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 alpha-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.     

Binding of L-selectin to its vascular and extravascular ligands is differentially regulated by pH

Ligands for L-selectin, a leukocyte adhesion molecule, are expressed in high endothelial venules (HEVs) in lymph nodes and extravascular tissues, such as renal tubules. Here, we report that the binding of L-selectin to its vascular and extravascular ligands is differentially regulated by pH. The optimal L-selectin-dependent binding of leukocytes to HEVs was observed at pH 7.4, a physiological pH in the blood. In contrast, the optimal binding of leukocytes to the renal tubules was observed at pH 5.6. Consistently, optimal binding of soluble recombinant L-selectin to a major vascular ligand, 6-sulfo sialyl Lewis X, was observed at pH 7.4. Binding to extravascular ligands, such as chondroitin sulfate (CS) B, CS E and heparan sulfate, occurred at pH 5.6. Under physiological shear stress ranging from 1 to 2 dynes/cm², maximal leukocyte rolling on vascular ligands was observed at pH 6.8 to 7.4, and no rolling was detected at pH conditions below 5.6. These findings suggest that the pH environment is one important factor that determines leukocyte trafficking under physiological and pathological conditions.     

CHST1 and CHST2 sulfotransferases expressed by human vascular endothelial cells: cDNA cloning, expression, and chromosomal localization

Sulfation is essential for the generation of functional vascular endothelial cell ligands for the leukocyte adhesion molecule, L-selectin. Therefore, human vascular endothelium cDNA libraries were screened to identify sulfotransferases homologous to chicken chondroitin 6-sulfotransferase (C6ST). Two sulfotransferases were identified: CHST2, a novel 530-amino-acid sulfotransferase with a carboxyl-terminal region that was 45 and 43% homologous with those of human and chicken C6ST, respectively, and CHST1, which was identical to human C6ST. Northern blot analysis showed that CHST2 was broadly expressed among tissues. The CHST2 gene mapped to human chromosome 3q24 close to 3q25. Thus, this study identified two sulfotransferases expressed by vascular endothelial cells that may contribute to the generation of L-selectin ligands during inflammatory responses.     

L-selectin ligands expressed by human leukocytes are HECA-452 antibody-defined carbohydrate epitopes preferentially displayed by P-selectin glycoprotein ligand-1.

Leukocytes express L-selectin ligands critical for leukocyte-leukocyte interactions at sites of inflammation. The predominant leukocyte L-selectin ligand is P-selectin glycoprotein ligand-1 (PSGL-1), which displays appropriate sialyl Lewis x (sLex)-like carbohydrate determinants for L-selectin recognition. Among the sLex-like determinants expressed by human leukocytes is a unique carbohydrate epitope defined by the HECA-452 mAb. The HECA-452 Ag is a critical component of L-selectin ligands expressed by vascular endothelial cells. However, HECA-452 Ag expression on human leukocyte L-selectin ligands has not been assessed. In this study, the HECA-452 mAb blocked 88-99% of neutrophil rolling on, or attachment to, adherent cells expressing L-selectin in multiple experimental systems. A function-blocking anti-PSGL-1 mAb also inhibited L-selectin binding to neutrophils by 89-98%. In addition, the HECA-452 and anti-PSGL-1 mAbs blocked the majority of P-selectin binding to neutrophils. Western blot analysis revealed that PSGL-1 immunoprecipitated from neutrophils displayed HECA-452 mAb-reactive determinants and that PSGL-1 was the predominant scaffold for HECA-452 Ag display. Leukocyte L-selectin ligands also contained sulfated determinants since culturing ligand-bearing cells with NaClO3 abrogated L-selectin binding. Consistent with this, human neutrophils expressed mRNA encoding five different sulfotransferases associated with the generation of selectin ligands: CHST1, CHST2, CHST3, TPST1, and HEC-GlcNAc6ST. Therefore, the HECA-452-defined carbohydrate determinant displayed on PSGL-1 represented the predominant L-selectin and P-selectin ligand expressed by neutrophils.     

Catch bonds govern adhesion through L-selectin at threshold shear

Flow-enhanced cell adhesion is an unexplained phenomenon that might result from a transport-dependent increase in on-rates or a force-dependent decrease in off-rates of adhesive bonds. L-selectin requires a threshold shear to support leukocyte rolling on P-selectin glycoprotein ligand-1 (PSGL-1) and other vascular ligands. Low forces decrease L-selectin-PSGL-1 off-rates (catch bonds), whereas higher forces increase off-rates (slip bonds). We determined that a force-dependent decrease in off-rates dictated flow-enhanced rolling of L-selectin-bearing microspheres or neutrophils on PSGL-1. Catch bonds enabled increasing force to convert short-lived tethers into longer-lived tethers, which decreased rolling velocities and increased the regularity of rolling steps as shear rose from the threshold to an optimal value. As shear increased above the optimum, transitions to slip bonds shortened tether lifetimes, which increased rolling velocities and decreased rolling regularity. Thus, force-dependent alterations of bond lifetimes govern L-selectin-dependent cell adhesion below and above the shear optimum. These findings establish the first biological function for catch bonds as a mechanism for flow-enhanced cell 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.     

Identification of L-selectin binding heparan sulfates attached to collagen type XVIII

L-selectin is a C-type lectin expressed on leukocytes that is involved in both lymphocyte homing to the lymph node and leukocyte extravasation during inflammation. Known L-selectin ligands include sulfated Lewis-type carbohydrates, glycolipids, and proteoglycans. Previously, we have shown that in situ detection of different types of L-selectin ligands is highly dependent on the tissue fixation protocol used. Here we use this knowledge to specifically examine the expression of L-selectin binding proteoglycans in normal mouse tissues. We show that L-selectin binding chondroitin/dermatan sulfate proteoglycans are present in cartilage, whereas L-selectin binding heparan sulfate proteoglycans are present in spleen and kidney. Furthermore, we show that L-selectin only binds a subset of renal heparan sulfates, attached to a collagen type XVIII protein backbone and predominantly present in medullary tubular and vascular basement membranes. As L-selectin does not bind other renal heparan sulfate proteoglycans such as perlecan, agrin, and syndecan-4, and not all collagen type XVIII expressed in the kidney binds L-selectin, this indicates that there is a specific L-selectin binding domain on heparan sulfate glycosaminoglycan chains. Using an in vitro L-selectin binding assay, we studied the contribution of N-sulfation, O-sulfation, C5-epimerization, unsubstituted glucosamine residues, and chain length in L-selectin binding to heparan sulfate/heparin glycosaminoglycan chains. Based on our results and the accepted model of heparan sulfate domain organization, we propose a model for the interaction of L-selectin with heparan sulfate glycosaminoglycan chains. Interestingly, this opens the possibility of active regulation of L-selectin binding to heparan sulfate proteoglycans, e.g. under inflammatory conditions.     

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.     

Sulphated endothelial ligands for L-selectin in lymphocyte homing and inflammation

Lymphocytes from the blood home to secondary lymphoid tissues through a process of tethering, rolling, firm adhesion and transmigration. Tethering and rolling of lymphocytes is mediated by the interaction of L-selectin on lymphocytes with sulphated ligands expressed by the specialized endothelial cells of high endothelial venules (HEVs). The sulphate-dependent monoclonal antibody MECA79 stains HEVs in peripheral lymph nodes and recognizes the complex of HEV ligands for L-selectin termed peripheral node addressin. High endothelial cell GlcNAc-6-sulphotransferase/L-selectin ligand sulphotransferase is a HEV-expressed sulphotransferase that contributes to the formation of the MECA79 epitope and L-selectin ligands on lymph node HEVs. MECA79-reactive vessels are also common at sites of chronic inflammation, suggesting mechanistic parallels between lymphocyte homing and inflammatory trafficking.     

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.     

L-selectin and its biological ligands

This review considers the leukocyte adhesive receptor known as L-selectin. This protein, belonging to the selectin family of cell-cell adhesion receptors, mediates adhesion by virtue of a C-type lectin domain at its amino terminus. The protein was discovered as a lymphocyte homing receptor involved in the attachment of lymphocytes to high endothelial venules (HEV) of lymph nodes. Its widespread distribution on all leukocyte populations underlies a more general role in a variety of leukocyte-endothelial interactions. In the HEV interaction, cognate HEV ligands for L-selectin have been identified as two sulfated, sialylated, and fucosylated glycoproteins, known as GlyCAM-1 and Sgp90. These ligands have mucin-like domains which confer important properties for their proposed adhesive function. The carbohydrate features of these ligands, essential for their interaction with L-selectin, are reviewed. The existence of extralymphoid ligands for L-selectin is also discussed.Presented at the XXXV Symposium of the Society for Histochemistry, 29 September 1993, Gargellen, Austria     

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.     

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.     

Constitutive expression of glyCAM-1 core protein in the rat cochlea

Glycosylation-dependent cell adhesion molecule-1 (GIyCAM-1) is a mucin-like glycoprotein previously identified on high endothelial venules (HEV) of lymph nodes and also in lactating mammary glands. A specifically glycosilated form ofGIyCAM-1 on HEV has been shown to be a ligand for a leukocyte L-selectin, which plays an important role in leukocyte rolling along the inflamed endothelium. Here we report that GIyCAM-1 is also expressed in the cochlea. Immunohistochemistry revealed the lateral wall of the cochlea, tectorial membrane, modiolus, organ of Corti, and spiral modiolar vein (SMV) to be strongly stained with polyclonal anti-GIyCAM-1 antibody. Moreover, RT-PCR of the cochlear tissue by the use of specific oligonucleotide primers for rat GIyCAM-1 generated a 378 bp product which was then verified by nucleotide sequencing to represent GIyCAM-1. Electron microscopic investigation revealed the presence of GIyCAM-1 over the entire lumenal surface of the vessels, and the basolateral infoldings in stria vascularis. However, soluble L-selectin or mAb MEGA-79 which recognizes a carbohydrate epitope on functional L-selectin ligands bound only to the spiral ligament, tectorial membrane and modiolus. These observations suggest that GIyCAM-1 expressed in the cochlear region is heterogenous in terms of its glycosylation.     

Further characterization of the interaction between L-selectin and its endothelial ligands.

L-Selectin is a lectin-like receptor on lymphocytes which mediates their attachment to high endothelial venules (HEV) within lymph nodes. Previous work has identified HEV-associated endothelial ligands for L-selectin as sialylated, fucosylated and sulphated glycoproteins of approximately 50 kDa and approximately 90 kDa (Sgp50 and Sgp90). The interaction of L-selectin with these ligands is carbohydrate directed, reflecting the involvement of its amino-terminal, calcium-type lectin domain. It has been reported, and we have confirmed, that anti-Ly22 blocks the adhesive function of L-selectin without reducing its binding to a carbohydrate- based ligand PPME (phosphomannan monoester core from Hansenula hostii). The epitope for this monoclonal antibody depends on the epidermal growth factor (EGF) domain of L-selectin. We demonstrate that anti-Ly22 inhibits the interaction of L-selectin with both of the Sgps, thus establishing that the interaction of L-selectin with HEV can be accounted for by the Sgps. Furthermore, the interaction of trypsin fragments of Sgp50 with L-selectin is inhibitable both by an antibody that maps to the lectin domain and by anti-Ly22. These findings raise the possibility that anti-Ly22 is affecting the function of the lectin domain of L-selectin rather than directly antagonizing the EGF domain. Toward a further characterization of L-selectin's carbohydrate specificity, we show that Sgp50 is partially inactivated by the linkage-specific Newcastle Disease virus sialidase (alpha 2,3 linkage). We additionally demonstrate that a sialyl Lewis x-related tetrasaccharide can interact with L-selectin, as has also been demonstrated for E-selectin and P-selectin.     

Differential requirements for core2 glucosaminyltransferase for endothelial L-selectin ligand function in vivo

L-selectin is a calcium-dependent lectin on leukocytes mediating leukocyte rolling in high endothelial venules and inflamed microvessels. Many selectin ligands require modification of glycoproteins by leukocyte core2 beta1,6-N-acetylglucosaminyltransferase (Core2GlcNAcT-I). To test the role of Core2GlcNAcT-I for L-selectin ligand biosynthesis, we investigated leukocyte rolling in venules of untreated and TNF-alpha-treated cremaster muscles and in Peyer's patch high endothelial venules (HEV) of Core2GlcNAcT-I null (core2(-/-)) mice. In the presence of blocking mAbs against P- and E-selectin, L-selectin-mediated leukocyte rolling was almost completely abolished in cremaster muscle venules of core2(-/-) mice, but not littermate control mice. By contrast, leukocyte rolling in Peyer's patch HEV was not significantly different between core2(-/-) and control mice. To probe L-selectin ligands more directly, we injected L-selectin-coated beads. These beads showed no rolling in cremaster muscle venules of core2(-/-) mice, but significant rolling in controls. In Peyer's patch HEV, beads coated with a low concentration of L-selectin showed reduced rolling in core2(-/-) mice. Beads coated with a 10-fold higher concentration of L-selectin rolled equivalently in core2(-/-) and control mice. Our data show that endothelial L-selectin ligands relevant for rolling in inflamed microvessels of the cremaster muscle are completely Core2GlcNAcT-I dependent. In contrast, L-selectin ligands in Peyer's patch HEV are only marginally affected by the absence of Core2GlcNAcT-I, but are sufficiently functional to support L-selectin-dependent leukocyte rolling in Core2GlcNAcT-I-deficient mice.     

Subsets of sialylated, sulfated mucins of diverse origins are recognized by L-selectin. Lack of evidence for unique oligosaccharide sequences mediating binding

Previous studies have shown that the mucin-type polypeptidesGlyCAM-1, CD34, and MAdCAM-1 can function as ligands for L-selectinonly when they are synthesized by the specialized high-endothelialvenules (HEV) of lymph nodes. Since sialylation, sulfation,and possibly fucosylation are required for generating recognition,we reasoned that other mucins known to have such componentsmight also bind L-selectin. We show here that soluble mucinssecreted by human colon carcinoma cells, as well as those derivedfrom human bronchial mucus can bind to human L-selectin in acalcium-dependent manner. As with GlyCAM-1 synthesized by lymphnode HEY, 2–3 linked sialic acids and sulfation seem toplay a critical role in generating this L-selectin binding.In each case, only a subset of the mucin molecules is recognizedby L-selectin. Binding is not destroyed by boiling, suggestingthat recognition may be based primarily upon carbohydrate structures.Despite this, O-linked oligosaccharide chains released fromthese ligands by beta-elimination do not show any detectablebinding to L-selectin. Following protease treatment of the ligands,binding persists in a subset of the resulting fragments, indicatingthat specific recognition is determined by certain regions ofthe original mucins. How ever, O-linked oligosaccharides releasedfrom the subset of non-binding mucin fragments do not show verydifferent size and charge profiles compared to those that dobind. Furthermore, studies with polylactosamine-degrading endoglycosidasessuggest that the core structures involved in generating bindingcan vary among the different ligands. Taken together, thesedata indicate that a single unique oligosaccharide structuremay not be responsible for high-affinity binding. Rather, diversemucins with sialylated, sulfated, fucosylated lactosamine-typeO-linked oligosaccharides can generate high-affinity L-selectinligands, but only when they present these chains in unique spacingand/or clustered combinations, presumably dictated by the polypeptidebackbone. L-selectin mucins sialic sialic acid sulfate adhesion     

Comparison of L-selectin and E-selectin ligand specificities: the L-selectin can bind the E-selectin ligands sialyl Le(x) and sialyl Le(a).

The L- and E-selectins are leukocyte and endothelial cell surface molecules which mediate leukocyte-endothelial cell adhesion by interacting with carbohydrate ligands. In the present study we find that L-selectin, like E-selectin, can interact with synthetic neoglycoproteins containing Sialyl Le(x) (Neu5Ac alpha 2-3Gal beta 1-4[Fuc alpha 1-3]GlcNAc beta-R), or Sialyl Le(a) (Neu5Ac-alpha 2-3Gal beta 1-3[Fuc alpha 1-4]GlcNAc beta-R). Additionally, both the E-selectin and L-selectin can bind the peripheral lymph node addressin, a high endothelial venule ligand for L-selectin. Despite overlapping interactions, the L- and E-selectins discriminate between their native ligands. The peripheral lymph node addressin is a preferential ligand for L-selectin; and furthermore, L-selectin expressing cells do not interact detectably with the cutaneous lymphocyte antigen, a native glycoprotein ligand for E-selectin found on a subset of lymphocytes associated with the skin.     

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 G(i)-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.     

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.