Regulation of Inducible Nitric Oxide Synthase Expression in IFN-γ-Treated Murine Macrophages Cultured under Hypoxic Conditions

Giovanni Melillo, Lynn S. Taylor, Alan Brooks, George W. Cox, Luigi Varesio

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We recently reported that a hypoxia-responsive element mediates a novel pathway of transcriptional activation of the inducible nitric oxide synthase (iNOS) promoter in murine macrophages treated with IFN-γ plus hypoxia (1 % O2). In this study, we investigated the expression of iNOS activity and the regulation of iNOS induction in IFN-γ-treated ANA-1 murine macrophages or thioglycollate-elicited peritoneal macrophages cultured under hypoxic conditions. We found that murine macrophages stimulated with IFN-γ plus hypoxia, despite a significant accumulation of iNOS mRNA, did not release nitrite into culture supernatant. However, cytosol from macrophages treated with IFN-γ plus hypoxia contained significant levels of iNOS protein and enzymatic activity. Experiments in which cells were treated with IFN-γ plus hypoxia and then cultured in normoxic conditions (20% O2) demonstrated that reoxygenation was required to achieve detectable accumulation of nitrite in the culture supernatant. Furthermore, we demonstrated that IL-4 inhibited IFN-γ plus hypoxia-dependent induction of iNOS mRNA expression, iNOS protein, and enzymatic activity. Experiments in which ANA-1 macrophages were transfected transiently with the full-length iNOS promoter linked to a chloramphenicol acetyltransferase reporter gene demonstrated that IL-4 also down-regulated the IFN-γ plus hypoxia-induced activation of the iNOS promoter. These data establish that hypoxia is a costimulus with IFN-γ for the induction of iNOS activity in ANA-1 macrophages as well as in murine peritoneal macrophages, and they provide the first evidence that IL-4 inhibits hypoxia-inducible gene expression. In addition, our results suggest that hypoxia, which occurs in many pathologic conditions, may play an important role in the activation of murine macrophages.

Original languageEnglish
Pages (from-to)2638-2644
Number of pages7
JournalJournal of Immunology
Issue number6
Publication statusPublished - Sep 15 1996


ASJC Scopus subject areas

  • Immunology

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