Interaction between ICAM-1 (CD54) and fibrinogen (fg) has been shown to enhance leukocyte adhesion, but its specific role in the process of migration across endothelial cell junctions remains unclear. To overcome the problem of multiple adhesion receptors found on endothelial cells, we have engineered stable Chinese hamster ovary cell lines expressing ICAM-1 (Chinese hamster ovary ICAM-1). The transfection of ICAM-1 alone in these cells is sufficient to recapitulate the entire process of neutrophil adhesion and transmigration. This phenomenon was mediated by fg-ICAM-1 interactions, as depletion of fg, as well as the use of an Ab that specifically inhibits ICAM-1-fg interaction (2D5), completely abolished the effect of ICAM-1 expression on PMN transmigration. In addition, this ICAM-1-mediated transmigration is clearly dependent on the occurrence of fg-ICAM-1 interactions on the monolayer, and not on neutrophils, as the preincubation of the PMN with the mAb was ineffective. Furthermore, PMN transmigration, but not adhesion, is totally abolished when the ICAM-1 cytoplasmic domain is deleted, indicating that signaling inside the cell is required to mediate the fg-ICAM-1 effect on transmigration. Using a specific inhibitor of the small GTP-binding protein Rho, we have obtained evidence that this signaling cascade is involved. Thus, our results clearly show that ICAM-1 plays a key role in the migration of leukocytes across cell junctions, and indicate that this phenomenon is not a direct consequence of the enhanced adhesion mediated by the expression of ICAM-1.

Although cadherins appear to be necessary for proper cell-cell contacts, the physiological role of VE-cadherin (vascular endothelium cadherin) in adult tissue has not been clearly determined. To shed some light on this question, we have disturbed the adhesive function of VE-cadherin in human endothelial cell culture using a polyclonal anti-VE-cadherin antibody. This antibody disrupts confluent endothelial cell monolayers in vitro and transiently generates numerous gaps at cell-cell junctions. The formation of these gaps correlates with a reversible increase in the monolayer permeability. We present evidence that destruction of the homotypic interactions between the extracellular domains of VE-cadherin induces a rapid resynthesis of VE-cadherin, leading to restoration of endothelial cell-cell contacts. The expression of new molecules of VE-cadherin correlates with a modest but significant increase in VE-cadherin mRNA synthesis. Altogether, these results establish a critical role for VE-cadherin in the maintenance and restoration of endothelium integrity.

ICAM-1 is involved in both adhesion and extravasation of leukocytes to endothelium during inflammation. It has been shown that the ICAM-1 cytoplasmic domain is important for transendothelial migration of leukocytes but the precise molecular mechanisms involving the intracytoplasmic portion of ICAM-1 is not known. To characterize precisely the molecular scaffolding associated with ICAM-1, we have used the yeast two-hybrid system, and we have identified six different proteins interacting with the ICAM-1 cytoplasmic domain. In this study, we report that the two forms of nonmuscle α-actinin (i.e., α-actinin 1 and α-actinin 4) associate with ICAM-1, and that these interactions are essential for leukocyte extravasation. These interactions were further confirmed by coimmunoprecipitation and immunofluorescence in endothelial cells and in ICAM-1-transfected Chinese hamster ovary cells. The function of these interactions was analyzed by point mutation of charged amino acids located on ICAM-1 cytoplasmic domain. We have identified three charged amino acids (arginine 480, lysine 481, and arginine 486) which are essential in the binding of α-actinins to the ICAM-1 cytoplasmic tail. Mutation of these amino acids completely inhibited ICAM-1-mediated diapedesis. Experiments with siRNA inhibiting specifically α-actinin 1 or α-actinin 4 on endothelial cells indicated that α-actinin 4 had a major role in this phenomenon. Thus, our data demonstrate that ICAM-1 directly interacts with cytoplasmic α-actinin 1 and 4 and that this interaction is required for leukocyte extravasation.

Integrins are alpha beta heterodimers that play a major role in cell-cell contacts and in interactions between cells and extracellular matrices. Identification of structural domains that are critical for the expression of such receptors at the cell surface in a functional conformation is one of the major issues that has not yet been resolved. In the present study, the role of the cytoplasmic and transmembrane domains of each of the subunits has been examined using platelet GPIIb/IIIa as a prototypic integrin. GPIIb/IIIa (alpha IIb/beta 3) is a member of the integrin family and functions as a receptor for fibrinogen, fibronectin, von Willebrand factor, and vitronectin at the surface of activated platelets. Human megakaryocyte GPIIb and GPIIIa cDNAs were used to create a GPIIb mutant coding for the extracellular GPIIb heavy chain alone (GPIIb delta 1) and a GPIIIa mutant lacking the transmembrane and cytoplasmic domains (GPIIIa delta m). Full length and mutant cDNAs were subcloned into the expression vector pECE and used to transfect COS cells. The formation of heterodimers and their cellular localization was analyzed by immunoprecipitation and immunofluorescence labeling using anti-platelet GPIIb/IIIa antibodies. We show here that the extracellular domains of alpha and beta subunits are able to form a heterodimer, although with a lower efficiency, in the absence of the transmembrane and cytoplasmic domains. The presence of the cytoplasmic and transmembrane domains in the alpha subunit is, however, necessary for expression at the surface of the cell whereas the corresponding domains of the beta subunit are not required.

Platelet glycoproteins aIIb and p3 are membrane proteins that associate to form a Ca'+-dependent heterodimer which constitutes an inducible member of the integrin family at the surface of the cell. To produce a soluble form of this complex, aIIb and p3 were both deleted of their transmembrane and cytoplasmic domains and were expressed in COS cells. Production of the truncated subunits and their mode of assembly were examined by immunoprecipitation experiments and compared to those of wildtype aIIbfl. Synthesis and processing of the truncated heterodimer proceeded via a pathway similar to that observed for the wild-type aIIbp3 in COS cells or in human megakaryocytes. The truncated p3 subunit associated with the Pro-truncated form of the aIIb subunit. This precursor form was not secreted. After proteolytic cleavage of the Pro-truncated aIIb, the mature heterodimer was secreted into the culture supernatant. To quantify the molar ratio of the various secreted soluble forms, an immunocapture assay was designed. All secreted tr-aIIb subunits associated with tr-p3. In contrast, tr-p3 was produced and secreted in excess as the free form. Immunoreactivity of the wild-type and soluble truncated complexes was identical since all the monoclonal antibodies used reacted with surface-located epitopes on both complexes. This indicated that the soluble truncated heterodimer adopted a native conformation. To purify this soluble heterodimer, tr-aIIbp3-containing culture supernatant was adsorbed on an RGDW-affinity column and eluted with a solution of the free peptide RGDW. In the RGD-eluted material, the amount of each subunit was stoichiometric, suggesting that the complex was not disrupted during purification. The capacity of the wild-type and truncated RGD-eluted complexes to interact with soluble fibrinogen was compared using a solid-phase immunocapture assay. tr-aIIbp3 and platelet aIIbp3 exhibited similar fibrinogen-binding capacity. For both complexes, these interactions were mediated by RGD and y fibrinogen signals.Keywords. Integrin, aIIbp3 ; soluble form; aIIbp3 integrin.Platelet glycoprotein IIbIIIa is a member of the p3 integrin subfamily which functions as a receptor for adhesive proteins at the surface of platelet. The molecule has a loose specificity as it interacts with various multivalent adhesive proteins including fibrinogen [l,21, von Willebrand factor [3], fibronectin [4] and vitronectin [5]. The glycoproteins IIb (or aIIb) and IIIa (or 83) subunits are synthesized separately in the megakaryocyte which is the precursor cell of a platelet in the bone marrow. The p3 subunit is produced in excess and stored, while the aIIb subunit is produced as a precursor form and is immediately associated with the p3 subunit prior to its proteolytic maturation and its expression at the surface of the cell [6]. Neither of the subunits alone is surface-exposed or secreted. They form a calcium-dependent heterodimer and are exclusively implicated in platelet adhesion and aggregation.aIIb/33 exemplifies a typical integrin which is ...