title: Hyperoxia synergistically increases TNF-alpha-induced interleukin-8 gene expression in A549 cells.
abstract: Interleukin (IL)-8 is an important mediator of acute lung injury. Hyperoxia induces IL-8 production in some cell types, but its effect on IL-8 gene expression in respiratory epithelium is not well described. In addition, IL-8 gene expression resulting from the combined effects of hyperoxia and proinflammatory cytokines has not been well characterized. We treated cultured respiratory epithelial-like cells (A549 cells) with hyperoxia alone, tumor necrosis factor (TNF)-alpha alone, or the combination of TNF-alpha and hyperoxia and evaluated IL-8 gene expression. Hyperoxia alone had a minimal effect on IL-8 gene expression, and TNF-alpha alone increased IL-8 gene expression in a time-dependent manner. In contrast, the combination of TNF-alpha and hyperoxia synergistically increased IL-8 gene expression as measured by ELISA (TNF-alpha alone for 24 h = 769 +/- 89 pg/ml vs. hyperoxia + TNF-alpha for 24 h = 1, 189 +/- 89 pg/ml) and Northern blot analyses. Experiments involving IL-8 promoter-reporter assays, electromobility shift assays, and Western blot analyses demonstrated that hyperoxia augmented TNF-alpha-mediated activation of the IL-8 promoter by a nuclear factor (NF)-kappaB-dependent mechanism and increased the duration of NF-kappaB nuclear translocation after concomitant treatment with TNF-alpha. Additional reporter gene assays demonstrated, however, that increased activation of NF-kappaB does not fully account for the synergistic effect of hyperoxia and that the NF-IL-6 site in the IL-8 promoter is also required for the synergistic effect of hyperoxia. We conclude that hyperoxia alone has a minimal effect on IL-8 gene expression but synergistically increases IL-8 gene expression in the presence of TNF-alpha by a mechanism involving cooperative interaction between the transcription factors NF-kappaB and NF-IL-6.
authors.affiliation: Division of Critical Care Medicine, Children's Hospital Research Foundation, Cincinnati, Ohio 45229, USA.
authors.forename: G L
authors.initials: GL
authors.lastname: Allen
authors.forename: I Y
authors.initials: IY
authors.lastname: Menendez
authors.forename: M A
authors.initials: MA
authors.lastname: Ryan
authors.forename: R L
authors.initials: RL
authors.lastname: Mazor
authors.forename: J R
authors.initials: JR
authors.lastname: Wispé
authors.forename: M A
authors.initials: MA
authors.lastname: Fiedler
authors.forename: H R
authors.initials: HR
authors.lastname: Wong
mesh_terms: D001665:Binding Sites / Q000235:genetics; D051743:CCAAT-Enhancer-Binding Protein-delta; D022762:CCAAT-Enhancer-Binding Proteins; D002467:Cell Nucleus / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000502:physiology; D015870:Gene Expression / Q000187:drug effects*; D017930:Genes, Reporter / Q000235:genetics; D006801:Humans; D018496:Hyperoxia / Q000235:genetics*; D016209:Interleukin-8 / Q000235:genetics* / Q000378:metabolism; D008156:Luciferases / Q000235:genetics / Q000378:metabolism; D009154:Mutation / Q000502:physiology; D016328:NF-kappa B / Q000378:metabolism / Q000502:physiology; D009687:Nuclear Proteins / Q000502:physiology; D010957:Plasmids; D011401:Promoter Regions, Genetic / Q000187:drug effects / Q000235:genetics / Q000502:physiology; D012333:RNA, Messenger / Q000378:metabolism; D014157:Transcription Factors; D014162:Transfection; D014407:Tumor Cells, Cultured; D014409:Tumor Necrosis Factor-alpha / Q000494:pharmacology*
chemical_list: D022762:CCAAT-Enhancer-Binding Proteins; C497252:CEBPD protein, human; D004268:DNA-Binding Proteins; D016209:Interleukin-8; D016328:NF-kappa B; D009687:Nuclear Proteins; D012333:RNA, Messenger; D014157:Transcription Factors; D014409:Tumor Necrosis Factor-alpha; D051743:CCAAT-Enhancer-Binding Protein-delta; D008156:Luciferases