SUPEROXIDE FORMATION AND MACROPHAGE RESISTANCE TO NITRIC OXIDE-MEDIATED APOPTOSIS

RAW 264.7 macrophages, when challenged with a combination of lipopolys accharide (10 mu g/ml) and interferon-gamma (100 units/ml), respond wi th endogenous NO. formation, which ultimately results in apoptotic cel l death. Apoptosis is detected morphologically by chromatin condensati on. Concomitantly we noticed the accumulation of the tumor suppressor protein p53. NO.-derived apoptosis was blocked by the NO.-synthase inh ibitor N-G-monomethyl-L-arginine. Repetitive treatment of RAW 264.7 ma crophages with lipopolysaccharide/interferon-gamma, followed by subcul turing viable cells, allowed us to select resistant macrophages which we called RES. RES cells still produced comparable amounts of nitrite/ nitrate in response to agonist treatment but showed no apoptotic marke rs, i.e. chromatin condensation or p53 accumulation. However, RES macr ophages undergo apoptosis in the presence of exogenously supplied NO., released from the NO-donors S-nitrosoglutathione or spermine-NO. Asse ssment of cytochrome c reduction established that RES cells released t wice the amount of superoxide compared to RAW 264.7 macrophages under both resting and stimulated conditions. We linked increased superoxide production to cellular macrophage resistance by demonstrating decreas ed apoptosis after simultaneous application of S-nitrosoglutathione or spermine-NO and the redox cycler 2,3-dimethoxy-1,4-naphthoquinone. Ou r results suggest that macrophage resistance toward NO.-mediated apopt osis is, at least in part, due to increased superoxide formation. Ther efore, the balance between reactive nitrogen and reactive oxygen speci es regulates RAW 264.7 macrophage apoptosis.