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-Galbeta1alpha4(Fucalpha1alpha3)-6-O-SO3-Glcbeta+ ++-OR) and 3',6'-disulfo Lewis x(Glc) (3, 6-di-O-SO3-Galbeta1alpha4(Fucalpha1alpha3)Glcbeta-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.     

Deciphering the catalytic machinery in a universally conserved ribosome binding ATPase YchF

The limited efficacy of monocyte-derived dendritic cell (mo-DC)-based vaccines is primarily attributed to the reduced mo-DC migratory capacity. One undefined aspect is the initial binding of mo-DCs to endothelial cells and vascular selectins. In this study, we investigated the role and modulation of the selectin binding determinant sialyl Lewis(x) (sLe(x)) in selectin-dependent mo-DC binding. Our data reveal that sLe(x) is required for maximal binding of mo-DCs to tumor necrosis factor (TNF)-α-activated endothelial cells under static conditions, as evidenced by the use of sialidase. Sialidase treatment also abrogated mo-DC cell tethering to immobilized, purified P-, L-, or E-selectin under flow. The requirement of sLe(x)-dependent binding of mo-DC to selectins was further substantiated by using sLe(x) free sugar and anti-sLe(x) antibody, which significantly suppressed mo-DC-selectin binding. P-selectin glycoprotein ligand-1 is required for mo-DC binding to both P- and L-selectin, but it is dispensable for E-selectin recognition. Interestingly, the extent of mo-DC tethering was maximal on P-selectin, followed by E- and L- selectin. Accordingly, L-selectin mediated faster mo-DC rolling than E- or P-selectin. Interferon (IFN)-γ induces a significant increase in mo-DC surface sLe(x) expression, which is probably due to the enhanced synthesis of C2GnT-I. These findings may contribute to improving mo-DC-based vaccination protocols.     

Monomeric and Multimeric Blockers of Selectins: Comparison of <Emphasis Type="Italic">in vitro</Emphasis> and <Emphasis Type="Italic">in vivo</Emphasis> Activity

The potency of the oligosaccharides SiaLe(x), SiaLe(a), HSO(3)Le(x), and HSO(3)Le(a), their conjugates with polyacrylamide (PAA, 40 kD), and other monomeric and polymeric selectin inhibitors has been compared with that of the polysaccharide fucoidan. The following assay systems were used: 1) a 96-well assay based either on the use of recombinant E-, P-, and L-selectins or an analogous assay with natural P-selectin isolated from human platelets; 2) a platelet-based P-selectin cell assay; and 3) a rat model of peritoneal inflammation. IC(50) values for the neoglycoconjugate SiaLe(a)-PAA were 6, 40, and 85 microM for recombinant E-, P-, and L-selectins, respectively; all monomeric inhibitors were about two orders of magnitude weaker. PAA-conjugates, containing as a ligand tyrosine-O-sulfate (sTyr) in addition to one of the sialylated oligosaccharides, were the most potent synthetic blockers in vitro. Compared with fucoidan, the most potent known P- and L-selectin blocker, the bi-ligand glycoconjugate HSO(3)Le(a)-PAA-sTyr displayed similar inhibitory activity in vitro towards L-selectin and about ten times lower activity towards P-selectin. All of the tested synthetic polymers displayed a similar ability to inhibit neutrophil extravasation in the peritonitis model (in vivo) at 10 mg/kg. The data provide evidence that monomeric SiaLe(x) is considerably more effective as a selectin blocker in vivo than in vitro, whereas the opposite is true for fucoidan and the bi-ligand neoglycoconjugate HSO(3)Le(a)-PAA-sTyr.     

Cell-free rolling mediated by L-selectin and sialyl Lewis(x) reveals the shear threshold effect

The selectin family of adhesion molecules mediates attachment and rolling of neutrophils to stimulated endothelial cells. This step of the inflammatory response is a prerequisite to firm attachment and extravasation. We have reported that microspheres coated with sialyl Lewis(x) (sLe(x)) interact specifically and roll over E-selectin and P-selectin substrates (Brunk et al., 1996; Rodgers et al 2000). This paper extends the use of the cell-free system to the study of the interactions between L-selectin and sLe(x) under flow. We find that sLe(x) microspheres specifically interact with and roll on L-selectin substrates. Rolling velocity increases with wall shear stress and decreases with increasing L-selectin density. Rolling velocities are fast, between 25 and 225 microm/s, typical of L-selectin interactions. The variability of rolling velocity, quantified by the variance in rolling velocity, scales linearly with rolling velocity. Rolling flux varies with both wall shear stress and L-selectin site density. At a density of L-selectin of 800 sites/microm(2), the rolling flux of sLe(x) coated microspheres goes through a clear maximum with respect to shear stress at 0.7 dyne/cm(2). This behavior, in which the maintenance and promotion of rolling interactions on selectins requires shear stress above a threshold value, is known as the shear threshold effect. We found that the magnitude of the effect is greatest at an L-selectin density of 800 sites/microm(2) and gradually diminishes with increasing L-selectin site density. Our study is the first to reveal the shear threshold effect with a cell free system and the first to show the dependence of the shear threshold effect on L-selectin site density in a reconstituted system. Our ability to recreate the shear threshold effect in a cell-free system strongly suggests the origin of the effect is in the physical chemistry of L-selectin interaction with its ligand, and largely eliminates cellular features such as deformability or topography as its cause.     

Selectin-carbohydrate interactions in shear flow

Hydrodynamic shear creates mechanical stresses on selectin bonds, modulating affinity and kinetic parameters. Chemical modification of sialyl Lewis(x) increases the strength of L-selectin bonds without altering recognition, suggesting that mechanical and biorecognition characteristics are separable. L-selectin bond formation rates may be strongly influenced by sulfate orientation in sulfo sialyl Lewis(x).     

Selectin–carbohydrate interactions in shear flow

Hydrodynamic shear creates mechanical stresses on selectin bonds, modulating affinity and kinetic parameters. Chemical modification of sialyl Lewis(x) increases the strength of L-selectin bonds without altering recognition, suggesting that mechanical and biorecognition characteristics are separable. L-selectin bond formation rates may be strongly influenced by sulfate orientation in sulfo sialyl Lewis(x).     

L-selectin dimerization enhances tether formation to properly spaced ligand

Selectin counterreceptors are glycoprotein scaffolds bearing multiple carbohydrate ligands with exceptional ability to tether flowing cells under disruptive shear forces. Bond clusters may facilitate formation and stabilization of selectin tethers. L-selectin ligation has been shown to enhance L-selectin rolling on endothelial surfaces. We now report that monoclonal antibodies-induced L-selectin dimerization enhances L-selectin leukocyte tethering to purified physiological L-selectin ligands and glycopeptides. Microkinetic analysis of individual tethers suggests that leukocyte rolling is enhanced through the dimerization-induced increase in tether formation, rather than by tether stabilization. Notably, L-selectin dimerization failed to augment L-selectin-mediated adhesion below a threshold ligand density, suggesting that L-selectin dimerization enhanced adhesiveness only to properly clustered ligand. In contrast, an epidermal growth factor domain substitution of L-selectin enhanced tether formation to L-selectin ligands irrespective of ligand density, suggesting that this domain controls intrinsic ligand binding properties of L-selectin without inducing L-selectin dimerization. Strikingly, at low ligand densities, where L-selectin tethering was not responsive to dimerization, elevated shear stress restored sensitivity of tethering to selectin dimerization. This is the first indication that shear stress augments effective selectin ligand density at local contact sites by promoting L-selectin encounter of immobilized ligand.     

The cooperative effect of L-selectin clusters and velocity-dependent bond formation that stabilizes leukocyte rolling

The role of clustered of L-selectin receptors on the leukocyte surface is discussed in connection with the postulated velocity-dependent formation of selectin-ligand tether bonds to interpret the mechanism of leukocyte tethering to, and rolling along, the vascular endothelium. The distinct feature of this step-wise process is a weak dependence of leukocyte rolling velocity on the hydrodynamic forces of ambient flow due to the increased number of selectin bonds with increasing flow shear rate and also their clustering. The contact zones on the leukocyte surface are separated by distances with distribution which corresponds to the distribution of distances of the observed L-selectin clusters. It suggests that the localization of L-selectin receptors to clusters and the way of their approach to the ligand molecules creates such conditions for binding of L-selectin and ligand molecules that resulting number of bonds stabilizes rolling velocity.     

Shear-dependent capping of L-selectin and P-selectin glycoprotein ligand 1 by E-selectin signals activation of high-avidity beta2-integrin on neutrophils

Two adhesive events critical to efficient recruitment of neutrophils at vascular sites of inflammation are up-regulation of endothelial selectins that bind sialyl Lewis(x) ligands and activation of beta(2)-integrins that support neutrophil arrest by binding ICAM-1. We have previously reported that neutrophils rolling on E-selectin are sufficient for signaling cell arrest through beta(2)-integrin binding of ICAM-1 in a process dependent upon ligation of L-selectin and P-selectin glycoprotein ligand 1 (PSGL-1). Unresolved are the spatial and temporal events that occur as E-selectin binds to human neutrophils and dynamically signals the transition from neutrophil rolling to arrest. Here we show that binding of E-selectin to sialyl Lewis(x) on L-selectin and PSGL-1 drives their colocalization into membrane caps at the trailing edge of neutrophils rolling on HUVECs and on an L-cell monolayer coexpressing E-selectin and ICAM-1. Likewise, binding of recombinant E-selectin to PMNs in suspension also elicited coclustering of L-selectin and PSGL-1 that was signaled via mitogen-activated protein kinase. Binding of recombinant E-selectin signaled activation of beta(2)-integrin to high-avidity clusters and elicited efficient neutrophil capture of beta(2)-integrin ligands in shear flow. Inhibition of p38 and p42/44 mitogen-activated protein kinase blocked the cocapping of L-selectin and PSGL-1 and the subsequent clustering of high-affinity beta(2)-integrin. Taken together, the data suggest that E-selectin is unique among selectins in its capacity for clustering sialylated ligands and transducing signals leading to neutrophil arrest in shear flow.     

An activated L-selectin mutant with conserved equilibrium binding properties but enhanced ligand recognition under shear flow

Selectins mediate the initial tethering and rolling of leukocytes on vessel walls. Adhesion by selectins is a function of both ligand recognition at equilibrium and mechanical properties of the selectin-ligand bond under applied force. We describe an EGF domain mutant of L-selectin with profoundly augmented adhesiveness over that of native L-selectin but conserved ligand specificity. This mutant, termed LPL, was derived by a substitution of the EGF-like domain of L-selectin with the homologous domain from P-selectin. The mutant bound soluble carbohydrate L-selectin ligand with affinity comparable with that of native L-selectin but interacted with all surface-bound ligands much more readily than native L-selectin, in particular under elevated shear flow. Tethers mediated by both native and mutant L-selectin exhibited similar lifetimes under a range of shear stresses, but the rate of bond formation by the mutant was at least 10-fold higher than that of native L-selectin toward distinct L-selectin ligands. Enhanced rate of bond formation by the mutant was associated with profoundly stronger rolling interactions and reduced dependence of rolling on a threshold of shear stress. This is the first demonstration that the EGF domain can modulate the binding of the lectin domain of a selectin to surface-immobilized ligands under shear flow without affecting the equilibrium properties of the selectin toward soluble ligands.     

Sialyl Lewis(x)/E-selectin-mediated rolling in a cell-free system.

Selections mediate transient adhesion of neutrophils to stimulated endothelial cells at sites of inflammation by binding counter-receptors that present carbohydrates such as sialyl Lewis(x). We have developed a cell-free adhesion assay using sialyl Lewis(x)-coated microspheres and E-selection-IgG chimera-coated substrates to investigate the premise that rolling primarily results from functional properties of selection-carbohydrate bonds, whereas cellular morphology and signaling act as secondary effects. Sialyl Lewis(x)-coated microspheres attach to and roll over E-selectin-IgG chimera-coated substrates between the physiological wall shear stresses of 0.7 and 2 dynes/cm2. Rolling velocities vary with time and depend on E-selectin-IgG chimera site density and wall shear stress. Our results show that sialyl Lewis(x) is a minimal functional recognition element required for rolling on E-selectin under flow.     

The three members of the selectin receptor family recognize a common carbohydrate epitope, the sialyl Lewis(x) oligosaccharide

The selectins (lectin-EGF-complement binding-cell adhesion molecules [LEC-CAMs]) are a family of mammalian receptors implicated in the initial interactions between leukocytes and vascular endothelia, leading to lymphocyte homing, platelet binding, and neutrophil extravasation. The three known selectins, L-selectin (leukocyte adhesion molecule-1 [LECAM-1]), E-selectin (endothelial-leukocyte adhesion molecule-1 [ELAM-1]), and P-selectin (GMP-140) share structural features that include a calcium-dependent lectin domain. The sialyl Lewis(x) carbohydrate epitope has been reported as a ligand for both E- and P-selectins. Although L-selectin has been demonstrated to bind to carbohydrates, structural features of potential mammalian carbohydrate ligand(s) have not been well defined. Using an ELISA developed with a sialyl Lewis(x)-containing glycolipid and an E-selectin-IgG chimera, we have demonstrated the direct binding of the L-selectin-IgG chimera to sialyl Lewis(x). This recognition was calcium dependent, and could be blocked by Mel-14 antibody but not by other antibodies. Recognition was confirmed by the ability of cells expressing the native L-selectin to adhere to immobilized sialyl Lewis(x). These data suggest that the sialyl Lewis(x) oligosaccharide may form the basis of a recognition domain common to all three selectins.     

L-选择素在白细胞活化中的作用

白细胞沿着血管内皮滚动、稳定黏附,最终到达炎症部位是一个复杂的、多步骤的过程,该过程需要众多分子协同完成。选择素家族分子对于白细胞沿着血管内皮的滚动起重要作用。L-选择素是选择素家族的一员,组成性的表达在白细胞微绒毛顶端,在白细胞沿血管内皮起始黏附过程中起主要作用。除具有黏附作用外,L-选择素还作为信号分子在黏附事件中发挥作用。该文结合作者的研究工作,综述了L-选择素在白细胞活化过程中的功能。     

Hydrodynamic shear regulates the kinetics and receptor specificity of polymorphonuclear leukocyte-colon carcinoma cell adhesive interactions.

The ability of tumor cells to metastasize hematogenously is regulated by their interactions with polymorphonuclear leukocytes (PMNs). However, the mechanisms mediating PMN binding to tumor cells under physiological shear forces remain largely unknown. This study was designed to characterize the molecular interactions between PMNs and tumor cells as a function of the dynamic shear environment, using two human colon adenocarcinoma cell lines (LS174T and HCT-8) as models. PMN and colon carcinoma cell suspensions, labeled with distinct fluorophores, were sheared in a cone-and-plate rheometer in the presence of the PMN activator fMLP. The size distribution and cellular composition of formed aggregates were determined by flow cytometry. PMN binding to LS174T cells was maximal at 100 s(-1) and decreased with increasing shear. At low shear (100 s(-1)) PMN CD11b alone mediates PMN-LS174T heteroaggregation. However, L-selectin, CD11a, and CD11b are all required for PMN binding to sialyl Lewis(x)-bearing LS174T cells at high shear (800 s(-1)). In contrast, sialyl Lewis(x)-low HCT-8 cells fail to aggregate with PMNs at high shear conditions, despite extensive adhesive interactions at low shear. Taken together, our data suggest that PMN L-selectin initiates LS174T cell tethering at high shear by binding to sialylated moieties on the carcinoma cell surface, whereas the subsequent involvement of CD11a and CD11b converts these transient tethers into stable adhesion. This study demonstrates that the shear environment of the vasculature modulates the dynamics and molecular constituents mediating PMN-tumor cell adhesion.     

Molecular dynamics of the transition from L-selectin- to beta 2-integrin-dependent neutrophil adhesion under defined hydrodynamic shear.

Homotypic adhesion o2 neutrophils stimulated with chemoattractant is analogous to capture on vascular endothelium in that both processes depend on L-selectin and beta 2-integrin adhesion receptors. Under hydrodynamic shear, cell adhesion requires that receptors bind sufficient ligand over the duration of intercellular contact to withstand hydrodynamic stresses. Using cone-plate viscometry to apply a uniform linear shear field to suspensions of neutrophils, we conducted a detailed examination of the effect of shear rate and shear stress on the kinetics of cell aggregation. A collisional analysis based on Smoluchowski's flocculation theory was employed to fit the kinetics of aggregation with an adhesion efficiency. Adhesion efficiency increased with shear rate from approximately 20% at 100 s-1 to approximately 80% at 400 s-1. The increase in adhesion efficiency. Adhesion efficiency increased with shear rate from approximately 20% at 100 s-1 to approximately 80% at 400 s-1. The increase in adhesion efficiency with shear was dependent on L-selectin, and peak efficiency was maintained over a relatively narrow range of shear rates (400-800 s-1) and shear stresses (4-7 dyn/cm2). When L-selectin was blocked with antibody, beta 2-integrin (CD11a, b) supported adhesion at low shear rates (< 400 s-1). The binding kinetics of selectin and integrin appear to be optimized to function within discrete ranges of shear rate and stress, providing an intrinsic mechanism for the transition from neutrophil tethering to stable adhesion.     

Sialylated, fucosylated ligands for L-selectin expressed on leukocytes mediate tethering and rolling adhesions in physiologic flow conditions

Interaction of leukocytes in flow with adherent leukocytes may contribute to their accumulation at sites of inflammation. Using L- selectin immobilized in a flow chamber, a model system that mimics presentation of L-selectin by adherent leukocytes, we characterize ligands for L-selectin on leukocytes and show that they mediate tethering and rolling in shear flow. We demonstrate the presence of L- selectin ligands on granulocytes, monocytes, and myeloid and lymphoid cell lines, and not on peripheral blood T lymphocytes. These ligands are calcium dependent, sensitive to protease and neuraminidase, and structurally distinct from previously described ligands for L-selectin on high endothelial venules (HEV). Differential sensitivity to O-sialo- glycoprotease provides evidence for ligand activity on both mucin-like and nonmucin-like structures. Transfection with fucosyltransferase induces expression of functional L-selectin ligands on both a lymphoid cell line and a nonhematopoietic cell line. L-selectin presented on adherent cells is also capable of supporting tethering and rolling interactions in physiologic shear flow. L-selectin ligands on leukocytes may be important in promoting leukocyte-leukocyte and subsequent leukocyte endothelial interactions in vivo, thereby enhancing leukocyte localization at sites of inflammation.     

Regulation of PSGL-1 interactions with L-selectin, P-selectin, and E-selectin: role of human fucosyltransferase-IV and -VII

P-selectin glycoprotein ligand-1 (PSGL-1) interactions with selectins regulate leukocyte migration in inflammatory lesions. In mice, selectin ligand activity regulating leukocyte recruitment and lymphocyte homing into lymph nodes results from the sum of unequal contributions of fucosyltransferase (FucT)-IV and FucT-VII, with FucT-VII playing a predominant role. Here we have examined the role of human FucT-IV and -VII in conferring L-selectin, P-selectin, and E-selectin binding activities to PSGL-1. Lewis x (Le(x)) carbohydrate was generated at the CHO(dhfr)(-) cell surface by FucT-IV expression, whereas sialyl Le(x) (sLe(x)) was synthesized by FucT-VII. Both human FucT-IV and -VII had the ability to generate carbohydrate ligands that support L-selectin-, P-selectin-, and E-selectin-dependent rolling on PSGL-1, with FucT-VII playing a major role. Cooperation was observed between FucT-IV and -VII in recruiting L-, P-, or E-selectin-expressing cells on PSGL-1 and in regulating cell rolling velocity and stability. Additional rolling adhesion assays were performed to assess the role of Thr-57-linked core-2 O-glycans in supporting L-selectin-, P-selectin-, and E-selectin-dependent rolling on PSGL-1. These studies confirmed that core-2 O-glycans attached to Thr-57 play a critical role in supporting L- and P-selectin-dependent rolling and revealed that additional binding sites support >75% of E-selectin-mediated rolling. The observations presented here indicate that human FucT-IV and -VII both contribute and cooperate in regulating L-selectin-, P-selectin-, and E-selectin-dependent rolling on PSGL-1, with FucT-VII playing a predominant role in conferring selectin binding activity to PSGL-1.     

Quantitative Characterization of E-selectin Interaction with Native CD44 and P-selectin Glycoprotein Ligand-1 (PSGL-1) Using a Real Time Immunoprecipitation-based Binding Assay

Selectins (E-, P-, and L-selectins) interact with glycoprotein ligands to mediate the essential tethering/rolling step in cell transport and delivery that captures migrating cells from the circulating flow. In this work, we developed a real time immunoprecipitation assay on a surface plasmon resonance chip that captures native glycoforms of two well known E-selectin ligands (CD44/hematopoietic cell E-/L-selectin ligand and P-selectin glycoprotein ligand-1) from hematopoietic cell extracts. Here we present a comprehensive characterization of their binding to E-selectin. We show that both ligands bind recombinant monomeric E-selectin transiently with fast on- and fast off-rates, whereas they bind dimeric E-selectin with remarkably slow on- and off-rates. This binding requires the sialyl Lewis x sugar moiety to be placed on both O- and N-glycans, and its association, but not dissociation, is sensitive to the salt concentration. Our results suggest a mechanism through which monomeric selectins mediate initial fast on and fast off kinetics to help capture cells out of the circulating shear flow; subsequently, tight binding by dimeric/oligomeric selectins is enabled to significantly slow rolling.     

Endoglycan,a member of the CD34 family,functions as an L-selectin ligand through modification with tyrosine sulfation and sialyl Lewis x

During lymphocyte homing to secondary lymphoid organs and instances of inflammatory trafficking, the rolling of leukocytes on vascular endothelium is mediated by transient interactions between L-selectin on leukocytes and several carbohydrate-modified ligands on the endothelium. Most L-selectin ligands such as CD34 and podocalyxin present sulfated carbohydrate structures (6-sulfated sialyl Lewis x or 6-sulfo-sLex) as a recognition determinant within their heavily glycosylated mucin domains. We recently identified endoglycan as a new member of the CD34 family. We report here that endoglycan, like the two other members of this family (CD34 and podocalyxin) can function as a L-selectin ligand. However, endoglycan employs a different binding mechanism, interacting with L-selectin through sulfation on two tyrosine residues and O-linked sLex structures that are presented within its highly acidic amino-terminal region. Our analysis establishes striking parallels with PSGL-1, a leukocyte ligand that interacts with all three selectins, mediating leukocyte-endothelial, leukocyte-leukocyte, and platelet-leukocyte interactions. Since the distribution of endoglycan includes hematopoietic precursors and leukocyte subpopulations, in addition to endothelial cells, our findings suggest several potential settings for endoglycan-mediated adhesion events.