Protection of insulin-producing RINm5F cells against cytokine-mediated toxicity through overexpression of antioxidant enzymes

Nitric oxide (NO) and reactive oxygen species (ROS) are crucial elements in cytokine-mediated beta-cell destruction. In insulin-producing RINm5F cells , overexpression of cytoprotective enzymes provides significant protection against the synergistic toxicity of NO and ROS. We therefore examined wheth er overexpression of catalase (Cat), glutathione peroxidase (Gpx), and Cu/Z n snperoxide dismutase (SOD) can provide protection for bioengineered RINm5 F cells against cytokine-mediated toxicity. A 72-h exposure of RINm5F contr ol cells to interleukin-1 beta (IL-1 beta) alone or a combination of IL-1 b eta, tumor necrosis factor-alpha, and gamma-interferon resulted in a time- and concentration-dependent decrease of cell viability in the 3-[4,5-dimeth ylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide (MTT) cytotoxicity assay. Although IL-1 beta alone caused only a moderate reduction of viability in t he range of 25%, the cytokine mixture induced a significant loss of viabili ty of >75%. This increased toxicity of the cytokine mixture compared with t hat of IL-1 beta alone could be explained by a higher rate of NO generation within the early 24-48 h incubation period that would favor the toxic syne rgism of NO and oxygen free radicals. Overexpression of Cat, Gpx, and Cu/Zn SOD protected against toxicity of the cytokine mixture but not against tha t of IL-1 beta alone. The reduction of cytokine-mediated toxicity was evide nt also because of an increased proliferation rate and a drastic decrease i n the cell death rate. The improved antioxidant defense status did not prev ent the activation of iNOS after cytokine exposure. However, RINm5F cells o verexpressing cytoprotective enzymes showed a significantly lower level of ROS-damaged protein residues. Thus, protection through Cat, Gpx, and Cu/Zn SOD overexpression was apparently because of an inactivation of ROS generat ed in the signal cascades of the cytokines. Overexpression of cytoprotectiv e enzymes thus represents a feasible strategy to protect insulin-producing cells against cytokine-mediated cytotoxicity.