Topographic requirements and dynamics of signaling via L-selectin on neutrophils

Chad E. Green, David N. Pearson, Nadine B. Christensen, and Scott I. Simon

Department of Biomedical Engineering, University of California, Davis, California 95616


    ABSTRACT
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Cross-linking of L-selectin on leukocytes signals phosphorylation of mitogen-activated protein kinases (MAPKs) leading to activation of CD18 function and enhanced transmigration on inflamed endothelium. We examined how alterations in the topography of L-selectin correlate with the dynamics of CD18 activation and phosphorylation of MAPK. Simultaneous ligation of humanized antibodies DREG55 and DREG200 provided a strategy for regulating the extent of cross-linking. Triggering of CD11b/CD18 upregulation and adhesion required clustering of L-selectin to microvillus-sized patches of ~0.2 µm2. Immunofluorescence revealed that L-selectin was colocalized with high-affinity CD18. Anti-L-selectin-coated protein A microspheres indicated that a single site of contact to a 5.5-µm bead, or multiple contacts to 0.94- or 0.3-µm beads, elicited maximum neutrophil activation. Adhesion signaled via L-selectin coincided with the kinetics of MAPK phosphorylation and was inhibited by blocking p38 or p42/44 activity. These data demonstrate the capacity of L-selectin to transduce signals effecting rapid (~1 s) neutrophil adhesion that is regulated by the size and frequency of receptor clustering.

protein kinases; adhesion molecules; antibodies; cellular activation


    INTRODUCTION
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

NEUTROPHILS CONSTITUTE the first line of defense against invading microorganisms and are the major cellular component of an acute inflammatory reaction (5, 44). They circulate in a passive state and become tethered to the vessel wall after the expression of the selectin family of cell surface glycoproteins (18). All three selectins share a similar mosaic structure consisting of three extracellular domains, an NH2-terminal lectin domain followed by an epidermal growth factor domain, and complement regulatory repeat elements (19). L-selectin (CD62L) is constitutively expressed on leukocytes and supports their capture on activated endothelium as well as in homotypic neutrophil aggregation (37). All three selectins bind with relative high affinity to the sialyl Lewisx (sLex) tetrasaccharide and related structures expressed on a variety of glycoprotein surface receptors (18). The best-characterized selectin ligand is P-selectin glycoprotein ligand (PSGL)-1, a heavily glycosylated receptor expressed as a homodimer on leukocytes (24). L-selectin ligands that are expressed in the vasculature are less well characterized (30). One well-defined L-selectin ligand is GlyCAM-1, a heavily glycosylated polysaccharide shown to activate lymphocytes through L-selectin by presenting sulfated sLex ligands in a multivalent manner (11, 15). Other potential L-selectin counterstructures include E-selectin (17, 47), CD34, sulfatides, and heparin sulfate proteoglycans (14, 26, 27).

Capture of neutrophils at vascular sites of inflammation by selectins provides a dynamic contact zone that is thought to facilitate ligation of G protein-linked chemotactic receptors including interleukin (IL)-8 and platelet-activating factor (PAF) (29, 46). This in turn leads directly to the rapid activation of beta 2-integrins (CD11/CD18) that function to decelerate rolling neutrophils and facilitate the transition to cell arrest by forming stable bonds with intercellular adhesion molecules (ICAMs) also upregulated on the endothelium (39). This multistep pattern of molecular recognition and intracellular signaling broadly defines the transition from selectin-dependent cell capture to shear-resistant firm adhesion mediated by activated integrins (36, 46).

Recent published data suggest that selectins function as both adhesion molecules and transmembrane signaling receptors as leukocytes become tethered and roll on inflamed endothelium (35). Clustering L-selectin on the surface of neutrophils with antibody or sulfated polysaccharide mimetics is synergistic with chemotactic stimuli in activating beta 2-integrin (42). Recent studies also suggest that transmembrane signaling via L-selectin enhances the microvascular sequestration of neutrophils at sites of inflammation by inducing rapid cell shape change and reduction in cell deformability that correlate with F-actin assembly and enhanced adhesion via beta 2-integrins and colocalization of L-selectin with CD18 (33, 34). A contribution of L-selectin to neutrophil recruitment in the microcirculation of mice, independent of its function in mediating primary capture to inflamed endothelium, was recently reported. Intravital microscopy revealed that neutrophil attachment and emigration from the vasculature of the cremaster muscle of L-selectin-deficient mice was significantly reduced after stimulation with chemotactic cytokines (12). Moreover, the distance of extravascular migration of activated neutrophils was substantially diminished in L-selectin knockout mice, despite equivalent numbers of rolling leukocytes in knockout and wild-type animals. Finally, chemotaxis of neutrophils into inflamed tissue was shown to involve p38 mitogen-activated protein kinase (MAPK) activity (6). Together, these studies suggest that L-selectin has the capacity to rapidly transduce signals that alter cell mechanical properties and the rate and extent of CD18 adhesion. By hastening the transition from cell rolling to arrest, L-selectin appears to regulate the efficiency of neutrophil recruitment to inflamed tissue. However, a detailed picture of how L-selectin ligation and membrane redistribution are associated with these functions is lacking.

In this study, we pursued the hypothesis that signal transduction via L-selectin is regulated by the dynamics and extent of receptor clustering and, moreover, that activation of beta 2-integrins is spatially associated with sites of L-selectin clustering. The strategy we employed was to systematically increase the frequency and extent of L-selectin clusters and to correlate this with the rate of beta 2-integrin surface upregulation and adhesion. We then examined the role of p38 and p42/44 MAPKs in signaling and correlated the dynamics of phosphorylation with triggering of beta 2-integrin function.


    MATERIALS AND METHODS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
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Isolation of neutrophils. Venous blood was collected from healthy adult donors into a sterile syringe containing 10 U/ml heparin. Neutrophils were isolated with a one-step Ficoll-Hypaque gradient medium from Amersham (Piscataway, NJ). The neutrophil-containing band was isolated from the gradient with an intravenous catheter and washed once with Ca2+-free HEPES buffer (HHB; in mM: 30 HEPES pH 7.4, 110 NaCl, 10 KCl, 1 MgCl2, 10 glucose). Cells were resuspended and kept at 4°C in a Ca2+-free HHB containing 0.1% human serum albumin (HSA). The isolated cells were used immediately after isolation. CaCl2 was added to a final concentration of 1.5 mM in the buffer before use. Cells isolated by this method yield >90% pure neutrophil suspension that is ~95% viable.

Agonists, inhibitors, and monoclonal antibodies. Humanized IgG4 forms of DREG200 and DREG55 antibodies (7) were generously provided by Protein Design Labs (Mountain View, CA). The DREG antibodies bind to distinct epitopes within the lectin domain of L-selectin (10). These IgG molecules were bound to neutrophils and cross-linked with either polyclonal goat anti-human IgG (H+L) F(ab')2 antibody or polyclonal goat anti-mouse IgG (H+L) F(ab')2 antibody to the Fc domains (KPL, Gaithersburg, MD). Anti-L-selectin antibody Lam1.3 was kindly provided by Cell Genesys (Foster City, CA). Lam1.3 binding has been mapped to the lectin domain of L-selectin (41). Anti-CD43 (Dako, Carpinteria, CA), a sialoglycoprotein constitutively expressed on human neutrophils, was used as a control binding antibody. Lam1.3 and anti-CD43 were immobilized on protein A-coated microspheres of 0.3-, 0.94-, or 5.5-µm diameter (Bangs Laboratory, Fishers, IN). The p38 MAPK inhibitor SB-202190 (Biomol Research Laboratories, Plymouth Meeting, PA) and the p42/44 kinase inhibitor PD-98059 (Calbiochem, La Jolla, CA) were dissolved in DMSO and stored frozen. The ligand-coated protein A beads were prepared by first washing the beads three times with PBS buffer followed by sonication for dispersal. Lam1.3 monoclonal antibody (MAb) was added at the indicated concentrations in a 100-µl volume and bound to beads by incubation for 60 min at 20°C with continuous mixing. The beads were then washed three times with HHB and dispersed by sonication.

Antibody coating of protein A beads. Quantum Simply Cellular Beads (QSCBs) (Bangs Labs) were used to quantify the antibody-binding capacity of anti-L-selectin on the surface of the protein A beads. QSCBs consist of five distinct bead populations with discrete binding capacities of goat anti-mouse IgG on their surface ranging from ~0 to 200,000 sites/bead. QSCBs are used to generate a standard curve of the antibody-binding capacity relative to the mean fluorescence intensity (MFI). Singlet QSCBs are gated on the FACScan, and the MFIs for all five bead populations are determined. The antibody-binding capacity is determined by regression of the MFI for each of the bead populations with the known binding capacity for each of the bead populations to generate the antibody-binding capacity standard curve. To determine the antibody site density on the surface of the 5.5-µm and 0.95-µm protein A beads, anti-L-selectin was bound at the indicated concentration (see Fig. 4A) and the MFI for the bead populations was determined. The antibody site density on the surface on the protein A beads was determined with the QSCB standard curve as described in the manufacturer's protocol.

Neutrophil activation. Upregulation of surface CD11b/CD18 (Mac-1) was detected by the binding of phycoerythrin (PE)-conjugated anti-human Mac-1 antibody (2LPM19C; Dako, Carpinteria, CA) and fluorescence flow cytometry (FACScan; Becton Dickinson, San Jose, CA). Mac-1 upregulation is expressed as fold increase from cells incubated in buffer alone at 37°C. Mac-1-dependent adhesion was quantitated with a bead adhesion assay as previously described (33). Briefly, 2-µm carboxylated fluorescent latex beads (Molecular Probes, Eugene, OR) were washed three times with HHB buffer and incubated in HHB with 0.25% HSA and 1.5 mM CaCl2 at 20°C for 60 min with periodic sonication. Cell suspensions were prepared at a 40-to-1 ratio of albumin-coated latex beads (ACLBs) to cells. Neutrophils (5 × 105) were sheared with 2 × 107 ACLBs in a final volume of 0.25 ml of HHB, 0.1% HAS, and 1.5 mM CaCl2 with a 5-mm magnetic stir bar as previously described (33).

HuDREG55 and -200 antibodies were used to activate human neutrophils by systematically increasing the valency of binding and cross-linking L-selectin. Treatment with either HuDREG55 or HuDREG200 alone results in a combination of monovalent and bivalent antibody binding to L-selectin, as each arm of the whole IgG is capable of binding a single target epitope per receptor. When HuDREG55 and HuDREG200 are added in combination, the potential for both antibodies to bind the same L-selectin molecule results in multivalent MAb binding and a moderate level of L-selectin clustering. Maximal clustering of L-selectin is achieved by adding secondary polyclonal anti-human F(ab')2 fragments to neutrophils bound with single HuDREG IgG or both HuDREG55 and HuDREG200 in combination. In each case, the secondary anti-human F(ab')2 fragments cross-link individual HuDREG antibodies, creating L-selectin clusters, as identified by highly fluorescent regions on the neutrophil membrane.

To stimulate neutrophil adhesion, anti-L-selectin antibodies were first bound to the neutrophil at room temperature for 10 min. Cells were then centrifuged to remove unbound ligand. L-selectin cross-linking was initiated by the addition of goat anti-human F(ab')2 antibodies (10 µg/ml) as described above followed by incubation at 37°C. Human IgG (10 µg/ml; Ancell, Bayport, MN) alone and goat anti-human F(ab')2 alone were used as antibody controls. Cell-bead collisions were induced by shearing the cell suspension in a test tube at a rotation rate of ~300 rpm corresponding to a shear rate of 100 s-1 and an estimated average shear stress of <1.0 dyn/cm2. The mixing assembly of magnetic stir bar and test tube was placed upstream from the sample inlet of a FACScan flow cytometer to measure the kinetics of neutrophil-bead adhesion in real time. In experiments using chemotactic stimulation, IL-8 or N-formylmethionyl-leucyl-phenylalanine (fMLP) was added at time zero just before application of shear in the mixing tube. In the MAPK inhibition studies, neutrophils were pretreated with SB-202190 (10 µM) or PD-98059 (100 µM) at 37°C for 45 min (concentrations chosen based on titration studies to obtain a optimal inhibition). The pretreated cells were then stimulated as described for the ACLB assay, but with kinase inhibitor present throughout the kinetic assay. The final concentration of DMSO in the reaction was <= 0.1%.

Neutrophils and beads were differentiated by forward light scatter and fluorescence emission in the green or FL1 wavelength. A live gate was set on a forward vs. side scatter dot plot to limit data acquisition to neutrophils and neutrophil/bead events, excluding unbound beads. 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. Neutrophils devoid of bound beads emitted a background level of green fluorescence. The mean number of beads bound per neutrophil was detected as quantal increases in green fluorescence and calculated as previously described (43).

Western analysis of p38 and p42/44 MAPK. Neutrophil suspensions (2 × 107 cells) were first incubated with HuDREG55 at 2,000 × Kd (80 nM) for 10 min at 24°C to saturate binding sites. At time zero on the x-axis of Fig. 6B, cells were washed and stimulated by addition of secondary goat-anti-human F(ab')2 antibody fragments at 37°C. For comparison, cells were also stimulated with TNF-alpha (1 ng/ml). A 90-µl aliquot containing 2 × 106 cells was removed at the indicated time points, centrifuged, aspirated, frozen in liquid N2, and then stored at -70°C. The cell extracts were prepared by thawing the cells on ice in the presence of lysis buffer containing 50 mM HEPES, pH 7.5, 1% (vol/vol) Triton X-100, 2 mM sodium orthovanadate, 10 mM NaF, 1 mM EGTA, 2× protease cocktail inhibitors (Boehringer Mannheim, Indianapolis, IN), and 1 mM phenylmethylsulfonyl fluoride (PMSF) for 1 h with periodic mixing. Cell debris was pelleted at 18,000 g for 15 min at 4°C. The clarified supernatant was transferred to a fresh tube and kept frozen at -70°C. The protein content of the extracts was determined with a Bradford-based protein assay kit (BioRad, Hercules, CA) with HSA as the standard. The protein extracts were separated by 1-mm-thick 10% SDS-PAGE, with each lane containing equal quantities of protein extract from each treatment (15-60 µg). After electrophoresis, protein was transferred to polyvinylidene difluoride (PVDF) membrane in 25 mM Tris (pH 8.3), 192 mM glycine, and 20% (vol/vol) methanol at 100 V for 1 h with cooling. After transfer, the membrane was rinsed with distilled water and air dried. Nonspecific binding to the membrane was eliminated by overnight incubation in Tris-buffered saline, pH 7.5 and 0.1% (vol/vol) Triton X-100 (TTBS), 0.2% (wt/vol) I-Block (Applied BioSystems, Foster City, CA), and 0.01% sodium azide at 4°C. The membrane was probed with 1:5,000 and 1:1,000 dilutions of 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, overnight at 4°C in blocking buffer. The membranes were washed again with TTBS at 20°C with shaking and then probed with biotinylated secondary goat anti-rabbit IgG at a dilution of 1:20,000 for 60 min at 20°C in blocking buffer. After antibody incubation, membranes were washed with TTBS followed by a 30-min incubation with Avidix-APTM (Applied BioSystems) at 1:50,000 in blocking buffer. The membranes were then developed for chemiluminescent detection with the Western-Light Plus protocol from Applied BioSystems. The developed film was digitized before densitometry image analysis with NIH Image 1.62.

Immunofluorescence. Neutrophils (5 × 105/ml) were preincubated at 24°C for 10 min with anti-CD45-FITC (Caltag Laboratory, Burlingame, CA), anti-CD11c-PE (Caltag Laboratory), anti-CD18 (R15.7; generously provided by Robert Rothlein, Boehringer Ingelheim, Ridgefield, CT) or, in combination, 1,000 × Kd (40 nM) HuDREG55-FITC and 1,000 × Kd (40 nM) HuDREG200-FITC. After incubation, cells were washed and resuspended in HEPES buffer (containing 0.1% HSA and 1.5 mM CaCl2) and placed on ice. Cross-linking of primary HuDREG or control antibodies was achieved by incubating samples with either secondary goat-anti-human or goat anti-mouse F(ab')2 (10 µg/ml) antibody fragments for 10 min at 37°C. High-affinity CD18 was detected with MAb 327C-Alexa (generously provided by Don Staunton, ICOS, Bothell, WA), a monoclonal antibody that recognizes an epitope exposed on the I-like domain of activated CD18. For two-color imaging of L-selectin and 327C colocalization, a PE-conjugated anti-CD62L antibody (DREG56; Immunotech, Marseille, France) was used in conjunction with nonlabeled HuDREG antibodies. CD11b/CD18 (Mac-1) was detected with a PE-conjugated anti-human CD11b antibody (2LPM19C; Dako). Cell suspensions were labeled on ice for 10 min and then washed and fixed in HEPES buffer containing 1.0% paraformaldehyde. Labeled cells were imaged with a Nikon TE200 inverted microscope employing a ×60 oil-immersion Plan-Apo objective with the appropriate band-pass filters for FITC or PE labels. Images were captured with a charge-coupled device (CCD) camera (Dage-MTI, Michigan City, IN) and then analyzed with Image Pro Plus v4.5 software (Media Cybernetics, Silver Spring, MD). We define a cluster as a localized region of the membrane where pixel intensity is at least threefold greater than background fluorescence intensity. With the analysis software, pixel maps are obtained for each cell visualized. Pixel intensity values are unitless and range from 0 to 255. A threshold intensity value based on these values and the image of the cell is chosen and set, which represents the average background intensity over the surface. This value typically ranges from 80 to 120, with clusters achieving off-scale values that are assigned a maximum intensity value of 255 by default. After thresholding, the number of fluorescent clusters, surface area (µm2) per cluster, and percent colocalization (if appropriate) were determined as reported in Table 1. Images and values represent averages from 60-90 cell observations per treatment from three separate experiments.

                              
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Table 1.   Topographic analysis of L-selectin and CD45 clustering in response to anti-L-selectin selectin antibody binding

Electron microscopy of neutrophils and beads. After the preparation of Lam1.3-coated beads bound to neutrophils, neutrophils were postfixed by suspending in a 2% glutaraldehyde solution overnight at 4°C. Samples were then resuspended in agarose and fixed in 2% buffered OsO4 and dehydrated in steps with ethyl alcohol. Spurrs resin was used as the embedding medium. A transmission electron microscope (H-600; Hitachi, Tokyo, Japan) was used to image the interface at the neutrophil-bead margin at ~×5,000 magnification.

Determination of neutrophil/microsphere contact area. Neutrophils were incubated with maximal dose anti-L-selectin-coated microsphere. Cells with attached microspheres were observed under bright-field phase-contrast microscopy with a ×60 oil-immersion lens, as depicted in Fig. 4B. The contact area was then determined by measuring the length of the cell-microsphere interface from 25 digitized images with reference to a calibrated reticule slide. The contact area was calculated with the formula
SA = <FR><NU>&pgr;</NU><DE>2</DE></FR><IT> D</IT><FENCE><IT>D−</IT><RAD><RCD><IT>D</IT><SUP>2</SUP><IT>−L</IT><SUP>2</SUP></RCD></RAD></FENCE>
where D is the microsphere diameter and L is the length of the cell-microsphere contact. This calculation of surface area (SA) is based on the assumption of a circular and continuous area of contact. Furthermore, it is assumed that the area of microsphere contact is based on the presumption that L is measured in the central plane (e.g., maximum length of contact). The assumption of continuous membrane contact between the microsphere and neutrophil was confirmed by transmission electron microscopy (TEM) analysis of >25 cells in which >90% revealed close apposition (Fig. 4B). For the 5.5-µm beads, L was 1.64 µm with an SE of 0.06. For the 0.94-µm beads, L was 0.5 µm with an SE of 0.03. To estimate the measurement error in contact area, the mean value Lavg was computed at the upper and lower values of the SE. Given these values, SA is estimated to be 2.15 ± 0.16 µm2 for the 5.5-µm beads and 0.21 ± 0.03 µm2 for the 0.94-µm beads.

Statistical analysis. Analysis of data was performed with GraphPad Prism version 3.0 software (GraphPad Software, San Diego, CA). All data are reported as means ± SE. Nonparametric grouping of data was analyzed by ANOVA and secondary analysis for significance with a Tukey or Newman-Keuls multiple-comparison test. Gaussian-distributed mean values were analyzed by Student's t-test. Group comparisons were deemed significant at P < 0.05.


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Binding kinetics of anti-L-selectin HuDREG antibodies on neutrophils. We first characterized the binding of humanized monoclonal anti-L-selectin (HuDREGs) antibodies to L-selectin on the surface of human neutrophils. HuDREG55 and HuDREG200 recognize distinct epitopes within the lectin domain of L-selectin and can bind simultaneously without displacement or steric repulsion. A sigmoidal curve fit the binding of antibody as a function of concentration to neutrophils (Fig. 1). Fluorescent conjugated HuDREG MAbs bound with a Kd of 0.04 and 0.05 nM for HuDREG55 and HuDREG200, respectively, as computed with nonlinear regression analysis of the dose response.


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Fig. 1.   Binding of anti-L-selectin antibodies on the surface of neutrophils. Increasing concentrations of fluorescein-conjugated HuDREG antibodies were incubated with 1 × 106 neutrophils/ml at 20°C. Cells were analyzed by flow cytometry at 37°C after removal of unbound antibody. Each data point is the mean fluorescent intensity (MFI) of bound antibody from 5,000 cells collected from 3 separate experiments. Curves were fit to binding isotherms with GraphPad Prism version 3.0 software. MAb, monoclonal antibody.

Mac-1 upregulation in response to increased clustering of L-selectin. We next examined the correlation between clustering of L-selectin by HuDREGs and neutrophil activation as quantitated by upregulation of surface Mac-1 (Fig. 2). Neutrophils were incubated with increasing concentrations of either a single HuDREG or HuDREGs added in combination up to 2,000 × Kd. Although this dose of HuDREG corresponds to ~500-fold above that required for ~99% receptor occupancy, it is in the range typically used to effectively block L-selectin-dependent capture and rolling on activated endothelium or in a soluble sLex binding assay (2, 4). Treatment with a single HuDREG even at 2,000 × Kd did not elicit significant membrane upregulation of Mac-1 above the baseline detected in response to addition of a humanized IgG isotype control (Fig. 2A). In contrast, a dose-dependent increase in Mac-1 expression was achieved by saturation of L-selectin with a single HuDREG (e.g., HuDREG55 at 1,000 × Kd) and titration of the second over a 100-fold range from 10 up to 1,000 × Kd. Treatment with combined HuDREG55 and HuDREG200 at 1,000 × Kd induced the maximum increase of ~50% upregulation of Mac-1 over the isotype control.


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Fig. 2.   Upregulation of CD11b/CD18 stimulated by anti-L-selectin. Neutrophils (1 × 106/ml) were incubated for 10 min at 24°C with the indicated concentration of HuDREG55 and/or HuDREG200. Antibody concentrations are given as fold increase relative to the Kd for HuDREG binding (see Fig. 1). After removal of excess HuDREG by centrifugation, neutrophils were incubated without (A) or with (B) secondary polyclonal antibody (cross-linker at 10 µg/ml) for 10 min at 37°C to initiate cell activation. Neutrophils were then assayed for CD11b/CD18 upregulation by flow cytometry with the anti-Mac-1 antibody 2LPM19C-phycoerythrin (PE). Human IgG alone and cross-linker alone were used as controls for A and B, respectively. Bars indicate fold increase in CD11b/CD18 expression over neutrophils incubated in buffer alone (control) at 37°C. Statistical significance between samples as shown was determined from 3 separate experiments.

To achieve activation by ligation of L-selectin with a single HuDREG it was necessary to cross-link the primary MAb with a goat-anti-human polyclonal secondary antibody against the heavy and light chains of human IgG. Cross-linking L-selectin yielded a dose dependence in Mac-1 upregulation that was significant at 1 × Kd of HuDREG binding and increased with receptor occupancy, reaching maximum response at 100 × Kd (Fig. 2B). Cross-linking HuDREGs added in combination proved to be the most efficient means of activation, because 30-75% more Mac-1 was upregulated compared with cross-linking a single HuDREG. The efficacy of this treatment is revealed by the observation that only 0.5 × Kd of combined and cross-linked HuDREGs triggered CD18 upregulation that was equivalent to binding a single HuDREG at 1,000 × Kd. Maximum activation at 1,000 × Kd of combined HuDREGs achieved activation comparable to that obtained with the chemotactic stimuli IL-8 or formyl peptide (Fig. 2B). Together, the data reveal a threshold in L-selectin ligation necessary to trigger neutrophil activation and amplification as a function of the extent of antibody cross-linking.

Topography of L-selectin after binding and cross-linking with anti-L-selectin. We next visualized the membrane distribution of L-selectin on neutrophils after treatment with fluorescein-conjugated HuDREGs in the presence and absence of secondary cross-linker by fluorescence microscopy. Treatment with HuDREG55 revealed a uniform circumferential distribution of L-selectin punctuated with small submicrometer domains of fluorescence clusters (Fig. 3a; Table 1). Subsequent cross-linking of HuDREG55 with secondary polyclonal antibody resulted in a fourfold increase in the area, but not the frequency, of L-selectin clusters (Fig. 3b; Table 1). An identical result was observed for HuDREG200 (data not shown). Incubation of neutrophils with combined HuDREGs elicited a redistribution of L-selectin into clusters that were on average threefold greater than single HuDREG (Fig. 3c; Table 1). Cross-linking combined HuDREGs with secondary polyclonal antibody resulted in a doubling of the frequency and area of L-selectin clusters (Fig. 3d; Table 1). As a control, neutrophils were treated with anti-CD45, which exhibited a uniform fluorescence distribution with no significant clustering (Fig. 3e). Cross-linking of CD45 with goat anti-mouse polyclonal antibody triggered redistribution into patches of fluorescence that were significantly larger than cross-linked L-selectin and less uniformly distributed on the neutrophil surface (Fig. 3f; Table 1). In contrast to L-selectin, clustered CD45 did not result in cellular activation as determined by CD11b/CD18 upregulation (33).


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Fig. 3.   Topography of L-selectin in response to cross-linking. Immunofluorescence microscopy was used to detect the distribution of L-selectin in freshly isolated neutrophils after labeling with HuDREG55-FITC (1,000 × Kd; a); HuDREG55-FITC at (1,000 × Kd; b) followed by cross-linking with goat anti-human F(ab')2; HuDREG55-FITC and HuDREG200-FITC (1,000 × Kd each; c); HuDREG55-FITC and HuDREG200-FITC followed by cross-linking with goat-anti-human F(ab')2 (d); control anti-CD45-FITC (e); and CD45-FITC cross-linked with goat anti-mouse F(ab')2 (f). Average number of fluorescent clusters and average area per cluster for each treatment were calculated based on 60-90 cell images from 3 separate experiments with Image Pro Plus 4.5 analysis software (see Table 1).

Anti-L-selectin-coated beads activate neutrophils. Analysis of L-selectin surface topography confirmed a correlation between the frequency and extent of L-selectin clustering and triggering of neutrophil activation. We further examined this by clustering L-selectin on the neutrophil surface by contact with protein A microspheres (diameter 0.3, 0.9, or 5.5 µm) coated over a range of anti-L-selectin concentrations. The number of antibodies bound per bead was determined for the 5.5- and 0.9-µm beads by comparison with a calibration set of beads containing defined numbers of antibody binding sites (Fig. 4A). Neutrophil suspensions were shear mixed with antibody-coated beads, and the average number bound per neutrophil was measured by phase-contrast microscopy, while the extent of Mac-1 upregulation was detected by flow cytometry. A dose-dependent increase in upregulation was elicited by the binding of beads, regardless of bead diameter. Stimulation increased most rapidly over the lower range of anti-L-selectin coating concentrations (0-100 nM) for the 0.9-µm and 5.5-µm beads compared with the 0.3-µm beads (Fig. 4B). This is the range in which MAb bound to beads increased most rapidly with coating concentration. At the highest anti-L-selectin coating concentration (~350 nM), Mac-1 upregulation was not statistically different between each bead diameter. At this concentration, neutrophils bound on average a single 5.5-µm bead, whereas they bound eight of the 0.9-µm beads and many more 0.3-µm beads (estimated to be >10 beads per cell) (Fig. 4C). Beads (0.9 and 5.5 µm) coated with anti-CD43 did not significantly upregulate CD18 beyond baseline at the maximum coating concentration (data not shown).


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Fig. 4.   Dose response for activation of CD11b/CD18 upregulation by anti-L-selectin coated microspheres. A: antibody binding capacity of 5.5-µm and 0.9-µm protein A-coated beads over the indicated range of antibody coating concentration was determined with Quantum Simply Cellular Beads (QSCBs) as described in MATERIALS AND METHODS. B: protein A-conjugated microspheres of 0.3-, 0.9-, and 5.5-µm diameter were coated with anti-L-selectin (LAM1.3) at various concentrations ranging from 0 to 700 nM. After removal of excess antibody, the coated beads were mixed with neutrophils at a bead-to-neutrophil (PMN) ratio of 20:1 for 10 min at 37°C. CD11b/CD18 upregulation was quantitated by flow cytometry and expressed relative to that obtained with beads coated by a control IgG. Data points represent means ± SE from 2-5 separate experiments, and curves were fit to the data with nonlinear regression analysis. * Significantly higher mean value for the 5.5-µm beads over the 0.3-µm beads (P < 0.05). C: no. of anti-L-selectin-coated beads bound to neutrophils as a function of bead diameter. Beads were coated with anti-L-selectin MAb at 700 nM, corresponding to maximum upregulation of CD11b/CD18. The average number of beads (mean ± SE) of each diameter bound per neutrophil was determined with a ×60 phase-contrast microscopy. Representative images showing adherent beads, as well as a transmission electron microscopy (TEM) image of the contact site between a 5.5-µm bead and a neutrophil, are shown for comparison. Beads attached to neutrophils were enumerated on 25-50 cells from 3 separate experiments.

These data indicate that a single site of membrane contact to a 5.5-µm bead was sufficient to elicit maximal neutrophil activation. We confirmed by TEM that beads formed a continuous area of contact with the neutrophil over an area of 2.15 ± 0.16 µm2 as measured by phase-contrast microscopy. Alternatively, multiple sites of contact to 0.9-µm beads over an area of 0.21 ± 0.03 µm2 was also effective at activating Mac-1 upregulation. The specificity of the response was confirmed by coating beads with a control isotype-matched antibody, which did not yield significant upregulation above control levels obtained in presence of beads without bound antibody (data not shown). Together, the data indicate that transmembrane signaling via L-selectin increases with site density of MAb ligation at a single site of contact but also in proportion to an increase in the frequency of bead contacts.

Kinetics of CD11b/CD18 activation and MAPK activity after clustering of L-selectin. A sensitive method for assessing the adhesive function of beta 2-integrin is real-time detection of the capture of ACLBs by neutrophils under fluid shear with flow cytometry. By using this assay it is possible to correlate the rate at which both constitutively expressed and upregulated CD11b/CD18 participate in adhesion function (31). We previously reported (35) that Mac-1 is activated to bind ACLBs after cross-linking of L-selectin at 2,000 × Kd of HuDREG. Here we compare the dose dependence of Mac-1 adhesion in response to binding and cross-linking over a wide concentration range of HuDREG (Fig. 5A). Treatment of cells with HuDREG55 elicited a dose-dependent increase in bead adhesion, but only on addition of secondary cross-linking antibody. As with upregulated expression of CD11b/CD18, a significant increase in bead capture was achieved at antibody concentrations corresponding to only 1 × Kd in the presence of cross-linker.


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Fig. 5.   CD11b/CD18-dependent neutrophil adhesion to albumin-coated latex beads (ACLBs) in response to cross-linking L-selectin. A: neutrophils were stimulated by incubation with the indicated dose of agonist and then washed and resuspended in buffer with () or without () goat anti-human F(ab')2 cross-linker (10 µg/ml). CD11b/CD18 upregulation was detected by continuously monitoring the capture of 2-µm ACLBs in a sheared cell suspension by flow cytometry. Data points represent the mean ± SE no. of adherent beads/neutrophil after 10 min of shear and stimulation. Each point represents 2-4 separate experiments. * Significant difference (P < 0.05) between samples in the presence and absence of cross-linker. B: kinetics of ACLB capture was monitored continuously for 6 min after neutrophil stimulation with the indicated agonist. For L-selectin cross-linking, neutrophil suspensions were preincubated with 2,000 × Kd HuDREG55 for 10 min at 20°C. Neutrophils were then stimulated at t = 0 by N-formylmethionyl-leucyl-phenylalanine (fMLP; 0.1 µM), interleukin-8 (IL-8; 1 nM), or HuDREG55 cross-linked with goat-anti-human F(ab')2 (10 µg/ml). Stimulated neutrophil-bead suspensions were immediately placed in the shear chamber upstream of the flow cytometer. Samples were recorded continuously, with each data point representing the average number of beads bound to ~150 neutrophils over a 5-s sampling interval. Data points represent means ± SE from 3 independent experiments. C: expansion of adhesion kinetics (see Fig. 6B) over the initial 100 s after application of shear and stimulation. Linear regression analysis was used to compare the rate of bead capture between stimuli. * Statistically greater rate of bead capture after fMLP stimulation (P < 0.001).

We next compared the dynamics of activation in response to binding of anti-L-selectin or chemoattractants in sheared cell suspensions. Neutrophils and ACLBs in suspension were exposed to stimulus and fluid shear while bead capture was continuously recorded on the flow cytometer (Fig. 5B). Stimulation with IL-8, fMLP, or HuDREG was most rapid over the initial minute of stimulation and resulted in a sustained increase in CD11b/CD18-mediated bead capture that reached a plateau by ~10 min. Stimulation with IL-8 (1 nM) or cross-linked HuDREG55 (2,000 × Kd) elicited similar kinetic rates of capture over the first 100 s (Fig. 5C). In contrast, stimulation with fMLP (1 µM) activated a twofold increase in the rate of capture over IL-8 or HUDREG55.

Previous work by our laboratory (35, 40) demonstrated a central role for p38 MAPK in signaling of neutrophil degranulation and adhesion via beta 2-integrin in response to L-selectin cross-linking. To delineate the relative role of the p38 and p42/44 MAPKs in the dynamics of CD11b/CD18 adhesion triggered via clustered L-selectin, neutrophils were pretreated with the specific p38 inhibitor SB-202190 or the p42/44 inhibitor PD-98059 and the rate of bead capture was assessed (Fig. 6A). To control for the potential toxic effects of the inhibitors on cell function, we pretreated cells and assessed bead capture in response to fMLP stimulation. As expected, the inhibitors did not affect G protein-mediated signaling of adhesion, suggesting that they were nontoxic at the doses applied. In contrast, stimulation by cross-linking anti-L-selectin was blocked to baseline (e.g., ~85% inhibition) with treatment by either the p38 or the p42/44 inhibitor. Combined pretreatment with both inhibitors failed to further decrease the rate of bead capture observed for the individual inhibitors alone (data not shown).


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Fig. 6.   L-selectin signals neutrophil adhesion via mitogen-activated protein kinase (MAPK). A: neutrophils were preincubated for 45 min at 37°C with either p38 inhibitor SB-202190 (10 µM) or p42/44 kinase inhibitor PD-98059 (100 µM). Cells were then stimulated with fMLP (0.1 µM) or HuDREG55 (2,000 × Kd) with goat anti-human F(ab')2 (10 µg/ml) cross-linker. Data are expressed as %inhibition of bead capture over the first 100 s compared with an untreated fMLP (0.1 µM) control. Each point represents data from 2-4 independent experiments. B: time course of p38 and p42/44 MAPK phosphorylation after neutrophil stimulation. Western blot analysis was used to detect the dual phosphorylated form of p38 and p42/44 MAPK after neutrophil stimulation with cross-linked HuDREG55 (2,000 × Kd) or TNF-alpha (1 ng/ml). HuDREG55 antibody was prebound to neutrophils by incubation for 10 min at 20°C. Cells were then activated at 37°C (t = 0) with goat anti-human F(ab')2 cross-linker (10 µg/ml) or the indicated agonist. The effect of MAPK inhibition on phosphorylation was examined at t = 3 min by cell preincubation for 45 min with either p38 inhibitor SB-202190 (10 µM) or p42/44 kinase inhibitor PD-98059 (100 µM) before cross-linking of HuD55. Relative intensity of p38 and p42/44 MAPK signal was quantitated by densitometry for all time points and expressed as fold increase normalized by the IgG control. Data represent the average pattern of phosphorylation observed in 3 separate experiments.

Given the rapid kinetics by which cross-linking L-selectin signaled neutrophil adhesion, we next examined the time course of p38 and p42/44 MAPK phosphorylation after stimulation. Phosphorylation of threonine and tyrosine residues on p38 (Thr180/Tyr182) and p42/44 (Thr202/Tyr204) was quantitated by densitometry of Western blots from lysates of neutrophils treated with cross-linked anti-L-selectin, TNF-alpha , or cross-linked human IgG control (Fig. 6B). Specific rabbit polyclonal antibodies that report on the active, phosphorylated forms of p38 and p42/44 produced bands at the expected molecular weights. As a positive control, neutrophils were stimulated with TNF-alpha , previously shown to be a potent stimulus of p38 phosphorylation and Mac-1 upregulation. Addition of secondary cross-linker to HuDREGs at time zero elicited a rapid rise in both p38 and p42/44 phosphorylation that peaked at three- to fourfold above the baseline activity by 1 min and stayed high up to 3 min (Fig. 6B). Stimulation with TNF-alpha elicited a comparable increase in p38 and p42/44 phosphorylation that was sustained beyond 3 min. The inhibitor PD-98059 reduced p42/44 phosphorylation fourfold at 3 min; however, SB-202190, which is known to only inhibit the alpha - and beta -isoforms of p38, did not inhibit total p38 phosphorylation.

Clustered L-selectin colocalizes with activated beta 2-integrin. We next examined the mechanisms by which clustered L-selectin could signal CD18 adhesion dynamics on par with that elicited through chemotactic stimulation. As shown in the two-color immunofluorescence images of Fig. 7, cross-linking HuDREGs induced clusters of L-selectin that colocalized with 327C. This antibody binds to an epitope exposed on the I-like domain of CD18 and reports on the active ligand-binding conformation (1, 23). In the absence of secondary cross-linking, L-selectin cluster area was small and coincided with minimal CD18 activation and no colocalization between L-selectin and 327C (Fig. 7a). However, on cross-linking, 60 ± 12% of activated CD18 colocalized with clustered L-selectin (Fig. 7b). Further analysis indicated that there were 3.0 ± 0.94 clusters of 327C per cell with an area of 0.37 ± 0.21 after L-selectin cross-linking. Colocalization was found to be specific to activated CD18, because cross-linking L-selectin did not elicit colocalization of CD11c expressed on neutrophils (Fig. 7c). Moreover, activation of CD18 was specific to L-selectin, because cross-linking CD18 or CD11c did not elicit significant 327C expression (Fig. 7, d and e). We next assessed whether CD18 activation and colocalization with cross-linked L-selectin involved signal transduction via p38 MAPK. Pretreatment of neutrophils with SB-202190 effectively eliminated expression of 327C in response to cross-linked L-selectin (Fig. 7f). By comparison, we confirmed that blocking p38 activity did not inhibit CD18 activation or clustering in response to stimulation with IL-8 (data not shown).


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Fig. 7.   Colocalization of high-affinity CD18 with clustered L-selectin. Neutrophils were activated as indicated by preincubation at room temperature for 10 min with HuDREG55/200 (1,000 × Kd of each antibody), anti-CD11c (10 µg/ml), or anti-CD18 (10 µg/ml) and then activated at 37°C with F(ab')2 cross-linker (10 µg/ml). Cell suspensions were then labeled on ice with anti-L-selectin-PE (a, b) and 327C-Alexa (c) HuDREG55/200-FITC and anti-CD11c-PE (d), 327C-Alexa and anti-CD11c-PE 327C-Alexa (e), and 327C-Alexa in presence of p38 MAPK inhibitor SB-202190 (10 µM; f). Cell suspensions were immediately fixed and visualized at ×60 on an inverted fluorescence microscope equipped with filters appropriate for FITC and PE. Color overlay and colocalization parameters were determined with Image Pro Plus analysis software. Cells shown are representative of at least 25 individual cells.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

This study demonstrates a direct correlation between the extent of L-selectin clustering and transmembrane signaling of neutrophil adhesion. With a combination of two humanized anti-L-selectin MAbs in development as anti-inflammatory agents (7), a threshold in the area of L-selectin clustering required to trigger neutrophil activation was revealed. Cross-linking with two bivalent anti-L-selectin antibodies was necessary and sufficient to induce membrane patching that correlated with sites of CD18 activation and rapid induction of CD11b/CD18-dependent adhesion with kinetics that paralleled the phosphorylation of p38 and p42/44 MAPK. The data establish a hierarchy in the relative number and site density of clustered L-selectin associated with signaling adhesion of neutrophils.

A common in vitro strategy used to simulate ligand-induced receptor clustering and cellular activation via L-selectin is by ligation and cross-linking of MAbs. Previous reports demonstrated that binding of L-selectin with a single anti-L-selectin was insufficient to trigger Mac-1 function even at doses several thousandfold in excess of that required to saturate L-selectin receptors (33). The current study extends these findings by showing that simultaneous binding of HuDREG55 and HuDREG200 exceeded this threshold for neutrophil activation but only at excessive concentrations of MAb. Evidence correlating the level of cellular activation with the extent of clustering was the finding that secondary cross-linking of HuDREGs with polyclonal antibody decreased the concentration required to trigger activation by 1,000-fold to a level corresponding to just ~50% of L-selectin saturation. This applied to HuDREG bound individually at 1 × Kd or combined at 0.5 × Kd. Remarkably, combined HuDREGs amplified Mac-1 activation by 100% over single MAb treatment and, at maximum stimulation, elicited Mac-1 upregulation on par with chemotactic stimulation by IL-8 or fMLP.

On the basis of the immunofluorescence of L-selectin clustering, we speculate that the large excess of MAb necessary to trigger neutrophil activation reflects a requirement for continuous receptor occupancy of HuDREG55 and -200 to their respective epitopes on L-selectin. Given the affinity and valency of HuDREGs, addition of a single HuDREG at saturation results in dimerization by binding of each Fab on the IgG to a single lectin domain of L-selectin (Fig. 8). Bivalent ligation is apparently insufficient to elicit significant receptor clustering or neutrophil activation. Rather, further constrainment and aggregation of receptors by simultaneous occupancy of both HuDREGs recognizing distinct epitopes on the lectin domain was necessary and sufficient to trigger activation. Immunofluorescence data confirmed that combined HuDREGs elicited a redistribution of L-selectin from a uniform punctate expression in the presence of single MAb to one of dense clusters of fluorescence. This correlated with a threefold increase in the area of L-selectin clusters and increased neutrophil activation (Table 1). Further patching of L-selectin with secondary cross-linker doubled again the area and frequency of clusters that correlated with potent activation. Patching of receptors in response to the clustering of a primary MAb by a secondary polyclonal antibody is not unique to L-selectin, as shown by the patching of fluorescent anti-CD45. However, the correlation between the extent of patching and amplification in transmembrane signaling appears to be a distinct property of L-selectin on neutrophils.


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Fig. 8.   Amplification in signaling neutrophil adhesion via CD18 correlates with extent of L-selectin clustering. Proposed model for L-selectin-mediated neutrophil activation is based on correlations between CD18 activation and the topography of clustered L-selectin. Both unbound L-selectin and that bound by a single HuDREG antibody fail to exceed the threshold of clustering necessary to trigger neutrophil activation. Combining HuDREGs at saturating doses of antibody is sufficient to cluster small patches of L-selectin and induce moderate colocalization with activated CD18 receptor. Secondary cross-linking of HuDREGs increases the size and frequency of L-selectin clusters, stimulates extensive L-selectin colocalization with high-affinity CD18, and culminates in the amplification of neutrophil adhesion involving signal transduction via phosphorylation of p38 and p42/22 MAPKs.

A second piece of data revealing a direct relationship between the extent of L-selectin engagement and signaling was detection of Mac-1 upregulation in response to the binding of microspheres presenting anti-L-selectin. Figure 4 reveals a direct relationship between anti-L-selectin presented on beads and activation elicited by binding over the range of bead diameters tested. Activation increased most rapidly over the lower range of anti-L-selectin coating concentrations, correlating to the increase in the number of antibody sites presented on the beads. At the highest concentration of anti-L-selectin, maximum activation was equivalent for each bead diameter. We conclude that amplification in signaling via clustered L-selectin is effectively transduced at a single site of contact or through multiple smaller sites of membrane contact.

A single site of membrane contact to a 5.5-µm latex bead over an area of ~2.0 µm2 was sufficient to trigger maximum upregulation of Mac-1. We estimate that this contact area could accommodate engagement of up to ~10 microvilli on the surface of a neutrophil over an area that is on the same order of magnitude measured for a leukocyte rolling on microvascular endothelium (20). Alternatively, neutrophils bound to a 0.9-µm bead over an area at ~0.2 µm2 could accommodate contact with a single microvillus (32). This area is consistent with the immunofluorescence data that revealed L-selectin in clusters of ~0.2 µm2, which was sufficient to trigger activation. Amplification in signaling also correlated with an increase in cluster frequency and area after addition of the secondary polyclonal cross-linker. However, maximum activation correlated with the binding of ~10 of the 0.9-µm beads, compared with a single 5.5-µm bead. Given the site density of anti- L-selectin on beads, we estimate that ligation of as few as ~100 sites at a single area of contact was sufficient to trigger neutrophil activation. The capacity of L-selectin to amplify transmembrane signals in this manner is distinct from receptor-mediated activation of T cells. For example, it was reported that activation via major histocompatibility complex I presented on 4-µm microspheres required a threshold contact area, but amplification in signal was not achieved by increased frequency in binding of smaller-diameter microspheres (25).

Sites of clustered L-selectin were fourfold more likely to overlap with expression of CD18 subunits activated to adopt the ligand-binding conformation. The high-affinity CD18 colocalized with L-selectin constituted ~20% of expressed CD18 (~40,000 sites) (33). We estimate that in a contact region of 2.0 µm2, on the order of 600 high-affinity CD18 receptors are associated with L-selectin. We recently reported that neutrophil activation by IL-8 also elicits rapid expression of activated CD18. A similar fraction of membrane CD18 (~15%) was found to adopt a clustered topography, which was the minimum required for efficient neutrophil capture to ICAM-1 in shear flow (23).

Recently, we reported (40) that signal transduction as a result of clustering L-selectin by addition of combined HuDREGs on neutrophils utilizes p38 MAPK to effect shape change, integrin activation, and release of secondary, tertiary, and secretory granules. A second MAPK associated with signaling in neutrophils is p42/44 ERK, which is phosphorylated in response to engagement of either PSGL-1 or L-selectin (13, 40). Here we analyzed the rate of p38 and p42/44 MAPK phosphorylation in response to the more potent stimulus of cross-linking anti-L-selectin with polyclonal antibody. Treatment of neutrophils at 2,000 × Kd with a single HuDREG alone was insufficient to signal MAPK activity. However, within a minute of addition of the secondary goat anti-human antibody, MAPK activity was boosted greater than tenfold. In neutrophils, it appears that both MAPK pathways are activated by the binding of L-selectin, as demonstrated by the equivalent and near total functional inhibition observed with SB-202190 and PD-98059. We confirmed that blocking p42/44 with PD-98059 indeed abrogated phosphorylation of Thr202/Tyr204; however, SB-202190 specific for p38 did not block its kinase activity. This is not entirely surprising given that p38 is made up of four isoforms of which only alpha  and beta  are blocked by SB-202190. The conclusion is that the intact p38 phosphorylation activity presumed to be generated by the gamma - and delta -subunits is not sufficient for activation of CD18.

There is also evidence suggesting that p38 and p42/44 converge at a common regulatory point on activation. These two MAPKs have been demonstrated to form a molecular complex in HeLa and HEK2y93 cells on stress activation (45). Formation of a complex involving both kinases on signal transduction would account for the near complete inhibition by either p38 or p42/44 inhibitor after L-selectin clustering in neutrophils. However, we confirmed that the p38 and p42/44 signaling pathways are independent after activation, in that inhibitors to each did not affect phosphorylation of the other kinase. We are currently pursuing the question of whether each kinase is capable of triggering distinct cellular functions that superpose to effect optimal activation of beta 2-integrin adhesion.

Several mechanisms may provide for multivalent binding and clustering of L-selectin as it recognizes and is bound by distinct vascular ligands. L-selectin is expressed on the tips of microvilli and membrane ruffles in a conformation that constitutively recognizes sLex expressed on ligands presented on inflamed endothelium and bound to extracellular matrix (3, 8). sLex present on L-selectin is also bound with high affinity by E-selectin and has been shown to be one of only a few leukocyte surface receptors that can be isolated with an E-selectin affinity column (16, 28, 47). Moreover, E-selectin binding to L-selectin forms bonds of sufficient strength to facilitate slow rolling of neutrophils (47). We previously reported (35) that neutrophils are activated during rolling on E-selectin in a parallel plate flow chamber. We are currently pursuing the hypothesis that E-selectin upregulated on inflamed endothelium binds multivalently to L-selectin and other sLex-presenting receptors, resulting in the rapid signaling of firm adhesion of neutrophils in shear flow (Pearson et al., unpublished observations).

There is also recent evidence that indicates L-selectin may undergo a conformational change in response to specific epitope binding that exposes a high-avidity site within the lectin domain (21). This in turn triggers association of L-selectin with the cytoskeleton and an increase in the avidity of adhesion to physiological ligands. Signaling via L-selectin has also been induced through dimerization of its cytoskeletal domains in a transfected lymphoblastoid cell line (22). This resulted in increased binding of carbohydrate ligands, an increase in the strength of lymphocyte adhesive interactions with vascular endothelium, and constitutive induction of intercellular aggregation (9). In this regard, we have demonstrated here that ligation of L-selectin by bivalent IgGs at distinct epitopes elicited membrane patching. Thus one mechanism for signal transduction may involve clustering of L-selectin by engagement of vascular counterreceptors that bind multivalently to O-linked sLex, that in turn trigger cytoskeletal association and assembly of signal-promoting elements. A second mechanism that can promote clustering of L-selectin as leukocytes interact with vascular ligands is its colocalization with other receptors in the plane of the membrane. We previously reported (34) on the colocalization between L-selectin and CD18 on the surface of neutrophils after anti-L-selectin antibody binding and cross-linking, and others have demonstrated the association of urokinase plasminogen activator receptor with the lectin domain of L-selectin (38). Here we show preferential association of high-affinity CD18 at sites of L-selectin clustering.

The physiological implication is that the extent of L-selectin engagement by extensively sialylated carbohydrate ligands or multivalent recognition and clustering by vascular lectinlike receptors at sites of inflammation provides for amplification in transmembrane signaling of beta 2-integrin adhesion function. Moreover, this mechanochemical transduction through L-selectin can occur at a single site of membrane contact involving a few microvilli expressing a few hundred receptors, thereby providing a means for the local and rapid recruitment of activated beta 2-integrin and the efficient conversion from neutrophil rolling to arrest.


    ACKNOWLEDGEMENTS

We thank Nick Landolfi from Protein Design Labs and Don Staunton of ICOS Corporation for their generous provision of reagents and technical expertise.


    FOOTNOTES

This work was supported by National Institute of Allergy and Infectious Diseases Grant AI-47294. S. I. Simon is an Established Investigator of the American Heart Association. C. E. Green is supported by National Institute of General Medical Sciences Training Fellowship T32-GM-08799-01A1.

Address for reprint requests and other correspondence: S. I. Simon, Dept. of Biomedical Engineering, One Shields Ave., Davis, CA, 95616 (E-mail: sisimon{at}ucdavis.edu).

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

First published November 13, 2002;10.1152/ajpcell.00331.2002

Received 16 July 2002; accepted in final form 8 November 2002.


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ABSTRACT
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RESULTS
DISCUSSION
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