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DNAM-1 and PVR regulate monocyte migration through endothelial junctions.

Reymond N, Imbert AM, Devilard E, Fabre S, Chabannon C, Xerri L, Farnarier C, Cantoni C, Bottino C, Moretta A, Dubreuil P, Lopez M - J. Exp. Med. (2004)

Bottom Line: In the present paper, we show that PVR and Nectin-2 are expressed at cell junctions on primary vascular endothelial cells.Moreover, the specific binding of a soluble DNAM-1-Fc molecule was detected at endothelial junctions.This binding was almost completely abrogated by anti-PVR monoclonal antibodies (mAbs), but not modified by anti-Nectin-2 mAbs, which demonstrates that PVR is the major DNAM-1 ligand on endothelial cells.

View Article: PubMed Central - PubMed

Affiliation: Institut National de la Santé et de la Recherche Médicale UMR599, Institut de Cancérologie de Marseille, IFR 137, 27 Bd. Lei-Roure, 13009 Marseille, France.

ABSTRACT
DNAX accessory molecule 1 (DNAM-1; CD226) is a transmembrane glycoprotein involved in T cell and natural killer (NK) cell cytotoxicity. We demonstrated recently that DNAM-1 triggers NK cell-mediated killing of tumor cells upon engagement by its two ligands, poliovirus receptor (PVR; CD155) and Nectin-2 (CD112). In the present paper, we show that PVR and Nectin-2 are expressed at cell junctions on primary vascular endothelial cells. Moreover, the specific binding of a soluble DNAM-1-Fc molecule was detected at endothelial junctions. This binding was almost completely abrogated by anti-PVR monoclonal antibodies (mAbs), but not modified by anti-Nectin-2 mAbs, which demonstrates that PVR is the major DNAM-1 ligand on endothelial cells. Because DNAM-1 is highly expressed on leukocytes, we investigated the role of the DNAM-1-PVR interaction during the monocyte transendothelial migration process. In vitro, both anti-DNAM-1 and anti-PVR mAbs strongly blocked the transmigration of monocytes through the endothelium. Moreover, after anti-DNAM-1 or anti-PVR mAb treatment, monocytes were arrested at the apical surface of the endothelium over intercellular junctions, which strongly suggests that the DNAM-1-PVR interaction occurs during the diapedesis step. Altogether, our results demonstrate that DNAM-1 regulates monocyte extravasation via its interaction with PVR expressed at endothelial junctions on normal cells.

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PVR is the major DNAM-1 ligand on endothelial cells. (a) Inhibition of DNAM-1–Fc binding by different anti-Nectin mAbs. HUVECs, either untreated or preincubated with anti-PVR (L95) and anti–Nectin-2 (L14) mAbs used alone or in combination, were analyzed by two-color immunofluorescence and FACS® analysis for DNAM-1–Fc binding used at 1.5 μM. FITC-conjugated goat anti–mouse or PE-conjugated goat anti–human were used as second reagents. (b) Similar experiments were performed by immunofluorescence microscopy on confluent HUVECs. 1.5 μM DNAM-1–Fc binding at endothelial junctions was not affected by preincubation with anti–Nectin-1 (R1.302) or anti–Nectin-2 (L14) mAbs. Anti-PVR (L95) mAb preincubation strongly affects the DNAM-1–Fc binding at endothelial junctions. mAbs were used at 20 μg/ml. (c) HUVECs, preincubated with the different anti–Nectin-2 (gray bars) or anti-PVR (black bars) mAbs, were analyzed by FACS® analysis for DNAM-1–Fc binding used at 0.15 μM followed by FITC-conjugated goat anti–human second reagents. Untreated HUVECs stained with N4vtr-Fc were used as negative control (white bars). The value 100% corresponds to DNAM-1–Fc binding in the presence of the irrelevant anti–Nectin-1 (R1.302) mAb. (d) The different anti-PVR mAbs were mapped by ELISA using PVR-Fc molecules. PVR-vcc-Fc is constituted by the full-length ectodomain of PVR, whereas PVR-v-Fc is only constituted by the V domain of PVR.
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fig5: PVR is the major DNAM-1 ligand on endothelial cells. (a) Inhibition of DNAM-1–Fc binding by different anti-Nectin mAbs. HUVECs, either untreated or preincubated with anti-PVR (L95) and anti–Nectin-2 (L14) mAbs used alone or in combination, were analyzed by two-color immunofluorescence and FACS® analysis for DNAM-1–Fc binding used at 1.5 μM. FITC-conjugated goat anti–mouse or PE-conjugated goat anti–human were used as second reagents. (b) Similar experiments were performed by immunofluorescence microscopy on confluent HUVECs. 1.5 μM DNAM-1–Fc binding at endothelial junctions was not affected by preincubation with anti–Nectin-1 (R1.302) or anti–Nectin-2 (L14) mAbs. Anti-PVR (L95) mAb preincubation strongly affects the DNAM-1–Fc binding at endothelial junctions. mAbs were used at 20 μg/ml. (c) HUVECs, preincubated with the different anti–Nectin-2 (gray bars) or anti-PVR (black bars) mAbs, were analyzed by FACS® analysis for DNAM-1–Fc binding used at 0.15 μM followed by FITC-conjugated goat anti–human second reagents. Untreated HUVECs stained with N4vtr-Fc were used as negative control (white bars). The value 100% corresponds to DNAM-1–Fc binding in the presence of the irrelevant anti–Nectin-1 (R1.302) mAb. (d) The different anti-PVR mAbs were mapped by ELISA using PVR-Fc molecules. PVR-vcc-Fc is constituted by the full-length ectodomain of PVR, whereas PVR-v-Fc is only constituted by the V domain of PVR.

Mentions: DNAM-1–Fc binding was performed on endothelial cells. To discriminate between PVR, Nectin-2, or still-unidentified junctional ligands expressed by these cells, anti-PVR and anti–Nectin-2 mAbs were tested for their ability to block DNAM-1–Fc binding on HUVECs. Thus, HUVECs, either preincubated with anti-PVR or anti–Nectin-2 mAbs and used alone, in combination, or with control mAbs, were analyzed by double fluorescence for DNAM-1–Fc binding used at 1.5 μM (Fig. 5 a). DNAM-1–Fc binding to HUVECs is not significantly reduced by anti–Nectin-2 mAb preincubation, and is similar to controls without mAb or with the irrelevant anti–Nectin-1 mAb. On the contrary, anti-PVR mAb alone nearly induces a full inhibition (97%) of DNAM-1–Fc binding. Using immunofluorescence microscopy, we show that DNAM-1–Fc binding to endothelial cell junctions was not modified by anti–Nectin-1 and anti–Nectin-2 mAbs, but was strongly inhibited by anti-PVR mAbs. This strengthens the specificity of DNAM-1 binding to PVR, expressed at interendothelial junctions (Fig. 5 b). To extend and confirm these observations, five different anti-PVR mAbs were tested for their ability to interfere with DNAM-1–Fc binding on HUVECs. As shown in Fig. 5 c, all of them efficiently inhibit DNAM-1–Fc binding. The various anti-PVR mAbs were mapped by ELISA assay (Fig. 5 d). PV.404, M5A10, and M2C24 react with the IgC-like domain of PVR, whereas D171 and L95 are directed against the IgV-like domain of the molecule. These results suggest that both V and C Ig-like domains of PVR are involved in the interaction with DNAM-1. Altogether, our results demonstrate that DNAM-1 interacts directly and specifically with PVR on endothelial cells, and that this interaction occurs at endothelial cell junctions.


DNAM-1 and PVR regulate monocyte migration through endothelial junctions.

Reymond N, Imbert AM, Devilard E, Fabre S, Chabannon C, Xerri L, Farnarier C, Cantoni C, Bottino C, Moretta A, Dubreuil P, Lopez M - J. Exp. Med. (2004)

PVR is the major DNAM-1 ligand on endothelial cells. (a) Inhibition of DNAM-1–Fc binding by different anti-Nectin mAbs. HUVECs, either untreated or preincubated with anti-PVR (L95) and anti–Nectin-2 (L14) mAbs used alone or in combination, were analyzed by two-color immunofluorescence and FACS® analysis for DNAM-1–Fc binding used at 1.5 μM. FITC-conjugated goat anti–mouse or PE-conjugated goat anti–human were used as second reagents. (b) Similar experiments were performed by immunofluorescence microscopy on confluent HUVECs. 1.5 μM DNAM-1–Fc binding at endothelial junctions was not affected by preincubation with anti–Nectin-1 (R1.302) or anti–Nectin-2 (L14) mAbs. Anti-PVR (L95) mAb preincubation strongly affects the DNAM-1–Fc binding at endothelial junctions. mAbs were used at 20 μg/ml. (c) HUVECs, preincubated with the different anti–Nectin-2 (gray bars) or anti-PVR (black bars) mAbs, were analyzed by FACS® analysis for DNAM-1–Fc binding used at 0.15 μM followed by FITC-conjugated goat anti–human second reagents. Untreated HUVECs stained with N4vtr-Fc were used as negative control (white bars). The value 100% corresponds to DNAM-1–Fc binding in the presence of the irrelevant anti–Nectin-1 (R1.302) mAb. (d) The different anti-PVR mAbs were mapped by ELISA using PVR-Fc molecules. PVR-vcc-Fc is constituted by the full-length ectodomain of PVR, whereas PVR-v-Fc is only constituted by the V domain of PVR.
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Related In: Results  -  Collection

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fig5: PVR is the major DNAM-1 ligand on endothelial cells. (a) Inhibition of DNAM-1–Fc binding by different anti-Nectin mAbs. HUVECs, either untreated or preincubated with anti-PVR (L95) and anti–Nectin-2 (L14) mAbs used alone or in combination, were analyzed by two-color immunofluorescence and FACS® analysis for DNAM-1–Fc binding used at 1.5 μM. FITC-conjugated goat anti–mouse or PE-conjugated goat anti–human were used as second reagents. (b) Similar experiments were performed by immunofluorescence microscopy on confluent HUVECs. 1.5 μM DNAM-1–Fc binding at endothelial junctions was not affected by preincubation with anti–Nectin-1 (R1.302) or anti–Nectin-2 (L14) mAbs. Anti-PVR (L95) mAb preincubation strongly affects the DNAM-1–Fc binding at endothelial junctions. mAbs were used at 20 μg/ml. (c) HUVECs, preincubated with the different anti–Nectin-2 (gray bars) or anti-PVR (black bars) mAbs, were analyzed by FACS® analysis for DNAM-1–Fc binding used at 0.15 μM followed by FITC-conjugated goat anti–human second reagents. Untreated HUVECs stained with N4vtr-Fc were used as negative control (white bars). The value 100% corresponds to DNAM-1–Fc binding in the presence of the irrelevant anti–Nectin-1 (R1.302) mAb. (d) The different anti-PVR mAbs were mapped by ELISA using PVR-Fc molecules. PVR-vcc-Fc is constituted by the full-length ectodomain of PVR, whereas PVR-v-Fc is only constituted by the V domain of PVR.
Mentions: DNAM-1–Fc binding was performed on endothelial cells. To discriminate between PVR, Nectin-2, or still-unidentified junctional ligands expressed by these cells, anti-PVR and anti–Nectin-2 mAbs were tested for their ability to block DNAM-1–Fc binding on HUVECs. Thus, HUVECs, either preincubated with anti-PVR or anti–Nectin-2 mAbs and used alone, in combination, or with control mAbs, were analyzed by double fluorescence for DNAM-1–Fc binding used at 1.5 μM (Fig. 5 a). DNAM-1–Fc binding to HUVECs is not significantly reduced by anti–Nectin-2 mAb preincubation, and is similar to controls without mAb or with the irrelevant anti–Nectin-1 mAb. On the contrary, anti-PVR mAb alone nearly induces a full inhibition (97%) of DNAM-1–Fc binding. Using immunofluorescence microscopy, we show that DNAM-1–Fc binding to endothelial cell junctions was not modified by anti–Nectin-1 and anti–Nectin-2 mAbs, but was strongly inhibited by anti-PVR mAbs. This strengthens the specificity of DNAM-1 binding to PVR, expressed at interendothelial junctions (Fig. 5 b). To extend and confirm these observations, five different anti-PVR mAbs were tested for their ability to interfere with DNAM-1–Fc binding on HUVECs. As shown in Fig. 5 c, all of them efficiently inhibit DNAM-1–Fc binding. The various anti-PVR mAbs were mapped by ELISA assay (Fig. 5 d). PV.404, M5A10, and M2C24 react with the IgC-like domain of PVR, whereas D171 and L95 are directed against the IgV-like domain of the molecule. These results suggest that both V and C Ig-like domains of PVR are involved in the interaction with DNAM-1. Altogether, our results demonstrate that DNAM-1 interacts directly and specifically with PVR on endothelial cells, and that this interaction occurs at endothelial cell junctions.

Bottom Line: In the present paper, we show that PVR and Nectin-2 are expressed at cell junctions on primary vascular endothelial cells.Moreover, the specific binding of a soluble DNAM-1-Fc molecule was detected at endothelial junctions.This binding was almost completely abrogated by anti-PVR monoclonal antibodies (mAbs), but not modified by anti-Nectin-2 mAbs, which demonstrates that PVR is the major DNAM-1 ligand on endothelial cells.

View Article: PubMed Central - PubMed

Affiliation: Institut National de la Santé et de la Recherche Médicale UMR599, Institut de Cancérologie de Marseille, IFR 137, 27 Bd. Lei-Roure, 13009 Marseille, France.

ABSTRACT
DNAX accessory molecule 1 (DNAM-1; CD226) is a transmembrane glycoprotein involved in T cell and natural killer (NK) cell cytotoxicity. We demonstrated recently that DNAM-1 triggers NK cell-mediated killing of tumor cells upon engagement by its two ligands, poliovirus receptor (PVR; CD155) and Nectin-2 (CD112). In the present paper, we show that PVR and Nectin-2 are expressed at cell junctions on primary vascular endothelial cells. Moreover, the specific binding of a soluble DNAM-1-Fc molecule was detected at endothelial junctions. This binding was almost completely abrogated by anti-PVR monoclonal antibodies (mAbs), but not modified by anti-Nectin-2 mAbs, which demonstrates that PVR is the major DNAM-1 ligand on endothelial cells. Because DNAM-1 is highly expressed on leukocytes, we investigated the role of the DNAM-1-PVR interaction during the monocyte transendothelial migration process. In vitro, both anti-DNAM-1 and anti-PVR mAbs strongly blocked the transmigration of monocytes through the endothelium. Moreover, after anti-DNAM-1 or anti-PVR mAb treatment, monocytes were arrested at the apical surface of the endothelium over intercellular junctions, which strongly suggests that the DNAM-1-PVR interaction occurs during the diapedesis step. Altogether, our results demonstrate that DNAM-1 regulates monocyte extravasation via its interaction with PVR expressed at endothelial junctions on normal cells.

Show MeSH
Related in: MedlinePlus