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Am J Physiol Lung Cell Mol Physiol 278: L253-L260, 2000;
1040-0605/00 $5.00
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Vol. 278, Issue 2, L253-L260, February 2000

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

Geoffrey L. Allen1, Ingrid Y. Menendez1, Marnie A. Ryan1, Robert L. Mazor1, Jonathan R. Wispé2, Michael A. Fiedler3, and Hector R. Wong1

Divisions of 1 Critical Care Medicine, 2 Pulmonary Biology, and 3 Pulmonary Medicine, Children's Hospital Research Foundation, Cincinnati, Ohio 45229

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)-kappa B-dependent 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 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-kappa B and NF-IL-6.

respiratory epithelium; inflammation; chemokines; tumor necrosis factor-alpha ; nuclear factor-kappa B; nuclear factor-interleukin-6


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