CanadaDuring an inflammatory response, endothelial cells undergo morphological changes to allow for the passage of neutrophils from the blood vessel to the site of injury or infection. Although endothelial cell junctions and the cytoskeleton undergo reorganization during inflammation, little is known about another class of cellular structures, the focal adhesions. In this study, we examined several focal adhesion proteins during an inflammatory response. We found that there was selective loss of paxillin and focal adhesion kinase (FAK) from focal adhesions in proximity to transmigrating neutrophils; in contrast the levels of the focal adhesion proteins b1-integrin and vinculin were unaffected. Paxillin was lost from focal adhesions during neutrophil transmigration both under static and flow conditions. Down-regulating endothelial paxillin with siRNA blocked neutrophil transmigration while having no effect on rolling or adhesion. As paxillin dynamics are regulated partly by FAK, the role of FAK in neutrophil transmigration was examined using two complementary methods. siRNA was used to down-regulate total FAK protein while dominant-negative, kinase-deficient FAK was expressed to block FAK signaling. Disruption of the FAK protein or FAK signaling decreased neutrophil transmigration. Collectively, these findings reveal a novel role for endothelial focal adhesion proteins paxillin and FAK in regulating neutrophil transmigration. IntroductionNeutrophils are essential mediators of host defense. During inflammation, neutrophils leave the bloodstream and traffic into the tissue in a well-described series of steps [1,2]. The molecular interactions governing the initial stages of tethering, rolling and firm adhesion have been well studied [1,2], whereas a clear picture of the latter steps underlying the passage of the leukocytes through the blood vessel has been slower to emerge [3][4][5].During transmigration endothelial cells undergo rapid changes in their structural organization [5,6]. For example, the junctional protein vascular endothelial cadherin (VE-cadherin) is phosphorylated and redistributes during neutrophil transmigration [7][8][9]. This results in the formation of transient gaps that 436allow for the passage of neutrophils [9]. Platelet endothelial cell adhesion molecule-1 (PECAM-1), another junctional protein, is regulated by the action of the kinesins and requires an intact microtubule cytoskeleton to enable PECAM-1 recycling from membrane compartments to the lateral junctions [10]. In the absence of PECAM-1 recycling, leukocyte transmigration is arrested [10]. Cortactin-mediated rearrangement of the actin cytoskeleton [7] and intermediate filaments [11] is also critical for successful transmigration. Overall, these results reveal that endothelial cells are active participants in transmigration.The importance of cytoskeletal and junctional proteins during leukocyte transmigration led us to ask whether another class of cellular structures, the focal adhesions, also play a role during this process. Focal a...

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.