The dual role of CD44 as a functional P-selectin ligand and fibrin receptor in colon carcinoma cell adhesion

Selectins and fibrin(ogen) play key roles in the hematogenous dissemination of tumor cells, and especially of colon carcinomas. However, the fibrin(ogen) receptor(s) on colon carcinoma cells has yet to be defined along with its relative capacity to bind fibrinogen versus fibrin under flow. Moreover, the functional P-selectin ligand has yet to be validated using intact platelets rather than purified selectin substrates. Using human CD44-knockdown and control LS174T cells, we demonstrate the pivotal involvement of CD44 in the P-selectin-mediated binding to platelets in shear flow. Quantitative comparisons of the binding kinetics of LS174T versus P-selectin glycoprotein ligand-1 (PSGL-1)-expressing THP-1 cells to activated platelets reveal that the relative avidity of P-selectin-CD44 binding is more than sevenfold lower than that of P-selectin-PSGL-1 interaction. Using CD44-knockdown LS174T cells and microspheres coated with CD44 immunoprecipitated from control LS174T cells, and purified fibrin(ogen) as substrate, we provide the first direct evidence that CD44 also acts as the major fibrin, but not fibrinogen, receptor on LS174T colon carcinoma cells. Interestingly, binding of plasma fibrin to CD44 on the colon carcinoma cell surface interferes with the P-selectin-CD44 molecular interaction and diminishes platelet-LS174T heteroaggregation in the high shear regime. Cumulatively, our data offer a novel perspective on the apparent metastatic potential associated with CD44 overexpression on colon carcinoma cells and the critical roles of P-selectin and fibrin(ogen) in metastatic spread and provide a rational basis for the design of new therapeutic strategies to impede metastasis.     

The Molecular Mechanics of P- and L-Selectin Lectin Domains Binding to PSGL-1

A laser trap was used to compare the load-dependent binding kinetics between truncated P- and L-selectin to their natural ligand, P-selectin glycoprotein ligand-1 (PSGL-1) over the predicted physiological range of loading rates. Human PSGL-1 was covalently coupled to polystyrene beads. Chimeric selectins were adsorbed to nitrocellulose-coated glass beads on a coverslip. A PSGL-1 bead was held in a laser trap and touched to a vertical surface of the glass bead, allowing a bond to form between selectin and ligand. The surface was moved away from the microsphere, applying load at a constant rate until bond rupture. Rupture force for both selectins increased with loading rate, but significant differences in rupture force between P- and L-selectin were observed only above 460 pN/s. These data are best represented as two energy barriers to unbinding, with the transition from the low to high loading rate regime at 260–290 pN/s. The data also allow the first estimate of a two-dimensional specific on-rate for binding of these two selectins to their natural ligand (1.7 μm²/s). These data suggest that P- and L-selectin lectin domains have very similar kinetics under physiological conditions.     

Selectin ligand expression regulates the initial vascular interactions of colon carcinoma cells: the roles of CD44v and alternative sialofucosylated selectin ligands

Selectin-mediated binding of tumor cells to platelets, leukocytes, and vascular endothelium may regulate their hematogenous spread in the microvasculature. We recently reported that CD44 variant isoforms (CD44v) on LS174T colon carcinoma cells possess selectin binding activity. Here we extended those findings by showing that T84 and Colo205 colon carcinoma cells bind selectins via sialidase-sensitive O-linked glycans presented on CD44v, independent of heparan and chondroitin sulfate. To assess the functional role of CD44v in selectin-mediated binding, we quantified the adhesion to selectins of T84 cell subpopulations sorted based on their CD44 expression levels and stable LS174T cell lines generated using CD44 short hairpin RNA. High versus low CD44-expressing T84 cells tethered more efficiently to P- and L-selectin, but not E-selectin, and rolled more slowly on P- and E-selectin. Knocking down CD44 expression on LS174T cells inhibited binding to P-selectin and increased rolling velocities over P- and L-selectin relative to control-transfected cells, without affecting tethering and rolling on E-selectin, however. Blot rolling analysis revealed the presence of alternative sialylated glycoproteins with molecular masses of approximately 170 and approximately 130 kDa, which can mediate selectin binding in CD44-knockdown cells. Heparin diminishes the avidity of colon carcinoma cells for P- and L-selectin, which may compromise integrin-mediated firm adhesion to host cells and mitigate metastasis. Our finding that CD44v is a functional P-selectin ligand on colon carcinoma provides a novel perspective on the enhanced metastatic potential associated with tumor CD44v overexpression and the role of selectins in metastasis.     

Replacing a lectin domain residue in L-selectin enhances binding to P-selectin glycoprotein ligand-1 but not to 6-sulfo-sialyl Lewis x

Selectin-ligand interactions (bonds) mediate leukocyte rolling on vascular surfaces. The molecular basis for differential ligand recognition by selectins is poorly understood. Here, we show that substituting one residue (A108H) in the lectin domain of L-selectin increased its force-free affinity for a glycosulfopeptide binding site (2-GSP-6) on P-selectin glycoprotein ligand-1 (PSGL-1) but not for a sulfated-glycan binding site (6-sulfo-sialyl Lewis x) on peripheral node addressin. The increased affinity of L-selectinA108H for 2-GSP-6 was due to a faster on-rate and to a slower off-rate that increased bond lifetimes in the absence of force. Rather than first prolonging (catching) and then shortening (slipping) bond lifetimes, increasing force monotonically shortened lifetimes of L-selectinA108H bonds with 2-GSP-6. When compared with microspheres bearing L-selectin, L-selectinA108H microspheres rolled more slowly and regularly on 2-GSP-6 at low flow rates. A reciprocal substitution in P-selectin (H108A) caused faster microsphere rolling on 2-GSP-6. These results distinguish molecular mechanisms for L-selectin to bind to PSGL-1 and peripheral node addressin and explain in part the shorter lifetimes of PSGL-1 bonds with L-selectin than P-selectin.     

P-selectin glycoprotein ligand-1 and E-selectin ligand-1 are differentially modified by fucosyltransferases Fuc-TIV and Fuc-TVII in mouse neutrophils

P-selectin glycoprotein ligand-1 (PSGL-1) and E-selectin ligand-1 (ESL-1) are the two major selectin ligands on mouse neutrophils. Transfection experiments demonstrate that each ligand requires alpha1,3-fucosylation for selectin-binding. However, the relative contributions made by the two known myeloid alpha1, 3-fucosyltransferases Fuc-TVII or Fuc-TIV to this alpha1, 3-fucosylation are not yet clear. To address this issue, we have used mice deficient in Fuc-TIV and/or Fuc-TVII to examine how these enzymes generate selectin-binding glycoforms of PSGL-1 and ESL-1 in mouse neutrophils. Selectin binding was analyzed by affinity isolation experiments using recombinant, antibody-like forms of the respective endothelial selectins. We observe essentially normal binding of E- or P-selectin to PSGL-1 expressed by Fuc-TIV-deficient neutrophils but find that PSGL-1 expressed by Fuc-TVII-deficient neutrophils is not bound by E- or P-selectin. By contrast, E-selectin binds with normal efficiency to ESL-1 on Fuc-TVII-deficient neutrophils but exhibits an 80% reduction in its ability to bind ESL-1 isolated from Fuc-TIV-deficient neutrophils. The same specificity with which Fuc-TVII and Fuc-TIV generate selectin-binding forms of PSGL-1 and ESL-1 was found in transfection experiments with CHO-Pro(-)5 cells. In contrast, each fucosyltransferase alone could generate selectin-binding glycoforms of each of the two ligands in CHO-DUKX-B1 cells. Our data imply that in mouse neutrophils and their precursors, Fuc-TVII exclusively directs expression of PSGL-1 glycoforms bound with high affinity by P-selectin. By contrast, Fuc-TIV preferentially directs expression of ESL-1 glycoforms that exhibit high affinity for E-selectin. This substrate specificity can be mimicked in CHO-Pro(-)5 cells.     

Low force decelerates L-selectin dissociation from P-selectin glycoprotein ligand-1 and endoglycan

Selectin-ligand interactions mediate the tethering and rolling of circulating leukocytes on vascular surfaces during inflammation and immune surveillance. To support rolling, these interactions are thought to have rapid off-rates that increase slowly as wall shear stress increases. However, the increase of off-rate with force, an intuitive characteristic named slip bonds, is at odds with a shear threshold requirement for selectin-mediated cell rolling. As shear drops below the threshold, fewer cells roll and those that do roll less stably and with higher velocity. We recently demonstrated a low force regime where the off-rate of P-selectin interacting with P-selectin glycoprotein ligand-1 (PSGL-1) decreased with increasing force. This counter-intuitive characteristic, named catch bonds, might partially explain the shear threshold phenomenon. Because L-selectin-mediated cell rolling exhibits a much more pronounced shear threshold, we used atomic force microscopy and flow chamber experiments to determine off-rates of L-selectin interacting with their physiological ligands and with an antibody. Catch bonds were observed at low forces for L-selectin-PSGL-1 interactions coinciding with the shear threshold range, whereas slip bonds were observed at higher forces. These catch-slip transitional bonds were also observed for L-selectin interacting with endoglycan, a newly identified PSGL-1-like ligand. By contrast, only slip bonds were observed for L-selectin-antibody interactions. These findings suggest that catch bonds contribute to the shear threshold for rolling and are a common characteristic of selectin-ligand interactions.     

Characterization of glycoprotein ligands for P-selectin on a human small cell lung cancer cell line NCI-H345.

P-selectin (CD62P) is a cell adhesion molecule expressed on stimulated endothelial cells and on activated platelets. It interacts with PSGL-1 (P-selectin glycoprotein ligand-1; CD162) on leukocytes and mediates recruitment of leukocytes during inflammation. P-selectin also binds to several types of cancer cells in vitro and facilitates growth and metastasis of colon carcinoma in vivo. Here we show that P-selectin, but not E-selectin, binds to NCI-H345 cells, a cell line derived from a human small cell lung cancer. EDTA or P7 (a leukocyte adhesion blocking mAb to P-selectin), but not PL5 (a leukocyte adhesion blocking mAb to PSGL-1), can inhibit this binding. P-selectin affinity chromatography can precipitate a approximately 110-kDa major band and a approximately 220-kDa minor band from [3H]-glucosamine-labeled NCI-H345 cells. No expression of PSGL-1 protein and mRNA can be detected in NCI-H345 cells. Taken together, these results suggest that NCI-H345 cells express glycoprotein ligands for P-selectin that are distinct from leukocyte PSGL-1.     

Enhancement of L-selectin, but not P-selectin, bond formation frequency by convective flow

L-selectin-mediated leukocyte rolling has been proposed to require a high rate of bond formation compared to that of P-selectin to compensate for its much higher off-rate. To test this hypothesis, a microbead system was utilized to measure relative L-selectin and P-selectin bond formation rates on their common ligand P-selectin glycoprotein ligand-1 (PSGL-1) under shear flow. Using video microscopy, we tracked selectin-coated microbeads to detect the formation frequency of adhesive tether bonds. From velocity distributions of noninteracting and interacting microbeads, we observed that tether bond formation rates for P-selectin on PSGL-1 decreased with increasing wall shear stress, from 0.14 ± 0.04 bonds/μm at 0.2 dyn/cm² to 0.014 ± 0.003 bonds/μm at 1.0 dyn/cm². In contrast, L-selectin tether bond formation increased from 0.017 ± 0.005 bonds/μm at 0.2 dyn/cm² to 0.031 ± 0.005 bonds/μm at 1.0 dyn/cm². L-selectin tether bond formation rates appeared to be enhanced by convective transport, whereas P-selectin rates were inhibited. The transition force for the L-selectin catch-slip transition of 44 pN/bond agreed well with theoretical models (Pereverzev et al. 2005. Biophys. J. 89:1446-1454). Despite catch bond behavior, hydrodymanic shear thresholding was not detected with L-selectin beads rolling on PSGL-1. We speculate that shear flow generated compressive forces may enhance L-selectin bond formation relative to that of P-selectin and that L-selectin bonds with PSGL-1 may be tuned for the compressive forces characteristic of leukocyte-leukocyte collisions during secondary capture on the blood vessel wall. This is the first report, to our knowledge, comparing L-selectin and P-selectin bond formation frequencies in shear flow.     

Nano- to microscale dynamics of P-selectin detachment from leukocyte interfaces. I. Membrane separation from the cytoskeleton

We have used a biomembrane force probe decorated with P-selectin to form point attachments with PSGL-1 receptors on a human neutrophil (PMN) in a calcium-containing medium and then to quantify the forces experienced by the attachment during retraction of the PMN at fixed speed. From first touch to final detachment, the typical force history exhibited the following sequence of events: i), an initial linear-elastic displacement of the PMN surface, ii), an abrupt crossover to viscoplastic flow that signaled membrane separation from the interior cytoskeleton and the beginning of a membrane tether, and iii), the final detachment from the probe tip by usually one precipitous step of P-selectin:PSGL-1 dissociation. In this first article I, we focus on the initial elastic response and its termination by membrane separation from the cytoskeleton, initiating tether formation. Quantifying membrane unbinding forces for rates of loading (force/time) in the elastic regime from 240 pN/s to 38,000 pN/s, we discovered that the force distributions agreed well with the theory for kinetically limited failure of a weak bond. The kinetic rate for membrane unbinding was found to increase as an exponential function of the pulling force, characterized by an e-fold scale in force of approximately 17 pN and a preexponential factor, or apparent unstressed off rate, of approximately 1/s. The rheological properties of tether growth subsequent to the membrane unbinding events are presented in a companion article II.     

P-selectin glycoprotein ligand-1 mediates rolling of human neutrophils on P-selectin

Neutrophils roll on P-selectin expressed by activated platelets or endothelial cells under the shear stresses in the microcirculation. P- selectin glycoprotein ligand-1 (PSGL-1) is a high affinity ligand for P- selectin on myeloid cells. However, it has not been demonstrated that PSGL-1 contributes to the rolling of neutrophils on P-selectin. We developed two IgG mAbs, PL1 and PL2, that appear to recognize protein- dependent epitopes on human PSGL-1. The mAbs bound to PSGL-1 on all leukocytes as well as on heterologous cells transfected with PSGL-1 cDNA. PL1, but not PL2, blocked binding of 125-I-PSGL-1 to immobilized P-selectin, binding of fluid-phase P-selectin to myeloid and lymphoid leukocytes, adhesion of neutrophils to immobilized P-selectin under static conditions, and rolling of neutrophils on P-selectin-expressing CHO cells under a range of shear stresses. PSGL-1 was localized to microvilli on neutrophils, a topography that may facilitate its adhesive function. These data indicate that (a) PSGL-1 accounts for the high affinity binding sites for P-selectin on leukocytes, and (b) PSGL- 1 must interact with P-selectin in order for neutrophils to roll on P- selectin at physiological shear stresses.     

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.     

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.     

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.     

Molecular dynamics simulation of shear- and stretch-induced dissociation of P-selectin/PSGL-1 complex

By mediating the tethering and rolling of leukocytes on vascular surfaces, the interactions between P-selectin and the P-selectin glycoprotein ligand 1 (PSGL-1) play crucial roles during inflammation cascade. Tensile stretch produced by rolling leukocytes and shear stress exerted by blood flow constitute the two types of mechanical forces that act on the P-selectin/PSGL-1 bond. These forces modulate not only dissociation kinetics of this bond, but also the leukocyte adhesion dynamics. However, the respective contribution of the two forces to bond dissociation and to the corresponding microstructural bases remains unclear. To mimic the mechanical microenvironment, we developed two molecular dynamics approaches; namely, an approach involving the shear flow field with a controlled velocity gradient, and the track dragging approach with a defined trajectory. With each approach or with both combined, we investigate the microstructural evolution and dissociation kinetics of the P-LE/SGP-3 construct, which is the smallest functional unit of the P-selectin/PSGL-1 complex. The results demonstrate that both shear flow and tensile stretch play important roles in the collapse of the construct and that, before bond dissociation, the former causes more destruction of domains within the construct than the latter. Dissociation of the P-LE/SGP-3 construct features intramolecular destruction of the epidermal-growth-factor (EGF) domain and the breaking of hydrogen-bond clusters at the P-selectin-lectin/EGF interface. Thus, to better understand how mechanics impacts the dissociation kinetics of the P-selectin/PSGL-1 complex, we propose herein two approaches to mimic its physiological mechanical environment.     

Comparison of PSGL-1 microbead and neutrophil rolling: microvillus elongation stabilizes P-selectin bond clusters

A cell-scaled microbead system was used to analyze the force-dependent kinetics of P-selectin adhesive bonds independent of micromechanical properties of the neutrophil's surface microvilli, an elastic structure on which P-selectin ligand glycoprotein-1 (PSGL-1) is localized. Microvillus extension has been hypothesized in contributing to the dynamic range of leukocyte rolling observed in vivo during inflammatory processes. To evaluate PSGL-1/P-selectin bond kinetics of microbeads and neutrophils, rolling and tethering on P-selectin-coated substrates were compared in a parallel-plate flow chamber. The dissociation rates for PSGL-1 microbeads on P-selectin were briefer than those of neutrophils for any wall shear stress, and increased more rapidly with increasing flow. The microvillus length necessary to reconcile dissociation constants of PSGL-1 microbeads and neutrophils on P-selectin was 0.21 microm at 0.4 dyn/cm2, and increased to 1.58 microm at 2 dyn/cm2. The apparent elastic spring constant of the microvillus ranged from 1340 to 152 pN/microm at 0.4 and 2.0 dyn/cm2 wall shear stress. Scanning electron micrographs of neutrophils rolling on P-selectin confirmed the existence of micrometer-scaled tethers. Fixation of neutrophils to abrogate microvillus elasticity resulted in rolling behavior similar to PSGL-1 microbeads. Our results suggest that microvillus extension during transient PSGL-1/P-selectin bonding may enhance the robustness of neutrophil rolling interactions.     

Glycosulfopeptides with O-glycans containing sialylated and polyfucosylated polylactosamine bind with low affinity to P-selectin

P-selectin glycoprotein ligand-1 (PSGL-1), a dimeric mucin on leukocytes, is the best characterized ligand for selectins. P-selectin binds stereospecifically to the extreme N terminus of PSGL-1, which contains three clustered tyrosine sulfates (TyrSO3-) adjacent to a Thr residue with a core 2-based O-glycan expressing sialyl Lewis x (C2-O-sLe(x)). GSP-6, a synthetic glycosulfopeptide modeled after the N terminus of PSGL-1, containing three TyrSO3- residues and a short, monofucosylated C2-O-sLe(x) bound to P-selectin with high affinity (K(d) approximately 650 nm). However, PSGL-1 from human HL-60 cells contains higher levels of O-glycans that are sialylated and polyfucosylated polylactosamines (PFPL). Furthermore, studies with fucosyltransferase-deficient mice suggest that sialylated PFPL structures contribute to binding to P-selectin. To resolve whether sialylated PFPL O-glycans participate in binding of PSGL-1 to human P-selectin, we synthesized glycosulfopeptides, designated GSP-6' and GSP-6", with three TyrSO3- residues and either difucosylated polylactosamine (C2-O-Le(x)-sLe(x)) or trifucosylated polylactosamine (C2-O-Le(x)-Le(x)-sLe(x)). Binding of the GSPs to P-selectin was measured by affinity chromatography, fluorescence solid-phase assays, and equilibrium gel filtration. Unexpectedly, both GSP-6' and GSP-6" bound to P-selectin with low affinity (K(d) approximately 37 microm for GSP-6' and K(d) approximately 50 microm for GSP-6"). Binding of GSP-6' and GSP-6" to P-selectin required fucosylation and, to a lesser extent, sialylation as well as the sulfated peptide backbone of GSP-6' and GSP-6". These results demonstrate that contrary to expectations, a core 2 O-glycan containing sialylated PFPL does not promote high affinity binding of PSGL-1 to P-selectin.     

Direct observation of membrane tethers formed during neutrophil attachment to platelets or P-selectin under physiological flow

Adhesion and subsequent aggregation between neutrophils and platelets is dependent upon the initial binding of P-selectin on activated platelets to P-selectin glycoprotein ligand 1 (PSGL-1) on the microvilli of neutrophils. High speed, high resolution videomicroscopy of flowing neutrophils interacting with spread platelets demonstrated that thin membrane tethers were pulled from neutrophils in 32 +/- 4% of the interactions. After capture by spread platelets, neutrophil membrane tethers (length of 5.9 +/- 4.1 microm, n = 63) were pulled at an average rate of 6-40 microm/s as the wall shear rate was increased from 100-250 s(-1). The average tether lifetime decreased significantly (P < 0.001) from 630 to 133 ms as the shear rate was increased from 100 s(-1) (F(bond) = 86 pN) to 250 s(-1) (F(bond) = 172 pN), which is consistent with P-selectin/PSGL-1 bond dynamics under stress. Tether formation was blocked by antibodies against P-selectin or PSGL-1, but not by anti-CD18 antibodies. During neutrophil rolling on P-selectin at 150 s(-1), thin membrane tethers were also pulled from the neutrophils. The characteristic jerking motion of the neutrophil coexisted with tether growth (8.9 +/- 8.8 microm long), whereas tether breakage (average lifetime of 3.79 +/- 3.32 s) caused an acute jump in the rolling velocity, proving multiple bonding in the cell surface and the tether surface contact area. Extremely long membrane tethers (>40 microm) were sometimes pulled, which detached in a flow-dependent mechanism of microparticle formation. Membrane tethers were also formed when neutrophils were perfused over platelet monolayers. These results are the first visualization of the often hypothesized tethers that shield the P-selectin/PSGL-1 bond from force loading to regulate neutrophil rolling during inflammation and thrombosis.     

Carcinoembryonic antigen and CD44 variant isoforms cooperate to mediate colon carcinoma cell adhesion to E- and L-selectin in shear flow

Selectin-mediated adhesion of tumor cells to platelets, leukocytes, and endothelial cells may regulate their hematogenous dissemination in the microvasculature. We recently identified CD44 variant isoforms (CD44v) as functional P-, but not E- or L-, selectin ligands on colon carcinoma cells. Moreover, an approximately 180-kDa sialofucosylated glycoprotein(s) mediated selectin binding in CD44-knockdown cells. Using immunoaffinity chromatography and tandem mass spectrometry, we identify this glycoprotein as the carcinoembryonic antigen (CEA). Blot rolling assays and flow-based adhesion assays using microbeads coated with CEA immunopurified from LS174T colon carcinoma cells and selectins as substrate reveal that CEA possesses E- and L-, but not P-, selectin ligand activity. CEA on CD44-knockdown LS174T cells exhibits higher HECA-452 immunoreactivity than CEA on wild-type cells, suggesting that CEA functions as an alternative acceptor for selectin-binding glycans. The enhanced expression of HECA-452 reactive epitopes on CEA from CD44-knockdown cells correlates with the increased CEA avidity for E- but not L-selectin. Through the generation of stable knockdown cell lines, we demonstrate that CEA serves as an auxiliary L-selectin ligand, which stabilizes L-selectin-dependent cell rolling against fluid shear. Moreover, CEA and CD44v cooperate to mediate colon carcinoma cell adhesion to E- and L-selectin at elevated shear stresses. The novel finding that CEA is an E- and L-selectin ligand may explain the enhanced metastatic potential associated with tumor cell CEA overexpression and the supportive role of selectins in metastasis.     

HCELL is the major E- and L-selectin ligand expressed on LS174T colon carcinoma cells

Engagement of vascular E-selectin and leukocyte L-selectin with relevant counter-receptors expressed on tumor cells contributes to the hematogenous spread of colon carcinoma. We recently demonstrated that the LS174T colon carcinoma cell line expresses the CD44 glycoform known as hematopoietic cell E-/L-selectin ligand (HCELL), which functions as a high affinity E- and L-selectin ligand on these cells. To define the contribution of HCELL to selectin-mediated adhesion on intact tumor cells, we measured the binding of LS174T cells transduced with CD44 short interfering RNA (siRNA) or with vector alone to 6-h interleukin-1beta-stimulated human umbilical vein endothelial cells (HUVEC) and to human peripheral blood mononuclear cells (PBMC) under physiological flow conditions. LS174T cell attachment to HUVEC was entirely E-selectin-dependent, and PBMC tethering to tumor cell monolayers was completely L-selectin-dependent. At physiological shear stress, CD44 siRNA transduction led to an approximately 50% decrease in the number of LS174T cells binding to stimulated HUVEC relative to vector alone-transduced cells. CD44 siRNA-transduced cells also rolled significantly faster than vector-transduced cells on HUVEC, indicating prominent HCELL participation in stabilizing tumor cell-endothelial adhesive interactions against fluid shear. Furthermore, HCELL was identified as the principal L-selectin ligand on LS174T cells, as PBMC binding to CD44 siRNA-transduced tumor cells was reduced approximately 80% relative to vector-transduced cells. These data indicate that expression of HCELL confers robust and predominant tumor cell binding to E- and L-selectin, highlighting a central role for HCELL in promoting shear-resistant adhesive interactions essential for hematogenous cancer dissemination.     

Fluid shear- and time-dependent modulation of molecular interactions between PMNs and colon carcinomas

This study compares the effects offluid shear on the kinetics, adhesion efficiency, stability, andmolecular requirements of polymorphonuclear leukocyte (PMN) binding totwo colon adenocarcinoma cell-lines, theCD54-negative/sLe^x-bearing LS174T cells and theCD54-expressing/sLe^x-low HCT-8 cells. The efficiency ofPMN-colon carcinoma heteroaggregation decreases with increasing shear,with PMNs binding HCT-8 more efficiently than LS174T cells at low shear(50-200 s¹). In the low shear regime, CD11b issufficient to mediate PMN binding to LS174T cells. In contrast, bothCD11a and CD11b contribute to PMN-HCT-8 heteroaggregation, with CD54 onHCT-8 cells acting as a CD11a ligand at early time points. At highshear, only PMN-LS174T heteroaggregation occurs, which is initiated byPMN L-selectin binding to a sialylated, O-linked, protease-sensitiveligand on LS174T cells. PMN-LS174T heteroaggregation is primarilydependent on the intercellular contact duration (or shear rate),whereas PMN-HCT-8 binding is a function of both the intercellularcontact duration and the applied force (or shear stress). Cumulatively, these studies suggest that fluid shear modulates the kinetics andmolecular mechanisms of PMN-colon carcinoma cell aggregation.