Adamson G M, Billings R E
Department of Surgery, School of Medicine, University of Nevada, Reno 89557.
Toxicol Appl Pharmacol. 1993 Mar;119(1):100-7. doi: 10.1006/taap.1993.1048.
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.
γ-干扰素（IFN-γ）已被证明在体内会加剧肿瘤坏死因子α（TNFα）诱导的肝毒性，并且在多种生物学作用中与TNFα协同发挥作用。在本研究中，我们检测了IFN-γ与TNFα的相互作用以及一氧化氮合酶（NOS）活性在分离的小鼠肝细胞内氧化应激产生中的作用。单独暴露于IFN-γ或TNFα均可显著增加NOS活性。联合使用时，TNFα和IFN-γ显著增加NOS活性，其增加程度超过了单纯相加效应的预期。IFN-γ增强了TNFα对肝细胞谷胱甘肽池的影响，增加了谷胱甘肽（GSH）耗竭和氧化型谷胱甘肽（GSSG）外流的程度。此外，IFN-γ加剧了TNFα诱导的三磷酸腺苷（ATP）耗竭。同时暴露于TNFα和IFN-γ会导致肝细胞产生显著的细胞毒性，而单独使用这两种细胞因子均未产生任何毒性。IFN-γ增强了用1,3-双（氯乙基）-1-亚硝基脲（BCNU，一种谷胱甘肽还原酶抑制剂）预处理的肝细胞中TNFα诱导的细胞毒性。当用抗氧化剂甘露醇处理肝细胞时，TNFα/IFN-γ诱导的GSSG外流受到抑制。此外，甘露醇降低了ATP耗竭的程度以及BCNU处理或未处理的肝细胞中TNFα和IFN-γ诱导的细胞毒性。相比之下，甘露醇消除了单独暴露于TNFα的BCNU处理细胞中的细胞毒性。因此，甘露醇对IFN-γ和TNFα诱导的有害氧化作用提供了显著的保护。然而，IFN-γ似乎也至少部分地通过增加氧自由基生成以外的机制增强了TNFα的有害作用。使用精氨酸的甲基化类似物NG-单甲基-L-精氨酸抑制NOS活性，结果表明TNFα/IFN-γ诱导的ATP耗竭、GSSG外流和细胞毒性并不依赖于NOS的激活。此外，直到暴露于任何一种细胞因子4小时后NOS活性才显著增加，而GSSG外流和ATP耗竭在孵育的最初4小时内就已发生。综上所述，这些结果表明IFN-γ与TNFα协同作用，导致细胞内氧化应激增强、能量代谢抑制和细胞毒性。然而，这些事件似乎与NOS活性的增加无关。
