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

Authors

  • Marta Llovera,

    1. Centre for Cardiovascular Biology & Medicine, School of Biomedical Sciences, King's College London, Guy's Campus, London SE1 1UL
    Search for more papers by this author
    • 3

      Current address: INSERM Unit 344, 156 rue de Vaugirard, 75730 Paris, France

  • Jeremy D Pearson,

    Corresponding author
    1. Centre for Cardiovascular Biology & Medicine, School of Biomedical Sciences, King's College London, Guy's Campus, London SE1 1UL
    Search for more papers by this author
  • Carlos Moreno,

    1. Department of Immunology, School of Medicine, King's College London, Denmark Hill Campus, London SE5 9PJ
    Search for more papers by this author
  • Valentina Riveros-Moreno

    1. Centre for Cardiovascular Biology & Medicine, School of Biomedical Sciences, King's College London, Guy's Campus, London SE1 1UL
    Search for more papers by this author

Centre for Cardiovascular Biology & Medicine, School of Biomedical Sciences, King's College London, Guy's Campus, London SE1 1UL. E-mail: jeremy.pearson@kcl.ac.uk

Abstract

  • 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

Ancillary