Macrophage killing of S180 cells is independent of activation of NOSII. Macrophages were purified from SR/CR and WT mice and analyzed for the production of RNS in response to challenge with S180 cells. (A) Macrophages stimulated with LPS and IFN-γ served as positive controls for RNS production. Macrophages were stimulated with S180 cells to determine if RNS was produced above baseline levels. All stimulations were carried out at 39°C for 24 h. Production of RNS was determined by incubation with Greiss reagent according to the manufacturer’s protocol. (B) 5 mM NMMA, 50 µM 1400W and 500 µM L-NIL were used as inhibitors of NOSII activation and RNS production in macrophages. (C) SR/CR macrophages were pre-incubated with NOSII inhibitors and then cultured with S180 cells in standard in vitro killing assay conditions to compare the level of S180 cell killing to that by uninhibited control macrophages. Results are reported as the mean and standard deviation of at least 3 experiments. (D) SR/CR macrophages were pre-incubated with 1.5 µg/ml LPS overnight and then cultured with S180 cells as described above in the presence of LPS. S180 cell killing was compared to that in a control without LPS. Results are reported as the mean and standard deviation of at least 4 separate experiments. The statistical significance of inhibition of killing as compared to uninhibited control samples was determined by the Student’s t-test. Statistically significant values were defined as P less than or equal to 0.05, and are denoted by a *.