Cytokines induce apoptosis in beta-cells isolated from mice lacking the inducible isoform of nitric oxide synthase (iNOS(-/-))

Prolonged exposure of rodent beta-cells to combinations of cytokines induce s the inducible form of nitric oxide synthase (iNOS) expression and Fas exp ression, nitric oxide (NO) production, and cell death. It also induces the expression of potential "defense" genes, such as manganese superoxide dismu tase (MnSOD) and heat shock protein (hsp) 70. NO is a radical with multifac eted actions. Recent studies have shown that NO, in addition to having cyto toxic actions, may 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 nonspecific effects of these agents. In this study, we examined the effects of cytokines on gene expression, determined by reverse transcriptase-polymerase chain reaction (RT-PCR), and viability, determine d by nuclear dyes, of pancreatic islets or fluorescence-activated cell sort er (FACS)-purified beta-cells isolated from iNOS knockout mice (iNOS(-/-), background C57BL/6x129SvEv) or their respective controls (C57BL/6x129SvEv). The combination of cytokines used was interleukin-1 beta (50 U/ml) plus ga mma-interferon (1,000 U/ml) plus tumor necrosis factor-alpha (1,000 U/ml). The lack of cytokine-induced iNOS activity in the iNOS(-/-) islet cells was confirmed by RT-PGR 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(-/-) is lets. Prolonged (6-9 days) exposure of WT islets to cytokines leads to an 8 0-90% 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. A fter 9 days, the apoptosis index was similarly increased in WT (39 +/- 3%) and iNOS(-/-) (33 +/- 4%) beta-cells. On the other hand, cytokines increase d necrosis in WT (20 +/- 4%) but not in iNOS(-/-) (7 +/- 3%) beta-cells. Fr om these data, we concluded that 1) NO is required for cytokine-induced hsp 70 mRNA expression but not for Fas and MnSOD expression, 2) cytokines indu ce both apoptosis and necrosis in mouse beta-cells, and 3) cytokine-induced apoptosis is mostly NO-independent, whereas necrosis requires NO formation .