Ezrin, radixin, and moesin (ERM) regulate cortical morphogenesis and cell adhesion by connecting membrane adhesion receptors to the actin-based cytoskeleton. We have studied the interaction of moesin and ezrin with the vascular cell adhesion molecule (VCAM)-1 during leukocyte adhesion and transendothelial migration (TEM). VCAM-1 interacted directly with moesin and ezrin in vitro, and all of these molecules colocalized at the apical surface of endothelium. Dynamic assessment of this interaction in living cells showed that both VCAM-1 and moesin were involved in lymphoblast adhesion and spreading on the endothelium, whereas only moesin participated in TEM, following the same distribution pattern as ICAM-1. During leukocyte adhesion in static or under flow conditions, VCAM-1, ICAM-1, and activated moesin and ezrin clustered in an endothelial actin-rich docking structure that anchored and partially embraced the leukocyte containing other cytoskeletal components such as α-actinin, vinculin, and VASP. Phosphoinositides and the Rho/p160 ROCK pathway, which participate in the activation of ERM proteins, were involved in the generation and maintenance of the anchoring structure. These results provide the first characterization of an endothelial docking structure that plays a key role in the firm adhesion of leukocytes to the endothelium during inflammation.

Cell-to-cell junction structures play a key role in cell growth rate control and cell polarization. In endothelial cells (EC), these structures are also involved in regulation of vascular permeability and leukocyte extravasation. To identify novel components in EC intercellular junctions, mAbs against these cells were produced and selected using a morphological screening by immunofluorescence microscopy. Two novel mAbs, LIA1/1 and VJ1/16, specifically recognized a 25-kD protein that was selectively localized at cell–cell junctions of EC, both in the primary formation of cell monolayers and when EC reorganized in the process of wound healing. This antigen corresponded to the recently cloned platelet-endothelial tetraspan antigen CD151/PETA-3 (platelet-endothelial tetraspan antigen-3), and was consistently detected at EC cell–cell contact sites. In addition to CD151/PETA-3, two other members of the tetraspan superfamily, CD9 and CD81/ TAPA-1 (target of antiproliferative antibody-1), localized at endothelial cell-to-cell junctions. Biochemical analysis demonstrated molecular associations among tetraspan molecules themselves and those of CD151/ PETA-3 and CD9 with α3β1 integrin. Interestingly, mAbs directed to both CD151/PETA-3 and CD81/ TAPA-1 as well as mAb specific for α3 integrin, were able to inhibit the migration of ECs in the process of wound healing. The engagement of CD151/PETA-3 and CD81/TAPA-1 inhibited the movement of individual ECs, as determined by quantitative time-lapse video microscopy studies. Furthermore, mAbs against the CD151/PETA-3 molecule diminished the rate of EC invasion into collagen gels. In addition, these mAbs were able to increase the adhesion of EC to extracellular matrix proteins. Together these results indicate that CD81/TAPA-1 and CD151/PETA-3 tetraspan molecules are components of the endothelial lateral junctions implicated in the regulation of cell motility, either directly or by modulation of the function of the associated integrin heterodimers.

The integrin ␣4␤1 (VLA-4) not only mediates the adhesion and transendothelial migration of leukocytes, but also provides costimulatory signals that contribute to the activation of T lymphocytes. However, the behavior of ␣4␤1 during the formation of the immune synapse is currently unknown. Here, we show that ␣4␤1 is recruited to both human and murine antigen-dependent immune synapses, when the antigen-presenting cell is a B lymphocyte or a dendritic cell, colocalizing with LFA-1 at the peripheral supramolecular activation complex. However, when conjugates are formed in the presence of anti-␣4 antibodies, VLA-4 colocalizes with the CD3-chain at the center of the synapse. In addition, antibody engagement of ␣4 integrin promotes polarization toward a T helper 1 (Th1) response in human in vitro models of CD4 ؉ T cell differentiation and naïve T cell priming by dendritic cells. The in vivo administration of anti-␣4 integrin antibodies also induces an immune deviation to Th1 response that dampens a Th2-driven autoimmune nephritis in Brown Norway rats. These data reveal a regulatory role of ␣4 integrins on T lymphocyte-antigen presenting cell cognate immune interactions.A fter the recognition of antigens (Ag) presented by dendritic cells (DCs), naïve T lymphocytes proliferate and differentiate into T helper (Th) 1 or 2 effector cells. These effector lymphocytes are characterized by distinct patterns of cytokine production and homing behavior. Th1 cells mainly produce IFN-␥ and IL-2 and have a key role in the cellular immune responses. Conversely, Th2 cells produce IL-4, IL-5, IL-6, and IL-10 and promote the humoral immune responses (1). DCs are the only Ag-presenting cells (APCs) involved in the priming of naïve Th cells and their polarization toward Th1 or Th2 differentiation. To acquire this capacity, DCs must undergo a maturation process characterized by the loss of their Ag-capturing capacity and the increase of their expression of costimulatory and adhesion molecules, including ␣4␤1 integrin (2). However, other APCs (e.g., B lymphocytes) are also involved in regulating the cytokine profiles of Th cell responses, indicating the importance of postpriming events (3).The interaction between T cells and APCs plays an important role in directing Th cell polarization. The strength of antigenic stimulation, the duration of T cell receptor engagement, the presence of different cytokines, and the participation of distinct costimulatory molecules are critical in determining the phenotype of differentiated T cells. The cytokine IL-12, high doses of Ag, and CD28͞B7-1 interaction promote Th1 differentiation, whereas an environment enriched in IL-4, low doses of Ag, and CD28͞B7-2 or inducible costimulator (ICOS)͞ICOS ligand participation promote Th2 responses (4).Integrins are a large family of ␣␤ heterodimeric transmembrane proteins that mediate cell-cell and cell-extracellular matrix adhesion. Several integrins, lymphocyte functionassociated (LFA-1; ␣ L ␤ 2 ), very late activation antigen-4 (VLA-4; ␣4␤1), and VLA-1 (␣1␤1) have ...