Impaired response to interferon-gamma in activated macrophages due to tyrosine nitration of STAT1 by endogenous nitric oxide

1 Inducible NO synthase (iNOS) expression and activity were measured in the mouse macrophage cell line J774 after exposure to bacterial lipopolysaccharide (LPS) with or without interferon-gamma (IFN-gamma). 2 Inhibition of NOS activity by concomitant N-G-monomethyl-L-arginine (L-NMMA) treatment further increased iNOS protein levels, with a substantial increase in iNOS half-life. 3 Western blotting and ELISA demonstrated that several cell proteins were tyrosine-nitrated when iNOS activity was high. 4 Rapid IFN-gamma -induced phosphorylation of STAT1 was reduced by about 40% when cells were pretreated to induce iNOS, unless L-NMMA was present during the pretreatment period. 2D gel electrophoresis demonstrated the presence of nitrotyrosine in STAT1 after iNOS induction, and confirmed the reduction in phospho-STAT1 on subsequent stimulation with IFN-gamma for 15 min and its partial restoration when L-NMMA was present during the pretreatment period. 5 We did not detect tyrosine nitration of the upstream kinase JAK2 in LPS+IFN-gamma pretreated cells, but JAK2 activity was also impaired, and was partially restored by concomitant L-NMMA pretreatment. 6 We conclude that endogenous production of NO induces feedback inhibition of signalling pathways activated by IFN-gamma, at least in part by nitrating tyrosine residues in STAT1 which prevents phosphorylation.