1 Department of Chemical and Biomolecular Engineering, Johns Hopkins University, Baltimore 21218; and 2 Division of Clinical Immunology, Department of Medicine, Johns Hopkins University School of Medicine, Baltimore, Maryland 21224
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ABSTRACT |
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This study examined the binding kinetics and molecular requirements of eosinophil adhesion to surface-anchored platelets in shear flow. P-selectin glycoprotein ligand-1 (PSGL-1) binding to platelet P-selectin initiates tethering and rolling of eosinophils to platelets under flow. These primary interacting cells assist in the capture of free-flowing eosinophils through homotypic tethering (secondary interactions) mediated via L-selectin-PSGL-1 interactions. Differences between eosinophils and neutrophils in PSGL-1 and L-selectin expression levels predict the pattern and relative extent of their adhesive interactions with immobilized platelets under shear, as well as the relative magnitude of their average rolling velocities. The majority of tethered eosinophils become rapidly stationary on the platelet layer, a process that is predominantly mediated via eosinophil PSGL-1 binding to platelet P-selectin and has an absolute requirement for intact cytoskeleton. Only a small fraction of these stationary eosinophils develop shear-resistant attachments mediated by CD18 integrins. However, stimulation of eosinophils with eotaxin-2 converts PSGL-1-P-selectin-dependent stationary adhesion to CD18-mediated shear-resistant stable attachment. These studies provide insights for designing strategies based on blocking of eosinophil-platelet interactions to combat thrombotic disorders in hypereosinophilic patients.
eosinophil; platelet; shear stress; P-selectin; CD18-integrins
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INTRODUCTION |
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LEUKOCYTE ADHESION to activated platelets represents a key event in the sequence of thrombus formation, as demonstrated in vitro (19) and observed in vivo after arterial injury (30) and during propagation of venous thrombosis (42). Several lines of evidence also suggest that enhanced leukocyte-platelet adhesion occurs in the circulation of patients with acute myocardial infarction (35) or stroke (23) or after coronary angioplasty (31). These heterotypic adhesive interactions are thought to promote thrombosis and vascular occlusion, thereby impairing blood flow and exacerbating ischemia (13).
To date, most work has focused on delineating the molecular mechanisms
by which neutrophils interact with activated platelets, because
neutrophils represent the largest leukocyte subpopulation in blood
(10, 14, 25, 26, 36, 39, 50). As a result, very little is
known about the molecular constituents mediating attachment of other
leukocyte subpopulations to platelets. For instance, eosinophils,
although they normally comprise <4% of circulating leukocytes in
blood, are dramatically increased in certain disease states and can
account for 20% of the total leukocyte population. Several reports
have noted the occurrence of thrombotic disorders in hypereosinophilic
patients that, in certain cases, was accompanied by occlusion of
arteries and small blood vessels (33, 38). In particular,
58% of patients with idiopathic hypereosinophilic syndrome (HES),
characterized by persistent eosinophilia and organ damage, develop
cardiovascular disease, often with associated mural thrombi
(51). In addition, neurological complications caused by
thromboemboli either of cardiac origin or locally produced within
cerebral vessels are frequently detected in HES (51). The
pathogenesis of eosinophil-mediated organ (e.g., cardiac) damage is
currently unknown but is thought to involve both the presence of
increased number of eosinophils and other as yet ill-defined stimuli
for recruitment and/or activation of these leukocytes. It has been
suggested that eosinophils may undergo a respiratory burst to generate
oxidative products that, alone or in concert with eosinophil
peroxidase, may cause oxidant-mediated damage (51).
Earlier work demonstrated that activated platelets induce superoxide
anion release by monocytes and neutrophils (34). It is
therefore likely that enhanced eosinophil-platelet adhesive interactions in the microcirculation of eosinophilic patients result in
eosinophil activation and release of oxidants that may precipitate
and/or exacerbate thrombotic disorders in these patients. Consequently, elucidation of the detailed molecular basis underlying eosinophil attachment to platelets may provide insights for the rational development of novel therapeutic agents, based on the blockade of these adhesive interactions, to effectively combat thrombotic disorders in eosinophilic patients.
Prior work has shown that thrombin-activated platelets interact with
isolated eosinophils in a Ca2+-dependent manner under
stationary conditions (9). Antibody blocking experiments
have revealed a role for platelet CD62P (P-selectin) in this process.
However, the molecular determinants (other than P-selectin) and the
detailed sequence of events involved in this heterotypic interaction
remain unknown. In contrast, a multistep, sequential process of
adhesive interactions has been elucidated for neutrophil recruitment to
immobilized platelet layers. In particular, platelet P-selectin
interacts with CD162 (P-selectin glycoprotein ligand-1; PSGL-1) to
mediate the initial tethering and rolling of neutrophils under dynamic
flow conditions (10, 36, 39). As neutrophils roll along
immobilized platelets, they are exposed to activating signals,
including agents presented on the platelet surface such as
platelet-activating factor (PAF) (36, 50), that upregulate
the binding affinity of integrins. Activation-dependent attachment of
the integrin receptor CD11b/CD18 (Mac-1) on neutrophils (10, 26,
50) to platelet-associated fibrinogen presented by CD41/CD61
(IIb
3) (26, 50) has been suggested to convert transient rolling interactions into stable neutrophil adhesion. The transition from selectin-mediated rolling to
CD11b-dependent arrest may be facilitated by the engagement of the
neutrophil CD18 integrin receptor CD11a, which binds to platelet CD102
(ICAM-2) (50). Once firmly adherent on activated platelets, neutrophils are able to migrate across the platelet layer,
primarily via the CD18 integrin receptor CD11b and to a lesser extent
via CD11a (10). However, several issues still remain
controversial. For instance, some studies have failed to confirm the
involvement of platelet-
IIb
3 in this
adhesion process (36, 39). Moreover, although some reports
have suggested that nearly all rolling neutrophils become firmly
adherent via CD18 integrin involvement within seconds of the initial
platelet P-selectin-mediated binding (10, 50), others have
noted that only ~50% of rolling cells stably adhere to
thrombin-treated platelet layers under flow (36, 43). In
the absence of stimulation of immobilized platelets with either
thrombin or ADP, previous work has indicated that the transition from
rolling to stable attachment requires exogenous neutrophil activation
(43).
This study was undertaken to elucidate the precise molecular constituents mediating adhesion of free-flowing eosinophils to immobilized, thrombin-treated washed platelet layers under controlled kinematic conditions. In particular, we examined whether the general neutrophil paradigm of rolling followed by stable adhesion is applicable for eosinophil adhesion to immobilized platelets in shear flow and whether eosinophil activation by exogenous stimuli, such as the CCR3-active chemokine eotaxin-2, is prerequisite for CD18 integrin-mediated stable attachment. In light of the well-established differences between eosinophils and neutrophils in the expression levels of PSGL-1 (i.e., about twice as much on the eosinophil surface), and CD62L (L-selectin) (8, 22, 44) responsible for homotypic leukocyte interactions in shear flow (6, 24), we wanted to compare the pattern and extent of eosinophil binding to immobilized platelets with those of neutrophils.
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MATERIALS AND METHODS |
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Reagents.
The IgG murine monoclonal antibodies (MAbs) 7E4 (blocking anti-CD18),
HP2/1 (blocking anti-CD49d), and D1G10VL2 (blocking anti-fibrinogen;
Ref. 5) were purchased from Immunotech (Westbrook, ME).
The blocking anti-CD29 MAb 4B4 was from Coulter (Miami, FL). The
blocking MAbs KPL-1 [anti-CD162 (anti-PSGL-1)], HI111 (anti-CD11a), ICRF44(44) (anti-CD11b), and HIP1 [anti-CD42b
(anti-GPIb)] were obtained from BD-Pharmagen (San Diego, CA). A
blocking anti-CD62P/E (anti-P-/E-selectin) MAb (EP5C7), which does not
affect CD62L function (47), was generously provided by Dr.
Nicholas F. Landolfi (Protein Design Labs, Fremont, CA). The
function-blocking anti-CD62L (anti-L-selectin) MAb LAM1-116 and
anti-CD11d (anti-d) MAb 240I were generously provided by
Dr. Thomas F. Tedder (Duke University Medical Center, Durham, NC) and
Dr. Pat Hoffman (ICOS, Bothell, WA), respectively. The nonpeptide
small-molecule platelet-
IIb
3 antagonist
XV454 (1) was a kind gift of Dr. Shaker A. Mousa (DuPont Pharmaceuticals, Wilmington, DE). The Fab
anti-
IIb
3 MAb c7E3 was from Centocor
(Malvern, PA). Human eotaxin-2 was kindly provided by Dr. John White
(GlaxoSmithKline, King of Prussia, PA). Formalin was purchased from
Richard Allan Scientific (Kalamazoo, MI), whereas latrunculin A was
obtained from Calbiochem (San Diego, CA). Citrate-phosphate-dextrose
(CPD) solution, 3-aminopropyltriethoxysilane (APES), thrombin,
prostaglandin (PGE1), cytochalasin B, chymotrypsin, glycyrrhizin, BSA, and isotype-matched IgG MAbs were from Sigma (St.
Louis, MO). A red blood cell agglutination reagent (Red-out) and
polymorphonuclear neutrophil (PMN) isolation media were purchased from
Robbins Scientific (Sunnyvale, CA).
Isolation of human granulocytes. Protocols involving human subjects were performed in accordance with the "Guiding Principles for Research Involving Animals and Human Beings" of the American Physiological Society. Human eosinophils were isolated from EDTA-anticoagulated venous blood of donors with mild allergic rhinitis or asthma by 1.090 g/ml Percoll density-gradient centrifugation at room temperature (RT) and removal of CD16-positive cells (neutrophils) with immunomagnetic beads (46). Eosinophil purity (based on the examination of Diff-Quik-stained cytocentrifugation preparation) was >96%, and viability (by erythrosin B dye exclusion) was nearly 100%. Eosinophils (5 × 106 per ml) were kept in assay buffer (RPMI 1640 medium containing 1 mM sodium pyruvate, 10 mM HEPES, 4.5 g/l glucose, and 0.1% BSA) at 4°C for no longer than 4 h before use in adhesion assays. In selected experiments, human neutrophils were isolated from CPD-anticoagulated venous blood of healthy volunteers by centrifugation through PMN isolation medium (16) and held (107 neutrophils/ml) at 4°C for up to 4 h before being used in flow-based adhesion assays. Before flow experiments, eosinophils (or neutrophils) were allowed to equilibrate at 37°C for 2 min and then were diluted to a cell concentration of 0.5 × 106/ml in assay buffer at 37°C and perfused over platelet-coated surfaces at prescribed wall shear stresses.
Immobilization of platelet layers on glass slides.
Platelet-rich plasma (PRP) was prepared by centrifugation (160 g for 15 min) of sodium citrate (0.38%
wt/vol)-anticoagulated human blood of healthy volunteers
(28). PRP specimens were subjected to a further
centrifugation (1,100 g for 15 min) in the presence of 2 µM PGE1, and the platelet pellet was resuspended in
HEPES-Tyrode buffer (in mM: 129 NaCl, 9.9 NaHCO3, 2.8 KCl,
0.8 K2PO4, 0.8 MgCl2 · 6H2O, 1 CaCl2, 10 HEPES, 5.6 dextrose) containing 5 mM EGTA and 2 µM PGE1 (14). Thereafter, platelets
were washed once via centrifugation (1,100 g for 10 min),
resuspended at 2 × 108/ml in HEPES-Tyrode buffer
(14), and kept at RT for no longer than 4 h before
use in flow assays. Before the perfusion experiments, purified
platelets were allowed to bind to 4% APES-treated coverslips (24 × 50 mm; Corning) for 30 min at 37°C in a humid environment (28). Under these conditions, a confluent layer of
platelets was formed, as evaluated by light microscopy for each
experiment (Fig. 1). The density and
confluence of platelet layers were not affected during the flow
experiment.
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Hydrodynamic flow assays. Leukocyte adhesion to immobilized platelets was quantitated under dynamic flow conditions with a parallel-plate flow chamber (1, 7, 28, 32). A platelet-coated coverslip was assembled with a flow chamber and mounted on the stage of an inverted microscope (Nikon TE300) equipped with a 10× phase objective (Nikon, Melville, NY), a 0.55× projection lens (Nikon), and a CCD100 camera (Dage-MTI, Michigan City, IN) connected to a VCR and a TV monitor. Surface-adherent platelets were then incubated with 1 U/ml thrombin (unless otherwise stated) in the presence of 0.1% BSA for 10 min at 37°C. After the platelet layer was washed with D-PBS-0.1% BSA for ~2 min, leukocytes were perfused through the chamber for 3 min at appropriate flow rates to obtain wall shear stresses of 0.5-3 dyn/cm2, thereby mimicking the fluid mechanical environment of the microcirculation and postcapillary venules. Leukocyte binding to surface-anchored platelets was visualized in real-time by phase-contrast videomicroscopy. A single field of view (0.55 mm2) was monitored during the 3 min of the attachment assay, and at the end five fields of view (each 0.55 mm2) were monitored for 10 s each. During all experiments, the entire flow system was maintained at 37°C in a warm air box surrounding the microscope.
Data analysis of attachment assays in flow. Five parameters were quantified in the analysis: 1) the number of primary interacting cells per square millimeter during the entire 3-min perfusion experiment, 2) the number of secondary interacting cells per square millimeter during that period, 3) the number of stationary interacting cells per square millimeter after 3 min of shear flow; 4) the percentage of total (primary + secondary) interacting cells that were rolling after 3 min of shear flow, and 5) the average rolling velocity (µm/s) of interacting leukocytes (1, 28). Cells that tethered directly to the platelet layer in the absence of any interaction with previously bound leukocytes were defined as primary interacting cells (3). Primary interacting leukocytes that tethered upstream of and continued to translate into the field of view were distinguished from those that initially tethered directly to the platelets within the field of view. Cells that attached to the substrate after first forming homotypic tethers with leukocytes already bound to the platelet surface were defined as secondary interacting cells (3). The number of primary and secondary interacting cells was determined manually by reviewing the videotapes. Stationary interacting cells were considered as those that moved <1 cell radius within 10 s at the end of the 3-min attachment assay. To quantify their number, images were digitized from the videotape recorder with a Scion frame grabber and a personal computer and processed with OPTIMAS 6.5 software package (Argis-Schoen Vision Systems, Alexandria, VA; Ref. 28). Rolling velocities were computed as the distance traveled by the centroid of the translating cell divided by the time interval (1, 28) with OPTIMAS 6.5 software. Only cells that rolled without stopping during the entire acquisition period were included in the analysis.
Data analysis of controlled detachment assays in flow. To assess the strength of attachment, in selected experiments leukocytes were allowed to tether to the platelet surface at a wall shear stress of 2 dyn/cm2 for 3 min, followed by the perfusion of buffer or actin polymerization buffers for 1 min, after which the flow rate was doubled every 30 s to achieve shear stress levels of 4, 8, 16, and 32 dyn/cm2. The number of leukocytes remaining stationary at the conclusion of 3 min of perfusion (2 dyn/cm2) was recorded, which served as the normalization basis with which to generate the percentage of cells that remained stationary throughout the experiment. The percentage of eosinophils remaining stationary was reevaluated after the perfusion of either flow buffer or actin polymerization inhibitors for an additional 1 min at 2 dyn/cm2, as well as being quantified after 20 s at each shear stress level tested. Rolling velocities were calculated as described in the attachment assays (1, 28).
Cell treatment with MAbs, enzymes, and eotaxin-2.
For some inhibition studies, leukocytes were pretreated for 30 min at
4°C with saturating concentrations of function-blocking MAbs that
were kept present during the perfusion assays. For other studies,
surface-adherent platelets were preincubated with MAbs (50 µg/ml,
unless otherwise stated), the small-molecule
IIb
3 antagonist XV454 (150 nM), or the
RGD-containing peptide GRGDSP (4 mg/ml) for 10 min at 37°C during the
thrombin-BSA incubation. Saturating concentrations of EP5C7 MAb, XV454,
and GRGDSP were also maintained in the flow buffer during the flow
assays. The extent of leukocyte binding to platelet layers, as well as
the strength of these adhesive interactions as determined in controlled detachment assays, were unaltered by the presence or absence of the
appropriate isotype-matched control MAbs (data not shown).
Statistics. Data are expressed as means ± SE. Statistical significance of differences between means was determined by one-way ANOVA. If means were shown to be significantly different, multiple comparisons by pairs were performed by the Tukey test. Probability values of P < 0.05 were selected to be statistically significant.
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RESULTS |
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Surface-anchored platelets support eosinophil adhesion under flow.
To study the pattern and extent of eosinophil adhesive interactions
with platelets in shear flow, eosinophils were perfused through a
parallel-plate flow chamber, whose lower plate was coated with
platelets, at prescribed wall shear stresses ranging from 0.5 to 3 dyn/cm2. Our data indicate that immobilized platelets
formed a highly efficient surface for eosinophil capture (Fig. 1). The
majority of eosinophils that tethered from the fluid stream were
observed to roll ~1-3 cell diameters before becoming stationary.
Treatment of platelet layers with thrombin increased the number of
stationary eosinophils and concomitantly decreased the percentage of
rolling cells (240 ± 9 vs. 148 ± 10 stationary
cells/mm2; 9.5 ± 2.4 vs. 37.0 ± 0.5% of
tethered cells rolling in the presence and absence of thrombin,
respectively, after 3 min of perfusion at 2 dyn/cm2).
Therefore, to investigate the mechanisms by which platelets recruit and
efficiently stabilize free-flowing eosinophils, platelets were
pretreated with thrombin in all experiments reported hereafter. A
progressive decrease in the number of stationary eosinophils was
detected between 0.5 and 3.0 dyn/cm2 (Fig.
2A). Concomitantly, an
increasing proportion of tethered eosinophils were observed to
translocate slowly along the platelet layer. The percentage of rolling
eosinophils increased from <4% at a wall shear stress of 0.5 dyn/cm2 to ~35% at 3 dyn/cm2 (Table
1).
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Relative contribution of selectins and selectin ligands to
eosinophil binding to surface-adherent platelets under flow.
Ensuing experiments focused on the elucidation of the molecular
pathways involved in the adhesive interactions between eosinophils and
immobilized, thrombin-treated platelets at a wall shear stress of 2 dyn/cm2. Treatment of eosinophils with neuraminidase, an
enzyme that cleaves sialic acid residues from the cell surface
(16), dramatically reduced the number of stationary
eosinophils on the platelet layer as well as secondary homotypic
eosinophil interactions (Fig.
4A). Similar results were
obtained when eosinophils were incubated with chymotrypsin, a protease
that cleaves both L-selectin and the highly sialylated PSGL-1 (Fig.
4A; Ref. 16). Nevertheless, eosinophils treated
with either neuraminidase (0.1 U/ml) or chymotrypsin (1 U/106 cells) alone were observed to tether directly and
roll on the platelet surface (Fig. 4A), albeit significantly
faster than untreated cells (Table 2). It
is noteworthy that, whereas 275 ± 26 primary interacting
neuraminidase-treated eosinophils/mm2 were observed to
tether in the field of view (Fig. 4A), another 357 ± 46 cells/mm2 were observed to roll into the field of view
after having previously tethered upstream. Similarly, another 58 ± 15 chymotrypsin-treated eosinophils/mm2 rolled into the
field of view from upstream. In contrast, in matched control samples
all eosinophils tethered directly to platelets within the field of
view, and no eosinophils were observed to enter the field of
observation by rolling from upstream. Flow cytometric analysis of the
eosinophil cell surface revealed that treatment with neuraminidase
reduced the sLex expression level by 94%, whereas
treatment with chymotrypsin cleaved L-selectin and reduced the PSGL-1
expression level by 35% (data not shown). It is noteworthy that the
combination of these enzymes completely blocked eosinophil interactions
with immobilized platelets under dynamic flow conditions (Fig.
4A). The inability of neuraminidase or chymotrypsin alone to
effectively abrogate the primary heterotypic adhesive interactions may
be due to the presence of residual sLex and PSGL-1
expression levels on the cell surface after enzyme treatment. However,
the sLex mimic glycyrrhizin (18, 37), which
has been shown to block selectin binding to sLex both in
vitro and in vivo, essentially abolished eosinophil attachment to
platelets at 2 dyn/cm2 (Fig. 4A). Together,
these data are suggestive of the potential involvement of sialylated
PSGL-1 and L-selectin in eosinophil accumulation on platelet layers in
shear flow.
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Role of CD18 integrins in eosinophil adhesion to immobilized
platelets in absence and presence of exogenous stimulation by
eotaxin-2.
Several lines of evidence suggest that initial tethering and rolling of
neutrophils on activated platelet layers is mediated by platelet
P-selectin and that subsequent involvement of neutrophil CD18 integrins
converts these transient adhesive interactions into firm adhesion
(10, 26, 50). We therefore wished to examine whether CD18
integrins mediate stationary adhesion of eosinophils to immobilized
platelets under dynamic flow conditions. Our data indicate that the
treatment of eosinophils with the function-blocking anti-CD18 integrin
MAb 7E4 failed to reduce the number of stationary interacting
eosinophils at a wall shear stress of 2 dyn/cm2 (Fig.
5A). Moreover, treatment of
eosinophils with function-blocking MAbs specific for eosinophil CD29
(1 integrins), CD49d (
4 integrins), or CD11d
(
d integrins) had no effect on the extent of stationary adhesion (data not shown).
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Role of eosinophil cytoskeleton in eosinophil-platelet interactions
under flow.
Our data show that PSGL-1 binding to platelet P-selectin not only
initiates the majority of eosinophil tethering and rolling but also
plays a predominant role in mediating stationary adhesion to
surface-bound platelets in shear flow in the absence of any exogenously
added stimulus. The ability of PSGL-1 to support eosinophil stationary
adhesion to platelet P-selectin may be regulated by its interaction
with the leukocyte actin cytoskeleton (40). We therefore
wanted to investigate how disruption of the eosinophil actin
cytoskeleton affects the pattern of eosinophil-platelet interactions.
To rule out any possible effects of the actin polymerization inhibitors cytochalasin B or latrunculin A on the platelet
cytoskeleton, experiments were performed with thrombin-treated,
formalin-fixed immobilized platelets. Treatment of platelets with
formalin for 15 min before the perfusion of eosinophils reduced, but
did not eliminate, the ability of eosinophils to arrest on the platelet surface, which is in accord with previous studies on
neutrophil-platelet interactions (40). At a shear stress
level of 2 dyn/cm2, >60% of eosinophils were observed to
roll on the fixed platelet surface. As shown in Fig.
6, eosinophils that developed stationary interactions with fixed platelets at 2 dyn/cm2 detached on
exposure to increasing shear forces in a similar yet enhanced manner
compared with unfixed platelets.
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DISCUSSION |
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The major findings of this work are as follows. 1) Eosinophils, tethered either directly to surface-anchored platelets via PSGL-1 binding to platelet P-selectin or through homotypic secondary interactions mediated by L-selectin-PSGL-1 tethering, become rapidly immobilized on the platelet layer at low shear. The majority of these stationary interactions are dependent on the high degree of eosinophil PSGL-1 binding to platelet P-selectin and have an absolute requirement for intact eosinophil cytoskeleton. Only a small fraction of these stationary eosinophils develop shear-resistant attachments mediated by CD18 integrins. 2) Exogenous stimulation of eosinophils with the CCR3-active chemokine eotaxin-2 converts PSGL-1-P-selectin-dependent stationary adhesion to CD18-mediated stable attachment.
Eosinophil tethering, rolling, and stationary adhesion to immobilized platelets in shear flow are mediated by PSGL-1-P-selectin interactions. In concert with previous studies using neutrophils (10, 36, 39), PSGL-1 binding supports eosinophil primary tethering and rolling along surface-bound platelets under flow. However, significant differences are observed in the adhesive interactions of these two leukocyte subpopulations with immobilized platelets in shear flow. More specifically, the number of eosinophils tethered directly to the platelet surface is higher than that of neutrophils. Furthermore, the relative percentage of tethered cells rolling along the platelet layer and their respective average rolling velocity are markedly diminished for eosinophils compared with neutrophils. Direct comparison of these interactions with respect to cell surface ligand expression must be avoided because eosinophils and neutrophils were purified via different isolation methods from different donors. However, our observations are in accord with previously published data in purified P-selectin, P-selectin-expressing Chinese hamster ovary cells, or fixed platelets (12, 18, 20). The aforementioned discrepancies may be attributed to qualitative differences in the molecular structure of PSGL-1 on eosinophils from that on neutrophils (29, 44) and/or the higher PSGL-1 expression levels detected on the eosinophil relative to the neutrophil surface (8, 12, 44). Interestingly, as shown in Table 3, the average rolling velocity of neutrophils at a given shear stress level (e.g., 7.0 ± 0.7 µm/s at 4 dyn/cm2) is essentially equal to that of eosinophils expressing nearly twice as much PSGL-1 (8) at twice the level of shear (e.g., 7.1 ± 0.7 µm/s at 8 dyn/cm2). This finding further supports the concept that the biomechanics of cell rolling depends on both the ligand density and the fluid shear at a given receptor-site density.
Previous studies suggested that nearly all tethered neutrophils become stably arrested via CD18 integrin involvement within seconds of the initial PSGL-1-P-selectin-mediated binding (10, 50). This finding is in clear contrast to other reports (36, 43) showing that nearly 50% of the tethered neutrophils roll continuously along immobilized platelets in shear flow and the remaining ~50% become firmly adherent via CD18-dependent binding. Our data indicate that the majority of eosinophils and neutrophils tethered to the platelet surface become rapidly stationary at a wall shear stress level of 2 dyn/cm2 in a P-selectin-dependent manner. However, the percentage of eosinophils (or neutrophils) remaining stationary decreases dramatically with increasing shear, with <25% of cells forming shear-resistant attachments mediated by CD18 integrins at a shear level of 32 dyn/cm2. It is noteworthy that increasing shear caused eosinophils (and neutrophils) to begin to roll on the platelet surface rather than abruptly detaching. A subsequent reduction in the shear stress level to 2 dyn/cm2 caused these "rolling" cells to become stationary again (data not shown). Cumulatively, our data suggest that immobilization of free-flowing, resting eosinophils (or neutrophils) to platelet layers at low shear results predominantly from the eosinophil (or neutrophil) PSGL-1 binding to high site densities of platelet P-selectin. Along these lines, eosinophils (and neutrophils) were observed to tether, roll and develop stationary adhesive interactions when perfused at 2 dyn/cm2 over high levels of immobilized, purified P-selectin (0.5 µg/ml), in contrast to rolling interactions that were exclusively observed at lower P-selectin concentrations (Eotaxin-2-induced eosinophil activation is required for shear-resistant CD18 integrin-mediated adhesion to immobilized platelets. The activation of leukocytes represents a key component in their adhesion cascade to vascular endothelium, platelets, or other leukocytes and upregulates the binding affinity of integrins via both conformational changes and altered interactions with the cytoskeleton (6, 24). Previous studies showed that activation of tethered neutrophils via platelet-derived activating agents such as PAF converts neutrophil rolling to shear-resistant, CD18-mediated firm adhesion to immobilized platelets (36, 50). The percentage of tethered neutrophils activated by agents generated by or through platelets varies from 25-50% (36) up to nearly 100% (50). In the current study, only ~25% of stationary eosinophils remained adherent to the platelet surface on exposure to a wall shear stress of 32 dyn/cm2 and ~60% of these adhesion events were eliminated by the use of the function-blocking anti-CD18 MAb 7E4. These data suggest that a small fraction of stationary eosinophils may have been activated by locally platelet-secreted agents such as PAF or RANTES, which was previously shown to be released by thrombin-stimulated platelets (17).
Selective leukocyte recruitment is the result of the orchestrated events involving cell surface receptors, their respective ligands, and chemokines. The CCR3-active chemokines such as eotaxin-2 selectively regulate eosinophil adhesion in a mitogen-activated protein kinase-dependent manner while having no chemotactic effect on neutrophils (4). Along these lines, addition of eotaxin-2 in the perfusion buffer induced rapid shear-resistant eosinophil firm adhesion to platelet layers via eosinophil CD18-integrins, as evidenced by the dramatic reduction of adhesion by the use of an anti-CD18 MAb. Previous studies suggest that neutrophil firm adhesion to immobilized platelets is predominantly mediated via CD11b/CD18 integrin binding to platelet-associated fibrinogen presented by ![]() |
ACKNOWLEDGEMENTS |
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We thank Drs. Thomas F. Tedder, Pat Hoffman, Shaker A. Mousa, Nicholas Landolfi, and John White for providing valuable reagents, Dr. Denis Wirtz (Johns Hopkins University) for helpful discussions, and Carol Bickel, Sherry A. Hudson, Daniel Plymire, Andy Jun, and Parag Pawar for technical support.
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FOOTNOTES |
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This work was supported by National Science Foundation Grant BES 9978160 and a Mid-Atlantic American Heart Association Grant-in-Aid.
Address for reprint requests and other correspondence: K. Konstantopoulos, Dept. of Chemical and Biomolecular Engineering, Johns Hopkins Univ., 3400 N. Charles St., Baltimore, MD 21218-2694 (E-mail: kkonsta1{at}jhu.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 January 15, 2003;10.1152/ajpcell.00403.2002
Received 30 August 2002; accepted in final form 9 January 2003.
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REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
1.
Abulencia, JP,
Tien N,
McCarty OJ,
Plymire D,
Mousa SA,
and
Konstantopoulos K.
Comparative antiplatelet efficacy of a novel, nonpeptide GPIIb/IIIa antagonist (XV454) and abciximab (c7E3) in flow models of thrombosis.
Arterioscler Thromb Vasc Biol
21:
149-156,
2001
2.
Alon, R,
Chen S,
Fuhlbrigge R,
Puri KD,
and
Springer TA.
The kinetics and shear threshold of transient and rolling interactions of L-selectin with its ligand on leukocytes.
Proc Natl Acad Sci USA
95:
11631-11636,
1998
3.
Alon, R,
Fuhlbrigge RC,
Finger EB,
and
Springer TA.
Interactions through L-selectin between leukocytes and adherent leukocytes nucleate rolling adhesions on selectins and VCAM-1 in shear flow.
J Cell Biol
135:
849-865,
1996[Abstract].
4.
Bandeira-Melo, C,
Herbst A,
and
Weller PF.
Eotaxins. Contributing to the diversity of eosinophil recruitment and activation.
Am J Respir Cell Mol Biol
24:
653-657,
2001
5.
Bombeli, T,
Schwartz BR,
and
Harlan JM.
Adhesion of activated platelets to endothelial cells: evidence for a GPIIbIIIa-dependent bridging mechanism and novel roles for endothelial intercellular adhesion molecule 1 (ICAM-1), alphavbeta3 integrin, and GPIbalpha.
J Exp Med
187:
329-339,
1998
6.
Burdick, MM,
McCarty OJT,
Jadhav S,
and
Konstantopoulos K.
Cell-cell interactions in inflammation and cancer metastasis.
IEEE Eng Med Biol
20:
86-91,
2001[ISI][Medline].
7.
Burdick, MM,
Schnaar RL,
Collins BE,
Bochner BS,
and
Konstantopoulos K.
Glycolipids support E-selectin-specific strong cell tethering under flow.
Biochem Biophys Res Commun
284:
42-49,
2001[ISI][Medline].
8.
Davenpeck, KL,
Brummet ME,
Hudson SA,
Mayer RJ,
and
Bochner BS.
Activation of human leukocytes reduces surface P-selectin glycoprotein ligand-1 (PSGL-1, CD162) and adhesion to P-selectin in vitro.
J Immunol
165:
2764-2772,
2000
9.
De Bruijne-Admiraal, LG,
Modderman PW,
Von dem Borne AE,
and
Sonnenberg A.
P-selectin mediates Ca2+-dependent adhesion of activated platelets to many different types of leukocytes: detection by flow cytometry.
Blood
80:
134-142,
1992[Abstract].
10.
Diacovo, TG,
Roth SJ,
Buccola JM,
Bainton DF,
and
Springer TA.
Neutrophil rolling, arrest, and transmigration across activated, surface-adherent platelets via sequential action of P-selectin and the beta 2-integrin CD11b/CD18.
Blood
88:
146-157,
1996
11.
Dong, C,
and
Lei XX.
Biomechanics of cell rolling: shear flow, cell-surface adhesion, and cell deformability.
J Biomech
33:
35-43,
2000[ISI][Medline].
12.
Edwards, BS,
Curry MS,
Tsuji H,
Brown D,
Larson RS,
and
Sklar LA.
Expression of P-selectin at low site density promotes selective attachment of eosinophils over neutrophils.
J Immunol
165:
404-410,
2000
13.
Entman, ML,
and
Ballantyne CM.
Association of neutrophils with platelet aggregates in unstable angina. Should we alter therapy?
Circulation
94:
1206-1208,
1996
14.
Evangelista, V,
Manarini S,
Rotondo S,
Martelli N,
Polischuk R,
McGregor JL,
de Gaetano G,
and
Cerletti C.
Platelet/polymorphonuclear leukocyte interaction in dynamic conditions: evidence of adhesion cascade and cross talk between P-selectin and the beta 2 integrin CD11b/CD18.
Blood
88:
4183-4194,
1996
15.
Finger, EB,
Bruehl RE,
Bainton DF,
and
Springer TA.
A differential role for cell shape in neutrophil tethering and rolling on endothelial selectins under flow.
J Immunol
157:
5085-5096,
1996[Abstract].
16.
Jadhav, S,
Bochner BS,
and
Konstantopoulos K.
Hydrodynamic shear regulates the kinetics and receptor specificity of polymorphonuclear leukocyte-colon carcinoma cell adhesive interactions.
J Immunol
167:
5986-5993,
2001
17.
Kameyoshi, Y,
Dorschner A,
Mallet AI,
Christophers E,
and
Schroder JM.
Cytokine RANTES released by thrombin-stimulated platelets is a potent attractant for human eosinophils.
J Exp Med
176:
587-592,
1992[Abstract].
18.
Kim, MK,
Brandley BK,
Anderson MB,
and
Bochner BS.
Antagonism of selectin-dependent adhesion of human eosinophils and neutrophils by glycomimetics and oligosaccharide compounds.
Am J Respir Cell Mol Biol
19:
836-841,
1998
19.
Kirchhofer, D,
Riederer MA,
and
Baumgartner HR.
Specific accumulation of circulating monocytes and polymorphonuclear leukocytes on platelet thrombi in a vascular injury model.
Blood
89:
1270-1278,
1997
20.
Kitayama, J,
Fuhlbrigge RC,
Puri KD,
and
Springer TA.
P-selectin, L-selectin, and alpha 4 integrin have distinct roles in eosinophil tethering and arrest on vascular endothelial cells under physiological flow conditions.
J Immunol
159:
3929-3939,
1997[Abstract].
21.
Kitayama, J,
Mackay CR,
Ponath PD,
and
Springer TA.
The C-C chemokine receptor CCR3 participates in stimulation of eosinophil arrest on inflammatory endothelium in shear flow.
J Clin Invest
101:
2017-2024,
1998
22.
Knol, EF,
Tackey F,
Tedder TF,
Klunk DA,
Bickel CA,
Sterbinsky SA,
and
Bochner BS.
Comparison of human eosinophil and neutrophil adhesion to endothelial cells under nonstatic conditions. Role of L-selectin.
J Immunol
153:
2161-2167,
1994
23.
Konstantopoulos, K,
Grotta JC,
Sills C,
Wu KK,
and
Hellums JD.
Shear-induced platelet aggregation in normal subjects and stroke patients.
Thromb Haemost
74:
1329-1334,
1995[ISI][Medline].
24.
Konstantopoulos, K,
Kukreti S,
and
McIntire LV.
Biomechanics of cell interactions in shear fields.
Adv Drug Delivery Res
33:
141-164,
1998[ISI][Medline].
25.
Konstantopoulos, K,
Neelamegham S,
Burns AR,
Hentzen E,
Kansas GS,
Snapp KR,
Berg EL,
Hellums JD,
Smith CW,
McIntire LV,
and
Simon SI.
Venous levels of shear support neutrophil-platelet adhesion and neutrophil aggregation in blood via P-selectin and beta2-integrin.
Circulation
98:
873-882,
1998
26.
Kuijper, PHM,
Gallardo Torres HI,
Lammers J,
Sixma JJ,
Koendrman L,
and
Zwaginga JJ.
Platelet associated fibrinogen and ICAM-2 induce firm adhesion of neutrophils under flow conditions.
Thromb Haemost
80:
443-448,
1998[ISI][Medline].
27.
Lim, YC,
Snapp K,
Kansas GS,
Camphausen R,
Ding H,
and
Luscinskas FW.
Important contributions of P-selectin glycoprotein ligand-1-mediated secondary capture to human monocyte adhesion to P-selectin, E-selectin, and TNF-alpha-activated endothelium under flow in vitro.
J Immunol
161:
2501-2508,
1998
28.
McCarty, OJ,
Mousa SA,
Bray PF,
and
Konstantopoulos K.
Immobilized platelets support human colon carcinoma cell tethering, rolling, and firm adhesion under dynamic flow conditions.
Blood
96:
1789-1797,
2000
29.
McEver, RP,
and
Cummings RD.
Role of PSGL-1 binding to selectins in leukocyte recruitment.
J Clin Invest
100:
S97-S103,
1997[ISI][Medline].
30.
Merhi, Y,
Guidoin R,
Provost P,
Leung TK,
and
Lam JY.
Increase of neutrophil adhesion and vasoconstriction with platelet deposition after deep arterial injury by angioplasty.
Am Heart J
129:
445-451,
1995[ISI][Medline].
31.
Mickelson, JK,
Lakkis NM,
Villarreal-Levy G,
Hughes BJ,
and
Smith CW.
Leukocyte activation with platelet adhesion after coronary angioplasty: a mechanism for recurrent disease?
J Am Coll Cardiol
28:
345-353,
1996[ISI][Medline].
32.
Mousa, SA,
Abulencia JP,
McCarty OJ,
Turner NA,
and
Konstantopoulos K.
Comparative efficacy between the glycoprotein IIb/IIIa antagonists roxifiban and orbofiban in inhibiting platelet responses in flow models of thrombosis.
J Cardiovasc Pharmacol
39:
552-560,
2002[ISI][Medline].
33.
Mukai, HY,
Ninomiya H,
Mitsuhashi S,
Hasegawa Y,
Nagasawa T,
and
Abe T.
Thromboembolism in a patient with transient eosinophilia.
Ann Hematol
72:
93-95,
1996[ISI][Medline].
34.
Nagata, K,
Tsuji T,
Todoroki N,
Katagiri Y,
Tanoue K,
Yamazaki H,
Hanai N,
and
Irimura T.
Activated platelets induce superoxide anion release by monocytes and neutrophils through P-selectin (CD62).
J Immunol
151:
3267-3273,
1993
35.
Neumann, FJ,
Marx N,
Gawaz M,
Brand K,
Ott I,
Rokitta C,
Sticherling C,
Meinl C,
May A,
and
Schomig A.
Induction of cytokine expression in leukocytes by binding of thrombin-stimulated platelets.
Circulation
95:
2387-2394,
1997
36.
Ostrovsky, L,
King AJ,
Bond S,
Mitchell D,
Lorant DE,
Zimmerman GA,
Larsen R,
Niu XF,
and
Kubes P.
A juxtacrine mechanism for neutrophil adhesion on platelets involves platelet-activating factor and a selectin-dependent activation process.
Blood
91:
3028-3036,
1998
37.
Rao, BN,
Anderson MB,
Musser JH,
Gilbert JH,
Schaefer ME,
Foxall C,
and
Brandley BK.
Sialyl Lewis X mimics derived from a pharmacophore search are selectin inhibitors with anti-inflammatory activity.
J Biol Chem
269:
19663-19666,
1994
38.
Rauch, AE,
Amyot KM,
Dunn HG,
Ng B,
and
Wilner G.
Hypereosinophilic syndrome and myocardial infarction in a 15-year-old.
Pediatr Pathol Lab Med
17:
469-486,
1997[ISI][Medline].
39.
Sheikh, S,
and
Nash GB.
Continuous activation and deactivation of integrin CD11b/CD18 during de novo expression enables rolling neutrophils to immobilize on platelets.
Blood
87:
5040-5050,
1996
40.
Sheikh, S,
and
Nash GB.
Treatment of neutrophils with cytochalasins converts rolling to stationary adhesion on P-selectin.
J Cell Physiol
174:
206-216,
1998[ISI][Medline].
41.
Simon, DI,
Chen Z,
Xu H,
Li CQ,
Dong J,
McIntire LV,
Ballantyne CM,
Zhang L,
Furman MI,
Berndt MC,
and
Lopez JA.
Platelet glycoprotein Ib is a counterreceptor for the leukocyte integrin Mac-1 (CD11b/CD18).
J Exp Med
192:
193-204,
2000
42.
Stewart, GJ.
Neutrophils and deep venous thrombosis.
Haemostasis
23, Suppl 1:
127-140,
1993[ISI][Medline].
43.
Stone, PC,
and
Nash GB.
Conditions under which immobilized platelets activate as well as capture flowing neutrophils.
Br J Haematol
105:
514-522,
1999[ISI][Medline].
44.
Symon, FA,
Lawrence MB,
Williamson ML,
Walsh GM,
Watson SR,
and
Wardlaw AJ.
Functional and structural characterization of the eosinophil P-selectin ligand.
J Immunol
157:
1711-1719,
1996[Abstract].
45.
Tachimoto, H,
and
Bochner BS.
The surface phenotype of human eosinophils.
Chem Immunol
76:
45-62,
2000[ISI][Medline].
46.
Tachimoto, H,
Burdick MM,
Hudson SA,
Kikuchi M,
Konstantopoulos K,
and
Bochner BS.
CCR3-active chemokines promote rapid detachment of eosinophils from VCAM-1 in vitro.
J Immunol
165:
2748-2754,
2000
47.
Tsurushita, N,
Fu H,
Melrose J,
and
Berg EL.
Epitope mapping of mouse monoclonal antibody EP5C7 which neutralizes both human E- and P-selectin.
Biochem Biophys Res Commun
242:
197-201,
1998[ISI][Medline].
48.
Walcheck, B,
Moore KL,
McEver RP,
and
Kishimoto TK.
Neutrophil-neutrophil interactions under hydrodynamic shear stress involve L-selectin and PSGL-1. A mechanism that amplifies initial leukocyte accumulation of P-selectin in vitro.
J Clin Invest
98:
1081-1087,
1996
49.
Wallace, PJ,
Packman CH,
Wersto RP,
and
Lichtman MA.
The effects of sulfhydryl inhibitors and cytochalasin on the cytoplasmic and cytoskeletal actin of human neutrophils.
J Cell Physiol
132:
325-330,
1987[ISI][Medline].
50.
Weber, C,
and
Springer TA.
Neutrophil accumulation on activated, surface-adherent platelets in flow is mediated by interaction of Mac-1 with fibrinogen bound to alphaIIbbeta3 and stimulated by platelet-activating factor.
J Clin Invest
100:
2085-2093,
1997
51.
Weller, PF,
and
Bubley GJ.
The idiopathic hypereosinophilic syndrome.
Blood
83:
2759-2779,
1994
52.
Yarmola, EG,
Somasundaram T,
Boring TA,
Spector I,
and
Bubb MR.
Actin-latrunculin A structure and function. Differential modulation of actin-binding protein function by latrunculin A.
J Biol Chem
275:
28120-28127,
2000