PEROXYNITRITE-INDUCED THYMOCYTE APOPTOSIS - THE ROLE OF CASPASES AND POLY (ADP-RIBOSE) SYNTHETASE (PARS) ACTIVATION

The mechanisms by which immature thymocyte apoptosis is induced during negative selection are poorly defined. Reports demonstrated that cros s-linking of T-cell receptor leads to stromal cell activation, express ion of inducible nitric oxide synthase (iNOS) and, subsequently, to th ymocyte apoptosis. Therefore we examined, whether NO directly or indir ectly, through peroxynitrite formation, causes thymocyte apoptosis. Im muno-histochemical detection of nitrotyrosine revealed in vivo peroxyn itrite formation in the thymi of naive mice. Nitrotyrosine, the footpr int of peroxynitrite, was predominantly found in the corticomedullary junction and the medulla of naive mice. In the thymi of mice deficient in the inducible isoform of nitric oxide synthase, considerably less nitrotyrosine was found. Exposure of thymocytes in vitro to low concen trations (10 mu M) of peroxynitrite led to apoptosis, whereas higher c oncentrations (50 mu M) resulted in intense cell death with the charac teristics of necrosis. We also investigated the effect of poly (ADP-ri bose) synthetase (PARS) inhibition on thymocyte apoptosis. Using the P ARS inhibitor 3-aminobenzamide (3-AB), or thymocytes from PARS-deficie nt animals, we established that PARS determines the fate of thymocyte death. Suppression of cellular ATP levels, and the cellular necrosis i n response to peroxynitrite were prevented by PARS inhibition. Therefo re, in the absence of PARS, cells are diverted towards the pathway of apoptotic cell death. Similar results were obtained with H2O2 treatmen t, while apoptosis induced by non-oxidative stimuli such as dexamethas one or anti-FAS antibody was unaffected by PARS inhibition. In conclus ion, we propose that peroxynitrite-induced apoptosis may play a role i n the process of thymocyte negative selection. Furthermore, we propose that the physiological role of PARS cleavage by apopain during apopto sis may serve as an energy-conserving step, enabling the cell to compl ete the process of apoptosis.