Nitric oxide production and Fas surface expression mediate two independent pathways of cytokine-induced murine beta-cell damage.

Urs Zumsteg 0 Simona Frigerio 0 Georg A. Hollnder 0 0 From the Department of Research, University Children's Hospital, Basel, Switzerland. University Children's Hospital , Romergasse 8, CH-4005 Basel , Switzerland Activated T-cells and macrophages infiltrate pancreatic islets early in the pathogenesis of type 1 diabetes. Their secretion of different pro-inflammatory cytokines such as interleukin (IL)-1 , interferon (IFN)- , and tumor necrosis factor (TNF)- affects -cell function. Here we report that a combination of these cytokines inhibits insulin release, stimulates inducible nitric oxide synthase (iNOS), and upregulates the surface expression of Fas in NIT-1 -cells and intact mouse islets. Using iNOS-deficient and Fas-deficient islets, respectively, we investigated the relative contribution of NO and Fas upregulation in cytokineinduced -cell damage. Interestingly, inhibition of insulin release did not occur in the absence of NO production. However, de novo expression of Fas-specific mRNA and Fas cell surface expression were detected and thus appear to be NO-independent. The lack of NO production partially protected islets from cytokineinduced apoptosis but had no effect on cell death induced by cell surface cross-linking of Fas with soluble Fas ligand (FasL). The absence of FasL on -cells and the degree of apoptosis observed in Fas-deficient islets exclude the possibility of cytokine-induced fratricide. In conclusion, pro-inflammatory cytokines exert a cytotoxic effect on -cells via an NO-dependent pathway and, in parallel, render -cells susceptible to Fas:FasL-mediated, NO-independent cell death triggered by activated T-cells. Diabetes 49:39-47, 2000 - Tdestruction of insulin-producing -cells within the ype 1 diabetes is the result of autoimmune islets of Langerhans (13). The precise cellular and molecular mechanisms effective for -cell death have yet to be defined. Histologic examination of pancreatic tissue early in the pathogenesis of type 1 diabetes has revealed an infiltration of islets with macrophages and T-cells. Macrophages are the first inflammatory cells to infiltrate the islets, where they may engulf -cells completely (4). A pathogenic role for macrophages in type 1 diabetes has been suggested by the use of agents such as silica, which inhibit macrophage function and prevent the development of diabetes in rodent models (5). The importance of T-cells for the pathogenesis of type 1 diabetes has been demonstrated in NOD/SCID mice, where the generation of mature T-cells was blocked (6,7) or, alternatively, where T-cell function was inhibited by the immunosuppressive agent cyclosporin A (8). Macrophages and T-cells both secrete pro-inflammatory cytokines. The critical role of these molecules in the pathogenesis of type 1 diabetes was first detailed when crude cytokine preparations derived from activated mononuclear cells were demonstrated to mediate functional and structural damage to isolated murine islets of Langerhans (9). Further studies with rat islets revealed that their exposure to the inflammatory cytokine interleukin (IL)-1 was sufficient to inhibit -cell function (10) and that other cytokines such as tumor necrosis factor (TNF)- and interferon (IFN)potentiated this cytotoxic effect (11). Despite their identification as soluble effectors, the -cell specific molecular changes induced by these cytokines remain incompletely understood. Toxic free radicals have been suggested as important mediators of -cell destruction (12). IL-1 , alone or in combination with TNF- and IFN- , induces in rodent and human islets the transcription of the inducible nitric oxide synthase (iNOS) gene (11,13,14). This enzyme catalyzes the generation of nitric oxide (NO) from L-arginine, and importantly, NO production appears to correlate with the inhibition of insulin secretion (13,15,16). The molecular mechanisms operational in this effect include a loss of optimal oxidative phosphorylation, a decrease in glycolysis, and consequently an impairment of ATP concentrations (17). The cellular source of oxygen radicals, however, was not specifically determined in experiments where isolated pancreatic islets were exposed to a mixture of cytokines (18). NO production has also been correlated with DNA fragmentation and subsequent cell death in macrophages and mesangial cells (19). Despite these observations, inhibition of NO synthesis in isolated pancreatic -cells (18) and in vivo (20) achieved only a partial protection from cytokine-mediated cytotoxicity. In addition to NO, other intra- and intercellular mechanisms have been demonstrated to initiate programmed cell death. One important mechanism is the interaction of the cell surface receptor Fas (CD95/APO-1) with its specific ligand, FasL (21). Fas is expressed on a wide variety of cells and, upon cross-linking, generates an apoptotic signal via activation of cysteine proteases called caspases. In contrast, FasL expression is much more limited and is found typically on the surface of activated T-cells and natural killer (NK) cells. Inappropriate Fas expression in the pathogenesis of autoimmune thyroiditis has been proposed by some (22) but not others (23). Although normal human pancreatic -cells do not express Fas on their surface, exposure of these cells to IL-1 induces Fas expression (24,25). Moreover, the production of NO may link cytokine exposure to Fas expression on human -cells (26), which provides a rational basis for therapeutic strategies using free radical scavengers (e.g., nicotinamide) to prevent the occurrence of programmed cell death. To test whether inflammatory cytokines induce changes in -cell function and viability via mechanisms dependent on inducible NO production, we analyzed insulin secretion, Fas expression, and induction of apoptosis in pancreatic -cells from wild-type animals and mice deficient for iNOS or Fas. We demonstrate that NO radicals are critical for the decrease in insulin secretion by -cells, but that their Fas cell surface expression is NO-independent and that pro-inflammatory cytokines promote a certain degree of apoptosis independently of NO and Fas. RESEARCH DESIGN AND METHODS Mice. OF1 and C57BL/6-lpr/lpr mice (5 to 7 weeks old) were purchased from Biological Research Laboratories (Fllinsdorf, Switzerland), and iNOS/ mice (27) (5 to 7 weeks old) were obtained from the Jackson Laboratory (Bar Harbor, ME) and bred in the animal facility at the Department of Research. All animal experiments were approved by the animal welfare committee, and mice were kept according to federal guidelines. Reagents. Recombinant human IL-1 (PeproTech, Rocky Hill, NJ), recombinant mouse IFN- (Genzyme, Cambridge, MA), and recombinant human TNF(Biogen, Cambridge, MA) were obtained commercially. Nw-nitro-L-arginine methylester (L-NAME) was obtained from Fluka Chemie AG (Buchs, Switzerland). For flow cytometric analysis of surface molecules, biotin-conjugated hamster antimouse Fas antibodies (Jo2) (28) an (...truncated)