Nitric oxide and TNF-α are involved in the antitumor effects of anti-CD40 Ab-activated macrophages against murine melanoma

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CD40 ligation has been shown to induce antitumor effects mediated by cytotoxic T cells and NK cells. More recently, in a model of murine melanoma, our findings suggested a role for antitumor macrophages following anti-CD40 Ab therapy. In this study, we investigated the cytotoxic effector mechanisms of anti-CD40 Ab-activated macrophages with particular attention to nitric oxide (NO) and tumor necrosis factor-α (TNF-α), known macrophage cytotoxic molecules. An initial time-course study showed that in vivo anti-CD40 Ab-activated macrophages from C57BL/6 mice exhibited the greatest degree of tumoristatic activity in vitro against B16 melanoma five days after anti-CD40 Ab treatment, which correlated with increased production of NO. In vitro inhibition of NO production by the NO synthase inhibitor, L-nitro-arginine-methyl esterase (L-NAME) resulted in reduced NO release and tumoristatic activity by day 5 anti-CD40 Ab-activated macrophages. This finding was consistent with our observation of a significant reduction in tumoristatic activity by anti-CD40 Ab-activated macrophages from inducible NO synthase knockout (iNOS KO) mice compared to macrophages from anti-CD40 Ab-activated control mice. However, NK cell killing following anti-CD40 Ab activation was not decreased in iNOS KO mice. Nevertheless, anti-CD40 Ab treatment of subcutaneous B16 tumor-bearing iNOS KO mice resulted in significant antitumor effects, which were not dependent on T cells and NK cells (based on in vivo depletion studies). To further clarify the cytotoxic effect of anti-CD40 Ab-activated macrophages, the addition of neutralizing TNF-α mAb was found to reduce, but not abrogate, the tumoristatic activity of anti-CD40 Ab-activated macrophages from control and iNOS KO mice. Additionally, the in vitro antitumor activity of anti-CD40 Ab-activated macrophages from TNF-α deficient mice was reduced but not eliminated. Taken together, our results suggest that both NO and TNF-α are involved in, but not solely responsible for, the cytotoxic effect of macrophages after their activation by CD40 ligation.