Cytokine toxicity and induction of NO synthase activity in cultured mouse hepatocytes

Interferon-gamma (IFN-gamma) has been shown to exacerbate tumor necrosis factor alpha (TNF alpha)-induced hepatotoxicity in vivo as well as act synergistically with TNF alpha in a variety of biological actions. In the present study we have examined interactions of IFN-gamma with TNF alpha and the role of nitric oxide synthase (NOS) activity in the generation of an intracellular oxidant stress in isolated mouse hepatocytes. Exposure to either IFN-gamma or TNF alpha significantly increased NOS activity. In combination, TNF alpha and IFN-gamma markedly increased NOS activity beyond that expected for a merely additive effect. IFN-gamma potentiated TNF alpha-induced effects on the hepatocyte glutathione pool, increasing the extent of GSH depletion and GSSG efflux. Furthermore, IFN-gamma exacerbated TNF alpha-induced ATP depletion. Exposure to both TNF alpha and IFN-gamma resulted in significant cytotoxicity in hepatocytes, whereas neither cytokine alone produced any toxicity. TNF alpha-induced cytotoxicity in hepatocytes pretreated with 1,3-bis (chloroethyl)-1-nitrosourea (BCNU, a glutathione reductase inhibitor) was potentiated by IFN-gamma. TNF alpha/IFN-gamma-induced GSSG efflux was prevented when hepatocytes were treated with the antioxidant mannitol. Furthermore, mannitol reduced the extent of ATP depletion as well as cytotoxicity induced by TNF alpha and IFN-gamma in either BCNU- or non-BCNU-treated hepatocytes. In contrast, mannitol abolished cytotoxicity in BCNU-treated cells exposed to TNF alpha alone. Thus, mannitol provides significant protection against deleterious oxidative effects induced by IFN-gamma and TNF alpha. However, IFN-gamma also appears to potentiate the deleterious effects of TNF alpha, at least in part, by mechanisms other than an increase in oxygen radical generation. Using the methylated analog of arginine, NG-monomethyl-L-arginine, to inhibit NOS activity, it was demonstrated that TNF alpha/IFN-gamma-induced ATP depletion, GSSG efflux, and cytotoxicity were not dependent upon the stimulation of NOS. Furthermore, significant increases in NOS activity did not occur until after 4 hr of exposure to either cytokine, whereas GSSG efflux and ATP depletion occurred during the first 4 hr of incubation. Taken together, these results indicate that IFN-gamma acts synergistically with TNF alpha, resulting in the potentiation of an intracellular oxidative stress, inhibition of energy metabolism, and cytotoxicity. However, these events do not appear to be related to an increase in NOS activity.