In the current study, we have defined a novel mechanism through which a bacteria-derived toxin, ET, may indirectly, through the counter-regulation of the endothelial paracellular pathway, impair extravasation of PMNs into tissues. Results ET protects against IL-8-stimulated transendothelial migration (TEM) of PMNs Since ET directly

stimulates ECs to increase cAMP [7], which in turn, enhances endothelial barrier integrity [11, 27–32], we asked whether ET might decrease TEM of PMNs. Pretreatment of monolayers of human microvascular endothelial cells of the lung (HMVEC-Ls) with ET decreased IL-8-stimulated TEM by ~ 60% (Figure 1A). Neither EF nor PA alone were able to reproduce the ET effect (Figure 1B). For these calculations, total fluorescence associated with PMNs placed in each upper compartment represented 3-deazaneplanocin A concentration 100% migration while % migration was calculated as fluorescence in the lower compartment/fluorescence in the upper compartment × 100%. Figure 1 Effect of ET on click here the TEM of PMNs. (A) Human microvascular endothelial cells from the lung (HMVEC-Ls) cultured to confluence in assay chambers were exposed for 4 h to either increasing concentrations of ET at the indicated doses each of EF and PA (EF:PA) or medium alone. (B) HMVEC-L monolayers cultured to confluence in assay chambers were exposed for 4 h to medium, ET (1000 ng/mL:1000

ng/mL), EF (1000 ng/mL), or PA (1000 ng/mL). These same HMVEC-L monolayers were then inserted into the wells of 24-well plates containing either IL-8 (10 ng/mL) or medium alone, after which calcein-AM-labeled PMNs were added to the upper compartment of each chamber. After 2 h, each lower compartment was fluorometrically

assayed. Each vertical bar represents mean (+/- SEM) TEM of PMNs (%). The n for each group is indicated in each bar. * indicates significantly increased compared to the simultaneous medium controls at p < 0.05. ** Phosphoprotein phosphatase indicates significantly decreased compared to the IL-8 stimulus alone at p < 0.05. ET acts at the level of the EC to decrease IL-8-driven TEM of PMNs Since ET decreased the TEM of PMNs (Figure 1A), we asked whether it acted directly on PMNs or indirectly via the EC response. When PMNs were co-incubated with ET in the absence of ECs, ET at the same concentration that impaired TEM (1000 ng/mL:1000 ng/L) did not decrease IL-8-driven PMN chemotaxis compared to medium controls (Figure 2A). These data indicate that the ability of ET to diminish TEM of PMNs cannot be explained through a direct effect on PMNs. Since these PMNs were preloaded with the fluoroprobe, calcein-AM, a known intracellular Ca2+-binder [34], and the host response to ET is calmodulin- and Ca2 + -dependent [1, 2, 8, 22], we asked whether calcein-AM might diminish PMN responsiveness to ET. The impact of ET on IL-8 driven chemotaxis of unlabeled PMNs was assessed. In these studies, IL-8 increased PMN chemotaxis ~ 1.4-fold compared to the simultaneous medium controls (Figure 2B).