We determined the role of the heterophilic interaction of vβ3 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 vβ3 integrin to its ligand RGD peptide, the binding of which reached a maximum at 10 minutes after the stimulation, and the CD31-induced vβ3 integrin activation on HUVECs was inhibited by inhibitors of protein kinase C and phosphatidylinositol 3 kinase (PI3-kinase). Furthermore, anti-vβ3 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-vβ3 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 4β1 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 vβ3 integrin on endothelial cells, that the heterophilic vβ3 integrin/CD31 interaction induces β1 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.

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 αvβ3 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 β1 integrin on T cells10 and CD34+ hematopoietic progenitor cells21 and adhesive function of β2 integrin on monocytes, neutrophils,22 and natural killer cells,23suggesting that leukocyte CD31 may act as a signaling molecule. However, the functional importance of the heterophilic interaction of αvβ3 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 αvβ3 integrin/CD31 interaction in eosinophil adhesion to interleukin-4 (IL-4)–stimulated vascular endothelial cells. We also studied the role of CD31 in αvβ3 and α4β1 integrin activation of endothelial cells and eosinophils, respectively. Our results indicate that the heterophilic αvβ3 integrin/CD31 interaction forms an important amplifier loop for β1 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.

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) α-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 αvβ3 MoAb LM609 (IgG1)12 and antihuman αvβ5 MoAb P1F6 (IgG1) were purchased from Chemicon International Inc (Temecula, CA). Mouse antihuman VLA-5 α-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 of51Cr-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-αvβ3 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-αvβ3 integrin MoAb (10 μg/mL) or GRGDSP peptide (or GRGESP peptide; 5 to 500 μg/mL). To further determine whether αvβ3 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 αvβ3 integrin to RGD peptide.

To determine whether CD31 stimulation of endothelial cells increases the adhesive function of αvβ3 integrin to its ligand, we performed the binding assay of αvβ3 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 αvβ3, αvβ5, and α5β1 integrins on endothelial cells recognize the Arg-Gly-Asp (RGD) sequence present in extracellular matrix proteins such as vitronectin and fibronectin,27 28we first examined the effect of anti-αvβ3 integrin, anti-αvβ5 integrin, and anti-α5β1 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-αvβ3 integrin MoAb, anti-αvβ5 integrin MoAb, or anti-α5β1 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 αvβ3 integrin activation on endothelial cells.51Cr-labeled HUVECs were preincubated with staurosporine (10−8 mol/L)29,30 or wortmannin (10−7 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 α4β1 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-αvβ3 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. Pvalues less than .05 were considered significant.

Role of CD31 and αvβ3 integrin in eosinophil adhesion to vascular endothelial cells.

To determine the role of CD31/CD31 homophilic and αvβ3 integrin/CD31 heterophilic bindings in the adhesive interaction between eosinophils and vascular endothelial cells, we examined the effect of anti-CD31 MoAb and anti-αvβ3 integrin MoAb on eosinophil adhesion to IL-4–stimulated cultured vascular endothelial cells. In our preliminary experiments, CD31, but not αvβ3 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 αvβ3 integrin were expressed on cultured HUVECs, as described previously.2-4,12 33 In addition, the distribution of αvβ3 integrin on HUVECs was not significantly affected by CD31 stimulation with anti-CD31 MoAb (Fig 1).

Fig. 1.

Immunofluorescence staining of CD31 and vβ3 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-vβ3 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.)

Fig. 1.

Immunofluorescence staining of CD31 and vβ3 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-vβ3 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 αvβ3 integrin on endothelial cells in mediating the eosinophil adhesion to IL-4–stimulated HUVECs was then examined by using anti-CD31 MoAb and anti-αvβ3 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; Fig3). 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-αvβ3 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 αvβ3 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.

Fig. 2.

Effect of anti-CD31 and anti-vβ3 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-vβ3 integrin MoAb LM609 (10 μg/mL), or control mouse IgG (10 μg/mL) for 10 minutes at 37°C, washed, and then incubated with51Cr-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).

Fig. 2.

Effect of anti-CD31 and anti-vβ3 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-vβ3 integrin MoAb LM609 (10 μg/mL), or control mouse IgG (10 μg/mL) for 10 minutes at 37°C, washed, and then incubated with51Cr-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 of51Cr-labeled eosinophils to HUVECs was performed. Data are the means ± SD for 3 to 6 experiments.

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 of51Cr-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,38-41we 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.

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).

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 αvβ3 integrin to its ligand.

Because it has recently been shown that CD31 stimulation of leukocytes increases the adhesive function of β1 and β2 integrins on the surface,10,21-23,42 we determined whether CD31 stimulation of endothelial cells increases the adhesive function of αvβ3 integrin to its ligand Arg-Gly-Asp (RGD) peptide.12 27Incubation 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-αvβ3 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-αvβ5 integrin MoAb or anti–VLA-5 α-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 αvβ3 integrin on HUVECs incubated with anti-CD31 MoAb by flow cytometry. Incubation with anti-CD31 MoAb had no significant effect on the expression of αvβ3 integrin on HUVECs (data not shown). These data indicate that CD31 stimulation of endothelial cells selectively increases the adhesive function of αvβ3 integrin on the surface.

Fig. 5.

CD31 stimulation of endothelial cells increases the adhesive function of vβ3 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-vβ3 integrin MoAb, anti-vβ5 integrin MoAb, or anti-5β1 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).

Fig. 5.

CD31 stimulation of endothelial cells increases the adhesive function of vβ3 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-vβ3 integrin MoAb, anti-vβ5 integrin MoAb, or anti-5β1 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 αvβ3 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).

Fig. 6.

Time course of endothelial CD31-induced increase in the adhesive function of vβ3 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.

Fig. 6.

Time course of endothelial CD31-induced increase in the adhesive function of vβ3 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.

Close modal
αvβ3 integrin/CD31 interaction mediates endothelial CD31-induced enhancement of eosinophil adhesion to IL-4–stimulated endothelial cells.

We then determined whether αvβ3 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-αvβ3 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-αvβ3 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 αvβ3 integrin/CD31 heterophilic interaction between endothelial cells and eosinophils mediates the VLA-4–dependent eosinophil adhesion to IL-4–stimulated endothelial cells.

Fig. 7.

Effect of anti-vβ3 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 of51Cr-labeled eosinophils to HUVECs was performed in the presence or absence of anti-vβ3 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).

Fig. 7.

Effect of anti-vβ3 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 of51Cr-labeled eosinophils to HUVECs was performed in the presence or absence of anti-vβ3 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).

Close modal
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 of51Cr-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).

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 of51Cr-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).

Close modal
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 of51Cr-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).

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 of51Cr-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).

Close modal
CD31 stimulation of eosinophils increases the adhesive function of α4β1 integrin (VLA-4) to its ligand.

To determine whether CD31 stimulation of eosinophils increases the adhesive function of α4β1 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; Fig10). 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).

Fig. 10.

CD31 stimulation of eosinophils increases the adhesive function of 4β1 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).

Fig. 10.

CD31 stimulation of eosinophils increases the adhesive function of 4β1 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).

Close modal

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-αvβ3 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).

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 of51Cr-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).

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 of51Cr-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).

Close modal
Protein kinase C and PI3-kinase are involved in CD31-induced αvβ3 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 β1 integrin activation through the receptors,43-46 we determined whether these kinases are involved in the signal transduction for CD31-induced αvβ3 integrin activation on endothelial cells. Staurosporine (protein kinase C inhibitor; 10−8 mol/L)29,30 and wortmannin (PI3-kinase inhibitor; 10−7 mol/L)31 32inhibited 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 αvβ3 integrin activation on endothelial cells.

Fig. 12.

Effect of staurosporine and wortmannin on endothelial CD31-induced increase in the adhesive function of vβ3 integrin to RGD peptide. 51Cr-labeled HUVECs were preincubated with staurosporine (Staur; 10−8 mol/L), wortmannin (Wort; 10−7 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).

Fig. 12.

Effect of staurosporine and wortmannin on endothelial CD31-induced increase in the adhesive function of vβ3 integrin to RGD peptide. 51Cr-labeled HUVECs were preincubated with staurosporine (Staur; 10−8 mol/L), wortmannin (Wort; 10−7 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).

Close modal

In this study, we show that CD31 stimulation activates αvβ3 integrin on endothelial cells and that the increased heterophilic interaction between αvβ3 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 αvβ3 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-αvβ3 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-αvβ3 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 α4β1 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 αvβ3 integrin/CD31 interaction forms an important amplifier loop for β1 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.

αvβ3 integrin has been implicated in angiogenesis, in which αvβ3 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 αvβ3 integrin is a ligand for CD31 using adhesion assays to recombinant CD31.15 16 However, the functional importance of the heterophilic αvβ3 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 αvβ3 integrin/CD31 interaction that αvβ3 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 αvβ3 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 αvβ3 integrin on HUVECs to RGD peptide coated on the plates may be more sensitive to be detected than the binding of αvβ3 integrin on HUVECs to CD31 on eosinophils.

There has been the controversy over whether αvβ3 integrin can bind CD31. Sun et al49 reported that anti-αvβ3 integrin antibody failed to inhibit the binding of CD31 proteoliposomes to resting HUVECs. This inconsistency seems to be because CD31 can bind αvβ3 integrin on HUVECs with a high affinity only when αvβ3 integrin is activated (Figs 5 and 6). Furthermore, besides αvβ3 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 αvβ3 integrin.52 Therefore, because a selective antagonist that specifically blocks αvβ3 integrin/CD31 interaction is not yet available, there is still a small possibility that other interactions that involve either αvβ3 integrin or CD31 but do not involve the specific interaction of αvβ3 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 αvβ3 integrin on endothelial cells. Previous studies showed that CD31 stimulation induced β1 and β2 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 αvβ3 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 β 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 β1 integrin activation and the thrombin-mediated activation of platelet integrin.44,45 53 

We also show that CD31 stimulation activates α4β1 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 β1 integrin activation on T cells10 and CD34+ hemopoietic progenitor cells21 and, to a lesser extent, β2 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 β1 integrin-mediated adhesion to fibronectin and purified VCAM-1, indicating that CD31 can act as a signaling molecule for β1 integrin activation. However, the molecular mechanism of leukocyte CD31-mediated inside-out signaling for β1 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 αvβ3 integrin/eosinophil CD31 interaction.

In summary, we have shown that CD31-mediated inside-out signaling activates αvβ3 integrin on endothelial cells and that the increased heterophilic interaction between αvβ3 integrin on endothelial cells and CD31 on eosinophils induces β1 integrin-mediated adhesion of eosinophils to endothelial cells. These results indicate that the heterophilic αvβ3 integrin/CD31 interaction forms an important amplifier loop for β1 integrin-mediated adhesion of eosinophils to endothelial cells and thus is involved in the selective eosinophil recruitment in allergic inflammation.

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.

1
Newman
PJ
Berndt
MC
Gorski
J
White
GC
II
Lyman
S
Paddock
C
Muller
WA
PECAM-1 (CD31) cloning and relation to adhesion molecules of the immunoglobulin gene superfamily.
Science
247
1990
1219
2
Muller
WA
Ratti
CM
McDonnell
SL
Cohn
ZA
A human endothelial cell-restricted externally disposed plasmalemmal protein enriched in intercellular junction.
J Exp Med
170
1989
399
3
Albelda
SM
Oliver
PD
Romer
LH
Buck
CA
EndoCAM: A novel endothelial cell-cell adhesion molecule.
J Cell Biol
110
1990
1227
4
Albelda
SM
Muller
WA
Buck
CA
Newman
PJ
Molecular and cellular properties of PECAM-1 (endoCAM/CD31): A novel vascular cell-cell adhesion molecule.
J Cell Biol
114
1991
1059
5
Ohto
H
Maeda
H
Shibata
Y
Chen
RF
Ozaki
Y
Higashihara
M
Takeuchi
A
Tohyama
H
A novel leukocyte differentiation antigen: Two monoclonal antibodies TM2 and TM3 define a 120-kd molecule present on neutrophils, monocytes, platelets, and activated lymphoblasts.
Blood
66
1985
873
6
Goyert
SM
Ferrero
EM
Seremetis
SV
Winchester
RJ
Silver
J
Mattison
AC
Biochemistry and expression of myelomonocytic antigens.
J Immunol
137
1986
3909
7
Stokiniger
H
Gadd
SJ
Eher
R
Majdic
O
Schreiber
W
Kasinrerk
W
Strass
B
Schnabl
E
Knapp
W
Molecular characterization and functional analysis of the leukocyte surface protein CD31.
J Immunol
145
1990
3889
8
Ashman
LK
Aylett
GW
Expression of CD31 epitopes on human lymphocytes: CD31 monoclonal antibodies differentiate between naive (CD45RA+) and memory (CD45RA−) CD4-positive T cells.
Tissue Antigens
38
1991
208
9
Torimoto
Y
Rothstein
DM
Dang
NH
Schlossman
SF
Morimoto
C
CD31, a novel cell surface marker for CD4 cells of suppressor lineage, unaltered by state of activation.
J Immunol
148
1992
388
10
Tanaka
Y
Albelda
SM
Horgan
KJ
van Seventer
GA
Shimizu
Y
Newman
W
Hallam
J
Newman
PJ
Buck
CA
Shaw
S
CD31 expressed on distinctive T cell subsets is a preferential amplifier of β1 integrin-mediated adhesion.
J Exp Med
176
1992
245
11
Muller
WA
Berman
ME
Newman
PJ
DeLisser
HM
Albelda
SM
A heterophilic adhesion mechanism for platelet/endothelial cell adhesion molecule 1 (CD31).
J Exp Med
175
1992
1401
12
Cheresh
DA
Human endothelial cells synthesize and express an Arg-Gly-Asp-directed adhesion receptor involved in attachment to fibrinogen and von Willebrand factor.
Proc Natl Acad Sci USA
84
1987
6471
13
Maxfield
SR
Moulder
K
Koning
F
Elbe
A
Stingl
G
Coligan
JE
Shevach
EM
Yokoyama
WM
Murine T cells express a cell surface receptor for multiple extracellular matrix proteins. Identification and characterization with monoclonal antibodies.
J Exp Med
169
1989
2173
14
Moulder
K
Roberts
K
Shevach
EM
Coligan
JE
The mouse vitronectin receptor is a T cell activation antigen.
J Exp Med
173
1991
343
15
Piali
L
Hammel
P
Uherek
C
Bachmann
F
Gisler
RH
Dunon
D
Imhof
BA
CD31/PECAM-1 is a ligand for αvβ3 integrin involved in adhesion of leukocytes to endothelium.
J Cell Biol
130
1995
451
16
Buckley
CD
Doyonnas
R
Newton
JP
Blystone
SD
Brown
EJ
Watt
SM
Simmons
DL
Identification of αvβ3 as a heterotypic ligand for CD31/PECAM-1.
J Cell Sci
109
1996
437
17
DeLisser
HM
Newman
PJ
Albelda
SM
Molecular and functional aspects of PECAM-1/CD31.
Immunol Today
15
1994
490
18
Muller
WA
Weigl
SA
Deng
X
Phillips
DM
PECAM-1 is required for transendothelial migration of leukocytes.
J Exp Med
178
1993
449
19
Vaporciyan
AA
DeLisser
HM
Yan
HC
Mendiguren
II
Thom
SR
Jones
ML
Ward
PA
Albelda
SM
Involvement of platelet-endothelial cell adhesion molecule-1 in neutrophil recruitment in vivo.
Science
262
1993
1580
20
Bogen
S
Pak
J
Garifallou
M
Deng
X
Muller
WA
Monoclonal antibody to murine PECAM-1 (CD31) blocks acute inflammation in vivo.
J Exp Med
179
1994
1059
21
Leavesley
DI
Oliver
JM
Swart
BW
Berndt
MC
Haylock
DN
Simmoms
PJ
Signals from platelet/endothelial cell adhesion molecule enhance the adhesive activity of the very late antigen-4 integrin of human CD34+ hemopoietic progenitor cells.
J Immunol
153
1994
4673
22
Berman
ME
Muller
WA
Ligation of platelet/endothelial cell adhesion molecule 1 (PECAM-1/CD31) on monocytes and neutrophils increases binding capacity of leukocyte CR3 (CD11b/CD18).
J Immunol
154
1995
299
23
Berman
ME
Xie
Y
Muller
WA
Roles of platelet/endothelial cell adhesion molecule-1 (PECAM-1, CD31) in natural killer cell transendothelial migration and β2 integrin activation.
J Immunol
156
1996
1515
24
Yan
H-C
Pilewski
JM
Zhang
O
DeLisser
HM
Romer
L
Albelda
SM
Localization of multiple functional domains on human PECAM-1 (CD31) by monoclonal antibody epitope mapping.
Cell Adhesion Commun
3
1995
45
25
Jaffe
EA
Nachman
RL
Becker
CG
Minick
CR
Culture of human endothelial cells derived from umbilical veins. Identification by morphologic and immunologic criteria.
J Clin Invest
52
1973
2745
26
Hansel
TT
Pound
JD
Pilling
D
Kitas
GD
Salmon
M
Gentle
TA
Lee
SS
Thompson
RA
Purification of human eosinophils by negative selection using immunomagnetic beads.
J Immunol Methods
122
1989
97
27
Smith
JW
Cheresh
DA
Integrin (αvβ3)-ligand interaction. Identification of a heterodimeric RGD binding site on the vitronectin receptor.
J Biol Chem
265
1991
2168
28
Hynes
RO
Integrins: Versatility, modulation, and signaling in cell adhesion.
Cell
69
1992
11
29
Tamaoki
T
Nomoto
H
Takahashi
I
Kato
Y
Morimoto
M
Tomita
F
Staurosporine, a potent inhibitor of phospholipid/Ca++ dependent protein kinase.
Biochem Biophys Res Commun
135
1986
397
30
Seynaeve
CM
Kazanietz
MG
Blumberg
PM
Sausville
EA
Worland
PJ
Differential inhibition of protein kinase C isozymes by UCN-01, a staurosporine analogue.
Mol Pharmacol
45
1994
1207
31
Yano
H
Nakanishi
S
Kimura
K
Hanai
N
Saitoh
Y
Fukui
Y
Nonomura
Y
Matsuda
Y
Inhibition of histamine secretion by wortmannin through the blockade of phosphatidylinositol 3-kinase in RBL-2H3 cells.
J Biol Chem
268
1993
25846
32
Okada
T
Sakuma
L
Fukui
Y
Hazeki
O
Ui
M
Blockade of chemotactic peptide-induced stimulation of neutrophils by wortmannin as a result of selective inhibition of phosphatidylinositol 3-kinase.
J Biol Chem
269
1994
3563
33
Conforti
G
Dominguez-Jimenez
C
Zanetti
A
Gimbrone
MA
Jr
Cremona
O
Marchisio
PC
Dejana
E
Human endothelial cells express integrin receptors on the luminal aspect of their membrane.
Blood
80
1992
437
34
Thornhill
MH
Kyan-Aung
U
Haskard
DO
IL-4 increases human endothelial cell adhesiveness for T cells but not for neutrophils.
J Immunol
144
1990
3060
35
Thornhill
MH
Haskard
DO
IL-4 regulates endothelial cell activation by IL-1, tumor necrosis factor, or IFN-γ.
J Immunol
145
1990
865
36
Schleimer
RP
Sterbinsky
SA
Kaiser
J
Bickel
CA
Klunk
DA
Tomioka
KB
Newman
W
Luscinskas
FW
Gimbrone
MA
Mcintyre
BW
Bochner
BS
IL-4 induces adherence of human eosinophils and basophils but not neutrophils to endothelium. Association with expression of VCAM-1.
J Immunol
148
1992
1086
37
Elices
MJ
Osborn
L
Takada
Y
Crouse
C
Luhowskyj
S
Hemler
ME
Lobb
RR
VCAM-1 on activated endothelium interacts with the leukocyte integrin VLA-4 at a site distinct from the VLA-4/fibronectin binding site.
Cell
60
1990
577
38
Dobrina
A
Menegazzi
R
Carlos
TM
Nardon
E
Cramer
R
Zacchi
T
Harlen
JM
Patriarca
P
Mechanisms of eosinophil adherence to cultured vascular endothelial cells. Eosinophils bind to the cytokine-induced endothelial ligand vascular cell adhesion molecule-1 via the very late activation antigen-4 integrin receptor.
J Clin Invest
88
1991
20
39
Walsh
GM
Mermod
JJ
Hartnell
A
Kay
AB
Wardlaw
AJ
Human eosinophil, but not neutrophil, adherence to IL-1-stimulated human umbilical vascular endothelial cells is α4β1 (very late antigen-4) dependent.
J Immunol
146
1991
3419
40
Weller
PF
Rand
TH
Goelz
SE
Chi-Rosso
C
Lobb
RR
Human eosinophil adherence to vascular endothelium mediated by binding to vascular cell adhesion molecule 1 and endothelial leukocyte adhesion molecule 1.
Proc Natl Acad Sci USA
88
1991
7430
41
Bochner
BS
Luscinskas
FW
Gimbrone
MA
Newman
W
Sterbinsky
SA
Derse-Anthony
CP
Klunk
D
Schleimer
RP
Adhesion of human basophils, eosinophils, and neutrophils to interleukin 1-activated human vascular endothelial cells: Contributions of endothelial cell adhesion molecules.
J Exp Med
173
1991
1553
42
Piali
L
Albelda
SM
Baldwin
HS
Hammel
P
Gisler
RH
Imhof
BA
Murine platelet endothelial cell adhesion molecule (PECAM-1)/CD31 modulates β2 integrins on lymphokine-activated killer cells.
Eur J Immunol
23
1993
2464
43
Shimizu
Y
van Seventer
GA
Ennis
E
Newman
W
Horgan
KJ
Shaw
S
Crosslinking of the T cell-specific accessory molecules CD7 and CD28 modulates T cell adhesion.
J Exp Med
175
1992
577
44
Shimizu
Y
Mobley
JL
Finkelstein
LD
Chan
ASH
A role for phosphatidylinositol 3-kinase in the regulation of β1 integrin activity by the CD2 antigen.
J Cell Biol
131
1995
1867
45
Kinashi
T
Escobedo
JA
Williams
LT
Takatsu
K
Springer
TA
Receptor tyrosine kinase stimulates cell-matrix adhesion by phosphatidylinositol 3 kinase and phospholipase C-γ1 pathways.
Blood
86
1995
2086
46
Kolanus
W
Seed
B
Integrins and inside-out signal transduction: converging signals from PKC and PIP3.
Curr Opin Cell Biol
9
1997
725
47
Brooks
PC
Montogomery
AMP
Rosenfeld
M
Reisfeld
RA
Hu
T
Klier
G
Cheresh
DA
Integrin αvβ3 antagonists promote tumor regression by inducing apoptosis of angiogenic blood vessels.
Cell
79
1994
1157
48
Drake
CJ
Cheresh
DA
Little
CD
An antagonist of integrin αvβ3 prevents maturation of blood vessels during embryonic neovascularization.
J Cell Sci
108
1995
2655
49
Sun
Q
DeLisser
HM
Zukowski
MM
Paddock
C
Albelda
SM
Newman
PJ
Individually distinct Ig homology domains in PECAM-1 regulate homophilic binding and modulate receptor affinity.
J Biol Chem
271
1996
11090
50
Prager
E
Sunder-Plassmann
R
Hansmann
C
Koch
C
Holter
W
Knapp
W
Stockinger
H
Interaction of CD31 with a heterophilic counterreceptor involved in downregulation of human T cell responses.
J Exp Med
184
1996
41
51
Deaglio
S
Morra
M
Mallone
R
Ausiello
CM
Prager
E
Garbarino
G
Dianzani
U
Stockinger
H
Malavasi
F
Human CD38 (ADP-ribosyl cyclase) is a counter-receptor of CD31, an Ig superfamily member.
J Immunol
160
1998
395
52
Ebeling
O
Duczmal
A
Aigner
S
Geiger
C
Schollhammer
S
Kemshead
JT
Moller
P
Schwartz-Albiez
R
Altevogt
P
L1 adhesion molecule on human lymphocytes and monocytes: Expression and involvement in binding to αVβ3 integrin.
Eur J Immunol
26
1996
2508
53
Zhang
J
Zhang
J
Shattil
SJ
Cunninggham
MC
Rittenhouse
SE
Phosphoinositide 3-kinase γ and p85/phosphoinositide 3-kinase in platelets. Relative activation by thrombin receptor or β-phorbol myristate acetate and roles in promoting the ligand-binding function of αIIbβ3 integrin.
J Biol Chem
271
1996
6265
54
Gleich
GJ
The eosinophil and bronchial asthma: Current understanding.
J Allergy Clin Immunol
85
1990
422
55
Bousquet
J
Chanez
P
Lacoste
JY
Barneon
G
Ghanavian
N
Enander
I
Venge
P
Ahlstedt
S
Simony-Lafontaine
J
Godard
P
Michel
FB
Eosinophilic inflammation in asthma.
N Engl J Med
323
1990
1033
56
Nakajima
H
Sano
H
Nishimura
T
Yoshida
S
Iwamoto
I
Role of vascular cell adhesion molecule 1/very late activation antigen 4 and intercellular adhesion molecule 1/lymphocyte function-associated antigen 1 interactions in antigen-induced eosinophil and T cell recruitment into the tissue.
J Exp Med
179
1994
1145
57
Moser
R
Fehr
J
Bruijnzeel
PLB
IL-4 controls the selective endothelium-driven transmigration of eosinophils from allergic individuals.
J Immunol
149
1992
1432

Author notes

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@intmed02.m.chiba-u.ac.jp.

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