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J Biol Chem, Vol. 273, Issue 43, 27768-27771, October 23, 1998
§,, and
From the In this paper we show that the engagement of the
platelet-endothelial cell adhesion molecule-1 (PECAM-1/CD31)
up-regulates the adhesion of human neutrophils to the EA.hy926
endothelial cell line through a phosphoinositide 3-kinase
(PI3K)-dependent pathway. Indeed, LY294002 and wortmannin
prevented the effect of PECAM-1/CD31 cross-linking on cell adhesion, at
concentrations known to inhibit PI3K without affecting other kinases.
Both compounds blocked neutrophil binding to murine fibroblasts
transfected with human ICAM-1, to purified ICAM-1 protein, or to
fibronectin, suggesting that PECAM-1/CD31-mediated up-regulation of
It is widely accepted that the process of leukocyte extravasation
and tissue localization depends on several adhesion systems that are
tightly regulated and connected one to each other (1-4). In
particular, adhesion of leukocytes to blood vessel wall, crawling across endothelial cells, and progression through the subendothelial matrix are regulated by changes in the avidity state of We have recently reported that, in NK cells, cross-linking of
PECAM-1/CD31 leads to increased adhesion to extracellular matrix and
endothelial cells, mostly mediated via Cells and Antibodies--
Peripheral blood neutrophils (PMN)
were isolated from healthy donors (buffy coats, kindly provided by the
Blood Transfusion Department of our institute) by dextran sedimentation
and density gradient centrifugation, followed by hypotonic lysis of
erythrocytes (10). The neutrophil pellet was harvested, washed in DPBS
without calcium and magnesium (Biochrom, Berlin, Germany) and
resuspended in ice-cold HEPES-Tyrode's buffer containing 129 mmol/liter NaCl, 9.9 mmol/liter NaHCO3, 2.8 mmol/liter KCl,
0.8 mmol/liter KH2PO4, 0.8 mmol/liter
MgCl2-6H20, 5.6 mmol/liter glucose monohydrate, 1 mmol/liter CaCl2, 10 mmol/liter HEPES, and 0.3 units/ml
aprotinin, leupeptin, and antipain (Sigma). Cells were >98%
neutrophils by Wright-Giemsa staining and >98% viable by exclusion of
trypan blue. Cross-linking of the CD31 molecules expressed by PMN was obtained as described (11). Briefly, cells (107/ml) were
stained for 20 min at 4 °C with saturating amounts (10 µg/ml) of
the anti-CD31 mAb M89D3, or mouse Ig (Sigma) as a control, washed and
treated with 1 mg/ml of rabbit anti-mouse (RAM) immunoglobulins (Sigma)
for 20 min at 4 °C, followed by incubation at 37 °C for 30 min.
PMN were then washed and used in functional or biochemical assays.
Purified and biotinylated anti-CD31 (11) or anti-CD11a (American Type
Culture Collection) mAb and the F(ab')2 of anti-CD31 mAb
were obtained as described (11).
Adhesion Assay--
The EA.hy926 cell line was kindly provided
by C. Edgell (13). ICAM-1 transfectants were obtained using the murine
NIH/3T3 fibroblast cell line as described (14). Adhesion of
radiolabeled (51Cr, as sodium chromate, 1 µCi/106/cells, NEN Life Science Products) PMN to
confluent EA.hy926 cells or ICAM-1 transfectants was performed as
described (14), before or after cross-linking of CD31 molecules. In
other experiments adhesion was assayed using plastic plates coated with
soluble ICAM-1 (p-ICAM-1, 10 µg/ml in DPBS, pH 9.3), purified by
affinity chromatography from lysates of ICAM-1 transfectants (15) or fibronectin (FN, Sigma, 10 µg/ml in DPBS, pH 9.3). In some samples, cells were pre-incubated for 30 min at room temperature with 100 nmol/liter wortmannin, or 50 µmol/liter Ly294002 or 25 ng/ml
rapamycin (all from Sigma) and washed twice, prior to the onset of the
adhesion assay. After 30 min, nonadherent cells were washed, and
adherent cells were lysed with 100 mmol/liter Tris buffer containing
1% Triton X-100. The radioactivity of the samples was measured in a
Enzyme-linked Immunosorbent Assay--
Flat bottom microwell
plates were coated with the anti-CD31 or the anti-CD11b mAb (10 µg/ml
in DPBS, pH 9.3) and blocked with 3% bovine serum albumin in DPBS
overnight at 4 °C; PMN were added to the coated plates
(105 cells/well), for 20 min at 4 °C followed by 30 min
at 37 °C. Adherent cells were lysed in 50 mmol/liter Tris buffer
with 0.5% CHAPS (Sigma). After washing, plates were stained with
purified biotinylated anti-CD31 or anti-CD11b mAbs (1:1000 dilution)
followed by avidin-horseradish-peroxidase (HRP) (1:10,000 dilution,
Sigma) or with the anti-p85 subunit of PI3K rabbit polyclonal antiserum (Upstate Biotechnology Inc., Lake Placid, NY) followed by HRP-goat anti-rabbit antiserum (GAR 1:100,000 dilution, Sigma). Samples were
then developed with diaminobenzidine (Sigma), and optical density was
determined at 405 nm (15).
Western Blot--
PI3K association to PECAM-1/CD31 was also
analyzed by Western blot in PMN lysates after PECAM-1/CD31
cross-linking and immunoprecipitation. PI3K activity was tested
indirectly by analyzing the autophosphorylation of serine/threonine
kinase p70 S6 kinase (S6K), a signaling protein downstream of PI3K.
After PECAM-1/CD31 cross-linking, performed on intact PMN as above,
cells were lysed (0.5% CHAPS), and PECAM-1/CD31 and PI3K or S6-K were
immunoprecipitated from 300 µg of protein/sample (protein dosage
performed with the Detergent-Compatible Bio-Rad kit based on the
colorimetric Lowry method, Bio-Rad) were run on a 10% SDS-PAGE and
electrotransferred (Hybond ECL, Amersham Italia S.r.l., Milan, Italy)
as described (16). Filters were blocked overnight with 10% non-fat dry
milk in phosphate-buffered saline and then incubated 1 h with the
anti-p85 PI3K rabbit polyclonal antiserum, with the anti-PECAM-1/CD31
mAb, with an anti-Ser(P) mAb (PSR-45, Sigma), or with an anti-S6K
rabbit polyclonal antiserum (C-18, Santa Cruz, CA), all at 1:1,000
dilution, followed by HRP-conjugated RAM (1:10,000 dilution, Sigma) or
HRP-conjugated GAR (1:100,000 dilution, Sigma). The immunoreactive
bands were revealed by luminol reaction (ECL, Amersham).
Ligation of PECAM-1/CD31 on Human Neutrophils Up-regulates
Integrin-mediated Cell adhesion through a PI3K-dependent
Pathway--
Occupancy of PECAM-1/CD31 on the surface of leukocytes
triggers a signal leading to integrin activation and engagement of their endothelial counter-receptors (7-11). To identify the
intracellular mediator(s) of such a signal transduction, we analyzed
the binding of PMN to the EA.hy926 cell line, upon PECAM-1/CD31
cross-linking, before and after PMN exposure to different kinase
inhibitors. Fig. 1 shows that ligation of
PECAM-1/CD31 on human neutrophils increases by 85% cell adhesion to
this endothelial cell line, whereas cross-linking of unrelated
molecules with mouse Ig followed by RAM or with RAM alone has no
effect. Similar results were obtained using the F(ab')2 of
the anti-PECAM1/CD31 mAb (not shown). Interestingly, wortmannin, a
fungal metabolite that inhibits PI3K (17), prevents the effect of
PECAM-1/CD31 ligation, at variance with rapamycin, which does not
affect PI3K (18) (Fig. 1). At the concentrations used (100 nmol/liter)
wortmannin fully inhibits PI3K, although it has no significant effect
on other kinases such as myosin light chain kinase (19) or
phosphoinositide 4-kinase (20). Further evidence for the involvement of
PI3K in the process of PECAM-1/CD31-induced PMN endothelial cell
adhesion comes from the finding that LY294002, another compound that
blocks this enzyme with a different mechanism as compared with
wortmannin (21), can also prevent the effect of PECAM-1/CD31 engagement
(Fig. 1). Because wortmannin is a potent inhibitor of
activation-dependent increases in integrin adhesiveness (17), we analyzed the involvement of Laboratory of Cardiovascular
Pathophysiology,
ABSTRACT
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Abstract
Introduction
Procedures
Results & Discussion
References
2 and 1 integrin-mediated adhesion is PI3K-sensitive. We also
provide evidence for the association of PECAM-1/CD31 to PI3K, because
PI3K was detectable in neutrophil lysates after PECAM-1/CD31
cross-linking and immunoprecipitation. PECAM-1/CD31-dependent recruitment of PI3K was suggested by
the finding that the serine/threonine kinase p70 S6 kinase (S6K), a
signaling protein downstream of PI3K, is activated in neutrophils upon
PECAM-1/CD31 cross-linking, based on the appearance of serine phosphorylation in S6K immunoprecipitates. In turn, S6K is not directly
involved in the up-regulation of integrin function because rapamycin,
which can inhibit S6K independent of PI3K, did not block
PECAM-1/CD31-induced adhesion of neutrophils to 1 and 2 integrin
substrates. In conclusion, PECAM-1/CD31 appears to be one of the
molecules functionally coupled to PI3K, suggesting that this enzyme may
represent a common pathway of integrin and adhesiveness regulation in
leukocytes.
INTRODUCTION
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Abstract
Introduction
Procedures
Results & Discussion
References
2 and 1
integrins (3, 4). Several molecules can modulate integrin function, up-
or down-regulating their affinity to specific ligands (3, 4), thus
allowing leukocyte transendothelial migration. The platelet-endothelial
cell adhesion molecule-1
(PECAM-1/CD31)1 participates
in this process both directly, as an adhesion molecule, and indirectly,
as a modulator of integrin functions (5, 6). Indeed, engagement of
PECAM-1/CD31 by specific monoclonal antibodies (mAbs) or by homophilic
interactions leads to up-regulation of 1 and 2 integrin
functions, including cytoskeletal association and exposure of high
affinity binding sites, in T lymphocytes, natural killer (NK) cells,
platelets, and neutrophils (7-10). These data point to a mechanism
whereby oligomerization of PECAM-1/CD31 on the cell surface results in
the triggering of specific signals capable of modulating integrin
affinity. However, the molecular basis of such signals is still
unclear.
1 and 2 integrin through a
cAMP-sensitive mechanism (11). In platelets, PECAM-1/CD31 becomes
tyrosine phosphorylated during aggregation in an
integrin-dependent process that results in the association
of the SHP-2 phosphatase with tyrosine residues of the PECAM-1
cytoplasmic domain (12). In this paper, the relationship between
PECAM-1 engagement and integrin activation in human neutrophils has
been addressed. We provide evidence for a functional association
between PECAM-1/CD31 and phosphoinositide 3-kinase (PI3K). In addition,
our data support the hypothesis that engagement of PECAM-1/CD31
promotes downstream activation events, including increased 1- and
2-mediated adhesion to specific substrates, which are regulated via
a PI3K-dependent pathway.
EXPERIMENTAL PROCEDURES
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Abstract
Introduction
Procedures
Results & Discussion
References
counter (Packard, Sterling, VA). Cell adhesion was calculated as
described (14). Results are expressed as the percentage of increase (or
decrease) in adherent cells.
RESULTS AND DISCUSSION
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Abstract
Introduction
Procedures
Results & Discussion
References
1 and 2 integrins in the
process of PECAM-1/CD31-mediated up-regulation of PMN endothelial cell
adhesion. To this aim, binding experiments were performed using murine
fibroblasts (NIH/3T3) transfected with human ICAM-1, one major ligand
for 2 integrins (3), or FN, an extracellular matrix protein that
binds to most 1 integrins (4). As shown in Fig.
2A, ligation of PECAM-1/CD31
induced a significant increase in PMN adhesion to ICAM-1 transfectants,
which was sensitive to wortmannin and LY249002 but not rapamycin
treatment. Similar results were obtained when PMN adhesiveness was
assayed using p-ICAM-1 (Fig. 2B) or FN (Fig. 2C).
Wortmannin has been reported to block 1 integrin-mediated increases
in T cell adhesiveness induced by CD2, CD28, or CD7 ligation (17, 22,
23); likewise, there is evidence for PI3K involvement in the modulation
of integrin activation in B and NK cells (17). Thus, the function of
several different integrin regulators can be blocked by pharmacological inhibition of PI3K activity. PECAM-1/CD31 appears to be one of the
adhesion molecules functionally coupled to PI3K, suggesting that this
enzyme may represent a common pathway of integrin and adhesiveness
regulation in leukocytes.
View larger version (19K):
[in a new window]
Fig. 1.
Ligation of PECAM-1/CD31 up-regulates
neutrophil adhesion to endothelial cells involving PI3K. Adhesion
of radiolabeled PMN to confluent EA.hy926 cells was evaluated before or
after cross-linking of CD31 molecules obtained by treatment with 10 µg/ml of the anti-CD31 mAb, followed by 1 mg/ml of RAM Ig and
incubation at 37 °C for 30 min. Unrelated mouse Ig were used as a
control. In some samples, cells were pre-treated for 30 min at room
temperature with 100 nmol/liter wortmannin, 50 µmol/liter Ly294002,
or 25 ng/ml rapamycin and washed twice, prior to the onset of the
adhesion assay. After 30 min, nonadherent cells were washed, and
adherent cells were lysed. The radioactivity of the samples was
measured in a counter (Packard, Sterling, VA). Results are
expressed as the percentages of increase (or decrease) in adherent
cells (means ± S.D. from eight independent experiments). Basal
PMN adhesion, calculated as described (17), was 10%. Wart,
wortmannin; LY, Ly294002.
View larger version (32K):
[in a new window]
Fig. 2.
Ligation of PECAM-1/CD31 up-regulates
integrin-mediated neutrophil adhesion through a
PI3K-dependent pathway. Adhesion of radiolabeled PMN
to confluent ICAM-1 transfectants (A), p-ICAM-1
(B), or FN (C) was evaluated before or after
cross-linking of CD31 molecules, obtained as described in the legend to
Fig. 1. In some samples, cells were pre-treated for 30 min at room
temperature with 100 nmol/liter wortmannin, 50 µmol/liter Ly294002,
or 25 ng/ml rapamycin and washed twice prior to the onset of the
adhesion assay. After 30 min, nonadherent cells were washed, and
adherent cells were lysed. The radioactivity of the samples was
measured in a counter (Packard, Sterling, VA). Results are
expressed as the percentages of increase (or decrease) in adherent
cells (means ± S.D. from eight independent experiments). Basal
PMN adhesion was 10%. Wart, wortmannin; LY,
Ly294002.
PECAM-1/CD31 Associates with PI3K in Human Neutrophils-- Because PI3K is a central signaling molecule for integrin regulators (17), we asked whether PECAM-1/CD31 is associated to PI3K in PMN. To address this question, co-precipitation experiments were performed using microwell plates coated with the anti-PECAM-1/CD31 mAb and PMN, which were lysed after cross-linking. As shown in Fig. 3, PI3K could be selectively detected in the immunoprecipitates obtained with the anti-PECAM-1/CD31 mAb after cross-linking of the molecule (Fig. 3A for enzyme-linked immunosorbent assay detection; Fig. 3B, upper panel, for Western blot; Fig. 3B, lower panel, shows the same blot hybridized with the anti-PECAM-1/CD31 mAb). Conversely, CD11b does not associate PI3K (Fig. 3A).
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and the FK506-binding
rapamycin-associated protein (17, 23). The precise sequence of events
downstream PI3K and the resulting cellular behavior need to be
elucidated. It is tempting to speculate that differential ligation of
PECAM-1/CD31 on neutrophils by distinct endothelial-specific ligands
(either homo- or heterophilic) leads to activation of selected kinase
cascades, resulting in a finely tuned modulation of integrin-mediated
leukocyte binding to endothelial cells.
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ACKNOWLEDGEMENTS |
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We thank C. Rugarli for support. We are also grateful to A. Poggi and A. Rubartelli for criticisms and suggestions.
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FOOTNOTES |
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* 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.
§ To whom correspondence should be addressed: Lab. of Cardiovascular Pathophysiology, Dept. of Cardiology, Scientific Institute San Raffaele, Via Olgettina 60, I-20132 Milan, Italy. Tel.: 39-2-2643-2284; Fax: 39-2-2643-2611; E-mail: pellegatta.fabio{at}hsr.it.
The abbreviations used are: PECAM-1/CD31, platelet-endothelial cell adhesion molecule-1; FN, fibronectin; HRP, horseradish peroxidase; mAb, monoclonal antibody; PI3K, phosphoinositide 3-kinase; PMN, polymorphonuclear neutrophil(s); S6K, serine/threonine kinase p70 S6 kinase; PAGE, polyacrylamide gel electrophoresis; NK, natural killer; RAM, rabbit anti-mouse; DPBS, Dulbecco's phosphate-buffered saline; ICAM-1, intercellular adhesion molecule-1; p-ICAM-1, purified ICAM-1; CHAPS, 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid; GAR, goat anti-rabbit.
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References
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= 405 nm (means ± S.D. from three
independent experiments). B, Western blot of lysates from
PMN recovered at different time points from CD31 cross-linking and
immunoprecipitation. After SDS-PAGE (10%) and electrotransfer,
nitrocellulose filters were hybridized with the anti-p85 subunit of
PI3K antiserum (upper panel) followed by HRP-conjugated GAR
Ig. The same blot was re-hybridized with the anti-CD31 mAb (lower
panel) followed by HRP-conjugated goat anti-mouse Ig. The
immunoreactive bands were revealed by luminol reaction. One
representative experiment of three is shown.
