Tumor necrosis factor-α-dependent production of reactive nitrogen intermediates mediates IFN-γ plus IL-2-induced murine macrophage tumoricidal activity

We have previously established that IFN-γ plus IL-2 induces murine macrophage tumoricidal activity. The purpose of this study was to identify the effector molecules that account for the IFN-γ plus IL-2-induced macrophage cytotoxicity against P815 mastocytoma cells. ANA-1 macrophages and normal thioglycollate-elicited mouse peritoneal macrophages produced little or no detectable nitrite (NO₂^-) after incubation with IFN-γ alone or IL-2 alone; however, IL-2 synergized with IFN-γ for the production of NO₂^-. IFN-γ plus IL-2 did not induce NO₂^- production or tumoricidal activity in ANA-1 macrophages that were cultured in medium devoid of L-arginine or in ANA-1 macrophages that were incubated with N^G-monomethyl-L-arginine. As observed previously with ANA-1 macrophage tumoricidal activity, IL-4 inhibited IFN-γ plus IL-2-induced, but not IFN-7 plus LPS-induced, NO₂^- production. IL-4 also selectively decreased the ability of IFN-γ and/or IL-2 to augment TNF-α mRNA expression in ANA-1 macrophages. Lastly, incubation of ANA-1 macrophages with anti-TNF mAb selectively inhibited the ability of IFN-γ plus IL-2 to induce NO₂^- production and tumoricidal activity. These results indicate that IFN-γ plus IL-2-induced tumoricidal activity is dependent upon the metabolism of L-arginine to reactive nitrogen intermediates, and they establish a role for TNF-α as a required intermediate for IL-2-dependent NO₂^- production and tumoricidal activity.