Endothelial cell paxillin is remodeled during leukocyte adhesion and is essential for transmigration

Abstract

The focal adhesion protein paxillin plays a critical role in focal adhesion remodeling and signaling and as such was examined within endothelial cells (EC) during in vitro neutrophil transmigration. We perfused isolated human neutrophils across TNF‐treated EC in a parallel plate flow chamber and investigated paxillin remodeling by immunostaining. Immunofluorescence data showed that there was a localized loss in EC paxillin staining following neutrophil adhesion under flow conditions. Imaging exogenous paxillin‐YFP in EC, we observed endothelial paxillin loss and remodeling in real‐time. In flow experiments in which we perfused fixed neutrophils or a neutrophilic cell line, both of which adhere but do not transmigrate, we found that paxillin loss was transmigration‐dependant. A functional role for EC paxillin was examined by utilizing siRNA. Using flow cytometry and western blotting, we showed more than ninety percent transfection efficiency and down regulation of paxillin. Downregulation of EC paxillin had no effect on the total number of neutrophils recruited to TNF‐stimulated EC; however, transmigration was blocked by more than 40%. This was not due to changes in expression of E‐selectin, IL‐8, or ICAM‐1 in paxillin downregulated EC. These data suggest that focal adhesion remodeling occurs during leukocyte recruitment and is essential for maximal transmigration.Research funded by The Canadian Institutes of Health Research.