NITRIC-OXIDE INDUCES APOPTOSIS IN MOUSE THYMOCYTES

Nitric oxide (NO) produced at high concentrations by the inducible NO synthase is an important effector molecule involved in immune regulati on and defense. We have examined whether NO represents a signal for tr iggering apoptosis in thymocytes. Freshly isolated thymocytes were inc ubated with different chemical NO donors for various intervals. Apopto sis was determined by detection of DNA strand breaks with in situ nick translation. All NO donors induced thymocyte apoptosis with 30% posit ive thymocytes vs 10% in controls after 8 h. Apoptosis was prevented b y addition of ZnSO4. Short-term pre-exposure to NO resulted in protect ion from apoptosis induced by glucocorticoids comparable with the prot ective effect of heat shock. Flow cytometry revealed that NO treatment as well as heat shock or dexamethasone incubation is accompanied by r eduction in the CD4(+)CD8(+) thymocyte subpopulation. Apoptosis induct ion was accompanied by increased expression of p53, as detected by PCR analysis 2 h after NO donor addition. In vivo treatment of mice with endotoxin results in increased thymic apoptosis. Focal apoptosis was f ound to occur in close proximity to blood vessels 18 h after LPS treat ment. Capillary endothelium and dendritic cells adjacent to apoptotic foci were found to stain strongly for inducible NO synthase expression . Furthermore, in an in vitro experiment using cocultures of thymocyte s with LPS/cytokine-activated endothelial cells expressing inducible N O synthase, a significantly increased rate of thymocyte apoptosis was found, and this could be prevented completely by inhibiting NO product ion. Addition of dexamethasone to these cocultures did not lead to a f urther increase in the percentage of apoptotic thymocytes, underlining the protective effect of NO on dexamethasone-induced apoptosis.