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Blood, Vol. 94 No. 4 (August 15), 1999:
pp. 1319-1329
Ligation of CD31 (PECAM-1) on Endothelial Cells Increases Adhesive
Function of  v 3 Integrin and Enhances 1 Integrin-Mediated
Adhesion of Eosinophils to Endothelial Cells
By
Ryuichi Chiba,
Noriaki Nakagawa,
Kazuhiro Kurasawa,
Yoshiya Tanaka,
Yasushi Saito, and
Itsuo Iwamoto
From the Department of Medicine II, Chiba University School of
Medicine, Chiba, Japan; and the Department of Medicine, University of
Occupational and Environmental Health, Kitakyushu, Japan.
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ABSTRACT |
We determined the role of the heterophilic interaction of  v 3
integrin on endothelial cells with CD31 on leukocytes in mediating leukocyte-endothelial cell interactions. Preincubation of interleukin-4 (IL-4)-stimulated human umbilical vein endothelial cells (HUVECs) with
anti-CD31 monoclonal antibodies (MoAbs) enhanced eosinophil adhesion to
the IL-4-stimulated HUVECs, and the endothelial CD31-induced enhancement of eosinophil adhesion to IL-4-stimulated HUVECs
was prevented by anti-vascular cell adhesion molecule-1
(VCAM-1) MoAb and anti-very late activation antigen-4
(VLA-4) MoAb, but not by anti-intercellular adhesion
molecule-1 (ICAM-1) MoAb, anti-lymphocyte function-associated antigen-1 (LFA-1) MoAb,
anti-P-selectin MoAb, or anti-E-selectin MoAb. CD31 stimulation of
HUVECs increased the adhesive function of v3 integrin to its
ligand RGD peptide, the binding of which reached a maximum at 10 minutes after the stimulation, and the CD31-induced v3 integrin
activation on HUVECs was inhibited by inhibitors of protein kinase C
and phosphatidylinositol 3 kinase (PI3-kinase). Furthermore,
anti-v3 integrin MoAb and RGD peptide as well as soluble CD31
inhibited endothelial CD31-induced enhancement of eosinophil adhesion
to IL-4-stimulated HUVECs. However, anti-v3 integrin MoAb had no
significant inhibitory effect on the eosinophil adhesion to
IL-4-stimulated or unstimulated HUVECs without CD31 stimulation of
HUVECs. Finally, CD31 stimulation of eosinophils increased the adhesive
function of 41 integrin (VLA-4) to its ligand fibronectin and
their adhesion to IL-4-stimulated HUVECs in a VLA-4-dependent manner.
These results indicate that CD31-mediated inside-out signaling
activates v3 integrin on endothelial cells, that the heterophilic
v3 integrin/CD31 interaction induces 1 integrin-mediated
adhesion of eosinophils to endothelial cells, and that the heterophilic
interaction itself is not significantly involved in firm adhesion of
eosinophils to endothelial cells.
© 1999 by The American Society of Hematology.
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INTRODUCTION |
CD31 (PECAM-1) IS A CELL adhesion
molecule of the Ig gene superfamily1 expressed on vascular
endothelial cells, platelets, monocytes, neutrophils, and certain
subsets of T lymphocytes.2-10 CD31 has both homophilic
(CD31-CD31) and heterophilic (CD31-X) adhesive
interactions,4,11 and it has recently been shown that
 v3 integrin, which is expressed by endothelial
cells12 and subsets of T cells,13,14 is a
heterophilic ligand for CD31.15,16 CD31 has been implicated
in many physiological events, especially leukocyte-endothelial cell
interactions.17 It has been shown that CD31 is required for
transendothelial migration of monocytes and neutrophils across cultured
vascular endothelial cells in vitro.18 In vivo animal model
studies have also shown that, using anti-CD31 antibodies, CD31 is
involved in monocyte and neutrophil recruitment into sites of
inflammation.19,20 Furthermore, several studies have shown
that stimulation of CD31 with anti-CD31 antibodies increases adhesive
function of 1 integrin on T cells10 and CD34+ hematopoietic progenitor cells21 and
adhesive function of 2 integrin on monocytes,
neutrophils,22 and natural killer cells,23 suggesting that leukocyte CD31 may act as a signaling molecule. However, the functional importance of the heterophilic interaction of
v3 integrin on endothelial cells with CD31 on leukocytes has not
yet been clarified in mediating leukocyte-endothelial cell interactions.
Therefore, to elucidate this issue, we studied the role of the
heterophilic v3 integrin/CD31 interaction in eosinophil adhesion to interleukin-4 (IL-4)-stimulated vascular endothelial cells. We also
studied the role of CD31 in v3 and 41 integrin activation of endothelial cells and eosinophils, respectively. Our results indicate that the heterophilic v3 integrin/CD31 interaction forms
an important amplifier loop for 1 integrin-mediated adhesion of
eosinophils to endothelial cells, but that the heterophilic interaction
itself is not significantly involved in firm adhesion of eosinophils to
endothelial cells.
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MATERIALS AND METHODS |
Materials.
All the reagents were purchased from Sigma Chemical Co (St Louis, MO),
unless otherwise stated. MCDB131 medium was purchased from Kurorera
Kogyo Co (Tokyo, Japan). Percoll was purchased from Pharmacia Fine
Chemicals (Uppsala, Sweden). Recombinant human IL-4 was obtained from
Ono Pharmaceutical Co (Osaka, Japan).
Mouse antihuman CD31 monoclonal antibodies (MoAbs) NIH31-1 (IgG1) and
NIH31-2 (IgG1)10,24 were a kind gift from Dr Y. Tanaka (University of Occupational and Environmental Health, Kitakyushu, Japan). Mouse antihuman vascular cell adhesion molecule-1
(VCAM-1) MoAb BBA6 (IgG1), antihuman intercellular
adhesion molecule-1 (ICAM-1) MoAb 84H10 (IgG1), and
antihuman very late activation antigen-4 (VLA-4) -chain
MoAb HP2.1 (IgG1) were purchased from Cosmo Bio Co (Tokyo, Japan).
Mouse antihuman P-selectin MoAb AK4 (IgG1) and antihuman E-selectin
MoAb 68-5H11 (IgG1) were purchased from Pharmingen (San Diego, CA).
Mouse antihuman v3 MoAb LM609 (IgG1)12 and antihuman
v5 MoAb P1F6 (IgG1) were purchased from Chemicon International
Inc (Temecula, CA). Mouse antihuman VLA-5 -chain MoAb KH/33 (IgG1)
was purchased from Seikagaku Co (Tokyo, Japan). A mouse MoAb (IgG1)
that recognizes keyhole limpet hemocyanin was purchased from Becton
Dickinson Immunocytometry Systems (San Jose, CA) and used as a negative
control. Recombinant human CD31 was purchased from R&D Systems
(Minneapolis, MN).
Arg-Gly-Asp (RGD) peptide (ProNectin F), Gly-Arg-Gly-Asp-Ser-Pro
(GRGDSP) peptide, and Gly-Arg-Gly-Glu-Ser-Pro (GRGESP) peptide were
purchased from Iwaki Glass (Chiba, Japan). Human fibronectin was
purchased from Seikagaku Co. [51Cr]sodium chromate was
purchased from Dupont NEN Research Products (Boston, MA). MACS
microbeads conjugated with mouse antihuman CD16 MoAb were purchased
from Miltenyi Biotec GmbH (Bergisch Gladbach, Germany). Transwell cell
culture chamber inserts (catalogue no. 3421) were purchased from Costar
Corp (Cambridge, MA).
Culture of vascular endothelial cells.
Human umbilical vein endothelial cells (HUVECs) were isolated from
fresh umbilical cords by trypsin digestion according to the method
described by Jaffe et al.25 Briefly, the umbilical vein was
rinsed with 50 mL of phosphate-buffered saline (PBS) and filled with
0.2% trypsin in PBS at room temperature for 15 minutes. Isolated
HUVECs were cultured in MCDB131 medium supplemented with 10% fetal
calf serum (FCS), basic fibroblast growth factor (10 ng/mL), heparin
(90 µg/mL), penicillin (100 µg/mL), and streptomycin (100 U/mL) in
a gelatin-coated 75-cm2 tissue culture flask at 37°C in
a humidified atmosphere of 5% CO2 in air. The primary
cultured HUVECs were harvested by trypsin digestion and seeded into a
gelatin-coated 24-well tissue culture plate for use of adhesion assay.
HUVECs were then cultured to confluence. Endothelial cells were
identified by their typical cobblestone morphology and the presence of
factor VIII-related antigen as detected by indirect immunofluorescence.
Confluence was verified by phase-contrast microscopy inspection before
use in experiments.
Purification of eosinophils.
Human eosinophils were purified from 100 mL of heparinized venous blood
from normal individuals by Percoll density gradient centrifugation and
a magnetic cell sorting using MACS anti-CD16 microbeads as described by
Hansel et al.26 Briefly, after erythrocytes were sedimented
in 1% (vol/vol) dextran in 0.9% NaCl, the leukocyte-rich cell
suspension (5 × 107/mL) was overlayered on an
isotonic Percoll solution (1.082 g/mL) and centrifuged at
1,000g for 30 minutes at 4°C. Granulocytes were obtained
from the bottom of the Percoll solution and washed twice in cold
Hanks' balanced salt solution (HBSS) containing 2% FCS. Cells were
then incubated with 100 µL of anti-CD16 MoAb MACS microbeads for 30 minutes at 4°C with occasional gentle mixing. Cells were then
suspended in 500 µL of cold HBSS containing 2% FCS and applied to a
separation column in a magnetic field. Pure eosinophils (purity
>99%) were obtained by negative selection, because eosinophils are
CD16 and pass through the column, and were suspended
in HBSS.
Radiolabeling of eosinophils and HUVECs.
Purified eosinophils or HUVEC (3 × 107) were
incubated with 0.4 mCi of sodium [51Cr] chromate in 1 mL
of HBSS containing 5% FCS at 37°C for 30 minutes. Excess isotope
was then washed off 3 times with HBSS containing 5% FCS and cells were
resuspended in HBSS containing 5% FCS.
Adhesion assay.
HUVEC monolayers grown on the well of 24-well tissue culture plates
were stimulated with IL-4 (100 ng/mL) for 8 hours at 37°C in 200 µL of MCDB131 medium containing 5% FCS in a humidified atmosphere of
5% CO2 in air. As a control, HUVECs were incubated with
culture medium alone for 8 hours. After HUVECs were thoroughly washed
with HBSS containing 5% FCS, 3 × 105 of
51Cr-labeled eosinophils were incubated with HUVECs for 10 minutes at 37°C in 200 µL of HBSS containing 5% FCS. HUVECs were
then gently washed 3 times with HBSS containing 5% FCS to remove the unbound cells. Adherent eosinophils were then lysed by the addition of
1 mL of HBSS containing 1% Triton X-100, and radioactivity of the
cells was counted in a gamma counter. Each experiment was performed in
triplicate, and the average of 3 determinations was used for subsequent
calculations. Adherence of eosinophils to HUVECs was expressed as the
percentage of radioactivity of total eosinophils added to each well.
In the first series of experiments, IL-4-stimulated or unstimulated
HUVECs were preincubated with anti-CD31 MoAb (10 µg/mL), anti-v3 integrin MoAb (10 µg/mL), or control mouse IgG (10 µg/mL) for 10 minutes at 37°C, then washed with HBSS containing
5% FCS, and used for adhesion assay. In the second series of
experiments, after IL-4-stimulated HUVECs were preincubated with
anti-CD31 MoAb or control mouse IgG (10 µg/mL each) for 10 minutes at
37°C and were then washed with HBSS containing 5% FCS, the
adhesion assay of 51Cr-labeled eosinophils to HUVECs was
performed in the presence of anti-VCAM-1 MoAb, anti-VLA-4 MoAb,
anti-ICAM-1 MoAb, anti-lymphocyte function-associated antigen-1
(LFA-1) MoAb, anti-P-selectin MoAb, or anti-E-selectin
MoAb (10 µg/mL each). In the third series of experiments, after
IL-4-stimulated HUVECs were preincubated with anti-CD31 MoAb or
control mouse IgG for 10 minutes at 37°C and were then washed with
HBSS containing 5% FCS, the adhesion assay of 51Cr-labeled
eosinophils to HUVECs was performed in the presence of anti-v3
integrin MoAb (10 µg/mL) or GRGDSP peptide (or GRGESP peptide; 5 to
500 µg/mL). To further determine whether v3 integrin/CD31 interaction between endothelial cells and eosinophils is involved in
the endothelial CD31-induced enhancement of eosinophil adhesion to
IL-4-stimulated endothelial cells, we also examined the effect of
soluble recombinant CD31 on endothelial CD31-induced enhancement of
eosinophil adhesion to IL-4-stimulated HUVECs. In these experiments, after IL-4-stimulated HUVECs were preincubated with anti-CD31 MoAb or
control mouse IgG for 10 minutes at 37°C and were then washed with
HBSS containing 5% FCS, the adhesion assay of 51Cr-labeled
eosinophils to HUVECs was performed in the presence or absence of
soluble recombinant CD31 (1 to 50 µg/mL).
Binding assay of v3 integrin to RGD
peptide.
To determine whether CD31 stimulation of endothelial cells increases
the adhesive function of v3 integrin to its ligand, we performed
the binding assay of v3 integrin on HUVECs to its ligand
Arg-Gly-Asp (RGD) peptide.12,27 Briefly, wells of 24-well tissue culture plates were coated with 5 µg/mL ProNectin F or 1%
bovine serum albumin (BSA; control) in PBS at room temperature for 2 hours. The wells were washed and subsequently coated with 1% BSA in
PBS at room temperature for 1 hour to block nonspecific binding sites.
After the wells were washed with HBSS, 51Cr-labeled HUVECs
were added to the well at 5 × 105 cells/well in 200 µL of HBSS containing 1% BSA and were incubated in triplicate for 10 minutes at 37°C. Unbound cells were then washed off, and bound
cells were lysed by the addition of 1 mL of HBSS containing 1% Triton
X-100; radioactivity of the cells was counted in a gamma counter.
Because v3, v5, and 51 integrins on endothelial cells
recognize the Arg-Gly-Asp (RGD) sequence present in extracellular matrix proteins such as vitronectin and fibronectin,27,28
we first examined the effect of anti-v3 integrin, anti-v5
integrin, and anti-51 integrin MoAbs on the binding of HUVECs to
RGD peptide. After 51Cr-labeled HUVECs were preincubated
with anti-CD31 MoAb (10 µg/mL) or control mouse IgG (10 µg/mL) for
10 minutes at 37°C and were then washed with HBSS containing 1%
BSA, the binding assay of 51Cr-labeled HUVECs to RGD
peptide was performed in the presence of anti-v3 integrin MoAb,
anti-v5 integrin MoAb, or anti-51 integrin MoAb (10 µg/mL each).
We also determined whether protein kinase C and phosphatidylinositol 3 kinase (PI3-kinase) are involved in the signal transduction for
CD31-induced v3 integrin activation on endothelial cells. 51Cr-labeled HUVECs were preincubated with staurosporine
(108 mol/L)29,30 or wortmannin
(107 mol/L)31,32 for 10 minutes at
37°C and were then preincubated with anti-CD31 MoAb (10 µg/mL) or
control mouse IgG (10 µg/mL) for 10 minutes at 37°C. After
washing with HBSS containing 1% BSA, the 51Cr-labeled
HUVECs were used for binding assay to RGD peptide.
Binding assay of VLA-4 to fibronectin.
To determine whether CD31 stimulation of eosinophils increases the
adhesive function of 41 integrin (VLA-4) to its ligand, we
performed the binding assay of VLA-4 on eosinophils to its ligand
fibronectin. Briefly, wells of 24-well tissue culture plates were
coated with 10 µg/mL fibronectin or 1% BSA (control) in PBS at
4°C overnight. The wells were washed and subsequently coated with
1% BSA in PBS at room temperature for 1 hour to block nonspecific binding sites. After the wells were washed with HBSS,
51Cr-labeled eosinophils were added to the well at 5 × 105 cells/well in 200 µL of HBSS containing 1%
BSA and were incubated in triplicate for 10 minutes at 37°C.
Unbound cells were then washed off, bound cells were lysed by the
addition of 1 mL of HBSS containing 1% Triton X-100, and the
radioactivity of the cells was counted in a gamma counter. In these
experiments, after 51Cr-labeled eosinophils were
preincubated with anti-CD31 MoAb (10 µg/mL) or control mouse IgG (10 µg/mL) for 10 minutes at 37°C and were then washed with HBSS
containing 1% BSA, the binding assay of 51Cr-labeled
eosinophils to fibronectin was performed in the presence or absence of
anti-VLA-4 MoAb (10 µg/mL).
Immunofluorescence.
After IL-4-stimulated HUVECs were incubated with anti-CD31 MoAb or
control mouse IgG (10 µg/mL each) for 10 minutes at 37°C and then
washed with HBSS containing 5% FCS, HUVECs were fixed in acetone for
10 minutes at 4°C, dried, and rinsed with PBS. After blocking with
4% FCS in PBS, HUVECs were stained with anti-CD31 MoAb-fluorescein
isothiocyanate (FITC; 10 µg/mL), anti-v3 integrin MoAb-FITC (10 µg/mL), or control mouse IgG-FITC (10 µg/mL) for 60 minutes at room
temperature, followed by washing with PBS.
Data analysis.
Data are summarized as the mean ± SD. The statistical analysis of
the results was performed by the analysis of variance using Fisher's
least significant difference test for multiple comparisons. P
values less than .05 were considered significant.
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RESULTS |
Role of CD31 and v3 integrin in
eosinophil adhesion to vascular endothelial cells.
To determine the role of CD31/CD31 homophilic and v3
integrin/CD31 heterophilic bindings in the adhesive interaction between eosinophils and vascular endothelial cells, we examined the effect of
anti-CD31 MoAb and anti-v3 integrin MoAb on eosinophil adhesion to IL-4-stimulated cultured vascular endothelial cells. In our preliminary experiments, CD31, but not v3 integrin, was expressed on human eosinophils by flow cytometry to the same levels as human neutrophils and monocytes had (data not shown), whereas both CD31 and
v3 integrin were expressed on cultured HUVECs, as described previously.2-4,12,33 In addition, the distribution of
v3 integrin on HUVECs was not significantly affected by CD31
stimulation with anti-CD31 MoAb (Fig 1).
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| Fig 1.
Immunofluorescence staining of CD31 and v3 integrin
on IL-4-stimulated vascular endothelial cells. HUVEC monolayers grown
on the well of 24-well tissue culture plates were stimulated with IL-4
(100 ng/mL) for 8 hours at 37°C. IL-4-stimulated HUVECs were then
incubated with anti-CD31 MoAb NIH31-1 (right panels; B, D, and F) or
control mouse IgG (left panels; A, C, and E) (10 µg/mL each) for 10 minutes at 37°C, fixed in acetone, and stained with anti-CD31
MoAb-FITC (C and D), anti-v3 integrin MoAb-FITC (E and F), or
control mouse IgG-FITC (A and B) (10 µg/mL each) for 60 minutes at
room temperature. (Original magnification × 1,000.)
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Eosinophil adhesion to unstimulated HUVECs was low (8.2% ± 1.2%
of total eosinophils added, mean ± SD, n = 6 experiments; Fig 2). In contrast, eosinophil adhesion to
HUVECs significantly increased after 8 hours of preincubation
of HUVECs with IL-4 (100 ng/mL; 15.3% ± 1.8% of total
eosinophils added, n = 6, P < .001; Fig 2), which was
accompanied by the selective VCAM-1 expression on HUVECs (data not
shown).34-36 Possible involvement of CD31 and v3
integrin on endothelial cells in mediating the eosinophil adhesion to
IL-4-stimulated HUVECs was then examined by using anti-CD31 MoAb and
anti-v3 integrin MoAb. Unexpectedly, preincubation of
IL-4-stimulated HUVECs with anti-CD31 MoAb (NIH31-1) significantly enhanced eosinophil adhesion to the IL-4-stimulated HUVECs by 62%
(24.9% ± 2.8% of total eosinophils added, n = 6, P < .001; Fig 2). In addition, anti-CD31 MoAb NIH31-1 and NIH31-2 (1 to 100 µg/mL) both dose-dependently increased eosinophil adhesion to
IL-4-stimulated HUVECs (n = 3 to 6; Fig
3). In contrast, preincubation of unstimulated HUVECs with anti-CD31
MoAb had no significant enhancing effect on the eosinophil adhesion to
the unstimulated HUVECs (n = 6; Figs 2 and 3). On the other hand,
anti-v3 integrin MoAb (LM609) had no significant effect on the
eosinophil adhesion to IL-4-stimulated or unstimulated HUVECs (n = 6 each; Fig 2). These results indicate that neither CD31/CD31 homophilic
nor v3 integrin/CD31 heterophilic interaction between endothelial
cells and eosinophils is directly involved in eosinophil adhesion to endothelial cells and that CD31 stimulation of endothelial cells with
anti-CD31 MoAb enhances eosinophil adhesion to IL-4-stimulated endothelial cells.
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| Fig 2.
Effect of anti-CD31 and anti-v3 integrin antibodies
on eosinophil adhesion to IL-4-stimulated vascular endothelial cells.
HUVEC monolayers grown on the well of 24-well tissue culture plates
were stimulated with or without IL-4 (100 ng/mL) for 8 hours at
37°C. IL-4-stimulated ( ) or unstimulated ( ) HUVECs were
preincubated with anti-CD31 MoAb NIH31-1 (10 µg/mL), anti-v3
integrin MoAb LM609 (10 µg/mL), or control mouse IgG (10 µg/mL) for
10 minutes at 37°C, washed, and then incubated with
51Cr-labeled eosinophils (3 × 105) for 10 minutes at 37°C. After HUVECs were gently washed, adherent
eosinophils were lysed by the addition of HBSS containing 1% Triton
X-100 and the radioactivity of the cells was counted in a gamma
counter. Data are the means ± SD for 6 experiments. *Significantly
different from the mean value of the control response to unstimulated
HUVECs (*P < .001). **Significantly different from the mean
value of the control response to IL-4-stimulated HUVECs (**P < .001).
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| Fig 3.
Dose-dependent effect of anti-CD31 antibodies on
eosinophil adhesion to IL-4-stimulated vascular endothelial cells.
IL-4-stimulated () or unstimulated () HUVECs were preincubated
with anti-CD31 MoAb NIH31-1 (31-1) or NIH31-2 (31-2) (1, 10, and 100 µg/mL) or control mouse IgG (Cont; 10 µg/mL) for 10 minutes at
37°C. After washing HUVECs, the adhesion assay of
51Cr-labeled eosinophils to HUVECs was performed. Data are
the means ± SD for 3 to 6 experiments.
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We also examined the effect of F(ab')2 and Fab of
anti-CD31 antibody on the CD31-induced enhancement of eosinophil
adhesion to IL-4-stimulated HUVECs. F(ab')2 of
anti-CD31 MoAb similarly increased eosinophil adhesion to
IL-4-stimulated HUVECs by 66% (n = 3), whereas the Fab fragments had
no significant effect, indicating that dimerization of CD31 is required
for the endothelial CD31-induced enhancement of eosinophil adhesion to
IL-4-stimulated endothelial cells.
CD31 stimulation of endothelial cells enhances eosinophil adhesion to
IL-4-stimulated endothelial cells through VCAM-1/VLA-4 interaction.
Because IL-4 selectively induces the expression of VCAM-1 on vascular
endothelial cells34-36 and VLA-4 is a counter-receptor for
VCAM-137 and is expressed on eosinophils,38-41
we then examined whether VCAM-1/VLA-4 interaction is involved in the
endothelial CD31-induced enhancement of eosinophil adhesion to
IL-4-stimulated endothelial cells. Eosinophil adhesion to
IL-4-stimulated HUVECs was inhibited by the presence of anti-VCAM-1
MoAb and anti-VLA-4 MoAb by 45% and 51%, respectively (n = 5, P < .001; Fig 4). Endothelial CD31-induced enhancement of eosinophil adhesion to IL-4-stimulated HUVECs was abolished in the presence of anti-VCAM-1 MoAb or
anti-VLA-4 MoAb to the same levels that anti-VCAM-1 MoAb and
anti-VLA-4 MoAb decreased eosinophil adhesion to IL-4-stimulated,
CD31-unstimulated HUVECs (n = 5, P < .001; Fig 4). However,
the presence of anti-ICAM-1 MoAb, anti-LFA-1 MoAb, anti-P-selectin
MoAb, or anti-E-selectin MoAb did not significantly affect the
CD31-induced enhancement of eosinophil adhesion to IL-4-stimulated
HUVECs (n = 5; data not shown). Therefore, endothelial CD31-induced
enhancement of eosinophil adhesion to IL-4-stimulated endothelial
cells is mediated through VCAM-1/VLA-4 interaction, but not by ICAM-1,
LFA-1, P-selectin, or E-selectin.
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| Fig 4.
Effect of anti-VCAM-1 and anti-VLA-4 antibodies on
endothelial CD31-induced enhancement of eosinophil adhesion to
IL-4-stimulated vascular endothelial cells. IL-4-stimulated HUVECs
were preincubated with anti-CD31 MoAb or control mouse IgG (10 µg/mL
each) for 10 minutes at 37°C. After washing HUVECs, the adhesion
assay of 51Cr-labeled eosinophils to HUVECs was performed
in the presence or absence of anti-VCAM-1 MoAb or anti-VLA-4 MoAb (10 µg/mL each). Data are the means ± SD for 5 experiments.
*Significantly different from the mean value of the control response
(*P < .001). **Significantly different from the mean value of
the corresponding control response (**P < .001).
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CD31 stimulation of endothelial cells increases the adhesive function
of v3 integrin to its ligand.
Because it has recently been shown that CD31 stimulation of leukocytes
increases the adhesive function of 1 and 2 integrins on the
surface,10,21-23,42 we determined whether CD31 stimulation of endothelial cells increases the adhesive function of v3
integrin to its ligand Arg-Gly-Asp (RGD) peptide.12,27
Incubation of unstimulated HUVECs with anti-CD31 MoAb
significantly increased HUVEC binding to RGD peptide by 142%
(control mouse IgG, 9.8% ± 1.3% v anti-CD31 MoAb, 23.8% ± 3.0% of HUVECs added, n = 6 experiments, P < .001; Fig 5), whereas incubation of
unstimulated HUVECs with anti-CD31 MoAb had no significant effect on
HUVEC binding to BSA-coated wells (control mouse IgG 2.7% ± 0.6%
v anti-CD31 MoAb, 3.0% ± 0.4% of HUVECs added, n = 4).
The presence of anti-v3 integrin MoAb completely inhibited
CD31-induced increase in the HUVEC binding to RGD peptide (11.3% ± 1.5% of HUVECs added, n = 6, P < .001; Fig 5). However, the
presence of anti-v5 integrin MoAb or anti-VLA-5 -chain MoAb
had no effect on the CD31-induced increase in HUVEC binding to RGD
peptide (n = 6; Fig 5). In addition, we examined the expression of
v3 integrin on HUVECs incubated with anti-CD31 MoAb by flow
cytometry. Incubation with anti-CD31 MoAb had no significant effect on
the expression of v3 integrin on HUVECs (data not shown). These
data indicate that CD31 stimulation of endothelial cells selectively
increases the adhesive function of v3 integrin on the surface.
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| Fig 5.
CD31 stimulation of endothelial cells increases the
adhesive function of v3 integrin to RGD peptide.
51Cr-labeled HUVECs were preincubated with anti-CD31 MoAb
or control mouse IgG (10 µg/mL each) for 10 minutes at 37°C and
were then washed. 51Cr-labeled HUVECs (5 × 105) were then added to the RGD peptide-coated well of
tissue culture plates and were incubated for 10 minutes at 37°C in
the presence or absence of anti-v3 integrin MoAb, anti-v5
integrin MoAb, or anti-51 integrin MoAb (10 µg/mL each). After
unbound cells were washed off, bound cells were lysed by the addition
of HBSS containing 1% Triton X-100 and the radioactivity of the cells
was counted in a gamma counter. Data are the means ± SD for 6 experiments. *Significantly different from the mean value of the
control response (*P < .001). **Significantly different from
the mean value of the control response of anti-CD31 MoAb-pretreated
HUVECs (**P < .001).
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Time course of CD31-induced increase in the adhesive function of
v3 integrin on HUVECs to RGD peptide was then examined. CD31-induced HUVEC binding to RGD peptide rapidly increased and reached
a peak at 10 minutes after the incubation of HUVECs with anti-CD31 MoAb
(Fig 6), which was 2.4-fold greater than
that of control MoAb-induced HUVEC binding at 10 minutes.
After that, the CD31-induced HUVEC binding to RGD peptide gradually
decreased and returned to the baseline at 40 minutes (Fig 6).
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| Fig 6.
Time course of endothelial CD31-induced increase in the
adhesive function of v3 integrin to RGD peptide.
51Cr-labeled HUVECs were preincubated with anti-CD31 MoAb
or control mouse IgG (10 µg/mL each) for 10 minutes at 37°C.
After washing the cells, the binding assay of 51Cr-labeled
HUVECs to RGD peptide was performed for 10 to 60 minutes at 37°C.
HUVEC binding to RGD peptide is expressed as the ratio of the binding
of anti-CD31 MoAb-pretreated HUVECs to that of control IgG-pretreated
HUVECs. Data are the means ± SD for 3 experiments.
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v3 integrin/CD31 interaction mediates
endothelial CD31-induced enhancement of eosinophil adhesion to
IL-4-stimulated endothelial cells.
We then determined whether v3 integrin/CD31 interaction between
endothelial cells and eosinophils is involved in the endothelial CD31-induced enhancement of eosinophil adhesion to IL-4-stimulated endothelial cells. As shown in Fig 7, the
presence of anti-v3 integrin MoAb significantly inhibited
endothelial CD31-induced enhancement of eosinophil adhesion to
IL-4-stimulated HUVECs by 94% (anti-CD31 MoAb, 25.9% ± 2.5%
v anti-CD31 MoAb and anti-v3 integrin MoAb, 15.8% ± 1.8% of total eosinophils added, n = 6, P < .001). GRGDSP
peptide (50 to 500 µg/mL), but not GRGESP peptide, also completely
inhibited endothelial CD31-induced enhancement of eosinophil adhesion
to IL-4-stimulated HUVECs (n = 4, P < .005; Fig 8). Furthermore, soluble recombinant
CD31 (1 to 50 µg/mL) significantly inhibited endothelial CD31-induced
enhancement of eosinophil adhesion to IL-4-stimulated HUVECs by 77%
to 87% (anti-CD31 MoAb, 26.1% ± 3.3% v anti-CD31 MoAb
and soluble CD31 at 50 µg/mL, 14.9% ± 2.0% of total eosinophils
added, n = 4, P < .005), whereas soluble CD31 had
no significant inhibitory effect on the eosinophil adhesion to
IL-4-stimulated HUVECs without CD31 stimulation of HUVEC
(Fig 9). These results indicate that the
increased v3 integrin/CD31 heterophilic interaction between
endothelial cells and eosinophils mediates the VLA-4-dependent
eosinophil adhesion to IL-4-stimulated endothelial cells.
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| Fig 7.
Effect of anti-v3 integrin antibody on endothelial
CD31-induced enhancement of eosinophil adhesion to IL-4-stimulated
vascular endothelial cells. IL-4-stimulated HUVECs were preincubated
with anti-CD31 MoAb or control mouse IgG (10 µg/mL each) for 10 minutes at 37°C. After washing HUVECs, the adhesion assay of
51Cr-labeled eosinophils to HUVECs was performed in the
presence or absence of anti-v3 integrin MoAb (10 µg/mL). Data
are the means ± SD for 6 experiments. *Significantly
different from the mean value of the control response (*P < .001). **Significantly different from the mean value of the control
response of anti-CD31 MoAb-pretreated HUVECs (**P < .001).
|
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| Fig 8.
Effect of RGD peptide on endothelial CD31-induced
enhancement of eosinophil adhesion to IL-4-stimulated vascular
endothelial cells. IL-4-stimulated HUVECs were preincubated with
anti-CD31 MoAb or control mouse IgG (10 µg/mL each) for 10 minutes at
37°C. After washing HUVECs, the adhesion assay of
51Cr-labeled eosinophils to HUVECs was performed in the
presence of GRGDSP peptide () or GRGESP peptide (control peptide;
 ; 5, 50, and 500 µg/mL each). Data are the means ± SD for 4 experiments. *Significantly different from the mean value of the
control response (*P < .005). **Significantly different from
the mean value of the control response of anti-CD31 MoAb-pretreated
HUVECs (**P < .005).
|
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| Fig 9.
Effect of soluble recombinant CD31 on endothelial
CD31-induced enhancement of eosinophil adhesion to IL-4-stimulated
vascular endothelial cells. IL-4-stimulated HUVECs were preincubated
with anti-CD31 MoAb or control mouse IgG (10 µg/mL each) for 10 minutes at 37°C. After washing HUVECs, the adhesion assay of
51Cr-labeled eosinophils to HUVECs was performed in the
presence () or absence () of soluble recombinant CD31 (1, 5, and
50 µg/mL). Data are the means ± SD for 4 experiments.
*Significantly different from the mean value of the control response
(*P < .005). **Significantly different from the mean value of
the control response of anti-CD31 MoAb-pretreated HUVECs (**P < .005).
|
|
CD31 stimulation of eosinophils increases the adhesive function of
41 integrin (VLA-4) to its ligand.
To determine whether CD31 stimulation of eosinophils increases the
adhesive function of 41 integrin (VLA-4) on the surface and
thereby enhances eosinophil adhesion to IL-4-stimulated endothelial cells, we examined the adhesive function of VLA-4 on eosinophils to its
ligand fibronectin. Stimulation of eosinophils with anti-CD31 MoAb
significantly increased eosinophil binding to fibronectin by 71%
(control mouse IgG, 19.6% ± 2.5% v anti-CD31 MoAb, 33.5% ± 2.9% of total eosinophils added, n = 4, P < .005; Fig 10). Anti-VLA-4 MoAb abolished
the CD31-induced increase in eosinophil binding to fibronectin (20.1% ± 2.4% of total eosinophils added, n = 4, P < .005; Fig
10). On the other hand, stimulation of eosinophils with anti-CD31 MoAb
had no significant effect on eosinophil binding to BSA-coated wells
(control mouse IgG, 1.9% ± 0.4% v anti-CD31 MoAb, 2.2% ± 0.3% of total eosinophils added, n = 4). In addition, anti-CD31
MoAb had no effect on the expression of VLA-4 on eosinophils (data not
shown).
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| Fig 10.
CD31 stimulation of eosinophils increases the adhesive
function of 41 integrin (VLA-4) to fibronectin.
51Cr-labeled eosinophils were preincubated with anti-CD31
MoAb or control mouse IgG (10 µg/mL each) for 10 minutes at 37°C
and were then washed. 51Cr-labeled eosinophils (5 × 105) were then added to the fibronectin-coated well of
tissue culture plates and were incubated for 10 minutes at 37°C in
the presence or absence of anti-VLA-4 MoAb (10 µg/mL). After unbound
cells were washed off, bound cells were lysed by the addition of HBSS
containing 1% Triton X-100 and radioactivity of the cells was counted
in a gamma counter. Data are the means ± SD for 4 experiments.
*Significantly different from the mean value of the control response
(*P < .005). **Significantly different from the mean value of
the control response of anti-CD31 MoAb-pretreated eosinophils
(**P < .005).
|
|
Stimulation of eosinophils with anti-CD31 MoAb also significantly
increased eosinophil adhesion to IL-4-stimulated HUVECs by
77% (control mouse IgG, 15.2% ± 1.7% v anti-CD31 MoAb,
26.9% ± 3.2% of total eosinophils added, n = 6, P < .001; Fig 11). Anti-VLA-4 MoAb and anti-VCAM-1 MoAb inhibited CD31-induced increase in
eosinophil adhesion to IL-4-stimulated HUVECs by 96% and 95%,
respectively (n = 6, P < .001; Fig 11). These results
indicate that CD31 stimulation of eosinophils increases the adhesive
function of VLA-4 on the surface and thereby enhances eosinophil
adhesion to IL-4-stimulated endothelial cells. In addition, the
presence of anti-v3 integrin MoAb or GRGDSP peptide did not
significantly affect the eosinophil CD31-induced enhancement of
eosinophil adhesion to IL-4-stimulated HUVECs (data not shown).
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| Fig 11.
CD31 stimulation of eosinophils increases eosinophil
adhesion to IL-4-stimulated endothelial cells through VLA-4/VCAM-1
interaction. 51Cr-labeled eosinophils were preincubated
with anti-CD31 MoAb or control mouse IgG (10 µg/mL each) for 10 minutes at 37°C. After washing the cells, the adhesion assay of
51Cr-labeled eosinophils to IL-4-stimulated () or
unstimulated () HUVECs was performed in the presence or absence of
anti-VCAM-1 MoAb or anti-VLA-4 MoAb (10 µg/mL each). Data are the
means ± SD for 6 experiments. *Significantly different from the mean
value of the control response to unstimulated HUVECs (*P < .001). **Significantly different from the mean value of the control
response to IL-4-stimulated HUVECs (**P < .001).
***Significantly different from the mean value of the corresponding
control response (***P < .001).
|
|
Protein kinase C and PI3-kinase are involved in CD31-induced
v3 integrin activation in endothelial
cells.
Finally, because it has been shown that, in hematopoietic cells,
protein kinase C and PI3-kinase are involved in inside-out signaling of
1 integrin activation through the receptors,43-46 we
determined whether these kinases are involved in the signal transduction for CD31-induced v3 integrin activation on
endothelial cells. Staurosporine (protein kinase C inhibitor;
108 mol/L)29,30 and wortmannin
(PI3-kinase inhibitor; 107 mol/L)31,32
inhibited CD31-induced increase in HUVEC binding to RGD peptide by 53%
and 45%, respectively (n = 8 each, P < .001; Fig 12). In addition, the combination of
staurosporine and wortmannin inhibited CD31-induced increase in HUVEC
binding to RGD peptide by 71% (n = 6, P < .001; Fig 12).
These results indicate that protein kinase C and PI3-kinase are
involved in the CD31-mediated signaling for v3 integrin
activation on endothelial cells.
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| Fig 12.
Effect of staurosporine and wortmannin on endothelial
CD31-induced increase in the adhesive function of v3 integrin to
RGD peptide. 51Cr-labeled HUVECs were preincubated with
staurosporine (Staur; 108 mol/L), wortmannin (Wort;
107 mol/L), or the combination of them for 10 minutes at
37°C and were then preincubated with anti-CD31 MoAb (CD31) or
control mouse IgG (Cont) (10 µg/mL each) for 10 minutes at 37°C.
After washing the cells, the binding assay of 51Cr-labeled
HUVECs to RGD peptide was performed for 10 minutes at 37°C. Data
are the means ± SD for 6 to 8 experiments. *Significantly different
from the mean value of the control response of anti-CD31
MoAb-pretreated HUVECs (*P < .001). **Significantly different
from the mean value of the responses of anti-CD31 MoAb-pretreated
HUVECs in the presence of staurosporine or wortmannin alone,
respectively (**P < .05).
|
|
| |
DISCUSSION |
In this study, we show that CD31 stimulation activates v3
integrin on endothelial cells and that the increased heterophilic interaction between v3 integrin on endothelial cells and CD31 on
eosinophils mediates firm adhesion of eosinophils to endothelial cells
through VLA-4/VCAM-1 interaction. We found that preincubation of
IL-4-stimulated HUVECs with anti-CD31 MoAb enhanced eosinophil adhesion to the IL-4-stimulated HUVECs (Figs 2 and 3) and that the
endothelial CD31-induced enhancement of eosinophil adhesion to
IL-4-stimulated HUVECs was prevented by anti-VCAM-1 MoAb and anti-VLA-4 MoAb (Fig 4), but not by anti-ICAM-1 MoAb, anti-LFA-1 MoAb, anti-P-selectin MoAb, or anti-E-selectin MoAb. We also found that CD31 stimulation of HUVECs increased the adhesive function of
v3 integrin to its ligand Arg-Gly-Asp (RGD) peptide (Fig 5), the
binding of which reached a maximum at 10 minutes after the stimulation
(Fig 6). Furthermore, we found that anti-v3 integrin MoAb and
GRGDSP peptide as well as soluble CD31 inhibited endothelial
CD31-induced enhancement of eosinophil adhesion to IL-4-stimulated
HUVECs (Figs 7 through 9). However, anti-v3 integrin MoAb had no
significant inhibitory effect on the eosinophil adhesion to
IL-4-stimulated or unstimulated HUVECs without CD31 stimulation of
HUVECs (Fig 2). Finally, CD31 stimulation of eosinophils increased the
adhesive function of 41 integrin (VLA-4) to its ligand
fibronectin (Fig 10) and their adhesion to IL-4-stimulated HUVECs in a
VLA-4-dependent manner (Fig 11). Thus, it is indicated that the
heterophilic v3 integrin/CD31 interaction forms an important
amplifier loop for 1 integrin-mediated adhesion of eosinophils to
endothelial cells, but that the heterophilic interaction itself is not
significantly involved in firm adhesion of eosinophils to endothelial cells.
v3 integrin has been implicated in angiogenesis, in which
v3 integrin on the surface of endothelial cells transmits signals for the survival, migration, and differentiation of endothelial cells.47,48 It has recently been shown that v3
integrin is a ligand for CD31 using adhesion assays to recombinant
CD31.15,16 However, the functional importance of the
heterophilic v3 integrin/CD31 interaction between endothelial
cells and leukocytes has not yet been elucidated. We provide the first
evidence for the functional role of the heterophilic v3
integrin/CD31 interaction that v3 integrin, when activated, on
endothelial cells is a signaling ligand for CD31 on leukocytes, but
that the adhesive property of the heterophilic interaction is not
significantly involved in firm adhesion of leukocytes to endothelial
cells. The difference in binding of v3 integrin to RGD peptide
and CD31 on eosinophils may be due to the difference in the
experimental conditions of the binding assays. That is, the binding of
v3 integrin on HUVECs to RGD peptide coated on the plates may be
more sensitive to be detected than the binding of v3 integrin on
HUVECs to CD31 on eosinophils.
There has been the controversy over whether v3 integrin can bind
CD31. Sun et al49 reported that anti-v3 integrin
antibody failed to inhibit the binding of CD31 proteoliposomes to
resting HUVECs. This inconsistency seems to be because CD31 can bind
v3 integrin on HUVECs with a high affinity only when v3
integrin is activated (Figs 5 and 6). Furthermore, besides v3
integrin, a 120-kD ligand50 and CD3851 have
been reported as counter receptors for CD31 and L1 adhesion molecule
has been reported as another ligand for v3
integrin.52 Therefore, because a selective antagonist that
specifically blocks v3 integrin/CD31 interaction is not yet
available, there is still a small possibility that other interactions
that involve either v3 integrin or CD31 but do not involve the
specific interaction of v3 with CD31 contribute to the
amplification of VLA-4/VCAM-1 binding between eosinophils and
endothelial cells.
We also demonstrate that CD31-mediated signaling activates v3
integrin on endothelial cells. Previous studies showed that CD31
stimulation induced 1 and 2 integrin activation on a variety of
leukocytes.10,21-23,42 Thus, our results indicate that CD31 also acts as a signaling molecule for integrin activation in
nonhematopoietic cells. We have also found that, using specific kinase
inhibitors, protein kinase C and PI3-kinase are involved in mediating
the CD31-induced inside-out signaling for v3 integrin activation on endothelial cells (Fig 12). In hematopoietic cells, 2 major pathways
for integrin activation have been shown to be mediated by activation of
protein kinase C and PI3-kinase.43-46 Protein kinase C may
directly phosphorylate integrin cytoplasmic domains, but it has
been argued that no good correlation exists between the phosphorylated
state and activation of integrin46; rather, it is likely
that the immediate target of protein kinase C is some intracellular
mediator acting upstream of the integrins. Recently, PI3-kinase has
also been suggested to mediate integrin activation in hematopoietic
cells, as indicated by the observations that the selective PI3-kinase
inhibitor wortmannin blocks both 1 integrin activation and the
thrombin-mediated activation of platelet integrin.44,45,53
We also show that CD31 stimulation activates 41 integrin (VLA-4)
on eosinophils and thereby is importantly involved in the firm adhesion
of eosinophils to endothelial cells through VLA-4/VCAM-1 interaction.
It has been reported that CD31 stimulation induces 1 integrin
activation on T cells10 and CD34+ hemopoietic
progenitor cells21 and, to a lesser extent, 2 integrin
activation on monocytes, neutrophils,22 and natural killer
cells.23 For example, Tanaka et al10 showed
that, using CD8+ T cells, cross-linking of CD31 with
anti-CD31 MoAb increased 1 integrin-mediated adhesion to fibronectin
and purified VCAM-1, indicating that CD31 can act as a signaling
molecule for 1 integrin activation. However, the molecular mechanism
of leukocyte CD31-mediated inside-out signaling for 1 integrin
activation is still mostly unknown and remains to be elucidated.
Eosinophil infiltration into the tissue is a characteristic feature of
allergic inflammation such as asthma.54,55 Recent studies
have shown that the selective adhesion of eosinophils to vascular
endothelial cells and its transendothelial migration are mediated by
VCAM-1/VLA-4 interaction.38-41,56 VLA-4 is expressed on
eosinophils but not on neutrophils.38-41 In addition, IL-4
selectively induces VCAM-1 but not ICAM-1 expression on endothelial
cells34-36 and increases the adherence of eosinophils to
endothelial cells.36,57 Therefore, our results provide a
novel regulatory mechanism of VCAM-1/VLA-4 interaction that is
controlled by endothelial v3 integrin/eosinophil CD31 interaction.
In summary, we have shown that CD31-mediated inside-out signaling
activates v3 integrin on endothelial cells and that the increased
heterophilic interaction between v3 integrin on endothelial cells
and CD31 on eosinophils induces 1 integrin-mediated adhesion of
eosinophils to endothelial cells. These results indicate that the
heterophilic v3 integrin/CD31 interaction forms an important amplifier loop for 1 integrin-mediated adhesion of eosinophils to
endothelial cells and thus is involved in the selective eosinophil recruitment in allergic inflammation.
| |
FOOTNOTES |
Submitted September 3, 1998; accepted April 13, 1999.
Supported in part by grants from the Ministry of Education, Science and
Culture, Japan.
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 Itsuo Iwamoto, MD, Department of Medicine
II, Chiba University School of Medicine, 1-8-1 Inohana, Chiba City,
Chiba 260, Japan; e-mail: iwamoto{at}intmed02.m.chiba-u.ac.jp.
| |
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ABSTRACT
INTRODUCTION