Of the several families of adhesion receptors involved in leukocyte-endothelial cell interactions, only the selectins have been shown to initiate leukocyte interaction under physiologic shear; indeed, beta 2 (CD18) intergrins responsible for neutrophil arrest are unable to engage without prior selectin-mediated rolling. In contrast, alpha 4 (CD49d) integrins are shown here to initiate lymphocyte contract ("tethering") in vitro under shear and in the absence of a selectin contribution. The alpha 4 integrin ligands MAdCAM-1 and VCAM-1 support loose reversible interactions including rolling, as well as rapid sticking and arrest that is favored following integrin activation. Moreover, alpha 4 beta 7 mediates L-selectin (CD62L)-independent attachment of blood-borne lymphocytes to lamina propria venules in situ. Scanning electron microscopy of alpha 4 beta 7hi lymphoid cells reveals that, like L-selectin, alpha 4 beta 7 is highly concentrated on microvillous sites of initial cellular contact, whereas the beta 2 integrin LFA-1 is excluded from villi. Thus, alpha 4 but not beta 2 integrins can initiate leukocyte adhesion under flow, a capacity that may be in part a function of topographic presentation on microvilli.

SummaryThe chemokine RANTES is a chemoattractant and activating factor for T lymphocytes. Investigation of the signal transduction mechanisms induced by RANTES in T cells revealed tyrosine phosphorylation of multiple protein species with prominent bands at 70-85 and 120-130 kD. Immunoprecipitation and Western analyses revealed that a protein of 125 kD was identical to the focal adhesion kinase (FAK) pp125 FAK. R.ANTES stimulated phosphorylation of FAK as early as 30 seconds, and immunoblots using antiphosphotyrosine monoclonal antibodies revealed that there was consistent phosphorylation of a 68-70 kD species in the pp125 FAK immunoprecipitates. Immunoblotting and kinase assays showed this to be two separate proteins, the tyrosine kinase zeta-associated protein (ZAP) 70, and the focal adhesion protein paxillin. These results indicate a potentially important role for RANTES in the generation of T cell focal adhesions and subsequent cell activation via a molecular complex containing FAK, ZAP-70, and paxiltin.

The Duffy blood group antigen-erythrocyte chemokine receptor has been shown to bind to chemokines of both the C-X-C and C-C classes and to the malarial parasites Plasmodium vivax and Plasmodium knowlesi. We performed experiments to evaluate the binding properties of this receptor for the newly appreciated "C" and "non-ELR C-X-C" classes of chemokines. Binding to mouse erythrocytes was also evaluated for the first time. Whereas ELR C-X-C and C-C chemokines bound to human erythrocytes with high affinity, differences in the ability of non-ELR chemokines to act as competitive inhibitors were noted. While non-ELR chemokines were unable to displace C-X-C chemokines on human cells, they exhibited a low affinity interaction with the C-C chemokine binding site. The newly discovered C chemokine, lymphotactin, was unable to displace either C-X-C or C-C chemokines. On mouse erythrocytes, non-ELR chemokines exhibited a low affinity for both the C-X-C and C-C chemokines binding sites; again lymphotactin failed to bind. Binding competition studies using an anti-Duffy monoclonal antibody and chemokines suggested a common binding domain. These data show that the chemokine superfamily has at least four functional subdivisions, each interacting differently with the Duffy antigen-erythrocyte chemokine receptor. In addition the chemokine binding function is conserved between mouse and man. Unlike other proteins in the superfamily C and non-ELR C-X-C chemokines do not efficiently bind red blood cells, thus their role may not require clearance from circulation.

The chemokine RANTES is a potent chemoattractant and activator of T lymphocytes. Mechanisms underlying the RANTES-induced activation of T lymphocytes leading to adhesion and migration have not been fully analyzed. We investigate here the function of RANTES in the regulation of T cell adhesion, specifically the induction of homotypic aggregation. RANTES induced the expression of many important cell surface adhesion and activation receptors in a normal human T cell clone and peripheral blood T lymphocytes, including members of the beta 1 and beta 2 integrin family, CD44, CD50, and CD28. Up-regulation of these markers correlated with RANTES-stimulated homotypic adhesion of T cells. This homotypic aggregation event was RANTES dose-dependent, prolonged, and pertussis toxin-independent, but herbimycin A-sensitive, suggesting that it involves signaling through alternative (G alpha i protein-independent) pathways. Using specific monoclonal antibodies, the homotypic aggregation event was shown to be lymphocyte function-associated antigen-1 (LFA-1)-dependent, with no observable interaction through alpha 4 or beta 1 integrins. Intercellular adhesion molecule-3 (ICAM-3) and possibly ICAM-1 participate as LFA-1 ligands. Additionally, RANTES phosphorylated the beta chain of LFA-1 1-2 min following stimulation. These results imply a specific role for the chemokine RANTES in T cell activation and intercellular adhesion.