Mechanisms of cytokine-induced death of cultured bovine luteal cells

Tumour necrosis factor α (TNF-α) and gamma-interferon (IFN-γ) are cytotoxic to bovine luteal cells in vitro and may contribute to cell death during luteolysis in vivo. In this study, the mechanism by which luteal cells are killed by TNF-α and IFN-γ was investigated. Luteal cells were cultured for 7 days in the presence or absence of TNF-α and IFN-γ. Inhibitors of arachidonate metabolism or scavengers of free radicals were included in the culture media. In addition, the effect of IFN-α on the viability of cytokine-treated luteal cells was tested. Lastly, untreated and cytokine-treated cells were subjected to single cell gel electrophoresis for quantification of DNA fragmentation. Neither indomethacin nor nordihydroguaiaretic acid, which are inhibitors of cyclooxygenase and lipoxygenase, respectively, were able to prevent cytokine-induced cell death. Similarly, both the phospholipase A₂ inhibitor arachidonyltrifluoromethyl ketone and the nitric oxide synthase inhibitor N^G-monomethyl-L-arginine, were largely without effect. In contrast, while vitamin C did not significantly affect viability, superoxide dismutase plus catalase increased viability of cytokine-treated cells (P < 0.05), and IFN-α prevented cell death (P < 0.05). Finally, while control cells remained free of DNA damage, TNF-α plus IFN-γ induced significant amounts of DNA damage by 48 h after initiation of treatment (P < 0.05). In conclusion, reactive oxygen species, but not arachidonate metabolism or nitric oxide, contribute to cytokine-induced luteal cell death in vitro, and the process of cell death may be via apoptosis. Furthermore, IFN-α may confer protective effects against cytokine-induced cell death in bovine luteal cells.