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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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Analysis of the cell surface expression of DNAM-1, PVR, and Nectin-2 on monocytes and primary vascular endothelial cells. (a) Fresh PBMCs were gated on monocytes on the basis of both size and granularity. Cells were analyzed by two-color immunofluorescence and FACS® analysis with anti-CD14 mAb in combination with anti–DNAM-1 (FS.123), anti-PVR (PV.404), or anti–Nectin-2 (R2.477) mAbs followed by FITC- or PE-conjugated goat anti–mouse second reagents. (b) HUVECs were analyzed by one-color immunofluorescence and FACS® analysis with anti–DNAM-1 (FS123), anti-PVR (PV.404), anti–Nectin-2 (R2.477), or anti–PECAM-1 (JC/70A) mAbs followed by FITC-conjugated goat anti–mouse second reagents. Gray profiles indicate cells incubated with the second reagent only. We previously controlled that trypsin treatment does not affect cell surface expression of Nectins. Similar results were obtained when HUVECs were detached with 0.02% (wt/vol) disodium EDTA in PBS. (c) Localization of Nectin-2 and PVR on primary endothelial cells. HUVECs were fixed; incubated with 2 μg/ml of anti–Nectin-2 (R2.477), anti-PVR (PV.404), and anti–PECAM-1 (JC/70), followed by Alexa-488–labeled goat anti–mouse second reagent; and analyzed by immunofluorescence microscopy. Staining was similar on live cells (not depicted). (d) HUVECs were fixed, permeabilized, and stained with the anti–Nectin-2 (R2.477) mAb and the anti–β-catenin rabbit antiserum followed by Alexa-488–labeled goat anti–mouse antibody and TRITC-labeled goat anti–rabbit second reagents, respectively. Cells were analyzed by immunofluorescence microscopy.
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fig1: Analysis of the cell surface expression of DNAM-1, PVR, and Nectin-2 on monocytes and primary vascular endothelial cells. (a) Fresh PBMCs were gated on monocytes on the basis of both size and granularity. Cells were analyzed by two-color immunofluorescence and FACS® analysis with anti-CD14 mAb in combination with anti–DNAM-1 (FS.123), anti-PVR (PV.404), or anti–Nectin-2 (R2.477) mAbs followed by FITC- or PE-conjugated goat anti–mouse second reagents. (b) HUVECs were analyzed by one-color immunofluorescence and FACS® analysis with anti–DNAM-1 (FS123), anti-PVR (PV.404), anti–Nectin-2 (R2.477), or anti–PECAM-1 (JC/70A) mAbs followed by FITC-conjugated goat anti–mouse second reagents. Gray profiles indicate cells incubated with the second reagent only. We previously controlled that trypsin treatment does not affect cell surface expression of Nectins. Similar results were obtained when HUVECs were detached with 0.02% (wt/vol) disodium EDTA in PBS. (c) Localization of Nectin-2 and PVR on primary endothelial cells. HUVECs were fixed; incubated with 2 μg/ml of anti–Nectin-2 (R2.477), anti-PVR (PV.404), and anti–PECAM-1 (JC/70), followed by Alexa-488–labeled goat anti–mouse second reagent; and analyzed by immunofluorescence microscopy. Staining was similar on live cells (not depicted). (d) HUVECs were fixed, permeabilized, and stained with the anti–Nectin-2 (R2.477) mAb and the anti–β-catenin rabbit antiserum followed by Alexa-488–labeled goat anti–mouse antibody and TRITC-labeled goat anti–rabbit second reagents, respectively. Cells were analyzed by immunofluorescence microscopy.

Mentions: We demonstrated recently that PVR and Nectin-2 are cell surface ligands of DNAM-1 that specifically induce NK cell–mediated cytolytic activity against tumor cell targets (2). In the present paper, we investigated the role of the interactions between DNAM-1 and its ligands on normal cells. Most leukocytes were shown to express DNAM-1 (1). PVR and Nectin-2 are cell adhesion molecules specifically expressed at E-cadherin–based AJs in epithelial cells. AJs are structurally and functionally similar in endothelial cells because VE-cadherin is the major component of endothelial AJ-mediating homophilic interaction between adjacent cells. In a first set of experiments, we analyzed the expression of DNAM-1, PVR, and Nectin-2 on leukocytes and endothelial cells. As shown in Fig. 1 a, low surface densities of PVR and Nectin-2 were detected on monocytes. Nectin-3 and Nectin-4 were not detected on monocytes (unpublished data). According to previous data, different leukocyte subsets, including monocytes (Fig. 1 a), highly express DNAM-1 (1). The expression of DNAM-1, PVR, and Nectin-2 was assessed on primary endothelial cells by both flow cytometry and immunofluorescence microscopy. DNAM-1 is not or just faintly expressed on HUVECs, whereas PVR and Nectin-2 are highly expressed at levels similar to PECAM-1 (Fig. 1 b). Moreover, both PVR and Nectin-2 are mostly expressed at endothelial cell junctions on HUVECs, similar to PECAM-1 (Fig. 1 c). They show an extremely similar distribution at cell borders compared with the VE-cadherin cytoplasmic partner β-catenin (Fig. 1 d). Finally, PVR and Nectin-2 as well as PECAM-1 are expressed on endothelial cells of placental blood vessels (Fig. 2 a, black arrows). In particular, in some sections and in accordance with their intercellular localization on HUVECs (Fig. 1 c), staining is observed at cell-to-cell contacts (Fig. 2 a, white arrows). PVR and Nectin-2 are also expressed in vessels from normal skin (Fig. 2 b). These data show that PVR and Nectin-2 are two new junctional molecules expressed at vascular endothelial cell junctions, whereas their counter receptor, DNAM-1, is expressed on monocytes.


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)

Analysis of the cell surface expression of DNAM-1, PVR, and Nectin-2 on monocytes and primary vascular endothelial cells. (a) Fresh PBMCs were gated on monocytes on the basis of both size and granularity. Cells were analyzed by two-color immunofluorescence and FACS® analysis with anti-CD14 mAb in combination with anti–DNAM-1 (FS.123), anti-PVR (PV.404), or anti–Nectin-2 (R2.477) mAbs followed by FITC- or PE-conjugated goat anti–mouse second reagents. (b) HUVECs were analyzed by one-color immunofluorescence and FACS® analysis with anti–DNAM-1 (FS123), anti-PVR (PV.404), anti–Nectin-2 (R2.477), or anti–PECAM-1 (JC/70A) mAbs followed by FITC-conjugated goat anti–mouse second reagents. Gray profiles indicate cells incubated with the second reagent only. We previously controlled that trypsin treatment does not affect cell surface expression of Nectins. Similar results were obtained when HUVECs were detached with 0.02% (wt/vol) disodium EDTA in PBS. (c) Localization of Nectin-2 and PVR on primary endothelial cells. HUVECs were fixed; incubated with 2 μg/ml of anti–Nectin-2 (R2.477), anti-PVR (PV.404), and anti–PECAM-1 (JC/70), followed by Alexa-488–labeled goat anti–mouse second reagent; and analyzed by immunofluorescence microscopy. Staining was similar on live cells (not depicted). (d) HUVECs were fixed, permeabilized, and stained with the anti–Nectin-2 (R2.477) mAb and the anti–β-catenin rabbit antiserum followed by Alexa-488–labeled goat anti–mouse antibody and TRITC-labeled goat anti–rabbit second reagents, respectively. Cells were analyzed by immunofluorescence microscopy.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2211807&req=5

fig1: Analysis of the cell surface expression of DNAM-1, PVR, and Nectin-2 on monocytes and primary vascular endothelial cells. (a) Fresh PBMCs were gated on monocytes on the basis of both size and granularity. Cells were analyzed by two-color immunofluorescence and FACS® analysis with anti-CD14 mAb in combination with anti–DNAM-1 (FS.123), anti-PVR (PV.404), or anti–Nectin-2 (R2.477) mAbs followed by FITC- or PE-conjugated goat anti–mouse second reagents. (b) HUVECs were analyzed by one-color immunofluorescence and FACS® analysis with anti–DNAM-1 (FS123), anti-PVR (PV.404), anti–Nectin-2 (R2.477), or anti–PECAM-1 (JC/70A) mAbs followed by FITC-conjugated goat anti–mouse second reagents. Gray profiles indicate cells incubated with the second reagent only. We previously controlled that trypsin treatment does not affect cell surface expression of Nectins. Similar results were obtained when HUVECs were detached with 0.02% (wt/vol) disodium EDTA in PBS. (c) Localization of Nectin-2 and PVR on primary endothelial cells. HUVECs were fixed; incubated with 2 μg/ml of anti–Nectin-2 (R2.477), anti-PVR (PV.404), and anti–PECAM-1 (JC/70), followed by Alexa-488–labeled goat anti–mouse second reagent; and analyzed by immunofluorescence microscopy. Staining was similar on live cells (not depicted). (d) HUVECs were fixed, permeabilized, and stained with the anti–Nectin-2 (R2.477) mAb and the anti–β-catenin rabbit antiserum followed by Alexa-488–labeled goat anti–mouse antibody and TRITC-labeled goat anti–rabbit second reagents, respectively. Cells were analyzed by immunofluorescence microscopy.
Mentions: We demonstrated recently that PVR and Nectin-2 are cell surface ligands of DNAM-1 that specifically induce NK cell–mediated cytolytic activity against tumor cell targets (2). In the present paper, we investigated the role of the interactions between DNAM-1 and its ligands on normal cells. Most leukocytes were shown to express DNAM-1 (1). PVR and Nectin-2 are cell adhesion molecules specifically expressed at E-cadherin–based AJs in epithelial cells. AJs are structurally and functionally similar in endothelial cells because VE-cadherin is the major component of endothelial AJ-mediating homophilic interaction between adjacent cells. In a first set of experiments, we analyzed the expression of DNAM-1, PVR, and Nectin-2 on leukocytes and endothelial cells. As shown in Fig. 1 a, low surface densities of PVR and Nectin-2 were detected on monocytes. Nectin-3 and Nectin-4 were not detected on monocytes (unpublished data). According to previous data, different leukocyte subsets, including monocytes (Fig. 1 a), highly express DNAM-1 (1). The expression of DNAM-1, PVR, and Nectin-2 was assessed on primary endothelial cells by both flow cytometry and immunofluorescence microscopy. DNAM-1 is not or just faintly expressed on HUVECs, whereas PVR and Nectin-2 are highly expressed at levels similar to PECAM-1 (Fig. 1 b). Moreover, both PVR and Nectin-2 are mostly expressed at endothelial cell junctions on HUVECs, similar to PECAM-1 (Fig. 1 c). They show an extremely similar distribution at cell borders compared with the VE-cadherin cytoplasmic partner β-catenin (Fig. 1 d). Finally, PVR and Nectin-2 as well as PECAM-1 are expressed on endothelial cells of placental blood vessels (Fig. 2 a, black arrows). In particular, in some sections and in accordance with their intercellular localization on HUVECs (Fig. 1 c), staining is observed at cell-to-cell contacts (Fig. 2 a, white arrows). PVR and Nectin-2 are also expressed in vessels from normal skin (Fig. 2 b). These data show that PVR and Nectin-2 are two new junctional molecules expressed at vascular endothelial cell junctions, whereas their counter receptor, DNAM-1, is expressed on monocytes.

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