Advances in Immunology Volume 9

CONTRIBUTORS TO THIS VOLUME JOHN

BIENENSTOCK

CHARLES G . COCHRANE

ZANVIL A. COHN B. J. HEYLER

J. B. H o r n THOMASB. TOMASI, JR.

ADVANCES I N

Immunology EDITED BY

F. J. DIXON, JR.

HENRY G. KUNKEL

Diviiion of Experimental Pathology

The Rockefeller University

Scripps Clinic and Research Foundation la lolla, California

New York, New York

VOLUME 9 1968

ACADEMIC PRESS

New York and London

COPYRIGHT@ 1968,

BY

ACADEMIC PRESS,INC.

ALL RIGHTS RESERVED. NO PART OF THIS BOOK MAY BE REPRODUCED IN ANY FORM,

BY PHOTOSTAT, MICROFILM, OR ANY OTHER MEANS, WITHOUT WRTITEN PERMISSION FROM THE PUBLISHERS.

ACADEMIC PRESS, INC. 111 Fifth Avenue, New York, New York 10003

United Kingdom Edition published by

ACADEMIC PRESS, INC. (LONDON) LTD. Berkeley Square House, London W.l

LmMm OF

CONGRESS CATALOG CARDNUMBER:61-17057

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

JOHN

BENENSTOCK, State Unioersity of New York, Buffalo, New York (1)

CHARLES G . COCHF~ANE, Department of Experimental Pathology, Scripps Clinic and Research Foundation, La Jolla, California (97)

ZANVIL A. COHN,Department of Cellular Immunology, The Rockefeller University, New Yo&, New York (163) B. J. HELYER, Department of Pathology, Otago University Medical School, Dunedin, New Zealand (215)

J. B. HOWIE,Department of Pathology, Otago University Medical School, Dunedin, New Zealand (215)

THOMAS B. TOMASI, J R . , State University of New York, Buffalo, New York ( 1 )

V

PREFACE Each issue of the Advances in Immunology invariably presents some new frontier of the ever-burgeoning field of immunology. Volume 9 is no exception, and a wide diversity of subjects is included, The science of immunity has traveled far since TopIey wrote: “It wanders, a little uneasily between departments of pathology, bacteriology and hygiene.” One very new and rapidly expanding area is the immunology of the external secretions. This is covered by Thomas B. Tomasi, Jr. and John Bienenstock in the first chapter. The surprising discovery by Dr. Tomasi a few years ago that yA-globulin represents the dominant immunoglobulin of external secretions initiated the development of this field. It is of special interest that these yA-globulins are attached to another type of protein, secretory piece, that appears to protect them from enzymatic degradation. Some of the diverse and complex phenomena involved in immunological tissue injury are described in the second chapter. Particular emphasis has been placed on the neutrophilic leukocyte because of growing evidence for an important role for this cell type. Much of this evidence has been gained from the work of the author, Dr. Charles G. Cochrane, who has succeeded in dissecting the relative role of cellular factors as opposed to plasma factors in the mediation of certain types of immunological tissue injury. Particular attention has been paid to the significance of the various mechanisms to human disease. The subject of the monocytes and macrophages, dealt with in the third chapter, represents a much older area of interest to immunologists. It is surprising, however, how little is understood concerning the exact relationship to the immune response. The widely heId theory that they play a major role in the processing of antigen for the initiation of antibody formation has come under attack recently. Much more is known about their structure and phagocytic function, in considerable part due to the work of Dr. Zanvil A. Cohn. His chapter not only covers these subjects in detail but reviews the many controversial areas as well. The last chapter deals with the NZB strain of mice that has attracted the attention of investigators throughout the world as an experimental model for human connective tissue disease. Doctors Howie and Helyer, who have written this paper, first described, with Dr. Bielschowsky in 1959, the unusual disease of these mice. The similarity to human systemic vii

viii

PREFACE

lupus erythematosus is extraordinary, and information pertinent to the human disease is appearing at a rapid rate. Of particular significance have been the data concerning various modes of therapy which have been so difIicult to obtain in man. We are indebted to the authors for their diligence and perseverance in writing these informative chapters. Our editorial task was greatly facilitated by the close cooperation of Academic Press.

HENRYG.KUNKEL FRANK J. DIXON September, 1968

Contents of Previous Volumes Volume 1 Transplantation Immunity and Tolerance

M. H A ~ E K A., LENGEROV~, AND T. HRABA

Immunological Tolerance of Nonliving Antigens

RICHARD T. SMITH Functions of the Complement System

ABRAHAM G. OSLER In Vitro Studies o f the Antibody Response hRAM

B. STAVITSKY

Duration of immunity i n Virus Diseases

J. H. HALE

Fate and Biological Action

of Antigen-Antibody Complexes

WILLIAM0. WEIGLE

Delayed Hypersensitivity to Simple Protein Antigens

P. G. H. GELLAND B. BENACERRAF

The Antigenic Structure o f Tumors

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

FRED KARUSH Heierogeneity o f y-Globulins JOHN

L. FAHEY

The Immunological Significance o f the Thymus

J. F. A. P. MILLER,A. H. E. MARSHALL, AND R. G. WHITE

Cellular Genetics o f Immune Responses

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

CHARLES G. COCHRANE AND FRANK J. DIXON Phagocytosis

DERRICK ROWLEY xi

xii

CONTENTS OF PREVIOUS VOLUMES

Antigen-Antibody Reactions in Helminth Infections

E. J. L. SOULSBY

Embryological Development of Antigens

REED A. FLICKINGER AUTHOR INDEX-SUB JECT INDEX Volume 3 In Vifro Studies of the Mechanism

K. FRANK AUSTENAND

of Anaphylaxis

JOHN

H. H U M P ~ Y

The Role of Humoral Antibody in the Homograft Reaction

CHANDLER A. STETSON

Immune Asdherence

D. S. NELSON Reaginic Antibodies

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

DANH. CAMPBELLAND JUSTINES. GARVEY

Blood Groups in Animals Other Than Man

W. H. STONEAND M. R. IRWIN

Heterophile Antigens and Their Significance in the Host-Parasite Relationship

C. R. JENKIN AUTHORINDEX-SUB JECT INDEX Volume 4 Ontogeny and Phylogeny of Adoptive Immunity

ROBERTA. GOODAND BEN W. PAPERMASTER Cellular Reactions in Infection

EMANUELSUTERAND HANSRUEDY RAMS-

Ultrastructure of Immunologic Processes

JOSEPHD. FELDMAN

Cell Wall Antigens of Gram-Positive Bacteria

MACLYNMCCARTY AND STEPHEN I. MORSE

Structure and Biological Activity of Immunoglobulins

SYDNEYCOHENAND RODNEYR. PORTER

CONTENTS OF PREVIOUS VOLUMES

Autoantibodies and Disease

H. G. KUNKELAND E. M. TAN

Effect of Bacteria and Bacterial Products on Antibody Response

J. MUNOZ AUTHOR INDEX-SUB JECT INDEX Volume 5 Natural Antibodies and the Immune Response

STEPHENV. BOYDEN Immunological Studies with Synthetic Polypeptides

MICHAELSELA

Experimental Allergic Encephalomyelitis and Autoimmune Disease

PHILIPY. PATERSON

The Immunology of Insulin

C. G. POPE

Tissue-Specific Antigens

D. C. DUMONDE AUTHOR INDEX-SUB JECT INDEX Volume 6 Experimental Glomerulonephritis: Immunological Events and Pathogenetic Mechanisms

EMILR. UNANUEAND FRANK J. DIXON

Chemical Suppression of Adaptive Immunity

ANN E. GABRIELSON AND ROBERTA. GOOD

Nucleic Acids as Antigens

OTTOJ. F’LESCIA

AND

WERNER BRAWN

In Vifro Studies of Immunological Responses of Lymphoid Cells

RICHARDW. DWITON

Developmental Aspects of Immunity JAROSLAV

Si-mzL

AND

ARTHURM. SILVERSTEIN

Anti-antibodies

PHILLPG. H. GELL AND ANDREWS. KELUS

Conglutinin and lrnmunoconglutinins

AUTHOR INDEX-SUB JECT INDEX P. J. LACHMANN

xiii

xiv

CONTENTS OF PRtzVIOUS VOLUMES

Volume 7 Structure and Biological Properties of Immunoglobulins

SYDNEYCOHENAND CESARMILSTEIN

Genetics of immunoglobulins in the Mouse

MICHAELPOTTERAND ROSE LIEBERMAN

Mimetic Relationships between Group A Streptococci and Mammalian Tissues JOHN

B. ZABRISKIE

lymphocytes and Transplantation Immunity

DARCY B. WILSONAND R. E. BILLINGHAM

Human Tissue Transplantation

P. MERRILL AUTHORINDEX-SUB JECT INDEX JOHN

Volume 8 Chemistry and Reaction Mechanisms of Complement

HANSJ. M~LER-EBERHARD

Regulatory Effect of Antibody on the Immune Response JONATHAN

W. UHRAND

GORAN MOLLEX

The Mechanism of Immunological Paralysis

D. W. DRESSER AND N. A. MITCHISON

In Vifro Studies of Human Reaginic Allergy

ABRAHAMG. OSLER,LAWRENCE M. LICHTENSTEIN, AND DAVID A. LEVY AUTHORINDEX-SUB JECT INDEX

Secretory Immunoglobulins’ T H O M A S B . TOMASI. JR., AND J O H N BIENENSTOCK State Universify of New York. Buffalo. New York

.

I I1 111 IV

. . . V.

VI .

VII .

Introduction . . . . . . . . . . . . . Historical Aspects of Secretory Immunoglobulins and Local Immunity Evidence for the Existence of a Secretory Immunoglobulin System . Technical Problems Encountered in the Analysis of Secretory . . . . . . . . . . . Inununoglobulins . Secretions Involved in the Secretory Immunoglobulin System . . A . Digestive Tract . . . . . . . . . . . B. Respiratory Tract . . . . . . . . . . . C . Genital Tract . . . . . . . . . . . D. Urinary Tract . . . . . . . . . . . E . Mammary Gland . . . . . . . . . . . Chemical and Immunological Characteristics of Secretory . . . . . . . . . . . Immunoglobulins . A. Isolation . . . . . . . . . . . . B . Immunological Properties . . . . . . . . . C. Chemical Properties of Secretory Immunoglobulins . . . D. Three-Dimensional Conformation . . . . . . . E . Isolation and Characteristics of the Secretory “Piece” . . . Sites of Synthesis of Secretory Immunoglobulins . . . . . A. Immunological Studies . . . . . . . . . B Immunofluorescent Studies . . . . . . . . . C. In Vioo Radioactive Tracer Studies . . . . . . . D. Tissue Culture . . . . . . . . . . . E . Possible Mechanisms of Secretion of yA . . . . . . Biological Properties of Secretory yA . . . . . . . A. “Natural” Secretory Antibodies . . . . . . . . B . Secretory Antibodies Following Immunization . . . . C . Complement Fixation . . . . . . . . . . Secretory Immunoglobulins in Disease . . . . . . . A. Antibody Deficiency States . . . . . . . . B. Gastrointestinal Diseases . . . . . . . . . C. Rheumatoid and Antinuclear Factors . . . . . . D. Secretory Immunoglobulins in Respiratory Allergies . . . Secretory Immunoglobulins in Animals . . . . . . . A. Rabbit . . . . . . . . . . . . . R . Cow and Sheep . . . . . . . . . . . C . Dog . . . . . . . . . . . . .

.

VIII .

IX.

X.

’ Supported

by U S . Public Health Service Grant AM 10419.

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2 2 6 8 12 12 17 20 21 22 24 24 26 28 33 34 39 39 41 43 44 45 48 48 51 61 62 62 67 76 77 79 81 82 82

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THOMAS B. TOMASI, JR. AND JOHN BIENENSTOCK

D. Mouse . E. Conclusions XI. Summary . References .

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87

Introduction

Early work in immunology was devoted in large part to investigations involving the serological responses of man and animals to infections, and immunization with a variety of antigens. This serological emphasis has to some extent persisted so that serum titers of antibody are commonly considered to be representative of the efficacy of immunization. Although in some cases there appears to be a good correlation between serum antibody and resistance to infection, certain observations, which will be reviewed later in some detail, suggest that factors other than the level of serum antibody may be involved in recovery from and resistance to infections. These observations, as well as others, have suggested the concept of a local immune response and its possible importance in the resistance of the organism to infection. This review will attempt to summarize current knowledge concerning the antibodies found in various nonvascular secretions and their potential relationship to local immunity. Attention will, on the whole, be given to those secretions in which some characterization of antibodies involved has been undertaken. Although an enormous body of literature now exists concerning various antibacterial properties of whole secretions, in many cases the antibacterial activity has not been clearly shown to be due to antibody, and these reports will not be reviewed here. II.

Historical Aspects of Secretory Immunoglobulins and local Immunity

Besredka, in 1919, as a result of studies on experimentally induced oral infections by enterobacteriaceae and skin infections with Bucillus unthrucis, postulated that local immunity could be established independently of systemic immunity and that this was of great importance in the general resistance of the organism to infections originating in the gastrointestinal (GI) tract and skin. Davies, in 1922, demonstrated that specific antibodies could be found in the feces of patients suffering from dysentery, and similar findings on experimentally induced infections in animals suggested an important protective role for local GI immunity and fecal antibody in infections. In the late 1940’s, Burrows and coworkers (1947; Burrows and Havens, 1948) in a classic series of experiments investigating the effect of cholera vaccine in guinea pigs,

SECRETORY IMMUNOGLOBULINS

3

demonstrated a correlation between local fecal antibody ( coproantibody) and protection against experimental infection. They were unable, however, to find a good correlation between serum antibody titers and resistance to oral infection. Those animals having high serum titers following parenteral immunization showed systemic immunity as demonstrated by resistance to intracerebral challenge with the organism but were not resistant to oral infection unless coproantibody was present. In addition, these investigators showed that coproantibody and urinary antibody titers appeared to be independent of serum titers and, therefore, were probably not derived from serum directly by simple transudation. The independence of serum and local antibody is well demonstrated in Fig. 1. Studies in irradiated animals further highlighted the independent behavior of serum and fecal antibodies by demonstrating that following an appropriate dose of irradiation, fecal antibody response to cholera vaccine was inhibited whereas serum antibody levels remained essentially unchanged (Burrows et al., 1950a,b). Antibody mediated immunity in saliva was demonstrated by the presence of diphtheria antitoxin in the saliva both of naturally immune individuals and following immunization with toxoid (Sugg and Neill, 1931). In this report it was concluded that the antitoxin concentration in saliva was directly related to the concentration in the blood. Schubert ( 1938), however, in systematic studies involving both man and rabbits, concluded that the salivary antitoxin was independent of that in the serum as shown in Fig. 1. Moreover, Schubert (1938) found that the concentration of antitoxin in the saliva was of suEcient magnitude to be of potential importance in immunity against diphtheria and that the presence of antitoxin in human saliva correlated well with the Schick test-only Schick-negative individuals had significant concentrations of salivary antitoxin. Bull and McKee, in 1929, called attention to the possible participation of local antibody (mucoantibody) in the resistance of the respiratory tract to infection by reporting that rabbits could be rendered resistant to pneumococcal respiratory infections by prior intranasal immunization with the same strain. Resistance occurred in some animals in whom serum antibody could not be detected, and these authors concluded that this constituted “another instance of the resistance of tissues apparently local to invasion of bacteria in the absence of demonstrable antibodies in the blood serum.” Walsh and Cannon (1936, 1933) confirmed these findings and, in addition, showed that animals in whom serum antibody could not be detected, nevertheless, had acquired significant immunity. Extensive studies on the origin of bronchial mucoanti-

4

THOMAS B. TOMASI, J R . AND JOHN BIENENSTOCK

r 200

20001

a w

1500-

'150

z z

2

I-

1000- FECAL

'100

500-

-50 J

3

-I

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TOXOID

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FIG. 1. Studies illustrating independence of serum and secretory antibody titers. (Top) Serum and fecal cholera agglutinins in guinea pigs; (bottom) Diphtheria antitoxin content of serum and saliva of immunized rabbits. [Cholera data redrawn from Burrows et al. (1950a); saliva data redrawn from Schubert (1938).]

bodies have been carried out by Fazekas de St. Groth and his colleagues (1951; Fazekas de St. Groth, 1951) on experimentally induced influenza virus infection in mice. These workers showed that following intranasal instillation of the virus there was a simultaneous appearance of antibody in both serum and mucus and that the proportion of mucoantibody to serum antibody was over 10 times that produced by subcutaneous immunization. By the intranasal inoculation of an unrelated virus or

SECRETORY IMMUNOGLOBULINS

5

certain astringents (e.g., tannic acid), a high proportion of bronchial antibody could also be induced in mice vaccinated via the intraperitoneal route. This phenomenon called “pathotopic potentiation” is thought to result from a local increasc in capillary or tissue permeability caused by the inflammatory effect of these agents. This important work emphasized that a rise in mucoantibody titer may reflect either local antibody production or the extravasation of humoral antibody due to alterations in capillary permeability. There has been great interest in veterinary medicine in regard to the diagnostic significance of antibodies which appear in the secretions of the reproductive tract. Most extensively investigated have been local immunity to the three major infectious diseases causing infertility in cattle (trichomoniasis, vibriosis, and brucellosis). Byrne and Nelson (1939) showed in a study of experimental trichomoniasis in rabbits that acquired resistance to reinfection did not coincide with circulating antibody and that intravenous immunization, which was followed by the appearance of humoral antibody, did not confer immunity. Similarly, in cattle, antibodies to Trichomonas foetus in vaginal mucin ( mucoantibodies ) occur in the absence of circulating antibodies, whereas parenteral administration of antigen produced circulating but no mucoantibodies (Kerr and Robertson, 1953). In the studies of Kerr ( 1955) Brucella abortus antibodies were absent in the vaginal and uterine secretions in animals with circulating titers as high as 1:2560 when immunization was carried out by parenteral administration of antigen. However, the intrauterine instillation of Brucella abortus vaccine resulted in vagina1 titers of 1:280 with serum titers 1 : l O or less. Batty and Warrack ( 1955) using the Oakley technique (Oakley et al., 1955) have also shown the local production of diphtheria and tetanus antibodies in the vagina and uterus of rabbits. The appearance of antibody was correlated with the accumulation of plasma cells in the vaginal mucosa. The studies discussed above, as well as other early reports reviewed in more detail by Pierce ( 1959) and Tomasi ( 1965), have suggested the following. In certain infections, circulating antibody has relatively little significance or is only indirectly related to resistance to infection. Local immunity and mucoantibodies may be of primary importance in the defense against certain infections particularly those which are noninvasive and confhed to the mucous surfaces. In some cases, mucoantibodies may have a diagnostic significance which is superior to that of serum antibodies. The effectiveness of immunization may not always be assayed by measurement of humoral antibody.

6

THOMAS B. TOMASI, JR. AND JOHN BIENENSTOCK

More recent developments in the concept of local immunity and the existence of a secretory system have depended on the recognition of the heterogeneity of antibodies and the definition of the various immunoglobulin classes (for review, see Cohen and Milstein, 1967). Most important was the discovery by Heremans et al. (1959) of the yA class of antibodies which is now known to be the predominant class in external secretions. Gugler et al. (1958) appear to have first described by immunoelectrophoresis the occurrence of yA in the external secretion, colostrum. The systematic studies of Hanson (1961) clearly demonstrated the complexity of the immunoglobulins in human colostrum and the quantitative predominance of yA in this fluid. The observation that many different external secretions contain by quantitative measurement a marked predominance of yA (Chodirker and Tomasi, 1963), together with the fhding of a predominance of yA-containing plasma cells locally in a secretory gland (Tomasi et al., 1965), formed the basis for the suggestion of the existence of a more or less distinct immunological system characteristic of external secretions ( Tomasi et al., 1965). Other observations pertinent to this thesis are reviewed below. 111.

Evidence for the Existence of a Secretory Immunoglobulin System

Secretions can be conveniently classified into two groups-internal and external according to their immunoglobulin content ( Fig. 2). External secretions bathe the mucous membrane surfaces which are in communication with the external environment, such as the secretions of

YM Internal

YG

YA Y M Externol

FIG. 2. Relative concentrations of immunoglobulin classes-serum on left and external secretion on right. In external secretions the ratios of yG/yA vary with different secretions but are generally less than 1. In internal secretions the ratios of yG/yA are similar to that of serum (approx. 6 : l ) . yM is either undetectable or present in small amounts relative to serum. Secretions, characterized by immunoglobulin content, are listed below. Those secretions in which secretory yA was identified are starred. INTERNAL aqueous humor, cerebrospinal fluid, synovial, amniotic, pleural, peritoneal

EXTERNAL 'parotid, 'submaxillary, 'lacrimal, 'nasal, * tracheo-bronchial, 'gastric, 'small intestinal, large intestinal, bile, seminal, 'urine

SECRETORY IMMUNOGLOBULINS

7

the GI and respiratory tracts. Internal secretions have immunoglobulin contents showing a quantitative distribution similar to that seen in normal serum with a ratio of yG/yA of approximately 6: 1. In this group are included the aqueous humor of the eye, cerebrospinal fluid, synovial fluid, and pleural, amniotic, and peritoneal fluid, all of which occur in cavities having no direct continuity with the outside environment. The external secretions are produced by or bathe mucous membranes and are, therefore, mostly related to cuboidal and columnar-type epithelia. In contrast with the internal secretions, the immunoglobulin which predominates in the external secretions is yA with the result that the rG/yA ratio approaches unity and is commonly less than 1. The relative predominance of immunoglobulins in normal external secretions is mainly in the order: yA, yM, yG. However considerable variation in the relative amounts of yM vs. yG are found in different fluids. A major characteristic of all external secretions examined to date has been the predominance of yA in a special secretory form that differs in several of its physicochemical characteristics from the yA of serum. Under external secretions are now included parotid and submaxillary secretions, colostrum, lacrimal fluid, nasal secretions, bronchial secretions, intestinal fluids ( gastric, small and large intestinal), bile, urine, and seminal plasma. Justification for considering the secretory system as more or less distinct from that responsible for the production of circulating antibody is based on evidence which is briefly summarized below; a detailed discussion of the individual points will be Ieft for subsequent sections. The evidence for the existence of the secretory systems stems primarily from the folIowing observations. 1. In certain external secretions such as saliva, the albumin-toglobulin ratio differs markedly from that of serum, suggesting that plasma proteins are not present simply as a result of transudation from serum. 2. In the majority of external secretions thus far studied, yA is the predominant immunoglobulin class ( approximately 60-1W of the total immunoglobulins present), whereas this immunoglobulin is a minor component (10-20%) in serum. 3. The yA of external secretions differs chemically and antigenically from serum yA. 4. There is evidence that in certain external fluids, secretory yA is produced locally. The evidence for this comes primarily (although not solely) from studies involving the fluorescent antibody technique and the demonstration that y A-producing plasma cells predominate in the area just below the mucous membranes of the respiratory and GI tracts.

8

THOMAS B. TOMASI, JR. AND JOHN BLENENSTOCK

5. The apparent lack of correlation between the concentration of yA in serum and secretions in certain pathological conditions and during development following birth. 6. The frequent finding of a dissociation between both the levels and class of antibody activity in serum and secretions of healthy individuals and following infections or immunization. Recovery from certain infections, such as viral respiratory diseases, is better correlated with the levels of local antibody than with serum antibody. Likewise, evidence is available that resistance to certain infections following immunization is best correlated with secretory rather than serum antibody.

IV.

Technical Problems Encountered in the Analysis of Secretory Immunoglobulins

The concentration of an immunoglobulin in a secretion is best expressed in milligrams per 100 ml. of unconcentrated fluid. In many secretions the total protein concentration varies widely within the same individual, depending both upon the rate of fluid secretion as well as other factors. It is desirable, therefore, to measure the total protein content of a secretion as well as the volume collected over a given time period. Immunoglobulin concentration can then be compared with the total concentration of protein in the sample, or expressed, as in the case of the urine, as milligrams excreted per 1000 ml. or per 24 hours. Measurement of total protein on whole secretions by most of the commonly used techniques involving colorimetric reactions (such as the Folin or biuret) does not give absolute concentrations because of the technical difficulties encountered with color development ( Kabat and Mayer, 1987) and also because of the standards employed. Since secretions are complex mixtures of proteins, many of which have not been isolated, use of a single standard such as albumin or y-globulin leads to results which are at best relative. A technique based on staining of paper strips, partially avoids some of these difficulties (Heremans, 1958). In some fluids such as bile the inherent color of the fluid prevents the use of colorimetric procedures, and other techniques such as the paper strip or refractive index methods must be used. It is important to realize, therefore, that none of the available methods provide absolute values for the total protein content of a secretion. Most of the more commonly used immunological techniques for quantitating immunoglobulins in secretions are not sufficiently sensitive to be used directly on the unconcentrated fluid and, therefore, preliminary concentration is often necessary. Many of the methods available, such as the collodion bag negative pressure dialysis, pre-evaporation, and

SECRETORY IMMUNOGLOBULINS

9

lyophilization techniques, incur considerable loss at the surface of the membrane or can result in significant denaturation of proteins. Secretory yA antibody activity does, however, at least partially survive lyophilization and myeloma yA preparations can be lyophilized and still retain the majority of their antigenicity. Concentration procedures currently thought to be most effective in dealing with secretions are negative pressure dialysis in Visking s/&-in. membranes or the use of artificial membranes and positive pressure.2 Quantitation of immunoglobulins is most frequently performed by quantitative radial diffusion in which ring diameters obtained with the concentrated fluids (usually 10-50 times) are compared with the diameters produced by purified standards. For quantitation of yA in secretions, it is necessary to use a homogeneous, 11 S, secretory yA as a standard. Marked differences result if a 7 S serum yA standard is used. For example, a salivary specimen, which gives a ring diameter on radial diffusion of 8 mm. when read from an 11S secretory yA standard curve, gives a concentration of 18 mg.% With a serum 7 s yA standard the concentration is 5.4 mg.% Although use of an 11S yA standard is considerably more accurate in view of the fact that this is the predominant molecular size in most external secretions, absolute figures for yA concentrations are not obtained since, as discussed below, many secretions contain 7 S and polymeric (18-20 S ) yA in varying proportions. Complement fixation techniques for measuring yA in large part avoid the problem of size heterogeneity, but other technical problems particularly anticomplementary activity are encountered with certain fluids. A new technique, electroimmunodiffusion, has been successfully applied to the quantitation of immunoglobulins in secretions by Merrill et al. (1967). This procedure also requires an 11S yA standard but has the advantage that it is sufficiently sensitive to be used directly on unconcentrated fluids such as saliva. Accurate quantitation of *the other immunoglobulins such as yG and yM is compIicated by the presence of degradation products of these molecules due to proteolytic activity. For example, GI secretions, urine, as well as other secretions, frequently contain Fc and F’c fragments. Thus, the standard used cannot be representative of the whole range of molecular sizes to be found in these fluids which are still reactive with the antisera. If the standard used has a molecular size corresponding to the largest size to be found in that particular biological fluid, then the

’ Amicon

Corp., 21 Hartwell Ave., Lexington, Massachusetts.

10

THOMAS B. TOMASI, JR. AND JOHN BIENENSTOCK

concentration obtained can be considered to be representative of the maximum possible amount of the immunoglobulin present. Other technical problems using precipitation techniques involve the presence of nonspecific precipitin lines not due to specific immunological reactions. Gastrointestinal secretions and bile often give several nonspecific rings on radial diffusion plates. If normal serum is diffused against normal concentrated GI juice, two or three precipitin lines can sometimes be detected. Such reactions can cause great difficulty in quantitation and in other studies involving antigenic analysis on these fluids. In studies on biological fluids using antisera raised against the concentrated whole secretion and absorbed with normal human serum, claims have often been made of the demonstration of secretion-specific antigens not found in normal human serum. A major pitfall in this kind of interpretation results if the substance is present in small amounts in serum but in relatively high concentration in the secretion. An example of this is free L chains which can be readily detected in urine since they are selectively concentrated by the kidney, but are very difficult to detect in normal serum, although recent evidence has clearly shown their presence ( Berggard, 1961) . A major problem in immunological studies of the secretory system has been the preparation of specific secretory yA antisera. Preparations of 11s secretory yA which are homogeneous by all criteria applied, including Ouchterlony analysis with potent antiwhole serum and antiwhole secretion antisera, when injected into animals often result in antisera containing antibodies against one or more secretion-specific components. We have examined some twelve different secretory yA antisera and all, except one bleeding from one animal, contained antibodies against contaminants when carefully analyzed against highly concentrated secretions. The most common contaminants are lactoferrin and the macromolecular component ( MMC) shown in Fig. 3. Such antisera, unless properly absorbed to render them specific for secretory yA, are useless for fluorescent and other studies involving sensitive techniques (such as complement hation) for the detection of secretory YA. Another problem, which arises in attempting to evaluate in various diseases whether an antibody is produced locally or is derived from serum, involves the effect of the inflammation itself on the extravasation of antibody from serum-so-called pathotopic potentiation. It is necessary in attempting to distinguish between the local formation of antibody and pathotopic potentiation to measure the antibody activity in the

11

SECRETORY IhlMUNOGLOBULlNS MMC

Secretory y A

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+

FIG. 3. Schematic representation of some commonly encountered immunological reactions with secretion specific antigens. Antisera against apparently homogeneous preparations of secretory yA often contain antibodies against “impurities” such as lactoferrin and macromolecular component (MMC) or both in addition to anti-0-chain and antisecretory “piece” antibodies.

secretion against an unrelated “marker” antigen and, in the case of local immunization studies, to administer two antigens, as suggested by Oakley et al. (1955). It seems likely that, in the presence of high serum titers, pathotopic potentiation may obscure a relatively small contribution from local formation. Evidence for antibody activity in a given immunoglobulin class in external secretions has frequently been obtained by the use of indirect methods. For example, the distribution of antibody activity has been shown to parallel the distribution of a particular immunoglobulin class by gel filtration or density gradient ultracentrifugation. The demonstration of antibody activity in an apparently homogeneous immunoglobulin preparation, unless in very high titer compared with the whole secretion, is not su5cient evidence in itself to establish the immunoglobulin class of the antibody. The inactivation of antibody activity by reducing agents, such as P-mercaptoethanol, has often been used as a measure of yM antibodies. However, since serum yA polymers and in some species other immunoglobulins (Onoue et a?., 1966; Franklin, 1962) are sensitive to reduction, this criterion is not absolute. Secretory yA antibody activity is relatively resistant to reduction (see Section VI). These types of indirect evidence when taken together is what is referred to in Table VIII as suggestive evidence that an antibody activity is in a given immunoglobulin class. More direct evidence can be obtained employing indirect hemagglutination techniques utilizing antisera directed against immunoglobulin heavy chains, or b y absorption studies with specific antisera showing the removal of antibody activity subsequent to absorption of a particular immunoglobulin from thc secretion. Other direct methods involve the use of the indirect immunofluorescent technique and radioimmunoelectrophoresis. In the latter methods the antigen is L I S L I ~labeled ~ with I3’I, although other isotopes may be used. For

12

THOMAS B. TOMASI, JR. AND JOHN BIENENSTOCK

example, in the ingenious experiments of Hodes et al. (1964) the poliovirus, which is apparently small enough to diffuse through the agar gel, was internally labeled with 32Pincorporated into the viral ribonucleic acid (RNA). All the above mentioned “direct” techniques involve the use of specific antisera to demonstrate the immunoglobulin class of the antibody. In addition to utilizing immunoglobulin H-chain-specific antisera one can also use secretory “piece” (SP) -specific antisera in order to demonstrate whether SP is involved in a given antibody activity. This type of experiment is demonstrated in Fig. 14. V.

Secretions Involved in the Secretory Immunoglobulin System

A. DIGESTIVE TRACT In 1960, Gab1 and Pastner demonstrated the presence of several serum proteins including 7-globulins in saliva. Similar studies were reported in 1960 by Ellison, Mashimo, and Mandel. Kraus and Sirisinha, in 1962, suggested that an individually regulated transfer of serum proteins occurred into the oral cavity and based this hypothesis on a quantitative comparison of 7-globulins in saliva and plasma. A more recent study (Kraus and Konno, 1963) demonstrated a lack of quantitative correlation between titers of antibody in plasma and saliva. Investigations of the types of y-globulin found in saliva produced evidence that in common with other external secretions the yG/yA ratio in parotid secretions was less than unity ( Chodirker and Tomasi, 1963). Moreover, the fact that the 7-globulin/albumin ratio was 6 times that found in normal human serum (Tomasi and Zigelbaum, 1963) suggested that transudation was not the sole mechanism of secretion. Further confirmation that the yA immunoglobulin class predominated in human saliva was produced by Simons et al. (1964). Tomasi et al. in 1965 demonstrated that the salivary yA has a sedimentation coefficient of 11S and possesses unique antigenic properties. In retrospect, Patton and Pigman who in 1959 performed analytical ultracentrifugal studies on parotid, submaxillary, and sublingual secretions were the first to demonstrate the 11S component in all of these secretions, subsequently shown to be secretory yA. Parotid fluid has been the salivary secretion most studied because of the ease of obtaining samples free of contamination from other secretions. Mean concentrations of yA in parotid saliva of approximately 3 mg.% have been reported (Tomasi and Zigelbaum, 1963), however, the standard used for calibration was serum 7 S yA which would underestimate ( approximately threefold) that actually present. More recently,

SECRETORY IMMUNOGLOBULINS

13

Claman et (11. ( 1967) have reported a median value of 9.5 mg.% in normal healthy adults and have demonstrated little clifftwnce between the concentrations of salivary yA in samples taken sirnultaneously frorn right and Icft parotid glands in the same intlivitlual. However, considerable variation exists from day to day in the same individual and depends in part on whether collections are obtained postpraiidially and the type of stimulation employed. Mixed submaxillary and sublingual samples contain a Iittle more than half the concentration of yA found in parotid secretions. The concentration of salivary yA found in whole saliva, which represents the normal admixture of parotid, submaxillary, sublingual, buccal, and gingival secretions, is apparently similar to that found for parotid secretions (Lehner et aZ., 1967). However yG can be regularly detected in whole saliva, whereas the concentration of yG in most normal parotid fluids is very low, and like yM and yD can only be found if extreme concentration of samples is undertaken ( Claman et al., 1967). Immunofluorescent studies of salivary glands (Tomasi et al., 1965) have demonstrated the predominance of ykcontaining plasma cells in the interstitial areas between the glandular acini (see Table I ) . The average volume of whole saliva produced per day in a 70-kg. man is approximately 1000-1500 ml. (Best and Taylor, 1966). Assuming a yA concentration of 5 to 15 mg.X, then the amount of yA secreted per day in whole saliva is between 50 and 250 mg. This represents approximately 5 1 0 %of the total yA synthesized per day. It has been recognized by oral physiologists that the gingival pocket surrounding the tooth contributes a small volume of fluid to the oral cavity and, therefore, to whole saliva. This crevicular fluid has been shown to contain predominantly yG and the yG/yA ratio in the secretion is 8 : 1 similar to that found in normal serum (Brandtzaeg, 1965). The crevicular fluid should, therefore, not be considered as a typical external secretion although it does communicate with the oral cavity. It is pertinent, however, that the epithelium lining the gingival pocket is squamous in type and not the typical mucous membrane as seen with most other external secretions. In addition, the subepithelial plasma cells around the gingiva have been shown by the immunofluorescent studies of Brandtzaeg and Kraus (1965) to stain mainly with anti yG antisera. Immunoelectrophoretic studies of gastric fluids have demonstrated the presence of yG and yA immunoglobulins (Hirsch-Marie and Burtin, 1964; Tenerova et al., 1961; Hurlimann, 1963). Hirsch-Marie and Burtin showed that the principal y-globulins of gastric juice are yG and yA; y M was not found in this study. The yA in gastric juice was thought to be derived from saliva, and studies with antigastric juice antiserum ab-

TABLE I TISSUELOCALIZATION OF SECRETORY IMMUKOGLOBULINS AND SECRETORY “PIECE”BY IMMUNOFLUORESCESCE

Structure

Relative cellular predominance

Quant itat.ive ratios Distribution of ?Acontaining plasma yA y M yG cells Interstitial bet,ween acini

Parotid gland

yh

yM

yG

Submaxillary gland Gingival tissue

yA

yM

yG

yG

yA

no yM

Nasopharyngeal tonsil

yG

yA

yM = yD

Nasalmucosa

yA

yG

yM

3-8

Bronchial mucosa

?A

= yG

yM

5

Epithelial yA a

Epithelial ‘‘piece”b

-

+

Tomasi et nl. (1963)

+

Gelzayd et al. (1967a) Rossen et al. (196ic)

Occasional acinar

+

-

Lymphoid follicles Occasional stained with all antisew 1 Submucosal in Faint intercellular glandular areas and along secretory ducts

5

1

+-

Serous acinar

Reference

Brandtzaeg and Kraus (1965) Crabbe and Heremans (1967a) Brandtzaeg et al. (196’7)

+ + +

Rossen et al. (1967~) Martinez-Tello and Blanc (1968) Itossen et al. (1967~)

-

Lamina propria

Faint, intercellular Duodenum mid jejunum

yA

Appendix

yA

=

yh1

yG

yG

yM

22

3 . 3 1 Lamina propria

-

-

23

Lymphoid follicles stained with all antisera 1 . 1 1 Lamina propria Lamina propria

15.6 1 . 7

Lacrimal gland a

yA

yM

yG

?A

yM

yG

1 Lamina propria

Lamina propria

Determined by use of fluorescent antiserum yA antisera. fluorescent antisecretory “piece” specific antisera.

* Determined by use of

Apical epithelial

+ mucus

Apical epkhelinl mucus

+

Jeffries and Sleisenger (1965); Crabb6 and Heremans ( 1 9 6 6 ~ ) Brandtzaeg et nl. (1967) Rubin et al. (1965); Crabbe et al. (1965); Crabbe and Heremans (1966r) Crabbe (1967)

Crabbe and Heremans ( 1 9 6 6 ~;) Gelzayd et al. (1967b) Gelzayd et al. (1967a) Crabbe and Heremans ( 1 9 6 6 ~;)Gelxayd et al. (1967aj Gelzagd et al. (1967s)

Brandtzaeg et al. (1967)

16

THOMAS B. TOMASI, JR. AND JOHN BENENSTOCK

sorbed with normal human serum demonstrated a precipitin arc in the yA region on immunoelectrophoresis of saliva and gastric juice. In retrospect, these findings were probably due to the specific secretory yA determinants now known to be characteristic of this molecule. Because of the difficulties in distinguishing the salivary vs. gastric origin of the immunoglobulins found in gastric juice, studies have been carried out on tissue extracts from gastric mucosa. Rapp et al. (1964) found a quantitative predominance of yA in gastric extracts. Havez et al. ( 1966b) have reported the identification of an antigenically unique form of yA in gastric extracts, probably representing secretory yA, although further studies are necessary to establish this with certainty. The predominance of yA-containing plasma cells in the lamina propria of the gastric mucosa (see Table I ) is also consistent with the hypothesis that gastric juice is part of the secretory system, in keeping with similar findings with the large and small intestine. Adult, small intestinal fluids have been reported to have a rG/ yA ratio of 3.811 (Chodirker and Tomasi, 1963). This ratio was obtained using a serum 7s yA standard. Moreover, because of the presence of H-chain fragments of y G (Fc- and F’c-like material) the concentration of yG is probably overestimated (see Section IV). For these reasons the ratio of yG/yA is probably lower than 3.811, although its true value remains to be established. The nature, origin, and biological significance of the immunoglobulin fragments found in normal intestinal juice have not been investigated. Presumably the majority of the fragments arise from proteolytic degradation in the lumen following secretion and are of little biological significance. However, it has not been ruled out that the more resistant Fab- and F(ab),-like units with their antibodycombining sites do possess important biological activities. In all investigations of fluids from the GI tract, efforts must be made to retard proteolysis and subsequent degradation of immunoglobulins in order to obtain accurate estimates of the quantities of intact immunoglobulins present. Alkalinization, collection in ice, and addition of trypsin inhibitors have met with only partial control of proteolysis. Plaut et al. (1968) have reported that heating of GI juice to 56°C. for 30 minutes appears markedly to inhibit subsequent proteolysis and degradation of exogenous added immunoglobulins. In normal small intestinal juice ( Plaut, 1968) and cholera stool, Northmp et al. (1968) have shown that yA predominates as determined by immunoclectrophoresis. Secretory yA can be identified although a significant fraction of the yA detected has a sedimentation coefficient lower than 11 S. Apparently intact yG and y M can be identified in small amounts in these secretions. Likewise, the intestinal

SECRETORY IMMUNOGLOBULINS

17

secretions of children have been shown to contain yA, yM, and yG ( Nusslt. et al., 1962) although their absolute concentrations have not been determined. Gastrointestinal secretions have also been shown to contain specific yA polio antibody and yA anti-Escherichia coli antibody often in higher titers and with a different distribution among the immunoglobulin classes than found in serum (Berger et al., 1967; Tourville et al., 1968a). Immunofluorescent studies have also demonstrated the predominance of YA-containing plasma cells in the lamina propria of the small intestine (see Table I ) . The major immunoglobulin class found in colonic and rectal washings appears to be yA (Bull, Bienenstock, and Tomasi, unpublished), and villous tumors of the rectum secrete a fluid rich in yA (Masson et al., 1966). Immunofluorescent studies of the plasma cells of colon and rectum support these findings. Several investigators have demonstrated the presence in human bile of yG immunoglobulin (Hardwicke et al., 1964; Russell and Burnett, 1963; Rawson, 1962). Chodirker and Tomasi (1963) reported a yG/yA ratio for bile of 3:1, and Schultze and Heremans (1966) state that the concentration of yA is as high or higher than that of yG in many, although not all, samples of bile. However, it has been suggested that, on the basis of the studies of Hardwicke et al. (1964) and from the demonstration by Rouiller (1956) of communications between the space of Disse and bile canaliculi, the majority of the plasma proteins found in bile originate in the serum. Further studies involving careful quantitation of immunoglobulins and determination of the physicochemical nature of the biliary yA and its cellular origin are needed.

B.

RESPIRATORY

T R A ~

Although the capacity of human nasal secretions to inactive viruses was described as long ago as 1917 by Amoss and Taylor, the first demonstrations that such activity was due to specific antibody was furnished by Francis (1943; Francis et al., 1943). Remington et al. (1964) demonstrated that the yA immunoglobulin class predominated in nasal fluid and that yG was present in only trace amounts in normal secretions. Similar conclusions were reported by Artenstein et al. (1964) who also showed by absorption studies with specific antisera that there were both yA and yG antibodies to several types of viruses in nasal secretions. Ample confirmation and extension of the studies quoted above have been furnished by the careful investigation of nasal secretions by Rossen et al. ( 1965, 1966a,b) and Bellanti et al. (1965). These studies have demonstrated that about 80%of the yA found in nasal secretions is of the 11 S secretory type both by sedimentation and anti-

18

THOMAS B. TOMASI, J R . AND JOHN BIENENSTOCX

FIG.4. Immunofluorescent studies on bronchial biopsies. (A) Bronchial glands stained with an anti serum yA antiserum (low power); ( B ) bronchial glands stained with an anti secretory “piece” antiserum (low power); ( C ) high-power view of interstitial plasma cells stained with an anti serum yA antiserum [A and B from Tourville, Bienenstock, Adler, and Tomasi (unpublished ); C from Martinez-Tello and Blanc (1968).1

SECRETORY IMMUNOGLOBULINS

19

genic nnalysis. Approximatt.ly 20% of nasal yA appcws to have a sedimentation coefficient of 7 S and is aiitigenically identical to serum 7 S yA. A surprising feature of one of these studies (Rossen et a]., 1966b) was the apparent demonstration that nasal yA was negative for InV factors despite the presence of K light chains. This finding does not correspond to data obtained for colostral yA (Tomasi et al., 1965) showing both K and h chains and InV determinants. Rossen et al. (1966b) have also suggested that there may be differences in yG subclass distribution between serum and nasal washings. However, because of small amounts of yG present and thc. technical d w i e s involved in quantitating subclasses, these results are difficult to interpret and further studies are necessary before significant differences in the distribution of subclasses can be accepted. In keeping with the other glandular structures so far discussed, Brandtzaeg et al. ( 1967) have demonstrated a predominance of plasma cells containing yA particularly marked along the nasal glandular secretory ducts. The plasma cell yA/yG ratio varied according to the section studied (Table I ) but the proportions of plasma cells containing yA and yG were in good accord with the ratios of these immunoglobulins in nasal secretions. There is uniform agreement in the literature that yA is the major immunoglobulin class to be found in bronchial fluids ( Anzai et al., 1963a; Ibayashi et al., 1963; Keimowitz, 1964; Dennis et al., 1964; Masson et al., 1965; Chodirker and Tomasi, 1963). Keimowitz, Chodirker, Masson and colleagues demonstrated that yA was the major type of immunoglobulin in tracheobronchial washings, whereas the other investigators studied sputum which of necessity is contaminated by saliva. Using antisera raised against bronchial mucus or tracheobronchial washings, none of these investigators were able to show specific antigenic characteristics of the yA molecule. This, however, may be due to technical problems since SP determinants have been recently demonstrated (Hanson and Johansson, 1967; Havez et al., 1966a) in fluids derived from the bronchial tree in normal individuals and in patients lacking serum yA. Moreover, fluorescent studies of normal bronchial mucosa show a predominance of yA cells (Martinez-Tello and Blanc, 1968) and staining of mucosal glandular epithelial cells occurs with SP-specific antisera (Fig. 4). Dennis et al. (1964) demonstrated that with infection and during acute asthmatic attacks as well as in pulmonary tuberculosis, the yG/yA ratio of sputum more nearly approached that of serum, whereas, in normal individuals it is approximately 1. Anzai et al. (1963a) conclusively demonstrated that yA is part of the insoluble miicous gel of sputum and resists limited hydrolysis. Masson and Heremans (1966) showed that the

20

THOMAS B. TOMASI, J l i . AND JOHN BIENENSTOCK

sedimentation coefficient of yA from sputum corresponded to that of saliva, i.e., 11.3 S; however, the sputum was probably contaminated to a small extent by saliva. Investigations on lacrimal fluids have shown that yA is the major immunoglobulin class found in this secretion (Chodirker and Tomasi, 1963; Josephson and Lockwood, 1964; Settipane et aZ., 1965); yG is found inconsistently in trace amounts and y M can usually not be detected by Ouchterlony analysis. Passage of several serum proteins not normally found in tears into lacrimal fluid occurs upon mild irritation of the conjunctiva produced, for example, by rubbing the eye firmly several times (Josephson and Lockwood, 1961). Antigenic analysis of lacrimal yA has shown it to be antigenically identical to secretory yA in parotid and nasal secretions (Rossen et aZ., 1966b). These workers have also shown that the majority of lacrimal yA has a sedimentation coefficient of 11 s. C. GENITALTRACT Much of the work that has been done with regard to immunoglobulins and antibody activity in the reproductive tract has been carried out in animals and is reviewed briefly in Section 11 (also see Pierce, 1959). Isohemagglutinins have been described in the cervical mucus with no significant correlation between secretions and serum titers (Solish et uZ., 1961). The occurrence of isohemagglutinins in cervical mucus was found in this study to be unrelated to blood group secretory status. In cervical mucus, yG immunoglobulin has been detected (Moghissi et al., 1960), and immunoglobulins of all three classes, yA, yG, and yM, have been shown to be present on immunoelectrophoresis of cervical mucus by Moghissi and Neuhaus (1962) and Anzai et al. (1963b). The former investigators concluded that, on the basis of inspection of immunoelectrophoretic patterns, yG was present in greater amounts than the other two classes of immunoglobulins. However, accurate quantitation of the immunoglobulin classes in cervical secretions does not appear to have been performed. Fennell and Vazquez ( 1960), in an immunohistochemical study of the human and rabbit vagina, have showed 7-globulin staining in the intermediate zone of the stratified squamous epithelial cells and also in exfoliated cells. Further studies on the cervix and endometrium (Fennell and Vazquez, 1962) led the authors to suggest that the cytoplasmic staining was due to nonspecific absorption of serum proteins diffusing from the capillary bed through the epithelium. No fluorescence of endometrial glands was noted. There appears to be little information available on immunoglobulins in human tubular fluid or

SECRETORY IMMUNOGLOBULINS

21

uterine secretions apart from the studies on the cervical mucus. Hervk et al. (1965) in an investigation of cervical mucus have noted differences in immunoelectrophoretic patterns depending on the stage in the ovarian cycle. Little or no protein was detected in normal cervical mucus. However, at ovulation on day 14 of the cycle, a pattern similar to that of serum was obtained. Analysis of fluid from a ruptured Graafian follicle suggested to these authors that the follicular fluid was similar in protein content to normal serum. In patients with obstruction of their Fallopian tubes, “plasma” proteins were absent in midcycle. Only yG was identified with certainty in this study. However, Schumacher et al. (1965) found y c in all samples of cervical mucus regardless of the stage of the cycle; yA was also found but only between the fifteenth and eighteenth days. Further study is evidently needed in this area of investigation especially regarding the possible influence of hormones on the protein content of secretions of the female genital tract. Little definitive work on immunoglobulins in prostatic and seminal vesicle secretions has appeared in the literature. Leithoff and Leithoff (1961) have demonstrated yG, yA, and yM immunoglobulins in seminal plasma, and Chodirker and Tomasi (1963) reported that prostatic fluid obtained by massage showed no difference in y C f y A ratios compared with that of serum. However, because these studies were performed on patients with prostatic disease the amount of yG might be expected to increase as serum components appeared secondary to the inflammation. Acharya et al. (1968) have recently demonstrated that yA appears to be the predominant immunoglobulin class in normal seminal plasma and that it might be in high-molecular-weight (11S ) form although this was not conclusively proven.

D. URIIVARYTRACX The yA in both male and female urine is of high molecular weight (Tomasi and Zigelbaum, 1963; Turner and Rowe, 1964, 1967) having a sedimentation coefficient of 11S and is antigenically indistinguishable from the secretory yA molecule characteristic of other external secretions (Bienenstock and Tomasi, 1968). A yG/yA ratio of 3 : l has been reported. However, because of the technical problems encountered with quantitation of yG fragments this probably represents a maximum figure and 11 S yA and 7 S yG are probably present in approximately equal concentrations in normal u1inc (about 1 to 2 mg./24 hours). Special problems are encountered in iiivcstigation of the urine which are not met in the other secretions. Large volumes of urine are produced and only about 80 mg. of protein per day is found in normal bladder urine.

22

THOMAS B.

TOMASI,

JR. AND JOHN BIENENSTOCK

Of this only about 1 mg. is yA (Bienenstock and Tomasi, 1968). Slight variations in glomerular permeability, or in tubular reabsorption or secretion mechanisms, or even alterations lower in the urinary tract can cause considerable differences both quantitatively and qualitatively in the proteins found in bladder urine. In normal urine, yM is not found although yD can be detected depending on the degree of concentration. Immunoglobulin fragments, such as L chains and Fc and F’c fragments, are routinely found in normal urine, and small fragments with a molecular weight of about 12,500 have been repeatedly reported to be present in urine and have biological activity against a variety of immunizing antigens (Merler, 1966; Hanson and Tan, 1965; Merler et al., 1963).

E. MAMMARYGLAND Interest in the immune globulins in colostrum has attracted the attention of a large number of investigators, and some of this work has been reviewed briefly in Section 11. Similar work in the cow, goat, rabbit, sheep, dog, and mouse will be reviewed in Section X. Gugler at al. (1958) appeared to have first described the predominance of yA in human colostrum and this was confirmed by Havez and Biserte (1959) and Montreuil et al. (1960). Further careful immunological studies suggested not only the predominance of yA but a very complex pattern of immunoglobulins in human colostrum including the presence of yM and yG (Hanson, 1961; Hanson and Berggard, 1962). The possibility of specific antigenic determinants present on colostral yA molecule not shared by serum yA was mentioned by Hanson (1961). In retrospect this may have represented secretory yA, but since definitive studies were not done, it is difficult to say with certainty. Chodirker and Tomasi (1963) showed by quantitative measurement the predominance of yA in colostrum. Tomasi et al. (1965) isolated yA from colostrum and found that approximately 80% was of the secretory type and was identical immunologically with that isolated from other secretions; 20% had a sedimentation coefficient of 7 S and was identical immunologically with serum yA. Investigations by Havez et al. (1967) have confirmed the secretory nature of yA in colostrum. A secretory yA molecule has been identsed in rabbit colostrum very similar in most parameters so far investigated to human secretory yA (Sell, 1967; Cebra and Robbins, 1966; Cebra and Small, 1967). This is the only animal which has so far been conclusively shown to have a secretory yA system quite analogous to man. As pointed out in Table 11, witches’ milk, the mammary secretion of newborns, does not contain yA but free unattached secretory

23

SECRETORY IMMUNOGLOBULINS

Parotid yA-Deficiency states (includes newborns, first 1 P 2 0 days) Early childhood (1-12 mo.), some adults Most normal adults Lacrimal Nasal Tracheobronchial yA-Deficiency states Normal adults Colostrum yA-Deficiency states, Witches’ milk Normal Gastric Small intestinal Urine yA-Deficiency states Normal adults Sweat,

+ + -

+ +

+ + + +

T r Tr Tr Tr -

+ +

-

+

+

+

+

+

-

+ + + -

+ +

+ + + + + + +

-20%

+ + + - + - +

“piece” is present ( Hanson and Johansson, 1967). In these respects this fluid resembles other external fluids of the newborn as well as the secretions of patients with dysproteinemias characterized by a deficiency of yA. From the above discussion concerning the character of the immunoglobulins of fluids bathing the mucous membranes it seems quite clear that there is some type of relationship (as yet undefined) between the epithelial cell and the yA immunoglobulin system. Although the morphological appearance of the epithelial cell may vary from organ to organ the secretory system seems thus far to be confined to those organs having either cuboidal or columnar-type epithelial cells in association with mucous membranes or secreting glands such as the salivary acini. The only fluid examined to date which seems to be related to stratified squamous epithelium is the gingival or crevicular fluid. This does not have the characteristics of other external secretions, and fluorescent studies of the gingival tissue (Brandtzaeg and Kraus, 1!365) do not show the characteristic predominance of yA-containing plasma cells. It will be of some interest, therefore, to study fluids bathing other stratified squamous

24

THOMAS B. TOMASI, J R . AND JOHN BIENENSTOCK

epithelia such as the vagina, although here some difficulty niay be encountered in determining the origin of such fluids; whether they are derived from the vaginal wall andlor from the cervical secretions. Pertinent in this context is the recent report, as yet unconfirmed (Page and Remington, 1967), that human sweat has a yG/yA ratio of 3 : l and contains SP determinants, although these were not dekitely shown to be attached to yA. If this interesting observation is confirmed it will suggest a unique circumstance for this secretion, since it is thought to be formed primarily by transudation and the epithelial surface which this fluid bathes consists of stratified squamous epithelium. VI.

Chemical and Immunological Characteristics of Secretory Immunoglobulins

In this section emphasis will be placed primarily on the methods of isolation and the chemical, physical, and immunological properties of secretory yA. Very little work has been reported with the other secretory immunoglobulins, primarily because of the low concentration of yG and yM in external secretions, and the resulting technical problems involved. Studies of these immunoglobulins may, however, be a fruitful area for future investigations. A. ISOLATION The technique necessary to isolate apparently homogeneous preparations of secretory immunoglobulins varies for different secretions. Since the concentration of yA is considerably higher (about 0.5 to 1.0 gm.%) in colostrum, this fluid is preferred when significant amounts of secretory ./A are required. It should be pointed out, however, that the concentration of yA relative to other immunoglobulins varies not only from sample to sample but also depends upon the period in lactation. Early samples taken within the first 48 hours contain the highest relative concentrations of yA, whereas later samples show lower y-globulin levels and increasing amounts of yG. One useful scheme for the isolation of secretory yA from colostrum and saliva is presented in Fig. 5. The yields from colostrum using this method are approximately 25%. In the case of parotid saliva using a two-step procedure [stepwise diethylaminoethyl ( DEAE ) elution followed by Sephadex G-2001, the yields approach 40% but due to the relatively small concentration of yA in this fluid this procedure requires large volumes of starting material. Similar schemes have been employed for obtaining both y G and yA from nasal secretions (Rossen et al., 1966a). Various methods have been suggested for the isolation of

25

SECRETORY Ihlh~UNOGLOBULINS

7 1 Colostrum

Serurn([rcitic

p H 4.6 with acetic acid

Supernale

fluid)

50% ( NH,l

ppt

Neutrolize starch block electrophoresis

t in

&

d

l

SO,

saline

f 0.05M ZnSO, pH 7.0

yA Containing fractions I

V pH 614 0.10 M

t

Sephadex G 200 Eluted with 0.14M soline

I

YA

FIG.5. Scheme for isolation of yA from human serum, ascitic fluid, colostrum, and saliva.

-,A from serum (Heremans et al., 1962; Vaerman, 1966), but none of the techniques is entirely satisfactory and yields are small (of the order of 10 to 25%). Because of this, ascitic fluid, which is available in large amounts, from patients with cirrhosis of the liver has been used as a convenient starting material. Chemical and immunological studies on ascitic %uid yA reveal it to be apparently identical to yA isolated from serum (Tomasi et al., 1965) from which it is undoubtedly derived by transudation. When fresh, normal, human serum is examined by density gradient ultracentrifugation, no more than 515%of the yA is distributed in areas having sedimentation coefficients greater than 7 S (Tomasi et al., 1965). It is not known with certainty whether the 7s yA which is found in colostrum and nasal fluids (about 20%of the total yA in each secretion) represents protein derived directly from serum or from the secretory molecule which has undergone dissociation or failed to complex with the SP, although some evidence has been presented that it is derived from serum (Butler et al., 1967). The available evidence points to the fact that the higher polymers of yA (approximately 18 S ) which are found in many fluids represent polymers of the secretory molecule (Tomasi et al., 1965). Intermediate sizes of yA (9-14 S ) which are found in small amounts in serum are also present in ascitic fluid and these lack the

26

THOMAS €3. TOMASI, JR. AND JOHN BIENENSTOCK

specific secretory determinants. Higher polymers (18 S and above) cannot be detected in ascitic fluid although they are present in small amounts in normal serum. It should be pointed out that in testing for the purity of immunoglobulin preparations, samples which appear to be homogeneous by ultracentrifugation, zone electrophoresis, and by antigenic analysis often show impurities which can be detected. by immunization of rabbits or other animals. For example, there is a particular secretion-specific protein, MMC, with physical characteristics very close to those of secretory yA which is highly antigenic and is often detectable only upon immunization. Likewise lactoferrin often contaminates secretory yA preparations, and antisecretory antisera frequently contain antibodies to both MMC and lactoferrin (Fig. 3 ) . In this regard, tests for purity should always include the use of multiple antisera including those made against whole secretions. B. IMMUNOLOGICAL PROPERTIES Using antisera made against serum yA or isolated yA myeloma proteins, no immunological differences have been reported between the serum and secretory yA. Thus, from this standpoint, there is no evidence that the a chains of secretory yA differ from those of serum. However, using certain antisera made against secretory yA isolated from either saliva, colostrum, or nasal fluids, immunological specificity for the secretory yA can be detected. This is shown in Fig. 6 by the spurring of secretory yA over serum yA, suggesting the presence in the 1 1 s yA of an antigenic determinant not found in serum. It should be clearly emphasized that the demonstration of this type of unilateral or single spur is crucial evidence for the existence of antisecretory yA-specific antibodies in an antiserum. Following absorption of an antisecretory yA antiserum with normal (lyophilized) serum, activity remains only against the secretory molecule. The absorbed antiserum, if it is truly specific, will not react with myeloma sera containing high concentrations of yA polymers ranging from 9 to 1 4 s or with sera from patients with systemic lupus erythematosus ( SLE ), Laennec’s cirrhosis, and Sjogren’s syndrome who have relatively large amounts of polymeric yA but unlike the myeloma polymers have a broad range of electrophoretic mobilities. Also these antisera do not react with preparations containing 11s yA anti-B antibodies isolated from serum by specific precipitation with blood group B substance (Tomasi et al., 1965). The secretory molecules isolated from the saliva, colostrum, as well as those found in urine, nasal fluids, tears, bronchial washings, and GI

SECRETORY IMMUNOGLOBULINS

27

FIG.6. Ouchterlony plate showing antigenic relationships for yA from normal human serum ( N H S ) , colostrum (Col.), urine (yA,,) and saliva (Sal.). The center well contains an antiserum which reacts both with secretory “piece” and yA determinants.

fluids are immunologically identical when examined with antisecretory antisera (Tomasi et al., 1965; Rossen et al., 1966b; Bienenstock and Tomasi, 1968; Hanson and Johansson, 1967). The extra antigenic determinant present in the secretory yA which is lacking in serum yA has been shown to be due to the presence of the SP, also called transport “piece,” T component, and T chain. This can be demonstrated, as shown in Fig. 7, using isolated SP preparations obtained either from agammaglobulinemic urine (or saliva) and SP obtained from dissociation of the intact secretory molecule by reductive cleavage (see Section VI,E ) . In investigations of SP reactive material in a given whole secretion using an SP-specific antiserum ( antisecretory yA antisera absorbed with normal human serum), it is essential that Ouchterlony analysis be performed in such a manner as to demonstrate the immunological identity of the precipitin arc given by the whole secretion and that given by a homogeneous secretory yA preparation. Otherwise it is impossible to determine with certainty whether a given precipitin arc obtained with a whole secretion is due to SP, either free or attached to yA, or to a contaminant. Immunoelectrophoresis is also helpful in identifying the characteristic mobility of 11S secretory yA and free SP and may assist in distinguishing these from each other as well as other contaminating components (Fig. 3 ) . Evidence that the antigenic specificity of the secretory molecule

28

THOMAS B. TOMASI, JR. AND JOHN BIENENSTOCK

FIG. 7. Gel diffusion experiment showing immunological relationships of secrctory yA, secretory “piece” (SP), and serum yA. Well 1-reduced and alkylated colostral yA containing both a chains and SP; well 2-SP from agammaglobulinemic urine; well %SP isolated from secretory yA; well 4-normal human saliva; wells 5 and Gnormal human serum; well 7-anticolostral yA absorbed with yC.

is associated only with higher polymers of yA is suggested by immunological studies on the yA fractions isolated from colostrum. The 7 s colostral yA is identical immunologically to the 7 s serum yA, whereas both the 11 S and 18 S colostral polymers spur over both of the 7 S proteins (Tomasi et d.,1965). Similar results have been obtained with nasal fluid 7 S and 11 S yA proteins (Rossen et al., 1966b). C. CHEMICAL PROPERTIES OF SECRETORY IMMUNOGLOBULINS Some of the differential chemical properties of serum vs. secretory 111. The major component in the various secretions appears to be an 11 S molecule having a molecular weight of 385,000 although as mentioned above, higher polymers ( 16-20 S ) do occur in most secretions. The molecular weight obtained by the equilibrium sedimentation techniques of Yphantis ( 1864) is similar to that obtained by Cebra and Small (1967) on rabbit colostral yA. The higher molecular weight of serum y A (170,000) compared to yG (150,000) has been shown to be due primarily to the large molecular size of the ,a chain (65,OOO) compared with the y chain (50,000)(Cohen and Milstein, 1967). The molecular weight of isolated H chains from human colostral y A has not been determined, but rabbit colostral 01 chains have a molecular weight of 64,000 + 3000 (Cebra and Small, 1967). Since yA are outlined in Table

29

SECRETORY IMMUNOGLOBULINS

TABLE COMPARISON OF PROPERTIES OF A -,

III FROM

Human serum y.4

Property

SERIJMAND SECRETIONS" Human secretory yA

Rabbitb colostral yA

~

Sedimentation coefficient (so?"w) Molecular weight Carbohydrate Hexose (%) Hexosamine (%) Fucose (%) Sialic acid (%) Moles disulfide per 170,000 p. N-terminal amino acids Gm factors InV factors Extinction cvefficiertt Partial specific volume ( V )

6 SS 170,000 5 2 9 0 22 18 15 Asp, (:luc -

+

h'&k,,

11.4 S

~~

xs

6.2

3 2

4

3 2

Id

0.73" 0 65 16 Asp, Glu,' Lys" -

+

13 4 ) I 0 72!P

10

370,000

:385,000

13 9

n

13.5 0 703

~

Data from Tomasi et al. (396.5, IOSS), unless otherwise specified. Cebra and Robbins (1966) aiid Cebra and Small (19137). Heimburger et nl. (1964). Hanson and Johanssoii (1967). Bernier et al. (1965). Axelsson et al. (1966). 0 Havez el al. (1967). * Schultre and Heremans (1966). a

human colostral yA shares H-chain antigenic determinants with serum N chains presumably the H chains of colostral yA are similar or identical to those of serum yA. This is supported by immunological studies (see Section V1,B) which thus far have failed to demonstrate any differences between the CY chain of colostral yA and those of serum. However, urea, starch gel electrophoresis at acid pH suggests differences in mobility between colostral and serum yA H chains (Fig. 8) (Cederblad et al., 1966; Mehta and Tomasi, unpublished) although the significance of this observation has not yet been clarified. The L chains of human colostral yA show a diffuse zone on acid urea gels similar to serum yA and yG. In alkaline urea gels, multiple banding is seen in the L-chain area similar to serum yA and yG but in addition a unique anodally fast band is seen (Fig. 8 ) , which represents SP (Cederblad et cd., 1966; Hanson and Johansson, 1967; Rejnek et al., 1966). A similar fast band representing rabbit T chain is shown by reduced and alkylated, rabbit colostral yA on disc electrophoresis in urea (Cebra and Small, 1967).

30

THOMAS B. TOMASI, JR. AM) JOHN BJENENSTOCK Acid pH

YM

yAcol.

Alkaline p H yAser.

Light chain area Heavy chain

area

Origin-

4

U

FIG.8. Schematic representation of relative mobilities of reduced and alkylated secretory and serum immunoglobulins in acid and alkaline, urea starch gel electrophoresis. At alkaline pH the secretory “piece” moves as a fast anodal band.

Relatively little data are available concerning the carbohydrate content of the secretory molecule. The total hexose content is similar to that of serum yA. The figure for sialic acid content may be somewhat low since determinations were performed on colostral samples that had been exposed to acid pH conditions which are known to remove part of this moiety. As with serum yA, changes in mobility can be demonstrated following treatment of the secretory molecule with neuraminidase. The amino acid composition data on the secretory molecules isolated from saliva and colostrum are compared with serum yA and rabbit colostral yA in Table IV. Secretory yA from colostrum and saliva are very similar and probably identical within the limits of the experimental methods. However, there are significant differences in amino acid content between the human secretory and serum yA proteins. The halfcystine content determined as carboxymethyl cysteine by amino acid analysis is similar for serum and secretory yA, and essentially identical values for these human proteins have been obtained by sulfhydryl titrations using the Ellman technique (Tomasi et at., 1968). The SP is characterized by a high content of glycine and methionine and low proline compared with serum yA. It should be pointed out that the figures reported in the literature for the amino acid content of serum yA vary widely probably, in part, because of the difficulties in obtaining highly purified preparations of serum yA.

L4b11N0 . - h D COMPOSITION

OF

TABLE IV HVM.AN SERUM, SALIVARY A N D COLOSTRAL yA,

AND

RABRIT

Amino acid

IIumair snl. y.4

Humnu col. y.4

Human ser. yA

Col. y-4/ sal. y.4

Ser. ?A/ 881. yA

Human secretory “piece”

Human ser. yA

Lysine Histidine Arginine Aspartic acid Threonine Serine Glutamic acid Proline Glycine Alanine Half-cystine Valine Methionine Isoleucine Leucine Tyrosine Phenylalanine

54.7 21.8 50.6 93.4 107.2 132.2 129.0 111.0 96.0 81.3 30.7 8i.7 7.4 25.5 109.4 39.7 35.5

55.9 20.6 49.9 93 . 4 108.5 127.4 119.5 99.0 88.6 81.6 32.5 89.5 7.2 26.7 109.2 37.3 36.6

63.5 22.0 48.5 93.5 117.0 140.0 124.5 104.0 82.0 81.0 33.5 95.0 8.5 23.0 113.5 41.5 42.5

1.02 0.94 0.99 1.00 1.01 0.96 0.93 0.89 0.92 1.00 1.06 1.02 0.97 1.05 1.00 0.94 1.03

1.16 1.01 0.96 1.00 1.09 1.05 0.97 0.94 0.85 1.00 1.09 1.08 1.15 0.90 1.04 1.05 1.20

4.0 1.4 4.0 7.6 8.0 9.2 8.3 6.6 15.2 5.7 2.4 6.9 1.4 2.1 8.3 4.0 4.7

5.1 1.8 3.9 7.6 9.5 11.3 10.1 8.4 6.6 6.6 2.7 7.7 0.7 1.9 9.2 3.4 3.4 ~

Data from Tomasi (1965) and Tomasi et al. (1968). Cebra and Robbins (1966).

yAa

% Moles

Residues per 160,000 gm.

a

COLOSTRAL

~

Human col. yA 4 1 4 7 9 10 10 8 7 6 2 7 0 2 9 3 3

~~~~

7 7 2 9 2 8 1 4 5 9 7 6 6 2 2 2 1

Ha1hith col. 7.k 3 7 1 4 ‘77 9 6 10 6 9 0 10 R 9 6 b h

6 3 6 0 2

2 2 9 4 4

i i 3 4 3.0

32

THOMAS B. TOMASI, JR. AND JOHN BIENENSTOCK

Salivary yA lacks the Gin genetic determinants which are characteristic only of the YG molecule, but does contain the InV genetic determinants present on the K type of light chain. However, four out of five nasal fluid yA samples lacked the InV allotypes although all possessed K light chains (Rossen et a,?., 1966b). The significance of this apparent difference between parotid and nasal fluid yA remains to be elucidated. Both the K and types of L chains are present in the colostral, salivary, and nasal fluid yA, although the ratios have not been accurately quantitated (Tomasi et al., 1965; Rossen et a,?., 196613). The electrophoretic mobility of human secretory and serum yA are similar in agar, polyacrylamide, and cellulose acetate electrophoresis ( Tomasi, unpublished). Only a few studies have been done on the enzymatic degradation of the secretory molecule. Using the proteolytic enzymes papain, trypsin, and chymotrypsin C, serum 7 S yA and 11S yA myeloma polymers are degraded to 3.8s Fab-like subunits; no Fc or F’c material is apparent (Tomasi and Czenvinski, 1968; Cederblad et al., 1966). The secretory molecule under similar conditions of enzyme concentration and incubation appears to be considerably more resistant to proteolysis with these enzymes and only a small fraction is degraded; the majority of the protein remains as an 11S molecule as determined by analytical ultracentrifugation ( Tomasi and Czerwinski, I968 ) . However, in these studies, proteolysis may actually have occurred; the 11S conformation being maintained by covalent and/ or noncovalent forces. Similar types of experiments involving incubation of purified preparations of immunoglobulins (yG, yM, serum, and secretory yA) with whole GI juice derived from the jejunum indicate that secretory yA is more resistant to proteolysis than the other immunoglobulins (Plaut et al., 1968). However, other studies (Cederblad et al., 1966) report that, although serum and secretory yA both appear to be more resistant to proteolysis than yG, the secretory molecule can be split by proteolysis in vitro by both papain and trypsin. The product is a 3.5s Fab-like fragment, and again no evidence of Fc material was found. The secretory molecule also appears to be quite resistant to reductive cleavage (Tomasi et al., 1965). Following treatment with high concentrations of P-mercaptoethanol (up to 0.5 M ) , no observable dissociation can be detected in the ultracentrifuge. In some cases, partial degradation of secretory yA is found following alkylation of the reduced molecule, but this occcurs irregularly and is dependent on the concentrations of reducing agent used. Following reduction and alkylation and treatment with concentrations of urea as low as 2 M , dissociation regularly occurs suggesting

30

SECRETORY IMMUNOGLOBULINS

that although disulfide bonds have been split the molecule is held together by noncovalent forces. Since serum or myeloma ll S polymers are sensitive to both enzymatic and reductive treatment, it is assumed that the relative resistance of the secretory molecule to dissociation is related to the presence of the SP. The possible biological significance of the resistance to enzymatic and reductive dissociation is discussed in a later section. D. THREE-DIMENSIONAL CONFORMATION Table V shows the optical rotatory dispersion (ORD) data obtained on yA proteins in an aqueous solvent and in 2-chloroethanol (Tomasi, 1965). For comparison, data obtained on 7G are included. The immunoglobulins in certain respects are unusual in their ORD behavior. Although they lack significant amounts of helices as determined by their b,, value in the classic Moffitt equation, they do show a specific rotation at 546 mp suggesting the presence of som2 type of organized structure (for a review, see Urnes and Doty, 1961). Both TABLE L' OPTICALROTATORYDISPERSION DATAOF Protein

rc

Serum 7 S 7.A Salivary 11 S y A Reduced yG 1:edured 11 S -,Ll

Solver1t

[fflalf,

Water Water Water 2-Chloroet h a i d 2-Chloroet haiiol

-14 -46 - 40 - 17 -2.5

ya"fh

1)"

% Helix

0

0

0

0 0 41

1)

-264 -269

41

a Optical rotatory dispersion data on yC; and serum and salivary ?A in aqueous arid organic ("-chloroetli:ttiol) solvelits. Proleiris reduced wilh 0.2 M BME for 6 hours at 20°C. * Data from Tomasi (1965).

serum and secretory yA are very similar to yG in their rotatory behavior. Certain organic solvents such as 2-chloroethanol allow more optima1 conditions for helix formation and many proteins in this solvent have close to 100%of their residues invoIved in heIix formation. Both yG and yA form a maximum of only 40%helix in this solvent. Cleavage of disulfide bonds, in an attempt to remove any possible restrictive influence of this linkage, does not significantly affect the final helical content in 2-chloroethanol. Whether the low helical content of the immunoglobulins is related to the presence in these molecules of a high content ( al?out 40%) of those amino acids (swine, threonine, proline, valine, and

34

THOMAS B. TOMASI, JR. AND JOHN BIENENSTOCK

isoleucine) that, as sugggested by Blout (1962), do not readily participate in helix formation is a matter of speculation. The type of threedimensional conformation which gives rise to the optical properties presented above is a matter of debate. However, the ORD data as well as studies using polarized infrared light are consistent with the presence of significant amounts of cross-p structure in these proteins. Essentially no information is available concerning either the tertiary or quaternary structure of the secretory yA molecule.

E. ISOLATION AND CHARACTERISTICS OF T H E SECRETORY ‘‘PE~” One technique used for the isolation of SP is outlined in Fig. 9. Following reduction, ablation, and chromatography in 1N propionic acid, it is important to take cuts from the Sephadex G-100 chromatograph (the shaded area in Fig. 9 ) which exclude L-chain determinants ( measured immunologically). These fractions are contaminated with H-chain determinants, but subsequent chromatography on

U

Homogeneous y A (colostral) .

0.2M BME 25OC, 12 hours. 0 . 3 M iodoacetomide

I H choin

Sephadex G 100 in 0.5N propionic acid

II

Sephodex 6200 in 0.14M NoC I

/Piece”

J\

FIG. 9. Schematic outline of the isolation of secretory “piece” from colostral yA (see text for details ).

Sephadex G-200 yields SP preparations which are homogeneous as tested by ultracentrifugation, disc electrophoresis, and immunological methods. Free SP is also present in secretions of patients with various types of dysproteinemias characterized by absence of serum yA. The SP isolated from agammaglobulinemic fluids is immunologically identical with that isolated by the above technique from the intact molecule (Fig. 7). Some of the properties of the SP isolated from colostral yA are outlined in Tahle VI.

35

SECRETORY IMMUNOGLOBULINS

The mode of linkage of the SP to the yA portion of the molecule is a matter of debate. Hong et al. (1966) have reported that SP can be dis; sociated using 6 M guanidine suggesting a noncovalent linkage. This is consistent with the reports of Cebra and Small (1967) on rabbit colostral yA. However, other studies (Tomasi and Czerwinski, 1968) suggest that only a fraction (approximately 20%) of the SP is bound noncovalently, the majority being linked by disulfide bonds. The evidence for this is based on the following studies. Chromatography of secretory yA on Biogel PlOO in 1N propionic acid or 6 M guanidine (containing 0.02 M iodoacetamide ) separates a small second peak containing unattached 1ABLE VI PROPERTIES OF SECRETORY PIECE"^ Sedimentation coefficient Molecular weight Carbohydrate (%) Moles of disulfide per mole Partial specific volume (P)

4.2 8 58,000

9.5 5 0,726

Data from Tomrtsi et (11. (1968).

SP. However, examination of the major peak eluted in the void volume indicates that SP determinants are still attached to yA. Rechromatography in 6 M guanidine results in a single peak containing intact secretory yA, which on treatment with small concentrations of reducing agents, releases the SP. In this regard recent studies of the binding of albumin to certain yA myeloma proteins suggest that this bonding is also through disulfide bonds ( Mannik, 1967). Noncovalent interactions undoubtedly also occur as suggested by the radiolabeling experiments discussed below, and in this respect the binding of SP resembles that of H and L chains in which both types of force are involved. The mobility of SP (isolated from secretory y A ) on immunoelectrophoresis is shown in Fig. 10. Free “piece” present in the secretions of agammaglobulinemic subjects has a similar mobility although minor variations in mobility are noted. As shown in Fig. 10, no reaction occurs between SP and an anti-L-chain antiserum. Immunodiffusion studies in our laboratory have likewise failed to demonstrate any antigenic relationship between SP and L chains; findings which are in accord with those of Hanson and Johansson (1967). The results of Hong et al. (1966) suggesting the existence of common antigenic determinants between SP and L chains may have been due to contamination of SP preparations

36

THOMAS B. TOMASI, JR. AND JOHN BIENENSTOCK

FIG.10. Immunoelectrophoresis of colostral yA (upper well) and isolated secretory “piece” (lower well). Upper trough-antiserum yA antiserum; center trough-anticolostral yA antiserum; lower trough-anti-L-chain antiserum. Anode on right.

with L chains or the formation of mixed dimers between L chains and SP. The molecular weight of isolated SP of 58,000 is not entirely compatible with the chromatographic position on Sephadex of the free SP present in agammaglobulinemic secretions or normal urine. Free SP has an elution volume less than that of albumin which is more consistent with a molecular weight of approximately 80 to 100,OOO by this technique. This suggests that the chromatographic behavior may be related to asymmetry although further work is necessary to clarify this point. It was also suspected that in agammaglobulinemia, the free “piece” might be a dimer, particularly in view of the findings with rabbit colostral yA suggesting that SP, with a molecular weight of 50,000, is composed of two 25,000 monomeric subunits linked by disulfide bonds (Cebra and Small, 1967). However, no evidence could be obtained with human SP of either covalent ( disulfide) or noncovalent associations between monomeric subunits, on treatment of free SP, isolated from agammaglobulinemic saliva, with reducing agents and 6 M guaindine ( Tomasi and Czenvinski, unpublished). Experiments with radiolabeled SP indicate that the in vitm complexing of SP is relatively specific for yA (Tomasi and Bienenstock, 1968). Addition of lS1I-1abeled SP to whole serum or isolated preparations of immunoglobulins shows that complexing occurs only with serum yA and not with yG or y M (see Fig. 11).The finding that reduced and alkylated SP obtained from dissociation of colostral yA will bind to serum yA suggests that noncovalent interactions are involved. Similar experiments with labeled SP obtained from agammaglobulinemic urine also demonstrate a specific interaction with yA. The site of linkage of the SP to the yA portion is unknown. However, since SP appears to have specific affinity for yA, it might be expected

37

SECRETORY IMMUNOGLOBULINS

FIG. 11. Radiolabeling experiments showing specific coniplexing of I3’Ilabeled secretory “piece” with serum yA. Complexing does not occur with yG, yM, transferrin, or other senmi proteins. Stained immunoelectrophoretic patterns on left, corresponding radioautographs on right. ( NHS-normal human serum. )

:hat the covalent and noncovalent associations occur with the chain. Likewise the number of SP molecules per mole of secretory yA has not been accurately determined. From the molecular weights one would expect a dimer of 7 S yA plus one SP molecule or one 7 S yA plus three or four SP molecules. The former seems more likely on the basis of approximate yields of SP, L, and H chains following reduction, but until the relative concentrations of SP and L chains are accurately determined, this question cannot be definitely answered. A tentative schematic model based on one SP per secretory yA molecule is shown in Fig. 12. The biological functions of SP are not fully understood. It appears from the data presented above that the secretory molecule is quite resistant to proteolysis and reductive cleavage. This is probably a result of the presence of SP in the 11 S secretory molecule, since myeloma and serum ./A polymers of 11 S size do not show a comparable degree of resistance under similar conditions. Teleologically, such stability would be of considerable biological advantage to an antibody molecule of which the activity is confined to complex fluids containing proteolytic enzymes such as external secretions. The available evidence indicates that the SP is synthesized in epi(Y

38

THOMAS B. TOMASI, JR. AND JOHN BIENENSTOCK

FIG. 12. Schematic model of secretory yA. Disulfide bonds represented by solid bars, and secretory “piece” by the triangular area.

thelial cells, and it has been suggested that SP is added to the yA protein in the process of transport across the mucous membrane. However, it is possible that complexing of SP and yA occurs in the secretion following transport and there is, therefore, no good evidence that SP is in any way involved in facilitating transport of yA. Since yA has a tendency to complex with a variety of proteins (Heremans, 1960; Mannik, 1967; Levitt and Cooperband, 1967; Ganrot, 1967), it is conceivable that its combination with SP is nonspecific and has little biological significance. This thesis has certainly not been excluded, but the specificity of the reaction of SP for yA in the radiolabeling studies outlined above, the occurrence of SP and yA together in all external fluids thus far examined, and the stability conferred on the secretory molecule by SP, suggest that it may have important and specific functions. The question of an enzymatic activity for SP has not been investigated except that recent work in our laboratory using antihuman amylase antisera have excluded amylase activity in SP (Bienenstock and Tomasi, unpublished). Further work in this area seems warranted particularly in view of the possibility suggested by the work of Adinolfi et al. (1966b) that secretory yA, together with lysozyme and complement, may have a lytic action on Escherichia coli.

SECRETORY IMMUNOGLOBULINS

VII.

39

Sites of Synthesis of Secretory Immunoglobulins

A. IMMUNOLOGICAL STUDIES

.

From quantitative studies of immunoglobulin levels in parotid saliva and serum in health and in a variety of disease states, it is apparent that there is often little correlation between yA levels found in saliva and serum (Claman et al., 1967; South et al., 1966). In patients with hypergammaglobulinemia of varied origin which was associated with elevated levels of serum yA, there is little correlation between salivary concentrations of this immunoglobulin and those found in serum (Claman et al., 1967; see also Table VII). No increase in salivary yA concentration was noted (Tomasi et al., 1965) in 8 patients with yA myeloma, 2 of whom had a high concentration of 11 S yA polymers. In 1 myeloma patient, density gradient ultracentrifugation was performed and it was shown that the salivary yA was predominantly 1 1 s in type and possessed secretory antigenic determinants, whereas the serum myeloma was predominately 7 S. In the parotid saliva of neonates, salivary yA can be found in the absence of measurable serum yA using a quantitative complement fixation technique which will detect as little as 0.25 pg. of yA per milliliter of serum ( Sullivan and Tomasi, unpublished). Similar results have been reported by South et al. (1967) and by Selner et al. (1967), the latter using a sensitive electroimmunodiffusion technique. However, apart from neonatal secretions, secretory yA has not been found in the absence of serum yA provided sensitive Ouchterlony or complement fixation techniques are used to detect serum yA. There is one discordant report from McFarlin et al. (1965) that in patients with ataxia telangiectasia and absent serum yA, plasma cells containing yA could be demonstrated in the parotid gland and bone marrow and that, in addition, yA could be detected in concentrated parotid saliva. These findings have so far not been confirmed by other investigators. However, Swanson et al. (1968) have reported one patient with a malabsorption syndrome and absent serum yA (less than 5.0 mg3) in whom fluorescent studies of the GI tract revealed a normal number of yA-staining plasma cells. However, as shown in Table VII, certain patients may have markedly depressed serum levels of yA (one-tenth of normal) and yet show essentially normal levels of salivary y A. As seen in Table 11, free or unattached secretory “piece” can be found in the concentrated saliva of all neonates and most children (South et at., 1967), and it is occasionally found in salivary samples from adults. Free SP is also found in most colostral samples and in all normal

40

THOMAS B. TOMASI, JH. AND JOHN BIENENSTOCK

urine ( Sullivan and Tomasi, unpublished; Bienenstock and Tomasi, 1968) . In addition, in patients with agammaglobulinemia, ataxia telangiectasia, and in healthy subjects who lack yA, free SP has been demonstrated in saliva, colostrum, bronchial washings, and urine (South et d., 1966; Bienenstock and Tomasi, 1968; Tomasi and Czenvinski, 1968; Hanson and Johansson, 1967). One possibility to account for the relative predominance of yA in external secretions is that a preferential secretory mechanism exists in the organ systems involved for transporting yA from serum to secretion TABLE V I I COMPARISON OF ?A LEVELSIN SERUM A N D RALIVA'~ Patients ~~

18 Normal N. C. L. s.

s.

M.

A. v. N. T.

M. H. M. M. R. I. M. F. J. V.

rA h4yelonia (7 8 ) ?A Myeloma (7 S) rA Myeloma (11 S) ?A Myeloma (11S) SLE SLE SLE Ragweed allergy Ataxia telang. Ataxia telang.

160

> 1000 > 1000 > 1000 > 1000

570 550 620 21 0 0

6.3 3.6 5.6 3.9 4.3 2.3 2.3 4.3 4.6 0 0

Patielits selected to illustrate the dissociation between serum and Ralivary levels of Data from Tomasi et al. (1965) and unpublished. b SLE-systemic lupus eiythematosus; te1ang.-telangiectasia. 11 S ?A standard.

7.4.

perhaps similar to that responsible for the selective transport of yG across the placental barrier (see Brambell, 1966). A specific recognition site might then be expected to be present on the yA molecule to account for facilitated transport. In this regard, South et al. (19f36), as a result of studies involving infusions of whole normal plasma into agammaglobulinemics, demonstrated transport of yA into the saliva of 1 and possibly 2 out of the 5 patients studied. The transport mechanism appeared to be specific for the yA molecule since yG and yM were absent from the saliva despite the high levels of these immunoglobulins in the serum. The quantitative aspects of transport were not determined, nor was it demonstrated whether the yA which was found in the saliva following the infusions was secretory (11S ) in nature. However, two

SECRETORY IILIMUKOGLOBULINS

41

recent studies (Selner et al., 1967; Haworth and Uilling, 1966) following excliangc transfusions in newborns with cqdiroblastosis have failed to demonstrate any transport of yA from scmiin to saliva. This was not related to the levels of serum yA achieved, since higher concentrations of yA were found following some of the exchange transfusions than in the infusion experiments of South et al. (1966). The findings following exchange transfusions are consistent with the in vivo 1311-labeledyA studies in adults (see Section VI1,C) which also failed to demonstrate transport of yA into saliva. Some 7 s yA occurs in normal colostrum, nasal secretions, and 1965; Bienenstock and Tomasi, urine (Rossen et ul., 1966a; Tomasi et d., 1968). The 7 S yA found in secretions is immunologically identical to serum yA. Whether this represents yA transported from serum as previously suggested (Butler et nl., 1967) or degradation of the 11S yA to “monomeric” 7 S form remains to be proven.

B. IMMUNOFLUORESCENT STUDIES Immunofluorescent techniques, although useful in investigations of sites of synthesis, can, however, give only indirect evidence that the synthetic source of a spec& immunoglobulin in a secretion is, indeed, the cells stained by the corresponding antiserum. Other difficulties, particularly relating to the secretory system, are encountered. For example, it has been found exceptionally difficult both in our laboratory and that of other investigators (Hanson and Johansson, 1967) to make monospecific antisera which will react only with SP, since when the antisera are tested against whole secretions, reactions are found in the majority of cases with nonimmunoglobulin secretion-specific components (see Section IV). Despite these problems, immunoffuorescent studies have shed considerable light on the probable source of immunoglobulins in several organ systems and such studies are summarized in Table I. From these results it can be seen that there is good accord between the quantitative predominance of a particular immunoglobulin class in a secretion and the distribution of plasma cells staining with specific antisera in that tissue. Thus, the yh-containing cells predominate in the lamina propria along the whole of the GI tract, in the parotid and submaxillary glands, nasal and bronchial mucosa, as well as the lacrimal glands. In gingival tissue, however, yG cells appear to predominate and in the lamina propria of the appendix the numbers of yA- and yGcontaining plasma cells are approximately equal. In the latter organ, lymphoid follicles stain approximately equally with all antisera used,

42

THOMAS B. TOMASI, JR. AND JOHN BIENENSTOCK

and a similar finding has been demonstrated with nasopharyngeal tonsils. There are no reports as yet of a systematic investigation into the Peyer’s patches and similar lymphoid collections of the GI tract. The morphological type of cells stained with immunofluorescent antisera in these studies are most often described as members of the plasma cell series, However, most investigators stress that these c d s have wide variations in morphological character, though many of them are identifiable as typical plasma cells with eccentric nuclei. Crabbe et al. (1965; Crabbb 1967) assumed a globular shape for nucleus and cell alike and calculated an average nucleocytoplasmic ratio of 0.7 for cells containing immunoglobulins in the lamina propria of the intestinal tract. Little difference was found for this ratio between cells stained with different antisera. The studies of Tomasi et d.(1965) demonstrated staining of plasmalike cells and cells of uncertain morphological type in the interstitium of the parotid gland with antisera directed against the secretory yA molecule and also with antisera specific for 7 S serum yA. No staining of the epithelial cells was found with a specific anti-7S yA antiserum. However, staining of the epithelial cells occurred with an antisecretory yA antiserum and with an antisecretory yA antiserum that was absorbed and, thus, reacted only with the secretory molecule and was presumably directed against SP alone. As a result of this work, as well as other evidence discussed later in this section, it was suggested that local production of yA might be occurring in interstitial plasma cells and that SP might be added to the yA molecule at the epithelial cell level in transit to the lumen. Rossen et al. ( 1 9 6 7 ~ ) have reported that both SP and 7 S yA staining occurred in serous acinar cells of the submaxillary salivary gland and in the epithelial cells of the bronchial mucosa. These authors have also described the existence of SP and yA determinants in cells beneath the epithelial layer and suggested that SP and yA were produced by the same cell, probably a plasmalike cell. However, other studies also performed on bronchial tissues ( Martinez-Tello and Blanc, 1968; and see Fig. 4 ) have not confirmed these findings. Moreover, it is now well established that SP and yA can be independently synthesized since in the newborn and agammaglobulinemic, SP is found in the absence of immunoglobulins and in the apparent absence of plasma cells. If Rosqen’s observations are valid, one would also have to hypothesize that there were two completely different types of +A-producing cells, since y A-containing plasma cells in peripheral and splenic lymphoid tissue do not stain for SP. Eidelman (personal communication) has

SECRETORY IMMUNOGLOBULINS

43

shown in a study of rectal biopsies of neonates the almost complete absence of plasma cells in the lamina propria until about 7 to 10 days of life when this structure rather suddenly receives a full complement of plasma cells similar in numbers to that found in the adult. Immunofluorescent staining of these tissues shows no staining in the lamina propria in the absence of plasma cells and an appropriate predominance of yA-containing plasma cells without SP folIowing colonization with plasma cells. Gelzayd et al. (1967a), in immunofluorescent studies of the rectum and colon, found fluorescence with anti-yA antisera in the apical portion of the epithelial cells and also in the mucus on the luminal side. Epithelial yA fluorescence might represent transported yA from the lamina propria, and several investigators (summarized in Table I ) have shown faint or occasional staining of the epithelial layer with antiserum yA antisera. However, as mentioned above and as shown in Fig. 4, little or no yA epithelial fluorescence was found in the parotid or bronchial mucosal glands with an antiserum yA antiserum although striking luminal fluorescence was observed. Moreover, Crabbe (1967) in his investigations on the GI tract did not find epithelial staining with antiserum yA antisera anywhere along the GI tract except for occasional staining of the epithelial layer of the nasopharyngeal tonsil, These discordant results need further clarification.

C . I n Vivo RADIOACTIVE TRACER STUDIES The metabolic behavior of serum yA labeled with l3II and yG labeled with I Z a I was studied (Tomasi et al., 1965) in 1 normal and 4 subjects with various diseases. No evidence of transport of intact yG or yA from serum to saliva was found. McFarlin et al. (1965) detected transfer of only trace amounts of labeled 7 s yA in 2 out of 3 cases studied and none in a third. Butler et al. (1967) found that labeled 11S yA isolated from nasal secretions was not transported into nasal secretions when administered intravenously. However, homologous and autologous 7s yA did appear in the nasal secretions apparently unchanged. These findings suggested that serum 7 S yA might be a source of the 7 s yA found in nasal secretions, but that the major portion of the ./A which was of the 11S secretory type was synthesized de no00 locally in nasal tissues. In the turnover studies of Tomasi et al. (1965) with labeled 7 S yA, no protein-bound radioactivity was found in the urine. This is of interest in regard to the derivation of urinary secretory yA (Bienenstock and

44

THOMAS B. TOMASI, J R . AND JOHN BIENENSTOCK

Tomasi, 1968) since, if the urinary yA were derived from serum by glomerular filtration or selective secretion, protein-bound radioactivity would have been expected.

D. TISSUECULTURE Incorporation of ’“-labeled amino acids into yA has been demonstrated by radioimmunoelectrophoresis with mammary gland tissue obtained from humans, rabbits, and monkeys (Hochwald et aE., 1964). Labeling of the other immunoglobulin classes was not found. Similar studies with human parotid tissue (Tomasi et al., 1965) demonstrated labeling of 11 S secretory yA in tissue culture and only trace amounts of yG were found to be labeled and, then, only after prolonged exposure of the radioactive slides to the sensitive film, Whether the small amount of y G synthesized was produced by white cells present in blood contaminating the tissue fragments or by plasma cells indigenous to the gland could not be determined. Analogous findings were reported by Hochwald et al. (1964) in tissue culture studies of monkey submandibular glands. In the two studies mentioned above, no attempt was made to determine whether incorporation of label occurred into yA, SPYor both. Thus, the only conclusion which is justified on the basis of this work is that at least one of the components of the secretory molecule was being synthesized by the tissue fragments. The possibility that only the SP is synthesized locally and that the yA portion is transported from serum cannot be excluded from these studies. Asofsky and Small (1967) used a similar technique and demonstrated incorporation of 14C-labeled amino acids into rabbit colostral yA. However, fractionation of the harvested culture fluid showed that the majority (about 75%)of the radioactive label was incorporated into the T chain and little into the yA heavy-chain or light-chain components. The results suggested to these investigators that the major source of the yA portion of the secretory molecule in rabbit colostrum originates from serum yA rather than from local synthesis by yA-producing plasma cells in the gland. Alternative explanations for these results considered unlikely by the authors, including a sizable pool of 7 s serum yA in the mammary tissue at the start of the study and an unusual distribution of amino acids in the SP. It is also possible that there is a disproportionate local rate of synthesis of yA and SP although in this case one might expect more incorporation of label into the yA portioii tlraii demonstrated in these studies, The work of Askonas et a2. (1954) would support the conclusion of Asofsky and Small. These workers, on the basis of the kinetics of incorporation into serum and colostral proteins of labeled amino acids given parenter-

SECRETORY IMMUNOGLOBULINS

45

ally, concfndcd that thv colostrd ~-gloltuliiisin goats and Inbl>its were derived primarily from serum. Similar ronclusions regarding the origin of milk y-globulins in the cow were reported by Dixori et a / . (1961) and Pierce and Feinstein (1965). It seems likely, therefore, that the mammary gland at least in certain species is able to transport immunoglobulins from serum. The transport mechanisms must be highly selective since it apparently involves predominantly yA in the rabbit and a fast yGglobulin in the sheep and cow (see Section X). Moreover, the mechanism whereby serum yA ( 7 and 9 S forms in the rabbit) becomes converted to 11 S secretory yA in the gland is still obscure but probably involves complexing with SP as in the human. Asofsky and Thorbecke (1961), by using the technique of organ culture, demonstrated labeling of yG and yA in cultures of human ileum. Labeling of yM occurred less frequently and cultures of one sample of appendical tissue was found to label only yG. In labeling experiments with monkey tissues, similar results were found with monkey ileum which incorporated labeled amino acids into both yG and yM. No antisera were available specifically directed against monkey yA. Surprisingly, monkey kidney was shown to incorporate label into yG and not yM. Antibody synthesis of unknown immunoglobulin classes has also been shown to occur with in ~ i t mtissue culture explants of rabbit vagina by Bell and Wolf (1967). Only rabbits immunized with diphtheria toxoid by the local vaginal route were shown to produce specific vaginal antibody. Immunization of the uterus and intravenous or footpad immunizations with Freunds adjuvant, produced high serum titers of antibody but no antibody production could be detected in vaginal tissue cultures. Neither the immunoglobulin class nor the type of cells responsible for immunoglobulin production were investigated.

E.

POSSIBLE

MECHANISMS OF SECRETION OF yA

The above observations are most consistent with the following interpretation. In the human salivary glands and probably in the GI and respiratory tracts, a significant fraction of the immunoglobulins present in the secretions are synthesized locally in tissue plasmalike cells which are found in close anatomical relationship to the glandular and mucous membrane epithelia. The possibility of some transport from serum cannot be excluded particularly if inflammation of the mucous membrane occurs for any reason. In agammaglobulinemia, if high enough serum levels of yA are produced by infusion, some yA may reach critical transport sites and be secreted. However, this is an inconstant

46

-

THOMAS B. TOMASI, JR. AND JOHN BIENENSTOCK

llSyA

External secretions

(7syA), +“piece”

Glandu lor plasma cell

E pi t he1 ia I cel Is

FIG. 13. Schematic representation of a postulated mechanism for the formation of secretory yA.

finding and does not normally occur to a significant extent, perhaps in part because of failure of yA to permeate capillaries or a “diffusion advantage” of locally produced yA, or for some other more complex reason. The locally produced yA which is assumed (but not proven) to be identical to serum yA, then traverses the mucous membrane or glandular epithelium by some as yet unknown mechanism. The SP, a nonimmunoglobulin glycoprotein with apparent specificity for the yA molecule, is synthesized by the epithelial cells. It complexes firmly with the yA portion, probably by both disuIfide as well as noncovalent forces, to form the intact 11 S secretory molecule. Where the SP and yA portions unite, whether inside the epithelial cell, on one of its surfaces, or in the lumen is not known. This hypothesis is summarized schematically in Fig. 13. It has been suggested (Tomasi et al., 1965; South et al., 1966) that SP might in some way facilitate the transport of yA and for this reason it has been referred to as transport “piece.” However, although an attractive possibility, no good evidence is presently available to support this thesis. In the case of mammary tissue of certain animals the available evidence suggests that the major portion of the colostral immunoglobulins are derived by transport from serum. Transport is, however, highly selective and only certain serum immunoglobulins are secreted (yA in the rabbit). The SP in the rabbit is synthesized locally in the mammary gland but its function, if any in facilitating transport, is unknown. Whether the human mammary gland is similar in these respects to those of animals and selectively transports immunoglobulins from serum or whether it synthesizes yA and perhaps other immunoglobulins locally as seems likely for other external secretions remains to be determined. Although the interpretations outlined above seem most likely on the basis of currently available information, other possibilities should be

SECRETORY IMMUNOGLOBULINS

47

entertained. The possibility of cellular transport by migrating lymphocytes through the epithelial surface to the lumen must be considered. In both man and animals the presence of lymphocytes within the epithelial cells of intestine, trachea, and urinary tract are well established (Trowell, 1958; Andrew, 1965; Andrew and Collings, 1946; see also Darlington and Rogers, 1966). These epithelial lymphocytes of man show considerable morphological similarity to the globule leukocytes described in animals in response to nematode infestation (see Dobson, 1966a,b,c; Whur, 1967). It is possible that both the epithelial lymphocytes and globular leukocytes do not actually lie inside epithelial cells but rather in the natural intercellular channels described by Tormey and Diamond (1967), which are apparently common to most epithelial structures involved in absorptive processes. These channels are firmly closed by a tight junction on the apical side of the cell but are open with gaps in the basement membrane on the basal side. Thus, the various studies (outlined in Table I ) showing fluorescence of epithelial cells might represent yA being transported in lymphocytes. In this regard, some fluorescence has been detected in intercellular positions by Brandtzaeg et al. (1967). However, Gelzayd et al. (1967a) detected yA fluorescence primarily in the apical portion of the epithelial cells, whereas epithelial lymphocytes are seldom found in this region since usually they lie in the basal portions beneath the cell nucleus. Furthermore, the bulk of evidencz (Darlington and Rogers, 1966) suggests that actual migration of lymphocytes into the lumen rarely occurs. The observation that yA staining is primarily apical would also be difficult to explain if yA transport occurred primarily by a mechanism involving reverse pinocytosis by epithelial cells as is often assumed for the transport of other large molecules. Another hypothesis which has been considered (Tomasi, 1965) involves the transudation or secretion of serum proteins with selective degradation by the proteolytic enzymes present in these fluids, the yA being more resistant to proteolysis than the other proteins. In this regard some evidence for an increased resistance to proteolysis of both serum and particularly secretory yA, compared with y G and yM, has been presented (Plaut et al., 1968; Tomasi and Czerwinski, 1968). However, this mechanism can be excluded in the case of parotid saliva. Previous work has shown that parotid fluid lacks proteolytic activity (Chauncey, 1961). Addition of yG to parotid fluid and subsequent incubation did not reveal significant degradation of the y G molecule ( Tomasi, 1965). Moreover, this thesis would leave unexplained the vast predominance of YA-type plasma cells in the organs of the externaI secretory system.

48

THOMAS B. TOMASI, JR. AND JOHN BIENENSTOCK

Biological Properties of Secretory yA

VIII.

A. “NATURAL” SECRETORY ANTIBODIES 1. Isohemugglutinins Serum isohemagglutinins have been described with intermediate sedimentation coefficients (approximately 11S ) (Rockey and Kunkel, 1962; Rawson and Abelson, 1964; Ishizaka et al., 1965), and these for the most part appear to be due to polymeric serum yA. Serum yA polymers can be differentiated from the 11s secretory yA by the absence of the specific SP determinants and also by the fact that serum 11s yA dissociates on treatment with reducing agents with loss of agglutinating activity, whereas secretory yA is unaffected. Tomasi et d. (1965) demonstrated that salivary and colostral secretory yA in both 11s and 18 S polymer ranges contained isoagglutinin activity, and similar results have been obtained by Adinolfi et aZ. (1966a). The polymeric form of serum yA is approximately 7 times more efficient in hemagglutination than the monomeric form on a weight basis (Ishizaka et al., 1965). However, no information is available on the number of antibodycombining sites and the relative efficiency of the 11s secretory yA molecule. Adinolfi et al. (1966a) in a study of colostral isohemagglutinins demonstrated antibody activity both in the yM and yA classes but not in the yG. In addition, they were able to show in several cases that the titer of colostral yA antibody exceeded that found in serum. Although isosgglutinins have been described in several other external secretions, the immunoglobulin class of these antibodies has in most cases not been investigated, with the exception of urine. Isoagglutinins could not be found in normal concentrated urine (Hanson and Tan, 1965; Prager and Bearden, 1965) However, on immunization of such individuals with blood group substances, isoagglutinins of the y G class have been described by both Prager and Bearden (1965) and Hanson and Tan (1965). Cross-reactivity between blood group substances and microorganisms have been well described, and it seems likely that the isohemagglutinins found in serum are a result of exogenous stimulation by cross-reacting antigens present in bacteria and viruses (Springer, 1967). For example, a single oral feeding of blood group-active Escherichia coli 086 to germfree animals (having no anti-B antibodies) produces high serum anti-B titers. Feeding experiments in man have given similar results. Likewise, myxoviruses such as influenza and fowl-plague virus contain significant blood group A and Forssman antigen specificity as components of their a

49

SECRETORY IMhlUNOGLOBULINS

virus coat. The isoagglutinins in external secretions are probably also formcd as a result of cross-reactions nit11 microorganisms but this has not been directly demonstratcd. 2. Viral and Bacterial Antibodios “Natural” antibodies apparently formed in the absence of overt infection or immunization, against a variety of bacteria and viruses, have been found in nasal secretions, saliva, small intestinal secretions (Fig.

FIG. 14. Immunofluorescent stuclies of Escherichh culi incubated with normal serum ffluorescent anti-7 S small intestinal secretions and serum. ( A ) E . c d i yA; ( B ) E. culi f intestinal secretions -/- fluorescent anti-7 S yA; ( C ) E . coli f intestinal secretions serum fluorescent antisecretory “piece” (SP); ( D ) E . coli ffluorescent anti-SP. [Data from Tourville et al. ( 1968a).]

+

+

+

CHARACTERIZATION

TABLE VIII SECRETORY A4NTIBODIES

OF “NATURAL”

Antibody class. Secretion Saliva

antigen

Nasal Small intestinal Colostm1

Blood group subs. Escheriehiu coli Lactobacillus, Streptococcus salivarius, Streptococcus mitis Salmonella typhosa, Shigella dysenteriae Polio I, Coxsackie A9, ECHO 28, Parainfluenza 3 Eseherichia coli Blood group subs.

Urine

Eseheriehia coli Blood group subs. Eseheriehia coli

yG

yh

y M

Reference

+*

Tomasi et al. (1965) Tourville etal. (1968b) +I Green (1966) +t Evans and Mergenhagen (1965) Artenstein et al. (1964) Tourville et al. (1968a) Tomasi et al. (1965) +t Adinolfi et al. (1966a) +t Adinolfi et a!. (1966b) - Prager and Bearden (1965) Hanson and Tan (1965) - Tourville et al. (1968b)

+ +* +

+ +

+- +* + +* + -

+ +*

a Key to symbols: (+) Evidence of strong activity in given immunoglobulin class; (-) evidence that activity not, in given immunoglobulin class; (*) good evidence for secretory ?A activity; (t) suggestive evidence for secretory ?A activity.

SECRETORY IhlMUNOGLOBULINS

51

14 ) , colostrum, and urine. Investigations concerning the characterization of these antibodies are summarized in Table VIII where appropriate references for more detailed information are given. In Section VI, there is a critical discussion of the methods (direct and indirect) of determining antibody activity in a given immunoglobulin class. B. SECRETORY ANTIBODIESFOLLOWING IMMUNIZATION 1 . Active Zininunization

,

Parenteral immunization in many instances can endow protection upon the individual providing a significant humoral antibody response is mounted. However, it is well established in the case of polio (Salk) vaccine that although protection can be demonstrated on successful immunization, prevention of the carrier state is not necessarily accomplished. This suggests that, although the stage of viremia can be prevented by circulating antibody, colonization of the GI tract and replication of the virus locally is not necessarily inhibited by serum antibodies. Since the portals of entry of many pathogenic viruses and bacteria are the GI and respiratory tracts, study of the role of secretory antibody and the procedures by which local immune mechanisms can be more effectively stimulated, could be of considerable importance in developing more effective immunization programs. a. Gastrointestinal Antibodies. Besredka ( 1927) appears to have first postulated a mechanism of local intestinal immunity against cholera. Burrows and Havens in 1948 demonstrated the effectiveness of oral live vaccination against cholera in both man and guinea pig and showed that protection could be correlated with the presence of coproantibody and not with the level of serum antibody. Neither urinary nor fecal antibody appeared to be derived from serum, at least by direct transudation. Oral immunization in man by dietary proteins routinely ingested may also stimulate production of serum antibody in apparently healthy people (Gunther et al., 1960; Peterson and Good, 1963; Saperstein et al., 1963; Rothberg and Farr, 1965). Rabbits immunized orally with low concentrations of bovine serum albumin will eventually develop serum antibody levels indistinguishable from those of animals immunized by intravenous or subcutaneous routes ( Rothberg et al., 1967). After oral immunization in man with attenuated poliovirus, or living or dead bacteria, specific antibodies can also be detected in the serum (Sabin et al., 1961; Buser and Schar, 1961; Thomson et al., 1948; Freter, 1962). [For a review of similar work relating to coproantibody, see Freter ( 1%2), Pierce (1959), and Freter and Gangarosa (1963).] Koshland and Burrows (1950) and

52

THOMAS B. TOMASI, JR. AND JOHN BIENENSTOCK

Koshland (1953) suggested that the mode of immunization may well affect the eventual production of coproantibody in animal experiments. The use of Freund's adjuvant in conjunction with intraperitoneal instillation of vaccine did not result in any coproantibody formation despite high levels in the serum, However, these investigators were detecting antibody activity by complement fixation techniques, and noncomplement-fixing antibodies (such as secretory y A ) would have been missed by the techniques employed. Since it is recognized that in the guinea pig the production of yl and y. antibodies can be predetermined by the mode of immunization, these results are perhaps not surprising. However, these experiments point out the possibility that adjuvants such as alum often used in immunization procedures in man might modify the type of antibody eventually produced at a local site. In a comparative study of live oral vaccination (Sabin) and killed parenteral (Salk) vaccination with poliovirus in children, Ogra et d. (1968) showed that the serum antibody responses and their distribution in the three major immunoglobulin classes were approximately the same with either route of immunization using the sensitive radioimmunodiffusion technique employing "P-labeled poliovirus ( Fig. 15). However, after parenteral immunization with killed poliovirus, no secretory antibody could be detected in nasal washings or duodenal juice. Oral vaccination with live poliovirus did produce predominantly a y A immunoglobulin response in the secretions studied (no yG or yM antibody could be detected), whereas serum antibody measured simultaneously could be found in all three classes but was present in highest concentrations in yG. Investigations were not carried out on the antibody response t0,oral killed poliovirus in order to determine whether the use of live vs. killed vaccine is of importance in the local response. Hodes et aZ. ( 1964) have demonstrated that colostrum contains predominantly yA polio antibody and the same group of investigators (Berger et al., 1967) demonstrated only yA antibody to poliovirus in saliva and duodenal juice, whereas, in urine, yG antibody was found in addition to yA. The mode of immunization was not recorded in these studies. Oral killed cholera vaccine can produce local or coproantibody production ( Freter, 1962; Freter and Gangarosa, 1963) with minimum serum antibody levels. However, daily doses of vaccine had to be given for 4 weeks to produce satisfactory titers and subsequent weekly doses of oral vaccine were necessary to maintain coproantibody levels. Recruitment of local immune competent cells either in the wall of the gut or mesenteric lymph nodes might account for the local production of antibody. This suggestion is supported by the cell transfer experiments

53

SECRETORY IMMUNOGLOBULINS

of Thind (196f3). Cells obtained from the mesenteric lymph nodes or peripheral lymphoid tissues ( axillary or popliteal nodes) of rabbits, immunized either intravenously with cholera vaccine or fed the same vaccine intragastrically, were transferred to normal recipients. Mesenteric lymph node cells when obtained from animals immunized by either

&

128-

0

m t2

a 32v) 3

g > P

B -1 90

=

0 u

Y

8-

2-

Lu 0:



? Origin

5

10

15

20

25

29

Anode

Pevikon Fractron

FIG. 10. Chemotaxis of rabbit neutrophiles in wit70 by electrophoretically separated ( p H 7.3) fractions of the triethylaminoethyl (TEAE)fraction activated by EAC' def. stromata. Chemotactic activity is found in fractions containing both C'5 and C'6; C'7 was associated with C'5. plpC'5; /3,,4'3. (From Ward et aZ., 1985.)

119

MEDIATION OF IMMUNOLOGIC TISSUE IN JURY

separated in the same fractions containing these components (Fig. 10), when the same electrophoresis was performed at pH 8.6, C’5 separated from C’3 and C’6 (C’7 was not measured) and the chemotactic activity was not found in any of the eluted fractions. However, when fractions rich in C’6 and C’5 were pooled (and most probably C’7 also, as found in data later obtained), chemotactic activity was restored (Fig. 11). 400 V, 30 hrs Barbital Buffer 8.6 Chemotaxis

E

C’6

PIF

C’~*PIF

(fract 16, (fract 26, 501) IOOA)

aJ

f

2 -,oo c

a - 50

0

0,I

QIV”

5

10

I5

20

25

30

Fraction (Tube Number)

FIG. 11. Electrophoretic separation (pH 8.6) of the triethylaminoethyl (TEAE) fraction following activation by incubation with EAC’ def. complexes. Most of the /3m-globulin is fast moving and widely separated from C’0, although a small zone of slower moving C’5 is also present. /31~C’5;/ & A ‘ S . (From Ward et al., 1905.)

As observed by Nilsson and Miiller-Eberhard ( 1967), C’5, C’6, and C’7 function as a complex of the three molecules in immune hemolysis of red blood cells. The trimolecular complex apparently reacts with the erythrocytes and the first four reacting components of complement but becomes disengaged immediately from the cell membrane. The cell is, thus, prepared for the hemolytic action of C’8 and C’9 and the trimolecular complex, C’5-6-7, is released into the surrounding medium. Employing the sequential activation of purified components of human complement, experiments were then performed in which the identity of the chemotactic factor as the C’5-6-7 complex was established. The experimental protocol and results are given in Table I11 (Ward et al., 1966). The addition of the terminal complement components caused inhibition of chemotaxis, a finding for which an explanation is not yet available. Employing the purified components of guinea pig complement (of Dr. Robert Nelson), similar results were obtained. Supporting data relating the activated C’5-6-7 complex and the chemotactic factor were obtained by density gradient centrifugation in which chemotactic activity and the C’54-7 complex sedimented together.

120

CHARLES G. COCHRANE

TABLE I11 CHEMOTAXIS OF NEUTROPHIIXS BY INTERMEDIATE COMPLEXES OF C’ Intermediate complex Human C’ EAC’1,4,2” EAC’1,4,2,3 EAC’1,4,2,3,5 EAC’1,4,2,3,5,6 EAC’1,4,2,3,5,6,7 EAC’1,4,2,3,5,6,7

+ ternlinal ctrrnponeiits‘

CoiiLrols EAC’1,4,2,3,6 EAC‘1,4,2,3,5,7 EAC‘I ,4,2,3,7 Lysis control EAC’1,4,2 Ag-Ah in 10% rabbit serum 10% rabbit serum

Chemotartic valuesb Not tested 35 21 283 -

-

38

-

144 11

8 8 79 59 456 -

244 41

36 69 3 260 4

36 1x0 18

16 19 12

-

-

Sensitized erythrocytes (5 x 107) containing the first three reacting components of human C’. * Each vertical column of figures represents a single experiment. c Terminal components probably representing two final reacting components (C’8 and C’9; Nilsson and Miiller-Eherhard,personal communication, 1967). 0

Of great interest was the finding that the synthetic peptide N-carbobenzoxy ( CBZ ) -glutamyl-L-tyrosine was found capable of dissociating the C’5-6-7 complex. Mixing this peptide with the active chemotatic agent during incubation in the chamber reduced greatly the migration of neutrophiles. Both this peptide and N-CBZ-glycyl-L-phenylalanine prevented activation of the C’5-6-7 complex in whole serum by immune precipitates. Removal of the synthetic peptide inhibitors prior to activation of the serum with immune reactants allowed generation of the chemotactic factor. Thus the synthetic peptides did not apparently alter normal serum proteins and components of complement prior to activation of the complement sequence. Although such findings underscore the synonymity of chemotatic factor and activated C’5-6-7 complex, they also open the door to a possible rational therapeutic approach to immunologic disease. A second chemotactic factor has been generated from the complement system more recently. The incubation of highly purified human plasminogen together with streptokinase and human or rabbit C’3 resulted in the formation of a material chemotactic for neutrophiles (Taylor and Ward, 1967). Physicochemical studies of this factor have

MEDIATION OF IMMUNOLOGIC TISSUE INJURY

121

revealed a molecular weight of approximately 6000 by gel filtration, rapid anodal migration in electrophoresis at pH 8.6, and slow sedimentation of the factor in a sucrose density gradient (Ward, 1967). This plasminderived fragment of C’3 is distinguishable by these properties from C’3 anaphylatoxin which is also obtained by hydrolytic cleavage of the parent C’3 molecule (Dias da Silva et al., 1967; Cochrane and MiillerEberhard, 1968). The plasmin-split product also failed to contract guinea pig ileum (Ward, 1967). However, studies in this laboratory have indicated recently that F(a ) C’3 is capable of inducing chemotaxis of neutrophiles. In addition, a similar split fragment from guinea pig and human C’5 also possesses both chemotactic and anaphylatoxin activities ( Shin et at., 1968; Ward, 1968). The relative role played by these various chemotactic factors derived from the complement components will have to be ascertained by quantitative comparison in the future. Keller and Sorkin have conducted a series of experiments on the chemotaxis of neutrophiles in vitro (Keller and Sorkin, 1965a,b). They employed chambers similar to those used in the studies just described, except that a pore size of 3 p rather than 0,65 p separated neutrophiles from chemotactic agents in the chambers. In addition to complementrelated chemotactic agents, these investigators described the formation of chemotactic factors in fresh serum that appeared in the absence of significant complement fixation. The Agg HGG exposed to acid pH conditions and aggregated bovine y-globulin ( Agg BGG) acted in this capacity. The authors concluded that no correlation existed between complement fixation in the serum and the formation of chemotactic factor. More recently, serum activated with antigen and antibody or Agg BGG was subjected to gel filtration on Sephadex G-200 (Keller and Sorkin, 196713) in order to identify the complement-independent chemotactic factor. Two chemotactically active zones of elution were observed, one in the void volume which the authors attributed to C’M-7 complex and another eluting in the final protein peak of the serum. Molecular sizes were not determined for the second peak of chemotactically active material, but it would appear to be in a molecular weight range of less than 100,000. The authors suggest that this second chemotactic agent is the noncomplement-dependent substance studied by them previously ( Keller and Sorkin, 1965b).

2. Directional Migration of Neutrophiles in a Concentration Gradient of Claemotactic Factor With the implications of the experimental results on chemotaxis in vitro in mind, investigations in several laboratories were undertaken to

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assess the true directional quality of the neutrophile migration toward increasing concentrations of the chemotactic factor. Keller and Sorkin (1966), in both chemotaxis chambers and glass slides using photomicrographic tracing of neutrophile migration, showed that neutrophiles moved toward areas of greater concentration of chemotactic factor. Reversing the gradient prevented the established directional migration. It was noted that random migration of the neutrophiles was enhanced by exposure

FIG. 12. Schematic representation of the cheniotaxis of a neutrophile (PMN) by activated C‘5-6-7. The concentration of activated C’5-6-7 is greatest at the point of complement interaction with the antibody. A (C’5-67); A (C’Fi6-7); PMN Chemotactic factor.

to activated (chemotactic) serum. Similarly, Ward et al. ( 1966) found that by adding relatively purified, activated C’5-6 (and C’7, although its presence was not realized at the time) complex to the cell suspension prevented migration of the neutrophiles through the micropore filter. Comely (1966) found it possible to reverse the migration of neutrophiles in a chemotaxis chamber (i.e., within the micropore filter) by reversing the gradient of chemotactic material at varying times of incubation. A diagram of the chemotactic attraction of neutrophiles by the C’5-6-7 factor is given in Fig. 12. The finding that neutrophiles can be prevented from migrating away from the generating source of chemotactic factor offers a second relationship between the in vivo and in vitro studies. A positive force may be exerted by the chemotactic factor, inducing neutrophile migration to-

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ward the source of activation both in vitro and in vivo, and, in addition, once accumulated the neutrophile is unable to migrate away from the higher concentration of the chemotactic factor.

3. Zmmune Adherence and Neutrophik Accumulation In addition to the chemotactic attraction exerted on neutrophiles by the complement system, another complement-mediated mechanism may play an important role in the accumulation of these cells at sites of antigen and antibody deposits in duo-the mechanism of immune adherence. Once antigen and antibody have interacted and have activated the complement sequence, many different formed elements of the blood adhere to the immune reactants. Neutrophiles, platelets, macrophages, and red blood cells have all been shown to adhere (Nelson, 1962; Siqueira and Nelson, 1961; Henson, 1967). The adherence results from the binding of these cells to C’3 that has reacted with C’l, 4 and 2 and has become &ed to antibody and neighboring tissue and cellular membranes (Nishioka and Linscott, 1963; Linscott and Nishioka, 1963). The mechanism of the binding is poorly understood. Henson (1967) found that neutrophiles of twelve species studied (man, baboon, rabbit, rat, mouse, guinea pig, horse, ox, sheep, goat, cat, pig) exhibit immune adherence with autologous or homologous complement. It is readily conceivable that with the fixation of C’l, 4,2, and 3 to bound antibody and of C’3 to neighboring membranes and tissues, the mechanism of immune adherence could be instrumental in the accumulation of neutrophiles. Depletion of complement by the various methods noted above or inducing immune reactions with antibodies that fix mammalian complement poorly would result in little C’3 binding and marked inhibition of immune adherence clumping of the passing neutrophiles. This phenomenon may explain, in part, the neutrophile accumulation noted in Arthus reactions of C5-deficient mice and C’6-deficient rabbits. At present, however, the importance of this phenomenon in various immunologic reactions in normal animals has not been determined. 4. Neutrophile Accumulation and Chewtaxis with Certain Other Substances Among the many substances of varied nature that have been found to induce the accumulation of neutrophiles in tissues, mention should be made of certain positively charged, vasocative materials that have received much recent interest. These all induce an increase in vascular permeability in low doses (about 1 to 2 pg. or less intradermally) and are highly basic, Some of these act as stimulants of neutrophile ac-

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CHARLES G. COCHRANE

cumulation when injected in animals. Bradykinin in highly purified form has been found to induce neutrophile accumulation in guinea pigs when applied to the mesentery (presented by Lewis, 1963; Graham et al., 1965). In other studies (Graham et al., 1965), Hageman factor, after activation on glass, was found to induce not only a permeability reaction in skin of guinea pigs but also the accumulation of neutrophiles. It is reasonable to suppose that part of this activity at least is attributable to the formation of bradykinin in vivo from the injected activated Hageman factor. Other substances with neutrophile-accumulating activity may be derived from cells. Frimmer and Hagener (1963) found that basic histones of calf thymus upon injection in animals caused increased vascular permeability and neutrophile accumulation. In addition, substances released from neutrophiles themselves when incubated in simple medium attract neutrophiles upon local administration to animals ( Moses et al., 1961). Purification of basic proteins from the granules of neutrophiles was carried out by Golub and Spitznagel (1966) and by Janoff and Zweifach (1964) using the method of precipitation with ethanol at 20% described by Frimmer and Hagener (1963). These basic proteins were active in increasing vascular permeability, as well as inducing neutrophile accumulation, and may account for much of the activity in the supernatants of incubated neutrophiles described by Moses et al. ( 1964). It is tempting to relate the capacity of these proteins to induce neutrophile accumulation with their basic quality. Indeed, synthetic basic peptides share this capacity (Stein et al., 1956). Janoff and Zweifach (1964) have suggested that the basic proteins with their positive charge may act as ligands between cells such as neutrophiles and endothelial cells that have negatively charged surfaces (Bangham and Pethica, 1959) and normally repel one another. Studies carried out in vitro in chemotaxis chambers in this laboratory and by others have not offered strong support for an important role of basic proteins in the chemotaxis of neutrophiles. Tests of bradykinin, kallidin ( lysyl bradykinin) , and the 20%ethanol-precipitated basic protein obtained from polymorph granules in doses of 20 pg/ml. yielded only 10%or less of the chemotactic activity of the complement-derived chemotactic factor (Ward et al., 1966). Injection of the kinins and neutrophile cationic protein into the skin of rabbits also induced a fraction of the neutrophile accumulation obtained with immune reactants. Cornely ( 1966), in testing lysates of neutrophile granules in chemotactic chambers, found definite chemotactic activity, hut this formed a small percentage of that activity obtained by serum previously treated with

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zymosan. In the latter study, the addition of serum to the neutrophile granule lysate did not augment the chemotaxis. In summary, there appears to be a neutrophile-accumulating quality of basic proteins, some of which are most certainly released at the site of immunologic reactions. Although quantitative comparisons between the amounts and activities of these proteins and the C’5-6-7 chqmotactic factor have not been rigorously performed, it would appear from the present data that the C’54-7 complex is considerably more potent. A relationship may exist between the ability of basic proteins to induce neutrophile accumulation and the complement chemotactic agent, in that basic proteins inactivate complement (Arnon et al., 1965). Indeed, Carpenter and Gill (1966) found that polylysine (mol. wt. 105,000) at concentrations of 100 pg./ ml. or less rapidly inactivated hemolytic complement activity and converted the active C’3 or PlC globulin to PlG, i.e., identically to the effect of immune complexes. It is not unreasonable to suppose that the C’5-6-7 complex was activated in the process. In addition, the basic proteins of rabbit neutrophiles also inactivate hemolytic complement activity ( Cochrane and Aikin, 1966). Hence injection of basic proteins may stimulate the activation of complement chemotactic factors.

5. Fuctors Intrinsic to Neutrophibs Afecting Their Chemotactic Response The chemotactic response of neutrophiles is a complex action involving several stages. Up to this point the discussion has centered on the various factors that induce migration of the neutrophile. These being rather specific in nature, such as the C’5-6-7 complex or the C’3-split fragment, one would strongly suspect rather specific and sensitive receptor mechanisms on or near the surface of the cells. And, since active locomotion is required energy sources would probably be called upon. Hence a search into the basic functions of neutrophiles is certainly warranted to obtain information about this extremely important phenomenon. Some knowledge is already available. Regarding energy requirements, Ward (personal communication ) has found the utilization of oxygen rises about 25% when neutrophiles are exposed to activated C’5-6-7 complex. Chemotactic migration was M ) (Ward, 1966), but this not inhibited by dinitrophenol (DNP) ( is not surprising considering the quantity of stored adenosine triphosphate (ATP) and the unique capacity of the neutrophile to function anaerobically. Recently two enzymes having esterase activity have been recognized

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CHARLES G . COCHRANE

in rabbit neutrophiles, each of which is in sonic way essential to the chemotactic process. These have been characterized through the use of organophosphorus inhibitors, the p-nitrophenylethyl phosphonate esters. These compounds inactivate enzymes in the class of serine esterases. Their capacity to inactivate the esterases is dependent upon the type of side chain attached to the phosphate group on the inhibitor. Variation of the number of carbon atoms in the side chain leads to changes in the capacity to inhibit a particular esterase. Thus a profile of inhibition may be experimentally determined that characterizes a given esterase. A different profile of inhibition would be obtained with different esterases using the same group of phosphonate inhibitors. Several classes of phosphonate inhibitors were tested for their ability to inhibit the chemotactic response of rabbit neutrophiles to the C‘S-6-i complex. In each class, inhibition was obtained when chemotactic factor was added while the cells were in the presence of inhibitor. However, if the cells were first exposed to phosphonate inhibitors and then washed prior to testing, only one group of inhibitors was still effective in preventing chemotactic migration of the neutrophiles (Ward and Becker, 1967). This was interpreted as indicating that there were two different esterase enzymes, one that required activation by the C’S6-7 complex, and another that existed in an inhibitable state in the cell without need of activation. Both enzymes were found to differ from many common enzymes by their patterns of inhibition, e.g., from trypsin, chymotrypsin, acetylcholine esterase, and C’1 esterase and the esterases in guinea pig and rat mast cells that are activated during the release of histamine. The inhibition profiles of these latter enzymes were previously determined with phosphonate inhibitors by Becker and Austen (1964, 1966; Austen and Becker, 1966). Further characterization of the esterase that was inhibitable without addition of C’5-6-7 complex, was obtained by protecting the esterase from the phosphonate action with a group of synthetic esters. These simple esters compete with the phosphonate for its site of interaction on the esterase molecuIe. A high degree of specificity was found in that to block phosphonate action the ester had to be an acetate, other structures being inactive (Becker and Ward, 1967). In addition, more recent studies have indicated that neutrophiles no longer respond to chemotactic stimuli if first exposed to large doses of C’5-6-7 complex. This deactivation can be inhibited with certain aromatic amino acid derivatives, this forming the basis of another method of characterizing the esterase enzymes (Ward and Becker, 1968). The role that these two esterases play in the recognition of a concentration gradient of chemotactic factor by neutrophiles is unknown.

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Their interdependence is also unknown. However, they play essential roles in the process of directional migration toward the C ’ 5 6 7 complex and an understanding of their function will undoubtedly be central to unravelling the problem of chemotaxis. The physical stability of neutrophilic membranes is also of importance in the capacity of these cells to undergo directional migration. Factors that were known to stabilize lysosomal membranes, such as hydrocortisone, methyl prednisolone, and chloroquine, inhibited the chemotactic response to immunologically generated complement factors ( Ward, 1966). Additionally, administration of hydrocortisone and chloroquine to guinea pigs inhibited the accumulation of neutrophiles at Arthus sites, thus drawing another parallel between chemotaxis in vitro and immunologic neutrophile accumulation in vivo. IV.

The Role of Neutrophiles and Other Cells in the Mediation of Acute Immunologic Reactions

In 1951, the essential role of neutrophiles in the development of immunologic reactions was first appreciated. Increasing attention has been given this cell subsequently, and it is now generally accepted that in many forms of acute immunologic injury, the neutrophile plays the central role. Its participation leads to severe structural alteration in tissues, fibrin deposition, and scarring. If successful and nonhazardous methods of preventing its participation were developed, the permanent loss of function of the involved area or organ would be far less. On the other hand, in keeping with the anticipated role of these cells in acute inflammation, the neutrophile also serves a useful purpose in that it engulfs and catabolizes the deposited complexes of antigen, antibody, and complement (Cochrane et al., 1959; Daems and Oort, 1962; Movat et aZ., 1963; Uriuhara and Movat, 1966). In the absence of neutrophiles, the deposited complexes persist for prolonged periods (Cochrane et aZ., 1959). Considering these reactions from this standpoint, the neutrophiles are brought into the site of deposited immune reactants by a rather complex and sophisticated mechanism, the purpose of which is to rid the individual of the offensive invader. As if by accident, as will be recorded below, the very constituents of these cells that are used to destroy the invader are turned against the surrounding tissue structures.

A. INHIBITIONOF ACUTE IMMUNOLOGIC REACTIONSFOLLOWING DEPLETION OF NEurmrmi.Es The essential role of neutrophiles was first demonstrated in the Arthus vasculitis. Nitrogen mustard or specific antineutrophile antisera have been employed to eliminate neutrophiles from the circulation in

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several species of animals ( Stetson, 1951; Humphrey, 1955a,b; Cochrane et al., 1959). Marked depletion of these cells was found essential for strong inhibition, levels in the circulation being less than 150 per cubic millimeter. More recently, these observations have been extended to encompass two distinctly different immunologic reactions (as noted in Section I1 above), the acute glomerulonephritis of nephrotoxic nephritis and the arteritis of serum sickness. In the Arthus phenomenon, depletion of neutrophiles did not affect the deposition of antigen, antibody, and (2’3, . but without neutrophile accumulation little or no disruption of vascular structures was observed. TABLE IV SICKNESS CARDIOVASCULAR HISTOLOGIC LESIONSI N SERUM IN NORMAL AND TREATED RABBITS

Lesion

Untreated control (%;29 animals)

Arterial endothelial proliferation Arterial medial necrosis Arterial fibrinoid deposit,s

65 52 38

Sheep Ant.ineutrophile (%;23 animals)

Normal sheep globulin (%; 20 animals)

Sheep Antilymphocyte (%; 12 animals

17

68 58 53

67 42 33

0 0

Little increased permeability resulted, no extrusion of red cells occurred, and edema of the surrounding tissues was markedly or completely diminished. In serum sickness of rabbits, when neutrophiles were removed just prior to development of the lesions the usual necrotizing arteritis did not appear ( b i k e r and Cochrane, 1965). Disruption of the arterial lamina elastica, i d u x of cells, and fibrinoid necrosis were all prevented by this measure (Table IV). The glomerulitis, normally seen in serum sickness, was not affected by neutrophile removal, but the possibility must be considered that effective elimination of the granulocytes could not be achieved for the required period of time. Marked or complete inhibition of glomerulonephritis in acute nephrotoxic nephritis of rats ancl rabbits was effected by removal of neutropliilrs immediately prior to injec-tion of nephrotoxic siwini ( Coclirnne et d., 1965) (Table V ) . Antibody was found Iodized along with C’3 on the glomerular basement membrane, but, in the absence of neutrophiles, damage to the basement membrane and proteinuria could be prevented

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TABLR V PROTEINURIA IN FIRST24 HOURSAFTER INJECTION OF MODERATEDOSAGE OF NEPHROTOXIC SERUM ~~~~

~

~

Normal Animal

Rats Rabbits

Neutrophile-depleted

No. animals

mg./24 hr.

No. animals

mg./24 hr.

8 6 5

246 50 1843

5

59 6 0.2

7

5

in great measure. However, a dose-response relationship became apparent when larger amounts of antibody were administered. Two mechanisms of injury manifest themselves by this measure, the first being neutrophile-dependent and requiring quite small amounts of complement-fixing antibody, and the second inducing basement membrane injury.in the absence of neutrophiles and requiring far greater amounts of antibody. A discussion of the polymorph-independent mechanism of injury appears below (Section V,C).

B. Smucrurms IN BLOODVESSELS AND TISSUES DAMAGED BY NEUTROPHILES Attention has recently been directed toward an analysis of the structures in blood vessel walls that serve as critical targets of the neutrophile attack and of the substances in neutrophiles responsible for the observed injury. These structures in Blood vessel walls and other tissues may be considered substrates of the reaction. In order to analyze the substances within neutrophiles responsible for injury and development of the reactions, it is essential to understand first the principal structural units attacked by the neutrophiles. In this way the mechanism of injury in hypersensitivity lesions will eventually be reduced to a series of chemical equations. Listed here are the beginnings. 1. The ArteriaE Znternul Elastic Lamina In the arteritis of serum sickness, one of the key structures that was found to be disrupted under the bo ardment of neutrophiles was the internal elastic lamina (Kn&er and C chrane, 1965). The earliest lesion in this arteritis appeared to be a lifting af the intimal cells away from the internal elastic lamina leaving a fine space which often contained a few round cells. The elastic lamina remained intact until neutrophiles entered into the vicinity. Once this occurred, discontinuities developed

T

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CHARLES G . COCHRANE

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131

in the elastic lamina and the zone of inflammatory cells proceeded into

the: media (Fig. 7 ) .

2. Vascular and Glomerular Basement Membrane

In smaller vessels and glomeruli the basement membrane appears to be a critical target or substrate of the neutrophile attack. This membrane acts as a filter or barrier to passage of macromolecular substances from the bloodstream into the extravascular spaces or urine. On morphologic grounds the basement membrane serves some of the same functions as the internal elastic lamina of large arteries. Neutrophiles are attracted and accumulate in the immunologic reactions, alteration of the basement membrane occurs, and macromolecules, red cells, and other formed elements pass freely through the vessel wall (Cochrane and Aikin, 19f36). A similar sequence of events occurs in inflammatory reactions incited by agents other than antigen-antibody complexes (Hurley, 1964b; Marchesi, 1964; Hurley and Spector, 1965; Cotran, 1965; Cotran et al., 1965). Destruction of basement membrane has also been noted in the presence of neutrophiles by a loss of antigenicity of the membrane and structural fragmentation as shown by electron microscopy (Cochrane and Aikin, 1966) ( Fig. 13). Endothelial cells were distorted and vacuolation was frequently observed. However, damage to basement membrane in areas where little alteration of endothelial cells was observable suggested that the former structure was a primary target. In glomerulonephritis, recent evidence has indicated that during the accumulation of neutrophiles, structural alteration of the basement membrane occurs. As illustrated in Fig. 5, in nephrotoxic nephritis, the neutrophiles frequently gain intimate contact with the glomerular baseFIG. 13. Upper photo: Electron photomicrograph of a normal venule from rabbit urinary bladder. A normal intact basement membrane may be observed along the external surface of the endothelial cell. end-endothelial cell; L-lumen; BMbasement membrane; IS-interstitial space. Magnification: X11,400. Middle photo: Electron photomicrograph of a venule taken from a 2-hour Arthus reaction in a rabbit. Neutrophilic leukocyte (PMN ) accumulation has begun in this affected vessel, but no damage to the basement membrane is observable. Magnification: X16,500. Lower photo: Electron photomicrograph of a &hour Arthus reaction in rabbit urinary bladder. Marked disruption of the basement membrane has occurred ( x ) . While PMN’s are not apparent in the photograph, several were seen in the interstitial spaces immediately adjacent. Fibrin deposition may be seen and the endothelial cell shows multiple evaginations. Magnification: X 11,400. (From Cochrane and Aikin, 1986.)

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CHARLES G. COCHRANE

ment membrane by dissecting between this structure and the overlying endothelial cell. At the time this occurs, proteintiria is noted and recent observations show that ( I ) basement membrane fragments can be found in the urine and ( 2 ) that large protein molecules clear the filtering membrane (Hawkins and Cochrane, 1968). During the course of these studies it became apparent that two basement membrane molecules appear in low concentration in urine of normal rabbits. During the neutrophile attack these normally excreted, basement membrane fragments appear in the urine in much greater concentration, and, in addition, unique basement membrane fragments are also found. At the TABLE VI RENAL PROTEIN CLEARANCE I N ACUTE NEPHROTOXIC NEPHRITIS-WE INFLUENCE OF NEUTROPHILE INJURYOF THE GLOMER~JLIIS

No. of rabbits

Circulating neutrophiles

Average albumin excretiona

9 13

Normal Depleted

15.60 1.56

a

Average IgG Albumin/globulin clearance ratio excretion" 8.80 0.53

1.8/1 2.9/1

Expressed as percent of creatinine cleared.

same time, large molecules such as IgG are cleared from the circulating plasma across the glomerular filter in relatively large amounts. A comparison of the clearances of albumin and IgG, expressed as a ratio in neutrophile-dependent and independent glomerular injury, are shown in Table VI. As will be discussed below, comparison of the albumin/ globulin clearance ratio is made between two sheep antisera, one that requires neutrophiles (No. 1) and another that is only moderately dependent on the accumulation of these cells to induce injury. In each case the accumulation of neutrophiles in glomeruli leads to more extensive disruption of the basement membrane as evidenced by the greater clearance of IgG molecules. C. CONSTITUENTS OF NEUTROPHILIC LEUKOCYTES THATARE RESPONSIBLE FOR IMMUNOLOGIC TISSUE DAMAGE

1. Lysosoms and Their Function In his classical sudies of inflammation, Metchnikoff (1887) drew attention to the capacity of microphages, or neutrophilic leukocytes, to engulf and degrade invading microorganisms. De Duve and his Mworkers ( 1955) described a new cytoplasmic organelle with sedimenta-

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tion properties diftering from those previously studied. The new organelles contained hydrolases that had an acid pH optimum and were capable of digesting nucleic acids, lipids, and carbohydrates. In deference to their content of lytic enzymes, the organelles were called lysosomes. A large group of enzymes and other proteins have been associated with lysosomes; these are shown in Table VII. Not all have been found in neutrophile lysosomes, but many of these constituents have been associated with the neutrophile lysosomal granules by Cohn and Hirsch ( 1960a). Lysosomal materials specifically related to neutrophile injury of tissue structures are discussed in detail in the next section. TABLE VII ENZYMES AND OTHER SUBSTANCES ASSOCIATED WITH LYSOSOMESG Acid phosphat,ase Acid ribonuclease Acid deoxyribonuclease Cathepsins B, C, D, E Phosphoprotein phosphatase Phosphatidic acid phosphatase Organophosphateresistantesterases 8-Glucuronidase B-Galactosidase j3-N-ace tylglucosaminidase a-bf ucosidase a-1,Pglucosidase a-mannosidase a-N-acetylglucosaminidase a-N-acet ylgalactosaminidase

Hyaluronidase Lysozyme Collagenase Aryl sulfatases A and B Phospholipase Acid lipase Phagocytin and related bactericidal proteins Endogenous pyrogen Plasminogen activator (? urokinase) Hemolysin(s) Mucopolysaccharides and glycoproteins Basic proteins: (a) Mast-cell-active; (b) Permeabdity-inducing, independent of mast cells

Adapted from Weissmann (1967).

a. Alteration of Lysosome Membrane Stability. The ability to alter the stability of lysosomal membranes has added considerably to our knowledge of the mechanism of injury induced by cellular constituents. Agents that effect an increased lability of lysosomal membranes include vitamin A, streptolysins 0 and S, a large group of neutral steroids such as etiocholanolone and progesterone, endotoxin, antibodies to these membranes, and ultraviolet irradiation. The action of these agents has been reviewed by Weissmann ( 1964), and the inflammatory effect of these agents will be illustrated with only a few examples. Administration of excess vitamin A to animals or living cell cultures has led to loss of cartilage matrix (Fell and Thomas, 1960; Thomas et al., 1960). Lysosomes in animals with induced hypervitaminosis A con-

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CHARLES G. COCHRANE

tain less than normal levels of acid hydrolases while greater than normal levels are found in the circulation (Weissmann and Thomas, 1963; Janoff and McCluskey, 1962; Weissmann et al., 1963). This has suggested that the loss in cartilage matrix results from the escape of hydrolytic enzymes from chondrocyte lysosomes. The appearance of the cartilage is similar to that following administration of papain (Thomas et al., 1960). An inhibitory effect of induced hypervitaminosis A on the development of Arthus reactions has been recorded (Weissmann et al., 1963). However, the cutaneous reactions to simple vasoactive substances that do not require the mediation of lysosomal agents were also inhibited. This suggested that the inhibitory effect of hypervitaminosis A was possibly not related to a depletion of lysosomal contents prior to induction of Arthus reactions. A rather dramatic illustration of the importance of stability of lysosoma1 membranes occurs when streptolysin 0 is added to rabbit neutrophiles in vitro. Within minutes the granules undergo extensive lysis, the cytoplasm becomes clear, and the nuclear lobes fuse (Hirsch et al., 1963; Zucker-Franklin, 1965). The marked alteration of the neutrophiles probably results from the release of hydrolases intracellularly ( Weissmann et al., 1963). To test the possible pathogenic significance of the lysis of lysosomal membranes, streptolysin S was injected repeatedly intraarticularly in rabbits ( Weissmann et al., 1965). Shortly after the first injection, a large neutrophile response occurred, which after 24 hours was converted, in part, to a mononuclear cell response. Repeated injections over a period of days induced a chronic proliferative arthritis, with formation of villi containing inflammatory cells, lymphoid germinal centers and plasma cells in the synovial tissues, pannus formation, and disintegration of the underlying cartilage. After five injections, the reactions became selfsustaining and a complement-fixing antibody to lysosomal membranes appeared. Similarly, injections of several polyene antibiotics into the skin and joint spaces of rabbits induced acute and chronic inflammation (Weissmann et al., 1967). The polyene antibiotics have been shown to disrupt membranes by reacting with discrete lipid components, phospholipids, and/or cholesterol. The inflammatory reaction in the joint was inhibited by simultaneous intraarticular administration of cortisone. AS was mentioned by the authors, it is difficult to be certain which cells within the joint spaces are primarily affected by injections of the lysosome-labilizing agents and whether the affected cells of greatest importance in producing the injury were cells already present such as synovial lining cells or the masses of neutrophiles that rapidly accumu-

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late at the site. The inhibition of reaction by cortisone acetate could be explained not only on the basis of its ability to decrease the release of enzymes from lysosomes, but also on its ability to inhibit neutrophile chemotaxis and accumulation (Ward, 1967). The injection of streptolysin S or polyene antibiotics into joint spaces of neutropenic rabbits might answer this question. In a final example of injury induced by factors affecting lysosomes, the importance of neutrophile accumulation has been determined. Studying the mechanism of urate microcrystal injury in gouty arthritis, McCarty et al. (1966) and Phelps and McCarty (1966) infused mate crystals into the joint spaces of dogs. Marked inflammation ensued with formation of an inflammatory exudate consisting predominantly of neutrophiles. The urate crystals were known to be engulfed or, when too large, surrounded by neutrophiles ( McCarty, 1962; Rajan, 1966). Administration of the crystals into the joint spaces of neutropenic dogs, however, did not result in inflammation and articular tissue injury (Phelps and McCarty, 1966). Thus it is clear that lysosomal disrupting agents injected in uiuo will induce a severe inflammatory response. Three questions remain unanswered: first, what the primary injury is that induces the exudation of inflammatory cells; second, whether the major portion of injury results from disrupted lysosomes in fixed cells or exudative neutrophiles; and, third, whether the presence of a labilizing agent of lysosomes is essential once the neutrophiles have accumulated. Stabilization of lysosomes has an inhibitory effect on the development of inflammatory reactions. An analysis of these agents and their effect in cells has been recently published ( Weissmann, 1966). Among these are glucocorticoids and their synthetic analogs, chloroquine, gold salts, phenylbutazone colchicine, and salycilates. Each of these has wellknown antiinflammatory effects and each inhibits in one way or another the release of lysosomal contents of cells. In addition, several tested to date have been found to inhibit chemotaxis of neutrophiles (Caner, 1965; Ward, 1966), thus offering a different and, perhaps, supplementary explanation of their inhibitory action in inflammation. Attention was also called by de Duve (1959) to the potential capacity of the lysosomes to participate in the normal engulfing and digestive functions of the cells, and this prediction has been born out. Following ingestion of particles such as zymosan or bacteria, the lysosomes adjacent to the engulfed particle suddenly and rapidly disappear. Vacuoles develop around the ingested particles and the latter then undergo structural alteration and dissolution ( Robineaux and Frederic,

136

CHARLES G . COCHRANE 50 %

7

... ..,.......

_.-.-

40 %

-

SONIC VIBRATION KILLED LEUKOCYTES M A T KILLED LEUKOCYTES

-

LIVING LEUKOCYTES

30%-

4 8 12 INCUBATION

-

16 20 24 HOURS

FIG.14. The catabolism of immune complexes [using 13’1 bovine serum albumin ( B S A ) as antigen] after phagocytosis by neutrophiles in rabbits. The neutrophiles were removed, washed, and placed in uitro. At various times samples were removed and the supernatant fluid tested for nonprotein-bound Y. The majority of the 1 3 1 ~ activity was found in the form of monoiodo- and diiodotyrosine by paper electrophoresis. (From Cochrane, 1967.)

1955; Hirsch and Cohn, 1960; Cohn and Hirsch, 1960b; Sbarra et al., 1961; Hirsch, 1962). The electron microscope has revealed that the rapid disappearance of the lysosomal granule is explained by a process of fusion of lysosomal membranes with those of the digestion vacuole. The lysosomal contents are then emptied into the digestion vacuole which contains the engulfed particle or immune precipitate, the cytoplasm being cleared of one granule in the process (Movat et al., 1963; ZuckerFranklin and Hirsch, 1964). Ingestion of immune precipitates by neutrophiles also occurs during the development of the vasculitis of the Arthus reaction (Cochrane et al., 1959; Daems and Oort, 1962; Movat et al., 1963; Uriuhara and Movat, 1966). This is associated with a loss of neutrophilic granules as noted above. During the process, immune precipitates are catabolized, releasing peptides and amino acids of the catabolized antigen into the surrounding milieu (Cochrane et al., 1959) (Fig. 14). This degradation of offending antigen is in keeping with the prime function of neutro-

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137

philes. At a time that phagocytosis of immune precipitates occurs, constituents, including proteases, are released in greater amounts into the surrounding milieu (Movat et al., 1964). This may owe in part to the increase in glycolytic formation of lactic acid known to occur during phagocytosis (Cohn and Morse, 1960; Sbarra and Karnovsky, 1959; Strauss and Stetson, 1960). An increase in hydrogen ion concentration favors instability of lysosomal membranes (Cohn and Hirsch, 1960a).

2. The Role of Proteolytic Enzymes (Cuthepsins) of Neutrophiles in the Injury of Tissue Structures a. Hydrolysis of Basement Membranes. Attention was drawn to the role of lysosomes of neutrophiles in the Arthus vasculitis by Thomas ( 1964) , who injected isolated neutrophilic lysosomes in antigen-antibody-prepared sites of neutropenic rabbits. Inflammatory reactions developed that were not found when the lysosomes were injected into virgin sites, an observation not clearly explained. Golub and Spitznagel (1966) injected fractions of neutrophilic lysosomes into rabbit skin and observed an increased permeability (to be discussed in detail below), and Uriuhara et al. (1965) and Burke et al. (1964) administered large quantities of neutrophilic extracts into rabbit skin and observed an increase in vascular permeability and hemorrhagic reaction. The permeability was partly inhibited when the extracts were treated with protease inhibitors. With the acid pH requirement for the rabbit neutrophilic hydrolase activity (Cohn and Hirsch, 1960a; Lapresle and Webb, 1962; Cochrane and Aikin, 1966), it is not certain what factor in the extracts induced the hemorrhagic lesion. Rabbit neutrophilic proteases upon purification have been found not to induce a hemorrhagic reaction following intradermal injection ( Cochrane and Aikin, 1966). In light of the observation that vascular and glomerular basement membranes appeared to serve as substrates of the neutrophile attack during acute injury, extracts of the granules of rabbit neutrophiles were incubated with purified glomerular basement membrane of rabbit kidneys under various conditions. As noted in Fig. 15, a release of peptides was observed when the incubation was performed at an acid pH (Cochrane and Aikin, 1966). Basement membrane fragments could be identified in the same supernatants. Of the four detectable basement membrane fragments, three showed complete identity with a single molecule that could be eluted by saline extraction of large quantities of basement membrane. Subsequent fractionation of the neutrophile extracts on a column of diethylaminoethyl ( DEAE ) cellulose and incubation of the fractions with basement membrane revealed that the active

138

CHARLES G. COCHRANE

FIG. 15. Paper electrophoresis of supernatants of reaction mixture containing basement membrane and neutrophile lysate. From left to right: (1) Migration of normal serum; ( 2 ) supernatant of trypsin-treated basement membrane; ( 5 ) supernatant of basement membrane plus distilled water a t p H 2.5; ( 4 ) supematant of neutrophile lysate incubated a t pH 2.5; and ( 5 ) supernatant of neutrophile lysate incubated with basement membrane, p H 2.5. (From Cochrane and Aiken, 1966.)

materials in the neutrophile extracts were acid hydrolases (Fig. 16). Their elution from DEAE cellulose with further purification by gel filtration ( Fig. 17), together with other characteristics, revealed that they were cathepsins D and E. These two enzymes were originally isolated from rabbit neutrophiles by Lapresle and Webb (1962). Their pH optima were found to be approximately 3.4 for cathepsin D and 2.5

139

MEDIATION OF IMMUNOLOGIC TISSUE INJURY .860,

.800,750-

.loo,650-

,600-

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FIG.18. Separation and activities of lysate of neutrophiles obtained from rabbit peritoneal cavities. The elution was carried out by a continuous salt gradient from 0 to 0.3 M ; pH was maintained constant at 8.0. Protease activity ( - - ) was deProtein (folin). termined by hydrolysis of hemoglobin substrate at pH 2.5. (-) (From Cochrane, 1967.)

--

for cathepsin E, values confirmed in the writer's laboratory (Fig. 18). The protease activity noted by Wasi et al. (1966) at neutral to slightly alkaline pH has not been found in other laboratories (Lapresle and Webb, 1962; Cohn and Hirsch, 1960a; Cochrane and Aikin, 19f36). As would be anticipated from their acid pH requirements, injection of each of these enzymes isolated from the neutrophile failed to induce a lesion in rabbit skin. However, basic proteins in the fractions excluded by the DEAE celhlose were highly active in provoking increased vascular permeability (see below, Section IV,A,3). It appeared from these data that under acid conditions, the two proteases are capable of damaging basement membrane at least in uitro. The vasoactive basic proteins, eluting immediately from DEAE cellulose, do not cause gross disruption of vascular basement membrane in uiuo, underscoring the importance of the hydrolases in the hemorrhagic Arthus reaction. Other enzymes of neutrophiles eluted from DEAE cellulose were

140

CHARLES G . COCHRANE

75 100 Volume fluate [ml]

50

125

FIG.17. Tests of purified neutrophile protease after elution from Sephadex G200. Background values of protease protein and protein released from basement membrane incubated without protease were previously subtracted from values given in vertical bar plots. Protease activity determined with bovine hemoglobin substrate p H 2.5. (From Cochrane and Aiken, 1W6.)

also tested on isolated basement membrane. The lipases, phosphatases, and lysozyme were all inactive in this regard. Direct measurements of the pH in vivo along the membranes at the point where disruption occurs has not been possible. However, the high hydrogen ion requirement for activity of the rabbit neutrophile cathepsins is most probably supplied in part by the neutrophile itself. Evidence of low pH within leukocytes after phagocytosis of particles was obtained

500-

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PH FIG. 18. pH Optima of the two proteolytic enzymes obtained from rabbit Cathepsin 1-cathepsin D; ( - - - - ) neutrophiles as shown in Fig. 16. (-) cathepsin 2-cathepsin E. Tests carried out with 6%bovine hemoglobin as substrate.

MEDIATION OF IMMUNOLOGIC TISSUE IN JURY

141

by Rous (1925), who observed an acid reaction to bromophenol blue within leukocytic granules, indicating a pH of 3.0 or less. Subsequently, considerable evidence has indicated that a heightened production of lactate occurs during phagocytosis and under anaerobic conditions (Sbarra and Karnovsky, 1959; Cohn and Morse, 1960; Strauss and Stetson, 1960). Sequestration of neutrophiles in uiuo would certainly be associated with anaerobic conditions. In addition, measurements of pH in joint fluid (buffered by the transudative proteins) in which neutrophile accumulation is occurring, showed a fall in pH over a period of time, as a function of the numbers of neutrophiles present (McCarty et al., 1966). In recent studies, Janoff (1968) has examined the capacity of human neutrophiles to injure isolated rabbit glomerular basement membrane. Extracts of these cells cause proteolytic breakdown of the basement membrane at a neutral pH, i.e., corresponding to the pH optimum of the cathepsins in these cells, Injection of the extracts into rabbit skin induced hemorrhagic reactions, evidence of severe injury of the vessel walls, For several years Hayashi and co-workers have implicated a proteolytic enzyme in the development of the Arthus reaction that could be extracted from Arthus sites or from cultures of mononuclear cells (Hayashi et al., 1958, 1980, 1962a,b; Tokuda et al., 1960; Hayashi and Nitta, 1961). It should be noted that the protease studied by these workers is derived from mononuclear cells and is extractable from Arthus sites at a time when neutrophiles are scant or absent, i.e., after the neutrophiles have left the site or have been destroyed and removed. The mononuclear cell enzyme activity studied by Hayashi is dependent upon SH groups and has a pH optimum of 7.1 to 7.4. Both of these properties distinguish it from neutrophilic proteases. Thus, the importance of this enzyme in the pathogenesis of the lesions discussed above (the Arthus being the prototype) is uncertain. b. Hydrolysis of Protein Polysaccharides of Cartiluge. Extracts of leukocytes, cartilage and liver are capable of degrading crude proteinpolysaccharides obtained from cartilage (Ziff et al., 1960; Ali, 1964; Fell and Dingle, 1962; Barland et al., 1966). The lysosomal hydrolases have been implicated in this reaction (Fell and Dingle, 1962; Barrett, 1966). More recently Weissmann and Spilberg (1968) have found that proteases from isolated neutrophilic lysosomes are capable of hydrolyzing the protein polysaccharide obtained from bovine cartilage. Degradation occurred at both acid and neutral pH and was inhibitable by €-aminocaproic acid.

142

CHARLES G. COCHRANE

3. lniuy Produced by Basic Proteins Obtained from Neutrophiles A second group of substances contained in polymorphs has also received considerable attention. These substances, the basic proteins, have several activities, including bactericidal, permeability, and mastcell-membrane activating. In studies of inflammation, Moses et al. ( 1984) analyzed a granulocytic substance (GS) released from rabbit neutrophiles after severaI hours incubation in saline at 37°C. This substance was capable of increasing vascular permeability, inducing a febrile response when injected intravenously in rabbits, and causing leukocytic sticking and emigration in small vessels. The permeability effect of the GS generally required 6&90 minutes to reach a maximum in their hands, but in the writer’s laboratory reactions developing in 15 minutes have been repeatedly observed. The GS was also found by Moses et al. (1964) to induce a febrile response that was monophasic in character. This activity was indistinguishable from leukocytic pyrogens studied by others. The GS pyrogen apparently was separable from endotoxin by its short latent period, its heat lability, and its ability to cause temperature elevation in endotoxin-tolerant rabbits. Other studies of a permeability factor stem from the work of Frimmer and Hagener (1963), who analyzed substances obtained from calf thymus capable of increasing vascular permeability. By extracting neutrophilic granules of rabbits with dilute H,SO, and then precipitating with ethanol at 20%concentration, Golub and Spitznagel (1966), Janoff and Zweifach (1964), and Janoff et al. (1965) have studied a neutrophilic cationic protein with permeability-inducing properties. This also caused increased vascular permeability and neutrophile accumulation when injected in vivo. It was found by Janoff et al. (1965) to bring about degranulation of rat mast cells, and its action in rats could be inhibited by administration of antihistamine. From the work of Janoff and Zweifach (1964), this permeability factor appeared to be separable from the granulocytic substance of Moses et al. (1964) by its lack of pyrogenicity and its presence in unincubated cells. Other studies, however, have indicated that the 20%alcohol fraction of the acid extract of polymorph granules contains pyrogenic activity ( Cochrane and Aikin, 1966). The crude preparations undoubtedly contain proteins with many different properties. The antibacterial activity of neutrophile basic proteins has been recognized for some time. Skarnes and Watson (1956) described an arginine-rich, lysine-poor protein, leukin, in rabbit neutrophiles with bactericidal properties. Leukin has more recently been found to consist

MEDIATION OF IMMUNOLOGIC TISSUE INJURY

143

of two low-molecular-weight proteins ( Skarnes, 1967). This substance bears a strong resemblance to phagocytin described by Hirsch (1960). Zeya and Spitznagel ( 1963, 1966a,b), working with neutrophilic granule basic proteins obtained by precipitation at 20%ethanol concentration, have studied the bactericidal properties of these extracts. By separating the basic proteins on cellulose acetate paper, these workers were able to separate their bactericidal activities from lysozyme, ribonuclease ( RNase) , and deoxyribonuclease ( DNase) . And of great interest, each protein appeared to have a distinct spectrum of bactericidal activity when tested against different microorganisms ( Zeya and Spitznagel, 1966a,b). These findings suggest a relationship between the bactericidal nature of these proteins and their biologic properties in mammalian tissues. Studies on the mechanism of action may well profit from both avenues of interest. By isolating the basic proteins of lysosomes in rabbit neutrophiles using different methods, i.e., with DEAE cellulose followed by gel filtration on Sephadex G-50 and, finally, preparatory polyacrylamide gel electrophoresis, four different vasoactive basic proteins have been identified (Ranadive and Cochrane, 1967; Cochrane and Aikin, 1966). These all bring about a permeability reaction reaching a maximum within 30 minutes. The activity of these proteins is, therefore, distinct from that studied by Golub and Spitznagel (1966). The permeability proteins were called bands 1, 2, 3, and 4 proteins after their order of migration in analytical polyacrylamide electrophoresis (Fig. 19). Band 2 protein appeared to be identical to that studied by Seegers and Janoff (1966), in that it was capable of degranulating rat mast cells and thereby releasing histamine, and its permeability activity in rabbit skin was blocked by pretreatment with antihistamine. This protein failed to release histamine from rabbit platelets in confirmation of the findings of Janoff (1967), and, in addition, was fully active in platelet-depleted rabbits. In contrast, the three other cationic proteins, bands 1, 3, and 4 proteins did not release histamine or degranulate rat mast cells, and pretreatment of rabbits with antihistamine did not inhibit their permeability activity. To date the mechanism of their action on blood vessels is unknown but is of considerable interest in that they may serve to alter blood vessel membranes in sites of immunologic injury, such as the glomerulus, where susceptible reservoirs of vasoactive amines are not available. Further biologic and physicochemical characterization of the neutrophilic basic proteins has been achieved recently. Seegers and Janoff (1966) employed gel filtration to purify the protein capable of releasing

144

CHARLES G . COCHRANE

AMINOACID COMPOSITION OF P M N CATIONIC PROTEINS (moles/100 moles recovered)

GeoF r I1 E (band 4) Lysine Histidine Arginine Aspartic acid Threonine Serine Glutamic acid Proline G1ycine Alanine Half cystine Valine Methionine Isoleucine Leucine Tyrosine Phenylalanine

8.6 2.0 6.6 13.8 5.9 5.9 6.8 4.8 7.5 9.5 4.0 5.2 0.7 5.2 7.9 4.7 1.2

-

Total

100.3

+

GS0F r 111E (band 1) 16.4 5.7 10.3 4.3 3.7 8.0 5.7 5.1 10.7 12.6 5.2

-

2.9 5.5 2.1 1.9 100.1

GsaF r Ill E GSOFr Ill E (band 2) (band 3)a 1.6 2.5 18.2 2.8 5.6 7.7 4.1 3.7 12.5 4.8 14.9 5.2 3.2 7.6 0.5 5.1

100.0

I

~~~

a

3 1

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~~

Permeability Activity Mastocytolytic Activity

1.6 2.1 6.8 4.4 19.0 13.9 4.0 19.3 6.0 2.9 3.0 5.3 1.9 2.0 99.9

+

c

1

7.7

+-

2

+-

+ +

+-

Amino acid analysis of band 3 subject to minor modifications.

FIG. 19. Demonstration of proteins 1, 2, 3 and 4-obtained from rabbit neutrophilic lysosomes. Polyacrylamide gel electrophoresis, amino acid composition, permeability activity in rabbit skin, and the capacity of each to release granules and histamine from rat mast cells shown.

MEDIATION OF IMMUNOLOGIC TISSUE IN JURY

145

histamine from rat mast cells. On unidirectional electrophoresis in starch gel, a major and two minor components were observed, although when separated in urea starch gel, only a major and one minor component appeared. The active material eluted from Sephadex G-25 in the molecular weight range of 1200 to 2400. Band 2 protein (histamine-releasing) and bands 1and 3 proteins, on the other hand, have a molecular weight of approximately 5000 as determined by gel filtration techniques (Ranadive and Cochrane, 1968). Amino acid composition data yielded similar values of molecular weight. The molecular weight of band 4 protein by both gel filtration and analytical ultracentrifugation was determined to be approximately 12,000. Amino acid analysis of each of the basic proteins isolated from preparative polyacrylamide gel electrophoresis and appearing as single bands in analytical polyacrylamide gel electrophoresis are shown in Fig. 19. The composition of band 2 protein is seen to be distinctive by its high content of arginine, strongly suggesting it to be identical or closely related to the protein obtained from Sephadex G-25 by Seegers and Janoff. It is apparent that the bands 1, 2, 3, and 4 proteins differ from each other. It will be of considerable interest to carry out further analyses of these basic proteins. Based on the equal ability of poly-L-lysine and arginine-rich histone to degranulate mast cells, it appeared that the strong positive charge of these molecules alone was sufficient to bring about alteration of mast cell membranes and release of granules (Janoff and Schaeffer, 1967). However, the finding of bands 1 and 3 proteins having the same molecular weight as the mast-cell-active, band 2 protein and migrating immediately on either side of band 2 protein in polyacrylamide gel would suggest something more than overall electrostatic charge to be involved in the ability of the protein to affect mast cell membranes. By the same token, it is of great interest that bands 1, 3, and 4 proteins are fully capable of inducing vascular permeability in the absence of a histamine effect while the permeability activity of band 2 protein is dependent on histamine release. Attention might, therefore, be drawn to certain amino acid groupings or perhaps a characteristic structure in each of these proteins that could explain these bioIogic properties. In attempts to analyze the mechanism of action of histamine release from mast cells by band 2 protein, it has been found that degranulation and release of histamine is blocked by treatment of the cells during activation with 2,4-DNP, diisopropyl fluorophosphate, and certain p-nitrophenyl phosphonates ( Ranadive and Cochrane, 1988) . These

146

CHARLES G. COCHRANE

latter agents inhibit certain serine esterases within the mast cell (Becker and Austen, 1964). Neutrophilic granules of other species have also been found to contain basic proteins capable of degranulating rat mast cells and/or inducing increased vascular permeability. Guinea pig and rat neutrophiles were found by Scherer and Janoff (1968) to contain a mast-celldegranulating, basic protein although the rat protein acted only on isolated mast cells and was present in low concentration. Similar results with rat neutrophiles were obtained by different techniques of separation in the writer’s laboratory, and, in addition, several permeability proteins that act without degranulation of mast cells were isolated from rat neutrophiles ( Ranadive and Cochrane, 1968).

4. Neutrophiles and Sloro-Reacting Substance ( SRS-A) Recent observations on the release of SRS-A in rats have indicated that the presence of neutrophiles is essential for maximal immunologic release. Rats depleted of neutrophiles by nitrogen mustard or antineutrophile serum showed almost complete suppression of immunologic SRS-A release (Orange et al., 1967, 1968). Treatments with antimast cell or antithymic lymphocyte serum were without effect. Augmenting the quantities of neutrophiles in the peritoneal cavity increased the amount of SRS-A released. The SRS-A containing preparations of fluid obtained from peritoneal cavities were active in inducing increased permeability, but it was unclear whether this activity was due to SRS-A or to other permeability factors released with the SRS-A. Recently, however, extraction of highly purified SRS-A containing fractions from the peritoneal fluid have been found to be vasoactive (Orange, Valentine, and Austen, unpublished observations).

5. Kinin-Forming Activity of Neutrophile Constituents Neutrophile fractions have been found capable of generating kinin activity from plasma kininogens. With rabbit neutrophiles the kinin formation occurred in the acid pH range (Greenbaum and Kim, 1967), whereas human neutrophiles generated kinin activity from plasma kininogen at a neutral pH (Melman and Cline, 1967). These are the same pH ranges required for digestion of isolated vascular basement membrane by the proteases of rabbit and human neutrophiles (see Section IV) , It is plausible from these data that even another mechanism of injury may be evoked simultaneously with those already described above, at a time when neutrophiles have accumulated at the site of antigen-antibody-complement interaction.

MEDIATION OF IMMUNOLOGIC TISSUE I N JURY

V.

147

Other Mediation Systems Associated with NeutrophileMediated Immunologic Injury

Along with the interaction of antigen with antibody, the activation of C’5-6-7 complex, the accumulation of neutrophiles, and the structural injury of tissue structures, other pathways of mediation are also stimulated. These include the release of anaphylatoxin, the clumping of platelets and release of their injurious contents, and certain permeability reactions independent of histamine that take place in the absence of neutrophiles. There has been little definition of the importance of these mediation systems in the injury of structures as outlined in Section 11.

A. DEGRANULATION OF MASTCELLSAND RELEASEOF VASOAC~IVE AMINES During immunologic injury and, in particular, in the anaphylactic reaction, the membranes of tissue mast cells undergo a striking alteration leading to loss of the cytoplasmic granules. As the granules escape the confines of the cell membrane, their content of vasoactive amines is given off into the surrounding milieu, inducing increased vascular permeability and smooth muscle contraction. The immunologic genesis of the reaction, involving a foreign antigen, may be of two types. The first involves homocytotropic antibody, which reacts at the mast cell surface with the foreign antigen. This, in general, does not involve as a requisite the fixation of complement and, therefore, does not properly belong in the present review. A second pathway involving mast cells is one in which antigen reacts with host antibody, fixes complement, and releases anaphylatoxin as a hydrolytic cleavage product of C’3 and C’5. The anaphylatoxin then acts on mast cells inducing degradation and release of histamine. Since complement is a requisite (Osler et al., 1959; Jensen, 1967; Dias da Silva and Lepow, 1967; Dias da Silva et al., 1967; Cochrane and Muller-Eberhard, 1968), this system is probably associated quite commonly with the complement-neutrophile pathway. The completion of its sequence depends on the availability of mast cells or, perhaps, basophiles, a factor that limits the diversity of this mechanism. In arteries, for example, mast cells and basophiles are not found and, thus, a target for the anaphylatoxin is wanting. Interaction of antigen with complement-fixing antibodies in vivo leads to the activation of C’3 and the formation of the inactive product, PlG (C’3i). On the basis of studies in vitro, the inactive C’3 has been termed F(b)C’3 (Dias da Silva et al., 1967). Its electrophoretic mobility is anodal compared with native C’3 protein which is explained by the

148

CHARLES G . COCHHANE

loss of a second, highly basic fragment, F(a)C’3. Fragments F(a)C’3 and F(b)C’3 result from the enzymatic cleavage of the native molecule. The F( a)C’3 exhibits the various properties of anaphylatoxin. The presence of F(b)C’3 so far cannot be considered clear evidence that anaphylatoxin activity has been manifested, although it does indicate that F( a)C’3 (anaphylatoxin) has been liberated. By the same token, when C’5 in human serum reacts with C’3, a cleavage product, F( a ) C 5 , that possesses anaphylatoxin activity as well is liberated (Cochrane and Muller-Eberhard, 1968). The finding of the altered molecule F(b)C’5 also suggests the active fragment F( a)C’5 has been liberated but cannot be considered synonymous with the manifestation of anaphylatoxin participation in the reaction. Direct evidence of mast cell degranulation in vivo, brought about by anaphylatoxin in iminunologic reactions, is lacking. In addition, such evidence will be difficult to obtain since the activation of the complement components is generally associated with the accumulation of neutrophiles. These cells, as noted above, contain the mast-cell-degranulating, band 2 protein. So far it is impossible to distinguish mast cell degranulation of anaphylatoxin from that of band 2 protein. In the passively induced, dermal, antigen-antibody reactions in rats studied by Lovett and Movat (1966) and Movat et al. (1966), neutrophile accumulation was observed together with mast cell disruption. The mechanism of the disruption is not clear, but it may have resulted either from the formation of anaphylatoxin or from the release of basic protein from neutrophiles. The effect of degranulation of the mast cells in the particular reaction remains to be ascertained, although the reaction apparently was not altered by antagonists of histamine and serotonin.

B. CLUMPING OF PLATELETS AND RELEASE OF VASOACTIVE MATERIALS

A distinctive feature of the participation of complement in the antibody-antigen reaction is the large number of molecules of C’3 reacting and binding at the activation site (Muller-Eberhard et d., 1966). Among the facets of C’3 activation that are biologically important is the immune adherence clumping of red blood cells, leukocytes, and platelets of certain species ( Siqueira and Nelson, 1961; Nelson, 1962). The species variation in immune adherence activity of these cell types has been documented experimentally ( Henson, 1967) (Table VIII). This mechanism may account at least in part for the accumulation of platelets seen in Arthus reactions (Uriuhara and Movat, 1966). As stressed recently by Mustard ( 1967 review), during the immune aggregation of platelets both in vitro and in uivo, certain constituents are

MEDIATION OF IMMUNOLOGIC TISSUE IN JURY

149

released such as serotonin, histamine, and a protein of less than 10,000 mol. wt. that induces histamine release from other cells. The imniunologically induced release of vasoactive amines from platelets has been a subject of recent investigation ( Humphrey and Jaques, 1955; Barbaro, 1961; Gocke and Osler, 1965; Gocke, 1965; Henson and Cochrane, 1968). Platelets are known to contain lysosomal granules and are able to phagocytize immune complexes, incorporating the complexes into phagosomes (Movat et al., 1965). They, therefore, share many functions with TABLE VIII IMMUNE ADHERENCE OF PLATELETS TO SENSITIZED SHEEPERYTHROCYTES AND HOMOLOQOUS C’1,4;2,3 Species whose platelets gave positive adherence reactions Guinea Pig Rabbit Rat Mouse Horse cat

Species whose platelets gave negative adherence reactions

ox

Goat Sheep Pig Baboon Man

neutrophiles. In addition, exogenous or endogenously released adenosine diphosphate ( ADP) brings about clumping of platelets and release of certain constituents (see Mustard, 1967). Infusion of ADP into the renal artery of rabbits over a 24-hour period induced platelet clumping in the small vessels including those in the glomeruli, and brought about “focal injury to glomeruli and proximal collecting tubules, with numerous casts in the collecting tubules in the cortex and medulla” (Mustard, 1967). Studies described in the previous section in which depletion of neutrophiles by administration of either nitrogen mustard or specific antineutrophile antibody resulted in marked or complete inhibition of the acute nephritis, arteritis, or vasculitis in Arthus reactions mitigate against an essential role of platelets. Levels of circulating platelets were little affected by either of these treatments, although Mustard (1967) has indicated that nitrogen mustard inhibits platelet clumping by ADP. Depletion of platelets, in addition, had no apparent effect on the development of acute nephrotoxic glomerulonephritis or Arthus reactions (Kniker and Cochrane, 1968). Nevertheless, in the development of immunologic reactions in which complement takes part, an additive effect of platelets and neutrophiles would seem likely. Certainly at times in Arthus reactions, platelets can be found in large numbers along with

150

CHARLES G. COCHRANE

neutropliilcs in the lesions ( Uriuhara and Movat, 1964, 1966). These platelcts, together with the neutrophiles, are most probably releasing injurious materials.

CELLULAR

INJURY NOTREQUIRING C. IMMUNE VASCULAR PARTICIPATION

If considerable amounts of antibody are employed to induce Arthus reactions or nephrotoxic glomerulonephritis, vascular and glomerular injury are manifested in the absence of neutrophile participation (Cochrane et al., 1965; Cochrane and Ward, 1967; Tucker et al., 1968). The quantitative relationships of antibody involved in the neutrophiledependent and neutrophile-independent injury of glomerulonephritis have recently been determined (Tucker et al., 1968). While roughly 15 pg. antibody bound in glomeruli of two kidneys is required to induce 600 I

*I mi

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0

< 200

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100

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-

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/

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Rabbll

FIG. 20. Dose-response curves of sheep antirabbit basement membrane and the quantity of proteinuria resulting in the first 24 hours after injection of the antibody in rabbits. The neutrophile-independent injury requires considerably more Normal rabbits; ( - - - - ) PMN-depleted rabbits. antibody. (-)

neutrophile accumulation and threshold injury, 35-40 pg. is required to initiate glomerular injury and proteinuria in the absence of neutrophiles ( Fig. 20). The slope of the dose-response curve of neutrophile-mediated injury is strikingly steep, suggesting that a few neutrophiles, perhaps owing to their relatively immense size, are able to induce severe injury. The slope of the dose-response curve obtained in the absence of neutrophiles is more gradual, increasing amounts of bound antibody inducing progressively greater injury. Depletion of circulating complement to date has not prevented the permeability reaction and noncomplement-

MEDIATION OF IMMUNOLOGIC TISSUE INJURY

151

fixing avian antibody has proven highly efficacious in bringing about permeability in both nephrotoxic glomerulonephritis and Arthus reactions (see Unanue and Dixon, 1967). The participation of complement is, therefore, highly suspect. Permeability-inducing substances may be generated in fresh serum by immune precipitates. The formation of this activity was linked to the complement system by Davies and Lowe (1960, 1962). On the other hand, permeability activity could be generated as well by washed duck antibody-antigen precipitates that did not deplete hemolytic complement levels detectably. In addition, serum from (2’5-deficient B10-D2 old-line mice was as capable as serum from C’5-sufFicient B10-D2 newline mice ( Cochrane, 1967). Chelation of divalent cations with ethylenediaminetetraacetate ( EDTA) prevented generation of the factor. Since all tests of permeability factor were performed in guinea pigs pretreated with antihistamine, the likelihood of the generation of a histaminereleasing substance appears low. A relationship has been drawn between the generation of the permeability factor in normal serum by immune precipitates and the kinin system ( Movat, 1967; Movat and DiLorenzo, 1968a,b). This possibility appears distinct in view of the findings of Brocklehurst and Lahiri (1962) and Brocklehurst and Zeitlin (1967) that during anaphylaxis in guinea pigs, bradykinin activity appears and a decrease in plasma kininogens is observed. Permeability activity in fresh rabbit and guinea pig serum that was induced with immune precipitates could be inhibited by M ) and the protease inhibitor, diisopropylfluorophosphate (DFP) ( Trasylol. The permeability was enhanced tenfold if activation was carried out in the presence of diethyldithiocarbamate, an agent known to interfere with kininase activity. Serum so treated induced contraction of guinea pig ileum in the presence of antihistamine and lowered the blood pressure of a rabbit pretreated with antihistamine. In addition, weaker generation of the permeability activity was obtained in Hageman-deficient human serum, the generating capacity being augmented with addition of purified Hageman factor. Separation of the antigenantibody activated serum has been carried out showing the presence of permeability agents in several fractions of serum (Movat and DiLorenzo, 1968b). VI.

Summary of Neutrophile-Dependent Immunologic Injury

A summary of the mediators studied to date that are associated with the development of acute immunologic injury in experimental models as described herein may be seen on the following page.

152

CHARLES G. COCHRANE

Antigen-antibody interaction

I

Fixation of complement components C’l,4, 2, 3 Activation and release of the complex C’5-6-7

1

Accumulation of neutrophiles and platelets

I

Phagocytosis of offending immune complex and neutrophile degranulation Release of cathepsins D and E and basic proteins 1, 2, 3, and 4 I

1

1

Disruption of glomerular

Release of basement membrane fragments into the urine

I Nonselective proteinuriu I

VII.

-

Dissolution of

Peptide release with basement membrane disruption

I

Extravasat,ion of plasma and hemorrhage

I

Neutrophile-Mediated Injury in Human Connective Tissue Disease

Evidence of immunologically induced, neutrophile-mediated injury in human disease is circumspect at present. Direct evidence implicating antigens and antibodies as inciting agents in human disease is beginning to accumulate, e.g., in the pathogenesis of various forms of glomerulonephritis. In addition to this, information implicating various mediation sequences is accumulating, the nature of the information being comparative, i.e., identification of factors in human disease known to be pathogenic in experimental situations. Several exampIes of this follow. In acute and chronic human glomerulonephritis the deposition of y-globulin and complement has been well-recorded (Lachmann et al., 1962; also see Kunkel and Tan, 1964; Kof3er and Paronetto, 1965). y-Globulin, together with complement, has more recently been found in human glomerulonephritis in which the antibody is apparently directed against glomerular basement membrane (Michael et al., 1966; Lerner et al., 1967). Neutrophile accumulation in glomerulonephritis during acute episodes is common, although the relationship of the presence of neutrophiles to the onset or exacerbation of renal injury has not been determined. Neutrophiles accumulated in glomeruli during acute injury may be found, as in experimental models of glomerulonephritis, forcing aside endothelial cells to gain intimate contact with the basement membrane (Fig. 21). Presumably, repeated acute neutrophilic insults could

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FIG.21. Electron photomicrograph of a glomerulus from a case of poststreptococcal glomerulonephritis in a young adult. The neutrophiles (PMN) fill the capillary lumen and have stripped the endothelial cell away from the basement membrane ( B M ) . Note the deposits ( D ) along the basement membrane. Human IgG and C3 were found in a granular distribution along the basement membrane with fluorescent antibodies. Ep-epithelial cell; US-urinary space. ( Photo courtesy of Dr. J. D. Feldman.) in time render permanent injury. A single 12-hour neutrophile attack in experimental animals resulted in damage to the glomerulus that persisted over the entire experimental period (Cochrane et al., 1965). In polyarteritis nodosa (Mellors and Ortega, 1956; Paronetto and Strauss, 1902), y-globulin and complement have been found in concentrations within the lesion sites. Neutrophile accumulation and fibrinoid necrosis are a common feature of these diseases and of hypersensitivity angiitis as well (Zeek, 1952). In the systemic vasculitis observed in cases of rheumatoid arthritis, a strong correlation was found among

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the incidence of lesions, low hemolytic complement levels, and high immunoglobulin levels. In these studies, low complement levels were also found in patients with systemic lupus erythematosus having a systemic vasculitis (Mongan et al., 1966). In acutely forming subcutaneous nodules of rheumatoid arthritis, Sokoloff ( 1964) found the central lesion to be an acute arteritis. The intimal proliferation and neutrophile accumulation, together with a striking disruption of the internal elastic lamina, reflected a close similarity to the features of serum sickness arteritis. Concentrations of y-globulin were previously found in centers of rheumatoid nodules (Vazquez and Dixon, 1957). Several cases of cryoglobulinemia have recently been reported (Miescher, 1966) in which deposition of y-globulin and complement were associated with neutrophile accumulation in blood vessel walls producing an acute vasculitis. The lesions closely resembled the Arthus vasculitis in these features. In rheumatoid arthritis, another close parallel may be drawn to the neutrophile-dependent immunologic injury. Hemolytic complement activity in the joint fluid of patients with active rheumatoid arthritis has been found markedly diminished while activity in the serum was relatively normal (Pekin and Zvaifler, 1964; Hedberg, 1964; Fostiropoulos et al., 1964). Comparisons were made among joint fluids from cases of rheumatoid arthritis, osteoarthritis, gout, and Reiter’s syndrome ( Pekin and Zvaifler, 1964). Large concentrations of leukocytes and, in particular, neutrophiles in joint fluids of patients with rheumatoid arthritis and other forms of acute joint disease have been repeatedly observed. Hollander and co-workers (1965) and Rawson et al. (1965) drew attention to vacuoles within the leukocytes of joint fluids in cases of rheumatoid arthritis and certain related conditions. These were termed RA cells. The granules of RA cells were found by fluorescent antibody techniques to be rich in yM- and yG-globulin (Rawson et al., 1965). Additional evidence supporting the finding of yM- and yG-globulincontaining neutrophiles and mononuclear leukocytes in affected joint fluids using fluorescent and electron microscopy and agar diffusion techniques has been presented subsequently ( Zucker-Franklin, 1965; Astorga and Bollet, 1965). Another similarity between the mediators of human rheumatoid arthritis and experimental immunologic models has been observed by Ward and Zvaifler (1968), who detected two chemotactic factors in synovial fluid of over half the patients with rheumatoid arthritis. One of the factors appeared to be the C’5-6-7 complex by ultracentrifugal and electrophoretic analyses, while the second as yet unidenaed material sedimented more slowly. Inactive C’3, &-globulin,

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was also identified in the fluid, indicating complement utilization. In terms of possible substrates of neutrophile constituents in the joint tissues, evidence indicates that neutrophiles are able to induce hydrolysis of mucopolysaccharides. In acute attacks of gout, one of the constant features is a marked accumulation of neutrophiles and other leukocytes in the joint spaces, together with the formation of microcrystals such as sodium urate ( McCarty, 1962). The neutrophiles were shown to engulf the microcrystals. Neutrophiles also ingest microcrystals in uitro, at which time granule lysis occurs (Rajan, 1966). As discussed above, the inflammatory arthritis in dogs induced by infusion of microcrystals was prevented by systemic neutrophile depletion (Phelps and McCarty, 1966), a finding that allowed the authors to draw a parallel between the mediation of experimentally induced gouty arthritis and the Arthus reaction. Another parameter of the complement-fixing reaction that may be employed as a test of previous complement fixation is the formation of immunoconglutinin ( Lachmann, 1967). Immunoconglutinins are immunoglobulins of predominantly the 19 S class but also the 7s class, which react with C’3 and to a lesser extent C‘4 bound to a cell or membrane surface. The immunoconglutins react with fixed C’3 or C’4 only after activation by C’l, 4, and 2 and, by the same token, are produced in response to the formation of fixed C’3 and C’4. Elevated levels of immunoconglutinins have been reported in cases of acute nephritis, ankylosing rheumatoid arthritis, systemic lupus erythematosus, rheumatic fever, mitral stenosis, Hashimoto’s disease, and several others (reviewed by Lachmann, 1967). The relationship of the formation and depletion of immunoconglutinins to the development of immunologic reaction of the type reviewed herein will require further study. These findings, therefore, draw strong parallels between the various factors known to be important in experimental neutrophile-dependent injury and acute inflammatory vasculitis, glomerulonephritis, arteritis, and arthritis of human beings. Therapeutic agents used in these diseases have multiple effects, but information in this regard has offered some insight into the role of various suspected mediators in human diseases. Corticosteroids, reviewed by Weissmann, 1964), chloroquine, ( Weissmann, 1964; Rajan, 1966; Malawista and Bodel, 1966) and, possibly, salicylates, the last being known to inhibit developinelit of the Arthus reaction (Smith aiicl Humphrey, 1949), have a stabilizing effect on lysosomes and presumably inhibit the release of injurious constituents of neutrophiles ( hliller and Smith, 1966). Hydrocortisone and choroquine also inhibit neutrophile chemotaxis

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in vitro and their accumulation in Arthus sites of guinea pigs. These agents also inhibited phagocytosis of particulate material by neutrophiles in uitro (Ward, 1966), and at least cortisone is a well-known inhibitor of antibody formation. Colchicine appears to inhibit the degranulation of lysosomes following particle ingestion ( Malawista and Bodel, 1966) as well as to stabilize lysosomal membranes (Rajan, 1966), although Weissmann (personal communication) has not been able to substantiate the latter finding. Colchicine also reduces the chemotactic capacity of neutrophiles ( Caner, 1965). Gold sodium thiomalate, used in the treatment of rheumatoid arthritis, inhibits the activity of acid phosphatose, P-glucoronidase, and malic dehydrogenase of macrophage lysosomes ( Persillin and Ziff, 1966). Indomethacin, a drug clinically effective in the treatment of gouty arthritis, also inhibits neutrophile chemotactic migration (Phelps and McCarty, 1966). By virtue of the lack of specificity of these antiinflammatory agents, it is difEcult to derive information relative to specific essential facets of the mediation pathway. Their overall effectiveness, on the other hand, is probably related to the inhibitory capacity of each on the various components of the mediation system. Hopefully, inhibitors will be developed in the future that block a single point in the mediation pathway, such as perhaps CoF which appears to act at the C’3 level. At that time, we will be much closer to therapeutic specificity, and will learn a great deal about the mediation of human disease as well.

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Sbarra, A. J., Bardawil, W. A., Shirley, W., and Gilfillan, R. F. (1961). Exptl. Cell Res. 24, 609. Scherer, J., and JanoA’, A. (1968). Lab. Inuest. 18, 196. Seegers, W., and Janoff, A. (1966). 1. Exptl. Med. 124,833. Shin, H. S., Pickering, R. J., Mayer, M. M., and Cook, C. T. (1968). J. Immunol. (In press ). Silverstein, A. M. (1960). Blood 16, 1338. Siqueira, M., and Nelson, R. A. (1961). J . Immunol. 86, 516. Skarnes, R. C. (1967). Nature 216, 806. Skames, R. C., and Watson, D. (1956). J . Exptl. Med. 104,829. Small, P. A,, and Baxter, J. H. (1965). J. lmmunol. 95, 282. Smith, W., and Humphrey, J. H. (1949). Brit. J. Exptl. Pathol. 30, 560. Sokoloff, C. (1964). ln “The Peripheral Blood Vessels” (J, L. Orbison and 0. E. Smith, eds. ), p. 294. Williams & Wilkins, Baltimore, Maryland. Stavitsky, A. B., Heymann, W., and Hackel, D. B. (1956). J. Lab. Clia. Med. 47, 349. Stein, O., de Vries, A,, and Katchalski, E. (1956). Arch. Intern. Pharmucodyn. 107, 243. Stetson, C . A. (1951 ). J. Exptl. Med. 94, 349. Strauss, B. S., and Stetson, C. A. (1960). J. Exptl. Med. 112, 653. Taranta, A., and Franklin, E. C. (1961). Science 134, 1981. Taranta, A,, Badalamenti, G., and Cooper, N. S. (1963). Nature 200, 273. Taylor, F. B., Jr., and Ward, P. A. (1967). J. Exptl. Med. 126, 149. Thomas, L. (1964). Proc. SOC. Exptl. Btol. 115, 235. Thomas, L., McCluskey, R. T., Potter, J., and Weissmann, G. (1960). J . Exptl. Med. 111, 705. Tokuda, A,, Hayashi, H., and Matsuba, K. (1960). J. Exptl. Med. 112, 249. Tucker, E., Hawkins, D., and Cochrane, C. G . (1968). Federation Proc. 27, 544. Unanue, E. R., and Dixon, F. J. (1984). J. Exptl. Med. 119, 965. Unanue, E. R., and Dixon, F. J. (1967). Aduan. Immunol. 6, 1 . Uriuhara, T., and Movat, H. Z. (1964). Lab. Invest. 13, 1057. Uriuhara, T., and Movat, H. Z. (1966). Exptl. Mol. Pathol. 5, 539. Uriuhara, T., Macmorine, D. R. L., and Franklin, A. E. (1965). Federation Proc. 24, 368. Vazquez, J. J., and Dixon, F. J. (1957). Lab. Inuest. 6, 205. Ward, P. A. (1966).J. Exptl. Med. 124, 209. Ward, P. A. (1967). J. Exptl. Med. 126, 189. Ward. P. A. (1968). J. lmmunol. (In press.) Ward, P. A., and Becker, E. L. (1967). 1. Exptl. Med. 125, 1001. Ward, P. A., and Becker, E. L. (1968). J . Exptl. Med. 127, 693. Ward, P. A., and Cochrane, C. G. (1965). J . Exptl. Med. 121, 215. Ward, P. A., and Zvaifler, N. J. (1968). J. Clin. Inuest. (In press.) Ward, P. A., Cochrane, C. G., and Miiller-Eberhard, H. J. (1985). J. Exptl. Med. 122, 327. Ward, P. A., Cochrane, C. G., and Miiller-Eberhard, H. J. (1966). Immunology 11, 141. Wasi, S., Murray, R. K., Macmorine, D. R. L., and Movat, H. Z. (1966). Brit. J . Exptl. Path. 47, 411.

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The Structure and Function of Monocytes and Macrophages

.

ZANVil A COHN Department o f Cellulor Immunology. The Rockefeller Universify. N e w York. N e w York

I . Introduction . . . . . . . . . . . I1. Life History . . . . . . . . . . . A. Origin of the Monocyte . . . . . . . B. Turnover and Intravascular Life Span of Blood Monocytes C . Emigration of Monocytes from the Intravascular Pool . D Origin and Life History of the Inflammatoiy Macrophage . E . Origin and Turnover of the Tissue Macrophage . I11. Morphology . . . . . . . . . . . A . Monocyte-Light and Phase Microscopy . . . . B. Monocyte-Electron Microscopy . C. Macrophage-Light and Phase Microsciopy . . . D. Macrophage-Electron Microscopy . . . . . . . . . IV . Maturation of Mononuclear Phagocytes . A. Maturation in Vivo . . . . . . . . B . Maturation in Vitro . . . . C . Mouse Peritoneal Phagocytes . . . . . . . . . . . . . . . . V . Metabolism . A . Energy Metabolism . . . . . . . . B . Synthesis of Macromolecules . . . . . . . . . . . . . . . . VI . Phagocytosis . A . Cell-Dependent Phenomena . . . . . . B . Attachment and Ingestion Phases . . . . . C . Influence of Humoral Factors . . . . . . D. Postphagocytic Events . . . . . . . . . . . . . . . . . VII . Pinocytosis . A. Metabolic Requirements . . . . . . . B. Induction of Pinocytosis . . . . . . . . . VIII . Intracellular Digestion and Bactericidal Properties . A. Bactericidal Propzrties . . . . . . . . B . Modification of the Intracellular Environment . . . C . Digestion of Bacteria and Bacterial Products . . . D . Interaction with Virus Particles . . . . . . E . Antigen-Antibody Complexes . . . . . . F. Proteins Pinocytized by Macrophages . . . . C . Macrophage Involvement in Morphogenesis . . . H . Inborn Errors of Metabolism . . . . . . . . . . . . . . . IX . Cellular Immunity A. Acquired Antibacterial Immunity . . . . . 163

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Anticelliilar Iinmnnity . . . . . . . . . C . Delayrtl Hypersensitivity . . . . . . . . . X. The Role of the Macrophage in the Iiiiniune Response . . . . A. Fate of Antigens within Macrophages . . . . . . B. Transfer of Imniunological Reactivity with Macrophages or . . . . . . . . . Macrophage Extracts . XI. Conclusions . . . . . . . . . . . . . References . . . . . . . . . . . . .

B.

I.

202 203 204 204 206 208 209

Introduction

The mononuclear phagocytes which comprise the reticuloendothelial system represent a large, widely distributed and morphologically heterogeneous group of cells. Originating from the mesoderm during embryogenesis they form a part of all tissues and are particularly prominent in the bone marrow, liver, spleen, connective tissues, serous cavities, and blood. Here they are involved in diverse functions, related in large measure to their endocytic activities and to the intracytoplasmic digestion which is a consequence of their heterophagic function. Certain of their detailed properties are beyond the scope of this review, and it will suffice to remind the reader of their importance in bone, lipid, and erythrocyte metabolism ( Jee and Nolan, 1963; Byers, 1960; Jandl, 1967). This review will attempt to cover the more recent advances in the biology and chemistry of the blood monocytes and tissue macrophages and to point out, where possible, mechanisms of immunological importance, Since it will not represent a comprehensive coverage of the literature, prior review articles will serve to orient the reader into more specific areas of interest. The general biology (Jacoby, 1965; Cohn, 1965), relationship to endocytic activity (Cohn and Hirsch, 1965; Cohn and Austen, 1963), cellular immunity (Mackaness and Blanden, 1967), phagocytosis and clearance mechanisms (Rowley, 1962; Suter and Ramseier, 1964; Karnovsky, 1962), lysosomes ( de Duve and Wattiaux, 1966), and relationship to delayed hypersensitivity ( Dumonde, 1967) have recently been reviewed. These articles contain most of the pertinent literature in this general area and serve as a basis for the following discussion. Although many of the properties of the mononuclear phagocytes are relatively clear, there are a number of aspects concerning their ultrastructure, life history, synthetic capabilities, turnover, and relationships to other cells in the immune system which have remained obscure. It is only recently that their origin has become clarified, and considerable work still remains in determining their kinetics in a variety of acute and delayed inflammatory situations. When approached at a more cellular level, it is also apparent that we know very little about the machinery that controls the reactivity of their plasma membranes and that influences

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their role as effective mediators of host defense. These questions also apply to other cells of the body so that the investigation of the macrophage can be considered a multidisciplinary exercise in cell biology which can be approached at various levels of interest. It.

life History

For the purposes of this discussion we shall separate the mononuclear phagocytes into two main classes of cells. The first is the monocyte which can be considered an immature member of the system and which is present in the circulating blood. By the ground rules of this review, only the intravascular component which meets certain morphological criteria will be termed a monocyte. The second class, which is larger and more heterogeneous, is composed of the extravascular cells or tissue macrophages. Since there is good evidence that the monocyte differentiates or matures into the tissue macrophage, this classification although somewhat arbitrary is less confusing than the host of synonyms employed in the past literature.

A. ORIGINOF

THE

MONOCYTE

The monocytes of the blood comprise, in most species, a small percentage of the circulating white blood cells and represent 3-7% of the total leukocytes. This relatively small number and the problems in identifying these cells with some security have long inhibited exacting studies on their origin. The more recent use of tritiated thymidine as a specific nuclear label stimulated studies on the kinetics of many circulating leukocytes, but it was only in the past 3 years that research on the monocyte has appeared. The two prevailing hypotheses considered the monocyte to originate either from a lymphoid cell in the spleen and lymph nodes or from a bone marrow precursor. The question was answered definitely by the studies of Volkman and Gowans (1965a) in the rat. By employing a skin window or subcutaneous cover slip preparation to immobilize blood-borne macrophage precursors, these authors investigated the labeling properties of macrophages with tritiated thymidine. Two types of experiments strongly indicated that the bone marrow was probably the most important source of the blood monocyte. In the first instance, rats were irradiated with 750 rads, a dose which inhibits the formation of labeled cells. The labeling pattern could be restored to normal levels by shielding the tibia1 marrow. The second involved the infusion of labeled cells from various sources into syngeneic recipients. These results indicated that labeled monocytes appeared only after the administration of labeled marrow cells and to a much smaller extent with spleen cells. In contrast, lymphocyte depletion by means of thoracic

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duct drainage or the use of smalIer doses of irradiation (400 rads) failed to influence the percentage of labeled cells. Results similar to these were reported in other systems. For example, Ballner (1963) and Goodman (1964) showed that the free peritoneal macrophages of radiation chimeras were all donor type, 6 weeks after receiving allogeneic bone marrow. From these data it appears that the most important source of the blood monocyte is from a rapidly dividing precursor which is primarily located in the hone marrow. The actual contribution of the spleen is still obscure although it is quite certain that splenectomy does not appreciably reduce the number of labeled monocytes. The previous information (Volkman and Gowans, 1965a,b), as well as subsequent studies of others (Spector et al., 1965; Whitelaw, 1966), and unpublished observations by Van Furth in this laboratory, make it extremely unlikely that the vast majority of “small lymphocytes” contribute to or are capable of transforming into blood monocytes. The major evidence relates to the striking discrepancies in the labeling pattern of the two cell types. Whereas, as many as 50%of the circulating monocytes are labeled after the administration of a single injection of thymidine, less than 3%of lymphocytes contain detectable isotope. In addition, the infusion of labeled thoracic duct cells (Volkman and Gowans, 1965b) into syngeneic recipients did not result in the formation of labeled monocytes and macrophages. The cultural observations of Rabinowitz and Schrek (1957) support this thesis.

B. TURNOVER AND INTRAVASCULAR LIFESPAN OF BLOODMONOCYTES Few studies have addressed themselves to the detailed kinetics of monocyte turnover, intravascular life span, and extravascular longevity. TO date, only Whitelaw (1966) has published on this subject using the rat as the experimental animal. After labeling for a period of 8 days almost all the circulating monocytes contained tritiated thymidine. The injection of label was then stopped and regression curves plotted semilogarithmically of the percent labeled cells over the next 10 days. These data suggest that both labeled and unlabeled monocytes leave the circulation in accordance with an exponential function, the half-time being approximately 3 days. Using a value for the absolute number of circulating monocytes, based upon peroxidase positive cells, the turnover was in the neighborhood of 3.6 X loGcells per day. Other information derived from this unalysis suggested that the monocytes leave the circulation at random, have an average generation time of 24 hours or less, and are derived from a precursor pool which divides about 3 times before yielding the circulating blood monocyte.

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It is apparent that considerably more information is required before a coherent picture of monocyte kinetics becomes clear. Certainly, studies in normal animals will have to be supplemented with those in which increased emigration due to inflammatory situations is taking place. What is of great interest, however, is the rapidity with which monocytes are labeled with thymidine, at times when other blood leukocytes remain unlabeled. This can obviously be exploited in studies of delayed sensitivity and graft reactions. Subsequent studies may, in addition, clarify the precursor cell in the bone marrow, delineate the factors that govern its replication, and evaluate the intramarrow maturation phase. C. EMIGRATION OF MONOCYTES FROM

THE

INTRAVASCULAR POOL

Under normal conditions it appears that monocytes leave the vascular system in a random fashion and may accumulate in a wide variety of tissues. The egress of these cells presumably occurs by a mechanism similar to that described for the granulocyte (Ebert and Florey, 1939; Marchesi and Florey, 1960). Following adherence to the vascular endothelium, primarily in postcapillary venules, the cells insert pseudopods between adjacent endothelial cells and migrate through the intercellular space. The basement membrane is then penetrated and the cells escape into the extravascular connective tissue. Although directed movement or chemotaxis is said to occur in in vitro systems (Harris, 1960), this phenomenon has not been observed in vim. The stimulus that directs emigration is currently unknown but at least during early intlammatory events may be similar to that responsible for granulocyte efflux. This is suggested from the fact that both granulocytes and monocytes enter the tissues at the same time ( Spector and Willoughby, 1963). Their presence during these early stages is often obscured by the preponderance of neutrophiles which accumulate in the tissues. The recent observations on in vitro polymorphonuclear leukocyte chemotaxis (Ward et al., 1965) in which C’5,6,7 appear to be involved and which appears to govern the accumulation of granulocytes in immune nephrotoxic states, have not been extended to the monocyte. In view of the marked difference in the inflammatory cell population observed in acute and delayed hypersensitivity reactions, it might well be expected that separate mechanisms which evoke continued monocyte migration will be found.

D. ORIGINAND LIFE HISTORY OF

INFLAMMATORY MACROPHAGE The best and most complete evidence concerning the origin of the tissue macrophages comes from experiments which have evaluated the macrophage population accumulated at inflammatory sites. Starting from THE

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the direct in vivo observations of Ebert and Florey (1939), it seemed clear that the majority of these cells were derived from the blood monocyte. Using rabbit ear chambers. it was possible to follow the emigration of individual blood monocytes froin the microcirculation into the surrounding connective tissue. These cells were then seen to mature into larger macrophages. Through the use of particulate labels it was then apparent that individual macrophages remained at the site of inflammation, did not divide to any significant extent, and could be identified for periods up to 54 days at the original tissue site. More recent studies employing tritiated thymidine-labeled, blood monocytes have reached the same conclusions (Volkman and Gowans, 1965a,b; Volkman, 1966; Spector et ul., 1965). These experiments performed at different tissue sites ( a ) epidermal skin windows, ( b ) subcutaneous tissues, and ( c ) peritoneal cavity have all indicated that the vast majority of labeled cells responding to physical or chemical imtants are blood monocytes which enter the tissues and then mature into labeled macrophages. Some authors have suggested that the macrophage is capable of division in the tissues. The data are not completely convincing at this stage in our knowledge and, in any event, probably represent a minimal mitotic response and cannot account for the majority of macrophages in the lesions. In contrast, however, Forbes and Mackaness (1963) have reported a more vigorous mitotic response following specific antigenic challenge. It is uncertain, however, whether this is indicative of the mitotic potential of monocytelike cells in general or the specific properties of a more immature cell which accumulates in response to an immunological reaction. The long-term history of the macrophages which accumulate at sites of tissue injury is not clear. Certainly, from the previously cited investigations, these cells do remain within local sites and are actively engaged in endocytic functions. In most instances, however, the ultimate fate and turnover of these cells are unknown other than the qualitative observations of dye-loaded macrophages in tattoo sites. There are, however, instances when the accumulated macrophages form a more organized tissue such as in a granulomatous reaction. Under these circumstances inflammatory macrophages become tightly interdigitated and take on the appearance of typical epithelioid cells. These events have been studied in humans with granulomatous disease during the development of specific Kveim reactions in active cases of sarcoidosis (Hirsch et al., 1966). In this instance, the injection of Kveim antigen mixed with particulate markers such as carbon or colloidal gold, leads to the uptake

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of the label by macrophages. This material is sequestered in large membrane-bounded phagosomes and these cells then are incorporated into the enlarging granulomatous nodule. Examination of epithelioid cells at intervals up to 60 days after injection shows the existence of highly labeled cells and suggests their continued presence in the lesion for at least this period of time. In addition, visual examination of the nonbiopsied nodules indicates label for periods of 6 months. Whether this is typicaI of a nonorganized group of tissue macrophages is uncertain. E. ORIGIN AND TURNOVER OF THE TISSUE MACROPHAGE There are in many organs, large numbers of cells with the endocytic properties of macrophages. Examples of these are the Kupffer cell in the liver, the sinusoidal and “dendritic” macrophages of the spleen, the glia of the central nervous system, and the alveolar macrophages or “dust cells” of the lung. Under normal, physiological conditions these cells play an important role in body economy and represent a relatively stable population of individual tissues. Little is known concerning their origin, turnover, and life span. Fragmentary evidence is available for the Kupffer cell and alveolar macrophage and little or none for the other tissues. The report by Howard (1964) suggests one origin of the Kupffer cell from cells contained in the thoracic duct. Since this source contains a number of cell types, the progenitor is still uncertain. Other studies performed with the parenteral injection of th~midine-~H indicate that approximately 1%of typical Kupffer or “littoral” cells become labeled within a 24 hour period (Kelly et al., 1962). These studies, although of little help in defining the precursor cells, do indicate a relatively slow turnover under normal conditions. Similar studies performed with regenerating liver and with a simultaneous monitoring of blood monocytes would be of great interest. A similar situation exists with alveolar macrophages in which, using karyotypic markers, it was suggested that approximately 50% of these cells come from the peripheral circulation (Pinkett et al., 1966). In general, the use of karyotypes for the study of mononuclear phagocytic kinetics has the deficiencies of not being able to examine the morphology of the cell in detail and depends upon the mitotic activity in situ which in many instances is quite low. Our lack of information in this area leaves open the possibility that the macrophages of the tissues may have more than one origin and may be derived from cells other than the blood monocyte. This dilemma is reflected in the lack of generalizations which we can make about the specific functional properties of this cell type.

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Ill.

Morphology

The distinction between various classes of lymphocytes, monocytes, and macrophages is in large measure based upon simple morphological criteria. For this reason, the unique features of the mononuclear phagocytes will be discussed in detail and the reliability of these structural features compared under different methods of fixation and staining. In general, it is usually possible to differentiate these cells with some security either at the light or electron microscopic level. A. MONOCYTE-LIGHT AND PHASE MICROSCOPY The structural features of the blood monocytes have been reviewed in the past (Cohn, 1965; Low and Freeman, 1958). Employing the usual Romanowsky stains and methanol fixation the cell exhibits an indented nucleus and a moderate amount of bluish-gray cytoplasm. Peroxidase staining has been used to distinguish the monocytes from larger lymphocytes. However, it is not clear what proportion of blood monocytes exhibit positively stained granules in the cytoplasm. This cytochemical technique appears to differentiate a portion of the monocytes from lymphoid cells but may not adequately reflect the total number of monocytes in the circulating blood. A more satisfactory method is to prepare living mounts and observe the surface membranes under phase contrast microscopy. Under conditions in which monocytes adhere to glass surfaces, they spread out and have distinctive ruffled membranes which are continually in motion and which are involved in the formation of phase lucent pinocytic vacuoles. Lymphoid cells under the same conditions are quite different in appearance, without ruffled membranes, and contain mitochondria which are considerably thicker than in the monocytic series. Additional structural features under phase contrast microscopy are the modest number of spherical phase-dense, cytoplasmic granules in monocytes and their absence in lymphoid cells. The distinctions between “mononuclear cells” becomes much more difficult in paraffin-embedded tissue sections. Although nuclear morphology and chromatin pattern of the nuclei are occasionally of benefit, no clear-cut distinction can be made in the usual preparation such as that employed for delayed reactions. Only when the monocytes have matured into the larger histiocytes is the task somewhat easier. Little is known about the structure of the precursor population in the bone marrow. It is apparent, however, that cells with the potential of maturing into macrophages do exist, can be cultured, and incorporate

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thymidine in vitro. Their identification during the early phases of bone marrow cultivation has, however, not been made.

B. MONOCYTE-ELECXRON M~CROSCOPY The examination of blood monocytes from a variety of species has been performed by means of electron microscopy (Low and Freeman, 1958; Sutton and Weiss, 1966). (See Fig. 1,) The basic architectural features are similar for both man and lower forms and will be reviewed as a unit. The nucleus contains one or two nucleoli with condensed peripheral chromatin. Little glycogen is seen in the cytoplasm. There is a moderate amount of rough-surfaced endoplasmic reticulum (ER) with attached ribosomes. Some free ribosomes are also normally observed although polysomes are unusual. The cisternae of the rough ER are flat and do not contain electron-dense material. Scattered through the cytoplasm are slim mitochondria which are not localized in any particular area. Little or no organized smooth-surfaced reticulum is seen other than the lamellae of the Golgi complex. The Golgi zone is well developed in the “hof” of the nucleus, is associated with centrioles, and composed of one or two stacks of lamellae. In addition, there are often numerous smooth-membrane bounded vesicles among the lamellae which appear in some cases to be budding from the stacks of membranes. The transitional zone between rough and smooth ER is usually not well demarcated, and continuities between the smooth reticulum and Golgi lamellae are not seen. One occasionally observes linear arrays of fibrils in the perinuclear region, the nature and function of which remain unknown (de Petris et ul., 1962). In addition, using appropriate fixation, microtubules are seen in the peripheral cytoplasm. The other important cytoplasmic organelle is the membrane-bounded dense body. These structures, although more prominent in macrophages, are seen to a greater or lesser degree in circulating monocytes. They are bounded by a limiting unit membrane and usually contain a homogeneously dense matrix. They are thought to be lysosomal in nature. The cell surface contains microvilli and in most cases there are electron-lucent micropinocytic vesicles in the cell periphery. The pinosomes are occasionally surrounded by spiked projections and have been termed *‘coated vesicles.”

C. MACROPHAGE-LIGHT AND PHASEMICROSCOPY

The tissue macrophage is a much more heterogeneous cell in terms of its morphology (Bennett, 1966). When compared to the monocyte the macrophages are larger ( 2 0 5 0 p ) and contain greater numbers of cyto-

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ZANVIL A . COHN

FIG. 1. Electron niicrograph of a human blood monocyte obtained from the buffy coat. This cell illmtrates the presence of mitochondria ( M ) , Golgi complex ( G o ) , membrane-bounded dense bodies or lysosomes ( L ) , perinuclear fibrils ( F ) , and nucleus ( N ) .

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plasmic dense bodies and mitochondria, In stained sections their nuclei are less condensed than lymphoid cells and vary from spherical to deeply indented structures. The cytoplasm is light blue or even slightly eosinophilic and ingested debris are often seen. A similar picture is obtained when stained smears are prepared from exudates. A more informative examination can be made with isolated cells which are allowed to spread on glass surfaces and viewed with phase contrast microscopy. When observed in the living state (Robineaux and Pinet, 1960; Cohn and Benson, 1965d) the activity of the peripheral membrane is marked and of considerable importance in identifying the cell type. This is typified by slow undulations and ruffles which are continuous and result in a wavelike action which moves in a centripetal fashion. Such ruffles can also be seen in glutaraldehyde-bed preparations and appear to be the origin of many phase-lucent, pinocytic vesicles. Long slim mitochondria extend into the pseudopods as well as being intermingled with spherical dense granules in the perinuclear region. This area contains an assortment of dense granules and lucent vacuoles, which may be scattered randomly or arranged in a rosette about the Golgi complex. Lipid droplets are often present in the cytoplasm and large phagosomes are common. The phagocytic vacuoles contain an array of particulates ranging from crystalloids to relatively intact cells. Much of the heterogeneity in structure appears to be related to previous endocytic events and the resulting sensitivity of ingested materials to intracellular hydrolysis.

D. MACROPHAGE-ELECTRON MICROSCOPY A number of papers have appeared in the past 5 years which describe the ultrastructure of macrophages from various tissue sites. These include the Kupffer cell (Novikoff and Essner, 1960), alveolar macrophage ( Karer, 1958 ), peritoneal macrophage ( North, 1966), and cells from spleen and lymph nodes ( Weiss, 1964). The overall structure is basically similar to the monocyte but varies in terms of the mass and contents of the cytoplasm. In general, the Golgi apparatus is more highly developed and contains multiple stacks of lamellae which are arranged in the nuclear "hof." These are associated with tiny smooth-surface vesicles which are interspersed between the cisternal elements and are more numerous than in the more immature monocyte. The rough ER is found most abundantly at the cell periphery and on that side of the nucleus opposite to the Golgi complex. There is often marked variation in the amount of rough ER contained in cells from different sources and even among the same population. As mentioned previously, the dense cyto-

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ZANVIL A. COHN

FIG. 2( A ) . Highly stimulated BCG-induced rabbit alveolar macrophage. This cell illustrates the rosette of heterogeneous dense bodies or lysosomes ( L ) which surround the numerous tiny, smooth Golgi vesicles ( G o ) .

plasmic granules are usually more prominent and may be quite variable in composition. Examples of these structures are shown in Fig. 2. Many if not all of the dense granules are thought to be secondary lysosomes and are probably derived from endocytic vacuoles. The multivesicular bodies and myelin figures may have two possible origins. The first is a heterophagic event in which mcmbrane-containing structures are cngulfed by the cell and thc second is an autophagic process (de Duve and Wattiaw, 1966). The latter mechanism occurs when a portion of the macrophage cytoplasm, including mitochondria, endoplasmic reticu-

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FIG.2 ( B ). A portion of a mouse peritoneal macrophage. This cell was obtained 4 days after the intraperitoneal administration of 10 p g . of bacterial endotoxin and shows the large numbers of perinuclear lysosomes ( L ) ; Golgi complex ( G o ) and nucleus ( N ) .

lum, etc., is enveloped by a pinocytic vacuole or lysosome, and is surrounded and subsequently degraded within a membrane-bounded structure. In other cases it appears that small invaginations of the vacuole wall occur, pinch off, and result in multivesicular bodies. It, therefore, appears that both heterophagic and autophagic phenomena can lead to the same morphological end result. Unless one can employ appropriate markers and follow the life history of these organelles, it is difficult if not impossible to state their origin with certainty. Epithelioid and giant cells arise from inflammatory macrophages. During the course of a granulomatous response or under in uitro culture conditions ( Sutton and Weiss, 1966), the highly phagocytic macrophage undergoes nuclear and cytoplasmic changes ( Dumont and Shelden, 1965; Gusek, 1964; Wanstrup and Christensen, 1966). In many cases the nucleus becomes elongated-a response which may reflect the tight packing of cells in the granuloma and is related to the elongation of the cytoplasm. Slim cytoplasmic extensions are formed and these are tightly intertwined with adjacent epithelioid cells. In addition, there seems to

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be ii change in the relative distribution of cytoplasmic organelles with more mitochondria being apparent and fewer dense bodies. This may be the result of a decrease in endovytic activity and/or a more vigorous formation of mitochondria. The giant cell of the granuloma appears to result from the fusion of preexisting macrophages. Following fusion the nuclei become oriented in a peripheral fashion and the cytoplasmic organelles are mixed in the central zone (Hirsch et al., 1966). This results in multicentric Golgi complexes and a corresponding increase in mitochondria and other cytoplasmic organelles. The factors involved in giant cell formation are not resolved at this time nor have specific functions been related to the syncytium. IV.

Maturation of Mononuclear Phagocytes

Much of the heterogeneity of monocyte and macrophage populations can be related to stages in the maturation of this cell type. In this section we shall deal with the differentiation of monocytelike cells in terms of their morphology, cytochemistry and biochemistry, and functional properties. The terms maturation or differentiation will be used interchangeably and will denote the increased size, complexity, and functional activity of this group of cells. A. MATURATION in Vivo

It had long been known by the earlier cytologists that macrophage elements which entered an inflammatory site increased in size and cytoplasmic complexity and took on the properties of a tissue histiocyte. Direct observations by Ebert and Florey (1939) made it clear that blood monocytes could undergo such a conversion, presumably under the influence of local factors. This morphological transformation then served as the basis for many of the techniques in which blood-borne cells accumulated on implanted cover slips and took on the properties of macrophages ( Rebuck and Crowley, 1955). Similar phenomena were observed in a variety of sterile and infectious inflammatory states, although in many of these instances it was not clear whether this represented the “activation” of local histiocytes or the emigration and maturation of a blood-derived monocyte (Lurie, 1964). In any event the sequence of events was similar to those observed in vitro and serve to complement these studies. These experiments, taken as a whole, indicate that certain agents which increase nonspecific resistance to infection are associated with an increased capacity of these cells to ingest and in certain cases inactivate specific microorganisms (Lurie,

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1939; Halpern, 1957; Shilo, 1959; Rowley, 1960; Howard et al., 1958). Cells obtained from stimulated animals were shown to contain higher levels of dehydrogenases (Allison et al., 1962) as well as a variety of hydrolytic enzymes (Grogg and Pearse, 1952; Allison et al., 1962; Suter and Hulliger, 1960; Thorbecke et al., 1961; Dannenberg et al., 1963; Dannenberg and Bennett, 1964; Myrvik et al., 1962; Heise et al., 1965; Saito and Suter, 1965). Additional findings by Cohn and Weiner ( 1963a) indicated that different populations of macrophages obtained from the rabbit contained elevated levels of hydrolases, a finding which correlated with an increased number of cytoplasmic granules with the properties of Iysosomes. The correlation among lysosomal enzymes, increased numbers of dense bodies, and cytoplasmic mass was also apparent following the introduction of a bacterial lipopolysaccharide into the mouse peritoneal cavity (Cohn and Benson, 1965a). B. MATURATION in Vitro Perhaps the most rigorous proof that blood monocytes and monocytelike cells from serous cavities differentiate or mature into macrophages comes from the in vitm cultivation of these cells. In many instances the in vitro results mirror the events in inflammatory lesions.

Blood Monocytes During the early stages of the development of tissue culture methodology, the cells of the peripheral blood served as a readily available source. Starting with the work of Lewis and Lewis (1925) and subsequent studies of Carrel and Ebeling (1926) and Heatherington and Pierce (1931), b d y coat cells were explanted and maintained in culture for many days. Under these conditions, granulocytes, platelets, and other short-lived leukocytes disintegrate leaving a more or less uniform population of adherent monocytes (Rabinowitz, 1961). The monocytes are usually quite active in phagocytizing cell debris and, in a short time, increase in cytoplasmic mass and in the number of cytoplasmic granules stainable with vital dyes. This process may continue for days, usually without evidence of division and with the progressive enlargement of the monocytes into typical macrophages. With more prolonged culture, giant cell formation is evident, which from strictly morphological evidence is the result of cell fusion. This well-organized series of in vitro events was not studied in greater detail until the cytochemical observations of Weiss and Fawcett ( 1953). Employing chicken blood, buffy coat, the authors obtained homogeneous populations of macrophages after 5 to 6 days of cultivation in autologous serum. During the development of

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these cells there was an increase in the number of lipid-containing organelles in the perinuclear region. These same inclusions, some of which could have represented ingested material also stained positively by the periodic acid-Schiff (PAS) reaction, an observation often noted in tissue macrophages. In addition, these authors comment on the occurrence of acid phosphatase in the cultivated cells, whereas no enzyme was detectable in the original monocytes by a cytochemical method. More recently, Sutton and Weiss ( 1966) have performed an electron microscopic study of chicken blood monocytes maintained in uitro. They document the maturation of these cells by means of serial electron micrographs, taken during the course of cultivation for a period of 70 days. The overall sequence of changes illustrated the transformation of the monocyte into a phagocytic macrophage and finally to epithelioidlike cells and multinucleated giant cells. The most striking ultrastructural changes were related to the increased number of dense granules which the authors refer to as lysosomes. These were obvious in the intermediate macrophages but as cultivation continued these organelles were lost and the mitochondria increased in number and formed the most prominent cytoplasmic component. Other important changes related to the increase in size of the Golgi complex and its close association with lysosomes. It is not clear whether the loss of dense granules with prolonged cultivation reflects a diminution of endocytic activity. A combined morphological and functional study has recently been performed on the isolated horse blood monocyte maintained in culture (Bennett and Cohn, 1966). Starting with peripheral blood it was possible to obtain relatively pure populations of monocytes which could then be examined biochemically under defined conditions. The procedure utilized centrifugation in a dense albumin gradient which resulted in the flotation of monocytes and a proportion of lymphocytes as a pellicle to the top of the tube. The second purification step employed the adherence of the monocytes to a glass surface, washing away the lymphoid cells, and leaving a homogeneous monocyte population. The cultivation of these cells in the presence of autologous serum or calf serum resulted in an increase in cytoplasmic volume without evidence of division. Within 24 hours the monocytes exhibited increased numbers of acid phosphatasepositive perinuclear granules which had the properties of lysosomes. Quantitative studies indicated the accumulation of lysosomal enzymes and a twofold increase in the specific activity of the mitochondria1 enzyme cytochrome oxidase. This latter finding was in keeping with the larger numbers of niitochondria observed by phase contrast microscopy. Additional biochemical results showed that glycolytic activity expressed

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either as glucose utilization or lactic acid production increased with the length of cultivation. These chemical parameters were associated with an increased capacity of the cultivated cell to ingest bacteria and to engulf particles of colloidal gold. The general impression from this study being that the maturation of the blood monocyte was expressed in both functional and biochemical indices of activity. C . MOUSEPFXUTONEAL PHAGOCYTES

The mouse peritoneal cavity normally contains a large resident population of cells about half of which are immature mononuclear phagocytes and the remainder lymphoid cells. Such cells represent a unique source of phagocytes, not requiring a prior peritoneal inflammatory stimulus, and have been used in a number of studies of host-parasite interactions (Rowley, 1960; Mackaness, 1962; Cohn, 1962a; Fauve et al., 1966) (see Fig. 3). They also serve as an excellent population for in uitro culture experiments in which the morphological and biochemical concomitants of macrophage functions have been evaluated. For this reason a more detailed review of their properties during cultivation will be described. 1. Morphology and Cytochemisty

Cells obtained from the unstimulated peritoneal cavity rapidly attach to and spread out on the surface of culture vessels. Following a short period of attachment the lymphoid elements may be washed away and one is left with a homogeneous population of mononuclear phagocytes (Cohn and Benson, 1965a). Although there is some heterogeneity in size and in the rapidity with which these cells attach and spread out, the majority illustrate the following series of events. The initial cell population has many of the morphological attributes of the blood monocyte in terms of size, nuclear morphology, and cytoplasmic organelles (Cohn and Benson, 1965a; Cohn et al., 1966b; North and Mackaness, 1963). It differs in that it usually contains a few more dense granules suggesting prior endocytic activity in the peritoneal cavity. As the cell spreads out on the glass surface, pseudopods begin to form and mitochondria enter the advancing process. Within 24 hours there is an increase in the length of mitochondria and a more marked increase in dense granules arranged about the nucleus. These granules which are phase- and electron-dense contain easily demonstrable acid phosphatase activity at the light microscope level and also avidly take up vital dyes such as neutral red. With more prolonged in uitro cultivation there is a progressive increase in cell size and the number of acid phosphatase-positive granules or lysosomes. At the ultrastructural level

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the newly formed lysosomes may exhibit a homogeneously dense matrix or contain a variety of vesicular elements within them. The other important alteration seen with the electron microscope is the enlargement of the Golgi complex with increased numbers of Golgi lamellae and vesicles (Cohn et al., 1966b). These changes, indicative of new lysosome formation and Golgi activity, now appear to be a common feature of the maturation of mononuclear phagocytes from either blood or tissue sources.

2. Biochemistry The previous cytochemical changes are corraborated by examining the enzyme content of cells undergoing in vitro maturation. Studies of three typical lysosomal enzymes-acid phosphatase, P-glucuronidase, and cathepsin-indicated that the cell content of these enzymes increased. The most striking enzyme elevation occurred with acid phosphatase in which a 4050-fold increase could be obtained in a period of 3 days. No appreciable cell division occurred during this period, although the protein content of cells rose to levels threefold greater than initial values and was in keeping with the presence of larger cells. Wiener (1967) and Wiener and Levanon (1968) have reported the production of an esterase which is largely released into the medium as well as the intracellular accumulation of acid phosphatase. 3. The Origin of Macrophage Lysosomes The previous results suggested that the cell maturation in vitro or in vivo was associated with the formation of new membrane-bounded organelles with the properties of lysosomes. Additional information obtained with inhibitors of protein synthesis indicated that these agents suppressed both the formation of dense cytoplasmic granules and the accumulation of acid hydrolases which occurred upon in vitro cultivation (Cohn and Benson, 1965b). Additional studies indicated that environmental factors to which the cells were exposed also played a large FIG.3. Top. Unstimulated mouse peritoneal phagocyte shortly after spreading out on a glass surface (phase contrast microscopy). Short mitochondria and spherical dense bodies surround the nucleus. FIG.3. Middle. Mouse phagocyte cultivated for 24 hours in 50%newborn calf serum. Large numbers of phase dense granules or lysosomes are present and long mitochondria extend into the pseudopod (phase contrast microscopy ). FIG.3. Bottom. A portion of the peripheral pseudopod of a cultivated mouse macrophage. Phase-lucent pinocytic vacuoles were arising from the tip and flowing cenbipetally among the mitochondria (phase contrast microscopy).

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role in the formation of macrophage lysosomes. One of the most important was the level of newborn calf serum in the medium. Cultures exposed to low levels of serum formed few dense granules and little or no enzyme, whereas high levels of serum resulted in the more rapid and extensive production of granules and lysosomal hydrolases (Cohn and Benson, 1 9 6 5 ~ ) . Examination of cells exposed to high levels of serum, by means of time-lapse cinematography, elucidated the origin of the dense granule (Cohn and Benson, 1 9 6 5 ~ )Shortly . after explantation and exposure to the medium the cells began to pinocytize actively. Vesicles were seen to form at the ruffled membrane and streamed in a unidirectional fashion into the perinuclear regions. Within a short time, large numbers of clear, phase-lucent pinocytic vacuoles had accumulated about the Golgi complex in the "hof" of the nucleus. Here, they underwent a transition in density, shrinking somewhat in the process, and becoming typical dense granules within a period as short as 90 minutes. Whereas the initial lucent vacuoles were uniformly negative for acid phosphatase, the dense granules were strongly positive, suggesting the acquisition of this enzyme during the formation of the dense granules. It is, therefore, apparent that the dense granules or lysosomes arose from pinocytic vacuoles. It was of interest that the influence of calf serum in stimulating dense granules and enzyme formation was also mirrored in its ability to stimulate the flow of pinocytic vesicles (Cohn and Benson, 1 9 6 5 ~ ) .

4. Formation

of Macrophage Lysosomes

The dense granules which originated from pinocytic vacuoles can be considered to be a secondary lysosome. Namely, a digestive body which contains constituents of the cell's environment or substrate as well as endogenously synthesized hydrolytic enzymes. This was evident from studies in which extracellular markers such as fluoresceinated proteins or colloidal gold (Fig. 4) were employed (Cohn et al., 1966a). After pinocytic activity these substances were strictly segregated within the dense granules and did not escape into the general cytoplasm. The next question to be answered, therefore, was how did newly synthesized macrophage enzymes enter the pinocytic vacuole? The best evidence suggests that newly synthesized protein and, presumably, hydrolytic enzymes are packaged within Golgi vesicles and are carried to the pinosome. By means of electron microscopic radioautography it could be shown that new protein is synthesized in the rough ER, subsequently transferred to the Golgi apparatus, and eventually ends up in the dense granule. This intracellular flow of newly

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FIG.4. Electron micrograph of a cultivated mouse peritoneal macrophage which has been exposed to colloidal gold for 3 hours. Occasional particles of gold are seen

at the cell surface. Most of the gold is present within electron-lucent pinocytic vacuoles (Pv) or electron-dense lysosomes ( L ) . The dense, preexisting lysosomes are labeled nonuniformly by fusion with new pinocytic vesicles. The large, clear pinocytic vacuoles contain uniformly dispersed colloid. A portion of the Golgi complex is present and is unlabeled.

synthesized material wits in keeping with ultrastructural observations in which Golgi vesicles were seen to accumulate about new pinocytic vacuoles and fuse with these structures. The presence of acid phosphatase activity in the Golgi complex was suggestive evidence that

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hydrolases followed a similar sequence. The importance of the Golgi complex has been stressed by Novikoff et al. (1964). These data suggest that the primary lysosome of the macrophage is the tiny, smooth-surface, Golgi vesicle; namely, a package of enzyme which has not come in contact with substrate. On the other hand, the secondary lysosome, containing both substrate and enzyme, is a demarcated cytoplasmic locus in which intracellular digestion is constantly taking place.

5. Turnover of Macrophage Lysosomes The continued presence of secondary lysosomes within macrophages is also dependent upon continued endocytic activity. Cells that have accumulated these organelles will subsequently lose them if placed in an environment in which pinocytic activity is depressed (Cohn and Benson, 1965d). The loss of both dense granules and assayable enzymes does not take place by means of extrusion into the medium. Rather, it seems that when both the input of substrate and the stimulus to enzyme formation are turned off, the organelle shrinks in size by means of intralysosomal digestion. During this phenomenon, lysosomal enzymes themselves seem to be digested as well. The reversibility of macrophage lysosome formation may have its counterpart in vivo since the environment may be continually changing and the stimulus for endocytosis may wax and wane. Such reversible “activation” would also be in keeping with Metchnikoff‘s early concepts of a “resting-wandering” cell. In any event, the response of the macrophage to its environment has the properties of an adaptive process in which hydrolase formation is the response to increased substrate interiorization. V.

Metabolism

Much of the work on the metabolism of the monocytes and macrophages represents ancillary information obtained through the study of the functional attributes of the cell.

A. ENERGY METABOLISM 1. Monocytes Relatively few investigators have examined the properties of the blood monocyte because of the difficulties of obtaining large enough samples. Bore1 et al. (1959) using mixed human leukocytes have calculated the intrinsic activities of the monocyte. Their information suggests that on a per cell basis the aerobic glycolysis of monocytes is more active

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than that of either the neutrophile or lymphocyte. More recently, Bennett and Cohn ( 1966) using isolated horse monocytes have demonstrated active aerobic glycolysis. Since the monocyte has little in the way of glycogen stores it is more dependent upon exogenous substrates than the neutrophile. 2. hlacrophage The more mature macrophage derives energy from both glycolytic and respiratory pathways. Aerobic glycolysis is active (Stahelin et al., 1956), and a Pasteur effect has been demonstrated by Harris and Barclay (1955) in exudate macrophages. A more detailed examination by Oren et al. (1963) of guinea pig alveolar macrophages indicates the presence of an important respiratory pathway with oxygen consumption values tenfold greater than for polymorphonuclear (PMN) leukocytes. This is in keeping with the large number of mitochondria in macrophages and their content of typical mitochondria1 enzymes. A significant “Pasteur effect” could be obtained with the alveolar cell, whereas no demonstrable “Crabtree effect” was noted.

B. SYNTHESIS OF MACROMOLECULES

1. Deoxyribonucleic Acid Evidence obtained from the use of tritiated thymidine indicates that the blood monocyte and tissue macrophage do not incorporate appreciable amounts of this isotope under most conditions (Van Furth and Cohn, unpublished). Much of the deoxyribonucleic acid ( DNA) synthesis of these cells occurs during maturation in the marrow and little in the periphery. The repression of DNA synthesis in the mature macrophage may not, however, be absolute. In this regard, Harris et al. (1966) have reported the incorporation of thymidine in heterokaryons formed from rabbit peritoneal macrophages and replicating cells. In this instance the cytoplasm of the dividing member of the heterokaryon apparently turns on macrophage DNA synthesis. Heterokaryons made with nondividing cells had no such influence. There are instances in which extensive incorporation of thymidine as well as mitotic figures have been observed in macrophage populations. The most striking was reported by Khoo and Mackaness (1964) in the peritoneal cavity of mice previously sensitized with albumin in adjuvant, The second injection of antigen, 2 weeks later, resulted in the proliferation of peritoneal cells which were described as macrophages. A similar conclusion was reached by other investigators using a microspectro-

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photometric method for macrophage DNA ( Rowley and Leuchtenberg, 1964) . 2. Ribonudeic Acid

Most macrophages obtained from stimulated or unstimulated serous cavities incorporated precursors into their ribonucleic acids ( RNA's) The turnover of rabbit peritoneal macrophage RNA was examined by Watts and Harris (1959). A rapid turnover was found, and it appeared that under these conditions most of the degradation products were reutilized by the cell. In the same series of experiments no synthesis or turnover of macrophage DNA could be detected. Using tritiated uridine and radioautographic techniques, cultivated mouse macrophages behave like many other mammalian cells (Cohn, unpublished). Initially, uridine is incorporated into the nucleus and is found over nucleoli, whereas later in the wash-out period there is migration of the label into the cytoplasm. over regions containing ribosomes. The nature of macrophage RNA and its relationship to informational exchange has been an active subject and will be discussed in a later section. Gottlieb et al. (1967) have recently reported on the incorporation of uridine into macrophage RNA in short-term experiments.

.

3. Proteins

Extensive protein synthesis occurs in macrophages cultivated in vitro. Many of the products have not been identified although from enzymatic studies reported in previous sections it appears that constitutive enzymes are formed. The presence of rough-surfaced ER and free ribosomes are obviously involved, although it is not clear to what extent synthetic products are retained by the cell or excreted into the environment. A few examples of biologically active molecules which are presumably synthesized by macrophages will be described in this section. a. Interferons. A number of observations have now clearly documented the fact that macrophages from different species and sources produce large amounts of interferon. Using mouse peritoneal cells, Glasgow and Habel (1963) showed that vaccinia virus which did not undergo a complete infectious cycle, stimulated the production of an interferonlike protein. More recently, Glasgow ( 1965), Smith and Wagner ( 1967a), and Acton and Myrvik (1966) reported the production of interferon by macrophages obtained from the mouse and rabbit. It seems likely that the production of interferon by human blood cells is related in part to the activity of surviving monocytes ( Wheelock, 1966). Smith

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and Wagner (1967b) also demonstrated that Newcastle disease virus (NDV) as well as bacterial endotoxin stimulated rabbit peritoneal macrophages to produce interferons which differed in molecular weight when examined on Sephadex G-100 columns. These products varied in molecular weight from 34,000 to greater than 134,000 and their synthesis was inhibited both by puromycin and actinomycin D. b. Endogenous Pyrogens. The formation of endogenous pyrogen has formerly been thought to be the sole property of PMN leukocytes. Recent studies of Atkins et aZ. (1967) and Hahn et al. (1967) have now demonstrated that rabbit macrophages obtained from both the lung and peritoneal cavity can be stimulated to produce large amounts of pyrogen. Although this material has never been isolated in pure form it is thought to be a protein. c. Serum Proteins. Studies by Stecher and Thorbecke (1967) have shown that macrophages from a number of rodent species and sources are able to incorporate 'Glabeled amino acids into serum proteins. This was demonstrated through the use of the radioautography of immunoelectrophoretic patterns. It was stated that the macrophage was much more active in the production of p l c and transferrin than other cell types examined. Macrophages obtained from primates were also shown to produce ple. VI.

Phagocytosis

A vast literature now exists on the phagocytic process in isolated cells and the clearance of particulates from the blood stream (Hirsch, 1965). In the latter case this has often been equated with the ingestion of particulates by elements of the reticuloendothelial system. For the purposes of this review we shall confine our remarks to more general aspects of the uptake of particulates, whereas in the next section the uptake of soluble molecules will be discussed. Both pinocytosis and phagocytosis are basically similar and depend upon the interiorization of the limiting membrane to form a phagosome or pinocytic vesicle.

A. CELL-DEPENDENT PHENOMENA Within the past decade it has become obvious that phagocytosis requires the expenditure of energy on the part of the macrophage and that it also depends upon as yet poorly defined properties of its limiting membrane. Both of these parameters are apparently related to the maturity of the mononuclear phagocyte population under study and under in vitro conditions can bc divorced from humoral elemcnts.

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1. Metabolic Requirements Initial studies on the biochemical basis of phagocytosis were performed with PMN leukocytes and have more recently been extended to macrophages. The pertinent literature has been reviewed by Karnovsky (1962). Experiments by Oren et al. (1963) suggest that guinea pig peritoneal macrophages depend upon glycolytic energy to accomplish the process of particle ingestion. Inhibitors such as fluoride and iodoacetate effectively inhibit phagocytosis, whereas inhibitors of respiration and oxidative phosphorylation have little effect. These same requirements appear to hold for the cultivated mouse peritoneal macrophages (Cohn, unpublished) and for the isolated blood monocytes (Bennett and Cohn, 1966). However, the guinea pig alveolar macrophage appears to have a greater dependence upon energy derived from respiratory mechanisms (Oren et al., 1963). In general, glycolysis is of central importance in the ingestion of particulates in most macrophages studied and also in the PMN leukocyte. These determinants are substantially different from those involved in the pinocytic activity of macrophages. 2. Phagocytic Potential in Relation to Cell Maturity Considerable evidence has accumulated indicating that the prior administration of bacteria, endotoxin, and inert materials yield a heightened state of phagocytosis by the reticuloendothelial system ( Rowley, 1966). Many of these experiments have been conducted with blood clearance methods and are difficult to interpret because of alterations in humoral factors and increased numbers of cells. There have, however, been instances in which the in vitro activity of macrophages has been stimulated. Perhaps the first of these was reported by Rowley (1960) and involved the exposure of mouse peritoneal cells to bacterial endotoxins. More recently, evidence has accumulated in which both morphological and functional properties of the cells have been investigated. Employing isolated horse blood monocytes (Bennett and Cohn, 1966) marked increases were noted in the phagocytic ability of cultivated cells when compared to the initial preparation. Similarly Perkins et al. ( 1967), using sensitive techniques for the study of erythrophagocytosis, have clearly demonstrated enhanced ingestion by mouse peritoneal cells. This was obtained by stimulating cells in vivo and also by maintaining them in culture. In both instances the enhanced phagocytic activity expressed either as the percentage of ingesting cells or the number of particles taken up was reflected in the state of macrophage differentiation. The mechanism underlying this phenomenon has not been defined. It may

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merely reflect the increased cytoplasmic mass of the cell with greater possibilities for particle contact or possibly enhanced activity of the macrophage membrane, It seems likely, although by no means completely certain, that similar changes take place in uiuo. In this regard, Evans and Myrvik (1967) have reported that the intravenous injection of Bacillus Calmette-GuCrin (BCG) is followed by a threefold increase in the phagocytic activity of isolated alveolar macrophages. 3. Macrophuge Membrune

Thus far no concerted effort has been made to isolate and study the properties of the limiting membrane of macrophages. The analysis of the plasma membrane would be of great interest in a cell that is so actively engaged in endocytic activities. To date, however, our knowledge consists of indirect experiments in which proteolytic enzymes have been used to modify the membrane. These suggest that a trypsin-sensitive coat is present which is of importance in the attachment of aged and chemically modified erythrocytes (Vaughn and Boyden, 1964; Vaughn, 1965). B. ATTACHMENTAND INGESTION PHASES

It has now become clear that the overall process of phagocytosis can be divided into two steps which appear to have separate determinants. The first has been called the attachment step, in which the particle is firmly bound to the surface of the phagocyte, but interiorization does not necessarily proceed (Bloom, 1927; Ledingham, 1912; Mudd and Mudd, 1933). The second or ingestion step encompasses the complex process by which the surface membrane invaginates and surrounds the particle. A clear separation of these stages using mouse peritoneal macrophages has been described by Rabinovitch (1967a,b). In this case glutaraldehyde-treated red cells exhibited specific attachment only to macrophages and no appreciable binding was noted with lymphocytes and granulocytes. Attachment to the macrophage did not require the presence of serum factors or exogenous divalent cations and was less sensitive to ambient temperature than the ingestion phase. In contrast, the ingestion phase was initiated by serum or specific antibodies directed against red cell antigens or protein antigens bound to the red cells. This step required divalent cations. These requirements may depend upon the nature of the particle since the attachment of effete or opsonized red cells appears to require divalent cations (Vaughn and Boyden, 1964; Lee and Cooper, 1966). The attachment of bacteria to the macrophage surface has also been investigated (Auzins and Rowley, 1963).

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C. INFLUENCE OF HUMORAL FACTORS The greater information obtained on the nature of immunoglobulins and complement components has also been expressed in terms of the phagocytic process.

1. Cytophilic Antibody From the studies of Boyden (1964) it appears that certain immunoglobulins are able to bind to the macrophage surface. These cytophilic antibodies are then capable of interacting with added erythrocytes with the formation of typical rosettes of red cells about the coated macrophage. In general, the production of cytophilic antibody requires the use of complete Freund's adjuvant in the immunization schedule (Boyden, 1964; Jonas et al., 1965; Nelson and Mildenhall, 1967). The nature of the immunoglobulin as prepared in guinea pigs has been examined (Jonas et al., 1965; Berken and Benacerraf, 1966). Apparently only 7 S y z is cytophilic for macrophages, whereas yl had little or no activity. In both instances the cytophilic antibody was only bound to macrophages and did not influence the surface of the PMN leukocyte. Of interest was the fact that this antibody is easily eluted from the macrophage surface at 37°C. This raises the possibility that eluted antibody might also be modifying the surface of erythrocytes prior to their attachment to macrophages. The use of IgG fragments indicated that the Fc segment was required (Berken and Benacerraf, 1966). It is possible from the studies of Uhr (1965) that other immunoglobulins when combined with antigen will also bind to the macrophage surface and influence the subsequent ingestion of the complex. 2. Relatiw Opsonidng Potential of Immunoglobulins The extensive documentation that specific antibody facilitated the ingestion of microorganisms was performed prior to our knowledge of the various classes of immunoglobulins. To date there have been relatively few articles which approach the subject and which have employed reasonably pure reagents with adequate physical and immunological controls. An additional complication is the question of whether the different immunoglobulin species are directed against the same antigenic determinant. Employing an in uiuo assay, Rowley and Turner ( 1966) and Rowley (1966) described the superior opsonizing ability of yM for the removal of gram-negative bacilli from the peritoneal cavity. Similar results suggesting the greater opsonizing capacity of IgM over IgG have appeared (Robbins et al., 1965; Solomon, 1966). In contrast,

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other investigators have reached the opposite conclusion and state that IgG is more effective in promoting phagocytosis than the macroglobulin ( Gerlings-Petersen and Pondman, 1965; Smith et al., 1967; Rabinovitch, 1967c,d). Although the nature of the particle may influence the relative activity of the globulins, these results also suggest that yM has greater activity in promoting opsonization in vivo, whereas yG is more effective in vitro. This could in part be related to the presence of complement components which might be a critical factor in facilitating the opsonization of particulates with y M ( Gerlings-Petersen and Pondman, 1965). The experiments of Rabinovitch ( 1967c,d) are particularly revealing. Using erythrocytes which were already attached to the surface of the macrophage, yM antibody in the absence of C’ had no stimulatory effect on the ingestion phase, whereas yG antibody produced prompt particle uptake. The lack of opsonization of yM was not the result of reduced binding to the erythrocyte surface, since adequate amounts could be detected on the red cell surface through the use of antiglobulin sera.

3. Complement The earlier work on complement components and its stimulatory effects on phagocytosis has been reviewed (Austen and Cohn, 1963; Boyden et al., 1965). Little additional information has been reported in regard to the macrophages although the relationship between immunoadherence and opsonization has been extensively discussed ( Nelson, 1963, 1965).

D. POSTPHAGOCYTIC EVENTS The ingestion of particulates by macrophages initiates a complex series of both morphological and biochemical events. In this section the interaction of the newly formed phagosome with cytoplasmic constitutents will be evaluated. 1. Morphological and Cytochemical Events The particles attached to the phagocyte surface are subsequently enveloped by the plasma membrane which then fuses with itself to form the phagocytic vacuole or phagosome. The formation of the phagosome completes the initial step in heterophagy and signals the onset of the digestive phase. As far as can be ascertained, the newly formed phagosome is devoid of hydrolytic enzymes and moves centripetally into the cell body to interact with cytoplasmic organelles. From studies employing alveolar macrophages (Cohn and Wiener, 1963b), Kupffer cells ( Novikoff and Essner, 1960; Essner, 1960), and guinea pig, peritoneal

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exudate macrophages (North, 1966), it seems quite certain that the phagosome fuses with preexisting lysosomes in the macrophage cytoplasm. This results in the resegregation of acid hydrolases within the phagocytic vacuole and the conversion of this structure into a digestive body or secondary lysosome. Then follows the depletion of preexisting lysosomes and the appearance of enzymes, i.e., acid phosphatase, in the phagosome (Strauss, 1964). It also is possible that Golgi vesicles, which can be considered the primary macrophage lysosome, also fuse with the new phagosome and contribute to the transfer of digestive enzymes. No obvious alterations at the light or electron microscopic level have been noted in other cytoplasmic constituents. The electron microscopy of phagocytosis by alveolar macrophages has been reported by Leake and Myrvik ( 1966). 2. Biochemical Alterations

a. Metabolism. As in the granulocyte, the macrophage also exhibits metabolic consequences following the uptake of particles. In some instances these are not as striking in view of the greater base-line activity of the cell; the events have been outlined by Oren et al. (1963) and more recently by Elsbach (1965) in alveolar macrophage populations. Alterations in carbohydrate and lipid metabolism have been noted and an increase in the hexose monophosphate shunt pathway described (Evans and Myrvik, 1967). b. Hydrolase Redistribution. The morphological change noted after particle uptake, expressed as degranulation, also has its biochemical counterpart. Cohn and Wiener ( 1963b) have employed rabbit alveolar macrophages and studied the intracellular distribution of lysosomal enzymes following the uptake of bacteria. Enzymes which are found primarily in a sedimentable, granule-bound form prior to phagocytosis are subsequently present in a soluble fraction after phagocytosis. During the course of the experiment, total enzymatic activity remained constant and the increase in soluble enzyme could be accounted for by a concomitant decrease in granule enzyme. This intracellular redistribution can be explained by the degranulation phenomenon and by the increased fragility of the newly formed digestive body which is ruptured during cell homogenization. VII.

Pinocytosis

Pinocytosis represents another mechanism by which cells may accomplish the quanta1 transport of environmental molecules. First described by w. H. Lewis (1931) in cultured macrophages almost four

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decades ago the process has been most extensively investigated in Amoeba. The elegant studies of Holter and his colleagues, reviewed by Chapman-Andresen (1963), serve a s a basis for studies in mammalian cells. The process is basically similar to phagocytosis differing only in that soluble molecules are incorporated and by the fact that the resulting vesicle is smaller in size. In this section we shall review studies with the macrophage although other cell types of the body may be actively engaged (Palade, 1953; Rose, 1957) in this form of endocytosis. The relationship of pinocytosis to antigen uptake has been stressed ( Robineaux and Pinet, 1960). A.

METABOLIC REQUIREMENTS

As yet the only cell studied in detail has been the cultivated mouse peritoneal macrophage ( Cohn, 1966). Employing a microscopic assay in which newly formed pinocytic vesicles are enumerated under phase contrast, it is possible to study the influence of both inhibitors and inducing molecules. The stimulus for pinocytosis in these experiments was 50%newborn calf serum which maintains vesicle flow at relatively high levels. Under these conditions it was found that inhibitors of respiration including anaerobiosis, cyanide, and antimycin A reduced vesicle counts to low levels. Similarly the inhibition of oxidative phosphorylation by either 2,4-dinitrophenol (2,4-DNP) or oligomycin had a marked inhibitory effect, thereby suggesting the ultimate participation of adenosine 5'-triphosphate ( ATP). Of some interest was the fact that inhibitors of protein synthesis such as p-fluorophenylalanine and puromycin could block pinocytic activity. This suggests that the synthesis of new plasma membrane is probably required for continuing membrane interiorization. Additional studies with actinomycin D indicated that the pinocytic activity of the macrophage was quite sensitive to this agent (0.1-0.003 pg./ml.) but only after a lag of about 2 hours. These data suggested but did not conclusively show a dependence upon DNA directed RNA synthesis. The ambient temperature also played a critical role, and a 10°C. reduction to 27°C. resulted in a 70%inhibition of vesicle formation. It seems apparent, therefore, that pinocytosis is a temperature- and energy-dependent reaction and one in which ongoing protein synthesis is required.

B. INDUCTION OF PINOCYTOSIS A variety of molecules stimulate the macrophage to increase its level of pinocytosis. These fall into three categories which can be sepawted on the basis of specificity and the nature of inducer involved.

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1. Induction by Anionic Molecules

Examination of a number of proteins revealed that only two-bovine plasma albumin and fetuin-both constituents of calf serum were able to stimulate effectively vesicle flow in cultivated mouse macrophages (Cohn and Parks, 1967a). Both proteins had low isoelectric points and were more active than cationic agents such as histone, protamine, and lysozyme. The stimulatory effect of albumin could be reduced by removing bound fatty acids and restored by the addition of either oleic or linoleic acid to the molecule at fatty acid-albumin molar ratios of 1:1-3:1. In the case of fetuin, removal of sialic acid by acid hydrolysis or neuraminidase digestion resulted in loss of activity. A similar stimulatory effect was obtained with a wide variety of anionic molecules including homopolymers of glutamic acid, acidic polysaccharides, nucleic acids, and other, smaller, negatively charged agents. In general, neutral and cationic molecules were much less effective and in many instances had no inducing effect whatsoever. There appeared to be little specificity in terms of the anionic moieties, and molecules containing carboxyl, phosphate, and sulfate groupings were all effective. In most cases, the minimum effective dose was a function of molecular weight -the larger the agent, the smaller the dose, Although the mechanism underlying these effects is unclear, it appears likely that anionic molecules interact with the plasma membrane, perhaps with cationic phospholipid moieties, and somehow induce the energy-dependent interiorization process. Dougherty et al. (1966) report the stimulation of pinocytosis in fibroblasts by heparin. The agents responsible for the increased pinocytic activity of macrophages in inflammatory sites has not been identified.

2. Nucleosides and Nucleotides A more specific example of chemical induction was noted in studies of nucleosides and nucleotides (Cohn and Parks, 196%). In this instance adenosine and its 5’-phosphates proved to be extremely potent inducers. Among this group, ATP was most active on a molar basis. The 2’ and 3’ isomers of adenosine monophosphate (AMP) as well as the nucleotides of inosine, guanosine, cytidine, and uridine were much less effective. Within the nucleosides only adenosine proved to be stimulatory. The mechanism behind these results is unknown. d

3. Immunological lnducers

Perhaps the most potent inducer present in bovine sera was a macroglobulin with the properties of an interspecies antibody (Cohn and

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Parks, 1 9 6 7 ~ ) .This material was absent in fetal serum and reached progressively higher titers in more ndult cattle. When separated from adult bovine serum it had the properties of agglutinating mouse erythrocytes and stimulated the pinocytic activity of mouse macrophages. Apparently both cell types share common antigens as revealed by absorption studies. In the presence of hemolytic complement, the antibody produced hemolysis of the mouse erythrocyte and cytolysis of the macrophage. The addition of macroglobulin to nonantibody-containing, fetal, calf serum resulted in the enhanced pinocytic activity of cultivated macrophages, increased numbers of lysosomes, and higher levels of acid hydrolases. The removal of antibody by absorption had the opposite effects. It seemed, therefore, that antibody directed against a macrophage membrane antigen stimulated pinocytosis and the morphological and enzymatic sequelae. Whether antibody directed against antigens artificially bound to the macrophage membrane has a similar effect is unknown. Further evaluation of the immunological activation of macrophages in vivo might be of considerable interest. One also wonders whether antibodies against the membrane antigens of other immunologically important cells would facilitate pinocytic activity and the uptake of antigenic molecules. VIII.

lntracellular Digestion and Bactericidal Properties

Since the time of Metchnikoff it has been realized that macrophages were capable of digesting a wide variety of particulate components of their environment. These ranged from intact or fragments of tissue cells, erythrocytes, bacteria, and Protozoa. Most of the earlier evidence stemmed from morphological observations in which the staining properties of the ingested organism were altered and structural dissolution ensued. In this section we shall briefly review the more recent evidence in which the fate of macromolecules have been examined in more detail. A. BACTERICIDAL PROPERTIES Although numerous in vitro studies have demonstrated that macrophages can inactivate a wide variety of gram-positive and -negative bacteria (Whitby and Rowley, 1959; Mackaness, 1960), almost nothing is known concerning the responsible mechanism. The acidic conditions of the phagocytic vacuole have been invoked, yet recent evidence indicates that this may not play a major role (Looke and Rowley, 1962). The situation in the macrophage is complicated by the fact that cell populations may vary widely in their bactericidal properties (Pavillard and Rowley, 1962). This form of heterogeneity is also stressed in studies on the maturation of the reticuloendothelial system (RES) in which the

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bactericidal capacity of the fixed mncrophilges was increased sharply in the neonatal period (Karthigasu et nl., 1965). In contrast, the fetal rat and chick were unable to kill gram-negative bacteria, even in the presence of specific antibody and adequate phagocytosis. Whether lysosomal enzymes play a role in the bactericidal properties of macrophages is unknown, although lysozyme-sensitive organisms would no doubt be readily killed. The situation differs markedly from the granulocyte in which one can easily extract bactericidal cationic proteins and study them in cellfree systems. No evidence exists for hydrogen peroxide as a mediator of macrophage bactericidal activity. OF THE INTRACELLULAR ENVIRONMENT B. MODIFICATION There is one example in the literature in which it seems likely that a molecule, taken up by macrophages and segregated within the lysosome, aids in the intracellular destruction of a microorganism. This stems from the initial observations of Cornforth et al. ( 1951) that mice treated with Triton WR-1339 were more resistant to challenge with tubercle bacilli. Subsequent work by Mackaness (1954) showed that treatment of cultured macrophages with Triton increased their capacity to destroy tubercle bacilli. These studies were difficult to interpret until Wattiaux et al. (1963) demonstrated that Triton was segregated within liver lysosomes and altered their density. This suggested that Triton, when segregated within a phagosome, is able to alter the surface of the bacillus, and facilitate the action of normally occurring bactericidal materials and hydrolytic enzymes. This form of synergism between chemotherapeutic agents and the machinery of phagocytes will no doubt be exploited in the future.

C. DIGESTION OF BACTERIA AND BACTERIALPRODUCTS The digestion of bacteria by phagocytes has been followed through the use of isotopically labeled organisms. Stahelin et al. (1956) employed 14C-labeled tubercle bacilli and showed the oxidation of small quanSubsequent work (Cohn, 1963a) tities of bacillary material into 14C02. used organisms uniformly labeled with either carbon or phosphorus and studied their fate within both granulocytes and macrophages. Following phagocytosis, extensive degradation of bacterial macromolecules occurred with the formation of acid-soluble products. This was preceded by a short period in which the organism lost its pool of small molecular weight intermediates-suggesting an initial defect in the bacterial plasma membrane. The majority of the degraded material was subsequently liberated into the medium. In the case of phosphorus-labeled organisms,

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approximately half of the degradation products were found as inorganic phosphate. From this type of analysis it appeared that bacterial protein, lipid, and RNA were more readily digested than bacterial DNA. Observations with the fluorescent antibody technique (Gill and Cole, 1965) and other labeled bacteria (Hill and Marcus, 1960) have been described. Certain reagents appear to inhibit the intracellular digestion of engulfed microorganisms. Both Gelzer and Suter (1959) and Cohn (196313) noted that the opsonization of microorganisms with specific antibody decreased the rate of degradation. In the latter case the inhibition was temporary and was followed by control kinetics. Perhaps, globulin coating the bacteria must first be hydrolyzed before bacterial substrate is attacked. As yet our knowledge of the enzymatic armamentarium of the macrophage is too limited to allow a description of its degradative potential. It is, nevertheless, clear that a wide variety of macromolecules can be hydrolyzed. The persistence of certain bacterial polysaccharides within members of the RES may reflect deficiencies in the respective glycosidases.

D. INTERACTION WITH VIRUSPARTICLES Very little is known about the fate of virus particles within elements of the RES. From studies already performed with other cell types (Dales, 1963; Silverstein and Dales, 1968), it seems that lysosomal enzymes are in some cases involved in the uncoating of the viral genome. The genome, which in the case of reovirus is a ribonuclease-resistant, double stranded RNA, somehow escapes from the lysosomal membrane and initiates a replicative cycle in the cytoplasm. Similar studies with macrophages would be of great interest in terms of the possibility that other large molecules escape into the cell cytoplasm and thereby interact with the machinery for protein synthesis. COMPLEXES E. ANTIGEN-ANTIBODY The formation of antigen-antibody complexes near equivalence leads to the formation of amorphous aggregates which are readily ingested by both granulocytes and macrophages. By employing iodonated albumin, Sorkin and Boyden (1959) compared the uptake of complexes and showed that those prepared near equivalence were more extensively ingested. Evidence was also prwcntetl that the peritoneal macrophage was then ablc to drgrade the niatc.ria1 to low-molecular-weight products. The degradation of complexes by granulocytes at Arthus sites is well established ( Cochrane et al., 1959), and in vitro observations have been

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reported (Patterson et al., 1962). Very little is known concerning the fate of complexes within mononuclear phagocytes although by virtue of their lysosomal enzyme content they wouId be expected to be relatively efficient in the digestion of proteins. Again, this may depend upon the maturity of the cell population.

F. PROTEINS PINOCYTIZED BY MACROPHAGES Observations have recently been made on the uptake and fate of soluble proteins by homogeneous macrophage populations ( Ehrenreich and Cohn, 1967). Using iodonated human serum albumin (HSA), it could be shown that albumin was taken up by pinocytosis and eventually segregated within macrophage lysosomes. The rate of uptake was relatively slow when compared to the phagocytosis of aggregated albumin.

Once within the lysosome it was rapidly degraded and, using pulsed cells, it was shown that intracellular degradation was correlated with the appearance of extracellular acid-soluble isotope. The nature of the excretion products was examined in detail by paper and gel chromatography. The results indicated that the only digestion product which was released by the macrophage into the medium was iodotyrosine. Such a product would not be expected to have importance in information transfer. The results with intact cells were quite different than those obtained when macrophage lysates were allowed to digest albumin (Ehrenreich, unpublished). In this case, large fragments were formed, similar to those obtained by the activity of cathepsin on albumin (Lapresle and Webb, 1962). This points out the probable role of lysosomal peptidases and the difficulty in extrapolating information from cellfree systems to the intact cell. The continual uptake and digestion of large molecules by pinocytosis may have physiological significance in uiuo. Although di5cult to study, this mechanism may play a role in the normal turnover of plasma proteins by members of the RES.

G . MACROPHAGE INVOLVEMENT IN MORPHOGENESIS The scavenger function of macrophages in cleaning up traumatized and in3ammatory tissue sites is associated with the ingestion and digestion of tissue cells and intercellular products (Woessner, 1965). This is a rapid and efficient mechanism of great importance to host economy. This role of macrophages also functions in certain forms of amphibian metamorphosis in which large tissue masses are removed in the transition to more adult forms. Perhaps, the best example is supplied by Weber (1962, 1964) in his work on the thyroxine-induced regression of

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the tadpole tail. In this sitiiation there is a major loss of tail protein as the organ shrinks in size. The levc4 of a c i d hydrolases may increase yielding a largcb inrrrasci in the specific activity of the enzymes. Examination of the regressing tail by electron microscopy sliows the presence of many active macrophages which are the principal site of acid phosphatase activity. Although it is not certain what evokes the initial cellular damage, it seems likely that a new population of tissue histiocytes is involved in the subsequent destruction of the tail. This presumably occurs through the process of phagocytosis and intracellular digestion. These and other related findings also point out the contribution of phagocyte lysosomal enzymes to the total enzyme pool of inflamed tissues.

H. INBORNERRORS OF META~OLISM In a number of genetically determined metabolic diseases, macromolecular products accumulate within affected tissue cells. In some instances, such as the lipidoses and inborn errors of polysaccharide metabolism, these same products are often seen within the macrophages of the RES. As yet, macrophages from such patients have not been examined in detail. It might be expected, however, that these cells show a similar metabolic defect and may, in fact, lack specific hydrolases which normally aid them in the degradation of stored molecules. These materials may either be synthesized by the macrophage or else ingested by endocytic activity. IX.

Cellular immunity

The term “cellular immunity” will be discussed in terms of two distinct but related sets of phenomenology. The first has a more extensive history (Metchnikoff, 1905) and relates to the enhancement of the bactericidal properties of mononuclear phagocytes, whereas the second will examine the role of macrophages in antitissue and antitumor immunity. An excellent review of this field has recently appeared (Mackaness and Blanden, 1967) which also covers the relationship to delayed hypersensitivity. A. ACQUIREDANTIBACTERIAL IMMUNITY The classic studies of Lurie (1964) on the response of experimental animals to tubercle bacilli indicated that reinfection was associated with heightened host resistance, This was thought to be related to cellular factors and in particular to the activity of epithelioid cells found in the infectious granuloma ( Lurie, 1942). This speculation was corroborated within in vitro culture systems (Suter, 1953; Pomales-Lebron and Stine-

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bring, 1957; Holland and Pickett, 1958) in wliich macrophages obtained from infected animals were able to inhibit the multiplication of a number of facultative intracellular bacterial parasites. These experiments clearly established the central role of the macrophages as the effector cell and paved the way for an examination of mechanisms. The clearest example of antibacterial cellular immunity came from the studies of Mackaness (1962) employing Listeria rmnocytogenes in the mouse. Following an intravenous challenge the organism multiplied within the liver and spleen reaching high population densities. This took place for the first 3 days and was then followed by a dramatic period of bacterial inactivation which began on day 4. This immunity at the organ level was then evaluated on the cellular level by examining the peritoneal macrophage population under in uitro conditions. Whereas these cells supported the intracellular growth of Listeria at the onset of infection, resistant cells appeared on about the fourth day. This population of macrophages could now inactivate more than 90%of ingested Listeria, an alteration which coincided with events at the organ level. This system has since been exploited and its success is in part related to a clear-cut bactericidal effect whereas most previous authors were dealing with growth inhibition. 1. Nature and Specificity of Acquired Antibacterial Cellular Immunity

A large body of information now exists which clearly indicates that the newly acquired antibacterial properties of macrophages are not immunologically specific. Thus, infection with one organism results in a macrophage alteration which is expressed against a wide variety of antigenically unrelated species (Fong et al., 1957; Elberg et al., 1957; Howard, 1961; Mackaness, 1964a,b; Blanden et al., 1966). Such experiments have used BCG and strains of Salmonella, Brucellu, and Listeria. One can conclude, therefore, that the stimulation of the mononuclear phagocyte, irrespective of mechanism, is expressed as an immunologically nonspecific antibacterial effect. Further studies on the underlying mechanisms have been hindered by our lack of knowledge concerning the intrinsic bactericidal properties of macrophage cytoplasm. There are suggestions, however, that the maturation of macrophage populations with the formation of new lysosomal hydrolases, etc., may be involved. These stem from observations (Mackaness and Blanden, 1967; Cohn, unpublished) of mouse peritoneal cells, stimulated with agents as diverse as calf serum and bacteria'l endotoxin,

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and results in a larger, lysosome-rich cell, which kills microorganisms much more readily than unstimulated phagocytes. From the foregoing discussion it seems likely that whatever the mechanism for macrophage “activation,” this effect is widespread throughout the host and may involve the fixed cells of the liver and spleen as well as the wandering macrophages of the peritoneal cavity. This is related to the fact that acquired cellular immunity is critically dependent upon an infection with viable, multiplying organisms and has been difficult to reproduce with dead vaccines (Braun et al., 1962). The suggestion has been made that a large, persistent pool of antigen is required for the continual stimulation of the phagocyte population (Mackaness, 1964a,b). This may be required not only for its influence on existing cells but also as a continued stimulus for the bone marrow to increase monocyte production, thereby enlarging the pool of effector cells. In addition, it has been noted that the injection of antigen into appropriately infected animals results in the mitotic activity of peritoneal macrophages (Khoo and Mackaness, 1964), which would not only add to the local population, but represent an immunologically specific parameter of this form of immunity. 2. Transfer of Antibacterial Cell Zmmunity

The immunity to a number of intracellular parasites has been successfully transferred by means of cells (Sever, 1960; Allen, 1962; Saito et al., 1962; Miki and Mackaness, 1961). Similar studies with the serum obtained from infected animals has been uniformly unsuccessful. The adoptive transfer studies have employed cells from both lymph nodes and spleen as well as exudate populations containing macrophages. It is probably safe to say, however, that the cell type involved in this type of transfer is unknown. This raises the question as to the contribution of competent lymphoid cells which could produce antibody in the recipient. In addition, studies have been performed with cell fractions, ribosomes, and extracts containing RNA which report the transfer of cellular immunity (Fong et al., 1961, 1963). The underlying mechanism and purity of the RNA preparations is unclear. Although serum is incapable of transferring immunity per se, it does have effects on the in vitro interaction between macrophages and parasites (Fong et al., 1957; Gelzer and Suter, 1959). In both instances the use of immune serum protected the macrophage from the cytotoxicity of intracellular parasites and was additive with the immunity expressed by the. crlls. Thc, niiturc of ;i serum f:ictor which is bound to the m;icro-

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phages from immune animals has been studied (Rowley et al., 1964; Turner et al., 1964) and found to be a 19 S yM type of immunoglobulin. Such a factor could also play a role in the rate of the ingestion phases as well. B. ANTICELLULARIMMUNITY The second aspect of cellular immunity relates to the cytotoxic activity of cells obtained from immune donors which results in the death of other tissue cells. This includes the phenomena of tumor immunity (Alexander and Fairley, 1967) and graft-vs-host reactions. In most cases, these are complex phenomena in which the relative effects of the microcirculation, circulating antibody, and other humoral factors as well as the responsible effector cell or cells is still under active scrutiny. Most of the interest in this field has been confined to the lymphocyte as the cytotoxic mediator, This literature has recently been reviewed by Dutton (1967) and will not concern us at this time. There are, howevcr, very interesting experiments which amplify the role of mononuclear phagocytes in these processes. The influence of peritoneal macrophages on a target cell population was examined by Granger and Weiser (1964; Weiser, 1963). Cells obtained from immunized animals were seeded over a monolayer and resulted in the prompt adhesion of the macrophages. Within a 12-hour period, destruction of the target cell had occurred resulting in macroscopically visible plaques. It also appeared that macrophages were also damaged during the reaction, From observations at the light microscope level the cytolytic effect did not seem to involve the ingestion of the target cell. Further studies indicated that this was an antigenically specific interaction but that the addition of immune serum to normal macrophages did not result in a comparable effect. The presence of exogenous complement components was not required. Subsequent studies (Granger and Weiser, 1966) indicated the presence of a hemagglutinin extractable from immune peritoneal cells, which was thought to be responsible for specific adherence. Furthermore, plaques could be inhibited by the incorporation of either actinomycin D or chloramphenicol into the medium. From the work and studies on lymphoid cells, it seems that intimate cell contact is required for a subsequent cytotoxic effect on target cells. This may or may not be the result of adsorbed specific globulins. The mechanism of cell damage is unknown and will require more exacting cytological evaluation. It is interesting that a somewhat similar phenomenon has been reported in the interaction between blood monocytes and erythrocytes (Lo Buglio et a/., 1967).

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Here, erythrocytes coated with IgG bind to blood monocytes and lymphocytes and undergo sphering as a result of the attachment process. This represents a form of cell injury without overt phagocytosis and in which complement does not play a major role. Macrophage populations are also involved in certain aspects of antitumor immunity. Experiments performed within the peritoneal cavity (Baker et al., 1963) and with in vitro systems (Old et at., 1963; Bennett, 1965) report the destruction of neoplastic cells by macrophage populations. The mechanisms involved in vivo are far from clear although immunization with a mixed peritoneal cell population evokes immunity; the presence of isoantibody is required. Under tissue culture conditions, however, sensitized macrophages readily phagocytize and destroy tumor cells. The role of macrophage phagocytosis both in this form of immunity and in graft rejection situations has not been clarified.

C. DELAYED HYPERSENSITIVITY During the induction of acquired antibacterial cellular immunity (Mackaness, 1964a) it has been noted that delayed hypersensitivity to microbial components is expressed at the same time that activation of the macrophages takes place. Thereafter, when an extensive destruction of bacteria occurs in parenchymatous organs, and bacterial antigenic mass is dwindling, cellular immunity wanes whereas delayed sensitivity may remain for longer periods. These temporal relationships have been discussed within a framework of common mechanisms (Mackaness, 1967) and in terms of the histological reaction (Spector, 1967). The role of the macrophage has also been examined with in vitro systems which mimic the specificity of the delayed reaction (George and Vaughn, 1962; David et al., 196ia,b,c; David, 1965). By employing peritoneal cells from sensitized animals, these authors could demonstrate the effect of antigen in inhibiting the migration of macrophages from capillary tubes. The inhibition appeared to be specific for the immunizing agent and did not occur under conditions resulting in Arthus reactivity. Of interest in terms of later observations, was the finding that only a small number of sensitized cells could confer reactivity upon cells from a normal donor. Treatment of sensitized cells with proteases, such as trypsin and chymotrypsin, abolished their reactivity to added antigen. Furthermore, active cell metabolism was involved since the inhibition of migration was bIocked by the addition of actinomycin D and puromycin. This interesting but somewhat complicated series of phenomena has been partially chrified by the studies of Bloom and Bennett

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( 1966) and David ( 1966). Both groups have suggested that the inhibition of migration depends upon two cell types. Using partially purified preparations of lymphocytes and macrophages, it was shown that antigen probably interacts with the lymphoid elements. When sensitized lymphocytes are incubated with antigen they release a nondialyzable factor into the medium, the production of which is blocked with inhibitors or protein synthesis. The addition of this uncharacterized material to either sensitized or normal macrophages then results in the inhibition of in ~ i t r omigration, These results suggest that the immunological specificity of the system resides in the lymphocyte and its antigeninduced product-possibly an immunoglobulin. The macrophage plays a less active role, is altered nonspecifically by the lymphocyte product, and may then become an effector cell. In terms of macrophage physiology it would be of great interest to perform detailed cytological and biochemical studies on such altered phagocytes. X.

The Role of the Macrophage in the Immune Response

It is generally agreed that macrophages are incapable of synthesizing specific immunoglobulins (Ehrich et al., 1946) and suspected that they do play some role in the immune response. This involvement has been the center of intensive investigation in many laboratories but is not as yet clarified. In this section we shall review what is known concerning the fate of antigens within mononuclear phagocytes and the ability of macrophages and macrophage extracts to initiate an immune response. FATEOF ANTIGENSWITHIN MACROPHAGES The injection of labeled antigens into intact animals leads to sequestration by the RES, possible intravascular destruction, and excretion by renal mechanisms. The kinetics of clearance usually illustrate a two- or three-compartment system, and, depending upon the immune status of the animal, the soluble or particulate nature of the antigen, etc., the labeled compound leaves the intravascular pool in short order. After a number of days, small quantities of isotope are often found in the liver and spleen and even less in the lungs, bone marrow, and lymph nodes (Miller and Nossal, 1964; Nossal et aZ., 1965). Such studies have indicated that persistence of label, in minute quantities and this depends upon the nature of the isotope employed. In general it has been difficult to examine the state of the labeled compound and whether or not it is still attached to the injected molecule. The reutilization of label is particularly important in interpreting the results and has not been resolved in most instances. In certain cases,' immunogeneity is retained A.

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suggesting a relatively intact molecule ( Kruse and McMaster, 1949), and by microscopy a portion is present within Kupffer cells. The intracellular localization of persisting antigen has been examined by Franzl ( 1962). Using homogenization and differential centrifugation techniques it was possible to localize the bulk of the immunogen to a “light” mitochondrial fraction which was rich in lysosomes. Presumably, red cells phagocytized by Kupffer cells are segregated within phagosomes and remain within this vacuolar system. It is also possible that small amounts of antigen enter liver parenchymal cells as well and contribute to the persisting pool. Many of these in vitro experiments have been reviewed by Campbell and Garvey (1963). These authors have also reported the presence of labeled, persisting antigen in fractions which also contain ribonucleic acid. In view of the widespread and heterogeneous distribution of antigens following parenteral administration, the quantitative difficulties in assaying and localizing the persisting molecule, the turnover of antigencontaining cells, and the uncertainty that this pool of immunogen contributes to the immune response, it is not surprising that clear-cut results have not been obtained. The sensitive and controlled systems of Humphrey et al. (1967) offer great promise. Similar studies have been performed in vitro with relatively homogeneous populations of macrophages. Although these studies present less formidable technical problems, they are more difficult to interpret in terms of events in the intact animal. The fate of ill-defined bacterial agglutinogens have been examined in two systems (Walsh and Smith, 1951; Cohn, 1962b, 1964). Following the ingestion of Escherichia coli by rabbit phagocytes the intracellular destruction of an agglutinogen was followed temporally and evaluated by assaying the agglutinin response of injected mice. Whereas the rabbit granulocyte rapidly destroyed the ability of this organism to evoke an antibody response, the peritoneal macrophage failed to alter the immunogeneity of the organism for a period of 5 hours. This was not the failure of macrophages in general since a highly stimulated BCG-induced alveolar macrophages was quite efficient in the degradation of immunogen. Apparently macrophage populations differ in their degradative potential, a finding which may correlate with their lysosomal enzyme content as well as with the nature of the antigen. Subsequent studies in which erythrocytes were used as immunogen have been reported (Perkins and Makinodnn, 1965; Morita and Perkins, 1965; Perkins et al., 19%). Under carefully controlled conditions, macrophages were capable of destroying the immunogeneity of the phago-

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cytized red cell. This parameter of cell activity was only slightly i d u enced by supralethal doses of ionizing irradiation. The fate of a soluble antigen has been examined with cultivated mouse peritoneal macrophages ( Ehrenreich and Cohn, 1967). Following pinocytosis and lysosomal segregation of iodonated albumin and yglobulin, in a 2O-hour period the macrophages degraded about 90% of the protein to acid-soluble products. Most of these were excreted into the medium in the form of labeled amino acids. No larger fragments of possible immunogenic importance could be found in the environment of the cells. From these studies it is apparent that macrophages have the ability to destroy the immunogeneity of a number of soluble and particulate antigens. Although this capacity seems clear it is, nevertheless, true that destruction never goes to completion in these in vitro systems, and evidence of persistent antigen also exists in vivo. In interpreting these results one may stress either degradation or persistence of antigen, in terms of the immune response and one’s own personal bias. It would be of considerable interest to examine the persistent macromoleculeits localization and the mechanisms protecting it from enzymatic destruction. One might expect to find it in a “residual body,” namely a lysosome which has lost its complement of hydrolases but which is still bounded by a semipermeable membrane (de Duve and Wattiaux, 1966).

B. TRANSFER OF IMMUNOLOGICAL REACXIVITY WITH MACROPHAGES OR MACROPHAGE EXTRACTS Most of the studies relating macrophages to the immune response center about the processing of antigen and the subsequent passage of information to an immunocompetent cell. The proponents of this view suggest that a two-cell system is involved in the inductive phase of antibody formation and base their views upon experiments that demonstrate the transfer of immunity with macrophages or macrophage extracts. Much of this work was stimulated by the experiments of Fishman (1961; Fishman and Adler, 1963a,b; Fishman et al., 1965), in which T2 phage was incubated with rat peritoneal exudate cells in vitro. Following 30 minutes of interaction, a phenol extract was then tested in a number of systems, i.e., lymph node cells, lymphoid cells in Millipore chambers, and lymph node fragments. The lymphoid cells exposed to the phenol extract produced small amounts of anti-T2 antibody. The interpretation of these results was made difficult by the background of natural antibody, the mixed exudate cell population, and the small amounts of antibody synthesized by the in vitro systems. Since the activity of the phenol

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extract was sensitive to ribonuclease and insensitive to the proteases tested, it was suggested that macrophage RNA was the active principle in the transfer experiments. Subsequent experiments examined the nature of the RNA species involved in relation to the type of immunoglobulin synthesized ( Fishman and Adler, 1963b). The light RNA fraction ( 4 7 S ) was related to the initial formation of 19 S antibody, whereas the heavier RNA (15-30 S ) was responsible for the later 7 S response. More recently, Adler et al. (1966) have reported that RNA from exudate cells of rabbits directs the allotypic specificity of the IgM antibody produced by lymph node fragments. Data consistent with an important role for RNA in the exchange of information come from the use of phenol extracts obtained from immune cells which stimulate the production of antibody by normal cells (Friedman, 1964; Cohen and Parks, 1964). One of the many questions raised by the experience of Fishman and his colleagues was whether the phenol extracts contained RNA alone or were mixtures of RNA and antigen. This point was examined in the T2 phage system by Friedman et al. (1965) who demonstrated a number of phage proteins in the phenol extracts by means of complement fixation. More extensive studies using 1311-labeledhemocyanin were reported by Askonas and Rhodes (1965). Here too, antigen was present in the RNA preparation although in a state which was more highly immunogenic than the hemocyanin molecule alone-suggesting the presence of a “superantigen.” These authors also found that the addition of hemocyanin to exudate cells, followed immediately by extraction, yielded an RNA fraction containing the antigen. This latter experiment points out a possible artifact present in all the foregoing experiments. Namely that homogenization of a cell containing antigen or in the presence of antigen results in a nonspecific binding of antigen with certain species of RNAa situation which may not exist in the intact cell. From what we now know of the cytological events of antigen uptake, large molecules are segregated within membranes and do not easily come in contact with cytoplasmic RNA, most of which is ribosomal in nature. It would, therefore, be of interest to collect antigen-containing lysosomes by gradient centrifugation and examine a purified fraction of intact organelles for RNA or highly immunogenic antigen. Recent evidence of Gottlieb et al. ( 1967) also corroborates the presence of an RNA-protein complex with immunogenic activity. Examination of the species of RNA revealed it to be of small size (4-6 S ) and to be present within the macrophage prior to the addition of antigen. In addition, the treatment of the complex not only with ribonuclease but also with pronase, a proteolytic enzyme, could destroy its immunogeneity.

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Although these authors refer to the RNA moiety as “immunogenic RNA,” it might as well fit into the category described as a “superantigen.” Since this RNA has very little specificity one wonders whether its function is merely to protect antigen from destruction or else to facilitate its upake by immunocompetent cells. The ability of macrophages to induce the formation of antibody to Shigella antigen in irradiated recipients has been reported by Gallily and Feldman ( 1967). The interaction of antigen with lymphocytefree macrophage suspensions was followed by their injection into X-irradiated recipients. The macrophage-Shigella complex resulted in agglutinating antibody, whereas the transfer of Shigellu alone or in the presence of lymphoid cells did not result in antibody formation. These experiments are also complicated by the lack of information concerning the mass of antigen transferred, the state of antigen within macrophages, the fate of transferred cells, and the specificity of the response. Certain other data are contradictory to the dictum that a two-cell system is required to mount an antibody response. These stem from the in vitro technique of Bussard (1966) in which the addition of sheep erythrocytes to a mixed mouse peritoneal cell population results in complement-dependent plaque formation in a viscous medium. This has been described as a primary in vitro response. Employing this method, Bendinelli and Wedderburn ( 1967) have examined the plaque-forming potential of both the peritoneal macrophages and lymphoid cells. The results indicated that the incubation of lymphoid cells plus sheep erythrocytes results in as many plaques as the mixed peritoneal cell population. In contrast, macrophages purified by a glass adherence method yielded few if any plaques. Such an experimental system, using more highly purified suspensions, may yield important information concerning the direct interaction of particulate and soluble antigens with lymphoid cells in vitro. XI.

Conclusions

This summary although slanted toward more immunologically oriented topics has illustrated the widespread involvement of the mononuclear phagocytes in the body economy. Many questions concerning the physiology of these cells remain to be answered. Some of the more obvious include: ( a ) the kinetics of monocyte turnover and the identification of the bone marrow progenitor, ( b ) the origin and turnover of the macrophages of parenchymatous organs, ( c ) the role of the macrophage in states of delayed hypersensitivity, ( d ) the detailed fate of antigenic molecules within macrophages, ( e ) the functional attributes

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of persisting intracellular antigen or of the RNA complex and its role both in vitro and in vivo. These are interdisciplinary tasks which should appeal not only to the immunologist but to the cell biologist and biochemist as well.

ACKNOWLEDGMENTS The author is indebted to Dr. Martha E. Fedorko and Dr. James G. Hirsch for supplying the electron micrographs.

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The Immunology and Pathology of NZB Mice' J.

6. HOWIE AND 6. J. HELYER

Deparfmanf of Pafhology, Otago University Medical School, Dunedin, New Zeaiond

I. Introduction

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B. The ( N Z B X N Z W ) F I Hybrids . . . . . C. The NZB Mice and Hybrids as Experimental Models III. The Experimental Usage of the Mice . . . . . A. Hybridization and Genetic Studies . . . . B. Transmission of the Disease by Spleen Cells . . C. Thymectomy and Thymus Grafts . . . . . D. Splenectomy . . . . . . . . . E. Therapeutic Studies . . . . . . . IV. Concluding Remarks . . . . . . . . References . . . . . . . . . .

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Introduction

The New Zealand Black (NZB) strain of mice was developed by Dr. Marianne Bielschowsky at the Otago University Medical School, as an inbred black-coated strain of mice for use in cancer research. It was noticed early in the development of these mice as an inbred strain that they died prematurely with anemia, hepatosplenomegaly, and sometimes jaundice. The pathogenesis of this syndrome was first elucidated and published in 1959 by Bielschowsky et al. It was shown that the mice developed an autoimmune type of hemolytic anemia with autoantibody production, and as such they were the first documented strain of experimental animals to develop autoimmune disease spontaneously. Subsequently Helyer and Howie, in a series of hybridization experiments showed that the autoimmune phenomena of the NZB strain appeared in all F, hybrids. In some of the hybrid strains, the autoimmune disorder maintained the NZB characteristic of erythrocyte autoantibody production. But in certain others, the nature of the process changed to 'This work lias been supported by the Medical ReseArcIi Council of New Zealancl, the United States Public Health Service (grant No. AM-07268), and the J. R. McKenzie Trust. 215

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one of antinuclear antibody production and renal disease in a form closely resembling lupus nephritis. The diseases which the mice develop, closely resemble their human counterparts, so that the NZB mice with hemolytic disease, and the hybrids with lupus nephritis, form experimental models of two rather digerent types of autoimmune processes. In clinical immunology, they provide opportunities for studies on etiology and pathogenesis and offer considerable advantages in the study of the therapeutic effectiveness of immunosuppressive agents, I n experimental immunology they have considerable potential for the study of fundamental immunological principles and of the distortion of these principles in autoimmunity. One of the major aims of this review is to describe where possible the patterns of development of the disease processes in these two groups of mice. Before such groups of animals can be used as experimental models the natural histories of their disorders, the temporal dynamics of their disease markers, and a full elucidation of the varying influence of genetic, environmental, and nonautoimmune factors is essential. This can best be achieved by two complementary and independent systems of study. Animals may be studied throughout their life-span using various clinical pathology markers such as anemia, various antibodies, proteinuria, and blood urea. The animals are killed when they become moribund. From this, the range of survival times is determined, and the terminal morbid anatomical features such as organ weights and histological changes can be ascertained. Furthermore, the identity of autoimmune and nonautoimmune diseases present at the end of the life-span can be decided. From such life-span studies, patterns of development of laboratory markers can be built up, but morbid anatomical data tend to represent the end or latter stages of the processes concerned. Furthermore many of these processes concerned show rapid changes over the terminal period of deteriorating health, so that the synthesis of an integrated pattern of disease processes over the life-span of the animal presents dif6culties. The death of the animals so often represents the effects of many, concurrently acting, pathological processes, so that to understand their individual effects and interactions this data must be complemented by an additional approach. Animals with stable weight patterns and considered to be clinically healthy are electively killed at intervals encompassing the range of survival of the strain. This provides clinical, pathological, and morbid anatomical data, representative of the state of animals before they become moribund. These data have shortcomings, because, as the electively killed program enters the period during which deaths of animals occurs, there is an increasing selection for longevity.

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From such comlhied studies, it is possible to unravel thc patterns of development of the disease syndromes characteristic of the strains, the variability of life-span, thc patterns of laboratory markers of the disorders, the influence of such facets as sex, breeding history, body weight, and other variable factors which may induce consistent modifications of the observed patterns of the disorders. II.

The Natural life Histories of the Mice

A. THENZB STRAIN The features of the natural history of this strain have been described by Bielschowsky et al. (1959), Helyer and Howie (1963b), Holmes and Burnet ( 1963a ) , Howie and Helyer ( 1965) , Mellors ( 1965, 1966a,b,c,d ) , Siegler (1965), East et al. ( 1965), Giltinan et al. (1965b), East and de Sousa (1966), Miyasato et al. (1967), and de Vries and Hijmans (1966, 1967). The accounts from different laboratories and from many different parts of the world show a considerable degree of agreement. As was clearly indicated by Bielschowsky et al. (1959) and again by Helyer and Howie (1963b), there are three distinct classes of animalsmales, virgin females, and breeder females. These three sex groups show considerable differences in all aspects of the strain studied. This has not been generally recognized and it accounts to a major degree, for many of the discrepancies among different published descriptions of the strain. Differences are in terms of details of variance in time of onset of the disorders, survival, and the incidence of the various laboratory markers. These differences in some instances are due to the relatively small samples of animals studied. They may also be attributable to differing conditions of environment, diet, and care, or to varying criteria and techniques in the demonstration of markers. Rather than attempt to annotate all the minor variations observed by the various workers in the field, the major part of the account of the natural history of the NZB strain will be based on the detailed study of more than 2000 mice of this strain, carried out in Dunedin over the last decade. The earliest and most consistent abnormality occurring in the NZB strain is the development of an autoimmune type of hemolytic anemia. However, this dominant feature is associated with several other disorders which may or may not be of an autoimmune nature. Proteinuria associated with renal disease increases in frequency and severity with age and may terminate in renal failure. Immunoproliferative disorders manifest as hyperplastic and neoplastic changes in lymphoid tissue occur, and, in the lungs, peribronchial aggregations of lymphoid cells may

218

J. B. HOWLE AND B. J. HELYEX

lead to bronchial and bronchioh stenosis and to pulmonary parenchymal complications. Peptic ulceration of the gastroduodenal region also increases in frequency with age and may he complicated by alimentary tract hemorrhage. Thus the health of the animals, as measured by body weight, and survival, as expressed by life-span, are determined by the interaction of at least five different syndromes,

1. Survival and Body Weight The mean survival time for males is 467 days, virgin females 431 days, and breeder females 418 days. The curves for distribution of age at death (Fig. 1 ) , show a relatively symmetrical curve for virgin females with a peak at 18 months, whereas the curves for males and breeder females are skewed and have peaks at 21 months. The mean survival

vI

U

30

-

O--OMM d ed se s O--O

25

3 6

L

9 12 15 18 21 24 27 30 Lunar months

FIG. 1. The distribution of age at death in NZB mice.

figures quoted by Holmes and Burnet (1963a) are in close agreement with those given above, but in the series of East et al. (1965) the mean survival times of 239 days for males and 310 days for females are quite remarkably different. Their more recent figures (East and de Sousa, 1966) of 342 for males and 340 for females are still very different. The average body-weight curves for the three sex classes of healthy NZB mice (Fig. 2 ) consist of three phases: a steep rise up to 6 months representing the initial growth of the animal; a plateau persisting until 12 to 15 months; and a subsequent secondary rise. The three phases are similar and more definite in males and virgin females, although the males are 4-5 gm. heavier. The secondary rise is unusual and is at present unexplained. It would be of considerable interest if it could be shown to have some relationship to the autoimmune disorders. Body weight ap-

THE IMMUNOLOGY AND PATHOLOGY OF NZB MICE

219

pears to have some relationship to longevity. If animals of 3 to 4 months of age are divided into heavy and light body weight groups, then the heavier animals have a significantly longer average survival. There is also a relationship between body weight and the markers of hemolytic

40

-

-Moles

0--0 Virgin females O.---O Breeder females

.C 30

2

3

>I

n

20

- - - - - t51

-

0 '

3

6

9

12

15

18

21

Lunar months

FIG.2. Average body weight curve of electively killed NZB mice.

disease, because heavier animals have more strongly positive antibody tests, more severe anemia, and greater hepatosplenomegaly. These variations in survival and in the expression of hemolytic disease in relation to body weight indicate that animals for experimental purposes should be matched for body weight.

2. Hemolytic Disease a. Autoantibody. The first indication of the development of autoimmune hemolytic anemia in these animals is the appearance of erythrocyte autoantibody. Initially it is an incomplete warm-type antibody and can be detected as erythrocyte-bound antibody by the direct antiglobulin test, or as free unbound antibody by using erythrocytes sensitized by ficin or papain, or by the indirect antiglobulin or albumin technique. The bound and unbound antibodies have identical characteristics. The antibody has maximal activity over the temperature range 18"37"C., although minimal activity can be detected at +4"C. It does not require complement. A weak complete antibody develops in some animals with advanced hemolytic disease and is responsible for inducing autoagglutination in freshly druwn samples of blood. It has been shown that the incomplete antibody is a 7 S 7-globulin ( Norins arid Holmes. 1961b), and the complete antibody 19 S (Mellors, 1965). The antibody has specificity for all mouse red cells, irrespective of strain, and shows a

220

J. B. HOWIE AND B. J. HELYER

weak cross-reaction with papain-treated rat cells. It has no reaction with rabbit, guinea pig, sheep, or human cells (Long et al., 1963). In addition to the mouse-specific erythrocyte autoantibodies, Holborow et al. (1965) have shown that the NZB strain also develops nonspec8c antibodies which react with stromal antigens of human and mouse red cells. Norins (1965) has shown that the strain lacks the complement antigen Hcl. The prevalence of serological positivity as determined by the presence of either bound or free erythrocyte autoantibody, first appears in a small proportion of each class at 3 months of age and reaches maximal incidence by 18 months of age (Fig. 3). In males and virgin females the curves of 10%. In breeders

4

Lunar months

FIG. 3. The prevalence of erythrocyte autoantibody in electively killed NZB mice. The presence of antibody indicates that it has been detected either as bound antibody by the direct antiglobulin technique or as free serum antibody by a ficin enzyme technique.

the curve is flatter and fails to reach 100%.These curves represent the incidence in healthy electively killed animals. If data from animals in declining health are included, then all three curves rise more steeply with similar slopes and approximate 100%by 12 months. These differences in patterns arise because the incidence of positive tests is higher in moribund than in healthy animals of the same age group. The time of onset of positive tests varies from 3 to 18 months. If animals are subdivided into groups accordiiig to the duration of survival, then it is apparent that the slope of increment of positive serological tests is the same in all groups. It would, thus, appear that the

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221

age at which erythrocyte autoantibody production commences does not influence sunrival. Once an animal Iwcomes scrolngically positive the antibody increases in strength. Also individual animals show considerable variations in the binding capacity of the antibody as measured by the presence and relative strengths of bound and unbound antibody. In males and breeders, bound antibody can usually be detected 1-2 months before free antibody is present, whereas in virgin females the two forms appear simultaneously. As animals fail in health and death approaches, changes in antibody strength and binding capacity cause alterations in the patterns of bound and unbound antibody in the three classes of animal. In males the binding capacity diminishes toward the time of death, with the strength of free antibody increasing and of bound antibody diminishing. In virgin females there is little alteration in binding capacity, whereas in breeder females there is a considerable increase in antibody production with a marked increase in prevalence and strength of both bound and unbound antibody. Thus, with approaching death the strength of free antibody increases in all animals, but bound antibody becomes weaker in males, remains unchanged in virgins, and gains considerably in strength in breeders. b. Anemia. A drop in hematocrit below the lower limit of normal of 40% (Helyer and Howie, 1963b), develops subsequent to the appearance of autoantibody. This is associated with and may be preceded by a reticulocytosis which in the majority of animals reaches 25%,but which may reach levels of loo%, particularly in males and virgin females. Polychromasia is consequently a consistent feature of the peripheral film. High reticulocyte levels are invariably associated with strongly positive antiglobulin tests and abundant free antibody. There is a general tendency for the reticulocyte percentage to rise as the hematocrit drops, but this varies widely in animals of the same sex group and also in individual animals throughout the course of the hemolytic disorder. Spherocytosis is commonly seen in animals with established disease and at least half of such animals have an increase in osmotic fragility. The erythrocyte survival time has been shown to be greatly reduced, and both liver and spleen participate in the increased rate of red cell destruction (Lindsey et al., 1966; Donaldson, 1967). Anemia does not develop invariably during the life-span of NZB mice, and 12.9%of virgin females, 231%of males, and 44% of breeders are not anemic at death. This is more common in animals dying in the younger age groups and becomes less likely with increasing age. The

222

J. B. HOWIE AND B. J. HELYER

highest incitlciicc of scvere ancmia is in virgin females. The prevalence of anemia in healthy animals can IIC, seen i n Fig. 4. A small proportion of males and virgins are anemic about thr timtx that autoantibody qyears. The considerably higher incidence of anemia in breeders about this time is attributable to the effects of pregnancies. The curve of prevalence of anemia rises from 9 months and reaches its maximum at 18 to 24 months. It has a very similar slope of increment to that of autoantibody but are also the same for follows it by an interval of 3 months. The slopes all three sex groups. 100

$ 80 C -0 -

01 0

o c

-Moles

*--a Virgin femoles *--*Breeder females

40-

Lunor

rnon t hs

FIG. 4. The prevalence of anemia in electively killed NZB mice. Anemia has been defined as a hematocrit reading below 40%).

Thus, anemia appears in the colony by 3 months of age, and over the first 9 months of life it is more common in breeders than in males and virgins. In healthy animals beyond 12 months of age, the proportion with anemia increases steeply at much the same rate in all three sex groups and reaches a maximum prevalence between 18 to 24 months. The severity of the anemia gradually increases with time, but as the health of the animals deteriorates, so the rate of progression of anemia becomes accelerated. This acceleration is particularly marked in virgins so that at death this group has the greatest incidence of severe anemia. C. Splenomegaly. Splenomegaly is a striking feature of both the clinical and postmortem examination of some NZB mice with established hemolytic disease. It is by no means a universal feature because 36%of virgins, 522 of males, and 62%of breeders have spleens within normal weight limits at death. Splenomegaly is more common in animals dying in the older age groups and gross enlargement is also more prevalent in

223

THE IMMUNOLOGY AND PATHOLOGY OF NZB MICE

virgins and males. Curves of the prevalence of various grades of splenomegaly in electively killed animals (Fig. 5 ) indicate that splenic enlargement commences between 9 to 12 months of age and increases with age to reach a maximum at 18 to 21 months. It is also more frequently present and develops to a greater extent in virgins than in males and breeders. The enlargement is mainly due to expansion of the splenic pulp by both extramedullary hemopoietic tissue and lymphoid hyperplasia with abundant immature and mature plasma cells and Russell bodies. Cortical

spleens 175. mgrns /

I*.,

,.?o

..

I

spleens 3 5 0 , rng m s I0\

spleens 7 0 0 . mgms

3

6

9

12

15

18

21

I

I

I

I

I

I

6

9

12

15

18

21

Lunar months FIG.5. The prevalence of grades of splenomegaly in electively killed NZB mice. The upper limit of normal spleen weight has been defined as 175 mg.

hyperplasia with the presence of germinal centers in the lymphocytic nodules is also sometimes present. Intracellular and extracellular hemosiderin is consistently present, and erythrophagic cells may be identified. Mellors (1965) has demonstrated the presence of immunoglobulins in the areas of medullary hyperplasia. H e has also shown that immunoglobulins specifically reactive with mouse erythrocytes can be extracted from the spleen and that this extraction results in loss of immunofluorescence of the tissue. He suggests that the spleen is the main site of formation of carythrocytc autoantibodics. Howevrr, in older animals with c~st;tl)lishcdhemolytic discwc it is an importaiit lremopoietic organ ;IS has lxmi shown by the rapid drop in hematocrit following splenectomy in such animals (Helyer and Howie, 196331,).

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J. B. HOWLE AND B. J. HELYER

d. Hepatomegaly. Hepatomegaly is also a feature of some animals with established hemolytic disease, but the degree of enlargement is much less than that seen in the spleen. At death, livers within the normal weight limits occur in 37.6%of virgins, 60.3% of males, and 60.8% of breeder females. Hepatic enlargement in healthy animals is first seen in animals of 6 months of age and reaches a maximal incidence by 18 to 21 months of age (Fig. 6 ) . It is maximal in prevalence and degree in virgin females and least in breeders. 100

80

8

60

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0

40

>

f 20

a I l l o

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t .males

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O---O 0.

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......* breeder fcrnoler.

6o

40

20

0

6

9

12

15

18

21

6

9

12

15

18

21

Lunar months

FIG. 6. The prevalence of grades of hepatomegaly in electively killed NZB mice. The upper limit of normal liver weight has been defined as 1405 mg. in virgin females, 1514 mg. in breeder females, and 1580 mg. in males.

The most consistent histological change in the liver is the accumulation of large amounts of hemosiderin in the Kupffer cells, and pigment stones commonly occur in the gallbladder of animals with florid active hemolytic disease. Focal areas of hepatic necrosis are seen occasionally in animals with strongly positive antibody tests and severe anemia. These areas are sometimes quite extensive and involve large segments of liver lobes. They have the appearance of areas of ischemic necrosis, and it has been suggested that they may be related to the occurrence of autoagglutination of erythrocytes in the hepatic sinusoids. The occurrence of areas of hepatic necrosis may cxaggeratc the degree of clinical jaundice terminally. ‘The occurrc~~icc of Kupffer cell hyperplasia and local lymphoid infiltrations in the ceiitrilobular and periportal regions of the Iiver lobules has been described by de Vries and Hijmans (1967).

THE IMMUNOLOGY AND PATHOLOGY OF NZB MICE

22s

B. Correlrtion of hlarkers of Heinolytic Disctise. The most florid markers of hemolytic disease are seen in t h c b virgin female (Fig. 7 ) . Autoantibody production commences in a few animals of this group at 3 months of age and affects all animals by 18 months. The appearance of an anemia of a hemolytic type occurs within 3 months of autoantibody production. By this time a strongly positive antiglobulin test is present 100 - U A u t o o n t i b o d y O--OHT

3

below 40%

6

9

12

15

18

21

24

Lunar months

FIG. 7. The prevalence of markers of hemolytic disease in electively killed virgin female NZB mice. (HT = hematocrit.)

and hepatosplenomegaly is starting to develop. In males the development of autoantibody and anemia occurs at much the same time as in virgin females, but the development of splenomegaly is delayed 23 months after the development of anemia. In breeder females the appearance of autoantibody starts in some animals about the same time as in virgins but reaches its maximal incidence and strength much more slowly. Also the appearance of splenomegaly is significantly delayed.

3. Renal Disease Renal disease, with an insidious onset and a fluctuating subclinical course, occurs in virtually all NZB mice. This has been described by Helyer and Howie (1963b), Howie and Helyer (1965), Howie et al. ( 1967), Holmes and Burnet ( 1963a), Mellors ( 1965, 1966a,b,c,d), Hicks and Burnet (1966), Miyasato et al. (1967), and de Vries and Hijmans ( 1967). It commences in some mice as early as 3 months of age, and in the early stages is associated with some degree of proteinuria and cylindruria. It remains clinically latent for the greater part of the lifespan, although some animals develop a type of nephrotic syndrome with ascites, hypoalbuminemia and hypercholsterolemia. Others develop a

226

J. B. HOWIE AND B. J . HELYER

inore active type of rcnal synclromr~of acute or subacutc renal failure very similar to tliat seen in the NZH x NZW hybrids. It has been suggested by Holmcs ancl Huriiet (1963a) nnd by Mellors (1965, 1966c), that the most common cause of death in adult NZB mice is chronic renal failure, and certainly a good proportion have evidence of renal impairment at death. However, this is usually associated with one or more other aspects of the disorders which occur in the strain. MelIors (1965) has distinguished two types of renal disease in these mice-chronic membranous glomerulonephritis and a lupuslike nephropathy. It is doubtful whether these may be identified readily during life, although the terminal course of the two syndromes may show distinctive characteristics. a. Clinical Patlzolugy. Mellors (1965, 1966c,d) has indicated that significant proteinuria (2+ to 3 + ) occurs rarely during the first 7 months of life and not prior to 4 months of age. He states that at 11 to 13 months, 57%of females have significant proteinuria, and a rare female has massive proteinuria. By 11 to 13 months, 22% of males have significant proteinuria. In the authors’ experience of more than 1000 electively killed animals, proteinuria is first seen in animals of 2 to 3 months of age, and its incidence in healthy animals throughout the life-span of the strain is shown in Table I. In males, proteinuria of 100 mg.3 or more occurs TABLE I THEINCIDENCE OF PROTEINURIA IN ELECTIVELY KII.LED NZB MICE Proteinuria (%) 100 mg.-% o r inore

300 mg.-% or more

Mic.e

3-12 months

12 months plus

3-12 months

12 months Plus

Males (438) Virgin females (270) Breeder females (220)

46.1 7.4 29.2

47.4 32.1 24.4

9.6 0 3.8

8.1 16.5

7.7

in 50% of all animals beyond 3 months of age and does not increase in incidence with age. Levels of proteinuria of 300 mg.3 or more are much less common but are still evenly distributed throughout the life-span. Breeder females show a lower incidence of proteinuria but as in males, this is evenly distributed in animals of all ages. Contrastingly, in young virgin females, proteinuria is uncommon and reaches only 7.4%in the

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227

first 12 months of life but increases to 32.1%in the subsequent half of the life-span. In more than half of these the proteinuria is 300 mg.-%or more. At death in terminally killed animals, azotemia with blood urea levels above 80 mg.-% is present in 55.8% of virgin females, 41.7%of males, and only 23.5%of breeder females. In each group the animals with renal failure are distributed throughout the death incidence curve. It would appear that of the three groups of animals, the virgin females are the most severely affected by the renal disorder and that, in females, reproductive activity in some way alters the pattern and diminishes the acuity of the renal dysfunction. The urinary proteins excreted in membranous glomerulonephritis (Mellors, 1966d) are mainly serum albumin, but with progression of the renal disease also include al-,p-, and yglobulins. Tests for occult blood in the urine are usually negative (Mellors, 1966d), but are positive in about 5% of the NZB females in which the disease runs a fulminating

FIG. 8. A glomrrulus i n iiii NZR f c . ~ r i i i l e iiioiiw showing tmtlotlirlial and mesangial cell proliterntion, n ~ i dmininial iiicsaiigial liyaline changes. ( H & E, mignification: X 900.)

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J. B. HOWLE AND B. J . HELYER

FIG. 9. A glomerulus in an NZB female mouse showing moderate hyaline changes in the mesangium and relative sparing of the peripheral capillary loops. ( H & E, magnification: X 780.)

course resembling lupus nephritis. Cylindruria, with both hyaline and granular casts and lipid bodies ( Mellors, 1966d), accompanies the proteinuria. b. Renal Histopathology. Three types of gross change have been described in NZB kidneys (Howie and Helyer, 1965). In a relatively small proportion of animals which appear to develop particularly severe hemolytic disease and where death results principally from profound anemia, the kidneys are chocolate-colored and of normal dimensions and texture. These show abundant hemosiderosis of the proximal tubules, and only minimal glomerular changes may be seen. More commonly the kidneys are enlarged and pale and show what has been described as chronic membranous glomerulonephritis (Mellors, 1965). This is associated with the appearance of hyaline material in the mesangial region and basement membrane of the glomerular tuft. This appears first in some animals between 3 to 4 months of age and may be associated with mild focal areas of endothelial and mesangial cell proliferation (Fig. 8 ) . The hyaline change appears to localize particularly in the mesangial

THE IMMUNOLOGY AND PATHOLOGY OF NZB MICE

229

FIG. 10. A glomerulus in an NZB female mouse showing advanced hyaline obliterative changes. ( H & E, magnification: X 470.)

region and, at first, spares the more peripheral capillary loops of the tuft (Fig. 9). It gradually extends to produce an irregular nodular subepithelial beading and thickening of the basement membrane of the loops ( Mellors, 1965) and progresses to hyalinization and disorganization of the whole glomerular structure ( Fig. 10). This predominantly mesangial lesion is not associated with capsular cell proliferation or fibroblastic obliteration of the capsular space, and focal necrosis and wire loop formation are not features. The hyaline material is strongly periodic acidSchiff (PAS) positive and, although in the more advanced stages it bears a superficial resemblance to amyloid as seen with conventional histological stains, it does not give characteristic staining reactions, and the architectural distribution in the glomerulus is not typical. The localization of the hyaline material a s diffuse and focal homogeneous expansions of tho lxisement inc~inl~rnnc~ has lwc?n confirmed by c~lcctron microscopy ( Mcllors, 1965; I licks ;111d Htlrnct, 1966; ‘l~iornson et ( I / . , 1967) and described as “basement-niembraiie-like” material. A striking feature of this hyaline glomerular disorder is that it may develop to a

230

J. B. HOWIE AND B. J . HELYER

remarkably advanced degree of obliteration and structural disorganization with little clinical disturbance beyond a mild to moderate proteinuria. The extent of the histopathological change is out of all proportion to the interference with renal function. In this type of glomerulonephritis, Mellors (1965, 1966a) has shown by immunofluorescent techniques that mouse immunoglobulins are extractable and can be recombined with extracted autologous glomeruli or isologous kidneys showing comparable glomerular disease. These extracts, however, do not recombine with normal isologous glomeruli, suggesting that the immunoglobulin may be deposited in the kidney in the form of antigen-antibody complexes rather than directly fixed to normal glomerular antigenic structures. In some animals, particularly virgin females, the kidneys are dusky pink or cafk au lait in color, and may be normal in size, slightly enlarged, or contracted. They sometimes show numerous subcapsular petechial hemorrhages and are finely granular. This type of renal change appears to develop much later in life than the membranous glomerulonephritis and shows histological changes typical of lupus nephritis. These include focal endothelial cell proliferative changes, foci of fibrinoid necrosis of the basement membrane, and capsular cell proliferation. They are identical with the renal lesions uniformly seen in the NZB x NZW hybrid which will be described in detail subsequently. In both types of lesions there is widespread involvement of glomeruli, although the most florid lesions are seen in the juxta-medullary glomeruli. Extensive lymphoid aggregations with abundant plasma cells and Russell bodies occur about the interlobular arteries, and fibrinoid necrotic lesions of the smaller renal arteries and arterioles are seen (Hicks, 1966), particularly in the granular, lupus type of kidney. The tubules show abundant casts, and, in advanced stages of renal disease, tubular atrophy and interstitial sclerosis become apparent. No account has been published correlating the clinical pathology of the urine and the renal histopathology with age and sex groups of the animals, and it would appear on the present data that the extent and progression of the renal lesions is not readily predictable during life in the majority of animals. c. Antinuclear Antibodies. Reports on the occurrence of antinuclear antibodies in NZB mice have shown considerable variability. LUPUS c.rythematosus ( L E ) cc4 tests were positive in 5% of mice at death ( Helyer and IIowie, 196311), and 6%ot adult NZB mice ( Mellors, 1966d). Miyasato et ul. ( 1967), using an indirrct LE cell technique, reported 14%positive tests in mice over 12 months of age. Antinuclear factors were

THE IMhiUNOLOGY AND PATHOLOGY OF NZR MICE

231

detected by an immunofluoresc.eiit technique in 6%of animals by Mcllors (1965, 19660) and in 28%of mice over 40 weeks of age by Miyasato et ~ 2 .(1967). Holborow et nl. (196s) demonstratd antinuclear antibodies in 20% of mice between 24 to 48 weeks of age but noted a higher incidence in an independent strain of mice without autoimmune disease. Using a different immunofluorescent technique, Norins and Holmes (1964a) demonstrated antinuclear antibodies in 42%of NZB mice but also found positive tests in 85%of healthy outbred mice. Using a latex nucleoprotein slide test (Hyland), East et al. (1965) found only a positive reaction in 1 sick female aged 51 weeks of 45 mice tested between 11 to 59 weeks of age. In the authors’ series of electively killed mice, positive latex nucleoprotein tests were only very occasionally positive in healthy animals but reached levels of 16.7%in females and 5.4% in males, killed terminally. The studies of Holborow et al. (1965) give an indication of the incidence of antinuclear factor according to the age of the animal and show that antinuclear factor does not appear to correlate with antierythrocyte autoantibody. No comprehensive study has been published relating antinuclear antibodies to renal disease in the NZB mice. In the authors’ experience antinuclear antibodies are not commonly present until the later stages of the life-span and are most common in virgin females. It would appear very likely that they correlate more satisfactorily with the more active and terminal stages of the renal disease.

4. Lymphoproliferutiue Disorders a. Hyperplasia. All authors who have described the basic features of the NZB strain, have noted the occurrence of marked hyperplasia of lymphoid structures such as in the spleen and lymph nodes. It also occurs in the lymphoid tissue in the lnngs, kidney$, liver, and bone marrow. The changes have been studied in some detail by East et al. (1965), who have described two phases of lymphoproliferative change. The first takes place over the first 3 to 11 months of life and consists primarily of the extensive development of large lymphoid follicles, containing multiple germinal centers in the white pulp of the spleen and the cortex of lymph nodes. These changes may be seen even in the absence of positive autoimmune markers. They gradually shade into a second phase of extreme hyperplasia of plasma cells and reticulum cells in the medullas of both spleen and lymph nodes and lymphoid tissue elsewhere. This phase is consistently associated with overt autoimmune disease. Burnet (1962a,b), Burnet and Holmes (1962, 1964a,b), and Holmes and Buniet (1963a) were the first to describe the occurrence of lymph-

232

J. 13. HOWLE AND B. J. HELYER

oid hypc.rplusin and lymph follicle formation ill the thymic medulla. In these puhlications they recorded a 1 er!. high incidence of such changes particularly in female niictl and indicated that they First a p p c ~ e d between 3 to 9 months of agc. Thcsc authors also cmphasizcd an absence of Hassall’s corpuscles. They postulated that the thymus was primarily involved in the initiation of the autoimmune process as the source of “forbidden” autoimmune clones of cells, although it was admitted that the occurrence of thymic lesions did not correlate with the development of markers of autoimmune disease. Bumet (1965) also reported that mast cells are more numerous in the NZB thymus glands than in standard strains, but the significance of this observation is not clear. East et al. (19%), Siegler ( 1965), and subsequently de Vries and Hijmans (1966, 1967) confirmed the presence of focal proliferative changes in the thymic medullas of NZB mice and interpreted them as being part of a widespread lymphoproliferative change. Contrary to Burnet’s report, all three groups described the presence of Hassall’s corpuscles. East et al. (1965) also noted that there was no correlation between the changes in the thymus and the incidence of overt autoimmune disease, and Siegler (1965) described similar histological changes in the thymus glands of noninbred Swiss albino ICR mice which do not develop autoimmune phenomena, Although lymphoproliferative changes occur in the thymic medullas of NZB mice as well as in other lymphoreticular structures, it is unlikely that Burnet’s thymic medullary follicles are of primary pathogenic significance. However, de Vries and Hijmans (1966, 1967) have drawn attention to cytopathic changes and a reduction in number of the medullary epithelial cells in the thymus glands of NZB mice in the first few weeks following birth. These they interpret as being consistent with Burnet’s concept of forbidden clones of lymphoid cells being eliminated in the thymus, and these views are in general agreement with those of East et al. (1965), who suggest that in NZB mice there is thymic dysfunction with inadequate thymic control of proliferation of lymphoid cells. In this respect the latter authors propose that NZB mice are immunologically hyperactive. Diener ( 1966) and Playfair ( 1967) have shown that young, Coombs-negative, NZB mice showed an enhanced response to antigenic stimulation by foreign red cells as an antigen. Diener also showed that this diminished with the advent of erythrocyte autoantibody production and particularly so in relation to increasing splenomegaly. Similarly, an increased susceptibility to Salrnanellu infection has been demonstrated by Kaye and Hook (1964) in NZB mice with established heinolytic disease.

THE IMMUNOLOGY AND PATHOLOGY OF NZB MICE

233

Studies on changes in thymic weight (East et d.,1965; Siegler, 1965; Miyasato et ol., 1967) have not suggested any striking differences from weight patterns of control strains. In thc prescwt authors’ material, there has been a definite tendency for the decrease in thymic weight associated with increasing age, to show a sustained arrest commencing at about 6 months, which correlates with the period when medullary hyperplasia is becoming apparent. b. Neophia. There is considerable variation in the reported incidence of lymphoid neoplasms in NZB mice. Bielschowsky and Bielschowsky ( 1962) reported an incidence of 5 in 137 animals ( 4 in 76 males and 1 in 61 females). Helyer and Howie (1963b) reported an incidence of 6%of animals showing lymphadenopathy at postmortem, of which onethird were considered to be definitely neoplastic. Holmes and Burnet (1963a) reported an incidence of “hyperplasia or tumour” in 18 of 55 males (37%)and 49 of 99 females (49.5%). East et al. ( 1965) reported an incidence of 7 thymomas in relatively young animals of a group of 43 mice (l6%), whereas Mellors (1966b) described 4 malignant lymphomas in 20 selected female mice between 9 to 11 months of age of which 2 were reticulum cell sarcomas, 2 “mixed-cell” tumors, and 1 a plasmacytoma. De Vries and Hijmans (1967) reported 7 lymphosarcomas in 35 mice of which 2 occurred in the thymus and 5 were extrathymic. Some of the variation is possibly due to the relatively small or selected nature of some of the series, but, as was clearly expressed by Holmes and Burnet (1963a) in their designation of hyperplasia or tumors, there is considerable difficulty and uncertainty in these mice, in distinguishing between profound hyperplasia as part of the autoimmune process and genuine neoplasia. Damashek ( 1965), in attempting to unify lymphoproliferative disorders, has pointed out that the lymphoid cell proliferation accompanying autoimmune disease may be a step toward lymphoid neoplasia, and Mellors’ mixed-cell tumor ( 1965, 1966b,d) may represent a series of intergrades between hyperplasia and genuine neoplasia. Bielschowsky and Bielschowsky ( 1962) showed that the incidence of lymphoid neoplasms in NZB mice could be increased fivefold by the administration of the carcinogen, 2-aminofluorene. Recently, Casey ( 1967, 1 9 6 8 ~ has ) shown that the administration of the immunosuppressive drug, azathioprine, to young NZB mice leads to the development of malignant lymphomas of lymphosarcomatous type. It could be that in NZB mice, the lymphoreticular system and the thymus gland in particular are unusually susceptible to carcinogenic influences and that variations in the environmental background of these influences in different laboratories, may be responsible, in part, for the varying incidences

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J. B. HOWIE AND B. J. HELYER

of neoplasms reported. Neoplasms unrelated to lymphoreticular structures are rare. c. Serum Proteins. In a strain of mice with autoimmune hemolytic anemia, renal disease with proteinuria, and with such marked immunoproliferative activity, it might be expected that considerable changes in serum protein pattern may take place. Mellors (1965) described an elevated total protein level in some NZB mice at 6 months of age and a subsequent increase in 7-globulin which usually persisted. He also noted that in addition yM-globulins were sometimes observed. Subsequently he reported (Mellors, 1966a,b) the presence in a small proportion of these animals of a hypergammaglobulinemia of unusual quality developing in association with mixed-cell lymphomatous neoplasms. East et al. (1965) showed a significant increase in al-macroglobulins in a small series of mice between 16 to 56 weeks of age. These increases were sustained throughout life, and bore no relationship to the presence of thymic lesions, lymphoid hyperplasia of follicular or medullary type, or the markers of hemolytic or renal disease. Kamm et al. (1966) in a more comprehensive study showed that NZB mice had elevated serum y-globulin levels at 3 and 9 months of age which increased further by 18 months. They found, however, that nonautoimmune control strains of mice had significant but less marked increases in 7-globulin with age, and pointed out that no data are available on the effect of increasing age on the serum proteins in mice. Considerable serum protein changes have been noted in those NZB mice which develop a form of nephrotic syndrome complicating their renal disease. In this respect Kamm et al. (1966) have noted that in older NZB mice, there is a relationship between y-globulin levels and the degree of proteinuria. d . Pulmonary Lesions. The most consistent and strikingly significant abnormality occurring in the lungs of NZB mice, is the peribronchovascular aggregations of lymphoid tissue. This occurs particularly in the hilar region and may be relatively sparse and infrequent in young animals, but beyond the age of 12 months, 80%of all NZB mice are affected. Histologically the infiltrate consists of lymphocytes of differing types, plasma cells, and frequent Russell bodies. It may be associated with a considerable degree of hyperplasia of the bronchial mucosa resulting in obstruction of the bronchial and bronchiolar lumina. Also as the infiltrate may be extensive enough to occupy a considerable volume of lung parenchyma, the lesion may cause marked respiratory embarrassment, The infiltrate is not associated with basement membrane changes, collagen necrosis, or sclerosis. It may represent a specific im-

235

THE IMMUNOLOGY AND PATHOLOGY OF NZB MICE

munological reaction to constituents of pulmonary tissue or, alternatively, it may just be a pulmonary component of the general immunoproliferative changes characteristic of the strain.

5 . Peptic Ulceration Howie and Helyer (1965) originally reported that 15%of NZB mice develop chronic peptic ulcers and that in some instances these were complicated by gastrointestinal hemorrhage which may contribute to the death of the animal. In a more comprehensive study, Wynn Williams et al. (1967) reported that ulcers first appeared between 2 to 4 months of age and in healthy animals reached a maximum incidence of 80%by 24 to 26 months (Fig. 11). The overall incidence in moribund animals

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was 56.4%and rose with increasing age at death to 100%in mice dying between 24 to 26 months. The ulcers occurred most commonly immediately distal to the pyloric ring and had striking histological similarity to chronic peptic ulcers in man. Raised, circumscribed, mucosal plaques of hyperplastic glands also occurred in the duodenum. In both healthy and moribund nnimals, the distributioi~curve of prevalence of plaques (Fig. 11) preceded the curve for rilceration. This suggested some sequt*ntial relationship lwtwtwi the two lesions. The ulcers showed no correlntion with the markcrs of hcmolytic or renal disease and at present

236

J. B. ElOWlE AND 13. J. HELYM

the etiology and pathogenesis of the ulcers is uncertain. The ulcers enlarge considerably during the administration of corticosteroids to the mice, and such therapy may be complicated by severe gastrointestinal hemorrhage. As a model of chronic peptic ulceration the NZB mouse is virtually unique, and it is tempting to speculate whether or not autoimmunity plays some part in the pathogenesis of these lesions.

B. THE (NZB X NZW)F, HYBRIDS In their initial hybridization studies on the transmission of the autoimmune disorders of the NZB strain, Helyer and Howie (1961) described how the original pattern of hemolytic disease in the parent NZB mice was modified to one of florid renal dysfunction and failure in the (NZB x NZY)F, hybrids. These hybrid mice were found to develop changes remarkably similar to, if not identical with lupus nephritis. Later a further F, hybrid, NZB x NZW, was reported (Helyer and Howie, 1963a), which also developed lupus nephritis. Because of the short life-span of the female NZB x NZW mouse and its relatively uniform behavior, it offered considerable advantages as an experimental model in the study of autoimmune renal disease and has been studied subsequently by a number of workers. Papers on the natural course of the disease in the NZB x NZW hybrid have been published by Howie and Helyer (1965), Burnet and Holmes (1965b), Dubois et al. ( 1966), Howie et al. (1967), Miyasato et al. (1967), and de Vries and Hijmans ( 1967). No further work on the NZB x NZY hybrids has been reported in the literature, so that the (NZB x NZW)F, hybrids will be used to illustrate the characteristics of the lupus nephritis hybrids. Within the limitations of the different numbers of animals used and different methods of study, the published accounts of the NZB x NZW mice are remarkably uniform. In Dunedin the authors have studied the natural history of the disease in 500 females and 157 male animals by both life-span and elective killing programs. The following account is based largely on these studies, some of which have been published recently (Howie et al., 1967). 1. Survival and Body Weight

The life-span of both male and female NZB x NZW hybrids is considerably reduced ( Fig. 12 ) , the iirithinctical incan survival time for virgin females being 279.8 days, a i d for inales 438.8 days. Both the virgin and breeder females sliow a strikingly iiniforni behavior compared with the NZB strain, and 87%of animals die within a 6 months period.

"7

T l l E lh4MUNOLOGY AND I'ATIlOLO(~Y OF NZH MICE

The curve for breeder females shows a virtually identical shape but is shifted 2 months toward an older age period. In males the distribution is less uniform and has a bimodal form suggesting the presence of two main groups, one of shorter survival comparable with breeder females, and the other significantly longer. The average body weight curves (Howie and Helyer, 1966) reach a maximum by 8 months in both males and females. However, in females and less frequently in males, edema and serous effusions complicating renal failure, may mask the true body +Male

R

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- virgins

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FIG. 12. The distribution of age at death in (NZB X NZW)F, hybrid mice. (Froni Howie ct uZ., 1967, with pennission of S. Karger, Basel.)

weight and result in a continued gain in weight until death. The survival times of reciprocal crosses are identical, and no significant differences in the patterns of the disease processes have been detected. The relatively uniform behavior of NZB x NZW virgin females makes them most satisfactory models for studying the incidence of various laboratory markers accompanying their disease processes, and they have been used to illustrate the temporal prevalence and interrelationships of these variates in the following sections. 2

RLJ)ILI/ Diwciae

a. iltitiirrcc.lcwr Z~cirtor.~.Tlrcsc ~ ~ 1 1lw 1 first detected between 2 to 4 months in a small proportion ot animals, either by antinuclear immuno-

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J. B. I-IOWIE AND B. J. HELYER

fluorescent or by latex nucleoprotein flocculation techniques, and reach 100%prevalence by 12 months of age (Fig. 13). The distribution of prevalence of positive tests by the two techniques is very similar. The LE cell test does not give positive results until 2 months later, and, although the actual prevalence does not rise above virtually all

a%,

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Lunar Months

The prevalence of antinuclear factors in female (NZB X NZW)FI hybrid mice. (N.P. = nucleoprotein, L.E. = lupus erythematosus, and A.N.F. = immunofluorescent antinuclear factors.) (From Howie et al., 1967, with permission of S. Karger, Basel.)

animals have positive tests at some time during their life. This delay in onset and the lower incidence of antinuclear factors measured by the LE cell technique may be indicative of a different type of antinuclear factor but, alternatively, may just indicate a lower level of sensitivity of the test. In males, not only does antinuclear factor develop 2 months later, but it also has a slower buildup in strength. b. Renal Dysfunction and Failure. Significant mild proteinuria with cylindruria first occurs in some female NZB x NZW hybrids between 4 to 6 months of age, and within 1 to 2 months exceeds 100 m g . 3 and may exceed 1000 mg.-%. At the higher levels, it is closely associated with the commencement of renal failure (Fig. 14). The curve for proteinuria resembles that for antinuclear factors but is 2 months later and is virtually identical with that for strongly positive immunofluorescent antinuclear antibody (Fig. 1 5 ) . In males, proteinuria has the

THE IhfXlUNOLOGY AND PATHOLOGY OF NZB MICE

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FIG. 15. The prevalence of inimunofluorescent antinuclear factor and proteinuria in female ( NZB X NZW )Fl hybrid mice. ( A.N.F. = antinuclear factors.) (From Howie et d.,1967, with permission of S. Karger, Basel.)

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1. B. HOWIE AND B. J . HELYER

simc nssoriatioii with strongly posiitivc i~iitinucle;ir factor aud, c'onscquently, appears 3 to 1 months later than in fcmalrs. In the majority of females, renal failure progresses rapidly, with blood urea levels rising steeply often in excess of 250 mg.-%.Death soon supervenes in females, but in males there is normally a significant interval between the development of azotemia and death. Indeed, a proportion of males continue to live in a relatively slowly progressive state of renal decompensation, and this partly accounts for the prolonged tail of the male survival curve. In addition to the occurrence of abundant hyaline and granular casts throughout the course of deveIopinent of the renal disease, red cells may appear in the more terminal stages. Sharard (1967), in an immunoelectrophoretic study of the urinary and serum proteins of the NZB x NZW mice in animals with heavy proteinuria, reported that a,-,/I-, and 7-globulins occurred in the urine in addition to albumin. This is in agreement with the immunofluorescent studies of the glomeruli of these mice by Nairn et al. (1966) and McGiven and Hicks (1967) who described accumulations of albumin, a l - , /I-, and 7-globulin, and a possible component of mouse complement in the glomerular lesions. During the course of the development of the renal disorder, a proportion of animals develop edema and the clinical features of the nephrotic syndrome, and Dubois et al. (1966) reported 18%of animals with gross ascites. In addition, hypoalbuminemia, elevation of serum a,-globulin, and hypercholeqterolemia were recorded by these authors. Miyasato et ul. (1967) recorded a marked increase in ,&globulin level in older mice. c. Hematologicul Changes. Leukopenia and, in particular, granulocytopenia occur along with the development of the renal disorder. Positive antiglobulin tests occur in less than 50%of females and in a lesser proportion of males. These are characteristically transient and only persist for a significant period in a minority of animals. Anemia commences when azotemia first occurs and increases in association with advancing renal failure. Burr cells and mild reticulocytosis may develop as the anemia progresses, and, although an autoimmune hemolytic element may be present in some animals with persisting antiglobulin tests, the anemia is predominantly one of renal failure. d . Morbid Anatomical Changes. Striking changes are seen in the kidneys. These are often grossly enlarged, and typically are cafe' au hit in color with numerous subcapsular petechial hemorrhages. In the more acute forms of th'k dizease, the kidneys may be swollen and shiny but in

TIIE IhlMUNOLOCY AND PATHOLOGY OF NZB MICE

24 1

animals with loiig~rsiirvival. varying tlcgrcrs of griinularity dc~vclop which arc maxini;il in malr~swith partially cornpcnsatc~clrenal failure. Aarons ( 1964) first showed that iiiimunoglobulin was deposited in an irregular granular manner on the basement membrane of NZB x NZW

glomeruli, and this has been confirmed subsequently by Channing et al. ( 1965), Dubois et (11. ( 1966), Nairn et al. (1966), and McGiven and Hicks ( 1967). Glomerular immunofluorescence commences between 4 to Uornerular lmmunofluorescence

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FIG. 16. T h e prevalence of glomevular iniinunofluorescence, proteinuria, and azotemia in female (NZB X NZW)F, hybrid mice. (From Howie et al., 1967, with permission of S. Karger, Basel.)

6 months of age, and its prevalence rises steeply in close relation to the slope of prevalence of antinuclear factor. As is shown in Fig. 16, this precedes proteinuria and strongly positive immunofluorescence, and coincides with the development of azotemia. The structural changes have been described in varying detail by Helyer and Howie (1963a), Howie and Helyer (1965), Burnet and Holmes (1965b), Hicks and Burnet ( 1966), and Miyasato et al. ( 1967). The histological sequence of events in the development of the renal lesion has been described by Howie et nl. (1967).

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J . B. HOWIE AND B. J. HELYER

The earliest histological changv is a glomerulitis which appears as focal areas of endothelial cell change in the peripheral capillary loops of the tuft and which may develop to form nests of endothelial cells distending and unfolding the individual glomerular lobules (Fig. 17). Inflammatory cells and karyorrhexis of nuclei may be present. This glomerulitis is associated with the deposition of immunoglobulin in a

FIG. 17. A glomerulus from a female (NZB X NZW)Fl hybrid mouse showing profound endothelial cell proliferation and no basement membrane changes. (Tripas, magnification: X 850.)

THE IhihfUNOLOGY AND PATHOLOGY OF NZR hIICE

243

fine granular line in the region of the baseincnt membrane and which consists of /3- and 7-globulin (McCivcn and Hicks, 1967). After an interval of 1 to 2 months, this progresses to a gloint.riilonephritis with focal and irregular liyaline thickening and deformity of the basement membrane (Fig. 18), which appears a s irregular coarse deposits of immunofluorescent material in relationship to the areas of thickening

FIG. 18. A glomerulus from a feiiiale (NZB X NZW)F, hybrid inouse showing endothelial cell proliferation, early fibrinoid change in the basement inembrane and some minor cilpstlliir adhesions. ( Masson, magnification: X 850. )

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B . HOWIE AND B. J. HELYER

FIG. 19. A glomerulus from a feiiiale (NZB X NZW)K hybrid iiiouse stained by imrnunoflnorescent technique, showing granular drposits in relation to the basement membrane. ( Magnification: X 890.)

(Fig. 19). The basement membrane changes may become more florid and clearly fibrinoid and necrotic over the next 2 months and involve portions or even the whole of the glomerular tufts. Such gross basement membrane changes when present tend to be associated with proliferation of capsular cells to form adhesions or crescents (Figs. 20 and 21). The gross basement membrane changes and the formation of crescents are represented by extensive, fusing masses of immunofluoresceiit material of coarse granular texture (Fig. 22). These have been shown by Nairn et al. (1966) to contain albumin, al-,8-, and y-globulin, fibrinogen, and a possible component of mouse complement. The fibrinoid-necrotic changes are more common in females than in males. In the latter the basement membranes show less fibrinoid change and are frequently more evenly hyaline. They tend to progress to wire loop formation with minimal involvement of capsular cells. Finally, the whole tuft may undergo disorganization and obliterative sclerosis, with consequent loss of immunottuorescence. It is of interest that only Dubois et al. (1966) have described the occurrence of hematoxylin bodies in the glomeruli, During the development of the glomerular lesions, tubular casts are universally present and are associated with PAS-positive droplets in the

‘HIE IMMUNOI,OGY AND PATHOLOCY OF NZB MICE

245

FIG. 20. A glonlerulrts of a female ( N Z B X NZW)F1 hybrid nlouse showing advanced fibrinoid change of the tuft and capsular cell proliferation. ( H & E, magnification: Y 850.)

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T. B. HOWIE A N D B .

J. HELYER

Frc. 21. A glomerulus of a female (NZB X NZW)F1 hybrid mouse showing fibrinoid change, capsular proliferation, and advanced disorganization. ( Tripas, magnification: X 850. )

epithelial cells of the proximal convoluted tubules. Both the casts and the epithelial cell droplets show immunofluorescenc'e. Animals may die at any stage along the sequence of development of the renal lesion and show varying combinations and grades of severity of the various changes. Also lesions of varying stages of development may be seen in the same kidney or even in the same glomerulus,

'IIIE IhIhlUI\C>LO(:Y A N D I'.i'lIIOLOGY OF NZFJ MICE

247

FIG.22. A glomerulus of a female ( N Z B X NZW) F1 hybrid mouse stained b y ininiunofluorescent technique showing granular deposits in relation to the basement membrane and a n extensive inass in relation to an area of fibrinoid necrosis. (Magnification: X 890.)

suggesting that the process inducing gIoinerular injury is both continuous and remitting. Chaniiing et al. ( 1965) reported electron-microscopic studies on 3 aging NZB x NZ\\' hybrids clrarly in advanced stages of the disease. Thcy describrd distortion : i d nurowing of cnpillary loops by rnesangial endothelial ~ n epithelial d cell swelling, and the deposition of electrondense materiiil within, arid 01 L both sides of the capillary basement mcm1)raiw. The. clinngrs \\"Y consiclcrrd to lw ic1cntic:al with those seen

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J. R. HOWIE AND B. J . IIELYEH

in human lupus nephritis. These findings wese confirmed by Hicks and Burnet ( 1966). More recently, McGiven and Lynraj-en ( 1968) have described glomerular deposits of electron-dense material, occurring on the intracapillary epithelial aspects of the glomerular basement membrane in NZB x NZW mice of 1 month of age. They showed that these were clearly present before the changes of both light and immunofluorescent microscopy could be detected. Necrotizing lesions occur in the medium-sized and small arteries of both NZB mice and NZB NZW hybrids. These lesions have been studied in some detail by Hicks (1966), who found them in the kidneys of 10% of NZB and 20%of NZB X NZW mice. He proposed that such animals would provide excellent models for the study of the pathogenesis of necrotizing arteritis. These lesions are found elsewhere in the body and may be readily seen in the myocardium, spleen, thymus, lymph nodes, pancreas, and gastrointestinal tract. They consist of diffuse or focal areas of “fibrinoid necrosis of the arterial wall and are associated with focal or circumferential aggregations of lymphocytes and plasma cells. Aneurysm formation may follow the disruption of the muscle coats. It is uncertain whether these vascular lesions represent areas of damage to the arterial wall analogous to that taking place in the glomerular basement membrane in lupus nephritis or whether they are related to the hypertension which has been shown to develop in a proportion of both these groups of mice (Hicks et al., 1965). It is important in this respect that in the present authors’ material, necrotizing arterial lesions in NZB X NZW mice occur with greater frequency in males than in females. They are seen in animals with less acute glomerular lesions, and whose disease runs a more prolonged course of compensated renal insufficiency. These animals might be considered more likely to develop a renal form of hypertension. e. Correlation of Features of Renal Disease. The sequence of clinical pathology tests in NZB x NZW females is shown in Fig. 23. Either antinuclear factor or glomerular immunofluorescence appears first and is associated with the development of endothelial proliferative activity in the glomeruli. Within 1 to 2 months, basement membrane thickening and deformity commence, and are associated with the commencement of proteinuria. As more severe basement membrane changes develop, proteinuria exceeds 100 mg.-W, and glomerular immunofluorescenw has \,ccome strong. At this stagc azotemia with progsessive anemia b c w ~ n e sapparent, and withiii :Lbrief period of tiine death superveues. I n inalcs, iir wddition to thc delay of ;it lcnst 2 inoiiths ill tlre onsc~tof the

x

TIiE IhlhlUNOLOGY AND PATHOLOGY OF NZB MICE

249

disorder, the rate of progress of the sequence of events is also significantly slower. f . Extrarenul Ckanges. Renal disease is the principal factor determining the survival of the lupus hybrids, and very few consistent extrarenal pathological features have been described. Burnet and Holmes ( 196513) have described conspicuous lesions in the thymus after 6 months of age characterized by active expansion of the medulla and aggregations of pyroninophilic cells in the cortex. These are similar to Giornerular lmmunofluorescence 0

80

lmmunotluorescent A N F

A Protelnurio

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m 0

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U I Q U

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2

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Lunar Months

FIG. 23. The prevalence of various clinical pathological tests and their relationship to the cuiiiulative incidence of death in (NZB X NZW)F, hybrids. (A.N.F. = antinuclear factors.) (From Howie et d.,1967, with permission of S. Karger, Basel. )

those described in the NZB strain but are more conspicuous and appear earlier in the females than in the males. Circumscribed aggregations of lymphocytes in the medulla of the same type as seen in the NZB mice are present but are considered to occur less frequently. These observations have been confirmed by Casey (1966) and Miyasato et al. (1967). They were extended by de Vries and Hijmans (1966, 1967) who described a striking hyperplasia of the large epithelial cells of the thymic medulla, increased numbers of Hassall's corpuscles, and invasion of these epithelial struc.tul-c>sby lymphoid cc.11.;. Tlwy notid that this latter plwiioinenoii oc.c.iiIb to ii I r r s i ~caxtciit iir ~ ~ o i i ~ t ~ ~ t o i i i i i n strains. unc. Splcnonicy& nntl g c w d i z 4 IyinpIi iiodi, c.iilargtmcnt is iincom-

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mon in NZB x NZW hybrids, although striking hyperplasia of the renal lymph nodes and large perivascular cuffs of lymphoid cells about the larger renal vessels are consistently associated with the renal disease. Dubois et al. (1966) has reported, in 8 of 33 animals, the changes of acute and chronic hepatitis with focal necrosis and polymorphonuclear and lymphocytic infiltration. They suggest that the changes are reminiscent of early human lupoid hepatitis. These changes have not been reported by other workers, and the present authors have not seen these liver changes in their material. C. THENZB MICEAND HYBRIDS AS EXPERIMENTAL MODELS The natural history of the autoimmune disorders of NZB mice is complicated and variable, because the health and survival of these animals is influenced by hemolytic, renal, immunoproliferative, pulmonary, and alimentary diseases. A thorough appreciation of the natural patterns of the prevalence of the markers of hemolytic disease, and of the complex influences of other disease syndromes, is essential if the NZB mice are to be used as experimental models of autoimmune hemolytic anemia. Both sex and reproductive activity greatly influence the mode of expression of these syndromes, and it is surprising to find reports in the literature in which no distinction is made between virgin and breeder females or even between males and females. Such reports have limited value. The hemolytic disease which the NZB strain develop, resembles remarkably closely the warm antibody type of autoimmune hemolytic anemia in man (Dacie, 1962), and it is the clinically dominant autoimmune manifestation during the life-span of the strain. All mice develop erythrocyte autoantibody in bound or free form, and the direct antiglobulin test has been widely used as the principal laboratory marker of the disease. Holmes and Burnet have used the term “Coombs conversion” to indicate the change from a negative to a positive test, and this is a reasonably reliable indication of the presence of autoimmune disease in the strain. Circulating antibody may appear before bound antibody can be detected, and variations in binding capacity may revert a “converted antiglobulin test to negative, so that in accurate studies of autoantibody production, antibody should be sought for in both bound and unbound forms. In contrast with the NZB mice, “Coombs conversion” in such lupus hybrids as NZB x NZW is a totally unreliable marker of the presence of autoimmune disease, because antinuclear antibody production is dominant in these animals, and erythrocyte autoantibody may be evanescent in occurrence or may even be absent.

THE IMMUNOLOGY AND PATHOLOGY OF NZB MICE

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Even within individual sex groups of NZB mice, there is marked variation in the actual age of the animal when autoantibody first appears, and during the life-span of many animals, anemia and splenomegaly may be absent or so mild that hemolytic disease has only a minimal influence on health and survival. Basic body weight exerts an important iduence on the character of antibody, on the level of anemia, and on the extent of hepatosplenomegaly, and these markers may show rapid changes during the period of deteriorating health preceding death. Consequently, in making comparisons among the markers of hemolytic disease in different groups of animals, the sex and reproductive history, the basic body weight, and the state of health must be matched. The temporal dynamics of some of the markers of hemolytic disease are now reasonably well documented, but much remains to be discovered regarding the sequence of the accompanying histological changes. The natural histories of the syndromes other than hemolytic disease are by no means complete, and the complex influences of these must be thoroughly understood if the NZB mouse is to be effectively used as an experimental model of autoimmune hemolytic anemia. MelIors (1965) suggests that the NZB mouse is a suitable experimental model of membranous glomerulonephritis, and undoubtedly the occurrence of autoimmune renal lesions during the course of the hemolytic disease is of great importance in the study of the autoimmune phenomena occurring in this strain. The renal disease is insidious and slowly progressive and cannot readily be predicted by laboratory tests during the major part of its course. With increasing age the renal lesions come to play a role of increasing significance in determining the survival and mode of death of the animal. This in itself limits the value of the animal as a model of hemolytic disease, because the clinical features associated with the course of the autoimmune disorders in these animals result from an infinitely variable mixture of hemolytic and renal components. Although the strain may be acceptable, particularly in the earlier periods of the life-span, as a model of autoimmune hemolytic disease, it has many serious shortcomings as an experimental model of autoimmune renal disease. Hicks and Burnet (1966) make no real distinction between the renal disease in NZB and in NZB x NZW mice and infer that they represent varying grades of the same process. Mellors (1965) distinguishes clearly the hyaline lesions seen in the majority of NZB mice as membranous glomerulonephritis, from the “lupuslike” nephropathy of the NZB x NZW hybrids and a small minority of NZB mice. Miyasato et aZ. (1967) make the same distinction as Mellors, but use the terms “glomerulitis”

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and “glomerulonephritis” indicating that they consider both lesions to be variants comparable with types of renal manifestations of human systemic LE as described by Pollak et al. ( 1964). There are good reasons at present for maintaining a distinction between the renal disorders of the two groups of animals. In the NZB mice the membranous glomerulonephritis commences early in life and insidiously runs its inexorable course of glomerular disorganization over much of the life-span of the animals. It is probably not associated with significant antinuclear antibodies and it induces minimal clinical disturbances, so that the extent of the renal disease present cannot be predicted during life, The lupus nephropathy when it occurs in the NZB strain is commonly grafted upon a background of chronic membranous change and is rarely seen in pure form. The two lesions are by no means mutually exclusive, In a considerable proportion of animals with advanced membranous change, focal areas of endothelial cell proliferation, fibrinoid necrosis, and capsular proliferative change occur terminally and may be associated with the concurrent development of antinuclear antibody activity. In contrast, the renal lesion of NZB x NZW hybrids is a pure form of lupus glomerulonephritis, and the hyaline membranous lesions, so characteristic of NZB mice are not seen. In these hybrids, the disease develops in close relationship to the presence of potent antinuclear factors, and the clinical and laboratory manifestations of the disease correlate closely with the renal changes. Thus, the course and extent of the disorder can readily be predicted from laboratory tests during life. The relationship between these two types of renal lesion, the hyaline lesion of membranous glomerulonephritis and the focal lesions of lupus nephritis, is not clear. They could be two independent processes, but it is also reasonable to suspect that they could represent the impact of a single immunological process of different grades of severity. In the NZB mouse the low-grade effect of this process may produce chronic hyaline membranous change, and only in the latter stages of the disorder may sufficient potential be gained in some groups of animals to induce overt lupus glomerulonephritis. In comparison, in the NZB x NZW hybrid, the potential for immunological damage must be much higher and reaches the tempo of lupus nephritis from the outset. The authors have no doubt that the female NZB X NZW hybrid is an excellent experimental model of systemic LE. This opinion may be criticized on the grounds that they do not develop serositis, arthritis or skin lesions and do not reproduce precisely the full range of expression of the human analog. The hybrid is derived from two highly inbred

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strains of mice and presents a high Ievel of genetic homogeneity which contrasts sharply with the Iieterogeneity of the human race. Tt has already lwen established 1)y liyhridizatioii tqwrimtmts ( Howit* and Helyer, 1965 ) that the varying rxpression of tlic autoimmune disorders in the NZB hybrids is influenced by their genetic constitution, so that it would be more appropriate to make a comparison between the variety of forms of human autoimmune diseases and those of an infinite range of NZB hybrids. In the NZB x NZW hybrid, the close correlation of the development of antinuclear factor with glomerular immunofluorescence and morphological changes in the glomeruli provides circumstantial evidence suggesting a pathogenetic relationship between antinuclear antibodies and the renal lesion, The presence of immunoglobulin in the glomerular lesions supports the proposal that the renal disorder is immunological in origin, and the granular nature of these deposits is consistent in appearance with the deposition of antigen-antibody complexes (Unanue and Dixon, 1967). These features, combined with the results of both histological and electron-microscopic studies make the resemblance of the disease in the NZB x NZW hybrid to human lupus nephritis quite remarkable. It is of great interest in this respect that Lambert and Dixon (1968) have shown that in (NZB x NZW)F, hybrid mice the typical renal lesions are characterized by the deposition in the glomeruli of 7G- and p,c globulins, together with deoxyribonucleic acid (DNA) and possibly other nuclear antigens. They showed that the YG-globulin in the glomeruli was largely rG2,-iype antibody to soluble nuclear antigen and concluded that the deposited material constituted antigen-antibody complexes deposited in a granular lumpy pattern along the capillary walls and in the mesangia. They were able to enhance the antinuclear response of young (NZB x NZW)F, mice and hasten the development of the glomerular lesions by active immunization with DNA-methylated bovine serum albumin. Furthermore, injection of soluble DNA into animals with minimal renal disease but with circulating anti-DNA antibodies caused a rapid progression of the glomerular lesions. Ill.

The Experimental Usage of the Mice

A. HYBRIDIZATION AND GENETIC STUDIES As has already been indicated, all animals of the NZB strain develop autoimmune disease. Fitzgerald (1962) studied the chromosomes of two adult male NZB mice and found them to be normal. He considered that

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the abnormalities of chromosome number and structure present did not determine the inheritance of this strain and that they were not associated with the pathogenesis of the hemolytic anemia. Two groups of workers have made hybridization studies to determine whether the disorder is genetically determined and to investigate the nature of the genetic differences responsible for the abnormality. In their initial studies Helyer and Howie (1961, 1963b) reported that, in NZB X NZY hybrids, the prevalence of erythrocyte autoantibody was irregular and inconstant. They showed that in these hybrids the clinical expression of the disorder had changed to renal disease, that positive LE cell tests were frequent, and that renal changes remarkably similar to lupus nephritis were invariably present. They subsequently reported on the NZB X NZW hybrids (Helyer and Howie, 1963a) in which a similar pattern of disease developed. Subsequently a number of NZB hybrids were described by Howie and Helyer (1965). Some developed a syndrome of hemolytic anemia which closely resembled that of the NZB parent, but in others the mode of expression of the disorder was in the form of lupus nephritis. In both classes of hybrid, the age of onset of the disorder and the average survival time varied from one group of hybrids to another. The disorders were not linked to coat color, and no differences could be demonstrated between reciprocal crosses so that the disorder was not sex-linked. They concluded that, although the autoimmune characteristics were expressed in the heterozygous state and were dominant, the expression was modified in the form of the disease, in the age of onset, and in the average survival time by the genetic contribution of the nonautoimmune parent. They reported that autoimmune disease also occurred in F, hybrids. Bielschowsky and Bielschowsky (1964) reported that in NZB x NZC hybrids the incidence of hemolytic anemia was 100%in F, mice and 74%in F, mice. They also noted that in reciprocal crosses of two of three groups of hybrids there was a reduction in fertility associated with the NZB male, which was related to a reduced number of implantation sites. Detailed genetic studies on hybridization of NZB mice with the C3H, AKR, C57BL, T6, and NZW strains have been reported by Holmes and Burnet (1964a, 1966a; Burnet and Holmes, 1965a). They confirmed the occurrence of autoimmune markers in the F, hybrids, and also the variable time and nature of the expression of the disorder. In studies with backcrosses between F, mice and NZB and nonautoimmune parents, they found that the autoimmune character was not segregated and the backcross mice were intermediate in character between the F, and the parent.

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The observations of Holmes and Burnet (1966a) on the NZB X AKR hybrids, with the complex interrelationships of the lymphomatous neoplasms of the AKR strain and the autoimmune disorders of the NZB strain, suggest an inverse relationship between the development of neoplasia and the development of autoantibody. In the NZB strain and in nearly all hybrids, the autoimmune manifestations are expressed earlier and are more severe in the females. The only hybrids described by Howie and Helyer (1965), in which autoantibody appeared earlier and was of greater strength in the male, were the NZB X NZCW mice. It is of interest that virtually all female mice of this parentage develop lymphoid neoplasms usually of the thymus (Howie and Helyer, unpublished observation). The studies on the genetic representation and transmission of the autoimmune disorders of NZB mice are by no means conclusive. Although the disorders would appear to be genetically determined, the mode of representation within the genome is not clear. Even if they are represented by a single gene, it is subject to considerable control in its mode of expression by the remainder of the genome. It also cannot be denied that the autoimmune process could result from the modification of the somatic genome by the incorporation of a virus. This would result in an inherited somatic change in which the virus genome was transmitted vertically by sperm as well as ovum, affecting hybrids of varying genotypes differently, and so inducing in the animals the different expressions of the autoimmune phenomena. OF THE DISEASE BY SPLEEN CELLS B. TRANSMISSION Holmes et nl. (1961) induced positive Coombs tests in young NZB mice by the injection of spleen cells from mice of 10 to 12 months of age with established hemolytic disease. The tests became positive within 3 weeks of inoculation and remained positive in a proportion of animals. In a subsequent publication, Holmes (1965) extended these observations and showed that cell suspensions from lymph nodes, thymus, bone marrow, and buffy coat failed to transfer autoantibody production to recipients. She also showed that the transfer occurred in splenectomized recipients and that prior immunization of the donors resulted in a secondary response in the recipients when the latter were stimulated by the original antigen. East et nl. (1965) failed to transfer the disorder with spleen cells, but the original findings of Holmes et a). (1961) were confirmed by Kaye and Hook ( 1904) and by Holborow et uZ. (1965). Lindsey and Woodruff ( 1967) have repopulated heavily irradiated, Coombs-positive, NZB mice with lymphoid cells from young NZB mice

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x

and F, (NZB CBA/T,) hybrids. They showed that mice receiving isogenic cells became Coombs negative and reverted to positive some months later, at a time predictable from the age of the donor cells. Those receiving nonisogenic F, cells became Coombs negative and remained so, and all dividing cells in spleen, lymph nodes, and 90% of those in the bone marrow were of donor origin. Mellors ( 1 9 6 6 ~ reported ) on the induction of membranous glomerulonephritis in young NZB mice, injected with splenic material from NZB mice with advanced renal disease. The changes were present 6-8 weeks after the transplant and, although only 1 of 6 animals showed a positive Coombs test, all developed proteinuria and typical renal changes. On the evidence of these experiments it would appear that immunologically competent cells have been transferred, which, in the recipients, have produced autoantibodies capable of inducing autoimmune disease. It has been suggested earlier that in the natural history of NZB mice, erythrocyte autoantibody production becomes established first and that antinuclear antibody production develops later in the more severe stages of renal disease. It is likely that the spleen cells of Holmes’ experiments were derived from mice in which erythrocyte autoantibody production was dominant and thus hemolytic disease was induced in the recipients. If it is accepted that antinuclear antibodies have a pathogenetic relationship to the renaI lesions of NZB mice, then in Mellors’ experiments, the splenic material selected from animals known to have advanced renal disease has probably been producing antinuclear antibodies and has induced renal disease in the recipients. Holmes et al. (1961) failed to induce positive Coombs tests in recipients when they used spleen cell suspensions which had been frozen and thawed 2 or 3 times and heated to 50°C.for 30 minutes. However, Mellors and Chen Ya Huang (1966) showed that a filtrable agent, derived from the transplantabIe malignant lymphomas of NZB mice, induced lymphoid cell hyperplasia, hypergammaglobulinemia, proteinuria, and membranous glomerulonephritis in preweanling NZB mice. The renal lesions were similar to those occurring spontaneously in older NZB mice. Virus particles resembling typical “C-type, murine, oncogenic virus were identified by electron microscopy in both the primary and transplanted tumors and in other tissues of both tumor-bearing and nontumor-bearing NZB mice. Recently, East and de Sousa (1966) and East and Prosser (1967) showed that serial intraperitoneal transfer of cell suspensions from enlarged spleens and lymph nodes of NZB donors to young NZB or nonisogenic BALB/ c mice, induced reticulum cell neoplasms of increasing

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virulence in the recipients. Coombs positivity was not induced. Viruslike particles resembling murine leukemia virus were detected in the tissues of both untreated NZB mice and recipients, but were found in the serum or plasma of the latter. The exact nature of the changes in the lymph nodes and spleens of the donor animals was not defined. In both the papers of Mellors and Chen Ya Huang (1966) and of East and Prosser ( 1967), the circumstantial relationship between autoimmune disease, malignant lymphoid neoplasm, and the presence of virus particles is of great interest but a pathogenetic linkage of the three factors as yet lacks proof. Helyer and Howie (unpublished observations ) attempted to transfer autoimmune disease to preweanling NZC mice by the intraperitoneal and intracerebral injection of cell-free filtrates derived from the enlarged spleens of NZB mice with established haemolytic disease. The injected mice failed to develop clinical pathological markers or morbid anatomical changes of autoimmune disease. In Dunedin, Middleton (personal communication) failed to isolate cytopathic agents or mycoplasmas (P.P.L.O.) from the thymus glands of one-day old NZB mice or from the ovaries or testes of mature animals. Middleton used test systems employing monolayer tissue cultures of CBA/ TG fibroblasts, primary human amnion cells and diploid human embryo fibroblasts, and P.P.L.O. agar plates. He also failed to induce viral synthesis by proflavine treatment and ultraviolet light irradiation of cell cultures derived from neonatal NZB thymus or adult NZB testis or ovary. C. THYMECTOMY AND THYMUS GRAFTS The thymus has been shown to have an important immunobiological function. The presence of germinal follicles and lymphoid hyperplasia in the thymic medulla of the NZB mouse (Burnet, 1962a,b; Burnet and Holmes, 1962, 1964a,b; Abbot and Burnet, 1964; Holmes and Burnet, 1963a, 1964a,b) was initially put forward as morphological evidence of the primary origin in the thymus of clones of immunologically competent cells responsible for autoantibody production. It was suggested that a causal relationship might exist between the thymic changes and the autoimmune disease. It would now appear to be more likely that the changes are part of a general lymphoid hyperplasia taking place in the spleen, lymph nodes, and elsewhere in the body. There is, liowcver, some experimental work on NZB mice and their hybrids wliich indicates that the thymus may play somc part in thc pathogenesis of autoimmune processes. Helyer and Howie ( 1963c; Howie and Helyer, 1966) reported that neonatal thymectomy within 24

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hours of birth in both NZB mice and NZB X NZW hybrids does not prevent the onset of the disorder and that following this procedure the autoimmune process develops precociously and with more acute manifestations, They also showed that the immediate grafting of a neonatal thymus of a nonautoimmune strain at the time of thymectomy failed to prevent the disease and that it restored the time of onset and the grade of severity of the manifestations to normal. It was concluded that if autoimmune processes were initiated in these animals, by the development of forbidden clones of immunocytes either within or under the influence of the thymus, then such clones must develop and undergo extrathymic dissemination during intrauterine life. Also during extrauterine life the thymus, either autoimmune or normal, appeared to exert at least some inhibitory influence on the development of autoimmune disease. East and de Sousa (1966) and East et al. (1967a) confirmed that neonatal thymectomy did not prevent the onset of autoantibody production in NZB mice and that it appeared in some instances to accelerate its onset, They also described the distinctive features of wasting disease as it occurs in NZB mice and emphasized the development of strikingly enlarged and active germinal centers in the lymphoid organs. High levels of IgM developed and persisted in the thymectomized animals so that the macroglobulinaemia characteristic of the strain was unaltered. In contrast, Holmes and Burnet (1964a; 1966b), although confirming that neonatal thymectomy does not prevent Coombs conversion in NZB mice, indicated that it induced 2 to 3 months delay in the onset of the change. Their animals were, in general, considerably older at thymectomy than those of Helyer and Howie ( 1 9 6 3 ~ and ) East et al. ( 1967a) and this may account for these divergent findings. Also as both Howie and Helyer (1966) and East et al. (1967a) have indicated, there is a very high preweaning loss and a high incidence of wasting disease in NZB mice, so that the surviving thymectomized animals are highly selected. Indeed, it is possible that thymectomy may select for survival animals that are fundamentally more robust or those which will develop autoantibody later in life than average. Holmes and Burnet (1964a) also demonstrated that isologous thymus grafts, to both thymectomized and intact NZB mice, developed characteristic medullary lesions. They also failed to show any acceleration of the onset of positive antiglobulin tests in both thymectomized and intact F,( NZB X C3H) hybrids grafted with NZB thymus glands. Helyer and Howie (1963~;Howie and Helyer, 1986) further showed that normal mice of a nonautoimmune strain neonatally thymectomized,

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and immediately grafted with thymus glands from newborn NZB mice or from NZB x NZW hybrids, tlevelop strongly positive antoimmune markers and renal changes characteristic of hipus nephritis. The neonatal thymus in autoimmune mice thus appears to be capable of transmitting the disease to normal mice. Such grafts may exert their autoimmune influence in a number of ways. It is unlikely that donor cells capable of autoantibody production could have been established from the graft ( Wakonig-Vaartaja and Metcalf, 1963; Miller, 1963). The grafted thymus may be incompetent and be unable to eliminate forbidden clones of cells from the host; or it may have induced the development of forbidden clones of immunocytes in the host by a positive humoral influence. The thymus in intact autoimmune mice may act as a primary source of dissemination of abnormal clones of immunocytes during some undefined period of intrauterine life. It is not, however, essential for the development of these clones during the postnatal period. During this period of time it appears to behave in much the same manner as a normal thymus in moderating the development and expression of the autoimmune disorder. The evidence of transfer of the disorder to normal animals by thymic grafts from autoimmune strains also suggests that the graft may harbor a transmissable viral agent. Evidence supporting humoral or viral transmission is put forward by Masters and Spurling (1967) who induced positive antiglobulin tests in normal mice by NZB thymus grafts enclosed in Millipore capsules. The observations that neither thymectomy nor a neonatally grafted normal thymus prevents the expression of the disorder (Howie and Helyer, 1966) would be consistent with the prenatal extrathymic dissemination of a virus. Furthermore the enhancing effect of thymectomy could result from the removal of a damping down or controlling influence of the thymus on a generalized established virus infection. These observations on the role of the thymus in the development of autoimmune disease in mice are far from conclusive, and their relevance to human disease is uncertain. It does, however, seem likely that beyond the intrauterine and neonatal period, the part played by the thymus in the pathogenesis of autoimmune disease differs little from that of lymphoid tissue generally in the body.

D. SPLENECTOMY Helyer and Howie (1963b) reported that splenectomy in adult NZB mice with established florid hemolytic disease and gross splenomegaly usually results in a rapidly progressive anemia and early death. In such

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animals the red cell survival would be profoundly reduced, and the grossly enlarged spleen is apparently an essential site of red cell production. In animals older than 6 months of age but without splenomegaly, the removal of the spleen had no significant effect, whereas in animals under 3 months of age the manifestations of hemolytic disease and the strength of autoantibody were diminished. Holmes and Burnet (196313) showed that splenectomy between 1to 4 months of age delayed Coombs conversion and other manifestations of hemolytic disease in males but not in females. They demonstrated, however, that the incidence of severe renal disease was increased in both sexes but that this reduced the survival time significantly only in females. They also confirmed that once the hemolytic disease had become established, splenectomy had no significant influence on the pattern of the disorder. Burnet and Holmes (1965b) reported that splenectomy at 8 weeks in NZB x NZW hybrids had no influence on the mortality of the animals. No studies on neonatal splenectomy in either group of animals have been reported.

E. THERAPEUTIC STUDIES It was noted by Helyer and Howie (1963b) that the administration of adrenocorticotropic hormone ( ACTH) to NZB mice with established hemolytic disease induced a striking clinical remission. This was accompanied by a reduction in the level of anemia and reticulocytosis, in the strength of autoantibody, and a marked reduction in the size of spleen and liver and in the degree of hemosiderosis. A relapse occurred when the treatment was stopped. The same effect was reported in greater detail by Casey and Howie (1965) using the corticosteroid drug, betamethasone phosphate, and by Giltinan et uZ. (1965a) using cortisone acetate. The latter group also indicated that the therapy prevented the development of autoantibody in younger mice, but that the susceptibility of the treated animals to infections precluded any comment on the influence of the treatment on the incidence of renal lesions. Casey (1968a) reported that 6-mercaptopurine administered to NZB mice had no beneficial influence on the development of autoantibody in young animals or on its strength once hemolytic disease was established, The drug had a serious deleterious effect on erythropoiesis in animals with established hemolytic disease causing bone marrow depression and a rapid and profound exaggeration of the level of anemia. Russell et aZ. (1966) and Russell and Hicks (1968) reported that the development of the renal lesions in NZB X NZW mice could be suppressed by the administration of cyclophosphamide. This resulted in a

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greatly increased life-span of the animals. When treatment was started before the development of the nephritis, the renal lesions were markedly suppressed but not entirely prevented. Also, in animals with established disease the renal changes were arrested and were seen to undergo healing but did not resolve. Casey (1966, 1968b) reported that both the activity and severity of the renal lesions in NZB x NZW hybrids were reduced by the use of large doses of betamethasone phosphate. This was accompanied by a reduction in incidence of albuminuria and of positive tests for antinuclear factor. Casey (1967, 1 9 6 8 ~ has ) reported the effects of azathioprine (Imuran) on both NZB mice and NZB x NZW hybrids. There was marked suppression of the renal lesions in the hybrids, but in NZB mice the drug did not prevent the onset of hemolytic disease or have any beneficial influence on the severity of anemia or on the antibody level once hemolytic disease had been established. However, in both groups of animals, the administration of the drug was associated with a strikingly high incidence in both sexes of highly malignant thymic neoplasms, some of which were associated with leukemia. Finally in view of the proposition by Hicks and Burnet (1966) and Hicks (1966) that a major component of the glomerular and vascular lesions of both NZB and NZB X NZW mice was the deposition of fibrin, McGiven (1967) treated young NZB x NZW mice with the anticoagulant, warfarin. He failed to show any influence of this drug on the incidence or nature of the renal histopathology. The results of the therapeutic use of corticoids in the autoimmune strains of mice are very similar to those observed in human autoimmune hemolytic anemia and lupus nephritis, To date the immunosuppressive agents such as cyclophosphamide and azathioprine have been remarkably effective in influencing the development and course of lupus nephritis in the NZB X NZW hybrids but surprisingly ineffective in influencing the course of hemolytic disease in the NZB strain. The value of these two groups of mice as model test systems for studying the efficiency and mode of action of therapeutic agents is apparent. The effect of betamethasone or cortisone provides a standard for measuring the effect of alternative therapeutic agents such as antimetabolites and other immunosuppressive agents, used either individually or in combination with corticosteroids. IV.

Concluding Remarks

It is nearly a decade since the NZB strain of mice and their lupus hybrids were described, and time has confirmed that it was a reasonable conclusion that they suffer from a basic disturbance of their immuno-

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logical mechanisms. Much is already known of the natural histories of the two groups of animals, although many of the disorders seen in the NZB stock are even now, incompletely described and understood. It became apparent at an early stage that variations in the hormonal environment, resuIting from differences in sex and breeding activity, influenced the pattern of disease, probably by exerting some control over the disordered immunological activity. These are important natural modifying influences. Both groups of animals have been used as experimental models. Although the majority of workers have elected to use the NZB strain, in the authors’ opinion, the NZB x NZW hybrid strain with its hybrid vigor, its clear-cut laboratory markers, and its accurately predictable pattern of disease, is a much more satisfactory model. The two groups of animals represent two rather different types of autoimmune disorder, so that from the viewpoint of experimental immunology they are complementary, and the use of both would provide a broader approach to many of the immunological problems being studied. It was shown that the autoimmune trait was inherited and that the details of inheritance were undoubtedly complicated. Although the disorders are transmitted in a dominant manner to hybrid progeny, the form of expression is modified by the genetic characteristics of the nonautoimmune parent. The lupus hybrids were an unexpected bonus from the hybridization studies. Transfer experiments showed that the disorders could be transmitted by isogenic grafts of immunologically competent cells from the spleen, thus identifying such cells as the executive clones responsible for, and irreversibly determined on, autoantibody production. Also postirradiation repopulation experiments have shown that autoantibody production ceases when these forbidden clones are replaced by immunocytes from Coombs-negative animals. As yet there is no acceptable explanation of just how self-recognition fails in these mice and what sort of disturbance results in autoantibody-producing clones being selected and permitted to proliferate. The recent paper of East et al. (1967b), describing how the development of autoantibody and the level of serum immunoglobulin were unaffected in germfree NZB mice, at least indicates that isolation of the strain from exogenous antigenic stimuli has no influence on autoantibody production. A burgeoning interest in the central immunological role of the thymus was developing about the time that the first reports on the mice appeared, and it was inevitable that a keen interest should have been taken in the morphology and function of the NZB thymus. The finding of IymphoreticuIar hyperplasia in the thymic medulla developing con-

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currently with the earliest development of positive autoimmune markers led to the conclusion that the thymus was the primary source of the forbidden clones. A more thorough study of the evidence did not support this hypothesis. a i d the incdiillary changes have now been relegated to a status in common with thc lymphoid hyperplasia developing elsewhere in the body of these animals. On the evidence of thymectomy and thymus grafting, the postnatal influence of the thymus on autoimmune disease may be regulatory or inhibitory, but not promotional. The prenatal and perinatal activities of the NZB thymus have not yet been studied adequately. There is some evidence of morphological abnormalities of the reticular cells in the thymus glands of these mice during the neonatal period. If this organ is the center for the primary generation and subsequent dissemination of rogue clones of immunocytes, this must take place prenatally. Thus the neonatal morphological changes may represent the continuing effects of prenatal disturbance rather than the initial stages of thymic dysfunction. There is clear evidence from grafting experiments that the neonataI autoimmune thymus has the capacity of transmitting or inducing autoantibody-producing cells in normal recipients. However, it is not certain whether this is by cellular transmission or by an inductive or instructive humoral influence. Although the evidence of induction of autoimmune disease by autoimmune thymus grafts has been considered from an immunological viewpoint, the transmission could be brought about by a virus. Initially the possibility that the autoimmune disorders of the NZB mice could be viral in origin was considered to be unlikely, although the example of the virus-induced arteritis of Aleutian mink (Henson et al., 1963; Padgett et al., 1967) was a reminder that this group of etiological agents should not be dismissed. The transplacental or transmammary transmission of such an agent was ruled out by the occurrence of the disorder in identical fashion in both sets of hybrids from reciprocal crosses. A genetic type of vertical transmission of a virus in which the virus has become incorporated in the genome of the strain must be considered as a possibility. The hypothesis of viral etiology is supported by the work of Hotchin (1962) who has indicated that a disorder with many features suggestive of autoimmune disease can be induced in mice chronically infected with lymphocytic choriomeningitis virus. More recently the finding in young NZB mice of viruslike particles resembling murine leukemia virus, and their association with transplantable lymphoreticular neoplasms, has raised the question of the sequential and pathogenetic relationships of the viruslike particles, the autoimmune disorders,

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and the lymphoid neoplasms. The fact that in the NZB strain an enhanced incidence of lymphoid neoplasms may be induced by a recognized carcinogenic agent and that the administration of an immunosuppressive alkylating agent has resulted in a high incidence of thymic neoplasms is of considerable importance. These observations could be consistent with the unmasking of a resident oncogenic virus in these mice, and it will be important to establish whether the viruses believed to be present in at least two NZB colonies are, in fact, universally present in NZB mice. There is also the suggestion that the renal disease may be transmitted by cellfree filtrates, but much additional work designed to study the possible linkage between resident viruses and autoimmune disease needs to be done. If it is ultimately shown that the autoimmune disorders of NZB mice and their hybrids are undoubtedly induced by viral infection, then a study must be made of how widely these particular infective agents are distributed in the murine world. If the virus or viruses are not unique to the strain, then it still remains to be elucidated, what features peculiar to the NZB mice determine their striking immunopathological response to the infection. Whether a virus infection is of primary etiological sigdcance or not, the immunological sequence of events leading to failure of self-recognition and uncontrolled autoantibody production still remains ill-understood, and the NZB mice and their hybrids still remain excellent experimental models for the study of human autoimmune disorders.

ACKNOWLEDGMENTS The authors are grateful to Dr. I. Aarons for Figs. 19 and 22 and to Mr. L. 0. Simpson and Mrs. B. Elliot for their assistance in preparing the manuscript.

REFERENCES Aarons, I. (1964). Nature 203, 1080-1081. Abbot, A., and Bumet, F. M. (1964). 1. Pathol. Bacteriol. 88, 243-245. Bielschowsky, M., and Bielschowsky, F. (1962). Nature 194, 692. Bielschowsky, M., and Bielschowsky, F. ( 1964). Australian J . Exptl. Biol. Med. Sci. 42, 561-568. Bielschowsky, M., Helyer, B. J., and Howie, J. B. (1959). Proc. Univ. Otngo Med. School 37, 9-11. Bumet, F. M. (1962a). Brit. Med. J. 11, 807-811. Bumet, F. M. (1962b). Australasian Ann. Med. 11, 79-91. Burnet, F. M. (1965). J. Pathol. Bacteriol. 89, 271-284. Burnet, F. M., and Holmes, M. C. (1962). Nature 194, 146. Bumet, F. M., and Holmes, M. C. (1964a). 1. Pathol. Bacteriol. 88, 229-241. Burnet, F. M., and Holmes, M. C. (1964b). In “The Thymus in Immunobiology” (R. A. Good and A. E. Gabrielsen, eds.), pp. 656-867. Harper & Row (Hoeber), New York.

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AUTHOR INDEX A Aarons, I., 225, 236, 237, 238, 239, 241, 249, 264, 266 Abbot, A., 257, 264 Abelson, N. M., 48, 93, 154 160 Acharya, U. S. V., 21, 81 Acton, J. D., 186, 209 Ada, C. L., 204, 213 Adinolfi, M., 38, 48, 50, 61, 87 Adler, F. L., 206, 207, 209, 211 Afonsky, D., 67, 87 Aikin, B. S., 125, 131, 137, 138, 139, 140, 142, 143, 157 Ainbender, E., 12, 17, 52, 56, 88, 91 Akiyama, T., 201, 213 Al-Askari, S., 203, 210 Aldrich, H., 20, 87 Alexander, P., 202, 209 Alford, R. H., 17, 24, 41, 94 Ali, S. Y., 141, 156 Allen, W. P., 201, 209 Allison, F., 108, 156 Allison, M. J., 177, 209 Alouf, J. E., 179, 210 Amoss, H. L., 17, 87 Anderson, D. W., 51, 94 Anderson, J. R., 69, 89 Andrew, W., 47, 87 Anzai, T., 19, 20, 87, 91 Appelmans, F., 132, 157 Arbesman, C. E., 77, 78, 79, 87, 90 Amon, R., 125, 156 Aronson, S. B., 16, 93 Artenstein, M. S., 17, 50, 55, 56, 66, 67, 87, 88

Aschaffenburgh, R., 51, 91 Ashbach, N. E., 84, 88 hkonas, B. A., 44, 81, 88, 205, 207, 209, 211

Asofsky, R., 44, 45, 81, 83, 88, 92 Astorga, G., 154, 156, 158 Atkins, E., 187, 209 Atwater, E. C., 154, 159

Austen, K. F., 114, 126, 146, 154, 156, 157, 159, 160, 164, 191, 209, 210 Austin, c. M., 204, 213 Auzins, I., 189, 205, 209, 211 Axelsson, H., 29, 88

B Bachniann, R., 65, 88 Badalamenti, C., 114, 161 Baker, H. J., 17, 91 Baker, P., 203, 209 Ballner, H., 166, 209 Bandhuin, P., 196, 214 Bannister, G. L., 83, 92 Barandun, S., 17, 93 Barclay, W. R., 185, 211 Barnes, R. D. S., 220, 231, 255, 265 Bangham, A. D., lN,156 Barbaro, J. F., 149, 156 Bardawil, W. A., 136, 161 Barland, P., 141, 156 Barnett, J. A., 191, 213 Barrett, A. J., 141, 156 Barth, E. E., 75, 88 Barth, W. F., 83, 90 Batty, I., 5, 11, 59, 62, 83, 88, 93 Bauer, C. H., 74, 89 Baxter, J. H., 114, 156, 161 Bearden, J., 48, 50, 56, 93 Becher, B., 134, 162 Beck, J. S., 69, 89 Becker, E. L., 78, 94, 126, 146, 156, 161

Behrman, S. J., 20, 94 Bell, E. B., 45, 88 Bellanti, J. A., 17, 50, 54, 55, 56, 63, 66, 67, 87, 88, 94 Benacerraf, B., 114, 156, 160, 177, 190, 209, 214

Bendinelli, M., 208,209 Ben-Efraim, S., 116, 156 Bennett, B., 171, 203, 209, 213 Bennett, W. E., 177, 178, 185, 188, 209 267

268

AUTHOR INDEX

Bennich, H., 58, 77, 88, 92, 95 Benson, B., 173, 177, 179, 181, 182, 210 Berdal, P., 78, 88 Berger, R., 12, 17, 52, 56, 88, 91 Berggard, I., 10, 22, 58, 88, 91 Berken, A,, 190, 209 Berman, K. S., 68, 90 Bernheimer, A. W., 134, 158, 168 Bernier, G. M., 29, 88 Besredka, A., 2, 51, 75, 88 Best, C. H., 13, 88 Bettey, M., 17, 93 Bielschowsky, F., 233, 254, 264 Bielschowsky, M., 215, 217, 233, 254, 264 Bienenstock, J., 16, 21, 22, 27, 29, 30, 31, 32, 35, 36, 40, 41, 43, 47, 50, 58, 70, 76, 88, 93, 95 Biro, C. E., 115, 156 Biserte, G., 16, 22, 29, 91 Biswas, E. R. I., 81, 88 Blanc, W. A., 14, 18, 19, 42, 62, 92 Blandau, R. J., 203, 209 Blanden, R. V., 164, 199, 200, 209, 212 Bloch, K. J., 34, 75, 80, 88, 114, 154, 156, 157, 160 Bloom, B. R., 189, 209 Bloom, W., 209 Blout, E. R., 88 Bodel, P., 155, 156, 159, 187, 209 Bokelman, G., 6, 22, 91 Bollet, A. J., 154, 156 Bond, V. P., 206, 211 Bonte, M., 22, 29, 91 &el, C., 184, 209 Borsos, T., 61, 89, 91 Bortnick, L., 68, 94 Boyden, S., 117, 157 Boyden, S. V., 189, 190, 191, 197, 209, 213, 214 Boyse, E. A., 203, 213 Brambell, F. W. R., 40, 88 Bran, M. A., 142, 158 Brandt, W. E., 55, 56, 88 Brandtzaeg, P., 13, 14, 15, 19, 23, 47, 68, 69, 88 Braun, D., 92 Braun, W., 201, 209 Brine, K. L., 124, 142, 159

Broberger, O., 72, 73, 88, 93 Brocklehurst, W. E., 151, 157 Brown, A. L., 64, 91 Brown, B. A., 169, 212 Brown, 1.’ M., 17, 90 Buckley, I. K., 108, 157 Buescher, E. L., 17, 50, 55, 56, 63, 66, 67, 87, 88 Bull, C. G., 3, 88 Bull, D., 17, 49, 50, 64,65, 89, 95 Bullen, J. J., 59, 88 Burke, J. S., 137, 157, 160 Burnet, F. M., 217, 218, 220, 225, 226, 229, 231, 232, 233, 236, 241, 248, 249, 251, 254, 255, 256, 257, 258, 260, 261, 264, 265, 266 Burnett, W., 17, 94 Burnstine, R. C., 74, 89 Burrows, W., 2, 3, 4, 51, 57, 59, 60, 76, 89, 92 Burtin, P., 13, 16, 91, 93 Buser, R., 51, 89 Bussard, A. E., 208, 209 Butler, W. T., 17, 19, 20, 24, 25, 27, 28, 32, 41, 43, 55, 56, 57, 58, 89, 90, 93, 94 Byers, S. O., 164, 209 Byrne, H. J., 5, 89

C Calcott, M. A., 148, 160 Caldwell, R. A,, 57, 93 Calvanico, N., 29, 30, 31, 35, 95 Campbell, D. H., 59, 89, 205, 209 Campbell, P. N., 44, 81, 88 Caner, E. Z., 135, 156, 157 Cannon, P. R., 3, 96 Carbonara, A. O., 15, 25, 42, 43, 89, 91 Cardwell, J. D., 13, 68, 92 Carlier, C., 19, 91 Camel, R., 69, 70, 71, 89 Carpenter, C. B., 112, 125, 157 Carpenter, G. M., 19, 20, 87, 91 Carrel, A., 177, 209 Casey, T. P., 225, 233, 236, 237, 238, 239, 241, 249, 260, 261, 265 Cass, R., 154, 159 Castro-Murillo, E., 154, 158

269

AUTHOR INDEX

Cate, T. R., 17, 55, 56, 89, 93, 94 Cattan, D., 64, 89 Cebra, J. J., 22, 28, 29, 31, 35, 36, 80, 81, 89 Cederblad, G., 29, 32, 89 Chalkina, 0. M., 54, 94 Channing, A. A., 241, 247, 265 Chanock, R. M., 54, 55, 56, 94 Chapman-Andresen, C., 193, 209 Char, D. C., 187, 211 Chauncey, H. H., 47, 89 Chen, D., 200, 201, 211 Chen Ya Huang, 256, 257, 266 Chdirker, W. B., 6, 12, 16, 17, 19, 20, 21, 22, 89 Chosson, A., 22, 93 Christensen, H. E., 175, 214 Cinader, B., 115, 116, 156, 157 Citron, P., 39, 65, 66, 95 Claman, H. N., 9, 13, 39, 41, 89, 92, 94 Clarke, D. A., 177, 214 Clarry, E. D., 13, 68, 92 Clem, L. W., 84, 89 C h e , M. J., 146, 159 Cochrane, C. G., 101, 102, 103, 105, 111, 112, 114, 116, 117, 118, 119, 121, 122, 124, 125, 127, 128, 129, 131, 132, 136, 137, 138, 139, 140, 142, 143, 145, 146, 147, 148, 149, 150, 153, 157, 158, 159, 160, 161, 167, 197, 209, 214 Cohen, E. P., 207, 209 Cohen, S., 6, 28, 89 Cohn, Z. A., 133, 136, 137, 139, 141, 157, 158, 164, 170, 173, 177, 178, 179, 181, 182, 185, 188, 191, 192, 193, 194, 196, 197, 198, 205, 206, 209, 210

Cole, R. M., 197, 211 Collings, C. K., 47, 87 Collins, F. M., ux),209 Colten, H. R., 61, 89 Connell, J. T., 20, 78, 94 Cook, C. T., 121, 161 Cooke, W. T., 72, 94 Coombs, R. R. A., 51, 74, 91, 93, 190, 212

Cooper, G. N., 189, 212

Cooper, M. D., 39, 40, 41, 46, 61, 62, 65, 95 Cooper, N. S., 114, 161 Cooperband, S., 38, 92 Comely, H. P., 122, 124, 157 Cornforth, J. W., 196, 210 Cotran, R. S., 131, 157, 202, 212 Couch, R. B., 17, 55, 56, 58, 89, 90, 93, 94

Couchman, K. C., 69, 95 Cowdrey, C. R., 169, 213 Cox, E. V., 72, 94 Crab&, P. A., 14, 15, 42, 43, 64, 72, 74, 89 Crandall, C. A., 81, 89 Crandall, R. B., 81, 89 Crisler, C., 116, 157 Crowley, J. H., 176, 213 Crozier, D., 55, 56, 88 Cruikshank, B., 72, 92 Czenvinski, D., 29, 30, 31, 32, 35, 40, 47, 95

D Dacie, J. V., 250, 265 Daems, W. T., 127, 136, 157 Datwyler, A., 6, 22, 91 Dagg, J. H., 69, 89 Dale, A. C., 68, 94 Dales, S., 197, 210, 213 Dalmasso, A. P., 148, 160 Damashek, W., 233, 265 Dangerfield, H. G., 55, 56, 88 Dannenberg, A. M., 177, 210 Danon, F., 64, 89 Darlington, D., 47, 89 David, J. R., 203, 204, 210 Davidson, M., 74, 89 Davies, A., 2, 89 Davies, G . E., 151, 157 Davis, S. D., 62, 66, 90 Debray, C., 64,89 de Duve, C., 132, 135, 157, 164, 174, 206, 210

Degaiid, P., 19, 91 Delannay, A., 117, 157, 179, 210 Deniopoulos, H. B., 236, 240, 241, 244, 247, 250, 265 Dennis, E. G., 19, 78, 89, 91

270

AUTHOR INDEX

de Petris, S., 171, 210 de Soma, M. A. B., 217, 218, 231, 232,

233, 234, 255, 256, 258, 265 Deupree, N. G., 3, 4, 89 de Vries, A., 124, 161 de Vries, M. J., 217, 224, 225, 232, 233, 236, 249, 265 Diamond, 3. M., 47, 95 Dias da Silva, W., 121, 147, 157 Dickinson, W., 72, 90 Diener, E., 232, 265 Dilling, L., 41, 91 DiLorenzo, N. L., 151, 159 Dingle, J. T., 141, 157 Dive, C.,17, 92 Dixon, F. J., 45, 82, 89, 101, 103, 104, 105, 107, 111, 113, 114, 127, 128, 136, 150, 151, 152, 153, 154, 157, 158, 159, 161, 197, 209, 253, 266 Dixon, H. M., 107, 159 Dobson, C., 47, 75, 89 Dobson, T. L., 169, 212 Dolovich, J., 77, 78, 79, 87, 90 Dmaldson, G. W. K., 221, 265, 266 Dong, L., 101, 160 Doniach, D., 69, 90, 95 Donnelley, M., 4, 90 Doty, P., 33, 95, 186, 207, 211 Dougherty, T. F., 194, 210 Douglas, R. G., 17, 19, 20, 27, 28, 32, 55, 56, 58, 89, 90, 94 Dray, S., 71, 90, 207, 209 D m m o n d , K. N., 152, 159 Dubiski, S., 115, 157 Dubois, E. L., 236, 240, 241, 244, 2-47, 250, 265 Dumonde, D. C., 164, 210 Dumont, A., 175, 210 Dushenski, L. A., 77, 78, 87 Dutta, N. K., 60, 90, 93 Dutton, R. W., 202, 210 Dwyer, C. M., 168, 176, 211 Dyce, B., 39, 65, 66, 95

Ebeling, A. H., 177, 209 Ebert, R. H., 124, 142, 158, 159, 167, 168, 176, 210 Edelman, G. M., 80, 84, 92 Ehrenreich, B. A., 198, 206, 210 Ehrich, W. E., 101, 157, 204, 210 Eidelman, S., 62, 66, 90 Eigelsbach, H. T., 57, 91 Eisele, J. W., 121, 147, 157 Elberg, S. S., 200, 201, 210, 211 Elliott, M. E., 2, 60,76, 89 Ellison, S. A., 12, 67, 90 Elsbach, P., 192, 210 Essner, E., 173, 184, 191, 210, 213 Evans, C. A., 203, 209 Evans, D. G., 189, 192, 210 Evans, R. T., 50, 68, 90

F

Fahey, J. L., 58, 83, 90, 94 Fairley, G. H., 202, 209 Famham, A. E., 185, 188, 192, 213 Farr, R. S., 51, 71, 74, 77, 90,93, 94 Fauci, A. S., 15, 72, 94 Faulkner, S. M., 191, 209 Fauve, R. M., 179, 210 Fawcett, D. W., 177, 214 Fazekas de St. Groth, S., 4, 90 Fedorko, M. E., 168, 176, 179, 181, 182, 210, 211 Feeley, J. C., 60, 90, 92 Feick, P., 71, 94 Feinstein, A., 45, 81, 82, 90, 93 Feinstein, D., 39, 65, 66, 95 FeIdman, M., 208, 211 Fell, H. B., 133, 141, 157 Felsenfeld, A. D., 53, 76, 90 Felsenfeld, O., 53, 76, 90 Fennell, R. H., 20, 90 Fernando, N. V. P., 127, 136, 160 Finkelstein, R. A., 60, 90 Finland, M., 22, 58, 92 Finstad, J., 84, 88 Fisher, J. M., 69, 70, 71, 90 Fishman, M., zO6, 207, 209, 211 E F i t 4 F. W., 14, 15, 43, 47, 73, 90 Easley, C. W., 29, 88 Fitzgerald, P. H., 253, 265 East, J., 217, 218, 231, 232, 233, 234, Fiellanger, J., 14, 15, 19, 47, 88 255, 256, 257, 258, 262, 265 nexner, S., 112, 157

271

AUTHOR INDEX

Florey, H. W., 108, 157, 167, 168, 176, 210, 212 Iiong, J., 200, 201, 210, 211 Forbes, I. J., 168, 211 Ford, C. E., 185, 211 Forman, C. W., 101, 157 Fostiropoulis, G., 154, 157 France, N. E., 74, 93 Francis, L., 187, 209 Francis, T., 17, 90 Frank, M. M., 116, 157 Franklin, A. E., 137, 161 Franklin, E. C., 11, 90, 113, 114, 156, 161 Franzl, R., 205, 211 Frederic, J., 135, 160 Freeman, J. A., 170, 171, 212 Frei, J., 184, 209 Fresh, J. W., 60, 90 Freter, R., 51, 52, 54, 60,76, 90 Friedman, H., 207, 211 Frimmer, M., 124, 142, 157 Fudenberg, H., 48, 91

G Gabl, F., 12, 90 Gallily, R., 208, 211 Ganrot, P. O., 38, 90 Garcia, G., 115, 156 Gamey, J. S., 59, 89, 205, 209 Geller, J. H., 68, 90 Gelzayd, E. A., 14, 15, 43, 47, 73, 90 Gelzer, J., 197, 201, 211 George, M., 203, 211 Gerhgs-Petersen, B. T., 191, 211 Gershowitz, H., ZO, 94 Gewurz, H., 111, 114, 158 Gianetto, R., 132, 157 Gibbons, R. J., 68, 90, 94 GiEIIan, R. F., 136, 161 Gill, F. A., 197, 211 Gill, T. J., 111, 112, 125, 157 Giltinan, P. J., 217, 248, 260, 265 Girardet, P., 17, 93 Gitlin, D., 22, 58, 92 Gjeruldsen, S. T., 14, 15, 19, 47, 88 Glasgow, L. A., 186, 211 Glassock, R. J., 152, 159

Glisin, V. R., 186, 207, 211 Glynn, A. A., 38, 50, 61, 87 Glynn, h4. F., 149, 160 Gocke, D. J., 149, 158 Goldberg, A., 69, 89 Goldman, A. S., 51, 94 Golub, E. S., 124, 137, 142, 143, 158 Good, R. A., 35, 39, 40, 41, 46, 51, 61, 62, 65, 91, 93, 95, 111, 114, 152, 158, 159 Goodman, J. R., 101, 160 Goodman, J. W., 166, 211 Goodman, M., 21, 87 Gordon, R. S., 74, 96 Gorham, J. R., 263, 265, 266 Gorrie, J., 255, 256, 266 Gottlieb, A. A., 186, 207, 211 Gowans, J. L., 165, 166, 168, 214 Grabar, P., 16, 93 Grasbeck, R., 12, 94 Graham, D. M., 4, 90 Graham, R. C., 124, 142, 158, 159 Granger, G. A., 202, 211 Gray, K. G., 69, 89 Green, G. E., 50, 67, 68, 91 Greenbaum, L. M., 146, 158 Greenough, W. B., 60,91 Greer, W. E., 53, 76, 90 Gribetz, H. J., 141, 162 Cr'ogg, E., 177, 211 Gryboski, J. D., 74, 92 Guerin, F., 16, 91 Guerin, L. F., 83, 93 Gugler, V. E., 8, 22, 91 Cunther, M., 51, 91 Gurner, B. W., 190, 212 Gusek, W., 175, 211 Gustafsson, B. E., 73, 93

H Habel, K., 186, 211 Hackel, D. B., 101, 113, 158, 161 Hagener, D., 124, 142, 157 Hahn, H. H., 187, 211 Halpern, B. N., 177, 211 Hammer, D. K., 111, 158 Hammarstrom, S., 73, 93 Hammerstrom, R. A., 206, 211 Hanson, L. A., 6, 19, 22, 23, 27, 29, 35,

272

AUTHOR INDEX

40, 41, 48, 50, 56, 58, 65, 70, 77, Hodes, H. L., 12, 17, 52, 56, 88, 91 Hoffman, H. N., 64, 91 91, 93 Holborow, E. J., 220, 231, 255, 262, 265 Hardwicke, J., 17, 91 Harris, H., 106, 107, 109, 158, 167, 185, Holland, J. J., 200, 211 Hollander, J. L., 154, 158, 160 186, 211, 214 Holm, A., 82, 94 Harris, T. N.,204, 210 Holm-, L. C., 217, 260, 265 Hamson, W. J., 73, 91 Holmes, M. C.,217, 218, 219, 220, 225, Hart, P. D.,196, 210 226, 231, 232, 233, 236, 241, 249, Hartley, T. F., 9, 13, 39, 89, 92 254, 255, 256, 257, 258, 280, 264, Haupt, H., 29, 91 265, 266 Havens, I., 2, 51, 57, 59, 80,76, 89 Hong, R., 35, 39, 40, 41, 46, 61, 62, 65, Haverback, B. J., 39, 84, 66,95 91, 95 Havez, R., 16, 19, 22, 29, 91, 93 Hood, J. E., 19, 91 Hawkins, D.,102, 132, 150, 158, 161 Hook, E. W., 232, 255, 266 Haworth, J. C., 41, 91 Hornbrook, M. M., 19, 77, 78, 89, 91, 92 Hayashi, H., 141, 158, 161 Hornick, R. B., 57, 91 Heatherington, D. C., 177, 211 Horowitz, R. E., 236, 240, 241, 244, Hedberg, H., 154, 158 247, 250, 265 Heide, K., 29, 91 Horvath, G., 184, 209 Heimburger, N., 29, 91 Hoskins, D. W., 92 Heimer, R., 70, 76, 77, 91 Hotchin, J., 263, 266 Heise, E. R., 177, 211 Helyer, B. J., 215, 217, 221, 223, 225, Howard, J. G.,169, 177, 200, 211 228, 230, 233, 235, 236, 237, 238, Howie, J. B., 215, 217, 221, 223, 225, 228, 230, 231, 235, 236, 237, 238, 239, 241, 249, 2.53, 254, 255, 257, 239, 241, 249, 253, 254, 255, 257, 258, 259, 260, 264, 265, 266 258, 259, 260, 261, 264, 265, 266 Henson, J. B., 263, 265, 266 Hsu, K. G., 14, 42, 94 Henson, P. M.,123, 148, 149, 158 Huizenga, K. A.,64, 91 Herbert, V., 69, 70, 71, 89 Heremans, J. F., 6, 8, 14, 15, 17, 19, Hulliger, L., 177, 214 25, 29, 38, 42, 43, 84, 65, 72, 74, Humphrey, J. H., 44, 77, 81, 88, 95, 128, 149, 155, 158, 161, 205, 211 89, 91, 92, 93, 94 Hurley, J. V., 108, 109, 131, 158, 160 Heremans, M. T., 6, 25, 91 Hurlimann, J., 13, 91 Hermanns, P. E., 64, 91 Hyde, L., 19, 87, 91 Herskovic, T., 74, 92 Hervb, R., 21, 91 Hevizy, M. M., 12, 17, 52, 56, 88, 91 I Heymann, W., 101, 113, 158, 161 Ibayashi, J., 19, 87, 91 Hicks, J. D., 225, 229, 230, 240, 241, Inoue, T., 141, 158 242, 248, 251, 260, 261, 265, 266 Ironside, P. N. J., 240, 241, 244, 266 Hijmans, W., 217, 224, 225, 232, 233, Ishizaka, K., 19, 48, 61, 77, 78, 89, 91, 236, 249, 265 92, 113, 158 Hill, B. M., 147, 160 Ishizaka, T., 48, 61, 77, 91, 92, 113, 158 Hill, G. A., 197, 211 Ito, K., 141, 158 Hirsch, J. G.,133, 134, 136, 137, 139, 143, 157, 158, 162, 184, 168, 176; J 179, 181, 182, 187, 210, 211 Hirsch-Marie, H., 13, 91 Jacobson, E. B., 44, 91 Hochwald, G. M.,44, 91 Jacoby, F., 184, 211

273

AUTHOR INDEX

Jacques, R., 149, 158 Jacquet, H., 258, 262, 26.5 Jandl, J. H., 164, 202, 212 Janis, R., 141, 156 Janoff, A., 124, 134, 141, 142, 143, 145, 146, 158, 161 Jensen, J., 147, 158 Jarrett, W. F., 75, 88 Jee, W. S. S., 164, 212 Jeffries, G. H., 15, 69, 70, 72, 92, 94 Jennings, F. W., 75, 93 Jenkin, C. R., 196, 202, 212, 213, 214 Jhala, H. I., 60, 93 Johansson, B. G., 19, 23, 27, 29, 32, 35, 40, 41, 88, 89, 91 Johansson, S. G. O., 77, 92, 95 Johnson, J. S., 80, 82, 92 Jonas, W. E., 190, 212 Josephson, A. S., 20, 92 Julita, J., 203, 209

K Kabat, E. A., 8, 92 Kaser, H., 17, 93 Kamm, D. P., 234, 266 Kapral, F. A., 177, 210 Kapsimalis, D., 68, 90 Karer, H. E., 173, 212 Karlsbad, G., 171, 210 Karnovsky, M. L., 137, 141, 160, 185, 188, 192, 196, 212, 213, 214 Karthigasu, K., 196, 212 Kanon, D. T., 52, 53, 56, 57, 63, 93 Kasel, J. A., 55, 92 Kasuga, T., 241, 247, 265 Katchalski, E., 124, 161 Katz, J., 74, 92 Kaye, D., 232, 255, 266 Keimowitz, R. I., 19, 92 Keller, H. U., 107, 121, 122, 159 Kelly, L. S., 169, 212 Kenny, K., 190, 213 Kerr, W. R., 5, 92 Kessel, R. W. I., 201, 209 Khambla, A,, 21, 87 Khoo, K. K., 185, 201, 212 Kim, K. M., 146, 158 Kirsner, J. B., 15, 72, 73, 90, 92 Klein, P., 112, 160

Klemperer, M. R., 114, 159 Kniker, W. T., 51, 94, 103, 128, 129, 149, 159 Knoetter, P., 68, 90 Kochwa, S., 12, 52, 56, 91 Koffler, D., 152, 159 Konno, J., 12, 68, 92 Koons, M., 141, 158 Korinek, J., 13, 95 Koshland, M. E., 51, 52, 92 Kostka, J., 29, 93 Kotynek, O., 29, 93 Kourilsky, F. M., 157 Kraft, S. C., 14, 15, 43, 47, 51, 72, 73, 74, 90, 92, 94 Kraus, F. W., 12, 13, 14, 23, 68, 88, 92 Kruse, H., 205, 212 Kugler, M. M., 74, 89 Kumate, J., 114, 159 Kunkel, H. G., 48, 65, 93, 152, 159 Kurtz, H. M., 111, 159 Kuze, T., 141, 158

L Lachmann, P. J., 152, 155, 159 LaGattuta, M., 131, 157 Lagercrantz, R., 73, 93 Lagunoff, D., 62, 66, 90 Lahiri, S. C., 151, 157 Lambert, P. H., 253, 266 Lapresle, C., 137, 138, 139, 159, 198, 212 Laster, L., 74, 96 Lawrence, H. S., 203, 210 Leachtenberg, C., 186, 202, 213 Leader, R. W., 263, 265 Leake, E. S., 177, 211, 212 Ledingham, J. C. G., 189, 212 Lee, A,, 189, 212 Lee, E. H., 48, 91 Lehner, T., 13, 68,92 Lehrich, J. R., 55, 92 Leithoff, H., 21, 92 Leithoff, I., 21, 92 Lepow, I. H., 121, 147, 157 Lerner, R . A., 152, 159 Levanon, D., 181, 214 Levin, F. M., 70, 76, 77, 91

274

AUTHOR INDEX

Manaligod, J. R., 217, 225, 230, 231, 232, 236, 240, 241, 249, 251, 266 Mandel, I. D., 12, 67, 90 Mandel, M. A., 83, 92 Mannik, M., 35, 36, 92 Marchalonis, J., 80, 84, 92 Marche, C., 64,89 Marchesi, V. T., 131, 159, 167, 212 Marcus, S., 197, 211 Margolis, S., 15, 72, 94 Markowitz, H., 64, 91 Martinez-Tello, F. J., 14, 18, 19, 42, 62, 92 Mashimo, P. A., 12, 90 Masson, P. L., 17, 19, 92 Masters, J. M., 259, 266 Mathews, R. H., 51, 91 Matsuba, K., 141, 158, 161 May, R. P., 191, 213 Mayer, M. M., 8, 92, 121, 161 Meier, R., 107, 116, 117, 159 Mellors, R. C., 153, 159, 217, 219, 223, M 225, 226, 227, 228, 229, 230, 231, 233, 234, 251, 256, 257, 266 McCarty, D. J., 135, 141, 154, 155, 156, Mehan, K. L., 146, 159 158, 159, 160 McCluskey, R. T., 115, 133, 134, 158, Mergenhagen, S. E., 50, 68, 90, 111, 114, 158 160, 161 Merler, E., 22, 58, 92 McCutcheon, M., 107, 159 Merrill, D. A,, 9, 13, 39, 41, 89, 92, 94 MacDonald, J. M., 68, 94 Metcalf, D., 259, 266 McDonnell, G. N., 111, 159 Metchnikoff, E., 132, 159 McFarlane, A. S., 58, 94 Merteens, E., 204, 210 McFarlin, D. E., 39, 43, 66, 92 MacGillivray, M., 52, 53, 56, 57, 63, Metchnikoff, E., 199, 212 Meynell, M. J., 72, 94 93 McGiven, A. B., 240, 241, 242, 244, 248, Michael, A. F., 152, 159 Michaels, R. H., 51, 94 261, 266 Mackaness, G. B., 164, 188, 179, 185, Miescher, P., 154, 159 195, 196, 199, 200, 201, 203, 209, Miki, K., 201, 212 Mildenhall, P., 190, 191, 212 211, 212 Miller, J. F. A. P., 259, 266 McIntyre, 0. R., 80, 69, 92 Miller, J. J., 204, 212 McKee, C. M., 3, 88 McMaster, P. D., 205, 212 Miller, W. S., 155, 159 Macmorine, D. R. L., 137, 139, 157, Milne, C. M., 38, 50, 61, 87 Milofsky, E., 185, 188, 192, 213 160, 161 Milstein, C., 6, 28, 89 Maier, P., 114, 162 Minden, P., 77, 93 Majno, G., 131, 157 Mitchell, C. A., 83, 92, 93 Makinodan, T., 188, 205, 213 Miyasato, F., 217, 225, 230, 231, 232, Malawisla, I. E., 155, 156, 159 Malik, G. B., 72, 92 234, 236, 240, 241, 249, 251, 266 Levinhar, H., 125, 156 Levitt, M., 38, 92 Lewis, G. P., 124, 159 Lewis, M. R., 177, 212 Lewis, W. H., 177, 192, 212 Lieberman, R., 83, 93 Lietze, A., 17, 78, 93 Lilly, F., 203, 213 Lindsay, M., 38, 50, 61, 87 Lindsey, E. S., 221, 255, 266 Linscc'tt, W. D., 116, 117, 123, 159, 160 Lo Buglio, A. F., 202, 212 Lockwood, D. W., 20, 92 Long, G., 220, 266 Looke, E., 195, 212 Lospahto, J., 141, 162 Lovett, C. A,, 148, 159, 160 Low, F. N., 170, 171, 212 Lowe, J. S., 151, 157 Lurie, M. B., 176, 177, 199, 209, 212 Lynraven, G. S., 248, 266

275

AUTHOR INDEX

Miyoshi, M., 141, 158 Modem, F. W. S., 101, 160 Moeller, H. C., 73, 93 Moghissi, K. S., 20, 93 Mollison, P. L., 48, 50, 61, 87 Mondal, S. P. D., 76, 90 Mmgan, E. S., 154, 159 Montreuil, J., 22, 93 Montri, S., 184, 209 Moore, D. E., 3, 4, 89 Moore, R., 101, 160 Morgan, C., 14, 42, 94 Morita, T., 188, 205, 212, 213 Momson, J.T., 51, 95 Morse, S. I., 137, 141, 157 Moses, J. M., 124, 142, 158, 159 Movat, H. Z., 127, 136, 137, 139, 148, 149, 150, 151, 157, 159, 160, 161 Muckerheide, M., 71, 94 Mudd, E. B. H., 189, 212 Mudd, S., 189, 212 Miiller-Eberhard, H. J., 112, 115, 117, 118, 119, 120, 121, 122, 124, 147, 148, 150, 152, 157, 159, 160, 161 Muh, J. P., 16, 19, 22, 29, 91 Mukerjee, S., 54, 93 Mukherjee, T. M., 229, 266 Muller-Eberhard, H. J., 167, 214 Mullet, S., 22, 93 Mulbgan, W., 75, 93 Muralt, G. V., 6, 22, 91 Murray, D., 72, 92 Murray, R. K., 139, 161 Myrvik, Q. N., 177, 186, 189, 192, 209, 210, 211, 212 Muschel, L. H., 111, 114, 158 Mustard, J. F., 148, 149, 160

N Nairn, R. C., 240, 241, 244, 266 Nakano, M., 201, 213 Naylor, C. R. E., 57, 93 Neil], J. M., 3, 95 Nelson, D. S., 190, 191, 212 Nelson, J. T. M., 75, 93 Nelson, P. M., 5. 89 Nelson, R. A., 112, 1.23, 148, 160, lfif Nettesheim, P., 188, 205, 213 Neuhaus, 0.W., 20, 93

Nilssm, U., 115, 119, 120, 160 Nishioka, K., 123, 159, 160 Nitta, R., 141, 158 Noguchi, H., 112, 157 Nolan, P. D., 164, 212 Norins, L. C., 217, 219, 220, 231, 240, 241, 244, 265, 266 Norris, H. T., 60, 90 North, R. J., 173, 179, 191, 192, 209, 212 Northrup, R., 16, 93 Nossal, G. J. V., 2Q4, 212, 213 Novikoff, A. B., 173, 184, 191, 213 Nowell, P. C., 169, 213 Nusslk, D., 17, 93

0 Oakley, C. L., 5, 11, 62, 93 Ogilvie, B. M., 75, 93 Ogra, P. L., 52, 53, 56, 57, 83, 93 Old, L. J., 177, 203, 213, 214 Onoue, K., 11, 81, 93 Oort, J,, 127, 136, 157 Orange, R. P., 146, 160 Oren, R., 185, 188, 192, 213 Ortega, L. T., 153, 159 Oshima, S., 177, 212 Osler, A. G., 147, 149, 158, 160 Ovary, Z., 114, 147, 156, 160 P Padgett, G. A,, 263, 266 Page, C. O N . , 24, 93 Pages, J., 117, 157 Palade, G., 193, 213 Panagoitis, N. M., 194, 210 Panse, M. V., 60, 93 Parish, W. E., 51, 74, 91, 93 Parks, E., 194, 195, 210 Parks, J. J., 207, 209 Paronetto, F., 152, 153, 159, 160 Parrott, D. M. V., 217, 218, 231, 232, 233, 234, 255, 258, 265 Pasieka, A. E., 83, 93 Pastner, D., 12, 90 Patterson, R., 198, 213 Patton, J. R., 11, 93 Pnvillard, E. R., 195, 213 Pearse, A. G. E., 177, 211

276

AUTHOR INDEX

Pearson, H. E., 17, 90 Pekin, T. J., 154, 160 Pelon, W., 51, 94 Perkins, E. H., 188,205,212,213 Perlmann, P., 73, 88, 93 Permis, B., 171, 210 Persellin, R. H., 156, 160 Peterson, R. D., 51, 93 Pethica, B. A., 124, 156 Phelps, P,, 135, 141, 155, 156, 159, 160 Phillips, R. A., 54, 93 Pickering, R. J,, 111, 114, 121, 158, 161 Pickett, M. J., 200, 211 Piel, C. F., 101, 160 Pierce, A. E., 5, 20, 45, 51, 82, 93 Pierce, E. J., 177, 211 Pigman, W., 12, 93 Pinet, J., 173, 193, 213 Pinkett, M. D., 169, 213 Pirani, C. L., 252, 266 Plaut, A., 16, 32, 47, 93 Playfair, J. H. L., 232, 266 Pollak, V. E., 217, 225, 230, 231, 232, 234, 236, 2A0, 241, 249, 251, 252, 266 Pollara, B., 35, 84, 88, 91 Pouey, M. J., 48, 50, 61, 87 Pomales-Lebron, A., 199, 201, 209, 213 Pondman, K. W., 191, 211 Postel, W. B., 187, 211 Potter, J., 133, 134, 151 Potter, M., 83, 93 Prager, M. D., 48, 50, 56, 93 Pras, M., 134, 162 Preisig, R., 17, 91 Prendergast, R. A., 6, 12, 13, 14, 19, 22, 25, 26, 27, 28, 29, 31, 32, 39, 40, 41, 42, 43, 44, 46, 48, 50, 65, 70, 78, 77, 95 Pressman, B. C., 132, 157 Pressman, D., 11, 81, 93, 114, 162 Prignot, J., 19, 92 Prosser, P. R., 2-56,257, 258, 262, 265 Pruzansky, J., 198, 213 Purcell, R. H., 54, 94 Putnam, F. W., 29, 88 Pye, T., 204, 213, 248, 265 Pyensen, J,, 135, 141, 159

Q Quintana, N., 184, 213

R Rabinovitch, M., 189, 191, 213 Rabinowitz, Y., 166, 177, 213 Rajan, H. T., 135, 155, 156, 160 Ramseier, H., 214 Ranadive, N., 143, 145, 148, 160 Randall, H. G., 147, 160 Rankin, J. G., 17, 91 Rapp, H. J., 61, 89, 91 Rapp, W., 16, 93 Ratnoff, 0. D., 124, 158 Rawson, A. J., 17, 48, 93, 154, 160 Raynaud, M., 179, 210 Reade, P. C., 196, 212 Rebuck, J. W., 176, 213 Rees, C., 69, 70, 71, 90 Rees, R. J. W., 196, 210 Reid, R. T., 77, 93 Rejnek, J., 29, 93 Remingtoa, J. S., 17, 22, 24, 56, 58, 78, 92, 93, 96 Reyes, V., 60, 90 Rhodes, J. M., 207, 209 Richards, C. B., 74, 93 Rider, J, A,, 72, 93 Riesman, R. E., 77, 78, 87 Righthand, F., 52, 53, 50, 57, 63, 93 Rihm, J. S., 51, 94 Ritz, H., 112, 160 Robbins, J. B., 22, 29, 31, 80, 81, 89, 190, 213 Robertson, M. D., 5, 92 Robey, M., 21, 91 Robineaux, J., 135, 160 Robineaux, R., 173, 193, 213 Rockey, J. H., 48, 65, 93 Rodhain, J. A., 25, 91 Rogers, A. W., 47, 89 Roitt, I. M., 69, 77, 90, 95 Rose, G . G . , 193, 213 Rose, H. M., 14, 42, 94 Rose, J. M., 48, 50, 61, 87 Rosen, F. S,, 114, 159 Rosenberg, L., 134, 162 Rosenthal, E., 68, 94

AUTHOR MDEX

Rossen, R. D., 14, 17, 19, 20, 24, 25, 27, 28, 32, 41, 42, 43, 55, 56, 57, 58, 89, 90, 92, 93, 94 Rothberg, R. hl., 51, 72, 74, 94 Rother, K., 115, 160 Rother, V., 115, 160 Rouiller, C., 17, 94 RouIeau, C., 39, 65, 66, 95 Rous, P., 141, 160 Raussel, P., 19, 91 Rovelstad, G. H., 68, 90 Rowe, D. S., 21, 56, 58, 95 Rowley, D., 164, 177, 179, 186, 188, 189, 190, 195, 202, 209, 211, 212, 213, 214 Rubin, C. E., 62, 66, 90 Rubjn, W., 15, 72, 94 Russell, I. S., 17, 94 Russell, P. J., 260, 266 Ryan, G . B., 108, 160 Rymo, L., 29, 32, 88, 89

277

Seegers, W., 143, 161 Seifter, J., 101, 157 Sela, M,,125, lfj6, 205, 211 Seligmann, M., 64, 89 Sell, S., 22, 81, 94 Selner, J. C., 39, 41, 94 Sergent, P., 21, 91 Settipane, G. A., 20, 78, 94 Sever, J. L., 201, 213 Sewell, P., 72, 94 Shapland, C.,69, 95 Sharard, A., 240, 266 Sheldon, H., 175, 210 Sherman, W. B., 20, 78, 94 Shilo, M., 177, 213 Shin, H. S., 121, 161 Shirley, W., 136, 161 Shrivastava, D. L., 76, 90 Siegler, R., 217, 232, 233, 266 Silver, R. H., 69, 92 Silverstein, A. M., 114, 161 Silverstein, S. C., 197, 213 Simons, K., 12, 94 Sabin, A. B., 51, 94 Simpson, L. O., 235, 266 Saito, K., 164, 177, 185, 188, 201, 213 Siqueira, M., 123, 148, 161 Samter, M., 78, 94 Sirisinha, S., 12, 92 Skarnes, R. C., 142, 143, 161 Sandford, J. P., 191, 213 Sandson, J., 141, 156 Sleisenger, M. H., 15, 69, 70, 72, 92, 94 Small, P. A., 22, 28, 29, 35, 36, 44, 80, Saperstein, S., 51, 94 81, 84, 88, 89, 114, 156, 161 Sarnecki, J., 84, 88 Smith, C. A., 205, 214 Sauthoff, R., 112, 160 Sbarra, A. J., 136, 137, 141, 160, 161 Smith, C. B., 54, 55, 56, 94 Smith, E. L., 82, 94 Schade, S. G., 71, 94 Smith, J. G., 155, 159 Schaeffer, S., 142, 145, 158 Smith, J. W., 191, 213 Schar, B., 107, 116, 117, 159 Smith, K., 72, 90 Schar, M., 51, 89 Smith, M. R., 108, 156 Schecter, I., 205, 211 Smith, T. J., 186, 213 Scherer, J., 142, 146, 158, 161 Smith, W., 155, 161 Schiller, K. F. R., 69, 94 Smithers, S. R., 75, 93 Schilling, R. F., 71, 94 Smorodintsev, A. A., 54, 94 Schinders, F., 115, 160 Socransky, S. S., 68, 94 Schneebeli, G. L., 194, 210 Sokoloff, C., 154, 161 Schneider, P., 200, 201, 210, 211 Solish, G. I., 20, 94 Schoefl, C . I., 185, 211 Schrek, R., 166, 213 Soaomon, A., 6, 12, 13, 14, 19, 22, 25, Schubert, O., 3, 4, 94 26, 27, 28, 29, 31, 32, 39, 40, 41, Schultze, H. E., 6, 17, 29, 91, 94 42, 43, 44, 46, 48, 50, 58, 65, 70, Schumacher, G . F. B., 21, 94 76, 77, 94, 95 Schwartz, F. D., 252, 266 Solomon, J. M., 190, 207, 211, 213

s

278

AUTHOR INDEX

Sorkin, E., 107, 121, 122, 159, 197, 213 Soulsby, E. J, L., 75, 95 South, M. A., 39, 40, 41, 46, 61, 62, 65, 95 Spargo, B., 101, 162 Spector, W. G., 108, 131, 158, 166, 167, 168, 203, 213, 214 Spilberg, I., 141, 162 Spigland, I., 51, 94 Spiro, H. M., 74, 92 Spitznagel, J. K., 124, 137, 142, 143, 158, 162 Spray, G. H., 69, 94 Springer, G. F., 48, 95 Sprinz, H., 61, 95 Spurling, C. L., 259, 266 Stahelin, H., 185, 196, 214 Stanworth, D. R., 77, 95 Stavitsky, A. B., 113, 161, 207, 211 Steblay, R., 101, 162 Stecher, V. J., 187, 214 Stein, O., 124, 161 Steinberg, A. G., 17, 19, 20, 27, 28, 32, 56, 94 Stetson, C. A., 128, 137, 141, 161 Stevenson, C. S., 20, 93 Stiel, M., 12, 94 Stinebring, W. R., 199, 213 Stobo, J., 66, 67, 95 Stock, J. A., 196, 210 Straus, E. K., 21, 58, 94, 95 Strauss, B. S., 137, 141, 161 Strauss, L., 153, 160 Straws, W., 192, 214 Strober, W., 39, 43, 66, 92 Stuchlikwa E., 13, 95 Sugahara, T., 113, 158 Sugg, J. Y., 3, 95 Sullivan, A. L., 82, 95 Sullivan, E. R., 17, 90 Sullivan, L. W., 69, 92 Sunderman, F. W., 76, 90 Suter, E., 164, 171, 175, 177, 178, 190, 196, 197, 199, 201, 211, 213, 214 Sutton, J. S., 171, 175, 178, 214 suzko, I. M., 198, 213 swanson, v., 39, 65, 66, 95 Szwed, C. F., 17, 93

T Taichmw, hl. S., 148, 160 Tan, E. M., 6, 12, 13, 14, 19, 22, 25, 26, 27, 28, 29, 31, 32, 39, 40, 41, 42, 43, 44, 46, 48, 50, 56, 58, 65, 70, 76, 77, 91, 95, 152, 159 Taranta, A., 113, 114, 161 Taylor, E., 17, 87 Taylor, F. B., Jr., 120, 161 Taylor, K. B., 69, 70, 71, 72, 90, 95 Taylor, N. B., 13, 88 Tenerova, M., 13, 95 Teplitz, R., 236, 240, 241, 244, 250, 265 Terry, R. J., 75, 93 Thind, K. S., 53, 95 Thomas, L., 133, 134, 137, 157, 161, 162, 203, 210 Thompson, D. L., 69, 95 Thomson, A., 59, 95 Thomson, D., 51, 95 Thomson, K. J., 229, 266 Thomson, R., 51, 95 Thorbecke, G. J., 44, 45, 88, 91, 177, 187, 214 Todd, J. E., 92 Tokuda, A., 141, 158, 161 Tomasi, T. B., 5, 6, 12, 13, 14, 16, 17, 19, 20, 21, 22, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 36, 39, 40, 41, 42, 43, 44, 46, 47, 48, 49, 50, 64, 65, 66, 67, 70, 76, 77, 78, 79, 82, 87, 88, 89, 90, 93, 95 Tominaga, K., 29, 88 Tormey, J. M., 47, 95 Torrigiani, G., 77, 95 Toshimura, M., 141, 158 Tourville, D., 17, 49, 50, 95 Trowell, 0. A., 47, 95 Truelove, S. C., 69, 72, 95 Tucker, E., 150, 161 Tuffrey, M., 220, 231, 255, 265 Turner, K. J., 190, 202, 213, 214 Turner, M. W., 21, 56, 58, 95

U Uhr, J., 190, 214 Uhr, J . W., 134, 162 Ukada, K., 141, 158

AUTHOR INDEX

279

Wasi, S., 139, 161 Watkins, J. F., 185, 211 Watriaux, R., 132, 157 Watson, D., 142, 161 Watson, W. C., 72, 92 Wattiaux, R., 164, 174, 196, 206, 210, 214 Watts, J. W., 186, 214 Webb, T., 137, 138, 139, 159, 198, 212 Weber, R., 198, 214 V Weber, T., 12, 94 Wedderburn, N., 208, 209 Vaerman, J. P., 25, 91, 95 Wehr, R. E., 51, 94 Valentine, M., 146, 160 Vannier, W. E., 17, 19, 20, 24, 27, 28, Weigle, W. O., 105, 127, 128, 136, 157 Weigle, W. O., 45, 82, 89, 197, 209 32, 41, 56, 58, 94 Weiser, R. S., 202, 203, 209, 211, 214 Vanotti, A., 184, 209 Weiser, W. J., 127, 136, 149, 160 Vasquez, J. J., 20, 90 Weiss, L., 171, 173, 175, 177, 178, 214 Vassalli, P., 115, 160 Vaughan, J. H., 80, 82, 92, 154, 159, Weissmann, G., 133, 134, 135, 141, 155, 203, 211 158, 161, 162 WeIlensiek, H. J., 112, 160 Vaughn, R. B., 189, 214 Wheelock, E. F., 186, 214 Vazquez, J. J., 45, 82, 89, 154, 161 Vernier, R. L., 152, 159 Whitby, J. L., 195, 214 Whitelaw, D. M., 166, 214 Versage, P. M., 60, 90 Whur, P., 47, 96 Visek, W. J., 59, 95 Wibo, M., 196, 214 Volkman, A., 165, 166, 168, 214 Wicher, K., 77, 78, 87 Vosti, K. L., 17, 56, 58, 78, 93, 96 Wied, G. L., 21, 94 Wiedermann, G., 134, 162 W Wiener, E., 177, 181, 191, 192, 210, 214 Wagner, R. R., 186, 187, 213 Williams, A. L., 29, 30, 31, 35, 95 Wakonig-Vaartaja, R., 259, 266 Walberg, H. E., 188, 213 Willmghby, D. A., 166, 167, 168, 214 Waldmann, T. A., 25, 39, 41, 43, 58, Wilson, J. F., 74, 96 Winemiller, R., 101, 162 66, 74, 89, 92, 94, 96 Walker, R. V. L., 83, 92 Witschi, H. P., 17, 93 Walsh, T. E., 3, 96, 205, 214 Wochner, R. D., 39, 43, 66, 74, 92, 96 Walter, P. C . , 177, 210 Woessner, J., 198, 214 Walters, M. N., 166, 168, 214 Wolf, B., 45, 88 Wangel, A. G., 69, 94 Wolff, S. M., 56, 57, 94 Wanstrup, J., 175, 214 Wollheim, F. A., 39, 40, 41, 48, 61, 82, Ward, P. A., 111, 114, 117, 118, 119, 65, 95 120, 121, 122, 124, 125, 126, 127, Wood, W. B., 108, 156, 187, 211 135, 154, 156, 156, 157, 161, 167, WOOdNff, M. F. A., 221, 255, 266 214 Woo(lwort11, H. C., 114, 159 Wardlaw, A. C., 115, 157, 177, 211 Work, T. S., 44, 81, 88 Warrack, G. H., 5, 11, 62, 83, 88, 93 Wartman, W. B., 107, 159 Wright, R., 69, 72, 94, 95 Warwick, W. J., 39, 95 Wynn Williams, A., 229, 235, 266 Unanue, E. R., 101, 104, 111, 113, 114, 128, 150, 151, 153, 157, 161, 253, 266 Uriuhara, T., 127, 136, 137, 148, 150, 157, 160, 161 Umes, P., 33, 95 Urquhart, G. M., 75, 88, 93 Ushiba, D., 201, 213

280

AUTHOR INDEX

Y Yagi, Y., 11, 81, 93, 114, 162 Yphantis, D. A., 28, 96

Z Zappasodi, P., 177, 209 Zeek, P. M.,153, 162 Zeitlin, 1. J., 151, 157

Zepp, H. D., 12, 52, 58, 88, 91 Zeya,

H. I., 143, 162

Ziff, M.,141, 156, 160, 162 Zigelbaum, S., 12, 21, 95 Zimmerman, A. L., 17, 78, 93 Zucker-Franklin, D., 134, 138, 154, 162 Zvaifler, N. J., 154, 160, 161 Zweifach, B. W.,124, 142, 158

SUBJECT INDEX A Agammaglobulinemia, secretory immunoglobulins, in, 64-65 Allergies, respiratory, see Respiratory allergies Amines, vasoactive, see Vasoactive amines Anaphylatoxin, release, i n tissue injury, 147 Anemia, pernicious, see Pernicious anemia Animals, secretory immunoglobulins in,

Brucellosis, antibodies in reproductive tract in, 5

C

Caries, secretory immunoglobulins in, 6769 Cartilage, protein polysaccharides, hydrolysis by neutrophile enzymes, 141 Cathepsins, of neutrophiles, in immunologic tissue injury, 137-141 Celiac disease, secretory immunoglobu79-84 lins in, 71-72 Antibodies, in reproductive tract, diagCellular immunity, macrophages and, nostic significance, 5 199-204 viral and bacterial, as secretory imCervical mucus, secretory immunoglobumunoglobulins, 49-51 lins in, 20-21 Antigens, fate in macrophages, 204-206 Antiinflammatory agents, in immuno~ogic Cholera, secretory antibodies in, 75-76 tissue injury, 155-156 Cobra venom factor, in immunologic tisAntinuclear factom, secretory i-unosue studies, 109, 111, 112-113 globulins in, 7 5 7 7 Colitis, ulcerative, secretory immunoglobulins, 72-74 Appendix, secretory immunoglobulins in, Colon, secretory immunoglobulins in, 15 15 Arterial internal elastic lamina, neutro- Colostrum secretions, immunoglobulins of, 6, 22-23, 34, 50, 56 phile damage by, 1291131 Arteritis and glomerulonephritis, imamino acid composition, 31 mune complex-induced, immunologic tissue injury in, 102104, 152 Arthus phenomenon, vasculitis, ii-unologic tissue injury in, 1op105, 110, 127-128, 150, 152 Ascitic fluid, secretory immunoglobulins in, isolation, 25

B

25

properties, 29 effect on phagocytosis, 191 role in neutrophile chemotaxis, 116127 Connective tissue disease, neutrophilemediated 152-156 Cow, secretory immunoglobulins in, 82 Cryoglobulinemia, neutrophile-mediated injury in, 154

Bacteria, phagocyte digestion of, 196-197 D Bacterial antibodies, as “natural” secreDeoxyribonucleic acid, synthesis, by tory antibodies, 49-51 Basement membranes, hydrolysis by neumonocytes and macraphages, 185trophile enzymes, 137-141 186 vascular and glomerular, neutrophile Digestive tract, secretory immunoglobulins of, 12-17 damage of, 131-132 Diphtheria antitoxin, 3 Bile, secretory immunoglobulins in, 17 Bronchial mucosa, secretory immuno- Dog, secretory immunoglobulins in, 8283 globulins in, 14, 18, 19 281

282

SUBJECT INDEX

Duodenum, secretory immunoglobulins in, 15, 56

E Electroimmunodiffusion, in analysis of secretory immunoglobulins, 9

G yA Antibodies, 6 Gastric fluids, secretory immunoglobulins in, 13-14, 16, 23 Gastritis, secretory immunoglobulins in, 6!4-71 Gastrointestinal allergy, secretory immunoglobulins in, 74 Gastrointestinal antibodies, as secretory antibodies, 51-54 Gastrointestinal diseases, secretory immunoglobulins in, 67-71 Genital tract, secretory immunoglobulins in, @ 212Genitourinary antibodies, as seeretory antibodies, 57-59 Gingival tissue, secretory immunoglobulins in, 14 y-G globulin, deposition in connective tissue disease, 152-154 Glomerulonephritis of nephrotoxic nephritis, immunologic tissue injury in, 101-103, 110 renal protein clearance in, 132, 152.153 Gout, neutrophile-mediated tissue injury in, 155

H Hydrolase, redistribution in phagocytosis, 192

I Immune response macrophage role in, 204 Immunity, local, in disease defense, 5 historical aspects, 2 Immunoconglutinin, elevated levels, in disease, 155 Immunofluorescent studies of secretory immuno~gIobulins,41-43 Immunoglobulins, secretory, see Secretory immunoglobulins

Immunologic tissue injury, by neutrophilic leukocytes, 97 in Arthiis phenomenon vasculitis, 104105 in glomerulonephritis of nephrotoxic nephritis, 101-103 mast-cell degradation and, 147-148 Inborn errors of metabolism, macrophages in, 199 Interferons, synthesis by macrophages, 186-187 Intestinal fluids, secretory immunoglobulins in, 16-17, 23, 50 Isohemagglutinins as secretory immunoglobulins, 48-49

J Jejunum, secretory immunoglobulins in, 15

K Kinin, formation by neutrophiles, 146

1 Lacrimal gland, secretory immunoglobulins in, 15, 16, 23 Leukocytes, neutrophilic, see Neutrophilic leukocytes Lysozymes, in immunologic tissue damage, 132137 enzymes of, 133 function of, 132-137 membrane, alteration of stability, 13% 137

M Macrophage( s ) , 183-214 in anticellular immunity, 202-203 antigen-antibody complexes, and, 197198 antigen fate in, 204-206 in cellular immunity, 199-204 in delayed hypersensitivity, 203-204 electron microscopy, 173-176 energy metabolism of, 185 in immune response, 204-208 in inborn errors of metabolism, 199 inflammatory, origin and life history, 167-169 interaction with virus particles, 197

SUBJECT INDEX

intracellular digestion and bacterididal properties, 195-199 light and phase microscopy, 171-173 lysosomes, formation, 182-184 origin of, 181-182 turnover of, 184 macromolecule synthesis, 185-187 in morphogenesis, 198-199 phagocytosis by, 187-192 pinocytosis in, 192-195 proteins pinocytized by, 198 tissue, origin and turnover of, 169 Mast cells degradation, immunologic injury and, 147-148 Mice, NZB, see NZB mice secretory immunoglobulins in, 83 Monocyte ( s ) , 163-214 blood, maturation, 177-179 electron microscopy, 171 emigration from intravascular pool, 167 energy metabolism of, 184-185 light and phase microscopy of, 170171 macromolecule synthesis, 185-187 morphology, 170-173 origin of, 165-166 turnover and intravascular life span, 166-167 Mononuclear phagocytes. See also Macrophages and Monocytes maturation, 176184 in uitro, 177-179 in oioo, 178-177 Mucoantibodies, 3 bronchial, 3 4 , 5 in disease resistance, 5

N Nasal mucosa, secretory immunoglobulins in, 14, 17, 19, 23, 50 Nasopharyngeal tissue, secretory immunoglobulins in, 14 Neutrophilic leukocytes, accumulahon, in nonspecific inflammation, 105-109 accumulation of, 105-127 in animals fixing complement poorly, 113-1 14 in cases of genetic deficiency of complement, 114-116 in nonspecific inflammation, 105-109

283

at sites of immunologic reactions in U ~ U O , 109-113

basic proteins of, tissue injury by, 142146 cathepsins, in tissue injury, 137-141 chemotaxis, 105-127 directional migration, 121-123 mechanisms in uitro, 118-127 immune adherence and, 123-125 factors affecting, 125-127 in connective tissue disease, 152-157 constituents responsible for immunologic tissue damage, 132-146 Iysozymes, 132-137 proteolytic enzymes, 137-141 depletion, effect on acute immunologic reactions, 127-129 immunologic tissue injury mediated by, 97-162 kinin-fodng activity of, 146 role in mediation of acute immunologic reactions, 127-146 slow-reacting substance and, 146 structures in blood vessels and tissues damaged by, 129-132 arterial internal elastic lamina, 129131 vascular and glomerular basement membrane, 131-132 NZB mice, diseases of, 216 as experimental models, 250-281 experimental usage of, 253-261 in hybridization and genetic studies, 253-255 in therapeutic studies, 260-261 in thymectomy and thymus grafts, 2.57-259 in transmission of disease by spleen cells, 255-257 in splenectomy, 259-280 immunology and pathology of, 215 hemolytic disease, 219-225 anemia, 221-222 autoantibody, 219-221 hepatomegaly, 224 splenonragaly, 222-22.5 lymphoproliferative disorders, 231-235 hyperplasia, 231-233 neoplasia, 233-234

284

SUBJECT INDEX

peptic ulceration, 235-236 pulmonary lesions, 234-235 serum proteins, 234 natural life history of, 217-253 renal disease, 225-231 antinuclear antibodies, 230-231 clinical pathology, 228-228 renal histopathology, 228-230 survival and body weight, 218-219 (NZB x NZW) F, hybrid mice, 236-250 as experimental models, 250-253 renal disease, 237-250 survival and body weight, 236-237

P Parasitic infestation, secretory immunoglobulins in, 74-75 Parotid fluid, secretory immunoglobulins, in, 12-13, 14, 23 Pathoptic potentiation, 5 Periodontal disease, secretory immunoglobulins in, 67-69 Pernicious anemia, secretory immunoglobulins in, 69-71 Phagocytes, mouse peritoneal, 179-184 biochemistry, 181 macrophage lysosomes, origin, 181182 morphology and cytochemistry, 179 Phagocytosis, 187-192 attachment and ingestion phases, 189 cell-dependent phenomena of, 187-189 cell maturity and, 188-189 influence of humoral factors, 190-191 complement, 191 cytophilic antibody, 190 opsonizing potential of immunoglobulins, 190-191 macrophage membrane and, 189 metabolic requirements, 188 postphagocytic events, 191-192 biochemical alterations, 192 morphologica1 and cytochemical, 191-192 Pinocytosis, 192-195 induction of, 19,3-195 metabolic requirements of, 193 Platelets, clumping, release of vasoactive materials and, 148-150

Polyarteritis nodosa, neutrophile-mediated injury in, 153-154 Prostatic fluid, secretory immunoglobulins in, 21 Protein( s ) pinocytized by macrophages, 198 synthesis by macrophages, 18S187 Pyrogens, endogenous, synthesis by macrophages, 187

R Rabbit, secretory immunoglobulins in, 81-82 Rectum, secretory immunoglobulins in, 15 Respiratory allergies, secretory immunoglobulins in, 77-79 Respiratory antibodies, as secretory antibodies, 54-57 Respiratory tract, secretory immunoglobulins, 17-19 Rheumatoid arthritis, neutrophile-mediated injury in, 154 Rheumatoid factors, secretory immunoglobulins in, 76-77 Ribonucleic acid, synthesis by macrophages, 186

s Saliva, secretory immunoglobulins in, 12, 50, 50

amino acid composition, 31 isolation, 25 levels, 40 Secretions, external, 6-7 internal, 6-7 Secretory immunoglobulins, 1-96 amino acid composition, 31 analysis, technical problems in, 8-12 in animals, 79-84 antibodies following immunization, 5161 active immunization, 51-59 passive immunization, 59-61 biological properties of, 48-62 chemical and immunological characteristics, 24-38 coinplex with proteins, 38 complement fixation and, 61-62 of digestive tract, 12-17 in disease, 62-79

285

SUBJECT INDEX

antibody deficiency states, 62-67 gastrointestinal type, 67-76 electrophoresis of, 30 evidence for existence of, 6 8 in genital tract, 20-31 historical aspects, 2-6 isolation, 24-26 in mammary gland, 22-24 as “natural” secretory antibodies, 4% 51 isohemagglutinins, 48-49 viral and bacterial antibodies, 49-51 of respiratory tract, 17-20 schematic mode, 38 secretion mechanisms, 45-47 secretory “piece” of, isolation and characteristics, 3 4 3 8 sites of synthesis, 39-47 immunofluorescent studies, 41-43 immunological studies, 3 9 4 1 in vivo radioactive trace studies, 4344 tissue culture, 44-45 three-dimensional conformation, 3,534 tissue localization, 14-15 in urinary tract, 21-22 Seminal plasma, secretory immunoglobulins in, 21-22 Serum, secretory immunoglobulins in, 25 amino acids, 31 isolation, 2.5 levels, 40 properties, 29 Serum proteins, synthesis by macrophages, 187 Serum-sickness arteritis, immunologic tissue injury in, 152 Sheep, secretory immunoglobulins in, 82

Slow-reacting substance (SRS-A), neutrophiles and, 146 Sprite, secretory immunoglobiilins in, 65 Spiitum, secretory immunoglobulins in, 1s20 Stomach, secretory immunoglobulins in, 15 Submaxillary gland, secretory immunoglobulins in, 14 Sweat, secretory immunoglobulins in, 2325

T Tears, secretory antibodies, 56 Telangiectasia, secretory immunoglobulins in, 66-67 Tracheobronchial fluid, secretory immunoglobulins in, 23 Trichomoniasis, antibodies in reproductive tract in, 5

U Urinary tract, secretory immunoglobulins in, 21-22 Urine, secretory immunoglobulins in, 2122, 23, 50, 56

V Vascular injury, immune, nonneutrophile type, 15Cb151 Vasoactive amines, release, immunologic injury and, 147-148 Vibriosis, antibodies in reproductive tract in, 5 Viral antibodies, as secretory immunoglobulins, 49-51 Viruses, macrophage interaction with, 197