The production of IFN-gamma by IL-12/IL-18-activated macrophages requires STAT4 signaling and is inhibited by IL-4.

Macrophages release IFN-γ on combined stimulation with IL-12 and IL-18, but the signaling requirements of this process and its regulation by other cytokines are unknown. Here, we demonstrate that STAT4 is indispensable for IL-12/IL-18-induced production of IFN-γ by mouse peritoneal macrophages. Type 2 NO synthase (NOS2), which we previously found to be a prerequisite for IL-12-induced IFN-γ production in NK cells, was not required for IFN-γ production by these macrophages. IL-12 alone already induced the expression of IFN-γ mRNA, but nuclear translocation of STAT4, the release of IFN-γ protein, and the subsequent production of NO was strictly dependent on the simultaneous presence of IL-18. NF-κB, which mediates IL-18 effects in T cells, was only weakly activated by IL-12 and/or IL-18 in macrophages. Known inhibitors of macrophage functions (e.g., IL-4 and TGF-β) also suppressed macrophage IFN-γ production and the subsequent production of NOS2-derived NO. The inhibitory effect of IL-4 was paralleled by nuclear translocation of STAT6, which in EMSAs was able to bind to the same DNA oligonucleotide as STAT4. These results further define the production of IFN-γ by macrophages and point to a diversity in the signals required for IFN-γ production by various cell types.