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Blood, Vol. 91 No. 8 (April 15), 1998:
pp. 3028-3036
A Juxtacrine Mechanism for Neutrophil Adhesion on Platelets Involves
Platelet-Activating Factor and a Selectin-Dependent Activation Process
By
Lena Ostrovsky,
Alison J. King,
Samantha Bond,
Debra Mitchell,
Diane E. Lorant,
Guy A. Zimmerman,
Robert Larsen,
Xiao Fe Niu, and
Paul Kubes
From the Department of Medical Physiology and Medicine, University of
Calgary, Calgary, Alberta, Canada; the Nora Eccles Harrison
Cardiovascular Research and Training Institute, and Department of
Internal Medicine, University of Utah School of Medicine, Salt Lake
City, UT; and Glycomed Inc, Ameda, CA.
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ABSTRACT |
The aim of this study was to identify the molecular mechanisms
involved in neutrophil adhesion to immobilized platelets with particular focus on the possible existence of a juxtacrine system for
neutrophil-platelet interactions. Platelets were immobilized onto
collagen (type I)-coated coverslips that were placed in a flow chamber
and neutrophils were perfused across these confluent monolayers at a
shear stress of 1 to 4 dynes/cm2. Neutrophils rolled, and a
significant proportion (25% to 50%) adhered to platelet monolayers.
P-selectin was expressed in very large quantities on the surface of
platelets and mediated all of the rolling, whereas the
 2-integrin mediated firm adhesion. An activation
mechanism for adhesion was necessary inasmuch as fixed neutrophils
continued to roll on immobilized platelets, but did not adhere.
Platelets adherent to collagen produced significant levels of
platelet-activating factor (PAF). Accordingly, the firm adhesion of
neutrophils to platelets was significantly inhibited by a PAF receptor
antagonist (WEB 2086). Treatment of only the platelets with
acetylhydrolase, which converts membrane-associated PAF to lyso-PAF,
prevented 60% of the adhesion. These data suggest that PAF, on the
surface of platelets, mediated a significant portion of the adhesive
interaction. Addition of some selectin-binding carbohydrates (fucoidan
or soluble SLEx analogs but not dextran sulfate) to the
platelets caused rolling neutrophils to immediately adhere, an event
that was not observed on histamine or thrombin-treated endothelium or
P-selectin transfectants. These data support the view that a juxtacrine
activation process exists on immobilized platelets for neutrophils.
This process can be greatly enhanced on platelets and may involve a
signaling mechanism through P-selectin.
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INTRODUCTION |
THE RECRUITMENT OF neutrophils from the
mainstream of the vasculature to the extravascular space is an early
and essential requisite in acute inflammation. This is a multistep
cascade of events in which the initial interaction is mediated by
P-selectin and results in the rolling of neutrophils along the length
of the endothelium.1,2 Pro-inflammatory molecules such as
platelet-activating factor (PAF) are rapidly synthesized and
coexpressed with P-selectin on the surface of the endothelium in
response to such mediators as histamine or thrombin.3,4 PAF
interacts with its receptor on the surface of neutrophils and functions
to activate the CD11/CD18 glycoprotein complex.5 This
adhesion molecule then supports the stable or firm adhesion of
neutrophils to counterreceptors on the surface of endothelium. The
tethering of neutrophils by P-selectin to the surface of endothelium is
thought to be essential in allowing PAF to interact with its receptor
and thereby induce neutrophil activation/adhesion. This cascade of
events has been termed the juxtacrine system for neutrophil
adhesion.5
In addition to endothelium, neutrophils interact with other cell types
including platelets. Platelets stimulated with soluble stimuli, such as
thrombin, will also express P-selectin and use this adhesion molecule
to adhere to neutrophils.6 This close cell-cell interaction
permits platelets to activate neutrophils and also permits for
transcellular biosynthesis of certain mediators (eg, leukotrienes) that
would not be produced in significant quantities by either cell
alone.7 When thrombin-activated platelets are immobilized
to glass, under flow conditions, these cells can also tether
neutrophils to their surface via P-selectin and perhaps through
L-selectin.8,9 This interaction has been described in vivo
for atherosclerosis, thrombosis, and inflammation and is likely to
occur when platelets adhere to either injured endothelium or exposed
extracellular matrix. The platelet-induced neutrophil recruitment may
be particularly important in high shear situations in which leukocytes
may tether to platelets but not to endothelium.9 In
addition to rolling along platelets, some investigators have reported
that rolling neutrophils will adhere particularly if an exogenous
chemotactic stimulus, such as N-formyl-methionyl-leucyl phenylalanine
(fMLP) or a phorbol ester, phorbol myristate acetate (PMA), was
applied.9-11 The fMLP- or PMA-stimulated neutrophils adhere
to platelets via CD11b/CD18, suggesting that there are as yet
unidentified ligands on platelets for the
B2-integrin.9-11 The spontaneous
conversion from rolling to adhesion of neutrophils on platelets, in the
absence of exogenous stimuli, has been observed.8,10 However, the endogenous activating mechanism(s) involved is entirely unknown.
The first objective of this study was to establish that the
collagen-induced contact-activation of platelets was sufficient (in the
absence of exogenous stimuli) to recruit neutrophils to roll and firmly
adhere to the platelet monolayer. The second objective was to identify
the molecular mechanisms underlying the neutrophil recruitment, with
particular emphasis on the possibility that a juxtacrine system for
neutrophil adhesion exists on platelets to cause rolling neutrophils to
adhere. We observed that adherent platelets possessed similar
characteristics to that described by Zimmerman et al4 for
activated endothelium; P-selectin-dependent neutrophil rolling was
spontaneously converted to CD18-dependent adhesion via endogenous PAF
production. However, we also identified a critical difference, ie,
administration of selectin-binding carbohydrates to the platelets
caused rolling neutrophils to immediately adhere, an event not observed
on histamine or thrombin-treated endothelium or P-selectin
transfectants. This latter point may suggest that binding of selectin
ligands to platelet P-selectin directly enhances neutrophil adhesion to
this substratum.
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MATERIALS AND METHODS |
Neutrophil isolation.
Human neutrophils were harvested from
acetate-citrate-dextrose-anticoagulated (ACD) venous blood collected
from healthy donors, as previously described,12 with minor
modification. All isolation steps were performed at room temperature.
Briefly, neutrophils were purified by dextran sedimentation (Dextran
250 000; Spectrum Chemicals, Gardena, CA) followed by
centrifugation through a density gradient (6.07% Ficoll Type 400;
Sigma, St Louis, MO) with 10% hypaque sodium
(Winthrop-Breon, Markham, Ontario, Canada). Isolated neutrophils were resuspended in Hanks' balanced salt solution (HBSS; with Ca2+ and Mg2+) at a
concentration of 1 × 106 neutrophils/mL. This yielded
neutrophils that were 97% pure and 95% viable.
Platelet isolation and monolayer preparation.
Platelets were isolated from donors by spinning the whole blood at 900 RPM for 10 minutes. The supernatant (approximately 10 mL from 30 mL
whole blood) was reconstituted to 50 mL with 2 mL of ACD anti-coagulant
and phosphate-buffered saline and spun for 10 minutes at 2,100 RPM. The
supernatant was discarded and the pellet resuspended in 5 mL of HBSS.
The platelets were diluted to a final concentration of 2 × 108 platelets/mL with HBSS, placed on ice, and used within
60 minutes of isolation.
Glass coverslips were incubated with collagen type-I for 1 hour at
37°C. One milliliter of the platelet suspension was then placed on
the collagen-treated coverslips and allowed to settle for 1 hour at
37°C. This approach generated confluent monolayers of platelets.
The coverslips were then gently rinsed in HBSS and incorporated into
the laminar plate flow chamber.
Protocol.
A Perspex parallel-plate flow chamber similar to the one described by
Lawrence et al13 was used. The platelet monolayers were
used as the chamber's bottom plate. The chamber was placed in a
thermoregulated plexiglass box that was maintained at 37°C by an
electric heating element. The neutrophil suspension was placed in a
37°C water bath 5 minutes before perfusion and was drawn through
polypropylene tubing into the chamber via a syringe pump. The
experiment was visualized by an inverted phase contrast microscope,
which allowed the neutrophils to be seen without fluorescent labeling.
A camera was attached to the microscope and its output was directed
through a VCR to a television monitor. The experiments were recorded
for playback analysis. Neutrophils were perfused over the platelet
monolayers and the neutrophil-platelet interactions were documented
over 20 minutes. As well, neutrophils were perfused at 1, 2, and 4 dynes/cm2 and, based on these results, all subsequent
experiments were performed at 2 dynes/cm2.
Molecular mechanisms underlying the neutrophil-platelet interactions
were assessed. The monoclonal antibody (MoAb) G1 (generously provided
by Dr R.P. McEver, University of Oklahoma, Oklahoma City, OK) directed against P-selectin was used at a
concentration of 2 µg/mL. Fucoidan, a selectin-binding carbohydrate
known to inhibit P-selectin function, was used at a concentration of
100 µg/mL (higher concentrations activated neutrophils). In fucoidan
posttreatment experiments, untreated neutrophils were allowed to
interact with the monolayer for 10 minutes. After 10 minutes, fucoidan
was added to the neutrophil suspension and perfused across the platelet monolayers. Commercially available fucoidan contained high levels of
endotoxin, and so fucoidan was cleaned before use. Briefly, the
fucoidan (250 mg) was dissolved in 12.5 mL of distilled water and the
pH was adjusted to 7.0 with 0.1 mol/L NaOH and an equal volume of 1 mol/L NaCl was added. The fucoidan salt solution (10 mg/mL fucoidan in
0.5 mol/L NaCl) was treated with 0.5 g of activated charcoal and
filtered using Whatman no. 1 paper (Whatman, Maidstone, UK) to remove the activated charcoal. The activated
charcoal treatment/filtering was repeated twice more. This solution was
filtered through a 0.2-µm cellulose acetate filter to remove residual
activated charcoal. The fucoidan solution was exhaustively dialyzed
against water, lyophilized, and dissolved at 10 mg/mL in distilled
water. The final solution was filtered through a 0.2-µm cellulose
membrane and then lyophilized. This procedure generated 175 mg of
product, which had less than 10 endotoxin units/mg. This method is
based on methods to remove endotoxin from commercial heparin. Polymixin B was not used because it did not remove endotoxin from the fucoidan in
part because of the highly negatively charged properties of the
carbohydrate.
In other experiments, we used SLEx with an aliphatic
aglycone attatched in -glycosidic linkage to the reducing sugar (500 µmol/L; Glycomed Inc, Alameda, CA), as previously
described.14 To assess the firm adhesion, MoAb
IB4 (anti-CD18 MoAb) was used at a concentration of 20 µg/mL and was incubated with the neutrophils at 37°C for 10 minutes before perfusion. In additional experiments, an RGD peptide
that binds to CD41 (GP IIb/IIIa) was used at 100 µmol/L (RGDS;
Peninsula Laboratories Inc, Belmont, CA). In a complementary experiment, platelets from a Glanzmann's thrombasthenia patient (devoid of GP IIb/IIIa) were used to assess the importance of GP
IIb/IIIa (CD41) in the neutrophil-platelet interaction.
To determine whether PAF or interleukin-8 (IL-8) were inducing the
rolling neutrophils to adhere, the PAF receptor antagonist, WEB 2086 (20 µg/mL; Boehringer, Indianapolis, IN) or the MoAb against IL-8, MoAb 208 (10 µg/mL; R&D, Minneapolis, MN)
were used. WEB 2086 or MoAb 208 was incubated with the neutrophil
suspension for 5 minutes at 37°C before perfusion. Additionally,
recombinant PAF acetylhydrolase (kindly provided by Dr G.M. Peterman,
ICOS Corp, Seattle, WA), which de-acetylates PAF to lyso-PAF, was used (6 µg/mL). Platelet monolayers were pretreated with PAF-AH for 30 minutes at 37°C and washed, and then neutrophils were perfused over
the platelet monolayer as previously described.
To compare neutrophil rolling and adhesion on platelet monolayers to
neutrophil-endothelial interactions, some experiments were performed on
histamine-treated human umbilical vein endothelium (HUVEC) and on
P-selectin-transfected CHO cells (generously provided by Dr R.P.
McEver). HUVEC were isolated as previously described, and only primary
or first-passaged endothelium was used as further passage failed to
express P-selectin.15 Histamine (25 µmol/L; Sigma) was
added to induce rolling on HUVEC, and fucoidan was administered as
previously described for platelet monolayers. To determine whether more
effective conversion from rolling to adhesion could be induced on
endothelium with fucoidan, in some experiments lower shear stress (1 dyne/cm2) was also used.
In some experiments, neutrophils were fixed by exposing the neutrophil
suspension to a 1% formalin solution for 15 minutes at 4°C. The
cells were then spun at 1,100 RPM for 5 minutes and resuspended in
HBSS. In other experiments, the platelets were fixed with 1% formalin.
Analysis of neutrophil interactions.
The number of rolling neutrophils was determined by counting the number
of neutrophils per minute that moved slower than free-flowing neutrophils across a predetermined length of monolayer. An adherent neutrophil was defined as one that stayed stationary on the monolayer for 10 seconds or more. The last 10 seconds of a given minute were
analyzed for adherent neutrophils. It should be noted that neutrophils
that adhered for 10 seconds on platelet monolayers generally remained
adherent for the next 10 minutes. Experiments were recorded at
200× magnification and the field of view was calculated to be
0.11 mm2.
Extraction, separation, and quantification of radiolabeled PAF.
The lipids were extracted from platelets, which were layered on
different substrata, using a modification15 of the method of Bligh and Dyer.16 The platelets were first laid down on
different substrata (ie, fibrinogen, gelatin, or collagen) and allowed
to incubate for 1 hour. They were then scraped and resuspended before the extraction step. Because endogenous PAF is rapidly degraded by
acetyl hydrolase, in some experiments we used phenylmethanesulphonyl fluoride (PMSF), a serine hydrolase inhibitor, to inactivate the intracellular acetyl hydrolase activity.17 In these
experiments, platelets were incubated with 2 mmol/L PMSF for 15 minutes
at 37°C. In flow chamber experiments, PMSF was not used because
platelets were not lysed. The scraped platelets in buffer were mixed
with the extraction solvent (as previously described). To this
combination, monophase sodium acetate and chloroform were
added, creating a biphasic mixture. The lower chloroform layer was
washed with an aqueous wash to remove any unreacted acetate. PAF, in
the chloroform phase, was then separated from other lipids on
thin-layer chromatography (TLC) plates, using a solvent system as
described by Mueller et al.18 The samples were then scraped
from the TLC plates in a narrow zone based on their comigration with
authentic PAF. The radioactivity of the samples from the appropriate
zone of the TLC plate was quantified by liquid scintillation counting,
with a counting efficiency of approximately 0.45.
Enzyme-linked immunosorbent assay (ELISA) for P-selectin expression.
Briefly, platelets were incubated in collagen-coated well plates,
fixed, and blocked with 1% bovine serum albumin. They were then
labeled with 2 µg/mL S12 (a nonblocking P-selectin MoAb; generously
donated by Dr R.P. McEver) by incubating for 30 minutes at 37°C and
washed, and then a peroxidase-labeled goat antimouse IgG (1 µg/mL;
Kirkegaard and Perry Laboratories, Gaithersburg, MD) was added for 30 minutes at 37°C. The platelets were then washed and color-developed
with a TMB one-step substrate (Dako, Carpinteria, CA) system. The color
reaction was stopped with 0.18 mol/L H2SO4, and
color was read on a plate reader at 450 nm.
Statistics.
All flow chamber data are reported as the mean ± SEM run 3 to 8 separate times in duplicate. Means were compared using the Mann-Whitney
U-test. Statistical significance was set at P < .05.
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RESULTS |
Neutrophils roll and adhere under flow conditions to immobilized
platelets via P-selectin.
Figure 1A demonstrates a continuous
increase in the number of rolling neutrophils on platelet monolayers
with time of perfusion. Initial tethering interactions of neutrophils
to platelet monolayers were followed by a rolling response, with the
neutrophils maintaining their rounded appearance. However, with time, a
significant number of the rolling cells stopped (varied from 25% to
50% of rolling cells), shape-changed, and spread on the monolayer
surface (Fig 1B). The firm adhesion increased rapidly over the first 5 to 10 minutes and then the number of adhering cells seemed to plateau. Based on this pattern, experiments were generally run for 10 to 20 minutes. All of the experiments in Fig 1 and in the other figures were
performed at 2 dynes/cm2. This was based on the fact that
this shear promoted both neutrophil rolling and adhesion, whereas, at 1 dyne/cm2, only a few cells could be seen rolling (the
majority of cells were adherent), and at 4 dynes/cm2, no
neutrophil-platelet interactions were noted (data not shown). Pretreatment of the platelet monolayers with G1 (2 µg/mL), an anti-P-selectin antibody, completely prevented the neutrophil rolling
(Fig 1A). Subsequent adhesion was also entirely abolished (Fig 1B),
consistent with the view that rolling is a prerequisite for adhesion.
There was a large amount of P-selectin expression on the cell
surface of platelets immobilized on collagen relative to histamine-
(Fig 2) or thrombin-treated endothelium
(data not shown).
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| Fig 1.
Accumulation of neutrophils over 20 minutes. Shown is the
number of rolling (A) and adhering (B) neutrophils on collagen (type I)
immobilized platelets. Over the first 20 minutes, approximately 25% to
50% of the rolling cells adhered at 2 dyn/cm2.
Pretreatment of platelet monolayers with the anti-P-selectin antibody
(G1; 2 µg/mL) prevented any neutrophil-platelet interactions. An
isotype-matched control antibody did not affect neutrophil rolling or
adhesion (data not shown).
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| Fig 2.
P-selectin expression. Shown is P-selectin expression on
platelets relative to P-selectin on HUVEC. Platelets were incubated on
collagen, as described in the Materials and Methods, and an ELISA was
performed to measure cell-surface P-selectin expression. HUVEC were
stimulated with histamine (25 µmol/L) for 10 minutes, and then the
P-selectin expression was measured. ( ) Nonspecific binding; ( )
P-selectin expression.
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Neutrophils adhere via CD18 but not CD41 on platelet monolayers.
Despite the high density of P-selectin expression on the platelet
surface, it is unlikely to be sufficient to support firm neutrophil
adhesion. An antibody directed against the CD18-glycoprotein (IB4; 20 µg/mL) significantly reduced the firm adhesion
of neutrophils to platelet monolayers
(Table 1). The GP IIb/IIIa integrin (CD41) on platelets was not involved in mediating adhesion. An RGDS peptide did not reduce the adhesion and platelets from a Glanzmann's
thrombasthenia patient, which lacked CD41 maintained adhesive
interactions comparable to a normal control (Table 1). It should be
noted that this experiment was only run once in duplicate due to very
limited access to the patient. None of the manipulations in Table 1
affected leukocyte rolling (data not shown).
PAF contributes to the neutrophil adhesion on platelet monolayers.
After the induction of neutrophil rolling on platelet monolayers, the
neutrophils began to change shape and subsequently firmly adhere. This
is not observed when neutrophils roll on P-selectin-transfected CHO
cells or on immobilized P-selectin (personal
observations).19 Additionally, fixed
neutrophils perfused across platelet monolayers rolled at control
levels; however, the adhesion was entirely eliminated (Table 1). All of
these observations suggest that the neutrophils are being activated
after contact with the platelet monolayer. To test whether PAF was
involved, we incubated neutrophils with the PAF receptor antagonist WEB
2086 (20 µg/mL). As seen in Fig 3A, WEB
2086 reduced neutrophil adhesion to platelet monolayers by 60% (from
45 to 18 cells/field of view). This was specific for adhesion, because
very significant numbers of neutrophils continued to roll on the
platelet monolayer, often exceeding the number of untreated rolling
cells (data not shown). The platelet surface was the likely source of
PAF, because selective treatment of the platelet monolayers with PAF-AH
also inhibited neutrophil adhesion by approximately 60%, whereas the
placebo (solvent in which PAF-AH was dissolved) had no effect on the
platelet-neutrophil interactions (Fig 3B). Additionally, fixation of
the platelet monolayer, which inhibits further synthesis or release of
PAF but does not affect membrane incorporated PAF, did not prevent adhesion (data not shown). However, some reduction in adhesion did
occur, suggesting either that PAF synthesis/degradation was an ongoing
process or that fixation blocked the synthesis and release of some
other pro-adhesive factors. Indeed, supernatants from platelet
monolayers had pro-adhesive activity that could not be inhibited by the
PAF receptor antagonist (data not shown).
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| Fig 3.
Neutrophil adhesion in the presence of (top) a PAF
receptor antagonist (WEB 2086; 20 µg/mL) or after (bottom) platelet
monolayer treatment either with PAF-acetylhydrolase or the solvent
(placebo). The placebo and acetylhydrolase group were compared with an
untreated monolayer, and the data are presented as a percentage of
adhesion relative to the 10-minute value for untreated platelet
monolayers. *P < .05 relative to placebo value. (A) ( ) WEB
2086-treated neutrophils; ( ) untreated neutrophils. (B) ( )
Placebo-treated monolayers; ( ) PAF-acetylhydrolase-treated
monolayers.
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Because 40% of the adhesion could not be prevented by WEB 2086 and
IL-8 has been reported to be released by platelets,20 we
tested the possibility that this cytokine also contributed to
neutrophil adhesion. An anti-IL-8 MoAb (10 µg/mL; R&D), at concentrations that prevent IL-8-induced neutrophil adhesion to endothelium,21 had no effect on neutrophil adhesion to
platelet monolayers (Table 1).
Platelets produce PAF when immobilized on collagen.
All of the data obtained thus far suggests that the platelets are the
likely source of PAF. Indeed, we measured significant amounts of PAF on
platelets immobilized on collagen (Fig 4).
Interestingly, other substrata did not produce PAF to the same degree.
PAF-AH had to be blocked by PMSF; otherwise, during the scraping and lysing step, PAF would be deacetylated by the liberated stores of
PAF-AH. This serves as further evidence that we were indeed studying
PAF. It is of further interest that platelets could be stimulated to
produce greater amounts of PAF when additionally stimulated with a
calcium ionophore (A23187; data not shown).
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| Fig 4.
PAF production by platelets. Platelets were layered on
gelatin, fibrinogen, and collagen, and the amount of PAF produced was measured after 1 hour of incubation. Platelets were either incubated in
buffer or with 2 mmol/L PMSF for 15 minutes at 37°C to degrade the
endogenous acetyl hydrolase activity. () Untreated platelets; ()
PMSF-treated platelets.
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Signaling via soluble selectin ligands enhances adhesion on
platelets but not on P-selectin-expressing endothelium.
Fucoidan, a selectin-binding carbohydrate, rapidly reduced
neutrophil rolling and prevented further neutrophil rolling from being
initiated (Fig 5A). However, rather than
detaching the majority of rolling cells, essentially all of the rolling
cells adhered firmly immediately upon fucoidan administration (Fig 5B).
This increased adhesion required platelet activation, because fixation of platelets completely inhibited the fucoidan-induced adhesion. Neutrophils were capable of adhering to fixed platelets in the absence
of fucoidan (7.0 ± 2.9 neutrophils/min/field of view), suggesting
that fixation did not remove surface adhesive molecules. We do not
believe that endotoxin was the mechanism of action for the following
reasons. First, the endotoxin-free fucoidan (<10 EU/mg; as described
in the Materials and Methods) inhibited rolling and induced neutrophil
adhesion to the same degree as endotoxin-contaminated fucoidan. Second,
endotoxin (10 EU/mL; Escherichia coli 0127:B8; Sigma) alone did
not induce the adhesive response (Fig 6).
Third, when an SLEx analog, with undetectable levels of
endotoxin was administered to rolling neutrophils, a similar pattern of
conversion from rolling to adhesion was noted (Fig 6). Finally,
uncleaned dextran-sulfate, at a concentration that completely inhibited
rolling, did not induce an increase in adhesion (Fig 6).
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| Fig 5.
Fucoidan posttreatment. Shown is the effect of the
selectin-binding carbohydrate, fucoidan, on rolling neutrophils (A) and adhering neutrophils (B). Fucoidan (100 µg/mL) was administered after
10 minutes of neutrophil perfusion (time = 0), because this was the
time when neutrophil adhesion plateaued. *P < .05 relative to
untreated neutrophils. 0P < .05 relative to time
0 value. () Untreated neutrophils; () fucoidan posttreated
neutrophils.
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| Fig 6.
Demonstrates the increase in neutrophil adhesion after
the addition of SLEX, fucoidan, fucoidan and WEB 2086, lipopolysaccharide (LPS), or dextran sulfate. In each of
these experiments, rolling and adhesion was observed for 10 minutes
before the administration of the reagent and then for an additional 10 minutes. The experiment was also performed on fixed platelets.
*P < .05 relative to untreated value. 0P < .05 relative to time 0 value.
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We do not believe that the fucoidan was activating the neutrophils
directly. When platelets were fixed and fucoidan was added to the
neutrophils, fucoidan detached rolling cells but did not increase
neutrophil adhesion, suggesting that activation of platelets was a
prerequisite (Fig 6). Additionally, when fucoidan was administered to
neutrophils rolling on thrombin (not shown) or histamine-stimulated human umbilical vein endothelium (a P-selectin-dependent event), the
rolling was completely abolished due to neutrophil detachment; adhesion
was not increased (Fig 7). Because optimal
adhesive interactions occur at 1 dyne/cm2 on HUVEC, we also
examined whether fucoidan would increase adhesion of neutrophils to
endothelium under this condition. The addition of fucoidan to
endothelium even at 1 dyne/cm2 still failed to induce the
rapid increase in adhesion that was observed on platelets (adhesion
increased by 14% on endothelium v more than 100% on
platelets). Interestingly, posttreatment with the P-selectin antibody
(G1; 2 µg/mL) rapidly detached the rolling neutrophils from platelets
and there was no detectable conversion of neutrophil rolling to
adhesion (Fig 8). Therefore, the rapid adhesion of rolling neutrophils to the platelet monolayer, seen after
fucoidan or SLEx administration, is unique to these
molecules.
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| Fig 7.
Fucoidan does not increase neutrophil adhesion on
histamine-treated HUVEC. Endothelium was treated at 6 minutes with
histamine (25 µmol/L) and neutrophil rolling (A) and neutrophil
adhesion (B) were determined before and after fucoidan administration
(added at 20 minutes).
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| Fig 8.
G1 posttreatment of platelet monolayers. After 10 minutes
of neutrophil-platelet interaction, the anti-P-selectin antibody (G1;
2 µg/mL) was administered, and neutrophil rolling (A) and neutrophil
adhesion (B) were assessed. *P < .05 relative to time 0 value. () Untreated neutrophils; () G1 posttreated neutrophils.
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| |
DISCUSSION |
It is now well documented that neutrophils tether, roll, and adhere on
activated endothelium and that the key event in the translation between
rolling and adhesion is dependent on critically localized chemotactic
agents produced directly on the surface of endothelium to act as a
juxtacrine activating process.4 In this study, we show that
contact-activated platelets, in the absence of any exogenous
pro-inflammatory stimuli, can support an identical multistep cascade of
adhesive interactions. This suggests that platelets not only produce
adhesion molecules that can capture (tether) neutrophils to initiate
rolling, but also produce chemoattractants to stage the critical
conversion from rolling to firm adhesion. Consistent with this view are
the data provided by us and others8-10 that the initial
rolling is P-selectin-dependent and the firm adhesion is dependent
on CD11b/CD18, because neutrophils in the presence of
anti-CD18 or anti-CD11b antibodies or neutrophils deficient in CD18
(LAD-1 cells) were unable to firmly adhere to platelet monolayers. In
the present study, we have further extended the understanding of the
cascade of events underlying the platelet-neutrophil interactions by
identifying platelet-derived PAF as an important molecule functioning
to activate and cause neutrophils to firmly adhere to the platelet
monolayer surface. However, we also highlight, for the first time, a
critical difference between the multistep process on endothelium and
platelets; soluble ligands for selectins can rapidly and greatly
enhance the efficiency of the transition from rolling to firm adhesion
but only on platelets.
In accordance with the view that PAF was a significant signal for
neutrophil adhesion was the observation that the PAF receptor antagonist (WEB 2086), at a concentration that inhibits 90% to 100%
of PAF-induced neutrophil adhesion to the endothelium,22,23 prevented 60% of rolling cells from adhering to the platelet
monolayers. Further support for the importance of PAF is highlighted by
the fact that 60% of the adhesion could be inhibited with
PAF-acetylhydrolase (PAF-AH), an enzyme specific for deacetylation of
PAF to inactive lyso-PAF. The PAF-AH experiments provide additional
insight into the source and location of the PAF. First, only the
platelet monolayers were exposed to PAF-AH, suggesting that the source
of PAF is likely the platelet. Second, exogenous PAF-AH is not
internalized and, therefore, degradation of PAF indicates that the PAF
activity is on the outside of the membrane. This strategy has been used previously to determine the sidedness of PAF in endothelial
monolayers.24 Finally, we were able to detect PAF activity
from platelets that had adhered to collagen, but not other substrata.
Based on these results, and borrowing from the insights provided by
Lorant et al5 in their study on neutrophil adhesion to
thrombin-treated endothelium via PAF, we propose the following
scenario. P-selectin functions to tether neutrophils to a monolayer of
platelets and, once in close proximity, PAF located on the platelet
surface ligates its receptor on neutrophils and mediates juxtacrine
activation of the PMN to cause firm adhesion via CD18.
Although this juxtacrine activation is reminiscent of the
chemoattractant-induced adhesion of neutrophils to endothelium, there
are a number of critical differences. There were greater levels of
P-selectin on platelets than on endothelium, but greater levels of PAF
on endothelium (data not shown) than platelets. However, the end result
was similar amounts of adhesion on both substrata, perhaps because the
increased amount of P-selectin may slow rolling cells so that they
adhere in the presence of lower concentrations of PAF. Indeed, in vivo
work has demonstrated that slow rolling cells (induced with
LTC4) responded to low concentrations of exogenous PAF by
adhering, whereas fast rolling cells (induced by histamine) were
unresponsive to the same concentration of PAF.25 An
alternative explanation may be that contact-activated platelets express
other pro-adhesive molecules than just PAF. Indeed, WEB 2086 and PAF-AH
only inhibited 60% of the adhesion. Finally, it is possible that
ligation of P-selectin on platelets causes these cells to express
additional activation mechanisms to signal neutrophils to adhere.
The latter contention deserves some attention in light of the striking
response of rolling neutrophils to immediately adhere on platelet
monolayers exposed to two soluble P-selectin ligands, an
SLEx analog or fucoidan, a fucosylated, sialylated
carbohydrate polymer. Although contaminants such as endotoxin within
the fucoidan or SLEx analog preparations could conceivably
activate neutrophils to adhere, the adhesion consistently occurred on
platelet monolayers but not on histamine-treated endothelium or
P-selectin transfectants. Moreover, fucoidan at 100 µg/mL
did not cause any detectable shape-change in neutrophils and did not
enhance adhesion on endothelium under static conditions in the presence
or absence of activating agents.26 Furthermore, we did not
see differences between endotoxin contaminated (>300 EU/mg) and
endotoxin-free (<10 EU/mg) fucoidan, and endotoxin per se failed to
cause neutrophils to adhere on platelet monolayers. The only obvious
difference between the experiments on endothelial and platelet
monolayers is the substratum, suggesting that soluble selectin binding
molecules activate platelets to either increase the production of
additional chemotactic agents or increase adhesive ligands for CD18. In
this study, we can rule out increased production of PAF as the mediator
responsible for fucoidan-induced adhesion due to lack of effect of WEB
2086. However, the possibility that increased expression of ligands for
CD18 (fibrinogen, ICAM-2, etc) cannot be dismissed.
Previous work has shown that selectins binding to their ligands can
activate target cells or alter their functional states. For example,
both membrane associated and soluble E-selectin can activate
neutrophils as well as endothelium.27 Cross-linking of
L-selectin has been demonstrated to be sufficient to enhance the
activation of neutrophils.28,29 These data suggest that selectins, including L-selectin and E-selectin, do have the capacity to
activate neutrophils. Our study, for the first time, raises the
possibility that binding of selectin ligands to P-selectin on
platelets, but not to P-selectin on endothelium, activates the target
cell to increase its adhesivity for rolling neutrophils. It is
intriguing that SLEx analog and fucoidan but not P-selectin
increased adhesion on platelets. The lack of increased adhesive
response to the P-selectin antibody may be related to the need to
cross-link the antibody and P-selectin before this adhesive response
may occur. Further definition of the mechanism will require
identification of the CD18 ligand on the platelet. We now know that the
CD18 ligand on platelets does not involve ICAM-210 or CD41
(this study), but may involve fibrinogen, which is secreted along with
PAF and P-selectin upon platelet activation.30 Whether
fibrinogen is responsible for the increased adhesion is clearly an
important future venue.
| |
FOOTNOTES |
Submitted February 19, 1997;
accepted December 1, 1997.
P.K. is supported by a grant from the Medical Research Council (MRC)
and is an Alberta Heritage Foundation for Medical Research (AHFMR)
senior scholar and MRC scientist. G.A.Z. is supported by Grant No. R01
HL44525 from the National Institutes of Health (NIH). D.E.L. is
supported by the Physician Scientists Award HL 02726 from the NIH.
Address reprint requests to Paul Kubes, PhD, Immunology
Research Group, Dept. of Medical Physiology, Faculty of Medicine, University of Calgary, Calgary, Alberta, T2N 4N1 Canada.
The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" is accordance with 18 U.S.C. section 1734 solely to indicate this fact.
| |
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Abstract
Introduction
Abstract