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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 and Nectin-2 directly bind to DNAM-1. (a) CHO/K cells either nontransfected or transfected with PVR or Nectin-2 cDNAs were analyzed by FACS® analysis with anti-PVR (L95) or anti–Nectin-2 (L14) mAbs, followed by a PE-conjugated goat anti–mouse second reagents (left). CHO/K: gray line, isotype matched mAb; black line, L95 mAb; and dashed line, L14 mAbs. CHO/K transfectants: gray line, isotype matched mAb and black histogram, L95 and L14 mAbs. Binding of DNAM-1–Fc at 1.5 μM on CHO/K cells was revealed by PE-conjugated goat anti–human second reagents (right): (gray line) incubation with the second reagent.; (black histogram) incubation with 1.5 μM DNAM-1–Fc. (b, left) PVR and Nectin-2 directly bind to Nectin-3. (right) To analyze PVR and Nectin-2 interactions with DNAM-1, biotinylated PVR-Fc and Nectin-2–Fc binding was measured by ELISA on wells coated with BT3-Fc (unrelated protein), Nectin-3–Fc (positive control), or DNAM-1–Fc. PVR-Fc and Nectin-2–Fc directly bind to DNAM-1–Fc and as expected with Nectin-3–Fc.
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fig4: PVR and Nectin-2 directly bind to DNAM-1. (a) CHO/K cells either nontransfected or transfected with PVR or Nectin-2 cDNAs were analyzed by FACS® analysis with anti-PVR (L95) or anti–Nectin-2 (L14) mAbs, followed by a PE-conjugated goat anti–mouse second reagents (left). CHO/K: gray line, isotype matched mAb; black line, L95 mAb; and dashed line, L14 mAbs. CHO/K transfectants: gray line, isotype matched mAb and black histogram, L95 and L14 mAbs. Binding of DNAM-1–Fc at 1.5 μM on CHO/K cells was revealed by PE-conjugated goat anti–human second reagents (right): (gray line) incubation with the second reagent.; (black histogram) incubation with 1.5 μM DNAM-1–Fc. (b, left) PVR and Nectin-2 directly bind to Nectin-3. (right) To analyze PVR and Nectin-2 interactions with DNAM-1, biotinylated PVR-Fc and Nectin-2–Fc binding was measured by ELISA on wells coated with BT3-Fc (unrelated protein), Nectin-3–Fc (positive control), or DNAM-1–Fc. PVR-Fc and Nectin-2–Fc directly bind to DNAM-1–Fc and as expected with Nectin-3–Fc.

Mentions: We analyzed the binding of DNAM-1–Fc relative to the expression of PVR or Nectin-2 molecules ectopically expressed in CHO cells (Fig. 4 a). We observed that 1.5 μM DNAM-1–Fc strongly binds to PVR-expressing cells, but binds less efficiently to Nectin-2–expressing CHO cells, whereas it does not bind to control CHO cells. These data suggest that, under these conditions, DNAM-1 preferentially binds to PVR. Of note, DNAM-1–Fc binding was never detected on other Nectin-expressing CHO cells (i.e., Nectin-1, -3, and -4; unpublished data). Even though these data strongly suggest a direct interaction, binding was analyzed by means of recombinant molecules. Both PVR-Fc and Nec2-Fc strongly bind to coated DNAM-Fc and not to the irrelevant control (Fig. 4 b). Of note, both PVR-Fc and Nec2-Fc also bind to coated Nec3-Fc as described previously (Fig. 4 b and reference 7). Interestingly, PVR-Fc binding to DNAM-Fc is stronger than Nec2-Fc, which confirms our FACS® analysis. Altogether, these data highlight for the first time the direct DNAM-1–PVR and DNAM-1–Nectin-2 interactions.


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 and Nectin-2 directly bind to DNAM-1. (a) CHO/K cells either nontransfected or transfected with PVR or Nectin-2 cDNAs were analyzed by FACS® analysis with anti-PVR (L95) or anti–Nectin-2 (L14) mAbs, followed by a PE-conjugated goat anti–mouse second reagents (left). CHO/K: gray line, isotype matched mAb; black line, L95 mAb; and dashed line, L14 mAbs. CHO/K transfectants: gray line, isotype matched mAb and black histogram, L95 and L14 mAbs. Binding of DNAM-1–Fc at 1.5 μM on CHO/K cells was revealed by PE-conjugated goat anti–human second reagents (right): (gray line) incubation with the second reagent.; (black histogram) incubation with 1.5 μM DNAM-1–Fc. (b, left) PVR and Nectin-2 directly bind to Nectin-3. (right) To analyze PVR and Nectin-2 interactions with DNAM-1, biotinylated PVR-Fc and Nectin-2–Fc binding was measured by ELISA on wells coated with BT3-Fc (unrelated protein), Nectin-3–Fc (positive control), or DNAM-1–Fc. PVR-Fc and Nectin-2–Fc directly bind to DNAM-1–Fc and as expected with Nectin-3–Fc.
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Related In: Results  -  Collection

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fig4: PVR and Nectin-2 directly bind to DNAM-1. (a) CHO/K cells either nontransfected or transfected with PVR or Nectin-2 cDNAs were analyzed by FACS® analysis with anti-PVR (L95) or anti–Nectin-2 (L14) mAbs, followed by a PE-conjugated goat anti–mouse second reagents (left). CHO/K: gray line, isotype matched mAb; black line, L95 mAb; and dashed line, L14 mAbs. CHO/K transfectants: gray line, isotype matched mAb and black histogram, L95 and L14 mAbs. Binding of DNAM-1–Fc at 1.5 μM on CHO/K cells was revealed by PE-conjugated goat anti–human second reagents (right): (gray line) incubation with the second reagent.; (black histogram) incubation with 1.5 μM DNAM-1–Fc. (b, left) PVR and Nectin-2 directly bind to Nectin-3. (right) To analyze PVR and Nectin-2 interactions with DNAM-1, biotinylated PVR-Fc and Nectin-2–Fc binding was measured by ELISA on wells coated with BT3-Fc (unrelated protein), Nectin-3–Fc (positive control), or DNAM-1–Fc. PVR-Fc and Nectin-2–Fc directly bind to DNAM-1–Fc and as expected with Nectin-3–Fc.
Mentions: We analyzed the binding of DNAM-1–Fc relative to the expression of PVR or Nectin-2 molecules ectopically expressed in CHO cells (Fig. 4 a). We observed that 1.5 μM DNAM-1–Fc strongly binds to PVR-expressing cells, but binds less efficiently to Nectin-2–expressing CHO cells, whereas it does not bind to control CHO cells. These data suggest that, under these conditions, DNAM-1 preferentially binds to PVR. Of note, DNAM-1–Fc binding was never detected on other Nectin-expressing CHO cells (i.e., Nectin-1, -3, and -4; unpublished data). Even though these data strongly suggest a direct interaction, binding was analyzed by means of recombinant molecules. Both PVR-Fc and Nec2-Fc strongly bind to coated DNAM-Fc and not to the irrelevant control (Fig. 4 b). Of note, both PVR-Fc and Nec2-Fc also bind to coated Nec3-Fc as described previously (Fig. 4 b and reference 7). Interestingly, PVR-Fc binding to DNAM-Fc is stronger than Nec2-Fc, which confirms our FACS® analysis. Altogether, these data highlight for the first time the direct DNAM-1–PVR and DNAM-1–Nectin-2 interactions.

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