Discrimination between NK and LAK cytotoxic activities of murine spleen cells by MTT assay: differential inhibition by PGE(2) and EDTA.

In the present study we propose a mathematical approach to improve the analysis of NK and LAK activities measured by MTT assay adapted for murine cells. We found that to calculate NK activity, high E:T ratios should be used (up to 50:1) and the phenomenon fits to a linear least-squares analysis. However, 5-fold less effector cells (10:1, E:T) should be used to detect LAK activity and the phenomenon has a nonlinear exponential behavior. Using this approach, we showed that EDTA inhibits LAK but not NK activity whereas PGE(2) inhibits NK but not LAK activity. In conclusion, this analytical approach allowed the discrimination between NK and LAK activities and exposed differences between these two cytotoxic activities.

Mycoplasma arginini enhances cytotoxicity of thioglycollate-elicited murine macrophages toward YAC-1 tumor cells through production of NO.

Bacterial products stimulate macrophage tumoricidal activity through release of tumor necrosis factor (TNF) and nitric oxide (NO). We show here that thioglycollate-elicited macrophages acquire cytotoxic activity when cocultured with Mycoplasma arginini-infected YAC-1 tumor cells and release TNF and NO. Fixed mycoplasma-infected cells, supernatants from infected-cell cultures, or purified heat-killed mycoplasma obtained from cell-free cultures were all able to induce TNF and NO production. Thus, the mycoplasma per se and not a product of infected cells induce the release of these molecules. Addition of prostaglandin E2 (PGE2) to the cocultures, which reduced TNF release, or antibodies to TNF, did not affect macrophage cytotoxicity nor NO release. Inhibition of NO production by L-NAME or aminoguanidine reduced the cytotoxicity, and treatment with a NO donor was toxic to YAC-1 cells. These results indicate that M. arginini activates thioglycollate-elicited murine macrophages for NO and TNF release increasing their cytotoxic activity toward YAC-1 cells and that this activity is dependent on NO but not TNF release.