (1986). A cytosulic phospholipase in human neutrophils that hydrolyzes arachidonoyl-containing phosphatidylcholine. Biochim. Biophys. Acra 878, 273-280. Auger, K. R., Sevunian, L. A,, Soltoff, S . P., Libby, P., and Cantley, L. C. (1989). PDGFdependent tyrosine phosphorylation stimulates production of novel polyphosphoinositides in intact cells. Cell 57, 167-175. Balsinde, J., Diez, E., and Mollinedo, F. ( 1988). Phosphatidylinositol-specificphospholipase D: A pathway for generation of a second messenger. Biochem. Biophys. Res. Commun. 154, 502508. Bellavite, P., Corso, F., Dusi, S . , Grzeskowiak, M., Della-Bianca, V., and Rossi, F. (1988). Activation of NADPH-dependent superoxide production in plasma membrane extracts of pig neutrophils by phusphatidic acid. J . B i d . Chem. 263, 8210-8214. Bennett, J. P., Cockcroft, S . , Caswell, A. H., and Gomperts, B. D. (1982). Plasma-membrane location of phosphatidylinositol hydrolysis in rabbit neutrophils stimulated with formylmethionyl-lleucylphenytalanine.Biochern. J . 208, 801 -808. Benidge, M. J., and Irvine, R . F. (1984). Inositol trisphosphate, a novel second messenger in cellular signal transduction. Nurure (London) 312, 315-321. Billah, M. M., and Siegel, M. I . (1987). Phosphulipase A> activation in chemotactic peptidestimulated HL60 granulocytes: synergism between diacylgiycerol and Ca+ in a protein kinase C-independent mechanism. Biochem. Biophvs. Res. Commun. 144, 683-691. Bjerve, K. S . , Daae, L. N. W., and Bremer, J. (1974). The selective loss of lysophospholipids in some commonly used lipid-extraction procedures. A n d . Biochem. 58, 238-245. Bokoch, G. M., and Gilman, A. G . (1984). Inhibition of receptormediated release of arachidonic acid by pertussis toxin. Cell 39, 301-308. Bokoch, G. M., and Reed, P. W. (1981). Stimulation of arachidonic acid metabolism in the polymorphonuclearleucocyte by an n-forniylated peptide; comparisons with ionophore A23 187. J . B i d . Chem. 255, 10223-10227. +

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ALEXIS E. TRAYNOR-KAPLAN

phosphatidylinusitol transfer protein from bovine brain as studied by steady-state and timeresolved fluorescence spectroscopy. Binrhim. Biophys. Acfa 898, 172- 180. Verghese, M. W., Smith, C. D., and Snyderman, R. (1985). Potential role for a guanine nucleotide regulatory protein in chemoattractant receptor mediated polyphosphoinositide metabolism, mobilization and cellular responses by leukocytes. Biochem. Biophys. Res. Commun. Ca 127, 450-457. Victor, M., Weiss, J., Klempner. M. S . . and Elsbach, P. (1981). Phospholipasc A2 activity in the plasma membrane of human polymorphonuclear leukocytes. FEBS Left. 136, 298-302. Volpi, M . , Yassin, R., Naccache, P. H . . and Sha’afi, R. I. (1983). Chemotactic factor causes rapid decreases in phophatidylinositol4,5-bisphosphate and phosphatidylinositol4-monophosphatein rabbit neutrophils. Biochem. Biophys. Res. Commun. 112, 957-964. Volpi. M . , Molski. T. F. P., Naccachc, P. H.. Fcinstein, M. B . , and Sha’afi, R. 1. (1985). Phorbol 12-n1yristatc, 13-acetate potentiates the action of the calcium ionophore in stimulating arachidonic acid release and production of phosphatidic acid in rabbit neutrophils. Biochem. Biophys. Res. Commrm. 128, 594-600. Waite, M. (1987). The phospholipases. Handb. Lipid Rcs. 5 . Waite. M., DeChatelet. L. R . , King. V.. and Shirley, P. S . (1979). Phagocytosis-induced release of from human neutrophils. Biochem. Biophys. Res. Commun. 90, 984-992. M., Thomas, M. J., and DeChatelet, L. R. (1981). Release and metabolism of in human neutrophils. J . B i d . Chern. 256, 7228-7234. Walsh, C. E., Dechatelet. I-. R . , Chilton. F. H . , Wykle, R . L., and Waite, M .(1983). Mechanism of arachidonic acid rclcasc in human polymorphonuclcar leukocytes. Biochim. Biophys. Acta 750, 32-40. Whitman, M., Kaplan, D. K.,Schafihausen, B., Cantley, L., and Roberts, T. M. (1985). Association of phosphatidylinositol kinase activity with polyoma middle-T competent for transforniation. Nature (London) 319, 239-242. Whitman, M., Kaplan, D., Roberts, T.,and Cantley, L. (1987). Evidence for two distinct phosphatidylinositolkinases in fibroblasts. Biochcm. J . 247, 165-1 74. Whitman, M., Downes, C. P., Keeler, M., Keeler, T., and Cantley, L. (1988). Type I phosphatidylinositol kinase makes a novel inositol phospholipid, phosphatidylinositol-3-phosphate. Niiturr (London) 332, 644-646. Wiederhold, M. D., Anderson, K . M., and Harris, J. E. (1988). Labelling of lipids and phopholipids with [3H]arachidonic acid and the biosythcsis of cicosanoids in U937 cells differentiated by phorbol ester. Biochim. Biophvs. Actu 959, 296 304. Yano, K . , Nakashima, S . . and Noznwa. Y . (1983). Coupling of polyphobphoinositide breakdown with calcium efflux in f’ormyl-mcthionyl-leucyl-phenylalanine-st~mul~tedrahhit neutrophils. FEES Let?. 161, 296-300. Zoeller. R. A . , Wightman, P. D., Anderson. M. S., and Raetz, C. R. H. (1987). Accumulation of lysophosphatidylinositol in RAW 264.7 macrophage tumor cells stimulated by lipid A precursors. J . B i d . Cheni. 262, 17212-17220. +

+

CURRENT TOPICS IN MEMRRANES AND TRANSPORT, VOLUME 35

Chapter 13 The Role of Arachidonic Acid Metabolites in Lymphocyte Activation and Function MARK L. JORDAN Division of Urology School of Medicine University of Pittsburgh Pittsburgh, Pennsylvania 15213

1.

Introduction Effects of Arachidonic Acid Metabolites on Lymphocyte Activation and Function A . CyClooXygendse Products B . Lipoxygenase Products 111. Lymphocyte Synthesis of Arachidonic Acid Metabolitcs A . Cyclooxygenase Products B . Lipoxygenasc Products IV. The Effects of Lipoxygcnasc Inhibitors on Lymphocyte Activation and Function V. Conclusions Refercnces

JJ.

1. INTRODUCTION Cellular activation occurs as a result of the binding of activating ligands to specific receptors on the cell membrane. As a result, the cell membrane undergoes molecular reorganization leading to transmembrane signaling and release of a variety of intracellular second messengers. During this process, stimulation of phospholipid hydrolysis of phospholipases results in the elaboration of a 20 : 4 polyunsaturated fatty acid, arachidonic acid (AA), which itself undergoes oxygenation to a series of biologically active products collectively referred to as the eicosanoids. AA produced during this process may be metabolized by one of two major pathways (Fig. I). The cyclooxygenase (CO) pathway converts AA into prostaglandins (PGs), thromboxanes, and prostacyclin; the lipoxygenase (LO) 333 Copynght 0 1990 by Academic Prmr, Inc All right? of reproduction In any form rcxrvcd

334

MARK L. JORDAN Phospholipids

1

Phosopholipascs

Other Lipoxygenases 4

Arachidonic Acid 5-Lipoxygenase

5-HETE

-

5-Hydroperoxyeicosatetraenoic Actd (5-HETE)

I

LTC,,

LTD4, LTE,

I -

LTA,

ASA

Prostacyclin Thr ornbo xane s

Synthetase

Leukotriene A, (LTA,)

I

LTB4 S y n t h e t a s c

Leukotriene B, (LTB,)

' NDGA also inhibits Cyclooxygeiiase to Some FIG. 1 .

d,!qrt'iT

Major nictabulic pathways uf arachidonic acid

pathway yields a group of hydmperoxy-( HPETEs) and hydroxy-(HETEs) metabolites. Further metabolism of the 5 - L o product 5-HPETE results in the production of sulfidopeptide metabolites, the leukotrienes (LTs). PGs are synthesized by a wide variety of cell types, whereas LTs are derived principally from cells involved in host defense, including neutrophils, mononuclear phagocytes, and platelets (Kennedy er al., 1980; Goetzl, 1983; Goldyne, 1984; Aderem et al., 1986). Both PGs and LTs may have potent effects on lymphocyte activation and function. There is conflicting evidence that normal lymphocytes themselves synthesize AA products, as will be discussed. However, since lymphocytes are often intimately associated with phagocytes and other AA metabolizing cells at sites of inflammation, there is ample opportunity for modulation of their activation and subsequent function by these mediators. Furthermore, certain subsets of lymphocytes bear LT receptors (Payan et al., I984), and there has been evidence that lymphocytes may provide other cell types with esterified AA for subsequent metabolism to biologically active products (Goldyne, 1984). Despite this evidence, there is sufficient controversy regarding the synthesis by and effects of AA metabolites on lymphocytes to preclude definite conclusions about their role

13. ARACHIDONIC ACID METABOLITES IN LYMPHOCYTES

335

in immunoregulation. The potential in vivo effects of AA metabolites on lymphocytes have not been adequately studied; this is in part due to the rapid clearance of these molecules as well as uncertainty concerning their cellular source, rates of synthesis, and physiologic levels at sites of immunologically initiated inflammation. These factors should be borne in mind when the pharmacologic properties and effects of AA metabolites on in vitro lymphocyte function are described. Measurement of AA metabolite levels in the in vivo inflammatory milieu together with the functional responses of lymphocytes in this environment will help to address this, as will be discussed. The purpose of this review is to focus on the mechanisms by which AA metabolites affect lymphocyte activation and function and to evaluate their immunoregulatory potential.

11. EFFECTS OF ARACHIDONIC ACID METABOLITES ON LYMPHOCYTE ACTIVATION AND FUNCTION A. Cyclooxygenase Products Prostaglandins of the E series have long been associated with inhibition of both lymphocyte activation and subsequent lymphocyte functional responses. These effects include inhibition of both antigen- and mitogen-induced T cell activation (Ting and Hargrove, 1984), interleukin 2 (IL2) production, and generation of cytolytic T cells (Wolf and Droege, 1982). These inhibitory responses have been shown to be accompanied by increases in intracellular levels of cyclic AMP (Strom et a / ., 1972), but the molecular mechanisms involved in this activation process are not well understood. More recent studies (Chouaib et al., 1987) have suggested that the inhibitory effects of PGE, on human T cell activation are accompanied by inhibition of PHA-induced increases in cystosolic calcium concentration ([Ca*+],). [Ca2+], mobilization was restored by the calcium ionophore A23187 in the presence of PGE, with only partial restoration of T cell function (IL-2 production and proliferation). In contrast, phorbol esters, which activate protein kinase C (PKC), completely restored mitogen-induced T cell proliferative responses in the presence of PGE,. Both phorbol ester and ionophore were required for restoration of 1L-2 production. Thus, the suppressive effects of PGE, on T cell activation appeared to be mediated primarily through down-regulation of PKC activity, but were also [Ca2+ I, dependent. Once T cells have become activated, the effects of PGs on their functional responses is less clearly defined. The nature of the activating stimulus and the specific lymphocyte population under study seem to be important. Despite the traditional view of PGE, as a suppressor of T cell-mediated responses, there has been sporadic evidence that PGE, may actually enhance the responses of certain T cell populations characterized by cell density or other nonfunctional param-

336

MARK L. JORDAN

eters (Goodwin and Ceuppens, 1983). Low density human T cells have been reported to exhibit blastogenic responses to PGE, (Stobo et al., 1979). Enhanced PHA rcsponsiveness of preincubated human T cells (specifically the OKTX+ subset) and stimulation of suppressor cell activity have also been described (Goodwin and Ceuppens, 1983). These discrepancies may be partly explained by the prevailing use of bulk preparations of lymphocytes, with the overall response reprcscnting that of the predominant cell type in the responding population. In view of the potential importance of PGE, as an immunoregulator, we have sought to clarify its rolc by studying the functional responses of a series of well characterized allosensitized niurine T lymphocyte clones. The accumulated evidence from our studies of lymphocyte clone locomotion, antigen- and IL2stimulated proliferation, and cytotoxicity indicated that the inhibitory effects of PGE, were in fact exerted on helper but not cytotoxic T cells (Jordan ef al., 1986a, 1987a). These “subset-specific” effects on T cell function occurred at physiologic concentrations (10-100 ng/nil) of PGE,, which can be detected at sites of inflammation in vivo. This led us to hypothesize that allosensitized lytic

10

20

30

60

TIME (MIN)

FIG. 2. Differential effects of PGEz on cyclic AMP production by T lymphocyte clones. 2.5 X 10“ resting (day 7 ) helpcr (clone 199-1.5) or cytotoxic (CTL, clone 199-4) cells were incubated either in medium (helper: W; CTL: n)or 100 ngiml PGEl (helper: a;CTL: El). After 10, 20, 30, or 60 niin, thc cells were lysed with 0.5 M perchloric acid, centrifuged at 2000 rprn for 10 min, and the cell-free supernatant analyzed for cyclic AMP content by radioimmunoasaay. Results are expressed as pmolcs cyclic AMP per 2.5 x: lob cells.

337

13. ARACHIDONIC ACID METABOLITES IN LYMPHOCYTES

T cells (CTL), once provided with sufficient growth factors, may continue to proliferate, migrate, and kill even in the presence of concentrations of PGE, that inhibit helper cell (T,) function. Indomethacin (lop6 M),either alone or in combination with inhibitory concentrations of PGE,, had no effect on these responses, indicating that endogenous lymphocyte PGE, production did not contribute to these subset-specific effects. Additional studies in our laboratory have revealed that these subset-specific effects were not due to PGE, receptor sites on T, which were absent on CTL (Jordan et al., 1987b). We subsequently found that PGE, induced higher levels of intracellular cyclic AMP (CAMP)in T, than CTL (Fig. 2); however, various agents that either directly increase cAMP production (dibutyryl-CAMP) or prevent its degradation by phosphodiesterase (theophylline), inhibit both T, and CTL function (Jordan et al., 1987b). Thus, the subset-specific effects of PGE, appear to be exerted on a step prior to induction of adenylate cyclase without involving differential receptor binding of PGE, to T, and CTL, but resulting in higher levels of cAMP in T, but not CTL. More recently, we have found that activators of PKC (such as phorbol esters) abrogate the inhibitory effects of PGE, on established T, function (Fig. 3).

CTL + I'MA

**

HELPER + PMA

**

CTL

HELPER

FIG.3 . Phorbol myristate acetate (PMA) abrogates the eflects of PGEz on T cell clone migration. Day 7 helper (clone 199.1 1) or cytotoxic (CTL, clone 199-4) cells were washed 3x and resuspended in medium (0.25% BSA-RPMI) with the indicated conccntration of PGEz with or without PMA (0.01 pglml). Cells were allowed to migrate for 60 min in a modified Boyden chamber containing a 5 micron pore filter. Cell viability (assessed by trypan blue exclusion) exceeded 95% after 60 niin incubation in the highest concentration of each additive. Data represent the mean from 3 filters. *, p < 0.01 v e r s u ~medium alone: **, p < 0.01 versus the corresponding concentration of PGEz alone.

338

MARK L. JORDAN

Further evaluation of the role of second messengers (including [CaZ+],)in the subset-specific effects mediated by PGE, on T cell function will help to further clarify these mechanisms. Although PGE, is only one factor that may regulate T cell function, its distinct effects on T, but not CTL may be a powerful mechanism by which the inflammatory response may confer additional specificity to the immune response.

B. Lipoxygenase Products In contrast to the copious literature on prostaglandins and the immune response, the role of AA lipoxygenase (LO) products in immunoregulation is still not well understood. Much of the early literature on LO metabolism focused on leukotriene B, (LTB,) as a potent inflammatory mediator, promoting neutrophil adherence, aggregation, lysozomal enzyme release, and chemotaxis (Goetzl and Pickett, 1980). More recently, LTB, has been found to enhance human monocyte IL- 1 production (Rola-Pleszczynski and Lemaire, 1985). By comparison, the effects of LO metabolites on T cell activation and function are not yet clear. Several lines of evidence indicate that LTB, stimulates T suppressor cells. Payan et al. (1984) reported that LTB, enhanced activation of OKT8+ cells. RolaPleszczynski et al. (1982) found that low levels (10- M to 10- M ) of LTB, induced human suppressor cell activity, with no effects at higher concentrations. LTD, had no such effects. Further studies indicated that suppressor activity on ConA-induced mononuclear cell proliferation required the presence of monocytes, was inducible by preincubation of T cells with LTB,, and could be reversed to enhance proliferation by indomethacin (Rola-Pleszczynski, 1985). ’This suggested a role for monocytes and cyclooxygenase products in LTB,induced suppressor activity. T4+ cells preincubated with LTB, required the subsequent presence of monocytes to exert suppressor effects, whereas T8+ cells were active even in the absence of rnonocytes. It was also suggested that T8cells could be induced to become phenotypically T8 + by prolonged incubation with LTB,. Reversal of this suppressor effect into a “helper” phenomenon in the presence of CO inhibition (Rola-Pleszczynski, 1985) was consistent with subsequent observations that LTB, augmented monocyte 1L 1 production (RolaPleszczynski and Lernaire, 1985), which is known to be endogenously downregulated by monocyte PGE, synthesis (Kunkel et ul., 1986). LTB, has also been reported to enhance natural killer (NK) cell activity (RolaPleszczynski et ul.. 1983). Johnson and Torres (1984) have shown that LTB,, LTC,, and nanomolar concentrations of AA induce gamma interferon (IFN-y) production by murine lymphocytes. This has also been confirmed in human T cells (Roia-Pleszczynski et al., 1986). Evidence also indicates that LTB,-pulsed human T4+ cells modulate monocyte IL-I production by secreting IFN-y,

13. ARACHIDONIC ACID METABOLITES IN LYMPHOCYTES

339

(Rola-Pleszczynski et ul., 1987). Thus, depending upon the responding T cell population, LTB, can exert either positive or negative effects. Our laboratory has found that LTB, enhances the locomotion of a variety of murine T cell clones (helper, cytotoxic, and helper-independent cytotoxic) over a narrow (0.1- I ng/ml) concentration range (Jordan et al., 1986a). In subsequent studies, we demonstrated that LTB, also potentiates both IL-2 and secondary mixed leukocyte culture supernatant (2" MLC SN) dependent proliferation of these clones (Jordan el al., 1986b). In contrast to our previously observed subset-specific effects of PGE, (Jordan rt al., 1986a), LTB, was stimulatory regardless of lymphocyte clone effector function. The use of T cell clones abrogates many of the problems inherent in studying the functions of bulk lymphocyte populations (such as those described previously), in which overall responses cannot be directly attributed to specific lymphocyte subsets, other than by selection or depletion techniques, which invariably result in some contamination by other cells (such as monocytes) with CO or LO synthetic capacity. In further work, we have been unable to detect any in vitro immunoregulatory effects of a variety of other LO products, including LTC,, LTD,, LTE,, 5- and 15-hydroperoxyeicosatetranoic acids (HPETEs), or 5 - and 15-hydroxyeicosatetranoic acids (HETEs) on T cell clone function. Payan and Goetzl (1983) also reported that LTC,, LTD,, and LTE, lacked effects on T cell activity. 5 - , 8-, 9-, 1 I-, and 12-HPETEs were found to suppress murine splenocyte responses to PHA or ConA by Gualde et ul. (1983); however, this was thought to occur by incorporation of the HPETEs into the lymphocyte cell membranes with subsequent loss of membrane conformational change induced by ConA. 15HPETE has been reported to inhibit the mitogen response of bulk human T cell populations and OKT4+ cells but to stimulate OKT8+ cell proliferation (Gualde et al., 1985). Despite its stimulatory effects on activated murine T cell clones, we have found no effect of LTB, on unactivated murine T cells, both in assays of lymphocyte locomotion (Jordan et al., 1986a) and mitogen-induced proliferation (Jordan et al., 1986b). These data suggest that cellular activation may be necessary before sensitivity to LTB, is acquired. Although its mechanisms of action on T cell function are as yet unclear, LTB, has been reported to act as a calcium ionophore (Serhan et al., 1982), to stimulate 1L-2 production by T4+ cells (Rola-Pleszczynski et al., 1986), to be necessary for mitogen-induced T cell proliferation (Goodwin et al., 1986), and to bind to receptors on different subsets of human T cells (Payan et al., 1984). Whether LTB, is an endogenous regulator of T cell activation has been the subject of some controversy. This is because (1) most reports have provided only indirect evidence for the role of LTs in T cell activation by using blockers of the LO pathway and ( 2 ) there is a lack of confirmation whether lymphocytes themselves are capable of synthesizing LTs. This will be addressed in more detail later.

340

MARK L. JORDAN

111.

LYMPHOCYTE SYNTHESIS OF ARACHIDONIC ACID METABOLITES

Because of the potent immunoregulatory properties identified for a variety of AA metabolites, it is crucial to specifically identify their cellular sources as accurately as possible if we are to understand and possibly therapeutically modify their role in the immune response. Unfortunately, for some of the reasons mentioned earlier, it has been cxtremely difficult to ascertain whcther or not lymphocytes are truly capable of AA metabolism.

A. Cyclooxygenase Products Although earlier studies suggested the presence of an active cyclooxygenase in lymphocytes (Ferraris and DeRobertis, 1974; Webb and Osheroff, 1976; Goodwin et al., 1977), most investigators now agree that contamination by nonlymphocyte populations accounted for these earlier observations. Many subsequent studies of separated lymphocyte populations and lymphocyte cell lines have uniformly failed to detect lymphocyte-derived CO products. Synthesis of 6keto-PGF,,,, PGE,, and PGF,, by fetal murine thymic cell cultures has recently been reported to affect endogenous regulation of thymic growth and Thy-I expression (Shipman et ul., 1984); however, these studies were done with whole organ homogenates and definite conclusions regarding thymocyte CO activity cannot be drawn. Abraham et al. ( 1 986) reported that an appropriately stimulated T cell line (HT-2) generatcd PGF,, PGE,, and PGD, and that this could be blocked by indornethacin.

6. Lipoxygenase Products The capacity of lymphocytes to synthesize Lo products is still subject to debate. Lymphocytes do contain esterified AA which can be released as free AA upon appropriate stimulation (Goldyne, 1984); this could provide nearhy monocyte-macrophages or even other lymphocytes with substrate from which biologically active products may be synthesized. Whether lymphocytes themselves metabolize AA to LO products has been unresolved, generally because of the difficulty in completely removing contaminating platelets, neutrophils, and monocytes from the ccll preparations. The use of T cell lines, highly purified T ccll preparations, or 'I' cell clones could potentially resolve this controversy. Poubelle et al. (1987) have studied human peripheral blood rnononuclear cells purified by counter flow elutriation to remove contaminating monocytcs, neutrophils, and platelets (99% pure lymphocytes by flow cytometry). No L'I'B, could be detected after stimulating these cells with calcium ionophore ( 1 pM), exogenous AA ( 5 FM),or PHA (5 kg/ml). The same authors were unable to detect LTB, synthesis (using RIA or reverse phase HPLC) by murine thy-

13. ARACHIDONIC ACID METABOLITES IN LYMPHOCYTES

341

mocytes, IL-2-dependent CTLL2 cytotoxic T cells, EL4 thymoma cells, or human Jurkat cells after exposure to several stimuli including calcium ionophore A23187, phytohemagglutinin, phorbol esters, IL-I, or IL-2, either in the presence or absence of exogenous AA. Other studies have reported LT production from distinct T cell populations. Ambrus et al. ( 1 988) produced a human T8 + TT hybridoma by fusing normal T cells with a non-LT-producing T4+ lymphoma line. Supernatants from cloned hybridoma cells were then tested for their ability to suppress immunoglobulin (Ig) production by pokeweed mitogen (PWM)stimulated normal human PBMC. Of 180 clones, 3 were found to suppress Ig production (both IgG and IgM). When added to the clone culture media, hydrocortisone, but not indomethacin, inhibited the generation of this suppressor activity. LTC, (3-10 ng/ml) was detected in the suppressor hybridoma clone supernatant but not in nonsuppressor hybridoma supernatant. No LTB,, PGE,, PGF,,, or PGF,, was detected in any supernatants. Anti-LTC, antisera removed the suppressor activity from the supernatant. Synthetic LTC, at 5 ng/ml produced equal amounts of suppression of Ig production as the native supernatant, and the suppressive activity derived from the supernatant eluted from reverse phase HPLC at the same retention times as synthetic LTC,. Another study using an IL-2-dependent cell line (HT-2) stimulated with the bee venom peptide melittin identified both CO (PGF,,, PGE,, and PGD,) as well as LO ( 5 ,12-, 15HETE) production by these cells (Abraham et al., 1986). However, no detectable CO or LQ production occurred when these cells were stimulated with calcium ionophore. Furthermore, when exogenous phospholipase A, activity was removed from the melittin by heat inactivation, very little eicosanoid production occurred. However, these experiments showed that once provided with an appropriate stimulus for AA release, HT-2 cells were fully capable of synthesizing a spectrum of eicosanoids. In the preceding studies, therefore, only transformed human T cells (Ambrus et al., 1988) and an IL-2-dependent murine T cell line (Abraham et ul., 1986) were found to synthesize eicosanoids. Mitogen stimulation of a variety of “normal” T cell populations failed to produce detectable AA metabolites (Poubelle et ul., 1987). Whether normal T cells produce eicosanoids is therefore still open to question. We have derived a series of allosensitized C57BL/6 (H-2b) anti-DBA (H-2d) murine T cell clones which have been phenotypically and functionally defined as possessing helper or cytotoxic function (Table 1). By treating these clones with anti-H-2Kd plus complement (to eliminate any residual DBA stimulator cells), we have preliminary evidence that some of these clones synthesize small, but detectable, amounts of LTB, in response to calcium ionophore, as determined by radioimmunoassay (Table 11). No PGE, was detected. LTB, was synthesized by both resting (nonproliferative phase) helper and cytotoxic clones 7 days after restimulation with IL-2 and antigen. These data support the concept that endogenous LO products may be important regulators of T cell alloactivation. However, more direct evidence for the involvement of AA metabolites in T

342

MARK L. JORDAN TABLE I FIINCTIONAI C I i A K A C I k l l l S I K S OF C57BLi6 ANTI-DBAI2 LYMPHOCYTF Cl ONTS" Proliferation in response

Cytotox ic I ty

to irradiated splenocytesh

@)

C57BLi6 (H-29

Clone

DBAi2 (H-2")

EL4 (H-Zh)

PX15 (H-2d)

Phenotype"

Helper 805 2 68 467 2 116 130 ? 17 175 + 106 597 2 58 176 t 42

55,980 5 6,409 15,627 2 2,859 72,261 2 8,800 65,065 t 1,287 85,994 2 9,660 135,635 5 2.330

319 t 17 238 2 21 311 t I0

671 -C 5

176-13 199-4

199-8

125

176-24 190-5 199-5 199-9 199-18 199-11

I 0

0 0

0 0

0 I -3

2 3 2

Lyt I + 2Lyt 1+ 2 L3T4 Lyt I + 2 - , L3T4 not tested

Lyt I + 2-, L3T4

cytotoxic

193-7

2

28

Cytotoxic (Icctin only) 195-9 204 2 32

Helper-independent cytotoxic 184-13 244 k 53

477

2

120

1,082 5 109

233 t 106 784

2 70

I 8 0 0 6 (no PHA) 59 (PHA)

71 66

74 65

Lyt 1 2' LYt 2 i , L3T4 LY t - 2 + , L3T4 LYt 2 + , L3T4 ~

~

3 (no PHA) Lyt

2+

(''HIT') 7,261 2 217

0

68

Lyt 1

2'

Clones are derived hy limiting dilution analysis (0.25 celliwell) of cells from ;L CS7BLi6 anti-DBAi2 rnixcd leukocyte culture (day 5 8 of culture). b I X 104 lyinphocytc\ are cultured in the presence of I X 10" irradiated (2000R) qplenocytes without exogenoub growth factors. At 48 hr 2 pCi of 3HTdR arc addcd per well and thymidine uptake determined after a 6.5 hr incuhatinn (mean ? S.D. ot triplicate wells). I.ymphiKytcs are incuhared with 2 X IO? "Cr~labeledtumor targets at I W : 1 (effector. target ratio) for 3 5 hr. after which the % 5 0 r e l e a s e isdetermined. Cliincs 176-24. 190-5. 199-15, IYY-9, IY9-8, and 199-11 are also not cytotoxic in the presence of Iectin. Lymphocytes are incubated with monoclonal Thy 1.2, Lyt 1.2, or Lyt 2 . 2 antisera, washed, and incubated with Iluorescein-ciinjugated IgG fraction of goat anti-mouse immunoglobulin. B, fluorescence is determined with a FACS IV Fluoresccnce levels helow 10% are arbitrarily considered negative. Where indicated. thc 13T4 phenotype has also been determined.

cell activation and function is necessary before any valid conclusions may be drawn.

IV. THE EFFECTS OF LIPOXYGENASE INHIBITORS ON LYMPHOCYTE ACTIVATION AND FUNCTION Thus far, the evidence for eicosanoid involvement in T cell activation and function is indirect. This is because most studies have relied on the use of

LTB,

AND

TABLE I1 PGE, PRODUCTION BY T LYMPHOCYTE CLONESO Treatment

LTB, (pgIlO6 cells -t SEM) Clone

None

199-1 1 (Helper) 199-15 (Helper) 199-4 (CTL) 199-8 (CTL) C57BL/6 PEC's (H-2Kh) DBAI2 PEC's (H-2Kd)

7.2 t 0.7 10.1 -+ 0.8 5.9 t 0.5 8.4 ? 0.6 4.4 t 0.2 3.0 ? 0.3

anti-H-2Kd + CaI

CaI 17.8 t 24.6 t 12.7 t 18.9 ? 7.8 ? 5.0 ?

PGE, (pg/106cells 2 SEM)

2.2 3.0 1.5 2.1 2.2 0.9

15.6 ? 22.4 t 13.0 2 19.4 ? 8.0 ? 0.2 -c

1.4 2.8 1.2 1.8 1.4 0.1

Cal

0

0

0

0

0

0 0

0 0

0 0

24.6 ? 2.5 31.5 2 2.8

25.7 t 3.0 1.2 2 0.4

12.8 18.2

?

1.0

&

1.7

anti-H-2Kd

+ Cal

None

0

Lymphocyte clones (C57BL/6 anti-DBA/2) were freshly harvested from culture, washed 3 X in RPMI-1640, then passed over Lympholyte-M separation medium (Cedarlane Inc.) with over 85% recovery. After 3 further washes, lymphocytes were treated twice with complement with or without monoclonal rat anti-mouse anti-H-ZKd (IgM, Litton, Inc.). The specificity of the monoclonal antibody was tested against C57BLi6 and DBAI2 splenocytes in a separate assay (not shown). The lymphocytes (greater than 90% recovery after MOAB) were then incubated in RPMI for 30 min at 37°C in 5% C 0 2 with or without 2 p,M calcium ionophore A23187 (Cal). Peritoneal exudate cells (PEC's) (over 95% macrophages by esterase staining) from C57BLi6 (H-2Kh) or DBAi2 (H-2Kd) mice were treated identically and used as controls for the LTB, and PGE, production and monoclonal antibody treatment. LTB, and PGE, content of the centrifuged cell-free supernatants were determined directly by radioimmunoassay (Seragen, Inc.). Results are mean ? SEM of three experiments. Q

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inhibitors of AA metabolism without simultaneous study of the metabolic pathways in question. For example, various nonspecific LO inhibitors have been found to inhibit several T cell functions, including proliferation (Leung et a l . , 1982), IL-2 and IFN-y production (Farar and Humes, 1985), and locomotion (Jordan er a / ., 1987~). Dornand et af. (1987) studied the effccts of LO inhibition on 1L-2 production and intracellular signaling in Jurkat and El4 cell lines. Nordihydroguaiaretic acid (NDGA), butylated hydroxyanisole (BHA), and caffeic acid (all nonspecific LO inhibitors) inhibited PHA- or anti-CD3-induced 1 L 2 production by PMA-treated Jurkat cells. NDGA and BHA also suppressed PHA- or anti-CD3-triggered [Ca2+Ii increases in Jurkat cells at concentrations that suppressed I L 2 production from these cells; however, the LO inhibitors had no effect on changes in [Ca2 Ii induced by exogenous calcium ionophore A23 187. LO inhibitors also had no effect if added after PHA- or A23187-induced increases in [Ca2+J i had occurred. A23 187-induced IL-2 production by PMA-treated Jurkat cells was also blocked by NDGA and BHA, but at higher concentrations than those required for PHA- or anti-CD3-induced 1L-2 production. IL-2 synthesis by the EL4 cell line could be induced by PMA alone, but this synthesis, unlike that for Jurkat cells, was not accompanied by an increase in lCaZ+ li. LO inhibitors suppressed PMAinduced IL-2 production by the EL4 cells. Since LO inhibitors suppressed A23 187-induced Jurkat IL-2 synthesis without affecting 1Ca2+Ii, it was suggested that LO inhibitors might interact at the level of PKC. This is supported by the fact that PMA-induced EL4 IL-2 production, which does not require an increase in [Ca2 li, was also blocked by LO inhibition. Although the evidence is still indirect, these studies suggest that endogenous LO metabolism may be required for T cell activation and IL-2 synthesis, perhaps via a PKC-dependent mechanism. We have studied the effects of in vivo systemic administration of NDGA on the in vivo generation of ( 1 ) allospecific and natural killer (NK) effector cells and (2) AA metabolite synthesis from sponge matrix allografts in mice (Jordan e t a / . , 1987d). In control animals, both allospecific and NK activity increased progressively up to 12 days after grafting. At 12 days, cells derived from the sponge allografts synthesized both LTB, and PGE,. Recipient NDGA treatment (50 mg/kg/day) impaired both NK activity and development of allospecific cytotoxic cells within the graft. Simultaneously, sponge derived cells from recipients of NDGA produced significantly less LTB, (55 +- 10 pg/lOh cells, p < 0.01) when compared to control animals ( I 196 ? 30 pg/106 cells). However, NDGA treatment did not affect sponge cell synthesis of PGE, (340 lr 25 vs. 255 f 22 pg/ loh cells, p = NS). This suggests that NDGA was acting as a specific LO inhibitor and was also an effective immunosuppressant in this model. Our laboratory has early evidence that PMA (a potent stimulator of PKC) enhances the in vitro locomotion of unactivated murine lymphocytes. Furthermore, both PMA and LTB, stimulate the locomotion of allosensitized T cell clones +

+

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(regardless of effector function), and both PMA and LTB, partially abrogate the inhibitory effects of PGE, on T, locomotion. These data may indicate that LTB, may act via PKC and suggest that the inhibitory effects of PGE, o n helper function may be mediated by down-regulation of PKC. Further work is currently in progress in our laboratory to determine whether endogenous eicosanoids are important in T cell activation and function and whether LO inhibition may be an effective approach to immunosuppression.

V.

CONCLUSIONS

The specific role assumed by arachidonic acid metabolites in the process of lymphocyte activation is still unclear. Evidence is accumulating that these mediators may exert positive and negative effects on lymphocyte function, depending upon the source of the mediator (CO vs. LO pathways), the nature of the activating lymphocyte agent, and the particular lymphocyte subset in question. Since eicosanoids are known to be released in quantity by several other cell types involved in the inflammatory response, there is great potential for significant immunoregulation by these metabolites. Inhibition of LO metabolism is an intriguing selective approach to immunosuppression that may have clinical application. The existence of endogenous lymphocyte eicosanoid synthesis, and whether this is important in lymphocyte activation, has not been fully elucidated. Further studies using specific eicosanoid synthesis inhibitors and antagonists in purified lymphocyte populations will help to critically evaluate the significance of endogenous eicosanoid metabolites in the immune response. REFERENCES Abraham, R. T., McKinney, M. M., Forray, C., Shipley, G . D., and Handwerger, B. S . (1986). Stimulation of arachidonic acid release and eicosanoid biosynthesis in an interleukin 2-dependent T cell line. J . Immunopharmacol. 8, 165-204. Aderem, A . A,, Cohen, D. S., Wright, S. D., and Cohen, Z. A. (1986). Bacterial lipopolysaccharides prime macrophages for enhanced release of arachidonic acid metabolites. J . Exp. M e d . 164, 165-179. Ambrus, J. L., Jr., Jurgensen, C. H.. Witzel, N. L.. Lewis, R. A , , Butler, J. L., and Fauci, A. S. (1988). Leukotriene C4 produced by a human T-T hybridoma suppresses Ig production by human lymphocytes. J . Immunol. 140, 2382-2388. Chouaib, S., Robb, R. J., Welte. K . , and Dupont, 6 . (1987). Analysis of prostaglandin E2 effect on T lymphocyte activation. J. Clin. Invest. 80, 333-340. Domand, J . , Sekkat, C . , Mani, J. C., and Gerber, M. (1987). Lipoxygenase inhibitors suppress IL-2 synthesis: relationship with rise of [Ca+ + 1, and the events dependent on protein kinase C activation. Intmunol. Lett. 16, 101-106. Farrar, W. L . , and Humes, J. L. (1985). The role of arachidonic acid metabolism in the activities of interleukin I and 2. J . Immunol. 135, 1153-1 159. Ferrdris, V. A , . and DeRobertis, F. F. (1974). Release of prostaglandins by rnitogen and antigen stimulated lymphocytes in culture. J . Clin. Invest. 54, 378-386.

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Goetzl, E. J. (1983). Leukocyte recognition and metabolism of leukotrienes. Fed. Proc. 42, 31283131. Goetzl, B. J., and Pickett, W. C. (1980). The human PMN leukocyte chemotactic activity of complex hydroxyeicosatetranoic acids (HETEs). 1. Immunol. 135, 3958-3961 . Goldyne, M. E. ( 1984). Meterogeneity in lcukocyte preparations: Effects on defining eicosanoid metabolism by human lymphocytes and monocytes. J. Allergy Clin. Immunol. 74, 331-337. Goodwin, J. S . , and Ccuppens, J. (1983). Spccial article: Regulation of the immune response by prostaglandins. J. Clin. Immunol. 3, 295-3 15. Goodwin, J. S . , Bankhurst, A. D., and Mcssner, R. P. (1977). Suppression of human T-cell mitogencsis by prostaglandins. Existence of a prostaglandin-producing suppressor cell. J . Exp. Med. 146, 1719-1734. Goodwin. J. S . , Atluru, D., Sierakowski, S . , and Lianos, E. A. (1986). Mechanism of action of glucucorticord-induced immunoglobulin production: role of lipoxygenase metabolites of arachidonic acid. J. Clin. Invest. 77, 1244-1250. Gualde, N., Chable-Rabinovitch, H., Motta, C . , Durand, J., Beneyout, J. L.. and Rigaud, M. ( I 983). Hydroperoxyeicosatetraenoic acids potent inhibitors of lymphocyte responses. Biuchim. Biophys. Acto 750, 429-433. Gualde, N., Atluru, D., and Goodwin, J. S . (1985). Effect of lipoxygenase metabolites of ardchidonic acid on proliferation of human T cells and T cell subsets. J. Immunol. 134, 1125- 1 129. Johnson, H. M., and Torres, B. A. (1984). Leukotrienes, positive signals for regulation of gamma interferon production. J. Immunol. 132, 413-416. Jordan, M . L., Hoffman. R. A,, Debe, E. F., and Simnions, R. L. (1986a). In vitro locomotion of allosensitized T lymphocyte clones in response to metabolites of arachidonic acid is subset specific. J. Immunol. 137, 661-668. Jordan, M. L., Hoffman, R. A , , and Simmons, R. L. (1986b). Leukotrienc B4 (LTB) augments IL2dependent proliferation of T lymphocyte clones. Transplan/. Proc. 18, 224-227. Jordan, M. L., Hoffman, R. A . , Debe, E. F., West, M. A., and Simmons, R. L. (1987a). Prostaglandin E2 mediates subset-specific effects on the functional responses of allosensitized T lymphocyte clones. Transplantation 43, 117-123. Jordan, M. L., Hoffman, R. A., and Simmons, R. L. (l987b). Further characterization of the subsctspecific effccts of prostaglandin E, on T lymphocyte clone function. Transplant. Proc. 19, 307309. Jordan, M. L., Hoffman, R. A., and Simniona, R. L. (1987~).Thc role of the lipoxygenase pathway in T lymphocyte clone function. Transplanr. Proc. 19, 333-334. Jordan, M. I . . , Carlson, A,, Hoffman. R. A , , and Simmons, R . L. (1987d). Lipoxygenase pathway inhibition impairs the allograft rebponse. Surgery 102, 248-255. Kennedy, M. S . , Stobo, I. D., and Goldyne, M. E. (1980). In vitro synthesis of prostagLandins and related lipido by populations of human peripheral blood mononuclear cells. Prosragfundins 20, 135- 145. Chensue, S. W., and Phan, S . H. (1986). Prostaglandins as endogenous mediators of Kunkel, S. I>., intcrlcukin I production. J . Immunol. 136, 186-192. Leung, K . H., Ehrke, M. J., and Mihick, E. (1982). Modulation of the development of cell mediated immunity: possible role of the products of the cyclo-oxygenase and the lipoxygenase pathways of arachidonic acid metabolism. Inr. J. Immunopharmarol. 4, 194-204. Payan, D. G., and Coetzl, E. J . (1983). Specific suppression of human T lymphocyte function by leukotriene R4. J. Immunol. 131, 551-5.53. Payan, D. G . , Missirian-Bastian, A., and Goetzl, E. J. (1984). Human T-lymphocyte subset specificity of the regulatory effects of leukotriene B4. lmmunofogy 81, 3501-3505, Poubelle, P. E., Borgeat, P., and Rola-Pleszczynski, M. (1987). Assessment of leukotriene B4

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synthesis in human lymphocytes by using high performance liquid chromatography and radioimmunoassay methods. J Immunol. 139, 1273- 1277. Rola-Pleszczynski, M. (1985). Differential effects of leukotrienc B4 on T4+ and T8+ lymphocyte phenotype and immunoregulatory functions. J . Immunol. 135, 1357- 1360. Rola-Pleszczynski, M.. and Lemaire, 1. (1985). Leukotrienes augment interleukin 1 production by human monocytes. J . Immunol. 135, 3958-3961. Rola-Pleszczynski, M., Borgeat, P., and Siroia, P. (1982). Leukotriene B4 induces human suppressor lymphocytes. Biochem. Biophys. Res. Commun. 108, 1531- 1537. Rola-Pleszczynski, M., Gagnon, L., and Sirois, P. (1983). Leukotriene B4 augments human natural cytotoxic cell activity. Biochem. Biophys. Res. Commun. 113, 531-537. Rola-Pleszczynski, M . , Chavaillaz, P. A , , and Lemaire, 1. (1986). Stimulation of interleukin 2 and interferon gamma production by leukotriene B4 in human lymphocyte cultures. Prosruglandins Leukotrienes Med. 23, 207-210. Rola-Pleszczynski, M., Bouvrette, L., Gingras, D., and Girard, M. (1987). Identification of interferon-gamma as the lymphokinc that mediates leukotriene B4-induced immunoregulation. J . Immunol. 139, 513-517. Serhan, C. N . , Fridovich, J . , Goetzl, E. J., Durham, P. B., and Weissman, G. (1982). Leukotriene B4 and phosphatidic acid are calcium ionophores. Studies employing arsena 111 in liposomes. J. B i d . Chem.257, 4746-4752. Shipman, P. M., Schmidt, R. R., and Chepenik. K. P. (1984). Relation between arachidonic acid metabolism and development of thymocytes in fetal thymic organ cultures. J. Immunol. 140, 2714-2720. Stobo, J. D., Kennedy, M . S . , and Goldyne, M. E. (1979). Prostaglandin E modulation of the mitogenic response of human T cells. Differential response of T cell subpopulations. J. Cfin. Invest. 64, 1188-1203. Strom, T. B., Deisseroth, A , , Morganroth, J., Carpenter, C. B., and Merrill, J. P. (1972). Alteration of the cytotoxic action o f sensitized lymphocytes by cholinergic agents and activators of adenylate cyclase. Proc. Nut/. Acad. Sci. U.S.A. 69, 2995-2999. Ting, C. C . , and Hargrove, M. E. (1984). Regulation of the activation of cytotoxic T lymphocytes by prostaglandins and antigens. J . Immunol. 133, 660-666. Webb, D. R., and Osheroff, P. L. (1976). Antigen stimulation of prostaglandin synthesis and control of imniune responses. Proc. Nay/. Acud. Sci. U.S.A. 73, 1300-1304. Wolf, M., and Droege, W. (1982). Inhibition of cytotoxic responses by prostaglandin E2 in the presence of interleukin 2. Cell. Irnmunol. 72, 286-293.

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CURRENT TOPICS IN MEMBRANES AND 'TRANSPORT. VOLUME 35

Chapter 74

Mechanisms Regulating the Production of Arachidonate Metabolites in Mononuclear Phagocytes RONALD J . UHING, * MATTHEW S. COWLEN," AND DOLPH 0. ADAMS*f Departments of ?Microbiology, flmmunology, and *Pathology Laboratory of Cell and Moleculur Biology of Leukocytes Duke University Durham, North Carolina 27710

I.

Introduction Involvement of Eicosanoid Production in Host-Defense Mechanisms Biochemical Mechanisms Involved in Eicosanoid Production A. Phospholipase Az B. Diacylglycerol Lipase C. The Cyclooxygenase Pathway of Arachidondte Metabolism D. The 5-Lipoxygenase Pathway of Arachidonate Metabolism E. Regulation of Eicosanoid Production by Anti-Inflammatory Drugs IV. Potential Transductional Mechanisms Involved in the Stimulation of Macrophage Eicosanoid Production V. Platelet Activating Factor as an Autocrine Component of Eicosanoid Production VI. The Relationship of Eicosanoid Production to Macrophage Development VII. Summary References 11. 111.

1.

INTRODUCTION

The cellular metabolism of arachidonate results in an increase in both the intracellular concentration and the secretion of arachidonate and the subsequently formed eicosanoids. Arachidonate metabolism is stimulated in a variety of cells in response to external stimuli. The metabolized arachidonate is either derived 349

Copyright 0 1990 by Academic Press. Inc. All nghts of reproduclion In any form reserved.

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RONALD J. UHING ET AL.

from cellular glycerolipids or acquired from the extracellular environment. An extracellular role for various eicosanoids has been indicated from a variety of data, including the identification of spccific cell surface receptors. A potential intracellular role has been most clearly described for arachidonatc as an activator of protein kinase C. The rcalization of the importance of stimulated eicosanoid production for extracellular signalling resultcd from studies of its importance as a mediator of inflammation. Eicosanoid production has been indicated to be involved in the actions of a variety of circulating cells. Of particular importance is the suggested involvement of eicosanoid production by infiltrating phagocytic leukocytes at sites of inflammation. Specific importance for stimulated eicosanoid production by mononuclear phagocytes has been indicated from studies of several inflammatory disorders. In addition, mononuclear phagocytes have been demonstrated to contain more esterified arachidonate in comparison with other leukocytes, with a large amount apparently available for stimulated eicosanoid production. This review summarizes current information on transductional sequences involved in stimulated eicosanoid production in mononuclear phagocytes. Due to our own interests, particular emphasis is placed on studies in murine peritoneal macrophages.

II. INVOLVEMENT OF EICOSANOID PRODUCTION IN HOST-DEFENSE MECHANISMS The mobilization of arachidonate and its metabolism into biologically active eicosanoids are intimately involved in the initiation and regulation of immune system responses. Prostaglandins and leukotricnes act as autocrine and paracrine agents that modulate the function and activity of the cellular components of the immune system, including lymphocytes, polymorphonuclear leukocytes, and mononuclear phagocytes (Davies et ul., 1984; Lagarde et a / ., 1989; Bonney and Davies, 1984). These eicosanoids also contribute to the manifestation of numerous physiological effects associated with inflammation, such as erythema, fever, pain, and edema (Davies et ul., 1984; Goldstein, 1988); influence the development of inflammatory diseases, such as rheumatoid arthritis (Harris, 1988), asthma (Barnes et al., 1988; Bigby and Nadel. 1988), gout (Gordon et al., 1988), and adult respiratory distress syndrome (Simon and Ward, 1988); and contribute to tissue injury associated with hydronephrosis and myocardial infarction (Spaethe and Needleman, 1987). Many therapeutic agents used to treat inflamtnatory disorders act by inhibiting the mobilization of arachidonate and the formation of eicosanoids. Arachidonate is stored almost exclusively as an ester in the sn-2 position of glycerophospholipids in the membranes of unstimulated cells (Irvine, 1982).

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Stimulation of these cells by agents such as calcium mobilizing agonists results in the release of arachidonate and initiates the cascade of events that leads to the production of biologically active metabolites of arachidonate. The most common mechanism involved in the liberation of arachidonate appears to be the activation of phospholipase A,, which hydrolyzes arachidonate from membrane phospholipids (Irvine, 1982). A second mechanism involves the phosphodiesterasemediated formation of diacylglycerol, which then serves as a source of arachidonate through the action of diacylglycerol lipase (Irvine, 1982). Regardless of the source, free arachidonate can be converted to biologically active eicosanoids by cyclooxygenase and lipoxygenase pathways (Fig. 1). Products of the cyclooxygenase pathway include prostaglandins and thromboxanes, whereas products of the lipoxygenase pathway include leukotrienes and peptidoleukotrienes. Cyclooxygenase products were first recognized as constituents of rabbit aorta contracting substance, which was shown to contain prostaglandins (Svenson et ul., 1975; Piper and Vane, 1969). Lipoxygenase products were first described as the slow reacting substance of anaphylaxis, which was found to be a mixture of peptidoleukotrienes (Morris et ul., 1980; Lewis et al., 1980). Among mononuclear phagocytes, mouse peritoneal macrophages produce primarily prostaglandin E,, prostaglandin I, ( prostacyclin), which is detected as 6-ketoprostaglandin F,,, and leukotriene C, (Humes et al., 1985; Riches et al., 1988; Bonneyet al., 1985). Mouse peritoneal macrophages also produce leukotriene B, and thromboxane A,, which is detected as thromboxane B, (Riches et al., 1988; Bonney et al., 1985; Bonney and Humes, 1984). Human monocytes also produce prostaglandin E, and leukotrienes B, and C,, as well as thromboxane A, and prostaglandin F,, (Riches et al., 1988). The specific arachidonate metabolites produced by mononuclear phagocytes are dependent on the particular external stimuli to which the cells are exposed. Immunological responses are tightly regulated by a complex system of immunogenic and immunosuppressive compounds that are produced by, and act on, the cells of the immune system. Eicosanoids are important components of this cell-cell communication system. For example, leukotriene B, can be produced by stimulated neutrophils and macrophages and induces chemotaxis of these cells, thereby augmenting the infiltration of both polymorphonuclear and mononuclear phagocytes to the site of inflammation (Davies et al., 1984; Goldstein, 1988; Harris, 1988). In contrast, E-series prostaglandins exert anti-inflammatory effects on leukocytes, including mononuclear phagocytes, such as inhibition of chemotaxis, decreased adherence to endothelium, inhibition of phagocytosis, and attenuation of the oxidative burst (Goldstein, 1988). The cell-cell interaction facilitated by eicosanoids also includes transcellular metabolism. Arachidonate and eicosanoids can serve as biochemical precursors to be further metabolized to active eicosanoids by other cells of the same type or of a different type (Lagarde et ul., 1989; Marcus, 1988). For example, mac-

Prostaglandi n Endoperoxide Synthose

[ (Cycloorvgenose) J. Prostaglandin

,

5-Liporygenose

ARACHIDONATE

J.

GP

5-Hydroperoxyetcosotetroenoic

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I Prostoglandin Endop&roxide

I

5 - t I pox ygenose (5-HPETE Dehvdrase. LTA, Synthosd

Synthose

Prostoglandin

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Ismrase

Prostaglandin H,

Endoperoxide

Prostaglandin E2

E

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Acid (5-HPETE)

Thrornboxone Synthose

+Thrombaxone

.1

Prosto~londln Endoperoxide Reductow

Prostaglandin I, (Prostacylin)

I

Prostaglandin

A,

Thrmboxone

Fza

Leukotriene A4

B, Hydrolose Leukotriene

54

Leukotriene C4 Synthose [Glutathlone tmnsfemse) Leukotriene C4

4 6- keto-Prostoglondin F,,

FIG. 1. Cyclooxygenase and 54ipoxygenase pathways for the metabolism of arachidonate. A schematic representation of suggested substrates, enzymes, and products involved in eicosanoid formation in mononuclear phagocytes

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rophages have been reported to synthesize eicosanoids from arachidonate derived from T lymphocytes (Goldyne and Stobo, 1983) and platelets (Smith et al., 1987). Eicosanoids can also modulate arachidonate release and metabolism by activating receptors that are coupled to regulatory transmembrane signaling pathways (Lagarde el al., 1989; Marcus, 1988). For example, leukotriene C , stimulates arachidonate mobilization and prostaglandin formation in macrophages (Bonney and Davies, 1984). These and other autocrine, paracrine, and metabolic functions of eicosanoids are involved in the ability of the immune system to mount an effective and tightly regulated response to pathogenic microorganisms, tumor cells, and tissue injury.

111.

BIOCHEMICAL MECHANISMS INVOLVED IN EICOSANOID PRODUCTION

The initial and rate limiting step in the formation of cyclooxygenase and lipoxygenase products is the liberation of arachidonate from the sn-2 position of membrane glycerolipids. Although the identity of the enzymes responsible for arachidonate release for eicosanoid formation in response to inflammatory stimuli is uncertain, phospholipase A,- and diacylglycerol lipase-dependent arachidonate release have becn suggested for mouse peritoneal macrophages.

A. Phospholipase A, Several distinct phospholipase A, activities have been reported in peritoneal macrophages. One has a pH optimum of 4.5 and is calcium independent (Wightman et al., 1981). This phospholipase A, activity appears to be associated with lysosomes and is secreted from macrophages in response to an appropriate stimulus such as zymosan particles (Riches et al., 1988; Bonney and Humes, 1984; Wightman et al., 1981). An acidic phospholipase A, activity associated with plasma membranes isolated from mouse peritoneal macrophages also has been described (Shibata et af., 1988a). However, the enzyme most likely to be involved in the release of arachidonate for eicosanoid formation resulting from the activation of transmembrane signaling pathways appears to be a basic phospholipase A, that is strictly calcium dependent. A membrane-bound, calciumdependent phospholipase A, with a pH optimum between 7.5 and 9.5 has been solubilized and partially purified from the P388DI macrophage-like cell line (Ulevitch et al., 1987), and a phospholipase A, activity with these characteristics has been identified in homogenates of mouse peritoneal macrophages (Wightman et al., 1981) and in plasma membranes isolated from guinea pig peritoneal macrophages (Shibata et al., 1988a). In addition, a cytosolic phospholipase A, has been purified from the RAW 264.7 macrophage cell line (Leslie et al., 1988).

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This phospholipase activity was optimal at pH 8.0 and was dependent on the presence of calcium. Evidence suggests that phosphatidylcholine, phosphatidylethanolamine, and the inositol phospholipids can be utilized as substrates for phospholipase A,-mediated arachidonate hydrolysis in macrophages (Wightman et a / . , 1981; Ulevitch et al., 1987; Leslie et al., 1988; Shibata et al., 1988b; Emilsson and Sundler, 1984, 1985, 1986). Phospholipase A, is relatively inactive in unstimulated cells but becomes rapidly activated by specific receptor-mediated signals. Several molecular mechanisms may be involved in the regulation of phospholipase A, activity in stimulated cells. The intracellular concentration of calcium appears to be critical in modulating the activity of phospholipase A, (Van Der Bosch, 1980). In macrophages, calcium-dependent phospholipase A, can be activated to release arachidonate during exposure of cells to agents that increase intracellular calcium concentration, such as calcium ionophores or agonists that stimulate the turnover of polyphosphoinositides. Intracellular pH may also influence the activity of phospholipase A,. In platelets, phospholipase A, activity and arachidonate release appear to be regulated by a mechanism that requires Na /H antiporterdependent cytosolic alkalinization (Baron and Limbird, 1988; Sweatt et al., 1985, 1986). Evidence suggests thal the rise in cytosolic pH increases the sensitivity of phospholipase A, to the stimulatory efTect of calcium. Whether a similar mechanism exists in mononuclear phagocytes is unknown. Lipocortin may also be important for the regulation of phospholipase A, activity (Flower, 1988; Dennis et ul., 1978; Hirata, 1987). This endogenous protein inhibits the activity of phospholipase A,, possibly by forming complexes with the enzyme and thereby decreasing the affinity of the enzyme for calcium or by forming complexes with phospholipid substrates (Hirata, 1987). The synthesis of lipocortin is augmented by glucocorticosteroids, suggesting the possibie involvement of lipocortin-mediated inhibition of phospholipase A, activity in the anti-inflammatory effects of these therapeutic steroid hormones. +

+

6. Diacylglycerol Lipase The release of arachidonate from diacylglycerol through the action of diacylglycerol lipase has been described for platelets. This pathway appears to be a two-step process involving phosphodiesterase-mediated formation of diacylglycerol, followed by the deacylation of diacylglycerol by diacylglycerol lipase. The extent to which diacylglycerol lipase activity contributes to stimulus-evoked arachidonate acid mobilization in mononuclear phagocytes remains to be determined. It has been suggested that a phospholipase C-diacylglycerol lipasemediatcd pathway for ardchidonate release occurs in mouse peritoneal macrophages stimulated with zymosan (Moscat er al., 1986a). However, no evidence was provided to demonstrate that diacylglycerol was indeed the source of the frce

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arachidonate formed in response to zymosan. Moreover, these authors provided evidence suggesting that, in macrophage homogenates, diacylglycerol lipase hydrolyzed fatty acids from the sn- 1 position of exogenous diacylglycerol and that a monoglyceride lipase was at least partially responsible for arachidonate hydrolysis (Moscat et al., 1986a). Similarly, calcium ionophore was shown to induce the generation of diacylglycerol from inositol phospholipids in intact peritoneal macrophages, and the diacylglycerol formed was deacylated at the sn-1 position without loss of arachidonate moieties (Emilsson and Sundler, 1985). Further experimentation will be required to assess the contribution of diacylglycerol lipase to receptor-mediated release of arachidonate in mononuclear phagocytes.

C. The Cyclooxygenase Pathway of Arachidonate Metabolism Free arachidonate can be metabolized into prostaglandins and thromboxanes through the action of prostaglandin endoperoxide synthase (Samuelsson et al., 1978; Needleman et al., 1986). This enzyme has intrinsic cyclooxygenase activity which converts arachidonate into prostaglandin G,. Prostaglandin endoperoxide synthase also possesses a peroxidase activity which converts prostaglandin G, into prostaglandin H,. These two distinct enzymatic activities associated with prostaglandin endoperoxide synthase reside in a single protein localized in the endoplasmic reticulum (Samuelsson et a l . , 1978; Needleman et ul., 1986; Rollins and Smith, 1980). Both the cyclooxygenase and peroxidase activities undergo self-deactivation that appears to result from the formation of oxidative intermediates (Hemler and Lands, 1980; Kent et al., 1983; Adams Brotherton and Hoak, 1983; Egan et al., 1979). The cyclooxygenase activity of prostaglandin endoperoxide synthase is also inhibited by nonsteroidal anti-inflammatory drugs, such as aspirin, ibuprofin, and indomethacin, and is therefore an important target in the therapeutic intervention of inflammation (Mizuno et al., 1982). Prostaglandin H, can be further metabolized into several other types of biologically active eicosanoids. Prostaglandin endoperoxide E isomerase, associated with microsomal membranes, can convert prostaglandin H, into prostaglandin E, (Ogino et ul., 1977), a major eicosanoid product of mouse peritoneal macrophages stimulated with bacterial lipopolysaccharide, zymosan, calcium ionophore, phorbol ester (Bonney and Humes, 1984; Humes et al., 1982; Scott et al., 1980; Aderem et al., 1986a), or immune complexes (Bonney et al., 1979; Rouzer et ul., 1982; Aderem et al., 1986a). Prostacyclin synthase (prostaglandin endoperoxide I isomerase) metabolizes prostaglandin H, to prostacyclin (prostaglandin I,) (Samuelsson et al., 1978; DeWitt and Smith, 1983), a major eicosanoid product of endothelial cells. The inactive prostacyclin metabolite 6-

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ketoprostaglandin F,, is produced by mouse peritoneal macrophages in response to zymosan (Bonney et a / . , 1978; Scott et al.. 1980, 1982; Aderem et ul., I986a), calcium ionophore (Aderem et a/. , 19864, phorbol ester (Bonney et a/., 1980; Aderem et d . , 1986a), or immune complexes (Bonney et al., 1979; Aderem et ul., 1986a). Thomboxane synthase (prostaglandin endoperoxide: thromboxane A isomerase) converts prostaglandin H2 into thromboxane A, (Samuelsson et u / . , 1978; Needleman et al., I986), which is a major arachidonate metabolite of platelets. Thomboxane B,, an inactive metabolite of thromboxane A,, has been detected in mouse peritoneal macrophages exposed to phorbol ester (Brune et d.,1978), zymosan (Scott et ul., 1982), or immune complexes (Brune et a/., 1978) and in mouse bone marrow macrophages and human monocytes stimulated with zymosan or calcium ionophore (Weideman et ul., 1978; Pawlowski et al., 1983). Prostaglandin endoperoxide D isomerase, a cytoplasmic enzyme, converts prostaglandin H, into prostaglandin D, (ChristHazelhof and Nugteren, 1979), a product of zymosan- and BCG-stimulated alveolar macrophages (Hsueh, 1979; Hsueh et a / ., 1979). Prostaglandin FZu, which has been detected in mouse bone marrow macrophages and human monocytes exposed to zymosan or calcium ionophore (Weideman et al., 1978; Pawlowski et a / ., 1983). is produced from prostaglandin H, by prostaglandin endoperoxide rcductase (Samuelsson et ul., 1978).

D. The 5-Lipoxygenase Pathway of Arachidonate Metabolism Free arachidonate can also be metabolized into biologically active eicosanoids through the action of lipoxygenase enzymes. Leukotrienes and peptidoleukotrienes are major eicosanoid products of stimulated mononuclear phagocytes, and formation of these compounds is initiated by the conversion of arachidonate to 5-hydroperoxyeicosatetraenoic acid (5-HPETE) through the action of 5-lipoxygenase (Lewis and Austen, 1988; Rokach and Fitzsimmons, 1987). The formation of 12-HPETE and 15-HPETE has also been described in mononuclear phagocytes, although information on potential transductional mechanisms involved in their formation is limited. The 5-lipoxygenase resides predominantly in the cytosol in unstirnulated cells but is rapidly translocated to cell membranes upon calcium ionophore stimulation of intact human leukocytes (Rouzer and Kargman, 1988) and rat basophilic leukemia cells (Wong et al., 1988). The association of 5-lipoxygenase with membranes was also shown to be calcium dependent in a cell-free system (Rouzer and Kargman, 1988; Wong et a l . , 1988). lonophore dose-response curves and time courses demonstrated a good correlation between membrane association of 5-lipoxygenase and formation of leukotrienes in human leukocytes (Rouzer and Kargman, 1988). However, membrane-associated 5lipoxygenase was found to be inactive, presumably as a result of suicide inactiva-

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tion rather than proteolytic degradation. Thus, 5-lipoxygenase appears to be activated by a calcium-dependent translocation from the cytosol to cell membranes. Membrane-bound 5-lipoxygenase then initiates leukotriene formation prior to becoming inactivated. The arachidonate metabolite resulting from 5-lipoxygenase activity, 5-HPETE, can be converted to the labile epoxide leukotriene A, by a dehydrase activity (LTA, synthase) associated with 5-lipoxygenase (Lewis and Austen, 1988; Rokach and Fitzsimmons, 1987). Leukotriene A, can be subsequently metabolized to leukotriene B, through the action of leukotriene A, hydrolase (Ridmark et al., 1980, 1984). Lcukotriene B, is produced in mouse peritoneal macrophages in response to zymosan (Bonney ef ul. , 1985; Humes et al., I982), in rabbit alveolar macrophages in response to zymosan and calcium ionophore (Hsueh and Sun, 1982), and in human monocytes exposed to immune complexes (Ferreri et al., 1986). Leukotriene A, can also be converted into peptidoleukotrienes. Leukotriene C, synthase, which possesses glutathione transferase activity, converts leukotriene A, to leukotriene C, by facilitating the covalent binding of the tripeptide glutathione to the carbon backbone of leukotriene A, (Bach et al., 1984; Yoshimoto et al., 1985). Peritoneal macrophages produce leukotricnc C, in response to zymosan, calcium ionophore, and immune complexes (Bonney and Humes, 1984; Humes et u l . , 1982; Rouzer et al., 1982; Scott et a / . , 1982).

E. Regulation of Eicosanoid Production by Anti-Inflammatory Drugs Nonsteroidal and steroidal anti-inflammatory drugs are w i d y used as invaluable components of therapeutic protocols to treat a great variety of inflammatory maladies and as scientific tools for research involving the study of eicosanoids. In general, the anti-inflammatory action of these drugs is a virtue of their ability to inhibit eicosanoid formation within the various cellular components of the immune system, including mononuclear phagocytes. Nonsteroidal anti-inflammatory drugs (NSAID) include salicylates, such as aspirin, the prototype NSAID, indoleacetic acids, such as indomethacin and sulindac, and phenylacetic acids, such as ibuprofin (Goldstein, 1988). These compounds directly inhibit the cyclooxygenase activity of prostaglandin endoperoxide synthase, which results in decreased formation of cyclooxygenase products (Goldstein, 1988; Mizuno et al., 1982). As a result, these drugs are anti-inflammatory, analgesic, and antipyretic. Aspirin, indomethacin, and ibuprofin were shown to inhibit prostaglandin E, formation in mouse peritoneal macrophages stimulated by zymosan, lipopolysaccharide, phorbol esters, or calcium ionophores (Bonney and Humes, 1984; Humes et al., 1983; Brune et al., 1984). In general, NSAIDs do not inhibit 5-lipoxygenase activity at relevant concentrations, and NSAIDs, including as-

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pirin, indomethacin, and ibuprofin, did not inhibit leukotriene C, production in macrophages stimulated with zymosan or ionophore (Bonney and Humes, 1984; Humes et al., 1983; Brune et a/., 1984). However, sulindac has been shown to inhibit the formation of both cyclooxygenase and 5-lipoxygenase products in macrophages, although the effect of sulindac on leukotriene production occurred only at high concentrations (Bonney and Humes, 1984). Several other agents inhibit leukotriene formation in mouse peritoneal macrophages, including BW755C, eicosatriynoic acid, nafazatrom, and nordihydroguaiaretic acid, although these compounds also affect the formation of prostaglandins (Bonney and Humes, 1984; Humes et al., 1983; Brune et al., 1984). Adrenal corticosteroid hormones and their pharmacologic derivatives are powerful anti-inflammatory agents. Although the precise mechanisms by which these drugs suppress the formation of cyclooxygenase and 5-lipoxygenase products are unclear, it appears that the anti-inflammatory effects of adrenal corticosteroids are at least partially due to their ability to inhibit phospholipase A, activity and arachidonate release (Goldstein, 1988). Dexamethasone has been shown to inhibit the release of arachidonate and the formation of leukotriene B, and thromboxane A, in mouse peritoneal macrophages and to suppress superoxide release, phagocytosis, and tumoricidal activity in these cells (Fuller el ill., 1984; Becker et al., 1988; Schultz ef al., 1985). A possible mechanism by which adrenal corticosteroids inhibit arachidonate release appears to be through an increase in steroid hormone receptor-mediated synthesis of the protein lipocortin, an endogenous inhibitor of phospholipase A, activity (Goldstein, 1988; Flower, 1988; Dennis et al., 1987; Hirata, 1987). However, in the case of glucocorticosteroid-dependent inhibition of macrophage tumoricidal activity, evidence suggested a mechanism independent of the inhibition of phospholipase A, activity (Schultz rt d.,1985). Future investigations using steroidal and nonsteroidal anti-inflammatory drugs may lead to a better understanding of the regulation and function of eicosanoids in mononuclear phagocytes and of the involvement of eicosanoids in the manifestation of inflammatory diseases.

IV. POTENTIAL TRANSDUCTIONAL MECHANISMS INVOLVED IN THE STIMULATION OF MACROPHAGE ElCOSANOlD PRODUCTION The above summary of the biochemical pathways involved in macrophage production of the various eicosanoids suggests that regulation in response to external stimuli is complex and is dependent on several transductional sequences that are initiated by the individual stimulus. As noted previously, eicosanoid production in macrophages can be induced in response to a variety of well characterized external stimuli and pharmacologic

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agents. These include immune complexes, particulate stimuli (e.g., zymosan), chemoattractants, lipopolysaccharide, pharmacologic activators of protein kinase C, and calcium ionophores. The variety of transductional sequences initiated by the above stimuli suggest complex regulation of both the original de-esterification of arachidonate and its subsequent metabolism. The ability of the above stimuli to cause eicosanoid production is further regulated by the activation state of the macrophage. The molecular events involved in the regulation of eicosanoid production by macrophage development and the relative involvement of cellular transductional sequences resulting in the production of the various eicosanoids in response to the different external stimuli remain to be defined in detail. The best described transductional sequences which can result in enhanced eicosanoid production are those which are initiated by the cell surface binding of leukocyte chemoattractants (for reviews see Uhing et al., 1988; Dillon et al., 1978; Omann et af., 1987). As noted previously, the potential transductional events involved in chemoattractant-activated eicosanoid production are likely to include the elevation of intracellular calcium, the activation of protein kinase C, or stimulation of Na / H exchange. The chemoattractants that stimulate eicosanoid production in niononuclear phagocytes include, among others, the arachidonate metabolite LTB, and platelet activating factor (Koo et al., 1988; Prpic et ul., 1988). An initial molecular sequence initiated by chemoattractants is the activation of a polyphosphoinositide phosphodiesterase via a receptor-coupled, bacteria toxin-sensitive GTP-binding protein. The resultant elevation of intracellular calcium is likely to be involved in the initial activation of phospholipase A,. The elevation of intracellular calcium in response to chemoattractants involves both the mobilization of intracellular stores and a more sustained elevation due to stimulated calcium influx (Uhing et al., 1988; Dillon et at., 1987; Anderson et al., 1586). The duration of the calcium response is likely to be reflected in the total eicosanoid production as well as in the relative production of the 5-lipoxygenase vs. the cyclooxygenase pathways (Tripp et a/., 1985). In addition to G protein activation of a polyphosphoinositide phosphodiesterase with resultant calcium-mediated activation of phospholipase A, and production of 1-acyl-2-arachidonylglycerolas a potential substrate for diacylglycerol lipase, data have indicated the potential involvement of an additional G protein for a more direct receptor-coupled stimulation of phospholipase A, activity in other cells (Burch et al., 1986; Jelsema, 1987; Kim et al., 1989). This phenomenon has further been indicated to be involved in the ability of guanine nucleotides to cause subsequent activation of a cell surface K + channel (Kurachi et ul., 1989; Kim et al., 1989). Currently, data is unclear regarding whether activation of the K channel is mediated via the a or Py subunits of the relevant G protein (Birnbaumer et al., 1988; Neer and Clapham, 1988). It has been suggested that K -channel (and thus prior phospholipase A,?) activation may be +

+

+

+

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mediated via Gi,-3 (Birnbaumer et uf., 1988), which is a major pertussis toxin substrate in leukocytes (Uhing et al., 1987; Goldsmith et al., 1988). The relative involvement of a direct, receptor-coupled activation of a phospholipase A, vs. that of a phospholipase C remains to be defined. Chemoattractants also cause the activation of protein kinase C via the synergistic action of calcium and diacylglycerol, which is produced from both stimulated PI metabolism and other precursors (Uhing et al., 1988, 1989; Dillon et ul., 1987; Omann et al., 1978; Truett et al., 1988). Bascd on the actions of pharmacologic activators of protein kinase C , the enzyme is suggested to regulate both the de-esterification of arachidonate and, synergistically with elevated cytosolic calcium, the relative involvement of the 5-lipoxygenase pathway (Tripp et a / ., 1985). The effect of phorhol esters on the de-esterification of arachidonate may involve rapid protein synthesis (Bonney et af., 1980). An additional potential mechanism involved in protein kinase C-mediated regulation of eicosanoid production is through the prior activation of the Na / H antiporter. Studies in platelets indicated that inhibitors of the antiporter attenuated the ability of several stimuli to provoke de-esterification of arachidonate (Sweatt ef ul., 1985). Prior studies, including those in macrophages (Aderem et al., 1984), had indicated that eicosanoid production in response to some stimuli was attenuated by the removal of extracellular sodium. Chemoattractant addition to leukocytes results in cytosolic alkalinization primarily due to the activation of the Na+ / H + antiporter (Sirnchowitz and Cragoe, 1986). Since this response is mimicked by pharmacologic stimuli of protein kinase C , the chemoattractantinduced response may involve prior activation of this enzyme. This summarizes the transductional sequences initiated by chemoattractants that are likely to be involved in the production of the various eicosanoids. Although the molecular sequences initiated by chemoattractants are the best understood relative to other stimulants of mononuclear phagocyte eicosanoid production, the relative involvement of the different intracellular messengers for both the initial de-esterification of arachidonate and its subsequent metabolism remains to be defined in detail for the various chemoattractants. Far less information is available on potential transductional mechanisms involved in eicosanoid production by other stimulants of mononuclear phagocytes. Information regarding the transductional sequences involved in the ability of immune complexes to cause eicosanoid production is unclear. Macrophage receptors for IgG havc been suggested to cause calcium mobilization, to serve as ion channels, and to be directly associated with phospholipase A, activity (Unkeless et al., 1988). Zymosan stimulates the production of eicosanoids in macrophages with resultant increases in both cyclooxygenase and 5-lipoxygenase products (Table I). Delineation of the transductional sequences involved in the actions of zymosan is complicated by the multiple modes for its recognition by mononuclear pha+

+

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TABLE I SUGGESTED SrlMULANTS IN

Stimulant A23 187

Phorbol esters Zymosan LPS Immune Complexes PAF and other chenloattrdctants

ElCOSANOlD PRODUCTION MURINE PERITONEAL MACROPHAGESO

A N D INTRACELLULAR S I G N A L S FOR

Major eicosanoids

Regulatory signals

CaZ+ PKC CaZ+,Na+ / H + Caz+ (weak), PKC, N a + / H + CaZ+(?). N a + / H + ( ? ) Ca2+, PKC, Na+/H+

PGE,, PGE,, PGE,, PGE, PGE,, PGE?,

PGI,, LTC, POI,, TXA, PGI,, LTC,, LTB,, TXA, PGI,, LTC,, TXA, PGI,, LTC,, LTB,

0 Abbreviations: PGE,, prostaglandin E,; PGI,, prostaglandin I? (prostacyclin); LTB,. leukotnene B,; LTC,, leukotriene C,; TXA,, thrornboxane A,, PKC, protein kinase C. LPS, bacterial lipopolysaccharide; PAF, platelet activating Factor.

gocytes (Riches et al., 1988). Phagocytosis of zymosan is not required for the stimulation of eicosanoid metabolism (Rouzer et u l . , 1980). Recognition of the a-mannan moiety of zymosan has been suggested to be at least partially responsible for its stimulatory effects on arachidonate metabolisni (Aderem and Cohn, 1986). A role for calcium in zymosan-induced eicosanoid production is indicated from the observation that its removal attenuates the response (Moscat et al., 1986b) and the ability of zymosan to stimulate PI metabolism (Emilsson and Sundler, 1984). A major role for Na +/H+ exchange is indicated from the observation that sodium removal from the extracellular medium attenuates eicosanoid production induced by zymosan (Aderem et al., 1984). In addition, inhibitors of the Na+/H+ antiporter attenuate the ability of zymosan to induce eicosanoid production (Dieter et al., 1987). A detailed characterization of the stimulation by zymosan is further complicated by the apparent requirement for rapid protein synthesis for its effects on the production of eicosanoids (Bonney et a l ., 1980). Bacterial lipopolysaccharide (LPS) is a weak stimulant of eicosanoid metabolism in peritoneal macrophages with resultant increases primarily of cyclooxygenase products (Humes et al., 1982). LPS treatment of macrophages potentiates the ability of subsequent stimuli to promote eicosanoid production (Aderem et ul., 1986b). Information has accumulated regarding transductional sequences involved in the action of LPS. Both LPS and its active moiety, lipid A, stimulate PI metabolism and calcium mobilization in peritoneal macrophages (Prpic et al., 1987). Data have also been presented indicating an ability of LPS to cause activation of protein kinase C (Weiel et al., 1986). In addition, we have observed that LPS addition to macrophages results in activation of the Na+ / H + antiportcr as evidenced by the amiloride sensitivity of both pH, elevation and enhanced 22Na+ uptake (Prpic et ul., 1989~). The priming effect of LPS for eicosanoid production is observed for a variety

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of stimuli, including zymosan, immune complexes, phorbol ester, and calcium ionophores (Aderem et d., I986b), as well as for the macrophage chemoattractant platelet activating factor (V. Prpic, unpublished observations). The priming effect occurs after a lag of -10 min and is maximal within 60 min. Aderem and colleagues have suggested that the ability of LPS to stimulate protein myristoylation (Aderem et d., 198hb, 1988) serves a causative role in its ability to prime for enhanced cicosanoid production.

V.

PLATELET ACTIVATING FACTOR AS AN AUTOCRINE COMPONENT OF ElCOSANOlD PRODUCTION

Platelet activating factor (PAF), originally identified as an activator of histamine production from platelets, has subsequently been suggested to be involved in numerous inflammatory responses and has been demonstrated to induce a response in a variety of cells involved in the repair of tissue injury and protective mechanisms against invading organisms (Braquet et al., 1987; Snyder, 1985). PAF is produced from platelets, a variety of leukocytes, and endothelial cells. The ability of PAF to serve as an intercellular mediator of inflammatory responses is attenuated by an acetylhydrolase in serum and by the rapid uptake and metabolism of PAF by responsive and possibly also nonresponsive cells. is Platelet activating factor [ I -alkyl-2(R)-acetylglycero-3-phosphorylcholine~ originally derived from 1-alkyl-2-acylglycerophosphorylcholine.This component of cellular phosphatidylcholine comprises nearly one-half of total cellular phosphatidylcholine in mononuclear phagocytes, with arachidonate as the major fatty acid in the 2 position (Albert and Snyder, 1983; Sigiura et al., 1983). Since phosphatidylcholine represents approximately 40% of the total phospholipid pool in mononuclear phagocytes, one can readily envision the potential magnitude of stimulated production of this intercellular mediator. The initial step in the synthesis of PAF is the action of phospholipase A,. As noted previously, this cellular activity has been indicated to be regulated as a consequence of macrophage stimulation and the resultant alterations in either intracellular calcium or pH, or both. The rate limiting step for synthesis of PAF in macrophages appears to be the activity of a acetyl CoA-dependent acetyltransferase (Wykle et al., 1980). The activity of this enzyme is regulated by levels of intracellular calcium. PAF is degraded by a reversal of these events by the sequential actions of acetyl hydrolase and acyl transferase activities. The involvement of phospholipase A, for the initiation of PAF synthesis as well as thc involvement of arachidonate in the re-acylation of lyso-PAF indicate the involvement of PAF metabolism as a component of stimulated eicosanoid production by mononuclear phagocytes.

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Recently, attention has also focused on the responses of mononuclear phagocytes initiated by PAF. PAF has been identified as a chemoattractant for human monocytes (Goetzl et al., 1980) and murine peritoneal macrophages (Prpic et al., 1988). As described for other leukocyte chemoattractants, we have found that PAF addition to peritoneal macrophages results in the rapid production of inositol 1,4,5-trisphosphate subsequent to the binding of PAF to a cell surface receptor (Prpic et ul., 1988) (Fig. 2). The initial inositol 1,4,5-trisphosphate is metabolized to a variety of inositol phosphate isomers. Concomitant with the stimulation of PI metabolism, PAF addition causes a marked increase in the concentration of intracellular calcium. The elevation of intracellular calcium involves both the mobilization from intracellular stores and a more sustained dependency on extracellular calcium. PAF addition also results in the alterations of other intracellular ions. Changes in pH, are rapidly evident upon addition of PAF to peritoneal macrophages (Prpic e t a / ., 1 9 8 9 ~ )Initially, . pHi is decreased, a phenomenon that has been described for other leukocyte chemoattractants. Subsequently, pH, increases with a resultant cytosoljc alkalinization. The amiloride sensitivity of both the intracellular alkalinization and the rapid effect of PAF for an accelerated uptake of 22Na.+-indicates that this lipid mediator causes a rapid activation of the Na /H antiporter. We have also indicated that PAF-induced activation of Na /H exchange is responsible for its subsequent stabilization of JE mRNA (Prpic et al., 1 9 8 9 ~ ) PAF . addition to peritoneal macrophages also +

+

+

+

PI PC PE

1Arch

FIG.2. Transmembrane signaling pathways initiated by PAF. A schematic representation of intracellular mediators of PAF-induced arachidonatc mobilization in mononuclear phagocytes. Abbreviations: PAF, platelet activating factor; G,, PAF receptor-associated GTP-binding protein; PIP,, phosphatidylinositol4,5 ,-hisphosphate;PDE, phosphodicsterasc; DAG, sn- 1,2-diacylglycerol;PKC, protein kinase C ; 11.4.sP3,inositol 1,4,5-trisphosphate;11.3.4.sP4,inositol 1,3,4,5-tetrakisphosphate; PI, phosphatidylinositol; PC, phosphatidylcholine; PE, phosphatidylethanolamine; Arch, arachidonate; PLA2, phospholipase A:.

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results in enhanced XhRb influx via enhanced N a + / K + antiporter activity (unpublished observations), suggesting that clevated intracellular K + occurs either as a consequence of prior N a + / H + exchange or as a direct consequence of protcin kinase C activation. We havc also observed that addition of PAF to peritoneal macrophages results in the secretion of eicosanoids (Prpic et a/., 1989b), suggesting that the production of PAF results in autocrine regulation of eicosanoid metabolism in macrophages. The ability of PAF to cause eicosanoid sccretion is attenuated by removal of extracellular calcium, indicating the involvement of elevated intracellular calcium for this effect of PAF. PAF stimulates the production of both cyclooxygenase and 5-lipoxygenase products from [3H]arachidonate-labeled macrophages, with PGE, being the major secreted eicosanoid. PAF addition to peritoneal macrophages results in the activation of protein kinase C as evidenced by 32P incorporation into a characteristic set of proteins (Prpic et af.,1988). PAF stimulation of the accumulation of the putative cellular protein kinase C activator sn- I ,2-diacylglycerol is kinctically distinct from PAF stimulation of PI metabolism, with the second, more pronounced accuniulation occurring subsequent to the activation of protein kinase C (Uhing et al., 1989). Our results also suggest that phosphatidylcholine is a likely precursor for the protein kinase C-mediated DAG accumulation. When the radyl composition of the sn- I ,2-diacylglycerol kinase (which phosphorylates alkyl, alkenyl, and acyl containing 1,2-diradylglycerols) substrates induced at 10 min by PAF in peritoneal macrophages is compared to that obtained after quantitative hydrolysis of macrophage phospholipids, a marked similarity to phosphatidylcholine is observed (R. J. Uhing et al., unpublished observations). Similarly, PAF addition to peritoneal macrophages results in the production of aqueous label from I3HH]cho1inelabeled macrophages (Uhing et a / ., 1989). Interestingly, although, as noted previously, macrophage PC is composed of a significant proportion of 1-alkylcontaining isomers, the PAPinduced diacylglycerol kinase substrate is primarily diacyl containing. These results suggest that either (1) the different diradyl forms of PC are compartmentalized in the cell or ( 2 ) phospholipase A, and the PAFstimulated phosphodiesterase action toward macrophage PC exhibit different substrate specifities.

VI.

THE RELATIONSHIP OF ElCOSANOlD PRODUCTION TO MACROPHAGE DEVELOPMENT

Production of eicosanoids by mononuclear phagocytes not only is regulated directly by external stimuli but also is dependent on the activation state of the cell. The sequential activation of macrophages has been most clearly defined for

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the functional acquisition for tumoricidal competence (for reviews see Adams and Hamilton, 1984, 1988). Macrophages, at various stages for this activation, can be induced in vivo by the appropriate inflammatory agents or in vitro by welldefined activating signals. Murine peritoneal macrophages, in particular, have been extensively studied with regard to both the in vivo and the in vitro activation. Production of eicosanoids in response to zymosan, phorbol ester, and a calcium ionophore exhibits an inverse relationship to in vivo macrophage activation (Humes et al., 1980; Lewis et a / . , 1986; Lewis and Adams, 1986). The reduction of the stimulated production of total eicosanoids is greatest for activated macrophages (elicited with Bacillus calmette-guerin) and intermediate for responsive macrophages (casein-elicited) when compared to resident peritoneal macrophages. Since total eicosanoid production is reduced as a consequence of in vivo activation, a site of regulation is likely to be either the original deesterification or the re-esterification of arachidonate. In addition, for activated macrophages, 5-lipoxygenase products represent a significantly lower percentage of the total eicosanoid products than for resident macrophages (Lewis et al., 1986), indicating an additional regulation by in vivo macrophage activation on this pathway. The regulation of eicosanoid production by in vivo macrophage activation is apparently not reflected by defined in vitro activation. The best defined system for in v i m macrophage activation is the development of elicited macrophages from responsive to activated by the sequential addition of interferon-y and bacterial lipopolysaccharide (Adams and Hamilton, 1984, 1988). Interestingly, both these signals prime for subsequent production of eicosanoids by some external stimuli. As noted in a preceding section, LPS potentiates eicosanoid production in response to several stimuli of macrophages. In contrast, interferon-y priming for eicosanoid production is apparently more selective. Interferon-y potentiates eicosanoid production in response to phorbol esters (Hamilton et al., 1985a) and PAF (R. J. Uhing el a/., unpublished observations), but not in response to zymosan or immune IgG complexes. The specificity of the priming ability of interferon-y may suggest a potential involvement of protein kinase C. Interferon-y has been indicated to increase the activity of macrophage protein kinase C without similar alterations of phorbol ester affinity (Hamilton et al., 1985b; Becton et a l . , 1985). Interferon-y also potentiates the production of the putative protein kinase C activator diacylglycerol in response to several macrophage stimuli, including phorbol myristate acetate (Sebaldt et al., 1989). Phorbol esters and other activators of protein kinase C in macrophages cause the accumulation of diacylglycerol from phospholipid precursors other than phosphatidylinositol (Uhing et al., 1989). The ability of interferon-y to potentiate diacylglycerol accumulation and phosphatidylcholine hydrolysis in response to PMA and PAF (Sebaldt et al., 1989), as well as to increase eicosanoid production in response to

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the same stimuli, suggests that its priming ability for eicosanoid production could involve a component of increased diacylglycerol lipase activity. In addition to the regulation of eicosanoid production by macrophage activation, the eicosanoids that are produced, in turn, regulate the development of macrophages. As noted previously, a major cyclooxygenase product formed in response to a variety of macrophage stimuli is PGE,. PGE, has been demonstrated to bind to a cell surface receptor on macrophages with resultant activation of adenylate cyclase via receptor-coupled G,. The resultant increase in CAMPby YGE, and other elevating agents has been indicated to result in the suppression of a variety of macrophage responses. A CAMP-suppressed response that has been extensively characterized is the surface appearance of class 11 major histocompatibility complex molecules (la) during the development of macrophages from responsive to primed. Induction of surface Ia in response to interferon-y is inhibited by PGE, and by other CAMP-elevating agents, including forskolin, cholera toxin, and isobutyl-methylxanthine, as well as by direct addition of CAMP analogs (Snyder et al., 1982; Figueiredo et id.,1989). Inhibition of interferon-y-induced la is evident at the levels of surface, mRNA, and interferon-y-induced transcription (Figueiredo et a l . , I989), suggesting that this inhibition may involve attenuation of an early transductional event initiated by interferon-y. In the case of Ia, this transductional event may be stimulated Na /H+ exchange. We have reported that the addition of interferon-y to murine peritoneal macrophages results in the rapid activation of the Na' / H + antiporter and that the resultant ion fluxes are likely to be involved in the induction of la in response to interferon-y (Prpic et a/., 1989a). Since PGE, is a major product of stimulated arachidonatc metabolism in macrophages in response to a variety of stimuli (Table I), eicosanoid production is likely to serve an autoregulatory role for their regulation of mononuclear phagocytes. Currently, little information is available on the extent of autoregulation in response to these various stimuli of macrophage eicosanoid production. Addition of PGE, or CAMP-elevating agents to macrophages inhibits the ability of PAF to cause eicosanoid production (V. Prpic, unpublished observations). This inhibition is apparently not via attenuation of the initial hydrolysis of phosphatidylinositol4,5-bisphosphatesince inositol 1,4,5trisphosphate production is not affected. Attention in phagocytic leukocytes has focused on the inhibition of other potential transductional sequences involved in eicosanoid production. Increases of intracellular CAMP have been indicated to attenuate the influx of calcium (Takenawa et ul., 1986). The inhibitory actions of CAMP in phagocytic leukocytes are unlikely to be explained totally by actions on transductional sequences ascribed to calcium-mobilizing agonists since, as noted previously for interferon-y, inhibition of the actions of other macrophage stimuli is also observed. +

14. ARACHIDONATE METABOLITES IN PHAGOCYTES

VII.

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SUMMARY

Current information indicates that multiple transductional sequences initiated by stimuli of mononuclear phagocytes are involved in the production of eicosanoids. These are likely to include direct receptor-coupled activation of enzymes involved in the de-esterification of the arachidonate contained in cellular phospholipids. In addition, alterations of the intracellular concentrations of several “second messengers” in response to external stimuli regulate both total eicosanoid production and the relative production of individual classes of eicosanoids (i.e., cyclooxygenase products vs. the leukotrienes). These regulators of eicosanoid production include intracellular calcium, intracellular pH, and the activation of protein kinase C. Investigations of the molecular mechanisms involved in stimulus-induced eicosanoid production are complicated by the apparent requirement for protein synthesis for the actions of several macrophage stimuli. In addition, eicosanoid formation is both regulated by and a regulator of macrophage development. Eicosanoid production by mononuclear phagocytes thus serves a paracrine function for the regulation of other inflammatory cells as well as an autoregulatory role for the response of macrophages to inflammatory mediators. ACKNOWLEDGMENT Supported in part by USPHS Grants CA16784, ES02922, and CA29589. REFERENCES Adams, D. O., and Hamilton, T. A. (1984). The cell biology of macrophage activation. Annu. Rcw. Immunol. 2, 283-318. Adams, D. 0..and Hamilton, T. A. (1988). Phagocytic cells: Cytotoxic activities of macrophages. I n “Inflammation: Basic Principles and Clinical Correlates” (J. I. Gallin, I. M. Goldstein, and R. Snyderman, eds.), pp. 471-492. Raven, New York. Adams Brotherton, A. F., and Hoak, J. C. (1983). Prostaglandin biosynthesis in cultured vascular endothelium is limited by deactivation of cyclooxygenase. J. Clin. Invest. 72, 1255- 1261. Aderem, A. A., and Cohn, Z . A. (1986). Bacterial lipopolysaccharides modify signal transduction in the arachidonic acid cascade in macrophages. Biochem. Mucrophages, Ciba Found. Symp. No. 118, 196-210. Aderem, A. A , , Scott, W. A , , and Cohn, Z . A . (1984). A selective defect in arachidonic acid release from macrophage membranes in high potassium media. J . Cell Biol. 99, 1235-1241. Aderem, A. A , , Cohen, D. S . , Wright, S . D., and Cohn, Z. A. (1986a). Bacterial lipopolysaccharides prime macrophages for cnhanced release of arachidonic acid metabolites. J . Exp. M e d . 164, 165-179. Aderem, A. A,, Keum. M. M., Pure, E., and Cohn, Z . A. (1986b). Bacterial lipopolysaccharides, phorbol myristate acetate, and zymosan induce the myristoylation of specific macrophage proteins. Proc. Null. Acad. Sci. U.S.A. 83, 5817-5821. Aderem, A. A . , Albert, K. A . , Keum, M. M . , Wang, J. K. T., Greengard, P., and Cohn, Z. A. ( 1 988). Stimulus-dependent myristoylation of a major substrate for protein kinase C. Nutiire (London) 332, 362-364.

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Shibata, Y., Abido, Y., and Takiguchi, H. (l988b). Phospholipase A2 in macrophage plasma membrane releases arachidonic acid from phosphatidylinositol. Biochim. Biopfiys. Acru 971, 121126. Simchowitz, L., and Cragoe, E. J . , Jr. (1986). Inhibition of chemotactic factor-activated Na+ /H exchange in human neutrophils by analogues of amiloride in structure-activity relationships in the amiloride series. M o l . Phurmucol. 30, 112-120. Simon, R. H., and Ward, P. A. ( I 988). Adult respiratory distress syndrome. In “Inflammation: Basic Principles and Clinical Correlates” (J. 1. Gallin, 1. M. Goldstein, and R. Snyderman, eds.), pp. 815-828. Raven, New York. Smith, E. F., Wise, W. C . , Halushka, P. V., and Cook, J. A. (1987). Macrophage eicosanoid formation is stimulated by platelet arachidonic acid and prostaglandin endoperoxide transfer. Biochim. Biophys. Acta 923, 355-361. Snyder, D. S . , Beller, D. I . , and Unanue, E. R. (1982). Prostaglandins modulate macrophage Ia expression. Nature (London) 299, 163-165. Snyder, F. (1985). Chemical and biochemical apects of platelet activating factor: a novel class of acetylatcrd ether-linked choline phospholipids. Med. Res. Rev. 5 , 107-140. Spaethe, S. M., and Ncedleman, P. (1987). Arachidonic acid metabolism in tissue injury. In “Cellular and Molecular Aspects of Inflammation” (C. Poste and S. T. Crooke, eds.), pp. 153-170. Plenum, New York. Sugiura, T., Nakajima, M.. Sekiguchi. N., Nakagawa, Y., and Waku, K . (1983). Different fatty chain compositions of alkeylacyl, alkyacyl, and diacyl phospholipids in rabbit alveolar macrophages: high amounts of arachidonic acid in ether phospholipids. Lipids 18, 125- 129. Svenson, J., Hamberg, M . , and Samuelsson, B. (1975). Prostaglandin endoperoxides. IX. Characterization of rabbit aorta contracting substance (RCS) from guinea pig lung and human platelets. Acta P h y i o l . Scand. 94, 222-228. Sweatt, J. D . , Johnson, S. L., Cragoe, E. J., and Limbird, L. E. (1985). Inhibitors of N a + / H + exchange block stimulus-provoked arachidonic acid release in human platelets. J . Biol. Chem. 260, 129 10- 12919. Sweatt, J. D., Connolly, T. M . , Cragoe. E. J . , and Limbird, L. E. (1986). Evidence that N a + / H + exchange regulates receptor-mediated phospholipase A2 activation in human platelets. J . Biol. Chem. 261, 8667-8673. Takenawa, T., Ishitoya, I . , and Nagai, Y . (1986). Inhibitory effect of prostaglandin E2, forskolin, and dibutyryl CAMPon arachidonic acid release and inositol phospholipid metabolism in guinea pig neutrophils. J . B i d . Chem. 261, 1092-1098. Tripp, C. S., Mahoney, M., and Needleman, P. (1985). Calcium ionophore enables soluble agonists to stimulate macrophage 5-lipoxygenase. J . B i d . Chem. 260, 5895-5898. Truett, A . P., 111, Verghesc, M. W., Dillon, S . B., and Snyderman, R. (1988). Calcium influx stimulates a second pathway for sustained diacylglycerol production in leukocytes activated by chemoattractants. Proc. Narl. Acud. Sci. U.S.A. 85, 1549- 1553. Uhing, R. J., Polakis, P. C., and Snyderman, R. (1987). Isolation of CTP-binding proteins from myeloid HL60 cells: Identification of two pertussis toxin substrates. J . B i d . Chem. 262, 1557515579. Uhing, R . J . , Dillon, S . B . , Polakis, P. G . , Truett, A . P., 111, and Snyderman, R. (1988). Chemoattractant receptors and signal transduction processes. In “Cellular and Molecular Aspects of Inflammation” (C. Poste and S . T. Crooke, edb.), pp. 355-379. Plenum, New York. Uhing, R. J., Prpic, V., Hollenbach, P. W., and Adams, D. 0. (1989). Involvement of protein kinase C in platelet-activating factor-stimulated diacylglycerol accumulation in murine peritoneal macrophages. J . Biol. Chem. 264, 9224-9230. Ulevitch, R. J., Watanabe, Y., and Sano, M. (1987). Solubilization, purification, and characteriza+

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tion of a membrane-bound phospholipase A2 from the P388D, macrophage-like cell line. J . B i d . Chem. 263, 3079-3085. Unkeless, J. C., Scigliano, E., and heedman, V. H . (1988). Structure and function of human and niurine receptors for IgC. Annu. Rev. Imrnunol. 6, 251-281. Van Der Bosch, H. (1980). Intracellular phospholipases A. Biochirn. Biophys. Acru 604, 191-246. Weideman, M . J . , Peskar, B. A . , Wrogemann. K . , Reitschel, E. T. H . , Stdudinger, H., and Fischer, H . (1978). Prostaglandin and throniboxane synthesis in a pure macrophage population and the inhibition, hy E-typc prostaglandins, of chemiluminesccnce. FEBS Lerr. 89, I3h-140. Weiel, J . E., Hamilton, T. A., and Adams, D. 0. (1986). LPS induces altered phosphate labelling of proteins in murine pcritoneal mdcrophages. J . Immunol. 136, 3012-3018. Wightman, P. D., Humes, J. L., Davies, P., and Bonney, R. L. (1981). Identification and characterization of two phospholipabe A2 activities in resident mouse peritoneal macrophages. Biachem. J . 195, 427-10260. Yoshimoto, T., Soberman, R . J., Lewis, R. A., and Austen, K . F. (1985). Isolation andcharacterization of leukotriene C4 synthetase of rat basophilic leukemia cells. Proc. Nod. Acud. Sci. V.S.A. 82. 8399-8403.

CURRENT TOPICS I N MEMURANES AND TRANSPORT. VOLUME 35

Chapter 75 Role of Cyclic Nucleotides in Lymphocyte Activation VOLKHARD KAEVER AND KLAUS RESCH Division of Molecular Pharmacology Department of Pharmacology und Toxicology Medical School Hannover 0-3000 Hannover 61, Federal Republic of Germany

I.

Introduction Cyclic Nucleotides as Potential Activation Signals A. General Considerations B. Antigen Receptor-Dependent Phase of Lymphocyte Activation C. Lymphokine Receptor-Dependent Phase of Lymphocyte Activation 111. Modulatory Effects of Cyclic Nucleotides in Lymphocyte Activation A. T Lymphocytes B. B Lymphocytes IV. Interrelation of Cyclic Nucleotides with Other Signal Transduction Pathways: Possible Mechanisms of Cross-Talk V. Conclusions References 11.

1.

INTRODUCTION

Cyclic nucleotides are well-known cellular messengers of many rapid activation processes as can be observed after binding of hormones to their specific receptors. The role of CAMP and cGMP in lymphocyte activation, however, has been a matter of considerable controversy throughout the past years (see Hadden and Coffey, 1982; Kaever and Resch, 1985; Nel et al., 1987, and references cited therein). Without exogenous stimuli, lymphocytes are small resting cells with little cytoplasm, which exhibit low metabolic activity. On encountering their specific antigen, they start to grow and proliferate and assume their specific functions. 375 Copynghi 0 1990 by Academic Pres, Inc All right$ of reproduction in any form r e w v c d

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The activation of lymphocytes proceeds in at least two discrete sequential steps. In the initial phase, the cells are stimulated by their respectivc antigen, which in the case of I’lymphocytes must be recognized in the context of MHC molecules on the surface of antigen-presenting accessory cells, thereby being “pushed” from the G , into the G , phase of the cell cycle. Receptors for growth and differentiation factors are expressed at the cell surface in this phase, and these factors are lymphokines synthesized by T lymphocyte subsets. Helpcr T cells secrete the T cell mitogen interleukin 2 (IL-2), which seems to be under the regulatory control of thc nionokine interleukin I (IL-I) (Mizel, 1987) and the B ccll growth and differentiation Factors. Binding of these lymphokines to antigenpreactivated lymphocytes in the subsequent second phase leads to clonal proliferation and terminal differentiation (Cambier rt d.,1987b; Hadden, 1988; Knudsen et al., 1987). In T lymphocytes, the antigen receptor consists of an a i p heterodimer, which is exclusively responsible for antigen recognition (Ti), and a complex of five different associated proteins (T3 or CD3 complex) that have to date been implicated in signal transduction (Resch and Szaniel, 1988). The initial activation phase is characterized by increascd RNA and protein synthcsis and the development of enlarged blastoid cells (Hadden, 1987). In T lymphocytes these effects can also be achieved by various mitogens, such as concanavalin A (ConA) or phytohemagglutinin (PHA), which may bind to the T cell receptor complex, and monoclonal antibodies directed against Ti/T3 (Imboden et al., 1985) or other cell surface molecules such as T11 /CD2 (Bismuth et ul., 1988). In B lymphocytes a similar model of activation has been suggested, with an antigen-specific step leading to expression of surface la and interleukin receptors (Cambier et ul., 1987b; Muraguchi et al., 1984a). To examine signal transduction pathways via intracellular second messengers in this early phase of B cell activation, plant lcctins or monoclonal antiimmunoglobulin G antibodies (anti-k) havc been used as model substanccs (Coggeshall and Cambier, 1984). Proliferation and subscquent differentiation into antibody-secreting cells are directed by different interleukins and B cell stimulating factors such as IL-4, IL-5, and IL-6. Polyclonal B cell activators such as lipopolysaccharide (DeFranco et al., 1987; Jakway and DeFranco, 1986) can also lead to cell division but are not as effective as T cell products in causing terminal differentiation with the associated Ig class switching. As will be discussed in this review, it seems conceivable that neither CAMP nor cGMP serve as positive proliferation signals but may display modulatory roles in the different stages of T and B lymphocyte activation. The CAMP system, especially, in many cases provides an inhibitory signal via different mechanisms (Kammer, 1988).

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11. CYCLIC NUCLEOTIDES AS POTENTIAL ACTIVATION SIGNALS A. General Considerations Cellular responses induced by external stimuli are mediated by various intracellular molecules serving as second messengers. Among these, cyclic nucleotides, particularly CAMP, have been known for more than 30 years. Figure 1, in a very simplified manner, depicts the regulatory components of cyclic nucleotide synthesis, degradation, and action. 1. cAMP SYSTEM cAMP is formed from ATP by the action of a membrane-bound adenylate cyclase which consists of a complex composed of different receptors, regulatory

cellular response

FIG. 1 . Cyclic nucleotide aystem. Binding of an external ligand to stirnulatory (R,) or inhibitory (R,) receptors leads to the activation or inhibition of the membrane-bound adenylate cyclase (Acyclase) via atimulalory (G,) or inhibitory (G,)guanine nucleotide binding proteins. Newly synthesized cAMP stimulates a CAMP-dependent protein kinase (PKA), thereby inducing various cellular responses. In the case of cGMP, ii membrane-bound guanylate cyclase (G-cyclase), tightly coupled to its receptor, or a cytosolic isoenzyme (G-cyclase,) is responsible for the generation of this cellular messenger. In accordance with the CAMP system, a cGMP-dependent protein kinase (PKG) exerts the cellular response. Both cyclic nucleotides are inactivated by different phosphodiesterases (PDE). (3, stirnulatory effect; k, inhibitory effect.)

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guanine nucleotide-binding proteins (G proteins), and a catalytic subunit (Kammer, 1988; Williamson and Hansen, 1987). Binding of ligands to either stimulatory receptors R, (e.g., P-adrenergic agonists, histamine, prostaglandin E) or to inhibitory receptors Ri (e.g., a,-adrenergic or M,-muscarinergic agonists) results in activation or inhibition of cAMP formation, respectively. These receptor-mediated changes in adenylate cyclase activity are controlled by stimulatory (G,) or inhibitory (Gi) G proteins which are present in the plasma membrane (Neer and Clapman, 1988). Ligand-receptor interaction leads to dissociation of the a-subunit from the G protein apy-heterotrimer accompanied by exchange of a-bound GDP by GTP. a,li-GTP and possibly free P-y subunits are both effectors of enzyme activity (Casey and Gilman, 1988). Signals are terminated by an intrinsic GTPase activity of the a-subunit, leading to reassociation of the inactive GDP-apy complex. Cellular responses are mediated by type I and type I1 isozymes of a CAMP-dependent protein kinase (PKA), which dissociates after binding of cAMP into a complex of two regulatory subunits with bound cAMP and into two active catalytic subunits (Corbin et ul., 1988). Whereas the catalytic subunits exert their effects by phosphorylation of various substrates, the regulatory subunits-CAMP complex may function as the transport vehicle for CAMP, thus altering nuclear gene expression or cytoplasmic posttranscriptional events (Kammer, 1988). cAMP is readily hydrolyzed by different phosphodiesterases (PDE). Among these, calcium-calmodulin-dependent, cGMP-stimulated, low K,, and nonspecific CAMP-PDE can be distinguished (Beavo, 1988). Most interestingly, activated PKA causes stimulation of the CAMP-PDE, by that means decreasing the agonist-induced signal generation (Corbin et al., 1988).

2. cGMP SYSI'EM

In contrast to CAMP, the role of cGMP as biological mediator is much less clear, although findings assume its importance in the regulation of retinal function, vascular smooth muscle tone, and action of the atrial natriuretic factor (ANF) (Tremblay et a / . , 1988). cGMP can be produced by two different enzymes, either a membrane-bound or a cytoplasmic guanylate cyclase (Goldberg and Haddox, 1977). Maximal basal activities in vitro are achieved with manganese as cofactor for both enzymes, although calcium or magnesium can also be used (Hadden e t a / . , 1979). The soluble enzyme is stimulated by NO-containing compounds, [e.g., sodium nitroprusside (SNP)] (Biihme et ul., 19841, fatty acids (Hadden and Coffey, I982), and the endothelium-derived relaxing factor (EDRF) (Murad, 1988). The particulate guanylate cyclase is tightly coupled to the receptor for ANF and possibly other hormones. Like its PKA homologue, the cGMPdependent protein kinase (PKG) is thought to act mainly by substrate protein

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phosphorylation. cGMP is degraded by cGMP-specific as well as by nonspecific PDE (Beavo, 1988). 3. CAMP-cGMP SYSTEM I N LYMPHOCYTES Nearly all components of the cyclic nucleotide system described previously are present in lymphocytes. Lymphocytes possess surface receptors for a variety of hormones linked to the adenylate cyclase via G,. For example, P-adrenergic, histamine H,, and prostaglandin E receptors have recently been characterized on cloned mouse T cells (Dailey et al., 1988). On the other hand, an inhibitory adenosine Ri appears to be lacking on human T lymphocytes (Kammer, 1988). As in other cell types, no adenylate cyclase activity was found in the cytosol, whereas the highest specific activity was found in the plasma membrane of thymocytes (see Kaever et al., 1984, and references cited therein). Known effectors of the hormone-sensitive adenylate cyclase, such as manganese, molybdate, GTP or its nonmetabolized analogue guanosine 5’-[ Py-imido]trisphosphate(5’-guanyl imidobisphosphate) (GMP-PNP), sodium fluoride (NaF), and forskolin, were also active in these cells. In the case of NaF and GMP-PNP the maximal enzyme velocity was increased, whereas the affinity of the enzyme to its substrate was almost unchanged (Kaever et al., 1984). The presence of a soluble as well as of a particulate guanylate cyclase has been reported in T and B lymphocytes (Cille et a / . , 1983; Deviller e t a / . , 1975; Kaever et al., 1984). The plasma membrane-bound guanylate cyclase was efficiently stimulated by the nonionic detergent Triton X- 100 and by high concentrations of lysophosphatidylcholine, whereas the cytosolic guanylate cyclase could be strongly activated by sodium nitroprusside. Lymphocytes express cyclic nucleotide-dependent protein kinase activities (Grove and Mastro, 1987; Kammer, 1988; Russell, 1978). The existence of various phosphodiesterases hydrolyzing CAMP and cGMP has also been reported in these cells (Coffey et al., 1981; Coffey and Hadden, 1983; Deviller et al., 1975; Epstein et al., 1980; Takemoto et al., 1979). In our experiments, low K,-PDE activities were demonstrated in the cytoplasm (about 75% of total activity) and in the membrane fraction of calf thymocytes. Whereas the soluble enzymes metabolized both nucleotides with high maximal velocities, the enzyme activities in purified plasma membranes were significantly lower (V. Kaever and K. Resch, unpublished observations).

B. Antigen Receptor-Dependent Phase of Lymphocyte Activation One problem in analyzing initial events after activation of lymphocytes is the choice of the cell type. Most experiments reported were performed using human

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pcripheral blood lymphocyte (HPBL) and animal thyniocytes. For the initial attempt, such mixed cell populations, representing physiological conditions, are advantageous. Only if a biochemical response has been measured in those cell populations that are able to proliferate and differentiate on addition of activating ligands, docs the examination of isolated subpopulations or T and B cell lines seem advisable. After addition of an activating ligand to isolated lymphocytes, the permeability of the cell membrane for different ions, sugars, nucleosides, and amino acids will be increased. In addition to a reorganization of plasma membrane receptors, changes in the activities of various enzymes become detectable within minutes. Many of these initial processes (e.g., alterations in membrane potential, monovalent ion transport, cytosolic calcium and pH, phosphoinositide turnover, arachidonic acid metabolism, protein phosphorylation, and oncogene expression) are discussed in detail in other chapters of this volume. During the past years, considerable efforts have been made to understand the molecular mechanisms by which intracellular messengers, generated by specific receptor-ligand interaction, induce competence for subsequent cell progression. Cyclic nucleotides have been assumed to he implicated in lymphocyte activation by various authors (see Section 11, B, 1, this chapter). Elevated levels of cAMP as well as cGMP could lead to activation of the appropriate protein kinase and thereby initiate mitogcncsis by phosphorylation of specific nuclear proteins or cnzymes (Johnson and Hadden, 1975).

I . CHANGES I N C u c ~ r cNUCLEOTIDE LEVELS There are considerable contradictory data concerning rises of cyclic nucleotides in the early phase of lymphocyte activation. Some investigators reported increases in cGMP within minutes after stimulation of T lymphocytes with mitogens (Coffey et a/., 1977; Hadden e r a / ., 1972, 1976; Hui and Harmony, 1980; Krishnaraj and Talwar, 1973; Largen and Votta, 1983; Schumm el al., 1974; Webb et ul., 1975; Whitfield et al., 19741, whereas others, in apparently well controlled experiments, failed to detect reproducible changes in cGMP levels during the initial activation process (Atkinson et u l . , 1978; DeRubertis and Zcnser, 1976; Kaever and Kesch, 1985; MacManus ef al., 197.5; Watson, 1976; Wedner et a/., 197.5). Unchanged levels of cGMP after mitogenic activation were reported in B lymphocytes (Goodman and Weigle, 1981). On the other hand, increases in cAMP have been described after the activation of lymphocytes with optimal or supraoptimal concentrations of different mitogens (see Abell and Monahan, 1973, and references cited therein; see also Berridge, 1975; DeRubertis et al., 1974; Hadden et a / . , 1976; Smith et al., 197 I ;Takigawa and Waksman, 1981; Wang el ul., 1978; Watson, 1976; Wedner et a / ., 1975; Whitfield et al., 1974). These results led to the assumption that cAMP could putatively serve as

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the proliferation signal of activated lymphocytcs (Parker et ul., 1974; Parker, 1978). In Fig. 2 the effects of stimulating human peripheral blood lymphocytes (HPBL) with ConA for 15 min on cyclic nucleotide levels are shown. After addition of a mitogenic concentration of ConA, which would finally lead to cell proliferation (less than 20 kg/ml under the conditions employed), small increases in cAMP were detected, but no significant changes in cGMP levels could be measured. Similar results were obtained when lymphocytes were treated for increasing time periods of up to 30 min with 5 pg/ml ConA. In five experiments done with cells from different donors, cAMP always increased about 1.5- to 2fold, whereas in no case were cGMP levels significantly changed during this time period (Kaever and Resch, 1985). The inability of many investigators to measure elevated cGMP concentrations was interpreted to be a consequence of high cell densities, the use of different methods to stop the incubation, or the presence of an immunoinhibitory factor (“PDE blank”) or other interfering substances, which should have been removed before radioimmunoassay (Coffey et al., 1977; Largen and Votta, 1983). Therefore, the validity of the detection method for cAMP and cGMP is of crucial importance (Wedner et al., 1975). In our hands, spiked standard amounts of cyclic nucleotides were totally recovered (>95%) in resting as well as in activated cells. Elevated levels of cAMP or cGMP after

cGMP ln -

/ & L J

1

5 10 20 50 100 CmA[pg/rnl]

1

111111~

5 10 20 50 100 ConA[pg/rni]

FIG. 2 . Initial changes in cyclic nucleotide levels after addition of concanavalin A to human peripheral blood lymphocytes. Cclls (2 X 10h/200 (*I) were incubated for 15 min at 37°C at the indicated concentrations of concanavalin A (ConA). Cyclic nucleotides were determined by radioimmunoassay (0,CAMP; 0 , cGMP). (Adapted from Kacvcr and Resch, 1985, Fig. 1, p. 219.)

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addition of selective activators of the corresponding nucleotide cyclases (e.g., NaF, forskolin, SNP) could accurately be determined without additional chromatographic purification steps by using highly specific antibodies. There were no indications for the occurrence of interfering factors in ConA-activated lymphocytes (Kaever and Resch, 1985). In contrast to ConA, addition of hormones resulted in distinct increases in cyclic nucleotide concentrations. A selective elevation of cAMP was achieved by use of the P-agonist isoproterenol, whereas an increase in cGMP could be detected after addition of the a-agonist phenylephrine. From these data the concept of cGMP being a positive activation signal in the initial phase of lymphocyte activation has to be seriously questioned. At supraoptimal concentrations of ConA (more than 20 kg/ml), a dramatic increase in cAMP and simultaneously a slight decrease in cGMP were found (Fig. 2). Succinyl-ConA, however, was also able to activate resting lymphocytes but did not induce any cAMP increase (Hadden et a l . , 1976; Takigawa and Waksman, 1981). A prerequisite of lymphocyte activation is receptor crosslinking either by chemical means or by a specific, at least, divalent ligand (Kamrner, 1988). In contrast to the divalent succinyl-ConA, the tetravalent ConA molecule, in addition to activation, leads to an enhanced agglutination of the cells which may be responsible for the cAMP increase (Watson, 1976). Optimal proliferation can only be achieved by the occupancy of less than 20% of the mitogen binding sites (Allan and Crumpton, 1973). Further evidence indicates that this CAMP enlargement may represent a nonspecific reaction rather than a specific signal function. High concentrations of the nonmitogenic lectin wheat germ agglutinin, which does not induce lymphocyte proliferation, also led to a significant increase in cAMP (Greene et al., 1976; V. Kaever and K. Resch, unpublished observations). Findings indicate that ConA and PHA induce their mitogenic activity via CD3 (T3) and CD2 ( T l l ) cell surface molecules, respectively (Isakov and Altman, 1986). Accordingly, anti-CD3 antibodies (Ledbetter et al., 1986) and anti-CD2 antibodies (Carrera et a l . , 1988) have been shown to induce cAMP elevation in the human lymphoma cell line JURKAT (originated from helper T lymphocytes) or in HPBL without any change in cGMP. This prompted us to re-evaluate the effects of different anti-T cell receptor antibodies on cyclic nucleotide levels in these cells. Although JURKAT cells are autonomously proliferating tumor cells, they respond to activation through the T cell receptor by synthesizing and secreting IL-2 and offer the advantage of a homogeneous cell population absolutely free of contaminating monocytes. Initial events, such as phosphoinositide turnover and intracellular calcium increase, could be induced by using either a monoclonal anti-CD3 antibody (BMA 030) or a monoclonal anti-Ti aP heterodimcr antibody (BMA 031) in JURKAT cells (Sornmermeyer and Resch, 1989). As can be seen in Fig. 3, in JURKAT cells, hormonal stimulation by prostaglan-

383

15. CYCLIC NUCLEOTIDES IN LYMPHOCYTE ACTIVATION 1500

1000

s 500

100

FIG.3. Initial changes in cyclic nucleotide levels aftcr addition of various agents or monoclonal anti-T cell receptor antibodies to JURKAT cells. Cells (2 X 1061200 *I) were incubated for the M ,,). sodium nitroprusside (SNP, indicated times at 37°C with prostaglandin E2 (PGE,, M , +), concanavalin A (ConA, 10 pg/ml, M), different monoclonal anti-T cell receptor antibodies (BMA 030,O.1 Fgiml, A;BMA 031,j.O pgiml, V),or no additions (control, 0).Cyclic nucleotides were determined by radioimmunoassay as described (Kaever and Resch, 1985). Basal lcvcls (2.56 0.24 pmol cAMPi10’ cells, 0.29 2 0.03 pmol cGMP/107 cells) were set as 100%.

*

din E, evoked a dramatic and short-lasting increase in CAMP, whereas SNP increased cGMP during the entire incubation period. In contrast, neither ConA nor monoclonal anti-T cell receptor antibodies significantly augmentated CAMP or cGMP during 1 hr of incubation. Antibodies against the surface molecules CD5 and Tp44 have been shown to induce enhanced cGMP production in antiCD3 antibody-preactivated JURKAT cells, although only the former monoclonal increased the intracellular calcium level (Ledbetter et al., 1986).

2. CHANGES IN

THE

CORRESPONDING ENZYME ACTIVITIES

Increases in the intracellular concentrations of cyclic nucleotides depend on an activation of the corresponding nucleotide cyclases induced by the receptorligand interaction. In the case of cGMP, either the plasma membrane-bound or the soluble guanylate cyclase could be affected. Increased guanylate cyclase activities in the early phase of lymphocyte activation by mitogens have been described (Coffey and Hadden, 1981; Deviller et al., 1975; Hadden et al., 1979). Elevated cGMP levels by activating the cGMP-dependent protein kinase (Carpentieri et d.,1981; Largen and Votta, 1983) were assumed to phosphorylate

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specific nuclear proteins, thus initiating mitogenesis (Johnson and Hadden, 1975). An incrcasc in RNA synthesis in isolated nuclei (Ananthakrishnan ei a l . , 198 1) induced by an activation of RNA polymerases types I and 111 (Hadden and Coffey, 1982) has been interpreted to be a result of PKG stimulation by cGMP. In our hands, neither the particulate guanylate cyclase in highly purified plasma membranes nor the soluble isozyme was stimulated within 4 hr of mitogen addition to thymocytes (Kaever and Resch, 1985). Similar data were also reported by others (DeRubertis and Zcnser, 1976). Unchanged activities of the soluble or membrane-bound cGMP-dependent PDE have been measured after addition of PHA to HPBL (Coffey ei ul., 1981). In accordance with the described augmentation of CAMPby various mitogens, enhanced activities of adenylate cyclase have been reported (DeKubertis er al., 1974; Kaevcr and Resch, 1985; Krishnaraj and Talwar, 1973; Smith e l ul., 1971). This stimulation lasted for about 1 hr in rabbit thymocytes, but 2 hr after ConA addition, differences in adenylate cyclase activities between resting and activated cells were no longer obvious (Kaever and Kesch, 1985). One explanation could be that the permanent influence of the lectin desensitizes the adenylate cyclase system, as known from hormonal stimulation. Data on the induction of the PKA activity by mitogenic agents are also rather conflicting. An increase in cytosolic PKA type I without change of the membrane-associated PKA type I1 activity by ConA has been reported (reviewed in Kammer, 1988). In contrast, stimulation of PKA type 11, and not type I, was found within minutes (Grove and Mastro, 1987) or within 4 hr (Russell, 1978) of lymphocyte activation. Alterations in CAMP-dependent PDE were not detected during the initial activation phase, but, at the earliest, after 4 hr of mitogen addition. Maximal effects were measured after 1-3 days (Epstein et ul., 1980; Takemoto et ul., 1979).

C. Lymphokine Receptor-Dependent Phase of Lymphocyte Activation Pure cytokines have become available as recombinant material only very recently. In addition, none of the cytokine receptors has been fully characterized so far. Thus, the amount of information on signal transduction is much more limited than that known from the antigen receptor-mediated phase. In the late phase of lymphocyte activation, consisting of proliferation and terminal differentiation, second messenger systems similar or identical to those described in the early phase could be operative, with only a different ligand binding to a newly expressed receptor. Alternatively, distinct mechanisms of signal transduction could be used (Nel et u/., 1987). One serious problem in considering the role of cyclic nucleotides in this phase is the presence of accesso-

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385

ry cells that may also produce cAMP and cGMP in comparable amounts to lymphocytes. Compared to the antigen receptor-mediated phase of lymphocyte activation very little data are available. In the case of T lymphocytes, effects of IL-1 and IL-2 on cyclic nucleotide levels have been described. IL I , which may possibly exert its action via the enhanced induction of IL-2 synthesis and IL-2 receptor expression, has been reported to increase cGMP levels 12-20 hr after its addition to pre-activated cells (Coffey and Hadden, 1985; Hadden et at., 1979; Katz et al., 1978; Wagshal and Waksman, 1978). Accordingly, I L 2 led to a slight elevation of cGMP within minutes (Hadden et al., 1987; Knudsen et ul., 1987). These results could provide evidence for a second messenger role of cGMP in the lymphokine-dependent phase of T cell activation (but see also Section 111, this chapter). The role of cAMP in the late phase of T lymphocyte activation may be even more complicated. As a rise and subsequent fall of cAMP seem to be necessary shortly before the onset of cellular DNA synthesis (Takigawa and Waksman, 1981 ; Wang et d., 1978), it has been suggested that the function of IL-2 could be to remove the CAMP-induced proliferation block by lowering the cellular cAMP content (Beckner and Farrar, 1986; Kammer, 1988). On the other hand, addition of IL-2 to PHA-pretreated human T lymphocytes resulted in a cAMP increase (Wickremasinghe et a[., 1987). Additionally, it had previously been noted that IL-1 led to a cAMP elevation in murine thymocytes within 10 min (Shirikawa et al., 1988). There are no data available concerning cyclic nucleotide levels or corresponding enzyme activities in pure B lymphocytes after addition of different lymphokines such as IL-4, 1L-5, and IL-6.

111. MODULATORY EFFECTS OF CYCLIC NUCLEOTIDES IN LYMPHOCYTE ACTIVATION According to Sutherland’s criteria (Robison e f al., 1971), cyclic nucleotides could only be regarded as true second messengers if the exogenous addition of the putative messenger results in comparable cellular changes as induced by the activating ligand. Due to their low membrane permeability, high concentrations (up to M ) of cyclic nucleotides have been used to significantly increase the intracellular level of cAMP or cGMP. In such an experimental design, one cannot exclude the possibility that adenosine or guanosine, which themselves can stimulate receptor-mediated events, may be formed by hydrolyzing enzymes released from damaged cells. In most cases, chemical modifications of cAMP or cGMP have been used, therefore, to facilitate cell membrane penetration. In the case of dibutyryl-CAMP (db-CAMP) or db-cGMP, free butyrate may be present in the incubation medium and directly influence different enzyme activities.

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VOLKHARDKAEVERANDKLAUSRESCH

Other approaches to artificially increase the cellular cyclic nucleotide content include the addition of hormonal agonists, PDE inhibitors such as isobutylmethylxanthine, or the utilization of specific effectors of the corresponding nucleotide cyclases. Forskolin, as direct activator of adenylate cyclase (Daly, 1984), led to a prompt cAMP increase in HPBL, whereas SNP induced activation of the soluble guanylate cyclase and thereby a risc in cGMP (Kaever and Resch, 1985).

A. T Lymphocytes The addition of cAMP or analogues in low concentrations has been reported to slightly increase thymidine incorporation into cellular DNA (Averner et al., 1972; Cross and Ord, 1971; Dumont e t a / . , 1989; Krishnaraj and Talwar, 1973; MacManus and Whitfield, 1969). In most cases, especially when cAMP or derivatives were used in higher concentrations, DNA synthesis was significantly decreased (Abell and Monahan, 1973; DeRubertis and Zenser, 1976; Hirschhorn et al., 1970; Krishnaraj and Talwar, 1973; May rt al., 1970; Smith er al., 1971; Watson, 1976; Webb et al., 1975). Such a reduced ability of cell proliferation was also followed by elevating the cAMP level through prostaglandins or PDE inhibitors (Berridge, 1975; Friedman, 1976; Parker, 1979). When added to resting HPBL in concentrations that elevated cAMP levels of these cells at least during the first hour of incubation, forskolin did not influence cell activation, shown by the incorporation of 13H]uridine into RNA (Fig. 4). However, activation of lymphocytes by ConA was largely diminished by increasing concentrations of forskolin, with a maximal inhibition of RNA synthesis of about 67% at a forskolin concentration of 0. I mM (Fig. 4). When forskolin was added at different times after the onset of ConA stimulation (up to 20 hr), the inhibition of ["luridhe incorporation was reduced but still measurable (data not shown). From these data, it seems reasonable that cAMP does not have an activating function in T lymphocytes but, more likely, in addition to inhibition of early activation steps, reveals direct inhibitory effects on cellular RNA synthesis or the modulation of processes following the initial activation phase (Abell and Monahan, 1973; Watson, 1976; Wang et ul., 1978). In order to confirm the cAMP antiproliferative potency, further experiments were performed with a murine T helper cell clone (Weiss et al., 1986). Figure 5 shows that ConA-induced DNA synthesis could be nearly totally blocked by prostaglandin E, (PGE,) added at the beginning of mitogenic activation. In contrast to resting control cells, CAMP was significantly enhanced in PGE,treated cells during the first 24 hr of incubation (Fig. 5). In these pure T cells only about 40% of DNA synthesis stimulated by IL2 could be inhibited by PGE, (data not shown). This is in agreement with earlier observations showing an inhibition of 1L-2 production and IL-2 action in T lymphocytes or T cell hybridomas by elevated cAMP levels (Averill et al., 1988; Aussel et al., 1988; Chouaib et al., 1985;

-8

-7

-6

-5

-4

log[MI FIG. 4. Influence of cyclic nucleotide-elevating substances on basal or concanavalin A-induced RNA synthesis in human peripheral blood lymphocytes. Cells (4 X 1051200 pI) were incubated for 24 hr with forskolin or sodium nitroprusside (SNP) at the indicated concentrations in the presence or absence of concanavalin A (10 pgiml). ['HIUridine was added during the last 4 hr of the incubation time. A , forskolin; 0 forskolin + ConA; A, SNP; 0 , SNP + ConA. (Adapted from Kaever and Resch, 198.5, Fig. 4, p. 222.)

1 -12

FIG.5 . Effect of prostaglandin E, on proliferation and cAMP synthesis in a T helper cell clone. Proliferation was determined after stimulating the cells (2 X 104/200pl) with concanavalin A (2 basal pgiml) for 44 hr in the presence of prostaglandin EZ at the indicated concentrations (0, [3H]thymidine incorporation; 0 , ConA-stimulated [3H]thymidine incorporation). [3H]Thymidine was added during the last 20 hr of the incubation time. cAMP was measured by radioimmunoassay (see Kaever and Resch, 1985) after incubation of the cells ( I X 105/200PI) with prostaglandin Ez (10-7 M ) for the indicated times.

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VOLKHARD KAEVER AND KLAUS RESCH

Gilniore and Weiner, 1988, Hayari et ul., 1985; Iwaz e t a / . , 1986; Makoul et u / . , 1985; Mary et al.. 1987, Tracey et al., 1988; Woogen et al.. 1987). On the other hand, it has previously been reported that addition of very low concentrations of CAMP (pM instead of mM) resulted in enhanced IL-2 receptor expression (Shirikawa et u / . , 1988). In cytotoxic T lymphocytes (CD8 iLy2 ), elevation of the intracellular CAMP level led to an inhibition of T cell-mediated cytotoxicity. (Gray et al., 1988; Saitoh e t a / ., 1988; Zhang et al., 1987). In addition, I L 2 receptor gene expression was diminished by CAMPin these cells (Farrar et d.,1987). Natural killer cell activity was inhibited, too, by db-CAMP without disturbing target cell binding (Steele and Brahmi, 1988) or by monomeric IgG, leading to a CAMP increase (Bancu et al., I988), although cAMP was also reported to induce IL-2 rcceptor gene activation in these cells (Narumiya et a / . , 1987). The induction of suppressor cell activity has also been proposed to be mediated by cAMP increases (Almawi et al., 1987; Parnham and Englberger, 1988). Taken together, there is clear evidence that CAMP at higher concentrations, which, however, can easily be induced by physiological stimuli such as various hormones, exerts inhibitory effects. Although evidence is scarce, one cannot completely ignore that low increases of CAMP levels promote activation. Exogenously added cGMP, db-cGMP, or 8-bromo-cGMP did not initiate the onset of cell proliferation of pure T lymphocytes (Hadden, 1977; Knudsen e f al., 1987; Krishnaraj and Talwar, 1973; Wedner et al., 1975; Weinstein et al., 197.5). Addition of prostaglandin F,,, which is believed to induce cGMP production in lymphocytes, did not affect IL-2 production or T cell proliferation (Chouaib e f d . , 1985; Tracey et al., 1988). In contrast, db-cGMP was reported to inhibit I L 2 dependent proliferation (Knudsen c’t ul., 1987; Maca, 1984). Another possibility for specifically increasing cGMP in lymphocytes, without changing the CAMP level, consists of the stimulation of the soluble guanylate cyclase by SNP. In our experiments and those reported by others (Atkinson et al., 1978), concentrations of SNP that led to marked cGMP increases did not initiate the activation of resting HPBL and did not influence cellular activation caused by ConA (Fig. 4). Accordingly, carbamylcholine augmented the lymphocyte cGMP level without being mitogenic (DeRubertis and Zenser, 1976). Immunosuppression induced by metabolites from the lipoxygenase pathway has been assumed to result from the generation of suppressor cells following an augmentation of lymphocyte cCMP (Mexmain et a/., 198.5). +

+

B. B Lymphocytes Similar to T lymphocytes, the role of CAMP in B cell proliferation, terminal differentiation, and subsequent antibody secretion has been controversial. B cellspecific cytokines have been isolated and cloned only very recently, and the molecular structures of their receptors are not well defined. In experiments with

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unseparated HPBL or spleen lymphocytes, agents enhancing CAMP concentrations have led to a decrease in LPS-, anti-p-, or lymphokine-induced proliferation (Muraguchi et ul., 1984b; Woogen et al., 1987) and to generation of immunoglobulin secreting cells (Simkin el al., 1987). Isolated B cells, in the presence of antigen and 1L-1-1L-2, synthesized significant amounts of antibodies only after addition of CAMP, which caused translocation of the protein kinase C (PKC) to the nucleus (Cambier et ul., 1987a). In the presence of T cells, antibody production was greatly diminished due to the CAMP-induced inhibition of helper cell functions (Gilbert and Hoffmann, 1985; Hoffmann, 1987; Kammer, 1988). It has been reported that IL-1 and CAMP are synergistic in inducing B cell proliferation, whereas IL-4 and cAMP diminish proliferation, but in both cases enhanced antibody production was obvious (Hoffmann, 1988). On the other hand, in a B cell line, early increases in CAMP (within 2 hr) have been correlated with inhibition of antibody secretion (Shearer et al., 1988b). High concentrations of cGMP or 8-bromo-cGMP (more than M ) marginally stimulated RNA or DNA synthesis in mouse B lymphocytes (Diamantstein and Ulmer, 1975a; Weinstein et a/., 1974). The action of cGMP in this cellular system was not exclusively a direct one but, at least in part, was mediated by adherent cells probably via IL-I (Diamantstein and Ulmer, 1975b). In addition, the mitogenic activity of 8-bromo-cGMP was much lower than that of 8-bromo-GMP or 8-bromoguanosine, which did not raise cGMP levels (Goodman and Weigle, 198 1 ; DeRubertis and Zenser, 1976).

IV. INTERRELATION OF CYCLIC NUCLEOTIDES WITH OTHER SIGNAL TRANSDUCTION PATHWAYS: POSSIBLE MECHANISMS OF CROSSTALK As pointed out previously, it seems unlikely that CAMPand cGMP are involved as intracellular messengers in the induction of lymphocyte proliferation, both in T and B lymphocytes. On the basis of available evidence, changes in cGMP levels do not appear to affect activation, whereas cAMP evidently is able to modulate the extent of activation. Several effects of CAMP have been described in lymphocytes, among which one or more could be responsible for the specific cellular effects. Elevated CAMP levels inhibited T cell receptor (O’Shea et al., 1987), IL-2 receptor (Goulton and Eardley, 1986), and other protein (Chaplin et al,. 1980) phosphorylation in antigen- or mitogen-activated cells. Additionally, modulation of potassium channels (Choquet et a / ., 1987), increase in the mobility of surface antigens (CD3, CD4, CD8) (Kammer et al., 1988), decrease in the T cell receptor a and 6 mRNA lcvels (Martinez-Vdldez et a / . , 1988), and inhibition of IL-2 synthesis (Aussel et al., 1988, Mary et al., 1987) and macromolecular events, such as gene expression and enzyme production stimulated by 1L2 (Farrar et al,. 1987, 1988), have been correlated with enhanced cAMP levels. Decreased protein

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phosphorylation and IL-2 synthesis could be restored by the PKA inhibitor H-8 (Averill et ul., 1988). cAMP interacts with the activation of phosphoinositide turnover induced by ligand-receptor interaction, by that means decreasing phosphatidylinositol 4,5-bisphosphate breakdown and subsequent increase in intracellular free calcium levels and PKC activity in T and B cells (Bismuth er al., 1988; Chouaib rt ul., 1985, 1987; Coggeshall and Cambier, 1984; Kaibuchi et al., 1982; Lerner et al., 1988; Mire-Sluis et al., 1987; Otani et al., 1984; Patel et ul., 1987; Shearer et al., 1988a; Takayama et al., 1988). All effects described could therefore be mediated by a CAMP-PKA-mediated abrogation of this signal transduction pathway. The precise point of interference (PUS, PKC, or calcium release) can be investigated by using PKC activators such as the phorbol ester TPA together with calcium ionophore, thereby circumventing the initial PLC activation. If under those experimental conditions the CAMP-mediated effect is still obvious, direct inhibition of PKC activity or calcium release has to be assumed. Unfortunately, contradictory results have been reported in the literature. Whereas in several cases effects of TPA-calcium ionophore were not influenced by CAMPelevating agents (Bismuth et ul., 1988; Chouaib et al., 1987; Shenker and Matt, 1987; Simkin e t a f . , 1987), inhibition of cellular activation by these agents has also been shown (Takayama rt ul., 1988).Stimulation of PLC by aluminum fluoride or GTPyS was also lessened by the simultaneous generation of CAMP (Mire-Sluis rt a / . , 1987). At least in the human T cell line JURKAT, an additional mechanism of CAMPinduced inhibition of the PLC-mediated signal transduction pathway, not involving PKA activation, may be operative. Cholera toxin, an irreversible activator of the adenylate eyclase stimulatory G, protein, inhibited anti-T cell receptor antibody-induced increases of inositol trisphosphate and of cytoplasmic free calcium. An increase in the cAMP level by forskolin, a direct activator of the adenylate cyclase catalytic subunit, however, did not mimic the cholera toxin effect (Imboden et al., 1986). In our experiments (Sommermcyer and Resch, 1989) these results could be confirmed. PGE,, also increasing adenylate cyclase activity via G,, however, had effects similar to cholera toxin (data not shown). As PUS activity is also regulated by so far ill-defined G proteins, from these data a cross talk between the adenylate cyclase and the PLC system at the G protein level seems reasonable. On the premise that similar or even identical G proteins are used in both systems, stimulation of one system could diminish the response of the other one due to a decreased availability of activated G,-GTPor Py subunits. Additional work will still be necessary to further elucidate such possible interactions.

V.

CONCLUSIONS

From the data discussed in this review it has to be assumed that neither cAMP nor cGMP are involved as second messengers in the antigen receptor-mediated

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initiation of lymphocyte activation. Increases in cAMP after mitogenic activation of T cells result from additional agglutinating effects of multivalent lectins, whereas divalent ligands induce early cellular events and competence for the action of cytokines without cAMP elevation. Dramatic rises in cGMP levels within minutes after addition of plant lectins or monoclonal antibodies directed against surface molecules to T cells have been reported, but these findings could not be reproduced by all investigators. The concept of cGMP being a positive activation signal in the early phase of lymphocyte activation is also questionable because intracellular rises of cGMP through specific activation of the guanylate cyclase do not influence basal or mitogen-induced RNA synthesis. Only few data concerning changes in cyclic nucleotide levels after binding of interleukins to their newly expressed receptors in the late phase of lymphocyte activation have been reported. Further experiments will clearly be necessary to clarify the significance of these findings. On the other hand, there is good evidence that cAMP mediates the effects of regulatory hormones, such as prostaglandin E, or P-adrenergic agonists, and thereby modulates various cellular functions. At least in high concentrations existing for prolonged times, which can also be found in physiological situations, cAMP exerts mostly inhibitory effects in the early as well as in the lymphokine receptor-dependent phase of lymphocyte activation. However, it still remains possible that peak increases only at short and definite time points promote the onset of cell cycle progression. In B lymphocytes, CAMP, too, does not appear to be involved in proliferation. Several reports, however, point to an important role in the differentiation into antibody-secreting cells. The CAMP-induced inhibition of immunoglobulin production observed in mixed cell populations obviously depends on inhibitory effects of cAMP on T helper cell functions. At the molecular level there is initial insight into the mechanisms of cAMP interference with other signal transduction pathways. Many of the cAMP effects, for example, the abrogation of specific ligand-induced phosphoinositide turnover, are mediated via activation of the CAMP-dependent protein kinase. At least in JURKAT cells, an interference of the cAMP signal transduction pathway with the phospholipase C system takes place at levels proximal to cyclic nucleotide formation, probably at the regulatory G protein level. ACKNOWLEDGMENTS The help of all our collaborators is gratefully acknowledged, particularly H. Sommermeyer for thc contribution of unpublished data and inany helpful discussions. We would like to thank Dr. R. Kurrle, Behring Werke, Marburg, F. R. G., for providing us with monoclonal anti-T cell receptor antibodies BMA 030 and BMA 03 I . REFERENCES Abell, C . W., and Monahan, T. M. (1973). The role of adenosine 3',5'-cyclic monaphosphate in thc regulation of mammalian cell division. J . Cell B i d . 59, 549-558.

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Allan, D., and Crunipton, M. J. ( 1973). Phytohemagglutinin-lyrnphocyte intcraction. Characterization of binding sites on pig lymphocytes for “’J-labelled phytolicriiagglutinin. E t p Cell Rcs. 78, 271-278. Almawi, W. Y.,Murphy, M. G.,Ogbaghehriel, A.,andPope,B. L. (1987). Cyclic AMPasthcsecond messenger for prostaglandin E, in modulating suppressor cell-activation by natural suppressorlcytotoxic cells. Int. J . Immunopharmuc,ol. 9, 697-704. Ananthakrishnan, R . , Cofiey, R. G.. and Hadden, J. W. (1981). Cyclic GMP and calcium in lymphocyte activation by phytohcinagglutinin. Lymphocyte Difer. 1, 183- 196. Atkinson, J. P., Kelly, J. P., Weiss, A.. Wcdner, H. J., and Parker, C. W. (1978). Enhanced intracellular cGMP concentrations and lectin-induced lymphocyte transformation. J . Immunul. 121, 2282-2291. Aussel, C.. Mary, D., Peyron, J.-F.. Pclassy, C., Ferma, B . , and Fehlmann. M. (1988). Inhibition and activation of interleukin 2 bynthesis by dii-cct modification of guanosine triphosphatc-binding proteins. J . Immunol. 140, 215-220. Averill, L. E., Stein, R. L., and Kammer, G. M. (1988). Control of human T-lymphocyte interleukin-2 production by a CAMP-dependent pathway. Cell. ImmrtnoL. 115, 88-99. Averner, M. J., Brock, M. lz., and lost, I. P. (1972). Stirnulation of ribonucleic acid synthesis in horsc lymphocytes by exogcnous cyclic adenosinc 3’,5’-n~onophosphate.J. B i d . Chem. 247, 4 13-4 17. Bancu, A. C . , Gherman, M., Sulica, A , , Goto, T., Rrrar, W., and Herberman, R. B. (1988). Regulation of human natural cytotoxicity by IgG. 11. Cyclic AMP as a mediator of monomeric 1gG-induced inhibition of natural killer cell activity. Cell. Imtnunol. 114, 246-~256. Bcavo, J. A. (1988). Multiple isozymes of cyclic nucleotide phosphodiesterase. In “Advances in Second Meshenger and Phosphorylation Research” (P. Greengard and G . A. Robison, eds.), Vol. 22, pp. 1-38. Raven, New York. Beckner, S K . , and Rrrar, W. L. (1986). Interleukin 2 modulation of adenylatc cyclase. Potential role of protein kinase C. J. B i d . Chem. 261, 3043-3047. Bcrridge, M. J. (1975). The interaction of cyclic nucleotides and calcium in the control of cellular activity. A h . Cyclic Nuc/eu/i& RL‘s. 6, I-9X. Bismuth, G . . Theodorou, I., Gouy, H., LeGouvcllo, S . , Bernard, A., and Debrk, P. (1988). Cyclic AMP-mediated alteration of the CD2 activation process in human T lymphocytes. Preferential inhibition of the phosphoinositide cycle-relatcd transduction pathway. Eur. J . Immunol. 18, 13.51- 1357. Bohmc, E., Grossmann, G . , Herz, I., Miilsch, A., Spies, C . , and Schultz, G . (1984). Regukdtion of cyclic GMP formation by soluble guanylate cyclase: Stimulation by NO-containing compounds. Adr: Cyclic Nitdeotide Pro/cin Fhospliorylrition Ros. 17, 259-266. Cambier, J. C., Newell, M. K., Justement, L. B., McGuire. J. C., Leach, K. L., and Chen, Z. Z. ( I987a). Ia binding ligands and CAMP stimulate nuclear translocation of PKC in B lymphocytes. Nature (London) 327, 629-632. Camhier, J. C., Justcment, L., Newell, M., Chen, Z., Harris, L., Sandoval, V., Klemsz, M., and Ransom, J. ( I987b). Transmembrane signals and intracellular “sccond messengers” in the regulation of quiescent B-lymphocyte activation. Irnrnunol. Rev. 95, 37-57, Carpentieri, U . , Minguell, J. J., and Gardner, F. H. (1981). Adenylate cyclase and guanylate cyclase activity in normal and leukemic human lymphocytes. Blood 57, 975-978. Carrera, A . C., Rincbn, M . , De Landizuri, M. O., and L6pcz-Botet, M. (1988). CD2 is involved in rcgulating cyclic AMP levels in T cells. Eur. J . Immutiol. 18, 961-964. Casey, P. J . , and Gilman, A. G.(1988). G protein involvement in receptor-effector coupling. J . R i d . Chrm. 263, 2577-2580. Chaplin, D. D., Wedner, H. J., and Parker, C. W. (1980). Protein phosphorylation in human peripheral blood lymphocytes: Mitogen-induced increases in protein phosphorylation in intact lymphocytes. ./. Immrmol. 124. 2390-2398.

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in immature thymocytes and mitogen-primed T-lymphocytes. I n f . J . Immunopharmacol. 9, 85 1-857. Hayari, Y . , Kukulansky, T., and Globerson. A. (1985). Regulation of thymocyte proliferative rcsponsc by macrophage-derived prostaglandin E, and interleukin I . Eur. J . Immunol. 15,43-47. Hirschhorn, R., Grossman, J., and Weissmann, G. (1970). Effect of cyclic 3',5'-adenosine monophosphate and theophylline on lymphocyte transformation. Pruc. Soc. Exp. B i d . Med. 133, 1361 1365. Hoffmann, M. K. (1987). An alternative B cell activation mechanism mediated by macrophages through the release of cyclo-oxygenase pathway products and interleukin 1. Lymphokine Res. 6, 299-308. Hoffmann, M. K. (1 988). The requirement for high intracellular cyclic adenosine monophosphate concentrations distinguibhes two pathways of B cell activation induccd with lymphokines and antibody to immunoglobulin. J . Immunol. 140, 580-582. Hui, D. Y., and Harmony, I. A. K. (1980). Inhibition by low density lipoproteins of mitogenstimulated cyclic nucleotide production by lymphocytes. J . B i d . Chem. 255, 1413-1419. Imboden, J. B., Weiss, A., and Stobo, J . D. (1985). Transmcmbrane signalling by the T3-antigen receptor complex. Immunol. Today 6, 328-331. Imboden, J. B., Shoback, D. M., Pattison, G., and Stobo. 1. D. (1986). Cholera toxin inhibits theTcell antigen receptor-mediated increases in inositol trisphosphate and cytoplasmic free calcium. Proc. Narl. Acad. Sci. U.S.A. 83, 5673-5677. Isakov, N., and Altman, A. (1986). Lymphocyte activation and immune regulation, Immunol. Today 7, 155-157. Iwaz, J., Lafont, S., and Revillard, J. P. (1986). Elevation of cyclic 3'5'adenosine monophosphate levels by cholera toxin inhibits the generation of interleukin 2 activity. Cek Immunol. 103, 455-461. Jakway, 3. P., and DeFranco, A. L. (1986). Pertussis toxin inhibition of B cell and macrophage responses to bacterial lipopolysaccharide. Science 234, 743-746. Johnson, E. M., and Hadden, J . W. (1975). Phosphorylation of lymphocyte nuclear acetic proteins: Regulation by cyclic nucleotides. Science 187, 1198-1200. Kaevcr, V., and Resch, K . (1985). Are cyclic nucleotides involved in the initiation of mitogenic activation of human lymphocytes'? Biochim. Biophys. Acra 846, 216-225. Kaever, V., Szamel. M., Goppelt, M., and Resch, K. (1984). Characterization and subcellular localization of nucleotide cyclases in calf thymus lymphocytes. Biochim. Biophys. Acra 776, 133- 143. Kaibuchi, K., Takai, Y., Ogawa, Y . , Kimura. S., Nishizuka, Y., Nakamura, T., Tonomura, A., and Ishikawa, A . (1982). Inhibitory action of adenosine 3'S-monophosphate on phosphatidylinosito1 turnover. Biochem. Biophys. Res. Commun. 104, 105-1 12. Kammer, G. M . (1988). The adenylate cyclase-CAMP-proteinkinase A pathway and regulation of the immune response. Immunol. Today 9, 222-229. Kammer, G. M., Boehm, C. A., Rudolph, S . A . , and Schultz, L. A. (1988). Mobility of the human T lymphocyte surface molecules CD3, CD4. and CDX: Regulation by a CAMP-dependent pathway. Proc. Natl. Acad. Sci. U.S.A. 85, 792-796. Katz, S. P . , Kierszenbaum, F., and Waksman, B. H. (1978). Mechanism of action of "lymphocyteactivating factor" (LAF). 111. Evidence that LAF acts on stimulated lymphocytes by raising cyclic GMP in G , . J . Immunol. 121, 2386-2391. Knudsen, T. E., Larsen, C. S., and Johnson, H. E. (1987). A study of cyclic nucleotides as second messengers after interleukin 2 stimulation of human T lymphocytes. Scand. J . Immunol. 25, 527-53 I . Krishnaraj, R., and Talwar. G. P. (1973). Role of cyclic AMP in mitogen induced transformation of human peripheral leukocytes. J . Immunol. 111, 1010-1017. -

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Lagen, M. T., and Votta, B. (1983). lmmunocytocheniical evidence for 3’,5’-cGMP and 3 ‘ 3 cGMP-dependent protein kinase involvement in lymphocyte proliferation. In ”Journal of Cyclic Nucleotide and Protein Phosphorylation Research” (G. Rrooker and R. W. Butcher, eds.), Voi. 9, pp. 231-244. Raven, New York. Ledbettcr, 1. A., Parsons, M.. Martin, P. J., Hanscn. J. A , , Rabinovitch, P. S., and June, C. H. (1986). Antibody binding to CD5 (Tp67) and Tp44 T cell surface molecules: EEects on cyclic nucleotides, cytoplasmic free calcium, and CAMP-mediated suppression. J . Immunol. 137, 3299-3305. Lerncr. A.. Jacobson, B . , and Miller, R. A. (1988). Cyclic AMP concentrations modulate both calcium flux and hydrolysis of phosphatidylinositol phosphates in mouse T lymphocytes. J. Immunol. 140, 936-940. Maca, R. D. (1984). The effects of cyclic nucleotides on the proliferation of cultured human Tlymphocytes. Irnmunopharmacolugy 8, 53-60, MacManus, J. P., and Whitfield, J. F. (1969). Stimulation of DNA synthesis and mitotic activity of thymic lymphocytes by cyclic adenosine 3’,5’-monoptiosphate. E,rp. Cell Rex 58, 188- 191. MacManus, J. P., Whitfield, J. F., Boynton, A. L.. and Rixon, R. H. (1975). Role of cyclic nucleotides and calcium in the positive control of cell proliferation. Adv. C.yc/icNucleotide Res. 5 , 7 1 9 734. Makoul, G . T.. Robinson, D. R . , Bhalla, A. K., and Glimcher, L. H. (1985). Prostaglandin E2 inhibits the activation of cloned T cell hybridomas. J. Immunol. 134, 2645-2650. Martincz-Valdez. H., Thompson. E., and Cohen, A. (1988). Coordinate transcriptional regulation of a and 6 chains of the T-cell antigen receptors by phorbol esters and cyclic adenosine 5’monophosphate in human thymocytes. J . B i d . Chem. 263, 9561-9564. Mary. D., Aussel, C . , Ferrua, B . , and Fehlinann, M. (1987). Regulation of interleukin 2 synthesis by CAMP in human T cells. J. Immunol. 139, 1179-1 184. May, C. D., Lyman, M., and Alberto, R. (1970). Effects of compounds which inhibit lymphocyte stimulation on the utilization of glucose by leukocytes. J. ANergy 46, 21-28. Mcxniain, S., Cook, J., Aldigier, J.-C., Gualde, N., and Rigaud, M. (1985). Thymocyte cyclic AMP and cyclic GMP response lo treatment with metabolites issued from the lipoxygenase pathway. J. Immunol. 135, 1361-1365. Mire-Sluis, A. R . . Hof€brand, A. V.. and Wickremasinghc, R . G. (1987). Evidence that guaninenucleotide binding regulatory proteins couple cell-surface reccptors to the breakdown of inositol-containing lipids during ‘r-Lymphocyte mitogenesis. Biochem. Biophys. Kes. Conintun. 148, 1223-1231 Mizel, S . B . (1987). Interleukin 1 and T-cell activation. Immunol. Toduy 8 , 330-332. Murad, F. (1988). Round table on cyclic GMP and atrial natriuretic factor. In ‘’ Advances in Second Messenger and Phosphoprotein Research” (R. Adelstein, C. Klee, and M. Rodbell, eds.). Vol. 21, pp. 101-103. Raven, New York. Muraguchi, A . , Kehrl, J. H., Butler, J. L., and Fauci, A. S . (1984a). Regulation of human B-cell activation, proliferation, and differentiation by soluble factors. J . Clin.Immunol. 4, 337-347. Muraguchi, A., Miyazaki, K . , Kehrl, J. H., and Fauci. A. S . (1984b). Inhibition of human B cell activation by dilerpinc forskolin: Interference with R cell growth factor-induced G, to S transition of the H cell cycle. J. Imrnunol. 133, 1283-1287. Narumiya, S.,Hirata. M., Nanba, T., Nikaido, T., Taniguchi, Y . , Tagaya, Y., Okadd, M., Mitsuya, H., and Yodoi, J. (1987). Activation of interleukin-2 receptor gene by forskolin and cyclic AMP analogues. Biochem. Biophys. Res. Commun. 143, 753-760. Necr, E. J . , and Claphani, D. E. (1988). Roles of G protein subunits in transmcmbrane signalling. Nature (Londonj 333, 129-134. Nel, A. E., Wooten, M. W., and Galbraith, R. M. (1987). Molecular signaling mechanisnis in Tlymphocyte activation pathways: A review and future prospects. Clin. Imrnunol. Immunpathol. 44, 167-186.

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O’Shca, J . J., Urdahl, K. B . , Luong, H. T., Chused, T. M., Samelson, L. E., and Klausner, R. D. (1987). Aluminum fluoride induccs phosphatidylinositol turnover, elevation of cytoplasmic free calcium, and phosphorylation of the T cell antigen receptor in murine T cells. J. fmrnunul. 139, 3463-3469. Otani, S.,Matsui, I . , Kuramoto, A , , and Morisawa, S . (1984). Induction of ornithinc decarboxylase in guinea-pig lymphocytes and its relation to phospholipid metabolism. Eiochim. Eiophys. Actu 800, 96-101. Parker, C. W. (1978). CAMP and lectin-induced mitogencsis in lymphocytes: Possible implications for neoplastic cell growth. Adv. Cvclic Nurleoiidr RES. 9, 647-660. Parker, C. W. (1979). Rolc of cyclic nucleotides in regulating lymphocytes. Ann. N . Y . Acud. Sri. 332, 255-261. Parker, C. W., Sullivan, T. J.. and Wedner. H. J. (1974). Cyclic AMP and the immune response. Adv. Cyclic Nudrotide Res. 4, 1-80. Parnham, M. J., and Englberger, W. (1988). Lipid mediators and lymphocyte function. Hundb. Exp. Phur-macol. 85, 385-414. Patel, M. D., Samelson, L. E., and Klausner, R. 0 . (1987). Multiple kinases and signal transduction. Phosphorylation of the T cell antigen receptor complex. J . Biol. Chem. 262, 583 1-5838. Resch, K . , and Szamel, M. (1988). Signal transduction in lymphocyte activation. In “Lymphocyte Activation and Differentiation” (J. C. Mani and J. Domand, eds.), pp. 659-667. de Gtuyter, Berlin. Robison, C . A , , Butchcr. R . W., and Sutherland, E. W. (1971). “Cyclic AMP”. Academic Press, New York. Russell, D. H. (1978). Type I cyclic AMP-dependent protcin kinase as a positive effector of growth. Adv. Cyrlic Nurleoride Rrs. 9 , 493-506. Saitoh, R., Minami, Y., Kawata, S., Miyoshi, S., Tajima, K . , Mashita, K . , Moriwaki, K., and Tarui, S. (1988). The inhibitory cffect of forskolin on antibody-dependent cell-mediated cytotoxicity using chang liver cells as target cells. L(/r Sci. 42, 239-245. Schumm. I>. E . , Morris, H. P., and Webb, T. E. (1974). Early biochcmical changes in phytohemagglutinin-stimulated pcripheral blood lymphocytes from normal and tumor-bearing rats. Eur. J . Cunrw 10 107-1 13. Shearer, W. T., Gilliam, E. B., Rosenblatt. H. M., and Onon, F. M. (1988a). Anti-p antibody stimulates the phosphatidylinositol cycle and immunoglobulin secretion in a human lymphoblastoid R-cell line, LA350. Cell. Immunol. 111, 296-315. Shearer, W. T., Patke, C . I . . , Gilliam, E. B., Roscnblatt, H. M . , Barron. K. S., and Orson, F. M. (1988b). Modulation o f a human lyniphoblastoid B cell line by cyclic AMP. Ig secretion and phosphatidylcholine metabolism. J . frnrnunol. 141, 1678- 1686. Shcnker, R . J., and Matt, W. C. (1987). Suppression of human lymphocyte rcsponsiveness by forakolin: Reversal by 12-0-letradccanoyl phorbol 13-acetatc, diacylglycerol and iononiycin. Immuiiophurniu~o/u~?. 13, 73-86. Shirikawa, F., Yamashita, U., Chedid, M . , and Mizel, S. B. (1988). Cyclic AMP-an intracellular second messenger for interleukin 1. Proc. Null. Acud. Sci. U.S.A. 85, 8201-8205. Simkin, N. J., Jelinek, D. F . , and Lipsky, P. E. (1987). Inhibition of human B cell responsiveness by prostaglandin EZ. J . /minuno/. 138, 1074- 108 I . Smith, J. W., Steiner, A. L., and Parker, C. W. (1971). Human lymphocyte metabolism: Effects of cyclic and noncyclic nucleotidcs on stimulation by phytohemagglutinin. J. Clin. Invest. 50, 442-448. Somnicrmeyer, H.,and Rcsch. K . (1989). Pertussis t o x i n 6-subunit-induced Ca2+-fluxes in JURKAT human lymphoma cells: The action of long-term prctrcatment with cholera and pertussis holotoxins. C d l . Signdling, in press. ompanies early activation Stecle, T. A., and Brahmi, Z . (1988). Phosphatidylinositol metabolism events in tumor targct cell-stimulated human natural killer cells. Cell. Immunol. 112, 402-413.

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Takayama, H., Trenn, G., and Sitkovsky, M. V. (1988). Locus of inhibitory action of CAMPdependent protein kinase in the antigen receptor-triggered cytotoxic T lymphocyte activation pathway. J . B i d . Chem. 263, 2330-2336. Fakernoto, D. J., Kaplan, S. A , , and Appleman, M. M. (1979). Cyclic guanosine 3’,5‘-monophosphate and phusphudiesterase activity in mitogen-stimulated human lymphocytes. Biochem. Biophys. Res. Commun. 90, 49 1-497. Takigawa. M.. and Waksman, B. H. (1981). Mechanisms of lymphocyte “deletion” by high concentrations of ligand. Cell. Immunol. 58, 29-38. Tracey, D. E . , Hardee, M. M., Richard, K . A,, and Paslay, J W. (1988). Pharmacological inhibition of intcrlcukin- 1 aclivity on T cells hy hydrocortisone, cyclosporinc. prostaglandins, and cyclic nuclcutidcs. Im,tiu,io/)hnrmut.olo#?; 15, 47 -62. Treniblay, J . , Gerzer, R., and Hamet. P. (1988). Cyclic CMP in cell function. In “Advances in Second Messenger and Phosphoprotcin Research” (P. Greengard and G. A. Robison, eds.), Vol. 22, pp. 319-383. Raven, New York. Wagshal, A. B., and Waksman, B. H. (1978). Regulatory substances produccd by lymphocytes. VIII. Cell cycle specificity of inhibitor of DNA synthesis (IDS) action in lymphocytes. J . Immrmol. 121, 966-972. Wang, T., Sheppard, J. R., and Foker, J. E. (1978). Rise and fall of cyclic AMP required for onset of lymphocyte DNA synthesis. Sr-ipnc.e 201, 155- 157. WdtSon, J. ( I 976). The involveiiicnt of cyclic nucleotide metabolism in the initiation of lymphocyte proliferation induced by mitogens. J . Immunol. 117, 1656- 1663. Wcbb, D. R . , Belohradsky, B., Hancs, D., Stites, D. P.. Perlnian, J . D., and Fudenberg, H. H. ( 1975). Control of mitogen-induced lymphocyte activation. 11. Analysis of cell populations and metabolic events involved in cyclic AMP-mediated recovery of DNA synthesis suppressed by mitogens. Clin. Immrtnol. Immrrnpu/hol. 4, 226-240. Wedner, H. J . , Dankner, R., and Parker, C. W. (1975). Cyclic GMP and lectin-induced lymphocyte activation. 1. Imrnunol. 115, 1682-1687. Weinstein, Y., Chambers, D. A . , Bourne, H. R., and Melmon, K . L. (1974). Cyclic GMP stimulates lymphocyte nucleic acid synthesis. Nutirrc (LondonJ 251, 352-353. Weinstein, Y., Segal. S.,and Melmon, K . L. (197.5). Specific mitogenic activity of 8-brumoguanosine 3‘,5’-monophosphate on B-lymphocytes. J . Immunol. 115, 112- I 17. Weiss, J . , Schwinzer, B., Kirchner, H., Gemsa, D., and Resch, K . (1986). Effects of cyclosporine A on functions of specific murine T-cell clones: Inhibition of proliferation, lymphokine secretion and cytotoxicity. ImmunubiuloXv (S/icttgnrr) 171, 234-25 I . Whitfield, J . P., MacManus, J. P., Boynton A . L . , Gillan, D. J., and Isaals, R . J. (1974). Concanavalin A and the initiation of thymic lymphoblast DNA synthesis and proliferation by a calcium-dependent increase in cyclic GMP level. J . Cell. Phvsiol. 84, 445-457. Wickremasinghe, R. C.,Mire-Sluis, A. R., and Hoffbrand. A . V. (1987). Interleukin-2 binding to activated human T lymphocytcs triggers generation of cyclic AMP but not of inositol phusphatcs. FEBS Lett. 220, 52-56, Williamson, 1. R., and Hansen, C. A. (1987). Signalling systems in stimulus-response coupling. In “Biochemical Actions of Hormones” ( G . Litwack, ed.), Vol. 14, pp. 29-79. Academic Press, New York. Woogen, S. D., Ealding, W., and Elson, C. 0.(1987). Inhibition of murine lymphocyte proliferation by thc B subunit of cholera toxin. J. Immunol. 139, 3764-3770. Zhang, Y. H., Mak, N. K . . Lcung, K. N., and Hunt, N. H. (1987). Modulation of T cells by cyclic AMP in murine influenza virus infection. I . In virro inhibition of cytotoxic T lymphocytes by exogenous cyclic AMP analogues and by agents which increase intracellular cyclic AMP concentrations. Immunophurmucolo~,v13, 3 7 4 5

CURRENT TOPICS IN MI..MBRANkS A N D TRANSPORT, VOLUME 15

Chapter 16 Alterations in Cyclic Nucleotides and the Activation of Neutrophils JOAN REIBMAN, * KATHLEEN HAINES, f AND GERALD WEISSMANNZ Depurtment of Medicine *Department of Pulmonury Medicine fDepurtment of Pediatrics $Division of Rheumatology New York University Medical Center New York, New York 10016

I. Introduction 11. Receptor-Mediated Activation of Adenylate Cyclase in Neutrophils 111. Cyclic AMP in Activated Neutrophils IV. Effect of Elcvated CAMP on Neutrophil Responses A. Degrdnulation B. Cheniotaxis C. Superoxide Anion Generation D. Expression of Receptors V. Effect of CAMP on lntracellular Signals A. Membrane Depolarization B. Intracellular Ca2+ C. Phospholipid Remodeling VI. Altered Cyclic Nucleotide Responses in Disease VII . Cyclic GMP in Neutrophils A. Degranulation B. Cyclic GMP in Activated Cells C. Guanylate Cyclase D. Effect of cGMP on Intracellular Signals VI11. Conclusion References

1.

INTRODUCTION

The role of cyclic nucleotides in modifying the behavior of neutrophils has been studied for over 20 years; nevertheless, and despite an enormous literature, 399 Copynght 0 1990 by AcddemiL Press. Inc All rights of reproduction in m y lorn K S C N C ~

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the exact means whereby cyclic nucleotides altcr neutrophil functions remain obscure. This chapter will review rcccptor interactions that lead to increases in cyclic adenosine 3',S'-monophosphate (CAMP), and cyclic guanosine 3' ,5'monophosphate (cGMP). We will review the functional effects of increased cyclic nucleotide concentrations and the evidence that they do. in fact, play an important role in signal transduction. Finally, we will consider the association of abnormal cyclic nucleotide responses with asthma and cystic fibrosis.

II. RECEPTOR-MEDIATED ACTIVATION

OF ADENYLATE CYCLASE IN NEUTROPHILS Neutrophils, like other circulating leukocytes, possess P-adrenergic receptors. These receptors are of the pz class sincc cngagement by ligands activates adenylate cyclase according to the Pz series of agonists; isoproterenol (K,,, = 0.7 pM) > epinephrine ( K , , , = 8.5 pM) > norepinephrine (K;,,t = 90 pM) (Galant et al., 1978; Dulis and Wilson, 1980; Galant and Allred, 1980; Galant and Britt, 1984). Most investigators havc used the ligand ['Hldihydroalprenolol (I3H]DHA), which binds rapidly to neutrophils (t!, < 1 min) (Galant et ul., 1978), to study the p receptor of neutrophils. As shown -in Table I, estimates for receptor density and affinity for ['HIDHA vary depending on the mode of membrane preparation. Estimates for receptor density range from 476 to as high as 1700 sitedcell. Using membranes prepared by polytron lysis, Davies and Lefkowitz estimate that normal neutrophils have a receptor density of 66.0 t 2.4 fmol/mg protein or a receptor density (site/cell) of 476 2 23 (mean 7t SEM) (Davies and Lefkowitz, 1980). The K , for I3H]DHA binding for these cells is 0.572 nM. These measurements were not altered by the age of the donor or by use of tobacco, coffee, or alcohol. Indeed, measurements of receptor density and affinity remained constant upon repeat determinations from each donor. Whereas thc structure of thc P-receptor of neutrophils has not been directly determined, there is no reason to suspect that it should differ from that determined of other cells. The receptor contains seven hydrophobic membrane spanning regions, with the amino terminus of the protein exposed extracellularly and the carboxy terminus facing the cytosol (Lefkowitz and Caron, 1988). The niolecule accommodates binding of specific adrenergic ligands at residues within the hydrophobic core of the protein (Strader rt d., 1989). Transmission of the extracellular signal to a guanine nucleotide-binding protein in the interior of the cell is mediated by an intracellular region that forms an amphiphilic cy. helix in the third intracellular loop (Fig. 1 ) (Strader et a / ., 1989). Presumably, desensitization of the P-adrenergic receptor of neutrophils results from its phosphorylation by various kinases (Lefkowitz and Caron, 1988). Although phosphorylation of the p receptor of neutrophils has not been demonstrated, the receptor under-

SUMMARY OF Membrane preparation homogenized sonication intact cells polytron lysis polytron lysis sonication sonication nitrogen cavitation

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Receptor density (fmol/mg)

Receptor density (siteicell)

K, L3H]DHA

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0.38 0.57 2.4 0.43

Galant et a/. (1978) Galant et a/. (1980) Dulis and Wilson (1980) Davies and Lefkowitz (1980) Davis et a / . (1983) Galant and Britt (1984) Galant and Allred (1980) Mueller er al. (1988)

870 10-15 [1H]DHA [3H]DHA ['HIDHA [3H]DHA [ 3HIDHA [ 1Z51]pindolol

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12 14-41

1770 476 1462 997

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JOAN REIBMAN ET AL.

0 ligand binding Gs c~upling sequestration

FIG. I . Model for the transmenihranc topology of the (3-adrenergic I-eceptor. Horizontal lines rcprcscnt the plasma menibrane with the top of' the diagram corresponding to the extracellular space. The receptor has seven transmembrane helices. The residues shown in bold circles are proposed to interact with the ligand. The verticle bars in thc third intracellular loop mark residues critical for G protein coupling. (Reprinted with permission from Strader er a / . , 19x9.)

goes both homologous and heterologous desensitization after isoproterenol incubation. Preincubation of neutrophils with isoproterenol results in a 35-40% loss of binding sites (Davies and Lefkowitz, 1983; Galant and Britt, 1984). This is associated with an 80% loss of specific adenylate cyclase activity in response to isoproterenol; isoproterenol desensitization results in p receptor uncoupling and a loss of high affinity p receptors (Galant and Britt, 1984). However, neutrophils also undergo heterologous desensitization of p receptors. After prolonged incubation of neutrophils with isoproterenol (3 hr), engagement of recep-

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tors by histamine or PGE, fails to provoke maximal stimulation of adenylate cyclase (Galant and Britt, 1984). In most cells, adenylate cyclase is under the dual control of guanine nucleotide-binding proteins G, and Gi. (Casey and Gilman, 1988). Whereas G, mediates stimulation of adenylate cyclase activity, Gi mediates its inhibition. The (Y subunit of each protein has a site for NAD-dependent ADP-ribosylation catalyzed by cholera toxin or pertussis toxin for G, and Gi, respectively. These G proteins can be activated by nonhydrolyzable analogues of GTP, by fluoroaluminate, or by vanadate (Casey and Gilman, 1988). The receptor interaction site of G proteins lies at the COOH-terminal region of the (Y subunit, while the site of interaction with Pr is at the NH,-terminal region of the (Y subunit (Holbrook and Kim, 1989). However, both the COOH-terminal and NH,-terminal regions are in spatial proximity to the membrane face of the protein. Binding of GTP to the nucleotide regulatory proteins results in their dissociation into (Y and Pr subunits. Subsequently the free (Y subunit, via an effector binding site that remains to be localized, alters the function of the catalytic subunit of adenylate cyclase, G, stimulating and Gi inhibiting adenylate cyclase (Casey and Gilman, 1988). However, GTP-binding proteins function differently in neutrophils compared to other cells. As in other cells, cholera toxin ADP-ribosylates the 41 kDa subunit of G , in neutrophils (Lad et a/., 1984a; Verghese et ul., 1986) and activates adenylate cyclase, generating an increase in CAMP(Bourne et af., 1973; Lad et al., 1984a; Bokoch and Gilman, 1984; Verghese el al., 1985a). In contrast, Bokoch and Gilman (1984) have demonstrated that pertussis toxin ADP-ribosylates a 41 kDa membrane protein in neutrophils, yet exposure of neutrophils to pertussis toxin does not elicit a rise in CAMP. Therefore, the classic pertussis toxin-sensitive G protein (Gi) is not functionally linked to adenylate cyclase in neutrophils, rather it has been demonstrated to be coupled to phosphatidylinositol phospholipase C (Verghese et al., 1985a, Smith et ul., 1985; Snyderman and Uhing, 1988). These proteins will be more thoroughly reviewed in another chapter. P-adrenergic receptors of neutrophils are tightly coupled to adenylate cyclase. Indeed, the efficiency of the coupling of the receptor to the enzyme has been calculated by measuring receptor binding and adenylate cyclase activity in the same neutrophil sonicates (Galant and Allred, 1980). In these studies, the apparent K , for isoproterenol binding was 2.82 pM with a K,,, of 0.47 pM. This calculates to a KJK,,, ratio of 6.5, suggesting high coupling efficiency. As in other cells, coupling efficiency of the receptor to adenylate cyclase requires guanine nucleotides as cofactors. However, coupling efficiency of the receptor to adenylate cyclase can also be altered by the mode of preparation of membranes. Fluoride ion and Gpp(NH)p stimulate adenylate cyclase activity in membranes prepared via nitrogen cavitation, while, despite the presence of GTP, isoproterenol and PGE, lose their ability to do so (Lad et ul., 1984b). In contrast, all

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these agents can stimulate adenylate cyclase in membranes prepared via sonication (Lad rt ( i f . , 1984b). Thus when asscssing both receptor and adenylate cyclasc activity, the mode of membrane preparation must be takcn into account. Whereas neutrophils have (J-adrenergic receptors that are coupled to adenylatc cyclase, engagement of these receptors by p agonists stimulates only a modest increment in cyclic AMP, and phosphodiesterase inhibitors are usually required for this determination. Baseline measurements of cAMP in intact neutrophils range from 0.7 to 12 pmol/107 cells and isoproterenol elicits a 2- to 3-fold rise above basal levels (Davies and Lefkowitz, 1983; Rivkin et ul., 1975; Zurier et a / . . 1974; Busse and Sosman, 1984; Nielson, 1987; Lad et al., 1985a). Paradoxically, the increase in cAMP in response to engagement of @ receptors in neutrophils is significantly less than that demonstrated for other circulating leukocytes (Bourne et a l . , 1973; Parker and Smith, 1973; Marone et a / . , 1980). The reason for this is unclear as receptor number as well as receptor affinity for isoproterenol are similar in both lymphocytes and granulocytes (Davies and Letkowitz, 1980; Davis et d.,1983). One suspects that the role of phosphodiesterases and the spatial clustering of receptors that elevate cAMP in these cells may differ. Adenylate cyclase in neutrophils can be activated by ligands that do not engage P-adrenergic receptors. Human polymorphonuclear leukocytes contain a histamine receptor (Busse and Sosman, 1976; Anderson et al., 1977; Seligmann rt ul., 1983). This receptor belongs to the H, class as detcrmined by the order of relative potencies of the H,and H, agonists (Busse and Sosman, 1976; Gespach and Abita, 1982). Exposure of cells to histamine (EC,, = 10 M )also results in increased [CAMP] in the presence of a phosphodiesterase inhibitor (Gespach and Abita, 1982; Anderson et a / ., 1977; Zavoico and Feinstein, 1984). Prostaglandins of the E scries, PGE, and PGE,, elicit a small but rapid increase in cAMP in both broken and intact cell preparations (Bourne et ul., 197 I ; Victor et al., 198 I; Rivkin et al., 1975; Smolen and Weissmann, 198 1). In the presence of theophylline, PGE, (100-250 F M ) elicits a 2- to 3-fold increase in [CAMP] from basal levels of 6.7 pmol/107 cells (Smolen and Weissmann, 198 I). Moreover, treatment of neutrophils with prostaglandins enhances the increase in cAMP produced by other stimuli in a synergistic manner (Zurier et a / . , 1974; Smolen and Weissmann, 1981). As demonstrated in Fig. 2, PGE, alone increases cAMP from resting levels of approximately 6 pmol/ lo7 cells to 17, and in the presence of zymosan, to 40 (Zurier et al., 1974). In most cell types, adenosine, acting through the A, receptor, also causes an increase in intracellular cAMP (Cronstein et a/., 1988). In the presence of the phosphodiesterase inhibitor RO-20-1724, adenosine or adL,iosine agonists such as NECA (5’-N-ethylcarboxamidoadenosine) elicit an increase from basal levels of approximately I2 pmol/ lo7 cells to 43 2 8 pmol (Cronstein et al., 1988). This increase peaks a( 2 rnin and rapidly returns to baseline. Indeed, without the

405

16. CYCLIC NUCLEOTIDES AND NEUTROPHIL ACTIVATION

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/h 60

Minutes

FIG.2. Stimulation of cAMP in human neutrophils. Neutrophils were treated in the presence of theophylline (SO0 pV) with PGE, (0,2.8 X 10-JM. 30 niin), isoproterenol (0, IO-bM. 15 min), or no additions (0). They were then exposed to zymosan. (Reprinted with permission from Zurier ef ul.. 1974.)

presence of a phosphodiesterase inhibitor, no increase in cAMP can be demonstrated at all (Marone et al., 1980; Cronstein et at., 1988). The accumulation of CAMP in neutrophils is also regulated by intracellular trafficking. An inverse correlation has been established between microtubule assembly and intracellular levels of cAMP (Weissmann et al., 1975a; Rudolph et al., 1977). Treatment of cells with colchicine, which inhibits microtubule assembly, induces an increase in CAMP; colchicine ( l o p 6 to 10V5 M) elicits a 3-fold increase in basal levels of cAMP in the presence of the phosphodiesterase inhibitor isobutylmethylxanthine (IBMX) (Rudolph rt al., 1977; Malawista et al., 1978). Moreover, the ability of @-adrenergic agonists (isoproterenol, 2 PGE,, and histamine to increase cAMP is also potentiated in the presence of colchicine (Rudolph et nl., 1977, 1979). That this effect is mediated via an effect on microtubules is supported by the similar response demonstrated in the presence of other agents that inhibit microtubule assembly, such as vinblastine and oncodazole. Moreover, the effect is not seen in the presence of lumicolchine, an isomer of colchicine that cannot prevent microtubule assembly (Rudolph et al., 1977). Colchicine does not itself directly stimulate adenylate cyclase in isolated membranes. Thus microtubules may modify intracellular levels of CAMP, per-

w),

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JOAN REIBMAN ET AL.

haps by directing intracellular trafficking of compounds required for its synthesis (Rudolph and Malawista, 1980).

111.

CYCLIC AMP IN ACTIVATED NEUTROPHILS

The role of cAMP as a second messenger for neutrophil activation has been of profound interest for over 20 years. This interest was piqued by the observation that treatment of neutrophils with chemotactic factors elicited a rise in cAMP on their own (Zurier et al., 1974; Herlin et al., 1978; Smolen and Weissmann, 1981; Smolen et ul., 1980). Exposure of neutrophils to the chemotactic peptide Nformyl-methionyl-leucyl-phenylalanine(MLP), immune complexes, C5a, leukotriene B, (LTB,), or acetylglycerylether phosphorylcholine (PAF) causes a small but consistent increase in intracellular levels of cAMP (Zurier et al., 1974; Herlin et al., 1978; Smolen and Weissmann, 1981; Smolen et al., 1980; Simchowitz et al., 1980; Hopkins et al., 1983). The increase in [CAMP] caused by chemoattractants is rapid (<5 sec with a maximum at 10-20 sec) and transient (by 60-120 sec [CAMP]has returncd to resting levels) (Smolen and Weissmann, 1981). The calcium ionophore A23187 also causes a very small increase in cAMP (Smolen and Weissmann, 1981). The effect of these ligands on the rise in CAMP is potentiated by preincubation of cells with PGE, (Zurier rt al., 1974; Smolen and Weissmann, 1981). In contrast, concanavalin A and phorbol myristate acetate do not elicit a rise in CAMP in neutrophils either in the presence or absence of PGE, (Smolen and Weissmann, 1981; Smolen et al., 1980). An increase in CAMP has also been associated with phagocytosis of zymosan by neutrophils (Pryzwansky et d.,198 1). Using immunocytochemical techniques, Pryzwansky et al. have demonstrated that resting neutrophils stain throughout their cytoplasm for both CAMPand cGMP (Pryzwansky et al., 1981). After exposure to zymosan, CAMP is distributed near the cell surface and is found in association with the forming phagosome. Moreover, staining for the regulatory subunit of type 1 CAMP-dependent protein kinase reveals it to be in a similar distribution. In contrast, this alteration in distribution is not seen in neutrophils stained for cGMP. These studies suggest that total measurement of cAMP in cells may not be true reflections of alterations in this compound; alterations in cAMP may be localized. Moreover, both distribution of the cyclic nucleotide as well as its total intracellular concentration may determine its role in cell signaling. While the secretagogue fMLP elicits an increase in CAMP, this increase is not altered by preexposure of neutrophils to theophylline (Smolen rt ul., 1980). This raises the following two questions: what is the mechanism by which fMLP provokes increments in the concentration of CAMP, and what controls the adeny-

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late cyclase. Verghese et al., using isolated membranes, have demonstrated that neutrophils utilize two pathways to increase levels of cAMP (Verghese et al., 1985b). In plasma membrane preparations, sodium fluoride stimulates adenylate cyclase in the absence or presence of GTP; the guanine nucleotide-binding protein is intact. Moreover, PGE, and isoproterenol (100 activate adenylate cyclase in plasma membranes in the presence of GTP; receptor coupling to adenylate cyclase is intact. However, the chemoattractant fMLP and the calcium ionophore A23 187 do not stimulate adenylate cyclase in isolated membranes, suggesting that these agents do not exert their effects via the same G protein. Furthermore, calcium has differential effects on the ability of membranes to generate cAMP depending on the stimulus used; depletion of Ca2+ decreases cAMP generation elicited by ionophore and f'MLP but not that elicited by PGE, and isoproterenol (Verghese et al., 1985b). Finally, both Smolen et al. and Verghese et al. concur that the inhibition of a phosphodiesterase does not enhance the increase in cAMP elicited by fMLP while that of PGE, and isoproterenol is enhanced. Thus accumulation of cAMP results from both Ca2 sensitive and Ca2 -insensitive pathways. Chemoattractants cause an increase in cAMP by a calcium-sensitive pathway that does not involve direct activation of membrane-bound adenylate cyclase. This view is supported by Lad et al. (1984a) who prepared vesicles from neutrophils that were enriched in adenylate cyclase. Both isoproterenol and PGE stimulated adenylate cyclase in these vesicles in a GTP-dependent manner. Moreover NaF activated the cyclase in the absence of GTP. In contrast, neither fMLP nor C5a was able to stimulate cyclase activity, again suggesting different mechanisms for the increase in CAMP. Ishitoya and Tadaomi (1987) also examined the mechanism for the synergy seen between PGE, and fMLP and Ca2+. The synergistic increase of cAMP in neutrophils treated with PGE, and fMLP or Ca2+ ionophore was blocked when calcium influx was prevented by EGTA or TMB-8. At high doses, the calmodulin antagonist W-7 also inhibited the synergy between PGE, and fMLP and Ca2+ ionophore. Addition of an activator of protein kinase C did not enhance synergy, suggesting that the effect of W-7 was via calmodulin. The synergy between PGE, and fMLP remained despite the inclusion of a phosphodiesterase inhibitor, suggesting that the Ca2 -calmodulin dependence was not due to suppression of a phosphodiesterase. Thus, in whole cells, the synergistic effect of PGE, and chemoattractants to increase cAMP is calcium dependent; calcium depletion decreases the effect. The activity of adenylate cyclase itself is, however, inhibited by Ca2 . Stolc examined the calcium and magnesium dependency of adenylate cyclase derived from sonicates of neutrophils (Stolc, 1977). As reported previously, Mg2+ was required for adenylate cyclase activity (Bourne et al., 197 1 ; Stolc, 1977). In contrast, increasing concentrations of Ca2 inhibited the stimulation of adenylate cyclase by ATP, Gpp(NH)p, histamine, or iso-

w)

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+

,

+

+

+

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JOAN REIBMAN ET AL.

proterenol. Thus in neutrophils, cAMP is increased by two mechanisms: a direct activation of adenylate cyclase or an as yet unidentified Ca2 -sensitive mechanism. Altered levels of intracellular CAMP may also be due to phophodiesterase activity. Cyclic nucleotide phosphodiesterase activity has been isolated from human neutrophils and primarily resides in the soluble subcellular fraction (Grady and Thomas, 1986).The phosphodiesterase activity exists in both a high affinity (K, = 1.9 pM) and a low affinity state (K,,, = I12 F M ) . The chemoattractant fMLP may increase CAMP by inhibition of this native enzyme; addition of phosphodiesterase inhibitor does not enhance thc increase in cAMP elicited by tMLP (Smolen ef al., 1980;Verghese rt ul., 1985).Exposure of cells to fMLP also elicits an increase in the high affinity phosphodiesterase found in both the sonicates and soluble fraction (Grady and Thomas, 1986). In summary, these studies suggest that there arc multiple mechanisms for raising intracellular levels of CAMP in neutrophils. While PGE, and isoprotercnol act via a transmembranc guanine nucleotide regulatory protein, chemoattractants act via a calcium-dependent mechanism that may involve thc inhibition of a native phosphodiestcrase. The ability of secretagogues such as fMLP, immune complexes, and Ca2+ ionophores to elicit an increase in CAMP raised the question of whether CAMP was an early intracellular messenger, activating neutrophils. Indeed, the increase in CAMPelicited by secretagogucs prccedes cell activation, suggesting a role, at least temporally, for CAMP as an intracellular messenger (Smolen et al., 1980). However, Simchowitz ef al. have demonstrated that whereas the increase in CAMP elicited with fMLP can be inhibited with 9-(tetrahydro-2-furyl)adenine (SQ 22,536),a putative inhibitor of adenylate cyclase, the generation of superoxide anion is not affected (Simchowitz et al., 1983).Thus the transient increase in CAMP elicited by chemoattractants can be separated from activation of neutrophils. Moreover, the response of neutrophils to exogenous cAMP or to agents that cause an increase in cAMP also makes the theory that cAMP acts as an intracellular messenger unlikely. In general, addition of exogenous CAMP or exposure of neutrophils to agents that increase CAMP leads to a decrease in cell activation. +

IV. EFFECT OF ELEVATED cAMP ON NEUTROPHIL RESPONSES A. Degranulation Almost 20 years ago, Weissniann et a / . reported that agents that raise intracellular levels of CAMP, such as PGE,, exogenous CAMP, and theophylline, decrease the release of acid hydrolases elicited by zymosan or latex particles

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409

(May et al., 1970; Weissmann e t a / . , 1971). The release of P-glucuronidase and acid phosphatase elicited by immune complexes formed from rheumatoid factor and aggregated IgG was also inhibited by dibutyryl cAMP and PGE, in the absence or presence of theophylline (Weissmann et al., 197 1b). These studies were the earliest demonstrations of inhibitory effects of cyclic nucleotides in neutrophil activation. Subsequent studies confirmed that PGE, and PGA, reduced enzyme release from neutrophils stimulated by zymosan in the absence or presence of theophylline (Zurier er ul., 1973). The inhibition elicited by prostaglandins was maintained in the presence of cytochalasin B (i.e., in the absence of phagocytosis). In contrast, PGF,, did not inhibit enzyme release. Ignarro et al. (1974) employed zymosan treated with rheumatic sera to stimulate release of (3glucuronidase from neutrophils. In this system as well, isoproterenol, epinephrine, and dibutyryl CAMP all significantly inhibited enzyme release in the absence or presence of theophylline (Ignarro et al., 1974; Ignarro and George, 1974). Propranolol almost completely reversed the inhibition elicited by epinephrine. Theophylline M ) employed in these experiments did not inhibit degranulation on its own. Results of subsequent studies employing opsonized zymosan and isoproterenol in the presence of phosphodiesterase inhibitor have been similar (Busse and Sosman, 1984). P agonists have also been reported to inhibit degranulation elicited by engagement of receptors other than those of complement or Fc receptors. Pre-incubation with isoprenaline, fenoterol, and salbutamol for 10 min inhibited the release of P-glucuronidase elicited by fh4LP (Fantozzi et al., 1984). In the absence of a phosphodiesterase inhibitor, maximal inhibition was demonstrated for all agents at a dose of l o p s M . The effect of the agonists was potentiated by the inclusion of the phosphodiesterase inhibitor IBMX at a dose that did not inhibit degranulation on its own. The presence of a phosphodiesterase inhibitor adds complexity to many studies as theophylline at high doses (100 or IBMX (<0.4 mM) inhibits degranulation after ligands in the absence of p agonists (Marone et al., 1980; Fantozzi et a l . , 1984). Therefore Marone et (11. (1980) studied the effect of P agonists on degranulation in the absence of a phosphodiesterase inhibitor. In contrast to earlier studies, Marone et ul. (1980) did not detect an inhibition in granule enzyme release (P-glucuronidase, histaminase) by isoproterenol (lo-' M ) in neutrophils stimulated with serum-treated zymosan in the absence of a phosphodiesterase inhibitor. Theophylline ( 10 4 , I O w 3 M ) inhibited P-glucuronidase (18% and 65% inhibition, respectively). Simultaneous measurements of intracellular CAMP demonstrated that a 5 or 10 min preincubation with isoproterenol (10 M ) did not elicit an increase in cAMP in neutrophils in the absence of a phosphodiesterase inhibitor. Using the chemotactic peptide fMLP as a stimulus, subsequent investigators also did not report a significant inhibition of P-glucuronidase nor an increase in cAMP with P agonists unless a phos-

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JOAN REIBMAN ET AL.

phodiesterase inhibitor was included in the reaction mixture (Lad et al., 1985b; Reibman et al., 1986). Inhibition was achieved with PGE, even in the absence of a phosphodiesterase inhibitor (Lad et d., 1985b). An explanation for this may lie in the studies of Tecoma et al. (1986). These authors also do not demonstrate an increase in cAMP when neutrophils have been pre-incubated with isoproterenol (10- M )before the addition of stimulus. However, when isoproterenol is added 30 sec before addition of the stimulus, an increase in cAMP is seen. Isoprotereno1 may be rapidly oxidized or metabolized in the absence of a phosphodiesterase inhibitor, thus limiting its effect on adenylate cyclase. The role of CAMP-dependent inhibition of degranulation is also confused by the effect of cholera toxin. Cholera enterotoxin, acting via G,, elicits an increase in cAMP in most cells (Casey and Gilman, 1988). Cholera toxin also elicits a delayed increase in cAMP in neutrophils in the absence or presence of a phosphodiesterase inhibitor (Bourne et al., 1973). However, cholera enterotoxin inhibits degranulation as measured by the release of P-glucuronidase only in the presence of a phosphodiesterase inhibitor (Bourne et ul., 1973; Zurier et al., 1974). These studies serve to question the role of cAMP as the agent which inhibits degranulation per se. In summary, while most investigators have reported that agents that elicit a rise in cAMP inhibit degranulation, this effect has not been universally demonstrated. The differences among the studies discussed raise the question as to whether the inhibitory effects exerted by P agonists and phosphodiesterase inhibitors are due to their direct effects on adenylate cyclase or to as yet undefined mechanisms. Moreover, the rapid in vitro oxidation or metabolism of some agonists such as isoproterenol may attenuate their real effects on adenylate cyclase. Differences in the extent and duration of the elevation of cAMP may alter its role in inhibition of cell responses. Finally, the intracellular compartmentalization of cAMP may also be crucial in determining its ability to inhibit neutrophil degranulation.

B. Chemotaxis Chemotaxis is altered by agents that increase cAMP levels in neutrophils. Rivkin ef al. ( 1957) demonstrated that epinephrine and isoproterenol inhibited chemotaxis of rabbit neutrophils stimulated by culture filtrates from Escherichia coli but that the inhibition was variable. As in other cells, purified E . c d i enterotoxin increases cAMP in neutrophils when measured 2-hr after incubation (Bergman et al., 1978). Moreover, filtrates of enterotoxigenic E. coli are less chemotactic for neutrophils than filtrates of nonenterotoxigenic strains (Bergman et ul., 1978). Purified E . coli enterotoxin decreased chemotaxis by 32%, an effect that was increased in the presence of 0.1 mM IBMX. However, in rabbit neutrophils, cholera toxin increased cAMP after a lag of 30 min, at which time a

16. CYCLIC NUCLEOTIDES AND NEUTROPHIL ACTIVATION

41 1

decrease in chemotaxis and spontaneous motility was also noted (Rivkin et ul., 1975). Paradoxically, the dose of cholera toxin required to decrease chemotaxis was much greater than that required for an increase in cAMP (50 ng/ml vs. 2 ng/ml). Moreover, pre-incubation with 10 ng/ml of cholera toxin elevated cAMP to the same level as 50 ngiml, yet inhibition of chemotaxis was not noted. Histamine (10 3-5 x 10 M ) increases chemokinesis, or random movement, at doses that cause an increase in cAMP (Anderson et d . , 1977; Seligmann et ul., 1983). The increase in chemokinesis can be blocked by pretreatment of cells with the H, receptor antagonist metiamide (Anderson et at., 1977; Seligmann et al., 1983). However, the effect is stimulus-specific (i.e., no alteration in chemotaxis or chemokinesis was noted when phorbol myristate acetate or the calcium ionophore A23 187 was employed as a stimulus) (Seligmann et al., 1983). These data support, but again d o not prove, an association of cAMP with chemotaxis. For chemotaxis as well as degranulation, the duration of the elevation of intracellular cAMP may be crucial for an effect. ~

~

C. Superoxide Anion Generation Activation of the NADPH-oxidase of neutrophils generates oxygen metabolites including superoxide anion. The intracellular messengers responsible for activation of this enzyme complex may differ from those required for degranulation (Haines et ul., 1988; Korchak et al., 1988a,b). Binding of P-adrenergic agonists to their receptors on neutrophils inhibits the ability of neutrophils to generate superoxide anions (Wong and Freund, 198 1; Fantone and Kinnes, 1983; Busse and Sosman, 1984; Tecoma et al,. 1986; Reibman et al., 1986; Mack et al., 1986; Nielson, 1987). This inhibition by P agonists can easily be demonstrated in the absence of a phosphodiesterase inhibitor. Superoxide anion production is quite sensitive to engagement of the P-adrenergic receptor; blockade of only half of the P receptors with the irreversible agonist isoproterenol is sufficient to inhibit the respiratory burst fully (Mueller et a l . , 1988). Moreover, inhibition of the 0 , ~response to engagement of the P-adrenergic receptor is rapid as isoproterenol ( 2 3 x lOW7 M ) shuts off oxidant production within seconds (Mueller et al., 1988). The inhibition of 02- elicited by agents that increase cAMP is dependent on the stimulus employed. Whereas PGE, inhibits superoxide anion generation elicited by chemoattractants such as fMLP, LTB,, or the ionophore A23187, no inhibition in the rate or quantity of superoxide anion generated is demonstrated in the presence of synthetic diacylglycerol or phorbol myristate acetate (Fantone and Kinnes, 1983; Mack et al., 1986; Nielson, 1987). Thus cAMP cannot be inhibiting assembly of the NADPHoxidase complex but rather must have an effect on a preceding signal which cannot be dependent on G proteins (viz A23187).

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D. Expression of Receptors Whereas engagement of P-adrenergic rcceptors does not affect expression of the fMLP receptor, the reversc is true for the Fc receptor (Teconia el al., 1986). Spagnuolo et al. (1987) have examined the effect of CAMP on the expression of Fc receptors by measuring the ability of neutrophils to form rosettes with IgGcoated erythrocytes. Dibutyryl CAMP, isoproterenol, and IBMX all reduccd the percentage of neutrophils forming Fc-rosettes. Paradoxically, cholera toxin (1010,000ng/ml) and PGE, and PGE, (0.3-3.0 pM) did not reduce the percentage of Fc-rosetting neutrophils. Therefore, the role of CAMP in this process is unfortunately as obscure as its role in degranulation. These findings are in contrast to those demonstrated for lymphocytes, again suggesting that the function and role of adenylate cylcase are not the same in these two types of cells (Spagnuolo et NI., 1987). These studies also suggest that the effect of P agonists on Fc receptor expression may not be mediated via an effect on CAMP; P agonists may act via alternative mechanisms.

V.

EFFECT OF cAMP ON INTRACELLULAR SIGNALS

One popular theory proposes that activation of neutrophils after engagement of a receptor proceeds via a GTP-binding protein (GP,J which elicits phospholipid remodeling. The products of lipid remodeling generated in response to chemoattractants include inositol trisphosphate, 1,2-diacylgIcyerol, as well as phosphatidic acid (Berridge and Irvine, 1984; Berridge, 1983, 1984; Serhan et al., 1982, 1983; Cockroft rt al., 1984; Bradford and Rubin, 1985; Verghese et ul., 1985b; Korchak et al., 1988a,b; Smolen and Weissmann, 1981). Phospholipid remodeling results in an increase in [Ca2+Ii and activation of kinases including protein kinase C (Snyderrnan and Uhing, 1988). The roles of other kinasessuch as CAMP-dcpendent kinases, Ca2+-calmodulin and tyrosine kinase remain to be fully clarified. However, were CAMPor agents that elicit an increase in CAMPto inhibit neutrophil activation, some effect would be expected on other intracellular messengers. A. Membrane Depolarization Several investigators have noted a stimulus-specific effect of cAMP on neutrophil rcsponses; increased lcvels of CAMP inhibit oxygen consumption stimulated by fMLP but not by phorbol myristate acetate (Nielson, 1987; Mack et al., 1986; De Togni et al., 1984). Membrane depolarization elicited by the addition of tMLP was also prevented by exogenous CAMP, while phorbol myristate

16. CYCLIC NUCLEOTIDES AND NEUTROPHIL ACTIVATION

41 3

acetate-induced depolarization remained normal in its presence (De Togni et al., 1984).

B. lntracellular Ca2+

fMLP and Ca2 ionophore act via Ca2 -dependent pathways to activate PKC while phorbol myristate acetate does so directly. Therefore, it would be reasonable to question whether altered levels of intracellular cAMP affect [Ca2’-Ii. Furthermore, experiments in platelets suggest that agents that cause a rise in cAMP “inhibit” increases in [Ca2+Ii (Feinstein et u l . , 1983; Zavoico and Feinstein, 1984; Knight and Scrutton, 1984). Using the fluorescent probe quin2, De Togni et af. (1984) reported that pre-incubation of neutrophils with dibutyryl cAMP (5 min) in the presence of theophylline did not decrease the rate or extent of the initial rise in [Ca2+Iielicited by fMLP. Reibman et al. (1986) reported similar results; isoproterenol inhibited superoxide anion generation in the absence of a phosphodiesterase inhibitor without any effect on [Ca2+Ii.Using the fluorescent probe fura-2, resting levels of [Ca2+Iiwere 58.4 k 4 nh4 in control cells vs. 63.8 k 15 nM in cells pre-incubated with isoproterenol (5 p M , 5 min). After exposure to fMLP ( I F M ) , peak levels reached 176 L 27 nM in the absence and 176 2 24 nM in the presence of isoproterenol (J. Reibman et al., unpublished observations). Ca2+ uptake as measured by 4sCa2+ was inhibited by isoproterenol only in the presence of a phosphodiesterase inhibitor (Reibman et ul., 1986). Tecoma et al. note a small decrease in [Ca2 Ii only at low concentrations of fMLP (0.1-0.01 nM), suggesting that cAMP may have a small but incomplete effect on calcium homeostasis (De Togni et al., 1984). Thus it seems unlikely that altered [Ca2+li accounts for the functional effects elicited by isoproterenol. +

+

+

C. Phospholipid Remodeling Since phospholipid remodeling is an integral part of signal transduction in neutrophils, the effect of cAMP on phospholipid remodeling has been studied by a variety of systems. Tou et (11. examined the effect of dibutyryl cAMP on the incorporation of [ Y - ~ ~ P ] Aor T Pmyo-[2-3H]inositolinto various phospholipids in resting cells. The incorporation of [-p3*PIATP into phosphatidic acid, phosphatidylinositol, diphosphoinositide, and triphosphoinositide was slightly decreased in the absence of cytochalasin B (83, 67, 79, and 86% of control, respectively) (Tou and Maier, 1976). A minimal effect was demonstrated for the uptake of myo-inositol into the phosphoinositides. Thus, basal lipid turnover is only slightly decreased in the presence of exogenous CAMP. Using cells labeled with [7H]glycerol, Della Bianca et ul. demonstrate altered

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phospholipid metabolism in cells stimulated with fMLP in the presence of both dibutyryl cAMP and theophylline (Fig. 3). The accumulation of phosphatidic acid and phosphatidylinositol 4-phosphate normally seen in response to N L P was decreased, and the stimulated hydrolysis of phosphatidylinositol was also decreased (Della Bianca ef al., 1986). In contrast, the rapid hydrolysis (seen within 30 sec of stimulation) of phosphatidylinositol bisphosphate (PIP,) continued, although the relabeling was inhibited. The accumulation of the water soluble inositol phosphates, inositol monophosphate, inositol bisphosphate, as well as inositol trisphosphosphate, was also decreased by the presence of exogenous cAMP and theophylline. As reported by Tecoma et al. (1986), the effect of isoproterenol (1 @ on phosphatidic I) acid and phosphatidylinositide formation was maintained even in the absence of a phosphodiesterase inhibitor. Again the hydrolysis of PIP, was not altered even when isoproterenol was added 30 sec before stimulus. Upon exposure of neutrophils to fMLP, rapid and sustained

P t d Ins

z

1

i-- T ,, 0

30

1

60

120

0

sec

u .-

30

120

60 sec

>

I

T

L

0

1

30

60

1

sec

120

0

30

60

se c

120

FIG. 3 . EKect of dihutyryl cAMP on [lH]glycerol-labeled phosphoinositide turnover. Neutrophils were incubated in the absence (0) or presence (0)of theophylline (2 mM) and dibutyryl cAMP (1 mM) and then exposed to fMLP (50 nM). (Reprinted with permission from Della Bianca et

d., 1986.)

41 5

16. CYCLIC NUCLEOTIDES AND NEUTROPHIL ACTIVATION

increases are found in 1,2-diacylglyceroI, which is derived both from the hydrolysis of PIP, as well as other sources (Rider and Niedel, 1987; Reibman et al., 1988; Korchak et al., 1988a). Pre-incubation of cells with isoproterenol ( 5 pM) inhibited the generation of superoxide anion but did not decrease the formation of diacylglcyerol (Fig. 4) (J. Reibman et ul., unpublished observations). These studies demonstrate that rises in [CAMP] alter stimulus-induced phospholipid remodeling; the formation of phosphatidic acid is diminished while neither the formation of 1,2-diacylglycerol nor the hydrolysis of PIP, is greatly affected. These effects are consistent with our suggestion that phosphatidate is an important intermediate in signal transduction. In most cells, cyclic nucleotides activate specific protein kinases. Initial attempts to isolate a CAMP-dependent protein kinase from neutrophils were difficult due to the abundance of proteolytic enzymes in neutrophils and an inhibitor found in lysosomes (Tsung and Weissmann, 1973). However, a CAMP-dependent kinase has been isolated from neutrophils and resides primarily in the cytosol (Tsung et ul., 1972, 1975; Tsung and Weissmann, 1973). The kinase is stimulated by cAMP as well as cyclic IMP with an apparent K , of 62 and 88 nM, respectively. Activity of the kinase requires divalent cations (Co2 > Mg2 > Mn2+).The holoenzyme has a molecular mass of 66 kDa, but in the presence of CAMP, the protein dissociates into subunits of 45 and 30 kDa, consistent with a regulatory and a catalytic subunit. The target proteins of this kinase remain to be determined. No clear target proteins have been visualized with one-dimensional polyacrylamide gel electrophoresis in cells pretreated with dibutyryl cAMP (Andrews and Babior, 1984). Three proteins have been described that are phosphorylated in response to exogenous cAMP in rabbit peritoneal neutrophils (Huang et al., 1983). These +

200 -

+

, FMLP+Isa

W

m +I

150t

L

0 0

8

100 -

LL 0

I

60

1

I

I

I

12C 180 240 300 Time (seconds)

FIG. 4. Effect of isoproterenol o n I .2-diacylglycerol formation in neutrophils. Neutrophils wcre labeled with I3H]arachidonic acid and then incubated with (A)and without (0)isoproterenol (5 p M . 5 min) before addition of FMLP (I0 - ' M ) . Cells exposed to isoproterenol alone are shown (0). (From .I.Reibman et a/.,unpublished observations.)

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include a protein of M , 43,000, p15.7 and thought to be actin, and acidic proteins of M , 135,000, 130,000. and I10,000, the identities of which are yet unknown (Huang et al, 1983). Cholera toxin and cyclic adenosine 3',S'-nionophosphate induce the human leukemic cell line HL60 to display the myeloid phenotype. This phenotypic alteration is associated with an increase in expression of type I CAMP-dependent protein kinase (Fontano et al., 19x9). Moreover, phosphorylation of proteins of 170. 108, and 183 kDa is noted. Thesc proteins also require further identification.

VI.

ALTERED CYCLIC NUCLEOTIDE RESPONSES IN DISEASE

The association of altered P-adrenergic receptor function and concentrations of intracellular cAMP with disease has also been studied for many years. Indeed, it has now been two decades since Szentivanyi first proposed that atopic asthma was mediated by an abnormal P-adrenergic response (Szentivanyi, 1968). Other investigators demonstrated a decrease in the formation of cAMP in response to P-adrenergic agonists in lymphocytes and mixed leukocytes (Parker and Smith, 1973; Parker et ul., 1973). However, these and subsequent studies have demonstrated that the suboptimal ability of neutrophils from asthmatic patients to activate adenylate cyclase is not due to an intrinsic defect in the cells but to downregulation and uncoupling of the P-adrenergic receptor as a result of previous exposure of the cells to p agonists (Galant and Britt, 1984). Neither the number nor affinity of binding sites for [3HJdihydroalprenolo1is reduced in asthmatics who have not received adrenergic therapy (Galant et al., 1980). Moreover, the activity of adenylate cyclase in broken cell preparations is also not reduced (Galant et al., 1980). In contrast, patients receiving p agonists have a 70% reduction in the number of binding sites for [3H]DHA (Galant et al., 1980). Of further interest, corticosteroids diminish the ability of p agonists to desensitize the adrenergic receptor (Davies and Lefkowitz, 1980). Treatment of cells with hydrocortisone does not change the number of P-receptors but rather decreases the uncoupling of receptors elicited by P-adrenergic agonists (Logsdon et al., 1972; Davies and Lefkowitz, 1983; Parker et al., 1973). Thus the defect in the response to P agonists demonstrated in neutrophils from patients with asthma is a result of treatment rather than an intrinsic cellular defect. In contrast to asthma, patients with cystic fibrosis may have an intrinsically abnormal response to P-adrenergic stimulation. Neutrophils from patients with cystic fibrosis display a decreased ability to produce cAMP in response to engagement of the @ receptor (Davis et uf., 1983). This defect is demonstrable in heterozygotes as well as homozygotes, suggesting that there is a genetic component to the defect. Neither the number of receptors nor their binding properties

16. CYCLIC NUCLEOTIDES AND NEUTROPHIL ACTIVATION

41 7

are reduced in these patients compared to controls (Davis et al., 1983). Moreover, the response of cells to PGE, is normal, suggesting that there is not an intrinsic defect in adenylate cyclase. Cystic fibrosis may prove to be a human model for a coupling defect between the receptor and adenylate cyclase.

VII.

CYCLIC GMP IN NEUTROPHILS

Although the literature is replete with studies on the role of cyclic adenosine 3’ 3‘,-monophosphate in neutrophils, there are fewer studies on the effect of cyclic guanosine 3’,5’-monophosphate. Indeed we have proposed that cAMP and cGMP have reciprocal ( “Yin/Yang”) effects on neutrophil function; whereas increased levels of cAMP inhibit release of lysosornal enzymes, cGMP enhances release (Weissmann et al., 1975a,b). This proposal has been confirmed experimentally.

A. Degranulation Muscarinic agonists, which elicit an accumulation in cGMP, also enhance degradation of the neutrophil. In studies utilizing opsonized zymosan in the presence of cytochalasin B , carbachol elicited a dose-dependent increase in the release of P-glucuronidase (Zurier et a / ., 1974). Carbachol (lo-* and l o p 6M) also increased intracellular levcls of cGMP. The increase was rapid, with maximal effects noted at 5 min. lgnarro et d.1974 also demonstrated that cGMP as well as the cholinergic agents acetylcholine and carbamylcholine enhanced lysosomal enzyme release triggered by aggregated IgG. These effects were blocked by atropine. The ability of carbachol and acetylcholine to enhance enzyme release suggests that neutrophils have muscarinic receptors. However, the properties of this receptor in neutrophils have not been examined in detail.

B. Cyclic GMP in Activated Cells Were cGMP to function as a second messenger, one would expect stimuli that elicit degranulation to elicit increases in cGMP. This has been demonstrated for zymosan-treated serum and a calcium ionophore (Smith and Ignarro, 1975). Smith and Ignarro demonstrate that zymosan-treated serum increases resting levels of cGMP from 0.48 2 0.14 pmol/106 cells to 31.7 pmol. Calcium ionophore has a similar effect, increasing cGMP to 38.6 pmol/106 cells. Acetylcholine enhances this effect to 52.2 pmol in the presence of zymosan-treated serum. Lad et ul. ( 1 985a) also report an increase in cytosolic guanylate cyclase activity in response to fMLP. Hatch ef at. (1977) report that zymosan-treated serum as well as the synthetic peptide formylmethionylalanine elicit an increase

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in cGMP from resting levels of 22.4 fmol/106 cells. Unfortunately, the measurement of cGMP by these two groups differs by orders of magnitude. Phorbol myristate acetate also causes an increase in guanylate cyclase activity to 180% of control values, suggesting an interaction with protein kinase C (Coffey et al., 1988). Granulocyte-macrophage colony-stimulating factor (GM-CSF) acts on mature neutrophils to inhibit random migration and to augment degranulation and superoxide anion generation (Coffey et al., 1988). Low doses of GM-CSF (1 U/ml) produced a rapid increase in [cGMP] within seconds. At higher doses (100 U/ml), the increase was biphasic. The increase in activity is associated with an increase in guanylate cyclase activity in the cytosol (Coffey et al., 1988). Thus it appears likely that many of the effects of GM-CSF are mediated via an effect on cyclic nucleotide regulation.

C. Guanylate Cyclase Little is known about guanylate cyclase in neutrophils. Guanylate cyclase activity can be measured in both membrane and cytosol fractions with the predominant activity found in the cytosol (Lad et al., 1985a; Coffey et al., 1988). Cyclase activity requires Mn2+ with a K, of 0.022 mM, although Mg2+ can also support activity ( K , of 0.91 mM) (Lad et al., 1985a; Coffey ef al., 1988). Maximum velocities of guanylate cyclase are 1.35 pmol cGMP/min*mg- protein in the presence of Mg2+ and 3.55 pmol cGMP/min.mg-l protein in the presence of Mn2+ (Coffey et ul., 1988). While Ca2 ' augments the activity of guanylate cyclase, the augmentation requires Mg2+ (Lad et a f . , 1958a). Sodium nitroprusside, azide, and hydrogen all stimulate guanylate cyclase.

D. Effect of cGMP on lntracellular Signals The literature on the effect of cGMP on intracellular messengers of neutrophils is likewise sparse. Acetylcholine increases uptake of 43CaC1,, suggesting that cGMP increases Ca2+ influx (Smith and Ignarro, 1975). Using the fluorescent probe quin2, Coffey et al. (1988) were unable to measure any effect of GM-CSF on [Ca2+J i . Moreover, GM-CSF did not increase levels of inositol phosphates labeled with [ 3H]inositol (Coffey et a / . , 1988). Granulocyte-macrophage colony-stimulating factor also did not cause a translocation of protein kinase C from the cytosol to membrane on its own as is observed with stimulation of neutrophils by phorbol myristate acetate (Wolfson et ul., 1958). Thus GM-CSF elicits a rise in cGMP but does not alter [Ca2+Ii, inositol phosphates, or the translocation of protein kinase C.As with CAMP,most cells have cGMP-dependent kinases. The importance and role of these in neutrophils are almost totally unknown. Prelimi-

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nary studies examining for phosphorylated target proteins have been unrevealing; in the presence of cGMP, no difference in phosphorylation could be detected by one-dimensional polyacrylamide gel electrophoresis (Andrews and Babior, 1984).

VIII. CONCLUSION The cyclic nucleotides have been a source of investigation in neutrophils for over 20 years. We have progressed little from the initial studies that proposed that CAMP was a negative effector and cGMP was a positive one. Whereas these simplistic formulations are by no means sufficient, we have come up with no satisfactory alternatives. One hopes that our newer understanding of the structure of adrenergic and muscarinic receptors, of cyclases, and of cyclic nucleotideregulatory proteins will permit this field to elevate itself from the descriptive level. REFERENCES Anderson, R., Glover, A , , and Rabson, A. R. (1977). The in vitro effects of histamine and metiamide on neutrophil motility and their relationship to intracellular cyclic nucleotide levels. J . Immunol. 118, 1690-1696. Andrews, P., and Babior, B. M. (1984). Phosphorylation of cytosolic proteins by resting and activated human neutrophils. Blood 64, 883-890. Bergman, M. J., Guerrant, R. L., Murad, F., Richardson, S . H . , Weaver, D., and Mandell, G. L. (1978). Interaction of polymorphonuclear neutrophils with Escherichia coli. Effect of enterotoxin on phagocytosis, killing, chemotaxis, and cyclic AMP. J. Clin. Invest. 61, 227-234. Berridge, M. J. (1 983). Rapid accumulation of inositol trisphosphate reveals that agonists hydrolyse polyphosphoinositides instead of phosphatidylinositol. Biochem. J. 212, 849-858. Bemidge, M . J. (1984). lnositol trisphosphate and diacylglycerol as second messengers. Biochem. J . 220, 345-360. Berridge, M. J., and Irvine, R. F. (1984). Tnositol triphosphate, a novel second messenger in cellular signal transduction. Nature (London) 312, 315-32 I . Bokoch, G . M., and Gilman, A. G . (1984). Inhibition of receptor-mediated release of arachidonic acid by pertussis toxin. Cell 39, 301-308. Bourne, H. R., Lehrer, R. I . , Cline, M. J., and Melmon, K . L. (1971). Cyclic 3’,5’-adenosine monophosphate in human leukocyte; synthesis, degradation, and effects on neutrophil candidacidal activity. J . Clin. Invest. 50, 920-929. Bourne, H. R., Lehrer, R. I., Lichtenstein, L. M., Weissniann, G . , and Zurier, R. (1973). Effects of cholera enterotoxin on adenosine 3’ ,5’-monophosphateand neutrophil function. J . Clin. Invest. 52, 698-708. Bradford, P. G., and Rubin, R. P. (1985). Characterization of formylmethionyl-leucy-phenylalanine stimulation of inositol triphosphate accumulation in rabbit neutrophils. Mol. Pharmacol. 27, 74-78. Busse, W. W., and Sosman, J. (1976). Histamine inhibition of neutrophil lysosomal enzyme release: an H, histamine receptor response. Srience 194, 737-738.

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CURRENT TOPICS IN M h M B R A N h S A N D T R A N S W R T . VOLUME 35

Chapter 77

Induction of Protein Phosphorylation during Leukocyte Activation WILLIAM L. FARRAR," DOUGLAS K . FERRIS,? DENNIS F. MICHIEL,* AND DIANA LINNEKIN" *Laborutory of Molecular lrnmunoregulation Cytokine Mechunisms Section Nutionul Cancer Institute-Frederick Cancer Research Facility Frederick, Maryland 2 I701 tProgrum Resources, Inc. Frederick Cuncer Research Fucility Frederick, Maryland 2 1701

I. Introduction Protein SerineiThreonine Kinaseb A. Protein Kinase C (PKC) B. Ca* + iCalmodulin (CaM)-Dependent Kinases C. Cyclic Nucleotide-Dependent Kindses and S6 Kinase 111. Protein Tyrosine Kinases IV. Lymphoid Cells and Phosphorylation A. T Cell Activation and Growth B. B Cell Activation V. Myeloid Cell Activation A. The Colony Stimulating Factors B. CSF Regulation of Phosphorylation C. Differentiation D. Functional Activation VI. Summary and Perspectives References 11.

1.

INTRODUCTION

Protein phosphorylation is now recognized as the major mechanism by which protein function is controlled by external physiological stimuli. The regulation of 425 Cupynghl 0 IY!N hy Academic P r e s Inc All right, nl rcproduction m m y lurm rc5cNed

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WILLIAM L. FARRAR ET AL.

diverse cellular responses can be controlled by a relatively limited repertoire of protein kinases and phosphatases. Evolutionarily well conserved, the members of protein kinase families may affect completely different cellular responses even though common kinase systems are regulated in a similar manner in diverse tissues. Here, we will attempt to review the proposed roles of various members of the major protein kinase families that have been suggested to play a role in leukocyte hematopoiesis and in the activation of cells participating in the mature antigen-specific immune response. Prior to embarking on a specific discussion of protein kinases in leukocyte development and activation, we will update some of the general findings of the major enzyme families believed to participate in the regulation of leukocyte biology.

II. PROTEIN SERINE/THREONINE KINASES A. Protein Kinase C (PKC) Originally described as a phospholipid and Ca2 regulated enzyme (Nishizuka, 1984), much attention has been given to lipid mediators other than diacylglycerols in the regulation of PKC activity. For example, it has been shown that sphingosine and lysosphingolipids inhibit PKC activation, suggesting that these metabolites of membrane sphingoglycolipids may function as negative effectors for processes involving PKC (Hannum and Bell, 1987; Hannun et al., 1986). Unsaturated fatty acids (such as oleic and arachidonic acid) have been shown to activate PKC isozymes in the presence or absence of CaZ+ and phospholipid (McPhail ut ul,, 1984). These are important new observations that suggest the existence of other pathways that regulate PKC. The significance of PKC activation by hydrolysis of phosphatidylcholine (PC) is still a matter of conjecture. It is conceivable that a phosphatidylcholine pathway consisting of lyso-PC and other fatty acids might be at least as important as the diacylglycerolinositol 1,4,5-trisphosphate (IP,) system (Fig. I ) . Given that the membrane content of PC is much higher than phosphotidylinositol (PI), PC regulation of PKC activity may be more important than the better characterized inositide pathways. Methodologies which have employed the use of radiolabeled inositol traces would, of course, not detect signal mechanism(s) using PC hydrolysis. Although an attractive hypothesis, this remains untested in leukocyte systems. Another interesting aspect of current PKC research is the molecular heterogeneity of PKC cDNA clones (Parker et al., 1986). Analysis has revealed that PKC isozymes are a family of closely related enzymes encoded by at least three distinct genes (Coussens et al., 1986). Further analysis by Northern and Southern hybridization experiments suggests additional complexities and even alternative splicing of the genes (Ono et ul., 1987). The three isoforms of PKC, designated +

427

17. PROTEIN PHOSPHORYLATION IN LEUKOCYTES Stimulant

/ \ PC

Sphingolipids

PIP2-PI

PLA,

PLC

3 Lyso PC

DAG

FA

IP,

t

Ca2 + Mobilization

(-1

(+)

CaMICa2+ -Dependent Enzymes cDNA __ CY

P I , PI1 Y

FIG. I .

I sozyme Type 111 Type I1 Type

I

Regulation of PKC activity.

types I, 11 and 111, with cDNAs a,p and y, respectively, are found in rat and primate brain and are classified based on the order of elution from hydroxylapatite columns. The isozymes exhibit similar physical properties and regulation by C a 2 + , phospholipids, or phorbol esters. They differ, however, in their autophosphorylation, immunoreactivity, tissue distribution, and probably substrate specificity (Huang et ul., 1986; Huang et ul., 1987). The two major structural domains in PKC, the phospholipid-diacylglycerolphorbol ester (regulatory) binding region and the ATP-substrate binding site (catalytic), can be predicted by the deduced amino acid sequences (Parker et al., 1986; Coussens et ul., 1986). Fortuitously, these domains may be separated into two functional components by the action of a Ca2 -dependent protease. Peptic cleavage of PKCs generates a 30 kDa regulatory domain and a 50 kDa catalytic domain which is catalytically active. The presence of disassociated 30 kDa and 50 kDa fragments in cells is likely to have as much physiological consequence as the intact (80 kDa) regulated PKC. Whether the conversion of 80 kDa PKC isozymes to the 50 kDa catalytic form occurs under conditions of physiological stimuli is unknown. The distribution of PKC isozymes in various tissues has been examined to a +

428

WILLIAM L. FARRAR ET AL.

limited extent. The majority of studies have been with rat brain (Ohno et ul., 1987; Kitano et a l . , 1987). These initial observations have suggested differential gene expression in various regions and tissues of brain, making likely the diRerential expression of PKC isozymes in the hematopoietic system. Although the specific roles of these isozymes in developing tissues are undefined, the unique presence of isozymes with tissue specificity suggests an involvement in developmental regulation. B. Ca2 /Calmodulin (CaM)-Dependent Kinases +

Essentially, five types of Ca2 /CaM kinases have been characterized in mamnialian systems (for reviews see Stull ct ul., 1986; Nairn et ul., 1985) (Table I). Although certain Ca2 /CaM kinases are thought to be activated in lymphocytes in response to the rise in intracellular Ca2+ ([Ca2+Ii)during mitogen or immunoglobulin cross-linking, none has been clearly characterized. Phosphorylase kinase, the enzyme that phosphorylates glycogen phosphorylase, appears to be a skeletal muscle enzyme composed of four types of subunits (a,p, y, 6). lsozymes differing in subunit structure are found in other tissues; other than muscle, however, these forms have not been characterizcd thoroughly. Myosin light-chain kinases are apparently related, though distinct enzymes are found in skeletal, cardiac, and smooth muscle. The major differences in these enzymes appear to be their differential phosphorylation by cyclic AMP (CAMP)dependent protein kinases. +

TABLE 1 MOLECULAR PROPERTIES OF Ca2+IC~M-DEPENUENT PROTEIN KINASW~ Enzyme

Structure ( M , )

Myosin light-chain kinase Phosphorylasc kinase

80.000- 150,000 Native, 1.3 X lo6 (a,(3, 7 , 8) Subunit u , 145,000 Subunit p. 128,000 Subunit y. 45,000 Subunit 8 , 17,000 Native, 48,000 Subunit 37,000-42,000 Native, 250,000-600,000 Subunit u . 50.000 Suhunit p, 60,000 Native, 140.000

Ca' ' icalmodulin kinase I

Ca*+/calmodulin kinase 11

Ca2+/calmodulinkinase 111

As summariled by Blackshear er

(I/.

198K)

Substrate Myosin P light chain Phosphorylasc

Synapsin I (site 1) Protein 111 Synapain I (sitc 11) Glycogen synthasc, MAP-2 Tyrosinc hydroxylase Elongation factor 2

17. PROTEIN PHOSPHORYLATION IN LEUKOCYTES

429

CaM kinase I has been purified to homogeneity from bovine brain using synapsin I as a substrate affinity matrix (Nairn and Greengard, 1987). CaM kinase I is widely found in other tissues and is known to phosphorylate synapsin I and protein I11 in neurons. Other substrates are unknown. CaM kinase I is activated in neurons in response to increases in [Ca2+Ii. The phosphorylation of synapsin I is believed to regulate neurotransmitter release through its interaction with synaptic vesicles and cytoskeletal components, such as actin. CaM kinase 11, also termed multifunctional CaM kinase, is a family of related kinases with native molecular weights ranging from 250,000 to 650,000 and subunits M , 50,000-60,000 (Cohen, 1988; Schulman, 1988). Similar to the PKC family, brain shows distinct distribution patterns for various CaM kinase 11 isozymes. Additionally, the p subunit may also undergo alternative mRNA splicing. The CaM kinase I1 system is distinct from the other CaM-dependent kinases in that it has a broader range of known substrates. These include microtubulesynapsin 1, microtubule-associated protein 2 (MAP-2), glycogen synthetase, smooth muscle myosin light-chain, and tyrosine hydroxylase. CaM kinase I11 is the most recently identified of the Ca2 /CaM-dependent kinases. Although awaiting complete characterization, CaM kinase 111 appears to phosphorylate only one protein, elongation factor 2 (EF-2) (Ryazanov, 1987), a protein which catalyzes the translocation of peptidyl-tRNA on the ribosome. In cultured cells, CaM kinase I11 is regulated by many factors, including growth factors and mitogens. For example, stimulating fibroblasts with bradykinin causes a transient rise in [Ca2+],, which activates CaM kinase 111 and results in the transient phosphorylation of EF-2 (Palfrey et al., 1987). +

C. Cyclic Nucleotide-Dependent Kinases and S6 Kinase Cyclic AMP-dependent protein kinase (A-PK) isolated from bovine heart has been resolved into CAMP-free (peak I) and CAMP-bound (peak 11) enzyme (Cobb et ul., 1987). The peak I1 enzyme is catalytically inactive. Catalytic activity rather than CAMPbinding has been determined to be the critical element in transducing signals regulating gene expression. The coexpression of plasmids encoding an active fragment of PKA inhibitor (PK-I) substantially inhibits the activation of CAMP-dependent genes, such as enkephalin (Grove et al., 1987). Not surprisingly, cyclic GMP-dependent kinases (G-PK) also have cGMP-free (peak I) and cGMP-bound (peak 11) forms. Although their biological roles are still unknown, the elevated presence of the cyclic nucleotide-bound (peak 11) forms of A-PK and G-PK has been associated with increase sensitivity of tissues to these “second messenger” stimuli. The S6 protein of the 40 S ribosomal complex has received considerable attention since its phosphorylation is regulated by a number of stimuli, including

430

WILLIAM L. FARRAR ET AL.

phorbol esters, growth factors, IL-2, insulin, and the tyrosine-specific pp6OV-”’”’ oncogene product (Blenis et al., 1987; Evans and Fanar, 1987). Interestingly, although EGF receptor, the insulin receptor, and are tyrosine-specific kinases, the S6 protein is phosphorylated on serine residues, suggesting that interkinase regulation occurs. The S6 kinase activity has been purified from various sources, and it appears to be homologous to other serine kinases. Activation of the S6 kinase appears to be closely associated with cellular proliferation, but its regulation by many diverse stimuli suggests that multiple pathways exist for regulating the S6 kinase.

111.

PROTEIN TYROSINE KINASES

We have briefly summarized findings pertaining to a number of the serine kinases that may be relevant to leukocyte biology. ’The protein kinases are a large family of enzymes, the number of described members now approaching 100 (for review see Hanks et a / ., 1988). Tyrosine kinases represent a remarkable family that consists of growth factor receptors as well as mutant proteins with oncogenic transforming activity (v-,c-oncogenes). There have been numerous demonstrations of the transforming potential of the tyrosine kinase family. These studies have included cells of hematopoietic lineages. These exciting observations have led to an explosion of information concerning the structure and function of protein tyrosine kinases. It is not possible to summarize the many excellent studies relevant to tyrosine kinases, so we will endeavor only to characterize some of the major areas of current interest and relate what is known about their participation in leukocyte biology. Table 11 lists the various subfamily members of the protein tyrosine kinases. Members of the subfamilies are found in relative levels among the hematopoietic lineages. For example, although there is little to no detectable c-src protein in lymphocytes, T cells can express high levels of the src-related proteins FYN, LYN, and LCK. Myeloid lineages produce HCK, frsifps, and fms and little of the other kinases found in T lymphocytes. The expression and potential roles of these kinases in hemopoietic development have only recently been examined. However, tyrosine kinases are being intensively invcstigated in other tissues. The important issues under study include (1) the relationship of autophosphorylation to kinase activity; ( 2 ) the relationship of tyrosine kinase activity to activation of the phosphatidylinositol kinase activity; (3) the participation of tyrosine kinases in receptor-mediated signal transduction, and (4)the coupling of receptors devoid of kinase activity to tyrosine kinases. A complete discussion of the roles of serine and tyrosine kinases in biological systems far exceeds the scope of this chapter. Following the brief introduction to the kinases generally believed to participate in receptor-mediated signaling, we will now focus on summarizing some of the critical work on kinase systems

431

17. PROTEIN PHOSPHORYLATION IN LEUKOCYTES TABLE I I PROTEIN TYROSINE KINASE FAMILY MEMBERV

A. .src subfamily src: cellular homolog of oncogene product from Rous avian sarcoma virus yes: cellular homolog of oncogene product from Yamaguchi 73 avian sarcoma virus fgr: cellular homolog of oncogene product from Gardner-Resheed feline sarcoma virus FYN: putative protein tyrosine kinase related to fgr and yes LYN: putative protein tyrosine kinase related to LCK and yes LCK: lymphoid cell protein tyrosine kinase HCK: hematopoietic cell putative protein tyrosine kinase dsrr64: Drosophila gene product related to src: polytene locus 648 dsrc28: Drosophila gene product related to src; polytene locus 28C B. abl subfamily abl: ccllular homolog of oncogene product from Abelson murine leukemia virus ARC: putative protein tyrosine kinase related to ubl-human genomic DNA (partial) dash: Drosophila gene product related to abl nub/: nematode gene product related to ab/ fesifps: cellular homolog of oncogene products from Gardner-Amstein and Snyder-Theilen feline sarcoma viruses and Fujinami and PRCIl avian sarcoma viruses C. Epidermal growth factor receptor subfamily ECFR: epidermal growth factor receptor; cellular homolog of oncogene product (v-erb-B) from AEV-H avian erythroblastosis virus neu: cellular oncogene product activated in induced rat neuroblastomas (also called erb-B2 or HER2) dw: Drosuphila gene product related to ECFR D. Insulin receptor SUbfdmlly 1NS.R: insulin receptor IGFIR: insulinlike growth factor I receptor DILR: Drosophila gene product related to 1NS.R LTK: leukocyte tyrosine kinase ros: cellular homolog of oncogene product from UR2 avian sarcoma virus 7less: Drosophila sevenless gene product essential for R7 photoreceptor cell development trk: colon carcinoma oncogene product activated by genetic recombination (MNNG)-induced oncogene product met: N-methyl-N’-nitro-N-nitrosoguanidine E. Platelet-derived growth factor receptor subfamily PDGFR: platelet-derived growth factor receptor CSFIR: colony-stimulating factor-type I receptor; cellular homolog of oncogene product (v:fms) from McDonough feline sarcoma virus kir: cellular homolog of oncogene product from Hardy-Zuckcrman 4 feline sarcoma virus ret: cellular oncogene product activated by recombination F. Other receptorlike protein tyrosine kinases T K R l I : putative protein tyrosine kinase TKR 16: putative protein tyrosine kinase ~

0

A\ condcnyed from Hanks er a/ (19x8)

432

WILLIAM L. FARRAR ET AL.

found to be modulated during hcmatopoiesis and activation of lymphoid and myeloid cell lineages.

IV. LYMPHOID CELLS AND PHOSPHORYLATION

A. T Cell Activation and Growth I . COMPETENCE A N D PROGRESSION

T lymphocyte growth is characterized by two distinct phases of cellular stimulation. The first stage, often referred to as activation or competence, is accomplished by the stimulation of resting lymphocytes by foreign antigen, usually presented in complex with histocompatjbility structures by cells of the macrophage-monocyte lineage. This physiological mechanism can be mimicked by mitogenic plant lectins, antibody cross-linking of the T3/Ti T cell receptor complex, or the addition of phorbol ester analogs with ionophores (Imboden and Stobo, 1985). The resulting response of lymphocytes is a wave of de lzovo gene transcription, including protooncogenes (Reed ct al., 1986; Cleveland et a / ., 1987), transferrin receptors, and the two critical components of T cell growth, interleukin 2 (IL-2) and the IL-2 receptor a subunit (Kronke rt al,. 1985; Reed et a / . , 1986). Once the activation or competence state is achieved, the progression from G , into S phase is based on the interaction of secreted IL-2 and high affinity receptors on the surface of the activated cells. The current model of the high affinity IL-2 receptor is that the high affinity state occurs when at least two proteins associate to form a binding site with an apparent K, of approximately to 10-l2 M . One protein member of the complex, a 55 kDa subunit, has been characterized and molecularly cloned. Termed IL-2Ra, this component has a very low affinity for 11,-2 ( K d lo-' to l o p 8 M ) (Greene, 1987; Waldmann, 1986), and no substantial evidence exists that the IL-2Ra protcin mediates 1L-2 signal transduction. Alternatively, the recently discovered IL-2RP subunit (7075 kDa) (Sharon et cil., 1986; Robb et al., 1987; Tcshigawara rt al., 1987) has a higher affinity (10 K d ) and apparently may mediate signal transduction (Ishii et al., 1988) as well as significant biological responses (Ottaldo et d.,1984). Both subunits appear to be required to form the high affinity binding site characterized on most proliferating T cells (Robb et a / . , 1987). 2. T CELLANTIGENRECEPTOR The T cell antigen receptor (TCK) complex is made of a and p chains which recognize the antigen (Marrack and Kappler, 1986; IIendrick et al., 1984; Chien et d., 1984; Saito et al., 1984) and the y. 6 , E, and 5 chains of the CD3 antigen

17. PROTEIN PHOSPHORYLATION IN LEUKOCYTES

433

(Samelson et al., 1985; Oettgen et a l . , 1986; Baniyash et ul., 1988a). With some cells, activation by a specific antigen can be simulated by lectin mitogens or by mitogenic antibodies directed against the T cell receptor complex. Binding to the T cell antigen receptor stimulates several second messengers, including phosphatidylinositol hydrolysis, increases in internal calcium concentration, and activation of protein kinase C. PKC phosphorylates a variety of cellular proteins including the y and E chains of the T cell antigen receptor (Samelson et al., 1987; Patel er al., 1987; Klausner et a/., 1987). It also appears that a protein tyrosine kinase is stimulated, which results in the phosphorylation of the 6 chain of the T cell antigen receptor as well as several other proteins (Baniyash et al., 1988b; Klausner et al., 1987). Therefore, the stimulation of the antigen receptor leads to the activation of at least two distinct kinase systems, one tentatively identified as a PKC isozyme (the specific isozyme is unknown) and the other, an unknown tyrosine kinase (Table 111). The interaction of PKC and TK in T cell activation can be uncoupled, since direct activation of PKC by phorbol ester does not result in activation of the tyrosine kinase (Weisman et ul., 1988). Additionally, antibodies to the CD2 molecule, which activate T cells, do not stimulate tyrosine kinase activity. Thus, activation of T cells can be achieved in the absence of tyrosine kinase activation. T cells can also be activated in vitro by the addition of antibodies that bind the antigen receptor, the Thy-] molecule, or a surface determinant such as CD2. Three ligands, antigen, or antibody against the E chain of the TCR or Thy-1 all activate phosphatidylinositol hydrolysis, PKC, and the tyrosine kinase responsible for both serine and tyrosine phosphorylation of the p21 subunit of the TCR complex (Klausner et al,. 1987). Stimulants of CAMP, which inhibit IL-2 syn-

TABLE I11 ACTIVATION PATHWAYS FOR THE T CELLANTIGENRECEPTOR" Stimulus Antigen

Monoclonal antibodies to anti-CD3-Eianti-Thy- I

Anti-CD2 antibodies

Effects 1. 2. 3. 4. 1.

2. 3. 1. 2.

Phosphatidylinositol hydrolysis Increased intracellular Ca*+ Activation of protein kinase C Activation of a protein tyrosine kinase Phosphatidylinositol hydrolysis Serine phosphorylation of CD3-y chain Tyrosine phosphorylation of CD3-[ chain T cell proliferation CD3-c chain is not phosphorylated on tyrosine

Inhibition Inhibited by cAMP

Not inhibited by cAMP

434

WILLIAM L. FARRAR ET AL.

thesis and T cell activation, inhibited the activation of phosphatidylinositol breakdown and TCR serine phosphorylation produced by all three stimuli (Klausner er al., 1987). In contrast, only antigen-stimulated tyrosine kinase activation is sensitive to CAMP, while antisera to Thy-I and the E chain of the TCR activate the tyrosine kinase in a manner insensitive to CAMP inhibition. These results suggested that although antigen or ci TCR antibody stimulate the same receptor, diffcrences in the details of the signal transduction process may occur, reflecting some plasticity even in the same receptor (Table 111). The apparent depletion of PKC activity in the 2B4 T cell hybridoma by high dose phorbol ester treatment eliminates antigen-receptor stimulated serine phosphorylation but not tyrosine kinase activation (Patel et a/., 1987). These experiments suggested that the activation of the tyrosine kinase associated with the TCR is not a result of PKC activation and probably represents an independent regulation. To date, there has been no identification of the tyrosine kinase associated with the TCR. It is also important to note that the activation of tyrosine kinase activity is not mandatory for T cell activation since ci CD2 antibody clearly activates T cells in the absence of stimulating the p21 tyrosine phosphorylation. Initial studies have strongly implicated that phosphatidylinositol hydrolysis is critical in T cell activation. Recently, this notion has been challenged. Mutants of the Jurkat T cell line, when stimulated with monoclonal antibody, show increases in [Ca2 Ii and early Ca2 mobilization but fail to synthesize IL-2 (Goldsmith and Weiss, 1988). Furthermore, antigen-specific CTL do not activate Ca2+ mobilization or inositol phosphate synthesis when stimulated with target cells (O'Rourke and Mescher, 1988). Additionally, external calcium is not required for CTL recognition and cytolysis. Both studies have suggested that the phosphatidylinositol pathway may not be sufficient for T cell activation and, in some cases, undetectable. Since the tyrosine kinase associated with the TCR may also be bypassed by ci CD2, questions remain regarding the potential additional undescribed signals in T cell activation. Recently, it has been demonstrated that the lck protooncogene has a role in T cell activation (Marth et al.. 1987). Veillette e l al. (1988a) showed that the T lymphocyte-specific tyrosine kinase termed LCK is functionally and physically associated with CD4 and CD8 T cell antigens. LCK coprecipitated with antibodies to either CD4 or CD8, and antibody-mediated cross-linking of either antigen modulated the tyrosine kinase activity of LCK. Activation of T cells with anti-TCR or mitogens also modulated LCK. These stimuli produced additional serine phosphorylations rather than increasing the catalytic tyrosine kinase activity (Veillette rt al., 1988b). Thus, antisera against CD4 or CD8 stimulated the tyrosine kinase activity of LCK while phorbol ester or TCR stimulants produced serine phosphorylation of LCK, presumably by PKC. The biological significance of the modulation of LCK catalytic activity in T cells is unknown; however, the +

+

17. PROTEIN PHOSPHORYLATIONIN LEUKOCYTES

435

mRNA and protein levels of LCK promptly decline upon T cell activation (Marth et al., 1987). In addition, two other human lymphocyte tyrosine kinases distinct from LCK, termed TPK I ( M , 70,000-100,000) and TPK I1 (M,35,00040,000), are also down-regulated following mitogenic stimulation (Hall et al., 1987). Table IV summarizes the kinases and substrates reportedly modulated during T cell activation and IL-2 stimulation.

3. INTERLEUKIN 2 Investigations into the mechanisms of growth factor actions are a major focus of current research. Many hormones, neurotransmitters, and growth factors activate distinct and often multiple classes of protein kinases. I L 2 is no exception, since work has demonstrated that both serine as well as tyrosine kinases are activated in T lymphocytes stimulated with IL-2. Ishii et al. (1986, 1987, 1988), Gaulton and Eardley (1986) and our laboratory (Evans and Farrar, 1987; Evans et al., 1987) have found that IL-2 stimulates the phosphorylation of a number of substrates on serine residues. Similar substrates are phosphorylated after phorbol ester stimulation. These observations suggest that IL-2 triggers a kinase system similar or identical to phorbol ester activated protein kinase C isozymes, thus regulating the serine phosphorylation of substrates seen at 67-69 kDa in size (Ishii et al., 1986, 1987; Evans et al., 1987). Additional complexities have emerged. The only I L 2 regulated phosphosubstrate identified to date is the S6 protein of the 40 S ribosomal complex (Evans and Farrar, 1987). Although the S6 protein is phosphorylated upon phorbol ester stimulation, we isolated a Mg2 dependent enzyme distinct from protein kinase C that apparently acted as the effector enzyme for the phosphorylation of the S6 protein, directed by either IL-2 or phorbol ester stimulation. Thus, a potential for kinase cascades and cross-talk has emerged when examining the kinase systems regulated by IL-2. I L 2 has been shown to regulate one or more enzymes with tyrosine kinase activity, Saltzman et al. (1988) have shown, using ID SDS-PAGE and antiphosphotyrosine immunoblotting, that IL-2 stimulation of lymphocytes increases the tyrosine phosphorylation of several proteins. We have characterized phosphotyrosyl proteins regulated by I L 2 in normal human T lymphocytes by twodimensional nonequilibrating pH gradient gel electrophoresis (NEPHGE) and phosphoamino acid analysis (Fig. 2). In addition, using cell lines that contain only the IL-2RP chain, we have found that the 70-75 kDa subunit of the IL-2 receptor increased phosphorylation of proteins ranging from 180 to 40 kDa (Ferris et al., 1989). These proteins efficiently bound to MAIG2 antisera coupled to Sepharose and were effectively competed with phenylphosphate. Furthermore, an examination of the phosphoamino acid ratios of individual proteins revealed that most contained both phosphotyrosyl and phosphoseryl residues. Among the phosphoproteins analyzed, three different characteristics emerged. Some phos+

TABLE 1V PROTEINS PHOSPHORYLATED DURING T CELLACTIVATION Protein (kDa)

Identity

Amino acid

Kinase

Stimulus Antigen, mitogen. or stimulating antibody (anti-CD3. anti-Thy-1) not stimulated by PMA Antigen Antigen PHA and ConA

p2 I

cchain of CD3

Tyrosine

NItl

pl15 p36-38 P66

N1 Lipacortin TPP-66

Tyrosine Tyrosine Tyrosine

NI N1

N1

Tyrosine

N1

NI N1

Tyrosine Tyrosine + Serine

Nl

NI

Tyrosine

+ Serine

Nl

p120, p100. p84, ps7, P38 pao-85 p180, p100, p92, p70, P o p66, p55-60, p.12 m p92

N1

NI

PP60c-sr"

Serine

PKC

P56

pp56lCA

Serine

PKC

p2 1

ychain of CD3

Serine

PKC

p2 I

rchain of CD3

Serine

PKC

CD4. CD8

NI

CD4, CD8, CDS,

NI

CD7, CD43[&pl15), CD3. LFA-chain

IL-2 on CTLL2 cells (1L-2 dependent) IL-2 on 32DilL-2 cells 1L-2 on human T cells 1L-2 on cell lines expressing 1L-2 receptor p chain Antibodies to CD3 or PMA on Jurkat cells PMA on T cells and LSTRA T cell lymphoma Antigen, mitogen. PMA. antibodies to CD3 or Thy- 1 Antigen, mitogen, PMA antibodies to CD3 or Thy-l PMA or via CD3-Ti or CDI on cloned T cells PMA on human PBMC and T cells

Reference Baniyash

el

a / . (I988b)

Patel el a). ( I 987) Patel er a/. (1987) Wedner and Bass ( 1 986) Saltzman et al. ( 1988) Morla et u l . (1988) Ferris er a / . (1989) Farrar and Ferris (1989) Ledbetter er a / . ( 1987) Casnellie and Lambens (1986) Patel e t a / . (1987)

Patel et al. (1987) Blue e r a / . (1987) Chatila and Geha ( I 988)

Serine

PKC

PKC

NI

NI

p67. p56, p45

NI

N1

NI

P65 P67

NI NI

P67

NI

P63

4 nuclear and cytosolic proteins pl from 5.3 to 6.1 pl 6 . 3

Serine

NI

Sensitive to base

NI

NI

NI

PKC

Ribosomal S6 protein NI

NI NI

S6 Kinase PKC

Serine

N1

Serine Threonine NI

NI NI NI

Tac subunit of IL-2 receptor

P55

P82

wp w

P70

p92, p82, p65, p61, p55, p28 P3 1 ~105-115, 990, p66, p58. p55, p40, p34 p55-62

y chain of

T cell re-

NI Threonine Serine Serine

+

NI NI NI

ceptor ~ 2 7 P26 P78

N1, not indicated

NI NI NI

PMA on human T cells and HUT102B2 leukemia cell line PMA + PHA + Caz+ ionophore on human lymphocytes PMA + PHA + Ca’+ ionophore on human lymphocytes ConA, PHA. A23187 on PBL DAG, 1L-2 and CSFs on several cell lines PMA or IL-2 on PHA-stimulated PBL and IL-2-dependent lines PMA or IL-2 on PHA-stimulated PBL and IL-2-dependent lines PMA. PHA, and anti-CD3 agarose on human T cell hybrid (1123) PDBu on resting human T cells OAG or IL-2 PMA or IL-2 on PHA-activated T cells IL-I on cloned cytotoxic T cell line IL-2 on quiescent murine T cells IL-2 on anti-Tac immunoprecipitates from Jurkat and PBLs

Shackelford and Trowbridge (1986) Kaibuchi et a / . (1985)

Kaibuchi et a/. (1985)

Chaplin et a / . (1980) Evans er u/. 1987) lshii e r a / . (1986, 1987)

lshii

ef

a / . (1988)

Swift et a / . (1988)

McCrddy er a/. (1988) Evans and Farrar ( 1987) Caulton and Eardley ( 1986) Lieberman et a/. ( I 986) Kohno et al. ( 1986) Benedict et a/. ( I 987)

A

Acidic

Basic

C

KDa 200

-

116-

9266

-

*

lb

45dr

31FIG 2. Two-dimensional analysis of immunoaffmity purified phosphotyrosyl proteins from control and IL-2-stimulated human T lymphocytes. Quiescent 1L-2dependent human T cells were equilibrated with [3’P]orthophosphate and either mock st~mulated(A) (control) or treated with recombinant IL-2 for 20 min (B). Panel C is a duplicate of panel B except phenylphosphate was added as a competitor during immunoprecipitation. The cells were lysed and the phosphotyrosine containing proteins affinity purified and analyzed by two-dimensional NEPHGE and autoradiography. Positions and molecular masses of standards in kilodaltons are indicated at left. Proteins that were modulated by IL-2 stimulation are indicated with arrows and approximate molecular masses in kilodaltons.

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phoproteins were coordinately phosphorylated on serine and tyrosine residues (e.g., pp42). In others, serine phosphorylation appeared to decrease while phosphotyrosine increased ( pp70). Also, one protein examined, pp92, contained only phosphotyrosine during the time course tested. These changes in phosphorylation on IL-2 stimulation suggested that one or more tyrosine kinases as well as serine kinases and protein phosphatases may be activated and regulate the phosphorylation status of individual substrates. These initial observations make it clear that the kinase activation pattern is complex. The use of leukemic T cell lines expressing only one subunit of the IL-2 receptor complex allowed us to determine whether the IL2RP subunit could transduce signals for tyrosine kinase activation. Although some increase in background tyrosine phosphorylation was seen, in both the gibbon T cell line MLA-144 and the human YT-2C2 cell line, IL-2 stimulated tyrosine phosphorylation (Farrar and Ferris, 1989). Of particular note was the JL2-induced phosphorylation of pp92 in normal T lymphocytes (Ferris et al., 1989). This protein was phosphorylated exclusively on tyrosine. Many of the phosphotyrosyl proteins in which we observed changes have similar molecular weights to those found by Saltzman et af. (1988) using 1 D immunoblotting. The finding that IL-2 activates both serine and tyrosine kinases suggests a complex signaling mechanism. The IL-2Ra subunit has not been shown to mediate biological responses or signal transduction by IL-2. Nor does the structural information available for the IL-2Ra suggest any clues to a mechanism for IL-2-mediated signal transduction. However, the IL-2RP chain appears to mediate receptor complex internalization (Robb and Greene, 1987), stimulation of serine kinase activities (Ishii et al., 1988), biological effects (Siege1 et af., 1987), and here, the activation of tyrosine kinase activity. Although relatively small, the 70 kDa IL-2RP subunit may contain intrinsic kinase activity or, at least, be capable of coupling to both serine and tyrosine kinases. One report by Benedict et af. (1987) found that in the presence of ATP, IL-2 stimulated phosphorylation of a 55 kDa protein in a p55 (IL2Ra) immunoprecipitates. Phosphorylation of an 85 kDa protein was also reported in response to IL-2 in a myeloid cell line (Morla et af., 1988). Although 1L-2 may stimulate the 85 kDa protein in a IL-2Ra precipitates, we have no evidence that this occurs in situ since IL-2 stimulation does not induce phosphorylation of proteins precipitated by 01 IL-2Ra (Tac) (W. L. Farrar et al., unpublished observations). Currently, no evidence has directly indicated kinase activity intrinsic to the p70-75 kDa IL-2RP chain, although clearly coupling to serine and tyrosine kinases occurs. The molecular cloning of IL-2RP may provide insights into its mechanism of kinase activation. The ability to resolve individual substrates regulated by IL-2 activate serine and tyrosine kinases will allow investigation of the effector kinases as well as of the functional significance of these posttranslational modifications. Among the diverse results regarding IL-2 activation of protein kinases are two

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consistent featurcs found by several laboratories: ( I ) IL-2 stimulates serine phosphorylation of some substrates in a manner similar to phorbol esters; and (2) IL-2 stimulates serine and tyrosine kinase activation via the IL-2RP chain. The involvement of PKC has been challenged by several investigations that have attempted to functionally deplete PKC isozymes by high dose phorbol ester treatment. These studies have found that IL-2 still stimulates proliferative response in these “PKC depleted” cells (Mills et al., 1988), thus, questioning the requirement of PKC isozymes for the 1L2 driven response. Valge et d . (1988) also showed that in phorbol ester-treated PKC depleted cells, phorbol ester could not induce de novo gene expression whereas 1L-2 still could. In our own experiments, high dose phorbol ester indeed decreases some PKC isozymes, phorbol ester binding sites, and PMA regulated gene expression. It did not, however, decrease IL-2 stimulated tyrosine kinase activity and regulation of c-myc expression (Farrar and Ferris, 1989). Although the effects of high dose phorbol ester treatment delete some newly initiated phorbol ester induced events, it is unclear whether this has altered the ratio of Ca*+, phospholipid regulated PKC to PKM, the unregulated catalytic subunit. T cell clones have been reported to proliferate in response to phorbol esters and IL-2; the deletion of one pathway may favor the shunting of signals to the other proliferative pathway. 6. B Cell Activation 1. COMPETENCE, PROLIFERATION, A N D DIFFERENTIATION

Activation of B lymphocytes can be categorized into acquisition of competence, proliferation, and differentiation into immunoglobulin secreting cells. Stimulation of B cells with antigen, antisera against membrane-bound immunoglobulin (mlg), IL-4 (also called B cell stimulating factor), or LPS results in transition of cells from G, to a cycling state. Additionally, 1L-5and lL-6 modulate proliferation and antibody secretion. This section will discuss the role of PKC and tyrosine kinases in these events. 2. SERINE KINASES PMA produces B cell proliferation (Nel et d ,1985a) and is associated with phosphorylation of B lymphocyte proteins (Hornbeck and Paul, 1986). Nel et af.,(1985a, 1986) reported phosphorylation of B cell cytosolic proteins of molecular weights 94,000 66,000, 60,000, 56,000, 50,000, 43,000, 38,000, 35,000, 28,000-30,000, 20,000-23,000, and 15,000-18,000 as well as a Triton-soluble protein of 28,000 in response to PMA. With the exception of p63 and p50, the PMA-induced phosphorylation of all the cytosolic substrates was inhibited by polymyxin B , a PKC inhibitor. Stimulation of B cells with anti-lg results

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in increases in inositol 1,4,5-trisphosphate and diacylglycerol (reviewed in Cambier and Ransom, 1987). Interestingly, studies using LPS indicate that IP, generation is not mandatory for B cell transition from Go to S phase (Betel et al., 1974). However, LPS does initiate redistribution of B lymphocyte PKC from cytosolic to membrane fractions (Grupp and Harmony, 1985). PKC translocation from the cytosol to membrane was observed after anti-lg stimulation as well as in response to PMA treatment (Nel et al., 1986). Hombeck and Paul (1986) reported membrane proteins with molecular weights of 47,000, 55,000, 62,000, 68,000, 68,000, and 65,000-70,000 were phosphorylated after PMA treatment, while anti-Ig elicited phosphorylation of p55 and several forms of p62, p68, and p65-70 proteins. Additional evidence suggesting PKC involvement in signal transduction through membrane-associated immunoglobulin is the similarity in peptide mapping of phosphoproteins resulting from PMA and anti-Ig stimulation as well as the reduction of anti-Ig-induced phosphorylation responses after PKC depletion by prolonged PMA exposure (Hornbeck and Paul, 1986). IL-4 stimulation of intact B lymphocytes failed to induce PKC translocation, phosphoinositide hydrolysis, Ca2+ mobilization, or membrane depolarization (Mizuguchi et al., 1986; Justement et al., 1986); however, incubation of B cell membrane fractions with 1L4 produced phosphorylation of a 44 kDa protein. Addition of anti-Ig to the membrane preparation failed to produce similar results leading Justement et al. (1986) to conclude that PKC was not involved in the response. The nature of the kinase mediating these actions has yet to be defined. Interestingly, IL-4 stimulated tyrosine phosphorylation of p170 and pl10 in the myeloid cell lines IC-2.9 and 32D (Morla et al., 1988) while studies using IC-2, the parent line of IC-2.9 found IL-4 induced no tyrosine phosphorylation (Koyasu et al., 1987). 3. TYROSINE KINASES The association of tyrosine kinases and lymphocytes was suggested by the reports of relatively high levels of tyrosine kinase activity associated with extracts of both thymus (Zioncheck et al., 1986) and spleen (Swarup et al., 1983; Earp et al., 1984). T and B lymphocytes contain distinct tyrosine kinases which phosphorylate different substrates and are observed under different experimental conditions (Earp et al., 1984). Splenic lymphocytes bearing surface immunoglobulin as well as the B lymphoblastoid line Raji were found to contain tyrosine phosphoproteins p61 and p55 after exposure to either vanadate or various concentrations of Triton X- 100. Tryptic peptide analysis indicated these proteins were related forms and distinct from p58 and p64, alkali-resistant phosphoproteins present in T cell extracts (Earp et al., 1985). In contrast to T cells, phosphorylation of B cell specific substances was resistant to inhibition by N-ptosyl-L-lysine chloromethylketone (TLCK) (Earp et al., 1985).

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Anti-Ig stimulates tyrosine phosphorylation of B cell cytoskeletal associated proteins between 55 and 61 kDa (Nel et al., 1985b). PMA stimulation of normal human B cells produced tyrosine phosphorylation of p75, p66, p43, and p28 in Triton-soluble fractions (i.e., membrane proteins), while cytoskeletal proteins (Triton insoluble) of 56-60 kDa were phosphorylated in response to PMA. There was considerable similarity of tyrosine phosphorylation patterns between normal B cells and those obtained from chronic lymphocytic leukemia patients. Little is known about the nature of the phosphoproteins associated with signal transduction in B lymphocyte activation. Two substrates of B cell phosphotransferase systems which have been identified are CD20 and lamin B. CD20 is a cell surface antigen expressed early in B cell differentiation. Tedder and Schlossman (1988) demonstrated that phosphorylation of CD20 on the 33 and 35 kDa isoforms occurs in proliferating B cells as well as constitutively in transformed B cell lines such as Raji, Daudi, SB, Bjab, and Namalwa. Stimulation of B cells with antisera to CD20 resulted in enhancement of CD20 phosphorylation. Pretreatment of B cells with PKC inhibitor H-7 produced a decrease of PMA, DAG, and anti-CD20 induced CD20 phosphorylation. Lamin B, a 67 kDa intermediate filament localized to the nucleus, has also been identified as a PKC substrate in B lymphocytes stimulated with either PMA or anti-lgM (Hornbeck et al., 1988).

V. MYELOID CELL ACTIVATION

A. The Colony Stimulating Factors Cells of the immune system are dcrived from a pluripotential stem cell from the regenerative compartment in bone marrow. ‘The ontogeny of myeloid cell production and control of myeloid cell proliferation and differentiation is determined by various hematopoietic growth factors. Additionally, some of these factors are capable of enhancing or eliciting functional responses such as migration, phagocytosis, and bacterial killing. Granulocyte macrophage colony stimulating factor (GM-CSF) and interleukin 3 (IL3) stimulate multilineage progenitor cells, while granulocyte colony stimulating factor (G-CSF) and macrophage colony stimulating factor (M-CSF) act on single lineage progenitors. Three major means of receptor-mediated signal transduction have been identified. These involve changes in CAMP concentrations, activation of a receptor encoded tyrosine kinase, and activation of PKC by diacylglycerol, an event often, but not always, associated with IP, increases. Each of these events has been associated with increases in second messengers, such as cyclic nucleotides or calcium, and subsequent activation of various classes of protein kinases. Very little is known of the signal transduction events involved in hematopoietic cytokine activation. Receptors specific for GM-CSF, G-CSF, IL-3, and M-CSF

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have been identified on the cells of the bone marrow as well as on their more mature progeny in studies of both mice and human cells. The receptor for GMCSF is estimated to be 85 kDa in size and approximately 200 receptors exist on the target cell. The receptor for G-CSF is 150 kDa and also distributed in low numbers on target cells. The M-CSF receptor has a molecular weight of 170,000, is the product of the protooncogene c-fms, and is the best characterized receptor in this family of factors. Ligand binding initiates activation of a tyrosine kinase encoded within the M-CSF receptor. This is a similar activation scheme as identified for insulin, platelet-derived growth factor, and epidermal growth factor. The receptor for IL-3 was originally identified in cross-linking studies as having a molecular weight of between 50,000 and 70,000, but recent work has suggested these may be proteolytic products of a 140 kDa form (Isfort et al., 1988a). The small size of the GM-CSF, G-CSF, and possibly the IL-3 receptor makes it improbable that there is a functional tyrosine kinase encoded in these molecules. Therefore, the mechanism of activation of these factors remains unresolved. Proliferation, differentiation, and elicitation of functional parameters, such as cell migration, degranulation, and the generation of the respiratory burst, are important components of cellular activation. The following section will address the relationship between phosphorylation and each of these events in myeloid cells.

B. CSF Regulation of Phosphorylation I . SERINEKINASEACTIVATION Farrar er a!. ( I 985) were the first to establish that protein kinase C translocation was associated with stimulation of a myeloid cell line by IL3. Treatment of the myeloid cell line FDC-P1 with 100 units of IL-3 elicited an increase in membrane-associated PKC as well as a decrease in cytosolic PKC activity. These responses were observed as rapidly as 1 min after 1L3 exposure, peaked at 20 min, and approached control levels by 60 min. Subsequent studies utilizing both one- and two-dimensional electrophoresis established that stimulation of FDC-P1 cells with either IL-3 or diacylglycerol resulted in the phosphorylation of 68 kDa and 20 kDa proteins on threonine residues (Evans et al., 1986). The role of PKC in these events was further confirmed by the phosphorylation of a p68 substrate with a similar charge-mass ratio as that in whole cell lysates in cytosolic fractions of FDC-PI cells by purified PKC in a cell free system. Interestingly, p68 was found to be phosphoryiated in response to lL-3 and G-CSF in the NSF-60 and NSF-60.8 cell lines and in response to IL-2 by the CT6 cell line (Evans et al., 1987). These observations paralleled the capacity of each cell line to proliferate in response to the factor being tested with the exception of NSF-60.8 where GM-

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CSF elicited proliferation yet failed to stimulate phosphorylation of p68. lshii et al. (1987) have observed the serine phosphorylation of a cytosolic protein with a molecular weight of 67,000 in IL-2-stimulated peripheral blood leukocytes and ILT-Mor cells, an 1L-2-dependent human T cell line. These results suggest remarkable conservation in signal transduction events distal to receptor binding in cell lines of both myeloid and lymphoid origin as well as among the growth factors IL-2, G-CSF, and IL-3. Studies of the myeioid cell lines 123 and subclones (rac. 1 and rB5) of the AC2 line in response to crude IL-3 preparations in WEHI conditioned media as well as to recombinant 1L-3 found a 33,000 molecular weight protein which was rapidly phosphorylated on serine residues in response to the growth factor (Garland, 1988). Further evidence for the involvement of PKC in the signal transduction of hematopoietic growth factors comes from studies demonstrating a maintenance of viability and limited proliferation responses of the IL-3-dependent FDC-Mix 1 cell line cultured with 100 ng/ml of phorbol myristate acetate (PMA) and 100 ng/ml of calcium ionophore in the absence of IL-3 (Whetton, 1986). Additionally, at submaximal concentrations of I L 3 , PMA or calcium ionophore was shown to cause a synergistic increase in proliferation responses of these cells, while PMA and calcium ionophore together with IL-3 had an additive effect. At maximal concentrations of IL-3, these effects were not observed. These data are consistent with PKC activation involved in survival and proliferative responses mediated by IL-3. Physiologically, PKC activation is thought to occur in response to diacylglycerol production. Ligand induced activation of phospholipase C results in the cleavage of phosphatidylinositol 4,5-bisphosphate and yields diacylglycerol and inositol 1,4,5-trisphosphate in a diverse number of cell receptor systems (Berridge and lrvine, 1984). There is also evidence that DAG can be generated from other sources such as phosphatidylcholine (Besterman et ul., 1986). Studies by Whetton et al. (1988) have shown that IL-3 does not cause increase in inositol 1,4,5-trisphosphate concentrations in FDC-P1 cells, further supporting the possibility that the DAG is coming from sources other than membrane phosphoinositides. 2. TYROSINE KINASE ACTIVATION Tyrosine kinase activation has been shown in response to growth factors such as insulin, PDGF, EGF, IGF- I , and NGF in appropriate cell lines. A role for tyrosine phosphorylation in signal transduction of hematopoietic growth factors is suggested by several lines of study. First, IL-3-dependent cell lines (Cook e l al., 1985), as well as mast cells (Pierce et at., 1985), were rendered I L 3 independent when transformed by Abelson virus. Northern blot analysis indicated that neither cell type was producing IL-3, thus demonstrating the absence

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of an autocrine mechanism of stimulation. Additionally, there were no changes in IL-3 receptor expression, further suggesting that the v-abl transforming protein was responsible for rendering these cells factor-independent. The second type of study indicating that tyrosine phosphorylation may be involved in myeloid cell growth utilized vanadate, an essential metal ion which inhibits tyrosine phosphatases. Vanadate sustained viability as well as induced proliferation of the murine 1L-3-dependent cell line IC-2 (Tojo et al., 1987). Culture of these cells with 12.5 pM of vanadate potentiated proliferation in response to submaximal concentrations of 1L-3. Indeed, activation of a variety of inyeloid as well as lymphoid cell lines has been associated with stimulation of tyrosine kinase(s). Koyasu et al. (1987) reported the tyrosine phosphorylation of a 150 kDa membrane-associated protein in response to IL-3 by both the IC-2 and DA-I cell lines. Interestingly, although GM-CSF and IL-4 produced proliferation of IC-2 cells, stimulation with GMCSF or IL-4 did not result in phosphorylation of p150 in these studies. In contrast, others found that IL-4 stimulated the phosphorylation of a 170 and 110 kDa protein in 1C-2.9 and 32D/IL-2 cells (Morla et al., 1988). Differences between the findings of these groups may have resulted from technical differences in the two studies. One group used irnmunoprecipitation (Koyasu et uE., 1987) with antibodies directed against phosphotyrosine, while the other group immunoblotted with antibodies against phosphotyrosine (Morla et al., 1988). Isfort et al. (1988a,b) have reported the phosphorylation of a 140 kDa protein by FDC-PI cells as well as DA-I, NFS-60, and DA-3 cells in response to IL-3. Figure 3 shows the effects of interleukin 3 on tyrosine phosphorylation in 32D cells. Shown are a number of tyrosylphosphoproteins including pp140. Accumulating evidence indicates that the 140 kDa protein is the I L 3 receptor or is tightly associated with the IL-3 receptor. This protein has been shown to be membrane associated and phosphorylated as rapidly as 30 sec after exposure of factor-deprived cells to IL-3 if p150 reported by Koyasu et al. (1987) is the same as the p140 described by Isfort et al. (1988a). Studies performing affinity crosslinking of IL-3 to its receptor have shown IL-3 associated with p140 as well as a 64 kDa molecule (Isfort et af., 1988a). The 64 kDa protein is the size of the IL-3 receptor reported in the original studies characterizing the IL-3 binding protein (May and Ihle, 1986; Parke et al., 1986; Nicola and Peterson, 1986; Sorenson et al., 1986). Isfort et al. ( I 988a) suggest that p65 may be a proteolytic product of a 140 kDa IL-3 receptor based on the observation that the ratio of 65 kDa to 140 kDa showed considerable interexperiment variation. The association of IL-3 and p140 has been further supported by immunoprecipitation of p140 with antisera to IL3 after a 10 min incubation with an excess of IL-3. Anti-phosphotyrosine sera has also been shown to precipitate a 140,000 molecular weight protein after cross-linking to IL-3. Thus far p140 has not been demonstrated to have intrinsic tyrosine kinase activity.

kDa

200-

116-

92 66 -

45 -

31FIG. 3 . Two-dimensional analysis of IL-3-stimulated tyrosine phosphorplation in a murine myeloid cell line 32D. The cells were washed free of IL-3 and equilibrated in ["P]orthophosphate for 3 hr. Following equilibration, the cells were either mock stimulated ( A ) or stimulated for 20 min with IL-3 (B). The cells were lysed and the phosphotyrosine containing proteins affinity purified and analyzed by two-dimensional NEPHGE and autoradiography. Positions and molecular masses of standards in kilodaltons are indicated at left. Proteins that were modulated by IL-3 stimulation are indicated with arrows and approximate molecular masses in kilodaltons.

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Ferris et al. (1988) described phosphorylation of p70 1 min after lL-3 stimulation of FDC-PI cells and p50 within 10 min. Isfort et al. (1988b) also reported phosphorylation of cytosolic proteins of molecular weight 70,000 56,000 and 38,000 in FDC-PI cells after 1L-3 stimulation. Additionally, others have shown IL-3 induced phosphorylation of proteins of 95,90,70, and 55 kDa in these same cells. Stimulation of IC-1.9 and 32D/IL-2 cells produced similar tyrosine phosphorylation as the FDC-PI cells and the 1C-2.9 line had an additional phosphoprotein of 160,000 molecular weight. This protein was cytosolic in origin, so it is not the same as p140/p150 previously described. There is less information pertaining to the effects of other hematopoietic growth factors on tyrosine phosphorylation in myeloid cell lines. GM-CSF stimulation of IC-2.9 cells produced phosphorylation of p150, p92, and p72 (Morla et al., 1988). Interestingly, IL-4 stimulated phosphorylation of a p170 and pl10 in IC-2.9 cells, although there was no proliferation response. 3. KINASECROSS-TALK Although the data regarding PKC and tyrosine kinase activation in myeloid cell proliferation have been presented separately here, there is abundant evidence that these phosphotransferase systems may be interacting. PKC has been shown to phosphorylate the EGF receptor (Shoyab et a l . , 1979) causing a lower affinity for binding of EGF. Treatment of chick embryo fibroblasts (CEF) with either PMA or diacylglycerol resulted in tyrosine phosphorylation of a 42 kDa polypeptide (Villa and Weber, 1988). Villa and Weber (1988) also found that PKC depletion of CEF cells reduced the capacity of EFG to stimulate tyrosine phosphorylation of p42, while phosphorylation of other EGF substrates was not affected. In contrast, down-regulation of PKC did not affect IL3, IL-4, and GMCSF induced phosphorylation in 32D cells, although phosphoamino acid hydrolysis of the substrates was not performed to identify tyrosine vs. serine-threonine phosphorylation (Morla el al., 1988). The 70 kDa phosphoprotein reported by this laboratory has a 6 : 4 ratio of phosphoserine to phosphotyrosine, while the 50 kDa protein was 90% phosphotyrosine (Ferris et al., 1988). The p150 reported in IC-2 and DA- 1 cells was phosphorylated on tyrosine and serine residues in a 6 : 4 ratio (Koyasu et al., 1987), while all of the IL-3 stimulated phosphoproteins found in FDC-PI cells by Isfort et al. (1988b) had approximately equal amounts of tyrosine and serine phosphorylation. 4. CONTROL OF CELLULAR VIABILITY The role of growth factor-induced phosphorylation of characteristic substrates in maintenance of viability as well as elicitation of the proliferation response is largely unknown. Earlier work has indicated that survival and growth of FDC-PI

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cells in response to 1L-3 may be coupled to glucose transport and subsequent maintenance of ATP levels. Whetton and colleagues (1984; Whetton and Dexter, 1983) have found that WEHI-3 conditioned medium (WCM), a potent source of IL-3, stimulated uptake of 2-deoxyglucose (2-DOG) 3- to 4-fold over controls. The survival of cells in the presence of mitochondria1 inhibitors indicated the TCA cycle was not a major source of ATP, and the increase in lactic acid production during proliferation indicated the glycolytic path was the most likely energy source. FDC-PI cells cultured 24 hr in the absence of WCM had a viability of 13% of control cultures maintained in WCM. Addition of certain glycolytic intermediates resulted in viability 40-50% of control cultures. The activities of several of the major glycolytic enzymes (hexokinase, phosphoglucoisomerase, phosphofructokinase, and pyruvate kinase) were tested in cells cultured in the presence and absence of WCM, and withdrawal of IL-3 caused no significant decreases in activity when compared to controls. Observations that ATP generating systems as well as ATP maintained viability of FDC-PI cells supported the hypothesis that 1L-3 stimulates or allows the maintenance of intermediate metabolism. Interestingly, PKC has been shown to phosphorylate the glucose transporter in cell free assays as well as in vivo (Witters et id., 1985). In summary, although phosphorylation is a rapid result of growth factor stimulation, the role of phosphorylated substrates in myeloid hematopoietic cell proliferation remains largely undefined. One approach to discern phosphorylation targets crucial to proliferation signaling has been the identification of proteins constitutively phosphorylated in factor-independent strains of cell lines. As discussed, several groups have demonstrated abrogation of IL-3 dependence after infection with retroviruses containing the oncogenes v-ubl (Pierce er a!. , 1985; Cook et al., 1985; Mathey-Prevot et a / . , 1986) or v-trk (Isfort et ul., 1988b). Infection of an IL-3-dependent lymphoid line with AMuLV produced a factorindependent progeny f mathey-Prevot et d., 19861, further suggesting conservation of growth factor signal transduction events between myeloid and lymphoid cells. Factor-independent cell lines have also been found to constitutively phosphorylate proteins normally phosphorylated in response to 1L-3. FDC-P1 cells infected with the trk oncogene grew in the absence of IL-3 (Isfort et al., 1988b) and had a similar phosphorylation profile as the parental line after IL-3 stimulation, although substrates unique to the transformants were also reported. Spontaneously derived factor-independent lines have also been shown to constitutively phosphorylate proteins. Stimulation of AC2 with 1L-3 produces a rapid phosphorylation of p33. Garland (1988) reports phosphorylation of p33 in the AC cell line, an IL-3 independent clone of AC2. The 150 kDa substrate found in IC-2 and DA- 1 cells is phosphorylated in growth factor-independent variants of each of these lines (Koyasu et a / ., 1987). Interestingly, the p I40 demonstratcd in FDC-P1 cells is not phosphorylated by the trk transformants unless first exposed to IL-3 (Isfort et ul., 1988b). Proliferation of the 32D myeloid cell lines trans-

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fected with the EGF receptor in response to EGF has further demonstrated the conservation of events distal to ligand-receptor interaction in promotion of growth (Pierce et al., 1988). These studies suggest that oncogenes produce unregulated growth through utilization of regulatory paths stimulated by growth factors and either produce a constitutive "on" signal or remove cell growth termination signals.

C. Differentiation 1. MODELS OF STUDY

The role of phosphorylation in cellular differentiation has been studied primarily using transformed cell lines that are developmentally arrested. Studies of fresh bone marrow have been hampered by limited availability, cell population heterogeneity, and limited conditions producing survival and differentiation in vitro. Therefore, cells such as HL60, U937, K562, and KG-1 have received widespread use by investigators studying events of differentiation. HL60, a promyelocytic leukemic cell line, can be induced to differentiate into granulocytic, monocytic, or eosinophilic phenotype in vitro depending on the nature of the stimuli. This multipotential model has provided investigators the means through which to evaluate phosphorylation events common to differentiation of myeloid cells as well as those unique to granulocytic or monocytic lineages. KINASESA N D TERMINAL DIFFERENTIATION 2. TYROSINE Although tyrosine kinase activation was originally associated with cellular growth and transformation, more recent work has found increased levels of tyrosine kinase activity occurring with differentiation as well. Work by Tuy et al. (1983) identified up to 30% phosphotyrosine in phosphoamino acid preparations isolated from platelets and red blood cells, both terminally differentiated cells. The protooncogene c-src has been shown to have increased expression in neural tissue and to be activated during differentiation of HL60 to either granulocytic or monocytic lineage (Barnekow and Gessler, 1986). Gee et al. (1986) found that HL60 and U937 cells induced to differentiate with PMA increased expression of pp60c-"rc. Further support for a generalized role of c-src in myeloid differentiation was the enhanced c-src autophosphorylation observed in response to a number of agents promoting differentiation, including GM-CSF, 1,25-dihydroxyvitamin D,, and DMSO. Bone marrow cells differentiating in vim were also found to have increases in c-src activity as determined by autophosphorylation. It has yet to be established if c-src activation and expression have a role in the actual induction of differentiation or if c-src is expressed or activated as a result of cell maturation.

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The gene product of c-jpsf,fes has been shown to be expressed in high concentrations in chicken myelocytic cells (Samarut et al., 1985). Glazer and coworkers have demonstrated an increase in the f p s f k s protein in HL60 cells induced to differentiate into either monocytes (Glazer et al., 1986) or granulocytes (Yu et d . , 1988). Inhibition of DMSO induced HL60 differentiation by tyrosine kinase substrates at concentrations similar to respective K , values supports the role of the p93 tyrosine kinase encoded by c-jes in differentiation. These investigators suggest the ,fey gene product as a potential component of myeloid growth factor signal transduction. Comparison of phosphoamino acid hydrolysis of total cellular protein between parental HL60 and progeny induced to granulocytic (Frank and Sartorelli, 1986) or monocytic (Frank and Sartorelli, 1988) lineage found a net decrease in the phosphotyrosine component of the phosphoprotein. Interestingly, tyrosine kinase activity was increased during differentiation of both lineages; however, there was a concomitant increase in phosphotyrosine phosphatase activation. Similar changes in phosphotyrosine metabolism were noted in WEHI-3B D+ cells induced to differentiate by anthracycline compounds (Frank and Sartorelli, 1986). Increases in tyrosine phosphorylation have also been demonstrated in U937 cells differentiated in response to PMA (Grunberger et al., 1948). 3. SERINEKINASESA N D DIFFERENTIATION The induction of monocytic differentiation by PMA has lead to studies of the role of PKC in these events. PMA-induced phosphorylation of various HL60 proteins has been reported by numerous investigators (Anderson et al., 1985; Lord et al., 1988; Morin P Z al., 1987; Faille e l al., 1986; Kiss rt al., 1987a). Summarized here will be studies in which at least preliminary characterization of the relation between substrate phosphorylation and HL60 digerentiation has been performed. Terminal differentiation of HL60 cells is associated with a downregulation of transferrin receptors. May et al. ( 1984, 1985; May and Tyler, 1987) have demonstrated that PMA-induced phosphorylation of the transferrin receptor results in internalization. These investigators have suggested these events as an early signal in PMA-induced differentiation of HL60. Cooper and co-workers (Feuerstein and Cooper, 1983, 1984; Feuerstein et al., 1985) have identificd a 17-20 kDa protein, pl 5 . 5 , and a 27 kDa protein with a pI of 5.5 which are phosphorylated on serine within 15 min of PMA addition to HL60 cells. The role of the 17 kDa protein in differentiation was further supported by the PMA-induced phosphorylation of p17 in A431 cells, a line in which proliferation is inhibited by PMA. In contrast, cells in which PMA was mitogenic had minor increases in p I7 phosphorylation in response to the phorbol ester. Although phosphorylation of these proteins was inhibited by the PKC inhibitor trifluoroperazine (Feuerstein and Cooper, 1984), cell free studies indi-

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cated that pi7 may not be a direct substrate of PKC but is phosphorylated through interaction of PKC with another kinase. Protein kinase A (PKA) was found to phosphorylate p 17 in vitro; however, peptide mapping studies indicated the sites of phosphorylation were different than that found after PMA in vivo stimulation. Although correlated with PMA-induced differentiation (Faille et a / ., 1986; Feurstein et al., 1985), the identity of p17 and functional significance have yet to be defined. Glazer and co-workers have suggested that PMA-induced differentiation of HL60 is mediated by M-kinase, the 50 kDa calcium and phospholipid independent product of PKC proteolysis (Zylber-Katz and Glazer, 1958). PMA treatment of HL60 also causes characteristic changes in expression of the protooncogenes c-fos and c-myc. c-fos RNA is increased within 10-15 min after stimulation, while c-myc is rapidly increased and then down-regulated. These alterations in gene expression suggest a direct effect of PKC at the level of the nucleus or else PKC initiation of a kinase cascade which terminates at the nucleus. Indeed, studies have identified phosphorylation of nuclear proteins by PKC. Sahyoun et al. (1986) demonstrated phosphorylation of topoisomerase I1 by purified PKC in cell free conditions. Calmodulin-dependent protein kinase also phosphorylates topoisomerase 11, though at higher K,, than PKC, while PKA did not produce topoisomerase I1 phosphorylation. The importance of the PKC-mediated phosphorylation of topoisomerase I1 was further supported by inhibition of PMA-induced HL60 differentiation by topoisomerase inhibitors. A nuclear matrix protein of 80,000 molecular weight has been identified as a PKC substrate when HL60 cells are treated in vivo with PMA (Kiss et al., 1987a; MacFarlane, 1986). Additionally, PMA and I ,25-dihydroxycholecaIciferal,both inducers of monocytic differentiation in HL60 cells, resulted in phosphorylation of histone protein 2B, while granulocytic differentiation by DMSO and retinoic acid did not increase phosphorylation of histone proteins (DeBord and Baxter, 1988). The mechanism of PMA-induced HL60 differentiation is still not certain. PMA is thought to bind to PKC and produce a calcium and phospholipid dependent activation of the enzyme. There are, however, a number of conflicting reports in the literature concerning the effects of various PKC activators and inhibitors in HL60 monocytic differentiation (Kiss et a/., 1987b; Morin et al.. 1987; Kraft et a/., 1986). Induction of HL60 granulocytic differentiation by agents such as DMSO and retinoic acid has been associated with phosphorylation of certain substrates. Studies by Faille et al. (1986) indicated that HL60 cells treated for 6 days with retinoic acid or DMSO induced a net dephosphorylation in the 18 phosphoproteins identified in the parental cell line. Interestingly, a 46-48 kDa phosphoprotein appeared after differentiation. The role of this phosphoprotein in differentiation was supported by its absence in mutants incapable of differentiating in

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response to DMSO and retinoic acid. Additionally, a 17 kDa phosphoprotein similar to that reported by Fernstein and Cooper was seen in monocytic HL60 progeny, while this protein was absent in granulocytic forms. Another group (Yamamoto et a / . , 1988) identified a 22 kDa protein phosphorylated by the granulocytic inducers G-CSF, DMSO, retinoic acid, and PGE,. Although there is a body of evidence suggesting stimulants of granulocytic differentiation increase cellular CAMP concentrations and subsequently activate PKA (Fontana et ul., 1984), there are also data conflicting with those studies (Chaplinski and Niedel, 1986). In summary, the mechanism of action of these compounds is as yet unresolved.

D. Functional Activation 1. NEUTROPHIIS A N D MONOCYTES

Functional activation of myeloid cells has been suggested to be associated with phosphorylation of characteristic substrates. The studies to be discussed herein will relate primarily to macrophage and neutrophils isolated from the peripheral blood. Similarities between mature forms of macrophage-monocytes and polymorphonuclear neutrophils (PMNs) include development from the granulocytemacrophage colony forming unit (GM-CFU), capacity of mature cells to migrate through gradients of chemoattractants, phagocytosis of opsonized bacteria, and antimicrobial actions through release of lysosomal granules and production of hydrogen peroxide. The appropriate elicitation of migration and antimicrobial responses in inflammatory cells is crucial to host defense. PMNs are short-lived cells with a half-life in the blood of 7-9 hr. No specific recognition is required for activation, so these cells are considered to be the major effector cell of nonspecific immunity. Macrophages have a multitude of roles in the immune response beyond those dealing with direct bactericidal actions. Macrophages are primed for cytotoxicity by gamma interferon and are fully activated by LPS.

2 . SERINEKINASES I N PRIMING A N D ACTIVATION Stimulation of monocytes with PMA results in rapid redistribution of PKC from cytosol to the membrane (Myers et al., 1985). Further, the dose-response curves for PMA elicitation of superoxide release and PKC activation were similar, and the structure-activity relation for various phorbol derivatives was identical for the two responses (Myers et al., 1958). Stimulation of murine peritoneal macrophages with either PMA or lipopolysaccharide resulted in substantial increases in four phosphoproteins of molecular weight 67,000, 37,000, 33,000, and 28,000 (Weiel et ul., 1986). The similarities in response to PMA and LPS suggested a role for PKC in LPS action. Aderem ef al. ( 1 988) have also reported

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LPS-induced phosphorylation of a 67 kDa cytosolic protein in peritoneal macrophages antigenically related to the 80-87 kDa PKC substrate in mouse brain. Limited proteolysis with Staphylococcus aureus V8 generated identical phosphopeptide maps for these two phosphoproteins. Gamma interferon enhanced LPS-mediated phosphorylation of p67, p37, p33, and p28, supporting the important role this lymphokine has in priming macrophage responses (Weiel et al., 1986). The role of protein kinase C in immune interferon-mediated activation of macrophages has not been rigorously defined. A recent report demonstrates gamma interferon-mediated translocation of PKC from the cytosol to the membrane of U937 cells (Fam et al., 1988), while increases in total activity of PKC in peritoneal macrophages were found after gamma interferon treatment. These increases in enzyme activity resulted from an increase in V,, but were not paralleled by changes in subcellular distribution (Hamilton et al., 1985; Becton et al., 1985). Little is known of the events distal to receptor binding of ILl-mediated activation of monocytes. Pretreatment of mononuclear cells from peripheral blood with glucocorticoids increases the number of IL-1 receptors on the cell surface from approximately 100 to 2000 per cell (Matsushima et al., 1987). Using this model, Matsushima et al. (1987) have reported the serine phosphorylation of a 65 kDa cytosolic protein in response to I L 1 which is dose and time dependent. I L l induced a 1.5-fold increase in phosphorylation of p65 in the absence of prednisolone and an approximate 2.5-fold increase after a 5 hr pretreatment with the glucocorticoid. The compounds HA1004 and W-7, inhibitors of cyclic nucleotide activated kinases and calmodulin activated kinase, respectively, decreased the IL- 1-induced phosphorylation of p65, while H-7, a protein kinase C inhibitor, had no effect on p65 phosphorylation. These authors suggest that signal transduction of I L l involves activation of a serine kinase in mononuclear cells, distinct from PKC. In studies of the T lymphoma line LBRM-33, Abraham et al. (1987) also concluded that IL-1 does not cause translocation of PKC; however, in Jurkat cells, IL-1 has been suggested to stimulate diacylglycerol production and subsequent PKC activation by a non-receptor-mediated mechanism (Rosoff et al., 1988). A valuable model utilized in investigation of phosphorylation and the respiratory burst has been the study of stimulus-induced phosphorylation of proteins in PMNs obtained from patients with chronic granulomatous disease (CGD). CGD is a family of disorders characterized by the inability of PMNs from these patients to produce superoxide anion in response to stimuli. A family of 48 kDa proteins with isoelectric points between 6.8 and 7.8 have been identified whose presence has been correlated with the capacity to generate a respiratory burst. PMNs from patients with two forms of the autosomal recessive and one form of the X-linked disease do not phosphorylate one or more of the 48 kDa proteins after PMA stimulation (Babior, 1988). Although the role of these proteins has not

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been defined in relation to the function of the NADP-oxidase responsible for formation of reactive oxygen intermediates, the correlation between these components is striking. Phosphorylation and acquisition of the capacity to generate a respiratory burst in HL60 maturation have also been evaluated. Gaut and Carchman (1987) reported a correlation between onset of phosphorylation of p76 and superoxide anion production in PMA-stimulated HL60 cells during DMSO-induced differentiation. Proteins with molecular weights of 212,000 and 134,000 were also found to be phosphorylated after PMA treatment in maturing HL60 cells. PMA stimulated phosphorylation of p28, p55, p61, and p66 in human monocytes, while opsonized zymosan treatment resulted in a 1.5- and 2.1-fold increase in labeled phosphate incorporation of p61 and p66, respectively (Kelly and Carchman, 1987). PMA and opsonized zymosan are stimulants of degranulation, suggesting that p61 and p66 may have an important role in degranulation responses. PMA stimulation of PMNs produced phosphorylation of proteins of molecular weight 40,000, 50,000, 55,000, 64,000, 70,000, and 90,000 in studies by White et al. (1984), while exposure to the chemotactic peptide fMet-LeuPhe resulted in phosphorylation of a 50 kDa protein as well as of 60 and 67 kDa proteins (Huang et al., 1984). Phosphorylation of p50 was inhibited by trifluorperazine at concentrations which had been found to inhibit PKC (White et al,. 1948). Factors chemotactic for PMNs and monocytes have been shown to stimulate IP, and DAG release. PKC is activated by DAG and subsequently translocates to the membrane. Pontremoli et al. (1986a) have reported phosphorylation of identical proteins in PMA or fMLP stimulated PMNs. Membrane phosphoproteins included p130, p78, p46, p40, and p34, while cytosolic proteins of 65,000, 55,000, 48,000, 38,000, 36,000, 30,000, and 22,000 were identified. These investigations have suggested that the membrane-bound form of PKC, which is calcium and phospholipid dependent, mediates the respiratory burst, while a 65 kDa serine kinase, which is calcium and phospholipid independent, phosphorylates cytosolic substrates initiating degranulation (Pontremoli et al., 1986a,b, 1987, 1988; Melloni et al., 1986).

3 . TYROSINE PHOSPHORYLATION IN PMNs Although tyrosine phosphorylation has been investigated extensively in relation to cell proliferation, there is evidence for tyrosine kinase activity in terminally differentiated cells (Tuy et al., 1983). One recent report demonstrates tyrosine phosphorylation of proteins with apparent molecular weights of 62,000 and 125,000 in intact PMNs stimulated with fMLP but not PMA (Huang et al., 1988). Membranes from PMNs also phosphorylated p62 and p125, as well as a protein of M , 40,000 with similarity to lipomodulin. A provocative observation of these studies was the inhibition of p62 phosphorylation in PMNs pretreated

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with pertussis toxin. The authors suggest that activation of the tyrosine kinase is distal to the N L P receptor yet requires signals independent of or in addition to the PKC component of formylpeptide signal transduction (Huang et al., 1988). The HcK gene product is a 59 kDa protein which is expressed in high quantities in PMNs and monocyte (Quintrell et al., 1987; Ziegler et al., 1987). Ziegler et al. (1988) have demonstrated an increase in HcK mRNA and protein expression in macrophages activated by LPS. Treatment of macrophages with gamma interferon as a priming stimulus did not produce increases in HcK mRNA but did synergize in LPS-mediated responses, indicating this gene product is associated with full cellular activation. Huang et al. (1988) have suggested the p62 substrate phosphorylated in PMNs in response to fMLP may be the HcK gene product based on partial characterization of tyrosine kinase activity associated with PMN membrane fractions and recognition of p62 by antisera against a sequence common to members of the src family. 4. CYCLICNUCLEOTIDE ACTIVATED KINASES AND CELLULAR FUNCTION

There has long been recognized a relationship between cytoplasmic concentrations of cyclic nucleotides and modulation of biological responses (George et al., 1970). In the early seventies, Goldberg (reviewed in Goldberg and Haddox, 1977) proposed the Yin-Yang hypothesis of cell regulation. This theory was based on the opposing effects of cAMP and cGMP on cellular function. PMN migration (Estensen et al., 1973) and degranulation (Smith and Ignarro, 1975) are generally enhanced by agents which increase cGMP concentrations intracellularly, while high concentrations of cAMP seem to inhibit migration, degranulation and phagocytosis. GM-CSF has been shown to prime PMN migration and superoxide production. Interestingly, GM-CSF activates PMN guanylate cyclase in a time frame corresponding to enhanced cellular responses (Coffey et al., 1988). Unfortunately, no relationship between phosphorylation, cyclic nucleotide-dependent kinase activation, and cellular function has been established to better understand the mechanisms responsible for these events.

VI. SUMMARY AND PERSPECTIVES The receptors that control leukocyte function can be generally separated into three structure-functional categories. Class I receptors may be directly coupled to “classical” second messenger systems, such as phosphoinositol hydrolysis or CAMP. The best examples of these include the TCR complex or surface Ig of T and B lymphocytes, respectively. These receptors apparently couple via G proteins to P L C activation. Class I1 receptors have a protein kinase activity intrinsic

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to the ligand binding protein, almost exclusively a tyrosine kinase. Among the many lymphohematopoietic growth factors, only the CSF- 1 receptor has been characterized in this category. Although tyrosine kinase activation is regulated by the TCR, no evidence to date can be found to identify catalytic activity with identified protein chains of the TCR complex. Class 111 receptors, probably the most enigmatic, are those which couple to serine and tyrosine phosphotransferase systems without any apparent kinase domain intrinsic to the receptor structure and, possibly, a lack of sufficient evidence to indicate second messenger generation. The I L 2 receptor system and possibly members of the CSF family such as IL3 and GM-CSF will probably fall into this category. No evidence exists for either the IL-2R (Y or f3 chains to possess intrinsic kinase activity. Nevertheless, IL-2 and IL-3 clearly activate both serine and tyrosine kinases. Whether other cytokines, such as TNF, and IL-1 thru IL-8, also follow these observations remains to be tested. The class I11 receptors present a unique paradox not well characterized in biological systems. Growth factor receptors such as EGF, PDGF, IGFl , FGF, and CSF-1 clearly fall into the class I1 intrinsic kinase receptor category. Although these receptors trigger autophosphorylation of cytoplasmic tyrosine associated with the binding protein, little is known about the cytoplasmic substrates. Moreover, even the growth factors that trigger class I1 receptors stimulate a vast array of seryl phosphorylation. The general belief is that the seryl phosphorylations are controlling the regulation of nuclear proteins and gene expression. As more information and reagents become available, we should be able to dissect the protein kinases associated with the more complex systems such as the TCR and the class I11 receptors such as IL2R and IL3R. The production of anti-receptor chain antibodies should greatly facilitate investigators’ efforts. The major serine kinase activity implicated in TCR, Ig and possibly cytokine signaling has been the PKC isozyme phosphotransferase system. The discovery of the isozyme gene family for PKC has made many past and new data interpretations difficult. The overwhelming use of phorbol esters as a discretionary tool for the PKC isozyme system probably suggests some caution in view of complexities in PKC isozyme regulation. For that matter, the PKC isozyme system is under the regulation of other phospholipid metabolism pathways unrelated to the PI hydrolysis system. Alternative signaling to the PKC system has been suggested even for the TCR and Ig receptors and cytokine regulation of growth. Most of these signaling systems are detected under conditions where the PKC system is extraordinarily stressed, leading to a degradation of the PKC isozyme (Valge et al., 1988). Under these conditions, I L 2 can still stimulate proliferation and gene expression. One suggestion may be via the tyrosine kinase system shown here and elsewhere (Ferris et al., 1989). Similarly, lack of T cell activation can occur in the presence of PKC activation and vice versa; activation can occur in the

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absence of PI hydrolysis and PKC activation (Goldsmith and Weiss, 1988). What both the cytokine and antigen-receptor systems may be telling us is that there appear to be optional multiple kinase pathways to control the biological response. Each receptor system may have also evolved a plasticity to couple to independent kinase systems to initiate vital signals critical for the survival of the cell. The apparent “stress” or deletion of one pathway may favor the “plastic response” or the use of alternative signaling pathways. Under conditions of physiological stimulation, the pathway used may depend on the state of differentiation of the cell or the tissue type in which the receptor is expressed. For instance, the IL2R may signal differently in a T cell than in a B cell. IL-1 receptors found in lymphocytes may significantly differ in signaling mechanisms in monocytes, fibroblasts, and, for that matter, neural cells. The tyrosine kinases have been more recently observed in relation to the TCR complex, CD4 and CD8 surface molecules, and the action of I L 2 and other CSFs. These observations have been made attractive by the findings that members of the tyrosine kinase v-onc family may transform cytokine-dependent myeloid cells; although an exciting area of investigation, v-onc tyrosine kinases have failed to growth factor abrogate primary cultures of bone marrow cells or lymphocytes. T lymphocytes have been difficult to transform with tyrosine kinase v-oncogenes, and the relative kinase activity of lymphocytes decreases upon activation (Marth et a / . , 1987). Activation of a major tyrosine kinase pp56 LCK by CD4 or CD8 cross-linking does not induce cellular proliferation, nor does activation of the tyrosine kinase associated with the TCR. Although the CSFs and IL-2 have been shown to stimulate one or more tyrosine kinases, the activation of tyrosine kinase activity can be equally associated with differentiation signals in both myeloid and lymphoid cells. We have summarized many of the known phosphorylation events associated with lymphohemopoietic differentiation and proliferation. Many of the kinases activated by the numerous extracellular stimuli of leukocytes are unknown. For that matter, very little is known about the potential substrates of specific kinases in hemopoietic tissues. While research into the mechanisms of simple polypeptides such as insulin has persisted for three decades, the availability of cloned cell lines and reagents to analyze receptor function will provide scientists with some of the tools necessary to examine immune cytokine signaling. The absence of detectable second messengers and intrinsic structural kinase domains with a number of cytokine receptors suggests an exciting era of research that may discover new mechanisms of signal transduction previously unrecognized. ACKNOWLEDGMENT This project has been funded at least in part with Federal funds from the Department of Health and

Human Services under contract number N01-CO-74102 with Program Resources, Inc. The content

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of this publication does not necessarily reflect the views or policies of the Department of Health and Human Services, nor does mention of trade names, commercial products, or organizations imply endorsement by the U.S. Government REFERENCES Abraham, R. T., Ho, S . N., Barna, T. J., and McKean, D. J. (1987). Transmembrane signaling during interleukin I-dependent T cell activation. J. Biol. Chem. 262, 2719-2728. Aderem, A. A,, Albert, K. A., Keun, M. M., Wang, J. K. T., Greengard, P., and Cohn, Z . A. (1988). Stimulus-dependent myristoylation of a major substrate for protein kinase C. Nature (London) 332, 362-364. Anderson, L., Gemmel, M. A,, Coussens, P. M., Murao, S . , and Huberman, E. (1985). Specific protein phosphorylation in human promyelocytic HL60 leukemia cells susceptible or resistant to induction of cell differentiation by phorbol-12-myristate-13-acetate.Cancer Res. 45, 49554962. Babior, B. M. (1988). Protein phosphorylation and the respiratory burst. Arch. Biochern. Biophys. 264, 361-367. Baniyash, M., Garcia-Morales, P., Bonifacino, J. S . , Samelson, L. E., and Klausner, R. D. (1988a). Disulfide linkage of the 5 and N chains of the T cell receptor. 1. B i d . Chem. 263, 9874-9878. Baniyash, M., Garcia-Morales, P., Luong, E., Samelson, L. E., and Klausner, R. D. (1988b). The T cell antigen receptor 5 chain is tyrosine phosphorylated upon activation. J. Biol. Chem. 263, 18225- 18230. Bamekow, A,, and Gessler, M. (1986). Activation of the pp60c-s'c kinase during differentiation of monomyelocytic cells in vitro. ElcfBO J. 5, 701-705. Becton, D. L., Adams, D. O., and Hamilton, T. A. (1985). Characterization of protein kinase C activity in interferon y treated murine peritoneal macrophages. J . Cell. Physiol. 125,485-491. Benedict, S . H., Mills, G. B., and Gelfand, E. W. (1987). Interleukin 2 activates a receptorassociated protein kinase. J. Immunol. 139, 1694-1697. Berridge, M. J., and Irvine, R. F. (1984). Inositol trisphosphate, a novel second messenger in cellular signal transduction. Nature (London) 312, 3 15-321. Besteman, J. M., Duronio, V., and Cuatrecasas, P. (1986). Rapid formation of diacylglycerol from phosphatidylcholine: A pathway for generation of a second messenger. Proc. Nurl. Acad. Sci. U.S.A. 83, 6785-6789. Betel, I . , Martijnse, J., and Van Den Berg, K. (1974)). Absence of an early increase of phospholipidphosphate turnover in mitogen-stimulated B lymphocytes. Cell. Immunol. 14, 429-434. Blackshear, P. J., Naim, A. C . , and Kuo, J. F. (1988). Protein kinases 1988: A current perspective, FASEB J . 2, 2951-2969. Blenis, J., Kuo, C. J., and Erikson, R. L. (1987). Identification of a ribosomal protein S6 kinase regulated by transformation and growth-promoting stimuli. J . Biol. Chem. 262, 1437314376. Blue, M. L., Hafler, D. A,, Craig, K. A., Levine, H., and Schlossman, S. F. (1987). Phosphorylation of CD4 and CD8 molecules following T cell triggering. J. Immunol. 139, 3949-3954. Cambier, J. C., and Ransom, J. T., (1987). Molecular mechanisms of transmembrane signaling in B lymphocytes. Annu. Rev. Immunol. 5 , 175-199. Casnellie, J. E., and Lamberts, R. J. (1986). Tumor promoters cause changes in the state of phosphorylation and apparent molecular weight of a tyrosine protein kinase in T lymphocytes. J. Biol. Chem. 261, 4921-4925. Chaplin, A. D., Wedner, H . J., and Parker, C. S . (1980). Protein phosphorylation in human peripheral blood lymphocytes: Mitogen-induced increases in protein phosphorylation in intact lymphocytes. J. Immunol. 124, 2390-2398.

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Effect of phorbol 12-myristate 13-acetate and its analogue 4a-phorbol 12.13-didecanoate on protein phosphorylation and lysosomal enzyme release in rabbit neutrophils. J. A i d . Chem. 299, 8605-861 I . Witters, A , , Vater, C. A . , and Lienhard, G. E. (198.5). Phosphorylation of the glucose transporter in virro and in vivo by protein kinase C. Nuture (London) 315, 777-778. Yamamoto, M . , Nishimura, J . , Ideguchi, H., and Ibayashi, H. (1988). Specific phosphorylation of 22-KD proteins by various inducers for granuloid differentiation in myeloid leukemia cells. Leuk. Res. 12, 71-80. Yu, G . , Grant, S., and Glazer, R. I. (1988). Associalion of ~ 9 3 c - ' ~ tyrosine I protein kinase with granulocyticimonocyticdifferentiation and resistance to differentiating agents in HL60 leukemia cells. Mol. Pharmuc,ol. 33, 384-388. Ziegler, S. F., Marth, J. D., Lewis, D. B., and Perlmutter, R. M. (1987). Novel protein-tyrosine kinase gene (hck) preferentially cxpressed in cells of hematopoietic origin. Mol. Cell. Biol. 7 , 2276-228.5. Ziegler, S . F., Wilson, C. B., and Perlmutta, R. M . (1988). Augmented expression of a myeloidspecific protein tyrosine kinase genc (hck) after macrophage activation. J. Exp. Med. 168, 1801- 18 10. Zioncheck, T. F., Harrison, M. L., and Geahlen, R. L. (1986). Purification and characterization of a protein-tyrosine kinase from bovine thymus. J. Biol. Chern. 261, 15637-15643. Zylber-Katz, E., and Glazer, R . I. (1985). Phospholipid and Ca2 dependent protein kinase activity and protein phosphorylation patterns in the differentiation of human promyelocytic leukemia line HL60. Cancer Res. 45, 5159-5164.

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( ' I I R K I - " ~ TOPI<'S IN Mt.MHKANt,S A N D 'I KANSPOKT. VOLUME 35

Chapter 18

The Role of Phosphorylation in Phagocyte Activation ALFRED I. TAUBER,* ANAND B . KARNAD, 7 AND IRENE GINIS" *Departments qf' Medicine and Pathology Boston University School of Medicinr Boston, Mussuchusetts 02118 fDepurtment of HemutologylOn~,~)lo~~) East Tennessee State University Johnson City,Tennessee 37614 Introduction Neutrophil Activation by PK-C A . Phorbol Ester Binding Studies €3. PK-C Inhibition Studies C. A Putative Role of Diacylglycerol D. PK-C Mobilization 111. Phosphorylation Studics IV. PK-C Activation of NADPH-Oxidase V. Conclusion References 1. 11.

1.

INTRODUCTION

That phosphorylation is an important mechanism of biochemical control is well established, but a defined role in the activation of the phagocyte has yet to be characterized. Certainly there is active phosphorylation-dephosphorylation when these cells are stimulated by a variety of agonists, but the actual assignment of a particular kinase/phosphatase protein substrate pair in the mapping of an activation cascade has not been satisfactorily accomplished. This is not to say that evidence for such a mechanism might not be inferred from existing data, only that in our treatment of the subject, a conservative interpretation is warranted. Our review is then guided by this cautionary proviso in an attempt to place in perspective the rapid progress that has been made in this area of pha469 Copyright 0 1990 by Acadcriii~Preaa. Inc All righi$ of reproduction In any form rr5erved.

470

ALFRED I. TAUBER ET AL.

gocyte cell biology. Several reviews may be consulted for more comprehensive consideration of certain aspects ( I -7), for in this treatment, we will only examine the most compelling evidence for phosphorylation control that has bccn cstablishcd in the human neutrophil for activation of the NADPII-oxidase, the enzymatic activity of the respiratory burst. Of the various soluble and particulate agonists that stimulate the oxidase, for our purposes the phorbol ester, phorbol myristate acetate (PMA), is of most relevance, for its receptor, protcin kinase C (PK-C), represents an important phosphorylating activity that has offered a functional assessment of oxidase activation in intact cells. PMA initiates pleiomorphic neutrophil functions, but the best characterized is its stimulation of the respiratory burst. The oxidasc generates superoxide (0,- ) as the initial oxidativc product of the burst, and oxidase defects in structure or activation account for the well-characterized clinical disorder chronic granulomatous discasc (CGD) (8- 10). Patients with classically described CGD have not been reported to have abnormal receptor affinities as inferred by normal chcmotactic and phagocytic responses (8,9) or phorbol ester receptor binding (11). but certain patients have demonstrated abnormal responses to sclcctcd stimuli and thus exhibit a defect in the activation apparatus (12-14). CGD, which includes all defects of the oxidative metabolic effector systcm, may be divided into two types: abnormal activating mechanisms and abnormalities of NADPH-oxidase (8-10). In no case has PK-C activity been deficient or abnormal. Thus CGD offers a useful model for examining the normal process of oxidase expression, and its study has been crucial in cstablishing the role of cytosolic factors in the activation of the plasma membrane-bound oxidase. This review will first examine the role of PK-C in NADPH-oxidase activation from a functional perspective, and then we will consider the progress made in decipherment of actual phosphorylated target proteins in an attempt to establish the role of phosphorylation in NADPH-oxidase expression. In bypassing many associated complex metabolic activities, thc PK-C system is a promising avenue for elucidating oxidase activation at a site just proximal to the final expression of the respiratory burst enzyme. Howevcr, this PK-C-dependent pathway is not the sole cascade for oxidase activation (2). Definition of parallel and crossover points in the complex hierarchy of the activation process has yet to be discerned. Our purpose then is restricted to considering the role of PK-C in the activation of the NADPH-oxidase as a mechanism of defining how phosphorylation might rcgulatc phagocyte activation. The evidence supporting the role of PK-C in neutrophil activation processes may be conveniently considered by the following criteria: (1) correlation of phorbol ester binding to biological effect, ( 2 ) PK-C inhibitor studies, (3) the role of the natural PK-C cofactor diacylglycerol, (4) correlation of PK-C translocation and neutrophil responsiveness, ( 5 ) phosphorylation data, and (6) reconstitution

18. PHOSPHORYLATION IN PHAGOCYTE ACTIVATION

471

studies employing PK-C and partially purified NADPH-oxidase. Each of these criteria will now be considered.

II. NEUTROPHIL ACTIVATION BY PK-C A. Phorbol Ester Binding Studies The activation of the human neutrophil by PMA leads to a spectrum of responses, including aggregation, stimulation of the respiratory burst, depolarization of the plasma membrane, enhanced phospholipid turnover, and degranulation of specific granules (1-3). The demonstration that PMA and certain other phorbol esters substitute for sn- I ,2-diacylglycerol (liberated by the action of phosphol ipase or phosphodiestrase) in activating PK-C allowed assessment of these various responses relative to PK-C ( 15). An important criterion is to establish a close correlation between ligand binding and biochemical expression. Such receptor-binding linkage to NADPH-oxidase activation by phorbol esters has been made ( 1 1,16,17). Showing close approximation of the phorbol esters’ affinity characteristics to activation of the burst established that PMA (k, = 5nM) and its analogue, phorbol- 12,13-dibutyrate (PDBu) (k, = 38 M),stimulated NADPH-oxidase through a specific rcceptor ( I 1). Extension of studies with intact cells to the subcellular assessment of the oxidase demonstrated a similar dose-response activation profile ( I 1 ). Characterization of the phorbol ester receptor showed that it was not susceptible to sulthydryl reactive reagents, stilbene disulfonic acids, or neuraminidase, which explains the earlier findings of a lack of effect of these agents on the generation of 0,- in PMA-stimulated cells, in contrast to cells stimulated with opsonized zymosan (OZ) (18,19). The stimulus specificity demonstrated that the inhibition acted on distinct plasma membrane receptors and not an oxidase exposed to the extracellular medium. PK-C in the prestimulated neutrophil is predominantly localized to the cytosol, as discussed below in Section II,D.

8. PK-C Inhibition Studies The second approach to define the role of PK-C utilizes inhibition studies. If a kinase inhibitor blocked neutrophil effector function, one might conclude that at some point in the initiation of that response a phosphorylation event was crucial. In the case of PMA stimulation, various kinase inhibitors have been employed to demonstrate the sensitivity of a particular function with a given stimulus, and then the drug has been employed to determine if other agonists show the same kjnase-sensitivity in their respective activation cascade. This second line of

472

ALFRED I. TAUBER

ET AL.

investigation has been controversial because the drugs employed lack specificity, but this approach remains potentially rewarding. Inhibition of the PMA-stimulated respiratory burst with a variety of kinase inhibitors has been clearly demonstratcd: I-(5-isoquinolinylsuIfonyl)-2-methylpiperazine (H-7) (20-22), staurosporine (23), 1-(5-isoquinolinyIsulfonyl)piperazinc (C-I) (24) and in the case of sphingoid long-chain bases, contested (2528). Sphinganinc and sphingosinc, which are potent inhibitors of PK-C, arc also cytotoxic in the concentrations employed when dissolved in dimethyl sulfoxide (26),but when equimolar bovine serum albumin is added to the mixture, cytotoxicity is eliminated (27). Once this methodnlogic problem was clarified, the sphingoids have becn of particular interest in demonstrating the role of PK-C activation in cells stimulated with physiological agonists, the most important being FMLP. A cautionary note must be made however: none of these inhibitors i s specific for PK-C; the piperazines and staurosporine inhibit other kinases, and sphingosine inhibits calmodulin-dependent enzymes (29). Thus, the use of a specific pseudosubstrate for PK-C (30) may be of particular help in clarifying the activity of PK-C in stimulated neutrophils if means are developed for its entry into the ccll. PMLP represents a particularly useful agonist in defining the neutrophil activation cascade. A natural product of gram negative bacteria (31), FMLP stimulates chemotaxis, depolarization, 0, production, and degranulation responses through the receptor-ligand coupled system best characterized in the human neutrophil (32J3). The pertussis toxin-scnsitive guanine nucleotide binding protein, G,, regulates phospholipase C , which is activated to hydrolyze phosphatidylinositol 1,4-bisphosphate (PIP,) to inositol 1,4,5-trisphosphate (IP,) and diacylglycerol (DAG). IP, serves as an agent to mobilize intracellular calcium, and with DAG, the cofactors arc thus generated which are then required for PK-C translocation to the membrane; PK-C binding with phosphatidylserine, or a like phospholipid, completes the activation process of this phase. As we will discuss, a second phase ensues upon the activation of calpain (also as a result of a rise in cellular free calcium), a neutral protcinase that cleaves PK-C (membrane bound) to a free cytosolic form, PK-M, that is calcium-phospholipid independent (34,35).Substantial effort then has been made to establish that FMLP stimulation fulfills this schema by showing both that DAG is generated appropriately for PKC activation requirements (discussed below) and that PK-C inhibitors in fact interrupt FMLP-induced functions. When H-7, C-I, or staurosporine has been used in studies assessing FMLPinitiated rcspiratory burst activation, no significant inhibition was seen at drug concentrations that inhibited PMA-stimulated responses (21,23,24,36-38). Each of these agents competes at the ATP binding site of the kinase, while sphinganine competes at the DAG-phorbol ester binding site (25), which may account for the effectiveness of the sphingoids to inhibit FMLP-stimulated 0, production (28). ~

~

473

18. PHOSPHORYLATION IN PHAGOCYTE ACTIVATION

The mechanism to explain these results is speculative (28): (1) PK-C exists in several isoenzymatic forms (39,40), potentially exhibiting differential inhibition or stimulation. [The data on which isoenzymes are expressed in the neutrophil are controversial, as discussed below (41-43).] Functional studies employing synthetic DAGs with variable length fatty acids suggested that differential expression of PK-C-either by isoenzyme structure or compartmentalizationmight account for differences between evoked degranulation and 0, - responses (44) (see below). (2) H-7 and C-1 might block only an earlier step (e.g., agonist binding, translocation) in the activation cascade for PK-C, which might be bypassed by FMLP stimulation. (3) FMLP stimulation might alter certain PK-C properties (e.g., membrane attachment) and affect interaction with these drugs. (4) There may simply be a methodologic problem. A preliminary report showed that when cells were prewarmed to 37°C and the inhibitor H-7 was then added, similar inhibitory effects are seen with PMA and FMLP (45). Which of these, or any other, explanations is correct remains problematic, but an approach to its resolution is suggested by the similar discrepancy noted with PMA-induced degranulation. Again, neither H-7 (25,42), C-I (46), nor staurosporine (Table I) inhibits PMA stimulated degranulation (specific granules), whereas sphingosine inhibited PMA-initiated degranulation totally and half of the secretion evoked by FMLP (47). As noted previously, a differential effect on PK-C activation may be explored with synthetic DAGs. Cox et al. demonstrated selective activation of the oxidase with synthetic diacylglycerols (44). Of a series of synthetic sn- I ,2-diacylglycerols, didecanoylglycerol (diC, o) induced lysosomal enzyme secretion but failed to stimulate the respiratory burst. Unlike other diacylglycerols that competed for all phorbol ester binding, diC,, competed for only 45% of the TABLE I INHlBlllON

OF

PK-C

DEGRANUI ATION AND 0,SUSPENDED NEUTROPHILS

MrDlATED IN

PRODUCTION

0,- p r o d u c t i o n h (nmolimini10~cells)

Control PMA H-7 (50 p M ) PMA + H-7 S t a u r o s p o r i n e ( I 0 nM) PMA a n d Staurosportne

3 2 2.6 17.6 2 0.2 0.5 t 0.8 17.9 2 1.7 0 2 0 21 2 3.5

*

0.2 78

2

0.2

2 0.5

*

0.1 0.2 4.1 2 0.4 0 . 1 2 0.1 2.3 2 0.4

Lysxyiiie releawd (mean k S . D . )at 5 rnin incuhation as detailed in Borregaard and Tauber ( I2 I ) SOD-inhibitable O 2 gcneratioii (mean ? S . D . ) as dctailed in Borregaard and Tauher ( I2 I ).

',

474

ALFRED I. TAUBER ET AL.

binding sites. The data showcd a correlation of selective PK-C binding with a limited cellular response and further suggcstcd that a discrete pool of PK-C was responsible for activation of the NADPH-oxidase. Alternatively, diC,, could have caused selective partitioning to specific granule membranes (for granule release) and not to the plasma mcmbrane (for oxidase activation). Thcsc findings are of particular interest in regard to the identification of multiple, distinct forms of PK-C by cloning techniques (39,40). The specificity and regulation of response have thus acquired a new molecular complexity. Determination of typespecific PK-C isoenzymes, with possibly differing activation characteristics and target proteins, promises to add a sophisticated dimension to the study of PK-C. Finally, an endogenous PK-C inhibitory factor (nonprotease, nonphosphatase) associated with the specific granules has been partially characterized (48). Its role, the substrates of the calpain-generatcd PK-M (34,35) system, the putative selective target pools of PK-C, and the subcellular localization of activated PK-C require extensive coordinated study to define physiological control mechanisms of phorbol ester stimulation.

C. A Putative Role of Diacyglycerol Diacylglycerol, like PMA, increases the affinity of PK-C for calcium and phosphatidylserine that permits activation of the enzyme at resting levels of intracellular calcium (15). The requirement for calcium in the intact neutrophil can only be seen when cells are depleted of calcium and then normal function may be restored as calcium is titrated back to ambient levels (49). 0,- generation has significantly lower requirements than depolarization, but in no case do fluorescent dyes demonstrate in PMA-stimulated cells the transicnt calcium rise seen with most other agonists, that is, FMLP or opsonzied zymosan (32,33). The role of DAG was first inferred from studies with cell-permeable DAGs that stimulated thc respiratory burst. The best studied, 1-oleoyl-2-acetylglycerol (OAG), and various synthetic DAGs (e.g., sn- 1,2-dioctanoylglyceroI) are similar to PMA in their activation profile (50-52), although OAG has an apparent calcium dependence that PMA lacks (53), and OAG exhibits an enhancement with calcium and arachidonate products that PMA lacks (52,54). [As discussed below, our own work employing OAG to stimulate 0, production in a reconstituted system of membrane containing NADPH-oxidase and PK-C is suggestive, but not definitive (55).] Hydrolysis of phosphoinositides generates DAG and IP, in FMLP-stimulated neutrophils (32,33), and while the role of IP, has been well Characterized as an intracellular messenger to mobilize calcium stores and stimulate the neutrophil, the evidence supporting a role of released endogenous DAG in the activation of the NADPH-oxidase has only lately been established.

18. PHOSPHORYLATION IN PHAGOCYTE ACTIVATION

475

Several studies have measured the mass of DAG generated in neutrophils stimulated with FMLP, ionomycin, or concanavalin A (ConA) to correlate kinetics and magnitude of the DAG response to 0, production (56-60). In Neidel’s study, employing a lipid extraction assay, DAG production paralleled 0,- generation, neither preceding the burst nor differing in magnitude with the extent of 0,- detected (56). The inability to detect early DAG formation in these studies, that is, coincident with IP, generation, hampers assigning a role of DAG to initiation of the burst (56,57). But more sensitive methods, employing radiolabeled glycerol and arachidonic acid, revealed a DAG rise of 35-60% within 2-5 sec of FMLP stimulation; ConA induced a slower and more modest rise (59,60). [Note that the platelet doublcs its DAG content within 5 sec of stimulation, peaking at 15-30 sec (61,62).] Whether the relatively small increase in DAG noted in these neutrophil studies is sufficient for PK-C activation or occurs in a critical locale are issues still to be defined. One explanation of these results is that DAG is rapidly converted to phosphatidic acid (PA) catalyzed by DAG kinase and possibly to arachidonate through the action of diacylglycerol lipase (63). Phosphatidic acid in fact is elevated 3-fold by FMLP or ConA stimulation (60), and although phosphatidic acid may activate the NADPH-oxidase in a cellfree system (64), a consensus is emerging that the minimal signaling requirements for ligand-induced respiratory burst stirnulation are the generation of increased intracellular calcium (from either intra- or extracellular sources) and diacylglycerol (60). Although the DAG kinase inhibitor R59022 failed to reveal augmented DAG production ( S S ) , the drug potentiates 0, production in FMLP-stimulated neutrophils; the augmentation is susceptible to the PK-C inhibitors H-7 (65), retinal (66), and gossypol (66) and further potentiated by leupeptin (65), which interferes with the conversion of PK-C to its soluble PK-M form (discussed later). Another avenue of investigation that itnplicates DAG in FMLP-stimulated 0, production has been shown with the manipulation of priming, where a low dose of one agonist (insufficient to elicit effector function) is followed by full dose stimulation with a second (or the same priming) agonist, resulting in an enhanced response (67). In PMA-priming human neutrophils, DAG production was increased twofold, correlating with augmented 0, - production, while the rise in cytosolic calcium was decreased by half, due in part to lowered levels of IP, (28). The authors postulate that since PMA augmented phosphatidic acid production, the increase in DAG was not due to inhibition of diglyceride kinase, suggesting that the source of DAG switched from phosphatidylinositol, 4,5-bisphosphate to an alternative lipid(s). Finally, the role of cytochalasin B in augmenting 0, - production in FMLP-stimulated neutrophils, while potentially having several mechanisms (including disruption of microfilaments, discussed below in regards to a possible oxidase-cytoskeleton relationship), may be related to its -

~

476

ALFRED I. TAUBER ET A t .

ability to generate DAG in pretreated neutrophils (68). These results would then support a role for PK-C activation mediated by DAG in FMLP-stimulated neutrophils and are important aspects of assigning PK-C a central role in physiological activation.

D. PK-C Mobilization That PK-C i s translocated from the cytosol to the plasma membrane upon neutrophil stimulation is well documented for a variety of stimuli, including PMA (69-72), calcium ionophores (35,69,72), FMLP (35,69,70,72), PAF (73,74), latex beads (75), opsonized zymosan (72,75), OAG (39,72), bacteria (76), and leukotriene B, (74). Monocytes follow the same pattern (77), and as shown in a diverse variety of cell types, this translocation property of activated PK-C is characteristic of its behavior (IS). A preliminary report found that although a,(3, and y PK-C isoenzymes are each found in the neutrophil, only the a species is translocated in the presence of calcium in the disruption buffer (42). The y isoenzyrne remained in the cytosol, and the p specics was divided between cytosol and membrane irrespective of the presence of calcium. These results, using polyclonal, anti-peptide antibodies, differ from the findings employing RNA hybridization techniqucs in PMA-stimulated cells that detccted only the a isoenzyme (using oligonucleotide probes) (4 1 ) and type I1 (p) isoenzyme using monospecific antibodies (43). These discrepancies are not resolvable based on the preliminary nature of the reports, but the definition of the PK-C constitucnts is of obvious irnportancc in assigning potentially different rolcs for the respective isoenzymcs. In the case of micromolar concentrations of calcium (a physiological rise in Ca2+ induced by many agonists), the cytosolic form of PK-C and a CaZ+requiring thiol proteinase, calpain, become associated with the neutrophil rncmbrane (78). The activation of calpain converts PK-C to a proteolytically modified Ca2 -phospholipid-independent form, both in broken cell preparations and in intact cells stimulated with PMA (34). Once the translocated PK-C has undergone proteolytic cleavage to a 65-kDa spccies (from 85 kDa), it is releascd to the cytosol in an activc form, presumably accessible to other protein substrates. The data thus far suggest that PK-C may be activated by two separate systems with different substrate targets, adding a second level of complexity to the control modulation of PK-C-mediated functions. Furthermore, the stability of PK-C binding to the membrane appears to vary with the stimulus used (35). Oxidase activation appears to require membrane-bound PK-C at least in the initiation phase. We deduce this conclusion from three types of studies: ( I ) PK-C translocation occurs on oxidase activation with FMLP (35,69,70,72) and PMA (69-72,79); (2) as we discuss later, PK-C, in the presence of calcium and phospholipid (SS),and PK-M effectively activate NADPH-oxidase. PK-M, in the +

477

18. PHOSPHORYLATION IN PHAGOCYTE ACTIVATION

ROI F. OF PK-C

IN

TABLE 11 0,- PRODUCTION 01: ADHERENT NEUTROPHILY

Prcincubation

Incubation

buffer

bufferh

0, production (nmoli 10” cells/ I5 min)

Value<

PBSL PBS and EGTA PBY+ PBS2’ PBS’+

3.0 f 1.0 3.6 t 3.0 0.97 ? 0.6 0.84 ? 0.3 0.99 2 0.5

0.79 0.016 0.019 0.0218

PBS2+ PBS’+

+

PBS and EGTA PBSZ+ and H-7 (100 p M ) PBS*+ and Leupeptin (1 P M ) ~

P

‘J1. Gini.\ and A . 1. Tauher (unpublished observation\).

P B S + conrains 0.9 mM Ca’ ’ PRS has no added Ca’+. p Values are calculated against control condition of PBS’ alone. d PK-C activity, measured at 5 inin, distrihutcd 23.8 i 6.68 in cytosol (and 76.2 -+ 6.6% in particulate fractions). The control cclls, left in \uspension, had 72.3 i 6.0% 01 PK-C in cytosol. which translocated lo the menihrane upon PMA \timulation. 79 0 i 4.09. 1,

~

9

+

reconstituted system, is effective only in the presence of phosphatidylserine, which we assume is required for its membrane attachment (80); (3) functional studies of intact neutrophils have revealed two phases of PK-C participation: an early PK-C (calcium dependent) and a later PK-M participation. In unreported studies, we have found that human neutrophils initiate 0, - production subsequent to their adherence (alone) that is Ca2 -dependent and H-7 sensitive. However, sustained 0,- stimulation is characteristic of PK-M activity in that it is C a 2 + independent but susceptible to leupeptin, an inhibitor of calpain (Table 11). We note with interest that ConA, a lectin which induces a robust phagocyte respiratory burst, releases PK-C from the human monocyte membrane to the cytosol, independent of its ability to mobilize intracellular calcium (8 1). Aside from demonstrating the distinctiveness of the PMA and ConA activation pathways, emphasizing the diversity of routes by which the respiratory burst may be stimulated, the data suggest a dissociation may be seen between oxidase activation and PK-C translocation. In a preliminary report, ionomycin was titrated in human neutrophils to show that the initial rise in calcium translocated PK-C to the membrane, and only higher levels of calcium were associated with an increase in total PK-C activity (82). +

111.

PHOSPHORYLATION STUDIES

Rapid progress has been made in elucidating the specific role of phosphorylation in the activation of the NADPH-oxidase, largely by defining phosphorylation defects in CGD. This disorder, characterized by an inability to express a respiratory burst, is due to structural defects in either the NADPH-oxidase or its

478

ALFRED I.TAUBER ET AL.

activating mcchanism. The Scripps classification divides CGD into four groups. The presence or absence of a low potential cytochrome b is further divided by autosomal recessive or X-linked inheritance (Table 111) ( 1 , 10,83). The recent cloning of the cytochrome b-related protein by Dinauer and Orkin (84) has identified likely structural defects in three forms of CGD, while Curnutte has demonstrated that missing or defective cytosolic factors critical for oxidase activation are thc cause of types I1 and V CGD (both are cytochrome b positiveautosomal recessive) (9, 10,83). The cytosolic factor(s), discovcred by several groups (85-88), was defined in studies that activated the NADYH-oxidase in a cell-free system. Plasma membranes, the site of the oxidase proper, could be stimulated to produce 0,- by arachidonic acid (AA) or sodium dodecyl sulfate (SDS) only in the presence of added cytosol. Wc showed that these agents did not activate the oxidase through PK-C ( 2 I ,89) and that the mechanism by which A A and SDS work is still unclear. [A recent report has shown that phosphatidic acid activates the oxidase in a cell-free system independent of cytosol (90), adding a new level of complexity to this issue. 1 Curnutte has defined four cytosolic factors termed C 1-4 by preparative isoclectric focusing; a fifth cytosolic component is inferred since reconstitution with the four factors was an insufficient substitute for unfractionated cytosol (83). C4 restored activity in two type I1 CGD patients, one of which was shown directly to have normal quantities of C1-3. A third patient's cytosol was partially corrected by C2 and termed type V CGD. With this classification of CGD, we may now address work relating defects of phosphorylation to the etiology of the disease and thereby chart a path by which the normal activation cascadc will ultimately be defined. Various studies have noted that a group of 48 kDa proteins were phosphorylated on neutrophil stimulation (9 1-97) and dephosphorylated as the oxidase activity returned to a resting state (95).t Concomitant absence of this phosphorylation pattern in both X-linked (most likely type I) (95,106) and autosomal recessive (most likely type 11) (94,97,106) CGD suggestcd the importance of defining these defects in light of the functional abnormalities noted previously. Okamura et al. observed three 48 kDa proteins, designated pp48/6.8, pp4U7.3, and pp48/7.8, phosphorylated on PMA stimulation that correlated in time and extent of labeling with rates of 0, production (106). [In rabbit neutrophils, ~

'Note that these proteins have bccn designated 47 kDa by other groups working with human ncutrophils (96,97), 46 kDa in guinea pig (9X,Y9) and 47 kDa in rabhit neutrophils (100). Horccker's group found a 46 kDa protein in the membranes and a 48 kDa species in the cytosol (101). The sanie 47 kDa protein found in platelets has been noted in difierentiated HL60 cells (102), but when correlation of HL60 phosphorylated proteins to 0 2 - expression was sought, pp212, pp134, and pp76 were considered the most likely candidates as playing a role in oxidase activation (103). In vitamin D3-differentiated U937 cells (a monoblartic phagocytic precursor cell line), a 48 kDa protein was implicated in PMA-stimulated oxidative metabolism (104) but was not seen in normal human monocytes (105).

479

18. PHOSPHORYLATION IN PHAGOCYTE ACTIVATION

_ _ _ ~

__

Scripps clasaiticdtion

X Linked ( X Cytochrome h absent

Cytochrome h present

)

I

Autosomdl (A )

111

X-Linkcd ( X + )

IV

Autosonial ( A + ) recessive

I1 V

_

_

_

~

~

Biochemical defect 91 kDa cytochrome h subunit. secondary loss of 22 kDa subunit 22 kDa cytocnrome b subunit, secondary loss of 91 kDa subunit 7 Point mutation in 91 kDa cytochrome h subunit CytOsOllc fdctor component c 4 (kinetic “S” factor) Cytosolic factor component C2

* The cytosolic lactor components have been defined by Curnutte er a/ ( 8 3 ) with isoelectric focusing: C1, pl 2.5-3.7; C2, pI5.7-6.2: C3. pI 6.8-7.3; C4, pl 8.5- 10.3. (Table provided by J . T . Curnutte, Scripps Clinic. La Jolla, CA.)

Sha’afi and co-workers noted only one protein with a molecular weight of 47,000 and a PI of 4.9 (loo).] The three proteins appear to be closely related, since only phosphoserine was identified as a phosphorylation product, and each protein yielded nearly identical one-dimensional phosphopeptide maps on proteolytic digestion. Preliminary studies from this group suggest that these proteins, identified by isoelectric focusing, differ only in the extent of their phosphorylation, with the more acidic forms representing the maximal extent of phosphorylation (107). In type 1 CGD neutrophils, pp48/6.8 and pp4817.3 were not phosphorylated, while in type 11 none of the three proteins were phosphorylated (106). In a separate study, these workers showed that the rare type 111 CGD neutrophils had the same defects exhibited by type I (the two more acidic proteins were not phosphorylated), while type IV CGD cells had no phosphorylation defect (108). It should be noted that these latest studies do not localize these proteins to cytosol or plasma membrane and represent cells solubilized in urea and Nonidet P-40 detergent, thus the relation of the phosphorylation defect to a structural defect of the membrane oxidase or the cytosolic component is not discerned by this work. Earlier studies showed the presence of the same or similar proteins in both the cytosolic and plasma membrane compartments (95); in type 1 CGD neutrophils, neither cytosolic nor plasma membrane proteins were phosphorylated. McPhail and co-workers in a more recent study ( I 09), employing one-dimensional electrophoresis, showed that pp48 was distributed in both plasma membranes and the cytosol, a finding also reported in neutrophil cytoplasts (97,108). It was selectively not phosphorylated by the reconstituted cell-free system activated with SDS. In functional studies, membranes from type I1 CGD neutrophils were restored to normal phosphorylation of a 48 kDa species when activated with

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cytosol from normal ncutrophils and SDS. Conversely, cytosol from the CGD cells could not reconstitute either oxidase activity or phosphorylation of pp48. The study also determined that PK-C activity was normal in the CGD neutrophils, confirming earlier binding studies ( 1 1) and AA reconstitution studies ( I lo), and suggesting that pp48, not the kinase expression (in PMA-stimulated cells), was abnormal. Koos and co-workers proved that the substrate could be phosphorylated by PK-C by excising the 47 kDa protein from gels and showing its phosphorylation by purified PK-C (97). PK-C has also been reported to phosphorylate the guinea pig 46 kDa protein in a cell-free system (97). In our own (unpublished) studies that examined thc phosphorylation of resting human neutrophil plasma membrane proteins with purified PK-C, there arc five predominant phosphorylated species, but none correlate precisely to pp48 (Fig. 1). (It is likely that it localizes to the cytosol in our membrane separation technique). These studies are obviously relevant to correlate the phosphorylation induced by PMA, but as discussed below, other agonists (i.e., FMLP) might dissociate oxidase activation and phosphorylation, and the entire issue of the relevance of thc pp48 phosphorylation for oxidase expression is not settled. The function of pp48 was recently hypothesized to be a structural role. In a preliminary report (107), Babior el t i / . reported that pp4W6.8 and pp4817.3 were associated with the cytoskeleton, while the more basic proteins were found in the detergent (Triton X- 100) soluble extract. They suggested, based on the finding that cytochrome b-deficient CGD neutrophils lacked the same cytoskeleton pp48 proteins, that the phosphorylated proteins might have the role of somehow linking the oxidase to the cytoskeleton, which under these experimental conditions associated in the same locale. To explain the oxidase-cytoskeleton association, one hypothesis is that pp48 may be part of the enzyme and extensive phosphorylation (lo pl 7.3) may initiate attachment to the cytoskcleton. But opposite to these results is the earlier study by Pontremoli ct ul., who showed that a 48 kDa cytoskeleton protein, phosphorylated in PMA-stimulated neutrophils, is released to the cytosol upon activation ( 1 1 I). Both phosphorylation and release to the cytosol were blocked by leupeptin, which inhibits calpain, the activity responsible for the conversion of PK-C to PK-M. That PK-M was responsible for phosphorylation of the 48 kDa species was strengthened by the finding that purified PK-M was sixfold more effective in its phosphorylation of the target protein than native PK-C (1 11). Another species of 20 kDa, probably myosin light chain, undergoes parallel conversion, and the authors postulate that cytoskeletal reorganization involving the 20 and 48 kDa components may be associated with requisite changes of the degranulation response. Of course the overlap between activation of the respiratory burst and degranulation of specific granules is well accepted, but the two functions may be dissociated (1 12), and as discussed previously, there is strong evidence that even PMA invokes distinctive activation pathways to elicit these responses. That is not to say that overlap does

48 1

18. PHOSPHORYLATION IN PHAGOCYTE ACTIVATION

- 90 - 69 -46 - 30

m

n u Y

Control

-90 -69

- 46 - 30

Protein Kinase C FIG. I . Phosphorylation of human neutrophil light mcinbranes by purified PK-C. The control sample contained human neutrophil light (plasma) InembrdneS (harvested from resting cells) ( 5 5 ) , phosphatidylserine, calcium, and [Y-~*P]ATP. whereas the PK-C sample contained phorbol dibutyrate (70 nM) and purified (to homogeneity) mouse brain cytosolic PK-C (55). The gels were run according to thc 2D PAGE protocol of Ames and Nikaido ( 1 32). Five distinct proteins are specifically phosphorylated by PK-C under these conditions: pp5117. IS, ppS117.26, pp3917.7, pp3116.7, and pp3216.4. (B. Warren, P. M. Blumberg, and A . I . Tauber, unpublished observations).

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not exist, but the map has yet to bc drawn in order to trace the basis of thcir respective interactions. Further support of a relation of the oxidase to the cytoskeleton is suggested by inhibitor studies, where microfilament disruption [e.g., cytochalasin ( 1 13) or Clostricfium botulinum toxin ( I 14)] inhibits FMLP-stimulated oxidative metabolism; scvcral sites of action are possible, i.e., Jesaitis and co-workers believe these effects are mediated through the cytoskelctal trafficking of the FMLP receptor, and the effect of cytochalasin on diacylglycerol metabolism represents another proximal site of action (68). This is only to suggest that the specificity of these inhibitor studies or their restrictive interpretation is problematic, and whilc there is tantalyzing speculation of a structural relationship between the oxidase and the cytoskeleton, it is at this point still speculative. Finally, it is intcrcsting to note that the platelet has a PK-C substrate of 47 kDa, which is prominently phosphorylated upon activation. Kecently, using a polyclonal antibody to the platelet protcin, HL6O cells wcrc found to inducc this protein upon differentiation (102). The identity of this protein has not been established, although lipocortin I ( I IS) and IP,-5-phosphatase ( 1 16) have been nominated. Unpublished data show inconsistency with lipocortin’s electrophoretic mobility, so thc phosphatase might be considered a possible candidate. Similar immunological studies with CGD neutrophils may be of interest in this regard. The resolution of three proteins at the 48 kDa level resolves the controversy with Segal, who found no phosphorylation defect in X-linked CGD (94). Using only one-dimensional gel electrophoresis, the single pp48/7.8 appears to represent a normal phosphorylation pattern, while the other two species are silent; in the autosomal variant, none of the three species are phosphorylated, and the onedimensional gel detects the dcfcct. As to whcthcr thc proteins arc missing, defectively phosphorylated (due to abnormalities of pp48 or its kinase), or degraded excessively, it appears that pp48 is missing in type I1 CGD (83). How the phosphorylation event is related to the activation of the oxidase is of crucial intcrcst. A purified oxidase preparation purportedly contains a similar peptide (48 kDa) (117), suggesting that this component may be regulated by phosphorylation. Evidence from Rossi’s laboratory (I18, I 19) supports this hypothesis. However, in a review (120), Babior doubts that the 48 kDa protein is an oxidasc cotnponcnt bccausc this protein(s) is not detected by silver staining of the gels, while the oxidase is quantitatively abundant (N. Okamura and B. M. Rabior, unpublished observations). An altcrnativc hypothesis is that these 48 kDa proteins regulate the oxidase indirectly through a mediator. Discernment of the actual function of the cytosolic factors (i.e., type II CGD) may serve as an example of such a regulatory function for the 48 kDa protein. The structural defect defined by Orkin and co-workers for type I CGD (9 1 kDa subunit of the b cytochrorne) by cloning techniques would argue for a structural defect of the oxidase itself. However, that structural defect docs not explain the absence of

483

18. PHOSPHORYLATION IN PHAGOCYTE ACTIVATION

normal phosphorylation: is the 48 kDa protein linked via the cytochrome and its lack of phosphorylation refleers abnormal oxidase (cytochrome) expression in type I CGD, or is the phosphorylated protein part of the structural component identified by cloning? Most troubling in constructing a consistent hypothesis for all these data is that phosphorylation of the 47 kDa proteins is blocked by H-7 in FMLP-stimulated rabbit neutrophils, and yet oxidase activation proceeds normally (100). Using guinea pigs, Ohtsuka rt ul. showed that a corresponding 46 kDa protein (PI 7.0 and 6.4) was well phosphorylated by OAG (10 pM), which had little effect on 0 2 - production. These results are at variance with studies performed with human cells and either reflect species differences or provide the experimental design to discern whether the phosphorylation of the 48 kDa protein(s) is related in a parallel fashion to activation of the NADPH-oxidase but is not a causal regulatory process. This is an important issue to resolve but requires further definition of the oxidase and its activating constituents. CGD appears to be a heterogeneous disorder, and various specific defects are likely to be identified within each broad classification; the power of identifying cytosolic factors complements the molecular biological advances made on the enzyme complex itself. In this regard, phosphorylation defects in cytosolic proteins harvested from CGD neutrophils promises to help elucidate the normal activation cascade of the respiratory burst.

IV.

PK-C ACTIVATION OF NADPH-OXIDASE

In nitrogen-cavitated human neutrophils, the NADPH-oxidase harvested from PMA-stimulated cells is found in the light membrane fraction (121), and almost 95% of the specific ['HIPDBu binding is found in the cytosolic fraction of unstimulated cells (55). Based on the findings that the addition of AA to a cytosolic factor reconstitutes an active oxidase, we extended those studies to characterize a PK-C pathway of oxidase activation. PMA alone was unable to stimulate 0, - activity in disrupted neutrophils when tested at concentrations corresponding to -SO-fold the Kd for the phorbol ester receptor; however, under specific conditions appropriate for PK-C activation, namely inclusion of PMA, phospholipid, ATP, and a cytosolic factor, the NADPH-oxidase was activated (55). Each component of a reconstituted PK-C system was required for NADPHoxidase expression. The dependence of oxidase activation on particular phospholipids paralleled that for PK-C studies in other tissues. The Ca2 dependence of the reaction could only be demonstrated with the addition of EGTA. Residual Ca2 in the cytoplasm and leached membrane Ca2 + was presumably sufficient for reconstitution of activity. The dose-response curve for PMA revealed a halfmaximally effective dose of 1.1 nmol/liter, which was consistent with the high affinity of PMA for PK-C (122) and the measured amount of PK-C in the cytosol +

+

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preparations. When purified PK-C obtaincd from mouse brain and its cofactors wcrc substituted for cytosol, the NADPH-oxidase was similarly activatcd. Quantitatively, the levels of PK-C in thc cytosol corresponded with the added purified cnzyme. Parallcl studics have been performed with purified PK-M prepared from mouse brain PK-C (80). Activities of NADPH-oxidase activation obtained were similar to those rcportcd with PK-C (e.g., 8-9 nmol O,-/min-mg-’ membrane protein). The requirement for calcium and phosphatidylserine remained with PK-M reconstitution, presumably because of membrane partition requirements. Attempt to solubilize the oxidase and thus obviate the need for phospholipid were unsuccessful, but we may conclude that the phosphorylation site that regulates oxidase activation in this system is not readily accessible to the fluid phase and requires juxtaposition of kinase and target in the membrane milieu. Addition of cytosol to membranes and PK-M alone was not sufficient for activation, suggesting that the PK-M substrate is not a cytosolic component in this system. As noted, activation of NADPH-oxidase has been achieved in broken cell preparations by adding AA (87,88), oleic acid (86), or SDS (85) to the membrane-bound oxidase. In these studies, an additional soluble factor was required, but it does not appear to be PK-C, as originally suggested. AA, oleate, or othcr oxygenated fatty acid derivatives activatc PK-C directly (123-125), and AA inhibits specific [13H]PDBu binding to the cytosolic neutrophil phorbol ester receptor, with a Ki of 38 pmol/litcr and an ID,, of 190 pmol/liter (P. M. Blumberg and A . I . Tauber, unpublished observations), values similar to those obtained using partially purificd PK-C from mouse brain (126). The mechanism for NADPH-oxidase activation by arachidonate in the in v i m setting docs not correlate with these obscrvations, howcver, and suggests that either another metabolic pathway is used or stimulation occurs by a nonenzymatic process. In both AA and SDS NADPH-oxidase broken-cell-stimulated systems, the PK-C inhibitor H-7 had no significant effect (127,128), and depletion of cytosolic ATP had no effect on AA stimulation ( 1 28, I29), results that are strongly supportive that AA and SDS oxidase activation are independent of PK-C. There are two key problems with the PK-C reconstitution studies: (1) inconsistent preparations and ( 2 ) low specific activity. In regards to the first issue, only Clark’s group (130) has confirmed our finding that PK-C could substitute for the cytosolic factor and stimulate the NADPH-oxidase with low specific activities. Since our initial studies, we and others have had difficulty in establishing a protocol that reliably allows harvesting a cytosol and membrane preparation that will consistently respond to PK-C reconstitution. The basis of this difficulty is unknown but may reside in the degree of preactivation of the “resting” ncutrophil [i.e., to the extent that the cell is partially activated (primed) before subjected to disruption and subcellular fractionation]. This issue is under current investigation in our laboratory and remains a stubborn obstacle to further pro-

18. PHOSPHORYLATION IN PHAGOCYTE ACTIVATION

485

gress with this system. The second issue, low specific activity, may well be related to the preparative problem. In our initial PK-C studies, oxidase activity obtained only 15% of the levels stimulated with AA or SDS (127), and these values are significantly lower than the activities currently obtained with the AA or SDS systems (129,13 I). The fact that we are accounting for less than 3% of the intact cell 0, -generating activity, while fulfilling the criteria for NADPHoxidase by pH and NADPH K,,, characteristics, has been a persistent problem in any attempt to compare PK-C reconstitution studies with those done with AA or SDS. Whether the low activity is due to suboptimal experimental conditions or represents an intrinsically different modality of activation is unresolvable with the current data, but is likely due to both explanations. We would argue that the evidence is compclling that AA in the cell-free system bypasses PK-C, and that because PK-C is able to activate the oxidase independent of the cytosolic factors (unlike AA), the two approaches are fundamentally different. Note that AA stimulates the intact cell respiratory burst in a distinct fashion from the cell-free AA system (89), thus we are left with an open question as to how the cell-free AA system might mimic intact cell activation of the oxidase, and thus the relevance of each approach is yet to be assigned. ~

V.

CONCLUSION

There are several reviews that offer worthy and reasoned schemata where PKC has a central role in neutrophil activation (e.g., 1,4,7,33). We concur that the data are compelling, and the role of a phosphorylating activity that controls NADPH-oxidase expression is well demonstrated in PMA-stimulated cells. However the phorbol esters are not physiological stimuli, and while they mimic natural agonists, we must recall that they serve as facile cofactors. PMA certainly stimulates a vigorous (one might wonder super-physiological) response, but is this model truly a reflection of the natural setting? As Sha’afi and Molski note, there are important differences between PMA and agonists like FMLP that raise intracellular calcium ( 1 ): translocated PK-C is likely to remain for an uncharacteristically long period in the membrane, giving both prolonged membrane stimulation and less PK-M generation; coupled with the differences noted in kinetics and rise in intracellular calcium, the two agonists demonstrate fundamentally different modes of cell stimulation that will have predictably different impacts on various activities. Aside from these cautions, there are others. First, the cells in virtually all of these studies are in suspension, whereas the phagocyte usually fulfills its responsibilities in the tissues, adherent to a surface. Our own unpublished research in this regard suggests complexity of PK-C function not discerned by cell suspension studies, and the issue requires further attention. Second, are studies performed with resting or partially activated (primed) neu-

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trophils? What we assume as a resting cell by the lack of 0, detection may well already be in a stimulated state as assessed by more sensitive assays, that is, actin polymerization (T. Howard personal communication). This will have obvious cffects as reflected in priming studies that both implicate and ignore PK-C participation (67). Third, are the sphinganine inhibition studies truly selective at the PK-C levels (29)? It is the only inhibitor reported effective against both FMLP and PMA stimulation. Now that a pseudosubstrate for PK-C is available (30), more specific inhibitor studies should be attempted. Finally, and most germane, are thc phosphorylation defects found in CGD causally relatcd to oxidase activation or simply abnormalities found in parallel to another defect‘? The data in this area are quickly being compiled and represent the best option at defining a phosphorylation cvent that may be analyzed as a controlling activity of NADPMoxidase cxpression. Again PMA has been employed in these studies and inconsistencies with other agonists have been described. Methodologic difficulties remain with the PK-C reconstitution system, and the AA cell-free system has not established its link to the biocheniistry of oxidase activation in the intact cell. Thus while these approaches offcr powerful insight, they are minimal systems that must be reconstructed back into the unperturbed neutrophil. But rcsolution of this issue, using purified components, appears to offer the best approach at defining a rolc for phosphorylation in phagocyte activation. ACKNOWLEDGMENTS The authors gratefully thank Drs. J. Curnutte and B . Babior for their critical review, and Ann Marie Happnie for hcr expert secretarial assistance in the preparation of this manuscript. This work was supported in part by National Institutes of Health grants A120064, HLB33565 and training grant HLO7501. REFERENCES I . Sha’afi, R. I . , and Molski. T. F. P. Activation of the neutrophil. P ~ J XAllergy42, :. 1-64 (1988). 2. Tauber, A. I. Protein kinase C and the activation of the human neutrophil NADPH-oxidase. Blood 69, 711-720 (1987). 3 . Gcnnaro, R., Scaggiantc. B., and Romeo, D. Protein phubphorylation and neutrophil activation. I n “Clinical Immunology: Human Intlarnn~aioryDiseases” (G. Marone, L. M. Lichtensrein, M. Condorelli, and A. S . Fauci, eds.). B. C. Decker, Toronto, 1987. 4. Bellavite, P. The superoxide-forming enzymatic system of phagocytes. Free Rudicals B i d . M d . 4, 225-261 (1988). 5 . Kaibuchi, K., Sawamura. M . , Tsuda, T. Hoshijima, M., and Tahai, Y. Protein kinase C and calcium in transmembrane signaling. In “Macrophage Biology” (S. Reichard and M. Kojma, eds.), pp. 221-230. Alan R. Liss, New York, 1985. 6. Korchak. H. M . , Vicnne, K., Wilkenfeld, C . , Robcrts, C., Rich, A. M . , and Weissmann, C. The first seconds of neutrophil activation: phoaphoinositides, protein kinase C, and calcium movements. Tran.v. Assc. Am. Physiciuns 98, 224-232 ( I 985). 7. Sadler, K . L., and Badwey, I. A. Second mcsscngers involved in superoxide production by ncutrophils: Function and metabolism. Hrmufol. Oncol. Clin. North Am. 2 , 185-200 (1988).

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8. Tauber, A . I . , Borregaard, N., Simons. E., and Wright, J. Chronic granulomatous disease: A syndrome of phagocyte oxidase deficicncics. Medicine 62, 286-309 (1983). 9. Curnutte, J. T., and Babior, 8 . M . Chronic granulomatous disease. Adv. Hum. Genet. 16, 229297 ( I 987). 10. Curnutte, J. T. Classification of chronic granulomatous disease. Hemarol. Oncol. Clin. North Am. 2, 241-252 (1988). I I . Tauber, A. I . , Brettler, D. B . , Kennington, E. A , , and Blumberg, P. M . Relation of human neutrophil phorbol ester receptor occupancy NADPH-oxidase activity. Blood 60, 333-339 (1982). 12. Harvath, L . , and Andersen, B . R. Defective initiation of oxidative metabolism in polymorphonuclear leukocytes. N . Engl. J. Med. 300, 1130-1 135 (1979). 13. Newburger. P. E.. Robinson, J.. Pryzwansky, K. B . , Rosoff, P. M . , Greenburger, J. S., and Tauber, A . I. Human neutrophil dysfunction with giant granules and defective activation of the respiratory burst. Blood 61, 1247- 1257 (19x3). 14. Weening, R. S.. Roos, D.. Weemas, C. M . R . , Horman-Muller, J. W. T., and Schaik, M . L. J. Defective initiation of the metabolic stimulation in phagocytizing granulocytes: A new congenital defect J. Lab. Clin. Mud. 88, 757-768 (1976). 15. Kikkawa, U., and Nishizuka, Y. The role of protein kinase C in transmembrane signalling. Annu. Rev. Cell B i d . 2, 149-178 (1986). 16. Lehrer, R. 1.. and Cohen, L. Receptor mediated regulation of superoxide production in human neutrophils stimulated with phorhol myristate acetate J. Clin. Invesf. 68, 13 14- 1320 (1981). 17. Nishihira, J.. and O’Flaherty, J. T. Phorbol myristate acetate receptors in human polyrnorphonuclear neutrophils. J. Immunol. 135, 3439-3447 (1985). 18. Tauber, A . I . , and Goctrl, E. J . Inhibition of complement-mediated functions of human neutrophils by impermeant stilbene disulfonic acids. J. frnmunol. 126, 1786- I789 (1981). 19. Fduber. A. I . , and Goetzl. E. J . Structural and catalytic properties of the solubilized superoxidegenerating activity of human polymorphonuclear leukocytes. Solubilization, stabilization in solution, and partial characterization. Biochentistrv 18, 5576-5584 (1979). 20. Gaudry, M., Perianin, A., Marquetly, C . , and Hakim, J. Negative effect of a protein kinase C inhibitor (H-7) on human polymorphonuclear neutrophil locomotion. fmmunology 63,7 15-7 I9 (1988). 21. Maridonnau-Parini, I . , Tnngale, S. M., and Tauber, A. I . Identification of distinct activation pathways of the human neutrophil NADPH-oxidase. J. Immunol. 137, 2925-2929 (1986). 22. Sha’afi, R . I., Molski, T. F. P., Huang, C. K., and Naccache, P. H . The inhibition of neutrophil responsiveness caused by phorbol esters is blocked by the protein kinase C inhibitor H-7. Biochem. Biophys. Res. Commun. 137, 50-60 (1986). 23. Sako, T., Tauber, A . I . , Jeng, A. Y., Yuspa, S. H., and Blumberg, P. M . Contrasting actions of staurosporinc, a protein kinase C inhibitor, on human neutrophils and primary mouse epidermal cells. Cancer Res. 48, 4646-4650 (1988). 24. Gerard, C . , McPhail, L. C., Marfat, A , , Stimler-Gerard, N . P., Buss, D . A . , and McCall, C. E. Role of protein kinaaes in stimulation of human polymorphonuclear leukocyte oxidative metabolism by various agonists. J. Clin. Invest. 77, 61-65 (1986). 25. Wilson, E., Olcott, M . L., Bell, R . M . , Menill, A . H., and Lambeth, J. D. Inhibition of the oxidative burst in human neutrophils by sphingoid long-chain bases. Role of protein kinase C in activation of the burst. J . B i d . Chem. 261, 12616-12623 (1986). 26. Pittet, D., Drause, K. H . , Wollheim, C. B., Bruzzone, R., and Lcw, D. P. Non-selective inhibition of neutrophil function by sphinganine. J. B i d . Chem. 262, 10072-10076 (1987). 27. Lambeth, J. D . , Burnham, D. N., and Tyagi, S. R. Sphinganine effects on chemudttractant induced diacylglycerol generation, calcium fluxes, superoxide production and on cell viability in the human neutrophil. J. B i d . Chem. 263, 3818-3822 (1988).

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2 8 . Tyagi, S . R . , Tamura, M . . Burnham, D. N . , and Lambeth, J. D. Phorbol myristate acetate (PMA) augments chemoattractant-induced diglyceride generation in human neutrophils hut inhibit phosphoinositide hydrolysis. J. R i d (‘hem. 263, 13191-13198 (1988). 29. Jclt’erson, A . H . , and Schulman, H . Sphingoainc inhibits calniodulin-dependent enzymes. J. B i d . Chem. 263, 1524i-15244 (l9XX). 30. House, C.. and Kemp, B. E. Protein kinaae C contains a pseudosubstrate prototope in its regulatory domain. Science 238, 1726- 1728 (1987). 3 1 . Marawo, W. A , , Phan, S. H . , Krutzach, H . , Showcll, H J . , Feltner, D.E., Nairn, R., Becker, E. L., and Wirrd, P A . Purification and identification of formyl leucyl-phciiylalaninc as the major neutrophil cheniotaclic factor produced by Esc-hrrichia coli. .I. B i d . Chem. 259, 54305439 (1984). 32. Allen, R. A , , Traynor, A. E., Omann, G . M . . and Jesaitis, A. J. The chemotactic peptide receptor. A model for future understanding of chemotactic disorders. Hemntol. Oncol. Clin. North Am. 2, 33-59 (19x8). 33. Oinann, G . A , , Allen, R . A , , Bokoch. Ci. M . , Painter, R. G . . Traynor, A. E . , and Sklare, L. A . Signal transduction and cytoskeletal activation in the neutrophil. Physiol. Rrv. 67, 285-322 (1987). 34. Mclloni, E., Pontremoli, S.,Michetti, M., Sacco, 0.. Sparatorc, B.,and Hurecker, R . 1.. The involvement of calpain in the activation of protein kinase C in neutrophils stimulated by phorbol myristic acid. J . B i d . Chem. 261, 4101-4105 (1986). 35. Pontremoli, S . , Melloni, E., Michetti. M., Salamino, P., Sparatore, B . , Sacco, O., and Horecker, A . I-. Differential mechanism of translocation of protein kinase C to plasma membranes in activatcd human neutrophils. Aiocliem. Biophys. Res. Commun. 136, 228-234 ( 1986). 36. Grinstein. S . , and Furuya, W. Receptor mediated activation of electropernieahilized neutrophils: Evidence for a Ca2+ and protein kinase C-independent signalling pathway. J. Biol. Chem. 263, 1779- I783 ( 1988). he, P. H., Molski, M . M., and Sha’afi, R . 1. Polymyxin B inhibits phorbol 12-myriatate I3-acetate, but not chemotactic factor induccd cficcts in rabbit neutrophils. FEBS Lett. 193, 227-230 (1985). 38. Cooke. E . , and Hallett, M. B. The rule of C-kinase in the physiological activation of the neutruphil oxidase. Evidence from using pharmacological Inanipulation of C-kinase activity in intact cells. Biochem. J. 232, 323-327 (19x5). 39. Cousscns, L., Parker, P. J., Rhee, L., Yang-Rng, T. L., Chcn, E . , Waterfield. M. D., Pranche, U., and Ullrich, A . Multiple, distinct forms of bovine and human protein kinase C suggest diversity in cellular signaling pathways. Science 233, 859-866 ( 1986). 40. Onu, Y . , Fujii, ‘r.%Ogita, K . , Kikkawa, U . , Igarashi, K . , and Niahimka, Y. The structure. expression, arid propcrtics of additional memhers of the protein kinase C family. J. B i d C’hrm. 263, 6927T6932 ( 1988). 41. Gerard, C., Lord, P., and McCall, C . Differential expression of protein kinasc C genes in human neutrophils. Clin. Res. 36, 565A (1988). 42. Fujiki, T., Rossi, M. W., Phillips, W. A . , Johnston, R . B . , Jr., and Korchak, H. M . Protein kinase C isotype distribution and selective isotype lranslocation with Ca2 in human neutrophils and cytoplasts. J. Leukocyte Biol. 44,274 (1988). Ahstr. 43. Yoshida, Y., Huang, F. L., Nakabayashi, H . . and Huang, K . P. Tissue distribution and developmcntal expression of protein kinase C isoenzyrnes. J. Biol. L’hem. 263, 9x68-9873 (1988). 44. Cox, C . C . , Dougherty, R. W., Ganong: 13. R . , Bell, R M . , Nicdel, J . E., and Synderman, R. DiHcrcntial stimulation of the respiratory burst and lysosumal enzyme secretion in human

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45.

46.

47.

48.

49.

50.

51.

52.

53.

54.

55. 56. 57. 58.

59.

60.

61.

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polymorphonuclear leukocytes by synthetic diacylglycerols. J. fmmunol. 136, 461 1-4616 (1986). Nath, J . , and Powledge, A. Involvement of protein kinase C (PK-C) in the activation of m e t leu-phe (FMLPj-mediated respiratory burst in human neutrophils. J. Leukocyte R i d . 44,27 1 (1988). Abstr. Salzer, W., Gerard, C . , and McCall. C. Effect of an inhibitor of protein kinase-C on human polymorphonuclear leukocyte degranulation. Biochem. Biophys. Res. Commrtn. 148, 747-754 ( 1987). Wilson, E., Rice, W. G . , Kinkade, J. M . , Merrill, A. H., Arnold, R. R . , and Lambeth. J. D. Protein kinase C inhibition hy sphingoid long-chain bases: Effects on secretion in human neutrophils. Arch. Bioc.hem. Ric~phys.259, 240-214 (1987). Balazovich, K . J . , Smolen, J. E . , and Boxer, L. A . Endogenous inhibitor of protein kinasc-C: Association with human peripheral blood ncutrophils but not with specific granule-deficient neutrophils or cytoplasts. J. fmmunol. 137, 1665-1673 (1986). Karnad, A. B., Hartshorn, K. L . , Wright, J.. Myers, J. B., Schwartz, J. H., and Tauber. A. I . Priming of human neutrophils with N-formyl-nlethionyl-leucylphenylalaninc(FMLP) by a calcium-independent, pertuaaia toxin-insensitive pathway. Blond (in Press) ( I 989). Fu.jita, I . , Irita, K., Takcshige. K . , and Minakan~i, S. Diacylglyccrol, I-oleolyl-2-acetylglycerol, stimulates superoxide-generation from human neutrophils. Biochem. Biophys. Res. Commun. 120, 318-324 (1984). Dewald, B., Payne, T. G., and Baggiolini, M. Activation of NADPH oxidase of human neutrophils. Potentiation of chemotactic peptide by a diacylglycerol. Biochem. Riophys. Res. Commun. 125, 367-373 (1984). O’Flaherty, J. T., Schmitt, J. D., and Wykle, R . 1. lntcractionsof arachidonate metabolism and protein-kinase C in mediating neutrophil function. Biochem. Biophys. Res, Commun. 127, 916-923 (1985). Ozaki, Y . , Kume, S . , Ohashi, T.. and Niwa, Y. Partial dependancy of I-olcolyl-2-acetylglycerol-induced superoxide production by human neutrophils on calcium ions and cytochalasin-B. Biochem. Biophys. Rex. Commun. 134, 690-697 (1986). Penfield, A , , and Dale, M. M. Prostaglandins El and E2 enhance the stimulation of superoxide release by I -oleolyl-2-acetylglycerol from human ncutrophils FEBS Letr. 181, 335-337 (1985). Cox, J. A , , Jeng, A. Y . , Sharkey, N. A., Blumberg, P. M., and Tauber, A. I . Activation of the human neutrophil NADPH-oxidase by protein kinase C. J. Clin. Invest. 76, 1932-1938 (1985). Rider, L. G., and Niedel, I. E. Diacylglycerol accumulation and superoxide anion production in stimulated human neutrophils. J. Riol. Chem. 262, 5603-5608 (1987). Cockcroft, S . , and Allan, D. The fatty acid composition of phosphatidylinositol, phosphatidate and 1,2-diacylglycerol in stimulated human neutrophils. Biochem. 1. 222, 557-559 (1984). deChaffoy de Courcelles. D., Roevens, P., and Van Belle, H. R59022, a diacylglycerol kinase inhibitor. J. Biol. Chem. 260, 15762-15770 (1985). Takenawa, T., Ishitoya, J.. and Nagai, Y. Inhibitory effect of prostaglandin E2, forskolin, and dibutryl CAMP on arachidonic acid release and inositol phospholipid metabolism in guinea pig neutrophils. J . Riol. Chem. 261, 1092-1098 (1986). Korchak, H. M., Vosshall, L. B . , Zagon, G . , Ljubich, P., Rich, A. M., and Weissmann, G. Activation of the neutrophil by calcium-mobilizing ligands. J . B i d . Chern. 263, I 1090-1 1097 (1988). Preiss. J. E., Loomis, C. R., Bishop. W. R . , Stein, R., Niedel, J. E . , and Bell, K. M. Quantitative measurements of sn- 1 , 2-diacylglycerols present in platelet hepatocytes, and ras and sis-transformed normal rat kidney cells. J. Biol. Chem. 261, 8597-8600 (1986).

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62. Rittentiousc-Simiiions, S. Production of diaglyceride from phosphatidylinositol in activated human platelets. J. Cliri. Invest. 63, 580-587 (1979). 63. Bcrridgc. M. J . Inositol trisphosphatc and diacylglycerol are second messengers. Biochern. .I. 220, 345 360 ( 1984). 64. Bcllcvitc. P . . Corso, F.. Dvsi. S . . Qr7eskowiak, M . , Della-Bianca, V., and Rossi, F. Activation of NADPH-dependent superoxide production in plasma menlbrdne extracts of pig ncutrophils by phosphatidic acid. J. H i d . Chem. 263, 8210-8214 (1988). hS, Gomez-Cambronero. J . , Molski, T. F. P., Beckcr, E. L., Sha’ati, K. I. lhc diacylglycerol

kinasc inhibmr KSY022 potentiates superoxide production but not secretion induced by fMctLeu-Phe: Erects of lcupcptin and the protein kinase C inhihitor H-7. Biochvn. Biophys. Res. Comrnun. 148, 38-46 ( 1987).

66. Cooke, E.,Al-Mohanna, F. A.. and Hallctt, M. B . Diacylglycerol kinase inhibitor, R59022, potentiates neutrophil oxidase activation by Ca2 ’ dcpeiidcnt stimuli. Biochcm. Pharmcrcol. 36, 3459-3462 (1987).

67. Tauher, A . I., Karnad, A . B . , Hartshom, K . L., Myers, J. B., and Schwartz, J. H . Parameters of neutrophil activation: Models of priming and deactivation. Irr “Biochemistry of the Acute Allergic Reaction” ( A . I. Tauher, B. U. Wintrobe, and A. S . Simon, e d York, pp. 297-310. (1989). 68. Honeycut, P.J . , and Niedel, J. E. Cytochalasin B enhancement of the diacylglycerol response i n formyl peptide-stimulated neutrophils. J. Biol. Chem. 261, 15900-15905 (1986). 69. Nishihira, J., McPhail, L. C. and O’Flaherty. J. T. Stimulus dependent mobilization of protein kinase C. Riochem. Biophvs. Res. Commitn. 134, 587-594 (1986). 70. Pikc. M C’., Jakoi, L . , McPhail. L. C., and Snyderman, R . Chemoilttractant~mediatedstimulation of ttic respiratory burst in human polymorphonuclear leukocytes may require appearance of potential kinase C activity in the cell’s particulate fraction. Blood 67, 909-913 (1986). 71. Wolfson, M . , McPhail, L. C.,

Ilah, V. N., and Snydemian, R. Phorbol niyristate acetate mediates rcdiatribotiun of protein kinnse:C in human neutrophils: Potential role in the activation of the respiratory burst enzyme. J. Immitnol. 135, 2057-2062 (1985). 72. Chrihtianscn, N . 0 . .Larsen, C. S . . Johl, H . , and Esmann, V. Membrane associated protein kinase C activity in superoxide producing human polymorphonuclear leukocytes. J . I.eukoryte Biol. 44, 33-40 ( I W X ) . 73. Gay, J. C . , and Stitt, E. S. Platelet activating factor induced protein kinase activity in the particulate fraction of human neutrophils. Blood 71, 159- 165 (1988). 74. O’Flahcrty, J. T., and Nishihira. J. Arachidonate metabolites, platelet-activating factor, and the mobilization of protein kinase C in human polymorphonuclear neutrophils. J. Immunol. 138, 1889-1895 (1987) 75. Deli, E., Kiss, Z . , Wilson, E . , Lambcth, J. D., and Kuo, J. F. lmmunocytochemical localization of protein kinase C in resting and activated human neutrophils. FEES L e / / . 221, 365-369 (1987). 76. Christiansen, N . O., Peterslund, N . A , , Lars.cn, C . S., and Esmann, V Evidence that bacteria

inducc translocation of protein kinase C activity in human polymorphonuclear leukocytes. Biochcm. Diophvs. Res. Cornmun. 147, 787-7Y3 ( IYX7). 77. Myers. M .A . , McPhail, L. C.. and Snyderman, R. Redistribution of protein kinase-C activity in human monocytes: Correlation with activation of the respiratory burst. J. Immuriol. 135, 341 1-3416 (1985). 78. Melloni, E., Pontrcnioli, S., Michctti, M., Sacco, 0.. Sparatore, B., Salamino, F.. and Horecker, R . L. The involvement of calpain in the activation of protein kinase C in neutrophils stimulated by phorhol myristic acid. Proc. Narl. Acad. Sci. U.S.A. 82, 6435-6439 (1985).

79. Pnntremoli, S . . Melloni. E., Michetti. M., Sacco, O.,Salamino, F., Sparatore, B . , and

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Horecker, B. L. Biochemical responses in activated human neutrophils mediated by protein kinase C and a Ca2+ requiring proteinase. J . B i d . Chem. 261, 8309-8313 (1986). 80. Tauber, A. I . , Cox, J. A . . Curnutte, J. T., Carrol, P. M . . Nakakuma, H., Warren, B., Gilbert. H., and Blumberg, P. M. Activation of human neutrophil NADPH-oxidane rn virro by the catalytic fragment of protein kinase C. Bioc,hem. Biophvs. Res. Cornmun. 158, 884-890 ( I 989). 81. Costa-Casnellie, M . R . , Segal. G . R . . and Lichtman, M. A. Signal transduction in human monocytes: Relationship between superoxide production and the level of kinase C in the membrane. J . Cell. Phjsiol. 129, 336-342 (1986). 82. Balazovich, K . J . , and Boxer, L. A. Protein kinase C translocation can be dissociated from its activation. Fed. Pruc. 46, 987 (1987). 83. Curnutte, J. T., Scott, P. J . . and Mayo, L . A. The cytosolic components of the respiratory burst oxidasc: Resolution of four componcnts, two of which are missing in complementary types of chronic granulomatous disease. Pruc. Nut/. Acad. Sci. U.S.A. in press (1989). 84. Dinauer, M . C.. and Orkin, S. H. Chronic granulomatous disease: Molecular genetics. Hemuto/. Onrol. Clin.Norrh A m . 2, 225-240 (1988). 85. Bromberg, Y., and Pick, E. Activation of NADPH-dependent superoxide production in a cell free system by sodium dodecyl sulfate. Cell. Immunol. 88, 213-221 (.1984). 86. Heyncman, R. A , , and Vercauteren, R. E. Activation of a NADPH oxidase from horse polymorphonuclear leukocytes in a cell free system. J. Leukocyte Biol. 36, 751-759 (1984). 87. Curnutte, J. T. Activation uf human neutrophil NADPH-oxidase by arachidonic acid in a cellfree system. J. Clin. Invesr. 75, 1740-1743 (1985). 88. McPhail. L. C . , Shirley, P. S . , Clayton, C. C . , and Snydernian. R. Activation of the respiratory burst enzyme from human neutrophils in a cell-free system: Evidence for a soluble cofactor. J . Clin. Invest. 75, 1735- 1739 (1985). 89. Maridonneau-Parini, I . , and Tauber, A. I. Activation of NADPH-oxidase by arachidonic acid involves phospholipase A2 in intact human ncutrophils hut not in the cell-frcc system. Bloc-hem. B i u p h y ~ Res. . Commun. 138, 1099-1 105 (1986). 90. Bellavite, P., Corso, F., Dusi, S., Grzeskowiak, M . , Della-Bianca, V., and Rossi, F. Activation of NADPH-dependent superoxide production in plasma membrane extracts of pig neutrophils by phosphatidic acid. J. Biol. Chem. 263, 8210-8214 (1988). 91. Okarnura, N., Ohashi, S., Nagdhisa, N., and Ishihashi, S. Changes in protein phosphorylation in guinea pig polyinorphonuclear leukocytes by treatment with membrdne-perturbing agents which stimulate superoxide anion production. Arch. Biochem. Biophys. 228, 270-277 (1984). 92. White, J. R . , Huang, C. K . , Hill, J. M., Naccache, P. H., Becker, E. L . , and Sha’afi, R. 1. Effect of phorbol 12-myristate- 13-acetate and its analogue 4-phorbol 12, 13-didecanoate on protein phoaphorylation and lysosoinal enzyme release in rabbit neutrophils. J . B i d . C h m . 259, 8605-861 I (1984). 93. Gennaro. R., Florio, C . , and Romeo, D . Co-activation of protein kinase C and NADPH oxidase in the plasma menibrane of neutrophil cytopl . FEES Len. 180, 185-190 (1985). 94. Segal, A . W., Heyworth, P. G . , Cockroft, S., and Barrowman, M. M. Stimulated neutrophils from patients with autosoi~~al recessive chronic granulomatous disease fail to phosphorylate a Mr-44,000 protein. Narure (London) 316, 547-549 ( I 985). 95. Hayahawa, T., Suzuki, K . . Suzuki, S . , Andrews, P. C . , and Babior, B. M. A possible role for protein phosphorylation in the activation of the respiratory burst in human neutrophils. Evidence from studies with cells from patients with chronic granulomatous disease. J. B i d . Chem. 261, 9109-9115 (1986). 96. Heyworth, P. G . , and Segal, A . W. Further evidence for the involvement of a phosphoprotein in the respiratory burst oxidase of human neutrophils. Biochrm J . 239, 723-73 I ( 1986). 97. Krdmer, 1. M., Verhoeven. A . J., van der Bend, R. L., Weening, R . S . , and Roos, D. Purified

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protein kinasc C phosphorylates a 47-kl)A protein in control neutrophil cytoplasts hut not in ncutrophil cytoplasts from patients with the autosomal form of chronic granulomatous disease. J . R t o l . C'hrm. 263, 2352Z2357 (1988). '1%.Olitsuka, T., Ozawa, M . , Katayama. T., Okamura, N., and Ishihashi, S . Syncrgiani of phosphorylation uf 46K protein(s) and arachidonate release i n thc induction of superoxide anion production in guinea pig polynlorphonuclcar leukocytes. Arch. Biochrtn. /lioph.v.\. 260, 22623 I ( 1988). 99. Ohtsuka. T., Okamura, N.. and Iqhibashi. S. Involvement of protein kinase C in the phosphorylation of 48 kDA protcins which arc phosphorylated in parallel with activalion of NADPH oxidnse in intact guinea-pig polymorphonuclcar. Riochmm Biophys. Acru 888. 332-337 ( 1986). 100. Sha'afi, K. I . . Molski, T. F, P.. Gomez-Camhronero, T., and Huang, C. K . Dissociation of the 47-kilodalton protein phosphorylation from degranulation and superoxide production in ncutrophils. J . f.rnkor,ye B i d . 43, 18- 27 (1988). 101. Pontremoli, S . . Mclloni. E., Salamino, E., Sparatore, B . . Michctti, M . , Sacco, O., and Horecker. B. L. Ditrerential mechanism of translation of protein kinase-C to plasma nicmbrdneS in activated human neutrophils. Arch. Biochern. Biophys. 250, 23-29 (1986). 102. Tyers, M., Rachubinski, R. A , , Sartori, C . S . , Harley, C. B . , and Haslam. R. J. Induction of the 47 kDA platelet substrate of protein kinase C during differentiation of H L 6 0 cells. Biochetti. J . 243, 249-2.23 (1987). 103. Gaul, J. R . , and Carchman. R. A. A correlation between phorbol diester-induced prbtein phosphorylation and supcroxide anion generation in H L 6 0 cclls during granulocyte maturation. J. B i d . Chem. 262, 826 (1987). 104. Sniper, R . G . , Ways, K . , D'Amico, C. N . , Siram, C i . , Gray, T. K . , and Cohen, M. S. The metabolitc u 25-dihydroxy vitamin DR enhances lyrnphokine mediated activation of the oxidative metabolism of the UY37 cell line and phosphorylation of a 48-kD respiratory burstassociated protein. Blood 70, 1639 -1644 (19x7). 105. Kclla, B. A . , and Carchman, R. A. The relationship between lysosomal cnrymc release and protein phnsphorylation in human monocytes stimulated by phorbol esters and opsoni7,ed zyiiiosan. J B i d . C hm . 262, 17404-1741 I (1987). 106. Okumura. N.. Cumuttc, J. T., Roberts, R. L., and Babior, B . M . Relationship of protein phosphorylation to the activation of the respiratory burst in human neutrophils. J . B i d . C'hem. 263, 6777-6782 (1988) 107. Babior, B. M., Cumutte, .I.T.. and Okamura, N. The respiratory burst oxidase and certain members of thc 4XK phusphoprotein family are associated with the ncutrophil cytoskeleton. B l n r ~ d72, 141a (1988). 108. Okamura, N . , Malawista. S . E., Roberts, R. L., Rosen, H.. Ochs, H. D., Babior, B. M.. and Curnuttc. J T. Phoaphorylation of the oxidase-related 48K phosphoprotein family in the unusual autosomal cytochronie-negative and X-linked cytochrome-positivc types of chronic granulomatous disease. Blood 72, 811-816 (1988). 109. Caldwcll, S. E., McCall, C. E.. Hendricks, C. L., Leone, P. A , , Bass, D. A , , and McPhail, L. C. Correlation of NADPH oxidase activation and phosphorylation of a 48-kD protein(s) by a cytosolic factor defective in autosomal recessive chronic granulomatous disease. .I. Clin. Invesf. 81, 1485 -1496 (1988). 110. Cumutte, J . T., Berkow, R. L., Roberts, R. I>., Shurin, S. B., and Scott, P. J. Chronic granulomatous disease due to a defect in the cytosolic factor required for nicotinamide adenine dinucleotide phosphate oxidase activation. 1. Clin. Invest. 81, 606-610 (1988). I I I . Pontremoli, S . , Melloni, E., Michetti, M . , Sparatore, B., Salamino, F., Sacco, O., and Horecker, B. L. Phosphorylation and proteolytic modification of specific cytoskeletal proteins in human neutrophils stimulated by phorhol 12-myristatc 13-acetate. Proc. Nut!. Arud. Sci. U . S . A . 84, 3604 3608 (1987).

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112. Badwey, J. A , , Robinson, J. M., Lazdins, J. K., Briggs, R. T., Karnovsky, M. J., and Karnovsky, M. L. Retinoids stimulate the release of superoxide by neutrophils and change their morphology. J . Cell P1zysiol. 127, 223-228 (1986). 113. Jesaitis, A. J., Tolley, J. O., and Allen, R . A. Receptor-cytoskeleton interactions and membrane traffic may regulate chenioattractant-induced superoxide production in human granulocytes. J. B i d . Chem. 261, 13662- 13669 (1986). 114. Al-Mohanna, F. A.. Ohishi, 1.. and Haliett, M. B. Botulinum C toxin potentiates activation of the neutrophil oxidasc. FEES Le//. 219, 40-44 (1987). 115. Touqui, L . , Rothhut, B . , Shaw, A. M . , Radin, A.. Vargaftig, B. B., and Russo-Marie, F. Platelet activation-a rolc for a 40K anti-phospholipase A2 protein indistinguishable from lipocortin. Nature (London) 321, 177- 180 ( I 986). 116. Connolly, T. M . , Lawing, W. J., and Majerus, P. W. Protein kinase C phosphorylates human platelet inositol trisphosphate 5'-phosphomonoesterase, increasing the phosphatase activity. Cell 46, 95 1-958 ( I 986). 117. Glass, G. A . , DeLisle. D. M . , DeTogni. P., Gabig, T. G . , Magee, B. H., Markert, M., and Babior, B. M. The respiratory burst oxidase of human neutrophils. 1. B i d . Cfiem.261, 13247I325 I ( 1 986). 118. Papini, E., Grzeskowiak, M., Bellavite. P., and Rossi, F. Protein kinas& phosphorylates a component of NADPH oxidase of ncutrophils. FEES Lett. 190, 204-208 (1985). 119. Bellavite, P., Papini, E., Zeni, L . , Della Bianca, V., and Rossi, R. Studies on the nature and activation of 0, forming NADPH oxidase of leukocytes. Identification of a phosphorylated component of the activc enzyme. Free Radicals Res. Commun. 1, 11-29 (1985). 120. Babior, B. M. Protein phosphorylation and the respiratory burst. Arch. Biochem. Biophys. 264, 361-367 (1988). 121. Borregaard, N., and Tauber, A. 1. Subcellular localization of the human neutrophil NADPHoxidase: b-cytochrome and associated flavoprotein. J. B i d . Chem. 259, 47 (1984). 122. Leach, K. L., James, M. L . , and Blumberg, P. M. Characterization of specific phorbol ester aporeceptor in mouse brain cytosol. f r o r . Nut/. Acad. Sci. U.S.A. 80, 4208-4212 (1983). 123. McPhail, L. C . , Clayton, L. C . , and Snyderman, R. A potential second messenger role for unsaturated fatty acids: Activation of Ca2 dependent protein kinase. Science 224, 622-624 ( I 984). 124. Murakami, K., and Routtenberg, A. Direct activation of purified protein kinase C by unsaturated fatty acids (oleate and arachidonate) in the absence of phospholipids and Ca2+. FEES Lett. 192, 189-193 (1985). 125. Hansson, A , , Serhan, C . N. Haeggstrom, J . , Ingelman-Sundberg, M . , and Samuelsson, B. Activation of protein kinase C by lipoxin A and other eicosanoids. Intracellular action of oxygenation products of arachidonic acid. Biochem. Biophys. Res. Commun. 134, 1215-1222 (1986). 126. Leach, K. L . , and Blurnberg, P. M. Modulation of protein kinase C activity and ['HI phorbol 12,13-dibutryate binding by various tumor promoters in mouse brain cytosol. Cancer Res. 45, 1958- 1963 (1985). 127. Cox, J. A , . Jeng, A. Y . , Blumberg. P. M., and Taubcr, A. I . Comparison of subcellular activation of the human neutrophil NADPH-oxidase by arachidonic acid, sodium dodecyl sulfate (SDS), and phorbol myristate acetate (PMA). J . Immunol. 138, 1884-1888 (1987). 128. Seifert, R . . and Schultz, G. Fatty acid-induced activation of NADPH oxidase in plasma membranes of human neutrophils depends on neutrophil cytosol and is potentiated by stable guanine nucleotides. Eur Biochem. 162, 563-969 (1987). 129. Curnutte, J. T., Kuvcr, R., and Scott, P. J. Activation of neutrophil oxidase in a cell free system: Partial purification of components and characterization of the activation process. J. Biol. Chem. 262, 5563-5.560 (19x7). 130. Clark, R. A , , Leidal, K. G . , Pearson. D. W.. and Nauseef, W. M. NADPH oxidase of human -

+

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ncutrophils: Subcellular localization and characterization of an arachidonate activatable superoxide gcncrating system. J . B i d . C h m . 262, 4065-4074 ( I 987). 131. Curnuttc. J. T., Kuver, R . , and Babior, B . M. Activation of the respiratory burst oxidasc in a fully soluble systcm from human ncutrophils. J . B i d . Clwni. 262, 6450-6452 (1987). 132. Ames, F. F., and Nikaido, K. Two dimcnaional gel electrophorcsis of membrane proteins. Di<>rhrmistTv15, 616-623 ( 1976).

CURRENT TOPICS IN M t M B K A N k S AND TRANSPORT. VOLUME 35

Chapter 19

Activation of Lymphocytes by Lymphokines GORDON B . MILLS Oncology Research Toronto General Hospital Toronto, Ontario MSG 2C4, Canada

1.

Introduction

11. Lymphokines Are Nonspecific in Activity yet Function within a Specific Immune System 111. Pleiomorphic Function of Lymphokines IV. Protein Structure

V. VI.

VII. VIII.

IX.

X. XI.

Gene Structure Lymphokine Receptors A. IL-l Receptor B. IL-2 Receptor C. IL-6 Receptor D. IFN-y Receptor Lymphokines as Initiation and Progression Factors Cell Activation A. General Principles B. Signal Transduction Pathways Transmembrane Signaling by Lymphokines A. Antigen Reccptors and Lymphokines Likely Signal through Different Pathways B. Phospholipid Metabolism C. Protein Kinases D. N a + / H + Antiport E. G Proteins F. Cyclic Nuclcotides G . Receptor Internalization H . Oncogene Expression Cross-Talk between Antigen and Lymphokinc Rcccptors Summary References

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Copyright 0 1990 by Acadcniic Precx. Inc All rights of reproduction in any form rescrvcd.

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1.

INTRODUCTION

Following damage or activation, cclls rclcase a series of polypeptide hormones involved in intercellular communication. These cytokines stimulate the cell of origin (autocrine) or nearby cells by diffusing through the interstitial space (paracrine) or stimulate distant cells by traveling through the bloodstream (endocrine). Cytokine receptors appear to be expressed on every cell type within the body, and virtually all quiescent cells can be induced to proliferate or differentiate through thc action of cytokines. The cytokines that transfer information between cells of the immune system have been designated lymphokines or interleukins (1L). The lyniphokincs include IL- 1 through IL-7, colony stimulating factor for granulocytes and macrophages (GM-CSF), and interferon y (IFN-y). There are also a series of additional nioleculcs that modulate immune functions that, following further characterization, will likely be included in this family of “immunotransmitters.” The availability of purificd rccombinant materials, specific antibodies, and cDNA probes has made it clear that the lymphokines are not produced solely, or even primarily, by immune cclls. In addition, their actions are not restricted to lymphocytes but, instead, affect many different cells involved in the response to injury, inflammation, and infection.

II. LYMPHOKINES ARE NONSPECIFIC IN ACTIVITY YET FUNCTION WITHIN A SPECIFIC IMMUNE SYSTEM The immune system effectively clears a myriad of foreign antigens from the host. This is accomplished with a relatively low incidence of damage to host structures. The specificity of the immune system, which ensures that host cells will not hc dcstroycd, is generated through recognition of foreign antigen by specific cell surface receptors. Surface immunoglobulin and the T cell receptor (TCR) function as the specific antigen receptor for B and T lymphocytes, respectively. Activation of antigen receptors results in the transient expression of high affinity receptors for each of thc lymphokincs. Thcrcforc only antigcn-stiniulatcd lymphocytes will be able to respond to lymphokines. The only exception is the IL- 1 receptor which is expressed on resting lymphocytes; however, ILI function requires that cells be concurrently stimulated by antigen, thus ensuring that only antigen-stimulated cells will rcspond to lyniphokincs (Dower et ul., 1986; Hurme et ul., 1987). Once a lymphocyte has been stimulated to express lymphokine receptors, the duration and rnagnitudc of thc immunc rcsponsc is governed by the action of lymphokines (Cantrell and Smith, 1984). The ultimate response is dictated by the intracellular machincry of thc cclls stimulated through thcir antigen and lymphokine receptors. Lymphokines are mainly produced within a complex composed of antigen,

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antigen-presenting cells, and T and B lymphocytes. This complex is held together by binding of antigen to antigen receptors combined with interaction of cell surface adhesion molecules with their ligands. Adhesion molecules include CD4 and CD8 which react with major histocompatibility Class 11 and Class I, respectively, and CD2 which reacts with lymphocyte function associated antigen 3 (LFA-3). Although systemic levels of lymphokines are generally very low, within the antigen-recognition complex, lymphokine concentrations are likely very high. Lymphokines can be preferentially secreted towards stimulating cells (Po0 et al., 1988), thus ensuring focused delivery of high local concentrations to responding cells. Diffusion of lymphokines out of the binding complex is decreased by internalization and degradation of lymphokines bound to receptors. Systemic concentrations are also decreased by dilution in interstitial fluid and serum, combined with rapid degradation in liver and kidneys. High concentrations of the IL-2Ra chain of the IL-2 receptor complex are released into the serum following cell activation (Rubin et ul., 1985). These chains bind I L 2 and further decrease the concentration of free 1L-2. The concentration of released receptor is sufficiently high in some disease states, such as in bum patients (Teodorczyk-Injeyan et al., 1989), that it may contribute to immunosuppression. Therefore, most lymphokines function in an autocrine or paracrine fashion either on the cell of origin or on cells which are components of a cognate antigen recognition complex rather than as endocrine factors on distant cells. 1L-1 and 1L-6 can be readily detected in the circulation at concentrations sufficient to activate their receptors. IL-6 appears to be tightly bound to a2macroglobulin and perhaps to soluble IL-6 receptors, resulting in free hormone concentrations which are below those required to bind to significant numbers of receptor molecules (Matsuda et al., 1989). In contrast, IL-1 does not appear to be bound to a carrier protein in the circulation and thus likely functions as a true endocrine hormone. Within the human immune system, IL-I is most effective as a cell-surface molecule and is relatively inactive in its secreted form (Arnold et at., 1985; Hurme, 1987). Thus IL-I appears to function as a lymphokine primarily in its cell-associated form and functions as a endocrine hormone in its secreted form outside of the immune system.

111.

PLEIOMORPHIC FUNCTION OF LYMPHOKINES

Like many other biologic systems, the lymphokines are functionally redundant. Several different lymphokines can induce differentiation or proliferation of a series of different cells. In addition, most lymphocytes express an array of receptors for different lymphokines and can be stimulated to proliferate or differentiate through several of these receptors. The ability of several different lymphokines to stimulate a single cell type to perform the same function could be

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mediated by similar signals being transmitted by various receptors or represent a convergencc in activation pathways. The expression of an array of receptors allows for fine-tuning of responses with lymphokincs transmitting both positive and ncgative regulatory signals. Lymphokines which transmit a differentiation signal to a lymphocyte at one stage of diffcrcntiation can transmit a proliferative signal to the same lymphocyte at a different stage of difierentiation. Thus the same lymphokine could be involved both in clonal expansion and in increasing effector mechanisms.

IV. PROTEIN STRUCTURE

Several of the lymphokincs have been cloned, and although they are not structurally related members of a superfamily, thcy have several features in common. Most lymphokines are relatively small secreted glycopeptides with sizcc ranging from 12 to 30 kDa. With the exccption of 1L-1,lyniphokines are synthesized with a hydrophobic leader sequence which facilitates their secretion from the cell. IL-1 does not have a hydrophobic leader sequence, which may relate to its primary role in the immune system as a cell surface-associated molccule (Dower rt ul., 1986; Hurme el al., 1987). Although most lymphokines are glycosylated, this glycosylation is not necessary for function, at least in vitru, as recombinant products produced in Escherichiu coli are as active as the native hormone. Since dcglycosylated GM-CSF is actually 4- to 6-fold more active than the native form, glycosylation may intcrfere with receptor binding and decrease the spccific activity. In vivo, glycosylation may play a role in mediating stability or clearance. A common motif identified by the X-ray crystallography of IL-2 along with circular dichroism and computer modeling suggests that 1L-2, 1L-3, IL-4, IL-6, G-CSF, and GM-CSF, but not IL-1 and 1L-5,have a framework of four left hand core helices which may be required for stabilization of the molecules (Cohen et al., 1986; Brandhuber et ul., 1987; F. E. Cohen, personal communication).

V.

GENE STRUCTURE

Although the coding sequences of the lymphokines are not closely related, they may havc had a conimon origin. This is suggested by similarities in exonintron organization with most lymphokines having four or five exons (Miyajima et al., 1988), similar sequences in their upstream and downstream regulatory regions (Miyajima et al., 1988; Taniguchi, 1988), and spatial localization of several lymphokincs to a single section of human chromosome I I and murine

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chromosome 5 (Yang et al., 1988). The homology of upstream sequences likely relates to conservation of signaling processes and leads to coordinate regulation of mRNA for families of lymphokines in response to cell activation. Despite widely divergent coding regions, the regulatory regions of G-CSF and 1 L 6 have approximately 90% homology (Taniguchi, 1988). IL-2, IL-3, GM-CSF, and IFN-y are secreted following stimulation of murine THI cells, whereas stimulation of TH2 cells leads to production of IL3, IL-4, GM-CSF, and IFN-y (Taniguchi, 1988), suggesting that the upstream regions of these groups of lymphokine genes share common motifs. Similar sequences are found upstream of the genes for IL-2 and GM-CSF and of one of the two IL-2 binding chains (1L2Ra), indicating that similar signaling processes can induce both lymphokine secretion and increases in receptor expression. This is particularly apparent in that the tat protein of the adult T cell leukemia virus (HTLV- 1) increases production of both lymphokines and lymphokine receptors. This appears to be the result of activation of a single endogenous DNA binding protein which normally regulates expression of these genes. Cyclosporin A, a fungal derivative used to facilitate organ transplantation, inhibits the transcription of mRNAs for a series of lymphokines, including I L 2 , 1L-3, IL-4, IL-5, IFN-y, and GM-CSF. Whether this represents a direct effect of cyclosporin A on a common group of endogenous DNA binding proteins or on a common signal transduction pathway has not been determined. Nevertheless, it suggests that the regulatory processes for the lymphokine genes are highly conserved. Several of the lymphokine mRNAs, in particular those of IL- I , IL-2, GMCSF, and IFN-y, contain a common AU rich region in their 3' untranslated region (Caput ef al., 1986; Miyajima et al., 1988). This AU rich region likely renders the lymphokines sensitive to degradation by specific RNases and may contribute to the short half-life of lymphokine mRNAs. The enzymes involved in degradation appear to be relatively specific for the lymphokine family, thus providing a mechanism for altering mRNA survival time. Although many of the lymphokine genes are scattered throughout the genome, there is an intriguing cluster of genes located on the long arm of chromosome 5 in the human and on chromosome 11 in the mouse. These include those for the growth factors GM-CSF, IL-3, IL-4, IL-5, CSF-1, and acidic fibroblast growth factor. In addition, the P-adrenergic receptor and the receptors for CSF-l and PDGF are located near the lymphokine and growth factor genes. In the human, GM-CSF and I L 3 are linked in tandem with only 9 kb of DNA separating the genes (Yang et al., 1988). This suggests that coordinate regulation could easily occur through common enhancers and also that the genes may be related as distant members of a gene family. This relationship is even more remarkable for the PDGF and CSF- 1 receptors wherein the distance between the two molecules

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is approximately 800 bases and a separate regulatory region for the CSF- 1 receptor has not been identified. The intron and exon organization for these two genes also suggests that they arose through gene duplication.

VI.

LYMPHOKINE RECEPTORS

Lyrnphokine receptors can be dividcd into two broad groups: those that require activation for expression and those that are constitutively expressed. The IL-1 receptor is the only one which falls solely into the second group, and yet even IL-1 receptor levels are regulated with 1L-1 binding resulting in a transient decrease in receptor levels and glucocorticoids markedly increasing receptor levels (Matsushima et al., 1986; Akahoshi et ul., 1988). The IL-2 receptor falls in both groups with the intermediate affinity IL-2 receptor (IL-2RP) being expressed on a subset of resting CD8 lymphocytes, LGL, LAK, and NK cells (Nakamura et ul., 1989; Sharon et al., 1988). Expression of IL2RP renders the cells responsive to high concentrations of 1L-2 (Mills and May, 1987; Tsudo et [ J [ ., 1987; Kobb et al.. 1987; Smith, 1988). However, high affinity IL2 receptors do not appear to be present on most lymphocytes and only appear as a consequence of cell activation (Greene and Leonard, 1986; Smith, 1988). A single affinity binding site has been identified for IL-1 (Matsushima et al., 1986), IL-3 (May and Thle, 1986), IL-4 (Park et al., 1987), and IFN--y (Petska and Langer, 1987; Aguet eral., 1988) (K,of 10 - y to IO-"'M). In contrast, two binding affinities have been observed for IL-5, IL-6, and IL-7 ( K d of approximately 10- and 10-'M) (Yamasaki et al., 1988). Remarkably, three different IL 2 binding affinities are generated through 1L-2 interacting with two independent IL-2 binding molecules (IL-2Ra and IL-2RP) or with a complex of thc two molecules (Kd of 10 x, and lo-" M ,respectively; Greene and Leonard, 1986; Smith, 1988). Despite the presence of low numbers of IL-1, IL-3, and IL-6 receptors (Kd of lo-" M to lo-" M), cellular responses to these lymphokines can be detected at to 10 I s M (Shirakawa ct (]I., 1988; Kosoff et al., 1988), indicating that either a high affinity state also exists or other as yet unidentified binding molecules also exist. These binding molecules could either act like IL-2Ra and increase the affinity of the receptor complex or be part of an independent receptor complex as has been idcntified for PDGF (Matsui er al., 1989). In addition to binding to ligand-specific receptors, I L 3 and GM-CSF may also share an additional common receptor. Some lymphokine functions appear to be mediated through the high affinity state, while other may be transduced through the low affinity receptor. Engagement of the high affinity receptor for IL-2 is sufficient to induce cell proliferation, whereas much higher concentrations of 1L2 are required to differentiate B cells, N K , and LAK cells. Similarly, IL-6 induces proliferation of some cell lines at ~

19. ACTIVATION OF LYMPHOCYTES BY LYMPHOKINES

50 1

approximately 1000-fold lower concentrations than are required to alter immunoglobulin secretion and to induce acute phase responses, suggesting that different receptors may be involved. As yet, cloning of lymphokine receptors has not provided significant insight into their signal transduction mechanisms. The receptor for IL-1 and IL-6 are members of the immunoglobulin superfamily (Sims et al., 1988; Yamasaki et al., 1988). Both IL-2 binding chains and the IFN-y receptor have also been cloned (Greene and Leonard, 1986; Aguet et al., 1988). The intracellular portion of the receptors for IL- I , IL-2, IL-6, and IFN-y do not show significant homology with components of any known signal transduction pathways, which suggests that the ligand binding molecule likely associates with a signal transduction molecules or complex. Therefore, it seems likely that the lymphokine binding molecules function as a part of a receptor complex and that ligand binding and signal transduction are mediated by independent molecules. Each of the receptors that have been cloned will be discussed separately below. The others have only been identified by cross-linking studies. IL-3 binds to two different proteins of 65 and 140 kDa (May and Ihle, 1986; Isfort et al., 1988). The relationship of these two molecules to each other and to the single class of IL-3 binding sites remains to be determined. IL-4 binds to a 140 kDa molecule on human cells and to a 75 kDa molecule on murine cells (Park et al, 1987). IL-5 binds to two molecules of 50 and 110 kDa, which correlates with the observation of two binding affinities. Each of these molecules is expressed in low numbers on a wide range of cells, which correlates with the activity of these lymphokines on a variety of cell types both within and outside of the immune system.

A. 11-1 Receptor There are at least two different genes for IL- 1, IL- l a and IL- 1p, which encode proteins with less than 26% amino acid identity. In addition, posttranslational processing generates a series of active molecules ranging in size from 3 to 30 kDa. Yet only one 80 kDa receptor molecule has been identified on T cells, and this molecule binds 1L-1 produced from either gene locus with equal affinity (Dower et al., 1986). However, cross-linking studies suggest that the T cell and fibroblast IL- 1 receptors have different sizes perhaps as the result of differential posttranslational modification. There appear to be low numbers (< 1000 recepto 1 0 - l o M ) on tors/cell) of one major high affinity binding site (Kd of most lymphocytes. However, there are reports of a higher affinity binding site on some cells (Kd of 10- M ) . The IL-1 receptor contains a consensus sequence PKC phosphorylation site (Sims et al., 1988), and phosphorylation of other receptors by PKC alters binding affinity. Therefore, phosphorylation of the IL1 receptor may explain the different binding affinities. Responses to 1L-1 can be detected at as low as 10- I s M (Shirdkawa et a / . ,

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1988; Rosoff et al., 1988) on cells that do not express detectable IL-I receptors as measured by cross-linking (Rosoff et ul., 1988), indicating that an additional 1 L 1 receptor molecule or a much higher affinity form of the low affinity receptor must exist. Alternatively, it has been proposed that IL I may mediate some of its effects through a receptor-independent mechanism, The ILI receptor is constitutively expressed in low numbers on a wide range of cells, which is consistent with its pattern of activity. The level of receptor is regulated upon incubation with 1L- I , inducing a transient decrease in expression likely through internalization of occupied receptors (Matsushima et al., 1986). In addition, incubation with glucocorticoids can increase IL- 1 receptor expression by at least 10-fold (Akahoshi et al.. 1988). Cloning of the gene for the murine IL-1 receptor has demonstrated that this molecule is a member of the immunoglobulin superfamily with three extracellular immunoglobulin-like domains (Sims et al., 1988). However, the intracellular and transrnernbrane domains do not exhibit similarity with other molecules and have not shed light on the method of signal transduction. The peptide backbone codes for a 65 kDa protein with seven potential N-glycosylation sites. The cytoplasmic portion is longer than many members of the immunoglobulin superfamily with 217 amino acids. Expression of the receptor results in a single binding affinity of approximately lO-’M, which is similar to that observed on T lymphocytes.

B. 11-2 Receptor The high affinity IL-2 binding site on the surface of lymphocytes consists of a complex of at least two independent IL2 binding molecules along with a series of accessory molecules (Greene and Leonard, 1986; Tsudo e l al., 1986; Benedict et ul., 1987; Robb et ul., 1987; Teshigawara et al., 1987; Saragovi and Malek, 1987, 1988; Edidin et al., 1988; Herrnman and Diamantstein, 1988; Sharon et ul., 1988; Smith, 1988). The gene for the 55 kDa IL-2 binding molecule (IL-2Rol), also called TAC or P55, has been cloned and extensively characterized (Greene and Leonard, 1986; Smith, 1988). In contrast, less is known about the molecular structure of the 75 kDa 1L-2 binding molecule (IL-2RP), and although it has been cloned, the cDNA sequence has not shed any light on its signal transduction mechanism. In cross-linking studies, IL-2RP appears to be a doublet with molecular weight estimates ranging from 65,000 to 77,000 (Robb c’t d . , 1987; Teshigawara et al., 1987; Saragovi and Malek, 1987, 1988; Edidin et al., 1988; Sharon et al., 1988; Smith, 1988; Mills et al., 1989a). Cross-linking of IL-2 to cells engineered to express the cDNA for IL2RP also results in a doublet. Whether the doublet is the result of differential posttranslational modification or cross-linking of additional cellular proteins to the binding molecule is unknown. Three different binding affinities for 1L-2 can be observed on the same cell.

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These binding affinities are likely the result of IL-2 binding to the two independent binding molecules combined with formation of a tertiary complex of IL-2Ra, IL-2RP, and 1L-2 (Robb ct a / . , 1987; Teshigawara et al., 1987; Saragovi and Malek, 1987, 1988; Edidin et al., 1988; Sharon et al., 1988; Smith, 1988). IL-2Ra binds IL-2 with a relatively low affinity (Kd M ) and has a much slower rate of association and dissociation with IL-2 (Robb et a / . , 1987; Smith, 1988). IL-2RP has a slightly higher affinity for IL-2 ( K , of lopyM ) and has a much slower rate of association and dissociation with IL-2 (Robb et al., 1987; Smith, 1988). A tertiary complex of IL-2 with IL-2Ra and IL-2RP appears to form the high affinity complex ( K , 10- I ' M ) with its characteristic fast association and slow dissociation rates (Smith, 1988). In murine systems, IL-2Ra and P associate in the absence of 1L-2 (Saragovi and Malek, 1988); however, whether these molecules associate in the absence of IL-2 in the human is not clear (Robb et a / . , 1987; Teshigawara et a / . , 1987; Sharon et a / . , 1988; Smith, 1988). The number of high affinity sites on the cell surface appears to be dictated by whichever of IL-2Ra or P is expressed in the lowest amount. The role played by each of the IL-2-binding molecules in I L 2 function has not been fully delineated. Both IL-2Ra and IL-2RP cross the membrane once and have intracellular chains with no obvious tyrosine kinase activity nor similarity to other signal transduction molecules (Greene and Leonard, 1986; Smith, 1988). They are therefore unlikely to act directly as signaling molecules and likely function as part of a signaling complex. Although very high, nonphysiological concentrations of IL-2 are required, many of the functions of IL-2 can be performed by IL-2RP in the absence of IL-2Ra (Robb and Greene, 1987; Bich-Thuy et al., 1987; Kehrl et a / . , 1987; Siegel et ul., 1987; Tsudo et al., 1987; Smith, 1988). In cells that express IL2RP in the absence of IL-2Ra, interaction of IL-2 with IL-2RP is sufficient to induce IL-2 internalization (Fujii et al., 1986; Robb and Greene, 1987), activation of the Na / H antiport (Mills and May, 1987), phosphorylation of intracellular proteins (Ishii et al., 1988), and tyrosine phosphorylation of several substrates, including the p chain of the IL-2 receptor (Mills e t a / ., 1989a,b). Addition of sufficient 1L2 to saturate IL-2Ra in cells that express IL2Ra in the absence of IL-2RP is without effect in each of these systems. This suggests that IL-2RP, but not IL2RaU,interacts with the signal transduction component of the IL-2R complex. Some resting NK cells and lymphocytes appear to express IL-2RP in the absence of IL-2Ra and respond to IL-2 with increases in natural killer activity, oncogene expression, and expression of the 55 kDa IL-2 binding molecule, once again suggesting that the IL-2RP is sufficient for signal transduction (Robb and Greene, 1987; Bich-Thuy et al., 1987; Siegel et ul., 1987; Tsudo et a/., 1987; Smith, 1988). Whether IL2RP is able to induce cell proliferation is controversial; however, two studies have presented evidence that binding of IL-2 to IL-2RP is sufficient for cell proliferation (Smith, 1988; Tanaka et a / . , 1988). It is likely that it is the high affinity

-

+

+

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GORDON 6.MILLS

1L-2R complex that is responsive to the low quantities of IL-2 present in vivo, and IL-2Ra is required to form the high affinity complex. However, it is possible that concentrations of I L 2 sufficicntly high to bind to and activate IL-2Rp in the absence of IL2Ra are present within thc activation complexes described below.

C. 11-6 Receptor The IL-6 receptor is also a member of' the immunoglobulin supcrfamily. The protein is heavily glycosylated, giving a final size of 80 kDa (Yamasaki et d,, 1988). The intracellular portion of the molecule is approximately 82 amino acids and docs not show any homology with other signal transduction systems, Transfection of a single cDNA into Jurkat cells results in expression of two binding affinities of l o p y and 10- I I M , which is similar to that on normal cells (Yamasaki et a / . , 1988). Therefore, the two different binding affinities likely arise as thc result of posttranslational modification, such as phosphorylation, of a single binding molecule. Following binding of IL-6 to its receptor, it associates with a 130 kDa molecule which is likely involvcd in signal transduction. This interaction does not require eithcr the intracellular o r transmembrane domain of thc IL6 receptor, and soluble receptors bind to this putative signal transduction molecule in the presence of IL6. A complex of the secreted IL-6 binding moleculc, IL-6, and the 130 kDa molecule inhibits the growth of some cells, suggesting that the complex is functional (T. Kishinioto, personal communication).

D. IFN-y Receptor Cross-linking studies with IFN-y have identified a varicty of binding proteins with molecular siLes from 70 to 161) kDa (Petska and Langer, 1987). Studies with anti-receptor antibodies indicate that the receptor is approximately 90,000 Da and that a smaller degradation product may be present (Novick et af., 1987). Cloning of the cDNA for the IFN-y receptor suggests its protein backbone encodes 54 kDa and that the remainder of the 90 kDa is made up of sugar residues at multiple N- and 0-linked glycosylation sites (Aguet et al., 1988). The intracellular domain consists of 222 amino acids, which could play a role in signal transduction. Similar to the other lymphokinc receptors, the cDNA sequence has not provided information as to the mechanism of action of the IFN-y receptor. Transfection of the cDNA for human IFN-y reccptor into murine cells resulted in a single class of high affinity receptors; however, the receptor was not functional, suggcsting that there may be species-specific mcmbcrs of the receptor complex (Aguet et a / . , 1988). Only a single class of receptors is identified in binding studies with an aRinity of 10-lo to 10- M . Most cells express low

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levels of the receptor with less than 1000 sites on resting cells; however, some tumor and cell lines exhibit higher levels.

VII. LYMPHOKINES AS INITIATION AND PROGRESSION FACTORS Most of the cells within the body are in the Go or resting stage of the cell cycle. In order to divide, cells must first enter G , and then receive signals allowing them to enter S phase and transit the cell cycle. In addition, there seem to be several points within the G , phase of the cell cycle wherein the cell can respond to exogenous signals and progress, rest, or return to Go. The length of the G, and G , phases of the cell cycle are variable; however, once the cell is committed to S phase, transit of the cell cycle proceeds with a relatively constant interval. Thus there are two major regulatory stages within the cell cycle: entry in Go and entry into S phase. Cells that are resting in Go express receptors for a limited number of ligands or growth factors. Following stimulation with the appropriate ligand, cells become “competent” to respond to a second series of factors by expressing a new set of cell surface receptors. Activation of these newly expressed receptors induces “progression” through the cell cycle. In the immune system, a resting lymphocyte is stimulated by binding of antigen either to its T cell receptor complex or to surface immunoglobulin. In some cases, lymphokine receptors are present on the surface of resting cells and can interact with signals transmitted through the antigen receptor to modulate the eventual response. These initiation signals lead to the synthesis of lymphokines and lymphokine receptors on stimulated cells. The progression signal is then normally given by lymphokines. IL-2, under some circumstances, may induce both competence and progression. High concentrations of 1 L 2 can bind to and stimulate IL-2RP, which is present on some resting cells (Sharon et ul., 1988; Nakamura et al., 1989; Smith, 1988). This is sufficient to increase NK and LAK activity and to induce cell migration. In addition, stimulation of IL-2RP increases expression of I L - ~ R ~resulting L, in formation of high affinity binding complexes. In the continuing presence of low concentrations of IL-2, the activated cells will proliferate (Smith, 1988). In order to allow for more definitive regulation of cell division, it is likely that the competence and progression signals are mediated by somewhat different pathways. In addition, at each step, it is likely that cross talk between positive or negative regulatory signals fine-tune the eventual response. Much attention has been focused on the immediate early events following the binding of lymphokines to their receptors. It is clear that activation of lymphokine receptors rapidly transmits transmembrane signals which can induce

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GORDON B. MILLS

changes in intracellular processes within seconds and alterations in gene expression within minutes. Nevertheless, the ligand, at least in the case of IL-2, IL-3, mitogenic lectins, and even phorbol esters and ionophores, which bypass cell surface events, must be present outside the cells for a period of hours (Cantrell and Smith, 1984; Sekaly et al., 1984; Ihle and Weinstein, 1986; Kumagai et d,,1987). This suggests that a cell that enters an area of high lyrnphokine concentration, such as that occurring in an activation complex, will not be activated to proliferate unless it remains in contact with the lymphokine for a number of hours. This decreases the possibility that cells that recognize one antigen, perhaps a host antigen, will respond inappropriately to lymphokines produced in response to a second antigen. Prolonged incubation with I L 2 is required to commit the cells to proliferate, indicating that IL-2 acts as a progression factor (Sekaly et a l . , 1982, 1984; Cantrell and Smith, 1984). Earlier studies suggested that an 8-10 hr incubation with 1L2 was sufficient to induce cell cycle progression. However, when IL-2 is removed from the cells by washing at low pH, the duration of incubation in IL-2 required to commit cells to L3H]thymidine incorporation is extended to at least 12 hr (Zhang and Mills, 1989). JL-2 must be present throughout the 12 hr incubation period. Even a 15 niin incubation without 11-2 returns the cells to the quiescent state, and they require a furthcr 12 hr incubation to initiate ["]thymidine incorporation (N. Zhang and G. B. Mills, unpublished observations). This suggests that IL-2 must transmit a continuous signal to cells and that accumulation of this signal is required to cause the cells to enter S phase. It also suggests that mechanism exist that rapidly terrninatc the activation process and return the cell to the resting state.

VIII. CELL ACTIVATION

A. General Principles Binding of ligands to their cell surface receptors rewlts in a conformational change which transmits information across the cell membrane, This information must then be transmitted as second messengers to the nucleus to initiate the processes required for cell proliferation and differentiation. In some receptors, such as those for EGF, CSF-I, and PDGF, the same molecule appears to be involved in binding of ligand and in intracellular second messenger generation. In others, such as the insulin receptor, the ligand binding domain does not even extend into the cell membrane, and the transmembrane signal is transmitted by an associated molecule. The lymphokine receptors so far characterized fall exclusively into this second group in so far as ligand binding and signal transduction appear to be encoded by different molecules.

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6. Signal Transduction Pathways Quiescent cells, of many different types, can be stimulated to proliferate by a variety of hormone-like polypeptide growth factors which bind to specific cell surface receptors (Deuel, 1987; Yarden and Ullrich, 1988). One of the earliest consequences of receptor activation is an increase in phosphorylation of tyrosine residues (Deuel, 1987; Yarden and Ullrich, 1988). Several growth factor receptors possess a growth factor-activated tyrosine kinase domain, while in others the tyr kinase domain resides in a molecule associated with the receptor (Deuel, 1987; Hanks et a / . , 1988; Yarden and Ullrich, 1988). In addition, many oncogenes encode proteins that have a tyrosine kinase domain (Hunter, 1984; Bishop, 1985; Hanks et al., 1988). In both growth factor receptors and oncogenes, the tyrosine kinase activity is required for cellular proliferation or transformation, indicating that tyrosine phosphorylation plays an important role in regulating cell proliferation (Yarden and Ullrich, 1988; Chou et al., 1987; Glenney et al., 1988; Chen et al., 1988). Several tyrosine kinases, including the insulin receptor, ABL, FGR, LCK, LTK, SRC, TCK, and FYN, are expressed in lymphocytes (Ben-Neriah and Bauskin, 1988; Koga et ul., 1988; Reed and Nowell, 1988; Veillette et a/., 1988). LTK is preferentially expressed in lymphokine-responsive lymphocytes, suggesting it may play a role in lymphokine signal transduction (Ben-Neriah and Bauskin, 1988). Some growth factor receptors also activate intracellular serine/threonine kinases including the family of phosphotransferases collectively designated protein kinase C (PKC) (Bell, 1986; Kikkawa and Nishizuka, 1986; Berridge, 1987; Nishizuka, 1988). These kinases were initially classed as PKC because of a common dependence on diacylglycerol, phospholipid, and calcium for activity (Kikkawa and Nishizuka, 1986). Some subforms of PKC are relatively calcium insensitive, and others can be activated by arachidonic acid in the presence or absence of calcium (Nishizuka, 1988). Activation of PKC alters cell proliferation, and overexpression of PKC either by intracellular injection or by genetic engineering induces characteristics of malignant transformation (Housey et al., 1988; Persons et al., 1988). In addition, many activators of PKC are potent tumor promoters (Kikkawa and Nishizuka, 1986). Phosphatidylinositol hydrolysis by phospholipase C (PLC) is also implicated as a rapid, primary event in the activation of cells by growth factors (Berridge, 1987). Activated PLC hydrolyzes membrane phosphatidylinositols (PI), producing diacylglycerol (DAGj and inositol phosphates (1P). Both products are putative second messengers, with DAG activating PKC and IP derivatives triggering release of Ca2 from intracellular stores and perhaps opening membrane calcium channels (Berridge, 1987). Increases in cytosolic free calcium ([Ca2+ I i ) stimulate the calcium/calmodulin-dependentprotein kinases and increase the affinity of PKC for diacylglycerol, thus indirectly increasing serine and threonine +

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GORDON B. MILLS

phosphorylation (Bell, 1986; Kikkawa and Nishizuka, 1986; Berridge, 1987; Nishizuka, 1988). Many rcccptors, including some with an intrinsic tyr kinase domain, interact with a family of signal transduction molecules called G proteins (Gilman and Casey, 1988; Neer and Clapham, 1988). These G proteins are activated by GTP and inhibited by conversion of GTP to GDP by intrinsic GTPase activity. The function of G proteins has been probed in intact cells with pertussis and cholera toxins, which inhibit or activatc specific G proteins through ADP-ribosylation, and in permcabilized cells with nonhydrolyzable GTP and GDP analogs, which stimulate or inhibit, respectively, all known G proteins (Gilman and Casey, 1988; New and Clapham, 1988). G proteins have been dernonstrated to regulate adenylatc cyclase, phospholipase C, and some membrane ion channels (Cilman and Casey, 1988; Neer and Clapham, 1988). In addition to the immediate consequence of activation of growth factor receptors at the cell surfacc, following ligand binding many of the receptors are internalized at an increased rate. This could act as a signal termination mcchanism, resulting in fewer receptors on the cell surfacc. However, in some cases, receptor internalization is required for function. Clearly this is the case for the transferrin receptor which functions to internalize transfcrrin and iron. This may also be the case in other systems where, for example, the insulin and epidermal growth factor reccptor kinascs remain active following internalization and phosphorylate specific substrates within the cell (Cohen and Fava 1985; Klein et ul., 1987).

IX. TRANSMEMBRANE SIGNALING BY LYMPHOKINES

A. Antigen Receptors and Lymphokines Likely Signal through Different Pathways In the immune system, the initiation signal for proliferation is provided to resting lymphocytes through activation of their antigen receptors. As described in othcr chapters in this volume, this appears to be a highly conserved proccss with both surface immunoglobulin and the T cell receptor activating phospholipasc C, which results in increased production of inositol phosphates and DAG. These second messengers, in turn, increase [Ca2+Ii and activate PKC. Although phospholipase C activation appears to play a major role in T cell activation, phospholipase C, [Ca2+ 1,. and PKC-independent pathways of T cell activation also exist (Gelfand et ul., 1985, 1986a,b, 1987a,b; Mills e t a ! . , 1985a, 1989c,d; Stanley et al., 1989). In addition to the stimulation of phospholipase C, activation of surface immunoglobulin and the T cell receptor increases phosphorylation of a series of intracellular substrates. Activation of the T cell receptor in murine systems (Samelson et ul., 1986, 1987) and in human systems (Fig. I),

- 200

- 92

I

-

68

-

43

- 29

FIG. 1 , Western blotting of PBL blasts with monoclonal anti-ptyr antibodies. Human peripheral blood lymphocytes (PBL) were rendered IL-2 sensitive by activation with PHA for 3 days followed by culture for 8 days in IL-2. Cells were returned to a resting state by culture in thc absence of 1L-2 for 2 days. As indicated in Table I , these cell( can be induced to proliferate either by IL-2 o r PHA. Cells were incubated with either IL-2 (1000 U) or PHA (10 bgiml) for 10 min and lysed by addition of boiling Laemli buffer supplemented with SO pkfZnCl and 100 Mvanadate. Cells were separated by SDS-PAGE and transferred t o nitrocellulose. The nitrocellulose was probed with the PY20 monoclonal antiphosphotyrosine (anti-ptyr) antibody (kind gift of M. Kamps) as described (Kamps and Sefton, 1989). Essentially identical results were obtained with a polyclonal anti-ptyr antibody that was affinity purified in this laboratory from serum of rabbits immunized by Drs. Lettwin and Pawson as described (Kamps and Sefton, 1988). The detection of proteins in the western blots is inhibited by addition of phosphotyrosinc, but not phosphoserine or phosphothreonine, to the anti-ptyr incubation mixture. It is also ATP dependent and reversed hy incubation of cell lysates with alkaline phosphatase, which indicates that the antibodies are detecting increases in tyrosine phosphorylation.

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GORDON B. MILLS

as well as the immunoglobulin receptor (G. B . Mills unpublished observations), incrcases tyrosine phosphorylation of a series of intracellular substrates. Kinase activity, in particular tyrosine phosphorylation, may represent one of the potential phospholipase C-independent activation pathways. In contrast, activation of lymphokine receptors does not appear to lead to activation of phospholipase C and resultant production of inositol phosphates or diacylglycerols through breakdown of membrane phosphatidylinositols. Furthermore, lymphokinc action is not dependent on changes in [CaL Iias appears to be the case fur the antigen receptors. This is not surprising, in that lymphokine signals collaborate with signals transmitted through antigen receptors and therefore would be expected to utilize different intracellular signal transduction pathways. This is also supported by the observation that although mitogenic lectins and lyrnphokines both stirnulatc MYC expression, different promoters are utilized, suggesting that different signaling pathways and endogenous binding proteins are stimulated (Broome et d.,1987). +

B. Phospholipid Metabolism I . P~IOSPIIATIDYLINOSITOL IIYDROLYSIS A N D INCRF.ASES I N [CaZ+Ii

In most reports, IL-l (Abraham e t a / . . 1987; Georgilis et al., 1987; Rosoff et a / . , 1988; Kester et d.,1989; Mills et al., 1989e), IL-2 (Weiss et al., 1984; O’Flynn el c d . , 1985; Mills et al., 1985b, 1986, 1988a; Larsen rt u / . , 1986; Dunn et al., 1987; Gelfand et ul., 1985, 1986a,b, 1987a,b; Kozumbo et a / . ,

1987; LeGrue, 1987, 1988; Otten et al., 1987; Wickremasinghe et al., 1987; Wolff, 1987; Harris et al., 1988; Tigges et ul, 1989), 11.-3 (Whctton et al., 1986), IL-4 (Justement et d., 1986; Mixguichi e f a / . , 1986), IL-5 (J. ‘hkatsu, personal communication). I L 6 (T. Kishimoto, personal communication), and IFNs (Mills et a / . , 1985~)do not activate phospholipase C as indicated by increases in phosphatidylinositol turnover, phosphoinositol production, or [Ca2 Ii. However, several reports have suggested that IL-1 (Godfrey rt a / . , 1987; Wijelath e l ul., 1988) and IL2 (Gearing et t i / . , 1985; Farrar et a l . , 1986; Bonvini et u l . , 1987; Utsunomiya et al., 1986; Rossio et al., 1986; Bourguignon et a / . , 1988) may activate this pathway under some circumstances. IFN-y has also been reported to increase diacylglyccrol and inositol phosphate production which would be compatible with activation of phospholipase C; however, increases in [Ca2+l i were not detectable in the same cells (Yap et al., 1986). The reasons for the discrepancy between the different laboratories are not clear, but they may relate to contaminants in the lymphokine preparations or the use of different responding cells and culture conditions. Nevertheless, it is clear that lymphokine-induced proliferation can occur in the absence of activation of PLC, changes in PI hydrolysis, production of phosphoinositol and DAG, and changes in ICa2+Ii. +

19. ACTIVATION OF LYMPHOCYTES BY LYMPHOKINES

51 1

The role of changes in [Ca2 1, has been investigated by depleting extracellular calcium and preventing increases in [Ca2+Ii.When increases in [Ca2+Ii are prevented, 1L-2 continues to induce cell proliferation (Weiss et al., 1984; Mills et a/., 1985b; Larsen et ul., 1986; Dunn et al., 1987) and IL-4 is able to increase Ia expression (Mizuguchi et ul., 1986; Dennis et al., 1987). IFN-y-induced cytotoxicity is also not dependent on extracellular calcium. However, depleting [Ca2+Iito low levels by chelating the intracellular calcium by loading cells with quin2 in low calcium medium does prevent this effect of IFN-y (Celada and Schreiber, 1986). This is likely due to a decrease in [Ca2+Ii rather than a result of preventing increases in [Ca2 Ii. IL-2 increases association of Ca2 with the cell membrane without inducing an increase in [Ca2 Ii (Larsen et al., 1986; Mills et al., 1988a). IL-2 has also been reported to increase 45Ca2+ association with the cell, although it was not determined whether this was due to membrane association or cellular uptake (Johnson el al., 1985). Nevertheless, preventing association of Ca2+ with the cell membrane does not alter cell proliferation (Weiss et al., 1984; Mills et ul., 1985b; Larsen et d., 1986; Mills t't al., 1988a), indicating that this process is the result of cell activation rather than an initiating event. +

+

+

+

2. HYDROLYSIS OF O T H E R MEMBRANE LIPIDS 1L-1 has been demonstrated to increase diacylglycerol production without concomitant increases in phosphoinositol production, indicating that the diacylglycerol originates from a lipid other than phosphatidylinositol (Rosoff et a/., 1988; Kester et al., 1989). Two reports disagree on the origin of diacylglycerol, suggesting that phosphatidylcholine (Kosoff el a / . , 1988) or phosphatidylethanolamine (Kester et a ( . , 1989) is the source of diacylglycerols. DAG production from phospholipids other than phosphatidylinositol may explain the activation of PKC observed with 1L-I and other lymphokines in the absence of phosphatidylinositol hydrolysis and increases in [Ca2'I,. IL- I (Godfrey et al., 1987; Levine and Xiao, 1985), but not I L 2 (Chang et al., 1986; Mills et al., 1985a, 1988a), IL-3 (Chang et al., 1986), or IFN-y (Boraschi et al., 1986), induces rapid and marked increases in arachidonic acid release from cells. I L I has been reported to induce arachidonate release from phosphatidylcholine, phosphatidylserine, and phosphatidylinositol, suggesting that it activates phospholipase C as well as phospholipase A, (Godfrey et a / ., 1987). In other reports, IL- 1 activates phospholipase A,, increasing PGE, production primarily from phosphatidylethanolamine (Chang et al., 1986). Since arachidonic acid can activate PKC (Nishizuka, 1988), this may explain, in part, the reported effects of these lymphokines on PKC. IFN-y markedly inhibits arachidonic acid release and formation of lipoxygenase products while increasing the release of cyclooxygenase products (Boraschi et al., 1985). Both 1L-1 and IL-2 increase production of 5 and 15 monohydroxyeicosatetrae-

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GORDON 8.MILLS

noic acids (HETE), lipoxygenase derivatives of arachidonic acid (Farrar and Humes, 1985). Inhibition of lipoxygenase decreased 1L 1-induced 1L-2 production and IL2-induced proliferation and interferon secretion, suggesting that lipoxygenase metabolites are required for these processes (Johnson and Torres, 1984; Farrar and Humes, 1985). Leukotrienes, which are products of the lipoxygenase pathway, can induce interferon production but not proliferation of IL-2dependent cells (Johnson et al., 1985; Farrar and Humes, 1985), which suggest that different pathways may be involved in IL2-induced production of interferon and proliferation. PGE, markedly inhibits 1L2 induced proliferation and has opposite efiects on CAMPproduction from IL-2, suggesting that this prostaglandin may bc a physiological inhibitor of IL-2 function (Beckner and Farrar, 1986). However, IL-2 has been demonstrated to increase production of both PGE, and thromboxane, which is difficult to reconcile with the inhibition of IL-2-induced proliferation by PGE,.

C. Protein Kinases Many of the lyinphokines have been reported to increase phosphorylation of intracellular substrates, which m y constitute the main cell activation pathway that mediates the effects of lymphokines. The role of phosphorylation in gencral and, in particular, phosphorylation of specific substrates in lymphokine-induced cell proliferation has not yet been deterniined. 1 . SERINE-'rHKEONINE

PHOSPHORYLATION

IL-1 (Matsushima et d . ,1987; Bird and Saklatvala, 1989), IL-2 (Mire et al., 1985; Farrar et al., 1986; Kohno et ul., 1986; lshii ct uZ., 1986, 1987, 1988; Gaulton and Eardley, 1986; Evans et ul., 1987a; Evans and Farrar, 1987a,b; Benedict et d., 1987; Le Gros vt al., 1987; Mills et d . , 198th; Kayhel et al., I988), 1L-3 (Evans ct uI., 1987a), J L 4 (Justement Pt a / ., 1986), IFN-y (Krust et al., 1984; Samuel et al., 1984), and GM-CSF (Evans et al., 1987a) induce phosphorylation on serine or thrconine of various substrates in human, murine, and rat cells. 1L-2-induced phosphorylation of intracellular substrates can be mediated by the IL-2RP (Ishii et ul., 1988), suggesting, once again, that this molecule, in the absence of IL-~Ku,is sufficient for signal transduction. Although most of the substrates for serine-threonine phosphorylation have not been identified, IL- 1 induces phosphorylation of the epidermal growth factor receptor (Bird and Saklatvala, 1989); IL-2 induces phosphorylation of the S6 ribosomal protein (Evans and Farrar, 1987a) and the cx (Gaulton and Eardley, 1986) and p chains of the 1L-2 receptor (K. Sugamura, personal communication); and IFN-y induces phosphorylation of P , , a ribosome-associated protein, and eiF-2u (Samuel C I d . , 1984).

19. ACTIVATION OF LYMPHOCYTES BY LYMPHOKINES

513

2. PROTEINKINASEC u. Lymphokines M ~ 7 yActivute PKC. IL-2 (Farrar and Anderson, 1985) and IL-3 (Farrar et ut., 1985; Whetton et al., 1988) have been reported to induce a rapid translocation of protein kinase C from the cytosol to the cell membrane, a process that is required for its activation. However, other studies have failed to detect 1L-2-induced PKC translocation (Takeshita et ul., 1988; G . B. Mills, unpublished observations). IL-4 does not appear to induce translocation of PKC (Justement et al., 1986). 1L-1 does not induce rapid translocation of PKC (Abrdham et al., 1987) but, similar to IFN-y, may induce a delayed translocation (Ostrowski et al., 1988). IFN-y has been reported to induce a rapid, transient increase in diacylglycerol production (Yap ct ul., 1986); however, this is not associated with a rapid increase in PKC translocation (Ostrowski et al., 1988). IFN-y may increase total cell concentrations of PKC which would also increase PKC activity (Hamilton et ul., 1985). Inhibitors of PKC or chelating calcium to low levels, which would inhibit some forms of PKC (Nishizuka, 1988), also interferes with IFN-y activity (Celada and Schreiber, 1986). b. Lymphokines und PKC Activutors Have Similar Effects on Cells. In several systems, phorbol esters and lymphokines have been reported to induce similar or overlapping effects on biochemical events within the cell. For example, phorbol esters and 1L-2 induce phosphorylation of similar substrates in membrane preparations (Evans and Farrar, 1987a,b) and in intact cells (Gaulton and Eardley, 1986; Ishii et al., 1986, 1987, 1988; Kohno et al., 1986), and phorbol esters and IL-3 induce similar patterns of phosphorylation (Evans et al., 1987a). Both 1 1 2 and phorbol esters also decrease phosphorylation of a similar substrate (Rayhel et al., 1988). IL-2 and phorbol esters induce phosphorylation of a series of nonoverlapping proteins, suggesting at least some of their effects are unique (Gaulton and Eardley, 1986; Evans and Farrar, 1987a; Rayhel et al., 1988). Both IL-2 and phorbol esters decrease CAMPproduction and inhibit agonist-induced increases in CAMP(Beckner and Farrar, 1986, 1987). Purified PKC can directly inhibit agonist-induced increases in CAMPin membrane preparations, indicating that PKC may be sufficient for such activity (Beckner and Farrar, 1986), and IL-2 only inhibits CAMPproduction under conditions wherein PKC can be activated. c. PKC Activators Cun Stirnulute Lymphokine-Sensitive Cells. Phorbol esters can replace 1L-1 in the stimulation of IL-2 production in several systems (Abraham et al., 1987; Mills et ul., 1989d). Some IL-2 and 114-dependent lines can be induced to proliferate transiently by activators of PKC (Kim et al., 1986; Mills et a/., 1988a). In contrast, 1L-%dependent lines are inhibited by phorbol esters (Ihle and Weinstein, 1986). Although the 1L-2-dependent lines will grow indefinitely in the presence of IL-2, phorbol esters will, in most cases, only

514

GORDON B. MILLS

support transient proliferation. In addition, several 112-dependent cell lines do not proliferate in response to phorbol esters (Albert et al., 1985; Kim et al., 1986; Koyasu et al., 1987; Mills et a/., 1988a). At least one cell line will proliferate for prolonged periods of time in response to either 1L2 or phorbol esters (Takeshita et ul., 1988). Prolonged phorbol ester treatment of this cell line results in a marked depletion of PKC activity, and transient incubation with phorbol esters induces translocation of PKC from a cytosolic fraction to a membrane-associated fraction. In contrast, I L 2 does not induce PKC translocation nor does it decrease total cellular PKC activity in this cell line (Takeshita et a/., 1988). This suggests that PKC activation and I L 2 activation are stimulating cells through independent pathways. That 1L-2 and phorbol esters stimulate cells through different mechanisms is also supported by the inhibition of phorbol esterbut not 112-induced proliferation by increases in intracellular CAMPconcentrations (Takeshita et a / . , 1988). It is possible that the increased proliferative response to phorbol esters is not due to activation of PKC but rather to the depletion of PKC (Takeshita et ul., 1988) and a resultant deercase in PKC activity. This possibility is supported by evidence that PKC activation limits rather than stimulates T cell function (Mills et ul., 1989d). Other studies have also suggested that phorbol esters and l L 2 may stimulate proliferation through different mechanisms. lL-2 at most stimulates a transient translocation and activation of PKC (Farrar and Anderson, 1985), whereas phorbol esters induce marked and prolonged translocation and degradation of PKC. However, although prolonged activation of PKC by poorly metabolized phorbol esters can induce proliferation of some 1L-2-dependent cell lines, diacylglycerol analogs, which, similar to IL-2, induce transient activation of PKC, do not induce proliferation of the same lines. In addition, in at least some systcms, phorbol esters do not induce proliferation directly but rather increase the endogenous production of lymphokines which then signal through activation of their receptors (Albert et al. , 1985; Kim et al., 1986; lsakov et a1. , 1987; Kumagai et u/., 1988). However, at least in some cells, phorbol esters induce cell proliferation without inducing lymphokine production (Isakov et al., 1985; Takeshita et ul., 1988). d . PKC Activation Is Not Obligatory f o r Lymphokine Function. Several studies have attempted to test whether lymphokines induce cell proliferation through activation of PKC and whether PKC activation is required for lymphokineinduced cell proliferation. An initial study indicated that H-7, an inhibitor of PKC, inhibited IL-2-induced cell proliferation (Clark el al., 1987). Higher concentrations of H-7 are required to inhibit PKC activity in intact cells than are required to inhibit cell proliferation, suggesting that H-7 is likely inhibiting cell proliferation through non-PKC mediated effects (Mills et ul., 1989d). In contrast, IL-2 induces proliferation in cells treated with sphingosine and

515

19. ACTIVATION OF LYMPHOCYTES BY LYMPHOKINES

staurosporine, potent inhibitors of PKC, wherein PKC was demonstrated to be inhibited and phorbol esters no longer induced cell proliferation (M. Hill and G . B . Mills, unpublished observations). In addition, IL- 1- and IFN-y-induced activation of the N a+/ H+ antiport is not prevented by inhibitors of PKC ( 0 s trowski et al., 1988). PKC can be functionally depleted from cells by incubation with high concentrations of phorbol esters. This does not prevent 1L1-induced IL-2-production (Mills et al., 1989c), IL-Zinduced cell proliferation (Mills et af., 1988a; Valge et a/., 1988), or 1L-1- or IFN-y-induced activation of the Na / H antiport (Ostrowski et al., 1988). Depletion of PKC also does not prevent IL I-induced phosphorylation and decreased binding by the epidermal growth factor receptor (Bird and Saklatvala, 1989). In somatic cell mutants that lack functional PKC, IL-2 (Mills et al., 1988a,b) and IL-4 (Paetkau and Mills, 1989) can induce cell proliferation, and IL-2 activates the N a + / H + antiport (Mills et a / . , 1988a,b). Since these cells do not require use of inhibitors or incubation with high concentrations of phorbol esters both of which may have effects on other cell processes, it appears clear that activation of PKC is not required for IL-2- or IL4-induced cell proliferation. Nevertheless, this does not suggest that activation of PKC may play a role in other actions of these lymphokines nor does it suggest that PKC does not modify some of the effects of these lymphokines. Taken together, the evidence suggests that although lymphokines and phorbol esters may have similar effects on some cells, these effects are mediated through different biochemical pathways. In addition, although some lymphokines may activate PKC, this activation is not an obligatory requirement for cell function, at least as far as 1L1, IL-2, IL4, and IFN-y are concerned. +

+

3. TYROSINE PHOSPHORYLATION As described previously, tyrosine phosphorylation plays an important role in cell proliferation and differentiation induced by a number of growth factors and oncogenes. Although none of the characterized lymphokine receptors contains a tyrosine kinase domain, IL-2 (Fig. 1; Bourguignon et a / . , 1988; Saltzman e t a / . , 1988; Morla et al., 1988; Mills et al., 1989a,b; Paetkau and Mills, 1989), IL-3 (Ferris et a / ., 1988; Morla et al., 1988), and 1L-4 (Morla et al., 1988; G . B. Mills unpublished observations) induce tyrosine phosphorylation of a series of cellular proteins. In a cell line that can proliferate in response to IL-2, 1L3,or IL-4, these ligands induced tyrosine phosphorylation of an overlapping series of proteins, suggesting that both parallel and converging pathways are involved (Morla eta/., 1988). In contrast to serine and threonine phosphorylation where phorbol esters and IL-2 demonstrate overlapping effects, phorbol esters do not induce tyrosine phosphorylation of substrates in the IL-2-sensitive cells studied (Saltzman et a/.,

516

GORDON B. MILLS

1988; Mills et al., 1989b). We have not been able to detect interferon- or I L I induced tyrosine phosphorylation (G. B . Mills, unpublished observations), suggesting that these lyniphokines likely signal through a diRerent pathway. As demonstrated in Fig. 1, incubation of human T lymphocytes, which have been rendered sensitive to IL-2 by activation with PHA and culture in 1L-2, with purified recombinant 1L-2 (Cetus) increases tyrosine phosphorylation of substrates of 92 and 78 kDa and a broad band between 70 and 75 kDa as indicated by western blotting with either polyclonal or monoclonal anti-phosphotyrosine antibodies. This pattern is quite different from that reported in other studies (Bourguignon et al., 1988; Saltzman ef al., 1988; Morla et ul., 1988) likely as the rcsult of the use of human cells in contrast to murine cells and the use of diffcrcnt anti-phosphotyrosine antibodies which recognize phosphotyrosine in different cnvironments. Phytohemagglutinin, likely through stimulation of the T cell receptor complex, increases tyrosine phosphorylation of substrates of 100, 90, 7 5 , 70, 68, 35 (Fig. I), and 26 (not illustrated) kDa. This suggests that activation of lymphocytes by 1L-2 and PHA activates different intraccllular kinases. Although neither the IL-2 receptor nor the TCR encode a tyrosine kinase, they may be associated with an intracellular tyrosine kinase similar to the association of CD4 and CD8 with the LCK tyrosine kinase (Rudd et al., 1988; Veillette ef ul., 1988). However, it is also possible that the IL-2 receptor and the TCR are inactivating specific intracellular tyrosine phosphatases such as the T200 protein. IL-2 induces a similar pattern of phosphorylation in YT 2C2 cells, which express IL-2KP in the absence of IL-2Ra, indicating that II.-2RP is sufficient to mediate tyrosine phosphorylation (Mills et ul., 1989a,b; Paetkau and Mills, 1989). Addition of IL-2 to immunoprecipitated 1 L 2 receptor complexes induces phosphorylation of a 78 kDa molecule (Benedict et al., 1987; Mills et al., 1988a; Paetkau and Mills, 1989), which is the same size as the predominant IL-2tyrosine phosphorylation substratc in intact cells (Fig. l), and suggests that the kinase activity may be part of the 1L2 receptor complex. Insulin, epidermal growth factor, CSF- 1 , and platelet-derived growth factor induce tyrosine phosphorylation of thcir receptors. As indicated in Fig. 1, 1L-2 increases phosphorylation of a broad band between 70 and 75 kDa, which appears similar in size and pattern to IL2RP as identified by cross-linking radiolabeled IL-2 to its receptor (Fig. 2, band at 90 kDa which represents 15 kDa of 1 L 2 cross-linked to a 75 kDa protein). When cell lysates are immunoprecipitated by anti-phosphotyrosine antibodies under denaturing conditions (SDS), IL-2RP is immunoprecipitated, and under nondenaturing conditions, IL-2RP and a portion of IL-2Ra are immunoprecipitated (Fig. 2; Mills et al., 19894. In both cases, pretreatment of the cell lysates with alkaline phosphatase prevents immunoprecipitation by anti-ptyr antibodies (Fig. 2) as does addition of phosphotyrosine, but no phosphoserine or phosphothrconine, to the cell lysates. As indicated in Fig. 2, only a portion (10-50%) of radiolabeled I L 2 cross-linkcd to TL-2RP is

517

19. ACTIVATION OF LYMPHOCYTES BY LYMPHOKINES

Lysis

SDS

Alkphos

-

I Pt

-

+ _

RlPA

+ + + ++++--

-

-

-

92

-

68

--

43

FIG.2. Immunoprecipitation of 'L51-labeled 1L-2 cross-linked to PBL with anti-ptyr antibodies. PBL blasts were prepared as described in Fig. I . f2sI-labeled IL-2 was cross-linked to the blasts under high affinity conditions with DSS. Cells were lyscd either with boiling 2% SDS supplcmcntcd as described previously or with RIPA-Vanadate buffer (Kamps and Sefton, 1988). Cell lysates were treated with bovine alkaline phosphatasc as recommended by the manufacturer (Pharmacia). Cell lysates were immunoprecipitated with the polyclonal anti-ptyr antibodies described in Fig. 1 and Separated on SDS-PAGE. Essentially identical results were obtained with the PY20 monoclonal antiptyr antibody.

immunoprecipitated by thc anti-phosphotyrosine antibodies. Whether this indicates that only a small number of receptors are tyrosine phosphorylated or that cross-linking decreases the number of phosphorylated molecules remains to be determined. In similar studies, at least a portion of cellular IL-3 receptors are also immunoprecipitated by anti-phosphotyrosine antibodies, indicating that the 140 kDa IL-3 binding molecule is tyrosine phosphorylated (Isfort et al., 1988). Although lymphokines induce tyrosine phosphorylation, direct evidence for the role of this tyrosine phosphorylation in lymphokine function is not currently available. However, transfection of the v-abl tyrosine kinase oncogene can by-

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GORDON B. MILLS

pass the effects of I L 2 (Cook el al., 1987), 1L-3 (Pierce et a l . , 1985; Cook et n l . , 1985; Ollif et ul., 1985; Mathey-Prevot, ct al., 1986), and GM-CSF (Cook et al.. 1985) and induce proliferation in cell lines that are normally dependent on exogenous lymphokines. v-src has similar effects on IL-3-dependent cell lines but not IL2 or GM-CSF sensitive cells (Watson et d . , 1987). No evidence tbr autocrine growth factor production was observed, which suggests either that these lymphokines induce cell proliferation through tyrosine phosphorylation of substrates which are also phosphorylated by the ubl oncogene or that ubl stimulates these cells through an alternative activation pathway that is not normally utilized by the lymphokines.

D. Na+/H+ Antiport Activation of the amiloride-sensitive Na+ / H + antiport with its resultant increase in cytosolic pH has been iniplicated in the function of polypeptide hormones. Most of the agents that have been demonstrated to activate the Na+/Il+ antiport have also been demonstrated to activate intracellular kinases and to increase phosphorylation of intracellular substrates on serine and threonine or tyrosine. IL-2 (Mills et al.. 1985d, 1988a; Gelfand ef al., 1987b), but not 1L-1 (Kester c’t uI. , 1989; Mills ct ul., 1 9 8 9 ~ or ) IFN-y (Mills ct ul., I985c), rapidly activates the Na+ /H antiport, resulting in an increase in intracellular pH within minutes of addition of ligand. IL-I and IFN-y have been reported to induce a slow or delayed activation of the antiport, resulting in an increase in intracellular Na+ and pI1 hours after addition of ligand (Stanton d . , 1986; Calalb ~1 a l . , 1987; Ostrowski et al., 1988). However, blocking the N a + / H + antiport or preventing the rise in intracellular pH does not inhibit IL- I -induced IL-2 production (Mills et al. 1989e), IgM cxpression (Calalb er al., 1987), or IL-2-induced cell proliferation (Mills et ul., 1985d, 1988a; Dornand el a/., 1987). Activation of PKC by phorbol esters or diacylglycerol analogs induces activation of the N a + / H + antiport with a resultant increase in cytosolic pH; however, IL-I and IFN-y can increase intracellular pH in cells in which PKC is inhibited or depleted (Ostrowski rt UI., 1988). Similarly, lL-2 induces iin increase in cytosolic pH in somatic cell mutants that do not contain functional PKC (Mills et d., 1988a,b). This suggests that although under some circumstances activation of PKC may play a role in activation of the antiport, alternative PKC-independent pathways for lymphokine-induced activation of the antiport clearly exist. +

~

E. G Proteins Since the characterized lymphokine receptors do not contain recognizable signal transduction units, the mechanisms that link lymphokine receptors to

19. ACTIVATION OF LYMPHOCYTES BY LYMPHOKINES

519

signal transduction are unknown. In many systems, G proteins function as signal transducing agents, which has led to the hypothesis that they play a role in lymphokine action. IL-2 has been reported to stimulate GTP binding and GTP hydrolysis in membrane preparations, which would be compatible with activation of G proteins (Evans et al., 1987b). This was inhibited by incubation of the membranes with pertussis toxin, which would also suggest that IL-2 activated G proteins (Evans et al., 1987b). The validity of the observation has been questioned (Bourne, 1987), in particular in that IL-2-induced cell proliferation is not pertussis toxin sensitive (Gelfand et a / . , 1988) as would be predicted if a pertussis toxin-sensitive G protein were involved in mediating the IL-2-induced GTPase activity. 112-induced cell proliferation (Gelfand et al., 1985, 1986a,b, 1987a,b), 1L-I-induced IL-2 production (Mills et a / ., I989e), and interferoninduced growth inhibition (G. B. Mills, unpublished observations) are independent of ADP-ribosylation of G proteins by pertussis or cholera toxin, which would indicate that if a G protein is involved in signaling by these lymphokines, it is not sensitive to these toxins. In contrast, IL-3-induced cell proliferation is inhibited by pertussis toxin, suggesting a possible role for G proteins in IL-3 signaling (He et a / ., 1988).

F. Cyclic Nucleotides A role for changes in cyclic nucleotide metabolism has been difficult to establish in the activities of many lymphokines. However, recent studies with IL-1 suggest that 1L-1 increases cAMP concentrations and that inducing increases in CAMPcan replace IL- 1 in IL-2R expression and thymocyte mitogenesis (Mizel et al., 1987). However, this is difficult to reconcile with other studies that indicate that increases in cAMP inhibit ligand-induced production of IL-2 (Farrar et a / ., 1987; G. B. Mills, unpublished observations). IL-2 has been reported to increase (Beckner and Farrdr, 1986, 1987; Wickremasinghe et al., 1987) or to have n o effect on (Knudsen et a/., 1987) cAMP production. Increasing intracellular cAMP concentrations decreases IL-2-induced cell proliferation (Beckner and Farrar, 1986, 1987; Knudsen et al., 1987), 1L-2-induced phosphorylation (Gaulton and Eardley, 1986), and IL2-stimulated expression of MYC (Farrar et al., 1987). However, in some IL-2-sensitive cells, increasing CAMP concentrations does not inhibit 1L-2-induced cell proliferation (J. B. Stanley and G. B . Mills, unpublished observations). IL-2 increases cGMP concentrations (Hadden and Coffey, 1982; Knudsen et al., 1987); however, increasing cGMP concentrations does not alter IL-2-induced cell proliferation or initiate proliferation of 1L-2-dependent cells (Knudsen et al., 1987). cGMP can replace the IL-2 requirement for IFN-?/ production in some cells (Johnson et al., 1982), suggesting that IFN-?/ production and cell proliferation may be mediated through different pathways.

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GORDON B. MILLS

G. Receptor Internalization Most of the lymphokine receptors are internalized within a period of minutes (Robb et al., 1981; Cantrell and Smith, 1984; Fujii et a l ., 1986; Lowenthal et al., 1986; Matsushima et al., 1986; Mizel et al., 1987; Robb and Greene, 1987; Petska and Langer, 1987; Duprez et al., 1988). In most cases this results in a decrease in receptor expression (up to 90% receptor loss with IFN-y, Petska and Langer, 1987; 80% with IL-1, Matsushima et ul., 1986) and may mediate a decrease in signal transduction. However, this is difficult to reconcile with the observation that prolonged receptor occupancy is required for the effects of 1L-2 and likely other lymphokines (Cantrell and Smith, 1984; Sekaly e t a l . , 1984; lhle and Weinstein, 1986). The majority of the internalized lymphokines are rapidly degraded (Robb et al., 1981; Fujii et al., 1986; Lowenthal ef al., 1986; Matsushima e? al., 1986; Petska and Langer, 1987). However, with 1L-1, 1L-2, and IFN-y (Fujii et al., 1986; Mizcl et al., 1987; Petska and Langer, 1987; Quarnstrom et al., 1988) there is evidence for localization to the nucleus, which has presented the possibility that the lymphokine may have specific receptors in thc nuclcus. It is possible that internalization is required for cell activation, and this would be compatible with the observation of localization to spccific intraccllular sites. Preventing internalization of 1L-2 either with receptor antibodies, agents which inhibit internalization, or by steric hindrance also abrogated the ability to IL-2 to induce cell proliferation without altering its ability to bind to receptors (Kumar c ~ ul., t 1987). Interestingly, intracellular injection of IFN-y or accumulation of IFN-y inside the cytoplasm of cells transfected with a truncated IFN-y cDNA devoid of a signal sequence was able to bypass the cell surface receptor and mediate IFN-y effects across species barriers (Sanceau et al., 1987). This suggests that internalized IFN-y may be active independent of its rcccptor and that the receptor functions solely to internalize IFN-y.

H. Oncogene Expression Activation of IL-1 receptors (Hagiwara ef al., 1987; Colotta et al., 1988; Kovacs et al., 1986) and 1L-2 receptors (Reed Ct ul., 1985, 1987; Heckford et d.,1986; Stern and Smith, 1986; Broome et al., 1987; Farrar et ul., 1987; Pauza, 1987; Mills et al., 1988a) can increase the production of mRNA for the nuclear oncogenes,fos, myc and myh. In some IL2-sensitive cells, fos expression is increased, without a change in myc expression, while in other cells the converse occurs (Kaczmarck, 1985; Rced et al.. 1987; Mills et ul., 1988a). In addition, jos and myc expression return to baseline levels within 1-2 hr of stimulation of cells with IL-2 (Stem and Smith, 1986), which is prior to the time required to commit cells to proliferate. Therefore, it is not possible to corrclatc induction of either of these oncogenes with IL-2-induced proliferation. myb

19. ACTIVATION OF LYMPHOCYTES BY LYMPHOKINES

521

expression is delayed and correlates somewhat more closely with IL-2-induced cell proliferation (Stern and Smith, 1986; Pauza, 1987). Transfection of v-myr into IL-2- and 1L-3-dependent cell lines can induce growth factor-independent proliferation (Rapp er a / . , 198.5). A further role for myc in lymphokine-induced proliferation is provided by the observation that spontaneously arising lymphokine-independent mutants of CT6 cells all constitutively express myr at high levels (Cleveland et ul., 1987). However, in some IL-2-dependent cells, 1L-2 does not induce myc expression (Kaczmarek et a / ., 198.5; Reed et ul., 1987; Mills et ul., 1988a), questioning once again whether myc is an obligatory component on the IL-2-induced proliferation pathway or whether it is involved in an alternative pathway. As described previously, transfection of tyrosine kinase oncogenes such as v-ab/ and v-src can induce proliferation of cells normally dependent on IL-2 (Cook et a/., I987), IL-3 (Pierce et a/., 1985; Cook et ul., 1985; Ollif et al., 1985; Mathey-Prevot el al., 1986; Watson et a / ., 1987), or GM-CSF (Cook et a / ., 1985; Watson er a l . , 1987) without inducing autocrine lymphokine secretion. This suggests either that the lymphokincs stimulate cells through tyrosine phosphorylation and the oncogenes mimic this effect or, alternatively, that the oncogenes induce cell proliferation through an independent pathway.

X. CROSS-TALK BETWEEN ANTIGEN AND LYMPHOKINE RECEPTORS Activated T lymphocytes can express antigen receptors and lymphokine receptors simultaneously and can be restimulated by activation of either receptor. In some of these cells, activation of the IL-2 receptor decreases T cell receptorinduced proliferation, and activation of the T cell receptor decreases IL-2-dependent proliferation (Table I; Webb and Sprent, 1987; Otten etul., 1986, 1987; Nau et al., 1987). Therefore, it seems likcly that signals produced by the IL-2 receptor and likely other lymphokine receptors interact with those generated by the T cell receptor to regulate T cell proliferation. In addition, expression of the IL-2R and T cell receptor appears to be reciprocally related, and activation of each receptor appears to regulate expression of the other receptor (Cantrell and Smith, 1984; Nau et a / ., 1987). Activation of T cell receptor stimulates phospholipase C (PLC), which hydrolyzes membrane phospholipids to produce diacylglycerol and inositol phosphates (Gelfand er al., 1987a,b; Berridge, 1987). These second messengers then lead to an increase in [Ca2+1, and activation of protein kinase C (PKC), either of which may alter IL-2-induced proliferation. As indicated in Table I, the calcium ionophore ionomycin, but not the phorbol ester TPA, which activates PKC, mimics the effect of activation of the T cell receptor and inhibits IL-2-induced proliferation of PBL blasts. The effect of PHA and ionophores does not require

522

GORDON 8.MILLS

[ 3Hlthymidine

incorporation (cpm) Additions

PRI, blasts

PI .9. I

Medium 1172 PHA PHA IL-2 lonomycin lonomycin and 1L-2 TPA TPA and 11.-2 TPA and ionornycin

350 13,000 4.400 4,400 320 7,000 6,300 1h,000 1 1,000

5,800 4 I ,000 5,700 18,000 7,300 20,000 6,300 40,000 6.000

+

Human peripheral blood lyrnphocytes were rendered IL-?-sensitive hy incubation with phytuhe~nagglutinin (PHA) liir 3 days followed by exparisinn in 11.2 lor 8 days. PI .9. I is a murine IL-2-sensitive T cell line that lacks liinctional PKC (Mills r f crl , 1988a.b).Cells wctc cultured with IL-2 (100 U/ml), PHA (I0 pgiml). ionomycin (0.5 p M ) , or TPA (10- X M ) as indicatcd. Following a 24 hr i n r u b a ~ tion, cell\ were pulsed tor 4 hr with [3H]thymidine, hatvcated. and counted on il scintillation counter. '1

activation of PKC in that PHA and ionophores decrease 112-induced proliferation in PI .9.1 cells (Tablc I), which lack functional PKC (Mills et crl., 1988b). This suggests that changes in [Ca2+Ii,but not activation of PKC, induced by stimulation of the T cell receptor may inhibit IL-2-induced proliferation. Activation of PKC appears to limit IL-2 production and inhibits signaling through the T cell receptor (Mills et ul., 1989~).In addition, phosphorylation of other growth factor receptors by PKC decreases receptor number, ligand affinity, and tyr kinase activity, which may alter the response to other lymphokines. Therefore, activation of PKC may function as a feedback inhibitory pathway and limit T cell responses. Pre-incubation with IL-2 decreases TCR-induced changes in [Ca2 Ii in some helper T cells and may explain the ability of IL-2 to inhibit activation of cells through the T cell receptor (Otten et ul., 1987). In contrast, activation of cells through the CD2 receptor is augmented by IL2 and this correlates with increased changes in [Ca2 ' J i induced by cross-linking of the CD2 receptor. The mechanism whereby activation of the IL-2 receptor decreases signaling through the T cell receptor is not known. Phospholipase C, which mediates the TCR-induced increases in [Ca*+ J,, is a substrate for the EGF receptor-mediated tyr kinase +

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523

(Wahl et al., 1988) and may be a substrate for the 1L-2 or T cell receptor-induced tyr kinases. Therefore, phosphorylation of phospholipase C as a result of activation of the IL-2 receptor complex may inhibit T cell receptor-induced increases in [Ca2 Ii. +

XI.

SUMMARY

Resting lymphocytes do not express significant numbers of high affinity receptors for lymphokines (with the exception of IL-IR). Following activation of antigen receptors, cells are induced to express high affinity receptors for an array of lymphokines. The eventual response is determined by interaction of the lymphokines with their specific receptors on the surface of target cells. The response of the individual cell is determined by its particular pattern of lymphokine receptor expression and by its intracellular machinery. Since several different lymphokines can induce similar responses in a single cell, some lymphokine receptors may signal cells through conserved pathways. However, different lymphokines can also induce disparate effects on the same cell; therefore, it is likely that in addition to overlapping signals, the lymphokines can also induce specific signals. Over the last 10 years the identification, cloning, and expression of the genes for lymphokines and their receptors has provided insight into their structure. Identification of the regulatory regions within the genome is also allowing characterization of the processes that control the expression of these genes. However, the cloning of the receptor genes has so far provided little insight into how the lymphokine signal is transmitted across the cell membrane and eventually to the nucleus. As detailed herein, a number of biochemical pathways that have been shown to be important in activation of other cell types have been investigated. The results are often contradictory, likely because of the use of different lymphokine-sensitive cells and also because of the possibility of interaction of several signaling pathways within a single cell. In general, it appears that in contrast to antigen receptors, lymphokine receptors do not activate phospholipase C with resultant increases in [Ca2+Iiand activation of PKC. In addition, these processes do not appear to be necessary for lymphokine function. The recent observation that many of the lymphokines induce tyrosine phosphorylation and that artificially increasing tyrosine phosphorylation by transfection of tyrosine kinases into lymphocytes can bypass their lymphokine requirements suggests that this may be an important consensus pathway for lymphokine function. ACKNOWLEDGMENTS Supported by grants from the National Cancer lnstitutc of Canada and the Medical Research Council of Canada. The author would like to thank Martha McGill, Mary Hill, and Chris May for technical assistance and E. Anetta for secretarial assistance.

524

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Tigges, M. A , , Casey, L. S . , and Koshland, M. E. (1989). Mechanisms of Interleukin-2 signaling: Mediation of different outcomes by a single receptor and transduction pathway. Science 243, 78 1-786. Tsudo, M.. Kozak, R . W., Goldman, C . K., and Waldman, T. A. (1986). Demonstration of an new non-TAC peptide that binds interleukin 2: a potential participant in a multichain interleukin-2 receptor complex. Proc. Natl. Acud. Sci. U.S.A. 83, 9694-9701. ’kudo, M., Goldman. C. K . , Bongiovanni, K . F., Chan, W. C.. Winton, E. F., Yagita, M., Grimm, E. A., and Waldmann, T. A. (1987). The p75 peptide is the receptor for interleukin 2 expressed on large granular lymphocytes and is responsible for the interleukin 2 activation of these cells. Proc. Nut/. Acud. Sci. U.S.A. 84, 5394-5401. Utsunomiya. N., Tsuboi. M . , and Nakanishi, M. (1986). Interleukin 2 increases T lymphocyte incinbranc mobility lxfore the rise in cytosolic calcium concentration. Biochemistry 25, 25822584. Valge, V. E.. Wong, J. G. P., Datlof, B . M., Sinskey, A . J., and Ran, A. (1988). Protein kinase C is required for responses to T cell receptor ligands but not to Interleukin-2 in T cells. Cell 55, 101112. Veillelte, A . , Bookman, M. A , , Hordk, E., and Bolcni, J . B. (1988). The CD4 and CD8 T cell surface antigens are associated with the internal membrane tyrosine-protein kinase pS61~k.Cell 55, 301-308. Wahl, M. I., Daniel, T. O., and Carpenter, G . (1988). Antiphosphotyrosine recovery of phospholipasc C activity after EGF treatment of A-431 cells. Sriencr 241, 968-971. Watson, J. D., Eszes, M., Overall, R . , Conlon, P., Widmer, M., and Cillis, S . (1987). Effect of infection with murine recombinant retroviruses containing the v-src oncogene on interleukin 2and interleukin 3-dependent growth states. J . fmmunol. 139, 123- 129. Webb, S . , and Sprent, J. (1987). Down regulation of T cell response by antibodies to the T cell rcccptor. J . Exy. Med. 165, 584-589. Weiss, M. J., Daley, J. F., Hodgdon, J. C . , and Reinher, E. L. (1984). Calcium dependency of antigen-specific (T3-Ti) and alternative (TI I ) pathways of human T cell activation. f’ruc. Nail. Acud. Sci. U.S.A. 81, 6836-6840. Whetton, A . D., Monk, P. N., Consalvey. S . D., and Downes, C. P. (1986). The haemopoietic growth Iactora interleukin 3 and colony stimulating factor-l stirnulatc proliferation but do not induce inositol lipid breakdown in murine bone-marrow-derived macrophages. EMBO J . 5, 328 1-3286. Whetton. A . D., Monk, P. N . , Consalvey, S . D., Huang, S . J., Dexter, T. M., and Downes, C. P. (1988). Interleukin 3 stimulates proliferation via protcin kinasc C activation without increasing inositol lipid turnover. Proc. Nutl. Acud. Sci. U.S.A. 85, 3284-3288. Wickremasinghe, R . G., Mire-Sluis, A . R . , and Hoffbrand, A. V. (1987). Interleukin-2 binding to activated human T lymphocytes triggers generation of cyclic A M P but not of inositol phosphates. FEES Lett. 220, 52-56. Wijelath, E. S . , Kardasz, A. M., Drummond, R . , and Watson, J. (1988). Interleukin-one induced inositol phospholipid breakdown in m u r k macrophages: Possible mcchanism of receptor activation. Biorhem. Biophys. Res. Commun. 152, 392-397. Wolff, C. H. J . (1987). Kinetics of Ca2+ uptake into lectin-induced secondary lymphocytes during reactivation with concanavalin A or Interleukin 2. Scund. J . Immunol. 26, 7-10. Yainasaki, K . , Taga, T., Hiratd, Y., Yawata, H., Kawanishi, Y., Seed, B., Taniguchi, T,, Hirano, T., and Kishimoto, T.(1988). Cloning and expression of the human Interleukin-6 (BSF-2/1FNB 2) rcccptor. Srirnc-r 241, 82S-X28. Yang, Y.-C., Kovacic, S., Kriz, R., Wolf, S . , Clark, S. C., Wellcms, T. E., Nicnhuis, A., and

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Epstein, N . (1988). Thc human genes for GM, CSF and It 3 are closely linked in tandem on chromosome 5. Blood 71, 958-961. Yap, W. H . , Teo, T. S . , and Tan, Y. H. (1986). An early event in the interferon-induced transmembrane signaling process. Science 234, 355-358. Yarden, Y., and Ullrich, A . (1988). Growth factor receptor tyrosine kinases. Annu. Rev. Biochem. 57, 443-465.

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C'URKENT TOPICS IN MEMBRANES AND TRANSPORT, VOLUME 35

Chapter 20

Role of Cytokines in Leukocyte Activation: Phagocytic Cells MICHAEL A . WEST Department of Surgery Hennrpin County Medicd Center Minnecrpolis, Minnesota 55415

I. 11.

111.

IV.

V.

VI.

Introduction Phagocyte Activation A. Neutrophil Activation B. Macrophage Activation Major Cytokines A. Interleukin 1 B. Tumor Necrosis Factor C. Interleukin 2 D. Interferon y E. Colony Stimulating Factors F. Interleukin 6 G. Interleukin 4 H, Transforming Growth Factor p Role of Cytokines in Neutrophil Activation A. Cytokine Effects on Neutrophil Production, Margination, and Differentiation B . Effect of Cytokines on Neutrophil Adhesion to Endothelial Cells C. Effect of Cytokines on Neutrophil Chcmotaxis D. Effect of Cytokines on Neutrophil Oxygen Metabolism and Microbicidal Mechanisms E. Summary: Cytokine Effects on Ncutrophil Activation Role of Cytokines in Macrophage-Monocyte Activation A. Cytokine Effects on Macrophage-Monocyte Production and Differentiation B. Cytokine Effects on Macrophage Recruitment, Adhesion, and Chemotaxis C. Cytokine Effccts on Macrophage Microbicidal Mechanisms D. Cytokine Effects on Macrophage Tumor Cytotoxicity E. Cytokinc Effects on Macrophage Arachidonic Acid Synthesis F. Cytokine Effects on Macrophage Cytokine Synthesis G. Cytokinc Effects on Macrophage Arginine-Dependent Effector Mechanism H. Effective Cytokines on Macrophage Oxidative Mechanisms 1. Summary: Cytokine Effects on Macrophage Activation Summary: Cytokine Effccts on Activation of Phagocytic Cells References

537 Copyright Cl 1090 by Academic Presc. Inc All rights nf reproduction In any foriii rerervcd

538

MICHAEL A. WEST

I.

INTRODUCTION

Until very recently, investigators have been unable to decipher the language used to exchange information between different types of cells at diverse sites within the body. It is now clear that soluble factors, known as cytokines, serve as the most important transmitters of intercellular communication. Specifically, cytokines are proteins produced by one cell which alter the function of another cell ( I ) . Cytokines are now known to be produced by virtually all types of cells, but production by leukocytes has been most extensively studied (1-3). Cytokines have varied effects on a multitude of different cells from tumor cells to cells of the immune system. Phagocytic cells, particularly neutrophils and macrophages, make up the effector limb of the host immune system (43.Phagocytes function to identify, localize, destroy, and eliminate a wide variety of foreign invaders. Phagocytic cells have evolved the capacity to destroy neoplastic cells and cells infected with viruses, fungi, bacteria, and parasites. Cytokincs facilitate a coordinated and appropriate inflammatory response by controlling many aspects of immune reactions (3). It has also become clear that polypeptide cytokine messengers may be important physiological regulators responsible for activation of leukocyte phagocytcs (4-8). The focus of this review will be limited to the effects of cytokines on functions which have been associated with activation of phagocytic cells.

II. PHAGOCYTE ACTIVATION Phagocyte uctivation is a general term that refers to the degree of cellular stimulation or activity. Activation of phagocytes is not an all or none phenomenon but rather represents a spectrum of functional changes above those of unstimulated cells. Unfortunately, the specific functional changes which occur following activation of phagocytic cells are poorly defined in most instances. The concept of activation of phagocytes arose entirely from noting the functional changes that occurred following in vitro exposure to test stimuli. The term activation connotes the acquisition of specific functional capacities. Different cellular capacities are turned on or off when neutrophils and macrophages are exposed to various stimuli (7,8). Any discussion of phagocytic cell activation should be predicated with the understanding that specific in vitro test stimuli have generally defined the activated functions. To understand the effect of cytokines on phagocytic cell function, it is useful to examine various individual functions of neutrophils and macrophages associated with activated, or stimulated, functional states. To the extent that cytokines stimulate or inhibit these same individual phagocytic cell functions, they alter the acrivation stute of the phagocytes. In contrast to the

20. CYTOKINES IN PHAGOCYTE ACTIVATION

539

biochemical stimuli that defined these functional alterations, cytokines may represent stimuli that have in vivo physiological relevance.

A. Neutrophil Activation In the bloodstream, granulocytic phagocytes predominate, and among these, neutrophils are quantitatively and qualitatively most important (4). Stimulation of eosinophils and basophils will not be addressed in the ensuing discussion. Participation of neutrophils in the host defense system necessitates that they first be attracted to the endothelial surface adjacent to an inflammatory site (9). Leukoadhesion is mediated by changes in the neutrophil itself as well as changes in the properties of the endothelial cells and is followed by neutrophil movement to the site of inflammation (4). Knowledge of the mechanism of neutrophil activation was obtained by studying the in vitro response of neutrophils to various inflammatory stimuli. In vitro exposure of neutrophils to activated complement components, C3a, C5a (10,ll) and formylmethionyl peptides (e.g., formylmethionylleucylphenylalanine,FMLP) ( 12,13) results in directed locomotion, chemotaxis (14). Once a neutrophil has arrived at an inflammatory site, it eliminates foreign invaders by phagocytosis followed by intracellular destruction (9). In vitro studies have shown that phagocytosis occurs via dissociable steps, with initial attachment followed by engulfment of the foreign particle (4). This process is facilitated by opsonization with immunoglobulin, the C3b fragment of complement, and other opsonins (4,14), which permit sequential interactions between opsonic ligands and receptors on the neutrophil membrane (4). The neutrophil respiratory burst is initiated by attachment of opsonized microbes to the cell membrane or by soluble mediators such as C5a (10) or FMLP (15). The oxidative burst results in production of short-lived active oxygen intermediates which destroy phagocytized microbes (16,17). Such oxygen metabolites include hydrogen peroxide, superoxide, singlet oxygen, and hydroxyl radical (9,16,17). In vitro studies of neutrophil responses to inflammatory stimuli have defined various neutrophil functions which are stimulated and which are associated with activated functional states. These functions are summarized in Table 1. Highly stimulated neutrophils typically demonstrate enhanced chemotaxis (14), increased phagocytosis (4), enhanced bacterial killing (16), and an augmented respiratory burst (9- 17). Antibody-dependent, cell-mediated cytotoxic activity (18) is also a functional capacity associated with neutrophil activation.

B. Macrophage Activation A fraction of mononuclear phagocytes, termed monocytes, also circulates in the bloodstream; however, the larger component of mononuclear phagocytes is fixed within tissues, where they are known as macrophages (5,19). Specialized

540

MICHAEL A. WEST TABLE 1 PROPERTIESOF ACTIVATEDNEUTROPHILS lrrcrcased phegocytosis Enhanced random migration Increased chemotaxis Increascd rnyeloperoxidase activity Increased reactive oxygen intermediate production lncrcascd microbicidal activity Increased cytotoxicity Lysosomal enzyme release

tissue mononuclear phagocytes have specific namcs at some sites, for example, Kupfier cells within the liver, alveolar macrophages in the lungs, and peritoneal macrophages in the peritoneal cavity. Despite their diverse sites and subtle diffcrcnces in function, all macrophages are derived from circulating monocytes, and ultimately both cell types are derived from a common bone marrow precursor (20,21). Differences in macrophage function at different sites as well as the function of circulating peripheral blood mononuclear cells (PBMNC) can be undcrstood in terms of differenccs in the degree of macrophage activation (68,19). In the case of macrophages, activation likewise refers to the degree of stimulation of various biochemical and functional paramctcrs observed after treating macrophages with test stimuli in v i m (6-8,22). Unfortunately, the jargon regarding these altered functional states is confusing (5-8 ). Macrophages which have not been specifically stimulated are generally referred to as resident macrophages (8). When resident macrophages are treated with vcry potent biochemical stimuli, they develop the ability to lyse tumor cells and destroy intracellular parasites (6). By convention, the stimulated state which is associated with significant tumor cytolysis or destruction of intracellular parasites has been accepted as a definition of the fully activutd macrophage (6-8). Between the extremcs of resident and fully activated macrophages, many other functions are gained or lost, depending upon the specific stiniulatory substances utilized, as well as the timing and order of macrophage stimulation (19). Macrophagcs that have intermediate states of altered functional capacitics above those of resident cells are variously referred to as elicited, primed, inflammatory, cxudate cells, and suboptimally primed (6-8). The functional changes seen with stimulation of monocyte and macrophages are summarized in Table 11. Unstimulated, resident macrophages are difficult to obtain and study experimentally because many of the isolation procedures, including attachment to glass or plastic, stimulate these cells (6). In v i m and in vivo treatment of macrophages with thioglycolate, zymosan, or muramyl dipeptide (MDP) stimulates mac-

20. CYTOKINES IN PHAGOCYTE ACTIVATION

54 1

TABLE I1 ACTIVATED MACROPHACES-MONOCYTES PROPERTIES OF

Decreased motility Increased cell surface antigen expression Increased Ia expression Plasminogen activator synthesis Increased tumor cytoxicity Enhanced killing of intracellular parasites Enhanced cytokine production Increased prostaglandin-leukotnerie synthesis Increased reactive oxygen intermediate production Increascd reactive nitrogen intermediate production Increased procoagulant activity

rophages to a higher level referred to as inflammatory (6,7) or elicited (8) macrophages. In vitro treatment with stimuli such as lipopolysaccharide (LPS) (22), phorbol myristate acetate (PMA) (23), or calcium ionophore A23187 (23,24) under appropriate conditions results in stimulation of macrophages to very high levels. Macrophages so stimulated in vitro are described as fully activated macrophages (6-8,19). Macrophage-mediated tumor cytotoxicity and destruction of intracellular parasites such as Toxoplusma and Leishmania require complete macrophage activation (6-8). As macrophage stimulation increases, other biochemical properties of these cells are altered; there is decreased expression of cell surface transferrin receptors (25,26), increased expression of cell surface Ia antigen (27), enhanced respiratory burst activity (8,28), decreased chemotaxis (6,7), and release of lysosomal enzymes (28). Activated macrophages also synthesize and secrete various biochemical messenger molecules as they become more stimulated, including prostaglandins, leukotrienes, and various protein cytokines (3,5,19,22).

111.

MAJOR CYTOKINES

In vitro stimulation with biochemical probes has been useful to begin to understand and dissect the mechanisms and pathways involved in neutrophil and macrophage activation. Such biochemical substances probably play a minor role in mediating alterations in macrophage function in vivo, however. The explosive growth of molecular biology in the past decade has permitted identification, isolation, and synthesis of highly active proteins that appear to coordinate communication between different cells. Originally, these polypeptide messengers were thought to be produced predominantly by lymphoid cells or monocytes and

542

MICHAEL A. WEST

were referred to as lymphokines (3) or monokines (2,5), respectively. However, it has become clear that many other cell types produce identical proteins (1). These protein messenger substances are now referred to using the generic term cyrokine (1,3). The well-characterized cytokines that have effects on phagocytic cclls includc intcrleukin la (IL-la) (29,30), interleukin 1 p (IL-Ip) (29,31), tumor necrosis factor a (TNFa) (22,32,33), tumor necrosis factor p (TNFP) (33,34), interleukin 2 (IL-2) (3,35), interleukin 6 (IL-6) (36,37), granulocyte macrophage-colony stimulating factor (GM-CSF) (38-40), colony stimulating factor-I (M-CSF, CSF-1) (41,42), interferon a (IFN-a) (43, 44), interferon y (IFN-y) (3,45), and transforming growth factor p (TGFP) (46,47). The cells producing these cytokines as well as the major target cells are summarized in Table 111.

A. Interleukin 1 Interleukin 1 (IL-1) is a cytokine whose production has been extensively studicd (3,29,48). Originally, IL-I was thought to bc produced predominantly by monocytes and tissue macrophages (3). As with most cytokines, the effect of IL- 1 was initially defined by using a bioassay, induction of thymocyte proliferation (29). Earlier studies of 1L1-like activity were undoubtedly contaminated with many other cytokines (3). Subsequently, molecular biological techniques resulted in the isolation of two nucleotide sequences that produced closely related TABLE 111 CELLULAR SOURCES A N D TARGETSOF MAlOK CYTOKINES" Cytokinc IL-Iu

LIP 1122

IL-4 IFN-a IFN-PI IFN-P, (1L-6) IFN-y M-CSF. CSF-I GM-CSF TNFu TNFP

m-p MDNCF

Source

Target

M+. EC, Fibroblasts M+ T Cells T Cells Lymphocytes MQ, I-ihroblnits, MQ, T Lymphocyte\ M+ Fibroblasts MQ,.bihrohlnsts Lymphocytes, M+ T Lymphocytes M+

Lymphocytes, EC. HC. M+? Fibroblasts, Others Lymphocytes, EC, HC, M+, Fibroblasts, Others I.ymphocytes, M+, Others B Cells, T Cells, Others Multiple Nucleated Cells Multiple Nucleated Cells HC, Lymphocytes, Othcrs M+, Lymphocytes, Others Bone Marrow, M+, Fibroblasts M+, PMN Multiple Cells Multiple Cells Fibroblasts. M+ PMN

'1 Abbreviations: IPN. interferon: TNF, tumor necrusis raactor: IL, interleukin; CSF. colony \timulaling Factor; GM GI-granulocyte , macrophage-colony stimulating tactor: 1 W p . rranstorniing growth tactor p: M+. macrophage: PMN. I)"lytn~)rphonucleIlrneuirophil. EC, endolhclial cell. HC. hepakocyies.

20. CYTOKINES IN PHAGOCYTE ACTIVATION

543

17 kDa proteins referred to as 1L-la (30) and IL-IP (31). There are subtle, but distinct, differences in the amino acid sequences of these two forms of IL-I. IL-la has 153 amino acids and has a pl of 5.0, whereas L I P is a 159 amino acid protein with an extracellular pl of 7.0. In addition to monocytes and macrophages, a wide variety of other cell types have been shown to produce IL-1, including keratinocytes, synovial cells, fibroblasts, neutrophils, and endothelial cells (29,47). Production by these diverse cell types of I L 1 was triggered by stimuli such as LPS (49), calcium ionophore, PMA (SO), and a combination of cytokines (5 I). I L 1 has been shown to activate T lymphocytes, stimulate fever, activate osteoclasts, inhibit adipocyte lipoprotein lipase, stimulate hepatic acute phase protein synthesis, and have a wide range of other functions (3,29,48).

B. Tumor Necrosis Factor Tumor necrosis factor a (TNFa) is a 17 kDa protein containing 157 amino acids produced predominantly by mononuclear phagocytes (33). It was originally defined by its ability to cause lysis of L929 fibroblasts treated with actinomycin D (52). TNFa is identical to another macrophage secreted factor which altered lipoprotein lipase activity in cultured adipocytes, which was known as cachetin (53). A closely related molecular form, tumor necrosis factor P (TNFP), is produced by T lymphocytes and was formerly referred to as lymphotoxin (LT) (31). Besides causing lysis of susceptible tumor cells, both species of TNF have been found to have a wide variety of effects on other cells, including induction of some aspects of the hepatic acute phase response (54-56), mitogenic activity for fibroblasts (57), induction of procoagulant activity by endothelial cells (58), and many other effects (33).

C. Interleukin 2 Interleukin 2 (IL-2) is a 15 kDa protein produced by activated T cells (1,3,35). IL-2 participates in the clonal expansion of activated T lymphocytes (35). IL-2 appears to act via association with specific high affinity cell surface receptors (59). IL-2 is a potent stimulus for lymphocyte production of other cytokines by Thelper cells (3).

D. Interferon y Interferon y (IFN-y) is a glycoprotein dimer weighing 45 kDa which consists of two protein chains of 20 kDa and 25 kDa which are glycosylated to varying degrees (3,45). IFN-7 functions primarily as an immunomodulatory cytokine, in contrast to other interferons which have substantial antiviral activity (45). IFN-y has prominent effects on cell surface expression of Ia antigens (27). IFN-y also

544

MICHAEL A. WEST

appears to be necessary for expression of IL-2 receptors by T lymphocytes (45). As will be discussed in more detail, IFN-7 is a potent stimulator of several rnacrophage functions (3,22,45).

E. Colony Stimulating Factors Granulocyte-macrophage colony stimulating factor (GM-CSF) is a 22 kDa glycoprotein produced by several different cell types, including activated lymphocytes, macrophages, endothelial cells, and fibroblasts (38-40). It has significant colony stimulating activity on myelomonocytic and erythroid stem cells (60). The effects of GM-CSF arc mediated through binding to high affinity cell surface receptors (6 1 ). Macrophage colony stimulating factor (M-CSF, also referred to as CSF-I) was originally described for its ability to induce proliferation of mvnocyte bone marrow progenitors (62) and is produced predominantly by lymphocytes (3,62).

F. lnterleukin 6 lnterleukin 6 (IL-6) is now thc accepted designation for a 26 kDa protein produced by T cells and fibroblasts (36,37). This cytokine was previously known as interferon P2 (IFN-P,) because of its antiviral activity (36). Other designations of this molecule included B cell differentiation factor and hepatocyte stimulatory factor (HSF) (54).It is now appreciated that IL-6 has littlc in common with other interferons. IL-6 inhibits fibroblast proliferation (37) and is the most important cytokine regulating hepatic acute phase protein synthesis (63).

G. lnterleukin 4 Interleukin 4 (IL-4) is another cytokine produced by activated T lymphocytes and was formerly known as B cell stimulating factor-1 (BSF-I) (3). IL-4 stimulates cytolysis by 'r lymphocytes and stimulates growth of activated B cells by interacting with a specific cell surface receptor (64).

H. Transforming Growth Factor

p

Transforming growth factor p (TGFP) is a 25 kDa protein dimer produced by fibroblasts that is held together by disulfide bonds (46-47). Like other cytokines, TGFP interacts with specific, high affinity cell surface receptors (65). TGFP inhibits T cell proliferation in response to IL-2 and IL- 1, B cell proliferation in response to growth factors, and expression of IL-2 receptors (65). From the preceding discussion, it is apparent that many of the cytokines that activate monocytcs-macrophages and ncutrophils are produced by these same

20. CYTOKINES IN PHAGOCYTE ACTIVATION

545

cells, particularly macrophage-like cells. In the remainder of this review, discussion will focus specifically on the effects of cytokines on stimulating various functions of neutrophils and macrophage-like cells which have been associated with activation of these phagocytic cells.

IV. ROLE OF CYTOKINES IN NEUTROPHIL ACTIVATION If cytokines are important mediators responsible for physiological stimulation or inhibition of neutrophils, effects similar to those obtained with biochemical or microbiological stimuli should be demonstrable after neutrophil exposure to cytokines. In the ensuing discussion, functional attributes associated with activation of neutrophils will be grouped by function and the effect of cytokines on individual functions reviewed.

A. Cytokine Effects on Neutrophil Production, Margination, and Differentiation Relatively little information concerning the effects of cytokines on neutrophil production and differentiation is available. The primary cytokines with clear-cut effects on neutrophil production and differentiation are IL-1 (3,29) and colony stimulating factor (40-42). GM-CSF has a major role in the differentiation of granulocyte progenitor cells into mature neutrophils (41 ). GM-CSF (40) and CSF-I (62) may also be involved in the granulocytosis which accompanies inflammatory reactions. Treatment of animals and humans with IL-1 resulted in a rapid increase in the number of circulating neutrophils (29). This neutrophilia resulted from demargination of neutrophils as well as increased production of neutrophils by bone marrow stem cells (29). IL-1 was also found to cause mobilization of mature neutrophils from the bone marrow into the peripheral blood (3,29).

B. Effect of Cytokines on Neutrophil Adhesion to Endothelial Cells The initial step in neutrophil migration to sites of acute inflammation involves adherence to endothelial cells (4). IL- 1 pretreatment of cultured human umbilical vein endothelial cell monolayers resulted in an 18-fold increase in neutrophil adhesion, whereas pretreatment of leukocytes did not affect adhesion (66). Enhancement of endothelial cell neutrophil adhesion required RNA and protein synthesis (66). Both 1L-la and IL-lp have been shown to increase PMN adhesion to endothelial cells (67). IL- 1 (Y treatment of endothelial cells also increased production of the phospholipid platelet activating factor (PAF) (67), which may

546

MICHAEL A. WEST

mediate some of the 1L-1 effects. I n v i m treatment with TNFa was also found to enhance endothelial cell adhesiveness for neutrophils (55,68,69). I n vivo studies revealed that injection of either IL-I or TNFa subcutaneously in rabbits resulted in acute inflammation and microvascular injury similar to the Shwartzman reaction ( 5 5 ) . TNFa and 1L-1 were shown to interact synergistically to achieve maximal PMN infiltration (68,69). Infiltration of neutrophils requires that they not only stick to the endothelial cells but also move beyond the endothelial cell barrier out into the interstitium. A cytokine, human leukocyte inhibitory factor ([,IF), that decreases adherence of "Cr-labeled neutrophils has been described (70,71). Other investigators described a leukocyte adhesion inhibitor (LAI) which accumulates in conditioned medium from IL-1-, TNF-, or LPS-treated human endothelial cell cultures (72). The two cytokines may be a single factor. Although many cytokine effects on neutrophil adhesion are mediated via the endothelial cells, direct effects of IL-la and TNFa on neutrophil adherence have also been examined (73). Markedly different effects of IL1 and TNF were observed in these studies. Treatment with TNFa resulted in enhanced neutrophil adherence, whereas 1Lla suppressed adherence (73). Furthermore, the stimulatory effects of the chemotactic peptide FMLP could be abrogated with IL-I but were augmented by TNFa. Studies in more complex in vivo systems showed that 1L-l a and IL- I p both resulted in marked neutrophil accumulation in skin (68) or the peritoneal cavity (69). Following injection of recombinant cytokines into the peritoneal cavity of mice, peak neutrophil infiltration was seen within 2 hr. Recombinant IL-la or IL-lp resulted in the greatest influx of neutrophils, with markedly lowered responses seen with TNFa or TNFP (69). Intraperitoneal injection of other cytokines, including IL-2, IFN-y, IFN-a, IFN-P, GM-CSF, or G-CSF, had no significant effects (74). The mechanisms of IL- 1 - or TNF-mediated effects on neutrophil infiltration were not due to stimulation of prostaglandin or leukotriene mediators because specific inhibitors of the respective metabolic pathways did not inhibit neutrophil migration (69). When TNFcYand 1L1 were injected in combination into a rabbit skin model, synergistic neutrophil recruitment was observed (68). The role of cytokine regulation of transendothelial neutrophil migration has been examined (75). Short-term preincubation of human umbilical vein endothelial cell monolayers with I L 1 or TNF resulted in increased numbers of endothelial cell-associated neutrophils and penetration of endothelial monolayers by neutrophils. Transendothelial neutrophil migration could be blocked by actinomycin D (75),suggesting a requirement for de nova protein synthesis. Transmigration mediated by this protein was prevented by low-dose, short-term trypsin digestion. Transendothelial neutrophil migration was enhanced by treatment of endothelial cells with TNF, but not I L 2 , 1FN-y, or the potent chemotaxin LTB, (75).

20. CYTOKINES IN PHAGOCYTE ACTIVATION

547

C. Effect of Cytokines on Neutrophil Chemotaxis Information on the mechanisms of neutrophil chemotaxis was obtained using C5a ( 1 1 ) and FMLP (12,13), which interact with specific cell surface receptors (14). Studies with earlier preparations containing ILI-like activity showed enhanced neutrophil chemotaxis (76). Studies with recombinant IL-1 have failed to show any significant chemotactic effect (77). Furthermore, it was noted that IL- 1P did not alter intracellular free calcium concentrations, which were markedly elevated by chemotaxins such as FMLP, LTB,, and calcium ionophore (77). Nonetheless, IL- 1 clearly stimulates neutrophil infiltration and has been shown to be a potent inducer of macrophage cytokine production (5,29). It has been hypothesized that 1L1 effects on neutrophil recruitment may be mediated via induction of other cytokines. In support of this mechanism, a neutrophil chemotactic factor has been isolated from activated macrophages (71,76,78). This factor is a 6-7 kDa protein which shares some sequence homology with platelet factor 4 (PF-4) and P-thromboglobulin (P-TG). This macrophage-derived neutrophil chemotactic factor (MDNCF) (7 1,76) has chemoattractant efficacy on neutrophils similar to that of FMLP, but it has no chemoattractant effect on monocytes (76).

D. Effect of Cytokines on Neutrophil Oxygen Metabolism and Microbicidal Mechanisms Neutrophils exposed to microorganisms, calcium ionophore, activated complement, or FMLP demonstrate an increased capacity for production of active oxygen intermediates, enhanced microbicidal activity, increased phagocytosis, and heightened neutrophil cell-mediated cytotoxicity. Recently, several different cytokines have been shown to produce a similar spectrum of functional alterations. Treatment of neutrophils with recombinant tumor necrosis factor has been shown to result in a rapid, dose-dependent reduction of nitroblue tetrazolium (NBT), indicating enhanced superoxide anion release (9,16,79,80). Treatment of neutrophils with TNFP increased latex bead phagocytosis (8 l), hydrogen peroxide production (82), and lysosomal enzyme release (82). Phagocytosis by neutrophils was increased by treatment with recombinant IFN-y (83). GM-CSF was likewise found to augment neutrophil phagocytosis, and this effect was postulated to be mediated via increased expression of Fc receptors (84). In other experiments, TNF had a weak direct effect on neutrophil oxidative metabolism but synergized with a wide variety of other inflammatory stimuli to stimulate oxidative pathways (82). Tumor necrosis factor may play a physiological role in PMN-mediated de-

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MICHAEL A. WEST

struction of microorganisms. Circulating TNF was detectable in the bloodstream of patients with acute infections (85) and correlated with the severity of the infection. TNF was also found in lung lavage fluid obtained from Legionellu pneumophiliu infected mice (86). TNF added to neutrophils in vitru resulted in a significantly augmented killing of L . pneunzuphilia (86). Lymphokine treated rat PMNs demonstrated increased tumor cell cytostasis and cytolysis in v i m (87), although the actual cytokine responsible has not been identified. Using human recombinant cytokines, other investigators have shown enhanced neutrophilincdiated antibody-dependent cellular cytotoxicity following treatment with interferon y and TNFP at all effector target cell ratios testcd (81,88,89).

E. Summary: Cytokine Effects on Neutrophil Activation The effects of major cytokines on neutrophils functions that have been associated with activated states are summarized in Table IV. TNF, IL- 1, and 1L-2 have promincnt eRects on neutrophil recruitment, whereas most cytokines have mininial effects on chemotaxis. 1L-1, TNF, IFN-y and GM-CSF probably have a physiological role in stimulating neutrophil phagocytosis, oxidative mechanisms, and overall microbicidal or cytocidal mechanisms. The effects of many

TABLE IV

EFFECTO F MAJORCYTOKINES

ON NEIITHOPHIL

Recruitrriciit

Cytokinc' Il.-la

IL-Ip IL-2 I I .-4 IFN-(U IFN-Pi IFN-8, (IL-6) IFN-y

M-CSF, CSF-I GM-CSF TNFa

TNFP TGFP MDNCF

adhesion

Chemotaxis

+++

++ or No A No A No A No A No A No A

+ I + ti+

'?

No A )

?

No A No A No D

++ +

$?

I

,

No A No A No A No A No A ? or +

+++

ACTIVAIION","

Oxidative iriechanism

+++

Phagocytosis t

3

1

I

?

I

7

'? I

,? +it 1

?

+++

,? i t

++ ++ ++i

?

I

I

? I

See text for \pecil'ic references. ((I ---; No A , n o d t r c t 01 cytokine; ?. insulricicnt dalil. Stiiiiulation shown a h + to + I t : inhibition. ' Abhrcvialions: IFN, inlerl'eron; TNF, tunior necrosis l'ackii. IL, interlcukin, CSF, colony stimulating F x l o r , GM-CSP. granulocyte macrophagr-colony stimulating tactur: TGF. transtorinmg growth factor: MDNCF, Inacrophdgc derived neutrophil chemotactic factor.

549

20. CYTOKINES IN PHAGOCYTE ACTIVATION

cytokines on neutrophil function remain unknown at present but will likely be the subject of continuing investigations in the near future. From the information available at the present time, it seems likely that cytokines are important regulators that are responsible for stimulation of neutrophils in vivo.

V.

ROLE OF CYTOKINES IN MACROPHAGEMONOCYTE ACTIVATION

Until recently, it was thought that macrophage-like cells were important sources of cytokines but that cytokines played a minor role in macrophage activation. Early studies demonstrated that supernatant from stimulated lymphocytes contained soluble factors that could participate in activation of macrophages (3). This activity was referred to as macrophage a r t i v a h g ,fuc.tor (MAF) and was probably due largely, but not exclusively, to IFN-(r (1,45). It has now become clear that virtually all major cytokines have some effects on macrophage functions. The macrophage functions (see Table 11) that have been associated with macrophage activation include (4-8,22) production, differentiation, recruitment, adhesion, microbicidal mechanisms, tumor cytolysis, synthesis of arachidonic acid metabolites, cytokine production, oxidative metabolism, the arginine-dependent effector pathway, and increased procoagulant activity. Macrophage activation is thought to occur via a two-stage mechanism (90) depicted in Fig. 1 . In the first stage, resident, unstimulated cells are primed or elicited by treatment with stimuli such as low-dose LPS, thioglycolate, comple-

?GM~CSF

Cytokine Synthesis Microbicidal Activity

L

I

--

Oxidative Mechanisms

Arachidonic Acid Metabolism

Sesident I Maicrophage

*tPrimed" CSF-1 Macrophage

"Activated" Macrophage

1Arcrinine Dewndent -Efiector Meihanism

FIG. 1 . Schematic depiction of the role of cytokincs in the two-stage hypothesis of macrophage activation. Unstimulated cells are primed by treatment with low-dose LPS, IFN-y, and possibly IL-2. Primed macrophages can be triggered to an activated state by many other cytokines. Activation of macrophages has classically been defined by demonstrating augmented effector functions as shown.

550

MICHAEL A. WEST

ment, lymphokine, or IFN-y. The primed macrophage shows alterations of cell surface receptors and increased metabolic activity and is susceptible to triggering agents that further stimulate it to an activated state. Various inflammatory stimuli have been used in vitro to trigger primed macrophages to states of complete activation. In thc ensuing discussion, data will be reviewed showing that many cytokines are also potent triggering agents. Nonetheless, triggering agents are generally unable to activate macrophages unless preceded by macrophage priming. The following discussion will attempt to review the current state of knowledge of the effects of cytokines on these activated macrophage functions.

A. Cytokine Effects on Macrophage-Monocyte Production and Differentiation Early studies indicated that IL- 1, or supernatants containing IL-1-like activity, enhanced bone marrow production of monocytic cells (29). With the availability of recombinant cytokine probes, the specific role of direct 1L1 stimulation of bone marrow was investigated. Recombinant cytokines that clearly stimulate bone marrow monocyte production include the colony stimulating factors (3841,91). In one study, intravenous treatment of mice with CSF-1 resulted in a tenfold increase in the circulating population of mature monocytes (91). Increased numbers of differentiated macrophages within the liver and in the peritoneal cavity were also seen following CSF- 1 treatment. Treatment with GMCSF was also found to result in enhanced bone marrow production of monocytic cells (91). IL1 (92) and TNF (93) also have been found to stimulate fibroblast production of GM-CSF. On the other hand, CSF treatment of macrophages enhanced IL- 1 production (68). Thus, a self-perpetuating feedback loop could be induced by IL-1 treatment in the presence of both cell types or following treatment in vivo. In light of these findings, it is possible that carlier results with crude IL- 1 may have been due to the presence of contaminating colony stimulating factors or 1L-1-mediated induction of CSF production. TNFa and TNFP were found to suppress the GM-CSF and CSF stimulation of bone marrow monocyte production (94). In addition to their role in monocyte production, cytokines control several aspects of monocytic differentiation and expression of cell surface antigens. CSF-1 treatment stimulated proliferation and differentiation of macrophages from monocytic precursors (94). CSF- I treatment also resulted in increased macrophage expression of Fc receptors and la antigen. These effects could be mimicked by IFN-PI or IFN-y (94), suggesting that CSF-1 -mediated macrophage differentiation may occur via release of macrophage interferons (94). Interferon-y treated macrophages displayed increased expression of Fc receptors (95), la antigen (45),and lymphocyte function associated antigen- 1 (LFA- I ) (95). IFN-y also resulted in amplification of IL-2 receptor (IL-2R) expression

20. CYTOKINES IN PHAGOCYTE ACTIVATION

551

(96,97) and inhibition of monocyte migration (96). Treatment of macrophages with recombinant IL-2 produced conflicting results. One study showed some induction of IL-2R on human lung macrophages and blood monocytes (Y7), whereas other investigations showed no significant IL-2 induced expression of IL-2R on human peripheral blood monocytes (59). IL-2 may enhance the number of IL-2R via production of other cytokines, possibly macrophage-derived interferon ( I ). Monocyte differentiation has also been examined using the human monocyte cell line UY37 (98,99). Cytokines have been found to be required to inhibit U937 proliferation and to permit these cells to acquire the differentiated functions of macrophage-like cells (98). The cytokine responsible for this differentiation has not been identified with certainty but is apparently distinct from IFN-y, TNFa, or GM-CSF (100). Various combinations of these cytokines were ineffective at arresting UY37 proliferation, whereas a 45-55 kDa heat-resistant T cell derived factor arrested UY37 growth for up to 5 days (100). The nature of this 45-55 kDa factor is undetermined, but it may be related to other growth inhibiting cytokines. In summary, the various colony stimulating factors have the greatest effects on macrophage production and differentiation. IL- I and TNF may also play a role although they may ultimately act via production of CSFs. Interferons may have physiologically important roles in macrophage differentiation, but the effects of several other major cytokines remain unknown at present.

B. Cytokine Effects on Macrophage Recruitment, Adhesion, and Chemotaxis Tissue macrophages exist at sites of potential infections and therefore do not need to be recruited to these sites. Like neutrophils, circulating monocytes must first adhere to the vascular endothelium and then migrate out into the tissue. Cytokines that promote this adhesion and monocyte recruitment include TNF (101), 1L-l (29, I O I ) , and 1L2 (102,103). While the mechanisms of cytokinemediated macrophage recruitment have not been completely elucidated, cytokine effects on endothelial cells predominate (101, 104). Endothelial cells exposed to endotoxin synthesize large quantities of IL-la and IL-IP (1) and express messenger RNA for these molecules (101). Treatment of endothelial cells with either IL-I or TNF resulted in expression of a 100-120 kDa polypeptide that was associated with increased cell adhesion (105). Induction of this cell surface antigen was dose-dependent, transient, and appeared to be induced independently by either cytokine. TNFa and TNFP also induced endothelial cell procoagulant activity and increased adhesiveness for lymphocytes, monocytes, and neutrophils ( 104). These TNF-mediated effects required endothelial cell RNA synthesis and were inhibited by IL- la or IL- 1 p ( 1 04). TNF itself has been shown

552

MICHAEL A. WEST

to stimulate cndothelial cell IL-I synthesis (94). The balance of I L I and TNF regulatory effects may thus deterniine endothelial cell adhesive properties. Several other cytokines also affect monocyte recruitment and motility. IFN-y is a potent stimulus of macrophage migration inhibition activity (106). Inhibition of macrophage movement required a two-stage treatment with low-dose IFN-y or exposure to IFN-y followed by endotoxin (106). Migration inhibition has previously correlated with significant degrees of macrophage stimulation (5). IL-2 resulted in a marked influx of monocytes and PMNs following injection into the peritoneal cavity (102). The mechanism of 1L-2 phagocyte recruitment is unknown but may involve I L 2 triggered synthesis of other cytokines. Finally, IFN-y has been shown to influence the barrier function of epithelial cells (107). Monolayers of intestinal epithelial cells treated with IFN-y displayed a markedly decreased electrical resistance, presumably due to IFN-y-mediated modifications of tight junction permeability. No effect on epithelial monolayer tight junction permeability was demonstrable with several other cytokines, including IL- 1, 1L-2, or TNF (107). Several cytokines have very prominent effects on monocytc-macrophage adhesion to endothelial cells and rccruitment. Particularly marked effects were seen with IL-2, but TNF and 1L-1 had effects as well. These cytokines acted via changes in the macrophages themselves as well as effects on endothelial cells. IFN-y was found to have only minor effects on these functions. Unfortunately, there is no good information concerning the responses obtained with less well studied cytokines on macrophage recruitment, chemotaxis, or adhesiveness.

C. Cytokine Effects on Macrophage Microbicidal Mechanisms Monocytes and macrophages play a prominent role in the host defense against microorganisms (5,19). One definition of complete macrophage activation has traditionally been macrophage-mediated destruction of intracellular parasites such as Leishmania (108) and Trypanosoma (109). Macrophage activation for destruction of intracellular parasites required high levels of endotoxin in the absence of cytokines. Several cytokines have been shown to induce macrophage microbicidal mechanisms, even in the absence of endotoxin. Induction of microbicidal mechanisms in macrophages by IFN-y has been more thoroughly investigated than other cytokines (109,110). Treatment of macrophages with IFN-y resulted in marked growth inhibition of several strains of Mycohacterium fuberculosis (1 lo), but other strains were resistant to IFN-yactivated macrophage destruction. Treatment of peritoneal macrophages with IFN-y induced fungicidal activity against both Candidu albicans and Blastomyces dermatitis (1 1 I). In contrast, IFN-y treatment of pulmonary alveolar macrophages did not yield significant canctictacidal activity ( 1 1 1). The presence

20. CYTOKINES IN PHAGOCYTE ACTIVATION

553

of IFN-)I was also found to be necessary in order for macrophages to kill the intracellular parasite Tuxuplusma gondii ( I 12). Treatment of macrophages with lymphokine-rich supernatant had previously been shown to involve a two-stage activation mechanism (1 13). More recent investigations have revealed that IFN-y was a necessary component of this lymphokine supernatant but, by itself, was insufficient for complete macrophage activation (1 14). Priming with IFN-y resulted in complete macrophage activation when followed by treatment with GMCSF, I L 2 , and IL-4 (109). Not all cytokines can trigger IFN-y primed cells, however; IL-I, IL-3, CSF-I, and IFN-a or p were unable to trigger IFN-yprimed macrophages ( 109). In contrast, treatment with high-dose GM-CSF alone completely activated macrophages for Leishmania fropica destruction ( 108). Macrophage microbicidal inhibition or destruction may involve generation of active oxygen intermediates (28,115),reactive nitrogen intermediates (1 16), or tryptophan degradation products (1 17). Tryptophan degradation is catalyzed by indolamine 2,3-dioxygenase (IDO). Treatment of peripheral blood monocytes with IFN-y, 1L-2, or IFN-P resulted in significant induction of monocyte I D 0 activity (1 17). The presence of LPS was required for maximum monocyte I D 0 activity by IFN-f3 ( 1 17). Intracellular infections of mononuclear phagocytes with viruses occur in addition to infections with bacteria or parasites, and cytokines affect intracellular viral replication. In vitru treatment of monocytes with GM-CSF increased production of human immunodeficiency virus type 1 (HIV-1) (118). In contrast, treatment with IFN-y resulted in inhibition of viral replication within the mononuclear cells. Differential effects of IFN-y were observed depending upon the timing of IFN-y treatment relative to HIV infection. Prominent IFN-y mediated inhibition of viral replication occurred when administered after HIV- 1 infection, whereas IFN-y pretreatment resulted in a slight enhancement of viral replication ( I 18). Intracellular parasites have evolved mechanisms that circumvent some macrophage microbicidal mechanisms. Destruction of these microorganisms is a prototypic function that defined complete macrophage activation. Several cytokines, including GM-CSF, 1L-2, IL-4, and to a lesser extent, TNF, can trigger macrophages to destroy microorganisms. IFN-y is probably the physiologically most important cytokine that primes macrophages, but GM-CSF, and possibly IL-2, may also function similarly. No significant inhibitory effects on microbicidal functions have been noted with cytokines. D. Cytokine Effects on Macrophage Tumor Cytotoxicity Macrophage-mediated tumor cytotoxicity represents another function which historically defined complete macrophage activation (6-8). The two-state theory of macrophage activation (90) was most clearly delineated by assaying tumor cell

554

MICHAEL A. WEST

cytotoxicity. Treatment of macrophages with supernatant from activated lymphocytcs was originally shown to be required for macrophage tumor cytotoxicity (3). The macrophage activating factor(s) (MAF) present in lymphokine-rich supernatant has been shown to be mediated by various cytokines (3). IFN-y is the macrophage activating factor which has been the subject of the most extensive investigation (45). IFN-y treatment of macrophages renders then1 sensitive to second signals, such as bacterial lipopolysaccharide (1 19) and various cytokines described subsequently. A specific receptor for IFN-y is present on macrophages and presumably triggers other second messenger molecules within the macrophage (120). Macrophage treatment with IFN-y caused elevation of cellular protein kinase activity and phosphorylation of cytoplasmic proteins ( I2 1 ). Phorbol esters and calcium ionophore A23 187 also elevated cellular protein kinase activity and intracellular calcium and have been shown to mimic the effects of IFN-y (50). Furthermore, intracellular calcium chelators inhibit the e&ct of IFN-y priming of macrophages (50). These data suggest that IFN-y binds to specific cell surface receptors and ultimately stimulates macrophages via second messenger pathways involving protein kinases. In most systems studied, IFN-y was a necessary, but inadequate, stimulus for complete macrophage activation ( I 22,123). Treatment of endotoxin unresponsive C3H/HeJ mice with IFN-y rendered them cytotoxic when triggered with endotoxin ( 123). IFN-y treatment enhanced macrophage oxidative metabolism, which may be one mechanism of tumor cytolysis (124). Although IFN-y was ablc to primc macrophages, other cytokines were again found to be necessary to trigger the IFN-y primed macrophages. IFN-a, and IFN-P had no effect when administered alone but completely activated macrophages primed with IFN-y (120) If."-a exists in a variety of active forms with a broad spcctrum of biological activities ( 1 25). Some typcs of IFN-a inhibited IFN-y-mediated macrophage activation ( 125). IFN-a inhibition of IFN-y macrophage priming depended upon the timing of IFN-y stimulation. Some IFN-a subtypes administered as long as 4 hr al'ter IFN-y inhibited tumor cytolysis, while IFN-a treatment 24 hr after IFN-y had no inhibitory effect. Prostaglandin E, (PGE,) was also found to inhibit macrophages activated for tumor cytolysis with endotoxin ( 1 26), but PGE, was unable to inhibit macrophages primed with IFN-y or IFN-a and triggered with endotoxin (127). High doses of IFN-a alone completely activated macrophages and maintained this activated state in the presence of PGE, (127). 1L-1 and TNF also triggered IFN-y primed macrophages for tumor cytolysis (2,29). TNF and IL-l are cytokines released by macrophages in response to LPS triggering (2,22,29). It was postulated that all thrcc cytokines may need to be present to induce tumor cytotoxicity by macrophages ( 128). Treatment with either TNFa or TNFP alone did not induce significant macrophage cytoxicity ( 129). However, either species of TNF could substitute for LPS to fully activate macrophages. Treatment of macrophages with IFN-y and TNF in the presence of

20. CYTOKINES IN PHAGOCYTE ACTIVATION

555

monoclonal antibodies to IL-1 resulted in markedly decreased cytotoxicity (128) vs. the results with no anti-IL-I. Treatment of macrophages with IFN-y and recombinant IL- 1 also resulted in high levels of tumor cytotoxicity, but minimal cytotoxicity was seen with the same treatment in the presence of monoclonal antibodies to TNF (128). Thus, all three cytokines may be required for induction of complete macrophage activation and tumor cytotoxicity. Treatment with cytokines such as IFN-y, TNF, or IL-I has pronounced effects on macrophage cytokine synthesis and release (see Section V,F). Macrophage priming with IFN-y has been examined extensively, but other cytokines can substitute for IFN-y to prime macrophages for tumor cytotoxicity. These cytokines include IL-4 (64), M-CSF (130), CSF-1 (130), and GM-CSF (127). CSF-1 was found to be the least potent stimulus and required very high triggering levels of lipopolysaccharide to confer significant tumor cytotoxicity (1 30). CSF- 1 was found to synergize with IFN-y in the presence of LPS (1 3 I). Enhanced tumor cytotoxic activity with M-CSF pretreatment was associated with induction of macrophage TNF synthesis ( 1 32). Treatment with M-CSF alone was incapable of activating macrophages for tumor cytotoxicity, whereas treatment with GM-CSF resulted in significant tumor cytotoxicity even in the absence of LPS (130,131). The effects of cytokines on macrophage tumor cytotoxic capacity have been extensively studied. Tumor cytolysis, like destruction of intracellular parasites, has been a function of stimulated macrophages that has traditionally defined a state of complete cellular activation. Most cytokines investigated are capable of triggering primed cells to destroy tumor cells in vitro, but particularly significant responses have been noted with IL-I. Interferons, particularly IFN-y, and the CSFs, probably function to prime mononuclear phagocytes in vivo. Whether cytokines actually participate in vivo in tumor cell destruction is unknown but probably occurs. Several clinical trials have shown significant tumor regression after treatment with recombinant IL-2 or IFN-y.

E. Cytokine Effects on Macrophage Arachidonic Acid Synthesis Activated macrophages secrete large quantities of arachidonic acid metabolites, both prostaglandins and leukotrienes (1 33). The specific arachidonic acid metabolites released vary with the stimulus utilized ( 133). Relatively little information concerning the effects of cytokines on monocyte-macrophage production of arachidonic acid metabolites is available. Treatment of monocytes with either recombinant TNFa or recombinant IL- 1 markedly increased prostaglandin E, synthesis in a dose-dependent manner ( 134). In other investigations, IL- 1/3 was found to be as potent as LPS in triggering release of both PGE, and thromboxane B, (TxB,) by monocytes (135). Cytokines may also play a role in regulation of

556

MICHAEL A. WEST

arachidonic acid synthesis. IFN-y trcatment was found to inhibit prostaglandin release in response to IL- I ( I 35) and LPS ( 136). IFN-y has not been shown to stimulate macrophage arachidonic acid metabolite secretion in the absence of other stimuli ( 135). Nonethelcss, IFN-y served as a potent priming stimulus to macrophage prostaglandin secretion in rcsponse to subsequent PMA triggering (135). However, IFN-y had no significant priming cffccts for arachidonic acid synthesis in response to zymosan, calcium ionophore A23 187, or antibody complexes ( I 35). The effects of many cytokines on macrophage synthesis and release of prostaglandins and leukotrienes remain unknown. IL- 1 and TNF stimulate arachidonic acid metabolism, and in general, IFN-y appears to have prominent inhibitory effccts. The role of cytokines in arachidonic acid metabolism by macrophages is complicated by the fact that both types of mediators are often produced simultaneously. Future studies should clarify the mechanisms involved and better dcfine the role of cytokincs in macrophage arachidonic metabolism.

F. Cytokine Effects on Macrophage Cytokine Synthesis Treatment of monocytes or macrophages with stimuli such as bacterial products and lipopolysaccharides stimulatcs these cells to releasc various cytokines (2,5,22). When macrophages were treated in vitrt, with LPS, there was a coordinated secretory protein response (22, I37), with synthesis of some secretory proteins increased and others simultaneously depressed. Synthesis of dozens of proteins was coordinately regulated following LPS treatment, but the functions of only a small number of these are known at present (22,137). It is possible that many of these secrcted proteins are cytokines whose function in intracellular communication is not yet known. Many investigations of macrophage activation have utilized the C3H/HeJ endotoxin-resistant strain of mice (9,138). Macrophages from these mice d o not synthcsize cytokines such as TNF or 1L-I in response to LPS. It was found that IFN-y-trcated C3H/HeJ mice secreted TNF and 1L- I and manifested tumor cell cytotoxicity in response to endotoxin (138,139). In vivu administration of LFN-y also increased TNF production in normal mice (140). Aerosol administration of IFN-y resulted in increased IL-1 production by alveolar macrophages and circulating blood monocytes (141). In most in vitro studies, IFN-y treatment of macrophages alone was insutficient for IL-I or TNF secretion (2,142) but may play a counter-regulatory role in cytokine production. In vitru treatment of PRMNC with IFN-y markedly inhibited LPS stimulated 1L-1 production (143). lFN-y was also found to inhibit IL-la-induced IL- I p production and I L I fiinduccd 1L-la release (144). These effects of IFN-y were not mediated via induction of prostaglandin synthesis because no effects of cyclooxygenase inhibitors were noted ( I 44).

20. CYTOKINES IN PHAGOCYTE ACTIVATION

557

1L-2 is a cytokine produced by T lymphocytes in response to 1L-1 stimulation ( 3 ) . It is now clear that IL-2 has significant effects on monocyte-macrophage cytokine production (2,s). Treatment of PBMNC with recombinant human IL-2 resulted in production of TNFa within 2-3 hr and TNFP within 8 hr (145). In other investigations IL-2 treatment of PBMNC was found to increase synthesis of IL-la and IL-1P but had a relatively minor effect on TNFa production (51). Pretreatment with IFN-y markedly enhanced 1L-2 stimulated monocyte synthesis of both TNF (145) and IL- 1 (5 1). The synergistic effects of IL-2 and IFN-y may have taken place via IFN-y stimulation of IL-2 receptor expression (51,96). 1L-2 treatment of PBMNC also induced supernatant activity which stimulated acute phase protein synthesis by hepatoma cells (146). This I L 2 triggered effect was blocked with anti-IL- Ij3 antisera, whereas anti-IL- Icu and polyclonal anti-TNF had no effect (146). It is now clear that IL-I (36) and TNF (36,147) increase fibroblast and monocyte production of IFN-P,, the major cytokine stimulating hepatic acute phase protein synthesis (36,37). The precise cytokine responsible for effects observed in vivo is often difficult to delineate. In the preceding example, IL-2-mediated effects on hepatocyte acute phase protein synthesis could be due to IL-2 stimulated production of L I P , which in turn stimulated fibroblast or macrophage production of IFN-P,. IFN-P, might then be the final cytokine responsible for hepatic acute phase protein synthesis (37). In vivo treatment of humans with IL-2 was found to enhance production of TNF and IL-2 (148). In a similar manner, in vivo treatment o f mice with IFN-y, IFN-a, and IFN-P also stimulated TNF production (145). Treatment of monocytes with recombinant TNF-a in vitro stimulates IL- I synthesis and secretion ( 134). Both TNFa and TNFP have been shown to induce IL-1 gene transcription (149). In vivo administration of TNFa stimulated IL-1 production by both circulating blood mononuclear cells and alveolar macrophages (141). In addition to IL-1 induction, TNFa and P induced transcription and production of GM-CSF and M-CSF (149). TNF administration to animals under certain conditions has been associated with a high mortality (150). These lethal effects of TNF could be blocked with antibody to TNF (150). Subsequent investigations have shown that in vivo treatment with recombinant TNF in low doses desensitizes animals to the lethal effects on both TNF and LPS (151). Similar effects were seen following in vivo desensitization to I L l a (15 1). On the other hand, IL-la stimulates monocyte synthesis, and release of IL- l p and IL-IP was found to increase secretion of IL-la (144). This IL-1 stimulated PBMNC synthesis of IL-1 could be inhibited by IFN-y treatment (144). The effects of other cytokines on macrophage cytokine production have not been studied in this same detail. IFN-a was found to augment LPS-induced IL-I production by thioglycolate elicited peritoneal exudate macrophages (152) but had no effect on IL-1 synthesis in the absence of LPS. TGFP treatment of murine monocytes significantly inhibited TNFa production (65).

558

MICHAEL A. WEST

In summary, cytokines have significant dose and timing dependent effects on macrophage activation and cytokine production. Several cytokines appear to function via a classical negative feedback loop to inhibit their own synthesis. In many cases, synergistic stimulation of macrophage activation state and cytokine production occurs following treatment with combinations of cytokines. IL-2 and IFN-y stimulate production of most cytokines when macrophages are subsequently triggered with appropriate stimuli. IFN-y also has prominent inhibitory effects, particularly on TNF and 11,-I production. TGFP may also function to inhibit macrophage cytokine synthesis. It is very likely that cytokines, in conjunction with other inflammatory stimuli, play a significant physiological role in regulating macrophage cytokine synthesis and release.

G. Cytokine Effects on Macrophage Arginine-Dependent Effector Mechanism A novel macrophage cytotoxic cffcctor mechanism has been described which requires high levels of macrophage activation in the presence of 1.-argininc ( 152). This cffcctor mechanism resulted in cytotoxicity for certain susceptible turnor cell lines and cytostasis of other tumor targets (152). A characteristic series 0 1 metabolic changes was induced in target cells via this rncchanisni which includcd inhibition of mitochondria1 respiration (153), inhibition of the citric acid cycle enzyme aconitase (152), and inhibition of DNA synthesis (152). Expression of this macrophagc-dependent effector mechanism required the presence of r<-argininein the microenvironment. Cytostasis or cytotoxicity via this mechanism could be competitively inhibited with an arginine analogue, NG-monomethyl-~-arginine(N"-MMA) (152). This arginine-dependent effector niechanism results in synthesis of short-livcd reactive nitrogen intermediates, demonstrated by increased production of nitrites and nitrates from activated macrophages ( I 16). Lymphokines and IFN-y were both found to stimulate increased macrophage nitrite and nitrate synthesis ( 1 16). IFN-y enhanced macrophage nitrite-nitrate synthesis to a greater extent than LPS or heat killcd Bucillus culmette-Ruerin (BCG) ( I 16). Various other cytokines, including IFN-a, IFN-P, TNFa, TNFP, GM-CSF, M-CSF, IL-lP, 1L-2, 1L-4, and TCFP, had no effect on release of reactive nitrogcn intermediates by mouse peritoneal macrophages (154). As with other macrophage functions, IFN-7 was found to be necessary, but by itself was an insufficient stimulus, for L-arginine-dependent tumor cell cytotoxicity (1 53). The presence of both IFN-7 and LPS was needed for significant arginine-dependent rnacrophage tumor cytotoxicity. Induction of the L-arginine-dependent effector mechanism was transferable with supernatant from activated macrophages (155). In addition to its effect on tumor cell growth, the L-arginine-dependent

20. CYTOKINES IN PHAGOCYTE ACTIVATION

559

effector mechanism may also influence the function of normal parenchymal cells. Inhibition of hepatocyte protein synthesis (156,157) by activated Kupffer cells was shown to be mediated by the arginine-dependent effector mechanism (158). Earlier experiments had shown that IFN-y and IL-2 synergized with LPS to activate Kupffer cells for inhibition of hepatocyte protein synthesis ( 159). In contrast, IL-I and TNF were found to have no effect on stimulation of Kupffer cells to inhibit hepatocyte protein synthesis in the presence or absence of LPS ( 160). Thus, IFN-y is the only cytokine found to significantly augment the argininedependent effector mechanism. In contrast to other functions of stimulated macrophages, the effect of most recombinant cytokines on the arginine-dependent effector mechanism has been examined but was found to be insignificant. The role of the arginine-dependent mechanism in vivo is unknown at present.

H. Effective Cytokines on Macrophage Oxidative Mechanisms Activated macrophages have enhanced oxidative metabolism and secrete increased quantities of reactive oxygen intermediates, including hydrogen peroxide and superoxide radical ( 1 15). Immune complexes (161), complement (115), and LPS have been shown to increase in vitro macrophage oxidative metabolism. Cytokines have now also been found to have significant effects on macrophage oxidative metabolism. IFN-y augmented monocyte chemiluminescence in response to other inflammatory stimuli (1 6 1 ) and triggered resident peritoneal macrophages to release increased quantities of hydrogen peroxide ( 154). Hydrogen peroxide release by resident macrophages was also increased by treatment with TNFa, TNFP, and GM-CSF (154). Other cytokines were not able to stimulate hydrogen peroxide release by resident cells, but all resulted in enhanced hydrogen peroxide release by elicited peritoneal macrophages ( 154). In other investigations, superoxide production by alveolar macrophages was markedly increased following exposure to IL-I or TNF ( 1 15). In these experiments, cytokines synergized with immune complex stimulation to maximally activate the alveolar macrophages. As with other macrophage functions, IFN-y is a potent primer of macrophage oxidative metabolism. Most cytokines have minimal direct effects on macrophage oxidative metabolism, but virtually all cytokines can trigger appropriately stimulated cells, especially at high concentrations. TNF has the most marked effects and therefore may have a role in stimulating this pathway in vivo. No cytokines have been noted to inhibit mononuclear phagocyte oxidative pathways, and therefore cytokines probably do not function as important negative regulatory mediators.

TABLE V EFFECTOF MAJORCVTOKINES O N MACROPHAGE-MONOCYTE .~CTIVATION~' ~~

~

Cytokine synthesis

Recruitment Cytokine

adhesion

-

IL-la IL- 1p IL-2 IL-4 IFN-a IFN-f3 I IFN-P2 (IL-6) IFN-y M-CSF, CSF-1 GM-CSF TNFa TNFp TCFB

Microbicidal

Tumor

mechanisms<

cytotoxicitj

Oxidative mechanismsh

Arginine rnechani\m

Arachidondte

synthesis

~

+++ +++ +++ ?

++ ++ ++t ?

No h No h

+++ (T) +++ (T)

?

+ + (T)

?

+ + (P)

++ (T) or - -

No h No h No A

++ (TI

No . ! I

No A No A No h

?

9

9

?

+ or -

+ + (TI No A No A

?

?

?

+

+t+

1

?

?

1

+++ >

?

++

+ ++ ++

_-

or

---

+++ (PI No h + + (TI

+++ (Pj + 07

+ + (P) + + (TI

+

+ + (TI ++ (TI

7

?

t

No h o r

+

?

No D

NoAor

+++ (P)

+++

No h No A

No No No No No

+ + + (T) + + + (T)

No h

++

++ ++ ? ? ?

A h

h h h

See text for specific references. Stimulation shown as + to + + +; inhibition, - to ---; N o A, no effect of cytokine: '?,insufficient data. Abbrekiations: IFN. interferon: TKF, tumor necrosis Cacror; IL. interleukin; CSF. colony stimulating factor; GM-CSF. granulocyte macrophage-colony stimulating factor. TGFP. transforming growth factor p. T indicates triggering activity; P indicates priming activity. 0

h

20. CYTOKINES IN PHAGOCYTE ACTIVATION

561

1. Summary: Cytokine Effects on Macrophage Activation The role of cytokines in stimulating various macrophage functions continues to be the subject of intensive investigation. The effects of cytokines on various functions associated with activated macrophage states are summarized in Table V. Note that there are still significant gaps in our understanding of cytokine effects on macrophage functions. Cytokines have been found to both stimulate and inhibit many macrophage functions in vitro. Thus, cytokines can appropriately be considered to be physiologically relevant regulators of the in vitro activation state of macrophages. Evidence suggests that cytokines may be the actual mediators responsible for complete macrophage activation, rather than direct stimulation by microbes or microbial components. All macrophage functions examined in vitro, previously associated with elevated functional states above those of unstimulated cells, have now been found to be altered by cytokines. The ultimate role cytokines play during in vivo macrophage activation remains unknown.

VI. SUMMARY: CYTOKINE EFFECTS ON ACTIVATION OF PHAGOCYTIC CELLS It is now appreciated that cytokines are ubiquitous messenger molecules, produced not only by inflammatory cells but also by a wide variety of different cell types throughout the body. Cytokines have been shown to have effects on most in v i m functions of both neutrophils and monocytes-macrophages. Typically, the effector functions of phagocytic cells require stimuli that activate these cells. This review has attempted to review information known about the effects of cytokines on phagocytic cellular functions associated with activation. Cytokines represent a more physiological stimulus for phagocytic cell activation compared to the biochemical stimuli used in vitro to define phagocyte activation states. Cytokines have been shown to both stimulate and inhibit activation of phagocytes. Furthermore, the timing, dose, or combinations of cytokines to which phagocytic cells are exposed markedly influence the type, duration, and degree of phagocytic cell effector response. Cytokines represent an important part of the language of the inflammatory response, and additional studies should more clearly define the precise role cytokines play in controlling in vivo phagocytic cell function. REFERENCES

I . Smith, E. M. (1988). Hormonal activities of lymphokines, monokines, and other cytokines. P r q . Allergy 43, 121-139.

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opment of macrophage cytotoxic activity requires conipletion of a sequence of short-lived intermediary reactions. J . Imrnunol. 121, 2035-2042. 114. Davis, C. E . , Belosevic, M., Meltzcr, M. S . , and Nacy, C . A. (1988). Kegulation of activated macrophage antimicrobial activities. Cooperation of lymphoines for induction of resistance to infcction. J. Immiinol. 141, 627-635. 115. Warren, J. S . , Kunkel, S . L., Cunningham, T. W., Johnson, K. J.. and Ward, P. A. (1988). Macrophage-derived cytokines amplify immune complex-triggered O2 responses by rat alveolar mdcrophagcs. Am. J . Pulhol. 130, 489-495. 116. Stuehr, D. J., and Marletta, M. A. (1987). Induction of nitriteinitrate synthesis in murine macrophages by BCC infection, lymphokines, or interferon-,. J . Immitnol. 139, 51 8-525. 117. Carlin. J. M . , Borden, E. C., Sondel, P. M., and Byme, C. I . (1989). Interferon-induced indoleaminc 2,3-diuxygenase activity in human mononuclear phagocytes 45. 29- 34. 118. Koyanagi, Y . , O’Brien, W. A,, Zhao, J. Q . , Golde, D. W., Gasson, J. C . , and Chen, I . S . Y. ( I 988). Cytokines alter production of HIV- 1 from primary mononuclcar phagocytes. Science 241, 1673-1675. 119. Pace, J. L., and Russell, S . W. (1981). Activation of mouse macrophages for tumor cell killing: I. Quantitative analysis of interactions between lymphokine and lipopolysaccharide. J . Immuno/. 126, 1863-1867. 120. Sheehan. K . C. F., Calderon, J., and Schreiber, R. D. (1988). Generation and ChardctCri.LdtioII of monoclonal antibodies specific for the human IFN-, receptor. J. Irnmunol. 140,4231-4237. 121. Hamilton, T. A., Becton, D. L., Somcrs, S. D., Gray, P. W., and Adams, D. 0. (1985). Interferon-, modulates protein kinase C activity in murine peritoneal macrophages. J. B i d . Chem. 260, 1378-1381. 122. Pace, J. L . , Russell, S. W., LeBlanc. P. A , , and Murasko, D. M. (1985). Comparative eKects of various classes of mouse interferons on macrophagc activation for tumor cell killing. J . Immunol. 134, 977 - 98 1 . 123. Chapes, S . K . , Killion, J. W., and Morrison, D. C. (1988). Tumor cell killing and cyto C3HIHeJ macrophages activated in vi/ru by lipid A-associated protein and interferon gamma. J . 1.rukocyre R i d . 43, 232-237. 124. Nathan, C. F., Murray, H. W., Wiebe, M. E., and Rubin, B. Y. (1983). Identification of interferon-y as the lymphokine that activates human macrophage oxidative metabolism and antimicrobial activity. J . Exp. Med. 158, 670-689. 12.5. Russell, S . W., and Pace, J. L. (1984). Both the kind and magnitude of stimulus are impurtant in overcoming the negative regulation of macrophage activation by PGE2. J. Leukocvre B i d . 35, 291-301. 126. Taffet, S . M., Pace, J. L., and Russell, S . W. (1981). Lymphokine maintains macrophage activation for tumor cell killing by interfering with the negative regulatory effect of prostaglandin E,. J. Immunol. 127, 121-124. 127 Browning, I. L. and Riholini. A. (1987). Interferon blocks interleukin I-induced prostaglandin release from human peripheral monocytcs. J . Irnmunopl. 138, 2857-2863. 128. Chen, I.. , Suzuki, Y., and Wheelock, E. F. (1987). Interferon-, synergizes with tumor necrosis factor and with interleukin-I and requires the presence of hoth monokines to induce antitumor cytotoxic activity in macrophages. J . Immunol. 139, 4096-4101. 129. Hcidenrcich, S . , Wcycrs, M., Congg, J.-H.. Sprenger, H., Nain, M., and Gemsa, D. (1988). Potentiation of lymphokinc-induced macrophage activation hy tumor necrosis factor-a. J . Immunol. 140, 151 1-1588.

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130. Sampson-Johannes, A . , and Carlino, J. A. (1988). Enhancement of human monocyte tumoricidal activity by recombinant M-CSF. J. Inimrrnol. 141, 3680-3686. 131. Falk, L. A., Hogan, M. M., and Vogcl, S . N . (1988). Bonc marrow progenitors cultured in the presence of granulocyte-macrophage colony-stimulating factor versus macrophage colonystimulating factor diffcrcntiate into macrophages with distinct tumoricidal capacities. J . Leukocyre Biol. 43, 47 1-476. 132. Grabstein, K. H . , Urdal, D. L . , Tushinski, R . J . , Mochizuki, D. Y . , Price, V. L., Cantrell, M. A , , Gillia, S., and Conlon, P. I. (1986). Induction of macrophagc tumoricidal activity by granulocyte-macrophage colony-stimulating Factor. Science 232, 506-508. 133. Bonney, R. J., and Humes, J. L. (1984). Physiological and pharmacological regulation of prostaglandin and leukotriene production by macrophages. J . Leukocvte B i d . 35, 1 - 10. 134. Dinarello, C. A , , Cannon, J. G.. Wolff, S . M . . Bernheini. H. A , , Beutler, B., Ceranii, A , . Figari, 1. S., Palladino, M. A , , Jr.. and O’Connor, 1. V. (1986). Tumor necrosis factor (cachectin) is an endogenous pyrogen and induces production of interleukin 1 . J . Exp. Med. 163, 1433-1450. 135. Browning, J. L., and Ribolini, A . ( 1987). Interferon blocks interleukin I-induced prostaglandin release from human periphcral monocytes. J . Imrnunol. 138, 2857-2863. 136. Hamilton, T. A., Rigabee, J. E . , Scott, W. A , , and Adams, D. 0. (1985). y-Interferon enhances the secretion of arachidonic acid metabolites from murine peritoneal macrophages stimulated with phorbol diesters. J. Immunol. 134, 263 1-2636. 137. Largen, M. T., and Tannenbaum. C. S. (1986). LPS regulation of specific protein synthesis in murine peritoneal macrophages. J. Immunol. 136, 988-993. 138. Beutler, B., Tkacenko, V., Milsark, I., Krochin, N., and Cerami, A. (1985). effect of y inteferon on cachectin expression by mononuclear phagocytes. Reversal of the Ips“ (endotoxin resistance) phenotype. J . Exp. Med. 164, 1791-1796. 139. Hogan, M. M.. and Vogel. S . N. (1988). Production of tumor necrosis factor by rlFNy-primed C3HIHeJ (Lps”) macrophages requires the presence of lipid A-associated proteins. J. Immimol. 141, 4196-4202. 140. Satoh, M., Inagawa, H., Shimddd, Y., Sum&, G . , Oxhima, H . , and Mizuno, D. (1987). Endogenous production of tumor necrosis factor in normal mice and human cancer patients by interferons and other cytokinea combined with biological response modifiers of bacterial origin. J . Biol. Response M o d i j 6, 512-524. 141. Debs, R. J., R c h s , H. J., Philip, R . , Montgomery, A. B., Brunette, E. N., Liggitt, D., Patton, J. S., and Shellito, J. E. (1988). Lung-specific delivery of cytokines induces sustained pulmonary and systemic imniunomodulation in rats. J . Immunol. 140, 3482-3488. 142. Gerrard, T. L . , Siegel, J. P., Dyer, D. R., and Zoon, K. C. (1986). Differential effects of interferon-a and interferon-y on interleukin 1 secretion by monocytes. J . Immunol. 138,25352540. 143. Brandwein, S. R. (1986). Regulation of interleukin I production by mouse peritoneal macrophages. J. B i d . Chem. 261, 8624-8632. 144. Ghezzi, P., and Dinarello, C. A. (1988). IL-l induces IL-I 111. Specific inhibition of IL-1 production by IFN-y. J. Immtmol. 140, 4238--4244. 145. Ncdwin, G . E., Svedcrsky, L. P., Bringman, T. S . . Palladino. M. A , , Jr., and Goeddcl, D. V. (1985). Effect of interleukin 2, interferon-y, and mitogens on the production of tumor necrosis factors Q and p. J . Imniunol. 135, 2492-2497 146. Mier, J. W., Dinarello, C. A., Atkins, M. B., Punsal. P. I . , and Perlmutter, D. H. (1987). Regulaton of hepatic acute phase protein synthesis by products of interleukin 2 (IL 2)-stimulated human peripheral blood mononuclear cells. J . Immunol. 139, 1268-1272. 147. Kohase, M . , Henriksen-DeStefano, D., May L. T.. Vilcek, J . , and Sehgal. F? B. (1986). Induction of &-interferon by tumor necrosis factor: A homeostatic mechanism in the control

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of cell proliferation. Cell 45, 649-666. 148. Michie, H. R . , Eberlein, T. J . , Spriggs, D. R . , Manogue, K. R., Cerami, A., and Wilmore, D. W. (1988). Interleukin-2 initiates metabolic responses associated with critical illness in humans. Ann. Surg. 208, 493-503. 149. Kaushansky, K . , Broudy, V. C., Harlan, J. M., and Adamson, J. W. (1988). Tumor necrosis factor-a and tumor necrosis factor+ (lymphotoxin) stimulate the production of granulocytemacrophage colony-stimulating factor, macrophage colony-stimulating factor, and IL-1 in vivo. J . Immunol. 141, 3410-3415. 150. Beutlcr, B., Milsark, 1. W., and Ccrami, A. C. (1985). Passive inimunication against cachectin/tumor necrosis factor protects mice from lethal effects endotoxin. Science 225, 869-87 I. 151. Wallach, D., Holtmann, H., Engelniann, H., and Nophar, Y. (1988). Sensitization and desensitization to lethal effects of tumor necrosis factor and IL-I. J . Immunol. 140, 2994-2999. 152. Hibbs. J. B . . Jr., Vavrin. Z..and Taintor, R. R . (1987). L-arginine is required for expression of the activated macrophage effeclor mechanism causing selective metabolic inhibition in target cells. J . Immunol. 138, 550-565. 153. Drapier, J. C . , and Hibbs, J. B., Jr. (1988). Differentiation of murine macrophages to express nonspecific cytotoxicity for tumor cells results in 1.-arginine-dependent inhibition of mitochondrial iron-sulfur enzymes in the macrophage effector cells. J . Immunol. 140, 2829-2838. 154. Ding, A. H., Nathan, C. F., and Stuehr, D. J. (1988). Release of reactive nitrogen intermediates and reactive oxygen intermediates from mouse peritoneal macrophages. J. Immunol. 141, 2407-2412. 155. Amber, 1. J., Hibbs, H. B., Jr., Taintor, R. R., and Vavrin, 2. (1988). The 1.-arginine dependent effector mechanism is induced in inurine adcnocarcinoma cells by culture supernatant from cytotoxic activated macrophages. J. Leukocyte B i d . 43, 187- 192. 156. West, M. A . , Keller, G . A., Cerra, F. B., and Simmons, R. L. (1985). Killed Escherichiu coli stimulates macrophage-mediated alterations in hepatocellular function during in vitro coculture: a mechanism of altered liver function in sepsis. Infecr. Immun. 49, 563-570. 157. Wcst, M. A , , Billiar, T. R . , Curran, R. D., Hyland, B. I . , and Simmons, R. L. (1989). Evidence that rat Kupffer cells stimulate and inhibit hepatocyte protein synthesis in witm by different mechanisms. Gustroenterology 96, 1572-1582. 158. Billiar, T. R., Curran, R. D., Stuehr, D. J., West, M. A , , Bentz, B. G . , and Simmons, R. L. (1989). An L-arginine dependent mechanism mediates Kupffer cell inhibition of hepatocytc protein synthesis in vitro. J . Exp. Med. 169, 1467-1472. 159. Curran, R . D . , Billiar, T. R., West, M. A , , Bentz, R . A,. andSimmons, R. L. (1988). Effect of interleukin-2 on Kupffer cell activation. Interleukin-2 primes and activates Kupffer cells to suppress hepdtocyte protein synthesis in vivo. Arch. Surg. 123, 1373- 1378. 160. West, M. A . , Billiar, T. R . , Mazuski, J. E., Curran, R. J . , Cerra, F. B., and Simmons, R. L. (1988). Endotoxin modulation of hepatocyte secretory and cellular protein synthesis is mediated by Kupffer cells. Arch. Surg. 123, 1400-1405. 161. Jungi, T. W., Lerch, P. G . , and Brcic, M. (1987). The effect of recombinant interferon-, on human monocyte-derived macrophages. Eur. J . Immunol. 17, 735-738.

CURRENI' TOPICS IN MEMBRANES AND TRANSPORT, VOLUME 35

Chapter 21 Protooncogene Expression following Lymphocyte Activation ROGER M. PERLMUTTER AND STEVEN F. ZIEGLER' Howard Hughes Medical Institute and Departments of Immunology, Medicine, and Biochemistry University of Washington Seattle, Washington 98195

I.

Introduction Alterations in Oncogene Expression in Stimulated T Lymphocytes A. c-abl B . 6~1-2 c. c-fos D. c-myb E. c-myc F. Other Protooncogenes 111. Alterations in Protooncogene Expression in Stimulated B Lymphocytes IV. Lymphocyte Activation Modulates Expression of the lck Gene V. Future Directions: The Underlying Complexity of Lymphocyte Activation References 11.

1.

INTRODUCTION

Investigation of the mechanisms responsible for regulating lymphocyte activation has focused on evaluating the pattern of expression of sets of protooncogenes whose products are believed to positively regulate cell growth in other systems. The oncogenes were originally identified through study of acutely transforming retroviruses that have transduced host sequences. Aberrant expression of these host genes under the control of viral transcriptional regulators results in neoplastic transformation (Varmus, 1984). The importance of the protooncogenes from which these activated viral elements were derived has been reinforced by 'Present address: Immunex Corporation, Seattle, WA 98101 571

Copyright 0 1990 by Academic Press, Inc. All nghrs of reproduction in any form reserved.

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ROGER M. PERLMUTTER AND STEVEN F. ZIEGLER

the finding that many of these genes are positioned at the breakpoints of chromosomal translocations in human tumors (e.g., uhl, myc) or are activated by point mutations in human malignancies (e.g.. r m ) . In addition, the viral oncogenes include elements derived from genes encoding growth factors (e.g., sis), growth factor receptors (e.g., erbB), or transcriptional regulators (e.g., erh”, .jun). The initial set of viral oncogenes has expanded to include a large number of elements that exhibit transforming activity when transfected into NIH 3T3 indicator cells. In addition. study of heritable human malignancies associated with interstitial chromosomal deletions has identified a new set of recessive oncogenes ( “anti-oncogenes”) that can suppress features of the transformed phenotype. Table I provides a classification of currently recognized protooncogenes. Impetus for the study of protooncogene expression in purified lymphocytes has derived from two sources. First, lymphocytes provide a plentiful supply of Go cells that can be readily induced to initiate DNA synthesis using defined stimuli. Since it is expected that most mammalian cells will share a common cell cycle circuitry, analysis of oncogene expression in stimulated lymphocytes has been pursued by cell biologists studying the control of proliferation. At the same time, immunologists interested in dissecting the lymphocyte activation sequence have used oncogene probes with the hope of gaining insight into the mechanism whereby antigen recognition stimulates lymphokine production and blast transformation. Interpretation of measurements of protooncogene mRNA abundance in populations of lymphoid cells before and after exposure to various stimulating regimens is subject to a variety of caveats which must be emphasized at the outset. Measurable changes in steady-state transcript levels of a variety of protooncogenes emerge from such studies. However, these changes are rarely, if ever, correlated with a known biological function, and hence such nieasurements cannot easily be placed in a framework that relates variations in protooncogene mRNA abundance to physiological responses. Some investigators, pursuing a TABLE I CLASSIFICATION OF PROTOONCOGENES Growth Factors:

s i s . int-2, hst

Growth Factor Receptors:

erb,.fms, ros. met, r d , ret, ncir, kit, sea, mas, tek Ha-rus, Ki-rcis. N-rus f g r , f Y n . hck, Irk. iyti* src. ycs.

GTP-Binding Proteins: Membrane-Associated Tyrosine Kinases: Scrim-Threonine Kinases: Nuclear Proteins: Other:

ubl, chl. t i k . pirw-l most niillruf

erhA\,my..tnyh. s k i bus, efs, crk, p53, pim-2, bcl-1,

fi~s* j i r r i ,

hcl-2

573

21. PROTOONCOGENE EXPRESSION IN LYMPHOCYTES

more general strategy, have used RNA subtraction approaches to isolate cDNA clones that define transcripts which increase in abundance during lymphocyte activation (Zipfel et al., 1989). This approach may indeed provide the tools for a systematic study of the genetic control of lymphocyte stimulation; however, it may prove difficult to identify which of these genes represent important regulators of the activation sequence or to define the functions of the products of these genes. Furthermore, although gene expression is frequently controlled at the level of mRNA abundance, a variety of translational and posttranslational modifications can dramatically affect the representation of active protein derived from a given amount of mRNA. Thus the catalogue of changes in oncogene mRNA abundance in activated lymphocytes provides only a first rudimentary glimpse of the complexity of the activation sequence in lymphocytes. In the following pages, we summarize what is known about oncogene expression in lymphocytes exposed to a variety of activating stimuli and attempt to deduce general patterns from these studies, which have been pursued in very heterogeneous experimental systems. We next illustrate the hazards in directly interpreting changes in oncogene mRNA abundance by considering changes in Ick mRNA and p5@" protein expression in activated T cells. These observations lead to a summary view of the underlying complexity of lymphocyte activation.

II.

ALTERATIONS IN ONCOGENE EXPRESSION IN STIMULATED T LYMPHOCYTES

Readers of other chapters in this volume will know that the process of lymphocyte activation has been most thoroughly dissected in the case of T lymphocytes, which can be readily propagated in vitro. Two distinct signals are required to provoke lymphokine expression and proliferation in purified T lymphocytes. The first of these is normally derived from the T cell antigen receptor and can be mimed using lectin mitogens, e.g., phytohemagglutinin (PHA) and concanavalin A (ConA). Provision of this stimulus results in rapid increases in intracellular free Ca2 concentrations. Although calcium ionophores will satisfactorily substitute for antigen in most cases (Imboden et al., 1985; Weiss et al., 1986), there is evidence that increases in intracellular Ca2 concentrations are not necessary to permit lymphokine expression in some T cell hybridomas (Sussman et al., 1988). Thus both antigen and calcium ionophore are probably triggering other responses in cultured T lymphocytes. T cells stimulated in vivo receive a second signal derived from antigen presenting cells. This signal can be replaced in vitro by addition of phorbol esters, which directly activate the Ca2 -phospholipiddependent protein kinase C (PKC). In analyzing the effects of lymphocyte activation on oncogene expression, it is important to consider the nature of the cell population being stimulated, as well as the details of the stimulation protocol. In +

+

+

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ROGER M. PERLMUTTER AND STEVEN F. ZIEGLER

addition, lymphokines, particularly 1L2, will stimulate accumulation of a presumably distinct set of transcripts in cultured lymphocytes. Thus oncogene expression manifested several hours after stimulation may result from a late effect of the initial activation sequence or may be provoked by autocrine stimulation from secreted lymphokines (Crabtree, 1989). A critical listing of changes in protooncogene expression resulting from lymphocyte activation follows and is summarized in Table 11.

A. c-abl The c-ah1 gene encodes a 150 kDa membrane-associated but wholly intracellular protein tyrosine kinase of unknown function (Witte et al., 1979). Originally identified in mutant form as the transforming element in the Abelson murine leukemia virus, translocation of c-ah1 to a site within the bcr gene is the characteristic lesion in human chronic myelogenous leukemia, resulting in the expression of a kinase product with altered structural and kinetic properties (Konopka et al., 1984). In the mouse, infection of bone marrow cells with Abelson virus characteristically provokes the development of B cell malignancies (Baltimore et al., 1979). Interestingly, the c-abl gene is expressed at relatively high levels in thymocytes (Witte et ul., 1979). Analysis of c-abl expression in stimulated lymphocytes has not been pursued to any great extent; however, Reed et al. (1986) demonstrated that c-ubl mRNA levels increased severalfold within 2 hr after PHA stimulation of peripheral blood mononuclear cells. The pattern of increase in c-uhl mRNA levels closely paralleled the increase in levels for a housekeeping gene, phosphoglycerate kinase. Hence, despite the efficacy of v-abl in transforming lymphoid progenitors, there is little evidence for specific regulation of c-abl transcript abundance in activated lymphoid cells.

B. bd-2 The majority of human B cell follicular lymphomas contain a t(14q32; 18q21) chromosomal translocation that juxtaposes the immunoglobulin heavy chain TABLE I1 T Cells TCR

Protooncogenc c-ubl

hcl-2 C -fos

c-myh c-my

IL-2R

-

ff

t t t t

early, stable immediate, transient in presence of IL-2 immediate, transicnt

B Cells

t t

early, stable inimediate, transient

t

immediate, stable

t

algM

+ BCCF

uBp3S

-

-

-

-

c)

t

t

-

T

21. PROTOONCOGENE EXPRESSION IN LYMPHOCYTES

575

locus with the bcl-2 transcriptional unit, resulting in the expression of high levels of bcl-2 transcripts. The bcl-2 gene encodes a putative 239 amino acid polypeptide that has no known catalytic activity. Nevertheless, overexpression of bcl-2 transcripts in the lymphocytes of transgenic animals results in follicular lymphoproliferation (McDonnell et al., 1989). Thus, there is reason to believe that the bcl-2 gene product may assist in regulating lymphocyte proliferation. Reed et al. (1987b) observed that transcription of the bcl-2 gene increases at least 15-fold after stimulation of peripheral blood lymphocytes with PHA, beginning at about 1 hr and reaching a peak level at about 6 hr after stimulation. This increased transcription rate is associated with comparable increases in steady-state levels of bcl-2 mRNA, reaching maximal levels at about 6-hr and remaining at this level for up to 5 days. Similar increases in bcl-2 transcript levels were also induced in T cell blasts made quiescent by IL-2 starvation and subsequently treated with I L 2 (Reed et al., 1987b). Two distinct promoter elements have been defined in the bcl-2 gene, the 5’-most element contains at least seven S p l promoter motifs and is separated by 370 kb from the second start site that contains a typical immunoglobulin variable region decanucleotide element (Seto er al., 1988). Experiments performed with protein synthesis blockade suggest that both positive and negative transcriptional regulators act to control the level of bcl-2 mRNA. It is not known whether sub-mitogenic stimulation of purified T lymphocytes with mitogens augments bcl-2 levels. In summary, while these experiments do not permit firm conclusions regarding the importance of bcl-2 in lymphocyte activation, they suggest that the bcl-2 gene product assists in promoting lymphocyte growth.

c. c-fos The c-fos gene encodes a nuclear protein that is believed to behave as a transacting transcriptional regulator (Turner and Tjian, 1989; Gentz et al., 1989). Considerable biochemical evidence supports the view that fos-encoded protein forms part of a complex with several other transcription factors including AP-1 (itself the product of the c-jun oncogene) that mediates a significant proportion of the transcriptional activation induced by PMA (Lee et al., 1987; Angel et al., 1988a). Early studies of c-fos expression demonstrated that serum-stimulation of quiescent fibroblasts induced transient increases in c-fos transcript levels that peaked within 1 hr (Curran and Morgan, 1987). These results prompted examination of c-fos expression in stimulated lymphocytes. Resting T cells have no detectable c-fos mRNA. Not surprisingly, stimulation of peripheral blood T cells or T cell lines via their antigen receptors causes very rapid accumulation of c-fos mRNA (detectable within 30 min). This stimulation of c - - i s expression is in most cases transient, lasting 1-2 hr (Reed et al., 1986, 1987a). Kaufman et al. (1987) reported high-level accumulation of c-fos transcripts in an alloreactive murine T cell hybridoma induced either by antigen or by

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ROGER M. PERLMUTTER AND STEVEN F. ZIEGLER

treatment with anti-Thy- 1 , an alternative activating regimen. In contrast to normal resting T cells, such hybridomas can typically be fully activated using a single stimulus. Accumulation of c-fos transcripts represents an immediate response to activation in the sense that it occurs within 30 min and does not require new protein synthesis. In fact, in lymphocytes, as in other cells, cycloheximide actually superinduces fos expression (Reed et a / . , 1986; Curran and Morgan, 1987). In the experiments of Kaufman et al. (1987), both cyclosporine A and anti-LFA- I (agents that block activation) suppressed the c-fos response. Shipp and Reinherz ( 1987) used combinations of stimulatory monoclonal antibodies to the human T cell receptor or the CD2 molecule to demonstrate that accumulation of c-fos transcripts results from signals that are not in themselves mitogenic and that do not require calcium influx. Similar results were also obtained for the early accumulation of c-tnyc transcripts. Thus the weight of evidence supports the view that a direct signaling mechanism at the cell membrane acts to stimulate accumulation of c-fos transcripts within minutes after exposure to an activating agent. Thefus-encoded protein may then act to stimulate cxpression of other genes in the activation sequence. One attractive candidate forfus-directed stimulation is the IL-2 gene, the cnhancer region from which is known to contain an AP-1 binding site (Crabtree, 1989). PMA, which is required for IL-2 induction in cloned T cells in v i m , is known to activate AP-1 posttranslationally (Angel et a l . , 198%) and also stimulates c-fos mRNA accumulation (Grausz et al., 1986; Grinstein el ul., 1988). Thus both the c-fos gene product and activated AP-I would be present in appropriately stimulated cells.

D. C-myb The c-myb gene behaves somewhat differently from c-fos: accumulation of c-myb transcripts is only induced by mitogenic stimuli. Like c_fos, c-myb encodes a nuclear protein that is thought to participate in transcriptional regulation (Klempnauer and Sippel, 1986). The c-myb gene was originally identified as the cellular homologue of the transforming element of the avian myeloblastosis virus and is expressed constitutively in thymocytes (Thompson PZ at., 1986), probably as a result of expression in immature thymoblasts (Yokota et ul., 1987). Transcripts from the c-myb gene begin to accumulate 6-hr after stimulation of human peripheral blood mononuclear cells with mitogen and reach peak levels after 24 or 48 hr (Reed et al., 1986). It is probable that most of this increase reflects stimulation by IL2, which itself provokes accumulation of c-myb transcripts in both peripheral blood lymphocytes (Reed et ul., 1986) and in a variety of cloned IL-2-responsive T cell lines (Dautry et al., 1988; Heckford et ul., 1988; Reed et al., 1987a; Stern and Smith, 1986). ConA treatment does not induce c-myb expression in these same cells in the absence of I L 2 . Combinations of PMA and calcium ionophore depress levels of c-myb mRNA in murine thymocytes and in

21. PROTOONCOGENE EXPRESSION IN LYMPHOCYTES

577

immature CD4 CD8 lymph node cells from mice with an inherited lymphoproliferative disease (Yokota et ul., 1987). This effect presumably results from maturation of these immature populations. +

E.

+

C-~YC

The c-myc oncogene encodes yet another nuclear protein of unknown function. Translocation of the c-myc gene into the immunoglobulin heavy chain locus is characteristically seen in the majority of human Burkitt lymphomas and murine plasmacytomas (reviewed in Cory, 1986). In addition, transgenic animals bearing a c-myc gene under the control of the immunoglobulin heavy chain enhancer develop B cell lymphoproliferative abnormalities (Adams et al., 1985). Hence, as in the case of the hcl-2 gene, there is reason to believe that the myc gene product assists in regulating lymphocyte proliferation. Increased expression of c-myc transcripts is seen in many cell populations that have been induced to proliferate, including serum-starved fibroblasts treated with exogenous growth factor and resting peripheral blood lymphocytes (Kelly and Siebenlist , 1988). There are two distinct pathways for c-myc induction in T-cells; stimulation of T cells by reagents that act via the T cell antigen receptor or via the I L 2 receptor results in increased accumulation of c-myc transcripts. A variety of studies have demonstrated that lectin-stimulated human peripheral blood cells exhibit a tenfold or greater increase in steady-state levels of c-myc transcripts that begins within 30 min and peaks 1-2 hr after stimulation (Kronke et al., 1985; Reed et al., 1985, 1986). A similar induction, albeit with a slightly delayed time course, is seen in T cell clones stimulated with ConA (Heckford et al., 1986, 1988; Reed ef al., 1987a). In purified human peripheral blood T lymphocytes, increases in c-myc transcript abundance were observed after treatment with mitogenic lectins, ionomycin, or phorbol esters (Kern et al,. 1986; Kumagai et al., 1987). In general, there is a good correlation between the effectiveness of the mitogenic stimulus and the increase in c-myc mRNA abundance (Grausz el af., 1986; Morris et al., 1988); however, stimuli that induce c-myc expression are not by themselves sufficient to provoke replication (Kumagai et a l ., 1987). Thus a nonmitogenic antibody directed against the CD3 component of the T cell antigen receptor satisfactorily stimulated accumulation of both c-myc and c-fos transcripts, as did cross-linking of the CD2 molecule in cells made nonresponsive (in terms of replication) through modulation of surface CD3 (Shipp and Reinherz, 1987). Induction of c-myc expression via lectin treatment is insensitive to, and in fact is augmented by, treatment with cycloheximide (Kelly and Siebenlist, 1986; Heckford et al., 1988). Steady-state levels of c-myc mRNA are determined by relative transcription rates from two closely spaced promoters (Kelly and Siebenlist, 1986), by a variable block to transcription elongation between the first and second exons (Bentley and Groudine, 1986; Nepveu and Marcu, 1986), and by

578

ROGER M. PERLMUTTER AND STEVEN F. ZIEGLER

changes in mRNA stability (Kelly and Siebenlist, 1986). Although the results obtained in different systems vary somewhat, it appears that ConA stimulation affects all of these processes but probably affects transcription most strongly (Kelly and Siebenlist, 1986, 1988; Heckford et al., 1988; Kronke et ul., 1985). Interestingly, although c-myc expression may not be sufficient to promote replication, studies with antisense oligonucleotide-mediated inhibition of c-myc translation suggest that it is necessary (Heikkla rt ul., 1987). Cells treated in this way and then stimulated with mitogenic lectin developed morphologic features of blast transformation but did not undergo DNA synthesis. The induction of c-myc by IL-2 is both rapid and stable, being detectable at 30 min and persisting for at least I2 hr (Reed et al., 1986, 1987a; Heckford et al., 1986, 1988). In cloned cell lines, cycloheximide completely inhibited the accumulation of c-myc transcripts provoked by IL2, while inhibiting c-myc transcription to a much smaller extent (Heckford et al., 1988). This observation suggests that induction of c-myc by 1L2 stimulates synthesis of an mRNA stabilizing protein.

F. Other Protooncogenes Expression of at least three other oncogenes (pS3, N-rus, and c-yes) has been found to increase in PHA-activated human peripheral blood mononuclear cells; however, these effects were modest, and the heterogeneity of the cell population studied renders the results difficult to interpret. A fourth protooncogene, c-ets-l , is of somewhat more interest. The c-ets-1 gene was originally identified in chicken as a component of the E26 virus. It encodes a 54 kDa cytoplasmic protein of unknown function that is expressed at highest levels in thymocytes and in bursa1 lymphocytes (Ghysdael et al., 1986). PHA stimulation of human peripheral blood mononuclear cells provokes a modest increase in c-ets- l transcript abundance beginning after about 2 hr (Reed et al., 1986). Increases in c-ets-1 transcript abundance were also observed in a murine T cell hybridoma stimulated with antigen or with antibodies to Thy-I (Kaufman et ul., 1987). The latter effects were blocked by cyclosporin A. In light of the lineage specificity of the c-ers-1 gene product, the pattern of expression of this protooncogene in stimulated lymphocytes deserves additional attention.

111. ALTERATIONS IN PROTOONCOGENE EXPRESSION IN STIMULATED B LYMPHOCYTES Patterns of protooncogene expression in activated B lymphocytes generally recapitulate those previously described in activated T cells. In most cases, resting peripheral blood B cells are isolated by panning or complement lysis, and the

21. PROTOONCOGENE EXPRESSION IN LYMPHOCYTES

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resulting populations are principally (>95%) comprised of Go cells. Activation of these B cells requires two complementary signals. Treatment with anti-IgM antibodies results in increased phosphatidylinositol turnover and induces rapid increases in intracellular Ca2 concentrations (Kishimoto and Hirano, 1988); however, this signal by itself is insufficient to provoke mitogenesis. Instead, addition of a B cell growth factor (BCGF) preparation, typically IL-4 (BSFl), permits entry of stimulated Go, cells into S phase (Rabin et al., 1986). Stimulation of human B lymphocytes with polyclonal anti-p and BCGF results in increased transcription of the c-myc, K-ras, ets, p53, and c-sis protooncogenes, as judged by transcription runoff experiments (Smeland et al., 1988). The pattern of activation of these genes closely resembles that seen for optimally stimulated T lymphocytes. As in the case of T lymphocytes, most of the increase in steady-state levels of c-myc transcripts occurring after stimulation appears to result from de n o w transcription as opposed to decreased transcriptional attenuation (Kelly and Siebenlist, 1988; Smeland et al., 1988). The increase in c-myc transcription can be provoked by anti-y stimulation alone, by stimulation of hapten-specific B cells with antibodies (Snow et al., 1986), or by stimulation using monoclonal antibodies that act through alternative stimulation pathways, particularly those involving the B cell surface protein Bp35 (Smeland et al., 1985; Clark et al., 1985). These stimulation protocols all provoke a Go to G, transition in responding cells, but treatment with monoclonal antibodies to Bp35 does not render cells competent to proliferate in response to BCGF (Clark et al., 1985). Thus, alterations in myc transcript abundance are associated with the initial activation sequence, as in T lymphocytes, but not with proliferation per se. Similar observations have been made with respect to c-fos expression. In this case, fos transcript abundance increased within an hour after stimulation of mouse B lymphocytes with anti-immunoglobulin or with PMA (McCormack et al., 1984; Monroe, 1988). Once again, this phenomenon mimics the pattern seen in stimulated T lymphocytes. In contrast with studies of T lymphocytes, c-myb expression is not increased in activated B cells (Smeland et al., 1988). +

IV. LYMPHOCYTE ACTIVATION MODULATES EXPRESSION OF THE Ick GENE The lck gene encodes a lymphocyte-specific protein tyrosine kinase ( ~ 5 6 ' ' ~ ) that is implicated in the control of lymphocyte growth (Perlmutter et al., 1988b). Two murine lymphoid tumor lines have been described in which retroviral promoter insertion has activated the endogenous lck gene, suggesting that this activation process can contribute to the process of lymphoid neoplasia (Garvin et ul., 1988; Adler et al., 1988; Marth et al., 1985; Voronova and Sefton, 1986). The product of the lck gene is structurally quite similar to the oncoprotein

580

ROGER M. PERLMUTTER AND STEVEN F. ZIEGLER

encoded by v-src. In addition, introduction of a single point mutation into the lck gene yields an activated ~ 5 6 "protein '~ that can directly transform NIH 3T3 cells (Marth et al., 1088a; Amrein and Sefton, 1988). Thus the lck gene is a protooncogene that is normally exprcsscd exclusively in lymphoid cells (Perlmutter et al., 1988a,b). Evidence indicates that p561rk directly associates with CD4 and CD8 molecules on the T lymphocyte cell surface (Rudd et af., 1988; Veillette et a[., 19884. Moreover, cross-linking of surface CD4 molecules appears to increase the intrinsic kinase activity of ~ 5 6 " (Veillette '~ et al., 1989).Thus there is reason to believe that this membrane-associated protein tyrosine kinase may transmit proliferative signals from the T cell antigen receptor to the cell interior (Perlniutter et a / ., 1988b). It might therefore be expected that examination of the expression pattern of the lck gene in stimulated lymphocytes would provide insights into the lymphocyte activation sequence, and indeed, such alterations are seen. Examination of changes in Ick transcript abundance and of ~ 5 6 ~ ~ ' ~ expression following lymphocyte stimulation, however, illustrates the difficulties that result from attempting to infer involvement of a protein in an activation pathway using mRNA abundance as a guide to gene expression. Resting T lymphocytes contain 4-10 copies of the lck mRNA per cell, depending upon the tissue source (Marth et a l . , 1987). Stimulation of these T lymphocytes results in a dramatic reduction in lck transcript abundance to less than 1 copy per cell (Fig. 1). The pattern of this response mimics that of lymphokine release in that both effects require two independent stimuli (e.g., ConA and PMA). In addition, the disappearance of Ick transcripts occurs with the same time course as the appearance of lymphokine transcripts (Marth et a/., 1987). Alterations in Ick mRNA abundance are not provoked by stiniuli that simply induce proliferation. A simple interpretation of these data suggests that the Ick gene product must somehow be involved in regulating lymphokine expression, perhaps in a negative fashion. At the protein level, the Ick response is more complex. In fact, activation of T lymphocytes results in the rapid elimination of ~ 5 6 " and ' ~ the appearance of novel forms of lck-encoded proteins that migrate with apparent relative sizes of

0

2

4

6

8

12 24

FIG. I . Reduction in Ick mRNA abundance following lymphocyte activation. Purified human T cells were maximally activated with ConA (25 Kgiml) and PMA (50 ng/nil) for the indicated intervals (in hours) prior to isolation of total RNA. Shown is an RNA blot performed using a human Irk cDNA probe labeled with W. (Adapted from Marth e/ a / . , 1987.)

21, PROTOONCOGENE EXPRESSION IN LYMPHOCYTES

581

Cio~ r s P o s t - Stimula t ion 24 6 4 2 1 0 ConA plus PMA

p60L>

PMA

alone

p 56Ick+

FIG. 2. Lymphocyte activation provokca long-term modification of p56lCk.Membranes from lymphocytes stimulated as in Fig. 1 were iwlated and Ick-encoded protein visuali7ed hy immunoblotting o f inctnbrane proteins separated by SIF-PAGE. Conversion of p56lCkto pW''k can be achieved using PMA alone but is more complete when both ConA and PMA are used. Importantly, ~60''": is quite ahundant at a time when Ick transcripts have essentially disappeared (Fig. I ) . (Adapted from Perlmulter rt a!., 1988b.)

60 kDa (Marth et al., 1989; Veillette et u l . , 1088b). This change in the bchavior of the k k gene product probably results from increased serine phosphorylation catalyzed by a PMA-activatable kinase that has somewhat unusual properties (Marth et ul., 1989; Veillette et ul., 1988b). Importantly, despite the change in mRNA abundance after stimulation, thcrc is relatively little change in p60'ck abundance over a 24 hr period (Fig. 2; Perlmutter et tit., 1988b). Thus the analysis of' lck mRNA expression is of little help in predicting the pattern of expression of the Ick gene product. In addition, the lck mRNA contains 5' untranslated rcgion sequences that decrcasc translational efficiency in hcterologous cell systems (Marth ef nf., 1988b), suggesting that there may be additional mechanisms of translational regulation that assist in controlling the expression of p56/i'k.

V. FUTURE DIRECTIONS: THE UNDERLYING COMPLEXITY OF LYMPHOCYTE ACTIVATION The studies reviewed here plainly illustrate that, by itself, measurement of protooncogene transcript abundance in stimulated lymphocytes is unlikely to

582

ROGER M. PERLMUTTER AND STEVEN

F. ZIEGLER

shed light on the physiological control of lymphocyte activation. As reviewed elsewhere in this volume, lymphocytes interact with a large variety of cytokines and cell-associated regulatory molecules. The end result of the encounter of any individual T lymphocyte with an antigen-presenting cell probably depends on the sum of these modulatory signals, as wcll as the specific interaction of the T cell antigen receptor with MHC-associated ligand. Thus analyses of protooncogene expression during lymphocyte activation in general underestimate the complexity of lymphoid cell behavior. The study of protooncogenes has, however, increased interest in the role of specific transcription factors in directing the lymphocyte activation sequence. Lymphocyte-specific transcriptional regulators that control antigen receptor gene expression have been isolated (Clerc et al., 1988; KOet al., 1988; Herr et a., 1988), and additional factors that control lymphokine gene expression have been identified (Crabtree, 1989). With these proteins in hand, it should soon be possible to proceed in a retrograde fashion, linking changes in the activity of trans-acting factors to cross-linking of cell surface receptors. At the same time, analysis of cloned T cell lines has permitted the identification of activation mutants that cannot correctly couple the T cell antigen receptor to intracellular second messengers (Sussman et al., 1988; Goldsmith et al., 1988). In one case, independent mutants were shown to contain complementing genetic defects (Goldsmith et al., 1988). These results encourage the pursuit of a genetic analysis of T cell activation. Finally, detailed characterization of the T cell antigen receptor complex (Clevers e l al., 1988) and of the I L 2 receptor (Taniguchi et al., 1986) may soon provide a biochemically satisfactory view of the lymphocyte activation sequence. Since regulatory circuits that control proliferation are probably shared by most cell types, these studies will ultimately yield insight into mechanisms of oncogenesis and the functions of the protooncogenes. ACKNOWLEDGMENTS We thank our colleagues for helpful discussions and gratefully acknowledge support from the Howard Hughes Medical Institute and from the National Institutes of Health (CA-45682). REFERENCES Adams, J. M . , Hams, A. W., Pinkert, C. A . , Corcoran, L. M., Alexander, W. S., Cory, S., Palmiter, R. D., and Brinster, R . L. (1985). The c - m y oncogene driven by immunoglobulin enhancers induces lymphoid malignancy in transgenic mice. Nature (London) 318, 533-538. Adlet, H.T., Reynolds, P. J . , Kelley, C. M., and Sefton, B . M. (1988). Transcriptional activation of Ick by retrovirus promoter insertion between two lymphoid-specific promoters. J . Virol. 62, 41 13-4122. Amrein, K. E., and Sefton, B. M. (1988). Mutation of a site of tyrosine phosphorylation in the lymphocyte-specific tyrosine protein kinase, p56'ck, reveals its oncogenic potential in fibroblasts. Proc. Nut/. Amd. Sci. U.S.A. 85, 4241-4251, Angel, P., Allegretto, E. A,, Okino, S., Hattori, K., Boyle, W. 1.. Hunter, T., and Karin, M. ( I 988a). Oncogene jun encodes a sequence specific trans-activator similar to AP- I . Nature (London) 332, 166- 17 1 .

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Angel, P., Hattori, K . , Smeal, T., and Karin, M. (1988h). The j u n proto-oncogene is positively autoregulated by its product, JuniAP-I. Cell 55, 875-885. Baltimore, D., Rosenberg, N . , and Witte, 0. N. (1979). Transformation of immature lymphoid cells by A-MuLV. Immunol. Rev. 48, 3-27. Bentley, D. L., and Groudine, M. (1986). A block to elongation is largely responsible for decreased transcription of c-myc in differentiated HL60 cells. Nature (London) 321, 702-706. Clark, E. A , , Shu, G . , and Ledhetter, J. (1985). Role of the Bp35 cell surface polypeptide in human B-cell activation. f r o c . Nutl. Acnd. Sci. U.S.A. 82, 1766- 1770. Clerc, R. G., Corcoran, L. M., LeBowitz, H. H., Baltimore, D . , and Sharp, P. A. (1988). The Bcell-specific Oct-2 protein contains POU-box and homeo-box-type domains. Genes Dev. 2, 1570- 1581. Clevers, H., Alarcon, B., Wileman. T., and Terhorst, C. (1988).The T cell receptor/CD3 complex: a dynamic protein ensemble. Annu. Rev. Immunol. 6, 629-662. Cory, S. (1986). Activation of cellular oncogenes in hematopoietic cells by chromosomal translocations. Adv. Cancer Res. 47, 189-234. Crahtree, G. R. (1 989). Contingent genetic regulatory events in T lymphocyte activation. Science 243, 355-361. Curran, T., and Morgan, J. 1. (1987). Memories offos. Bioessuys 7, 255-258. Dautry, F., Weil, D., Yu, J., and Dautry-Varsat, A. (1988). Regulation of pim and myh mRNA accumulation by interleukin 2 and interleukin 3 in murine hematopoietic cell lines. J . B i d . Chem. 263, 17615-17620. Gamin, A. M., Pawar, S., Marth, J. D., and Perlmutter, R. M. (1988). Structure of the murine Irk gene and its rearrangement in a murine lymphoma cell line. Mol. Cell. Biol. 8, 3058-3064. Gentz, R., Rauscher, F. J., 111, Abate, C., and Curran, T. (1989). Parallel association of Fos and Jun leucine zippers juxtaposes DNA hinding domains. M o f . Cell. Biol. 9, 1695-1699. Ghysddel, J., Gegonne, A , , Pognonec, P., Dernis, D., Leprince, D., and Stehelin, D. (1986). Identification and preferential expression in thymic and bursa1 lymphocytes of a c-ets oncogeneencoded M, 54,000 cytoplasmic protein. Proc. Nnrl. Acud. Sci. U . S . A . 83, 1714-1718. Goldsmith, M. A , , Dazin, P. F.. and Weiss, A. (1988). At least two non-antigen-hinding molecules are required for signal transduction by the T-cell antigen receptor. froc. Nutl. Acud. Sci. U.S.A. 85, 8613-8617. Grdusz, J. E., Frandelisi, D., Dantry, F., Monier, R., and Lehn, P. (1986). Modulation of c+s and c-myc levels in normal lymphocytes by calcium ionophore A23 187 and phorbol ester. Eur. J. Immunol. 16, 1217-1222. Grinstein, S . , Smith, J. D., Onizuka, R., Cheung, R. K., Gelfand, E. W., and Benedict, S. (1988). Activation of NA + /H exchange and the expression of cellular protooncogenes in mitogenand phorbol ester-trcatcd lymphocytes. J . B i d . Chem. 263, 8658-8665. Heckford, S . E., Gelmann, E. P., Agnor, C. L., Jacobson, S., Zinn, S., and Matis, L. A. (1986). Distinct signals are required for proliferation and lymphokine gene expression in murinc T cell clones. J . Immunol. 137, 3652-3663. Heckford, S. E., Gelmann, E. P., and Matis, L. A. (1988). Distinct mechanisms of c-myc and lymphokinc gene expression in an antigcn specific T cell clone. Oncogene 3, 415-421. Heikkla, R., Schwab, G . , Wickstrom, E., Loke, S. L., Pluznik, D. H . , Watt, R . , and Neckers, L. M. (1987). A c-myc antisense oligodeoxynucleotide inhibits entry into S phase but nor progress from Go to G , . Nuture (London) 328, 445-449. Herr, W., Sturm, R . A., Clerc, R. G., Corcoran, L. M., Baltimore, D., Sharp, P. A , , Ingraham, H. A., Rosenfeld, M. G.. Finney, M., Ruvkun, G., and Horvitz, H. R. (1988). The POU domain: a large conserved region in the mammalian pii-I, oct-I, oct-2, and Cnenorhubditis eleguns unc-86 gene products. Genes Dev. 2, 1513-1516. Imhoden, J. B., Weiss, A., and Stobo, I. D. (1985). The antigcn receptor on a human T cell line initiatcs activation by increasing cytoplasmic free calcium. J . Immunol. 134, 663-665. +

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Kaufman, Y . , Silverman, T., Levi, B.-Z., and Ozato, K. (1987). Induction of c-eis and c+s gene cxprcssion upon antigcnic stiniulation of a T cell hybridoma with inducible cytolytic capacity. J. Exp. Med. 166, 810-815. Kelly, K., and Siebenlist. U . (1986). Thc rcgulation and expression orc-myc in normal and malignant cells. Annu. Rev. Immunol. 4, 317-338. Kelly. K., and Siebenlist, U . (1988). Mitogenic activation of normal T cells leads to increased initiation of transcription in the c - m y locus. J . B i d . Chem. 263, 4828-483 I . Kern. J. A., Reed, J. C., Daniele, K. P., and Nowell. P. C. (1986). Thc rule of the accessory ccll in mitogen-stimulated human T ccll gene expression. J . f i n m i m i l . 137, 764-769. Kishimoto. T., and Hirano, T. (1988). Molecular regulation of B lymphocyte response. Annu. Rev. Immunol. 6 , 485-512. Klempnauer, K.-H.. and Sippel, A. E. (1986). Subnuclear localization of proteins encoded by the oncogene v-myh and its cellular hoinolog c-myb. Mol. Cell. B i d . 4, 2843-2848. KO, H.-S., Fast, P., McBride, W., and Staudt, L. M. (1988). A human protein specific for the immunoglobulin octamer DNA motif contains a functional honicobox domain. Cell 55, 135144. Konopka, J. B., Watanabe. S. M., and Witte, 0. N. (1984). An alteration of the human c-ubl protcin in K562 leukemia cells unmasks associated tyrosine kinase activity. Cell 37, 1035- 1042. Kronke, M., Leonard, W. J., Depper. J. M., and Greene, W. C. (1985). Sequential cxprcssion of genes involved in human T lymphocyte growth and differentiation. J . Exp. Med. 161, 15931598. Kumagai, N . , Bencdict, S . H., Mills, G. B . , and Gelfand, E. W. (1987). Requirements for the simultaneous presence of phorbol esters and calcium ionophores in the expression of human T lymphocyte proliferation-related genes. J. Immunol. 139, 1393- 1399. Lee, W., Mitchell, P., and Tijan, R. (1987). Purified transcription factor AP-I interacts with TPAinducible enhancer elements. Cell 49, 741-752. McConnack, J. E., Pcpe, V. H . , Kent, R . B., Dean, M., Marshak-Rothstein, A,, and Sonenshein, G . E. (1984). Specific regulation of c - m y oncogene expression in a murine B-cell lymphoma. Proc. Nut/. Acud. Sci. U . S . A . 81, 5546-5550. McDonnell, T. J., Deane. N., Platt, F. M . , Nune7, G . , Jacger, IJ., McKearn, J. P. and Korsiiieyer, S, J. ( 1989). hcl-2 Irnrnunoglobulin transgenic mice demonstrate extended B cell survival and follicular lymphoproliferation. Cell 57, 79-88. Marth, J. D., Peet, R., Krebs, E. G . . and Pcrlmuttcr, R. M. (1985). A lymphocyte-specific proteintyrosine kinase gene is rearranged and overexpressed in the murine T cell lymphoma LSTRA. Cell 43, 393-404. Marth, J. D., Lewis, D. B., Wilson, C. B., Ccarn, M. E . , Krebs, E. C.. and Perlmutter, R. M. ( 1987). Regulation of pp56lck during T-cell activation: functional implications for the src-likc protein tyrosine kinases. EMBO J. 6, 2727-2734. Marth, J. D., Cooper, J. A , , King, C. S . , Ziegler, S . F., Tinker, D. A., Overell, R. W., Krebs, E. G . , and Perlmutter, R. M. (1988a). Neoplastic transformation induced by an activated lymphocytc-specific protein tyrosine kinase ( p . 5 6 9 ) .Mol. Cell. B i d . 8, 540-550. Marth, J. D., Overell, R. W., Meier, K. E., Krebs, E. G., and Perlmutter, R. M. (1988b). Translational activation of the lck proto-oncogene. Nature (London) 332, 171- 173. Marth. J. D.. Lewis. D. B., Cooke, M. P., Mellins, E. D., Gearn, M. E., Samelson, L. E . , Wilson, C. B., Miller, A. D., and Perlmutter, R. M. (1989). Lymphocyte activation provokes modification of a lymphocyte-specific protein tyrosine kinase ( p S W ) . J. Immunol. 142, 24302431 Monroe, J. C. (1988). Up-regulation of c-jos expression is a component of the mlg signal transduction mechanisms but is not indicative of competence for proliferation. J . Immunol. 140, 14541460.

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Morris, D . R., Allen, M. L., Rabinovitch, P. S . , Kuepfer, C. A., and White, M. W. (1988). Mitogenic signaling pathways rcgulating expression of c-mvc and omithine decarboxylasc genes in bovine T-lymphocytes. Biochemistry 27, 8689-8693. Nepveu, A , , and Marcu, K. B. (1986). lntragenic pausing and antisense transcription within the murine c-myc locus. EMBO J . 5 , 2859-2865. Perlmutter, R. M . , Marth, J. D., Lewis. D. B.. Peet, R.. Ziegler, S . F., and Wilson, C. B. (1988a). Structure and expression of /ck transcripts in human lymphoid cells. J . Cell. Biorhem. 38, 117126. Perlmutter, R. M., Marth, J. D., Ziegler, S . F., Garvin, A. M., Pawar. S . , Cooke, M. P., and Abraham, K . M. (1988b). Specialized protein tyrosine kinase proto-oncogenes in hematopoietic cells. Biochim. Biophys. Acru 948, 245-262. Rabin, E. M., Mond, J. J., Ohard, J . , and Paul, W. E. (1986). B cell stimulatory factor I (BSF-I) prepares resting B cells to enter S phase in response to anti-IgM and lipopolysaccharide. J . Exp. Med. 164, 517-531. Reed, J. C.. Nowell, P. C., and Hoover, R. G. (1985). Regulation ofc-myc mRNA levels in normal human lymphocytes by modulators of cell proliferation. Proc. Narl. Acad. Sci. U.S.A. 82, 4221-4224. Reed, J. C., Alpers, J. D., Nowell. P. C.. and Hoover, R. G. (1986). Sequential expression of protooncogenes during lectin-stimulated mitogenesis of normal human lymphocytes. Proc. Narl. Acad. Sci. U.S.A. 83, 3982-3986. Reed, J. C.. Alpers, J. D., Scherle, P. A , , Hoover, R. G., Nowell, P. C., and Prystowsky, M. B. ( 1987a). Proto-oncogene expression in cloned T lymphocytes: mitogens and growth factors induce different patterns of expression. Oncogene 1, 223-228. Reed, J. C., Tsujimoto, Y., Alpers, J. D., Croce, C . M . , and Nowell, P. C. (1987b). Regulation of hcl-2 proto-oncogene expression during nornial human lymphocyte proliferation. Science 236, 1295- 1299. Rudd, C. E., Trevillyan, J. M . , Dasgupta, J. D . , Wong, L. L., and Schlossmdn, S. F. (1988).The CD4 receptor is complexed in detergent lysates to a protein-tyrosine kinase (pp58) from human T lymphocytes. Proc. Narl. Acad. Sci. U.S.A. 85, 5190-5194. Seto, M., Jaeger, U.. Hockett, R. D., Grdninger, W., Bennett, S . , Coldman, P., and Korsmeycr, S . J. (1988). Alternative promoters and exons, somatic mutation and deregulation of the Bcl-2-Ig fusion gene in lymphoma. EMBO J . 7, 123- 131, Shipp, M. A , , and Rcinherz, E. L. (1987). Differential expression of nuclear protooncogenes in T cells triggered with mitogenic and nonmitogenic T3 and TI I activation signals. J . Immunol. 139, 2143-2148. Smeland, E., Godal, T., Ruud, E., Beiske, K., Funderud. S . . Clark, E. A , , Pfeifer-Ohlsson, S . , and Ohlsson, R. (1985). The specific induction of myc protooncogene expression in normal human B cells is not a sufficient event for acquisition of coinpetcnce to proliferate. Proc. Narl. Acad. Sci. U.S.A. 82, 62554259. Smcland, E. B., Blomhoff, H. K., Ohlsson, R . , Davies, C. D., Funderud, S . , and Boye, E. (1988). Transcription of protooncogenes during stimulation of normal human B lymphocytes. Eur. J . Immunol. 18, 1847- 1850. Snow, E. C., Fetherston, J. D . , and Zimmer, S. (1986). Induction of the c-myc protooncogene after antigen binding to hapten-specific B cells. J . Exp. Med. 164, 944-949. Stem, J. B . , and Smith, K. A . (1986). Interleukin-2 induction of T-cell G , progression and c-myh expression. Science 233, 203-206. Sussman, J . J . , Mercep, M., Saito, T., Germain, R. N . , Bonvini, E., and Ashwell, J. D. (1988). Dissociation of phosphoinositide hydrolysis and CA? tluxes from the biological responses of a T-cell hybridoma. Nature (London) 334, 625-628. Taniguchi, T., Matsui, H.,Fujita, T., Hatekayama, M . , Kashima, N ., Fuse, A,, Hamuro, J., Nishi+

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Takaoka. C., and Yamada, G. (1986). Molecular analysis of the interleukin-2 system. Imrnunol. Rev. 92, 121-134. Thompson, C. B., Challoner, P. B . , Neiman, P. E., and Groudine, M. (1986). Expression of the c-myb proto-oncogene during cellular proliferation. Nuiure (London) 319, 3744376. Turner, R., and Tjian, R . (1989,. Leucine repears and an adjacent DNA binding domain mediate the formation of functional cFOS-cJUN heterodimers. Mol. Cell. B i d . 9, 1689- 1694. Varmus, H. E. (1984). The molecular genetics of cellular oncogenes. Annu. Rev. Genet. 18, 553612. Veillette, A . . Bookman. M. A., Horak, E. M . , and Bolcn, J B . (1988a). The CD4 and CD8 T cell surface antigens are associated with the internal membrane tyrosine-protein kinase p56'"k. Cell 55, 301-308. Veillette, A,, Horak, 1. D., Horak, E. M., Bookman, M. A , , and Bolen, J. B. (1988b). Alterations of the lymphocyte-specific protein tyrosinc kinase p56lCkduring T cell activation. Mol. Cell. B i d . 8, 4353-4358. Veillette, A., Bookman, M. A., Horak, E. M . , Samelson, L. E., and Bolen, J. 6.(1989). Signal transduction through the CD4 receptor involves the activation of the internal membrane (London) 338, 257-259. tyrosine-protein kinase ~ 5 6 " Nuture ~. Voronova, A . F., and Sefton, B. M . (1986). Expression of a new tyrosine protein kinase is stimulated by retrovirus promoter insertion. Nature (London) 319, 682-685. Wciss, A . , Imhoden, J . , Hardy, K., Manger, B.,Terhorst, C . , and Stobo, J. (1986). The role of the T3iantigen receptor complex in T-cell activation. Annu. Rev. lmmunol. 4, 593-61 9. Witte, 0. N.. Rosenberg, N., and Baltimore, D. (1979). A normal cell product cross-reactive to the major Abclson inurine leukemia virus gene product. Nuridre (London) 281, 396-398. Yokota, S., Yuan, D., Katagiri, T., Eiscnberg, R . A,, Cohen, P. L., and Ting, 1. P.-Y. (1987). The expression and regulation of c-myb transcription in B6/lpr Lyt-2-, L3T4- T lymphocytes. J. Immune/. 139, 2810-2817. Zipfel, P. F., Irving, S. G., Kelly, K., and Siebenlist, U . (1989). Complexity of the primary genetic response to mitogenic activation of human T cells. M d . Cell. B i d . 9, 1041-1048.

CLIRRENI' Topics I N MEMBRANES AND TRANSPORT, VOLUME 35

Chapter 22 Early Gene Expression in the Activation of Mononuclear Phagocytes DOLPH 0. ADAMS,*f STEWART P. JOHNSON, f AND RONALD J . UHINGf Deparrments of *Microbiology, *Immunology, and $Pathology Laboratory of Cell and Molecular Biology of Leukocytes Duke University Medical Center Durham, North Carolina 27710

I.

Introduction

11. Early Genes 111. Molecular Mechanisms of Macrophage Activation A. Four Cascades of Signal Transduction

B. Gene Regulation in Macrophage Activation IV. Early Genes in Macrophage Activation V. Conclusions and Future Directions References

1.

INTRODUCTION

Over the past several years, it has become clear that the stimulation or activation of many cells is accompanied by the very rapid expression (i.e., within a few minutes) of proteins, which often are expressed only transiently (Olawshaw and Pledger, 1988; Sinkovics, 1988; Wingender, 1988; Lebovitz and Lieberman, 1988). Certain genes encoding these proteins, by analogy to phenomenologically similar genes of oncogenic retroviruses, have been termed protooncogenes (Reddy et al., 1988), but protooncogenes constitute only a subset of the larger group of early or immediate early genes (see, e.g., Lau and Nathans, 1987; Honess and Roizman, 1974). When competence for fibroblast division is induced by plateletderived growth factor (PDGF), activation of several such genes has been observed, and these have been termed competence genes (Stiles, 1983). These 587

Copyright 0 1990 hy Academic Pres. Inc All nghls of rcprvductiun in any furin reserved

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diverse genes, here collectively referred to as early genes, now appear to encode several families of important proteins, among which are ones that function in the nucleus to regulate the cell undergoing activation and ones that are secreted to serve either autocrinc or paracrine functions. The activation of mononuclear phagocytes by various stimuli represents an excellent modcl for studying the regulation and function of early genes (Adams and Hamilton, 1984, 1989). Although mononuclear phagocytes (like neutrophils) can be rapidly stimulated le.g., by chemotactic stimuli such as formylated peptides or platelet activating factor (PAF)I (see Chapter 14 by Uhing rt al. in this volume), physiological activation with regard to macrophages is generally reserved for the development of increased competence to complete complex functions, such as the destruction of facultative-intracellular microorganisms or tumor cells (Adams and Hamilton, 1984, 1989). This activation, which is complexly regulated by multiple inductive and suppressive stimuli, requires -24 hr for completion. Macrophage activation can be formally defined as the acquisition of competence to perform or complete a complex function, manifested by altered expression of separate and independently regulated gene products. This may scrvc as a useful definition for cellular activation in general and, indeed, has been applied to the activation of endothelial cells (Pober, 1988). A useful and widely applied model for analyzing the activation of mononuclear phagocytcs requires that the responsive macrophages are exposed sequentially to a priming signal such as interferon gamma (IFN-y) and subsequently to a triggering signal such as bacterial lipopolysaccharide (LPS) (Adams and Hamilton, 1984). These operationally defined stages of macrophage activation (i.e., responsive, primed, and fully activated macrophages) can be characterized by a library of quantitative, objective markers representing increases and decreases in surface and secreted proteins. Indeed, the precise protein alterations required for macrophage activation have now been delineated for a number of functions (Adams and Hamilton, 1984). These alterations in protein expression, in turn, are generally regulated by alterations in either the transcription of genes encoding the individual proteins or by posttranscriptional regulatory events (e .g., altered stability of message) (Adanis and Koerner, 1988). Macrophage activation can thus be viewed as a complex, stringently regulated, and tightly coordinated sct o f genetic events, which include both stimulatory and inhibitory regulation of gene expression. Over the past several years, this laboratory has focused its efforts on delineating some of the molecular events controlling macrophage activation (for reviews see A d a m and Hamilton, 1987; Hamilton and Adams, 1987; Uhing and Adams, 1989). Thc regulation of macrophage activation by surface activating signals such as IFN-y and LPS results from the initiation of at least four distinct cascades of' intraccllular second messengers. These in turn initiate or suppress various

22. EARLY GENE EXPRESSION IN PHAGOCYTES

589

genes that result in the activation of macrophages. Early genes in macrophage activation have been observed in at least three of these cascades. This chapter will consider the current extent of information on early genes in macrophage activation.

II. EARLY GENES Early genes, initiated by a wide variety of signals, can be observed in numerous cells including macrophages, neutrophils, lymphocytes, fibroblasts, and a wide variety of others (Olawshaw and Pledger, 1988; Sinkovics, 1988; Wingender, 1988; Lebovitz and Lieberman, 1988; Kawahara and Deuel, 1989; Ryder et a l . , 1988). Although many of the early genes initially observed and described were protooncogenes, some early genes do not fall into this category. At present, early genes comprise at least two large families: (1) nuclear regulatory proteins, and (2) proteins that serve autocrine and paracrine functions. The first family includes the protooncogenes c;fos, c-myc, and c-jun and nuclear transcription factors such as AP-I, NFAT-1, and NFK-B (Wingender, 1988). The second group, encoding proteins which are believed to regulate the behavior of the same and other cells, includes the A chain of platelct-derived growth factor, 9E3, gro, I6C8/EPA/TIMP, JE, and KC (for references see Kawahara and Deuel, 1989). Last, the current catalog of early genes encompasses both unidentified and incompletely identified genes as well as several additional protooncogenes with homology to cellular kinases, growth factor receptors, and G proteins (Reddy et al., 1988). The fundamental mechanisms of action by nuclear transcription factors are well beyond the scope of this chapter (for reviews see Maniatis et al., 1987; Dynan and Tjian, 1985; McKnight and Tjian, 1986; Ptashne, 1988). It is important to note, however, that many of these proteins are characterized by common structural motifs, which include the so-called leucine zipper, currently thought to promote formation of homo-/heterodimers between two molecules of transcription factor(s) (for review see Struhl, 1989). Formation of these complexes may be extremely important for cellular regulation. The heterodimer formed (via the leucine repeats) between the protein products of c-fos and c-jun binds with much higher affinity (i.e., -25-fold) to the AP- 1 recognition site than the homodimer of c-jun; in fact, evidence suggests that the homodimer of c-fos may not be able to initiate transcription at all (Curran and Franza, 1988; Turner and Tjian, 1989; Gentz et al., 1989; Halazonetis et al., 1988; Nakabeppu et al., 1988). Interestingly, effective initiation of transcription may not only depend on the protein or proteins involved but also depend upon prior modifications of the protein or protein complexes (for review see Robertson, 1988). These emerging observa-

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tions suggest at least two mechanisms whereby nuclear binding proteins may be regulated in order to control transcription (i .e., complex formation and covalent modification). The regulation of genes encoding early proteins in response to external stimuli is, in general, not well understood. Extensive emphasis has been placed on the regulation of the protooncogene cfos (for review see Verma and Sassone-Corsi, 1987). The upstream region controlling transcription of the gene and the critical areas within this have been delineated. The induction of c-fos can be attributed to numerous surface-active agents, and these act through multiple pathways, one of which depends on protein kinase C and one of which depends on elevations in intracellular cyclic AMP and subsequent stimulation of CAMP-dependent protein kinase; induction may also involve mechanisms independent of the previous two. This echoes an emerging theme derived from a number of diverse experimental systems that protein phosphorylation may regulate the function of transcription factors (Morrison et al., 1988; Prywes et al.. 1988; Magasanik, 1988; Yamamoto et al., 1988). Although phosphorylation may directly promote transcriptional activity, at least one model (e.g., that of the nuclear factor NFKP) has been proposed in which phosphorylation of a cytoplasmic inhibitor via protein kinase C releases NFKP from the bound inhibitor and permits its translocation to the nucleus (Baeuerle and Baltimore, 1988).

111. MOLECULAR MECHANISMS OF MACROPHAGE ACTIVATION

A. Four Cascades of Signal Transduction Macrophage activation, initiated by IFN-y and LPS, results from at least four distinct cascades of transductional events (Fig. I). Each of the cascades is functionally distinct from the others; the first three have all been shown to be essential for the initiation of macrophage activation (for review see Adams and Hamilton, 1987; Hamilton and Adams, 1987; Uhing and Adams, 1989). Of note, cascades 11, 111, and 1V are initiated by multiple signals. To date, TFN-y and only IFN-y initiates cascade I , while LPS initiates two cascades (I1 and 111). The events in cascade I , initiated by IFN-y, begin with a very rapid (i.e., within a few seconds) activation of the Na+ / H+ exchanger or antiporter, resulting in an influx o f N a + and intracellular alkalinization (Prpic et d., 1989b) (Fig. 2 ) . Subsequent to this, at 10-15 min, Ca2+ efflux occurs without a concomitant increase or decrease in intracellular [Ca2+], (Somers et d.,1986, and unpublished observations). Levels of specific mRNA for JE, initially described as an early gene for fibroblast competence initiated by PDGF (Stiles, 1983), are then heightened-along with the increased potential of protein kinase C (Prpic et

-

GASCADEN

Transalption of TNF gene

mRNAl banslatlon 01 Is mRNA

v

I

Q Iran$albn of TNF mRNA

+

lNF

1111

I

ALTERED FUNCTION

FIG. 1. Four signal cascades regulate gene expression in mononuclear phagocytes. Shown are the stimuli for the individual cascades and their regulation of class II major histocompatibility complex (Ia) and tumor necrosis factor (TNF) genes. Other abbreviations used are IFN, interferon y; PAF, platelet activating factor; LPS, bacterial lipopolysaccharide; a2-M, a2-macroglobulin; MAL-BSA, maleylated-bovine semm albumin; PGE2. prostaglandin E2.

CASCADE I

i

CASCADE /I

i

CASCADE /I/

i CASCADE IV

IMMEDIATE: 0-5 min

...-_ ....

IN TERMEDATE: 5 min 4 h i

-

......-.-

SLOW: 4 hi

- 24 hr

FIG.2. Transductional sequences involved in the activation of mononuclear phagocytes. Abbreviations not described in Fig. 1 are PKC, protein kinase C; PC,phosphatidylcholine; PIP,, phosphatidylinositol4,5-bisphosphate;PLC, phospholipase C: Gc and Gs, GTP-binding proteins

22. EARLY GENE EXPRESSION IN PHAGOCYTES

593

al, 1989a; Hamilton er al., 1985). Beginning at -6 hr, transcription of class I1 major histocompatibility complex (MHC) genes is observed, followed by heightened levels of message, translation, and ultimately increased surface expression of class I1 MHC molecules (Fertsch-Ruggio er a l . , 1988; Figueiredo et a / . , 1989a). The heightened transcription of class I1 MHC genes is related to activation of the N a + / H + antiporter (Prpic et al., 1989a). Current studies in our laboratory indicate that this can be ascribed to the binding of a nuclear regulatory protein to a defined sequence within the upstream promoter region of the Ia genes (Johnson et al., 1989). The events in cascade 11, which are initiated by LPS and platelet activating factor, center initially around the hydrolysis of polyphosphoinositides, followed by the formation of breakdown products of PIP,, such as I,,4,sP3and subsequently I , .3.4.sP4r the generation of diacylglycerol, and rapid elevations in [Ca2+Ii(Prpic et ul., 1987, 1988; R. Uhing, 1989 unpublished observations). Stimulation of protein kinase C is manifested by the phosphorylation of a defined set of proteins (Weiel et al., 1986). Subsequent to these events and appearing at a later time (i.e., 10-20 min) is the activation of the Na+ / H + exchanger (Prpic et al., 1989b). We have also associated with cascade I1 the transcription of genes encoding tumor necrosis factor (TNF) and the protooncogenes c-fos and c-myc and the heightened stability of message for JE (see Adams and Koemer, 1989, for review). The events in cascade 111, initiated by surface interactions of LPS, by various maleylated proteins interacting with the low affinity receptor for such molecules (Haberland er al., 1989), or by a,-macroglobulin-protease complexes, comprise essentially the synthesis of a defined set of early proteins, which are characterized by rapid appearance, transient synthesis, and short half-lives (Hamilton et al., 1987; Johnston et al., 1987). KC expression in macrophages is observed in this cascade (Introna et al., 1987). Although one would predict that the initiation of this cascade is mediated by an early second messenger (i.e., one appearing within seconds after application of stimuli), such a messenger has yet to be identified. Several of the early genes in cascade Ill have been cloned from macrophages (Hamilton et al., 1989; Tannenbaum et ul., 1989). A fourth cascade of events (cascade IV), characterized by elevations in intracellular levels of CAMP,has been evaluated for its inhibitory actions on the other three cascades. The elevation of intracellular cAMP has long been recognized as serving a negative immunomodulatory role (Bourne et al., 1974). The elevation of cAMP inhibits the actions of events in cascades I and I1 but apparently not cascade 111 (Figueiredo et al., 1989b; Tannenbaum and Hamilton, 1989). Elevation of CAMP can occur through direct receptor-coupled activation of adenylate cyclase (e.g., prostaglandin E, and P-adrenergic agonists) or indirectly (e.g., chemoattractants); information on the molecular mechanisms involved in the inhibitory actions of cAMP is limited (Uhing et al., 1988).

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It is particularly interesting to compare cascades I and 11. Both are qualitatively similar in that they involve activation of the Na+ /H exchanger but are quantitatively distinct in that the exchanger is rapidly activated in response to IFN-y and much more slowly activated in response to LPS (Prpic et al., 1989a,b). In response to either agonist, levels of message for JE increase as determined by Northern analyses. Thc stimulation can be blocked by inhibiting the N a + / H + exchanger with amiloride or its more specific analogs. Interestingly, the time course and the extent of activation of the Na /H exchanger by PAF or LPS are quite different even though both are acting through cascade 11. This difference is mirrored closely in their relative kinetics for inducing heightened mRNA levels of JE. The two cascades, however, differ qualitatively in that, in cascade 11, the breakdown of phosphatidylinositol4,5-bisphosphate, accompanied by stimulation of protein kinase C and increased [Ca2+Ii, accompanies the activation of the Na /H+ exchanger (Prpic et al., 1989b). These four cascades are interrelated in a number of ways. Prior treatment of the macrophages with IFN-y potentiates events occurring in cascade 11. To date, two mechanisms have been suggested to explain this. First, IFN-y heightens the potential activity of protein kinase C (Hamilton et al, 1985). Second, all of the putative protein kinase C activator sn-1,2-diacylglycerol in macrophages generated in response to LPS or PAF does not come from the breakdown of the polyphosphoinositides (Uhing et al., 1989). In fact, the majority appears to come from alternative sources such as the hydrolysis of phosphatidylcholine. This alternative pathway for the generation of diacylglycerol, which is subsequent to the stimulation of protein kinase C and elevations in [Ca2+Ii,is potentiated by prior treatment of macrophages with IFN-y (Sebaldt et ul., 1989). Activation of the Na /H exchanger is also potentiated by IFN-y (Prpic el al., 1989b). PAF addition to IFN-y-treated macrophages leads to a more rapid and extensive activation of the antiporter (Prpic et ul., 1989b). Pretreatment of macrophages also lowers the dose requirement of LPS required to initiate functional responses mediated through cascade I11 (Hamilton et al., 1986). In turn, stimulation of cascade IV can dampen transductional effects which are observed in cascades I and I1 (Figueiredo et al., 1989b; Tannenbaum and Hamilton, 1989). Initiation of cascade 1V inhibits the surface expression of class 11 MHC molecules. The inhibition of surface molecules is reflected in levels of both transcription and message. The mechanisms for suppressing class I1 MHC molecules by cascade IV differ from those observed when cascade 111 is stimulated (Figueiredo et al., 1989b). +

+

+

+

+

B. Gene Regulation in Macrophage Activation We have analyzed the regulation of seven model genes in macrophage activation (for review, see Adams and Koerner, 1989). These model genes include three

595

22. EARLY GENE EXPRESSION IN PHAGOCYTES

encoding proteins that are important to the functions of macrophages [i.e., class I1 MHC (immune-associated or la molecules), tumor necrosis factor (TNF), and interleukin 1 (IL-I)] and two molecules believed to be nuclear regulatory factors for a variety of cells (i.e., the products of protooncogenes c-fos and c-myc). Additionally, we have studied the regulation of two genes (i.e., JE and KC) that were originally suggested to be competence genes initiated by PDGF in fibroblast division; these have subsequently been shown to encode proteins belonging to a superfamily of inducible cytokines related to platelet a-granule proteins. (Kawhara and Deuel, 1989: Oquendo et ul., 1989). For each of the model genes, it was determined whether altered expression of protein can be attributed to heightened-attenuated transcription or to posttranscriptional mechanisms. These changes, in turn, were then related to one or more of the transductional cascades described here (see Section IV for details, see also Adams and Koerner, 1989). Surface expression of class I1 MHC molecules is an acquired property in macrophages essential for the effective presentation of antigen to bystanding T lymphocytes (Unanue and Allen, 1987). This process is stringently regulated by positive and negative signals in the environment of the macrophages. Potent expression is enhanced in macrophages in response to IFN-y and suppressed by LPS (Adams and Hamilton, 1984). The enhanced surface expression mirrors enhanced message levels and ultimately enhanced transcription as determined in nuclear run-on assays (Koerner et ul., 1987; Figueiredo et al., 1989a). Likewise, suppressed surface expression by LPS is ultimately reflected in suppressed transcription. Several lines of evidence indicate that the initiation of transcription by IFN-y can be attributed, at least in part, to activation of the Na+/H exchanger (Prpic et al., 1989b). For example, inhibition of Na+ / H + exchange by inhibitors of the antiporter blocks interferon-mediated induction of la. Suppression by LPS can be attributed to the activation of events in cascade I11 (see Section IV),but it should also be noted that there are alternative mechanisms for suppression of la via cascade IV (see previously). +

IV. EARLY GENES IN MACROPHAGE ACTIVATION The changes in specific mRNA levels for various genes during macrophage development and activation, including early genes, have been tabulated and reviewed (Adams and Koerner, 1989). The known early genes in macrophage activation and their inductive stimuli are listed here (Table I). Expression of the protooncogenes c-fgr, c-sis, and c-src has been observed in the dewelopment of monocytes to macrophages (see Adams and Koerner, 1989, for review). As in fibroblasts, this group comprises nuclear regulatory genes such as c-fos and c-myc, genes such as TNF, IL-1, JE, and KC which regulate the functions of other cells; and other, as yet, unidentified genes. Of particular interest, three of

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DOLPH 0. ADAMS ET AL.

SOME

EARLYGENES

TABLE I IN MACROPHAGE ACTIVATION

Gene

Stimulant

Reference

c-fos

LPS LPS LPS LPS LPS, IFN-y LPS, MAL-BSA LPS, IFN-7, IFN-P LPS, IFN-y, IFN-P LPS LPS, IFN (3 IFN-y LPS

Introna et al. (1986) lntrona et ul. (1 986) Collart ri a / . (1986) Koide and Steinman ( 1987) Introna CI ul. (19x7) Introna et ul. (1987) Hamilton et a/. (1989) Hamilton ct ul. (1989) Haniilton e/ ( I / . (1989) Hamilton ct ul. (1989) Luster rt d.( 1988) Davatelis a / . (1988)

C-Ifl,YC

TNF

IL- 1 JE KC

c7 D3 D5

D8 IP- 1 Macrophage Inflammatory Protein- I Monocyte-derived neutrophil chemolactic factoi

IL- I . T N F

Matsushimi

PI

ul. (1988)

the early genes identified initially in macrophages (i.e., C7, D3, and D8) are induced in fibroblasts treated with PDGF, suggesting the generality for expression of these early genes in a variety of cells (Tanncnbauni et a l . , 1989; Hamilton et a l . , 1Y8Y). We have begun defining the mechanisms involved in the regulation of early genes in macrophages (Adams and Koerner, 1989). We have analyzed the intracellular level of regulation (Table 11). All of these genes have enhanced levels of specific mRNA in response to various activating stimuli (Table 1). By use of nuclear run-on assays, it has been determincd that the activating stimuli enhance the transcription of some of these genes as well as levels of specific mRNA; for others, increases in specific mRNA are not accompanied by increased transcription, suggesting the involvement of posttranscriptional mechanisms such as enhanced stability of message. We have also related the changes in transcription or messagc levels to one of the four signal cascades by using several tools (Table 11). First, what signals induce the changes (e.g., mal-BSA induces KC and initiates only cascade III)? Second, can induction of mRNA be initiated by pharmacological mimicry of the cascade (e.g., JE can be induced by clamping the pH, in the alkaline to simulate activation of the Na+ / H + exchange system)? Third, what is the effect of inhibitors of one of the cascades (e.g., inhibiting the N a + / H + exchanger blocks the LPS- and the IFN-y-mediated induction of JE)? These results are summarized in Table 11. Although our knowledge of such regulation is obviously just beginning, some interesting observations have already emerged. For instance, the regulation of JE

597

22. EARLY GENE EXPRESSION IN PHAGOCYTES TABLE I1 REGULATION OF SOMEEARLY GENES I N MACROPHAGE ACTIVATION" ~~

Gene

Level of Regulationh

T T T

c-fos

c-my TNF IL- 1 JE

KC

T

r

TS TS TS mRNA mRNA T TS

Cascade(s)c I1

I1 11 11

I , IT 111

" Adapted from A d a m and Koemer (1989. and updated). TS, transcription; mRNA, increased level of message

',

without increased transcription. <SeeFig. 1

and KC in macrophages is distinct from that in fibroblasts. In fibroblasts, PDGF initiates transcription of JE and KC (Stiles, 1983). In macrophages, LPS or other stimulants initiate enhanced transcription of KC but not of JE, and the transcription of KC is not related to phosphatidylinositol metabolism (Introna et al., 1987). Induction of enhanced levels of mRNA for JE is induced by IFN-y, LPS, or PAF, and these three stimulants apparently act via the activation of the N a + / H + exchange system (Prpic e t a / . , 1989a,b). The role of early genes in nuclear regulation of mononuclear phagocytes is just beginning to emerge. For example, the role of c$os protein and its relationship to c-jun has yet to be explored in mononuclear phagocytes. Members of the group of genes initiated in cascade 111 remain to be characterized both as to sequence and as to which one(s) is pertinent to the suppression of transcription of class 11 MHC genes initiated by IFN-y. LPS and maleylated proteins both suppress such transcriptional activation (Figueiredo et a / . , 1989a). Such inhibition can be blocked by co-treatment of the macrophages for a few hours with cycloheximide, suggesting that the inhibitory effects are due to the synthesis of new proteins (Hamilton et al., 1987; Collart er a/., 1986). By contrast, the inhibitory effects mediated in cascade 1V cannot be suppressed by inhibitors of protein synthesis (Hamilton et al., 1987).

V.

CONCLUSIONS AND FUTURE DIRECTIONS

The molecular mechanisms which control macrophage activation are now beginning to emerge (Fig. 2). Multiple second messenger events initiated by potent regulatory signals such as IFN-y and LPS have now been delineated as

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DOLPH 0. ADAMS ET AL.

having the fundamental intracellular level of regulation of genes encoding cardinal functional proteins such as la, 1L-1, and TNF. The broad interrelationships between these cascades of events are now beginning to be defined, such that transcription of several genes can be ascribed to one or more of the four signal cascades defined. Macrophage activation, like the activation of other cells such as fibroblasts and lymphocytes, is accompanied by the expression of early genes, which are believed in part to regulate genomic expression and in part to serve autocrine and paracrine regulatory functions. The early genes, which are observed during macrophage activation, and their regulatory features are just now beginning to be cataloged (Table I) as is their regulation by internal signals (Table 11). At present, functional information about these proteins in macrophages is quite limited, but emerging evidence suggests that one or more of the proteins in cascade 111, members of which have now been cloned (Hamilton rt ul., 1989), may be involved in the suppression of IFN-y-mediated transcription of Ia genes. The role and importance of early gene expression in cellular biology is clearly an important and exciting, but still emerging, story. Some of the critical issues to be addressed will be how the transcription of these genes is regulated by external stimuli and second messengers and how the products of these genes regulate the expression of other genes. A particularly exciting possibility is the question of how, in macrophages and in other cells, similar stimuli can elicit quite distinct patterns of gene expression (for review in macrophages, see Adams and Koerner, 1989). One exciting possibility to explain such differential gene regulation is cooperation between products of two or more early genes as typified by interactions of the protein products of c-fos and c-jun. Macrophages provide a powerful and interesting model system for exploring these general issues because of the extensive information now available about second messengers and genes important to the function of these cells. ACKNOWLEDGMENTS The authors gratefully acknowledge the helpful advice and discussion of Dr. Thomas A. Hamilton as well as his generosity in providing pre-prints of a manuscript in press. Research supported in part by USPHS grants CA16784, ES02922, and CA29589. REFERENCES Adams, D. O., and Hamilton, T. A. (1984). The cell biology of macrophage activation. Annu. Rev. Immunof. 2, 283-318. Adams, D. O., and Hamilton, T. A. (1987). Molecular bases of signal transduction in macrophage activation induced by IFNy and by second signals. Immunol. Rev. 97, 5-28. Adams, D. O., and Hamilton, T. A. (1989). Macrophages as destructive cells in host defense. In “Inflammation: Basic Principles, and Clinical Correlates” (J. I . Gallin, 1. M. Goldstein, and R. Snyderman, eds.), pp. 471-492. Raven, New York. Adams, D. 0.. and Koerner, T. J. (1989). Gene regulation in macrophage development and activation. Year Immunol. 4, 159-180.

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Baeuerle, P. A , . and Baltimore, D. (1988). IKP: A specific inhibitor of the NFKPtranscription factor. Science 242, 540-546. Bourne, H. R., Lichtenstein, L. M., Melmon. K . L . . Henney, C. S . , Weinstein, Y., and Shearer, G. M. (1974). Modulation of inflammation and immunity by cyclic AMP. Science 184, 19-28. Collart, M . A,, Belin, D., decossodo, S., and Vassalli, P. (1986). y-Interferon enhances macrophage transcription of tumor necrosis factoricachectin, interleukin-I, and eurokinase genes which are controlled by short-lived repressors. J . Exp. Med. 164, 21 13-21 18. Curran, T., and Franza, R. B., Jr. (1988). Fos and jun: The AP-I connection. Cell 55, 395-397. Davatelis, G . , TeKamp-Olson, P., Wolpe, S . D., Hermsen, K . , Luedke, C., Gallegos, C., Coit, D., Menyweather, J., and Cerami, A. (1988). Cloning and characterization of a cDNA for murine macrophage inflammatory protein (MIP), a novel monokine with inflammatory and chemokinetic properties. J . Exp. Med. 167, 1939-1944. Dynan, W. S . , and Tjian, R. (1985). Control of eukaryotic messenger RNA synthesis by sequencespecific DNA-binding proteins. Nature (London) 316, 774-777. Fertsch-Ruggio, D., Schoenberg, D. R., and Vogel. S . N. (1988). Induction of macrophage Ia antigen expression by rIFN-y and down-regulation by IFN-aIP and dexamethasone are regulated transcriptionally. J. B i d . Chem. 141, 1582- 1589. Figueiredo, F., Koerner, T. J., and Adams. D. 0. (1989a). Molecular mechanisms regulating the expression of class 11 histocompatibility molecules on macrophages: Effects of inductive and suppressive signals on gene transcription. J . Immunol. in press. Figueiredo, F. Okonogi, K., Gettys, T., Uhing, R. J., Prpic, V., and Adams, D. 0. (1989b). Submitted for publication. Gentz, R., Roszer, F. J . , 111, Abate, C . , and Curran, T. (1989). Parallel association offos and jun leucine zippers juxtaposes DNA binding domains. Science 243, 1695- 1699. Haberland, M. E., Tannenhaum, C. S., Williams, R. E., Adams, D. O., and Hamilton, T. A. (1989). Role of the maleyl-albumin receptor in activation of murine peritoneal macrophages in vitro. J . Immunol. (in press). Halazonetis, T. D., Georgopolous, K., Greenberg, M. E., and Leder, P. (1988). C-jun dimerizes with itself and with c-fos, forming complexes of different DNA binding affinities. Cell 55,917924. Hamilton, T. A., and Adams, D. 0. (1987). Molecular mechanisms of signal transduction in macrophage activation. Immunol. Toduy 8, 151-158. Hamilton, T. A , , Becton, D. A., Somers, S. D., and Adams, D. 0. (1985). Interferon gamma modulates protein kinase C activity in murine peritoneal macrophages. J . B i d . Chem. 260, 1378-1381. Hamilton, T. A., Somers, S . D., Jansen, M. M . , and Adams, D. 0. (1986). Effects of bacterial lipopolysaccharide on protein synthesis in murine peritoneal macrophages: Relationship to activation for macrophage tumoricidal function. J . Cell. Physiol. 128, 9-17. Hamilton, T. A , , Gainey, P. V., and Adams, D. 0. (1987). Maleylated-BSA suppresses IFNymediated la expression in murine peritoneal macrophages. J . Immunol. 138, 4063-4068. Hamilton, T. A., Bredon, N., Ohmori, Y., and Tannanbaum, C. S . (1989). Interferon-y and interferon+ independently stimulate the expression of Iipopolysaccharide-induciblegenes in murine peritoneal macrophages. J . Immunol. 142, 2325-233 I. Honess, R. W., and Roizman, B. (1974). Regulation of herpesvirus macromolecular synthesis. 1. Cascade regulation of the synthesis of three groups of viral proteins. J. Virol. 14, 8-19. Introna, M., Hamilton, T. A , , Kaufman, R. E., Strassman, G . , Adams, D. 0.. and Bast, R. C. (1986). Functional activation of macrophages with bacterial LPS alters expression of c-myc and c-myc and c-fos oncogenes. J. Immunol. 137, 271 1-2715. Introna, M., Bast, R. C., Tannenbaum, C. S., Hamilton, T. A., and Adams, D. 0. (1987). The effect of LPS on expression of the “early” competence genes JE and KC in murine peritoneal macrophages. J . Immunol. 138, 3891-3896.

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Index A

lymphocytes and activation by lymphokines, 518 calcium changes, 168 intracellular pH, 208, 210, 213, 217-220 ion transporr, 108- I 12 phagocytes and, 594 phagocytic leukocytes and, 229, 230, 233, 235 Amino acids leukocytes and, 427 lymphocytes and activation by lymphokines, 501, 502, 504 cyclic nucleotides. 380 GTP-binding proteins, 53 phosphoinositide metabolism, 253, 273 protooncogene expression, 575 phagocytes and cytokines, 543 GTP-binding proteins, 73, 82, 85, 86 T cell receptor and, 2, 6, 8, 9, 11 Anti-inflammatory drugs, 355, 357, 358 Antibodies leukocytes and, 433, 434. 445, 456 lymphocytes and activation by lymphokines, 496, 504, 516, 520 calcium changes, 153, 154, 161, 165, I66 cyclic nucleotides, 370, 382, 383, 389, 391 GTP-binding proteins, 48, 52, 53 intracellular pH, 212, 214-216, 223 ion transport, 106, 109 protooncogene expression, 578, 579 monoclonal, see Monoclonal antibodies phagocytes and calcium, 186 chemoattractant regulation, 22 CTP-binding proteins, 83-86

Acctylcholinc. 417 Actin leukocyres and. 429 neutrophils and, 416 phagocytes and calcium, 194- 197 ion transport, 138 phosphoinositide metabolism, 317 phosphorylation, 486 phagocytic leukocytes and, 234-236 Adenylate cyclase arachidonate metabolites and, 366 CTP-binding proteins in phagocytic cells, 91, 92 low molecular weight, 73 neutrophil composition, 82, 84 receptor coupling, 70, 71 signal transduction, 87 lymphocytes and acivation by lymphokines, 508 arachidonic acid, 337 cyclic nucleotides, 377-379, 384, 390 CTP-binding proteins, 49, 5 I ion transport, 116 phosphoinositide metabolism, 262, 275277, 286 neutrophils and, 400, 402-408. 410, 416. 417 phagocytes and gene expression, 593 phosphoinositide metabolism, 3 18 P-Adrenergic receptors, 400. 41 1-414, 416 Alkalosis, 230, 231 Allogeneic cells, 21 I Amiloride arachidonate metabolites and, 361

603

604 phosphorylation, 476, 482 phosphoinositide metabolism in lymphocytes and, 250 l3 cells, 282 286 calcium, 268, 269 mitogens, 258 T cell proliferation. 262, 263. 272-279 T cells, 255-257 Antigen-presenting cells, lymphocytes and calcium changes. 154, 158 cyclic nucleotides. 376 phosphoinositide metabolism, 254, 256, 260, 281 Antigens lymphocyte activation by lymphokines and, 496, 497. 523 initiation, 505, 506 rcccptors. 52 1-523 transmembranc signaling, 508, 5 10 lymphocytes and aracliidonic acid, 335, 336, 341 calcium changes. 1.55, 1.58, 159. 161. lh4, 165 cyclic nucleotides, 375, 376, 379 385, 389, 390 C’IP-binding proteins, 50-53, 57, 59 intracellular pH. 212, 214 prutuuncogcnc cxprcssion, 572, 573, 575, 577, 578. 580, 582 phapocytcs and calcium, 186

and, 250. 252 cellular immunity, 279, 281 T cell proliferation, 258, 259. 270, 27227x T cella, 253, 254, 256, 257 protein phosphorylation in leukocytes and. 426, 457 lymphoid cells, 432-435. 440 myeloid cells, 453 T cell receptor and, I, 12 Arachidonate metaholites in phagocytes, 349, 350. 367 hiochcinical nicchanisnis, 353 anti-inllaninlatury drugs, 357, 358 cyclooxygenase. 355, 356 diacylglyccrol lipase, 354, 355 5-lipoxygenase pathway, 356, 357 phospholipase A ? . 353. 354

INDEX host-defense mechanisms, 350-353 macrophages, 364- 366 plalclel activating factor, 362-364 transduction, 358-362 Arachidonic acid lymphocytes and, 333-335, 345 cyclooxygcnase, 335-338, 340 lipoxygenasr, 338-345 lymphokines, 507, 51 I , 512 phagocytcs and cytokines, 549, 555. 556, 560 iihosplroryla~ion, 475, 480. 484-480 phosphoinositide metabolism and lymphocytes, 258 phagocytes, 310, 319-324 Arginine, 549, 558-560 Asthma. 400, 406 ATP leukocytes and, 427, 439, 448 lymphocytes and cyclic nuclcotidcs, 377 GTP-binding proteins, 53 inn tmnspciit. 104. 1 1 0 , 113 phosphoinositide metabolism, 26 I neutrophils and, 407, 413 phagocytes and calcium, 182. 183. 185. 195 GTP-binding proteins, 80 ion transport, 133 phosphoinositide nietabolism. 30.5. 309. 311, 316, 319 phosphorylation. 472, 4x3, 484 phagocytic leukocytes and, 228, 232, 233, 239 ATPasc lymphocytes and, 267 phagcicytic Icukocytcs and. 233. 239

B €3 cclls

leukocytes and, 440-442, 457 lymphocytes and activation by lymphokines and, 500 calcium changes. 153, 165-170 cyclic nuclcutides, 376. 380. 388-390 intracellular pH, 21 I , 216-222 inn transport, 106, 109. I I I , 112. 115 phosphoinositide metabolism, 250, 257, 282-287

INDEX

605

proliferation, 264, 274, 275. 277, 278 protooncogene expression, 574, 578, 579 phagocytcs and, 544 T cell receptor and, 2. 12 B lymphocytes GTP-binding proteins and, 45, 48, 50. 5358 T cell receptor and, 1 Bacteria arachidonatc metabolites and, 359, 365 leukocytes and, 442. 452 lymphocytes and, 46 phagocytes and chernoattractant regulation, 20, 33 cytokincs, 538, 539. 553 gene expression, 588 GTP-binding proteins, 65. 66, 90 ion transport, 138 phosphoinositide metabolism, 313, 319, 320, 322 phosphorylation, 472 phagocytic leukocytes and, 234, 237-239 BAPTA, lymphocytes and calcium changes, 156. 164, 166, 169 intracellular pH, 214 Bone marrow arachidonate metabolites and, 356 leukocytes and, 443, 449, 457 lymphocytes and phosphoinositide metabolism, 250 protooncogenc expression, 574 phagocytes and, 544. 545, 550 Bordelella pertusxi3

lymphocytes and, 46, 47, 51, 5 5 , 58 phagocytcs and, 68 Botulinuin toxin. 86

c C5a. phagocytes and chenioattractant regulation, 20. 21, 32-35 GTP-binding proteins, 66. 74 Calciosornes lymphocytes and, 267, 275 phagocytes and, 185- 187 Calcium ardchidonate metabolite5 and, 367 biochemical mechanisms, 353-357 host-defense mechanisms, 35 I macrophages, 362-364, 365

platelet activating factor, 362-364 transduction, 359-362 cytoplasmic, sc'e Cytoplasmic calcium in phagocyte activation GTP-binding proteins in phagocytic cells and luw molecular weight, 74 neutrophil activation, 76-78, 81 signal transduction, 87-89 ion transport in lymphocyte activation and, 119, 120 cation channels, 114 manion channels, 113 niembranc potenfjal changes, 106, 107 pH, 109, 110 ion transport in phagocytic activation and, 132, 147 conductance, 142- 145 macrophagcs, 133, 136-140 lymphocytes and activation by lymphokines, 507. 508, 510,511,521-523 arachidonic acid, 335, 338-342 cyclic nucleotides, 378, 382, 383, 390 GTP-binding proteins, 5 I intracellular pH, 209, 212, 214. 215, 218, 223 protooncogene cxprcssion, 573, 576, 579 neutrophils and, 407, 408, 41 I , 413, 417, 418 phagocytes and chemoattrdctant regulation, 25-27, 33 cytokines, 541, 543, 547, 554, 556 gene expression, 590, 593, 594 phosphorylation, 472, 474-476 phagocytic leukocytes and, 230, 235, 236 phosphoinositidc metabolism in lymphocytes and, 251, 287 B cells, 283-286 CAMP. 276, 277 ccllular immunity, 279-282 mitogens, 257-259 negative tignals, 279 protein kinasc C, 27 1, 272 responses, 266-269 T cells, 256, 261-263 phosphoinositide metabolism in phagocytes and, 304, 324 neutrophils, 311, 313-320 phosphatidic acid, 306, 308-310

606

INDEX

protein phosphorylation

in

leukocytes and,

426-429 lymphoid cells, 433,434,440,441 myeloid cells, 442,444,451,454 Calcium changes during lymphocyte activation, 153, 154,170 R lymphocytes depolarization, 169, 170 hyperpolarization, 168, 169 ligands. 16X. 170 receptors, 165-168 measurement, 154, I55 T lymphocytes hyperpolarization, 162, I63 ligands, 164,165 pH, 161,162 receptors, 155- I 6 I Calmodulin lymphocytes and activation by lyinphokincs. 507 cyclic nucleotides, 378 inIracellular pH, 214 ion transport, I14 ncutrophil~and, 407 phagocytes and, 472 Calmodulin kinases, 42X,429,451,453 Calpain, 472.476,477,480 Calscqucstrin. 186 Carboh ydrate lymphocytes and, 255 phagocyto and, 34 Catecholamines. 88 CD2 cells, 250,254,268,269 Cl)3 complcx. lymphocytes and CTP-binding proteins, 50,52, 57-59 phosphoinositide metabolism calcium, 266,268 mitogens, 2.58,2.59 1’ cells, 253. 254. 256,262,272-274 CD4 cells leukocytcs and, 434.457 lymphokincs and, 497.516 CDR cells leukocytes and. 434,457 lymphocytes and, 256,257,266,268,273 lyinphokines and, 497,516 cDNA leukocytes and, 426,427 lymphocytes and activation by lymphokines, 496,502,

504.520

phosphoinositide metabolism, 258,269 protooncogene expression, 573 phagocytes and, 85 T cell receptor and, 2-4 CHAPS, phagocytcs and, 25,32 Chemoattractant regulation of phagocytosis, SYC Phagocytosis, chemoattractant regulation of Chcmotaxis neutrophils and, 410 phagocytes and, 547,548,552 Cholera toxin lyniphokines and, 508,519 neutrophils and, 410-412.416 Cholesterol, 184 Chromatin, 21 3 Chromosomes lymphocytes and activation by lyniphokincs, 4YX.499 intracellular pH, 2I3 protooncogcnc expression, 572 T cell receptor and, 2,5 , 6,7,1 1 Chronic granulomatous disease leukocytes and, 453 phagocytes and, 470.477-480,482,483,

486 CI-HC03- exchange, 228,231,232.241-

243 Clones GTP-binding proteins iii phagocytic cells and, 92 low molccular weight, 73.74 neutrophil composition, 82,84-86 receptor coupling. 68, 69 leukocytes and, 426,432,448,457 lymphocytes and activation by lymphokines, 498,501.

502,523 arachidonic acid, 336, 339,341-344 calcium changes, 158. 160,165 cyclic nucleotides, 376,379,386,388 GTP-binding proteins, 52 ion transport, I17 protooncogene expression, 576,577 phagocytes and cytokincs, 543 gene expression, 593,598 phosphorylation, 474,483 phoaphoinositide metabolism in lymphocytes and, 250, 25 I, 253 B cells, 286

INDEX

607

CAMP, 277 phagocytes and cellular immunity, 279, 281, 282 calcium, 190, 191 mitogens, 258. 259, 278 gene expression, 590, 593 T ccll proliferation, 261. 266, 268. 271, GTP-binding proteins, 84, 87-89 272 phosphoinositide metabolism, 318 T cells, 253, 255-257 phosphoinositide metabolism in lymphocytes T cell receptor and. 4, 6, 12 and, 252, 287 Colony stimulating factors B cells, 284, 286 cytokines in phagocytes and, 542, 544, 546 cellular immunity, 280, 281 macrophages, 550,551,553,555. 557,559 T cells, 262, 275-278 neutrophils, 545, 547, 548 protein phosphorylation in leukocytes and, leukocytes and, 442-449, 455-457 428, 429, 455 lymphokines and. 496, 498-500, 506 lymphoid cells, 433, 434 transmembrane signaling, 5 12, 5 16, 5 18, myeloid cells, 452, 455 521 Cyclic GMP neutrophils and, 418 lcukocytcs and, 429, 455 phagocytes and, 27, 28, 90 lymphocytes and, 375, 376, 379-386, 388, Concanavalin A 389, 391 lymphocytes and neutrophils and, 400, 406, 417-419 arachidonic acid. 338, 339 Cyclic nucleotides in lymphocytes, 375, 376, calcium changes, 155, 159, 161 390, 391 cyclic nuclcotides. 376. 381, 382. 384, activation signals, 377 386, 388 antigen receptor, 379-384 intracellular pH. 210, 21 I , 214. 215 CAMP, 377-379 ion transport, 106, 109. 1 1 1 , 116, 117 cGMP, 378, 379 protooncogene expression, 573. 576-578 lymphokine receptor, 384, 385 neutrophils and, 406 niodulatory effects, 385, 386 phagocytes and B lymphocytes, 388, 389 calcium, 195 T lymphocytes, 386-388 ion transport, 141 signal transduction, 389, 390 phosphoinositide mctabolisni, 306, 31 7 Cyclic nucleotides in neutrophils, 399-401, phosphorylation, 475, 477 419 Conductance, phagocytes and. 133. 135. 139. adenylate cyclase, 400, 402-406 142- I47 CAMP, 406-408 CT. see Vihrio cholera toxin chcniotaxis, 410, 41 I Cyclic AMP degranulation, 408-410 arachidonate metabolites and, 366 intracellular signals, 412-416 lymphocytcs and receptors , 4 12 activation by lymphokines, 512, 513, 519 superoxide anions, 41 1 arachidonic acid, 335. 337 cGMP, 417-419 calcium changes, 167 disease, 416, 417 cyclic nucleotides, 375-391 Cyclooxygenase GTP-binding prutcins. 47, 5 I arachidonate metabolites and iontransport, 113. IIS, 118 biochemical mechanisms, 353, 355lyniphokines and, 519 358 neutrophils and, 400, 403-408, 417-419 host-defense mechanisms, 351, 352 chemotaxis, 410. 41 1 niacrophages, 366 degranulation, 408-410 transduction, 359-361 intraccllular signals. 412-416 arachidonic acid in lymphocytes and, 333 recepturs, 412 activation, 335-339 superoxide anions, 41 1 synthesis, 340

608 lymphocytes and, 5 I 1 phagocytes and, 556 Cycloaporin. I0 5 Cyatic fibrosis. 400, 416. 417 Cytcichalasins lymphocytes and calcium changes, 166 phosphoinosit idc mcta bol ism, 264, 265 neutrophils and, 409, 413, 417 phagocytes and calcium, 195 phosphninositide metabolism, 322 phosphorylation, 475. 482 Cytochromes. 478-~480.483 C ytokinca leukocytes and. 442, 456, 457 lymphocytca and activation by lymphokincs, 496 cyclic nucleotides, 384. 388, 391 intracellular pH, 208. 222 phosphoinositide metaboliam, 279 protooncogcne expression, 5x2 phagocytes and, 595 Cytokincs in phagncyrcs, 538. 541, 542, 561 activation, 538, 539 colony stimulating factors. 544 interleukin- I , 542, 543 intcrlcukin-2, 543 interleukin-4, 544 interleukin-6, 544 rnacrophagca, 539-541. 54')-552, 560. 561 arachidonic acid, 555, 556 arginine. SSX, 5.59 microbicidal mechanistns, 552, 5.53 oxidation, 5.59 synthrsis, 556-558 tuniors, ,753- 55.5 ncutrophils, 539, 545-549 transforming growth factor p, 544, 545 tumor necrosis factor. 543 Cytoplasm Icukocytcs and, 442 lymphocytes and activation by lymphokines, 502, 520 cyclic nucleotidcs. 375, 378, 379, 390 intracellular pH, 210 phosphoinositidc metabolism. 269. 27 I , 272 protooncogene expression, 578 ncutrophila and, 406

INDEX phagocytes and cytokincs, 554 gene expression, 590 phosphoinositide metabolism, 3 13, 3 16 phosphorylation, 483 T cell receptor and, 4, 6, 8, 9 Cytoplasmic calcium in phagocyte activation, 1x0, 197. 198 changes cell populations, 187-191 single cells, 191-193 homeostasis calciosonic, 185- 187 plasma membrane, 184, 185 rneasurenient. 180, 18 1 indicators, I8 I , I82 tripping, I X2- I84 second mesaengers metabolic responses, 193, 194 motility, 194-197 Cytoplasmic pH in phagocytic leukocytes activation, 234 function, 2 3 7 ~ ~ 2 3 9 microbicidal activity, 236, 237 migration, 234-236 proton extrusion, 239 differentiation, 239, 240 granulocytes, 240, 241 macrophagcs, 242, 243 monocytes, 242 mcchanismb, 227, 228 ATP-dependent Hi extrusion, 232, 233 CI-HC03- exchange. 232 Na+/H+ exchange, 228-230 Cytoskeleton leukocytes and, 236, 429, 442 phagocytes a n d , 4 7 5 , 4 8 0 , 4 8 2 Cytosol arachidonate metabolites and, 353-357, 360, 363 lymphocytes and activation by lymphokines, 507, 5 13, 514, 518 arachidonic acid. 335 calcium changes, 161, 165- 170 cyclic nucleotidea. 379 intracellular pH, 2 I 9 ion transport, 105 phosphoinositide metabolism, 25 I , 274, 275, 285

609

INDEX neutrophils and, 400, 415, 417, 418 phagocytes and GTP-binding proteins, 79, 83, 85. 86, 90 phosphoinositide metaholisrn, 308, 3 12314, 316. 317. 322, 323 phosphorylation. 471, 475, 476, 478480. 482-485 phagocytic leukocytes and, 229. 230 protein phosphorylation and lymphoid cclls, 440, 441 myeloid cells, 443, 444. 447, 453, 454

D Degranulation leukocytes and, 454, 455 neutrophils and, 408-410, 417, 418 phagocytes and, 471-473, 477, 480 phagocytic leukocytes and. 237. 239 Delaycd hyperscnaitivity, 250 Depolarization neutrophils and, 412 phagocytes and. 474 Diacylglycerol arachidonatc metabolites and, 35 I , 360, 364 lymphocytes and calcium changes, 160. 165 intracellular pH. 219 ion transport. I I0 lymphokines, 507, 508, 521 transmembrane signaling. 510. 511, 513, 514, 518 ncutrophils and. 41 I , 412, 415 phagocytes and calcium, 190. 191, 196 gene expression, 593, 594 GTP-binding proteins, 88 phoaphorylation. 471 -376. 482 phagocytic leukocytes and, 230 phosphoinositide metabolism in lymphocytes and. 251, 252. 287 B cclls. 284 cellular immunity, 280 ‘r cell prolifcretion, 257. 258. 261, 263, 271-273 phosphoinositide metabolisiu in phagocytes and neutrophils, 310, 313, 317-319, 321, 323, 324 phosphatidic acid, 306. 308. 310

protein phosphorylation in leukocytes and, 426, 427 lymphoid cells, 441. 442 myeloid cells, 442, 443, 447, 453, 4.54 Diacylglyccrol lipase biochemical mechanisms, 353-355 host-defense mechanisms, 351 macrophages, 366 transduction, 359 DIUS, leukocytes and, 231, 232, 241 Differentiation lcukocytes and, 449-452, 454, 457 phagocytic leukocytes and, 239-243 Diglyceride kinase. 305-308, 310. 314, 325 Dimcthylaniiloridc lymphocytes and, 208. 21 I , 215. 216 phagocytic lcukocytes and, 240, 241 DMSO leukocytes and, 450-452, 454 PhdgOcyteS and, 472 phagocytic leukocytes and, 240, 241 DNA lymphocytes and activation by lymphokincs, 499 cyclic nucleotides, 385, 386, 389 GTP-binding proteins, 57 intracellular pH, 208-212 protooncogene expression, 572, 578 phdgoCyteS and, 558 phosphoinositide metabolism in lymphocytes and, 252 B cells, 283, 285 T cells, 257, 260, 212-274, 277, 278 T cell receptor and, 4-7

E EDTA, phagocytes and, 313 Effector cells, 250, 251 EGTA lymphocytes and intracellular pH, 214 phosphoinositide metabolism, 28 I neutrophils and, 407 phagocytes and calcium, 183, 195, 197 phosphoinositide metabolism, 309, 3 I6 phosphorylation, 483 Eicosanoids

610 arachidunate metabolites and, 349, 350, 367 biochemical mechanisms, 353-358 host-defense mechanisms. 350-353 macrophagea, 364-366 platelet activating factor, 362-364 transduction, 358-362 arachidonic acid in lymphocytes and, 333, 341, 342. 345 EIPA, phagocytic leukocytes and, 240. 242 Enducytusis, phagocytes and, 138 Endoplasmic reticulum ardchidonate metabolites and, 355 lymphocytes and calcium changes, 156 phusphoinusitidc nictabolisni, 266. 267, 283 phagocytes and calcium. 1x6 phosphuinusitidc metabolism, 3 10, 3 I 1 Eiiduthelial cells, cytokines and, 539, 543, 544 macrophages, 55 I , 552 neutrophils, 545. 546 Enzymes arachidonatc metabolites and, 367 biochemical mechanisms, 353. 355, 356 host-defense mechanisms, 351 platelet activating factor, 362 transduction, 360 GTP-binding proteins in phagocytic cells and, 66 neutruphil activation. 75- 77 receptor coupling, 71 signal transduction. 87, 89 leukocytes and, 426, 42X, 429, 435, 448, 453 lymphocytes and activation by lymphukincs. 499 arachidonic acid, 338 cyclic nucleutidcs, 378-380. 383. 385, 389 CTP-binding pruteins, 46, 47, 50. 58 intracellular pH, 207-209, 214 ion transpun. 104, 110, I18 neutrophils and, 403, 408, 409. 415. 417 phagucytcs and chemuattractant regulation, 21, 31, 32, 34 cytokines, 541, 547, 558 phusphurylation, 470, 472, 474, 480, 482-484

INDEX phagocytic leukocytes and, 237 phosphoinusitide metabolism in lymphocytes and. 25 I , 287 B c c h , 286 cellular immunity, 280 T cells, 254, 261. 262, 271. 273-275 phosphoinusitide metabolism in phagocytes and. 303-305, 324, 325 neutrophils, 3 10-324 phosphatidic acid, 308, 310 T cell receptor and, 3-5, 7 Epidermal growth factor leukocytes and, 430, 431, 443, 444, 447, 449 lymphocytes and activation by lymphokines. 506. 508, 512. 515, 516.522 intracellular pH, 209 phosphoinositide metabolism, 252, 273 phagocytes and, 33, 36 Epinephrine, 409, 410 Epithelial cells lymphocytes and. 268 phagocytcs and. 552 Epitopes, lyrnphucytes and intraccllular pH. 2 15 phosphoinusitide metabolism, 258, 269 Erythrocytes lymphucytes and. 2 I S neutrophils and, 412 phagocytes and, 183, 187 Esrhrrirhia coli lymphocytes and. 498 neutrophils and. 410 Exocytosis lymphocytes and, 28 I phagocytes and GTP-binding proteins, 78, 91 phosphoinositide metabolism, 3 I7 Extracellular matrix, phagocytes and, 28

F Fatty acids ardchidonate metabolites and. 355, 362 leukocytes and, 426 lymphocyte\ and. 333 phagucytes and chemoattractant rcgulatiun, 20

INDEX phosphoinositide metabolism, 308, 32 I , 323 phosphorylation, 473, 484 phagocytic leukocytes and, 238 Fc receptors lymphocytes and, 255, 283 neutrophils and, 412 phagocytes and calcium, I95 cytokines, 547, 550 GTP-binding proteins, 74 ion transport, 144-147 Feedback lymphocytes and, 160 phagocytes and, 189- 191 Fibroblasts leukocytes and, 429, 457 lymphocytes and activation by lymphokines, 499. 501 intracellular pH, 209, 210 phosphoinositide metabolism, 252, 287 protooncogene expression, 575, 577 phagocytes and chemoattractant regulation, 36 cytokincs. 543, 544. 557 gene expression, 587, 589, 595-598 phosphoinositide metabolism, 304 Fluorescence lymphocytes and calcium changes, 154-158, 166 ion transport, 10.5, 121 phosphoinositide metabolism, 256. 266, 280, 283, 286 neutrophils and. 413 phagocytes and calcium, I8 1 - 183 ion transport, 129, 142 phosphorylation, 474 Fluorimetry, lymphocytes and, 155 FMLP leukocytes and, 454, 455 neutrophils and, 406-409, 41 1-414, 417 phagocytes and chemoattractant regulation, 25. 27-29, 32, 33, 35 cytokines, 539, 546. 547 ion transport, 138, 139, 142-144, 147 phosphorylation, 472-476, 480, 482-485 phagocytic leukocytes and, 230, 234-237 Forskolin

61 1 arachidonatc metabolites and. 366 lymphocytes and cyclic nucleotides, 386 phosphoinositide metabolism, 275-278 phagocytes and, 3 I8

G G proteins arachidonate metabolites and, 359 lymphocytes and, 45-50 activation by lymphokines, SOX, 518, 519 antigen receptor, 50-52 B lymphocytes, 53-57 calcium changes, 161 cyclic nucleotides, 378, 390, 391 future perspectives, 57-59 IL-2 receptor, 52, 53 neutrophils and, 400, 403, 407, 408, 41 I , 412 phagocytes and, 65-67, 91, 92 calciuni, 190 chemoattractant regulation, 25, 26, 32 gene expression, 589 low molecular weight, 72-74 neutrophil activation, 74-81 neutrophil composition, 81-87 phosphoinositide metabolism, 308, 3 13, 314, 317, 318, 320 phosphorylation, 472 receptor coupling, 68-71 regulatory cycle, 71, 72 signal transduction, 87-91 phagocytic leukocytes and, 230, 235 phosphoinositide metabolism in lymphocytes and, 253, 287 B cells, 285, 286 T cells, 261, 262, 276 GDP, lymphocytes and, 46, 48, 49 Gelsolin, phagocytes and, 195 Gene expression in phagocytes, 587-589, 597, 598 early genes, 589, 590, 595-597 macrophage activation, 590-595 Glycoprotein lymphocytes and GTP-binding proteins, SO phosphoinositide metabolism, 253, 256, 258

612

INDEX

phagocytcs and chemoattractant regulation, 29 cytokincs, 543. 544 Crlycosyl-phosphatidylinositol~254, 255 Glycosylation lymphokines and, 498, 502, 504 phagocytcs and. 543 T cell receptor and, 8 Golg~apparatus, phagocytes and chemodttractant regulation, 25 GTP-binding proteins, 73 GrdllUlocytCS cytokines in phagocytes and, 542, 544 macrophages. 551. 553, 555. 557. 5.59 neutrophils, 545, 547, 548 leukocytes and, 442-44.5. 447, 449-452, 455

lymphocytes and, 496, 498-500, 512, 518, 521

neutrophils and, 404, 418 phagocytes and. 27. 2X, 321 phagocytic leukocytes and, 228, 239-24 1 Growth factors lymphocytes and activation hy lymphokines, 499, 505, 507,515, 518. 522

arachidonic acid, 337 intracellular pH, 2OX-210, 213, 216 phosphoinositide metabolism, 252, 270, 273

protooncogene expression, 577 phagocytcs and, 589 phag(JCyIk kUkUcyft3 a d , 243 protCll1 phosphorylation in leukocytes and, 430, 456

lymphoid cells, 435 myeloid cells, 442, 444, 447-449 receptor, 73 GI‘P lymphocytes and activation by lymphokines, 508 calcium changes. 160 cyclic nucleotides, 378, 390 neutruphils and. 403. 407 phagocytes and, 25, 26, 32 GI?-binding proteins, see G proteins Guanylate cyclase leukocytes and. 45.5 lymphocytes and. 379. 383. 384. 386, 3x8, 39 I ncutrophils and, 417, 418

H Hematopoeisis, leukocytes and, 426, 428, 430, 432, 457

myeloid cells, 444, 447, 448 Histamine, neutrophils and, 404, 41 I lymphocytes and, 207, 210, 212, 223 ncutrophils and, 413 phagocytes and, 180, 1 x 4 ~187, 191 phayucytic leukocytes and, 228, 229 Hormones arachidonatc metabolites and, 354. 358 leukocytes and, 435 lymphocytes and activation by lyrnphokincs, 4Y6, 497, 507, 5 I 8 cyclic nucleotidcs, 375, 37X, 379. 382, 386. 388. 391 phosphoinositidc mctabalisni, 262 phagocytes and, 71, 72, 84, 87, 88 Host-defense mechanisms. arachidonate metaholites and, 350-353 HPBL. see Human peripheral blood lymphocyks Human peripheral blood lymphocytes, 379 381, 384, 386, 389 Hybridization leukocytes and. 426 phagocytes and, 476 ‘I’ cell receptor and, 2-8 Hybridomas tcukocyces and, 434 lymphocytcs and arachidonic acid. 341 cyclic nucleotides, 386 phosphoinositide metabolism, 254, 258, 277, 27X

protooncogene expression, 573, 575, 576. 578

Ilydrogen, see d s u N a + / H + antiport lymphocytes and, 161, 165, 168 phagocytic leukocytes and, 228, 232. 233 Hyperpolarization, phagocytic Icukocytcs and, 233

1

IBMX, see Isobutylnicthylxanthine Immunoglobulins

INDEX arachidonatc nictabolitcs and, 360, 365 leukocytes and, 428, 440-442, 455. 456 lymphocytes and arachidonic acid. 34 I calcium changes, 154, 165-167, 169 cyclic nucleotides, 376, 388, 389. 391 GTP-binding proteins, 53, 5 5 , 57-59 intraccllular pH, 2 1 I , 2 16, 2 18-222 ion transport, 107, 109 protooncogcnc expresbion, 574, 577, 579 ncutrophils and, 409. 417 phagocytes and calcium, 183, 187, 194. 195, 197 cytokines, 539 ion transport, 133, 142, 144. 145, 147 phosphoinositide mctabolism. 3 19 phosphoinositide metabolism in lymphocytes and, 250 B cclls. 282-287 cellular immunity, 279 T cells, 274, 278 T cell receptor and. I , 2, 4, 5 . 7, 9, 12 Jnflammatjon arachidonate metabolites and, 350, 367 biochemical mechanisms, 353, 354, 357, 358 host-defense mechanisms, 350, 35 I macrophages, 365 platelet activating factor. 362 cheinoattrdctant regulation of phagocytes and. 19-21 CSa, 35 IL- I , 36 oligopeptides, 23. 26, 28, 29, 31 cytokincs in phagocytes and, 538-54 I , 561 macrophages, 550, 558, 559 neutrophils, 545-547 leukocytes and, 452 lyrnphocytcs and activation by lymphokines, 496 arachidonic acid, 334-336, 338, 345 phagocytes and calcium, 187, 191 GTP-binding proteins, 66, 87 phagocytic leukocytes and, 234 Inhibition arachidonate metabolites and biochemical mcchanisms, 354, 355, 357, 358 host-defense mechanisms, 35 1 macrophagcs. 366

613 transduction, 361 cytokines in phagocytes and, 561 macrophages. 551, 552, 554, 556-559, 561 neutrophils, 546 GTP-binding proteins in phagocytic cells and, 66 neutrophil activation, 76-78 neutrophil composition, 82, 84 rcceptor coupling, 70, 71 signal transduction, 87-89, 91 ion transport in lymphocyte activation and, 119, 120 cation channels, 1 14- I 18 membrane potential changes, 105, 107 Na/K-ATPase, 1 1 1. 112 pH, 108 lymphocytes and activation by lymphokines, 499, 508, 522. 523 arachidonic acid, 335-338, 342-345 calcium changes, 160, 162, 163, 166, 170 cyclic nucleotides, 379, 388-391 GTP-binding proteins, 48, 51-53, 55-58 intracellular pH, 208, 210, 211, 218, 219 protooncogcne expression, 578 transmembrane signaling. 511-515, 518520 neutrophils and, 403, 404, 407-415, 417, 418 phagocytes and calcium, 184, 188, 190 chemoattractant regulation, 31-33, 35, 36 gene expression, 588, 590, 593-597 ion transport, 138, 140, 141, 143 phosphoinositide metabolism. 31 1, 3 13, 314, 316-319, 324 phosphorylation, 470-474, 477, 486 phagocytic leukocytes and, 229-233, 235, 238, 239, 241 phosphoinositide metabolism in lymphocytes and, 250, 252, 287 B cells, 285, 286 calcium, 268 CAMP, 275-277 cellular immunity, 281 mitogens, 278, 279 protooncogenes, 270 T cells, 256, 260, 262, 264 protein phosphorylation in lcukocytes and, 429

614

INDEX

lymphoid cell.\, 434.440,442 myeloid cells. 448.450,451,453-455 lnoaitol phosphates arachidonate metabolites and, 363 lcukocytes and, 434 lymphocytes and, 251 activation by lymphokines, 507,508,

510,521 B cella, 283-286 CAMP, 276. 277 cellular immunity, 280,282 mitogens, 258,259 negative signals, 279 protein kinaac C, 273 T cells, 256,260,261,263-269 phagocytes and, 304,31 1-317. 319, 325 Jnositol 1,4,5-tnsphosphate arachidonatc metabolites and, 363,366 lcukocytes and, 426,441.442,444,454 lymphocytcs and. 25 I, 252,287 H cells, 283 cellular immunity. 282 T cells, 257,259, 263 268,275 phagocytcs and. 304,313-317 phosphorylation. 472,474,475 Insulin leukocytes and. 430,431,443.457 lyniphocytes and activation by lymphokincs, 507.508,516 intracellular pH, 209 phosphoinositide metabolism, 254,255,

257,273 Interferon arachidonate metabolites and, 365,366 leukocytes and, 452,453 lymphocytes and activatiou by lymphokines, 496,499,

500,504 arachidonic acid, 338,344 intracellular pH. 216,218,219,221 phosphoinositide metabolism, 279 transmembrane signaling, 510-513, 516,

518-520 phagocytes and, 542-544 gene expression, 588,590, 594,595,

597.598 macrophago. 549-556,559 ncutrophils, 546-548 phagocytic leukocytes and, 242 Interleukin- 1

leukocytes and, 453.457 lymphocytes and activation by lymphokines, 496-499,523 arachidonic acid, 338,341 calcium changes, 168 cyclic nucleotides. 376,385,389 intracellular pH, 216,218-221 ion transport, 109 receptors, 500-502 transmembrane signaling, 510-512,515, 516, 518-520 phagocytea and, 542--544 chernoattractant regulation, 20,21,3.5. 36 gene expression. 595,598 macrophages, 550-553. 555 -559 neutrophils, 545-548 phosphoinositidc mctabolism in lymphocytes and, 252 R cells, 285 T cclls. 255,256,260,277 Interleukin-2 leukocytes and, 430,456,457 lymphoid cells, 432,434,435.439,440 Inyeloid cells, 434.443 lymphocytcs and arachidonic acid. 335. 336,339.341,344 calcium changes, 154,1%. 161, 162,

164,165 cyclic nucleotides, 376,382.385, 386,

388-390 GTP-binding proteins, 5 I - 53, 57 intracellular pH, 21 1 , 21s.216 ion transporl, 106-108,116, 117,119 protooncogene expression. 574-578,582 lymphokines and. 497-499,521-523 initiation, 505,506 receptors, 500-504 transmembrane signaling, 510 516,51852 1 phagocytes and, 542-544 macrophages, 550-553,555-559 neutrophils, 546.548 phosphoinositide metabolism in Iymphocytcs and, 252 B cells, 286 CAMP, 27.5,277 cellular immunity, 279 mitogens. 258,259 protein kinase C . 272-274 T cells. 257.268,269,279

INDEX

615

Interleukin-3 Isozymes, leukocytes and. 426-428, 433, 435, leukocytes and, 442-445, 447, 448, 456 440, 456 lymphocytes and activation by lymphokincs, 498-501, 506 phosphoinositide metabolism, 252 L transmcmbranc signaling, 510. 512, 513, 517-519,521 Laminin, phagocytes and, 28, 29. 31 phagocytes and, 553 Lectins Interleukin-4 leukocytes and, 432, 433 leukocytes and, 440, 441. 445, 447 lymphocytes and lymphocytes and activation by lymphokines, 506, 510 activation by lymphokincs, 498, 499, calcium changes, 154, 161-163 501,510-513,515 cyclic nucleotides, 382, 391 phosphoinositidc mctabolism, 285 intracellular pH, 207, 211. 213 protooncogene expression, 579 ion transport, 109 phagocytes and. 544, 553, 555, 558 phosphoinositide metabolism, 255, 258, Interleukin-5. 5 10 273 Interleukin-6 protooncogene expression, 573. 577. 578 lymphocytes and, 497-501, 504, 510 phagocytes and, 477 phagocytes and. 542, 544 Lcukocytcs lntracellular pH in lymphocyte activation, 207, arachidonate metabolites and, 350. 366, 367 208, 222, 223 biochemical mechanisms, 356 B cell differentiation, 21 6-222 host-defense mechanisms, 350, 35 1 nonlymphoid cell changes, 208-210 platelet activating factor, 362, 363 T lymphocytes transduction, 359 antibodies, 214, 215 arachidonic acid and, 339 calcium. 214 cytokines in phagocytes and, see Cytokines growth factors, 216 in phagocytes mitogcn stimulation, 210-212 neutrophils and, 400, 404, 416 oncogene expression, 2 12-2 14 phagocytes and Ion transport chemoattractant regulation, 20, 2 I , 3 1 lymphocyte activation and, see Lymphocyte 32, 34, 35 activation. ion transport in GTP-binding proteins, 65, 66, 78, 92 phagocytic activation and, see Phagocytic phagocytic. see Phagocytic activation, ion activation, ion transport in transport in lonomycin phagocytic. cytoplasmic pH in, see lymphocytes and Cytoplasmic pH in phagocytic activation by lymphokines, $22 leukocytes calcium changes, 161, 164, 167, 168 protein phosphorylation in, see Protein phosintracellular pH, 213, 214 phorylation in leukocytes ion transport, 107, 109 Leukotriene B, phagocytes and araehidonate metabolites and, 35 1 , 357, ion transport, 132, 136, 137 359 phosphoinositide metabolism, 3 16 lymphocytes and, 338-341. 343-345 phosphorylation, 475, 477 neutrophils and, 406, 41 I lsobutylmethylxanthine, neutrophils and, 405, phagocytes and 409, 410. 412, 415 calcium, 189 Isoenzymes, phagocytes and. 473, 474, 476 chemoattractant regulation, 20, 31-34 lsoproterenol, neutrophils and, 400, 402, 404 cytokines, 547 CAMP, 407-4 12 GTP-binding proteins, 66, 74

616

INDEX

phosphoinohitidc metaholisin. 3 17, 3 18 phosphorylation, 476 phagocytic lcukocytes and, 230, 234 Lcukotriencs ar:ichidonate rnctabolitcs and, 350, 35 I, 353, 356 358 lymphocytes and activation by lymphokincs, 5 12 arachidonic acid, 334. 339 phagocytes and, 541, 546, 5 5 5 , 556 L i ga nd s calcium changes during lymphocyte activation and. 154, 155, 170 R lymphocytcs, 16.5-170 T lyillphocytes, 156, 158-161, 163-165 GTP-binding proteins in phagocytic cells and, 66 neutrophil activation. 7 5 , 76, 80 receptor coupling, 72 hignal tiansduction, 90 leukocytes and, 433, 443. 444, 449, 456 lymphocytcs and cyclic nucleolidcs. 378, 383, 385, 389, 30 I

GTP-binding proteiiis, 46, 48, 49, 52, 53, 55. 57 intrdccllular pH. 207. 212, 213, 223 ion transport, 113, 121 protooncogcnc expression. 582 Iymphokine\ and, 497, 500, 522 cell activation. 506, 508 initiation, 505. 506 lran5nienibrane signaling, 518, 519 neutrophils and, 400 phagocytes and calcium. 191 chenioattractant regulation. 22, 24. 25. 31.3s

cytokines, 539 ion transport, 133, 142, 144, 145, 147 phosphoinositide metabolism, 3 I 7 phosphorylation, 471, 475 phagocytic leukocytes and. 237 phosphoinositide metabolisin in lymphocytes and, 252, 287 R cells, 284 cellular iminunity. 279 T cella, 254, 263. 265. 266, 270, 271 Lipids ardchidoiiatc metabolites and, 361, 363

leukocytes and, 426 lymphocytes and activation by lyinphukines, 5 I I , 5 I 2 calcium changes, 159 ion transport, 104 nculrophils and, 416 phagocytcs and chernoattractant regulation, 20, 21 ion transport, 144 phosphoinositide metabolism, 313, 323 phosphorylation, 475 abolites and, 355, 357, 359, 361, 362. 365 leukocytes and. 440, 441, 452, 453, 455 lymphocytes and cyclic nucleotides, 376, 389 cytokines, 541, 543, 546, 549, 554-559 intracellular pH, 216, 219-221 ion trailspoil, 100 phosphoinositide metabolism, 25 1 . 284286

phagocytes and calcium changcs. 166, 168 chemoattractanr regulation, 33 gcne expression, 588, 590, 593-595, 597 CTP-binding protcins, 90 ion transport. 138, 142 phagocytic leukocytes and. 243 5-Lipoxygenasc, 352, 356 360, 365 Lipoxygenase, lymphocytes and, 35 I activation by lymphokines, 511, 512 arachidonic acid, 333, 345 activation, 338, 339 inhibilors, 342, 344, 345 synthesis, 340 343 cyclic nuclcotides, 388 Lithium, phagocytic Ieukocytcs and, 229, ~

240

I P S , see Lipopolysaccharide Lymphocyte activation calcium changcs during, see Calcium changes during lymphocyte activation intraccllular pH in, .see lntracellular pH in lymphocyte activation Lymphocyte activation, ion transport in, 104, 105, 118-121

cation channels, I 13 118 manion channels, 112, I I 3 membrane potential changes. 105-107 ~

INDEX NaiK-ATPase, 110-1 12 pH changes, 108-1 10 Lymphocyte activation by lymphokines, 496. 497, 523 cell activation, 506-508 gene structure. 498-500 initiation, 505, 506 pleiomorphism. 497. 498 protein structure. 498 receptors, 500, 501 antigen, 521-523 IL- I , 501, 502 IL-2, 502-504 IL-6, 504 interferon, 504, 505 tranhmcmbrane signaling antigens, 508-510 cyclic nuclcotidcs, 5 19 G proteins, 518, 519 N a + / H + antiport, 518 oncogene expression. 520, 521 phospholipids. 510-512 protein kinases, 5 12-518 receptor internalization, 520 Lymphocyte derived chemotactic factor. 21 Lymphocyte-specific tyrosine kinasc, 434, 435, 457 Lymphocytes arachidonate inetabolitcs and, 350. 353 arachidonic acid in, set’ Arachidonic acid in lymphocytes chemoattractant regulation, 2 I cyclic nucleotides in. see Cyclic nuclcotidcs in lymphocytes cytokines and, 543. 544. 549 gene expression and, 589. 595, 598 GTP-binding proteins and, s e p GTP-binding proteins in lymphocyte activation ion transport and, 137, 138, 147 neutrophils and, 404, 416 phosphoinositide metabolism in, SPP Phosphoinositide metabolism protein phosphorylation and. 428, 430, 457 B cells, 440, 441 myeloid cells, 455 T cells. 432, 435, 439 protooncogene expression in, see Protooncogenc expression in lymphocytes Lymphoid cells, protein phosphorylation and, 445

617 H cclls, 440-442 T cells. 432-440 Lymphokincs cyclic nucleotides and, 376, 384, 385, 389, 39 I cytokines and, 542, 548. 550. 553, 554, 55x GTP-binding proteins and, 45, 50 lymphocyte activation by, see Lymphocyte activation by lymphokines phosphoinositide metabolism and, 259, 282 protein phosphorylation and, 453 protooncogene expression and, 572, 574, 580, 582 Lysolipids, phagocytes and, 32 1-324 Lysophosphotide acyltransferasc, phagocytes and, 310 Lysosomes arachidonate metabolites and, 353 neutrophils and, 4 I5 phagocytes and chemoattractant regulation, 2 I , 3 I , 32 cytokines, 541, 547 phosphoinositide metabolism, 3 19, 323 phosphorylation, 473 phagocytic leukocytes and, 237

M Macr(~ptiage-activatingfactor, 259 Macrophages arachidonate metabolites and, 367 biochemical mechanisms, 353-358 development, 364-366 host-defense mechanisms, 351, 353 platelet activating factor, 362-364 transduction, 358-362 cytoplasmic pH in phagocytic leukocytes and activation, 234, 236, 237, 239 mechanisms, 228, 230, 232, 233 leukocytes and, 442, 445 lymphocytes and, 496. 498-500, 512, 518 neutrophils and, 418 phagocytes and, 27. 28 calcium, 182-185, 191, 195-197 chemoattrdctant regulation, 20, 23. 25 cytokines, 538-561 gene expression, 588, 589, 593-598 GTP-binding proteins, 65, 79, 90

618

INDEX

ion transport, 127- 143, 145-147 phosphoinositide metabolism, 310, 314, 321-323 phagocytic leukocytes and, 242, 243 phosphoinositidc metabolism in lymphocytes and B cclls, 282. 285 T cells, 255, 2.57. 260 262 Mapncaiu in leukocytes and. 435 lymphocytes and cyclic nuclcotides, 378 yhohphoinositidc mctaibolism, 260, 261 neutrophila and, 407, 418 phagocytes and cheiiiuattractant regulation. 27 phosphoinositidc metaboliam, 306. 31 I , 31 1

Ma.jor histocunipatihility complex arachidonare metabolites and. 366 lymphocytes and activation by lyrnphokines, 447 calciuni. 158. 159 GTP-binding proteins, 50 protooncogene expression. 582 phagocytes and, 593. 595, 597 phosphoinositide metabolism in lymphocytes and. 250 B cells. 282, 286 T cells, 253, 255, 274 T cell receptor and, 12 Manganese lymphocytes and, 157. 166, 378 phagocytes and, 188, 189 Mast cclls, phosphoinositide inetaholism and, 2X I

Methylisohutylamiloride, 215. 217-2 I9 Microhicidal activity phagocytes and, 547-549, 553, 560 phagocytic lcukocytea and, 236, 237 Microfilaments, lyriiphocytes and, 264, 482 Microsomes lymphocytes and, 268 phagocytic leukocytes and. 233 Microtuhulea Icukocytcs and, 429 neutrophilb ;inn. 405 phagocytes and, 74 phagocytic leukocytes and, 237 Migration, phagocytic leukocytes and, 234236. 23X

Mitochondria leukocytes and, 448 Iyinphocytes and, 105, 107 phagocytes and calcium, 185, 186 chcnioattractant regulation, 20 phagocytic leukocytes and, 233 Mitogcnesis. lymphocyte activation and, 104, 119, 121 cation channels, 116-1 I X manion channels. 113 membrane potential changes, 106, 107 NaiK-ATPasc. I I I , I I? pH. 108-110 Mitogens arachidonic acid and, 335, 339, 341 cyclic nucleotides in lymphocytes and, 376, 389, 391 activation. 380, 381. 383, 384 modulatory effects, 386, 388 leukocytes and, 428, 432, 433, 435 lymphocytes and activation by lymphokines, 506, 510, 5111 calcium changes, 161, 163, 168, 170 intracellular pH, 210-214, 216, 223 protooncogene expression, 573, 575-579 phagocytes and calcium. 188, 189 cytokines, 543 phosphorylation, 483 phagocytic leukocytes and, 243 phosphoinositidc metabolism in lymphocytes and B cells, 282, 284 calcium, 266-269 cellular immunity, 2X I negative signals, 278, 279 T cell proliferation, 258 -261, 263, 264, 27 1-273 T cells, 254, 255. 257 Mitosis, lymphocytes and, 252 Moncnsin, lymphocytes and, 219-22 I Monoclonal antibodies lcukocytcs and, 433, 434 lymphocytes and cyclic nucleotidea, 376, 382, 383, 391 prolooncogene expression, 576, 579 phagocytea and calcium, 195 cytokines, 555 GTP-binding proteins, 85

INDEX phosphoinositide metabolism in lymphocytes and, 252 B cells, 284 calcium, 266. 268, 269 cellular immunity, 279, 280 mitogens, 258, 259 T cells, 255-257, 261, 274, 278 Monocytes arachidonate metabolitcs and. 356 arachidonic acid and, 338, 340 phagocytes and cytokines, 539-541, 543. 550-561 gene expression, 595 phosphorylation, 477 phagocytic leukocytes and, 228, 240, 242, 243 mRNA arachidonatc metabolites and, 363, 366 leukocytes and, 429, 435, 455 lymphocytes and activation by lymphokines, 499, 520 calcium changes. 154 cyclic nucleotides. 389 intracellular pH. 208. 212-214, 216-219, 221, 222 phosphoinositidemetabolism, 258,277,279 protooncogene expression. 572-578, 580, 58 1 phagocytes and calcium, 182 cytokines, 551 gene expression, 590. 594-596 Mutation leukocytes and, 430 lymphocytcs and activation by lyniphokinea, 515, 521 GTP-binding proteins, 48 intracellular pH, 210 ion transport, I 17 phosphoinositide metabolism, 254, 258, 259, 271 protooncogene expression, 580, 582 phagocytes and, 73 T cell receptor and. 2 Mycloid cells phagocytic leukocytes and, 228 protein phosphorylation and, 432, 457 colony stimulating factors, 442-449 differentiation, 449-452 functional activation, 452-455 Myosin, phagocytes and, 480

619 N Na+/H+ antiport arachidonate metabolites and, 354, 366 platelet activating factor, 363, 364 transduction, 359-361 lymphocytes and activation by lyrnphokines, 503. 515, 518 intracellular pH, 208-223 phagocytes and, 590, 593, 594, 596, 597 phagocytic leukocytes and activation, 235, 236 differentiation, 240-243 mechanisms, 228-231 Na/K-ATPase, lymphocytes and, 108- 112, 119 NADPH. phagocytes and, 307, 308 NADPH oxidase neutrophils and, 41 I phagocytes and calcium, 190, 194 GTP-binding proteins, 78, 79, 85 ion transport, 144 phosphorylation. 470, 471. 474-478. 483-485 phagocytic leukocytes and, 237, 243 Natural killcr cells arachidonic acid and, 338, 344 lymphocytes and activation by lymphokines, 500, 503, 505 cyclic nucleotides, 388 ion transport, 116 phosphoinositide metabolism, 279-28 1 Neomycin, lymphocytes and, 286 Ncutrophils arachidonate metabolites and, 35 I arachidonic acid and, 334, 338, 340 calcium and, 182-185, 188-191, 194-197 chemoattractant regulation and, 20. 21, 2428, 31-35 cyclic nucleotides in, see Cyclic nucleotides in neutrophils cytokines and, 538, 539, 543-549, 561 cytoplasniic pH and, 228, 230, 231, 234, 236, 238, 239 activation. 234, 236, 238. 239 mechanisms, 228, 230, 231 gene expression and, 589 GTP-binding proteins and, 66, 91 activation, 74-81 composition, 8 1-87

INDEX

low molecular weight. 74 receptor coupling, 69 signal transduction, 87-91 ion transport and, 127. 128, 132. 139, 140 conductances, 143, 144 inenihrane potential, 141 phosphoinositide metabolism and, 304, 324 activation. 310-324 phosphatidic acid, 305-310 phosphorylation and, 470-480, 482-486 protcin phosphorylation and, 452-455 Nitrogen ncutrophils arid. 403 phagocytes and, 553, 558 Norepinephrine, neutrnphil? and. 400 Nucleotides cyclic in lyrnphocytcs, w r Cyclic nuclcotides in lymphocytes in ncutrophils. ~ e Cyclic r nucleotidcs in neutrophils GTP-binding proteins i n phagocytic cells and, 67. 91 low molecular weight, 73 neutrophil activation. 7.5, 77-8 I neutrnphil composition. 82 rcccptor coupling, 72 signal transduction, 91 lymphocytes and calcium changes, I67 GTP-binding proteins. 52 phagocytes and calcium, I83 chcnioaltrac~antregulation. 2.5. 32 cytokines, S42 T cell receptor and. 3-5, 9, 11

0

Olcoylacctylglyccrol, phagocytcs and, 33. 90 Oligomycin. phagocytic Icukocytcs and. 233 Oligopeptides, phagocytes and, 20, 23-31, 35 Oncogenes cxprcscion in lymphocytes. .see Protooncogcnc expression i n lyriiphocytcs leukocytes and, 430. 448, 449, 457 lymphocytes and activation by lymphokinca. 503, 507 517, 518, 520, 521

GTP-binding proteins, 49 intracellular pH. 2 13 Ornithine dccarboxylasc. lyniphocyies and, S2, 26 1 Osniotic shock. lymphocytes and, 109 Osteoclasts, phagocytic leukocytes and, 233. 239 Ouabain, lymphocyte activation and, I 1 I Oxygen leukocytes and, 454 neutrophils and, 41 I , 412 phagocytes and niacrophages, 549, 553, 555, 559, 560 neutrophils. 547, S48 phosphorylation, 475, 477, 478, 485, 486 phagocytic leukocytes and, 236-238

P PBL, see Peripheral blood lymphocytes Perforina, lymphocytes and, 281 Peripheral blood lymphocytes. 2 I I , 2 12, 2 14. 216 Pertussis toxin arachidonate metabolites and, 360 GTP-binding proteins in phagocytic cells and. 91 neutrophil activation, 75-80 neutrophil composition, X I -83 signal transduction, 91 leukocytes and. 455 lylllphocytcs and activation by lymphokines, 508, 5 19 calcium changes, 167 GTP-binding proteins, 49 pho~plioinositidc iiictabolisni. 262. 268. 285, 286 neutrophils and. 403 phagocytes and calcium, 188, 196 chciiioattractant regulation, 25 phosphoinositide metabolism, 308, 3 17 320 phagocytic leukocytes and, 230, 235

PH arachidonate mctabulitcs and, 353, 3.54, 362, 367 cytoplasniic, in phagocytic leukocytes, srr Cytoplasmic pH in phagocytic leukocytes

INDEX lymphocyte activation and, see lntracellular pH in lymphocyte activation lymphocytes and activation by lymphokines, 506, 5 I8 calcium changcs, 161, 168 phosphoinositide metabolism. 26 I phagocytes and ion transport, 129. 140. 142, 143 phosphoinositidc rnctabolism, 3 14 phosphorylation, 483 PHA, see Phytohemagglutinin Phagoc ytes arachidonate metabolites in, see Arachidonate metabolites in phagocytes arachidonic acid and, 334 cyclic nuclcotides and. 406 cytokincs in, see Cytokines in phagocytes cytoplasmic calcitirn and. see Cytoplasmic calcium in phagocyte activation gene expression in, see Genc expression i n phagocytcs GTP-binding proteins in, s r e G proteins. phagocytes and phosphoinositide metabolism in, see Phosphoinositidc metabolism in phdgocytes phosphorylation in, seo Phobphorylation in phagocytes physiology, 19-2 I protein phosphorylation and. 442. 452 receptor, 21, 22 CSa, 34, 35 IL- I . 35, 36 LTB,, 31-34 oligopeptides, 23-3 1 Phagocytic activation, ion transport in. 127. 128, 147 ionic conductances, 142- 147 macrophages, 133- 140 membrane potential, 140, 141 ncutrophils, 139, 140 resting membrane potcntial, 128- I32 Phagocytic Icukocytcs, cytoplasmic pH in, SPC Cytoplasmic pH in phagocytic leukocytes Phenotype arachidonic acid and, 342 leukocytes and, 449 lymphocytes and ion transport, I17 phosphoinositide metabolism, 252, 255, 282 protooncogenr expression, 572

621 neutrophils and, 416 phagocytes and, 185 phagocytic leukocytes and, 241, 242 Phorbol esters arachidonate metabolites and, 356, 362, 365 arachidonic acid and, 33.5, 337, 341 leukocytes and, 427, 430, 432-435, 440 lymphocytes and activation by lymphokines, 506, 513515, 518 cyclic nucleotides, 390 intraccllular pH. 209, 211, 213, 215, 219, 220 phosphoinositide metabolism, 272, 280 protooncogene cxpression, 577 phagocytes and cytokines, 554 phosphoinositide metaholism, 306, 307, 318-320, 323, 324 phosphorylation, 470-473, 483-485 phagocytic leukocytes and, 230 Phorbol niyristate acetate arachidonic acid and, 344, 345 lymphocytes and. 575, 576, 579, 581 ncutrophils and, 406, 41 1-413, 418 phagocytes and calcium, 190, 197 cytokines, 541, 543, 556 GTP-binding proteins, 88, 89 ion transport, 138, 141 phosphoinositide metabolism, 3 18, 319, 323 phosphorylation. 470-473, 47.5-478. 480,483,485 phosphoinositide nietabolisrn in lymphocytes and. 252 B cells, 284, 285 cellular immunity, 281 protein kinase C, 271-274 T cells. 255, 257-260, 277, 278 protcin phosphorylation and lymphoid cells, 440-442 myeloid cells, 444, 447, 449-454 Phosphatidic acid, phagocytes and, 303-310. 324 neutrophils. 314, 321, 417 phosphorylation, 475. 478 Phosphatidylcholinc leukocytes and, 426, 444 lymphocytes and, 5 I1 Phosphatidylinositol

622 arachidonate metabolites and, 365 leukocytes and. 426. 433. 434. 457 lymphocytes and, 250-253, 287 activation by lymphokines, 507, 510. 51 1 B cclls, 283, 284. 286 CAMP. 275-277 cellular immunity, 28 I mitogcns. 259 protooncogcnc cxpreksion. 579 T cells. 255, 260. 262, 264, 270-272 phagocytes and, 303, 304 gene expression, 597 neutrophils. 31 I , 312. 314, 319-323 phosphatidic acid. 309, 310 Phosphatidylinositol 4,5-bisphosphate lymphocytes and. 25 1-253, 287 n cells, 2x3 286 CAMP, 276. 277 ccllular immunily, 279, 281, 282 cyclic nuclcotides, 390 negative signals. 279 T cells, 257-265, 269-274 phagocytes and, 303, 304 neutrophils, 311, 313-315, 317-319 phosphatidic acid, 308 Phosphatidylinositol biaphosphate, ncutrophils and. 414, 415 Pho~phatidylinositol kinases. phagocytes and, 311 -313, 319 Phosphatidylinositol 4-phosphate lyinphocyics and, 251, 258-260, 270-272. 776 phagocytes and, 303, 312. 313 Phosphatidylinositol trisphosphate. lyniphocytes and. 251 Phosphodiesterases lyniphocytes and, 378, 379. 384, 386 ncutrophils and. 404, 405. 407, 409-41 1, 414 Phosphoinositidc metabolism in lymphocytes, 24'1-253. 287 B cells, 2x2-287 cellular immunity, 279 cytotoxicity, 281, 2x2 NK cells. 279-281 T ccIIs, 253 257 calcium. 2hh- 269 CAMP. 27.5-278 mitogens. 257-259 negative signals, 278, 279

INDEX proliferation, 260-265 protein kinase C, 27 1-275 protooncogcnes, 269-27 I Phosphoinositidc metabolism in phagocytes, 303-305. 324, 325 ncutrophils phosphatidylinoaitol kinases, 3 I 1-3 13 phospholipase A?, 3 19-324 phospholipase C, 3 13-3 19 polyphosphoinositidc phosphatases, 3 13 phosphatidic acid, 305, 306 diglyceridc kinase, 306-308 lysophosphotide acyltransferasc. 3 10 phospholipase D, 308-3 10 Phosphoinositidcs leukocytes and, 441, 444 neutrophils and, 413 phagocytes and, 474 phagocytic leukocytes and, 230 Phospholipase Az arachidonate metabolites and, 35 I biochemical mechanisms, 353, 354, 3.58 platelet activating factor. 362. 364 transduction, 359. 360 arachidonic acid and, 341 lymphocytes and, 5 I I phagocytcs and, 319-325 Phospholipasc C arachidonate metabolites and, 354, 360 CTP-binding proteins in phagocytic cells and, 73, 91 neutrophil activation, 76 79 neutrophil compostition, X3 signal transduction, 89 leukocytes and, 444, 455 lyinphocytes and activation by lymphokines, 507, 508. 510, 521-523 calcium changes. 160, 167 cyclic nucleotides, 390, 3Y I GTP-binding proteins. 50, 51. 55, 57, 58 ncutrophils and, 403 phagocytcs arid calcium, 190, I96 chemoattractant regulation. 26. 34 phagocytic leukocytes and, 230 phosphoinositide metabolism in lymphocytes and, 287 5 cells. 286 T cells, 255. 259, 261, 262, 271, 276

INDEX phosphoinositide metabolism in phagocytes and, 304. 324. 325 neutrophils, 311, 313-319 phosphatidic acid, 307, 308 Phospholipase D, phagocytes and, 305, 307310, 316. 325 Phospholipids arachidonate metabolites and, 351, 354. 355, 362. 364, 365 leukocytes and, 426, 427, 440, 45 I , 454 lymphocytes and activation by lymphokines, 507. 510 512.521 arachidonic acid, 333 GTP-binding proteins, 5 I , 52, 55-58 phosphoinositide metabolism, 25 I , 260, 272 neutrophils and, 412-416 phagocytes and calcium, 190, 196 chemoattrdctant regulation, 27 GTP-binding proteins. 70. 72, 75, YO phosphoinositide metabolism, 305, 3 10313, 316. 318-323 phosphorylation, 471, 472, 476. 483 Phosphorylation lymphocytes and activation by lymphokines. 501, 504, 507, 508, 522, 523 cyclic nucleotidcs, 378, 380. 389 GTP-binding proteins, 46, 48 intracellular pH, 208 ion transport. 110, I13 protooncogenc expression, 58 1 transmembrane signaling, 5 12. 5 13, 5 IS519 neutrophils and, 400, 416. 419 phagocytes and, 469-47 I , 485, 486 calcium, 185 chemoattractant regulation. 33, 34 diacylglyccrol. 474-476 gene expression. 590, 593 GTP-binding proteins, 80, 89 NADPH-oxidase, 483-485 phorbol esters. 471 phosphoinositide metabolism, 304, 306, 31 1-313 protein kinasc C inhibition, 471-474 protein kinase C mobilization, 476. 477 studies, 477-483

623 phagocytic leukocytes and, 230 phosphoinositide metabolism in lymphocytes and, 251, 287 B cells, 285 T cell proliferation, 259, 260, 270-278 T cells, 256, 257 protein, in leukocytes, see Protein phosphorylation in leukocytes Phytohemagglutinin, lymphocytes and activation by lymphokines, 516, 521, 522 arachidonic acid and, 335, 336, 340, 341, 344 calcium changes, 155, 157-161, 165 cyclic nucleotides, 376, 382, 384, 385 intracellular pH, 21 1-215 ion transport, 106, 109, 1 1 1 , 116 phosphoinositide metabolism, 255-259, 266, 268, 273 protooncogene expression, 573, 575, 578 Plasma cells, lymphocytes and, 250, 282 Plasma membrane arachidonate metabolites and, 353 GTP-binding proteins in phagocytic cells and, 92 low molecular weight, 73 neutrophil activation, 78 neutrophil composition, 83 rcccptor coupling, 71 signal transduction, 89, 90 lymphocytes and calcium changes, 156, 159, 167. 170 cyclic nucleotides, 378-380, 383 GTP-binding proteins, 45-47, 52-55, 58 intracellular pH, 209, 210 ion transport, 104, 105, 110 neutrophils and, 407 phagocytes and calcium, 182, 184, 185, 189, 190 chemoattractant regulation, 24-26, 32, 34 ion transport, 136 phosphorylation, 470, 471, 474, 476. 478-480 phagocytic leukocytes and, 229, 231. 233, 238, 239 phosphoinositide metabolism in lymphocytes and, 251, 252, 287 B cells, 283, 285, 286 cellular immunity, 280 T cells, 256, 261, 266-268, 273-277

INDEX phosphoinositide metabolism in phagocytes and ncutrophils, 310, 311. 313, 317-320. 322 phoaphatidic acid, 305, 307. 308, 110 Platelet activating factor arachidonate metabolites and, 359, 362-365 phagocytcs and cytokinea, 545 gcnc cxprcssion, S X X . 593, 597 CTP-binding proteins, 66, 74 ion transport, I36 phosphoinositide metabolism, 32 I phagocytic leukocytes and. 235 Platclct-dci-ivcd growth factor leukocytes and, 43 I , 443. 444 lymphocytes and activation by lymphokines. 499, 500.

soh. s 1 x phoaphoinositide metabolism, 270 phagocytes and gene expression, 5x7, 589, 590, 596. 597 phosphoinositide metabolism. 304, 3 12 Plittclets. arachidonate nietabolites and, 354, 356 I’lcioniorphism. lymphocytes and, 497, 498 PMA, S E E Phorhol myristate acetate Pokeweed initogen. lyrnphocytez and. 21 1 Pol y merimtion leukocytes and, 1234-236 phagocytes and, 317, 486 Polymorphonuclear Icukocytcs, 2 I Lm,,32-3s oligopeptides, 23. 24. 26-29. 31 Polypeptldcs leukocytes and, 447, 457 lymphocytcs and activation by lymphokines, 496, 507, 518 GTP-binding proteins. 50 intracellular pH, 2 16 phosphoinositide metabolism. 253 phagocytcs and chemoattractant regulation, 24 cytokines. S3X, 541, 5.51 tiTP-binding proteins. 66 phosphoinosittde nietaholism. 313 T cell receptor and. 9 I’olyphosphatidyliiiosltldc phoq)hatascs, phapocytes and, 313 Potassium, see also NaiK ATPase arachidonatc nietabolites and, 359, 364

lymphocytes and calcium changes, 162, 163, 168, 169 cyclic nucleotides, 389 ion transport, 106, 107, 110-120 phosphoinositide metabolism, 268 phagocytes and, 130-132, 134-144, 147 Prostaglandins arachidonate metabolites and, 364, 366 biochemical mcchanisms. 355-358 host-defense mechanisms, 350-353 leukocytes and, 452 lymphocytes and activation by lymphokines, 5 I 1 , 5 12 arachidonic acid, 333-340, 343-34s cyclic nucleotides, 378, 379, 382, 383, 386-388, 390 phosphoinositide metabolism, 262, 275277. 281 neutrophils and, 403, 404, 406-412, 417 phagocytes and cytokinea. 541, 546, 554-556 phosphoinositide metabolism, 3 I8 Protein, see ulso bpecific protein arachidonate metabolites and, 354, 355, 360-362, 364, 367 cytokines in phagocytes and, 538, 541-544 m a c r o p h a p , 554, 5.56, 559 neutrophils, 545, 546 lymphocyte activation hy lymphokincs and cell activation, 507 gcnc structure. 499 rcccptors, 502, SO3 structure. 498 transniernhrenc signaling, S l O . 512. 51.5, 5 I6 lyniphocytcs and calcium changes, 158, 159 cyclic nucleotides, 376, 380, 384, 389 intraccllular pH, 208, 212, 218 ion transport. 104. 117 protooncogene expression, 573, 575580 neutrophils and, 400, 405, 406, 408, 409 phagocytes and calcium. 181. 184, 186, 187, 195 chenioattractant regulation. 20, 25, 26, 31-33 gene expression, 587-590, 593, 595, 597, 598

ion transport, 133, 138

INDEX

625

phosphoinositide metabolism. 304. 31 I, phagocytic leukocytes and, 230, 241 312. 314, 319 phosphoinositidc metabolism in lymphocytes phosphorylation, 469, 474, 476, 479, and, 287 480, 482-484 B cells, 285 phagocytic leukocytes and, 230 cellular immunity, 281 phosphoinositide metabolism in lymphocytes mitogens, 258, 259 and. 287 T cells, 257, 261, 263, 270-275 B cells, 285. 286 phosphoinositide metabolism in phagocytcs cellular immunity, 28 I and, 304 protcin kinase C, 27 1-275 neutrophils, 313, 318, 319, 324 T cell proliferation. 260. 262, 263. 270, phosphatidic acid, 306, 307, 310 277 protein phosphorylation and, 426-428, 456, T cells. 254-257 457 T cell receptor and, 2, 6, 12 lymphoid cclls, 433-435, 440-442 Protein kinasc myeloid cells, 442-444, 447, 45 1-455 lymphocytes and Protein kinase G, lymphocytes and, 378, 384 activation by lymphokines, 507, 512 Protein kinases, protein phosphorylation in leucyclic nucleotides, 383, 391 kocytcs and ncutrophils and, 415. 416 lymphoid cells, 435, 439 phagocytes and mycloid cells, 442, 445 chcmoattractant repul;ition, 21, 33, 34 Protein phosphorylation in leukocytes, 425, cytokines, 554 426. 4.55-457 gene expression, 590 lymphoid cells GTP-binding proteins. 88-90 B cells, 440-442 protein phocphorylation in leukocyte5 and, T cells, 432-440 426, 428, 456 myeloid cells Protein kinasc A colony stimulating factors, 442-449 leukocytes and, 45 I, 452 differentiation, 449-452 lymphocytes and. 378, 384 functional activation. 452-455 Protein kinase C protein kinaaes arachidonate metabolites and. 350, 367 calcium, 428, 429 macrophages, 365 calmodulin, 428, 429 platelet activating factor. 364 cyclic nuclotides, 429 transduction, 359-361 protein kinase C, 426-428 arachidonic acid and, 335, 337, 345 S6 kinase. 429, 430 lymphocytes and tyrosine kinasc, 430-432 activation by lymphokincs, 501, 507. Proteol ysis 508, 521-523 leukocytes and, 443, 445, 451, 453 calcium changes. 154, 161. 164, 166. neutrophils and, 415 I67 phagocytes and, 66, 82, 85, 476 cyclic nucleotides, 390 Protooncogenes gene expression, 590. 593, 594 expression in lymphocytes, 571 573 intraccllular pH, 208, 21 I, 219. 223 alteration, 573-578 ion transport, 110, 112, 119 B lymphocytes, 578, 579 protooncogene cxprcssion, 573 futurc directions, 581, 582 transmenibranc signaling, 5 1 1, 5 13-5 15 Ick gene, 579-581 neutrnphils and, 407, 412, 413, 418 expression in phagocytes and, 587, 590, phagocyte5 and 593, 595 calcium, 185, 189, 190, 196 phosphoinositide metabolism in lymphocytes phosphorylation, 470-478. 480-486 and, 269-271, 279, 286, 287 ~

626

INDEX

protein phosphorylation in leukocytes and, 434, 443, 451 fyr. see Bordetella pertussis toxin Ptdlns, J W Phosphatidylinositnl PtdTnsP?, see Phosphatidylinositol 4,shis phosphate Purified protein derivative, ‘I cell receptor and, 12

R Kcspiretory hurst lcukocytcs and. 236-23X. 243, 454 phagocytes and cytokines, 539 phosphorylation, 470-475, 477, 483 Kcstriction fragment length polymorphism, T cell receptor and. 4. 5 . 7, 9 Retinoic acid leukocytes and. 45 I , 452 phagocytes and, 313 phagNytiC leukocytcs and. 240-242 Rhodopsin lymphocytes and. 48 phagocytes and, 70, 80, 89 Ri hosonics leukocytcs and, 429, 435 lymphocytes and. 5 I 2 RNA leukocytes and, 451 lymphocytes and cyclic nucleotidcs, 376. 384, 386. 391 phosphoinositide metabolism, 284, 285 protooncogene expression, 572 phagocytcs and cytokines, 545, 551 phosphorylation. 476 T cell receptor and, 2 RNA polymerase, lymphocytes and, 384

S

S6 kinase, leukocytes and, 429, 430 Second messengers arachidonate metabolites and, 367 leukocytes and. 429, 433, 442, 445, 456, 457 lymphocytes and activation by lymphokines, 506, 508. 521

arachidonic acid, 333, 338 cyclic nucleotides, 377, 384, 385, 390 intracellular pH, 208 pliosphoinositide metabolism, 252, 293, 263, 286 protooncogene expression, 582 neutrophiis and, 406, 417 phagocytes and calcium, 187. 1x9, 193-197 cytokines, 554 gene expression, 588, 593, 597, 598 phagocytic leukocytes and, 235 Serine kinases, 456 lymphoid cells, 439-441 inyeloid cells, 443, 444, 447, 450-454 Signal transduction lymphncytes and cyclic nuclcotides, 384, 389-391 GTP-binding proteins, 46, 49, 52 phosphoinositide metabolism, 252, 269, 276 lymphokincs and. 499 cell activation, 506- 508 receptors, Sol-504 transmembrane signaling, 510, 512, SIX5 20 neutrophils and, 400, 413, 415 phagocytes and gene expression, 590-595 GTP-binding proteins, 71, 80, 87-92 ion transport, 133. 140. 142-147 phosphoinositide metabolism in phagocytcs and, 304, 305. 324 neutrophils, 319, 320, 322 phosphatidic acid, 306-308 protein phosphorylation in leukocytes and, 429. 430. 457 lynlphuid cells, 432. 434, 43Y, 441, 442 myeloid cells, 442,444,448,450,453,455 Sodium, see also Na+/H+ antiport; NaiK ATPasc ion transport in lymphocyte activation and. 110-1 13, 1 I7 cation channels, 118 membrane potential changes, 106, 107 lymphocytes and, 161, 163, 165, 168. 169 pliagocyres and calcium, 185 ion transport, 131, 132, 139-141, 143 phagocytic leukocytes and, 232, 233

INDEX

627

Sodium nitroprussidc, lymphocytes and, 378, 379, 382, 383, 386-388 Spleen, lymphocytes and, 260, 277 Substance P, phagocytcs and, 3 I7 Superoxide arachidonate mctabolites and, 358 leukocytes and, 452, 453, 455 neutrophils and, 41 I . 413, 415 phagocyter and cytokines, 539, 559 phosphoinosi tide metabolism, 306- 308, 313, 317, 324 phosphorylation, 470 Suppressor cells arachidonic acid and, 336 lymphocytes and cyclic nucleotides. 388 phosphoinositide metabolism, 250

T T cell receptor, I , 2, 12, 13 CY chain, 2-5 p chain, 5-7 T chain, 7-10 8 chain, 10-12 T cells arachidonic acid and, 335-342. 344, 345 leukocytes and, 430, 432-440. 444, 455457 lymphocytes and activation by lymphokines, 496, 501, 505, 521-523 calcium changes, 153-168, 170 cyclic nucleotides, 380, 385, 386, 38839 1 GTP-binding proteins, 57-59 intracellular pH, 21 I , 212, 214-216 ion transport, 107, 109, 112, 117, 118 protooncogene expression. 573-578, 580, 582 transmembrane signaling, 508, 514, 516 phagocytes and, 543, 544, 551 phosphoinositide metabolism in lyniphocytes and, 250, 252, 282-284, 287 calcium, 266-269 CAMP, 275-278 cellular immunity, 279-282 mitogens, 257-259

negativc signals, 278, 279 proliferation, 260-265 protein kinasc C, 271-275 protooncogenes, 269-27 1 receptor. 253-256 surface antigens, 256, 257 Theophylline. neutrophils and, 408, 409, 413, 414 Thrombin, lymphocytes and, 210 Thromboxancs arachidonate metabolites and, 351, 355, 356, 358 lymphocytes and, 512 phagocytes and, 555 Th ymocytes arachidonic acid and, 340, 341 lymphocytes and activation by lymphokines, 519 cyclic nucleotides, 379, 380, 384, 385 intracellular pH, 210, 211, 213-215 protooncogene expression, 574, 576, 578 phagocytes and, 542 phosphoinositide metabolism in lymphocytes and calcium, 266, 268, 269 cellular immunity, 282 T cells, 257, 261-264, 271 Thymus leukocytes and, 441 lymphocytes and, 250 TPA lymphocytes and, 160, 164, 166, 168 phagocytic leukocytes and, 230, 237, 238, 24 I , 242 Transcription arachidonate metabolites and, 366 lymphocytes and activation by lymphokines, 499 calcium changes, 165 intraccllular pH, 212, 213 protooncogene expression, 57 1-582 phagocytes and, 588-590, 593-598 phosphoinositide metabolism in lymphocytes and B cells, 286 T cells, 257, 258, 269, 270, 277 T cell receptor and, 2, 6 , 1 I Transducin, phagocytes and, 69, 81, 82, 89 Transduction

628 arachidonate metabolites and. 350. 356, 358-362, 366 signal, see Signal transduction Tranzfei-rin leukocytes and, 450 lymphocytes and, 257. 508 phagocytes and, 541 Transforming growth factor p,, phagocytcs and, 542, 544, 54S, 557, 558 Translocation arachidonate metabolites and, 357 leukocytes and, 429, 441, 443 lymphocytes and activation by lymphokines. 513. 514 cyclic nucleotides, 389 phosphoinositide metabolism, 274, 275. 277 protooncogene expression, 572 ncutrophils and. 418 phagocytes and gene expression, 590 CTP-binding proteins, 90, 91 phosphoinositide nietaholism, 307, 308 phosphorylation, 470, 476, 477, 485 phagocytic leukocyte$ and, 229, 231 Tunior arachidonate metabolites and, 353, 358, 365 lymphocytes and cyclic nucleotides, 382 GTP-hinding proteins, 5 I intracellular pH, 216 protooncogene expreaaion, 572, 579 phagocytcs and calcium, 182, 185, 187, 190 cytokincs, 538, 540. 541. 549. 553-555, 558, 560 phosphoinositide metabolism, 31 I phagocytic leukocytes and, 233, 239 Tumor necrosis factor lymphocytes and, 279 phagocytes and, 542, 543 chemoattractant regulation, 28 gene cxprcssion, 593, 595, 598 GTP-binding protein<, 91 mdcrophages, 550-552. 554-559 neutrophils, 546-548 Tyrosine kinase leukocytes and, 430, 431, 456, 457 lymphoid cclls, 433. 434, 440-442 myeloid cells, 443-447, 449, 450, 454, 455

INDEX lymphocytes and activation by lymphokines, 503. 507, 522. 523 phosphoinositide metabolism, 2.52, 263, 270-273. 277 protooncogene expression, 579, 580 transmenibrane signaling. 5 15-5 18. 521 phagocytes and GTP-binding proteins, 7 3 phosphoinositide metabolism, 304, 312, 313. 324

v Vihrio cholwa toxin. lymphocytes and. 46. 47, 51, 53, 57 Vihrio rholerue, phagocytes and. 68

W

WGA. see Wheat germ agglutinin Wheat germ agglutinin, lymphocytes and calcium changes, 155, 161 cyclic nuctcutides, 382 intracellular pH. 212, 215 phosphoinositide mctabolisni, 273

Y Ycast, phagocytes and calcium, 187, 195 GTP-binding proteins, 70, 73, 78

Z

Zymosan arachidonate metabolites and, 365 biochemical mechanisms, 353, 355-358 transduction, 059-362 leukocytes and, 454 neutrophils and, 404, 406, 408, 417 phagocytcs and calcium, 191 cytokines, 540, 556 ion transport. 139, 141 phosphoinositide metabolism, 32 1 , 323 phosphorylation, 471, 474

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