Endothelial cell cortactin coordinates intercellular adhesion molecule-1 clustering and actin cytoskeleton remodeling during polymorphonuclear leukocyte adhesion and transmigration.

Endothelial cell ICAM-1 interacts with leukocyte beta(2) integrins to mediate adhesion and transmit outside-in signals that facilitate leukocyte transmigration. ICAM-1 redistribution and clustering appear necessary for leukocyte transmigration, but the mechanisms controlling ICAM-1 redistribution and clustering have not been identified. We recently reported that Src kinase phosphorylation of endothelial cortactin regulates polymorphonuclear cell (PMN) transmigration. In this study, we tested the hypotheses that the Src family kinase-cortactin pathway mediates association of ICAM-1 with the actin cytoskeleton and that this association is required for ICAM-1 clustering and leukocyte transmigration. Cross-linking ICAM-1 induced cytoskeletal remodeling and a decrease in ICAM-1 lateral mobility, as assessed by fluorescence recovery after photobleaching. Cytoskeletal remodeling after ICAM-1 cross-linking was reduced by knockdown of cortactin by small interfering RNA, by expression of a cortactin mutant deficient in Src phosphorylation sites (cortactin3F), and by the Src kinase inhibitor PP2. Pretreatment of cytokine-activated human endothelial monolayers with cortactin small interfering RNA significantly decreased both actin and ICAM-1 clustering around adherent PMN and the formation of actin-ICAM-1 clusters required for PMN transmigration. Our data suggest a model in which tyrosine phosphorylation of cortactin dynamically links ICAM-1 to the actin cytoskeleton, enabling ICAM-1 to form clusters and facilitate leukocyte transmigration.

ICAM-1 regulates neutrophil adhesion and transcellular migration of TNF-alpha-activated vascular endothelium under flow.

In vivo, leukocyte transendothelial migration (TEM) occurs at endothelial cell junctions (paracellular) and nonjunctional (transcellular) locations, whereas in vitro models report that TEM is mostly paracellular. The mechanisms that control the route of leukocyte TEM remain unknown. Here we tested the hypothesis that elevated intercellular adhesion molecule-1 (ICAM-1) expression regulates the location of polymorphonuclear leukocyte (PMN) TEM. We used an in vitro flow model of tumor necrosis factor-alpha (TNF-alpha)-activated human umbilical vein endothelium cells (HUVECs) or an HUVEC cell line transfected with ICAM-1GFP (green fluorescent protein) and live-cell fluorescence microscopy to quantify the location of PMN adhesion and TEM. We observed robust transcellular TEM with TNF-alpha-activated HUVECs and ICAM-1GFP immortalized HUVECS (iHUVECs). In contrast, primary CD3+ T lymphocytes exclusively used a paracellular route. Endothelial ICAM-1 was identified as essential for both paracellular and transcellular PMN transmigration, and interfering with ICAM-1 cytoplasmic tail function preferentially reduced transcellular TEM. We also found that ICAM-1 surface density and distribution as well as endothelial cell shape contributed to transcellular TEM. In summary, ICAM-1 promotes junctional and nonjunctional TEM across inflamed vascular endothelium via distinct cytoplasmic tail associations.

Coordinated redistribution of leukocyte LFA-1 and endothelial cell ICAM-1 accompany neutrophil transmigration.

The leukocyte integrin lymphocyte function-associated antigen 1 (LFA-1) and its endothelial ligand intercellular adhesion molecule (ICAM)-1 play an important role in transmigration as demonstrated by in vivo and in vitro models of inflammation. Despite the prominent role, little is known concerning the distribution and dynamic behavior of these adhesion molecules during leukocyte transmigration. Therefore, we examined the spatial and temporal distribution of LFA-1 on neutrophils actively transmigrating tumor necrosis factor-alpha-activated human umbilical vein endothelial monolayers under shear flow. Upon neutrophil arrest, LFA-1 was evenly distributed. However, once neutrophils initiated transmigration, LFA-1 rapidly redistributed to form a ringlike cluster at the neutrophil-endothelial junctional interface through which transmigration occurred. As transmigration was completed, LFA-1 redistributed to the neutrophil uropod. Endothelial ICAM-1 and JAM-A both colocalized with the ringlike LFA-1 cluster. Further analysis of PMA-stimulated neutrophils, which increase mobility of LFA-1, showed a rapid redistribution of LFA-1 and ICAM-1, but not endothelial JAM-A. Thus, endothelial JAM-A does not appear to contribute to adhesion or transmigration in this system. This is the first demonstration that neutrophil LFA-1 rapidly redistributes to form a ringlike structure that coclusters with endothelial ICAM-1 as the neutrophil transmigrates.

Elastase release by transmigrating neutrophils deactivates endothelial-bound SDF-1alpha and attenuates subsequent T lymphocyte transendothelial migration.

Leukocyte trafficking to sites of inflammation follows a defined temporal pattern, and evidence suggests that initial neutrophil transendothelial migration modifies endothelial cell phenotype. We tested the hypothesis that preconditioning of human umbilical vein endothelial cells (HUVEC) by neutrophils would also modify the subsequent transendothelial migration of T lymphocytes across cytokine-stimulated HUVEC in an in vitro flow assay. Using fluorescence microscopy, preconditioning of HUVEC by neutrophils was observed to significantly reduce the extent of subsequent stromal cell-derived factor-1alpha (SDF-1alpha [CXCL12])-mediated T lymphocyte transendothelial migration, without reducing accumulation. In contrast, recruitment of a second wave of neutrophils was unaltered. Conditioned medium harvested after transendothelial migration of neutrophils or supernatants from stimulated neutrophils mediated a similar blocking effect, which was negated using a specific neutrophil elastase inhibitor. Furthermore, T lymphocyte transendothelial migration was inhibited by treatment of HUVEC with purified neutrophil elastase, which selectively cleaved the amino terminus of HUVEC-bound SDF-1alpha, which is required for its chemotactic activity. The reduction in T lymphocyte transendothelial migration was not observed using a different chemokine, ELC (CCL19), and was not reversed by replenishment of SDF-1alpha, indicating endothelial retention of the inactivated chemokine. In summary, transmigrating neutrophils secrete localized elastase that is protected from plasma inhibitors, and thereby modulate trafficking of other leukocyte subsets by altering the endothelial-associated chemotactic activities.