Advances in Immunology Volume 10

CONTRIBUTORS TO THIS

VOLUME

D. BERNARD AMOS

K. FRANK AUSTEN BARUJ BENACERRAF

HOWARDM. GREY KARL HABEL

ROBERTP. ORANGE OSCAR D. RAWOFF

GREGORY W. SISKIND

ADVANCES I N

Immunology EDITED B Y

F. J. DIXON, JR.

HENRY G. KUNKEL

Division of Experimenfal Pathology Scrippr Clinic and Research Foundation La lolla, Colifornia

The Rockefeller University New York, New York

VOLUME

10

1969

ACADEMIC PRESS

New York and London

COPYRIGHT

@ 1989,BY ACADEMIC PRESS, INC.

ALL RIGHTS RESERVED NO PART OF THIS BOOK MAY B E REPRODUCED IN ANY FORM,

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United Kingdom Edition published by ACADEMIC PRESS, INC. (LONDON) LTD. Berkeley Square House, London WIXGBA

LIBRARYOF CONGRESS CATALOG CARDNUMBER:61-17057

PRINTED I N THE UNITED STATES OF AMERICA

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

D. BERNARDAMOS, Division of Immunology, Duke University Medical Center, Durham, North Carolinu (2.51)

K. FRANKAUSTEN,Department of Medicine, Harvard Medical School at the Robert B. Brigham Hospital, Boston, Massachusetts (105)

BARUJ BENACEERRAF, Laboratory

of Immunology, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, Maryland, and the Department of Medicine, Cornell University School of Medicine, New York, New York (1)

HOWARD M.GREY, Department of Experimental Pathology, Scripps Clinic and Research Foundation, La Jolkr, California (51) KARLHABEL, Department of Experimental Pathology, Scripps Clinic and Research Founddion, La Jollu, California (229)

ROBERTP. ORANGE, Department

of Medicine, Haroard Medical School at the Robert B. Brighum Hospital, Boston, Massachusetts (105)

RATNOFF, Department of Medicine, Case Western Reserve University School of Medicine, and University Hospitals of Cleveland, Cleveland, Ohio (145)

OSCARD.

GREGORY W . SISIUND,Laboratory

of Immunology, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethsda, Maryland, and the Department of Medicine, Cornell University School of Medicine, New York, New York (1)

V

PREFACE Volume 10 of Adoances in Immunology contains six reviews which reflect the ever-widening boundaries of immunology and its increasing depth of penetration into biomedical research. The continuing growth of the subject makes it desirable and essential for the reader to profit from periodic authoritative summations of knowledge on discrete subjects of immunology. When the summation expresses the perspective of a leader in his field, the contribution is doubly valuable. We are greatly indebted to the authors of this volume for taking the time to prepare such reviews. The first chapter by Drs. Siskind and Benacerraf relates the essential features of the antibody response to the interaction of antigen with preformed, cell-bound, antibodylike receptors. The effect of this interaction on individual cells is determined by the affinity of the antigen cell-bound antibody combination and results in a recruitment or selection of cells and their activation. This process, describable in thermodynamic terms, is the central biological event which can explain or predict such features of the antibody response as the progressive increase in average binding affinity of antibody produced, the effect of antigen dose on amount and affinity of antibody, the mechanism of action of adjuvants, the essential role of specific cell proliferation stimulated by antigen, the interference of humoral antibody with antigenic selection of cells, the phenomenon of “original antigenic sin,” and the induction of tolerance. Dr. Grey, in the second chapter, offers a definitive review of the immunoglobulins of various species, a subject to which he has been an important contributor. These phylogenetic data are placed in perspective and used as a basis for understanding the development and present structure of the complex immunoglobulin systems of man and other vertebrate species. One of the important and hitherto poorly understood mediators of anaphylaxis, slow reacting substance, is described in the third chapter by Drs. Orange and Austen who have contributed greatly to progress in this field. The events involved in the formation and release of this mediator as well as the current knowledge of its chemical and pharmacological characteristics are presented. The biological implications of slow reacting substance for the entire subject of acute immunologic reactions and their pharmacologic manipulations are discussed and provide an enticing preview to advances which may be expected in this field in the next few years. vii

viii

PREFACE

Hemostasis is a complex process which is only recently attracting the attention of immunologists. The interesting parallels and, at times, direct interrelationships of hemostasis and serologic events initiated by antigenantibody reactions are now becoming apparent. In the fourth chapter, Dr. Ratnoff discusses the interdependency of the blood clotting process, fibrinolytic phenomena, inflammation, and immunologic reactions. This review provides both the basis for a clear understanding of the many elements of hemostasis and a perspective which views the several defense mechanisms as a well-integrated continuum. The contributions of immunology to the understanding of viral oncogenesis have and will continue to be of utmost importance. Fortunately, for the investigator and perhaps in some instances for the host himself, virus-induced tumors may bear the antigenic imprint of their inducers. The antigens of virus-induced tumors of animals and man are clearly and succinctly discussed in the Bfth chapter by Dr. Habel who is one of the outstanding contributors to this field. The origin and characteristics of the various types of antigens in viral tumors and their participation in spontaneous or induced immunologic responses of the host are elucidated. The implications of such immunologic responses for prevention or therapy of virus-induced tumors are also considered. While most fields of research grow in relationship to investigators’ interests and available techniques, occasionally an important practical problem demanding immediate attention is thrust upon the scientific community regardless of its state of readiness. Such a situation exists with respect to the pressing need for effective tissue typing in man, necessitated by the technical feasibility of organ transplantation. In the last chapter, Dr. Amos writes a clear statement of our current knowledge of the genetics and immunology of human histocompatibility. In addition, he provides a first-hand view of the difficulties and limitations as well as the achievements of tissue typing as it is practiced today. We wish to acknowledge the cooperation and assistance of the publishers who have done much to ensure the quality of this series of volumes.

rug, 1969

FRANK J. DIXON HENRYG. KUNKEL

Contents of Previous Volumes Volume 1 Transplantation Immunity and Tolerance

M. HASEX,A. LENGEROV~~, AND T. HRABA

Immunological Tolerance of Nonliving Antigens

R I w T. SMITH

Functions of the Complement System

ABRAHAMG. OSLER

In Vitro Studies of the Antibody Response

ABRAMB. STAVITSKY

Duration of Immunity i n Virus Diseases

J. H. HALE

Fate and Biological Action of Antigen-Antibody Complexes WILLIAM

0.WElGLE

Delayed Hypersensitivity to Simple Protein Antigens

P. G. H. GELLAND B. BFNACERRAF

The Antigenic Structure of Tumors

P. A. GORER AUTHOR INDEX-SUBJECT INDEX Volume 2

Immunologic Specificity and Molecular Structure

FRED KARUSH

Heterogeneity of y-Globulins JOHN

L.FAHEY

The Immunological Significance of the Thymus

J.F.A.P.MILLER,A.H.E.MARsHAu,ANDR.G.W~

Cellular Genetics of Immune Responses

G . J. V. NOSSAL

Antibody Production by Transferred Cells CHARLES G. C o m m AND FRANK J.

Phagocytosis

D m c g ROWLEV xi

DIXON

Xii

CONTENTS OF PREVIOUS VOLUMES

Antigen-Antibody Reactions in Helminth Infections

E. J. L. SOULSBY

Embryological Development of Antigens

REED A. F”GER AUTHOR INDEX-SUB j ~ c INDEX r 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. STONE AND M. R. IRWIN

Heterophile Antigens and Their Significance in the Host-Parasite Relationship

C.R. JE”

AUTHOR INDEX-SUBJE~ INDEX Volume 4 Ontogeny and Phylogeny of Adoptive Immunity

ROBERTA. GOODAND BEN W. PAPERMASTER

Cellular Reactions in Infection

RAMSEW EMANUELSUTEFIAND HANSRUEDY

Ultrastructure of Immunologic Processes JOSEPH D. FELDMAN Cell Wall Antigens of Gram-Positive Bacteria

MACLYNMCCARTYAND STEPHENI. MORSE

Structure and Biological Activity of Immunoglobulins

SYDNEY C o m a AND 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 AUTHORINDEX-SUB J

E

INDEX ~

Volume 5 Natural Antibodies and the Immune Response

STFPHENV. BOYDEN

Immunological Studies with Synthetic Polypeptides

MICHAEL SELA

Experimental Allergic Encephalomyelitis and Autoimmune Disease

PHILIP Y. PATERSON

The Immunology of Insulin

C. G. POPE

Tissue-Specific Antigens

D. C. DUMONDE

AUTHORINDEX-SUBJE~ 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

Orro J. PLESCU AXD WERSER BMUN

In Vitro Studies of Immunological Responses of Lymphoid Cells

RICHARDW. DWLTON

Developmental Aspects of Immunity JAROSWV

STERZL

AND

ARTHUR M. SILWRSTEIN

Anti-antibodies

PHILIPG. H. GELLAND ANDREWS. KELUS

Cong Iutinin and Immunoconglutin ins

P.J. L4-N

AUTHORINDEX-SUB J

E

INDEX ~

XiV

CONTENTS OF PmVIOUS VOLUMES

Volume 7 Structure and Biological Properties of Immunoglobulins SYDNEY COHEN AND CESAR MILSTEIN Genetics of Immunoglobulins in the Mouse

MICXAEL P

m AND ROSELIEBERMAN

Mimetic Relationships between Group A Streptococci and Mammalian Tissues

JOHNB. ZABmsm

lymphocytes and Transplantation Immunity

DARCY B. WILSON AND R. E. BILLINCHAM

Human Tissue Transplantation

JOHNP. MERRILL AUTHORINDEX-SUB j ~ c INDEX r Volume 8 Chemistry and Reaction Mechanisms of Complement

HANSJ. M~~LLER-EBERFIARD

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 Vitro Studies of Human Reaginic Allergy

ABRAHAMG. OSLER,LAWRENCE M. LICIITENSTEIN, AND DAVID A. LEVY AUTHORINDEX-SUBJECX INDEX Volume 9 Secretory lmmunoglobulins

THOMAS B. TOMASI,JR. AND JOHNBIENENSTOCK

Immunologic Tissue Injury Mediated by Neutrophilic leukocytes

CIWZLES G. C O C ~ U N E

The Structure and Function of Monocytes and Macrophages

ZANVILA. COHN

The Immunology and Pathology of NZB Mice J. B. H o w AND B. J. HELYER

AUTHOR INDEXSUB JECX I N D ~

Cell Selection by Antigen in the Immune Response GREGORY W. SlSKlND AND BARUJ BENACERRAF Laboratory o f Immunology, National Institute of Allergy and Infectious Diseases, Nafional InsfifUtes

o f Health, Bethesda, Maryland, and the Department of Medicine, Cornell University School of Medicine, New York, New York

.

I. Introduction . . . . . . . . . . . , . . . . . . . . . 11. Antibody-Binding Affinity A. Definitions and Concepts . . , . . . . . . B. Heterogeneity of Affinity . . . . . . . . . 111. Maturation of the Immune Response . . . . . . . IV. Commitment of Individual Plasma Cells and Sensitized Lymphocytes to the Synthesis of Immunoglobulins of a Single Class and Individual Specificity . . . . . . . . . . . A. General Considerations of Clonal Theory . . . . . . B. Plasma Cells . . . . . . . . . . . . C. Lymphocytes . . . . . . . V. Effect of Antigen Dose upon the Amount and Aanity of Serum Antibody . . . . . . . . . VI. Maturation of the Immune Response: The Selective Stimulation of the Proliferation of Those Cells That Produce Highest-AfEnity Antibody . A. General Considerations . . . . . . . . . B. The Selective Advantage of Anti-2,4-dinitrophenyl Cells Synthesizing K Molecules during Immunization of Guinea Pigs with . . . . . . 2,4DinitrophenyLProtein Conjugates C. Secondary Responses in Rabbits Immunized with 2,4-Dinitrophenyl-Protein Conjugates Elicited by 2,4Dinitrophenyl Coupled to Heterologous Proteins . . . . . . . . . D. The Relationship between the Electrical Charge of the Antigen and the Charge of the Corresponding Antibody . . . VII. Effect of Humoral Antibody on the Control of Antibody Synthesis . . . . . . VIII. “Original Antigenic Sin” . . . . . . . . . IX. Immunological Tolerance (Unresponsiveness) A. General Characteristics . . . . . . . . . B. Antibody Affinity and Tolerance Induction . . . . . C. Response of Tolerant Animals to Antigens Cross-Reactive with the Tolerated Antigen . . . . . . . . . X. Summary of Antigen Selection Hypothesis . . XI. Practical Conclusions and Further Problems to Investigate . References . . . . . . . . . . .

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1.

1 3 3 5

8 12 12 13 16

20

25 25 25 26 29

30 33

35 35 37

38 41 44 45

Introduction

We shall describe here certain basic phenomena characteristic of the immune response and analyze their mechanism at both the cellular and the molecular levels. .Among the characteristics of the immune response 1

2

GREGORY W. SISKZND AND BARUJ BENACERRAF

with which we shall deal in this paper are ( a ) the change in average binding afEnity of antibody for the antigenic determinant which occurs with time after immunization; ( b ) the effect of antigen dose on the concentration and average binding affinity of serum antibody; ( c ) the effect of adjuvants upon the concentration and afEnity of circulating antibody; ( d ) the commitment of individual plasma cells and lymphocytes to the synthesis of antibody of a single class, allotype, specificity, and affinity; ( e ) the crucial role of specific cell proliferation, resulting from antigenic stimulation, upon the process of selection of populations of immune cells producing antibodies of progressively higher &nity for the immunizing antigen; ( f ) the effect of humoral antibody upon the process by which antigen selects cells synthesizing antibodies of progressively higher affinity; ( g ) the phenomenon of “original antigenic sin,” that is, the selective effect of previous immunization on the population of cells which become available for stimulation by a structurally related antigen; (12) the specificity of tolerance induction as related to the energetics of the antigen-antibody interaction and the relationship of specific unresponsiveness to immunological selection and to the process of immune cell proliferation. These various phenomena will be explored in an attempt to formulate a unified concept of the immune response as an antigen-driven proliferation and selection of specific cells that are committed to the synthesis of specific immunoglobulin molecules prior to contact with antigen. An attempt will be made to define the conditions governing the selection aspect of this process. We have assumed a strictly clonal theory of antibody synthesis such as originally proposed by Burnet (1-3), in which cells become committed to the production of a specific antibody molecule in a random fashion prior to antigen exposure, presumably by a process of somatic mutation. We would suggest that such precommitted cells bear representations of antibody molecules having binding properties identical to those of the antibody synthesized by the particular cell, or its progeny, upon antigenic stimulation. Such antibody present on committed cells shall be referred to as “cell-associated antibody.” We would suggest that cells are specifically stimulated to proliferate and/or secrete antibody as a result of the interaction of cell-associated antibody with specific antigen (possibly in a “processed” form). A single cell so stimulated would be expected to produce a homogeneous antibody product. The heterogeneity of serum antibody would thus reflect a heterogeneity of the antibody-synthesizing cell population. In such a system, cells can be viewed as competing for available antigen. Thus, cells bearing antibody molecules of highest bind-

C0X“I‘ROL OF IMMbTE RESPONSE

3

ing affinity for the antigen would stand a better chance of binding antigen and, thus, of being stimulated to proliferate and/or secrete antibody. We view antigen (or processed antigen) as acting continuously, throughout the course of the immune response, to select those cells of highest antigen-binding affinity from a proliferating immune cell population. We have here an essentially evolutionary view of the immune response, in which availability of antigen serves as the crucial selective pressure. Thus, we suggest that one step in the pathway leading to antibody synthesis involves the interaction of antigen (or processed antigen) with preexisting antibody molecules presumably located on the surface of the cell that synthesized them. This selective step in the immune rcsponse can be considered in simple energetic terms, that is, antigen molecules would be most likely bound by those cells bearing antibody molecules of highest affinity for that antigen. Based upon such a theory the results of a wide variety of different experimental situations may be predicted. In summary, we shall review the evidence that one step, in some as yet undefined sequence of steps leading to antibody synthesis, behaves as if it involves the interaction of antigen with pre-existing antibody molecules in a manner predictable by simple energetic considerations. Based upon this interaction of antigen with cell-associated antibody, populations of antibody-forming cells can continuously be selected by antigen in a predictable fashion. The importance of the interaction of antigen with “cell-associated antibody” has been discussed by a number of workers including Eisen ( 9 5 u ) , Steiner and Eisen (as), Jerne ( 3 u ) , Mitchison ( 3 b ) , Talmage ( 3 c ) , Lenox and Cohn ( 3 d ) , and by Fazekas de St. Groth and Webster (139). II.

Antibody-Binding Affinity

A. DEFIhTI?ONS AND C O N C E m The term affinity refers to the “strength” of the interaction between the antibody molecult: and the antigenic determinant. With a high-affinity antibody, a strong bond is formed between the antibody and the homologous antigenic determinant. In essence this means that it would require a large amount of energy to pull apart the antibody-antigen complex. Affinity can be viewed as the measurement of antibody specificity (in the sense of conformational correspondence to the homologous determinant; not in the sense of a lack of cross reactivity, since the degree of cross reactivity is often greater with higher-affinity antibody), and as such may be expressed either as the equilibrium constant ( K )for the antibody-antigen interaction or as the standard free energy change ( AFO) for the reaction. For the general reaction:

4

GREGORY W. SISKIND AND BARUJ BENACERRAF

+

Ab H F ! AbH (1) where Ab is antibody, H is hapten, and AbH is antibody-hapten complex, the association constant ( K )can be written

The higher the equilibrium constant (in units of liters/mole), the stronger the antibody-hapten bond and the higher the relative concentrations of antibody-hapten complex present at equilibrium, i.e., the higher the affinity of the antibody. Affinity can also be expressed as the free energy change (AF) occurring in the course of the antibody-hapten interaction. However, since free energy ( F ) depends upon concentration, AF changes progressively during the course of the reaction and is, therefore, not an ideal function with which to express affinity. For convenience, one makes use of the so-called standard free energy ( F O ) which is the free energy of a substance in its standard state (1 mole of material, at 1 atm. pressure and 25OC; if in solution, at a concentration of 1 molal). Hence, A F O is independent of concentration as it represents the free energy difference between reactants and products in their standard state. Since the equilibrium constant ( K ) and the standard free energy change ( A F ” ) are both expressions of the tendency of the reaction to proceed and since both AFo and K essentially define equilibrium conditions, it is reasonable that some mathematical relationship should exist between these two functions. A classic relationship is known to exist:

-RT In K where R is the gas constant, T is the absolute temperature, and In K is the natural logarithm of the equilibrium constant. Thus, a high value for K (high affinity) corresponds to a very negative AFO. Thus, antibody affinity expressed either as K or as AFO represents a thermodynamic measurement of the strength of the antibody-hapten interaction. Another term frequently found in the immunological literature is “avidity.” This term generally refers to the ability of an antibody (usually under dilute conditions) to perform some function involving binding (e.g., neutralization of toxins or viruses). Avidity hopefully depends at least in part upon affinity but also may depend upon antibody valence and on a variety of nonspecific factors. Avidity is thus basically an operationally defined estimate of binding strength which may at times be useful in describing biological systems. Avidity is consequently defined only in terms of the experimental procedures used to carry out AF’

=

CONTROL OF IMMUNE RESPONSE

5

the measurement. Affinity, in contrast, refers to a thermodynamic expression of the binding energy. Investigations dealing with antibody affinity generally are carried out using haptens. In most cases it is only with such molecules that it is possible to study the interaction between antibody and a defined antigenic determinant under conditions in which nonspecific interactions between portions of the antibody and antigen molecules distant from the specific binding sites can be eliminated and complications due to aggregation can be avoided. For a more detailed discussion of the energetics of the antigen-antibody interaction and the types of chemical bonds involved, one can consult a recent review by Karush ( 3 e ) . B. HETEROGENEITY OF AFFINITY A variety of early studies have suggested that antibody present in sera from an individual animal is heterogeneous with respect to its antigen-binding properties ( 4 9 ) . With the use of haptenic determinants, it has become clear that the immune response of an individual animal to a simple haptenic determinant is characterized by the production of molecules differing widely in affinity. It was found that results of studies using equilibrium dialysis could not be interpreted in terms of a homogeneous population of molecules all having the same association constant for the haptenic determinant (10-14). The data obtained in such studies were shown to be consistent with the assumption of either a Gaussian (11,14) or a Sipsian (12) distribution of affinities in the population of antihapten antibody molecules in the serum of an individual animal. The Gaussian and Sipsian functions describe very similar symmetrical distribution curves. As a result of such observations, association constants obtained for the antibody-hapten interaction must be referred to as “average intrinsic association constants” (&) . Based on the approach described above, & would be the peak of a presumed normal distribution curve of antibody affinities. The constant 16 can be operationally defined, reasoning from the law of mass action [Eq. ( 2 ) ] as being equal to the reciprocal of the free hapten concentration present at equilibrium when haIf of the available antibody-combining sites are occupied by hapten. The extent of heterogeneity is generally expressed as a ‘%heterogeneity index” which describes the degree of spread of the normal distribution curve of affinities. It was shown by Nisonoff and Pressman (12) that heterogeneity indicies for antihapten antibodies produced against the same determinant by different rabbits are very variable. That is, the degree of heterogeneity of affinity appears to vary from animal to animal. Recently, Eisen and Siskind ( 15) have separated anti-e,.l-dinitro-

6

GREGORY W. SISKIN'D AND BARUJ BENACERRAF

TABLE I FRACTIONATION OF RABBITANTI-2,4-DINITROPHENYL ANTIBODYFOR AFFINITYBY SEQUENTIAL PRECIPITATION" Fraction no.

1 2 3 4 5 6 7 8 9 10

KO(litersjmole X 10-6) Rabbit A

Rabbit B

Rabbit C

16.0

> 1000.0

1.4 1.7 3.4 1.1 0.36 0.23 0.08 0.05 0.04

8.8 4.2

1.9

1.8 0.85

0.16

-

0.07 0.09

330.0 89.0 19.0 8.1 1.0 0.53 0.23 0.17 0.11

-

a Rabbits immunized with 5 mg. 2,4-dinitrophenyl (DNP)-bovine 7-globulin (BGG). Antibody fractions were prepared by 2,4dinitrophenol elution of specific precipitates formed upon addition of 25 pg. DNP-BGG/ml. serum. Precipitates formed were removed by centrifugation and additional antigen added to supernate. Antibody was purified from each precipitate and its affinity for cDNP-bIysine in 0.1 M tris-HC1, pH 7.6, at 30°-32"C. was determined by fluorescence quenching titration. Adapted from Eisen and Siskind (16).

phenyl (DNP) antibody from a single bleeding of an individual rabbit to a series of ten fractions differing 10,000-fold in average association constant (Table I ) . This separation was accomplished by a procedure of fractional precipitation employing a series of small additions of antigen. The precipitate formed following each successive addition of antigen was collected, the antibody eluted with hapten, and the average affinity of each antibody fraction for hapten determined. In this way heterogeneity of affinity was, for the first time, directly demonstrated and the very marked extent of this heterogeneity was emphasized. In addition to the heterogeneity of binding affinity, a perhaps analogous heterogeneity of "antibody-combining site size" has been demonstrated. Schlossman and Kabat (IS) were able to separate antidextran antibodies into a fraction, the precipitation of which with dextran could be maximally inhibited by trimer of glucose and a fraction maximally inhibited by the hexamer. Although most studies have indicated that specific antibody consists of a highly heterogeneous population of molecules, several recent reports have suggested that under special circumstances antibody relatively homogeneous in affinity may be obtained. Mamet-Bratley (17)reported

CONTROL OF IMMUNE RESPONSE

7

that antibody to tobacco mosaic virus was homogeneous in d n i t y . However it is not ccimpletely clear that the methods employed could have detected moderate degrees of heterogeneity. Krummel and Uhr ( 1 8 ) , studying antibody to phage +X-174using specially designed neutralization assays, concluded that the antibody was relatively homogeneous with regard to binding affinity. Under the conditions of their assay, approximately 2M of the antibody-combining sites were titrated without detecting any heterogeneity. No statement can be made concerning the nature of the remaining 80% of the antibody molecules. However, it appears probable from their results that such antiphage antibody, if not homogeneous, consists of a smaller number of subpopulations of antibody molecules than appears to be present in the usual antihapten antibody preparations. It has been reported by Kitagawa et al. (19) that very late in the immune response to the p-azobenzoate determinant, the antibody present is more homogeneous with respect to afEnity than the antibody formed early after immunization. The heterogeneity index obtained from equilibrium dialysis studies on very late antibody was found to be 1 (no heterogeneity), and fractional precipitation yielded a series of fractions all having the same association constant. It was reported by Nisonoff et al. ( 2 0 ) that one unique rabbit synthesized antibody to p-azobenzoate which crystallized spontaneously upon standing in the cold. This crystalline antibody did not exhibit detectable heterogeneity in equilibrium dialysis studies. The question can be raised whether the observed heterogeneity of affinity represents a basic property of the immune response or rather reflects a heterogeneity of antigenic determinants. Several attempts have been made to explore this issue using relatively more homogeneous antigens than the usual hapten-protein conjugate which has a large number of haptenic groups at different positions on the protein molecule. In the usual hapten-protein conjugates the varying environments of the digerent hapten groups might be responsible for an actual heterogeneity of antigenic determinants. Eisen et (11. (21 ) have found that anti-DNP antibody formed by rabbits immunized by mono-DNP-ribonuclease was essentially as heterogeneous as antibody raised against the usual DNP-protein antigens. Comparable results were also reported by Parker et a2. (44). More recently Haber et al. ( 2 2 ) found that using an octapeptide, angiotensin, as a hapten and poly-L-lysine as a carrier, antibody was obtained which bound angiotensin in an apparently homogeneous fashion. Furthermore, Richards and Haber ( 2 3 ) have reported that immunization with a DNP-polypeptide conjugate of defined sequence elicited the formation of antibodies of high afEnity early after immunization with no subsequent

8

GREGORY W. SISKIND AND BARUJ BENACERRAF

change in affinity. Pappenheimer et al. ( 2 4 ) has reported binding data for the interaction of the octasaccharide subunit of pneumococcal type 8 polysaccharide with horse anti3 8 antibody which are suggestive of a considerable degree of homogeneity of the antibody present. Thus, although the antibodies formed during the usual immune response to proteins and to protein-hapten conjugates are characterized by a marked degree of heterogeneity of binding affinity, several special circumstances of immunization have been reported which appear to favor the formation of a relatively more homogeneous population of antibody molecules. Finally, one must differentiate two distinct modes of describing heterogeneity of affinity. The first would represent the actual number of different species of molecules, each of a specific affinity, present in the population, irrespective of the fraction of the total population that each species represents or their dispersion from the mean. This view of heterogeneity is rarely discussed because we have thus far no way of measuring the actual number of types of molecules present. The second approach to antibody heterogeneity is to consider it the spread of a normal distribution curve of affinities. Here one is concerned not only with the number of classes but also with their percent representation in the total population and their individual spread from the mean considered in a statistical manner. In such a view, populations containing the same number of different types of molecules might differ in degrees of heterogeneity, depending upon how the different classes were quantitatively represented in the population. This is the approach which has been invariably adopted by workers in the field with data being reported as an average association constant (&), that is, the peak of an assumed normal distribution curve of antibody affinities and as an index of heterogeneity expressing the degree of spreading of the normal distribution curve about the mean; the actual number of different species of antibody molecules is not determined. It should further be borne in mind that a change in average affinity might represent either the appearance of new species of antibody molecules or merely a shift in the relative proportions of the different species of molecules present in the total population. It should be kept in mind that it has not been definitively proved that the population of antibody molecules is actually symmetrically distributed about a single peak average binding constant. A skewed or even bimodal distribution might be missed because of limitations of measurement techniques. Ill.

Maturation of the Immune Response

A number of workers using various methods for estimating avidity have noted a progressive change in the binding characteristics of antibody

9

CONTROL OF IMMUNE RESPONSE

with increasing time after immunization (5, 2 5 3 1 ) . We shall refer to this progressive increase in antibody avidity (or affinity) by the term “maturation of the immune response.” Jerne (29) found that antidiphtheria toxin antibody increased progressively in avidity as based upon comparisons of in vivo neutralizing activity and in vitro flocculation assay. Comparable observations have been reported with different antigens by Talmage and Maurer (30) and by F a n (31).An increase in afTinity with time for antibody to iduenza virus has been reported by Fazekas de St. Groth (32). A possible related phenomenon is the observations of Hooker and Boyd (33) that greater cross-reactivity is displayed by antisera produced late in the immune response to a haptenic determinant. Increasing cross-reactivity might result from increasing affinity under certain circumstances, especially where very similar determinants are being compared. Numerous ( 3 4 3 9 ) other workers have indicated increasing cross-reactivity following prolonged immunization; however, in these latter studies the complexity of the antigens or antigenic mixtures involved preclude any definitive conclusions. More recently, studies using defined haptenic determinants and unambiguous methods to determine average association constants (&ties) have established that under the conditions of these studies a progressive rise in affinity with time after immunization was consistently seen (Tables I1 and 111). Eisen and Siskind ( 1 5 ) showed that the affinity of anti-DNP antibody formed by individual rabbits immunized with DNP-bovine y-globulin (BGG) in Freund’s adjuvants increased progressively with time. These results were later c o n h e d and extended

TABLE I1 MATURATION OF AFFINITY OF RABBITANT1-2,4-DINITROPHENYL ANTIBODY WITH TIMEAFl7C.R IMlaUNIZATIOX‘~b Antigen dose

(w.1

0.05 0.5 5.0 50.0

Time after immunization (weeks)

2

3

4

6

-

9.88 (3) 10.3 (7) 9.70 (8) 8.06 (5)

10.0 (9) 11.2 (7) 10.2 (5) 8.52 (5)

11.1 (8) 12.7 (7) 11.0(5) 9.54 (4)

8.72 (6) 8.96 (17) 8.46 (6)

Affinity = -AF” (kcal./mole); numbers in parentheses = no. of animals. Animals immunized with Z14-dinitrophenyl(DNP)-bovine -,-globulin in complete Freund’s adjuvant and bled at times indicated. Affinity of purified antibody measured by fluorescence quenching with cDNP-blysine in 0.15 M NaC1, 0.01 M phosphate buffer, pH 7.5 at 21OC. Adapted from Siskind et al. (40). a b

10

GREGORY W. SISKIND AKD BARUJ BENACERRAF

TABLE I11 OR AFFINITY OF GUINEAPIG ANTI-2,4-DlNITROPHENYL MATURATION ANTIBODYWITH TIMEAFTER IMMUNIZATION' ~~~~~

~

Antigen dose (mg.)

2 Weeks after immunization

2 Months after immunization

mg./ml. (S.D.)

-AFa (S.D.)

mg./ml. (S.D.)

-AFa (S.D.)

0.05 1.0

2.47 (1.05) 1.47 (0.56)

7.93 (0.55) 7.93 (0.16)

1.08 (0.72) 0.21 (0.15)

ll.g(l.36) 8.36 (0.63)

Guinea pigs immunized with 2,4-dinitrophenyl (DNP)-bovine serum albumin in complete Freund's adjuvants and bled at the indicated times. Antibody concentration determined by quantitative precipitin reaction with DNP-bovine fibrinogen. Affinities of purified anti-DNP antibodies for cDNP-L-lysine were measured by fluorescence quenching titration in 0.15 M NaCl, 0.01 M phosphate buffer, pH 7.5 at 26°C. Adapted from Goidl et al. (41).

by Siskind et al. (40)within the same system. Similar observations were made in guinea pigs immunized with DNP-bovine serum albumin (BSA) by Goidl et aZ. (41).Little and Eisen ( 4 2 ) showed a similar increase in afEnity to r-trinitrophenyl (TNP)-lysine, and Klinman et al. ( 4 3 ) have reported an increase in afEnity with time in the response of a horse to p-aminophenyl-P-lactoside.Maturation of affiity with time has also been reported with different haptenic determinants by Parker et al. (44,44u), by Fujio and Karush (a), and by Zimmering et al. (46). It should be noted that all these observations were made using complete Freunds adjuvants for immunization, so that a strong immunizing and proliferative stimulus, as well as a persisting deposit of antigen were present. In some of the above studies (15, 40, 41) the effect of antigen dose upon the rate of maturation was also investigated. It was clearly shown that with decreasing doses of antigen the rate of maturation of antibody affinity increases progressively, In rabbits with very high doses of antigen (250 mg. DNP-BGG), almost no maturation was observed during an 8week period of observation following immunization (15), whereas with 0.5 mg. DNP-BGG there was an increase in A F O of approximately 4 kcal./mole between 2 and 6 weeks after antigen injection (40). In rabbits, 0.5 mg. of DNP-BGG was found to be an optimal immunizing dose (see Section V for a detailed discussion of dose effects). With lower doses of antigen, maturation of d n i t y was observed, but at a somewhat reduced rate (40,4 7 ) . With very low doses of antigen in adjuvants, relatively little serum antibody is produced but, nevertheless, some maturation (less than optimal) in affinity does occur. Measurements of the

CONTROL OF IMMUNE RESPONSE

11

affinity of the initial antibody formed upon boosting at various times after primary immunization demonstrated a progressive increase in affinity (47). It is of interest that over a 100-fold range of dosage the average affinity of the antibody present at 2 weeks after immunization was the same (40).Antigen dose-dependent differences in affinity were only observable later in the immune response. This progressive increase in affinity with time might result from either of two mechanisms: first, the large amounts of antigen present early after immunization (especially after large doses of antigen) bind high-affinity antibody, resulting in a selective removal of high-affinity antibody from the serum; or, second, high-affinity antibody molecules are synthesized in large amounts late after immunization which were not being synthesized (or were being synthesized in relatively smaller amounts) early in the response. That is, according to the first hypothesis, the antibody actually being synthesized is the same early and later after immunization, the difference in average affinity being the result of selective absorption of high-affinity serum antibody early in immunization. The second hypothesis states that the population of antibody molecules being synthesized changes with time, presumably corresponding to a shift in the cell population synthesizing antibody. Steiner and Eisen (48),in a very elegant series of studies, have clearly demonstrated that the second explanation ( a change in antibody being synthesized) is correct. They were able to measure the affinity of antibody snythesized during a brief incubation in an antigen-free culture system by lymphoid cells from immunized rabbits. Cultures from animals late in immunization produced higher-affinity antibody than did cultures from animals early in immunization. Furthermore, cultures from animals receiving lower doses of antigen produced higher-affinity antibody than did cultures of lymphoid cells from animals immunized with larger doses of antigen. It has been shown by McGuigan et at. (49) that amino acid differences exist between anti-DNP antibody synthesized early and late in the immune response. Bernstein et al. (50) compared peptide maps of Fab fragments of early and late antibody and of high- and low-affinity antiDNP antibodies obtained by fractionation of individual rabbit antisera. No differences in maps were observed, suggesting that the amino acid differences reported by McCuigan et at. (49) were localized on the variable portion of the Fab fragment. During the immune response of guinea pigs to DNP-protein conjugates, Nussenzweig and Benacerraf (51 ) observed a shift in the proportion of K to L molecules in the anti-DNP antibody population (see Section VI). This change was related to the timedependent increase in antibody affinity to the DNP determinant.

12

GREGORY W. SISKIND AND BARUJ BENACERRAF

In contrast to the general rule that antibody affinity increases with time following immunization, Richards and Haber (22,23) working with a relatively well-defined DNP-polypeptide antigen of regularly repeating structure obtained a very early, high-affinity antibody response which did not further increase in affinity with time. In summary, in the variety of studies mentioned above there is a progressive change in the character of the antibody present such that the average affinity of the population of antibody molecules becomes progressively greater. There is, furthermore, a change in the character of the antibody present with regard to its average amino acid composition ( 4 9 ) and light-chain composition ( 5 1 ) . We would suggest that such changes can best be understood as the result of a selection by antigen, on the basis of its interaction with prexisting antibody molecules, of those cells capable of synthesizing the highest-affinity antibody. Low-affinity cells failing to capture antigen are thus not stimulated to divide and, consequently, disappear from the antibody-forming cell population. These observations will be discussed more fully in Section VI. IV.

Commitment of Individual Plasma Cells and Sensitized Lymphocytes to the Synthesis of Immunoglobulins of a Single Class and Individual Specificity

A. GENERAL CONSIDERATIONS OF CLONAL THEORY Burnet’s original clonal selection theory (1) required as one of its principal postulates that, previous to antigen contact, individual immunocompetent cells be differentiated to produce a single immunoglobulin with individual specificity. The expansion of the clonal selection theory to explain the dynamic changes in antibody affinity observed in the course of immunization, and the relationship between antigen dose and antibody affinity, on the basis of the selection and stimulation by antigen of the cells producing the highest-affinity antibody, rests on the same postulate, i.e., that a differentiated individual immunocompetent cell synthesizes at most a restricted population and probably only a single immunoglobulin molecule of characteristic specificity and affinity. Furthermore, this postulate needs to be enlarged to include those specific cells that proliferate, in the course of the primary and secondary response, so as to enlarge the population of “memory cells” from which antibody-secreting plasma cells differentiate. The extent of this differentiation from memory cells into plasma cells appears to be in some manner controlled by antigen concentration. Motivated by these theoretical considerations, much effort has been

CONTROL OF IMMUNE RESPONSE

13

directed during the last decade to the study of the immunoglobulins synthesized by individual cells using a variety of ingenious techniques. As could reasonably be e.xpected, the most conclusive results have been obtained with plasma cells or with cells actively engaged in the synthesis and secretion of antibody, because the high immunoglobulin content of these cells has facilitated their study. Some limited information concerning precursor lymphocytes has also been obtained. In this discussion, the paradox has to be recognized that the easiest studied cell is the plasma cell, which is the least crucial cell as far as the clonal selection theory is concerned, whereas the most important cells to explore, the speciiic precursor cells, are the most difficult because of their low immunoglobulin content.

B. PLASMACEZLS 1. Class or Type The observations of numerous laboratories performed on many different species and with different techniques, all agree that individual plasma cells synthesize antibodies of a single immunoglobulin (Ig) class and subclass and also of a single light ( L ) -chain type ( A or K ) .This has been observed in man for different subclasses of IgG, for IgA, and for K and L molecules (52-56); and in guinea pigs for yl and y2 immunoglobulins (57). Some controversy still exists concerning IgM antibodies based on a report by Nossal (58) that the same rabbit cells can first synthesize IgM and then convert to I’gG production. This conclusion was reached by an analysis of the quality of anti-Salmonella antibodies produced in microdroplets containing single cells, before and after treatment with 2-mercaptoethanol which is assumed to destroy exclusively IgM antibody activity. It should be noted that this indirect method may result in serious errors. Merchant and Brahmi (59) recently devised an elegant modification of the Jerne plaque technique ( 6 0 ) , which permits the simultaneous analysis, with different reagents, of the produce of single cells synthesizing antibodies against sheep erythrocytes. This technique introduces the antibody-producing cells between two agar layers containing sheep red blood cells; these are then developed separately to reveal IgM and/or IgG antibody-producing cells. At no time, after primary or secondary immunization of rabbits with sheep red cells, were cells synthesizing both IgG and IgM detected. The evidence at present is clearly in favor of the conclusion that individual plasma cells synthesize antibodies of a single class and L-chain type.

14

GREGORY W. SISKIND AND BARUJ BENACEXWAF

2. Allotype An animal heterozygous for an immunoglobulin allotype locus contains in its serum, immunoglobulins of both allotypes in a fixed ratio. However, studies of immunoglobulin synthesis at the cellular level reveal the fascinating phenomenon of allelic exclusion. That is, individual plasma cells from heterozygous rabbits were shown to produce immunoglobulins bearing one or the other allotype but never both (61). It appears, therefore, that some somatic mechanism operates during the differentiation of immunocompetent cells, by which, in random manner, one of the allotypic genes is not expressed. Thus, the differentiated cell synthesizes an immunoglobulin- of a single class, type, and allotype. An understanding of the mechanism of allelic exclusion would greatly facilitate the solution of the difficult problem of the genetic control of immunoglobulin structure. A very important illustration of negative selection of immune cells based upon the phenomenon of allelic exclusion of immunoglobulin allotype is the observation of Dray and associates (62-65), confirmed by Dubisky and Fradette (66), by Dubisky (67), and by Mage (68, SQ), that immunization of a homozygous mother against the immunoglobulin allotype of the father results in the suppression, in the heterozygous offspring, of the synthesis of immunoglobulins of the father’s allotype and in the compensatory overproduction of the immunoglobulins of the mother allotype. 3. Specificity In spite of a few reports to the contrary, the weight of evidence indicates that individual antibody-secreting cells synthesize antibodies of a single specificity (70-77). Considering the crucial importance of this observation for a cell selection theory based on the selection and stimulation by antigen of specific cells, each synthesizing an antibody of defined specificity and affinity, we shall describe in detail several of the experimental systems used by different investigators to arrive at this conclusion. In evaluating the few dissenting reports, one should consider that errors most likely to occur in these technically difficult experiments will generally cause an erroneous detection of cells producing antibodies of two different specificities rather than the contrary result. Nossal (78, 79) and Nossal and Makela (8 0 ) immunized rats with two non-cross-reacting strains of SalmoneUa and examined thosuands of individual immune cells in microdrops for the presence of antibodies of both specificities. Only rare microdrops were found which contained antibodies of more than one specificity. When this occurred, the reaction

CONTROL OF IMMUNE RESPONSE

15

with one strain of Salmonella was strong, whereas the reaction with the second strain was very weak. Nossal ( 8 1 ) has recently suggested that these few apparent double producers were actually artifactual. Miikela (74) studied the production of antibodies by single cells from lymph nodes of rabbits up to 11months after immunization with two non-crossreacting phages (T2 and T5) and failed to detect any double antibody producers. These results were in sharp contrast with the observations of Attardi et al. (82,83) to be discussed below. Green et al. (70), using a combination of immunofluorescence and radioautography, studied the specificity of the plasma cells from guinea pigs and rabbits immunized with DNP-protein conjugates (antigens bearing two types of determinants on the same molecules). They examined 1569 immune cells, producing 7 S antibodies, and none were found to be producing antibodies against both the hapten and the antigenic determinants of the carrier. Gershon et d.(71) also studied the response of rabbits immunized with an antigen bearing two types of determinants. Rabbits were immunized with HSA to which two non-cross-reacting haptenic determinants ( polyalanyl and p-azobenzenearsonate) were conjugated. The individual haptens were coupled to separate fractions of sheep red blood cells, and the lymph nodes of the rabbits were assayed for cells releasing 19s antibody by a modiiication of the Jerne plaque technique. Examination of 27,845 antibody-producing cells did not reveal any double producers. The experiments of Biozzi et al. ( 7 2 ) and of Petersen and Ingraham ( 7 3 ) ,using still different techniques, yielded identical results. The only major study in the literature which is not in agreement with these observations is that of Attardi et al. (82, 83), who investigated the response of rabbits hyperimmunized with phage T2 and T5. They reported that a relatively high proportion of microdrops of lymph node cell suspensions believed to contain only a single immune cell, were capable of neutralizing both non-cross-reactingphages. There is no apparent explanation for these differences in results except that Attardi et al. ( 8 3 ) examined cells of rabbits immunized for 6 to 24 months, in contrast with Green et aZ. ( 7 0 ) and Gershon et aZ. ( 7 1 ) , who studied cells early in immunization. However, Miikela ( 7 4 ) ,using the same phages and in an 11-month period of immunization, failed to confirm the earlier observations of Attardi et al. ( 8 3 ) . It is therefore reasonable to conclude on the basis of evidence from many laboratories that the individual plasma cells of animals immunized simultaneously with several antigens, or immunized with antigens bearing several distinct determinants on the same molecule, produce antibodies of

16

GREGORY W. SISKIND AND BARUJ BENACERRAF

only a single specificity. That is, individual plasma cells from animals immunized with antigens bearing two types of determinants form antibodies specific for one or the other determinant and not for both. This is in spite of the fact that the antibody populations synthesized against each of the two specificities are markedly heterogeneous. In the present state of our understanding of protein chemistry, it appears that a difference in binding characteristics must imply a difference in tertiary structure of the antibody molecule, which can only result from a difference in primary structure (amino acid sequence). From this point of view, antibody molecules of varying binding properties for the same determinant are just as different from one another as are antibody molecules of different specifkities. Since antibodies against two different antigenic determinants on the same antigen molecule are known to be synthesized by different cells, it is reasonable to conclude that antibody molecules having different binding characteristics for the same determinant are also synthesized by different cells and that an individual plasma cell synthesizes a homogeneous immunoglobulin of characteristic Specificity and affinity. If this reasoning is correct, an antibody-producing plasma cell exhibits the same commitment as a malignant myeloma cell to produce what appears to be a homogeneous and, perhaps, a unique immunoglobulin molecule. It might be noted that each of the several different myeloma proteins with anti-DNP activity which have been recently studied has a distinctive association constant for the DNP-ligand and binds the ligand in a homogeneous manner with respect to affinity (83a).Data have been reported by MiikeTa indicating that individual antihapten antibody forming cells make antibodies of different binding properties (83b).It would be presumed that each cell produces a homogeneous antibody product and that the heterogeneity of serum antibody reflects a heterogeneity of antibody forming cells.

C. LYMPHOCYTES Direct studies of the class and specificity of the immunoglobulin produced by individual lymphocytes have proved to be much more difficult than similar experiments with antibody-secreting cells. Most studies concerning the types of immunoglobulin synthesized by lymphocytes and their immunological specificities have been based upon the phenomenon of blast transformation which immune lymphocytes display when exposed to antigen, This phenomenon is also observed when normal lymphocytes are treated with anti-IgG or antiallotype sera, as has been shown by Sell and Gel1 (84, 85). Blast transformation is believed to be triggered by an antigen-antibody reaction on the lymphocyte cell-membrane, and it can

CONTROL OF IMMUNE RESPONSE

17

be conveniently quantitated by measuring the incorporation of thymidir~e-~H into deoxyribonucleic acid (DNA). Gel1 (86) has shown that blood lymphocytes from an allotypically heterozygous rabbit, such as a 5,6 animal at the B locus, exhibit an increase in th~midine-~H incorporation when exposed to anti5 or anti-6 sera, When cells are treated with both antisera simultaneously the response appears to be largely additive. This observation would be compatible with the view that allelic exclusion is already present in the lymphocyte. Similar results have been recently reported by Sell (87). Concerning the class of immunoglobulins synthesized by individual rabbit lymphocytes, Sell (88, 89) observed that a large proportion of peripheral lymphocytes respond to both anti-IgG and anti-IgM antisera with blast transformation in a manner that could not be considered additive. This he interpreted as suggesting that the same lymphocytes can synthesize different classes of immunoglobulins. However, a lack of commitment of individual mature lymphocytes to a single immunoglobulin class cannot be considered established, since the specificity of the antisera used in these experiments is open to question. The antisera were rendered specific by absorption with soluble antigens and can thus be presumed to contain soluble immune complexes. Such complexes are potentially capable of dissociating and reacting with lymphocytes. Moreover, immune complexes by themselves have been shown by BlochShtacher et aZ. (go), under appropriate conditions, to stimulate blast transformation of lymphocytes. The response of sensitized lymphocytes to antigen in culture provides convincing evidence for the heterogeneity of the population of specific cells synthesizing antibody of a single specificity. Paul et aZ. (91),using the technique of stimulation of incorporation of thymidine=H by antigen (92, 93), studied the antigen dose-response curve of lymphocytes from lymph nodes of guinea pigs immunized with different concentrations of antigen in adjuvants (Fig. 1). The lymph node cells from guinea pigs immunized with 50 pg. of DNP-guinea pig albumin responded in yitro to antigen with a progressive increase in th~midine-~H incorporation over a 10,000-foldrange of antigen concentrations (from to lo+*pg./ml.). This indicates a considerable heterogeneity of the antigen receptor sites on these cells. In contrast to the response to specific antigens which increases progressively over a large range of antigen concentration, the dose-response curve of fractions of the same cell preparation treated with rabbit antilymphocyte serum is very sharp (94), progressing from no response to maximal response over a 10-fold range of concentration of the stimulating agent (Fig. 2).

18

GREGORY W. SISKIND AND BAHUJ BENACERRAF

._

I

10

400

f DNP-GPA Concentration in cultures (pg/ml)

FIG.1. Effect of the immunizing dose of antigen upon the increase in 'H-thymidine ( TdR) incorporation by sensitized lymph node cells stimulated by varying concentrations of antigen in tissue culture. Dose of 2,4-dinitrophenyl-guinea pig albumin used for immunization of guinea pigs indicated next to each curve. Each curve represents the combined data from seven or more individual experiments. Arrows indicate concentrations of antigen calculated to stimulate 50% of the maximum observed increment in thymidine incorporation. Taken from Paul et nl. (91).

Furthermore, it was shown that the character of the antigen d o s e response curve of immune lymphocytes depends upon the amount of antigen used for immunization. Lymph node cells of guinea pigs immunized with large doses of antigen (milligram doses) faiI to respond in uitro to low concentrations of antigens, suggesting the presence of a population of cells bearing low-affinity antibody, with few or no cells bearing high-affinity antibody. In contrast, cells from guinea pigs immunized with low doses of antigen (1pg.) show increased thymidine-*H incorporation in uitro upon exposure to low antigen concentrations (lo-' pg./ml.). The response of such a cell preparation plateaus very rapidly with increased antigen concentration, indicating that only cells bearing high-&nity antibody have been produced following immunization with this dose of antigen. This observation parallels results obtained in studies of the effect of antigen dose on serum antibody affinity (see Section V). The results are consistent with the view that individual immune lymphoid cells which respond to antigen by proliferation synthesize immunoglobulins of differing specificity and affinity. In summary, there is strong evidence that individual plasma cells and

19

CONTROL OF IMMUNE RESPONSE

probably also sensitized lymphocytes are committed to the synthesis of antibodies of characteristic specificity and affinity. These cells have arisen as a result of selective proliferation of specific precursor cells upon stimulation by antigen. Two questions remain: first, can one, based upon available evidence, conclude that immunocompetent cells also become committed prior to contact with antigen, as originally postulated by Burnet and, second, if so, by what mechanism is this commitment to the synthesis of a single immunoglobulin achieved-genetic control or somatic mutation? Two lines of arguments can be offered as evidence of commitment of specific immunocompetent cells prior to contact with antigen. 1. The data from tolerance experiments have shown that the antibody

1500 1400 0

c-

1300-

-

1200 e0 1100F! 8 1000c

.CC

900

;1 800

&

-

-

?

700 600 500 400 300

.E

200

$2

100-

nc

.+ ?I

E

-

0I

lo-' DNP2,-GPA:

I

I

lo-'

t

I

lo+' lot2 o-a

'01

p g / m l of culture medium

0.i

ALS: o/o

I

1

10

concentration in culture medium M

FIG.2. Comparative studies of the stimulation of incorporation of *H-thymidine by sensitized guinea pig lymph node cells with specific antigen 2,4-dinitrophenyl (DNP)-guinea pig albumin ( GPA) and with rabbit antiguinea pig lymphocyte antiserum, Taken from Foerster et al. (94).

20

GREGORY W. SISKIND AND BARUJ BENACERRAF

produced by partially tolerant rabbits is of extremely low a5nity for the tolerizing antigen (95), indicating that the high-affinity cells can be rendered selectively tolerant (discussed below) (95a, 117). 2. If an immunocompetent cell was multipotential and if its commitment to the synthesis of a single antibody resulted from contact with antigen, it is di5cult to understand how a cell would become committed by an antigen to synthesize a low-a5nity antibody when it is also capable of synthesizing an antibody with high affinity for the same antigenic determinant. The simultaneous formation of both low- and high-a5nity antibodies strongly suggests the previous commitment of the cells to the synthesis of a distinct antibody molecule of defined a5nity. A last problem to be discussed concerns the mechanism of commitment of individual immunocompetent cells to the synthesis of individual immunoglobulins. This question is identical to the problem of the generation of heterogeneity in the variable segments of the immunoglobulin chains. Numerous genetic and somatic mutation theories have been proposed to explain the available facts without, as yet, any evidence permitting a choice to be made from among the different theories. The assignment of a precise mechanism to this generation of diversity in immunoglobulin and to the commitment of specific cells to the synthesis of a unique immunoglobulin is not essential to the theory of cell selection by antigen proposed in this review. V.

Effect of Antigen Dose upon the Amount and AfFinity of Serum Antibody

Antigen dose can influence the character of the immune response and together with route of administration can affect the relative amounts of different classes of antibody synthesized. A number of workers (96-101) have shown that injection of animals with soluble or alum-precipitated antigen shortly before or simultaneously with immunization with antigen in Freund’s adjuvant will selectively depress delayed reactivity and the formation of antibodies of certain immunoglobulin classes. The use of complete, as compared with incomplete, Freund’s adjuvant has been shown to influence the character of the immune response with respect to the immunoglobulin classes of antibody formed (10.2-105). Uhr and Finkelstein (106) have reported that after very low doses of phage, only a 19s antibody response is observed, whereas after higher doses a sequence of 19s followed by 7s antibody occurs. The details of the processes that control the relative contribution of different classes of antibody to the immune response are not understood at present. The effect of varying antigenic doses upon the phenomenon of “origi-

21

CONTROL OF IMMUNE RESPONSE

nal antigenic sin” is discussed in a later section, where it is shown that these effects can be clearly understood in terms of a selectional theory such as that being elaborated here. Administration of excessive doses of antigen are known, at least under certain circumstances, to induce immunological tolerance. This topic and its relationship to a theory of antigen selection of populations of cells will also be discussed in a later section. Stenl (107)has shown that following a low dose of antigen for primary immunization, little antibody is synthesized but a marked secondary response occurs upon boosting. In contrast, following a large initial dose of antigen a marked primary response occurs but a very weak secondary response is seen upon boosting. This was interpreted by Stenl (107)as the induction of a “terminal” differentiation by high doses of antigen in contrast of a proliferative response to low doses of antigen. He pointed out the possible contribution of such a mechanism to the induction of immunological tolerance. Recently, extensive studies on the effect of antigen dose upon the amount and a5nity of anti-DNP antibody synthesized have been reported by Eisen and Siskind (15)and by Siskind et al. (40), working with rabbits and by Goidl et al., working with guinea pigs (41).These studies indicate (Tables 11-IV) that following a large dose of antigen there is initially a high concentration of serum antibody (40,41) which reaches peak concentrations relatively early in the immune response (40,41); serum concentrations then fall so that later in the response relatively low levels of low-affinity antibody are present (15,40, 41 ). In contrast, with TABLE IV

IMMUNE RESPONSE OF NORMAL RABBITS TO VARYING DOSESOF 2,4-DINITROPHENYGBOVINE &LOBULIN IN COMPLETE FREUND’S ADJUVANTS~.~ ~

~~

Antigen dose (mg.) 0.05 0.5 5.0 50.0

~

Time after immunization (days) 4

-

7

13

0.02 (12) 0.07 (3) 0.26 (8) 0.01 (10) 0.07(10) 1.06(25) 0.04 (6) 0.18(6) 1.78(6)

_-

20

0.08 (12) 0.61 (11) 1.16(18) 1.14(5)

27

41

0.44(9) 0.54 (9) 2.31 (6) 4 . 2 3 (7) l.SO(24) 1.98(7) 1.09(5) 1.36(4)

Antibody concentrations determined by quantitative precipitin reaction with 2,4-dinitrophenyl (DNP)-bovine fibrinogen. Taken from Siskind et a2. (40). b Values are anti-DNP concentration (mg./ml.); numbers in parentheses are no. of animala.

22

GREGORY W. SISKIND AND BARUJ BENACERRAF

lower doses of antigen, the serum antibody level early after immunization is lower than after high doses of antigen ( 4 0 ) ,but later in the immune response these lower doses of antigen lead to extremely high concentrations of very high-affinity antibody (40). If still lower doses of antigen are used, the amount of antibody synthesized and its affinity is decreased throughout the course of the immune response ( 4 7 ) . It should be noted that in these investigations immunization was, in all cases, by a single injection of antigen in complete Freunds adjuvant, so a depot of antigen was continually present in the animal. Thus, early in the immune response more antibody is present following high doses of antigen than following lower doses of antigen. In contrast, late in the immune response far higher concentrations of antibody are seen after relatively low antigen doses than after higher doses. Furthermore, the optimal dose of antigen with regard to the amount of antibody present late in the immune response is also the dose which leads to maximal affinity of circulating antibody ( 4 0 ) . This optimal dose is approximately 500 pg. of DNP-BGG in the rabbit ( 4 0 ) and probably near 50 pg. of the same antigen in the guinea pig (41) . Doses below 500 pg. in the rabbit result in decreased levels of circulating antibody and also lower-affinity antibody than after the optimal immunizing dose (40). Preliminary results of Werblin and Siskind ( 4 7 ) have indicated that upon boosting following a suboptimal primary dose, antibody of higher affinity is produced but not as high as that observed after a primary response elicited by an optimal antigen dose. Animals immunized with still lower primary antigen doses which resulted in relatively little antibody formation in the primary response were boosted at varying times after the primary injection. The affinity of the antibody present 1week after the boost was measured. The affinity was somewhat greater when the time interval between primary and secondary antigen injections was longer ( 4 7 ) . These results show that some maturation of affinity occurs with time after immunization even with very low antigen doses in the absence of significant levels of serum antibody. However, the degree of maturation in affinity seen after these extremely low doses of antigen, as evaluated from the response to boosting, is far less than the observed following a single optimal immunizing dose of antigen. This pattern of antigen dose effects can, to a great extent, be understood in terms of cell selection by antigen. With high doses of antigen, even low-affinity cells can capture antigen; thus, little selective pressure exists and little maturation in affinity occurs. With lower (more optimal) antigen doses, there is greater selective pressure from reduced amounts of antigen, only high-affinity cells capture antigen and proliferate and the affinity increases rapidly. This simple selective concept is not sufficient

CONTROL OF IMMUNE RESPONSE

23

to understand completely the reduced amounts of antibody seen later after high doses of antigen or the relatively low affiity of antibody following suboptimal antigen doses. Several additional factors must be considered to explain these findings which at first might appear inconsistent with the general hypothesis. Let us first consider the effect of supraoptimal doses of antigen. The reduced amount and affinity of antibody seen in these animals is comparable to the findings with partially tolerant animals ( 9 5 ) which will be discussed in detail in a later section. Thus one factor involved might well be an induction of tolerance in high-affinity antibody-forming cells by supraoptimal doses of antigen (95u, 117). Finally, the high concentrations of circulating antibody early after high doses of antigen and the findings of Sterzl (107) described above both suggest that a high dose of antigen stimulates cells to produce antibody without much proliferation so that a relatively small population of cells incapable of subsequent stimulation results. That is, under the influence of high concentrations of antigen, cells are stimulated to undergo some form of terminal merentiation into antibody-producing cells incapable of further division [Z cells in the terminology of Sercarz and Coons (lOS)]. With lower doses of antigen (especially with adjuvants), cell proliferation is stimulated and relatively few cells are driven into a terminal differentiation. Under these circumstances a large population of antibody-forming cells is present late in immunization and high antibody titers exist at that time. The marked degree of proliferation, as opposed to terminal differentiation, may well contribute in still an additional way to the high affinity of antibody seen later after an optimal immunizing dose of antigen. During the course of this cell proliferation, somatic mutation may occur. This could permit cells to arise that are capable of synthesizing antibody of higher a h i t y than any cells present at the onset of the response. This possibility is discussed in detail in a later section. Next, let us consider the reduced affinity of antibody formed after suboptima1 doses of antigen. On the basis of a purely selectional theory, one would presume that with very low antigen doses only the highestaffinity cells would be stimulated and an extremely high-affinity response would ensue. This, however, does not appear to be the case. Several factors probably combine to explain this apparent inconsistency. First, the belief that only the highest-affinity cells could be stimulated when antigen is limiting, is based upon the assumption that the system is operating at thermodynamic equilibrium. Upon careful consideration, however, it seems unlikely that in the intact organism thermodynamic equilibrium is actually achieved, e.g., all cells may not be equally accessi-

24

GREGORY W. SISKIND AND BARUJ BENACERRAF

ble to antigen at a given time, cell migration will lead to changes in the population of cells in a particular node at different times, and antigen molecules bound to a reasonably avid cell may, for practical purposes, be removed from the system and not be available to interact with a perhaps higher-affinity cell located at some different spot in the animal. Thus, reactions occurring in an intact animal with discrete reacting units (cells) located distinctly separate from one another cannot be considered to occur at thermodynamic equilibrium in the same manner as the reaction might take place in solution in a test tube. If one accepts that we are probably not operating under equilibrium conditions in the intact animal, then one is forced to conclude that statistical considerations must play a role in determining the outcome. That is, whether a particular cell is stimulated depends not only on its af6nity for antigen but also on its chance of coming in contact with one or more molecules of the antigen at an appropriate time and place. If one assumes that with respect to affinity there is a normal distribution of antibody-forming cells present in the animal at the onset of the immune response, then one must conclude that there are far more cells of average af6nity present than of very high affinity. This implies that on a purely chance basis, an antigen molecule is more likely to contact a cell of average affinity than one of high affinity. Thus, statistical factors would operate to maintain antibody affinity at some average value and would oppose any tendency toward increase in affinity. On the other hand, equilibrium considerations (competition of cells for available antigen) would tend to lead to a progressive increase in affinity. The result observed in the immune response would thus be the outcome of some balance between these two opposing factors. It appears likely that statistical factors would be most important when antigen concentration is extremely low. This is consistent with the experimental findings mentioned above. Second, with a very reduced dose of antigen there would be only a small population of cells stimulated to proliferate. The size of the initial population would be expected to affect the rate of maturation of affinity. Furthermore, the question of the role of mutation in a proliferating, specific cell population contributing to the rise in affinity through the appearance of cells synthesizing new antibody molecules, some of which might be of higher affinity than preexisting antibodies, must be considered. This theoretical possibility was mentioned above and will be discussed again, in somewhat greater detail, in Section X. Such a mutational mechanism would help to explain the rather striking coincidence that the dose of antigen leading to the highest obtainable concentration of antibody also leads to the production of antibody of highest affinity.

CONTROL OF IMMUNE RESPONSE

VI.

25

Maturation of the Immune Response: The Selective Stimulation of the Proliferation of Those Cells That Produce Highest-Affinity Antibody

A. GENERALCONSIDERATIONS

The increase in average affinity of antibodies during the immune response can be explained by the selective proliferation, stimulated by antigen, of those specific cells that are best able to bind antigen (or processed antigen ) because they bear antibody of higher affinity. These proliferating cells form an expanding and dynamically changing population of lymphoid cells from which plasma cells differentiate. As the antigen concentration decreases in the lymphoid tissues and as higher-affinity antibody is produced by some cells, the cells bearing lower-affinity antibody, being less able to bind antigen competitively, would tend to represent a progressively smaller fraction of this proliferating population. Having cstablished in a previous section that antibody-producing cells synthesize individual immunoglobulins of defined specificity and affinity, we shall now analyze several experimental immune systems which provide data in favor of this selective scheme. There is evidence that in the course of immunization the synthesis of antibody of highest affinity for the antigen is constantly favored. This results in the selection of antibody types and fractions best adapted to the immunizing antigen. Several immune systems illustrating this mechanism of maturation will be described.

B. THE SELECrrVE ADVANTAGE OF ANTI-2,4-DIh'mOPHENn C U S SYXTHESIZING K MOLECULES DURING IMMUNIZATION OF GUIXEA PIGS WITH ~,~-DINITROPHE~'YL-PROTEIN CONJUGATES Nussennveig and Benacerraf (109) have shown that guinea pig antiDNP yl and y2 antibodies are predominantly of the K type. Several weeks after immunization with DNP-BSA, less than 10% of the anti-DNP antibodies bear L-type chains, although in normal guinea pig 7-globulin the ratio of K/L molecules is 2 : l as in man. Immunization with DNP-BGG stimulates the formation of higher-affinity anti-DNP antibodies more rapidly than does immunization with DNP-BSA, and guinea pigs immunized with DNP-BGG, after 3 weeks, when antibody affinity is already very high, produce anti-DNP antibodies which are over 9!3% K molecules. Earlier in immunization with this antigen (about the ninth day), when low-affinity antibodies are still present, a small percentage of anti-DNP antibodies bearing X chains are found in the serum, but their relative proportion drops rapidly as the serum level of anti-DNP antibodies and their average affinity increase (Table V). These findings suggest that K molecules can form antibodies with higher affinity for the

26

GREGORY W. SISKIND AND BARUJ BENACERRAF

TABLE V L MOLECULES IN h T I - 2 , 4 - DI NI T RO PHEN Y L ANTIBODIES PURIFIED SERAOF GUINEAPIQS IMMUNIZED WITH DIFFERENT DOSESOF

PERCENTAGE OF FROM

2,4-DINITROPHENYL-BOVINE y-GLOBULIN AND

BLEDAT

Vmious TIMESAETER IMMUNIZATION"

L molecules in

Time after immunization (days)

purified antiD N P antibody

(mg.)

Anti-DNP antibody in serum (mg./d.)

0.05

7 10 14 29 7 10 14 29

h'D* 0.49 2.50 5.10 NDb 0.56 2.00 2.40

17.1 5.7 1.1 0.3 18.4 6.9 3.3 2.6

Dose of antigen

1.00

(7%)

KOX (liters/mole) -

12 430 -

3.2 7.1

Taken from Nussenzweig and Benacerraf (109).

* Not done because amounts of ant,ibody were too small. DNP determinant than can L molecules. This was verified experimentally (109). Anti-DNP antibodies from guinea pigs immunized with DNP-BSA were separated by serial precipitation with antigen to isolate antibody fractions with progressively lower affinity. The proportion of A-chain-bearing molecules was found to be inversely related to the average affinity in these fractions; no L molecules were present in the fraction with I?, above 10' (Fig. 3). These observations illustrate that although A-type anti-DNP antibodies can be formed by guinea pigs immunized with DNP-protein conjugates, the cells that synthesize them fail to capture antigen and do not proliferate. In contrast, the high-dnity-K-type anti-DNP cells are selected for and multiply to become the main cell type at the time when the average affinity of the serum antibody increases.

c.

RESPONSES I N RABBWSIMMUNIZED WITH 2,4-DIh71TFIOCONJUCATES ELICITJXIBY 2,4-DINITROPHENYL COUPLED TO HETEROLOCOUS PROTEINS SECONDARY

PHENYGhOTEIN

All cellular immune responses (delayed sensitivity reactions either in uiuo or in uitro and anamnestic responses) of animals immunized with hapten-protein conjugates show considerable carrier specificity ( 91,110115). We shall consider here only the thermodynamic consequences of

27

CONTROL OF IMMUNE RESPONSE

1

Time after immunization

0

7 0

0

A

11 D a y s 19 Days 28 Days

0

0 Y

0

A

-

0,

0

I

6

A

0

A 5

I 2

I

0

A

"

I I I I 4 6 8 1 0 1 2 70 of L molecules

FIG. 3. Re1ationshi.p between binding ffiities ( K O ) for e2,4-dinitrophenyl (DNP)-L-lysine and percentage of L molecules in fractions of anti-DNP antibodies specifically isolated by serial precipitations from sera of guinea pigs bled at several time intervals after iminunization with DNP-bovine serum albumin. Taken from Nussenzweig and Benacmraf ( 109).

carrier specificity. In these terms, carrier specificity in cellular reactions is believed to reflect, at least in part, the fact that the specificity of cellassociated antibodies (which is presumed to be identical to that of humoral antibodies) encompasses both the hapten and the carrier molecule and that the contribution of the carrier molecule to the binding is crucial (at least early in the response) for the elicitation of those responses that result from the reaction of specific cells with antigen. This high degree of specificity could be due to a high-energy requirement for the triggering of cellular immune reactions. As an illustration of carrier specificity, good anamnestic responses by rabbits immunized with DNP-BGG are not elicited by DNP-heterologous conjugates early in the immunization ( 1 1 3 ) . However, weeks or months after immunization, hapten-specific booster responses can be observed in these animals upon challenge with heterologous conjugates of the same hapten (952,116-118, 118a) (Table VI). These haptenspecific secondary responses are generally not as strong nor as depend-

28

GREGORY W. SISKIND AND BARUJ BENACERRAF

TABLE V1 EFFECT OF BOOSTING WITH HOMOLOQOUS OR HETEROLOGOUS CARRIERS UPON THE AFFINITYAND AMOUNTS OF ANTI-2,4-DINITROPHENYL ANTIBODY FORMED IN THE SECONDARY RESPONSE' Anti-DNP serum antibody No. rabbits

Boosting antigenb

Preboost (rng./ml.)

Postboost (mg./ml.)

A(AF') (kcal./mole)

2 4

DNP-BGG DNP-LiH

0.17 0.33

1.56 0.43

-2.95

0.00

a Rabbits boosted with 5 mg. of the indicated DNP-protein intravenously 5 months after primary immunization with 5 mg. DNP-BGG in complete Freund's adjuvants. Serum anti-DNP antibody levels were measured just prior to and 1 week after secondary immunization by precipitin reaction with DNP-bovine fibrinogen. Affinities of purified antibodies were determined by fluorescence quenching titration with 2,Cdinitrophenol. A(AF") = AF" for postboost antibody-hapten interaction - the AF' for preboost antibody-hapten interaction. Adapted from Paul et al. (116). DNP, 2,4dinitrophenyl; BGG, bovine -,-globulin; LIH, Limulus hemocyanin.

able as those elicited by the immunizing conjugates, and some heterologous conjugates are clearly better than others in their ability to elicit such reactions (116). The relative inefficiency of conjugates of hapten with heterologous carriers to elicit hapten-specific secondary responses may be explained in part by the fact that the affinity of the cellular antibodies for the hapten alone is insufficient to trigger the cellular response. Later in the immunization, when antibody affinity increases, enough cells have antibody with sufficient affinity for the hapten to bind hapten conjugates of heterologous carriers; these cells can then be stimulated and a secondary response elicited. These conclusions were strengthened by a comparative study of the average affinities of anti-DNP antibodies before and after the anamnestic responses of rabbits boosted with either the immunizing DNP-conjugate or with DNP coupled to a heterologous carrier (116). No significant difference in antibody affinity for the DNP-lysine determinant could be detected after boosting with the immunizing conjugate. In contrast, there was a marked increase in affinity of the antibody for the DNP-hapten (the only portion of the determinant common to both the immunizing and boosting antigen) after challenge with DNPheterologous conjugates. Furthermore, some rabbits that showed no Significant change in their serum levels of anti-DNP antibodies after such a heterologous challenge, exhibited significant increase in the affinity of their antibodies for DNP-lysine. Apparently, only cells bearing antibody with sufficiently high affinity for the DNP portion of the

COEJTROL OF IMMUNE RESPONSE

29

determinant, common to both antigens, were selected for in secondary responses by what can be considered cross-reacting antigens.

D. THE RELATIONSHIP BETWEEN THE ELECITUCAL CHARGE OF THE ANTIGENAND THE CHARGE OF THE CORRE~PONDING AATIBODY Studying antibodies produced by rabbits immunized with protein antigens of different charge, Sela and Mozes (119) noted a reverse relationship between. the charge of the antigens and that of the corresponding antibodies. The phenomenon was also extended to the mobility of anti-DNP antibodies elicited in rabbits by DNP conjugates of synthetic polyamino acids of digerent charge. This last observation was confirmed by Benacerraf et al. (120) who showed that the antiDNP antibodies procluced by rabbits immunized with a DNP conjugate of a copolymer of L-lysine and L-alanine were considerably more negatively charged than were most of the antibodies produced following immunization with a DNP conjugate of a copolymer of L-glutamic L-lysine, and L-alanine (with a high content of glutamic acid). These two anti-DNP antibodies differed markedly in their specificity for the carrier polymer used in immunization, as shown by fluorescence quenching titrations. These data can be explained, as was done with other systems, on the basis of the selection by antigens of the best fitting antibody molecules by means of a selective stimulation of the specific cells synthesizing these molecules. When highly charged antigens are used, it is reasonable to expect that the charge will play some role in the antigen-antibody binding (121-123) and, therefore, the selection of antibody molecules of opposite charge to that of the antigen is to be expected. However, it remains to be determined whether the binding contribution due to the opposite charge of the two molecules (antigen and antibody) is contributed solely by the antigenic determinant and by the antibody-combining site or whether these charge effects extend to interactions of portions of the molecules distant from the antibody-combining site. If the latter is the case, the reaction of antigen with antibody could be regarded as being able to be affected by the interaction of these two molecules at many points, and immunological specificity being determined by many cooperative forces could thus be defined precisely only in thermodynamic terms. That interactions between antigen and antibody molecules distinct from the interaction between specific combining sites can contribute to the overall energy of binding involved in the formation of a stable aggregate has been clearly demonstrated (124, 125). Such factors would be expected to contribute to the selective process.

30

GREGORY W. SISKIND AND BARUJ BENACERRAF

VII.

Effect

of Humoral Antibody on the Control of Antibody Synthesis

It is now well known that in a variety of different immunological systems, passively administered circulating antibody will specifically inhibit antibody synthesis to concomitantly administered antigens. This phenomenon, generally referred to as immunological suppression, has recently been extensively reviewed by Uhr and Moller ( 1 2 6 ) . We shall discuss only those specific aspects of suppression that operate in the maturation of antibody afsnity and appear pertinent to the general thesis of this paper. Numerous studies have shown suppression to be highly specific for the antigen against which the circulating antibody is directed [for reference, see Uhr and Moller ( 1 2 6 ) ] ,suggesting that circulating antibody brings about suppression by virtue of its ability to bind antigenic determinant and in this way prevents it from interacting with potential antibodyforming cells. In addition to this general mode of action of passive antibody in causing an “afferent block,” i.e., interfering with immunization by preventing antigen from reaching or triggering potential antibodyforming cells, an “efferent block” has been clearly demonstrated by Moller (127, 128) as also operative in tumor enhancement by passive antibody. In terms of the thesis being developed here, circulating antibody can be viewed as competing with cell-associated antibody for available antigen. Based on this hypothesis a variety of predictions can be made: ( a ) the higher the affinity of the circulating antibody the more effective it should be in bringing about suppression; ( b ) the greater the dose of circulating antibody the more effective it should be in suppression; ( c ) the higher the affinity of the potential antibody-forming cell population or the larger the cell population the more difficult it should be to obtain suppression with passive antibody; and ( d ) low-affinity antibody-forming cells should compete poorest for antigen and thus be most readily suppressed. A number of studies have indicated that the degree of suppression is related to the dose of passive antibody administered [for references and more detailed discussion, see Ref. ( 1 2 6 ) l . Walker and Siskind ( 1 2 9 ) , working with the DNP-haptenic system, have shown that high-affinity antibody is far more effective in producing suppression than is low-affinity antibody (Table VII) i.e., less antibody of high aflinity than of low affinity is required to produce a given degree of suppression. It has been reported by several workers (126,130) that antibody produced late in the immune response is more effective in producing suppression than is antibody formed early after immunization. In view of the well-known

31

CONTROL OF IMMUNE RESPONSE

TABLE VII ANTI-2,4-DlNlTROPHENYL ANTIBODYREQUIRED TO PRODUCE 60% SUPPRESSION OF ANTIBODY SYNTHESIS AT 2 WEEKSAFTER IMMUNIZATION AS A FUNCTION OF THE AFFINITYOF THE PASSIVE ANTIBODY'

d M 0 l . I N T O F PASSIVE

Affinity of passive antibody

(KO)

Passive antibody (mg.)

1 . 9 x 106 6 . 1 >( lo7

49 14

101'

6

a Passive antibody given intravenously, in varying doses 1 day before immunization with 5 mg. 2,4.-dinitrophenyl (DNP)-bovine yglobulin in complete Freund's adjuvant. Rabbits were bled a t 2 weeks and the concentration of anti-DNP antibody determined by quantitative precipitin reaction with DNP-bovine fibrinogen. Affinity of antibody is expremed as the association constant in liters/mole a t 21°C. for the reaction with e-DNP-clysine in phosphatebuffered saline. Original data from which values in table are calculated are given in Walker and Siskind (129).

increase in binding affinity with time after immunization ( 1 5 ) , it may be presumed that it is the higher affinity of the late antibody which permitted it to be more effective in suppression. Most workers have noted that to achieve efficient suppression, passive antibody must be administered shortly before, simultaneously with, or relatively soon after antigen injection [for references, see Ref. (126)l. Presumably as the affinity of the potential antibody-forming cell population increases with time after immunization, cells become increasingly effective in capturing antigen and, consequently, increasingly resistant to suppression. It has been reported by Wigzell (131) that suppression can be produced even long after immunization; however, the degree of depression was limited and relatively large amounts of passive antibody were required. Similar observations have been made in the DNP system by Siskind (132) who found that suppression of late 7 S antibody synthesis required relatively large doses of passive antibody and could, furthermore, only be effectively achieved with very high-affinity antibody. Furthermore, Uhr and Baumann (133) have shown that it is more d B cult to suppress a secondary than a primary response; suppression of the secondary response could be achieved, but only with relatively large amounts of passive antibody. This was related to the fact that smaller amounts of antigen are required to trigger a secondary than a primary response. We would interpret this as being due to the presence of a larger specific cell population of higher average affinity in the primed as compared to the virgin animal.

32

GREGORY W. SISKIND AND BARUJ BENACERRAF

Finally, it has been reported by Siskind et al. ( 4 0 ) that if partial suppression of specific antibody synthesis is achieved by repeated doses of passive antibody so as to maintain a constant low concentration of passive antibody, then the residual antibody synthesized by the animal 4 weeks after immunization is of higher affinity than normally found at that time for that dose of antigen. Thus, as predicted, low-affinity antibody-forming cells were more readily suppressed by passive antibody than were high-affinity cells, resulting in an increase in the average affinity of the antibody synthesized. However, if passive antibody is only given once just prior to injection of antigen in Freund's adjuvants, then the animals behave as if immunization is merely delayed for several days and produce, at 2 or 3 weeks, low titers of antibody of somewhat lower affinity than expected at that time in immunization (129). Antibody concentration and affinities increase in an essentially normal manner if one allows for a lag of 1to 2 weeks before onset of immunization. That is, the animals behave as if the passively administered antibody initially prevents immunization; however, as the passive antibody level falls with antigen still present (injected as a depot in Freund's adjuvants), an essentially normal immune response follows. Thus, suppression of antibody synthesis by passive antibody behaves as would be predicted from the hypothesis of competition of serum antibody with cells for available antigen. In this way circulating antibody might be expected to play a role in the control of the immune response. Several such control functions for circulating antibody have been suggested in the past: 1. Uhr and Baumann (134)suggested that circulating antibody might serve to limit the immune response and thus prevent uneconomical hyperimmunization. 2. Sahiar and Schwartz (135) have suggested that circulating 7 S antibody might be involved in the termination of 19 S antibody synthesis. These workers have shown that if the 7 S response is suppressed by treatment with 6-mercaptopurine the 19 S response persists longer than in untreated animals. Furthermore, they were able to terminate such a prolonged 19 S response by passive administration of 7 S antibody. Unfortunately the relative binding affinities of 19 S and 7 S antibodies, at appropriate times, in the system studied, are not known so evaluation of the data from an energetic point of view is not possible. Uhr and Finkelstein (106) have shown that after low doses of +X-174,19 S antibody is synthesized without any detectable 7 S response. Such a 19 S response is self-limited despite the absence of 7s antibody synthesis. Thus, other

CONTROL OF IMMUNE RESPONSE

33

factors in addition to suppression must be involved in the termination of 19 S antibody synthesis. 3. Brody et d.(136) presented data to suggest that where antigenic competition existed antibody formation to one antigenic determinant might depress subsequent responses to that determinant and result in a favoring of antibody synthesis to a second antigenic determinant. 4. On the basis of the effect of suppression on antibody affinity and the increased effectiveness of high-affinity antibody in causing suppression, Siskind et al. (40)suggested that circulating antibody contributes to the maturation of antibody affinity. That is, antibody synthesized by high-affinity cells tends to suppress low-affinity antibody synthesis and thus contributes to the progressive rise in average antibody affinity. As affinity of the antibody increases, it becomes increasingly better at causing suppression and, consequently, only higher and higher-affinity cells can capture antigen and be stimulated to divide and/or synthesize antibody. Circulating antibody, therefore, serves to exert an additional selective pressure toward higher-affiity antibody production which, in turn, results in even greater selective pressure in the same direction. VIII.

“Original Antigenic Sin”

“Original antigenic sin” refers to the effect of previous immunization on the response to stimulation with a structurally related antigen ( 1 36 4. This phenomenon has been most extensively explored in the elegant studies of Webster (137) and of Fazekas de St. Groth and Webster (32,138, 139) on the immune response to sequential infection with different strains of influenza v i r u s in ferrets, in rabbits, and in man. Animals or man immunized with one strain of influenza virus, upon subsequent exposure to a second, partially cross-reacting strain of influenza virus, produce a large amount of antibody which is totally cross-reacting and which is characterized by having a very high avidity for both the original and boosting virus strains. The avidity for the original virus of the antibody formed in response to stimulation with the cross-reacting virus was the same as the avidity of the antibody present in the serum just prior to boosting and was equal to the high avidity of the antibody formed early in a secondary response to the original virus. Antibody formed early in a normal primary response to either virus was shown to be of low avidity. The response to a cross-reacting virus in an immunized animal was shown to be similar to a secondary response with respect to its resistance to the depressive effects of X-irradiation. In terms of a selectional theory, one would expect that the cells pres-

34

GREGORY W. SISKIND AND BARUJ BENACERRAF

ent as a result of the primary immunization which are synthesizing antibody of high affinity for the cross-reacting antigen used in boosting would capture antigen preferentially and be stimulated to divide. The antibody formed under such circumstances would, of course, be totally crossreacting and of high affinity for both the original and the boosting antigens. In addition, since this antibody would be synthesized by a restricted population of cells as compared with the cell population involved in the usual primary or secondary response, one would expect the antibody formed to be more homogeneous than the antibody found in a normal primary or secondary response. Absorption studies (32, 138) suggested that the antibodies formed by an immunized animal challenged by a second virus strain were, in fact, more homogeneous with respect to avidity than were the antibodies formed in a normal primary or secondary response. With the original antigenic sin-type antibody, no change in avidity could be detected upon partial absorption, and the ratio of avidities for the two viruses remained constant upon absorption with either virus. This is in contrast to results of absorption studies on normal primary or secondary antibody in which a heterogeneity of binding characteristics was readily detected. The larger the cross-reacting immune cell population resulting from the primary immunization, the more effective it will be in capturing antigen and the less antigen will be available to stimulate low-affinity cells which usually contribute to the early primary response to the second antigen. Furthermore, it might be reasoned that if a very large dose of cross-reacting antigen were given for the booster response, the highaffinity cells would be unable to capture all of the antigen and some lowaffinity cells should then be stimulated. Such predictions were tested by Fazekas de St. Groth and Webster (139). Following a small dose of antigen for primary stimulation, only the earliest antibody formed after boosting with a second virus strain was of the totally cross-reacting, high avidity type characteristic of the original antigenic sin phenomenon. Later, antibody was formed which had binding characteristics of primary response antibody to the second virus strain. After a normal primary immunization, boosting with a relatively low dose of the second antigen results in antibody entirely of the original antigenic sin type. However, if the dose of the second virus is increased one obtains, in addition to the antigenic sin-type antibody, antibodies characteristic of a normal primary response to the second virus. In an animal that was primed with both viruses, boosting with one led to a secondary-type response to the boosting virus and no significant production of antigenic sin-type antibody. Fazekas de St. Groth and Webster (139) interpreted their results in terms

CONTROL OF IMMUSE RESPOKSE

35

of a trapping-type mechanism related to the avidity of cell-associated antibody and suggested that populations of antibody-forming cells can be selected by antigen on the basis of antibody-binding characteristics. This interpretation is completely compatible with that being developed in the present paper. Observations which could be interpreted as comparable to the original antigenic sin phenomenon discussed above have been reported by Paul et al. (116), who studied the responses of rabbits immunized with DNPprotein conjugates upon boosting with the hapten conjugated to a different protein carrier. (These results have been discussed in detail in Section V1,B.) Thus, the phenomenon of original antigenic sin can also be understood in terms of the selection by antigen, of a population of cells, on the basis of the energetics of the interaction of antigen with preexisting cell-associated antibody molecules. Cells bearing high-affinity cell-associated antibody preferentially capture antigen and are stimulated. When a large population of cells of a particular specificity is present due to previous immunization, an alteration in the pattern of response to a cross-reacting antigen may be observed as a result of the presence of an unusual distribution of cells capable of interacting with the second antigen. IX.

Immunological Tolerance (Unresponsiveness)

A. GENERAL CHARACTERISTICS Immunological tolerance refers to a specific depression of immunological reactivity as a result of previous exposure to the antigen. There have been a number of extensive reviews dealing with this topic (140]Pa), and no attempt will be made here to be all inclusive in our discussion, Rather, we shall deal selectively with those aspects that appear germane to the thesis 'being developed. In general, there are two distinct ways in which tolerance can be induced. These correspond to the concepts of high and low zone tolerance introduced by Mitchison (145) in his studies in mice with BSA. Highdose tolerance results from injection of much greater amounts of antigen than lead to optimal immunization. This form of tolerance appears to be more readily induced during the neonatal period. On the other hand, low-dose tolerance, as shown by Thorbecke and Benacerraf (146) results from the repeated injection of subimmunogenic doses of antigen, generally in increasing amounts, and preferably in a form that is relatively nonphagocytizable and nonimmunogenic. Thus, Dresser ( 147-149) has observed that if BGG is cleared of all aggregated protein by ultracentrifu-

36

GREGORY W. SISKIND AND BARUJ BENACERRAF

gation, very small doses will induce tolerance upon intravenous injection into mice. Nossal and Ada (150) have shown that soluble flagellin will induce tolerance in minute doses, whereas the aggregated, particulate flagella will immunize. Frei et al. (151) have extended this concept by showing that following an intravenous injection of BSA in rabbits the antigen still present in the serum hours later is very effective in inducing tolerance if injected into a second rabbit. That is, the relatively nonphagocytizable portion of the total antigenic mass is capable of inducing tolerance, and, presumably, the aggregated or slightly denatured, readily phagocytized portion is responsible for immunization. Low-dose tolerance with soluble antigen may be due to the presence of insufficient amounts of “immunogenic” (aggregated or denatured) antigen in the preparation of antigen at the dose used. Thus, tolerance can be induced either with excessive amounts of antigen or with subimmunogenic amounts. With low doses of antigen, highly soluble, relatively nonphagocytizable, antigen preparations tend to induce tolerance most readily. One additional general fact about tolerance which should be mentioned is that a number of workers have found that agents which nonspecifically enhance immune cell proliferation [e.g., appropriate doses of X-ray (152, 153), adjuvants (154), and endotoxin (155)] will prevent induction of tolerance or will tend to terminate prematurely an existing state of tolerance. On the other hand, conditions leading to a nonspecific depression of cell proliferation [e.g., X-irradiation-for references, see Ref. (156)-and antimetabolite drugs (157)] tend to facilitate tolerance induction. Finally, tolerance is of finite duration and at some time, depending upon the dose of antigen used for its induction, will spontaneously disappear. The duration of the tolerant state may be prolonged by periodic injections of antigen. A number of workers (158-161) have noted the spontaneous appearance of circulating antibody or have been able to demonstrate a spontaneous preparation to give a secondary-type response in mice upon loss of tolerance. These basic facts about tolerance have been interpreted as suggesting that when native antigen interacts directly with lymphoid cells tolerance results, whereas antigen that has undergone some form of processing or localization (perhaps in or on a macrophage) stimulates proliferation and/or antibody synthesis upon interaction with potential antibodyforming cells. ,That is, antigen presented in such a form or manner that it tends to interact directly with lymphoid cells without being processed or localized at appropriate sites will induce tolerance. Very large doses of antigen would “flood” the animal with antigen and thus lead to non-

CONTROL OF IMMUNE RESPONSE

37

processed antigen reaching potential antibody-forming cells. On the other hand, very low doses of antigen, especially in a soluble, nonphagocytizable form, would be inefficiently bound by macrophages but would still interact with the lymphoid cell bearing the specific combining site (especially if the combining site was of very high affinity). The outcome of antigen injection is thus the result of two competing processes,ne leading to tolerance and the other to immunity. Varying conditions will favor one or the other of these processes. It appears likely, therefore, that the mechanisms of high and low zone tolerance are basically the same: interaction of antigen dirmtly with the specific lymphoid cell without intervention of some processing or localizing step necessary for induction of an immune reaction. The existence of two zones separated by a zone of immunity is probably the consequence of the presence of two distinct forms of antigen in the injected material, one form being essentially immunogenic and the other form being primarily tolerogenic. With an antigen preparation, such as the ultracentrifuged BGG studied by Dresser (147-149)which has had the immunogenic fraction removed, two zones of paralysis would not be expected and are not seen. Similarly, pneumococcal polysaccharide, which has been shown by Howard and Siskind (162) not to contain a nonphagocytizable tolerogenic fraction such as seen with certain protein antigens (151),does not exhibit two separate dosage zones for tolerance induction (162, 163). The high and low zone phenomenon may thus be a consequence of the presence of two forms (immunogenic and tolerogenic) of antigen in the immunizing injection and two competing processes (immunity and tolerance induction), therefore, occurring simultaneously. As to the exact mechanisms by which tolerance is induced, two possibilities, not necessarily exclusive, must be considered: (1)The immunocompetent lymphoid cell, or its precursor, reacting directly with native antigen either dies or becomes unresponsive to processed antigen. ( 2 ) The immunocompetent cells are stimulated by nonprocessed antigen to differentiate into plasma cells without proliferating to form the large clone of specific cells which is essential for a sustained immune response. Both processes would result in the elimination of specific immunocompetent cells. There is some evidence that the latter process occurs in the course of the induction of paralysis to pneumococcal polysaccharides in normal or immunized animals (164,165). AFFIh?TY A h 9 TOLERANCE INDUCTION B. ANTIBODY If tolerance is the result of antigen (nonprocessed) interacting with

cell-associated antibody molecules on precommitted lymphoid cells, then tolerance induction should preferentially effect high-affinity antibody-

38

GREGORY W. SISKIND AND BARUJ BENACERRAF

E F F E r T OF PARTIAL

TABLE VIII TOLERANCE UPON

ANTIBODY ~~

Group

No. of rabbits

Antibody concentration (mg./ml.)

Normal Tolerant

9 10

1.02 0.54

AFFINITY" ~~~

-AF"

(kcal./mole) 8.67 6.61

a Rabbits made partially tolerant to 2,4-dinitrophenyl (DNP)-home serum albumin (HoSA) by neonatal injection of 45 mg. of antigen in divided doses over the first 12 days of life. Challenged, along with normal rabbits of the same age, with 5 mg. DNPHoSA in complete Freund's adjuvant and bled 3 weeks later. Antibody concentration determined by precipitin reaction with DNP-bovine fibrinogen. Affinity of the purified antibodies for c-DNP-clysine were determined by fluorescence quenching titration in 0.15 M NaCI, 0.01 M phosphate buffer, pH 7.5, a t 25°C. Adapted from Theis and Siskind (95).

forming cells. This prediction has been tested by Theis and Siskind (95) in newborn animals made tolerant with high doses of DNP-horse serum albumin ( HoSA) and by Theis et al. (166) in adult rabbits made tolerant to DNP-rabbit serum albumin ( RSA ) by repeated intravenous injections of low doses of antigen. In either case, upon subsequent immunization with the DNP-protein used to induce tolerance, the small amount of antihapten antibody synthesized by partially tolerant animals was of extremely low affinity as compared to that formed by control nontolerant animals (Table VIII). It was also noted that the affinity of the antibody synthesized by partially tolerant animals increased slowly with time after immunization so as eventually to achieve the range of affinities seen among normal animals much earlier in immunization. C. RESPONSEOF TOLERANT ANIMALSTO ANTIGENSCROSS-REACTIVE WITH THE TOLERATED ANTIGEN Certain characteristics of the response of tolerant animals to antigens cross-reactive with the tolerated antigens can be better understood in the light of the expanded cell selection theory proposed in this paper. An example of tolerance to partial determinants is afforded by the observation of Battisto and Chase (167, 168), subsequently confirmed by Coe and Salvin (169), that animals tolerant to a hapten-autologous protein conjugate would, nevertheless, synthesize antihapten antibody when challenged with a hapten-heterologous protein conjugate. This system has been further analyzed from the energy point of view by Werblin et al. ( 170). Preliminary results indicate that the antihapten antibody produced, even though in normal amounts, is of moderately reduced affinity

CONTROL OF IMMUNE RESPONSE

39

as compared to antibody produced by control animals. This indicates that the highest-affinity antihapten antibody-producing cells are preferentially rendered tolerant and remain tolerant in the face of challenge with a hapten bearing foreign protein. Weigle (171-173) made the very intriguing observation, subsequently confirmed by other workers (174, 175), that rabbits rendered unresponsive to a soluble protein, BSA, nevertheless could be induced to form antibodies that precipitated with BSA by immunization with cross-reacting foreign albumins or with hapten conjugates of the tolerated BSA. The anti-BSA antibodies produced were completely precipitated by the immunizing antigens and therefore were not directed to BSA determinants not present on the cross-reacting antigens. Weigle interpreted this phenomenon as a “break of tolerance.” The results of recent experiments by Paul et al. (176) and, independently, by St. Rose and Cinader (177) have helped to clarify the precise nature of this break of tolerance. Paul et al. (176) attempted to break the tolerance of rabbits rendered unresponsive to BSA as adults by the low-dose tolerance technique described by Thorbecke and Btmacenaf (146) (highest dose of BSA injected, 1 mg., and total dose less than 10 mg.). The tolerant rabbits were immunized with DNP-BSA (with an average of ten DNP groups per protein molecule) in incomplete adjuvants. The immunological specificities of the anti-BSA antibodies produced by these animals and by normal rabbits similarly immunized with DNP-BSA were compared. It was established that the anti-BSA antibodies produced by normal rabbits bound BSA and the immunizing antigen, DNP-BSA, almost equally well; in contrast, the anti-BSA antibodies produced by the tolerant animals bound DNP-BSA much better than BSA. Thus, the BSA-tolerant rabbits remained unresponsive to BSA determinants to which they were originally tolerant, even after immunization with DNP-BSA. The anti-BSA antibodies produced by these animals in response to DNP-BSA must be considered as specific for cross-reacting determinants of the DNP-BSA molecules, to which tolerance had never been established. That is, the anti-BSA antibody was produced by cells that were not rendered tolerant by the low-dose tolerance-inducing regimen used in these experiments, presumably because of their low affinity for BSA. According to the cell selection theory, the prediction can be made that, if rabbits were rendered tolerant with higher doses of BSA, more anti-DNP-BSA cells with low affinity for BSA would be rendered unresponsive. As a consequence, fewer cells would be able to respond to DNP-BSA upon subsequent challenge. This proved to be precisely the case (178). When rabbits were rendered tolerant with 100-times higher

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GREGORY W. SISKIND AND BARUJ BENACERRAF

doses of BSA than used in the earlier experiments, immunization with DNP-BSA resulted in very little or no antibody being produced capable of precipitating BSA. In addition the serum levels of anti-DNP-BSA antibodies produced by these animals were much reduced compared to those produced by normal rabbits immunized with DNP-BSA. These various experiments demonstrate that the specificity of tolerance, as the s p d c i t y of immunity, has to be considered in energetic terms taking into account the dose of antigen used in inducing specific unresponsiveness. The dose of antigen can be considered to determine the specificity and the breadth of the tolerance achieved, i.e., the extent to which cells bearing antibody with lower affinity for the tolerated antigen (and, in fact, more specific for cross-reacting antigens) are rendered unresponsive. The reduced affinity of antihapten antibody response to hapten-protein conjugates seen in animals rendered tolerant to the carrier molecule can be understood in similar terms. In both tolerance induction and immunization, high-anity cells capture antigen most efficiently. What determines whether the interaction of antigen with cellassociated antibody will lead to proliferation and antibody formation or to tolerance induction is not known at present. Certain characteristics (discussed above) of antigen preparations and procedures which tend to induce tolerance suggest that some macrophage-associated processing or localizing step may be involved in the pathway toward antibody formation, and that when this step is by-passed tolerance ensues. The nature of the hypothetical macrophage-associated processing step is unknown. In a sense, tolerance induction modulates the subsequent response to a cross-reacting antigen in much the same manner as immunization modifies the response to boosting with a cross-reacting antigen (original antigenic sin; see above). In both cases, an alteration in the character of the cell population has occurred as a result of the initial exposure. With immunization, a certain group of specific cells is caused to become an unusually large proportion of the total cell mass capable of responding to the second antigen. With tolerance induction, certain cells are deleted from the population of cells normally present which are capable of responding to the second antigen. In either case the response to the second antigen will be altered in a predictable manner. In summary, the fact that tolerance induction preferentially affects high-affinity antibody synthesis implies that some step in the process leading to tolerance involves an interaction of antigen with pre-existing antibody molecules. Thus, both immunization and tolerance induction behave as if high-affinity cells are selectively affected. This can only be under-

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stood if one assumes that there are precommitted cells that compete for antigen on the basis of the affinity of the antibody which they produce for the antigenic determinant. X.

Summary of Antigen Selection Hypothesis

We would propose that some process in the sequence of steps leading to antibody synthesis involves the interaction of antigen with preexisting antibody molecules presumably located on the surface of potential antibody-forming cells. Following interaction of cell-associated antibody with antigen, the cell is either stimulated to proliferate and/or secrete antibody or is rendered unresponsive, the distinction perhaps being determined by whether the antigen molecule has or has not undergone some as yet undefined processing or localizing step. It is suggested that it is on the basis of the step involving the interaction of antigen with cell-associated antibody that antigen is able to select, in a manner predictable by simple energetic considerations, a specific population of lymphoid cells. That cell populations of predictable character can indeed be selected by antigen has been clearly demonstrated by the in uitm studies on the increase in thymidine uptake upon exposure to antigen of cells taken from animals after priming with different doses of antigen (91). The maturation of the immune response with regard to binding affinity (15, 40, 41) can be understood as a competition of antibodyforming cells for available antigen. That is, decreasing antigen concentration exerts a selective pressure, in the evolutionary sense, for a progressive increase in antibody affinity, since cells bearing low-affinity antibody are inefficient in capturing antigen and are therefore not stimulated to divide and secrete antibody. Several other factors probably involved in the control of the immune response can be identified and are readily understood in terms of the general selection hypothesis: 1. Tolerance induction in high-affinity cells would tend to depress antibody affinity when supraoptimal doses of antigen are given. 2. Circulating antibody acts to compete with cells for available antigen thus increasing the selective pressure for a progressive increase in binding affinity. It has been shown that higher-affinity antibody is more effective in suppressing antibody formation (129). Therefore, as affinity increases the pressure toward further increase in affinity becomes greater, and only cells of still higher affinity remain capable of capturing antigen and being stimulated to proliferate and secrete antibody. 3. Statistical factors are probably involved, since it is unlikely that the reactions in vivo occur under strictly equilibrium conditions. Such

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statistical factors would tend to lead to the formation of “average” a5nity antibody, because an antigen molecule would be most likely to interact with a cell of the most common type ( average affinity). 4. The rate of cell proliferation is important in contributing to the maturation of the immune response and in the formation of a sufficiently large population of specific cells. It is only in a large proliferating population that the effects of selective pressure by antigen can operate. Therefore, conditions, or properties of antigen, which nonspecifically favor proliferation would be expected to increase the rate of maturation of &nity. Similarly, procedures that nonspecifically depress proliferation of lymphoid cells would be expected to depress the rate of maturation. In addition, the possibility must be considered that somatic mutation, considered by some to be responsible for the generation of diversity in immunoglobulin structure, continues to occur after introduction of antigen in the proliferating specific cell population. Random mutations occurring during proliferation could indeed provide additional cells of high affinity which would be selected to proliferate by antigen. Cells arising by mutation which are of low affinity would, of course, not be selected to proliferate. Thus, a variety of apparently disconnected immunological phenomena including the progressive increase in antibody affinity, the original antigenic sin phenomena, the effect of varying doses of antigen on antibody afEnity, the ratio of L to K chains in antibody synthesized, the behavior of passive antibody in the suppression of antibody synthesis, and the effect of partial tolerance induction on antibody affinity can be understood in terms of the selectional theory described above. The theory proposed is based essentially upon Burnet’s clonal selection theory (1-3) and assumes that the diversity of serum antibody molecules reflects a diversity of antibody-forming cells, each cell being capable of synthesizing one or a small number of distinct immunoglobulin molecules, the diversity of the cell population having arisen, as suggested by Burnet, by a process of random somatic mutation. It is further proposed here that somatic mutation might continue to play a significant role during the process of active immunization following the introduction of antigen. This last point deserves to be somewhat more fully elaborated. Two alternative possibilities exist as to the detailed composition of the antibody-producing cell population present early as compared with that present late in the immune response. First, all species of antibody molecules and the cells that synthesize them, which are present late in the immune response, are also present early in immunization, the difference in average affinity being totally attributable to changes in relative con-

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43

centrations of high- versus low-affinity antibody molecules and their respective cells, Second, new cells forming high-affinity antibody molecules are present late in the immune response which were not present in the animal early in immunization. According to this latter hypothesis, the change in affinity with time could be, at least in part, attributable to the synthesis of new antibody molecules of high affinity which were not present early in the response. Such high-affinity molecules might arise as a result of a process of random somatic mutations occurring in a rapidly dividing cell population, the rapid cell division having been induced by antigenic stimulation. Random mutations in this specific cell population might give rise to cells synthesizing antibody molecules of either higher or lower affinity than the original parent cell. Mutations capable of synthesizing antibody of higher affinity would preferentially capture antigen, proliferate, and, under the influence of the selective pressure of decreasing antigen concentration, tend to become a predominant fraction of the population. In wntrast, cells synthesizing low-affinity antibody are selected against, and tend to disappear from, the population of cells actively synthesizing antibody. The advantage of a system involving selection and concurrent mutation as compared with one involving merely simple selection is self-evident. The new variations in antibody structure which arise would increase the possibility of having available cells of very high affinity which would then be selected by antigen to proliferate. However, since mutation is a random and relatively infrequent event this process is probably rarely of major significance even if it exists. Finally, we must emphasize that, although we have stressed the role of a selection step involving interaction of antigen with preexisting cell-bound antibody, we do not want to imply that this is the only, or necessarily the first step, in the process leading to antibody synthesis. One additional word of caution must be extended. Thermodynamic measurements and concepts such as those frequently appealed to in this paper are critically only applicable to closed systems operating at equilibrium. The measurements of free energy of binding for the antibody-hapten interaction are thus formally true only in the “test-tube” in uitro system in which the measurements were carried out. We do not wish to leave the impression that we believe these measurements to be directly and literally transposable to the complex, nonequilibrium, in uiuo situation, where reacting units probably often exist in a membrane-bound state. We are merely using results obtained in studies on the properties of the end product (antibody) of a complex process to extrapolate backward so as to impute some of the properties of at least one step in the process leading to that product. That so many diverse experimental observations can be predicted on the basis of such energy considerations

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GREGORY W. SISKIND AND BARUJ BENACERRAF

implies that, in at least a rough manner, one step in the process leading to antibody formation is indeed influenced by the energetics of the antibody-antigen interaction as reflected in the energetics of the interaction of the final antibody product with specific antigen. Although the in uiuo process may very well not follow the laws of classical, closedsystem thermodynamics, the concepts of classical thermodynamics appear empirically useful in analyzing the biological problem. The evidence thus indicates that there exists a step in the immune response that obeys, at least by analogy, thermodynamic considerations which can be deduced from in uitro antibody-antigen interactions. XI.

Practical Conclusions and Further Problems to Investigate

This review has described a series of recent observations from many laboratories on the natural evolution of the immune response, which attempt to relate antigen-induced changes in specificity and affinity of antibodies with corresponding selective changes in the proliferating populations of precursor cells from which plasma cells producing these antibodies differentiate. Several practical conclusions can be drawn from the data and the ideas discussed in this review: 1. A sample of serum antibody obtained at a single arbitrary time after immunization cannot be considered as representative of the immune response to the antigen. 2. In the evaluation of the quality and adequacy of an immune response, it is much more relevant to consider the affinity of the antibody synthesized than the amount of antibody produced, because ( a ) a considerably greater range in antibody affinities can be observed than in amounts of antibody produced, and ( b ) antibody affinity in biological systems is extremely important (179, 180) and probably crucial for adequate protection because most biological phenomena depending upon antigen-antibody reactions operate in vim at very low antigen concentrations. 3. High-affinity antibodies and the cells synthesizing them develop through a process of continuous selection which has to be understood in order to obtain more efficient immunization. 4. Factors that influence the rate of change of affinity of antibody during immunization are now better understood. These include: ( a ) the use of the optimum dose of antigen, injected preferably as a depot-the optimum dose needs to be determined for each antigen and for each species, but, in general, it should be recognized that too much rather than too little antigen has usually been used in the past; ( b ) the physical

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45

state of the antigen--aggregated antigens are more immunogenic, and soluble antigens can selectively render high-affinity cells unresponsive and limit antibody synthesis to less desirable low-affinity antibody; ( c ) the effect of adjuvants that nonspecifically increase the proliferation of specific cells, and thus might improve conditions for selection; in contrast, agents that inhibit cell proliferation and decrease antibody synthesis might also reduce the rate of selection of a high-affinity cell population. In the course of this review, several problems were raised which have not been resolved and which are essential to a precise understanding of the mechanisms of the immune response and of cell selection by antigen. 1. How is the commitment of immunocompetent cells to the synthesis of immunoglobulins with different specificity achieved-by genetically controlled processes or by somatic mutation? At what stage in differentiation is this commitment realized? 2. What is the nature of the cell-associated receptors of committed specific cells? Are they immunoglobulins, identical with those to be synthesized by the progeny of these cells and do they have (as is postulated here) the same immunological specificity? 3. Does antigen need to be “processed by macrophages or bound to their cell surface to stimulate specific immunocompetent cells to proliferate and differentiate? And, alternatively, does native antigen render specific cells unresponsive or simply stimulate them to differentiate without forming proliferating clones? That is, what are the biochemical signals that direct a cell to produce antibody or to proliferate or to become tolerant?

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218a. Steiner, L. A., and Eisen, H. N. (1967). 1. Exptl. Med. 126, 1185. 119. Sela, M., and Mozas, E. (1966). Proc. Natl. Acad. Sci. US.55, 445. 120. Benacerraf, B., Nussenzweig, V., Maurer, P., and Stylos, W. (1969). Israel J. Med. Sci. 5, 171. 121. Grossberg, A. L., and Pressman, D. (1960). J. Am. Chem. SOC. 82, 5478. 122. Pressman, D,, Grossberg, A. L., Pence, L. H., and Pauling, L. (1946). J. Am. Chem. SOC. 68, 250. 123. Nisonoff, A., and Pressman, D. (1957). J. Am. Chem. SOC. 79, 1616. 124. Siskind, G . W. (1966). 1. Immunol. 96, 401. 125. Nisonoff, A,, and Pressman, D. (1959). J. Imrnrcnol. 83,138. 126. Uhr, J. W., and Moller, G. (1968). Adcan. Immunol. 8, 81. 127. Moller, C . (1963). 1. Natl. Cancer Inst. 30, 1205. 128. Moller, G. ( 1964). Transpkzntation 2, 405. 129. Walker, J. G., and Siskind, G. W. (1968). Immunology 14, 21. 130. Dixon, F. J,, Jacot-Ch~illarmod,H., and McConahey, P. (1967). J. Exptl. Med. 125, 1119. 131. Wigzell, H. (1966). J. E x p t l . Med. 124, 953. 132. Siskind, G. W. Unpublished observations. 133. Uhr, J. W., and Baumann, J. B. (1961). J. E x p t l . Med. 113, 959. 134. Uhr, J. W., and Bauinann, J. B. (1961).I . Exptl. Med. 113, 935. 135. Sahiar, K., and Schwartz, R. S. (1964). Science 145, 395. 136. Brody, N. I., Walker, J. G., and Siskind, G. W. (1967). J. E x p t l . Med. 126, 81. 136a. Francis, T., Jr., Davenport, F. M., and Hennessy, A. V. (1953). Trans. Assoc. Am. Physiciuns 66, 231. 137. Webster, R. G. (1966). J. Immunol. 97, 117. 138. Fazekas de St. Groth, S., and Webster, R. G. (1966). J. Erptl. Med. 124, 331. 139. Fazekas de St. Groth, S., and Webster, R. G. (1966). 1. Exptl. Med. 124, 347. 140. Brent, L. (1958). Progr. Allergy 5, 271. 141. Chase, M.W. (1959). Ann. Rev. Mtcrobiol. 13, 349. 142. Haiek, M., Lengerovh, and Hraba, T. (1961). Aduan. Immunol. 1, 1. 143. Smith, R. T. (1961).Aduan. Immunol. 1,67. 144. Dresser, D. W., and Mitchison, N. A. (1968). Aduan. Immunol. 8, 129. 145. Mitchison, N. A. (1955). P ~ o c Roy. . SOC. B161, 275. 146. Thorbecke, G. J., and Benacerraf, B. (1967). Immunology 13, 141. 147. Dresser, D. W. (1961). Immunology 4,13. 148. Dresser, D. W. (1962). Immunology 5, 161. 149. Dresser, D. W. (1962). Immunology 5, 378. 150. Nossal, G. J. V., and Ada, G. L. (1964). Nature 201, 580. 151. Frei, P. C., Benacerraf, B., and Thorbecke, G. J. (1965). Proc. Natl. Acad. Sci. us. 53, 20. 152. Fefer, A., and Nossal, G. J. V. (1962). Transpkznt Bull. 29, 443. 153. Makeh, O., and Nossal, G. J. V. (1962). J. Immunol. 88, 613. 154. Neeper, C. A,, and Seastone, C. V. (1963). J. Immunol. 91, 378. 155. Brooke, M. S. (1965). Nature 206, 635. 156. Humphrey, J. H. in “Immunological Diseases” (M. Samter, ed.), p. 100. Little, Brown, Boston, Massachusetts, 1965. 157. Schwartz, R. S. (196(3). Federntion Proc. 25, 185. 158. Terra, G., and Hughes, W. L. (1959). J. Immunol. 83,459. 159. Thorbecke, J. G., Siskind, G. W., and Goldberger, N. (1961). J. Immunol. 87, 147.

50 160. 161. 162. 163. 164. 165. 166. 167. 168. 169. 170. 171. 172. 173. 174. 175.

GREGORY W. SISKIND AND BARUJ BENACERRAF

Siskind, G . W., Patterson, P. Y.,and Thomas, L. (1963).1. Zmmunol. 90, 929. Mitchison, N.A. (1965).Immunology 9,129. Howard, J. G., and Siskind, G . W. (1969).J. Clin. Erptl. lmmunol. 4, 29. Siskind, G.W., and Howard, J. G. (1966).J . Exptl. Med. 124,417. Howard, J. G., Elson, J., Christie, G. H., and Kinsky, R. G. (in press). J . Clin. Exptl. lmmunol. Paul, W. E., Siskind, G. W., and Benacerraf, B. (in preparation). Theis, G. A., Thorbecke, G. J., and Siskind, G. W. (1968).Federation Proc. 27, 685. Battisto, J. R., and Chase, M. W. (1955).Bacteriol. Proc. 94. Battisto, J. R., and Chase, M. W. (1965).J. Erptl. Med. 121, 591. Coe, J. E., and Salvin, S. B. (1963).J. Erptl. Med. 117,401. Werblin, T., Siskind, G. W., Paul, W. E., and Benacerraf, B. Unpublished observations. Weigle, W. 0. ( 1961).1. Exptl. Med. 114, 111. Weigle, W. 0. (1962).1. Exptl. Med. 116,913. Weigle, W. 0. (1964).1. lmmunol. 92,791. Schechter, J., Bauminger, S., Sela, M., Nachtigal, D., and Feldman, M. (1964).Immunochemistry 1, 249. Nachtigal, D., Eschel-Zussman, R., and Feldman, M. (1965).lmmunology 9,

543.

176. Paul, W. E., Siskind, G. W., and Benacerraf, B. (1967).Immunology 13, 147. 177. St. Rose, J. E. M., and Cinader, B. (1967).I . Exptl. Med. 125, 1031. 178. Paul, W.E., Thorbecke, J. G., Siskind, G. W., and Benacerraf, B. (in preparation). 179. Siskind, G. W., and Eisen, H. N. (1965).J. I m m u d . 95,436. 180. Hurliman, J., and Ovary, Z.(1965).J. lmmunol. 95,765.

Phylogeny of Immunoglobulins1 HOWARD M. GREY2 Deportment of Experimental Pathology, Scripps Clinic and Research Foundotion, La lalla, Colifornia

I. Introduction

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11. The Question of Invertebrate Antibodies 111. Vertebrate Immunoglobulins

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Introduction

With the rapidly expanding knowledge of the detailed chemical structure of mammalian immunoglobulinsSwhich has been accumulating during the past 10-15 years, there has been a great deal of effort directed toward attempts to elucidate the genetic mechanisms involved in the formation of this unique class of proteins. As a result of this interest in the genetics of immunoglobulin formation, there has also been a renewed interest in evolutionary aspects of immunology and, particularly, in phylogenetic studies of iminunoglobulin structure. These studies on the structure of immunoglobulins should eventually provide valuable information that will help in understanding the evolution of this complex family of proteins and their peptide chains. The evolutionary relationships between a series of structurally similar proteins such as the immunoglobulins can be approached in two ways. The structure of the different proteins can be compared within a single, higher vertebrate species, i.e., a horizontal study. Alternatively, the structure of homologous proteins that are present in animals representative of the major taxonomic groupings can be stud'This is publication No. 321 from the Department of Experimental Pathology, Scripps Clinic and Research Foundation, La Jolla, California. * Established Investigator of the American Heart Association, Inc. 'Where possible, the nomenclature used in this article is that suggested by subcommittees of the World Health Organization.

51

52

HOWARD M. GREY

ied, i.e., a vertical study. Taken together, information regarding the degree of relatedness as well as the order of evolution of the different proteins should be obtainable. Both of these approaches are successfully being used at present in the study of immunoglobulin evolution. Interest in the phylogeny of immunity started in the nineteenth century, immediately following the basic characterization of the immune response as an important biological phenomenon in mammals. Unfortunately, this early work was performed before there was any detailed knowledge of the chemical structure of antibodies, and, therefore, much of it is difficult to interpret in light of our present knowledge of antibody structure, Because of this fact and since this early literature has previously been reviewed (Huff, 1940), this article will make only passing reference to many of the earlier studies despite their importance as the foundation on which much of the work done over the last several years in this field is based. Also, this article will limit itself to comparative studies dealing with the structural characteristics of immune molecules present in the body fluids of animals and will not attempt to review the literature concerning plant immunity. II.

The Question of Invertebrate Antibodies

The question of whether or not invertebrates produce antibodies in any way analogous to vertebrate immunoglobulins is at present unanswered. When considered in a broad sense it must be concluded that invertebrates possess an active immune system in that they are capable of successfully protecting themselves against potentially harmful foreign substances. To a large extent this is performed at a cellular level by phagocytic elements (Bang, 1961; Hilgard and Phillips, 1968; Reade, 1!368). However, humoral substances which are also active in invertebrate immunity have been described. The general subject of invertebrate immunity has been dealt with in detail previously (Huff, 1940),and will not be covered extensively here except to describe a few of the more recent experiments in this area to illustrate the difficulties inherent in the study of invertebrate immunity. The major problems arise in choosing the criteria by which to judge whether or not substances produced in invertebrates can be considered as antibodies. Electrophoretic studies of invertebrate vascular or coelomic fluid has shown the absence of proteins migrating as y-globulins (Woods and Engle, 1957).However, there appears to be no a priori reason for assuming invertebrate antibodies must have the mobility of mammalian y-globulin since it is now well appreciated that even mammalian immunoglobulins may be considerably more anionic than 7-globulin. It must be concluded, then, that electrophoretic

PHYLOGENY OF IMMUNOGLOBULINS

53

mobility is not a good criterion by which to evaluate the presence or absence of immunoglobulins. Another criterion which can be used to relate proteins of different species to one another and which will be dealt with in detail later, is based on their antigenic relationship. Antibody to human y-globulin made in the rabbit will also react with a variety of other mammalian y-globulins; the extent of the reaction of these heterologous mammalian y-globulins is a measure of the structural relationship between the heterologous y-globulin and the human y-globulin used for immunization. Such a study has been made to test the relationship of human y-globulin to invertebrate proteins using a human anti-y-globulin antibody (rheumatoid factor) which reacts with rabbit y-globulin and agglutinates erythrocytes coated with rabbit y-globulin. Serum obtained from the horseshoe crab (Limulus polyphemus) had the capacity to inhibit this agglutination (Cohen and Hermes, 1962). If nonspecific factors are not responsible for the results, this inhibition could suggest that the Limulus serum contained molecules that shared some structural features with rabbit y-globulin. Further studies must obviously be performed, however, before such an interpretation can be made with any assurance. Substances have been found in the blood and other body fluids of invertebrates which have the capacity to combine with structure( s ) on the surface of erythrocytes of certain species and cause agglutination of those erythrocytes. Since this agglutinin is found in normal animals and reacts in some cases with great specikity for certain types of red blood cells, it was very tempting for early workers in the field to draw the conclusion that these substances represented natural heteroagglutinins similar to the isoaggluthins found in man and other mammalian species. These heteroagglutinins have been found in several species of arthropods (Noguchi, 1903; Cantacuzdne, 1912,1919; Cohen et al., 1965; Marchalonis and Edelman, 1%8b), molluscs (Chahovitch, 1921; Johnson, 1964; Tripp, 1966), sipunculids, caterpillar, lobster, echinoderms, coelenterates, and annelids (Brown et al., 1968). Certain of these heteroagglutinins have been shown to be specific for the species of erythrocytes with which they react. Limubs heteroagglutinins react specifically with human erythrocytes (Cohen et al., 1W5);lobster heteroagglutinins show specificity for whale erythrocytes (Cushing et al., 1963); some caterpillar heteroagglutinins agglutinate human 0 erythrocytes, but not A or B cells. The specificity of these reactions is very similar in some instances to that shown by plant lectins for antigens of the ABO system, and, although they may exhibit exquisite stereospecificity, it is dubious whether they have any phylogenetic relationship to vertebrate antibody.

54

HOWARD hl. GREY

Besides having natural agglutinins or lysins, invertebrates have the capacity of an induced immunity in response to the injection of foreign material. The great majority of experiments in this area have involved the injection of live or dead microorganisms and subsequently following the appearance of substances in the body fluids of the injected animals that were capable of lysing or neutralizing the organisms previously injected. In contrast to the specificity of the reaction exhibited by some of the natural agglutinins, the specificity of the humoral substances responsible for induced immunity has not been well established. Gingrich studied the immune response to Pseudomom aeruginosa in the large milkweed bug, OncopeItus fasciatus (Gingrich, 1964). Four hours after an injection of the bacteria, lytic substances were found in the hemolymph. The peak production occurred 12-24 hours after the injection. The lytic factor was active against the immunizing organism as well as against Escherichia coli, but had no effect on Proteus vulgaris, Bacillus subtilis, or Mfmococcus pyogenes. On the other hand, animals injected with bovine serum albumin or human erythrocytes responded with the production of small but definite amounts of lytic factor which was active against Pseud o m m aeruginosa. The active substance was extremely stable in comparison to vertebrate antibody. Treatment with molar sodium hydroxide or hydrochloric acid or heating to 75°C. for 1 hour had no effect on the lytic activity of the hemolymph. Similarly, wax moth larvae injected with Pseudomom aeruginosa responded with a humoral immune substance within several hours of injection which was active against the immunizing organism as well as other species of Pseudomonas but was inactive against Proteus mirubilis (Stephens, 1959; Stephens and Marshall, 19s2). Snails ( Australorbis ghbratus ) injected with Schistosoma mansoni developed humoral substances capable of immobilizing the parasite; however, animals injected with Daubaylia potomuca also responded by producing substances capable of immobilizing the schistosomes. On the other hand, the production of the immobilizing factor was not totally nonspeciiic, since animals injected with albumin, polystyrene, echinostomes, and acid-fast bacilli did not produce miracidial immobilizing substances ( Michelson, 1963, 1964). Similarly, in other invertebrate species the production of biologically active substances capabla of inducing humoral immunity shortly following an injection of microorganisms has been demonstrated; the specificity of the immune bodies for the inducing organism was either not investigated or shown to be relatively nonspecific as in the examples cited above (Huff, 1940; Briggs, 1958; Bang and Bang, 1W2). Bernheimer, on the other hand, injected caterpillars with "2 coliphage, Escherichiu coli, and P-hemolytic streptococci, and did not detect the production of any immune substances (Bernheimer, 1952).

PHnOGENY OF IMMUNOGLOBULINS

55

From thc few examples cited above it is obvious that the relatively small amount of information available on both natural agglutinins and induced humoral substances does not conform in certain important aspects to what is known of vertebrate antibodies. The natural agglutinins show stereospecificity, but the titer of the agglutinin cannot be increased by injection of the specific ligand. The induced humoral immune substances, on the other hand, have demonstrated limited stereospecificity and display kinetics of production atypical for vertebrate antibody. These findings, however, do not preclude the possibility that these substances are structurally related to vertebrate antibodies and may represent primitive forms of immunoglobulins. Only detailed structural studies of these or other invertebrate proteins can hope to give the necessary data to answer this question. Such studies have been initiated. Evans and coworkers have begun an investigation of the structure of a heat-labile macromolecule produced in the spiny lobster after intracardiac injection of naturally occurring bacterial flora (Evans et al., 1968). Marchalonis and Edelman have examined the subunit structure of the natural hemagglutinin found in the horseshoe crab, Limulus polyphemus ( Marchalonis and Edelman, 1968b; Fernbdez-Morhn et al., 1%8). This hemagglutinin is a protein which migrates as a &globulin in starch block electrophoresis. It has an s,”,,, of 13.5 and a molecular weight of 390,000.It is composed of subunits bound to one another by noncovalent interactions. In the absence of calcium ions the molecule has an s rate of 10.6 and loses its hemagglutinating capacity. Under acid ( p H 2 ) or alkaline (pH 9.6) conditions, dissociation to 4 S subunits occurs, and with more drastic denaturants (8 M urea or 20%acetic acid), the protein dissociates into 1.5S subunits with a molecular weight of 22,700. Starch gel electrophoresis of the hemagglutinin in 8 M urea, at acid or alkaline pH, reveals rapidly moving bands. At acid pH the bands migrate in a similar position to human light chains, but at alkaline pH they migrate much more rapidly than human light chains. Under neither condition of pH do they exhibit the same heterogeneity as light chains. Data from the molecular weight determinations and amino acid composition would predict the presence of 17 tryptic peptides if the hemagglutinins consisted of a single subunit. Fingerprinting of the trypsin-digested protein gave a total of 20 spots. These data are somewhat suggestive of a single subunit but are also compatible with multiple subunits that have the same primary structure in some portions of the molecule but differ in others. If the number of subunit types is very large (greater than 5 ) , then those peptides that ditfer from one subunit to the next may not be detected by the fingerprint technique because they are present in concentrations too low for detection by the routine staining procedures. Regardless of the degree of heterogeneity in primary se-

56

HOWARD M. GREY

quence of the 23,000-molecular-weight subunit, the data of Marchalonis indicates that Limulus hemagglutinin with a molecular weight of 390,000 consists of approximately 18 subunits of equal size-a subunit structure markedly different from vertebrate immunoglobulins. However, the finding of a subunit the same size as vertebrate light chains must be considered of sufficient interest to warrant further studies of the primary structure of this type of protein. Ill.

Vertebrate Immunoglobulins

Antibodies with the specificity and inducibility of mammalian antibodies have to date been described only in vertebrates. It has been possible to demonstrate humoral antibodies in all the major classes of vertebrates (see Fig. l ) ,and with one exception in all species examined within these major classes. Good and co-workers (for review of this literature, see Good and Papermaster, 1964) have made extensive morphologic and serologic studies on the immune response in the lower vertebrates, They have studied the response of representatives of the two orders of living Cyclostomes, the lamprey (order Petromyzontia) and the California hagfish ( order Myxinoidea). Although they were capable of demonstrating antibody formation in the lamprey to keyhole limpet hemocyanin and T2 bacteriophage, they were unable to demonstrate an immune response in

FIG.1. Phylogenetic relationships of vertebrates. Living species of the major classes that have been studied with regard to immunoglobulin structure are listed at top.

PHYLOGENY OF IMMUNOGLOBULINS

57

Fa b

FIG.2. Schematic mode1 of vertebrate immunoglobdin molecule.

the hagfish to the same as well as other antigens (Papermaster et aZ., 1964). This failure to demonstrate antibodies in the hagfish has led these authors to conclude that antibody formation is a biological system restricted to vertebrates. Recently, however, the immune response in the hagfish has been reinvestigated, and it has been possible to demonstrate allograft skin rejection as well as humoral antibodies to coliphage in this species ( Hildemann, personal communication) . If these preliminary findings can be substantiated, then all vertebrate species tested will have been shown capable of mounting an immune response. This serves again to point out the necessity for detailed studies in invertebrates if the origins of immunoglobulin formation are to be ultimately understood.

A. THEY-GLOBUIJN MOLECULE Before discussing the phylogenetic data, it will be useful to describe in summary fashion the basic structure of mammalian immunoglobulin molecules. The y-globulins are a heterogeneous species of molecules that are functionally related in that they all exhibit antibody activity as well as share basic structural features. The fundamental structure of a mammalian 7 S yG immunoglobulin molecule is depicted in Fig. 2. The data supporting this structure have been obtained mainly from three speciesman, rabbit, and mouse. It is a symmetrical molecule consisting of two pairs of polypeptide chains held together by disulfide bonds as well as noncovalent interactions. The smaller pair of polypeptide chains, called light chains, have a molecular weight of 22,000-23,OOO. There are two basic types of light chains, K and h, which differ from each other structurally so that they are antigenically non-cross-reacting.

58

HOWARD hi. GREY

All immunoglobulins contain K- and A-type light chains regardless of the class to which they belong. The ratio of K to A differs between species; in humans approximately 60!4 of immunoglobulin molecules are K type. The region responsible for the antigenic and chemical typing of the light chains into K and A is located in the C-terminal half of the chain, and this portion of the chain is invariant from one light chain to another within the same type, except for minor genetic differences. On the other hand, the N-terminal half of the light chain is variable from one chain to the next within a given type. There are areas of the N-terminal half of the chain which are similar in several different species of light chains and allow light chains to be divided into subclasses, but thus far no two light chains have been found to be identical in this portion of the molecule. In most instances the light chains are bound to the other set of polypeptide chains, the heavy chains, by means of disulfide bonds located at the C-terminal end of the light chains. The heavy-chain structure also varies considerably and this variation is the basis of the classification of immunoglobulins. 7-Globulin can be divided into three major classes: yG, comprising about 70%or more of the total y-globulin; yA, comprising 1-2W;and yM which makes up about 1-10%of the total y-globulin. The heavy chains of these classes are referred to as y, a,and p chains, respectively, and have a molecular weight ranging from 50,OOO for the 7 chain to 70,000 for the p chain. In some species there are other immunoglobulin classes that make up only a very small fraction of the total immunoglobulin content. At least some, if not all, of the structural features that distinguish the heavy chains of the different classes are located in the C-terminal portion of the chain (Fc fragment). Most of the carbohydrate content of the molecule is located on the heavy chain, the quantitative values varying from one immunoglobulin class to another ( Abel et al., 19S8). When YG-globulin is digested with papain, it is split into functionally distinct fragments, with molecular weights of approximately 50,000. The Fc (crystallizable fragment) consists of the C-terminal half of both heavy chains and is responsible for much of the biological function of the molecule such as complement fixation and tissue binding. Two Fab (antigenbinding) fragments are generated by papain digestion. Each consists of a light chain and the N-terminal half of the heavy chain (Fd fragment). Although present knowledge is still incomplete, it appears that the Fd fragment of the heavy chain has a similar degree of variability to the Nterminal portion of the light chain. The exact location of the combining site is still in doubt but present evidence seems to favor the hypothesis that both light and heavy chains contribute directly toward the structure

PH.YLOGENY OF IMMUNOGLOBULINS

59

of the active site. According to this hypothesis the primary structure of the N-terminal portion of the two chains would be sufficiently unique to dictate the conformation of a particular combining site and that sufficient variation can occur in this primary structure to account for the very large number of different specificities known to exist. Pepsin acts in a manner similar to papain except that it degrades the Fc fragment into small peptides leaving a bivalent, 100,000-molecularweight F( ab’), fragment which can be easily reduced to univalent fragments. Pepsin leaves a slightly larger piece of heavy chain on the Fab fragment than does papain (Smyth and Utsumi, 1967; Givol and De Lorenzo, 1968). When y-globulin is reduced in the absence of dissociating solvents such as urea, three to five disulfide bonds are reduced. Two of these are interchain L-H disuEde bonds and at least one is an interchain H-H disulfide bond which holds together the bivalent pepsin Fab fragment. The number of H-€1 disulfide bonds varies between species and immunoglobulin classes within a species. Although the outline of the structure of y-globulin given above is based on studies of yG globulins, in its basic concept it appears to hold for the other immunoglobulins as well. The most striking differences between yG proteins and yA or yM is the capacity for the latter two immunoglobulins to polymerize. Gamma M consists of five 7s subunits, whereas yA proteins are polydisperse and range in size from 7 S to 17 S. The difference between these immunoglobulins will be discussed in greater detail in later sections.

B. FISHIMMUNOGLOBULINS 1. Cyclostm

Antibody formation can be induced in lampreys (Petromyxon murinus) to T2 bacteriophage (Papermaster et al., 1964), Brucella (Pollara et al., 1968a,b), and erythrocytes (Boffa et al., 1967). The antibodies are electrophoretically heterogeneous and have been described variously as yl,p, and a migrating proteins by zone electrophoretic analysis. By density gradient ultracentrifugation and gel filtration chromatography, there is evidence that the immunoglobulins are polydisperse. Boffa was able to localize antierythrocyte activity to protein fractions with s rates of 10.9 S and 6.6 S (Boffa et al., 1967). The animals used for these experiments also had natural agglutinins for the erythrocytes. Marchalonis and co-workers have described a similar natural agglutinin to horse erythrocytes with an s rate of 46 S (Marchalonis and Edelman, 1968a). Whether

60

HOWARD h l . GREY

either of the induced human erythrocyte agglutinins is related to this 46 S natural agglutinin is unknown at present. Antibody to 7'2 phage has also been shown to be polydisperse, with two peaks of neutralizing activity on sucrose gradient ultracentrifugation in the 14 and 7 S regions (Papermaster et al., 1964). Marchalonis and Edelman ( 1968) have reported the first detailed analysis of these immunoglobulins. Both 14 S and 7 S immunoglobulins were present in low amounts in the lamprey serum; the 7 S component was present at a concentration of approximately 0.3 mg./ ml., and the 1 4 s component at a concentration less than 0.1 mg./ml. Due to the low concentrations of 1 4 s immunoglobulins, it was not possible to purify this component, and detailed structural studies, were therefore, not performed. However, immunodiffusion studies utilizing pursed 7 S immunoglobulin and rabbit antilamprey 7 S immunoglobulin gave lines of identity between the 7 S and 14 S immunoglobulins. As will be discussed in later sections, similar findings have been observed in other vertebrate immunoglobulins, including man, and are highly suggestive that the 14 S and 7 S immunoglobulins are very similar or perhaps identical in primary structure, and that, as is the case with yA, they represent polymeric and monomeric forms of the same class of immunoglobulin. In higher vertebrate species, independent chemical evidence strongly supports this theory. However, further studies on the lamprey 14 S component must be done before this conclusion can be made with any assurance. Unlike most vertebrate immunoglobulins (certain yA proteins being the one other known exception), the lamprey light and heavy polypeptide chains appear to lack L-H disulfide bonds. Gel filtration analysis of unreduced lamprey 7 S immunoglobulin (mol. wt. = 188,000) in a dissociating solvent results in separation of heavy and light polypeptide chains. The light-chain peak obtained from the unreduced protein is somewhat diffuse and appears to peak slightly ahead of the L-chain peak obtained after reduction and alkylation. Molecular weight determinations indicate that the molecular weight for the heavy-chain preparation is 70,000 and for the reduced and alkylated light chain is 24,000. These molecular weights are the same as that found for mammalian immunoglobulin light chains and p heavy chains. The unreduced light chains appear to be made of two species; one with a molecular weight of 24,000 and one with a molecular weight of 40,OOO. Approximately 30%of the lamprey immunoglobulin is light chains. These data suggest that the lamprey 188,000-molecular-weight 7 S protein is composed of four polypeptide chains-two heavy and two light. The heavy and light chains are apparently noncovalently bound to one another as are the two heavy

PHYLOGENY OF IMMUNOGLOBULINS

61

chains to each other. The light chains may to some extent however, be bound to one another in the form of L L disulfide linked dimers as is suggested by the 40,000-molecular-weight determination obtained in 20% acetic acid. If this subunit structure proves to be correct, it would be strikingly similar to the results obtained with mouse and human yA proteins ( Abel and Grey, 1968; Grey et d., 1968a). Other information regarding structural features of lamprey immunoglobulins are ( 1 ) as judged by immunoelectrophoresis, papain treatment did not appear to alter the 7 S protein; (2) molecular weight determination of the intact 7 s protein at different protein concentrations vaned from approximately 70,000 at low concentrations to 150,000 at higher concentrations, suggesting that the polypeptide chains may have undergone concentration-dependent dissociation in the absence of denaturing reagents; (3) some light chains contained an N-terminal aspartic or asparagine residue (see Section 11,G); and (4)isolated light chains were antigenically unrelated to the intact protein, indicating that upon dissociation from the heavy chains the light chains underwent conformational changes which rendered them antigenically non-cross-reacting with the light chains in the intact molecule. This drastic change in antigenic determinants appears unique to lamprey light chains, although more subtle antigenic differences between free and bound light chains have been described in mammalian immunoglobulins (Epstein and Gross, 1964; Nachman and Engle, 1964; Grey et al., 1965; Prendergast et al., 1966; Terry and Roberts, 1966; Korngold, 1967; Mihaesco and Seligmann, 1968b). 2. E lusmobranch Several species of elasmobranchs have been studied for their capacity to produce antibodies. In those instances where it has been possible to study the immune response over a period of several months, it has been generally possible to distinguish two types of immunoglobulins on the basis of size-a 17-19s antibody which is formed for the first 3 to 6 months after immunization and a 7 s antibody which is &st detectable after several months of immunization but which makes up an increasingly larger proportion of the total antibody content in the later stages of immunization (Clem and Sigel, 1965; Sigel and Clem 1966; Clem and Small, 1967; Clem et al., 1967; Grey, 1963a; Suran et al., 1967). One of the more common methods used, for comparing high- and lowmolecular-weight antibodies has been a functional comparison based on the capacity of reducing agents to destroy antibody activity in different immunoglobulin classes. In mammals it has been used extensively to dis-

62

HOWARD M. GREY

tinguish yG from yM antibodies, since under certain circumstances yM appeared to lose antibody activity upon reduction, whereas y G did not (Fudenberg and Kunkel, 1957; Grubb and Swahn, 1958; Bauer et al., 1963). Subsequent studies have shown that the results obtained are dependent in large part on the particular test system used for detecting antibody activity. yM Antibodies are very efficient in hemagglutination compared with yG (Grey, 1964; Ishizaka et al., 1965a, 1965b). This high efficiency appears to be related to the fact that yM is a polymer consisting of five subunits in close proximity to one another. When the molecule is depolymerized upon reduction, the efficiency for detecting yM antibodies is thereby lowered and there is a drastic decrease in antibody activity as measured by these as well as certain other test systems. However, in other test systems, such as equilibrium dialysis, which rely solely on the primary interaction between antigen and antibody for the detection of antibody activity and not on secondary manifestations of antigen-antibody interaction, such as agglutination, it appears that treatment of yM antibody with reducing agents has no effect on its capacity to combine with its specific hapten ( Onoue et al., 1964,1965). This difference in the relative efficiency with which polymeric and monomeric forms of antibodies can be detected in various test systems helps explain some of the discrepancies in the results reported by different investigators on the susceptibility of elasmobranch 17 S and 7 S antibody to reducing agents. Although some data would indicate that 1 7 s and 7 S antibodies from sharks are both only partially inactivated (Sigel and Clem, 1966; Suran et al., 1967), other data have been reported using different antigens and different assay systems that would indicate the 17 S antibody activity is completely abolished by reduction (Papermaster et al., 1964; Clem and Small, 1967). Other factors, such as a change in affinity of antibodies, may also play a role in determining the apparent susceptibility of immunoglobulins to reducing agents ( Grey, 1967b). Regardless of which of these factors is more important in rendering an antibody susceptible to reduction in a given instance, it is now apparent that in itself reduction is not a reliable criterion for distinguishing between types of immunoglobulins, so that reference to its use for this purpose will be made only where there is independent data to indicate the type of immunoglobulin that is being effected. Structural studies on the two molecular species of shark immunoglobulins have been performed and are summarized in Table I. The molecular weights for the intact proteins and their constituent heavy and light chains are the same, within experimental error for the three species studied. The hexose determinations indicate relatively high sugar

63

PHYLOGENY OF IMMUNOGLOBULJNS

TABLE I STRUCTURAL CHARACTERISTICS OF ELASMOBRANCH ANTIBODY Molecular weight Species

s rate

Intact protein

Dogfish sharka

17

Nurse sharkb

982,000 198,000

19 7 19 7

7

Lemon sharke ~~

a

~~

800-900,000 160,000

Heavy chain

Light chain

71,600 73,400 70,000

20,100 20,500

71,000 71,000

22-23,000 22-23,000

22,OOO

Carbohydrate content

(%I

8.7 (anthrone) 7.6 3 . 5 (orcird) 3.6 3.7 (orcinol) 3.5

~

Marchalonis and Edtdman (1965). Clem et al. (1967). Clem and Small (1967).

content. Although a different method of quantitation was used for determining the hexose content in the dogfish immunoglobulins, it would appear that there may be some differences in carbohydrate content between this shark and the other two species studied. The striking finding, however, is that the heavy chains as well as the light chains from the 17-19 S proteins were identical in size to those of the 7 S proteins. Further studies were done to compare the heavy-chain structure of the two immunoglobulins. Amino acid analysis ( Marchalonis and Edelman, 1965) and peptide mapping (Marchalonis and Edelman, 1965; Clem and Small, 1967) of the tryptic peptides of the isolated heavy and light chains also suggested that the 17s heavy chain was very similar to the 7s heavy chain. These studies also showed the similarity between the light chain of the two immunoglobulins. Furthermore, antisera which recognized both heavy- and light-chain antigenic determinants could not distinguish between the 7s and 1 7 s immunoglobulins from the same species. These data indicate that, unlike the situation with mammalian 7 S yG and 19 S yM where, by all the criteria mentioned above clear-cut differences between the two immunoglobulins can be discerned, the 7 S and 17-19 S shark munoglobulins are indistinguishable from one another by all criteria except that of size. Whether this indicates that they represent monomer and polymer forms of the same protein or whether they are structurally closely related but distinct proteins which could not be distinguished by the criteria used to test them, is at present unknown; but it is clear from these studies that they do not represent separate immunoglobulin classes but rather very closely related proteins which may, in fact, be the product of the same genes.

64

HOWARD M. GREY

The high carbohydrate content of the heavy chain, its mobility in starch gel, as well as the molecular weight of the heavy chain and the intact protein, suggest that the elasmobranch 17-19 S immunoglobulin is homologous to mammalian yM, whereas the 7 S protein may represent a low-molecular-weight form of the same protein. Although highly suggestive, the data are not conclusive and would also be compatible with these proteins being homologous to yA or lacking homology with known mammalian immunoglobulins. Data obtained with mammalian yM and its reduced monomer suggest that the 1 9 s pentamer may have only five antibody-combining sites and that each subunit is univalent. The capacity of reduced, leopard shark, 1 9 s antibody and unreduced as well as reduced 7 s antibody to cause precipitation of antigen would suggest that the monomeric subunit of these proteins is multivalent (Suran et al., 1967). This is supported by the finding that 7 S antibody from the nurse and lemon shark can agglutinate red cells coated with specific antigen (Clem et al., 1967; Clem and Small, 1967). As will be discussed later, recent studies with mammalian yM using conditions which can detect relatively low-affinity combining sites suggest that mammalian yM subunits may also be bivalent and that the difference in results obtained between elasmobranch and mammalian macroglobulin may only be quantitative and represent differences in the affinity of combining sites rather than differences in the number of sites. 3. Holostean, Chondrostean, and Teleostean

Data on fish other than elasmobranchs is much more fragmentary but in general is in keeping with the findings discussed above. The goldfish (Uhr et al., 1962) and grouper (Clem and Small, 1968) have been shown to make a 19 S antibody early in the immune response and a 7 s antibody, as well, several months after the initial immunization. However, in other instances, only 1 9 s antibody has been found as long as 10 months after primary stimulation ( Clem and Sigel, 1966). Molecular weight determinations on the 19 S antibody of the paddlefish (a chondrostean) (Pollara et al., 1968c) and grouper ( a teleost) (Clem and Small, 1969, personal communication) indicate a molecular weight of approximately 900,000 for the intact protein, 70,000-75,000 for the heavy chain, and 20,0W23,000 for the light chains. No detailed analysis on the 7s antibody of the paddlefish has been made so that structural comparison between the 1 9 s and 7 s immunoglobulins are not possible at present. Recent studies on the grouper 7s antibody indicate that it has a molecular weight of about 120,000; the molecular weight of the light

PHYLOGENY OF IMMUNOGLOBULINS

65

chains being 20,000-22,000 and that of the heavy chains 40,000. Antigenically, the grouper 7 s antibody cross-reacts and is antigenically deficient to the 1 9 s antibody, whereas the 7 s antibody does not appear to have antigenic determinants not shared by the 1 9 s antibody. These findings, if substantiated, would indicate that there are two distinct classes of immunoglobulins in the grouper which are distinguished by heavy chains of grossly different molecular weights. Whether this proves to be the case or whether the short heavy chain is the result of catabolism of a larger-sized heavy chain must await further investigation. Obviously if the 40,000-molecular-weight heavy chain is synthesized de nouo, it will be of great interest to determine if other teleost 7s antibodies have similar heavy chains or whether this represents an immunoglobulin class peculiar to the grouper. Grouper 19 S and 7 S anti-2,4-dinitrophenol (DNP) antibodies have been analyzed for their valency (Clem and Small, 1968). The 19 S antibody was capable of precipitating a polyvalent antigen and had a valency of 5 when relatively low-affinity antibody was studied and a valency of approximately 10, with high-affinity antibody. The 7 s antibody was nonprecipitating and apparently univalent although highaffinity 7s antibody has not been studied as yet. These results are similar to those found with rabbit 1 9 s yM antibody and its 7 s subunits (Section III,F,3b). The apparent univalency of the grouper 7 S anti-DNP antibody is at variance with the finding of agglutinating and precipitating 7 S antibody in some elasmobranchs. These apparent differences, however, as discussed above, may reflect quantitative differences in the affinity of the combining sites rather than indicating real differences in the number of sites. C. AMPHIBIANIMMUNOGLOBULINS Structural studies have been carried out on antibodies formed in frogs ( Uhr et al., 1962; Trnka and Franek, 1960; Alcock, 1965), toads (Diener and Nossal, 1966), and salamanders (Ching and Wedgwood, 1966, 1967). It has been possible to demonstrate by ultracentrifugal analysis that the first antibody formed to a variety of antigens is a macroglobulin and that 7 S antibody is formed later in the immune response and is the major immunoglobulin 2 to 3 months following immunization (Uhr et al., 1962; Diener and Nossal, 1!366). Both 19 S and 7 S antibodies are at least partially inactivated by reducing agents. In the axolotl, antibodies are also heat inactivated at 56°C. The heat lability of other amphibian or fish antibodies has not been critically tested but should be, since heat in-

66

HOWARD M. GREY

activation may possibly be the cause for an inability to demonstrate an antibody response in certain lower vertebrates whose serum has been heated to 56°C. prior to testing for the presence of antibody. The polypeptide chain structure of frog 7 S and 19 S antibodies has been studied by Marchalonis and Edelman (1966). The polypeptide chains are disulfide-bonded to one another. Starch gel eletrophoresis of reduced and alkylated 19 S and 7 S frog immunoglobulins shows that the light chains are similar to one another and appear as two or more bands at acid pH unlike other vertebrate light chains. The heavy chains migrate differently from one another. The 19 S heavy chains migrate similarly to mammalian p- chains and those of the 7 S immunoglobulin migrate more slowly (but somewhat faster than human y- chains). The 1 9 s immunoglobulin contains 10.8%hexose as measured by the anthrone reaction, whereas the 7 s antibody contains only 2.1% hexose. The molecular weights of the light chains from the 19S and 7 S immunoglobulins were 20,000 and 22,000, respectively; those of the heavy chains were 72,000 for the 19 S heavy chains and 53,600 for the 7 S heavy chains, indicating that the heavy chains of the two immunoglobulins are structurally dissimilar. Antigenic analysis with a rabbit antiserum made against the 19 S protein also confirmed this conclusion in that this antiserum gave a line of partial identity between the 19s and 7 s proteins indicating that the 19 S protein contained antigenic determinants not present on the 7 S protein.

D. REPTILE IMMUNOGLOBULINS Antibody formation, as well as some structural features of the antibody produced, have been studied in tortoises (Maung, 1963; Lykakis, 1968), turtles (Grey, 1963a; 1968), lizards (Evans and Cowles, 1959; Evans et al., 1965), and alligators ( B a d et al., 1961; Lerch et al., 1967). In the tortoise and turtle, it has been possible to demonstrate heavy and light antibodies which, by gel filtration and density gradient, probably correspond to 19 S and 7 S antibodies. Direct s rate measurements of isolated proteins have not been made, however; nor is there anything known of the structure of their polypeptide chains. Studies have been made on the reIative avidity of turtle antibodies during the course of immunization. It has been previously shown in mammalian systems that the strength of the antigen-antibody bond increases for 2 to 3 months after the initial immunization of an animal, after which time it reaches a plateau of avidity which is maintained during the next several months (Jerne, 1951; Fan, 1958; Grey, 1964). The same phenomenon has also been studied in hapten-antihapten systems

67

PHYLOGENY OF IM&lUNOCLOBULINS

where it has been demonstrated that the intrinsic equilibrium constant increased with time (Eisen and Siskind, 1964). Although it is not absolutely certain that “avidity” measurements can be directly equated with the equilibrium constants obtained in hapten systems, it appears most likely that they are measuring the same parameter, namely the strength of antigen-antibody interaction ( Talmage, 1960). Because of the nature of the antigen employed in the turtle studies, it was necessary to use an indirect measure of avidity, i.e., the effect of dilution on the binding of antigen by antibody. This technique has been used in the past with mammalian antibody and has correlated with other more direct measurements of avidity such as dissociation rate measurements (Farr, 1958). The quantitative values obtained with low-avidity antibody are more affected by dilution of the reaction system than are those of high-avidity antibody. The results obtained with turtle and rabbit antiserum to keyhole limpet hemocyanin (KLH) at different times during the immune response are shown in Table 11. The early rabbit antiserum showed a 60% decrease in measurable precipitating antibody upon dilution of the test system from 1 to 8 ml, whereas the hyperimmune serum obtained 6 months after the first injection of KLH showed no effect of dilution. Two pools of turtle antisera, one obtained relatively early and one late in the course of immunization, were remarkable in two respects. First, the effect of dilution on these antisera was more marked than even the earliest rabbit antibody obtainable, suggesting that turtle antibody at all stages of immunization is of very low avidity compared with mammalian antibody. Second, there was no measurable change in the avidity of the antiserum during the course of immunization as is seen in mammalian systems. The significance of these differences in binding characteristics between turtle and mammalian antibodies is at present unknown since the mechanism by which the avidity of antibody changes during the course TABLE rI

EFFECT OF DILUTION O N ANTIBODY TITER N KLH4 precipitated per ml. of serum Serum source Turtle, day 37-68 pool Turtle, day 75-113 pool Rabbit, day 7, primary Rabbit, hyperimmune a

KLH

=

A (1-ml. volume) 8.5 14.2 2.5.0 94 5

giant keyhole limpet, hemocyanin.

B (8-ml. volume)

Effect of dilution, A/B

1.8 2.9 9.9 1020

0.21 0.20 0.40 1.08

(pg.)

68

HOWARD M. GREY

of immunization has not been elucidated. The two most likely explanations for this phenomenon are (1) that an antigen initially stimulates cells capable of making low- and high-avidity antibody and that there are more cells that make antibody capable of binding the specific antigen weakly than those capable of strong binding (but the cells producing high-avidity antibody proliferate at a more rapid rate than those producing low-avidity antibody so that during the course of immunization there is a gradual shift in the cell population in favor of cells producing high-avidity antibody, thereby accounting for the observed shift in the avidity of the antiserum), or (2) that the shift in avidity is due to subcellular mechanisms such as somatic mutations which occur during the course of immunization and which subtly alter the structure of the antibody produced by a cell and its progeny so that there is a progressive change in the binding characteristics of the antibody produced by an animal as more of these subcellular events accumulate. Whether or not either of these postulated mechanisms is responsible for the observed change in avidity following immunization, the results suggest that the turtle lacks some of the immunological potential of mammals and that studying the binding characteristics of lower vertebrate antibody may be another useful parameter by which to study the phylogeny of antibody. It has recently been possible to immunize fish with simple haptens coupled to macromolecular carriers (Clem and Small, 1968), so that data on the change of affinity as well as equilibrium constants for nonmammalian vertebrate antibodies should be available in the near future.

E. AVIANIMMUNOGLOBULINS 1. Chicken

Because of the relative ease with which birds can be immunized, immunologists have studied this nonmammalian class of animals more than any other. However, only two species of birds, chickens and ducks, have been investigated to any great extent with regard to the types of immunoglobulins present in the serum so that our knowledge of bird immunoglobulins is very fragmentary considering the enormous variety of species present in this class. Following immunization with certain antigens, chickens make a 19 S antibody which is antigenically distinct from the 7s antbody subsequently produced (Uhr et al., 1962; Benedict et al., 1963b; Benedict, 1967). The 19 S response is relatively small compared to the 7 S response when primary binding tests are used (Rosenquist and Gilden, 1963);

PHYLOGENY OF IMMUNOGLOBULINS

69

whereas hemagglutination gives a falsely high impression of the amount of 19 S antibody produced. Although detailed structural studies on this protein have not been performed, it has the biological characteristics, such as the temporal relationship of appearance, mercaptoethanol sensitivity (Benedict et al., 1963a), and electrophoretic behavior (Benedict et aZ., 1963c), of mammalian yM, and it has been assumed to be the counterpart of this immunoglobulin class. Obviously detailed structural studies will have to be performed to demonstrate more precisely whether this is a protein homologous to yM. The bulk of the antibody formed to a variety of antigens migrates electrophoretically in the slow as well as fast 7-globulin region, has an isoelectric point of 5.2 (Banovitz et al., 1959), and has an s rate separately reported at 7.1 (Dreesman and Benedict, 1965b) and an s;,~ of 7.73 (Tenenhouse and Deutsch, 1966) with a molecular weight determined by diffusion (Orlans et al., 196l),and light scattering measurements (Dreesman, 1965) of 175 to 180,OOO and by ultracentrifuge analysis of 206,000 (Tenenhouse and Deutsch, 1966). It has a reported hexose content of 3.1%which is 2-3 times that of mammalian yG proteins. Results obtained on the subunit structure of 7s chicken 7-globulin are similar to those obtained with mammalian yG with certain striking exceptions. Like mammalian yG, papain digestion of chicken y-globulin yields electrophoretically and antigenically distinct fragments. The electrophoretically slow fragment contains the antibody-combining site (Fab) and the fast fragment is crystallizable (Fc) ( Michaelides et al., 1W; Dreesman and Benedict, 1965; Gold et al., 1966). Chicken 7-globulin is apparently quite susceptible to the action of papain since 30450% of the total protein is degraded to dialyzable material after 15 hours of digestion. This amount of digestion is considerably greater than that found for rabbit yG and is slightly greater than that reported for human yG (Hsiao and Putnam, 1961). The size of the chicken Fab and Fc are similar, both having an s20,w equal to 3.8, and precipitin inhibition studies indicate that the Fab fragment is univalent as is the case with the Fab fragment of mammalian yG. Unlike mammalian yG, pepsin digestion results in a univalent Fab’ rather than a bivalent F( ab’),. Studies have also been performed on the polypeptide chains of chicken y-globulin that would suggest properties not found in mammalian yG (Dreesman and Benedict, 1965; Gold and Benedict, 1967). The H and L chains are bound to one another by disulfide bonds and there is a 3: 1 weight ratio of H: L, similar to that found in mammalian yG. Unlike mammalian yG, however, it was also found that approximately 50% of a partially reduced and alkylated preparation dissociates into H and L chains when placed on a Sephadex

70

HOWARD M. GREY

G-200 column in the absence of any dissociating reagent such as acid or urea (Gold and Benedict, 1967). Free light chains could also be demonstrated by immunoelectrophoresisof the reduced and alkylated 7-globulin. These results would suggest that the noncovalent interactions between the H and L chains are considerably weaker in the chicken than they are in mammals. However, it is also possible that labile intrachain &sulfide bonds in the H or L chains that are important for the native conformation of the chain were cleaved, thereby altering the conformation and reducing the noncovalent interactions between heavy and light chains. It would be of considerable interest to know which of these possibilities correctly explains this interesting observation. There is other evidence which indicates the presence of a wide spectrum of noncovalent binding energies between H and L chains of different species or within a species. In particular, the study of Cohen and Gordon (1965) showed that in the human, some light chains dissociated from heavy chains at pH 3.6 and with decreasing pH an increasing percentage of L chains were dissociated. In general h chains dissociated more readily than K chains so that at pH 3.3, 35%of h chains dissociated, whereas only 10% of K chains dissociated. The data obtained with chicken y-globulin would suggest that an even greater spectrum of binding energies between H and L chains may exist than is at present appreciated. On the basis of its relatively low isoelectric point, high sedimentation coefficient, and high hexose content, it has been postulated (Tenenhouse and Deutsch, 1966) that chicken y-globulin is more similar to mammalian yA than to yG. Studies of primary structure will have to be performed before this suggestion can be more adequately evaluated. Radioimmunoelectrophoresis of chicken antiserum has identified a third immunoglobulin in chicken serum which migrates in the yl region (Dreesman et al., 1965). It has been tentatively called yA but there is no structural evidence to substantiate this claim and efforts to isolate a yA protein by the zinc sulfate procedure of Heremans and co-workers have been unsuccessful ( Heremans et al., 1959). Much of the immunochemical interest in chicken antibody during the past 15 years has been due to anomalous findings observed when chicken antibodies are used in an immune precipitin reaction. When precipitin reactions are performed in high salt concentration (1.5 M NaCl), mammalian antisera give lower values of precipitating antibody than when performed at physiologic salt concentration. This is presumably due to the dissociating effect that the high salt concentration has on the antigenantibody bond. Contrary to this, chicken antisera give higher values of precipitating antibody when measured in high salt concentration than in

PHYLOGENY OF IMMUNOGLOBULINS

71

low (Goodman et d.,1951; Goodman and Wolfe, 1952). At least part of the differences observed at high and low salt concentration are due to the coprecipitation of a lipoprotein macroglobulin component from the serum of chickens. This appears to be a normal serum component which is not part of the complement system. The coprecipitation is more marked at high salt concentration and in aged serum (Gengozian and Wolfe, 1957; Van Orden and Treffers, 1968b). These macroglobulins not only act by adding their own weight to an immune precipitate but they may also be capable of cross-linking otherwise soluble complexes into larger, insoluble ones. This latter possibility appears likely from the observation that the macroglobulins can agglutinate latex particles coated with chicken y-globulin (Franklin, 1962). Another important effect of high salt concentration on chicken antigen-antibody reaction systems is that chicken 7-globulin undergoes dissociable aggregation at high salt concentrations (Hersh and Benedict, 1966; Van Orden and Treffers, 1968a). Ultracentrifuge analysis at low salt concentrations indicated there was little concentration ( c ) dependence of the sedimentation constant, the S L ,being ~ 7.0. At high salt concentration the s vs. c plot of the chicken y-globulin showed anomalous behavior which was typical of a concentration-dependent dissociating system. At relatively high protein concentration the s vs. c plot was not abnormal in that the observed s rate increased with decreasing protein concentration. An extrapolation of this portion of the curve to zero concentration would give an s&,, of 14 S for chicken yG in 1.5 M salt which is indicative of considerable aggregation of this protein and would be compatible with other data indicating a molecular weight of 540,000 for chicken 7-globulin at high salt concentrations (Orlans et al., 1964). However, at increasingly lower protein concentrations the sedimentation constant actually decreased to values compatible with unaggregated protein. These data suggest that at high protein concentrations the chicken 7-globulin was aggregated and that as the protein concentration decreased there was a shift in equilibrium between aggregated and nonaggregated protein so that at very low protein concentrations most of the material was unaggregated. Obviously the aggregation of chicken yG into larger molecular units with several combining sites per molecule could considerably increase the capacity of a chicken antiserum to precipitate with antigen. Other anomalous serologic reactions have been reported with chicken antisera which suggest that some chicken antibodies may contain a single antibody-combining site. Antigen-antibody precipitates dissolved in a large excess of antigen. have been analyzed in the ultracentrifuge for the limiting size of the complexes formed in the region of extreme antigen

72

HOWARD M. GREY

excess. Confiicting results have been reported with this system. Banovitz et al. (1964) found two types of complexesone compatible with the formula of Ag,Ab and one of AgAb. Under the conditions of antigen excess used the latter-sized complexes are suggestive of univalent antibody. Williams and Donermeyer (1962) concluded from similar studies that all chicken antibody is bivalent. A complicating factor in some of these experiments was the presence of the coprecipitating macroglobulin in the reaction mixture. It would be of considerable interest to have valency studies performed on purified chicken 7-globulin with a hapten-antihapten antibody system so that less equivocal data might be obtained concerning this important point.

et a2. (1959) and Orlans

2. Duck The other avian species in which data on immunoglobulin structure are available is the duck. Three major immunoglobulins have been characterized (Grey, 1963b, 1967a,b; Unanue and Dixon, 1965). These are a 19 S immunoglobulin which has the immunoelectrophoretic appearance and reducing agent sensitivity of mammalian yM; a y,-globulin with an so,, equal to 7.8; and a 7,-globulin with an sk,, equal to 5.7. These latter two low-molecular-weight immunoglobulins make up the bulk of the immune response to a variety of antigens. In the animal hyperimmunized to bovine serum albumin, approximately three-quarters of the total antibody is of the 5.7 S type, whereas early in the immune response approximately equal amounts of both antibodies are produced (Grey, 196%). The 5.7 S and 7.8 S immunoglobulins are antigenically related to one another but not identical. Rabbit antisera have been obtained which detect antigenic determinants specific for the 7.8 S protein but not for the 5.7 S protein so that the 7.8s protein spurs over the 5.7 protein when examined by immunodiffusion. Digestion of both proteins with papain yields electrophoretically and antigenically distinct fragments. The electrophoretically slow fragments of either protein are antigenically similar to the 5s fragment obtained by pepsin digestion. Also, the pepsin fragments of the 5.7s and 7.8s proteins give lines of identity in immunodiffusion. These results are similar to those obtained when human yG is digested with these enzymes and thereby allows identification of the slow papain fragment as the Fab and the fast fragment as the Fc. After partial reduction and alkylation the 7.8 S protein can be separated into heavyand light-chain components by gel filtration in 1M propionic acid. The pattern obtained is similar to that seen with mammalian yG. Under the same conditions, the 5.7 S protein gives a single broad peak (Fig. 3 ) . Antigenically, light chains identical to those obtained with the 7.8 S pro-

PHYLOGENY OF IMMUNOGLOBULINS

73

0.3 0.2

0.1

d 0

0.4i 5.7 S Globulin

Tube number

FIG.3. Separation of H and L chains from duck immunoglobulins on Sephadex G-100 in 1 A4 propionic acid. (0.D = optical density.)

tein are obtained from the descending limb of the peak, and heavy-chain material from the ascending portion of the peak. The heavy chains from the 5.7 S protein migrate in a similar position to the light chains on acid urea starch gel electrophoresis, whereas the heavy chains of the 7.8s protein migrate more slowly, in a position similar to that of mammalian heavy chains. Both the gel filtration and starch gel electrophoresis data suggest that the heavy chain from the 5.7s protein is smaller than that of the 7.8s protein or mammalian y chains. This would be in keeping with the low s rate of the intact protein compared with mammalian immunoglobulins. However, proof of this must come from molecular weight determinations of the intact protein and the component chains. When compared with mammalian immunoglobulins the 5.7 S protein has other anomalous features. After partial reduction and alkylation the s rate of this protein decreases to 3.5S . Antigenically the protein appears homogeneous, and there is no evidence of release of light chains as occurs with

74

HOWARD M. GREY

chicken y-globulin. This marked reduction in s rate without any evidence of fragmentation by antigenic analysis suggests that if the low s rate is due to fragmentation of the native protein the fragments thus produced are antigenically identical. This could only occur if the fragments represented L H half-molecules. Such half-molecules have been produced with rabbit yG (Palmer et al., 1963; Palmer and Nisonoff, 1964) after acidification of mildly reduced protein. The half-molecules thus produced are univalent. After mild reduction the 5.7 S antibody loses all hemagglutinating capacity, whereas its capacity to bind antigen is only slightly decreased. This preferential loss of agglutinating capacity would, of course, be compatible with the production of univalent half-molecules. Detailed data on other species of birds are lacking. Studies with turkeys indicate the presence of 19 S and 7 S immunoglobulins, but no other characterization has been made (Dreesman et aZ., 1963, 1967). Whether the unique findings in chicken and duck immunoglobulins are representative of the entire avian class or whether they are unique to the individual species studied will not be known until detailed studies on many more avian species have been undertaken.

F. MAMMALIAN IMMUNOGLOBULINS The great bulk of information available on immunoglobulin structure has been obtained in mammals. However, detailed structural information is limited to a relatively few species of eutherian (placental) mammals. No information is available to date on prototherian or metatherian mammals, although immunological studies have been started on the echidna, the Australian spiny anteater, so that data relative to immunoglobulin structure may be available for this prototherian in the future (Diener and Ealey, 1965; Diener et aZ., 1967a,b). Eutherian mammals are subdivided into four major categories: Unguiculata, in which the primate order is the only one for which detailed studies are available; Glires, which contains the rodents and rabbits; Mutica, which contains the aquatic mammals and on which no data are available; and Ferungulata, on which data are available and which includes the following species: dog, horse, pig, cow, and sheep (Young, 1962). 1. Human

The structure of human immunoglobulins has been intensively studied partly because of the considerable clinical interest in the biological activities of human antibodies and, perhaps more importantly, because of the availability of myeloma proteins. In the past 15 years much of our understanding of the chemical structure of the immunoglobulins has come from the study of myeloma proteins. These proteins are the prod-

PHYLOGENY OF IMMUNOGLOBULINS

75

ucts of plasma cells that have undergone neoplastic changes and, in most cases, probably represent the product of a single clone of cells. Individual proteins are obtainable in large quantities in a purified state; they are extremely homogeneous relative to normal 7-globulin or most purified antibody preparations and are, therefore, quite suitable for chemical study. Moreover, several myeloma proteins have recently been demonstrated to possess antibody activity to simple haptens (Eisen et al., 1967, 1968) as well as to protein and polysaccharide antigens (Kritzman et ul., 1961; Cohn, 1967; Metzger, 1967; Grey et ul., 1968b), so that they are also suited for study of the antibody-combining site as well. Multiple myeloma and related lymphomatous disorders are not uncommon in man and have been used to help delineate the classes and subclasses of immunoglobulins in this species. Plasmacytomas can be induced in certain strains of mice as well, and this experimental model has been successfully employed for the study of immunoglobulin structure in that species. Spontaneous occurrence of multiple myeloma has also been reported in the dog (Rockey and Schwartzman, 1967) and horse (Dorrington and Rockey, 1968), and it is anticipated that these proteins will be of increasing help in elucidation of immunoglobulin structure in species other than man and mouse where they have already been proven to be of the utmost value. With the aid of myeloma proteins it has been possible to delineate five classes of immunoglobulins on the basis of antigenic and biochemical differences between their heavy chains: yG, yA, yM, yD, and YE. Each of these immunoglobulin classes is associated with both of the major light-chain types, K and A. The relative concentrations of the immunoglobulin classes in norrnal serum as well as some of their physical-chemical distinguishing characteristics are shown in Table 111. Amino acid sequence analysis has been completed on several human light chains including proteins of both x and X type (Hilschmann and Craig, 19%). No complete sequence of a y chain has been completed as yet, although considerable data have accumulated on the amino terminal end of the heavy chain, the region of inter- and intrachain disulfide bonds, and the carboy terminal region. These data have been recently reviewed (Cohen and Milstein, 1967) and will be referred to only when pertinent for comparative analysis. Light chains, as well as being divisible into K and h types, can be further subdivided into subclasses based on structural relationships in the variable regions. Both K and h chains consist of three distinct subclasses (Hood et al., 1967; Milstein, 1967; Niall and Edman, 1%7; Hood and Ein, 1968; Langer et al., 1968). These subclasses are based on certain striking structural similarities found in particular light chains. When the proteins

76

HOWARD M. GREY

TABLE I11 HUMANIMMUNOGLOBULIN CLASSES

No. of subclasses

described Serum conc. (mg./%) s constant

Molecular weight

4

3

2

-

-

1200

150

75

0.3-30

.01-. 07

6.6-7.2 150-160,000

Carbohydrate yo (w/w)

2.9

7 30-150 150-160,000 900,000 160-180,000 (monomer) (pentamer) 8 12 12 7-17

18-20;

8

200,000 11

are grouped by these similarities the homologies in the variable region increase, compared to the degree of homology that one would find by comparing two light chains chosen at random. There are obvious difficulties in attempting to sort out similarities in structure that have as much variability as does the N-terminal region of light chains. However, the data thus far obtained would suggest that there are structural subclasses of K and light chains. This, taken with the singleness of the structure of the C-terminal region [with the exception of the single residue exchange related to the allotypic variation in K chains (Baglioni et aZ., 1966) and the Oz variability in chains (Ein, 1!368)] has been taken as evidence that the variable and constant regions are under separate genetic control. Classification of light chains into subclasses has been possible by analyzing the light chains antigenically as well (Stein et aZ., 1963; Solomon et al., 1965; Korngold and Madalinski, 196%). Since the data for light chain subclasses are dependent primarily on sequence data and since these are to a large extent fragmentary and limited to a relatively small number of proteins, the number and distribution of the light-chain subclasses may not be complete as yet. The situation in the heavy chains is somewhat different. Here, division of heavy chains has been done primarily on the basis of antigenic differences although sequence data are becoming increasingly available. It has been possible to examine large numbers of proteins and the classification into subclasses appears complete for yG proteins (Dray, 1960;Ballieux et al., 1964; Grey and Kunkel, 1964; Takatsuki and Osserman, 1964; Terry and Fahey, 1964). Less work has been done on the other classes, and it is quite likely that the number of subclasses will increase considerably for these classes in the future. Gamma G proteins are divisible into four subclasses. Whereas im-

PHYLOGENY OF IMMUNOGLOBULINS

77

munoglobulin classes are antigenically unrelated in their heavy chains, the subclasses have antigenically very closely related chains. The limited sequence data bear out their close relationship, as illustrated by the sequence of the C-terminal octadecapeptide as well as by the composition of the glycopeptide of the four subclasses (Grey and Abel, 1969). These data together with those of Milstein and his collaborators would suggest a sequence homology of 6 9 5 % or greater between the four subclasses. The degree of sequence homology shared between the different classes of heavy chains is at present unknown, but it will undoubtedly be less striking and may be similar to the 40%found for K and X light chains. Studies on the association of allotypic markers with proteins of the four subclasses has greatly aided the understanding of both the genetics of 7-globulin synthesis and the relationship of the subclasses to one another (Kunkel et al., 1964; Natvig et at., 1967). It has become apparent from these studies that the genes coding for the four yG subclasses are very closely linked to one another and that the known y-chain genetic markers distribute themselves among three of the four subclasses so that the Gm system does not describe a single locus with multiple alleles, but rather markers for three closely linked loci (pseudoalleles). As yet no genetic markers have been described for the yG4 subclass. Family studies have indicated that the crossover rate within this gene complex is very low but does occur. The available information obtained from these family studies would suggest that the linkage of genes associated with the four y-chain subclasses is in the order yl-y3-y%y4 ( Natvig et al., 1967). Considerably less is known of the detailed structure of the other immunoglobulin classes of man. Gamma A globulins appear in a 7s form, similar to yG, as well as in several different disul6de-linked polymeric forms ranging in sedimentation rate from 9 to 17s. The reason for these multiple molecular forms and whether primary structural differences exist between the different forms is largely unknown. No convincing structural differences have been observed between monomer and polymer forms of the same protein although differences in the fingerprint patterns have been noted (Ballieux, 1963). It appears, however, that when monomers are reduced and subsequently oxidized they are capable of polymerizing to the same extent as native polymers. This would suggest that the primary structure of the monomer is the same as that of the polymer, but that the cysteine residues involved in the polymerization are blocked and that they only become available for forming intersubunit disul6de bonds after reduction (Abel and Grey, 1968). A similar situation has been described for human serum albumin where it has been shown that the

78

HOWARD M. GREY

L H

5 0

H

b

FIG.4. Schematic model of the arrangement of the polypeptide chains in human yA immunoglobulins: ( a ) yAl; ( b ) y A 2 and yA3.

monomer has a cysteine residue that is disuKide linked to glutathione or cysteine, whereas in the dimer albumin this cysteine residue forms a disulfide bond with the cysteine on another albumin molecule (King, 1961) . As well as occurring in the serum, yA is the major immunoglobulin of external secretions such as colostrum, saliva, tears (Hanson, 1961;Tomasi and Zigelbaum, 1963). In these secretions it is found as a dimer in association with another polypeptide chain, the secretory piece (SP). The binding of SP to the yA appears to be through noncovalent forces as well as by disulfide bonds, although some molecules of yA may be only noncovalently bound to the SP. The function of the SP is unknown but one possibility is that it serves to protect the yA from proteolysis by the enzymes present in the secretions (Tomasi and Calvanico, 1968). Three antigenic subclasses of yA have been described (Kunkel and Prendergast, 1966; Vaerman and Heremans, 1966; Feinstein and Franklin, 1966; Grey et al. 1968a). The most striking structural difference between the subclasses is that y A l (the predominant subclass in normal serum) contains L - H and H-H disulfide bonds similar to other immunoglobulins, whereas yA2 and yA3 lack the L-H disulfide bonds, having instead L-L and H-H disulfide bridges. This is schematically illustrated in Fig. 4. In order to accommodate the G - L disulfide bond the light chains are positioned on the inner aspect of the heavy chain rather than on the outer aspect. Another structural distinction between the yA subclasses is found in the carbohydrate composition. Gamma A1 contains galactosamine as well as glucosamine and the other sugars usually found in immunoglobulins-galactose, mannose, fucose, and sialic acid, The amino sugars are positioned such that all the galactosamine is present in a single glycopeptide and all the glucosamine appears in one other position in the yA heavy chain. In yA2 and yA3, there is no galactosamine but instead two glucosamine glycopeptides are found (Abel and Grey, 1969). It is of considerable interest that the galactosamine containing peptide of 35 to 40 amino acids contains two interchain disulfide bonds as well as having an extremely high content of proline (30435%)(Abel and Grey, 1969; KO et al., 1967). This unique association of high proline content

PHYLOGENY O F IMMUNOGLOBULINS

79

and interchain disulfide bonds has been found in rabbit yG at the amino terminal end of the Fc fragment and has been called the “hinge” region because it is thought that the high proline content gives this peptide region a relatively unordered structure, thereby allowing the Fab and Fc fragments to move rather independently of one another about this peptide region (see Fig. 2). Rabbit yG is the only immunoglobulin besides yA in which galactosamine has been reported. In this species the galactosamine is present in this hinge region. Human YGand yM proteins lack galactosamine; however, recent studies indicate that a yD myeloma protein also contains a galactosamine peptide which is located at the amino terminal end of the F’c fragment (Spiegelberg et al., 1969). It would appear then that galactosamine, when it is present in an immunoglobulin, is associated with the hinge region. However, it should be noted that not all immunoglobulins have carbohydrate in this region; also, there is some evidence that certain immunoglobulins may have a glucosamine-containing carbohydrate moiety in this region (Mihaesco and Seligmann, 1968a; Davie and Osterland, 1968). High molecular weight antibody has been recognized in the human for over 20 years. It is easily distinguished from yG on the basis of its antigenic individuality, its molecular weight, and its carbohydrate content. Its distinction from yA is somewhat less clear-cut. Although these two immunoglobulins are also immunologically distinct, they both have a relatively high carbohydrate content as well as the ability to polymerize. Gamma A proteins aggregate to form polymers of different sizes varying from dimer to tebamer or pentamer, whereas yM forms pentamers as well as larger aggregates. Until recently it was not appreciated that like yA, yM can also be present in serum as a monomer (Rothfield et al., 1965; Stobo and Tomasi,1966; Solomon, 1967; Solomon and Kunkel, 1967; Klein et al., 1967; Hunter et al., 1968; Perchalski et al., in press). The serum concentration of this yM monomer is usually quite low relative to the 19 S yM. However, in certain disease states it is easily demonstrable and may be significant in certain autoimmune phenomena. A similar low molecular weight yM has also been described in the horse (Sandor, 1962; Sandor et al., 1964) as well as in lower vertebrates as has been previously discussed in Section III,B,2. Although there are very few data available at present on the amino acid sequence of yA and yM proteins, the available data support the suggestion that yA and y M are more closely related to one another than to YG.This is shown in the C-terminal sequences (Table IV). In general, both human yM and yA are more susceptible to proteol-

80

HOWARD M. GREY

TABLE IV

GTERMINAL SEQUENCES Chain Y

a P

OF

H U ~ yN, a, AND

p

CHAINS

C-Terminal

Gln-Lys-Ser-Leu-Ser-Leu-Ser-Pro-Gl y

Met-AlaGlu-Val-Asp-G1y-Thr-Cys-Tyra * Met-Ser-Asx-Thr-Ala-Gly-Thr-Cys-Tyrasc 0

Abel and Grey (1967). Prahl and Grey (in preparation). c Wikler et al. (1969). b

ysis than yG.Whereas both yield Fab fragments, it has not been possible to isolate the Fc fragments of yA, presumably due to extensive digestion even after short periods of incubation with enzyme. It has been possible to obtain low yields of Fc fragments from yM proteins, and by so doing it has been demonstrated that the disulfide bonds responsible for linking the monomeric subunits to one another are located in this fragment ( Mihaesco and Seligmann, 196810; Onoue et al., 1968a; Yakulis et al., 1968). The homology between the C-terminal region of the a- and p-chains with the C-terminus of light chains has led to the suggestion that the cysteine penultimate to the C-terminus is the active residue involved in the disulfide binding of subunits; however, no direct evidence for this postulate is available ( Doolittle et d.,1966; Abel and Grey, 1967). The yD is a minor component of normal serum and has been characterized solely through the study of myeloma proteins. It is the one immunoglobulin class in which antibody activity has not been clearly demonstrated. The reason for this failure is uncertain but it may be related to the very low serum concentration of this immunoglobulin; however, until antibody activity is demonstrated some reservation as to the biological function of this protein should be maintained. Structural studies on this immunoglobulin have been hampered by the rarity of myeloma proteins, of this type, and its extreme susceptibility to spontaneous degradation. There are certain features that are known, however. Perhaps most striking, is the finding that most yD myeloma proteins are of the A type (Rowe and Fahey, 1965; Fahey et al., 1968). This is in contrast to other immunoglobulin classes in which the majority of myeloma proteins as well as their normal serum counterparts are of K type. The significance of this finding is at present totaUy obscure. The yD has l2% carbohydrate and, as mentioned above, contains galactosamine in a region which appears homologous to the hinge region of yG. It contains only three inter-

PHYLOGENY OF IMMUNOGLOBULINS

81

chain disulfide bonds-two L H and one H-H; the locations of these bonds have not been determined but they do not appear to be in the hinge region, as is the case for yG1 (Spiegelberg et al., 1969). Attempts to investigate structural homology between this immunoglobulin class and others are in progress. The fifth immunoglobulin class, YE, although representing, as is the case with yD, a small fraction of the total immunoglobulin present in serum, is of great interest since it has been identified as the major source of human reagin, i.e., skin-sensitizing antibody. Structural studies on a yE myeloma protein as well as normal yE have indicated it has a molecular weight of 200,000 and an 8 S sedimentation coefficient giving a calculated weight of 75,000 for each heavy chain (Ishizaka and Ishizaka, 1966; Ishizaka et al., 1966; Bennich and Johansson, 1967; Johansson and Bennich, 1967). It has a carbohydrate content similar to that of yM and yD and can be digested into an Fc fragment of approximately 100,OOO molecular weight and two Fab fragments of 50,000 molecular weight. There is a total of 16 half-cystines per mole of heavy chain compared to values obtained with other irrimunoglobulin classes of 11-13 for yG, 10-12 for yD, 14 for yM, and 16-20 for yA. The distribution between inter- and intrachain disulfide bonds has not as yet been elucidated.

2. Other Primates The immunoglobulins of nonhuman primates have been studied predominantly to determine the extent of their structural relationships to human immunoglobulins. These studies have been limited largely to comparisons of antigenic structure and only recently have comparative biochemical studies been performed (Shuster et al., in press; Wang et al., 1969). Goodman (1962a,b, 1963a,b, 1967), using rabbit and chicken antisera to human as well as other primate proteins, has made a systematic comparison of the antigenic cross-reactions of several serum proteins. In general, his studies and those of others (Picard et al., 1963; Williams, 1964) agree with the currently accepted taxonomic classscation of the primates in that human 7-globulin is very similar to that of the anthropoid apes, especially chimpanzee and gorilla and somewhat less similar to the gibbon and orangutan. The fact that most antisera could not distinguish between chimpanzee arid human 7-globulin led Goodman to suggest that man, gorilla, and monkeys should be classed as Hominidae, with the other apes to be placed in the family Pongidae instead of the more common classification which places man as the sole species of Hominidae. The

82

HOWARD M. GREY

y-globulins of Old World monkeys (rhesus, baboon) were found to be less closely related to human y-globulin than that of the apes but more closely related to it than the 7-globulin of New World monkeys (spider monkey, marmoset ) , The New World monkeys, on the other hand, were more highly cross-reacting with human y-globulin than that of the prosimians (loris, bush baby). As mentioned above, this pattern of antigenic relatedness is in keeping with other morphological characteristics which form the basis of the taxonomic classifications. Picard et al. (1963) have performed similar studies using purified yG and yM as antigens and have shown similar results to those of Goodman for both immunoglobulin classes, In comparing the antigenic cross-reactions of the y-globulins with those of the albumins, Goodman found that the primate albumins appeared to be more closely related to one another than did the y-globulins. If these antigenic relationships are indicative of primary structure relationships, this finding would suggest that the y-globulins may have a greater rate of mutation than do the albumins. Antigenic studies have also been done to determine the presence of K and light-chain antigens, heavy-chain subclass antigens, as well as Gm and Inv genetic factors. Alepa and Terry (1965) found that chimpanzees contain K and A antigens as well as antigens specific for yG1, 2, and 3 (yG4 was not tested). Also both Invl and b were present. The incidence of these light-chain genetic markers in the population was similar to that found in man. The presence or absence of light-chain antigens in other primates has not been investigated. Shuster et al. (in press) have made a comprehensive, quantitative study of the presence of yG3 antigens in primates. Their findings with rabbit anti-yG3 antisera indicated that gorilla, orangutan, and chimpanzee yG3 were very similar to human, whereas the other member of the Hominoidae, the gibbon, did not contain yG3 antigens. Using a rhesus anti-yG3 antiserum, only human yG3 was detected indicating that multiple antigenic determinants were involved in the yG3 locus and that the monkey antisera recognized determinants present in human yG3 which were lacking in the apes. Representative species of Cercopithecoidea (Old World monkeys), Ceboidea (New World monkeys), and prosimians were also tested. The rabbit antiserum showed 3050%cross-reactivity with certain species of each of these major subdivisions. The rhesus antiserum demonstrated the presence of yG3 antigens in both Ceboidea and prosimians; as would be expected Cercopithecoidea, the family to which the rhesus belongs, did not react with this antiserum. A baboon anti-yG2 antiserum detected yG2 antigens in all Hominoidae except the gibbon. This antigen could not be detected in prosimians or Old World monkeys. Genetic factors specific

83

PHYLOGENY OF IMMUNOGLOBULINS

for the yG1 locus (Gm, a, z, f, y ) ; yG3 (Gm b and g) as well as yG2 (Gm n), have also been sought for in nonhuman primates (Litwin, 1967a,b; Boyer and Young, 1961; Podliachouk, 1959; Shuster et al., in press). The Gm b antigens were found in hominoids and Old World monkeys; Gm a, on the other hand, was found only in apes, whereas Gm z, another yG1 factor, was found in apes and Old World monkeys, but the reactions were weak and probably of a cross-reacting nature. The Gm y, g, and x antigens were only found in humans. These data in primates, although incomplete with regard to certain immunoglobulins, give a good indication of the relatedness of human y-globulin to other primates. The finding of “human” yG immunoglobulin antigenic determinants and genetic markers in the higher primates clearly indicates that the yG subclasses are not unique to humans but are present in other primates as well. Obviously these studies can only determine y-globulin antigens shared by humans and other primates. In the lower primates, where certain subclasses were not recognizable by the crossreaction with human proteins, nothing can be said about the presence or absence of yG subclasses since they might have been present but antigenically unrelated to their human counterparts.

3. Mammalian, Nonprimute Species Detailed analysis of the immunoglobulins of other mammalian species has been performed in only a few species. Table V summarizes the immunoglobulins of those species of mammals in which more than fragmentary data are available. All species appear to have proteins analogous to human yG and yM, although in some cases the characterization is

IMMUNOGLOBULIN ~~

Order Lagomorph Rodent

TABLE V CLASSES PRESENT I N MAMMALIAN

SPECIES

~

Species

-

Rabbit Mouse Rat Guinea pig Hamster Carnivore Dog Artiodactyl Pig cow Sheep Perissodactyl Horse

YM

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

Anaphylactic antibody Anaphylactic antibody Anaphylactic antibody 3 4 S y, 19 S yG 19 S yG

T component, 10 S y,

84

HOWARD M. GREY

based solely on sedimentation characteristics, yA has been well characterized in only three species. Other immunoglobulin classes or subclasses have been recognized by unique activities, specific antigenic determinants, or other structural features. a. yG. The major immunoglobulin class in all mammals appears to be a yG as characterized by its s rate ( 7 S), low carbohydrate content (approximately ZW), and slow electrophoretic mobility. In several species there are two electrophoretically and antigenically distinguishable types of yG which migrate in the fast region ( yl) and in the slow y region ( y z ) . Whether these two subclasses are homologous to any of the human subclasses is not known at present, but certain biological and biochemical characteristics suggest that yl may be homologous to human yG2 (and/or yG4) and yz to human yG1 (and/or yG3). The most detailed information regarding the structure of yG in these species is available in the rabbit. Rabbit yG is structurally very similar to human yG. H and L chains of both species are of similar size, and the intact molecule is digested by papain and pepsin in the same manner. Most molecules of rabbit yG have a single H-H interchain bond (Palmer et al., 1963, Palmer and Nisonoff, 1964), whereas human yG has two or more H-H interchain disulfides (Frangione and Milstein, 1968; Pink and Milstein, 1967a,b; Steiner and Porter, 1967). The amino acid sequence of the entire Fc fragment and a portion of the Fd fragment have been reported (Givol and Porter, 1965; Hill et al., 1966b; Cebra et al., 1968a,b). The fact that a single sequence for the Fc fragment could be obtained suggests that if yG subclasses exist in the rabbit, there must be one major subclass with the other subclasses present in such low concentrations that they were not detected by, and did not interfere with, the sequence determinations. It is possible to compare the amino acid sequence of rabbit Fc fragment with that of the human (Press et al., 1966; Milstein et al., 1967; Prahl, 1968; Rutishauser et al., 1968). There is 76% sequence homology in this fragment. This is somewhat less than the 85% homology between rabbit and human hemoglobin, which would suggest that the evolution of yG, or at least the Fc fragment may have differed in its rate of change from that of other serum proteins. This is in keeping with the antigenic data of Goodman on the differences between primate and human proteins (Goodman, 1962b) . Mainly through their work on mouse myeloma proteins, Potter and Fahey and their respective co-workershave elucidated the major immunoglobulins of the mouse. There are three 7 S immunoglobulins; yl (YF), yza (yG), and Y2b (yH) (Fahey et al., 1964a,b; Potter et al., 1965). The yl as mentioned previously, is a less basic protein than the yz proteins and

PHYLOGENY OF IMMUNOGLOBULINS

85

it differs to a greater extent antigenically from the yze and YZb immunoglobulins than the latter two proteins differ from one another. For these reasons it was originally designated as a separate class of immunoglobulin rather than a subclass of yG. With respect to the evolution of immunoglobulins, the distinction is rather artificial since the difference between classes and subclasses is merely a quantitative one based on degrees of antigenic relatedness. Proteins of different classes as well as dserent subclasses, represent products of distinct structural genes, the differences in degrees of homology being explainable on the basis that genes controlling different classes diverged and evolved from one another earlier than those controlling subclasses. As mentioned above, it is not known what the relationship is between the mouse immunoglobulins and those of the human, although on the basis of biological activity as well as certain biochemical characteristics, such as enzyme susceptibility, it would seem that yz, may be homologous to yG1 (the major immunoglobulin capable of fixing complement, fixing to heterologous skin, and having a relative long half-life); 7% may be homologous to yG3 (relatively susceptible to proteolysis, short half-life, complement fixing; yl may be homologous to yG2 or yG4 (relatively fast electrophoretic mobility and poor or no complement-fixing activity) ( Fahey and Sell, 1965; Miiller-Eberhard and Grey, unpublished observations; Spiegelberg and Grey, 1968; Nussenzweig et al., 1964; Ovary et al., 1965; Spiegelberg et al., 1968). It is also possible, as will be discussed later, that the subclasses in mouse and man evolved quite separately from one another, after the two species diverged from one another. The other species of rodents studied-rats, guinea pigs, and hamsters-also possess y and yz immunoglobulins (Banovik and Ishizaka, 1967; Coe, 1968; Nussenzweig and Binaghi, 1965; Yagi et al., 1962a,b; Benacerraf et al., 1963; Bloch et al., 1968). The immunoglobulins appear to be functionally related to one another in that, where it has been studied, the yl fixes to the skin of the homologous species and does not fix complement; whereas the y2 of all these species fixes to skin of heterologous species and is capable of fixing complement (Ovary et al., 1963; Bloch et al., 1963).Antigenically, it has been possible to recognize a second yz immunoglobulin in the rat as well as a more acidic immunoglobulin by means of radioimmunoelectrophoresis ( Banovitz and Ishizaka, 1967; Bloch et al., 1968) . This technique is extremely sensitive. However, false positive reactions have been described (Minden et al., 1967) so that it should not be used as the sole criterion for identification of an immunoglobulin. In the rat, dog, and rabbit, another species of low-molecular-weight

86

HOWARD M. GREY

immunoglobulin has been identified on the basis of its anaphylactic antibody activity for homologous species. These immunoglobulins appear to be homologous to human yE with respect to their biological activity, heat lability, low serum concentrations, and gel filtration characteristics. It has not, as yet, been possible to isolate these proteins in a purified state (Binaghi and Benacerraf, 1964; Mota, 1964; Rockey and Schwartzman, in press; Zvaifler and Becker, 1968;Patterson et al., 1963; Binaghi et al., 1964). The yl and yz immunoglobulins exist in the artiodactyls, such as sheep (Silverstein et al., 1963; Aalund et al., 1965; Pan et al., 1968),cow (Pierce and Feinstein, 1965; Murphy et al., 1965), and pig (Kim et al., 1966a,b) as well. It is of considerable interest that biologically the immunoglobulins in these species have somewhat different biological functions compared to the rodent yl and yz in that, in the rodents, yl does not fix complement, whereas in the cow and sheep it does (Feinstein and Hobart, personal communication). Also, in the cow and sheep, it is the yl which is selectively secreted into the colostrum, whereas in man, rabbit, and mouse, yA is selectively secreted (Pierce and Feinstein, 1965; Fahey and Barth, 1965; Murphy et d.,1965). The pig has a yl and y 2 immunoglobulin; and, in the newborn colostrum-deprived piglet, trace amounts of a 3.5-4s immunoglobulin which lacks antibody activity has aIso been described (Franek and Riha, 1964; Franek et al., 1961; Prokesova et d.,in press). Antigenically, this low-molecular-weight protein has both y heavy-chain and light-chain determinants and has a molecular weight of approximately 80,OOO. These data were considered suggestive that it represented half-molecules consisting of one H and one L chain. However, more work is necessary before the exact nature and significance of this trace serum component can be definitely established. It has also been claimed that the newborn piglet when immunized forms initially a 19S antibody which is antigenically identical to yG rather than yM (Kim et al., 1966a,b). The same has been claimed to be true in the cow (Hammer et al., 1968), although other investigators have not been able to confirm the observations in the piglet (Franek, 1962; Prokesova et al., in press). At present the published data do not allow a firm conclusion to be drawn on whether a unique 19 S yG immunoglobulin does indeed exist. If it does, it is of obvious interest with regard to the phylogeny as well as ontogeny of mammalian immunoglobulins, Two other mammalian species have been well characterized with respect to their immunoglobulins-the dog and the horse. Dog serum contains three antigenically distinct immunoglobulins in the yz regionpa,y2b, and yZc.There is a faster migrating 7 s yl immunoglobulin as well (Johnson and Vaughan, 1967; Johnson et al., 1967; Patterson et al., 1968).

PHYLOGENY OF IMMUNOGLOBULINS

87

In the horse there is comparable degree of rG heterogeneity (Weir and Porter, 1966; Rockey et at., 1964; Klinman et aZ., 1965, 1966; Montgomery et al., 1969). There are three 7 s yz immunoglobulins-y2,, Y 2 b and yZc. These proteins have molecular weights, carbohydrate’ content, and polypeptide chains very similar to other mammalian yG proteins. A 10s yl mobility immunoglobulin which has specific antigenic determinants has also been described. Another high-molecular-weight immunoglobulin found in hyperimmune equine antipneumococcus antisera has been described ( Zolla and Goodman, 1968). This latter immunoglobulin also has a fast electrophoretic mobility and is made up of noncovalently bound aggregates of a 7 S globulin ranging from 9 S to 15S. Whether the 10 S yl described by Rockey et al. (1964) is the same as this 9-15 S antibody of Zolla and Goodman, is unknown at present as is their possible relatedness to the 19 S yG of the cow and pig. For many years another immunoglobulin class has been recognized on the basis of its prominence as an antibody component in horses hyperimmunized to pneuinococcal polysaccharide as well as other antigens. This immunoglobulin has a ,&globulin mobility and has been referred to as the T component (Smith and Gerlough, 1947; Tiselius and Kabat, 1939; Van der Scheer et aZ., 1940, 1941; Jager et at., 1950). It has a sedimentation coefficient of 7 S, has antigenic determinants that distinguish it from other immunoglobulins, and has a high carbohydrate content (Weir and Porter, 1966; Klinman et d.,1966). These characteristics led to the conclusion that the T component was equivalent to the horse yA. Amino acid sequence analysis of the C-terminal octadecapeptide from the horse T component and yG, however, have led to a revision of this conclusion (Weir et al., 1966): As seen in Table VI, the T component and yG have extensive homology in this region and it bears no relationship to the C-terminal region of mouse and human yA (Table IV) . These data have been interpreted as indicating that the T component represents a subclass of horse yG rather than a distinct immunoglobulin class. These studies point out the dsculties in assigning a protein to an immunoglobulin class on the basis of gross features of homology such as carbohydrate content and electrophoretic mobility rather than on characteristics that reflect more critically the primary sequence of the protein. Table VI also shows the sequence data for yG octadecapeptides of other mammalian species. It has been possible to study this C-terminal stretch because in all these yG immunoglobulins a methionine residue is at position 19 from the C-terminus and the heavy chain can be cleaved at this position with cyanogen bromide. The small size of the C-terminal peptide relative to the other heavy-chain peptides obtained with this pro-

TABLE; VI SPECIESCOMPARISON OF C-TERMINAL SEQUENCES OF Species Human yla

19 Met

12

11

His Glu Ala Leu His Asn His

Tyr

Arg Arg

Phe Tyr

18

17

16

15

14

13

72

ya-Gm b w-Gm g

Y4

Guinea pig yrb Rabbit yGc HOW yGd Horse T c o w y,' c o w Y1

Thr

Val Glu

Prahl (1967).

Givol and Porter (1965). Weir et al. (1966). s Inouve and Givol (1967); Milstein and Feinstein (1968).

8

CHAINS

7

Gln Lys Ser

Ala

Val

* Turner and Cebra, personal communication (1969).

c

Sequence 10 9

y

LMet

6

5

4

3

2

Leu

Ser

Leu

Ser

Pro Gly

Ile Val Asn Val Thr Thr

1

Leu -4%

LY

His

LYS Lys

Ala Als

i!

4

PHYLOGENY OF IMMUNOGLOBULINS

89

cedure makes isolation of the C-terminal peptide relatively simple. Neither the yA nor yM proteins studied to date have a methionine in the same position; however, they do possess a methionine at position 9 from the C-terminus (Wikler et al., 1969: Prahl and Grey, unpublished observations). Most striking is the high degree of homology found between all the mammalian species of yG thus far studied. The largest divergence from the most commonly found sequence is 4 of 19 residues (the most divergent protein being the horse T component), whereas most of the proteins differ by only 1 or 2 residues representing a homology in the order of 90% or greater. These data have certain implications regarding the evolution of yG subclasses. If one accepts the postulate based on sequence homologies that there was at one time a primitive heavy-chain gene (and, as discussed in Section IV, before that a primitive light-chain gene) and that through the process of gene duplication followed by unrelated mutational events, the duplicated genes evolved independently so that their gene products were distinguishable from one another as Merent classes or subclasses of immunoglobulin, then the question can be posed : When in the course of evolution did yG subclasses appear? In particular, did subclasses appear before or after the mammalian species represented in Table VI diverged from a common ancestor? It would appear that some of the data available from the octadecapeptides would argue in favor of the postulate that subclasses arose after divergence of these species from a common ancestor. An example of the reasoning involved is illustrated by the types of variations seen at position 2 from the Cterminus. In all species except the cow (and one subclass of the human), proline is present at this position, whereas in both yl and yz of the cow, alanine is present. If subclasses were present in the common ancestor of these species, then it would be reasonable to assume that proline was present since it is the most frequent residue in this position. For both the cow yl and y? to have alanine in that position, it would require the same mutational event to have occurred twice during the evolution of the cowonce in the yl gene and once in the yz gene; whereas, in the other four species with the exception of the human yG4 subclass, the proline persisted. It would appear that a more likely explanation of the data would be that yG subclasses were not present in the common ancestor and that rather a single y-chain gene was present. In the course of speciation, the cow gene underwent a point mutation which resulted in the prolinealanine replacement. Following this event, gene duplication occurred resulting in both y genes having an alanine in position 2. The same argument would best explain the fact that the four human yG subclasses have leucine in position 6, whereas none of the other species do. If this

90

HOWARD M. GREY

argument is valid it would indicate that the subclasses appeared after the Cretaceous period (approximately 70 million years ago). b. yM. A 1 9 s immunoglobulin with the electrophoretic mobility of human yM has been described in each of the mammalian species listed in Table V. Besides having specific antigenic determinants and having mercaptoethanol sensitivity typical of yM antibody, little biochemical characterization of this immunoglobulin has been reported except in rabbit, mouse and horse. In 1937, Heidelberger and Pederson first demonstrated that a large fraction of horse antipneumococcal antibody was associated with 19s antibodies having a molecular weight of approximately 1million. Similar values were obtained for the macroglobulin antibodies in the cow and pig (Kabat, 1939). In the mouse, monoclonal yM proteins have been described in animals with leukemia (Clausen et al., 1960; Rask-Nielsen et al., 1960; Vaerman et al., 1963) as well as with plasma cell tumors ( McEntire et al., 1965). In the latter case the macroglobulin was associated (in the mouse) with the rare A-chain type. Whether this light-chain type is the predominant type for normal mouse yM is not known at present. As in the human, the mouse yM has a high carbohydrate content of about 12%,and the p chain has a molecular weight of approximately 70,000 as measured by gel filtration of completely reduced and alkylated protein (Grey, unpubl., 1968). Lamm and Small (1966) have studied the polypeptide chain structure of rabbit yM. Their results indicated a molecular weight of 850 to 900,OOO for the intact yM, and 70,000 for the p chain. The p heavy chain contained 9% hexose and 3.3% hexosamine (compared with 1.4% hexose and 1.5% hexosamine for y chains ). Rabbit yM antibody has also been extensively studied in an effort to determine the number of antibody-combining sites per molecule. Initial studies using purified rabbit antibody to p-iodobenzenearsonate indicated the presence of five binding sites per 19s pentamer and that each 7s monomeric subunit had a single combining site (Onoue et al., 1965). Similar studies by others have confirmed these findings (Frank and Humphrey, 1968; Metzger, 1967; Schrohenloher and Barry, 1968; Voss and Eisen, 1968). Since the physicochemical studies mentioned previously strongly suggest that yM subunits have a polypeptide chain structure similar to that of yG, being composed of two H and two L chains, and since similar studies with yG have indicated two combining sites per molecule, the dilemma has arisen of explaining a univalent yM subunit in the context of the four polypeptide chain models. Postulates based on the presence of a second site which is sterically hindered from expressing itself, or on the presence of a second site specific for an unrelated antigen

PHYLOGENY OF IMMUNOGLOBULINS

91

have been offered. More recently, however, there have been two studies which offered data indicating that yM antibodies have ten rather than five combining sites. Human yM anitbodies to Salmonellu 0 antigen studied by equilibrium dialysis using a tetrasaccharide as hapten, showed a valency of 10 (Merler et al., 1968). Onoue et al. (1968b) have also reinvestigated the valency of rabbit yM antibodies using an azonaphthalene sulfonate hapten. This hapten induced the formation of antibody with higher affinity than previously available, thereby allowing a greater degree of combining site saturation in the presence of excess hapten. These studies suggest the presence of two populations of combining sites, one of high affinity ( K lo7 litedmole) and one of low affinity ( K lo5 litedmole). Data were presented to suggest that there was one low and one high affinity site per subunit. If these experiments can be confirmed it would appear necessary to revise certain presently held concepts regarding the symmetry of the arrangement and/or primary sequence of the polypeptide chains present in individual immunoglobulin molecules. c. yA. The yA globulins have been identified with assurance in only a few nonhuman mammalian species. The major difficulties that have been encountered stem from the fact that yA may be present in the serum in extremely low concentration in some species and that, unlike yM, the physicochemical characteristics of the serum yA are not sdciently unique to permit easy isolation and characterization of the protein. A further complicating factor is that many species contain yl immunoglobulins which have an electrophoretic mobility very similar to that described for yA, so that identification of yA on the basis of its appearance in immunoelectrophoresis is impossible. The occurrence of myeloma proteins, as well as the selective secretion of yA into colostrum and other external secretions in certain species, has allowed the yA class to be identsed with assurance in three speciesmouse, rabbit, and dog. In the dog, an electrophoretically fast migrating 10s myeloma protein has been described which is similar to a protein present in the serum and colostrum of the dog. On the basis of these findings as well as other physical studies which are in keeping with yA proteins, dog yA has been identiiied (Rockey and Schwartzman, 1967). Independent of these studies and on the basis of antigenic cross-reactivity with human yA, dog serum and colostral yA have been identified by Vaerman and Heremans (1968). This latter method of interspecies antigenic cross-reactivity should be quite useful in identifying this, as well as other immunoglobulins in mammalian species in which more direct characterization is impossible.

92

HOWARD

at. GREY

In the rabbit, a protein has been isolated from the colostrum which is homologous to human colostral yA (Cebra and Robbins, 1966). Like the human protein, the rabbit colostral yA is an 11S polymer and is associated with an antigenically distinct secretory piece which is found in the colostrum but not in the serum. The heavy chains obtained from this protein have a molecular weight of approximately 65,000 (Cebra and Small, 1967). An antigenically related protein has been identified in rabbit serum in very low concentration. Mouse yA has been characterized by studies on myeloma proteins. Approximately one-half of plasmacytomas induced in Balb/C mice by the injection of mineral oil produce yA proteins. These proteins have the characteristic size heterogeneity that human yA myeloma proteins possess, with sedimentation constants of 7 to 15 S (Fahey et al., 1964a). Mouse yA has a carbohydrate content of about 8% which is similar to that of human yA, as well as having a very similar C-terminal tripeptide of Ile-Cys-Tyr ( See Table IV) . Antigenically related proteins have also been identified in normal mouse serum and colostrum. Unlike the human in which the normal serum yA is predominantly in the form of the 7 S monomer, approximately half of the yA in the mouse is present in polymeric form (Grey, unpublished observations). Also, unlike human yA, mouse yA can be split by papain into Fab and Fc fragments which are electrophoretically and antigenically distinct from one another ( Fahey, 1963; Askonas and Fahey, 1962). With regard to its polypeptide chain structure, mouse yA appears to be closely related to the human yA2 subclass in that like human yA2 (and yA3), mouse yA proteins lack L-H disuEde bonds and a large proportion of the light chains are present as L L disul6de-bound dimers, The molecular weight of the mouse 7 s yA monomer has been reported to be 12&130,000 (Eisen et al., 1968). The light chains are of the same size as human light chains (22-23,OOO) so that the molecular weight of the heavy chains should, according to these figures be approximately 40,000. Gel filtration of partially reduced and alkylated yA seemed to substantiate this in that mouse a chains were eIuted after human y chains. However, gel filtration of completely reduced and alkylated mouse a chains in 8 M urea suggest that mouse a chains are of the same size as human y chains (i.e., 5055,000) (Grey, unpublished observations; Seki et al., 1968) Obviously, further studies on the molecular weights of the intact protein as well as its polypeptide chains are required to clarify these apparent discrepancies. Although no yA subclasses have been identified in the mouse, a structural variant has been observed. A few myeloma proteins have been found which, although

.

PHYLOGENY OF IMMUNOGLOBULINS

93

antigenically related to yA, are structurally unique in that they have a 3.9 S sedimentation constant, and large quantities of these proteins are excreted in the urine. These proteins possess both L chains as well as H chains and are thought to represent L - H half-molecules (Lieberman et al., 1968; Seki et al., 1968). The heavy chains of one of these proteins were found to have ;a molecular weight of approximately 40,000 by gel filtration of completely reduced and alkylated protein. Also, fingerprint comparisons between the heavy chains of 3.9 S and 7 S yA proteins indicated that the 3 . 9 s N chains lacked several peptides found in all 7 s a chains. Unlike the 7 s yA, the L and H chains in the 3 . 9 s proteins are disulfide-bonded to one another. These data would all seem to suggest rather marked structural differences between these two types of yA proteins. Whether the 3.9 S yA variant represents a yA subclass which is also present in normal mouse serum is not known at present. Recently, several mouse yA proteins having antibody-like activity have been described (Cohn, 1967; Potter and Leon, 1968; Eisen et al., 1968; Schubert et al., 1968). Numerous ligands have been shown to react with these proteins, but most of them demonstrate binding of relatively low affinity, Studies on one high-a5nity anti-DNP yA myeloma protein have indicated that this protein has a single combining site. It has a binding constant of Z x lo7 liter/mole and shows no evidence of lowaffinity binding sites as described for rabbit yM antibodies (Eisen et al., 1968). Thus far only light chains have been shown to participate in the binding of the DNP ligand ( Metzger and Potter, 1968). G. VERTEBRATE I J G H T CHAINS Antigenic and peptide analyses and, subsequently, amino acid sequence studies have established the presence of two basic types of light chains, K and A, in human immunoglobulins. Although antigenic studies show no cross-reactions and peptide mapping reveals only one peptide that is shared between K and X chains, sequence analysis has demonstrated signscant hornology between the two types, strongly suggesting a common evolutionary source (Putnam et al., 1967). Approximately 40% of the residues are common to both light-chain types. This is similar to the degree of homology shared by the a and /3 chains of human hemoglobin, where 45% of residues are common. Besides the 40%identity of residues in K and X chains, their homology is also evident from the nearly identical positioning of the intra- and interchain disulfide bonds. The extent of homoIogy between K and X is similar in both parts of the chain, the variable N-terminal half and constant C-terminal half. There is antigenic as well as chemical evidence that K- and A-type

94

HOWARD M. GREY

light chains are present throughout the mammalian class and may be present in lower vertebrates as well. Serologic studies and peptide fingerprinting have demonstrated the presence of two types of light chains in nonhuman primates, mouse, rabbit, and guinea pigs (Doolittle and Astrin, 1 x 7 ; Nussennveig and Benacerraf, 1966; McIntire et aZ., 1965; Nussenzweig et al., 1966; Appella et aZ., 1968). Extensive chemical studies have been performed in @teen mammalian and three avian species by Hood and co-workers (Hood et al., 1966, 1967). These investigators analyzed the C-terminal and N-terminal regions of light chains isolated from normal pooled 7-globulin. Human h chains have a blocked N-terminal residue in the form of a cyclized glutamyl residue, whereas K chains contain aspartic or glutamic acid as amino terminal residue. Also in some instances it was possible to isolate and sequence the N-terminal peptide from those chains with blocked N-terminals so that the homology with the human chains could be more critically evaluated. The carboxyterminal peptides were also isolated in several instances, and the quantitative recovery of the C-terminal residues measured. (Cysteine would be expected and was found for those chains believed on the basis of Nterminal studies to be K type, except in the case of the pig where the C-terminal tripeptide was -Cys-Glu-Ala.) In the case of A-type chains, serine was the most common C-terminus (human, rabbit, guinea pig, dog, whale, and mule) with the pig and horse having alanine and proline, respectively. In all h chains studied, cysteine was the penultimate amino

Turkey Duck Chicken Rabbit Mouse Rat Guinea Pig BabwnHumon Dog Cat

Legomorphi

Rodents

Avianr

Mammals

1- ' et

FIG.5. Distribution of

al., 1967.)

Primate8

Mink

Pig Bovine Sheep Horse

A r liodw t yls Per i sso doc t y Is

Carnivores

K

Chains

X Chains Reptiles K

and 1 light chains among various species. (From Hood

95

PHYLOGENY OF IMMUNOGLOBULINS

acid residue. On the basis of these criteria it was possible to determine whether K and/or A light chains were present in a given species and what the relative proportions of the two chains were. These data are summarized in Fig. 5. In all species except mink and horse there was evidence for two types of light chains. The relative proportion of the two chains varied over a wide range from almost exclusive K chains to the reverse situation where only , i chains were found. In general, phylogenetically closely related species tended to have similar K : ratios. The reason for these drastic differences in the expression of the genes responsible for production of K and A chains is unknown. N-terminal sequences have been performed on two lower vertebrate species-the shark and the paddlefish. In the dogfish and nurse shark, blocked N-terminals predominate ( Hood, personal communication). In the leopard shark there was a 25%yield of aspartic and an 8% yield of glutamic acid, so that at least one-third of the light chains in this species are K type (Suran and Papermaster, 1967). In the paddlefish, only a 9% yield of glutamic acid was obtained, approximately 90%of light chains presumably having a blocked, &like, N-terminus (Pollara et al., 19S8). Table VII shows the sequence data obtained for both heavy and light chains of the shark and paddlefish in comparison to human K and p chains. The two most striking findings are (1) the high degree of homology between the fish light chains and human K chains and (2) the near identity in sequence of the H and L chains obtained from the fish immunoglobulins. On the other hand, the fish heavy chains show a much lower degree of N-TERMINAL SEQUEHCES OF H

TABLE VII L CHAINSFROM SHARKAND PADDLEFISH

AND

Leopard shark

L

ASP Glu

Ile

Val

Paddlefish

L

Asp

Ile

Val

Asp

Ile

Val Gln

Human K chain Leopard shark

H

Glu

Ile

Val

Paddlefish

H

Asp

Ile

Val

Human p

Glp

Ser

Val

Human

Glp

Vd

Gln Thr

y

Leu Val GlY Ile Leu

Leu Met Val Leu Ile Leu

Thr

Thr Thr

Gln

Thr

Glu

Thr

Ma

Leu

ASP Glu

Leu

Arg

Glu

96

HOWARD M. GREY

homology to either human p or y chains. The presence of aspartic acid at the N-terminus of these H chains represents the only instances in which immunoglobulin heavy chains have not been described as having a blocked N-terminal residue due to the presence of pyrolidone carboxylic acid. IV.

Concluding Remarks

Although much of the data is of fragmentary nature, there is enough available information to suggest a sequence of events involving multiple gene duplications that might have occurred during the evolution of the immunoglobulins. A phylogenetic tree based on these data is shown in Fig. 6. On the basis of the homology in primary sequence and disulfide linkages between the variable and constant regions of the light chains, it has been postulated that a primitive immunoglobulin gene coded for a peptide chain equal in length to one-half of a light chain (11-12,OOO mol. wt. (Singer and Doolittle, 1966; Hill et d.,1966a). Since the most primitive vertebrates extant have immunoglobulins consisting of fully developed heavy and light chains the origin of the primitive half light chain is placed in the prevertebrate era. Through partial gene duplication, a gene capable of coding for a peptide chain the length of a light chain evolved and, subsequent to this, other gene duplications occurred so that two light chain genes evolved which eventually diverged to form the K and X genes. By partial gene duplication, a gene capable of coding for a peptide chain equal to twice the length of a light chain was formed.

b.

\\v$ btPrimitive heavv (Z50,OOO M W protein)

"li I

chain

Primitive L chain ("25,000 M.W. protein) L chain precursor ("12,000 M.W protein)

FIG.6. Evolution of immunoglobulin genes.

F’HYLOGENY OF IMMUNOGLOBULINS

97

This was the primitive heavy-chain gene. The studies on cyclostome and shark immunoglobulins would suggest that this primitive heavy chain was homologous to what is recognized in mammals as the p chain. The available data would suggest that the gene duplication involving the divergence of light chains into K and h occurred by the Ordovician period or before. The first evidence of further gene duplication and divergence occurs in teleosts and amphibians where another heavy-chain gene emerged which may be homologous to mammalian yG. Since yM and yA appear to be more closely related to one another than to yG, it is postulated that yA was a result of a later gene duplication involving the p chain. There is not enough structural data available on the 8 or e chains to even guess at their phylogenetic relationships with the other heavychain classes, It is supposed on the basis of the data available on the y-chain subclasses that these evolved by gene duplication of the y chain after the divergence of the major mammalian orders, approximately 70 million years ago. This scheme does not provide any information regarding the evolution of the variability which is found in the N-terminal portion of heavy and light chains. The answer to this problem is the crux of the antibody problem. By what genetic mechanism can an individual make what appears to be an almost infinite variety of antibody molecules from the limited quantity of deoxyribonucleic acid at his disposal? Several schemes have been presented to answer this question (Cohen and Milstein, 1967; Lennox and Cohn, 1967). REFERENCES

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Slow Reacting Substance of Anaphylaxis

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ROBERT P ORANGE1 AND K FRANK AUSTEN

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Deporfmenf of Medicine. Horvard Medical School of the Robert B Brighom Horpifol. Borfon. Morrachureftr

I. Introduction . . . . . . . . . . . . . I1 Physical and Chemical Properties . . . . . . . . . A. Stability . . . . . . . . . . . . . B . Solubility Characteristics . . . . . . . . . C. Adsorption Characteristics . . . . . . . . . D. Electrophoretic Mobility . . . . . . . . . E . Chromatographic Separation . . . . . . . . . F Slow Reacting Substance of Anaphylaxis and Neuraminic Acid . . . . . . . . . . . . Glycosides 111. Pharmacology . . . . . . . . . . . . . A. Bioassay . . . . . . . . . . . . . B Characterization of Slow Reacting Substance of Anaphylaxis by Differential Bioassay . . . . . . . . . . C. Role of Slow Reacting Substance of Anaphylaxis in Antigen-Induced . . . . . . . . . . Bronchoconstriction D Permeability Studies . . . . . . . . . . E. Other Pharmacological Effects . . . . . . . . IV . Immunological Release of Slow Reacting Substance of Anaphylaxis intheRat . . . . . . . . . . . . . A . Introduction . . . . . . . . . . . . B Immunoglobulins Involved in the Antigen-Induced Release of Slow . . . . Reacting Substance of Anaphylaxis of the Rat . C. Cellular Elemenis Involved in the Immunological Release of Slow . . . . Reacting Substance of Anaphylaxis of the Rat . D In Vioo Inhibition of the Immunological Release of Slow Reacting . Substance of Anaphylaxis of the Rat by Diethylcarbamazine . E Dissociation of the Immunological Release of Slow Reacting Substance of Anaphylaxis of the Rat and Histamine . . . . F Passive Cutaneous Anaphylaxis in the Rat . . . . . . V. Immunological Release of Slow Reacting Substance of Anaphylaxis . . . . . . . . in Other Species Including Man . A . Guinea Pig . . . . . . . . . . . . B. Primates Including Man . . . . . . . . . . VI Concluding Comments . . . . . . . . . . . References . . . . . . . . . . . . .

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1M

108 109 109 110 111 112 112 112 114 116 118 119 119 119 121 126 128 132 136 137 137 139 140 141

* Helen Hay Whitney ]?ellow (formerly U.S.Public Health Service Postdoctoral Trainee supported by Training Grant AM-05076 from NIAMD) 105

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I.

Introduction

The chemical mediators involved in the pathogenesis of immediatetype hypersensitivity have been the subject of extensive investigations since the initial observations of Dale and Laidlaw (1910) concerning the release of histamine. However, almost 60 years later, our knowledge of the mechanisms of release, modes of action, and number of these mediators is still fragmentary. The chemical mediators of anaphylaxis presently recognized include the vasoactive amines, histamine and serotonin, the nonapeptide, bradykinin, and slow reacting substance of anaphylaxis (SRS-A). This latter mediator is the least well studied because its chemical structure is unknown and the available bioassay is both cumbersome and difficult to quantitate. The purpose of this review is to consider the evidence that slow reacting substance of anaphylaxis may have considerable biological significance in immunological tissue injury. The term “slow reacting substance” is a descriptive one referring to substances of unidentified chemical composition which possess certain common characteristics. These compounds produce a slow, prolonged contraction of only certain isolated smooth muscle preparations. In general, slow reacting substances are not stored in tissues in appreciable quantity and, apparently, must be both formed and released from tissues or cells following some form of eliciting stimulus. A further feature of these slow reacting substances is that they appear to be somewhat lipidsoluble. Since unknown chemical composition, certain pharmacological activities, a requirement for both formation and release from tissues, and lipid solubility are the accepted general characteristics of an SRS, the term probably refers to a variety of otherwise unrelated compounds, Feldberg and Kellaway (1938) originally coined the term SRS to describe a smooth muscle-contracting activity which appeared in the effluent of the perfused lungs of guinea pigs and cats following treatment with cobra venom. Kellaway and Trethewie (1940) noted the appearance of a substance possessing similar pharmacological characteristics in the effluent of sensitized guinea pig lungs following challenge with specific antigen in uitro. Later, Brocklehurst ( 1953) differentiated the latter substance from histamine by demonstrating that the contraction it produced on the isolated guinea pig ileum was not inhibited when an antihistamine was added to the organ bath. Over the next 10 years, the formation and release of SRS from tissues or isolated cells was described using three different releasing mechanisms: treatment with phospholipase A or bee and snake venoms rich in this enzyme (Vogt, 1958; Hogberg and Uvniis, 1960; Middleton and Phillips, 1964);challenge with compound 48/80, the condensation product of pmethoxyphenyl ethylmethylamine and for-

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maldehyde, or related natural and synthetic polymers (Uvnas,1963; Chakravarty et d.,19S9); and antigen-antibody interaction ( Austen and Humphrey, 1962; Brocklehurst, 1960; Chakravarty, 1960). Release has been studied in a variety of tissues including egg yolk (Vogt, 1958), guinea pig, rat, monkey, and human lungs (Brocklehurst, 1960), cats' paws (Chakravarty et al., 1959), and isolated rat mast cell suspensions (Uvnas, 1963). Brocklehurst (1960) suggested that the SRS recovered from shocked guinea pig lungs be designated slow reacting substance of anaphylaxis to differentiate this material from that recovered by nonimmunological mechanisms. The term SRS-A now generally refers to any slow reacting substance released immunologically. Orange and Austen (1969) have suggested that a suffix be used to denote the species of origin; thus the SRS released following antigen-antibody interaction in the rat peritoneal cavity is designated SRS-A'St. At present, there is no way of knowing whether the SRS-A recovered from different species is a single substance or whether it represents a family of closely related compounds. The term SRS could be extended to a variety of other lipid-soluble acids, including the prostaglandins, Darmstoff, irin, G-acid, and other possibly related substances. These biologically active lipids have been the subjects of extensive reviews (Vogt, 1958; Pickles, 1967; Bergstrom, 1966, 1967; Ambache, 1959), and they will not be considered here. Vogt (1969) has recently presented evidence to indicate that the slow reacting materials released by nonimmunological means consist of prostaglandins and nonspecifically oxidized fatty acids. This review will be concerned with the physicochemical and pharmacological properties of SRS-A and the immunological mechanisms involved in the formation and release of SRS-A in different species. II.

Physical and Chemical Properties

Although SRS-A has been a subject of investigation in several different laboratories for over 30 years, the precise chemical structure of this compound( s ) has still not been elucidated. Several difficulties have been encountered during attempts to isolate and characterize this material: most preparations of SRS-A are very labile and are lost on storage; the amount of SRS-A recovered from perfused, shocked lung preparations is too small to permit the utilization of common preparative techniques; SRS-A adsorbs or binds to proteins and phospholipids, and thus spot tests may yield misleading results; and until recently, the appropriate lipid chemistry techniques were not available. Further difEculty may arise from the fact that the fluid recovered from organs or cells subsequent to

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ROBERT P. ORANGE AKD K. FRANK AUSTEN

antigen-antibody interaction may contain several smooth muscle-contracting principles, and thus the product of every step in an isolation procedure may have to be rigorously tested on several different smooth muscle preparations to c o n h that the substance extracted is SRS-A. Nonetheless, the works of Chakravarty ( 1959),Brocklehurst ( 1962), and Anggard and his co-workers (1963)have more clearly defined some of the physical and chemical properties of SRS-A.

A. STAB~ITY Guinea pig SRS-A (SRS-Agp) as it appears in anaphylactic fluid is quite labile at room temperature; at pH 7.5-9.5, a 50%loss in activity is observed within 24 hours, the inactivation being somewhat less marked at lower pH (Chakravarty, 1959). Crude SRS-Aratstored at -70°C. in Tyrode's solution loses about 50%of its activity following a single freezing and thawing. When SRS-A'st is extracted in 8ofg ethanol, evaporated to dryness, and resuspended in distilled water at an acid or alkaline pH, subsequent boiling (Fig. 1) reveals lability in acid and stability in alkali. More than 75%of the activity is lost in 60 minutes in 0.05 N HCl whereas only a 10% loss occurs in 0.05 N NaOH. Chakravarty (1959) observed a similar pH effect with SRS-Agp and further noted that boiling in the presM glutathione) or an oxidizing ence of either a reducing agent ( M periodic acid) destroyed biological activity. agent (4.4 x Peroxides appear to destroy rapidly the activity of SRS-Am, but halogenation in the presence of a palladium catalyst is without effect (Brock100 X

00

60

40 20

I0

10

20

30

40

50

60

70

MINUTES

FIG. 1. Stability of slow reacting substance of anaphylaxis of the rat (SRSArst) in distilled water (open circles) or at acid (crosses) or alkaline (filled circles) pH to boiling.

SLOW REACTING SUBSTANCE OF ANAPHYLAXIS

109

lehurst, 1962). Although SRS-A appears to be quite labile on storage in solution, freeze-dried samples, or material extracted in 80%ethanol and evaporated to dryness, retain undiminished activity for several months. This observation has permitted the accumulation of sufficient material to initiate further steps in identification. Slow reacting substance of anaphylaxis has proved resistant to destruction by a variety of enzymes including trypsin, chymotrypsin, pepsin and activated papain ( Brocklehurst, 1962), carboxypeptidase, leucine aminopeptidase, phospholipases A, B, C, and D ( Anggard et al., 1963), and pronase (Orange and Austen, 1969). B. S O L U B I LCHARACTERISTICS ~ Slow reacting substance of anaphylaxis appears to be soluble in water or 80%methanol or ethanol, but only slightly soluble in propanol or watersaturated n-butanol (Chakravarty, 1959; Brocklehurst, 1962). The SRSArat extracted in 80%ethanol and evaporated to dryness appears to form a micellar suspension in distilled water; the suspension clears when an equal volume of pyridjne is added. Slow reacting substance of anaphylaxis is not soluble in acetone, chloroform, or chloroform-methanol 1 :1 or 1:2 (vol./vol.). When an ethanol-extracted preparation of SRS-Arat is dissolved in theoretical upper phase (Folch et al., 1957) and then washed with theoretical lower phase, over 80%of the SRS-Arat activity is found in the upper phase fraction, and the remainder is associated with the interphase. The lower phase containing most of the neutral lipid and phospholipid is without activity. Slow reacting substance of anaphylaxis behaves as a hydroxy acid in that following acidification to pH 23,it will pass into organic solvents, such as diethyl ether, and it may then be recovered from the ether phase by partition with alkaline water (pH 8.0) ( Chakravarty, 1959; Anggard et al., 1963).

c.

ADSORPTION CHARACTERISTICS

When the effluent of perfused, shocked, guinea pig lungs is treated with 20 volumes of acetone, all the SRS-AgP activity is removed from solution and it may be recovered from the precipitate by resuspending the latter in Tyrode's buffer (Brocklehurst, 1962). When perfusate containing SRS-AgP is placed in a dialysis sac and dialyzed against an equal volume of Tyrode's solution for a few hours, little SRS-Am is recovered from the dialyzate; however, if the perfusate is first treated with trypsin or 80%ethanol, the SRS-AgP becomes equally distributed inside and outside the sac (Middleton and Phillips, 1964; Brocklehurst, 1962). Figure 2 describes the time course of loss of activity of an ethanol-extracted prepara-

110

ROBERT P. ORANGE AND K. FRANK AUSTEN

0

10

20

30

40

50

60

70

MINUTES

FIG. 2. Apparent loss of slow reacting substance of anaphylaxis of the rat

(SRS-A"') activity upon incubation at 37°C.in buffer (open circles), 1% (filled

circles), 10%(triangles), or 100% (crosses) normal rat serum.

tion of SRS-Aratincubated at 37°C. with varying concentrations of normal rat serum. Almost all observed loss of SRS-Arar activity is noted within the first 5 minutes of incubation, especially at higher serum concentrations, with only a slight further reduction in activity over the next 55 minutes. This suggests loss by binding to some factor in normal rat serum rather than enzymatic degradation. Further, when SRS-A'"' is incubated at 37°C. for 60 minutes in 503 normal rat serum, the 80%reduction in activity observed is more apparent than real, since, when this sample is treated with 80%ethanol to precipitate the serum proteins, over 75%of the SRS-Arat activity is recovered in the supernatant. Comparison of euglobulin, pseudoglobulin, and albumin-rich fractions of normal rat serum revealed that the pseudoglobulin fraction was most active in binding SRS-Aratso as to mask its biological activity. Brocklehurst (1962) and Berry and Collier (1984)have utilized an adsorption method to obtain a preparation of SRS-AgP free of histamine and excessive salt. The SRS-AgP is adsorbed onto partially inactivated charcoal from which it is then displaced by water-saturated n-butanol or dilute alkali.

D. ELECTROPHORETIC MOBILITY Electrophoresis in a supporting medium has never been a successful preparative technique for the purification of SRS-A possibly because of adsorption problems. Brocklehurst ( 1962 ) using electrophoresis in a sucrosewater density gradient found that charcoal-purified SRS-A@

SLOW REACXINC SUBSTANCE OF ANAPHYLAXIS

111

migrated anodally at pH 8.0 in a single band. Uvnas (1963) purified SRS-AgP by silicic acid chromatography; he observed that, on further anion exchange chromatography or column electrophoresis, SRS-Am appeared to have acid properties. These very preliminary studies as well as the solubility characteristics of SRS-A suggest that the molecule may be a highly polar hydroxy acid. E. CHROMATOGRAPHIC SEPARATION The chromatographic isolation of SRS-A has been attempted using paper chromatography, silicic acid column chromatography, and more recently, thin layer chromatography. Chakravarty ( 1959) purified SRSAgp on one-dimensional paper chromatography using n-propanolammonia-water (60:30:10, vol./vol.) as the developing solvent in a nitrogen atmosphere. The active spot had an 23, value 0.6-0.7 and it did not stain for phosphate, The active spot was identified as an acid region by staining with bromthymol blue and, following acid hydrolysis, yielded a ninhydrin-positive reaction. Anggard et al. (1963) prepared ethanol-extracted SRS-AgP and, following several washes in acetone, applied the material to a silicic acid column. The elution was started with chloroform followed by chloroformmethanol, the concentration of methanol being increased in a stepwise fashion. The active material recovered was evaporated to dryness, washed with ether, and extracted into ether at pH 3.0. This material was rechromatographed on a second silicic acid column, the elution beginning with ether-chloroform ( 1:1, vol./vol. ) , then chloroform-methanol as before, and the active fractions were evaporated. The active material was then washed with chloroform to remove any phosphatides, the SRS-ASP remaining in the insoluble fraction. The final preparation was readily soluble in alkaline water and gave a negative spot test for phosphorus, but a positive ninhydrin reaction and a positive Ehrlich reaction for hexosamines. Passage of guinea pig lung perfusate containing SRS-AgP through Dowex 50 (H’ ion form) and Dowex 1-acetate columns is associated with a 9O!Z loss in the SRS-Am activity (Cirstea et al., 1967). Chromatographic separation of an ethanol-extracted preparation of SRS-knt has recently been achieved on thin layer chromatography (Orange et al., 1969a). Using “basic” silica gel G (Skipski et aL, 1962) as the adsorbent and a solvent system comprised of chloroformmethanol-aqueous 2.5 N ammonia (55:35:11, vol./vol.), a single spot with an 231 value 0.6-0.8 has been recovered possessing SRS-AraCactivity on bioassay. This fraction yielded a positive anthrone reaction for neutral hexoses (Roe, 1955); it had no sialic acid as determined by the thio-

112

ROBERT P. ORANGE AND K. FRANK AUSTEN

barbituric acid analysis (Warren, 1959),and it had negligible phosphorus as assessed by microanalysis (Lowry et al., 1954).When this fraction was reapplied to thin layer plates and the developing system, n-propanolammonia-water (6:3:1,vol./vol.) was used, almost 904: of the biologic activity recovered was found in an area ( R f = 0.5) quite separate from the glucose-containing fraction. Similar Rf values were obtained with both developing solvent systems for SRS-Asp and SRS-Amonkep (Orange et al., 1969~).

F. SLOWREXCI-INGSUBSTANCEOF ANAPHYLAXISAND NEURAMINIC ACID GLYCOSIDES Goadby and Smith (1962) observed that there was an appreciable loss of extractable lipids in guinea pig lungs subsequent to anaphylaxis in uivo or in vitro. These losses were largely prevented by pretreatment with ethanolamine (Smith, 1962). Smith (1962) also noted that the physicochemical characteristics of SRS-AgP were similar to those described for neuraminic acid-a substance found in high concentration in the mucoproteins of the nasal and bronchial secretions of man. Although N-acetylneuraminic acid had no direct pharmacological activity on the isolated guinea pig ileum, a stable methylglycoside of neuraminic acid, methoxyneuraminic acid, did produce an SRS-A-like contraction. Furthermore, there was enough total neuraminic acid in the perfusate of shocked guinea pig lungs to account for all the SRS-A= activity. The possibility that SRS-Am is a mixture of glycosides of neuraminic acid has been re-examined (Cirstea et al., 1967). It was observed that the perfusate of shocked guinea pig lungs rich in SRS-AgP contained about 2.0 p g , of free sialic acid and about 12.0 pg. of total (free plus bound) sialic acid. However, the threshold concentration of sialic acid or its derivatives required for smooth muscle contraction was more than 20 times greater than that found in the perfusate fluid containing appreciable quantities of SRS-Am. Furthermore, although the ratio of free to bound neuraminic acid was increased in the lung tissue after antigen challenge, there was no change in the total sialic acid concentration of the tissue. The postulate that SRS-A is a type of neuraminic acid glycoside is not supported by the present evidence. Ill.

Pharmacology

A. BIOASSAY Since the precise chemical structure of SRS-A is not known, the identification of this material in biological fluids is determined by bioassay on the isolated guinea pig ileum in the presence of 5 x lo-' M atropine and

SLOW RIUCITNC SUBSTANCE OF ANAPHYLAXIS

113

M mepyramine maleate (Brocklehurst, 1953; Austen, 19f39). Using an oxygenated 5.0-ml. organ bath .at 37"C., a 3.0-cm. ileal strip is suspended in Tyrode's solution and the organ bath is brought to a final volume of 5.0 ml. by the addition of test fluid containing SRS-A. This modification avoids the exposure of the smooth muscle preparation to air immediately prior to the addition of the test sample, affording a more constant baseline during the bioassay. A dose-response effect must be established for each sample and a reference sample of SRS-A should be retested at frequent intervals during the assay since the sensitivity of the assay may increase twofold with time. Tachyphylaxis is not observed with SRS-A; during long assays, the smooth muscle may become irritable. The concentration of SRS-A found on bioassay is usually expressed in terms of arbitrary "units" (Brocklehurst, 1960; Berry and Collier, 1964; Stechschulte et aZ., 1967). A unit of SRS-A usually refers to the concentration required to produce a contraction of the guinea pig ileum with an amplitude equivalent to 5.0 mpg. histamine base in that assay. With the availability of a stable ethanol-extracted preparation of SRS-A, a reference standard can be prepared. The typical contraction of the isolated guinea pig ileum produced by an ethanol-extracted preparation of SRS-A'"' is demonstrated in Fig. 3

FIG. 3. Kymograph recording comparing contractions produced by histamine and by slow reacting substance of anaphylaxis of the rat ( SRS-A''t). (From Orange et aZ., 196913.)

114

ROBERT P. ORANGE AND K. FRANK AUSTEN

and compared to that produced by histamine. The guinea pig ileum contracts abruptly on exposure to histamine with virtually no latent period and relaxation occurs with a single washing. The SRS-A's' produces a slow, prolonged contraction of the guinea pig ileum following a latent period of about 10 seconds. The contraction usually reaches a plateau within 1 to 3 minutes and complete relaxation of the ileum is observed only after repeated washings. The subsequent responses of the guinea pig ileum to histamine are potentiated for several cycles; this potentiation appears to be characteristic of a variety of hydroxy acids (Pickles, 1967; Bergstrom et d.,1968; Brocklehurst, 1960). The addition of 1PsM mepyramine maleate abolishes the histamine responses, whereas the contractions produced by SRS-A'"' persist without diminution. B. CHARACTERIZATION OF SLOWREWXKG SUBSTANCE OF ANAPHYLAXIS BY DIFFERENTIAL BIOASSAY Since several substances possessing smooth muscle-contracting activity may be recovered from tissues following antigen-antibody interaction, the identification of SRS-A requires bioassay with several different smooth muscle preparations and the use of selective pharmacological antagonists. For example, the perfusate of sensitized guinea pig lung following antigen challenge contains at least two slow reacting substances separable by silicic acid chromatography (Anggard et al., 1963). The activity eluting with chloroform produces an atypical contraction of the guinea pig ileum with tachyphylaxis and is very active on the rabbit duodenum. A less lipophilic material eluting with chloroform-methanol ( 1:1,vol./vol.) produces a typical SRS-Am contraction of the guinea pig ileum without tachyphylaxis and 400-600 times (by weight) more is required to contract the rabbit duodenum. The starting perfusate was equally active on both smooth muscle preparations. The selection of appropriate smooth muscle preparations for differentiating various chemical mediators has been previously reviewed ( Brocklehurst, 1962, 1967; Ambache, 1966; Weeks et al., 1969;Bergstrom, 1967). The biological activity of SRS-A may be differentiated from that of other chemical mediators by a series of bioassays (Table I ) . SRS-A is readily differentiated from histamine by its ability to contract the guinea pig ileum or human bronchial smooth muscle in the presence of a potent antihistamine. Serotonin contracts the guinea pig ileum with eventual tachyphylaxis, and its action is antagonized by methysergide or BOL-148, neither of which interferes with the action of SRS-A on the ileum; further, in contrast to SRS-A, serotonin contracts the estrous rat uterus but not the human bronchial smooth muscle. Slow reacting sub-

115

SLOW EEACIING SUBSTANCE OF ANAPHYLAXIS

TABLE I CHARACTERIZATION O F SLOW REACTlNQ SCBSTANCE~OF ANAPHYLAXIS BY DIFFERENTIAL BIOASSAY Smooth muscle preparationa

Test material ~

SRS-A Histamine Serotonin Bradykinin Prostaglandins a

+

Guinea pig ileum

+ + a3 + +

Estrous rat uterus ~

-

-

+ + +

Gerbil colon

-

+ +

~~~

Human bronchi

+ +@ -b

= contraction; - = no contraction; a3 = tachyphylaxis. Except the prostoglandin, PGF?,.

stance of anaphylaxis may be distinguished from bradykinin by its lack of activity on both the estrous rat uterus and on the ascending colon of the gerbil and by its resistance to inactivation by various proteolytic enzymes. The gerbil colon is very sensitive to the prostaglandins, but insensitive to histamine ( Ambache, 1966), serotonin, and SRS-A (Orange and Austen, 1969). The prostaglandins, PGE, and PGE2, produce relaxation of the human bronchial smooth muscle, whereas PGF2, produces a contraction of this smooth muscle but with associated tachyphylaxis. However, there is no crossed tachyphylaxis between PGF2, and a charcoal-purified preparation of SRS-Am (Sweatman and Collier, 1968). Hoinochlorcyclizine appears to be capable of reducing the spasmogenic activity of SRS-AgPon the guinea pig ileum in vitro (Kimura et ul., 1960), but it is even more active as an antagonist of serotonin. Although no specific antagonist of SHS-A has been described, SRS-A is readily distinguished from known mediators by its action on the guinea pig ileum in the presence of an antihistamine and its failure to act on the estrous rat uterus and gerbil colon; resistance to destruction by proteolytic enzymes and action in the presence of a serotonin antagonist are additional distinguishing features. The SRS-A recovered from different species may be compared in terms of their biological activity on selected smooth muscle preparations. The SRS-Amankey obtained from monkey lung slices passively sensitized in uitro with human atopic sera and challenged with either specific antigen or a rabbit antiserum directed against IgE (Ishizaka et uZ., 1W9) behaves on bioassay in an identical manner to SRS-A'"' (Table 11). The anaphylactic fluid containing SRS-Amonke' was extracted in 80% ethanol,

116

ROBERT P. ORANGE AND K. FRANK AUSTEN

TABLE I1 COMP.ARISON OF THE BIOLOGICAL ACTIVITY OF SLOW REACTING SUBSTANCE OF ANAPHYLAXIS OF THE RATAND OF THE MONKEY Smooth muscle preparationD ~~~

Material tested (conc./rnl.)

SRS-kst (units) SRS-Amonkey(units) Bradykinin (mpg) PGEI (rnpg) PGF,, ( m a )

~

Guinea pig ileum 1 1

5 50 40

Estrous rat uterus

>50 >50

0.05 5 20

Ascending gerbil colon

>50 >50 4

5 8

a All values refer to the concentrations of test compounds required to produce contractions of equivalent amplitude in that assay. Although 50 units of SRSX did not achieve a significant contraction of the estrous rat uterus or gerbil colon, a higher dose was not employed; the contractions produced by the remaining agents were comparable a t the concentration indicated. Modified from Orange and Austen (1969).

evaporated to dryness, and treated with chymotrypsin as described for SRS-Arat (Orange et aZ., 1969a) before bioassay. These preparations of SRS-A could be clearly distinguished from bradykinin and the prostaglandins, PGE, and PGF,,. C. ROLEOF SLOWREACTINGSUBSTANCE OF ANAPHYLAXIS IN ANTIGEN-INDUCED BRONCHOCONSTRICCION Although Brocklehurst (1962) observed that SRS-AgP did not contract guinea pig tracheobronchial smooth muscle in uitro, Collier (1968) using a slightly different preparation of SRS-Agp has produced evidence to the contrary. Furthermore, using as an experimental model normal and sensitized guinea pigs prepared by the method of Konzett and Rossler (1940) for recording air overflow volume, the possible contributions of histamine, bradykinin, SRS-AXP and the catecholamines to antigeninduced bronchoconstriction in the guinea pig in uiuo have been studied (Berry et aZ., 1963; Berry and Collier, 1964; Collier and James, 1967; Collier, 1968). When normal guinea pigs were injected intravenously (i.v.) with 40 to 160 units of a charcoal-purified preparation of SRS-Asp free of histamine, a marked increase in the resistance of the lungs to inflation was observed; this was not altered by pithing of the spinal cord and crushing of the sympathetic nerves and vagi of the guinea pig (Berry and Collier, 1964) or by treating the SRS-A'@preparation with chymo-

SLOW FEA(;TING SUBSTANCE OF ANAPHYLAXIS

117

trypsin ( Berry et at., 1963). Intravenous injections of histamine or bradykinin were also associated with an increased air overflow volume in the Konzett-Rossler preparation of guinea pigs (Berry and Collier, 1964). By the use of antagonists such as mepyramine maleate and meclofenamate and by the induction of bradykinin tachyphylaxis, it was concluded that histamine contributed more to anaphylactic bronchoconstriction in the guinea pig than did the kinins or SRS-An*. Each mediator appeared to have a characteristic time course of release (Collier and James, 1967). In the Konzett-Rossler model, endogenous catecholamine release may function to ameliorate anaphylactic bronchoconstriction. The i.v. administration of histamine, bradykinin, or SRS-AgP is associated with the release of adrenaline. Further, anaphylaxis in the guinea pig is associated with an increased blood level of adrenaline (Piper et at., 1967), and pretreatment of guinea pigs with P-adrenergic blockers, such as pronethalol or propanalol, potentiates the anaphylactic bronchoconstriction in the guinea pig (Collier and James, 1966, 1967). Although SRS-ASP may contribute to anaphylactic bronchoconstriction in the guinea pig, it does not appear to be the most important mediator in this species. Brocklehurst (1960) presented the first evidence of a possible role for SRS-Ah" in the mediation of antigen-induced bronchoconstriction in the human. The exposure of lung tissue from two asthmatic patients to specific pollen antigen in vitra was associated with the release of SRS-Ahu. When bronchial ring preparations from these patients were suspended in dtro in the presence of an antihistamine, the subsequent addition of the specific allergen resulted in a prolonged contraction of the bronchial ring and the release of SRS-AhUinto the suspending medium. More recently, the antigen-induced release of SRS-Ahufrom normal human lung tissue passively sensitized in vitro has been described (Sheard et al., 1967; Parish, 1967). Herxheimer and Stresemann ( 1963) noted that the inhalation of a crude aerosol preparation of SRS-AgP in a closed circuit spirometer was associated with a reduction in the vital capacity of asthmatic patients, but not in control subjects. Pretreatment of the asthmatic subjects with aspirin, phenazone, amidopyrine, flufenamic acid, or phenylbutazone did not significantly antagonize the effects of the SRS-AgP aerosol ( Herxheimer and Stresemann, 1966). The interpretation of these observations is rendered difficult because the asthmatic bronchiole is hyperactive to a variety of pharmacological agents (Curry and Lowell, 1948; Austen, 1965). However, the prolonged contraction of the human bronchial smooth muscle in vitro produced by SRS-Am in the absence of tachyphylaxis plus the exquisite sensitivity of this smooth muscle to

118

ROBERT P. ORANGE AND K.

FRANK AUSTEN

SRS-AgP (Brocklehurst, 1962) underline the possible significance of a comparable mediator in the bronchospastic symptoms of human asthma and anaphylaxis. Of particular relevance is the recent finding of Ishizaka et al. (1969) that the IgE antibodies present in human atopic sera are capable of sensitizing primate lung tissue in vitro for the subsequent immunological release of both histamine and SRS-Amonke'upon challenge with specific pollen antigen or specific anti-IgE antiserum.

D. PERMEABIL~~Y STUDIES Brocklehurst (1967) stated that large doses of a purified preparation of SRS-AgP produced an increased permeability in skin. Orange and Austen (1969) have studied the permeability-enhancing activity of an ethanol-extracted, chymotrypsin-treated preparation of SRS-Arat in the skin of different species. As shown in Table 111, SRS-Aratappears to effect a marked permeability change in the skin of guinea pigs pretreated with a combination of histamine and serotonin antagonists and the permeability effects appear to occur in a dose-response fashion. The permeabilityenhancing activity of SRS-kst serves as a further criterion for distinguishing this mediator. Pretreatment of guinea pigs with mepyramine maleate TABLE I11 PERMEABILITY STUDIES WITH SLOW REACTING SUBSTANCE OF ANAPHYLAXIS OF TRE RAP ~~

Compound tested SRS-kat (units)

Serotonin (mpg) Histamine ( w g ) Bradybinin (mpg) PGEi ( w g ) PGFk ( w g ) Extracted peritoneal fluidb (dilution)

Concentration per site 125 50 25 5 10 10 10 10 10

1:2

~~

Rat

Guinea pig

1+8X8 1+8X5 1+3X3 Tr4X3 Tr3 X 3 n 3 x 3 Tr3 X 3 1+4X4 1+4X5 1+3X3

4 + 2 0 X 17 3 18 X 15 3 12 x 12 2+9X10 T r l X l Tr2x2 Tr4X4 Tr2 X 3 TrlX2 Tr3x3

+ +

a Each result represents the mean value for 3 animals. All animals were pretreated with mepyramine maleate, 50 mg./kg., and methysergide, 4 mg./kg., i. p., 30 minutea before the experiment. All sites were injected intradermally with 0.1 ml. volumes of test compounds, and the animals were immediately injected i. v. with 1.0 ml. of 0.1% Evan's blue dye. Thirty minutes later, the animals were sacrificed in ether, and the skin was reflected and transilluminated, and the size (mm.) and intensity (04+) of the lesions recorded. Tr = trace. * Peritoneal fluid of rats injected i. p. with homologous antibody but not challenged with antigen. The fluid was extracted as for SRSArstdescribed in the text (Orange and Aueten, 1969).

SLOW REACTING SUBSTANCE OF ANAPHYLAXIS

119

and methysergide blocks the permeability activity of histamine and serotonin, and the prostaglandins, PGE, and PGF,,, do not have marked permeability effects. The presence of bradykinin in the SRS-Aratpreparation is unlikely following treatment with chymotrypsin. The permeability effects of S R S - P ' in the dermis of the rat are not striking. The dermis of monkeys appears to be even more sensitive than that of guinea pigs to SRS-A'"', and the intradennal injection of 10 to 20 units of SRS-A'"' may result not only in increased vascular permeability, but also in hemorrhagic necrosis of skin. The permeability activity of SRS-A'"' suggests a possible role for this chemical mediator in forms of immunological tissue injury other than antigen-induced bronchoconstriction; its role as a contributing permeability factor in passive cutaneous anaphylaxis in rats is presented in a later section.

E. OTHERPHARMACOLOGICAL EFFECTS Although the preparations of SRS-A studied in various laboratories are crude and the amounts available are limited, SRS-A does not appear to be a potent hypotensive agent. No reduction in the blood pressure of rabbits or cats is observed following the i.v. injection of SRS-Asp (Brocklehurst, 1962). The i.v. administration of SRS-AgD to anesthetized guinea pigs is associated with a transient fall in arterial blood pressure (Piper et aZ., 1967), and, in the rat, large doses may result in hemorrhage into the upper intestine but without hypotension (Brocklehurst, 1967). Perfusion of normal guinea pig lungs in vitro with SRS-AgP is associated with the appearance in the lung effluent of a marked increase in the concentration of the prostaglandin, PGEz (Piper and Vane, 1969). The release of prostaglandins in vitro from sensitized guinea pigs upon challenge with specih antigen may be initiated in part or in whole by the concomitant release of SRS-Asp (Piper and Vane, 1969). The interaction of SRS-A with other chemical mediators of anaphylaxis in terms of both release and potentiation of end-organ effects requires further study. IV.

Immunological Release of Slow Reacting Substance of Anaphylaxis in the Rat

A. INTRODUCTION Study of the antigen-induced release of SRS-A'"' was made feasible by the observation of Rapp (1961) that rats injected intraperitoneally (i.p. ) with hyperimmune rabbit antiserum, followed 24 hours later by the i.p. administration of specific antigen, released SRS-A"' intraperitoneally. No differences in the pharmacological properties of SRS-A'"' and SRS-Am (as supplied by W. E. Brocklehurst) were observed. Intraperitoneal histamine release was inconstant except when the latent period was very

120

ROBERT P. ORANGE AND K. FRANK AUSTEN

1

Inhibitors i . p . or i . v.

Normal rat +

"Sensitized" rat min peritoneal ++----* Harvest cell suspension Antigen i.p. or i . v .

_______ time

i.p. Antiserum or fractions

I I

-

_ _ _ bioassays _ _ _ -_ Histamine Serotonin Bradykinin Slow reacting substance (SRS-A)

I

I

t Supernatant

_ ---+

Cells (boiled)

FIG.4. Procedure for the antigen-induced release of chemical mediators into the peritoneal cavity of the rat.

brief, and no release of SRS-A'"' was observed in sensitized rats challenged with specific antigen 2-3 minutes after death. These observations prompted study of the humoral and cellular mechanisms involved in the immunological release of SRS-A'"' using the rat peritoneal cavity as an in uiuo test tube as outlined schematically in Fig. 4. Normal male Sprague-Dawley rats weighing 200-400 gm. are prepared for the antigeninduced release of SRS-Arntby the i.p. injection of heterologous (Rapp, 1961;Orange et aZ., 1967), homologous (Stechschulte et al., 1967; Orange et al., 1968a), or fractionated (Morse et al., 1W8) antisera. Following an optimal latent period of 2 to 4 hours (Stechschulte et aZ., 1967), the animals are challenged by the i.p. or i.v. administration of specific antigen. Exactly 5 minutes later, the rats are stunned, exsanguinated, and the abdominal wall incised and reflected. The peritoneal fluid is recovered using siliconized Pasteur pipettes, and the free peritoneal cells are sedimented by gentle centrifugation ( 150g x 4 minutes). The supernatants are decanted into iced polypropylene tubes, and the cell buttons are resuspended in 3.0 ml. of Tyrode's solution and boiled for 8 minutes to extract the residual cellular histamine. The residual cellular histamine concentration is a function of the presence and number of free peritoneal mast cells. The samples are then assayed on the isolated guinea pig ileum in the presence of atropine sulfate and mepyramine maleate for the presence of SRS-A'"'. Representative samples are assayed on the guinea pig ileum in the absence of mepyramine to determine the con-

SLOW NEACTING SUBSTANCE OF ANAPHYLAXIS

121

centration of histamine and on the estrous rat uterus in the presence and absence of methysergide to establish the levels of bradykinin and serotonin. Pharmacological agents tested for their ability to inhibit the antigen-induced release of SRS-Arat are administered 1 0 3 0 seconds before the antigen and by the same route as that employed for the antigen in that experiment. B. IMMUNOGLOBULINS INVOLVED IN THE ANTIGEN-INDUCED RELEASE OF SLOWREACTING SUBSTANCEOF ANAPHYLAXIS OF THE RAT The initial demonstration in the guinea pig (Ovary et al., 1963; White et al., 1963; Bloch et al., 1963) that physicochemically different homologous immunoglobulins, 7 S yl and 7 S yz, participate in distinctly different biological phenomena, i.e., passive cutaneous anaphylaxis (PCA ) and the Arthus reaction, respectively, led to an awareness of the differing biological properties of the various antibody classes. In the guinea pig, in uitro studies demonstrated that 7 S yl antibodies mediated histamine release (Baker et al., 1964), whereas 7 S y2 antibodies interacted with antigen so as to activate the complement system. In the rat, the homologous immunoglobulins that interact with specific antigen to activate the complement system were again differentiated from those involved in mediating passive cutaneous anaphylaxis (Binaghi et al., 1964) and histamine release (Austen et al., 19s5). Furthermore, the mast cell esterase activated and required for histamine release was distinguished by its inhibition profile using the phosphonate esters from the esteratic form of the activated first component of rat complement ( C l r n t ) (Becker and Austen, 1966). Further consideration of the biological properties of rat antibodies is contingent upon a more precise definition of the various rat immunoglobulin classes. It is now apparent that at least five classes of immunoglobulins may be identified in the sera of immunized rats; using immunoand radioimmunoelectrophoresis, four of these immunoglobulin classes are depicted in Fig, 5 and in an accompanying schematic diagram (Bloch et al., 1968). Following electrophoresis in agar gel, four precipitin bands were developed with a rabbit antiserum directed against rat 7-globulin, and these lines were termed rat IgGa, IgGb, IgA, and IgM. The addition of 1251-labeled hapten-conjugated antigen [ 2,4-dinitrophenyl ( DNP )bovine serum albumin (BSA)] to the troughs and subsequent radioautography revealed antigen-binding to all four immunoglobulin precipitin arcs. The protein designated I g M migrates with thc. /3-globulins on electrophoresis, appears in the 19 S peak on Sephadex (2-200 gel filtration, is eluted from DE5%cellulose columns with 2.0 hi NaCI, and demon-

122

ROBERT P. ORANGE AND K. FRANK AUSTEN

\ 0

IgGb

’

\

FIG.5. Immuno- and radioimmunoelectrophoretic analysis and schematic drawing of precipitin arcs developed by a rabbit antiserum directed against rat y-globulin. ( From Bloch et al., 1968.)

strates antigen-binding for about 5 weeks following immunization. The protein identified as IgA also has a “fast” electrophoretic mobility, is present in the 7 s peak of a Sephadex G-200 gel filtration column, is eluted from DE52-cellulose columns by buffers of higher ionic strength and lower p H than those required for the bulk of IgG, has a higher carbohydrate content than fractions containing IgG ( Binaghi and Sarandon de Merlo, 1966), and appears to be decreased or absent in rats undergoing neonatal thymectomy ( Arnason et al., 1964). The proteins designated IgGa and IgGb have a slower electrophoretic mobility, appear in highest concentration in the 7 S peak on Sephadex G-200 gel filtration, And are eluted in the first peak on DE52-cellulose chromatography, although the IgGb elution is slightly retarded. These immunoglobulins have a carbohydrate content similar to that of the IgG immunoglobulins of other species (Binaghi and Sarandon de Merlo, 1966), but they possess

SLOW REACIlNG SUBSTANCE OF ANAPHYLAXIS

123

some antigenic differences in their F, fragments (Nussenzweig and Binaghi, 1965). These four immunoglobulin classes have been identified in the sera of twelve different strains of rats (Bloch et al., 1968). A fifth immunoglobulin designated rat homocytotropic ( Becker and Austen, 1966) or "mast cell sensitizing" (Mota, 1M4) antibody is not seen in Fig. 5 and probably represents a unique immunoglobulin class analogous to human IgE (Ishizaka and Ishizaka, 1967). It is heat labile and sensitive to 2-mercaptoethanol (Bloch and Wilson, 1968). In the rat, homocytotropic (heat-labile) antibody is recognized only by its functional characteristics, namely, the mediation of passive cutaneous anaphylaxis with a 72-hour latent period (Mota, 1964; Bloch and Wilson, 1968) and the sensitization of peritoneal mast cells for subsequent antigen-induced histamine release ( Austen et al., 1965). This immunoglobulin appears to have a fast electrophoretic mobility and, on Sephadex G-200 gel filtration, is eluted after the 19 S and before the 7 S peak (Bloch and Wilson, 1968; Jones and Ogilvie, 1967). Rat homocytotropic antibody is eluted from diethylaminoethyl ( DEAE ) cellulose columns with the IgA fractions (Stechschulte et al., 1967; Bloch, 1967; Bloch and Wilson, 1968; Jones and Ogilvie, 1!367), but absorption studies indicate that this antibody is not a member of the IgA immunoglobulin class ( Austen et al., 1965). Studies concerned with the identification of the homologous antibodies involved in the antigen-induced release of SRS-A'"t were initiated by Stechschulte et al. (1967). These workers observed that rats injected with constant amounts of precipitating antibody from different antiserum pools differed considerably in the quantity of SRS-Arst released upon subsequent antigen challenge. This finding suggested that SRS-Arat release was not a function of the entire population of precipitating antibodies, but rather a property of some subpopulation. When rat antiserum was fractionated by starch block electrophoresis or by DEAE cellulose chromatography, only the fractions containing homologous IgG were capable of preparing rats for the antigen-induced release of SRS-Ar"'. Morse et al. (1968) studied the time course of appearance of the homologous antibodies capable of preparing rats for the immunological release of SRS-Arat following a single immunogenic stimulus. The responsible antibody was present in sera obtained 1week after immunization with DNP-bovine 7-globulin ( ByG) in complete Freund's adjuvant; it reached peak titers between weeks 2 and 4 and then declined to negligible levels by week 10. Serum pools obtained at different intervals following immunization were fractionated by stepwise elution on D E 5 2 cellulose columns, and a typical elution pattern for a rat anti-DNP

124

ROBERT P. ORANGE AND K. FRANK AUSTEN

FIG.6. Typical elution pattern obtained on stepwise elution from DE52-cellulose of whole rat antiserum. ( From Bloch et al., 1968.)

antiserum pool is outlined in Fig. 6. Immuno- and radioimmunoelectrophoresis of the seven protein peaks revealed that peak 1 contained predominantly IgGa and some IgGb; peak 1' contained fast IgGa and moderate amounts of IgGb; peaks 2 through 6 contained lesser amounts of these proteins. Peaks 4, 5, and 6 contained IgA with the highest concentration in peak 5; IgM was found only in peak 7. These fractions were concentrated to the starting serum volume applied to the column, dialyzed against 0.15 M NaCl for 18 hours, and then tested for their ability to prepare rats for the immunological release of SRS-A'"'. It appeared that only the fractions containing IgGa were capable of preparing rats for SRS-A'"' release upon subsequent exposure to specific antigen. This observation was corroborated by comparing the biological activity of fractions containing predominantly IgGa, IgGb, IgA, or IgM at equal hemolytic or hemagglutinating titers; only the fractions containing IgGa prepared rats for the immunological release of SRS-kat (Morse et aZ., 1968). Figure 7 involves a comparison of the SRS-A'"'-releasing and hemolytic activities of peaks 1and 1' obtained by DE52-cellulose chromatography of rat antisera acquired at weekly intervals following immunization with DNP-ByG. An excellent correlation was observed between the ability of peak 1 to prepare rats for the immunological release of SRS-A'"' and to sensitize antigen-coated, tanned erythrocytes for lysis in the presence of complement. Peak 1' failed to mediate a significant release of SRS-A'"', although there was an appreciable hemolytic titer associated with this fraction for several weeks. The association of the hemolytic and SRS-A'"'-releasing

125

SLOW REACTING SUBSTANCE OF AKAPHYLAXIS

activity of peak 1suggests that a single antibody population is responsible for both biological activities. The ability of fractions containing rat IgGa to prepare rats for the antigen-induced release of SRS-Arat was not altered by heating the fractions for 4 hours at 56°C. (Morse et d.,1969). It thus appears that a thermostable 7 S IgG antibody population associated with IgGa is a species of homologous immunoglobulin involved in the immunological release of SRS-Arn'. Although rat IgGa is capable of fixing complement, it is not clear whether complement activation is an essential requirement for immunological release of SRS-A'"'. Pretreatment of rats with a semipu&ed, nontoxic fraction of venom from the cobra (Naja @a) which markedly depletes the animals of C3 ( Muller-Eberhard, 1967; Nelson, 1966) was accompanied by an inability to release SRS-A'"' (Orange et al., 1967, 1968a). Furthermore, partial decomplementation of rats by pretreatment with heat-aggregated human y-globulin ( HAHyG) (Christian, 1958) was associated with a partial suppression of the immunological release of SRS-A'"' (Morse et al., 1969). However, these observations do not permit

2

1400

I

I*-

-0-Q

0

Mean SRS-A Release / R a t

il

"OI

A n n

-,-

___

j

I

\ d

3

4

3

6

7

8

-0

9

1280

10

WEEKS

FIG. 7. Comparison of slow reacting substance of anaphylaxis of the rat (SRS-A"')-releasing and hemolytic activities of peaks 1 and 1' obtained by DE52cellulose chromatography of rat antisera obtained at weekly intervals from rats immunized with 2,4-dinitrophenyl-bovine y-globulin in complete Freund's adjuvant, (From Morse et al., 1968.)

126

ROBERT P. ORANGE AND K. FRANK AUSTEN

a conclusion as to whether complement is required for the immunological release of SRS-Ara'. A slow reacting material has been released from guinea pig lung tissue following treatment with cobra venom (Feldberg and Kellaway, 1938; Middleton and Phillips, 1964), and, thus, pretreatment with the venom factor may effect substrate depletion when used in the rat. The HAHyG may not only decomplement rats but may also activate the pathway to the formation and release of SRS-A'"', thus producing substrate depletion. The role of complement in the antigeninduced release of SRS-A'"' is not established and will be later assessed when an in vitro system for the release of this mediator is available. (See Addendum I, on p. 144.) C. CELLULAR ELEMKITS INVOLVED IN THE IMMUNOLOGICAL RELEASE OF SLOWREACTINGSUBSTANCE OF ANAPHYLAXISOF THE RAT The cellular elements involved in the immunological release of SRS-A'"' have been investigated by pretreating rats with Werent biological or pharmacological agents so as to increase or decrease a specific cell population in the rat before preparation for the antigen-induced release of SRS-A'"' with heterologous (Orange et al., 1967) or homologous (Orange et al., 1968a) hyperimmune antiserum. Discordant evidence was available as to the role of the mast cell in the immunological release of SRS-A'"' (Uvnas and Thon, 1959; Boreus and Chakravarty, 1960; Austen and Humphrey, 1963). Preliminary experiments (Orange et al., 1967) confirmed the observation of Fawcett ( 1955) that the intraperitoneal injection of distilled water effected a disruption of the rat mesenteric mast cells as determined microscopically and the associated disruption of the free peritoneal mast cells was established by measurement of the total cellular histamine. When rats were pretreated with distilled water 5 days before being prepared for the antigen-induced release of SRS-Ara', no suppression of SRS-Ara' release was observed despite a virtual absence of the free and fixed peritoneal mast cells ( Orange et al., 1968a). This observation was corroborated by experiments involving pretreatment of rats with a rabbit antiserum directed against rat mast cells (Ra anti-RMC) (Valentine et al., 1967). Although depletion of rat peritoneal mast cells did not influence the subsequent antigeninduced release of SRS-Arat,it did prevent the homocytotropic antibodymediated release of histamine. Conversely, the induction of a profound leukopenia in rats by pretreatment with high doses of nitrogen mustard was associated with a marked suppression of the immunological release of SRS-A'"', but did not alter the homocytotropic antibody-mediated release of histamine. Rats rendered neutropenic by pretreatment with a

SLOW REACTING SUBSTANCE O F ANAPHYLAXIS

127

rabbit antiserum directed against rat polymorphonuclear leukocytes (Ra anti-RPMN) also demonstrated a marked suppression of SRS-M"' release. Pretreatment of rats with a rabbit antiserum directed against rat thymic lymphocytes (Ra anti-RTL) (Guttman et al., 1967) effected a greater than 80% absolute lymphopenia in rats without suppressing the subsequent antigen-induced release of SRS-Ara'. It thus appears that the RPMN leukocyte, but not the peritoneal mast cell or circulating lymphocyte, is a cellular prerequisite for the antigen-induced release of SRS-Arnt in rats prepared with whole hyperimmune antiserum, Experiments were next undertaken employing PMN leukocyte exudates induced in the peritoneal cavity of rats by the intraperitoneal injection of glycogen. When the number of PMN leukocytes in the rat peritoneal cavity was substantially increased by the induction of a peritoneal exudate, actively or passively sensitized rats demonstrated a two- to sevenfold increase in the antigen-induced release of SRS-A'"'. Further, the suppression of SRS-A'"' release associated with pretreatment of rats with Ra anti-RPMN is substantially reversed by the i.p. injection of PMN leukocytes recovered from the peritoneal exudates of normal unsensitized rats. Finally, the release of SRS-A'"' has been achieved in the peritoneal cavities of unsensitized rats following the passive transfer of peritoneal exudates from actively sensitized rats and subsequent antigen challenge; the cell-free supernatant alone was inactive. Thus, the PMN leukocyte is implicated in the immunological release of SRS-Arnt on the basis of inhibition of SRS-Ara' release by specific depletion of this cell type, partial restoration of release by repletion of this cell population, enhancement of release by increasing the number of PMN leukocytes intraperitoneally, and by preliminary passive transfer studies. The possible role of the rat eosinophilic leukocyte in the antigeninduced release of SRS-P"' was investigated by the induction in rats of peritoneal exudates consisting predominantly of neutrophilic or eosinophilic leukocytes (Archer and Hirsch, 1963) and comparing the SRS-A'"' release achieved upon antigen challenge. In the presence of a greater than tenfold increase in peritoneal eosinophiles, a 50%reduction in SRSArat release was observed whereas the neutrophilic exudate yielded the expected enhancement (Orange and Austen, 1969). Whether the observed suppression of SRS-Arat release is related to an effect of the eosinophile on the antibody, antigen-antibody complexes, or released SRS-APR'remains to be determined. The precise contribution of the neutrophile to the reaction sequence leading to the formation and release of SRS-A'"' is not known. There is no conclusive evidence that the rat neutrophile contains the SRS-A'"'

128

ROBERT P. ORANGE AND K. FRANK AUSTEN

substrate or precursor or that this cell type synthesizes SRS-A'"' de tu)tx) following antigen-antibody interaction. Whether the homologous IgGa immunoglobulins are "cytotropic" for rat neutrophiles or whether the neutrophiles ingest immune aggregates comprised of IgGa and specific antigen, a phenomenon enhanced in the presence of complement (Gigli and Nelson, 1968), will be better studied using isolated cells and immunoglobulin fractions in uitro. A wide variety of biologically active enzymes associated with neutrophilic lysosomes have been implicated in certain forms of immunological tissue injury, and these have been extensively reviewed elsewhere (Cochrane, 1967; Cohn and Hirsch, 1980; Seegers and Janoff, 1966). The release of a slow reacting material from neutrophiles following the phagocytosis of antigen-antibody complexes in vitro has been recently described, and this material has been designated SRSAPhh'g ( Macmorine et al., 1968; Movat et al., 1969). However, this material is quite active on the estrous rat uterus and may be extracted to some extent from normal neutrophilic leukocytes. Thus, whatever its final chemical characterization, SRS-Aphag does not possess the properties currently attributed to SRS-A. (See Addendum 11, on p. 144.)

D. In Viw, INHIBITIONOF THE IMMUNOLOGICAL RELEASE OF SLOW REACXINC SUBSTANCE OF ANAPHYLAXI~ OF THE RAT BY DIETHYLCARBAMAZINE As is often the case, a fortuitous, but certainly not illogical observation made in a clinical setting prompted a series of laboratory experiments. In this instance the studies were on the effects of diethylcarbamazine citrate (Hetrazan, Lederle) on the antigen-induced release of SRS-A'"'. Diethylcarbamazine is an effective chemotherapeutic agent against microfilarial infestations in animals and man (Hawking, 1950,1966; Santiago-Stevenson et al., 1948), and in addition has proved efficacious in the treatment of tropical eosinophilia (Danaraj, 1958). Mallen (1965) was also impressed with the efficacy of this drug in relieving the intractable bronchospasm associated with tropical eosinophilia, and this observation prompted him to conduct a preliminary clinical trial with this drug in patients with severe asthma without tropical eosinophilia. Fourteen of fifteen severely afflicted asthmatic patients responded satisfactorily to diethylcarbamazine within 24 hours of the onset of treatment and with only minimal side effects. Because of the aforementioned possibility that SRS-A might play a role in bronchospastic disease in man, Orange et al. (196813) investigated the effect of diethylcarbamazine on the in duo antigen-induced release of SRS-A'"'. Preliminary experiments using

SLOW REACTING SUBSTANCE OF ANAPHYLAXIS

129

heterologous antisera established that the administration of diethylcarbamazine in a dose of 20 mg./kg. i.v., 10 seconds before specific antigen i.v., effected about a 70%suppression of the antigen-induced release of SRS-A'"'. The inhibition of SRS-A release obtained with diethylcarbamazine appeared to occur in a dose-response fashion, and pretreatment with 5.0 mg./kg. diethylcarbamazine effected only about 25%suppression of the immunological release of SRS-A'"'. The inhibition was not due to an effect of diethylcarbamzine on the bioassay of SRS-A. Diethylcarbamzine is known to be a weak antihistaminic (Harned et al., 1948), and it was observed to have less than one-thousandth the antihistaminic activity of mepyramine maleate. Similar inhibition data were obtained in rats prepared with homologous hyperimmune antisera (Orange et al., 1968a). When rats were pretreated with diethylcarbamazine 15 minutes or longer before antigen challenge, there was no inhibition; thus, diethylcarbamazine did not produce some irreversible tissue alteration and had to be present in optimal concentrations at the time of antigen-antibody interaction in order to be inhibitory. The short duration of action of diethylcarbamazine was consistent with the published studies on the metabolism of this agent which indicated that the rat excreted diethylcarbamazine at a rate of 100 mg. per kilogram per hour (Harned et aZ., 1948). Diethylcarbamazine did not alter the in vivo white blood cell count, differential cell count, or serum whole complement level. Diethylcarbamazine did not interfere with antibody-antigen interaction in vitro as determined by precipitin analysis of heterologous antisera in the presence of a 20 mM concentration of this drug. Diethylcarbamazine did not alter the viability of RPMN leukocytes in vitro, nor did it affect the hemolytic activity of normal rat serum in vitro (Orange and Austen, 1968). Inhibition of the antigen-induced release of SRS-Aratis associated with tissue desensitization. Rats prepared with homologous antisera and challenged with specific antigen in the presence of diethylcarbamazine do not release appreciable amounts of SRS-kat, and repeat antigen challenge 2 hours later, when diethylcarbamazine has been presumably metabolized to noninhibitory levels, is not associated with SRS-A'"' release because of previous antibody utilization (Orange et al., 1968a). Recent studies (Orange et al., 1969d) employing isolated fractions rich in heat labile homocytotropic antibody have demonstrated that diethylcarbamazine is also capable of blocking SRS-A""' release mediated by this homologous immunoglobulin. From these observations the following inferences on the mechanism of action of diethylcarbamazine might be drawn: diethylcarbamazine does not interfere with antigen-antibody interaction, nor does it effect an

130

ROBERT P. ORANGE AND K. FRANK AUSTEN

TABLE IV INHIBITION OF THE ANTIQEN-INDUCED RELEASEOF SLOWREACTING SUBSTANCE OF ANAPHYLAXIS OF THE RAT WITH DIETHYLCARBAMAZINE AND ITS ANALOQS (20 MG./EG. I.v.) Analogs

Structure

Piperazine :

Mean percent inhibition

H

Piperazine

0

2,5 - Piperazinedione

0

1-Diethylcarbamyl-4methylpiperazine (Hetrazan)

H2y/N\yH2 H2C, ,CHZ N I CHS

66

Piperidine : nL-Pipecolic acid

12

Pipecolamide

82

Pyridine : Nicotinamide

31

0

/I

Isonicotinic acid hydrazide (Isoniazid)

C-NH--NH, I HC+‘,CH I

I/

HCQN,CH

67

131

SLOW REACTING SUBSTANCE OF ANAPHYLAXIS

TABLE IV (continued) Analogs

Mean percent inhibition

Structure

Pyridine (continued) 0

I1 I

C-NH-NH-CH H C A X

I

CHS

I

Iproniazid

7% 6

Benzene : 0

I1

$! -NH,

Benzamide

30

0

ll

q-NH-NH,

Benzhydrazide

12

NH 0 11 II C-NH-C-NH, I

N-Amidinobenzarnide

H

I

C

II

~

H

HC, ,CH 'C H

44

Nonring structures : Ethanolamine

HOCH,CH,NHz

0

1, I-Diethylurea

(C,H,),NCONH,

0

Choline chloride

[ HOCH,CH2N+(CHs),]C1 -

10

irreversible alteration in the serum and cellular factors contributing to SRS-A release; it does not antagonize the end-organ activity of SRS-ket in the bioassay; and it appears to act at some step in the reaction sequence leading to the formation and release of SRS-A subsequent to antigen-antibody interaction and prior to the elaboration of the mediator. A study of the chemical analogs of diethylcarbamazine was under:aken in an attempt to determine the subgroups within this molecule

132

ROBERT P. ORANGE A N D K. FRANK AUSTEN

required for optimal inhibitory activity. This data, though somewhat fragmentary, may prove useful in attempts to provide an agent with greater inhibitory activity and a longer duration of action. Substitutions at both the carboxamide grouping and piperazine ring were examined (Table IV). All chemicals tested for their ability to inhibit the antigeninduced release of SRS-A'"' were injected i.v. in a dose of 20 mg./kg., 30 seconds before i.v. challenge with specific antigen. Each chemical was tested in three rats in an experiment, and each experiment was repeated at least 3 times. The mean SRS-Arst release for treated animals was compared with the mean release for control animals in that experiment and the results were expressed as percent inhibition of SRS-Arat release. A mean inhibition of greater than 25%was considered to be signscant in this test system. Two piperazine analogs of diethylcarbamazine, piperazine and 2,s-piperazinedione lacking the carboxamide grouping proved ineffective in inhibiting the antigen-induced release of SRS-Arat. The requirement for a piperazine ring structure was investigated by assessing the activity of piperidine analogs. Although dGpipecolic acid was inactive, its carboxamide derivative, pipecolamide, was quite effectivein inhibiting the immunological release of SRS-A'"'. The unsaturated pyridine carboxamides also proved to be active inhibitors, but the benzene carboxamides had little or no activity. Nonring structures such as the substituted urea, 1,l-diethylurea, and ethanolamine and choline which were reported active in the guinea pig lung (Smith, 1961) were inactive in the rat test system. It thus appeared that the structural requirement for inhibition of the antigen-induced release of SRS-A'"' included a carboxamide grouping and a saturated or unsaturated ring containing nitrogen. None of the effective analogs, at the concentrations used, altered leukocyte viability in uitro or the hemolytic activity of normal rat serum in uifro, and they did not appear to interfere with the bioassay of SRS-A"' (Orange and Austen, 1968). In acting in the pathway to the release of SRS-A'"', these inhibitors appear to be both unique and selective, as will be discussed in the next section.

E. DISSOCIATION OF THE IMMUNOLOGICAL RELEASE OF SLOWREACTING SUBSTANCE OF ANAPHYLAXIS OF THE RAT AND HISTAMINE The characteristics of the physicochemically distinct homologous immunoglobulins involved in the antigen-induced release of histamine and SRS-A'"' in the rat are tabulated in Table V. These antibodies differ in their physicochemical characteristics and in their sensitization requirements, but have the same or different cellular prerequisites depending upon the chemical mediators released. Selective pharmacologi-

A

TABLE V ANTIBODIES MEDIATING THE RELEASE OF HISTAMINE AND SLOW REACTING SUBSTANCE OF ANAPHYLAXIS FROM THE RAT

COMPiiRISON OF THE OF

Characteristics Pharmacological agents released

Time of appearance during immunization of rat Sensitivity to heating (56°C.) Mercaptoethanol sensitivity Complement fixation Concentration in serum Mobility in gel electrophoresis Location on DE52-cellulose chromatography Location on Sephadex G-200 gel filtration Latent period for PCAo Persistence a t skin site Latent period for I.P. release: Histamine SRSArat Suppression of SRSArst release: Diethylcarbamazine Disodium cromog1ycat.e Suppression of histamine release: Diethylcarbamaeine Disodium cromoglycate 0

Passive cutaneous anaphylaxis.

Homocytotropic antibody

SRSAmt, histamine, and serotonin Early (1C14 days)

+ +-

Trace “Fast” (a) Peaks 4 4 ( 1 6 region)

>7

s, <19 s

Homologous IgGa

S R S k M thistamine, , and serotonin Early (14-28 days) and late (hyperimmune)

-

+

Appreciable “Slow” (y) Peaks 1-3 (IgG region)

7s

48-72 hours Long (days)

4 hours Short (hours)

2-72 hours

2 hours

+

+-

2 hours

+ -

+

2 hours

+

P 4

0

F

$

z9 0

3

134

ROBERT P. ORANGE AND K. FRANK AUSTEN

cal inhibition of the pathways to SRS-A'"' or histamine release has recently been achieved. As was described in the preceding section, diethylcarbamazine effectively inhibits in vivo the immunological pathway leading to the formation and release of SRS-A'"'. The inhibition observed appears to be selective in that diethylcarbamazine and its effective analogs do not suppress the homocytotropic antibody-mediated release of histamine ( Orange and Austen, 1968). A second pharmacological agent, disodium cromoglycate ( Intal, Fison's Pharmaceuticals ) appears to inhibit the homocytotropic antibody-mediated release of histamine in several species including the rat (Cox, 1967). Disodium cromoglycate appears to act at some step subsequent to antigen-antibody interaction and prior to release of the mediator itself and is not an antihistamine. The inhibitory activity of disodium cromoglycate is selective in that this agent does not inhibit the IgGa-mediated release of SRS-ArUt (Orange and Austen, 1968; Morse et al., 1969), although preliminary studies (Orange et ul., 1969d) indicate that this agent does inhibit the homocytotropic antibody-mediated release of SRS-Arat.These drugs are unique in that they represent nontoxic agents which selectively suppress in uivo spec%c immunological pathways leading to the release of chemical mediators involved in immediate-type hypersensitivity. The initial observations of Rapp ( 1961) and Stechschulte et al. (1967) suggested that only a variable amount of histamine was present in association with the SRS-Arat released into the peritoneal cavities of rats prepared with whole heterologous or homologous antiserum. However, the IgG fraction of homologous, hyperimmune antiserum did mediate the release of significant amounts of histamine as well as SRS-A'"' (Stechschulte et ul,, 1967). This observation was reinvestigated by comparing the ability of whole hyperimmune rat antiserum and its IgGa fraction to prepare the rat peritoneal cavity for the concomitant release of histamine and SRS-ArUt (Morse et aZ., 1969). It appeared that the IgGacontaining fraction was far more active in mediating the release of histamine than was the unfractionated antiserum, The release of histamine mediated by fractions containing homologous IgGa could not be attributed to the presence of homocytotropic antibody since the elution characteristics of IgGa on DEAE cellulose differed from those of homocytotropic antibody; the capacity of IgGa to mediate histamine release was not diminished by heating at 56°C. for 4 hours; and the optimal latent period for IgGa to mediate the intraperitoneal release of histamine was only 2 hours rather than the 48 hours required by heat-labile homocytotropic antibody. The mechanism of histamine release mediated by the heat-stable IgGa fraction did not seem to be PMN leukocyte-dependent and thus could not be attributed to the release of cationic proteins from PMN leukocyte lysosomes (Seegers and Janoff, 1966; Ranadive and

SLOW REACI"G

SUBSTANCE OF ANAPHYLAXIS

135

Cochrane, 1967; Cochrane and Miiller-Eberhard, 1968). The release of histamine did not appear to be complement-dependent, since no suppression of the release of this mediator was observed in rats decomplemented by pretreatment with cobra venom factor or HAHyG ( Morse et al., 1969). This observation also negated a critical role for the anaphylatoxins (Jensen, 1967; Dias da Silva and Lepow, 1967; Cochrane and Miiller-Eberhard, 1968) in this reaction. The histamine release mediated by the IgGa fraction was suppressed by pretreatment of rats with disodium cromoglycate suggesting that ihis reaction sequence and that initiated by rat homocytotropic antibody shared a similar or common step. The conclusion of Morse et a2. (1969) that the rat possesses two classes of antibodies capable of mediating the mast cell-dependent release of histamine-heat-labile homocytotropic antibody and heat-stable IgGa-raised the question of whether the latter also mediated the release of SRS-A'"'. Evidence that IgGa mediates the release of both histamine and SRS-APat includes the following: the time course of appearance following immunization of the heat-stable antibodies mediating the release of both histamine and SRS-Aratwas virtually the same; the chromatographic elution patterns and the electrophoretic mobilities appeared identical; the latent periods for sensitization were similar; no dissociation of the two activities could be achieved by starch block electrophoresis of the IgGa fraction; and the IgGa fraction appeared to be free of other rat immunoglobulins on the basis that it produced a single precipitin arc on radioimmunoelectrophoresis, a single peak on analytical ultracentrifugation, and elicited in guinea pigs an antibody that reacted with the Fabfragment of several rat immunoglobulins but only with the F, fragment of IgGa. A conclusion as to whether more than one antibody class is present in the IgGa fraction or whether this fraction represents a single class of heat-stable antibodies with three biological activities-complement h a tion, mast cell sensitization, and mediation of SRS-Arat release-must await further study. However, the observation that IgGa fractions are capable of sensitizing mast cells for histamine release leaves open the possibility that IgGa-mediated SRS-A'"' release may also be mast cell dependent, at least to some extent, whereas in whole hyperimmune serum, mast cells are not sensitized for histamine release and SRS-Arat release appears to be PMN-leukocyte dependent. For purposes of this discussion the critical points are that disodium cromoglycate blocks the antigen-induced release of histamine (Orange and Austen, 1968) following sensitization with either heat-labile homocytotropic or heatstable IgGa ( Morse et ul., 1969), whereas diethylcarbamazine selectively blocks the release of SRS-Aratmediated by hyperimmune antiserum or heat-labile homocytotropic antibody.

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ROBERT P. ORANGE AND K. FRANK AUSTEN

F. PASSIVE CUTANEOUS ANAPHYLAXIS IN THE RAT The homologous antibodies capable of preparing the rat peritoneal cavity for the release of SRS-Ararand/or histamine are also capable of preparing rat skin for PCA (Ovary, 1952; Mota, 19f34;Stechschulte et al., 1967). The heat-labile rat homocytotropic antibodies elicit a PCA reaction in rats following an optimal latent period of 48 to 72 hours (Bloch, 1967; Mota, 1964). The heat-stable homologous IgGa antibodies, or whole antisera containing these antibodies, mediate a PCA reaction in rats with an optimal latent period of about 4 hours (Stechschulte et al., 1967). Dermal blueing may also be evoked in rats by the intradermal injection of Ra anti-RMC (Orange and Austen, 1968; Austen and Valentine, 1968) which appears to release histamine and serotonin from the mast cells in the dermis of rats by a form of complement-dependent cytotoxic, immunological tissue injury (Valentine et al., 1967). The mediators involved in the elicitation of these three cutaneous reactions may be identified through the use of selective pharmacological inhibitors. The PCA reaction mediated by rat homocytotropic antibody may be prevented by inhibiting the pathway to the release of the vasoactive amines with disodium cromoglycate or by blocking the end-organ activity of these mediators using specific antagonists, such as mepyramine maleate and methysergide (Orange and Austen, 1968). Disodium cromoglycate does not inhibit the dermal blueing response elicited by Ra anti-RMC, demonstrating once more the selectivity of its inhibitory activity; this lesion is completely suppressed by pretreatment of rats with a combination of histamine and serotonin antagonists. The d-hour PCA reaction in rats mediated by heatstable IgG antibodies appears to be somewhat more complex in that only partial suppression is achieved with disodium cromoglycate or a combination of antihistamine and antiserotonin agents. Virtually complete suppression may be achieved by the addition of diethylcarbamazine to either regimen although this agent alone produces only slight suppression (Orange et al., 196813).It also is of interest that this PCA reaction appears to require the presence of PMN leukocytes (Lovett and Movat, 1966). A possible role for SRS-A'"' in the pathogenesis of this lesion may be implied on the basis of the slight permeability activity of SRS-Aratin the rat (Table I11) , the requirement of diethylcarbamazine for complete suppression of the lesion, the necessity of PMN leukocytes for full expression of the reaction, and the established biological properties of rat IgGa. Whether the SRS-A'"' released in this reaction actually induces a signscant permeability change itself or merely acts by potentiating the histamine response (Fig. 3 ) awaits further investigation.

SLOW REACTING SUBSTANCE OF ANAPHYLAXIS

V.

137

Immunological Release of Slow Reacting Substance of Anaphylaxis in Other Species Including Man

A. GUINEAPIC Although the immunological release of SRS-Am from sensitized guinea pig lung was described almost 30 years ago (Kellaway and Trethewie, 1940), the homologous immunoglobulins involved in this reaction sequence have only recently been studied by Stechschulte et al. (1967). Immunization of guinea pigs with a variety of immunogens emulsified in complete Freund's adjuvant results in the production of two major types of 7s antibodies directed against the same antigen but differing in electrophoretic mobility and certain biological properties (Ovary et al., 1963; Bloch et al., 1963; White et al., 1963; Baker et al., 1964). Homologous 7Sy, antibodies are capable of mediating passive cutaneous and systemic anaphylaxis, but they fail to fix complement in the presence of specific antigen. Conversely, guinea pig 7 S yz antibodies fix complement in the presence of antigen and mediate a reversed passive Arthus reaction, but not passive cutaneous or systemic anaphylaxis. Figure 8, taken from the observations of Stechschulte et d., ( 1967), compares the biological activities of the eluates obtained following starch block electrophoresis of hapten-specific guinea pig antiserum. It was observed that the eluates containing the guinea pig 7 S yl antibodies were capable of sensitizing chopped guinea pig lung slices in uitro for the release of histamine and SRS-Agp upon subsequent challenge with specific antigen, whereas the 7 S yz-containing eluates, although active in mediating passive immune hemolysis, were incapable of mediating the release of histamine or SRS-Agp. It is of interest that the homologous immunoglobulins mediating both histamine and SRS-A release in the guinea pig, 7 S yl, and in the rat, IgGa, are heat-stable. It appears that heterologous antibodies, present in hyperimmune rabbit serum, will prepare either species for the antigen-induced release of histamine and SRS-A (Austen and Brocklehurst, 1961b; Stechschulte et al., 1967). The nature of the target cell involved in the release of SRS-Am from perfused, sliced lung has not been investigated, although it is clear that the histamine is derived from the mast cell. The reaction sequence initiated by the interaction of specific antigen with homologous antibody in guinea pig lung may be blocked by competitive inhibitors of chymotrypsin, but not of trypsin, carboxypeptidase, nor leucine aminopeptidase (Austen and Brocklehurst, 1960, 1961a,b). The enzyme activated by the addition of specific antigen to sensitized guinea pig lung is quite sensitive to inhibition by diisopropylfhorophosphate (DFP); it is present in unchallenged lung in a DF'P-resistant pre-

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-

and

O.D. UNITS

PASSIVE H EM OLY SI S (-)

DI L UTlON TITER

t 22

20

18

16

y, Anti-DNP-ByG

800 r

12

10

6

8

-

y, Anti-DNP-ByG

1 4 HI STAMl NE

SRS-A units/

14

qm. 400

0

FIG. 8. Comparison of slow reacting substance of anaphylaxis (SRS-A) and histamine release from sliced guinea pig lung sensitized with either 7 S y, or 7 S ys guinea pig antibodies separated by starch block electrophoresis. Each division of right ordinate is equal to 0.02 0. D. units. PCA-passive cutaneous anaphylaxis; DNP2,Pdinitrophenyl; ByC-bovine gamma globulin. ( From Stechschulte et al., 1967.)

cursor form. The phosphonate ester inhibition profile of the antigenantibody-activated esterase involved in histamine release has been distinguished from that of the esteratic form of the first component of guinea pig complement (Becker and Austen, 1964). Similar studies on the nature of the esterase involved in SRS-Agp formation and release have not been conducted. Investigations are also lacking on the effect of ionic strength or anoxia (Austen and Brocklehurst, 1961c) on the antigen-induced release of SRS-AgP. It is noteworthy that studies on the enhancing effect of dibasic acids and inhibitory action of monobasic fatty acids revealed a parallel effect on histamine and SRS-AgP release. Two dibasic acids, succinate and maleate, strikingly enhance the antigen-induced release of histamine and

SLOW REACTING SUBSTANCE OF ANAPHYLAXIS

139

SRS-Asp from sensitized guinea pig lung by potentiating some step subsequent to antigen-antibody interaction ( Austen and Brocklehurst, 1961b). The activity of these dicarboxylic acids is highly selective in that the carboxyl groups must be separated by a 2-carbon chain and they must be free or fixed in the cis position, The observed enhancement cannot be attributed to an accelerated time course of release of the mediators, increased histamine formation, or an effect on the tricarboxylic acid cycle. Monobasic fatty acids inhibit the antigen-induced release of histamine and SRS-Ag*from sensitized guinea pig lung tissue in uitro, and the inhibition appears to occur at some antigen-antibody-activated step. The concentration of monobasic fatty acid required to produce 50% inhibition of histamine release decreases with increasing length of the carbon chain. Antigen-antibody interaction in guinea pig lung is associated not only with the release of histamine and SRS-AgP, but also with the release of the kinin-forming enzyme, kallikrein ( Brocklehurst and Lahiri, 1962a; Jonasson and Becker, 1966). A marked elevation in plasma kinin and reduction in plasma kininogen levels have been observed in uiuo in guinea pigs undergoing protracted anaphylaxis ( Brocklehurst and Lahiri, 1962b). The immunological activation of kallikrein in guinea pig lung does not appear to involve a cation-dependent step and thus differs from the mechanism of release of histamine and SRS-AgP; activation of kallikrein may involve initial activation of the Hageman factor (Jonasson and Becker, 1966). The immunoglobulin responsible for the antigen-induced activation of guinea pig kallikrein has not yet been determined.

INCLUDING MAN B. PRIMATES Brocklehurst ( 1960) originally described the in uitm antigen-induced release of SRS-A from the perfused lungs of rhesus monkeys actively sensitized with egg albumin and from the lungs of pollen-sensitive asthmatic humans. Passive sensitization of normal human lung tissue in vitru with whole antisera containing reaginic antibody ( IgE) has also resulted in the release of histamine and SRS-Ah" upon antigen challenge (Parish, 1967; Sheard et al., 1967). The activity of the atopic sera appears to be reduced or lost upon heating at 56°C. for 4 hours, suggesting, but not proving, that the heat-labile human IgE mediates the release of both chemical mediators. More recently, atopic sera have been used to passively sensitize norma1 monkey lung tissue in vitro for the antigen( Ishizaka et al., 1969). Preinduced release of histamine and SRS-AmonkeY liminary studies suggest that a single heat-labile immunoglobulin, IgE, is involved in the release of both mediators. (See Addendum III, on p. 144.)

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These preliminary observations are at variance with studies in the guinea pig (Baker et al., 1964; Stechschulte et al., 1967) and in the rat (Morse et al., 1968) which indicate that a heat-stable immunoglobulin mediates the release of SRS-A. The precise definition of the prerequisite cellular elements and effective pharmacological inhibitors of the immunological release of SRS-A from primate lung must await further investigations. It may be that, as with histamine, SRS-A release will be mediated by more than one immunoglobulin class with different classes predominating in different species or under different experimental circumstances. VI.

Concluding Comments

Slow reacting substance of anaphylaxis possesses potential biological signscance not only for antigen-induced bronchoconstriction but also for forms of immunological tissue injury relating to increased vascular permeability. Although chemical identification of this substance has not been achieved, pharmacological identification has been carried out, and it is readily distinguished from all other materials released by antigen-antibody interaction. Investigations concerned with the cellular and serum factors contributing to the immunological formation and release of this mediator have provided methods of enhancing the release of SRS-A (Austen and Brocklehurst, 1961b; Orange et al, 1968a). The availability of larger yields of SRS-A, stable on storage and free of other contractile elements, in addition to newer methods in preparative lipid chemistry, may afford an opportunity of chemically characterizing this highly active principle. Studies concerned with the immunoglobulins and cellular elements involved in the antigen-induced release of SRS-A and histamine have provided further insight into the mechanisms of immediate-type hypersensitivity. Certain prerequisite cellular elements and effective pharmacological inhibitors of histamine and SRS-A release have been identified in the rat. The term “anaphylaxis” must now be used in the clinical sense with the only implication being that the reaction is due to the action of chemical mediators. In the rat, for example, the lesion termed “passive cutaneous anaphylaxis” can be mediated by two distinctly different homologous immunoglobulins, involving the same as well as different target cells and mediators, and requiring somewhat different regimens to obtain pharmacological suppression ( Orange and Austen, 1968). In the rat, the inhibitory activities of disodium cromoglycate and diethylcarbamazine appear to be quite selective. Acting subsequent to antigen-antibody interaction and prior to the release of the mediator,

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these agents permit “desensitization”of tissues without inflammation since the antibody is utilized in a nonproductive reaction. These inhibitors, or analogs of greater potency and longer duration of action, may eventually be of considerable therapeutic significance for man.

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ADDENDA I. The recent observation that a heat labile human immunoglobulin, IgE, could from primate lung slices mediate the release of both histamine and SRS-Amonkey (Ishizaka et al., 1969), prompted a re-examination of the biologic activity of rat heat labile homocytotropic antibody. Rats prepared by the intraperitoneal injection of homologous antiserum containing homocytotropic antibody activity released only histamine upon challenge with specific antigen 72 hours later (Orange et al., 1969c), the same latent period optimal for mediation of passive cutaneous anaphylaxis by this immunoglobulin class. Since reagin-rich human serum prepared monkey lung slices for histamine and SRS-Amonkey release following a 1-2 hour latent period, the biologic capability of rat heat labile homocytotropic antibody was examined using a similar interval. Under these conditions (Orange et al., 1969c) rat homocytotropic antibody prepared the peritoneal cavity for the subsequent antigen-induced release of both histamine and SRS-kat. The evidence that two distinctly different homologous immunoglobulins can prepare the rat for the release of SRS-knt includes: different elution characteristics on DE-52 chromatography with heat stable antibody appearing in peak 1 and heat labile antibody being present in peak 5 (Fig. 6); different positions of the heat stable and heat labile antibodies mediating SRS-Mat release by Sephadex G-200 chromatography; and approximate sedimentation constants of 6.8 S for the heat stable IgGa antibody as compared to 8.2 S for the heat labile homocytotropic antibody. The characteristics of the heat labile antibody mediating SRS-Mat release are in agreement with those reported previously for this antibody when it was defined in terms of histamine release or the capacity to mediate the lesion of passive cutaneous anaphylaxis with a 48 to 72 hour latent period (Binaghi et al., 1964; Bloch and Wilson, 1968). The method of immunization (Morse et al., 1969) determines to a large extent whether the homologous immunoglobulin mediating the release of SRS-knt is of the heat labile homocytotropic or heat stable IgG class. 11. The very recent observation (Orange et al., 1969c) that the rat homocytotropic antibody is also capable of mediating the antigen-induced release of SRS-kat prompted investigation of the cellular prerequisites involved in this pathway. Preliminary results (Orange et al., 1969d) suggest that the rat peritoneal mast cell contributes significantly to the homocytotropic antibody-mediated release of SRS-Rat. Depletion of rat peritoneal mast cells by pretreatment with distilled water or Ra antiRMC results in a marked suppression of the immunological release of SRS-A'"'. It thus appears that the antibodies used to prepare the rat for the immunological release of SRS-kar dictate at least to some extent the cellular elements participating in the antigen-induced formation and release of this mediator. 111. Following passive sensitization in vitro with human serum rich in reaginic ( homocytotropic ) antibody activity, challenge with either specific pollen antigen (direct anaphylaxis) or guinea pig anti-human IgE (reversed anaphylaxis) results in the release of both histamine and SRS-AmonkeY (Ishizaka et al., 1969). Heating the antiserum at 58°C. for 4 hours destroys its capacity to sensitize primate lung fragments for the release of these mediators by either mechanism. Evidence that the responsible human immunoglobulin is IgE includes: loss of sensitizing activity on treatment of the reaginic serum with an IgE-immunosorbent; reverse-type release of SRS-Amonkey and histamine mediated by monospecific antisera against IgE but not IgG, IgA, IgM, or IgD; and sensitization of monkey lung fragments with purified E myeloma protein or its F, piece for the reverse release of both histamine and SRS-Am""keY.

Some Relationships among Hemostasis. Fibrinolytic Phenomena. Immunity. and the Inflammatory Response' OSCAR D. RATNOFF2 Deparfrnenf o f Medicine. Core Wesfern Reserve University School of Medicine. and Universify Hospitals o f Cleveland. Cleveland. Ohio

I . Introduction . . . . . . . . . . . . . I1. Hemostatic Mechanisms . . . . . . . . . . A The Platelets . . . . . . . . . . . . B. The Formation of Fibrin . . . . . . . . . C . Fibrinoid . . . . . . . . . . . . D. Fibrinogen and Chemotaxis . . . . . . . . E. Thrombin . . . . . . . . . . . . F. The Formation of Thrombin . . . . . . . . G. Procoagulant Properties of Trypsin, Snake Venoms. and Staphy. . . . . . . . . . . locoagulase . H. Inhibitors of Blood Clotting . . . . . . . . I11. Fibrinolytic Phenomena . . . . . . . . . . A . Substrates of Plasmin . . . . . . . . . . B. The Activation of Plasminogen . . . . . . . . C. Plasma Inhibitors of Plasmin . . . . . . . . IV. The Inflammatory Process . . . . . . . . . . A. Histamine as a Mediator of Inflammation . . . . . . B . Serotonin as a Mediator of Inflammation . . . . . . C . Polypeptide Kinins as Mediators of Inflammation . . . . D. Plasma Inhibitors of Kinins . . . . . . . . E . The Relationship between Kinin Formation and Blood Clotting . F. Plasmin and Kinin Formation . . . . . . . . C . Granulocytes and Kinin Formation . . . . . . . H . Plasma Permeability Factors . . . . . . . . I . Human Serum Necrotizing Factor . . . . . . . J. Kinins and Inflammation . . . . . . . . . K . Foreign Body Reactions . . . . . . . . . L. Arthritis . . . . . . . . . . . . V. Complement . . . . . . . . . . . . . A . C 1 Esterase Inhibitor . . . . . . . . . . . . . . . . . B. Hereditary Angioneurotic Edema VI. Blood Clotting and Antigen-Antibody Reactions . . . . . . . . VII . Anaphylatoxins and Related Mediators of Inflammation . VIII. Anaphylaxis . . . . . . . . . . . . .

.

146 147 147 149 151 151 151

152 157 158 159 160 161 163 163 164 165 166 169 170 172 172 173 175 176 176 177 179 182 183 186 188 192

' Otherwise unpublished studies were supported in part by Research Grant HE 01861 from the National Heart Institute. the National Institutes of Health. U.S. Public Health Service. and in part by a grant from the American Heart Association. Career Investigator of the American Heart Association. 145

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. IX. Endotoxins and the Shwartzman Phenomenon . A. The Effect of a Single Injection of Endotoxin . B. The Generalized Shwartzman Reaction . . C. The Local Shwartzman Reaction . . . X. Allograft Rejection . . . . . . . . . . XI. Clinical and Experimental Nephritis . XI. The Arthus Phenomenon . . . . . . XIII. Delayed Hypersensitivity . . . . . . XIV. Concluding Remark . . . . . . . References . . . . . . . . . I.

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196 196

198 201 203 204 205 206 206 206

Introduction3

In the study of biological processes, the accumulation of information is often accelerated by a narrow point of view. The fastest way to investigate the body’s defenses against injury is to look individually at such isolated questions as how the blood clots or how complement works. We must constantly remind ourselves that such distinctions are man-made In life, as in the legal clich6, the devices through which the body protects itself form a seamless web, unwrinkled by our artificialities. The present review concerns some of the ways in which the body responds to injury or to harmful agents. It makes no pretense at being an exhaustive review of the boundless literature concerning these subjects. Citations have been selected because of their historic significance, because they may lead to more detailed information, or because they reflect work done by the author or his colleagues. Without doubt an entirely Werent selection of references could have been made-an apology to those whose works I have seemingly ignored. This article tries to describe from the vantage point of the coagulationist some ways in which hemostasis, and its ally, fibrinolytic phen~rnena,~ influence inflammation and immune mechanisms.

’Abbreviations: AAME, N-acetyl-L-arginine methyl ester; ACTH, adrenocorticotropic hormone; ADP, adenosine diphosphate; ALTEE, N-acetyl-L-tyrosine ethyl ester; AMCA, aminomethylcyclohexane caproic acid; C’14’9,first through ninth components of complement; DEAE cellulose, diethylaminoethyl cellulose; DFP, diisopropylphosphofluoridate; EA, erythrocytes ( E ) sensitized with specific antibody ( A ) ; EACA, e-aminocaproic acid; EDTA, sodium ethylenediaminetetraacetic acid; 5HT, serotonin, 5-hydroxytryptamine; PF/Age, permeability factor in “aged” guinea pig serum; PF/ Dil,permeability factor in diluted plasma; PF/Nat, penneability factor in fresh undiluted plasma; PF/P, permeability factor released from guinea pig serum by antigenantibody complexes; PTA, plasma thromboplastin antecedent, Factor XI; PTC, Christmas factor, Factor IX; SBTI, soybean trypsin inhibitor; SRS-A, slow reacting substance-A; TAME, p-toluenesulfonyl-L-argininemethyl ester. ’The term “fibrinolytic phenomena” will be used generically to encompass proteolysis by plasmin (fibrinolysin) not only of fibrinogen and fibrin but of other natural substrates as well.

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I shall examine the seamless web piecemeal, but I shall try to emphasize the whole. II.

Hemostatic Mechanisms

In vertebrates, the blood circulates within a meshwork of closed tubes, the blood vessels. Many devices have evolved to meet the threat imposed by a leak in these conduits. Two are of immediate interest, the accumulation of platelets at the site of vascular injury and the coagulation of fluid blood, both the subject of numerous reviews (e.g., 63, 119, 316, 385, 508, 595). A. THE PLATELETS

When a blood vessel is injured, whether by physical trauma or incident to the inflammatory response, platelets, cytoplasmic fragments derived from megakaryocytes, quickly stick to the surface of the damaged endothelium, and a few are actually engulfed by the endothelial cells (384). The force determining adhesion of platelets arises, at least in part, from collagen or collagen-like material exposed by the interruption of the endothelial lining of the vessels (78, 265, 737). Fresh platelets pile up on top of those adherent to the vascular wall, a process thought to be brought about through the action of ADP released by the injured vessel or by the platelets themselves (199). The aggregates of platelets which form through the action of collagen and ADP are loosely bound and break up readily. But, as the aggregates accumulate, thrombin begins to form in their vicinity. This enzyme induces adhesion of platelets to each other, apparently through actions upon intracellular substances closely resembling fibrinogen ( Factor I ) (115, 700, 736) and fibrin-stabilizing factor (Factor XIII) (301). At the same time, the platelets seem to fuse, a process known as viscous metamorphosis, and they discharge their phospholipid-rich granules into the surrounding blood. This phospholipid and, perhaps more importantly, that from platelet membranes are important adjuvants in the formation of local thrombi (386). Platelets also contain antihemophilic factor (Factor VIII) (383, 599), proaccelerin (Factor V ) (255, 349, 599), and PTA (Factor XI) (260) which may contribute to the clot-promoting properties of these cells. In the test tube, viscous metamorphosis is accompanied by clot retraction, that is, shrinkage of the fibrin clot with extrusion of serum. This phenomenon requires the presence of intact (92) and viable (243) platelets and is an energy-requiring process (243). The sequence of events leading to retraction begins with the elaboration of thrombin in the

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vicinity of platelets and the release of fibrinopeptides A and B (see Section I1,B) by this enzyme from the fibrinogen-like protein of these cells ( 4 3 6 ) . Perhaps the interaction of thrombin and platelet fibrinogen is responsible for the increase in platelet permeability which occurs during clotting (242). Subsequent to these changes, thrombasthenin, a contractile protein of platelets similar to muscle actomyosin, participates in events leading to retraction ( 5 6 ) .Thrombasthenin is found in isolated platelet granules and membranes (456) and functions as a magnesiumdependent adenosine triphosphatase ( 5 7 ) . However thrombasthenin brings about retraction, it at the same time liberates ADP, enhancing platelet aggregation ( 4 3 6 ) . The way in which clot retraction aids hemostasis and the healing process is conjectural, but its mechanical force is considerable ( 577). The bleeding tendency of patients whose platelets cannot undergo retraction lends strength to the view that the process is of importance. Although there are marked species differences, platelets contain histamine ( 734), 5HT (511, 738), epinephrine, and norepinephrine ( 7 1 0 ) . In contrast to human platelets, those of rabbits are particularly rich in histamine and 5HT, making them uniquely suited for some experimental studies ( 2 6 8 ) .Most of the 5HT is tightly bound to platelet granules, but some is also found in the rest of the cell (126, 729). The 5HT is probably not synthesized or catabolized by platelets, but is taken up from the surrounding plasma through an energy-requiring process (75, 241, 270). It can be released by treating animals or human subjects with reserpine, which blocks its adsorption without altering its slow diffusion back into the plasma (264). In rabbits heavily treated with reserpine, the platelets' capacity to adhere to glass and to aggregate upon the addition of ADP is said to be diminished ( 7 4 3 ) .In rabbits, rats, guinea pigs (613),and man (245) the administration of reserpine does not alter the bleeding time. Nevertheless, a role for 5HT in hemostasis is suggested by the increased bleeding time observed when reserpine is given with anticoagulants (581) and by the induction of hemorrhage when reserpine-treated rabbits are subjected to the stress of an intraperitoneal injection of hypertonic saline solution (276). The discharge of histamine and 5HT from the platelet can be brought about by various agents which induce their aggregation, such as collagen, antigen-antibody complexes, and thrombin ( 269, 735). The release of 5HT from platelets occurs at the same time as that of ADP (220). As might be anticipated from these observations, histamine and 5HT are discharged by platelets during clotting (108, 268). In part, this release

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may be brought about by the formation of thrombin (269, 735), perhaps because this enzyme alters the membrane and allows the discharge of platelet granules (200). Release of histamine and 5HT also takes place in platelets incubated in normal serum (269), an event blocked by heparin (269, 666), EDTA (666), and SBTI (666), but not necessarily mediated by thrombin (666). Serotonin itself can aggregate platelets in oitro, (430, 480), but the importance of this response is unclear, since the aggregated platelets disaggregate and become refractory to further aggregation ( 36). Like other cells, platelets contain a large complement of enzymes (385). Among these are one or more proteolytic enzymes which are capable of digesting fibrinogen and casein but resemble cathepsin A rather than plasmin. These enzymes may be derived from platelet granules (455). The discharge of these enzymes when platelets disintegrate may well contribute to the local events accompanying thrombosis or inflammation. The adhesion and aggregation of platelets are apparently adequate to seal small wounds. Evidence for this is found clinically in the relative freedom from bleeding after trivial injury in most individuals with congenital afibrinogenemia. Such patients have no difficulty in forming thrombin, needed for platelet aggregation, but of course they cannot form the fibrin structure of the blood clot.

B. THE FORMATION OF F m m The platelet plug is insufficient to stanch the flow of blood from any but the smallest wounds. A second mechanism, found in all vertebrates studied, provides for transformation of blood into a solid gel which can seal vascular defects. Basically, this gel is composed of a meshwork of fibrin strands which entrap blood cells and serum. Fibrin is a fibrous protein derived, during clotting, from fibrinogen, a soluble precursor composed, in man, of three pairs of polypeptides, (A), p ( B ) , and y (68). The transformation of fibrinogen to fibrin is brought about by thrombin, a proteolytic enzyme evolved when blood is shed, which splits arginyl-glycyl bonds near the C-terminal end of the a ( A ) and /3 ( B ) chains (30, 69) ( Fig. 1).This hydrolysis releases two pairs of small polypeptide fragments, fibrinopeptides A and B respectively, constituting about 3% of the fibrinogen molecule. The remainder, now called “fibrin monomer,” polymerizes to form the insoluble fibrin clot. As will be noted subsequently, the fibrinopeptides are by no means inert by-products of coagulation; in the laboratory, at least, they seem intimately involved in the defense reactions of the body. (Y

150

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I

FIBRINOGEN ( I )

TH R 0 M B I N

ACCELERATOR

1

(?I

FIBRIN MONOMER t FIBRINOPEPTIDES (SOLUBLE) THROMBIN t FIBRIN STAB1 LI ZING FACTOR (XIII)

co*

FIBRIN POLYMER (INSOLUBLE)

FIG.1. The formation of fibrin.

Fibrin formed in mixtures of purified fibrinogen and thrombin has little tensile strength and is soluble in such agents as 5 M urea and 1% monochloroacetic acid. Although these observations suggest that the fibrin monomers are held together by hydrogen bonds, physicochemical evidence suggests that they are actually linked by readily dissoluble covalent bonds (171). In whole blood, however, the fibrin formed is normally of considerable tensile strength and is insoluble in urea and monochloroacetic acid. The chemical links that make this possible are created through the action of fibrin-stabilizing factor (Factor XIII) (317, 558). This enzyme, inert in circulating plasma, is changed to an active form by thrombin (360). Then, in the presence of calcium ions, it induces the formation of firm chemical bonds by transamidation between adjacent fibrin monomers. In this reaction, the 7-amides of glutamine residues serve as acceptors, and the €-aminogroups of lysine as donors (357). Hereditary disorders of fibrinogen and fibrin-stabilizing factor are instructive in relation to the defenses of the body. As mentioned earlier, individuals congenitally deficient in fibrinogen get along reasonably well unless injured or subjected to surgery or childbirth. Although they may suffer hemarthrosis after injury, permanent damage docs not ensue. Hereditary deficiencies of fibrin-stabilizing factor are associated with a bleeding tendency. In a few such cases, once a wound has stopped bleeding, it breaks down repeatedly, leading to poor healing and abnormal scar formation (155). Why this is not universally the case is not known. The failure of wound healing has been related to impaired growth of fibroblasts which is observed in tissue cultures containing plasma deficient in fibrin-stabilizing factor (39). A third type of hereditary abnormality in fibrin formation is synthesis

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151

of a structurally abnormal fibrinogen. About ten families have been discovered in which this abnormality exists; the fibrinogens in these families seem to differ from each other ( 4 1 0 ) .The functional defect appears to be an abnormality in aggregation of fibrin monomers or soluble polymers (194, 689), and, in three individuals, operative wounds have dehisced (194, 409), as if the fibrin were inadequate for wound healing. Several patients have had recurrent thrombosis, but how this comes about is not known ( 3 8 ) . C. FmmNoID In many pathological processes, material resembling fibrin in its staining properties is laid down extravascularly ( 1 0 ) . This “fibrinoid” differs in its composition in different situations (693). In the lesions of thrombotic thrombocytopenic purpura, renal cortical necrosis, or the experimental generalized Shwartzman reaction, for example, one constitutent of fibrinoid is chemically related to fibrin, as demonstrated immunologically ( 208, 685) and by electron microscopy ( 618). Conceivably, the deposited material contains fibrin itself or incompletely formed fibrin, chemically altered fibrin, or complexes of fibrinogen, fibrin monomer, or soluble fibrin polymers with each other or with other plasma or connective tissue constitutents. Similar material can be demonstrated in rabbit glomeruli after the injection of sodium polyanetholsulfonate (Liquoid), thromboplastin, or thrombin ( 6 8 4 ) .

D. FIBRINOGEN AND CIIEMOTAXIS Utilizing a skin window technique, Riddle and Bamhart (553) found that eosinophiles and neutrophiles migrated to the site of instillation of homologous fibrinogen. A similar, but more protracted “chemotactic” response was evoked by fibrin. Fibrin split products, derived by incubation of fibrin with plasmin (Section III,A), were even more effective (32). Spector and Willoughby ( 6 3 4 ) , using a grossly different method, also noted that bovine fibrinogen was slightly chemotactic in rat skin. If these results can be applied to the inflammatory response, the fibrin which forms an integral part of the reaction may, in turn, accelerate it. E. THROMBIN The formation of fibrin and the activation of fibrin-stabilizing factor are brought about by thrombin, an enzyme not found in freshly shed normal blood but evolving rapidly under appropriate conditions. Thrombin also induces the aggregation of platelets and clot retraction, and it alters antihemophilic factor and proaccelerin, enhancing their role in

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thrombin formation; whether these two factors can function in the absence of thrombin is not entirely clear. Thrombin is said to hydrolyze the oxidized B chain of insulin ( 160) and heat-denatured albumin (655), but it is doubtful that these actions are of physiological sigmficance. Thrombin also hydrolyzes a number of synthetic substrates, including esters of N-substituted arginine or lysine (160, 170,609),several pnitrophenyl esters ( 359, 39Q), and benzoylarginyl-pnitroanilide (523); of these, the most studied is TAME. Thrombin’s active center contains the sequence glycyl-aspartyl-seryl-glycyl-glutamyl-alanine, resembling several other proteases (210). It is inhibited by DFP (209, 425) and phenylmethylsulfonylfluoride ( 81, 596), which react with the serine residue at the active center, but not by SBTI (223). Among other inhibitors of thrombin, hirudin, a protein derived from leeches, and heparin are particularly useful in the laboratory.

F. THE FORMATION OF THROMBIN Thrombin is a split product of prothrombin (Factor 11), a soluble plasma protein. Much materia1 has been published concerning the problem of how this transformation comes about. Two major sequences have been elucidated. In one, the extrinn’c pathway, clotting is initiated by the injured tissue with which blood comes in contact (Fig. 2 ) . The tissue furnishes an ill-defined complex, tissue thromboplastin, which, in the test tube, produces an explosive formation of thrombin. Tissue thromboplastin is composed of at least two entities-a heat-labile protein, probably derived from microsomes (104, 727), and phospholipid (464). The protein component interacts with a plasma protein, Factor VII (precursor of

1

TISSUE THROMBOPLASTIN FACTOR (PRO-SPCA)

t

STUART F. ( X I

ACT. STUART F:

PROACCELERIN (V)

PROTHROMBINCONVERT I NG PRINCIPLE

PROTHROMBIN ( 1 1 )

THROMBIN

FIG. 2. The extrinsic pathway of thrombin formation. (SPCA=serum prothrombin conversion accelerator.)

HEMOSTASIS A N D OTHER DEFENSE MECHANISMS

HAGEMAN F.(XII)

153

n ACT. H'AGEMAN

RT.A.(XI)

F.

ACT. P.T.A.

m\

/

A.H.F (Vlll)ltilLDMe"

XMAS E (1x1 ACT. XMAS E *ALTERED A.H.F.

n

phospholipid

STUART E ( X ) PROACCELERIN (V) THRoMB"

ACT. STUART F:

bALTERED PROACCELERIN c a* phospholipid

'hPROTH RO M B IN CONVERTING PRINCIPLE

PROTHROMBIN ( I I ) THROMBIN

FIG.3. The intrinsic pathway of thrombin formation. (PTA = plasma thromboplastin antecedent.)

serum prothrombin conversion accelerator, autoprothrombin I ) and calcium ions to form a clot-promoting agent which then changes another plasma protein, Stuart factor (Factor X), into an active form (plasma thrombokinase, autoprothrombin C ) ( 4 6 3 ) . In all probability, activated Stuart factor itself splits prothrombin into thrombin and an inert fragment (427, 603). Its action requires the presence of calcium ions, proaccelerin, and phospholipid; the last is derived from tissue thromboplastin, from platelets, or from the plasma itself (178,248).Both activated Stuart factor and proaccelerin are bound to the phospholipid micelles and in this state serve as a "prothrombin converting principle" which liberates thrombin enzymatically (503). The efficiency of proaccelerin is greatly enhanced if it is first altered by thrombin; the same enzyme ultimately destroys it (505, 701 ) . The second sequence leading to the formation of thrombin, the intrinsic pathway, does not depend upon the presence of tissue thromboplastin (Fig. 3). Blood, drawn under conditions minimizing its contamination with tissue, clots readily upon contact with glass or certain other foreign surfaces. Bordet and Gengou ( 7 4 ) first showed that the clot-

154

OSCAR D. RATNOFF

promoting effect of these surfaces, originally described by Lister (356), depended upon a component of plasma. In 1956, Shafrir and deVries (600) and Margolis (389) reported that the clot-promoting effect of glass was not mediated by the clotting factors then generally recognized. Taking advantage of their techniques, Rosenblum and I (547) observed that glass reacted with a specific plasma protein, Hageman factor (Factor X I ) . This agent, the presence of which has been suspected earlier (537), is an enzymelike substance deficient in the plasma in a hereditary disorder, Hageman trait (531). Well over a hundred individuals with Hageman trait have now been recognized. The abnormality is inherited as an autosomal recessive characteristic (525). Predictably, the blood or plasma of patients with Hageman trait clots abnormally slowly in glass tubes. Carefully prepared native Hageman factor-deficient plasma, collected in silicone-coated apparatus without addition of an anticoagulant, is incoagulable, whereas whole blood ultimately clots, as if the cellular components can initiate clotting (531). Plasma diluted with anticoagulant and recalcified also clots in glass tubes, as though traces of Hageman factor were present, or glass initiates clotting through an action upon another plasma constituent. Strikingly, although the defect measured in the laboratory is as gross as that of many hemophiliacs, Hageman trait is not ordinarily associated with a bleeding tendency. A few patients have bled excessively after major challenges such as surgery, but even then usually only to a mild degree. These observations point to the possibility that mechanisms other than the activation of Hageman factor may initiate clotting in the body. Supporting this, several individuals with Hageman trait have suffered myocardial infraction (212,256). The reductw ad absurdem is the recent death of Mr. Hageman, the first patient studied, from pulmonary embolism subsequent to fracture of the pelvis (530). No unusual pathological features were noted at autopsy. Hageman factor, the agent deficient in the plasma of patients with Hageman trait, has been partially pursed from human (535, 636) and ) It is a protein which, in sucrose gradients, has a bovine ( 5 9 1 ~ plasma. sedimentation coefficient of 5 S (147), and its molecular weight has been . ( 5 9 1 ~believes ) estimated to be about 80,000 ( 1 4 7 , 5 9 1 ~ )Schoenmakers that bovine factor is a glycoprotein; we have not detected carbohydrate in our human preparations. The clot-promoting effect of glass appears to represent an “activation” of the Hageman factor molecule. The nature of this activation is not known, but it is associated with a molecular alteration such that activated Hageman factor sediments rapidly to the bottom of sucrose gradients, is

HEMOSTASIS AND OTHER DEFENSE MECHANISMS

155

excluded from columns of Sephadex G-200, and fails to penetrate acrylamidc gels upon disc electrophoresis (147). These observations are compatible either with the view that activation is a polymerization process, such as has been described for phosphorylase B (298), or that Hageman factor becomes hydrophobic. Innumerable insoluble substances, having in common a negative surface charge (263) initiate the clotting of platelet-poor plasma, presumably by activating Hageman factor. Among these agents are kaolin, diatomaceous earth, talc, silica, barium carbonate, and asbestos ( 5 2 5 ) . Some of these may be introduced into the body one way or another. Microcrystalline sodium urate, calcium pyrophosphate, and L-homocystine activate Hageman factor and may be responsible for some of the clinical features of acute gouty arthritis, pseudogout, and homocystinuria. Spider webs, a folk remedy for bleeding, have a negative surface charge (263) and activate Hageman factor (546). Recent experiments provide some insight into ways in which Hageman factor may be activated physiologically. Collagen fibers are thought to have the capacity to activate this clotting factor, although the evidence is not yet entirely convincing (120, 471, 728). Perhaps, when the continuity of a blood vessel is interrupted, the Hageman factor in plasma is activated by collagen-like protein to which it is exposed. Contact of blood with skin also activates Hageman factor; the stimulus seems to be the surface layer of sebum ( 475, 481 ) . Controversial evidence concerning the activation of Hageman factor by antigen-antibody aggregates is reviewed in Section VI. The clot-promoting agents described thus far are all insoluble. Two substances have been found which, in solution, activate Hageman factor. One of these, ellagic acid ( 4,4’,5,9,6,6’-hexahydroxydiphenicacid 2,6, : 2’,6’-dilactone) was found in a study of the clot-promoting properties of aged tannic acid solutions (533). In concentrations as low as M, ellagic acid accelerates clotting by activating Hageman factor. The properties of this compound may depend upon the presence of adjacent hydroxyl groups on each of its benzene rings and upon the lactone bridges which give the molecule an internal rigidity. The ready availability of ellagic acid has made it useful to study Hageman factor in the living animal. The second agent, cellulose trisulfate, has been investigated only recently (296); its structure only vaguely resembles that of ellagic acid, and it has no free hydroxyl groups. The procoagulant function of activated Hageman factor is the activation of a second substance, PTA (Factor XI). Rate studies suggest that the change in PTA is brought about enzymatically, but the bonds affected are unknown ( 536). Schoenmakers

156

OSCAR D. RATNOFF

(591) reported that bovine Hageman factor hydrolyzes N-benzoyl-Larginine ethyl ester, an effect blocked by DFP. In our own studies, human Hageman factor is not inhibited by DFP (535),and the slight esterolytic activity of our preparations is not blocked by specific antiserum, so that we are not yet able to point to a synthetic substrate of this agent (526). Other functions of activated Hageman factor, the activation of PF/Dil, plasma kallikreinogen, plasminogen, and C’1 esterase, will be discussed in subsequent sections. Plasma thromboplastin antecedent was first detected because its hereditary deficiency is accompanied by evidences of a bleeding tendency, usually mild (571). The plasma of affected individuals usually contains between 5 and 2% of the PTA activity of normal plasma, complicating experimental studies in which it is used. Since patients with Hageman trait do not bleed, mechanisms other than that mediated by Hageman factor must exist through which PTA is activated. In agreement with this, preparations of PTA derived from Hageman factor-deficient plasma gradually become active during purification (536). Plasma thromboplastin antecedent is a protein, only partially studied at this time. After many false starts, I have been able to purify it about 5000-fold compared to the plasma from which it was derived (528).Such preparations still contain two components, as detected by disc electrophoresis. More importantly, they possess at least four biological activities, the capacity to promote clotting, to increase vascular permeability, to release plasma kinins, and to induce fibrinolysis. At this writing, I do not know whether our preparations, however pure, are contaminated with agents acting like PF/Dil, plasma kallikrein, or an activator of plasminogen, or whether PTA possesses all these properties, an idea propagated in part by Margolis (395). But PF/Dil and plasma kallikrein are inhibited by SBTI, which is without effect on the procoagulant properties of PTA. Studies with much less purified preparations of PTA have suggested that this substance is an enzyme; it is inhibited by DFP, which appears to attach to a serine residue ( 3 0 2 ) . The procoagulant function of activated PTA is its activation of Christmas factor (Factor IX), an agent deficient in the plasma of patients with a hemophilia-like disorder, Christmas disease (534). Christmas factor has not yet been purified. Once activated, Christmas factor participates with antihemophilic factor (Factor VIII), the agent deficient in classic hemophilia, to form an agent which, like tissue thromboplastin and Factor VII, can convert Stuart factor to its activated form. The nature of this interaction is not yet known, but calcium ions and phospholipids must be

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present. The subsequent steps of the intrinsic and extrinsic pathways are identical. The fact that antihemophilic factor functions much more efficiently after it has been altered by small amounts of thrombin suggests a parallel with the formation of the prothrombin-converting principle (512). At first it was thought that activated Christmas factor was an enzyme that converted antihemophilic factor to an activated form ( 534). No convincing evidence that this takes place has been forthcoming, and I have been unable to demonstrate any alteration of bovine antihemophilic factor by activated Christmas factor (523). G. PROCOAGULANTPROPERTIES OF TRYPSIN,SNAKEVENOMS, AND STAPHYLOCOAGVLASE The formation of thrombin may come about through the action of enzymes not normally present in human plasma. Thus trypsin induces the generation of thrombin in uitro, both by activating Stuart factor and by proteolytic splitting of prothrombin (5). Although these effects may be of local importance in pancreatic disease, free trypsin probably never exists in the circulating blood in sufficient amount to induce coagulation since plasma possesses antitryptic activity. More pertinent to a review of the defenses of the body are the clotpromoting properties of various snake venoms. Not unexpectedly, the venoms of different species behave in grossly different ways. For example, the venom of Bothrops jararaca, a poisonous South American lance-head viper, clots fibrinogen directly, although in the process it splits off only fibrinopeptide A (67). The venom of the Malayan pit viper (Ancistrodon rhodostoma) and certain other snakes also clots fibrinogen, defibrinating the victim (550). The patient is none the worse for this experience unless he is otherwise wounded, in which case he bleeds like anyone else with afibrinogenemia. Defibrination of experimental animals with Malayan pit viper inhibits experimental thrombosis (398), a property which has been utilized in the treatment of patients with thrombotic disease. The venom of the American copperhead snake (Ancistrodon contortrix contortn'x) , a species similar to the Malayan pit viper, also clots fibrinogen weakly (80), but I am unfamiliar with studies in experimental animals. Tiger snake (Notechis scutatus scutatus) venom behaves in the same way as activated Stuart factor, converting prothrombin to thrombin if phospholipid, proaccelerin, and calcium ions are available (280), whereas that of the Russell's viper (Vipera msse2lii) has at least two properties-activating Stuart factor (375) and altering proaccelerin in a manner similar to thrombin (252, 504). Although Factor VII and Stuart factor are closely

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similar, Herzig and I (249) have not been able to discern an effect of Russell’s viper venom upon Factor VII. One additional way in which fibrin may form deserves notice, although from our parochial human point of view it can hardly be called a defense mechanism. Pathogenic strains of Staphylococcus aureus produce a toxin, staphylocoagulase, which initiates clotting in human blood by reacting with a plasma constituent, “staphylocoagulase-reactingfactor” (623, 649). This factor is either prothrombin itself, or a closely related substance ( 578, 650). Staphylocoagulase is presumably responsible for the thrombi that form in blood vessels near the site of staphylococcal infection, decreasing the blood supply to the infected area; perhaps local fibrin formation also protects the bacteria from phagocytosis. To attribute the localization of staphylococcal infections to staphylocoagulase is a recurrent attractive idea.

H. INHIBITORSOF BLOODCLOTTING As might be expected, circulating blood is rich in agents that inhibit

the clotting system and thus reduce the chance of inadvertent thrombosis.

As Margolis (389) &st noted, the clot-promoting activity which evolves when plasma comes into contact with foreign surfaces gradually disappears. Both activated Hageman factor and activated PTA lose activity. One agent responsible resides in the a-globulin fraction of plasma ( 4 7 6 ) . I was impressed that the destruction of clot-promoting activity was temperature-dependent and wondered whether inhibition was enzymatic (521), Although Nossel’s (476) observation that SBTI protected activated PTA seems to support t h i s view, our experiments are not in agreement with this observation (522). Activated Stuart factor, introduced into the bloodstream, is rapidly inactivated by the liver ( 6 2 5 ) .Additionally, plasma itself slowly inhibits this activated clotting factor (132). Serum also inactivates tissue thromboplastin, an activity requiring the presence of calcium ions (658). Whether the effect of serum is directly upon tissue thromboplastin or upon a complex of thromboplastin and Factor VII (253) is not known to me. Several mechanisms are available to inactivate thrombin. As fibrin forms, it adsorbs and inactivates thrombin, limiting its action (509). Moreover, plasma contains an inhibitory agent, antithrombin 111, which slowly inactivates this enzyme (507).The digestion products resulting from the action of plasmin upon fibrinogen also inhibit fibrin formation ( Section II1,A).

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The efficiency of these various inhibitory mechanisms is apparent from experiments in which large amounts of ellagic acid, a potent activator of Hageman factor, were infused intravenously. No evidence of thrombosis could be found unless a segment of artery or vein was isolated after the injection of ellagic acid. Under these conditions, a clot appeared in the static portion of blood ( 7 7 ) . These experiments emphasize the capacity of the body to protect itself against the effects of activated clotting factors, and the importance of stasis in the formation of thrombi. Heparin has not been demonstrated in the free state in human plasma. The addition of heparin to plasma inhibits the activation (534, 602) or action (479) of Christmas factor, Heparin also inhibits thrombin, a process potentiated by a plasma fraction designated antithrombin I1 but not clearly separable from antithrombin I11 ( 2 6 2 ) . Heparin originates principally in mast cells ( 2 5 7 ) .It is a mucopolysaccharide, esterified with sulfuric acid which, because of its strong negative charge, has many biological activities. Among these are the inhibition of the formation of PF/Dil and inactivation of this enzyme and of kallikrein, both of which participate in the elaboration of kinins. Conceivably, heparin, liberated from mast cells by many stimuli, may participate in the local response to injury. Ill.

Fibrinolytic Phenomena

Toward the end of the nineteenth century, Denys and de Marbaix (130) made the critical observation that canine fibrin dissolved rapidly in serum which had been treated with chloroform or ether. Shortly thereafter, Dastre (118) found that such fibrin, incubated in its own serum, would gradually dissolve even in the absence of an organic solvent. Delezenne and Pozerski ( 129 ) extended these observations, reporting that chloroform released an enzyme from dog serum which was capable of digesting gelatin and casein. Shortly thereafter, Hedin (247) localized the source of the proteolytic activity to the euglobulin fraction of plasma. Since these pioneer studies, a massive literature has accumulated, attempting to elucidate the nature of the plasma’s proteolytic properties and to link these to the defense mechanisms of the body. The fibrinolytic activity of plasma resides in the one or more enzymes known collectively as plasmin (102) or fibrinolysin (358), found in freshly drawn blood as an inert precursor, plasminogen or profibrinolysin. Clots prepared from normal human plasma by recalcihation or the addition of thrombin often undergo liquefaction when incubated at 37°C. under sterile conditions, a phenomenon attributed to formation of plasmin. Fibrinolysis is ordinarily slow; complete lysis, if it occurs, usually

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taking several days. But if the blood is drawn from an individual with chronic hepatic disease (216, 519) or from someone who has been subjected to such stresses as exercise ( 6 2 ) , anxiety (376), shock (651), or the injection of typhoid vaccine (234), lysis is more rapid and may take place within hours. The lysis of clotted whole blood is usually slower and often is not discernible. In contrast, the euglobulin fraction of plasma, clotted by recalcification or thrombin, often undergoes fibrinolysis in 6 or 8 hours, and under conditions of stress or in the presence of hepatic disease, within an even shorter time.

A. SUBSTRATES OF PLASMIN Plasmin in an enzyme of broad specificity, digesting not only fibrin, but fibrinogen (216), proaccelerin (4, 139, 350), antihemophilic factor ( 4 , 139, 694), prothrombin ( 4 , 597), and perhaps other clotting factors as well (139, 192). The digestion of fibrinogen or fibrin takes place at several intramolecular sites, so that products of varying size appear in digests of these proteins (387, 478). These “split products” are potent inhibitors of clotting, manifesting their effects in several ways. They block both the formation of thrombin (469,672) and of fibrin (192,468, 672). Fibrin formation is inhibited both by competition of split products for the action of thrombin (695) and by interference with the normal polymerization of fibrin monomers (8, 320, 673). The result is a grossly abnormal network of fibrin, demonstrable by electron microscopy ( 3 1 ) . Whether these actions of split products are important in localized areas of fibrin formation is not known, but these agents may contribute to the systemic bleeding tendency of patients undergoing fibrinolysis. The susceptibility to hemorrhage is compounded by adsorption of the split products to platelets, the aggregation of which by ADP and thrombin is thereby inhibited (310). The anticoagulant split products of fibrinogen appear to limit fibrinolysis, an example of product inhibition (460). The possibilities that fibrin split products serve as stimuli to the synthesis of fibrinogen ( 3 3 ) and are chemotactic for eosinophiles and granulocytes ( 3 2 ) have also been raised. Plasmin digests 7-globulin (274), ACTH (429), glucagon (429), somatotropin (429), and possibly, but not certainly, insulin (516). Besides these properties, it has at least seven functions related to the defense mechanisms of the body. It converts the C’ls subcomponent of C’1 to C’1 esterase (334, 542); it inactivates C’1 esterase (542) and C’4 ( 3 2 1 ) ; it releases chemotactic substances and permeability enhancing activity from C’3 (699);it forms biologically active polypeptide kinins by one or more mechanisms ( 2 8 ) ;and it digests the protein of the chondromucoproteins

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of cartilage (315). Many of these activities take place, in the test tube, at concentrations of plasmin similar to those potentially available in the body; fibrinolysis has received the most attention for historic reasons and because of the ease with which it is demonstrated. Besides the substrates that it might find in nature, a number of other substances are digested by plasmin, such as cascin (247), gelatin (247), and denatured hemoglobin (221). Additionally, azocoll ( collagen coupled to an azo dye) (669), P-lactoglobulin (101), and complexes of protamine with insulin ( 8 9 ) or heparin (304) are digested by plasmin. Plasmin also digests many synthetic amino acid esters and amides (674); although TAME has received the most attention, lysine esters are particularly susceptible ( 674), especially N-acetylglycyl-lysine methyl ester (459).

All these observations suggest that plasmin is similar in many ways to trypsin, and, indeed, it is inhibited by soybean (221, 428), pancreatic (102, 428), and lima bean (221) trypsin inhibitors, by Trasylol ( a polypeptide derived from beef parotid glands which may be identical with pancreatic trypsin inhibitor) (400), and by DFP (439). Although the active group in plasmin has not been identified, indirect evidence suggests that it may include the same aspartyl-seryl-glycl grouping present in trypsin and thrombin (568). B. THE ACTIVATION OF PLASMINOGEN Plasminogen can be converted to plasmin in many ways. Exposure of plasma or its euglobulin fraction to organic solvents, notably chloroform, gradually increases its fibrinolytic and proteolytic activity, perhaps by denaturation of plasmin inhibitors (129, 517). Activation by chloroform depends upon the presence of Hageman factor (482). Tillett and Garner (667) reported that bacteria-free filtrates of phemolytic streptococci dissolved fibrin; the active principle is now called “streptokinase.” Milstone (426) found that the streptococcal agent acted only in the presence of a “lytic factor” in plasma which Kaplan (288) and Christensen and MacLeod (100, 102) later identified as plasminogen. First thought to act enzymatically, streptokinase reacts stoichiometrically with plasminogen, forming a complex with proteolytic activity (123,517, 652). Once a small amount of such plasmin evolves, it further activates both human and bovine plasminogen, although the latter by itself is quite resistant to streptokinase. Other bacterial products also activate plasminogen, notably staphylokinase, derived from Staphylococcus aureus (205), but much less is known about their properties. It takes little imagination

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to invent roles for these bacterial products in the pathogenesis of inflammation. Plasminogen can also be changed to plasmin by exposure to various tissues (19,191 ) and body fluids. The activating properties of tissues are most clearly evident in vascular endothelium (668, 702). Urine contains a potent activator of plasminogen, urokinase, which behaves like a protease (96, 689, 726) and hydrolyzes various synthetic substrates, notably N-acetyl-L-lysine methyl ester ( 605). Since human urokinase is not antigenic for man (374), it is under intensive study as an agent to induce therapeutic fibrinolysis. None of these observations seems to bear upon the mechanisms by which clotted plasma or its euglobulin fraction undergoes fibrinolysis. This phenomenon has been attributed to the presence of a plasminogen activator in blood (348, 451, 590). The rate of fibrinolysis has also been correlated with the rate at which a plasma inhibitor of plasmin deteriorated in vitm (518). But the declining titer of inhibitor may reflect its binding to freshly formed plasmin and is, therefore, less of a clue to the pathogenesis of fibrinolysis than I once thought. The conversion of plasminogen to plasmin is related to blood clotting in several ways. Fantl and his associates (182,183) believed, contrary to other investigators, that fibrin was a more susceptible substrate of plasmin than fibrinogen. Mullertz (450) and I (520) interpreted experiments similar to Fantl's to mean that the clotting process or the presence of fibrin accelerated plasmin formation. One attractive explanation is that plasminogen is adsorbed to fibrin and physically separated in this way from plasma. There it is more readily activated and can attack its substrate, fibrin, without interference from circulating inhibitors ( 7 ) . These conjectures may explain why fibrin is digested more readily than fibrinogen, but not how clotting may initiate fibrinolysis. Niewiarowski and Prou-Wartelle (470) and Iatridis and Ferguson (271) related spontaneous fibrinolysis in the presence of kaolin to the presence of Hageman factor; the latter investigators believed that the effect of activated Hageman factor upon plasminogen was mediated through a plasma activator ( 2 7 2 ) . Ogston, Ogston, Forbes, and I (482), reexamining this problem, separated a potent activator of plasminogen from normal or Hageman factor-deficient plasma by adsorbing plasma with Celite, as previously reported by Mackay ( 3 7 7 ) . This material, when partially purified, was already in an activated form. In all probability it is inert in freshly drawn plasma. The steps we envision involve sequential interactions among Hageman factor and a foreign surface, one or more "plasminogen activators" (noncommittally named "Hageman factorcofactor"), and plasminogen (482). These experiments may help to ex-

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plain one way in which clotting initiates fibrinolytic phenomena, but at present no evidence exists that Hageman factor-cofactor is related to the plasminogen activators of plasma detected in liver disease and under conditions of stress. The activation of plasminogen can be inhibited by EACA and its congeners both in uitro ( 2 , 6, 138) and in viuo ( 6 0 8 ) . Unexpectedly, under some experimental circumstances, EACA enhances the activation of plasminogen which occurs spontaneously (141 ) or upon addition of chloroform ( 1 4 5 ) . r-Aminocaproic acid has many important effects upon the body's defense mechanisms which will be described subsequently, but it is unnecessary to assume that these are all related to inhibition of the activation of plasminogen. C. PLASMA INHIBITORSOF PLASMIN Circulating plasma contains several potent inhibitors of plasmin, which presumably serve to limit its function. In one study, three inhibitory fractions were differentiated by rather gross characteristics: one was labile at 56"C., one was destroyed by ammonia, hydrazine, and certain primary amines, and one resisted both of these treatments ( 5 4 0 ) .Another report separated plasma inhibitors of plasmin into two groups-one of which reacted rapidly and was localized to the a,-globulin fraction of plasma, the other of which inactivated plasmin slowly and resided in the a2-globulin fraction ( 4 7 4 ) . A third set of experiments demonstrated that C'1 esterase inhibitor (Section V,A) was an effective antiplasmin, but its relationship with the other plasmin inhibitors was not investigated ( 5 4 4 ) . These three studies are but samples of the confusion that exists in this area. Plasma is also rich in inhibitors directed against trypsin, but the bulk of antiplasmin activity can be differentiated from these substances ( 5 9 2 ) .One conclusion to be drawn is that careful studies in this area will be of great help in elucidating the functions of plasmin. The foregoing description of the fibrinolytic properties of plasmin is most abbreviated and is designed only to provide a point of orientation for its role in inflammation and immune processes. Several recent reviews may be helpful to the reader (133, 184, 607, 639, 690). Whatever its importance, plasmin may well turn out to be an enzyme essential for mammalian life, since not one instance of a hereditary deficiency of this enzyme has been found. IV.

The Inflammatory Process

Inflammation defines the local defense reactions to the presence of injurious stimuli ( 4 1 4 ) . The term encompasses the sweep of events from the earliest stages, in which increased vascular permeability, vasodilata-

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tion, and liquefaction of the extracellular matrix take place, through the final steps of healing at the site of the damage ( 5 2 7 ) .The nature of the response varies with the particular type of injurious agent, the site attacked, and the physiological state of the host. The concept that the early response to injury is mediated chemically received its first major support from Lewis ( 3 5 2 ) ,who proposed that the vasodilatation and increased vascular permeability which appeared immediately after trivial injury were attributable to the combined effects of a local axon reflex and the liberation of a histamine-like substance. Since Lewis’ day, innumerable chemical mediators have been described, as well as mechanisms for their release too numerous to encompass in a review many times larger than this. Instead, in this and succeeding sections, my aim will be to trace the history of knowledge linking the humoral mediation of inflammation with blood clotting, fibrinolysis, and immune mechanisms. The repetitious and overlapping nature of what follows reflects the thesis of this review that the processes of nature are not discrete but represent generalized reactions which we have categorized for our own convenience. MEDIATOR OF INFLAMMATION The humoral theory of inflammation derives from an experiment of utmost simplicity. Lewis (352) stroked the skin firmly with a ruler. After a few seconds, the line of the stroke reddened. A few seconds later, the line was surrounded by a dif€useblush, and about a minute after this, a wheal appeared along the line of the stroke. Sectioning cutaneous nerves abolished the blush but not the initial red reaction or wheal formation. Since histamine, pricked into the skin, reproduced this “triple response,” Lewis postulated that this amine or some similar “ Hsubstance was a mediator of the reaction to injury. Considerable evidence has accumulated that histamine may be released early during inflammation. For example, histamine has been identified in early inflammatory exudates (630) and in the lymph draining an experimentally injured limb ( 1 5 9 ) . Two phases can be discerned in the response to certain experimental injuries ( 4 2 0 ) . The early increase in vascular permeability after such stimuli as mild thermal bums (632, 722), the intrapleural administration of turpentine ( 631 ) , or the intracutaneous injection of antigen into a sensitized animal (246) is largely attributable to the release of histamine. Rut the second, delayed increase in vascular permeability is not altered by the administration of antihistaminics or of agents that deplete the tissues of histamine such as compound 48/80 or polymyxin B (631). Nor can histamine account for all manifestations of

A. HISTAMINE AS

A

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inflammation; thus, it does not induce the migration of leukocytes, a major feature of the inflammatory response ( 7 0 ) . The major source of histamine liberated during inflammatory reactions is the ubiquitous mast cell ( 5 5 4 ) . Histamine is stored within its granules; an early sign of inflammation is degradation of mast cells. After various stimuli, moreover, histamine is probably synthesized rapidly by mast cells, through decarboxylation of histidine ( 5 8 8 ) . Histamine is also present in platelets, which accumulate quickly at the point of vascular injury; the concentration varies remarkably from species to species. The degree to which the discharge of histamine from platelets contributes to the inflammatory response to antigen-antibody complexes is discussed subsequently (Section VI). A number of studies concern the microscopic events following the injection of histamine. Minute amounts bring about dilatation of muscular vessels (739), while fluid apparently seeps through tiny spaces which open at the junctions between the endothelial cells of the smallest venules (381, 382). Material then passes into the extracellular spaces through areas of thinning and actual breaks in the basement membrane ( 1 1 4 ) . An attractive hypothesis, that the increase in vascular permeability results from increased hydrostatic pressure brought about because histamine constricts veins (575), has not been confirmed ( 6 1 7 ) . Rather, the endothelial cells appear to contract so that there is a limited tearing apart of the endothelial cells ( 3 8 0 ) . After these initial changes, platelets and leukocytes adhere to the damaged wall, and the inner surfaces of the vessels are so altered that they are capable of engulfing intravenously injected colloidal carbon (740).A similar course of events may explain the actions of 5HT and polypeptide kinins.

B. SEROTONIN AS A MEDIATOROF INFLAMMATION In 1884, Stevens and Lee (645) reported that a vasoconstrictor substance was liberated during blood clotting. Later, Janeway, Richardson, and Park (275) demonstrated that the source of the vasoconstrictor was the platelet. A modem re-examination of these experiments identifies the vasoconstrictor in serum (513) and platelets (511) with 5HT. The extent to which 5HT is an inflammatory mediator varies from species to species. The mast cell granules of the rat are particularly rich in this agent ( 4 9 ) but this is not true in other mammals. In most species the platelets are a major depot of 5HT (Section II,A), and their disintegration releases 5HT into the surrounding medium. Serotonin is particularly important in the pathogenesis of inflammation in rats, in which it increases vascular permeability (576), contracts

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some smooth muscles ( 8 3 ) ,induces a modest migration of leukocytes to the site of its injection ( 6 3 4 ) , and enhances the inflammatory response around subcutaneously implanted polyvinyl sponges (589). Further, it can be demonstrated in early turpentine-induced pleural exudates in rats, but not at a later period ( 6 3 0 ) . In other species, the importance of 5HT in the mediation of idammation is less certain. Extracts of platelets and 5HT itself are extremely painful when applied to the base of a blister in human subjects ( 1 3 ) ,but pain is unaccompanied by edema; 5HT does not increase vascular permeability in species other than the rat (422, 628). Although 5HT constricts rat venules, its effect upon the vessels of other species is quite variable. It is a powerful constrictor of pulmonary vessels (579) and has been implicated as contributing to the endocardia1 fibrosis and valvular lesions in patients with carcinoid tumors (373),although t h i s is not established. In the rabbit, 5HT sensitizes muscular venules to constriction by catecholamines and alters capillary and venular endothelium so that it can phagocytize carbon particles ( 7 4 0 ) . In this species, the combined intracutaneous injection of 5HT and catecholamines results in a diffuse hemorrhagic reaction ( 7 4 0 ) . Despite this rather denigrating view of the role of 5HT in human inflammatory responses, I suspect that it is probably of considerable importance, arguing from its release from platelets under many experimental circumstances. C. POLYPEPTIDE KININS AS MEDIATOF~S OF INFLAMMATION The simplistic view that histamine is the mediator of inflammatory processes was sharply challenged by Menkin in a series of papers which presage much of present-day thinking. He (411) rejected the view that histamine is the primary agent responsible for the increase in vascular permeability characteristic of inflammation. Instead, Menkin attributed this change to the elaboration of a thermostable agent, which he named leukotaxine because it also seemed to attract leukocytes to the damaged area. Leukotaxine was thought to be a polypeptide (412, 413) of intracellular origin ( 4 1 4 ) . Menkin (414, 415) extracted a host of other active agents from inflammatory exudates, including necrosin ( a generic name for various proteoIytic enzymes ) , both thermolabiIe and thermostable pyrogens, a leukocytosis-inducing factor, and a second permeabilityenhancing substance, exudin. Menkin may have minimized the importance of histamine because the lesions he studied were too advanced. His experiments were subjected to sharp criticism (156), but his view that some of the events of idamma-

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tion were induced by polypeptides, released locally, has a modem sound. Two converging lines of evidence have supported his basic thesis, although the polypeptides Menkin isolated were probably not the most important. In 1909, Abelous and Bardier (1) made the important discovery that human urine contains a nondialyzable hypotensive agent. Similar hypotensive agents have been found in pancreatic juice (493) and saliva (721). Werle, Frey, and their associates attributed the fall in blood pressure induced by these excretory and secretory fluids to the presence of a group of agents to which they gave the name kallikrein (196). Pancreatic kallikrein appears to be synthesized as an inert precursor which can be activated by pancreatic trypsin ( 715). The kallikreins contract smooth muscle, such as guinea pig ileum and the estrous rat uterus, and increase vascular permeability. Their action requires the presence of plasma ( 714) ; kallikrein splits off a small molecule, kallidin ( lysyl-bradykinin) from a precursor globulin, kallidinogen, kininogen, or prokinin (714, 717, 719); it is this small molecule which brings about the biological effects of kallikrein (Fig. 4). The hydrolytic properties of pancreatic and salivary kallikrein, responsible for generating kallidin, are reflected in their capacity to hydrolyze synthetic amino acid esters, such as those of arginine ( 671, 707), and tyrosine ( 707), and by their inhibition by pancreatic trypsin inhibitor (708); SBTI lacks this function (346, 708, 716). Kinin-forming enzymes have also been found in cerebrospinal fluid (99), tears ( 346), synovial fluid ( 294), sweat (195), and leukocytic extracts ( 406, 733), as well as in various tissues. In 1928, Kraut (311) reported the exciting observation that plasma, too, contained kallikrein-or, more exactly, a precursor of kallikrein. In their early experiments, this precursor, kallikreinogen, was activated by treating plasma with acetone (312) or papain (311). Activation can also be brought about by incubating plasma with trypsin (193, 718) or plasmin (686), or by acidification (227, 312), or dilution (585). The role of blood clotting in the activation of kallikreinogen is discussed in Section BRADY K IN I N ARG-PRO-PRO-GLY-PHE-SER-PRO-PHE-ARG-OH LYSYL-BRADYKININ (KALLIDIN) LYS-ARG-PRO-PRO-GLY-PHE-SER- PRO-PHE-ARG-OH M E T H I O N Y L - L Y S Y L - BRADYKININ MET-LYS -ARG-PRO-PRO -GLY-PHE-SER -PRO -PHE-ARG- OH

FIG.4. The chemical structure of kinins.

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IV,E. Human plasma kallikrein is not activated by histamine, 5HT, nicotinic acid, or endotoxin ( 733). Like tissue kallikreins, plasma kallikrein is a proteolytic enzyme, splitting a nonapeptide, bradykinin, from its precursor in plasma, kininogen, ( 5 6 2 ) , and hydrolyzing amino acid esters, particularly those of arginine ( 707) and lysine (459, 606) but not of tyrosine ( 707). It is inhibited by DFP (707), SBTI, ovomucoid and pancreatic trypsin inhibitors ( 7 0 6 ) , and by Trasylol ( 6 7 0 ) , a pattern which differs from that of tissue kallikreins. Plasma kallikrein is also inhibited by serum C’1 esterase inhibitor (286, 544) (Section V,A), Indirect evidence suggests that urinary kallikrein is derived from plasma kallikreinogen ( 716). A second series of experiments approached the problem in a different way. Feldberg ( 1 8 5 ) ,in 1938, demonstrated that cobra venom incubated with dog lymph, induced the contraction of guinea pig ileum. The significance of this observation has been elaborated by Rocha e Silva and his colleagues. They demonstrated that one or more agents that lowered blood pressure and contracted smooth muscle were generated in mixtures of human plasma and the venom of Bothrops iararaca or Bothrops atrox, South American crotaline snakes (562). The active principle in venom is a protease which hydrolyzes amino acid esters (235) and is inhibited by DFP ( 2 2 4 ) , but not by SBTI ( 2 3 5 ) .The agent generated by venom is bradykinin, the same small polypeptide split off by plasma kallikrein, and named by Rocha e Silva because it contracted smooth muscle more slowly than did histamine. In retrospect, these results are not unexpected, for snake venom is a specialized form of saliva. Other venoms have similar properties (225,397). Little has been done to build upon Menkin’s original observations concerning leukotaxin. But an enormous literature has appeared, extending the studies of Frey and Rocha e Silva. Several different plasma enzymes have been identified which have either kallikrein-like properties or seem to be activators of plasma kallikreinogen (17, 164, 705). Similarly, several different biologically active polypeptides have been found which can be derived from plasma through the action of the kallikreins or trypsin; the existence of others is suspected. Called kinins generically, three have been chemically identified: bradykinin, a nonapeptide ( 72) ; lysyl-bradykinin (kallidin ) ( 497); and methionyl-lysyl-bradykinin (169) (Fig. 4 ) . Armstrong ( 1 7 ) has extracted a fourth polypeptide, kinin E, not yet analyzed, Donaldson et al. (150) have isolated what may be yet another, and Greenbaum (219) suggested that polymorphonuclear extracts release an unidentified kinin from human kininogen. Plasma also contains an aminopeptidase, perhaps kallikrein itself, which can convert lysyl-

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bradykinin to bradykinin (226, 708). Presumably, bradykinin can also be formed from methionyl-lysyl-bradykinin. The kinins are derived from two and perhaps more a,-globulins, kininogens, or prokinins (17,164,225,273,396,687,716). One kininogen, with a molecular weight of 197,000, is attacked readily by glandular or plasma kallikreins, whereas another, with a molecular weight of 57,000, is hydrolyzed in preference by glandular kallikrein. The two are probably closely related, since they cannot be distinguished immunologically (498). Evidence concerning their chemical structure has been summarized by Pierce (496). The kinins have important biological properties. I have already mentioned their hypotensive effects. Bradykinin contracts the isolated guinea pig ileum and estrous rat uterus, but relaxes the rat duodenum (168). Kinins increase vascular permeability (59, la),dilate small blood vessels (258), induce pain ( 1 6 ) , and provoke sticking of leukocytes to small blood vessels and migration of these cells into extravascular spaces (217, 347)-all components of the inflammatory response. Unlike histamine, bradykinin does not produce a flare in human skin (586). In some species, kinins cause bronchoconstriction (110), and t h i s property has been invoked to explain some of the symptomatology of human asthma. As is true of histamine, the increased vascular permeability is apparently related to disruption or separation of the junctions between venular endothelial cells (382). The dilatation of small blood vessels may account for the fall in blood pressure, increased coronary arterial flow ( M ) ,increased cardiac output ( 65, 4 7 ) , increased cerebrospinal fluid pressure, and headache (616). The effect of kinins upon pulmonary arterial pressure is variable (65). Not all of the systemic effects of kinins are directly attributable to these agents, for their injection leads to the release of catecholamines (186,322), whereas, in human subjects, the injection of bradykinin brings about transient venous constriction by reflex stimulation of the sympathetic nervous system (402). Most experimental work has been done with synthetic bradykinin. In fact, all kinins do not behave in the same way. For example, bradykinin is a much more potent bronchoconstrictor and hypotensive agent than lysyl-bradykinin (465) and is slightly more active in contracting the rat uterus (646), but it is less effective in enhancing cutaneous vascular permeability (149).

D. PLASMAINHIBITORS OF KININS Not unexpectedly, plasma has the capacity to inactivate kinins rapidly (714, 720). This inhibitory activity resides principally in an enzyme,

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carboxypeptidase N, which is a peptidase similar to but not identical to carboxypeptidase B, and is capable of splitting the C-terminal arginine from bradykinin or lysyl-bradykinin (175). This plasma ‘lcininase” provides a serious obstacle to the study of kinins. Fortunately, it can be inhibited by incubating plasma at pH 2 (261) or by exposing it to cysteine (682), dimercaptopropanol (BAL) (177), sodium diethyldithiocarbamate (187), EDTA (14),a-phenanthroline (175), or EACA or similar o-amino acids (64, 162). The optimal technique for inactivating kininases varies from species to species. Zinc, cadmium, or cobalt ions reverse the inhibitory effect of chelating agents (64). The peptidase nature of kininase has made possible an assay using hippuryl-L-lysine as the substrate (176). A second plasma kininase, which splits the dipeptide phenylalanyl-arginine from the carboxyl end of bradykinin has also been discerned (731). Its susceptibility to inhibitory agents is different from that of carboxypeptidase N.

E. THE RELATIONSHIP BETWEEN KININ FORMATION AND BLOODCLOTITNC In their original study of the effects of snake venom, Rocha e Silva, Beraldo, and Rosenfeld (562) suggested that some bradykinin might be released when blood clots. They based this view on the observation that blood contained less prokinin once it had clotted, and they ascribed the effects of coagulation to a kinin-releasing factor in platelets. Several years later, Armstrong and her colleagues ( 1 3 ) reported that normal human plasma, applied to the exposed base of a cantharidin-induced blister, was exceedingly painful. The pain could not be attributed to a histamine-like substance, since neither itching nor wheal formation was observed. The “pain-producing substance” developed only when plasma was exposed to glass and had characteristics resembling bradykinin, contracting smooth muscle in a similar way (15, 16). Similar kinin-like activity appears in rat or guinea pig plasma exposed to glass, whereas cat blood behaves as if it acquires 5HT-like properties, apparently derived from platelets; neither activity generates in dog or rabbit blood under these circumstances (14). Margolis (390, 391), fresh from his studies of the clot-promoting effects of glass, demonstrated that the elaboration of kinins in Armstrong’s experiments was due to the activation of Hageman factor which takes place when plasma is exposed to glass. He proposed that activated Hageman factor reacted with a second entity in plasma, “component A,” to produce an agent that brought about the formation of kinins. In agreement with this, Hageman factor-deficient plasma does not provoke pain when applied to a blister base nor evolve kinins when exposed to glass

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(391), whereas activated Hageman factor does initiate the formation of kinins (394). The importance of Hageman factor in the evolution of kinins has been confirmed in several ways. Webster and I (709) corroborated Margolis’ findings, demonstrating that neither diatomaceous earth nor acetone would induce the formation of kallikrein in Hageman factor-deficient plasma. The abnormality could be corrected by the addition of partially purified Hageman factor. Presumably, then, Margolis’ component A is comparable to or identical with plasma kallikreinogen. In contrast, the conversion of plasma kallikreinogen to kallikrein by trypsin proceeds normally in Hageman factor-deficient plasma. These studies have been corroborated in studies of guinea pig serum by Davies et al. ( 1 2 0 ) . In another approach, Eisen (162) observed that plasma euglobulin fractions, treated with kaolin, acquired esterolytic activity as measured with TAME; this property was not due to the formation of plasmin, since its generation was not blocked by EACA. Hexadimethrine bromide, however, which blocks either the activation or activity of Hageman factor (541), inhibited the generation of esterolytic activity and the activation of kallikrein ( 1 6 3 ) . Similar experiments have been reported by Sherry (606). The role of Hageman factor in the activation of plasma kallikreinogen was also demonstrated indirectly in vim by Gautvik and Rugstad ( 2 0 3 ) . These investigators injected rats with ellagic acid, a soluble activator of Hageman factor, and observed a sharp decrease in arterial blood pressure, confirming an earlier observation in dogs ( 77). Subsequent injections were progressively less effective, but the response to the injection of bradykinin or histamine was unaltered. Their data suggested that the repeated injections of ellagic acid had depleted the rats of kininogen, while leaving adequate amounts of Hageman factor and kallikrein. Presumably, the kininogen had been converted to kinin, which was then destroyed by plasma kininase ( Section IV,D above). Similarly, the injection into dogs of cellulose sulfate solution, which activates Hageman factor (296), causes transient hypotension and depletion of kininogen (572). The studies which I have selected have utilized glass, kaolin, diatomaceous earth, ellagic acid, and cellulose sulfate to activate Hageman factor. Any agent capable of activating this clotting factor might be expected to generate kinins through the same mechanisms. A second way in which the clotting process may participate in the inflammatory response is more obscure. Gladner and his associates (211, 484) noted that bovine fibrinopeptide B, one of the peptide fragments released from fibrinogen by thrombin, potentiated the effect of bradykinin upon uterine smooth muscle. Dias da Silva, Blomback, and I ( 1 3 6 )

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found this was also true for fibrinopeptides B derived from human, green monkey, pig, and reindeer fibrinogen, which differ from each other in amino acid composition and chain length, but all have the carboxyl terminal sequences alanyl-arginine or glycyl-arginine. Among fibrinopeptides A tested, only that of the rabbit, in which the carboxyl terminal sequence is glycyl-arginine, was effective. How fibrinopeptides work is not known. We could not obtain evidence that they potentiate the action of histamine or acetylcholine upon smooth muscle (136) nor of bradykinin upon vascular permeability in guinea pig skin ( 1 4 8 ) . Bovine fibrinopeptide B and human fibrinopeptide. A constrict pulmonary vessels and produce a fall in dynamic lung compliance, functional residual volume, and ventilatory conductance and an increase in respiratory rate and ventilatory volume in dogs, rabbits, and lambs (37). It is not clear if these effects are related to the potentiation of kinins, but they do indicate that fibrinopeptides may have a physiological function.

F. PLASMINAND KININ FORMATION The kinin-forming systems are activated by plasmin. Beraldo (SO) reported that kinin-like substances appeared in mixtures of plasmin and plasma globulin, a result corroborated by others (345,706).Both in vitro (28, 166, 378) and in viw (524), this release of kinins seems to be due to the action of plasmin upon kininogen. A second effect of plasmin may be the activation of kallikreinogen. Thus, the transient fall in blood pressure which follows the intravenous injection of plasmin (222) has been attributed to tffe formation of kallikrein ( 2 8 ) .The possibility that plasmin acts both upon kallikreinogen and upon kininogen is not surprising, since trypsin has both these activities. Whether plasmin’s role in the release of kinins has physiological importance is not certain. Concentrations of plasmin which increase vascular permeability are less than those which can be achieved by maximal activation of the plasminogen in normal human plasma, but whether the enzyme could overcome the natural inhibitors of plasmin is not certain ( 5 2 4 ) . The possibility exists that in hereditary angioneurotic edema the formation of plasmin may be a step in the elaboration of kinins, but the effect of plasmin may be mediated indirectly through its activation of C1 ( 1 4 2 ) . G. GRANULOCYTES AND KININ FORMATION Granulocytes contain kininases (406, 732) as well as agents capable of liberating kinins from plasma (406, 733). The kinin-forming enzymes

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are present both in the lysosomal and extralysosomal parts of the cells, whereas the kininases are localized to the extralysosomal fraction ( 2 1 9 ) . The kinin-forming activity is probably not kallikrein-like, for intact granulocytes liberate kinins only if the plasma in which they are suspended contains Hageman factor, as if their role were to activate this substance ( 407). The principle kinin generated when granulocytic fractions are incubated with kininogen seems distinct from bradykinin ( 2 1 9 ) .

H.

PLASMA

PERMEABILITY FA~ORS

In 1953, Mackay and her colleagues (379) reported that the intracutaneous injection of diluted guinea pig serum increases vascular permeability in this species. The capacity to enhance permeability increases for some minutes after the serum is diluted and then gradually recedes, presumably because serum also contains inhibitors of the responsible agents (421, 423). Conceivably, some or all of the inhibitory property resides in the fraction described as C’1 esterase inhibitor (see Section V,A 1. Similar permeability properties appear in diluted rat (724) and human (167) serum or plasma, but rabbit serum (724) is only rarely active. The permeability-enhancing property in guinea pig (379, 421) and human (167) plasma and serum is not antagonized by antihistaminic drugs, whereas that in rat serum is blocked by local injection of such agents ( 7 2 4 ) .The agent responsible for increasing vascular permeability which appears upon dilution of plasma or serum has been called PF/Dil or globulin permeability factor. In human plasma, it has been localized to a &-globulin fraction ( 4 2 3 ) . It is apparently a hydrolytic enzyme, since it hydrolyzes TAME (287) and is inhibited by SBTI (167, 421), pancreatic trypsin inhibitor ( 4 2 3 ) , and DFP ( 4 5 ) . Comparable experiments, with more erratic results, were performed by Kuroyanagai (313) in rabbits injected with diluted human serum. Shortly after PF/Dil was discovered, Spector (629) found that he could reproduce Mackay’s experiments only if the diluted plasma was first exposed to glass surfaces. Margolis (392, 393) deduced that the evolution of PF/Dil must therefore depend upon the presence of Hageman factor, activated by contact with glass. He found that plasma deficient in Hageman factor did not increase vascular permeability. Miles and I (541) confirmed his views by demonstrating that purified human Hageman factor itself increased vascular permeability in guinea pig skin, and, in minute amounts, allowed PF/Dil to form in diluted plasma devoid of this clotting factor. The elaboration of PFIDil is blocked by small amounts of

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hexadimethrine bromide, protamine sulfate, or Liquoid, but heparin is onIy slightly inhibitory ( 541 ) . The characteristics of PF/ Cil are, of course, immediately reminiscent of plasma kallikrein. But purified guinea pig PF/Dil does not form kinins in the absence of kallikreinogen (401). Kagen, Leddy, and Becker (287) separated two fractions of human plasma by chromatography upon columns of DEAE cellulose. One fraction, which did not adhere to the cellulose, increased vascular permeability and released kinins from plasma, whereas the other, which was adsorbed to the cellulose, enhanced permeability but did not generate kinins in the absence of kallikreinogen. Presumably, the first fraction contained plasma kallikrein, and the second contained PF/ Dil. Both fractions hydrolyzed TAME and were inhibited by SBTI and DFP ( 2 8 7 ) . Both were also inhibited by large amounts of heparin, although the effect of this agent was much more pronounced upon PF/Dil ( 3 6 7 ) . Similarly, C1 esterase inhibitor blocks both PF/Dil and plasma kallikrein but is more effective against the former (286, 544). Experiments of Mason and Miles (401) suggest that the activation of plasma kallikreinogen can be brought about by PF/Dil. Perhaps this explains the liberation of kinins which occurs when serum is diluted in a g h s muscle bath ( 5 8 6 ) . In agreement with their observations, Kagen (285) brought about the release of kinins by the addition of partially purified PF/ Dil to unheated plasma, containing kallikreinogen. These studies do not exclude the additional possibility that activated Hageman factor acts directly upon kallikreinogen. PF/Dil has usually been demonstrated in diluted plasma. McConnell and Becker (366) exposed undiluted plasma to glass and noted the development of permeability-enhancing activity, which they attributed to the presence of kallikrein and PF/Dil. An agent probably identical with PF/Dil has also been found in experimental inflammatory exudates ( 6 3 5 ) . PF/Dil and plasma kallikrein are by no means the only plasma permeability factors which have been described. When undiluted rat, rabbit ( 3 4 5 ) ,or human serum or plasma is injected into guinea pig skin, vascular permeability is increased ( 1 6 7 ) . The nature of this effect of heterologous plasma is not known, but the responsible agent, PF/Nat, is distinct from PF/Dil, for it is not inhibited by SBTI or by the inhibitor of PF/Dil found in guinea pig serum, and it has a more prolonged effect ( 1 6 7 ) . Elder and Wilhelm (167) suggested that PF/Nat may be identical with human serum necrotizing factor (Section IV,I). Yet another plasma permeability factor, designated PF/Age, is detected in undiluted guinea pig serum incubated at 2°C. for 3 to 5 days

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( 379). PF/ Age is not inhibited by antihistaminic agents. When guinea pigs are injected with their own serum, the lesions evolved are smaller than those induced by the serum of other guinea pigs (421). The increase in vascular Permeability induced by PF/Age takes place much more slowly than that brought about by the injection of PF/Dil, and is only poorly antagonized by SBTI ( 4 2 1 ) .Elder and Wilhelm (167) suggested that PF/Age is an agent corresponding to the PF/Nat of human serum, but this is uncertain. Davies and Lowe (121,122) incubated immune precipitates, prepared from egg albumen and specific rabbit antiserum, with guinea pig serum for 5 minutes at room temperature. The serum, separated from the precipitates by centrifugation, increased vascular permeability in guinea pig skin. The nature of the agent which had evolved is not clear. It was not antagonized by antihistaminic agents or SBTI. Davies and Lowe (122) established that the permeability factor released by antigen-antibody precipitates from guinea pig serum, which they designated PF/P, formed only if the first four components of complement were present. But PF/P is not a classic anaphylatoxin, since it is not a histamine liberator (121, 122). I have not exhausted the list of agents derived from plasma which enhance vascular permeability. Later sections will discuss C’1 esterase, anaphylatoxin, and other substances exhibiting this property.

I. HUMANSERUMNECROTIZING FACTOR When a small amount of human serum is injected intracutaneously into depilated guinea pigs, a characteristic necrotic lesion sometimes appears (127, 306, 361, 437). The site of injection turns blue or purple within a few minutes. Depending upon the severity of the response, within a day the discdoration deepens or frank necrosis takes place. In one-third or less of cases, serum prepared from normal blood evokes positive tests, whereas that of individuals with a rather incoherent spectrum of disorders, including fracture of the hip, rheumatoid arthritis, tuberculosis, and malignancy, for example, induces necrosis about two-thirds of the time (71, 306, 437). The factor or factors in serum responsible for the induction of lesions are heat-labile ( 71 ) and have been localized to globulin fractions (306, 437), but they have not otherwise been identified nor their mode of action discovered. The presence of thrombi in capillaries and veins in the injected area suggests that the necrosis is ischemic in origin (361). Antihistaminics (587) or EACA (437) do not prevent the formation of lesions. Interestingly, soybean, lima bean, and ovomucoid trypsin inhibitors enhance the activity of serum. The significance of these

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studies, particularly in relation to the formation of local thrombi, is not yet evident.

J. E m s AND INFLAMMATION Both clinical and experimental evidence indicates that kinins may be important in inflammatory reactions. Kinins have been demonstrated, for example, in experimental (633) and clinical ( 1 5 ) pleural exudates, human cantharidin or thermal burn blister fluid (13, 1 5 ) , rheumatoid ( 1 5 ) and gouty (408) synovial fluid, hydrocoele and ascitic fluids ( 1 5 ) , and nasal secretions in hay fever ( 1 3 8 ~ )The . injection of autologous plasma into human subcutaneous tissue releases kinins locally ( 7 3 3 ) . Kinins also accumulate at the site of injection of compound 48/80, an agent which releases histamine (733),and in the tissues of rats subjected to mild thermal injury ( 5 6 0 ) . In human subjects, kinins have been detected in perfusates of cutaneous areas of cold urticaria (128), ultraviolet ray-induced injury ( 1 7 2 ) , and wheal-and-flare allergic reactions ( 4 1 8 ) .In an exceptionally provocative study, Chapman and his associates ( 9 8 ) hypnotized normal individuals and suggested that one arm was hot, burning, and susceptible to injury. Both arms were then subjected to mild thermal injury. In most cases, the inflammatory response was greater in the susceptible arm than on the control side. A subcutaneous perfusate of the burned area on the susceptible side contained bradykinin-like material. Chapman’s studies emphasize the importance of the central nervous system in the response to injury, neglected in the rest of this review. K. FOREIGN BODY~ C I T O N S Some of the insoluble substances known to activate Hageman factor may be introduced into the body in one way or another. In animals, the subcutaneous injection of silica, asbestos, or kaolin (299) or the intraperitoneal instillation of silica (424) or talc (magnesium silicate) (206) results in localized inflammatory responses, beginning with edema and congestion and followed by the formation of granulomatous nodules of macrophages, fibroblasts, and fibrous tissue cells. Central necrosis may occur. The crystals may be engulfed by “foreign body” giant cells. These experimental situations are the counterpart of the events leading to granuloma formation after the introduction of talc into surgical wounds (188, 206,675). A similar response is seen in the lungs of “Blue Velvet” addicts. These individuals inject themselves intravenously with mixtures of concentrated paregoric and crushed tripelennamine ( Pyribenzamine ) tablets. After

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repeated injections, the addicts develop pulmonary hypertension, which may be lethal (712). Diffuse arterial and arteriolar thrombosis occurs, followed by the formation of foreign body granulomas in the vascular walls, The offending agent in the narcotic mixture is talc, a component of Pyribenzamine tablets, a conclusion sustained by experimental studies in rabbits (506). In two cases at University Hospitals of Cleveland, most of the peripheral veins had been sclerosed by repeated injections of Blue Velvet, and the patients resorted to injecting the external jugular veins. Similar lesions have been observed after the intravenous injection of starch, contained in oral secobarbital or adulterated heroin (584). A reasonable speculation is that these responses are mediated through Hageman factor (525). In support of this, Margolis (391) reported that the immediate inflammatory reaction to the intracutaneous injection of kaolin was abnormally weak in a patient with Hageman trait. Warren and Kellermeyer (703) produced foreign body granulomas in the lungs of mice by the injection of divinyl copolymer benzene beads, which activate Hageman factor. The inflammatory response was blocked by Liquoid ( polyanetholsulfonate ) , an inhibitor of Hageman factor, and significantly lessened if the mice were first injected with ellagic acid, which Gautvik and Rugstad (203) had shown would deplete the animals of kininogen. Similarly, Kellermeyer, Kellermeyer, and Warren (297), in unpublished studies, induced foreign body granulomas in the lungs of mice with silica particles, again suppressing the process by prior injection of ellagic acid. These various studies support the opinion I have expressed before that pneumonoconiosis may be linked to the activation of Hageman factor by the offending agents (525).

L. ARTHR~TIS The importance of kinin-producing mechanisms in the evolution of inflammatory reactions in man is most clearly seen in acute gouty arthritis, a condition in which crystals of sodium urate are present in the joint space (363). Melmon and his associates (408) demonstrated the presence of kinins in the synovial fluid in acutely inflamed gouty joints. These polypeptides, because they increase vascular permeability and induce pain, seem ideal mediators for the evolution of arthritic effusions. Kellermeyer and his colleagues have carried out a series of experiments demonstrating one route through which kinins might evolve in gout. Faires and McCarty ( 1 8 1 ) and Seegmiller et al. (598), reviving ideas first propounded by Garrod, induced acute arthritis by the intra-articular instillation of sodium urate. Kellermeyer and Breckenridge (292), intrigued by the analogy between glass and the glasslike crystals of sodium urate, demon-

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strated that microcrystalline sodium urate was an excellent activator of Hageman factor, Further, Hageman factor (293), a permeability factor perhaps identical with PF/Dil (290) and kallikreinogen (294) have been found in normal joint fluid; presumably, then, under appropriate circumstances sodium urate crystals can instigate the development of acute arthritis by activating Hageman factor which, in turn, leads to the elaboration of permeability factors and kinins. Kellermeyer and Graham (291, 295) have carried this speculation further. Kinins have relatively poor chemotactic activity for leukocytes, but the injection of activated Hageman factor into the rabbit ear chamber induces sticking of these cells to the endothelium of small blood vessels and their migration into extracellular tissues ( 2 1 7 ) . Perhaps, then, the activation of Hageman factor can contribute to the leukocytosis noted in arthritic joint effusions. The leukocytes, in turn, ingest urate crystals and disrupt, releasing lysosomal hydrolytic enzymes which are themselves capable of inducing an acute arthritis (598, 711). Granulocytes (406) and lysates of leukocytes (219, 733) also induce kinin formation (Section IV,G) , Moreover, Hageman factor is known to activate plasminogen which, in turn, can split chemotactic factors from C’3 (699), further increasing the local accumulation of leukocytes. And Hageman factor, directly or indirectly, can change C’1 to C’1 esterase, raising the possibility that it can stimulate the formation of anaphylatoxins (620) or kinins ( 143). Needless to say, this view of acute gouty arthritis does not account for all that is known about this condition. Thus Phelps and his associates (495) were not able to inhibit arthritis induced by urate crystals by local administration of carboxypeptidase B, which inactivates kinin polypeptides. Conversely, they could not induce arthritis in dogs rendered granulocytopenic by the administration of vinblastine (494). A role for complement in the pathogenesis of gouty joints has been suggested by the finding that urate crystals deplete serum of C’2, C’3, C‘4, and C5, an action not requiring the participation of Hageman factor (457). As yet, these experiments suggest that, whatever its role in the induction of acute gouty arthritis, Hageman factor cannot account for the whole pathological process. Much less work has been done concerning other arthritides. Calcium pyrophosphate, a crystal found in synovial fluids of patients with pseudogout (364), is an activator of Hageman factor (292), and induces synovitis when injected into human or canine joints (364). The synovial effusion in acute rheumatoid arthritis and psoriatic arthritis contains kinins (15, 408) as well as Hageman factor and kinin-forming agents (165). Such effusions also contain fibrinogen, fibrin, fibrin split products,

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and prothrombin ( 3 2 ) . Synovial leukocytes in effusions appear to engulf fibrin or its breakdown products ( 351 ), and protein immunologically similar to fibrin is found in the inflammatory tissue and synovia of the rheumatoid joint ( 2 0 8 ) .Barnhart ( 3 2 ) suggests that the fibrin is chemotactic, adding to the inflammatory response. Antigen-antibody complexes, such as might exist in rheumatoid joints, can bring about the elaboration of humoral mediators of inflammation, perhaps through an effect upon Hageman factor (122, 440). Macroglobulin from the serum of patients with rheumatoid arthritis, combined with aggregated normal human IgG, results in the formation of kinins when incubated with normal plasma ( 1 7 3 ) . The naturally occurring 22 S and 19 S globulins found in some rheumatoid serums also increase vascular permeability. Lack ( 314) has proposed that plasmin, activated by lysosomal enzymes, can digest the mucoproteins in cartilage. Conceivably, under appropriate circumstances, plasmin may contribute to the evolution of arthritic changes. Obviously, much remains to be done before we achieve an understanding of the rheumatoid joint. V.

Complement

Not unexpectedly, the biological phenomena brought about by the congeries of plasma proteins designated C’ are tangled with the hemostatic and fibrinolytic mechanisms. Much of what has been learned about the functions of complement has come from an examination of its role in the lysis of sheep erythrocytes sensitized with specific rabbit antiserum. At least nine components of complement are known to participate in the events leading to the hemolysis of such sensitized erythrocytes, designated in the order in which they are believed to act: C’l, C’4, (72, C’3, C’5, C’6, C’7, C’8, and C’9 ( 4 4 5 ) .The first identified reaction is the attachment of C’1 to the sensitized cell (EA). C’1 is a macromolecular complex of three subcomponents, C’lq (earlier called 11S), C’lr, and C’ls, apparently bound together by calcium ions and readily dissociated by the addition of sodium EDTA (332). In the reaction between C’1 and sensitized erythrocytes, it is the C’lq subcomponent which is fixed to the antigenantibody complex ( 4 5 8 ) , probably by its attachment to the antibody molecule ( 4 4 7 ) . Subsequent to the attachment of C’1 to the sensitized erythrocyte, C’4 is bound (499), either to the membrane (237) or to the antibody. In this reaction, C’4 is apparently changed to an activated form through the action of the c“1 attached to the sensitized cell (449) Next, C’2 is bound, but only if magnesium ions and C’1 are available (405).The site of attachment of C’2 may be on the C’4 molecule (647). After C’2

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becomes attached, C’1 is no longer needed and its inactivation at this point has no effect upon subsequent hemolysis. The sensitized cell, now designated as the intermediate complex EAC’1,4,2, is then hemolyzed as the result of a succession of reactions requiring the presence of C’3, C’5, C’6, C’7, C’8, and C’9, the nature of which has been the subject of several excellent reviews (329, 446, 462) and need not be reiterated here, In these reactions, C’3 becomes attached to the sensitized cell (448) and C’5, C’6, and C‘7 appear to act as a complex ( 4 7 3 ) . One route to our present understanding of the functional behavior of C’1 has come about through a study of the effects of plasmin upon complement. In unpublished studies in 1947, Thomas and I (548) observcd the puzzling fact that the addition of streptokinase to fresh serum had an “anticomplementary” effect. Some years later, it became clear that this property of streptokinase was due to its activation of plasmin, which rendered C’l, C’2, C’4, and, to a slight degree, C’3 hemolytically inactive ( 5 0 0 ) . Of course the possibility was entertained that human serum contained antibodies against streptokinase and that the inactivation of the various components was the result of banal complement fixation. This explanation, however, could not account for the fact that chloroformactivated bovine plasmin inactivated C’2 and C’4 in human serum, avoiding the issue of antistreptokinase antibodies. Further investigation of the effect of plasmin demonstrated that calcium ions potentiated the destruction of C’2 and C‘4 by plasmin ( 333). More excitingly, the inactivation of C’2 and C’4 by this enzyme took place only if C’1 was present; calcium ions seemed to stabilize this component ( 338). These various observations led to the hypothesis that C’1 was changed by plasmin from a proenzymatic to an enzymatic state, and that in this form, “activated” C’1 inactivated C‘2 and C’4. This conjecture drew a parallel to the events accompanying complement fixation by immune aggregates, in which C’l, C’2, and C’4 are fixed to the aggregates (499) if calcium ions are present (344). This view of the role of C’1 marked the revival of ideas expressed many years before, that the effects of antigen-antibody reactions were mediated by enzymatic means (281, 353, 656). It soon found support in Levine’s (343) discovery that DFP inhibited immune hemolysis, an effect localized by Becker (40, 41) to an action upon C’l; the effect of DFP can be partially reversed by nicotinohydroxamic acid (41 ). The implication that activated C’1 is a hydrolytic enzyme was strengthened by the discovery that crude C‘l, prepared by extraction of euglobulin precipitates of human serum at high ionic strength, rapidly

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lost hemolytic activity when the ionic strength was lowered, and at the same time acquired the capacity to inactivate C’2 and C 4 in solution and to hydrolyze TAME (336). An apparently identical enzyme could be eluted from antigen-antibody aggregates which had been exposed to serum (335).The eluate digested TAME, but even more avidly, ALTEE, a substrate chosen because Troll, Sherry, and Wachman (674) had noted that an enzyme hydrolyzing this substrate contaminated crude preparations of plasmin. Similar experiments were performed independently by Becker (4.57, who demonstrated that sensitized erythrocytes exposed to the first, second, and fourth components of complement ( EAC’1,4,2) hydrolyzed TAME. The enzyme derived from preparations containing C’1 was distinguished from other plasma enzymes that can hydrolyze TAME by its instability at 56”C., the failure of SBTI to block its action, and by its capacity to hydrolyze not only ALTEE, but N-acetyl-3,5-dinitro-~-tyrosine ethyl ester, N-acetyl-L-phenylanineethyl ester, and N-acetyl-L-tryptophan ethyl ester ( 5 3 8 ) . Definitive evidence of the identity of the esterolytic enzyme and an activated form of C’1 was a macromolecule which could be dissociated into three components, C’lq, C’lr, and C’ls by the addition of EDTA ( 3 3 2 ) . The subcomponents could be separated by chromatography upon columns of DEAE cellulose (332) and reaggregated by the addition of calcium ions (458). When highly purified preparations became available (230), it became clear that 01 esterase was derived from the C’ls subcomponent of C’l, for it could substitute for C’ls in the formation of EAC’1 if Clq and C’lr were present (231 ) . How C’1 is changed to C’1 esterase by fixation to antigen-antibody aggregates is not yet known, but once again studies of plasmin seem to point the way. The observation, already mentioned, that plasmin inactivated C’2 and C’4 only if C’1 is present was explained by the fact that plasmin, activated by streptokinase, can convert C’1 to C’1 esterase (334). The action of plasmin is localized to an effect upon the C’ls subcomponent ( 5 4 2 ) ,obviating the argument that the formation of C’1 esterase is the result of complement fixation ( 3 2 1 ) . In experiments with C l s , the plasmin tested was activated spontaneously or by urokinase, which is nonantigenic, to avoid problems arising from complement fixation. Belief that the formation of C’1 esterase from C’ls is a proteolytic step was strengthened by the discovery that trypsin can convert C’1 to C’1 esterase ( 5 4 2 ) .These studies led to the hypothesis that the formation of C’1 esterase by antigen-antibody aggregates is a proteolytic step, an opinion which had been expressed by Taylor and Fudenberg ( 6 5 2 ~ )An . earlier view, that the activation of C’1 esterase might be autocatalytic,

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could not be substantiated, but preparations containing C’lr are able to change C’ls to C’1 esterase by what appears to be an enzymatic process (459). C’lr has attributes of a hydrolytic enzyme, as it can digest several synthetic amino acid esters, particularly AAME (459). None of these experiments tells us why C’1 is inactive in serum, but becomes activated by adsorption to antigen-antibody aggregates. One clue presently under study derives from an examination of the effect of cations upon the reaction between C’lr and C’ls. Calcium ions, at concentrations such as exist in plasma, have no effect upon the hydrolysis of AAME by preparations containing C’lr, whereas at concentrations as low as O.OOO125 M ,they completely inhibit the reaction between C’lr and C’ls. Presumably, in circulating blood, the C’lr contained within macromolecular C’1 is held in an inert state by calcium ions. The addition of preparations containing Ctlq to a mixture of C’lr and C’ls completely bypasses the inhibition of calcium ions. One argument, then, is that C’lq is so altered by antigen-antibody aggregates that it can in some unknown way release C’lr from inhibition by calcium ions and, thus, allow it to act upon its substrate, C’ls (543). Studies testing this hypothesis are in progress. Besides its capacity to convert C’ls to C’1 esterase, plasmin appears to have other actions upon complement. Thus, it probably inactivates C‘4 directly (321) and diminishes the C’1 esterase activity which evolves in its presence (542).Plasmin also splits a small fragment from C3 which is chemotactic and increases vascular permeability (699).This fragment, a heat-labile polypeptide with a molecular weight of about 6000, represents about 4!Z of the molecular weight of C’3. It is distinguishable from the chemotactic agent released from complement by antigen-antibody reactions, which is derived from the C’5, C’6, C’7 complex (698), and from the anaphylatoxin derived from C’3. Whether any of these actions of plasmin upon complement are important in life is not yet evident. The experiments described in the preceding paragraphs demonstrate some of the ways in which plasmin may affect complement. The converse possibility is raised by experiments of Taylor and Miiller-Eberhard ( 653), who have demonstrated that antisera directed against C’3, C’4, and yM globulin inhibit the lysis of clots formed from diluted whole blood, perhaps through an effect upon platelets, which are known to contain plasminogen ( 385). A. C’1 ESTERASE INHIBITOR When serum is added to C’1 esterase, it inhibits this enzyme’s capacity to hydrolyze ALTEE (538). The inhibitory property has been highly

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purified (490, 491) and has been localized to a heat-labile wglobulin ( 491 ) which is identical with a,-neuramiuoglycoprotein (492). C‘1 esterase inhibitor blocks not only the esterolytic properties of C’1 esterase in free solution, but the functional activities of C1 adsorbed to antigenantibody complexes as well (251, 328, 330, 337). Once the complex EAC’1,4,2 has formed, however, C’1 esterase inhibitor will not block immune hemolysis ( 3 3 0 ) . These studies, then, fortify the view that in immune hemolysis C’1 is no longer needed once it has reacted with C’2. The action of C’1 esterase inhibitor is not limited to its effect upon C’1 esterase. It inhibits the formation of C’1 esterase (331, 334, 544); this property is localized to inhibition of Ctlr, since the esterolytic activity of this subcomponent is also blocked (544). Further, it inhibits a plasma kallikrein, the plasma permeability factor designated as PF/Dil (286, 544), activated PTA, and activated Hageman factor ( 1 9 2 ~ )And, . at concentrations similar to those existing in normal human plasma, it is an effective inhibitor of human plasmin ( 5 4 4 ) , although it has not yet been identified with other plasmin inhibitors in plasma. Perhaps the capacity of C’1 esterase inhibitor to neutralize plasmin explains the observation that streptokinase seems to inactivate C’1 esterase inhibitor (321, 538). Conceivably, the plasmin formed by the addition of streptokinase combines stoichiometrically with C’l esterase inhibitor, thus reducing its titer in pIasma. Once again, it is not yet apparent whether the plasmin-inhibitory properties of C’l esterase inhibitor have biological importance. Whether C’1 esterase inhibitor is identical with those plasma inhibitors of PF/Dil (421) and plasma kalIikrein (670) described much earlier is not certain. Some experiments with organic solvents suggest that this may be the case. Acetone and chloroform destroy inhibitors in plasma of kinin-forming factors (161). Similarly, ether (140) or chloroform (20) seem to destroy C’1 esterase inhibitor, since C’1 esterase forms readily in plasma treated with these agents.

B. HEREDITARY ANGIONEUROTIC EDEMA Hereditary angioneurotic edema is a familial disorder, inherited as an autosomal dominant trait and characterized by repeated episodes of sharply localized nonidammatory edema (152).The edema can occur in almost any location. When it compromises some vital area, such as the respiratory tract, death may result. The first inkling concerning the nature of hereditary angioneurotic edema came from studies of Landerman and his associates (318) in which the intracutaneous injection of a patient’s diluted serum enhanced vascular permeability in his own skin to a greater degree than did the

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injection of similarly diluted normal plasma. Injected into normal individuals, the patient’s serum did not behave differently than that of the controls. The increase in vascular permeability could be blocked by mixing the patient’s diluted serum with SBTI and, to a lesser extent, with EACA. Extending these observations, Landerman and his colleagues ( 319) found that the intracutaneous injection of plasma kallikrein increased vascular permeability in hereditary angioneurotic edema, suggesting that patients with this disease might lack inhibitory activity to plasma kallikrein or to a plasma permeability factor. In agreement with this, the serum of an affected individual was deficient in an inhibitor of one or both of these agents. Further, the inhibitory activity of serum against the esterolytic properties of preparations of plasma kallikrein was sharply diminished in hereditary angioneurotic edema, although the abnormality was less evident when plasma kallikrein was assayed by testing its hypotensive properties. An independent approach to the nature of hereditary angioneurotic edema by Donaldson and Evans (144) clarified the nature of the defective serum inhibitor. These investigators observed that in this disorder serum lacked inhibitory activity against C’1 esterase. In contrast, in acquired forms of angioneurotic edema, no such deficiency was demonstrable. The two views, that hereditary angioneurotic edema was associated with a deficiency of serum inhibitory activity against plasma kallikrein (or plasma permeability factor) or against C’1 esterase were reconciled by the demonstration that partially purified preparations of C’1 esterase inhibitor, prepared by Pensky (491), had the capacity to inhibit PF/Dil and, to a lesser extent, plasma kallikrein (286); these experiments have recently been confirmed with much purer preparations of C’1 esterase inhibitor (544). The question remained unsettled how the serum defect could lead to increased vascular permeability. Donaldson and Rosen (151) showed that during attacks free C’1 esterase was demonstrable in the serum, as evidenced by its capacity to hydrolyze ALTEE and by the inactivation of (72 and C’4. Serum obtained between attacks, unlike normal serum, readily generated C‘1 esterase activity, when incubated at 37°C. In agreement with this, Austen et al. (26, 27) reported that the concentration of C’2 in serum was diminished in proportion to the degree of edema in patients with this disorder and proposed that this was a reliable diagnostic test. The decrease in detectable C’4 in the patient’s serum was accompanied by a change in its electrophoretic pattern, the PIE(i.e., C’4) fraction of serum splitting into two or more components (151).

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With the demonstration that C’1 esterase increased vascular permeability in guinea pig skin by liberating a histamine-like substance ( 5 3 9 ) , the suggestion arose that the edema of patients with hereditary angioneurotic edema might be explained by this mechanism. In fact, however, it was well known that antihistaminic drugs are without benefit in this disease. Another mechanism seems much more likely to explain the increased vascular permeability characterizing attacks. Plasma, obtained from patients during an attack, strikingly increases vascular permeability in guinea pig skin (146, 150). Plasma obtained between attacks lacks this property, but it acquires the capacity to increase vascular permeability when incubated in EDTA. The evolution of the permeabilityenhancing agent or agents can be blocked by incubating nonattack plasma in SBTI (146, 150). A low-molecular-weight polypeptide can be extracted from plasma possessing permeability-enhancing activity. This substance is similar to, but possibly not identical with, bradykinin (150). Unlike bradykinin, the active principle is inactivated by trypsin and, upon injection into human subjects, enhances vascular permeability without evoking pain or erythema ( 150). These observations suggest that episodes of increased vascular permeability in hereditary angioneurotic edema are due to the local activation of a plasma kallikrein-like enzyme, with subsequent liberation of kininlike material. In agreement with this, Burdon ( 9 3 ) demonstrated that the plasma concentration of kininogen, the precursor of kinins, was diminished after an attack of edema. Moreover, the intracutaneous injection of C’1 esterase caused a local increase in vascular permeability both in normal individuals and in patients with hereditary angioneurotic edema, except at a time immediately after an attack ( 3 0 7 ) .The activity of the C’1 esterase preparation was not blocked by the administration of antihistaminics. Its effect depended upon the presence of C’2, but not C’3, as demonstrated by injection of C’1 esterase into individuals lacking these components (307). And kinin-like material has been demonstrated in fluid perfused through a wheal during an attack of edema (417). None of these experiments tells us what brings on an attack nor why the episodes are localized. Clinical experience suggests that the central nervous system may be of importance in the initiation of attacks, since these are likely to occur during periods of physical or emotional stress (152,362).Two clues to the way bouts are induced have been uncovered. Donaldson (143) has reported that the addition of ellagic acid, an activator of Hageman factor, to serum obtained from a patient in remission accelerated the evolution of C’1 esterase activity, an effect blocked by heparin or SBTI. She has also observed that urokinase, an activator of

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plasminogen, had a similar effect (142). Perhaps, then, suitable central nervous system stimuli, known to increase the rate of fibrinolysis in vitro, may bring about the activation of plasmin, and this enzyme, in turn, converts C’1 to C’1 esterase. The subsequent steps apparently require the participation of C’2, a plasma kallikrein, and a kininogen, but data supporting this speculation are not yet available. Lest this description of the pathogenesis of hereditary angioneurotic edema seem too pat, the possibility that histamine may yet be of importance must not be brushed aside. Recently, Granerus et ul. (218) demonstrated that urinary excretion of histamine was increased between attacks of hereditary angioneurotic edema, and in one patient, during an attack and for several days thereafter, urinary excretion of histamine and of its metabolite l-methyl-Cimidazole acetic acid was greatly increased. How these observations can be reconciled with the inability of antihistaminics to alter the course of the disease is not evident, but Becker (44u) has recently reported that antihistaminic agents may inhibit the cutaneous response of rabbits, rats, and mice to bradykinin, unlike their effectiveness in guinea pigs. The possibility that the activation of plasmin plays a decisive role in the initiation of attacks of edema h d s great support in studies by Nilsson (472) and Lundh (362), who prevented attacks by the administration of EACA or AMCA, agents inhibiting the activation of this enzyme. During treatment, the histamine metabolite, l-methylimidazole-5-aceticacid, disappeared from the urine. Puzzling, in another case, EACA was without benefit (362). VI.

Blood Clotting and Antigen-Antibody Reactions

When platelets are exposed to antigen-antibody complexes, they discharge ADP (442), clump (269,419,619), and undergo viscous metamorphosis ( 58). Further, they release phospholipid (platelet Factor 3 ) (259) and agents enhancing vascular permeability (453), including histamine (269), serotonin (269), and a third, as yet unidentified, heatstable factor which probably releases histamine and can contract smooth muscle ( 486). These various reactions suggest one important way in which immune reactions induce biological effects. The discharge of platelet phospholipid is probably one cause of the acceleration of clotting which takes place when whole blood is exposed to immune complexes (556, 612); no acceleration occurs in platelet-poor plasma (545, 641 ). The aggregation of platelets by soluble antigen-antibody complexes is apparently secondary to their adhesion to the platelet wall-a process requiring complement when the concentration of antibody is low (58,

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619) but perhaps not when it is relatively high (419, 442). Small antigen-antibody aggregates may be phagocytized by platelets ( 442), whereas larger aggregates evoke degranulation of these cells without phagocytosis ( 4 8 6 ) .Aggregation of platelets occurs subsequent to the adhesion of the antigen-antibody complexes, for, unlike platelet aggregation, adhesion is not blocked by pyrazole compounds such as phenylbutazone (452). Aggregation of platelets by antigen-antibody complexes cannot be attributed to the release of thrombin. It occurs in plasma adsorbed with barium sulfate, which removes prothrombin ( 2 6 9 ) ,and it is not inhibited by heparin (269, 611, 619) or hirudin ( 5 8 ) , which inhibit thrombin. The release of ADP when antigen-antibody complexes adhere to platelets may be responsible for the aggregation of these cells and for the release of permeability factors (453) and still more ADP ( 4 4 2 ) . The release of whereas that of ADP and serotonin is histamine is blocked by DFP (44), blocked by DFP and by salicylaldoxamine, an inhibitor of C’3 ( 2 1 3 ) ,but not by heparin ( 6 1 2 ) . Despite the implications of these observations, the aggregation of pIatelets and the subsequent release of their contents are not necessarily entirely a specific effect of antigen-antibody complexes. I have already noted that aggregation need not require complement (419, 442). Moreover, latex particles coated with 7-globulin bring about aggregation of platelets and the discharge of their contents ( 4 5 2 ) ; in the absence of 7-globulin, the particles adhere to the platelets without inducing aggregation, once again separating the processes of adhesion and aggregation. Other particles, such as kaolin, may cling to the platelet and release its phospholipid and 5HT ( 240,626). The release of vasoactive amines which takes place when platelets are incubated with a mixture of antigen and the plasma of sensitized rabbits is probably dependent upon the presence of calcium ions (269, 611 )-a point in dispute ( 4 5 4 ) .The discharge of 5HT from human platelets has been used as a sensitive test for circulating antibodies ( 9 5 ) . Movat (440, 441) provides grounds for the belief that Hageman factor may be involved in the interaction between immune aggregates and blood. In his hands, antigen-antibody complexes accelerated clotting if they had first been exposed to normal human serum, but not if they had first been exposed to serum lacking Hageman factor. Guinea pig serum, prepared in such a way that it had not been in contact with glass, when incubated with antigen-antibody aggregates evolved permeabilityenhancing activity, attributable in part to the evolution of kinins. Serum, treated with antigen-antibody aggregates in the presence of an inhibitor

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of kininase, induced contraction of the guinea pig ileum and rat uterus and decreased the blood pressure of rabbits. On this and other evidence, Movat implicated the activation of Hageman factor by antigen-antibody aggregates in the genesis of the biologically active agents he found. Although his experiments are persuasive, acceptance of his conclusions await a rationalization of his findings with our own observation that antigen-antibody aggregates do not accelerate the clotting of plateletpoor plasma which has been protected from contact with glass. The disruption of platelets brought about upon contact with antigenantibody reactions has led Packham and her associates (486) to speculate upon the analogy between this phenomenon and immune lysis of cells. In support of this concept, they found that washed human platelets, lysed by freezing and thawing, provided complement-like activity for the hemolysis of sensitized sheep erythrocytes. No evidence is available, however, suggesting that complement participates in the changes that occur in platelets during hemostasis; its relationship to fibrinolysis and platelets was discussed on p. 182. VII.

Anaphylatoxins and Related Mediators of Inflammation

When antigen-antibody aggregates are incubated in fresh serum, containing complement, the mixture can induce a lethal reaction which resembles anaphylaxis when injected into experimental animals ( 197, 198). Friedberger (197) named the agent responsible for this dramatic result “anaphylatoxin.” With the years, the term has acquired a generic meaning for any of a group of mediators of inflammation which may be derived from complement or other components of serum upon exposure to antigen-antibody aggregates ( 485) or certain other substances, notably kaolin (300), diatomaceous earth ( 1 3 7 ) , agar ( 7 3 ) , or starch ( 4 6 1 ) . Anaphylatoxins can also be generated by diluting serum with distilled water (477). The anaphylatoxins do not act directly upon a target organ, but only after the elaboration of a mediator such as histamine. Characteristically, repeated injections of anaphylatoxin appear to desensitize the target organ, as if some substrate upon which it acts has been exhausted (55). At present, anaphylatoxins are differentiated from kinin-releasing agents on rather dubious semantic grounds; it is hard to see an intrinsic distinction, for both release small biologically active molecules. Our understanding of the way in which antigen-antibody aggregates bring about the formation of an anaphylatoxin stems from the work of Humphrey and Jaques (269), who reported that serum, exposed to antigen-antibody complexes, increased vascular permeability in rabbit

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skin. Later, Osler et al. (485) found that rat serum which had been treated with immune aggregates contained an agent that induced contraction of guinea pig ileum and increased vascular permeability in guinea pig skin. They emphasized that complement was needed for its generation. These two observations suggested that histamine was liberated either in the serum or in the ileum, which is readily contracted by this amine. More recently, Lepow and I (539) found that C’1 esterase, which forms when antigen-antibody aggregates are incubated with complement, increased vascular permeability in guinea pig skin. The change in permeability was prevented by injection of the guinea pig with triprolidine, an antihistaminic agent, but not by SBTI. Electron microscopy localized the alteration to the postcapillary venule, the point which Majno and Palade (382) had demonstrated to be affected by this amine. Thus, C’1 esterase behaved as if it brought about the release of histamine. As might be expected, a slight increase in vascular permeability was also noted upon the injection of C’lq and C’lr, presumably because these could react with other components of C‘1 in the guinea pig skin. The mechanism through which histamine is elaborated by the injection of C’1 esterase has been the subject of a series of studies by Lepow, Dias da Silva, and their associates. The effect of C’1 esterase was enhanced by mixing it with guinea pig serum before injection, suggesting that the elaboration of the histamine-releasing agent required the interaction of C’1 esterase with components of serum (620).Moreover, a mixture of C’1 esterase with guinea pig or rat plasma acquired the capacity to contract guinea pig ileum, an effect inhibited by triprolidine ( 1 3 4 ) . The substrate in guinea pig ileum was readily exhausted, that is, this tissue demonstrated tachyphylaxis or densensitization to the toxic effects of the serum-C’l esterase mixture. The treated serum itself did not contain histamine, for the active agent was nondialyzable. Instead, it appeared to release this substance from the tissues, as demonstrated in lung slices, perhaps by an action on mast cells, for these decreased in numbers on application of the mixture to mesentery. In these studies, the property of serum responsible for the release of histamine was related to the presence of C’4, C’2, and C’3. In agreement with this, an agent that contracted guinea pig ileum, released histamine from rat peritoneal mast cells, degranulated guinea pig mesenteric mast cells and increased vascular permeability evolved in solutions containing the first four components of complement isolated in highly purified form (135).This agent, an anaphylatoxin, was localized to a small fragment, designated F( a)C’3, derived from C’3 ( 1 3 3 ) .The fragment had the characteristics of a polypeptide with a molecular weight of 6800 or less. These experiments pro-

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vide one explanation for the effect of C’1 esterase upon vascular permeability. Other mechanisms may be involved, since the increase in vascular permeability which occurs in human skin upon the injection of C’1 esterase does not appear to require the presence of C’3 nor involve the release of histamine ( 307). I am unfamiliar with studies in which the anaphylatoxin derived from C‘3 through the action of C’1 esterase has been produced by mechanisms in which Hageman factor or plasmin participate, although that this would be the case seems logical. Incubation of C‘3 with trypsin, however, leads to the generation of an anaphylatoxin that desensitizes the guinea pig ileum to the subsequent administration of F( a)C’3, suggesting trypsin separates the same or a similar fragment from C‘3 (107). Plasmin does release a chemotactic factor from rabbit (654) or human (699) serum or from rabbit or human C’3 (699). The chemotactic property has been localized to a fragment of C’3 with a molecular weight of about 6000. Although it has been stated that this fragment differs from F( a ) C’3, the observation that it increases vascular permeability in rat skin suggests that the differentiation between the two fragments should be re-examined. Thus far, it has not been possible to demonstrate that plasmin induces the release of other biologically active substances from complement. Jensen (279), however, found that trypsin released an anaphylatoxin from guinea pig C’5. In similar experiments, with human C’5, Cochrane and Muller-Eberhard (107) observed that this enzyme split a poIypeptide from human C’5, designated F(b)C‘5, which behaved as if it released histamine in tissues. This fragment acted in a different way from F( a)C’3, since exposure of the guinea pig ileum to the one did not desensitize this tissue to the other (107). A reasonable assumption is that these studies of complement are an explanation of the way anaphylatoxins are induced by antigen-antibody aggregates. In rats, the first five components of complement are needed for the release of histamine from sensitized peritoneal mast cells by rabbit antirat-y-globulin ( 2 2 ) . Similarly, Jensen (279) provided data supporting the concept that in guinea pigs the anaphylatoxin released by exposure of sensitized erythrocytes to components of complement was derived from C’5. The anaphylatoxic activity resides in a fragment of C‘5 with a molecular weight of 15,000 and has the additional property of attracting polymorphonuclear neutrophiles ( 610). The relationship of this agent to the chemotactic factor Ward (698) derived from human C4, (76, C’7 complex is not yet known, but it is clearly separable

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from the anaphylatoxin released from human C3 by antigen-antibody aggregates ( 1 3 3 ) . Endotoxic lipopolysaccharide, added to fresh serum, also brings about the formation of an anaphylatoxin-a process requiring the presence of complement, particularly C’3 and C‘5 (624). At the same time, a chemotactic agent appears in the mixture, which can be differentiated from that induced by the addition of plasmin to serum by its heat stability and its higher molecular weight, 15,00030,000 ( 3 ) . However anaphylatoxins are liberated by agar and similar substances, their effects are mediated at least partially through the release of histamine (228,229, 438, 559,561) from mast cells (438) and platelets ( 269). Evidence that complement is related to the formation of anaphylatoxins by such agents is less clear. Heating serum at 56°C.for 30 minutes before exposure to agar does not impede the generation of anaphylatoxin ( 561) . Large amounts of heparin inhibit anaphylatoxin formation, but this agent has such widespread effects that one can draw no conclusions from this observation. Bier ( 6 1 ) could not block anaphylatoxin formation in guinea pig serum in which complement had been inactivated by Congo red, while Becker ( 4 3 ) was equally unsuccessful with DFP, which inactivates C’1 esterase. On the other hand, other organophosphorus inhibitors were effective ( 4 4 ) . Ungar (678) has proposed that complement is important in the liberation of anaphylatoxin by such substances as agar. Exposure of rat serum to dextran causes partial inactivation of C’1 and almost complete inactivation of C’2 and C’4, a result suggesting that C’1 has been activated. Dias da Silva and Lepow (134) observed that whatever the mechanism through which agar releases anaphylatoxins in guinea pig plasma, the pathway through which they are liberated is coincident with that traversed to form those induced by C’1 esterase. The response of the guinea pig ileum to the one is exhausted by exposure of the muscle to the other. An early hypothesis linked the formation of anaphylatoxins to the liberation of a proteolytic enzyme (88, 282). This view is supported by the observation that plasma, treated with agar in such a way that it liberates histamine from platelets, can digest denatured hemoglobin (269). Further, agar-treated rat serum develops esterolytic activity against TAME ( 5 7 3 ) . Although these experiments suggest that agar activates plasminogen, one need not infer that plasmin is responsible for anaphylatoxin formation. Esterolytic activity against TAME might also be due to the activation of kallikrein, for example, and agar-treated plasma

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generates bradykinin, as if this polysaccharide liberated this enzyme (574). It is true that plasma, treated with trypsin or exposed to chloroform, can release histamine ( 2 6 9 ) , but plasmin itself is ineffective (71, 559). More likely the effect of chloroform is brought about through the generation of C’1 esterase (20) and is therefore not necessarily related to the generation of anaphylatoxins by agar and such substances. VIII.

Anaphylaxis

As knowledge concerning anaphylaxis accumulated, two principal schools of thought arose to explain its pathogenesis, paralleling the humoral and anatomic schools of pathology. One hypothesis, influenced by knowledge about anaphylatoxins, attributed anaphylaxis to the elaboration in blood or tissue fluids of toxic agents derived through the action of antigen-antibody complexes (197, 198). The second opinion, based upon studies of isolated tissues, held that the symptoms resulted from the direct effect of antigen-antibody reactions upon the cells of the target organs (116, 593). These two viewpoints now seem to express an archaic distinction, for there is nothing incompatible between ideas which were formerly the subject of great controversy. Whatever way one looks at anaphylaxis and related phenomena, the elaboration or release of one or more simple humoral agents is a vital step in pathogenesis. In species such as the guinea pig and man, in whom death may occur almost instantly, the lethal agent is probably liberated by the action of antigens upon antibody within or closely adherent to cells of the target organ. In those species in which anaphylaxis is a slower process, the effects of immune aggregates may be more widespread, and here the release of vasoactive substances from platelets, for example, may contribute to the evolution of symptoms. Vaughan (684a),was an early proponent of the idea that the injection of antigen into a sensitized animal leads to the activation of a specific enzyme which liberates toxic substances, i.e., anaphylatoxins, from protein substrates in plasma. This view, similar to that which envisions that agar and other similar substances form anaphylatoxins through the liberation of a protease, was supported by experiments such as those of Bronfenbrenner ( 8 8 ) and Jobling ( 2 8 2 ) . It finds modem expression in the work of Ungar and his associates who reported, as did Jobling ( 2 8 1 ) , that the antitryptic titer of plasma decreased during anaphylaxis (677) and that plasmin formed when the serum of a sensitized guinea pig was incubated with specific antigen (681). The activation of a fibrinolytic agent took place only if the guinea pig serum was fresh ( 6 8 0 ) . Ungar

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( 679) suggested that proteolytic activity released by antigen-antibody reactions led to the release of histamine. In this simple form, the hypothesis that plasmin is formed during anaphylaxis or, in vitro, by antigenantibody aggregates has not been sustained (23, 202, 278). Nonetheless, it is conceivable that plasmin, formed locally rather than in the circulation, may play a part in anaphylaxis, for Zweifach (742) found that EACA, an inhibitor of the activation of plasminogen, provided partial protection against anaphylaxis in mice. More likely, elaboration of plasmin during anaphylaxis is a phenomenon secondary to the tremendous stress imposed by this syndrome, Thus, Gans and Krivit (202) observed that, in rabbits, anaphylaxis brought about an increase in the concentration of a plasminogen activator in the plasma, but EACA had no effect upon the lethal outcome. The cellular hypothesis of anaphylaxis early envisioned that symptoms were due to the local release of histamine. Dale and Laidlow (117) drew a close analogy between the symptoms of anaphylaxis and those evoked by the intravenous injection of histamine. Dragstedt (153, 154) isolated a histamine-like substance from the bloodstream and thoracic duct of intact animals during anaphylaxis, and this agent has been demonstrated in the perfusate of an isolated guinea pig lung during anaphylactic shock (34, 637). Mongar and Schild (431, 432) liberated histamine by the addition of antigen to chopped lungs of sensitized guinea pigs, but only if the cells remained intact. The source of the histamine which is released is, at least in part, the mast cells, from which histamine can be released by antigen-antibody interaction in vitro ( 2 4 ) . Mongar and Schild observed that the release of histamine upon the addition of antigen to chopped sensitized guinea pig lung requires the . led to presence of calcium (433) and a heat-labile factor ( 4 3 2 ~ )This the view that the antigen reacted with antibody fixed to the cell and that the combination, in the presence of calcium, activated a heat-labile enzyme which released histamine from mast cells granules ( 4 3 4 ) . The nature of this tissue enzyme is not known, but Austen and Brocklehurst (25) provided evidence that it resembles C’1 esterase. The local release of histamine seems to explain many of the phenomena of anaphylaxis in guinea pig, dog, and man, but it is probably not responsible either for all symptoms observed in these species or for those that occur in some other species (21, 552, 583). Thus, in rats, the administration of antihistaminics or depletion of histamine stores by treatment with polymyxin B does not prevent anaphylaxis. Other candidates for the role of humoral mediators of anaphylaxis include acetylcholine, 5HT, plasma kinins, and slow reacting substance-A.

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Although Wenner and Buhrmester (713) reported that acetylcholine could be detected in rabbit’s heart blood during anaphylaxis, I was unable to duplicate their findings (515); it is improbable that this neuromediator is significantly involved in the pathogenesis of this syndrome. Whether the release of 5HT is important in some species is disputed. Fink (189) believed that she could demonstrate its release in the isolated mouse uterus during local anaphylaxis. Similarly, systemic anaphylaxis in sensitized rabbits is accompanied by an increase in the concentration of plasma 5HT, possibly released from platelets ( 691,692). But anaphylaxis is not prevented by depleting rats (583) or rabbits (190) of 5HT or administering 2-bromolysergic acid diethyl amide, a specific antagonist. Austen ( 2 1 ) has summarized evidence that 5HT may be of importance in anaphylaxis in rats and mice. The possibility exists that the 5HT detected during anaphylaxis is not of primary importance but reflects instead the secondary damage to platelets by antigen-antibody complexes. Yet another possible mediator is the agent known as SRS-A, described by Brocklehurst (82), so called because it produces a slow, sustained contraction of guinea pig ileum. Brocklehurst (84) detected this substance in the perfusate after local anaphylaxis in the isolated guinea pig, rabbit, or human asthmatic lung, and it has also been found after perfusion of rat lung (97). Although mast cells have been implicated in the release of SRS-A, in rats, at least, the source of this substance appears to be the polymorphonuclear leukocyte ( 4 8 3 4 . Slow reacting substance-A induces potent constriction of the bronchioles of guinea pigs, comparable to that which accompanies lethal anaphylaxis ( 5 2 ) ; a reasonable conclusion is that SRS-A is of particular importance in this species. Slow reacting substance-A has also been detected in nonimmunologic experimental exudates ( 633,635). The possibility that kinins are released during anaphylaxis has received relatively little attention. Beraldo (50) reported that bradykinin could be identified in the blood during anaphylactic shock in the dog, an observation confirmed in this species ( 2 9 ) and in guinea pigs, rats, and rabbits (85). Brocklehurst and Lahiri (86) and Jonasson and Becker (284) detected kallikrein after infusion of antigen into sensitized guinea pig lung. The infusion of ellagic acid into nonsensitized guinea pig lungs gave a similar result, leading these investigators to suggest that the initiating event in the activation of kallikrein by antigen-antibody reactions is the activation of Hageman factor. This view is attractive in the light of Movat’s (440, 441 ) and Donaldson’s (143) evidence that this clotting factor may be implicated in the mediation of antigen-antibody reactions. No evidence has been published implicating PF/Dil in anaphylactic shock (723).

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During anaphylaxis, the hemostatic mechanisms are profoundly altered. The number of circulating platelets drops precipitously (157, 510, 691). In dogs (60,157), rabbits (157,197), and guinea pigs (197), the clotting time is prolonged. The concentrations of fibrinogen, plasminogen ( 29, 202), proaccelerin ( 580), and prothrombin (29) decrease significantly. In rabbits, at least, thrombocytopenia precedes other coagulative changes (580). The importance of these hemostatic alterations in the development of symptoms of anaphylaxis probably differs depending upon the species and the severity of the episode. They are apparently secondary phenomena, rather than the primary events in the pathogenesis of shock. Thus, neither the administration of hirudin (197) or heparin (580), which inhibit thrombin, forestall anaphylactic shock. Evidence concerning EACA, which prevents the fall in fibrinogen (29, 202) is conbadictory (29, 53, 202, 742). In guinea pigs, the deliberate induction of thrombocytopenia before the administration of antigen is not protective (269), although in rabbits, the symptoms are ameliorated. Nonetheless, the fall in the platelet count after the administration of antigen is accompanied by release into the circulation of histamine (153) and 5HT (691) which may, of course, augment the animals’ symptoms. In dogs, the lengthened clotting time has been attributed the presence of heparin in the circulating blood (277); this has not been found in other species, but an inhibitor of the reaction between thrombin and fibrinogen, not identified as heparin, has been detected in rabbit plasma after anaphylaxis (157). The fall in the concentrations of fibrinogen, proaccelerin, and prothrombin suggests that intravascular coagulation may be an element of anaphylactic shock. Thrombuslike masses are found within small pulmonary vessels in rabbits (696), although evidence that these are true fibrin thrombi rather than precipitates of antigen and antibody is weak (372, 696). Salmon and his associates (580) have demonstrated deposition of fibrin in small blood vessels, especially in the glomeruli, in this species. Thrombocytopenia may well be the result of aggregation of platelets by antigen-antibody complexes; clumps of platelets, some of which are degranulated, are found in the blood vessels in anaphylaxis, intermingled with antigen-antibody aggregates, some of which are phagocytized by these cells (442). An alternative possibility, that thrombocytopenia is secondary to the release of histamine, cannot be supported experimentally (510). These various studies suggest that the hemostatic alterations of anaphylactic shock are due either to the damage induced by the reaction of antigen and antibody in tissues or, in the case, of platelets, to the coincident effect of immune complexes on these cells.

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IX.

Endotoxins and the Shwartzman Phenomenon

A. THE EFTECTOF A SINGLEINJECTION OF ENDOTOXIN A single intravenous injection of appropriate amounts of gram-negative endotoxin induccs lethal shock in experimental animals. At autopsy, little is found to explain the catastrophe (569). For example, in dogs extensive hemorrhagic necrosis of the small intestine is found, whereas the lungs, liver, and kidneys show “evidences of ( a ) stagnant anoxic process” (354). After sublethal doses of endotoxin, profound alterations can be demonstrated in the hemostatic mechanisms, but anatomic examination does not usually implicate thrombosis in the pathogenesis of the syndrome. In rabbits, an occasional thrombus is found in the small blood vessels of lungs, liver, spleen, and kidneys, and these only in a small proportion of animals (204,370,662).Hepatic vein thrombosis may be found in rats, but only if they are first fed a high fat, low protein diet ( 5 5 1 ) . The possibility that the effects of endotoxin are mediated through the blood-clotting mechanism has been investigated repeatedly. The addition of endotoxin to whole human (371) or rabbit (259, 557) blood shortens the clotting time in silicone-coated tubes. Robbins and Stetson (557) interpreted this clot-promoting effect as a reflection of the formation of antigen-antibody complexes between endotoxin and naturally occurring antibodies. The clot-accelerating properties of endotoxin are probably due to its action upon platelets. McKay (371) believed that endotoxin shortened the clotting time of platelet-poor plasma, but neither I (522) nor Horowitz (259) could confirm this. In uitro, endotoxin aggregates platelets (131, 549), a phenomenon requiring the presence of proaccelerin ( 5 4 9 ) . In the process, platelet phospholipids (platelet Factor 3), 5HT, and bactericidins directed against Bacillus subtilis are released into the plasma (131,309). The coagulative changes observed after a single intravenous injection of endotoxin into rabbits have been variable, reflecting differences in the nature and dosage of endotoxin, in the technique used to draw blood, and perhaps in the animals themselves. Within 5 minutes, the platelet count drops sharply (239, 259), but quickly increases again so that during the ensuing 24 hours, the number of platelets is only moderately depressed (341, 370, 514, 642, 730). The thrombocytopenia is apparently accompanied by clumping of platelets in vivo (179, 642) and results in the release into the plasma of clot-promoting phospholipids (259, 549) and 5HT (124), comparable to what is observed in uitro. Yamazaki (730)

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suggested that the clumping of platelets is mediated by an agent in plasma, again reminiscent of studies in uitro. As one might anticipate, the clotting time of whole blood is strikingly shortened within as brief a time as a minute after the injection of endotoxin ( 2 5 Q ) but , this effect is transient. In McKay’s (370) and Carozza’s ( 9 4 ) experience, the clotting time was still short after 4 hours; in these studies, blood was obtained by cardiac puncture and femoral arterial catheterization, respectively. In contrast, the clotting time of blood drawn through a catheter threaded into the internal jugular vein was abnormally long within 3 hours after the injection of endotoxin and remained so for at least 24 hours ( 5 1 4 ) , a finding previously reported by Volk and Losner ( 6 8 8 ) . Lengthening of the clotting time has also been observed by Hardaway (238) and Lillehei (355) in dogs and by Altura ( 0 ) in rats. In our own studies, 24 hours after the intravenous injection of endotoxin, the rabbit’s partial thromboplastin time was significantly prolonged, correlating roughly with the degree of lactescence which appears in the plasma of endotoxin-treated rabbits ( 514) because its total lipid content is sharply increased ( 3 3 9 ) .Why the clotting time lengthens has not been explained. The rate at which fibrinogen is removed from the circulation increases ( 3 4 0 ) , but the plasma concentration of this protein does not usually decrease appreciably, or it actually increases (201,340,341,370), presumably because fresh fibrinogen enters the circulation at an accelerated rate. The fibrinogen may be qualitatively altered, for if heparin is added to plasma (663) or to Cohn fraction I (601) prepared from blood drawn 4 hours after the injection of endotoxin into rabbits, a flocculent precipitate forms. This appears to be a complex of fibrinogen and fibrin monomers, as if only part of the fibrinopeptides had been released from fibrinogen ( 6 0 2 ) . The one-stage prothrombin time, a measure of the extrinsic pathway of coagulation, is either unaltered (341 ) or only slightly lengthened ( 2 3 9 ) . No evidence has been found of increased fibrinolytic activity (201, 323, 341) or of the appearance of anticoagulants directed against the formation of thrombin (341 ). The implication that alterations in hemostatic processes are important in the evolution of endotoxic shock finds support in the partial preventive effects of heparin (238) and TAME ( 7 4 2 ) in different species. EACA is inhibitory in dogs but not in rabbits ( 6 3 8 ) . Heparin, however, does not block the lethal effects of meningococcus toxin ( 2 1 5 ) .Inhibition of platelet aggregation by the administration of such agents as nitrogen mustard, aspirin, and phenylbutazone also inhibits endotoxic shock ( 1 7 9 ) . These various inhibitors have many biological effects, and caution should be used in interpreting their action. If changes in clotting are of importance,

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the effect need not be just from the induction of thrombosis which is, as I have noted, a minor part of the pathological process. The release of histamine (250,309) and 5HT (124,125,131), perhaps consequent to the disruption of platelets, is another way in which hemostatic changes may be an element in the pathogenesis of endotoxic shock. Further, kinin-like vasoactive substances are liberated into the blood (309, 467), contributing to the development of hypotension. The release of kinins is mirrored in a depletion of plasma kininogen (467, 670). The administration of Trasylol, which inhibits kallikreins (403), or prior depletion of plasma kininogen by the injection of cellulose sulfate (572), defers or prevents endotoxic shock. On the other hand, in the test tube, human plasma kallikreinogen is not activated by endotoxin ( 7 3 3 ) .These observations have led to the opinion that endotoxin activates Hageman factor (174, 657), but this hypothesis, however appealing, has not yet been established ( 259,529 ) . The changes induced by endotoxin described thus far need not account for all its effects. For example, endotoxins behave as if they were adrenergic (570, 741 ), and experimental endotoxic shock can be ameliorated by the administration of suitable adrenergic blocking agents ( 3 5 5 ) .

B. THEGENERALIZED SHWARTZMAN REACIION When a second, otherwise nonlethal intravenous injection of endotoxin is administered to rabbits 24 hours after the first, the animals fall ill within a few hours, develop diarrhea, and, within a day, usually die ( 662). This course of events, first observed by Sanarelli (582) is now described as the generalized Shwartzman reaction. The most striking pathological change is bilateral diffuse hemorrhagic necrosis of the renal cortex, but areas of hemorrhage and necrosis are also seen in many other tissues (12, 204, 582, 662). The glomerular capillaries are plugged with material which is either fibrin or some other derivative of fibrinogen (340, 368, 488, 685). Necrosis of the interlobular arteries may be found ( 2 0 4 ) . Thrombosis of small blood vessels in many organs by material resembling fibrin is a prominent feature of the reaction ( 11, 91,204,370). Cortisone ( 6 6 1 ) , thorium dioxide (Thorotrast) (214), trypan blue (214), colloidal iron (621), and carbon (621 ), the last four blockaders of the reticuloendothelial system, can be substituted for the initial, preparative dose of endotoxin in the induction of the generalized Shwartzman reaction. In pregnant rabbits, only one injection of endotoxin is needed to evoke the phenomenon ( 11,563,661 ) . Studies on the pathogenesis of the generalized Shwartzman reaction have led to a unifying concept, well expressed by Hjort and Rapaport

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(254). The injection of endotoxin initiates intravascular formation of fibrin monomers, soluble polymers, or fibrin itself. These are ordinarily removed from the circulation by the reticuloendothelial system. But in animals which have been “prepared by a previous injection of endotoxin or of agents known to block the reticuloendothelial system, fibrin or fibrin intermediates cannot be “cleared from the circulation, and instead deposit in small blood vessels. The deposition of this fibrin-like material is enhanced by alterations in plasma fibrinogen, perhaps induced by the liberation of acid mucopolysaccharides from granulocytes. Evidence supporting this chain of events will be reviewed briefly. In prepared rabbits, the injection of endotoxin is followed by accelerated disappearance of labelled fibrinogen (340) and a fall in the concentration of fibrinogen in plasma (370).At the same time, the concentration of clotting factors which might be expected to be consumed during fibrin formation decreases. Thus, the platelet count falls (341, 370, 563) and the concentrations of prothrombin (305, 341, 563), proaccelerin (305, 341, 563), and antihemophilic factor (113, 305) decrease. The platelets behave as if qualitatively altered by thrombin (704).These observations are consistent with the view that the generalized Shwartzman reaction is a special instance of the defibrination syndrome. In agreement with this, the reaction can be provoked by the intravenous injection of thrombin into pregnant rats (388) or in rabbits prepared by prior injection of Thorotrast (323).Thorotrast-prepared rabbits will also develop the generalized Shwartzman reaction when injected with purified fibrin (565) or with platelet phospholipid (566), although the latter observation is disputed (444). When thromboplastin or thrombin is injected into unprepared animals at a s&ciently slow rate, the circulating plasma may be completely defibrinated, yet few if any thrombi are demonstrable in small blood vessels (35, 532). Lee (323, 325) found that the fibrin or some other derivative of fibrinogen was phagocytized by the reticuloendothelial system. Evidence reviewed above that endotoxin induces intravascular clotting is strengthened by Lee’s (326) observation that fibrin was also present in reticuloendothelial cells after a single injection of endotoxin. The need for a preparatory injection of endotoxin, Thorotrast, or similar substances thus seems evident. In each case, the reticuloendothelial system is so burdened that a subsequent injection of a clot-promoting agent-endotoxin or thrombin-results in the formation of fibrin or fibrin intermediates that cannot be cleared from the circulation and are therefore deposited in small blood vessels, notably in the renal cortex (254, 325). These observations suggest that a proper alteration of the conditions

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used to induce intravascular coagulation should bring about the Shwartzman reaction without prior preparation of the rabbit or the use of endotoxin. When endotoxin is injected directly into the aorta, so that a high concentration reaches the tissues, lesions of the generalized Shwartzman reaction can be induced in the unprepared animal (238). Similar lesions can be produced by intra-aortic injection of thrombin in otherwise untreated animals (111, 555). The intravenous injection of thrombin into animals given EACA, Trasylol, or soybean, lima bean, or ovomucoid trypsin inhibitors induces the generalized Shwartzman reaction (48, 323). These considerations led investigators to wonder whether the phenomenon could be induced by activating the animal’s clotting mechanisms. The injection of phospholipid into “prepared rats or rabbits does not evoke the reaction (444). Nor does kaolin (662), an activator of Hageman factor, initiate the Shwartzman reaction when injected intravenously into prepared animals. Whether ellagic acid, a soluble activator of Hageman factor, will induce the reaction in Thorotrast-treated rabbits is disputed (76, 567), but the combination of ellagic acid, norepinephrine ( to stimulate a-adrenergic receptor sites), and EACA is effective (369), implicating the intrinsic pathway of clotting in the pathogenesis of the reaction. In prepared animals, the injection of endotoxin is followed by a transient fall in the white blood cell count (614, 642, 662). Leukocytes have been thought to be needed for the development of the generalized Shwartzman reaction. Thus, the induction of leukopenia by the administration of nitrogen mustard is preventive (622, 662). The significance of this observation is clouded by the fact that irradiation-induced leukopenia does not block the generalized Shwartzman phenomenon (283). Evidence for the importance of platelets in the protection afforded by nitrogen mustard is contradictory. This agent impairs platelet function (454), but the transfusion of platelets to rabbits treated with nitrogen mustard does not overcome its protective effect against the generalized Shwartzman reaction ( 6 2 2 ) . The possibility that acid mucopolysaccharides, liberated by leukocytes, may be implicated in the deposition of fibrin-like material in the glomeruli, suggested by Thomas (MO),gains support from studies with agents such as dextran sulfate or Liquoid. The injection of these agents into rabbits results in renal lesions resembling the generalized Shwartzman reaction (51, 90, 244, 697). Amounts of dextran or Liquoid, insufficient in themselves to incite renal damage, induce the phenomenon if injected either at the same time as EACA, which inhibits fibrinolysis (684), or 1 hour after a preparatory injection of endotoxin (660). Dex-

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tran, Liquoid, and similar agents act directly upon the clotting mechanism, Liquoid, for example, precipitates fibrinogen from plasma (648) and aggregates platelets (58, 684), while warfarin (which inhibits the synthesis of the vitamin K-dependent clotting factors) prevents renal cortical necrosis after the administration of Liquoid ( 1 8 0 ) . Still, the specificity of the effect of Liquoid is not certain, for it reacts with many plasma proteins, including some, such as C’lr and C’1 esterase inhibitor, which may be implicated in the generalized Shwartzman reaction (544). In any case, these studies exemplify the view that the generalized Shwartzman reaction describes a process which can be initiated in many ways and which includes in its elements the intravascular deposition of fibrin or related proteins. If the generalized Shwartzman reaction is an example of intravascular coagulation, one would anticipate that anticoagulant drugs should be inhibitory. Both heparin (215) and warfarin (604) do prevent the deposition of intravascular fibrin-like material. Similarly, one might expect that activation of the plasma’s fibrinolytic mechanisms would inhibit the reaction, and this is, indeed, the case (48,112,308,564).Conversely, inhibition of fibrinolysis should enhance the Shwartnan phenomenon by stabilizing the fibrin laid down in the small blood vessels. EACA, which blocks the activation of plasmin, permits the development of lesions upon the injection of thrombin (48, 323), ellagic acid ( 3 6 9 ) ,or Liquoid (369, 684). A number of investigators have confined their observations to determination that protease inhibitors do not block the reaction (341,742), although this is not universally agreed upon ( 1 0 3 ) . The data which I have assembled support the contemporary view that the generalized Shwartzman reaction is accompanied by intravascular deposition of fibrin or some other fibrinogen derivative. I have not attempted to review evidence that endotoxin has other, profound circulatory effects of importance in the evolution of shock and the development of the Shwartzman reaction. Phenoxybenzamine (Dibenzyline), a blocking agent for a-adrenergic receptor sites, prevents glomerular capillary thrombosis in most pregnant rats given an intravenous injection of endotoxin ( 4 4 3 ) . This observation emphasizes the role of catecholamines in the production of renal lesions. Yet to be learned is the extent to which kinin-forming systems participate in these circulatory changes.

C. THELOCALSHWARTZMAN REACTION When gram-negative endotoxin is injected intracutaneously into a rabbit, and, 24 hours later, a second injection is given intravenously, after a lapse of several hours a purpuric, hemorrhagic, and necrotic reaction

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occurs at the site of the intracutaneous injection (614). In sensitized animals, the injection of specific antigen can be substituted for the intracutaneous injection of endotoxin (614, 643), while agar ( 6 1 5 ) , glycogen (66S), kaolin (66S), or cortisone (661 ) can replace the intravenous injection of endotoxin. Blockade of the reticuloendothelial system with, for example, Thorotrast, overcomes the natural insusceptibility of some rabbits to the reaction ( 4 7 ) . The intradermal “preparatory” injection of endotoxin evokes a local inflammatory reaction, characterized by erythema and edema, visible within 6 to 8 hours and maximal about 24 hours after the injection ( 3 2 7 ) . The sequential changes which take place have been well described by Lee and Stetson ( 3 2 7 ) . The area is infiltrated with leukocytes, particularly polymorphonuclear neutrophiles, which accumulate about the adventitia of small veins (289, 641). Within 24 hours after the injection, mononuclear cells may also be prominent in the injected site. Paradoxically, in contrast to other inflammatory states, local vascular permeability is not increased ( 6 6 4 ) . Within half an hour after the intravenous “challenging” injection, the small arteries and arterioles in the dermal site are constricted, margination of leukocytes in venules and small veins is intensified, and capillaries and small veins are occluded by masses of leukocytes and platelets (327, 642). Fibrin and red blood cells are detected in the aggregates and may block the circulation to the area. Within the next 2 hours, progressive necrosis of all elements of tissue occurs, with disintegration of the leukocytes in the area and degeneration of blood vessel walls (289, 327). In the next few hours, the lesions becomes hemorrhagic. Ultimately, the necrotic tissue hardens into a dark eschar which is sloughed after a few days (327). Leukopenia, induced by the administration of benzene, X-irradiation, or nitrogen mustard, prevents the local Shwartzman reaction ( 4 6 ) . Clamping the abdominal aorta inhibits the action of nitrogen mustard by sparing the femoral marrow from its toxic effects ( 6 4 4 ) . How leukocytes participate in the pathogenesis of the reaction is not known, but Thomas (659) was able to prepare rabbit skin by the intracutaneous injection of neutrophilic granules. Mustard and Packham (454) have pointed out that the techniques used to induce leukopenia either reduce the number of platelets or damage these cells. Perhaps, then, leukocytes are not as essential an element of the local Shwartzman reaction as was originally thought. The ischemic nature of the fully developed lesion emphasizes the importance of local thrombosis in its pathogenesis. Supporting this, the

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phenomenon can be prevented by reducing the coagulability of the blood by the administration of Dicumarol (627) or heparin (215). Similarly, streptokinase is inhibitory, while paradoxically, SBTI and pancreatic trypsin inhibitor are said to be only partially inhibitory (103),and thrombocytopenia (342) or the injection of TAME or EACA do not block the local Shwartzman phenomenon (103); the latter statement has been contradicted ( 5 3 ) . Lee and Stetson (327, 643) have proposed that the local and general Shwartzman phenomena are mediated through an immune reaction between endotoxin and naturally occurring antibodies. As I noted, the injection of antigen into sensitized animals can prepare them for the local Shwartzman reaction. Further, in Thorotrast-prepared rabbits, antigenantibody aggregates induce renal cortical necrosis (324), while heparin prevents this change. The participation of humoral mediators of inflammation in the genesis of the localized Shwartzman reaction has been explored by Antopol and Chryssanthou (IOU, 103). These investigators prevented hemorrhage, vascular congestion, and edema by the administration of agents that antagonize the action of bradykinin and 5HT, without at the same time preventing leukocytic infiltration or thrombosis. Consonant with these observations, Trasylol, which inhibits kallikreins, prevents the local Shwartzman reaction ( 2 3 2 ) .These experiments emphasize the complexity of the responses involved in the development of the phenomenon. X.

Allograft Rejection

Fibrin is not usually present in the occluded blood vessels of cutaneous homografts undergoing rejection ( 87). But one of the early complications of human renal transplantation is occlusion of renal arterioles and glomerular capillaries with platelet thrombi (416, 502). Thrombosis of these vessels, resulting in cortical necrosis, is regularly found in man' and animals undergoing accelerated renal homograft rejection, presumably because they have a pre-existing sensitivity to the transplanted tissues (236, 501, 640, 725).In one study of kidneys undergoing rejection at an unspecified time after transplantation, material antigenically resembling fibrin was demonstrable immunologically in the blood vessels and glomeruli; in two cases, the fibrin-like material disappeared after the episode had . split products (Section II1,A) are said to subsided ( 3 7 1 ~ )Fibrinogen be found in the urine during the first 2 weeks after every renal transplant, and they reappear during reversible or irreversible rejection reactions, as if they were derived from fibrin, laid down in glomeruli, and digested there by plasmin ( 7 9 ) .Experimental homograft survival can be prolonged

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by the administration of EACA (54, 207), but the mechanisms involved are not obvious. The phenomenon of rejection has been interpreted in several ways. Starzl (640) and Kissmeyer (303) have likened accelerated renal homograft rejection to the generalized Shwartzman reaction. Packham and her colleagues (486) proposed that the arterioles and capillaries of allografts become coated with antibodies and that this surface then induces platelet aggregation. Alternatively, antigen-antibody complexes, reacting on the surface of the vessels, injure the endothelium, exposing subendothelial collagen which induces platelet aggregation. In support of this view, they quote unpublished studies of J. F. Mowbrey in which he inhibited platelet aggregation in renal vessels by administration of phenylbutazone, and in this way prevented rejection. A recent study of Lowenhaupt and Nathan (36la)bolsters the view that platelets are important in allograft rejection. In dogs, the earliest histological abnormality is the accumulation of platelets in the small blood vessels in the kidney to which the dog had been specifically sensitized before transplantation. XI.

Clinical and Experimental Nephritis

In light of the possibility that the generalized Shwartzman phenomenon is an example of antigen-antibody interaction, its resemblance to “Masugi” nephritis, induced by the intravenous injection into rabbits of antirabbit kidney serum, is noteworthy. In this form of experimental nephritis, fibrin is deposited within the capillaries and Bowman’s space (683). Both fibrin deposition and endothelial proliferation can be sharply reduced by the administration of warfarin, but this treatment does not prevent proteinuria. These observations suggest that Masugi nephritis is an example of localized intravascular coagulation, presumably brought about by the immune reaction between antibody and renal tissue. In agreement with this, heparin was found to protect rabbits against duck antikidney serum, but so much was used that other actions of this anticoagulant may have been involved (233). Blood-clotting mechanisms may also be involved in the renal lesions of experimental serum sickness. Cochrane, Hawkins, and Kniker (106) depleted rabbits of platelets by administering antiplatelet antibodies. Such treatment diminished the glomerular lesions and proteinuria induced by antigen-antibody complexes. Pretreating mice with heparin or urokinase also blocks the experimental nephritis initiated by the intravenous injection of soluble antigen-antibody complexes (2654. Remembering that antigen-antibody reactions induce aggregation of platelets, it is of interest that the injection of ADP into pigs and rabbits

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brings about immediate aggregation of platelets within kidney blood vessels; a day later, focal areas of glomerular injury are present (486). The sequential changes in the glomeruli are similar to those described in focal glomerulonephritis. Material antigenically like fibrinogen has been demonstrated in the glomeruli of patients with acute glomerulonephritis, where it is diffusely present throughout glomeruli, within and between the proliferating cells, and sometimes in areas along the basement membrane (365). Sometimes, fibrin-like material is also demonstrable in renal lesions in toxemia of pregnancy (435), lupus nephritis (208, 365), and membranous (365) or chronic glomerular (208) nephritis, particularly along the basement membranes; fibrin-like antigens are not found in lipoid nephrosis (208). XII.

The Arthus Phenomenon

In 1903, Arthus (18) described experiments in which he injected horse serum subcutaneously into rabbits at intervals of 6 days. This treatment resulted in intense local reactions, characterized by edema and hemorrhagic necrosis, first noted after the fourth injection. The Arthus phenomenon is the local response to the injection of antigen in sensitized rabbits; other species are less susceptible, but do react. Sensitization need not be through subcutaneous injection; other routes of injection of the sensitizing antigen are satisfactory (466). The Arthus phenomenon can also be induced passively, by intracutaneous injection of preformed homologous antibody and intravenous injection of antigen or vice uersa. The role of complement in the evolution of the phenomenon is uncertain, but in guinea pigs antibodies that do not fix complement are less effective ) the position than those which do ( 6 6 ) . Indeed, Cochrane ( 1 0 5 ~ takes that only complement-fixing (and precipitating) antibodies will bring about the Arthus reaction, conditions needed to induce the migration of polymorphonuclear leukocytes. As in many types of injury, such as thermal burns, the discharge of a histamine-like agent accounts for an initial phase of increased vascular permeability at the site of injection of antigen (246). This response is followed within an hour by a second increase in vascular permeability, attributed to a distinctive permeability factor found in the lesions and perhaps derived from injured cells (246). The second stage is associated with the local accumulation of polymorphonuclear neutrophiles and with systemic leukopenia, and can be abolished by inducing leukopenia with nitrogen mustard (266, 641 ). Material reacting antigenically like fibrinogen is demonstrable in or around damaged blood vessels (489). Ebert, Barclay, and Ahern (158) studied the inflammatory reaction character-

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istic of the Arthus phenomenon in the rabbit ear chamber and observed aggregation of platelets and leukocytes within the blood vessels, some of which were thought to have thrombosed completely. Opie (483) and Stetson (641) described similar thrombotic changes in capillaries and veins in typical skin lesions, followed by local hemorrhage. The local injection of neutrophilic granules enhances the reaction, as if acid hydrolases were implicated in the pathogenesis of vascular damage ( 6 5 9 ) . Although these observations suggest that ischemia secondary to local thrombosis is a fundamental component of the Arthus phenomenon, neither thrombocytopenia (267) nor the presence of large amounts of heparin (105, 266), EACA ( 7 4 2 ) , or TAME (742) inhibit the reaction sigdcantly. One must conclude that thrombosis may enhance local damage, but is not of primary importance. XIII.

Delayed Hypersensitivity

Thrombotic lesions are usually not described among the histological characteristics of experimental delayed hypersensitivity reactions ( 676). But fibrinogen is present in the vessels and adjacent connective tissue in cutaneous tuberculin reactions in sensitized guinea pigs (489). Heparin or warfarin diminish or prevent delayed hypersensitivity reactions; it is not clear how interference with clotting mechanisms brings this about (109). EACA and Trasylol, which might block local fibrinolytic reactions (53) are not inhibitory. In human subjects, a constant constituent of chronic granulomas is the presence of fibrinoid, which reacts immunologically with antifibrin antibody (208) (Section I1,C). Further studies in this area are needed to determine the way in which blood-clotting mechanisms participate in the delayed hypersensitivity reaction. XIV.

Concluding Remark

This lengthy review has attempted to bring together some of the information currently available concerning the relationships among the blood-clotting process, fibrinolytic phenomena, the inflammatory response, and immune mechanisms. Its goal has been achieved if it has demonstrated the interwoven nature of these reactions. The data assembled support the philosophic view that the defenses of the body may be dissected by the investigator to suit his experimental convenience, but that in nature they form a seamless web.

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Antigens of Virus-Induced Tumors' KARL HABELZ Deportment o f €xperimenfol Pathology, Scripps Clinic ond Rercorch Foundotion,

Lo Jollo, Colifornio

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A. In Vivo Techniques . . . B. In Vitro Techniques . . . T Antigens . . . . . . . . . Structural Virus Antigens . . . . . . A. Deoxyribonucleic Acid Virus Tumors . . . B. Ribonucleic Acid Virus Tumors . . . . Immunological Phenomena in Viral Oncogenesis . A. Age Factors . . . . . . . . B. Immunosuppression . . . . . . C. Immunological Tolerance . . . . . D. Tumors in Man . . . . . . . Significance of Virus-Coded Antigens in Oncogenesis A. T Antigens . . . . . . . . B. Transplantation Antigens . . . . . C. Prevention and Therapy . . . . . Human Tumors . . . . . . . . References . . . . . . . . .

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Introduction

Virus-induced tumors have always had a double potential from the standpoint of possible new antigens being present in the oncogenically transformed cells which are absent in their normal counterparts. Besides any new cellular antigens appearing in the tumor cell as an expression of the genetic changes responsible for oncogenicity, the possibility arose that antigens representing specific information brought by the transforming virus might also be found. This has been the case, and investigation of these virus-specified new antigens and the host's reaction to them have provided logical explanations for several well-known biological phenomena in virus-induced tumors. Ever since the earliest demonstration of structural viral antigens in the case of Rous sarcoma virus tumors (Zilber, 1961) and nonstructural ones in the case of polyoma v i r u s tumors (Habel, 1961; Sjogren et al., 1961a), 'This is publication No. 318 from the Department of Experimental Pathology, Scripps Clinic and Research Foundation, La Jolla, California. Supported by Grant CA 10,596 from the National Institutes of Health. 229

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the most significant fact has been that these new antigens are really viruscoded and, therefore, specific. In all systems thus far investigated all tumors induced by a given virus share the same new antigens. This has introduced into tumor biology a specificity relating original inducing agent with final tumor which previously has been so importantly lacking. The ever-increasing number of virus-tumor systems in which virus-specified new antigens can be demonstrated suggests that this is a universal phenomenon. Although work in this area is relatively recent, several reviews have been published (Old and Boyse, 1964; Sjogren, 1965; Klein, 1966; Habel, 1967). For that reason no attempt will be made here to cover again the entire development of our current knowledge of virus-induced tumor antigens. Rather, newer information published in the last few years will be presented against the background of earlier established facts, and some evaluation will be attempted on the significance of the experimental results to the field of virus oncogenesis. To do this the virus-induced tumor antigens will arbitrarily be divided into those identifiable as structural parts of the virus particle and those not found in the complete virion, and the latter will be subdivided into those on the cell surface involved in transplantation resistance and those that are intracellular. Although work continues on development of methods for demonstrating these antigens, in the last few years more effort has been aimed at three objectives: (1) attempts to see if the new virus-specified antigens serve any specific function, particularly as to the expression of oncogenic properties of the transformed cell; ( 2 ) the use of the antigens as tools in experiments aimed at finding the biological and biochemical events responsible for tumor induction by a virus; and ( 3 ) application of methods developed for experimental animal tumors in an attempt to find whether known viruses may induce tumors in man. These three aspects of the problem, therefore, will be given more emphasis than others in this presentation. II.

Transplantation Type or Surface Antigen

A. In Vivo TECHNIQUES The transplantation antigen in this review is considered as that new antigen, presumably on the cell surface and coded for by the viral genome present in the transformed cell, which is responsible for homograft type of transplant rejection of virus-induced tumors in isologous systems. The demonstration of this type of virus-specified antigen in cells trans-

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formed by polyoma virus was one of the earliest evidences of virusinduced tumor antigens (Sjogren et al., 1961b; Habel, 1961). Mice or hamsters inoculated with a single dose of infectious virus or multiply injected with allogeneic polyoma tumor cells were resistant to challenge with a variety of isogeneic polyoma tumors. It was in this early polyoma work that the importance of quantitative challenge with serial dilutions of counted tumor cells became obvious. The basic transplantation antigen phenomenon had been missed 2 years earlier in my laboratory because the then standard technique of nonquantitative challenge with a tumor fragment introduced through a trocar was used. Although the in uiuo nature of the test procedure was a severely limiting factor, a number of important characteristics of this “transplantation-type” antigen were delineated. The immunizing virus had to be viable but the challenging tumor could be virus free. The resistance was lymphocyte-mediated and not transferred by serum. Antiviral antibodies were not involved, and there was no evidence that the tumor cells contained any antigens present in the mature virus particle (Habel, 1962a). Yet the specificity of the transplantation resistance was determined by the virus, since it was the same no matter what species was used or to what histological type the tumor belonged or what was the organ of origin (Sjogren, 1964a). The antigen also was present in cells transformed in tissue culture (Habel, 1962d). First demonstrated in polyoma tumors, it was subsequently found in tumors induced by SV 40 virus (Habel and Eddy, 1963), Shope papillomas (Evans et al., 1962) and, more recently, in adenovirus tumors (Trentin and Bryan, 1966)-all caused by deoxyribonucleic acid (DNA) viruses. However, there is also evidence that tumors induced by ribonucleic acid ( RNA) viruses likewise contain such a transplantation-type antigen. Although lymphomas induced by the RNA viruses do show this rejection phenomenon (Sachs, 1962), it is difficult to rule out that viral envelope antigen on the cell surface may be responsible in the case of tumors in the natural host species. However, when ”virus-free” sarcomas are induced by avian leukosis virus in mammalian species, they still contain such an antigen (Jonsson and Sjogren, 1966). The same is true of the sarcomas produced on inoculation of the RNA mouse sarcoma virus (Fefer et al., 1967). Antiserum cytotoxic for Friend leukemia cells was found to retain its cytotoxicity after absorption with concentrated virus, suggesting the presence of a new surface antigen other than incorporated viral envelope (Steeves, 1968). The specificity of the transplantation-type antigen determined by the inducing virus is in sharp contrast to the lack of such specificity in tumors

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induced by the same chemical carcinogen (Klein et al., 1960).This specificity was shown by a cross-challenge experiment in hamsters using polyoma and SV 40 viruses as immunizing agents and the corresponding tumors for challenge (Habel and Eddy, 1963). However, in the case of adenovirus-induced tumors there is some cross protection against challenge between adenovirus types 12,18, and 7 (Sjogren et al., 1967). This is not surprising since their T antigens also cross react and they have biological characteristics in common ( Huebner, 1967).

. B. In Vitro TECHNIQUES The need for an in uitro test system to demonstrate the surface transplantation antigen of virus-induced tumors is just as great as in work on normal histocompatibility antigens. Such a system is needed to broaden studies to include human tumors and to test for antigens between species as well as in any attempts to isolate and purify the antigen itself. As might be expected, several techniques already used for histocompatibility antigens have been applied to experimental virus tumors. Most of these involve the interaction of specific antibodies with the corresponding antigen on the tumor cell surface, and the use of different criteria for such a reaction. Cell lysis when complement is present along with antibody has been demonstrated by vital dye staining (Slettenmark and Klein, 1962) and by release of W r (Haughton, 1965). This has been found most useful in the study of mouse leukemia cell antigens and, in fact, is difEcult to apply to systems where the target cell is other than of a lymphocytic nature. The effect of antibody on tumor cells could also be shown by reducing the ability af the cell to grow under quantitative cloning cultural conditions (Hellstrom and Sjogren, 1987). Here the disadvantage is the frequent low cloning efficiencies of tumor cells even in the absence of antibody. Fluorescent antibody (FA) staining of the cell surface, especially when the conjugated antiserum is used in a direct test on viable cells in suspension, has given positive results in SV 40 (Tevethia et al., 1965) and polyoma-transformed cells (Malmgren et al., 1968). The preparation of positive antisera here is a problem and requires hyperimmunization with tumors. In a test similar to FA staining, radioactive iodine has been used in double-labeling of antiserum and control serum (Harder and McKhann, 1968). So far the tendency for normal y-globulin to stick to the surface of cells has made the background high so that specific ratios are usually less than 2. The mixed cell agglutination is an ingenious technique for developing an antibody-cell surface antigen interaction, and it has been applied to virus-induced tumors with varying success (Bart% et al., 1967). This method requires careful balancing of quantitative fac-

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tors in the developing system which, as they are altered toward greater sensitivity, probably have less specificity. Fewer in uitro methods have appeared in which the sensitized or immune cell reacts with the antigen on the surface of the target tumor cell. One is the same as the antiserum clone reduction test except that lymphocytes are used (Hellstrom and Sjijgren, 1967). It has the same disadvantages, i.e., first, that the tumor has to be adapted not only to growth in culture separate from normal elements but to clonal growth and, second, that normal control lymphocytes frequently cause a substantial nonspecific reduction of growing clones. A second method using sensitized lymphocytes and target tumor cells involves the release of a substance that inhibits the migration of normal macrophages under tissue culture conditions (George and Vaughan, 1962). As yet this technique has had only supedicial application to tumor surface antigens and has the disadvantage of being poorly quantitated. The most damaging criticism of all these in oitm methods is that there is no definitive evidence that the surface antigens they are measuring are, indeed, of the transplantation type. However, even if they are not, so long as the reaction is specific and some new virus-induced antigen is measured, they would be useful in the virus-tumor field. When antiserum and sensitized lymphocytes are produced in a host xenogeneic to the tumor tissue, complications may arise due to normal antigens. This would be true especially if normal mucopolysaccharides or mucoprotein antigens might be hidden in the normal cell membrane but available in the transformed cell. Just as with the comparable normal cell surface histocompatibility antigens, there is no definitive evidence on the chemical nature of the transplantation tumor antigen although it is now assumed to be a lipoprotein. Ill.

T Antigens

The T or ‘heoantigens” are new antigens demonstrable in cells transformed by known animal viruses but are intracellular rather than on the surface and, although apparently virus-coded, they do not in general represent structural entities of the mature virus particle. Most of the work done on T antigens has involved the DNA tumor viruses (Huebner et al., 1963; Black et al., 1963; Habel, 1965), and, in fact, at the present time there is no clear-cut evidence that they are present in comparable form in RNA virus-transformed cells. As will be mentioned in the discussion of the structural virus antigens, these occur in RNA virus-transformed cells, and when intracellular may be counterparts of the T antigens in DNA virus-cell systems.

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The T antigens have been demonstrated by FA staining, complementfixing (CF), and gel diffusion methods, but the first two have been used most extensively. The FA test is more sensitive since it can indicate the presence of antigen in a single cell out of many in the study of a population, whereas the less sensitive CF test lends itself more readily to quantitative comparisons of test materials. In order to demonstrate the T antigens, it is necessary to have an antiserum containing specific antibodies against them. This has almost exclusively come from hamsters carrying the original or transplantable tumors induced by the DNA tumor viruses. The amount of antigen in the tumors and the level of antibody response to them vary from animal to animal but, in general, are lowest in the polyoma system. Here also, for FA staining of polyoma T antigen, a heat-labile factor in normal hamster serum is required (Takemoto et al., 1966). Prolonged presence of the tumor appears to be important for antibody production and levels drop after removal of tumor. T antigens are present in the nucleus of tumor cells and cells transformed in uitm by the DNA tumor viruses. Just as in the case of the transplantation type, the specificity of the T antigens is determined by the transforming virus no matter what the histological variety, organ, or species source. In the adenovirus group there are cross reactions by CF between the T antigens produced by different adenovirus types. However, the grouping of related T antigens follows the same pattern as that established on other biological and biochemical characteristics (Huebner, 1967). The T antigens appear to be different from the specific transplantation surface antigen in the DNA virusinduced tumors. The production of T antigens is not specific for the oncogenic transformation interaction of the DNA tumor viruses with cells; it is also seen in lytic and abortive infection (Sabin and Koch, 1964). Their appearance in the lytic infection cycle is one of the earliest specifically identifiable synthetic events and occurs even if viral DNA synthesis is inhibited (Rapp et al., 1965). Not only is the input DNA sufficient to initiate its synthesis but the incoming viral genome does not have to be intact. Virus quantitatively inactivated by ultraviolet radiation will lose its infectivity before losing its ability to induce T antigen (Carp and Gilden, 1!365), and this latter follows closely the loss of ability to transform cells in uitm (Benjamin, 1965). Production of T antigen in lytic systems can be separated from production of infectious virus by using metabolic inhibitors or by carrying out the lytic cycle at reduced temperatures (Kitahara and Melnick, 1965). In abortive infection where neither transformation nor lysis with

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progeny virus production take place, the appearance of T antigen may be the only evidence that dynamic interaction has occurred. With time and particularly with cell division, the T antigen gradually disappears and there is then no evidence of viral iduence ( Black, 1966). Because lytic and abortive infections as well as transformation call forth T antigen, this marker cannot be used regularly for direct and immediate quantitating cell transformation in tissue culture systems. However, it is only in transformed cells that T antigen persists after several cell divisions, and at that time may be useful as a quantitative transformation assay tool. Purification and chemical characterization of T antigens has been limited. It appears to be protein in nature, but some evidence suggests that in situ it is associated with RNA (Gilden et al., 1965). It is heatlabile and, in the case of adenovirus Type 12, the T antigen has been reported from 2.2s to 7.5s in size. However, there is some evidence that T antigen may not be a single entity since more than one line has been seen on column chromatography (Hollinshead et al., 1968) and polyacrylamide gel electrophoresis (Tockstein et al., 1968). Its localization in the nucleus of cells reacting with DNA viruses is consistent with the fact that this is the site for the replication of these viruses. By using labeled antibody and electron microscopy the T antigen appears to lie in bundles of fibrous structures (Levinthal et al., 1967). Because of the early appearance of T antigen in the lytic cycle the question has been raised as to whether it might be a specific virus-coded early enzyme involved in viral DNA synthesis. Evidence concerning identity with a specific thymidine kinase has been conflicting. Carp reported that antisera from SV 40 tumored hamsters would specifically inhibit the thymidine kinase activity in extracts of SV 40 transformed cells (Carp, 1967). On the other hand, Kit and co-workers have published a series of papers indicating no evidence for identity between T antigen and DNA-synthesizing enzymes including thymidine kinase (Kit et ul., 1967). An abnormal type of arginase which is antigenic has been demonstrated in Shope virus-induced rabbit papillomas (Rogers, 1959). The T antigens of DNA virus-transformed or lytically infected cells have been quite useful as specific markers in basic in uitro studies. In some situations, such as the experiments indicating a ‘Xybrid between SV 40 and adenovirus genomes, the SV 40 T antigen production was the only evidence of that part of the hybrid since SV 40 virus could not be isolated (Chanock et al., 1964). An especially interesting use of T antigens is their marker function in cell hybridization experiments where transformed cells are fused with normal cells in culture (Steplewski et al.,

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1968). Transfer of the ability to produce T antigen to the normal cell nucleus in a heterokaryon-fused hybrid cell occurs regularly even to nuclei of cells normally not susceptible to infection or transformation by the virus. Only with certain transformed cells is there an accompanying and much less efficient induction of infectious virus production in transformed, normal susceptible cell heterokaryons. The biochemical events occurring in these systems is not known but the factors involved in repression of expression of the whole virus genome integrated in transformed cells may thus be studied. IV.

Structural Virus Antigens

A. DEOXYRIBONUCLEIC ACID VIRUSTUMORS In general, once a DNA tumor virus transforms a cell in uitro or induces a tumor in d u o the virus as a demonstrably infectious entity disappears. We now know that at least a part of the virus genome persists in some sort of an integrated state through subsequent cell divisions and is transcribed to virus-specific mRNA, which, in turn, is probably translated to produce the antigens described in this chapter. In the absence of infectious virus production in the tumor cell, there is usually no evidence of antigenic proteins which are known to be structural parts of the virion particle. This need not necessarily be true since the cistrons required for structural antigen production may be present but repressed. In fact, this is now known to be true in certain cells transformed by SV 40 virus since whole infectious virus can be produced when these cells are fused with normal susceptible cells. It certainly is possible that some structural antigens are synthesized and others repressed. In some adenovirus type-12 hamster tumors the C antigen of the virus has been demonstrated. In early studies, Shope papilloma virus tumors which had become carcinomatous and were capable of continued transplant passage in rabbits called forth antiviral antibodies even though the tumors were free of demonstrable infectious virus. In those DNA virus-induced tumor cells in which infectious virus and, therefore, structural virus antigens are produced, the cell dies as in lytic infection. Even in Shope papillomas where, in cottontail rabbits, v i r u s is regularly produced, it is found in the dying, keratinized, superficial cells.

B. RIBONUCLEICACID Vmus TUMOFS In most instances when an RNA virus transforms a cell, both the cell and the virus survive and the complete infectious virus continues to be made in all subsequent cell generations. The efficiency of virus produc-

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tion varies between viruses and cells and at different stages of tumor growth or culture passage. With avian and murine leukosis viruses, not only the type-specific viral envelope antigen is present at the cell surface, where maturing virus is budding off into the extracellular environment, but a group-specific (gs) internal structural virus antigen is also produced in the cytoplasm (Kelloff and Vogt, 1966; Hartley et al., 1985). When the avian leukosis viruses induce tumors in mammalian species and, perhaps, under certain conditions in the natural species, the gs antigen is produced, but not envelope antigen or infectious virus. However, if these so-called "nonproducer" cells are cocultivated with susceptible cells, whole infectious virus appears although a "helper" virus may be required. Since the virus envelope antigen is present in the cell membrane, it probably can act like a transplantation-type antigen and can be demonstrated by the use of antiviral antibody with cytotoxic or FA staining techniques. Precipitable types of soluble gs viral antigen are found in the blood and milk of leukemic or preleukemic mice and in those infected with mammary tumor virus (Nowinski et al., 19s8). The murine gs antigen appears to cross react with a similar antigen in feline leukemia (Geering et al., 1968) but not with human, bovine, or canine leukemia cells. The fact that these gs and envelope virus antigens may be demonstrated in mice before there is gross evidence of leukemia, has been useful in studying the natural distribution of these agents in mouse strains ( Aoki et al., 1968a; Hartley et al., in press). In most instances, positive animals have no corresponding antiviral antibodies due probably to immunological tolerance. Strains of mice with a high incidence of spontaneous leukemia show type-specific Gross soluble antigen in plasma and tissues throughout their lifetime, whereas certain low-incidence strains are negative for antigen but develop specific antibodies. Presence of antigen or antibody was found to be related to H-2 genotype (Aoki et al., 1968b). Furthermore, gs antigen production in tissue culture has provided an indirect means of quantitating infectious murine leukemia v i r u s (Hartley et al., 1965). Immunological relationships and, therefore, classifications of the avian (Ishizaki and Vogt, 1966) and murine leukosis viruses (Old et al., 1964) have been readily accomplished through the use of the type-specific envelope antigens on the transformed cell surface. It is interesting that one cell surface antigen appears to be common to certain mouse leukemias and the virus-induced mammary tumor. However, the most intriguing facet of mouse leukemia antigens concerns one, the TL antigen, which as yet has not been demonstrated as viral in origin (Boyse et al., 1966).The normal thymocytes of certain strains of mice (T+ ) and leukemic cells of

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certain other strains (T-) have these antigens in common. Both in uitro and in uiuo there is a disappearance of the antigen from T+ cells in the presence of anti-T antibody but it reappears on removal of the antibody (antigenic modulation). Furthermore, the genes determining TL and H-2 antigens appear to be located in the same part of the chromosome and their expression interrelated. The sharing of antigens between the virus envelope and the transformed cell surface can probably work in both directions. There is a good deal of evidence indicating that myxoviruses and others, such as the leukosis agents maturing by budding at the cell surface, incorporate cell membrane material into their viral envelopes. This sharing of either normal or altered cell-membrane antigens may be of importance in determining susceptibility and resistance of certain genetic lines of cells and animals to infection and transformation with various strains of leukemia viruses (Lilly, 1967). In fact, with the avian leukosis viruses, the antigenic character of the envelope is directly related to its ability to make effective interaction with cells from genetically different chickens (Ishizaki and Vogt, 1986). V.

Immunological Phenomena in Viral Oncogenesis

A. AGE FACXORS

It has been characteristic of both RNA and DNA tumor viruses that tumor production in susceptible animals is age-dependent. Some viruses, such as polyoma and SV 40 fail to induce tumors in mature animals even with large doses of infectious virus. Even with some of the murine leukemias where adults are susceptible, the newborn develops tumors with a smaller inoculum and has a shorter incubation period. With polyoma and SV 40 viruses, when tumors are produced in young hamsters beyond the newborn stage, they tend to be fibrosarcomas at the site of inoculation rather than tumors of internal organs as seen in inoculated newborns. At least in the case of polyoma tumors in mice the evidence is fairly clear that this age dependence is directly related to the rapidly developing immunological competence in early life with the consequent recognition and rejection of the foreign transplantation type of antigen present in the animal's own virus-transformed cells ( Habel, 1962b). Even in inoculated newborns, especially those receiving smaller doses of virus, there appears to ensue a balance between the multiplying tumor cells and the development of immunological reactivity. Under the impact of what are probably multiple factors quantitatively influencing the immunological capability of an individual animal, as well as nonimmunological factors, the

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balance may go in either direction. This probably explains the shaded end points for titration of virus oncogenicity as compared to sharp ones for its infectivity in lytic systems. In the polyoma-mouse system, there is also evidence that inoculated newborns develop at least a short-lived tolerance to the tumor antigen. The fact that spontaneous tumor incidence in both experimental animals and man is high at the other extreme of life, old age, can be hypothetically related to diminishing immunological competence since homologous skin grafts will be more tolerated at that time and thymus function is reduced.

B. IMMUNOSUPPRESSION The importance of immunological capability in determining virus oncogenesis has been more dramatically demonstrated by the use of immunosuppressive measures in virus-inoculated adults where tumors now are induced. Whole-body X-irradiation (Law and Dawe, 1960), thymectomy (Malmgren et al., 1964), and more recently antilymphocyte serum (Allison and Law, 1968) have all been shown to make adult animals susceptible to moderate doses of polyoma virus. Many laboratory mouse colonies are naturally infected with polyoma virus but a naturally occurring polyoma tumor is never seen. Yet when newborn animals in such a colony are thymectomized they do develop their own polyoma tumors ( Law, 1965). Thymectomy of newborns, however, makes animals more resistant to challenge with leukemia (Gross, 1959) and mouse mammary tumor viruses (Martinez, 1964). In the case of leukemia this is apparently due to removal of the target cells. This ability of the animal to immunologically suppress the development of his own virus-transformed cells is undoubtedly the basis for the resistance of virus-inoculated adult animals to challenge with an isologous transplantable tumor originally induced by the same virus. Furthermore, this phenomenon must be kept in mind when evaluating negative results in animal tests for oncogenicity of a given virus. It is of interest when discussing the role of immunorejection in virusinduced tumors to recall that there is evidence that certain chemical carcinogens have an immunosuppressive effect (Linder, 19f32).This is especially pertinent in the present controversy between some virologists and oncologists as to whether all chemical carcinogen-induced tumors may be due merely to activation of latent tumor viruses. The fact that these tumors appear to contain new antigens which are not common, even when induced in littermates or the same animal by the same carcinogen, is evidence against their viral etiology. The immunological situation in virus-induced murine leukemias is of

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special interest. It would appear that most strains of mice are infected at birth, probably by vertical transmission from the mother, yet the eventual incidence of clinical leukemia varies from 100% to less than 5%. In some of the resistant strains, whole-body X-irradiation will increase this incidence. This has been interpreted as due to some inducing effect on target cells but would appear also to have immunological implications. However, in the various strains of mice there are prolonged periods during which virus is obviously multiplying in tissues but not producing apparent leukemia, and it is logical to assume that lymphatic-type cells or their precursors are involved since most viral leukemias are lymphocytic in nature. These same cells are necessary for antibody response and hypersensitivity reaction to foreign antigens so it was not surprising to find that immunological capability was reduced in leukemic animals. This may be manifested by reduced serum antibodies (Old et al., 1960), decreased numbers of antibody-producing cells ( Ceglowski and Friedman, 1968), and an inhibition of skin graft rejection (Dent et al., 1965). Furthermore, these immunosuppressive effects are present during the latent period of the leukemic process. When these phenomena are involved with the ability of the infected animal to react to the new antigens associated with his own virus-transformed cells, a cyclic enhancement of oncogenicity could result. In fact, one of the virus-specified antigens, the internal gs antigen, readily shown to be present in leukemic cells and in spleens during the latent period (Hartley et aZ., in press), fails to call forth antibodies in most instances. Since the mice are probably infected with the virus as embryos, there may well be an added inhibition of specific reaction to viral structural or virus-induced new cellular antigens due to immunological tolerance being established. C. IMMUNOLOGICAL TOLERANCE There is good evidence that immunological tolerance plays a significant role in viral oncogenesis in several RNA tumor virus systems. With the mouse mammary tumor, virus transmission to the offspring may not be by transplacental vertical transmission but through the mother’s milk, Yet these naturally infected newborns are not immunizable as adults when inoculated with v i r u s and later challenged with transplantable tumor. Similarly treated animals, not naturally infected, do develop resistance (Lavrin et al., 1966). Since the mammary tumor virus is an RNA myxolike virus, it is a question as to whether the antigen involved in this phenomenon is viral as such in the cell membrane or virus-spec& new cell surface antigen. Similar tolerance has been shown to operate in the avian lymphomatosis (Rubin et d.,1962) and murine leukemia virus systems (Axelrad, 1963).

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D. TUMORS IN MAN Even though there is no direct evidence that any human tumors (with the exception of the human wart) are induced by viruses, nevertheless there are impressive examples of situations where immunological factors appear to influence oncogenesis in man. Several of these involve leukemias and lymphomas which are prime candidates for viral etiology by analogy with animal leukemia systems. In a fine epidemiological study (Miller, 1968), it has been found that an excess of leukemia cases over the expected was found in children with the congenital diseases of Fanconi’s aplastic anemia, Down’s syndrome, and Bloom’s syndrome, all of which show chromosomal abnormalities, but in contrast, children with Bruton agammaglobulinemia, Chediak-Higashi syndrome, and ataxia-telangiectasia have an increased incidence of lymphoma. The latter group of congenital diseases is characterized by immunological defects. It has long been observed that in patients with multiple warts the necrosis of one due to treatment or trauma may be accompanied by regression of all the others. This can most logically be explained by immunological rejection after sensitization due to release of antigen. In Burkitt lymphoma cases the titers of antibody demonstrable by Klein’s FA staining test, to be discussed later, usually increase after tumors have regressed in response to chemotherapy, again suggesting an antigenic stimulus although an equally feasible explanation would be removal of antibody-absorbing antigenic cells. Finally, the rapidly increasing use of immunosuppressive regimens in human organ transplant recipients is apparently influencing the inadvertent successful transplantation of donor tumor cells (Martin et uZ., 1965). VI.

Significance of Virus-Coded Antigens in Oncogenesis

A. TANTIGENS Although the demonstration of the several different types of new, apparently virus-coded, antigens in virus-transformed cells has been an interesting and useful development, their functions, if any, remain obscure. In the case of T antigens it seems logical to hypothesize that they are enzymes and are somehow involved in the replication of viral DNA. Their production in lytic as well as transforming infection rules out their being directly or solely responsible for the transforming event. Furthermore, there have been reports that not all in vitro transformed cells or all tumors induced in animals by polyoma or SV40 viruses contain demonstrable T antigen (Hare, 1967; Diamandopoulos et ul., 1968).

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The amount of demonstrable T antigen can vary from tumor to tumor of the same transplantable line, and it is logical to assume that in some instances the amount is below the level of detectability. The findings of Westphal ( Westphal and Dulbecco, 1968) that the number of SV 40 and polyoma virus genome equivalents per transformed cell can vary from 5 to 60 in dif€erent cell systems might be reffected in the amount of gene product synthesized. There have been two reports of virus-transformed cells reverting in culture to cells no longer having certain characteristics. One was the RNA Rous sarcoma virus-transformed hamster cells, where the gs antigen was no longer demonstrable after “reversion” ( MacPherson, 1966) and in which oncogenicity was decreased. In the other case, polyoma mouse tumor cells lost certain transformed characteristics demonstrable by in vitro techniques but still retained their oncogenicity and T antigen (Rabinowitz and Sachs, 1968). Pertinent to this general question of the necessity for the new antigen in tumors in order to maintain their oncogenic nature are some in vitro cell hybridization studies. When a polyoma mouse cell was hybridized with a normal mouse cell the single hybrid nucleus contained polyoma T antigen, the cell surface had the polyoma transplantation antigen, and the hybrid cells produced tumors on inoculation of mice (Defendi et al., 1967). These experiments also strongly suggest that the virus-transformed cells’ tumor properties and specific antigen production were not due to deletion of some part of the genetic makeup of the normal cell but to a positive expression of virus genome. The intranuclear location of T antigens in DNA virus tumor cells makes it seem unlikely that they are involved in immunological control of tumor development. The use of T antigens as markers to identify a tumor or a transformed tissue culture cell line as having been specifically induced by a given virus has been one of their very practical applications. Since tumors arise and cell cultures become transformed “spontaneously,” it has always been a problem to relate the final event with the original exposure to virus, especially in systems where the event occurs infrequently and after a long latent period. In controlled experimental systems this application of antigens has been possible and in the case of transformation of human cells in culture the T antigen is the chief evidence of causal relationship (Habel et al., 1965). However, an important limitation to this marker function is the fact that cells already transformed or tumors already induced by other agents, whether virus (Stuck et al., 1964) or carcinogen (Sjogren, 1964a), can be “supertransformed on exposure to a different tumor virus. So the demonstrated presence of a particular virus-coded

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antigen does not necessarily identify the agent responsible for the original transforming event. Another complicating factor in using antigens as evidence of initial transformation is the rapidly developing evidence that myxoviruses, for which there is no evidence of oncogenic transforming ability, may chronically infect cells in vitro with continued production of viral envelope antigens in the cell membrane but with unimpaired cell growth and division. Not only nontumor myxoviruses may continue to replicate along with cell division, but infection with bacteria, mycotic agents, or especially mycoplasma may represent “new, foreign” antigens in cell lines or tumors, and the animal carrying such tumors or inoculated with such cell lines may well develop the corresponding antibodies.

B. TRANSPLANTATION ANTIGENS The evidence for a function of the virus-induced, new, transplantation type of antigen in tumors is more apparent, Since the immunologically competent animal can recognize the foreign nature of the new antigen in his own virus-transformed cells and react to them in a homograft-type reaction, the development of a grossly apparent tumor can be influenced. Theoretically, the more “foreign” the cell surface antigen and the more reactive the animal, the less chance that a given transforming event will result in a tumor. In the case of H-2 histocompatibility antigens, there is a significant variation in their concentration on the cell surface, and it has been found that late appearing SV 40 tumors and metastases tend to show reduced transplantation-type antigen ( Deichman and Kluchareva, 1966). Certainly, before concluding that a particular virus is not oncogenic, it should be tested not only in newborn animals but with specific procedures such as thymectomy or antilymphocyte serum to reduce immunological response. The inoculation of the test virus into immunologically privileged sites should also be considered. Just as in the case of T antigens the question has been raised as to whether the surface transplantation type of virus-induced tumor antigen is necessary to the oncogenic properties of the transformed cell. In general, these antigens appear to be a manifestation of a genetically stable characteristic of the DNA virus-induced tumor cell. Polyoma mouse tumors still contain these antigens after many transplant passages even through animals made relatively resistant by virus inoculation ( Sjogren, 196413). There is some evidence that hamster tumors on passage may decrease in their ability to be rejected in a transplantation antigen . strains of polyoma virus challenge type of test (Habel, 1 9 6 2 ~ )Certain have produced tumors that fail to demonstrate the transplantation antigen in challenge experiments, yet the same viruses in other species do (Hare,

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1967). Again, it is difficult to rule out quantitative reduction instead of absence of the antigen. The presence of new antigenic sites on the cell surface in the case of transplantation antigens and virus envelope antigens might certainly influence such cell phenomena as mobility, nutrition, and control of cell division. AND THERAPY C. PREVENTION The prevention and treatment of tumors in general including those induced by viruses is another aspect of tumor antigens significant in oncogenesis. In the case of virus-induced tumors there are two possible approaches to prevention by immunization. Specific viral vaccines might be used to prevent invasion and infection by the virus and the specific transplantation surface antigen might immunize against the tumor. Obviously this approach will be dBcult in those systems where vertical transmission or infection at birth are involved and immunological tolerance plays an important role. In experimental systems, vaccines have been demonstrated to be effective. Murine leukemia virus attenuated in tissue culture has immunized mice against challenge with virulent virus (Barski and Youn, 1965) as well as formalin-inactivated virus vaccine (Fink and Rauscher, 1964). In the SV 40-hamster system, animals inoculated at birth with virus and well into the latent period of viral oncogenesis can be protected against tumor development by inoculation of either live virus (Eddy et al., 1964) or viable tumor cells (Goldner et d.,1964). The use of passive immunization with prevention by antiserum against the virus or the tumor has also been effectivein some systems ( Mirand et al., 1966). However, in considering the possible use of vaccine and, more particularly, of antiserum against tumor, one must remember the enhancement phenomenon where tumor development may be increased by such procedures rather than prevented.

VII.

Human Tumors

When the virus-coded sbecific antigens, especially the easily-demonstrable T antigens, appeared on the scene there was great hope that such techniques could readily establish any possible etiological relationship between human tumors and aheady known viruses. This possibiIity became even more exciting when it was shown that many of the commonly occurring human types of adenovirus were oncogenic in newborn hamsters (Trentin et al., 1962). A two-prong attack was possible-specific antigen could be sought in human tumors using known positive sera produced in experimental animals, and specific antibody could be assayed

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in tumor patients’ sera using as antigen the specific animal tumors or in uitro transformed human cells containing the adenovirus T antigens. Furthermore, even without a specific virus at hand, if a series of human tumors were shown to contain a new abnormal antigen in common and not present in autochthonous normal tissue, this would strongly suggest a common etiology due to an as yet unknown tumor virus. The former approach has thus far given negative results but the latter has been positive in several human tumor types. The only system in which a major effort has been made to show specific antigen and antibody related to a known oncogenic virus has been with the adenoviruses. Using complement fixation and FA staining, large numbers of a variety of human tumors have been examined either as crude original tumor tissue or after growing in tissue culture and gave completely negative results. Although some preliminary findings were suggestively positive (Lewis et al., 1!367), further, more extensive studies have failed to show specific antibodies against adenovirus T antigens in sera of patients with a variety of tumors in any proportion greater than that seen in the general population at similar ages (Rowe and Lewis, 1968). When positive anti-T antibodies are found, it will be difficult to rule out a recent acute infection as their stimulus. When it was discovered that many batches of the Salk-type poliomyelitis vaccine administered to large numbers of children had contained substantial amounts of viable SV40 virus, again the question of virus induction of human tumors was in question. SV 40 virus is a naturally occurring agent in monkeys which is oncogenic in newborn hamsters and which is capable of multiplying in and transforming normal human cells in tissue culture. Its presence in Salk vaccine came from its natural occurrence in the kidneys of monkeys used as the tissue culture source for growing the poliovirus required for vaccine production. Follow-up epidemiological studies for as long as 10 years have failed to find any increased incidence of tumors of any kind in children who received Salk vaccine known to contain infectious SV 40 as a contaminant. However, it was shown that a low percentage of such children developed anti-SV40 antibodies, suggesting active infection with the virus. Rapidly progressing experimental work in the field of animal leukemia and lymphoma had firmly established that these diseases in avian, murine, and feline species were indeed caused by a group of RNA viruses very similar in their characteristics to the myxovirus group which cause such human diseases as influenza, mumps, and measles. The demonstration of common antigens, virus-speciiied and indeed probably actual virus structural antigens at the tumor cell surface, made the search for such common

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antigens in human leukemia and lymphoma a logical next step. The African lymphoma, known as Buikitt’s tumor, as well as lymphatic leukemia in children appeared to be the best material in which to seek such common antigens. When the Kleins used Burkitt patients’ sera and tested by FA staining of the cell surface of viable Burkitt tumor cells, they found evidence of a common antigen and a high incidence of cross-reacting antibody in all Burkitt cases (Klein et al., 1966). In the meantime a herpeslike virus was found in cells from Burkitt tumors established in tissue culture (Epstein et al., 1965).Subsequently, the Henles showed that Burkitt cases had serum antibodies against this virus demonstrated by FA staining of tumor cells in culture. Although practically 100%of Burkitt cases had such antibodies, a number of normal humans also showed them, their incidence increasing with age (Henle and Henle, 1966). Morphologically similar viruses have now been found not only in the leukocytes of leukemic patients but also in normal humans and chimpanzees (Gerber and Birch, 1967). The most intriguing new information concerning this virus is the Henles’ evidence that it is the cause of infectious mononucleosis in man (Henle et al., 1968). These herpeslike virus particles have been shown capable of infecting, multiplying in, and establishing permanent in vitro growth of normal human leukocytes on exposure to the virus in tissue culture (Henle et al., 1967). The new antigen-presumably the herpeslike virus capsid antigen-in Burkitt and leukemia cells (and occasionally in normal leukocytes) has now been demonstrated by CF (Gerber and Birch, 1967) and immunodiffusion (Oettgen et al., 1967) as well as by FA staining techniques. At the time of this writing, there is still no definitive evidence that this virus has any etiological role in Burkitt’s tumor or leukemia. The complexity of the situation with its demonstrated presence in normal humans and its apparent causal relationship to mononucleosis makes evaluation difficult. The unraveling of this complicated, multifaceted situation will probably be dependent upon more clear-cut experimental facts coming out of parallel systems in the avian and murine leukoses. A further interesting aspect of the Burkitt tumor antigen is the fact that precipitating antibodies reacting with extracts of some Burkitt cell cultures are also found in patients with carcinoma of the pharynx (Oettgen et al., 1967). Furthermore, similar appearing virus particles have been found associated with the Luckk-type adenocarcinoma of the kidney in Amphibia (Fawcett, 1956) and in Marek‘s disease of chickens (Churchill and Biggs, 1967). In fact, a recent report indicates similarity of virus antigen by agar gel diffusion between the Burkitt agent and the frog virus (Finket al.,1968).

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The Hellstroms have developed a clone reduction type of test where human tumor cells are exposed in tissue culture to autochthonous peripheral lymphocytes, and a quantitative inhibition of tumor cell growth occurs if the leukocytes are sensitized to tumor-cell surface antigens. This was an application of a test system previously shown to be effective with known tumor virus systems such as polyoma and SV40. The Hellstroms chose to work with neuroblastomas of children and have reported a high incidence of positive results using leukocytes from tumor patients and from their normal mothers (Hellstrom et al., 1968). As discussed previously (Section VI,B), this test requires not only growth of the human tumor cell in culture but its ability to be quantitatively cloned and this is not easy with any human tumor and, so far, impossible with many. The quantitative aspects of the method also leave something to be desired since normal leukocytes frequently inhibit clonal growth of the tumor cells to substantial levels. A very recent publication reports similar findings in human osteosarcomas (Morton and Malmgren, 1968) but here the test precedure, as in the Kleins’ Burkitt tumor studies, has been FA staining. This is of special interest since there has been a recent report of the production of osteosarcomas in hamsters after inoculation with cellular material from human osteosarcoma cases (Finkel et al., 1968). A preliminary report indicates that sera from a high proportion of melanoma patients give indirect FA staining of a pigmented melanoma cell established in suspended cell culture (Oettgen et al., 1968). There have been a number of other reports that certain types of human tumors contain identical new antigens not present in the corresponding normal tissues. However, these studies have all involved the use of antitumor or antinormal tissue sera produced in heterologous species and are difficult to interpret as compared to the previously discussed autochthonous systems.

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Oettgen, H. F., Aoki, T., Geering, G., Boyse, E. A., and Old, L. J. (1967). Cancer Res. 27, 2532-2534. Oettgen, H. F., Aoki, T., Old, L. J., Boyse, E. A., de Harven, E., and Mills, G. M. ( 1968). J. NatZ. Cancer Illst. 41,827-843. Old, L. J., and Boyse, E. A. (1964). Ann. Reo. Med. 15, 167-186. Old, L. J,, Clarke, D. A., Benacerraf, B., and Goldsmith, M. (1960). Ann. N.Y. A c d . Sci. 88, 264-280. Old, L. J., Boyse, E. A., and Stockert, E. ( 1964). Nature 201,201-202. Rabinowitz, Z., and Sachs, L. (1968). Nature 220, 1203-1206. Rapp, F., Butel, J. S., Feldman, L. A., Kitahara, T., and Melnick, J. L. (1965). J. Exptl. Med. 121, 935-944. Rogers, S. (1959). Nature 183, 1815-1816. Rowe, W. P., and Lewis, A. M., Jr. (1968). Cancer Res. 28, 131SL1320. Rubin, H., Fanshier, L., Cornelius, A., and Hughes, W. F. (1962). Virology 17, 143156. Sabin, A. B., and Koch, M. A. (1964). Proc. Nutl. Acad. Sci. U S . 52, 1131-1138. Sachs, L. ( 1962). J. Nutl. Cancer Inst. 29,759-764. Sjogren, H. 0. (1964a). J . Natl. Cancer Inst. 32, 361-374. Sjogren, H. 0. ( 1964b). J. NutZ. Cancer Inst. 32, 661-666. Sjogren, H. 0. (1965).Progr. ErptZ. Tumor Res. 6, 289-322. Sjogren, H. O., Hellstrom, I., and Klein, G. (1961a). Cancer Res. 21, 329-337. Sjogren, H. O., Hellstrom, I., and Klein, G. (1961b). Exptl. Cell Res. 23, 204408. Sjogren, H. O., Minowada, J., and Ankerst, J. (1967). J. Exptl. M e d . 125, 689-701. Slettenmark, B., and Klein, E. ( 1962). Cancer Res. 22, 947-954. Steeves, R. A. ( 1968). Cancer Res. 28, 338-342. Steplewski, Z., Knowles, B. B., and Koprowski, H. ( 1968). Proc. NatZ. Acad. Sci. U.S. 59, 769-776. Stuck, B., Old, L. J., and Boyse, E. A. ( 1964). Nature 202, 1016-1018. Takemoto, K. K., Malmgren, R. A., and Habel, K. (1966). Science 153, 11221123. Tevethia, S. S., Katz, M., and Rapp, F. (1965). Proc. SOC. Exptl. B i d . Med. 119, 896-901. Tockstein, G., Polasa, H., Pina, M., and Green, M. (1968).Virobgy 36, 377-386. Trentin, J. J., and Bryan, E. (1966). Proc. SOC. Erptl. B i d . Med. 121, 12161219. Trentin, J. J., Yabe, Y., and Taylor, G. (1962). Science 137,835-841. Westphal, H., and Dulbecco, R. (1968). Proc. Nutl. A c d . Sci. U.S. 59, 1158-1165. Zilber, L. A. ( 1961). J. NatZ. Cancer Inst. 26, 1311-1319.

Genetic and Antigenetic Aspects of Human Histocompatibility Systems D. BERNARD AMOS Division of Immunology, Duke University Medical Center, Durham, Norfh Carolina

I. Introduction . . . . . . . . . . . 11. Major Histocompatibility Systems . . . . . . 111. The H L A System of Man . . . . . . . . A. Development of the Concept of a Single Locus System . B. The Designated HL-A Specificities . . . . . C. Procurement of Alloantisera Defining New Specificities . IV. The Use of Typing for HL-A Factors in Donor Selection . A. Family Members . . . . . . . . . B. Nonliving Donors . . . . . . . . . V. Other Human Histocompatibility Antigens . . . . VI. Properties and Genetic Control of Histocompatibility Antigens VII. Possible Future Developments . . . . . . . VIII. Appendix: Techniques for the Detection of HL-A Specificities A. Leukoagglutination . . . . . . . . B. Leukocytotoxicity . . . . . . . . . C. Platelet Complement Fixation . . . . . . References . . . . . . . . . . . I.

.

. .

.

. .

.

. . . .

. .

. . . . . . .

. . . . . .

. . .

.

. .

. .

251 253 256 256 262 269 273 273 278 281 283 288 289 289 290 292 292

Introduction

Histocompatibility ( H ) antigens are substances which are associated with or included in the plasma membrane of tissue cells of some, but not all, members of a species. Tissue or cell suspensions carrying such antigens will induce an immune response when introduced into another member of the same species. The immunity can be measured in several ways, such as by the detection of antibodies in the serum of an individual rejecting a transplant, by a more rapid rejection of a second graft from the same donor, or by demonstration of increased immunological reactivity in lymphoid cells of the recipient. From immunological studies and from more limited chemical characterizations of antigens of different systems, it appears that a wide variety of substances can function as H antigens. In many respects, H antigens resemble blood group antigens, with the distinction that blood group antigens are by definition found on the red cell and may also be present on other cells and tissues, whereas H anti'This work was supported in part by U.S. Public Health Service Grants GM 12535,l PO1 A1 08897, MO1-FR-SO, and 5 KO6 A1 18399. 251

252

D. BERNARD AMOS

gens are found on tissues and may or may not be present on the red cell. The antigens of the ABO system function both as red cell and H antigens. The marked differences in immunogenicity between antigens of different blood group systems are well established; examples of iso- (or al1o)immune anti-M are extremely rare, whereas isoimmune anti-A is readily formed in 0 or B individuals. In red cell serology and in blood transfusion practice this has led to the concept of major and minor blood groups. A similar distinction between major and minor histocompatibility loci has also been proposed. This difference has been thought to be qualitative, and, based on evidence from man and from laboratory animals, a number of characteristics relating to major H systems may be summarized. (1) The major systems are extremely complex. There is, consequently, extreme polymorphism within a species with respect to these factors (Amos, 1964). (2)Antigens determined by the major locus are widely distributed throughout the various organs and tissues of the body (Basch and Stetson, 1962). (3)The amount of antigen present on a given organ or tissue is genetically determined and tends to be relatively constant from individual to individual (Amos et al., 1963a). ( 4 ) Antigens of the major locus are active in lymphocyte stimulation, as shown ( a ) in oitro, in tests such as the mixed lymphocyte culture reaction of Bain et al. (1964) and of Bach and Hirschhorn (1964) or ( b ) in uiuo, in tests such as the normal lymphocyte transfer test of Brent and Medawar (1963) or transfer reaction of Ramseier and Streilein (1965). (5)The immune response against antigens of the major system is more difficult to control by immunosuppression than the response against minor antigens (Silver et al., 1967) ( 6 ) Tolerance is more easily induced against antigens of the minor system (Uphoff, 1961). (7) Antigens of the major locus are effective stimulators of allogeneic lymphocytes in the absence of any evidence of prior immunization ( Albertini and Bach, 1968). All of these postulates have not been directIy tested in every species in which a major locus has been identified but indirect evidence has supported direct experimentation. As stated in ( I ) above, the major system (where identified) in each species controls many antigenic specificities; the alleles2 or haplotypes The word “allele” is often used to describe two different attributes. In the narrow sense, an allele is a variant form of a single gene. One of the clearest examples is the substitution of Val for Glu in hemoglobins, where allelic substitution presumably involves only a single nucleotide or nucleotide triplet. In a looser sense, allele has been used to describe variant forms of a more complex genetic unit such as Rh or H-2, where allele is more nearly equivalent to half the genotype; thus, H-2’ and H-2b are alleles in this sense. To avoid confusion occasioned by this misuse of the term,

HUMAN HISTOCOMPATIBILITY SYSTEMS

253

correspondingly differ greatly from individual to individual. Typical examples of major systems are H-2 of the mouse, AgB of the rat, the B system of fowls, and HL-A of man. II.

Maior Histocompatibility Systems

Isoantigens of the mouse were described by Gorer as early as 1937, and the major locus, H-2, was described by Gorer et al., 1948. It was not until 1955 that a reasonably comprehensive report of the H-2 system was published by Amos et al. Following the reports by Allen (1955) and by Amos and his colleagues (1955) of the first recombinants, other confirmatory evidence soon followed to show that the H-2 region of the ninth linkage group of the mouse occupied a finite length of chromosome (Gorer and Mikulska, 1959; Stimpfling and Snell, 1962; Stimpfling and Richardson, 1965). From an analysis of the antigenic constitution of the recombinants, it was apparent that at least five segments of chromosome carried genetic information determining the production of the H-2 specificities (Shreffler et al., 1966). Five genetic components would appear to be a minimal estimate, crossing-over not yet having been detected between the remaining determinants. In the mouse, the A strain has been characterized most intensively because it carries two specificities ( 4 and 11) which are thought to lie at the extremities of the genetic region. An A strain mouse may be crossed to a mouse of a second strain such as C57BL which lacks 4 and 11. The F, hybrid is then backcrossed to another C57BL. The offspring are tested simultaneously for the presence of antigens 4 and 11. Half of the backcross progeny should have both antigens, the remainder should have neither, If crossing-over has occurred within H-2, one of the specificities, 4 or 11, will probably be missing. The possible recombinant is again crossed to C57BL. Half of the offspring will carry the recombinant H-2 genetic unit, Two such animals are mated. Some of the progeny will be homozygous for the new character. The antigenic properties of the recombinant can then be tested in extenso. Detailed testing of the original heterozygote is impracticable because A and C57BL share several specificities such as 5 ( E ) and 6 ( F ) . Testing for recombination in other strains is more difficult since only mice of genotype H-2” carry both 4 and 11, markers for the limits of the region. The recombination frequency between 4 and 11 has been variously estimated at from approximately Ceppellini has proposed the term “haplotype.” A haplotype or haptogroup is the term given to a particular unit. It corresponds to the contribution made by one of a pair of homologous chromosomal units to the genotype.

254

D. BERNARD AhfOS

0.3 to 1.04;; recombination between 4 and 6 or 4 and 13 is less than 0.25% (no recombinants being found in 400 mice tested by Amos) but may occur as a rare event. Recombination between cistrons is likely to be much more frequent than recombination between the codons within a cistron. It seems likely that H-2 specificities 4, 6, and 13 are on the same cistron, 4 and 5 are on separate cistrons or subloci. The detection of recombination in man is much more difficult. The recombinant mouse can be selectively bred to provide informative offspring. For the positive identification of a recombinant in man, the individual would have to be a member of the second generation in a threegeneration family. Even then, characterization of the recombinant would be made difficult if the spouse carried many of the same antigens. In the absence of recombination the H-2 complex behaves as a single genetic unit in normal tissues; (tumors may be exceptional, as noted elsewhere). During embryonic development, H-2 antigens are not detectable on most tissues before the thirteenth day of intrauterine life although they are already present on the thymus (Schlesinger, 1964). All the antigenic specificities appear simultaneously as if they were controlled by the same operon. Together with the detection of the recombinants by Gorer, Hoecker, Allen, Stimpfling, Shreffler, and their associates, came a description of additional antigenic speciticities controlled by the H-2 system (Snell and Stimpfling, 1966). After some thirty or so determinants had been described, there seemed little point in cataloging the others that were known to exist, although sporadic reports have appeared and a major revision by Shreffler and others is now in progress. Impetus has recently been given by studies on wild mice and by the interest generated by problems not easily resolved in studies on the HL-A specificities ( Ivanyi et al., 1969). Development of information in the rat has been comparable to that of H-2. The original description of the A locus by Burhoe (1947) was, however, followed by a long period of inactivity, broken to some extent by the studies of Ode11 and his colleagues in 1957 and Owen in 1962 on the C and D blood group loci. More intensive studies by Palm (1964), Bach and Amos (1967), and by Czech investigators (Stark et al., 1968) have shown that rat AgB, human HL-A, and mouse H-2 are closely comparable in many respects. Studies in rodents were greatly simplified by the ready availability of inbred strains and by the coincidence that the antigens of the major locus were present on the red celI. It was relatively simple to immunize an animal of strain A against tissue from strain B and then to show a crossreaction with strains C and D. Absorption of the serum A anti-B with

HUMAN HISTOCOMPATIBILITY SYSTEMS

255

tissue from strain C would frequently remove all activity against C but not against B or D, absorption with D would remove activity for D but not C or B, absorption with C and D would leave activity only against B. It would then be argued that at least three antigens were present on B. Antigen 1 would be shared between B and C, antigen 2 shared between B and D, and antigen 3 would be peculiar to B. A fourth antigen present on B, C, and D could not be recognized unless tissues from additional strains were used for absorption. This is, indeed, what happened (Amos, 1962). As new strains were introduced, many of the reagents previously thought to be monospecific were found to contain a mixture of antibodies. A study of the antigens of a species having numerous inbred strains is relatively simple because all animals of the same strain react alike and are homozygous, at least with respect to the loci studied. Even so, the original analysis of H-2 was probably greatly oversimplified because of the restricted population available for study. Although there are dozens of inbred strains of mice, many of them are known to be descended from the same original stocks. In many instances, it is highly probable that there was common ancestry since the original stocks came from the restricted supply of mutants maintained by mouse fanciers and dealers in the nineteenth century. Consequently, certain alleles or antigenic combinations are found quite frequently, especially H-2a, H-2d, and H-2k, possibly because these combinations possessed certain advantages under conditions of inbreeding. Common ancestry is even more pronounced in the inbred strains of rat, since many rat strains originated from pen-bred stocks maintained at the Wistar Institute. Cohen, studying certain antigens of rabbits, has found that polymorphism tends to persist beyond the tenth generation, suggesting that homozygosity in that species is achieved only with considerable difficulty (Cohen and Tissot, 1965). Whereas both alleles of the inbred animal are alike, two alleles in an individual from an outbred population are usually different; therefore, the methods of analysis are of necessity considerably more complicated. In animals some degree of simplification is still possible if inbred strains are available for crossing. In analyzing the alleles of a wild mouse, it is sufficient to mate a male with homozygous inbred females from a wellcharacterized laboratory strain. Suppose a wild mouse with H-2 alleles H-2xlH-2~was mated with an animal of genotype H-2b/H-2b; all offspring would be either H-2b/H-2x or H-2blH-2~.Stock antisera prepared in H-2b animals against different inbred strains would probably react or cross-react with certain antigens of the H-2x hybrid; others would react with the H-2y antigens. The two classes of hybrid could thus be distinguished and two H-2blH-2~(or two H-2blH-2~)individuals mated. This

256

D. BERNARD AMOS

would give H-2b/H-2x, H-2b/H-2b, and H-2xJH-2x individuals in the ratio 2:l:l. By testing with anti-H-2b reagent that did not react with H - ~ xthe , homozygous H-2x individuals could be recognized and a line homozygous for H-2x could be established for detailed study after only two generations. The situation is entirely different in man. Most human populations are extremely heterogeneous in ancestry, either from assimilation following conquest or from migration and immigration. Homozygosity can occur in man but is uncommon. It could occur in ordinary outbred groups when brothers marry sisters, and cousins descended from these marriages themselves marry. We have, in fact, identified one pair of HL-A identical cousins. This is a rare event. More commonly both haplotypes carried by an individual are dissimilar, and the analysis becomes correspondingly more dBcult. The few isolated human populations with a high coefficient of inbreeding in which homozygosity for HL-A antigens would be frequently expected have not yet been studied. 111.

The HL-A System of Man

A. DEVELOPMENT OF THE CONCEPT OF A SINGLELocus SYSTEM The first substantial information about human tissue and leukocyte alloantigens was derived from studies on autoimmunity to leukocytes ( Moeschlin and Schmid, 1954; Dausset, 1954), alhough occasional studies had been reported earlier (e.g., Wichels and Lampe, 1928). Since autoantibodies were frequently present in certain types of anemia, it was thought that autoantibodies might also be present in leukopenia. Antibodies to leukocytes were soon found but were later shown to be allorather than autoantibodies and were formed as a response to the multiple transfusions given to the diseased patients ( Dausset, 1958). Difficulties in obtaining adequate volumes of serum over an adequate period of time from very sick patients complicated the original studies. Rapid progress has followed the finding of Payne, van Rood, and their colleagues in 1958 that antileukocyte antibodies are common in the serum of multigravid women. The early literature has been comprehensively reviewed by Walford ( 1960) and by Killmann (1960). Some idea of the complexity of HL-A can be obtained from an inspection of Fig. 1. This is an excerpt from some of our recent data in which volunteers were typed in preparation for a skin-grafting experiment. For this series, 41 subjects were tested on from two to four occasions with a variety of antileukocyte sera. Reactions obtained were compared in all

I I I I I I I I I I I I I I I I

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Ii i i II IIIIIiIIIIIIII IIIII

llllllllllllllllllr

1

i

-I

1

-

I -

-

FIG.1. Reactions of 64 sera with cells from 41 unrelated subjects. The sera are roughly grouped. From such reaction patterns a computer program can select related sera or identify phenotypic resemblances between subjects.

cl

258

D. BERNARD AMOS

possible combinations: subject 1would be compared with the remaining 40, subject 2 (having been compared with subject 1 already) would be compared with the remaining 39, subject 3 (having been compared with 1 and 2 ) would be compared with the remaining 38, and so on, giving a total of 820 comparisons between individuals. Of the 820 possible combinations, only one pair gave identical reactions and only four pairs gave agreement with over 95%of the sera used. For these investigations only 63 antisera were used; if our full panel of over 150 sera had been employed, the probability is that no two cells would appear alike. It can be readily understood that when van Rood (1962) first attempted this type of massive comparisons in testing a panel of 80 sera obtained from multiparous donors against cells from 100 unrelated subjects, the complexity of the pattern of reactions obtained was overwhelming. To enable him to compare the reactions of the Werent sera, he introduced a simple computer program designed to give a 2 x 2 comparison. In this type of program, now used by all leukocyte serologists, the reactions of each pair of sera is compared for each cell tested. If serum 1and serum 2 both react with cell number 1, the reaction is entered if serum 1reacts with cell 2 while serum 2 is negative, the comas -. Conversely, if serum 1 parison is scored in a separate column as fails to react with cell 3 while serum 2 is positive, this is scored in a third and if neither serum reacts, an entry is made in a fourth column as -, column as -, -. When all the reactions of the two sera against all cells have been entered, the entries in the four columns are added up. Van Rood found that a number of sera gave similar but not identical results. On reexamination of the data, a smaller group of sera which tended to give antithetical reactions to the first group was identified. That is, when the first group of sera failed to react with cells from a given subject, the second group of sera tended to give positive reactions and vice versa. Frequently both sets of sera were positive. This suggested that the first sera were detecting one member of a diallelic series called 4a and the second group detected its allele, 4b. Cells could be 4a4a4a4b or 4b4b, while 4040 was nonexistent or rare (van Rood, 1962). Further examination yielded another pair of alleles, 5a and 5b, and later specificities grouped together as 6a and 6b; 7a, 7b, 7c, and 7d; 8a; and 9a were described (van Rood et al., 1967). The 6 and 7 series were more complex than the 4 and 5 systems. A complicated interrelationship existed between antigens of “groups” 4, 6, 7, and 8. No such relationship was found between members of these systems and the 5 and 9 groups. Payne et d. (1964), using a different series of reagents and with different techniques (see Appendix), identified two factors, LA1 and LA2, which tended to show an allelic relationship. Since cells lacking both LA1 and LA2 were

+, +;

+,

+;

HUMAN HISTOCOMPATIBILITY SYSTEMS

259

quite common, it was thought that these might be the first two antigens of a multiallelic series. Later specificities LA3 and LA4 were described (Bodmer et al., 1966; Walford et al., 1967). It is rare for a cell to have three of these factors but cells possessing none of them are quite frequent. Additional antigens of this series are now being postulated (Dausset et al., 1969). Dausset et al. (1965), using sera obtained in his own laboratory and supplemented by other sera obtained from colleagues, identified a series of ten antigens, each of which had some degree of nonrandom association with each of the others. Antigen 1 of Dausset, the first of the series to be described under the designation “Mac,” was similar to LA2 of Payne and Bodmer and 8a of van Rood. Many of the antigens were also independently described by Terasaki (Vredevoe et al., 1966),Batchelor ( Batchelor and Sanderson, 1967), Walford ( Walford et al., 1967), Shulman (1965), and others (e.g., Amos, 1965). In each case, certain interrelationships between antigens were clearly recognized by the investigator working with each set of sera. Although interrelationships between specificities were recognized by all investigators, the simplicity of the genetic control was not completely appreciated until Dausset et al. (1965) postulated that each of the ten antigens they could recognize were under the control of a single locus. The truth of this hypothesis was proved by Amos and by Bach. The segregation of haplotypes was quite unmistakable in one family with eight children (Amos, 1967). Two haplotypes contributed by the father were detected by six and by seven different sera, respectively. The patterns were so obvious that certain rules for the analysis of genotypes could be applied. It was relatively easy to apply the same kind of analysis of pattern recognition in other large families. Details of the steps involved have been reported (Amos et al., 1967). A typical example taken from a more recent study is also given in Table I. Here the four HL-A haplotypes are designated by the symbols A, B, C, and D. These haplotypes are constant only within a family (so far we have been able to identify one haplotype through four generations and many others for three); they usually vary greatly between families. One haplotype ( A ) from the father determines specificities which react with ten sera including sera directed against HLA-3 and HLA-7; children 1, 2, 4, and 7 accordingly react with these sera. Sera recognizing HLA-2 react with products of the homologous haplotype, thus these three sera are positive with cells from the father and the remaining five children. Similarly, sera reacting with some components of 4a, with HLA-1, and with Walford’s Lc20, react with the mother and children 1, 2, 3, 4,5, 6, 7, and 8, identifying the inheritance of the C haplotype, whereas other sera reacting with other components of 4a react with products of the maternal D haplotype present in the other child,

TABLE I GENOTYPIC ANALY810 OF A TYPICAL LAROEFAMILYFamily 0117 Genotype Father Mother C1 c2 c3

c4

c5 C6

c7

C8 c9

AB CD AC AC BC AC BC BC AC BC BD

Haplotype A

B

+ +

+

+ -

+ +

1-

+ +

t

-

-

RB 1/8/67 RIL 7/65 RA/BeBb 1/67 RA P-1 GW/FC 7/65 GW 7/65 J D 6/65 CA8 Cutten Storm DAL 2/67 BH/RA

-

t

-

WR P-1 hlJ 9/67 Ma9

C

1)

-

+

-

+ + + -t + + + +

-

-

-

-

-

-

-

t-

-

DK 9/66

KH/BeB 1/67 Morrison Anderson DK/ROD 9/66

+

FS 1/67 FS P-1 ROY 6/65 LBu 11/66 SD P-3 SC 30 NW 11/65

P-1

~~

0

Eight sera gave irregular or incomplete patterns.

A + B

C+D

++ + + + + + + + +

+ + + + + + + + +

-

+

RA P-1 NW 3/65 B J I 12/66 KH 1/67 KH/FC 1/67 JT P-1

A + C

+ + + + + + + + + + -

CaR 7/66 BC 11/66 JPo 11/66

A + D

+ + +

+-

+-

++

I ~ + C n + D

+ + + + + + + + + + +

-

-t

t

++ +

-

-

-

-

-

JoC 11/66 11HP-1 AJ 5/67 H F P-1 COu 3/65 Willett EnN 12/65 Harris Thompson M I 1 P-1 BM 5/66 N W 3/65/ NH, RA McM 9/65 R B P-1 BH/JcA P-1 S A 1/67 DAL P-1 RB/AB 1/67

~~

b

+ +-

None

Letters after "/" are initials of donor oi cells used ior absorption.

tl

mm

i

3

>

z

%

HUMAN HISTOCOMPATIBILITY SYSTEMS

261

The genotypes of the children are indicated. Note that all children have inherited haplotypes AC, AD, BC, or BD and that certain sera react with antigens determined simultaneously with more than one haplotype. To date we have tested over 100 families. It is not always possible to genotype in this manner, especially where one parent is missing or the family is small. Frequently no sera are found to react only with the products of one haplotype, and it is surprising how much variation exists between the antigenic expression of the alleles. There are several ways that the marked difference between the apparent antigenicity of different haplotypes can be explained. One obvious possibility is that the alleles are not codominant. In this case one entire haplotype could be suppressed in a cell just as it is in antibody-producing cells. This appears to be unlikely. Even in the case of allotypic markers, a minority population producing the second series of antigens is usually identifiable. Even if it were possible for (say) the maternal C haplotype to fail to reach expression in the presence of a more dominant D, it would be somewhat surprising to find it suppressed in AC and BC children as well. In fact, when enough sera are used, it is generally possible to find at least one marker peculiar to that given haplotype within the family. In those few cases in which no specific marker is found, as for example when no sera react solely with the maternal C haplotype, it is usual to find that certain sera react strongly with the mother and all children, thus antigens controlled by both C and D haplotypes are present and the C genetic determinant is functional. Examples in which both parents are negative and some children are reactive have been found. They can usually be traced to an error, technical or serological, and are corrected upon subsequent testing, or the occult existence of the antigen can be revealed by absorption. Recessive antigens, like recessive haplotypes, appear to be uncommon. A second possibility is that the alleles of many of the specificities are amorphs, just as “ 0may be regarded as the amorphic allele of “A” in the ABO or ABH system. This seems highly probable in view of the difficulty experienced in detecting true alleles of many of the H-2 specificities. [This view is not shared by Dausset and others who believe they have identified the majority of the alleles in two apparent subloci (Dausset et al., 1969).] A third possibility (and one that is compatible with the second) is that the number of antigenic speciikities is greater than is generally conceded, so by chance, certain haplotypes cany specificities that cannot as yet be recognized. In support of this hypothesis has been our experience with skin graft rejection. In one family, the maternal C haplotype could only be detected through a weak reaction with one serum out of more than 100 tested, A graft from a donor carrying this

262

D. BERNARD AMOS

haplotype to a sibling having the maternal D and sharing the homologous paternal haplotype was rejected in 10 days. Evidently this haplotype, although very feebly antigenic by serological testing, carried strong transplantation antigens. In summary, each individual carries two homologous units of genetic information which determine the HL-A haplotypes. Within a family the same haplotypes recur; between families differences are great. The number of specificities peculiar to a given haplotype can vary greatly and the number of antigenic specificities that can be recognized is increasing rapidly.

B. THEDESIGNATED HL-A SPECIFICITIES As explained above, relatively few investigators were responsible for

the descriptions of the first specificities to be recognized. Most prominent were Dausset, van Rood, and Payne, together with their respective collaborators. Supplies of antisera were somewhat restricted, techniques differed considerably from laboratory to laboratory, and, consequently, exchange and standardization of reagents were difficult. Exchange of information, leading ultimately to agreement regarding six specificities, was greatly accelerated as a result of the three practical workshops that have been held. The first workshop, held in 1964,was on a very limited scale ( see “Histocompatibility Testing,” 1965). Experimentation was mainly confined to a demonstration of techniques. Each investigator was provided with six sera and with blood from a small panel of unrelated donors with which to demonstrate the details of his own procedures. Perhaps fortuitously, two investigators obtained almost identical results, and other investigators showed quite convincingly that they were detecting different antibodies present in the same sera. It was apparent that most of the procedures had considerable merit and that more than one method was needed for the detection of all the specificities that were then recognizable. The second workshop, held in 1965, was considerably more ambitious. Cells from 45 subjects were made available, and the investigators provided their own sera so a comparison between the reagents used in some 12 laboratories was achieved. Several laboratories obtained virtually identical results, showing that the same specificities were being detected (under different names) in different laboratories through very divergent procedures, including leukoagglutination, cytotoxicity, and complement fixation. It was, for example, concluded that Dausset’s Mac, Payne’s LA2 (Terasaki‘s 2 ) , and van Rood’s 8a were probably the same factor. A summary of the statistical association between factors was published (Bruning et al., 19s5).

HUMAN HISTOCOMPATIBILITY SYSTEMS

263

In planning the third workshop in 1967, advantage was taken of the 30 families previously tested by Ceppellini, van Rood, and their associates. Members of 11 selected families were bled and the cell panel was expanded to include 91 samples (including a pair of identical twins) by blood samples from unrelated donors. During this workshop it was possible to examine 44 haplotypes contributed by the 22 parents of 11 families (Curtoni et al., 1967a). All 44 haplotypes were different; few, if any, of the investigators had reagents that could identify unequivocally all of them. Most investigators identified haplotypes determining some of the common specificities, such as HLA-2, but several could only be identified by single sera which were not known to fall into any well-defined class or group. Although no formal analysis of the data obtained during the workshop has been published, a resum6 of the results has been presented in tabular form by Curtoni et al. (1967a) and in the form of a histogram and summaries of some of the haplotypes by Amos et al. (1969a). The agreement among the results obtained by most of the 16 participating teams was quite impressive. The segregation patterns as well as the reactions obtained with cells from random donors gave excellent agreement with respect to six specificities (Curtoni et al., 1967a). Following this demonstration, agreement was reached upon the locus designation (Amos, 1968) and an elected panel of 12 immunologists and geneticists recommended designations for certain of the individual specificities (Allen et al., 1968). These specificitiesare listed in Table II.!' During the last few years there has been free exchange of information 'As was the case with earlier tables of mouse H-2 specificities, exceptions and qualifications need to be considered if tables listing antigenic specificities are to be used intelligently. Most of the comparisons between sera from different laboratories that were used as the basis for the agreement were made during the third workshop in Turin. At that time, cells from a total of 41 unrelated subjects were tested (Curtoni et aZ., 1967a). The 41 unrelated subjects included the 22 parents of the families. The families were selected because they were informative with respect to HLA-2 and 4a. Cells from only six of the 41 subjects reacted with most samples of anti-HLA-8, and only two with anti-HLA-7. Few of the cells were tested in duplicate and even fewer were tested on separate occasions. Some of the published results are approximations of the results obtained with several sera. The error rate of leukocyte typing under ideal conditions vanes from about 0.5 to over 516, depending upon the state of the cell suspension and the peculiarities of the serum. The definition of monospecificity of a serum is still being discussed. Walford has suggested that in order to reach 95%confidence that two antigens not associated more than 90% of the time will be detected, a total of at least 30 absorptions must be performed. Although this condition has been met for many of the sera, nothing has been said about absorption with negatively reacting cells. We have recently detected absorption-positive, cytotoxicity-negative cells on several occasions. Specificities detected by absorption may or may not coincide completely with those detected by direct testing, so that, although the table of equivalents is useful as a guide, it cannot yet be taken as definitive.

TABLE I1

NEW HLA NOMENCLATURE A N D PREVIOUS DESIQNATIONS~.~ New HGA nomenclature HLA-1 HL-A2 or HLA Mac H LA-3 HLA-4. HLA-5 HLAa HLA-7 HLA-8

Amos Batchelor Ceppellini

Dausset

4

5 -

To-8 To-9 To-10

45

2.5

To-5

5

2 41

-

To-20 To-7

10

19

1

1

2

Kissmeyer- Payne Nielsen Bodmer van Rood Shulman

11 1 or Mac 12

LA1 LA2 LA3

LA1 LA2 LA3

LA1 8a LA3

-

-

Da5

-

4d 7d

7c 7d

8

-

Terasaki Walford

1 2 8

Lel

-

6

-

-

5 11

Lc-8 Lc-7

PIGrLyB1 Hill

-

Lc-2

Lc-3

~

From Allen d al. (1968). * A dash (-) indicates that no symbol has been allocated within the nomenclature concerned. c HLA4 will be reserved for one of the higher frequency 4. factors, and HGA6 for 4 b . Before assigning these specificities, an exchange of serum among collaborating laboratories will be necessary.

U

8

E b

5

HUMAN HISTOCOMPATIBILITY SYSTEMS

265

and of sera between many laboratories. The workshops have added greatly to the common pool of information regarding antigenic specificities; van Rood and certain other investigators have energetically characterized cells from many panels with their best sera; Terasaki has dispatched thousands of microtiter plates to other investigators; and the N.I.H. Collaborative Program has been responsible for the collection, cataloging, and dispatch of liters of serum, much of which had been carefully characterized before submission. This exceptional collaboration has led to the accumulation of reasonably detailed information relating to the specificities listed in Table 11. Spccificities HLA-1, HLA-2, and HLA-3 belong to an interrelated series which includes two further members awaiting formal recognition. The designation HLA-9 has been proposed for one of these (Walford Lcll, Bodmer/Payne LA4, Terasaki 4). The fifth has been described by Dausset et al. (1968) but the antiserum has not been submitted for consideration to the nomenclature committee. Specificities of these series tend to be mutually exclusive, only one member is usually present in a given haplotype, for this reason they are thought to be true alleles. There are strong associations between some of these factors and certain specificities not of the LA series; for example, there is a positive association between HLA-2 and antigens of van Roods 4a series. In general, excellent agreement has been obtained when sera reacting with one of the defined specificities were compared on test panels in different laboratories. There are, however, some interesting departures from expected results which can be commented on to illustrate some of the problems involved in the recognition of HL-A specificities. Reference will be made to HLA-2, HLA-3, and HLA-5. Twenty antisera primarily reacting with specificity HLA-2 were tested at the Turin workshop. The reactions of many of these sera were in exact agreement. However, cells from Turin donor No. 22 were exceptional in that no reaction was detected by eight of the sera, although the cells were clearly positive when tested with other samples of anti-HLA-2. Dausset had independently reported that of his anti-HLAS sera, one which gives consistent results in Caucasians reacts differently in Negroes ( Dausset et al., 1967), and he has also reported on a “long Mac” (serum No. 42) which reacts with cells that are HLA-2 negative (Dausset et al., 1965). From this and other evidence, it may be suggested that even such wellrecognized specificities as HLA-2 may be found in slightly variant forms. This possibility was taken into account by the panel on nomenclature in drawing up criteria for defining antigenic specificities. If two antigens resemble each other so closely that their correlation ( T ) is greater than

266

D. BERNARD AMOS

0.9 and yet they are clearly different in family studies, one shall be given a second number preceded by a point (Allen et al., 1968). Thus, if Turin cell 22 carries a true variant of HLA-2, the antigen could be designated 2.1; similarly, if Dausset’s antigenic variant detected on the cells of Negroes is different from HLA-2 and from HLA-2.1, it should be designated HLA-2.2. The close association between antigens having a similar distribution does not imply that they are alike in their chemistry or in their biological properties, although this may be so. To date, no fine distinctions have been made between subclasses of antigen, although some apparently meaningful differences appear when the reactions of sera that superficially react alike are examined more carefully. This is especially apparent with the specificities 4a and 4b which are so ill-defined that the committee merely set aside categories HLA-4 and HLA-6 for them. Specificity HLA-3 is interesting in that a “long” or more reactive form of antibody has been studied in detail (Amos and Yunis, 1969). During the Turin workshop a serum designated BC known to recognize Bodmer and Payne’s LA3 (now HLA-3 which we had called by the local designation ,404) was tested. It was erroneously thought that the serum contained two antibodies, one of which reacted with LA3 ( A o ~ )and , the other of which reacted against an unknown determinant which we called A014 By absorption, anti-A014 could apparently be removed, leaving anti-Ao4. Anti-4 could not be removed to leave anti-14. This is not an uncommon situation and antigens for which such a complex relationship exists are called “inclusions.” However, attempts at removal of anti-14 by absorption turned out to be rather disappointing and, although all samples obtained by absorbing with 4-14 positive cells had the general properties of an anti-4, there was variability in the reaction with certain cells with different absorptions. When one such sample of absorbed serum was tested in Turin, it reacted with all LA3-positive cells and with cells from three LA3-negative donors, whereas the unabsorbed serum reacted with these and with several additional cells. More recently, 17 different bleedings of this serum obtained over a period of 2 years and spanning four cycles of immunization have been subjected to controlled semiquantitative absorption by Dr. Yunis. Absorptions were carried out on each of the 17 samples at five different dilutions using five different concentrations of cells from over 20 subjects. First, it was noticed that the unabsorbed sera from various stages of immunization reacted differently with cells from three groups of donors. Lymphocytes from one group of donors (group 1) reacted strongly with all the antibody samples. Cells from another set of donors (group 2) reacted somewhat less intensively and gave negative

267

--Immunizing

1

60

100

,

140

First Immunizotion Cycle

f f 0

Donor

I

40

80

120

160

Third Immunization Cycle

200

21

Time of Cycle in Doys

FIG. 2. Cytotoxic reactions of sequential bleeclings from HLA-3 antibody producer BC against cells from three classes of donor. (From Amos and Yunis, 1969.)

results with certain weaker bleedings. Group 3 cells gave a weak positive reaction only with three of the very strongest samples and were negative with the other 14 (Fig. 2 ) . Upon absorption, group 1cells would remove all activity for themselves and for cells of the other classes; group 2 cells would remove activity for cells of group 2 and would lower the titer for group 1 cells. If the cell concentration was increased, or the serum was diluted one extra step, group 2 cells would remove all activity for group 1 cells as well. Similarly, group 3 cells could absorb activity for group 1 or 2 cells even from serum samples with which they did not react directly. For these absorptions a concentration of 1.2 x lo8 cells/ml. was incubated in two steps with serum dilutcd to two log 2 dilutions or more from the end point for the appropriate test cell (i.e., diluted to 0.25 when the end point was 0.06). Control absorptions did not show a loss of activity even if the cell conccntration was raised to 5 xlOScells/ml. of diluted serum. Group 1 cells and some cells from group 2 reacted when tested with samples of anti-HLA-3 sera from other laboratories. Group 3 cells rarely reacted directly with these sera but could readily absorb activity for known group 1 cells. Again, there was no absorption with cells used as negative controls for BC. We, therefore, believe that the HLA-3 antigen can exist in different

2f33

D. BERNARD AMOS

forms, in different concentrations, or in different spatial arrangements on the plasma membrane. Group 1 cells appear to have the greatest effective concentration of antigen, group 2 cells somewhat less, whereas group 3 cells have relatively little available antigen. If this view is correct, it is easy to understand how the generally accepted definition of HLA-3 has been arrived at and why it is erroneous, Cells of classes 1 and 2 appear to carry the greatest amount of antigen and are most easily detected. Monospecific sera-i.e., those giving the sharpest results with relative freedom from bothersome weak reactions from “contaminating extra antibodies”-are highly prized; therefore, every attempt would be made to select a serum giving reactions with the easily recognizable group 1 and group 2 cells. The serum BC gives an almost exact correlation with “known” antiHLA-2 sera when diluted to 0.5 or 0.25, i.e., when it still reacts very strongly with group 1 cells and moderately strongly with group 2 cells. Certain samples of BC obtained at different times after immunization also give an exact correlation with HLA-3 without dilution and without absorption. These samples, like the reference sera for HLA-3, fail to detect group 3 cells, although the antigen is clearly present when tested for by absorption. Unless careful absorptions are carried out at different dilutions with cells from negatively reacting donors and, whenever possible, serum samples obtained at different times are included in the test, the full expression of the antigen in the population is likely to be missed. A fourth class of cell appears to represent a peculiar form of anticomplementarity previously reported on by Tiilikainen (1967) and by Ferrone et al. (1967). This anticomplementarity may not be noticeable in the reactions with the majority of cells but is expressed reproducibly with cells from certain donors. Reactions remain negative unless the antibody is removed and the cells washed before complement is added; then a strong positive reaction develops. (The false negative reaction is not due to interference from anti-A or anti-B in the serum, but could be accounted for by the presence of some other antibody not detected by the cytotoxicity test.) The frequency with which this phenomenon is observed vanes from 0 to over 30%depending upon the serum used. Class 4 cells appear to be strongly antigenic in that they absorb strongly and appear to belong to the class of cells described as cytotoxicity negative, absorption positive (CYNAP) by Ceppellini and others. Another class of CYNAP reactions has been reported by Svejgaard and Kissmeyer-Nielsen (1968). Certain HLA-2 negative cells had the property of absorbing all activity from an antiserum apparently containing two distinct antibodies. Another anti-HLA-2 serum was not absorbed by these cells. Svejgaard and Kissmeyer-Nielsen attribute this to cross-reactivity between antigens.

HUMAN HISTOCOMPATIBILITY SYSTEMS

269

The designation HLA-5 was given to the specificity detected by six sera or groups of sera defining antigens Amos Ao45, Batchelor 25, Ceppelh i To5, Dausset 5, van Rood Da5, and Terasaki 6. This specificity appears to be included in van Rood's original 4a. The homogeneity of this group of six sera has not been exhaustively studied on other panels, and it may later be necessary to reclassify some of these specificities. The reviewer does not know the significance of apparently minor deviations in the distribution of specificities which otherwise share a similar distribution in the population. It may be merely that two different antibody donors react slightly differently when immunized with what is essentially the same antigen; it may be that the two factors differ only with respect to some very minor characteristic such as a slight difference in their conformation or it could be that the two factors are really quite unalike but show a fortuitous association in their distribution in the population being considered. Because evidence about the effective strength of the histocompatibility antigens is so fragmentary, it seems essential to have as precise as possible a definition of the individual specificities. The question of correct identification of a specificity has particular relevance to attempts at donor-recipient matching for transplantation and to the characterization of purified antigenic products. Suppose that a prospective donor reacted with a typical serum believed to detect HLA-3, whereas the recipient did not. The donor would almost certainly be a member of group 1 or 2. There is a high probability that the recipient would really belong to group 3 or group 4, the reaction going undetected. An apparent incompatibility for an important antigen would in fact be a compatibility. Worse, donor and recipient could both fail to react. If the negative reaction of the donor concealed a potential group 3 reactivity, an apparent compatibility could conceal a disastrous incompatibility. It was thought that the introduction of monospecific sera would eliminate typing or matching errors. This is only so if the peculiarities of the sera are appreciated and the full reactivity is obtained. In summary, HLA-1, HLA-2, HLA-3, HLA-5, HLA-7, and HLA-8 have so far been recognized. Since there is some doubt that the identities of at least some of these factors have been definitively established, an identification of the serum used, together with the bleeding date, should be given in all descriptions of these specificities.

C. PROCUREMENT OF ALLOANTISERADEFINING NEW SPECIFICITIES Most of the antisera now used are obtained from multipara or from volunteers immunized through skin grafting, by the injection of lymphocytes, or following blood transfusion.

270

D. BERNARD AMOS

Multiparous or multigravid women are bled in the clinic and their serum tested against cells from a donor panel. Sera from about 20 to 25% of these women contain leukoagglutinins; a somewhat lower proportion contain cytotoxins. Whenever possible, serum is obtained 16 weeks postpartum since Payne has reported that titers may drop off after this time (Payne, 1962). This is true only of a proportion of multipara, others are known to have continued antibody production for 20 years following the birth of their last child. It should be permissible to reimmunize postmenopausal multipara with leukocytes from their positively reacting children. Although sensitization can occur during pregnancy, the greatest stimulus appears to be given during parturition since the reactive strength of antibodies obtained from primigravida is low and the incidence of antibodies is greater in gravida I1 than in primigravid women (Zmijewski et uZ., 1967a). The specificity may change with time. One serum (SA) studied over a 10-year period in our laboratory-appeared to have retained its ability to react with certain highly responsive cells but to have changed with respect to more marginally reactive cells. Antibodies obtained by deliberate immunization appear to differ from antibodies obtained from multigravida. Antibodies obtained experimentally are not infrequently complemented by human or guinea pig serum, whereas multigravid sera are more apt to require rabbit complement. These differences have not been systematically explored. It is only known that most of the agglutinating or cytotoxic antibodies are IgG globulin. The ideal donor-recipient pair for planned immunization would be siblings who d 8 e r at one HL-A haplotype or parent-child pairs. Most frequently unrelated subjects must be used. As large a panel as is practicable is typed, and donor-recipient pairs are arranged so that the donor carries a particular antigen lacking from the recipient or, if the object is to obtain a completely new antibody, donor and recipient are selected to be phenotypically identical with respect to the antibodies available for typing. A purified, washed suspension of lymphocytes is injected intradermally into multiple sites on the forearm to a total of up to 5 x 10'. Inflammatory lesions develop at the injection sites, reaching a maximum within 48 hours and thereafter fading slowly, leaving a brown stain which persists for up to 2 months. A second set of injections of cells from the same donor is given 6-8 weeks later. Antibodies may appear within 10 days of the second injection. The antibody donor is then plasmaphoresed on one or more occasions and serum samples are taken at frequent intervals. The blood is collected during plasmaphoresis into heparin, the plasma is clotted by the addition of protamine, the serum is expressed from the clot, distributed into small vials, and stored at -80°C. In this

HUMAN HISTOCOMPATIBILITY SYSTEMS

271

form it is stable for at least several years. If no antibodies appear, injections are repeated at approximately monthly intervals for up to six injections. It is unusual to obtain antibodies with a titer greater than 0.12 after a primary immunization, and the titer may increase only slightly if further injections are given at this time. To obtain a high titered serum, the subject is allowed to rest for at least 1 month after all antibodies have disappeared from his circulation and is then reinjected with lymphocytes from the same donor. On occasion we have given less than 2 x lo6 cells as a booster and have obtained a dramatic secondary response; more commonly lo' to 2 x 10' lymphocytes are administered. The titer begins to rise within 5 days and reaches a peak within 10 to 14 days. Occasionally two peaks are separated by a zone. Titers are commonly in the order of 0.03 or higher following such a second immunization. Subjects vary greatly in their anamnestic response after a long inactive interval. Some donors have produced a strong antibody response on challenge after having been without circulating antibodies for 3 years, others have failed to respond as readily after a prolonged rest. Antibody producers, whether experimental or multiparous, should not be used as blood donors while they possess high titered antibody. Brittingham (1957) reported a shocklike response following transfusion from an antibody-bearing donor; however, no adverse reaction was observed when 250-ml. volumes of antibody were infused into leukemic subjects (Laszlo et al., 1968), and Terasaki and his colleagues have administered alloantibodies in an attempt to utilize an allogenic antilymphocyte serum. Antibody may also follow repeated skin grafting. This has been observed by Batchelor (1965) and Batchelor and Sanderson (1967), who found that up to four sequential grafts might be needed. Hammond et al. (1967) have given repeated transfusions of whole blood from the same donor and found the response to depend upon the volume of blood used rather than upon the number of transfusions given. For the characterization of new antisera, it is necessary to have a characterized cell donor panel. Reference sera for this purpose may be obtained from the N.I.H. serum bank4 and frequently from individual investigators. Cell donors should be established residents, Cells are then tested at least twice with each of the reference sera using only the testing procedure recommended for each. A serum that detects HLA-2 by the

'Chief, Collaborative Program on Transplantation and Immunology, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, Maryland.

272

D. BERNARD AMOS

cytotoxicity test may give completely digerent results by agglutination (Zmijewski and Amos, 1966). Once the panel has been typed, new antisera can be tested. The results obtained are compared with the known reactions. Values for x2 and r are obtained by computation, and the new sera are tested on families to show that the specificity detected segregates with known HL-A specificities. A decision is then made as to whether the specificity appears to be new or if it is similar to a known HL-A factor. If two sera appear to resemble each other except for the reactions of two or three cells, the determinations with those cells are repeated, with absorption if necessary. The two sera are tested on informative families. Ideal families are those in which one parent and some of the children react with the sera. The two sera being considered should detect the same haplotype. If in one or more families the second serum detects a different haplotype, the two sera cannot be considered to detect the same antigen, no matter how high the xz or T value obtained with the unrelated panel. If a new specificity is detected, it is customary to give a local designation. This may be the name of the serum and the bleeding date (e.g., Hipp 1/67) or a code designation for that laboratory is employed. A list of such specificities detected in our laboratory together with an explanation as to how they were recognized and their possible equivalents is given in Table 111. Such tables of “equivalents” have been published by several investigators (Allen et al., 1968; van Rood and Eernisse, 1968). Absorption is then carried out to check that the serum detects only one apparent specificity. The absorbing cells should be from both positively and negatively reacting donors and absorptions should be performed at varying cell concentrations with antibody diluted to within two log 2 steps of its end point. Failure to standardize the reactivity of the serum accounts for much of the difEculty experienced with absorption. Under these conditions 1.2 x lo8 buffy coat cells from positive donors will usually remove all activity, whereas no nonspecific absorption should be apparent with 2.4 x 105 cells. Provision has been made by the nomenclature panel for accredidation of sera recognizing new specificities (Allen et al., 19S8). Once the local comparison has been made and the appropriate absorptions made to show that the serum is apparently monospecific, a sample may be sent to one of the dozen or so collaborating laboratories. This laboratory will then compare the new serum on its own panel and will also observe the segregation of the new specificity in informative families. If the serum appears to define a completely new antigenic specificity, it will be sent to a second laboratory for conhmation. The information obtained will be relayed to

HUMAN HISTOCOMPATIBILITY SYSTEMS

273

the investigator and further absorptions may be required. All pertinent information will then be circulated by the acting secretary, Dr. Z. Tmka, to the full committee and, if agreement is reached, a new designation will be made and a sample of the serum will be kept as reference. If the serum defines a specificity which is very much like a known antigen but is clearly different in some respects, it will probably receive a subsidiary designation as in the example quoted earlier where new antigens in the HL-A series could be called HLA-2.1 or HLA-2.2. This system is capable of infinite expansion and, while drawing attention to similarity, makes no committment that two superficially similar factors are identical (see Allen et al., 1968). IV.

The Use of Typing for HL-A Factors in Donor Selection

A. FAMILY MEMBERS Since HL-A functions as a closely linked system, the inheritance of haplotypes within a single family is very simple. As explained in an earlier section, only four genotypes are possible in a sibship. A fifth child must inherit the same allele; as one of its siblings; so if there are five or more children, the family must include at least one HL-A identical pair. Skin grafts between such HL-A identical pairs persist for an average of 24 days (range 1 5 4 3 days) (Amos et al., 1969b). Siblings who differ with respect to both HL-A alleles ( AC to BC, AD to BC) may be no more alike with respect to HL-A than any unselected pair of unrelated individuals, and skin graft survival for grafts between such siblings is almost the same as survival times for grafts between unselected random subjects. All parent-child and 50% of sibling-sibling combinations differ with respect to one HL-A allele. If the antigenic information carried by that allele is strong, the resultant incompatibility is also strong, and a graft may be rejected in 7 days or less. Many of the alleles are strong, and the mean rejection time for a graft between family members differing at one allele is not very different from that of grafts from siblings differing at both alleles or between unrelated subjects (Amos et aZ., 1968, 1969b). However, although the range for two-allele graft rejection is relatively restricted, some of the one-allele grafts persist for as long as HL-A identical grafts (Dausset et al., 19139). In the absence of firm information about the immunogenicity of the individual specificities or the alleles, we prefer to carry out skin graft tests. Suppose the recipient is genotypically AC and he has an HL-A identical sibling who is not available as a kidney donor but who can participate in a skin graft series, both parents ( AB and CD), and a second sibling genotypically AD; skin from the parents and the AD sibling would be placed on the AC sibling. Rejection times of the

TABLE 111 ANTI~ENIC SPECIFICITIES Specihcity

Senrm

Date

Freq.

Aol A02 A03 A011 A04 A05 A06 A07 A08 A039 A052 A09 A010 A012

FAY

11/65 6/65 9/66 9/66 11/66 12/65 P-1 5/65 P-1

0.43 0.31 0.38 0.75 0.70

P- 1 P-1 2/67 9/66

0.13 0.31 0.63 0.21

A013 A015 A016 -4017.1 Ao17.2 Ao17.3 A019 A020 A021 A022

SA FS

0.46 0.19 0.37 0.30 0.24

McM CAR $110 JT

4/65 P-1 1/66 3/65 3/65 3/65 9/65 2/66 9/65

A023 A024 A027 A028 A029

RA RA KII THA JAC

RIL DK DK BC ENN AJ WB BH BLI BH DAL DAL SA

PIGG NW NW

Nw

P- 1

P-1

1/67 P-1 1/67 1/67 1/67

0.60

0.36 0.68 0.49 0.75 0.75

0.50

0.73 0.10 0.51

Abs. with 0

SB SD ROD 0

Identical

too

HLA-2 (a-g) HLA-7(e)

?

? LT RA ED JCA

HLA-7(a) New

0

JMat JM 0 0 0

HLA-B(c,e) Da5(e) C p l l ( e ) KNT12(e)

NH NH RA 1

+

0 0 0 0

BEB 0 BEB 0

New New

Related toa

"Allelic"

to9.b

8a 7Cb) Da4(e) HLA-ll(e) 4a(d) HLA-16.c) HLA-3(f.e) Lcl7(b) A o l 5 b ) 4ab)

A022 Ao56, HLA-S(b,c) Ao4Z(b) L c l 7 b ) Lc7(b) Ao24(b) 56(b)

HLA-11 (b,c)

Aol (b) Aol(c)

HLA-3b) (Aa15.Ao46)m) HLA-7(b,c) BoLa4(e) Da4(e) Bo4c(f) Aol6(c) Ao53(b) Te4(e) 4ak.b) Da4 (e) Cpl1 (e) Te4 (e) C p l l ( e ) KNT12(e) Vr4a(e) LAl(b) Ao20(b) (AoS.Aol)(h) Ao2(h) Aol9(h) (AoS.AolO)(b) Aol6(h) A019b) (Ao23.Ao24)(c) Lc7 (c) Lcl(c) Lc2O(c) 4b(a) HLA-l(b) H L A d ( b ) Da3(e) KN4al(e) 7 d b ) Lc7b)

AoW(c) Ao22(b)

Lc7(b) A o 4 l b ) Ao66(b) 4b(e) 7c(e) Te9(e) DalO(e) AoGO(b) (AoB. AolO)(b) Ao.!?$(b)

P

:: B

E

1/67 1/67 1/67 1/67 5/66 1/67 7/65 7/65 P-1

A03 1 A033 A034 A060

JOC RB RB

Ad6 A037 A040 A041

GW

A042 A049 A043 A045 A045 A046 A047 A051 A053 A054 A055 A056 A059

AG AG TOH JPO JPO JPO DAP SD HF BL Bhl ROY

1/67 1/67 P-1 11/66 11166 1 1/66 11166 10165 P-3 P- 1 10/65 5/66 6/65

A061

LBU

A062 A063 A064

A035

RB

BST

GW XP

MH

VER

P-1

0.07 0.21

AB

0.17 0.25

JM 0

0.34

FC ? BUL

0.05 0.23 0.36 0.60 0.34 0.71 0.71 0.11 0.13 0.23 0.19

MM

0 ?

JMat

Ao2(b) New

New New

0 ET 0

0

+ FC

ND 0 0 0

0.19 0.29

0

11/66

0.23

0

IIM

P-1

0.14

0

JD XH

6/65 10/65

0.31

0 0

0

?

HLA-Nb)

Lc3(b) LcS(b) Ao22(b) Ao56(b)

Lcl (b) AolS(b) AoBf (b)

Ao56(b) A022(b) Ao62(c)

(Aold.AolS)(b) Aol5(b) Aodf (b) Aol(b) HLA-H(b)

HLA4(b) 6b(a) 7c(a)

Ao66(b,c) 7d(b,o)

Ao56b) Lc7(b) Le20(b) Ao29(b) Lcl(b) HLA-Z(e) HLA-Z(c) Ds5(e) Lcl7(b) HLA-5(e) (AoS,A039,Ao52)(b) Aol6(c) A021 (c) (Ao12.A013)(c) Bt3 (e) A061 (a) Ao59(c) 4a(s) 7a'(a) (Ao23,Ao24)(b) &(h) 4 4 ) Ac33(c) Lc7(b,c) A04l(c) Aol5(b.c) Ao.W(b,c) A06b.c) Ao53(c) Ao59(h) Ao5301) Aol5(h) Ao64(h) A035(c) Aol6fi) Aol9(h) Aol5(h) (.4015,A046)(c) Lc3(c) Ao59(h) Ao6l(h) Ao.W(h) (4a,7a)(a)

(A033,A034,Ao6O)(h) (Ao23,Ao24)(h) (Ao9,AolO) (h) AoSS (b)

AoJGb) LcS(b) AodO(c) Ao5O(b) Ao55(h) Ao.W(h) (Ao23,Ao24)(h) AoZ(b) (Ao4,Ao14)(h) (Ao33.Ao34.Ao60)(b) A056 (h) (Ao23,Ao24)(h)

Test situations: (a) Duke 1-typed by van Rood: (b) Duke 2-8/68; (c) Duke 3-haplotypes 8/68; (d) Denver; (e) Turin; (f) San Franckco; (h) Madison. Italicrr indicate type 1 allele (two specificities do not occur together on Bame allele in haplotype data): boldface type indicates type 3 allele (individuals usually positive for one or other of two specificities-occasionally an individual is phenotypically positive for both specificities or negative for both); all others are type 2 alleles (two specificities very seldom occur together on same allele in haplotype data-high negative aseaciation). 0

b

276

D. BERNARD -4MOS

AD and CD grafts give an indication of the immunogenicity of the D haplotype, whereas rejection of the AB graft measures the immunogenicity of the B haplotype. In this case the interpretation is simple. If, say, the AB graft persists for 19 days whereas the AD and CD grafts are rejected in 10 days, the father would be an ideal donor since the B haplotype is evidently feebly immunogenic. If there is no HL-A identical test subject, analysis is indirect and sources of error are greater. Suppose again the patient was AC and he had both parents and a BD, a BC, and an AD sibling. The BD sibling would be an unlikely donor; the BC or AD sibling and the AB or CD parents would be considered. Grafts from the AD or BD child to the father would give some measure of the D haplotype; grafts from the BC or BD child to the mother would measure the B haplotype. This procedure is not as good as grafting to an HL-A identical subject, although it has some merit. Serious errors are introduced if the B and D haplotypes share strong antigens not present on A or c. Grafting skin from BD to AB or BD to CD subjects will not show these factors. Each haplotype can be tested in six different combinations within a family; for example, the A haplotype can be tested by the grafts AB + BC, AB + BD, AC + CD, AD -+ CD, AC + BC, and AD + BD. We have found by writing out the H-2 alleles in a mouse model that these six combinations can represent at most three different antigenic relationships. Each of the overlaps is duplicated. In the mouse model produced by mating the F, hybrid between C,H (H-2k) and YBR (H-2d) with the F , between DBA/l (H-2q) and C57BL (H2b), thus obtaining four classes of offspring, H-2k/H-2q, H-2k/H-2b, H-2d/H-2q, and H-2d/H-2b, two of the grafts would test H-2 antigens 4 and 31, two would test 4, 14, and 31, and two would test 4, 13, and 31. Where the three different combinations can be tested in this way, an estimate of the effective strength of a given specificity can be obtained by inference. From such studies in man and mouse, we should be able to find out if intra-allelic relations between specificities are effective in determining immunogenicity or if the individual specificities themselves are of paramount importance. For the reader interested in analyzing such a model, the antigenic configurations in the F, parents and the four classes of F, hybrids can be written out from the table given by Snell and Stimpfling (1966) and the antigenic differences tabulated. In practice the procedure works as well in the mouse model as it does in man, different antigens do have different effects. For example, in the mouse antigen 14 appears to be quite strong in determining skin graft survival. Andrus and Amos, in experiments in progress, have found that grafts incompatible for specificities 2, 14, and 22 are rejected in about 11 days, whereas grafts in-

HUMAN HISTOCOMPATIBILITY SYSTEMS

277

compatible for 2 and 22 or 4, 13, and 4, 13, and 31 are rejected in about 14 days. In each case the donors only differed by the products of one H-2 haplotype and the segregation of unrelated genes was random. In man the correlation between graft survival of over 14 days and good function of a kidney transplant predicted by Ceppellini et al. (1965a) appears to be valid. Bach and his colleagues are attempting to measure immunogenicity in a different way. Using unidirectional mixed lymphocyte culture, Albertini and Bach (1968) are comparing the dose-response to cells from different siblings differing at one allele. If successful, this approach would obviate the necessity for test skin grafting. Ultimately, it is hoped that serological procedures by themselves will be adequate for an evaluation of functional incompatibility. For the cadaveric transplant, serological tests appear to offer the only hope for matching within the time available. Living kidney donors in the Duke University series have been selected according to the criteria outlined above ( Stickel et al., 1967). The category class 1 is reserved for HL-A identical transplants. All but one of ten HL-A identical kidneys have functioned superbly. The tenth succumbed to a recurrence of glomerular disease not associated with rejection during his second year. Recipients of an HL-A identical kidney require very little immunosuppression. One such kidney has functioned perfectly for over 4 months although the recipient has received no immunosuppression in any form throughout this period. A second recipient of an HL-A identical kidney maintained excellent renal function for over a month without drugs. He was later returned to low dose of azathioprine as a safety measure, and was sent home on a total immunosuppressive regimen of less than 1 mg. azathioprine per kilogram. He continues to maintain excellent function. A reaction against an HL-A identical kidney can occur but appears to be exquisitely sensitive to prednisone. Category 2a include sibling-sibling and parent-child combinations differing by the products of a poorly immunogenic allele. The allelic strength is judged on the basis of skin grafting and of serological similarity. Some of these transplants are quite as successful as those in HL-A identical group 1 (Stickel et al., 1969), although a proportion of these subjects do take longer to regain full renal function. This trend is much more marked in subjects (class 2b) who received a kidney from a donor judged to have a relatively strongly immunogenetic incompatible haplotype. Subjects of class 2b have a considerable delay in obtaining good creatinine clearances. Even after 6 months, clearance values may not rise as high as in HL-A identical and class 2a recipients, whereas the incidence of attempted rejection requiring steroid treatment in the early postoperative period is much higher ( Stickel

278

D. BERNARD AMOS

TABLE I V RELATIONSHIP BETWEEN HISTOCOMPATIBILITY AND KIDNEYGRAFTSURVIVAL I N GENOTYPED FAMILIES' ~

~~~~~~~~~~~

~

Group

N

I 11 111

10 6 11

HL A class

Threatened rejections

Maximum BUNb with first rejection

1 2a 5-2b 6-2a or 2b

5/10 4/6 11/11

22-34 28-51 30-159

Onset of rejection

Deaths

After day 22 After day 22 Most within first 10 days

1 0 2

Average dose of prednisone (mg./day) days posttransplant Group

I I1

I11

0-10

10-20

2040

40-90

90-180

180-365

365-730

1 49 62

5 78 127

46 76

17

13 23 47

6 18

3 0 26

2 3

43

Average BUNb (mg./100 ml.) days posttransplant Group

0-10

10-20

20-40

40-90

90-180

180-365

365-730

I I1 I11

35 38 50

25 27 41

24 25 30

23 22 25

18 19 22

17 18 19

-

a

15 16

Data from Stickel et al., 1969. Blood urea nitrogen.

et al., 1969). Two of these patients have died with evidence of rejection complicating other contributory causes of death (Table IV). B. NONLIVING DONORS Only very rarely is there any close family relationship between donor and recipient. Frequently a dying potential donor can be typed in life and the reactions obtained compared with those of cells from a number of recipients maintained on conservative treatment or on dialysis. In some laboratories, typing is only carried out after death; matching is compared restrospectively for information only. Retrospective typing has been especially common with heart transplants where the lack of any attempt at selection is perhaps reflected in the continuing high rate of rejection. Instead of being able to establish a relationship based on genotype, these donor-recipient pairs are selected on the basis of phenotypic simi-

HUMAN HISTOCOMPATIBILITY SYSTEMS

279

larity. At present this is a relatively crude process and is complicated by four factors: 1. Errors introduced during typing are incorporated in the phenotype. (Errors in genotyping by contrast can often be recognized and corrected.) Such errors can make a very considerable difference in the HL-A phenotype. Quite frequently incompatibilities can be missed or false incompatibilities scored from this cause. It is notoriously difficult to obtain good cell suspensions from patients dying from trauma since they often have total leukocyte counts of over 25,000 of which less than 3%are lymphocytes. The numerous granulocytes often behave abnormally; they may adhere feebly to glass or other surfaces but yet tend to clump spontaneously. More reliable cell suspensions can be obtained by teasing out mesenteric lymph nodes collected at autopsy. 2. The second source of error is occasioned by the use of sera containing two antibodies. Multispecific sera can often be used successfully in genotyping families since such sera frequently react only with the products of one haplotype. In phenotyping, one component could react with cells of the donor and another with cells from the recipient. Unless the serum is absorbed with cells from each of the subjects, false compatible reactions can be quite common. 3. The third factor is that we still do not know how to weigh incompatibilities. This is somewhat of a problem in attempting to assess haplotype strength in matching family donors but is compounded by the simultaneous difference at both haplotypes in unrelated subjects. 4. Finally, it is highly likely that many antigens remain to be detected. This does not interfere with genotyping since the unknown antigens must travel with one of the four haplotypes and there are usually abundant markers to plot the haplotype distribution. That it is a real factor in graft donor selection among unrelated subjects is shown by the failure of any laboratory to give an accurate prediction of skin graft survival. Ceppelh i (Ceppellini et aZ., 1969), Dausset (Dausset et aZ., 1969), and others have noted a difference of approximately 2 days between the rejection times of grafts between phenotypically similar and dissimilar unrelated subjects. Our own experience has been comparable. Differences due to variability in immunological responsiveness of the subjects tend to obscure differences attributable to antigenic factors. This 2-day difference in mean survival must be contrasted with the difference between 11 and 24 days mean survival observed in the HL-A dissimilar and identical sibling series, and gives some idea of the present inadequacies in selecting for compatibility among unrelated subjects. Terasaki (Terasaki et al., 1968),Payne (Payne et al., 1967), van Rood

280

D. BERNARD AMOS

(van Rood and co-workers, 1969), and others have compared renal homograft survival with phenotypic matching. In general, kidneys from matched donors function better, provoke fewer episodes of acute rejection, and are tolerated longer than grafts from grossly mismatched donors. Obviously, even in its present pitifully inadequate state, matching has considerable value, and it is manifestly foolish to ignore obvious incompatibilities. As more effort is put into obtaining new reagents, existing ones are refined, and an estimate is made of the relative immunogenicity of single factors or factors acting in concert, phenotyping is likely to become more and more of a reliable guide. Several attempts have been made to unify the tests and to obtain meaningful data. One of these has been conducted by Terasaki who has established a typing service on an unprecedented scale (Terasaki et al., 1968). Terasaki and his colleagues also organized a city-wide scheme whereby potential recipients were typed, matched with donors as they became available, and the recipient or recipients who most closely approximated the donor were chosen to receive a transplant (Pate1 et al., 1969). This service has been extended to include typing for heart and liver transplants and, on occasion, subjects have been flown considerable distances to obtain the best available match. Ewotransplant was introduced by van Rood ( 1967) and his colleagues, and a similar but separate scheme is envisaged for Switzerland. As in other collaborative efforts, the recipients are typed electively and the results stored in a form available to a central computer. Notice of a potential donor is received, typing is carried out locally, the results fed into the computer, and the appropriate recipients selected. A citywide scheme is available in Boston and another, evolving in Richmond and surroundings, is based on the Medical College of Virginia. An exchange organized from Duke University is attempting to coordinate typing in an area of the Midwest, parts of the Eastern seaboard, and Canada. Within a few years when accurate predictions are possible, it may be necessary to amalgamate different regions to allow enough comparisons for proper selection. If it should be found that many of the individual specificities are important, it may be necessary to have a recipient panel of several hundred patients who can be matched with every donor. During the course of a year, many thousands of choices could be made if the donorrecipient area was extensive. Even in one limited area involving Richmond, Durham, Atlanta, and Charlottesville, it was calculated by Hume and his colleagues that over 60,000possible matches could be made in the course of a year. Although there are differences in the frequency with which certain antigens and certain combinations of antigen occur, it

HUMAN HISTOCOMPATIBILITY SYSTEMS

281

should be possible to find an acceptable match for any given patient. The costs for such a nationwide scheme would not be prohibitive, and the potential gain in terms of reduced hospitalization time and reduced mortality would more than compensate for the laboratory effort needed. V.

Other Human Histocompatibility Antigens

The A and B antigens of the ABH ( ABO) blood group system have proved to be extremely strong transplantation antigens under circumstances not fully understood. Their relevance was first clearly noted when a group B kidney was almost immediately rejected by a group 0 recipient (Hume et al., 1955). The significance of the blood group antigens in transplantation was not fully appreciated immediately and several other A or B kidneys, including several chimpanzee kidneys (Reemtsma et al., 1964) were transplanted into incompatible recipients (Starzl et al., 1964). Interestingly, not all were rejected and long-term survival has been reported (Gleason and Murray, 1967). The effect of incompatibility for blood group A was appreciated independently by Ceppellini and by Dausset and their colleagues (Ceppellini et al., 196%; Dausset et al., 1965). Visetti and his colleagues (1967) demonstrated that the A, antigen was more potent than Az and also that cross-sensitization occurred. Rapaport et al. ( 1968) reported white graft reactions in group 0 recipients presensitized with soluble A or B blood group substance or with A,B, A,, or B erythrocytes freed from leukocytes by repeated sedimentation. In this experiment white grafts were only reported when the skin graft was from an A,B donor even if A,B erythrocytes were used for sensitization. Although circulating antibody is presumably involved in the destruction of well-vascularized kidney homografts, cellular responses are implicated in the rejection of A, or B skin grafts since circulating antibody levels did not rise following the administration of 3 x 1O’O erythrocytes. Ceppellini et al. (1966) have noted white graft reactions in unsensitized individuals, but, conversely, other A grafts to group 0 recipients have persisted and undergone normal or even delayed first-set rejection (Ceppellini et al., 196%). Rapaport et al. (1968) speculate that pepsin used in preparation of bacterial vaccines may have sensitized many “normal” individuals. Although incompatibility for blood group A or B does not invariably lead to accelerated graft destruction, Ceppellini and his colleagues ( 1965b) have convincingly shown a highly significant association in family studies on unimmunized subjects. Visetti et al. (1967) and Rapaport et al. ( 1968) have found accelerated reactions in preimmunized recipients.

282

D. BERNARD AMOS

Ceppellini has reported a much weaker association between graft rejection times and incompatibility for P, incompatibility, and no apparent effect of MN or Rh incompatibility (Ceppellini et al., 19f36). Rapaport et al. (1968) found Rh, Leb, M, N, Fy", P, and S to have no apparent effect in sensitizing recipients when the antigen was administered intravenously or intradermally in the form of 4 x l O l o erythrocytes. With the exception of antigens of the HL-A system and A, B, and possibly P blood group factors, no known antigen has been found to be effective in stimulating homograft rejection. Undoubtedly there are many other H factors in man but it may be inferred that non-HL-A (or in the mouse, non-H-2) factors are very secondary in their effects. In the human series, ABO and HL-A identical grafts persist on average for twice as long as grafts differing at both HL-A haplotypes. This distinction would be obscured if strong histocompatibility antigens were segregating independently of HL-A. One of the &st antigenic groups to be detected by van Rood was the apparently diallelic five system, the two alleles being designated 5a and 5b. The five system is not related to HL-A. The antigen 5b is widely distributed on the tissues, being present in quantity on spleen, tonsil, kidney, liver, lung, and placenta; the organ distribution of 5a has been less studied because of the lower incidence of 5a+ subjects, but it is known to be present on spleen, kidney, and placenta (van Rood and Eernisse, 1968). Antigens 5a and 5b are both present on granulocytes and are usually detected by granulocyte leukoagglutination; 5b is present on lymphocytes (but in what relative concentration is not known) and has been detected by Rosenfield et al. (1967) on the red cell. van Rood and Eernisse (1968) examined tissue culture cells derived from a tumorous kidney from a 5a+, 5bf donor and found that 5b antigen persisted, whereas the 5a antigen was apparently lost. The 5b antigen was found by Bruning and his colleagues (Bruning et al., 1967) to be a heat-stable, trypsinand periodate-sensitive, phenol-resistant material, probably a polypeptide. The highly purified product, with high in oitro activity, was extracted from placenta. A second non-HL-A antigen ( a leukoagglutinogen) was designated 9a by van Rood (van Rood et al., 1965). Unfortunately, very few investigations have been made on this antigen, but van Rood has suggested it may have loose linkage with his antigen 7d (HLA-8) (van Rood et al., 1967).One antiserum capable of detecting 9a is known. Ceppellini studied a serum, BUF, which had very dissimilar properties in cytotoxicity and in agglutination tests (Ceppellini et al., 1965~). In cytotoxicity the reactivity closely resembled that of van Rood's anti-

HUMAN HISTOCOMPATIBILITY SYSTEMS

283

4a, and with this serum several “partagens” of 4a could be detected. (The different reactivities obtained with absorbed samples of BUF appear to parallel closely the class 1, 2, and 3 cells detected by our serum BC.) The agglutinin had a different population distribution from the cytotoxin and was later found to resemble a factor detected by Lalezari and Bernard‘s serum DeR (1965). The relevant antigen, now alternatively designated To1 by Ceppellini and NA1 by Lalezari, is apparently found only on granulocytes. It is only a transplantation antigen in the sense that it induces an antigranulocyte response and if formed during pregnancy, the antibody can cross the placenta to induce neonatal granulopenia ( Lalezari and Bernard, 1966a). It is noteworthy that antibodies to HL-A, although also 7 s immunoglobulins, rarely if ever cross the normal placenta, whereas anti-NAl can be found in cord blood. A second granulocyte antigen, NBl, reported by Lalezari, has been less intensively studied, its properties appear to be very similar to those of NA1 (Lalezari and Bernard, 196613). Zmijewski has tested sera from numerous multipara and has detected a battery of antigens which apparently lie outside the HL-A system. These have been roughly grouped into several interrelated systems by Zmijewski, e.g., E and H. No equivalents to many of the sera were detected by other investigators at the Turin workshop (Curtoni et al., 196%). All of the above antigens were detected by agglutination of leukocytes, platelets or red cells. They are apparently not detectable by cytotoxicity procedures. Shulman and his colleagues and van der Weerdt have carried out intensive investigations into the antigens of platelets. Several strong antigens can be detected by platelet complement fixation (Colombani et al., 1967). Some of these, e.g., KO and Zw, appear to be confined to platelets (van der Weerdt, 1965); other antigens, such as B1 of Shulman (Shulman et al., 1964),have been found on granulocytes and lymphocytes and have been found to coincide with certain HL-A specificities. VI.

Properties and Genetic Control of Histocompatibility Antigens

Kaliss, Day, and their colleagues attempted to extract the antigen responsible for the induction of antibodies capable of transferring immunological enhancement (Day et al., 1954; Kaliss and Day, 1954). They first obtained an aqueous extract with good biological activity and studied some of its physical and biochemical properties. The material was resistant to acetone precipitation and to enzymes such as amylase or hyaluro-

284

D. BERNARD AMOS

nidase. All activity was lost on filtration and the active principle readily adsorbed to any surface. In the light of later studies it seems unfortunate that their demonstration of solubilities in butenol and partial resistance to autolysis and treatment with papain were overlooked for several years. Kandutsch continued the studies begun by Day and his colleagues, again concentrating on a product that would induce immunological enhancement ( Kandutsch, 1957; Kandutsch and Reinert-Wench, 1957). Unfortunately, the establishment of enhancement was particularly time consuming. It takes several weeks from the time of immunization to the development of the enhanced state and perhaps as long as 2 more weeks before the existence of enhancement is indicated by continued growth of the tumor used in the test; thus up to 5 weeks was required before it could be shown that a particular fraction was active. Kandutsch and Stimpfling (1962) were able to obtain a highly concentrated product that could be “solubilized” as a micelle through the use of Tween. The antigenic product was later further refined by exposure to cobra venom (Kandutsch et al., 1%). Davies (1962), Haughton (1964), Manson (Manson et al., 1963), and others concurrently developed a variety of extraction procedures that yielded highly concentrated materials. The usual method of measuring antigenic activity in their studies was through inhibition of agglutination. In this rapid and simple test, product was mixed with appropriately diluted antibody (Amos et al., 1963b). If the product was active, the antibody would no longer agglutinate mouse red cells. The most active products usually existed in the form of a finely dispersed suspension rather than as a true solution and would inhibit at a concentration of approximately 2 pg. per milliliter. The greatest difficulty was experienced in freeing the preparation from lipid, and for some time it was thought that the active product was a lipoprotein (Kandutsch and Stimpfling, 1962). Rapid progress has been made following the introduction of proteolytic enzymes by Nathenson and Davies (1966). The original suspension of tumor, spleen, or tissue culture cells is sonicated or disrupted in a nitrogen bomb. The cellular, nuclear, and mitochondria1 debris is removed and a concentrate, often of the postmitochondrial fraction, or of an especially enriched plasma membrane preparation, is solubilized by autolysis or by exposure to papain, ficin, or other proteolytic enzymes (Nathenson and Shimada, 1968; Summerell and Davies, 1969). Manson and his colleagues (Manson et al., 1963) have used a lipoprotein concentrate as a starting material, whereas Kahan et al. (1967) have prepared a highly purified concentrate following sonication alone. The solubilized product is purified by column chromatography usually with

HUMAN HISTOCOMPATIBILITY SYSTEMS

285

diethylaminoethyl ( DEAE ) Sephadex and finally by discontinuous gel electrophoresis. The most highly purified preparations have an estimated molecular weight of less than 60,000 (Kahan and Reisfeld, 1969). Recent products contain virtually no lipid but do include amounts of carbohydrate estimated to run as high as 10%(Davies, 1968). Mann et aE. (1969) have reported finding two peaks on separation with Sephadex G-200 and polyacrylamide gel. Boyle finds at least three and possibly as many as five distinct fractions of HL-A differing in their antigenic activity ( 1969). Nathenson and Shimada ( 1968) reported the recovery of fractions of different molecular weights, while Davies et al. (1968) reported that most of the individual HL-A or H-2 specificities can be separated chromotographically on the basis of charge. Kahan and Reisfeld (1!369), using a different method of antigen separation, find only one band on discontinuous gel electrophoresis. There are two obvious possible explanations for the divergence in findings between the investigators obtaining single fractions and those obtaining two or more fractions. Davies, Nathenson, and Boyle may all have artificially split certain polypeptide residues from a higher molecular weight material, or Kahan and Reisfeld may have isolated an antigenic product which is not analogous to HL-A. The latter appears to be somewhat more likely. ( I ) The molecular weights obtained by the investigators reporting several fractions are roughly comparable (50,000-70,oOO)to those reported by Kahan, so the mouse antigenic products cannot be subfractions of a material analogous to the guinea pig antigen. ( 2 ) Antigens belonging to minor loci probably differ in their properties from those belonging to major systems. There is little direct evidence relating to this since few investigators have studied the chemistry of minor antigens. The only human non-HL-A antigen studied in detail is van Rood's 5b antigen studied by Bruning et a2. (1964). This material was readily solubilized from placenta (not a particularly good source for HL-A nor H-2), and the chemical and physical properties of the 5b product were very different from those of HL-A or H-2. In the mouse, H-6a could be readily separated from H-2 by Lilly and Nathenson (1968) and, although not fully characterized, H-6 appears to have a larger molecule with more carbohydrate than H-2. If analogies with red cell antigens have any value, the properties of ABO antigens appear to differ from those of other systems such as Rh. On balance, it appears quite possible that the specificities of the major systems can be separated into several polypeptide fractions, each having a slightly different charge but of similar molecular weight, This is consistent with the concept that the genetic determinant is compound.

286

D. BERNARD AMOS

Again, taking an analogy, the IgG molecule is thought to be the product of a complex of genes that have accumulated following repetitive unequal crossing over. Four such genes have been thought to control the heavy chain and two more the light chain. The genetic determinants for antigenic systems such as H-2 or HL-A could have arisen in the same manner. If so, recombination between the various subunits might be expected. Crossing over has been fully documented repeatedly in mice (Stimpfling and Richardson, 1965); nearly 20 possible examples of crossing over in HL-A have been recorded (Batchelor and Chapman, 1967; Terasaki, 1968; Ward et al., 1969). Although none of HL-A crossovers has been fully substantiated, at least two appear to be highly probable, since, in each, one group of antigens was gained while other specificities were lost from the haplotype (Ward et al., 1969). Barring errors in the determination-unlikely since the genotyping of the remainder of the family was unexceptional and the determinations were repeated several times-crossing over or mutation are the only likely causes. Both would be most easily explained if several cistrons, arranged like beads on a string, were responsibIe for the control of the individuaI specificities. Two other fragments of information may have some relevance to a consideration of the genetic control and the product synthesized: the first relates to “antigenic modulation,” and the second to a consideration of the specificity of the antibodies produced. Old and Boyse have reported modulation of antigens of the TL system (Boyse et al., 1967). The gene for TL is closely linked to H-2. The TL is not a transplantation antigen because when the cell meets antibody to TL, the antigen promptly disappears (is modulated) and does not reappear until there is no antibody in the environment. Boyse and Old found that the amount of H-2.4 apparently increased during modulation, but they were unable to modulate H-2 antigens directly. Recently, Bjaring and Klein have reported modulation of H-2.3 and H-2.5 (19S8), and Cohen and Amos have observed apparent modulation of several specificities of the H-2k allele. In agreement with Old and Boyse, Bjaring and Klein found no modulation of H-2.4 or H-2.11; this suggests that there may be differences in the chemical nature of the antigenic determinant. Antigens H-2.3 and H-2.5 might, for instance, be carbohydrate groupings attached to a polypeptide, H-2.4 and H-2.11 being components of the polypeptide chain. It is perhaps relevant that Ozer and Wallach (1967) reported differential solution of H.2.5 which Bjaring and Klein found to be modulated. It is particularly interesting that although H-2.4 or H-2.11 could be deleted, no examples could be found in which all H-2 antigenic activity was lost. This suggests heterogeneity in the chemical nature of the various ligands. The argument deriving from considerations of specificity of reactivity is

HUMAN HISTOCOMPATIBILITY SYSTEMS

287

a little more indirect. The author has been impressed by the consistency with which different antibodies against certain specificities such as HLA-1, HLA-2, and HLA-7 react with cells from the same panel members, whereas different antibodies thought to be directed against HLA-5 or 4a may, although resembling each other closely, consistently show points of difference. DifTerences in behavior of this nature might be expected if certain specificities, such as HLA-2, were carbohydrate in nature and others were more complex proteins. Genetic determination would be through the polypeptide sequence, but, although some of the specificities would be given directly by the amino acid backbone, others would be given by polysaccharide side chains. The addition of the polysaccharide ligand would be controlled by the availability of transferases already present in the cell. If this hypothesis is correct, mutants should occur in which the carbohydrate Specificities were absent or diminished in some but not necessarily all tissues. Again, H-2.5 may fit into this category since it is present in the genotype H-2b, is found in normal concentration in most tissues, but appears to be present in only trace amounts on the C57BL (H-2b) red cell (Amos et d.,1955). Harris and Zervas (1969) have reported that HL-A antigens are present on the reticulocyte though not on the mature red cell. Although some of the antigens appear to be present in amounts approaching those found on the lymphocyte, other HL-A specificities are apparently present in only trace amount. To what extent this variation is individual specific and to what extent it is antigen specific is undetermined. Williams (unpublished data) finds the antigenic representation of antigens to be more consistent on cells from kidney (biopsy fragments or tissue cultured cells) by immune adherence tests than on lymphocyte; thus, although still incompletely documented, it appears that there is considerable variation in the expression of the antigens on different cell types and individuals. Studies on the nature of the genetic determinants and on the structure of the antigenic product are closely interrelated. A comparison of the amino acid sequences of two antigenically distinct fractions from an inbred strain such as those isolated by Davies would be most instructive. One would expect certain homologous sequences, interrupted by variable portions. Weaver and Boyle (1969) have already found that xenoantibodies react with each of their subfractions, suggesting considerable homology between them. Information relating to the antigenic codguration of the purified fractions could be directly applied to provide an understanding of the genetic determinant; it will be most instructive when antigen derived from a true recombinant is compared with antigen from siblings carrying the corresponding normal haplotypes. In the meantime, the question as to how many genes are involved is being debated.

288

D. BERSARD AMOS

Dausset (Dausset et al., 1969), Svejgaard and Kissmeyer-Nielsen ( 1968), and Singal (Singal et al., 1969), for example, believe that two or three subloci control most of the specificities. According to this hypothesis, specificities LA1, 2, 3, 4, and 5 are members of an allelic series comprising the first sublocus. The hypothesis is founded upon the observations of several investigators that lymphocytes from the majority of subjects appear to carry only two of the LA specificities; for example, LA1 and LA3, LA2 and LA4. Further, only one specificity appears to be controlled by a single haplotype. However, cells possessing three of the LA series of antigens have been described on several occasions, and we have recently tested cells from a donor who appeared to possess all four. This could possibly be explained on the basis of gene duplication, but the exceptions do constitute a serious objection to the simplistic concept of a single sublocus controlling several specificities, and two of the recombinants studied by us appear to divide specificities controlled by this postulated sublocus (Ward et al., 1969). The sublocus concept appears to represent a statistical association rather than a genetic unit, and reference to the first sublocus may be similar to referring to the “D e n d of the H-2 region. It may be a useful simplification for purposes of descriptions of the gross antigenic constitution but should not detract attention from alternative possibilities. Each sublocus is probably as complex as the whole Rh system. Although in their chemistry and in the genetic control of the specificities, H-2 and HL-A appear to be very similar, certain known features of the mouse H-2 system have not been explored with respect to HL-A. Antigen H-2 appears to be in a chromosomal segment which is generally highly active. Close to the H-2 determinants are the genes for the TL series of antigens (Boyse et al., 1967), and associated with H-2 is resistance of susceptibility to the Gross leukemia virus and possibly the factors associated with hybrid resistance (Lilly, 1966; Cudkowicz and Stimpfling, 1964). Within the H-2 region are the genes regulating the Ss series of determinants (Shreffler, 1965) and close to H-2 is a determinant that controls the ability to respond to polypeptide antigens (McDevitt and Tyan, 1968). Possibly other chromosomal regions are equally active, but the flurry of genetic activity concentrated in the same segment of chromosomes as the H-2 complex at present appears to be very remarkable. VII.

Possible Future Developments

The immediate prospect is of continued slow but assured progress toward a better definition of the antigenic specificities. This is an essential prerequisite for other studies. As the antigens become better defined, the biochemist can, in turn, adequately characterize his products to know for

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certain which specificities are found on a given molecule. He can then hope to equate certain groupings with a particular activity. The purified products can also be used for the preparation of more specific antisera. Concomitant with this development will be studies on the actual synthetic pathways. The function of the antigens must be understood before intelligent attempts can be made to control their synthesis. If this antigen molecule is an essential structural component of the cell or if it has an essential part in cell function, then modulation or other means of antigen suppression may be fruitless. If the association with the cell membrane is coincidental and the function of the antigenic molecule has already been achieved, control of antigenicity becomes an intriguing possibility. Questions still to be answered include the following: Why are HL-A antigens on the lymphocyte or the macrophage so potent at inducing lymphocute transformation when the same antigen on a heat-killed or freeze-killed lymphocyte is ineffective? What determines the conditions for immunological enhancement? What is the relationship between tolerance and enhancement? How can tolerance to tissue transplants be induced by soluble extracts? Such questions, together with attempts at the elucidation of the genetic determinant, characterization of intermediate steps in antigen formation and of the structural relationship of the antigen to the membranes of the cell, and an understanding of the utility of polymorphism to the fitness of a species, are problems which will be intensively investigated but which are unlikely to be fully resolved for several years. VIII.

Appendix: Techniques for the Detection of HL-A Specificities

Relatively few of the many techniques available have gained widespread acceptance. Variant methods for determining leukoagglutination and cytotoxicity are used in almost all laboratories. Complement fixation, antiglobulin consumption, mixed agglutination, and immune adherence are used on occasion; platelet agglutination and inhibition of clot retraction have mainly historical interest. A. LEIJKOAGGLUTINATION

There are two major variants, depending upon the initial treatment of the blood. Dausset (1965),Payne ( 1965), Zmijewski (Zmijewski et al. 1967b), and others (e.g., Svejgaard et al., 1967) have used defibrinated blood. The blood is usually collected into a small Ehrlenmeyer flask containing up to ten glass beads and a magnetic stirrer bar. The flask is gently agitated, or the bar is slowly rotated for 15 minutes. A sedimenting agent, such as gelatin, dextran, or polyvinyl pyrrolidone, is added and the plasma containing the leukocytes is collected. The leukocytes may be

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washed, suspended to the desired concentration, and then added to the antibody, In semimicromethods, one or two drops of cell suspension and serum are incubated together in small tubes, sometimes in the presence of added complement and with other additives such as phenol. Acetic acid is commonly added to lyse red cells which may otherwise interfere with the microscopic reading. In the micromethods (Payne, 1968; Zmijewski et al., 1967b), 5pl. or less of the reagents are injected under a film of oil in a small dish, the dish is rotated for a few minutes, acetic acid is added, and the test is read microscopically. Dausset ( 1965),van Rood and van Leeuwen ( 1965), and others have used blood collected into ethylenediaminetetraacetate ( EDTA) The concentration and pH of the EDTA are critical. Sedimentation is carried out as with defibrinated blood; in one variant of the test, plasma is centrifuged and the cell button resuspended to allow resedimentation of any residual red cells from small volume. Platelets may be removed by differential centrifugation through a gradient (Amos and Peacocke, 1963). The concentration of the cell suspension is adjusted and the cells are incubated with serum in siliconized tubes. In van Rood’s procedure, acetic acid is added before microscopic reading on a special carrier; in our variant acetic acid is not needed. One of the most critical requirements is cleanliness-lint or dirt causes false agglutination. The EDTA and defibrinated techniques do not give equivalent results (Svejgaard et al., 1967). Both can be highly effective but sera have to be selected to suit the procedure used. Although labeled “agglutination,” the reaction is much more complex than hemagglutination (Zmijewski, 1965). Pure suspensions of lymphocytes are agglutinated with some difficulty and the mobility of granulocytes may be important (Payne, 1963). Antigenically negative cells are included in the clumps, but the basis is not the same as that of immune adherence since complement is not necessary (Zmijewski, 1965). The procedure of Lalezari differs sharply from the preceding methods (1965). The cells are prepared following red cell sedimentation by selected batches of Polybrene. The agglutination reaction is very slow but extremely intensive. The results are not easily comparable with EDTA or defibrination methods; this procedure has been used to define the NA series of antigranulocyte antibodies.

.

B. LEUKOCYTOTOXICITY The cytotoxicity test relies upon alterations in permeability induced by the action of complement on lymphocytes which have been exposed to

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antibody. Blood is defibrinated or collected into heparin. Red cells are removed by sedimentation, granulocytes are allowed to attach to nylon, glass, or cotton, and a purified suspension of lymphocytes is mixed with antibody. Complement is added immediately or after a preincubation period, after which excess antibody may be removed. Several different indicator systems are used. The original technique relied upon the ability of intact lymphocytes to exclude dyes such as eosin, Nigrosine, or trypan blue (Gorer and OGorman, 1956). Dead cells take up the dye. Dead cells also lose refractibility when examined under phase contrast microscopy (Mittal et al., 1968). A different indicator system introduced by Rottman and Papermaster (1966) has been used by Bodmer and others (Bodmer et al., 1967). Fluorescein diacetate is nonpolarized and will enter intact cells freely. Within the live cell, esterases split off the acetate ions and the now polarized fluorescein cannot leave the cell, so undamaged cells fluoresce. The dye can pass out from damaged cells; therefore, dead cells do not fluoresce but can be clearly seen under darkground illumination. The most quantitative indicator is provided by jlCr ( Sanderson, 1965). The original lymphocyte suspension is exposed to radioactive chromium, washed extensively, and then treated with antibody and complement. Chromium released into the supernatant is measured in an auto gamma counter. Unlike agglutination procedures, the various techniques give comparable results, differing mainly in their sensitivity. Interestingly, although many antibodies are cytotoxic in the presence of rabbit complement, relatively few are fully complemented by human serum. Mixed agglutination procedures are used extensively for the detection of antigen on cells grown in tissue culture (Metzgar and Flanagan, 1965; Milgrom and Kano, 1965). The cell monolayer is washed and exposed to antibody. Excess antibody is carefully washed off and a specially sensitized suspension of red cells is added. Group 0 Rhf antibodies are treated with a suitable anti-D and washed. The monolayer is treated with dilute antiglobulin and the indicator cells added. If antigen is present on the monolayer, the indicator cells adhere. Titers reached are often considerably higher than those reached by the same serum in agglutination, but the pattern of reactivity is comparable to the EDTA techniques. A variant recently developed in our laboratory is applicable to cells in suspension. A suspension of purifled lymphocytes is incubated with the alloantibody and washed. Chimpanzee antihuman serum at high dilution is then added to these cells. The Rhf cells sensitized with anti-D are added to the washed suspension. Then the cell mixture is allowed to sediment for 1 hour, the tubes are inverted twice, and the suspension ex-

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amined microscopically. Rosettes are formed when the alloantigen has combined with the lymphocyte. The procedure was developed specifically for the separation of cells from mosaics.

FIXATION C. PLATELETCOMPLEMENT Shulman et al. ( 1!364),van der Weerdt (1965),and Colombani et al. ( 1967) have elaborated this technique. Five microliters of diluted antisera are mixed with an equal volume of platelets, log per milliliter, and a known concentration (two 100%hemolytic units) of guinea pig complement. An equal volume of a dilute suspension of sensitized sheep red blood cells in veronal b d e r is added, the tubes are incubated at 37°C. for 30 minutes, centrifuged, and the degree of hemolysis determined. Only a limited number of sera are suitable for use in this system, but those sera often have a very high titer and give clear-cut results.

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HUMAN J3ISTOCOMPATIBILITY SYSTEMS

297

van Rood, J. J., van Leeuwen, A., Schippers, A. M. J., Pearce, R., van Blankenstein, M., and Voklers, W. ( 1967). In “Histocompatibility Testing-1967’’ ( E . S. Curtoni, P. L. Mattiuz, and R. M. Tosi, eds.), pp. 203-218. Munksgaard, Copenhagen. van Rood, J. J., van Leeuwen, A,, Pearce, R., and van der Does, J. A. (1969). Transplantation Proc. 1, 372. Visetti, M., Scudeller, G., Leigheb, G . , and Ceppellini, R. (1967). Mineroa Dennatol. 42, 563. Vredevoe, D. L., Mickey, M. R., Goyette, D. R., Magnuson, N. S . , and Terasaki, P. I. (1966). Ann. N.Y. A c d . Sci. 129, 521. Walford, R. L. (1980).“Leukocyte Antigens and Antibodies.” Grune & Stratton, New York. Walford, R. L., Shanbrom, E., Troup, G. M., Zeller, E., and Ackerman, B. (1967). In “Histocompatibility Testing-1967” (E. S . Curtoni, P. L. Mattiuz, and R. M. Tosi, eds. ), pp. 222-229. Munksgaard, Copenhagen. Ward, F. E., Southworth, J. G., and Amos, D. B. (1969). Transplantation Proc. 1, 352. Weaver, R. A., and Boyle, W. (1969). Biochim. Biophys. Acta 173, 377. Wichels, P., and Lampe, W. (1928). Klin. Wochnrchr. 7, 1741. Zmijewski, C. M. (1965). In “Histocompatibility Testing-1965” (H. Balner et al., eds.), p. 193. Munksgaard, Copenhagen. Zmijewski, C. M., and Amos, D. B. (1966). Vox Sang. 11,377. Zmijewski, C. M., Zmijewski, H. E., and Huneycutt, H. C. (1967a). Intern. Arch. Allergy 32, 574. Zmijewski, C. M., St. Pierre, R., Fletcher, J., Wilson, S . F., Cannady, W., and (E. S. Zmijewski, H. E. ( 1967b). In “Histocompatibility Testing-1987” Curtoni, P. L. Mattiuz, and R. M. Tosi, eds. ), p. 389. Munksgaard, Copenhagen.

AUTHOR INDEX Numbers in parentheses are reference numbers and indicate that an author’s work

is referred to, although his name is not cited in the text. Numbers in italics show the page on which the complete reference is listed.

Amos, D. B., 252, 253, 254, 255, 259, A 262, 263, 264, 266, 267, 271, 272, Aalund, O., 86, 97,102 273, 277, 278, 284, 286, 287, 288, Abel, C. A,, 58, 61, 77, 78, 80, 97, 100 290, 292, 295, 296, 297 Abelous, J-E., 167( l), 206 Amouch, P., 59, 98 Abernethy, w‘, 178(620)>189(620)> Anderson, E, E,, 277, 278: 2g6 224 Andersson, L., 186(472), 220 Abilgaard, C. F., 199(113), 210 Andrews, E. C., Jr., 195(372), 217 Ablondi, F. B., 163(2), 206 Angevine, D. M., 138(lo), 207 Achong, B., 246, 248 Anggard, E., 108, 109, 111, 114, 141 Ackeman, B., 259, 297 Anken, M., 235, 249 Action, R. T., 55, 99 Ankeney, J. L., 157(398), 218 Adas G . Lv 13(58), 36( 150), 47, 49 Aderst, J., 232, 250 Adair, N. E., 9( 39), 46 Anselmi, B., 169(616), 186(616), 224 Adkinson, N. F., 191(3), 207 Antopol, W., 201( 103), 203( 100, 103), Adler, S., 258, 293 207, 209 Ahern, J. J., 205( 158), 211 Aoki, T., 237, 246, 247, 247, 250 Ahmad-Zadeh, C., 235, 249 Apitz, K., 198(11, 12), 207 Alagille, D., 160(4), 207 Appel, W., 161(400), 218 Albertini, R. J., 252, 277, 292 AppeUa, E., 84, 92, 93, 94, 97, 103 Alcock, D. M., %,97 Arase, M., 165(49), 208 Alepa, F. P., 82, 97 Arbesman, C. E., 176(138a), 210 Alescio-Zonta, L., 76, 97 Archer, G. T., 127,141 Alexander, B, 157(5), 207 Armstrong, D., 166( 13), 168(17), 169 Alford, T. C., 235, 249 (14, 15, 16, 17), 170(13), 176(13, Alkjaersig, N., 160(8, 192, 320), 162 15), 178(15), 207 (7, 6051, 163(6, 607, 608), 168 (606), 171(606), 207, 212, 215, Armstrong’ 252’ 292 Arnason, B. G., 121, 122, 123, 141, 193 223 (241, 207 Allard, C.,196(551), 222 Allen, F. H., 263, 264, 266, 272, 273, Aronson7 A* I*’283’ 294 Aronson, M . , 191(561), 222 292 Arthus, M., 205( 181, 207 Allen, J. C., 76, 101 Asherson, G. L., 20( 100, 1011, 48 Allen, J. G., 199( 35),207 Askonas, B. A., 92,97 Allen, S . L., 2.53, 292 Asofsky, R. M., 90, 94, 101 Allison, A. C., 239, 247 Astrin, K. H., 94,99 Almodovar, L. R., 53,98 Alpers, H. S., 186(612), 187(611, Astrup9 T*p162(19)’ 207 Attardi, G., 15(82, 83), 47, 48 612), 224 Altura, B . M., 197(9),207 Attleberger, M. H., 66, 99 Ambache, N., 107, 114, 115, 141 Auditore, J. V., 148(242), 213 Ames, S. B., 157(512), 221 Auserwald, W., 163( 1381, 210 299

300

AUTHOR INDEX

Austen, K. F., 7(22), 12(22), 46, 85, Basinger, C., 199(35), 207 98, 107, 109, 111, 112, 113, 115, Batchelor, J. R., 259, 263, 264, 266, 271, 272, 273, 286, 292, 293 116, 117, 118, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 132, Battisto, J. R., 38(167, 168), 50 134, 135, 136, 137, 138, 139, 140, Bauer, D. C., 62, 98 141, 142, 143, 144, 179(647), 183 Bauer, E., 167(311), 215 (20), 184(26, 27), 190(22), 192 Baumann, J. B., 31(133), 32(134), 49 (201, 193(21, 23, 24, W), 194 Baumgartner, H. R., 149(36), 207 (21, 483a), 207, 220, 225 Bauminger, S., 14(71), 15(71), 39 Austin, C. M.,13(58), 47 (174), 47, 50 Axelrad, A. A,, 240, 247 Bayer, W. H., 179(351), 216 Axelrod, A. E., 152(399), 218 Bayler, T., 172(37), 207 Beck, E. A., 150(39), 151(38), 196 B (341), 197(341), 199(341), 201 Bach, F. H., 252, 254, 277, 292 (341), 208, 216 Bachrnann, R., 80,99 Becker, E. L., 86, 204, 121, 123, 138, Back, N., 160(28), 172(28), 176( 138a), 139, 141, 142, 168(286), 173(45, 194(29), 195(29), 207, 210 287), 174(286, 287, 366), 180(40, Baglioni, C., 76, 97 41), 181(42), 182(286), 183(318), Bailey, K., 149(30), 207 184(286, 319), 187(44), 190(22), Bailey, W. L., 196(124), 198(124, 125), 191(43, 44), 193(23), 194(284), 210

Bain, B., 252, 292 Baker, A. R., 121, 123, 140, 141, 193 (241), 207 Baldwin, D. S., 205(365), 217 Ballieux, R. E., 13(56), 47, 76, 77, 97 Balner, H., 282, 296 Bang, B. G., 54, 97 Bang, F. B., 52, 54, 97 Bang, N. U., 160(31), 207 Banovitz, J., 69, 72, 85, 98 Barclay, M., 111, 143 Barclay, W. R., 205( 158), 211 Bardier, E., 167( l ) , 206 Barge, A., 259, 273, 2.81, 282, 294, 29.5 Bad, E. F., 66,98 Barnhart, M. I., 151(32, 553), 160(32, 33), 179( 32), 207,222 Baronofsky, I. D., 195(510), 221 Barr, Y.,246, 248 Barry, C. B., 90,103 Barski, G., 244, 247 Bartel, A. H., 66, 98, 101 Barth, R. F., 232,247 Barth, W. F., 20( 105), 48, 85, 86, 99, 102

Bartosch, R., 193(34), 207 Basch, R. S., 252, 293

207, 208, 214, 215, 217

Becker, R. M., 202(46), 208 Bedler, F. K., 205(435), 219 Beeson, P. B., 202(47), 208 Beller, F. K., 200(48), 201(48), 208 Benacerraf, B., 10(41), 11(51), 12(51), 13(57), 14(70, 75), 15(70), 17 (91, 94), 18( 91), 19(94), 20( 103), 21(41), 22(41), 25(109), 26(91, 109, 110, 111, 112, 113), 27(109, 113, 116), 28(116), 29(120), 35 (116, 146), 36( 151), 37( 151, 165), 38(170), 39(146, 176, 178), 41 (41, 91), 46, 47, 48, 49, SO, 85, 86, 94, 98, 102, 121, 137, 141, 143, 144, 205( 66), 206( log), 208, 210, 240,250

Benacerraf, R. A., 102 Benditt, E. P., 165(49, 576), 208, 223 Benedict, A. A,, 68, 69, 70, 71, 74, 98, 99, 100

Benjamin, A,, 283, 292, 293 Benjamin, T. L., 234, 247 Bennett, C., 84, 103 Bennich, H., 80, 81, 98, 101 Benson, S., 59, 103 Bent, V. D., 282, 293

AUTHOR INDEX

Beraldo, W. T., 168(562), 170(562), 173(50), 194(50), 208, 222 Berek, U., 167(714), 169(714), 227 Bergstrom, K., 160( 695), 226 Bergstrom, S., 107, 114, 141 Berken, A., 200(51), 208 Bernard, G. E., 283, 295 Bernard, H., 5 ( 7 ) , 45 Bernheimer, A. W.,54, 98 Bernhisel, H., 87, 101 Bernier, G. M., 13(52, 53, 56), 47, 76, 97 Berquist, U., 108, 109, 111, 141 Berry, P. A., 110, 113, 116, 117, 141, 194(52), 208 Bertelli, A., 195(53), 203(53), 204 (54), 206( 53), 208 Besredka, A., 188(55), 208 Bettex-Galland, M., 148(56, 5 7 ) , 186 (58), 187(58), 201(58), 208 Bettleheim, F. R., 149(307), 207 Bhatia, H. M., 53, 98 Bhoola, K. D., 169(59), 208 Bialek, J., 2-58, 293 Biedl, U., 195( 60), 208 Bier, O., 191( e l ) , 208 Biggs, P. M., 246, 248 Biggs, R., 147(63), 160(62, 376), 208, 217 Bill, A. H., Jr., 247, 249 Billote, J. B., 20(97), 48 Binaghi, R. A., 85, 86, 98, 102, 121, 122, 123, 141, 143, 144 Biozzi, G., 14( 72), 15(72), 47 Birch, S. M., 246, 248 Bishop, E. A., 169(396), 170(64), 208, 217 Bishop, J. M., 169(65), 208 Biskis, B. O., 247, 248 Bjaring, B., 286, 293 Bjorkman, S. E., 186(472), 220 Black, P. H., 233, 235, 247 Blair, A. M . J. N., 117, 139, 143 Blair, P. B., 240, 249 Bloch, K. J., 20(103), 48, 85, 86, 98. 102, 113, 120, 121, 122, 123, 124, 125, 126, 134, 135, 136, 137, 138, 140, 141, 143, 144, 193(24), 205 (66), 207, 208

301

Bloch-Shtacher, N., 17(go), 48 Block, J. H., 196(354), 216 Blomback, B., 149(68, 69), 157(67), 171( 136), 172(136), 208, 210 Blomback, M., 149(69), 208 Bloom, W., 165( 70), 208 Blum, L., 180(500), 221 Boake, W. C., 175(71, 361), 192(71), 217 Bodmer, J., 258, 259, 291, 293, 295 Bodmer, W . F., 258, 259, 263, 264, 266, 272, 273, 291, 292, 293, 295 Bodnar, S. R., 172(222), 213 Boffa, G. A., 59, 98 Bohle, A., 199( 305), 215 Boissonnas, R. A., 168(72), 208 Bone, A. D., 148( 710), 226 Boor, A. K., 9(34, 35), 46 Booth, B., 66,99 Bordet, J., 153(74), 188(73), 209 Borel, Y., 20(99), 48, 205(489), 206 (489), 220 Borelli, J., 147( 736, 737), 148(735), 149(735), 227 Boreus, L. O., 126, 142 Born, G. V . R., 148(75), 149(36), 207, 209

Botti, R. E., 157(398), 159(77), 171 (77), 198(529), 200(76), 209, 218, 222 Botts, J., l61(652), 225 Bounameaux, Y., 147(78), 209 Bouthiller, Y., 14(72), 15(72), 47 Boyd, W. C., 5 ( 7 ) , 9(33, 36), 45, 46, 53, 98 Boyer, M. H., 151( 194), 212 Boyer, S. H., 83, 98 Boyle, W., 287, 293, 297 Boyles, P. W., l 6 l ( 205), 212 Boyse, E. A., 237, 242, 246, 247, 247, 248, 249, 250, 286, 288, 293 Bozicevich, J., 194(691, 692), 195(691), 226 Bradley, S. G., 86, 101 Brahmi, Z., 13(59), 47 Brannen, W. T., Jr., 152(359), 216 Braun, W. E., 203(79), 209 Brauns, G., 90, 103 Brdicka, R., 254, 296

302

AUTHOR INDEX

Breckenridge, R. T., 152(81), 153(503, 5051, 157( 80), 177(292), 178(292, 293), 209, 215, 221 Brent, L., 35(140), 49, 252, 293 Brenzl, B., 254, 296 Brettschneider, L., 203( 640), 225 Bridges, R. A,, 59, 95, 103 Briggs, J. D., 54,98 Brinkhous, K. M., 160(694), 226 Brittan, R-S.,281, 296 Brittingham, T. E., 271, 293 Brocklehurst, W. E., 106, 107, 108, 109, 110, 113, 114, 116, 117, 118, 119, 137, 138, 139, 140, 141, 142, 166 (83), 193(25), 194(82, 84,85,86), 207, 209 Brodie, B. B., 148(245, 264, 613), 213, 214, 224 Brody, G. L., 203( 87), 209 Brody, N. I., 33(136), 49 Bronfenbrenner, J., 191(88), 192(88), 209 Brooke, M. S., 36( 155), 49 Brown, R., 8(24), 46, 53, 98 Brown, R. J., 68, 69, 98 Brown, W. E., 114, 143 Bruce, S., 168(397), 218 Brunfeldt, K., 161(89), 209 Bruning, J. E., 262, 282, 285, 293 Brunson, J. G., 198(91), 200(90, 660), 209, 225 Bryan, E., 231,250 Bryant, B. F., 283,294 Bryant, R. E., M , 9 9 Buckley, C. E., 271, 295 Budtz-Olsen, 0. E., 147(92), 209 Budzyriski, A. Z., 160(310), 215 Buffet, R. F., 244,249 Buhrmester, C. C., 194(713), 226 Bullman, H. N.,%( 115), 48 Burdon, K., 185( 93), 209 Burhoe, S. O., 254, 293 Burnet, F. M., 2(1, 2, 3), 12(1), 37 (1, 21, 42(L 2, 31, 45 Bums, J. W., 200( ma),213 Burrowes, C. E., 128,143 Busse, R. J., Jr., 154(530), 222 Butel, J. S.,234,250 Byers, P. H., 178(457), 220

C Calaprice, N. L., 53, 99 Calcott, M. A., 179(447), 180(448), 219 Caldwell, M. J., 152(596), 223 Calle, J. D., 169(59), 208 Calvanico, N., 78, 104 Cannady, w., 290,297 Cantacuzhe, J., 53,98 Capps, W. I., 237, 240, 248, 349 Carbonara, A., 76,97 Carbone, P. P., 80, 99 Carlson, L. A., 114, 141 Carney, P. G., 232,239,249 Carozza, F. A., Jr., 197(94), 209 Carp, R. I., 234,235,248 Carpenter, C. B., 127, 142 Carrara, M. C., 168(219), 173( 219), 178(219), 213 Carroll, W. R., 160(387), 217 Casey, M. J., 235, 248 Caspary, E. A., 187( 95), 209 Castania, A., 171(572), 198( 572), 223 Cebra, J, J., 13(52, 53, 54), 47, 78, 84, 92, 98, 101 Ceder, E. T., 148(734), 227 Ceglowski, W. S., 240,248 Celander, D. R., 162(96), 209 Centis, D., 268, 294 Ceppellini, R., 263, 264, 266, 272, 273, 277, 279, 281, 292, 293, 297 Cerottini, J. C., 235, 249 Chahovitch, X., 53, 98 Chain, E., 166( 156), 211 Chakravarty, N., 107, 108, 109, 111, 126, 142, 194(97), 209 Chan, K. E., 157(398, 550), 218, 222 Chang, W. W. Y., 86,102 Chanock, R. M., 235,248 Chapman, B. A., 286,293 Chapman, L. F., 167(99), 176(98), 209 Chase, M. W., 35(141), 38(167, 165) 49, 50 Chatelanat, F., 151( 618 ), 224 Chiappino, G., 13(55), 14(61), 47 Ching, Y.-C., 65, 98 Christensen, H. E., 90, 98, 103

303

AUTHOR 1M)EX

Christensen, L. R., 159( 102), 161( 100, 101, 102), 209 Christian, C. L., 125,142 Christie, G. H., 37( 164), 50 Chryssanthou, C., 201( 103), 203( 10A, 103), 207, 209 Churchill, A. E., 246, 248 Cinader, B., 39( 177), 50 Cioli, D., 76, 97 Cirstea, M., 111, 112, 142 Citron, J., 187(626), 224 Clamp, J. R., 78, 101 Clark, M. C., 129, 142 Clarke, D. A,, 240, 250 Clarke, F. H . J., 87, 104 Clarke, N., 152(104), 209 Clausen, J., 90,98, 103 Clem, L. W., 61, 62, 83, 64, 65, 68, 79, 98, 103 Clemente, P., l 6 l ( 400), 218 Clements, J . A., 172(37), 207 Clifford, P., 246, 249 Cline, M. J., 167(406), 172(406), 173 (407), 178(406), 218 Cluff, L. E., 203( 342),216 Cochios, F., 158(132), 210 Cochrane, C. G., 128, 135, 142, 143, 190 (107), 204( 106), 205( 105A), 206 (105),209 Code, C. F., 148( 108), 209 Coe, J. E., 20(104), 38(167, 168), 48, 50, 85, 98 Cohen, C., 255, 293 Cohen, E., 53, 98 Cohen, I., 259, 273, 292 Cohen, S., 70, 74, 97, 99, 206( l w ) , 210 Cohn, M., 3(3d), 15(82, 83), 4S, 47, 48, 75, 93, 97, 99, 101, 103 C o b , Z.A., 128,142 Colberg, E., 13(54), 47 Collier, E. M., 283, 292, 296 Collier, H. 0. J., 110, 113, 115, 116, 117, 119, 141, 142, 143, 169(110), 194(52), 208, 210 Collins, R. D., 200( 111, 555, 622), 210, 222, 224 Colombani, A., 281, 282, 293

Colombani, J., 259, 261, 264, 265, 273, 279, 283, 285, 288, 292, 293, 294 Colombani, M., 285, 283, 292, 293, 294 Colopy, J. E., 154(531), 222 Comaish, J-S., 187(95), 209 Combridge, B. S., 162(377), 172(378), 217 Condie, R. M., 57, 59, 60,62, 102, 201 (112), 210 Conley, C. L., 147(243), 148(436), 199(532), 213,219, 222 Connor, W. E., 154(256), 214 Converse, J. M., 281, 282, 295 Conway, H., 204(207), 212 Coombs, R. R. A., 79, 101 Coons, A. H., 23( 108), 48 Cooper, N., 186(419), 187(419), 218 Cordes, S., 69, 100 Cornelius, A., 240, 250 Conigan, J. J., Jr., 199(113), 210 Costea, N., 80,104 Cotran, B. S., la(114), 210 Cowan, K. M., 180(344), 216 Cowles, R. B., 66, 99 Cox, J. S. G., 135, 142 Craft, M. K., 169(465), 220 Craig, L. E., 75, 100, 151(208), 179 (208), 198(368), 205(208), 206 (208), 212, 217 Crain, J . D., 160(274), 214 Creech, O., Jr., 281, 295 Crigler, C. W., 148(734), 227 Crosmier, J., 203( 236), 213 CrowIe, A. J., 20(96), 48 Crum, J. D., 155(533), 222 Cuddigan, B. J., 147(115), 210 Cudkowicz, G., 288, 293 Cunningham, B. A., 84,103 Cunningham, R. W., 129, 142 Curragh, E. F., 152(170), 211 Curry, J. J., 117, 142 Curtoni, E. S., 263, 271, 277, 279, 281, 282, 283,293, 294 Curtoni, M., 282, 293 Cushing, J. E., 53, 99

D Dale, 11. H., 106, 142, 192( 116), 193 (117), 210

304

AUTHOR INDEX

DaImasso, A. P., 180(448), 219 Damgaard, E.,192(680), 226 Danaraj, T.J., 128,142 Dastre, A., 159(118),210 Dausset, J., 256, 259, 261, 263, 264, 265,266,272,273, 279,281, 282,

de Torres, R. A., 235,249 Deutsch, H.F., 69, 70,104 de Vaux St.-Cyr, C., 122,141 deVries, A., 154(600), 223 Dewald, H.A., 169(465), 220 DeWitt, C . W., 281,295 283,285,288,290,292, 293,294, Deykin, D., 158(132),210 295 Diamandopoulos, G. Th., 241, 248 Davenport, F. M., 33(136a), 49 Dias da SiIva, W., 135,142, 163(133), Davidson, C . S., 160(El), 225 167(294), 168(15O), 171( 136), Davidson, E., l60(443), 201(443), 219 172(136), 178(294), l85( 150), Davie, E. W.,147(119), 154(535), 189(133,134,135), 191(133,134),

155(536), 156(534, 535), 157 (534), 159(534), 215,222 Davie, J. M., 79,99 Davies, D. A. L., 284, 285, 294, 295, 296 Davies, G . E.,155(120),171( 120), 175 (121,122), 179(122), 210 Davies, M.C., l6l(123),210 Davis, N. C., 160(429),219 Davis, R. B., 148(126), 196(1%), 198 (124,125), 210 Davis, R. L., 200(go), 209 Dawe, C . J., 239,249 Dawson, G., 78,101 Day, E.D., 283,294 De Boutaud, F.,61,63, 64,98 Decamp, G., 158(132),210 Decreusefond, C., 14( 72),15( 72),47 Defendi, V., 235,242,248 De Harven, E., 237,247,248,249,250 Deichman, G. I.,243,248 de Kruif, P. H., 188(477),220 Delaney, R.,84,96,100 Delaunay, A., 175(127), 210 DeLaus, F.V., 176(128),210 Del Bianco, P. L., 165(617), 224 Delezenne, C., 159(1291, 161( 1291, 210 De Lorenzo, F., 59,100 Demant, P., 254,294 Dent, P. B.,240,248 Denys, J., 159(130),210 DeRenzo, E.C., l6l( 1231,163(2),206, 210 Des Prez, R. M., 186(259), 196(131, %9), 197(2-59], 198( 131, 2591, 210, 214

210,211, 215

Diehl, V., 246,249 Diener, E., 65,74,99 DiLorenzo, N. L., 187(441), 194(441),

219

Dixon, F. J., 30(130),49,72, 104,151

(685),203(640),225,226

Doerr, R., 188(137),210 Doleschel, W., 163(138), 210 Dolovich, J., 176(138a),210 Donaldson, V. H.,154(147),155(147),

160(139), 163(141, 145), 168 (150), 169(149), 172( 142, 148), 178(143), 183(140, 152), 184 (151), 185(143, 146, 150, 152, 307), 186(142), 190(307), 194 (la),210, 211, 215 Donermeyer, D. D., 72,LO4 Doolittle, R. F.,80,94,96,99,103 Dormont, J,, 203(236),213 Dorrington, K. J., 75,99,102 Dossetor, J. B.,203(502),221 Douglas, A. S., 162(374),217 Dragstedt, C. A., 193(153, 154), 195 (153),211 Dray, S., 13(54), 14(62, 63, 64, 651, 47,76,99 Dreesman, G. R., 69,70, 74,99 Dreskin, 0.H.,156(571), 23 Dresse, A., 169(322), 215 Dresser, D. W., 35( 144,147,148,1491, 37(147,148,149),49 Dreyer, W. J., 75,94,100, 101 Dreyfus, P. M., 200(%4), 213 Drilhon, A., 59,98 Drummond, K. N.,203(416), 218 Drummond, M. C.,158(650), 225

AWI'HOR INDEX

Dry, R. M. L., 166(13),170(13),176 (13),207 Dubbs, D. R., 235,249 Dubiski, S., 14(66,67),47, 94,97 Duckert, F., 150(39, l55), 151( 689), 162(689), 208, 211, 226 Dulbecco, R., 242,250 Dunn, P., 9(40), 10(40), 11(40), 21

305

Elliott, R. B., 169(404), 218 Elmore, D. T., 152(170),211 Elson, J., 37( 164),50 Embleton, D., 18(656), 225 Enders, J. F., 241,248 Endres, G. F., 150(171), 211 Engle, R. L.,61,76,102, 104 Engle, R. L., Jr., 52,104 (40),22(40), 32(40),41(40),46 Englert, M. E., 161(123),210 Dutcher, T.F., 148(245), 213 Ephrussi, B., 242,248 Duthie, E. S., 166(156), 211 Epstein, J. H., 176(172), 211 Dutton, R. W., 17(92), 26(115),48 Epstein, L.B., 284,294 Dvorak, H.F., 20(97),48 Epstein, M., 246,248 Epstein, W. V., 61,99,179(173),211 Erdos, E. G., 170(175,176, 177,731), E 198(174,403), 211, 218, 227 Eady, J. D., 17(92), 48 Erlanger, B. F., 10(46),46 Ernst, M.,150(39),208 Eagle, H.,195( 157),211 Eschel-Zussman, R.,39( 175),50 Ealey, E. H.M., 74,99 Ebert, R. H., 169(217),178(217), 205 Esnouf, M. P., 153(178, 248), 157 (158),211, 213 (280),211, 213, 214 Espmark, J. A., 232,247 Eddy, B. E., 231, 232, 244, 248 Edelman, G . M., 53,55, 59,60,63,66, Evans, C . A., 231,248 84,99,101, 102, 103 Evans, E.E., 55,66,99 Evans, G., 196(179), 197(179), 211 Edery, H., 164(159),211 Evans, M.J., 55,99 Edman, P., 75,102, 149(69), 208 Evans, R. R., 184(144),211 Eernisse, J. G., 272,282,296 Eggstein, A. A., 180(281), 191(282), Evensen, S. A,, 201(180),212 192(281, 282), 214 F Ehrenpreis, S., 150(171), 152(160), 211 Fagraeus, A,, 232,247 Eichbaum, F., 203(627), 224 Fahey, J. L., 20(105),48, 76, 80,84, Ein, D., 75,76,99,100 85, 92, 99, 102, 103, 104, 147 Eisele, J. W., 163(133), 189(133), 191 (700),226, 285,295 (1331,210 Fahey, L. J., 92,97 Eisen, H. N.,3(48,95a), 5(10, 15), 6 Faires, J. S., 177(181), 178(364), 212, (15), 7(21), 9(15), lO(15, 42), 217 ll(48, 49), 12(49), 16(83a), 20 Fanciullacci, M., 165(617), 169(616), (95a,117),21(15), 23(95a, 117), 186( 616), 224 27(95a, 117, 118a), 31(15), 41 Fanshier, L.,240,250 (15),44(179), 45, 46,47, 48, 50, F a d , P., l82(182,183), l69( 182),212 67,75,90,92,93,99,104 Farr, A. L., 112,142 Eisen, V., 168(1sQ), le9(1&1), 170 Farr, R. S., 9(31), 46, 66,67,85,99, (162), 171(162, 163), 172(lee), 102 178(l65), 183(161), 211 Farrell, C., 247,248 Elder, J. M., 173(167, 423), 174(167), Fauconnet, M.,20( 99),48 175(167),211,219 Favre-Gilly, J. E., 158(509), 221 Elliott, D. F., l68(leg), 169(168),211 Fawcett, D. W., 126,142, 246,248

306

AUTHOR lNDEX

Fazekas d e St. Groth, S., 3(139),9(32), Flinner, R. L., 281, 295 W32,138,139),34(32,138,139), Foerster, J., 17(94), 19(94), 48 46, 49 Folch, J., 109,142 Fearnley, G. R., 163(184),212 Folk, J . E., 171(211),212 Fefer, A., 36(152), 49, 231,248 Forbes, C. D., 161(482), 182(482), 183 Feingold, N.,259, 281, 265, 273, 279, ( 192a),212, 220 288,294 Forell, M . M., 167(193,718), 212, 227 Feinstein, A,, 79,86,88,99, 101, 102, Forman, W . B., 151(194), 212 103 Forsyth, R. P., 198(467), 220 Feinstein, D., 78,99 Foschi, G. V.,284,295 Feldberg, W., 106,126,142, 168(185), Fox, F., 176(675), 225 169(186), 193(34), 207, 212 Fox, R. H., 167( 195),212 Feldman, L. A., 234, 250 Fradette, K.,14(86) 47 Feldman, M., 14(71), 15(71), 39(174, Franceschini, P.,281,293 175). 47, 50 Franchi, G., 185(617),224 Fellows, R. E., Jr., 84,96,100 Francis, T., Jr., 33( 136a),49 Ferguson, J. H., 147(349), leO( 350), Franek, F., 85, 86,99, 104 162(205,271,272,348), 212, 214, Frangione, B., 79,84,99, 102, 103 216 Frank, J. A., 195(898),226 Fernindez-Morh, H., 55,99 Frank, M.M., 90,99 Ferreira, S. H., 170(187),212 Franklin, E. C., 20( 103),48, 64,65,68, Ferris, B.,149(455),219 75, 78, 79, 85, 98, 99, 100, 103, Ferrone, S., 268,294 104 Fienberg, R., 176(188),212 Frauenberger, G., 10(43), 46, 87, 101 Findley, A., 204(207),212 Fray, A., 204(233),213 Fine, J . M., 59,98 Frederiks, E., 282,296 Fink, M . A., 194(189), 212, 244, 246, Freeman, M.J., 86,102 248 Freer, R., l68(219), 173(219), 178 Finkel, M. P., 247,248 (219),213 Finkelstein, M. S., 20( l06), 32( lm), Frei, P. C., 36( 151), 37(151), 49 48, 64,65,68,104 Frey, E.K., 167(196, 311,312), 215 Finstad, J., 57,59,60, 82,84, 95,102, Frick, P. G., 151(689), 162(689), 226 103 Fried, R., 175(308), 215 Fischer, E. H., 155(298),215 Friedberger, E., 188(197), 192(197), Fischer, P., 194(190), 212 195(197), 212 Fisher, L. M.,148(276), 214 Friedemann, U., 188(198), 192(198), Fishkin, B.G., 71,104 212 Fitzpatrick, M.,162(182), 169(182), Friedman, B. K., 148(738), 227 212 Friedman, E.,190(610), 224 Fjeldborg, O., 204(303),215 Friedman, H., 2-40, 248 Flanagan, J . F., 291,295 Friendly, D., 283, 294 Flax, M.H., 20( 97),48 Fritz, H., 168(870), 183(glO), 198 Fleisher, M.S., 182( 191),212 (670),225 Fletcher, -4. P., 160(8,31, 192, 3m), 162(7,605), 163(8,607,608), 168 Frontino, C., 204(54), 208 (606), 171(806), 207, 212, 215, Fudenberg, H . H., 82, 81, 100, 101, 103, 104, 181(852a), 225 224 Fujimoto, M. M., 147(260), 214 Fletcher, I., 290,297 Fujio, H.,10(45),46 Flick, J . A., 193(278),214

AUTHOR INDEX

307

Glynn, J. P., 231,248 Glynn, M . F., 187(213, 452), 212, 219 Gaarder, A., 147(199), 212 Goadby, P., 112,142 Gabrielson, F. C., 283,294 Goergen, X.,199(305),215 Gaintner, J. R., 149(200), 212 Catze, W., 167(719),227 Gales, S. B., 162(374),217 Goidl, E. A,, 10(41), 21(41), 22(41), Gallo, G., 205(365),217 41(41), 46 Gamble, C. N., 198(91),209 Gold, E. F., 69;70,100 Ganley, 0. H., 165(588), 223 Goldberger, N.,36( 159),49 Gans, H., 193(202), 195(202), 197 Goldfinger, D., 199(444),200(444),219 (201),212 Goldfinger, S. E., 176(408), 177(408), Gamer, R. L., 161(667), 225 178(408), 218 Gascon, L. A., 191(573), 192(574), 223 Goldner, H., 244,248 Gautvik, K. M., 171(203), 177(203), Goldsmith, M., 240, 250 212 Golubow, J., 152(399),218 Geering, G., 237, 246, 248, 249, 250 Good, R. A., 56,57,59,61,62, 64,95, Geever, E. F., 197(238), 200(238), 213 100, 102, 103, 179(215),196(662), Geiser, S.,84,103 198(214,621,661,662),200(662), Gell, P. G. H., 14(61), 16(84, 85), 17 201( 112, 215), 202(644), 203 (86),26(110, 112), 47, 48 (215, 412), 210, 212, 218, 224, Gengou, O., 153(74), 209 225, 240,248 Gengozian, N.,71,100 Goodell, H., 176(98),209 George, C., Jr., 159(358),217 Goodman, J. W., 87,104 George, M.,233,248 Goodman, M., 71,81,84,100 Gerber, I. E., 196(204), 198(204), 212 Goodpasture, E. W., 160( 216), 212 Gerber, P.,246, 248 Gordon, S.,70,99 Gerheim, E. B., 161(205),205 Gorer, P. A,, 253,287,291,292, 294 Gerlough, T.D., 87,103 Gorman, L. R., 231,248 German, W.M., 176(206), 212 Gott, S. M., 7(44), lO(44,44a),46 Goyette, D. R., 259,297 Germuth, F. G., Jr., 195(372), 217 Gershon, H.,14(71), 15(71), 47 Grace, J. T., Jr., 244,249 Gewun, H., 191(624), 207, 224 Graham, R. C., Jr., 169(217), 178(217), 213, 215 GighIi, I., 128,142 Granerus, G., 185(362), 186(218,3621, Gilden, R. V., 68,103, 234,235,248 213, 217 Gillette, R. W., 204(207), 212 Gray, M . E., 197(339), 216 Gingrich, R. E., 54,100 Gray, W . R., 75,94, 100, 101 Girardi, A. J., 244,248 Gitlin, D., 151(208), 179(208), 198 Green, A. A., 165(513),221 (368), 205(208), 206(208), 212 Green, I., 13(57), 14(70,751, 15(70), 217 47 Givol, D., 59,84, 87,88,98, 100, 101, Green, M., 235,250 Greenbaum, L. M., 168(219), 173 104 (219),178(219),213 Gladner, J. A., 152(209,210), 171(211, Grette, K.,148(220),213 484), 212, 220 Grey, H. M., 58,61,62,66,72,75,76, Glassock, R., 280,295 77, 78,80, 85, 97, 100, 101, 102, Gleason, R. E., 281,294 103, 104 Gleich, G. J., 176(418), 185(417),218 Grob, D., 161(221), 213 Glenn, J. F., 277,278,296 Gross, D., 61,99 Glueck, H . I., 154(212),212

G

308

AUTHOR INDEX

Gross, L., 239, 248 Grossberg, A. L., 5( 14), 7( 19), 29( 121, 122), 46, 49, 90, 102 Groszlan, S., 235, 249 Groth, C. G., 203( 640), 225 Groyon, R. M., 70, 99 Grubb, R., 62, 100 Grubbs, G. E., 244, 248 Guest, M. M., 152(223), 160(460), 162(96), 172(222), 209, 213, 220 Gunnells, J. C., 277, 278, 296 Gurewich, V., 196(549), 222 Guth, P. S., l6Q(28), 172(28), 194 (29), 195(29), 207 Guttman, R. D., 127,142 Guttman, S., 168( 72), 208

Hardy, W. D., Jr., 237, 248 Hare, J. D., 241, 243, 244, 248 Harned, B. K., 129, 142 Harris, P., 169(65), 208 Harris, R., 287, 294 Hartley, J. W., 237, 240, 248, 249 Haitmann, R. C., 147(245), 148( 242), 213, 222 Has’ek, M., 35( 142), 49 Hashimoto, K., 176(675), 225 Hattler, B. G., 273, 292 Haughton, G., 232, 249, 284, 292, 294 Haurowitz, F., 69, 100 Hausman, R., 200(244), 213 Haverback, B. J., 148(245), 213 Hawking, F., 128, 142 Hawkins, D., 204( 106), 209 H Hayashi, H., 164(246), 205(246), 213 Haanen, C., 154( 591a), 156(591), 223 Hayes, C. P., Jr., 277, 278, 296 Habel, K., 229, 2301, 231, 232, 233, 234, Hedin, S. G., 159(247), 161(247), 213 238,242, 243, 248,250 Heene, D., 152(596), 223 Heidelberger, M., 5(4, 5 ) , 9 ( S ) , 9 ( 25, Haber, E., 7(22, 23), 12(22, 23), 46 26), 21(4), 45, 46, 90, 100 Habermann, E., 167(227), 168(224, 22S), 169(225, 226), 213 Hektoen, L., 9( 34, 35), 46 Hellem, A., 147( 199), 212 Hagan, J. J., 163(2), 206 Heller, P., 80, 104 Hahn, F., 191(228,229), 213 Haines, A. L., 181(230, 231), 213 Hellstrom, I. E., 229, 231, 232, 233, 247, Haines, R. F., 203( 87), 209 249, 250 Hellstrom, X. E., 232, 247,249 Halberg, L., 186(218), 213 Helmreich, E., 69, 102 Hale, J. H., 158(623), 224 Halliday, S., 129, 142 Hemker, H. C., 153(2A8), 213 Hemker, P. W., 153(248), 213 Halmagyi, D. F. J., 168(397), 218 Halpern, B. N., 203(232), 204(233), Henle, G., 246, 249 Henle, W., 246, 249 213 Hennessy, A. V., 33(136a), 49 Ham, T. H., 160(234), 213 Henning, G., 86, 100 Hamberg, U., 168(235), 213 Henry, C., 13( 60), 47 Hambuechen, R., 169(720), 227 Heptinstall, R. H., 195(372), 217 Hamburger, J., 203( 236), 213 Heremans, J., 81, 82, 90, 98, 103 Hamilton, J., 9(39), 46 Heremans, J. F., 70, 78, 90, 91, 100, Hamilton, R. L., 197(339), 216 103, 104 Hammond, D., 271, 294 Heremans, M-T., 70, 100 Hammer, D. K., 86, 100 Hermann, G., 251, 296 Hanson, L. A., 78, 100 Hermes, P., 53, 98 Harboe, M., 179(237), 213 Hardaway, R. M., 196(239), 197(238, Hersh, R. T., 68, 69, 71, 98, 100 Hershey, S. G., 197(9), 207 239), 200(238), 213 Herxheimer, H., 117, 142 Harder, F. H., 232, 248 Hardisty, R. M., 148( 241 1, 187(240 1, Herzig, R., 157(80), 158( 249), 209, 213 213

309

AUTHOR INDEX

Hessel, B., 149(69),208 Hewitt, R. I., 128,143 Hewitt, R. L., 281, 295 Hiernaux, M., 195(580), 223 Hildemann, W. H., 100 IIilgard, H. R., 52,100 Hill, B. M.,163(474), 188(485), 189 (485),220 Hill, J. M., 154(636),224 Hill, R. L., 84,96,100 Hilleman, M.R., 244,248 Hills, J. D., 197(94),209 Hilschmann, N.,75,100, 101 Hilton, S . M., 167(195),212 Hinshaw, L. B., 198(250), 213 Hinz, C . F., Jr., 179(332), 181(332), 183(251), 213, 215, 216 Hirsch, J. G., 127,128,141, 142 Hirschhorn, K., 17(90), 48, 252, 292 Hirsh, J., 187(213),212 Hjort, P., 147(255), 157(252), 158 (253),193(252), 214 Hjort, P. F., 199(254),201(180), 212, 214 Hoak, J. C., 154(256),214 Hogberg, B., 106, 107, 108, 109, 111, 141, 142 Holden, H. F.,168(185), 212 Holgate, J. A., 116,117,141, l69(llO), 194(52),208, 210 Hollander, J. L., 177(363), 217 Hollinshead, A. C., 235,249 Holman, G., 155(120), 171(120), 210 Holmes, J. H., 281,296 Holmgren, J., 159(257), 214 Holt, E.,159(252),214 Holton, F.A., 169(258),214 Holton, P.,169(258),214 Hong, C . V . , 201( 112),210 Hood, L.E., 75,94,100, I01 Hook, E. W., 186(259), 196(131,%9), 197(%9), 198(131,%9), 210, 214 Hooker, S . B., 9(33,361,46 Horibata, K., 15(82, 83), 47, 48 Horn, R. G., u)O( 6%), 224 Hornbrook, M. M., 80,101 Homer, G. J., 168(397), 218 Horowitz, H. I., 186(259), 196(131,

2-59), 197(259), 198(131, 259), 210, 214 Horowitz, W. I., 147(260),214 Horton, E. W., 169(168), 170(261), 211, 214 Howard, J. G., 37(162,163,164), 50 Howe, A. C., 160(350), 216 Howell, R. R., 177(598), 178(598), 223 Howell, W. H., 159(252),214 Howland, J. W., 200(283), 214 Hraba, T.,35( 142),49 Hu, C . C., 20(96),48 Hubbard, D., 155(263), 214 Hudson, R. P., Jr., 203(725), 227 Huebner, R. J., 232,233,234,235,237, NO,247, 248, 249 Huff, C . G., 52,54,101 Huggins, S. E., 66,101 Hughes, F. B., 148(264), 214 Hughes, W. F., 240,250 Hughes, W. L., 36( 158),49 Hugues, J., 147(265), 214 Humair, L. M., 204(265a), 214 Hume, D.M., 203(725), 227, 281, 294 Hummel, F. P., 192(680),226 Humphrey, J. H., 36(156), 49, 90, 99, 107, 141, 148(268,269, 270), 149 (269), 186(269), 187(269), 188 (269), 191(269), 192(269), 195 ( 269), 205( 266), 206( 266, 2671,

214 Huneycutt, H. C., 270,289,297 Hunter, A., 79,101 Hurliman, J., 44(180),50 Husni, E. A., 197(238), 200(238), 213 Hutcherson, J. D., 197(339), 216 Hutton, R. A., 187(240),213 I

Iatridis, S. G., 162(271,272), 214 Ingraham, J. S., 14(73), 15(73), 47 Ingram, G. I. C., 148(75), 209 Inouve, H., 88,101 Iredell, J., 175(437),219 Irwin, J. W., 195(696),226 Ishizaka, K., 62, 81, 85, 98, 101, I.11, 115, 116, 118, 123, 139,142, 143,

144

310

AUTHOR INDEX

Ishizaka, T., 62, 81, 101, 111, 115, 116, 118, 123, 139, 142, 143, 144 Ishizaki, R., 237, 238, 249 Itano, H. A., 92,93,103 Jto, Y., 231, 248 Ivanovic, N., 152(596), 223 Ivanyi, D., 254, 259, 265, 281, 294 Ivanyi, P., 254, 259, 265, 281, 294

Johnston, C. G., 195( 157), 211 Johnston,C. L., 161(2U5), 212 Johnstone, D. E., 200(283), 214 Jonasson, O., 139, 142, 194(284), 224 Jones, J. W., u)3(87), 209 Jones, V. E., 123,142 Jonsen, J., 147( 199), 212 Jonsson, N., 231, 249 Jordan, M. M., 198(250), 213 J Jung, E., 150( 155), 211 Jackson, D. P., 148(242, 436), 149 Jupelle, F., 188(55), 208 (200), 212, 213, 219 Jureziz, R., 7(20), 46 Jacobsen, S., 169(273), 214 Jurgelait, H. C., 284, 294 Jacot-Guillarmod, H., 30(130), 49 K Jager, B. V., 87, 101 Kabat, E. A., 6(16), 46, 87, 90, 101, Jager, L. A., 87, 101 104 James, G. W. L., 116, 117, 142 Janeway, C. A., 151(u)8), 160(274), Kagen, L. J., 168(286), 174(285, 286, 179(208), 205( 208), 206( 208), 287, 367), 183(286), 184(286), 212, 214 214, 217 Kahan, B. D., 284,285,294 Janeway, T. C., 165(275), 214 Kaliss, N., 283, 294 Janoff, A., 128, 135, 142, 143 Kandukch, A. A., 284,294 Jaquenoud, F A . , 188( 72), 208 Jaques, L. B., 148(276), 195(277), 214 Kano, K., 203(725), 227, 291, 295 Jaques, R., 148(268, 269), 149(269), Kaplan, A. M., 86, 102 186(269), 187( 269), 188(269), Kaplan, M. H., 161(288), 214 191(209), 192(269), 195(269), Kark, S., 175(437), 219 Karli, L., 91,102 214 Kamovsky, M. L., 111, 116, 143 Jemski, J. V., 193(278), 214 Jenkins, G. C., 20(102), 48, 121, 137, Karsner, H . T., 202(289), 214 Karush, F., 5(3e, 10, l l ) , lO(43, 451, 143 45,46, 87,101,103 Jensen, F., 242, 248 Katz, M., 232, 250 Jensen, J., 135, 142, 190(279), 214 Jepson, J. B., 169( 16), 176(15), 178 Kay, D., 147( 115), 210 Keele, C. A., 166(13), 169(16), 170 (15), 207 (13), 176(13, 15), 178(15), 207 Jeremic, M., 201(180), 212 Jeme, N. K., 3(3a), 9(29), 13(60), Kellaway, C. H., 106, 126, 137, 142, 168(185), 212 45, 46,47, 66, 101 Kellermeyer, R. W., 167(294), 177(292, Jobe, A,, 93, 103 297, 703), 178(290, 291, 292, 293, Jobin, F., 153( 178), 157(280), 211, 294), 214,215,226 214 Jobling, J. W., 180(281), 191(282), Kellermeyer, W. F., Jr., 155(296), 171 (296), 177(297), 215 192(281, 182), 214 Kelloff, G., 237, 249 Johansson, K., 108, 109, 111, 141 Kelus, A. S., 14(61), 47 Johansson, S. G. O., 81, 98, 101 21 Johnson, D., 196(239), 197(239), 213 Kendall, F. E., 5(4, 51, 9(5, s), Tohnson, H. M., 53,101 (41, 45, 46 Kent, A. B., 155(298), 215 johnson; J. S., 86, 101 Johnson, M. C., 7(44), lO(44, Ma), 46 Kent. S. P.. 88.99 ~~

311

AUTHOR INDEX

Keppler, A., 167(719), 227 Kettle, E. H., 176(299), 215 Keysser, F., 188(300), 215 Kickhofen, B., 86, 100 Kiesselbach, T. H., 147(301), 215 Killingback, P. G., 117, 139, 143 Killmann, S. A., 256, 295 Kim, Y. B., 86,101 Kimura, E. T., 115, 142 King, G. S., 246, 248 King, T. P., 78, 101 Kingdon, H. S., 156(302), 215 Kinsky, R. G., 37( 164), 50 Kishimoto, T., 80, 91, 102 Kissmeyer, N., 204( 303), 215 Kissmeyer-Nielsen, F., 264, 268, 288, 290, 296 Kit, S., 235, 249 Kitahara, T., 234, 235, 249, 250 Kitayawa, M., 7( 19), 46 Kjelgard, M., 161(89), 209 Kjelgard, N. Q., 161(304), 315 Klein, E., 229, 232, 246, 249, 250 Klein, G., 79, 101, 230, 231, 232, 246, 249, 250, 286, 293 Kleinmaier, H., 199(305), 215 Klemperer, F., 175(308), 215 Klemperer, M. R., 185( 307), 190(307), 215 Kliman, A., 157(5), 201(308), 207, 215 Klinman, N. R., 10(43), 46, 87, 101, 103 Kluchareva, T. E., 243,248 Kneebone, G. M., 169(404), 218 Knicker, W. T., u)4(106), 209 Knight, K. L., 69, 100 Knowles, B. B., 235, 236, 250 KO, A., 78, I01 Kobold, E., 196(309), 198(3(@), 215 Koch, M.A., 234,250 Kohler, H., 80, 89, 104 Kohler, P. F., 75, 100 Kohn, G., 246,249 Kohn, N. N., 178(364), 217 Konigsberg, W. H., 84, 103 Konishi, K., 150(360 1, 217 Konzett, H., 116, 142 Koono, M., 164(246), 205(%6), 213

KopeC., M., 160(310), 215 Koprowski, H., 235, 236, 242, 248, 250 Korach, S., 79, 103 Komgold, L., 61,76, 101 Kourilsky, F. M., 85, 86, 98, 121, 137, 141, 144, 205(66), 208 Kowalski, E., leO(310, 468), 215, 220 Kraner, K. L., 86, 103 Kraus, R., 195(60),208 Kraut, H., 167(196, 311, 312), 212, 215 Krebs, E. G., 155(298), 215 Krecke, H. J., 199(305), 215 Kren, V., 254, 296 Kritznan, J., 75, 101 Krivit, W., 193( 202), 195(202), 197 (201), 212 Kriz, M., 169(273), 214 Krummel, W. M., 7( 18), 46 Kuff, E. L., 90,94,101 Kunkel, H. G., 61, 62, 75, 76, 77, 78, 79,100,101,102,103 Kuntzman, R., 148(613), 224 Kuroyanagi, T., 173(313), 215 Kyerbye, K. E., 290,296

1 Lack, C. H., 161(315), 179(314), 215 Lagrue, G., 204( 233), 213 Lahiri, S. C., 139, 142, 194(85, 86),209 Laidlaw, P. P., 106, 142, 193( 117), 210 Laki, K., 147(316), 150(317), 152 (209, 210), 171(484), 212, 215, 220 Laland, S., 147( 199), 212 Lalezari, P., 282, 283, 290, 295 Lambert, P. H., 195(580), 223 Lamelin, J. P., 17(94), 19(94), 48 Lamm, M. E., 11(50), 47, 90, 94, 101, 102 Lampe, W., 256, 297 Landerman, N. S., 183(318), 184( 3191, 215 Landsteiner, K., 5 ( 6, 91, 45 Lane, W . T., 233, 249 Lang, K., 201(648), 225 Langer, von B., 75, 101 Larson, C., 69,70,99 Larson, V . M., 244, 248

312

AUTHOR INDEX

Lasch, H. G., 199(305, 704), 215, 226 Laszlo, J., 271, 295 Latallo, Z. S., 160(310, 320, 469), 215, 220 Laureu, A-B., 160(321), 181(321), 182(321), 183(321), 185(362), 186(218, 362), 213, 215, 217 Lavrin, D. H., 240,249 Law, L. W., 239,247,249 Lawrence, H. S., 281, 282, 295 Leach, S. J., 152( l60), 211 Lebovitz, H. E., 84,96,100 Lebrun, J., 175( 127), 210 Lecompte, J., 169(322), 194( NO), 212, 215 Leddy, J. P., 173(287), 174(287), 214 Lee, E. H., 62,101 Lee, F. S., 165(645), 225 Lee, L., 197(323), 199(323, 325, 326), 200(323), 201(323), 202(327), u)3(324, 327), 215, 216 Lees, M., 109, 142 Legge, J. S., 74, 99 Legrand, L., 259, 281, 265, 273, 279, 288,294,295 Leigheb, G., 277, 279, 281, 282, 293, 297 Leiner, K. Y., 112, 142 Lengerovb, 35(142) 49 Lennox, E. S., 97, 101 Lenox, E., 3(3n), 15(82, 83). 4-5,47, 48

Levin, J., 196(341), 197(341), 199 (%1),201(341), 203(342), 216 Levine, B. B., 28( lll),48 Levine, L., 180(343, 344), 216 Levinthal, J. D., 235,249 Levinthal, M., 165(380), 217 Levy, L. R., 180(334), 181(334), 183 (334, 4911, 184(491), 216, 221 Lewis, A. M., Jr., 245, 249, 250 Lewis, G. P., 164(159), 167(346), 169 (168, 169, 186, 347), 172(345), 174(345), 211, 212 Lewis, J. H., 147(349), 160(350), 162 (348), 179(351), 216 Lewis, R., 175(437),219 Lewis, T., 164(352), 216 Liacopoulos-Briot, M., 14(72), 15(72), 47 Lichtenstein, M., 191(3), 207 Liebeman, R., 93,101 Lieberman, S., 10(46), 46 Liefmann, 180(353), 216 Lillehei, R. C., 196(354), 197(355), 198(355, 570), 216, 223 LiIIy, F., 237, 238, 249, 285, 288, 295 Linder, 0. E. A., 239, 249 Lindquist, R. R., 127, 142 Lindsley, D. L., 254, 295 Lipihki, B., l60( 310), 215 Lisowska-Bemstein, B., 11( SO), 47 Lister, J., 153(356), 216 Little, J. R., Jr., 7(21), 10(42), 46, 75,

217 Lopes, M. A,, 69, 100 221,222,224 Lopez, A., 158(132), 210 LeQuire, V-S.,197(339), 216 Lbrhd, L., 149(30), 150(317, 360), Lerch, E. G., 66, 101 152(359), 207, 215, 217 Lerner, R. A., 203( 640),225 Lerner, R. G., 197(340), 198(340), 199 Losner, S., 197(688),226 LoveU, R. R. H., 175(71, 361), 192 ( N O ) , 216 (71), 208, 217 LeRoy, E. C., 155(728), 227 Lovett, C. A., 136, 142, Levenson, S. M., 160(651), 225

AUTHOR INDEX

Lowe, J. S., 155(120), 171(120), 175 (121, 122), 179(122), 210 Lowell, F. C., 117, 142 Lowenhaupt, R., 204(361a), 217 Lowenstein, L., 252, 292 Lowry, 0. H., 112, 142 Lucas, G. L., 155(263), 214 Lukes, R. J., 86,103 Lundh, B., 160(321), 181(321), 182 (321), 183(321), 185(362), 186 (362), 215, 217 Liischer, E. F., 148(56, 57), 186(58), 187(58), 2U1(58), 208 Lykakis, J. J., 66,101 Lyman, S.. 253, 294

313

Macleod, C. M., 159( 102), 161(102), 209 Macmorine, D. R. L., 128, 143, 186 (453), 187(453), 219 McNicol, G. P., 162(374), 217 MacPherson, I., 242, 249 MacQueen, J. M., 259, 263, 273, 292 Madalinski, K., 76,101 Miikela, O., 14(74, 80), 15(74), 16 (83b), 36( 153), 47, 48, 49 Mage, R. G., 14(63, 64, 85, 68, 69>, 47, 94, 97 Magnuson, N. S., 259, 297 Maier, L., 167( 718), 227 Maier, P., 85, 104 Majno, G., l65( 114, 380, 381, 382), 169(382), 189(382), 210, 217 M Malawista, S . E., 177(598), 178(598), McCarty, D. J., Jr., 177(181, 363), 178 223 (364, 494), 212, 217, 221 Malessa, S., 199(704),226 Mallen, M. S., 128, 143 McCarthy, J. S., 20(97), 48, 75, 101 McCluskey, R. T., 199(326), 204(683), Mallett, D. L., 155(536), 222 205(109, 365, 435), 206(109), Mahgren, R. A., 232, 234, 239, 247, 249, 250 210, 215, 217, 219, 226 Malmquist, J., 160(321), 167(733), McConahey, P., 30( 130), 49 168(733), 172(732, 733), 176 McConnell, D. J., 174(388, 367), 217 (733), 178(733), 181(321), 182 McCoy, J. L., 231, 248 (321), 183(321), 198(733), 215, McCracken, B. H., 281, 295 227 McDevitt, H. O., 288,295 Macfarlane, R. G., 147(63), 153(248), Mamet-Bratley, M. D., 6( 17), 46 157(375), 160(62, 376), 208, 213, Mann, D., 285, 295 Mann, F. D., 147(383), 217 217 Mannik, M., 61, 100 McGovem, J. P., 185(93), 209 Manox, W. C., 196(354), 216 McGuigan, J. E., 11(49), 12(49), 47 Manson, L. A., 284,295 McIntire, K. R., 90,94,101 McKay, D. G., 162(370), 196(370, Marbaix, H., 159( 130), 210 371), 197( 370), 198(368, 370), Marchalonis, J. J., 53, 55, 59, 60, 63, 66, 99, 101, 102 199(370, 444), ZOO( 369, 444), 201 Marchesi, V . T., 147(384), 217 (308, 369, 443), 215, 217, 219 Mackay, M., 162(377), 172(378), 217 Marchioro, T. L., 203(502), 221, 281, 296 Mackay, M. E., 173(379, 724), 175 Marcinak, E., 152(596), 223 (379). 217, 227 Marcus, A. J,, 147(385, 386), 148(456, McKenzie, I. F. C., 203(371a), 217 729), 149(385), 217, 219, 227 McKhann, C. F., 232,248 Marcus, D. M., 193(23), 207 McKinnon, G. E., 195(372), 217 Marder, V . J., 160(387), 217, 283, 292, McKusick, V . A., 166(373), 217 296 McLaughlin, C. L., 20(105), 48 MacLean, L. D., 197(355), 198(355), Margaretten, W., 199(388, 444), u)o (444), 217, 219 216

314

AUTHOR INDEX

Margolis, J., 154(389), 156(395), 158 (3891, 168(397), 169(396), 170 (a, 390, 391), 171(391, 394), 173 (392, 393), 177(391), 208, 218 Margolius, A,, Jr., 147(599), 223 Mark, R., 253, 296 Markham, J. W., 166(13), 170(13), 176(13), 207 Marrach, J. R., 69,102 Marshall, J. H., 54, 104 Martin, C. J., 152(399), 218 Martin, D. C., 241,249 Martinez, C., 239, 249 Martinez, R. C.,179(S l ) , 216 Marx, R., 161(400), 218 Mason, B., 164(722), 174(401), 218, 227 Mason, D. T., l69( 402), 218 Massion, W. H., 198(403), 218 Masten, J. L., 157(398), 218 Masurel, M., 282, 293 Mathews, W., 177(712), 226 Mathies, M. J., 62, 98 Matsumoto, S., 91, 102 Matsumura, M., 164(246), 205( 246), 213 Mattern, P., 79, 101, 103 Mattiuz, P. L., 263, 271, 277, 279, 281, 282, 283,293, 294 Make, R., 154(591a), 156(591), 223 Maung, R. T., 66,102 Maurer, P. H., 9( a), 29( lu)), 46, 49 Maxwell, G. M., 189(404), 218 Maycock, W. #A,, 162(377), 172 (378), 217 Mayer, C. E., u)o(111),210 Mayer, M. M., 9(26), 46, 179(405, 647), 180(344), 190(610), 216, 218, 224, 225 Maynert, E. W., 149(200), 212 Medawar, P. B., 252,293 Meeker, W. R., 196(124), 198(124), 210 Meeker, W.R., Jr., 198(125), 210 Mellors, A., 190(610), 224 Mellors, R. C., 75,101 Melmon, K. L., 167(406), 169(402), 172(406), 173(407), 176(408),

177(408), 178(406, 408), 179 (173), 198(467), 211, 218, 220 Melnick, J. L., 234, 235, 249, 250 Mhnach6, D., 151(409, 410), 218 Menkin, V., 163(414), 166(411, 412, 413, 414, 415), 418 Merchant, B., 13(59), 47 Mergenhagen, S.-E., 191(624), 207, 224 Merler, E., 91, 102, 160(274), 214 Merrill, J. P., 127, 142, 203(79), 209, 281, 294 Merryman, C., 85, 102 Metzgar, R. S., 291,295 Metzger, H., 75, 80, 90, 93, 99, 102 Michael, A. F., Jr., 203(416), 218 Michaelides, M. C., 69, 102 Michel, B., 176(418), 185(417), 218 Michelson, E. H., 54, 102 Mickey, M. R., 2-59, 279, 280,288, 291, 295, 296, 297 Middlebrook, W. R., 149(30), 207 Middleton, E., 106, 109, 126, 143 Miescher, A., 2O5( 489), 206( 489), 220 Miescher, P., 186(419), 187(419), 205 (489), 206(489), 218, 220 Miescher, P. A., 20( 99), 48 Miggiano, V. C., 277, 281, 293 Mihaesco, C. O., 61, 79, 80, 102 Mikulska, Z. B., 253, 287, 292, 294 Miles, A. A., 164(420), 168(422), 171 (541), 173(45, 379, 421, 423, 724), 174(401, 541), 175(379, 421), 183 (421), 194(723), 208, 217, 218, 227 Milgrom, F., 203(725), 227, 291, 295 Mill, P. J., 173(423, 724), 194(723), 219, 227 Miller, D. F., 281, 294 Miller, J. W., 176(424), 219 Miller, K. D., 152(425), 219 Miller, M. C., 283, 292, 296 Miller, R. W., 241,249 Milliez, P., 204(233), 213 Mills, G. L., 168(17), 169(17), 207 Mills, G. M., 247,250 MiIIs, J. A., 17(93 1, 48 Milstein, C. P., 75, 84, 88, 97, 99, 102, 103

AUTHOR INDEX

Milstone, J. H., 153(427), 161(426), 219

Minden, P., 85, 102 Minowada, J., 232, 250 Mirand, E. A., 244,249 Mirsky, I. A., 160(429), 161(428), 219 Mishell, R., 84, 92, 99 Mist, S. H., 192(681), 226 Mitchell, J. R. A., 149(430), 219 Mitchell, P., 200(48), 201(48), 208 Mitchison, N. A., 3(36), 35( 144, 145), 36(161), 45, 50, 59 Mittal, K. K., 279, 280, 291, 295, 296 Miwa, I., 198( 174), 211 Miyoshi, H., 164(246), 205( 246), 213 Mizell, M., 246, 248 Mladick, E., 259, 292 Moller, G., 30( 126, 127, 128), 31( 126), 49

Moeschlin, S., 256, 295 Mongar, J. L., 193(431, 432, 432a, 433, 434), 219 Moore, R. W., 74, 99 Moran, W. H., 198(570), 223 Morard, J. C., 204(233), 213 Moritz, A. R., 202( 289), 214 Morris, P. J., 203( 725), 227 Morris, R. H., 205(435), 219 Morse, E. E.,148(436), 219 Morse, H. C., 111, 74, 85, 98, 113, 120, 121, 122, 123, 124, 125, 134, 135, 140, 141, 143, 144 Morter, R. L., 86, 102 Morton, D. L., 247,249 Moses, J. H., 169(217), 178(217), 213 Moskowitz, R. W., 175(437), 219 Mota, I., 86, 102, 123, 136, 143, 186 (44a), 191(438), 208, 219 Mounter, L. A., 161(439), 219 Mouton, D., 14(72), 15(72), 47 Movat, H. Z., 128, 136, 143, 179(440), 186(442, 453), 187(440, 441, 442, 453), 194(440, 441), 195(442), 219

Mozes, E., 29( 119), 49 Miiller-Berghaus, G., 199(444), 200 (369, 444), 201(369, 443), 217, 219

Miiller-Eberhard, H. J., 85, 102, 125,

315

135, 142, 179(445, 447, 449), 180 (446, 448, 473), 182(653), 190 ( 107), 219,220 224 Miillertz, S., 162(450, 451), 219 Munson, A. E., lsO(28). 172(28), 194 (29), 195(29), 207 Murase, H., 196(730), 227 Mulphy, F. A., 86, 97,102 Murphy, R. C., 153(701), 172(222), 213, 226

Murray, J. E., 281, 294 Murtaugh, P. A,, 171(211), 212 Mushinski, J. F., 93,101 Mustard, J. F., 186(442, 453, 486), 187 (213, 442, 452, 453, 454, 486), 196 (179), 197( 179), 200( 454), 202 (454), 205(486), 211, 212, 219, 220

Mybliwiec, M., 155(471), 220

N Nachman, R. L., 61, 76, 102, 104, 148 (456),149(455), 219 Nachtigal, D., 39(174, 175), 50 Naff, G. B., 160(542). 161(459), 163 (544), 168(459, 544), 174(544), 178(457), 179(458), 181(332, 458, 542), 182(459, 542, 543), 183 (331, 544), 196(514), 197(514), 201(544), 216, 220, 221, 222 Nagel, E., 193( 341), 207 Nagler, A. L., l65(740), 166( 740), 193 (742), 195(742), 197(742), 198 (741), 201( 742), 206(742), 227 Naimi, S., u)o(567), 222 Najarian, J. S., 279,295 Nanninga, L. B., 160(460), 220 Nathan, E., 188(461), 204(361A), 217, 220

Nathenson, S. G., 284, 285, 295 Natvig, J. B., 77, 102 Neeper, C. A., 36( 154), 49 Nelson, R. A., Jr., 125, 128, 142, 143, 180(462), 186(619), 187( 619), 220, 224

Nemerson, Y., 152(464), 153(484), 220 Neta, H., 128, 142 Niall, H., 75, 102 Nicks, J. P., 2-59. 292

316

AUl’HOR INDEX

Nicolaides, E. D., 169(465), 220 Nicolle, M., 205( 466), 220 Niculescu, V., 111, 112, 142 Nie-Khah, H., 69, 98 Niemetz, J., 158(476), 220 Nies, A-S., 198(467),220 Niewiarowski, S., 155(471), 160(468, 469), l62(470), 220 Nilsson, I. M., 186(472), 220 Nilsson, U. R., 180(473), 220 Nishizawa, E. E., 188(488), 187(452, 468), 188(486), 205(486), 219, 220 Nisonoff, A,, 5( 12, 13), 7(20), 29( 123, 125),46, 49, 74, 84, 102 Noguchi, H., 53, 102 Nordin, A. A., 13( 80),47 Norman, P. S., 20(98), 48, 163(474), 220 Norton, S., 71, 100 Nossal, G. J. V., 13(58, 153), 14(78, 79, 80), 15(81), 38( 150, 152), 47, 49, 65, 99 Nossel, H. L., 155(475, 728), 158 (476), 220, 227 Novy, F. G., 188(477), 220 Nowinski, R. C., 237, 249 Noyes, H. E., 197(238), 200(238), 213 Nuckolls, J. W., 200(622), 224 Nussennveig, R. S., 85, 102 Nussenzweig, V., 11( 51), 12(51), 13 (57), 14(70), 15(70), 25, 26, 27 (109), 29( 120), 47, 48, 49, 85, 94, 102, 123, 143, 160(478), 220

0

(382), 163(544), 168(544), 174 (544). 183(544), 184(544), 201 (544), 220, 222 Okon. M. E., 167(227), 213 Old, L. J., 229, 237, 240, 242, 246, 247, 248, 249, 250, 286, 288, 293 Oliver-Gonzalez, J., 128,143 Olmsted, F., 169(487), 220 Olsen, S., 204( 303), 215 O’Meara, R. A. Q., 152(104), 209 Onoue, K., 62, 80, 90, 91, 102 Opie, E. L., 206(483), 220 Oppenheim, J. J., 28(114), 48 Orange, R. P., 107, 109, 111, 112, 115, 116, 118, 120, 125, 126, 127, 128, 129, 132, 134, 135, 136, 139, 140, 142, 143, 144, 194(483a), 220 Orlans, E., 69, 71, 72, 102 Osbahr, A. J., 171(484), 172(37), 207, 220 Osebold, J. W., 86, 97, 102 Osler, A-G., 180(344), 188(485), 189 (485), 216, 220 Osserman, E. F., 78, 104 Osterland, C. K., 75, 79, 99 Ota, R. K., 185(510), 221 Ovary, Z., 20( 103), 26( 113), 27( 113, lie), 28( l l s ) , 35( lie), 44( 180), 48, 50, 85, 98, 102, 121, 136, 137, 141, 143, 188(485), 189(485),205 (66), 206( 109), 208, 210, 220 Owen, R. D., 254, 263, 284, 266, 272, 273,292, 295 Owren, P. A., 147(199, 255), 212, 214 Ozer, J. H., 286, 295

P Oates, J. A., 187(733), 188(733), 172 (732, 733), 176(733), 178(733), Packham, M. A., 188(486), 187(213, 452, 454, 486). 188(486), 200 198( 733 ) , 227 (454), 202(454), 205(486), 212, Oberdod, A,, 191(229), 213 219, 220 O’Brien, J. R., 149( 480), 159( 479 1, Pagano, J. S., 242, 248 220 Page, I. H., 165(513), 169(487), 197 Odell, T. T., Jr., 254,295 (eOl), 220, 221, 223 Oettgen, H. F., 248, 247,250 Page, L. B., 7(22), 12(22), 46 Ogilvie, B. M., 123,142 Painter, B., 55, 99 O’Gorman, P., 291, 294 Ogston, C. M., 155(481), 161(482), Pait, C.F., 5 ( 8 ) , 45 Palade, G.E., 165(381, 382), 169(382), l62( 482), 220 189(382), 217 Ogston, D., 155(481), 161(482), 162

AUTHOR INDEX

Palm, J., 252, 254, 284, 295, 296 Pahner, J. L., 66, 74, 84, 98, 102 Pan, I. C., 86, 102 Papermaster, B. W., 58, 57, 59, 60, 61, 62, 64, 95, 100, 102, 104 Papermaster, C. W., 291, 296 Pappas, G. D., 198(488), 220 Pappenheimer, A. hl., Jr., 8(24), 46 Parish, W. E., 117,139,143 Park, E. A., 165(275),214 Parker, C. W., 7(44), 10(44,44a), 46 Paronetto, F., 205( 489), 206( 489), 220 Patch, M. J., 157(512), 221 Pate, D., l60(694), 226 Patel, R., 279, 280, 295, 296 Patterson, P. Y.,36( 160), 50 Patterson, R., 88,102 Paul, M. H., 166(579), 223 Paul, W. E., 10(41), 17(91), 18(91), 21(41), 22(41), Ze(91), 27 (116), 28( lie), 35(l l 6 ) , 37( l a ) , 38( 170), 39( 176, 178), 41(41, 191), 46, 48,50 Pauhng, L., 5 ( 14), 29( 122), 46, 49 Payne, R., 258, 263, 284, 266, 270, 272 273, 289, 290, 292, 293, 295 Peacocke, N., 290, 292 Pearce, C. W., 281,295 Pearce, R., 258, 280, 282, 297 Pearl, M. A., 281, 295 Peart, W. S., 203( 502), 221 Pechet, L., 157(5 ) , 207 Pedersen, K. O., 90,100 Pence, L. H., 29( 122), 49 Pensky, J., 163(544), 168(544), 173 (332). 174(544), 179(458), 181 (458), 183(192a, 331, 332, 490, 491, 492, 544), 184(491, 544), 201 ( W ) ,212, 216, 220, 221, 222 Perchalski, J. E., 79, 103 Perisutti, G., 160(429), 219 Perkins, H. A,, 279,295 Permin, P. M., l62( 19), 207 Pernis, B., 13(55), 14(61), 47 Petersen, V. P., 204(303), 215 Petersen, W., 180(281), 191(282), 192 (281, 282), 214 Peterson, B. H., 14(73), 15(73), 47 Peterson, R. D. A., 240, 248

317

Peterson, R. F., 111,143 Petroff, J. R., 167(493), 221 Pettinger, W., 167(733), 168(733), 172 (733), 176(733), 178(733), 198 (733), 227 Phelps, P., 178(494,495), 221 Philips, M., 163(2), 206 Phillips, G . B., 106, 109, 126, 143 Phillips, J. H., 52, 100 Picard, J., 81, 82, 103 Picken, M. E., 183(251),213 Pickles, V. R., 107, 114, 143 Pierce, A. E., 86, 103 Pierce, G. E., 247, 249 Pierce, J. V., 167(707, 708), 168(497, 706, 707), 169(496, 498, 708), 172 (706), 221, 226 Piggot, P. J., 84, 103 Pillemer, L., 163(540), 179(499), 180 (333, 338, 499, 5 0 0 ) , 181(335, 336), 186(545), 216, 220, 221 Pilling, J., 160(62), 208, 217 Pina, M., 235, 250 Pinckard, R. N., 70, 99 Pink, J. R. L., 84,102, 103 Piper, P. J., 117, 119,143 Pless, J., 168(72), 208 Pletscher, A., 148(613), 224 Ploug, J., 161(304), 215 Podliachouk, L., 83, 103 Polasa, H., 235, 250 Pollara, B., 59, 64,95, 103 Pomerangz, J. R., 20( 98), 48 Pope, L., 155(546), 222 Porter, K. A., 203(501, 502), 221 Porter, R. R., 84, 87, 88, 90, 98, 100, 103, 104 Potter, M., 84, 92, 93, 94, 99, 101, 102, 103 Poulsen, J. E., l61(89), 209 Pozerski, E., 159( 129), 101( 129), 210 Prahl, J. W., 80,84,88,103 Prendergast, R. A., 61, 78, 101, 103 Prentice, C. R. M., 153(503, 505), 157 (504), 221 Press, E. M., 84, 103 Pressman, D., 5(12, 13, 141, 7(19), 16 (83a), 29( 121, 122, 123, 125), 46, 48, 49, 62, 85, 90, 102. 104

318

AUTHOR INDEX

Price, A. R., 277, 278, 296 Prockop, D. J., 178(495), 221 Prowwartelk, O., 162(470), 220 Pruett, R., 196(309), 198(309), 199 (309), 215 Pruzansky, J. J., 86,102 Pryce, D. M., 175(361), 217 Puro, H. E., 177(506, 712), 221, 226 Putnam, F. W., 13(50), 47, 76, 80, 89, 93, 97, 103, 104

Q

Queng, J. T., 185(93), 209 Quick, A. J., 147(508), 158(507, 509), 195(510), 221

R Rabinowitz, Z.,242,250 Rabson, A. S., 239, 249 Radema, H., 79, 101 Raffel, S., 5(8), 9(27, 28), 45, 46 Ramseier, H., 252, 295 Ranadive, N. S., 135,143 Rand, M., 148(511), 165(511), 221 Randall, H. G., 188(485), 189(485), 220 Rapaport, F. T., 259, 273, 281, 282, 294, 295 Rapaport, S. I., 147(255), 157(512), 197(340), 198(340), 199(254, 340), 214, 216, 221 Rapp, F., 232, 234, 241, 248, 250 Rapp, H. J., 119, 120, 134, 143 Rapport, M. M., 148(738), 165(513), 221, 227 Rasch, C., 198(514), 197(514), 221 Rask-Nielsen, R., 90, 98, 103, 104 Ratchffe, H. E., 183(318), 184(319), 215 Ratnoff, 0. D., 144(527), 147(119, 599), 151(194), 152(81, 523), 153 (503, 505), 154( 147, 525, 530, 531, 535, 547), 155(147, 525, 533, 536, 540), 156(302, 520, 528, 534, 5351, 157(504, 523, 534), 158(249), 159 (77, 521, 522, 534), 160(334, 516, 519, 538, 542), 161(459, 482, 517), 102(482, 518, 520), la(16, 5401, 165(517), 168(150, 459, 54), 169

(149, 217), 171(77, 136, 541, 709), 172(136, 148, 524), 173(541), 174(541, 544), 178(217, 020), 180 333, 500, 548), 181(334, 335, 336, 338, 538, 542), 182(459, 539, 542, 543), 183(192a, 334, 538, 544), 184(544), 185( 146, 150, 539), 186 (545), 189(539), 620), 196(514), 197(514), 198(529), 199(S 2 ) , 201(544), 209, 210, 211, 212, 213, 215, 216, 220, 221, 222, 223, 226 Rauscher, F. J., 244,248 Ravdin, I. S., 195(157), 211 Reade, P. C., 52,103 Ream, V. J., 196(549), 222 Reed, W. P., 8(24), 46 Reemtsma, K., 281, 295 Regan, E. E., 159(602), 223 Reid, G., 148(511), 165(511), 221 Reid, H. A., 157(550), 222 Reimer, S. M., 198(657), 225 Reinert-Wench, U., 284,294 Reisfeld, R. A., 94, 97, 284, 285, 294 Relyveld, E. H., 122, 141 Renaud, S., 196(551), 222 Rendall, J. M., 203(502), 221 Reuse, J. J., 222 Richards, C. B., 71, 72, 102 Richards, F. E., 7( 22, 23), 12(22, 23), 46 Richards, R. K., 115, 142 Richardson, A., 253,286,296 Richardson, H. B., 165(275), 214 Riddle, J. M., 151(553), 222 Ridgway, H., 154(036), 224 Riha, I., 86, 99 Riley, J. F., 165(554), 222 Rittenburg, M. B., 27(118), 48 Robbins, B., ux)(555), 222 Robbins, J,, 186(556), 196(557), 222 Robbins, J. B., 92,98 Robbins, K. C., 150(558), 222 Robbins, S. H., 200( lll), 210 Roberts, M., 80, 102 Roberts, M. S., 61,104 Roberts, N. R., 112, 142 Robineaux, R., 175(127), 210 Robinson, R. R., 277, 278, 296 Rocha e Silva, M., 168(235, 562), 170

319

AUTHOR INDEX

( 187, 562), 176(560), 191( 559, 56l), 192(559), 212, 213, 222 Rockey, J. H., lO(43). 46, 75, 86, 87, 91, 99,101, 102, 103 Rodnan, G. P., 179(351), 216 Rodriguez-Erdmann, F., 198( 563), 199 (5M, 565, 566), 200(567), 201 (564), 222 Roe, J. H., 111, 143 Roehll, W., Jr., 154(212), 212 Roepper, E., 165(49), 208 Rossler, R., 116, 142 Rogentine, G., Jr., 285, 295 Rogers, H. J., 161(315), 215 Rogers, S., 235,250 Rogowiaka-Gorzelak, I., 155(471), 220 Rolfs, M., 256, 295 Ronwin, E., 161(568), 222 Rose, A. W., 53, 98 Rose, M. E., 69, 71, 72, 102 Rosen, F. S., 160(274), l68( 150), 181 (336), 183( 152), 184( 151), 185 (150, 152, 307), 190(307), 196 (569), 211, 214, 215, 216, 223 Rosen, V. J., 241, 249 Rosenberg, J. C., 198( 570), 223 Rosenblum, J. M., 154(547), 222 Rosenfeld, G., 168(562), 170( 562), 222 Rosenfield, G., 203( 627), 224 Rosenfield, R. E., 282, 295 Rosenquist, G. L., 68, 103 Rosenthal, N., 156( 571 ) , 223 Rosenthal, R. L.,156(571), 223 Ross, A., 183(337), 216 Ross, M. H. P., 198(488), 220 Rothfield, N. F., 79, 103 Rothschild, A. M., 171(572), 191(573), 192(574), 198(572), 223 Roman, B., 291,296 Rouiller, C., 151(684), 200(684), 201 (884), 226 Rowe, D. S., 80, 99,103 Rowe, W . P., 233, 237, 240, 245, 247, 248, 249, 250 Rowley, D. A., 165(575, 5761, 223 Rubenstein, E., 148( 577), 223 Rubenstein, H. M., 159(578), 223 Rubin, B. A., 235,248

Rubin, H., 240,250 Rubini, M., 241, 249 Rubinstein, P., 282, 295 Rudolph, A. M., 166(579), 223 Rugstad, H. E., 171(203), 177(203), 212 Rule, N. G., 152( 359), 216 Rusovici, L., 111, 112, 142 Russell, P. S., 262, 296 Russell, T., 176(418), 218 Rutishauser, U., 84, 103 Ryder, A., 159(358), 217

s

Sabin, A. B., 234, 250 Sachs, L., 231,242,250 S&er, L. B., 148(456), 187(456), 219 Sahiar, K., 32( 135),49 St. Pierre, R., 290, 297 St. Rose, J. E. M., 39(177), 50 Salk, E., 172(378), 217 Salmon, J., 195(580), 223 Salmon, S., 160(274), 214 Salvin, S . B., 20( 104), 38(167, 168), 48, 50 Salzman, E. W., 148(581), 223 Sanarelli, G., 198(582), 223 Sanders, B. G., 75, 94, 101 Sanderson, A. R., 259, 271, 291, 293, 296 Sandor, G., 79, 103 Sandrin, E., 188(72), 208 Santiago-Stevenson, D., 128, 143 Sanyal, R. K., 193(583), 194(583), 223 Sapira, J., 177(584), 223 Sarandon de Merlo, E., 122, 141 Sarelis, A., 159(602), 223 Sawyer, W. D., 163(608), 224 Sayers, R. R., 176(424), 219 Schachter, M., 167(585), l69( 59, 110, 586), 173(379), 174(588), 175 (379), 208, 210, 217, 223 Schaefer, S., 142 Schaffer, D. E., 175(587), 223 Schayer, R. W., 165( 588), 223 Schechter, J., 39( 174), 50 Scheraga, H. A., 150( 171), 152( leO), 211 Scherbel, A. L., l66( 589), 223 Schiffman, S., 157(512), 221

320

AUTHOR INDEX

Schild, H. O., 193(431, 432, 432a, Shanberge, J. N., 159( 602), 196(371), 433, 434), 219 217, 223 Schippers, A. M . J., 258, 282, 297 Shanbrom, E., 259, 297 Schlegel, J. V., 281, 295 Shapiro, J., 177(712), 226 Schlesinger, M., 254, 296 Shapiro, S. S., 153(6031, 162(370), 196 Schlossman, S. F., 6( l 6 ) , 46 (370, 371), 197(370), 198(370), Schmale, J., 80, 104 199(370), 201( 604), 217, 224 Schmerling, D. H., 150(155), 211 Sharp, A. A., 149(430), 219 Schmid, E., 256, 295 Shaw, A. R., 5(13), 46 Schmitz, A., 162(590), 223 Sheard, P., 117, 139, 143 Schoeff, G. I., 165(382), 169(382), Sheffer, A. L., 184(26, 27), 207 189(382), 217 Sheon, R. P., 154(530). 222 Schoemakers, J., 154(591a), 156(591), Sherman, R., 69, 102 223 Sherry, S., 152(609), 160(8, 192, 320), Schrohenloher, R. E., 90,103 l61(674), 162(7, 605), 163(6), Schubert, D., 93, 103 168(606), 181(674), 207, 212, 215, Schulman, I., 199( 113), 210 224, 225 Shimada, A., 284,285,295 Schultz, F., 167(196), 212 Shimamoto, T., 196(730), 227 Schultz, J. R., 114,143 Schultz, W. H., 192(593), 223 Shin, H. S., 190(6lO), 224 Schultze, H. E., 70,100 Shinoda, T., 80, 89, 93,103,104 Shipley, B. A., 161(439), 219 Schwartz, R. S., 32( 135), 36( 157), 49 Shore, P. A., 148(245, 264, 613), 149 Schwartzman, R. M., 75, 86, 91, 103 Schwick, H. G., 183(492), 221 ( M a ) , 186(612), 187(611, 612), Scudeller, G., 279, 281, 293, 297 213, 214, 224, 225 Shorley, P. G., l69(llO), 210 Seastone, C . V . , 36( 154), 49 Shreffler, D. C., 253, 288, 296 Seegal, B. C., 223 Shulman, N. R., 160(387), 163(592), Seegers, W., 128, 135,143 217, 259, 203, 264, 266, 272, 273, Seegers, W. H., 147(595, 700), 152 283, 292, 296 (596), 153(701), 160(59'7), 172 Shuster, J., 81, 103, 104 (222), 213, 223, 226 SeegmiUer, J. E., 176(408), 177(408, Shwartzman, G., 200( 614), 202(614), 224 598), 178(408, 598), 218, 223 Sickles, G. M., 202(615), 224 Segel, N., 189(65), 208 Sicuteri, F., 165(617), l69( He), 186 Seibert, C., 66, 99 (616), 224 Seibert, R. H., 147(599), 223 Siemsen, J. K., 197(340), 198(340), Seigler, H. F., 273, 277, 278, 292, 296 199(340), 216 Seki, T., 92,93,103 Sela, M., 14(71), 15(71), 29(119), 39 Siew, S., 151(693), 226 Sigel, M. M., 61, 62, 63, 64, 98, 103 (174), 47, 49, 50 Seligmann, M., 61, 79, 80, 102, 160 Silver, W. K., 252, 296 Silverstein, A. M., 86, 103 (478), 220 Sell, S., l6(84,S), 17(87, 88, 89), 48, Simms, E. S., 7(21), 11(49), 12(49), l6( 83a), 46, 47, 48, 75, 92, 93, 99 85, 99 Simon, G., 151(618, 684), 200(884), Sknyi, A,, 187(453), 219 201( 684), 224, 226 Sercarz, E., 23( 108), 48 Simon, J., 186(58), 187(58), 201(58), Seymour, J. L., 184( 27), 207 208 Shafrir, E., 154(600), 223 Simon, S. E., 162(183), 212 Shainoff, J. R., 197(601), 223

AUTHOR INDEX

Singal, D. P., 279, 280, 288, 291, 295, 296 Singer, S. J., 69, 72, 80, 96, 98, 99, 103 Siqueira, M., 186(619), 187(619), 224 Siskind, G. W., 5 ( 15), 6( 15),9( 15, 40), lO(15, 40, 41, 47), ll(40, 47, 50), 17(91), 18(91), 20( 95), 21( 15, 40, 411, 22(40, 41, 47), 23(95), 28( 91), 27( lie), 28( 116), 29 (1241, 30(129), 31(15, 129, 132). 32(40, 129), 33(136), 35(H e ) , 36(159, 1601, 37(162, 163, 185), 38(95, 166, 170), 39( 176, 178), 41 (15, 40, 41, 129, 191), 44(179), 46, 47, 48, 49, 50, 67, 99 Sjogren, H. O., 229, 230, 231, 232, 233, 242, 243, 246, 249, 250 Skipski, V. P., 111,143 Skirgaudas, J., 177(506) Slettenmark, B., 232, 250 Sloane, E. M., 170(175, 176). 211 Sloane-Stanley, G. H., 109, 142 Small, P. A., 61, 62, 83, 64,65, 68, 98 Small, P. A., Jr., 79, 90,92, 98, 101, 103 Smink, R. D., Jr., 178(620), 189(620), 224 Smith, E. L., 87,101, 103 Smith, P. E., 281. 295 Smith,P. M., 283,294 Smith, R. T., %(la), 49, 197(663), 198(821), uw)(860), 224, 225 Smith, S. P., 200( 622), 224 Smith, W., 158(823), 224 Smith, W. G., 112, 132, 142, 143 Smyth, D. G., l68( 1691,211 Smyth, D. S., 59,103 Snell, G. D., 253, 254, 276, 294, 296 Snyderman, R., 190(610), 191(624), 224 Solomon, A., 78,79, 103 Soulier, J. P., 160(4), 207 Southworth, J. G., 273, 277, 278, 284, 286, 288,292, 294, 296, 297 Spaet, T. H., 158(625), 187(626), 224 Spanoudic, S., 203( 627 1, 224 Sparrow, E. M., 166(628), 173(724), 224, 227 Spector, W. G., 151(634), 184(631, 632), 166(630), 173(829), 174

321

(635), 176(633), 194(633, 635), 224 Speer, R. J., 154(636),224 Spencer, R. A,, 284, 292 Spero, J. A., 179(351), 216 Spiegelberg, H. L., 58, 79, 85, 97, 104 Spinelli, A., 193(637), 225 Spink, W. W., 197(638), 225 Spitzer. J. M., 197(340), 198(340), 199 (340), 216 Spragg, J., 7(22), 12(22), 46 Stacey, R. S., 148(241), 213 Stachurska, J., 160(469), 215, 220 Stacy, R. S., 148(75), 209 StafEord, J. L., ed., 163(839), 225 Stark, K., 175(306),215 Stark, O., 254, 296 Starzecki, B., 168(397), 218 Stanl, T. E., 203(640), 225, 281, 296 Stavitsky, H. B., 62,98 Stechschulte, D. J., 112, 113, 120, 123, 129, 134, 136, 137, 138, 140, 143, 144 Steeves. R. A., 231, 250 Stein, S., 76, 104 Steiner, L. A., 3(48), 7(21), 11(48), 27(118a), 46, 47, 49, 84, 98, 104 Steinmek-Kayne, M., 75, 101 Stelos, P., 62, 102 Stephens, J. M., 54, 104 Steplewski, Z., 235, 236, 250 Sterzl, J., 21(107), 23(107), 48, 86, 99, 103 Stetson, C. A,, 202(644, 864, 6651, 225, 252, 293 Stetson, C. A., Jr., 186(556, 841), 196 (557, 6421, 200(642), 202(327, 841, 643), 203(327, 843), 205 (841), 206(641), 216, 222, 225 Stevens, L. T., 165(845). 225 Stewart, J. W., l69( 16, 846), 176(15), 178( 15), 207, 225 Stickel, D. L., 277, 278,296 Stiffel, C., 14(72), 15(721, 47 Stimpfiing, J. H., 254,276,284,286,288, 293,294,296 Stinebring, W. R., 193(278), 214 Stjernswiird, J., 248,249 Stobo, J. D., 79,104

322

AUTHOR INDEX

Stockert, E., 237, 248, 250, 286, 288, 293 Stonington, 0. H., 281, 296 Streilein, J. W., 252, 295 Stresemann, E., 117, 142 Strobel, H., 188(55), 208 Stoff, J., 179( 173), 211 Stone, S. H., u)(101), 48 Stroud, R. M., 179(647), 225 Stuck, B., 242, 250 Stiiber, B., 201(648), 225 Stylos, W., 29( lu)), 49 Subra Row, Y.,129, 142 Suhaciu, Gh., 111, 112, 142 Summerell, J, M., 284, 296 Suran, A-A., 61, 62, 64, 95, 103, 104 Svejgaard, A., 268, 288, 290, 296 Swahn, B., 62, 100 Swanson, L. W., 154(%6), 214 Swart, A. C., 153(248), 213 Sweatman, W. J. F., 115, 143 Swisher, S. N., 86, 101 Szenberg, A., 13(58), 47 Szeto, I. C. F., 179(351), 216

T Tager, M., 158(649, %O), 225 Tagnon, H. J., 160(234, 651), 213, 225 Taguchi, F., 235, 248 Taichman, N . S., 186(442), 187(442), 195(442), 219 Takatsuki, K., 76,104 Takemoto, K. K., 232, 234, 249, 250 Takeuchi, Y., I%, 142,143 Talmage, D. W., 3(3C), 9(30), 45, 46, 67, 104, 281, 296 Tan, M., 179( 173). 211 Tarail, M. H., 61, 62, 64,104 Taushce, F. G., 254,295 Taylor, F. B., Jr., leO(651), 161(652), 181(652A), l82( 653), 190(654), 224, 225 Taylor, G., 244, 250 Tenenhouse, H. S., 69, 70, 104 Terasaki, P. I., 203(840), 225, 259, 263, 284, 268, 272, 273, 279, 280, 286, 298, 291, 292, 295, 296, 297 Terres, G., 38( 158),49

Terry, L. L., 148(245), 213 Terry, M. C., 5(8), 9(27, 28), 45, 46 Terry, W. D., 61, 75, 76, 82, 97, 100, 104 Tevethia, S. S., 232, 241, 248, 250 Thal, A., 196(309), 198(309), 215 Thean, P. C., 157(550), 222 Theis, G. A,, 20(95), 23(95), 38(95, let?), 48, 50 Thelin, G. M., 152(655), 225 Thiele, F. H., 180( 656), 225 Thomas, D. P., 198(657), 225 Thomas, L., 36(160), 50, 158(658), 179( 215), 180(548), 198(91, 214, 488, 621, 741), zoO(90, 660). 201 (215), 202(659, 661, 664), 203 (215), 206(659), 209, 212, 220, 222, 224, 227 Thomas, 0. C., 185(93), 209 Thon, I. L., 108, 109, 111, 126, 141, 143 Thorbecke, G. J., 35( 146), 36( 151, 159), 37( 151), 38(166), 39(178), 49, 50, 86,103 Thorn, G. W., 281, 294 Tidball, M . E., 149(666), 186(612), 224, 225 Tiilikainen, A., 268, 296 Tillett, W. S., 161(687),225 Tiselius, A., 87, 104 Tissot, R. G., 255,293 Titani, K., 93, 103 Tockstein, G., 235,250 Todd, A. S . , 161(669), 162(668), 225 Todd, E. W., 179(332), 216 Toh, C. C., 148(270), 214 Tomasi, T. B., 78, 79, 104 Tomich, E. G., 148(245, 613), 213, 224 Tominaga, K. T., 13(56), 47, 76, 97 Tosi, R. M., 263, 268,283, 293, 294 Trautschold, I., 167(671), 1@(670), 183(670), 198(670), 225 Travis, B. L., 161(205), 212 Treffers, H. P., 9(28), 46, 71, 104 Trentin, J. J., 231, 244, 250 Trethewie, E. R., 106, 137, 142 Triantaphyllopoulos, D. C., 160( 672, 673), 225

AUTHOR INDEX

323

Tridente, G., 281, 293 van Leeuwen, A., 258, 262, 280, 282, Tripp, M., 258, 291, 293, 295 285,290,293,296,297 Tripp, M. R., 53, 104 Van Orden, D. E., 71,104 Trnka, Z., 65, 104, 263, 264, 266, 272, van Rood, J. J., 256, 258, 262, 263, 264, 273, 292 266, 272, 273, 280, 282, 285, 290, Troll, W., 152(209), l 6 l ( 674), 181 292,293,295,296,297 (674), 193(742), 195(742), 195 Van Vunakis, H., 152(425), 219 (742), 197(742), u)1(742), 206 van Zwet,T. L., 79,101 (742), 224,225, 227 Vas, M.R., 252, 292 Vasquez, J. J., 14(77), 47 Troquet, J., 169(322), 215 Troup, G. M., 259,297 Vassalli, P., 13(57), 14(70, 75), 15 Trump, G., 53, 99 (70), 47, 151(684), 186(58), 187 Turner, H. C., 88, 233, 234, 247, 249 (58), u)0(684), 201(58, 684), 204 Tyan, M. L., 288, 295 (683), 205(365, a), 208, 217, 219,225, 226 Tye, M. J., 161(674), 176(675), 225 Vaughan, J. H., 86, 101, 233, 248 Vaughan, J. W., 192(684a), 226 U Vaughan, V. C., 192( 684a), 226 Udenfriend, S., 194(691, 692), 193 Vaughan, V. C., Jr., 1 9 2 ( W a ) , 226 ( 691), 226 Vazquez, J., 177(5O6), 221 Uhr, J. W., 7(18), 17(20), zO(lOS), Vazquez, J. J., 151(685), 226 30( l26), 31( 126, la),32( 106, Vernier, R. L., 203(416), 218 la),64, 65, 68, 46, 48, 49, 104, Vessey, R. E., 129, 142 206(676), 225 Vick, J. A., 197(638), 198(250), 213, Unanue, E. R., 72,104 225 Ungar, G., 191(678), 192(677, 680, Visetti, M., 279, 281, 282, 293, 297 681), 193(679), 225, 226 Viza, D. C., 285, 294 Uphoff, D. E., 252, 296 Vogt, P. K., 237, 238, 249 Uriuhara, T., 186(442), 187(442), 195 Vogt, W., 106, 107, 143, 167(686), 169 (442), 219 (687), 226 Utsumi, S., 59, 103 Vojtiskova, M., 254, 294 Uvniis, B., 106, 107, 108, 109, 111, 126, Voklers, W., 258, 282, 297 141, 142, 143, 194(97), 209 Volk, B. W., 197(688), 226 Von Felten, A., 151(689), l62( 689), V 226 Von Kaulla, K. N., 163(690), 226 Vaerman, J-P., 78, 90, 91, 104 Valentine, M. D., 113, 120, 125, 126, Von Korff, R., 197(663 1,225 128, 129, 136, 140, 141, 143, 194 von Liitzow, A., 163(138), 210 von Roden, P., 167(721 1,227 (483a), 220 Vooys, W. H., 282, 296 Vallotton, M.,7(22), 12(22), 46 Voss, E. W., 90, 104 Van Arman, C. G., 170(282), 226 Vredevoe, D. L., 259,297 van Blankenstein, M.,258, 282, 297 Vandebroek, G., 81, 82, 103 W vander Does,J. A,, 280,297 Waalkes, T. P., 194(691, 6921, 195 Vanderheiden, J. F., 199( 113), 210 (691), 226 Van der Scheer, J., 5 ( 6 ) , 45, 87, 104 van der Weerdt, Ch. M., 283, 292, 296 Wachman, J., l 6 l ( 674), 181(674), 225 Waddell, W. R., 281, 296 Vane, J. R.,117, 119, 143 Wagner, B. M., 151(693), 226 Van Holde, K. E., 74, 84, 102

324

AUTHOR INDEX

Wagner, R. H., 147(301), 152( 655), 160(894), 215,225,226 Walford, R. L., 256,259, 261, 283, 284, 266, 272, 273, 275, 279, 288, 292, 294, 297 Walker, J. G., 9(40), 10(40), 11(40), 21(40), 22(40), 30( 129), 31(129), 32(40, 129). 41(40, 129), 46, 47, 49 Wallach, D. F. H., 286,295 Wall&, P.,160(695), 226 Walter, J. B., 195(696), 226 Walton, K. W., 200(697), 228 Wmg, A-C., 81, 103,104 Ward, F. E., 263, 273, 277, 278, 286, 288, 292, 296, 297 Ward, P. A., 160(699), 178(699), 182 (698, 699), 190(654, 698, 699), 225, 226 Ware, A. G., 147(700), 152(223), 153 (701), 172(222), 213, 226 Warner, E. D., 154(256), 214 Warren, B. A., 162(702), 226 Warren, K. S., 155(296), 171(298), 177 (297, 703), 215, 226 Warren, L., 112,143 Wassermann, M., 188(300), 215 Waters, E. T., 195(277), 214 Watson, D. W., 86,101 Weaver, R. A., 287, 297 Weber, E., 199(704), 226 Webster, M. E., 168(497, 705, 706, 707), 169(498), 171(709), 172 (706), 176(408), 177(408), 178 (408), 184(319), 215, 218, 221, 226 Webster, R. G., 3( 139), 33( 137, 138, 139),34(138,139), 49 Wedgwmd, R. J., %,98 Weeks, J. R., 114,141, 143 Weigle, J., 258,295 Weigle, W. 0..39(171, 172, 173), 50 Weil-Malherbe, H., 148(710), 226 Weinheimer, P., 55,99 Weir, R. C.,87, 88,104 Weiser, R. S., 231, 248 Weiss, D. W., 240,249 Weissbach, H., 194(691, 692), 195 (691), 226

Weissman, G., 178(711), 226 Wendt, V. E., 177(712), 226 Wenner, W. F., 194( 713), 226 Werblin, T., 10(47), 11(47), 22(47), 38(170), 46, 50 Werle, E., 181(400), 167(312, 671, 714, 715, 716, 717, 718, 719, 721), 188 (670, 716), 169(714, 716), 183 (670), 198(870), 215, 218, 225, 226, 227 Wessler, S., 196(549), 198(657), 222, 225 West, G. B., 165(554), 193(583), 194 ( 583), 222, 223 Westphal, H., 242, 250 Wetterquist, H., 185(362), 186(218, 362), 213, 217 White, J. G., 148( 126), 210 White, R. G., 14(76), 20( 102), 47, 48, 121, 137, 143 Whittingham, S., 203( 371a), 217 Wichels, P., 256, 297 Wicker, R., 235, 249 Wiese, W. H., 245, 249 Wigzell, H., 31( 131), 49 Wikler, M., 80, 89, 93, 103, 104 Wilander, D., 159(257), 214 Wilhelm, D. L., 164(722), 166(422, 628), 173(45, 167, 421, 423, 724), 174( 167), 175( 167, 379, 421), 183 (421), 194(723), 208, 211, 217, 218, 219, 224, 227 Wilkinson, P. C., 20( 102), 48, 121, 137, 143 Williams, C. A., Jr., 81, 104 Williams, G. M., 203(725), 227, 297 Williams, H. E., 198(487), 220 Williams, J. R. B., 162(726), 227 Williams, J. W., 72, 104 Williams, W. J., 152(727), 227 Willoughby, D. A., 151(634), 164(630, 831, 832), 166(630, 6341, 174 (835), 176(633), 194(633, 6351, 224 Wilner, G. D., 155(7281,227 Wilson, D. B., 252, 290, 296 Wilson, R. J. M., 123, 141, 144 Winkelmann, R. K., 176(128, 172, 4181, 185(417), 210, 211, 218

325

AUTHOR INDEX

Winn, H. J., 262, 296 Wider, F. C., 53, 98 Wistar, R., 74, 99 Wohler, I. M., 170( 176), 211 Wohler, J. R., 170(177), 211 Wolf, P. L., 177(506, 712), 221, 226 Wolfe, H. R., 9(37, 38), 46, 69, 71, 72, 98, 100 Wolff, H. G., 167(99), 176(98), 209 Wolman, M., 200( 51), 208 Wolstencroft, R. H., 26( 114), 48 Wong, D., 186(44a), 208 Wong, R. L., 1%(49), 208 Woods, K. R., 52, 104 Worowski, K., 155(471), 220 Wu, M., 112,142 Wunderlich, J., 84, 92, 99 Wurz, L., 180(338), 216 Wunel, M., 148(729), 227 Wyckoff, R. W. G., 87,104

Y Yabe, Y., 244, 250 Yagi, Y., 7 ( 19), 46, 62, 85, 90, 102, 104 Yakulis, V.,80, 104 Yamamura, Y.,80, 91, 102 Yamazaki, H., 196( 7301,227 Yang, H. Y. T., 170(731), 227 Yoshida, M. C., 242, 248 Yoshinaga, M . , la(246), 205( 246), 213 Youn, J. K., 244,247 Young, G. O., 14(65), 47 Young, J. Z., 74, 104 Young, P. E., 115, 142 Young, R. D., 244, 248

Young, W. J., 83, 98 Yount, W. J., 61, 78, 100 Yuda, N. N., 129, 142 Yunis, E., 266, 267, 292

Z Zachariae, H., 167(733), 168(733), 172 (732, 733), 176( 733), 178( 733), 198(733), 227 Zappacosta, S., 7( u>), 46 Zeller, E., 259, 297 Zervas, J. D., 287,294 Zigelbaum, S., 78, 104 Zilber, L. A., 229, 250 Zilliken, F., 154(591a), E 6 (591), 223 Zimmering, P. F., 10(46), 46 Zimmennan, B., 198(570), 23 Zmijewski, C. M., 270, 272, 277, 278, 289, 290, 296, 297 Zmijewski, H. E., 270, 289, 290, 297 Zolla, S., 87, 104 Zon, L., 148(734), 227 Zucker-Franklin, D., 147( 386), 217 Zucker, M. B., 147(385, 736), 148(735, 738), 149(385, 735), 182( 385), 217, 227 Zumpft, M., 252, 292 Zunker, H. O., 199(388), 217 zur Hausen, H., 246, 249 Zvaifler, N. J., 86, 104 Zweifach, B. W., 148(729), 165(739, 740), 166(740), 193(742), 195 (742), 197(742), 198(741), 201 (742), 206( 742), 227 Zweifler, A. J., 148( 743), 227

SUBJECT INDEX A

ribonucleic acid virus tumors, 2 3 6 238 Antigen-antibody reactions, blood clotting and, 186-188 Antigen selection hypothesis, summary,

Age, viral oncogenesis and, 238-239 Allograft rejection, mechanism of,

20%2oQ

Amphibia, immunoglobulins of, 65-66 Anaphylatoxins, inflammation and,

18S192

41-44

Anaphylaxis, passive cutaneous, 136 pathogenesis of, 192-195 Anaphylaxis slow reacting substance, see Slow reacting substance of anaphylaxis Antibody, &nity, tolerance induction and, 37-38 control of synthesis, humoral antibody and, 30-33 electrical charge, relationship to antigen charge, 29 highest d n i t y , production of, 25-29 humoral, control of antibody synthesis and, 3&33 invertebrate, question of, 52-56 serum, antigen dose and, 20-24 synthesis of K type, selective advantage of, 25-26 Antibody-binding affinity, definitions and concepts, M heterogeneity of, 5-8 Antigen( s ) , cross-reactive, immunological tolerance and, 3 8 4 1 dose, effects on serum antibody, 20-24 electrical charge, relationship to antibody charge, 29 human histocompatibility, 251-292 T or neoantigens, virus induced tumors and, 233-238 transplantation type or surface, in uitro techniques, 232-233 in vivo techniques, 230-232 virus-coded, oncogenesis and, 241-244 virus structural, deoxyribonucleic acid virus tumors, 236

Arthritis, inflammation and, 177-179 Arthus phenomenon, mechanism of,

205-206

B Birds, immunoglobulins of, 68-74 Blood clotting, see Hernostasis

C cells, immmolo&al release of Slow reacting substance of anaphylaxis and,

126-128

Clonal theory, general considerations,

12-13

Complement, C 1 esterase inhibitor and, 182183 functions of, 179-182 hereditary angioneurotic edema and,

183-186

D Delayed hypersensitivity, blood clotting and, 206 Deoxyribonucleic acid virus tumors, structural vim antigens, 236 2,4-Dinitrophenyl-protein conjugates, im~~~unization to* 25-28 secondary responses 28-29

E Endotoxin, effect of single injection, 196-198 Shwartzman reaction and, 198-203 C1 Esterase, inhibitor of, 182-183

F Fibrin, formation, hemostasis and, 149-

151

Fibrinogen, chemotaxis and, 151

827

328

SUBJECT INDEX

Fibrinoid, hemostasis and, 151 Fibrinolysis, 159-160 plasma inhibitors of plasmin and, 163 plasminogen activation and, 161-163 plasmin substrates and, 160-161 Fish, immunoglobulins of, 59-65 Foreign body reactions, inflammation and, 176-177

G Genetics, histocompatibility antigens and, 283-288 y-Globulins, structure of, 57-59 GranuIocytes, kinin formation and, 172173 Guinea pig, immunological release of slow reacting substance of anaphylaxis in, 137-139

H Hemostasis, antigen-antibody reactions and, 186188 fibrin formation and, 149-151 fibrinogen and chemotaxis in, 151 fibrinoid and, 151 inhibitors of, 158-159 kinin formation and, 170-172 platelets and, 147-149 snake venoms and, 157-158 staphylocoagulase and, 157-158 thrombin, 151-152 formation of, 152-157 trypsin and, 157-158 Hereditary angioneurotic edema, complement and, 183-186 Histamine, inflammation and, 164-165 Histocompatibility antigens, detection, leukoagglutination, 289-290 leukocytotoxicity, 290-292 platelet complement fixation, 292 future developments, 288-289 human, other than HL-A, 281-283 properties and genetic control, 283288 Histocompatibility systems, human HL-A system,

concept of single locus system, 256262 designated specificities, 262-269 procurement of alloantisera defining new Specificities, 269-273 major, 253-256 typing for HL-A factors in donor selection family members, 273-278 nonliving donors, 278-281 Human, HL-A histocompatibility system, concept of single locus system, 256262 designated specificities, 262-269 procurement of alloantisera defining new specificities, 269-273 immunoglobulins of, 7 4 8 1 immunological release of slow reacting substance of anaphylaxis in, 139140 other histocompatibility antigens, 281283 serum, necrotizing factor in, 175-176 tumors, viruses and, 241, 244-247 Immune response, control, further problems, 4 4 4 5 maturation, 8-12 electrical charge of antigen and antibody, 29 general considerations, 25 secondary responses, 26-29 selective advantage of certain cells, 2526 Immunoglobulins, amphibian, 65-66 avian, 68-74 fish, 59-65 light chains of, 93-96 mammalian, human, 7 4 8 1 other primates, 81-83 nonprimates, 83-93 reptilian, 66-68 slow reacting substance of anaphylaxis and, 121-126 synthesis of single type,

329

SUBJECT INDEX

lymphocytes and, 16-20 plasma cells and, 13-16 Immunological factors, viral oncogenesis, age and, 238-239 immunological tolerance and, 240 immunosuppression and, 239-240 tumors in man,241 Immunological tolerance, general characteristics, 35-37 induction, antibody affinity and, 37-38 response to cross-reactive antigens and, 38-41 viral oncogenesis and, 240 Immunosuppression, viral oncogenesis and, 239-240 Inflammation, 163-164 anaphylatoxins and related mediators, 188-192 anaphylaxis and, 192-195 arthritis and, 177-179 foreign body reactions and, 176177 histamine and, 184-165 human serum necrotizing factor and, 175-176 kinins and, 176 plasma inhibitors of kinins and, 169170 plasma permeability factors and, 173-175 polypeptide kinins and, 165169 serotonin and, 165-166 Invertebrates, antibodies, question of, 52-56

K Kinin( s ) , formation, blood clotting and, 170-172 granulocytes and, 172-173 plasmin and, 172 inflammation and, 176 plasma inhibitors of, 169-170 polypeptide, inflammation and, 166169

1 Leukoagglutination, histocompatibility antigens and, 289-290

Leukocytotoxicity, histocompatibility antigens and, 290-292 Lymphocytes, commitment to single immunoglobulin synthesis, 16-20

M Mammals, immunoglobulins of, 74-93

N Nephritis, clinical and experimental, 204-205 Neuraminic acid glycosides, slow reacting substance of anaphylaxis and, 112

0 Oncogenesis, viral, immunological factors in, 238241 virus-coded antigens, T antigens, 241-243 prevention and therapy, 244 transplantation antigens, 243-244 Original antigenic sin, basis of, 33-35

P Plasma, kinin inhibitors, in, 169-170 permeability factors, inflammation and, 173-175 plasmin inhibitors in, 163 Plasma cells, commitment to single inimunoglobulin synthesis, 13-16 Plasmin, kinin formation and, 172 plasma inhibitors of, 183 substrates of, 160-161 Plasminogen, activation of, 161-163 Platelet( s), hemostasis and, 147-149 Platelet complement fixation, histocompatibility antigens and, 292 Primates, immunoglobulins of, 81-83 immunological release of slow reacting substance of anaphylaxis in, 139-140

330

SUBJECT INDEX

R Reptiles, immunoglobulins of, 86-88 Ribonucleic acid virus tumors, s t r ~ c tural virus antigens, 23C238

s

Serotonin, inflammation and, 165-186 Serum, human, necrotizing factor in, 175-178 Shwartzman reaction, generalized, 198-201 local, 201-203 Slow reacting substance of anaphylaxis, immunological release, 119-121 cellular elemenk involved, 126-128 dissociation from histamine, 132135 immunoglobulins involved, 121-126 in vivo inhibition of, 128-132 passive cutaneous anaphylaxis and,

136

species and, 137-140 pharmacology, bioassay, 112-114 characterization by differential bioassay, 114-118 other effects, 119 permeability studies, 118-119 role in antigen-induced bronchoconstriction, 116-118 physical and chemical properties,

107-108

adsorption characteristics, 109-110

chromatographic separation, 111-

112

electrophoretic mobility, 110-111 neuraminic acid glycosides and, 112 solubility, 109 stability, 108-109 Snake venoms, hemostasis and, 157-158 Staphylocoagulase, hemostasis and, 157-

158

T Thrombin, formation of, 152-157 hemostasis and, 151-152 Trypsin, hemostasis and, 157-158 Tumors, human, and, 241, 244247 virus induced, surface antigens, 230-233 T antigens, 233-236

U Unresponsiveness, see Immunological tolerance

V Vertebrates, immunoglobulins, 56-96 light chains, 93-96 Viruses, oncogenic, immunological factors and,

238-241

CUMULATIVE INDEX-VOLUMES A

1-1 0

B Adaptive immunity, Bacteria, chemical suppression of, 6, 91 effect on antibody response, 4, 397 ontogeny and phylogeny of, 4, 1 gram-positive, cell wall antigens of, 4, Allergic encephalomyelitis, and autoim249 Blood groups in animals, 3, 315 mune disease, 5, 131 Anaphylaxis, C in vitro studies of, 3, 1 slow reacting substance of, 10, 105 Cells, transferred, antibody production Animals, blood groups in, 3, 315 by, 2, 205 Anti-antibodies, 6, 461 Cellular genetics, of immune responses, 2, Antibodies, 163 nahiral, and immune response, 5, 1 Cellular reactions in infection, 4, 117 reaginic, 3, 181 Cell wall antigens, of gram-positive bacAntibody, teria, 4,249 effect on immune response, 8, 81 Chemical suppression of adaptive imhumoral, role in homograft reaction, 3, munity, 6, 91 97 Complement, chemistry and reactions, 8, Antibody production, by transferred cells, 1 Complement system, functions of, 1, 131 2, 205 Antibody response, Conglutinin, 6, 479 effect of bacteria on, 4, 397 D in t i t r o studies of, 1, 211 Antigen ( s ) , Disease, autoantibodies and, 4, 351 embryological development of, 2, 309 heterophile, in host-parasite relationE ship, 3, 351 Embryological development, of antigens, in immune response, cell seIection by, 2, 309 10, 1 nonliving, immunological tolerance of, F 1, 67 Fibrinolytic phenomena, and inflammanucleic acids as, 6, 231 tory response, 10, 145 of virus-induced tumors, 10, 229 retained, role in immune mechanisms, G 3, 261 Genetic aspects of human histocompatitissue-specific, 5, 245 bility, 10, 251 Antigen-antibody complexes, biological Genetics of immunoglobulins, in mouse, action of, 1, 283 7, 91 Antigen-antibody reactions, in helminth y-globulins, heterogeneity of, 2, 41 infections, 2, 265 Antigenetic aspects of human histocom- Glomerulonephritis, experimental, pathogenetic mechanisms, 6, 1 patibility systems, 10, 251 Antigenic structure of tumors, 1, 345 H Autoantibodies and disease, 4, 351 Autoimmune disease, allergic encephalo- Helminth infections, antigen-antibody reactions in, 2, 265 myelitis and, 5, 131 331

332

CUMULATIVE INDEX

Hemostasis, and idammatory response, 10, 145 Heterogeneity of y-globulins, 2, 41 Homograft reaction, role or humoral antibody in, 3, 97 flost-parasite relationship, heterophile antigens and, 3, 351 Human histocompatibility systems, genetic and antigenetic aspects, 10, 251 Human reaginic allergy, in uitro studies of, 8, 183 Human tissue transplantation, 7, 275 Hypersensitivity, delayed, to protein antigens, 1, 319

Insulin, immunology of, 5, 209 I n uitro immunological responses of lymphoid cells, 6, 253 I n uitro studies of anaphylaxis, 3, 1 In uitro studies of antibody response, 1, 211 In uitro studies of human reaginic allergy, 8, 183

1 Lymphocytes, and transplantation immunity, 7, 189 Lymphoid cells, immunological responses of, in uitro, 6, 253

I Immune adherence, 3, 131 Immune mechanism, retained antigen in, 3, 261 Immune response, cell selection by antigen, 10, 1 cellular genetics of, 2, 163 effect of antibody on, 8, 81 natural antibodies and, 5, 1 Immunity, and inflammatory response, 10, 145 developmental aspects of, 6, 337 duration in virus diseases, 1, 263 transplantation, and tolerance, 1, 1 Irnmunoconglutinins, 6, 479 Immunoglobulins, biological activity of, 4, 287 genetics of, in mouse, 7, 91 phylogeny of, 10, 51 secretory, 9, 1 structure, biological properties of, 7, 1 Immunologic processes, ultrastructure of, 4, 175 Immunologic specificity, and molecular structure, 2, 1 Immunologic tissue injury, by neutrophilic leukocytes, 9, 97 Immunological paralysis, mechanism of, 8, 129 Immunological studies, with synthetic polypeptides, 5, 29 hmunological tolerance, of nonliving antigens, 1, 67 1mmun0I0gy of insulin, 5, 209 Infection, cellular reactions in, 4, 117

M Macrophages, structure and function, 9, 163 Mammalian tissues, mimetic relationships between streptococci and, 7, 147 Molecular structure, immunologic specificity and, 2, 1 Monocytes, structure and function, 0, 163 Mouse immunoglobulins, genetics of, 7, 91

N Neutrophilic leukocytes, immunologic tissue injury mediated by, 9, 97 Nucleic acids as antigens, 6, 231 NZB mice, immunology and pathology of, 9, 215

0 Ontogeny, of adaptive immunity, 4, 1

P Phagocytosis, 2, 241 Phylogeny of, adaptive immunity, 4, 1 immunoglobulins, 10, 51 Polypeptides, synthetic, immunological studies with, 5, 29 Protein antigens, delayed hypersensitivity to, 1, 319

R Reaginic antibodies, 3, 181

CUMULATIVE INDEX

s Secretory immunoglobulins, 9, 1 Slow :reacting substance of anaphylaxis, 10,105 Streptococci, Group A, and mammalian tissues, mimetic relationships, 7, 147

T Thymus, immunological significance of, 2, 111 Tissue-specific antigens, 5, 245 Transplantation, human tissue, 7, 275 Transplantation immunity,

333

and tolerance, 1, 1 lymphocytes and, 7, 189 antigenic structure of, 1, 345 virus-induced, antigens Of* ''9 229

U Ultrastructure, of immunologic processes,

4, 175

V Virus diseases, duration of immunity in, 1, 263