THE CYTOTOXICITY OF TUMOR-NECROSIS-FACTOR DEPENDS ON INDUCTION OF THEMITOCHONDRIAL PERMEABILITY TRANSITION

Complete prevention of the killing of L929 fibroblasts by tumor necros is factor alpha (TNF) in the presence of 0.5 mu g/ml actinomycin D (Ac tD) was obtained with cyclosporin A (CyA), an inhibitor of the mitocho ndrial permeability transition (MPT), and aristolochic acid (ArA), a p hospholipase A(2) inhibitor, Peripheral benzodiazepine receptor (PBzR) agonists (PK11195, FGIN 1-27, or chlorodiazepam), agents known to pot entiate induction of the MPT, potentiated the cytotoxicity of TNF in t he absence of ActD, an effect prevented by CyA plus ArA, The MPT was d emonstrated independently of its effect on viability as the CyA-sensit ive loss of rhodamine 123 fluorescence from cells preloaded with the d ye, Treatment with TNF and ActD resulted in the loss of 80% of rhodami ne fluorescence within 6 h, a time prior to any loss of viability, CyA plus ArA completely prevented this effect of TNF, Potentiation of the cytotoxicity of TNF by PBzR agonists was associated with induction of the MPT, as assessed by the loss of rhodamine fluorescence, CyA plus ArA completely prevented the loss of rhodamine 123, Ceramide replaced TNF in killing L929 fibroblasts, an effect also prevented by CyA plus Ark Ceramide in the presence of ActD resulted in the loss of rhodamine fluorescence, an effect that was again prevented by CyA plus ArA. In addition, CyA plus ArA prevented the ability of PBzR agonists to poten tiate the cytotoxicity of ceramide, In the presence of each PBzR agoni st, ceramide caused the loss of rhodamine fluorescence, an effect comp letely prevented by CyA plus ArA. D609, an inhibitor of phosphatidylch oline-specific phospholipase C, completely prevented the killing by TN F, but not by ceramide, in the presence of ActD, D609 prevented induct ion of the MPT occurring with TNF, but not with ceramide. Inhibitors o f endocytosis, as well as lysosomotropic amines, prevented the cytotox icity of TNF, but not that of ceramide, It is concluded that the MPT i s causally linked to the genesis of irreversible cell injury with TNF, In the face of an inhibition of protein synthesis, the MPT occurs as a consequence of the formation of ceramide.