Regulation of peroxynitrite-induced thymocyte apoptosis by poly(ADP-ribose) synthetase (PARS)

Peroxynitrite, an oxidant formed in the reaction of nitric oxide (NO) and superoxide has been shown to be an important mediator of tissue injury in shock, inflammation and hypoxia. These effects are related in part to PARS activation. Since NO has previously been shown to play a role in the negative selection of thymocytes, here we have investigated whether NO directly, or indirectly through the formation of peroxynitrite, may be responsible for thymocyte apoptosis. Immunohistochemical detection of nitrotyrosine, a marker of peroxynitrite demonstrate the formation of peroxynitrite in vivo in the thymi of naive mice. Immunohistochemical double staining also revealed that nitrotyrosine-reactive cells co-localize with apoptotic cells in the corticomedullary junction. Exposure of thymocytes to low concentrations (20μM) of peroxynitrite led to apoptosis as indicated by DNA fragmentation, phosphatidylserine exposure and propidium iodide exclusion, whereas higher concentrations (100μM) of peroxynitrite resulted in an intense cell death with the characteristics of necrosis. Using PARS-deficient thymocytes or in the presence of the PARS inhibitor 3-aminobenzamide (3-AB) which inhibited ATP consumption by PARS, cells were diverted from necrosis toward apoptosis. Results similar to the ones seen with peroxynitrite were also obtained with H2O2 treatment, whereas the apoptosis induced by non-oxidative stimuli such as dexamethasone or anti-FAS antibody was unaffected by PARS inhibition. Our results indicate that peroxynitrite may be involved in the negative selection of thymocytes and peroxynitrite-induced ATP-depletion by PARS determines the mode of cell death.