Viruses, Autoimmunity and Diabetes Mellitus. Abner Louis Notkins, M.D., Takashi Onodera, Ph.D., Jo Satoh, M.D., and Bellur S. Prabhakar, Ph.D.

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1 Viruses, Autoimmunity and Diabetes Mellitus Abner Louis Notkins, M.D., Takashi Onodera, Ph.D., Jo Satoh, M.D., and Bellur S. Prabhakar, Ph.D. Laboratory of Oral Medicine National Institute of Dental Research National Institutes of Health, Bethesda, Maryland U.S.A. There are a large number of important human diseases of undetermined etiology which have an autoimmune component. In some of these diseases the autoimmune abnormalities are very broad; for example, in the case of lupus erythematosus autoantibodies are found against a variety of different cell types and antigens including DNA, RNA, red blood cells, platelets, lymphocytes, immunoglobulins, thyroglobulins, neurons, heart muscles and collagen. In other diseases, the autoimmune component is far more restricted and may be organ specific; for example, in the case of myasthenia gravis the autoantibodies are directed primarily against the acetylcholine receptor. What triggers these autoimmune responses? Could a virus infection sometimes be involved? To try to answer these questions we went to some of our animal models of virus-induced diseases1). These experiments, which were initially designed to study the role of viruses, have provided some surprising observations concerning the reactivity pattern of autoantibodies from both animals and humans. Let me begin the story by describing some of our studies with reovirus. The word "reo" is an acronym which stands for respiratory-enteric-orphan virus and it was so named because the virus was found in the respiratory tract and enteric tract of humans, but did not seem to produce any serious disease. Therefore, it was referred to as an "orphan" virus. In animals, the story is quite different. When inoculated into new-born mice, the virus can infect a number of different cell types, including cells in the pancreatic islets of Langerhans and the anterior pituitary. The infection results in a mild and transient form of diabetes characterized primarily by abnormal glucose tolerance tests2). Histologic examination of the islets reveals a mild inflammation and necrosis with infiltration of plasma cells. Using a fluorescein-labeled antibody to reovirus, some of the islet cells were found to contain viral antigens. By electron microscopy, viral particles were found in beta cells and occasionally in alpha and delta cells. During the course of this work we noted that a number of the virus-infected mice showed retardation of growth2). Between 40 and 50% of the animals showed lower weight which was 2 SDor more below the mean of uninfected animals (Fig. 1). Moreover, many of the animals displayed other clinical symptoms including oily hair, alopecia, and steatorrhea. Further experiments revealed a reduction in the concentration of growth hormone in the blood of some of these animals, and byimmunofluorescence and electron microscopy viral antigens and viral particles were found in the anterior pituitary. These studies showed that reovirus could produce a polyendocrine disease: the virus infects cells in the islets of Langerhans and in the anterior pituitary, and the animals show abnormal glucose tolerance tests and retardation of growth. It is well known that many patients with insulin-dependent diabetes mellitus and particularly patients with polyendocrine disease have autoantibodies in their circulation. To see if these mice with polyendocrine disease also had autoantibodies in their circulation, we tested' their sera by indirect immunofluorescence for autoantibodies that reacted with tissues from normal animals(fib. 2) By this

2 糖尿病 Fig. 1 26巻12号 (1983) Reovirus-infected mice showing growth retardation matched uninfected control mouse (center). along with age A B Fig. 2 Autoantibodies in the sera of reovirus-infected mice detected by indirect immunofluorescence. section of normal pancreas and pituitary incubated with sera from runted mice that had been infected with reovirus. (A) Pancreas showing cytoplasmic staining of cells in the islets of Langerhans with little if any staining of surrounding acinar cells. (B) Pituitary showing staining of cells in the anterior lobe of the pituitary (AP), but little if any staining in the intermediate (IP) and posterior (PP) lobes. 1178

3 Fig. 3 Titer of autoantibodies to growth hormone (A) and insulin (B) in the plasma of reovirus-infected and control mice as determined by ELISA. Each point represents an individual animal, ( ) uninfected mice; ( œ) infected mice. method autoantibodies that reacted with cells in the islets of Langerhans, anterior pituitary, and gastric mucosa were found. In comparison, relatively little or no autoantibody was found in the sera of uninfected mice. What is the nature of the antigen with which these autoantibodies react? By adsorption experiments we found that the antibody that reacted with cells in the islets of Langerhans could be adsorbed by incubation with insulin, and the antibody that reacted with cells in the anteriorpituitary could be adsorbed by incubation with growth hormone2). From these studies it was concludedthat these antibodies reacted with insulin and growth hormone, respectively. This was confirmed byan enzyme-linked immunoabsorbant assay (ELISA). By this latter method the time of appearance, duration, titer and percentage of animals having autoantibodies to growth hormone and insulin were determined (Fig. 3). During the course of this work we observed that animals infected with reovirus type 1 developed autoantibodies to growth hormone, whereas animals infected with a closely related virus, reovirus type 3, had little if any antibody to growth hormone. Studies were carried out to determine which of the ten reovirus genes were involved in the induction of these autoantibodies2)-4). In brief, one of these genes, known as the S1, seems to play a major role in triggering the autoantibodyresponse. This gene codes for a polypeptide on the surface of the virion, known as sigma 1, which appears to influence the tropism of the virus for certain cell types2) `4). These studies have raised a number of interesting questions. What is actually responsible for the development of the polyendocrine disease? Is it the direct destructive effect ofviruses on endocrine

4 Table 1 Reversal of reovirus-induced growth retardation by immunosuppression a) Treated with anti-lymphocyte serum cells, or is it the autoimmune response triggered by the viral infection, or is it a combination of the two? To try to answer these questions, we used immunosuppression techniques5). We infected mice with reovirus and then suppressed the immune response on the assumption that, if the autoimmunity played an important role, immunosuppression might prevent the development of the disease. Our experiments showed that immunosuppression with antilymphocyte serum or with antithymocyte serum or with cyclophosphamide largely prevented the development of the glucose abnormalities. The antilymphocyte serum also greatly inhibited the retardation in growth and the development of oily hair (Table. 1). From these studies we concluded that the host's autoimmune role in the development of this polyendocrine disease5). response plays a very critical Precisely how does the virus infection trigger this autoimmune response? The answer is not known, but there are several possibilities. First, as already indicated, the virus can damage cells and can release antigens, perhaps in a form that the immune system does not ordinarily see. Second, we have preliminary results that infection with reovirus may cause a decrease in the number of suppressor cells. Third, we also have preliminary results suggesting that the virus may cause a polyclonal B cell activation. These factors alone or in combination might trigger the autoimmune response. In order to study in greater detail the role that these autoantibodies actually play in the pathogenesis of the polyendocrine disease, we thought that it would be useful to obtain large quantitites of these autoantibodies in relatively pure form. To do this, we used hybridoma technology. In brief, we fused spleen cells from mice making autoantibodies with myeloma cells. We then screened the supernatant fluids of the hybrids for autoantibodies against a variety of tissues from normal uninfected mice using indirect immunofluorescence. By this method we have obtained a large number of monoclonal autoantibodies6). Some react only with cells in the periphery of the islets of Langerhans, presumbably against glucagon, and others react with cells only in the center of the islets, presumably insulin. Several monoclonal autoantibodies react with cells in the anterior pituitary and we have evidence that some of these monoclonals react with growth hormone. Monoclonal autoantibodies that react with nuclei of normal cells, single isolated cells scattered throughout thesmall intestines, and cells in the gastric mucosa also have been found. We have now obtained over 40 monoclonal autoantibodies (Fig. 4). Probably the most provocative observation is the demonstration that some of thesemonoclonal autoantibodies react with antigens in more than one orgen7). We refer to these multiple organ reactive autoantibodies as MOR antibodies. For example, we have a monoclonal autoantibody that reacts with cells in the anterior pituitary. The same antibody also reacts with cells in the pancreas, the small intestines, and the gastric mucosa. This suggests that the antibody is either reacting with the same molecule present in several different organs or is reacting with a common antigenicdeterminant on different molecules in multiple organs.

5 招 待 講 演I: Viruses, Autoimmunity and Diabetes Mellitus A 8 C D Fig. 4 Monoclonal autoantibodies reactive with normal mouse tissues. (A) Section of pancreas incubated with a monoclonal autoantibody showing cytoplasmic staining of cells in the periphery of the islets of Langerhans. (B) Monoclonal autoantibody reacting with nuclei (section of normal pancreas), but not cytoplasm. (C) Monoclonal autoantibody reacting strongly with cells in the gastric mucosa. (D) Section of pituitary stained with a monoclonal autoantibody that reacts with growth hormone. Brilliant fluorescence is seen in the anterior lobe of the pituitary (AP); no staining of the posterior lobe '(PP) is observed. Initially toantibodies some we thought that of this type. hybridomas obtained these Most from MOR of them antibodies belong the spleens were to the of normal 1181 quite rare, but now we are finding IgM class. Experiments mice also may synthesize in progress MOR many au- suggest that antibodies. The

6 糖尿病 26巻12号 (1983) A B C D Fig. 5 Immunofluorescent staining of normal human tissues with a human monoclonalmor autoantibody that react with multiple organs: (A) anterior pituitary; (B) thyroid follicular cells; (C) gastric mucosa; (D) pancreatic islets and unidentified cells scattered in theacinar tissue. nature of the antigen (s) with which these autoantibodies termined by use of immuno-affinity columns. sepharose beads, tissue extracts are prepared react is still not known, The monoclonal are being and then passed through the column.only (s) with which the autoantibody reacts binds to the column. column, concentrated and electrophoresed on polyacrylamide autoantigens autoantibodies but is being decoupled the These antigens are then eluted from the gels. By this procedure some of the are being isolated and identified7). What is of particular interest is that these MOR multiple organ autoiummunity beginning of my talk. antibodies might in some of the human autoimmune be a partial explanation diseases which I described A molecule in one organ might share common antigenic molecules in other organs. An antibody elicited against that antigenic determinant all organs containing that determinant, resulting in multiple organ autoimmnity. determinants would peripheral monoclonal lymphocytes autoantibodies that with hybridomas myeloma and human-mouse heterohybridomasby cells8). Many of these hybridomas react with antigens in multiple organs; that 1182 for at the with react with Finally, we have extended our findings from mice to humans. Over the last year wehave in preparing over a dozen human-human human to antigen succeeded fusing are synthesizing is, they belong to the

7 MOR type of antibodies. For example, one of these human monoclonal antibodies reacts with cells in the anterior pituitary, thyroid, gastric mucosa and pancreas(fig. 5). In conclusion, our studies show, first, that in mice, a viral infection can trigger an autoimmune polyendocrine disease. Second, by hybridoma technology it is possible to isolate monoclonal autoantibodies. Thrid, a number of these autoantibodies are of the MOR type, that is, they are multiple organ reactive. Fourth, by using hybridoma technology a number of human monoclonal autoantibodies have been obtained, and many of these also are of the MOR type. Fifth, by using these monoclonal antibodies to prepare immuno-affinity columns, it now should be possible to isolate and identify some of the autoantigens involved in human autoimmune diseases. References 1) Notkins, A.L.: The causes of diabetes. Scientific Am. 241(5): (1979) 2) Onodera, T., Toniolo, A., Ray, U.R., Jenson, A.B., Knazek, R.A., Notkins, A.L.: Virusinduced diabetes mellitus. XX. Polyendocrinopathy and autoimmunity. J. Exp. Med. 153: (1981) 3) Weiner, H.L., Drayana, D., Averill, D.R. Jr., Fields, B.N.: Molecular basis of reovirus virulence: role of the S1 gene. Proc. Natl. Acad. Sci. U.S.A. 74: 5744 (1977) 4) Weiner, H.L., Powers, L.M., Fields, B. N.: Absolute linkage of virulence and central nervous system tropism of reovirus to viral hemagglutinin. J. Inf. Dis. 141: (1980) 5) Onodera, T., Ray, U.R., Melez, K.A., Suzuki, H., Toniolo, A., Notikins, A.L.: Virusinduced diabetes mellitus: Autoimmunity and polyendocrine disease prevented by immunosuppression. Nature 297: (1982) 6) Haspel, M.V., Ondera, T., Prabhakar, B.S., Horita, M., Suzuki, H., Notkins, A.L.: Virusinduced autoimmunity: Monoclonal antibodies that react with endocrine tissues. Science 220: (1983) 7) Haspel, M.V., Onodera, T. Prabhakar, B. S., McClintock, P.R., Essani, K., Ray, U.R., Yagihashi, S., Notkins, A.L.: Multiple organreactive monoclonal autoantibodies. Nature 8) Satoh, J., Prabhakar, B.S., Haspel, M.V., Ginsberg-Fellner, F., Notkins, A.L.: Human monoclonal autoantibodies that react with multiple endocrine organs. N. Engl. J. Med.