APOPTOSIS OF MYELIN-REACTIVE T-CELLS INDUCED BY REACTIVE OXYGEN AND NITROGEN INTERMEDIATES IN-VITRO

Apoptosis is a major mechanism of T cell elimination during ontogeny a nd tolerance induction as well as in autoimmunity, To assess the possi ble involvement of reactive oxygen and nitrogen intermediates (ROI and NO.) in T cell apoptosis during autoimmune demyelination we investiga ted the effects of H2O2 and NO. in vitro on activated autoreactive CD4 (+) T cell lines capable of transferring experimental autoimmune encep halomyelitis (EAE) and experimental autoimmune neuritis (EAN), For det ection and quantitation of apoptotic cells, DNA fragmentation was asse ssed by in situ tailing with fluorescein-ddUTP and subsequent how cyto metric analysis, H2O2 applied directly to the cell cultures for 6 to 1 8 hr at concentrations of 10 to 300 mu M and ROI released by combinati on of hypoxanthine and xanthine oxidase (HX/XO) caused apoptosis in a dose-dependent manner in 13-33% of T cells of neuritogenic and encepha litogenic T cell lines, Apoptosis induction could be suppressed by the H2O2-neutralizing enzyme catalase, NO. released by the penicillamine derivative SNAP induced apoptosis to a similar extent as ROI, Maximum values were 38% in an encephalitogenic V beta 8.2-T cell receptor-bear ing T cell line and 26% in a neuritogenic T cell line, T cell lines wi th specificity to ovalbumin revealed slightly lower susceptibility to apoptosis induction by all three kinds of trigger, which is, however, most probably not due to the different antigen specificity, but rather a result of fewer in vitro restimulation cycles of these cells, In ne uritogenic cells high-dose (100 units/ml) exogenous interleukin-a (IL- 2) prevents H2O2-induced apoptosis. In conclusion, macrophage-derived reactive oxygen and nitrogen intermediates have the potency to limit i nflammatory demyelination by elimination of autoreactive and bystander T cells via apoptotic cell death, and IL-2 is a rescue factor. (C) 19 97 Academic Press.