Role of nitric oxide and viral products in pancreatic B-cell dysfunction and death

Type 1 diabetes mellitus (T1DM) is an autoimmune disease caused by progressive destruction of insulin-producing pancreatic beta-cells. Both viral infections and the cytokines interleukin-1beta (IL-1beta) and interferon-gamma (IFN-gamma) have been suggested as potential mediators of beta-cell death in early T1DM. Nitric oxide (NO) is a highly diffusible, short-lived free radical gas, which plays a significant role in several physiological processes in a diversity of tissues and organisms. Prolonged exposure of rodent or human pancreatic beta-cells to combinations of cytokines induces the expression of the inducible form of nitric oxide synthase (iNOS) and Fas, NO production, and cell death. It also induces the expression of potential "defense" genes, such as manganese superoxide dismutase (MnSOD) and heat shock protein (hsp) 70. Recent studies have shown that NO, in addition to having cytotoxic actions, may also regulate gene transcription. It remains unclear whether NO mediates cytokine-induced gene expression and subsequent beta-cell death. Previous studies using NO synthase blockers yielded conflicting results, which may be due to non-specific effects of these agents.

In the first part of our work, we examined the role of NO in beta-cell dysfunction and death by using an iNOS knockout mice (iNOS-/-, background C57BL/6x129SvEv). We evaluated the effects of cytokines on gene expression, as determined by reverse transcriptase-polymerase chain reaction (RT-PCR), and viability, as determined by nuclear dyes, of pancreatic islet cells or fluorescence-activated cell sorter (FACS)-purified beta-cells isolated from iNOS knockout mice or their respective controls (C57BL/6x129SvEv). The combination of cytokines used was interleukin-1beta (50 U/ml) + gamma-interferon (1000 U/ml) + tumor necrosis factor-alpha (1000 U/ml). The lack of cytokine-induced iNOS activity in the iNOS-/- islet cells was confirmed by RT-PCR and nitrite determination. Cytokines induced a > 3-fold increase in Fas and MnSOD mRNA expression in wild-type (wt) and iNOS-/- islets. On the other hand, hsp 70 was induced in wt but not in iNOS-/- islets. Prolonged (6-9 days) exposure of wt islets to cytokines lead to an 80-90% decrease in islet cell viability, whereas viability decreased by only 10-30% in iNOS-/- islet cells. To determine the mode of cytokine-induced cell death, FACS-purified beta-cells were exposed to the same cytokines. After 9 days, the apoptosis index was similarly increased in wt (39 +/- 3%) and iNOS-/- (33 +/- 4 %) beta-cells. On the other hand, cytokines increased necrosis in wt (20 +/- 4 %) but not in iNOS-/- (7 +/- 3 %) beta-cells. From these data, we conclude that: 1) NO is required for cytokine-induced hsp 70 mRNA expression, but not for Fas and MnSOD expression; 2) cytokines induce both apoptosis and necrosis in mouse beta-cells; 3) cytokine-induced apoptosis is mostly NO-independent, whereas necrosis requires NO formation.

In the second part of our work, we examined the role of the viral product double-stranded RNA (dsRNA) in beta-cell dysfunction and death. DsRNA is produced by many viruses during their replicative cycle. We investigated whether dsRNA (here utilized as synthetic poly IC (PIC)) modifies the effects of IL-1beta and IFN-gamma on gene expression and viability of rat pancreatic beta-cells and the role of NO in this process. FACS-purified rat beta-cells were exposed for 6-16 h (study of gene expression by RT-PCR) or 6-9 days (study of viability by nuclear dyes) to PIC and/or IL-1beta or IFN-gamma. PIC increased the expression of Fas and Mn superoxide dismutase mRNAs by 5-10-fold. IL-1beta and a combination of PIC + IFN-gamma(but not PIC or IFN-gamma alone) induced expression of iNOS and consequent NO production. Induction of iNOS expression by PIC + IFN-gamma requires NF-kappaB activation, as suggested by transfection experiments with iNOS promoter-luciferase reporter constructs into primary beta-cells. The PIC-responsive region in the Fas promoter is located between nucleotides -223 and –54. Site-directed mutations at the NF-kappaB and C/EBP binding sites prevented PIC-induced Fas promoter activity. Increased Fas promoter activity was paralleled by enhanced susceptibility of PIC + cytokine-treated beta-cells to apoptosis induced by FasL. Combinations of IL-1beta + IFN-gamma, PIC + IFN-gamma or PIC + IL-1beta induced a 2-3-fold increase in the number of apoptotic beta-cells. Blocking of iNOS activity decreased PIC + IL-1beta-, but not PIC + IFN-gamma-, induced apoptosis. beta-cell infection with an adenovirus encoding the NF-kappaB inhibitor AdIkappaB(SA)2 prevented both necrosis and apoptosis induced by PIC + IL-1beta or PIC + IFN-gamma. mRNAs for several chemokines and one cytokine were induced by PIC, alone or in combination with IFN-gamma, in pancreatic beta-cells. These included IP-10 (CXCL10), IL-15, MCP-1 (CCL2), fractalkine (CX3CL1) and MIP-3alpha (CCL20). There was not, however, induction of IL-1beta expression. PIC has an additive effect on cytokine-induced beta-cell death, by both NO-dependent (in the case of IL-1beta) and NO-independent (in the case of IFN-gamma) mechanisms.

To further elucidate the molecular mechanisms involved in PIC + IFN-gamma-effects, the global profile of genes modified by these agents was analysed by high-density oligonucleotide arrays representing 8000 probes in primary rat beta-cells. Following a 6 or 24h treatment with IFN-gamma, PIC or IFN-gamma and PIC, we observed changes in the expression of 51 to 189 genes. IFN-gamma modified the expression of MHC-related genes, and also of genes involved in beta-cell metabolism, protein processing, cytokines and signal transduction. PIC affected preferentially the expression of genes related to cell adhesion, cytokines and dsRNA signal transduction, transcription factors and MHC. PIC and/or IFN-gamma up-regulated the expression of several chemokines and cytokines that could contribute to mononuclear cell homing and activation during viral infection, while IFN-gamma induced a positive feedback on its own signal transduction. PIC + IFN-gamma inhibited insulin and GLUT-2 expression without modifying pdx-1 mRNA expression. Based on these findings, we propose an integrated model for the molecular mechanisms involved in dsRNA + IFN-gamma induced beta-cell dysfunction and death.

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