The Cytotoxicity of Tumor Necrosis Factor Depends on Induction of the Mitochondrial Permeability Transition

Abstract

Complete prevention of the killing of L929 fibroblasts by tumor necrosis factor alpha (TNF) in the presence of 0.5 microg/ml actinomycin D (ActD) was obtained with cyclosporin A (CyA), an inhibitor of the mitochondrial permeability transition (MPT), and aristolochic acid (ArA), a phospholipase A2 inhibitor. Peripheral benzodiazepine receptor (PBzR) agonists (PK11195, FGIN 1-27, or chlorodiazepam), agents known to potentiate induction of the MPT, potentiated the cytotoxicity of TNF in the absence of ActD, an effect prevented by CyA plus ArA. The MPT was demonstrated independently of its effect on viability as the CyA-sensitive loss of rhodamine 123 fluorescence from cells preloaded with the dye. Treatment with TNF and ActD resulted in the loss of 80% of rhodamine 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 ArA. 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 potentiate the cytotoxicity of ceramide. In the presence of each PBzR agonist, ceramide caused the loss of rhodamine fluorescence, an effect completely prevented by CyA plus ArA. D609, an inhibitor of phosphatidylcholine-specific phospholipase C, completely prevented the killing by TNF, but not by ceramide, in the presence of ActD. D609 prevented induction of the MPT occurring with TNF, but not with ceramide. Inhibitors of endocytosis, as well as lysosomotropic amines, prevented the cytotoxicity of TNF, but not that of ceramide. It is concluded that the MPT is 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.