Prevention of diabetes in nonobese diabetic mice by tumor necrosis factor ( TNF ) : Similarities between TNF-a and interleukin 1 ( insulitis / adoptive transfer / Ia expression / autoimmune disease ) CHAIM 0

The role of tumor necrosis factor a (TNF-a) in the pathogenesis of autoimmune diabetes mellitus was tested in the nonobese mouse (NOD) model system. The effects of TNF-a were assessed on three levels: (i) insulitis development, (ii) development of overt diabetes, (iii) adoptive transfer of diabetes by splenic lymphocytes. Spontaneous diabetes mellitus was blocked in NOD mice by long-term treatment with recombinant TNF-a. Treatment with TNF-a caused'a significant reduction in the lymphocytic infiltration associated with the destruction of the insulin-producing beta cells. Class II major histocompatibility complex Ia expression by islet cells was not up-regulated by TNF-a. Moreover, TNF-a was able to suppress the induction of diabetes in adoptive transfer of lymphocytes from diabetic female mice to young nondiabetic male NOD mice. These activities of TNF-a were shared by interleukin la in this system. These studies have implications for the pathogenesis and therapy of autoimmune diabetes mellitus. Nonobese diabetic (NOD) mice spontaneously develop diabetes remarkably similar to human autoimmune insulindependent diabetes mellitus. There is increasing evidence that the human and the NOD disease result from immune destruction of the insulin-producing beta cells in the islets of Langerhans (1). The disease is characterized by progressive lymphocyte infiltration into the islets (insulitis) prior to the expression of overt diabetes and by the appearance of anti-islet cell antibodies in the serum (2). A T-cell-mediated autoimmune pathogenesis is implicated because the disease can be passively transferred with lymphocytes into irradiated prediabetic mice (3) and prevented by treatment with antibodies directed against Thy-1.2 (4) and L3T4 (5) or by treatment with cyclosporin A (6). Diabetes can also be prevented by treatment with antibodies to class II Ia antigens (7). Untreated animals develop profound glucose intolerance and ketosis and die within weeks of the onset of overt diabetes. The role of lymphokines, especially interferon y (IFN-y), interleukin 1 (IL-1), and tumor necrosis factor a (TNF-a), in the pathogenesis of autoimmune diabetes has received increasing attention recently (8, 9). It was shown that IFN--y and TNF-a can induce the aberrant expression of class II major histocompatibility complex (MHC) molecules on pancreatic beta cells in vitro, suggesting a role for these lymphokines in the induction of the autoimmune process in diabetes (9). A different group of investigators has suggested that IL-1 is toxic to pancreatic beta cells in vitro and that TNF-a significantly enhances this toxicity (8). Recently we have suggested that TNF-a may play a significant role in preventing autoimmune nephritis in the (NZB3 x NZW)F1 lupus nephritis model system (10). Here we present evidence that TNF-a has a major effect in vivo by protectingNOD mice from developing autoimmune insulitis and diabetes. MATERIALS AND METHODS Recombinant murine TNF-a (0.53 mg/ml; 2.6 x 107 units/ml) was kindly provided by Genentech. Recombinant human IL-la (108 units/,ug) was provided by Steve Gillis (Immunex, Seattle, WA). Mice were injected i.p. with 200 ng of IL-la per injection according to Morrissey et al. (11) or with TNF-a, 3 ,tg. Assessment of Diabetes. Diabetes development was monitored by testing the mice for urinary glucose three times weekly. Diabetes development was confirmed by hyperglycemia (fasting glycemia, >3 g/liter) and by histological examination showing destruction of at least 50% of the islets. Diabetic mice usually died within 2-8 weeks after development of glucosuria with overt diabetic symptoms, including polyuria, polydypsia, and severe weight loss. Histopathology. Pancreas tissue was fixed in 10% buffered formalin and embedded in paraffin, and sections ofpancreatic tissue were stained with hematoxylin/eosin. For immunohistochemistry of pancreatic endocrine cells, the formalinfixed paraffin sections were deparaffinized and immunostained for insulin, glucagon, and somatostatin using an immunoperoxidase technique (DAKO PAP kit) according to Sternberg et al. (12). For immunohistochemistry of class II la expression in islets, pancreatic tissue was cut into small blocks, embedded in OCT compound (Tissue-Tek, Miles Scientific) and frozen in dry ice/isopentane without fixation. Cryosections were cut 4 ,m in thickness, fixed in 4% paraformaldehyde, and immunostained with biotinylated monoclonal antibodies 40N and MKD6. 40N is reactive with NOD-I-Ap molecules; MKD6 reacts specifically with I-AdI3 molecules (BALB/c) but not NOD. All slides were examined independently by two observers without knowledge of treatment or strain of mice from which the pancreas was taken. Adoptive Transfer of Diabetes. Adoptive transfer experiments were done according to Wicker et al. (3). Recipient male NOD mice, 8-12 weeks of age, were irradiated [775 R (1 R = 0.258 mC/kg) from a 137Cs source] and injected i.v. within 2 hr of irradiation with donor splenocytes suspended in 0.25 ml of phosphate-buffered saline (PBS). Diabetic donors were female NOD mice sacrificed 1-3 weeks after the onset of glycosuria. Single cell suspensions were prepared from 10-15 spleens and pooled for each experiment. Recipient mice were treated with TNF-a, IL-la, or PBS i.p. on different time schedules as indicated. TNF-a and IL-1 Production. Peritoneal exudate cells were obtained from NOD and SWR mice 4 days after injection of 3% thioglycolate medium. Following adherence to plastic, Abbreviations: NOD, nonobese diabetic; TNF, tumor necrosis factor; IL, interleukin; IFN, interferon; MHC, major histocompatibility complex. 968 The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact. Proc. Natl. Acad. Sci. USA 87 (1990) 969 macrophages were activated with 100 units of recombinant IFN-y per ml and 1 ,ug of lipopolysaccharide (LPS) per ml as described (10). TNF-a and IL-1 production was assayed in the supernatants by bioassays as described (13, 14). For Northern blot analysis, peritoneal exudate cells were harvested 4 hr after mice were injected i.p. with 500 ,ug of LPS. Total cellular RNA was isolated by guanidine isothiocyanate extraction followed by cesium chloride density gradient centrifugation. Northern blot analysis was carried out following standard protocols. Ten micrograms of total RNA per lane was hybridized to murine TNF-a (15), murine IL-1,B (16), or ,3-actin (17) probes.