Cytotoxic actions of cytokines on cultured mouse luteal cells are independent of nitric oxide

We investigated the cytotoxic effects of various cytokines secreted by macrophages or T lymphocytes on luteal cells, and the role of nitric oxide (NO) produced by luteal cells in cytotoxic actions of cytokines. Mouse luteal cells were cultured in serum-free medium with interferon-γ (IFN-γ), tumor necrosis factor-α (TNF-α) or interleukin-1β (IL-1β) alone, or with various combinations of these cytokines for 6 days. Cytotoxic actions of cytokines and NO production by luteal cells were evaluated by number of viable cells and the amount of nitrite and nitrate (stable metabolites of NO) in medium, respectively. IFN-γ (1000 U/ml), TNF-α (3000 U/ml), or IL-1β (30 U/ml) alone, and the combination of TFN-α and IL-1β (10 U/ml) did not decrease number of viable cells and was without effects on NO production. The combination of IFN-γ and IL-1β (10 U/ml) also did not decrease the number of viable cells, while it increased NO production a little but significantly. Combinations of INF-γ and TNF-α, and IFN-γ, TNF-α and IL-1β (10 U/ml) markedly decreased number of viable cells. The combination of IFN-γ and TNF-α increased NO production a little but significantly, and the combination of three cytokines (IFN-γ, TNF-α, and IL-1β) caused a greater increase in NO production. An NO synthase inhibitor, l- N G-monomethy- l-arginine (0.5 mM) or aminoguanidine (0.5 mM) abolished increases in NO production induced by combinations of IFN-γ and TNF-α, and IFN-γ, TNF-α and IL-1β completely without effects on number of viable cells. The present results indicate that combinations of cytokines including IFN-γ and TNF-α induce death of cultured mouse luteal cells, and that the cytotoxic actions of these cytokines are independent of NO production by luteal cells.