Oren Dwir

The LFA-1 integrin supports rolling adhesions on ICAM-1 under physiological shear flow in a permissive cellular environment.

The LFA-1 integrin is crucial for the firm adhesion of circulating leukocytes to ICAM-1-expressing endothelial cells. In the present study, we demonstrate that LFA-1 can arrest unstimulated PBL subsets and lymphoblastoid Jurkat cells on immobilized ICAM-1 under subphysiological shear flow and mediate firm adhesion to ICAM-1 after short static contact. However, LFA-1 expressed in K562 cells failed to support firm adhesion to ICAM-1 but instead mediated K562 cell rolling on the endothelial ligand under physiological shear stress. LFA-1-mediated rolling required an intact LFA-1 I-domain, was enhanced by Mg2+, and was sharply dependent on ICAM-1 density. This is the first indication that LFA-1 can engage in rolling adhesions with ICAM-1 under physiological shear flow. The ability of LFA-1 to support rolling correlates with decreased avidity and impaired time-dependent adhesion strengthening. A β2 cytoplasmic domain-deletion mutant of LFA-1, with high avidity to immobilized ICAM-1, mediated firm arrests of K562 cells interacting with ICAM-1 under shear flow. Our results suggest that restrictions in LFA-1 clustering mediated by cytoskeletal attachments may lock the integrin into low-avidity states in particular cellular environments. Although low-avidity LFA-1 states fail to undergo adhesion strengthening upon contact with ICAM-1 at stasis, these states are permissive for leukocyte rolling on ICAM-1 under physiological shear flow. Rolling mediated by low-avidity LFA-1 interactions with ICAM-1 may stabilize rolling initiated by specialized vascular rolling receptors and allow the leukocyte to arrest on vascular endothelium upon exposure to stimulatory endothelial signals.

Cytoplasmic anchorage of L-selectin controls leukocyte capture and rolling by increasing the mechanical stability of the selectin tether

L-selectin is a leukocyte lectin that mediates leukocyte capture and rolling in the vasculature. The cytoplasmic domain of L-selectin has been shown to regulate leukocyte rolling. In this study, the regulatory mechanisms by which this domain controls L-selectin adhesiveness were investigated. We report that an L-selectin mutant generated by truncation of the COOH-terminal 11 residues of L-selectin tail, which impairs association with the cytoskeletal protein α-actinin, could capture leukocytes to glycoprotein L-selectin ligands under physiological shear flow. However, the conversion of initial tethers into rolling was impaired by this partial tail truncation, and was completely abolished by a further four-residue truncation of the L-selectin tail. Physical anchorage of both cell-free tail-truncated mutants within a substrate fully rescued their adhesive deficiencies. Microkinetic analysis of full-length and truncated L-selectin–mediated rolling at millisecond temporal resolution suggests that the lifetime of unstressed L-selectin tethers is unaffected by cytoplasmic tail truncation. However, cytoskeletal anchorage of L-selectin stabilizes the selectin tether by reducing the sensitivity of its dissociation rate to increasing shear forces. Low force sensitivity (reactive compliance) of tether lifetime is crucial for selectins to mediate leukocyte rolling under physiological shear stresses. This is the first demonstration that reduced reactive compliance of L-selectin tethers is regulated by cytoskeletal anchorage, in addition to intrinsic mechanical properties of the selectin–carbohydrate bond.

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.

Inhibition of L-selectin-mediated Leukocyte Rolling by Synthetic Glycoprotein Mimics*

Synthetic carbohydrate and glycoprotein mimics displaying sulfated saccharide residues have been assayed for their L-selectin inhibitory properties under static and flow conditions. Polymers displaying the L-selectin recognition epitopes 3′,6-disulfo Lewis x(Glc) (3-O-SO3-Galβ1α4(Fucα1α3)-6-O-SO3-Glcβ-OR) and 3′,6′-disulfo Lewis x(Glc) (3,6-di-O-SO3-Galβ1α4(Fucα1α3)Glcβ-OR) both inhibit L-selectin binding to heparin under static, cell-free binding conditions with similar efficacies. Under conditions of shear flow, however, only the polymer displaying 3′,6-disulfo Lewis x(Glc) inhibits the rolling of L-selectin-transfected cells on the glycoprotein ligand GlyCAM-1. Although it has been shown to more effective than sialyl Lewis x at blocking the L-selectin–GlyCAM-1 interaction in static binding studies, the corresponding monomer had no effect in the dynamic assay. These data indicate that multivalent ligands are far more effective inhibitors of L-selectin-mediated rolling than their monovalent counterparts and that the inhibitory activities are dependent on the specific sulfation pattern of the recognition epitope. Importantly, our results indicate the L-selectin specificity for one ligand over another found in static, cell-free binding assays is not necessarily retained under the conditions of shear flow. The results suggest that monovalent or polyvalent carbohydrate or glycoprotein mimetics that inhibit selectin binding in static assays may not block the more physiologically relevant process of selectin-mediated rolling.

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.

Avidity enhancement of L-selectin bonds by flow: shear-promoted rotation of leukocytes turn labile bonds into functional tethers

L-selectin is a key lectin essential for leukocyte capture and rolling on vessel walls. Functional adhesion of L-selectin requires a minimal threshold of hydrodynamic shear. Using high temporal resolution videomicroscopy, we now report that L-selectin engages its ligands through exceptionally labile adhesive bonds (tethers) even below this shear threshold. These tethers share a lifetime of 4 ms on distinct physiological ligands, two orders of magnitude shorter than the lifetime of the P-selectin–PSGL-1 bond. Below threshold shear, tether duration is not shortened by elevated shear stresses. However, above the shear threshold, selectin tethers undergo 14-fold stabilization by shear-driven leukocyte transport. Notably, the cytoplasmic tail of L-selectin contributes to this stabilization only above the shear threshold. These properties are not shared by P-selectin– or VLA-4–mediated tethers. L-selectin tethers appear adapted to undergo rapid avidity enhancement by cellular transport, a specialized mechanism not used by any other known adhesion receptor.

Tumor cell MUC1 and CD43 are glycosylated differently with sialyl-Lewis a and x epitopes and show variable interactions with E-selectin under physiological flow conditions

The mucins secreted from the colon carcinoma cell line COLO 205 have the MUC1 and CD43 (leukosialin) as core proteins, where both carry sialyl-Lewis a and MUC1 sialyl-Lewis x epitopes. The adhesion of E-selectin expressing CHO cells to the coated mucins was analyzed in a flow system revealing that the MUC1 mucin adhered better than the CD43 mucin. One reason could be their different glycosylation, a difference that was explored by analyzing the biosynthesis of MUC1 and CD43 in COLO 205 cells. Both the MUC1 and CD43 mucins became sialyl-Lewis a reactive, but after different times as revealed by pulse-chase studies. However, only MUC1 became sialyl-Lewis x reactive. These differences suggest that MUC1 and CD43 are synthesized in different compartments of the cell. It was also observed that the mucins from colon carcinoma patients had MUC1-type mucins that carried both sialyl-Lewis a and x epitopes and CD43-type sialyl-Lewis a mucins with only low levels of sialyl-Lewis x epitopes. One could hypothesize that colon carcinoma derived MUC1 is decorated with potent E-selectin epitopes, and that this could be one of several reasons for the involvement of MUC1 in cancer development.

Endothelial Chemokines Destabilize L-selectin-mediated Lymphocyte Rolling without Inducing Selectin Shedding

Chemokines presented on specialized endothelial surfaces rapidly up-regulate leukocyte integrin avidity and firm arrest through Gi-protein signaling. Here we describe a novel, G-protein-independent, down-regulatory activity of apical endothelial chemokines in destabilizing L-selectin-mediated leukocyte rolling. Unexpectedly, this anti-adhesive chemokine suppression of rolling does not involve L-selectin shedding. Destabilization of rolling is induced only by immobilized chemokines juxtaposed to L-selectin ligands and is an energy-dependent process. Chemokines are found to interfere with a subsecond stabilization of selectin tethers necessary for persistent rolling. This is a first indication that endothelial chemokines can attenuate in situ L-selectin adhesion to endothelial ligands at subsecond contacts. This negative feedback mechanism may underlie the jerky nature of rolling mediated by L-selectin in vivo.

GLYCAM-1 SUPPORTS LEUKOCYTE ROLLING IN FLOW : EVIDENCE FOR A GREATER DYNAMIC STABILITY OF L-SELECTIN ROLLING OF LYMPHOCYTES THAN OF NEUTROPHILS

L-selectin plays a major role in leukocyte traffic through lymph node high endothelial venules (HEV). We have investigated the role of GlyCAM-1, a major L-selectin ligand produced by HEV, in mediating leukocyte rolling under in vitro flow conditions. Purified GlyCAM-1 was found to support tethering and rolling in physiological shear flow of both human and murine L-selectin expressing leukocytes at an efficiency comparable to the HEV-derived L-selectin ligands termed peripheral node addressin (PNAd). Major dynamic differences between L-selectin rolling of peripheral blood T lymphocytes and neutrophils expressing similar L-selectin level were observed on GlyCAM-1. Lymphocytes established slower and more shear resistant rolling than neutrophils and could roll on GlyCAM-1 at shear stresses lower than the threshold values required for L-selectin-mediated neutrophil rolling. Notably, high stability of L-selectin rolling of lymphocytes requires intact cellular energy, although initial lymphocyte tethering to L-selectin ligands is energy-independent. By contrast, L-selectin mediated rolling of neutrophils is insensitive to energy depletion. The distinct dynamic behavior and energy-dependence of L-selectin rolling in different leukocytes suggest that L-selectin adhesiveness in shear flow is regulated in a cell-type specific manner. The greater stability of L-selectin rolling of lymphocytes on surface-adsorbed GlyCAM-1 may contribute to their selective recruitment at peripheral lymph nodes.

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.