Brief hypoxia differentially regulates LPS-induced IL-1beta and TNF-alpha gene transcription in RAW 264.7 cells.
Episodes of tissue hypoxia and reoxygenation frequently occur during gram-negative 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)-alpha and interleukin (IL)-1beta 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-alpha and IL-1beta 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-1beta and TNF-alpha concentrations in cell lysates and culture supernatants were measured by ELISA, and steady-state mRNA was measured by Northern analysis. LPS-induced IL-1beta 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-alpha 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-1beta gene expression by hypoxia occurred transcriptionally. Allopurinol or catalase treatment did not alter modulation of LPS-induced IL-1beta expression by hypoxia, suggesting that this suppression was not caused by reactive oxygen species. Cycloheximide pretreatment suggested that hypoxia-induced downregulation of IL-1beta expression did not require de novo protein synthesis.
Ndengele
M M
MM
Department of Internal Medicine, Saint Louis University School of Medicine, MO 63110-0250, USA.
Bellone
C J
CJ
Lechner
A J
AJ
Matuschak
G M
GM
D000818:Animals; D002232:Carbocyanines / Q000494:pharmacology; D002374:Catalase / Q000494:pharmacology; D002460:Cell Line; D006706:Homeostasis; D000860:Hypoxia / Q000235:genetics*; D007375:Interleukin-1 / Q000037:antagonists & inhibitors / Q000235:genetics*; D008070:Lipopolysaccharides / Q000494:pharmacology*; D051379:Mice; D012333:RNA, Messenger / Q000037:antagonists & inhibitors / Q000378:metabolism; D014158:Transcription, Genetic; D014409:Tumor Necrosis Factor-alpha / Q000235:genetics* / Q000378:metabolism
D002232:Carbocyanines; D007375:Interleukin-1; D008070:Lipopolysaccharides; D012333:RNA, Messenger; D014409:Tumor Necrosis Factor-alpha; D002374:Catalase