In vivo and in vitro apoptosis of human thymocytes are associated with nitrotyrosine formation

Most thymocytes are deleted by thymic selection. The mechanisms of cell dea th are far from being clear. Peroxynitrite is a powerful oxidant produced i n vivo by the reaction of superoxide (O-2(.-)) With nitric oxide (NO.) and is able to mediate apoptosis. The aim of this study was to analyze whether NO and peroxynitrite could play a role in human thymocyte apoptosis, The re sults indicate that 3-(4-morpholinyl)-sydnonimine (SIN-1, an O-2(.-) and NO . donor) and chemically synthesized peroxynitrite, but not S-nitroso-N-acet yl-D,L-penicillamine (SNAP, an NO. donor), have a strong apoptotic effect o n human thymocytes (annexin V staining and TUNEL reaction). This effect was inhibited by exogenous superoxide dismutase (SOD), which interacts with O- 2(.-) and inhibits the formation of peroxynitrite. Because peroxynitrite fo rmation requires NO, thymic stromal cells were investigated to determine if they produced NO.. Inducible NOS was synthesized in cultured thymic epithe lial cells in certain conditions of cytokine stimulation, as shown by messe nger RNA levels, protein analysis, and nitrite production in the supernatan ts, SIN-1-treated thymocytes had high levels of tyrosine nitration, abolish ed by the addition of exogenous SOD, Tyrosine nitration was also detected i n thymus extracts and sections, suggesting the presence of peroxynitrite in situ, In thymus sections, clusters of nitrotyrosine-positive cells were fo und in the cortex and corticomedullary areas colocalized with cells positiv e in the TUNEL reaction. These data indicate an association between human t hymocyte apoptosis and nitrotyrosine formation. Thus, the results support t he notion of a physiologic role for peroxynitrite in human thymocyte apopto sis, (Blood, 2001;97:3521-3530) (C) 2001 by The American Society of Hematol ogy.