Brief hypoxia differentially regulates LPS-induced IL-1β and TNF-α gene transcription in RAW 264.7 cells

Ndengele, Michael M., Clifford J. Bellone, Andrew J. Lechner, and George M. Matuschak. Brief hypoxia differentially regulates LPS-induced IL-1b and TNF-a gene transcription in RAW 264.7 cells. Am J Physiol Lung Cell Mol Physiol 278: L1289–L1296, 2000.—Episodes of tissue hypoxia and reoxygenation frequently occur during gramnegative bacteremia that progresses to septic shock. However, few studies have evaluated modulation by hypoxia and reoxygenation of the proinflammatory cytokine gene expression that is normally induced by gram-negative bacteremia or endotoxemia. In buffer-perfused organs, hypoxia downregulates Escherichia coli-induced expression of tumor necrosis factor (TNF)-a and interleukin (IL)-1b in the liver but upregulates these cytokines in the lungs. To identify molecular mechanisms underlying these events, we investigated the effects of brief (1.5-h) hypoxia on TNF-a and IL-1b expression in cultured RAW 264.7 cells during their continuous exposure to lipopolysaccharide (LPS) endotoxin derived from E. coli (serotype 055:B5) for up to 24 h. IL-1b and TNF-a concentrations in cell lysates and culture supernatants were measured by ELISA, and steady-state mRNA was measured by Northern analysis. LPS-induced IL-1b synthesis was downregulated by hypoxia at both the protein and mRNA levels despite no change in cellular redox status as measured by levels of GSH. In contrast, LPS-induced TNF-a production was unaffected by hypoxia as assessed by cell lysate mRNA and lysate and supernatant protein levels. Nuclear runoff analysis showed that downregulation of IL-1b gene expression by hypoxia occurred transcriptionally. Allopurinol or catalase treatment did not alter modulation of LPS-induced IL-1b expression by hypoxia, suggesting that this suppression was not caused by reactive oxygen species. Cycloheximide pretreatment suggested that hypoxia-induced downregulation of IL-1b expression did not require de novo protein synthesis.