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The Journal of Immunology, 2004, 172: 7780-7790.
Copyright © 2004 by The American Association of Immunologists

Shear-Dependent Capping of L-Selectin and P-Selectin Glycoprotein Ligand 1 by E-Selectin Signals Activation of High-Avidity {beta}2-Integrin on Neutrophils1

Chad E. Green*, David N. Pearson*, Raymond T. Camphausen2,{dagger}, Donald E. Staunton{ddagger} and Scott I. Simon3,*

* Department of Biomedical Engineering, University of California, Davis, CA 95616; {dagger} Wyeth Research, Cambridge, MA 02140; and {ddagger} ICOS, Bothell, WA 98021


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Two adhesive events critical to efficient recruitment of neutrophils at vascular sites of inflammation are up-regulation of endothelial selectins that bind sialyl Lewisx 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 Lewisx 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.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Selectins are a family of C-type lectin glycoproteins that initiate recruitment of leukocytes from the blood stream on inflamed endothelium (1). L-selectin expressed on leukocytes and E- and P-selectin up-regulated on inflamed endothelium function as cell-tethering molecules by binding to sialylated and fucosylated oligosaccharides on the plasma membrane. The carbohydrate most commonly recognized by the lectin domain of selectins is the tetrasaccharide sialyl Lewisx (sLex) 4 (2). During the multistep process of leukocyte adhesion, selectin-mediated cell rolling is thought to promote ligation of endothelial-bound chemotactic molecules, leading to the rapid activation of {beta}2-integrins and cell arrest (3). Intravital microscopy and parallel-plate flow chamber (PPFC) studies corroborate that leukocyte recruitment occurs as a continuum of binding events initiated by rolling via selectins and braking by engagement of activated integrins. Conventional wisdom suggests that G-protein-coupled receptor engagement of inflammatory mediators (i.e., IL-8, platelet-activating factor) presented on the endothelial glycocalyx is the primary recognition event signaling stable adhesion of neutrophils via subsecond activation of {beta}2-integrins (4). Emerging evidence implicates a second means of leukocyte activation that involves selectins functioning as mechano-chemical transducers of signals that effect rapid activation of {beta}2-integrins (5, 6).

E-selectin up-regulated to the apical endothelial surface after cytokine activation remains dispersed until leukocyte-endothelial adhesive interactions induce association with the cytoskeleton and clustering at cell-cell junctions (7). Through binding to glycosylated lipids and sialylated carbohydrates on leukocytes, E-selectin supports rolling at rates on the order of a leukocyte diameter per second, an order of magnitude slower than that of L- or P-selectin (8, 9). These rolling interactions rapidly transition to CD18-dependent adhesion and directed migration of neutrophils on E-selectin substrates such as activated HUVECs (6, 10). E-selectin ligands identified on human neutrophils include L-selectin and P-selectin glycoprotein ligand 1 (PSGL-1), which are constitutively expressed on the tips of microvilli and are decorated with sialylated and fucosylated glycans, including sLex (11, 12, 13, 14). E-selectin ligand-1 has been characterized on murine neutrophils; however, a homologue on human neutrophils has yet to be identified (15). When bound by ligand, L-selectin and PSGL-1 also function as signaling receptors. A number of reports have demonstrated leukocyte activation in response to cross-linking of L-selectin, which triggers calcium release, activation of mitogen-activated protein kinase (MAPK) signaling pathways, neutrophil shape change, degranulation, and rapid membrane up-regulation of {beta}2-integrin expression and cell adhesion (16, 17, 18, 19). In particular, L-selectin ligation and clustering at a single site of membrane contact involving few microvilli provides for local and rapid recruitment of activated {beta}2-integrin and efficient conversion from neutrophil rolling to arrest in shear flow (16). Likewise, Ab cross-linking of PSGL-1 or ligation with soluble P-selectin potentiates CD18-dependent adhesion via activation of the Ras and MAPK signaling pathways in neutrophils (20).

We have recently reported that neutrophil rolling on cell monolayers expressing E-selectin and ICAM-1 activates {beta}2-integrins and neutrophil arrest, which was independent of signaling via G protein-coupled receptors (6). Signaling involved E-selectin recognition of L-selectin and PSGL-1, which triggered p38 and p42/44 MAPK activity. The detailed mechanism for this shear-dependent recognition and signaling remains unresolved. For example, it is unclear how E-selectin recognition of L-selectin and PSGL-1 alters their membrane distribution and initiates affinity and avidity shifts in CD18.

In the current study, we examine the molecular dynamics of adhesion receptors at the neutrophil membrane upon E-selectin binding. We show that E-selectin preferentially recognizes sLex on L-selectin and PSGL-1 and signals active transport of adhesion molecules, leading to rapid arrest of neutrophils in shear flow. Binding of E-selectin promoted colocalization of L-selectin and PSGL-1 into membrane clusters on microvilli, an event that temporally correlated with MAPK phosphorylation and focal clustering of high-affinity CD18. Phosphorylation of p38 and p42/44 MAPK was required for coclustering of L-selectin and PSGL-1, as well as clustering of high-affinity CD18. Together, the data reveal a mechanism by which neutrophil rolling on E-selectin and macromolecular assembly of L-selectin and PSGL-1 culminate in activation of CD18.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Isolation of neutrophils

Venous blood was collected from healthy adult donors by venipuncture into a sterile syringe containing heparin (Elkins-Sinn, Cherry Hill, NJ). Neutrophils were isolated from whole blood using a one-step Ficoll-Paque density gradient medium (Robbins Scientific, Sunnyvale, CA), as previously described (21). Cells were kept at 4°C in a Ca2+-free HEPES buffer (110 mM NaCl, 10 mM KCl, 10 mM glucose, 1 mM MgCl2, and 30 mM HEPES (pH 7.4)). CaCl2 was added to the buffer at 1.5 mM final concentration before analysis. By this method, neutrophils were found to remain viable and unactivated for ~4 h after separation.

Cell culture and reagents

Transfected L-cells expressing human E-selectin (L-E) or coexpressing human E-selectin and ICAM-1 (L-E/I), as previously described, were maintained in a modified RPMI 1640 medium (Invitrogen, San Diego, CA) (6). Transfected Chinese hamster ovary (CHO) cells expressing L-selectin ligand sulfotransferase, core 2 {beta}1,6-N-acetylglucosaminyltransferase, {alpha}1,3-fucosyltransferase, and CD34, as previously described and generously provided by Minoru Fukuda (The Burnham Institute, La Jolla, CA), were maintained in modified {alpha}-MEM (Invitrogen) (22). HUVECs were purchased from Cascade Biologics (Portland, OR) as proliferating cultures and were maintained in supplemented Medium 200 (Cascade Biologics). All cultures were grown in a 37°C humidified atmosphere containing 5% CO2, reaching confluence in 3–5 days. For static and shear adhesion assays, cells were passaged onto 35-mm glass coverslips (Belco, Vineland, NJ) and were grown for 2–3 days before adhesion assays.

Agonists, inhibitors, and Abs

Human IL-8 and TNF-{alpha} were from R&D Systems (Minneapolis, MN). Humanized IgG4 form of DREG55 was provided by Protein Design Labs (Mountain View, CA) (23). Mouse anti-PSGL-1 Abs KPL-1 (BD PharMingen, San Diego, CA) and PL-1 (SeroTech, Raleigh, NC) were used for all experiments. Recombinant chimeric human E-selectin (E-sel-IgG) and L-selectin (L-sel-IgG) were purchased from GlycoTech (Rockville, MD). Recombinant chimeric human P-selectin (P-sel-IgG) and chimeric human E-selectin forms E-LE-Fc, E-LE and Fc containing the lectin, epidermal growth factor-like domain (EGF), and/or IgG Fc domains were previously described (24, 25). FITC anti-CD43 and PE anti-L-selectin were purchased from Caltag Laboratories (Burlingame, CA). The specific inhibitors of p38 MAPK (SB-202190) and p42/44 MAPK (PD98059) were purchased from Biomol Research Laboratories (Plymouth Meeting, PA). The sialyl di-Lewisx mimetic inhibitor TBC 1269 (1,6-Bis[3-(3-carboxymethylphenyl)-4-(2-{alpha}-D-mannopyranosyloxy)phenyl]hexane) and the control compound TBC 1900 were gifts from Texas Biotechnology (Houston, TX) (26). PE-conjugated anti-human Mac-1 (2LPM19c) was purchased from DAKO (Carpinteria, CA). Alexa-488 (Molecular Probes, Eugene, OR) conjugated mAb 327C was previously described (27, 28). Monoclonal Ab 327C binds to a neoepitope up-regulated on activated neutrophils and correlates with expression of the high-affinity, ligand-binding state of the {beta}2-integrin I-domain.

Flow cytometric detection of neutrophil activation and adhesion

Neutrophils (106/ml) were preincubated with Fc (4.2 µM), E-LE (4.2 µM), E-LE-Fc (1.2 µM), or E-sel-IgG (33 nM) and then were stimulated by addition of a secondary goat anti-human IgG (H+L) F(ab')2 (20 µg/ml) or fMLP (1 µM) at 37°C. Cells were labeled with Alexa488-327C (10 µg/ml) and PE anti-Mac-1 (10 µg/ml) before flow cytometry (FACScan; BD Biosciences, Mountain View, CA). CD11b/CD18 adhesion was detected using an albumin-coated latex bead (ACLB) assay, as previously described (21). Briefly, 2-µm fluorescent latex beads (Molecular Probes) were coated with albumin and sheared with neutrophils at a ratio of 40:1 ACLBs to cells in a mixing chamber placed upstream from the flow cytometer (29). To stimulate neutrophil adhesion, cells were preincubated with 10 µg/ml anti-L-selectin and 10 µg/ml anti-PSGL-1 or E-sel-IgG and then were stimulated by addition of a secondary goat anti-human IgG (H+L) F(ab')2 (20 µg/ml) at 37°C. For chemotactic stimulation, 1 µM fMLP was added just before application of shear. Events were acquired continuously at a resolution of 100–200 particles/s at a flow rate of 15 µl/min for up to 10 min after stimulation. Neutrophil-bead adhesion was represented by quantal increases in fluorescence corresponding to populations of neutrophils binding increasing numbers of beads (29). Neutrophil-bead interactions were quantitated as the average number of beads per neutrophil according to the following equation:

where n represents the number of nonadherent neutrophils and NB represents the number of neutrophil-bead aggregates bound to between one and six or more beads.

Neutrophil adhesion assays

Neutrophil adhesion under flow conditions was measured in a PPFC, as described previously (30). Isolated neutrophils (106/ml) were passed over confluent HUVEC, L-cell, and CHO cell monolayers on 35-mm coverslips at a shear stress of 1.0 dynes/cm2 for a total interaction period of 1 min at 37°C. HUVECs were cultured on coverslips precoated with glutaraldehyde-cross-linked gelatin and were stimulated with 200 U/ml TNF-{alpha} 4 h before an adhesion assay. For sLex inhibition, L-E/I monolayers were preincubated with TBC1269 or TBC1900 at 225 µM for 10 min at 23°C before mounting on the PPFC. Digital image sequences were acquired using an enclosed inverted microscope (TE200; Nikon, Melville, NY) equipped with a 20x phase contrast objective (numerical aperture = 0.45), an analog charge-coupled device camera (Dage-MTI, Michigan City, IN), and a digital frame grabber (Scion, Frederick, MD). Image sequences were captured and analyzed using Image Pro Plus v4.5 (Media Cybernetics, Silver Spring, MD). A temperature-controlled Lucite box surrounding the microscope and flow chamber assured that all flow experiments were done at 37°C. Arrested neutrophils were defined as those neutrophils that moved less than one cell diameter in 30 s. Rolling cells were defined as having moved greater that one cell diameter in 30 s, with velocity computed as the distance rolled divided by the period of time a cell traversed the field of view. The fractions of rolling and arrested cells were determined by normalizing to the total number of neutrophils interacting with the cell monolayer, both adherent and rolling, present in the field of view. Interacting cells were defined as those moving with a velocity ~10-fold less than the free stream velocity in the same plane of focus as the substrate monolayer. Adhesion under static conditions was observed by pipetting neutrophil suspensions directly onto 35-mm glass coverslips coated with HUVEC, L-cell, or CHO cell monolayers at 37°C for 1 min before fixation in 1% paraformaldehyde.

Immunofluorescence microscopy

To observe colocalization of L-selectin and PSGL-1 on suspended cells, neutrophils (106/ml) were preincubated with 33 nM E-sel-IgG, 10 µg/ml L-sel-IgG, 10 µg/ml P-sel-IgG, 10 µg/ml human IgG and then were stimulated by incubation at 37°C for 1 min. Cells were labeled with PE-anti-PSGL-1 and FITC-humanized DREG55 at 4°C before fixation in 1% paraformaldehyde. No difference was observed between neutrophil suspensions labeled either before or after fixation. For MAPK inhibition, neutrophils (106/ml) were preincubated with either 10 µM SB202190 or 100 µM PD98059 at 37°C for 45 min. To observe colocalization of L-selectin and PSGL-1 in static and shear flow adhesion assays, coverslips were removed from the adhesion chambers and labeled at 4°C with FITC-humanized DREG55, PE-anti-PSGL-1, or Alexa488-327C, as denoted, before washing and fixation in 1% paraformaldehyde. Fixed coverslips were imaged by fluorescence microscopy using a Nikon TE2000-S inverted microscope using a 60x Plan Apo objective (numerical aperture = 1.4) under oil and a Sutter filter wheel (Sutter Instrument Company, Novato, CA) housing excitation filters appropriate for FITC, PE, and Alexa488 fluorophores. Images were captured with an ORCA digital charge-coupled device camera (Hamamatsu Photonics, Hamamatsu City, Japan) and Simple PCI acquisition software (Compix, Cranberry Township, PA). Confocal microscopy of adherent neutrophils was performed using a Zeiss LSM 510 Meta confocal microscope (Carl Zeiss, Thornwood, NY) using a 63x Plan Apo objective (numerical aperture = 1.2) under water and LSM 510 acquisition software (Carl Zeiss). All fluorescent and confocal images were analyzed and processed using Image Pro Plus v4.5 software (Media Cybernetics, Silver Spring, MD). For quantitation of receptor topography and colocalization, a cluster was defined as a localized region of the membrane in which pixel intensity is at least 3-fold greater than background fluorescent intensity. Pixel intensity values are unitless and range from 0 to 255. Based on these values and the image of the cell, a threshold intensity value was chosen that represents the average background intensity over the surface. This value typically ranges from 80 to 140, with clusters achieving off-scale values that are assigned a maximum intensity value of 255 by default. After thresholding on the background fluorescence, the number of fluorescent clusters (frequency), surface area per cluster, and percent colocalization were determined.

Western analysis of p38 and p42/44 kinases

Neutrophils (2 x 107 cells) were preincubated with E-Sel-IgG (10 µg/ml) or anti-L-selectin (DREG56) (10 µg/ml) and anti-PSGL-1 (PL-1) (10 µg/ml) and then were stimulated by addition of a secondary goat anti-mouse F(ab')2 or TNF-{alpha} (0.1 ng/ml) at 37°C for 7 min. Cells were removed, centrifuged, aspirated, frozen in liquid N2, and stored at –70°C. The cell extracts were lysed in buffer containing 50 mM HEPES (pH 7.5), 1% (v/v) Triton X-100, 2 mM sodium orthovanadate, 10 mM NaF, 1 mM EGTA, 2x protease mixture inhibitors (Boehringer Mannheim, Indianapolis, IN), and 1 mM PMSF for 1 h and then were centrifuged and frozen at –70°C. Protein extracts were separated by 10% (p38) and 12% (p42/44) SDS-PAGE loaded with 15–60 µg of protein. After electrophoresis, protein was transferred to polyvinylidene difluoride membrane and probed with rabbit antiserum (Cell Signaling Technology, Beverly, MA) against either total MAPK or the dual-phosphorylated forms of p38 (Thr180/Tyr182) and p42/44 (Thr202/Tyr204), respectively. The membranes were then developed using Avidix-APTM and Western-Light Plus (Applied Biosystems, Foster City, CA) before analysis using NIH Image (1.62).

Statistical analysis

Analysis of data was performed using GraphPad Prism version 4.0 software (Graphpad Software, San Diego, CA). All data are reported as mean ± SD. Nonparametric groupings of data were analyzed by ANOVA, the Newman-Kuel multiple comparison test, and the Tukey multiple comparison test. Group comparisons were deemed significant for p < 0.05.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Neutrophil arrest on HUVECs and E-selectin/ICAM-1 L-cells in shear flow

A PPFC assembled above an inverted light microscope was used to image neutrophils sheared at 1 dyne/cm2 over a monolayer cell substrate (Fig. 1a). Neutrophils rolled at a velocity of ~2 µm/s on E-selectin up-regulated on TNF-{alpha}-stimulated endothelial monolayers or expressed at equivalent site density on E-selectin transfected L-cells. In contrast, neutrophils rolled ~15-fold faster on CHO cells transfected to express the L-selectin mucosal ligand CD34 (22). In post-capillary venules during inflammation, rolling rapidly transitions to cell arrest via binding of activated {beta}2-integrins. This was recapitulated in the flow chamber as ~50% of rolling neutrophils transitioned to arrest within two to three cell diameters of capture on L-E/I or activated HUVEC monolayers (6). Arrest on the CD34 CHO cell monolayer did not increase above baseline observed with untransfected parent cells, despite the fact that equal numbers of neutrophils tethered and rolled on CD34, L-E/I, and HUVECs. The transition from rolling to arrest required activation of {beta}2-integrin and binding to ICAM-1 as shown by the absence of arrest in L-cells expressing E-selectin alone.



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  		FIGURE 1. Neutrophil roll and arrest on E-selectin after coclustering of L-selectin and PSGL-1 in shear flow. Neutrophils were injected into a PPFC and sheared over L-E, L-E/I, TNF-{alpha}-stimulated HUVECs or CHO-CD34 monolayers. The wall shear stress was maintained at 1 dyne/cm2 for 1 min. a, Rolling velocity data represent mean ± SD for 45 cells (n = 3). *, Significance between rolling velocity on L-E compared with rolling velocity on L-E/I (p < 0.001) and HUVECs (p < 0.01). b, Fractions of total neutrophils interacting with the monolayer that were either rolling or arrested. Data represent the mean ± SD for 300 cells (n = 3). c, Neutrophils were injected into either a static chamber or PPFC (1 dyne/cm2) and exposed either L-E/I, TNF-{alpha}-stimulated HUVECs, or CHO-CD34 monolayers for 1 min before immunofluorescent labeling. Micrographs are representative of 50 cells (n = 3). Clusters were analyzed for frequency, area, and percent colocalization of PE- and FITC-labeled Abs. Data presented as mean ± SD for 50 cells (n = 3). *, Significance between HUVEC cluster area and L-E/I cluster area (p < 0.001). PMN, Polymorphonuclear leukocyte.

 
We next examined the membrane distribution of the two known E-selectin ligands, L-selectin and PSGL-1, on neutrophils by immunofluorescence after rolling and arrest on each cell monolayer in the flow chamber. Directly conjugated fluorescent Abs to L-selectin and PSGL-1 did not exhibit significant concentration or colocalization on neutrophils allowed to sediment and contact HUVECs or L-E/I under static conditions (Fig. 1c). In contrast, in the presence of venular levels of fluid shear (i.e., 1 dyne/cm2), neutrophils that transitioned to arrested exhibited large concentrations or clusters of yellow fluorescence (i.e., clusters identified by image analysis as 3-fold above background intensity), indicating colocalization of green fluorescent anti-L-selectin and red fluorescent anti-PSGL-1. These coclusters were capped on the trailing edge of neutrophils adherent to activated HUVECs or L-E/I in shear flow (Fig. 1c). Image analysis of populations of arrested neutrophils revealed that 70–80% of membrane L-selectin and PSGL-1 redistributed to coclusters with equivalent frequency on HUVECs and L-E/I (Fig. 1c). Moreover, average cluster area was twice as large on neutrophils adherent to HUVECs (Fig. 1c). Under both static and shear conditions, neutrophils rolling on a CD34-expressing CHO cell monolayer did not elicit significant clustering of L-selectin or PSGL-1. As a control for specificity of glycoprotein recognition by E-selectin, we imaged the sialoglycoprotein CD43 receptor that is constitutively expressed on neutrophils and found insignificant colocalization of L-selectin or PSGL-1 under static or shear conditions (data not shown).

Confocal fluorescence microscopy of L-selectin and PSGL-1 on neutrophils rolling on E-selectin

To spatially map L-selectin and PSGL-1 on neutrophils transitioning from rolling to arrest on E-selectin in the flow chamber, optical slices were obtained beginning at the plane of contact with L-E/I and rising up the height of the neutrophils at 2.0-µm increments. The representative confocal images depicted in Fig. 2a clearly reveal the three-dimensional location of coclusters of L-selectin and PSGL-1. Consistent with the rim immunofluorescence detected in the light microscopic images, small coclusters of L-selectin and PSGL-1 are evident at the plane of adhesive contact (i.e., 0 µm) and are concentrated at ~2 µm above monolayer contact. A consistent observation was that coclusters were less frequent within the neutrophil contact area and at heights of 4 µm and above. To confirm that E-selectin recognition of sLex ligands on the neutrophils affected L-selectin and PSGL-1 coclusters, L-E/I monolayers were preincubated with a saturating concentration of a small molecule mimetic of sLex (TBC 1269) or with a control compound (TBC 1900), and neutrophils were sheared over the monolayers in the flow chamber as above. This treatment abrogated colocalization of L-selectin and PSGL-1 but did not affect immunodetection of these glycoproteins, as indicated by the discrete red and green fluorescence (Fig. 2a). Blocking the binding of sLex on E-selectin also affected neutrophil rolling and arrest on the L-E/I monolayer. Neutrophil interaction decreased nominally; however, tethered cells rolled twice as fast and arrest was inhibited by ~50% in response to blocking with the TBC 1269 (Fig. 2b). Taken together, these data show a direct correlation between E-selectin clustering of sLex presenting ligands on rolling neutrophils and the efficiency of transition to arrest in shear flow.



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  		FIGURE 2. Coclustering of L-selectin and PSGL-1 on E-selectin and neutrophil arrest are dependent on binding of sLex. a, Neutrophils were injected into a PPFC and passed (1 dyne/cm2 for 1 min) over an L-E/I monolayer pretreated with the sLex mimetic compound TBC1269 or control compound TBC 1900. The coverslip was removed and labeled with FITC-anti-L-selectin and PE-anti-PSGL-1 before fixation and confocal microscopy. Micrographs are representative of 20–30 cells (n = 3). b, Rolling velocity and the fractions of rolling and arrested cells represent the mean ± SD for ~500 cells (n = 5). *, #, and {ddagger} denote significance between TBC1269-treated L-E/I monolayers and TBC 1900-treated monolayers for rolling or arrest (p < 0.001). PMN, Polymorphonuclear leukocyte.

 
Binding of recombinant E-selectin to neutrophils in suspension

Studies were initiated to examine the relative binding affinity of recombinant E-selectin constructs to ligands expressed on human neutrophils in suspension. A full-length IgG chimera of E-selectin (E-sel-IgG), composed of two lectin domains, EGFs, and six complement-like repeats, exhibited a dense band at the predicted molecular mass of ~300 kDa (Fig. 3). A second, less dense band (i.e., <5% based on densitometry) detected at ~600 kDa corresponded to a fraction of aggregated E-sel-IgG (Fig. 3b). Flow cytometric analysis of E-selectin construct binding to neutrophils revealed that E-sel-IgG bound to unstimulated neutrophils with a half maximum saturation constant (EC50) of ~20 nM. A second IgG construct of E-selectin (E-LE-Fc), which contained an enterokinase site spliced into the region normally occupied by the complement-like repeats, had a molecular mass of 225 kDa (Fig. 3). Binding of this construct was 100-fold weaker than that of the E-sel-IgG construct (EC50 = ~2 µm) and was on the order of P-sel-IgG or L-sel-IgG binding (data not shown). Cleavage of E-LE-Fc at the enterokinase site yielded a single band at 60 kDa, corresponding to a monovalent E-selectin-EGF construct (E-LE) (Fig. 3). E-LE bound to neutrophils weakly and was not significantly different from the enterokinase liberated Fc domain (EC50 > 10 µm).



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  		FIGURE 3. Structural analysis and binding affinities of recombinant E-selectin constructs. a, The structure of the chimeric E-selectin constructs is illustrated where the complementary repeats, EGF, lectin domain (lectin), and enterokinase insertion site are represented for each molecule. EC50 values represent the concentration of construct at half saturation, as determined from flow cytometric experiments in which the binding of fluorescent E-selectin constructs was evaluated up to equilibrium on isolated neutrophils. EC50 values represent the mean of the binding curve for 10,000 cells (n = 3). b, Molecular mass was determined by PAGE analysis under nondenaturing and nonreducing conditions with BSA and urease trimer as standards.

 
Treatment of neutrophils in suspension with recombinant selectin-IgG provided a means of examining the redistribution of L-selectin and PSGL-1 on individual cells by immunofluoresce. Furthermore, by performing flow cytometry on populations incubated with selectin-IgG, it was possible to assess the dynamics of integrin activation in comparison with soluble chemotactic stimulus. Representative two-color immunofluorescence images depict a uniform punctate distribution of anti-L-selectin and anti-PSGL-1 in untreated neutrophils or in those treated with a nonspecific anti-human IgG (Fig. 4a). Exposure of neutrophils to the full-length recombinant E-sel-IgG at its EC50 (~20 nM) for 1 min induced between one and three large clusters of L-selectin and PSGL-1 that colocalized to membrane patches measuring 2–3 µm2 in area (Fig. 4, b and e). These patches also correlated to a majority (i.e., ~60%) but not all of the bound E-sel-IgG, as determined by secondary labeling of the humanized E-sel-IgG (data not shown). In contrast, immunofluorescent detection of membrane CD45 did not indicate any colocalization with either L-selectin or PSGL-1 upon E-sel-IgG binding (data not shown). Addition of L-sel-IgG or P-sel-IgG at their EC50 (i.e., 2 µm) resulted in clustering of PSGL-1, but to a lesser extent than with E-sel-IgG, and these clusters did not colocalize with L-selectin (Fig. 4, c and d). Image analysis of cluster frequency and area revealed that the control human IgG had no effect on the punctate distribution or induced clustering of L-selectin or PSGL-1 (Fig. 4e). The weaker binding dimeric E-LE-Fc added at its EC50 induced more, but smaller, clusters and an equivalent extent of L-selectin/PSGL-1 colocalization. The weakest binding monomeric E-LE construct did not cause significant clustering or colocalization. Taken together, these data indicate that full-length E-sel-IgG bound significantly tighter than did L- or P-selectin and exhibited a unique capacity to induce rapid colocalization and capping of L-selectin and PSGL-1 on neutrophils in suspension.



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  		FIGURE 4. Membrane redistribution of L-selectin and PSGL-1 after binding of selectin-IgG to neutrophils in suspension. The topography of L-selectin and PSGL-1 on neutrophils was visualized after binding of E-, L-, or P-selectin. Neutrophils were bound with control IgG (a), E-sel-IgG (b), L-sel-IgG (c), or P-sel-IgG (d) and labeled with FITC-anti-L-selectin and PE-anti-PSGL-1 before activation at 37°C before fixation. Micrographs are representative of 60–100 cells (n = 3). e, Cell micrographs were analyzed for cluster frequency, cluster area, and receptor colocalization using Image Pro Plus v4.5 (Media Cybernetics). Data are given as mean ± SD for 30–50 cells (n = 3). * and # denote significance between E-sel-IgG-treated neutrophils and those treated with L-sel-IgG, P-sel-IgG, and E-LE-Fc (p < 0.001 for cluster area and p < 0.01 for cluster frequency). {ddagger}, Significance between E-sel-IgG-treated neutrophils and those treated with L-sel-IgG and P-sel-IgG (p < 0.001 for percent colocalization).

 
Recombinant E-selectin up-regulates {beta}2-integrin expression and adhesive function

An early response of neutrophils to ligation of chemotactic factors is up-regulation of CD11b/CD18 expression and CD18 adhesive function. We assessed the capacity of E-selectin binding to neutrophils in suspension to activate CD18 as a function of its binding affinity and valency by binding E-selectin constructs and secondary cross-linking polyclonal Ab. Stimulation with the chemotactic agonist fMLP for 1 min up-regulated CD11b/CD18 expression by >2-fold and activated conversion to high-affinity CD18 by ~4-fold, as reported by mAb 327C (Fig. 5a). Treatment with the full-length E-sel-IgG construct at 20 nM elicited up-regulation of CD18 expression and the high-affinity conformation by ~2-fold. In comparison, addition of the lower affinity dimeric E-LE-Fc construct up-regulated CD18 expression and function only after cross-linking with a secondary polyclonal Ab. Neither the monomeric E-LE construct nor the liberated Fc exhibited the capacity to activate CD18, even at concentrations 100-fold higher than E-sel-IgG in the presence of a cross-linker.



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  		FIGURE 5. Binding of E-selectin-IgG signals CD18 membrane up-regulation and activation in suspension. a, Neutrophils were preincubated with Fc, E-LE, E-LE-Fc, or E-sel-IgG and then were stimulated with or without cross-linker or with 1 µM fLMP, as indicated. Cells were labeled with PE-anti-Mac-1 and Alexa488-327C before analysis on a FACScan flow cytometer. Data represent mean ± SD (n = 3). *, Significance between E-LE-Fc- and Fc-treated neutrophils for both mAb 327C and Mac-1 expression (p < 0.01). #, Significance between E-LE-Fc- and fMLP-treated neutrophils for both mAb 327C and Mac-1 expression (p < 0.001). b, Isolated neutrophils were preincubated with either E-sel-IgG or a combination of mouse anti-L-selectin (DREG56) and mouse anti-PSGL-1 (PL-1) for 10 min at 23°C to saturate binding sites. ACLBs were added at a ratio of 40:1 and sheared (~1 dyne/cm2) with neutrophils using a mixing chamber maintained at 37°C, located immediately upstream of the FACscan flow cytometer. The rate of CD11b/CD18-dependent capture was computed from the slope of the ACLB binding kinetics over the first minute of stimulation. Data represent the mean ± SD (n = 3). *, Significance between cross-linked mouse anti-L-selectin/mouse anti-PSGL-1 and cross-linked human anti-L-selectin/mouse anti-PSGL-1 (p < 0.001). #, Significance between cross-linked mouse anti-L-selectin and secondary goat anti-mouse F(ab')2 cross-linker control (p < 0.01). PMN, Polymorphonuclear leukocyte.

 
We next assessed the efficacy of E-sel-IgG to bind neutrophils and to activate CD11b/CD18-dependent adhesion in a sheared cell suspension. This adhesion assay provides for subsecond detection of the conversion of CD11b/CD18 to its high-avidity state, in that the collision frequency between ACLBs and neutrophils is on the order of 10 per second. Application of shear in the absence of stimuli induced neutrophil-bead collisions, but not ACLB adhesion (Fig. 5b). Remarkably, E-sel-IgG elicited the most rapid rate of bead capture, reaching peak adhesion over the first minute of shear and stimulus. Compared with chemotactic stimulation with fMLP, or cross-linked anti-L-selectin and anti-PSGL-1, E-sel-IgG elicited a 5-fold more rapid rate of ACLB capture (Fig. 5b). However, by 5 min of shear, the extent of neutrophil-ACLB adhesion induced by E-sel-IgG was on par with that stimulated by fMLP, IL-8, or co-cross-linked anti-L-selectin and anti-PSGL-1 (data not shown). It was also confirmed that clustering L-selectin and PSGL-1 simultaneously was critical in boosting CD11b/CD18 adhesion. Cross-linking both the mouse anti-human L-selectin and PSGL-1 monoclonals with polyclonal goat anti-mouse agent elicited twice the rate of CD18 adhesion than with either receptor alone. We next cross-linked neutrophils with a humanized anti-L-selectin along with the mouse anti-human PSGL-1 monoclonal, which served as a reagent control. This intervention stimulated ~70% less adhesion because the goat anti-mouse secondary did not recognize the humanized anti-L-selectin (Fig. 5b).

E-selectin activation of MAPK regulates coclustering of L-selectin/PSGL-1 and redistribution of high-affinity CD18

Because signaling via cross-linking of L-selectin and PSGL-1 has been previously shown to rapidly activate MAPKs (18, 20), we examined tyrosine phosphorylation of p38 and p42/44 MAPKs in response to binding of E-sel-IgG (Fig. 6a). Within minutes of addition to neutrophils in suspension, E-sel-IgG induced a >4-fold increase in phosphorylation of p38 and p42/44 MAPK compared with that elicited by incubation with a polyclonal human-IgG control. Kinase activity was on par with that induced by simultaneous cross-linking of anti-L-selectin and anti-PSGL-1 with secondary goat anti-mouse polyclonal Ab. In comparison, stimulation with TNF-{alpha} elicited a 10- to 15-fold increase in p38 and p42/44 activity, respectively, as previously reported (16).



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  		FIGURE 6. E-selectin signals coclustering of L-selectin/PSGL-1 and activation of CD18 via MAPKs. a, Neutrophils were preincubated with MAPK inhibitors and then treated with E-selectin-IgG or the combination of anti-L-selectin and anti-PSGL-1. Neutrophils were stimulated with cross-linker (anti-L-sel/anti-PSGL-1) or with TNF-{alpha} at 37°C. After fixation and Western blotting, the relative intensity of p38 and p42/44 MAPK signal was quantified by densitometry and was expressed as fold increase of neutrophils preincubated with a human-IgG control. Data represent the average phosphorylation pattern (n = 3). b, Inhibitors of MAPK-blocked E-selectin signaled redistribution of L-selectin and PSGL-1. Neutrophils were preincubated with MAPK inhibitors and labeled with FITC-anti-L-selectin and PE-PSGL-1 before activation with E-sel-IgG at 37°C. Micrographs are representative of 30–50 cells (n = 3) and were analyzed for cluster frequency, area, and percent colocalization using Image Pro Plus v4.5 (Media Cybernetics). Data are given as mean ± SD for 30–50 cells (n = 3). *, Significance between E-sel-IgG-treated neutrophils and those with p38 and p42/44 blocked (p < 0.001). c, E-selectin signals CD18 activation and redistribution via MAPK. Neutrophils were preincubated with MAPK inhibitors and then stimulated with E-sel-IgG or 1 nM IL-8 for 1 min at 37°C. Fluorescent images of Alexa488-327C are representative of 30–50 cells (n = 3). Monoclonal Ab 327C cluster frequency and area are given as mean ± SD for 30–50 (n = 3). * and # denotes significance between E-sel-IgG control and E-sel-IgG-treated neutrophils with inhibition of p38 and p42/44 MAPK (p < 0.05 and p < 0.001, respectively).

 
Pretreatment of E-sel-IgG-treated neutrophils in suspension with a soluble high-affinity blocker of p38 phosphorylation reduced the frequency of L-selectin/PSGL-1 coclusters by ~60% and the cluster area by ~80% (Fig. 6b). Inhibition of p42/44 activity with soluble blocker effectively diminished cluster area by ~70% but exerted less inhibition on L-selectin/PSGL-1 colocalization or cluster frequency. These results demonstrate that E-selectin-induced colocalization rapidly signals MAPK phosphorylation, which in turn drives active clustering and capping of L-selectin and PSGL-1.

Because redistribution of high-affinity CD18 into membrane clusters is critical to neutrophil capture on endothelium in shear flow, we examined the role of MAPK in this process on neutrophils in suspension (6, 16). Within minutes of IL-8 or E-sel-IgG stimulation of neutrophils, active CD18 was up-regulated and redistributed into clusters of 1–3 µm2 in area (Fig. 6c). These clusters did not coincide with sites of coclustered L-selectin/PSGL-1. Moreover, addition of a control human-IgG elicited no CD18 activation over the same time course (data not shown). Preincubating neutrophils with inhibitors to p38 and p42/44 MAPK virtually abrogated clustering of 327C clusters. However, expression of fluorescent 327C bound to E-sel-IgG stimulated neutrophils, as assessed by flow cytometry, indicating that integrated membrane expression was not significantly diminished due to blocking either p38 (72 ± 26% of max; p > 0.05) or p42/44 (94 ± 5% of max; p > 0.05). These results support a direct role for p38 and p42/44 MAPK phosphorylation both in the colocalization of L-selectin/PSGL-1 and in the redistribution of high-affinity CD18 into clusters previously reported to be critical for neutrophil capture in shear flow (31).


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
In the current study we demonstrate for the first time that E-selectin binding to neutrophils in suspension, or as they roll on a monolayer cell substrate in shear flow, results in the clustering and colocalization of L-selectin and PSGL-1. Coclustering of E-selectin ligands during neutrophil tethering and rolling on monolayers and inflamed endothelium was facilitated by fluid shear and was dependent on MAPK-regulated membrane transport processes. Within seconds of E-selectin binding, neutrophil CD18 shifted to a high-affinity ligand binding state, as imaged by mAb 327C, which revealed membrane clustering of active CD18. This redistribution was also signaled via p38 and p42/22 MAPK activity. These data reveal spatial and temporal regulation of signaling and adhesion receptor dynamics assembled in response to E-selectin engagement of neutrophils in shear flow.

Recognition of sLex on L-selectin and PSGL-1 by E-selectin activates CD18

E-selectin supports rolling velocities of leukocytes on the order of a cell diameter per second (6). Intravital study of leukocytes within the venules of cytokine-activated cremaster muscle of E-selectin–/– mice reveal rolling velocities that are ~3-fold faster than those in P-selectin–/– and wild-type mice (32). Correlating with slow rolling mediated by E-selectin are data supporting its cooperativity in leukocyte arrest, which was reportedly reduced ~80% in E-selectin–/– mice compared with wild type (33). Directly linking the efficiency of CD18-mediated arrest with E-selectin functions are data from Forlow et al. (34), which demonstrate that leukocyte arrest in TNF-{alpha}-activated venules of E-selectin–/–/CD18–/– double mutant mice was reduced ~5-fold compared with either CD18–/– or E-selectin–/– single mutants. Although all three selectins bind PSGL-1 during leukocyte rolling in vivo, as demonstrated by blocking each with a recombinant PSGL-1 construct, E-selectin also binds other sLex-presenting ligands (35). In this regard, we have reported that E-selectin binding to L-selectin and PSGL-1 mediates slow rolling of neutrophils and that blocking either ligand increased rolling velocity and reduced CD18-dependent cell arrest by ~3-fold (6, 11).

All three selectins recognize glycoproteins decorated with the sLex tetrasaccharide, a fucosylated and sialylated carbohydrate moiety (2). E-selectin is unique in that it binds monovalent sLex with 10-fold greater affinity than P-selectin and a 5-fold greater affinity than L-selectin (25, 36). Despite this difference, selectin binding to monovalent sLex is weak, with affinity in the millimolar range. Several lines of evidence indicate that leukocyte rolling is mediated through multivalent binding of clustered sLex-bearing glycoprotein ligands (37, 38). E-selectin binds readily to a wider array of sLex ligands and is sulfation independent, compared with L- and P-selectin, which require carbohydrate 6 sulfation and tyrosine sulfation, respectively (39). L-selectin and PSGL-1 are well-suited ligands for multivalent recognition by E-selectin, in that both are constitutively expressed on membrane microvilli and contain multiple N- and O-linked sLex (13, 14, 40, 41). In fact, the predominant receptor bound by human E-selectin in affinity chromatography of leukocyte cell lysates is L-selectin (12). Similarly, published data suggest that E-selectin mediates cell rolling through low-affinity binding to sLex expressed on N- and O-linked sites of PSGL-1 and other membrane glycolipids (8, 11, 25). Here, we report that E-selectin-mediated coclustering of L-selectin and PSGL-1 was dependent on lectin domain recognition of sLex. Remarkably, pretreatment of E-selectin with the di-sLex mimetic TBC1269, a small molecule currently being studied as an anti-inflammatory therapeutic, had a greater affect on the transition to arrest than did capture and rolling of neutrophils in the PPFC (26, 42, 43). Inhibition with TBC1269 increased the fraction of rolling neutrophils by ~25%, but decreased arrest by ~50%. The capacity of TBC1269 to competitively antagonize the sLex-selectin interaction is supported by data from Kogan et al. (26), who reported that preincubating E-selectin-IgG, L-selectin-IgG, or P-selectin-IgG with TBC1269 at the IC50 significantly reduced binding to sLex-bearing HL-60 cells.

A direct correlation was demonstrated between the rate of neutrophil adhesion via CD11b/CD18 activation and the binding affinity of the E-selectin constructs. Full-length E-sel-IgG bound most tightly (Kd = ~20 nM) and induced rapid (<60 s) clustering of L-selectin and PSGL-1 into domains colocalized with the construct itself. In contrast, dimeric E-selectin (E-LE-Fc) only elicited small punctate clusters of L-selectin/PSGL-1, whereas E-LE had no significant effect on ligand topography. These findings are in agreement with recent published data from our laboratory that indicate a threshold level of L-selectin membrane clustering is necessary to trigger neutrophil activation and CD18 adhesion (16). Likewise, a threshold level of recombinant E-sel-IgG molecules was required to trigger ligand redistribution and to transduce a dose-dependent functional response in neutrophils. This configuration resembles the clustered topography adopted by E-selectin on IL-1{beta}-stimulated endothelium or within the contact area of adherent leukocytes (7, 44).

E-selectin mechano-transduction via clustering of sialylated ligands on rolling neutrophils

Confocal and immunofluorescence microscopy of adherent neutrophils revealed that coclusters of L-selectin/PSGL-1 were capped at a height of ~2 µm up the trailing edge of rolling cells. In contrast, transition from rolling to arrest was achieved by concentration of active CD18-forming preferential high-avidity clusters within the contact region (C. E. Green and S. I. Simon, manuscript in preparation). This particular topographic redistribution required the presence of fluid shear and E-selectin recognition of sLex, because few coclusters appeared on neutrophils under no flow or in the presence of the sLex mimetic TBC1269. A simple model may explain the distribution of clustered E-selectin ligands. As neutrophils roll forward of the pivot of contact with the monolayer, torque is applied to membrane tethers at the trailing edge (45). Direct observation of this process has revealed that lipid tethers are extruded as tensile forces act through the bonds securing microvilli at the cell’s trailing edge (46). These tethers presumably release as selectin bonds rupture or by rupture and retraction of the lipid tether to the membrane at a position above the contact region as neutrophils rotate forward. Forces acting on a stably rolling or arrested neutrophil vary between 100 pN and 500 pN at venular shear rates (47). Because single membrane tethers formed through adhesion receptors can sustain tensile forces of ~45 pN, we estimate that as few as 10 membrane tethers could stabilize a neutrophil rolling at a constant velocity or arrested on the endothelium (48). Indeed, it was reported that up to nine L-selectin-decorated microvillous tethers were extruded from neutrophils rolling on a peripheral lymph node addressin-coated surface (45). The shear dependence of L-selectin and PSGL-1 coclustering may also be explained by the existence of a threshold requirement for efficient E-selectin engagement. Neutrophils settling under static conditions on E-selectin would limit the effective number of bonds formed to only the contact area, whereas, under conditions of shear, E-selectin engagement is promoted both by the presence of tensile forces on bonds and by the increase of effective contact area on the rolling neutrophil (49).

Our data support the contention that coclustering of L-selectin and PSGL-1 is not driven solely by diffusive capture of ligand upon binding of E-selectin, but rather is redistributed to these sites by an active transport process. For example, assembly of L-selectin and PSGL-1 from a uniform distribution in resting neutrophils into 3-µm2 clusters captured at focal sites of E-sel-IgG binding would require receptors to diffuse at ~10 µm2/min. This rate of diffusion is 10- to 100-fold greater than that previously measured for integral membrane proteins passively diffusing in a lipid bilayer (~0.1–1.0 µm2/min) (50). Active transport and coclustering of ligands observed in response to E-selectin were specific, as binding of P-selectin-IgG or L-selectin-IgG to neutrophils only elicited small punctate clusters of PSGL-1. Moreover, the area of L-selectin/PSGL-1 coclusters was diminished 80% by pretreatment with soluble blockers of p38 and p42/44 MAPK activity. One possible mechanism supporting this rapid recruitment of L-selectin and PSGL-1 is assembly within membrane rafts, a process recently shown to be blocked by inhibition of p42/44 MAPK (51).

E-selectin binding activates MAPKs and outside-in signaling

Previous reports have documented that MAPKs and their downstream targets are rapidly phosphorylated in neutrophils after ligation of L-selectin or PSGL-1 (18, 20). We have recently shown that multivalent cross-linking of L-selectin activates p38 and p42/44 phosphorylation that peaks within 1 min, a time frame consistent with that required for the activation and clustering of CD18 (16). E-sel-IgG binding to neutrophils in suspension was nearly as effective as Ab cross-linking of L-selectin and PSGL-1. This is the first report that p38 and p42/44 activity peaks in tandem after neutrophil activation. Furthermore, MAPK inhibition diminished coclustering of L-selectin/PSGL-1 and assembly of CD18 into high-avidity sites required for neutrophil arrest. This is supported by the observation that blocking either MAPK, decreased arrest of neutrophils rolling on an L-E/I monolayer yet had no significant effect on neutrophil rolling velocity, indicating that the primary role of L-selectin/PSGL-1 membrane caps is to transduce outside-in signals (6). Thus, MAPK activity serves to link incoming signals via E-selectin ligation and outgoing integrin effector functions. A superposition of signals derived from E-selectin and G-protein-coupled receptors has been reported to potentiate neutrophil activation and transmigration compared with either alone (29, 52). In this context, binding of E-selectin may serve to prime neutrophils before subsequent ligation of endothelial chemotactic agonists in a process that synergistically amplifies L-selectin/PSGL-1 coclustering and signaling of cell arrest on inflamed endothelium (53).

A molecular model that accounts for the assembly of leukocyte adhesion receptors catalyzed by ligand binding and driven by shear forces into membrane compartments enriched in signaling elements is emerging. Published data indicate that artificial cross-linking of L-selectin and PSGL-1 on the neutrophil membrane by Abs promotes their incorporation within membrane rafts (51, 54). Effector molecules such as Ras and MAPKs can initiate transduction of outside-in signals by localizing to the membrane raft complex (55, 56). Inside-out signaling may then proceed through Rho and Rho-associated kinase activity, which in turn regulates organization of LFA-1 into clusters within membrane rafts (57, 58). LFA-1 mobility and clustering within the membrane is further regulated by calpain, an enzyme downstream of Rho and MAPKs that functions in directional guidance of neutrophil chemotaxis and formation of focal adhesion complexes (59, 60, 61, 62, 63). Thus, Rho activity may be important in controlling the mobility of high-avidity {beta}2-integrin. We propose a model in which tensile loading of multivalently engaged E-selectin ligands induces their coclustering and transmembrane activation of Ras/Raf/MAPK kinase/MAPK, which in turn triggers the assembly of high-avidity {beta}2-integrin through activation of calpain and Rho. This sequence of events initiated by E-selectin binding ligands under shear force could provide a means of mechano-transduction and precise amplification in the rate and extent of integrin activation and high-avidity {beta}2-integrin binding ICAM-1.

The relative importance of E-selectin in leukocyte recruitment and signaling may vary among neutrophils from different animal species, as well as among human leukocytes such as monocytes and T cells. This is due to differential expression of fucosyltransferase-VII, fucosyltransferase-IV, and core 2 {beta}1–6-N-glucosaminyltransferase leading to disparities in the glycosylation of L-selectin and PSGL-1 as a function of leukocyte type (64, 65, 66, 67). However, emerging evidence supports a direct role for E-selectin recognition of sLex on rolling neutrophils, signaling a boost in the efficiency of {beta}2-integrin-mediated arrest.


   Acknowledgments
 
We thank Nick Landolfi from Protein Design Labs, Minoru Fukuda of The Burnham Institute, and Texas Biotechnology for their generous provision of reagents.


   Footnotes
 
1 This work was supported by National Institutes of Health Grant AI47294. S.I.S. is an Established Investigator of the American Heart Association. C.E.G. is supported by National Institute of General Medical Sciences Training Fellowship T32-GM08799-01A1. Back

2 Current address: Compound Therapeutics, Inc., 1365 Main Street, Waltham, MA 02451. Back

3 Address correspondence and reprint requests to Dr. Scott I. Simon, Department of Biomedical Engineering, University of California, One Shields Avenue, Davis, CA 95616-5294. E-mail address: sisimon{at}ucdavis.edu Back

4 Abbreviations used in this paper: sLex, sialyl Lewisx; PSGL-1, P-selectin glycoprotein ligand 1; MAPK, mitogen-activated protein kinase; L-E, transfected L-cells expressing human E-selectin; L-E/I, transfected L-cells coexpressing human E-selectin and ICAM-1; CHO, Chinese hamster ovary; E-sel-IgG, recombinant chimeric human E-selectin; L-sel-IgG, recombinant chimeric human L-selectin; P-sel-IgG, recombinant chimeric human P-selectin; EGF, epidermal growth factor-like domain; ACLB, albumin-coated latex bead; PPFC, parallel-plate flow chamber. Back

Received for publication January 15, 2004. Accepted for publication April 9, 2004.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

  1. Lawrence, M. B., T. A. Springer. 1991. Leukocytes roll on a selectin at physiologic flow rates: distinction from and prerequisite for adhesion through integrins. Cell 65:859.[Medline]
  2. Foxall, C., S. R. Watson, D. Dowbenko, C. Fennie, L. A. Lasky, M. Kiso, A. Hasegawa, D. Asa, B. K. Brandley. 1992. The three members of the selectin receptor family recognize a common carbohydrate epitope, the sialyl LewisX oligosaccharide. J. Cell Biol. 117:895.[Abstract/Free Full Text]
  3. Springer, T. A.. 1994. Traffic signals for lymphocyte recirculation and leukocyte emigration: the multistep paradigm. Cell 76:301.[Medline]
  4. Rainger, G. E., A. C. Fisher, G. B. Nash. 1997. Endothelial-borne platelet-activating factor and interleukin-8 rapidly immobilize rolling neutrophils. Am. J. Physiol. 272:H114.
  5. Hu, Y., J. M. Kiely, B. E. Szente, A. Rosenzweig, M. A. Gimbrone, Jr. 2000. E-selectin-dependent signaling via the mitogen-activated protein kinase pathway in vascular endothelial cells. J. Immunol. 165:2142.[Abstract/Free Full Text]
  6. Simon, S. I., Y. Hu, D. Vestweber, C. W. Smith. 2000. Neutrophil tethering on E-selectin activates {beta}2 integrin binding to ICAM-1 through a mitrogen-activated protein kinase signal transduction pathway. J. Immunol. 164:4348.[Abstract/Free Full Text]
  7. Yoshida, M., W. F. Westlin, N. Wang, D. E. Ingber, A. Rosenzweig, N. Resnick, M.A. Gimbrone, Jr. 1996. Leukocyte adhesion to vascular endothelium induces E-selectin linkage to the actin cytoskeleton. J. Cell Biol. 133:445.[Abstract/Free Full Text]
  8. Alon, R., T. Feizi, C. T. Yuen, R. C. Fuhlbrigge, T. A. Springer. 1995. Glycolipid ligands for selectins support leukocyte tethering and rolling under physiologic flow conditions. J. Immunol. 154:5356.[Abstract]
  9. Lawrence, M. B., T. A. Springer. 1993. Neutrophils roll on E-selectin. J. Immunol. 151:6338.[Abstract]
  10. Lo, S. K., S. Lee, R. A. Ramos, R. Lobb, M. Rosa, G. Chi-Rosso, S. D. Wright. 1991. Endothelial-leukocyte adhesion molecule 1 stimulates the adhesive activity of leukocyte integrin CR3 (CD11b/CD18, Mac-1, {alpha}m{beta}2) on human neutrophils. J. Exp. Med. 173:1493.[Abstract/Free Full Text]
  11. Patel, K. D., K. L. Moore, M. U. Nollert, R. P. McEver. 1995. Neutrophils use both shared and distinct mechanisms to adhere to selectins under static and flow conditions. J. Clin. Invest. 96:1887.
  12. Zollner, O., M. C. Lenter, J. E. Blanks, E. Borges, M. Steegmaier, H. G. Zerwes, D. Vestweber. 1997. L-selectin from human, but not from mouse neutrophils binds directly to E-selectin. J. Cell Biol. 136:707.[Abstract/Free Full Text]
  13. Bruehl, R. E., T. A. Springer, D. F. Bainton. 1996. Quantitation of L-selectin distribution on human leukocyte microvilli by immunogold labeling and electron microscopy. J. Histochem. Cytochem. 44:835.[Abstract]
  14. Moore, K. L., S. F. Eaton, D. E. Lyons, H. S. Lichenstein, R. D. Cummings, R. P. McEver. 1994. The P-selectin glycoprotein ligand from human neutrophils displays sialylated, fucosylated, O-linked poly-N-acetyllactosamine. J. Biol. Chem. 269:23318.[Abstract/Free Full Text]
  15. Levinovitz, A., J. Muhlhoff, S. Isenmann, D. Vestweber. 1993. Identification of a glycoprotein ligand for E-selectin on mouse myeloid cells. J. Cell Biol. 121:449.[Abstract/Free Full Text]
  16. Green, C. E., D. N. Pearson, N. B. Christensen, S. I. Simon. 2003. Topographic requirements and dynamics of signaling via L-selectin on neutrophils. Am. J. Physiol. Cell Physiol. 284:C705.[Abstract/Free Full Text]
  17. Waddell, T. K., L. Kialkow, C. K. Chan, T. K. Kishimoto, G. P. Downey. 1995. Signaling functions of L-selectin. J. Biol. Chem. 270:15403.[Abstract/Free Full Text]
  18. Smolen, J. E., T. K. Petersen, C. Koch, S. J. O’Keefe, W. A. Hanlon, S. Seo, D. Pearson, M. C. Fossett, S. I. Simon. 2000. L-Selectin signaling of neutrophil adhesion and degranulation involves p38 MAP kinase. J. Biol. Chem. 275:15876.[Abstract/Free Full Text]
  19. Steeber, D. A., P. Engel, A. S. Miller, M. P. Sheetz, T. F. Tedder. 1997. Ligation of L-selectin through conserved regions within the lectin domain activates signal transduction pathways and integrin function in human, mouse, and rat leukocytes. J. Immunol. 159:952.[Abstract]
  20. Hidari, K. I.-P. J., A. S. Weyrich, G. A. Zimmerman, R. P. McEver. 1997. Engagement of P-selectin glycoprotein ligand-1 enhances tyrosine phosphorylation and activates mitogen-activated protein kinases in human neutrophils. J. Biol. Chem. 272:28750.[Abstract/Free Full Text]
  21. Simon, S. I., A. R. Burns, A. D. Taylor, P. K. Gopalan, E. B. Lynam, L. A. Sklar, C. W. Smith. 1995. L-selectin (CD62L) crosslinking signals neutrophil adhesive functions via the Mac-1 (CD11b/CD18) {beta}2-integrin. J. Immunol. 155:1502.[Abstract]
  22. Hiraoka, N., B. Petryniak, J. Nakayama, S. Tsuboi, M. Suzuki, J. C. Yeh, D. Izawa, T. Tanaka, M. Miyasaka, J. B. Lowe, M. Fukuda. 1999. A novel, high endothelial venule-specific sulfotransferase expresses 6-sulfo sialyl Lewisx, an L-selectin ligand displayed by CD34. Immunity 11:79.[Medline]
  23. Co, M. S., N. F. Landolfi, J. O. Nagy, J. H. Tan, V. Vexler, M. Vasquez, L. Roark, S. Yuan, P. R. Hinton, J. Melrose, et al 1999. Properties and pharmacokinetics of two humanized antibodies specific for L-selectin. Immunotechnology 4:253.[Medline]
  24. Sako, D., X. J. Chang, K. M. Barone, G. Vachino, H. M. White, G. Shaw, G. M. Veldman, K. M. Bean, T. J. Ahern, B. Furie. 1993. Expression cloning of a functional glycoprotein ligand for P-selectin. Cell 75:1179.[Medline]
  25. Somers, W. S., J. Tang, G. D. Shaw, R. T. Camphausen. 2000. Insights into the molecular basis of leukocyte tethering and rolling revealed by structures of P- and E-selectin bound to SLeX and PSGL-1. Cell 103:467.[Medline]
  26. Kogan, T. P., B. Dupre, H. Bui, K. L. McAbee, J. M. Kassir, I. L. Scott, X. Hu, P. Vanderslice, P. J. Beck, R. A. Dixon. 1998. Novel synthetic inhibitors of selectin-mediated cell adhesion: synthesis of 1,6-bis[3-(3-carboxymethylphenyl)-4-(2-{alpha}-D-mannopyranosyloxy)phenyl]hexane (TBC1269). J. Med. Chem. 41:1099.[Medline]
  27. Lupher, M. L., Jr, E. A. Harris, C. R. Beals, L. M. Sui, R. C. Liddington, D. E. Staunton. 2001. Cellular activation of leukocyte function-associated antigen-1 and its affinity are regulated at the I domain allosteric site. J. Immunol. 167:1431.[Abstract/Free Full Text]
  28. Beals, C. R., A. C. Edwards, R. J. Gottschalk, T. W. Kuijpers, D. E. Staunton. 2001. CD18 activation epitopes induced by leukocyte activation. J. Immunol. 167:6113.[Abstract/Free Full Text]
  29. Tsang, Y. T. M., S. Neelamegham, Y. Hu, E. L. Berg, A. R. Burns, C. W. Smith, S. I. Simon. 1997. Synergy between L-selectin signaling and chemotactic activation during neutrophil adhesion and transmigration. J. Immunol. 159:4566.[Abstract]
  30. Abbassi, O., T. K. Kishimoto, L. V. McIntire, D. C. Anderson, C. W. Smith. 1993. E-selectin supports neutrophil rolling in vitro under conditions of flow. J. Clin. Invest. 92:2719.
  31. Lum, A. F., C. E. Green, G. R. Lee, D. E. Staunton, S. I. Simon. 2002. Dynamic regulation of LFA-1 activation and neutrophil arrest on ICAM-1 in shear flow. J. Biol. Chem. 277:20660.[Abstract/Free Full Text]
  32. Kunkel, E. J., K. Ley. 1996. Distinct phenotype of E-selectin-deficient mice: E-selectin is required for slow leukocyte rolling in vivo. Circ. Res. 79:1196.[Abstract/Free Full Text]
  33. Ley, K., M. Allietta, D. C. Bullard, S. Morgan. 1998. Importance of E-selectin for firm leukocyte adhesion in vivo. Circ. Res. 83:287.[Abstract/Free Full Text]
  34. Forlow, S. B., E. J. White, S. C. Barlow, S. H. Feldman, H. Lu, G. J. Bagby, A. L. Beaudet, D. C. Bullard, K. Ley. 2000. Severe inflammatory defect and reduced viability in CD18 and E-selectin double-mutant mice. J. Clin. Invest. 106:1457.[Medline]
  35. Hicks, A. E., S. L. Nolan, V. C. Ridger, P. G. Hellewell, K. E. Norman. 2003. Recombinant P-selectin glycoprotein ligand-1 directly inhibits leukocyte rolling by all 3 selectins in vivo: complete inhibition of rolling is not required for anti-inflammatory effect. Blood 101:3249.[Abstract/Free Full Text]
  36. Poppe, L., G. S. Brown, J. S. Philo, P. V. Nikrad, B. H. Shah. 1997. Conformation of sLex tetrasaccharide, free in solution and bound to E-, P-, and L-selectin. J. Am. Chem. Soc. 119:1727.
  37. Mehta, P., R. D. Cummings, R. P. McEver. 1998. Affinity and kinetic analysis of P-selectin binding to P-selectin glycoprotein ligand-1. J. Biol. Chem. 273:32506.[Abstract/Free Full Text]
  38. Stahn, R., H. Schafer, F. Kernchen, J. Schreiber. 1998. Multivalent sialyl Lewisx ligands of definite structures as inhibitors of E-selectin mediated cell adhesion. Glycobiology 8:311.[Abstract/Free Full Text]
  39. Kanamori, A., N. Kojima, K. Uchimura, T. Muramatsu, T. Tamatani, M. C. Berndt, G. S. Kansas, R. Kannagi. 2002. Distinct sulfation requirements of selectins disclosed using cells that support rolling mediated by all three selectins under shear flow: L-selectin prefers carbohydrate 6-sulfation totyrosine sulfation, whereas P-selectin does not. J. Biol. Chem. 277:32578.[Abstract/Free Full Text]
  40. Moore, K. L., K. D. Patel, R. E. Bruehl, L. Fugang, D. A. Johnson, H. S. Lichenstein, R. D. Cummings, D. F. Bainton, R. P. McEver. 1995. P-selectin glycoprotein ligand-1 mediates rolling of human neutrophils on P-selectin. J. Cell Biol. 128:661.[Abstract/Free Full Text]
  41. Picker, L. J., R. A. Warnock, A. R. Burns, C. M. Doerschuk, E. L. Berg, E. C. Butcher. 1991. The neutrophil selectin LECAM-1 presents carbohydrate ligands to the vascular selectins ELAM-1 and GMP-140. Cell 66:921.[Medline]
  42. Ramos-Kelly, J. R., L. H. Toledo-Pereyra, J. Jordan, F. Rivera-Chavez, T. Rohs, M. Holevar, R. A. Dixon, E. Yun, P. A. Ward. 2000. Multiple selectin blockade with a small molecule inhibitor downregulates liver chemokine expression and neutrophil infiltration after hemorrhagic shock. J. Trauma 49:92.[Medline]
  43. Ramos-Kelly, J. R., L. H. Toledo-Pereyra, J. A. Jordan, F. A. Rivera-Chavez, R. A. Dixon, P. A. Ward. 1999. Upregulation of lung chemokines associated with hemorrhage is reversed with a small molecule multiple selectin inhibitor. J. Am. Coll. Surg. 189:546.[Medline]
  44. Kiely, J. M., Y. Hu, G. Garcia-Cardena, M. A. Gimbrone, Jr. 2003. Lipid raft localization of cell surface E-selectin is required for ligation-induced activation of phospholipase C{gamma}. J. Immunol. 171:3216.[Abstract/Free Full Text]
  45. Chen, S., T. A. Springer. 1999. An automatic braking system that stabilizes leukocyte rolling by an increase in selectin bond number with shear. J. Cell Biol. 144:185.[Abstract/Free Full Text]
  46. Schmidtke, D. W., S. L. Diamond. 2000. Direct observation of membrane tethers formed during neutrophil attachment to platelets or P-selectin under physiological flow. J. Cell Biol. 149:719.[Abstract/Free Full Text]
  47. Schmid-Schönbein, G. W., Y. C. Fung, B. W. Zweifach. 1975. Vascular endothelium-leukocyte interaction: sticking shear force in venules. Circ. Res. 36:173.[Abstract/Free Full Text]
  48. Shao, J. Y., R. M. Hochmuth. 1996. Micropipette suction for measuring piconewton forces of adhesion and tether formation from neutrophil membranes. Biophys. J. 71:2892.[Medline]
  49. Lawrence, M. B., G. S. Kansas, E. J. Kunkel, K. Ley. 1997. Threshold levels of fluid shear promote leukocyte adhesion through selectins (CD62L,P,E). J. Cell Biol. 136:717.[Abstract/Free Full Text]
  50. Jacobson, K. A., S. E. Moore, B. Yang, P. Doherty, G. W. Gordon, F. S. Walsh. 1997. Cellular determinants of the lateral mobility of neural cell adhesion molecules. Biochim. Biophys. Acta 1330:138.[Medline]
  51. Phong, M. C., P. Gutwein, S. Kadel, K. Hexel, P. Altevogt, O. Linderkamp, B. Brenner. 2003. Molecular mechanisms of L-selectin-induced colocalization rafts and shedding. Biochem. Biophys. Res. Commun. 300:563.[Medline]
  52. Ruchaud-Sparagano, M. H., T. R. Walker, A. G. Rossi, C. Haslett, I. Dransfield. 2000. Soluble E-selectin acts in synergy with platelet-activating factor to activate neutrophil {beta}2-integrins: role of tyrosine kinases and Ca2+ mobilization. J. Biol. Chem. 275:15758.[Abstract/Free Full Text]
  53. Ley, K.. 2002. Integration of inflammatory signals by rolling neutrophils. Immunol. Rev. 186:8.[Medline]
  54. Handa, K., F. Jacobs, B. M. Longenecker, S. I. Hakomori. 2001. Association of MUC-1 and SPGL-1 with low-density microdomain in T-lymphocytes: a preliminary note. Biochem. Biophys. Res. Commun. 285:788.[Medline]
  55. Prior, I. A., C. Muncke, R. G. Parton, J. F. Hancock. 2003. Direct visualization of Ras proteins in spatially distinct cell surface microdomains. J. Cell Biol. 160:165.[Abstract/Free Full Text]
  56. Liu, P., Y. Ying, R. G. Anderson. 1997. Platelet-derived growth factor activates mitogen-activated protein kinase in isolated caveolae. Proc. Natl. Acad. Sci. USA 94:13666.[Abstract/Free Full Text]
  57. Rodriguez-Fernandez, J. L., L. Sanchez-Martin, M. Rey, M. Vicente-Manzanares, S. Narumiya, J. Teixido, F. Sanchez-Madrid, C. Cabanas. 2001. Rho and Rho-associated kinase modulate the tyrosine kinase PYK2 in T-cells through regulation of the activity of the integrin LFA-1. J. Biol. Chem. 276:40518.[Abstract/Free Full Text]
  58. Krauss, K., P. Altevogt. 1999. Integrin leukocyte function-associated antigen-1-mediated cell binding can be activated by clustering of membrane rafts. J. Biol. Chem. 274:36921.[Abstract/Free Full Text]
  59. Siljander, P., R. W. Farndale, M. A. Feijge, P. Comfurius, S. Kos, E. M. Bevers, J. W. Heemskerk. 2001. Platelet adhesion enhances the glycoprotein VI-dependent procoagulant response: involvement of p38 MAP kinase and calpain. Arterioscler. Thromb. Vasc. Biol. 21:618.[Abstract/Free Full Text]
  60. Glading, A., P. Chang, D. A. Lauffenburger, A. Wells. 2000. Epidermal growth factor receptor activation of calpain is required for fibroblast motility and occurs via an ERK/MAP kinase signaling pathway. J. Biol. Chem. 275:2390.[Abstract/Free Full Text]
  61. Lokuta, M. A., P. A. Nuzzi, A. Huttenlocher. 2003. Calpain regulates neutrophil chemotaxis. Proc. Natl. Acad. Sci. USA 100:4006.[Abstract/Free Full Text]
  62. Kulkarni, S., T. C. Saido, K. Suzuki, J. E. Fox. 1999. Calpain mediates integrin-induced signaling at a point upstream of Rho family members. J. Biol. Chem. 274:21265.[Abstract/Free Full Text]
  63. Stewart, M. P., A. McDowall, N. Hogg. 1998. LFA-1-mediated adhesion is regulated by cytoskeletal restraint and by a Ca2+-dependent protease, calpain. J. Cell Biol. 140:699.[Abstract/Free Full Text]
  64. Snapp, K. R., C. E. Heitzig, L. G. Ellies, J. D. Marth, G. S. Kansas. 2001. Differential requirements for the O-linked branching enzyme core 2 {beta}1–6-N-glucosaminyltransferase in biosynthesis of ligands for E-selectin and P-selectin. Blood 97:3806.[Abstract/Free Full Text]
  65. Dwir, O., F. Shimron, C. Chen, M. S. Singer, S. D. Rosen, R. Alon. 1998. GlyCAM-1 supports leukocyte rolling in flow: evidence for a greater dynamic stability of L-selectin rolling of lymphocytes than of neutrophils. Cell Adhes. Commun. 6:349.[Medline]
  66. Mitoma, J., B. Petryniak, N. Hiraoka, J. C. Yeh, J. B. Lowe, M. Fukuda. 2003. Extended core 1 and core 2 branched O-glycans differentially modulate sialyl Lewis X-type L-selectin ligand activity. J. Biol. Chem. 278:9953.[Abstract/Free Full Text]
  67. Maly, P., A. D. Thall, B. Petryniak, C. E. Rogers, P. L. Smith, R. M. Marks, R. J. Kelly, K. M. Gersten, G. Cheng, T. L. Saunders, et al 1996. The {alpha}(1,3)fucosyltransferase fuc-TVII controls leukocyte trafficking through an essential role in L-, E-, and P-selectin ligand biosynthesis. Cell 86:643.[Medline]



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