Symposium on Rheumatic Diseases
Immunologic and Viral Factors in Autoimmune Diseases Norman Talal, MD.*
"Is it too optimistic to suggest that we are close to understanding the pathogenesis of systemic lupus erythematosus? The rapid advance of knowledge in immunology and virology and the extensive study of autoimmunity in the New Zealand strains of mice may have brought us to this point. ''33 That statement, written seven years ago, is an appropriate introduction to a discussion of recent progress in understanding autoimmunity in relation to immunologic regulation and modulation of surface membrane antigens on lymphocytes. The general thesis presented is that surveillance of lymphocyte membrane antigens is an important element in normal immunologic regulation. This surveillance represents a physiologic form of self-recognition which is necessary for the proper functioning of the immune system, but which may lead to the development of autoimmunity under a wide range of circumstances. These circumstances include viral and other infections, drug ingestion, immunodeficiency states, and the aging process. Seen in this context, autoimmunity is a common event that occurs readily because of the inherent regulatory properties of the immune response. This article first reviews genetic and cellular aspects of immunologic regulation as they relate to autoimmunity. This leads into a discussion of the natural history of New Zealand Black (NZB) and NZB/NZW Ft mice, strains which are genetically predisposed to the spontaneous development of an autoimmune disorder resembling systemic lupus erythematosus. This mouse model for human lupus continues to provide important observations concerning viral and immunologic factors in disease. Recent experiments from our own laboratory on T cell regulation and sex hormone modulation of the antibody response to nucleic acids are discussed. Finally, a hypothesis is presented that attempts to integrate genetic, immunologic, and viral factors in a general scheme of pathogenesis. 'Professor of Medicine, University of California School of Medicine, San Francisco Supported by USPHS Grants AM16140 and Ca15486, and by research funds from the Veterans Administration
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GENERAL CONCEPTS OF IMMUNOLOGIC REGULATION Immunologic regulation depends upon a highly complex set of interactions occurring between antigen, antibody, immune complexes, complement components, lymphocytes, and macrophages. A network of cell membrane receptors, and factors capable of binding to such receptors, are responsible for controlling the many and diverse activities of the immune response. The immune system, like the nervous system, is highly dependent upon appropriate cellular interactions and upon the transmission of chemical signals across cell membranes. The physiologic control of the immune system involves a complex integration of genetic, cellular, and possibly viral components expressed on the cell membrane. When the immune response is functioning effectively, there is a harmonious appearance of normal reactivity expressed in the events of antibody formation, delayed hypersensitivity, and selfnonself discrimination. Regulation is apparent in the appropriate initiation and termination of immune responses involving T and B lymphocytes, in the differentiation of B lymphocytes into plasma cells, in the switch from IgM to IgG antibodies, and in the general utility of these responses to the host who is protected from infections and neoplasia. The major factors involved in immunologic regulation are outlined below. Genetic Aspects A. Immune response (Ir) genes are located within the major histocompatibility complex (MHC) B. Immune response associated (la) antigens and H, molecules are present on lymphocyte membranes. Immunologic Aspects A. Cellular cooperation involving lymphocytes and macrophages (T cellT cell, T cell-B cell, T cell-macrophage, B cell-macrophage). B. Primary role of T lymphocytes in immune regulation depends upon an equilibrium between helper and suppressor T cells. C. Regulatory T cells produce factors which contain la determinants and are antigen-specific (? express variable region genes of immunoglobulin heavy chains). D. Histocompatibility requirement for immune cell collaboration.
The separation of genetic and immunologic factors is highly artificial. Immune response (Ir) genes located within the major histocompatibility complex (H 2 in the mouse, HLA in man) play major roles in determining the magnitude and immunoglobulin class of the antibody response. The expression of immune response associated (la) and H, antigens on the lymphocyte and macrophage surface are important in cellular cooperation and signal recognition. The initiation and termination of an immune response, either humoral or cellular, probably depend upon several different kinds of cellular interactions involving T cells, B cells, macrophages, and possibly other cells in the lymphocyte series. The primary responsibility for control of these responses falls to the helper T and suppressor T cells. In mice, these appear to be separate T cell subpopulations expressing
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distinct membrane alloantigens. The equilibrium established by these regulatory cells may determine whether autoimmunity occurs or is prevented. For example, B lymphocytes with immunoglobulin receptors for autoantigens are present in healthy humans and normal strains of mice. Such autoantigen-binding cells have been clearly shown for thyroglobulin' and DNN in humans, and for RNA (polyadenylic acid)23 and certain erythrocyte antigens'3 in mice. In some circumstances, the numbers of such cells are only slightly greater in autoimmune individuals than in normal controls. The existence of autoantigen-binding lymphocytes in healthy humans and mice indicates a very real predisposition to autoimmunity in normals, and raises the question of why some individuals develop autoimmune disease and others do not. Suppressor T cells are thought to play a major role in preventing autoantibody production by these autoantigen-binding lymphocytes. The size of these autoimmune clones is kept small, and autoantibody production minimal, by the action of suppressor T cells. These potentially autoreactive cells may also explain the wide variety of circumstances and relative ease with which autoimmunity can be induced, e.g., by drugs, infectious agents, the aging process, and immunodeficiency. Autoimmunity is reversible in many of these situations, which may represent the organism's ability to restore physiologic control and abort the autoimmune state. An alternative way to induce autoimmunity, even in the face of adequate suppressor function, would be through the generation of excessive helper T cells. Helper T cells are generally required for B cells to progress in their differentiation pathway from IgM to IgG production. The sequential development of IgM and later IgG autoantibodies has been shown for antibodies to nucleic acids 19 , 27, 32 and is discussed in another section of this article. T cell regulation is mediated by the release of factors which may act on other T cells, on B cells, or on macro phages. Both nonspecific and specific factors have been described. Some factors enhance and others suppress immune responses. The factors contain la determinants as well as binding sites for specific antigens, suggesting that T cells may express variable region genes of immunoglobulin heavy chains. T cell membranes also contain idiotypic sites (unique individually specific immunoglobulin determinants) which are also present on secreted antibody and on B cell membranes (where they function as receptors for interaction with antigen). Thus, T cells may have surface receptors which at least partially resemble the immunoglobulin receptors on B cells. The presence of MHC gene products on T cells and T cell factors relates to the histocompatibility requirement for immune cell collaboration. Much of our understanding of immunologic regulation and autoimmunity comes from studies in animals, although evidence is now accumulating for suppressor cells and regulatory factors in humans as well. Observations in New Zealand Black (NZB) and related hybrid mice made over the last 20 years are presented as an example of information obtainable from animal models of human disease.
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SPONTANEOUS AUTOIMMUNE DISEASE IN NZB AND NZB/NZW MICE Abnormal regulation of the immune system is a feature of many human and experimental autoimmune disorders, and is well demonstrated in New Zealand Black (NZB) and NZB/NZW F1 (B/W) mice who spontaneously develop diverse autoantibodies, immune complex glomerulonephritis, defective T cell function, monoclonal macroglobulinemia, and malignant lymphomas. 1o , 30, 31 Genetic, immunologic, and viral factors all play a role in pathogenesis. The genetic contribution is poorly understood, although multiple autosomal genes are involved, The mice harbor C-type oncornaviruses 12 , 15 and make antibodies to viral antigens, particularly to glycoprotein (gp) 70 which is the major envelope glycoprotein of this virus. 36 Our laboratory has studied the control of immune reactivity and autoantibody formation in these mice, focusing on the role of T lymphocytes and sex hormones.32 A major defect in T cell regulation, modulated by sex hormones and possibly involving viruses, may be an important mechanism promoting autoimmunity and lymphoid neoplasia in these strains. New Zealand mice are not the only ones that develop these problems. The recently described MRL/1 strain becomes ill with massive lymphoproliferation and a severe autoimmune disease characterized by immune complex glomerulonephritis, hypergammaglobulinemia, formation of antinuclear factors and antibodies to DNA.l7 These mice are genetically unrelated to the NZB. A single mutant autosomal recessive gene determines the lymphoproliferation which is not clearly malignant and contains numerous T cells. Other murine strains (SW AN, PN) also develop autoimmune disease similar to the NZB and B/W. Thus, an expanding number of autoimmune models are becoming available for study. The key features of spontaneous disease and associated immunologic deficits in NZB and B/W mice are summarized as follows: (1) clinically normal at birth although immunologically hyperactive and prematurely competent; (2) decline in suppressor T cell activity, T cell tolerance, and serum thymic humoral factors at one to two months; (3) autoantibodies increase in tit er from three months onward, first in females; (4) hemolytic anemia and immune complex glomerulonephritis appear after five months; and (5) decreased cellular immunity, lymphoproliferation, and monoclonal macroglobulinemia after eight months. The mice appear clinically normal at birth although some aspects of immunologic hyperactivity (both humoral and cellular) are present even this early in life. Immune competence develops prematurely compared with normal strains/ suggesting an abnormality of immunologic regulation that first develops in utero. Suppressor T cell activity declines between one to two months of age in NZB mice,3 associated with an inability to develop tolerance to disaggregated foreign gammaglobulin 26 and a deficiency of thymic humoral factors. The administration of thymic factors at this age can restore suppressor activity;4 however, the injection of thymosin is without benefit thus far on the course of disease. B , 34
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The first clinical appearance of autoantibodies occurs at about two to three months of age, although they can be detected much earlier by more sensitive methods. Antibodies to erythrocytes and to lymphocyte surface antigens appear in almost every NZB mouse, whereas antibodies to nucleic acids are found in B/W mice. Antibodies cytotoxic to T lymphocytes occur commonly in young NZB mice,25 are characteristically IgM, and do not distinguish NZB T cells from other T lymphocytes. This antibody occurs less often in B/W mice, a strain in which individual animals may develop antibodies to nucleic acids without detectable serum levels of antibodies to T cells.9 There is a marked sex difference in B/W mice, females developing severe immune complex glomerulonephritis and dying approximately four months earlier than males. The renal deposits contain complement, antibodies to DNA,!! and antibodies to the major glycoprotein (gp 70) of murine C-type viruses. 36 The more accelerated disease of female B/W mice is associated with earlier appearance and greater amounts of IgG antibodies to DNA.19 The titers of autoantibodies rise progressively, associated with a general augmentation of humoral immunity. Adult NZB and B/W mice make excessive antibody responses to several experimental antigens, including foreign proteins, sheep erythrocytes, and synthetic nucleic acids. 32 . 33 The major manifestations of disease become clinically apparent after five months of age. lO NZB mice develop Coombs' positive hemolytic anemia; B/W mice develop proteinuria and uremia. Both strains develop marked lymphocytic and plasmacytic infiltrates in many different organs. This extensive lymphoproliferation may suggest malignancy.H,3! Mice that survive tl,le autoimmune disorder are susceptible to the development of malignant lymphomas, at times associated with the production of monoclonal IgM.29 The development of lymphoma is enhanced by the administration of immunosuppressive drugs, such as azathioprine and cyclophosphamide. These older mice often have marked deficiencies of cell-mediated immunity, as shown by impaired proliferative responses to phytomitogens, reduced capacity to induce graft-versus-host disease or to reject malignant tumors and skin grafts. The number of T cells in their peripheral lymphoid organs are decreased, and their ability to mount antibody responses to sheep red blood cells and other experimental antigens may decline to levels below those of normal strains. Disordered Immunologic Regulation and Autoimmunity in NZB and NZB/NZW Mice Experimental evidence suggests that B cell and helper T cell functions are increased in NZB and B/W mice, whereas suppressor T cell activity is decreased. The simplest explanation would be a defect in suppressor T cells or factors which then fail to regulate helper T cells and B cells. Much current work is attempting to define the precise mechanisms responsible for this failure. Antibodies to DNA and RNA can be detected in low titers as early
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as four weeks of age in B/W mice, using a sensitive filter radioimmunoassay and sucrose density gradient ultracentrifugation to fractionate serum. Antibodies to DNA are both IgM and IgG,'9. 32 whereas antibodies to RNA (polyadenylic acid, poly A) are exclusively IgM in these young mice. There is a striking difference between female and male animals in the Ig distribution of antibodies to DNA. Females have much more IgG anti-DNA than do males. Antibodies to DNA increase slowly from one to four months, as shown by progressively greater serum binding activity. The Ig distribution of anti-DNA antibodies remains relatively unchanged until five to six months when, in females, there is a sudden and marked increase in DNA binding by IgG antibodies. This same sequence of events is repeated in males, but offset in time by approximately four months. The males make predominantly IgM antibodies to DNA until nine months of age, when there is a great increase in IgG binding of DNA. The timing of the switch from IgM to greatly increased IgG antibodies to DNA correlates, in each sex, with the onset of severe glomerulonephritis leading rapidly to death. This switch appears to herald the onset of a severe exacerbation of disease. These results on anti-DNA responses are paralleled by studies on antibodies to poly A which are also produced spontaneously by B/W mice. These antibodies are only IgM in young mice, and then switch to IgG in older animals. The switch occurs first in females (at seven months), and later in males (at 11 months). The IgM to IgG switch in antibody synthesis is a major event in B cell differentiation. The two influences that promote this event are antigen and T cells, probably acting together. T cell control is mediated by factors (as already discussed) which contain antigen binding sites as well as la determinants. In normal mouse strains, the switch from IgM to IgG is highly T cell dependent. T cells or T cell factors can convert strains of genetically low responder status to high responder status, an event accompanied by an IgM to IgG switch. Nude mice or thymectomized mice react to thymicdependent antigens by producing predominantly IgM rather than the expected IgG antibodies. For these reasons, we presumed that the IgM to IgG switch for nucleic acid antibodies reflected the activity of regulatory T cells, and might provide an indirect but highly relevant way to study T cell function. The Ig switch could represent the conversion from a partial to a more complete regulatory defect with further impaired ability to suppress disease expression. Neonatal thymectomy significantly increased mortality in male B/W mice. 22 The thymectomized males developed an immediate and persistent increase in antibodies to DNA, associated with an accelerated switch to IgG which occurred at four rather than at nine months. These results are consistent with the elimination of a thymic suppressor influence in the males, resulting in an essentially female pattern of disease expression. By contrast, neonatal thymectomy of females had relatively little effect on antibodies to DNA, suggesting that this antibody response is already independent of a thymic suppressor mechanism at the time of surgery. This could occur because helper T cells for anti-DN A
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are relatively more peripheralized in newborn female compared with male B/W mice. Prepubertal castration of males (performed at two weeks) also resulted in significantly shortened survival, whereas castration had no effect in females. 21 Castrated males had greater amounts of antibodies to DNA and developed a premature switch to IgG . In these respects they resembled thymectomized males and female B/W mice. Thus castration of males, like thymectomy, caused a worsening of disease and an accelerated appearance of IgG antibodies to DNA. Castrated females had little change in antibodies to DNA. The effects of castration indicate that sex hormones (like the thymus) greatly influence the spontaneous antibody response to DNA and the severity of disease in B/W mice. The major influence appears to be a protective effect of male hormone. This protective effect of androgen probably explains why autoimmunity in general is more common in females or in males who fail to produce androgens (Klinefelter's syndrome). Our results indicate similarities between castration and thymectomy, and suggest a mechanism by which sex hormones influence these immune responses. The elimination of androgen was similar to removing a thymic suppressor mechanism, allowi,ng an IgG switch for antibodies to DNA. These effects on autoantibodies are consistent with the known regulatory effects of sex hormones on normal immune responses. For example, female mice are less susceptible to immunologic tolerance than males. Female mice of various normal strains have higher antibody responses to several different antigens when compared with males. Castrated male mice give an augmented "female" response to these antigens. These results in normal strains, like the results in B/W mice, suggest that sex hormones may modulate T cell regulation. Thus, sex differences in autoimmunity may be aberrant expressions of the normal physiologic effects that sex hormones exert on immune reactivity. In human lupus, antibodies to DNA and RNA may belong to either the IgM or IgG immunoglobulin class. Active lupus nephritis is associated in particular with serum IgG antibodies to DNA which deposit as immune complexes in the renal glomeruli. At the other end of the spectrum, asymptomatic relatives of patients with systemic lupus erythematosus (SLE) have a significant increase of antibodies to RNA but not to DNA.5 Four families demonstrated antinucleic antibodies both in the SLE probands and in asymptomatic family members. IgG antibodies to nucleic acids (both DNA and RNA) were present in the patients and correlated with active SLE. Antibodies to RNA in the asymptomatic relatives were present only in the IgM class.35 This may represent a limited form of the lupus diathesis, and raises the possibility that a switch from IgM to IgG antibodies to nucleic acids may occur in human SLE as well as in B/W mice. The general conclusions drawn from these studies are: 1. Autoantibody responses are regulated, but the regulation is disordered and inappropriate.
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2. Regulation is related to the thymus, probably to the action of helper and suppressor T cells. 3. Sex hormones modulate T cell regulation; androgens are protective and may help maintain suppressor T cell activity. 4. The IgM to IgG switch may be an indirect but relevant way to study regulation in murine lupus and possibly also in human lupus. These conclusions suggest that regulatory mechanisms exert influences upon autoantibody responses, but that the regulation promotes rather than suppresses autoimmunity. The possible utilization of androgens for the treatment of murine lupus is also suggested. Such experiments are currently under way in our laboratory.
VIRUSES AND IMMUNOLOGIC REGULATION Autoimmunity can be thought of as a disturbance in immunologic regulation in which the balance between helper and suppressor T cells is disturbed. This might arise from a deficiency or malfunction of suppressor T cells, an abnormal activation of helper T cells, a bypass of the requirement for T cell help, or by a combination of several mechanisms. Viruses could interfere with normal control mechanisms in a way that might create a regulatory imbalance and lead to autoimmunity. Much current evidence suggests a possible role for C-type oncornaviruses in the pathogenesis of lupus,t2 although definitive proof is lacking and questions have been raised about the adequacy of experimental design and specificity of serologic reagents. C-type viruses are named for their characteristic appearance in the electron microscope. They occur abundantly in NZB and B/W mice where they can be seen in many tissues and at all ages starting from fetal life. These viruses contain RNA in their virion which is transcribed by an RNA-dependent DNA polymerase and then incorporated into the host genome. As part of the genome, viral information can be expressed along with the host's own genetic information. These viruses bud from cell surfaces and viral antigens are expressed on cell membranes. One of these antigens (gp 70) is the major envelope glycoprotein of the C type virus. Antibodies to gp 70 are found in high titer in NZB and B/W mice, and immune complexes containing gp 70 are found in the kidney deposits in B/W mice. 36 Antigenic material cross-reactive with antisera to primate C-type viruses are also present in the tissues of lupus patients 31 and in the kidney deposits in lupus glomerulonephritis. 16 , 18 These represent the latest findings in a long line of research that has attempted to relate lupus to chronic or persistent virus infection. This subject has been well summarized elsewhere. 12 , 20 A possible way that viral antigens on cell membranes may interfere with immunologic regulation deserves further consideration. The lymphocyte membrane represents a common meeting ground where histocompatibility antigens, immune response associated antigens (la), and viral antigens can interact and influence each other (Fig. 1). A normal surveillance mechanism exists whereby new antigens expressed
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Io Figure 1. Schematic representation of the lymphocyte surface shows the presence of H2 antigens , viral antigens (gp 70), and immune response associated (la) antigens.
on the lymphocyte surface are recognized immunologically in association with histocompatibility antigens. This recognition has been demonstrated both for chemical and viral antigens, and may represent an important mechanism in immunobiology. For example, infection of mice with lymphocytic choriomeningitis virus (LCM) leads to the generation of an "altered self" surface antigen on lymphocytes that requires both H2 determinants and viral determinants for recognition. 37 The H2 molecules may serve as receptors and, by associating with new membrane antigens, promote an immune response to these new antigens. 6 It is possible that recognition of C-type viral antigens may occur through a similar mechanism. The fact that gp 70 and H2 molecules demonstrate functional interactions on the surface of a lymphoid tumor favors this hypothesis. 24 Thus, circumstances might arise in which viral antigens and self antigens on the lymphocyte surface might interact inappropriately in a way that could generate potentially harmful immune reactivity. If this occurred on the surface of helper or suppressor T cells, it could lead to modification of existing regulatory mechanisms and perhaps fa vor the generation of autoimmunity. Although still hypothetical, this postulate integrates genetic and viral factors in autoimmunity, and is in agreement with current concepts of membrane modulation and receptor triggering in many biological phenomena. 6
REFERENCES 1. Bankhurst, A. D., Torrigiani, G., and Allison, A. C.: Lymphocytes binding human thyro-
2. 3. 4. 5. 6.
globulin in healthy people and its relevance to tolerance for autoantigens. Lancet, 1 :226, 1973. Bankhurst, A. D., and Willia ms, R C.: Identification of DNA binding lymphocytes in patients with systemic lupus erythematosus. J. Clin. Invest. , 56:1378, 1975. Barthold, D. R. , Kysela, S., and Steinberg, A. D. : Decline in suppressor T cell function with a ge in female NZB mice. J. Immunol., 112 :9 , 1974. Dauphinee, M. J., Talal, N ., Goldstein, A. L., et aI.: Thymosin corrects abnormal DNA synthetic response of NZB mouse thymocytes. Proc. Nat. Aca d. Sci., 71 :2637, 1974. DeHoratius, R J., Pillarisetty, R, Messner, RP., et aI.: Anti-nucleic acid antibodies in systemic lupus erythematosus patients and their families. J. Clin. Invest., 56:11491154, 1975. Edelma n , G. M.: Surface modulation in cell recognition and cell growth: Some new hypotheses on phenotypic alteration and transmembranous control of cell surface receptors. Science, 192 :218-226, 1976.
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7. Evans, M. N., Williamson, W. G., and Irvine, W. J.: The appearance of immunological competence at an early age in New Zealand black mice. Clin. Exp. Immunol., 3 :375, 1968. 8. Gershwin, M. E., Steinberg, A. D., Ahmed, A., et al.: Study of thymic factors. 11. Failure of thymosin to alter the natural history of NZB and NZB/NZW mice. Arthritis Rheum., 19:862-866,1976. 9. Goldblum, R., Pillarisetty, R., and Talal, N.: Independent appearance of anti-thymocyte and anti-RNA antibodies in NZB/NZW FJ mice. Immunology, 28 :621, 1975. 10. Howie, J. B., and Helyer, B. J.: The immunology and pathology of NZB mice. Adv. Immunol., 9 :215-266, 1968. 11. Lambert, P. H., and Dixon, F. S.: Pathogenesis of the glomerulonephritis of NZB/NZW mice. J. Exp. Med., 127:507, 1968. 12. Levy, J.: Autoimmunity and neoplasia: The possible role of C-type viruses. Amer. J. Clin. Pathol., 62:258-280,1974. 13. Linder, E., and Edgington, T. S.: Immunobiology of the autoantibody response. 11. The lipoprotein-associated soluble HB erythrocyte autoantigen of NZB mice. J. Immunol., 110:53-62, 1973. 14. Mellors, R. C.: Autoimmune and immunoproliferative diseases of NZB/Bl and hybrids. Int. Rev. Exp. Pathol., 5:217,1966. 15. Mellors, R. C., and Huang, C. Y.: Immunopathology of NZB/Bl mice. V. Virus-like (filtrable) agent separable from lymphoma cells and identifiable by electron microscopy. J. Exp. Med., 124:1031-1038, 1966. 16. Mellors, R. C., and Mellors, J. W.: Antigen related to mammalian type-C RNA viral p30 protein located in renal glomeruli in human systemic lupus erythematosus. Proc. Nat. Acad. Sci., 73:233-237,1976. 17. Murphy, E. D., and Roths, J. B.: A single gene model for massive lymphoproliferation with immune complex disease in new mouse strain MRL. Proc. 16th Int. Congr. Hematol., Kyoto, Japan, Sept. 5-11, 1976. Amsterdam, Excerpta Medica. 18. Panem, S., Ordonez, N. G., Kirstein, W. H., et al.: C-type virus expression in systemic lupus erythematosus. New Engl. J. Med., 295:470-485,1976. 19. Papoian, R., Pillarisetty, R. J., and Talal, N.: Immunologic regulation of spontaneous antibodies to DNA and RNA. II. Sequential switch from IgM to IgG in NZB/NZW FJ mice. Immunology, 31 :121-125,1976. 20. Phillips, P. E.: The virus hypothesis in systemic lupus erythematosus. Ann. Intern. Med., 83 :709-715, 1975. 21. Roubinian, J. R., Papoian, R., and Talal, N.: Androgenic hormones modulate autoantibody responses and improve survival in murine lupus. (J. Clin. Invest., in press.) 22. Roubinian, J. R., Papoian, R., and Talal, N.: Effects of neonatal thymectomy and splenectomy on survival and regulation of autoantibody formation in NZB/NZW FJ mice. (J. Immunol., in press.) 23. Sawada, S., Michalski, J. P., Pillarisetty, R. J., et al.: Lymphocytes binding polyadenylic acid and synthesizing antibodies to nucleic acids in autoimmune and normal mice. (Manuscript in preparation.) 24. Schrader, J. W., Cunningham, B. A., and Edelman, G. M.: Functional interactions of viral and histocompatibility antigens at tumor cell surfaces. Proc. Nat. Acad. Sci., 72 :50665070,1975. 25. Shirai, T., and Mellors, R. C.: Natural cytotoxic autoantibody and reactive antigen in New Zealand Black and other mice. Proc. Nat. Acad. Sci., 68: 1412, 1971. 26. Staples, P. J., and Talal, N.: Relative inability to induce tolerance in adult NZB and NZB/NZW FJ mice. J. Exp. Med., 129:123,1969. 27. Steward, M. W., and Hay, F. C.: Changes in immunoglobulin class and subclass of antiDNA antibodies with increasing age in N/ZBW F J hybrid mice. Clin. Exp. Immunol., 26:363-370, 1976. 28. Strand, M., and August, J. T.: Type-C RNA virus gene expression in human tissue. J. Virol., 14:1584-1596, 1974. 29. Sugai, S., Pillarisetty, R. J., and Talal, N.: Monoclonal macroglobulinemia in NZB/NZW F J mice. J. Exp. Med., 138:989,1973. 30. Talal, N.: Animal models for systemic lupus erythematosus. Clin. Rheum. Dis., 1 :485496,1975. 31. Talal, N.: Autoimmunity and lymphoid malignancy in New Zealand black mice. In Schwartz, R. S. (ed.): Progress in Clinical Immunology. New York, Grune and Stratton, 1973, pp. 101-120. 32. Talal, N.: Disordered immunologic regulation and autoimmunity. Transplant. Rev., 31 :240-263, 1976. 33. Talal, N.: Immunologic and viral factors in the pathogenesis of systemic lupus erythematosus. Arthritis Rheum., 13:887-894,1970.
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34. Talal, N., Dauphinee, M. J., Pillarisetty, R. J., et al.: Effect of thymosin on thymocyte proliferation and autoimmunity in NZB mice. Ann. N.Y. Acad. Sci., 249:438,1975. 35. Talal, N., Pillarisetty, R. J., DeHoratius, R. J., et al.: Immunologic regulation of spontaneous antibodies to DNA and RNA. I. Significance of IgM and IgG antibodies in SLE patients and asymptomatic relatives. Clin. Exp. Immuno!., 25 :1-6, 1976. 36. Yoshiki, T., Mellors, R. C., Strand, M., et al.: The viral envelope glycoprotein of murine leukemia virus and the pathogenesis of immune complex glomerulonephritis of New Zealand mice. J. Exp. Med., 140:1011-1027,1974. 37. Zinkernagel, R. M., and Doherty, P. C.: Hz compatibility requirement for T cell-mediated lysis of target cells infected with lymphocytic choriomeningitis virus. J. Exp. Med., 141 :1427, 1975. Department of Medicine University of California School of Medicine Third Avenue and Parnassus Street San Francisco, California 94143
Immunologic and Viral Factors in Autoimmune Diseases Norman Talal, MD.*
"Is it too optimistic to suggest that we are close to understanding the pathogenesis of systemic lupus erythematosus? The rapid advance of knowledge in immunology and virology and the extensive study of autoimmunity in the New Zealand strains of mice may have brought us to this point. ''33 That statement, written seven years ago, is an appropriate introduction to a discussion of recent progress in understanding autoimmunity in relation to immunologic regulation and modulation of surface membrane antigens on lymphocytes. The general thesis presented is that surveillance of lymphocyte membrane antigens is an important element in normal immunologic regulation. This surveillance represents a physiologic form of self-recognition which is necessary for the proper functioning of the immune system, but which may lead to the development of autoimmunity under a wide range of circumstances. These circumstances include viral and other infections, drug ingestion, immunodeficiency states, and the aging process. Seen in this context, autoimmunity is a common event that occurs readily because of the inherent regulatory properties of the immune response. This article first reviews genetic and cellular aspects of immunologic regulation as they relate to autoimmunity. This leads into a discussion of the natural history of New Zealand Black (NZB) and NZB/NZW Ft mice, strains which are genetically predisposed to the spontaneous development of an autoimmune disorder resembling systemic lupus erythematosus. This mouse model for human lupus continues to provide important observations concerning viral and immunologic factors in disease. Recent experiments from our own laboratory on T cell regulation and sex hormone modulation of the antibody response to nucleic acids are discussed. Finally, a hypothesis is presented that attempts to integrate genetic, immunologic, and viral factors in a general scheme of pathogenesis. 'Professor of Medicine, University of California School of Medicine, San Francisco Supported by USPHS Grants AM16140 and Ca15486, and by research funds from the Veterans Administration
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GENERAL CONCEPTS OF IMMUNOLOGIC REGULATION Immunologic regulation depends upon a highly complex set of interactions occurring between antigen, antibody, immune complexes, complement components, lymphocytes, and macrophages. A network of cell membrane receptors, and factors capable of binding to such receptors, are responsible for controlling the many and diverse activities of the immune response. The immune system, like the nervous system, is highly dependent upon appropriate cellular interactions and upon the transmission of chemical signals across cell membranes. The physiologic control of the immune system involves a complex integration of genetic, cellular, and possibly viral components expressed on the cell membrane. When the immune response is functioning effectively, there is a harmonious appearance of normal reactivity expressed in the events of antibody formation, delayed hypersensitivity, and selfnonself discrimination. Regulation is apparent in the appropriate initiation and termination of immune responses involving T and B lymphocytes, in the differentiation of B lymphocytes into plasma cells, in the switch from IgM to IgG antibodies, and in the general utility of these responses to the host who is protected from infections and neoplasia. The major factors involved in immunologic regulation are outlined below. Genetic Aspects A. Immune response (Ir) genes are located within the major histocompatibility complex (MHC) B. Immune response associated (la) antigens and H, molecules are present on lymphocyte membranes. Immunologic Aspects A. Cellular cooperation involving lymphocytes and macrophages (T cellT cell, T cell-B cell, T cell-macrophage, B cell-macrophage). B. Primary role of T lymphocytes in immune regulation depends upon an equilibrium between helper and suppressor T cells. C. Regulatory T cells produce factors which contain la determinants and are antigen-specific (? express variable region genes of immunoglobulin heavy chains). D. Histocompatibility requirement for immune cell collaboration.
The separation of genetic and immunologic factors is highly artificial. Immune response (Ir) genes located within the major histocompatibility complex (H 2 in the mouse, HLA in man) play major roles in determining the magnitude and immunoglobulin class of the antibody response. The expression of immune response associated (la) and H, antigens on the lymphocyte and macrophage surface are important in cellular cooperation and signal recognition. The initiation and termination of an immune response, either humoral or cellular, probably depend upon several different kinds of cellular interactions involving T cells, B cells, macrophages, and possibly other cells in the lymphocyte series. The primary responsibility for control of these responses falls to the helper T and suppressor T cells. In mice, these appear to be separate T cell subpopulations expressing
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distinct membrane alloantigens. The equilibrium established by these regulatory cells may determine whether autoimmunity occurs or is prevented. For example, B lymphocytes with immunoglobulin receptors for autoantigens are present in healthy humans and normal strains of mice. Such autoantigen-binding cells have been clearly shown for thyroglobulin' and DNN in humans, and for RNA (polyadenylic acid)23 and certain erythrocyte antigens'3 in mice. In some circumstances, the numbers of such cells are only slightly greater in autoimmune individuals than in normal controls. The existence of autoantigen-binding lymphocytes in healthy humans and mice indicates a very real predisposition to autoimmunity in normals, and raises the question of why some individuals develop autoimmune disease and others do not. Suppressor T cells are thought to play a major role in preventing autoantibody production by these autoantigen-binding lymphocytes. The size of these autoimmune clones is kept small, and autoantibody production minimal, by the action of suppressor T cells. These potentially autoreactive cells may also explain the wide variety of circumstances and relative ease with which autoimmunity can be induced, e.g., by drugs, infectious agents, the aging process, and immunodeficiency. Autoimmunity is reversible in many of these situations, which may represent the organism's ability to restore physiologic control and abort the autoimmune state. An alternative way to induce autoimmunity, even in the face of adequate suppressor function, would be through the generation of excessive helper T cells. Helper T cells are generally required for B cells to progress in their differentiation pathway from IgM to IgG production. The sequential development of IgM and later IgG autoantibodies has been shown for antibodies to nucleic acids 19 , 27, 32 and is discussed in another section of this article. T cell regulation is mediated by the release of factors which may act on other T cells, on B cells, or on macro phages. Both nonspecific and specific factors have been described. Some factors enhance and others suppress immune responses. The factors contain la determinants as well as binding sites for specific antigens, suggesting that T cells may express variable region genes of immunoglobulin heavy chains. T cell membranes also contain idiotypic sites (unique individually specific immunoglobulin determinants) which are also present on secreted antibody and on B cell membranes (where they function as receptors for interaction with antigen). Thus, T cells may have surface receptors which at least partially resemble the immunoglobulin receptors on B cells. The presence of MHC gene products on T cells and T cell factors relates to the histocompatibility requirement for immune cell collaboration. Much of our understanding of immunologic regulation and autoimmunity comes from studies in animals, although evidence is now accumulating for suppressor cells and regulatory factors in humans as well. Observations in New Zealand Black (NZB) and related hybrid mice made over the last 20 years are presented as an example of information obtainable from animal models of human disease.
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SPONTANEOUS AUTOIMMUNE DISEASE IN NZB AND NZB/NZW MICE Abnormal regulation of the immune system is a feature of many human and experimental autoimmune disorders, and is well demonstrated in New Zealand Black (NZB) and NZB/NZW F1 (B/W) mice who spontaneously develop diverse autoantibodies, immune complex glomerulonephritis, defective T cell function, monoclonal macroglobulinemia, and malignant lymphomas. 1o , 30, 31 Genetic, immunologic, and viral factors all play a role in pathogenesis. The genetic contribution is poorly understood, although multiple autosomal genes are involved, The mice harbor C-type oncornaviruses 12 , 15 and make antibodies to viral antigens, particularly to glycoprotein (gp) 70 which is the major envelope glycoprotein of this virus. 36 Our laboratory has studied the control of immune reactivity and autoantibody formation in these mice, focusing on the role of T lymphocytes and sex hormones.32 A major defect in T cell regulation, modulated by sex hormones and possibly involving viruses, may be an important mechanism promoting autoimmunity and lymphoid neoplasia in these strains. New Zealand mice are not the only ones that develop these problems. The recently described MRL/1 strain becomes ill with massive lymphoproliferation and a severe autoimmune disease characterized by immune complex glomerulonephritis, hypergammaglobulinemia, formation of antinuclear factors and antibodies to DNA.l7 These mice are genetically unrelated to the NZB. A single mutant autosomal recessive gene determines the lymphoproliferation which is not clearly malignant and contains numerous T cells. Other murine strains (SW AN, PN) also develop autoimmune disease similar to the NZB and B/W. Thus, an expanding number of autoimmune models are becoming available for study. The key features of spontaneous disease and associated immunologic deficits in NZB and B/W mice are summarized as follows: (1) clinically normal at birth although immunologically hyperactive and prematurely competent; (2) decline in suppressor T cell activity, T cell tolerance, and serum thymic humoral factors at one to two months; (3) autoantibodies increase in tit er from three months onward, first in females; (4) hemolytic anemia and immune complex glomerulonephritis appear after five months; and (5) decreased cellular immunity, lymphoproliferation, and monoclonal macroglobulinemia after eight months. The mice appear clinically normal at birth although some aspects of immunologic hyperactivity (both humoral and cellular) are present even this early in life. Immune competence develops prematurely compared with normal strains/ suggesting an abnormality of immunologic regulation that first develops in utero. Suppressor T cell activity declines between one to two months of age in NZB mice,3 associated with an inability to develop tolerance to disaggregated foreign gammaglobulin 26 and a deficiency of thymic humoral factors. The administration of thymic factors at this age can restore suppressor activity;4 however, the injection of thymosin is without benefit thus far on the course of disease. B , 34
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The first clinical appearance of autoantibodies occurs at about two to three months of age, although they can be detected much earlier by more sensitive methods. Antibodies to erythrocytes and to lymphocyte surface antigens appear in almost every NZB mouse, whereas antibodies to nucleic acids are found in B/W mice. Antibodies cytotoxic to T lymphocytes occur commonly in young NZB mice,25 are characteristically IgM, and do not distinguish NZB T cells from other T lymphocytes. This antibody occurs less often in B/W mice, a strain in which individual animals may develop antibodies to nucleic acids without detectable serum levels of antibodies to T cells.9 There is a marked sex difference in B/W mice, females developing severe immune complex glomerulonephritis and dying approximately four months earlier than males. The renal deposits contain complement, antibodies to DNA,!! and antibodies to the major glycoprotein (gp 70) of murine C-type viruses. 36 The more accelerated disease of female B/W mice is associated with earlier appearance and greater amounts of IgG antibodies to DNA.19 The titers of autoantibodies rise progressively, associated with a general augmentation of humoral immunity. Adult NZB and B/W mice make excessive antibody responses to several experimental antigens, including foreign proteins, sheep erythrocytes, and synthetic nucleic acids. 32 . 33 The major manifestations of disease become clinically apparent after five months of age. lO NZB mice develop Coombs' positive hemolytic anemia; B/W mice develop proteinuria and uremia. Both strains develop marked lymphocytic and plasmacytic infiltrates in many different organs. This extensive lymphoproliferation may suggest malignancy.H,3! Mice that survive tl,le autoimmune disorder are susceptible to the development of malignant lymphomas, at times associated with the production of monoclonal IgM.29 The development of lymphoma is enhanced by the administration of immunosuppressive drugs, such as azathioprine and cyclophosphamide. These older mice often have marked deficiencies of cell-mediated immunity, as shown by impaired proliferative responses to phytomitogens, reduced capacity to induce graft-versus-host disease or to reject malignant tumors and skin grafts. The number of T cells in their peripheral lymphoid organs are decreased, and their ability to mount antibody responses to sheep red blood cells and other experimental antigens may decline to levels below those of normal strains. Disordered Immunologic Regulation and Autoimmunity in NZB and NZB/NZW Mice Experimental evidence suggests that B cell and helper T cell functions are increased in NZB and B/W mice, whereas suppressor T cell activity is decreased. The simplest explanation would be a defect in suppressor T cells or factors which then fail to regulate helper T cells and B cells. Much current work is attempting to define the precise mechanisms responsible for this failure. Antibodies to DNA and RNA can be detected in low titers as early
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as four weeks of age in B/W mice, using a sensitive filter radioimmunoassay and sucrose density gradient ultracentrifugation to fractionate serum. Antibodies to DNA are both IgM and IgG,'9. 32 whereas antibodies to RNA (polyadenylic acid, poly A) are exclusively IgM in these young mice. There is a striking difference between female and male animals in the Ig distribution of antibodies to DNA. Females have much more IgG anti-DNA than do males. Antibodies to DNA increase slowly from one to four months, as shown by progressively greater serum binding activity. The Ig distribution of anti-DNA antibodies remains relatively unchanged until five to six months when, in females, there is a sudden and marked increase in DNA binding by IgG antibodies. This same sequence of events is repeated in males, but offset in time by approximately four months. The males make predominantly IgM antibodies to DNA until nine months of age, when there is a great increase in IgG binding of DNA. The timing of the switch from IgM to greatly increased IgG antibodies to DNA correlates, in each sex, with the onset of severe glomerulonephritis leading rapidly to death. This switch appears to herald the onset of a severe exacerbation of disease. These results on anti-DNA responses are paralleled by studies on antibodies to poly A which are also produced spontaneously by B/W mice. These antibodies are only IgM in young mice, and then switch to IgG in older animals. The switch occurs first in females (at seven months), and later in males (at 11 months). The IgM to IgG switch in antibody synthesis is a major event in B cell differentiation. The two influences that promote this event are antigen and T cells, probably acting together. T cell control is mediated by factors (as already discussed) which contain antigen binding sites as well as la determinants. In normal mouse strains, the switch from IgM to IgG is highly T cell dependent. T cells or T cell factors can convert strains of genetically low responder status to high responder status, an event accompanied by an IgM to IgG switch. Nude mice or thymectomized mice react to thymicdependent antigens by producing predominantly IgM rather than the expected IgG antibodies. For these reasons, we presumed that the IgM to IgG switch for nucleic acid antibodies reflected the activity of regulatory T cells, and might provide an indirect but highly relevant way to study T cell function. The Ig switch could represent the conversion from a partial to a more complete regulatory defect with further impaired ability to suppress disease expression. Neonatal thymectomy significantly increased mortality in male B/W mice. 22 The thymectomized males developed an immediate and persistent increase in antibodies to DNA, associated with an accelerated switch to IgG which occurred at four rather than at nine months. These results are consistent with the elimination of a thymic suppressor influence in the males, resulting in an essentially female pattern of disease expression. By contrast, neonatal thymectomy of females had relatively little effect on antibodies to DNA, suggesting that this antibody response is already independent of a thymic suppressor mechanism at the time of surgery. This could occur because helper T cells for anti-DN A
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are relatively more peripheralized in newborn female compared with male B/W mice. Prepubertal castration of males (performed at two weeks) also resulted in significantly shortened survival, whereas castration had no effect in females. 21 Castrated males had greater amounts of antibodies to DNA and developed a premature switch to IgG . In these respects they resembled thymectomized males and female B/W mice. Thus castration of males, like thymectomy, caused a worsening of disease and an accelerated appearance of IgG antibodies to DNA. Castrated females had little change in antibodies to DNA. The effects of castration indicate that sex hormones (like the thymus) greatly influence the spontaneous antibody response to DNA and the severity of disease in B/W mice. The major influence appears to be a protective effect of male hormone. This protective effect of androgen probably explains why autoimmunity in general is more common in females or in males who fail to produce androgens (Klinefelter's syndrome). Our results indicate similarities between castration and thymectomy, and suggest a mechanism by which sex hormones influence these immune responses. The elimination of androgen was similar to removing a thymic suppressor mechanism, allowi,ng an IgG switch for antibodies to DNA. These effects on autoantibodies are consistent with the known regulatory effects of sex hormones on normal immune responses. For example, female mice are less susceptible to immunologic tolerance than males. Female mice of various normal strains have higher antibody responses to several different antigens when compared with males. Castrated male mice give an augmented "female" response to these antigens. These results in normal strains, like the results in B/W mice, suggest that sex hormones may modulate T cell regulation. Thus, sex differences in autoimmunity may be aberrant expressions of the normal physiologic effects that sex hormones exert on immune reactivity. In human lupus, antibodies to DNA and RNA may belong to either the IgM or IgG immunoglobulin class. Active lupus nephritis is associated in particular with serum IgG antibodies to DNA which deposit as immune complexes in the renal glomeruli. At the other end of the spectrum, asymptomatic relatives of patients with systemic lupus erythematosus (SLE) have a significant increase of antibodies to RNA but not to DNA.5 Four families demonstrated antinucleic antibodies both in the SLE probands and in asymptomatic family members. IgG antibodies to nucleic acids (both DNA and RNA) were present in the patients and correlated with active SLE. Antibodies to RNA in the asymptomatic relatives were present only in the IgM class.35 This may represent a limited form of the lupus diathesis, and raises the possibility that a switch from IgM to IgG antibodies to nucleic acids may occur in human SLE as well as in B/W mice. The general conclusions drawn from these studies are: 1. Autoantibody responses are regulated, but the regulation is disordered and inappropriate.
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2. Regulation is related to the thymus, probably to the action of helper and suppressor T cells. 3. Sex hormones modulate T cell regulation; androgens are protective and may help maintain suppressor T cell activity. 4. The IgM to IgG switch may be an indirect but relevant way to study regulation in murine lupus and possibly also in human lupus. These conclusions suggest that regulatory mechanisms exert influences upon autoantibody responses, but that the regulation promotes rather than suppresses autoimmunity. The possible utilization of androgens for the treatment of murine lupus is also suggested. Such experiments are currently under way in our laboratory.
VIRUSES AND IMMUNOLOGIC REGULATION Autoimmunity can be thought of as a disturbance in immunologic regulation in which the balance between helper and suppressor T cells is disturbed. This might arise from a deficiency or malfunction of suppressor T cells, an abnormal activation of helper T cells, a bypass of the requirement for T cell help, or by a combination of several mechanisms. Viruses could interfere with normal control mechanisms in a way that might create a regulatory imbalance and lead to autoimmunity. Much current evidence suggests a possible role for C-type oncornaviruses in the pathogenesis of lupus,t2 although definitive proof is lacking and questions have been raised about the adequacy of experimental design and specificity of serologic reagents. C-type viruses are named for their characteristic appearance in the electron microscope. They occur abundantly in NZB and B/W mice where they can be seen in many tissues and at all ages starting from fetal life. These viruses contain RNA in their virion which is transcribed by an RNA-dependent DNA polymerase and then incorporated into the host genome. As part of the genome, viral information can be expressed along with the host's own genetic information. These viruses bud from cell surfaces and viral antigens are expressed on cell membranes. One of these antigens (gp 70) is the major envelope glycoprotein of the C type virus. Antibodies to gp 70 are found in high titer in NZB and B/W mice, and immune complexes containing gp 70 are found in the kidney deposits in B/W mice. 36 Antigenic material cross-reactive with antisera to primate C-type viruses are also present in the tissues of lupus patients 31 and in the kidney deposits in lupus glomerulonephritis. 16 , 18 These represent the latest findings in a long line of research that has attempted to relate lupus to chronic or persistent virus infection. This subject has been well summarized elsewhere. 12 , 20 A possible way that viral antigens on cell membranes may interfere with immunologic regulation deserves further consideration. The lymphocyte membrane represents a common meeting ground where histocompatibility antigens, immune response associated antigens (la), and viral antigens can interact and influence each other (Fig. 1). A normal surveillance mechanism exists whereby new antigens expressed
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Io Figure 1. Schematic representation of the lymphocyte surface shows the presence of H2 antigens , viral antigens (gp 70), and immune response associated (la) antigens.
on the lymphocyte surface are recognized immunologically in association with histocompatibility antigens. This recognition has been demonstrated both for chemical and viral antigens, and may represent an important mechanism in immunobiology. For example, infection of mice with lymphocytic choriomeningitis virus (LCM) leads to the generation of an "altered self" surface antigen on lymphocytes that requires both H2 determinants and viral determinants for recognition. 37 The H2 molecules may serve as receptors and, by associating with new membrane antigens, promote an immune response to these new antigens. 6 It is possible that recognition of C-type viral antigens may occur through a similar mechanism. The fact that gp 70 and H2 molecules demonstrate functional interactions on the surface of a lymphoid tumor favors this hypothesis. 24 Thus, circumstances might arise in which viral antigens and self antigens on the lymphocyte surface might interact inappropriately in a way that could generate potentially harmful immune reactivity. If this occurred on the surface of helper or suppressor T cells, it could lead to modification of existing regulatory mechanisms and perhaps fa vor the generation of autoimmunity. Although still hypothetical, this postulate integrates genetic and viral factors in autoimmunity, and is in agreement with current concepts of membrane modulation and receptor triggering in many biological phenomena. 6
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globulin in healthy people and its relevance to tolerance for autoantigens. Lancet, 1 :226, 1973. Bankhurst, A. D., and Willia ms, R C.: Identification of DNA binding lymphocytes in patients with systemic lupus erythematosus. J. Clin. Invest. , 56:1378, 1975. Barthold, D. R. , Kysela, S., and Steinberg, A. D. : Decline in suppressor T cell function with a ge in female NZB mice. J. Immunol., 112 :9 , 1974. Dauphinee, M. J., Talal, N ., Goldstein, A. L., et aI.: Thymosin corrects abnormal DNA synthetic response of NZB mouse thymocytes. Proc. Nat. Aca d. Sci., 71 :2637, 1974. DeHoratius, R J., Pillarisetty, R, Messner, RP., et aI.: Anti-nucleic acid antibodies in systemic lupus erythematosus patients and their families. J. Clin. Invest., 56:11491154, 1975. Edelma n , G. M.: Surface modulation in cell recognition and cell growth: Some new hypotheses on phenotypic alteration and transmembranous control of cell surface receptors. Science, 192 :218-226, 1976.
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7. Evans, M. N., Williamson, W. G., and Irvine, W. J.: The appearance of immunological competence at an early age in New Zealand black mice. Clin. Exp. Immunol., 3 :375, 1968. 8. Gershwin, M. E., Steinberg, A. D., Ahmed, A., et al.: Study of thymic factors. 11. Failure of thymosin to alter the natural history of NZB and NZB/NZW mice. Arthritis Rheum., 19:862-866,1976. 9. Goldblum, R., Pillarisetty, R., and Talal, N.: Independent appearance of anti-thymocyte and anti-RNA antibodies in NZB/NZW FJ mice. Immunology, 28 :621, 1975. 10. Howie, J. B., and Helyer, B. J.: The immunology and pathology of NZB mice. Adv. Immunol., 9 :215-266, 1968. 11. Lambert, P. H., and Dixon, F. S.: Pathogenesis of the glomerulonephritis of NZB/NZW mice. J. Exp. Med., 127:507, 1968. 12. Levy, J.: Autoimmunity and neoplasia: The possible role of C-type viruses. Amer. J. Clin. Pathol., 62:258-280,1974. 13. Linder, E., and Edgington, T. S.: Immunobiology of the autoantibody response. 11. The lipoprotein-associated soluble HB erythrocyte autoantigen of NZB mice. J. Immunol., 110:53-62, 1973. 14. Mellors, R. C.: Autoimmune and immunoproliferative diseases of NZB/Bl and hybrids. Int. Rev. Exp. Pathol., 5:217,1966. 15. Mellors, R. C., and Huang, C. Y.: Immunopathology of NZB/Bl mice. V. Virus-like (filtrable) agent separable from lymphoma cells and identifiable by electron microscopy. J. Exp. Med., 124:1031-1038, 1966. 16. Mellors, R. C., and Mellors, J. W.: Antigen related to mammalian type-C RNA viral p30 protein located in renal glomeruli in human systemic lupus erythematosus. Proc. Nat. Acad. Sci., 73:233-237,1976. 17. Murphy, E. D., and Roths, J. B.: A single gene model for massive lymphoproliferation with immune complex disease in new mouse strain MRL. Proc. 16th Int. Congr. Hematol., Kyoto, Japan, Sept. 5-11, 1976. Amsterdam, Excerpta Medica. 18. Panem, S., Ordonez, N. G., Kirstein, W. H., et al.: C-type virus expression in systemic lupus erythematosus. New Engl. J. Med., 295:470-485,1976. 19. Papoian, R., Pillarisetty, R. J., and Talal, N.: Immunologic regulation of spontaneous antibodies to DNA and RNA. II. Sequential switch from IgM to IgG in NZB/NZW FJ mice. Immunology, 31 :121-125,1976. 20. Phillips, P. E.: The virus hypothesis in systemic lupus erythematosus. Ann. Intern. Med., 83 :709-715, 1975. 21. Roubinian, J. R., Papoian, R., and Talal, N.: Androgenic hormones modulate autoantibody responses and improve survival in murine lupus. (J. Clin. Invest., in press.) 22. Roubinian, J. R., Papoian, R., and Talal, N.: Effects of neonatal thymectomy and splenectomy on survival and regulation of autoantibody formation in NZB/NZW FJ mice. (J. Immunol., in press.) 23. Sawada, S., Michalski, J. P., Pillarisetty, R. J., et al.: Lymphocytes binding polyadenylic acid and synthesizing antibodies to nucleic acids in autoimmune and normal mice. (Manuscript in preparation.) 24. Schrader, J. W., Cunningham, B. A., and Edelman, G. M.: Functional interactions of viral and histocompatibility antigens at tumor cell surfaces. Proc. Nat. Acad. Sci., 72 :50665070,1975. 25. Shirai, T., and Mellors, R. C.: Natural cytotoxic autoantibody and reactive antigen in New Zealand Black and other mice. Proc. Nat. Acad. Sci., 68: 1412, 1971. 26. Staples, P. J., and Talal, N.: Relative inability to induce tolerance in adult NZB and NZB/NZW FJ mice. J. Exp. Med., 129:123,1969. 27. Steward, M. W., and Hay, F. C.: Changes in immunoglobulin class and subclass of antiDNA antibodies with increasing age in N/ZBW F J hybrid mice. Clin. Exp. Immunol., 26:363-370, 1976. 28. Strand, M., and August, J. T.: Type-C RNA virus gene expression in human tissue. J. Virol., 14:1584-1596, 1974. 29. Sugai, S., Pillarisetty, R. J., and Talal, N.: Monoclonal macroglobulinemia in NZB/NZW F J mice. J. Exp. Med., 138:989,1973. 30. Talal, N.: Animal models for systemic lupus erythematosus. Clin. Rheum. Dis., 1 :485496,1975. 31. Talal, N.: Autoimmunity and lymphoid malignancy in New Zealand black mice. In Schwartz, R. S. (ed.): Progress in Clinical Immunology. New York, Grune and Stratton, 1973, pp. 101-120. 32. Talal, N.: Disordered immunologic regulation and autoimmunity. Transplant. Rev., 31 :240-263, 1976. 33. Talal, N.: Immunologic and viral factors in the pathogenesis of systemic lupus erythematosus. Arthritis Rheum., 13:887-894,1970.
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34. Talal, N., Dauphinee, M. J., Pillarisetty, R. J., et al.: Effect of thymosin on thymocyte proliferation and autoimmunity in NZB mice. Ann. N.Y. Acad. Sci., 249:438,1975. 35. Talal, N., Pillarisetty, R. J., DeHoratius, R. J., et al.: Immunologic regulation of spontaneous antibodies to DNA and RNA. I. Significance of IgM and IgG antibodies in SLE patients and asymptomatic relatives. Clin. Exp. Immuno!., 25 :1-6, 1976. 36. Yoshiki, T., Mellors, R. C., Strand, M., et al.: The viral envelope glycoprotein of murine leukemia virus and the pathogenesis of immune complex glomerulonephritis of New Zealand mice. J. Exp. Med., 140:1011-1027,1974. 37. Zinkernagel, R. M., and Doherty, P. C.: Hz compatibility requirement for T cell-mediated lysis of target cells infected with lymphocytic choriomeningitis virus. J. Exp. Med., 141 :1427, 1975. Department of Medicine University of California School of Medicine Third Avenue and Parnassus Street San Francisco, California 94143
