Figure 1. PMN migration across P. aeruginosa treated lung epithelial barriers is diminished following inhibition of the HXA₃ synthetic pathway.

(A) Four distinct lung epithelial cell lines (Table I: A549, Calu-3, H292, and BEAS-2B) grown on Transwells to form epithelial barriers were treated with either P. aeruginosa strain PAO1 (black bars) or K12 E. coli strain MC1000 (gray bars). Monolayers incubated in HBSS alone serve as a negative control (white bars). A chemo-attractant gradient of fMLP (100 nM) was established to serve as a positive control for PMN movement (dashed bars). The symbol (*) represents a statistically significant difference compared to negative controls (HBSS and MC1000). (B & C) Transwells were pre-treated with pharmacological inhibitors CDC - 12-lipoxygenase inhibitor (50 μM), ONO-RS-082 - PLA₂ inhibitor (5 μM), RHC-80267 - DAG lipase inhibitor (50 μM), NS-398 -COX inhibitor (50 μM) or vehicle control (1:1000 DMSO) prior to infection. The number of PMNs that migrate across lung epithelial barriers H292 (B) and BEAS-2B (C) was assessed in response to infection with PAO1, MC1000, or uninfected controls (HBSS/fMLP) with or without pre-treatment of each pharmacological inhibitor. The symbol (*) represents a statistically significant difference within a treatment group comparing inhibitor to vehicle control. (D) The amount of PGE₂ released (ng/ml) by H292 cells in the presence or absence of PAO1 infection with or without pharmacological inhibitor pre-treatment was quantified by E.I.A.. The symbol (*) represents a statistically significant difference between uninfected (HBSS) and PAO1 infected within a treatment group. Statistical significance was determined using a two-tailed unpaired Student’s T-test (p < 0.05). Each data point represents the average of at least three separate wells. Each experiment was performed on at least three separate occasions yielding similar results.