Limits...
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.

Show MeSH

Related in: MedlinePlus

DNAM-1 recognizes junctional ligands expressed on endothelial cells. (a) HUVECs were analyzed by one-color immunofluorescence and FACS® analysis with Nec2-Fc, PVR-Fc, DNAM-1–Fc, and N4vtr-Fc (negative control) followed by FITC-conjugated goat anti–human second reagents. Gray profiles indicate cells incubated with the second reagent only. All soluble proteins were used at 20 μg/ml. Similar results were obtained when HUVECs were detached with 0.02% (wt/vol) disodium EDTA in PBS. (b) Immunofluorescence microscopy analysis of HUVECs using 20 μg/ml DNAM-1–Fc followed by FITC-conjugated goat anti–human second reagents. No staining was revealed with Nec2-Fc. The DNAM-1–Fc staining delineates the junctional systems between adjacent cells suggesting that DNAM-1 interacts with ligands localized at endothelial cell junctions. Similar results were obtained on live confluent HUVECs (not depicted).
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC2211807&req=5

fig3: DNAM-1 recognizes junctional ligands expressed on endothelial cells. (a) HUVECs were analyzed by one-color immunofluorescence and FACS® analysis with Nec2-Fc, PVR-Fc, DNAM-1–Fc, and N4vtr-Fc (negative control) followed by FITC-conjugated goat anti–human second reagents. Gray profiles indicate cells incubated with the second reagent only. All soluble proteins were used at 20 μg/ml. Similar results were obtained when HUVECs were detached with 0.02% (wt/vol) disodium EDTA in PBS. (b) Immunofluorescence microscopy analysis of HUVECs using 20 μg/ml DNAM-1–Fc followed by FITC-conjugated goat anti–human second reagents. No staining was revealed with Nec2-Fc. The DNAM-1–Fc staining delineates the junctional systems between adjacent cells suggesting that DNAM-1 interacts with ligands localized at endothelial cell junctions. Similar results were obtained on live confluent HUVECs (not depicted).

Mentions: Our data showed that DNAM-1, PVR, and Nectin-2 are expressed on monocytes. Potential interactions between these molecules with PVR and Nectin-2 on endothelial cells were assessed. To this end, chimeric molecules formed with the whole ectodomain of these molecules fused to the Fc fragment of human IgG1; namely NAM-1–Fc, PVR-Fc, and Nec2-Fc were analyzed for their binding on endothelial cells. Nec2-Fc and PVR-Fc do not significantly bind to HUVECs when compared with an irrelevant control (Fig. 3 a, N4vtr-Fc). Alternately, DNAM-1–Fc strongly binds to HUVECs. Confocal microscopy shows that DNAM-1–Fc, but not Nec2-Fc, specifically stains and delineates endothelial cell junctions, which suggests that DNAM-1 ligands are junctional molecules (Fig. 3 b). This is in agreement with the localization of PVR and Nectin-2 at cell junctions (Fig. 1).


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)

DNAM-1 recognizes junctional ligands expressed on endothelial cells. (a) HUVECs were analyzed by one-color immunofluorescence and FACS® analysis with Nec2-Fc, PVR-Fc, DNAM-1–Fc, and N4vtr-Fc (negative control) followed by FITC-conjugated goat anti–human second reagents. Gray profiles indicate cells incubated with the second reagent only. All soluble proteins were used at 20 μg/ml. Similar results were obtained when HUVECs were detached with 0.02% (wt/vol) disodium EDTA in PBS. (b) Immunofluorescence microscopy analysis of HUVECs using 20 μg/ml DNAM-1–Fc followed by FITC-conjugated goat anti–human second reagents. No staining was revealed with Nec2-Fc. The DNAM-1–Fc staining delineates the junctional systems between adjacent cells suggesting that DNAM-1 interacts with ligands localized at endothelial cell junctions. Similar results were obtained on live confluent HUVECs (not depicted).
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2211807&req=5

fig3: DNAM-1 recognizes junctional ligands expressed on endothelial cells. (a) HUVECs were analyzed by one-color immunofluorescence and FACS® analysis with Nec2-Fc, PVR-Fc, DNAM-1–Fc, and N4vtr-Fc (negative control) followed by FITC-conjugated goat anti–human second reagents. Gray profiles indicate cells incubated with the second reagent only. All soluble proteins were used at 20 μg/ml. Similar results were obtained when HUVECs were detached with 0.02% (wt/vol) disodium EDTA in PBS. (b) Immunofluorescence microscopy analysis of HUVECs using 20 μg/ml DNAM-1–Fc followed by FITC-conjugated goat anti–human second reagents. No staining was revealed with Nec2-Fc. The DNAM-1–Fc staining delineates the junctional systems between adjacent cells suggesting that DNAM-1 interacts with ligands localized at endothelial cell junctions. Similar results were obtained on live confluent HUVECs (not depicted).
Mentions: Our data showed that DNAM-1, PVR, and Nectin-2 are expressed on monocytes. Potential interactions between these molecules with PVR and Nectin-2 on endothelial cells were assessed. To this end, chimeric molecules formed with the whole ectodomain of these molecules fused to the Fc fragment of human IgG1; namely NAM-1–Fc, PVR-Fc, and Nec2-Fc were analyzed for their binding on endothelial cells. Nec2-Fc and PVR-Fc do not significantly bind to HUVECs when compared with an irrelevant control (Fig. 3 a, N4vtr-Fc). Alternately, DNAM-1–Fc strongly binds to HUVECs. Confocal microscopy shows that DNAM-1–Fc, but not Nec2-Fc, specifically stains and delineates endothelial cell junctions, which suggests that DNAM-1 ligands are junctional molecules (Fig. 3 b). This is in agreement with the localization of PVR and Nectin-2 at cell junctions (Fig. 1).

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