Department of Biomedical Engineering, University of California, Davis, California 95616
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ABSTRACT |
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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
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INTRODUCTION |
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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
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 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
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 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
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
2-integrin function.
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MATERIALS AND METHODS |
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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 sWestern 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- (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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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
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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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RESULTS |
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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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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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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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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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Kinetics of CD11b/CD18 activation and MAPK activity after
clustering of L-selectin.
A sensitive method for assessing the adhesive function of
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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Clustered L-selectin colocalizes with activated
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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DISCUSSION |
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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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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 and
are blocked by SB-202190. The conclusion is that the intact p38
phosphorylation activity presumed to be generated by the
- and
-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 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 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
2-integrin and the
efficient conversion from neutrophil rolling to arrest.
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ACKNOWLEDGEMENTS |
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We thank Nick Landolfi from Protein Design Labs and Don Staunton of ICOS Corporation for their generous provision of reagents and technical expertise.
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FOOTNOTES |
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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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