Peroxynitrite Protects Raw 264.7 Macrophage from Lipopolysaccharide/Interferon-γ-Induced Cell Death

Abstract

Peroxynitrite, formed by the interaction of superoxide with nitric oxide, has previously been implicated mostly as a cytotoxic agent. In contrast, its physiological and, possibly, beneficial effects are largely unknown. We have previously shown [Journal of Biological Chemistry, 1997,272,7253] that RAW 264.7 macrophages can be selected to be resistant toward lipopolysaccharide (LPS)/interferon-γ (IFN-γ)-induced cytotoxicity. Resistant cells produced comparable amount of nitric oxide, but showed increased formation of superoxide, which might lead to increased production of peroxynitrite. We utilized this well characterized cell model to seek evidence that peroxynitrite might cause protection of RAW cells from cytokine toxicity. Exogenous peroxynitrite (30-50 μM), applied to RAW cells before cytokine stimulation, dramatically reduced LPS/IFN-γ toxicity. Measurement of cell viability after overnight incubation with a mixture of LPS (10 μg/ml) and IFN-γ (100 U/ml), showed that pretreatment with 40 μM peroxynitrite completely reverted LPS/IFN-γ cytotoxicity. Differently, pretreatment of RAW cells with peroxynitrite (10-60 μM) did not prevent cytotoxicity induced by the nitric oxide-donors S-Nitroso-L-glutathione (0.2-1 mM), or spermine NONOate (0.2-2 mM), and by Actimomycin D (0.5-1 μg/ml), suggesting that the protective effect is specific for the LPS/IFN-γ pathway. These results were confirmed through extensive controlled studies aimed to optimize cell exposure to peroxynitrite, and showed that peroxynitrite protects macrophages from cytokine-induced cytotoxicity.