Advances in Immunology Volume 17

ADVANCES IN Immunology VOLUME 17 CONTRIBUTORS TO THIS VOLUME ELMERL. BECKEX SAM M. BEISER VINCENTP. BUTLER,JR. KEJ...

Author: Frank J. Dixon | Henry G. Kunkel

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ADVANCES IN

Immunology

VOLUME 17

CONTRIBUTORS TO THIS VOLUME ELMERL. BECKEX SAM M. BEISER VINCENTP. BUTLER,JR.

KEJTH M. COWAN PETERM. HENSON EUGENEM. LANCE

P. B. MEDAWAR

ROBERTN. TAUB

ADVANCES IN

Immunology E D I T E D BY

F. J. DIXON

HENRY G. KUNKEL

Division of Experimentof Pothology Scrippr Clink ond Reseorch Foundation lo Jollo, Colifornio

The Rockefeller Univerrity New York, New York

VOLUME 17 1973

ACADEMIC PRESS

New York and London

A Subsidiary of Harcourt Brace Jovanovich, Publishers

COPYRIGHT 0 1973, BY ACADEMIC PRESS, INC. ALL RIGHTS RESERVED. NO PART O F THIS PUBLICATION MAY BE REPRODUCED OR TRANSMITTED IN ANY FORM OR BY ANY MEANS, ELECTRONIC OR MECHANICAL, INCLUDING PHOTOCOPY, RECORDING, OR ANY INFORMATION STORAGE AND RETRIEVAL SYSTEM, WITHOUT PERMISSION IN WRITING FROM THE PUBLISHER.

ACADEMIC PRESS, INC.

111 Fifth Avenue, New York. New York 10003

United Kingdom Edition published by ACADEMIC PRESS, INC. (LONDON) LTD. 24/28 Oval Road, London NW1

LIBRARY OF CONGRESS CATALOO CARDNUMBER:61-17057

PRINTED IN THE UNITED STATES OF AMERICA

CONTENTS

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LIST OF CONTRIBUTORS PREFACE

CONTENTS OF PREVIOUS VOLUMES.

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Vii

ix

xi

Antilymphocyte Serum EUGENEM LANCE.P B MEDAWAR. AND ROBERT N TAUB

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. . . I . Introduction . . . . . . . . . . . . . I1. History . . . . . . . . . . . . . . I11. Preparation of Antilymphocytic Antisera . . . . . . . IV . Purification of Antilymphocytic Serum . . . . . . . V . Assays of Potency . . . . . . . . . . . . VI . Effect on Lymphoid Cells or Tissue . . . . . . . . . . . . . VII . Scope of Antilymphocytic Serum Action in Vioo VIII . Inimunogenicity of Antilyniphocytic Serum Immunoglobulin G . . IX . Discriminate Action of Antilymphocytic Serum on Cell-Mediated . . . . . . . . . . . . . Immunity X . Chronic Administration of Antilymphocytic Serum . . . . . . . . . . . . . . XI . Synergism with Other Agents XI1. Antilymphocytic Serum and the Induction of Immunological Tolerance . . . . . . XI11. Mode of Action of Antilymphocytic Serum XIV. Effects in Man . . . . . . . . . . . . XV. Projections for the Future . . . . . . . . . . References . . . . . . . . . . . . .

2 3 4 15 24 27 36 46 47 51 54 55 57 62 73 73

In Vitro Studies of Immunologically Induced Secretion of Mediators from Cells and Related Phenomena ELMERL BECXERAND PETER M HENSON

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I . Introduction . . . . . . . . . I1. General Characteristics of Secretory Process . . I11. Mediator Secretion from Isolated Tissues and Organs . . . . IV . Mediator Secretion from Mast Cells V. Mediator Secretion from Basophiles . . . . VI. Mediator Secretion from Platelets . . . . VII . Mediator Secretion from Neutrophiles . . . VIII . Phagocytosis by Neutrophiles . . . . . IX . Mediator Secretion from Monocytes and Macrophages X . Chemotaxis . . . . . . . . . XI . Lymphocyte Transformation . . . . . . XI1. General Summary . . . . . . . . References . . . . . . . . . V

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94 96 99 106 122 126 143 152 158 159 162 166 178

vi

CONTENTS

Antibody Response to Viral Antigens

KEITH M . COWAN

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I. Introduction I1 Virus Structure and Viral Antigens . . I11. Measurement of Antibody to Viral Antigens IV. The Antibody Response . . . . V . Concluding Reinarks . . . . . References . . . . . . .

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195 197 208 223 244 245

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255 256 261 297

311

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Antibodies to Small Molecules: Biological and Clinical Applications

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. . . . . . . . . I . Introduction . I1. General Principles . . . . . . . . I11. Specific Applications . . . . . . . References . . . . . . . . . VINCENT P BUTLER. JR.,

AND

SAMM BEISER

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SUBJECTINDEX.

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343

LIST OF CONTRIBUTORS Numbers in parentheses indicate the pages on which the authors’ contributions begin.

ELMERL. BECKER, Department of Pathology, University of Connecticut Health Center, Farmington, Connecticut ( 93) SAM M . BEISER,Departments of Medicine and Microbiology, College of Physicians and Surgeons, Columbia University, New York, New York (255)

VINCENTP. BUTLER,JR., Departments of Medicine and Microbiology, College of Physicians and Surgeons, Columbia University, New York, New York (255) KEITH M. COWAN,Plum Island Animal Disease Laboratory, Agricultural Research Service, U . S . Department of Agriculture, Greenport, New York (195)

PETERM . HENSON, Department of Experimental Pathology, Scripps Clinic and Research Foundation, La Jolla, California ( 9 3 ) EUGENEM . LANCE,Division of Surgical Sciences, Clinical Research Centre, Harrow, Middlesex, England ( 1 ) P. B. MEDAWAR, Division of Surgical Sciences, Clinical Research Centre, Harrow, Middlesex, England (1) ROBERT N . TAUB,Transplantation Immunology Laboratory, Mount Sinai Hospital, New York, New York ( 1 )

vii

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PREFACE The extent to which inmmnology continues to permeate new frontiers of biology and medicine is a constant source of amazement. It is certainly in part a consequence of the more diversified usages of immunological methodology which is clearly exemplified by the last article, by Drs. Butler and Beiser, on the extraordinary array of small molecules to which antibodies can now be obtained. More significantly, perhaps, it stems from the broad significance of the science of immunology itself to mammalian systems and their derangements. The scope of relevant subjects to be covered by reviews of this type is necessarily broadened, and this is clearly apparent in the diversification in Volume 17. In the first contribution, Drs. Lance, Medawar, and Taub present a very complete analysis of the question of antilymphocyte serum. These workers were pioneers in the experimental work with this material and this wide experience is very apparent in the exhaustive and thorough treatment afforded the subject. There is no doubt that the use of antilymphocyte serum, particularly preparations with defined specificities for single types of lymphocytes, will have broad usage in experimental immunology. Its place in human therapy remains unanswered, although impressive results have been obtained in certain centers. The authors review this work critically and completely. In the second article, Drs. Becker and Henson have attempted a very difficult task in trying to review the intricate and in most instances illdefined topic of mediators, not only those involved in immunological reactions but many others as well. Of necessity, it has to represent a progress report, but it will undoubtedly prove of considerable value to a wide audience and the bibliography is particularly complete. Certain common denominators become apparent in this review, such as the role of Ca and Mg ions and the cyclic AMP system, and these topics are well covered by the authors. The contribution by Dr. Cowan contains many examples of the use of immunological techniques in the dissection of the components of both the simple and the more complex viruses. Emphasis is placed on the picornavirus-type foot-and-mouth disease virus, where very advanced methodology has been utilized in the definition of a spectrum of different antigens. A number of practical and theoretical developments in radial immunodiffusion have evolved from the studies on this virus by Dr. Cowan and his associates at Brookhaven. A variety of other viral antigens including those of the influenza group are also reviewed. ix

X

PREFACE

The final article deals primarily with the biological and clinical applications of antibodies to various low molecular weight, biologically active molecules. There does not appear to be any limit to the type of molecule to which antibodies may be produced; the only limitation is the ingenuity of the chemist to conjugate a specific type to a suitable carrier, Important new examples are cyclic AMP and angiotensin. The diagnostic value of such antisera when combined with the newer methods of radioimmunoassay is abundantly clear. The therapeutic possibilities of antisera to such a substance as angiotensin in ameliorating disease manifestations are certainly intriguing but not as yet realized. Work on this review was one of the last activities of Dr. Beiser prior to his untimely death; his outstanding contributions to the field add greatly to this section. The complete cooperation of the publishers in the production of Volume 17 is gratefully acknowledged.

HENRYG. KUNKEL FRANK J. DIXON

Contents of Previous Volumes Volume 1 Transplantation Immunity and Tolerance AND T. HRABA M. HA~EK, A. LENGEROVL,

Immunological Tolerance of Nonliving Antigens

RICHARDT. SMITH Functions of the Complement System

ABRAHAM G. OSLER In Vifro Studies of the Antibody Response

ABRAMB. STAVITSKY Duration of Immunity in Virus Diseases

J. H. HALE Fate and Biological Action of Antigen-Antibody Complexes

WILLIAM0. WEICLE Delayed Hypersensitivity to Simple Protein Antigens

P. G. H. CELLAND B. BENACERRAF The Antigenic Structure o f Tumors

P. A. GORER AUTHORINDEX-SUB JECX INDEX Volume 2 Immunologic Specificity and Molecular Structure

FREDKARUSH Heterogeneity of y-Globulins JOHN

L. FAHEY

The Immunological Significance o f the Thymus

J. F. A. P. MILLER,A. H. E. MARSHALL, AND R. G. WHITE Cellular Genetics of Immune Responses

G. J. V. NOSSAL Antibody Production by Transferred Cells

CHARLESG. COCHRANE AND FRANK J. DIXON Phagocytosis

DERRICK ROWLEY xi

xii

CONTENTS OF PREVIOUS VOLUMES

Antigen-Antibody Reactions in Helminth Infections

E. J. L. SOUL~BY Embryalogical Development of Antigens

REED A. FLICKINCER AUTHORINDEX-SUB JECT INDEX Volume 3 In Vifro Studies of the Mechanism of Anaphylaxis

K. FRANKAUSTENAND JOHN H. HUMPHREY The Role of Humoral Antibody in the Homograft Reaction

CHANDLER A. STETSON Immune Adherence

D. S. NELSON Reaginic Antibodies

D. R. STANWORTH Nature of Retained Antigen and Its Role in Immune Mechanisms

DANH. CAMPBELL AND JUSTINE S. GARVEY Blood Groups in Animals Other Than Man

W.H. STONEAND M. R. IRWIN Heterophile Antigens and Their Significance in the Host-Parasite Relationship

C. R. JENKIN

AUTHORINDEX-SUB JECT INDEX Volume 4 Ontogeny and Phylogeny of Adaptive Immunity ROBERTA. GOODAND BENW. PAPERMASTER Cellular Reactions in Infection

EMANUEL SUTERAND HANSRUEDY RAMSEIER Ultrastructure of Immunologic Processes JOSEPH

D. FELDMAN

Cell Wall Antigens of Gram-Positive Bacteria

MACLYNMCCARTYAND STEPHENI. MORSE Structure and Biological Activity of Immunoglobulins

SYDNEYCOHENAND RODNEYR. PORTER

CONTENTS OF PREVIOUS VOLUMES

Autoantibodies and Disease

H. G. KUNKELAND E. M. TAN Effect of Bacteria and Bacterial Products on Antibody Response

J. Mmoz AUTHOR INDEX-SUB JECT INDEX Volume 5

Natural Antibodies and the Immune Response

STEPHEN V. BOYDEN Immunological Studies with Synthetic Polypeptides

MICHAELSELA Experimental Allergic Encephalomyelitis and Autoimmune Disease

P ~ I Y. P PATERSON The Immunology of Insulin

c. G. POPE

Tissue-Specific Antigens

D. C. DUMONDE

AUTHOR INDEX-SUB JECT INDEX Volume 6

Experimental Glomerulonephritis: Immunological Events and Pathogenetic Mechanisms

EMILR.UNANUE AND FRANK J. DIXON Chemical Suppression of Adaptive Immunity

ANN E. GABRIELSON AND ROBERT A. GOOD Nucleic Acids as Antigens

OTTOJ. PLESCU AND WERNER BRAUN In Vifro Studies of Immunological Responses of Lymphoid Cells

RICHARDW. DUITON Developmental Aspects of Immunity

JAROSLAV STERZLAND ARTHURM. SILVERSTEIN Anti-antibodies

PHILIPG. H. GELLAND ANDREWS. KELUS Conglutinin and lmmunoconglutinins

P. J. LACHMANN AUTHORINDEX-SUB JECT INDEX

xiii

xiv

CONTENTS OF PREVIOUS VOLUMES

Volume 7 Structure and Biological Properties of Immunoglobulins SYDNEY C O ~ AND N CESAR MrrsTEJN Genetics of Immunoglobulins in the Mouse

MICHAELPOTI-ERAND ROSELIEBERMAN Mimetic Relationships between Group A Streptococci and Mammalian Tissues JOHN

B. ZABRISKIE

lymphocytes and Transplantation Immunity

DARCY B. WILSON AND R. E. BILLINGHAM Human Tissue Transplantation

JOHNP. MERRILL AUTHORINDEX-SUBJECT INDEX Volume 8 Chemistry and Reaction Mechanisms of Complement

HANSJ. M~~LLER-EBERHARD Regulatory Effect of Antibody on the Immune Response JONATHAN

W. Urn AND GORANMOLLER

The Mechanism of Immunological Paralysis

D. W. DRESSER AND N. A. MITCHISON In Vifro Studies of Human Reaginic Allergy

ABRAHAMG. OSLER, LAWRENCE M. LICHTENSTEIN, AND DAVID A. LEVY AUTHORINDEX-SUBJECTINDEX Volume 9 Secretory Immunoglobulins

THOMAS B. TOMASI, JR.,

AND JOHN

BIENENSTOCK

Immunologic Tissue Injury Mediated by Neutrophilic Leukocytes

CHARLES G. COCHRANE The Structure and Function of Monocytes and Macrophages

ZANVIL A. COHN The Immunology and Pathology of NZB Mice

J. B. HOWIE AND B. J. HELYER

AUTHORINDEX-SUB JECT INDEX

CONTENTS OF PREVIOUS VOLUMES

Volume 10 Cell Selection by Antigen in the Immune Response

GREGORY W. SISKINDAND BARUJ BENACERRAF Phylogeny of Immunoglobulins

HOWARD M. GREY Slow Reacting Substance of Anaphylaxis

ROBERTP. ORANGE AND K. FRANKAUSTEN Some Relationships among Hemostasis, Fibrinolytic Phenomena, Immunity, and the Inflammatory Response

OSCARD. RATNOFF Antigens o f Virus-Induced Tumors

KARL HABEL Genetic and Antigenetic Aspects of Human Histocompatibility Systems

D. BERNARDAMOS AUTHOR INDEX-SUB j ~ c INDEX r Volume 11 Electron Microscopy o f the Immunoglobulins

N. MICHAELGREEN Genetic Control of Specific Immune Responses

HUGH0. MCDEVITTAND BARUJBENACERRAF The lesions in Cell Membranes Caused b y Complement JOHN

H. HUMPHREY AND ROBERTR. DOURMASHKIN

Cytotoxic Effects of lymphoid Cells In Vifro

PETERF’ERLMANNAND GORANHOLM Transfer Factor

H. S. LAWRENCE Immunological Aspects of Malaria Infection

IVOR N. BROWN AUTHOR INDEX-SUB j ~ c INDEX r Volume 12 The Search for Antibodies with Molecular Uniformity

h x ~ m M.KRAUSE Structure and Function o f y M Macroglobulins

HENRYMETZGER

xv

xvi

CONTENTS OF PREVIOUS VOLUMES

Transplantation Antigens

R. A. REISFELD AND B. D. KAHAN The Role of Bone Marrow in the Immune Response

NABIH I. ABWU

AND

MAXWELLRICHTER

Cell Interaction in Antibody Synthesis

D. W. TALMAGE, J. RADOVICH, AND H. HEMMINGSEN The Role of Lysosomes i n Immune Responses

GERALD WEISSMANN AND PETERDUKOR Molecular Size and Conformation of Immunoglobulins

KEITH J. DORRINGTON AND CHARLES TANFORD AUTHORINDEX-SUB JECT INDEX Volume 13

E HANSBENNICHAND S. GUNNAR 0. JOHANSSON

Structure and Function o f Human Immunoglobulin

Individual Antigenic Specificity of Immunoglobulins

JOHNE. HOPPERAND ALFREDNISONOFF In Vitro Approaches to the Mechanism of Cell-Mediated Immune Reactions

BARRYR. BLOOM Immunological Phenomena in Leprosy and Related Diseases

J. L. TURKAND A. D. M. BRYCESON Nature and Classification of Immediate-Type Allergic Reactions

ELMERL. BECKER AUTHORINDEX-SUB JECT INDEX Volume 14 lmmunobiology of Mammalian Reproduction

ALANE. BEERAND R. E. BILLINGHAM Thyroid Antigens and Autoimmunity

SIDNEY SHULMAN lmmunological Aspects of Burkitt’s Lymphoma

GEORGE KLEIN Genetic Aspects o f the Complement System

CHESTER A. ALPER AND FREDS. ROSEN The Immune System: A Model for Differentiation in Higher Organisms

L. HOODAND J. PRAHL AUTHORINDEX-SUB j ~ c INDEX r

CONTENTS OF PREVIOUS VOLUMES

Volume 15 The Regulatory Influence of Activated T Cells on B Cell Responses t o Antigen

DAVDH. KATZ AND BARUJBENACERRAF The Regulatory Role of Macrophages in Antigenic Stimulation

E. R. UNANUE Immunological Enhancement: A Study of Blocking Antibodies

JOSEPHD. FELDMAN Genetics and Immunology of Sex-linked Antigens

DAVIDL. GASSER AND WILLYS K. SILVERS Current Concepts of Amyloid

EDWARD C. FRANKLIN AND DOROTHEA ZUCKER-FRANKLIN AUTHOR INDEX-SUB JECT INDEX Volume 16 Human Immunoglobulins: Classes, Subclasses, Genetic Variants, and ldiotypes

J. B. NATVICAND H. G. KUNKEL Immunological Unresponsiveness

WILLIAM0. WEIGLE Participation of Lymphocytes i n Viral Infections

E. FREDERICK WHEELOCK AND STEPHENT. TOY Immune Complex Disease in Experimental Animals and Man

C. G. COCHRANE AND D. KOFFLER The lmmunopathology of Joint Inflammation in Rheumatoid Arthritis

NATHAN J. ZVAIFLER AUTHORINDEX-SUB j ~ c INDEX r

xvii

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An ti lymphocyte Serum

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EUGENE M LANCE. P B MEDAWAR. AND ROBERT N TAUB Division o f Surgicol Sciences. Clinical Reseorch Cenfre. Harrow. Middlesex. England. and Transplanfation Immunology Laborotory. Mount Sinai Hospifal. New York. N e w York

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I. Introduction I1. History . . . . . . . . . . . . . . I11. Preparation of Antilymphocytic Antisera . . . . . . . A. Source of Antigen . . . . . . . . . . . B Choice of Species . . . . . . . . . . . C Schedule of Immunization . . . . . . . . . D . Comment on the Preparation of Antilymphocytic Sera . . . . . . . . . . IV. Purification of Antilymphocytic Serum . A Absorption . . . . . . . . . . . . . B . Antilymphocytic Serum Fractions and Antibody Fragments . . C . Antibody Eluates . . . . . . . . . . . V Assays of Potency . . . . . . . . . . . . A . In Vivo . . . . . . . . . . . . . B . In Vitro . . . . . . . . . . . . . VI . Effect on Lymphoid Cells or Tissue . . . . . . . . A. In Vitro . . . . . . . . . . . . . B In Vioo . . . . . . . . . . . . . . . . . VII . Scope of Antilymphocytic Serum Action in Viuo . A Effect on Inflammation . . . . . . . . . . B. Effect on Cell-Mediated Immunity . . . . . . . . C. Humoral Immunity . . . . . . . . . . . D. Erasure of Memory . . . . . . . . . . . VIII. Imniunogenicity of Antilymphocytic Serum Immunoglobulin G . . IX . Discriminate Action of Antilymphocytic Serum on Cell-Mediated . . . . . . . . . . . . . Immunity . A. Suppression of Responses in Virgin Animals . . . . . . B . Effect in Sensitized Animals . . . . . . . . . . . . C . Effect of Antilymphocytic Serum on Viral Systems . D . Morphological Evidence . . . . . . . . . . X . Chronic Administration of Antilymphocytic Serum . . . . . XI. Synergism with Other Agents . . . . . . . . . . XI1. Antilymphocytic Serum and the Induction of Immunological Tolerance . . . . . . XI11. Mode of Action of Antilymphocytic Serum . A . Selective Action on Recirculating Lymphocytes . . . . . B Alternative Possibilities . . . . . . . . . . XIV Effects in Man . . . . . . . . . . . . . A . Parallelism between Clinical and Experimental Evidence . . . B . Special Aspects of Antilymphocytic Serum Production for Use in Man C. Administration and Side Effects . . . . . . . . D . Clinical Use of Antilymphocytic Globulin . . . . . . 1

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E. M. LANCE, P. B. MEDAWAR, AND R . N. TAUB

XV. Projections for the Future References . . .

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73 73

I. Introduction

It is always di5cult to be sure of perspective when viewing a subject at close range, yet, judging from the number of published papers and even reviews (James, 1968, 1969; Medawar, 1968; Lance and Medawar, 1970c; Lance et al., 1971; Denman, 1969; Taub, 1970a; Sell, 1969; Gum, 1969; Caron, 1968; Woodruff, 1969, 1971; van Bekkuni, 1969; Renoux and Mikol, 1967; Russell, 1968, 1969; Shanfield and McLean, 1969; Brendel, 1969), antilymphocytic serum ( ALS) has captured the imagination of the scientific community. There seem to be clear and adequate reasons for this enthusiasm, although the ultimate role of this agent both with respect to therapeutics and research remains to be clarified. Impelled by the success and growing demands of clinical transplantation, a wide variety of agents has been used to suppress the rejection response. The great majority of these “conventional” immunosuppressive agents share several features in common: ( a ) often they have been the side product of the search for drugs to control malignancy (immunosuppression is an indirect result of some very general antimetabolic effects, and a wide variety of tissues and organs are affected); ( b ) the dosage range for immunosuppression is close to or coextensive with the toxic range, and finally ( c ) these agents do not generally discriminate between the two arms of the immune response-cellular and humoral. In so far as they do discriminate they appear to be more effective in opposing humoral rather than cell-mediated immunity. For reasons which will be fully developed in this review, ALS promises to be a considerable advance. Although it is too early for a final judgment, yet in some ways this is a good time for a reappraisal of research in this field. For it may well be asked whether the early promise heralded in the publications of the 1960s has been fulfilled. For some time it appeared as if the biological usefulness of ALS ended with the evolution of Homo sapiens. Although remarkable and reproducible effects could be achieved in a wide variety of animal species, there was little convincing evidence that ALS was useful in man. Surgeons of considerable seniority and experience in the field of transplantation became sceptical, remarking when asked their opinion of ALS, “Oh! Yes . . . I have heard of it . . . Jolly useful in mice.” We believe that this is a good time to inquire carefully into the cause of this apparent discrepancy and examine the more recent clinical evidence which, to some extent, suggests that man is, indeed, not exceptional in his response to ALS. Antilymphocytic serum, in the context of this review, refers to the product obtained when lymphoid cells or cell fractions from animals of

ANTILYMPHOCYTE SERUM

3

one species are injected into animals of another species. Strictly speaking, this definition excludes sera produced by the injection of cells other than lymphoid cells. However, as will be pointed out below, this exclusion is an operational one, and in some cases homologous sera or sera raised against tissue other than lymphoid tissue may share the properties of heterologous ALS. Reference is made throughout this review to the potency or activity of ALS. Unless otheiwise stated we are referring to the power to prolong the survival of tissue allografts in vivo. This stipulation and restriction are necessary because many of the in vivo and in oitro properties of ALS appear to have little or nothing to do with the defining property: immunosuppression. II. History

The current intense interest in ALS stems from its remarkable properties as an immunosuppressive agent. Inderbitzin (1956) is credited with the first demonstration of this effect. While working in the laboratory of John Humphrey, he showed that such antisera could strongly inhibit the skin reactions of delayed hypersensitivity in the guinea pig. In 1961, Waksman et al. confirmed these findings and extended the range of effects to inhibition of autoimmune disease and the prolongation of skin homograft survival. The abrogation of the homograft response recorded in his experiments, although feeble, was undoubtedly significant, and it was not until the demonstration by Woodruff and Anderson (1963, 1964) of striking prolongation of the life of skin homografts in rats under the aegis of ALS that the present frenetic phase of investigation began. Although knowledge of the immunosuppressive action of ALS is barely 15 years old, similar sera have been used for almost 70 years as a tool in biological research. The first recorded application was by the great Russian zoologist Metchnikoff ( l899), who used ALS to investigate the cellular basis of inflammation. Over the ensuing decades, sporadic reports anticipated much of our current knowledge of the properties of these antisera. Besredka, in 1900, showed that ALS agglutinated and killed lymphocytes in vitro, that cytotoxicity was destroyed by heating the serum to 55°C. for 30 minutes, that these agents showed species specificity, and caused a leukopenia in vivo. The specificity of ALS for lymphocytes was first shown by Bunting in 1903. He reported relatively specific and immediate fall in blood lymphocytes after intraperitoneal ALS injection. Some of the sera used by early workers failed to discriminate among leukocytes ( Flexner, 1902; Christian and Leen, 1905; Besredka, 1900). However, Chew and Lawrence (1937) were able to establish that sera that discriminated rather specifically between lymphocytes and polymorphonuclear leukocytes could be raised. Cruickshank (1941) investigated the role of the spleen in the media-

4

E. M. LANCE, P. B. MEDAWAR, AND R . N. TAUB

tion of ALS-induced lymphopenia. In contrast with the immune hemolysis of erythrocytes, splenectoniy did not alter the rapid and profound lymphopenia induced by ALS. Moorhead (1905) was apparently the first to attempt to raise antithymocyte sera to investigate thymic function; however, his efforts were not rewarded with success. Ritchie (1907) and later Pappenheimer (1917) were able to raise antithymic sera that did not discriminate between lymphoid cells from various sources. The studies of Pappenheimer are of additional significance, since he showed that at least two populations of antibodies were raised by the injection of thymocytes-one directed toward lymphoid cells and another directed toward erythrocytes. Absorption with erythrocytes did not affect the titer of lymph agglutinins; however, these could be completely absorbed with thymocytes. Woodruff and Forman were among the first to recognize the immunosuppressive potential of ALS but were discouraged in their initial efforts (1960). They found that repeated injection of rabbit antirat lymph node antiserum into rats produced an initial but short-lived lymphopenia which was followed by a lymphocytosis despite continued administration of antiserum. Antilymphocytic serum-induced lymphopenia was a direct effect, since it was elicited in adrenalectomized rats. Initial attempts to prolong the survival of allografts in Wistar rats were unsuccessful. Woodruff and Anderson (1963) reopened the question by combining ALS treatment with thoracic duct drainage and were able to show striking skin graft prolongation. This demonstration was quickly followed by the work of Gray et al. (1964), of Monaco et al. (1967a), and of Levey and Medawar (1966a,b) establishing ALS as the most potent known inhibitor of the transplant-rejection reaction. Clinical application of ALS was pioneered by Starzl and his collaborators ( 1966, 1967a,b,c, 1968, 1969b) . After extensive experience in dogs, they used ALS as adjunctive immunosuppression to the usual programs of azathioprine and corticosteroids in humans. Although these clinical studies were not strictly controlled, there seemed to be a decrease in morbidity, a reduced requirement for corticosteroids, and a reduction in the loss of kidneys from rejection. I l l . Preparation of Antilymphocytic Antiserd

A. SOURCEOF ANTIGEN Potent antisera have been raised by using a wide variety of lymphoid cells. Indeed, it seems a general principle that any source of lymphoid tissue may be used. The types of lymphoid tissue that have been reported to yield potent antisera are summarized in Table I. A point of some theo-

ANTILY MPHOCY TE SERUM

5

TABLE I Souiiciss OF ANTICICN USICDTO RAISE ANTILYMPHOCYTE SERA Normal viable cells Thymus Lymph nodes Spleen Thoracic duct Peripheral blood Tonsil Preserved lymphoid cells Epidermal cells Embryonic fibroblasts Lymphoid cells from antilymphocytic serum-lreated animals Viable malignant cells Cells from patients with lymphomns L-cells Lymphoblastic rell lines Myeloma cells Nonviable cells or cell components Heat-killed lymphoid cells Lymphoid cells from cadavers Chemical extracts from lymphoid cells Subcellular component8 from lymphoid cells

retical interest is that potent antisera can be raised by using the residual lymph node population from ALS-treated animals ( Miller et d.,1970). At various times, claims for the superiority of one lymphocyte type as antigen over others have been made. Thymocytes have been especially favored, and there is some evidence that antithymocyte antisera may be better than, for instance, anti-lymph node antisera. The possibility that antithymocyte antisera may have special advantages because of a postulated antibody toward thymic humoral factors ( thymosin ) was raised by Nagaya and Sieker (1965, 1967, 1969a,b). Not only have others failed to confirm the superiority of antithymocyte antisera, but Wood and Vriesendorp (1969) found the exact opposite. The critical experiment, which would take the form of a comparison of the effects of antithymic and anti-lymph node antisera in both thymectomized and intact animals, has not yet been reported. The choice of antigen source is strongly conditioned by expedience and availability, especially in clincal settings. Lymphocytes from most sources are contaminated to a greater or lesser extent by other tissue components. Upon injection into animals, such crude preparations give rise to a number of antibodies that are not only irrelevant to the immuno-

6

E. M. LANCE, P. B. MEDAWAR, AND R . N. TAUB

suppressive effect of ALS but also are potentially toxic ( Woiwood et al., 1970). There are in general two possible approaches to this problem: purification of the antigen so that irrelevant antibodies do not arise or absorption of unwanted antibodies from a multivalent antiserum. The latter approach will be dealt with below. The first stage in antigen preparation is the production of a single cell suspension. When the starting material is blood or lymph this occasions no problem, but solid lymphoid organs must be disrupted either in mechanical homogenizers or preferably by passage through fine mesh screens. Great care must be taken to remove stromal elements, as these seem particularly prone to give rise to antibodies that react with basement membrane. The cells must, then, be thoroughly washed to eliminate unwanted serum proteins after which some attempt to remove contaminating erythrocytes is warranted. To this end perfusion of vascularized organs prior to disruption may be helpful (Iwasaki et al., 1967). Flash osmotic shock which will preferentially lyse erythrocytes can be applied to the cell suspension. At this point the cell suspension is more or less heterogeneous, depending upon the antigen source. Filtration through cotton or glass wool columns enriches such suspensions in small lymphocytes by preferentially retaining polymorphonuclear leukocytes and monocytes. When whole blood is used as the starting material, an additional processing step is required to remove platelets. From the point of view of ease of processing and homogeneity sources of lymphocytes would probably rank in the order: thoracic duct, thymus, lymph nodes, peripheral blood, and spleen. Accessibility and availability are two practical considerations when selecting an antigen source. In this respect it seems possible to raise suitable antisera by using neoplastic lymphoid tissue (Witz et al., 1968; Pichlmayr, 1970). The potential use of cultured lymphoblasts seems particularly attractive ( Najarian et al., 1969a,b,c; Perper et al., 1970a,b; Moore, 1969a,b) for a relatively unlimited supply of pure lymphocytes. A further stage in antigen purification involves the isolation of subcellular lymphocyte components. Levey and Medawar ( 1966b) were the first to approach this subject. They prepared by relatively simple manipulations a crude membrane fraction, a nucleoprotein fraction, and soluble extract. The crude membrane fraction was by far the most effective but shared several disadvantages with whole cells-namely, potency tended to fall off rather than increase with repeated immunization, and high titers of potentially toxic antibodies were also produced. Lance et al. (1968, 197Oa) and Zola et al. (1970) took this approach a step further (see also Grabar, 1970; Aschkenasy et al., 1970; Traeger et al., 1970a,b; Nagaya et al., 1970; Hayes et al., 1970; Knight et al.,

ANTILYMPHOCYTE SERUM

7

1970). Through the use of differential centrifugation and sucrose gradients, they isolated various subcellular fractions from mouse thymocytes that had been disrupted by intracellular cavitation in a nitrogen decompression apparatus. The fractions were identified by enzyme markers and by electron microscopy and consisted of supernatant, microsomal, plasma membrane, niitochondrial, and nuclear fractions. Rabbits were repeatedly immunized by intravenous inoculations of aliquots of these fractions, and the sera were collected at intervals. The resulting antisera were assayed for i n vivo potency and toxicity as well as for i n vitro lymphocytoxins and hemagglutinins. All subcellular fractions could raise active antisera, but the supernatant fraction was extremely feeble. The nuclear fraction (which also contained some undisrupted whole cells) gave rise to active antisera initially, but repeated immunization led to decline in potency. Antisera raised with mitochondria contained very high titers of hemagglutins. The membrane fraction led most consistently to formation of potent and nontoxic antisera and, moreover, could be combined with adjuvant. Antiserum raised in this way was much more potent than that resulting from membrane alone (Fig. l), remained nontoxic, and could be used without prior absorption. There was no tendency for decline in potency with repeated immunization. The membrane ingredient responsible for potency could be solubilized by a variety of treatments, including sonication, high salt concentration, 8 M urea, and deoxycholate. Treatment of solubilized membrane with a variety of proteolytic enzymes or by butanol extraction decreased or eliminated the efficacy of membrane antigen, whereas ribonuclease and deoxyribonuclease were without effect. These results were consistent with the possibility that the relevant antigens were lipoproteins. Further characterization of the relevant antigens or antigen must await more detailed fractionation and analysis. The facts that the antigens are widely distributed throughout all lymphoid tissue and that strain differences, if they exist, are minimal suggest an analogy with the MSLA antigens of the mouse, i.e., a differentiation antigen characteristic of the species lymphocyte. Boyse et al. (1968) studied the activity of ALS in relation to alloantigens of murine lymphocytes. They suggested that heterologous AES was directed against an antigenic configuration which included but which was larger than the LY series of antigens. Shigeno et al. (1968) concluded that ALS was not directed toward the 0, TL, or LY specificities, whereas Asakumah and Reif (1968) thought that a portion of ALS activity might, in fact, be directed against a species variety of 0 antigen ( a marker for thymus-derived lymphocytes in mice). This possibility is fully compatible with the observations that thymocytes carry cell surface antigens not shared by other lymphocytes (Grabar

8

E. M. LANCE, P. B. MEDAWAR, AND R. N. TAUB

A:Mmbrane + Freund's alone t antigen pulse iserum sample

0 B:hmkane

'1 0

t

ANTIGEN DOSE IN

250

750

250 20

30

t

ti

tl

10

400 MICROGRAMSOF PROTEIN

40

11

50

60

DAY OF EXPERIMNT

CYTOTOXIC TITERS

Group A all 4 bleeds 50% lysls >> 1120.480 Group B 50% lysls bleed 1 < 112560 2 115120 3 115120 4 < 115120

FIG. 1. Potency assays of group A and B antisera. New Zealand rabbits were immunized with aliquots of thymocyte membrane at the indicated times and dosages. The only difference between groups A and B was the incorporation of complete Freund's adjuvant with the first dose of membrane in group A which was injected into the footpads. All injections in group B and all subsequent injections in group A were intravenous. Note the consistently greater potency both in uioo and in oitro of the group A serum. In oivo potency was assayed by the ability to prolong the survival of A-strain skin homografts on CBA mice.

et al., 1965; Potworowski and Nairn, 1967; Grabar, 1970; Colley et al., 1970). The relevant antigens may not be possessed exclusively by lymphocytes. Levey and Medawar (1967b) reported that active antisera could be raised by using epidermal cells, L cells, and mouse embryo fibroblasts; these findings were confirmed in part by Barth et al. ( 1968). These observations suggest that the differentiation antigens of mouse lymphoid tissue may be to some extent represented in other tissues. The fact that the 6' antigen is found on brain (Boyse and Old, 1969) and the more

ANTILYMPHOCYTE SERUM

9

recent discovery of B on epidermal cells (M. Scheid, personal communication) make this a likely possibility. Neither fresh nor live cells are essential to the production of ALS. Cells taken from animals 24 hours after death have been demonstrated effective by Brent et al. (1968). Lymphoid cells killed by heating to 48.5"C. for 20 minutes were every bit as effective as live cells, thereby discounting the possibility that the ability to home to lymphoid organs played a role in immunization (Jooste et al., 1968). Of some importance is the fact that lymphoid cells may be stored, accumulated, and used at a later date (Nossa et al., 1969). Jooste et al. (1968) reported that mouse thymocytes equilibrated with dimethyl sulfoxide (DMSO) at a final concentration of between 10 to 15%and stored frozen at -79°C. were still effective although noticeably inferior to live cells. It may well be that thet best conditions for storage have not been achieved. Symes and Riddell ( 1966) have described the controlled cooling of lymphoid cells suspensions in DMSO with liquid nitrogen. Human spleen cells stored under these conditions ( -196°C.) for 50 days could still transform after thawing and stimulation with phytohemagglutinin (PHA) (Symes et al., 1966; Meek et al., 1967). More recently, thoracic duct cells stored in a similar fashion have been useful in raising ALS (Carraz et al., 1967; Traeger et nl., 1968c, 1969). Almost all the available evidence suggests that ALS does not possess strain specificity, so that within a species any source of lymphoid cells may be used. For example, Jooste et al. (1968) found that there were no systematic differences of potency among antisera raised in rabbits to the thymocytes of CBA, A, VS, Parkes, Albino, or miscellaneous outbred mice when tested in CBA mice. Sera that are active in CBA mice are also active in mice of other strains, e.g., C57/B16, A, Balb/c, C3H. Gowland et al. (1969) found that ALS raised against C3H cells was more effective when tested in CBA than in C3H mice. However, slight differences may exist: Brent et al. (1967) reported that rabbit anti-CBA antisera seemed somewhat more effective in CBA than in C57B1 mice, and Chard (1968) found that the F( a b ) ? fragment of anti-C57B1 antiserum would block the cytotoxicity of anti-C57B1 but not that of anti-CBA antisera, suggesting that some portion of the rabbit antiserum is directed toward strain-specific antigen. To what extent this is important with respect to immunosuppression remains unclear.

B. CHOICEOF SPECIES Heterologous ALS has been raised in a wide variety of species, but, surprisingly, the question of whether there are distinct advantages or disadvantages in the choice of species has not been investigated to any

10

E. M. LANCE, P. B. MEDAWAR, AND R. N. TAUB

great extent. It seems clear that within the mammalian order any recipient can give potent antisera. The range of recorded experience is summarized in Table 11. The species that have been most thoroughly studied have been the rabbit and the horse. More recently, the use of calves or pigs has been advocated by Binns et al. (1970). The degree of genetic disparity between antigen donor and antiserum producer may be of some importance. Balner and Dersjant (1967) reported that a monkey antimonkey ALS gave relatively feeble results in comparison to rabbit antimonkey TABLE I1 RANGEOF SPECIES CHOICEIN RAISING ANTILYMPHOCYTIC SERUM ~ _ _ _ _ _ _

Source of serum

Source of antigen

References.

Rabbit

Mouse Rat Guinea pig Hamster Dog Monkey Man Mouse Rat Rabbit Dog Pig Goat Human Monkey Mouse Dog Mouse Monkey Man Dog Mouse Dog Mouse Man Mouse Man

Levey and Medawar (1966a,b) Woodruff and Anderson (1963) Waksman et al. (1961) Wallace et al. (1971) Lance et aE. (1971) Balner and Dersjant (1967) Monaco et al. (1967a) James and Medawar (1967) James and Anderson (1967) Burde et al. (1971) Iwasaki et al. (1967) Lucke el al. (1968) Gunnarson et al. (1969) Iwasaki et al. (1967) Balner and Dersjant (1967) Jeejeebhoy (1967) Pichlmayr et al. (1967a) Binns et al. (1970) Lance and Medawar (1970a,b) Shorter et al. (1967) Halpern et al. (1969) Southworth et al. (1970) Pichlmayr el al. (1967s) Binns et al. (1970) Southworth et al. (1970) Binns et al. (1970) E. Simpson and R. M. Binns (personal communication) Binns et al. (1970) Binns et al. (1970) Binns rt al. (1970) Jooste et al. (1968)

Horse

Monkey Dog Sheep Goat

cow Pig

Duck

Dog Rat Chicken Rabbit

a A single reference only is given ahhough in most categories mult,iple references exist.

ANTILYMPHOCYTE SERUM

11

ALS. Moreover, with the exception of the report by Taub (1969), antisera raised within a species are regarded as impotent. Taub raised ALS in C57/ Ks mice against CBA thyniocytes which significantly prolonged the survival of A-strain grafts on CBA mice (mean survival time 17.5 clays), In general, intraspecific antisera ( alloantisera) are likely to exert their effects through enhancement, It has been suggested that alloantisera may be directed against the hypothetical receptor sites of a recipient’s lymphocytes ( Ramseier and Lindenmann, 1969). In contrast to heterologous ALS, the activity can be completely absorbed with nonlymphoid tissues (Taub, 1970a). Reports of a naturally occurring syndrome with cell-mediated, complement-dependent lymphopenia mimicking the functional deficient in ALS-treated animals ( Kretschmer et aZ., 1969) and the induction of “autoimmune” lymphopenia in dogs (Chiba et aZ., 1965) suggest lymphotoxic action of autologous antibodies ( Schwartz, 1969). Two theoretical factors bearing on ALS potency have received little attention. First, the half-life of r-globulin in the circulation varies considerably depending on the choice of donor and recipient ( Spiegelberg and Weigle, 1965). Since the duration of effect may depend on the continued availability of antibody in the immunosuppressed host, it would seem that pairs selected for this property might improve the efficacy of antisera. Second, if, as suggested by the work of R. N. Taub and M. Ruskiewicz (personal communication), different antigens on lymphocytes are “seen” by different species ( a result suggested by the ability of lymphocytes saturated with ALS antibodies of one species to absorb antilymphocytic antibodies from ALS produced by another species), then pooled antisera raised in different species might prove more effective than the use of an antiserum of single provenance. Although the choice of species within mammals seems fairly unrestricted, avians are a poor choice for the production of antisera intended for mammalian use (Jooste et al., 1968; Lance, 1968d; Riethmuller, 1967a,b; Riethmuller et al., 1968); the converse may not be true (Jankovic et al., 1970). Antisera raised in chickens and ducks to mouse thymocytes were totally inactive in uiuo. Duck anti-rabbit ALS could prolong the survival of skin allografts, although this activity was feeble. The lack of potency could not be attributed to a failure on the part of antigeninjected animals to produce antilymphocytic antibodies, for such antisera were strongly cytotoxic in vitro in the presence of avian sources of complement (Fig. 2 ) . Some activity could be demonstrated in the presence of rabbit complement, but guinea pig and mouse complement were virtually ineffective. Therefore, one could surmise that failure resulted from the known inefficiency with which antibodies produced

12

E. M.LANCE, P. B. MEDAWAR, AND R. N. TAUB I Duck ALS absorbed

I

Duck ALS unabsorbed

Chicken ALS absorbed c r D u c k 1:4 c. Rabbit .1:5 MG-pig 1:2 CI Mouse 1:l

15

160

10,240

1 5

160

lq240

15

160

14240

Reciprocalof serum dilution

FIG.2. Influence of complement source on in uitro cytotoxicity of avian antilymphocytic serum ( ALS ) on mouse lymphocytes. High titers are found when avian complement is used. Rabbit and guinea pig complement are less effective, and there is no demonstrable cytotoxicity with mouse complement.

in one species may interact with the complement of different species (Cushing, 1945; Rice, 1950). This inefficiency is not limited to mammalian-avian combinations and could, therefore, be of some importance in guiding the choice of species for the production of ALS. Apart from the preliminary report by Alexander et al. (1968, 1969) that goat ALS may not activate human complement effectively, little cognizance has been taken of this factor. Because patients may be immunized to heterologous proteins either before or during treatment, in clinical practice it may be advisable to have two different species of ALS available. Studies of Amemiya et al. (1970) suggest that cross-reactivity between inimunoglobulin G ( IgG) of different species might guide this choice.

C. SCHEDULEOF IMMUNIZATION The pattern of injection of serum donors falls into three general categories: a short course of antigen injection (two- or three-pulse sera) ; the repeated administration of antigen over relatively long periods of time (hyperimmune sera), and finally those schedules that incorporate the use of adjuvants. Levey and Medawar introduced the two-pulse method for raising rabbit antimouse thymocyte serum ( 1966a). Rabbits received two injections of lon murine thymocytes 2 weeks apart and were exsanguinated a week after the last injection. Such antisera were not toxic in a clinical sense and could be administered without absorption. Moreover, they were reliably potent, augmenting the survival of skin allografts across an H-2

ANTILYMPHOCYTE SERUM

13

barrier two- to threcfold. The distribution of median survival times of such allografts in a large number of such sera, reproduced from Jooste et al. ( 1968), is as follows:

15-19 20-24 25-29 3034 34-39 40-44

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

Levey and Medawar found that a single pulse of antigen produced generally weaker antisera, whereas the addition of a third pulse did not produce antisera significantly better than two. Indeed, the repeated injection of antigen pulses 4-6 actually resulted in the formation of less effective antisera. The short course schedule has been adopted by a number of workers ( Lance, 1968a,c,d; Berenbaum, 1967; Berenbaum et al., 1971; Prince, 1970; Levey et al., 1970; Shorter et al., 1967, 1968) whose experience generally confirms that of Levey and Medawar. Schedules of raising hyperimmune antisera have been employed by a number of workers (Woodruff et al., 1967a,b; Iwasaki et al., 1967; Pichlmayr et al., 1967a,b,c,d;Traeger et al., 1967; Carraz et al., 1967), but there is no evidence that such antisera are more potent than those raised by the short course (Thomas et al., 1970). There is, of course, the practical advantage that by repeated bleeding a greater yield of antiserum can be achieved per animal. However, this must be counterbalanced by the disadvantages: a rise in potentially toxic antibodies increasing the need for absorption (Edwards et al., 1970) and the progressive decline in potency often observed with such hyperimmune sera. This decay phenomenon has been widely reported and includes antisera raised in horses and rabbits (Woodruff et al., 1967a,b; Lance et al., 1968; B. Fisher et al., 1969; E. R. Fisher et al., 1969a,b; Jooste et al., 1968; Lee et al., 1964; Dormoiit et al., 1970; James et al., 1970; Levey and Medawar, 1966b). Moreover, the phenomenon is observed with antigens from a variety of sources including thymocytes, lymphocytes, splenic cells, epidermal cells, and crude membrane preparations. The phenomenon is not limited to the production of ALS but has been documented for the analogous situation in which the repeated immunization of rabbits with mouse erythrocytes produced a progressive fall in 19 S and 7 S hemolysins (Lee et al., 1964). The reason for decay is unknown, but it seems reasonable that the recipient may become preoccupied with lesser and irrelevant constituents of the antigenic mix that interfere with production of “active” antibodies. Alternatively, antilymphocytic antibodies may become local-

14

E. M.

LANCE,

P. B.

MEDAWAR,

AND R. N. TAUB

ized to a nonimniuiiosuppressive fraction of the immunoglobulins ( James et al., 1969a,b). An encouraging finding is that relatively pure membrane preparations may be repeatedly injected without decline in potency (Lance et al., 1968). The use of adjuvants in the production of ALS has been widely advocated. Most investigators have recommended emulsification of antigen in Freunds complete adjuvant (Gray et al., 1966; Nagaya and Sieker, 1965; Denman et al., 1!367a,b; Guttman et al., 1967a,b,c,d; Monaco et al., 1966a,b,c; Wood and Vriesendorp, 1969; Traeger et al., 1 9 6 8 ~ ) . There is no question that highly potent antisera can be raised in this way in rabbits and horses. Nevertheless, a note of caution comes from the findings of Jooste et al. (1968) which were subsequently confirmed (Koumans et al., 1971). They reported that the inclusion of adjuvants into the immunization procedure produced antisera that, although undoubtedly potent, were at the same time highly toxic. Such antisera injected into mice ( even after extensive absorption with erythrocyte, serum proteins, and suspensions of kidney and lung) caused interference with wound healing and pathological changes in the liver and kidney sometimes followed by wasting, paralysis, and death. They concluded that such antisera might be rendered useful but only after a very extensive program of purification. Wood and Vriesendorp (1969) found higher titers of hemagglutinins in sera raised with adjuvants than without; Pichlmayr ( 1970) found higher antiplatelet titers with adjuvant. There are few comparative studies of antigen dosage or route of administration. Jooste et al. (1968) investigated the dosage range of los10'O mouse thymocytes administered intravenously to rabbits, and it appeared that the intermediate dose lou was most effective. Gozzo et al. (1971) studied a wider range of antigen doses, again in the rabbit, and found that with the use of adjuvant a much lower dose of antigen was effective (see also Pichlmayr, 1970). Iwasaki et al. (1967) injected horses with large numbers of lymphoid cells to raise antisera; they favored 2 X 1O'O cells per inoculum. In general, choices of dose and route have been made empirically, and there is too little information on this score for further comment.

D. COMMENT ON

THE

PREPARATION OF ANTIL,YMPHOCYTIC SERA

Considering the obvious importance of this subject for prospective clinical use, the relevant literature reveals little that is systematic and comparative. It is unlikely that antihuman lymphocytic serum can be produced in the absence of accurate, predictive, in uitro assays considering the difficulty in evaluating the results from clinical practice. Therefore guide lines will have to be drawn from the results of animal experi-

ANTILYMPHOCYTE SERUM

15

mentation. On the basis of currcnt information the antigen sourcc of choice lies between thymocytes and thoracic duct lymphocytes ( cultured lymphoblasts remain a distinct possibility). The rabbit must represent the species of choice, regardless of the practical and logistic implications, because most evidence now establishes them as the best producers of predictably potent and nontoxic antisera. The horsc remains a second choice, whereas the possibilitics inherent in the pig, calf, goat, and sheep require further study. IV. Purification of Antilymphocytic Serum

A. ABSORPTION Whole unabsorbed ALS contains a mixture of antibodies some of which are antilymphocytic, whereas others are directed at various constituents of donor tissue, e.g., erythrocytes, serum proteins, platelets, and stromal antigens. The extent to which ALS is contaminated by this latter class of antibodies depends on the heterogeneity of the antigenic preparation and the schedule of immunization. Nonetheless, no known method of raising ALS produces a product free from these irrelevant antibodies which are potentially toxic to the recipient when present in moderate amounts. Most antisera, therefore, require absorption before use. The most frequent need seems to be the removal of hemagglutinins which can be accomplished by absorption with whole erythrocytes or with eiythrocyte stroma (Eyquem et al., 1970). An ingenious approach, recommended by Pichlmayr ( 1970), was injection of antierythrocyte antibodies simultaneously with antigen which lowered hemagglutinin titers, Induction of tolerance to erythrocytes in serum donors is another possible approach (Seiler et aZ., 1970). Monaco has suggested that erythrocyte stroma may be coupled to diethylaminoethyl ( DEAE )-Sepharose columns so that hemagglutinins are removed concurrently with protein fractionation (Latham et al., 1970). The advantage of this approach is that such columns may be regenerated and used repeatedly. Absorption procedures may sometimes take advantage of cross-reacting antigens of related species; for example, antimouse ALS may be absorbed with rat erythrocytes as an initial step prior to a final absorption with mouse erythrocytes. In man the use of outdated, stored, AB erythrocytes seems an ideal source of absorbing material. Absorption with serum proteins is recommended by Iwasaki et 01. ( 1967). Insoluble antigen-antibody complexes may be removed by relntively low-speed centrifugation, but it may also be desirable to remove the soluble complexes by high-speed ultracentrifugation as these are potentially damaging to the recipient.

16

E. M. LANCE, P. B. MEDAWAR, AND R. N. TAUB

The need for removal of antiplatelet or antibasement membrane antibodies is best avoided by selection of antigen, and preparations containing high titers of these antibodies might best be discarded. However, if for some reason it is considered desirable to use such antisera, techniques for their removal have been described (Starzl et al., 1970a,b). The suggestion that antithymocyte serum might be improved by absorption with splenic cells (Leuker and Tribble, 1969) cannot be taken seriously, unless tests of potency can be produced to substantiate these claims. B. ANTILYMPHOCYTIC SERUM FRACTIONS AND ANTIBODYFRAGMENTS After removal or reduction of contaminating antibodies the serum still contains antilymphocytic antibodies belonging to a variety of antibody classes and serum proteins of nonantibody character. Waksman et al. (1961) were the first to show that the immunosuppressive activity of ALS resided in the crude y-globulin fraction by using the technique of ammonium sulfate precipitation. This finding was soon confirmed by many others (Monaco et al., 1965a,b; Currey and Ziff, 1966). Formal studies of antisera fractionated by DEAE or Sephadex chromatography (James and Medawar, 1967; Lance, 1967, 1968d) of both horse and rabbit antimouse ALS have shown that the bulk if not all the activity resided within the 7 s IgG class of antibody. In these studies, careful comparison was made in a standard skin allograft assay' of the various fractions, and the activity of the native serum could be equalled by amounts of IgG equal to that contained in the test dose of whole serum. Moreover, treatment of whole serum or y-globulin fractions with 2-mercaptoethanol (2-ME) under conditions that destroyed the activity of immunoglobulin ( IgM ) antibodies did not appreciably alter potency. The restriction of ALS activity to the IgG fraction has been confirmed by a number of studies (Woodruff et al., 1967b); James and Anderson, 1967; Iwasaki et al., 1967; Monaco et al., 1967a,b; Betel et al., 1970). Some of the experimental evidence on this point is reproduced in Table 111. Aliquots of three rabbit antimouse pools were fractionated by column chromatography with Sephadex G-150, and the 1 9 s and 7 s components were restored to original serum concentration. Immunoelectrophoresis against specific antisera showed the 7 S fraction to be free of IgM and the 19 S fraction to be free of IgG. The relative abilities of these fractions to protect skin homografts in the standard assay procedure were compared to a simultaneous assay of the whole native serum pool from which they were prepared. The 1 9 s fraction did not demonstrably alter the fate of first-set A-strain skin grafts on CBA mice, whereas the 7 S fraction was in one

17

ANTILYMPHOCYTE SERUM

TABLE I11 ASSAYSOF POTENCY: COMPARISON OF 19 S A N D 7 S FRACTIONS O F RABBIT ANTILYMPHOCYTIC SERUM (SURVIVAL OF A-STRAINS K I N ON CBA MALEMICE) Rabbit serum pool

Fraction or fragment

Protein concentration (mg./ml.)

No. Animals tested

MEL'

S.D.

(100) 10 10

(11.6) 21.0 20.6

(1.3) 3.1 3.8

Whole serum Whole serum +2mercaptoethanol 7 s 19 s

10.5 5.8

10 17

22.8 11.8

2.7 1.6

B

Whole serum 7 s 19 S

13.0 5.4

10 10 10

28.0 22.9 11.2

5.6 3.2 1.8

C

Whole serum 19 s 19 S b

-

14.0' 14.0

20 8 7

26.4 12.4 16.0

3.5 2.6 2.1

A

' MEL

= Mean expectation of life. Animals received 0.5 ml. on days +2, $3, +4, +5, +6. Represents 3-5 times concentration over original serum content.

instance as potent as the whole serum (pool A ) and in the other only slightly less effective (pool B ) . Reduction of whole serum with 2-ME under conditions that degraded macroglobulins did not affect potency. Concentration of the 19s fraction to 3 to 5 times the original serum concentration (pool C ) proved ineffective. However, repeated administration at closely spaced intervals of this concentrated fraction did produce a feeble but definite effect. We shall return to the significance of this last observation. Since the 7 S fraction could be shown to contain IgA as well as IgG, the relative contributions of these two components were studied by preparing pure IgG fractions after chromatography on DEAE-cellulose columns. Immunoglobulin G, eluted from two pools of rabbit ALS and one batch of horse ALS, was restored to 10 mg./ml. and by immunoelectrophoresis was shown to be free of immunoglobulin A ( IgA) and in the case of horse, immunoglobulin T (IgT), a protein fraction of horse serum. The results of assays employing these preparations (Table IV) establish that the graft-protecting ability is largely if not wholly within the IgG fraction. If the relevant molecules are of IgG specificity, is the intact molecule necessary for the expression of activity or would molecular fragments

18

E. M. LANCE, P. B. MEDAWAR, AND R. N. TAUB

TABLE IV ASSAYS

O F POTENCY: IMMUNOGLOIIULIN

Serum pool

Prot,ein concentration Fraction or fragment (mg./ml.)

D horse

E rabbit adjuvant

5.6 7.2

-

10

43.0

4.7

7

40.2

3.6

10

27.0 25.3

6.1 5.4

10 10

15.8 11.5 10.7

2.7 1.3 0.4

10

23,s

9.4

10

22.3

6.7

6 4

29.2 32.5

5.6 4.4

(3

5

19.0

3.3

6 4.6d

5

16.5

2.1

50%*

Whole serum Whole serum F(ab)z

50% 50% 5

I rabbit

IgG IgG

+ F(abh

5 12

J rabbit

IgG IgG

+ Fab’

+

=

24.8 25.1 10,s

Whole serum

Whole serum F(ab)z Fab’

MEL

S.D.

6

10

H rabbit

MEL.

16 6 7

Whole serum IgG

G rabbit

No. of animals

10

10

F rabbit

G FKZCMICNTS

Whole serum I& Fab’

IgG

a

AND IMMUNOCLOIIULIN

6

6

10 10

1.4

Mean expectation of life.

* I n this assay, 0.25 ml. was given on days + 2

and +5 instead of t,he usual 0.5-ml. dose. c Given as a single injection on day + 4 in the afternoon. Dose of 2.3 mg. Fab’ given A.M. and P.M. on day + 3 and twice in A.M. on day $4.

bearing the antigen-combining sites do as well? This question has been investigated by preparing the F( a b ) z or Fab’ fragments from ALS-IgG through enzymatic digestion and comparing the ability of these products to protect skin allografts with equimolar amounts of the parent IgG (Table IV). None of the fragments prepared from either horse or rabbit ALS-IgG prolonged the survival of allografts over control levels. An additional check on this point was made when either F( ab)? or Fab‘ was given in combination with ALS-IgG to see whether synergy might occur or whether the fragments by binding with potential antigens on the lymphocyte surface would block the action of subsequently administered

ANTILYMPHOCYTE SERUM

19

intact molecules. Neither synergism nor iiiterfcrence was found with the possible exception of experiment J, where massive amounts of Fab’ given prior to ALS-IgG did seem to curtail graft survival. Since the IgM fraction contained antilymphocytic antibodies and the IgG antibody fragments contained the antigen-binding sites, why were these fractions and fragments ineffective in vivo? In the hope of identifying those properties essential to its action, an extensive comparison of the in vitro and in vivo properties of ALS fractions and fragments was undertaken (Lance, 1968d) to learn in what ways the intact ALS-IgG molecule differed from either intact ALS-IgM or -1gG fragments. These results are summarized in Table V. Alterations in morphology and function which have been attributed to the use of whole ALS are duplicated by the use of the isolated IgG fraction. In all test systems in which the interaction of antibody and cells is made to occur in vitru, the 1 9 s fraction is as effective as the 7s or IgG fraction. In the presence of complement, equal cytotoxic titers are developed. Exposure in the absence of complement induces equal alteration of the ability of lymphoid cells to migrate to lymphoid organs or to carry out immunological transactions. This is clearly a function of immune sera, as the corresponding fractions of normal rabbit sera do not lead to these changes. These findings leave no doubt that within the 1 9 s fraction of ALS, antibodies directed against lymphocytes exist in high titer. However, when the interaction between cells and antibody occurs in vivo, there are widely divergent results. The implication of these findings is that in vivo the access of 1 9 s antibody to these cells is restricted. A relatively simple explanation which may account for this discrepancy takes cognizance of the rapid elimination of 19 S antibodies from the body when contrasted with those of 7s size (half-life in the circulation for 19 S is 2-2.5 days, whereas that for 7 S is 5.6-7.0days). Thus at any given time after administration, 1 9 s antibodies are present in the circulation in lower concentration than the corresponding 7 S antibodies and are, therefore, at a relative disadvantage. Implicit in this argument is the assumption that apart from differences in clearance and tissue penetration the effect of the two fractions is similar. In vitro and some in vivo findings support this line of reasoning. The differences between 7 S and 19 S fractions are predominantly in magnitude and duration, i.e., quantitative rather than qualitative, which could be explained by the shorter biological half-life and, therefore, the shorter interval during which biologically effective concentrations of 19 S antibodics would be maintained in the recipient. If this explanation is true then it should be possible to duplicate the effects of ALS by repeated and frequent injections of 19 S antibodies, an

20

E. M. LANCE, P. B. MEDAWAR, AND R. N. TAUB

TABLE SURVEYOF BIOLOGICAL ACTIVITIESOF Effect on lymphocyte migration: treat,ment Material"

Immunosuppression

Cytotoxicity

Normal rabbit

Nil

Trace

serum WholeALS ALS 7 S

++++ ++++

1/10,000 1/5,000

ALS 19 S

Trace

1/5,000

ALS-IgG

++++

1/10,000

Nil Nil

1/500 Nil

ALS F(ab)*

ALS Fab'

In vitro

Cell recipients

Cell donor

Lymphopenia

Nil

Nil

Nil

Nil

++++ ++++ ++++ ++++ ++++ ++++ ++++ ++++ Sf++ 4Trace ++

++++ ++++ ++++ ++++ Nil ++ + Trace

Trace

Nil

Nil

Nil

~~

ALS, antilymphocytic serum; IgG, immunoglobulin G.

* GVH, graft versus host.

assumption confirmed by the results shown in Table 111, serum pool C. To insure the absence of 7 S contaminants this particular preparation had been passed through Sephadex G-150 twice. The total amount of protein given to each animal represented the 1 9 s content of approximately 10 ml. of whole ALS, i.e., 10 times as much as is given in the ordinary assay procedure. These results in Table I11 received support from the observations of Mandel and Asofsky (1968a,b) that synergism could be demonstrated between IgM and IgG fractions and also from the finding of Anderson et al. (1967, 1968) of a slight ability of 19 S ALS antibody to prolong skin allografts in rats. The biological properties of the enzymatically derived F( ab) and Fab' fragments of ALS IgG have been extensively studied with agreement that these fragments have no effect on the rejection of skin allografts (Anderson et aZ., 1967, 1968; Woodruff et al., 1967a; Riethmuller, 1.967a; Lance, 1967, 1968d). Loss of the Fc portion of the molecule is believed responsible for this lack of effect. The Fc portion of the niolecule mediates many diverse functions which include complement fixation and ability to cross the placental barrier and also determines the rate of

21

ANTILYMPHOCYTE SERUM

V ANTILYMPHOCYTIC SERUM FRACTIONS AND GVH reaction treatmentb

Zn vitro Nil

Cell donor Nil

FR.4GMlcNTS

Lymphocyte depletion histopathology Lymph node Nil

Spleen Nil

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

++++ ++++ ++++ ++++ Nil Nil ++++ Nil

Nil

Nil

Nil

Nil

Half-life in blood NRS-IgG 6-7 days -

Dist,ribution in vivo Parallele the blood distribution curve

-

ParalleLs the blood 6-7Daysin distribution curve tolerant recipient By 24 hours the percent2-2.5 Days age found in lymphoid organs was 36 that of the 7 S fraction. By 48 hours the distribution approximates background. 6-7Daysin tolerant recipient Less t,han 6 hours Less t'han 6 hours

excretion of the intact molecule (Porter, 1963). It has been assumed that the loss of ability to fix complement accounts for the failure of the fragments to duplicate the activities of intact ALS-IgG (James, 1967a,b). Woodruff and his colleagues have studied the fragments derived from horse and rabbit ALS (Woodruff et al., 1967a; James, 1967a,b) and have concluded that these fragments could not fix complement. The findings of Lance did not entirely support this contention, since rabbit F ( a b ) , was strongly cytotoxic in the presence of both guinea pig and rabbit complement although notably less efficient than the intact molecule. Schur and Becker ( 1963) also reported that immune rabbit F( ab), was 40% as efficient as the intact IgG molecule in complement fixation. A parallel exists concerning F( ab), and the 19 S fractions; namely, the discrepancy in results depends on whether the interaction with cells occurs in uiuo or in uitro. The clearance studies of the F( ab), fragment establish that the half-life of this material is extremely short (circa 6 hours). Spiegelberg and Weigle ( 1965) have shown the extreme rapidity with which fragments devoid of the Fc piece are eliminated from the body. Therefore, the F( ab), fragment is less effective than the parent

22

E. M. LANCE, P. B. MEDAWAR, AND R. N. TAUB

molecule for two reasons. First because it has a reduced ability to bind complement and, second, because it is eliminated from recipients SO rapidly, Both these deficiencies are attributable to destruction of the Fc portion of the molecule. The Fab’ fragment is totally devoid of activity and is absolutely incapable of causing complement fixation. In light of this interpretation, the seemingly contradictory findings of Guttman and his colleagues ( 1 9 6 7 ~ )become understandable. They have shown in a model system of organ transplantation that the F( ab)? fragment of ALS-IgG was able to protect renal allografts to some extent. The F( a b ) r fragment was however much less efficient than the parent IgG molecule and accordingly much larger quantities of the fragment were needed to achieve an effect ( Guttman et al., 1968). Our studies shed no light on their claim that the Fab’ fragment was as effective as the F( ab)? derivative, and, indeed, we remain dubious on this point. The administration of large quantities of Fab‘ seemed to interfere with rather than augment the action of ALS-IgG. This may have occurred because antigenic determinants were covered by an inactive fragment thereby protecting such cells from the action of the intact molecule. From the above the logical conclusion seems to be that raw ALS should be purified by the extraction of the IgG fraction, retaining the whole molecule for use. Advances in techniques for extracting IgG from raw serum should facilitate acceptance of this principle (Moberg et al., 1969; Perper et al., 1!367; Najarian et al., 1970a,b,c; Brummelhuis and Krijnen, 1970). The small amount of activity lost by discarding the IgM fraction is compensated for by being rid of immunogenic foreign proteins to which it would be difficult to induce tolerance (for significance, see below). Moreover there is the chance that, by discarding the IgM fraction, a fair amount of undesirable antibody, e.g., hemagglutinin is removed as well. Woodruff (1967a,b) noted that a large proportion of the antierythrocyte antibodies were contained in the 19 S fraction, a finding not entirely unquestioned (Iwasaki et al., 1967). Horse serum contains a protein fraction, IgT which is excluded in the preparation of the IgG. Although N. Klinman and E. M. Lance (unpublished data) could find no immunosuppressive activity in highly purified horse IgT prepared from two-pulse horse antimouse ALS, others have found antilymphocytic antibodies in this fraction (Funck, 1900; Starzl et al., 1970a,b). Kashiwagi et al. (1970) reported that ALS activity remained confined to the IgG fractions of rabbit and goat antisera but that many of the leukoagglutinins were found in the IgT fraction of hyperimmune horse serum. The possibility cannot be discounted, especially in hyperimmune horse ALS, that activity would be lost if the IgT fraction were discarded. More recent work suggests that the relevant IgG antibodies may be

ANTILYMPHOCYTE SERUM

23

restricted to an IgG subclass, and Perper et al. (1971) have found that IgG subclass antibodies may be actually antagonistic with respect to potency. Therefore, it may become desirnblc to recommcnd a further step in the purification of ALS.

c. ANTIBODY

ELUATES

Although the antibodies responsible in vivo for the in~munosuippressive action of ALS are of IgG specificity, only a small fraction of the total IgG fraction is effectively antilymphocytic. Lance ( 1967) and Woodruff et al. (196%) have estimated, after absorption studies of radioactive ALS-IgG, that the relevant antibodies comprise 15%of the total serum IgG. Woodruff (1968) purified ALS-IgG by acid elution from human lymphoid cell membranes. He tested the resulting preparation in vitro and found that the ability to bind with, agglutinate, and stimulate lymphocytes had been retained but that the cytotoxic properties were greatly reduced. Horse ALS-IgG was used and the acid elution step was carried out at 37°C. Lance (1969) purified rabbit 7 S ALS by absorption onto mouse thymocyte membranes and elution at pH 3.0 at 4°C. Although some denaturation was noted, the eluate retained in large part its biological activity and consisted of a relatively homogeneous solution of IgG molecules. Approximately 2.0% of the original IgG was recovered which could recombine with lymphocytes to the extent of about 75%. Gram for gram the eluate was 10 times as effective in killing lymphocytes in vitro and 50 times as effective in prolonging the survival of skin allografts as the parent IgG preparation. Moreover, the biological halflife of acid-treated molecules did not differ substantially from that of native IgG. This eluate, which accurately reflected the potency of whole ALS, was extremely valuable for studying the fates of relevant molecules in vivo but was not considered to have any practical application for clinical use. The tremendous effort required to obtain such eluates, especially in quantity, as well as the unavoidable denaturation more than offset the potential advantage of discarding the majority of irrelevant IgG molecules. This view may have to be modified since Wilson et al. (1971) have recently described rapid and efficient purification of antilymphocytic antibodies on an immunoadsorbent column. The methods available to extract and purify the IgG fraction from crude ALS have been summarized in detail by James (1968) and will not be elaborated here. Suffice it to say that by a variety of differential precipitations, by chromatographic techniques, or by selective electrophoresis, pure preparations of IgG may be obtained. The advantages of this purification step include discarding the great bulk of irrelevant and immunogenic serum proteins, eliminating some potentially noxious

24

E. M. LANCE, P. B. MEDAWAR, AND

n.

N. TAUB

antibodies with reduction in the requirement for absorption, and using an IgG fraction to which it is relatively easy to induce a state of immunological tolerance. These at the moment would seem to outweigh the disadvantages, namely, the time and cost of such procedures, the inevitable though small amount of denaturation, the possibility of introducing contaminants, and the possibility of discarding some immunoglobulins that may be relevant to potency. V. Assays of Potency

One of the most pressing problems for those who would apply ALS clinically is the search for a suitable and reliable assay of potency.

A. InVivo Medawar and his colleagues routinely assay mouse ALS by its ability to prolong the survival of skin allografts in a standardized system (Jooste et al., 1968). The advantage of this assay is that it measures directly the property in which we are most interested. Monaco et al. (1967b) have shown that antihuman ALS can prolong the survival of skin allografts in man, and Najarian and co-workers (1970a,b,c) have carried out careful dose-response evaluations in a similar system. However, it is difficult if not impossible to see how such tests could come to be applied as a matter of routine. In the search for a substitute, the potency of antihuman ALS has been appraised in surrogate hosts. Standardized doses have been administered to subhuman primates bearing skin allografts (Lance, 1968c; Balner et al., 1968b, 1969a,b,c; Lance and Medawar, 1970b; Bonneau et al., 1970; Darrow et al., 1971; Barnes et al., 1971). Although this procedure conflicts with the basic principle that ALS is species-specific, a good deal of cross-reactivity has been noted between the leukocyte antigens of man and other primates, especially the chimpanzee (Balner et al., 1967). Balner and colleagues (1969b) have shown that the chiinpanzee offers a more sensitive test system than do lower primates. Sera that are without demonstrable effect in the latter may prove to be effective in the former. However, the expense and limited availability of chimpanzees exclude the widespread use of this species for ALS assay. Lance and Medawar (1970b) suggested that the relative insensitivity of the monkey assay might not necessarily be a disadvantage, since in this way only the more potent antisera might be selected. This assumption has been widely accepted, and Balner (1969, 1970a,b) has adopted the monkey assay as a routine screening procedure for antihuman ALS. They have tested many antisera produced in different laboratories and

ANTILYMPHOCYTE SERUM

25

have shown that this assay system can give important information with respect to potential toxicity (Balner et al., 1970) and can also be used to rank antisera in order of potency. Yet no confirmation exists at present to allow direct reliable cxtrapolation. Indeed, it is slightly disquieting that antisera tested and found effective in a hunlan skin allograft system by Simmons et a/. (1971) were not considered very potent when screened by Balner in the monkey assay. Although it is agreed that intraspecies allograft assays constitute the most direct and, indeed, the definitive assay of potency, it is also true that the advent of a reliable replacement would be of great value. A variety of alternatives have been proposed and these will be considered briefly. The lymphopenia produced by injection of ALS has been used to assay potency (Jeejeebhoy, 1965a; Denman and Frenkel, 1968a). However, most reports have tended to emphasize the lack of correlation with allograft protection ( Jeejeebhoy and Vela-Martinez, 1968; Balner et al., 1968a,b; Starzl et al., 1967b). Treatment of lymphocytes in vitro or pretreatment of lymphocyte donors with ALS has been shown to reduce drastically the subsequent performance of these cells in a graftversus-host ( GVH) test (Levey and Medawar, 1967a; Ledney and van Bekkum, 1968; van der Werf et al., 1968; Mandel and Asofsky, 1968a,b; Brent et al., 1967, 1968),On this basis a potential assay can be formulated (Saleh et al., 1969a,b). Levey and Medawar (1966a) found that a relatively small dose of ALS inoculum could suppress the lymphocyte transfer response (Brent and Medawar, 1966) in guinea pigs and, therefore, could be used to assay anti-guinea-pig ALS. However this system takes advantage of the fact that inbred lines of guinea pigs are available and the intended recipient can be heavily irradiated. These requirements cannot be met or easily circumvented in outbred large mammals or in man. In mice, ALS given just prior to an injection of syngeneic lymph node lymphocytes that were labeled with radioactive chromium caused a marked alteration in the migration of these cells (Taub and Lance, 1968a). They were prevented from homing to lymphoid tissue, and the bulk of the radioactivity was recovered in the liver. It has been proposed that this test could be adapted in man (Lance, 1968c), and Martin (1969a,b,c,d) reported that human lymphocytes could be assayed by a variation of this test in mice. He showed that such cells exposed to ALS in vitro did not home to the spleens of recipients in normal numbers but were diverted to the liver. He proposed that this assay measured opsonizing antibody and found a correlation with the protection of skin allografts in mice (see also Lanioreux et al., 1970). If the problems inherent in what is essentially a xenogeneic migration assay such as occurs in the

26

E. M. LANCE, P. B. MEDAWAR, AND R. N. TAUB

presence of preformed heteroantibodies can be overcome, this might well be a promising test system. B. In Vitro There is no disagreement that a reliable in uitro test which could be rapidly and inexpensively performed with no risk to life or health would provide the best solution to the problem. Lymphocyte agglutination is used by some as the definitive assay (Iwasaki et al., 1967) but is not entirely satisfactory. For instance, avian ALS as well as F( ab) and 19 S antibodies agglutinate lymphocytes but are ineffective in uiuo ( Riethmuller, 1967a,b; Woodruff et al., 1967a,b,c; Jooste et ah, 1968; Mandel and Asofsky, 1968a,b; Lance 1968c; Naysmith and James, 1968). Tests based upon the measurement of lymphocyte activation are subject to the same criticism (Holt et al., 1966; Humphrey et al., 1967). There is no confirmation that this property is related to the in uiuo immunosuppression achieved by ALS, and, furthermore, the test is nonspecific in the sense that antiallotypic antibody (Gell and Sell, 1965; Sell and Gell, 1965) and ALS F( ab)z can activate lymphocytes strongly but do not produce immunosuppression (Monaco et al., 1966b; Levey and Medawar, 1966a; Anderson et al., 1967, 1968). Recently inhibition by ALS of antigen-induced blast transformation has been used ( Eijvoogel et al., 1970) and found to correlate with in uiuo measures of potency. The cytotoxic test has thus far not been an accurate predictive tool (Bach et al., 1967). Whereas all sera tested that are active in uiuo have been cytotoxic in uitro [see Govallo and Kosmiadi (1968) for a striking exception] the converse is not always true (Jooste et al., 1968; M. Ruszkiewicz, personal communication). Some of the possible sources of error may be enumerated. Tests on whole sera are misleading because the 19 S antibodies contribute to the in uitro titer out of proportion to their action in uiuo (James and Medawar, 1967; Lance, 1967, 1968c; Mandel and Asofsky, 1968a,b). Moreover, the IgG fraction may contain antibodies directed against irrelevant antigens. Antibodies against H-2 antigens are cytotoxic in uitro (Reif, 1963; Wigzell, 1965) but, when injected into the whole animal, are ineffective presumably because the great bulk of tissue, other than lymphocytes, which possesses these antigens absorbs them (Garver and Cole, 1961). Another source of error may be introduced when complement other than that of the intended recipient species is used in the titration (Cushing, 1945; Rice, 1950; Lance, 1968c; Jooste et al., 1968). It remains to be seen whether only IgG that has been fully absorbed and tested against the lymphocytes and complement of the intended recipient would enhance the value of the cytotoxic test. Studies specifically devoted to a search for a reliable in vitro assay have failed

ANTILYMPHOCYTE SERUM

27

to correlate significantly the various tests enumerated above with the promotion of allograft survival (Bach et al., 1967; Antoine et d.,1968; Wood and Vriesdorp, 1969; Jeejeebhoy and Vela-Martinez, 1968). Two relatively new test systems appear promising. The rosette inhibition test introduced by Bach and Antoine (1968) appears to measure a function that correlates reasonably well with skin allograft assays. Bach (1970) has introduced evidence to show that rosette-forming cells are thymus-derived lymphocytes. The correlation, although not exact, has been well worked out in murine systems (Bach et al., 1969a,b; Mosedale et al., 1970). Tentatively, results of surrogate skin allograft assays in monkeys and the rosette inhibition test appear to correspond in evaluations of antihuman ALS (Bach, 1970; J. F. Bach et al., 1970; Levey et al., 1970; Bach and Dormont, 1971). As currently performed the test employs whole unabsorbed antiserum and is subject to many of the same criticisms that were directed against the cytotoxic test. Perhaps the reliability of this assay could be enhanced by attention to these points. The opsonization tests performed in uitro by Greaves et al. (1969) or in uiuo by Martin (1969a,b; Martin and Miller, 1969) are appealing because they measure a function that is believed to be important in the mechanism of action of ALS (see below). The information at present available regarding its accuracy as a predictor of in uiuo potency (Clayman et al., 1969; Roitt et al., 1970; M. K. Bach et al., 1970a,b; Svehag and Manhem, 1970) suggests a relationship to graft survival. For the sake of completeness, we shall mention inhibition of macrophage migration ( Southworth et al., 1970), inhibition of blast transformation induced by contact of sensitized cells with antigen in uitro (Greaves et al., 1967), inhibition of the mixed lymphocyte reaction ( Brochier and Revillard, 1971), and promotion of allogeneic tumor growth (Deodhar et al., 1968) among test for potency of ALS. None of these possess obvious advantages over methods already available. In summation, no present in uitro test can replace the intraspecies allograft assay to predict in uiuo potency of ALS. The best alternatives appear to be a combination of the surrogate host assay with either the rosette inhibition or opsinization test or both. VI. Effect on Lymphoid Cells or Tissue

A. In Vitro Components of ALS coat lymphocytes ( Denman and Frenkel, 1968a,b) as demonstrated by immunofluorescence ( Levey and Medawar, 1966a; Woodruff et al., 1967d; Russell and Monaco, 1967), radioautography (Lance, 1969), and uptake of radiolabeled antibody (Woodruff

28

E. M. LANCE, P. B. MEDAWAR, AND R. N. TAUB

et al., 1 9 6 7 ~ ).A consequence of this binding is strong agglutination (Gray et al., 1964, 1966; Abaza et al., 1966; Abaza and Woodruff, 1966). Although high concentrations of antibody alone may prove cytotoxic to lymphocytes (Humphrey et al., 1967; Mosedale et al., 1968), this effect is usually not manifest until complement is added (Pappenheimer, 1917; Reif, 1963; Abaza and Woodruff, 1966; Gray et al., 1966; Amiel, 1969). Bitensky (1963) has shown that cytotoxic antibodies do not penetrate the cell in the absence of complement but react with cell surface antigens. Induced distortions of the cell surfaces have been studied by Clarke et al. (1970). Addition of complement causes blebs to form on the cell surface followed by cytoplasmic swelling and nuclear changes. There is no reason to think that ALS achieves its cytotoxic action on lymphocytes in a way different from that of other cytotoxic cell systems (Humphrey and Dourmashkin, 1965; Dumonde et al., 1965). Under suitable conditions, ALS can bring about blast transformation of lymphocytes (Grasbeck et al., 1963, 1964; Sell et al., 1965; Holt et al., 1966; Bach and Bach, 1970) or thymocytes (La Via et al., 1968; Claman and Brunstetter, 1968).This property is presumably related to the ability to bind to cell surface receptors and, perhaps, inflict microinjury, but the whole mechanism of cell transformation is at present poorly understood. The magnitude of this effect compares well with mitogens such as phytohemagglutinin ( PHA) . Antilymphocytic serum may either interfere with or enhance the mitogenic effect achieved in mixed lymphocyte reactions (Greaves et al., 1967; Mosedale et al., 1968; Revillard and Brochier, 1970) or by PHA (Ling et al., 1967). Moreover activation by ALS is sometimes more effective in the presence of small amounts of complement ( Ling et al., 1967). Treatment with ALS in vitro interferes with the subsequent immunological performance of lymphoid cells. This is true both when the function is assayed in vivo, i.e., the ability to cause GVH disease (van der Werf et al., 1967, 1968; Brent et al., 1968; Field and Gibbs, 1968; Ledney and van Bekkum, lW), to transfer immunity adoptively (James, 1968) or to home to the lymphoid tissues of syngeneic hosts (Martin and Miller, 1967; Taub and Lance, 1968a) and in vitro, i.e., mixed lymphocyte reactions (Greaves et al., 1967) or response to antigen by ribonucleic acid ( RNA) and deoxyribonucleic acid (DNA) synthesis (Greaves et al., 1967). Antilymphocytic serum can stimulate or interfere with the inhibitory effect that lymphoid cells exert on target cells in culture, possibly by coating recognition sites (Holm and Perlmann, 1969a,b, 1970; Lundgren and Moller, 1969, Lundgren, 1969a,b; Lundgren et al., 1968). Changes in electrical charge and cell metabolism after ALS treatment in vitro have been reported (Bert et al., 1970; Averdunk and Kirstaedter,

ANTILYMPHOCYTE SERUM

29

1969; Phondke et al., 1970). Brent and colleagues (1967, 1968) have provided powerful support for the coating notion and its reversibility by showing that competence can be restored to ALS-treated cells by removal of the protein coat with trypsin prior to testing. Studies with ALS serum fractions showed that the major portion of the lymphocyte-transforming ability was located within the IgG class of antibodies (James et al., 1969a,b; James, 1968). The F( ab)z but not the Fab’ derivative was also active in this system. The species specificity of ALS has been repeatedly documented (Cruickshank, 1941; Abaza and Woodruff, 1966; Gray et al., 1964, 1966), yet there is no question that some cross-reactivity, generally of a low order of magnitude, may occur ( Anigstein et al., 1965;Carraz et al., 1967; Iwasaki et al., 1967; Caspary et al., 1971). Cross-reactivity may be greater for leukemic lymphocytes than for normal cells (Schrek et al., 1969; Schrek and Preston, 1967). Crude unabsorbed ALS shows considerable cross-reactivity with tissues of nonlymphoid origin. Reactivity in vitro has been documented for erythrocytes and other lymphoid cells (Pichlmayr, 1967; Pichlmayr et al., 1967a,d), platelets ( Starzl et al., 1967b), polymorphonuclear leukocytes (Thorsby, 1967; Schroder and Schroder, 1969), mast cells (Guttman et al., 1967a,c), fibroblasts (Thorsby and Lie, 1968), hematopoetic stem cells (Field and Gibbs, 1968), and macrophages ( Hughes et al., 1971; Huber et al., 1969). These cross-reactions are probably to a large extent a reflection of antibodies directed toward histocompatability antigens and may be removed by absorption with nonlymphoid tissue without altering the immunosuppressive or lymphocyte cytotoxic properties of such antisera (Gray et al., 1966; Levey and Medawar, 1966a; McKenzie et al., 1969). Unabsorbed ALS forms precipitin lines with the saline extracts of many different nonlymphoid tissues (Sachs et al., 1964; Gray et al., 1966; Lawson et al., 1967). Some of this cross-reactivity may reflect the presence of serum proteins in these extracts, and absorption with serum proteins has been recommended on a routine basis (Lawson et al., 1967; Iwasaki et al., 1967). Perper et aZ. ( 1970a,b) have studied the tissue specificity of an antiserum raised to cultured lymphoblasts and found that there was a reduction in lymphocyte-specificantibodies and increased cross-reactivity with other tissues. These results raise serious questions about the use of cultured cells that may undergo dedifferentiation.

B. In Vivo Injection of ALS into intact animals causes prompt and profound lymphopenia (Gray et al., 1964, 1966; Iwasaki et al., 1967; Denman et al., 1968a,b; Twb, 1968a; Taub and Lance, 1968b). This is associated with

30

E. M.

LANCE, P. B. MEDAWAR,

AND R. N. TAUB

a drop in the lymphocyte content of the thoracic duct lymph (Agnew, 1968; Tyler et al., 1969; Lance, 1970c), although Martin and Miller (1967) could not demonstrate this. The kinetics of this response are to some extent conditioned by the route of injection (Besredka, 1900; Sachs et al., 1964); the effect develops more rapidly after intravenous inoculation than after injection through other parenteral routes. The rate of recovery of lymphocytes in the peripheral blood has been variously reported as relatively rapid (Cruikshank, 1941; Nagaya and Sieker, 1966; Levey and Medawar, 1966a; Iwasaki et al., 1967) or rather prolonged with full recovery requiring 3 4 weeks (Mandel and Asofsky, 1968a, Taub, 1968a; Taub and Lance, 1968b). There is also some conflict of opinion on the effect of repeated doses of ALS on the peripheral lymphocyte count. Some reports emphasize the persistence of low levels of lymphocytes with closely spaced injections (Pichlmayr, 1966; Monaco et al., 1966a,b,c; Pichlmayr et al., 1967a), whereas others have found a pharmacological tolerance with progressive rise of blood lymphocytes despite continued treatment ( Besredka, 1900; Nagaya and Sieker, 1966; Carraz et al., 1967; Starzl et al., 1967~).To a large extent these opposing reports may be reconciled by taking into account the host immunological response to the heterologous antibodies ( Lance, 1968a). There is general agreement that the level of lymphopenia does not correlate well with the alteration of immunological responsiveness ( Russe et al., 1965; Abaza et al., 1966; Starzl et al., 196713; Balner et al., 1968a,b; Jeejeebhoy and Vela-Martinez, 1968; Jeejeebhoy, 1965a,b; Levey and Medawar, 1966b; Woodruff and Anderson, 1963) raising the question whether or not the regenerant population of lymphocytes found in the blood after ALS treatment is qualitatively different from that found in untreated animals (Woodruff, 1967a; Lance, 1968a; Boak et al., 1968a,b, 1969). Denman et al. (1968b) hsave shown that when the number of blood lymphocytes has returned to normal or near normal levels the capacity to transact immune responses is still reduced. Tursi et al. (1969) have shown that lymphocytes from ALS-treated mice responded poorly to PHA, and furthermore recovery of immune competence could be monitored by following responsiveness to PHA of peripheral blood cells. The regenerant cell populations found after ALS treatment synthesize DNA to a far greater extent than do normal cells (Nagaya and Sieker, 1967; Denman and Frenkel, 1968a,b; Taub, 1968,a,b, 1970a,b; Denman et al., 1968a,b), implying either that they belong to a rapidly turning over pool of cells or are being formed in large numbers and, hence, are very young cells. The studies of lymphocyte kinetics in ALS-treated animals can only be understood in light of knowledge of the heterogeneity of lymphocyte populations and their kinetics. It has been estimated that the tissue

ANTILYMPHOCYTE SERUM

31

reservoir of small lymphocytes is 40 times that ordinarily found in blood (Cronkite et al., 1!264), and, therefore, the number of cells in the circulation at any given point in time may not reflect accurately the total availability of these cells in the entire animal. Moreover, there are at least two types of lymphoid cells in the peripheral blood, and these have markedly different life-spans (Everett et al., 1964). The kinetics and function of the short-lived peripheral blood lymphocyte has not as yet been fully characterized, but there is good evidcnce that after ALS treatment this population expands ( Denman et al., 1968a,b) at the expense of the long-lived lymphocytes ( Denman and Frenkel, 1968a,b; Denman et aZ., 1968a,b; Taub, 1969).

I . Alterations in Lymphoid Tissue The recorded alterations in lymphoid tissue cover the entire range of possibilities from gross hypertrophy and hyperplasia (Chew and Lawrence, 1937; Levey and Medawar, 1966a) to virtually complete destruction (Monaco et al., 1965a,b, 1966a,c; Gray et al., 1966; Lawson et al., 1967). The variation is due in large measure to differences in experimental conditions, i.e., the time of observation (Bunting, 1903), the use of antisera prepared by a variety of measures (Taub and Lance, 1968b), and the lack of systematic observation. However, difficulty also arises because whole serum contains many components that produce characteristic but varying effects. Taub and Lance have proposed a classification of morphological changes (Taub and Lance, 196Sb) and within this framework the lesions that arise may be attributed to the following factors. a. Serum proteins, which are irrelevant to the action of ALS but which are immunogenic, produce lesions after administration of both immune and nonimmune serum falling under the general heading of serum sickness, and include germinal center formation, medullary hyperplasia of the lymph nodes, analogous changes in the spleen, and the late development of arteritis and glomerulonephritis ( Iwasaki et al., 1967; Guttman et al., 1967b,c; Lance, 1968a; Balner et al., 1968a). b. “Irrelevant” antibodies may produce effects by direct action, e.g., immune hemolysis of erythrocytes leading, in turn, to marrow hyperplasia and foci of extramedullary hematopoiesis ( Flexner, 1902; Lance, ,1968a) or by a secondary effect, e.g., thymic atrophy as a consequence of stress (Taub and Lance, 1968b). c. The fraction of ALS responsible for immunosuppression causes a triad of findings, which seem to be an invariable and necessary concomitant to the injection of potent antisera. Within lymph nodes the characteristic sign is selective depletion of small lymphocytes from the

32

E. M. LANCE, P. B. MEDAWAR, AND R . N. TAUB

thymus-dependent or paracortical area (Parrott, 1967; Turk and Willoughby, 1967; Lance, 1968a; Simpson and Nehlsen, 1971a,b). The medulla, true cortex, and germinal centers are initially unaffected. In the spleen the characteristic lesion is a depletion of small lymphocytes from the peiiarteriolar area of the Malphigian bodies (Lance, 1968a; Taub and Lance, 196813). The third significant finding is a negative one, namely, the absence of significant alteration in the thymus even after prolonged treatment (Russe and Crowle, 1965; Iwasaki et al., 1967; Parrott, 1967; Lance, 196th). It should be pointed out that the existence of this triad of characteristic effects has not been universally accepted. Some workers have stressed the apparently gross and indiscriminate destruction of lymph node architecture after ALS injection (Monaco et al., 1965a,b, 1966a,c; Lawson et al., 1967; Denman and Frenkel, 1967, 196813). This lesion is associated with sera raised with adjuvants and may be mediated through the stress provoked. Shrinkage of the thymus cortex and atrophy of the thymus have also been reported (Nagaya and Sieker, 1965; Monaco et al., 1965a,b; Denman et al., 1967a). That some reduction in cortical thymocytes may occur cannot be categorically refuted, but the bulk of the evidence is against the necessary occui-rence of any major gross or microscopic change in the thymus. The lymphoid hyperplasia reported even in the earliest studies of ALS, and subsequently documented, may be ascribed to the immune response evoked by the immunogenicity of ALS-IgG (see Everett et al., 1970). These changes are found after “pathognomonic” lesions have developed, and their irrelevance is attested to by the fact that ALS-IgG given to animals preparalyzed to IgG either does not provoke these changes or elicits them to a greatly reduced degree without impairing the immunosuppressive action (Lance, 1967; Taub and Lance, 1968b; RodriguezParadisi et al., 1971). 2. In Vivo Fate of Antilymphocytic Antibodies Nava et al. (1969) found no significant difference in the distribution of lZ5I-labeledALS and normal rabbit globulin in mice. Moore (1959) found no difference in the affinity for lymphoid cells between antibody eluted from the kidney or from lymph nodes. Hintz and Webber (1965) reported that ALS-radiolabeled antibody showed the greatest affinity for the thymus. Significant localization was found in lymph nodes, bone marrow, muscle, and the gastrointestinal ( GI ) tract, whereas uptake in the spleen was paradoxically low. Denman and Frenkel (1968a) found heavy localization of fluorescein-labeled ALS-IgG in lymphoid organs but exclusion from the thymus. To a large extent the significance of these findings is obscured and interpretation hampered by the presence of

ANTILYMPHOCYTE SERUM

33

large amounts of nonantilyinphocytic antibody globulin. To overcome these difficulties, Lance ( 1969) prepared *251-labeled,specific, rabbit, antimouse thymus antibody which had been absorbed onto and subsequently eluted from mouse thymus membrane. This material is rapidly cleared from the bloodstream and eliminated from the body. The bulk of injected material is excreted within 24 hours and over 90%is gone in 48 hours. The plot of disappearance curves from the whole body is closely paralleled by the elimination from the various lymphoid and nonlymphoid organs as well. This finding strongly suggests that the direct or primary action of R single pulse of ALS must be quite evanescent however long its secondary biological effects may last. Both Pichlmayr et al. (19674 and R. N. Taub and M. Ruszkiewicz (personal communication) have followed the disappearance of cytotoxic antibody from the bloodstream after a single dose of ALS and have found that, within a matter of hours, cytotoxic antibody can no longer be detected. Traeger et al. (1970a) have proposed monitoring cytotoxic antibody in recipient serum as a means of judging adequate dose administration. This rapid elimination of antibody is compatible with the potency of such preparations, for when animals are intentionally preimmunized to ALSIgG, the elimination of a subsequent dose of IgG is extremely rapid, often being complete within 48 hours (Lance and Dresser, 1967; Clark et al., 1967). Nonetheless, even under these circumstances, ALS can exert a powerful immunosuppressive action (Lance, 1967; Levey and Medawar, 1966a). The mechanism for the very rapid clearance of active antibody eluate is unlikely to be due to an active immune clearance mechanism on the part of the host because it develops far too rapidly. The period of rapid clearance after injection of ALS-IgG into virgin animals does not begin for about 6 days (Lance and Dresser, 1967). Denaturation was also ruled out by internal controls of the elution procedure. The most likely explanation was felt to be that lymphocytes became coated with antibody in the circulation and these antigen-antibody complexes are rapidly cleared, degraded, and excreted. Humphrey has shown that antigen and antibody are simultaneously degraded after immune clearance of complexes (1965). If the clearance of eluted antibody is due to a passive mechanism, i.e., piggy back on circulating lymphocytes, then depletion of these cells should slow down clearance. E. M. Lance and S. V. Jooste ( unpublished observations) showed that the clearance of labeled eluate became slower (half-life 36 hours) in mice that had been previously treated with ALS. The interpretation is that the smaller number of lymphocytes resulted in a diminished number of binding sites for passive clearance of antibody.

34

E. M. LANCE, P. B. MEDAWAR, AND R. N. TAUB

The distribution of eluted ALS antibody was considerably different from that of nonantibody IgG. The uptake of eluate at 24 hours in lymphoid tissues exceeded that of normal IgG, whereas the converse was true for nonlymphoid tissue. Over the first 48 hours the distribution of normal IgG in the tissues was increasing, while the rate of disappearance of eluate was at a maximum. The elimination of eluate from lymphoid organs appeared to proceed at a more rapid rate and to a more complete degree than that from nonlymphoid organs. Finally the uptake by lymphoid organs of eluate was at all times only a minute fraction of the total injected. Radioautography showed labeled cells in the peripheral blood shortly after injection, In the lymph nodes the heaviest labeling was confined to the paracortex with sparing of the true cortex, germinal centers, and the medulla. The label in the spleen was initially seen around the periphery of the Malphigian follicles but later became diffuse over the white pulp. The thymus was lightly labeled at all times with more label in the cortex than the medulla. [For confirmation of thymic exclusion of label, see also Ranl@vet al. ( 1970) and Denman and Frenkel (1968a,b).] In the kidney, a dense accumulation of label was found in relation to the proximal tubules suggesting the route of excretion, whereas in the liver the label was clearly localized to the cytoplasm of the Kupffer cells and was often associated with phagocytosis of labeled leukocytes. This distribution of label within lymphoid tissues was felt to reflect the pathway of migration of cells in the recirculating pool (Gowans and Knight, 1964). In the thymus, lymph nodes, and spleen, some cells that were coated with antibody were not destroyed. Why this happened remains a matter of speculation. A number of factors may come into play. J. H. Humphrey and R. R. Dourmashkin (personal communication, 1968) have shown that for lysis of erythrocytes by IgG antibody, several thousand molecules must attach to the same cell. Woodruff et al. (1967b) have calculated that each lymphocyte can take up as many as 5.0 X lo6 molecules on its surface, but the exact number required for lysis is not known. Therefore, although lymphocytes in lymphoid organs are exposed to IgG antibody, the concentration may be insufficient for irreversible damage. Another limiting factor may be the local availability of complement. Waksman et al. (1961) documented the fall in circulating complement after ALS. If complement is necessary for the direct destruction of cells (Bitensky, 1963; Dumonde et al., 1965) then a limited availability of complement within lymphoid tissue might explain the relative protection that these cells receive. There is also the possibility that cells in the circulation that are opsonized by antibody are rapidly cleared and destroyed by cells of the reticuloendothelial system (Moller and MBller, 1966; Cajano,

ANTILYMPHOCYTE SERUM

35

1960) but that within lymphoid tissue these cclls are not exposed to phagocytes to the same extent. Finally there is the analogy to the markedly different effects antibody is known to exert against dissociated tumor cells in comparison to solid tissue growths (Gorer and Amos, 1956; Garver and Cole, 1961; Kaliss, 1 9 6 ) .

3. Fates of ALS Afected Lymphoid Cells With the experimental model developed by Bainbridge et al. (1966; Bainbridge and Gowland, 1966), the fates of lymphocytes labeled with chromiumdl, exposed to ALS in vitro, and reintroduced into normal syngeneic recipients were studied by Martin and Miller (1967) and by Taub and Lance (1968a). Instead of the expected distribution of such cells to the lymphoid compartment of the host, ALS-treated cells were rapidly and almost completely taken up by the liver. Their fates were identical to those of cells that had been killed by intentional exposure to heat. The high liver uptake reflects either the outright killing of ALScoated cells after interaction with host complement or opsonization or, most probably, a combination of these two mechanisms. Since exposure in vitro of the heterogeneous population of lymph node cells resulted in a uniform diversion, ALS antibody per se does not discriminate between populations. The fates of labeled cells given to ALS-treated recipients depended on the relative timing between administration of ALS and cells. Antilymphocytic serum, given at the time of or just before the cells, duplicated the effects of in vitro treatment (Taub and Lance, 1968a; Seifert and Brendel, 1969). As the interval between ALS and cell introduction was prolonged the effect became progressively less marked: ALS given to recipients after labeled cell injection was virtually ineffective. In view of the known short half-life of antilymphocytic antibody (see Section VI,B,2), these data suggest that the ALS-mediated cell damage in vivo is largely exerted on circulating cells at a time when they are external to lymphoid compartments. Treatment of cell donors with a short but intensive course of ALS resulted in a residual cell population that migrated differently from normal cells when introduced into untreated syngeneic recipients. This residual population was deficient in cells localizing in recipient lymph ngdes, whereas the spleen-seeking proportion was unaffected. This pattern of altered migration was comparable to that found in neonatally thyinectomized animals, animals thymectomized as adults and subjected to lethal whole-body irradiation, and animals that had been drained chronically through a thoracic duct fistula (Taub and Lance, 1971). These procedures are known to deplete the animal of the population of

36

E. M.LANCE, P. B. MEDAWAR, AND R. N. TAUB

recirculating small lymphocytes, and it is this population of cells that localizes preferentially in recipient lymph nodes (Lance and Taub, 1969; Zatz and Lance, 1971). Alternative possibilities to explain the altered distribution of cells from ALS-treated donors include poor viability of the cells or coating of the cells with antilymphocytic antibody. These are rendered unlikely because such cell suspensions did not show increased staining with trypan blue (Lance, 1968d), migration to the spleen was largely unaffected unlike the behavior of cells coated with ALS in uitro or killed by exposure to heat, no cytotoxicity could be developed by incubation of these cells with rabbit complement, and finally the disturbance in proportional migration, which extended in some cases for more than a month after the last serum injection, long outlasted the metabolic lifetime of ALS antibody in the host (Lance and Cooper, 1970). VII. Scope of Antilymphocytic Serum Action in Vivo

Antilymphocytic serum has been demonstrated to exert powerful immunosuppression in a wide variety of species, and there is no reason to believe that this effect is not an expression of a broad biological phenomenon. The list which is ever growing now extends to mice (Gray et al., 1964), rats (Woodruff and Anderson, 1963), guinea pigs (Waksman et al., 1961)) rabbits (Lance, 1968c), dogs (Abaza et al., 1966), pigs (Lucke et al., 1968), several species of monkeys (Balner and Dersjant, 1967), chimpanzees (Balner et al., 1968a), ,and man (Starzl et al., 1 9 6 7 ~ )There . is, however, one important exception. R. M. Binns and E. Simpson (personal communication) have raised bovine antipig ALS by using a regimen that has been successful against other species (Binns et al., 1970, 1971), but they were unable to demonstrate prolongation of skin allografts in the pig. The peculiar lymphoid anatomy of the pig and the unique response to allografts of liver suggest that lymphoid kinetics may be quite different in this species.

A. EFFECTON INFLAMMATION Some aspects of nonspecific inflammation may be depressed by ALS including those incited by nonspecific irritants (Turk and Polak, 1969; Turk et al., 1968; Waksman et al., 1961) and those that depend on the active production of antigen-antibody complexes ( Turk et al., 1968; Turk and Polak, 1969). On the other hand, in models of passive cutaneous anaphylaxis ( Waksman et al., 1961) and immune complex nephritis (Denman et d.,1966; Taub and Lance, 1968b), no suppression was noted. The extent to which these effects depend on the presence of antilymphocytic antibody or antibody to other cellular mediators of in-

ANTILYMPHOCYTE SERUM

37

flammation or, in the latter, case on the initiation of serum sickness, has not been determined, However, Perper et al. (1969) showed that the anti-inflammatory effect was not mediated by the adrenals and was separable from the immunosuppressive effect.

B. EFFECTON CELL-MEDIATED IMMUNITY 1. Delayed Hypersensitivity Since the time of the report of Inderbitzin (1956), there has been abundant confirmation of the ability of ALS to suppress the dermal manifestations of delayed hypersensitivity ( Waksman et al., 1961; Wilhelm et d., 1958; Russe and Crowle, 1965; Nagaya and Sieker, 1965, 1966; Lance, 1967, 1968; Turk and Willoughby, 1967; Brunstetter and Claman, 1968; Turk et al., 1968). This finding applies as well to primates ( Balner and Dersjant, 1967) including man (Iwasaki et al., 1967; Monaco et al., 1967a). Antilymphocytic serum administered at or just prior to challenge causes a marked reduction in responsiveness. Inderbitzin showed that the release of histamine was reduced and reported reduction in the mononuclear infiltrate at the site of challenge, a finding confirmed by Waksman et al. (1961). Turk and Willoughby (1967) have shown that ALS can block the central lymphoid changes characteristic of these responses. They as well as Waksman et al. pointed out that nonspecific inhibition of inflammation may play a role, but the most likely explanation for these effects is the reduction of potentially reactive cells at the challenge site. Despite this dramatic effect when ALS and challenge are concomitant, animals recover reactivity very rapidly after cessation of ALS treatment. This finding is in many ways analogous to the observations of Levey and Medawar (1966a, 1967a) in their studies of the lymphocyte transfer reaction. The response to challenge corresponds to a peripheral reaction susceptible to the action of ALS. Once treatment ceases, reactivity is restored by the release of competent cells from central lymphocyte stores into the periphery. Abrogation of central reactivity ( immunological memory) in presensitized animals has been extremely difficult to achieve, possibly because persistent antigen (retained for long periods when given in conjunction with Freunds adjuvant) served to reimmunize animals after the lapse of treatment (Russe and Crowle, 1965; Lance, 1967). Antilymphocytic serum can, however, prevent sensitization during the induction of immunity to contact sensitivity (Table VI). Guinea pigs were immunized to dinitrochlorobenzene ( DNCB ) . Two animals received 5 ml. of ALS on the day prior to sensitization, and 2.5 ml. on the first, second, and third days thereafter. A second group received ALS as above, and on the fourth day the sensitization site was surgically removed. The

38

E. M.

LANCE,

P. B.

MEDAWAR,

AND R. N. TAUB

TABLE VI EFFECTOF ANTILYMPHOCYTIC SERUMON INDUCTION OF IMMUNITY TO CONTACT SENSITIVITY I N THE GUINEAPIG

Group

Immunizationa

Serumb

Removal of sensitization site

A B C

DNCB DNCB DNCB

ALS ALS None

No Yes Yes

(I

Response to challenge

+++, ++++ 0, 0 +++, ++

DNCB, dinitrochlorobenxene. ALS, antilymphocytic serum.

third group received no serum but the sensitization site was removed. All animals were challenged 18 days after immunization. The animals receiving ALS in combination with removal of the sensitization site gave no reaction, i.e., they behaved as if unimmunized. The remaining groups showed vigorous reactivity. 2. Transplantation Immunity Antilymphocytic serum is the most powerful experimental agent for overcoming the allograft response. The rejection of both first and second set skin allografts can be prevented (Waksman et al., 1961; Monaco et al., 196!5a,b; Woodruff and Anderson, 1963, 1964; Levey and Medawar, 1966a,b; Brent et al., 1967; Gray et al., 1966; Balner et al., 1968a,b; Balner and Dersjant, 1969; Grabar and Chouroulinkov, 1970). The extent and duration of this effect seems to be limited only by the willingness of the experimenter to continue treatment and the ability of the animal to support the untoward sequelae (Lance, 1968a; Nehlsen, l970,1971a,b). Monaco et al. ( 1 9 6 6 ~ )felt that ALS was more effective if given prior to grafting, but Levey and Medawar (1966a) did not find this critical with respect to skin allografts in as much as ALS could reverse a rejection response which had already been allowed to start (see also Waltman et aZ., 1969, for similar findings with corneal allografts). The effect of ALS in overcoming rejection in presensitized animals (Levey and Medawar,. 1966a) as well as its ability to ablate memory of prior exposure distinguish it from all other immunosuppressive agents (Levey and Medawar, 1966b; Lance, 1968b). The results achieved with skin allografts extend to organ grafts as well, e.g., canine renal and hepatic transplants (Abaza et al., 1966; Monaco et al., 1966c; Starzl et al., 1966, 1967a;b,c; Pichlmayr et al., 1966, 1967a,b, 1968b,c; Atai and Kelly, 1967; Ellis et al., 1967; Lawson et al., 1967; Abbott et al., 1966, 1969; Braf et al., 1969; Shanfield et al., 1968;

ANTILYMPHOCYTE SERUM

39

Clunie et al., 1968), cardiac allografts (Halpern et al., 1969), lung allografts (Iwasaki et al., 1970), transplants of whole joints (Reeves, 1969) or whole limbs (Lance et al., 1971), and renal transplants in rats (Guttman et al., 1967a,b,c,d, 1968). In these systems it seems clear that the best results were achieved when ALS administration began prior to transplantation. A review of reports on organ transplants in large mammals leaves the distinct impression that the effects have not been as dramatic as those achieved with skin grafts in rodents. At present there is no need to invoke any basic biological differences to explain this discrepancy, which may be due to difficulties in working with outbred stock and the proportionally smaller doses of ALS usually employed. Nonetheless, the possibility that other factors are involved, e.g., damage inflicted upon organ grafts by humoral antibodies ( Porter, 1967), cannot be discounted. The degree of genetic disparity between donor and host often critical for other immunosuppressive regimens makes little difference to the effects of ALS. “Easy” allograft combinations are prolonged to about the same extent as difficult transplants across the major histocompatibility loci (Levey and Medawar, 1966b). Nowhere is this principle more obvious than in the survival of xenografts across the widest possible barriers. Xenografts of skin, considered before the advent of ALS an impossible transplant, survive for prolonged periods making the xenograft response amenable to study. Monaco et al. (1966a) were the first to show the acceptance of rat skin on mice treated with ALS. Lance and Medawar (1968) reported prolonged survival of both first-set and secondset xenografts of rat and guinea pig skin on ALS-treated mice, and human skin grafts survived for over 2 months without histological evidence of cellular reaction. Monaco (1970) confirmed the long survival of human adrenal tissue in ALS-treated thymectomized mice. Others have used human fetal transplants (Phillips and Gazet, 1969). In parallel with its effects upon normal tissues, ALS can promote the acceptance and survival of tumor allo- and xenografts ( Anigstein et al., 1965; Phillips and Gazet, 1967; Deodhar et al., 1968; Lewis et al., 1969; Stanbridge and Perkins, 1969; Beverley and Simpson, 1970; Wallace et al., 1971). 3. Graft-versus-Host Response

Numerous reports attest that ALS inhibits this reaction when the cell inoculum is exposed to ALS in vitro (van der Wed et al., 1967, 1968; Ledney and van Bekkum, 1968; Brent et al., 1968; James and Naysmith, 1968; Field and Gibbs, 1968). In mice and rats, pretreatment of cell donors with ALS reduces or ablates the GVH response (Levey and Medawar, 1967a; Ledney and van Bekkum, 1968; Brent et al., 1967, 1968;

40

E. M. LANCE, P. B. MEDAWAR, AND R. N. TAUB

Boak et al., 1967, 1968a,b; Boak and Wilson, 1968; Monaco et al., 196713; Schwartz et al., 1968; von Thierfelder et al., 1970; Thierfelder et al., 1971). These results have their parallel in the finding by Levey and Medawar (1967a) that splenic cells from ALS-treated donors were less well able to bring about the rejection of skin allografts when injected into lethally irradiated, syngeneic recipients than were cells from normal donors. Treatment of recipients with ALS is also effective (Nouza et al., 1971; Boak et al., 1968a,b; Ledney, 1969). The efficacy of donor pretreatment in abolishing GVH reactions does not apply to all species. Balner et al. (1968a,b, Balner, 1969) found this an ineffective approach in monkeys. In his model the best results were obtained by ALS treatment of recipients, but far greater doses were required than those necessary, for instance, to prolong allograft survival (van Bekkum et al., 1967). Difficulty in suppressing GVH reactions in chickens was reported by Tucker ( 1968). Levey and Medawar studied a model of the GVH reaction in guinea pigs ( 1966a, 1967a). Antilymphocytic serum completely abolished all components of the normal and immune lymphocyte transfer reaction. This could be achieved either by brief pretreatment of recipients or by more prolonged and intense treatment of cell donors. Conventional immunosuppressive agents were ineffective in opposing the first phase of this reaction.

4. Infectious Disease One of the most remarkable “clinical” (if that word can be applied to experimental animals) observations is the freedom from infection by common bacterial pathogens in ALS-treated animals. Susceptibility to infections of this genre is not enhanced by ALS treatment, although the acute inflammatory response to deliberate infection with staphylococci and Pseudomonus aeruginosa may be diminished (Morris and Burke, 1967; Grogan, 1969a,b). However, Grogan ( 1969a,b) reported increased mortality in P . ueruginosa-infected ALS-treated rats and resistance to mycobacterial infection ( Gaugas and Rees, 1968), mycoplasma (Allison, 1970), Protozoa (Spira et al., 1970; Barker and Powers, 1971), nematodes, and helminthic parasites is reduced (Doming0 and Warren, 1968; Kassai et al., 1968). Although relatively little attention has been focused on the modification of bacterial infections by ALS, a great deal is known about the effects of ALS on viral disease (Hirsch and Muiphy, 1968a,b). In general the potentiation of viral infection by ALS has been attributed to blocking of cell-mediated immunity, but titers of antiviral antibodies have not been affected. Reduction in interferon production has been reported

ANTILYMPHOCYTE SERUM

41

( Barth et al., 1969a,b). Antilymphocytic serum depresses the primary cellular response to a variety of viral agents: vaccinia (Hirsch et al., 1968; Hirsch et al., 1968), herpes simplex (Nahmias et al., 1968), yellow fever ( Hirsch and Murphy, 1967), mousepox ( Blandin, 1970), lymphocytic choriomeningitis (Gledhill, 1967; Hirsch et al., 1967, 1968; Lundstedt and Volkert, 1967), Rauscher leukemia (Hirsch and Murphy, 1968a,b), Moloney leukemia (Allison and Law, 1968), adenovirus-12 (Allison et al., 1967), and polyoma (Allison and Law, 1968; Gaugas et al., 1969; Nehlsen, 1971a,b). In vitro treatment with ALS of human lymphocytes causing blast transformation increased susceptibility to infection with vesicular stomatitis virus (Edelnian and Wheelock, 1968) and distemper virus (Poste, 1970). A warning that ALS may interfere with preexisting immunity to virus or potentiate latent disease is contained in the findings of Abaza et al. (1966) and van Bekkum et al. (1967). The former report distemper infections in ALS-treated dogs previously immunized with attenuated distemper vaccine, and the latter noted a number of deaths in ALStreated monkeys from presumably latent virus infections. Volkert and Lundstedt ( 1968) found that protracted ALS treatment could provoke latent lymphocytic choriomeningitis virus infection. On the other hand, Hirsch et al. (1968) found no difference between normal rabbit serum and ALS-treated mice which had been previously vaccinated and then challenged with intracerebral vaccinia. The reduction in cellular response to virus induced by ALS may have paradoxical effects. Thus, ALS protected against infection by low doses of yellow fever virus, although this effect became inapparent at high doses of virus (Hirsch and Murphy, 1967). No changes in clinical or morphological effects of influenza infection in mice were noted (Hirsch and Kaye, 1968). However, ALS treatment prevented the lethal effects of lymphocytic choriomeningitis viral infection ( Gledhill, 1967; Hirsch et al., 1967, 1968; Lundstedt and Volkert, 1967)-persisting viremia and high titers of circulating antibody were found, but encephalitis was obtunded or ablated. Clearly the immune response to this virus is of more potential damage than viral infection per se. In view of the above evidence, it is surprising that no one has reported the occurrence in man of viral disease potentiated by ALS therapy.

5. Autoimmune Disease The effect of ALS has been evaluated in a number of experimental models of autoimmunity, and the subject has been thoroughly reviewed by Dennian ( 1969). Allergic encephalomyelitis can be suppressed by

42

E. M. LANCE, P. B. MEDAWAR, AND R. N. T A D

ALS given concurrently with immunization or after several weeks (Waksman et al., 1961; Leibowitz et al., 1968a,b). Even when first treated, advanced neurological findings showed some amelioration, suggesting that established disease could be affected, a result reminiscent of the reversal of skin allograft rejection recorded by Levey and Medawar (1966a). Kalden et al. (1968) reported that ALS given prior to immunization completely inhibited the histological manifestations of experimental thyroiditis with suppression of antithyroglobulin antibodies. The manifestations of adjuvant arthritis were completely suppressed by ALS provided that treatment followed shortly after adjuvant administration. Late treatment was ineffective (Currey and Ziff, 1966, 1968). Similar observations pertain to autologous immune complex nephritis in rats (Barabas et al., 1969). The effects of ALS on the spontaneous autoimmune disease of NZB mice have been extensively studied by Denman and co-workers (1965, 1966, 1967b, 1968b; Denman and Denman, 1970; Holborow and Denman, 1968; Denman and Ziff, 1964). Antilymphocytic serum treatment could significantly retard the development of Coomb's positive hemolytic anemia which could be reinstated by the transfer of splenic cells from older affected mice. Once hemolytic anemia developed, ALS was powerless to oppose it. By contrast, ALS could not prevent the renal disease and appeared to accelerate its development. Immune complexes formed in response to ALS administration itself seemed an unlikely cause for this acceleration, because the same results were noted in animals rendered tolerant to ALS 7 S IgG. On the other hand, enhancement of renal disease in ALS-treated mice could be forestalled if they were reconstituted with allogeneic lymphoid cells. In Denman's view ( 1969), renal disease occurs when a delicate balance between virus and host lymphocytes is upset. Excessive depletion of small lymphocytes by ALS favors renal disease in mice, just as neonatal thymectomy (East et al., 1967). 6. Tumor Immunity

We have already mentioned that ALS treatment favors the take and growth of allogeneic and xenogeneic tumors and enhances the effect of oncogenic viruses. Antilymphocytic serum treatment alone increases susceptibility to tumor induction by adenovirus, polyoma, and Maloney virus (Allison et al., 1967; Allison and Law, 1968; Gaugus and Rees, 1968; Vredevoe and Hays, 1969; Nehlsen, 1971a,b), duplicating in many ways the effects of neonatal thymectomy. Thymectomy prevents the development of Moloney leukemia ( the thymus participates and is required) ;

ANTILYMPHOCYTE SERUM

43

however, thymectomized ALS-treated mice developed reticulum cell sarcoma at the site of ALS inoculation (Allison and Law, 1968). In the case of syngeneic transplantable tumors, B. Fisher et al. (1969) and E. R. Fisher et al. (1969a) have shown that ALS treatment increases take, growth rate, and the incidence of metastasis. An example of this effect is shown in Fig. 3 where a transplantable Balb/c myeloma grew faster and to a larger size in ALS-treated animals (E. M. Lance, previously unpublished observations; Mandel and DeCrosse, 1969). Tumor induction by chemical carcinogens is affected by ALS. Balner

TINT IN DAYS

FIG. 3. Growth curves of a syngencic myeloma (RPC 23) in Balb/c mice. Mice were treated with repeated injections of either antilyinphocyte sernni ( ALS ), antiplasma cell serum ( APCS), or nomial rabbit sernm (control), beginning day - 1 and continued on alternate days nntil day 17. On clay 0, 15 x lo* inyeloma cells were introduced subcntaneously in all animals. The APCS my have produced slight but insignificant inhibition of growth, whereas the growth curve of ALS-treated mice is significantly enhanced.

44

E. M. LANCE, P. R.

MEDAWAR,

AND R. N. T A D

and Dersjant (1969) found a more rapid rate of induction and growth of methylcholanthrene ( MCA) tumors in ALS-treated mice, although the final incidence of tumor was comparable to controls (see also Wagner and Haughton, 1971; Grant and Roe, 1969). Others have reported an increased incidence of MCA tumor induction in ALS-treated animals ( Cerilli and Treat, 1969; Rabbat and Jeejeebhoy, 1970). Nevertheless, Balner (1971) found the incidence of X-ray-induced leukemia to be reduced in animals treated with rabbit but not horse ALS. Nagaya and Sieker (1969a,b) found that ALS treatment did not influence the development of leukemia in AKR mice. C. HUMORAL IMMUNITY Antilymphocytic serum inhibits the primary response to a wide variety of antigens, both soluble and particulate (Gray et al., 1964; Denman et al., 1966; Berenbaum, 1967; Atai and Kelley, 1967; James and Anderson, 1967, 1968; James and Jubb, 1967; Pichlmayr et al., 1967c; Guttman et al., 1968; Barth and Southworth, 1968; Moller and Zukoski, 1968; Reithmuller et al., 1968; Muschel et al., 1968; Lance, 1967, 1970a). Those antigens most closely studied have been Salmonella antigens, bovine serum albumin (BSA), and sheep erythrocytes, and the effect includes not only a reduction in the quantity of antibody but also a delay in the kinetics of the response. In summary, the principal findings of these studies indicate that, although the primary response may be suppressed, the degree of inhibition depends on several factors, i.e., the amounts of antigen and ALS used and the timing between ALS and antigen exposure. The relative timing between ALS and antigen is critical, for ALS given prior to antigen is effective, whereas ALS given with or after antigen is largely ineffective. Increasing doses of ALS exert continuously greater inhibition upon the primary response, but at least for “strong” immunogens, even massive doses of ALS do not completely abort this response. The effect of ALS can be countered to some extent by increasing the dose of antigen. Not all antigens are equally susceptible to ALS. The response to keyhole limpet hemocyanin ( KLH ) or pneumococcal polysacchai-ide may, in fact, be augmented (Bauni et al., 19f39; Taub et al., 1969; Baker et al., 1970; Barth et al., 1971). The IgG component of the primary response is suppressed to a greater extent than is the IgM response (Lance, 1968d; Bauni et al., 1969), a finding reminiscent of the defect in thymectomized, irradiated animals (Taylor et al., 1967). The possibility that some of the effects of ALS are due to interference with macrophage function must be considered (Barth et al., 1969a,b; Marshall and Knight, 1969). Chare and Boak (1970) considered these effects to be a secondary feature of ALS administration.

ANTILYMPHOCYTE SERUM

45

The effect of ALS on the secondary response was far less than that on the primary response. In some cases slight immunosuppression was observed, but in others no suppression was found. It would appear that ALS affects primarily the antigen-sensitive cell. The crucial importance of timing of ALS injection on the primary response and the insensitivity of the secondary response provide the chief evidence for believing that this must be so, for once the chain of events launched by antigen recognition is under way, ALS exerts little or a greatly reduced effect. The studies of Martin and Miller (1968) and Barth ( 1969) strongly support this conclusion. The relative insensitivity to suppression by ALS of the primary response to so-called thymusindependent antigens and the ability to restore competence to thymusdependent antigens by the infusion of thymocytes (Miller and Mitchell, 1968; Leuchars et al., 1968; Martin and Miller, 1968) suggest that the thymus-derived antigen-sensitive cell is the chief target of action, whereas the antibody-producing cell is unaffected. Jeejeebhoy ( 1971) found both cell types to be affected. That the immunosuppressive action of ALS can be overcome by increasing the dose of antigen is siniilar to the effect observed by Sinclair and Elliot (1968) after neonatal thymectomy. If, as Mitchison (1970) proposes, the thymus-derived, antigen-sensitive cell cooperates with the antibody-producing cell by effecting a local concentration of antigen, then a rational explanation of the effect of antigen dosage in these two situations is readily available. Finally, ALS may bc used to abet the induction of tolerance to BSA ( Lance, 1970a).

D. ERASURE OF MEhfORY Levey and Medawar (1966b) reported that mice previously sensitized by skin allografts could be restored to “virgin” reactivity by treatment with ALS, but Russell and Monaco were unable to duplicate these results (1967). Lance studied this phenomenon in animals which had been sensitized to BSA and in others previously grafted with skin across the H-2 barrier (1968b). The dosage and schedule of administration of ALS after immunization were identical in both systems, With respect to BSA, all animals responded to a subsequent challenge dose with titers above those characteristic of a primary response, i.e., in no case had erasure of memory occurred. Although some of the animals treated with ALS gave secondary responses lower than those of normal rabbit serum ( NRS )-treated controls, sensitization was evident since the challenge dose (100 pg.) would not be expected to provoke a detectable response by the technique employed when given to mice de nouo (Mitchison, 1964).

46

E. M. LANCE, P. B. MEDAWAR, AND R. N. TAUB

Animals sensitized to transplantation antigens and treated with ALS thereafter rejected second skin allografts at a tempo characteristic of firstset rejection, i.e., as if they had not seen these antigens before. Memory in this system did not return even when long periods of time were allowed to elapse between ALS treatment and challenge. In a repeat of this experiment with a less potent ALS second-set rejection was modified but not ablated-these animals rejected the challenge allograft at a pace intermediate between first- and second-set rates. These results are important not only because they document an unique immunosuppressive attribute of ALS but also because they hold out hope that in certain clinical situations where immunosuppression is desired, already immune patients may be amenable to treatment. The way in which ALS achieves these effects is speculative, but the following explanation was offered. In skin allograft rejection, the effector cell is a circulating lymphocyte and is highly susceptible, therefore, to the action of ALS. The pace of rejection reflects the number of cells in the recirculating population capable of responding to graft antigens. Immunization increases the proportion of specifically reactive cells; ALS destroys this population, and the regenerant population reflects the original reactivity of the host. In humoral immune systems, the effector cell (or at least a considerable portion) remains sequestered in host lymph node or spleen where it enjoys a measure of protection from ALS treatment and, consequently, a second dose of antigen confronts a still enriched population of cells capable of specific response. VIII. lmmunogenicity of Antilymphocytic Serum Immunoglobulin G

The potential immunogenicity of ALS-IgG has both practical and theoretical implications. The subject has been most thoroughly studied in mice where the lack of immunogenicity of proteins of the IgG class is well-established (Dresser, 1962, 1965). By contrast the highly immunogenic character of ALS-IgG has been repeatedly documented (Lance and Dresser, 1967; Clark et al., 1967; Denman and Frenkel, 1967; Guttman et al., 1967b; James and Anderson, 1968; Iwasaki et al., 1967; Pichlmayr et al., 1968c; Lance and Medawar, 1968; Jasin et al., 1968; Kashiwagi et al., 1968, 1969) after administration to a wide variety of animal species including man. Recent experiments by Amemiya et al. (1972) suggest that immunogenicity may be reduced by treatment with Takaprotease; unfortunately potency was also severely reduced. However, a state of nonreactivity to ALS-IgG can be induced either by prior paralysis with NRS-IgG or by the%administrationover a long time of closely spaced doses of ALS (high zone paralysis) (Lance and Dresser, 1967; Lance and Medawar, 1968; Pichlmayr et al., 1968c;

ANTILYMPHOCYTE SERUM

47

Howard et al., 1968, 1969; Gewurz et al., 1971; Mergciihagen and Howard, 1970; Wood, 1970). The possibility that ALS might prevent reactivity against itself (Monaco et al., 1966c) has not been confirmed by the experience of others (Lance and Dresser, 1967; Currey and Ziff, 1966; Clark et al., 1967). The inimunogenicity of ALS-IgG might be explained in one of two ways: it could act as a nonspecific adjuvant by virtue of the “normal” 7-globulins or the adherence of IgG molecules to lymphocytes drawing the attention of iiiacrophagcs could be the decisive factor favoring immunity in contrast to the paralysis induced by normal (nonadherent) IgG (Howard et nl., 1968). The first possibility can be discounted, because doses of ALS-IgG that induced immunity to normal IgG still caused immunosuppression with respect to other antigens. The immunogenicity of ALS raises the question whether its in viva effects might not be due to antigenic competition (Adler, 1964). However, this possibility is eliminated by the finding that the prior induction of paralysis to IgG did not diminish but rather enhanced the therapeutic effect of a subsequent dose of ALS (Lance, 1967; Denman and Frenkel, 1967; Gaugas and Rees, 1968; Raju and Grogan, 1969). In contrast, either active or passive immunization to IgG curtailed the expression of potency of subsequently administered ALS (Lance, 1967; Judd et al,, 1969; Hardy et al., 1970). Under these circumstances ALS-IgG is rapidly cleared from the circulation ( Lance and Dresser, 1967), suggesting that the maintenance of a titer of circulating antibody is important for the expression of ALS activity and emphasizing the importance of preventing immunization to IgG in ALS recipients. Considering the very rapid rate of elimination of IgG in immunized animals, it is remarkable that under these circumstances ALS should be effective at all (Lance, 1967; Levey and Medawar, 1966a; Raju and Grogan, 1969). The clear implication of this result is that the critical reaction between lymphocyte and antibody can take place extremely rapidly and that prolonged persistence of ALS antibodies is not necessary. This review correlates well with the short biological half-life of the relevant, antilyinphocytic IgG ( ALg ) antibody molecules even in iionimmunized animals (see above). In practice the immunogenicity of ALS-IgG has two undesirable consequences: it creates the hazard of serum sickness and reduces effective immunosuppression. The clinical correlates will be fully developed below. IX. Discriminate Action of Antilymphocytic Serum an Cell-Mediated Immunity

In this section we shall summarize the evidence that ALS has a greater inimunosuppressive effect when directed toward cell-mediated

48

E. M. LANCE, P. B. MEDAWAR, AND R. N. TAUB

immunological reactions (rejection of transplants, delayed skin hypersensitivities, GVH reactions, etc. ) than those mediated by circulating antibodies. In as much as some of the evidence collated here has been presented elsewhere, detailed reference will be made only to new data. In fairness it must be mentioned that not everyone will accept the conclusions in this section (James and Anderson, 1968), although the evidence presented contra is not incompatible with these conclusions. From the outset it was apparent that ALS administration to experimental animals was remarkably free from infectious complications, suggesting that ALS did not interfere with defense mechanisms against common bacterial pathogens (presumably largely mediated by humoral antibodies and nonlymphoid cells) (see Medawar, 1968). However, because of the difficulty inherent in comparing directly two such different processes, it was hard to establish objective criteria of relative effects. Levey and Medawar (1967a) pointed out that the effects of ALS on cell-mediated and humoral mechanisms could be contrasted by reference to an external standard. They stated that a 0.25-ml. dose of ALS led to prolongation of skin allograft survival in mice equivalent to that obtained after 6OOR of whole-body irradiation. This same dose of ALS was virtually without effect on humoral immunity, whereas the suppression of humoral immunity by 600 R was profound. A. SUPPRESSION OF RESPONSES IN VIRGIN ANIMALS Although ALS can inhibit the primary response in both cell-mediated and humoral reactions it is interesting to contrast the requirements in these two types of immune response. Timing is critical for humoral immunities-for ALS to be effective it must be administered prior to or simultaneously with antigen. If ALS is given after the antigen, its suppressive effect is largely lost. On the other hand, one of the distinctive properties of ALS in transplantation systems is that it is capable of inhibiting rejection when first given after the reaction has already begun. A second contrast is that increasing the dose of antigen in humoral antibody systems appears to mitigate the effects of ALS. An analogous situation has not been observed in the suppression of cell-mediated immunities by ALS. Lance and Medawar (1969) have found that skin allografts of sizes over a tenfold range do not influence the tempo of rejection in ALS-treated mice. Perhaps the most striking evidence bearing on this point stems from the observations of responsiveness of mice chronically treated with low doses of ALS (Lance, 1968a). In this experiment, virtually indefinite survival of skin allografts across an H-2 barrier was achieved, while animals maintained normal responsiveness to heterologous serum proteins

ANTILYMPHOCYTE SERUM

49

and to primary immunization with Salmonella typhosa H antigen. Lance and Batchelor (1968) reported a very interesting dissociation of responsiveness in these animals which elaborated circulating antibodies to skin graft isoantigens but, at the same time, had neither gross nor histological evidence of a cellular response to the very same allograft. Beverley and Simpson (1970) found a similar dissociation in ALS-treated mice bearing hamster tumor xenografts. However, Monaco and Franco (1969) did not observe dissociation in their studies, Finally, the humoral response to some antigens (those completely or relatively thymusindependent) may not be suppressed at all, but enhanced; no parallel exists for cell-mediated immune reactions where transplantation reactions over the widest possible ranges are susceptible to suppression.

B. EFFECT m SENSITIZEDANIMALS The effect of ALS on the response to antigen in sensitized animals is even more strikingly divergent for the two types of immunity. Resistance of the secondary humoral response is heavily documented, and the susceptibility of the second-set reaction manifested most vividly in the abrogation of immunological memory is one of the outstanding characteristics of ALS. Lance (1969) studied the discriminative action of ALS by parallel assays of cell-mediated and humoral immune processes in the same animals. The results of one of these experiments is reproduced in Table VII. Two groups of CBA mice were immunized by S . typhosa H antigen and were also allowed to reject bilateral C57BL/ 6 skin grafts. After immunization, one group was treated with ALS and the other received comparable amounts of NRS. Large doses of cells (roughly 1O* cells/recipient ) prepared from the pooled lymph nodes and spleens of these animals were injected intravenously into either (CBA X C57BL/6) F, adult hybrids or CBA female mice which had been exposed to 600 R of whole-body irradiation the day before. Cells from the ALS-treated animals were markedly inferior in causing a GVH response when compared with cells from untreated or NRS-treated donors, On the other hand, no reduction was evident in the capacity to transfer adoptively the secondary response to H antigen. A second experiment was slightly different in design. Bovine serum albumin was substituted for H antigen and the IgG fraction of either ALS or NRS was used. The experimental animals were normal mice or mice primed to BSA or C57BL antigens. The humoral antibody response was measured in both intact animals and an adoptive cell transfer system. Cells from animals treated with NRS-IgG mounted a vigorous GVH response in hybrid recipients, and splenic enlargement was greater when the cell donors had been presensitized. The ALS-IgG treatment caused a drastic reduction

TABLE VII ADOPTIVETRANSFER OF CELL-MEDIATED AND HUMORAL ANTIBODYRESPONSES ~

(CBA X C57BL/6)F1

Cell donors

Treatr mento

Cells transferred

Cell recipients

ALS

2. CBA sensitized to C57BL/6 skin and S. typhosa H antigen

NRS

3. CBA sensitized to C57BL/6 skin

-

4.

a

125 Million spleen and lymph node cells/recipient 125 Million spleen and lymph node cells/recipient 125 Million spleen and lymph node cells/recipient

CBA (600 R) (H titer logz)

~_________

~

1. CBA sensitized to C57BLj6 skin and Salmonella typhosa H antigen

No.

Average Splenic spleen index weight (mg./gm.)

(CBA X C57BL/6)Fi

178

6.4

-

-

14.7

10.1

-

-

15.2 -

CBA (600 R)

10

(CBA X C57BL/6)Fi

10

CBA (600 R)

10

(CBA X C57BLj6)Fi

10

272

10.2

(CBA X C57BL/6)Fi CBA (600 R)

20 10

121 -

4.3

272

ALS, antilymphocytic serum. See text for details of timing and dosage. NRS, normal rabbit serum.

-

<4

ANTILYMPHOCYTE SERUM

51

in the degree of splenic enlargement and eliminated the difference between primed and virgin cells. The primary response to BSA was much reduced in ALS-IgG-treated animals, but only a slight effect was noted on the secondary response. The test system is a severe one since the GVH reaction between these two strains is vigorous, and large numbers of cells were used for testing. Nonetheless, marked reduction of reactivity was achieved; to the contrary, little reduction in the secondary response to either BSA or H antigen was observed. Since the cells in both systems were derived from the same hosts, they reflected the capacities of the cell donors. These accordingly must have also had dissociation of immune responsiveness induced by ALS.

C. EFFECT OF ANTILYMPHOCYTICSERUMON VIRAL SYSTEMS Although the cellular response to a wide variety of viral pathogens can be reduced or eliminated by ALS, yet the titer of circulating antibodies is not affected. Similar dissociation has been found in rats infected with malarial parasites ( Spira et al., 1970).

D. MORPHOLOGICAL EVIDENCE The lesions after ALS treatment are characterized by the loss of small lymphocytes from the paracortical areas of the lymph nodes and periarteriolar areas of the spleen. The cortex and medullary systems of lymph nodes as well as the peripheral portions of the splenic white pulp do not show this depletion. The former areas are thought to be primarily associated with the lymphocyte subpopulation that transacts cell-mediated reactions, whereas the latter are closely identified with the production of humoral antibodies. X. Chronic Administration of Antilymphocytic Serum

There are many situations in clinical practice where the need for immunosuppression extends over long periods of time. It is, therefore, pertinent to enquire what advantages and hazards might be expected in the chronic use of ALS. The continuous administration of ALS to inbred mice procures virtually indefinite survival of skin allografts (Lance, 1968a; Nehlsen, 1971a,b; Simpson and Nehlsen, 1971a,b). In the most extensive study of this subject, Nehlsen was able to suppress reactivity toward allografts, xenografts, and thymus-dependent antigens in CBA mice treated continuously with ALS from birth over an 18-month period of observation. These results could be attributed to immunosuppression rather than to other mechanisms such as tolerance or enhancement, since

52

E. M. LANCE, P. B. MEDAWAR, AND R. N. TAUB

discontinuation of treatment invariably led to rejection. Moreover, long after the initiation of treatment, reactivity to new histocompatibility antigens was as effectively abrogated as that, at the beginning of treatment (Lance, 1968a). On the other hand, the same approach in outbred mice or dogs (Nehlsen, 1971a,b; Lance et al., 1971) was not nearly so successful, and, with time, escape occurred in many animals. In part this escape may be attributed to difficulty in controlling reactivity toward the heterologous IgG. With regard to this prospect the “continuous” administration of ALS is much more effective than a “pulsed or intermittent schedule of administration even though the overall dosage given does not differ (Lance, 1968a). The adverse effects of chronic treatment may be considered under several headings: hematological, immunological, infectious, and neoplastic. Anemia and hemolysis have been frequently observed to follow ALS administration (Monaco et al., 1966a; Starzl et al., 1967a; Taub and Lance, 1968a; Denman and Frenkel, 1967). These effects are less marked if the antiserum is given subcutaneously rather than by intravenous or intraperitoneal routes ( Iwasaki et al., 1967). A possibility that these effects are due to bone marrow depression of stem cells has been raised by Field and Gibbs ( 1968) and DeMeester et al. (1968). Harris et al. (1969) have reported damage to bone marrow cells after ALS administration. However, Taub and Lance (1968a) found hyperplastic bone marrow in association with anemia, and Nehlsen (1971a,b) found that continuous administration of ALS fully absorbed with erythrocytes did not lead to anemia in her animals, Therefore, anemia is most probably the result of the continuous administration of antisera with low or moderate titers of hemagglutinins. The same appears to apply to platelet depression where chronic administration of absorbed antisera has not been associated with thrombocytopenia or bleeding. Clearly, the requirements for absorption are most stringent when antisera are intended for longterm use. Immunization to contaminating serum protein or to heterologous IgG itself leads to anaphylaxis, serum sickness, and immune complex nephritis (Starzl et al., 1967a,b; Iwasaki et al., 1967; Lance, 1968a; Taub and Lance, 1968a; Nehlsen, 1971a,b; Denman et al., 1967a). These reactions are not peculiar to ALS treatment as the same range of effects can be seen in animals treated with normal heterologous serum. Amyloid deposits associated with long-term treatment have also been observed ( Clerici et al., 1969; Nehlsen, 1971a; Simpson and Nehlsen, 1971a,b). The importance of using pure ALS-IgG fractions and the desirability of preventing immunization to IgG have been repeatedly emphasized

ANTILYMPHOCYTE SERUM

53

throughout this review. The initial injections of ALS frequently provoke a marked reaction, not unlike anaphylaxis, which disappears or diminishes with subsequent doses (Abaza et al., 1966; E. M. Lance, unpublished observations), Evidence has recently been presented * that this side effect may be akin to reverse anaphylaxis in which lymphocytes of the host act as the antigen and ALS as the antibody (Rosenberg et al., 1971). The implication here is for caution to be exercised when ALS is first administered, perhaps by spreading out the doses or by the simultaneous use of corticosteroids. Although two cases of coliform infection in dogs were reported by Iwasaki et al. (1967), increased incidence of infection with bacterial pathogens has not been a feature of chronic ALS administration. Apart from the purposeful introduction of viral agents into ALS-treated animals where lowered resistance is clearly demonstrable, spontaneous viral infection in ALS-treated animals, although uncommon, has been reported ( Abaza et al., 1966; Balner and Dersjant, 1967; Nehlsen, 1970, 1971a,b). Nehlsen also found two cases of fungal infection apparently carried onto ALS-treated animals by infected skin grafts. These infections did not become generalized. Of incidental interest are the facts that mice chronically treated with ALS were completely capable of normal reproduction and that their progeny were immunologically normal ( Nehlsen, 1970, 1971a,b; Jooste, 1968). Chronically ALS-treated mice grew and developed normally when compared to untreated controls. In view of the widespread notion that the natural function of cellmediated immunity is to prevent the development of malignant clones (immunological surveillance) and despite the clear evidence that ALS treatment allows tumor transplantation and supports the action of oncogenic viruses and chemical agents (see above), there is precious little evidence that treatment with ALS alone for prolonged periods of time influences the background rate of spontaneously arising tumors ( Russe and Crowle, 1965; Denman and Denman, 1970; Lance, 1968a; Taub and Lance, 1968a). Nehlsen ( 1970, 1971a,b; Sinipson and Nehlsen, 1971a,b) observed 61 tumors arising in a large number of chronically ALS-treated mice. Of these, 59 were attributed to polyoma virus inadvertently introduced through a contaminated source of rabbit antiserum; the remaining 2 were lymphoblastic lymphomas and may have arisen as the direct consequence of continuous immunosuppression by ALS. This dearth of evidence in experimental animals that ALS treatment potentiates spontaneous neoplasia may not be all that comforting when considering the situation in man. Here ALS may have to be used under complex circumstances which may in themselves predispose or synergize with ALS in tumorigenisis (see below).

54

E. M. LANCE, P. B. MEDAWAR, AND R. N. TAUB

XI. Synergism with Other Agents

Thoracic duct drainage or thyniectoniy, procedures that deplete recirculating lymphocytes or prevent their regeneration, synergize with ALS (Woodruff and Anderson, 1963, 1964; Jeejeebhoy, 1965a,b, 1967; Monaco et al., 1965a,b, 1969; Davis and Lewis, 1968; Gunn et al., 1970). Thymosin can enhance or curtail ALS action depending on whether treatment precedes or follows ALS treatment (Franc0 et al., 1970). Irradiation given prior to or various cytotoxic drugs given with or after ALS enhance the immunosuppressive effect ( Levey and Medawar, 1966a; Gunnarsson et al., 1969; Hoehn and Simmons, 1967; Perper et al., 1970a)b; R. L. Simmons et al., 1968; Jeejeebhoy et al., 1968; Floersheim, 1969; Weil and Simmons, 1968). On the other hand, radiation or adrenalectomy after ALS curtail potency (Levey and Medawar, 1966a). These results have been interpreted in terms of the rate of turnover of immunocompetent cells. Floersheim and Ruszkiewicz (1969) showed that ALS in conjunction with dimethyl Myleran allowed the take of allogeneic marrow with substantial and long-lasting chimerism. A remarkable synergism exists between ALS and corticosteroids of the hydrocortisone type (Levey and Medawar, 1966b; Lance et al., 1970b; Lance and Medaw,ar, 1969; Gunn et al., 1970; Lance and Cooper, 1970) . Small doses of hydrocortisone given after immunosuppression had been induced by ALS were as effective in maintaining this state as was continued treatment with ALS itself. Moreover, hydrocortisone given prior to ALS resulted in potentiation of immunosuppression (Table VIII). Lance et al. (1970b) and Lance and Cooper (1970) explored the changes in lymphoid subpopulations which occurred during synergy of this type. They concluded that the synergy could be explained in terms of the additive effects of these two agents on lymphocytes. AntilymphoTABLE VIII POTENTIATION OF ANTILYMPHOCYTIC SERUM BY PRIOR INJECTION OF HYDROCORTISONE~ HC dose

(pg.)

ALS dose (ml.)

Mean survival time ___

3 x 200 3 x 100 3 X 50 3 x 0 3 x 200 0

0.4 0.4 0.4 0.4 0 0

26.6 23.2 18.2

17.2 12.1 10.7

4 A + CBA; HC at days -1, 0, + l ; ALS at day +3: ALS, antilymphocytic serum; HC, hydrocortisone.

ANTILYMPIIOCYTE SERUM

55

cytic seruni brought about a rapid and profound fall in the numbcr of recirculating lymphocytes. Hydrocortisone had two effects : ( 1) with small doses, a marked depletion of thymocytes occurred largely at the expense of the immature cells, and ( 2 ) with larger doses, a direct effect on peripheral lymphocytes was noted in addition to the thymic effect. Synergy occurred, therefore, because low doses of corticosteroids produced a chemical thymectomy and, therefore, retarded the recovery of the recirculating cell population. The authors then showed that thymectomy and steroids were interchangeable agents in synergizing with ALS, both with respect to functional results and effects on lymphocyte subpopulations. Moreover, the use of both thymectomy and steroids with ALS simultaneously did not produce effects greater than either agent alone. This did not hold true for large doses of steroids as already indicated. Curtailment of ALS action by adrenalectomy could also be understood, since a more rapid regeneration of the recirculating population was documented in adrenalectomized ALS-treated animals. The synergism between Bordetella pertussis and ALS (Ptak et al., 1969) is best explained by the flushing out of lymphocytes from central reservoirs into the circulation where they were exposed to ALS action more effectively. XII. Antilymphocytic Serum and the Induction of Immunological Tolerance

Monaco et al. (1966a) were the first to show that ALS in conjunction with thymectomy could be used to set the stage for the induction of tolerance to transplantation antigens. Lance and Medawar ( 1969) extended these studies to show that specific tolerance could be achieved by using ALS as the sole immunosuppressive agent. These studies originally conducted in allografted mice have now been advanced to include tolerance induction in monkeys (Lance and Medawar, 1970b) and dogs (Lance et al., 1971) as well as xenografts (Lance et al., 1969; Cerilli et al., 1970a,b) and protein antigens (Lance, 19704. The important variables in these studies will be summarized briefly. The timing of the antigen injection relative to ALS treatment was critical. Antigen introduced prior to initiation or completion of ALS treatment curtailed graft survival ( sensitization), whereas antigen injected several days after the last ALS dose potentiated graft survival (tolerance) (see also Nisbet, 1969). The skin grafts in these studies were used as indicators of immunological reactivity. Recruitment into the tolerant state was directly proportional to the dosage of antigen (usually in the form of viable lymphoid cells). However, the effect could be elicited in mice with as few as 2 X loa cells. Monaco (1970) found a

56

E. M. LANCE, P. R. MEDAWAR, AND R. N. TAUB

threshold effect at the upper and lower limits of antigen dosage. Antigen introduced by a variety of routes at the appropriate time procured tolerance, but the intravenous route was by far the most efficient. Monaco et al. (1971) have also procured tolerance by intraorgan injection of viable marrow cells. Lymphoid cells from the spleen, thymus, or the peripheral blood as well as niyeloid cells seemed equally effective as a source of antigen, although some preference for niyeloid cells has been claimed (Wood et al., 1971; Monaco et al., 1971). Treatment with ALS must be adequate: when too little, no difference between control and experimental groups was found. In general the dose required for tolerance induction is of the same order of magnitude as that needed to erase immunological memory (i.e., about 1.5 to 2.0 ml./ mouse). Tolerance could be procured in sensitized animals. By using ALS and cyclophosphamide together, Turk ( 1970) reported analogous findings with respect to potassium dichromate, a skin-sensitizing chemical. Animals displaying nonreactivity to test skin grafts were negative for circulating antibodies to graft antigens. Furthermore, tolerant animals were chimeric for cells derived from the antigen inoculuni ( Seiler, 1970). The specificity of the nonreactive state was verified by rejection of thirdparty skin grafts at the expected rate and the selective loss of competence in GVH assays in appropriate, newborn, hybrid recipients. Although tolerance could be readily obtained by these methods, the duration was not protracted (generally lasting on average between 50 to 100 days). The repeated injection of antigen doses at intervals greatly extended the duration of tolerance, but indefinite survival in all but a small proportion of animals could not be achieved in this way. The use of cyclophosphamide or methylhydrazine derivatives ( Floersheim and Brune, 1971) in conjunction with ALS was moderately successful, but the most useful adjunct was small doses of whole-body irradiation (450 R ) just prior to cell injection. In experiments of this kind tolerance of indefinite duration could be achieved. Radiation was believed to assist ALS in two ways: creation of lehensraum for the cell inoculum and placement of host cells at a selective disadvantage during the competition between host and recipient cells after cessation of treatment. Graft-versus-host disease in recipients was circumvented in one of three ways: by the use of cells of limited immunological potential, e.g., thymocytes or bone marrow; by the use of cells from ALS-treated donors; and by the use of cell-free antigen extracts (see also Abbott et al., 1969). Brent and colleagues (Brent and Kilshaw, 1970; Brent et al., 1971) thoroughly explored the last possibility and found that in contrast to the use of living whole cells, the most advantageous time for antigen ex-

ANTILYMPHOCYTE SERUM

57

tract administration was prior to ALS treatment. This relationship suggests that enhancing antibody might play a role in their system; however, they could not identify any antibody to donor antigens in their unrespoiisive mice. Although treatment with ALS alone could procure tolerance on behalf of xenogeneic antigens (Lance et al., 1971), yet thymectomy in conjunction with ALS was required for impressive results. Antilyniphocytic serum action also conformed with tlie long-established law of tolerance, i.e., that induction becomes more difficult as histocompatibility differences between donor and recipient increase. XIII. Mode of Action of Antilymphocytic Serum

A. SELECTIVEACTION ON RECIRCULATING LYMPHOCYTES The most likely explanation for tlie range of effects achieved by ALS is selective depletion of the recirculating population of lymphocytes. When this hypothesis was first advanced (Lance, 1968a), it was based solely upon functional and morphological data. Since then new facts have emerged. Special mention must be made of the observations of Levey and Medawar (1967b), who were the first to point out the particular efficacy of ALS on opposing reactions in the periphery; the elegant labeling studies of Denman (Denman and Frenkel, 1968a,b; Denman et al., 1968a,b) and Taub (1969, 1970a,b), who showed that the residual population in ALS-treated animals was predominantly one that turned over rapidly, i.e., short-lived lymphocytes; and studies by Mai-tin and Miller ( 1967) and Leuchars et al. ( 1968), who indicated the selective cffect of ALS on thynius-derived, antigen-sensitive cells. The six lines of evidence that support this hypothesis have been the subject of a recent review by one of us (EML), and the arguments are summarized here. Many of the details have been described earlier in this review.

1. Functional Data The discrimination by ALS for cell-mediated immune processes and for the cooperative aspect ( Mitchison, 1970) of some humoral immunities suggests that tlie target of ALS action is the cell population that mediates these effects; namely, the recirculating, thymus-derived, small lymphocyte. The retention of humoral immunity to thymus-independent antigens, the lack of effect on the antibody-producing cells, and the dissociation seen in parallel assay systems make it clear that the population of cells involved with these reactions are spared.

58

E. M. LANCE, P. B. MEDAWAR, AND R. N. TAU3

2. Morphological Data The histological changes characteristic of ALS administration include depletion of small lymphocytes from those areas that are identified with the population of recirculating small lymphocytes, whereas areas associated with the population of sessile cells are spared. There is, therefore, a good correlation between the functional and morphological evidence.

3. Fate of Lymphocyte Affected by ALS Lymphocytes intentionally exposed to ALS are killed or opsonized and cleared from the circulation by the reticuloendothelial (RE) system of the liver. Lymphocytes introduced into the circulation of ALS-treated animals suffer the same fate and are prevented from migrating to lymphoid organs. Animals chronically treated with ALS show a selective removal from lymphoid organs of cells that home to the lymph nodes, whereas the spleen-seeking population remains normal or actually slightly increased. [For the evidence that lymph node migration is a property possessed solely by recirculating lymphocytes, see Lance and Taub (1969), Zatz and Lance ( 1970), and Taub and Lance ( 1971) .] 4. History of Relevant Antibody There are three chief points regarding the mode of action of the relevant antibody. ( a ) Antilymphocytic antibody is eliminated from recipients very rapidly-the bulk is cleared within 24 hours, and at 72 hours only a minute fraction remains. ( b ) There is a relatively poor penetration of lymphoid tissue. The greatest amount of activity is found in the bloodstream 4 hours after injection. The liver and kidneys also have high uptake, whereas that in the thymus at any time is extremely low. Lymph nodes and spleen contain small fractions of the injected dose, although, on a weight-for-weight basis, more antibody localizes in these organs than, for instance, an equal mass of skeletal muscle. ( c ) The pattern of in vivo localization suggests that a factor other than antigenic specificity determines the pattern of distribution. Radioautography shows that lymphocytes in the circulation are rapidly coated with antibody and cleared from the bloodstream by the Kuppfer cells of the liver. Within lymphoid organs the pattern of distribution corresponds to the traffic pattern of recirculating lymphocytes.

5. Kinetic Data The recovery of lymphocytes occurring in the peripheral blood at a time when animals are still immunologically suppressed seems difficult to reconcile with the selective depletion hypothesis, but peripheral blood

ANTILYMPHOCYTE SERUM

59

lymphocytes are known to be heterogcneous (Everett et al., 1964). Thoracic duct lymph, on the other hand, has a more homogeneous population, and a prediction of the hypothesis is that lymphocytes in this compartment should be rapidly depleted and restored only very slowly after ALS introduction. Moreover, the output of small lymphocytes in the thoracic duct should remain low even when the peripheral blood lymphocyte count has recovered. These predictions have been confiimed by direct study of the thoracic duct output in ALS-treated animals by Agnew (1968), Tyler et al. (1969), Gelfand et al. (1969), and Lance and Gowans (in Lance, 1 9 7 0 ~ ) . 6. Antigenic Data

Schlesinger and Yron (1969) found that the lymph node cells from ALS-treated animals became refractory to the cytotoxic action of anti-0 ( 0 being a marker for thymus-derived lymphocytes) but were fully responsive to anti-I-I-2 antisera. The reduction in 0-positive lymphocytes has been confirmed in both lymph nodes and spleens of ALS-treated animals by Raff (1969) and by Nehlsen ( 1970, 1971a,b). Based upon the foregoing evidence, the sequence of events by which ALS acts can be postulated. After injection, relatively high titers of specific antibody arise in the bloodstream where they contact and coat lymphocytes. These cell-antibody complexes bind complement and are consequently phagocytized by the RE cells of the liver. The importance of an intact RE system has been emphasized by Harris et al. (1971). [Some cells may be killed outright, but the finding by Cinader et al. ( 1971) that late-acting complement components are not required for immunosuppression suggests that cytotoxicity is not a necessary feature; see also Barth and Carroll (1970).] Cells removed from the bloodstream are replaced by cells from the central lymphoid compartments, since members of the recirculating pool are constantly immigrating to lymph from blood. This sequence of events continues until the titer of ALS falls below a critical level, Because the turnover time of the recirculating lymphocyte is relatively rapid, titers of circulating antibody would not have to be maintained long to produce a drastic depletion of this lymphocyte population. Since the majority of the members of this pool are long-lived, slowly regenerating lymphocytes, the defect in cell numbers is repaired only slowly. Thus, the immunosuppressive effect of ALS long outlasts its metabolic lifetime within the recipient. Cells that are sessile are protected because ALS penetrates their areas of residence poorly. Hence the preservation of germinal centers and medullary areas explains the relative inefficiency of ALS in opposing humoral immunity. Furthermore, the reappearance of lymphocytes in the

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peripheral blood would be due to the short-lived and rapidly turning over population of lymphocytes (Caffrey et al., 1962; Everett et al., 1964). As soon as the titer of ALS had fallen this population could quickly reemerge. After extensive treatment with ALS, recovery of competence takes place slowly, between 40 to 50 days (Lance, 1968a; Lance and Medawar, 1969). Russell and Monaco (1967) have shown a correlation between recovery of competence and recovery of lymph node histology which may not be completed for well over a month. These observations are consistent with the notion that the regeneration of recirculating lymphocytes takes place very slowly ( 1-2%/day) (Gowans, 1959; Caffrey et al., 1962). Since the thymus is essential for the regeneration of this population of lymphocytes (Miller and Osoba, 1967), it is not surprising that thymectomy delays the recovery of ALS-treated animals.

PossmnIms B. ALTERNATIVE Blindfolding, originally proposed by Levey and Medawar (1966a; Field et al., 1969), was subsequently rejected by them (Levey and Medawar, 1967a) when they observed persistent immunoincompetence after repeated cell division. Brent et al. (1967, 1968) claimed to be able to restore competence to lymphocytes from ALS-treated animals by treatment in vitro with trypsin. This result has not been confirmed and could not be duplicated by Lance and Medawar (Lance, 1968d). Enhancement can be readily dismissed. Antilymphocytic serum is largely species-specific yet can promote the survival in mice of xenografts from a wide variety of sources. Furthermore enhancement could not possibly apply to protein or bacterial antigens. Some role for enhancement has been claimed for the protection of renal allografts by Guttman et al. (1967b,c) and by Raju et al. ( 1969), who found that skin allografts incubated in ALS in vitro survived longer than did controls (see also Burde et al., 1971). However, Levey and Medawar (1967b) reported that rabbit anti-A-strain ALS was no more effective in prolonging the survival of A-strain skin on CBA mice than was rabbit anti-CBA. Finally tail skin or ovarian grafts taken from ALS-treated donors were rejected as rapidly as those from untreated donors ( S . V. Jooste, personal communication, 1968; Barnes and Crosier, 1969). Sterile activation would account for the presence of large numbers of lymphocytes in the circulation, the not infrequently obsei-ved hyperplasia of lymphoid tissue, the transformation of lymphocytes in vitro, and the presence of blast cells in the tissues of ALS-treated mice. Sterile activation proposes transformation and replication of lymphocytes without a specific immunological object but precluding reaction to an ini-

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munogen. Lymphoid depletion is, therefore, considered incidental and not essential to the action of ALS. The objections to this proposal include the fact that transformation by ALS takes place in the absence of complement, whereas in uiuo the presence of complement would be expected to lead to toxicity. Transforming ability is shared by a wide variety of agents; however none of these can compete with ALS as an immunosuppressive agent ( Landy and Chessin, 1969). Moreover transforming ability cannot be a sufficient cause of ALS action, since antibody fragments that can transform are inactive in uiuo. The increased number of blast cells after ALS treatment are mostly the consequence of immunization, and this proliferative response is reduced in animals tolerant to ALS-IgG (Taub and Lance, 1968b; Everett et al., 1970). Denman et al. (1968b) observed a marked drop in lymphocyte count in the peripheral blood (from 100,000 to 1,000) occurring within a few hours without evidence of blast transformation; therefore, transformation does not appear to be a necessary feature of ALS action. Antigen competition is ruled out by the finding that ALS is equally or more effective in the presence of an immunological reaction to the heterologous protein. Reference to the Liacopoulos phenomenon ( Liacopoulos and Goode, 1964; Liacopoulos, 1965; Liacopoulos et al., 1967, 1968) is inappropriate because of the very different requirement of timing and dosage found for ALS action. Action through a nonimmunological pathway is unlikely, since activity may be completely removed after absorption with lymphocytes (see also Judd et al., 1971). Interference with a thymic hormone can be discounted because of the known synergy between ALS and thyniectomy, the rapid elimination of ALS antibody from recipients, the fact that anti-lymph node antiserum is as effective as antithymus antiserum, and finally the fact that antigenic extracts from thymus, e.g., membrane, are as effective as whole thymus preparations in raising ALS. The possibility that ALS causes a gross and indiscriminate destruction of lymphocytes and that this is a prerequisite to its action must be considered because in uitro ALS action upon lymphocytes is indiscriminate and because ALS-treated animals may have massive and nonselective ablation of lymphocytes in the circulation and in the central lymphoid organs (Monaco et al., 1965a,b; 1966a,b; 1967a; Gray et al., 1966; Lawson et al., 1967). But this general depletion cannot be a requirement, since equal or greater immunosuppressive effects follow the use of sera which cause only a zonal and selective depletion in lymph nodes and spleen and which entirely spare the thymus. Sera raised with adju-

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vants are often highly toxic, and a stress effect mediated through host adrenals is the most likely explanation for this seeming discrepancy in observations. It should be mentioned that ALS effects do not depend upon adrenal participation, since ALS is effective in adrenalectomized hosts. Therefore the alternative proposals to the hypothesis of selective depletion of recirculating lymphoctyes are found wanting in one respect or another, yet the possibility of dual or multiple mechanisms cannot be completely excluded (Turk and Willoughby, 1969). XIV. Effects in Man

Initial attempts to evaluate the efficacy of ALS in human recipients were beset with difficulties which were not easy to surmount. The sequential trials by Starzl and his colleagues (1967a,b) in patients with renal allografts took place at a time when the results of renal transplantation were improving generally. It was, therefore, difficult to determine to what extent ALS administration was responsible for improved graft survival. Assessment in a setting of this type was and will continue to be troublesome because of the difficulty in controlling histocompatibility, because other immunosuppressive agents continue to be used, because the pattern of treatment must be changed in accord with the needs of each patient, and because the problems of transplantation are complicated by those of organ preservation. Moreover, it took a long time for clinicians to realize that various antisera produced in a wide variety of centers and all called “ALS” might vary considerably both in potency and toxicity. This problem was compounded by the lack of an appropriate assay and by the lack of systematic experimental guidance in the early sixties. These deficiencies were combined with an understandable reluctance to extrapolate directly the results obtained in small mammals to man.

A. PARALLELISM BETWEEN CLINICAL AND EXPERIMENTAL EVIDENCE At the present time there are several lines of evidence that ALS, in parallel with its effects in experimental systems, is immunosuppressive in man. The most convincing evidence comes from the studies of skin allograft rejections in patients or normal volunteers where ALS has been given without adjunctive immunosuppression ( Monaco et al., 1967a)b; Simmons et al., 1971).With the continuous administration of 4 mg./kg./ day of ALS-IgG, skin allograft survival was doubled, and with progressively higher doses a clear dose-response curve was obtained. This property has already been put to use in promoting the survival of skin allografts on burned patients (Traeger et al., 1970b). Delayed skin hypersensitivity reactions to tuberculin and other skin

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test antigens have been suppressed in patients receiving ALS for more than 5 days (Monaco et al., 1967a,b; Mannick et al., 1971; Brunstetter and Claman, 1965; Revillard and Brochier, 1971). Attempts to sensitize ALG recipients to DNCB and KLH during therapy failed (Mannick et al., 1971). Less convincing but probably significant are the reports which show that the use of ALS in conjunction with other immunosuppressive agents has improved the survival rates amongst patients receiving poorly matched kidney allografts (Monaco et al., 1970; Mannick et al., 1971; Sheil et al., 1971), that the number of rejection crises is reduced when ALS is included in the treatment protocol (Merkel et al., 1969; Najarian et al., 1970b,c; Monaco et al., 1970; Mannick et al., 1971), and that rejection crises can be reversed by raising the dose of ALS aIone (BrendeI and Land, 1969; Traeger et al., 1971). The effect of ALS on humoral antibody formation in man has not been adequately studied. The serum levels of the so-called natural antibodies (i.e., isoantibodies to the ABO group of erythrocyte antigens) are not reduced. As with experimental animals, lymphopenia in man is often modest and of short duration (Starzl et al., 1967b; Najarian and Simmons, 1971). On the other hand, Davis et al. (1971) have reported lymphopenia which persisted for 6 weeks after the start of ALS treatment (see also Mannick et al., 1971). These lymphocytes found in the blood of ALStreated patients showed a high base-line incorporation of tritiated thymidine, suggesting that they belonged to a rapidly turning over population (Melli et al., 1968; Revillard and Brochier, 1971). Whether this represents a situation analogous to that reported by Denman et al. (1968a,b) in mice or merely reflects an immune response to foreign protein remains uncertain. The ability of these cells to respond to PHA may not be reduced when tested in homologous serum (Revillard and Brochier, 1971). The PHA response was reduced when the cells were cultured in a.utologous serum presumably because of the inclusion of ALS antibody. There are several reasons why this finding in humans might be discrepant with studies in mice: ( 1 ) ALS treatment might not have been effective; ( 2 ) PHA stimulation, which is relatively specific in mice for recirculating thymus-derived cells (Tursi et al., 1969), may have a broader range of effects on human lymphocyte subpopulations; and ( 3 ) since these patients are treated with other immunosuppressive agents in addition to ALS, there may be a proportional reduction in the various lymphocyte populations. The test system only measures the reactivity of a given number of cells which under this circumstance might not be reduced and might not reflect the overall reactivity of the patient. Antigen-induced lymphocyte transformation is reduced in ALS re-

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cipients (Revillard and Brochier, 1971), and this may reflect a reduction in a specific subpopulation of antigen-sensitive cells. Moreover, a modification of the rosette inhibition assay has been used to follow ALS-treated patients, It was found that if the spontaneous rosette formation of peripheral blood cells was below 1/50,000 lymphocytes, no renal allograft rejection episode occurred (Munro et al., 1971). These findings support the idea that a specific antigen-sensitive cell, distinct from the PHA-responsive cell, is removed in man by ALS treatment.

B. SPECIALASPECTSOF ANTILYMPHOCYTICSERUM PRODUCTION FOR USE IN MAN Numerous facets of the problem of raising, purifying, and administering ALS have been extensively covered in this review. A few points are worth reemphasis. If one assumes that the dose-response relationship established in experimental murine systems applies as well to man, then roughly 3000 times as much antiserum is required to achieve the same effects. If, for example, mice require 0.5 ml. of antiserumIweek in order to retain skin allografts indefinitely as a consequence of ALS treatment, then a patient requires 1.5 liters/week or 75 liters a year. Even if we assume that these estimations are off by 50%, it is clear that for clinical use enormous quantities of antiserum will have to be raised. Scaling up production to this order may mean that theoretical advantages may have to be sacrificed to considerations of practical expediency.

1 . Choice of Antigen Although lymphocytes obtained from the thymus and thoracic duct seem ideal sources of antigen, it is unlikely that they will be available in quantities sufficient to satisfy the requirements of large-scale production. Lymph node lymphocytes by and large are open to the same objections. The spleen, a potential source of large numbers of lymphocytes has not proven satisfactory because of contamination by red cells and platelets (Deodhar et al., 1971; Munro et al., 1971; Mee and Evans, 1970). Townsend et al. (1969) have produced a clinically acceptable ALS by using as antigen splenic lymphocytes. Other promising candidates are cultured human lymphoblasts, production of which can be scaled up almost without limit. Math6 et al. (1970) have obtained large numbers of peripheral blood lymphocytes from normal subjects by use of the IBM continuous flow blood cell separator. These lymphocytes are contaminated less than 1%by granulocytes and erythrocytes. As inany as 1 O ’ O cells can be removed from a single donor without causing perceptible lymphopenia. Antisera may also be prepared against the lymphocytes from patients with chronic lymphatic leukemia (Dormoiit et al., 1970);

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however the theoretical hazard of transmitting a potential viral oncogen niakes this alternative unattractive. Purification of antigens by extracting lymphocyte membranes has been reported by Levey et al. (1970) and by Barnes et al. (1971). Potent antisera have been raised, and we believe their use should be explored further. Attempts are also being made to raise ALS by the use of soluble cellular antigens ( Haenen-Severyns et al., 1969; Lejeune et al., 1970). There has been little formal analysis of the superiority of one or another antigen source with regard to immunosuppressive properties in man. Simmons et al. (1971) compared horse ALS raised against human lymph node cells, thymocytes, or cultured lymphoblasts by their ability to extend the survival of skin allografts in volunteers with multiple sclerosis. Antithymocyte and antilymphoblast sera were of equal potency, whereas anti-lymph-node serum was slightly less effective.

2. Choice of Species Size apart, the rabbit seems an ideal animal for the production of heterologous antiserum because it is a potent antibody former. Its serum is minimally toxic and is less likely to cause allergic disturbance than others (Cerilli et al., 1970a,b; Davis et al., 1971; Darrow et al., 1971); antibody formation in this species has been most completely studied experimentally. Whereas the slaughter of myriad rabbits does not seem a probable solution to the enormous needs for clinical use, one can foresee occasions when a second species for ALS yield will be useful, e.g., in the patient sensitized to ALS from the major source or in need of reinforced treatment of a rejection crisis. Moreover rabbit antihuman ALS might serve as a useful reference standard against which to test other ALS sources. 3. Purification and Assay These aspects have been fully covered above and will not be elaborated further. At the monient the concensus of fact and opinion dictates the extraction of the IgG molecules from whole serum, careful absorption of noxious antibodies, and assay by a combination of the surrogate test in primates and rosette inhibition and opsonization tests in uitro.

C. ADMINISTRATION AND SIDE EFFECTS 1. Immediate and Local Efects Fever to 103" or 104°F. occurring 3-12 hours after injection (Starzl et al., 1967a,b; Trneger et al., 1971; Doak et al., 1969; Townsend et al., 1969; Sheil et al., 1971; R. N. Tauh, unpublished) may follow the first

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few intramuscular or intravenous doses of ALG. The fever is frequently heralded by chills and accompanied by arthralgias and lumbar pain. The pathogenesis of the fever is not clear, but may relate to destruction of leukocytes by infused antibody and complement, with the consequent release of endogenous pyrogens. The arthralgias and lumbar pain are reminiscent of transfusion reactions, although much less severe, and may be due to antigen-antibody complexes localizing in the kidney. In the experience of one of us (RNT), these symptoms were occasionally seen after infusions of large volumes of normal horse y-globulin, and may not necessarily have been due to antilymphocytic antibody. These reactions have been readily controllable with an antihistamine ( diphenhydramine) or small doses of corticosteroids. Intramuscular injection of ALS is often followed by severe enough pain at the site of injection to cause limitation of the daily dose (Starzl et al., 1967a,b,c,d; Mannick et al., 1971; Doak et al., 1969; Davis et al., 1971). Some observers did not note such pain (Shorter et al., 1970; Pirofsky et al., 1971). Intravenous injection of ALG has been accomplished without serious untoward effects (Traeger et al., 1971; Brendel, 1971; R. N. Taub, unpublished observations) and avoids the necessity for painful intramuscular injections. Prolonged intravenous ALS treatment produces mild phlebitis which usually resolves spontaneously. Some hazards in the use of intravenous ALG include occasional episodes of dyspnea, bradycardia, and hypotension (Traeger et al., 1971; Doak et al., 1969). It is not certain whether hypotension occurs as part of an anaphylactoid reaction or as a pharmacological response to horse y-globulin. Doak et al. (1969) were able to reduce the hypotensive effects of their antisera by dialysis against distilled water. Certain ALG preparations contain high titers of hemagglutinating antibody and may produce a hemolytic anemia requiring transfusion therapy (Starzl, 1967; R. N. Taub and R. E. Rosenfield, unpublished observations). The direct Coomb's test may be positive during or after a hemolytic episode. Thrombocytopenia has been noted by several observers after the use of ALS (Traeger et al., 1971; Starzl, 1967; Deodhar et al., 1971; Sheil et al., 1971). Acute thrombocytopenia caused by ALS is accompanied by sequestration of platelets in the spleen and the liver ( Andrassi et al., 1971). Splenic entrapment of antibody-coated platelets may be a prominent mechanism of thrombocytopenia; the one patient that Deodhar et al. (1971) described with thrombocytopenia still had an intact spleen. Some antibody found in ALG may show activity against glomerular basement membranes. These antibodies are particularly troublesome

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because they are very difficult to absorb out and may conceivably cause heterologous nephrotoxic nephritis, in addition to the autologous nephrotoxic nephritis which may be seen after any serum sickness reaction. The presence of these antibodies has been shown by imniunofluorescence in renal biopsies from human transplanted patients (Iwasaki et al., 1967; Traeger et al., 1971; Thiel et al., 1971). In one study, two large pools of equine antihuman thymocyte ALS were strongly nephrotoxic, causing uremia and tubular degeneration. It was impossible to remove this antibody by standard absorption procedures, It was suspected, although not formally proven, that the antigen provoking the formation of these nephrotoxic antibodies was contaminating reticulin and capillary basement membranes in the lymphoid organ homogenates used for immunization of horses. Possibly a nontoxic ALS would have been produced if pure suspensions of lymphoid cells were used (Taylor, 1970). 2. Prevention of Anaphylactoid Response to ALG Mild anaphylactoid reactions are frequently encountered in patients given ALG. Such reactions are easily controllable with small doses of antihistamine or prednisone in our own experience, but sometimes serum sickness and serious, rarely fatal anaphylactic reactions ensue ( Starzl et al., 1969a; Tmeger et al., 1971). It is possible that the more serious reactions could be due to relatively impure ALG preparations. Even with the use of relatively pure y-globulin, however, a majority of patients treated with ALG develop rapid immune elimination of equine IgG after 2 to 3 weeks of treatment (Butler et al., 1971; Weksler et al., 1969, 1970, 1971; Traeger et al., 1971), perhaps because, as Lance and Dresser (1967) showed, ALG is much more immunogenic than normal rabbit y-globulin. Taub et al. (1969) found that in a patient receiving no immunosuppressive therapy, prevention of rapid immune elimination and the induction of specific tolerance to ALG were achieved by a single welltolerated intravenous dose of 70 mg./ kg. of normal aggregate-free equine y-globulin. This finding was soon confirmed and extended by other groups (Brendel et al., 1969; Butler et al., 1969; Najarian et al., 1969a,b,c, 1970a,b,c; Weksler et al., 1971). The successful induction of high-zone immune tolerance to equine IgG probably requires dosages in the range of 50 mg./kg. Moberg et aE. (1970) used total doses of 100 to 200 mg. of horse IgG (1-2 mg./kg.). They found no evidence that tolerance consistently developed, and all their patients developed antibodies to ALG. Other factors that might influence the development of tolerance might be the immunogenicity

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and purity of the preparations used as well as the route of administration. The rationale for this maneuver for the induction of tolerance to ALG seems clearly established in experimental animals (Lance and Dresser, 1967). However, because of individual patient variation and the possibility of previous sensitization to horse proteins, pretreatment with deaggregated material combined with an immunosuppressive drug, such as cyclophosphamide, as Butler et al. (1970, 1971; Butler and Rossen, 1971) suggest, or combined with azathioprine and steroids, as advocated by Brendel (1971), might be advisable. Gewurz et al. (1971) pointed out that 4040%of patients treated with 4 to 20 mg./kg. ALG in daily intravenous doses for Iong periods of time became tolerant of IgG. This “high zone” tolerance has its parallel in the mouse (Lance and Dresser, 1967) but cannot be relied upon for consistent clinical results.

3. ALS Treatment and Neoplasia Antilymphocytic serum-treated animals are especially susceptible to the development of certain neoplasms; ALS facilitates the emergence of viral-induced neoplasms (Allison and Law, 1968; Gaugas et aZ., 1969) and may abet the metastasis of an established tumor (Deodhar and Crile, 1969). In man, protracted immunosuppressive therapy of organ transplant patients is accompanied by an increased incidence of neoplasia (Doak et al., 1969; Penn et al., 1969). Penn et al. (1971) listed 40 cases of malignancy developing in renal transplant patients, 11 of these after 236 consecutive transplants performed in Denver over an %year period (corrected incidence of 3.8%vs. 0.058%in a similar sample of the general population), They found an unusually high incidence of reticulum cell sarcoma invading the central nervous system ( 6 cases), a distinctly uncommon predilection for this tumor. Eight of the 40 patients had been given ALG; in one case reticulum sarcoma developed at the gluteal site of ALG injections. All patients had received intensive therapy with azathioprine, prednisone, radiation, and combinations of the three. There is no evidence that the incidence of malignancy among renal transplant patients has increased due to the addition of ALS to the immunosuppressive prograrr, (Starzl et al., 1970b; Penn et aZ., 1971). Starzl et al. (1972) recently reviewed tumor development in renal transplant recipients and collected 57 cases (15 from among their own). They pointed out that ALG-treated cases were not at higher risk, and if anything, the incidence of tumor formation was lower in these patients. It is unlikely that ALG is carcinogenic per se, since mice treated with ALS throughout their lifetimes do not show a higher incidence of tumors

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unless virus is present ( Nehlsen, 1971a,b). Whether ALG operates to curtail “immune surveillance” directly or to abrogate immunity to an oncogenic virus remains to be determined.

4. ALS Treatment and Infection Whether or not the addition of ALS to the immunosuppressive protocol increased the already high incidence of infectious complications is difficult to assess from the clinical reports. Monaco et aZ. (1970) reported a high incidence of fungal, protozoal, and viral infections in ALGtreated recipients of cadaveric renal transplants. Control figures are not given.

D. CLINICAL USE OF ANTILYMPHOCYTIC GLOBULIN 1. Kidney Transplantation The principal clinical use of ALG at present is as an adjunct to the immunosuppressive regimen of azathioprine and prednisone in the management of patients with allotransplanted kidneys. Although ALG is more powerful than any other agent in prolonging the survival of skin grafts in experimental animals, its effects have not been as dramatic in allografts of solid organs. In his early studies, Starzl et aZ. (1967a) found that the combination of ALG and azathioprine was much more effective than either alone in prolonging the survival of renal homografts in dogs. Since in experimental animals ALS is much more effective in inhibiting cellular than humoral immune responses, it is possible that ALS does not effectively counteract the antibody phase of renal graft rejection. A definitive assessment of the value of ALg in organ transplantation is not yet possible. Preparations of ALg used by various groups are not yet standardized; the treatment regimens employed differ from investigator to investigator, and ALG is not the only agent used, The parameters available for evaluation have been few and difficult to interpret, consisting of mortality, incidence of severe, irreversible rejection crises, maintenance of renal function as measured by the blood urea nitrogen or serum creatinine, and comparison with dosage requirements of other immunosuppressives,particularly prednisone. In his earliest studies, Starzl et al. (1967a, 1968) found that patients in an ALG-treated series outperformed previously transplanted, nonALG-treated patients with respect to lower 1 year mortality, lower incidence and reduced severity of rejection crises, and lower requirements for prednisone. These improvements were noted even in the face of the side effects of the antisplenic serum that was used. There was no evidence of either Masugi or serum sickness nephritis in any of the kidneys. Despite the multiplicity of dosage regimens and preparations used,

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subsequent investigators have corroborated that ALS has been a valuable addition to the immunosuppressive regimen in renal honiotransplantation (Doak et al., 1969; Breiidel and Land, 1969; Shorter et al., 1970; Traeger et al., 1968b,c; Fries et al., 1970). Deodhar et al. (1971) treated approximately 50 patients with a full course of ALG (2-3 nig./kg./day) for 3 weeks or more after transplantation, in addition to the usual dose of Imuran and steroids. In the live donor group, ALG-treated patients showed significantly better survival at the 2 year level than those not treated. In the cadaver donor group, the patient survival to 1 and 2 years was similar in the ALG- and nonALG-treated group. Birtch et al. (1971) treated cadaver donor transplant patients with a short (1-2 weeks) course of ALG beginning at the time of transplantation, A greater number of their ALG-treated patients were free of any rejection crisis during the first 3 months and during the first year; also the number of severe rejection crises was lower in this group. However, grafts threatening rejection were given ionizing radiation as well as ALS treatment. Sheil et al. (1971; Sheil and Rogers, 1969) performed a controlled clinical trial of goat ALg in doses up to 600 mg. daily intramuscularly in patients with cadaver renal allografts. There were significantly fewer graft failures in the ALGtreated group in both early and late postoperativc periods. Renal function was measurably better, and the incidence of acute rejection was lower in the ALGtreated group (see also Stevens et al., 1968a,b). Mannick et al. (1971) also evaluated the use of rabbit antihuman lymphocyte globulin in cadaver kidney transplantation. In the ALG-treated group, all acute rejection episodes occurred 6 weeks or more after transplantation (the usual time of such episodes is between 5 and 12 days) and were easily and promptly reversed by a temporary increase in the dose of steroids. It is difficult to determine if an intensive course of ALG might be useful in reversing impending or ongoing acute renal rejection. Mee and Evans (1970) found ALG to be relatively ineffective in combating established rejection. On the other hand, Traeger et al. (1971) felt that 10-20 mg./kg. of ALG per day did improve renal function in acute or subacute rejection crises (see also Brendel and Land, 1969). Most other investigators have not altered the ALG dose during the rejection crisis, but usually increase the dose of prednisone or Imuran to very high levels and often add other agents. 2. Liver Transplantation The advent of heterologous ALG has largely been responsible for long survivals of orthotopic liver grafts (Starzl et al., 1969a,b; Fortner et al.,

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1970, 1971). Usually Iargc dosages of ALG arc required for liver allografted patients because the dose of azathioprine is limited by potential hepatotoxicity. Large doses of ALG must also be used for prolonged periods during liver rejection crises which are more protracted than those seen after renal allotraiisplnntatioii. Liver allografts have been insufficiently studied to determine the magnitude of the immunosuppression obtained and the mechanism of action of large doses of ALG given in these situations. Starzl et al. (1972) (see also Starzl, 1971) point out that the majority of patients with hepatic transplants performed as treatment for hepatoma have succumbed to metast’atic disease, a sequela possibly related to immunosuppression. They now recommend transplantation under these Circumstances only in extreme circumstances.

3. Cardiac Transplantation It is generally felt that inadequate immunosuppression remains the major obstacle to improving the survival of cardiac grafts (Mesmer et al., 1969). The value of ALG added to the usual heavy immunosuppressive drug and radiation regimen in management of the transplant patients remains uncertain (mtbost and Cachera, 1968; Cooley et al., 1969; Stinson et al., 1971). Prolonged survival after cardiac transplant has been documented without ALG (Kahn et al., 1970); nevertheless, controlled studies have not been done to determine the effects of ALG. Usually, threatened cardiac graft rejection has been treated with an increased dosage of methylprednisone. Immunosuppressive doses ( 7 mg./ kg. or greater) of a powerful antiserum are now being tested by Stinson et al. ( 1971) .

4. Bone Marrorv Transplantation Antilymphocytic serum treatment facilitated induction of hematopoietic allogeneic chimerism in mice rendered pancytopeiiic with lethal doses of dimethyl Myleran ( Floersheim and Ruskiewicz, 1969). In man, Math6 et al. (1969, 1970, 1971) found that ALG given to patients with hypoplastic anemia similarly allowed a “take” of marrow from an H-LA nonidentical donor. It was especially remarkable that symptoms of secondary disease were minimal or absent; previously a11 such successful takes were soon followed inexorably by the fatal complications of a GVH reaction. Speck and Kissling (1971) have provided elegant confirmation of the benefits in using ALS to condition recipients for bone marrow transplantation. They studied an experimental model of drug-induced aplastic anemia in rabbits and treated these animals with ALS prior to bone marrow grafting. Untreated animals all developed severe GVH, whereas

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treated animals had excellent hemopoietic reconstitution but did not develop GVH. An explanation for this result probably lies in their observation that the PHA-responsive cells of such reconstituted animals remain of host type, whereas the heniopoietic cells were of donor type. Therefore, ALS treatment seems to have produced n dissociated take of B cells while eliminating T cells.

5. Autoimmune Disease Eiicouraging results have been obtained by several investigators in the treatment of various immunologically mediated or autoimmune diseases with ALS. However, it seems prudent to withhold final assessment of its value until the disease mechanisms themselves are better characterized and provide a clearer rationale for the use of ALS. Trepel et al. (1968) treated 2 patients with dermatomyositis and 1 patient with teniporal arteritis with ALS for 16 to 38 days and noted some improvement in all patients. Pirofsky et al. (1969) used ALS in the treatment of immunologically mediated renal disease. Two of 3 patients with lupus nephritis in whom steroid and Iniuran therapy had failed went into a prompt and good remission; the mechanisms involved are as yet incompletely understood. More recently, Pirofsky et al. (1971) have treated different types of autoimmune disease with goat antihuman ALS. A therapeutic response was obtained in 65%of a heterogeneous group which included patients with lupus nephritis, glomerulonephritis, Goodpasture’s syndrome, lupus arthritis, autoimmune hemolytic anemia, cerebritis, Sjogren’s syndrome, and rheumatoid arthritis. Eight patients with myasthenia gravis also appeared to show some improvement after ALS treatment. Objective criteria of improvement in these cases were not, however, cited and it is difficult to assess the mechanisms by which ALS may have brought about this improvement. Certain patients did not respond to ALS treatment including those with Guillain-Barre syndrome, Haniman-Rich disease, and lupoid hepatitis. Oberling and Hiebel (1971) reported that 2 patients with severe rectal colitis received approxiniately 300 ml. of equine ALS over a period of 3 to 4 weeks after which time both went in remission. Here, too, the mechanisms involved are obscure. Brendel and colleagues ( Brendel, 1971) have used ALG in conjunction with azathioprine, steroids, and thoracic duct drainage in a relatively large number of patients with so-called autoimmune disease. They emphasize the importance of the prior induction of tolerance to ALS-IgG and have found impressive evidence of efficacy (remission) in cases of dermatoniyositis, UPU US

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erythcmatosus, sympathetic ophthalmia, and multiple sclerosis. They cautiously advocate continued clinical trials with ALG in diseases of this kind. Tsirimbas et al. (1968) gave horse antihuman lymphocytic serum to 6 patients with chronic lymphatic leukemia for 12 to 18 days. There was no untoward reaction of any kind. Thirty minutes after the first dose of ALS, there was marked lymphopenia, and in 3 cases the lymphocyte count decreased to half its former value. Otheiwise little improvement was seen. Similar results had been obtained by Monaco et al. (1967a), Pfisterer et n2. (1968), and by Laszlo et al. (1968) with isologous antilymphocyte plasma. Reduction in the size of lymph nodes have been noted as well ( Laszlo et al., 1968; Sanguinetti et al., 1968). XV. Projections for the Future

The ultimate goal of a clinician who embarks upon a system of treatment requiring immunological intervention must be to bring the entirc immunological process under complete control, amplifying some elements in the immune process and suppressing others, as the need arises. Antilyniphocytic serum contributcs to the achievement of this ambition an agent that can selectively suppress the activities of a subclass of lymphocytes, namely that which is responsible for the so-called cellmediated immune reaction. The fact that ALS enjoys this property gives one good reason to hope that it will be possible to devise a conventional chemotherapeutic agent that enjoys this property also. Such an agent might bc a steroid, a mustard, or a member of some hitherto unknown class of immunosuppressive agents. Until it has been devised, ALG will no doubt be in continuous demand because its properties are unique. The imperative need at the moment is to institute an extensive series of searching clinical trials and to overcome the production difficulties which still stand in the way of making sufficient ALG available. These difficulties are not essentially unlike those that have beset every other attempt to scale up a laboratory enterprise to a clinically acceptable level. There is, therefore, no reason to doubt that they can be solved in the next year or two.

ACKNOWLEDGMENTS The authors would like to acknowledge the excellent assistance in preparation of the manuscript rendered by Mrs. Joy Heys and Miss Elizabeth Francis.

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van Bekkuni, D. W., Ledney, G. D., Balner, I%, van Putten, L. M., and de Vries, M. J. (1967). In “Antilymphocytic Serum” (G. E. W. Wolstenholme and M. O’Connor, eds. ), p. 97. Churchill, London. van der Werf, B. A., Monaco, A. P., Wood, M. L., and Russell, P. S. (1967). Surg. Forum 18,241. van der Werf, B. A., Monaco, A. P., Wood, M. L., and Russell, P. S. (1968). In “Advance in Transplantation” ( J . Dausset, J. Hamburger, and G. Math&, eds.), p. 133. Munksgaard, Copenhagen. Volkert, M., and Lundstedt, C. (1968). J. E x p . 1Med. 127, 327. von Ruttger, A., and Kirstaedter, H-J. ( 1969). Hoppe-Seyle/s 2. Physiol. Chem. 350, 1132. von Thierfelder, S., Werner, J., and Gotze, D. (1970). Blut 21, 384. Vredevoe, D. L., and Hays, E. F. (1969). Cancer Res. 29, 1685. Vreeken, J., van Aken, W. G., and Eijsvoogel, V. P. (1969). Lancet 2,594. Wagner, J., and Haughton, G. (1971). J. Nut. Cancer Inst. 46, 1. Waksnian, B. H., Arbuys, S., and Arnason, B. (1961). J. E x p . Med. 114, 997. Wallace, R., Vasington, P. J., and Petricciani, J. C. (1971). Nature (London) 230, 454. Waltman, S. R., Burde, R. M., and Faulkner, H. W. (1969). Transplantation 8, 147. Weil, R., and Simmons, R. L. ( 1968). J. Allergy 29,493. Weksler, M. E., Schwartz, G. H., Stenzel, K., and Rubin, A. I. (1969). Lancet 1, 1361. Weksler, M. E., Bull, G., Schwartz, G. H., Stenzel, K. H., and Rubin, A. I. (1970). J. Clin. Invest. 49, 1589. Weksler, M. E., Bull, G., Schwartz, G. H., Stenzel, K. H., and Rubin, A. I. (1971). Transplant. Proc. 3, 754. Wigzell, H. (1965). Transplantation 3, 423. Wilhelm, R. E., Fisher, J. P., and Cooke, R. A. ( 1958). J. Allergy 29, 493. Wilson, B. J., Malley, A., Cook, M. W., and Mackler, B. F. (1971). J. Immunol. 106,402. Witz, I., Yagi, Y., and Pressman, D. (1968). Proc. SOC. Exp. Biol. Med. 127, 562. Woiwood, A. J., Courtenay, J. S., Edwards, D. C., Epps, H., Knight, R. R., Mosedale, B., Phillips, A. W., Rahr, I., Thomas, D., Woodrooffe, J. G., and Zola, H. (1970). Transplantation 10, 173. Wood, M. L. (1970). Symp. Ser. Immunobiol. Stand. 16,247. Wood, M. L., and Vriesendorp, H. M. ( 1969). Transplantation 7, 522. Wood, M. L., Monaco, A. P., Gozzo, J. J., and Liegeois, A. (1971). Transplant. Proc. 3, 676. Woodruff, M. F. A. (1960). “Transplantation of Tissues and Organs.” Thomas, Springfield, Illinois. Woodruff, M. F. A. (1967a). J. Clin. Pathol. 20, 466. Woodruff, M. F. A. (1967b). In “Transplantation von Organen und Geweben” ( K . E. Seiffert and R. Geissendorfer, eds.), p. 93. Thieme, Stuttgart. Woodruff, M. F. A. (1968). Nature (London) 217, 821. Woodruff, M. F. A. (1969). Endeavour 28, 65. Woodruff, M. F. A. (1971). Transplant. Proc. 3, 34. woodruff, M. F. A., and Anderson, N. F. ( 1963). Nature (London) 200, 702. woodruff, M. F. A., and Anderson, N. F. (1964). Ann. N. Y. Acad. Sci. 120, 119. Woodruff, M. F. A., and Forinan, B. (1960). Quoted in Woodruff (1960). Woodruff, M. F. A., James, K., Anderson, N. F., and Reid, B. L. (1967a). In “Anti-

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In Vitro Studies of Immunologically Induced Secretion of Mediators from Cells and Related Phenomena

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ELMER 1 BECKER' AND PETER M H E N S O N ' Department o f Pothology. University o f Conneclicut Health Center. Farmingfon. Connecticut. and Department of €xperimental Pathology. Scripps Clinic and Research Foundation. La Jolla. California

I. Introduction . . . . . . . . . I1 General Characteristics of Secretory Process . . I11 Mediator Secretion from Isolated Tissues and Organs . . . . . . . A. Perfused Lung . B Lung Slices or Fragments . . . . . IV. Mediator Secretion from Mast Cells . . . . A. Nonimmunological Stimuli . . . . . B. Immunological Stimuli . . . . . . V. Mediator Secretion from Basophiles . . . . A. Human . . . . . . . . . B. Rabbit . . . . . . . . . VI . Mediator Secretion from Platelets . . . . . A Nonimmunological Reactions . . . . . B. Immunological Reactions . . . . . . C . Summary . . . . . . . . . VII . Mediator Secretion from Neutrophiles . . . . A. Nonimmunological Release by Leukocidin . . B. Immunological Release . . . . . . C. Summary . . . . . . . . . VIII . Phagocytosis by Neutrophiles . . . . . A. Adherence . . . . . . . . . B. Engulfment . . . . . . . . C Degranulation . . . . . . . . IX. Mediator Secretion from Monocytes and Macrophages X . Chemotaxis . . . . . . . . . XI . Lymphocyte Transformation . . . . . . XI1. General Summary . . . . . . . . References . . . . . . . . .

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Research was partially supported by U . S . Public Health Service Grant AI.09648 . This is publication No . 32 from the Department of Pathology. University of Connecticut Health Center. Farmington. Connecticut 06032 . 'This is publication No . 610 from the Department of Experimental Pathology. Scripps Clinic and Research Foundation. La Jolla. California 92037. Research is supported by U . S . Public Health Service Grant AI.07007. and the author recipient of U . S . Public Health Service Career Development Award 3.K04.GM-42. 567.0151 . 93

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I . Introduction

The major purpose of this review is to consider the noncytotoxic release of mediators from cells induced in vitro by immunological stimuli. To a much lesser extent, as a background, we shall also discuss the release of mediators from these same cells by nonimmunological means. One of the major emphases of this review will be to demonstrate the overall resemblance of the release of mediators by iminunologically induced, noncytotoxic reactions to secretory processes in general. In addition, consideration will be given to chemotaxis and phagocytosis by polymorphonuclear leukocytes; release of lysosomal enzymes from the latter cells by staphylococcal toxin; leukocidin; aggregation of platelets; and the specifically and nonspecifically induced transformation of lymphocytes. Here, the focus of attention will be not only on the characteristics of these reactions but on the resemblance of these phenomena to each other, to secretory processes in general, and to inimunologically induced, mediator secretion in particular. In the context of this review, the term mediators is restricted to substances whose release is directly or indirectly initiated by an immunological reaction and which are responsible for one or more of the manifestations of the subsequent allergic response (Becker, 1971a). The mediators with which we shall be concerned are those released during the course of antibody-determined (immediate-type ) allergic reactions. Although not all immediate-type allergic reactions have their major manifestations determined by mediators, many do, including diverse and important responses such as anaphylaxis, hay fever, some forms of asthma, the Arthus reaction, certain kinds of experimental and human glomerulonephritis (reviewed in Becker, 1971a). The mediators themselves may be divided into two classes: those of low and those of high molecular weight. The low molecular weight mediators include histamine, serotonin, the ltinins, slowly reacting substance of anaphylaxis (SRS-A), and eosinophile chemotactic factor of anaphylaxis ( ECF-A) . Some of the high molecular weight mediators are the lysosomal enzymes and cationic proteins of the polymorphonuclear leukocyte, heparin, the complement-derived factors such as anaphylatoxins, C3a and C5a, and the high molecular weight cheinotactic factor, Css? (reviewed in Becker, 1971a). Mediators may arise from purely humoral enzyme systems such as the kinin-forming system and the complement system or from specific cells, the so-called mediator cells. Only the mediators originating in cells are among the subjects of this review, thus, kinin formation or complement activation with the formation of biologically active fragments, although of great importance, will not be considered.

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There are two gcneral mechanisms of cellular mcdintor release: the cytotoxic and the noncytotoxic. Cytotoxic release is due to a generalized irreversible destruction of the permeability control of the mediator cell with the efflux of mediator being part of the nonselective loss of cytoplasmic constituents. As its name implies, death of the cell is the U S L outcome. Immunological cytotoxic release of mediators usually results from the action of the terminal components of complement activated either through the entire complement sequence or through the properdin system. Nonimmunological cytotoxic mediator release is represented by the cytolytic histnmine-releasing activity of n-decylamine or similar agents on mast cells generally, of high concentrations of compound 48180, a condensation product of N-methyl honioamisylamine and formalin, on guinea pig mast cells, of leukocidin on neutrophiles, or of high concentrations of thrombin on platelets. The cytotoxic mechanisms as such, important as they may be under a number of circumstances, will not be dealt with further. Noncytotoxic mediator release differs profoundly from cytotoxic release in a number of major respects. In the former, there is a highly selective release of the mediator or mediators. Although there may be a release of some of the other constituents of the granule within which the mediators are contained or the entire granule itself, there is little or no loss of other cytoplasmic constituents, and no resulting death of the cell. In what follows, we shall consider the results of recent in uitm studies of immunological, noncytotoxic, mediator release by organs and tissue slices and by isolated cells such as mast cells, basophiles, platelets, and neutrophiles. Little attention will be paid to the nature of the antibodies involved, except for their role in sensitization of target tissue and cells; the reader interested in these other aspects of antibodies is referred to recent reviews such as those of Bloch and Ohman (1971), Zvaifler et al. (1971), Vaz (1971), Bennich and Johansson (1971), and Ishizaka and Ishizaka ( 1971 ) . As early as 1958, Smith suggested that release of histamine from rat peritoneal mast cells by protamine sulfate or polymyxin does not require cell disruption and death, and wrote of the mast cell as “an endocrine cell which elaborates histamine and other products and secretes them when appropriately stimulated” (Smith, 1958). At about the same time, platelets were shown to release vasoactive amines and nucleotides in a noncytotoxic manner (Grette, 1962). Later, Becker (1968) and Lichtenstein ( 1968) independently suggested that the immunological release of mediators from the appropriate cells is noncytotoxic and may involve a mechanism analogous to secretion. The selective nature of the release from platelets, as well as other characteristics (see Section V ) have led a number of observers ( Holmsen et al., 1969; Stormorken, 1969;

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Davey and Liischer, 1968a; Mustard and Packham, 1970; Salzman, 1972) to consider the release reaction a form of secretion. The diversity of topics considered in the discussion of the general characteristics of the secretory process means that in most cases we have to cite reviews rather than primary references. In discussing the release of mediators by nonimmunological stimuli, we usually refer to the latest work on a given topic rather than cite all the references. In building up the necessary background for considering the newer work on immunologically induced secretion and related phenomena, considerations of space necessitate referring to reviews of the older literature rather than citing the original papers, although these older contributions are fundamental to our present knowledge. In addition, the vast amount of literature on the various topics makes it inevitable that we shall fail to cite some pertinent references. 11. General Characteristics of Secretory Process

Since one of the major tasks of this review is to emphasize the secretory nature of immunologically induced noncytotoxic mediator release, we shall start by considering the general characteristics of the secretory process. There are a large number of different kinds of cells that secrete many different kinds of substances. A few examples of this diversity are the secretion from the pancreas of insulin from /3 cells, glucagon from cells, and the enzymes, amylase, chymotrypsinogen, ribonuclease, etc., from the acinar cells, catecholamine secretion from the chromaffin cells of the adrenal medulla, the release of acetyl choline from nerve endings, the secretion of vasopressin and oxytocin from the neurohypophysis, and the various hormones from the anterior pituitary. All the secretory processes just mentioned, as well as others, have a number of features in common. In very many instances, in fact in all of the above cases, the secretory product or products are stored in membrane-bound granules or vesicles: insulin and glucagon are stored in and /3 cells of the pancreas, respectively; amylase, granules in the chymotrypsinogen, etc., are stored in zymogen granules of the pancreatic acinar cells; granules of the chroniaffin cells of tlie adrenal niedulla contain epinephrine and norepinephrine; nerve endings of tlie neurohypophysis contain granules in which are stored oxytocin and vasopressin; and tlie hormones of the anterior pituitary are also stored in cell granules. The process of secretion involves the movement of tlie granule to tlie cell membrane and a fusion of a smaller or greater number of the granules with it. In some instances, as in the case of insulin secretion, the fusion leads to “erosion” at the point of contact, and the intact p granules, devoid of their encasing membrane, are discharged extracellularly ( Lacey, (Y

(Y

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1970). Likewise, in the secretion of epinephrine (Poisner, 1970a), the entire granule may be discharged into the exterior, although the granule membrane is left in the cell (cf. mast cells in Section IV,A). The general process has variously been termed emiocytosis, reoerse pinocytosis, reuerse phngocytosis, exocytosis, as well as exoplasmosis. Several of the granule membranes, for example, that of the chromaffin granule and of the granule of the platelets, have been shown to have an adenosinetriphosphatase (ATPase) system, and in theories of the mechanism of membrane fusion, a role for this ATPase system as well as for Ca2+and adenosinetriphospliate ( ATP ) has been suggested ( Mathews, 1970; Poisner, 1970a; Woodin and Wieneke, 1970a; Poste and Allison, 1971). Secretion requires cellular metabolic energy. In many cases, as in the release of amylase from the pancreas (Schramm, 1967) the cells are dependent on oxidative phosphorylation for their supply. However, in the case of insulin secretion, inhibition by 2-deoxyglucose or mannoheptulose suggests glycolysis is the source ( Levine, 1970). There is also an absolute requirement for external Ca?+ in all the secretory processes mentioned so far, as well as in others (Rubin, 1970). In a number of instances, such as secretion from the neurohypophysis or adrenal medulla, or insulin from the pancreas, the requirement for external Ca2+is associated with an uptake of Ca2+when the cell is stimulated to secrete (Rubin, 1970). Whether or not this is absolutely general is made uncertain by the report of Bygdeman and Staijne (1971) that, although platelet aggregation and secretion induced by collagen are Ca*+-dependent, no associated Ca2+ uptake is demonstrable. However, Mg2+in the external medium plays a more variable role. In a number of instances, as in the release of epinephrine from the adrenal medulla (Douglas, 1966) or insulin from the pancreas (Malaise et al., 1970), Mg“ antagonizes the action of Ca2+. Another common feature in the secretory process is the important role played by 3’,5’-adenosine monophosphate ( cyclic AMP). Increased production of cyclic AMP through activation of the enzyme, adenyl cyclase, has been shown to be associated with secretion of hormones of the anterior and posterior pituitary, amylase release from the salivary gland, steroid secretion from adrenal cortex, acetylcholine secretion from nerve endings (Goldberg and Singer, 1969), and insulin and glucagon release from the pancreas, among others. Cyclic AMP in these instances is postulated to act as the “second messenger” ( Robison et al., 1971). The p-adrenergic drugs, such as isoproterenol and epinephrine, increase the adenyl cyclase activity of many cells. Norepinephrine and other a-adrenergic agents, under some circumstances, decrease adenyl cyclase activity. In 1961, Douglas and Rubin termed stimulus-secretion coupling the

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sequence of events from stimulation to secretion and considered Caz+as the agent concerned. This phrase was patterned on the term stimulus contraction coupling applied previously to muscle by Sandow and was coined to draw attention to the parallelism between secretion by cells of the adrenal medulla and the contraction of muscle. Since that time, evidence has accumulated that there is more than a parallelism: apparently secretion involves contractile processes of the secretory cells (for example, see Stormorken, 1969; Poisner, 1970a). The evidence at present is largely indirect, resting in part on the demonstration of the inhibition of secretory processes by agents such as colchicine and the vinca alkaloids, vinblastine, vincristine, etc., which affect microtubules. Colchicine and related products inhibit catecholamine release from the adrenal medulla (Poisner and Bernstein, 1971), insulin release (Lacey, 1970), and thyroid secretion (NBve et al., 1971). More recently, the mold product, cytochalasin B, which apparently acts on microfilaments in many cells ( Wessels et al., 1971), has been shown to inhibit thyroid secretion (Williams and Wolff, 1971) and the secretion of growth hormone (Schofield, 1971). The effects of colchicine (Gabbay and Tze, 1972; Trifar6 et al., 1972) and cytochalasin B (Estensen and Plagemann, 1972; Cohn et al., 1972; Haslam, 1972) are not restricted to microtubules and microfilaments, respectively, so that the interpretation of the results of their use must be viewed with caution. There is also the growing realization that very many diverse cells contain contractile protein systems similar in many aspects to actinomyosin of muscle, the so-called actinomyosinoid systems, and that the microtubules and microfilaments may well consist of such systems, These actinomyosinoid proteins have been shown to be present in bacteriophage, bacteria, fungi, plant cells, protozoa including ameba, echinoderm eggs and sperm, erythrocytes, platelets ( thrombosthenin), brain, liver mitochondria (Jahn and Bovee, 1969), and the neutrophile (Senda et al., 1969) among other cells. The presence of actinomyosinoid protein in the adrenal medulla has been cited as being consistent with the hypothesis of a contractile mechanism in catecholamine release (Poisner, 1970b). It is also of interest in this regard that purified microtubular protein is phosphorylated by a cyclic AMP-dependent kinase (Murray and Froscio, 1971). Despite the great advances in our knowledge and ideas of the secretory process, there is no generally accepted, detailed picture of the mechanism of stimulus-secretion coupling in cells. There is, however, general agreement that, although details may well differ from cell to cell, the secretion process is generally similar in different cells and bears a general resemblance to basic biological processes such as cell movement, muscle contraction, and nerve transmission. As already stated, one of

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the major purposes of this review is to demonstrate that diverse inimunological phenomena such as in uitro, immunological, mediator release from organs and tissues, from mast cells, basophiles, platelets, and neutrophiles; phagocytosis and chemotaxis in neutrophiles, transformation of lymphocytes, and, possibly, even antibody synthesis, share, to the extent that they have been studied, these similarities. Ill. Mediator Secretion from Isolated Tissues and Organs

The great bulk of the work on mediator release from isolated tissues and organs has utilized the perfused lung or lung slices, and so our attention will be wholly confined to these systems. The tissue mast cells probably provide the source of the histamine released when sensitized, guinea pig, lung slices or perfused, sensitized, guinea pig lungs are challenged with antigen, since the proportion of histamine released correlates very well with the number of mast cells in these tissues and the extent of their degranulation (reviewed in Mota, 1966). In human lung slices, sensitized with human heat-labile homocytotropic antibody (probably YE) and challenged with antigen, Parish (1967) was able to demonstrate a correlation of the release of histamine and SRS-A (see below) with changes in the mast cells. The inference that lung mast cells are the target of the antigen+ antibody or YE-anti-yE reactions is supported by the finding of Tomioka and Ishizaka (1971) that the major and possibly only cell type in monkey lung that binds YE imniunoglobulin is the mast cell. The evidence, however, is not yet sufficient to exclude the possibility that tissue cells other than mast cells are the target of YE antibody-determined reactions. Uvniis and Thon (1959) reported that low levels of SRS-A release are obtainable from rat peritoneal mast cells. Ishizaka et al. (1972a) were able to obtain suspensions of cells from monkey lungs containing 1-10% mast cells. Anti-IgE added to the cell suspension released not only histamine but small amounts of SRS-A. The authors considered that the results were suggestive but not conclusive that the mast cell was the source of both mediators. As the authors point out, their studies do not rule out the possibility that the mast cell is the source of an agent that acts on another cell to release SRS-A. Almost all in vitro studies of SRS-A release have used guinea pig, human, or inonkey tissue slices or perfused whole lung. Austen and his co-workers (Orange et al., 1969, 1970) using the rat peritoneum as an “in vivo test tube” have shown that heat-stable rat IgGa antibodies release histamine and SRS-A after a 2-hour latent period. The source of the histamine but not of the SRS-A is the mast cells of the peritoneal cavity. Peritoneal polymorphonuclear leukocytes and complement are required for the IgGa-mediated release of

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SRS-A in duo, and the polymorphonuclear leukocyte may be the source of the SRS-A. This, however, is not certain, since SRS-A release has not yet been demonstrable in vitro from these cells. Sensitization of the rat peritoneum with rat yE ( heat-labile, homocytotropic) antibodies followed by challenge with antigen after a 2-hour latent period leads to the release of both SRS-A and relatively small amounts of histamine. Under these circumstances, the release of both mediators depends on the presence of mast cells but is independent of the presence of neutrophiles (Orange et al., 1970). The optimal latent period for the release of SRS-A is 2 hours; on challenge with antigen at 12 hours, release of SRS-A is barely detectable. The capacity to mediate the release of histamine persists essentially unchanged for 48 hours. This persistence is consistent with the persistence of the fixation of YE antibody to the target cell, whereas, the limited duration of the ability of yE antibody to release SRS-A suggested to Austen and his co-workers that the action of an immunological complex was involved; however, other interpretations are possible. Thus, in summary, it is very probable that the mast cell is the source of histamine released from the isolated tissues or organs to be considered here. This cell may also be the source for SRS-A and ECF-A when the tissue is sensitized by YE antibody, although the evidence is far from compelling. The source of the SRS-A in the rat peritoneum sensitized with rat yGa antibody is not the mast cell; it may be the polymorphonuclear leukocyte, but the evidence for this is still indirect. In species other than the rat, the source of SRS-A and other mediators from tissues sensitized with the appropriate heat-stable homocytotropic or heterocytotropic antibody is even less well-known.

A. PERFUSED LUNG Histamine release accompanied by mast cell degranulation is obtained from unsensitized guinea pig lung perfused with soluble complexes prepared in excess antigen with either rabbit yG antibody or guinea pig yl or yz antibody. The last is of interest since guinea pig lung is not passively sensitized for antigen-induced histamine release by perfusion with guinea pig y 2 antibody but is sensitized with either guinea pig yl or rabbit antibody (Broder, 1969a). Further studies with lungs perfused with antigen-rabbit yG antibody complexes showed that histamine release is inhibited by lack of Ca2+in the perfusing fluid, or if the lung is heated to 45"C.,or following the addition of iodoacetate, N-ethylmaleimide, phenol, epinephrine, or theophylline, or on increasing the concentration of sodium chloride or sucrose in the perfusing fluid ( Broder and Taichman, 1971) . Succinate, maleate, or disodium chromoglycate enhance histamine release

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by soluble complexes. The effect of inhibitors and enhancers is the same as found previously with histamine release from sensitized, guinea pig, lung slices challenged with antigen or when whole lung is perfused with rabbit antiserum to guinea pig y-globulin (Broder and Baumal, 1972). Histamine release by perfusion of the lungs with soluble complexes is temperature-dependent. Perfusion at 20°C. gives no histamine release but a large degree of desensitization. This temperature-dependent desensitization is similar to but less than with whole lung passively sensitized with antibody and challenged with antigen. In general, the perfused, whole lung system is much more easily desensitized than the passively sensitized, chopped lung system. This difference between the whole lung and chopped lung systems is unexplained. Interestingly and also inexplicably, antigen-antibody complexes do not release histamine from chopped lung ( Mota, 1966). Anaphylatoxin prepared by reacting agar with fresh guinea pig or rat serum releases histamine when perfused through whole guinea pig lung. Factors influencing this release are similar to those affecting release by antigen-antibody complexes except that phenol or lowering the temperature does not inhibit, nor does succinate enhance, histamine release by anaphylatoxin ( Broder, 1970). Normal rabbit y-globulin but not bovine y-globulin inhibits the histamine-releasing activities of antigen-antibody complexes made with both rabbit and guinea pig antibody. The Lineweaver-Burke plots for the dose-response curves of soluble complexes in the presence and absence of rabbit y-globulin are compatible with the hypothesis that 7-globulin inhibits by competing for a receptor, presumably on the mast cell of the lung. This receptor is apparently common to complexes prepared with rabbit or guinea pig antibody (Broder, 196913). The inhibiting effect of 7-globulin in whole serum might explain why increasing the length of time of preliminary perfusion with buffer increases the amount of histamine released on subsequent challenge with antigen (Broder and Schild, 1965). It might also explain the inability to demonstrate degranulation or histamine release when antigen-antibody complexes are administered intravenously to guinea pigs ( Mota, 1966).

B. LUNGSLICESOR FRAGMENTS

1. Sensitization Slices or fragments from the lungs of guinea pigs, monkeys, and humans have proved very useful to study the nature of the antibody involved in passive sensitization, the mechanisms of passive sensitization, and the mechanisms of mediator release. Rabbit antibody passively

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sensitizes guinea pig lung slices for SRS-A and histamine release, and guinea pig yl but not y? antibody sensitizes the same tissue for the release of histamine, SRS-A (Baker et al., 1964), and ECF-A ( Kay et al., 1971). Human yE antibody sensitizes monkey (T. Ishizaka et al., 1970) as we11 as human lung slices (Orange et al., 1971a) for SRS-A and histamine release and for ECF-A release (Kay and Austen, 1971). Goat antihuman IgG and antihuman IgE both release histamine from washed, normal, human lung slices but the anti-IgE antibody was distinctly more reactive. Whether the activity of the anti-IgG antiserum was due to the specific antibody or to contamination with anti-lightchain or anti-IgE is not certain. Anti-IgA and anti-IgM antisera showed no histamine-releasing activity (Paul and Weir, 1969). Studies of the mechanism of sensitization of guinea pig lung slices illustrate the difficulties of interpretation attendant upon the use of such a highly heterogeneous material. Target mediator cells undoubtedly make up only an extremely small portion of the total cellular and noncellular mass of the slice, and the uptake of antibody is probably largely directed to nontarget structures. The latter is suggested by the findings of Brocklehurst and Colquhoun (1965) that the uptake of guinea pig yp antibody by uiiseiisitized lung slices is approximately the same as that of guinea pig y l antibody, even though only the yl sensitizes for histamine release. The degree of passive sensitization of guinea pig slices with rabbit antibody decreases greatly as the temperature of sensitization is lowered. This large temperature coefficient for passive sensitization, the nature of the inhibitory effect of iionantibody y-globulin, and the slow reversal of sensitization with time suggested to hlongar and Schild that antibody was attached to its cellular receptors at more than one point, and Mongar and Winne (1966) suggested a double attachment of antibody to cell receptors. The latter researchers pointed out, that their model is not unique, and this was further emphasized by Colquhoun (1968). Binaghi ( 1968) considered that the inhibition of sensitization obtained with nonspecific y-globulin is due not to competition with antibody for sites on the cell but to association of antibody with the nonspecific yglobuIin. E. Colquhoun (personal communication to E. L. Becker ) has pointed out that in order for Binaghi’s theory to fit his results, he has to assume that the putative antibody-y-globulin dinier dissociates to a negligible extent. This is unlikely. Work on the sensitization of mouse niast cells by mouse yl antibody (Vaz and Prouvost-Danon, 1969) and rat mast cells with rat yGa (Bach et al., 1971a) (see below) indicates a very rapid dissociation of these kinds of antibody from their respective cells which is independent of the time of contact of cell and antibody. One would also expect either rabbit

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rG antibody or thc analogous guinea pig 7 , antibody to dissociate rapidly from guinea pig, lung mast cells, Yet, the decrease of sensitization on washing with buffer in the passively sensitized lung system is slow and the rate of decrease falls with time of sensitization. As Brocklehurst aiid Colquhouii (1965) emphasize, the slow rate of reversal cannot be accounted for by diffusion through extracellular space. Spuzic et al. ( 1966), finding that the addition of nonspecific ./-globulin to purified rabbit antibody appeared to interfere much more with sensitization of guinea pig lung at 4°C. as compared to 37"C., suggested that the apparent high energy of activation of the sensitization process might largely reflect the energy required to overcome interference by nonspecific 7-globulin. However, the work of Sullivan et al. (1971) suggests that there is a temperature-dependent redistribution of antibody over the surface of human basophiles (see Section IV,B,l). If this is a general obligatory phenomenon in sensitization, it might also account for the high-temperature coefficient of sensitization.

2. Mechanism of Secretion u. Guinea Pig Lung. The earlier work reviewed by Austen and Humphrey (1963) and by Schild (1968) indicated that antigen-induced histamine and SRS-A release from sensitized guinea pig lung slices requires Ca?+, a heat-labile factor, and free sulfhydryl groups. Sr2+can replace Ca2+,and Ba2+and Mg2+antagonize the effects of Ca2+(Mongar, 1970). The release also requires the activation of a serine esterase, possibly having chymotrypsin specificity ( Becker and Austen, 1964), which differs from that responsible for antigen-induced histamine release from sensitized, rat, peritoneal mast cells ( Becker and Austen, 1966). This latter difference might well be another example of the functional variabiIity of mast cells from different species and different sites, discussed subsequently ( Section IV) . Histamine and SRS-A release is inhibited by increase in the ionic strength of the medium aiid is potentiated by its decrease. Ouabain inhibits histamine release, suggesting a possiblc role for the Na+ p i m p (Yarnasaki and Endo, 1965). Elimination of Na+ and substituting isotonic sucrose decreases histamine release but does not completely prevent it ( Mongar and Schild, 1958). Whether the decrease is due to the lack of Na+ or the presence of sucrose is not known. Mediator release is also inhibited by monobasic fatty acids and disulfide and sulfhydryl inhibitors and is increased by diabasic acids such as succinate, maleic, glutaric, and ketoglutaric. The enhancement by the latter group of compounds does not occur through the Krebs cycle as indicated by the lack of effect of fumarate and citrate (Austen and Humphrey, 1963; Schild, 1968). Inhibition by anoxia is reported to occur in the presence of

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glucose if the oxygen is rigorously excluded (Edman et al., 1964). This inhibitory effect of anoxia in the presence of glucose was attributed by the authors (see also Schild, 19fB) to the reduction of tissue disulfide bonds. This is quite possible; but in the absence of glucosc the requirement for complete anoxia for inhibition is not so stringent, and this inhibition is reversed by glucose ( Diamant, 1962 ) . Phlorizin inhibits glucose-dependent histamine release from anoxic tissue slices, but this action of phlorizin is reversed by pretreatment of the slices with insulin. Deoxyglucose prevents histamine release to a greater extent in the absence of oxygen than in its presence (Yamaski and Endo, 1965). In the presence of glucose, KCN and other cytochrome oxidase inhibitors have no effect on histamine release (see Austen and Humphrey, 1963). In the absence of glucose, very low concentrations of antimycin A, a specific inhibitor of the cytochrome chain, and also 2,4-dinitrophenol inhibit histamine release, and this is reversed by the addition of glucose (Prouvost-Danon, 1968). These results suggest that normally respiration may be the source of energy for mediator release in guinea pig lung, but anaerobic glycolysis can substitute when this source is blocked by O2 lack or inhibition of the respiratory chain. This would make the situation in the guinea pig lung similar to that in the rat and mouse peritoneal mast cell (Sections IV,A,l,A,2,B,l, and B,2) and to muscle contraction. Very low concentrations ( 10-8-10-9M ) of sympathominietic aniiiies inhibit antigen-induced histamine release from actively ( Assem and Schild, 1971a) and passively (Assem et al., 1970) sensitized, guinea pig lung slices, and this inhibitory action follows their order of activity as known p-adrenergic stimulants. Phosphodiesterase inhibitors, such as theophylline and ICI 30966, also inhibit histamine release and comiderably enhance the inhibitory activity of low doses of the p-adrenergic agents (Assem and Schild, 1971a). The inhibition by p-adrenergic agents is prevented by p-adrenergic blockers, such as propranolol, practolol, and butoxamine (Assem and Schild, 1971b). These results, as well as others in other cells and tissues (see below), are compatible with the hypothesis that an increase of intracellular cyclic AMP inhibits immunologically induced histamine release. It is also possible, although it does not necessarily follow, that histamine release is associated with a decrease in intracellular cyclic AMP. Mongar and Schild (reviewed in Schild, 1968) early found that chopped, sensitized guinea pig lung when reacted with antigen in the presence of inhibitors failed to release histamine after washing and rechallenge. They suggested that this desensitization was due to the decay of an activated intermediate, probably an enzyme. b. Human Lung. Orange et al. (1971b) have shown that histamine

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and SRS-A release from hun~anlung, passively Sensitized with y E antibody, is inhibited by deovyglucose in the absence of glucose, Suggesting a role for glycolysis as the energy source. Iodoacetic acid also inhibits mediator release, but can be prevented by pyruvate, suggesting that the inhibition by iodoacetate occurs on the pathway to the production of pyruvate. I t also suggests that pyruvate might be able to support histamine release through aerobic oxidation. There is no other evidence available as to whether or not aerobic oxidation in the presence of glucose inay also serve as an energy source, as just suggested for the guinea pig lung system. Diisopropyl fluorophosphate ( DFP ) inhibits both SRS-A and histamine release; preincubation of the lung with DFP and then washing does not result in prevention of antigen-induced histamine release but does prevent the release of SRS-A. This suggests that immunological histamine release from human lung involves an activatable esterase. It is possible but not certain that SRS-A release requires an activatable esterase and in adclition, an “activated’ esterase, as found for cheniotaxis (Soe Section X ) . Synipathoniimetic aniines inhibit antigen-induced histamine (Asseni and Schild, 1969) as well as SRS-A release (Orange et al., 1971a) from passively sensitized human lung. The inhibition is markedly enhanced by theophylline and prevented by propranolol and other ,8-adrenergic blockers (Orange et al., 1971b; Assem and Schild, 1971b). Diethylcarbaniazine inhibits the release of both histamine and SRS-A, and a marked synergism is observed with sympathomimetic amines ( Orange et al., 1971a) similar to that observed with the immunological release of histamine from monkey lung slices (Ishizaka et al., 1971b). Propraiiolol combined with either epinephrine or norepinephrine not only prevents the inhibitory activity of the catecholamines but the combination actually increases the release of SRS-A and histamine (Orange et al., 1971a). These results suggested to the authors that increases in cellular levels of cyclic AMP induced, for example, by ,8-adrenergic stimulation are inhibitory, whereas, decreases in cellular levels of cyclic A M P induced by n-adrenergic stimulation enhance the antigen-induced release of the mediators. This conclusion was supported by findings that the changes of cyclic AMP levels in human lung fragments after treatment with various agents directly and conipletely parallel the changes hypothesized to occur with mediator release (Kaliner et al., 1971; Orange et al., 1971~). However, because of the cellular heterogeneity of lung fragments, the changes in cyclic A M P levels were not measured on the same cells from which the mediators were released; thus, the congruence in results is strongly suggestive and tantalizing but not probative. Cholinergic stiniulation with carbamylcholine ( carbachol ) enhances

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the immunological release of histamine and SRS-A release, which is prevented by atropine. This cholinergic stimulation seems to be independent of the levels of cyclic AMP (Kaliner et al., 1972) and suggests that immunological release of mediators from human lung is not only under adrenergic but also cholinergic control. Antigen-induced histamine and SRS-A release from monkey lung fragments passively sensitized with antiserum containing y E antibodies is affected by agents that increase and decrease levels of cyclic AMP in a fashion similar to that just described for human lung (Ishizaka et al., 1971b). The effect of agents such as colchicine or cytochalasin B on secretion from sliced lung systems has not been reported. IV. Mediator Secretion from Mast Cells

At present, the mast cells of the peritoneal fluid of the rat and mouse are the only isolated mast cells suitable for the study of the mechanisms of their mediator secretion (see, however, Ishizaka et aZ., 1972b; Patterson et al., 1972). The peritoneal as well as the tissue mast cells of these rodents contain and can be stimulated to secrete both serotonin and histamine, unlike mast cells from other species which contain only histamine. This variation is in accord with the general finding that mast cells from different species or even in different sites within the same animal may differ functionally and morphologically. For example, compound 48/80 is a very effective histamine releaser for rat peritoneal mast cells (IV,A,l) but at low or even at moderately high doses has no effect on mouse peritoneal mast cells, guinea pig mast cells, human or rabbit basophiles, or rabbit platelets, and at very high concentrations acts as a general lytic agent for guinea pig mast cells (Mota, 1966; Haye and Schneider, 1966; Vaz and Prouvost-Danon, 1969), These and other differences are probably correlated with variations in the detailed mechanisms of induced secretion but, as the work under review will bring out, there are sufficient likenesses in the modes of mediator release from mast cells of different species and different tissues to give reasonable assurance that the general processes are similar. A. NONIMMUNOLOGICAL STIMULI A number of nonimmunological agents, polypeptides such as mellitin from bee venom, and lysosomal cationic protein from neutrophiles (band 2 protein), 48/80, polyniyxin B, d-tubocuraiine, morphine, stilbamidine, and ATP, degranulate and release histamine and serotonin from mast cells without any general change in the outward permeability of the cell (Diamant, 1967; Johnson and Moran, 1969a; Ellis et aZ., 1970).

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Moreover, the morphological and functional resemblance between the mediator release induced from rat mast cells by these agents and by one or another variety of noncytotoxic antigen-antibody reaction is sufficiently close to make the responses to these nonspecific mediator releasers useful models for study of the inimunological mechanisms ( Bloom and Haegermark, 1965; Bloom et al., 1967, 1970; Bloom and Chakravarty, 1970; Lagunoff, 1972a). The bulk of the information concerning the induced secretory response to these nonimmunological agents has been amassed using 48180, the cationic protein from neutrophile lysosomes (band 2 protein), and ATP. It is mostly the results obtained with these nonspecific agents which will be described in the following.

I , Compound 48/80 as a Stimulant for Mediator Secretion by Mast Cells Bloom and Haegermark (1965) and Bloom and Chakravarty (1970) have documented the sequence of morphological changes that rat mast cells undergo in response to both compound 48/80 and immunological stimulation. At the lowest concentration of 48/80, only a few cells are altered, and there is little or no extrusion of granules extracellularly. The number of altered granules, extruded granules, vacuoles formed, and the amount of histamine released increase with increasing concentration of 48/80. The perigranular membrane is left inside the cell following extrusion of the naked granule. The granule appears to be extruded following a propagated fusion of the perigranular membrane with the external cell membrane. Both Uvnas and Thon (1966) and Lagunoff (1966, 1972c) have advanced the theory that the binding of histamine and serotonin to the rat mast cell granule is purely electrostatic and their release from the granule is due to a simple ion exchange of the positively charged amines with the Na+ of the extracellular fluid. In this view, mediator secretion is essentially a two-stage process. The first stage is an active, energy-dependent extrusion of the granule to the outside of the cell, and the second is exchange of the amine ( s ) with the Na' of the extracellular fluid. This simple view was seemingly contradicted by the morphological studies alluded to above in which histamine release was associated with alteration of granules which were obviously intracellular and by the work of Carlsson and Ritzen (1969). They demonstrated that some of the intracellularly altered granules no longer contained serotonin and that the percentage loss of dry mass due to granule extrusion was less than the percentage loss of serotonin. These studies were compatible with the conclusion that mediators could be released intracellularly without extracellular granule extrusion.

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However, Uvnas and his co-workers (Rohlich et al., 1971; reviewed by Uvnas, 1971), using 48/80, and Lagunoff ( 1972a), employing polymyxin B, have “preserved the phenomenon” by presenting biochemical and inorphological evidence that granules are not only extruded into the extracellular fluid outside the domain of the cell but also into intracellular compartments in direct communication with and containing extracellular fluid. In the latter instance, the Na+ in this extracellular fluid is then exchanged with the serotonin or histamine of the granule so exposed to give rise to the “intracellularly” altered granules. Although, the detailed view of how the extracellular spaces are created within the cells differs between Uvnas (1971) and Lagunoff (1972a,b) the results they report and their overall interpretation agree very well. Whatever the detailed mechanism, there is now good reason to believe that the overall process for noncytotoxic mediator release in rat mast cells, in fact, is a two-stage process: the first stage is an energy-requiring process to bring the granule in contact with extracellular fluid either inside or outside the cell; the second stage is an ion exchange of histamine or serotonin for the Na+of the extracellular fluid. At present, the functional and morphological differences within and between mast cells of various species as well as the differences among mediator cells make this two-stage concept a hypothesis the generality of which is to be tested rather than a conclusion to be universally applied. Pertinent in this regard may be the finding of Mongar and Schild (1958) that omission of K+ or the substitution of sucrose for Na+ decreased antigen-induced release of histamine from sensitized, guinea pig lung slices but did not eliminate it. Pruzansky and Patterson (1968) found that antigen-induced histamine release from sensitized human leukocytes occurs in a low ionic strength medium-a medium that retains a high proportion of the histamine in the isolated intact granule. It is also of interest that during anaphylaxis in guinea pigs, electronmicroscopic studies of mast cells showed intracellular fragmentation of their granules but no extracellular expulsion ( Taichman, 1971). Mediator release induced by 48/ 80 or by immunological reactions does not result in the loss of the cytoplasmic enzyme, lactic dehydrogenase (LDH), nor of ATP and very little of the K+ of the cells ( Diamant, 1967; Johnson and Moran, 1969a), and these cells are still capable of excluding trypan blue (Johnson and Moran, 1969b). Moreover, mast cells are able to undergo repeated cycles of histamine release by 48/80 (Uvnas, 1968). These observations are in accord with the morphological findings cited above and together provide clear-cut experimental evidence that mediator release and mast cell degranulation under these circumstances is undoubtedly noncytotoxic and, thus, may very well be a

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true secretory phenomenon. These findings are also in accord with the two-stage concept of mediator secretion described above. Diisopropyl fluorophosphate inhibits histamine release by 48/80 (Van Arsdel and Bray, 1961). Whether this results from preventing the function of an activatable esterase, as seems operative in the release of histamine from rat mast cells by immunological and other nonimmunological means (Sections IV,A,2 and IV,B,l), is not clear but is obviously worthy of investigation. Rat peritoneal mast cells have a high rate of anaerobic and aerobic glycolysis in the presence of glucose ( Chakravarty, 1965).The respiratory rate although distinctly lower is still substantial (Chakravarty and Zeuthen, 1965). Saeki (1964) showed that KCN, 2,4-dinitrophenol, or anaerobiosis inhibits histamine release from isolated mast cells by 48180 in the absence of glucose but not in its presence. Glucose has no effect on the inhibition induced by iodoacetate. These results indicate that, as in other histamine-releasing systems, 48/80 requires energy for mediator release. They suggest that like the antigen-induced release of histamine from guinea pig lung tissue (Section II1,B) and in muscle contraction, the energy for mediator secretion by 48/80 in the presence of oxygen is supplied by an oxidative process, whereas, in its absence and in the presence of glucose, it can be supplied by glycolysis. The inability of phlorizin and deoxyglucose to inhibit degranulation of rat peritoneal mast cells by 48/80 under aerobic conditions in the absence of glucose, but their ability to do so under anaerobic conditions in the presence of glucose ( Saeki, 1964), is in accord with this concept. Van Arsdel and Bray ( 1961), Uvnas and Thon ( 1961), and Saeki (1964) have all reported that 48/80 acting on isolated mast cells gives as good or almost as good mediator release or degranulation in the presence of ethylenediaminetetraacetate (EDTA) as in its absence. However, calcium lack prevents degranulation of rat mesenteric mast cells by this same agent. Ouabain increases Na+ intake but has no effect on the histamine released by 48/80 (Slorach and Uvnas, 1969). Disodium cromoglycate inhibits degranulation and histamine release ( Orr et al., 1971) . Chymotrypsin and phosphatidase A have been reported to degranulate and release histamine from rat mast cells (Moran et al., 1962; Hogberg and Uvnh, 1960; Bach et al., 1 9 7 1 ~ )Both . substances require Cat+ for their action. Metabolic inhibitors, such as 2,Cdinitrophenol and sodium arsenite, and sulfhydryl inhibitors, such as allicin, inhibit the action of these agents as does anaerobiosis in the absence of glucose. The releasing action of these enzymes is also prevented by heating the cell to 45°C. Thus, the action of these enzymes is very similar to 48/80.

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The similarities of the action of these enzymes to those of 48/80 and other immunological and iioiiim1iiuiiologica1 stimuli provided the basis for the suggestion that these various nonenzymatic triggers induce mediator release by activating a chymotrypsin-like enzyme and/ or a phosphatidase A-like enzyme in the mast cell membrane (Uvniis and Antonsson, 1963; Bach et al., 1971~).The histamine-releasing activity of chymotrypsin is in accord with the hypothesis that the activation of a chymotrypsin-like esterase is an obligatory event in noncytotoxic mast cell changes triggered by various agents (Becker, 1968; Section IV,A,2, and BJ). However, there is serious question as to the significance of the evidence upon which the hypothesis of the participation of phosphatidase A in the sequence of reactions is based. None of the phosphatidase A preparations used was pure. Uvnas and his co-workers used a phosphatidase A preparation from bee venom, but Fredholm (1966) and Rothschild (1965) have both reported that the ability of bee venom is not associated with its phosphatidase A content but rather with the presence of a polypeptide, mellitin. Moreover, purification of snake venom phosphatidase A also removes its ability to release histamine (reported in Fredholm, 1966). The significance of the observation ( Amundsen et al., 1969) that phospholipase A enhances histamine release by glandular kallikrein is completely unclear. The phospholipase A was impure, and Trasylol, an inactivator of kallikrein, had no inhibitory effect nor did any of the metabolic inhibitors of histamine release. Padawer (1967a,b) demonstrated microtubules in rat mast cells and that these are disrupted by colchicine. He suggested the mast cells contain a contractile system concerned in granule extrusion (Padawer, 1969, 1970). Gillespie et al. ( 1968) showed that colchicine inhibits histamine release from rat mast cells by 48/80 and polyniyxin B but not by a lytic mast cell agent, n-decylaniine. Demecolcine, vinblastine, and giiseofulvin, all agents that disrupt microtubules, also inhibit histamine release. Deuterium oxide, which increases the occurrence of microtubules, causes histamine release and also enhances histamine release induced by 48/80. These results are in accord with the idea that a contractile system, at least one part of which is made up of microtubules, is involved in degranulation and histamine release (Padawer, 1969; Gillespie et al., 1968). Orr et al. (1972) showed that cytochalasin B inhibits histamine release from mast cells induced by either 48/80 or by the interaction of antigen with cell-bound antibody. In their hands, in contrast to Gillespie et al. (1968), high concentrations of colchiciiie were required to cause inhibition, and they postulated that the motive power for granule extrusion comes from contractile microfilaments and that, at best, micro-

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tubules niay providc the framework that guides the granule extrusion. On the basis of thc data of Gillespie ct al., it is possible, however, that the 20-minute incubation of colchicine with the mast cells, used by Orr et al., was not sufficient to bring out the maximum inhibitory potential of colchicine. Loeffler et a?. (1971) have shown that release from peritoneal mast cells by coinpound 48180 is inhibited by high concentrations of dibutyryl cyclic AMP (but not by 3,s’-cyclic AMP), ATP (in the presence of 0.9 mM Ca2+),ADP, AMP, adenosine, or pyrophosphate. Theophylline, reserpine, diethylaminoethylreseiyine, and perphenazine, all reported to be phosphodiesterase inhibitors, also depressed histamine release by 48/80. The last three named compounds were also histamine liberators. Prostaglandin E, (PGE,) was also found to be inhibitory. Johnson and Moran (1970) found that both a- and p-adrenergic agonists and antagonists inhibited histamine release from mast cells induced by 48/80 and by antigen and concluded that known adrenergic mechanisms were not involved in amine secretion. Isolated rat mast cells reacted with 48/80 develop an unresponsive state, but this is presumably solely due to combination of 48/80 with its receptor on the cell since washing the cell restored the responsiveness (Thon and Uvnb, 1967). Bloom et al. (1967) showed that mast cells exposed to 48/80 at 22°C. exhibited an activated state which decayed; however, no unresponsiveness ( desensitization) was found. 2. Release by Lysosomal Cationic Protein Band 2 protein, one of the basic proteins of the lysosomes of rabbit polymorphonuclear leukocytes, releases histamine from isolated rat peritoneal mast cells ( Seegers and Janoff, 1966; Ranadive and Cochrane, 19%). Release is greatly inhibited at 10°C. and completely at 0°C.; it is also inhibited by 2,4-dinitrophenol in the absence of glucose but not in its presence, by iodoacetate, and by heating the cells to 45°C. Just as cells that were reacted with 48/80, EDTA has little or no effect on histamine release by band 2 protein (Section IV,A,l and see below). These results suggest that like other nonimmunological and inimunological processes of histamine release, the reaction stimulated by band 2 protein is temperature-dependent, requires energy, and has a heat-labile step ( Ranadive and Cochrane, 1971) . Organophosphorus inhibitors, such as DFP or the p-nitrophenyl ethyl phosphonates, prevent histamine release when present at the time the band 2 protein is reacted with the mast cell. Pretreatment of either the cationic protein or the cell with organophosphorus inhibitor has no effect on subsequent histamine release. Moreover, inhibition profiles ( activity-structure relations) of p-nitro-

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phenyl ethyl phosphonates are the same as found in the noncytotoxic immunological release processes. These results indicate that the band 2 cationic protein induces histamine release by activating an activatable esterase which is the same as that activated by the several immunological triggers (Section IV,B,l). Coffey (1970) reported that histamine release induced by lysosomal cationic protein was inhibited by prednisone and isoproterenol. Inhibition by the latter was increased by theophylline. He considered these effects mediated by adenyl cyclase and by a Caz+-requiring ATPase (see Section IV,A,3). Cells reacted with band 2 protein either in the cold or at 37°C. in the presence of 2,4-dinitrophenol are prevented from releasing histamine but are, nevertheless, “activated,” since after they are washed free of external band 2 protein (and inhibitor) and incubated at 37”C., they do release histamine. The histamine release from activated cells is not prevented by DFP but is inhibited by EDTA, indicating that a temperature-sensitive step comes after esterase activation and before a step requiring divalent cations, Moreover, an energy-requiring step seemingly precedes or is simultaneous with the divalent cation-requiring step. This is among the clearest evidence that esterase activation is a very early step in the reactions in which it plays a role. The results with EDTA are paradoxical since EDTA inhibits histamine release from activated cells but not from cells reacted directly with band 2 protein at 37°C. (Ranadive and Cochrane, 1971) . Ranadive and Cochrane reported that the activated cell would “decay” and become “desensitized,” that is, unresponsive to further stimulation. Moreover, by manipulation of the experimental conditions, at least two different activated stages were defined.

3. Release by ATP Keller (1966) and Diamant and Kriiger (1967) demonstrated that 0.01-0.1 mM ATP induces histamine release from isolated mast cells. Compounds ADP, AMP, phosphocreatine, 2-phosphoenolpyruvic acid (PEP), and 3’,5‘-cyclic AMP are without effect; however, 2’-deoxyadenosine 5’-triphosphate is as active as ATP (Sugiyama, 1971). The release initiated by ATP requires Ca2+and is inhibited by Mg?+.Dinitrophenol inhibits in the absence of glucose, whereas, oligoniycin inhibits whether or not glucose is present (Diamant and Kriiger, 1967). Conipounds KCN, N-ethylmaleimide, p-chloromercuribenzoate, and a number of heavy metal ions also inhibit ( Sugiyama, 1971). Dianiant and Peterson (1971) demonstrated that histamine release by ATP is enhanced by glucose and mannose which the mast cell is able to metabolize by the

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Embden-Myerhoff pathway, but not by galactose or fructose which it caiiiiot metabolize. Lactate and pyruvate also enhance release by ATP. Iodoacetamide blocks the enhancing effect of glucose but not that of pyruvatc or lactate. Antiniyciii A (0.1 p M ) , an inhibitor of oxidative phosphorylatioii, completely inhibits ATP-induced histamine release even in the presence of glucose, lactose, or pyruvate. These results support the suggestion that an increase of the aerobic metabolic rate enhances histamine releasc by ATP (Diamaiit and Peterson, 1971). In addition, they suggest that the presence of sulfhydryl groups is essential. Ouabain is without effect on either ATP- or 48/80-induced histamine release. Adenosinetriphosphate in the presence of Ca2+causes an uptake of Ca?+by the mast cell (Dahlquist et nl., 1973a). Neither Ca2+nor ATP injected into the cell results in degranulation. Whether both of them injected together would have an effect was not tested (Tasaka et al., 1970a). In the absence of Ca2+,there is no histamine release or degranulation, but there is swelling of the cell with an influx of Na+ and efflux of K+ (Sugiyama, 1971; Dahlquist et nl., 1973a). These changes are prevented by Ca2+(Dahlquist et al., 1972a). Dahlquist and Diamant ( 1972) reported that, in the absence of Na+ and K , no histamine release is induced by ATP even though the uptake of Ca2+is markedly stimulated. The latter authors concluded that both Ca2+and Na+ uptake are required for histamine release to be stimulated by ATP. There apparently has been no study of the role of cyclic AMP in Ca-ATP-induced histamine release. First, histamine release by 48/80 and by ATP differ in so far as the latter is dependent on Ca2+and inhibited by Mg?+, whereas release by 48/80 is little if at all affected by these two ions. Second, glucose completely reverses the inhibition by oligoniyciii of histamine release by 48/80 but has little effect on inhibition by oligomycin or antimycin A of the release induced by ATP (Diamant and Kriiger, 1967). Peterson and Diamant (1972) suggest that inhibition of glycolysis induced by external ATP explains the observed differences in energy dependence for histamine release induced by 48/80 and ATP. Despite these differences there is a relation between the mechanisms of histamine release stiniulated by these two agents. Pretreatment of mast cells with ATP in the absence of Ca?+prevents 48/80 from inducing histamine release or degranulating the cell. Neither EDTA, antimycin, nor any of the other nucleotides tested has this effect (Dahlquist et al., (197313). Mast cells exposed to ATP in the presence either of Mg2+,Ca2+, Ba?+, or Sr2+ but not Be'+ are not inhibited in their later response to 48/80, and this protection is prevented by EDTA (Dahlquist et al., 1 9 7 3 ~ )Moreover, . in mast cells that have been inhibited in their response

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to 48/80 by prior treatment with ATP, the sensitivity can be restored by the addition of Mg?+.The depressed sensitivity to 48/80 induced by low concentrations of ATP is gradually reversed by prolonged incubation, and this restoration of sensitivity is enhanced by glucose but blocked by EDTA. “he “desensitization” by ATP in the absence of Ca2+to the histaminereleasing action of 48/80 might come about, as suggested by Mongar and Schild for other systems (Schild, 1968), through the induction by ATP of a short-lived intermediate required for the action of both ATP and 48/80. This short-lived intermediate can presumably be reconstituted by some cation-requiring and energy-dependent process. How this putative intermediate acts, either by counteracting “the configurational changes of the plasma membrane induced by ATP, so that it again becomes reactive with 48/80” (Dahlquist et al., 1972c) or in some other fashion, is not clear at present. The histamine-releasing effect of Caz+-ATP on the mast cell is reminiscent of the release of catecholamines from isolated chromaffin granules and of vasopressin from isolated granules of the neurohypophysis by ATP, although in the two latter instances Mg2+is the required cation rather than Ca2+(Poisner, 1970a). As has been done for the latter systems, Diamant and Kriiger (1967) have postulated the involvement of a mast cell membrane, Ca*+-dependentATPase in the histamine release induced by ATP. Similarly, Hadden et al. (1971a) have invoked a membrane ATPase in the transformation of lymphocytes (see Section XI ) . Adenosinetriphosphate enhances the antigen-induced release of histamine from basophiles (see Section V,A) . The effect of histamine release by Ca2+ and ATP on agents supposedly acting on the contractile mechanism of the cell has not been reported.

B. IMMUNOLOGICAL STIMULI There are many different ways by which antigen-antibody reactions induce mediator release from mast cells. The antigen may react with mast cells sensitized with strongly bound homocytotropic antibody such as rat or mouse mast cells sensitized with rat or mouse y E antibody. Alternatively, the antigen may react with mast cells sensitized with homologous weakly bound homocytotropic antibody such as mouse yG, or rat yG, antibody. These have been called antibody adherent reactions (Becker, 1971a). Mediator release may also occur when antibody reacts with antigen (usually immunoglobulin) on the cell; these are antigen adherent reactions ( Becker, 1971a). There are several different kinds of antigen adherent reactions, one of which is the reaction of antibody

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with antigen adsorbed to the suiface of a mediator cell. Examples of this type are histamine release obtained ( 1 ) by the reaction of human immunoglobulin on human leukocytes ( basophiles ) with rabbit antibody against human YE immunoglobulin (Ishizaka et al., 1969) or ( 2 ) by the combination of rat immunoglobulin on the surface of thc rat mast cells with relatively high concentrations of rabbit antirat y G-globulin ( Humphrey et al., 1963). Rabbit antirat yG-globulin can also react with rat mast cells in the presence of coinplenient to give noiicytotoxic histamine release (Austen and Becker, 1966). Another kind of antigen adherent reaction is the release of histamine following the reaction of antirat mast cell antibody in the presence of complement with antigens that are an integral part of the rat mast cell membrane (Valentine et al., 1967). Under most circumstances, the last reaction is a cytotoxic reaction and will not be considered further here ( however, see below). In addition to these antibody and antigen adherent reactions, isolated complexes of antigen with antibody also may react with mediator cells to give mediator release, the so-called aggregate release reactions (reviewed in Becker, 1971a). As already pointed out, studies employing isolated mast cells have been essentially confined to peritoneal mast cells of mice and rats, because until now, they have been the only isolated mast cells available (however, see, Ishizaka et al., 1972b). Although a great deal of knowledge has been obtained concerning cells from these sources, mast cells from various species and within a given species vary among themselves ( Section IV), and before a comprehensive picture of immunologically induced mediator secretion can be obtained, the base of the observations will have to be widened by work with isolated mast cells from other tissues and other species when these become available.

1 . Rat Mast Cells Work on the sensitization of tissues and organs must have as its foundation detailed knowledge of the sensitization process in homogeneous populations of isolated cells; we are only starting to obtain this. Enough work has been done on both rat and mouse mast cells to demonstrate marked differences in their sensitization by homologous, heatlabile, homocytotropic yE or YE-like antibody and by homologous, heat-stable, homocytotropic antibodies ( yl of the mouse and yGa of the rat). In the rat, all studies on sensitization until just recently have been done with the unfractionated peritoneal cell population of which the mast cells constitute only approximately 6%of the total cells. Very poor sensitization is obtained when whole antiserum containing yE antibodies is used to sensitize isolated, pui-ified, rat mast cells (Bach et al., 19714.

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This might be due to the loss of some substance from the cells during purification, as suggested by the findings that phosphatidylserine has the ability to enhance histamine release from mast cells by dextran (Goth et al., 1972) and that leukocyte extracts enhance, to a variable extent, antigen-induced histamine release from human basophiles ( Lichtenstein, 1968). For sensitization of rat mast cells with rat y E antibody, Bach et al. (1971a) found that a buffer of relatively low pH, containing Ca2+, A&( SO,),, and heparin was optimal. The enhancing effect of heparin on the sensitization of human leukocytes (basophiles) by human yE antibody has been reported previously (Osler et al., 1968), but there have been no reports of stimulatory effects of A13+ (or Zn2+,Cu2+,Fez+, or Ca2+)on the sensitization process. In fact, the sensitization of human leukocytes with human y E antibody is enhanced by EDTA (Osler et al., 1968). For optimal mediator release on challenge of cells already sensitized with yE antibody, it was found necessary to decrease the concentration of Na, K+, and Ca2+,and neither heparin nor heavy metal salts had any effect ( Bach et al., 1971a). The binding of yGa antibody to rat mast cells is obviously much weaker than the binding of y E antibody. Unlike sensitization with rat y E antibodies, sensitization of rat mast cells with rat yGa antibodies proceeds without a latent period and without a necessity for the antibody to bind firmly to the cell. Nevertheless, the interaction of y E and yGa antibodies apparently involves a common receptor and one or more common points in the sequence of reactions leading to a mediator release from the cell. This was shown by the findings that yGa antibody interferes with the capacity of y E antibody to prepare the mast cell for histamine release and that cells, desensitized by an antigen-yGa antibody reaction, are also desensitized to an antigen-yE antibody reaction ( Bach et al., 1971b). This finding was further confirmed when specifically purified antibody preparations containing rat yE antibodies were used to sensitize mast cells in vitro (Bach et al., 1972). The very large difference in strength of binding rat yGa and y E antibodies to rat mast cells implies that studies done on antigen-induced mediator release from isolated mast cells of actively sensitized rats involve yE antibody-mediated release. The latter assumption, of course, presumes that y E and yGa are the only classes of antibody with homocytotropic activity in the rat. Bach et al. ( 1 9 7 1 ~ )studied the nature of the putative receptor for rat y E antibody by comparing the effects of various enzymes on rat mast cells before and after sensitization with rat y E antibody. Of the eighteen enzymes studied, only two, Clostriditim perfringens neuraminidase (but

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not Vibrio sialidase ) and phospholipase C, produced significant inhibition of the reaction. They suggested that the receptor for yE antibody might contain sialic acid residues and the hydrophilic portion of membrane phospholipids. The indirect approach used presented difficulties that impeded a clean-cut interpretation. Not the least of these difficulties were those introduced by the use of impure preparations of some of the enzymes. Nevertheless, the similarity of these results to those of Cline and Warner (1972) using a mouse mastocytoma (Section IV,B,2) is striking. Antigen-induccd mediator release from rat mast cells is inhibited by disodium cromoglycate (Kusiier et al., 1972) by preheating the mast cell, by lack of Ca2+.Sr2+ can replace Ca2+and Mg2+antagonizes the effect of Ca2+ (Foreman and Mongar, 1972). The release is also inhibited by sulfhydryl blocking agents, by increase in ionic strength of the medium, by salicylaldoxime, by phenol, and by anoxia in the absence of glucose but not in its preseiicc (reviewed in Austen and Humphrey, 1963; Schild, 1968). The last observation suggests that, although in the absence of 02, anaerobic glycolysis may be able to provide the energy source for mediator release, in the presence of 02,the aerobic pathway is the energy source (Sections 111, IV,A,l, and IV,A,2). Mongar and Perera ( 1965), however, consider that aerobic oxidation is not involved; rather, the effect of O2 is to oxidize sulfhydryl groups to form disulfide bonds. In support of their contention, Perera and Mongar (1965) reported that antigen-induced histamine release did not lead to increased O2 consumption from the rat mast cells. However, by using a sensitive Cartesian diver technique, Chakravarty ( 1968) found an increase in the respiration of sensitized rat mast cells of approximately 30%accompanying antigeninduced histamine release. The stimulation of respiration lasted 15-20 minutes. Chakravarty considered that this finding supported the hypothesis of the importance of oxidative metabolism in mediator release in this situation. It is possible, however, that the increase of 0, consumption under these circumstances comes from a stimulation of the pentose ( hexose monophosphate) shunt mechanism as found in phagocytosis by leukocytes. The latter, as it occurs in phagocytosis by polyniorphonuclear Icukocytes, is not related to the primary engulfment phase but to subsequent events. Similarly, the increase in respiration of mast cells might not be directly related to histamine release. The use of p-nitrophenyl ethyl phosphonate inhibitors indicates that antigen-induced histamine release from rat mast cells sensitized with rat YE antibody requires the activation of a serine csterase (Bccker and Awten, 1966). This activatable esterase is the same as that required to be activated in the coniplcment-dependent noncytotoxic histamine release

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from rat mast cells reacted with rabbit antiserum against rat yG globulin on the cell ( Austen and Becker, 1966) and in histamine release by rabbit, band 2, lysosomal protein (Ranadive and Cochrane, 1971; Section IV,A,2). As already discussed ( Section IV,A,2), esterase activation with band 2 protein is an early step, occurring before the &?+-dependent and energy-requiring steps. This suggests that the immunologically induced secretion of mediators in mast cells goes through the same mechanisms as that induced by band 2 protein-a conclusion supported by the findings of Ranadive and Muir (1972) (see below). In other words, a reasonable hypothesis is that all the various immunological triggers of induced mediator secretion from rat mast cells act on the same receptors and that at least some of the nonimmunological stimuli do also. In light of this possibility, it would be of interest to test whether, in the antigeninduced histamine release from yGa-sensitized mast cells, there is a requirement for activation of a serine esterase, especially in view of the evidence just cited that yE and yGa antibody-mediated histamines go through a common receptor. In this regard, also, evidence that human yE antibody can sensitize rat mast cells for antigen-induced degranulation (Perelmutter and Liakopoulou, 1971) suggests that it would be of interest to see, if, as expected from their antigenic similarities (Kanyerezi et al., 1971), the y E antibodies of the two species act on the same rat mast cell receptor and through the same activatable esterase. There is excellent evidence that complement-dependent release of histamine by rabbit antirat yG globulin reacting with yG globulin on rat mast cells is noncytotoxic (Johnson and Moran, 196913). In view of the definite requirement for at least four components of complement and probably the fifth ( Austen and Becker, 1966), it is possible that histamine release under these circumstances arises from the generation of C5a, although C5a has been reported not to release histamine from rat mast cells (Lepow et al., 196s). Under some circumstances, histamine is released very well from rat mast cells by antirat yG globulin when rabbit serum genetically deficient in C6 is the source of complement; on other occasions, under what appear to be in the same circumstances, little or no histamine release is obtained with C6-deficient serum (Austen and Becker, 1966). This suggests the possibility that, although, in general, the complement-dependent histamine release by rabbit antirat y-globulin is noncytotoxic (Johnson and Moran, 1969b), requiring only the sequence of complement reactions through C5, under some ill-defined circumstances, the reactions may be cytotoxic, utilizihg the entire complement sequence. On the other hand, the complement-dependent release of histamine by rabbit antirat mast cell antiserum is certainly cytotoxic (Valentine et al., 1967), although it would appear that even here a non-

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cytotoxic release is possible (Johnson and Moran, 1969b). Saeki ( 1964) reported that the degranulation of rat mast cells induced by antirat serum is similar to that effected by 48/80, antigen, and by chymotrypsin in regard to the influence of pH, temperature, and Ca2+lack. In addition, phlorizin and 2-deoxyglucose have no effect on the degranulating action of 48/80, chymotrypsin, or antirat serum under aerobic conditions, but inhibit the glucose-dependent anaerobic degranulation, suggesting that even in the last reaction, aerobic oxidation and anaerobic glycolysis are both available as energy sources. Ranadive and Muir (1972) found that rat peritoneal mast cells made unresponsive to band 2 lysosomal protein (Section IV,A,2) also became unresponsive to antibody against rat y -globulin and complement. Similarly, cells made unresponsive to antirat y-globulin and complement became unresponsive to band 2 protein. They suggested that the two stimuli induce histamine release by the same pathway and that certain differences between the two processes are dependent on the different rates of activation.

2. Mouse Mast Cells Two classes of mouse homocytotropic antibodies are capable of sensitizing mouse mast cells. The first is the heat-stable, sulfhydryl-resistant, yl antibody which sensitizes mouse skin for a passive cutaneous anaphylaxis (PCA) with a short 2-3 hour latent period and persists in the skin for 24 to 36 hours. The second is the heat-labile, sulfhydryl-resistant antibody that sensitizes mouse skin for a PCA with a long (24-72 hour) latent period and persists in the skin for days and weeks (reviewed by Vaz and Prouvost-Danon, 1969). Most recently, A. Prouvost-Danon et al. (1972) have prepared a rat antiserum against mouse yE-like antibody by immunizing rats with rat peritoneal mast cells sensitized with the mouse antibody. The antiserum recognizes a new class of mouse immunoglobulin that differs from mouse yl, y?, IgA, and IgM immunoglobulins and which has been designated yE by them. Furthermore, these investigators have shown a strict association between yE antibody and the heat-labile homocytotropic activity of mouse antisera. Peritoneal cell suspensions from the mouse contain 0.24%mast cells depending on the strain used. The mouse mast cells occur in two types: one a large polyhedric cell, 2 5 3 0 pm. in diameter, and, the second, a round cell with a diameter of 10 to 13 pni. Histamine release from sensitized mouse mast cells on addition of antigen is associated with an alteration of the ultramicroscopic appearance of the specific granules similar to what has been observed in the rat mast cells. The alteration of the granule consists of swelling, loss of electron density, and a confluence between adjacent granules with a rupture of the perigranular membrane. There

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is little actual extracellular extrusion of the granule; however, as the authors point out, this might be a technical artifact (Nelson et al., 1968). Evidence is insufficient to decide whether the two-stage hypothesis (see above) holds for mediator release from mouse as well as froin rat mast cells. Antigen-induced mediator release from mouse peritoneal mast cells, sensitized with mouse, heat-labile, homocytotropic ( YE-like ) antibodies, has been studied with the cells either actively or passively sensitized in d u o because it is impossible to sensitize mouse mast cells in vitro even with antiserum containing very high titers of mouse, YE-like, reaginic antibody. Recently, however, Prouvost-Danon and Binaghi ( 1970) have succeeded in sensitizing mouse mast cells in uitro by using specifically purified YE-like antibody in place of crude antiserum. This illustrates the importance in sensitization of the autoinhibition by nonspecific 7-globulin and also suggests a trial of specifically purified antibodies in other similar instances where in uitro sensitization has failed, such as rabbit basophiles with rabbit yE antibody. As in the rat, mouse yE antibodies bind to the mouse mast cell quite firmly, whereas, the heat-stable, homocytotropic, mouse yl antibodies bind so loosely that one wash is sufficient to reverse completely their adherence to the mast cell ( Prouvost-Danon, 1968, p. 175). The sensitization by yE antibody is markedly temperature-dependent ( ProuvostDanon, 1971). Freshly formed complexes of antigen and yl antibody are quite efficient in giving histamine release from mouse mast cells, but this effectiveness falls as the interval between the formation of the antigenantibody complex and the addition of the cells is increased (Vaz and Ovary, 1968). Thus, it might be expected that the most efficient cytotropic mechanism is the one in which the yl antibody is bound, even though quite loosely, to the mast cells. Evidence similar to that developed for the rat yGa and yE antibodies (Section IV,B,l) is not available to decide whether mouse yl and mouse yE-like antibodies have the same or very similar receptors on the mouse mast cell although this is probable. Inhibition by antigen excess is very evident in mouse mast cells sensitized with yl antibody but much less evident in cells sensitized with yE antibody. Otherwise, the course of mediator release (both serotonin and histamine) seem similar whether it is triggered by antigen reacting with YE-like or yl antibodies (Vaz and Prouvost-Danon, 1969). Mediator release is rapid at 37°C. but prevented by lowering the temperature. In the absence of glucose, antimycin A, an inhibitor of the cytochrome system or 2,4-dinitrophenol, an uncoupler of oxidative phosphorylation, completely inhibits the antigeninduced release of serotonin or histamine from mast cells sensitized with

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yl or YE-like antibodies; glucose prevents the inhibition by these agents.

Succinate has no effect on mediator release (Vaz and Prouvost-Danon, 1969). As in the guinea pig lung system or rat mast cell, these results suggest that, in the presence of oxygen, oxidative metabolism is capable of playing a significant role in providing energy for mediator secretion, but if this is prevented, glycoylsis can substitute. In this connection, it would be of interest to know the effects of inhibitors of glycolysis, such as deoxyglucose or fluoride, in the presence and absence of glucose. Cells sensitized with yE-like antibody and reacted with antigen in the cold will not release histamine; after being washed in the cold and brought to 37°C. they do release histamine. Whether any reaction other than combination with antigen occurs in the cold is not clear. No desensitization apparently takes place when cells reacted with antigen in the cold are allowed to stand in the cold. Partially purified, well washed, mouse mast cells have been shown to have not only y , but also y Z R and Y ? h immunoglobulins on their surface (Tigelaar et al., 1971). Nevertheless, antibodies of the y z class are unable to sensitize the mouse mast ceIl for in uitro histamine release (Ovary et al., 1972). Histamine-containing mouse mastocytoma can seemingly bind mouse yE-like antibody, but no histamine release ensues following antigen challenge ( Minard and Levy, 1972). These findings provide additional evidence for the view already expressed that binding of antibody to the target cell is not sufficient in itself to prepare the cell for mediator release on the addition of antigen. Cline and Warner (1972) have demonstrated the presence of receptors for all known mouse IgG classes on the suiface of a mouse mast cell tumor grown in tissue culture but not for IgM, IgA, or light chains. Receptor activity was unaffected by treating the cell with phospholipase D, neuraminidase, or azide but was reduced by iodoacetamide and fluorescein isothiocyanate, and, in agreement with the results of Bach et al. ( 1 9 7 1 ~ )on rat mast cells, by phospholipase C. Cline and Warner concluded that the receptor is a stable phospholipid or phospholipoprotein which is protease resistant. Very low concentrations of rabbit, antimouse, y-globulin antibodies induce degranulation and histamine release from thoroughly washed mouse peritoneal mast cells in the absence of complement (ProuvostDanon et al., 1970). This is unlike the situation with the rat mast cell where thorough washing of the cell prevents the reaction, and, when low coiicentrations of rabbit antirat y-globulin are used, the reaction is complement-dependent ( Austen and Becker, 1966). Also, after sensitization with antimouse immunoglobulin, the mouse mast cell can be repeatedly washed. Except for the slowness of the release induced by rabbit antimouse immunoglobulin, the characteristics of histamine re-

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lease so far studied seem similar to the antigen-induced release of mediator from sensitized mouse mast cells. V. Mediator Secretion from Basophiles

A. HUMAN Antigen-mediated release of histamine from human blood leukocytes sensitized with human yE antibody has proved to be a very useful tool in studying the basic immunological mechanisms of human anaphylactictype reactions at the cellular level as well as the mechanisms of immunologically induced cellular secretion (Osler et al., 1968). Although it was originally argued on the basis of older work that approximately 50% of the histamine released from preparations of human leukocytes came from the eosinophiles and neutrophiles, newer studies make it probable that essentially all of it is derived from the basophile (Sampson and Archer, 1967; Pruzansky and Patterson, 1970; Kunske et al., 1971; Ishizaka et al., 1972b). This view is supported by the findings that the basophile is the only type of cell in blood that is demonstrably sensitized by y E antibody or its Fc fragment or that reacts with anti-yE antibody (K. Ishizaka et al., 1970b; Tomioka and Ishizaka, 1971; Sullivan et al., 1971) . Ishizaka et a2. ( 1971a) have demonstrated degranulation of human basophiles by rabbit antihuman yE employing the conditions used for histamine release by the same antibody suggesting that, as in other cells, degranulation and mediator release are related. Hastie ( 1971) has demonstrated by phase microscopy that antigenically stimulated basophiles from actively sensitized humans degranulate but continue to show motility for 3 hours after degranulation is complete i.e., the process is clearly noncytotoxic. Following the addition of antigen, there is a latent period of 5 to 10 minutes; this is followed by a loss of directed motility and a spreading of the cell. The long latent period may be in accord with the finding that immunological release of histamine from human leukocytes is usually a relatively slow process (Osler et al., 1968) in contrast to the mast cell in which degranulation and mediator release occur within seconds. The next change is the appearance of phase-pale vesicles which show a variable degree of coalescence with a concomitant reduction in the number of specific granules. With continued incubation, there is a disappearance of all phase-pale vesicles, and the cells recover some ameboid motility although not as much as they had before degranulation. Sensitization of human leukocytes ( basophiles ) by YE antibody (T. Ishizaka et al., 1969; K. Ishizaka et al., 1970a) is not enhanced by Caz+or Mg2+,but it is enhanced by heparin or EDTA and is time- and

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temperature-dependent (reviewed in Oslcr et al., 1968). There is a marked variation in the capacity of leukocyes from various donors to become sensitized, but, as K. Ishizaka et al. (1970b) and Sullivan et al. (1971) indicate, this is probably not due to an inability of these individuals to accept YE antibody on their basophiles. These latter studies emphasize again that sensitization, in the sense of preparing a cell for mediator secretion is more than a matter of simple adherence of antibody to the cell. Sullivan et al. (1971) showed by electron microscopy using a hybrid antibody technique that cells kept at 0°C. during sensitization with human yE immunoglobulin had YE bound in patches distributed around the entire circumference, whereas cells kept at room temperature had yE distributed asymmetrically over one pole of the cell. This is similar to the finding that lymphocytes treated with anti-immunoglobulin antibody demonstrate a temperature-dependent cap of antibody ( see Section XI). It is possible that this cap formation might play a role in the antigen-induced histamine response in YE-sensitized mast cells. Sullivan et al. (1971) made the tentative estimate that there could be as many as 4 X lo5 molecules of IgE bound per basophile, although it might well be less. By using a more precise technique, Ishizaka et al. ( 1 9 7 2 ~ found ) 220043,200 IgE molecules per human basophile, depending on the source of the basophile. Antigen-induced histamine release from human leukocytes is not obtained in the absence of Ca'+ in the medium; Mg2+must also be present for maximal release. Sensitized cells heated to 45°C.lose their ability to give histamine release. Lowering the temperature of the reaction decreases histamine release. Diisopropyl fluorophosphate causes an irreversible inhibition which is obtained in the absence of interaction of the cell with antigen. Thus, there is no evidence that an activatable esterase is required, although an activated esterase may be involved (reviewed in Osler et al., 1968); but, in view of the evidence that an activatable esterase is required in other systems, this last point requires further study. The sulfhydryl inhibitors, iodoacetamide and p-hydroxymercuribenzoate, inhibit histamine release, suggesting a need for sulfhydryl groups. Neither sodium cyanide, nor dinitrophenol, nor anoxia has any effect, suggesting that the oxidative pathway may not be operative at all as an energy source, However, it would be worth testing these substances both in the presence and absence of glucose. Fluoride or deoxyglucose are both inhibitory, indicating that the glycolytic pathway can be used. Adenosine triphosphate in the medium enhances antigenically induced histamine release but causes no release in the absence of antigen, suggesting an energy-requiring process at the cell surface. The antigeninduced reaction is also enhanced by a lipoprotein fraction from serum,

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and a fraction extracted from leukocytes (Lichtenstein, 1968). Colchicine blocks antigenically induced histamine release (Levy and Carlton, 1969). Deuterium oxide, an agent known to preserve microtubules, potentiates antigen-induced histamine release ( Gillespie and Lichtenstein, 1972). The potentiation occurs at the second or Ca2+-dependent stage (see below), Cytochalasin B enhances antigen induced release, seemingly acting on the second stage (Gillespie and Lichtenstein, 1972; Colten and Gabbay, 1972). Sensitized human leukocytes reacted with antigen at 37°C. for 2 minutes in a Ca2+-and Mg2+-freebuffer give no histamine release. They are, however, “activated,” since they give histamine release after they are washed to remove free antigen in the cold and then resuspended in buffer containing Ca2+ and Mg2+ at 37°C. (Lichtenstein, 1971). The activated state progressively decays to give a desensitized cell when the cells are kept at 37°C. in the absence of Ca2+.The activated (first stage) cells can be maintained for at least 30 minutes if they are kept at 4°C. The same sort of activation is obtained if the cells are reacted with antigen at 26” or 4”C., but the degree of activation is not as great as when the cells are reacted at 37°C.; however, the rate of deactivation is less at the lower temperature. Chlorophenesin inhibits the formation of activated cells at 37°C. but not at 4°C.; whereas, it inhibits the second stage of the reaction (release of histamine at 37°C. in the presence of Ca2+and Mg2+) of cells activated at 4°C. but not at 37°C. This suggests that there is a difference in first-stage cells produced at 4” and at 37°C. Deoxyglucose inhibits only the second ( Ca2+-dependent) stage, whereas diethylcarbamazine ( Hetrazan) inhibits both stages of the reaction equally. Lichtenstein and Margolis ( 1968) reported that catecholamines, methylxanthines, and dibutyryl cyclic AMP inhibit antigeninduced release from human leukocytes in a manner suggesting that increasing the intracellular cyclic AMP levels decreases histamine secretion. Bourne et al. (1973) have reported that cholera enterotoxin causes a delayed increase in adenyl cyclase activity of human leukocytes, which correlates with a delay in its inhibitory effects on histamine release. Lichtenstein and De Bernard0 ( 1971) showed that isoproterenol inhibits the first stage, preceding the Ca’+-dependent step, and the inhibition can be blocked by the p-adrenergic blocker, propranolol. Theophylline and the prostaglandins, PGE, and PGE2, act only on the first stage; the action of PGE, is not blocked by propranolol (Lichtenstein and Bourne, 1971). Theophylline and dibutyryl cyclic AMP act predominately on the first stage. Interestingly, histamine itself, an activator of adenyl cyclase in some tissues, blocks the release of histamine pri-

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marily at the first stage of the reaction (Lichtenstein and Bournc, 1971). Bourne et al. (1972) showed that the ability of catecholamines, prostaglandins, and histamine to stimulate accumulation of intracellular cyclic AMP in a mixed population of leukocytes correlated well with their ability to inhibit antigen-induced release of histamine from sensitized basophiles. They obtained evidence that in the leukocyte, the synthesis of cyclic AMP is controlled by at least three separate receptors-for catecholamines, histamine, and prostaglandins, respectively. Basophiles from allergic and most normal human donors release histamine (Ishizaka et al., 1969) and degranulate (Ishizaka et d.,1971a) when reacted with rabbit antihuman yE antiserum. They also release histamine when reacted with anti-yG antibody, but the amount of antibody required is several hundred times more than with anti-yE (Lichtenstein et al., 1970). Histamine release or degranulation by anti-yE antibody is inhibited by theophylline, dibutyryl cyclic AMP, and colchicine in the same fashion as when histamine release is obtained from sensitized cells reacted with antigen. This suggests that the mechanisms for the antigeninduced and for the antibody-induced histamine release might be the same. This is seemingly also true when histamine release is evoked by anti-yG antibody ( Grant et al., 1972).

B. RABBIT Antigen-induced histamine release from sensitized rabbit leukocytes (Greaves and Mongar, 196th; Siraganian and Osler, 1970) arises from the basophiles (Greaves and Mongar, 1968a; Siraganian and Osler, 1971b). Greaves and Mongar ( 196th) showed that contact of antigen with the sensitized leukocyte for 30 to 60 seconds is required in order to obtain histamine release. No histamine is released if the reaction is carried out at 20°C. or below; a temperature of 37°C. gives maximum release, and, at 41"C., marked inhibition is present which is complete between 44" to 47°C. (Greaves and Mongar, 1968b). Omission of Ca2+prevents the reaction; the presence of Mg2+is without effect (Greaves and Mongar, 1968b; Siraganian and Osler, 1970). N-Ethylmalimide, p-chloromercuribenzoate, and iodoacetatc inhibit the reaction as do disulfide-reducing agents, whereas, succinate and maleate enhance histamine release (Greaves and Mongar, 1968b). In addition to histamine, antigenically stimulated rabbit leukocytes also give rise to a factor, the platelet-activating factor (PAF), capable of releasing histamine from rabbit platelets. The release of this mediator as well as its action on rabbit platelets is discussed in detail in Section VI,B,3,a.

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VI. Mediator Secretion from Platelets

A large number of stimuli can induce mammalian platelets to aggregate and to liberate a variety of their constituents. Aggregation, although its mechanism is unknown, shows many similarities with cell processes such as secretion and phagocytosis, so that we shall briefly summarize its properties. Grette (in 1962) coined the term release reaction to describe the process by which thrombin acts on platelets to release vasoactive amines and certain constituents such as adenine nucleotides, cations, platelet protein, lysosomal enzymes, e.g., P-glucuronidase and acid phosphatase, as well as clotting and permeability factors (Mustard and Packham, 1970). Cytoplasmic factors or mitochondria1 enzymes are not generally released (Grette, 1962; Holmsen and Day, 1968). As will be shown, in addition to thrombin, a number of other nonimmunological and immunological stimuli can induce secretion, that is, the release reaction. In most species, the vasoactive amine released is serotonin; the rabbit platelet releases histamine in addition. As in other secretory processes, the substances released are stored in one or another type of platelet granule. The serotonin and, in rabbit platelets, histamine are stored within one type of granule, the so-called dense body (White, 1971) perhaps complexed with ATP (Berneis et al., 1969). Serotonin is accumulated from the blood probably by way of the canalicular system (White, 1971) and is maintained in the cell by energy-requiring processes (Pletscher, 1968). Lysosomal enzymes are located in another kind of granule, the so-called a-granule. Platelet reactions may be considered in two phases: The first is adherence (or adhesion) of the platelet to a particle or to an immunological complex leading to some release of constituents, such as ADP and ATP, the latter being converted to ADP after release. The liberation of ADP and possibly other materials from these adherent platelets then induces the second phase of aggregation (platelets binding to platelets) which also release constituents. In the following discussion “adherence” (or adhesion) and “aggregation” will, when possible, be used to describe these two phases. In addition to, or as a consequence of, aggregation and release of constituents, platelets exhibit some other activities. They participate in the coagulation process by providing or releasing clotting factors, notably phospholipids (platelet Factor 3). Again a potentially self-perpetuating system is set up since the thrombin generated during coagulation can itself activate platelets. Once the clot is formed, there occurs a process, in the past referred to as viscous metamorphosis of the platelets, which

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leads to morphological changes accompanied by release of constituents and subsequent retraction of the clot. In this clot retraction, the platelet contractile protein, thronibosthenin ( see below) has been clearly implicated. The process may also be inhibited by antisera to IgM, C4, and C3 (Taylor and Miiller-Eberhard, 1970), but the exact roles of IgM and these complement components are unknown. Platelets also play a role in maintaining capillary integrity by processes which are not clearly understood (Johnson, 1971). In considering the secretory mechanism of platelets, nonimmunological stimuli will be discussed first since they have been studied in more detail. Review of the immunological processes will then follow and be divided into three categories. The first is the noncytotoxic release induced by immunological complexes with or without complement, by aggregated immunoglobulins or by antiplatelet antibodies. The second category is the complement-dependent lysis of the platelet which may follow the above reactions. Although the lytic reaction is cytotoxic rather than secretory, it will be discussed briefly for the sake of completeness. Finally, cooperative reactions among sensitized basophiles, antigen, and platelets or among immunological complexes, neutrophiles, and platelets will be considered.

A. NONIMMUNOLOGICAL REACTIONS For a complete discussion of nonimmunological platelet aggregation and release, the reader is referred to reviews by Mustard and Packham (1970), Marcus (1969), Michal and Firkin ( 1969), Packham and Mustard (1971), Aledort (1971), and Salzman (1972). Some features applicable to the immunological reactions and to the general release processes under discussion are considered below. Aggregation and the release reaction, although ultimately separable (see below), are intimately and intricately interrelated. Adenosine diphosphate ( ADP) induces aggregation of platelets and release of vasoactive amines. It is not only a primary agent in this regard but, as already brought out, its release by a variety of agents makes it a secondary, but central agent in the action of these compounds on platelets. Because of this, ADP will be the first of the nonimmunological stimuli to be discussed, first in its effect on aggregation and then on release.

1. Aggregation and Release Induced by ADP

a. Aggregation. At low concentrations of ADP, the aggregation is entirely reversible, but the platelets become refractory to additional doses. Higher concentrations of ADP induce a release reaction that is usually

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manifested in platelet-rich plasma by a second wave of aggregation (see below). Platelet aggregation requires Ca'+ ions ( Mustard and Packham, 1970). Extracellular Mg?+can be inhibitory and a balance between the two ions may be necessary ( Ardlie, 1971; Herrmann et al., 1970). Removal of glucose prevcnts aggregation ( Kinlough-Rathbone et al., 1970a) and platelet energy processes (both glycolysis and oxidative phosphorylation) are required (Murer et al., 1967; O'Brien, 1966). A role for platelet esterases in aggregation has not been clearly demonstrated. Nevertheless, tosyl arginine methyl ester was found to be inhibitory ( Kilburn and Firkin, 1968). The effect of ADP on platelets involves reversible morphological changes in which microtubules encircle the cell organelles and cluster in the center of the platelet (White, 1968a, 1971; Hovig, 1968). At the same time, the disc shape is lost, the platelets assume an irregular outline with pseudopods, swell, and then aggregate. The changes in shape but not the aggregation can occur even in the absence of Ca'+ (Lloyd and Nishizawa, 1971), and, in fact, similar changes are induced by EDTA (White, 1968d). Also, a similar sequence of events follows thrombin treatment. The final result of the movement of canalicular system and organelles to the center of the platelet is their fusion and obliteration, leaving an amorphous, electron-opaque central area. A process of contraction has, therefore, been suggested to occur during aggregation ( Grette, 1962; White, 1971). However, the exact role of the contractile protein, thrombosthenin (first isolated by BettexGalland and Luscher ) , remains unclear, Thrombosthenin has Ca2+-and Mg2+-dependentATPase activity, resembles actomyosin in many properties, and has been demonstrated in the platelet membrane fraction (Nachman et al., 1967; Chambers et al., 1967); in the form of microfilaments ( Zucker-Franklin, 1970; Bettex-Galland et al., 1969), it has been seen just under the surface membrane and around the canalicular system. The relationship of these microfilaments and, thus, of thrombosthenin to the microtubules is uncertain. Colchicine causes disappearance of the microtubules but not of the microfilaments ( Zucker-Franklin and Bloomberg; 1969; White, 1968b), and only at very high concentrations does it prevent aggregation (White, 1969; Sneddon, 1971). Cytochalasin B apparently causes disruption of the microfilaments but does not prevent ADP aggregation, although it does inhibit clot retraction ( Wessels et al., 1971; Haslam, 1972). The microtubules probably serve a structural role in maintaining the asymmetric disc shape of the platelets ( Zucker-Franklin and Bloomberg, 1969; Behnke, 1970), whereas the microfilaments might represent the contractile elements of the cell.

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Platelet aggregation is prevented by sulfhydryl inhibitors, although not by ouabain. Thc required sulfhydryl groups appear to be on the platelet surface (Aledort et al., 1968), whereas those required for clot retraction appear to be internal, perhaps on thrombostheniii ( LevyToledano et a]., 1971). This difference provides a clear dissociation of the two activities. Adenosine diphosphate itself is reported to rcducc intracellular levels of cyclic AMP ( Salzman, 1972), although not by directly inhibiting adenyl cyclase. Cyclic AMP or its dibutyryl derivative prevent aggregation as do agents that are reported to increase intracellular cyclic AMP such as PGE, in extremely minute doses (Kloeze, 1967; Weeks et al., 1969) and methyl xanthines ( Ardlie et al., 1967) or both (Marquis et al., 1969). Low doses of PGE?, which reduce intracellular cyclic AMP, cause or enhance aggregation ( Salzman, 1972). Many drugs previously known to inhibit platelet aggregation are now known to affect phosphodiesterases (Vigdahl et aZ., 1971). It thus appears that, during both aggregation and the release reaction (see below) a reduction occurs in cyclic AMP levels. A great many mechanisms have been proposed for the aggregation of platelets. These include bridging by Ca2+, fibrinogen, or even by thrombosthenin itself ( Booyse and Rafelson, 1971) , Whether aggregation results from contraction (White, 1971) or relaxation of thrombosthenin (Davey and Liischer, 1968b; Salzman et al., 1969) is not yet decided. Any workable theory must account for the possible role of cyclic AMP, not in inducing the aggregation, but in maintaining the normal state and allowing shape changes and aggregation only after its levels have been reduced. Although, platelet aggregation and release reaction share a remarkable number of coniinon features and requirements, many inhibition studies (see Mustard and Packham, 1970) do suggest that there are differences. For example, some drugs like PGE, inhibit aggregation much more readily than they affect the release reaction, whereas, other materials only inhibit the release and have little or no effect on aggregation ( e.g., colchicine, aspirin, phosphatidylseriiie) ( O’Brien, 1968; Zucker and Peterson, 1968; Nishizawa and Mustard, 1971). It has been reported that release can be induced by bringing platelets into close contact with each other (Massini and Liischer, 1971). However, aggregation can be achieved without a subsequent release reaction (Jenkins et al., 1971), and many agents (see below) can induce release without necessarily causing aggregation, although if the platelets are stirred, the released ADP does produce aggregation. An interesting group of patients has recently been described (Weiss and Rogers, 1972) in whom the release

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reaction is apparently absent. Study of abnornialities should prove fruitful in clarifying these interrelated platelet functions. b. Release Induced by ADP. In concentrations higher than are required for aggregation, ADP induces a release reaction (Mills et al., 1968). This is usually manifested by a second wave of aggregation, resulting from the ADP released. Where studied, ADP-induced release appears to resemble the other release reaction of platelets (see below). It is inhibited by aspirin (Zucker and Petersen, 1968) and may be enhanced by PGE, (Shio and Ramwell, 1972). However, Packham et al. (1971) have suggested that the release, in fact, results from ADP-enhanced adherence of platelets to y-globulin (from the platelet-rich plasma) which has become bound to the wall of the test tube. This point needs further investigation.

2. Release Induced by Catecholamines and the Role of Cyclic A M P Epinephrine induces a calcium-dependent aggregation of platelets (Mitchell and Sharp, 1964) and release of the granule constituents mentioned earlier (Mills et al., 1968). The secretion is associated with a second wave of aggregation, probably induced by the ADP that is released. Species differ in this response, and most studies have been performed with human platelets ( Packham and Mustard, 1971). The aggregating and releasing activities of catecholamines on platelets are apparently due to interaction of the aniines with an a-adrenergic receptor on the platelets (Mills et al., 1968; Bygdeman, 1968), associated with a decrease in the activity of adenyl cyclase and, thus, a reduction in intracellular levels of cyclic AMP (Zieve and Greenough, 1969; Marquis et al., 1970; Salzman, 1972). Agents known to stimulate adenyl cyclase (Wolfe and Shulman, 1969) and, therefore, to increase levels of cyclic AMP, such as PGE, and the methyl xanthines, inhibit aggregation of platelets by epinephrine (Emmons et al., 1967; Ardlie et al., 1967) as well as that induced by ADP (Section VI,A,l). Addition of cyclic AMP to the platelets is also inhibitory. The mechanism by which cyclic AMP affects platelet aggregation or release is essentially unknown. It is unclear what role, if any, is played by the cyclic AMP-dependent protein kinase demonstrated in these cells ( Salzman, 1972). Apparently, however, in platelets, cyclic AMP may serve to maintain the status quo, Decrease in the nucleotide level is associated with aggregation and release, whereas increase or addition of the nucleotide results in inhibition. However, the presence in any one reaction of release and aggregation (see above) makes it difficult to be sure exactly where an agent is acting. This point needs further clarification. Moreover, as pointed out by Salzman (1972) the difficulties in

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accurately measuring trace levels of cyclic AMP in these cells are extreme, and great caution must be taken at this stage in concluding too much from data that inherently are highly variable.

3. Release lncluced by Thrombin

A variety of protcolytic enzymes including trypsin and thronibin, but not chyniotrypsin, can directly induce release of granule-associated constituents from platelets (see Mustard and Packham, 1970). As already described, cytoplasmic or mitochondria1 enzymes are not generally released. The morphological changes are similar to those seen in the other platelet reactions and may be, in part, induced by the released ADP. However, there is an increasingly cytotoxic effect of large amounts of the enzyme (Rodman and Mason, 1967; Hovig, 1968; Sneddon, 1972), and many of the controversies as to which constituents are released or whether or not platelets are lysed are explicable on this basis. A requirement for calcium for the release reaction has been shown (Sneddon, 1972) if low concentrations of thrombin are employed, although previous reports had been contradictory (see Mustard and Packham, 1970). Platelets can be incubated with thrombin in the presence of EDTA without release, whereupon, addition of Ca’+ immediately initiates secretion ( Markwardt, 1967). Thrombin also induces release of calciuni (Murer, 1969b; Detwiler and Feinman, 1973). Both the release and the clot retraction induced by thrombin are dependent on intact sulfhydryl groups ( Aledort et al., 1968). Thrombin induces increased glycolysis ( Mustard and Packham, 1970; Gross and Schneider, 1971), ATP utilization ( Karpatkin, 1967), and immediate consumption of 0,and oxidation of glucose by a KCN-insensitive process. This latter is probably by the hexose monophosphate pathway (Murer, 1968; Warshaw et al., 1966). Liberation of platelet constituents requires both glycolysis and oxidative phosphorylation ( Murer, 1969a) . Heating of platelets to 42°C. inactivates them so that they no longer undergo the release reaction, aggregation, nor clot retraction (White, 1968~). Since thrombin itself is a sei-ine esterase, it has not been possible to determine whether thrombin activates a platelet esterase. Indeed, the site of action of the enzyme has remained controversial (Davey and Luscher, 1967; Nachnian, 1968). Some studies suggest that thrombin acts directly on thrombosthenin (Cohen et al., 1969), whereas others have failed to confirm this ( Baenziger et al., 1971). Nevertheless, there is rapid cleavage of a membrane-associated protein in intact platelets. This protein is not

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itself adenyl cyclasc, but the activity of adenyl cyclase is decreased by treatment with thrombin (Brodie et al., 1972). The role of cyclic AMP appears similar to that in thc other platelet reactions. Thronibiii decreases cyclic AMP (sec Salziiian, 1972) and its release reaction is potentiated by a-adrenergic agents. Incrcasing the levels of cyclic AMP, on addition of the nucleotide, inhibits the release ( Kinlough-Rathbonc et al., 1970b). Similar effects were observed on clot retraction mediated by thronibosthenin ( Murer, 1971). High concentrations of colchicine inhibit the release induced by thrombin (Mustard and Packham, 1970), but levels that still cause complete disruption of microtubules do not prevent clot retraction (White, 1968b; Zucker-Franklin and Bloomberg, 1969). However, larger doses do delay the contraction ( Shepro et al., 1969). In contrast, cytochalasin B effectively prevents the clot retraction (Wessells et al., 1971), but its action on the release reaction is unknown.

4. Release Induced by Collagen An example of the release reaction caused by adherence of platelets to a particulate stimulus is that induced by collagen. Platelets adhere closely to collagen fibers, swell, appear to lose their organelles, and release granule-bound constituents (see Mustard and Packham, 1970), possibly by degranulation into vacuoles that communicate with the extracellular medium ( Bryon et al., 1970). Collagen also induces marked aggregation of platelets which appears to be largely due to the ADP released following the adherence reaction ( Haslam, 1967). Adherence of platelets to collagen does not require divalent cations (Hovig, 1964; Spaet and Zucker, 1964) in contrast to the subsequent release of ADP and serotonin and the subsequent aggregation produced by this ADP, both of which have been reported to require the presence of calcium (Hovig, 1964; Mustard and Packham, 1970). However, Ca?+ uptake could not be detected during the reaction (Bygdeman and Stjarne, 1971) . The adherence may involve intact sulfhydryl groups ( Al-Mondhiry and Spaet, as reported by Packham and Mustard, 1971). Recently a membrane-associated enzyme, collagen-glucosyltransferase, has been implicated in the reaction of platelets with collagen. Glucosyl groups are transferred specifically to collagen, probably by way of 9 lipid intermediate (Jamieson et aZ., 1971). Release requires platelet energy ( ATP) as evidenced by its inhibition in the absence of glucose (Mustard and Packham, 1970), and this energy may be supplied by glycolysis and oxidative phosphorylation (Ball et al., 1969). Release induced by collagen requires the activation of a serinc esterase ( Henson et al., 1973).

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Incubation of colchicine with platelets in high concentrations inhibits secretion of nucleotides induced by collagen ( Mustard and Packham, 1970 ) . The role of cyclic AMP in collagen-induced release appears similar to its role in the other reactions. The release is associated with decreased iiitracellular levels of cyclic AMP, is enhanced by a-adrenergic agents, and inhibited by drugs that increase cyclic AMP (Mustard and Packham, 1970; Salzman, 1972).

5. Platelet Phagocytosis Movat and co-workers have reported phagocytosis of polystyrene particles or of immunological complexes by platelets ( reviewed by Mustard and Packham, 1968). Particles were observed within platelets by electron microscopy, and similar inclusions have been seen in U ~ U O . From the work of Behnke (1967) and White (1972) it is probable that this process is not one of phagocytosis but is merely penetration of the canicular system. However, further study of the metabolic and environmental requirements for the uptake, as opposed to the subsequent aggregation and degranulation, is indicated.

B. IMMUNOLOGICAL REACTIONS It has been known since the turn of the century that platelets react with immunological complexes. Later experiments demonstrated release of histamine from platelets upon injection of antigen into immunized rabbits or on addition of antigen to blood from these animals (reviewed in Austen and Humphrey, 1963). This reaction has been extensively studied (see below and Osler and Siraganian, 1972; Pfueller and Luscher, 1972) and has been shown to be lytic. It has, however, provoked a great deal of interest in platelet release reactions and their role in anaphylaxis. Platelets from all species tested react with immune complexes. However, the mechanism varies, involving adherence to the complement which has become bound to the complex in some species and adherence to the immunoglobulin in others. In fact, mammalian platelets can be divided into two groups: ( 1 ) those that adhere to complement, e.g., mouse, rabbit, dog, and horse, and ( 2 ) those that do not, e.g., man, baboon, pig, sheep, goat, and ox (reviewed in Nelson, 1963; Henson, 1972a). Platelets from this latter group, although they do not exhibit immune adherence to bound complement do, nevertheless, adhere to immunological complexes, but in this case they react with the immunoglobulin in the complex. In either case, release of constituents results, but in the past these differences have provided many difficulties in the interpretation of results. To illustrate the various immunological release

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reactions, platelets from the rabbit and from man will be discussed as they provide examples of these two groups.

1 . Rabbit Platelets a. Release Following Adherence to Immunological Complexes. Rabbit platelets adhere to immunological complexes or particles that have fixed complement (reviewed in Lamanna, 1957; Nelson, 1963; Henson, 1972a). The complement component involved is probably C3, and the reaction is thus a form of immune adherence, The nature of the adherence bond is not known. The reaction with complement-coated zymosan particles (zymosan complement) proceeds in the cold, although more slowly than at 37"C., and can clearly be differentiated from ADP aggregation of platelets ( Henson, 1970b, 1972a). This immunological adherence is accompanied by secretion of platelet constituents. Washed rabbit platelets do not adhere to particles that have only antibody bound to them (Henson and Cochrane, 1969a; Henson, 1970b; Siqueira and Nelson, 1961), nor do they bind to surfaces coated with 7-globulin or to immunological complexes in the absence of complement (Mustard and Packham, 1970; Movat et al., 1965). Despite this evidence for a lack of immunoglobulin receptor on rabbit platelets, two studies in platelet-rich plasma have suggested that there may be direct adherence of complexes to platelets without the participation of complement (Marney and Des Prez, 1971; Miescher et al., 1960). However, in neither case was the lack of binding of complement to the complexes directly demonstrated. The platelets come into close contact with the complement-coated particle, lose their disc shape, swell, develop pseudopods, and the organelles cluster into the center of the cell and eventually disappear ( Henson, 1969b, 1970b). Vasoactive amines, nucleotides, and a small amount of p-glucuronidase but not LDH are released. The morphological changes in this immunologically induced secretion are thus similar to those described earlier for nonimmunological reactions. The adherence and secretion are independent of ADP but, since this nucleotide is released, can be augmented by it. Platelets are not lysed by the reaction, although if later-acting complement components are included, some lysis may then occur (see below). The release reaction is inhibited by selected chelating agents, by glucose deprivation, and even more markedly by 2-deoxyglucose, demonstrating a requirement for calcium and energy from platelet metabolism ( Henson, 1969b, 1970b). Whether oxidative phosphorylation is an additional source of energy is not known. The role of platelet serine esterases in this release has been clearly demonstrated ( Henson, 197Ob). Diisopropyl fluorophosphate inhibits the

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rclease, but not the adherence. Pretreatment of either platelets or zyniosan complement with DFP, followed by washing, has no effect upon the release when the two are subsequently mixed. This experiment strongly suggests that the adherence to C3 activates a platelet serine esterase, essential for the secretion process, which only then can be inhibited by DFP. Other examples of this phenomenon in platelets will be mentioned below. Release, but not adherence, is inhibited by PGE, and theophylline, potentiated by epinephrine, and inhibited by phentolamine (P. M. Henson, unpublished observations) and, thus, resembles other platelet secretory processes in which increased cyclic AMP inhibits, and decreased cyclic AMP, for example, by a-adrenergic stimulation, may enhance. Colchicine also prevents the release. b. Antiplatelet Antibody. A very similar release reaction to the one described above was demonstrated to occur following interaction of an IgG, fraction of sheep antiplatelet antiserum with washed rabbit platelets ( Henson, 1970a). Morphological changes include alterations in shape and loss of granules as before. Platelet clumping generally occurs, as first shown by Marino in 1905 for the reaction of heterologous antiplatelet antibody with platelets. The clumping is not necessary for the release, being at least in part due to agglutination, ie., antibody cross-linking rather than ADP-induced aggregation, The release is an active process requiring platelet energy metabolism and environmental calcium. Diisopropyl fluorophosphate-inhibitable serine esterase activity is again generated in the platelets by this stimulus and was shown to be essential for the release. In addition, divalent antibody is required; F( ab), being active, whereas F( ab) is not and is even inhibitory, This requirement for a multivalent stimulus may be compared to some of the reactions of neutrophiles, basophiles, and mast cells ( Section XII). When complement is included in the reaction, lysis of the platelets ensues (Henson, 1970b). c. Lysis of Rabbit Platelets by Immunological Aggregates. The reaction of antigen with antibody in the presence of plasma under conditions of antibody excess can lead to lysis of rabbit platelets, even though the antigen is in no way part of the platelet membrane. This reaction may be termed aggregate lysis (Becker, 1971a). Its cytotoxic nature was demonstrated morphologically by showing liberation of cytoplasmic as well as granule constituents and by its independence from platelet metabolism (Siraganian et al., 1968a,c; Henson, 196913, 197Ob). As mentioned above, the phenomenon has been extensively studied following the observation of histamine release from platelets in aggregate anaphylaxis in rabbits. Although the requirement for plasma was shown in the earliest

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studies, controversy arose as to whether this denoted a requirement for complement (Humphrey and Jaques, 1955; Gocke, 1965; Des Prez and Bryant, 1969) or not ( Barbaro, 1961a,b; Westerholm, 1965; Siraganian et al., 1 9 6 8 ~ )This . was resolved in favor of the obligatory role of complement by demonstration of the ineffectiveness of plasma genetically deficient in C6 or depleted of C3 with cobra venom factor ( CoF) (Henson and Cochrane, 1969a; Henson, 1970b; Marney, 1971). These experiments also served to show that anaphylatoxins were not involved. As immunological complexes develop in the plasma, platelets adhere to them, forming clumps (Henson and Cochrane, 1969a; Henson, 1970b). This adherence requires the fixation of complement only to the C3 or C5 step and occurs in C6-deficient plasma, although in such plasma no lysis and release of constituents results. It is not yet clear why under this latter circumstance adherence of platelets to the complexes does not induce secretion by the mechanism described for particulate antigens. Perhaps the complexes are too small to stimulate the platelet surface adequately. In complement-sufficient plasma, however, additional aggregation occurs, in part due to the ADP that is released, and the platelets lyse, thus reducing the light-scattering abilities of the suspension even further (Henson, 1970b). Clumping is not a prerequisite for the release since this may occur without visible aggregation (Gocke and Osler, 1965; Siraganian et al., 1968c), presumably as the result of very small complexes reacting with individual platelets. Heat-aggregated 7-globulin also induces aggregation and lysis of rabbit platelets (Davis and Holtz, 1969), and aggregation is potentiated by epinephrine, suggesting an effect of the ADP that is released. In explaining the mechanism of this reaction, Henson (1969a, 1970b, 1972a) postulated that platelets adhere to C3 on the complexes as they are in the process of activating complement components. The C%?' that is formed may transfer to the closely adherent platelet surface and, in the presence of C8 and C9, cause membrane lysis. A similar process has been recently demonstrated to occur with erythrocytes (Gotze and Muller-Eberhard, 1970). Recently, Des Prez and Marney ( 1971) suggested a similar explanation for this mechanism. For a while it was unclear whether Ca2+or Mg?+or both are required for the release (Siraganian et al., 196%; Henson and Cochrane, 1969~1; Marney and Des Prez, 1971). However, the elegant studies of Sandberg et al. (1970), Sandberg and Osler (1971), Gotze and Miiller-Eberhard (1971), and Goodkofsky and Lepow (1971) have now revealed the presence of an alternative pathway to the regular complement sequence ( C1423) which is magnesium-dependent. The discrepant results are thus explained, since complement activation by C14 and 2 requires Ca?+and Mg?+and that by the alternative pathway or properdin system requires

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only Mg". In fact, Oliveira et al. (1970) showed that the alternative pathway could, indeed, induce the platelet injury. The presence of two pathways of complement activation also explains the data of Des Prez and Bryant ( 1966,1969). (1. Effect of Enclotoxin on Platelets. The reaction of rabbit platelets with bacterial endotoxin may not be immunological in the sense of requiring antibody but, nevertheless, shows many similarities to the lytic immunological reaction just described. Injection of endotoxin into rabbits causes platelet aggregation and sequestration and, thus, an immediate and profound thrombocytopenia. Rabbit platelets are aggregated by endotoxin in platelet-rich plasma (Davis et al., 1961; Hinshaw et ul., 1961; Des Prez et al., 1961) and release their constituent amines. The reaction requires plasma factors and results in platelet adherence to the endotoxin followed by morphological changes, degranulation, and eventually lysis (Davis, 1966; Spielvogel, 1967). It has been suggested that the plasma factors are complement components ( Spielvogel, 1967) and that the adherence is to C3 which the endotoxin has fixed. Following such adhexence, the formation of C m could result in transfer of complement components to the platelets with their subsequent lysis by C8 and C9. It now appears that complement is readily activated by endotoxin (see Gewurz et al., 1968), by way of the properdin (alternative) pathway (Marcus et aZ., 1971). Involvement of this pathway would thus account for the cation requirements for the effect of endotoxin (Des Prez and Bryant, 1966) and for the finding that absorption of plasma with zymosan at 16"C. removes its ability to support endotoxin-induced release of amines (Des Prez, 1967; Des Prez and Bryant, 1969). Indeed, these authors suggested that the heat-labile factor in this system might bc properdin. The involvement of complement in this reaction also explains the relative inability of endotoxin to induce aggregation of human platelets in vitro or release of their constituents (Corn, 1966; Mueller-Eckhardt and Liischer, 1968d; Spielvogel, 1967; Nagayama et al., 1971), since human platelets do not adhere to C3 on immunological complexes. However, there have been reports that endotoxin can interact with human platelets (Ream et al., 1965; Des Prez and Marney, 1971) and does induce thrombocytopenia in primates ( Bennett and Cluff, 1957; Sutton et nl., 1969). This reaction possibly is by way of the coagulation system. If so, aggregation and release still ensue, but in this case they are induced by thrombin. 2. Human Platelets a. Release Following Adherence to Immunological Complexes. IYashed human platelets release constituents when they react with

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antigen-antibody complexes (Humphrey and Jaques, 1955; Movat et al., 1965; Mueller-Eckhardt and Liischer, 1968a), aggregated y-globulin ( Mueller-Eckhardt and Liischer, 1968a; Bettex-Galland et al., 1963; Henson and Spiegelberg, 1972), y-globulin-coated polystyrene or kaolin particles (Glynn et al., 1965; Movat et al., 1965; Mueller-Eckhardt and Liischer, 1968b; Mustard et al., 1967; Packham et al., 1968; Jobin et al., 1971), or y-globulin-coated glass surfaces (Evans and Mustard, 1968; Packham et al., 1968, 1969). Among the constituents released are nucleotides, serotonin, and permeability factors (Mustard et al., 1965; Packham et al., 1968; Mueller-Eckhardt and Liischer, 1968a,b) but not the cytoplasmic enzyme, LDH (Henson and Spiegelberg, 1973). It is clear that complement in the medium is not required for this reaction which is initiated by direct adherence of the platelets to the aggregated immunoglobulin. Aggregated immunoglobulins IgG1, IgG,, IgG,3, and IgG, are active but IgA, IgM, IgD, and IgE are not (Henson and Spiegelberg, 1973; Pfueller and Liischer, 1972). Serum is inhibitory for the release induced by IgG, and IgG,, suggesting that complement might inhibit by binding to the aggregates and preventing adherence of platelets. Platelets adhere to the Fc piece of the aggregated IgG molecules and are stimulated to release their constituents. The morphological changes are similar to those seen with other releasing stimuli, i.e., swelling, pseudopod formation, loss of organelles (Movat et al., 1965; Packham et al., 1968). Platelet phagocytosis has also been reported ( Section VI,A,5). As with the other immunological reactions of platelets, the process can be divided into adherence and ADP-induced aggregation. Both can be detected in the aggregometer (Davis and Holtz, 1969; Henson and Spiegelberg, 1973). They can be separated by inhibitors of the release reaction or of the aggregating effect of the ADP (e.g., adenosine or potato apyrase) . The releasing action of immunological complexes is not a result of thrombin activation ( Liischer, 1967; Henson and Spiegelberg, 1973). The requirement of calcium for the release is not clear since MuellerEckhardt and Liischer (1968a,b) found little inhibition with EDTA, whereas, Glynn et al. (1966a) and Mustard and Packham (1970) found that EDTA inhibits release, although not adherence. Mustard and Packham also report experiments indicating that metabolic energy is required. The aggregation of platelets by immunological complexes is inhibited by 1 mM iodoacetate, adenosine, AMP, and EDTA (Movat et al., 1965) and by PGE, (Mustard and Packham, 1970). MuellerEckhardt and Liischer (1 9 6 8 ~ )also demonstrated the inhibiting action of 0.1 mM iodoacetate and 1 mM N-ethylmaleimide, sulfhydryl-blocking agents that prevent many platelet functions (see Mustard and Packham, 1970). Diisopropyl fluorophosphate is reported to be inhibitory ( Glynn

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et al., 1966b), as are chymotiypsin substrates (Jobin et al., 1970). However, chymotrypsin itself does not induce release from platelets. Thus, the role of platelet esterases remains unclear. b. Antiplatekt Antibody and Drug-Induced Antiplatelet Factors. Heterologous antihuman platelet antisera, similar to heterologous antirabbit platelet antisera ( see above), induce release of nucleotides ( Mueller-Eckhardt and Liischer, 1968a). The reaction does not involve complement or thrombin, is partially inhibited by EDTA, and results in platelet aggregation as well as release. The release is sensitive to sulfhydryl inhibitors, Antiplatelet antibody also stimulates platelet metabolism (Karpatkin and Siskind, 1967). Isologous human antiplatelet antibodies directed against histocompatibility antigens are well known. Antiplatelet factors have also been implicated in many cases of idiopathic thrombocytopenic purpura ( Harrington et al., 1951; Shulman et al., 1964). However, direct demonstration of such antibody is extremely difficult and variable, although tests involving activation of platelet Factor 3 (Karpatkin and Siskind, 1969) or inhibition of uptake of serotonin (Kamoun and Hamburger, 1970) may prove somewhat more reliable. Circulating immune complexes may also become bound to the platelets and induce their destruction, thus mimicking the effect of an antiplatelet antibody (Myllyla et al., 1969,1971; Karpatkin and Siskind, 1969; Shulman et at., 1964). A variety of drugs is capable of inducing thrombocytopenia in man by imniunological mechanisms. Although direct investigations of the release mechanisms have not been made, some studies on this reaction, notably with Sedormid and quinidine, are important to consider briefly. Ackroyd ( 1949a,b, 1951) showed that addition of “sensitized’ plasma, platelets, Sedormid, and a complement source induce platelet aggregat’1011 and lysis. He suggested that the drug binds to the platelet and acts as a hapten. However, the drug binds poorly to washed platelets in the absence of antibody although better binding occurs when the platelets are in plasma, possibly because of uptake onto the plasma proteins adhering to the platelet ( Shulman, 1964). Shulman postulated an “innocentbystander” role for platelets in which the platelets bind the drugantibody complexes and are lysed by the complement that is activated. But the story may not be quite so simple since these particular drugs or this particular antibody seem to be of unique importance. Antibody and drug do not fix complement unless platelets are present. Moreover, addition of immunological complexes to human platelets in the presence of complement does not induce lysis of platelets (Henson and Spiegelberg, 1972). Perhaps the monovalent drug-antibody complex is too small to activate complement by itself, but when a number of complexes are

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concentrated together by binding to a platelet surface they can now activate complement and, under some circumstances, induce platelet lysis.

3. Cooperative Reactions of Platelets a. Basophile-Mediated Release from Platelets. The addition of antigen to washed leukocytes and platelets from an immunized rabbit results in release of vasoactive amines from the platelets ( Barbaro and Zvaifler, 1966; Schoenbechler and Barbaro, 1968; Schoenbechler and Sadun, 1968; Henson, 19f39b, 1970d; Siraganian and Osler, 1970). The release is immunologically specific and requires the presence of sensitized leukocytes, but the platelets can come from a nonimmunized animal. The nature of the leukocyte involved was for a while uncertain (Barbaro and Schoenbechler, 1970; Schoenbechler and Barbaro, 1968; Henson, 1970d), but it is now clear that the cell is the basophile (Siraganian and Osler, 1971b; Henson and Benveniste, 1971; Benveniste et al., 1972). Following the demonstration that the leukocytes can be passively sensitized in wivo by transfer of serum (Colwell et al., 1971; Benveniste et al., 1972), the sensitizing antibody was identified as being of the IgE class (Benveniste et al., 1972; Henson and Benveniste, 1971), in confirmation of earlier suggestions (Barbaro and Zvaifler, 1966; Henson and Cochrane, 1969b; Siraganian and Osler, 1970). The mechanism of action on the platelets involves the antigeninduced release from the basophiles of a soluble factor, PAF (Henson, 1969b, 1970d; Siraganian and Osler, 1971a), the nature of which has not yet been determined, although it is small in size and binds to serum albumin ( Benveniste et al., 1972). The release of PAF from basophiles is inhibited by glucose lack or by incubation with 2-deoxyglucose or DFP and requires extracellular calcium (Henson, 1970d; Benveniste et al., 1972). The liberation of PAF and of histamine from basophiles are both inhibited by high concentrations of agents that increase intracellular cyclic AMP (Osler and Siraganian, 1972). Like histamine, PAF is not released from cells incubated with antigen at 20°C. or below, a maximum is attained at 37"C., and the reaction falls off at higher temperatures. Storage of sensitized rabbit leukocytes in the cold in the absence of antigen leads to progressive loss of ability of the sensitized cell to respond to antigen (Barbaro and Schoenbechler, 1970). Whether this indicates involvement of an Na/Ksensitive ATPase or microtubules or some other factor is not known. Desensitization results when the sensitized cells are treated with antigen in the absence of Ca2+(Barbaro and Schoenbechler, 1970; Henson, 1970d). The results, although too limited to be definitive, suggest that the mechanisms of antigen-induced release of histamine and of PAF from the sensi-

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tized rabbit basophile are very similar if not identical. Recently, release of PAF from human leukocytes has been reported (Benveniste et d., 1972). The action of PAF on rabbit platelets is to induce their aggregation and release of vasoactive amines and nucleotides. This release does not result in liberation of cytoplasmic enzymes ( Henson, 1970d; Henson and Benveniste, 1971). Following the release, platelets appear morphologically intact, although swollen, irregularly shaped, and with internalized or inapparent organelles. Siraganian et al. ( 1971a) have described this as a cytotoxic reaction, based upon the release of previously incorporated *%b. However, the data on morphology, release of selected constituents, and inhibition of release do not confirm this, Apparently, the release of "Rb (and, therefore, potassium) in this reaction occurs even though the platelets remain intact and, in fact, still incorporate serotonin after the reaction (P. M. Henson, unpublished observations). The release reaction induced by PAF requires environmental calcium since it is inhibited by Mg EGTA. It is also prevented by 2-deoxyglucose, demonstrating a requirement for glucose metabolism and, thus, for platelet energy ( Henson, 1970d; Henson and Benveniste, 1971). Diisopropyl fluorophosphate is inhibitory but only if present while the PAF is added to the platelets (Henson et al., 1973). Pretreatment of either PAF or platelets with DFP does not inhibit. This again demonstrates that activation of a platelet esterase(s ) is an essential step in the release process. The cyclic AMP system seems to be involved here as in other release reaction of platelets. Prostaglandin El and theophylline are inhibitory, as is adenosine, whereas, epinephrine is stimulatory. Colchicine was also found to prevent the release (0.05 mM giving 50%inhibition), suggesting that microtubular integrity and, thus, platelet shape are important (P. M. Henson, unpublished observations). The aggregating effect of PAF may result in part from the release of ADP. However, aggregation is not required for release since it occurs when diluted suspensions of platelets are used without stirring, i.e., conditions in which platelet contact is minimized (Siraganian and Osler, 1971a). b. Neutrophile-Mediated Release from Platelets. The presence of small numbers of neutrophiles greatly enhances the release of histamine from rabbit platelets undergoing immunological release reactions ( Henson, 1 9 7 0 ~ ) .One such reaction involves peritoneal neutrophiles, platelets, soluble antigen, antibody, and plasma and appears to follow activation of thrombin in the plasma. Close contact between neutrophile and platelet is required, perhaps brought about by the joint adherence of both cells to the immunological complexes. Clot-promoting factors in the

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neutrophile combined with platelet clotting factors may serve to activate the coagulation sequence. The release of histamine is inhibited by hirudin and by other agents that prevent thrombin-induced release (see above). The adherence of neutrophiles and platelets to zymosan particles that have fixed C3 results in' active release of platelet constituents ( Henson, 1 9 7 0 ~ ) This . reaction does not require plasma. The release is inhibited by EGTA, 2-deoxyglucose, adenosine, and DFP, which is consistent with it being a secretory process. Although the nature of this augmentation by neutrophiles is not known, it was shown that it was not entirely due to the release of ADP from the neutrophiles; in fact, leukocytes have been reported to reduce ADP-induced reactions by metabolizing the nucleotide (Harrison et al., 1966). Other workers have also briefly noted potentiating effects of neutrophiles on immunological reactions of rabbit platelets ( Schoenbechler and Barbaro, 1968).

C. SUMMARY Many agents induce release of constituents from platelets, apparently by similar processes. The substances most readily released, vasoactive amines and nucleotides, come from the so-called dense bodies, granules that resemble in some ways those in mast cells. Whether the two-stage hypothesis (Section IV,A,l) applies to platelets as we11 as to mast cells, as suggested by Uvnas (1971), is not known. There is as yet little clear evidence of an actual extrusion of granules from the platelets (see also Holmsen et al., 1969). In fact, similar to the proposal of Libanska ( 1967), the secretion may merely result from an opening of the membrane surrounding the granule, with solubilization of its contents. This process has been demonstrated by French and Poole (1963) in platelets within a blood clot. The release may be stimulated by adherence of platelets to particles or immunological complexes or reaction with antibody. In a number of cases, the stimulus has been shown to result in activation of a platelet serine esterase. The substrate for this esterase is unknown but exogenous serine esterases such as thrombin and trypsin also induce release, perhaps through bypassing activation of the enzyme or perhaps by activating it themselves. Several of the stimuli decrease the levels of cyclic AMP, suggesting, by analogy with the effect of thrombin, a reduction in the activity of adenyl cyclase. Esterase activation precedes the step inhibited by increasing levels of cyclic AMP (Henson et al., 1973). It is thus possible that esterase activation is prior to the putative decrease in activity of adenyl cyclase. Epinephrine and adrenergic agents may more directly achieve this latter end. High levels of intracellular cyclic AMP may lead (Y

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to sequestration of intracellular calcium; lowering of the cyclic AMP levels could then result in mobilization of Ca?+(see Ardlie, 1971). This mobilization might also account for the calcium liberated during the release reactions. In the presence of calcium, a source of metabolic energy and, perhaps, of inicrotubules and of contractile elements, the release then occurs. The release reaction may involve a stage that is required in platelet aggregation followed by an additional stage necessary for the release itself. On the basis of this hypothesis, aggregation may be obtained without release ( Section VI,A,l,a ) by inhibiting this putative second stage, and release may result without aggregation (Section VI,B,3) by preventing platelet-platelet contact. The differences between the concentration of a drug, e.g., PGE, (Mustard and Packham, 1970), required to inhibit aggregation or release may reflect quantitative differences in the requirements of the two stages for release and aggregation. As a result of either immunological or nonimmunological stimuli marked morphological changes occur. The accumulation of organelles in the center of the platelets and the obliteration of the canalicular system has been postulated to be the result of a contractile process. The canalicular system is surrounded by microfilaments and alteration of their state of contraction may result in transient opening of the granulecontaining sacs, allowing inflow of cation, dissolution of the granule matrix, and, thus, extracellular release of the granule contents. VII. Mediator Secretion from Neutrophiles

Neutrophiles from all species so far studied have the ability to adhere to immunological complexes. This results from the presence on their surfaces of separate receptors for both C3 and immunoglobulin. Normally in the body or in uitm at 37”C., adherence results in phagocytosis of the complex by the neutrophile, assuming, that is, that the complex is of a suitable size for uptake to occur. The immunological complex or particle is taken into a phagocytic vacuole into which neutrophile granules then discharge their contents. The process of intracellular degranulation into the phagocytic vacuole is obviously one of secretion, in this instance, internal secretion, and resembles closely the secretory reactions of other cells. In addition, during the course of these events, some of the granule constituents gain access to the outside of the cell. Whether or not the extracellular release is usually accidental, the release to the outside is a direct consequence of the “degranulation” process ( Henson, 1971b, 1972d), is more easily studied than the “intracellular degranulation,” and is an important source of mediators such as proteolytic enzymes and permeability factors in inflammatory processes. Consequently, this release

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process will be considered in some detail and its relationship to degranulation explored. Recently, a more direct way has been developed of inducing extracellular granule discharge ( Hawkins, 1971; Henson, 19714, in which the immunological complexes are spread over surfaces too large to be taken up into the neutrophile ( i n uiuo parallels are known), Here direct exocytosis of granules occurs and its mechanism can be readily studied. Phagocytosis itself is a ccll function that resembles other cell processes discussed herein, and the spectrum of neutrophile activities, such as chemotaxis, adherence, phagocytosis, release, and digestion, are all interrelated and, therefore, will be considered. In addition, a few nonimmunological mechanisms of inducing release from neutrophiles are known, such as that following leukocidin treatment. Although leukocidin is actually cytotoxic, its mechanism has been well studied and has provided useful information on neutrophile function which is relevant to the immunological release processes. The effect of leukocidin will, therefore, be discussed in some detail.

A. NONIMMUNOLOGICAL RELEASEBY LEUKOCIDIN Since the beginning of the century, a product of staphylococci, called leukocidin, has been known to have cytotoxic effects on rabbit and human neutrophiles and monocytes, including an irreversible inhibition of cell respiration. Of importance to this discussion is that interaction of purified leukocidin with rabbit neutrophiles from peritoneal exudates release of granule constituents with little or no release of cytoplasmic enzymes (reviewed by Woodin, 1968; Woodin and Wieneke, 1970a). Although the ultimate effect of leukocidin is undoubtedly cytotoxic, the lysosomal enzyme release represents a secretory phase which either precedes or accompanies the cytotoxic effect. The detailed analysis to which this process has been subjected by Woodin has revealed processes that may be involved in the physiological secretion from neutrophiles. Addition of small amounts of leukocidin to neutrophiles causes an immediate rounding up and swelling of the cell, the granules exhibit random Brownian motion, become marginated, and eventually disappear (Gladstone and van Heyningen, 1957). The cells do not appear to lyse. By electron microscopy, some evidence of granule exocytosis was obtained and was supported by not finding any granule enzymes in the cell sap but only in the medium and in the remaining granules (Woodin, 1968). However, it should be noted that the electron micrographs shown did not clearly indicate this and the cells showed considerable degenerative changes. An important observation is that after release, the cells retain a number of vesicles which, it was proposed, result from discharge

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of granule contents to the outside. The membrane surrounding the granule is retained, along with ccrtain granule enzymes, such as acid and alkaline phosphatase and myeloperoxidase, calcium, and phosphate. Calcium is required. In EDTA, leukocidin induces cell swelling and Brownian movement of granules but no specific release; however, there is a gradual libcration of cytoplasmic constituents. Later addition of Ca'+ and ATP immediately induces liberation of granule enzymes. D ~ r i i i gthe release, large amounts of Ca'f accuniulate within the cell in the vesicles but not in the cytoplasm, indicating that the accumdation is not the result of a generalized increase in permeability. The release does not require cell energy metabolism. However, ATP does appear to be involved since calcium-deprived cells treated with leukocidin, after replacement of calcium, are stimulated to release only by the addition of ATP. Independence from glycolysis clearly differentiates the release from other secretory processes in this cell. The directly demonstrated requirement for ATP in leukocidin-induced release suggests that the requirement for energy metabolism in other release processes might be to furnish or maintain a source of available ATP. The site of action of leukocidin appears to be the cell membrane. It does not appear to bind to the cell but is altered and inactivated by intact cells or isolated membranes. It was suggested that the leukocidin reacts with triphosphoinositide in the membrane and that this is part of the potassium pump of the cell. A leakage of potassium results which is prevented by tetraethylammonium ions. Neutrophiles are unusual in having high levels of inkacellular Na+ and controlling cation concentration by K+ regulation (Elsbach and Schwartz, 1959; Woodin and Wieneke, 1970a). A K-sensitive phosphatase has been described in neutrophile membranes ( Woodin and Wieneke, 1WOb) which is activated by leukocidin and is possibly related to the putative potassium pump. Diisopropyl fluorophosphate increases the release of enzymes and the accompanying accumulation of calcium from neutrophiles treated with leukocidin. This action and that of the phosphonate esters is at an early stage of the process and may be on the potassium pump (Woodin and Wieneke, 197013). In this system, DFP probably acts as a detergent rather than as an esterase inhibitor ( Woodin and Wieneke, 1 9 7 0 ~ ) . Woodin and Wieneke (1970a,c) have drawn an analogy between these effects of organophosphorous inhibitors and their actions in chemotaxis. However, Becker ( 1 9 7 1 ~ )was able to show that two of the esterases essential for chemotaxis are unrelated to the potassium pump (Section X ) . Moreover, the effect on chemotaxis seemingly is due to phosphorylation by the organophosphorous inhibitors and is not a result of the detergent action of the components.

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Streptolysin 0 and vitamin A exhibit properties similar to leukocidin (Woodin and Wieneke, 1970a; Dingle, 1968, 1969) although differences do exist ( Zucker-Franklin, 1965). B. IMMUNOLOGICAL RELEASE

1. Secretion Znduced by Nonphagocytizable Surfaces The pathogenesis of some forms of neutrophile-mediated tissue injury involves the binding of neutrophiles to immune reactants along large surfaces such as the glomerular basement membrane. Under these circumstances, Hawkins and Cochrane (1968) detected the liberation of neutrophile enzymes and permeability factors into the urine. Neutrophiles adhere to immunological complexes spread over surfaces in vitro and release granule constituents; the cells are not lysed and do not liberate cytoplasmic enzymes. The technique provides a useful model for the in viuo situation and for the study of the secretoiy process in neutrophiles. The process is probably important in the release of mediators in vivo ( Henson, 1 9 7 2 ~ )Since . the granules are discharging along a stimulated portion of the cell membrane, which during normal phagocytosis bounds the phagocytic vacuole, the system may also provide a means of directly examining the mechanism of intracellular degranulation. The surfaces employed in vitro are collagen membranes (Hawkins, 1971, 1972a), micropore filters (Henson, 1971a) or the surfaces of petri dishes (Henson and Oades, 1973; Henson et al., 1972). By using micropore filters, Henson (1971b,c) showed that the process involved exocytosis of granules along the portion of the membrane of the neutrophile stimulated by the immunological complex and not along the nonadherent part of the membrane. Hawkins (1971) did not observe such an extrusion of granules, but he may not have looked early enough, and, moreover, the somewhat convoluted surface of the micropore filters provided a greater area for observation than the flat collagen membrane used by him. Immunological complexes on a surface stimulate neutrophile to release granule enzymes in a serum-free medium (Henson, 1971a,c, 1972b; Hawkins, 1971) , presumably through action on the immunoglobulin receptor of the cell. However, the presence of complement (C3) augments the release by acting on the C3 (immune adherence) receptor of the neutrophiles. Of the immunoglobulin subclasses in man, IgG,, IgG,, IgG,, and IgG, are active as well as IgA, and IgA,; but IgD, IgE, and IgM do not induce enzyme release (Henson et al., 1972). Chemotactic factors on surfaces have recently been shown to stimulate lysosomal secretion ( Showell et al., 1973).

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Rabbit neutrophiles adherent to immunological complexes on a nonphagocytizable surface release P-glucuronidase, P-galactosidase, acid phosphatase, and myeloperoxidase, enzymes that arise from the primary or azurophile granule, alkaline phosphatase that comes from the secondary or specific granule, and lysozyme arising from both. The intracellular origin of these enzymes has been described by Cohn and Hirsch (1960a), Baggiolini et al. (1969), Bainton and Farquhar (1968), and Spicer and Hardin (1969). Other granule materials, the exact location of which is not clear, are also liberated, e.g., cathepsins and small cationic permeability-inducing factors ( Hawkins, 1971; Henson, 1971a). Alkaline phosphatase behaves differently from the other materials, not being liberated into the medium but remaining bound to the external cell membrane at the site of secretion where it can be visualized histochemically by electron microscopy ( Henson, 1971b) . The secondary granules are released earlier and more rapidly than the primary granules, thus confirming previous reports of different rates of degranulation of the two kinds of granules (Bainton, 1970; Senda and Kishigami, 1962, quoted in Ohta, 1964)* The exocytosis requires energy metabolism and is inhibited by substituting 2-deoxyglucose for glucose in the medium (Henson and Oades, 1972b). Adherence of neutrophiles to the immune reactants on the surface stimulates the hexose monophosphate pathway of glucose metabolism concurrently with the stimulation of release. Close association of the hexose monophosphate stimulation and degranulation has been noted during phagocytosis ( Section VIII ) , However, by selectively inhibiting either the hexose monophosphate or the glycolytic pathways. Henson and Oades ( 1972b) demonstrated that the hexose monophosphate “shunt” is not supplying the energy for the exocytosis. In contrast, glycolysis is required and if it is inhibited, neither release nor hexose inonophosphatc pathway stimulation occurs. Removal of extracellular calcium is inhibitory to the exocytosis from rabbit neutrophiles ( Henson, 1972b ) but preincubation with chelating agents (e.g., 2 mM Mg EGTA) is required to prevent the secretion from human cells ( Henson, unpublished observations). Diisopropyl fluorophosphate also inhibits the rcleasc. Attempts to demonstrate activation of a DFP-inhibitable step in neutrophiles adhering to immune complexes on surfaces have so far been unsuccessful (Henson, 1972a; also unpublished observations). In these experiments, pretreatment of the neutrophiles with DFP was also inhibitory, presumably showing a requirement for an active esterase in the release, as previously demonstrated for phagocytosis (Pearlman et al., 1969; Section VII1,B). Indirect evidence has led to the suggestion of a role for cyclic AMP in neutrophile exocytosis ( Henson, 1972c; Hawkins, 1972b; Zurier et al.,

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1972a; Weissmann et al., 1972). Compounds PGE, and theophylline are both inhibitory. Prostaglandin El has been shown to stimulate adenyl cyclase and increase the levels of intracellular cyclic AMP in neutrophiles (Scott, 1970; Bourne et aZ., 1971). However, adenosine triphosphate ADP, and even AMP (1 mM) markedly inhibit the release of enzymes (Henson and Oades, 197213; cf. Section X ) . Dibutyryl cyclic AMP also prevents the release. Isoproterenol and epinephrine are ineffective, neither augmenting nor preventing the release of enzymes (Hawkins, 1972b; Henson, 1 9 7 2 ~ ).More direct evidence that cyclic AMP does not play a role in the release has been recently obtained using cholera enterotoxin to stimulate adenyl cyclase (Bourne et al., 1973) (see Section VII,B,2), Moreover, during the release from neutrophiles on surfaces, no changes in cyclic AMP levels were found when compared with control cells which were not releasing enzymes ( Henson, unpublished observations). Microtubules have been implicated in degranulation (see below), However, Henson (1 9 7 2 ~ )found that colchicine or vincristine did not significantly inhibit the release of granule enzymes from neutrophiles adherent to surfaces, and only a minor effect was reported by Hawkins (1972b). In contrast, Weissmann et al. (1972) found colchicine to be inhibitory. As yet, there is no explanation for these discrepancies (but see Section V111,C). Cytochalasin B inhibits uptake of particles by neutrophiles (Davis et al., 1971; Malawista, 1972; Malawista et al., 1971) but consistently enhances exocytosis on surfaces ( Henson and Oades, 1973; Hawkins, 1973; Weissmann et al., 1972). It was suggested that this agent alters the cell, perhaps by acting on the microfilaments, so as to allow increase of movement of granules within the cytoplasm and, thus increasing the chances for attachment of granules to the portion of the plasma membrane that adhered to the immunoreactants on the nonphagocytizable surface (Henson and Oades, 1973). However, cytochalasin B also inhibits hexose monophosphate stiniulatioii ( Henson and Oades, 1973; Hawkins, 1973; also see Malawista, 1972), apparently by inhibiting aptake of glucose (Zigmond and Hirsch, 1972). 2. Secretion during Phagocytosis

Phagocytosis by neutrophiles is considered in detail in Section VIII. One aspect of this process, the extracellular release of .constituents, is described here as an example of secretion from this cell. In their classic study of neutrophile degranulation, Cohn and Hirsch (1960b) found that during phagocytosis of bacteria by rabbit peritoneal neutrophiles, 104% or less of the granule enzymes escaped into the ambient fluid. Since

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then, many rcpoifs have appeared on the release of one or more neutrophile constituents from cells phagocytizing immunological complexes (Movat et al., 1964; Janoff and Zeligs, 1968; Tew et al., 1969; Martin et al., 1967; Hawkins and Peeters, 1971; Baehner et al., 1969; Crowder et al., 1969; Parish, 1969), endotoxin (Cline et al., 1968), starch (Pruzansky and Patterson, 1967; Baehner et aZ., 1969) bacteria (Wright and Malawista, 1972), or zymosan (Parish, 1969; May et al., 1970; Weissmann et al., 1971a,b; Henson, 1971a,b,c). Human and rabbit cells behave similarly, although a greater percentage of enzymes is liberated from rabbit neutrophiles. Among the materials released are inflammatory mediators such as permeability-inducing factors and proteolytic enzymes. However, cytoplasmic materials are not liberated ( Hawkins, 1972a; Weissmann et al., 1971a; Henson, 1971a; Wright and Malawista, 1972) and cell lysis does not occur during the 30-60 minute period of phagocytosis and release. It should be noted that endogenous pyrogen is also released from neutrophiles following phagocytosis ( Berlin and Wood, 1964). However, the release is much slower, does not appear to involve glycolysis, and has been shown in blood neutrophile to be prevented by actinomycin and puromycin (Bodel, 1970). The material cannot be extracted from neutrophiles and may have to be synthesized before release. The mechanism of its secretion, although of considerable interest, is, therefore, clearly different from those under discussion. Most of these experiments were performed in serum-containing media and the uptake probably results from complement fixation to the particles either by conventional or alternative pathways of activation. Immunological complexes, however, may stimulate neutrophile uptake and release directly ( Hawkins and Peeters, 1971; Henson, 1971c), although this reaction may be increased by the presence of complement, either by promoting larger aggregates and/ or more phagocytosis. Zymosan particles coated with either antibody or C3 are equally effective in stimulating uptake into and release from rabbit neutrophiles ( Henson, 1971a). Lysozyme, an enzyme probably present in more than one type of granule (Baggiolini et al., 1969), is released to a greater extent than are the other enzymes (Ohta, 1964; Crowder et at., 1969; Henson, 1971a,b). In contrast, cathepsins, not all of which may be segregated within the granules ( Baggiolini et at., 1969; Stiles and Fraenkel-Conrat, 1968), are not released to the same extent as P-glucuronidase ( Hawkins and Peeters, 1971; Henson, 19713). Other enzymes, such as acid phosphatase, may diminish in total activity during the course of the experiment (e.g., Hawkins and Peeters, 1971; Holmes et al., 1969; Mandell et al., 1970), thus preventing accurate analysis. Morphologically the release results from discharge of granules into a

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phagocytic vacuole which for a variety of reasons may still be open or may later become open to the exterior (Henson, 1971c, 1973; Weissmann et al., 1971a). This is clearly demonstrated by the behavior of alkaline phosphatase, which is neither released into the medium nor can be found on the externaI membrane of the cell, but remains bound to the vacuole membrane where it was liberated from the granule (Henson, 1971b). The larger the particles or aggregates phagocytized, the more readily extracellular release of granule enzymes is induced (Baehner et al., 1969; Hawkins and Peeters, 1971; Henson, 1971b; Henson et al., 1972). This progression in size culminates in nonphagocytizable surfaces (Section VII,B,l), the effect of which is to induce discharge of granules directly to the exterior. Except under very unusual circumstances, e.g., uptake of monosodium urate (see below), leakage of granule enzymes into or through the cytoplasm does not seem to occur. The release process requires metabolic energy, physiological temperatures, environmental calcium, and is inhibited by DFP (P. M. Henson, unpublished observations) and, thus, resembles the phagocytic process which it accompanies. Inhibitors have been used in an attempt to study the role of cyclic AMP. The release of p-glucuronidase from neutrophiles phagocytizing zymosan, but not the proportion of cells that take up particles, is inhibited by high levels of PGE, and theophylline and by both synergistically (May et al., 1970; Weissmann et al., 1971a,b). Dibutyryl cyclic AMP is also inhibitory. These results were confirmed by Henson (1972~)who also found that tenfold greater concentrations are required to inhibit release during phagocytosis than during exocytosis on nonphagocytizable surfaces. However, Bourne et al. (1973) found no inhibition of release when they used cholera enterotoxin to increase adenyl cyclase activity. There was no correlation between the inhibitory action of PGE, and theophylline on release and their ability to induce increases in cyclic AMP. As the authors pointed out, these findings cast considerable doubt on the idea that cyclic AMP regulates exocytosis in neutrophiles. From this work and the previously mentioned demonstration of the lack of any change in cyclic AMP levels of neutrophiles during exocytosis on nonphagocytosable surfaces (see above), it is clear that no role for cyclic AMP can be established on the basis of indirect evidence from inhibitors; direct correlation of intracellular levels of cyclic AMP is required for this purpose. Colchicine is reported to inhibit the extracellular release of enzymes during phagocytosis by neutrophiles ( Weissmann et al., 1972; Hawkins, 1972b), similarly to its effect on degranulation (Section VII1,C). Cytochalasin B increases the release to the outside (Weissmann et al., 1972; Hawkins, 1973; Henson and Oades, 1973; Zurier et al., 1972a). Although

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it prevents uptake of particles, cytochalasin B does not prevent their adherence ( Section VII1,B).

3. Phagocytosis of Alonosoclium Urate or Silica Crystals of uratc or silica particles are phagocytized by neutrophilcs (sec McCarty, 1970) possibly as a result of the proteins, e.g., inimunoglobulin and complement, they bind from the sei-um. Liberation of enzymes and other constituents follows this uptake. The release, although blocked by colchicine, is not a secretory process but results from lysis of the cell (Schumacher and Phelps, 1971; Weissmann et al., 1971a; Weissmann and Rita, 1972) and will not be discussed further.

4. Secretion Induced by Antineutrophile Antibody Heterologous antiiieutrophile antibody reacting with rabbit neutrophiles in the presence of complement induces an immediate massive release of lysosomal enzymes with a delayed release of cytoplasmic constituents ( Quie and Hirsch, 1964; Hawkins, 1972a). This process is eventually cytotoxic and, thus, resembles that seen with leukocidin and vitamin A (Section VI1,A). A similar phenomenon of release of lysosomal but not of cytoplasmic enzymes is seen during the incubation of embryonic bone rudiments in the presence of antibody and complement (Coombs and Fell, 1969). Complement components through C6 are required for the release (Lachmaim et al., 1969) which may indicate an ability of later-acting components to stimulate fusion of granules with cell membranes as is proposed for vitamin A (Dingle, 1968, 1969). This latter idea is strengthened by the finding that, in this bone-rudiment system, vitamin A induces a similar response to that of antibody and complement (Coombs and Fell, 1969). C. SUMMARY Secretion of neutrophile enzynics and mediators is intimately associated with the process of degranulation during phagocytosis and is probably a manifestation of the same process. Adherence of the neutrophile membrane to a surface or particle coated with immunoglobulin or complement stimulates the cell, a reaction that possibly involves an activatable esterase. The cell membrane is presumably altered and granules approach the membrane in a directed manner, fuse with it, and discharge their contents by processes that require cell energy ( supplied by glycolysis), calcium, and probably contractile elements, It is not yet clear what role cyclic AMP has in the secretory processes. Release of enzymes to the outside of the cell is secondary to the uptake of particles

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and degranulation into the vacuoles and occurs if the neutrophile is adhei-ing to particles on surfaces too large to be taken up or by a variety of mechanisms in which the phagocytic vacuole, almost accidentally, is open to the outside. VIII. Phagocytosis by Neutrophiles

Phagocytosis, the process by which cells, such as ncutrophiles, ingest particulate materials, may be divided into four phases : adherence, engulfment, degranulation, and digestion. In this review of processes related to secretion, nothing need be said about digestion and little about adherence, However, the engulfment stage may involve many cell activities similar to those required for release reactions, and the degranulation, which accompanies it, is itself a secretory process. These then will be briefly discussed (see also Section VII,B on immunological release from neutrophiles ) , Studies on phagocytosis in vitro have been extensive and results often conflicting. In many cases the differences result from vai-iations in technique, e.g., source and purity of neutrophiles (exudate or blood cells), differences in particles studied and the medium employed, as well as differences in incubation periods and in methods of assessment of uptake and degranulation (see Boyden et al., 1965). Many techniques for assessing engulfment, in fact, cannot distinguish it from adherence of the particle to the cell. Another difficulty is that an inhibitor may act on more than one stage of the process, thus making interpretation of its effect extremely difficult. Thus, under some circumstances, EDTA inhibits adherence, engulfment, and degranulation. There have been many reviews on the subject (e.g., Karnovsky, 1962, 1968; Karnovsky et al., 1972; Boyden et al., 1965; Rabinovitch, 1968; Spicer and Hardin, 1969; Sbarra et al., 1971; Nelson, 1965), and only those aspects of the process relevant to the cell functions under discussion will be considered here. A. ADHERENCE Undoubtedly, neutrophiles adhere by nonimniuiiological mechanisms to particles such as latex or glass surfaces, but this will not be discussed here. What will be considered is the more common immunological adherence where the neutrophiles adhere to the Fc piece of immunoglobulin (Lay and Nussenzweig, 1968; Quie et al., 1968; Henson, 1969a; Messner and Jelinek, 1970; Henson et al., 1972) or to complement (Lay and Nussenzweig, 1968; Henson, 1969a; Nelson, 1965). The receptors for these molecules are different from each other but adherence to either promotes phagocytosis. The immunoglobulin must be in the form of an

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aggregate or immunological coniplcx to promote adequate adherence and uptake and this probably results from cooperative binding effects since the bond between single imniunoglobulin niolecules and the neutrophile surface is weak ( Phillips-Quagliata et al., 1969). Here the most iniportant complement component is C3 (Gigli and Nelson, 1968), although C5 and C4 may also participate (Shin et al., 1969; Miller and Nilsson, 1970; Henson, 1972a). Binding of C3 to a particle or immunological complex is necessary, and a configurational change in C3 is required before adherence is promoted. Adherence ( e.g., of rabbit neutrophiles to erythrocytes coated with complement) may not be entirely passive, for, although it occurs with iodoacetate-treated cells where uptake is inhibited, it is, nevertheless, prevented by Mg?' deprivation and by lowered temperature (Lay and Nussenzweig, 1968; Henson, 1969a, 1972a). The nature of this reaction is as yet unknown.

B. ENGULFMENT As a result of the adherence of neutrophiles to particles such as immune complexes, the cell is triggered to undergo the engulfment step. There is some evidence that, up to a limit, the larger the size of the particle, the more readily it is taken up and stimulates cell respiration or degranulation. If very small, the particle may not be engulfed at all (Karnovsky, 1962; Baehner et al., 1969; Hawkins and Peeters, 1971; Henson and Oades, 1972b). This latter failure may result from poor adherence of very small particles, perhaps with little attached immunoglobulin and not much binding strength. Alternatively, a critical size may be required to initiate engulfment as proposed for Amoeba (Korn et al., 1967) on the surface of which small latex pai-ticles accumulate until they reach a sufficient mass and are then engulfed. As indicated above, the engulfment stage has not always been distinguished from the other phases of phagocytosis. Some aspects of the process have been reviewed by Rabinovitch ( 1968), who earlier (1967) dissociated attachment from uptake in niacrophages. A similar dissociation has been demonstrated in neutrophiles ( Pearlman et al., 1969; Henson, 1969a, 1972a). Serine esterases are involved in the engulfment of erythrocytes (EAC 1432) by neutrophiles just as they are involved in cheniotaxis (Section X ) . Pearlman et al. ( 1969) demonstrated that pretreatment of neutrophiles with p-nitrophenyl ethyl phosphonates or DFP prevented uptake. The profiles of inhibition by different derivatives of the phosphonates were almost identical to those shown for the activated esterase required for chemotaxis, suggesting that this enzyme might also be involved in en-

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gulfment. They pointed out, however, that cheniotaxis was more readily inhibited than phagocytosis and that inhibition in the former case was blocked by certain cstcrs. This difference niay result from differences in the nature of the two functions and the conditions used. Attempts to demonstrate the activation of an esterase during uptake did yield suggestive, although not conclusive, evidence for such a step. Thus, p-nitrophenyl ethyl 6-aniinohexylphosphonate and cyclohexyl butylphosphonofluoridate are more inhibitory if present duiing the reaction than if used to pretreat the cells. It has long been known that uptake into neutrophiles requires energy which in this cell is supplied by glycolysis. Thus, uptake occurs under anaerobic conditions and is largely prevented by inhibitors of glycolysis, such as iodoacetate, arsenite, and NaF (see Karnovsky, 1962, 1968), but is unaffected by KCN or aiitimycin A. In contrast, recent experiments of Bodel and Malawista (1969) have shown that 0.5 mM iodoacetate in their system did not inhibit uptake of bacteria. They did, however, obseive some inhibition with high multiplicities of particles and interpreted their findings as utilization of preformed metabolites by the cells to supply the requisite energy. Similarly, Brogan (1966) showed that iodoacetate inhibited uptake of starch in a serum-free medium but not if serum ( complement) was present presumably to supply a greater stimulus. In contrast to its action on engulfnient, iodoacetate was very effective in preventing degraiiulation and stimulation of the hexose nionophosphate shunt ( Malawista and Bodel, 1967; Bodel and Malawista, 1969). A marked increase in oxygen uptake also accompanies the process of uptake, but this respiratory stimulation, as will be discussed below, concerns degranulation as well as engulfment. Adenosine triphosphate in concentrations greater than 1 niM inhibits engulfment (Pearlman et al., 1969) just as it inhibits immunological enzyme release ( Henson, unpublished observations ) , release induced by leukocidin ( Woodin, 1968), and cell movement and chemotaxis ( Rivkin and Becker, 1972). The role of cations in the engulfment process is unclear. Pirt of the difficulty niay be that cations, especially magnesium, are required in adherence of neutrophiles (e.g., Allison and Lancaster, 1964; Bryant et d., 1966) iiicluding adherence of rabbit neutrophiles to complement-coated the reports particles (Lay and Nussenzweig, 1968; Henson, 1 9 6 9 ~ )Thus, . that phagocytosis is inhibited by removal of calcium and magnesium do not agree ( Wilkins and Banghani, 1964; Allison and Lancaster, 1964; Henon and Delaunay, 1966; Kvarstein, 1970), and it has often been found that Mg?' will restore the activity as well as calcium. Allison and Lancaster (1964) showed a difference between human and rabbit neu-

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trophiles, phagocytosis by the latter being completely inhibited by EDTA, whereas 30% of the phagocytic activity persisted in human cells so treated. Deionization of the medium was much less effective tlian EDTA. By using zymosan coated with liuman complenieiit or rabbit antibody, P. M. Henson ( impublished obscrvatioiis ) confirmed these results and was only able to completely prevent uptake into human neutrophiles by preiiicubating them with 5 mhl Mg EGTA. Adherence of neutrophiles, however, still took place. Kvarstein ( 1969) showed that calcium was required for uptake of polystyrene. Possibly intracellular calcium suffices for engulfment, and to show an effect, this too must be depleted, e.g., by preincubation with chelating agents. This area needs further study. Drugs that increase neutrophile cyclic AMP do not affect engulfment. Thus, PGEI, theophylline, cyclic AMP, and dibutyryl cyclic AMP were reportedly without significant effect on the uptake of zymosan (May et al., 1970; Weissmann et al., 1971b), whereas Bourne et al. (1971), by using a quantitative measure of uptake of Candida, found only a 30% reduction in engulfnient with high concentrations of dibutyryl cyclic AMP ( 3 mM), theophylliiie ( 3 mM), or PGE, (0.01 mM). Park et al. ( 1971) reported that cyclic AMP levels increased in phagocytizing leukocytes. However, Stossel et al. (1970) and Manganiello et al. (1971) did not observe such an increase when they used purified neutrophiles. Colcliicine and vinblastine have been reported not to affect the engulfment of bacteria ( Malawista, 1971b; Malawista and Bodel, 1967; Malawista and Bensch, 1967), only the subsequent degranulation and stimulation of respiration. However, sufficiently high concentrations of colchicine may reduce uptake ( R. B. Zurier, personal communication ) . Recently, cytochalasin B has been shown to prevent uptake of particles into neutrophiles (Davis et nl., 1971; Malawista, 1972; Malawista et al., 1971). Although this agent may work by interfering with contractile fibers (Wessels et al., 1971), this may not be its only mode of action (Sanger and Holtzer, 1972; Estensen and Plagemann, 1972; Cohn et al., 1972; Section VII,B,l). Contact with the immunoglobulin or C3 on the particle or iniinune complexes stimulates the cell, perhaps by way of an activatable esterase, resulting in the extrusion of pseudopods-a process that probably requires energy, calcium, and contractile fibers. An actinomyosin-like contractile protein has recently been isolated from neutrophiles (Senda et al., 1969; Shibata et d., 1972). Further stimulus to the extending portion of the pseudopod in contact with the particle would eventually lead to enclosure of the particle. It is clear that a requirement for a minimum particle size for ingestion would follow from this suggestion and is con-

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sistent with the data, and this would be related to the density of receptor sites on the cell for C3 or immunoglobulin. However, it is apparent that differences exist between the engulfment process and the mechanisms of cheniotaxis and cell movement (see Section X) as will be discussed below ( Section XII).

C. DEGRANULATION Once the particle is taken into the cell and the phagocytic vacuole has formed, the neutrophile granules fuse with the vacuole membrane and discharge their contents into the vacuole. This was first observed by Robineaux and Frederic in 1955 and was extensively and elegantly studied morphologically and biochemically ( Hirsch and Cohn, 1960; Cohn and Hirsch, 1960b) and eventually in the electron microscope (Lockwood and Allison, 1963; Zucker-Franklin and Hirsch, 1964; Horn et al., 1964). Cinematography revealed that only those granules that are situated near the vacuole degranulate into it ( Hirsch, 1962). Extrusion of granules around the border of the cell, unrelated to the vacuoles, was not observed. There appears, therefore, to be some change i n the vacuole membrane allowing granule membranes to interact with it. The nature of this change is unknown, although calcium may be involved in the binding (Woodin and Wieneke, 1970a). The ability of lysolecithin to induce membrane fusion raises the possibility of its local involvement in this type of phenomenon (Lucy, 1970, 1971). Hawiger et al. ( 1969a,b, 1972) found that neutrophile granules reacted with artificial phospholipid spherules prepared with lecithin or even more avidly with those prepared with inositol phosphatide. Contact between granule and phospholipid occurred resulting in granule disruption and activation of granule enzymes. These authors suggest that exposure of free phospholipid on the inner surface of the neutrophile (vacuole) membrane might allow interaction with cationic proteins on the neutrophile granules. The process was inhibited by pretreating the spherules with protamine sulfate, and the reaction was apparently performed in the absence of calcium. Although experiments with isolated granules are of interest, there is, as yet, no evidence that this process occurs in the cell. The difficulty of probing within the cell to analyze the biochemical events involved in degranulation has precluded development of a clear understanding of the process and has led to a number of conflicting suggestions. Hopefully, the development of systems in which degranulation occurs to the outside of the cell (Section VII,B,l) or in which the vacuole and its contents may be isolated (Stossel et al., 1971) may help elucidate the mechanism. Metabolic changes that accompany phagocytosis are numerous and

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complicated and have been extensively reviewed ( e.g., Karnovsky, 1962, 1968; Sbarra et al., 1971; Karnovsky et al., 1972). They include an immediate increase in glycolysis, O2 uptake, and glucose oxidation by the hexose monophosphate pathway. These activities are largely insensitive to cyanide, but stimulation of the hexose monophosphate pathway is prevented by anaerobiosis. The mechanism by which these functions are stimulated has been the subject of controversy (Sbarra et al., 1971). Increase in respiration appears in large part to result from a direct effect involving reduced nicotinamide adenine dinucleotide ( NADH ) oxidase and reduced nicotinamide adenine dinucleotide phosphate ( NADPH ) oxidases (Zatti and Rossi, 1965) the activities of which increase during phagocytosis ( Sbarra et al., 1971). Although a number of workers have demonstrated a close correlation of increased hexose monophosphate pathway activity with the degranulation process (Hirsch and Cohn, 1960; Malawista and Bodel, 1967; Selvaraj and Sbarra, 1966; Hohnes et al., 1969), stimulation of the hexose monophosphate pathway occurs before discernible degranulation ( Zatti et al., 1965). Moreover, evidence that hexose monophosphate oxidation does not supply the energy for degranulation comes from the understanding that, even though this pathway is greatly increased, most of the glucose is still metabolized by glycolysis ( Karnovsky, 1962), and, indeed, degranulation occurs under anaerobic conditions ( Selvaraj and Sbarra, 1966) or when NADH oxidation is inhibited by hydrocortisone (Mandell et al., 1970) or in leukocytes deficient in glucose-6-phosphate dehydrogenase ( Cooper et al., 1972). Recently, Henson (unpublished observations ) have completely inhibited the increase in hexose monophosphate oxidation without affecting the exocytosis of granules on nonphagocytizable surfaces. Inhibition of glycolysis, however, prevents both release and hexose monophosphate stimulation. There are indications, too, that the hexose monophosphate pathway may be stimulated without significant uptake or degranulation by soluble immune complexes or aggregates (Strauss and Stetson, 1960; Henson, unpublished observations), endotoxin (Cohn and Morse, 1960), nor antirieutrophile antibody ( Rossi et aZ., 1971 ). These various observations may be important since the pyridine nucleotide oxidases are in large part granule enzymes, and according to one theory, stimulation of the hexose monophosphate pathway follows some degranulation ( Karnovsky, 1962, 1968). Zatti et al. (1965) suggested that early changes in granule membrane permeability might suffice to provide the enzyme activities, but there is little evidence for this and the two processes, degranulation and stimulation of the hexose monophosphate pathway, although associated, may not be causally related to each other in either direction.

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The role of calcium in degranulation is difficult to study in the phagocytizing cell. However, its renioval does prevent exocytosis of granules on nonphagocytizable surfaces ( Henson, 1972c; Section VII,B,l ) . Colchicine and vinblastine, at concentrations that disrupt niicrotubules, have been reported to inhibit, although not completely, the process of degranulation (Malawista and Bensch, 1967; Malawista and Bodel, 1967; Malawista, 1971b). As mentioned above, these workers found little effect in their system on uptake. On nonphagocytizable surfaces, however (see above), the effect of colchicine is controversial. Although it is apparent that at least in some systems, such as in the uptake of bacteria, microtubules may play some role in degranulation they may not always be important. At present, any explanation of the role of microtubules flounders on our ignorance of the processes that bring granules to vacuoles, the influences of vacuole size, or the distance between granule and vacuole. Since cytochalasin B appears to prevent uptake (Section VIII,B), its role on degranulation into the vacuole in phagocytosis has not been determined (but see Section VII,B,l ) . Although the morphological processes of intracellular degranulation are apparent, the biochemical mechanisms are still IargeIy unknown. The process does, however, appear to be a prime candidate for inclusion with other secretory mechanisms. IX. Mediator Secretion from Monocytes and Macrophages

In contrast to the information available for the other cells, that for macrophages is quite limited. Monocytes and macrophages are granulecontaining phagocytic cells that adhere to immunological complexes and nonimmunological materials, engulf them, and liberate lysosomal enzymes into the ensuing phagocytic vacuole. The cells also have a pinocytotic capacity and can take up soluble protein and fluid droplets. Macrophages, unlike most of the other cells considered in this review, differ in this capacity and in their ability to synthesize new granule enzymes in response to stimuli such as phagocytosis (Axline, 1970; North, 1970). Although the cells come from a common bone marrow precursor, they vary in metabolic and other biochemical properties, depending on their eventual localization within the body (e.g., blood, lungs, or peritoneum). Adherence to immunological complexes results from receptors for C3 or immunoglobulin on the cell surface (Huber et al., 1968). Phagocytosis appears to be under similar control to that in other cells and, in peritoneal macrophages, depends on glycolysis (see Axline, 1970; Rabinovitch, 1968; Cohn, 1972). This should be contrasted with pinocytosis in these cells which involves oxidative metabolism.

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Extracellular release of granule but not cytoplasmic constituents has been reported during phagocytosis in vitro (Colin and Wiener, 1963; Weissmann et al., 1971b,c) and perhaps in vivo (Treadwell, 1965), although the nature of the particle to be taken up may influence this . analysis of the mechanism effect ( Weissmann et al., 1 9 7 1 ~ ) Detailed has not yet been undertaken, but it does appear to resemble the secretory processes in the other cells. As with release during phagocytosis in neutrophiles, it is difficult to distinguish the processes of phagocytosis from those of release in macrophages. Release of granule enzymes but not uptake of particles is inhibited by cyclic 3',5'-AMP, dibutyiyl cyclic AMP, and cyclic guanosine monophosphate ( GMP) ( Weissmann et al., 1971b,c). However, cyclic 2',3'AMP is also effective. Weissmann et al. showed a biphasic effect of cyclic nucleotides on the digestion of aggregated albumin, presumably resulting from degranulation. Thus, low levels stimulated, whereas higher concentrations inhibited. However, noncyclic nucleotides were also partially effective, and the question of biphasic effects of cyclic AMP on degranulation is unresolved ( see Section VII1,C). According to Weissmann et al. ( l W l c ) , the release requires divalent cations but is prevented by colchicine and vinblastine, although at concentrations that inhibit uptake. In contrast, Allison et aZ. (1971) have reported that colchicine does not prevent phagocytosis of bacteria or pinocytosis but does stop saltatory movements of the vacuoles and directional movement of the whole cell. Cytochalasin B prevents uptake and stops all cell movement. X. Chemotaxis

Like the other reactions discussed in this review, the chemotactic response undoubtedly is a complex, multistep, and probably multibranched process. The various steps can be assigned to two general stages: the first stage consists of the sequence or sequences by which the chemotactic stimulus signals the cell to move, that is, it consists of those steps that are specific for the cheniotactic response; and the second stage consists of the sequence or sequences of reactions by which the cell movement occurs (Becker, 1971b). Unfortunately, except in a few instances, the information necessary to separate the two stages is lacking. Recently, Showell et al. (1973) have shown that a bacterial chemotactic factor and C5a induce secretion of lysosomal enzymes, and in recent unpublished observations this has been extended to C3a and Cm. The ability of a cell to give release is not, however, correlated with its chemotactic responsiveness.

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The chemotactic response of rabbit polymorphonuclear leukocytes to C m (Ward and Becker, 1967, 1968, 1970a; Becker and Ward, 1969) and to C3a, C5a, and bacterial factor (Becker, 1972) involves the obligatory activation of a cell-bound serine esterase, proesterase 1. Activation of proesterase 1is not only a general requirement for chemotactic activity but the degree of such activation correlates with the level of chemotactic activity attained (Becker, 1972). This esterase exists in two forms in the cell: in an enzymatically inert proesterase, proesterase 1, incapable of being inhibited by phosphonates; and in an already activated form, esterase 1. Esterase 1 is capable of being inhibited by phosphonate esters and of hydrolyzing acetyl m-phenylalanine P-naphthyl ester ( Becker and Ward, 1969; Ward and Becker, 1970a; Becker, 1972). It is found in the microsomal fraction (Davies et al., 1970; E. L. Becker and S. Kegeles, unpublished observations), and a part of the activity is associated with the membrane portion of this fraction (E. L. Becker and S. Kegeles, unpublished observations). The nature and mechanism of action in the cell of the activated proesterase form of esterase 1 are presently unknown. Kallikrein is chemotactic for neutrophiles (Kaplan et al., 1972). Whether kallikrein is chemotactic by activating proesterase 1, by substituting for proesterase 1, or by other mechanisms remains to be determined. Another serine esterase, the “activated esterase,” different from the activated form of esterase 1, has been defined on the basis of the irreversible inhibition of chemotaxis by relatively elevated concentrations of phosphonate esters (Becker and Ward, 1967). The function of this esterase is unknown. Rabbit polymorphonuclear leukocytes incubated with the complement-derived chemotactic factors, C567, C5a, or C3a, show a progressive loss of their ability to respond to these same factors or to crude bacterial factor. The cells are “desensitized or “deactivated.” Cells incubated with crude bacterial factor are not deactivated to either bacterial factor or to the complement-derived factors (Ward, 1972). There is evidence that the inability of crude bacterial chemotactic factor to deactivate is due to its lesser efficiency in activating proesterase 1 (Becker, 1972). The bacterial chemotactic factor or C5a induced an increase in cell volume, presumably through inducing an influx of water (Showell et al., 1973). There is a statistically significant correlation between the volume increase and the chemotactic responsiveness of the cell. Both Ca?+and Mg” in the medium are required for maximal chemotactic response to bacterial factor or to C5a. Mg2+alone in sufficient concentration will sustain maximal spontaneous motility, although Ca2+ reduces the concentration of Mg2+required for maximal motility (Becker

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and Showell, 1972). These results suggest that external Ca?+might play a role in stage 1, the cheniotactic stage, whereas Mg2+apparently plays a role in the spontaneous motility of the cell. Ouabain inhibits the chemotactic response of the neutrophilc to CE7, and this is reversed by K+, suggesting the possible involvement of an ion pump (Ward and Becker, 1970b) , Factor (2x7 induces an increase in 0, consumption of the polymorphonuclear leukocyte ( P. A. Ward, unpublished work). Whether this effect is directly related to the chemotactic response or whether, just as the similar increase found in phagocytosis, it bears on events subsequent to the response is not known. The absence of glucose has no effect on the chemotactic response of human neutrophiles (Lotz and Harris, 1956; Carruthers, 1967). Insulin restores the chemotactic activity of the leukocytes of diabetics (Mowat and Baum, 1971). Dinitrophenol gives only slight inhibition of chemotaxis at 1 to 10 mM (Ward, 1966; Carruthers, 1967) but enhances the cheniotactic response and cell motility at lower concentrations ( Ward, 1966). These findings together with the denionstration of the inhibitory activity of iodoacetate (Carruthers, 1966) are all compatible with the idea that anaerobic glycolysis, but not the oxidative pathway, is the energy source involved in cell motility and chemotaxis. Steroids and chloroquine inhibit chemotaxis (Ward, 1966), and hydrocortisone inhibits ameboid motion of neutrophiles ( Ketchel et al., 1958). These results together with the enhancing action of vitamin A has led Ward (1966) to suggest the possibility that lysosomes may be involved in the chemotactic response. Streptolysin 0 inhibits chemotaxis and cell movement of human neutrophiles (Andersen and Van Epps, 1972). The microtubular disaggregating agents, colchicine ( Caner, 1964) and vincristine, and vinblastine and Colcemid (Ward, 1971) inhibit chemotaxis, and colchicine has been shown to depress cell motility (Caner, 1964). Cytochalasin B reversibly inhibits the chemotactic response of rabbit and human neutrophiles and depresses the motility of rabbit neutrophiles. Subinhibitory concentrations of cytochalasin B stimulate the chemotactic response of human and rabbit neutrophiles but have no demonstrable stimulatory effect on their motility ( Becker et al., 1972). When placed in the bottom compartment of the chamber, cyclic AMP is weakly chemotactic for rabbit polymorphonuclear leukocytes ( Leahy et al., 1970; Rivkin and Becker, 1972) as is dibutyryl cyclic AMP, but ATP, ADP, and AMP are not (Rivkin and Becker, 1972). Prostaglandin E, is also slightly chemotactic under the same circumstances (Kaley and Weiner, 1971). Dibutyryl cyclic AMP, ATP, ADP, and cyclic AMP but not AMP inhibit chemotaxis when placed in the upper compartment.

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Dibutyryl cyclic AMP and to a lesser and variable extent cyclic AMP enhance the spontaneous motility of the leukocytes, whereas, ATP and ADP inhibit this motility. There is evidence that ATP inhibits chemotaxis and spontaneous motility by the same mechanism and that the mechanisms of inhibition of chemotaxis and of spontaneous movement by ADP are also similar, but the inhibitory activities of ATP and ADP are exerted through different mechanisms. Epinephrine or isoproterenol mixed with the cells in the upper chamber are equally active in inhibiting chemotaxis and cell motility, but norepinephrine is without effect. The degree of inhibition is quite variable from one cell preparation to another. Prostaglandins El and E2 inhibit chemotaxis and spontaneous motility. Theophylline also inhibits chemotaxis and cell motility and exerts a synergistic effect on the inhibition of chemotaxis by epinephrine. The inhibitory effect of epinephrine is reversed by the p-adrenergic blocker, propranolol. These results suggest but do not prove that chemotaxis is under the modulating influence of cyclic AMP, with an increase of cyclic AMP decreasing the chemotactic responsiveness of the cell, which might occur through decreasing the ability of the cell to move in response to the chemotactic stimulus. A hypothesis (Becker, 1971b, 1972; Becker and Showell, 1972) that attempts to explain some of these results is that chemotactic factor( s ) stimulate the cell to put out pseudopods in the area where the factor attaches to its receptor. This extrusion of a pseudopod involves the contractile machinery of the cell, presumably both the microtubules and the microfilaments. The latter postulate explains the inhibitory actions of colchicine and of cytochalasin B. A further concept is that the contractile apparatus has much the same nature and is under similar control to actomyosin of muscle. In line with this suggestion, activation of proesterase 1 might induce membrane changes leading to the influx of extracellular Ca2+or a translocation of intracellular Ca?+from a bound to an unbound site or both. The Ca2+, in this view provides the excitationresponse coupling postulated or found to occur in the secretory contractile systems discussed in Section 11. Xi. Lymphocyte Transformation

Lymphocytes are stimulated to transform and to divide by

R

number

of agents. Some of these are apparently nonimmunological, such as phytohemagglutinin ( PHA) , pokeweed mitogen, staphylococcal exotoxins, and streptolysin S; others are specific, such as antigens to which the lymphocytes are sensitized or antibody to various immunoglobulins or other antigens on the surface of the cell. The receptors for PHA and for allogeneic lymphocytes seem to differ ( Lindahl-Kiessling and Peterson,

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1969). In the mouse and human, only thymus-derived lymphocytes ( T cells) are stimulated by soluble PHA (Doenhoff et al., 1970)) whereas both T- and bone marrow-derived mouse lymphocytes ( B cells) are stimulated by pokeweed mitogen ( Greaves and Bauminger, 1972). Endotoxin and specifically the lipid A portion stimulates only B cells (Chiller et al., unpublished observations). To what these differences are attributable is unknown. Taylor et al. (1971) have shown that antibody reacting with immunoglobulin on the surface of lymphocytes induces the antigen-antibody complexes which are formed to gather over one pole of the cell containing the Golgi apparatus. This so-called cap formation is completely inhibited by coId sodium azide and 2,4-dinitrophenol, and by high concentrations (10-15 pglml.) of cytochalasin B but not by Colcemid. They reported that cap formation is followed by pinocytosis. Taylor and his co-workers suggest the possibility that cap formation and subsequent pinocytosis may be involved in triggering lymphocyte transformation. Both Karnovsky et al. (1972) and dePetris and Raff ( 1972) believe that cap formation requires cell movement, but the former consider it a mere passive concomitant of such movement. Phytohemagglutinin and another mitogen, concanavalin A, accumulate over one pole of the lymphocyte surface. Cytochalasin B, which partially inhibits cap formation on lymphocytes, markedly inhibits pinocytosis in these same cells and depresses transformation of lymphocytes by PHA, purified protein derivative (PPD), and allogeneic cells (unpublished work of D. Webster and A. C. Allison, cited by Taylor et al., 1971). The finding that insoluble phytomitogens can transform lymphocytes, however, is difficult to reconcile with the thesis that pinocytosis of the mitogen is a requirement for transformation (Greaves and Bauminger, 1972), but is compatible with the idea that aggregation of ,receptors is the trigger. Moreover, Karnovsky et al. (1973) believe that the effect of cytochalasin B on cap formation is a toxic one. K. Yoshinaga and B. H. Waksman, in unpublished work, have shown that sufficiently high concentrations of cytochalasin B inhibit the synthesis of DNA induced in rat lymph node cells by specific antigen, PHA, or concanavalin A. Lower concentrations of cytochalasin B enhance the synthesis of DNA stimulated by PHA or concanavalin A, but enhancement was not seen in cells stimulated with specific antigen. The half-maximal binding of PHA to lymphocytes requires only 7.5 minutes (Mendelsohn et al., 1971), but the role of PHA in the initiat'ion of transformation is not complete until at least 6 hours after addition (Kay, 1970). Within the first few minutes to 0.5 hour after addition of PHA or

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streptokinase-streptodornase to unsensitized lymphocytes or of antigen to sensitized lymphocytes, there is a stimulation of the incorporation of phosphate into phospholipids, presumably membrane phospholipids (Lucas, 1970), and an uncovering of the phosphate groups of DNA. By 15 minutes there is increased acetylation of arginine-rich histones and increased RNA synthesis (reviewed in Cooper, 1970). After 30 minutes there is a stimulation of the uptake of y-aminoisobutyric acid by PHAstimulated lymphocytes (Mendelsohn et al., 1971), and, after an hour of exposure, there is an increase in the uptake of K+ (Quastel and Kaplan, 1970). There is an increased incorporation of amino acids into protein starting 2-3 hours after PHA stimulation. The increase in DNA occurs only 24436 hours after contact with PHA. Within 2 to 4 hours after stimulation of the lymphocytes by PHA or antigen, hydrolases of the lysosomal granule become distributed from the granule to the less sedimentable fraction of the cells and there is increased pinocytosis. Stimulation of lymphocytes leads to increased endocytosis (Hirschorn et al., 1968). It has been suggested that the accompanying shifts in lysosomal hydrolases might lead to changes in the nucleus possibly by a proteolytic removal of repressor protein from the DNA (reviewed in Weissmann and Dukor, 1970). The protease inhibitors, caminocaproic acid ( EACA), tosyl arginine methyl ester ( TAME), tosyl lysyl chloromethyl ketone ( TLCK ), and tosylamide phenylethyl chloromethyl ketone ( TPCK ) inhibit leukocyte transformation ( Hirschoni et al., 1971). Although these findings are in accord with the latter hypothesis, Weissmann and Dukor (1970) point out that there is still no direct experimental evidence for it. Within 2 hours or less following the addition of PHA to human peripheral lymphocytes, there is an increase in lactic and pyruvate production and a decrease in ATP and ADP. After 2 hours, an increase is induced in the level of ATP and ADP, glucose l,g-diphosphate, and 2,3phosphoglycerate. Iodoacetate prevents all changes induced by PHA; NaF and deoxyglucose prevent the increase in lactic production, but not the decrease induced in ATP and ADP; and KCN does not affect the stimulation of lactic production by PHA. From these results and others not described here, Roos and Loos (1970) concluded that PHA stimulation induces an energy demand that is met initially by an increase in glycolysis, and after about 2 hours Krebs cycle activity is increased. The latter might be the cause of the increase in respiration following PHA stimulation, although part of the increase may come from stimulation of the hexose monophosphate shunt mechanism. Diethyl maleimide, p-chloromercuribenzoate and 2,4-dinitrophenol inhibit DNA and RNA synthesis of PHA-stimulated lymphocytes (reviewed by Quastel et al., 1970), and

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reducing agents enhance transformation ( Fanger et al., 1970). The relation of the changes in carbohydrate metabolism described above to the transformation is not clear; however, inhibition of transformation by 2,4dinitrophenol suggests that aerobic oxidation is the energy source for the transformation; whether glycolysis plays a role or can substitute for respiration is not evident. Cations Ca2+ and Mg?' are required for the transformation, as is apparently Fez' and Zn?+( Alford, 1970). One suggested role for divalent cations is that they are specifically required for the initial interaction of PHA and lymphocytes (Kay and Austen, 1971). Phytohemagglutinin induces uptake of Ca2+within 38 minutes of contact with the mitogen with lymphocytes, and one possibility is that this uptake is an obligatory step in the cellular transformation induced by PHA (Allwood et al., 1971) . Ouabain inhibits transformation and this is reversed by increasing the levels of K+ in the medium. This has been ascribed to inhibition of the Na+/K pump of the cell preventing the Kt uptake induced by PHA ( Quastel and Kaplan, 1970). Although ouabain not only inhibits transformation by PHA but by specific antigen as well, the stiniulatory effects of PHA resume immediately after removal of ouabain but are not so reserved in cells stimulated by specific antigen (Wright et al., 1972). Smith et al. (1971a) showed that 1-2 minutes contact with PHA and prostaglandins produces large increases in intracellular levels of cyclic AMP of human leukocytes, whereas isoproterenol and aininophylline produce lesser increases. These increases are associated with increase in the adenyl cyclase activity of the cell. Low concentrations of added cyclic AMP produce slight stimulation of thymidine incorporation into DNA in the absence of PHA. However, stimulatioii by PHA of the incoiporation of thymidine, uridine, and leucine into the DNA, RNA, and protein of the lymphocyte is inhibited (not stimulated) by isoproterenol, aminophylline, and the prostaglandins (Smith et al., 1971b; Estes et al., 1971). The inhibition occurs at a rclatively early step in the sequence of reactions. Dibutyryl cyclic AMP produces maximal inhibition if present during the first hour after PHA addition. Other cyclic nucleotides as well as the tri-, di-, and monophosphates of adenosine and guanosine are also effective, and Hadden et al. (1972) have suggested that guanosine 3'5' cyclic MP is a possible intracellular mediator of transformation. Similar findings were obtained in the antigen-induced transformation of sensitized lymphocytes. Smith et al. (1971b) point out that their "data fail to provide evidence that cyclic AMP alone, increased as a result of stimulation of lymphocyte adenyl cyclase by PHA, can initiate the complex series of metabolic

166

ELMER L. BECKER AND PETER M. HENSON

alterations which culminate in lymphocyte transformation. . . .” Sustained elevation of lymphocytc cyclic AMP levels result in an inhibition of synthesis of macromolecules. The cyclic AMP levels of blood lymphocytes from severe asthmatics failed to increase when stimulated with isoproterenol, whereas increases did occur in cells from nonatopics and patients with hay fever (Smith and Parker, 1970). Hadden et a2. (1971b) showed that in the presence of low concentrations of hydrocortisone, norepinephrine increases glucose uptake, anaerobic glycolysis, and thymidine uptake in PHA-stimulated human lymphocytes. The effect of norepinephrine is inhibited by phentolamine, an a-adrenergic blocker. They concluded that a-adrenergic stimulation enhanced lymphocyte transformation by PHA. This is in contrast to the inhibition of PHA-stimulated transformation by p-adrenergic agonists in the work of Smith et al. and others (see above). Coffey et a2. (1971, 1972) reported that a-adrenergic stimuli raised membrane ATPase levels and that asthmatics have especially high ATPase levels which are brought to normal by corticosteroids. Franks et al. (1971) demonstrated that PGA, and PGE,, but not PGF,, or PGF,, stimulate the adenyl cyclase of thymic lymphocytes with a resultant increase in the intracellular level of cyclic AMP. Those prostaglandins that increase intracellular cyclic AMP also increased the rate of thymic cell proliferation; those that did not cause an increase in cyclic AMP had no effect on lymphocyte mitosis. From work on bacteria (decrombrugghe et al., 1971) it is clear that cyclic AMP may act on transcription and possibly on the translocation process as well. Whether a similar mechanism of action is operative in the effects of cyclic AMP in lymphocyte tramformation remains to be elucidated ( see Cooper, 1970). XII. General Summary

The work just reviewed, which is partly summarized in Table I, makes it clear that the characteristics of the immunologically induced, noncytotoxic mediator release and the related phenomena taken up here show distinct resemblances to secretory processes in general as outlined in Section 11. In all instances considered in Table I, where the point has been investigated, external Ca’+ is found to be required or to enhance the given response. The only exceptions noted are the release of histamine from rat peritoneal mast cells by compound 48/80 and by band 2 lysosomal protein (Sections IV,A,l and 2 ) . With histamine release by band 2

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167

protcin, there is the paradox that unactivated cells do not require Ca2+ for release but activated cells do. This suggests that, with these findings in mind, a restudy of the Ca" requirements of 48/80-stimulated cells might be worthwhile. In regard to 48/80 stimulation, it is also of interest that, although there is little if any Ca'+ requirement for release of histamine from rat peritoneal mast cells, rat mast cells in fixed tissues, such as the mesentery, do require Ca'+ for their response when stimulated by 48/80 or antigen (Section IV,A,l). Only in the transformation of lymphocytes by PHA (Section X I ) and the secretion induced from polyniorphoiiuclear leukocytes by leukocidin (Section VI1,A) has the requirement for Ca" been shown to be associated with an uptake of Ca". In view of the findings in secretoiy processes in general (Section 11), and of the concept of Ca2+being the agent responsible for stimulus-secretion coupling as well as the analogies with muscle contraction that have been drawn, it is obvious that the study of the uptake of Ca2+in the various systems described in Table I would be of great value. In regard to muscle contraction and the requirement for external Ca?', two situations have been described: ( 1 ) In skeletal muscle, contraction can occur in the absence of external Ca2+, but there is a mobilization of internal Ca2+ through the sarcoplasm reticular system. ( 2 ) With cardiac muscle, external Ca2+ is required; stimulus leads to an uptake of external Ca2+and this, in turn, is associated with a mobilization of internal Ca?+ bound in the sarcoplasmic reticulum. From the almost universal requirement for external Ca2+in the responses considered in this review, it is possible that these functions resemble more the action of cardiac than of skeletal muscle. As is evident from Table I, only in antigen-induced histamine release from sensitized human basophiles is Mg?' required ( Section V,A), although Mg2+enhances chemotaxis and spontaneous movement and adherence by polymorphonuclear leukocytes ( Sections VII1,A and X ) . In all the other instances studied, Mg2+has either no effect or is inhibitory. The role of other ions has been investigated even less. From the analogy with muscle contraction, one might expect an influx of Na+ and an efflux of K+ on stimulation. Histamine release from rat peritoneal mast cells by Ca?+and ATP has been shown to be associated with an influx of Na+ (Section IV,A,3), but the stimulation of lymphocytes by PHA has been shown to be associated with an uptake of K+ (Section X I ) . Release of K+ has been reported from platelets stimulated both nonimmunologically ancl immunologically ( Sections VI,A and B ) . Small amounts of K+ are released from rat peritoneal mast cells following stimulation by 48/80 or by antigen (Sections IV,A and BJ), but this has been attributed to a release of K' from the granule. The alternative

168

ELMER L. BECKER AND PETER M. HENSON

SUMMARY OF

CHARACTERISTICS OF

IMMUNOLOGICALLY AND

TABLE NONIMMUNOLOGICALLY

Requirement for System

I. Histamine release A. Isolated tissues and organs 1. Guinea pig lung perfused with Ag-Ab complexes 2. Sensitized guinea pig lung slices 3. Sensitized human lung slices

Ca2+

Mg2+

+

N.R.

SH

Activatable Activated esterase esterme

€3. R a t Mast Cells 1. By 48/80

0

0

+ + + +

2. Band 2 prot,ein

0

0

N.R.

+

0

3. ATP/Caz+

+ yE antibody 5. Ag + TGa antibody 4. Ag

6. C-dependent noncytotoxic C. Mouse mast cells 1. sensitized with yE antibody 2. Sensitized with yl antibody D. Basophiles 1. Human

2. Rabbit 11. Histamine or serotonin release from platelets A. Human 1. Collagen 2. Thrombin 3. Epinephrine 4. ADP aggregation

5. After adherence to immunological complexes 6. Antiplatelet antibody

+ +

0 0

N.R.

+ +

N.R. 0 0

N.R.

N.R.

+" + +

0

N.R.

N.R.

N.R.

N.R.

+

+

0

N.R. N.R.

N.R. N.R.

N.R. N.R.

N.R.

+

N.X.

N.R.

N.R.

N.R.

N.R.

N.R.

N.R.

N.R.

N.R.

N.R.

N.R.

+ +

+

+ +

0

+

N.R.

N.R.

N.R.

N.R.

N.R.

N.R.

N.R.

N.R..

N.R.

N.R.

N.R.

N.R.

N.R.

N.R.

+ + + + + +

0

N.R.

N.R.

+ N.R. N.R.

+ + + + + +

0

169

IMMUNOLOGICALLY INDUCED MEDIATOR SECRETION

I INDUCED

nIEDlATOn SECRETION

Nature of energy source

Aerobic oxidation or anaerobic glycolysis Aerobic oxidation or anaerobic glycolysis Anaerobic glycolysis or aerobic oxidation Aerobic oxidation or anaerobic glycolysis Aerobic oxidation or anaerobic glycolysis Aeiobic oxidation Aerobic oxidation or anaerobic glycolysis N.R. N.R. Aerobic oxidation or anaerobic glycolysis Aerobic oxidation Anaerobic glycolysk

N.R.

Glycolysis and aerobic oxidation Glycolysis and aerobic oxidation N.R.

AND

Heat. lability

+ +

RELATED PIIENOM13NAa Necessity for contractile system

Desensitisation

Enhancement by dicarboxylic acid

+ + +

Intracellular cyclic AMP

N.R.

N.R.

+ + +

+ + + +

+

0

+

N.R.

+

N.R.

Increase inhibits N.R.

N.R.

N.R.

+

N.R.

N.R. N.R.

N.R.

+

T o 48/80 action

Increase inhibi t,sn Increase inhibits Increase inhibits

N.R. N.R.

N.R. N.R.

N.R. N.R.

N.R.. N.R.

Increase inhibits N.R. N.R..

N.R.

N.R.

+

0

N.R.

N.R.

N.R.

N.R.

0

N.R.

+ +

+

+ +

+ +

Increase inhibits N.R.

N.R.

N.R..

+

N.R.

N.R.

N.R.

N.R.

?

+

N.R.

+ + + +

N.R.

+ +

Glycolysis and aerobic oxidation N.R.

N.R.

N.R.

N.R.

N.R.

N.R..

N.R..

N.R.

N.R.

N.R.

Increase inhibits Increase inhibits Increase inhibits Increase inhibits Increase inhibits N.R. (Continued)

170

ELMER L. BECKEX AND PETER M. HENSON

TABLE I Requirement for System

B. Rabbit 1. After adherence to immunological complexes 2. Antiplatelet antibody 3. PAF-induced release 4. Neutrophile-induced release 111. Leukocidin-induced lysosomal release IV. Phagocytosis-associated granule release V. Chemotaxis by neutrophiles

VI. Phagocytosis by neutrophiles VII. Lymphocyte transformation

Caa+

+ + + + + + + + +

nIg2+

SH

Activatable Activated esterme esterase

N.R.

+

+

0

N.R. N.R.

N.R.

+

+ +

0 0

N.R.

N.R.

N.R.

N.R.

0

0

N.R.

t

+

+

0

+

N.R.

+

N.R..

N.R.

N.R.

+

+

+ N.R.

N.R.

Abbreviations: Ag, antigen; Ab, antibody; ATP, adenosine triphosphate; ADP, adenosine “Increase inhibits,’’ in general, merely means that agents known or believed to increase c Cation Ca2+ required when cell is activated. 0

explanation, that this is associated with and reflects a putative membrane depolarization, is well worth exploring, as are the effects of the various stimuli on membrane potentials of the cells under consideration. The requirement for sulfhydryl bonds is apparently universal (Table I ) but this is such a generalized characteristic that its significance is quite unclear and probably differs from one situation to another. The role of cell surface ATPase in these release reactions and related phenomena is unclear. Ouabain is generally, although not invariably (Sections III,B, X, and XI), ineffective as an inhibitor, and, although sulfhydryl reagents are effective, they may act on other molecules in the cell beside the ATPase. Nevertheless, ATP itself will initiate release from mast cells and is inhibitory to neutrophile and platelet reactions. The “ectoATPase” activity has been invoked to explain ATP-induced histamine release from mast cells as well as some features of platelet aggregation. The platelet ectoATPase activity is inhibitable with antibody against thrombosthenin. It has been suggested that ADP inhibits an

IMhZUNOLOGICALLY INDUCED MEDIATOR SECRETION

171

(Continued)

Nature of energy source

Heat, lability

Necessity for contractile system

Desensitizntion

Enhanccment by dicarboxylic acid

+

N.R.

N.R.

N.R. N.K

N.R.

+

N.R.

+

N.R. N.R.

0

N.R. N.R.

N.R. N.R.

N.R. 0

N.R. N.R.

Anaerobic glycolysis

N.R.

N.1:.

N.R.

Anaerobic glycolysis

N.R.

+

N.R.

Anaerobic glycolysis

N.R.

Aerobic oxidation

N.R.

N.R.

N.R.

N.R. N.R. N.R.

+ + + +

N.R. N.R.

N.R.

Intracellular cyclic AMP Increase inhibib N.R. Increase inhibits N.11. N.R. Increase inhibits Increase inhibits 0 Increase inhibits

diphosphate; PAF, platelet-activating factor; N.R., no report. intracellular cyclic AMP inhibit the reaction.

ouabain-insensitive cctoATPase in platelets and allows relaxation and aggregation ( Salzinan et al., 1966). However, there is no direct evidence for this, and Guccione et al. (1971) have shown that ADP is converted to ATP at the platelet membrane. Although not emphasized because of space limitations, it is clear that immunological stimuli to cells are generally multivalent. The stimulation of platelets by antiplatelet antibody, of basophiles by antigen or anti-IgE, and of neutrophiles or platelets by immunological complexes requires that the antibody be at least divalent. This may be related to the “aggregation” of immunoglobulin receptors on lymphocytes by anti-immunoglobulin or antigen, as described by Taylor et al. (1971). This aggregation and/or configurational alteration of receptors capable of moving in a fluid lipid niembrane (Singer and Nicolson, 1972) may initiate the activation process. Nevertheless, small molecules also stimulate release, e.g., C5a, and band 2 protein on mast cells. However, the valency or mode of binding of these small activators is not known, and, although

172

ELMER L. BE-

A N D PETER M. HENSON

their positive charge suggests an electrostatic mechanism, other cationic proteins similar to band 2 protein do not activate mast cells. Table I1 lists the systems for which there is evidence that the activation of a serine esterase is required. This requirement is not known to be one of the general characteristics of secretory processes described in Section 11. To what degree this reflects a lack of investigation of this point is not known. As seen in Table 11, there are a number of different responses, chemotaxis, phagocytosis, and release of mediators, for which there is some evidence for an activatable esterase being required. These responses are induced by quite variegated stimuli, from cells as widely different as polymorphonuclear leukocytes, mast cells, and platelets. In certain situations, such as antigen-induced histamine release from human basophiles (Section V,A) or the killing of cells by sensitized lymphocytes (Ferluga et al., 1972), the requirement for an activatable esterase has been looked for but not found. It is not known whether these negative instances reflect a lack of involvement of this kind of enzyme or that our present admittedly crude techniques (largely the use of organophosphorous inhibitors, see below) are inadequate to detect the enzyme except under the most favorable circumstances. However, in view of the number and variety of responses for which there is evidence that activation of a serine esterase is an obligatory step, we deem it likely that this will be found to be a general requirement in most, if not all, the phenomena reviewed here and might possibly have an even more general role. TABLE I1 SYSTEMSFOR WHICHTHERE Is EVIDENCE FOR A REQUIREMENT OF AN ACTIVATABLE ESTERASE

1. Chemotactic response of rabbit polymorphonuclear leukocytes to C5a, C3a, C567, and bacterial factor. 2. Erythrophagocytosis of EAC423 by rabbit polymorphonuclear leukocytes. 3. Antigen-induced histamine release from rat peritoneal mast cells sensitized by rat homocytotropic antibody. 4. Complement-dependent, noncytotoxic histamine release from rat peritoneal mast cells induced by rabbit antirat YG-globulin. 5. Histamine release from rat peritoneal mast cells induced by band 2 lysosomal protein from rabbit polymorphonuclear leukocytes. 6. Antigen-induced histamine release from sensitized guinea pig lung slices. 7. Antigen-induced histamine release from human lung sensitized with yE antibody. 8. Histamine release from rabbit platelets by zymosan containing bound complement. 9. Noncytotoxic histamine release from rabbit platelets by antiplatelet antibody. 10. Release of serotonin from rabbit platelets by platelet-activating factor obtained from rabbit basophiles. 11. Release of serotonin from rabbit platelets by collagen.

IMMUNOLOGICALLY INDUCED MEDIATOR SECRETION

173

In the chemotactic response of polymorphonuclear leukocytes, all the chemotactic stimuli studied, C3a, C5a, (2567; or bacterial factor, activate the same esterase, proesterase 1 ( Section X ) . Similarly, antigen-induced histamine release from rat peritoneal mast cells sensitized with rat yE antibody ( Section IV,B,l ) , noncytotoxic complement-dependent histamine release by rabbit antirat y-globulin (Section IV,B,l), or histamine release by band 2 lysosomal protein (Section IV,A,B) all require activation of the same proesterase. In view of the fragmentary evidence that esterase activation is one of the very early steps following combination of the agonist with its receptor, esterase activation may be a “common initial pathway” in the sequence of reactions for the various responses. In all responses listed in Table 11, except the chemotaxis of polymorphonuclear leukocytes, the evidence for the involvement of a serine esterase is indirect. It consists of the demonstration that organophosphorous inhibitors, such as DFP or phosphonate esters, interfere with the response when they are present at the same time that the cell is stimulated, but treatment of the stimulating agent or the cell separately has no effect, and the necessary assumption that the organophosphorous inhibitors only act irreversibly. However, as reviewed in Section X, there is now clear-cut, direct biochemical evidence that the activatable esterase involved in the chemotactic response of polymorphonuclear leukocytes is an amino acid esterase, proesterase 1.This lends some indirect support to the idea that the various activatable esterases are proteases. Possible additional support to this concept are the findings that proteases, such as chymotrypsin, release histamine from rat peritoneal mast cells (Section IV,A,l); thrombin and trypsin are release agents for platelets (Section VI,A,3), and kallikrein is chemotactic for polymorphonuclear leukocytes (Section X). Whether in each instance these enzymes substitute for the activatable esterase, or stimulate the activatable esterase concerned, or involve some other, quite unrelated mechanism is not known but obviously is well worth investigating. The evidence for the requirement of an activated esterase is also indirect existing only of the irreversible inhibition suffered when cells are treated separately with organophosphorous inhibitors. Since not much more is known concerning the nature of these putative esterases, their significance and role are equally unclear. The energy source may be primarily anaerobic glycolysis in responses such as phagocytosis or chemotaxis by polymorphonuclear leukocytes. Histamine release from mast cells under various conditions apparently depends on aerobic oxidation, although, like muscle contraction, if oxygen is not available or the aerobic oxidation sequence is poisoned, release can be sustained by anaerobic glycolysis. The possibility that the

174

ELMER L. DECKER AND PETER M. HENSON

two mechanisms of energy release can bc utilized has been insdficiently investigated in a number of cases, and several systems might well bc restudied from that point of view. In many instances, platelets seem to require both pathways simultaneously for optimal activity. Where looked for, the various responses have been shown to depend on a heat-labile factor present in cells (Table I ) . Whether this putative, heat-labile factor is a heat-labile enzyme, as suggested many years ago by Mongar and Schild ( Section IV,A), is not known. The present evidence that a contractile system is implicated in the various responses considered in this review is indirect, being almost wholly evidence of inhibition of the given response by microtubular disaggregating agents, such as colchicine, or by cytochalasin B, an agent, supposedly, specifically affecting microfilaments. The evidence is not only indirect and equivocal (Section I ) but, as seen in Table I, is frequently lacking. In general, where it has been looked for, the evidence has been found, as in the release of histamine by mast cells, release from platelets, inhibition of spontaneous motility and chemotaxis by polymorphonuclear leukocytes, and inhibition of the stimulatory effect of PHA on DNA synthesis. Cytochalasin B has been reported to inhibit engulfment of bacteria, but colchicine does not reduce phagocytosis except in high concentrations ( Sections VII1,B and IX) . The degranulation of polymorphonuclear leukocytes accompanying phagocytosis of zymosan particles has been reported to be inhibited by colchicine, whereas, using nonphagocytizable surfaces, there is controversy as to its effect on release of granule enzymes (Section VII,B,l ) , Cytochalasin B enhances degranulation in the latter system. Furthermore, chemotaxis of polymorphonuclear leukocytes (Section X ) and the stimulating effect of PHA on DNA synthesis of lymphocytes (Section XI) are both enhanced by subinhibitory concentrations of cytochalasin B; in the latter two instances, however, unlike the case with degranulation, higher concentrations are inhibitory. The cause of the enhancement in any of the three circumstances is unknown. It might indicate that in these systems microfilaments exert a restraining influence on the given aspect of the responses being enhanced or that, as suggested in other connections, microfilaments are not the only structures affected by cytochalasin B. Despite the present unsatisfactory state of the evidence, the concept that mobilization of one part or another of the contractile system of the cell is a major step in stimulus-response coupling is not only an important unifying and heuristic hypothesis in considering the general secretory processes but promises to be the same for the specific responses reviewed here. A feature of many of these cell responses is that they may be localized

IMMUNOLOCICALLY INDUCED MEDIATOR SECRETION

175

to one portion of the ccll, only the part of the cell meinbralie that is stimulated being involvcd. Mast cells may be stimulated by local application of 48/80 and will degranulate on that side only (Tasaka et d . , 1970b ) . Neutrophiles cshibit directional migration and extrude granules only into the phagocytic vacuoles or along the portion of the cell membrane in contact with the iminunological reactants. I t is possible that granules exhibit directional migration to the parts of the cell that are stimulated. This, perhaps, may be along microtubules, as inferred from tissue culture cells by Freed and Lebowitz (1970) and as suggested for macrophages by Allison et al. (1971). The motive process then may involve contraction of microfilanients, as demonstrated by the movement of granules in melanocytes by Malawista ( 19714. Directional movement of the whole cell could be under similar control. As pointed out by Schild ( 1968), “desensitization is a descriptive omnibus word denoting lack of response to a second dose of antigen” (or other stimulating agent). As further pointed out by Schild, desensitization can have, and, in various instances, undoubtedly has various causes, including exhaustion of free receptors, exhaustion or decay of required enzyme or state of an enzyme, and depletion of stores of metabolites or mediators. Although desensitization when looked for has been demonstrated in almost all the phenomena discussed in this review (Table I ) , in no case have the factors concerned been identified in molecular terms, although, in the case of deactivation ( desensitization) of polymorphonuclear leukocytes by complement-derived chemotactic factors, the desensitization has been closely associated with activation of proesterase 1 to esterase 1 (Section X). However, it has yet to be proved that it is the activation that is responsible for desensitization in this or any other instance. The importance of desensitization is that it is one of the ways of breaking into and separating the complex sequence of biochemical reactions involved into several discrete steps. For the same reason a similar and even greater importance is to be attributed to the study of the related, although superficially opposite, phenomenon of activation. In order for a cell or tissue to be termed activated, it must react with the stimulatory agent under circumstances that do not permit a completed response; the free or loosely bound stimulating agent is washed away, and the activated cell when restored to favorable conditions is shown to be able to complete the response. Depending on the cell or tissue and the attendant circumstances, the activated cell may be stopped or caught at any place or step in the sequence of reactions from the initial combination of stimulating agent and receptor to the penultimate step, whatever that may be. The studies of the several activated stages demon-

176

ELMER L. BECKER AND PETER M. HENSON

strated in the histainine release from mast cells induced by band 2 protein (Section IV,A,2) and in antigen-induced histamine release from sensitized human basophiles (Section V,A) show the promise of this approach. Enhancement of histamine release from various systems by succinate (Table I ) is another phenomenon that, although apparently not general, is sufficiently common to be worthy of note. Unfortunately, other than the statement that this effect apparently does not occur through stimulation of the Krebs cycle, nothing more can be said about it. Cyclic AMP seems to be involved in regulation of the release from the basophile, mast cell (especially those of the human or guinea pig lung), and the platelet. This is in contrast to the accumulating data for the release from the neutrophile and macrophage. The difference in this regard between these two groups of cells is paralleled by differences in the nature and contents of the granules. In almost all the cases cited in Table I, involvement of cyclic AMP has been inferred from the effects on the given response of agents known to affect intracellular cyclic AMP in other cells. Among these are the catecholamines, the prostaglandins, and xanthine derivatives such as theophylline and caffeine. Only for the stimulation of lymphocytes by PHA and for the stimulation of platelets by several nonimmunological and immunological agents have these inferential effects on intracellular cyclic AMP been confirmed by actual measurements of the nucleotide in the cells concerned. In a few instances, measurements have been made of the effect of these various agents on the level of cyclic AMP in the tissue or mixed cell populations containing the given mediator cell. Obviously, direct measurements of intracellular cyclic AMP levels should be performed on homogeneous cell populations in all systems where this is possible. In the systems mentioned above, an increase in cyclic AMP levels leads to inhibition of the given response and, in some instances, there is evidence that decrease of cyclic AMP leads to or enhances the response. This is the reverse of the situation with most secretory processes where increases in cyclic AMP leads to or are associated with an increased response. The significance of this difference is not clear. However, certain smooth muscles such as the taeniae coli of the guinea pig (reviewed in Robison et al., 1971) and the smooth muscles of the bronchus (Vulliemoz et al., 1971) are relaxed by P-adrenergic agents that increase levels of cyclic AMP. An apparent paradox is that in at least two instances, that of lymphocyte transformation and of chemotaxis, an increase of extracellular cyclic AMP can give the same response that an increase of intracellular cyclic AMP apparently inhibits. The explanation is not immediately evident.

IMMUNOLOGICALLY INDUCED MEDIATOR SECRETION

177

The mode of action of cyclic AMP is not known for any of the systems studied here. It is not even known whether cyclic AMP exerts a niodulating or permissive effect on a given response or is one step in the direct sequence leading from a given stimulus to a given response. These same complaints or confessions of ignorance, however, can be made about most phenomena in which cyclic AMP has been implicated and are reasons for further work rather than expressions of despair. In much of the above we have emphasized the similarities in the characteristics of the various responses. However, it is clear that different functions even within one cell may exhibit different requirements. The aggregation of platelets and their release reaction show remarkable similarities but do not always accompany each other. Moreover, colchicine and similar acting compounds will inhibit release but not aggregation. Similarly, neutrophile phagocytosis and degranulation seem to be initiated by the same stimuli. Nevertheless, the latter, but not the former, is inhibited by colchicine (although not perhaps the degranulation on nonphagocytizable surfaces ) , but the drug has little effect on engulfment. Exocytosis of granules is enhanced by cytochalasin B, whereas engulfment is inhibited. PGE, inhibits degranulation but does not alter engulfment. Chemotaxis and engulfment are probably closely related processes; they are both inhibited by increased concentrations of cytochalasin B, but oiily chemotaxis is inhibited by colchicine and PGE,. Whether these different responses are programmed by the stimulation of different receptors is as yet unknown. Nevertheless, one can speculate that a process that is inhibited by cytochalasin B is required in the engulfment stage and in chemotaxis and cell movement but not in secretion. A second process is inhibited by colchicine and is involved in chemotaxis and cell movement but not in engulfment and probably not in secretion. It is, of course, open to question whether one can interpret the results of these inhibition studies in so literal a fashion, but the interpretation just advanced does stress that it is not necessary to consider movement of the cell or of cell organelles as necessarily a unitary process. Rather it is probable that the various functions dependent on movement of the cell or cell organelles may have only certain steps in common. In essence, at this time, the differences as well as the similarities in the various responses reviewed here can only be noted; the explanation of any one of them in terms of the detailed molecular and biochemical events occurring in and on the involved cells awaits further thought and further work. It is hoped that in this further work and further thinking, a useful and enlightening concept will be that the reactions and functions, which have heretofore been considered specifically immunological, can

178

ELMER L. BECKER AND PETER M. HENSON

be thought of in terms used for general biological and physiological processes such as muscle contraction, cellular secretion, and cell movement. ACKNOWLEDGMENTS We wish to express our appreciation to friends and colleagues who have kindly and generously allowed us to quote their unpublished work. We also wish to express our deep appreciation to Miss Deborah Lundy for her industry and patience in typing and retyping the manuscript.

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Antibody Response to Viral Antigens KEITH M. COWAN Plum Island Animal Disease laboratory, Agriculfural Research Service,

U. S.

Department o f Agriculture, Greenport, N e w York

I. Introduction . . . . . . . . . . . 11. Virus Structure and Viral Antigens . A. Simple Viruses . . . . . . . B. Complex Viruses 111. Measurement of Antibody to Viral Antigens . . A. Neutralization . . . . . . . B. Hemagglutination Inhibition . . . . C. Complement Fixation . . . . . . D. Enzyme Inhibition . . . . . . E. Precipitation . . . . . . . . F. Labeled Antigens and Antibodies . . . G. Passive or Indirect Agglutination Tests . . IV. The Antibody Response . . . . . . . . . . . A. Virions as Antigens . B. Response to Viral Antigens Other Than Virion C. Antibody Specificity . . . . . . . . . . . . V, Concluding Remarks . . . . . . . . . References .

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I . Introduction

The antibody response of animals exposed to viruses may be considered from many points of view dependent upon the particular interests and inclinations of the investigator. Those interested in the kinetics of antibody formation, the regulation of the antibody response, and the nature of antigen-antibody interactions have found animal and bacterial viruses to be extremely valuable model antigens because of their high antigenicity and the availability of precise and extremely sensitive, plaqueinhibition assay procedures for the detection and measurement of antibody. Other investigators will be concerned with the antibody response for purposes such as diagnosis, vaccine evaluation, epidemiological surveys, and the significance of the occurrence of antibody and its relationship to immunity. Those interested in virus structure, virus replication mechanisms, pathogenesis, and cellular sites of viral antigen synthesis or localization may be primarily interested in the antibody response for purposes of developing component-specificreagents for their analytical studies. These considerations provide only a partial indication of the diversity of interests in the antibody response to viral antigens. 195

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The bulk of studies on the antibody response to viral antigens has been concerned with the intact virus particles (virions ) , and considerably less to their isolated subunits, incomplete particles, and the various “soluble” and virus-associated antigens. These nonvirion antigenic components are of considerable interest for a variety of reasons. The preparation of effective vaccines from viral subunits would provide an obvious safety factor by eliminating the viral genome and its potential for infection and cell transformation, and would also minimize adverse reactions produced by some vaccines containing the intact virion. Considerable efforts are being made to isolate and identify the immunogenic components from disrupted virus particles, and the excellent monograph by Neurath and Rubin (1971) should be referred to for details. Additionally, the nonvirion antigens may also be of concern in many diagnostic aspects of virus diseases, and play a vital role in the nature of the disease. Their isolation and characterization are also important for establishing their structural and functional roles in virus architecture and mode of replication. Investigations on the antibody response of animals exposed to viruses are obviously complicated by the multiplicity of antigenic components involved, the differing activities of the various classes of antibodies with which they interact, and the manifestations of these antibody-antigen interactions. Any meaningful evaluation of the antibody response to a virus necessitates a thorough dissection of the system into its component parts, Once embarked upon such a project, the investigator soon finds himself on the proverbial carrousel where it is essential to learn more about the antigens being dealt with in order to comprehend the significance of the response being determined. Similarly, analysis of the viral antigens necessitates a detailed knowledge of the structural and functional characteristics of the antibodies used in such studies. It is clearly impossible to consider all antigens of all viruses in this review, just as it is impossible to consider all aspects of the antibody response to viral antigens. The humoral antibody response will be primarily considered since excellent reviews on the secretory system may be referred to (Dayton et a,?.,1969; Ogra and Karzon, 1971; Rossen et a,?., 1971), but cellular aspects of immunity to viruses are beyond the scope of this review. The approach taken is intended to be somewhat practical in terms of establishing the general nature of viral antigens, what is being determined by seriological procedures applied to virus systems, the appraisal of such data for diagnostic and vaccine evaluation purposes, and finally, the nature of the antibody response and some potential implications to basic immunological and virological problems. The viruses of foot-and-mouth disease (FMD) and influenza will be

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mainly considered as they may serve as models for the structurally and antigenically simple and complex viruses, respectively. Other viruses will be considered for comparative purposes, but the failure to cite some of the excellent studies done on several exceedingly important groups of viruses should be attributed to the limitations of the author rather than to the lack of importance of the investigations. II. Virus Structure and Viral Antigens

Before considering the antibody response to viral antigens, it is necessary to consider some of the physical, chemical, and biological characteristics of the antigens involved. The nature of the relatively simple picornaviruses and the complex myxoviruses will be considered. A. SIMPLEVIRUSES

1. Structural Characteristics Foot-and-mouth disease virus (FMDV) belongs to a vast group of serologically distinct, small, single-stranded, ribonucleic acid ( RNA ) viruses ( picornaviruses ) . Picornaviruses have been isolated from virtually all animal species examined and, although many of the viruses are recognized pathogens, innumerable isolates have been made from human and nonhuman animal sources where no disease was evident. The picornaviruses may be divided into two general groups based on their sensitivity to acid pH. The enteroviruses and most cardioviruses are stable at acid pH and include polioviruses, Coxsackie viruses, and echoviruses of human origin. The acid-labile viruses include the rhinoviruses, of which there are some ninety different serotypes (Kapikian, 1971), and FMDV with seven serotypes (Types A, 0, C, SAT-1, SAT-2, SAT-3, and Asia 1 ) , but there are approximately sixty serological subtypes among the different serotypes. The structure and characteristics of FMDV have been comprehensively reviewed ( Bachrach, 1968), and a recent excellent review of picornavirus structure (Rueckert, 1971) should be consulted for details. Briefly, the virus of FMD is fairly characteristic of the other picornavinises with a diameter of B O A . , a sedimentation coefficient ( s rate) of 140 S, a buoyant density of 1.43 gm./ml. in CsCl, and a molecular weight of about 8.4 X lo6 daltons. The FMDV contains 31%single-stranded RNA, with a molecular weight of 2.6 X loBdaltons, and 69%protein of approximately 5.8 X 100 daltons. The protein coat (capsid) presents a surface pattern consisting of 32 capsomers, although the problem of differentiating a 32- from a 42-capsomer structure has been considered (Breese et al., 1965). The FMDV has a somewhat lower s rate (140 S ) than other

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picornaviruses ( 150 or 160 S ) , and a higher buoyant density in CsCl than the rhinoviruses ( 1.38-1.41 gm./ml. ) or the acid-stable picornaviruses (1.321.36 gm./ml). A model for the protein composition of FMDV has recently been presented (Talbot and Brown, 1972) which is similar to that demonstrated by other picornaviruses. Electrophoresis on polyacrylamide gels in the presence of sodium dodecylsulfate and mercaptoethanol showed four virus polypeptides (VP,-VP,) with molecular weights of 34, 30, 26, and 13.5 X lo3 daltons in the ratio of 1:1:1:0.5. An additional component (VP,) having a molecular weight of 39,500 is often found at low concentrations and is considered to be a precursor of VP, and VP, (Vande Woude et al., 1972a). This model of the polypeptide composition of FMDV is in general accord with that of other investigators (Wild et al., 1969; LaPorte, 1969, 1970; Vande Woude and Bachrach, 1971; Burroughs et al., 1971) as well as for other picornaviruses (e.g., Maizel and Summers, 1968; Rueckert et al., 1969; Johnston and Martin, 1971).The relevance of the multiple polypeptides of picornaviruses to their antigenic characteristics will be considered subsequently. A consideration of the antigenic components of FMDV necessitates a brief mention of the noncapsid virus-specific proteins produced during virus synthesis. It has been shown with several picornaviruses that viral messenger RNA is translated to produce large molecular weight protein products that are cleaved during, or subsequent to, translation into a number of smaller proteins (Jacobson and Baltimore, 1968a,b; Summers and Maizel, 1968; Kiehn and Holland, 1970; Butterworth et al., 1971; Vande Woude and Ascione, 1972). Some of these are incorporated into the virus capsid as the VP,-VP, polypeptides, whereas others do not become part of the virus structure. A number of these noncapsid virus proteins (NCVP) are demonstrable; however, their association with any specific viral function has not generally been established. The potential antigenic activity of the NCVP components will be considered in the following section.

2. Viral Antigens and Their Characterization Concentrated fluids from cell cultures (BHK-21 cells) infected with FMDV give three precipitin bands in double immunodiffusion reactions when tested against sera from animals convalescing from FMD (Fig. 1). By using purified antigens, it was found that one band was due to virus (140 S antigen), one to protein subunits of the virus ( 12 S antigen), and the third to a NCVP termed the virus infection-associated (VIA) antigen ( Cowan and Graves, 1966). These three antigenic components could also be demonstrated in fluids obtained from vesicles produced in animals

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Fic. 1. Agar gel precipitin reactions with guinea pig anti-foot-and-mouth disease virus (FMDV)-A12 serum (center well) and a crude concentrate of FMDV-A,,infected baby hamster kidney cell culture fluid ( B H K ) , purified virus ( 140 S ) , virus protein subunit ( 12 S ) , and virus infection-associated (VIA) antigen preparations. (From Cowan and Graves. 1986.)

infected with FMDV. The virus (140s antigen) used in such reactions was concentrated by precipitation with methanol ( Bachrach et aE., 1964) or polyethylene glycol (Wagner et al., 1970), purified by ultracentrifugation into isodense CsCl (Trautman and Breese, 1982), and characterized by various physicochemical (Bachrach et d.,1964) and immunological (Wagner et al., 1970) procedures. The precipitin band attributed to 140 S antigen could be further established by demanstrating the presence of single-stranded RNA in the band by acridine orange staining (Cowan and Graves, lW), Treatment of purified 140 S antigen at acid pH resuIts in the breakdown of the virion to RNA and 12 S protein subunits having a diameter of approximately 70A. (Bradish et al., 1960). The 12s component derived from 140 S antigen may be further purified by chromatography on diethylaminoethyl ( DEAE )-cellulose ( Cowan, 1968) . The immunological relationship between the 140s antigen and the 12s component derived from it is of particular interest as immunodiffusion studies indicate they are antigenicdy distinct (see Fig. 1) ( Brown and Crick, 1958; Ceglowski, 1965). The apparent lack of antigenic relationship between virions and their protein subiinit has been demonstrated for several viruses such as

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turnip yellow mosaic virus (TYMV) and tobacco mosaic virus (TMV) ( Rappaport et al., 1965), as well as QB bacteriophage ( Hung et al., 1969). The nature of antigenic determinants and the dependence of their specificity on secondary, tertiary, and quaternary structure have been considered at length for viruses (Jerne, 1960; Knight, 1961; Van Regenmortel, 1966; Neurath and Rubin, 1971) as well as for a variety of nonviral antigens (Benjamini et al., 1972). It is evident that conformational factors may play a vital role in deteimining the antigenic characteristics of several viruses and the apparent lack of antigenic relationship to their protein subunits. The antigenic distinctiveness of FMDV 140s particles and the 1 2 s antigen has been indicated, but it would seem that the crossing precipitin bands observed in immunodiffusion analyses (Fig. 1) may be misleading. Absorption of antiserum with 12 S antigen results in an evident decrease in antibody reactive with the 140 S antigen in spite of the apparent antigenic distinctiveness shown in immunodiffusion assays ( Camright, 1962; Cowan, 1968). Similarly, serum prepared by immunizing guinea pigs with 12 S antigen results in the production of antibody reactive with both the 12 and 140 S antigens. Upon absorption of such serum with 140 S antigen, the 140 S reactivity was eliminated and some decrease in antibody to 12 S antigen could also be noted (Cowan, 1968). It seems likely that the 140 and 12 S antigens possess a common antigenic determinant( s ) , but each also has distinctive antigenic sites. The crossing precipitin bands obtained with the two antigens would then be attributed to a “double” Type IV reaction described by Ouchterlony ( 19f38) due to multideterminant antigens containing common as well as distinctive antigenic determinant sites. The excellent studies relating FMDV structure to antigenic characteristics by Brown and his co-workers (Wild et d., 1969; Brown and Smale, 1970; Rowlands et al., 1971; Burroughs et al., 1971; Talbot and Brown, 1972) provided a structural model which could be in accord with the previous observations. The model proposed by Talbot and Brown (1972) indicated the virus was composed of sixty subunits, each containing one molecule of VP,, VP2, and VP3, as earlier suggested for MausElberfeld virus by Rueckert et at. (1969). Trimers of these subunits form the 12 S subunits which represent the twenty triangular faces of the icosahedral structure of the virus. The remaining VP, polypeptide was suggested to be located at the apposition of the faces of the icosahedron which would satisfy the occurrence of one-half the molar concentration of this polypeptide as compared to the concentrations of the others. Treatment of FMDV with trypsin was shown to reduce infectivity, reduce its ability to induce neutralizing antibody when used as an im-

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niunogen (Rowlands et al., 1971), and apparently modify the binding site for antibody at the vertices of the icosahedron (Wild and Brown, 1967; Brown and Smale, 1970). Tiypsin only cleaved VP, (Burroughs et al., 1971); therefore, it was proposed that VP, occurred at the vertices of the icosahedron (Talbot and Brown, 1972) and that it was concerned with adsorption of virus to susceptible cells and was the primary site of action of neutralizing antibody ( Rowlands et al., 1971). However, the ability of trypsin-treated virus to induce the formation of some neutralizing antibody in immunized animals suggested the occurrence of an additional antigenic determinant site. Thus, three antigenic determinant sites on the virus were indicated: ( 1 ) 12 S sites at the triangular faces of the icosahedron; ( 2 ) trypsin-sensitive VP, sites at the vertices of the icosahedron; and ( 3 ) a trypsin-stable site that also probably occurs at the vertices. The earlier discussion indicating that only a portion of the antibody reactive with 1 2 s antigen was capable of reacting with 140s antigen would be in accord with the 140s model where 1 2 s antigen is at the faces of the icosahedron. However, the failure of all of the anti-12 S antibody to react with 140s indicates the presence of an antigenic determinant site specific for 12 S antigen that does not occur on the surface of the intact virus. This 12 S-specific determinant could occur as the result of exposing determinant sites that are masked in the intact virion (cryptotopes) or of conformational changes of the 1 2 s subunit upon degradation of the virus particle resulting in the formation of new antigenic sites (neotopes). According to the model of Talbot and Brown (1972), the 140 Sspecific site would occur at the vertices of the icosahedron and would primarily involve VP,. However, immunization of animals with 12 S antigen, which contains VP1, results in the production of relatively little antibody reactive with 140s. Thus, if VP, is responsible for the 140sspecific determinant site, its conformation in the 12 S particle may be different from that in the intact virion, or else the 140 S-specific determinant may be the result of the quaternary arrangement of the VP, polypeptides at the vertices. As suggested earlier, it would appear that tertiary and quaternary structure play a vital role in establishing specific antigenic determinants on FMDV as well as an other picornaviruses. Relative to the discussion of the 140 and 12 S antigens of FMDV, it is necessary to consider antigenic particles occurring with certain serotypes of FMDV that are RNA-free capsids (empties). The empties have approximately the same diameter as the virus or 140 S antigen, an s rate of about 75 S, and a density of 1.31 gm./ml. (Graves et al., 1968; Cowan and Graves, 1968; Breese, 1968). The 75 S particles have been reported to contain all polypeptides occurring in the virion, but unlike virions,

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VP, is the major constituent (Vande Woude et al., 1972a). Antigenically the 7 5 s particle would appear to be closely related to both the 12 and 140 S particles. Although 75 S produced precipitin bands in immunodiffusion analyses which suggested coalescence with both 12 and 140 S antigens, the regions of coalescence were vague and did not give the impression of strict identity ( Graves et al., 1968). The occurrence of RNA-free capsid particles has been reported for a number of picornaviruses, such as poliovirus (Le Bouvier, 1955, 1959; Mayer et al., 1957), Coxsackie virus (Schmidt et al., 1963), echoviruses (Forsgren, 1968), and a number of plant and bacterial viruses. There is considerable interest in these particles because of their relevance to virus structure and synthesis, diagnosis, vaccine preparation, and the antibody response. Poliovirus has been studied in more detail than other picornaviruses in this regard and will be considered as the model system. Poliovirus preparations contain two virus-sized particles which appear to be antigenically distinct-one being the RNA-containing virions ( D or N antigen), and the other the RNA-free or empty capsids ( C or H antigen) (Mayer et al., 1957; Hummeler et al., 1962). The D antigen may be converted to C antigen by various denaturing procedures such as heating at 56°C. or exposure to ultraviolet light (Le Bouvier, 1955, 1959; Roizman et al., 1959); however, this antigenic conversion is not necessarily accompanied by the release of RNA if shorter treatment periods are used. It has been suggested that the interaction between viral RNA and capsid protein produces a specific surface conformation which is changed when the RNA is released, resulting in the D-to-C antigenic conversion (Katagiri et al., 1967). This would be similar to a proposal presented by Knight (1961) who suggested the possible importance of viral nucleic acid in determining quaternary structure of the viral capsid proteins. However, it has been shown that the conversion of virions to empty capsids results in the loss of VP, as well as the RNA, and this led to the suggestion that VP, may be an internal protein involved in stabilization of genome configuration (Maize1 et al., 1967). It has also been suggested that VP, of a bovine enterovirus occurs inside the capsid (Martin and Johnston, 1972). A recent study demonstrated that the RNA released by heat treatment of poliovirus and rhinovirus was associated with protein, but the identification of the protein was not established ( McGregor and Mayor, 1971). Although the above discussion would suggest that VP, is an internal protein, Breindl (1971) has provided evidence that VP, is a capsid surface component and is responsible for the binding of D-reactive antibodies. Therefore, whether the D reactivity of the virion is attributable to a specific capsid surface polypeptide (VP,) or to the confoimation of the

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multipolypeptide capsid surface (VP,, VP,, and VP,) is still in doubt. That conformation is extremely important is further suggested by electrophoretic experiments with poliovirus demonstrating that the interaction of a virion with even a single molecule of antibody will change its electrophoretic characteristics ( Mandel, 1971; Kriss and Mandel, 1972). It was suggested that the interaction of a molecule of antibody with one subunit resulted in a change of every other subunit by cooperative or allosteric transition. If such an interaction induced evident changes in the electrophoretic characteristics of the virion, alterations of the antigenic characteristics might also be anticipated.

3. Nonstructural Antigens Early studies with FMDV indicated the presence of a heat-labile complement-fixing antigen which could not be demonstrated to be related to either the 140 or 12 S antigens (Planterose et al., 1963). Subsequently, by immunodiffusion and complement-fixation ( CF ) procedures, a third antigenic component which was antigenically distinct from the 140 and 12 S antigens (Cowan and Graves, 1966), was demonstrated. Failure to demonstrate this antigen as a virus structural component and failure of animals immunized with purified inactivated virus to produce antibody to the antigen, indicated its nonvirion nature. Antibody to this antigen only occurred in the serum of infected animals. These findings led to calling it the virus infection-associated (VIA) antigen (Cowan and Graves, 1966). Sera from animals infected with the different serological types of FMDV reacted with VIA antigen prepared from Type A virus, but no reaction was obtained with sera from animals infected with viruses other than FMDV. It was concluded that VIA antigen was specific for FMD infection, but it was not type-specific. The inability to demonstrate VIA antigen as a structural component led to the suggestion that it could be a virus-induced component, such as an enzyme involved in virus replication (Cowan and Graves, 1966). Viral, RNA-dependent, RNA polymerase appeared to be a likely candidate as this enzyme was functionally short-lived and had to be synthesized continually for continued viral RNA synthesis ( Baltimore and Franklin, 1963; Baltimore et al., 1963; Eggers et al., 1963; Caliguiri et al., 1965). A virus-induced enzyme such as this would reasonably be recognized as a foreign protein by the host and would possibly be produced in amounts sufficient to induce a demonstrable antibody response. A subsequent study demonstrated that antibody to VIA antigen was able to inhibit the cell-free synthesis of virus-specific RNA by FMDV RNA polymerase ( Polatnick et uZ., 1967), and this provided strong evidence for a relationship of VIA antigen with the viral, RNA-dependent, RNA polymerase.

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Although it was suggested that VIA antigen was an internal antigen of the virion (Rowlands et al., 1969), recent coelectrophoresis studies on acrylamide gels clearly demonstrated the lack of a polypeptide in a virion that corresponded with the molecular weight (57,000 daltons) of the VIA antigen (Vande Woude et al., 197213). This study also demonstrated that VIA antigen coelectrophoresed with NCVP, and provided presumptive evidence of biological activity for one of the virus-specific noncapsid proteins.

B. COMPLEXVIRUSES Unlike relatively simple picornaviruses, viruses such as the myxoviruses, poxviruses, adenoviruses, herpesviruses, and others, are both structurally and antigenically complex. In addition to expected protein and nucleic acid constituents, they may also contain significant amounts of lipids and carbohydrates. Many of these viruses are composed of multiple layers which are antigenically distinct, and a given structural layer may be composed of functionally and immunologically distinct components. Influenza viruses will be briefly considered to illustrate some of these points. Excellent review articles on influenza virus are available and should be consulted (Hoyle, 1968; Robinson and Duesberg, 1968; Pereira, 1969; Kingsbury, 1970; Webster and Laver, 1971; Compans and Choppin, 1971 ) .

I. Structural Characteristics Influenza viruses are somewhat variable in size and morphology but are generally spherical having a diameter of 800 to 12OOA. Filamentous forms of a similar diameter and up to 7 pm. in length are frequently observed with freshly isolated strains (Choppin et al., 1960). The virion composition is 60-75% protein, 1837%lipid, 5-7%nonnucleic acid carbohydrate, and 0.8-1% RNA (Ada and Perry, 1954; Frommhagen et al., 1959). Unlike the picornaviruses of which the viral RNA is a singlestranded molecule, the RNA of influenza virus appears to consist of five or six smaller molecules giving a summed molecular weight of approximately 2.5 X lo6 daltons (Duesberg, 1968; Pons and Hirst, 1968). The virion consists of a helical nucleocapsid enclosed in an envelope covered with spikes. The nucleocapsid contains the viral RNA and protein, some of which is termed the internal soluble (S) or ribonucleoprotein ( RNP) antigen ( Duesberg, 1969; Kingsbury and Webster, 1969). The influenza viruses are divided into Types A, B, and C, based on the immunological distinctiveness of the RNP antigens in C F tests. The envelope contains three distinct components-the hemagglutinin and the enzyme neuraminidase, which occur on morphologically different types

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of spikes (Laver and Valentine, 1969; Webster and Darlington, 1969), and a host-cell antigen ( Harboe, 1963). Examination of purified influenza virus by electrophoresis on acrylamide gels demonstrated seven distinct polypeptides-three were carbohydrate-freepahd four were glycoproteins ( Compans et al., 1970; Schulze, 1970). Treatment of virus with proteolytic enzymes resulted in removal of the spikes, also resulting in the loss of hemagglutinating and neuraminidase activity as well as the four glycoproteins. Thus, the nucleocapsid contained three noncarbohydrate-containing proteins, whereas the four glycoproteins were associated with the envelope. A somewhat different result was reported by Skehel and Schild (1971) who found only three glycoproteins in the envelope, and four nonglycosylated proteins were internal nonenvelope constituents. The hemagglutinin subunit is about 140 A. long and is apparently composed of two distinct glycoproteins (Laver, 1971; Skehel and Schild, 1971; Schulze, 1972). Laver (1971) indicated that these polypeptides have molecular weights of 60,000 and 12,000 daltons, a molecule of each is joined by disulfide bonds to form dimers, and the hemagglutinin subunit, in turn, is composed of two dimers. Webster and Laver (1971) have calculated that a virus particle contains approximately 620 hemagglutinin subunits which is considerably less than the 1900 subunits estimated earlier by Fazekas de St. Groth and Webster (1963). However, the latter estimate was made by antibody uptake measurements, prior to the recognition that the hemagglutinin and neuraminidase constituted antigenically distinct sites. The neuraminidase subunit was also reported to consist of two polypeptides having molecular weights of about 58,000 daltons ( Webster, 1970). Similar findings were presented by Skehel and Schild (1971) who indicated that at least one of the proteins was glycosylated, and that the molecular weights of the two proteins were 70,000 and 80,000 daltons. The molecular weight of the neuraminidase subunit has been estimated at approximately 150,000 daltons (Laver and Valentine, 1969; Webster and Darlington, 1969); therefore, it would appear to be composed of two or three molecules. Webster and Laver (1971) calculated that there are approximately 120 neuraminidase subunits per virion. There is general agreement that the proteins of the nucleocapsid are carbohydrate-free, but the number of proteins is in doubt. Three proteins were demonstrated by Compans et al. ( 1970) and Schulze (1970), whereas Skehel and Schild (1971) indicated four proteins, and also found that the RNP antigen activity was associated with their species 5 polypeptide. Schulze (1970, 1972) provided evidence that the nucleoprotein core of the virus was separated from the envelope proteins by a con-

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tinuous layer of lipid which protected the internal proteins from the effect of proteolytic enzymes. Within the lipid coat, the viral RNA was associated with VP, to form the RNP, which, in turn, was surrounded by a layer of protein VP,. The nature of the third nucleoprotein (VP, ) is suggested by recent studies of Chow and Simpson (1971) who found the RNA-dependent RNA polymerase in the nucleoprotein core of the virion, Virus particles stripped of the envelope glycoproteins retained their RNA polymerase activity providing evidence for the internal 10cation of the enzyme. It was not established with which core nucleoprotein the enzyme activity was associated, but Schulze (1972) suggested it could be VP,. That such may be the case is further suggested by studies of Hirst and Pons (1972) on the biological activity of influenza RNP in recombination experiments. The model of influenza virus recently presented by Schulze (1972) and Klenk et al. (1972) provides a rational proposal relating structure with the biological functions of the different protein constituents (for further details, see above-mentioned reports ) . 2. Antigens

The antigenic complexity of influenza viruses is readily anticipated in view of their structural complexity. As indicated previously, the envelope contains three distinct antigens-the hemagglutinin and the neuraminidase, which occur as separate subunit spikes, and a host-cell antigen that is composed primarily of carbohydrate and appears to be attached to viral proteins ( Harboe, 1963; Laver and Webster, 1966). The hemagglutinin and neuraminidase antigens are used to classify the influenza Type A viruses into the various subtypes. Unlike the picornaviruses, in which disruption of the virions generally induces marked alterations in the antigenic characteristics of the subunits, influenza viruses may be disrupted to provide subunits that retain their biological and antigenic characteristics, Thus, active hemagglutinin may be isolated by disrupting purified virus with sodium dodecylsulfate followed by electrophoresis on cellulose acetate strips (Laver, 1964; Laver and Webster, 1968; Laver and Valentine, 1969). The hemagglutinin subunit is of particular interest as it is the means by which the virus attaches to specific glycoprotein receptor sites on the cell and it is responsible for inducing antibody that neutralizes virus infectivity (Webster et al., 1968; Schild, 1970). Schild demonstrated that sera from rabbits immunized with purified hemagglutinin had a high level of virus-neutralizing and hemagglutination-inhibiting activity, but were free of antibody to neuraminidase or the internal RNP antigen. The ability of ether or detergent-disrupted virus as well as hemagglutinin subunits to induce the formation of neutralizing antibody is of consideraI

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ble interest, These treatments abolish the pyrogenic activity of intact virus vaccines (Davenport et al., 1964; Fazekas de St. Groth et al., 1969) permitting the preparation of a more desirable product. Although hemagglutinin subunits may be isolated from the virion and retain activity, treatment with denaturing agents results in complete loss of activity as well as inability to block the hemagglutination-inhibiting activity of antisera (Webster, 1970). Therefore, it was suggested that the antigenic determinants were not attributable to adjacent amino acids on the polypeptide chain but, rather, to folded areas on a single chain or areas resulting from multichain associations. However, Eckert ( 1966, 1967, 1969) degraded virus with urea and dithiothreitol and obtained a low molecular weight material that had no hemagglutinating activity but was able to block antibodies to the hemagglutinin. However, the antibody-blocking activity of the protein was veiy low, so that the role of protein conformation must still be considered. The enzyme neuraminidase constitutes the other “spike” antigen on the virus surface. It hydrolyzes terminal sialic acid ( N-acetylneuraminic acid) from the viral hemagglutinin glycoprotein receptors on the cell surface, permitting the elution of the virus particles ( Gottschalk, 1966). Antibody to neuraminidase does not prevent the infection of susceptible cells but does cause an apparent neutralization of the virus (Seto and Rott, 1966; Webster and Laver, 1967; Kilbourne et al., 1968). This is apparently due to the inhibition of the neuraminidase activity by the antibody with a resultant failure of the virus to be released from the infected cells, Neutralization of influenza viruses containing large numbers of neuraminidase sites also appears to occur with antibody to neuraminidase, but this has been attributed to steric blocking by the neuraminidase antibody of the hemagglutinin sites (Webster et al., 1968; Kilbourne et al., 1968). Although antibody to neuraminidase lacks true neutralizing capabilities, it may play a significant role in protection to the disease ( Schulman et al., 1968; Schulman, 1969). The remaining envelope antigen of the influenza virus is the host component (Knight, 1944). This component was found to be a carbohydrate that is apparently covalently linked to surface proteins of the virus (Harboe, 1963; Laver and Webster, 1966). The role of this carbohydrate antigen in virus structure and its significance as an antigen is not known. The internal RNP antigens have been considered previously and are primarily of concern for type differentiation. Some differences have been found between the RNP antigens of different strains of Type A virus by CF assays (Davenport et al., 1960), but it is evident that these are minor. Antibody to influenza virus, RNA-dependent, RNA polymerase was

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demonstrated in the sera of chickens convalescent from fowl plague virus infection, but serum from rabbits immunized with the RNP antigen did not inhibit this enzyme system (Scholtissek et al., 1971). However, studies of Chow and Simpson (1971) demonstrated the viral, RNAdependent, RNA polymerase in purified virions and subviral particles (nucleoprotein cores), indicating that the enzyme was an internal component. If such is the case, it might be anticipated that antibody to the RNA polymerase could be induced by immunization with intact virions. The failure to induce antibody to the RNA polymerase with RNP antigens indicated that the enzyme was not associated with RNP antigen or else it was denatured in the process of isolation. Alternatively, the RNA polymerase could be a distinctly different protein such as the VP, suggested by Schulze ( 1972). The true antigenic complexity of influenza viruses is only partially suggested by the demonstration of a number of antigenically distinct components. Gradual antigenic variation of influenza A viruses may occur as a consequence of minor changes in the hemagglutinin or neuraminidase, or both. More dramatically, genetically stable, antigenic hybrids may develop with the hemagglutinin derived from one parent while the neuraminidase is from another. However, it is beyond the scope of this review to consider the compounding antigenic complexity of the Type A influenza viruses as a consequence of antigenic variation. The recent excellent review of Webster and Laver (1971) should be consulted for a full appreciation of the antigenic intricacies of these viruses and the necessity to correlate their structural and immunological characteristics with their behavior in nature. I l l . Measurement of Antibody to Viral Antigens

The foregoing considerations of the complexity of viral antigens and their differing biological activities indicate that any attempts to measure antibody to these antigens must take into account the nature of the assay procedures applied. Unless suitable precautions are taken to establish what is being measured by the assay technique, the significance of the determinations may be dubious. It is of interest to consider some of the assay procedures used in virology for the measurement of antibody so as to appreciate both the power and limitations of these techniques.

A. NEUTRALIZATION The most obvious and frequently used procedure for measuring antibody to viruses is based on the ability of antibody to neutralize virus infectivity in susceptible hosts. A variety of animals, embryonated eggs, tissue culture cells, etc., may serve as indicators of virus infectivity, but

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the development of the plaque assay technique for animal viruses on cell monolayers ( Dulbecco, 1952) provided a powerful quantitative procedure for studies of virus neutralization (Dulbecco et d., 1956). Similarly, the development of the equilibrium filtration method provided another powerful tool for influenza virus neutralization studies ( Fazekas de St. Groth and Webster, 1961),The development of such niethodologies made possible detailed studies on the antibody response to animal viruses and the characterization of virus-antibody interactions ( e.g., see Svehag, 1968; Webster, 1965, 1968a,b ) . Although virus neutralization procedures provide a reasonable assmmice that a virus antigen is involved, it is obvious that only antibody to specific, critical, surface antigenic components or determinant sites on infectious virions will be measured. Antibodies reactive with viral antigens, such as empty capsids, soluble and internal antigens, or surface antigenic components not involved in neutralization, will not be determined. For example, neutralization studies with influenza viruses would not detect antibody specific for the internal RNP antigen or RNA polynierase or to the surface neuraminidase antigen. An additional problem in using virus neutralization techniques for antibody measurement concerns the differing biological activity of the various classes of antibody and their changing characteristics with time following induction. I t has generally been found that the avidity of both the immunoglobulin M ( IgM) and immunoglobulin G ( IgG) antibodies to viruses increased with time (Svehag, 1965; Finkelstein and Uhr, 1966; Webster, 1968b), although Webster found the early 19 S antibody to have high avidity. The neutralizing efficiency of antibodies has been attributed to their avidity which is dependent upon the affinity of the antibody combining sites, the number of combining sites (valence), and the conformational characteristics of the antibody molecules ( Blank et al., 1972). It was suggested, therefore, that conventional viral neutralization may not be used as quantitative assay for determining antibody concentration ( Haimovich and Sela, 1966; Blank et al., 1972). A number of viruses, such as lactic dehydrogenase virus, herpes simplex virus, and lymphocytic choriomeningitis virus, may combine with antibody but are not neutralized or are neutralized to only a limited extent. However, addition of specific anti-immunoglobulin serum (Notkins et al., 1966, 1968; Ashe and Notkins, 1966) or complement (Yoshino and Taniguchi, 1965; Hampar et a]., 1968a; Berry and Almeida, 1968) results in neutralization. These observations are of interest in studies of the mechanism of virus neutralization, and of the involvement of in vivo virus-antibody-complement complexes in the immunopathology of various viius diseases (Oldstone and Dixon, 1969). The role of anti-immuno-

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globulin or complement in these reactions is generally attributed to an increase in the bulk of protein on the virion surface which hinders absorption, etc.; however, Wallis and Melnick (1971) suggest it is due to aggregate formation. The virus of African swine fever (ASF) provides a more extreme situation where no neutralization is obtained with antibody even when anti-immunoglobulin or complement are added ( D e Boer et al., 1969). Antibody to viral antigens was readily demonstrated by CF and precipitin procedures, and ferritin-tagged antibody was shown to react with the virion (Breese et al., 1967). The fact that swine inoculated with attenuated ASF virus may resist infection upon challenge with virulent virus is of interest with regard to the mechanism of this protection. The neutralization of most viruses by antibody provides an invaluable method for antibody determination. However, it should be appreciated that these procedures may only provide a relative measure of the activity of the antibody and not necessarily an actual measure of the amount of antibody. The antibody determined will be restricted to that directed toward specific critical antigenic sites, and other virus-specific antigenic components will not be detected. An obvious powerful application of the critical site-antibody reaction is that the isolation of antigenic subunits involved in the neutralization reaction may be identified and evaluated by their ability to inhibit the neutralizing activity of antibody.

B. HEMAGGLUTINATION INHIBITION A number of viruses, such as adenoviruses, reoviruses, influenza, parainfluenza, mumps, Newcastle disease, arboviruses, vaccinia-variola, and certain enteroviruses, agglutinate erythrocytes in vitro. Hemagglutination may also be obtained with noninfectious virus or soluble components as well as with infectious virus. The demonstration by Hirst (1942) that the hemagglutination reaction with influenza virus was specifically inhibited by homologous antiserum provided the hemagglutination-inhibition (HAI) test for antibody measurement. The test was subsequently applied to a great number of different viruses. Hemagglutination and HA1 assays have been of particular value in determining the structure of adenoviruses and the biological characteristics of their structural subunits ( Norrby, 1971). The use of the HA1 test is, of course, limited to hemagglutinating viruses, and the antibody measured will be that directed against the hemagglutinating components, As with neutralization procedures, this is advantageous in that it limits the antibody being measured to specific antigenic components but, obviously, will not measure those directed against the nonhemagglutinating antigens. The HA1 activity of antiserum

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is dependent upon the quantity and avidity of the antibody (Fazekas de St. Groth, 1962; Webster, 1965), and therefore, the same limitations as are indicated for the use of neutralization as an antibody-measuring procedure should probably be taken into account.

C. COMPLEMENT FULATION The complement fixation (CF) technique provides one of the most extensively used, and possibly misused, procedures in virology. It is simple to perform, amenable to quantitation, and is relatively sensitive for detecting both antigen and antibody activity. It is unlike the neutralization and hemagglutinin-type tests in that it detects most antigen-antibody interactions and is not restricted to selected types of antigenic components. Therefore, CF may detect and measure the interaction of antibody with both infectious and noninfectious virions, structural and nonstructural soluble viral antigens, as well as nonviral components. As a consequence, the virtues of CF procedures may be obscured by the multiple antigens generally present in crude virus preparations and the corresponding antibodies occurring in the antisera. Any meaningful attempt to apply CF procedures to the measurement of viral antibody necessitates the use of well-characterized and, preferably, purified antigens. This was dramatically illustrated by Le Bouvier (1955) and Mayer and his co-workers ( Mayer et al., 1957; Roizman et al., 1959) in studies using the C (virion) and D (empty capsid) antigens of poliovirus to examine the antibody response by CF assays. Acute phase sera gave greater reactivity with the C antigen than with D antigen, whereas convalescent sera reacted better with D antigen. This indicated that antibodies of two different specificities were produced and that their temporal patterns differed. Thus, the antibody response determined was obviously dependent upon the antigen used in the CF assays. The more obvious situation involves the use of antigen preparations containing several different antigens. As with most viruses, crude antigen preparations of FMDV contain several different antigenic components, i.e., 140 S, 12 S, and VIA antigens. Unless isolated antigenic components are used in CF assays or the parameters concerning their CF characteristics established, it is unlikely that the investigator will know which antibody to which antigen is primarily being determined (Cowan and Trautman, 1967). Similar considerations exist for the measurement of antigen where it would generally necessitate the use of component-specific antisera ( Cowan, 1968). Thus, the meaningful interpretations of CF reactions necessitate the use of isolated and purified antigenic components and/ or antisera specific for a given component. An even greater limitation of CF procedures for virus antibody

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measurement is due to differences in the CF capabilities of the various classes of immunoglobulins and the apparent inability of the antibody from certain species of animals to fix complement in conventional assays. An illustration of the former situation concerns the failure of the IgM antibody of various species of animals to fix complement with a number of viruses. This was demonstrated for Russian spring-summer virus in humans ( Wiederman et al., 1963), FMDV in guinea pigs (Graves et al., 1964; Cowan and Trautman, 1965), cattle (Cowan, 1!366), and pigs ( McKercher and Giordano, 1967a), arboviruses in guinea pigs (Bellanti et al., 1965), and Coxsackie virus in humans and monkeys (Schmidt et al., 1968).The reason for the failure of IgM to fix complement with these viruses has not been established, but it has been found that the molecular volume of the antigen and the determinant spacing on the antigen molecules play a vital role in determining the fixation of complement by IgM ( Cunniff and Stollar, 1968). The CF characteristics of other classes of immunoglobulins with viral antigens has not been explored to any extent, but it may be anticipated that viruses may be similar to other antigens in this regard. Guinea pig 7s y2 antibody was shown to be responsible for CF, but no fixation occurred with 7 S yl antibody (Bloch et al., 1963). The reverse situation appears to occur with ruminants where the faster migrating electrophoretic class of IgG (IgG,) of cattle had greater CF activity with FMDV than did IgG, (Cowan, 1966), as did also that of sheep, using ferritin as antigen (Feinstein and Hobart, 1969). In the case of human immunoglobulins, it was shown that IgG1, IgG,, and IgGn fixed complement, whereas IgG1, IgA, IgD, and IgE did not (Ishizaka and Ishizaka, 1969). However, it has been demonstrated that complement may be utilized through an alternative pathway by guinea pig 7 S 7, (Sandberg et al., 1971) and aggregated IgE ( Ishizaka et al., 1972). The failure of antibody from bovine and avian species to fix guinea pig complement with viral antigens has been recognized for many years (e.g., Traub and Mohlmann, 1943). As a consequence, indirect C F tests were developed for the detection of antibody in these species (Rice and Brooksby, 1953). However, it has been found that the addition of fresh normal chicken serum to avian systems (Brunifield and Pomeroy, 1959) and normal bovine serum to bovine systems (Boulanger and Bannister, 1960; Knight and Cowan, 1961; Cowan, 1966) resulted in perfectly satisfactory CF reactions. The possibility that fixation of complement components may occur through the alternative pathway has not been determined for these species. With respect to humans, it has been observed that non-coniplenient-fixing antibodies occurred in virus infections

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(Schmidt and Harding, 1956), but the nature of this failure has not been explored. Complement fixation procedures provide an exceedingly powerful tool for the examination of virus systems. However, it is essential to appreciate their limitations and to establish definitively what is being measured. Although antigens may be measured in absolute units by CF, antibody measurements must be considered to be relative-a measure of antibody activity rather than the quantity of antibody.

D. ENZYMEINHIBITION Myxoviruses and paramyxoviruses contain as an antigenically distinct structural component on their surfaces the enzyme neuraminidase which E ydrolyzes the terminal N-acetylneuraminic acid from glycoproteins. The demonstration that antibody to influenza virus specifically inhibited the neuraminidase activity of the virus provided an assay procedure for measuring antibody to this enzyme (Ada et al., 1963). The procedure described by Laver and Kilbournc (1966) serves to illustrate the nature of this assay. A predetermined amount of intact virus is combined with the substrate fetuin, and the liberated N-acetylneuraminic acid is measured spectrophotometrically at a wavelength of 549 nm. For antibody inhibition assays, the amount of virus is adjusted to liberate sufficient N-acetylneuraminic acid to give an optical density of 0.300 to 0.900, and the dilution of antiserum that causes 50%inhibition of the enzyme activity provides the titer of the serum. Viral and virus-induced enzymes of extreme interest are those involved in the synthesis of virus components. The demonstration by Baltimore and Franklin (1962) that extracts of cells infected with Mengo virus could incorporate radioactive nucleotides into acid-insoluble virusspecific RNA in the presence of the four ribonucleotide triphosphates, Mg2+,and an ATP-generating system provided evidence for viral, RNAdependent RNA polymerase and a cell-free system for its assay. Subsequently, similar enzyme activity was demonstrated for animal virus systems such as poliovirus ( Baltimore, 1964), encephalomyocarditis ( E M C ) virus (Horton et al., 1966), and FMDV (Polatnick and Arlinghaus, 1967). With these viruses, thc RNA polymcrase was not considered to be a component of the virion. The availability of a cell-free, FMDV, RNA-dependent, RNA polymerase assay system made it possible to exaniinc the proposal that possibly the noncapsid FMDV VIA antigen was enzymatically inactive RNA polymerase ( Cowan and Graves, 1966). It was readily demonstrated that scrum or the y-globulin fraction containing antibody to VIA antigen

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inhibited the cell-free synthesis of virus-specific RNA in the FMDV RNA polymerase system (Polatnick et al., 1967). No inhibition occurred with serum from normal animals nor from ones immunized with purified inactivated virus. The failure of serum from animals immunized with purified inactivated virus to inhibit the enzyme system provided further evidence that the RNA polymerase was not a virus constituent and that antibody to the virion (140s) or its protein subunits ( 1 2 s ) did not interfere with enzyme activity. A number of animal viruses have now been described of which the nucleotide polymerases are shown to exist in the virion. Deoxyribonucleic acid (DNA) vaccinia virus was shown to contain DNA-dependent RNA polymerase (Munyon et al., 1967) as do the poxviruses (Kates and McAuslan, 1967). The double-stranded RNA reoviruses ( Borsa and Graham, 1968; Shatkin and Sipe, 1968), the single-stranded RNA vesicular stomatitis virus (Baltimore et al., 1970), and influenza viruses (Chow and Simpson, 1971) contain RNA-dependent RNA polymerase in the virion. With the influenza viruses (fowl plague virus), it has been demonstrated that the serum from infected birds would inhibit the RNA polymerase activity (Scholtissek et al., 1971); however, these studies did not establish whether the enzyme is a virion or a nonvirion constituent. The recent exciting demonstration of RNA-dependent DNA polymerase as a constituent of RNA tumor viruses (Baltimore, 1970; Temin and Mizutani, 1970) led to studies of the inhibition of this enzyme by antibody (Gerwin et al., 1970; Aaronson et al., 1971; Scolnick et al., 1972b). Antisera to the C-type RNA tumor viruses inhibited the viral RNAdependent D N A polymerase systems and provided a highly sensitive procedure for differentiating C-type viruses isolated from different species. By utilizing antisera to these different isolates in enzyme inhibition studies, it was possible to demonstrate that the enzyme from primate viruses was immunologically different from that in the viruses isolated from birds and lower mammals (Scolnick et al., 197213). These observations are of extreme interest as they will assist in identifying the species source of isolated RNA tumor viruses and their possible incrimination as cancer agents of humans. The measurement of antibody by inhibition of viral nucleotide systems is a relatively complicated procedure and extreme care must be taken to assure that the polymerase systems measured is virus-induced and not of host-cell origin, The occurrence of ribonuclease (RNase) in serum can obviously interfere with the RNA-dependent RNA polymerase inhibition assays, and chromatographic procedures were used in the FMDV studies to eliminate RNase from the globulin fractions examined ( Polatnick et al., 1967).

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It may be anticipated that antibody-inhibition studies of viral enzymes may provide valuable information relating function and structure of virion and viral-induced components. Similarly, the recognition of these immune systems may provide valuable, new, diagnostic procedures and new insight into the pathogenesis of many viral diseases.

E.

PRECIPITATION

1. Precipitin Reactions in Fluid Medium The use of quantitative precipitin assays for the estimation of the antibody content of sera has been used extensively in plant (Van Regenmortel, 1966) and bacterial (e.g., Rowlands, 1967; Rohrmann and Krueger, 1970; Rappaport, 1970) virology, but the application of the procedure to animal virology has been extremely limited. Fluid precipitin and flocculation reactions with animal viruses have been reviewed ( Smith, 1958), and viruses, such as vaccinia ( Ledingham, 1931), poliovirus (Smith et al., 1956; Schmidt and Lennette, 1959), and influenza as well as other myxoviruses (Belyavin, 1955) were shown to give such reaction. The obvious reason why such procedures have not been used to any extent is the problem of obtaining quantities of purified viral antigens sufficient to perform such assays. Advances in virus production and purification methodologies ( Bachrach and Breese, 1968; Wagner et al., 1970) provided sufficient FMDV ( 140 S ) and its 12 S protein subunits to perform limited preliminary quantitative precipitin analysis for antibody determinations ( Cowan, 1968). Later attempts to develop a quantitative assay for antibody to FMDV antigens by single radial immunodiffusion techniques ( Cowan and Wagner, 1970) necessitated additional precipitin tests in order to obtain standard calibrated sera of known antibody content. These experiments will be briefly considered as they illustrate both the advantages and limitations of quantitative precipitin analyses of complex immune systems such as encountered in virology. Pooled serum from guinea pigs infected with FMDV was examined for antibody to the 140 and 12 S antigens. The serum contained primarily 7 S y 2 antibodies to the 140 S, 12 S, and VIA antigens; however, previous studies demonstrated that VIA antigen was antigenically distinct from 140 and 12 S antigens and would not be expected to interfere with antibody determinations to the virion antigens (Cowan and Graves, 1966). Purified 140 and 1 2 s antigens were used to perform these assays by conventional procedures ( Kabat and Mayer, 1961 ) where the protein content of the precipitates was determined by Folin-Ciocalteu analysis

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utilizing purified 140 S, 12 S, and guinea pig 7 S y 2 as standards (Cowan and Wagner, 1972). Quantitative precipitin tests were performed first with 140 and 1 2 s antigens, and antibody excess, equivalence, and antigen excess zones could be readily demonstrated. On the basis of the 1 2 s experiment, the serum was absorbed with an optimal amount of 12 S antigen, and then a quantitative precipitin test was performed using 140 S antigen. The results are summarized in Table I. The 140 S antigen precipitated 265 pg. antibody protein per milliliter of unabsorbed serum, and the 12 S antigen precipitated 185 pg. antibody. This would be a total of 450 pg. of anti140 and anti-12 S antibody. However, when the 12 S absorbed serum was tested with 140s antigen, only 145 pg. of antibody was precipitated. This discrepancy in the amount of antibody precipitated by 140 S antigen from unabsorbed and 12 S-absorbed serum would suggest that 12 S had precipitated 120 pg. of antibody (265 less 145 pg.) that was also reactive with 140 S antigen. Thus, of the 185 pg. of antibody precipitated by 12 S antigen, 120 pg. also had anti-140 S activity, and only 65 pg. was specific for 12 S antigen. These findings indicate that the 140 and 12 S antigens possess distinct antigenic determinants, but they also have at least one common determinant. The antiserum used in this study could then be TABLE I ANTIBODY PRECIPITATED FROM GUINEAPIG ANTISERUM TO FOOT-AND-MOUTH DISEASEVIRUSBY VIRUS(140 S) AND VIRUSPROTEINSUBUNIT(12 S) ANTIQENS, AND THE EFFECTOF ABSORPTION OF SERUMWITH 1 2 s ANTIGENON THE AMOUNTOF ANTIBODY PRECIPITATED B Y 140 S ANTIQEN 12 S-Absorbed seruma

Starting serum’ Antigen added (lg./ml. serum)

(pg. Ab/ml. pptd. by 140 S)

(rg. Ab/ml. pptd. by 12 S)

(rg. Ab/ml. pptd.

25 50 75 100 150 200 300 400 500 600 800

110 220 235 265 255 255 260

94 141 165 185 80

-

a-

=

not done; Ab = antibody.

-

-

-

by 1 4 0 s ) 50 90 -

123 145 83 -

-

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estimated to contain 145 pg. of antibody uniquely specific for 140s antigen, 65 pg. specific for 1 2 s antigen, and 120 pg. reactive with Common 140 and 12 S determinants, for a total of 330 pg. of antiviral antibody. This is clearly quite different from the 450 pg. estimated by the initial precipitin tests with 140 and 12 S antigens. These quantitative precipitin studies also demonstrated that the 140 S particle reacted with a maximum of fifty to sixty molecules of 7 S antibody in extreme antibody excess, and with approximately twenty molecules at the equivalence zone of the precipitin curve. Although quantitative precipitin assays such as described are laborious and extremely costly in terms of viral antigens, they do provide valuable insight for the evaluation of other serological assays. For example, Fig. 1 showed crossing precipitin bands with 140 and 12 S antigens, indicating they are antigenically distinct. However, close examination of the precipitin bands revealed an abrupt decrease in intensity of both the 140 and 12 S bands upon crossing, i.e., on the spur portion of the bands, The quantitative precipitin data would support the earlier contention ( Section II,A,2) that the crossing of the 140 and 12 S precipitin bands was due to a type IV reaction (Ouchterlony, 1968), i.e., antigens containing common as well as distinct antigenic determinants. Quantitative precipitin assays are obviously too costly for routine antibody assays; however, they do provide calibrated antisera for use as standards in alternative serological procedures. As a result, they provide additional insight into what is being measured by alternative techniques and permit a more rational evaluation of the data obtained. 2. Precipitin Reactions in Semisolid Media The development of immunodiffusion methods by Oudin (1946), Ouchterlony (1948), and Elek (1948) led to their early application to studies of influenza virus (Jensen and Francis, 1953) and, subsequently, to that of a great variety of different viruses. The Ouchterlony-type double-diffusion method has been most frequently used, primarily for the qualitative examination of viral antigen preparations. It has been applied in a semiquantitative manner with viruses such as arboviruses where dilutions of antiserum are placed in the reservoirs surrounding the antigen well, and the highest dilution of serum giving an evident precipitin band was considered the serum end point or titer (Clarke, 1964). Immunodiffusion techniques, which are more amenable to quantitation, have generally been used for characterization of antigens in animal virus systems. A modification of the double-diffusion procedure in tubes ( Oakley and Fulthorpe, 1953) was used by Polson and Hampton (1960)

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to determine the diffusion coefficient of poliovirus. The modified OakleyFulthorpe technique of Preer (1956) has also been applied to determine the approximate equivalence zone for poliovirus precipitin analyses (Scharff and Levintow, 1963; Horwitz and Scharff, 1969). Although the procedure is clearly amenable to antibody quantitation, it has not been exploited, perhaps due to the lack of suitable amounts of viral antigens and the tedious nature of the technique. Single radial immunodiffusion ( SRID) techniques ( Mancini et al., 1964, 1965) have been applied to many immune systems but only to very few animal virus systems. Fiber antigen of adenovirus was quantitatively measured by the conventional or forward SRID method (antiserum in agar and antigen diffused from the reservoirs) to determine recoveries during purification procedures (Pettersson et al., 1968). Both forward and reversed (antigen in agar and antiserum diffused from the wells) SRID procedures were studied with FMDV antigens to determine their sensitivity and applicability for measuring both antibodies and antigens (Cowan and Wagner, 1970). The highest sensitivity for detecting either reagent was obtained by incorporating it into the agar and diffusing the reciprocal reagent from the wells. Thus, forward SRID was most sensitive for antibody detection, and the reversed method for antigen detection. The quantitation of antibody by forward SRID is based on the demonstration that the area or volume of the precipitin ring is inversely proportional to the concentration of antibody incorporated into the agar (Vaerman et al., 1969). The advantages of forward SRID for antibody determinations are that higher sensitivity is obtained and, if the antiserum being tested contains antibody to several different antigens as may be the case with viruses such as FMDV, the antibody content may be determined for each antigenic component if they are available in a purified form and a suitably calibrated antiserum is available as a standard (Cowan and Wagner, 1970). In practice, varying dilutions of antisera are incorporated into agar layers, and multiple wells are cut in the agar which receive the different purified antigen preparations (Fig. 2 ) . In addition to antibody quantitation studies, the technique is extremely useful for screening sera for antibody to a variety of antigens (Wagner et al., 1972) and for determining antigenic relationships among FMDV subtypes (Lobo, 1972). The data from SRID assays of FMDV and other immune systems has been analyzed, and computer programs have been developed for rapid and efficient processing (Trautman et al., 1971). In evaluating quantitative antibody determinations, the limitation cited in the previous section must be considered. The reversed SRID procedure has recently been adapted to studies of influenza viruses (Schild et al., 1972). Virus was incorporated into the

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219

Serum 1/9/68

FIG. 2. Radial immunocliffusion reactions of two guinea pig antisera to footand-mouth disease virus A,?( 119 ). Varying dilutions of serum were incorporated in the agar layer, and 10 p l . each of purified virus ( 140 S ) at a concentration of 500 pg./ml., virus protein subunit ( 1 2 S ) at 200 pg./inl., and a 1 : 6 dilution of a stock virus infection-associated ( V I A ) antigen placed in the wells. The position of the antigens in all plates was as indicated in the upper left-hand photograph. Serum 1/9/68 served as the standard and contained 160 pg. antibody protein/ml. serum to 140 S, and 137 pg./ml. to 12 S as detemiined by quantitative precipitin analysis. Seruni 10/31/69 was calculated to contain 273 pg, and 189 pg. antibody/ml. to 140 S and 12 S antigens, respectively. Subsequent quantitative precipitin analysis indicated 265 pg. and 184 pg. antibody/ml. to 140 S and 12 S , respectively. (From Cowan and Wagner, 1970.)

agar and antiserum was diffused from wells cut in the agar. Antibodies to both hemagglutinin and neuraminidase could be measured in a single assay as double rings were formed. A dense central ring was due to the hemagglutinin system, and n less dense outer halo to the neuraminidase reaction. This application of the reversed SRID procedure is of particular interest as it was possible to quantitate antibody where the antigen used was too large to diffuse effectively in the gel which would be necessary for forward SRID analyses. The immunoelectroosmophoresis or counterelectrophoresis procedure should be mentioned although it has not been used extensively for antibody quantitation. The procedure is used extensively in serum hepatitis diagnosis for the detection of either Australia antigen or its antibody (Taylor, 1972). The method may be used for detecting antibody or antigen if the antigen under examination moves toward the anode, enabling it to react with the cathodally migrating antibody. The procedure has recently been applied to African swine fever virus, and it provided a

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KEITH M. COWAN

highly sensitive technique for detecting antibody to viral antigens ( Pan et al., 1972).

F. LABELEDANTIGENSAND ANTIBODIES Antiviral antibodies or antiglobulin antibodies labeled with radioactive markers, ferritin, fluorescing compounds, and enzymes such as peroxidase are generally used in qualitative procedures for the identification and localization of viral antigens. The measurement of antibodies by these procedures has generally involved the use of labeled antiglobulins ( indirect method) as indicators of antigen-antibody interaction. For example, antibodies to Burkitt's lymphoma were titrated by flooding smears of Burkitt's lymphoma cell lines with dilutions of suspect human sera and demonstrating the attachment of antibody to the cells with fluorescent antihuman globulin (Henle and Henle, 1966). The intensity of staining observed was related to the dilution of the serum. A direct precipitin test was used with influenza virus and the "1-labeled globulin fraction of anti-influenza sera to provide an estimate of the antibody content of sera (Fazekas de St. Groth and Webster, 1963). In another fluid precipitin test for measuring human antibody to influenza virus, virus and sera were combined, and the complexes sedimented and washed by high-speed centrifugation ( Daugharty, 1971). The resuspended virus-antibody complex was then combined with **51-labeled anti-IgG and the labeled complex separated from unbound anti-IgG by sucrose density-gradient centrifugation. The amount of radioactivity incorporated in the virus-antibody complex provided a relative measure of the amount of antiviral antibody. Both of these procedures would measure antibody to the hemagglutinin and neuraminidase antigens, and it may be anticipated that the titers determined by these methods and by HA1 would probably not correlate well. Radioimmunoassays using radionctive-labeled virus in combination with antiserum to serum globulins or to specific immunoglobulins are being used for antibody detection and measurement, antigen detection and identification, and for determining the immunoglobulin class of antibody occurring in serum and secretions. Most of these assays are based on that of Gerloff et al. (1962) who described a specific and highly sensitive test for antibody determination in which "P-labeled purified poliovirus was incubated with antiserum and the immune complex and uiicomplexed globulins were precipitated with rabbit antiglobulin serum. The amount of radioactivity precipitated provided a relative measure of antibody-virus reaction, and it was virion-specific in that purified virus was used as antigen.

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22 1

The tremendous potential of this general procedure as a highly sensitive assay for the detection and measurement of antiviral antibodies is indicated by a recent single issue of a journal in which three different virus systems were described. Type A arboviruses were grown in the presence of "C- or "-labeled amino acids, purified, and then used as antigen for the assay (Dalryniple et al., 1972). Rabies virus ( Wiktor et at?., 1W2) and the VP, ( 6 s ) antigen from mammalian Type-C viruses (Scolnick et al., 1972a) were purified and labeled with '?Tfor use in the radioimmunoassay for antibodies. The procedure of Gerloff et al. (1962) also provides a powerful technique for determining the rate of synthesis of the individual poliovirus protein during the replicative cycle (Scharff et al., 1964). Highly specific antisera to the desired antigen were prepared by immunization of animals with purified materials. The specific antiviral sera were then added to radioactively labeled cell Iysates or to other antigen-containing preparations, and the resultant, labeled, antigen-antibody complexes were precipitated with antiglobulin serum. The measurement of radioactivity provided a determination for specific antigenic components, and when sequential samples were analyzed, the rate of synthesis of the different viral antigens could be established duiing the viral replicative cycle. A related procedure has been described for measuring antibody to herpes simplex virus ( HSV) (Notkins et al., 1971). Rate-zonal ultracentrifugation was performed with a mixture of HSV labeled with thymidine-?H and anti-HSV antibody, either with or without added antiglobulin serum. The position in the tube of the radioactive viius zone was related to the concentration of antibody used to sensitize the virus, and it was denionstrated that antibody-sensitized virus particles, either infectious or inactive, could readily be separated from particles that had not reacted with antibody. Iiiimunodiffusioii techniques, utilizing radioactively labeled polio virus or echovirus and specific antisera to human IgG, inimunoglobuliii A ( IgA), and IgM, have been used to determine the amounts and classes of antibodies occurring in serum and other secretions following exposure to viruses (Ainbender et al., 1965; Ogra et al., 1968; Ogra, 1970). Dilutions of the serum under test were diffused against antisera specific for the different immunoglobulins, and after the precipitin bands were formed, the slides were washed to eliminate unprecipitated proteins. The virus labeled with was then diffuscd, the slides again washed, and radioactivity determined by radioautography ( Ogra et al., 1968). The labeled viiiis binds with the immunoglobulin precipitin arcs containing antiviral antibody activity, and titcrs wcre expressed as the highest dilution of serum giving an arc detectable by radioautography. This

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KEITH M. COWAN

procedure provides a relative measure of antibody in the different immunoglobulin classes for the 32P-labeledvirus. The various applications of radioactively labeled viral antigens and antibodies have provided highly sensitive tests for the detection of either reactant. Unless purified, well-characterized and standardized reagents are used, the procedures may suffer the same limitations as those of the serological procedures discussed previously in that one may not know precisely what is being measured. In most instances, quantitation was only in relative terms and not absolute ones. The extreme sensitivity of radiolabeled reagents obviously necessitates the uses of refined methodologies and extensive control mixtures to validate the results obtained.

G. PASSIVE OR INDIRECT AGGLUTINATION TESTS Agglutination of particulate antigens by antibody is generally accepted as an extremely sensitive procedure for detecting antibody. As a consequence, many different viruses and viral antigens have been adsorbed to a variety of inert carrier particles for passive agglutination assays. Viruses, such as Newcastle disease (Burnet and Anderson, 1946), Japanese encephalitis, and dengue (Hale and Pillai, 1960), attach to untreated erythrocytes, and, when the procedure is suitably standardized, the viral antigen-sensitized red cells are agglutinated by antiviral sera. Several different viruses have been complexed to erythrocytes with tannic acid (Casals, 1967), and glutaraldehyde has also been used (Tokuda and Warrington, 1970). The sensitivity for detecting antibody with antigen-sensitized red blood cells is well established, but, as is the case with other serological tests applied to viruses, it is not always clearly evident what antigenic components and corresponding antibodies are being measured. The necessity for identifying reactants in order to avoid the misinterpretation of passive hemagglutination results was clearly demonstrated with Rauscher murine leukemia virus (RLV) (Sibal et al., 1971). These authors demonstrated that tannic acid-treated sheep red blood cells (SRBC) sensitized with purified virus or ether-treated virus reacted with immune monkey serum but not with immune mouse serum. However, SRBC sensitized with virus treated with detergent-ether mixtures reacted with both monkey and mouse antiserum. Resolution of these observations was obtained with HA1 studies of gel-filtration chromatographic fractions isolated from purified virus. It was found that SRBC sensitized with intact or ether-treated virus resulted in virus particles where the internal or viral nucleoid antigen (the group-specific antigen) was manifest. The failure of mouse immune serum to agglutinate the

ANTIBODY RESPONSE TO VIRAL ANTIGENS

223

virus- or ether-virus-scnsitized SRBC was due to the lack of antibody in mouse serum to this group-specific antigen, whereas monkey immune serum did contain such antibody. The use of detergent-ether-treated virus-sensitized cells made available the type-specific antigen, and antibody reactive with it occurred in both mouse and monkey antisera. Purified intact virus had been used for sensitization of SRBC in earlier studies to measure thc antibody response to RLV (Sibal et al., 1967), and these recent findings of Sibal et aZ. (1971) now suggest that only antibody to the group-specific antigen was being determined. It is evident from these very finely detailed studies that a thorough dissection and characterization of both antigen and antiserum reagents are essential for a proper evaluation of the immune response. A final consideration of passive agglutination procedures for measurement of antibody concerns the differing agglutinating characteristics of IgG and IgM antibodies. Frecman and Stavitsky (1965) found that IgM was far superior to IgG in agglutination reactions. Thus, a comparison of agglutinin titers of sera collected early and late after exposure to an antigen may have little relationship to the actual amount of antibody occurring in the sera at these different times. IV. The Antibody Response

A. VIRIONSAS ANTIGENS A generalized version of the antibody response (Fig. 3) to viruses has been characterized as biphasic where an initial peak of antiviral activity occurs 5-8 days after exposure, which is then followed by another peak at 15 to 20 days (e.g., Fazekas de St. Groth and Webster, 1964; Svehag and Mandel, 1964a). The biphasic nature of this response curve may be attributed to the generally accepted sequential appearance of the 19 ( IgM) and 7 S ( IgG) classes of antibodies. The sequential change in the physicochemical characteristics of antibodies to viruses was initially dcrnonstrated by Brown and Graves (1959) and Brown (1960) with cattle and guinea pigs infected with FMDV. It was shown that the antibody present at 7 days after infection had pglobulin electrophoretic characteristics, whereas that occurring after 14 days had slow y-globulin mobility. Ultracentrifugation studies were not done to establish the sedimentation classes of these antibodies, but subsequent studies clearly identified the @-globulinas IgM or 19s antibody, and the slow y-globulin as IgG or 7 S antibody (Graves et al., 1964; Brown et al., 1964; Cowan and Trautman, 1965; Cowan, 1966; McKercher and Giordano, 1967b). However, the studies of Bauer and Stavitsky

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KEITH M. COWAN

D A Y S AFTER INOCULATION

FIG.3. The generalized antibody response to virus as determined by neutralization ( Neut. ) or hemagglutination-inhibition ( HA1 ) and complenient-fixation ( CF) procedures. The response deterniined by neutralization or HA1 procedures is usually biphasic. The CF response is monophasic and lags behind the other responses clue to the failure of 19 S antibody to fix complement.

(1961) served as the stimulus to investigate this phenomenon with a variety of antigens. Both bacterial and animal viruses proved to be extremely valuable antigens for such studies because of their high antigenicity and because of the availability of highly Sensitive and quantitative plaque-inhibition assay techniques. As little as 0.1 pg. of +X174 bacteriophage inoculated into guinea pigs induced sufficient antibody to be demonstrable by immunodiffusion (Uhr, 1964), and 1 pg. of poliovirus inoculated intravenously into rabbits induced neutralizing antibody within 24 hours (Svehag and Mandel, 1964a). As a consequence, detailed studies on the antibody response to bacteriophage (Uhr and Finkelstein, 1963, 1967; Uhr, 1964) and poliovirus (Svehag and Mandel, 1964a,b; Svehag, 1964a,b, 19%) confirmed the finding that 19 and 7 S antibodies developed sequentially. These studies also determined the kinetics of appearance of the antibodies and established many factors influencing their induction in both the primary and secondary responses. The findings of these investigators and others (e.g., Fink et al., 1961; Stelos et al., 1961;

ANTIBODY

nL.srowx

TO VIRAL ANTIGENS

225

Beiiedict et al., 1962) on the specifics of the antibody response to viral and nonviral antigens will not be considered further as they have been reviewed in detail (Uhr, 1964; Uhr and Finkelstcin, 1967; Svehag, 1967, 1968). Sequential appearance of 19 and 7 S antibody has been the general finding with viruses, and it occurs upon infection of animals as well as upon immunization with nonreplicating virus vaccines. Thus, in addition to the viruses already cited, humans infected with Russian spring-sunimer encephalitis virus ( Wiedernian et al., 1963); influenza virus-immunized mice (Berlin, 1963, 1966) and rabbits (Webster, 1968b); arbovirusinfected guinea pigs (Bellanti et al., 1965); HSV-immunized guinea pigs (Tokumaru, 1967), RLV-immunized monkeys (Sibal et al., 1967); measles virus-immunized guinea pigs ( Heffner and Schluderberg, 1967); and Coxsackie virus-infected monkeys and humans (Schmidt et al., 1968) are some of the instances where this has been demonstrated. Immunoglobulin M antibody appears rapidly and reaches a maximal level between 5 to 10 days after infection or immunization and then declines to relatively low levels by 30 or 40 days. However, IgM antibody persisted in some rabbits for 5 to 6 months following immunizatioii with poliovirus ( Svehag, 1967). Similarly, some swine infected with FMDV had demonstrable IgM antibody 6 months later ( McKercher and Giordano, 1967b). It would appear that there may be considerable individual variation with respect to IgM antibody persistence. In the case of humans immunized with yellow fever 17D live virus vaccine, the conventional early IgM response occurred, but the IgM generally remained at a higher level than the IgG over a prolonged period of time (Monath, 1971). It was suggested that the persistence of IgM antibody was due to recuiTeiit antigenic stimulation through the development of a latent asymptomatic infection by the attenuated virus. Immunoglobulin G antibody becomes readily detectable a day or 2 after IgM and reaches a peak level 15-20 days after exposure to virus. The time course of appearance of IgG antibody was generally accomplished by treatment of early immune sera with 2-mercaptoethanol in order to inactivate the IgM antibody. However, it was shown by Berlin ( 1963) that 2-mercaptoethanol could also decrease the neutralizing activity of IgG antibody to influenza virus. In a number of instances, it has also been our experience that this treatment reduced about 25% the IgG precipitating antibody to FMDV. Consequently, the time course of IgG antibody appearance that has generally been reported could be in error and particularly so if the early and late appearing IgG antibody vary in susceptibility to mercaptoethanol treatment. It is possible that C F procedures would provide a more reliable assay

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KEITH M. COWAN

for the kinetics of appearance of IgG antibodies, since it has been found that IgM antibody does not fix complement in many virus systems (Wiederman et al., 1963; Graves et al., 1964; Cowan and Trautman, 1965; Bellanti et al., 1965; Schmidt et al., 1968). Thus, the delay in appearance of C F antibody activity (Fig. 3) as compared to neutralizing or HA1 activity is due to the failure of the early appearing IgM antibody to fix complement, and the appearance of CF activity would correspond to the development of IgG antibody. However, the use of CF for determining the IgG response is complicated by findings previously discussed concerning the C F characteristics of the IgG subclasses. Although it is evident that IgM antibody predominates in the early antibody response to viruses, whether or not it actually precedes the development of IgG is still subject to question, The application of highly sensitive radioimmunoassay procedures demonstrated the simultaneous appearance of IgM and IgG antibodies to antigens such as serum albumin and 7-globulin (Freeman and Stavitsky, 1965; Osler et al., 1966). Failure to demonstrate this in earlier studies was attributed to the use of passive hemagglutination assay procedures which favored the detection of IgM antibody. The possible difference in the antibody responses to soluble antigens as compared to larger antigens, such as viruses, will be considered subsequently. The above considerations of the limitations of passive hemagglutination and C F procedures for measuring the actual antibody response are not peculiar for these techniques alone. The more frequently used neutralization and HA1 procedures used in virus antibody response studies are also subject to many limitations. As considered previously ( Section III,A), these procedures would only measure the antibody response to certain antigenic subunits or “critical” antigenic determinants occurring on the virus surface. Thus, the antibody response to the neuraminidase subunits on influenza viruses would not be determined by conventional HA1 or neutralization procedures. In the case of poliovirus, it has been indicated that the peptide VP, is the reactive site for neutralizing antibody activity (Breindl, 1971). If so, the use of the neutralization test for determining the antibody response would again limit the antibody measured to that directed to the VP, site. The previous considerations (Section II1,A) of the differing avidity of the various antibody classes and their change with time may also influence the nature of the antibody response determined. The term avidity is to indicate the relative binding strength of the antibody molecules to an antigen. Recent investigations indicate that the avidity of antibody is determined by the affinity of the antibody-combining site, the number of sites, as well as the conformational characteristics of the

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antibody moIecules ( Blank et nl., 1972). Thus, high avidity antibody could be a result of high-affinity combining sites on niono- or bivalent molecules (Rosenstein et al., 1971) or multivalent molecules, such as IgM which may have low-affinity combining sites (Finkelstein and Uhr, 1966; Sarvas and Mlkell, 1970; Makela et al., 1970). In view of these considerations, it was concluded that virus neutralization assays would not provide an accurate procedure for measuring reIative serum antibody concentrations (Haimovich and Sela, 1969; Blank et al., 1972). This leads one to assume that early studies on the kinetics of antibody formation using neutralization assays may be misleading in that what was being measured was a combination of the rate of antibody formation and the developing avidity of the antibody. The various considerations of the neutralization of viruses by antibody up to this point have invariably involved plaque-inhibition assay techniques or other tissue culture procedures. However, vii.us neutralization assays may also be performed in susceptible host animals in which rather uncontrollable host factors may play a determining role in the fate of virus-antibody complexes. It may be expected that factors such as host complement activity and the phagocytic processes may markedly influence the apparent neutralization reaction. It is further evident that the manner in which such in uiuo neutralization reactions are performed may influence the results obtained (Cunha and Honigman, 1963). These assays may be performed in two general ways-( I ) by a conventional neutralization test in which virus and antiserum are combined and then inoculated into a susceptible host or ( 2 ) by the passive protection procedure according to which serum is first inoculated into the animal and then virus is administered after a suitable interval. Although assays of this type may lack the quantitative features of tissue culture procedures, they may provide a more reliable index of the resistance status of animals for purposes of vaccine evaluation and the determination of the effective immune status of populations. In the FMDV studies, the mouse neutralization-type test has been generally adopted for the ineasurement of neutralizing antibody developed in immunized or infected cattle (Skinner, 1953). Although this assay has generally provided a reliable index of the immune status of the animal under examination in our laboratory, numerous instances occurred where immunized animals did not resist challenge with virulent virus in spite of high levels of neutralizing antibody (P. D. McKercher, personal communication, 1972; P. Sutmoller and J. W. McVicar, personal coniniunication, 1972). However, when protection-type tests were performed in mice, sera from these animals were found to have relatively low protective antibody levels as compared to those from animals able to

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KEITH M. COWAN

resist challenge. Thus, the protection test provided a more reliable indicator of the immune status of animals than did the neutralization test. The sera under consideration were collected relatively late following immunization when antibody would be of the IgG class, although the subclass of IgG is not known. The occurrence of subclasses of IgG has been recognized for most species of animals, and it has also been demonstrated that they may vary considerably in their biological activity. An example of this is the CF activity of guinea pig 7 S y2 compared to the lack of activity of 7 S yl (Bloch et al., 1963) and the reversal of their activities in inducing passive cutaneous anaphylaxis (Ovary et al., 1963). It is of further interest that the manner of immunization of guinea pigs was found to influence the relative amounts of 7 S yl and 7 S y2 antibodies produced (Benacerraf et d.,1963). Immunization with soluble antigens emulsified in complete oil adjuvant resulted in the production of both antibodies, of which the 7 S y z appeared earlier. However, immunization by the intraperitoneal route without adjuvant resulted primarily in the formation of 7 S yl antibody. In the case of rabbits immunized intravenously with poliovirus, 7 S yl antibody appeared prior to 7 S y 2 ( Svehag, 1964a). Guinea pigs immunized with HSV developed 7 S y z antibody to a somewhat higher level than 7 S yl following primary immunization, but, following a booster inoculation, the 7 S yl antibody content was increased to a greater extent than the 7 S y 2 (Tokumaru, 1967). A somewhat similar situation has been reported for humans infected with varicella-zoster virus (Leonard et al., 1970). In the primary infection (varicella), both IgM and IgG were produced, but the IgG was primarily of the slow electrophoretic class. Upon reinfection (herpes zoster), both fast and slow IgG antibodies became evident. It was suggested that the primary response was “incomplete” in that slow IgG was primarily produced and that a second infection was required to complete the response. The possibility that the different subclasses of IgG may be different in their protective capabilities under in vivo conditions was demonstrated by Tokumaru (1967). In this study it was found that guinea pig 7 S yl antibody was 3 times more effective in protecting guinea pigs against herpes virus challenge than was an equivalent neutralizing amount (tissue culture determined) of 7 s y 2 antibody. Thus, sera having equivalent neutralizing activities would not necessarily have equal protective capacities if they contained relatively different amount of 7 S yl and 7 S y2 antibodies. Whether or not actively induced, fast and slow IgG subclasses of antibodies differ in their ability to protect an immunized susceptible host is not known. It will be of interest to establish this as it could influence the methods of compounding and administering virus

ANTIBODY RESPONSE TO VIRAL ANTIGENS

229

vaccines in order to preferentially induce the IgG antibody subclass desired. These considerations of the antibody response to viruses indicate that the different antibody classes and subclasses may differ in their ability to neutralize viruses, just as they may differ in other biological activities. In order to establish definitively the relative activity of the different antibody classes to viruses in neutralization and protection reactions, as well as in other serological reactions, we felt it desirable to examine the FMDV immune systems by quantitative immunochemical procedures. With this approach, we feel it may be possible to evaluate virus-antibody interactions at a molecular level rather than by conventional relative determinations. The previously considered quantitative precipitin tests performed with FMDV provided antiserum reagents containing known weights of antibody. These were used for calibrating the SRID procedure for the measurement of antibody (Cowan and Wagner, 1970) and for developing computational methods for data processing ( Trautman et al., 1971). Although these experiments are still in progress, it is of interest to examine the antibody response of cattle infected with FMDV as determined by various serological procedures.

D A Y S AFTER

INOCULATION

FIG. 4. Antibody response to virion (140 S ) antigen of a steer infected with foot-and-mouth disease virus A,, as deterniiiiecl by single radial iinmunodiffusion analysis.

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KEITH M. COWAN

Four steers were infected by intradermalingual inoculation of virus; 1 received Type A virus, 2 Type 0, and 1 Type C. Blood was collected daily for the first 17 days and at varying intervals up to day 62. All sera were examined for antibody content by SRID and CF assays, using purified virus as antigen, but only a limited number of the sera have yet been tested for neutralizing activity by the mouse test. The antibody response as measured by SRID of the steer infected with Type A virus is presented in Fig. 4.The responses of cattle infected with Type 0 and Type C viruses were very similar, although the Type C-infected animal responded to a higher level. Antibody was first detected on day 4 at a concentration of 6 pg. antibody ( Ab) protein per milliliter of serum. The antibody content rose to about 200 pg. Ab/ml. by day 7 which was followed by a relatively slow increase to day 9, and then abruptly increased to a peak at day 13 at a concentration of 620 Fg. Ab/ml. A similar shoulder was also evident with Type O-infected cattle between about 7 and 9 days. With the Type C-infected animal, an actual decrease in antibody occurred from day 8 until day 11, but then it rose sharply reaching a peak level at 15 days. The antibody content decreased quite rapidly in all of the animals, and by day 34 was approximately one-third the 13to 15-day peak levels. The antibody response curve presented in Fig. 4 may appear somewhat unusual for viruses, but it should be recognized that a linear scale was used. More familiar-appearing response curves are presented in Fig. 5 where SRID, CF, and neutralization results are presented on a log,, scale. There was an evident delay in the appearance of CF activity due to the failure of the early appearing IgM antibody to fix complement. By all three assays, the maximum antibody level occurred at day 13 or 14, and then proceeded to decline at approximately the same rate. The precipitin data developed make it possible to provide preliminary estimates of the mass of antibody required in the CF and neutralization reactions (Table 11).Sera collected between the eleventh and seventeenth day required about 0.6 pg. Ab/ml. to fix 4 of 5 (50%hemolytic) units of complement, whereas the 9- and 10-day samples required a greater total mass of antibody because only a portion was complement-fixing IgG antibody. The values obtained for sera collected after 17 days are rough estimates as these sera have yet to be assayed by quantitative C F procedures. However, it is suggestive that greater amounts of late antibody were required for CF than for the earlier sera, and this may indicate the later sera contained a relatively greater proportion of the poorer complement-fixing IgG, antibody, The antibody mass estimates are also very rough for the neutralization data as more precise neutralization assays, e.g., plaque-inhibition procedures, will be required to

231

ANTIBODY IIESI’ONSE TO VIRAL ANTIGENS

I

0

2

4

I

6

8

10

12

14

16

18

20

22

24

26

28

30

32

34

D A Y S A F I E R INOCULATION

FIG.5. Antibody response of a steer infected with foot-and-month disease virnsA?, as deterniined by single radial ininiunodiffusion ( SRID; ), complement fixation (CF; A),and neutralization (Neut.; ) procedures. The units of antibody for SRID are in microgmnis of antilmly per iiiilliliter of serum; for CF, it is the reciprocal of the dilution of serinn fixing 4 of 5 50% hemolytic units of complement; and for neutralization, it is the reciprocal of the dilution of serum protecting one-half of the suckling mice against 100 LDooof virus.

provide the quantitative information necessary. Regardless, it is suggestive that a lesser mass of antibody is required with the later sera (IgG antibody) than for 7-day serum ( IgM). This may indicate that, on the one hand, IgG antibody is more effective on a mass basis than IgM, but, on the other hand, taking into account molecular weight differences, the IgM would be more efficient on a molar basis. Although these studies are still in a preliminary stage, it may be anticipated that the application of quantitative immunochemical techniques may provide new informat’ion on the true antibody response to viral antigens.

B. RESPONSETO VIRALANTIGENSOTHERTHANVIRION Earlier discussions indicated several instances where an apparent lack of antigenic relationship between intact virus particles and their protein subunits or RNA-free empty capsids occurred. Although early investi-

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KEITH M. COWAN

TABLE I1 RELATIONSHIP ~ E T W E E N PRECIPITATING, COMPLEMENT-FIXING, AND NEUTRALIZING ANTIUODY ACTIVITIESOF SERACOLLECTED .IT VARIOUSTIMES FROM A STEERINFECTED WITH FOOT-AND-MOUTH DISEASEVIRUS.TYPIC A-24 Complement fixation Ab/ml. fixing 4 of 5 CHm)

Neutralization

(pg.

Days after infection

Precipitind (pg. Ab/ml.)

The+

4 5 6 7 8 9 10 11 12 13 14 15 16 17 20 27 34 41 48 55 62

6 50 120 190 209 223 300 401 513 623 536 558 49 1 480 400 300 185 140 127 70 52

<5 <5 <5 <5 <5 45 130 700 886 920 898 872 848 862 480 480 240 160 120 60 60

Titep

2.54 4.96 2.31 0.57 0.58 0.68 0.60 0.64 0.58 0.57 0.83 0.63 0.77 0.88 1.06 1.20 0.87

3.5 3.17 3.1 2.54 2.81 2.75 2.61 2.63

(pg. Ab/ml. protecting 50% of mice)

0.55

-

0.16 0.27 0.24 0.53 0.22 0.23 0.17 0.12

Single radial immunodiffnsion determinations. Ab-antibody. of serum dilution fixing 4 of 5 50% hemolytic units of complement (CH,) with an optimal amount of purified virus antigen. Fixat,ion was for 1.5 hours a t 37°C. c Loglo of the reciprocal of the serum dilut,ioii protecting one-half of the suckling mice inoculated with 100 LD, of virus.

* Reciprocal

gation of the virions and empty capsids of TYMV did not reveal any difference in their antigenic structure (Markham et al., 1948), it was noted that rabbits immunized with as little as 0.01 nig. of purified nucleoprotein (virions ) produced much higher levels of precipitating antibody than if immunized with an equivalent amount of empty capsids. A subsequent study by Marbrook and Matthews (1966) confirmed these earlier findings with both TYMV and TMV antigen preparations in both rabbits and mice. It was further noted that the addition of isolated TYMV RNA

ANTIBODY RESPONSE TO VIRAL ANTIGENS

233

did not enhance the inimunogenicity of the empty capsids. Although the point was not stressed, detectablc antibody tended to appear later in animals immunized with empty capsids than in those immunized with virus. It was suggested that viral RNA enhanced ininiunogenicity, but the authors were unable to provide a suitable explanation for the phenomenon. Similar findings were made by Loor (1967) with TMV, its protein subunits, and reaggregated protein subunits. It was also found that “Clabeled protein subunits and reaggregated subunits were eliminated more rapidly than whole virus from lymph nodes. It was proposed that the greater immunogenicity of virions can be attribufed to greater deposition in antibody-forming tissue because of their particulate nature and to slower degradation by proteolytic enzymes. It was pointed out that although reaggregated protein subunits may be similar in size to virions, they may be readily dissociated at physiological pH and would probably be equivalent to subunits once inoculated into the animal. It was further found that antibody persisted for a longer time following immunization with virus, and this was attributed to the greater persistence of virus as compared to subunits. Strictly comparable experiments on the relative immunogenicity of animal viruses, their subunits, or empty capsids have not been described; however, there are several indications in the literature that some animal viruses may behave similarly to the plant viruses cited above. Influenza virus disrupted by ether treatment was found to induce HAT and neutralizing antibody, and was also less pyrogenic than intact virions (Davenport et al., 1964). A comparison of the relative inimunogenicity in rabbits of intact virus with ether- or detergent-treated virus demonstrated that untreated and ether-treated viruses were generally more immunogenic and induced a more rapid antibody response than the detergent-treated preparations ( Webster and Laver, 1966). The possibility that the difference in time course of appearance of antibody was due to differing ability of intact and disrupted viruses to induce IgM antibody was explored by determining the susceptibility of early antisera to mercaptoethanol treatment. One of the virus strains (SW) disrupted with detergent apparently failed to induce IgM, whereas ether-treated virus did. Disrupted virus vaccines prepared from the AS and BEL strains also induced IgM antibody. However, similar studies by Fazekas de St. Groth et al. (1969) indicated that the delayed appearance of antibody in the primary response to subunit vaccines was due to the production of little or no IgM antibody. Based on earlier studies of Nossal et al. (1964) with flagella and flagellin antigens, it was suggested that relatively large antigens, e.g., intact virus or flagella, were required for

234

KEITH M. COWAN

macroglobulin antibody stimulation, whereas small antigens, such as viral subunits or flagellin, would only induce 7 S antibody formation. Experiments demonstrating that empty capsids ( C antigen) of poliovirus actually induced the formation of neutralizing antibodies ( Hinuma et al., 1970) also provided evidence that mammalian virus antigens, with physical characteristics similar to those of TYMV, induced antibody responses similar to those demonstrated by Marbrook and Matthews (1966). Intact virus ( D antigen) in rabbits induced higher levels of neutralizing antibody than empty capsids, and a more rapid antibody response was also obtained. Higher levels of CF antibody were also produced by virus than by empty capsids indicating that a greater mass of antibody was induced. Investigations on the relative immunogenicity of the different antigens of FMDV also indicate that virions ( 1 4 0 s ) induce more antibody than do equivalent amounts of empty capsids ( 7 5 s ) or subunits (12 S ) (Cowan, 1972). The 140, 75, and 12 S antigens of FMDV (type A, subtype 24) were purified, treated with acetylethyleneimine to assure their noninfectivity, and the protein content established by Folin-Ciocalteu analysis. Guinea pigs were immunized with 5 pg. of protein of each preparation incorporated into incomplete Freunds adjuvant. Guinea pigs from each group were bled 7, 14, 21, 28, and 35 days later, and the serum pools were tested for antibody to each antigen by the radial immunodiffusion procedure (Cowan and Wagner, 1970) and for neutralizing activity by the mouse test. The results of these experiments are summarized in Table 111. Immunization of guinea pigs with 140s antigen resulted in the induction of sufficient antibody to 140 and 75 S to be measurable by the seventh day after inoculation, but antibody to 12 S was not evident until day 21. Antibody to 140 and 75 S was not demonstrable until day 21 following inoculation with 75 S and did not induce as much antibody as the 140 S immunization. However, antibody to 12 S appeared on day 14 and eventually reached a considerably higher level than obtained by immunization with either 140 or 12 S antigens. Immunization with a 5-pg. dose of 1 2 s antigen resulted in antibody neither to 140 nor 75S, and only low levels of antibody to 12 S were produced, but not until about the twenty-eighth day. Although studies are still in progress on the characterization of the antibodies induced by the different viral antigens, the antibody demonstrable at 7 days in the 140 S-immunized animals was clearly of the IgM class, whereas immunization with the 75 and 1 2 s antigens resulted in the production of primarily 7 S class antibody and no or very small amounts of IgM antibody. Attempts were made to modify the time course and amount of antibody induced by 1 2 s by incorporating vary-

TABLE I11 ANTIBODY RESPONSE OF GUINEAPIGSIMMUNIZED WITH FOOT-AND-MOUTH DISEASE VIRUS (140 S), EMPTY C-IPSID(75 S), OR V I R U S PROTEIN S U B U N I T (12 s) INCORPORATED IN INCOMPLETE O I L ADJUVANT TO 140 s, 75 s, AND 12 s AXTIGENS

t

2

2

3

Immunizing antigen (5 K . of protein) 140 s

75 s

12 s

Antibody to:

Antibody to:

.4ntibody to:

J

0 Days after immunization 7 14

21 28 3s

140

Neut. PDw 0.871.70 2.80 2.81 3.50

140 s

s (m./ml.) 3b 4

31 77 133

75 s (w./ml.) 2 3 42 95 129

12 s Gp./ml.)

Neut. PDsa

-

<0.3 0.44 1 .s4 1.35 2.58

43 183

214

g

140 S

(pg./ml.)

75 s (pg./ml.)

12 s (pg./ml.)

Xeut. PDm

-

-

-

<0.3 <0.3 <0.3 0.5 <0.3

17 67

51

1s 64 56

40 188 327 337

a

Log10 of the reciprocal of the serum dilution protecting 50% of the suckling mice inoculated with 100 LDro of virus.

b

Radial immrinodiffusiondeterminations.

(pg./ml.) -

-

7.5 S (%./rnl.)

-

12 R (pg./ml.) -

43 -

61

+I 0

c 3

*r r z

3

Irl

z

cn

M

w

CR

236

KEITH M. COWAN

ing amounts of the synthetic double-stranded polynucleotide, polyinosinic-polycytidilic acid ( poly I :C ) into the vaccine, but no enhancement occurred. In general, the 140s virion induced higher levels of antibody more rapidly than the equivalent-sized RNA-free 75 S particles, and, in turn, the 75 S particle was superior to 12 S antigen as an immunogen. It would appear that the presence of viral RNA as well as the size of the antigen both played a role in determining the antibody response to these FMDV antigens. The greater immunogenicity of whole virus as compared to viral subunits has also been demonstrated with adenoviruses (Neurath and Rubin, 1971) . In order to induce in guinea pigs an amount of antibody sufficient to neutralize 100 median tissue culture infective dose (TCID,") of virus, approximately 100 times more monomeric hexon antigen was required than with whole virus, and, in turn, aggregated hexon was considerably more effective than monomeric hexon antigen. It was concluded that the state of aggregation of the hexon antigen was extremely important in determining immunogenicity as had been shown with flagellin antigen (Nossal et al., 1964). Even so, the hexon antigen was shown to be exceedingly immunogenic since 1 ng. in incomplete Freund's adjuvant elicited precipitating, complement-fixing, and neutralizing antibody in guinea pigs ( Haase et al., 1972). The greater immunogenicity of whole virus as compared to disrupted virus does not seem to be the case with all viruses. Vesicular stomatitis virus disrupted by Tween-ether, Nonidet, or sodium deoxycholate was reported to be more immunogenic than intact virus (Brown et al., 1967; Cartwright et al., 1970). The preparation of viral subunits of rabies virus for immunization is of considerable current interest, and the successful immunization of animals with soluble components has been reported by several investigators (Crick and Brown, 1969, 1970; Schlumberger et al., 1970; Schneider et al., 1971). Schlumberger et al. ( 1970) noted that intact virus induced antibody more rapidly and to a higher level than the soluble fraction; however, a booster inoculation of soluble antigen resulted in antibody levels essentially equivalent to that obtained with intact virus. Schneider et al. (1971) isolated the hemagglutinin from saponin-disintegrated pure rabies virus and found it to be as immunogenic as intact virus when adjusted to equivalent weight concentrations. Thus, with viruses, such as vesicular stomatitis virus and rabies, the soluble antigenic components would appear to have immunogenicity equivalent to that of the intact virus. A difference in the time course of appearance of antibody to the different antigenic components of FMDV has been observed in infected animals as well as in those immunized with noninfectious antigens. Both

ANTIBODY RESPONSE TO VIRAL ANTIGENS

237

cattle and guinea pigs infected with FMDV could be shown to have produced precipitating antibody to virus (140 S antigen) by the seventh day after inoculation, whereas antibody to the 12 S protein subunit antigen could not be demonstrated until about 14 days (Brown, 1960). Similar observations with guinea pigs have been made in our laboratory (K. M. Cowan, unpublished); however, the use of the highly sensitive, radial immunodiffusion procedure demonstrated antibody to 140 S antigen as early as 4 days after infection of cattle, but antibody to the 12 S and VIA antigens was not detected until 6 and 8 days, respectively (Cowan and Graves, 1972). In general, it would appear that whole virus preparations are superior immunogens to their soluble or subunit antigenic components. The differing immunogenicity of whole virus and viral subunits may be attributed to differences in size or state of aggregation and/or the presence of nucleic acid associated with the antigen. With viral subunits, such as monomeric hexon and aggregated hexons of adenovirus ( Neurath and Rubin, 1971), the greater immunogenicity of the latter would evidently be attributed to a difference in the size of the immunogenic particles. In the case of the empty capsid (75 S) and protein subunit ( 12 S ) antigens of FMDV, the earlier appearance and higher levels of antibody induced by 75 S as compared to 12 S may also be related to the sizes of the antigenic components. The differences in immunogenicity found for virions and the RNAfree empty capsids of viruses, such as poliovirus, TMV, TYMV, and FMDV, suggest an adjuvant effect of viral RNA. It has been shown with several different nonviral antigens that they become considerably more potent immunogens when associated with RNA ( Askonas and Rhodes, 1965; Friedman et al., 1965; Roelants et al., 1970), and these have even been referred to as superantigens (Goodman, 1972). Both RNA and DNA were found to have an adjuvant effect for bovine y-globulin, and they also stimulated a more rapid appearance of antibody (Merritt and Johnson, 1965). Similarly, enhancement of immunogenicity with synthetic double-stranded polynucleotides has been demonstrated with several nonviral antigens (Braun and Nakano, 1967; Turner et al., 1970; Schmidtke and Johnson, 1971) as well as influenza virus (Woodhour et al., 1969). The enhanced immunogenicity of viral antigens, whether due to aggregation or association with viral RNA, would appear to be correlated with their ability to induce a satisfactory IgM antibody response (Webster and Laver, 1966; Fazekas de St. Groth et al., 1969). The controversies concerning the sequential appearance of IgM and IgG following antigen administration may, in part, be due to the types of

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KEITH M. COWAN

antigens utilized for such invcstigation. The early investigations used antigens such as bacteriophage ( Uhr, 1964), poliovirus ( Svehag and Mandel, 1964a), and FMDV (Brown, 1960), and the combination of particle size as well as their nucleic acid component induced an Igkl response that preceded IgG. With antigens, such as albumins or yglobulins, the IgM and IgG appeared to be produced simultaneously (Freeman and Stavitsky, 1965; Osler et al., 1966), and it was suggested that the failure to demonstrate this in the earlier studies was due to the assay procedures applied. However, similar conclusions may well have been reached with viral antigens if disrupted viruses, empty capsids, or protein subunits had been used instead of intact virus particles. These considerations of the antibody response to viral antigens have been concerned only with the primary response. In essentially all of the instances considered, a booster dose of disrupted virus or protein subunits resulted in excellent anamnestic responses that gave antibody levels almost equivalent to those obtained with the intact viruses. Thus, the differing immunogenicity of virions and their subunits was generally more readily evidenced in the primary than in the secondary response ( Webster and Laver, 1966; Fazekas de St. Groth et d.,1969; Schlumberger et al., 1970; Neurath and Rubin, 1971). Although subunit viral vaccines may be less efficacious in inducing a primary response for immunization purposes than whole virus vaccines, the reduction in adverse reactions and the elimination of viral genomes may more than compensate for this deficiency. However, an obvious limitation of the subunit viral vaccines would pertain to those viruses in which virion integrity is apparently essential for maintaining antigenic determinants that are dependent on conformation at the tertiary or quaternary levels and which are involved in the neutralization process.

C. ANTIBODYSPECIFICITY The diagnostic applications of immunological procedures for identification of viruses, as well as the antibodies reacting with them, are of vital concern in disease control and surveillance. The successful application of serological techniques in virology is obviously dependent on the specificity of these reactions. Factors influencing the preparation of either antibody or antigen reagents having the desired specificity are of interest to consider not only because of the obvious practical diagnostic significance but also as an attempt to explain the changing specificity frequency observed with antiviral antibody during the immune response. Prior to a consideration of the question of specificity of serological reactions, it is probably important to define the term when applied to antigen-antibody interactions as compared to diagnostic procedures. We

ANTInODY

IlESPOKSE TO VIRAL ANTIGENS

239

think of aiitigeii-antibody interactions as being specific in that antibody will combine cither with the homologous antigen or with antigens that are chemically very closely related to it. Thc latter are true cross-rcactions, but are quite specific aiid may providc valuable insight into the chemical iiaturc of the cross-reacting antigens. However, it has been indicated that the latter reactions are the microbiologist’s migraine in that they becloud his diagnostic capabilities ( Heidelbcrger, 1967). Thus, the diagnostician will generally view specificity as the ability to identify a virus as to its particular group, subgroup, or type. The inability to accomplish this because of extensive cross-reactions may be viewed as nuisance reactions by the diagnostic technician, whereas the inimunologist may consider them as valuable indicators of chemical relationships. Obviously, the question of specificity of antigen-antibody interactions is dependent on the point of view of the beholder. The occurrence of multiple antigens in virus preparations is probably one of the more frequent reasons for cross-reactions, and the extent of this niay reasonably be influenced by the assay technique utilized. Individual antigenic components may vary with respect to their specificity for a given virus, and the particular antigenic components detected by a given assay procedure may determine the specificity or lack of specificity obtained by that technique. Similarly, the nature of the antigen and aiitiseruni reagents employed in the reactions may determine the specificity of the reaction. Although these considerations are self-evident, the resolution of the antigenically complex virus systems niay be a formidable task. However, the isolation of the individual antigenic constituents and their physical, chemical, biological, and immunological characterization may be a prerequisite for the development of rontine and meaningful diagnostic procedures. A simpIe illustration of the above considerations can be made with the FMDV immune system. As indicated earlier, a crude harvest of tissue culture or an animal sourcc of FMDV will contain 140 S antigen as well as 12 S and VIA antigens, and some Type A virus preparations niay also contain 75 S antigen. Serum collected from animals infected with FMDV will contain antibodies reactive with all of these. Consequently, an assay technique such as CF performed on such a mixture of antigens and antibodies may provide evidence of antigen-antibody interactions, but it may give little information about what is being measured either in terms of antigens or antibodies. With regard to the type specificity of the different antigenic components, the 140 S antigen is considered to be type-specific, the 12 S antigen is somewhat cross-reactive ( Bradish and Brooksby, 1960), and the VIA antigen I., not type-specific and niay react cqually well with heterologous aiid homologous sera

240

KEITH M. COWAN

(Cowan and Graves, 1966). Such a mixture of type-specific and non-typespecific reactants in the CF assay can obviously lead to confusion, and either type-specific or nonspecific results may be obtained, depending on the test condition or reagents used in the assay (Cowan and Trautman, 1967). The use of reagents such as purified antigens and/or component-specific antisera prepared by absorption techniques ( Cowan, 1968) enables one to develop highly specific assays or ones of broad reactivity if desired. The detection of antibody to the VIA antigen of FMDV for epizootiologic surveys ( McVicar and Sutmoller, 1970) provides an instance where broad reactivity was desired in order to circumvent the requirement of testing individual serum for antibody to several different serological types of the virus. Once an animal was identified as having antibody to VIA antigen, subsequent 140 S-specific assays could be performed to establish the serological type of the infecting FMDV. The FMDV type specificity could be assayed by neutralization procedures, which would involve only the 140s antigen, or by CF or immunodiffusion procedures using purified 140 S preparations as sources of antigens. The occurrence of multiple antigens and antibodies in poliomyelitis may result in specific or nonspecific C F reactions following infection of humans. This is dependent on the nature of the antigen used in the CF assays, i.e., virion ( D antigen) or empty capsids ( C antigen), and on the time of collection of the serum sample (Schmidt and Lenette, 1956; Le Bouvier, 1955; Roiman et al., 1959). The C antigen was more crossreactive than the D antigen and also reacted more frequently with early sera, whereas the D antigen reacted better with sera collected later during convalescence. It is evident from this that when viral systems containing multiple antigens and antibodies are examined, the nature of the antigen preparation utilized could determine the relative type specificity of the'reaction as well as the apparent time course of appearance and disappearance of antibody. The Coxsackie virus provides a complex situation with regard to specificity involving multiple antigenic components, various assay procedures, as well as different classes of antibody. Both neutralization and HA1 procedures were found to be highly type-specific in initial infections as well as in a subsequent infection with heterologous viruses (Schmidt et al., 1965). However, the CF and precipitation reactions became increasingly cross-reactive upon subsequent infections. With respect to precipitin reactions, it had been demonstrated that two precipitin bands could occur in immunodiffiision reactions: one band was type-specific and involved the intact, infectious virus particles, whereas the other band was not type-specific but group-reactive and involved a less dense, non-

ANTInODY RESPONSE TO VIRAL ANTIGENS

24 1

infectious viral particle (Schmidt et al., 1963). It had also been noted that the type-specific precipitin band persisted for a relatively short time following infection ( Schmidt et al., 1965). Subsequent studies demonstrated the early serum antibody was largely 1 9 s in character and was followed by a characteristic conversion to 7 S antibody during convalescence ( Schmidt et al., 1968). The demonstration that the neutralizing and HA1 responses were parallel and type-specific was reasonably attributed to the interaction of the type-specific virion particles and antibody. However, the CF assay would measure both the type-specific and group-reactive antigens resulting in less type specificity than either neutralization or HA1 assays. The immunodiffusion precipitin reactions were of particular interest in that it was found that 1 9 s antibody appeared to react only with the type-specific virion antigen, whereas the 7 S antibody reacted with the group antigen preparation (Schmidt et al., 1968). Thus, the short duration of the type-specific precipitin reaction was attributed to 19 S antibody and virion particles whereas the persisting group reaction was due to 7 S antibody and the noninfectious group antigen. The preceding provided examples of situations in which the specificity of serological reactions with viral antigens would be influenced by the nature of the antigen preparation and the method of assay. The Coxsackie viruses provided an additional complication in that the physicochemical class of antibody influenced the specificity of the reactions as well. The possible difference in the specificity of IgG and IgM antibodies to viral antigens has been investigated and has yielded conflicting results. It is of interest to consider some of the studies not only because of their potential importance in the development of better and more specific virus typing procedures but also for the implications concerning possible differences in the combining characteristics of antibodies of the different physicochemical classes. Early immunodiffusion studies with FMDV demonstrated that sera collected from cattle 7 days after infection cross-reacted strongly with heterologous subtypes of FMDV, whereas sera obtained later after infection were much more specific (Graves, 1960). It was then shown that the early appearing cross-reacting antibody had p-globulin electrophoretic mobility ( 1 9 s or IgM antibody), whereas the later appearing specific antibody had ./-globulin ( 7 S or IgG) characteristics (Brown and Graves, 1959; Brown et al., 1964). The differences in specificity of the IgM and IgG antibodies determined in these studies could not be attributed to the detection of different antigenic components as it was shown that the 140 S antigen (virion) was the relevant antigen by both immunodiffusion and neutralization procedures.

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The greater specificity of IgG as compared to IgM antibodies has also been reported for influenza viruses ( Webster, 196813). Serum obtained from rabbits 5 days after vaccination contained high levels of IgM antibody having high avidity and low specificity. However, the early occurring IgG antibody had low avidity and high specificity, whereas late IgG antibody resembled the IgM antibody by having high avidity and low specificity. Thus, high specificity of the antibody was associated with low avidity, and low specificity with high avidity. To explain these observations, a model in which the antigenic determinant was considered to consist of several distinct chemical groupings ( subdeterminant groupings ) was proposed, and, for purposes of illustration, six such groupings were indicated. The early IgG antibody was proposed to possess antibodycombining sites that were complementary to only two of the six antigenic subdeterminant groupings. Thus, high specificity could occur, but the avidity would be low because of limited complementation. In contrast, the late occurring IgG had “matured” and the antibody-combining site was then complementary for all six antigenic subdeterminants resulting in high avidity reactions. However, the specificity of this highly avid antibody would be lessened because of the increased likelihood that the antibody could react with a common subdeterminant grouping occurring on heterologous viruses. The high avidity and low specificity of IgM antibody was considered by Webster (1968b), and two alternatives were proposed-one was the high affinity of individual antibody-combining sites, and the other was the multiple attachment of low-affinity combining sites. It was indicated that either mechanism could explain the cross-reactivity of IgM antibody, but that the resolution of this question would depend on affinity determinations of isolated combining sites. Contrary to the above-cited findings, IgM antibody has been reported to be more specific than IgG antibody for differentiating closely related viruses. Monkeys that had been immunized with a niyxovirus (DA virus) produced antibody of the 7 s class that reacted with the homologous virus as well as with the serologically related mumps virus (HefFner and Schluderberg, 1967). However, upon challenge with mumps virus, 19 S antibody, specific for only mumps virus, was induced and the 7 S antibody level to DA virus was boosted by this challenge inoculation. Thus it appeared that 7 S antibody was cross-reactive whereas the 19 S antibody was specific for the homologous challenge virus. Studies of the Group B arboviruses demonstrated that rabbit and guinea pig 19 S antibody was more specific than 7 S antibody for differentiating these closely related viruses by HA1 procedures (Westaway, 1968a,b). It was also found that IgG2 antibodies tended to be less cross-

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reactive than IgG, antibodies. The suggestion was made that 19 S antibodies react predominantly with a virus-specific antigen, whereas 7 S antibodies react with a group-specific antigen. The determination of minor antigenic differences between HSV strains (Hampar et al., 1968b, 1969, 1970; Stevens et al., 1968) and simian cytomegalovirus strains (Hampar et al., 1969) was more readily established with late occurring, rabbit 1 9 s antibody than with either early appearing, 19 S antibody or early or late, 7 S antibodies by neutralization assays. This appears to be explained by the finding that these antibodies react with different virus-associated antigens ( Miyamoto et al., 1971; Hampar et al., 1971). The late 7 S antibody has specificity for the viral envelope surface and may differentiate interspecies strains of herpesvirus into distinct serotypes. However, it will not readily differentiate either intra- or interspecies herpesvirus variants that are closely related. The early and late 19 S and the early 7 S antibodies react mainly with subsurface structural antigens, but the late 19 S antibody appears to react with those glycoproteins most notably reflecting antigenic differences between the closely related strains of HSV. It was indicated previously with FMDV that the 19s antibody was more cross-reactive than 7 S antibody (Brown and Graves, 1959; Brown et al., 1964). However, recent studies with guinea pig 19 and 7 S antibodies demonstrated that 19 S antibody had greater specificity in that it could distinguish between antigenic variants of virus, a distinction not possible with 7 S antibody (Cowan, 1969, 1970; Wagner and Cowan, 1971). To explain this it was proposed that the antigenic determinant sites on the virus particles were composed of several smaller subdeterminant groupings, e.g., an ab determinant site was composed of subdeterminants a and b. It was suggested that one of the FMDV variants isolated contained ab determinant sites, whereas the determinant sites of another isolate consisted of only b subdeterminants. The evidence presented by several investigators (Kaplan and Kabat, 1966; Frank and Humphrey, 1968; Franks and Liske, 1968; Moreno and Kabat, 1969) indicating that 19 S antibody had smaller combining sites than 7 S antibody led to the proposal that 7 s antibody had a sufficiently large combining site to react with the FMDV a19 determinant as a whole, but the restricted size of the 1 9 s combining site permitted it to react only with the subdeterminant groupings ( Cowan, 1970). Thus, 7 S antibody was considered to have anti-ah activity and would react with either 140 S-ab or 140 S-2, particles, but would not differentiate between them. However, the 19 S antibodies had distinct anti-a or anti-b specificities and could readily differentiate between these variants. The possible pertinence of thesc considerations to the relative speci-

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ficity of early and late antisera to FMDV was suggested by immunodiffusion and absorption studies performed with cattle sera collected early and late after infection with FMDV (Cowan, 1972). In accord with the findings of Brown and Graves (1959) and Brown et al. (1964), the early sera ( 7 or 8 DPI) reacted with the homologous infecting Type C virus and also with heterologous Types A and 0 viruses, whereas the late serum (approximately 45 DPI) reacted only with the homologous virus. Absorption experiments were performed in an attempt to explain the extensive cross-reactivity of the early antiserum. Absorption with homologous virus (Type C ) inhibited the antibody reactive with both homologous and heterologous viruses. However, absorption with heterologous Type A virus inhibited anti-A activity, but not anti-0 nor anti-C activity. Similarly, Type 0 virus inhibited anti-0, but not anti-A nor anti-C antibody. Thus, the early serum appeared to contain antibodies of high specificity for different antigenic determinants on the homologous Type C virus that were related to ones on the heterologous A and 0 viruses. This suggests an incongruous situation in which the extensive crossreactivity of the early antiserum was due to the occurrence of several highly specific antibody moieties capable of reacting with minor antigenic determinants ( subdeterminants? ) that were either not recognized or were not able to react with 7s antibodies, perhaps as a consequence of steric or conformational factors, The question of the relative specificity of early and late antibodies would then depend on the particular virus in question, and whether the site responsible for type specificity resided in a subdeterminant grouping or in the total determinant site. V. Concluding Remarks

This review of the antibody response to viral antigens has probably devoted more attention to the methodologies.applied to the measurement of antibodies than to the actual antibody response. This was intentional as it was hoped to stress the point that the antibody assay employed may influence the nature of the response determined. This should be evident in view of multiple antigenic materials that may be present in crude virus preparations, the antigenically distinct structural units occurring in the complex viruses, and the mosaic of distinct antigenic determinant groupings that may be present on the surface of even the “simple” viruses. Similarly, the different biological activities of the various classes of immunoglobulins, as well as the changing reactivities of a given class of immunoglobulin, may determine the nature of the response obtained with the different assay procedure. Thus, the information sought in a given situation should determine the types of assay procedures selected

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for the study, and this may depend on the availability of suitable antigen or antiserum reagents. Although these approaches are self-evident, too frequently it is not known precisely what information is desired except in rather broad terms, This is particularly the case in the preparation and evaluation of viral vaccines and in the determination of the immune status of an individual or a population of individuals. Vaccines are applied to induce the immune state, and the information sought for control purposes is that which will predict the efficacy of the vaccine prior to its application under field conditions. Recent controversies concerning the standardization of human viral vaccines suggest that current methodologies do not always provide this information. We may wonder whether or not the assays applied are measuring the antigenic components in the vaccines responsible for inducing immunity and whether or not the procedures applied to evaluate the antibody response to such vaccines are measuring antibody to the relevant antigens or antigenic determinants. Although in recent years tremendous progress has been made in characterizing viral antigens, the practical application of this knowledge has been extremely limited. The viral vaccines used in both human and veterinary medicine generally consist of such a conglomeration of viral and nonviral immunogens that the average immunologist or immunochemist may be reluctant even to administer them to his laboratory animals. In particular, these obstacles become evident if it is desired to obtain meaningful information on the antibody response to a single constituent of the vaccine mixture, The increasing implication of viruses in a great variety of pathological conditions indicates a greater necessity to establish the chemical and biological properties of viral components and to determine the significance of the antibody response to these constituents. REFERENCES

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Antibodies to Small Molecules: Biological and Clinical Applications' V I N C E N T P. BUTLER, JR., AND SAM M. BEISER Departments o f Medicine and Microbiology, College o f Physicians and Surgeons, Columbia University, N e w York, N e w York

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I. lntrodulction

It is well known that low molecular weight compounds, when linked as haptens to protein or polypeptide carriers, will elicit the production of antibodies capable of reacting specifically with the hapten used (Landsteiner, 1945). The development of sensitive methods of detecting and quantitating reactions between hapten and antibody and the perfection of techniques for obtaining highly purified preparations of antihapten antibodies have played a significant role in the development of modern immunological concepts. Antihapten antibodies have proved particularly useful in the study of antigenic determinants, structural requirements for immunogenicity, the nature of antigen-antibody reactions, and the chemical, physical, and biological properties of antibody (Beiser et al., 1!368). In recent years, haptens of biological importance have been studied with increasing frequency, and this review will deal with the pro-

'These studies have been supported by research grants ( HL-10608, HL-05741, and AI-06860) from the U. S . Public Health Service and by grants-in-aid from tlie New York Heart Association, the American Heart Association (72-853), and Burroughs Wellcome & Co. Dr. Butler is tlie recipient of an Irma T. Hirschl Career Scientist Award; he fomierly was the recipient of a Research Career Development Award (HL-11,315) from the U. S . Public Health Service. 255

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duction, detection, characterization, biological properties, and clinical applications of antibodies with specificity for such haptens. It. General Principles

A. METHODSOF ELICITINGANTIBODIES In order to elicit antibodies specific for substances with molecular weights of 1000 or less, these substances must be conjugated covalently as haptens to protein or synthetic polypeptide carriers. Because of their ready availability, low cost, excellent immunogenicity, high degree of solubility, and relative resistance to denaturation under the somewhat rigorous chemical conditions of some hapten conjugation procedures, the serum albumins of various species have been frequently employed as the carrier proteins. Various serum globulin fractions, fibrinogen, ovalbumin, and hemocyanin have also been used widely. Recently, synthetic polypeptides have often been employed as carriers of biologically active haptens. Although less immunogenic and more expensive than the serum albumins, these carriers are totally free of natural products and, thus, do not elicit antibodies to albumins and other proteins that may impair the usefulness of the resulting antisera in certain biological studies. It is of interest that homologous serum albumins also can be used as effective carriers for haptens with minimal or no antibody response to the carrier. A low molecular weight substance must possess at least one functional group that can be used for attachment to the available functional groups (Table I ) of the carrier under chemical conditions which neither cause significant structural alterations to the introduced substance nor produce sufficient.denaturation of the carrier to render it insoluble. Low TABLE I OF CARRIER PROTEINS AND POLYPEPTIDES FUNCTIONAL GROUPS TO WHICH HAPTENS MAY BE CONJUGATED Functional group Amino Carboxyl Phenolic Imidazo Sulfhydryl Indolyl Guanidino

Amino acid N-Terminal Lysine C-Terminal Aspartic acid Glutamic acid Tyrosine Histidine Cysteine Tryptophan Arginine

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molecular weight substances have been most frequently conjugated to protein carriers through carboxyl, amino, and hydroxyl groups. A detailed discussion of the various conjugation procedures used has been published recently ( Beiser et al., 1968). Methods of interest for particular haptens will be described briefly in the appropriate sections below. After removal of unconjugated hapten and other chemical by-products of the conjugation procedure, hapten-carrier conjugates may be used to immunize experimental animals. Ordinarily, conjugates are suspended in complete Freund’s adjuvant mixture and 1 mg. or less is injected weekly; somewhat longer periods of time (as long as 8 to 16 months, in some instances) than are employed for protein antigens may be required to obtain antihapten antibody of satisfactory titer, specificity, and affinity (Smith et al., 1970; Jaffe et al., 1971). If the available quantity of hapten or conjugate is small, a recently described alternative method of immunization, which employs a single primary immunizing dose together with Bordetella pertussis vaccine ( Vaitukaitis et al., 1971), may be useful.

B. DETECXION OF ANTIBODIES In most instances, animals immunized with hapten-protein or haptenpolypeptide conjugates form antibodies with specificity for the carrier as well as antibodies specific for the introduced haptenic group. The method chosen for detection of antihapten must, therefore, be one in which antibodies specific for the carrier will not also interact. Haptens coupled to homologous albumins are immunogenic, and such conjugates usually do not elicit significant production of antibody to the carrier, Antibodies to a heterologous carrier protein can be removed by prior absorption of antiserum with unconjugated carrier protein or, alternatively, by prior purification of hapten-specific antibodies by means of an insoluble immunoadsorbent or by other immunochemical procedures. It is of interest that, in some instances of conjugates prepared by the carbodiimide method, it has been found that native albumin does not completely remove antibody to the carrier, but albumin polymerized with carbodiimide effectively removes such interfering antibodies ( Adler and Liu, 1971). Fortunately, it is usually possible to detect antihapten antibodies by techniques that do not require prior removal of antibodies to carrier proteins. One may employ classic precipitin, complement fixation, or passive hemagglutination techniques to demonstrate the interaction of antihapten antibody with conjugates in which the hapten is attached to a protein or polypeptide carrier which is antigenically unrelated to the carrier used for immunization. Similarly, one may detect hemagglutination of erythrocyte-hapten conjugates or inactivation of hapten-conjugated bacteriophage. Direct demonstration of hapten-antibody inter-

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actions has been made possible by the ready availability of radioactively labeled haptens and hapten derivatives. The binding of labeled hapten to antibody may be determined by equilibrium dialysis. In addition, there are methods that can effect separation of antibody-bound hapten from free hapten; these include eIectrophoresis, gel or membrane filtration, coprecipitation of hapten with antibody (by the ammonium sulfate or double antibody method), and dextran-coated charcoal. Whatever method is employed, it is important to ascertain that the observed interaction is not present in control sera (from nonimmunized animals and from animals immunized with unrelated antigens) and that this interaction with antibody is specifically inhibited by purified nonradioactive hapten. C. DETERMINATION OF SPECIFICITY OF ANTIBODIES The specifkity of antibodies for a hapten is usually determined by comparing the capacity of various haptens to compete with a standard hapten (usually the homologous hapten) for antibody-combining sites. Such tests are generally termed hapten inhibition tests, and, in the past, the ability of haptens to inhibit precipitation or complement fixation resulting from the reaction of antibody with hapten-protein conjugates was quantified. Recently, the ready availability of radioactively labeled haptens and the development of relatively simple procedures for separating hapten-antibody complexes from free hapten, has made possible the direct measurement of the binding of hapten by antibody. Here, too, specificity may be determined by a type of hapten inhibition test, in this case the ability of unlabeled hapten to inhibit (or compete with) the binding of the labeled hapten by antibody (cf. Butler and Chen, 1967). In attempting to obtain antibodies of greatest specificity, it is important to keep in mind the heterogeneity of the immune response and to study sera of individual immunized animals separately. It is not unusual to find an occasional animal that responds with an antibody of the desired specificity; for example, T-even phage deoxyribonucleic acids ( DNA's ) are unusual in containing glucosylated hydroxymethylcytosine. Phage T2 DNA contains a-ghcosylated hydroxymethylcytosine, whereas T4 DNA contains glucose in both a- and /3-linkages. Occasionally a rabbit responds to T4 DNA producing antibody specific almost exclusively for the p-glucosylated hydroxymethylcytosine. Such antisera can distinguish clearly between T2 DNA and T4 DNA ( Murakami et al., 1962). The specificity of antibodies to haptens appears to be directed primarily against that portion of the molecule furthest from the site of conjugation to the carrier. For example, antibodies to digoxin are produced following immunization with the cardiac glycoside conjugated to a pro-

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tein through its carbohydrate moiety and with its steroidal aglycone located distally from the protein. These antibodies cross-react weakly with the related glycoside, digitoxin, which has the same carbohydrate as digoxin but a steroid that differs only in the lack of a hydroxyl group at the C-12 position. However, a glycoside, deslanoside, with a different carbohydrate but with the same steroid portion as digoxin, reacts almost as well as does digoxin with the antidigoxin antibodies. The aglycone, digoxigenenin, completely lacking the carbohydrate portion, also reacts well with the antibodies ( Butler and Chen, 1967; Smith et al., 1970). A similar result has been obtained in studies of the steroid system. Antibodies formed in response to testosterone-17-bovine serum albumin, in which the conjugation is through the C-17 position of the D ring, cross-react extensively with many other steroids, e.g., progesterone, cortisone, aldosterone, that have A rings similar to the one found in testosterone. Antibodies to testosterone-3-bovine serum albumin, coupled through the A ring, are less affected by the similarities in the A ring and differentiate among the steroids primarily on the basis of the D ring and the accompanying side chain ( Lieberman et al., 1959). Africa and Haber (1971) followed this line of reasoning by immunizing with a conjugate coupled through the A ring to obtain antibodies with greater specificity for aldosterone than had been obtained previously with conjugates coupled through the D ring. A corollary of the observations noted above demonstrating that antibodies specific for different portions of a hapten can be elicited is that a single specificity may not be most effective for all purposes. It is entirely conceivable that antisera with the specificities most desirable for radioimmunoassay might not be suitable for physiological studies because they do not neutralize the biological activity of the hapten. There is probably much to be learned from investigating the effects of antibodies directed against different parts of a steroid hormone, for example, on its tissue-binding properties and on its biological activities.

D. IMMUNOASSAY METHODS The immunoassay methods that have been developed for the detection and measurement of low molecular weight haptenic substances are all based upon the ability of these substances to inhibit the immunological reaction between specific antihapten antibodies and the corresponding hapten-carrier conjugate or the corresponding radiolabeled hapten. In actual practice, increasing concentrations of a known standard solution of the substance to be assayed are incubated with constant predetermined amounts of antihapten antibody and of hapten-carrier conjugate, or of radiolabeled hapten, under conditions of antigen (hapten) excess.

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A standard curve can then be constructed upon which decreasing amounts of antigen-antibody interaction (or increasing percentages of inhibition of that reaction) can be shown to correspond with increasing concentrations of the low molecular weight test compound. If the biological fluid being assayed ( I ) does not interfere with the assay procedure, ( 2 ) does not degrade or metabolize the test substance, and ( 3 ) does not contain substances that cross-react significantly with the antihapten antibody employed, the concentration of the test substance in that biological fluid can be determined from the degree to which it inhibits the reaction between antibody and the conjugate or radiolabeled hapten, when compared to a simultaneously performed standard curve (Yalow and Berson, 1964; Hunter, 1967; Berson and Yalow, 1971a). Although inhibition of precipitation, complement fixation, passive hemagglutination, and hapten-conjugated bacteriophage inhibition have been utilized to measure low molecular weight substances, the greatest degrees of sensitivity and precision have been obtained with immunoassay methods that employ radiolabeled haptens or hapten derivatives. If the test substance is a tyrosine-containing peptide or possesses a phenolic group, it can readily be labeled with radioactive iodine by the method of Hunter and Greenwood (1962) for use in a radioimmunoassay procedure (Hunter, 1967). If the test substance lacks a phenol group but is available in radioactive form with a high specific activity, the inhibition of the interaction between the radiolabeled hapten and specific antibody is ordinarily used as the basis for the immunoassay procedure. If higher specific activity is required, however, it may be necessary to conjugate to the hapten a radioactive group (Spragg et al., 1966; McGuigan, 1967) or a moiety, such as tyrosine or a tyrosine derivative (Oliver et al., 1968; Goodfriend and Ball, 1969; Steiner et al., 1969; Melani et al., 1970; Nossel et al., 1971) which can subsequently be radioiodinated. A variant of the latter procedure is the conjugation of the hapten to a protein or tyrosine-containing synthetic polypeptide carrier (unrelated antigenically to the carrier originally used for immunization), which can later be radio-iodinated with high specific activity for use in radioimmunoassay procedures (Midgley et al., 1969; Newton et al., 1970; Levine and Van Vunakis, 1970). Ideally, immunoassay procedures are carried out with untreated serum, plasma, or other biological fluid. However, in some instances, prior treatment or separatory procedures are necessary to inactivate or remove substances that degrade or metabolize the test substances. In other instances, prior extractions or separatory procedures are necessary to concentrate the test substance, to remove it from a normal binding site on

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a plasma protein in the test serum, or to separate it froin structurally related compounds also capable of inhibiting the reaction between antibody and the radiolabeled hapten or hapten derivative. Many methods are currently available and suitable for the separation of free, unbound, radioactive hapten from antibody-bound radioactivity in radioimmunoassay procedures, but only two have been extensively used, namely the dextran-coated charcoal and the double-antibody methods. The dextran-coated charcoal method, originally developed by Herbert et aZ. ( 1965, 1968), involves the almost instantaneous adsorption of low molecular weight compounds by charcoal particles which have been coated by dextran in such a way that larger substances such as antibody molecules cannot be adsorbed to any significant degree. Following addition of dextran-coated charcoal, immediate centrifugation results in precipitation of non-antibody-bound radioactivity with the charcoal pellet, while antibody-bound radioactivity remains in the supernatant fluid. The convenience and rapidity of this technique make it the method of choice for use with any hapten-antibody system to which it is applicable. Some radiolabeled haptens and hapten derivatives, notably radioiodinated hapten-protein or hapten-polypeptide conjugates, are not readily or completely adsorbed to dextran-coated charcoal. In such instances, the addition of an antiserum to the y-globulin of the species from which the antihapten antibody was derived will result in the precipitation of the antihapten antibody together with bound radiolabeled hapten (or hapten derivative), while the free or unbound radioactivity remains in the supernatant fluid after centrifugation. This double-antibody method usually requires 24-48 hours and employs large volumes of anti-y-globulin serum, which must ordinarily be obtained from a large animal (goat, sheep, horse, or cow) at considerable expense. In all other respects, however, it is a highly satisfactory and a very useful method (Morgan and Lazarow, 1963; Hunter, 1967), The ammonium sulfate precipitation method (Minden and Farr, 1967; Farr, 1971) may also be used to precipitate immunoglobulin-bound radioactive hapten but, in general, it is less precise and reproducible than the coated charcoal or double-antibody radioimmunoassay techniques. 111. Specific Applications

In Table I1 are listed those low molecular weight compounds of biological importance to which hapten-specific antibodies have been elicited. The remainder of this review will be devoted to a discussion of the production, properties, and applications of these antibodies,

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TABLE I1 Low MoLRcuLm WEIGHTSUDSTANCES OF BInLoGICIL INTEREST T O WHICHANTIBODIESH A V K BICEN ELlCITI;.D Hormones: peptide Adrenocorticotropic hormone (ACTH) Angiotensins I and I1 Bradykinin Calcitonin Gastrin Glucagon a-Melanocyte-stimulating hormone (a-MSH) Oxytocin Pancreoz ymin-cholecystokinin (PZ-CCK) Secretin Thyrotropin-releasing hormone Vasopressin Hormones: nonpeptide Cyclic adenosine-3’,5’-monophosphate Cyclic guanosine-3’,5‘-nionophosphate Histamine Prostaglandins Serotonin Steroid hormones : Aldosterone Cortisol Cortisone Deoxycorticosterone Estradiol-17S Estriol Estrone 2-Hydroxyestrone 17-H ydroxyprogesterone Pregnenelone Progesterone Testosterone Thyroid hormones: Thyroxine Triiodothyronine Coenzymes and vitamins Folic acid Pyridoxal Vitamin A Drugs Acet,ybnlicylic acid

Barbiturates Cardiac glycosides : Digi toxin Digoxin Ouabain Chloramphenicol Cytotoxic drugs: Methotrexate tphenylalanine mustard Diethylstilbestrol Hallucinogenic drugs : D-Lysergic acid (LSD) Mescaline Hydralazine Medroxyprogesterone acetate Morphine Penicillin Procaine nmide Sulfonamides: Sulfanilamide Sulfanilic acid Sulfapyridine Sulf athiazole Tetracycline d-Tubocurarine Toxins Arsenicals Carcinogens : Acetylaminofluorene Aminofluorene An thracene p- An thracene Benzanthracene Benzpyrene Dimet hylaminoazobeiuerle Ilimethylaminos tilbene Methylbenzanthracene Methyldimethylaminostilbene l12-Naphthoquinone p-Napht hylamine Genist ein Insecticides: DIIT Malathion Paralytic shellfish poison Strychnine

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Peptidw derived from proteins C-Peptide of proinsulin Fibrinopeptides A and B “Loop” peptide of lysoxyme Tobacco mosaic virus peplides Miscellaneous Carbohydrates: Cellobiose Cellobiuronic acid Colitose Gentiobiose Geutiobiuronic acid Galactose Galacturonic acid Glucose Glucuronic acid Isomaltonic acid Isomaltotrionic acid Lactose Laminaribiose Maltose Panose

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Phosphai itlyliriositonianriosides Sophorose Caterholamiries: Epinephrine Normetanephrine Cholesterol Lipids : Ceramide lactoside Phosphatidylinosi to1 Sulfatides Nucleic acid constituents: Dinucleoside phosphates Minor bases Nucleosides Nucleotides Oligonucleotides Purines Pyrimidines Transfer ribonucleic acid Plant hormones: Gibberellic acid 3-Indoleacetic acid

A. HORMONES: PEPTIDE 1 . Adrenocorticotropic Hormone ( ACTH) The anterior pituitary hormone, ACTH, is a small polypeptide of 39 amino acid residues. Antibodies can be elicited in cxperimental animals with unconjugated ACTH (Berson and Yalow, 1968). However, because of its relatively small molecular size and because it stimulates corticosteroid hormone production with resultant inhibition of antibody synthesis, difficulty has been encountered in regularly obtaining antisera of sufficient titer and affinity when unconjugated ACTH is used as an inimunogen. Accordingly, ACTH has been conjugated to protein carriers by the carbodiimide ( McGuire et al., 1965) and glutaraldehyde (Reichlin et al., 1968) methods. Rabbits immunized with ACTH-protein conjugates form antibodies to ACTH which can be used, together with radioiodinated hormone, in the assay of circulating ACTH in the plasma of patients with pituitary and adrenocortical disorders. 2. Angiotensins I and I1 Angiotensin I is an inactive decapeptide produced by the proteolytic action of the renal enzyme, renin, on a plasma cu-globulin substrate.

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Angiotensin I is rapidly converted in lung and Dlasnia to the active octapeptide hormone, angiotensin 11, a potent pressor agent and an important physiological stimulus to the secretion of aldosterone by the adrenal cortex (Haber, 1969; G. W. Boyd et al., 1972). Unconjugated angiotensin I1 is weakly antigenic ( Dietrich, 1966); antibodies can also be elicited by immunization with angiotensins I or I1 adsorbed to charcoal particles (Boyd et d., 1967; Boyd and Peart, 1968; Hollenians et d., 1969). However, the most useful antisera to angiotensins I and I1 have been obtained from animals immunized with angiotensin-protein or angiotensin-polypeptide conjugates. Several methods have been used to conjugate angiotensin to protein and polypeptide carriers. By its reaction with p-nitrobenzoylchloride, angiotensin I1 was converted to its p-aminobenzoyl derivative; after diazotization of the p-amino group, the peptide derivative could be coupled to protein carriers (Deodhar, 1960). Goodfriend et al. (1964) conjugated angiotensin I1 to albumin carriers with water-soluble carbodiimides, which formed peptide bonds between the carboxyl and amino groups of the peptide and corresponding groups on the protein carrier. Haber et al. (1965) conjugated angiotensin I1 via its carboxyl-terminal end to a synthetic poly-L-lysine carrier by the carbodiiniide method and also conjugated this peptide to the same carrier via its amino-terminal end with the use of m-xylylene diisocyanate. Angiotensins I (Haber et al., 1969) and I1 (Stason et al., 1967) have been conjugated to succinylated poly-L-lysine by the carbodiimide method. Animals immunized with angiotensin-protein or angiotensin-polypeptide conjugates form antibodies to angiotensin. Using antibodies to angiotensin I1 and the radio-iodinated peptide, a number of radioimmunoassay methods for the measurement of angiotensin I1 in human plasma have been developed (Vallotton et al., 1967; Boyd et al., 1967; Catt et al., 1967a, 1969; Goodfriend et al., 1968; Gocke et al., 1968, 1969; Page et al., 1969). Plasma levels of angiotensin I1 provide a reasonable reflection of renin activity (Gocke et al., 1968; Page et aZ., 1969), but there are a number of methodological problems inherent in the measurement of angiotensin I1 in plasma. For example, sample-tosample variations in the conversion of angiotensin I to I1 by a plasma angiotensin-converting enzyme may be a source of error. In addition, degradation products of angiotensin 11, notably a hexapeptide fragment, may cross-react with the antibody (Catt and Coghlan, 1967; Catt et al., 196713; Cain et al., 1969) and a proteolytic enzyme of plasma may degrade radio-iodinated angiotensin I1 during the assay procedure ( Pagc et al., 1971). Similar radioimmunoassay methods for angiotensin I have been more

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recently describcd (Haber et al., 1969; Hollenians et al., 1969; Giese et al., 1970; Stockigt et al., 1971; Cohen et al., 1971; Menard and Catt, 1972; Sealey et al., 1972). The assay of angiotensin I also presents certain technical problems; for example, another plasma protein with a molecular weight of 118,000 may interfere with the immunoassay of angiotensin I by an unknown mechanism (Page et al., 1971). Nevertheless, since generation of angiotensin I from renin substrate in plasma is a reflection of renin activity, the angiotensin I immunoassay, performed at timed intervals on a patient’s plasma, provides a more direct assay of renin activity than can be obtained with the measurement of circulating angiotensin 11. Plasma renin activity, as measured by the generation of angiotensin I under standard conditions, correlates very well with plasma renin activity as determined by laborious bioassay techniques ( Haber, 1969; Haber et al., 1969; Giese et al., 1970; Stockigt et al., 1971; Cohen et al., 1971; Sealey et al., 1972; Menard and Catt, 1972). For example, plasma angiotensin I release, like renin activity, is low in patients with aldosterone-secreting adenomas ( Stockigt et al., 1971), whereas plasma angiotensin I release, like renin activity, is increased in sodium-restricted subjects (Haber et al., 1969) and patients with hyperplastic hyperaldosteronism (Stockigt et al., 1971). Differential renal vein renin measurements determined by radioimmunoassay of angiotensin I have been useful in the diagnosis of surgically remediable hypertension due to unilateral renal artery disease ( Stockigt et al., 1972). The development of methods to measure plasma renin activity by radioimmunoassay has facilitated the performance of renin determinations in large numbers of patients with primary or essential hypertension and has made it practical to divide such patients into groups with high, low, or normal plasma renin activity (Laragh et al., 1972). This division may have considerable clinical and therapeutic implications. For example, hypertensive patients with low renin activity appear to be less susceptible to cardiac and cerebral vascular complications than are hypertensive patients with normal or high renin (Brunner et al., 197213). In another study, propranolol, a 8-adrenergic blocking agent that suppresses renin secretion, has been found to be a more effective antihypertensive drug in high renin than in low renin patients (Buhler et al., 1972). Angiotensin radioimmunoassay techniques have recently been used to demonstrate that plasma concentrations of angiotensin I, but not angiotensin 11, are significantly higher in the renal vein than in the renal artery, thus supporting the concept that angiotensin I is formed in the renal vasculature (Itskovitz and Odya, 1971). Antibodies to angiotensin I1 are capable of neutralizing the pressor effect of the peptide (Boyd and Peart, 1968; Hedwall, 1968; Oken and

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Biber, 1968; Christlieb et al., 1969; Goodfriend et al., 1970; Eide and Aars, 1969, 1970; Bing and Poulsen, 1970; Macdonald et al., 1970; Brunner et al., 1972a). It has been found that the amount of antibody required to block the pressor response of rats to exogenous angiotensin I1 is enhanced in renal (twofold), deoxycorticosterone,or genetic (fourfold) hypertension in comparison with normal animals. The suggestion has, therefore, been made on the basis of these immunological observations that changes in the affinity of aiigiotensin receptors may be critically involved in hypertensive disease, even when circulating aiigiotensin I1 levels are normal (Brunner et al., 1972a). Despite its ability to block the pressor effect of exogenous angiotensin, immunization of rabbits against angiotensin I1 does not prevent the development of renal hypertension nor does it lower preexisting hypertension, thus strongly suggesting that angiotensin is not the sole or even the major factor in either the initiation or maintenance of renal hypertension (Hedwall, 1968; Eide and Aars, 1969, 1970; Louis et al., 1970; Macdonald et al., 1970). Recently, it has been reported that rats passively immunized against angiotensin I1 were significantly protected from acute renal failure induced by glycerol, and this finding has been used in support of the hypothesis that angiotensin is involved in the pathogenesis of acute ischemic renal failure ( Powell-Jackson et al., 1972).

3. Bradykinin The nonapeptide hormone, bradykinin, has been conjugated to protein or synthetic polypetide carriers by the carbodiimide ( Goodfriend et al., 1964) or toluene diisocyanate (Spragg et al., 1966; Talamo et al., 1968) methods. Animals immunized with bradykinin-containing conjugates have formed antibodies to the nonapeptide. These antibodies have been detected by complement fixation inhibition (Goodfriend et al., 1964) and by the binding of radiolabeled bradykinin derivatives (Spragg et al., 1966; Rinderknecht et al., 1967; Goodfriend and Ball, 1969). Since bradykinin does not contain a tyrosine residue, direct radio-iodination is not possible, and radioimmunoassay methods have employed tritiumlabeled acetyl derivatives of bradykinin (Spragg et al., 1966, 1967; Rinderknecht et aZ., 1967), synthetic bradykinin containing proline-l'c ( Spragg et al., 1968), or, more recently, radio-iodinated desaminotyrosine (Goodfriend and Ball, 1969) or tyrosine derivatives (Talanio et al., 1969; Mashford and Robeits, 1971) . Radioimmunoassay methods employing radio-iodinated tyrosine-bradykinin derivatives have been utilized in the development of radioimmunoassay methods for the n~easuren~ent of bradykinin in human plasma, synovial fluid (Talamo et al., 1969), and

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urine ( Mashford and Roberts, 1971) and have been used in a study of the kinetics of the release of bradykinin caused by kallikrein, when this latter enzyme is added to or activated in normal human plasma (Colman et al., 1972). Antibradykinin antibodies may potentiate the contraction of rat uterus caused by the nonapeptide hormone, perhaps by protecting it from enzymatic degradation (Goodfriend et al., 1970). In contrast, guinea pigs immunized with a bradykinin-protein conjugate did not exhibit the increase in pulmonary air-flow resistance normally produced by the intrapleural administration of bradykinin ( Davis and Goodfriend, 1969).

4. Calcitonin Calcitonin (formerly called thyrocalcitonin ) is a hypocalcemic, hypophosphatemic polypeptide hormone, with a molecular weight of approximately 3600, which is synthesized in the thyroid gland. A knowledge of serum and urinary calcitonin levels is particularly useful in the search for asymptomatic patients with medullary carcinoma of the thyroid gland, a disease which is often familial and which is ordinarily accompanied by excessive secretion of calcitonin (Clark et al., 1969; Tashjian et al., 1970; Melvin et al., 1971; Baylin et al., 1972). Most radioimmunoassays for this polypeptide have employed antibodies elicited in response to unconjugated hormone ( Arnaud et al., 1968; Deftos et al., 1968; Tashjian et al., 1970; Deftos, 1971). However, antibodies to human calcitonin have been elicited in rabbits immunized with the hormone adsorbed to finely divided carbon (Clark et al., 1969) and antibodies to porcine calcitonin have been elicited in animals immunized with calcitonin-protein or calcitonin-polypeptide conjugates prepared by the carbodiimide method (Hargis et al., 1966; Tashjian, 1969). Such antibodies have been used to localize calcitonin in the thyroid gland by the immunofluorescent technique ( Hargis et al., 1966). 5. Gastrin A peptide hormone, 17 amino acids in length, derived from the cells of the pyloric antrum, gastrin is a potent stimulator of the secretion of acid by the stomach. Although antibodies satisfactory for use in radioimmunoassays can be elicited in experimental animals with unconjugated gastrin (Schneider et al., 1967, Ode11 et al., 1968; Yalow and Berson, 1970; Ganguli and Hunter, 1972), most immunochemical studies have employed conjugated gastrin. This hormone and several amino acid sequences contained in this hormone, ranging from 4 to 15 amino acids in length, have been synthesized and conjugated to protein carriers by the toluene diisocyanate ( McGuigan, 1967), dichloromethoxytriazine

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( McGuigan, 1970c), and carbodiimide methods ( McGuigan, 1967, 1968a,b; Young et al., 1969b; Hansky and Cain, 1969; Jaffe et al., 1970a,b; McGuigan and Thomas, 1972). Animals immunized with these conjugates have formed antibodies that react with native or synthetic gastrin. Antibodies have also been elicited with gastrin complexed to polyacrylate latex particles ( Stremple et al., 1967; Stremple and Meade, 1968). Antibodies to the carboxyl-terminal tetrapeptide, the sequence which is responsible for the physiological activity of gastrin ( McGuigan, 1967), are capable of inhibiting the gastric acid secretory effects of exogenous (Jaffe et al., 1969) and endogenous (Jaffe et al., 1970a) gastrin. However, since pancreozymin-cholecystokinin, another peptide hormone, has the identical carboxyl-terminal amino acid sequence ( McGuigan, 19704, antitetrapeptide antibodies cross-react with this peptide hormone, thus precluding the use of antitetrapeptide antibodies in a specific immunoassay for gastrin ( McGuigan, 1968b, 1969, 1970d). In contrast, antibodies to the amino terminal residues 1-13 of synthetic human gastrin do not . cross-react with cholecystokinin-pancreozymin ( McGuigan, 1 9 7 0 ~ )Antibodies to conjugates containing intact or virtually intact gastrin molecules have been used together with radio-iodinated gastrin in the development of sensitive and specific radioimmunoassay methods capable of detecting as little as 1 pg. of gastrin in tissues or biological fluids ( McGuigan and Trudeau, 1970a; McGuigan, 1970b,d). Gastrin radioimmunoassay methods have been extremely useful in the clinical diagnosis of the Zollinger-Ellison syndrome ( severe upper gastrointestinal ulcer disease associated with non-/3-islet cell pancreatic tumors that secrete gastrin); in this disorder, fasting serum concentrations of immunoreactive gastrin have ranged from 600 to 300,000 pg./ml. ( McGuigan and Trudeau, 1968; Stremple and Meade, 1968; Trudeau and McGuigan, 1969; Friesen et al., 1970; McGuigan, 1970d) in contrast to a mean level of 165 ( k 2 8 S.E.) pg./ml. in control subjects (Trudeau and McGuigan, 1970). Serial gastrin determinations by radioimmunoassay have been shown to have prognostic value in predicting the response to surgical treatment in patients with the Zollinger-Ellison syndrome (Friesen et al., 1970). Trudeau and McGuigan ( 1969) have used the immunoassay procedure to demonstrate a stimulatory effect of hypercalcemia on gastrin secretion in a patient with the Zollinger-Ellison syndrome. In most patients with pernicious anemia and in many patients with chronic gastritis and achlorhydria, serum gastrin levels are also elevated (McGuigan and Trudeau, 1970b; Ganguli et al., 1971; Hansky et al., 1971a; Korman et al., 1971a). This finding is in keeping with the observation of Trudeau and McGuigan (1971) that there is, in general, an inverse relation between fasting serum gastrin concentrations and

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rates of basal or stiniulated gastric hydrochloric acid secretion in man. Although elevated gastiin levels have been encountered in some patients with gastric (Trudeau and McGuigan, 1971; Korman et al., 1972d) or duodenal (Byrnes et al., 1970; Reeder et al., 1970) ulcer, hypergastrineniia has clearly been shown by immunoassay methods not to be a prominent feature of peptic ulcer disease. The role, if any, of gastrin in this disease process has not yet been elucidated (Trudeau and McGuigan, 1970; McGuigan, 1970d; Berson and Yalow, 1971b); in fact, Korman et a2. (1971~) have found lower fasting serum gastrin concentrations in duodenal ulcer patients than in normal subjects and have noted (1972a) rises in fasting serum gastrin levels following surgical procedures for duodenal ulcers ( 1972a,c). Serum immunoreactive gastrin levels have been shown to be elevated in many patients with renal insufficiency-a finding that suggests that the kidney plays an important role in the degradation or excretion of gastrin ( Korman et al., 1972b). Gastrin radioimmunoassay methods have been used to study the physiology and pharmacology of gastrin release under a variety of clinical and experimental conditions (Berson and Yalow, 1972). The serum gastrin radioimmunoassay method has been used in dogs to study the release of endogenous gastrin into the portal circulation ( McGuigan et al., 1970), to study the metabolic clearance of gastrin from canine serum, and to correlate serum gastrin concentrations with gastric acid secretion ( McGuigan et al., 1971) . Gastrin radioimmunoassay methods have been employed to demonstrate the inhibitory effect of secretin on gastrin secretion (Hansky et al., 1971c) and to assess the stimulatory effect of vagal nerve stimulation (Jaffe et al., 1970c), of various foodstuffs (McGuigan and Tiudeau, 1970a; Korman et al., 1971b,c), of insulin-induced hypoglycemia ( Hansky et al., 1971b), of hypercalcemia ( Reeder et al., 1970), and of catecholamines (Hayes et al., 1972) on gastrin secretion. Serum gastrin measurements by radioimmunoassay have failed to show a significant effect, inhibitory or otherwise, of atropine on gastrin secretion (Korman et al., 1971c; Walsh et al., 1971). In other studies with antibodies to gastrin-protein conjugates, fluorescein-labeled and peroxidase-labeled antigastrin antibodies have been used to identify specific gastrin-containing epithelial cells of the antral mucosa of the hog and of man (McGuigan, 1968c; Pearse and Bussolati, 1970; Bussolati and Pearse, 1970; McGuigan and Greider, 1971; McGuigan et al., 1972) . Using fluorescein-labeled antigastrin antibodies, gastrin-containing cells have been demonstrated in delta cells of human pancreatic islets ( Grieder and McGuigan, 1971). Recently, Lipshutz et al. (1972) have presented evidence that gastrin antiserum: ( a) specifically antagonized the response of lower esophageal

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sphincter circular muscle to gastrin in uitro; ( b ) diminished the response of this sphincter to endogenous release and to exogenous administration of gastrin; and ( c ) markedly reduced the resting level of lower esophageal sphincter pressure in experimental animals. On the basis of these biological studies with antihapten antibodies, the authors have concluded that endogenous gastrin is the major determinant of resting lower esophageal sphincter pressure.

6. Glucagon A small polypeptide hormone, glucagon is secreted by the pancreas; it contains 29 amino acids and has a molecular weight of 3485. Unconjugated glucagon is weakly antigenic (Unger et al., 196l), but more satisfactory antisera can be obtained by immunization of rabbits with glucagon-protein conjugates prepared by the carbodiimide ( Assan et al., 1965; Young and Kraegen, 1968; Senyk et al., 1972), glutaraldehyde (Frohman et al., 1970), or bisdiazotized benzidine (Senyk et al., 1972) methods. Antisera to glucagon elicited by glucagon-protein conjugates can detect as little as 0.05 ng. glucagon by a double-antibody radioimmunoassay method, employing radio-iodinated glucagon. Antiglucagon antisera prepared in this manner also abolish rises in serum insulin and glucose induced by glucagon in the rat, as well as blocking the increase in hepatic phosphorylase activity and glucose output produced by glucagon during perfusion of isolated rat livers (Frohman et al., 1970). Antiserum to porcine pancreatic glucagon, obtained using the carbodiimideglucagon polymer technique of Hedding (1969), has been employed in the localization, by an immunofluorescent technique, of cells containing glucagon-like immunoreactivity in the gastric fundus and jejunum of the dog (Polak et al., 1971a).

7. a-Melunocyte-Stimulating Hormone The anterior pituitary, a-melanocyte-stimulating hormone (a-MSH ), a polypeptide with a molecular weight of 1665, has been conjugated to serum albumin by the carbodiimide method. Rabbits immunized with a-MSH-albumin conjugates formed antibodies that reacted with a-MSH but not with P-MSH or with ACTH, as determined by complement fixation ( McGuire et al.,1965). 8. Oxytocin

The nonapeptide posterior pituitary hormone, oxytocin, is weakly antigenic (Gilliland and Prout, 1965), but more satisfactory antibodies have been obtained by immunization with oxytocin adsorbed to carbon particles (Chard et al., 1970) or by immunization with a polymer of

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oxytocin, produced by treatment with a carbodiimide reagent ( Gusdon, 1967). Antioxytocin antibodies are capable of inhibiting oxytocin-induced contraction of postpartum rat mammary gland strips (Gusdon, 1967) and are also capable of inhibiting the oxytocin-induced ejection of milk from the rat mammary gland (Vorherr and Munsick, 1970). Since the other posterior pituitary hormone, vasopressin, also possesses milkejection activity, oxytocin activity in posterior pituitary extracts can be measured as that amount of milk-ejection activity which is specifically inhibited by antioxytocin and not by antivasopressin serum (Vorherr and Munsick, 1970). Recently, a radioimmunoassay for oxytocin has been developed (Chard et al., 1970) and has been used to determine the excretion rate for exogenous and endogenous oxytocin in human urine ( N . R. H. Boyd et al., 1972); this method has also been applied to the study, in the goat, of oxytocin release during parturition ( McNeilly et al., 1972b) and in response to other physiological stimuli ( McNeilly et al., 1972a). Fluorescein-labeled antioxytocin antibody has been utilized to localize oxytocin in human uterine muscle ( Gusdon, 1967).

9. Pancreozymin-Cholecystokinin Pancreozymin and cholecystokinin are actually a single peptide hormone ( PZ-CCK ) synthesized by duodenal endocrine cells. This hormone, which stimulates pancreatic and gall bladder secretion, has been conjugated to rabbit serum albumin by the carbodiimide method. Rabbits immunized with the PZ-CCK-albumin conjugate formed antibodies that reacted with radio-iodinated PZ-CCK. Since these antibodies did not crossreact with gastrin ( which contains the same carboxyl-terminal tetrapeptide), they could be used in the development of a radioimmunoassay procedure for the determination of PZ-CCK concentrations in human serum (Young et al., 1969a). With this radioimmunoassay procedure, it has been demonstrated that PZ-CCK is released into the circulation after fatty meals, protein meals, and large oral glucose loads (Young et al., 1968b, 1969a). 10. Secretin A peptide hormone, secretin is made up of 27 amino acids. Its principal action is stimulation of alkaline secretion by the pancreas. Rabbits immunized with secretin-albumin conjugates prepared by the carbodiimide method form antibodies capable of binding radio-iodinated secretin. Antibodies to porcine secretin prepared in this manner have been utilized in the development of a radioimmunoassay €or secretin in human serum (Young et al., 1968a). This procedure has led to a better understanding of the role of secretin in normal gastrointestinal physiology

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and carbohydrate metabolism ( Berson and Yalow, 1972). For example, the radioimmunoassay method has been used to demonstrate that secretin release can be caused by oral (but not intravenous) glucose, by intravenous gastrin, or by direct infusion of hydrochloric acid, or glucose into the duodenum (Young et al., 1968a; Chisholm et al., 1969, 1971). By using the radioimmunoassay procedure, evidence has been obtained that secretin released by these mechanisms is responsible for the augmentation of insulin release observed after an oral glucose load, when the release of insulin is both earlier and greater than would be expected from the oral glucose alone (Chisholm et al., 1969, 1971; Kraegen et al., 1970). Antisecretin antibodies have also facilitated the identification, by an immunofluorescent technique, of this peptide hormone in small granular S cells in the transitional zone of the canine duodenal mucosa (Polak et al., 1971b).

11. Thyrotropin-Releasing Hormone Thyrotropin-releasing hormone ( TRH ) is a recently recognized tripeptide hormone which is secreted in the hypothalamus and causes release of thyrotropin ( thyroid-stimulating hormone or TSH ) from the anterior pituitary gland (Boler et al., 1969; Burgus et al., 1969). Syn) has been conjuthetic TRH ( L-pyroglutamyl-L-histidyl-L-prolineamide gated, presumably via the imidazole group of its histidine residue, to bovine serum albumin with the use of the bifunctional diazonium compound, bisdiazotized benzidine ( BDB) . Rabbits immunized with TRHalbumin conjugate formed antibodies capable of specific binding of radioiodinated TRH, enabling the development of a sensitive and highly specific double-antibody radioimmunoassay method capable of detecting subnanogram quantities of TRH. Preliminaiy attempts to utilize this method in detecting the native tripeptide hormone in human serum have not revealed detectable TRH ( Bassiri and Utiger, 1972),

12. Vasopressin Arginine vasopressin ( AVP ), the nonapeptide antidiuretic hormone of the posterior pituitary gland of man (and most other mammals) is, like oxytocin, weakly antigenic in man (Roth et al., 1966) and in experimental animals (Robertson et al., 1970; Edwards et al., 1972)- Several laboratories have reported the production of antivasopressin antibodies following immunization of experimental animals with vasopressinmacromolecule conjugates. In these studies, AVP or lysine vasopressin (LVP; found in most members of Suina) have been conjugated to protein or synthetic polypeptide carriers by the carbodiimide (Perniutt et al.,

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1966; Miller and Moses, 1969a; Wu and Rockey, 1969a,b; Vallotton, 1971), glutaraldehyde (Johnston, 1972), or toluene diisocyanate (Permutt et al., 1966; Edwards et al., 1972) method. Antivasopressin antibodies inactivate vasopressin ( Miller and Moses, 1969b; Vorherr and Munsick, 1970), and their ability to bind radio-iodinated vasopressin has enabled the recent development of radioimmunoassays for vasopressin in pituitary extracts (Miller and Moses, 1969a), urine (Miller and Moses, 1971, 1972; Oyama et al., 1971), and extracts of plasma (Robertson et al., 1970; Beardwell, 1971; Skowsky and Fisher, 1972; Johnston, 1972).

B. HORMONES : NONPEPTIDE 1. Cyclic Adenosine-3’,S-monophosphate and Cyclic

Guanosine-3‘,S-monophosphate Adenosine-3‘,5’-cyclic phosphate ( cyclic AMP) acts as the mediator of the cellular effects of many biologically active substances in a variety of tissues. Cyclic AMP has been succinylated and the succinyl derivative has been conjugated to protein carriers. After synthesizing a radioiodinated tyrosine methyl ester derivative of succinyl cyclic AMP, antibodies to cyclic AMP could be demonstrated by their reaction with the radio-iodinated cyclic AMP derivative, as detected by the double-antibody method (Steiner et al., 1969, 1972a). The fact that cyclic AMP specifically inhibits the binding of the radio-iodinated cyclic AMP derivative has enabled the development of a sensitive, specific, and precise radioimmunoassay method for the measurement of this cyclic nucleotide in tissues and biological fluids (Ferrendelli et al., 1970, 1972; Steiner et al., 1970, 1972b,c; Jarett et al., 1972; Wehmann et al., 1972). Another radioimmunoassay method, employing cyclic AMP-3H and Millipore membrane filtration, has recently been described ( Weinryb et al., 1972). By using antibodies to guanosine-3’,5’-monophosphate ( cyclic GMP ) and a radio-iodinated tyrosine methyl ester derivative of cyclic GMP, a sensitive, specific, and precise radioimmunoassay for cyclic GMP in tissues and body fluids has also been reported (Ferrendelli et al., 1970, 1972; Steiner et al., 1970, 1972a,b; Jarett et al., 1972). With the ammonium sulfate precipitation method and an lT-tyrosinated derivative of cyclic GMP together with the ”‘I-tyrosinated cyclic AMP derivative, cyclic GMP and cyclic AMP can be simultaneously measured in plasma, urine, or tissue ( Wehmann et al., 1972). Using the immunofluorescent technique, antibodies to cyclic AMP have been used to localize this cyclic nucleotide in cerebellar neurons (Bloom et al., 1972). Recently, specific antibodies and radioimmunoassay methods for cyclic inosine-3‘,5‘-monophosphate and for cyclic uridine-3’,5’-monophosphate

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have been described, employing methods similar to those utilized with cyclic AMP and cyclic GMP (Steiner et al., 1970,1972a).

2. Histamine Histamine-protein conjugates were initially synthesized by condensing histamine with p-nitrobenzoyl chloride, reducing the nitro group to an amino acid group with ferrous sulfate and ammonia, then diazotizing and coupling to protein carriers (Fell et al., 1943). Rodney and Fell ( 1943) also coupled an isocyanate derivative of histamine to protein by a carbamido linkage. Rabbits immunized with either of these histamineprotein conjugates formed histamine-specific antibodies, and guinea pigs immunized with these conjugates displayed a marked resistance to the symptoms of anaphylaxis (Fell et al.,1943; Rodney and Fell, 1943). Humans immunized with histamine azoprotein form antibodies that are capable of neutralizing histamine in vitro and in vivo (Cohen and Friedman, 1943 ) , An early report expressed optimism that immunization with histamine azoprotein might be beneficial in patients with allergic disorders (Sheldon et al., 1941), but definitive evidence for the efficacy of such immunization has never been published. More recently, histamine has been conjugated to protein carriers by the carbodiimide method. Guinea pigs and rabbits immunized with histamine-protein conjugates form antibodies that react with histamine (Davis and Meade, 1970; Burtin-Laborde, 1970). Guinea pigs immunized with histamine-protein conjugates are significantly protected against the undesirable effects of a toxic dose of histamine (Davis and Meade, 1970); passive immunization with antihistamine antibodies also protects against the action of histamine in experimental animals (Went and Kesztyus, 1951 ) .

3. Prostaglandins The prostaglandins are 20-carbon aliphatic carboxylic (fatty) acids with a cyclopentane ring. Although they are widely distributed in nature and have diverse physiological effects, no simple sensitive method for their assay exists. Accordingly, various prostaglandins (including PGA1, PGA?, PGE1, and PGF,,) have been conjugated to polylysine or protein carriers by the carbodiimide or mixed anhydride methods. Rabbits inimunized with prostaglandin-polylysine ( complexed with succinylated hernocyanin ) or prostaglandin-protein conjugates have formed antibodies to prostaglandins. These antibodies can be detected by their capacity to bind 3H-prostaglandins or radio-iodinated conjugates of prostaglandins with a synthetic polypeptide ( containing lysine, glutamic acid, alanine, and tyrosine), as demonstrated by the ammonium sulfate precipitation or

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double-antibody method. Unfortunately, antibodies to a specific prostaglandin hapten usually cross-react with heterologous prostaglandins, thus hampering the development of radioimmunoassays that are highly specific for individual prostaglandins ( Levine and Van Vunakis, 1970; Caldwell et al., 1971; Jaffe et al., 1971; Levine et al., 1971a; Jubiz et al., 1972; Kirton et al., 1972). However, antibodies specific for prostaglandin Bl havc been used to measure serum prostaglandin A, isomerase that converts PGA, to PGB, (Polat and Levine, 1971), and recently methods for the radioinimunoassay of PGF,, in the peripheral serum of man (Caldwell et al., 1971; Cernosek et al., 1972) have been described.

4. Serotonin Serotonin( 5-hydroxytiyptamine ) is found mainly in the mucosal layer of the gastrointestinal tract and, to a lesser extent, in brain tissue. Its occurrence in other tissues varies markedly from species to species. Serotonin has been implicated as a significant mediator of anaphylaxis in the mouse and rat, but its role in central nervous system function and in gastrointestinal physiology is still not clear. Ranadive and Sehon ( 1 9 6 7 ~ )coupled serotonin to bovine serum albumin by the Mannich formaldehyde reaction. The resulting conjugate elicited antibodies specific for serotonin which were capable of inhibiting cutaneous reactions evoked in mice by intradermal injections of serotonin. 5-Hydroxyindole-3-acetic acid (HIAA) did not combine with the antiserotonin antibodies elicited in this manner, nor did HIAA coupled to proteins by the Mannich reaction produce antibodies that combined with serotonin. The same investigators ( Ranadive and Sehon, 1967a,b) found, however, that HIAA could be coupled to proteins with carbodiimides to yield a conjugate that elicits antibodies that combine with serotonin and are capable of inhibiting the effect of exogenous serotonin on capillary permeability in mice, and which also partially inhibit passive cutaneous anaphylactic reactions in mice. The data reported by Ranadive and Sehon, in addition to their significance in demonstrating antibody formation against a small molecule of biological importance, provide another example of the importance of the coupling procedure. The HIAA, when coupled to proteins through position 1 on the indole ring by the Mannich reaction, does not elicit antibodies that cross-react with serotonin nor do the antibodies inhibit the biological activity of serotonin. On the other hand, the carbodiimide reaction yields a HIAA-protein conjugate that elicits antibodies capable of combining with and inhibiting the effects of serotonin. Filipp and Schneider (1964) also reported the production of anti-

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bodies specific for serotonin capable of inhibiting the activity of serotonin in rats. These investigators coupled 5-hydroxytryptamine-creatinine sulfate to protein by a complex series of chemical reactions to prepare the conjugated antigen.

5. Steroid Hormones By using carboxyl-containing derivatives, either the hemisuccinate or the O-carboxymethyl oxime, steroid hormones were first conjugated to protein carriers by the mixed anhydride technique. Steroid hormones coupled in this manner included cortisone (Erlanger et al., 1957), testosterone (Erlanger et al., 1957, 1967; Nieschlag and Loriaux, 1972), deoxycorticosterone, progesterone, estrone ( Erlanger et al., 1959, 1967), pregnenolone (Erlanger et al., 1967), 2-hydroxyestrone (Yoshizawa and Fishman, 1971), cortisol (Neri et al., 1964), and aldosterone (Neri et al., 1964; Mayes et al., 1970; Ito et al., 1972; Farmer et al., 1972); more recently, the carbodiimide method has been utilized to link carboxylcontaining derivatives of testosterone ( Dufau et al., 1972), progesterone (Abraham et al., 1971a; Lindner et al., 1972), aldosterone (Bayard et al., 1970a), estriol (Tulchinsky and Abraham, 1971; Lindner et al., 1972), and estradiol-17p ( Lindner et al., 1972) to protein carriers. Goodfriend and Sehon (1958) prepared a 17-amino derivative of estrone and converted it by phosgenation to estrone-17-isocyanate, which was readily coupled directly to albumin under mildly alkaline conditions. Gross et al. ( 1968, 1971) have coupled diazotized p-aminobenzoic acid to estrogens through the phenolic A ring of the steroid hormones; these derivatives have then been conjugated to protein carriers with a carbodiimide reagent. Africa and Haber (1971) prepared a hydrazone of aldosterone to couple it via its A ring to a carrier protein. With steroid-protein conjugates, it has been possible to elicit the formation of antibodies to cortisone, deoxycorticosterone ( Beiser et al., 1959; Lieberman et al., 1959), testosterone (Beiser et al., 1959; Lieberman et al., 1959; Vaitukaitis et al., 1971; Nieschlag and Loriaux, 1972; Dufau et al., 1972), progesterone (Beiser et al., 1959; Lieberman et al., 1959; Abraham et al., 1971a; Lindner et al., 1972), estrone (Beiser et al., 1959; Lieberman et al., 1959; Goodfriend and Sehon, 1960, 1961a), 2-hydroxyestrone ( Yoshizawa and Fishman, 1971) , estradiol-17p (Gross et al., 1968; Jeffcoate and Searle, 1972; Lindner et al., 1972), estriol (Tulchinsky and Abraham, 1971; Gross et al., 1971; Lindner et al., 1972), and aldosterone (Mayes et al., 1970; Bayard et al., 1970a; Africa and Haber, 1971;Farmer et al., 1972; It0 et al., 1972). Immunochemical studies have revealed widespread cross-reactions among these steroids, particularly among those with similar A rings

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coupled to the carrier through the D ring (Beiser et al., 1959; Lieberman et al., 1959; Goodfriend and Sehon, 1961a; Niswender and Midgley, 1970; Gross, 1970; Midgley et al., 1971). The extent of cross-reactivity can be significantly diminished if one immunizes with conjugates coupled through the A ring, as has been demonstrated with testosterone (Lieberman et al., 1959; Niswender and Midgley, 1970), progesterone (Niswender and Midgley, 1970), estradiol-17p ( Gross et al., 1968), estriol (Gross et al., 1971), and aldosterone (Africa and Haber, 1971). Antibodies with different specificities have been obtained by immunization with conjugates linked via the B or C rings, as has been done with estradiol-17p (Jeffcoate and Searle, 1972; Lindner et al., 1972), estriol, and progesterone (Lindner et al., 1972). By using tritiated steroids, radio-iodinated steroid-protein conjugates, or radio-iodinated tyrosine derivatives of steroid hormones ( Midgley et al., 1969, 1971; Midgley and Niswender, 1970), a number of radioimmunoassay procedures for the determination of steroid hormones have been developed, and a recent text has been devoted to this subject (Peron and Caldwell, 1970). Because of problems connected with the cross-reactivity of antibodies and with the presence of steroid-binding proteins in human plasma, most of these assay procedures have been carried out with plasma extracts, prepared by a variety of techniques ( Murphy, 1970). Such radioimmunoassay procedures have been reported for the measurement of estradiol-17p (Abraham, 1969; Jiang and Ryan, 1969; Mikhail et al., 1970a,b,c; Abraham et al., 1970; Abraham and Odell, 1970; Hotchkiss et al., 1971; Emment et al., 1972), estriol (Tulchinsky and Abraham, 1971; Gurpide et al., 1971), estrone (Mikhail et al., 1970a,b,c; Emment et al., 1972), 2-hydroxyestrone (Yoshizawa and Fishman, 1971), progesterone (Abraham et al., 1971a; Furuyama and Nugent, 1971), 17-hydroxyprogesterone (Abraham et al., 1971b), testosterone (Furuyama et al., 1970; Ismail et al., 1972; Nieschlag and Loriaux, 1972; Dufau et al., 1972), deoxycorticosterone (Arnold and James, 1971), and aldosterone (Mayes et al., 1970; Bayard et al., 1970a,b; Wahlen et al., 1970; Farmer et al., 1972; Ito et al., 1972; Katz and Romfh, 1972); the simultaneous radioimmunoassay of progesterone, 17-hydroxyprogesterone, and estradiol-17p has also been described (Abraham et al., 1 9 7 1 ~). These radioimmunoassay methods have been employed in a number of physiological and clinical studies. For example, variations in circulating levels of estradiol-17p (Shaikh and Abraham, 1969; Abraham and Klaiber, 1970; Hotchkiss et al., 1971; Abraham et al., 1972; Henricks et al., 1972; Johnson et al., 1972), estriol (Tulchinsky and Abraham, 1971; Gurpide et al., 1971), progesterone (Abraham et al., 1971a, 1972), 17-hydroxyprogesterone (Abraham et al., 1971b, 1972), testosterone ( Ismail et al.,

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1972), and aldosterone (Beitins et al., 1972; Katz and Romfh, 1972) have been studied by radioimmunoassay in humans and in experimental animals in various phases of the menstrual cycle, during and after pregnancy, and after the menopause. Radioimmunoassay methods have also been used to measure testosterone production in response to gonadotropins in the rat testis in uitro (Dufau et al., 1972) and to detect estradiol-17p and estrone in the adrenal effluent plasma of ovariectomized monkeys ( Resko, 1971). Estradiol-17p secretion by human, simian, and canine testes has been determined by radioimmunoassay of peripheral and spermatic vein plasma samples. With this approach, elevated spermatic vein estradiol concentrations ( postgonadotropin ) were demonstrated in 2 patients with the incomplete form of the feminizing testes syndrome; this observation, coupled with a precipitous fall in the peripheral plasma estradiol concentration after orchiectomy, provided direct evidence that the human testis secretes estradiol (Kelch et al., 1972). Recently, radioimmunoassay for aldosterone in peripheral and adrenal vein plasma has been found to be highly useful in the diagnosis and localization of adrenal adenomas associated with primary aldosteronism ( Horton and Finck, 1972; Melby, 1972). Antibodies to steroid hormones are capable of inhibiting the biological effects of exogenous and endogenous steroids in passively immunized rats and mice. For example, antibodies to testosterone, but not antiestrone antibodies, inhibit the androgenic effect of testosterone on seminal vesicle weights of castrated rats. Similarly, anticortisol antibodies inhibit the eosinopenic response to cortisol in adrenalectomized mice, and antibodies to aldosterone inhibit the effect of this mineralocorticoid hormone on the urinary excretion of sodium and potassium in adrenalectomized rats (Lieberman et al., 1959; Neri et al., 1964). Antibodies to estrone inhibit the uterotropic effect of estrone in immature mice and rats (Lieberman et al., 1959; Goodfriend and Sehon, 1961b; Neri et al., 1964). Antibodies to estradiol-17p specifically inhibit tissue uptake of estradiol17/3-3H and prevent uterine weight increases, endometrial stimulation, and vaginal cornification due to exogenous estradiol or to endogenous ( gonadotropin-stimulated) estrogens in immature or ovariectomized mice (Ferin et al., 1968). In immunological studies of the female reproductive cycle, antiestradiol-17p antibodies have been shown to inhibit the release of luteinizing hormone ( L H ) in ovariectomized ewes given progesterone and estradiol-17p in a manner analogous to their normal endogenous release during the normal estrous cycle of an intact ewe (Caldwell et al., 1970); these antibodies also inhibit ovulation induced by endogenous LH in immature rats given pregnant mare serum, thus suggesting a positive feedback role of estrogens in LH release (Ferin

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et al., 1969a). Antibodies to progesterone do not inhibit ovulation, but do block the uterine effects of progesterone (Ferin et at., 1969b). More recently, the specific inhibitory effects of these antibodies have been utilized to define the temporal aspects of the physiological requirements for estradiol-17p and progesterone on nidation and pregnancy in rats ( Raziano et al., 1972). Fluorescein-labeled antibodies to steroid hormones have been utilized to localize estrone in the mature rat ovary (Goodfriend et al., 1961) and to localize testosterone in granules of epithelial cells of the seminal vesicles of rats (Haferkamp et at., 1968) and in the testes of rats and fowl (Woods and Domm, 1966). 6. Thyroxine and Triiodothyronine Churchill and Tapley ( 1964) have conjugated tetraiodothyropropionic acid to bovine serum albumin by the mixed anhydride method and utilized the conjugate to elicit in rabbits the formation of antibodies capable of binding thyroxine and its analogs. Although the immunogenic haptenic determinants have not been identified, antibodies capable of binding triiodothyronine and thyroxine have been elicited in animals immunized with thyroglobulin (Premachandra et al., 1963; McKenzie and Haibach, 1967; Beall and Solomon, 1968; Roitt et al., 1968; Margherita and Premachandra, 1969; Pogoriler et al., 1971; Chopra et al., 1971a,b,c). More recently, antibodies with a high degree of specificity for triiodothyronine have been elicited in rabbits immunized with synthetic antigens prepared by conjugating triiodothyronine to succinylated poly-L-lysine or to human serum albumin by the carbodiimide method (Brown et al., 1970a,b; Gharib et al., 1970). The development of radioimmunoassay methods for the measurement of triiodothyronine was hampered by the fact that triiodothyronine is bound by the thyroxine-binding globulin of serum. Initially, triiodothyronine was extracted from serum prior to assay (Brown et al., 1970b) but, more recently, a variety of compounds, including tetrachlorothyronine, sodium salicylate, diphenylhydantoin ( Dilantin), and 8-anilino-1-naphthalene sulfonic acid, have been used to inhibit the binding of triiodothyronine by thyroxine-binding globulin, thus making it possible to measure triiodothyronine by radioimmunoassay in unextracted human serum (Mitsuma et al., 1971a,b, 1972; Larsen, 1971, 1972; Gharib et al., 1971; Chopra et al., 1971c; Lieblich and Utiger, 1972). The development of precise, sensitive, and simple radioimmunoassay methods has enabled more accurate measurement of serum triiodothyronine concentrations than was previously possible with chemical methods and will probably eventually supplant these methods in routine clinical usage. Thyroxine has also been measured in extracted (Chopra

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et al., 1971b) and unextracted (Mitsunia et al., 1972) hunian serum by the radioimmunoassay method; in the latter instance, thyroxine and triiodothyronine are rapidly and simultaneously measured in an assay procedure that employs thyroxine-’”I and triiodothyronine-lZ5I ( Mitsunia et al., 1972). AND VITAMINS C. COENZYMES

1 . Folic Acid

Folic acid ( pteroylglutaniic acid) has been conjugated to protein and synthetic polypeptide carriers by the carbodiimide method. Experimental animals immunized with folic acid-containing conjugates have formed antibodies against folic acid (Ricker and Stollar, 1967; Jaton and UngarWaron, 1967; Rothenberg et al., 1969; Rubenstein and Little, 1970; Rothenberg and Gizis, 1971). These antibodies protect folic acid from enzymatic reduction, by folate reductase, to tetrahydrofolic acid ( Rothenberg et al., 1969) and have enabled the development of a coated charcoal radioimmunoassay method, employing folic a ~ i d - ~ for H , the measurement of immunoreactive folate in serum and serum extracts (da Costa and Rothenberg, 1971).

2. Pyridoxal Pyridoxal (vitamin B,) can be linked to the free amino groups of protein carriers by forming a Schiff base; the labile double bond thus obtained can be reduced with sodium borohydride, to form a stable pyridoxal-protein conjugate ( Fischer et al., 1958; Churchich, 1965). Rabbits immunized with pyridoxal-protein or pyridoxal-polypeptide conjugates form antibodies specific for pyridoxal ( Ungar-Waron and Sela, 1966; Cordoba et al., 1966, 1970). Purified antipyridoxal antibodies exert a marked inhibitory effect on glutamic-oxalacetic transaminase ( Laspartate :2-oxoglutarate aminotransferase; E.C. 2.6.1.1. ) , an enzyme in which the pyridoxal phosphate coenzyme moiety is thought to be bound in a Schiff base linkage through an c-amino group of a lysine residue ( Ungar-Waron and Sela, 1966). 3. Vitamin A Retinoic acid (vitamin A acid) has been Conjugated to protein carriers by the mixed anhydride method. Animals immunized with retinoic acid-protein conjugates form antibodies capable of binding vitamin A,3H(r e t i n ~ l - ~ Has) determined by the dextran-coated charcoal method. As little as 10 pmoles of unlabeled retinol caused significant inhibition of the binding of r e t i n ~ l - ~by H antibody, suggesting that antibodies to retinoic

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acid may prove useful in the development of a sensitive radioininiunoassay for the detection and measurenient of vitamin A (Conrad and Wirtz, 1972) .

D. DRUGS 1. Acetylialicylic Acid Chloride and azide derivatives of acetylsalicylic acid ( aspirin) have been conjugated to protein carriers. Animals immunized with aspirylprotein conjugates form antibodies that will react with acetylsalicylic acid (Weiner et al., 1963; Wiclier et nl., 1968) and which partially protect febrile rats from the antipyretic effect of aspirin (Butler et al., 1940). Aspiryl-protein conjugates have also been employed in a passive hemagglutination technique to detect antibodies in the sera of individuals who are clinically sensitive to aspirin or to p-aniinosalicylic acid ( Weiner et al., 1963; Girard et al., 1969). Aspirin anhydride, a frequent contaminant of commercial aspirin preparations, has also been shown to react readily with protein to form conjugates that elicit antiaspiryl antibodies in guinea pigs and rabbits (de Weck, 1971).

2. Barbiturates A barbiturate-protein conjugate has been prepared by coupling a barbituric acid derivative to bovine u-globulin using the carbodiimide method, Rabbits immunized with barbiturate-protein conjugates prepared in this inanner form antibodies capable of binding pentobarbital-'"C or barbital-14Cas determined by the ammonium sulfate precipitation method. These antibodies have been used in the development of a radioimmunoassay capable of detecting as little as 0.5 ng. of various barbiturates in plasma, urine, or brain honiogenates. This radioimmunoassay method has been employed in the study of the plasma half-lives of barbital and pentobarbital in the rat ( Spector and Flynn, 1971; Flynii and Spector, 1972). 3. Cardiac Glycosides a. Digitoxin. Digitoxigenin, the steroidal aglycone of digitoxin, will react with succinic anhydride to form 3-0-succinyl digitoxigenin. The presence of the carboxyl group in this succinyl derivative of the aglycone permits its conjugation to protein carriers either by the mixed anhydride method or by the carbodiimide technique. Rabbits immunized with succinyl digitoxigenin-protein conjugates form antibodies that react with the aglycone and also react quite well with digitoxin. With the radio-

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AND SAM M. BEISER

iodinated tyrosine methyl ester derivative of succinyl digitoxigenin ( also prepared by the mixed anhydride method), these antibodies have been used in the development of a sensitive and specific double-antibody radioimmunoassay method, capable of detecting as little as 1 ng./ml. of digitoxin in the serum of patients receiving this drug (Oliver et al., 1968). This assay is performed upon chloroform extracts of patients’ sera and involves an overnight incubation step; thus, it is somewhat less convenient and less rapid than more recently developed methods. It, nevertheless, represents the first clinically useful immunoassay method for cardiac glycoside concentrations, and, furthermore, the method used for the synthesis of the radio-iodinated tyrosine methyl ester derivative of the succinylated aglycone has been widely used in more recently developed assay methods for digoxin as well as for digitoxin. In studies of one potent antidigoxin serum, it has been found that, although the average intrinsic association constant (KO)for these antibodies for digitoxin is thirty-two-fold lower than the KO for digoxin, the & for digitoxin is quite high (5.3X los M - I ) , indicating a significant affinity of these antidigoxin antibodies for digitoxin (Smith et al., 1970). Smith (1970) took advantage of this high affinity in using this antidigoxin serum together with digit~xin-~H of high specific activity in the development of a dextran-coated charcoal radioimmunoassay method, requiring only 1 hour to perform and capable of detecting 2 ng. of digitoxin in 1 ml. of unextracted serum. b. Digoxin. Digoxin has been conjugated to protein carriers by the periodate oxidation method, and rabbits immunized with the resulting digoxin-protein conjugates form digoxin-specific antibodies ( Butler and Chen, 1967). Because of their specificity and high affinity for this cardiac glycoside (Smith et al., 1970), these antibodies have been used together with d i g ~ x i n - ~of H high specific activity in the development of a rapid, convenient, and specific dextran-coated charcoal immunoassay method capable of detecting 0.2 ng./ml. or less of digoxin (well below the usual therapeutic range of 0.5 to 2.0 ng./ml.) in the unextracted serum or plasma of patients receiving this drug (Smith et al., 1969). Although there is some overlap in serum digoxin concentrations between digoxin-toxic and nontoxic patients, and, although an elevated serum digoxin concentration is never specifically diagnostic of digoxin toxicity, the results of serum digoxin radioimmunoassay determinations provide evidence that there is a good correlation between the serum concentration and the clinical response to the drug. Serum digoxin concentrations in excess of 2 ng./nil. are frequently associated with toxic manifestations, whereas somewhat lower concentration ranges (0.5-2 ng./ml.) are usually encountered in adult subjects who appear to be having a good clinical response to

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digoxin. Serum digoxin levels less than 0.5 ng./ml. are frequently associated with an inadequate clinical response to the drug. A knowledge of the serum digoxin concentration is most helpful clinically in the following groups of patients: digoxin-treated subjects who, because of electrocardiographic, gastrointestinal, visual, or neurological abnormalities, are suspected of having digoxin intoxication; digoxin-treated patients who, because of renal insufficiency, old age, or changing dosage requirements, are at high risk for the development of digoxin toxicity; patients in whom the clinical history of digoxin ingestion is questionable, unreliable, or unobtainable; and, finally, in patients whose therapeutic response to an apparently adequate dosage of digoxin is unsatisfactory (Smith et al., 1969; Evered et al., 1970; Chamberlain et al., 1970; Butler, 1970; Smith and Haber, 1970; Beller et aZ., 1971; Evered and Chapman, 1971; Hoeschen and Proveda, 1971; Oliver et al., 1971; Doherty, 1971; Smith, 1971, 1972a; Butler, 1972; Calverley, 1972). The availability of the serum digoxin radioimmunoassay has facilitated studies of the clinical pharmacology of this drug, notably of its intestinal absorption (White et al., 1971; Heizer et al., 1971; Lindenbaum et al., 1972) and biological availability (Lindenbaum et al., 1971; Shaw et al., 1972; Huffman and Azarnoff, 1972). In this latter connection, significant and potentially hazardous differences in the extent of digoxin absorption from tablets prepared by different manufacturers have been demonstrated by the radioimmunoassay method (Lindenbaum et al., 1971; Shaw et al., 1972). Digoxin-specific antibodies have interesting biological properties. They are capable of removing digoxin from rat renal cortical cells and from human erythrocytes (Watson and Butler, 1972). They are capable of reversing the inhibitory effect of digoxin on the influx of potassium into erythrocytes (Watson and Butler, 1972) and of reversing the positive inotropic (Curd et al., 1971; Skelton et al., 1971) and toxic electrophysiological (Mandel et al., 1972) effects of digoxin on isolated cardiac muscle preparations. Rabbits that have been immunized with digoxin-protein conjugates and whose sera contain digoxin-specific antibodies develop no electrocardiographic abnormalities when given a dose of digoxin which is uniformly lethal in control animals (Schmidt and Butler, 1971a). It has also been demonstrated that the passive administration of antidigoxin antibodies will rapidly reverse the clinical and electrocardiographic manifestations of severe digoxin intoxication in dogs which had previously received lethal doses of this cardiac glycoside (Schmidt and Butler, 1971b). In the hope of decreasing the immunogenicity of heterologous antibody and of accelerating the excretion of digoxin in humans with potentially lethal digoxin intoxication, sheep antidigoxin antibody

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has been purified by an immunoadsorbent technique; the purified antibodies and their Fab fragments are capable of reversing digoxin toxicity in the dog (Curd et al., 1971; Smith et al., 1971). It has been recently noted that, when injected into digoxin-immunized rabbits, unconjugated digoxin persists in the circulation in complex with y-globulin for a year or more, in contrast with its 7-10-day disappearance rate in control rabbits. This observation suggests that hapten-antibody complexes, unlike protein-antibody complexes, are not deposited in the glomeruli and blood vessels of immunized animals, and raises the possibility that haptens and other small antigenic determinants may be capable of preventing or reversing immune complex deposition in experimental animals and man ( Schmidt et al., 1972). c. Ouabain. Ouabain has been conjugated to protein carriers by the periodate oxidation method ( Ciofalo and Ashe, 1971; Smith, 1972b). Rabbits immunized with ouabain-protein conjugates have formed ouabain-specific antibodies which have enabled the development of a radioimmunoassay method for the study of ouabain pharmacokinetics in experimental animals and man (Selden and Smith, 1972). Like digoxinspecific antibodies, the antibodies to ouabain are biologically active, as evidenced by the fact that they reverse the positive inotropic and toxic electrophysiological effects of ouabain (Gold and Smith, 1971) and by the report that 3 of 5 rabbits immunized with ouabain-protein conjugates suffered no toxic effects from a lethal dose of ouabain (Ciofalo and Ashe, 1971). Ouabain-specific antibodies have also been shown to be capable of reversal of ouabain-induced inhibition of canine, myocardial, microsomal, Na+-, and K+-activated adenosinetriphosphatase ( ATPase) (Smith, 1972b).

4. Chloramphmicol Following reduction of its nitro group to an amine, chloramphenicol has been converted to the diazonium derivative and coupled to protein carriers. Rabbits immunized with chloramphenicol-protein conjugates form chloramphenicol-specific antibodies capable of neutralizing the effect of the antibiotic on Escherichia coli growth and which have been used in the detection and measurement of chloramphenicol in the nuclear and ribosomal fractions of cells grown in tissue culture in the presence of the antibiotic (Hamburger, 1966; Hamburger and Douglass, 1969). Radio-iodinated chloramphenicol-protein conjugates prepared in the course of these studies have been used to detect specific antichloramphenicol antibodies in human serum by the ammonium sulfate precipitation technique ( Orgel and Hamburger, 1971).

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5. Cytotoxic Drugs a. Methotrexate. A folic acid antagonist, methotrexate has been conjugated via its carboxyl group to the free amino groups of the synthetic polypeptide, poly-DL-alanyl-poly-L-lysine,by the carbodiimide method. Rabbits immunized with methotrexate-polypeptide conjugates formed antimethotrexate antibodies ( Jaton and Ungar-Waron, 1967). b . L-Phenylalanine Mustard. An alkylating agent, L-phenylalanine mustard ( Alkeran) will react directly with the amino groups of human y-globulin at pH 6.6. Rabbits immunized with the resulting conjugates form antibodies to L-phenylalanine mustard. Malignant ependymomas in mice treated by giving the animals an injection of L-phenylalanine mustard, followed in 30 minutes by specific antibody, showed less growth than in control animals in which the mustard exerted no significant inhibition of tumor growth. In these experiments, the antihapten antibody was shown by fluorescent techniques to localize in the region of the cell membrane of the mustard-treated tumors (Burke et al., 1966). 6. Diethylstilbestrol A synthetic compound, diethylstilbestrol consists of two peripheral phenolic groups linked by two carbon atoms, each of which has an ethyl substituent. Although structurally it is not a steroid, diethylstilbestrol exerts estrogenic activity. Purified antiazobenzoyl estradiol-17p antibodies bind diethylstilbestrol and may prove useful in the immunological assay of this synthetic nonsteroidal estrogen (Gross and Grant, 1970).

7 . Hallucinogenic Drugs D-Lysergic acid (LSD) has been conjugated to poly-L-lysine by the carbodiimide reaction. Rabbits immunized with electrostatic complexes of LSD-poly-L-lysine with succinylated hernocyanin have formed antibodies that react with LSD and cross-react with related ergot alkaloids, as determined by complement fixation (Van Vunakis et al., 1971). Mescaline ( 3,4,5-trimethoxyphenylethylamine) and the related amine, 3,4-dimethoxyphenylethylamine, have been conjugated to polyglutamic acid by the carbodiimide method. Rabbits immunized with electrostatic complexes of each of these conjugates with methylated bovine serum albumin have formed antibodies with specificity for mescaline or for 3,4dimethoxyphenylethylamine, respectively, as determined by complement fixation. Antibodies directed toward 2,5-dimethoxy-4-methylamphetamine have been elicited in rabbits immunized with conjugates (synthesized by the glutaraldehyde method) of rabbit serum albumin with the dimethoxymethylamphetamine (Van Vunakis et al., 1969).

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8. Hydralazine The antihypertensive drug, hydralazine, has been conjugated to erythrocytes with BDB, a bifunctional diazonium compound. The hydralazine-erythrocyte conjugates have been used to detect antihydralazine antibodies in the sera of patients with hydralazine-induced systemic lupus erythematosus (Heine and Friedman, 1962; Hahn et al., 1972) and in the sera of rabbits that received multiple injections of unconjugated hydralazine in Freund's adjuvant mixture over a 6-month period (Friedman and Heine, 1963).

9. Medroxyprogesterone Acetate A synthetic compound, medroxyprogesterone acetate is an orally active steroidal progestin. It has been conjugated to bovine serum albumin, and a goat immunized with the resulting conjugate has formed antibodies capable of binding medroxyprogesterone a ~ e t a t e - ~ Has, determined by the double-antibody method. These antibodies have been used in the development of a sensitive radioimmunoassay method capable of detecting 100 pg. of the unlabeled progestin in 0.1 ml. of serum. This assay method has been employed in clinical pharmacological studies of medroxyprogesterone acetate in the serum of women receiving this compound (Cornette et al., 1971).

10. Morphine An antigenic conjugate was prepared by Spector and Parker (1970) by coupling 3-O-carboxymethylmorphine to bovine serum albumin. The resultant antibodies, in a radioimmunoassay using the Farr technique, were capable of detecting as little as 0.5 ng. of morphine. Codeine ( morphine 3-methyl ester) resembles more the carboxymethylmorphine group in the immunizing antigen than does morphine itself and competes better with the labeled hapten, dihydr~morphine-~H, for antibody-combining sites than does morphine. Nalorphine cross-reacts to some extent, but methadone is essentially inactive in this system. Adler and Liu (1971) also employed a bovine serum albumin conjugate of carboxymethylmorphine to induce antimorphine antibodies. By using antisera absorbed with methadone and with bovine serum albumin polymerized with carbodiimide, a hemagglutination inhibition test sensitive to 1ng. of morphine per milliliter was reported. The same hapten derivative used in the previous studies was coupled to polylysine by Van Vunakis et al. (1972). The conjugate was complexed to succinylated hemocyanin for immunization, The antibody produced bound codeine, dihydrocodeine, morphine, heroin, hydromor-

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phone, and nalorphine, in descending order of effectiveness. These authors, using a radioimmunoassay sensitive at the picomole level, examined sera obtained at autopsy from 35 cases of sudden death in which drug abuse was suspected. Twenty-two of the sera reacted with the antiserum to morphine. A recent abstract (Ryan et al., 1971) reported that 40%of y-globulin preparations from sera of heroin addicts demonstrated binding of morphine-"H. Immunoglobulin M, although often elevated in such sera, did not bind labeled morphine. A radioimmunoassay for morphine in which nitrocellulose membranes were used to separate hapten-antibody complexes from free hapten has been reported. This method can detect as little as 10 ng./ml. of morphine in unextracted urine or blood ( Gershman et al., 1972). Another procedure reported recently for preparing morphine-protein conjugates involves the use of the mixed anhydride method to couple morphine-3-hemisuccinate to bovine serum albumin ( Wainer et al., 1972). Antibody production was measured by demonstrating binding of labeled morphine.

11. Penicillin A number of penicillin derivatives and breakdown products have been conjugated to protein or polypeptide carriers. These conjugates, which are immunogenic in experimental animals (Schneider and de Weck, 1970), have been extremely useful in the detection of antipenicillin antibodies and in studies of the mechanisms and specificities of allergic reactions to penicillin in man. These studies have been extensively discussed in review articles (Parker, 1965; Levine, 1965, 1W; Schwartz, 1969; de Weck et al., 1968; Levine and Zolov, 1969; Shaltiel et al., 1971). Recently, penicillin antibodies have been used to develop immunological assays for the detection and measurement of penicillin in biological specimens ( Karchmer et al., 1972; Wiederman et al., 1972). 12. Procaine Amide Procaine amide is an antiarrhythmic drug whose prolonged use is often associated with the development of antinuclear antibodies, sometimes associated with clinical syndromes with many of the features of systemic lupus erythematosus ( Blomgren et al., 1972). Procaine amide has been diazotized and conjugated to bovine serum albumin and to sheep erythrocytes. Rabbits immunized with procaine amide-albumin conjugates form antibodies that agglutinate procaine amide-coated erythrocytes. By using procaine amide-coated red cells to detect circulating antibodies to the antiarrhythmic agent, such antibodies were found in the sera of 19 to 35% of patients with various diseases and in 30%

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of normal adult sera, but in none of 24 neonatal sera studied. The most striking finding in this study was the fact that, although 50% of patients receiving procaine amide without adverse effects had antibodies to the drug, only 1 of 18 patients with procaine amide-induced systemic lupus erythematosus had detectable antibodies to procaine amide ( Russell and Ziff, 1968). 13. Sulfonamides Sulfanilamide, sulfapyridine, sulfathiazole, and sulfanilic acid have been diazotized and conjugated to protein carriers. Rabbits immunized with sulfonamide-protein conjugates form antibodies with primary specificity for the individual sulfonamide present in the particular conjugate employed, but significant cross-reactivity of antibodies with other sulfonamides is usually observed (Wedum, 1942; Rubin and Aasted, 1971; Rubin, 1972). 14. Tetracyclines The injection into rabbits of oxytetracycline, without prior conjugation to a carrier protein, in complete Freund's adjuvant mixture has been reported to have elicited the formation of antibodies that will react with oxytetracycline and cross-react with other tetracycline antibiotics, as determined by a passive hemagglutination technique. In this study, however, a trace protein impurity common to all the tetracycline preparations studied has not been conclusively excluded as an alternative cause of the hemagglutinating antibodies elicited by the oxytetracycline preparation ( Queng et al., 1965). 15. d-Tubocurarine A quaternary ammonium base, d-tubocurarine is obtained from a curare, Chondrodendron tomentosum, derived from the western Amazon region of South America. Clinically, d-tubocuranine has uses as a skeletal muscle relaxant in the practice of anesthesia. Rabbits immunized with d-tubocurarine-bovine serum albumin conjugates form antibodies that recognize the unconjugated alkaloid. With these antibodies and d-tuboc~rarine-~H, a sensitive radioimmunoassay for the determination of blood levels of this drug in man has been developed (Horowitz and Spector, 1972 ) .

E. TOXINS 1. Arsenicals The diazonium derivative of p-aminophenylarsenic acid has been conjugated to ox globulin. Rabbits immunized with this synthetic

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arsenical-globulin conjugate formed antibodies that appeared to confer some protective effect against the toxic effects of resorcinol-trisazophenyl-p-arsenic acid in mice (Singer, 1942).

2. Carcinogens The isocyanate derivatives of a number of carcinogenic substances have been coupled to protein carriers via a carbamido linkage. These substances have included anthracene, 1,2,5,&dibenzanthracene ( Creech and Franks, 1937), l,2-benzanthracene (Creech and Jones, 1940), 3,4benzpyrene, lO-methy1-1,2-benzanthracene,8-anthracene ( Creech and Jones, 1941a,b) , %acetylaminofluorene, 4-dimethylaminostilbene, 2 methyl-4-dimethylaminostilbene ( Creech and Peck, 1952; Peck et al., 1953), and 4-dimethylaminoazobenzene ( Baldwin, 1962) . Other carcinogenic compounds, p-naphthylamine ( Korosteleva and Skachkov, 1964) and 2-aminofluorene (Kitagawa et al., 1966), have been diazotized and then conjugated to protein carriers; the noncarcinogenic isomer of the first of these compounds, a-naphthylamine, has also been conjugated to a protein carrier by the diazonium method (Korosteleva and Skachkov, 1964). Rabbits immunized with these carcinogen-protein conjugates have formed antibodies with specificity for the introduced carcinogenic group (Creech et al., 1947a,b,c, 1953, 1955; Creech, 1952; Baldwin, 1962; Korosteleva and Skachkov, 1964; Kitagawa et al., 1966). Immunization with a carcinogen-protein conjugate appears to confer some protection against the carcinogenic effects of the specific hapten used. Immunization of mice with dibenzanthranyl-carbamido-protein conjugates gives evidence of reducing their susceptibility to carcinogenesis from injected dibenzanthracene, in comparison with untreated control mice (Franks and Creech, 1939). In rats immunized with a 2-anthrylcarbamido-protein conjugate, significant tumor inhibition was observed following a single intragastric feeding of 2-anthrylamine, a potent carcinogenic compound, Fewer immunized animals developed persistent, palpable tumors; in addition, immunized animals developed fewer tumors, and induction was delayed (Peck and Peck, 1971). Antibodies to the 4-dimethylaminoazobenzene ( DMAB) group have been used to demonstrate the presence of the DMAB prosthetic group in rat liver antigens during the early stages of DMAB carcinogenesis ( Baldwin, 1962). Antibodies specific for the 2-azofluorenyl group have been used to investigate the behavior and localization of hepatocellular components capable of binding 2-acetylaminofluorenein rat livers during chemical carcinogenesis. The results of these experiments, employing fluorescein-conjugated antibodies, indicate that carcinogen-binding components are present at a particularly high concentration at the cell boundary and in the perinuclear zone and that these components are

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AND SAM M. BEISER

partially deleted in cells of hyperplastic nodules, which appear at the later stage of carcinogenesis, and are almost completely deleted in hepatomatous cells ( Kitagawa et al., 1966; Tanigaki et al., 1967). 1,2-Naphthoquinone reacts directly with protein without the need for diazotization or other chemical manipulation. Since this compound has been implicated as a possible mediator of the action of @-naphthylamine in producing bladder tumors, 1,2-naphthoquinone-protein conjugates have been synthesized and have been shown to be capable of eliciting antinaphthoquinone antibodies in rabbits. After conjugation with fluorescein, it is hoped that these antibodies will be useful in the localization of naphthoquinone or related compounds in the urinary bladder of dogs in various stages of p-naphthylamine treatment ( Ollodart and Rose, 1962). A number of carcinogenic compounds, including 3-methylcholanthrene, 3,4-benzpyrene, 9,10-dimethyl-l,2-benzanthracene(Old et al., 1963) , azobenzene, p-aminoazobenzene, 4-dimethylaminoazobenzene, and a number of related carcinogenic azo dyes (Gordon, 1964), are capable, in unconjugated form, of eliciting delayed hypersensitivity responses in guinea pigs.

3. Genistein The aglycone of a plant glycoside, genistein ( 5,7,4'-trihydroxyisoflavone), has been isolated from the plasma of sheep grazing in clover pastures. Genistein and related isoflavones exert an estrogen-like effect in mice and sheep, and excessive ingestion of these isoflavones has been held responsible for serious infertility in grazing sheep and cattle. Genistein-specific antibodies, which do not cross-react with estradiol, have been elicited in rabbits immunized with a synthetic conjugate prepared by coupling genistein-2-carboxylicacid to a polyamino acid carrier by the carbodiimide method. It has been proposed that a synthetic vaccine to isoflavones might protect grazing animals against the harmful effects of estrogenic pastures, without interfering with the activity of the endogenous steroidal estrogens. It has also been suggested that antibodies to isoflavones may be useful for the detection of these so-called phytoestrogens in blood by a radioimmunoassay technique (Bauminger et al., 1969). 4. Insecticides ( D D T and Malathion) Two widely used insecticides are DDT, a chlorinated hydrocarbon, and Malathion, an organophosphate. Neither can be directly conjugated to protein carriers, but derivatives of both compounds, containing carboxyl groups, have been synthesized. Anhydrides and acyl chlorides of

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these carboxyl derivatives have then been prepared by allowing them to react with acid anhydrides or with thionyl chloride. The final anhydride and acyl chloride derivatives of the two insecticides will react readily with amino groups of protein carriers to form insecticide-protein conjugates. Rabbits immunized with these conjugates form antibodies that react with DDT or with Malathion. It is hoped that these antibodies may serve in the localization of pesticide derivatives in tissues and that insecticide-protein conjugates may prove useful in the study of insecticide allergy in man ( Haas and Guardia, 1968; Centeno et al., 1970).

5 . Paralytic Shellfish Poison Paralytic shellfish poison (PSP) is a potent toxin which is produced by the dinoflagellate Gonyaulax catenella and which is concentrated with no ill effects in clams and mussels that use the Gonyaulax as a food source. However, human ingestion of PSP results in paralytic poisoning and occasionally death. Since the mouse bioassay test used for detection of PSP is not specific, an immunological assay has been developed (Johnson and Mulberry, 1966). Although PSP has the empirical formula C,,H,,N7O,.2HC1 (Schantz et al., 1957; Mold et aL, 1957), its precise chemical structure is not known. Nevertheless, using formaldehyde as a coupling agent, PSP can be conjugated to bovine serum albumin. Rabbits immunized with PSP-albumin conjugates formed antibodies to the toxin, as demonstrated by passive hemagglutination and mouse protection tests (Johnson et al., 1964). By using inhibition of passive hemagglutination of PSP-coated red cells or inhibition of flocculation of PSP-coated bentonite particles, it is now possible to detect PSP in contaminated shellfish (Johnson and Mulberry, 1966).

6. Strychnine A monoamino derivative of strychnine has been diazotized and conjugated to hernocyanin. Rabbits immunized with monoaminostrychnineazohemocyanin conjugates formed antibodies to strychnine, but the titers of antistrychnine antibody were too low to neutralize the lethal effect of strychnine in mice ( Hooker and Boyd, 1940).

F. PEPTIDEFRAGMENTS OF PROTEIN MOLECULES With the carbodiimide method, peptides derived by chemical or enzymatic means from larger parent protein molecules can be conjugated to protein or synthetic polypeptide antigens. Immunization of experimental animals with these synthetic conjugates results in the formation of antibodies specific for such peptides; in certain instances, these anti-

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bodies will also react with the protein molecule from which the peptide was originally derived.

1. C-Peptide of Proinsulin During the biosynthesis of insulin in the beta cells of the pancreas, proinsulin is cleaved proteolytically to yield insulin and the so-called C-peptide. Rabbit serum albumin-C-peptide conjugates prepared by the carbodiimide method have elicited antibodies to C-peptide in rabbits. C-Peptide, which lacks tyrosine, was tyrosylated with N-carboxytyrosyl anhydride and then radio-iodinated enabling the development of a sensitive radioimmunoassay for C-peptide. Since the antibodies to C-peptide recognize proinsulin as well as C-peptide, these molecules must be separated by gel filtration before they can be measured in human serum. The C-peptide radioimmunoassay has been used to document the fact that Gpeptide and insulin are released from beta cells in equimolar amounts when glucose is administered to fasting human subjects (Melani et al., 1970). 2. Fibrinopeptides A and B Fibrinopeptides A and B are the first two peptides released from fibrinogen by the action of thrombin, in the process of blood coagulation. Fibrinopeptide A has been conjugated to protein carriers by the carbodiimide method. Rabbits immunized with protein-fibrinopeptide A conjugates form antibodies that react specifically with fibrinopeptide A; these antibodies do not react with fibrinopeptide B and their crossreaction with native fibrinogen is minimal. Since fibrinopeptide A does not contain a tyrosine residue, derivatives containing tyrosine or desaminotyrosine have been synthesized and radio-iodinated, enabling the development of a sensitive and specific radioimmunoassay method for the measurement of fibrinopeptide A. This assay method is being applied to the study of coagulation in uiuo and in uitro ( Nossel et at., 1971; Qureshi and Nossel, 1972). Rabbits immunized with fibrinopeptide B combined with acrylic plastic particles have been reported to form antibodies specific for fibrinopeptide B ( Berglund, 1965). 3. “Loop” Peptide of Lysozyme A synthetic conjugate, prepared by covalent binding of an egg-white lysozyme fragment (sequence 6483, denoted ‘loop” peptide) to a synthetic polypeptide, elicited anti-loop-peptide antibodies in rabbits. These antibodies react with native lysozyme. Like antibodies with similar specificity from antilysozyme sera isolated by an immunoadsorbent technique, anti-loop-peptide antibodies are capable of distinguishing between the

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loop peptide containing a disulfide bridge and the open-chain peptide derived from it. This capacity suggests that such antibodies are directed against a conformation-dependent determinant, both in native lysozyme and in the synthetic conjugate ( Arnon and Sela, 1969). 4. Tobacco Mosaic Virus Peptides In related studies the N - ( p-aminobenzoyl) derivatives of the carboxylterminal amino acid and of the carboxyl-terminal di-, tri-, tetra-, penta-, and hexapeptides homologous to the carboxyl-terminal amino acid sequence of tobacco mosaic virus proteins have been synthesized, diazotized, and conjugated to protein carriers. Antisera to these peptides and even to the carboxyl-terminal amino acid were capable of neutralizing the homologous strain of tobacco mosaic virus ( Anderer, 1963; Anderer and Schlumberger, 1965, 1966). These observations led to the finding that antisera to artificial antigens containing the aminobenzoyl derivatives of different L-amino acids as haptenic groups may be utilized in a serological screening technique to detect free carboxyl-terminal amino acids in virus coat proteins ( Anderer et al., 1967). G. MISCELLANEOUS 1. Carbohydrates A number of small carbohydrate molecules have been coupled to protein or polypeptide carriers by means of azophenyl or azobenzyl linkages. Carbohydrate haptens rendered antigenic in this manner have included monosaccharides, such as glucose ( Avery and Goebel, 1929; Tanenbaum et al., 1961), galactose ( Avery and Goebel, 1929; Westphal and Feier, 1956; Beiser et al., 1960; Rude et al., 1966), glucuronic acid ( Goebel, 1940; Zolla and Goodman, 1967), galacturonic acid ( Goebel, 1940), colitose (Luderitz et al., 1960); disaccharides, such as maltose (Goebel et al., 1934), lactose (Goebel et al., 1934; Karush, 1957; Yariv et al., 1962), cellobiose (Goebel et al., 1934; Goebel, 1938, 1939; Gleich and Allen, 1965), gentiobiose (Goebel et al., 1934; Goebel, 1940), sophorose (Allen et al., 1967) , laminaribiose (Allen et al., 1970), cellobiuronic acid (Goebel, 1938, 1939, 1940), gentiobimonic acid (Goebel, 1940); and the trisaccharide, panose (Martineau et al., 1971). The mixed anhydride method has been used to prepare. antigenic protein-carbohydrate conjugates containing glucuronic acid, galacturonic acid ( Borek et al., 1963), and isomaltonic and isomaltotrionic acids ( Arakatsu et al., 1966). Glucose has also been coupled to a polypeptide carrier by making use of 0-(tetraO-acetyl-p-D-glucopyranosyl) -N-carboxy-L-serine anhydride ( Rude et d., 1966). Recently, phosphatidylinositomannosides derived from Mrjcobnc-

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terium tuberculosis have been rendered immunogenic by coupling them electrostatically to methylated bovine serum albumin ( Khuller and Subrahmanyam, 1971). Animals immunized with each of the conjugates described above have formed antibodies with specificity for the corresponding chemically introduced carbohydrate moiety. That antibodies to carbohydrate haptens are biologically active was first shown by Goebel (1938, 1939), who demonstrated not only that antibodies to cellobiuronic acid precipitated Type I11 pneumococcal polysaccharide (which contains this carbohydrate as a major constituent) but also that anticellobiuronic acid antibodies agglutinated Type I11 pneumococci and protected mice against otherwise fatal infections with these virulent organisms; interestingly, antibodies to a cellobiose antigen conferred no protective effect against a virulent strain of Type I11 pneumococci (Goebel, 1939). In an extension of these studies, Goebel ( 1940) later showed that anticellobiuronic acid antibodies as well as antibodies to gentiobiuronic and glucuronic acids confer passive immunity on mice infected with multiple lethal doses of Type I1 pneumococci. In another study of the biological properties of antibodies to carbohydrate haptens, it was demonstrated that antibodies to colitose agglutinate strains of Salmonella and of Escherichia coli that contain this sugar as an end group of their O-antigenic polysaccharides ( Luderitz et al., 1960). 2, Catecholamines Antibodies reactive with adrenaline, a sympathetic amine, were described by Went and Kesztyus (1939) in animals immunized with a synthetic adrenaline-azoprotein conjugate but, to our knowledge, biological studies with these antibodies have not been reported. Normetanephrine is an important metabolic product of the sympathetic neurotransmitter, noradrenaline. Normetanephrine has been rendered antigenic by coupling it to macromolecular carriers with glutaraldehyde. Rabbits immunized with the resulting conjugates formed antibodies that react with normetanephrine. These antibodies have enabled the development of a radioimmunoassay method, utilizing a radio-iodinated albumin-normetanephrine conjugate and the doubleantibody technique, capable of detecting 10 ng. of normetanephrine. Unfortunately, metanephrine reacts as well as normetanephrine in this assay system. It is anticipated, however, that these antibodies can be employed for normetanephrine determinations in cerebrospinal fluid, where interference from metanephrine should be negligible (Peskar et al., 1972).

3. Cholesterol The bifunctional reagent, sebacyl dichloride, has been used to conjugate cholesterol to protein carriers. In rabbits given high cholesterol

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diets and immunized with cholesterol-protein conjugates, significant decreases in serum cholesterol levels and significant reductions in the severity of atherosclerotic lesions in the thoracic aorta have been reported (Bailey et al., 1963; Bailey and Butler, 1967). 4. Lipids

Antiserum against phosphatidylinositol has been obtained in rabbits immunized with a complex of the hapten and methylated bovine serum albumin ( Kataoka and Nojima, 1970). It has been reported that ceramide lactoside (Tal et al., 1967) or sulfatides (cerebroside sulfuric esters) derived from spinal cord (Niedieck, 1967), when mixed in proper proportions with carrier proteins, will form complexes which, when injected into experimental animals, will elicit antibodies that react with ceramide lactoside or sulfatides, respectively.

5. Nucleic Acid Constituents The immunogenicity of purified nucleic acids is very weak or perhaps even nonexistent. Antibodies that react with denatured DNA have been elicited in animals immunized with synthetic hapten-protein or haptenpolypeptide conjugates containing purines (Butler et al., 1962, 1965), pyrimidines ( Tanenbaum and Beiser, 1963), ribonucleosides and ribonucleotides (Erlanger and Beiser, 1964; Lacour and Harel, 1965; Sandberg and Stollar, 1966; Rosenberg et al., 1972), nucleoside 5’-carboxylic acids (Sela et al., 1964; Sela and Ungar-Waron, 1965; Ungar-Waron et al., 1967; Karol and Tanenbaum, 1967), dinucleoside phosphates (Wallace et al., 1971), deoxyribonucleotides, and oligonucleotides ( Halloran and Parker, 1966a,b). Although the complexes are not covalently linked, anti-DNA antibodies have also been elicited in animals immunized with electrostatic complexes of methylated bovine serum albumin with oligonucleotides (Plescia et al., 1965) or with DNA (Plescia et al., 1964; Stollar and Sandberg, 1966; Forsen et al., 1970). The production and properties of these anti-DNA antibodies have been extensively reviewed (Beiser and Erlanger, 1966; Levine and Van Vunakis, 1966; Beiser et al., 1967; Plescia and Braun, 1967, 1968; Levine and Stollar, 1968; Erlanger et al., 1972). Similar methods have been used to obtain antibodies with specificity for synthetic polyribonucleotides ( Seaman et al., 1965; Stollar, 1970), ultraviolet-irradiated DNA (Levine et al., 1966; Tan, 1968), photooxidized nucleotides ( Van Vunakis et al., 19SS), transfer RNA (Bonavida et al., 1970), methylated bases (Sawicki et al., 1971; Levine et al., 1971b), inosine (Inouye et aZ., 1971; Bonavida et al., 1971), 5-bromouracil, and 5-iodouracil ( Sawicki et al., 1971) . Antibodies to nucleosides have been applied to the study of a number of biological problems. For example, antibodies to 6-methyladenosine and

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VINCENT P. BUTLER, JR., AND SAM M. BEISER

to 5-bromouracil, respectively, have been used to detect each of these minor bases in denatured DNA (Sawicki et al., 1971). Fluoresceinlabeled antinucleoside antibodies are capable of localizing denatured, single-stranded DNA in the nuclei of L cells during the period of maximum DNA synthesis ( Klein et al., 1967) ; these fluoresceinated antibodies have also been used to detect denatured DNA in the glomeruli of patients with active systemic lupus erythematosus nephritis ( Andres et d., 1970) and in the renal lesions of autoimmune disease in NZB/NZW F, mice (Seegal et al., 1969). More recently, fluorescein-labeled antinucleoside antibodies have been applied to the study of chromosome structure (Freeman et al., 1971; Dev et al., 1972). Antibodies to 5-methyluridine have been used together with iod~deoxyuridine-~~~I in the development of a radioimmunoassay method for the measurement of serum thymidine (Christine et al., 1972). Antibodies to purines, pyrimidines, and nucleosides have also been shown to possess biological activity. Antibodies specific for thymine and guanine nucleosides inhibit the ability of calf thymus DNA to serve as a primer for DNA polymerase (Wallace et al., 1969). Antipurinoyl antibodies appear to induce the regression of transplanted Ehrlich ascites tumors in mice (Sinai et al., 1965; Lachman and Cohen, 1970). Antipurinoyl and antipyrimidine antibodies can enter the fertilized sea urchin egg and inhibit its embryonic development ( Rosenkranz et al., 1964). Recently, it has been shown that antithymidine antibodies will inhibit the growth of methylcholanthrene-transformed Chinese hamster lung cells but do not affect the multiplication of normal lung cells in tissue culture, suggesting that such antibodies may be capable of selective inhibition of the multiplication of malignant cells ( Liebeskind et al., 1971).

6. Plant Hormones The plant hormones, 3-indoleacetic acid and gibberellic acid, have been conjugated to protein carriers by the carbodiimide method. Rabbits immunized with either of these protein-hormone conjugates form antibodies specific for the corresponding plant hormone as determined by inhibition of precipitation, complement fixation, and hapten-modified bacteriophage inactivation. By using inhibition of inactivation of modified phage as the assay system, micromolar concentrations of these plant hormones can now be conveniently and reliably determined (Fuchs and Fuchs, 1969). ACKNOWLEDGMENT We are grateful for many helpful discussions with Dr. Bernard F. Erlanger, Department of Microbiology, Columbia University, during the preparation of this manuscript.

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AUTHOR INDEX Niinihers in italics refer to the pages on which the complete referenccs are listed. Anieniiya, H., 12, 46, 74 A Amend, J. R., 6, 10, 13, 15, 16, 22, 26, Aai-onson, S. A., 214, 245, 248, 251 29, 30, 31, 32, 37, 46, 52, 53, 67, Aars, €I., 266, 300 81, 82 Aasted, B., 288, 308 Abaza, H. M., 27, 28, 29, 30, 36, 38, 41, Amiel, J. L., 28, 64, 71, 74, 84 Amos, D. B., 10, 27, 35, 73, 79, 83, 88 53, 73, 92 Abbott, W. M., 14, 30, 31, 38, 47, 56, Anisbaugh, D. F., 44, 75 Amundsen, E., 110, 178 74, 85 Ances, I. G., 278,298 Abelmann, W. H., 283, 298 Anderer, F. A., 293,297 Ablashi, D. V., 243, 248 Abraham, C. E., 276, 277, 297, 308, 309 Andersen, B. R., 161, 178 Anderson, J. T., 46, 74 Abramoff, P., 268,309 Anderson, N. D., 52, 77 Abrudeanu, O., 9, 85, 86 Anderson, N. F., 3, 4, 10, 13, 16, 20, 21, Accinni, L,, 296, 297, 308 26, 27, 30, 36, 38, 44, 46, 48, 54, Ackroyd, J. F., 139, 178 74, 81, 91, 92 Ada, G. L., 204, 213, 233, 236, 245, 250 P., 108, 190 Anderson, Adams, H. R., 116,182 Anderson, S. G., 222, 246 Adamson, A. R., 264,298 Anderson, T. F., 202, 249 Adler, F. L., 47, 74, 257, 286, 297 Anderson, W., 166, 181 Africa, B., 259, 276, 277, 297 Agate, F. J., Jr., 259, 276, 277, 278, 298, Andrassi, K., 74 Andre, J., 289, 300 304, 306 Andres, G. A., 16, 22, 68, 89, 296, 297, Agnew, H. D., 30,59,74 308 Agundis, C., 280, 300 Andrzejewski, C. Z., 279, 302 Aikawa, S., 234, 248 Angelotti, R., 291, 303 Ainbender, E., 221,245 Anigstein, D. M., 29, 39, 74 Aladjem, F., 266, 307 Aledort, L. M., 127, 129, 131, 178 Anigstein, L., 29, 39, 74 Alexander, J. W., 12, 74 Antoine, B., 26, 27, 74 Alexandre, G. P. J,, 88 Antonsson, J., 110, 192 Alford, R. H., 165, 178 Aoki, J., 7, 76 Allen, C. R., 47, 81 Appelbaum, D. M., 266,309 Allen, 0. N., 289, 300 Appleman, A., 16, 75 Allen, P. Z., 293, 297, 301, 305 Arakatsn, Y., 293, 297 Allison, A. C., 40, 41, 42, 43, 68, 74, 97, Arbesnian, C. E., 281, 310 110, 159, 175, 178, 188, 189 Arbuys, S., 10, 16, 34, 36, 37, 38, 42, Allison, E., 154, 178 44, 91 Allison, F., 156, 186 Archer, G. T., 122, 190 Allwood, G., 165, 178 Archer, J. F., 52, 80 Almeida, J. D., 209, 246 Archimbaud, J.-P., 9, 13, 29, 30, 70, 77, Alspaugh, M., 295,301 Altemeier, W. A., 12, 74 90 311

312

AUTHOR INDEX

Bachmann, F., 137, 191 Bachrach, H. L., 197, 198, 199, 202, 215, 245, 246,252 Bachvaroff, R., 74 Baeliner, R. L., 149, 150, 153, 157, 178, 181 Baenziger, N. L., 131, 132, 178, 180 Baer, L., 265, 298,304 Bagby, M. K., 286,302 Baggiolini, M., 147, 149,179 Bailey, J. M., 295, 297 Bailey, W. L., 137, 181 Bainbridge, D. R., 35, 75 Bainton, D. F., 147, 179 Baker, A. R., 102,179 Baker, P. J., 44, 75 Baldwin, R. W., 289, 295 Ball, D. L., 260, 264, 266, 302 Ball, G., 132, 179 Balner, H., 10, 16, 24, 25, 27, 30, 31, 36, 37, 38, 40, 41, 43, 44, 53, 70, 74, 75, 79, 84, 85, 91 Baltimore, D., 198, 203, 213, 214, 246, 247, 249 Ban, J., 269, 307 Banerjee, S. D., 98,155, 180 Bangham, A. D., 154, 193 Bannister, G. L., 212, 246 Bansillon, V., 9, 70, 90 Barabas, A. Z., 42, 75 Barbaro, J. F., 140, 142, 179, 181, 190 Barber, A. J., 132, 184 Bard, R. H., 265, 298 Bardana, E. J., Jr., 66, 72, 87 Barker, L. R., 40, 75 Barnes, A. D., 24, 60, 65, 75 Barnett, E. V., 295,301 Baron, J. H., 269, 302 Barr, K. L., 304 Barrett, C. D., 207, 233, 247 Barrett, I., 15, 83 B Barth, R. F., 8, 41, 44, 45, 59, 75, 88 Bartholomew, K., 275, 303 Babers, F. H., 293, 302 Bassenge, E., 11, 77 Bach, F. H., 28, 74 Bassiri, R., 272, 297 Bach, J. F., 26, 27, 74 Bach, M. K., 27, 28, 35, 74, 102, 109, Batchelor, J. R., 49, 82 Battenberg, E. R., 273, 298 110, 115, 116, 121, 178

Ardill, J., 269, 303 Ardlie, N. G., 128, 129, 130, 143, 178 Arlinghaus, R. B., 203, 213, 214, 250 Arnason, B., 10, 16, 34, 36, 37, 38, 42, 44, 91 Arnaud, C. D., 267, 297, 302 Arnold, M. L., 264, 277, 297, 298 Arpels, C., 7, 88 Asakumah, R., 7, 74 Aschkenasy, A., 74 Ascione, R., 198, 204, 252 Asfis, N., 80 Ash, J. F., 98, 128, 132, 155, 192 Ashe, H., 284, 299 Ashe, W. K., 209,245,250 Asherson, G . L., 55, 87, 165, 172, 178, 182 Ashford, T: P., 129, 190 Ashwell, G., 293, 297 Askonas, B. A., 237,245 Asofsky, R., 20, 25, 26, 30, 84 Assan, R., 270,297 Assem, E. S. K., 104, 105, 178 Atai, M., 38, 44, 74, 85 Atkinson, F. F. V., 271, 272, 310 Atkinson, L. E., 277, 303 August, C. S., 11, 82 Aust, J. C., 6, 67, 85 Austen, K. F., 99, 100, 102, 103, 104, 105, 106, 109, 110, 113, 115, 116, 117, 118, 121, 133, 160, 165, 178, 179, 184, 185, 191, 192, 260, 266, 309 Austen, W. G., 102, 105, 188 Austin, C. M., 233, 236, 250 Averdunk, R., 28, 74 Avery, 0. T., 293,297,302 Axline, S. G., 158, 178 Ayengar, R., 225, 246 Azarnoff, D. L., 283, 303

AUTHOR INDEX

Bauer, D. C., 223, 246 Bauer, H., 95, 193 Baum, J., 44,75,161,187 Baumal, R., 101, 180 Bauminger, M. F., 163, 182 Bauminger, S., 290, 297 Baussillon, V., 70, 79 Bayard, F., 276, 277, 278, 297, 298 Baylin, S. B., 267, 297 Bayracki, C., 66, 72, 87 Bazzi, C., 63, 84 Beall, A. C., 67, 76 Beall, G. N., 279, 297, 299 Beardwell, C. G., 273, 298 Beathard, G. A., 64,65, 90 Beattie, E. J., Jr., 70, 79 Beaven, M. A., 267,297 Becht, H., 206, 208, 214, 249, 251 Beck, G., 29, 38, 86 Beck, N. P., 189 Becker, E. L., 21, 88, 94, 95, 103, 110, 113, 114, 115, 117, 118, 121, 135, 145, 146, 147, 153, 154, 159, 160, 161, 162, 172, 178, 179, 182, 189, 190, 192 Becker, J. A., 130, 187 Begeman, H., 72, 90 Behnke, O., 128, 133,179 Beil, E., 32, 40, 87, 90 Beiser, S. M., 255, 257, 259, 276, 277, 278, 293, 295, 296, 297, 298, 299, 300, 301, 304, 306, 307, 308, 309, 310 Beitins, I. Z., 276, 277, 278, 297, 298 Belamarich, F. A., 132, 190 Bell, P., 67, 89 Bellanti, J. A., 212, 225, 226, 246 Beller, F. K., 137, 188 Beller, G. A., 283, 298 Belyavin, G., 215, 246 Benacerraf, B., 212, 228, 246, 250, 290, 306 Benda, P., 285, 310 Bendich, A,, 295, 299 Benedict, A. A., 225, 246 Benjamini, E., 200, 246 Bennett, I. L., Jr., 137, 170 Bennett, R., 276, 277, 302

313

Bennich, H., 95, 115, 122, 125, 179, 184 Bensch, K. G., 148, 155, 158, 186 Benveniste, J., 140, 141, 179, 183 Beranger, G., 24, 76 Berenbaum, M. C., 13, 44, 75 Berger, R., 221, 245 Berglund, G., 292, 298 Berlin, B. S.,225, 246 Berlin, R. D., 149, 179 Berrnan, L., 41, 42, 74 Berneis, K. H., 126, 179 Bernhardt, J. P., 9, 13, 29, 30, 70, 77, 90 Bernstein, J., 98, 189 Berrios, J. H., 10, 60, 76 Berry, D. M., 209, 246 Berson, S. A,, 260, 263, 267, 269, 272, 298, 310 Bert, G., 28, 75 Berthoux, F., 6, 62, 63, 65, 66, 67, 70, 90 Besredka, A., 3, 30, 75 Betel, I., 16, 25, 75 Bettex-Galland, M., 128, 138, 179 Betuel, H., 9, 70, 75, 79, 90 Beverley, P. C. L., 39, 49, 75 Bewick, M., 64, 65, 85 Biber, T. U. L., 266, 299, 306 Bielski, R. K., 30, 87 Bigger, J. T., Jr., 283, 305 Bigley, N. I., 267, 308 Biglieri, E. G., 265, 309 Billote, J. B., 14, 82 Binaghi, R. A,, 102, 119, 120, 179, 189 Bing, J., 266, 298 Bing, R. J,, 11, 77 Binns, R. M., 36, 76 Birtch, A. G., 70, 76 Bitensky, L., 28, 34, 76, 78 Blackstone, M. O., 283, 305 Blanchard, H., 46, 82 Blandin, R. V., 41, 76 Blank, S. E., 209, 227, 246 Bleicher, S. J., 261, 303 Blennerhassett, J. B., 38, 88 Bloch, K. J., 95, 102, 103, 109, 110, 115, 116, 118, 121, 178, 179, 185, 191, 192,212,228,246, 250

AUTHOR INDEX

Blomgren, S. E., 287,298 Blonde, L., 273, 310 Bloodwell, R. D., 71, 77 Bloom, F. E., 273,298 Bloom, G. D., 107, 111,179 Bloom, S., 270, 272, 307 Bloomberg, N., 128, 132, 193 Blough, W. M., Jr., 265, 299 Blumenthal, H. T., 279,307 Boak, J, L., 30, 40, 44, 76, 77 Bodel, P., 149, 154, 179 Bodevin, E., 129, 186 Bohak, Z., 131, 180 Boler, J., 272, 298 Bolinger, R. E., 268, 301 Bolland, H., 73, 86 Bomel, J., 38, 86 Bonavida, B., 295, 298 Bonneau, M., 24, 76 Bonner, J. T., 161, 186 Bonnet, P., 9, 13, 29, 30, 70, 77, 90 Booyse, F. M., 129, 179 Borduas, A. G., 25, 82 Borek, F., 276, 293, 298, 300 Borsa, J., 214, 246 Bouersox, B. E., 6, 29, 54, 86 Boulanger, P., 212, 246 Boullenne, C., 65, 83 Boulton-Jones, M., 64, 65, 85 Bourne, H. R., 124, 125, 148, 150, 155, 180, 186 Boussac-Aron, Y., 119, 189 Bovee, E. C., 98, 184 Bowden, J. P., 291, 306,308 Bowers, C. Y.,272, 298 Boyd, G. W., 264, 265, 266, 267, 298, 299, 305 Boyd, N. R. H., 271,298 Boyd, W. C., 291,303 Boyden, S. U., 152,180 Boyse, E. A., 7, 8, 76, 88 Bradish, C. J., 199, 239, 246 Bradley, M. O., 98, 128, 132, 155, 192 Bradvica, H., 285, 310 Braf, Z. F., 38, 76 Brandon, F. M., 209, 233, 247 Brantigan, C. O., 46, 82 Brasfield, D. L., 260,282, 306 Brashlear, J. R., 109, 110, 116, 121, 178

Brashler, J. R., 27, 35, 74 Braun, W., 237, 246, 295, 307 Bray, R. E., 109, 192 Breese, S. S., Jr., 197, 199, 201, 210, 215, 245, 246, 252 Breindl, M., 202, 226, 246 Brendel, W., 2, 10, 29, 30, 33, 38, 46, 63, 86, 67, 68, 70, 72, 73, 76, 82, 86, 87, 90 Brent, L., 9, 13, 25, 28, 29, 35, 38, 39, 56, 60, 75, 76 Brettschneider, L., 4, 16, 22, 46, 66, 67, 68, 69, 70, 82, 86, 89 Bright, R., 295, 297 Brisson, G., 97, 186 Brittinger, G., 164, 184 Brochier, J., 6, 9, 13, 27, 28, 29, 30, 62, 63, 64, 70, 76, 77, 87, 90 Brock, W. A,, 275, 299 Brocklehurst, W. E., 102, 103, 180 Brocteur, G., 65, 83 Broder, I., 100, 101,180 Brodie, G. N., 131, 132, 178, 180 Brogan, T. D., 154, 180 Brooks, D. K., 67, 76 Brooksby, J. B., 212, 239, 246, 251 Brown, B. L., 279, 298 Brown, F., 198, 199, 200, 201, 203, 204, 223, 236, 237, 238, 241, 243, 244, 246, 247, 250, 251, 252, 253 Brown, J. J., 266, 307 Brown, K. J., 15, 83 Brown, R. A., 202, 249 Brown, R. J., 225, 246 Brown, S. M., 44, 67, 89 Brumfield, H. P., 212, 246 Brummelhuis, H. G. J., 22, 76 Brune, K., 56, 79 Bmnner, H. R., 265, 266, 298, 304 Brunstetter, F. H., 28, 37, 63, 76, 77, 83 Bryant, C. J., 61, 81 Bryant, R. E., 136, 137, 154, 180, 181 Bryon, P. A,, 9, 13, 29, 30, 70, 77, 90, 132, 180 Buchanan, K. D., 269,303 Buchniann, A. E., 73, 88 Buckley, C. E., 111, 73, 83 Buescher, E. L., 212, 225, 226, 246 Buffe, D., 7, 79

AUTHOR INDEX

Buhler, F., 67, 90 Buhler, F. R., 265, 298, 304 Bui-Mong-Hung, 10, 39, 80 Bull, G., 67, 91 Bultmann, B., 279, 302 Bunting, C. H., 31, 76 Burde, R. M., 10, 38, 60, 76, 91 Burger, G. M., 8, 75 Burgus, R., 272, 299 Burke, G. C., 293,298,309 Burke, J. F., 14, 40, 82, 85, 285, 299 Burke, J. S., 149, 187 Burleson, R., 13, 27, 65, 83 Burnet, F. M., 222, 246 Burroughs, J. N., 198, 200, 201, 246, 253 Burroughs, M. A. K., 243, 248, 252 Burstein, S., 275, 299 Burtin-Laborde, C., 274, 299 Bussolati, G., 269, 299, 307 Butcher, R. W., 97, 176, 190 Butler, G. C., 281, 299 Butler, J., 295, 297 Butler, V. P., Jr., 255, 257, 258, 259, 260, 282, 283, 284, 292, 295, 298, 299, 305, 306, 308, 309, 310 Butler, W. T., 67, 68, 76 Butterworth, B. E., 198, 246 Byfield, P. G. H., 267, 299 Bygdeman, S., 97, 130, 132, 180 Rymes, D. J., 268, 269, 299, 310

C Cachera, J.-P,, 10, 39, 71, 78, 80 Caen, J., 129, 186 Caffrey, R. W., 31, 59, 60, 76, 78 Cain, M. C., 264, 299 Cain, M. D., 264, 268, 299, 302 Cajano, A., 34, 77 Calderon, J., 280, 300 Caldwell, B. V., 275, 277, 278, 299, 307 Caliguiri, L. A., 203, 205, 246, 247 Calverley, R. K., 283, 299 Cameron, J. S., 64, 65, 85 Campbell, D. H., 276, 277,302 Campbell, J. E., 291, 303 Canales, C. O., 64, 65, 90 Caner, J. E. Z., 161, 180 Canfield, R. E., 260,292,306

315

Carbonara, A. O., 218, 250 Card, J. L., 199, 215, 252 Cardiff, R. D., 221, 247 Carlsson, S. A,, 107, 180 Carlton, J. A., 124, 186 Carnaghan, R. B. A., 27, 28, 79 Caron, G. A,, 2, 77 Carpenter, C. B., 14, 22, 29, 31, 39, 60, 70, 76, 80 Carraz, M., 6, 9, 13, 25, 29, 30, 33, 70, 77, 82, 90 Carroll, G. F., 59, 75 Carruthers, B. M., 161, 180 Carswell, E., 290, 306 Cartwright, B., 200, 203, 204, 236, 241, 243, 244, 246, 247, 250, 251 Casals, J., 222, 247 Caspary, E. A., 29, 77, 78 Castermans, A., 65, 80 Catt, K. J., 264, 265, 276, 277, 278, 299, 300, 305 Cattam, A., 71, 84 Cavallo, T., 63, 66, 70, 84 Ceglowski, W. S., 199, 247 Centeno, E. R., 291, 299 Cerilli, G. J., 4, 25, 29, 30, 38, 44, 52, 55, 62, 63, 65, 66, 77, 89 Cemosek, R. M. G., 275, 299, 304 Chakrabarty, M., 243, 248 Chakravarty, N., 107, 109, 117, 179, 180 Chamberlain, D. A,, 283, 299, 310 Chambers, D. A., 128, 129, 171, 180,190 Chan, S. P., 237, 252 Chandra Sekhar, N., 129,192 Chang, P., 266, 298 Chanock, R. M., 196,247 Chao, F. C., 132, 190 Chapman, C., 283, 301 Chard, T., 9, 77, 270, 271, 272, 273, 298, 299,300,305 Chare, M. J., 44, 77 Charlesworth, J., 63, 65, 66, 70, 88 Charney, J., 202, 250 Charters, A. C., 267, 306 Chase, L. R., 132, 180, 273, 309 Chayen, J., 28, 34, 78 Cheevers, F. S., 289, 300 Chen, B., 166, 181

316

AUTHOR INDEX

Chen, J. P., 258, 259, 282, 299 Chessin, L., 61, 83 Chesterman, F. C., 41, 68, 79 Chew, W. B., 3,31, 77 Chiba, C., 11, 77 Chirigos, M. A., 237, 252 Chisholm, D. J., 268, 269, 271, 272, 299, 304,310 Choay, J., 64, 71, 84 Choppin, P. W., 204, 205, 24% Chopra, I. J., 279, 299 Chouroulinkov, I., 38, 79 Chow, N., 206,208,214,247 Christian, C. L., 296, 297, 308 Christian, H. A., 3, 77 Christine, M., 296, 299 Christlieb, A. R., 266, 299 Chung, H. W., 277, 306 Churcher, G., 215, 252 Churchich, J. E., 280,299 Churchill, W. H. J., 279, 299 Cinader, B., 59, 77 Cinotti, G. A., 296, 297 Ciofalo, F., 284, 299 Claman, H. N., 28, 37, 63, 76, 77, 83 Clark, J. G., 20, 26, 33, 46, 47, 74, 77 Clark, M. B., 267, 299 Clarke, D. H., 217, 247 Clarke, M., 28, 77 Clayman, J. A., 27, 77 Clem, L. W., 209, 227, 246 Clendinnen, B. G., 269, 307 Clerici, E., 52, 77 Cleton, F. J., 25, 30, 31, 36, 38, 40, 75 Cline, M. J., 117, 121, 148, 149, 155, 180 Cluff, L. E., 137, 179 Clunie, G. J. A., 38, 77 Cochrane, C. G., 111, 112, 118, 134, 136, 140, 141, 146, 179, 183, 189, 190 Cochrum, K. C., 22, 86 Coffey, R. C.,112, 114, 166, 180, 182 Coghlan, J. P., 264,299 Cohen, C., 27, 77 Cohen, E. L., 265,299 Cohen, I., 131, 180 Cohen, M. B., 274,299

Cohen, S., 269, 295, 296, 299, 304, 305, 308 Cohn, R. B., 5, 84 Cohn, R. H., 98, 155, 180 Cohn, Z. A., 147, 148, 156, 157, 158, 159, 180, 181, 184 Cole, L. J., 26, 35, 79 Colley, D. G., 8, 77 Collier, B., 98, 192 Collier, E. M., 139, 191 Collins, R. D., 156, 183, 265, 309 Collins, W. P., 277, 300 Collste, L., 28, 83 Colman, R. W., 267,300 Colobert, L., 75 Colquhoun, D., 102, 103, 180, 181 Colten, H. R., 124, 181 Colucci, J., 279, 280, 306 Colwell, E. J., 140, 181 Compans, R. W., 204, 205, 247 Condemi, J. J., 287, 298 Conn, J. W., 265,299 Conrad, D. H., 281, 300 Cook, M. W., 23,Ql Cooke, R. A., 37, 91 Cooley, D. A,, 67, 71, 76, 77, 84 Coombs, R. R. A., 151,181,185 Cooney, R. M., 15, 83 Cooper, H. L., 164,166,181 Cooper, M. R., 157,181 Cooper, N. S., 134,187 Cooper, S., 36, 54, 82 Cooperband, S. R., 63, 65, 66, 70, 77, 84 Cora-Figueroa, E., 295, 307 Cordoba, F., 280, 300 Corn, M., 149, 181 Cornette, J. C., 286, 300,304 Corson, J. M., 30, 40, 76 Cottier, H., 31, 77 Couch, R. B., 196, 251 Coulling, I., 270, 272,307 Coupland, G. A. E., 269,304 Courtenay, J. S., 6, 91 Courtenay, T. H., 9, 25, 28, 29, 38, 39, 60, 76, 79 Cowan, K. M., 198, 199, 200, 201, 202, 203, 204, 211, 212, 213, 215, 218, 219, 223, 226, 229, 234, 237, 240, 243, 244, 247, 248, 249, 250, 252

AUTHOR INDEX

317

Davis, R. C., 39, 54, 63, 65, 66, 70, 77, 83, 84 Davis, T. R. A., 267, 274, 300 Davis, W. C., 149, 180 Day, H. J., 95, 126, 142, 184 Dayton, D. H., 196, 247 Debabis, P., 25, 82 De Bakey, M. E., 67, 76 De Bernardo, R., 124, 186 De Boer, C. J., 210, 220, 246, 247, 250 De Bruyere, M., 88 DeChatelet, L. R., 157, 181 Dechayannes, M., 132, 180 decrombrugghe, B., 166,181 DeCrosse, J. J., 43, 84 de Duve, C., 147, 149, 179 Deftos, L. J., 267, 300 DeGiovanni, G., 65, 80, 83 de la Chapelle, A,, 28, 79 de la Chapelle, P. S., 28, 79 Delaunay, A., 154, 183 DeMeester, T. R., 52, 77 Dempster, A. P., 129, 190 Denman, A. M., 2, 14, 25, 27, 29, 30, 31, D 32, 34, 36, 41, 42, 44, 46, 47, 52, 53, 55, 57, 61, 63, 77, 78, 80, 87 da Costa, M., 280,300 Denman, E. J., 14, 29, 30, 31, 36, 42, 44, Dagher, R. K., 30, 40, 76 53, 57, 61, 63, 77, 78 Dahlquist, R., 113, 114, 181 Dennis, J., 202, 212, 225, 226, 241, 251 DAlesandro, P. A., 224, 252 Deodhar, S. D., 27, 39, 64, 66, 68, 70, Dalrymple, J. M., 221, 247 78, 264, 300 Dalton, N. T., 65, 66, 68, 70, 78 De Petris, S., 159, 163, 171, 175, 178, Dameshek, W., 44, 67, 89 181, 191 Danielson, G. K., 66, 70, 88 De Prada, M., 126, 179 Dardenne, M., 26, 27, 74 de Riel, K., 287, 304 Dardiri, A. H., 210, 247 Dersjant, H., 10, 24, 25, 30, 36, 37, 38, Darlington, R. W., 205, 253 40, 43, 75 Darrow, C. C., 24, 65, 77 Daugharty, H., 220, 247 Descotes, J., 38, 86 Davenport, F. M., 207, 233, 237, 238, Desiderio, D., 272, 299 247, 248 De Sousa, M. A., 42, 78 Davey, M. G., 96, 129, 131,181 Des Prez, R. M., 131, 134, 136, 137, 154,180,181,187 Davey, M. J., 165, 178 Davidsohn, I., 13, 83 Dessaulles, E., 264, 265, 307 Davidson, W. D., 267, 269, 306, 307 Detwiler, T. C., 131, 181 Davies, A. J. S., 45, 57, 83, 163, 181 Dev, V. G., 296,300 Davies, P., 159, 160, 175, 178, 181 de Vassal, F., 71, 84 Davis, A. T., 148, 155, 161, 179, 181 DeVries, A., 131, 180 Davis, J. O., 277, 303 de Vries, M. J., 24, 25, 30, 38, 40, 41, 75, 91 Davis, R. B., 136, 137, 138, 181, 189

Cowles, C. A., 223, 225, 252 Cox, J. S. G., 109,188 Cramer, R., 157, 190 Cran, E., 264, 299 Creech, H. J., 289,300, 301, 307 Crepin, Y.,10, 39, 80 Crick, J., 199, 236, 246, 247 Crile, G., Jr., 27, 39, 68, 78 Cronkite, E. P., 31, 77 Crosier, C., 60, 75 Crowder, J. G., 149, 181, 187 Crowe, V. G., 128, 184 Crowle, A. J., 32, 37, 53, 87 Cruickshank, A. H., 3, 29, 30, 77 Cullen, D. R., 268, 301 Cumberland, V. H., 269,304 Cunha, R. G., 227,247 Cunniff, R. V., 212,247 Cunningham, G. J., 28, 34, 78 Curd, J. G., 283, 284, 300, 309 Currey, H. L. F., 16, 42, 47, 77, 81 Cushing, J. E., Jr., 12, 26, 77

318

AUTHOR INDEX

de Weck, A. L., 281, 287, 300, 308 De Weerd, J. H., 13, 66, 70, 88 Deykin, D., 137, 190 Diamant, B., 104, 106, 107, 108, 112, 113, 114, 179, 181,189 Dias Da Silva, W., 118, 186 Dickerson, C., 24, 65,75 Dickey, J. F., 277, 303 Dietrich, E. B., 67, 76 Dietrich, F. M., 264, 300 Dietz, A. A., 29, 88 Dingle, J. T., 146, 151, 181, 185 Dionigi, R., 12, 74 Dismukes, W. E., 287, 304 Dixon, F. J., 209, 221, 250, 253 Doak, P. B., 65, 66, 68, 70, 78 Dobbelstein, K., 38, 86 Dodd, M. C., 267, 308 Doenhoff, M. J., 163, 181 Doherty, J. E., 283,300 Dolais, J., 270, 297 Domingo, E. O., 40,78 Domm, L. V., 279,310 Donaldson, D. M., 149,191 Dong, E., Jr., 71, 89 Doniach, D., 279,307 Dormont, J., 13, 15, 27, 64, 74, 78 Dougherty, S. F., 47, 80 Douglas, W. W., 97, 181 Douglass, J. H., 284, 302 Dourmashkin, R. R., 28, 81 Dow, D. S., 164, 189 Dresser, D. W., 33, 44, 46, 47, 87, 68, 78, 82, 90 Drings, P., 74 Drouet, J., 270, 297 Dubernard, J. M., 70, 79 Dubnick, B., 117, 185 du Bois, M. J. G. J., 26, 78 Dubost, C., 71, 78 Ducharme, D. W., 129,192 Duesberg, P. H., 204,247,251 Dufau, M. L., 276, 277, 278, 300 Duffus, W. P. H., 163, 171,191 Duhart, E., 73, 88 Dukes, C. D., 288,307 Dukor, P., 149, 150, 155, 159, 164, 192 Dulbecco, R., 209,247 Dumonde, D. C., 28, 34,78 Dumont, J. E., 98, 188

Duncan, G. W., 286,300 Dunker, A. K., 198,200,251 Dunn, E., 26, 28, 81 Dunn, T. F., 272, 299 Duriu, A., 6, 33, 90

E East, J., 42, 78 Eckert, E. A., 207, 247 Edel, F., 276, 300 Edelin, J. B., 88 Edelman, R., 41, 78 Edman, K. A. P., 104,181 Edsall, G., 15, 63, 69, 83, 85 Edwards, C. R. W., 272, 273, 300 Edwards, D. C., 6, 13, 30, 76, 78, 90, 91 Edwards, R., 277, 297 Eggers, H. J., 203,246,247 Eide, I., 266, 300 Eijsvoogel, V. P., 26, 78, 91 Eisele, J. W., 118, 186 Eisentraut, A. M., 270, 309 Ekins, R. P., 279, 298 Elek, S. D., 217, 247 Elliot, E. V., 45, 88 Ellis, F. G., 64,65, 85 Ellis, H. V., 111, 106, 182 Ellis, L. R., 29, 31, 38, 61, 78, 83 Ellis, S. M., 279, 298 Ellison, E. H., 268, 309 Elsbach, P., 145, 182 Elwin, K., 113, 181 Embling, P. H., 29, 30, 31, 57, 63, 78 Emment, Y.,277, 300 Emmons, P. R., 130, 142, 182, 183 Endahl, G. L., 267,308 Enderlin, F., 67, 90 Endo, K., 103, 104, 175, 191, 193 Enlitz, M., 32, 87 Enzmann, F., 272, 298 Epps, H. B. G., 6, 13, 78, 81, 90, 91 Erlanger, B. F., 255, 257, 259, 276, 277, 278, 295, 296, 297, 298, 299, 300, 301, 304, 306, 307, 308, 310 Estensen, R. D., 98, 148, 155, 161, 179, 181, 182 Estes, G., 138, 165, 182 Eulitz, M., 38, 46, 87 Evans, D. B., 64, 70, 84

AUTHOR INDEX

319

Fish, J. C., 64, 65, 90 Fisher, B., 13, 43, 60, 78 Fisher, D., 273, 308 Fisher, E. R., 13, 43, 60, 78 Fisher, J. P., 37, 91 Fishman, J., 276,277, 310 Fitch, F. W., 287, 310 Fitzgerald, M. G., 55, 88 Flax, M. H., 30, 31, 38, 47, 85 Flexner, S., 3,31, 78 F Floersheim, G. L., 54, 56, 71, 78, 79 Fabre, L. F., Jr., 276,277,301 Flynn, E . J., 281, 301, 309 Fanger, M. W., 165,182 Folkers, K., 272, 298 Faris, T. D., 4, 38, 89 Ford, P., 6, 13, 14, 82 Farley, D. B., 264, 302 Foreman, J. C., 117, 182 Farmer, R. W.,276, 277, 301 Forman, B., 4, 91 Farquhar, M. G., 147,179 Forsen, N. R., 295, 301 Farr, R. S., 261, 301, 306 Forsgren, M., 202, 248 Farrow, J. T., 285, 310 Forsling, M. L., 271, 272, 273, 298, 300, Fasnian, G. D., 264, 266, 302 305 Fateh-Moghadam, A., 38, 46, 82, 86, 87 Fortner, J., 70, 79 Faulkner, B. M., 152, 180 Foster, G. V., 267, 299 Faulkner, H. W., 38, 91 Fouillet, Y.,9, 13, 29, 30, 77 Favour, C. B., 161, 185 Fowler, R., 55, 88 Favre, H., 281, 301 Fox, M., 30, 40, 76 Fazekas de St. Groth, S., 205, 207, 209, FraenkeI-Conrat, J., 149, 191 211, 220, 223, 233, 237, 238, 247, Frailey, J., 275, 303 Francis, T., Jr., 217, 249 248 Feier, H., 293, 310 Franco, D. J., 49, 54, 79, 85 Feinman, R. D., 131, 181 Frank, H., 293, 297 Feinstein, A,, 212, 248 Frank, M. M., 243,248 Fell, H. B., 151, 181, 185 Franklin, E. C., 228,246 Franklin, R. M., 203, 213, 246 Fell, N., 274, 301, 307, 308 Felstead, P. H . A., 28, 85 Franks, D., 243, 248 Ferin, M., 277, 278, 279, 301, 306, 307 Franks, W. R., 289, 300, 301 Fredholm, B., 107, 110, 111, 179, 182 Ferlnga, J., 172, 182 Fredholm, J. C., 182 Ferrendelli, J. A,, 273, 301, 309 Freed, J. J., 175, 182 Festenstein, H., 55, 87 Freeman, J. S., 70, 89 Field, E. J., 29, 77, 78, 81 Freeman, M. J., 223, 226, 238, 248 Field, E. O., 28, 29, 39, 52, 78 Figarola, F., 2, 10, 39, 52, 55, 57, 82 Freeman, M. V . R., 296,301 Freeman, N. K., 204,248 Filipp, G., 275, 301 French, J . E., 142, 182 Fillipone, D. R., 29, 30, 87 Frenkel, E. P., 14, 25, 27, 30, 31, 32, 34, Finck, E., 278, 303 42, 44, 46, 47, 52, 57, 75, 77, 78 Fink, C. W., 224,248 Frey, P. A,, 291,303 Fink, M . A., 222, 223, 225, 252 Finkelstein, M. S., 209, 224, 225, 227, Frick, G., 87 Fried, J., 287, 310 248, 252 Friedlander, A., 276, 277, 305 Firkin, B. G., 127, 128, 185, 187 Friedman, A., 237, 253 Fischer, E. H., 280,301

Evans, G., 138,182,189 Evans, W . H., 155, 186 Evered, D. C., 283, 301 Everett, N. B., 30, 31, 32, 40, 59, 60, 61, 76, 78, 88, 90 Eyquem, A., 13, 15, 64, 78 Eysvogel, V . P., 25, 30, 31, 36, 38, 40, 75

320

AUTHOR INDEX

Giebenhain, M. E., 277, 302 Friedman, E., 59, 79 Giese, J., 265, 301 Friedman, H., 286, 301,303 Gigli, I., 153, 182 Friedman, H. J., 274, 299 Gilchrist, C., 41, 68, 79 Friedman, H. P., 237,248 Giles, G., 4, 16, 22, 68, 70, 89 Friedman, R. M., 41,44, 75 Fries, D., 6, 9, 13, 29, 30, 33, 62, 63, 65, Gillespie, E., 110, 124, 182 Gilliland, P. F., 270, 301 66, 67, 70, 77, 79, 90 Giordano, A. R., 212, 223, 225, 249 Friesen, S. R., 268, 301 Giradet, R., 59, 79 Frohman, L. A,, 270,301 Frommhagen, L. H., 202, 204, 241, 248, Girard, J. P., 281, 301 Girey, C. S . D., 267,300 251 Gizis, F., 280, 308 Froscio, M., 98, 188 Fuchs, S., 293, 295, 296, 298, 301, 303, Gjika, H., 285, 310 Gladstone, G. P., 144, 182 308 Glasgow, A. H., 63, 65, 66, 70, 77, 84 Fuchs, Y.,296, 301 Glass, E. A., 152, 157, 163, 185 Fudenberg, H. H., 29, 80, 158, 184 Gledhill, A. W., 41, 79 Fulthorpe, A. J., 217, 250 Gleich, G. J., 293, 301 Fulwood, M., 132, 179 Glenn, E. M., 37, 86 Funck, M., 79 Clew, G., 129, 130,178 Furuyama, S., 276, 277, 301, 305 Glick, S. M., 272, 273, 307, 308 Glynn, M . F., 138, 182, 188, 189 G Gocke, D. J., 136, 182, 264, 302 Gaebel, W. F., 293, 294, 297, 302 Gabbay, K. H., 98, 124, 181, 182 Gijtze, O., 136, 182, 212, 251 Gabriele, G., 29, 83 Goffman, J., 209, 250 Gagnon, F., 138, 184 Gaines, C., 40, 51, 88 Gold, H. K., 284, 302 Gallo, R. C., 295, 304 Goldberg, A. L., 97, 182 Ganguli, P. C., 267, 268, 301 Goldberg, N. D., 165, 183 Goldfinger, S. E., 283, 303 Gardner, C. A., 286,302 Goldstein, I. J., 293, 297, 305 Carver, R. M., 26, 35, 79 Golstein, I . R., 149, 150, 151, 192 Gaugas, J. M., 40, 41, 42, 47, 68, 79 Gonzalez, C., 280, 300 Gay, V. L., 277, 306 Good, R. A,, 6, 67, 85, 149, 157, 166, Gazet, J.-C., 39, 86 183,184,189 Gecelter, L., 46, 82 Gee, J. B. L., 148, 155, 186 Goode, J. H., 61, 83 Gelfand, M. C., 59,79 Goodford, P. J., 165, 178 Cell, P. G. H., 26, 28, 78, 88, 293, 298 Goodfriend, L., 276, 277, 278, 279, 302 Gerloff, R. K., 220, 221, 248 Goodfriend, T. L., 260, 263, 264, 266, Gershengorn, M., 279, 308 267, 270, 300, 302, 305 Gershman, H., 287, 301 Goodkofsky, I., 136, 182 Gerten, J. ( Gerten-Banes, J.), 264, 265, Goodman, J. W., 237, 248, 250, 270, 293, 302, 308 308, 310 Genvin, B. I., 214, 248 Goodwin, F. T., 265,298 Gewurz, H., 6, 10, 22, 24, 25, 47, 54, 62, 63, 65, 67, 68, 79, 80, 84, 85, 88, Gorden, P., 272, 273, 307 Gordon, J. J., 25, 82, 87, 290, 302 137, 182 Corer, P. S., 35, 79 Gharib, H., 279,301 Goth, A., 116,182 Gibbs, J. E., 28, 29, 39, 52, 78 Gottesman, M., 166, 181 Gideon, L., 55, 65, 77

321

AUTHOR INDEX

Gottlieb, C. W., 261, 303 Gottschalk, A,, 207, 248 Gotze, D., 32, 40, 87, 91 Gould, D., 132, 183 Govallo, V. I., 26, 79 Gowans, J. L., 34, GO, 79 Gowland, G., 9, 25, 28, 29, 35, 38, 39, GO, 75, 76, 79 Gozzo, J. J., 14, 56, 79, 85, 91 Grabar, P., 7, 8, 38, 74, 79 Grace, J. T., Jr., 214, 250 Graf, L., 293, 310 Graham, A. F., 214, 246 Grant, G. A,, 44, 79 Grant, J. A,, 125, 182 Grant, J. D., 276, 277, 285, 302 Grasbeck, R., 28, 79 Graves, J. H., 198, 199, 201, 202, 203, 212, 213, 215, 223, 226, 237, 240, 241, 243, 244, 246, 247, 248, 250 Grawe, L., 269, 305 Gray, H. M., 54, 79 Gray, J. G., 4, 14, 26, 28, 29, 30, 36, 44, 61, 79, 85

Greaves, M. F., 27, 28, 30, 63, 79, 87, 90 Greaves, M. W., 125, 163, 182 Green, K., 113, 114, 181 Greenough, W. B., 111, 130, 193 Greenwood, F. C., 260, 303 Greider, M. H., 269, 302, 305 Grette, K., 95, 126, 128, 182 Griepp, R. B., 71,89 Griffiths, F. B., 268, 310 Grim, C. E., 265,299 Grimley, P. M., 103, 122, 123, 191 Grogan, J. B., 40, 47, GO, 79, 87 Gross, R., 131, 182 Gross, S. J., 276, 277, 285, 302 Grossman, J., 164, 184 Groth, C. G., 4, 46, 68, 69, 71, 82, 89 Grumbach, M. M., 278, 304 Guardia, E. J., 291, 302 Guccione, M. A., 130, 171, 182, 189 Guiberleau, M., 47, 81 Cuillemin, R., 272, 299 Gunn, A., 2, 54, 79, 82 Gunnarsson, A., 10, 22, 54, 80, 84 Gurewich, V., 137, 190 Gurpide, E., 277, 302 Gusdon, J. P., 271, 302

Gushfson, L., 44, 85 Gutersohn, J., 287, 300 Guttman, R. D., 14, 22, 29, 31, 39, 46, GO, 80

Guyer, R. B., 265,299 Gwilliam, J. M., 109, 188

H Haas, G. J., 291, 302 Haase, A. T., 236, 248 Haber, E., 257, 259, 260, 264, 265, 266, 267, 276, 277, 279, 281, 282, 283, 284, 297, 298, 299, 300, 301, 302, 303, 307, 308, 309, 310

Hadden, E. M., 114, 165, 166, 180, 182, 183

Hadden, J. W., 114, 165, 166, 180, 182, 183

Haddox, M. K., 165, 183 Haegermark, O., 107, 111, 179 Haenen-Severyns, A. M., 65, 80, 83 Haferkamp, O., 279, 302 Hagen, P. O., 110,178 Hager, F. B., 70, 76 Hahn, B. H., 286, 302 Haibach, H., 279, 305 Haimovich, J., 209, 227, 248 Hale, E. M., 200, 249 Hale, J. H., 222, 248 Halgrimson, C. G., 16, 22, 68, 71, 86, 89 Hall, D. E., 109, 110, 188 Hall, L., 198, 246 Hallenbeck, G. A,, 10, 13, 66, 70, 88 Hallman, G. L., 71, 77 Halloran, M. J., 295, 302 Halpern, M. B., 10, 39, 80 Hamada, K., 98, 155, 190 Hamburger, J., 139, 185 Hamburger, R. N., 284, 302, 306 Hamer, J., 283, 308 Hammerling, U., 7, 88 Hammerschlag, E., 295, 304 Hamniond, W., 68, 86 Hnmpar, B., 209, 243, 248, 250, 252 Hampers, C. L., 70, 76 Hampton, J. R., 130, 182 Hampton, J. W. F., 217, 250 Haning, R., 276, 277, 303 Hansen, J. A., 56, 76

322

AUTHOR INDEX

Hansky, J., 268, 269, 302, 304 Harboe, A., 205, 206, 207, 248 Hardin, J. H., 147, 152, 191 Harding, H. B., 213, 251 Hardt, F., 34, 87 Hardy, D., 28, 83 Hardy, J. D., 60, 87 Hardy, M. A., 47, 54, 63, 69, 79, 80, 85 Harel, L., 295, 304 Hargis, G. K., 267,302 Harington, C. R., 281,299 Harrell, B. E., 61, 83 Harrington, J. T., 63, 65, 66, 70, 77, 84 Harrington, W; J., 139, 183 Harris, H., 161, 186 Harris, N. S., 59, 80 Harris, P. F., 52, 80 Harrison, M. J. G., 130, 142, 182, 183 Hart, D. A,, 165, 182 Hart, I. C., 271, 305 Harvey, J. J., 41, 68, 79 Haselkorn, R., 200, 251 HBskovcova, H., 40, 86 Haslam, R. J., 98, 128, 132, 183 Hastie, R., 122, 183 Hatton, D., 55, 65,77 Haughton, G., 91 Hausen, P., 11, 44, 87 Havas, H. F., 289,300 Haverback, B. J., 266,307 Havizy, M., 221, 245 Hawiger, A,, 183 Hawiger, J., 156, 183 Hawker, R. J., 24, 65, 75 Hawkins, D., 144, 146, 147, 148, 149, 150, 151, 153,183 Hayat, M., 64,71, 84 Haye, K. R., 106,183 Hayes, C. R., 6, 80 Hayes, J. R., 269, 303 Hays, E. F., 42, 91 Hayter, C. J., 283, 301 Head, L. R., 106,189 Hedding, L. G., 270,303 Hedwall, P. R., 265, 266, 303 Heffner, R. R., Jr., 225, 242, 248 Hegg, M. E., 12, 74 Heide, K., 15, 88

Heidelberger, M., 239, 248 Heine, W. I., 286, 301, 303 Heizer, W. D., 283, 303 Hellem, A. J., 128, 188 Henderson, W. M., 199, 246 Henle, G., 220, 248 Henle, W., 220, 248 Hennessy, A. V., 207, 233, 247 Hknon, M., 154, 183 Henricks, D. M., 277, 303 Henry-Aymard, M., 218, 251 Henson, P. M., 132, 133, 134, 135, 136, 138, 139, 140, 141, 142, 143, 144, 146, 147, 148, 149, 150, 152, 153, 154, 158,179,183 Herbert, V., 261, 303 Heremans, J. F., 218, 250, 252 Herlem, G., 61, 83 Herren, R., 138, 182 Herrmann, R. G., 128,184 Hersh, E. M., 67, 76 Herzig, D. J., 117, 185 Hess, S. M., 273, 310 Hess, W. M., 149, 191 Hess, W. R., 210, 220, 246, 247, 250 Hicklin, M. D., 41, 80 Hiebel, G., 72, 86 Hilleman, M. R., 237, 253 Hiller, M. C., 139, 191 Hinshaw, L. B., 137,184 Hintz, B., 32, 80 Hinuma, Y.,202, 234, 248, 249 Hirsch, J. G., 147, 148, 149, 151, 156, 157, 179, 180, 184, 189, 193 Hirsch, M. S., 40, 41, 68, 79, 80 Hirschhorn, K., 164, 184 Hirschhorn, R., 164, 184 Hirsh, J., 138, 182 Hirst, G. K., 204, 206, 210, 248, 250 Ho, H. H., 240, 241, 251 Ho, R. S., 279, 299 Hobart, M. J., 212, 248 Hockert, T., 279, 301 Hodes, H. L., 221,245 Hodges, C. V., 29, 31, 38, 61, 78, 83 Hogberg, B., 109, 184 Hoglund, S., 218, 250 Hoehn, R. J., 54, 80, 88

323

AUTHOR INDEX

Hoeschen, R. J., 283, 303 Hoffer, B. J., 273, 298 Hoffstein, S., 147, 148, 150, 192, 193 Holborow, E. J., 14, 29, 30, 31, 36, 42, 44, 57, 61, 63, 78, 80 Holland, J . J., 198, 249 Hollander, C. S., 279, 280, 306 Hollemans, H. J. G., 264, 265, 303 Hollingsworth, J. W., 139, 183 Hollingsworth, S., 271, 298 Hollis, V. W., Jr., 222, 223, 252 Holm, G., 28,80 Holmes, B., 149, 157, 184 Holmes, G. E., 212, 225, 226, 246 Holmes, J. T., 70, 79 Holmsen, H., 95, 126, 142, 184 Holt, L. J., 26, 28, 80 Holt, P. J. L., 28, 83 Holtz, G. C., 136, 138, 181 Holtzer, H., 155, 190 Honigman, M. N., 227,247 Honour, A. J., 130, 182 Hood, W. B., Jr., 283, 298 Hook, E. W., 149, 157, 181, 186 Hooker, S. B., 291, 303 Hopf, U., 67,76 Hopper, K., 277, 297 Horn, R. G., 156, 183, 184 Hornung, B., 73, 86, 90 Horowitz, H. I., 181 Horowitz, P., 288, 303 Hors, J., 13, 64,78 Horton, M . S., 213, 248 Horton, R., 276, 277, 278, 303 Horvat, J., 11, 81 Honvitz, M. S., 218, 248 Horzinek, M., 236, 251 Hotchkiss, J,, 277, 303 Hovig, T., 128, 131, 132, 184 Howard, M. R., 283, 299, 308, 310 Howard, R. J., 47, 80, 84 Howard, R. R. S., 80 Howes, H. A., 274,281, 308, 310 Howland, B. G., 267,309 Howland, W. S., 70, 79 Hoyer, B. H., 220, 221, 248 Hoyle, L., 204, 249 Hsu, K. C., 243, 250, 296, 297, 304, 308

Hsu, L. S., 146, 159, 160, 190 Huang, A. S., 214,246 Hubay, C. A,, 27, 77 Huber, H., 29, 80, 158, 184 Huffnian, D. H., 283, 303 Hughes, D., 29, 78, 81 Hughes, W., 269, 305 Hughes, W. L., 296,299 Hulnie, B., 30, 76 Hume, D. M., 29, 30,87 Hummeler, K., 202, 249 Humphrey, J. H., 28, 33, 81, 103, 104, 115, 117, 133, 136, 138, 178, 184, 243, 248 Humphrey, L. J., 26, 28, 81 Hunez, D., 134,187 Hung, P. P., 200, 249 Hunt, A. C., 10,36, 83 Hunter, R. L., 41, 44, 75 Hunter, W. M., 260, 261, 267, 301, 303 Hurwitz, E., 293, 295, 308, 309 Hurwitz, R., 16, 22, 68, 89 Hutchison, D. E., 66, 89 Hyslop, N. E., Jr., 287, 304

I Idelson, B. A., 63, 65, 66, 70, 77, 84 Ikegami, N., 203, 246 Immelman, E. J., 10, 36, 83 Inderbitzin, T., 3, 37, 81 Inglis, A. E., 2, 10, 39, 52, 55, 57, 82 Inouye, H., 295,298,303 Ireland, D. M., 132, 179 Irvin, W. S., 286, 302 Irvine, W. J., 42, 81, 268, 301 Isaza, J., 269, 305 Ishida, N., 202, 249 Ishizaka, K., 95, 99, 102, 105, 106, 115, 122, 123, 125, 182, 184, 186, 191, 212, 249 Ishizaka, T., 95, 99, 102, 105, 106, 115, 122, 123, 125, 184, 212, 249 Ismail, A. A. A., 277, 303 Isokavic, K., 11, 81 Ito, T., 276, 277, 303 Itskovitz, H. D., 265, 303 Iwasaki, H., 39, 81 Iwasaki, Y., 4, 6, 10, 13, 15, 16, 22, 25,

324

AUTHOR INDEX

26, 29, 30, 31, 32, 36, 37, 38, 46, 52, 53, 62, 63, 65, 66, 67, 69, 81, 89 Iyer, R. N., 293,297,305

Jackson, B. M., 269, 307 Jackson, D. B., 271, 298 Jacobs, A. A,, 152, 157, 190 Jacobs, R., 22, 82 Jacobson, M. F., 198, 249 Jacoby, G. A,, 264,302 Jaffe, B. M., 257, 260, 268, 269, 275, 303,305,306 Jahn, T. L., 98,184 James, K., 2, 10, 13, 14, 16, 20, 21, 23, 26, 27, 28, 29, 33, 34, 38, 39, 42, 44, 46, 47, 48, 74, 75, 77, 81, 86, 91, 92 James, V. H. T., 264, 277, 297,298 James, V. S., 13, 14, 29, 81 Jamieson, G. A,, 132,184 Janeway, C. A., 11, 82 Jankovic, B. D., 11, 81 Janoff, A., 111, 149, 184, 190 Jansen, C. R., 31, 77 Jaques, R., 136, 138, 184 Jarett, L., 273, 303 Jasin, H. E., 16, 81 Jasmin, C., 64, 71, 84 Jaton, J. C., 118, 185, 280, 283, 284, 285, 295,300,303,309 Javierre, M. Q., 121, 189 Jeejeebhoy, H. F., 10, 25, 27, 30, 44, 45, 54, 59, 77, 81, 87 Jeffcoate, S. L., 276, 277, 303 Jelinek, J., 152, 187 Jenkins, C. S. P., 129, 184 Jenkins, D. E., 67, 76 Jenner, M. R., 278, 304 Jensen, K. E., 217, 249 Jerne, N. K., 200, 249 Jesseph, J. E., 267, 308 Jetzer, T., 22, 24, 67, 85 Jiang, N. S., 277, 303 Jobin, F., 138, 139, 184 Johansson, S. G. O., 95, 115, 122, 125, 175, 184 Johnson, A. G., 211, 237, 250, 251

Johnson, A. R., 106, 108, 118, 119, 182, 184, 185 Johnson, B., 221, 250 Johnson, H. B., 132, 141, 142, 146, 150, 183 Johnson, H. M., 291, 303 Johnson, J. A,, 277, 303 Johnson, J. R., 63, 65, 66, 70, 88 Johnson, L., 47, 68, 79 Johnson, S. A,, 111, 127, 185 Johnson, W. J., 13, 88, 291, 299 Johnston, C. I., 273, 303 Johnston, J. H., 274, 308 Johnston, M. D., 198, 249 Johnston, W. E., 277, 303 Jones, R. N., 289,300 Jones, W. R., 81 Jooste, S. V., 9, 10, 11, 13, 14, 24, 26, 53, 81 Jordan, M. M., 137,184 Jorgensen, L., 131, 184 Jorgensen, M., 265, 301 Jouvenceaux, A., 9, 13, 29, 30, 70, 77, 90 Jubb, V. S., 81 Jubiz, W., 275, 303 Judd, K. P., 47, 61, 67, 68, 76, 81

K Kabat, E. A., 215, 243, 249, 250, 293, 297 Kagan, A., 273,307 Kahn, D. R., 71, 81 Kalden, J., 42, 81 Kaley, G., 161, 185 Kaliner, M. A., 104, 105, 106, 185, 188 Kaliss, N., 35, 81 Kalowski, S., 63, 65, 66, 70, 88 Kamen, B., 280, 308 Kamoun, P. P., 139, 185 Kantor, 0. S., 286, 302 Kanyerezi, B., 118, 185 Kapikian, A. Z., 197, 249 Kaplan, A. P., 160, 185 Kaplan, E. L., 267, 297 Kaplan, J. G., 164, 165, 189, 193 Kaplan, M. E., 243, 249 Kaplan, S. L., 278, 304 Kapur, B., 22, 82

AUTHOR INDEX

Karchmer, A. W., 287, 304 Karnovsky, M. J., 149, 150, 152, 153, 157, 163, 178, 185, 191 Karnovsky, M. L., 149, 150, 152, 153, 154, 157, 163, 178, 185 Karol, M. H., 295, 304 Karpatkin, S., 131, 139, 185 Karush, F., 293, 304 Karzon, D. T., 196, 221, 250 Kasel, J. A., 196, 251 Kashiwagi, N., 4, 12, 16, 22, 30, 36, 38, 46, 66, 67, 74, 82, 89 Kassai, T., 40, 82 Katagiri, S., 202, 234, 248, 249 Kataoka, T., 295,304 Katchalski, E., 131, 180 Kates, J. R., 214, 249 Katz, F. H., 277, 278, 304 Katz, H. I., 120, 188 Kauffman, M. H., 26, 28, 81 Kaufman, B. M., 284, 308 Kaufman, H. E., 196, 247 Kawano, M. H., 70, 79 Kawano, N., 70, 79 Kay, A. B., 102, 160, 165, 185 Kay, J. E., 163, 165, 185 Kaye, H., 41,80 Kayhoe, D. E., 24, 65, 77 Keehn, M. A., 209, 243, 248 Keiser, H. R., 267, 297 Kelch, R. P., 278, 304 Keller, R., 112, 185 Kelley, G. E., 63, 65, 66, 70, 88 Kelly, W., 38, 44, 74 Kelly, W. G., 277, 302 Kem, D. C., 265, 276, 277, 299, 305 Kennedy, L. A., 42, 83 Kennedy, T. L., 269, 303 Kent, A. B., 280, 301 Kenyon, J. R., 30, 76 Kesztyus, L., 274, 294, 310 Ketchel, M. M., 161, 185 Keutel, H., 70, 89 Khuller, G. K., 294, 304 Kiehn, E. D., 198,249 Kilbourne, E. D., 206, 207, 213, 249,253 Kilbum, E. P., 128, 185 Kilburn, K. N., 6, 85 Kilshaw, P. J., 13,56, 75, 76

325

Kimnra, A. Y., 264, 307 Kimura, J., 72, 90 Kingsbury, D. W., 204, 249 Kinlough-Rathbone, R. L., 128, 129, 132, 171, 182, 184, 185 Kinscherf, D. A,, 273, 301 Kipnis, D. M., 260, 273, 274, 301, 303, 309 Kirchheim, D., 29, 31, 38, 61, 78, 83 Kirkham, J. B., 199, 246 Kirstaedter, H. J., 28, 74, 91 Kirton, K. T., 286, 300, 304 Kissling, M., 71, 88 Kitagawa, M., 289, 290, 304, 309 Kitau, M. J., 270, 271, 272, 273, 299, 300 Klaiber, E. L., 277, 297 Klein, L. A., 272, 273, 307, 308 Klein, M., 26, 27, 74 Klein, S. W., 202, 250 Klein, W. J., Jr., 296, 304 Klenk, H.-D., 205, 206, 247, 249 Kliman, B., 264,265,302 Kloeze, J., 129, 185 Kloosterziel, W., 264, 265, 303 Knedel, M., 38, 86 Knight, C. A., 200, 202, 204, 207, 248, 249 Knight, E. J., 34, 79 Knight, G. J., 212, 249 Knight, R. R., 6, 44, 82, 84, 91 Knight, S., 28, 83 Knight, V., 67, 68, 76 Knobil, E., 277,303 Knoohuizen, M., 116, 182 Kochwa, S., 44, 67, 89 Koenig, M. G., 156,183 Koerner, T., 264, 265,302 Koh, S. W., 59, 77 Kommerell, B., 74 Konioto, S., 113, 191 Kondo, K., 98, 155, 190 Konomi, K., 64, 66,70, 78 Kooistra, J. B., 6, 29, 54, 86 Koopman, W. J., 105,185 Koprowski, H., 221, 236, 238, 251, 253 Korman, M. G., 268, 269,302,304 Kom, E. D., 153,185 Kornfeld, S., 163, 164, 187 Korosteleva, T. A., 289, 304

326

AUTHOR INDEX

Kosmiadi, G. A., 26, 79 Koumans, R. K. J., 14,82 Kourilsky, F. M., 212, 228, 246 Kowarski, A., 276, 277, 278, 297, 298 Kraegen, E. W., 270, 272, 299, 304, 310 Kraft, D.,287, 310 Krakaner, K., 160, 181 Krakoff, L. R., 265, 298 Kramer, J . H., 41, 80 Krebs, E. G., 280,301 Kretschmer, R., 11, 82 Krijnen, H. W., 22, 76 Kriss, S. L., 203, 249 Kriiger, P. G., 112, 113, 114, 181 Krueger, R. G., 215,251 Kugler, J. H., 52, 80 Kunske, R. D., 122,185 Kunz, C., 212, 225, 226, 253 Kuruvila, K. D., 64, 66, 70, 78 Kusner, E. J., 117, 185 Kvarstein, B., 154, 155, 185

1 Lacefield, W. B., 128, 184 Lacey, P. E., 96, 98, 185 Lachman, C., 296, 304,308 Lachmann, P. J., 151, 185 Lacombe, M., 10, 39,80 Lacour, F., 295, 304 Ladaga, L. G., 38,88 LaFontaine, G. S., 24, 65, 77 Lagg, S., 264,265,307 Lagunoff, D., 107, 108, 185, 186 Lajolo di Cossano, D., 28, 75 Lamanna, C., 134,186 Lamourenx, G., 25, 82, 87 Lamy, R., 13, 15, 64, 78 Lancaster, M. G., 154, 178 Lance, E. M., 2, 6, 9, 10, 11, 13, 14, 16, 19, 20, 23, 24, 25, 26,27, 28, 29, 30, 31, 32, 33, 35, 36, 37, 38, 39, 44, 45, 46, 47, 48, 49, 51, 52, 53, 54, 55, 57, 58, 59, 60, 61, 67, 68, 79, 81, 82, 89, 92 Land, W., 38, 46, 63, 67, 70, 76, 82, 87 Landon, J. C., 47, 80, 264, 270, 271, 272, 273,298, 299,300

Landor, J. H., 269,305 Landsteiner, K., 255, 304 Landy, M., 61,83 Lannigan, R., 42, 75 Lapointe, F., 138, 184 LaPorte, J., 198, 249 Laragh, J. H., 264, 265, 266, 298, 302, 304, 308 LaRaia, P. J., 105, 185, 188 Larsen, P. R., 279,304 Laster, L., 131, 192 Lastowecka, A., 98, 191 Laszlo, J., 73, 83 Latham, W. C., 15, 63, 69, 83, 85 Latour, M., 24, 76 Latzina, A., 63, 69, 85 Lau, K. S., 261, 303 Laver, M. C., 269, 304 Laver, W. G., 204, 205, 206, 207, 208, 213, 233, 237, 238, 245, 249, 253 Lavergne, M., 13, 64, 78 La Via, M. F., 28, 83, 157, 181 Law, L. W., 41, 42, 43, 68, 74 Lawrence, J. S., 3, 31, 77 Lawson, R. K., 29, 31, 38, 61, 78, 83 Lay, W. H., 152, 153, 154, 186 Lazarow, A., 261,306 Lazarus, L., 269, 271, 272, 299, 304, 310 Leachman, R. D., 67, 71, 76, 77, 84 Leahy, D. R., 161, 186 Lease, G. O., 207, 233, 247 Lebacq-Verheyden, A. M., 218, 252 LeBouvier, G. L., 202, 211, 240, 249 Lebowitz, M. M., 175, 182 Ledingham, J. C. C., 215, 249 Ledney, G. D., 25, 28, 39, 40, 41, 83, 91 Lee, C. L., 13, 83 Lee, E. H., 122,184 Lee, L. H., 215,252 Lee, M. R., 267, 300 Leeman* 263, 270, 305 Leen, T.F.,39 77 Legros, J. 271, 305 Lehrer, H., 258, 306 Lehrer, R. I., 124, 148, 150, 155, 180 Leibowitz, S . , 42, 83 Lejeune, G., 65, 80, 83

’*,

327

AUTHOR INDEX

Le Minor, L., 293,294,305 Lennette, E. H., 202, 212, 215, 225, 226, 228, 240,241, 249,251 Leonard, L. L., 223,249 Lepow, I. H., 118, 136, 182, 186 Leskowitz, S., 285, 299 Leslie, G. A., 209, 227, 246 Lessof, M. H., 42, 83 Leuchars, E., 45, 57, 83, 163, 181 Leuker, D. C., 16,83 Leventhal, M., 185 Lever, A. F., 266, 307 Levey, R. H.,4, 6, 8, 9, 10, 11, 12, 13, 14, 24, 25, 26, 27, 29, 30, 31, 33, 37, 38, 39, 40, 41, 42, 45, 47, 48, 54, 55, 57, 60, 65, 74, 81, 82, 83 Levin, W. C., 64, 65, 90 Levine, B. B., 149, 150, 153, 155, 187, 189, 287, 304 Levine, L., 70, 76, 258, 260, 264, 266, 275, 285, 286, 287, 294, 295, 299, 301, 302, 304, 306, 307, 308, 310 Levine, R. J., 97, 110, 182, 186 Levintow, L., 218, 221, 251 Levitsky, L., 278, 298 Levy, D. A., 116, 121, 122, 123, 124, 125, 186, 187, 188 Levy-Toledano, S., 129, 186 Lewis, E. J., 63, 69, 85 Lewis, J. L., 39, 54, 77, 83 Lewis, K. H., 291,303 Leznoff, A., 279,302 Liabeuf, A., 25, 82 Liacopoulos, P., 61, 83 Liakopoulou, A., 118, 189 Libanska, J., 186 Lichtenstein, L. M., 95, 106, 115, 116, 122, 123, 124, 125, 148, 150, 180, 182, 184,186,188 Lie, S., 29, 90 Lieberman, S., 259, 276, 277, 278, 298, 300,301, 304,306 Liebermann, G., 44, 75 Liebeskind, D. S. P., 296, 304 Lieblich, J., 279, 304 Liegeois, A., 56, 85, 91 Lilly, J., 16, 22, 68, 89 Lind, P. E., 213, 245

Lindahl-Kiessling, K., 162, 186 Lindenbaum, J., 283,305 Lindenmnnn, J., 11, 87 Lindner, H. R., 276, 277, 290, 297, 305 Lindquist, R. R., 14, 22, 29, 31, 39, 60, 80 Ling, N. R., 26, 28, 80, 83 Linscott, W. D., 158,184 Lipshutz, W., 269, 305 Liske, R., 243, 248 Littauer, U. Z., 295, 303 Little, J. R., 280, 308 Littledike, T., 267, 297 Liu, C. T., 257, 286, 297 Liu, S.-L., 213, 248 Livingston, D. M., 221, 251 Lloyd, J. V., 128, 186 Lobo, C., 218, 249 Lockwood, W. R., 156, 186 Loeffler, J., 111, 186 Logan, E., 14, 29, 81 Logsdon, P. J., 166, 180 Lomax, P., 276, 277, 302 Loor, F., 233, 249 Loos, J. A,, 164, 190 Loriaux, D. L., 276, 277, 306 LoSpalluto, J., 224, 248 Lotz, M., 161, 186 Louis, W. J., 266, 305 Lourie, S. H., 16, 81 Lovenberg, W., 111, 186 Lucas, C. P., 265,299 Lucas, D. O., 13, 27, 65, 83, 164, 186 Lucke, J. N., 10, 36, 83 Lucy, J. A., 156, 186 Luderitz, O., 293, 294, 305 Luduena, M. A,, 98, 128, 132, 155, 192 Liischer, E. F., 96, 128, 129, 131, 133, 137, 138, 179, 181, 186, 187, 189 Lukens, D., 202, 250 Lund, J. O., 265, 301 Lundgren, G., 28, 83, 84 Lundstedt, C., 41, 91 Lynch, J. M., 291, 306

M Macadam, R. F., 266, 307 McAuslan, B. R., 214, 249

328

AUTHOR INDEX

McCall, C. E., 157, 181 McCall, M. S., 270, 309 McCardle, R. J., 4, 38, 89 McCoy, H. V., 12, 74 McCune, C. L., 223, 225,252 McDevitt, H. O., 237, 251 Macdonald, G. J., 266,305 McDonough, E., 63, 69, 85 McDougal, D. B., Jr., 273, 301 McGill, R., 263, 270, 305 MacGillivray, M., 221, 250 McGovern, J. P., 288, 307 MacGregor, J., 266, 307 McGregor, S., 202, 249 McGuigan, J. E., 260, 267, 268, 269, 301, 302, 303, 305, 306, 309 McGuire, J. S., 263, 266, 267, 270, 302, 305 McKenzie, I. F. C., 29, 83 McKenzie, J. M., 279,305 McKenzie, W. N., 6, 85 McKercher, P. D., 212, 223, 225, 249 Mackler, B. F., 23, 91 McLaren, L. C., 220, 221,248 McLean, E. R., Jr., 161,186 MacLean, L. D., 2, 25, 38, 87, 88 McManus, J. P., 166, 182 Macmorine, D. R. L., 138, 149, 187, 188 McNeilly, A. S., 271, 305 McVicar, J., 218, 240, 249, 252 Madison, L. L., 270, 309 Madoff, M. A., 15, 63, 69, 83, 85 Makela, O., 227, 249, 251 Mage, M. G., 209, 243, 248, 250 Mahar, S., 209, 250 Mahlich, H., 67, 90 Maize], J. V., Jr., 198, 202, 221, 249, 251, 252 Majerus, P. W., 131, 132, 178, 180 Malaise, W. J., 97, 186 Malaise-Lagae, F., 97, 186 Malakian, A., 8, 77 Malawista, S. E., 110, 148, 149, 154, 155, 157, 158, 175, 179, 182, 186, 193 Malley, A., 23, 91 Malmgren, R. A., 41, 44, 75, 88 Mamelle, C., 70, 79

Mancini, G., 218, 250 Mandel, B., 203, 223, 224, 238, 249, 250, 252 Mandel, M. A., 20, 25, 26, 30, 43, 84 Mandel, W. J., 283, 305 Mandell, G. L., 149, 157, 186 Manganiello, V., 155, 186 Manhem, L., 27,89 Mannick, J. A., 63, 65, 66, 70, 77, 84 Manuel, L., 64,65, 85 Manuel, Y., 9, 13, 29, 30, 77, 90 Marbrook, J., 232, 234, 250 Marchioro, T. L., 4, 6, 10, 13, 15, 16, 22, 25, 26, 29, 30, 31, 32, 36, 37, 38, 46, 52, 53, 62, 63, 65, 66, 67, 69, 81, 89 Marcu, A,, 9, 85, 86 Marcus, A. J., 127, 128, 186, 188 Marcus, R. L., 137, 187 Marder, V. J., 139,191 Margherita, S. S., 279, 305 Margolis,'S., 124, 186 Mark, V. H., 285, 299 Markham, R., 232, 250 Markwardt, F., 131, 187 Marney, S. R., Jr., 134, 136, 137, 181, 187 Marquis, N. R., 129, 130, 187, 192 Marsh, D., 125, 182 Marshall, D. E., 274, 301 Marshall, V. R., 44, 84 Martin, D. P., 24, 65, 77 Martin, E. M., 213, 248 Martin, M. J., 271, 305 Martin, R. R., 149, 181, 187 Martin, S. J., 198, 202, 249, 250 Martin, W. J., 25, 27, 28, 30, 35, 45, 57, 84 Martineau, R. S., 293, 305 Martos, L. M., 243,248 Mashford, M. L., 266,267, 305 Mason, R. G., 131, 190 Mason, R. J., 155, 156, 186, 191 Massini, P., 129, 187 Math&,G., 64, 71, 84 Mathews, E. K., 97, 187 Matthews, R. E. F., 232, 234, 250 Maulitz, R. M., 283, 305

AUTHOR INDEX

Maurer, J. E., 291, 306 Mautner, V., 236, 248 May, C. D., 149, 150, 155,187 May, J., 63, 65, 66, 70, 88 Mayberry, W. E., 279,301 Mayer, E. A., 215, 249 Mayer, M. M., 137, 187, 202, 211, 240, 250, 251 Mayes, D. M., 276, 277, 301, 305 Mayor, H . D., 202,249 Mazow, J. B., 67, 68, 76 Mazzei, D., 28, 63, 75, 84 Meade, K . M., 274,300 Mende, R. C., 268,309 Mears, D., 63, 65, 66, 70, 88 Medawar, P. B., 2, 4, 6, 8, 9, 10, 11, 12, 13, 14, 16, 24, 25, 26, 27, 29, 30, 31, 33, 37, 38, 39, 40, 42, 45, 46, 47, 48, 54, 55, 57, GO, 76, 79, 81, 82, 83, 84 Mee, A. D., 64,70, 84 Meek, E. S., 9, 84, 89 Meeker, W . R., Jr., 137, 181 Meinzel, E., 73, 86 Melani, F., 260, 292, 305 Melby, J. C., 278, 305 Melli, G., 63, 84 Mellow, M. H., 283, 305 Melmon, K. L., 125, 148, 149, 155, 180 Melnick, J. L., 210, 252 Melvin, K. E . W., 267, 297, 305, 309 Mempel, W.,40, 90 Menard, J., 265, 305 Mendelsohn, J., 163, 164, 187 Mendoca, M., 10, 39, 80 Meneghelli, V., 157, 193 Merchant, B., 61, 83 Meredith, J., 65, 66, 68, 70, 78 Mergenhagen, S. E., 47, 80, 84, 137, 182 Merino, G . E., 59, 80 Merkel, F. K., 6, 22, 24, 63, 67, 84, 85, 86 Merrill, J. P., 14, 22, 29, 31, 39, GO, 80 Merritt, K., 211, 237, 250 Mesmer, B. J., 71, 84 Messiner, K., 38, 86 Messner, R. P., 152, 187, 189 Metchnikoff, E., 3, 84

329

Metzger, H., 103, 122, 123, 191 Michal, F., 127, 187 Michel, I. M., 273, 310 Michlniayr, G., 29, 73, 80, 86 Middleton, E., Jr., 166, 180, 183 Midgley, A. R., Jr., 260, 277, 303, 306 Miescher, P., 34, 187 Migeon, C. J., 276, 277, 278, 297, 298 Mikaeloff, P. H., 38, 86 Mikhail, G., 277, 306 Mikol, C., 2, 87 Milam, J. D., 71, 77 Milgroni, F., 281, 310 Miller, A., 55, 65,77 Miller, D. A., 296, 300 Miller, G. L., 289, 307 Miller, H. H., 267, 305 Miller, J., 5, 84 Miller, J. F. A. P., 27, 28, 30, 35, 45, 57, GO, 84 Miller, M., 273, 306 Miller, M. E., 153, 187 Miller, 0. J., 295, 296, 300, 301 Miller, W., 224, 248 Mills, D. C. B., 130, 187 Minard, P., 121, 187 Minden, P., 261, 306 Minnich, V., 139, 183 Mitchell, G. F., 45, 84 Mitchell, G. W., 152, 157, 190 Mitchell, J. R. A., 130, 142, 182, 183, 187 Mitchison, N. A,, 45, 57, 84 Mitnick, H., 193 Mitsuma, T., 279, 280, 306 Miyamoto, K., 243, 248, 250 Miyazawa, M., 7 , 76 Mizrahi, R., 287, 308 Mizutani, S., 214, 252 Moberg, A. W., 6, 10, 22, 24, 25, 47, 54, 62, 63, 65, 67, 68, 79, 80, 84, 85, 86, 88 Mocerelli, P., 52, 77 Mohlmann, 212, 252 Moller, E., 34, 84 Mdler, G., 28, 34, 44, 83, 84 Mojovic, M., 10, 39, 80 Mold, J. D., 291, 306, 308

330

AUTHOR INDEX

Monaco, A. P., 4, 10, 14, 15, 16, 24, 26, 27, 28, 29, 30, 31, 32, 36, 38, 39, 40, 44, 45, 47, 49, 52, 55, 56, 60, 61, 62, 63, 69, 73,

25, 37, 54, 74,

79, 80, 83, 85, 87, 91 Monath, T. P. C., 225, 250

Mongar, J. L., 102, 103, 104, 108, 117, 125, 175, 181, 182, 187, 189 Mongin, J., Jr., 129, 192 Monovich, R. E., 6, 23, 29, 37, 54, 86 Montgomerie, J. Z., 65, 66, 68, 70, 78 Moore, C. V., 139, 183 Moore, G. E., 6, 22, 24, 63, 67, 84, 85, 86 Moore, K.,32, 85 Moorhead, T. G., 4, 85 Moppert, J., 67, 90 Moran, N. C., 106, 108, 109, 118, 119, 182,184,186,187

Moreno, C., 243, 250 Morgan, C., 243, 250 Morgan, C. R., 261, 306 Morgen, R. O., 67, 68, 76 Morisette, M., 139, 184 Morrill, L. M., 275, 299 Morris, P. J., 29, 40, 83, 85 Morse, S. I., 157, 180 Mosedale, B., 6, 13, 27, 28, 78, 82, 85, 90, 91, 92

Moses, A. M., 273, 306 Mota, I., 99, 101, 106, 187 Movat, H. Z., 134, 138, 149, 182, 187, 188

Mowat, A. G., 161,187 Miiller-Eberhard, H. J., 127, 136, 158, 182, 184, 191

Mueller-Eckhardt, C., 137, 138, 139, 187 Miirer, E. H., 128, 131, 132, 188 Muir, J. D., 118, 119, 190 Mulberry, G., 291, 303 Mulligan, J. J., 226, 238, 250 Munck, O., 265,301 Munniz, F., 64, 65, 90 Munro, A,, 64, 65, 85 Munsick, R. A., 271, 273, 310 Munyon, W. H., 214, 250 Murad, E., 155, 191 Murakami, W. T., 258, 306 Muresan, T., 9, 85, 86 Murphy, B. E. P., 277, 306

Murphy, E. A., 138, 182 Murphy, F. A., 40, 41, 80 Murphy, J. S., 204, 247 Murray, A. W., 98,188 Murray, J. E., 70, 76 Muschel, L. H., 44, 85 Mustard, J. F., 96, 126, 127, 128, 129, 130, 131, 132, 133, 134, 138, 143, 171, 184, 185, 187, 188, 189 MyllylS, G., 139, 188

N Nabseth, D. C., 63, 65, 66, 70, 77, 84 Nachman, R. L., 128, 131,188 Nagaya, H., 5, 6, 14, 30, 37, 39, 81, 85 Nagayama, M., 137, 188 Nahas, G. G., 176,192 Nahmias, A. J., 41, 80 Nairn, R. C., 8, 87 Najarian, J. S., 6, 10, 22, 24, 25, 47, 54, 59, 62, 63, 65, 67, 68, 79, 80, 84, 85, 86, 88

Nakamoto, S., 64, 66, 70, 78 Nakano, M., 237,246 Nava, C., 13, 32, 86, 88 Naysmith, J. D., 26, 39, 81, 86 Nehlsen, S. L., 10, 32, 36, 38, 41, 42, 51, 52, 53, 54, 59, 69, 75, 76, 79, 82, 86, 88

Nelson, D. S., 133, 134, 152, 188 Nelson, J. C., 279, 299 Nelson, R. A., Jr., 134, 153, 182, 188, 191 Nelson, R. L., 120, 188 NBmec, M., 40,86 Neri, L. L., 128, 129, 171, 180, 190 Neri, R. O., 276,278, 306 Neurath, A. R., 196, 200, 236, 237, 238, 250

NBve, P., 98, 188 Neveu, T., 26, 27, 74 Newberry, W. M., Jr., 165, 191 Newman, J. E., 29, 81 Newman, J. F. E., 241, 243, 244, 246 Newton, M. A., 265,298 Newton, W. T., 259, 260, 268, 269, 275, 303, 305,306

Nicolson, G. L., 171, 191 Niedieck, B., 295, 306 Nielsen, M. D., 265, 301

331

AUTHOR INDEX

Nieschlag, E., 257, 276, 277, 306, 310 Nihei, N., 279, 306 Nilsson, U. R., 153, 187 Nisbet, N. W., 55, 86 Nishizawa, E. E., 128, 129, 138, 186, 188 Nisonoff, A., 165, 182, 227, 251 Nissley, P., 166, 181 Niswender, G. D., 260, 277, 303, 306 Nitecki, D. E., 270, 308 Noakes, C. A,, 265, 309 Nojima, S., 295,304 Nolan, B., 28, 30, 36, 38, 41, 53, 73, 77 Nora, J. J., 67, 71, 76, 77, 84 Nordman, C., 28, 79 Norrby, E., 210, 250 North, J. D. K., 65, 66, 68, 70, 78 North, R. J., 152, 158, 180, 188 Norton, W. L., 165,182 Noseworthy, J., 152, 157, 163, 185 N o w , L., 9, 86 NossaI, G . J. V., 233, 236, 250 Nossel, H. L., 260, 292, 306, 307 Notkins, A. L., 47, 80, 209, 221, 245, 248, 250 Nouza, K., 40, 86 Novicky, R., 236, 251 Nugent, C. A., 276, 277, 301, 305 Nussenzweig, V., 152, 153, 154, 186

Old, L. J., 7, 8, 76, 88, 290, 306 Oldstone, M. B. A,, 209, 250 Oliveira, B., 136, 137, 188, 190 Oliver, G. C., Jr., 260, 282, 283, 306 Ollodart, R., 290, 306 Olsson, C. A., 63, 65, 66, 70, 77, 84 Ono, K., 16, 22, 82, 89 Oppenhoff, I., 264,302 Orange, R. P., 99, 100, 102, 104, 105, 106, 184, 185, 188 Orgel, H. A., 284,306 Orr, T. S. G., 109, 110, 188 Ortaldo, J. R., 140, 181 Ortolani, C., 63, 84 Osler, A. G., 116, 122, 123, 125, 133, 135, 136, 137, 140, 141, 182, 188, 190, 191, 212, 226, 238, 250, 251 Osoba, D., GO, 84 Osterland, C. K., 286, 302 Otherson, H. B., 14, 30, 31, 38, 47, 74, 85 Ouchterlony, O., 200, 217, 250 Ondin, J., 217, 250 Ovary, Z., 120, 121, 188, 191, 192, 212, 228,246, 250 Overby, L. R., 200, 249 Owen, K., 30, 76 Oyama, S. N., 273,307 Oyer, P. E., 260, 292, 305 Ozerkis, A. L., 54, 88

0 Oades, Z. G., 132, 146, 147, 148, 150, 153, 183 Oakley, C. L., 217, 250 Oberling, R., 72, 86 O’Brien, J. R., 128, 129, 188 O d d , W. D., 267, 276, 277, 297, 306 Odya, C., 265, 303 Ofstad, E., 110, 178 Ogg, C. S., 64, 65,85 Oginsky, E. L., 289,300 Ogra, P. L., 196, 221, 250 Ohanian, S. H., 10, 27, 88 Ohnian, J. L., 95, 179 Ohta, H., 147, 149,188 Okamnra, Y., 155, 190 O’Kane, H. O., 13, 32,86,88 Oken, D. E., 265,266,306 Okinoto, J. T., 22, 86

P Packham, M. A., 96, 126, 127, 128, 129, 131, 132, 133, 134, 138, 143, 171, 182, 184, 185, 188, 189 Padawer, J., 110, 189 Page, L. B., 264, 265, 302, 307, 310 Pagliara, A. S., 273, 309 Palczuk, N. C., 295, 307 Palutke, M., 53, 87 Pan, I. C., 220, 250 Paoletti, E., 214, 250 Pnppenheimer, A. M., 4, 28, 86 Parish, W. E., 99, 149, 189 Park, B. H., 149, 157, 184, 189 Parke, J. A. C., 27, 28, 85 Parke, P., 13, 27, 65, 83 Parker, B. M., 260, 282, 283, 306

332

AUTHOR INDEX

Parker, C. W., 165, 166, 191, 257, 260, 272, 273, 274, 275, 282, 283, 286, 287, 295, 298, 302, 303, 306, 307, 308, 309 Parks, W. P., 214, 221, 245, 251 Parrott, D. M. V., 32, 42, 78, 86 Pastan, I., 166, 181 Patriarca, P., 157, 190 Patterson, R. J., 106, 108, 122, 149, 185, 189 Paul, B. B., 152, 157,190 Paul, W., 102,189 Pavlovsky, S., 73, 88 Pearlman, D. S., 147, 153, 154, 189 Pearse, A. G. E., 268, 269, 270, 272, 299,301, 307 Peart, W. S., 30, 76, 264, 265, 266, 298, 305 Pecco, P., 28, 75 Peck, E. B., 289, 307 Peck, R. M., 289, 300,307 Peeters, S., 149, 150, 153, 183 Peixoto, J. M., 121, 189 Pellizzari, E. D., 276, 277, 301 Penn, I., 4, 16, 22, 67, 68, 70, 71, 86, 89 Penttinen, K., 139, 188 Pereira, H. G., 204, 218, 236, 248, 250, 251 Perel, E., 276, 277, 290, 297, 305 Perelmutter, L., 118, 189 Perera, B. A. V., 117, 187, 189 Perkins, F. T., 39, 88 Perkins, W. D., 32, 61, 78, 191 Perlmann, P., 28, 80 Permutt, M. A,, 272, 273, 307 Peron, F. C.,277, 307 Perper, R. J., 6, 10, 22, 23, 27, 29, 35, 37, 53, 54, 74, 80, 86, 87 Perrament, M. F., 61,83 Perrin, J., 9, 13, 29, 30, 70, 77, 90 Perry, B. T., 204,245 Perry, D. W., 130,189 Perry, H. M., 286, 302 Pert, W. S., 76 Peskar, B. A., 294, 307 Peskar, B. M., 294,307 Petersen, M. J., 272, 308 Peterson, C., 112, 113, 181, 189

Peterson, J., 129, 130, 193 Peterson, R. D. A,, 162, 186 Petricciani, J. C., 10, 39, 91 Petrovic, S., 11, 81 Pettersson, U., 218, 250 Peuchot, C. H., 73, 88 Pfisterer, H., 73, 86, 90 Pflueller, S. L., 133, 138,189 Phelps, P., 151, 190 Philipson, L., 218, 250 Phillips, A. W., 6, 82, 91 Phillips, B., 39, 86 Phillips, B. A., 202, 249 Phillips-Quagliata, J. M., 153, 189 Phondke, G. P., 29,86 Picache, R., 4, 70, 89 Pichlmayr, I., 86 Pichlmayr, R., 6, 10, 14, 15, 29, 30, 33, 38, 46, 72, 73, 82, 86, 87, 90 Pickup, P. M., 104,178 Pillai, K., 222, 248 Pincus, T., 243, 252 Pirofsky, B., 66, 72, 87 Plagemann, P. G. W., 98, 155, 182 Plan, M., 6, 33, 90 Plan, R., 24, 76 Planinsek, J., 289, 290, 304 Planterose, D. N., 203, 250 Plate, J. M., 10, 27, 88 Playfair, J. H. L., 27, 30, 63, 79, 87, 90 Plescia, 0. J., 295, 307 Pletscher, A., 126, 179, 189 Pogoriler, C., 279, 307 Poisner, A. M., 97, 98, 114, 189 Polak, J. M., 270, 272, 307 Polak, L., 36, 90 Polat, H., 275, 307 Polatnick, J., 203, 204, 213, 214, 250, 252 Pollard, T. D., 156, 191 Polley, M. J., 158, 184 Polson, A,, 217, 250 Pomeroy, B. S., 212, 246 Pons, M. W., 204, 206, 248, 250 Poole, J. C. F., 142,182 Porter, G. A,, 72, 87 Porter, K.'A., 4, 6, 10, 13, 15, 16, 22, 25, 26, 29, 30, 31, 32, 36, 37, 38, 39,

AUTHOR INDEX

46, 52, 53, 62, 03, 65, 66, 67, 08, 81, 87, 89 Porter, R. R., 21, 87 Poste, G., 97, 189 Potts, J. T., Jr., 267, 300 Potworowski, E. F., 8, 87 Poulsen, K., 266, 298 Powell, A. E., 27, 77 Powell-Jackson, J. D., 266, 307 Powers, E., 287, 301 Powers, K. G., 40, 75 Preer, J. R., Jr., 218,251 Premachandra, B. N., 279, 305, 307 Pressman, D., 6, 91, 289, 290, 304, 309 Preston, F. W., 29, 88 Preussner, S., 87 Prevost, R., 13, 90 Prhvot, J., 9, 13, 29, 30, 70, 77, 90 Prince, G. H., 13, 87 Procupez, T., 260, 292, 306 Prout, T. E., 270,301 Prouvost-Danon, A., 102, 104, 106, 119, 120, 121, 189,192 Proveda, V., 283, 303 Pruzansky, J. J., 102, 122, 149, 185, 189 Ptak, W., 55, 87 Pullar, D. M., 13, 14, 29, 81 Purdy, J. M., 277,297 Purnode, A,, 264, 265, 302,307 Putnam, C. W., 4, 12, 16, 22, 46, 67, 68, 69, 70, 71, 74, 89 Puttavituria, A., 53, 87

333

Rahr, L., 13, 78, 81, 90 Rai, K., 31, 77 Raju, S., 47, GO, 87 Raniseier, H., 11, 87 Ramsey, N., 22, 86 Ramwell, P., 130, 190 Ranadive, N. S., 111, 112, 118, 119, 189, 190, 275, 307 Ranl$v, P., 34, 87 Rao, P., 137, 191 Rapp, H. J., 115,184,202,250 Rappaport, I., 200, 215, 251 Rapport, M. M., 293,310 Rassat, J. P., 38, 86 Ray, A. K., 279, 307 Raynaud, M., 13, 15, 64, 78 Raziano, J., 279, 307 Ream, V. J., 137,190 Reeder, D. D., 269, 307 Reemtsma, K., 70, 89 Rees, R. J. W., 40, 41, 42, 47, 68, 79 Reeves, B., 39, 87 Reichert, L. E., Jr., 277, 306 Reichlin, M., 263, 270, 301, 307 Reid, B. L., 13, 16, 20, 21, 23, 26, 27, 34, 74, 91, 92 Reif, A. E., 7, 26, 28, 74, 87 Reinhardt, F., 212, 225, 226, 253 Reisberg, M. A., 67, 68, 76 Reith, W. S., 279, 298 Remmers, A. R., 64, 65,90 Rennels, E. G., 29, 39, 74 Renoux, M., 2, 87 Renzini, V., 266, 305 Q Resko, J. A., 278, 307 Quastel, M. R., 164, 165, 189, 193 Rethy, L., 40, 82 Queng, J. T., 288, 307 Revillard, J. P., 6, 27, 28, 33, 62, 63, 64, 76, 87, 90 Quie, P. G., 148, 151, 152, 155, 161, 179, 181, 189 Revol, L., 75, 132, 180 Quint, J., 47, 54, 63, 69, 79, 80, 85 Reynolds, E. W., 71,81 Qureshi, G. D., 292, 307 Rhodes, J. M., 237, 245 Rice, C. E., 12, 26, 87, 212, 251 Richard, G. B., 75 R Ricker, R., 280, 307 Rabbat, A., 44, 54, 59, 77, 81, 87 Riddell, A. G., 9, 84, 89 Rabinovitch, M., 152, 153, 158, 189 Riegel, B., 291, 306, 308 Rabson, A. S., 41,44, 75 Rieke, W. O., 31, 59, 60, 76, 78 Rafelson, M. E., 129, 179 Riel, F. J., 291, 308 Raff, M. C., 59, 87, 163, 171, 181, 191 Riethmuller, D., 11, 44, 87 Rahr, I., 6, 82, 91 Riethmuller, G., 11, 20, 26, 44, 87

334

AUTHOR INDEX

Righthand, F., 221, 250 Rinderknecht, H., 266, 307 Rita, G. A., 151, 192 Ritchie, W. T., 4, 87 Ritz, E., 74 Ritzen, M., 107, 179, 180 Ritzmann, S. E., 64, 65, 90 Rivera, P., 280, 300 Rivkin, I. R., 154, 161, 190 Roane, P. R., Jr., 202, 211, 240, 251 Robb, I. A., 130, 187 Robbins, J. B., 257, 276,310 Roberts, G. C. K., 130, 187 Roberts, M. L., 266, 267, 305 Robertson, G. L., 272, 273, 307 Robertson, J. I. S., 266, 307 Robinson, J. O., 95, 193 Robinson, W. W., 204,251 Robison, G. A,, 97, 176, 190 Rochas, S., 119, 189 Rochelle, D. G., 67, 71, 76, 77 Rockey, J. H., 273,310 Rodey, G., 149, 157,184 Rodman, N. F., 131,190 Rodney, G., 274,301,307 Rodriguez, E., 226, 238,250 Rodriguez-Paradisi, E., 32, 40, 87, 90 Roe, F. J. C., 44,79 Rohlich, P., 108, 190 Roelants, G. E., 237, 251 Rogers, D. E., 154, 180 Rogers, J., 129, 192 Rogers, J. H., 63, 65, 66, 70, 88 Rohrmann, G. F.,.215,251 Roitt, I. M., 27, 28, 30, 63, 79, 87, 90, 279, 307 Roizman, B., 202, 211, 240, 250, 251 Rollet, A,, 9, 70, 90 Romberg, B., 264, 299 Romfh, P., 277,278,304 Roos, D., 164,190 Rose, N. R., 290, 306 Rosen, F. S., 11, 82 Rosenberg, B. J., 295,307 Rosenberg, J. C., 53, 87 Rosenblatt, M., 11, 77, 281, 310 Rosenfeld, C., 71, 84 Rosenfield, M. R., 98, 155, 180 Rosenkranz, H. S.,296, 308 Rosenstein, R. W., 227, 251

Rosenthal, J., 221, 250 Ross, G. T., 39, 83, 257, 276, 310 Rosselin, G., 270, 297 Rossen, R. D., 67, 68, 76, 196, 251 Rossi, F., 157, 190, 193 Rossing, N., 34, 87 Roth, J., 272, 273, 307, 308 Rothberg, R. M., 287, 310 Rothenberg, S. P., 280,300, 308 Rothschild, A. M., 110, 190 Rott, R., 206, 207, 208, 214, 247, 249, 251 Roup, W. G., Jr., 276, 277, 301 Rowe, D. S., 28, 88 Rowlands, D. J., 198, 200, 201, 204, 215, 246, 251 Rozenberg, M. C., 128,188 Rubenstein, A. H., 260, 292, 305 Rubenstein, W. A., 280, 308 Rubin, A. D., 44, 67, 89 Rubin, A. I., 67, 91 Rubin, B., 288, 308 Rubin, B. A., 196, 200, 236, 237, 238, 250 Rubin, R. P., 97, 190 Rubin, W., 149, 157, 186 Rude, E., 293, 308 Rueckert, R. R., 197, 198, 200, 246, 251 Rugarli, C., 63, 84 Ruoslahti, E., 227, 249 Ruppelt, W., 73, 86 Rim, S. B., 212, 225, 226, 246 Russe, H. P., 30, 32, 37, 41, 53, 80, 87 Russell, A. S., 288, 308 Russell, P. K., 221, 247 Russell, P. S., 2, 4, 10, 14, 16, 24, 25, 26, 27, 28, 29, 30, 31, 32, 36, 37, 38, 39, 40, 44, 45, 47, 52, 54, 55, 56, 60, 61, 62, 63, 73, 74, 79, 85, 87, 91 Ruszkiewicz, M., 6, 9, 10, 11, 13, 14, 24, 26, 36, 54, 55, 71, 75, 76, 79, 81, 82 Ryan, J. J., 287, 308 Ryan, R. J., 277,279,301,303

S Sachs, J. H., 29, 30, 87 Sadun, E. H., 140,190

AUTHOR INDEX

Saeki, K., 109, 119, 190 Safier, L. B., 128, 188 Saha, J. R., 283, 305 St. John, D. J. B., 268, 302 Saleh, W. S., 25, 87 Salisbury, G., 28, 77 Salzman, E. W., 96, 127, 128, 129, 130, 132, 133, 171,180,190 Sampson, D., 122, 190 Sandberg, A. L., 136, 137, 188, 190, 212, 251,295, 308,309 Sangar, D. V., 198, 200, 201, 246, 251 Sanger, J. W., 155, 190 Sanguinetti, F. A,, 73, 88 Sardes, V. M., 53, 87 Sarles, H. E., 64, 65, 90 Sarvas, H., 227, 251 Sater, J., 149, 157, 184 Saubier, E., 9, 13, 29, 30, 70, 77, 90 Sawicki, D. L., 295, 296, 308 Sbarra, A. J., 152, 157, 190 Scaramuzzi, R. J., 278, 299 Schafer, W., 207, 247 Schaffer, C. F., 52, 77 Schaffer, F. L., 202, 250 Schally, A. V., 272, 298 Schambelan, M., 265, 309 Schantz, E. J., 291,306, 308 Scharff, M. D., 218, 221, 248, 251 Schechter, Y., 295, 308 Scheele, C., 209, 250 Schiff, P., 29, 83 Schild, G. C., 205, 206, 218, 251, 252 Schild, H. O., 101, 103, 104, 105, 108, 114, 117, 175, 178, 180, 181, 187, 190 Schlesinger, M., 59, 88 Schlesinger, R., 63, 69, 85 Schluderberg, A., 225, 242, 248 Schlumberger, H. D., 236, 238, 251, 293, 297 Schlumberger, J. R., 64, 71, 84 Schmidt, D. H., 283, 284, 291, 308 Schmidt, N. J., 202, 212, 213, 215, 225, 226, 228, 237, 240, 241, 249, 251 Schmidt, R., 67, 89 Schmidtke, J. R., 237,251 Schmitt, G. W., 63, 65, 66, 70, 77, 84 Schmittdiel, E., 29, 38, 86 Schneider, C. H., 287, 300, 308

335

Schneider, D. R., 267, 308 Schneider, H., 275, 301 Schneider, L. G., 236, 251 Schneider, M., 64, 71, 84 Schneider, R., 106, 183 Schneider, W., 131, 182 Schnure, J. J., 263, 307 Schoenbechler, M. J., 140, 142, 179, 181, 190 Schofield, J. G., 98, 190 Schofield, P. F., 27, 39, 78 Scholtissek, C., 208, 214, 251 Schramm, M., 97, 190 Schrek, R., 29, 88 Schroder, E., 266,309 Schroder, H., 29, 88 Schroder, J., 29, 88 Schroter, G. T., 16, 22, 68, 89 Schulman, J. L., 207,249,251 Schultz, B. C., 129, 130, 178 Schulze, I. T., 205, 206, 208, 251 Schumacher, H. R., 151, 190 Schur, P. H., 21, 88 Schwartz, C. J., 129, 130, 178 Schwartz, G. H., 67,91 Schwartz, I. L., 145,182 Schwartz, M. A., 281, 287, 308, 310 Schwartz, R. S., 11, 88 Schwarz, M. R., 30, 32, 40, 59, 61, 78, 88, 90 Schwarzenberg, L., 64,71, 84 Schwerdt, C. E., 202,250 Schwick, H. G., 15, 88 Scibienski, R. J., 200, 246 Scolari, L., 218, 252 Scolnick, E. M., 214, 221, 245, 248, 251 Scott, P. R., 269, 304 Scott, R. E., 148, 190 Sealey, J. E., 265, 266, 298, 304, 308 Sealy, W. C.,39, 81 Seaman, E., 295, 304, 308, 310 Searle, J. E., 276, 277, 303 Secchi, A. G., 135, 136,191 Seegal, B. C., 296,297,308 Seegers, W., 111, 190 Sehon, A. H., 275, 276, 277, 278, 279, 291,299,302,307 Seifert, J., 38, 46, 67, 76, 82, 87 Seiler, F. R., 15, 88

336

AUTHOR INDEX

Seiler, M. J., 56, 88 Sejkorova, J., 40, 86 Sela, M., 209, 227, 248, 280, 287, 293, 295, 297, 298, 303, 308, 309 Selden, R., 284, 308 Sell, K. W., 24, 65, 77 Sell, S., 2, 26, 28, 79, 88 Sellers, E. A,, 279, 307 Selvaraj, R. J., 157, 190 Senda, N., 98, 155, 190 Senitzer, D., 295, 301 Senyi, A,, 138, 188 Senyk, G., 270,308 Seppala, I. J. T., 227, 249 Sessa, G., 159, 192 Setlow, P., 295,310 Seto, J. T., 207, 251 Shaikh, A. A., 277, 308 Shaltiel, S., 287, 308 Shanfield, I., 2, 38, 88 Shanks, R. G., 269, 303 Sharman, R., 9, 10, 11, 13, 14, 24, 26, 81 Sharp, A. A., 130, 187 Sharp, G. C., 286,302 Sharp, J. T., 61, 81 Shatkin, A. J., 214, 252 Shavel, J. S., 291, 308 Shaw, T. R. D., 283,308 Shea, N., 283,305 Sheagren, J. N., 88 Sheffield, F. W., 215, 252 Sheil, A. G. R., 63, 65, 66, 70, 88 Sheldon, J. M., 274, 308 Shelton, E. R., 98, 155, 180 Shenkman, L., 280,306 Shepro, D., 132, I90 Sherer, D., 22, 82 Sherwood, L. M., 264,302 Shibata, N., 155, 190 Shigeno, N., 7, 88 Shin, H. S., 136, 137, 153, 182, 187, 190 Shinomoto, T. T., 240, 241, 251 Shio, H., 130, 190 Shiu, M. H., 70, 79 Shorter, R. G., 10, 13, 32, 66, 70, 86, 88 Showell, H, J,, 146, 159, 160, 162, 179, 190

Shulman, N. R., 130, 131, 139, 190, 191, 192, 193 Shumway, N. E., 71,89 Sian, C. M., 123,184, 212,249 Sibal, L. R., 222, 223, 225, 252 Sichuk, G., 70, 79 Siegel, A., 200, 251 Sieker, H. O., 5, 6, 14, 30, 37, 39, 81, 85 Siggins, G. R., 273, 298 Siguenza, R. F., 243, 248 Silverman, P. H., 40, 51, 88 Silverstein, A. M., 293, 298 Simmons, M. J., 55, 88 Simmons, R. L., 6, 14, 22, 24, 25, 30, 31, 38, 47, 54, 62, 63, 65, 67, 68, 74, 79, 80, 84, 85, 86, 88, 91 Simmons, S., 152, 157, 163, 185 Simon, G., 138, 179 Simpson, E., 10, 32, 36, 39, 49, 51, 52, 53, 75, 76, 88 Simpson, R. W., 206, 208, 214, 247 Sinai, Y.,296, 308 Sinclair, N. R., 45, 88 Singer, E., 289, 308 Singer, J. J., 97, 182 Singer, S. J., 171, 191 Sipe, J. D., 214, 252 Siqueira, M., 134, 191 Siraganian, R. P., 125, 133, 135, 136, 137, 140, 141, 188, 191 Siskind, G. W., 139, 185 Sjoerdsma, A., 111, 186 Skachkov, A. P., 289,304 Skehel, J. J., 205, 252 Skelton, C. L., 283, 308 Skinner, A., 163, 164, 187 Skinner, H. H., 227, 252 Skowsky, R., 273, 308 Slorach, S. A., 109, 191 Sloss, A., 27, 77 Smale, C. J., 200, 201, 236, 246, 247 Small, P. A., 196, 247 Smellie, W. A. B., 38, 76 Smeraldi, E., 52, 77 Smith, D. E., 95, 191 Smith, E. J., 12, 24, 65, 74, 77 Smith, F. R., 153, 185 Smith, J. W., 165, 166, 191, 257, 275, 303

AUTHOR INDEX

Smith, K. M., 232, 250 Smith, M. R., 153, 190 Smith, R. M., 273, 303 Smith, T. W., 257, 259, 282, 283, 284, 298, 299, 300, 302, 303, 308, 309, 310 Smith, W., 215, 252 Sneddon, J. M., 128, 131, 191 Snipe, C. R., 31, 77 Snyder, E. R., 280, 301 Sokal, G., 88 Sokal, J. E., 270, 301 Soliman, O., 13, 43, 60, 78 SOU, R., 22, 24, 25, 62, 63, 65, 67, 86, 88 Solomon, D. H., 279, 297, 299 Solomon, J. M., 237,248 Solomon, S. S., 165, 182 Soloway, A. H., 285, 299 Somnier, H., 291, 308 Somnierville, I. F., 277, 300 Sonnenblick, E. H., 283, 308 Southworth, J. G., 8, 10, 27, 41, 44, 75, 88 Soveny, C., 268, 269, 302, 304 Spaet, T. H., 132, 191 Speck, B., 71, 88 Spector, S., 281, 286, 288, 301, 303, 309 Spencer, R. J., 10, 13, 88 Speroff, L., 275, 299 Spicer, S. S., 147, 152, 156, 184, 191 Spiegelberg, H. L., 11, 21, 88, 132, 138, 139, 141, 142, 146, 150, 183 Spieler, P. J., 149, 150, 151, 192 Spielvogel, A. R., 137, 191 Spira, D. T., 40, 51, 88 Spitler, L., 270, 308 Spooner, B. S., 98, 128, 132, 155, 192 Spragg, J., 260, 266, 309 Spurr, C. L., 157, 181 Spuzic, I., 103, 191 Sri Ram, J., 260, 277, 306 Stampfer, M., 214, 246 Stanbridge, E. J., 39, 88 Stanger, D. W., 291, 306, 308 Stanworth, D. R., 26, 28, 80, 83 Starkie, S. J., 16, 22, 68, 89 Starzl, T. E., 4, 6, 10, 12, 13, 15, 16, 22, 25, 26, 29, 30, 31, 32, 36, 37, 46,

337

52, 53, 62, 63, 65, 66, 67, 68, 69, 70, 71, 74, 81, 82, 86, 88, 89 Stashak, P. W., 44, 75 Stason, W. B., 264, 309 Staub, A. M., 293,294,305 Stavitsky, A. B., 223, 226, 237, 238, 246, 248 Stechschulte, D. J., 99, 100, 102, 116, 178, 185, 188 Stein, H., 11, 44, 87 Steiner, A. L., 165, 191, 260, 273, 274, 298, 301, 303, 309, 310 Steiner, D. F., 260, 292, 305 Stelos, P., 224, 252 Stemberger, H., 287, 310 Stenzel, K. H., 67, 91 Stem, D., 98, 191 Stetson, C. A., Jr., 157, 191 Stevens, D. A., 243,252 Stevens, J. E., 36, 37, 90 Stevens, L. E., 70, 89 Stewart, J. H., 63, 65, 66, 70, 88 Stewart, J. M., 266, 309 Stich, W., 73, 86, 90 Stiles, M. A., 149, 191 Stinson, E. B., 71, 89 Stjarne, L., 97, 132, 180 Stockigt, J. R., 265, 309 Stollar, B. D., 212, 247, 280, 295, 296, 299, 304, 307, 308,309 Stoltzfus, C. M., 198, 200, 246, 251 Stone, S. S., 210, 246 Storey, B. G., 63, 65, 66, 70, 88 Stormorken, H., 95, 98, 142, 184, 191 Stossel, T. P., 155, 156, 186, 191 Straws, R. R., 152, 157, 190 Stremple, J. F., 268, 309 Strickland, R., 209, 247 Strickland, R. G., 304 Sturgis, S. H., 161,185 Subrahmanyam, D., 294, 304 Sugiyama, K., 112, 113, 191 Sullivan, A. L., 103, 122, 123, 191 Summers, D. F., 198, 202, 249, 252 Sundaresan, K., 29, 86 Sundaresan, P., 29, 86 Suszko, I. M., 106, 189 Sutherland, E. W., 97, 176, 190 Sutherland, S. K., 29, 83 Sutmoller, P., 240, 249

338

AUTHOR INDEX

Thierfelder, E., 38,86 Sutton, D., 137,191 Thierfelder, S. T., 32,38,40, 73,86,87, Suzuki, K.,266,309 90 Svehag, S.-E., 27,89,209,223,224,225, Thomas, D., 6,13,78,82,90,91,92 228,238,252 Thomas, H. F.,268,305 Swaney, J. B., 198,202, 252 Thompson, A. F., 30,76 Swerdloff, R., 276,277,297 Thompson, J. C., 267,269,306,307 Symes, M.O., 9,10,36,83,84,89 Thompson, J. S., 30,87 Szepes, G., 40,82 Thompson, K.,200,246 Thon, I. L., 99,107,109,111, 191,192 T Thorneycroft, 1. H., 278,299 Tadjebakche, H., 7,79 Thorsby, E., 29,90 Taichman, N.S., 100,108,134,138,180, Thouverez, J. P., 132,180 187,191 Tidow, G., 29,86 Takahashi, T., 13,83 Tigelaar, R. E.,121,191 Taketomi, T., 295,309 Tillson, S. A., 278,299 Tal, C., 295,309 T h e y , N.L.,70,76 Talamo, R. C., 266,267, 300, 309 Titterington, D.M., 266,307 Talbot, P., 198,200,201,246, 252 Todaro, G. J., 214,245,248,251 Taliaferro, L.G., 224,252 Tokuda, C.,222,252 Tallent, M. B.,22,24,63,67,86 Tokumaru, T., 225,228,252 Tamm, I.,203,204,246,247 Tolksdorf, S., 276,278,306 Tan, E. M., 295,309 Tomar, R., 295,297 Tanaka, T., 155,190 Tomasi, T. B., Jr., 196,247 Tanenbaum, S. W., 293,295,296,298, Tomioka, H., 99,106,115,122,123,125,

299,304,308,309

184, 191

Tanigaki, N.,290,309 Taniguchi, S., 209,253 Tanner, D., 5,84 Tapley, D. F.,279,299 Tasaka, K.,113,175,191 Tashjian, A. H., Jr., 267,297,305,309 Tatsumi, N.,98,155,190 Taub, R. N.,2,9,10,11, 13,14,24,25,

Tonutti, E., 279,302 Torisu, M., 46,74 Torrigiani, G., 27, 30, 63, 79, 87, 90,

26, 28,29, 30,31,32, 35,36,44, 52,53,57,58,61,67,81,82,89 Tavormina, P. A., 129,187 Taylor, E. L.,98,128,132,155,192 Taylor, F.B.,Jr., 127,191 Taylor, H. E., 67,89 Taylor, P.E., 219,252 Taylor, R. B., 44,90,163,171,191 Tchobroutsky, G., 270,297 Temin, H.M.,214,252 Tenenhouse, A,, 267,302 Teramoto, A. Y.,221,247 Terasaki, P. 1.,!4, 16,22,68, 69,89 Tew, J. G.,149,191 Thiel, G., 67,90

Townsend, C. M., 22,64,65,67,82,90 Traeger, J., 6,9,13,29,30,33, 62,63,

279,307 Toth, G., 40,82 Touraine, J. L.,6,62,63,65,66,67,70,

79,90

65,66,67,70,77, 79,90 Traeger-Fouillet, Y., 9, 13,29,30,77,90 Traub, E.,212,252 Trautman, R., 197, 199,201, 202, 211,

212, 218, 223,226,229,240,246, 247,248,252 Treadwell, P. E., 159,191 Treat, R. C., 44,77 Tremblay, F., 139,184 Trentin, J. J., 47,61,67,68,76,81 Trepel, F.,72,90 Trian, R., 24,76 Tribble, J. L., 16,83

AUTHOR INDEX

339

van Bekkum, D. W., 2, 24, 25, 28, 30, 38, 39, 40, 41, 75, 83, 90, 91 Vance, V. K., 263,307 van der Hart, M., 26, 78 Van Der Meer, J., 264,265,303 van der Werf, B. A., 16, 24, 25, 28, 39, 40, 62, 63, 85, 91 Vande Wiele, R. L., 277, 278, 279, 301, 306, 307 Vande Woude, G. F., 198, 202, 204, 252 Van Epps, D. E., 161,178 van Gorder, T. J., 23, 86 van Heyningen, W. E., 144, 182 van Leeuwen, A., 24, 75 van Loghem, J. J., 26,78 van Maanen, J. H., 279,307 van Oss, C., 268,309 van Putten, L. M., 24, 25, 30, 38, 40, 41, 75, 91 Van Regenmortel, M. H. V., 200, 215, 252 van Rood, J. J., 24, 75 Van Vunakis, H., 258,260,275,285,286, 287, 295, 301, 304, 306, 308, 310 U VanWay, M. H., 154, 180 Uhr, J. W., 153, 189, 209, 224, 225, 227, Vasington, P. J., 10, 39, 91 238, 248, 251, 252 Vassalli, P., 138, 179 Unanue, E. R., 185, 191 Ungar-Waron, H., 280, 285, 295, 303, Vatter, A. E., 28, 83 Vaughan, E. D., 265, 298, 304 308, 309 Vaughan, J. H., 287,298 Unger, R. H., 270, 309 Vaughan, M., 155, 156, 186, 191 Urban, C. L., 132,184 Vaz, N. M., 95, 102, 106, 119, 120, 121, Uriuhara, T., 134, 137, 187 188, 191, 192 Utiger, R. D., 260, 272, 273, 279, 297, Vela-Martinez, J. M., 25, 27, 30, 54, 81 304, 307, 309 Uvnls, B., 99, 107, 108, 109, 110, 111, Verbi. W., 27, 87 Verosky, M., 176, 192 142, 184, 187, 190, 191, 192 Vesikari, T., 139, 188 Veysseyre, C., 9, 13, 29, 30, 70, 77, 90 V Vick, J. A,, 137, 184 Vaerman, J. P., 218, 250, 252 Vieth, F. J., 2, 10, 39, 52, 55, 57, 82 Vaheri,' A., 139, 188 Vigdahl, R. L., 129, 130, 187, 192 Vaitukaitis, J., 257, 276,310 Villa, M. L., 52, 77 Valentine, M. D., 115, 118, 192 Vischer, T., 67, 90 Valentine, R. C., 205, 206, 249 Vogt, M., 209,247 Vallotton, M. B., 264, 273, 309, 310 Volkert, M., 41, 91 van Aken, W. G., 91 von Ruttger, A., 91 Van Arsdel, P. P., Jr., 109, 192 van Thierfelder, S., 40, 91

Trifnrci, J. M., 98, 191 Triner, L., 176, 192 Troland, P., 64, 71, 84 Troll, W., 164,184 Troop, S. B., 129, 131,178 Trudeau, W. L., 268, 269, 305, 309 Tryon, M., 289,300 Tsao, H. S., 267, 297 Tseng, L., 277, 302 Tsirimbas, A. D., 73, 86, 90 Tsuruhara, T., 276, 277, 278, 300 Tucker, D. F., 40,90 Tulchinsky, D., 276, 277, 297, 309 Turk, J. L., 32, 36, 37, 62, 90 Turner, W., 237, 252 Tursi, A., 27, 30, 63, 79, 87, 90 Tyler, F. H., 277, 310 Tyler, R. S., 40, 88 Tyler, R. W., 30, 32, 59, 61, 78, 90 Tytell, A. A., 237, 253 Tze, W. J., 98, 182

340 Vorherr, H., 271, 273, 310 Vredevoe, D. L., 42, 91 Vreeken, J,, 91 Vriesendorp, H. M., 5, 14, 27, 91 Vujic, D., 11, 81 Vulliemoz, Y., 176, 192 Vyvial, T. M., 64, 65, 90

AUTHOR INDEX

Wedner, H. J., 273, 298 Wedum, A. G., 288,310 Weed, R. I., 129, 131,178 Weeks, J. R., 129,192 Weetall, H. H., 276, 277, 302 Wehmann, R. E., 273,310 Weibel, E. R., 128, 179 Weigle, W. O., 11, 21, 88 Weil, R., 54, 91 W Weiner, L. M., 281, 310 Wagner, E., 38, 46, 86,87 Weiner, R., 161, 185 Wagner, G. G., 199, 215, 218, 219, 229, Weinryb, I., 273, 310 234,243, 247,252 Weinstein, R., 278, 304 Wagner, J., 91 Weir, D. M., 102, 189 Wahlen, J. D., 277, 310 Weisman, R. A., 153, 185 Wainer, B. H., 287, 310 Weiss, H. J., 129, 192 Waite, M. A., 266, 307 Waksman, B. H., 8, 10, 16, 34, 36, 37, Weissmann, G., 124, 147, 148, 149, 150, 151, 155, 159, 160, 164, 180, 181, 38, 42, 44, 77, 91 184, 187, 192, 193 Wallace, R., 10, 39, 91 Weksler, M. E., 67, 91 Wallace, S. P., 295, 310 Wells, G. A,, 243, 248 Wallace, S. S., 296, 310 Wells, J. V., 165, 182 Wallach, R., 266, 298 Wallis, C., 210, 252 Went, I., 274, 310 Wallis, V., 163, 181 Werner, H. P., 287, 310 Wallis, V. J., 45, 57, 83 Werner, J., 40, 91 Wallon, C., 13, 84, 78 Wessells, N. K., 98, 128, 132, 155, 192 Walsh, J. H., 269, 310 Wessler, S., 137, 190 Waltman, S. R., 10, 38, 60, 76, 91 West, C. D., 277, 310 Wakon, J. A., 71, 81 E. G., 242, 253 Westaway, Warburton, D., 296, 300 B., 109, 136, 187, 192 Westerholm, Ward, P. A., 147, 153, 154, 160, 161, Westphal, O., 293, 294, 305, 308, 310 179,189,192 Wetzel, B. K., 156, 184 Warner, N. L., 117, 121, 180, 188 Wlieelock, E. F., 41, 78 Warren, J., 9, 79 Warren, K. S., 40, 78 White, A., 149, 181, 187 Warrington, R. E., 222, 252 White, J. G., 126, 128, 129, 132, 133, 192, 193 Warshaw, A. L., 131,192 White, R. J., 283,299, 310 Wasserman, E., 286, 310 Watchi, J. M., 26, 27, 74 Whitfield, J. F., 166, 182 Watson, J. F., 283, 310 Wicher, K., 281,310 Watt, J. G., 28, 30, 36, 38, 41, 53, 73, Wiederman, G., 212, 225, 226, 253, 287, 310 77 Webber, M. M., 32,80 Wieneke, A. A,, 97, 144, 145, 146, 156, 193 Weber, H., 67, 90 Wiener, E., 159, 181 Webster, M. E., 266, 267, 302 Webster, R. G., 204, 205, 206, 207, 208, Wigzell, H., 26, 91 209, 211, 220, 223, 225, 233, 237, Wiktor, T. J., 221, 236, 238, 251, 253 238, 242, 247, 248, 249, 252, 253 Wild, T. F., 198, 200, 201, 253

341

AUTHOR INDEX

Wilhelm, R. E., 37, 91 Wilkins, D. G., 154, 193 Willard, L. F., 6, 80 Willenis, C., 98, 188 Williams, C . A., 267, 302 Williams, C . M., 38, 76 Williams, H. E., 149, 180 Williams, J. A,, 98, 193 Williams, L. F., 63, 65, 66, 70, 77, 84 Williams, R. C., Jr., 152, 189, 287, 308 Williamson, W. G., 42, 81 Willoughby, D. A., 28, 32, 36, 37, 62, 77, 90 Wilner, G. D., 260, 292, 306 Wilson, B. J., 23, 91 Wilson, C. B., 70, 79 Wilson, R. E., 6, 30, 40, 70, 76, 80 Wilson, S. D., 268, 309 Winne, D., 102, 187 Wirtz, G. H., 281, 300 Witz, I., 6, 91 Woiwood, A. J., 6, 30, 76, 91 Wolf, P. L., 11, 77 Wolfe, S. M., 130, 193 Wolf€, J., 98,193 Wong, S. L. R., 276,277,302 Woo, J., 276, 277,303 Wood, A. J., 14, 29, 64, 65, 81, 90 Wood, M. L., 4, 5, 10, 14, 16, 24, 25, 26, 27, 28, 29, 30, 31, 32, 37, 38, 39, 40, 47, 52, 54, 55, 56, 61, 62, 63, 73, 74, 79, 85, 91 Wood, W. B., Jr., 149, 153, 179, 190 Woodhour, A. F., 237, 253 Woodin, A. M., 97, 144, 145, 146, 154, 156, 193 Woodrooffe, J. G., 6, 27, 82, 85, 91 Woodruff, M. F. A,, 2, 3, 4, 10, 13, 16, 20, 21, 22, 23, 26, 27, 28, 29, 30, 33, 34, 36, 38, 41, 46, 47, 53, 54, 73, 74, 77, 80, 91, 92 Woods, J. E., 279, 310 Work, T. S., 213, 248 Wortis, H. H., 44, 90 Wren, S. F. G., 38, 88 Wrenn, J. T., 98, 128, 132, 155, 192 Wright, P. G., 149, 165, 193 Wu, C. H., 277, 306

W u , W. H., 273, 310 Wylcr, R. S., 291, 306, 308

Y Yagi, Y.,6, 91, 289, 290, 304, 309 Yalow, R. S., 260, 263, 267, 269, 272, 293,298,310 Ynmada, K. M., 98, 128, 132, 155, 192 Yamakawa, T., 295, 309 Yamamaka, J., 11, 77 Yamasaki, H., 103, 104, 113, 175, 191, 193 Yariv, J., 293, 310 Yates, P., 132, 179 Yokoyama, T., 46, 74 Yoshino, K., 209, 253 Yoshizawa, I., 276, 277, 310 Young, J. D., 268, 269, 270, 271, 272, 299, 304, 310 Younger, L. R., 260,292, 306 Yron, I., 59, 88 Yu, T. Z., 6, 29, 54, 86 Yuill, M. E., 281, 299

Z Zamir, R., 27, 28, 79 Zatti, M., 157, 190, 193 Zatz, M., 36, 58, 92 Zawadski, A., 13, 43, 60, 78 Zech, P., 9, 70, 90 Zeiss, C. R., Jr., 106, 189 Zeitlin, A., 276, 277, 305 Zelickson, A. S., 120, 188 Zeligs, J. D., 149, 184 Zenehergh, A., 88 Zenker, R., 10, 29, 30, 33, 38, 86 Zepp, H. D., 221,245 Zeuthen, E., 109, 180 Zeve, V., 214, 248 Zieve, P. D., 130, 193 Ziff, M., 14, 16, 32, 42, 47, 52, 77, 78, 81, 288, 308 Zigmond, S., 148, 193 Zinimering, P. E., 278, 279, 301 Zimmet, P. Z., 264, 299 Zola, H., 6, 82, 91, 92

342

AUTHOR INDEX

Zolla, S., 293, 310 Zollinger, I. N . , 267, 308 Zolov, D. M., 287, 304 Zucker, M. B., 129, 130, 132, 137, 188, 191, 193 Zucker-Franklin, D., 128, 132, 146, 156, 193

Zuhlke, V., 6, 10, 13, 15, 16, 22, 26, 29, 30, 31, 32, 37, 46, 52, 53, 67, 81 Zukoski, C., 44, 84 Zurier, R. B., 124, 147, 148, 149, 150, 151, 155, 159, 180, 192, 193 Zvaifler, N. J., 95, 140, 179, 193

SUBJECT INDEX Antilymphocyte serum, A assays of potency, Agglutination, in vitro, 26-27 passive, antibody measurement and, in oivo, 24-26 222-223 chronic administration effects, 5153 Antibodies, effects in man, to small molecules, administration and side effects, 65detection, 257-258 69 determination of specificity, 258-259 clinical use of antilyinphocytic immunoassay methods, 259-261 glol~ulin,69-73 methods of elicitation, 256-257 parallelism between clinical and exspecific applications, 261-263 perimental evidence, 62-64 coenzymes and vitamins, 280-281 projections for future, 73 drugs, 281-288 special aspects of serum production, miscellaneous, 293-296 64-65 nonpeptide hormones, 273-280 effect on lymphoid cells or tissue, peptide hormones, 263-273 in uitro, 27-29 protein fragments, 291-293 in uivo, 29-36 toxins, 288-291 historical, 3-4 viral antigens, measurement, in~munoglobulinG, immunogenicity, complement fixation, 211-213 enzyme inhibition, 213-215 46-47 hemagglutination inhibition, 210immunological tolerance and, 55-57 211 mode of action, labeled antigens and antibodies, 220alternative possibilities, 60-62 222 recirculating lymphocytes and, 57neutralization, 208-210 60 passive or indirect agglutination, preparation, 222-223 choice of species, 9-12 precipitation, 215-220 comment on, 14-15 Antibody, schedule of immunization, 12-14 fragments, antilymphocyte serum and, source of antigen, 4-9 16-23 Antibody response, purification, antibody specificity, 238-244 absorption, 15-16 viral antigens other than virion, 231antibody eluates, 23-24 238 serum fractions and antibody fragvirions as antigens, 223-231 ments, 16-23 Antigen, scope of action in uivo, source, antilymphocyte serum and, 4-9 cell-mediated immunity, 37-44 viral, erasure of memory, 45-46 antibody measurement, 208-223 humoral immunity, 44-45 complex, 206-208 inflammation, 36-37 simple, 198-204, synergism with other agents, 5 4 5 5 343

344

SUBJECT INDEX

B Basophiles, mediator secretion, human, 122-125 rabbit, 125

C Chemotaxis, mediator secretion and, 15% 162 Coenzymes, antibodies to, 280-281 Complement fixation, antihody measurement and, 211-213

D Drugs, antibodies to, 281-288

E Enzyme ( s ) , inhibition, antibody measurement and, 213-215

H Hemagglutination, inhibition, antibody measurement and, 210-211 Hormones, nonpeptide, antibodies to, 273-280 peptide, antibodies to, 263-273 Human, antilymphocyte serum effects, administration and side effects, 6569 clinical use of antilymphocytic globulin, 69-73 parallelism between clinical and experimental evidence, 62-64 projections for future, 73 special aspects of serum production, 64-65

I Immunity, cell-mediated, antilymphocyte serum and, 3 7 4 4 , 47-48 morphological evidence, 51 sensitized animals, 49-51 viral systems, 51 virgin animals, 4 8 4 9

huinoral, antilymphocyte serum and, 44-45 Iimiiunoglobuliii G, antilymphocyte serum, inimunogenicity, 4 6 4 7 Immunological tolerance, antilymphocyte serum and, 55-57 Inflnnuiiation, antilymphocyte serum and, 36-37

L Leukocidin, niediator release and, 144146 Lung, mediator secretion, 99-100 perfused, 100-101 slices or fragments, 101-106 Lymphocytes, recirculating, antilymphocyte serum and, 57-60 transformation, mediator secretion and, 162-166

M Macrophages, mediator secretion, 15% 159 Mast cells, mediator secretion, immunological stimuli, 114-122 noniiiiiiiunological stimuli, 106-114 Mediator secretion, basophiles, human, 122-125 rabbit, 125 chemotaxis and, 159-162 general characteristics, 96-99 isolated tissues and organs, 99-100 lung slices and fragments, 101-106 perfused lung, 100-101 lymphocyte transformation and, 162166 mast cells, immunological stimuli, 114-122 nonimniunological stimuli, 106-114 monocytes and macrophages, 158-159 neutrophiles, 143-144, 151-152 immunological release, 146-151 nonimmunological release, 144-146 platelets, 126-127, 142-143

345

SUBJECT INDEX

iminunological reactions, 133-142 nonimm~~i~ological reactions, 127-133 hlemory, erasure, antilymphocyte serum and,

Protein ( s ), peptide fragments, antibodies to, 291-

293

T

45-46 hlonocytes, mediator secretion, 158-159

Toxins, antibodies to, 288-291

N Neutralization, antibody ineasnrenient and, 208-210 Neutrophiles, mediator secretion, 143-144, 151-152 imnnmological release, 146-151 nonimninnological release, 144-146 phagocytosis, adherence, 152-153 degranulation, 1 5 6 1 5 8 engulfment, 153-156

P Phagocytosis, neutrophiles and, adherence, 152-153 degranulation, 156-158 engnlfment, 153-156 Platelets, mediator secretion, 126-127, 142-143 immunological reactions, 133-142 nonimmunological reactions, 127-133 Precipitation, antibody measnrement and,

215-220

V Virion( s ) , antibody response, 223-231 Virus( es), antigens, measurement of antibody to, complement fixation, 211-213 enzyme inhibition, 213-215 hemagglutination inhibition, 210-

211 labeled antigens and antibodies,

220-222 neutralization, 208-210 passive or indirect agglutination,

222-223 precipitation, 215-220 antigens other than virion, antibody response, 231-238 complex, antigens, 206-208 structure, 204-206 simple, antigens, 198-204 structure, 197-198 Vitamins, antibodies to, 280-281

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