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 ...

The compartmentalization of plasma membrane proteins has a key role in regulation of lymphocyte activation and development of immunity. We found that the proline-rich tyrosine kinase-2 (PYK-2/RAFTK) colocalized with the microtubule-organizing center (MTOC) at the trailing edge of migrating natural killer (NK) cells. When polyclonal NK cells bound to K562 targets, PYK-2 translocated to the area of NK–target cell interaction. The specificity of this process was assessed with NK cell clones bearing activatory or inhibitory forms of CD94/NKG2. The translocation of PYK-2, MTOC, and paxillin to the area of NK–target cell contact was regulated upon specific recognition of target cells through NK cell receptors, controlling target cell killing. Furthermore, parallel in vitro kinase assays showed that PYK-2 was activated in response to signals that specifically triggered its translocation and NK cell mediated cytotoxicity. The overexpression of both the wt and a dominant-negative mutant of PYK-2, but not ZAP-70 wt, prevented the specific translocation of the MTOC and paxillin, and blocked the cytotoxic response of NK cells. Our data indicate that subcellular compartmentalization of PYK-2 correlates with effective signal transduction. Furthermore, they also suggest an important role for PYK-2 on the assembly of the signaling complexes that regulate the cytotoxic response.

A screen for natural products bearing pharmacological properties has yielded a secretion of the mollusk Cryptomphalus aspersa (SCA), which possesses skin-regenerative properties. In this report, we outline some of the cellular and molecular effects underlying this observation. First, we found that SCA contained antioxidant SOD and GST activities. In addition, SCA stimulated fibroblast proliferation and rearrangement of the actin cytoskeleton. Additional mechanisms involved in the regenerative effect of SCA included the stimulation of extracellular matrix assembly and the regulation of metalloproteinase activities. Together, these effects provide an array of molecular mechanisms underlying SCA-induced cellular regeneration and postulate its use in regeneration of wounded tissue.

CD69 and CD23 are leukocyte receptors with distinctive pattern of cell expression and functional features that belong to different C-type lectin receptor subfamilies. To assess the functional equivalence of different domains of these structurally related proteins, a series of CD69/CD23 chimeras exchanging the carbohydrate recognition domain, the neck region, and the transmembrane and cytoplasmic domains were generated. Biochemical analysis revealed the importance of the neck region (Cys68) in the dimerization of CD69. Functional analysis of these chimeras in RBL-2H3 mast cells and Jurkat T cell lines showed the interchangeability of structural domains of both proteins regarding Ca2+ fluxes, serotonin release, and TNF-α synthesis. The type of the signal transduced mainly relied on the cytoplasmic domain and was independent of receptor oligomerization. The cytoplasmic domain of CD69 transduced a Ca2+-mediated signaling that was dependent on the extracellular uptake of Ca2+. Furthermore, a significant production of TNF-α was induced through the cytoplasmic domain of CD69 in RBL-2H3 cells, which was additive to that promoted via FcεRI, thus suggesting a role for CD69 in the late phase of reactions mediated by mast cells. Our results provide new important data on the functional equivalence of homologous domains of these two leukocyte receptors.

The relocation of kinases in T lymphocytes during their cognate interaction with APCs is essential for lymphocyte activation. We found that the proline-rich tyrosine kinase-2 (Pyk2) is rapidly translocated to the T cell-APC contact area upon T cell-specific recognition of superantigen-pulsed APCs. Stimulation with anti-CD3-coated latex microspheres was sufficient for Pyk2 reorientation, and the coengagement of CD28 boosted Pyk2 redistribution. Nevertheless, Pyk2 translocation did not result in its recruitment to lipid rafts. Two results support that Pyk2 translocation was independent of its kinase activity. First, Lck activity was required for TCR-induced Pyk2 translocation, but not for TCR-induced Pyk2 activation. Second, a kinase-dead Pyk2 mutant was equally translocated upon TCR triggering. In addition, Lck activity alone was insufficient to induce Pyk2 reorientation and activation, requiring the presence of at least one intact immunoreceptor tyrosine-based activation motif (ITAM). Despite the dependence on functional Lck and on phosphorylated ITAM for Pyk2 translocation, the ITAM-binding tyrosine kinase ζ-associated protein 70 (ZAP-70) was not essential. All these data suggest that, by translocating to the vicinity of the immune synapse, Pyk2 could play an essential role in T cell activation and polarized secretion of cytokines.

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