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-γ (IFN-γ).
Inhibition of NOS activity by concomitant NG-monomethyl-L-arginine (L-NMMA) treatment further increased iNOS protein levels, with a substantial increase in iNOS half-life.
Western blotting and ELISA demonstrated that several cell proteins were tyrosine-nitrated when iNOS activity was high.
Rapid IFN-γ-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-γ for 15 min and its partial restoration when L-NMMA was present during the pretreatment period.
We did not detect tyrosine nitration of the upstream kinase JAK2 in LPS+IFN-γ pretreated cells, but JAK2 activity was also impaired, and was partially restored by concomitant L-NMMA pretreatment.
We conclude that endogenous production of NO induces feedback inhibition of signalling pathways activated by IFN-γ, at least in part by nitrating tyrosine residues in STAT1 which prevents phosphorylation.
British Journal of Pharmacology (2001) 132, 419–426; doi:10.1038/sj.bjp.0703838