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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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Blocking PVR–DNAM-1 transinteractions arrest monocytes over intercellular junctions at the apical surface of endothelial cells. Staining was performed after a 1-h migration run on transwell filters. (a) Monocyte migration was run in the absence (control) or the presence of anti-PVR mAb (PV.404) at 20 μg/ml. Cells were fixed, stained with 10 μg/ml anti-CD146 (previously conjugated to the 564-nm–labeled IgG1 isotype-specific Zenon Fab) and 2 μg/ml anti-CD14 (FITC-conjugated) mAbs to homogeneously stain endothelial cells and visualize monocytes, respectively. A series of images was recorded simultaneously in the XY and XZ planes. The results are representative of three independent experiments. (left) Monocytes untreated (control) are ending their transmigration and can be seen through the filter. (right) Upon anti-PVR treatment, monocytes are blocked over the endothelial cell monolayer. (b) Migration was run in the presence of 20 μg/ml of anti-PVR mAb (PV.404). Cells were fixed and stained with 10 μg/ml anti–VE-cadherin (previously conjugated to the 564-nm–labeled IgG1 isotype-specific Zenon Fab) and 2 μg/ml anti-CD14 (FITC-conjugated) mAbs to visualize endothelial junctions and monocytes, respectively. (top) White arrows indicate the position of blocked monocytes over the endothelial cell junctions in the XY plane. (bottom) XZ plane cross-section view according to the dotted line arrow in the XY plane. White arrowheads indicate the junctional staining of VE-cadherin. Red arrow marks the protruding monocyte membrane close to junctional VE-cadherin. (c) Monocyte transmigration through the HUVEC monolayer was performed as in b. Anti–DNAM-1 mAb (FS123) was used at 20 μg/ml. XZ plane cross-section view of monocytes arrest over the endothelial cell junctions as described in panel a. The results are representative of three independent experiments.
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fig7: Blocking PVR–DNAM-1 transinteractions arrest monocytes over intercellular junctions at the apical surface of endothelial cells. Staining was performed after a 1-h migration run on transwell filters. (a) Monocyte migration was run in the absence (control) or the presence of anti-PVR mAb (PV.404) at 20 μg/ml. Cells were fixed, stained with 10 μg/ml anti-CD146 (previously conjugated to the 564-nm–labeled IgG1 isotype-specific Zenon Fab) and 2 μg/ml anti-CD14 (FITC-conjugated) mAbs to homogeneously stain endothelial cells and visualize monocytes, respectively. A series of images was recorded simultaneously in the XY and XZ planes. The results are representative of three independent experiments. (left) Monocytes untreated (control) are ending their transmigration and can be seen through the filter. (right) Upon anti-PVR treatment, monocytes are blocked over the endothelial cell monolayer. (b) Migration was run in the presence of 20 μg/ml of anti-PVR mAb (PV.404). Cells were fixed and stained with 10 μg/ml anti–VE-cadherin (previously conjugated to the 564-nm–labeled IgG1 isotype-specific Zenon Fab) and 2 μg/ml anti-CD14 (FITC-conjugated) mAbs to visualize endothelial junctions and monocytes, respectively. (top) White arrows indicate the position of blocked monocytes over the endothelial cell junctions in the XY plane. (bottom) XZ plane cross-section view according to the dotted line arrow in the XY plane. White arrowheads indicate the junctional staining of VE-cadherin. Red arrow marks the protruding monocyte membrane close to junctional VE-cadherin. (c) Monocyte transmigration through the HUVEC monolayer was performed as in b. Anti–DNAM-1 mAb (FS123) was used at 20 μg/ml. XZ plane cross-section view of monocytes arrest over the endothelial cell junctions as described in panel a. The results are representative of three independent experiments.

Mentions: Endothelial junctions have been described as key regulators of blood vessel permeability. Consistent with their distribution at endothelial cell junctions, PVR and Nectin-2 may thus participate in the events that regulate leukocyte extravasation through the endothelium. Having demonstrated that PVR is the major ligand of DNAM-1 on endothelial cells, we investigated the potential role of the PVR–DNAM-1 interaction during the TEM of monocytes. We controlled that anti-PVR mAbs readily access to the junctions of live HUVECs (not depicted), and we showed that anti-PVR mAbs do not modify the endothelial permeability (not depicted) and the endothelial integrity (see Fig. 7). Their ability to block monocyte transmigration in an in vitro TEM model was explored. Anti-PVR mAbs induced a significant blocking in monocyte transmigration when compared with isotype-matched irrelevant antibodies (Fig. 6 a, anti-CD34 and anti–Nectin-4 mAbs). Inhibition of monocyte transmigration was observed with five different anti-PVR mAbs (blockade between 60 and 80%) and was similar to that obtained with anti-CD99 mAb (Fig. 6 a, 12E7) and anti–LFA-1/CD11a (Fig. 6 b, 25.3.1), taken as positive controls (24, 25). Targeting of DNAM-1 with two different mAbs also strongly inhibits monocyte transmigration to 80%. Fab fragments of anti-PVR (mAbs) and anti–DNAM-1 (mAbs) efficiently inhibited TEM, as they blocked monocyte transmigration to the same extent as mAbs (Fig. 6 a). This rules out the possibility that inhibition may be due to Fc binding of mAbs to monocyte Fc receptors. The inhibition of monocyte TEM upon anti-PVR or anti–DNAM-1 mAb treatment is due to a blockade and not a slow down, as the same blockade was observed after a 3-h transmigration run. When incubating anti-PVR mAbs only on HUVECs, monocyte TEM was strongly blocked, which was not the case with anti–DNAM-1 mAb (Fig. 6 b). Because DNAM-1 is not or just faintly expressed on HUVECs, these results strongly suggest that DNAM-1 on monocytes is involved in this process. These results also suggest that anti-PVR mAbs block monocyte at the level of TEM and not by interfering with chemotaxis. To definitively demonstrate that mAbs did not inhibit chemotaxis, anti-PVR and anti–DNAM-1 Fabs were tested in a chemotaxis assay. No inhibition was noted when monocytes were incubated with both Fabs (Fig. 6 c). Altogether, our results demonstrate that endothelial PVR interacts with DNAM-1 on monocyte to ensure monocyte TEM.


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)

Blocking PVR–DNAM-1 transinteractions arrest monocytes over intercellular junctions at the apical surface of endothelial cells. Staining was performed after a 1-h migration run on transwell filters. (a) Monocyte migration was run in the absence (control) or the presence of anti-PVR mAb (PV.404) at 20 μg/ml. Cells were fixed, stained with 10 μg/ml anti-CD146 (previously conjugated to the 564-nm–labeled IgG1 isotype-specific Zenon Fab) and 2 μg/ml anti-CD14 (FITC-conjugated) mAbs to homogeneously stain endothelial cells and visualize monocytes, respectively. A series of images was recorded simultaneously in the XY and XZ planes. The results are representative of three independent experiments. (left) Monocytes untreated (control) are ending their transmigration and can be seen through the filter. (right) Upon anti-PVR treatment, monocytes are blocked over the endothelial cell monolayer. (b) Migration was run in the presence of 20 μg/ml of anti-PVR mAb (PV.404). Cells were fixed and stained with 10 μg/ml anti–VE-cadherin (previously conjugated to the 564-nm–labeled IgG1 isotype-specific Zenon Fab) and 2 μg/ml anti-CD14 (FITC-conjugated) mAbs to visualize endothelial junctions and monocytes, respectively. (top) White arrows indicate the position of blocked monocytes over the endothelial cell junctions in the XY plane. (bottom) XZ plane cross-section view according to the dotted line arrow in the XY plane. White arrowheads indicate the junctional staining of VE-cadherin. Red arrow marks the protruding monocyte membrane close to junctional VE-cadherin. (c) Monocyte transmigration through the HUVEC monolayer was performed as in b. Anti–DNAM-1 mAb (FS123) was used at 20 μg/ml. XZ plane cross-section view of monocytes arrest over the endothelial cell junctions as described in panel a. The results are representative of three independent experiments.
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fig7: Blocking PVR–DNAM-1 transinteractions arrest monocytes over intercellular junctions at the apical surface of endothelial cells. Staining was performed after a 1-h migration run on transwell filters. (a) Monocyte migration was run in the absence (control) or the presence of anti-PVR mAb (PV.404) at 20 μg/ml. Cells were fixed, stained with 10 μg/ml anti-CD146 (previously conjugated to the 564-nm–labeled IgG1 isotype-specific Zenon Fab) and 2 μg/ml anti-CD14 (FITC-conjugated) mAbs to homogeneously stain endothelial cells and visualize monocytes, respectively. A series of images was recorded simultaneously in the XY and XZ planes. The results are representative of three independent experiments. (left) Monocytes untreated (control) are ending their transmigration and can be seen through the filter. (right) Upon anti-PVR treatment, monocytes are blocked over the endothelial cell monolayer. (b) Migration was run in the presence of 20 μg/ml of anti-PVR mAb (PV.404). Cells were fixed and stained with 10 μg/ml anti–VE-cadherin (previously conjugated to the 564-nm–labeled IgG1 isotype-specific Zenon Fab) and 2 μg/ml anti-CD14 (FITC-conjugated) mAbs to visualize endothelial junctions and monocytes, respectively. (top) White arrows indicate the position of blocked monocytes over the endothelial cell junctions in the XY plane. (bottom) XZ plane cross-section view according to the dotted line arrow in the XY plane. White arrowheads indicate the junctional staining of VE-cadherin. Red arrow marks the protruding monocyte membrane close to junctional VE-cadherin. (c) Monocyte transmigration through the HUVEC monolayer was performed as in b. Anti–DNAM-1 mAb (FS123) was used at 20 μg/ml. XZ plane cross-section view of monocytes arrest over the endothelial cell junctions as described in panel a. The results are representative of three independent experiments.
Mentions: Endothelial junctions have been described as key regulators of blood vessel permeability. Consistent with their distribution at endothelial cell junctions, PVR and Nectin-2 may thus participate in the events that regulate leukocyte extravasation through the endothelium. Having demonstrated that PVR is the major ligand of DNAM-1 on endothelial cells, we investigated the potential role of the PVR–DNAM-1 interaction during the TEM of monocytes. We controlled that anti-PVR mAbs readily access to the junctions of live HUVECs (not depicted), and we showed that anti-PVR mAbs do not modify the endothelial permeability (not depicted) and the endothelial integrity (see Fig. 7). Their ability to block monocyte transmigration in an in vitro TEM model was explored. Anti-PVR mAbs induced a significant blocking in monocyte transmigration when compared with isotype-matched irrelevant antibodies (Fig. 6 a, anti-CD34 and anti–Nectin-4 mAbs). Inhibition of monocyte transmigration was observed with five different anti-PVR mAbs (blockade between 60 and 80%) and was similar to that obtained with anti-CD99 mAb (Fig. 6 a, 12E7) and anti–LFA-1/CD11a (Fig. 6 b, 25.3.1), taken as positive controls (24, 25). Targeting of DNAM-1 with two different mAbs also strongly inhibits monocyte transmigration to 80%. Fab fragments of anti-PVR (mAbs) and anti–DNAM-1 (mAbs) efficiently inhibited TEM, as they blocked monocyte transmigration to the same extent as mAbs (Fig. 6 a). This rules out the possibility that inhibition may be due to Fc binding of mAbs to monocyte Fc receptors. The inhibition of monocyte TEM upon anti-PVR or anti–DNAM-1 mAb treatment is due to a blockade and not a slow down, as the same blockade was observed after a 3-h transmigration run. When incubating anti-PVR mAbs only on HUVECs, monocyte TEM was strongly blocked, which was not the case with anti–DNAM-1 mAb (Fig. 6 b). Because DNAM-1 is not or just faintly expressed on HUVECs, these results strongly suggest that DNAM-1 on monocytes is involved in this process. These results also suggest that anti-PVR mAbs block monocyte at the level of TEM and not by interfering with chemotaxis. To definitively demonstrate that mAbs did not inhibit chemotaxis, anti-PVR and anti–DNAM-1 Fabs were tested in a chemotaxis assay. No inhibition was noted when monocytes were incubated with both Fabs (Fig. 6 c). Altogether, our results demonstrate that endothelial PVR interacts with DNAM-1 on monocyte to ensure monocyte TEM.

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