Hyperoxia synergistically increases TNF-alpha-induced interleukin-8 gene expression in A549 cells

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 ex pression in respiratory epithelium is not well described. In addition, IL-8 gene expression resulting from the combined effects of hyperoxia and proin flammatory cytokines has not been well characterized. We treated cultured r espiratory epithelial-like cells (A549 cells) with hyperoxia alone, tumor n ecrosis factor (TNF)-alpha alone, or the combination of TNF-alpha and hyper oxia and evaluated IL-8 gene expression. Hyperoxia alone had a minimal effe ct on IL-8 gene expression, and TNF-alpha alone increased IL-8 gene express ion in a time-dependent manner. In contrast, the combination of TNF-alpha a nd hyperoxia synergistically increased IL-8 gene expression as measured by ELISA (TNF-alpha alone for 24 h = 769 +/- 89 pg/ml vs, hyperoxia + TNF-alph a for 24 h = 1,189 +/- 89 pg/ml) and Northern blot analyses. Experiments in volving IL-8 promoter-reporter assays, electromobility shift assays, and We stern blot analyses demonstrated that hyperoxia augmented TNF-alpha-mediate d activation of the IL-8 promoter by a nuclear factor (NF)-kappa B-dependen t mechanism and increased the duration of NF-kappa B nuclear translocation after concomitant treatment with TNF-alpha. Additional reporter gene assays demonstrated, however, that increased activation of NF-kappa B does not fu lly account for the synergistic effect of hyperoxia and that the NF-IL-6 si te in the IL-8 promoter is also required for the synergistic effect of hype roxia. We conclude that hyperoxia alone has a minimal effect on IL-8 gene e xpression but synergistically increases IL-8 gene expression in the presenc e of TNF-alpha by a mechanism involving cooperative interaction between the transcription factors NF-kappa B and NF-IL-6.