Revital Shamri

Lymphocyte arrest requires instantaneous induction of an extended LFA-1 conformation mediated by endothelium-bound chemokines.

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.

Chemoattractant Signals and β2 Integrin Occupancy at Apical Endothelial Contacts Combine with Shear Stress Signals to Promote Transendothelial Neutrophil Migration

Lymphocyte transendothelial migration (TEM) is promoted by fluid shear signals and apical endothelial chemokines. Studying the role of these signals in neutrophil migration across differently activated HUVEC in a flow chamber apparatus, we gained new insights into how neutrophils integrate multiple endothelial signals to promote TEM. Neutrophils crossed highly activated HUVEC in a β2 integrin-dependent manner but independently of shear. In contrast, neutrophil migration across resting or moderately activated endothelium with low-level β2 integrin ligand activity was dramatically augmented by endothelial-presented chemoattractants, conditional to application of physiological shear stresses and intact β2 integrins. Shear stress signals were found to stimulate extensive neutrophil invaginations into the apical endothelial interface both before and during TEM. A subset of invaginating neutrophils completed transcellular diapedesis through individual endothelial cells within <1 min. Our results suggest that low-level occupancy of β2 integrins by adherent neutrophils can mediate TEM only if properly coupled to stimulatory shear stress and chemoattractant signals transduced at the apical neutrophil-endothelial interface.

Novel chemokine functions in lymphocyte migration through vascular endothelium under shear flow

The recruitment of circulating leukocytes at vascular sites in target tissue has been linked to activation of Gi‐protein signaling in leukocytes by endothelial chemokines. The mechanisms by which apical and subendothelial chemokines regulate leukocyte adhesion to and migration across endothelial barriers have been elusive. We recently found that endothelial chemokines not only stimulate integrin‐mediated arrest on vascular endothelial ligands but also trigger earlier very late antigen (VLA)‐4 integrin‐mediated capture (tethering) of lymphocytes to vascular cell adhesion molecule 1 (VCAM‐1)‐bearing surfaces by extremely rapid modulation of integrin clustering at adhesive contact zones. This rapid modulation of integrin avidity requires chemokine immobilization in juxtaposition with the VLA‐4 ligand VCAM‐1. We also observed that endothelial‐bound chemokines promote massive lymphocyte transendothelial migration (TEM). It is interesting that chemokine‐promoted lymphocyte TEM requires continuous exposure of lymphocytes but not of the endothelial barrier to fluid shear. It is noteworthy that lymphocyte stimulation by soluble chemokines did not promote lymphocyte TEM. Our results suggest new roles for apical endothelial chemokines both in triggering lymphocyte capture to the endothelial surface and in driving post‐arrest events that promote lymphocyte transmigration across endothelial barriers under shear flow.

The CD81 Tetraspanin Facilitates Instantaneous Leukocyte VLA-4 Adhesion Strengthening to Vascular Cell Adhesion Molecule 1 (VCAM-1) under Shear Flow

Leukocyte integrins must rapidly strengthen their binding to target endothelial sites to arrest rolling adhesions under physiological shear flow. We demonstrate that the integrin-associated tetraspanin, CD81, regulates VLA-4 and VLA-5 adhesion strengthening in monocytes and primary murine B cells. CD81 strengthens multivalent VLA-4 contacts within subsecond integrin occupancy without altering intrinsic adhesive properties to low density ligand. CD81 facilitates both VLA-4-mediated leukocyte rolling and arrest on VCAM-1 under shear flow as well as VLA-5-dependent adhesion to fibronectin during short stationary contacts. CD81 also augments VLA-4 avidity enhancement induced by either chemokine-stimulated Gi proteins or by protein kinase C activation, although it is not required for Gi protein or protein kinase C signaling activities. In contrast to other proadhesive integrin-associated proteins, CD81-promoted integrin adhesiveness does not require its own ligand occupancy or ligation. These results provide the first demonstration of an integrin-associated transmembranal protein that facilitates instantaneous multivalent integrin occupancy events that promote leukocyte adhesion to an endothelial ligand under shear flow.

Chemokine Stimulation of Lymphocyte α4Integrin Avidity but Not of Leukocyte Function-associated Antigen-1 Avidity to Endothelial Ligands under Shear Flow Requires Cholesterol Membrane Rafts

VLA-4 and LFA-1 are the major vascular integrins expressed on circulating lymphocytes. Previous studies suggested that intact cholesterol rafts are required for integrin adhesiveness in different leukocytes. We found the α4 integrins VLA-4 and α4β7 as well as the LFA-1 integrin to be excluded from rafts of human peripheral blood lymphocytes. Disruption of cholesterol rafts with the chelator methyl-β-cyclodextrin did not affect the ability of these lymphocyte integrins to generate high avidity to their respective endothelial ligands and to promote lymphocyte rolling and arrest on inflamed endothelium under shear flow. In contrast, cholesterol extraction abrogated rapid chemokine triggering of α4-integrin-dependent peripheral blood lymphocytes adhesion, a process tightly regulated by Gi-protein activation of G protein-coupled chemokine receptors (GPCR). Strikingly, stimulation of LFA-1 avidity to intercellular adhesion molecule 1 (ICAM-1) by the same chemokines, although Gi-dependent, was insensitive to raft disruption. Our results suggest that α4 but not LFA-1 integrin avidity stimulation by chemokines involves rapid chemokine-induced GPCR rearrangement that takes place at cholesterol raft platforms upstream to Gi signaling. Our results provide the first evidence that a particular chemokine/GPCR pair can activate different integrins on the same cell using distinct Gi protein-associated machineries segregated within defined membrane compartments.

RhoA Is Involved in LFA-1 Extension Triggered by CXCL12 but Not in a Novel Outside-In LFA-1 Activation Facilitated by CXCL9

Chemokines presented on endothelial tissues instantaneously trigger LFA-1-mediated arrest on ICAM-1 via rapid inside-out and outside-in (ligand-driven) LFA-1 activation. The GTPase RhoA was previously implicated in CCL21-triggered LFA-1 affinity triggering in murine T lymphocytes and in LFA-1-dependent adhesion strengthening to ICAM-1 on Peyer’s patch high endothelial venules stabilized over periods of at least 10 s. In this study, we show that a specific RhoA 23/40 effector region is vital for the initial LFA-1-dependent adhesions of lymphocytes on high endothelial venules lasting 1–3 s. Blocking the RhoA 23/40 region in human T lymphocytes in vitro also impaired the subsecond CXCL12-triggered LFA-1-mediated T cell arrest on ICAM-1 by eliminating the rapid induction of an extended LFA-1 conformational state. However, the inflammatory chemokine CXCL9 triggered robust LFA-1-mediated T lymphocyte adhesion to ICAM-1 at subsecond contacts independently of the RhoA 23/40 region. CXCL9 did not induce conformational changes in the LFA-1 ectodomain, suggesting that particular chemokines can activate LFA-1 through outside-in post ligand binding stabilization changes. Like CXCL9, the potent diacylglycerol-dependent protein kinase C agonist PMA was found to trigger LFA-1 adhesiveness to ICAM-1 also without inducing integrin extension or an a priori clustering and independently of the RhoA 23/40 region. Our results collectively suggest that the 23/40 region of RhoA regulates chemokine-induced inside-out LFA-1 extension before ligand binding, but is not required for a variety of chemokine and non-chemokine signals that rapidly strengthen LFA-1-ICAM-1 bonds without an a priori induction of high-affinity extended LFA-1 conformations.

Membranal Cholesterol Is Not Required for L-Selectin Adhesiveness in Primary Lymphocytes but Controls a Chemokine-Induced Destabilization of L-Selectin Rolling Adhesions

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.