Mechanisms of cytokine-induced death of cultured bovine luteal cells

Tumour necrosis factor alpha (TNF-alpha) and gamma-interferon (IFN-gamma) a re cytotoxic to bovine luteal cells in vitro and may contribute to cell dea th during luteolysis in vivo. In this study, the mechanism by which luteal cells are killed by TNF-alpha and IFN-gamma was investigated. Luteal cells were cultured for 7 days in the presence or absence of TNF-alpha and IFN-ga mma. Inhibitors of arachidonate metabolism or scavengers of free radicals w ere included in the culture media. In addition, the effect of IFN-alpha on the viability of cytokine-treated luteal cells was tested. Lastly, untreate d and cytokine-treated cells were subjected to single cell gel electrophore sis for quantification of DNA fragmentation. Neither indomethacin nor nordi hydroguaiaretic acid, which are inhibitors of cyclooxygenase and lipoxygena se, respectively, were able to prevent cytokine-induced cell death. Similar ly, both the phospholipase A, inhibitor arachidonyltrifluoromethyl ketone a nd the nitric oxide synthase inhibitor NC-monomethyl-L-arginine, were large ly without effect. In contrast, while vitamin C did not significantly affec t viability, superoxide dismutase plus catalase increased viability of cyto kine-treated cells (P < 0.05), and IFN-alpha prevented cell death (P < 0.05 ). Finally, while control cells remained free of DNA damage, TNF-alpha plus IFN-gamma induced significant amounts of DNA damage by 48 h after initiati on of treatment (P < 0.05). In conclusion, reactive oxygen species, but not arachidonate metabolism or nitric oxide, contribute to cytokine-induced lu teal cell death in vitro, and the process of cell death may be via apoptosi s. Furthermore, IFN-alpha may confer protective effects against cytokine-in duced cell death in bovine luteal cells.