Nitric oxide regulates Th1 cell development through the inhibition of IL-12 synthesis by macrophages

We have previously reported that mice lacking inducible nitric oxide syntha se (NOS2) developed enhanced Th1 cell responses. We now investigated the me chanism by which NO modulates Th1 cells differentiation. Peritoneal macroph ages from NOS2-deficient mice infected with Leishmania major in vivo or sti mulated with IFN-gamma or lipopolysaccharide (LPS) in vitro produced signif icantly higher levels of IL-12 than those from heterozygous or wild-type mi ce. A macrophage cell line, J774, produced significant amounts of IL-12 fol lowing activation with LPS, or LPS plus IFN-gamma. This could be markedly e nhanced by the NOS inhibitor L-N-G monomethyl arginine (L-NMMA), but profou ndly inhibited by the NO-generating compound S-nitroso-N-acetyl-penicillami ne (SNAP). The effect of NO in this system is selective, since SNAP enhance d and L-NMMA decreased TNF-alpha synthesis by LPS-activated J774 cells. The differential effect of NO on IL-12 and TNF-alpha is at the transcriptional level and is activation dependent. Since IL-12 is a major inducer of Th1 c ells which produce IFN-gamma that can activate macrophages to produce IL-12 , our data demonstrate that NO can be an inhibitor of this feedback loop, p reventing the excessive amplification of Th1 cells which are implicated in a range of immunopathologies.