These data confirm those generated in our studies with calcein-AM-labeled PMNs (Figure 2A) and further support exclusion of a direct ET effect on PMNs. Figure 2 ET effect on IL-8-driven TEM of PMNs is due to a direct effect on ECs. (A) Naked filters mounted on chemotaxis chambers were placed into wells containing either medium or IL-8 (10 ng/mL), after which calcein-AM-labled PMNs, suspended in medium containing ET (1000 ng/mL:1000 ng/mL) or medium alone, were added to each upper compartment. After 2 h, the contents of each lower compartment were fluorometrically assayed. Each vertical bar represents mean (+/- SEM) chemotaxis of

PMNs (%). (B) Naked filters were mounted in modified Boyden chemotaxis chambers in which the lower compartment contained either medium or IL-8 (10 ng/mL). PMNs, suspended in medium containing PF-02341066 mw ET or medium alone, were added to each BAY 73-4506 upper compartment. After 0.5 h, the filter was removed, fixed, GSK1210151A washed, stained with crystal violet, washed, and the top surface of each filter scraped free of cells. The crystal violet was then extracted and absorbance measured at 560 nm. Each vertical bar represents mean (+/- SEM) absorbance at 560 nm. (C) HMVEC-Ls were seeded at a density of 1.0 × 105 cells/assay chamber and cultured

overnight prior to treatment for 6 h with either medium or increasing concentrations of ET. Each vertical bar represents mean (+/- SE) transendothelial 14 C-BSA flux. (D) HMVEC-Ls cultured to confluence in assay chambers were treated for 6 h with medium, TNF-α (100 ng/mL), TNF-α in the presence of ET (1000 ng/mL:200 ng/mL), LPS (100 ng/mL), or LPS + ET (1000

ng/mL:200 ng/mL). Each vertical bar represents mean (+/- SEM) transendothelial flux of 14 C-BSA. The n for each group is indicated in each bar. * indicates significantly increased compared to the simultaneous medium controls at p < 0.05. ** indicates significantly decreased compared to the simultaneous medium control at p < 0.05. *** indicates significantly decreased compared to either Epothilone B (EPO906, Patupilone) TNF-α or LPS alone at p < 0.05. To establish whether the ability of ET to decrease IL-8-driven TEM of PMNs was mediated indirectly through the EC response, we measured the effect of ET on movement of a permeability tracer across the endothelia. In a subconfluent HMVEC-L monolayers, where the average baseline transendothelial 14 C-albumin flux was 0.0256 (+/- 0.0147) pmol/h, ET, at increasing concentrations, dose-dependently decreased mean (+/- SEM) transendothelial 14 C-albumin flux compared to the simultaneous medium controls (Figure 2C). ET concentrations as low as 100 ng/mL:100 ng/mL diminished transendothelial 14 C-albumin flux. These data indicate that ET restricts passage of macromolecules through the same endothelial paracellular pathway through which PMNs migrate.