Regulation of inducible nitric oxide synthase expression in IFN-gamma-treated murine macrophages cultured under hypoxic conditions.
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-gamma plus hypoxia (1% O2). In this study, we investigated the expression of NOS activity and the regulation of NOS induction in IFN-gamma treated ANA-1 murine macrophages or thioglycollate-elicited peritoneal macrophages cultured under hypoxic conditions. We found that murine macrophages stimulated with IFN-gamma plus hypoxia, despite a significant accumulation of iNOS mRNA, did not release nitrite into culture supernatant. However, cytosol from macrophages treated with IFN-gamma plus hypoxia contained significant levels of iNOS protein and enzymatic activity. Experiments in which cells were treated with IFN-gamma 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-gamma 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-gamma plus hypoxia-induced activation of the iNOS promoter. These data establish that hypoxia is a costimulus with IFN-gamma 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.
Melillo
G
G
Laboratory of Experimental Immunology, National Cancer Institute-Frederick Cancer Research and Development Center, MD 21702, USA.
Taylor
L S
LS
Brooks
A
A
Cox
G W
GW
Varesio
L
L
D000818:Animals; D002460:Cell Line; D005786:Gene Expression Regulation / Q000187:drug effects / Q000276:immunology; D000860:Hypoxia / Q000201:enzymology* / Q000276:immunology*; D007371:Interferon-gamma / Q000494:pharmacology*; D015847:Interleukin-4 / Q000494:pharmacology; D008264:Macrophages / Q000166:cytology / Q000187:drug effects* / Q000201:enzymology*; D017737:Macrophages, Peritoneal / Q000187:drug effects / Q000378:metabolism; D051379:Mice; D008810:Mice, Inbred C57BL; D019001:Nitric Oxide Synthase / Q000037:antagonists & inhibitors / Q000096:biosynthesis*
D015847:Interleukin-4; D007371:Interferon-gamma; D019001:Nitric Oxide Synthase