It is widely believed that rolling lymphocytes require successive chemokine-induced signaling for lymphocyte function-associated antigen 1 (LFA-1) to achieve a threshold avidity that will mediate lymphocyte arrest. Using an in vivo model of lymphocyte arrest, we show here that LFA-1-mediated arrest of lymphocytes rolling on high endothelial venules bearing LFA-1 ligands and chemokines was abrupt. In vitro flow chamber models showed that endothelium-presented but not soluble chemokines triggered instantaneous extension of bent LFA-1 in the absence of LFA-1 ligand engagement. To support lymphocyte adhesion, this extended LFA-1 conformation required immediate activation by its ligand, intercellular adhesion molecule 1. These data show that chemokine-triggered lymphocyte adhesiveness involves a previously unrecognized extension step that primes LFA-1 for ligand binding and firm adhesion.

The capacity of integrins to mediate adhesiveness is modulated by their cytoplasmic associations. In this study, we describe a novel mechanism by which α4-integrin adhesiveness is regulated by the cytoskeletal adaptor paxillin. A mutation of the α4 tail that disrupts paxillin binding, α4(Y991A), reduced talin association to the α4β1 heterodimer, impaired integrin anchorage to the cytoskeleton, and suppressed α4β1-dependent capture and adhesion strengthening of Jurkat T cells to VCAM-1 under shear stress. The mutant retained intrinsic avidity to soluble or bead-immobilized VCAM-1, supported normal cell spreading at short-lived contacts, had normal α4-microvillar distribution, and responded to inside-out signals. This is the first demonstration that cytoskeletal anchorage of an integrin enhances the mechanical stability of its adhesive bonds under strain and, thereby, promotes its ability to mediate leukocyte adhesion under physiological shear stress conditions.

VLA-4 (␣ 4 ␤ 1 ) is a key integrin in lymphocytes, interacting with endothelial vascular cell adhesion molecule 1 (VCAM-1) on blood vessels and stroma. To dissect the contribution of the two cytoskeletal VLA-4 adaptor partners paxillin and talin to VLA-4 adhesiveness, we transiently knocked them down in Jurkat T cells and primary resting human T cells by small interfering RNA silencing. Paxillin was required for VLA-4 adhesiveness to low density VCAM-1 under shear stress conditions and was found to control mechanical stability of bonds mediated by the ␣ 4 subunit but did not affect the integrin affinity or avidity to VCAM-1 in shear-free conditions. Talin 1 maintained VLA-4 in a high affinity conformation, thereby promoting rapid VLA-4 adhesion strengthening to VCAM-1 under both shear stress and shear-free conditions. Talin 1, but not paxillin, was required for VLA-4 to undergo optimal stimulation by the prototypic chemokine, CXCL12, under shear stress conditions. Interestingly, talin 1 and paxillin played the same distinct roles in VLA-4 adhesions of primary T lymphocytes, although VLA-4 affinity to VCAM-1 was at least 200-fold lower in these cells than in Jurkat cells. Collectively, our results suggest that whereas paxillin is a mechanical regulator of VLA-4 bonds generated in the absence of chemokine signals and low VCAM-1 occupancy, talin 1 is a versatile VLA-4 affinity regulator implicated in both spontaneous and chemokine-triggered rapid adhesions to VCAM-1.

Cholesterol-enriched lipid microdomains regulate L-selectin signaling, but the role of membrane cholesterol in L-selectin adhesion is unclear. Arrest chemokines are a subset of endothelial chemokines that rapidly activate leukocyte integrin adhesiveness under shear flow. In the absence of integrin ligands, these chemokines destabilize L-selectin-mediated leukocyte rolling. In the present study, we investigated how cholesterol extraction from the plasma membrane of peripheral blood T or B cells affects L-selectin adhesions and their destabilization by arrest chemokines. Unlike the Jurkat T cell line, whose L-selectin-mediated adhesion is cholesterol dependent, in primary human PBLs and in murine B cells and B cell lines, cholesterol depletion did not impair any intrinsic adhesiveness of L-selectin, consistent with low selectin partitioning into lipid rafts in these cells. However, cholesterol raft disruption impaired the ability of two arrest chemokines, CXCL12 and CXCL13, but not of a third arrest chemokine, CCL21, to destabilize L-selectin-mediated rolling of T lymphocytes. Actin capping by brief incubation with cytochalasin D impaired the ability of all three chemokines to destabilize L-selectin rolling. Blocking of the actin regulatory phosphatidylinositol lipid, phosphatidylinositol 4,5-bisphosphate, did not affect chemokine-mediated destabilization of L-selectin adhesions. Collectively, our results suggest that L-selectin adhesions are inhibited by actin-associated, cholesterol-stabilized assemblies of CXCL12- and CXCL13-binding receptors on both T and B lymphocytes. Thus, the regulation of L-selectin by cholesterol-enriched microdomains varies with the cell type as well as with the identity of the destabilizing chemokine.

Cell adhesion is a complex event dictated by the properties of individual adhesion molecules. It is desirable to link their individual properties to the adhesive behavior of a whole cell. Here, we examine with atomic force microscopic (AFM) and flow chamber experiments how the exogenous activation of the major T cell integrin VLA-4 affects the adhesiveness of T-lymphocytes to the endothelial VLA-4 ligand, VCAM-1. The atomic force measurements are performed on substrates with different VCAM-1 densities to compare the properties of single adhesive bonds on low-density ligands with the effect of multiple bonds on higher ligand densities. We determine the basal off-rate and the potential width of the single adhesive VLA-4-VCAM-1 bond by Monte-Carlo simulations. We show that activating VLA-4 with magnesium increases the potential width of the bond from 0.18 nm to 0.44 nm, but hardly affects the basal off-rate of single VLA-4-VCAM-1 bonds measured at low VCAM-1 density, lowering it only from 1.2 s À1 to 0.9 s À1 . At higher VCAM-1 densities, the apparent off-rate of high avidity VLA-4-VCAM-1 interactions is significantly reduced by magnesium to <0.1 s À1 . In the flow chamber, both the frequency of T cell attachments to VCAM-1 and their lifetime rise after VLA-4 activation by magnesium. By extrapolating the single-molecule properties to whole cells, we suggest that exogenous stimulation with magnesium strongly increases the rate of VLA-4 rebinding to VCAM-1 and only slightly decreases the off-rate of single VLA-4-VCAM-1 bonds.