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Blood, Vol. 93 No. 3 (February 1), 1999:
pp. 876-885
Platelet/Polymorphonuclear Leukocyte Interaction: P-Selectin Triggers
Protein-Tyrosine Phosphorylation-Dependent CD11b/CD18
Adhesion: Role of PSGL-1 as a Signaling Molecule
By
Virgilio Evangelista,
Stefano Manarini,
Rita Sideri,
Serenella Rotondo,
Nicola Martelli,
Antonio Piccoli,
Licia Totani,
Paola Piccardoni,
Dietmar Vestweber,
Giovanni de Gaetano, and
Chiara Cerletti
From the Istituto di Ricerche Farmacologiche Mario Negri, Unit of
Biology of Cell Interactions, "Giulio Bizzozero" Laboratory of
Platelet and Leucocyte Pharmacology; Laboratory of Tumor and Vascular
Cell Biology, Department of Vascular Medicine and Pharmacology,
Consorzio Mario Negri Sud, Santa Maria Imbaro, Italy; and Institute of
Cell Biology, ZMBE, University of Muenster, Muenster, Germany.
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ABSTRACT |
Polymorphonuclear leukocyte (PMN) adhesion to activated platelets is
important for the recruitment of PMN at sites of vascular damage and
thrombus formation. We have recently shown that binding of activated
platelets to PMN in mixed cell suspensions under shear involves
P-selectin and the activated  2-integrin CD11b/CD18. Integrin activation required signaling mechanisms that were sensitive to tyrosine kinase inhibitors.1 Here we show that mixing
activated, paraformaldehyde (PFA)-fixed platelets with PMNs under shear
conditions leads to rapid and fully reversible tyrosine phosphorylation
of a prominent protein of 110 kD (P~110). Phosphorylation was both
Ca2+ and Mg2+ dependent and was blocked by
antibodies against P-selectin or CD11b/CD18, suggesting that both
adhesion molecules need to engage with their respective ligands to
trigger phosphorylation of P~110. The inhibition of P~110
phosphorylation by tyrosine kinase inhibitors correlates with the
inhibition of platelet/PMN aggregation. Similar effects were observed
when platelets were substituted by P-selectin-transfected Chinese
hamster ovary (CHO-P) cells or when PMN were stimulated with
P-selectin-IgG fusion protein. CHO-P/PMN mixed-cell aggregation and
P-selectin-IgG-triggered PMN/PMN aggregation as well as P~110 phosphorylation were all blocked by antibodies against P-selectin or
CD18. In each case PMN adhesion was sensitive to the tyrosine kinase
inhibitor genistein. The antibody PL-1 against P-selectin glycoprotein
ligand-1 (PSGL-1) blocked platelet/PMN aggregation, indicating that
PSGL-1 was the major tethering ligand for P-selectin in this
experimental system. Moreover, engagement of PSGL-1 with a nonadhesion
blocking antibody triggered 2-integrin-dependent genistein-sensitive aggregation as well as tyrosine phosphorylation in
PMN. This study shows that binding of P-selectin to PSGL-1 triggers
tyrosine kinase-dependent mechanisms that lead to CD11b/CD18 activation in PMN. The availability of the 2-integrin to
engage with its ligands on the neighboring cells is necessary for the tyrosine phosphorylation of P~110.
© 1999 by The American Society of Hematology.
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INTRODUCTION |
THE BINDING OF polymorphonuclear
leukocytes (PMN) to activated platelets in mixed-cell suspensions under
high-speed rotatory motion can be modeled as an adhesion cascade
involving a P-selectin-dependent recognition step followed by an
adhesion-strengthening interaction mediated by CD11b/CD18.1
As platelet-PMN adhesion was prevented by tyrosine kinases inhibitors,
an intermediate tyrosine kinase-dependent signal regulating
2-integrin adhesiveness was postulated.1 The
multistep adhesion cascade was first characterized for PMN adhesion to
the endothelium.2,3 In this model, the selectin-mediated
rolling movement culminates in the firm adhesion sustained by the
binding of intercellular adhesion molecules (ICAMs) expressed on the endothelial surface to 2-integrins on
PMN.2,3 At variance with the adhesive molecules involved in
the rolling step that do not require an activation signal to recognize
the ligand, 2-integrins require a functional
upregulation to become competent to bind their
counter-receptor.4 This implies that an intermediate
2-integrin-activating signal is delivered from the
endothelial cell surface to PMN in the few seconds between rolling
movement and firm attachment. To explain this signaling two
possibilities not excluding each other have been reported in the
literature. The first is that tethering by selectins allows a
juxtacrine-activating signal by lipid autacoids and chemoattractants bound on endothelial cell membrane.5 The second possibility is that selectin binding to PMN may, per se, induce Mac-1
activation,6-8 although conflicting results have been
reported.9
In addition to PMN adhesion to the
endothelium,2,3 a similar, well-characterized, multistep
model has been recently shown for adhesion of flowing PMN over
surface-adherent platelets.10-14 This phenomenon may have
pathophysiological relevance, because platelets activated at the site
of vascular damage could play an important role in leukocyte
recruitment in a growing thrombus,15,16 where accumulated
leukocytes can contribute to increase fibrin deposition.15
Activated platelets adhering to the surface of a damaged vessel could
substitute endothelial cells in allowing recruitment and migration of
leukocytes through the vessel wall.13 These events on one
hand may contribute to the maintenance of the vascular and tissue
integrity and on the other may play a pathogenetic role in inflammatory
and thrombotic disease. Indeed, activated platelets express not only
P-selectin but also different 2-integrin ligands
including fibrinogen17,18 and ICAM-2.19 Moreover, activated platelets can release platelet-activating factor
(PAF), adenine nucleotides, and the CXC chemokines ENA-78, GRO- ,20 and the neutrophil-activating peptide-2
(NAP-2).21,22 All these platelet-derived products are
potent PMN agonists and some of them may be involved in
2-integrin-dependent arrest of PMN on surface-adherent
platelets.14 Previous experimental evidence shows that
P-selectin, alone23-25 or in combination with
chemokines,26,27 is able to stimulate different responses
by leukocytes, suggesting that some of these responses are directly
triggered by the adhesive molecules whereas others require integration
with additional signals elicited by chemokines. Experimental evidence
includes the observation that P-selectin may per se stimulate
CD11b/CD18-dependent phagocytosis by PMN,28 although
conflicting results have been reported.29 The functional
responses elicited by P-selectin on leukocytes could be prevented by
specific antibody to the cloned30,31 P-selectin
glycoprotein ligand-1 (PSGL-1), indicating that this adhesive receptor
is able to transduce an "outside-in" signal when engaged by the
ligand.27 This concept was recently strengthened by the
observation that engagement of PSGL-1 on PMN, per se, induced protein-tyrosine phosphorylation, activated MAP kinases, and stimulated interleukin-8 secretion.32 More recently Blanks et al
showed that PSGL-1 engagement on mouse but not on human PMN induces
LFA-1 and Mac-1-dependent adhesion to ICAM-1.33
Altogether these observations left open the question whether P-selectin
is directly or indirectly involved in the mechanisms allowing activated
platelets to stimulate the tyrosine kinase-dependent adhesiveness of
Mac-1 that mediates aggregation of PMN and platelets. In the present
study using activated platelets, P-selectin expressing Chinese hamster
ovary cells (CHO-P) and soluble recombinant P-selectin, we addressed
the question whether P-selectin was able to trigger protein-tyrosine
phosphorylation in PMN as well as the tyrosine kinase(s)-dependent
function of the 2-integrin Mac-1. Finally, we
investigated the possible role of the cloned P-selectin receptor, PSGL-1, in this phenomenon.
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MATERIALS AND METHODS |
Chemicals.
Hydroethidine (HE) was purchased from Molecular Probes Europe (Leiden,
The Netherlands); 2',
7'-bis-(2-carboxyethyl)-5(6)-carboxy-fluorescein triacetoxy
methyl ester (BCECF-AM), fluorescein isothiocyanate (FITC)-conjugated
antimouse IgG (whole molecule), FITC-conjugated anti-human CD11b
monoclonal antibody (clone 44), F(ab)" fragments of sheep anti-mouse
IgG, n-formyl-Methyl-Leucyl-Phenylalanine (fMLP), prostaglandin
E1 (PGE1), phorbol 12-myristate, 13 acetate (PMA), N-2 hydroxyethyl piperazine-N 1-2-ethanesulfonic acid (HEPES) ethylene glycol-bis (b-aminoethyl ether)-N, N, N',
N',-tetraacetic acid (EGTA), thrombin from human plasma (2,000 National Institute of Health [NIH] U/mg of protein), the kinases
inhibitors genistein, erbstatin A, and
1-(5-isoquinolinylsulfonyl)2-methylpiperazine (H-7) were purchased from
Sigma Chemical Co (St Louis, MO), and deionized water was purchased
from Merck (Milano, Italy). Paraformaldehyde (PFA) was purchased from
Fluka (Milano, Italy). Dextran T500 and Ficoll-Hypaque were from
Pharmacia Fine Chemicals (Uppsala, Sweden) and Aldrich Chimica srl
(Milano, Italy). fMLP was dissolved in dimethyl sulfoxide (DMSO) at
concentrations of 100 mol/L, stored at  20°C, and diluted in
isotonic saline just before use. Thrombin was dissolved in saline at
concentrations of 50 U/mL and stored at 20°C until use.
BCECF-AM and HE were dissolved in DMSO at concentrations of 1 mg/mL and
8 mg/mL, respectively, stored at 20°C, and used within 4 weeks. The MEK inhibitor PD98059 is from Calbiochem (La
Jolla, CA). The R-PE conjugate anti-human CD62P (clone CBL 474P) was
purchased from Serotec (Milano, Italy). The recombinant horseradish
peroxidase-conjugated anti-phosphotyrosine monoclonal antibody (MoAb)
RC20 was from Transduction Laboratories (Exeter, UK). Enhanced
ChemiLuminescence Western blotting system (ECL-kit) was from Amersham
Life Science (Little Chalfont, Buckinghamshire, UK). Reagents for
electrophoresis and Western blot analysis were pure grade.
Antibodies.
The following MoAbs were kindly provided: the anti-P-selectin WAPS
12.234 and the anti-L-selectin DREG-56 and
DREG-20035 by Dr Eugene C. Butcher (Foothall Research
Center, Stanford University, CA), the anti-ICAM-2 B-T1 and
B-R736 by Dr Carl G. Gahmberg (Department of Bioscience,
University of Helsinky, Helsinky, Finland), the anti-PSGL-1 PL1 and
PL237 by Drs Kevin L. Moore and Roger P. McEver (WK Warren
Medical Research Institute, University of Oklahoma, Oklahoma City), the
anti-CD18, 60.338 by Dr John Harlan (University of
Washington, School of Medicine, Seattle), P18 and P2 recognizing
v3 and IIb3,39 respectively, by Dr John L. McGregor (INSERM, Unité 331, Lyon, France), the anti-CD11b Ab 44 by Dr Nancy Hogg (Imperial Cancer Research Fund, London, UK), and
LPM19c, an anti-CD11b,40 by Dr Karen Pulford (University of
Oxford, Oxford, UK).
The anti-CD11a, TS1/22,41 the anti-CD18, IB4
(American Tissue Type Culture Collection, Manassas, VA),
and the anti-CD11c, HC1.142 (kindly provided by Dr Carmelo
Bernabeu, Centro de Investigationes Biologicas, Madrid, Spain) were
purified from mouse ascites or hybridoma cell supernantant using
protein G-sepharose affinity column.
The construction of the P-selectin-IgG fusion protein has been
described.43
Preparation of PMN leukocytes and of platelets and culture of CHO
cells.
Blood was collected from healthy volunteers who had not received any
medication for at least 2 weeks. Approval was obtained from the
Institutional Review Board for these studies. Volunteers were informed
that blood samples were obtained for research purposes and that their
privacy would be protected. Platelet-rich plasma (PRP) was prepared by
centrifugation of citrated blood at 200g for 15 minutes. PMN,
isolated from the remaining blood by Dextran sedimentation followed by
Ficoll-Hypaque gradient and hypotonic lysis of erythrocytes, were
washed and resuspended in ice-cold HEPES-Tyrode buffer (pH 7.4)
containing 129 mmol/L NaCl, 9.9 mmol/L NaHCO3, 2.8 mmol/L
KCl, 0.8 mmol/L KH2PO4, 5.6 mmol/L dextrose, 10 mmol/L HEPES. Immediately before the experiment 1 mmol/L
MgCl2 and 1 mmol/L CaCl2 were added to the
cells. All the procedures for PMN isolation were performed at 4°C.
Cellular suspensions contained 95% of PMN and an average of 1 platelet/30 PMN. For adhesion experiments PMN were stained with the
vital red fluorescent dye HE (20 µg/ 5 × 107
PMN/mL) for 30 minutes at 4°C as previously reported.1
PFA-fixed resting, thrombin-activated unloaded, or BCECF-loaded
platelets were prepared as previously described.1
Wild-type CHO or CHO stably transfected with the cDNA encoding for
human P-selectin (CHO-P) were kindly provided by Genetics Institute,
Cambridge, MA, and cultured as previously reported.44 Immediately before the experiments, CHO and CHO-P cells were detached by incubating the monolayer with 5 mmol/L of both EGTA and EDTA for 10 minutes, washed twice in Hepes Tyrode, and resuspended in the same
buffer at the concentration of 107/mL.
Cytofluorimetric analysis of P-selectin expression on platelets and
CHO cells.
P-selectin expression on platelets and CHO cells was evaluated by
indirect immunoflurescence using WAPS 12.2. As already
reported,1 in our experimental conditions about 50% of
unstimulated washed platelets express very low levels of P-selectin (MF
20) as compared with thrombin-activated platelets (100% positive with
a MF of 185). CHO-P cells express high amount of P-selectin (83 ± 5% of the total population showed a mean fluorescence intensity of 388 ± 110 [mean ± SEM, n = 5]). Wild-type CHO cells did not bind
the anti-P-selectin antibody.
Experimental conditions.
All the experiments have been performed in the following standard
conditions: PMN alone or mixed with platelets (ratio 1/5 or 1/10) or
with CHO cells (ratio 5/1) were incubated in a final volume of 500 µL
in siliconized glass tubes (internal diameter 6 mm; ChronoLog, Mascia
Brunelli, Milano, Italy). The tubes were placed in an aggregometer
(Platelet Ionized Calcium Aggregometer, PICA, ChronoLog, Mascia
Brunelli) at 37°C with stirring (1,000 rpm) obtained by an iron bar
(4 mm long) rotating under a magnetic field. Although the shear rate
produced by this stirring speed cannot be precisely quantified, it
should approximate 250/s.45
This experimental condition allowed clear highlighting of the essential
role of the 2-integrin CD11b/CD18 in mediating
PMN/platelet adhesion.1
For blocking studies, antibodies were preincubated at saturating
concentration (20 µg/mL) with the desired cell fraction for 15 minutes at 4°C.
Protein kinase inhibitors or the diluent (DMSO) were preincubated with
PMN 2 minutes at room temperature before mixing with platelets, CHO
cells, or P-selectin chimeras.
Double-color cytofluorimetric assay of PMN adhesion to platelets or
CHO cells in suspension.
The previously described methodology1 was used to evaluate
PMN-platelet adhesion. Briefly, BCECF-loaded platelets and HE-PMN mixed-cell population were incubated in standard conditions. The interaction was stopped at different times by addition of one volume of
PFA 2%, and samples were kept at 4°C in the dark and analyzed by
flow cytometry within 1 hour.
PMN adhesion to CHO cells was evaluated in similar manner. HE-loaded
CHO cells were incubated with unloaded PMN in standard conditions. The
interaction was stopped at different times by addition of one volume of
PFA 2%, and cells were fixed for 30 minutes. After fixation,
mixed-cell populations were incubated for additional 30 minutes with
FITC-conjugated anti-CD11b antibody, which does not bind CHO cells, to
stain the PMN and kept at 4°C in the dark before flow cytometry.
Flow cytometry.
To evaluate PMN/platelet adhesion, PMN were identified on the basis of
forward and side scatter alone or in combination with the specific red
fluorescent marker. Gating on events identified as PMN was performed to
exclude single platelets. For each experiment a sample in which
thrombin-activated platelets were mixed with PMN in the presence of 10 mmol/L EGTA was used to set a threshold on the green fluorescence scale
(FL1; 90% of events below the threshold) to identify PMN showing the
platelet green marker fluorescence.
The percentage of PMN showing the platelet marker, ie, above the
threshold, represents the percentage of PMN binding platelets [PMN(+)%].
Platelet adhesion to PMN was also quantified by evaluating the relative
number of platelets bound to 100 PMN (PLT/100 PMN).1
To evaluate PMN/CHO cells adhesion, CHO cells were identified on the
basis of the specific red fluorescent marker. Gating on events
identified as CHO cells was performed to exclude single PMN. For each
experiment a sample in which CHO cells were mixed with PMN in the
presence of 10 mmol/L EGTA was used to set a threshold on the green
fluorescence scale (FL1; 90% of events below the threshold) to
identify CHO cells showing the PMN green marker fluorescence.
The percentage of CHO cells showing the PMN marker, ie, above the
threshold, represents the percentage of CHO cells binding PMN
[CHO(+)%]. In preliminary experiments the CHO cells adhesion to PMN
was also evaluated by optical microscopy counting the number of CHO
cells carrying PMN and the number of free, nonadherent PMN. The latter
number gives a precise value of the percentage of PMN involved in the
formation of mixed-cell aggregates. PMN incubated with CHO-P cells in
standard conditions formed mixed aggregates in which single CHO-P
appeared surrounded by several PMN. Sometimes more than one CHO-P cells
formed mixed aggregates with PMN. Similarly to what was observed with
platelets, PMN adhesion to P-selectin expressing CHO cells was
transient, reaching a maximum at 3 minutes after the start of stirring.
At this time about 60% of CHO-P cells and 70% of PMN were involved in
forming mixed aggregates. The interaction of wild-type CHO cells with
PMN was negligible.
PMN homologous aggregation.
PMN aggregation represents an homologous cell-cell adhesion that is
essentially mediated by 2-integrins, particularly by CD11b/CD18.46,47 For this reason, PMN aggregation can be
considered a specific index of the functional upregulation of this
integrin. Accordingly, in this study we evaluated the effect of soluble P-selectin-IgG chimera as well as the effect of the engagement of
PSGL-1 with MoAb on PMN aggregation.
PMN (5 × 106/mL) were incubated in standard
conditions with the P-selectin-IgG chimera. The reaction was stopped
at different times by adding one volume of the cells to one volume of
PFA 2%. After fixation, PMN aggregation was evaluated by counting the number of free, nonaggregated PMN (single PMN) by optical microscopy. In preliminary experiments, a dose-response curve showed that maximal
PMN aggregation was induced by 10 µg/mL of P-selectin-IgG chimera.
For this reason this concentration was used for all subsequent experiments. At this concentration the chimera did not aggregate PFA-fixed PMN, excluding that multimers of the protein agglutinate the
cells in an activation-independent manner. P-selectin-IgG chimera
contains the Fc portion of the human IgG. To exclude a possible effect
of this portion mediated by the Fc receptor on PMN, we used nonimmune
human IgG at concentration of 50 µg/mL as control material. Because
human IgG has no effect, we decided to challenge PMN with the
P-selectin chimera in the presence of human IgG to compete for the Fc receptors.
The effect of antibody-mediated engagement of PSGL-1 on PMN aggregation
was evaluated by preincubating for 15 minutes at 4°C, 5 × 106 PMN with 20 µg of the noninhibitory anti-PSGL-1
monoclonal antibody PL2. After preincubation PMN were rapidly
centrifuged to remove unbound antibody, resuspended, and incubated in
standard conditions in the absence or in the presence of 10 µg/mL of
anti-mouse F(ab)2 fragments. In these experiments the
anti-CD11c antibody HC1.1 was used as control. The reaction was stopped
at different times by adding one volume of the cells to one volume of
PFA 2%. After fixation PMN aggregation was evaluated by counting the
number of free, nonaggregated PMN.
Tyrosine phosphorylation experiments.
PMN were incubated alone or with platelets (ratio 1:5), CHO cells
(ratio 5:1), P-selectin-IgG chimera (10 µg/mL), or with anti-PSGL-1
antibodies in the absence or presence of anti-mouse F(ab)2
fragments or with fMLP (1µmol/L) exactly as described for adhesion
experiments. The reaction was stopped at different times after
initiation of stirring at 37°C by adding one volume of the cells to
an equal volume of 2× reducing Laemmli's buffer, added with 2 mmol/L sodium orthovanadate, 5 mmol/L EGTA, 5 mmol/L EDTA, 10 mmol/L
sodium pyrophosphate, 10 mmol/L iodoacetic acid, 1 mmol/L phenylmethylsulfonyl fluoride, 10 mmol/L sodium fluoride, 10 µg/mL leupeptin and aprotinin, 1 mg/mL trypsin-chymotrypsin inhibitor. Samples were boiled for 10 minutes and centrifuged for 10 minutes at
7,000g. Aliquots of 100 µL, corresponding to 1.25 × 106 total PMN lysate, were loaded into 7.5% to 12.5%
gradient sodium dodecyl sulfate-polyacrylamide gel (SDS-PAGE). Proteins
were transferred onto nitrocellulose sheets and nonspecific sites
blocked using 1% bovine serum albumin (BSA) in Tris-buffered saline
overnight at room temperature on a horizontal shaker. The
nitrocellulose sheets were then incubated with the recombinant
horseradish peroxidase-conjugated anti-phosphotyrosine antibody RC20
(0.1 µg/mL, 30 minutes at 37°C). Detection was performed by
chemiluminescence using ECL-kit. Phosphotyrosine bands were visualised
by autoradiography. For each experiment, basal level of
protein-tyrosine phosphorylation was assessed on unstimulated PMN.
Selected autoradiograms were analyzed using a UltroScan XL densitometer
(LKB Instr, Stockholm, Sweden), and values in arbitrary
units were corrected for background.
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RESULTS |
Adhesion to activated platelets stimulates protein-tyrosine
phosphorylation in PMN.
PMN and PFA-fixed resting or thrombin-activated platelets were
incubated alone or combined at 37°C and 1,000 rpm stirring. In
parallel, PMN were challenged with 1 µmol/L of fMLP for comparison. The interaction was stopped at different times by adding cells to
boiling, SDS containing gel-loading buffer and cell lysates was
processed for the analysis of protein-tyrosine phosphorylation. Antiphosphotyrosine immunoblotting of whole cell lysates from activated
platelet/PMN mixed-cell suspensions showed rapid and completely
reversible tyrosine phosphorylation of a protein of molecular weight
between 97 and 116 kD (P~110). Increased tyrosine phosphorylation of additional proteins with relative molecular masses
around 80 and 40 kD were also occasionally observed. Accordingly, we
decided to evaluate tyrosine phosphorylation of this protein as a
marker of tyrosine kinase(s) activation in PMN triggered by platelet
adhesion. As expected, lysates from PMN or activated platelets incubated alone did not show any change in the pattern of
tyrosine phosphorylated proteins, whereas stimulation of PMN by fMLP resulted in increased tyrosine phosphorylation of
different proteins and in the appearance of a tyrosine
phosphorylated protein at 110 kD (Fig
1). This shows that the tyrosine-phosphorylated P~110, found in
activated platelet/PMN mixed-cell populations, belongs to PMN and
undergoes tyrosine phosphorylation as a consequence of cell-cell
interaction.
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| Fig 1.
Activated platelets induce protein-tyrosine
phosphorylation in PMN. PFA-fixed thrombin-activated platelets alone
(P-activated), PMN alone or combined with PFA-fixed resting
(P-resting), or thrombin-activated (P-activated) platelets at a ratio
of 1:5 were incubated for different times at 37°C and stirring at
1,000 rpm (standard conditions). In parallel experiments PMN were
challenged with fMLP (1 µmol/L) for comparison. The figure shows the
Western blot of samples from a representative experiment and the graph
reports the optical density (in arbitrary units) of the major
tyrosine-phosphorylated protein showing a relative molecular mass
between 97 and 116 kD (P~110) in the corresponding samples (symbols
in brackets). Values are means ± SEM, n = 3.
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Protein-tyrosine phosphorylation is required for activated
platelet/PMN adhesion.
The functional relationship between protein-tyrosine phosphorylation
and cell-cell adhesion was investigated using tyrosine kinase(s)
inhibitors. As shown in Fig 2A, the
dose-dependent inhibition by genistein of P~110 tyrosine
phosphorylation clearly correlates with inhibition of adhesion. Another
tyrosine kinase inhibitor, erbstatin A, significantly reduced the
formation of mixed platelet/PMN conjugates (Fig 2B). In contrast, the
broad-based serine threonine protein kinases A, C, and G inhibitor H-7
and the specific MEK inhibitor PD98059 did not significantly modify
adhesion (not shown). This confirms that protein-tyrosine
phosphorylation in PMN is specifically involved in enabling
platelet/PMN firm adhesion.
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| Fig 2.
Protein-tyrosine phosphorylation is required for
platelet/PMN adhesion. (A) PMN were preincubated for 2 minutes at
37°C with different concentrations of genistein or an equivalent
amount of DMSO before addition of PFA-fixed thrombin-activated
platelets. In parallel, HE-loaded PMN were preincubated for 2 minutes
at 37°C with different concentrations of genistein or an equivalent
amount of DMSO before addition of PFA-fixed BCECF-loaded
thrombin-activated platelets. Coincubation in standard conditions was
stopped at 2 minutes and samples processed for the evaluation of
protein-tyrosine phosphorylation and cell-cell adhesion. The figure
shows the Western blot of samples from a representative experiment, and
the graph reports the optical density of the tyrosine-phosphorylated
P~110 in parallel with the number of platelets bound by 100 PMN.
Values, reported as percentage of control, are means ± SEM, n = 3. (B) HE-loaded PMN were preincubated for 2 minutes at 37°C with
erbstatin A (10 µg/mL; black triangles), genistein (10 µg/mL; black
squares), or DMSO (control; white squares) before addition of PFA-fixed
BCECF-loaded thrombin-activated platelets and incubation in standard
conditions. The reaction was stopped at different times and samples
processed for fluorescence-activated cell sorter (FACS) analysis. Data
report PLT/100 PMN and are expressed as percentage of the peak level
(at 1 minute) of the DMSO-treated samples. At this time, 65 ± 8% of PMN bound 361 ± 150 platelets (means ± SEM, n = 3).
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The functional availability of both P-selectin and CD11b/CD18 is
required for platelet-induced tyrosine phosphorylation P~110 in PMN.
Platelet/PMN adhesion in stirred mixed-cell population requires
P-selectin and CD11b/CD18.1 To investigate the role of these adhesive molecules in the platelet-dependent tyrosine
phosphorylation in PMN, we first investigated the ion requirement for
this phenomenon. As shown in Fig 3A,
platelet-induced P~110 tyrosine phosphorylation in PMN could only be
observed when both Ca2+ and Mg2+ were present,
suggesting that, as already observed for adhesion, the functional
integrity of both P-selectin and the 2-integrin is
required for P~110 tyrosine phosphorylation to occur. This interpretation is furthermore supported by experiments in which P-selectin or the or chain of Mac-1 were blocked by specific MoAbs. Indeed, anti-P-selectin, anti-CD18, and anti-CD11b antibodies strongly reduced activated platelet-induced P~110 tyrosine
phosphorylation in PMN (Fig 3B). Anti-CD11b/CD18 antibodies that did
not inhibit adhesion did not modify P~110 tyrosine phosphorylation
(not shown).
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| Fig 3.
P-selectin and CD11b/CD18 are both required for
platelet-induced protein-tyrosine phosphorylation in PMN. (A) PMN were
coincubated with thrombin-activated platelets in standard conditions in
the absence or in the presence of 1 mmol/L Ca2+ and
Mg2+ alone or together. The reaction was stopped at 2 minutes and samples processed for protein-tyrosine phosphorylation. The
figure shows the Western blot of samples from a representative of two
different experiments. (B) PMN and thrombin-activated platelets were
preincubated for 15 minutes with the corresponding antibodies.
Mixed-cell suspensions were coincubated in standard conditions, the
reaction stopped at 2 minutes, and samples processed for analysis of
protein-tyrosine phosphorylation. The figure shows the Western blot of
samples from a representative experiment and bars report the optical
density of the tyrosine-phosphorylated P~110. Values are means ± SEM, n = 3 or 4.
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To completely exclude any possible effect of additional molecules
coexpressed with P-selectin on the surface of activated platelets,
first the latter were substituted with CHO cells transfected with
P-selectin encoding cDNA and second PMN were challenged with soluble,
recombinant P-selectin-IgG chimera.
P-selectin expressed on CHO cells triggers genistein-sensitive
2-integrin-dependent adhesion and
stimulates protein-tyrosine phosphorylation.
In initial experiments we evaluated PMN adhesion to wild-type (CHO) or
P-selectin-expressing CHO cells (CHO-P) in suspensions (PMN:CHO ratio
5:1) at 37°C and 1,000 rpm stirring. As shown in Fig 4A, CHO-P cells transiently adhered to
PMN forming mixed-cell conjugates that were strongly reduced, as
expected, by an anti-P-selectin antibody. Moreover, PMN adhesion to
CHO-P was also inhibited by an anti-CD18 antibody as well as by
genistein, indicating, in agreement with previous
observations,48 that CHO-P cells express in addition to
P-selectin a 2-integrin ligand. Experiments in which
wild-type CHO cells were incubated with PMN in the presence of
Mn2+ in a Ca2+- and Mg2+-free
medium showed that under this condition, PMN were able to form mixed
conjugates with CHO cells that could be inhibited by different
anti-CD18 as well by the anti-CD11b antibody LPM19c (not shown). This
indicates that Mac-1 recognizes a ligand constitutively expressed on
CHO cells. In the absence of exogenous activation, binding of this
ligand by the 2-integrin requires coexpression of
P-selectin and an intact tyrosin kinase(s) activity in PMN. Taken
together these results indicate that to firmly adhere to P-selectin-expressing CHO cells in suspension at high shear rate, PMN
use an adhesive machinery similar to that mediating adhesion to
activated platelets. Similarly to what was observed with platelets, CHO-P/PMN adhesion was accompanied by the induction of P~110 tyrosine phosphorylation (Fig 4B).
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| Fig 4.
P-selectin expressed on CHO cells triggers
genistein-sensitive 2-integrin-dependent adhesion and
stimulates protein-tyrosine phosphorylation in PMN. (A) HE-loaded
wild-type CHO or P-selectin expressing CHO (CHO-P) cells were incubated
in suspension with PMN at a final ratio of 1:5 in standard conditions.
The anti-CD18 antibody (IB4) was preincubated with PMN and the
anti-P-selectin antibody (WAPS12.2) was preincubated with CHO-P for 15 minutes. PMN were pretreated with genistein (10 µg/mL; GEN) as in Fig
2. The interaction was stopped at different times and samples processed
for FACS analysis. Data report the percentage of CHO cells binding PMN.
Values are means ± SEM, n = 3 to 5. (B) PMN and CHO-P were
coincubated as reported above. The interaction was stopped at the
indicated times or at 2 minutes, when the effect of antibodies was
investigated. The figure shows the Western blot from a representative
of three different experiments and the corresponding optical density of
P~110 (means ± SEM, n = 3).
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P-selectin-IgG fusion protein triggers genistein-sensitive
2-integrin-dependent homologous PMN aggregation and
stimulates protein-tyrosine phosphorylation.
PMN aggregation represents homologous cell-cell adhesion, which is
essentially mediated by the 2-integrin
Mac-1.46,47 For this reason PMN aggregation can be
considered a specific index of the functional upregulation of this
integrin. Accordingly, in this study we decided to evaluate the effect
of soluble P-selectin-IgG chimera on PMN aggregation as a functional
index of the ability of soluble P-selectin to trigger the activation of
Mac-1. As reported in Fig 5A, P-selectin
chimeras induced PMN aggregation, which was prevented by the anti-CD18
antibody. Moreover, according to the inhibition of platelet/PMN or
CHO-P/PMN aggregation, P-selectin-induced PMN/PMN aggregation was also
blocked by genistein, reinforcing the hypothesis of an important
regulatory role for protein-tyrosine phosphorylation in
P-selectin-triggered Mac-1 function. The P-selectin-IgG chimera was
also able to induce a strong tyrosine phosphorylation of P~110
protein (Fig 5B).
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| Fig 5.
P-selectin-IgG chimera triggers genistein-sensitive,
2-integrin-dependent PMN aggregation and stimulates
protein-tyrosine phosphorylation. (A) PMN were incubated in standard
conditions in the presence of 50 µg/mL of nonimmune human IgG in the
absence (control) or in the presence of 10 µg/mL of P-selectin-IgG
chimera (P-sel). PMN were preincubated with the anti-CD18 antibody
(IB4) or with genistein (GEN) as reported in Figs 2 and 3. The reaction
was stopped, and PMN remaining single, nonaggregated were counted by
optical microscopy in a Burker chamber. Values (means ± SEM, n = 3 or 4) are reported as percentage of the basal level. (B) PMN were
challenged with P-selectin-IgG chimera as reported above. The reaction
was stopped at different times. The figure shows the Western blot from
a representative of three different experiments and the
corresponding optical density of P~110 (mean ± SEM, n = 2 or
3).
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These results together with those obtained with CHO-P cells show that
P-selectin interaction with its receptor(s) on PMN is sufficient to
switch on the tyrosine phosphorylation-dependent signal that
upregulates the 2-integrin function.
Engagement of the 2-integrin with the
ligand is required for P-selectin-triggered P~110 tyrosine
phosphorylation in PMN.
Similar to what was already observed with activated platelets,
triggering of P~110 tyrosine phosphorylation by CHO-P cells or by
P-selectin-IgG chimera required the functional availability of the
2-integrin. Indeed, as reported in Fig 4B, the
adhesion-blocking anti-CD18 antibody IB4 blocked CHO-P-induced P~110
tyrosine phosphorylation. Similar results were obtained when PMN were
challenged with recombinant P-selectin. P-selectin-triggered P~110
tyrosine phosphorylation was abolished by the aggregation blocking
anti-CD18 antibodies IB4 (Fig 5B) and 60.3.
PSGL-1 triggers tyrosine phosphorylation-dependent CD11b/CD18
adhesion.
The role of PSGL-1 as major P-selectin receptor on PMN was first
investigated by evaluating the effect of anti-PSGL-1 MoAbs on
activated platelet/PMN adhesion. As reported in
Fig 6, blockade of PSGL-1 with the
inhibitory antibody PL1 strongly reduced the formation of mixed-cell
conjugates, indicating a major role of PSGL-1 as P-selectin ligand in
our system.
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| Fig 6.
PSGL-1 mediates activated platelet/PMN adhesion.
HE-loaded PMN were preincubated for 15 minutes at 4°C in the
absence (control) or in the presence of the noninhibitory (PL2), the
inhibitory (PL1) anti-PSGL-1, or with the anti-CD18 antibody (IB4)
before addition of PFA-fixed BCECF-loaded thrombin-activated platelets.
Data report the number of platelets bound by 100 PMN (PLT/100 PMN) and
are expressed as percentage of the peak level (at 1 minute) of the
untreated sample. At this time, 65.7 ± 5.3% of PMN bound 393 ± 55 platelets (means ± SEM, n = 2 to 5).
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Because it was recently reported that engagement of PSGL-1 by MoAb is
able to stimulate protein-tyrosine phosphorylation in PMN,32 we investigated whether the effects of P-selectin on PMN could be reproduced by PSGL-1 engagement with specific MoAbs. As
reported in Fig 7A, incubation of PMN with
the noninhibitory anti-PSGL-1 antibody PL2 induced PMN aggregation.
This effect was more pronounced when PL2 was crosslinked with a
secondary anti-mouse F(ab)2 fragment. PMN aggregation
induced by PL2 cross-linking was inhibited by the anti-CD18 antibody
IB4 and by genistein, indicating that engagement of PSGL-1 enables a
tyrosine kinase(s)-dependent 2-integrin-mediated
homologous adhesion. Furthermore, in agreement with previous
data,32 we also found that ligation of PSGL-1 with PL1 or
PL2 alone resulted in increased tyrosine phosphorylation of different
proteins, as well in the appearance of a tyrosine-phosphorylated protein of about 110 kD. This effect was potentiated after
cross-linking (Fig 7B). Taken together these results indicate that
PSGL-1 represents not only the tethering molecule linking PMN to
P-selectin-expressing cells, but it also triggers the tyrosine
phosphorylation-dependent signal upregulating the adhesive function of
Mac-1.
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| Fig 7.
Engagement of PSGL-1 by the monoclonal antibody PL2
triggers genistein-sensitive, 2-integrin-dependent PMN
aggregation and stimulates protein-tyrosine phosphorylation. (A) PMN
were preincubated at 4°C with PL2 (anti-PSGL-1), rapidly washed to
remove unbound antibody, and incubated in the absence
[PL2-F(ab)2] or in the presence
[PL2+F(ab)2] of 10 µg/mL of rabbit anti-mouse IgG
F(ab)2 fragments in standard conditions. The anti-CD18
antibody (IB4) was preincubated with PMN together with PL2 for 15 minutes at 4°C before cross-linking. PMN were treated with
genistein (10 µg/mL; GEN) for 1 minute after preincubation with PL2
and before cross-linking. In control experiments, PMN were preincubated
at 4°C with HC1.1 (anti-CD11c), washed to remove unbound antibody,
and incubated in the presence of 10 µg/mL of anti-mouse
F(ab)2 fragments in standard conditions
[HC1.1+F(ab)2]. PMN aggregation was evaluated as in Fig
6. Values are means ± SEM (n = 3). (B) PMN were treated and
incubated exactly as for aggregation experiments. The figure shows the
Western blot of a representative of three different experiments and the
corresponding optical density of P~110 (means ± SEM, n = 2 to
3).
|
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| |
DISCUSSION |
Like endothelial cells,2 platelet
monolayers10-14 or platelets aggregated in a growing
thrombus 15,16 sustain accumulation of flowing PMN. This
suggests that activated platelets express the complete adhesive and
signaling machinery necessary for the multistep adhesion cascade
essential for PMN recruitment under shear conditions. This adhesive
machinery is initiated by P-selectin-dependent attachment and rolling
of PMN. The velocity of rolling PMN rapidly slows down, and they arrest
within few seconds after initial tethering on the platelet
surface.13 Arrest of rolling PMN can be blocked by
anti-CD11b/CD18 antibodies, indicating that it is based on the binding
of this molecule to a counter-receptor on platelets.13,14 At variance with P-selectin binding to its counter-receptor, the 2-integrin CD11b/CD18 is not constitutively able to
recognize the ligand but requires functional
upregulation.49,50 The requirement for a platelet-induced
activation of PMN arrest on a platelet monolayer was first suggested by
Yeo et al.10 In a recent study,1 we confirmed
and extended this concept showing that activated platelet/PMN adhesion
in mixed-cell suspensions subjected to high shear rate can be modeled
as a two-step adhesion cascade requiring P-selectin and the
2-integrin CD11b/CD18. In this model, platelet/PMN mixed
conjugates did not form in the presence of tyrosine kinase inhibitors,
suggesting a functional cross talk between P-selectin receptor and the
2-integrin that involves protein-tyrosine phosphorylation.
These observations opened the question whether P-selectin is directly
or indirectly involved in the mechanisms allowing activated platelets
to stimulate the tyrosine kinase(s)-dependent adhesive function of
Mac-1.
In the present study, we showed for the first time that binding of
activated platelets to PMN triggers protein-tyrosine phosphorylation, particularly that of a major protein of about 110 kD. This shows that
activated platelets stimulate intracellular signal(s) able to activate
tyrosine kinase(s) in PMN. Moreover, the dose-dependent inhibition of
the tyrosine phosphorylation of this protein by genistein correlates
with its ability to inhibit platelet/PMN adhesion, supporting a
functional relationship between these two events. The effect of
activated platelets could be mimicked by P-selectin either expressed on
transfected cells or in solution. In fact, P-selectin either expressed
on CHO cells or as soluble recombinant P-selectin-IgG chimera were
able to trigger the tyrosine kinase(s)-dependent adhesive function of
the 2-integrin. Binding of PMN to P-selectin transfected
CHO cells as well as PMN/PMN aggregation were both mediated by the
2-integrin Mac-1 and ligands expressed on CHO cells or
PMN. PMN adhesion to CHO-P as well as stimulation of PMN by soluble
P-selectin-IgG chimera resulted in tyrosine phosphorylation of a
protein with molecular mass of about 110 kD.
An important role in transmitting the P-selectin-induced signal is
probably played by PSGL-1. In fact, an inhibitory anti PSGL-1
monoclonal antibody strongly reduced platelet/PMN adhesion, indicating
that PSGL-1 is the most important receptor tethering activated
platelets to PMN in our system. Moreover, the engagement of PSGL-1 with
a noninhibitory MoAb resulted in a 2-integrin-, tyrosine kinase(s)-dependent homologous PMN aggregation. This shows
that PSGL-1 engagement triggers functional upregulation of Mac-1.
Moreover, in agreement with Hidari et al,32 we also observed that engagement of PSGL-1 by specific MoAbs increased protein
phosphorylation of different proteins in PMN. Altogether the results of
our experiments argue in favor of the interpretation that the binding
of P-selectin to PSGL-1 on PMN is able to transmit signals able to
activate the 2-integrin Mac-1. However, this interpretation does not exclude that additional molecules, acting in a
juxtacrine fashion and requiring engagement of PSGL-1, could still be
playing an important role during platelet-PMN and PMN-PMN interaction.51 The latter interpretation is also in
agreement with data from Lorant et al52 showing that
P-selectin enhances 2-integrin activation by other agonists.
P~110 is the major protein undergoing tyrosine phosphorylation during
cell-cell interaction in our model, suggesting that this protein could
play a role in regulating the 2-integrin adhesiveness. This would imply that first an initial weak interaction of the integrin
with its ligand allows tyrosine phosphorylation of P~110 and that
this, in turn, would be necessary for the 2-integrin to
acquire the full adhesive phenotype. Indeed, in agreement with previous
studies,53, 54 we found that antibody-mediated
cross-linking of the 2 chain results in tyrosine
phosphorylation of different proteins, including P ~ 110 (data not
shown). In this scenario, P-selectin interacting with its receptor on
PMN may directly trigger the initial integrin ligand interaction
and/or it may contribute to the activation and recruitment of
tyrosine kinase(s) necessary for phosphorylation of key protein(s),
possibly P~110.
To clarify the identity of this protein requires further work and will
allow the role of protein-tyrosine phosphorylation in
2-integrin function to be elucidated.
The P-selectin-Mac-1 cross-talk, regulated by protein-tyrosine
phosphorylation, that clearly emerges from our study may also occur
when PMN interact with activated endothelial cells via P-or E-selectin
in flow conditions.
Knowledge of the mechanisms and molecules that regulate these phenomena
may help design drugs for novel anti-inflammatory and antithrombotic
pharmacological intervention.
| |
ACKNOWLEDGMENT |
We thank Dr Paolo Pertile for fruitful discussion; Drs E.C. Butcher,
C.G. Gahmberg, K.L. Moore, R.P. McEver, J. Harlan, J.L. McGregor, N. Hogg, K. Pulford, and C. Bernabeu for their kind gift of antibodies; Dr
B. Furie and the Genetics Institute of Cambridge, MA, for kindly
providing P-selectin-transfected CHO cells; and the Art Department and
the Gustavus A. Pfeiffer Memorial Library staffs for their contribution
in editing the figures and the manuscript.
| |
FOOTNOTES |
Submitted July 14, 1998; accepted September 25, 1998.
Supported by the Italian National Research Council (Convenzione
CNR Consorzio Mario Negri Sud and "Altri interventi 1998") and
by the Fondation Segré, Geneva, Switzerland.
The publication costs of this
article were defrayed in part by
page charge payment. This article
must therefore be hereby marked
"advertisement"
in accordance with 18 U.S.C. section
1734 solely to indicate this fact.
Address reprint requests to Virgilio Evangelista, MD, Unit of Biology
of Cell Interactions, Consorzio "Mario Negri Sud," Via Nazionale,
66030 Santa Maria Imbaro, Italy; e-mail: evangeli{at}CMNS.MNEGRI.IT.
| |
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Top
Abstract
Introduction