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Signalling events involved in interferon‐γ‐inducible macrophage nitric oxide generation

Nitric oxide (NO) produced by macrophages (Mφ) in response to interferon‐γ (IFN‐γ) plays a pivotal role in the control of intracellular pathogens. Current knowledge of the specific biochemical cascades involved in this IFN‐γ‐inducible Mφ function is still limited. In the present study, we evaluated the participation of various second messengers – Janus kinase 2 (JAK2), signal transducer and activator of transcription (STAT) 1α, MAP kinase kinase (MEK1/2), extracellular signal‐regulated kinases 1 and 2 (Erk1/Erk2) and nuclear factor kappa B (NF‐κB) – in the regulation of NO production by IFN‐γ‐stimulated J774 murine Mφ. The use of specific signalling inhibitors permitted us to establish that JAK2/STAT1α‐ and Erk1/Erk2‐dependent pathways are the main players in IFN‐γ‐inducible Mφ NO generation. To determine whether the inhibitory effect was taking place at the pre‐ and/or post‐transcriptional level, we evaluated the effect of each antagonist on inducible nitric oxide synthase (iNOS) gene and protein expression, and on the capacity of IFN‐γ to induce JAK2, Erk1/Erk2 and STAT1α phosphorylation. All downregulatory effects occurred at the pretranscriptional level, except for NF‐κB, which seems to exert its role in NO production through an iNOS‐independent event. In addition, electrophoretic mobility shift assay (EMSA) analysis revealed that STAT1α is essential for IFN‐γ‐inducible iNOS expression and NO production, whereas the contribution of NF‐κB to this cellular regulation seems to be minimal. Moreover, our data suggest that Erk1/Erk2 are responsible for STAT1α Ser727 residue phosphorylation in IFN‐γ‐stimulated Mφ, thus contributing to the full activation of STAT1α. Taken together, our results indicate that JAK2, MEK1/2, Erk1/Erk2 and STAT1α are key players in the IFN‐γ‐inducible generation of NO by Mφ.