Defining criteria for autoimmune diseases (Witebsky's postulates revisited)

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Defining criteria for autoimmune diseases (Witebsky's postulates revisited) Noel R. Rose and Constantin Bona With new knowledge gained from molecular biology and hybridoma technology, as well as the original Witebsky postulates, we propose that three types of evidence can be marshalled to establish that a human disease is autoimmune in origin. They include direct evidence from transfer of pathogenic antibody or pathogenic T cells; indirect evidence based on reproduction of the autoimmune disease in experimental animals; and circumstantial evidence from clinical clues. In 1957, at the dawn of the modern e~a of research on autoimmunity, some rational steps were drawn up to establish the autoimmune etiology of human diseasesL They were consciously modelled on Koch's postulates, and required that an autoimmune response be recog nized in the form of an autoantibody or cell-mediated immunity; that the corresponding antigen be identified, and that an analogous autoimmune response be induced in an experimental animal. Finally, the immunized animal must also develop a similar disease. Stringent as they are, these steps still form a good basis for defining a human autoimmune disease. Now, 35 years later, a great deal of information has been gathered concerning the immune response and qutoimmunity, and the time has come to re-evaluate these original postulates in the light of current knowledge, particularly that acquired using molecular biology and hybridoma technology. On the basis of these developments, recent additional postulates have been put forward to define pathogenic iymphocytes mediating autoimmune diseases 2. This is especially timely since the original concept that autoreactivity is pathological has been challenged by numerous findings which demonstrate that self reactive lymphocytes represent a normal population of the immune system cell repertoirej, with only a small fraction of these representing pathogenic lymphocytes. Thus, the difference between 'physiological' and 'pathological' autoimmunity is now more sharply focused. The types of self react_we lymphocyte clones that induce autoimmunity can be classified into several major categories based on the type of autoantibody produced and not necessarily on the phenotype of the cell.

B-cell clones producing polyreactive antibodies These naturally occurring antibodies bind with low affinity to multiple self and foreign antigens. They are generally encoded by unmutated germline genes and their major characteristic is a high degree of connectivity3. The beneficial effect of such antibodies is not well documented but could play a physiological role in neutralizing toxic metabolites4, in early defense against microorganismss, or in elimination of senile or malignant cells3.

B-cell clones producing antibodies witb exquisite specificity [or autoantigens Hybridoma technology allows for immortalization of precursor cells able to produce monospecific autoantibodies from both normal patients and subjects prone to autoimmune disease 6,7. It has not been possible to show, however, that these antibodies cause disease.

B-cell clones producing pathogenic autoantibodies These antibodies are responsible for the onset of disease and tissue damage following natural or experimental ~ransfer.

T cells responsible for autologous mixed lymphocyte reactions Such T cells appear to be an unanticipated consequence of positive selection in the thymus since they are specific for major histocompatibility complex (MHC) antigens. These cells are activated in the absence of any identifiable foreign antigen by reaction with MHC class II antigenss.Q.

T cells specific for autoantigens (ound in healthy and diseased individuals T-cell clones able to recognize self peptides have been isolated from peripheral blood lymphocytes of human healthy subjects l°. for example, T-cell clones specific for type !! collagen are found in healthy subjeers I' as well as in patients with rheumatoid arthritis ~2.

Pathogenic T cells able to transfer autoimmune disease For logistical reasons, such T cells can be demonstrated only in experimental systems employing genetically identical, inbred animals. We propose that three types of evidence can be marshalled to establish that a human disease is actually autoimmune in origin: direct proof, indirect evidence and circum.~tantial evidence. While direct transfer can prove the au~oimmune origin of an autoantibodymediated disease, for cell-mediated diseases we generally depend upon indirect evidence. Moreover, we suggest that there are many other diseases that are not autoimmune in the strict sense but which, nonetheless, involve immunological reactions, such as the

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viewpoint overproduction of particular cytokines. In addition, we recognize that environmental factors, including infection, frequently initiate autoimmune responses. which may or may not be pathogenic. Direct proof The most straightforward evidence for the autoimmune etiology of a human disease stems from instances where the disease is reproduced in a normal recipient by direct transfer of autoantibody. Obviously such instances are rare. In a classical experiment, Harrington injected himself with plasma from a patient with idiopathic thrombocytopenia t3. The manoeuver produced platelet depletion and a severe bleeding disorder. We are not aware of any similar attempts to reproduce a human autoimmune disease by deliberate antibody transfer because of the obvious risks involved. A few diseases are sometimes transferred as experiments of nature. They result from transplacental transmission of pathogenic IgG au:oantibody from an afflicted mother to the fetus. Neonatal myasthenia gravis ~4, Graves' disease ts and polychondritis ~6 exemplify this situation. Sometimes an autoantibody that is not even recognized as pathogenic in the mother can produce disease in the infant. Such is the case in the fetal heart block syndrome produced by anti-Ro antibody lr and hyperthyroidism produced by anti-thyroidstimulating hormone receptor antibody, placentally transferred from the mother ~8. As an alternative to human-to-human transfer, patient's serum or, even better, purified immunoglobulin, can be infused into experimental animals. Although rare, exact duplicates of the human disease lesions in the animal may show the major pathological manifestations of the human lesion, Two such examples can be cited, injections of immunoglobulin (Ig) from patients with pemphigus into new-born mice causes blisters exhibiting the histopathological characteristics of human skin lesions t. The injection of antibodies specific for the acetylcholine receptor (AchR) into animals sometimes causes myasthenia and induces myograms characteristic of muscle weakness 2°. Unfortunately, the effects of such transfers of human serum to experimental animals are often unpredictable. All of the above examples of serum transfer pertain only to diseases that are due directly to pathogenic autoantihody. Another approach to demonstrate the pathogenicity of an autoantibody is in vitro destruction of cells carrying the corresponding antigen. For example, autoantibodies from patients with paroxysmal cold hemoglobinuria can lyse the erythrocytes from the affected subject, as well as from normal subjects who express the appropriate ailoantigens 2'. Many autoimmune diseases are caused by auroantigen-specific T cells rather than antibody. Since ceil transfer requires matching MHC, human-to-human or human-to-animal transfers are not usually feasible. However, there are now opportunities to carry out such transfers using immunoiogicatly crippled severe combined immune deficient (SCID) mice. Volpg et al. 2" showed that thyroiditis can be produced by fragments of infiltrated human thyroid implanted under the kidney capsule of SCID mice. Duchosal et al. 23 described

lupus-like immune complex lesions in the kidneys of SCID mice infused with peripheral blood lymphocytes from lupus patients. We believe the approach of using immunologically incompetent animal recipients of human cells will increase in the future and will provide an excellent tool to demonstrate pathogenic T cells. Indirect evidence In several experimental models, the role of pathogenic T cells can be demonstrated by transfer: experimental allergic encephalomyelitis (EAE) with myelin basic protein (MBP)-specific CD4* T cellsZ4; uveitis with S-antigen-specific T cellsZS; arthritis with T cells specific for type 1I collagenZ6; diabetes with non-obese diabetic bone marrow into F1 recipients -'7 or skin fibrosis with tight skin mice (TSK) bene marrow cells into C57Bl/6'pa/pa (Ref. 28). Since such cell-transfer experiments cannot be carried out with human subjects, alternative strategies must be used. Reproduction of autoimmune disease in experimental animal models

As opportunities for direct transfer of human autoimmune disease are limited, indirect evidence is more feasible. The time-honored strategy is to identify the offending antigen in the human disease, isolate the equivalent antigen in an experimental animal, and reproduce the essential features of the disease by experimental immunization. The advantage of this approach is its wide applicability. During the past decade, progress has been made in characterizing individual epitopes of autoantigens. Initially this was performed using autoantibodies as probes. With the advent of molecular biology, the cloning of genes; the preparation of short fusion proteins ,,~ defined sequences g~l l iaw 1~:_~u.. . . . . cnrre~pcmding . . . . . . . . . . tu i l |¢.1.1 l y , the utilization of synthetic peptides, allow for the identification of epitoTes on autoantigens recognized by either B or T cells. These tools allow definition of pathogenic epitopes of autoantigens and demonstrate that some of these epitopes are conserved during phylogeny. The best example is the Ache from many distant species which are recognized by autoantibodies of myasthenic patiet~ts ~9. Similarly, we recently showed that monoclonal antibodies from scleroderma patients and from TSK mice (which develop a scleroderma-like syndrome) exhibit the same fine specificity for topoisomerase i, since they bind to the same fusion protein 3°. These findings provide a rational basis for further efforts to induce an autoirnmune disease in experimental animals with a given autoantigen, although several pitfalls are also apparent. In contrast to a spontaneous autoimmune disease, experimental immunization with autologous antigens often requires the use of a potent adjuvant which may affect the pathological manifestations of disease. Different animal species, even different strains of the same species, vary greatly in their susceptibility to experimentally induced autoimmune disease. This reactivity is often related to genetic factors, mainly MHC antigens, since the recognition of self peptide is restricted by M H C antigens3L Therefore, many species and strains may need to be tested before a successful model is developed. Finally, human

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viewpoint Table 1. Evideace of autoimmunity in clinical disease Direct proof

Indirect proof

Transfer of disease by Ab

Transfer of disease Induction of disease in by cells to SCID animals by autoantigen mice

X X

X

X X X

X X X

X

X X

X X

X

X

X

X

X

{X) X

X

X X

X X X X

X

X X

X

X

X X X X X

X X X X

X X X X X X X X X X

X

X

c v t n n ~ n in

X X X

X X

Systemic diseases Autoimmune hemolytic anemia Idiopathic thromboGoodpasture's syndrome Rheumatoid arthritis Sjbgren's syndrome SLE Scleroderma Polymyositis

Autoantibodies or selfGeretic reactive T models cells

AB T cells

Exper- Maternal To imental animals Organ specific diseases Myasthenia gravis Graves' disease Chronic thyroiditis Insulin dependent diabetes Pernicious anemia/atrophic gastritis Addison's disease Azoospermia Polychondritis Uveoretinitis Pemphigus Vitiligo Primary biliary cirrhosis Multiple sclerosis Myocarditis

Transfer of Identifidisease by cation lymphocytes within in expcrim. lesions~ of: models

X

X

Y

X X X X X X X

X X X X

X represents a positiveindication.Ab: antibody;SCiD: severecombined immunedeficienr;SLE:systemiclupus erythematosis. (X) represents the inductionof experimentalallergic encephalitiswhich acts as a model for multiplesclerosis. autoimmune disease is a complex affair involving several autoantigens. This includes the autoantigens responsible for the offset of autoimmune disease as well as the autoantigens released subsequent to primary injury which are responsible for the activation of additional lymphocyte clones that amptif7 autoimmune reactions. At least four experimental diseases are reasonable replicas of human disorders that can reliably be classified as organ-specific, immunization with thyroglobulin (Tg) produces chronic thyroiditis'; with AchR produces myasthenia gravis2°; with uveal S antigens, uveitis2t; and with sperm, orchitis3z. There are some problem areas where an experimental model has not vet been related to a human disease. EAE has many points of similarity with MS,

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but the etiological role of MBP in MS has not yet been established. Immunization of mice with type II collagen produces a synovitisz6, but the role of this type of collagen has not been established with a human rheumatoid arthritis; this is in spite of the finding that by highly sensi-tive techniques such as enzyme-linked immune-spot (ELISPOT) cells producing antibody to collagen I! are found in many synovial fluids from cases of rheumatoid arthritis 33. The pathogenetic antigen has not yet been identified, although many T cells reactive with a heat shock protei¢ have been described 34. When the suspected pathogenic antigen of human disease is not known, an alternative strategy is to use an anti-idiotype carrying the internal image of self antigen as an immunizing agent 33.

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viewpoint Genetically induced disease models With a few exceptions, models of experimentally induced autoirnmunity in animals have been found only for organ-specific autoimmune diseases. To replicate the systemic autoimmune diseases, a number of genetically determined animal models are available. The best studied examples are the New Zealand black (NZB) mice that develop a form of Ctmmbs-positive hemolytic anemia that closely resembles human autoimmuEe (acquired) hemolytic anemia. Autoantibodies obtained from NZB hybridomas injected in normal mice cause severe hemolytic anemia 3s and a fraction of transgenic mice bearing V genes encoding Coombs antibodies develop autoimmune hemolytic anemia ~6. Furthermore, the vast majority of these transgenic mice develop anemia after injection of bacterial lipopolysaccharide (LPS), which is known to impair B-cell tolerance. The F1 hybrids, NZB x NZW, spontaneously develop a multisystem disease closely resembling human SLE. Using cell transfers it has been shown, for instance, that the fundamental abnormality resides in the cells of the immune system, including the bone marrow, thymus and B cells rather than in one of the affected organs j~. At least three other mtmse models produce many of the manifestation of lupus and related connective tissue diseases, such as BXSB, MRL/Ipr and gld mice4°. They have also proved useful for investigating the immunopathogenesis of the diseases. Similarly in NOD mice, which represent an experimental model of type I diabetes 4~, the injection of bone-marrow cells into Fi hybrids causes an ovelt disease after a prodromal period ot a few months 27. Many other genetically determined models of human auroimmune disease have been described. They include NOD mice39 and biobreeding (BB) ra~s4°, which spontaneously develop both auroimmune diabetes and thyroiditis; OS chickens and BUF rats, which develop autoimmune thyroidiri#l; and TSK mice42 and UDC line 200 chickens4~, which develop a sclerodermalike disorder. Isolation of autoantibodies or autoreactive T cells indirect proof to demonstrate the autoimmune origin of a disease may be based on the isolation of autoantibodies or self reactive T cells from the organs which represent the major target of autoimmune attack. As examples, anti-erythrocyte antibodies or anti-platelet antibodies can be elured from erythrocytes or thrombocytes of patients with autoimmune hemolytic anemia or idiopathic thrombocytopenic purpura, respectively. Autoimmune DNA antibodies can be eluted from the kidney of patients with lupus and anti-glomerular basement membrane antibodies from patients with Goodpasture's disease. Cytotoxic T-cell clones specific for thyrocytes have been isolated from thyroids of patients with Graves' syndrome although their pathological significance is not yet established44. The isolation of autoantibodies or self reactive T cells from the organs which represent the major target of autoimmune disease represents an important new approach to constructing a chain of evidence to establish the autoimmune origin of a given human disease.

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A summary of the present status of a number of human diseases is given in Table 1. Circumstantial evidence A number of human diseases are frequently designated 'autoimmune' even though they do not meet the criteria described in the previous two sections. This suspicion is raised by distinctive clinical clues. (1) Association with other autoit~,amune diseases in the same individual or the same family. (2) Lymphocytic infil~ration of target organ, especially if there is a restriction in V gene usage. (3) Statistical association with a particular MHC haplotype or aberrant expression of MHC class II antigens on the affected organ. (a) Favorable response to immunosuppression. This circumstantial evidence by itself cannot define an autoimmune disease, but is able to provide an incentive for future research. Noel R. Rose is at the Dept o[ Immunology and Infectious Diseases, The ]ohns Hopkins University, 615 North Wol[e Street, Baltimore, MD 21205, USA; Constantin Bona is at the Dept of Microbiology, Mount Sinai Scbool of Medicine, Annenberg Building 16-60, 1 Gustave Levy Place, New York, N Y 10029, USA. References 1 Witebsky, E., Rose, N.R., Terplan, K., Paine, J.R. and Egan, R.W. (1957) J. Am. Med. A.~oc. 164, 1439-14-;7 2 Bona, C.A. (19911Autoimmunity 10, 169-172 3 Avrameas, S. t1988) Int. Rev. lmmunol. 3, 1-15 4 Grabar, P. t1983) lmmunol. Today 4, 337-340 5 Coombs,R.R.A., Coombs, A.M. and Ingrain, D.C. 11961) The Serology of Conghttinm and its Relation to Disease, CC Thomas 6 Madaio, M.P., Scharmer, A., Shattner, M, and Schwartz, R.S. (1986) ]. lmmto~ol. 137~2535-2540 7 Liblau, R., Tournier-Lasserve, E., Maciazek, J. et al. (1991) Eur. J. lmmunol. 21, 1391-1395 8 Glimcher,L.H, and Shevach, E.M, ~1982)J. Exp. Med. 156, 640-645 9 Quartin, R,S., Monestier, M., Moran, T.M. et aL t1987) Cell. lmmunoi. 110, 163-175 10 Rees, A.D.M., Lombardi, G., Scoging,A. et aL 11989) Int. lmmunoL 1, 124--129 i 1 Lacour,M., Rudolfi, U., Schlesier, M. and Peter, H.H. (1990) Eur. J. hnmunol. 20, 931--934 12 Londei, M., Verhoef, A., DeBerardinis,P. et al. (1989) Proc. Natl Acad. $ci. USA 86, 8502-8506 13 Harrington, W.J., Minnich, V., Hollingsworth,J.W. and Moore, C.V. (1990) ], Lab. Clin. IV.ed. ! 15, 636-645 14 Lefverr,A.K. {1988} in Anti-idiotype Antibodies m Myasthema Gravis in Biological Application o[ Antiidio~'pes (Bona, C., ed.), pp. 20-25, CRC Press 15 Davies, T. and DeBernardo, F. !1983) Thyroid Autoantibodies atld Diseases: An Overview in Autoimmune Endocrine Disease (Davies, T., ed.L pp. 127, J. Wiley & Sons 16 Arundel, F.W. and Has.qrick,J.R. 0960) Arch. Dermatol. 82, 439--440 17 Atspaugh, M. and Maddison, P.L. (~979) Arthritis Rheum. 22, 796-798 18 Hoffman, W.H, Sahasrananan, P., Ferandos, S.S., Burek, C.L. and Rose, N.R. (1982} ]. Clin. Endocrinol. Metab. 54, 354- 356 19 Anhalt, GJ., Labib, R,S., Voorhees,J.J., Beals, T.F. and Diaz, L.A. (1982) New Engt. J. Med. 306, 1189-1196

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viewpoint 20 Gomez, C.M. and Richman, D.P. (1987)]. lmmunoL 139, 73-76 21 Donath, J. and Lansteiner, K. (1904) Muench. Med. Wocbscbr. 51, 1590-1593 22 Volpd, R., Kasuga, Y., Akasu, F. et al. (1993) Clin. lmmunol, and lmmunopatboL 67, 93-99 23 Duchosal, M.A., McConahey, F.j., 7.~L2...... , ~.A. ~ d Dixon, F.J. (1990}]. Exp. Med. 172, 985-988 24 Zamvil,S., Nelson, P., Trotter, J. et alo (1985) Nature 317, 355-358 25 Hu, L.H., Redmond, M.T., Sanui, H. et al. (1989) Cell. Immunol. 122, 251-261 26 Trentham, D.E., Dynesius,R.A. and David, J.R. (1978) ]. Clin. Invest. 62, 359-366 27 Serreze, D.V., Leiter, E.H., Worthen, S.M. and Sbultz~ L.D. (1988) Diabetes 37, 252-255 28 Phelps, R.G., Daian, C., Shibata, S., Fleischmajer,R. and Bona, C.A.J. Autoimmunity (in press} 29 Lennon,V.A. and Lambert, G.H. (1980) Nature 285, 238-240 30 Muryoi, T., Kasturi, K.N., Kafina, M.J. et al. (1992} J. Exp. Med. 175, 1103-1109 31 Todd, J.A., Acha-Orbea, H., Bell, j.I. et al. (1988) Science 240, 1003-1009 32 Tung, K.S.K. and Menge, A.C. (1985) in The

Autoimmune Diseases (Rose, N.R. and Mackay, I.R., eds), ppo 537-590, Academic Press 33 Tarkovski, X. et al. (1989) Arthritis Rheum. 32, 1087 34 Thompson, S.J., Rook, A.W., Brealey, R.J., Vender Zee, R. and Elson, C.J. (1990} Eur. J. lmmunol. 20, 2479-2484 35 Reininger, L., Shibata, T., Ozaki, S. et al. (1990) Eur. J. ImmunoL 20, 771-777 36 Okamoto, M., Murakami, M., Shimizu, A. et al. (1992) ]. Exp. Med. 175, 71-79 37 Sekigawa, I., lshida, Y., Hirose, S., Sato, H. and Shirai, T. (1986)J. hnmunol. 136, 1247-1252 38 Theofilopoulos, A.Ig. and Dixon, F.J. (1985) Adv. lmmunol. 37, 269-390 39 Makino, S., Kunimoto, Y., Muraoka, Y. et al. (1980} Exp. Anita. 29, 1-13 40 Nakhooda, A.F., Like, A.A., Chappel, C.I., Murray, F.T. and Marliss, E.B. (1977) Diabetes 26, 100-112 41 Rose,N.R., Kong, Y.M. and Sundick, R.S. (1980) Clin. Exp. lmmuno[. 39, 545-550 42 Green, M.C., Sweet, H.O. and Bunker, LE. (1976) Am. J. Pathol. 82, 493-512 43 Haynes, D.C. and Gershwin, M.E. (!984) 1. Clin. Invest. 73, 1t57-1568 44 McKenzie,W.A. and Davis, T.F. (1987} immunology 61,101-103

A three-tiered view of the role of IgA in mucosal defense Mary B. Mazanec, John G: Nedrud, Charlotte S. Kaetzel and Michael E. Lamm Mucosal lgA has generally been viewed as an immune barrier to prevent the adherence and absorption o f antigens. Recent studies employing polarized epithelial monolayers have suggested two additional functions for mucosal IgA. One is to neutralize intracellular microbial pathogens, such as viruses, directly within epithelial cells. The second is to bind antigens in the mucosal lamina propria and excrete tbem through the adjacent epithelium into the lumen, thereby ridding the body o f locally formed immune complexes and decreasing their access to the systemic circulation. The immune system has traditionally been divided into cellular and humoral arms, with the former assigned to protecting against intracellular and the latter against extracellular agents. With regard to humoral immunity, antibodies in mucosal secretions have features that distinguish them from the antibodies in the blood ~. For example, mucosal antibodies are svnthe,fized locally and are predominantly of the immunoglobulin A (IgA) isotype. Furthermore, far from being a minor class of immunoglobulin, as was initially surmized from the lower concentration of IgA than IgG in the serum, it is now recognized that the body synthesizes more lgA than all other isotypes of immunoglobulin combined2. Obviously, IgA, and more particularly mucosal IgA (since most IgA is locally synthesized for export from the body), must be functionally important.

lgA as an immunological barrier The majority of the IgA produced enters the mucosal secretions via epithelial transcytosis mediated by the polymeric Ig receptor (also known as the transmembrane secretory component) ~, Polymeric IgA binds to the receptor on the basolateral surface of mucosal epithelial cells and is endocytosed and transported to the apical surface. At this surface, proteotytic cleavage between the membrane-spanning and extracellular domains of the polymeric lg receptor results in the release into the lumen of secretory lgA, a compiex of polymeric lgA and secretory component (the soluble extracellular domain of the polymeric Ig receptor). Many instances of resistance to infection, both clinical and experimental, can be correlated with specific titers of secretory IgA antibodies4-n. There has been

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