Abstract 206 Cytokines: Intracellular Signaling Platform, Tuesday, 5/4

Acute Lung Injury (ALI) remains a significant cause of morbidity in critically ill patients. Although O2 is a key supportive therapy for these patients, high levels of O2 (hyperoxia) have been shown to exacerbate ALI by mechanisms which are incompletely understood. We hypothesize that O2, in the presence of pro-inflammatory stimuli, may enhance gene expression of certain cytokines. To test this hypothesis, we have developed an in vitro model of human pulmonary epithelial cells (A549), in which TNFα-induced IL-8 production is synergistically enhanced in response to hyperoxia (95% O2). ELISA assays performed on the supernatant of cells treated with TNFα (2ng/ml) demonstrated increased IL-8 (789 ± 89 pg/ml) compared to unstimulated controls (23 ± 4 pg/ml). Hyperoxia alone minimally increased IL-8 (110 ± 13), but treatment with TNFα + O2 resulted in more IL-8 (1189 ± 43 pg/ml) than the TNFα alone group. This increase in IL-8 with TNFα + 95% O2 is greater than would be anticipated if the effects of O2 and TNFα were simply additive. IL-8 mRNA levels were subsequently evaluated by Northern blot. IL-8 mRNA in cells treated with 95% O2 alone was undetectable and cells treated with TNFα + 95% O2 had greater mRNA levels than those in the TNFα alone group. To investigate hyperoxia's effect on transcriptional control of the IL-8 gene, we performed transient transfections using three different promoter-luciferase constructs. The first promoter was 200bp segment of the native IL-8 promoter. The second (mutant) promoter contained a mutated NFκ-B binding site in the native 200bp segment and the third was a synthetic promoter construct containing three NFκ-B binding sites in tandem. Transfected cells were treated with TNFα (2ng/ml) ± 95% O2 or 95% O2 alone for 4 hrs and luciferase activity was measured. Cells transfected with the native promoter construct demonstrated a ∼2 fold induction of luciferase activity in response to TNFα and no induction in response to 95% O2 alone. TNFα + 95% O2, however, resulted in a ∼4 fold induction of luciferase activity. Cells transfected with the mutant promoter failed to show induction of luciferase activity in response to TNFα ± 95% O2 or 95% O2 alone. Finally, transfections with the synthetic NFκ-B promoter demonstrated a ∼3 fold induction after treatment with TNFα, but no augmentation of this response with the addition of 95% O2. We conclude that oxygen has a key signaling role in augmenting TNFα-induced transcriptional activation of IL-8. Furthermore, NFκ-B activation is requisite for IL-8 gene expression in response to TNFα, but its activation alone is not sufficient to explain the augmentation seen with hyperoxia. Further studies are necessary to elucidate the complete mechanism of hyperoxia's role in IL-8 gene regulation. These discoveries could lead to better understanding of oxygen's role in the pathophysiology of ALI and potential novel therapies.