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Vascular adhesion protein 1 (VAP-1) mediates lymphocyte subtype-specific, selectin-independent recognition of vascular endothelium in human lymph nodes.

Salmi M, Tohka S, Berg EL, Butcher EC, Jalkanen S - J. Exp. Med. (1997)

Bottom Line: Distinct subsets of mononuclear cells bind to high endothelial venules (HEVs) in different lymphoid organs to a different extent, but the molecular mechanisms behind this selectivity have remained poorly characterized.Finally, intravital microscopy revealed that VAP-1 is involved in initial interactions between human lymphocytes and endothelial cells in inflamed rabbit mesenterial venules in vivo.In conclusion, VAP-1 is a novel contact-initiating ligand that discriminates between different subpopulations of mononuclear cells and is an appealing target for selective modulation of adhesion of CD8- and CD16-positive effector cells.

View Article: PubMed Central - PubMed

Affiliation: National Public Health Institute, and MediCity Research Laboratory, Turku University, 20520 Turku, Finland. marko.salmi@utu.fi

ABSTRACT
Interactions between lymphocyte surface receptors and their ligands on vascular endothelial cells regulate the exit of lymphocytes from the circulation. Distinct subsets of mononuclear cells bind to high endothelial venules (HEVs) in different lymphoid organs to a different extent, but the molecular mechanisms behind this selectivity have remained poorly characterized. Here we show that vascular adhesion protein-1 (VAP-1) mediates subtype-specific binding of CD8-positive T cells and natural killer cells to human endothelium. VAP-1-dependent, oligosaccharide-dependent peripheral lymph node (PLN) HEV adhesion under shear was independent of L-selectin, P-selectin glycoprotein ligand 1, and alpha4 integrins, the known lymphocyte receptors involved in the initial recognition of endothelial cells. PLN HEV adhesion was also critically dependent on peripheral lymph node vascular addressins (PNAds), but lymphocyte L-selectin was absolutely required for PNAd binding. Most lymphocytes relied on both PNAd and VAP-1 in HEV binding. The overlapping function of L-selectin ligands and VAP-1 in PLN introduces a new control point into the lymphocyte extravasation process. Finally, intravital microscopy revealed that VAP-1 is involved in initial interactions between human lymphocytes and endothelial cells in inflamed rabbit mesenterial venules in vivo. In conclusion, VAP-1 is a novel contact-initiating ligand that discriminates between different subpopulations of mononuclear cells and is an appealing target for selective modulation of adhesion of CD8- and CD16-positive effector cells.

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VAP-1 mediates initial interactions between human lymphocytes and inflamed vascular endothelium in vivo. (a) An anti–human  VAP-1 mAb 5B11 recognizes VAP-1 in HEVs in frozen sections of rabbit  mesenterial lymph node. (b) Negative control staining with mAb 4D7.  An HEV is pointed out by an arrow. (c) Rabbit VAP-1 is expressed on  the luminal surface of inflamed mesenterial vessel. After intravenous injection of mAb 5B11, the in vivo–bound mAb was immunohistochemically detected in the mesenterial section with a second-stage peroxidase-conjugated anti–mouse Ig. (d–o) Intravital microscopy was used to study  the effect of a control mAb 4D7 and an anti–VAP-1 mAb 5B11 on binding of fluorescently labeled human tonsillar lymphocytes with inflamed  mesenterial vessels in rabbits as detailed in Materials and Methods. (d) A  micrograph of one segment of the vessel under study. L, lumen of the  vessel; T, connective tissue of the mesenterium; white triangles, the walls of  the vessel; white arrows, rolling rabbit granulocytes. (e–i) A tethering lymphocyte. The same segment as in d viewed under stroboscopic epiillumination after injecting fluorescently labeled human cells. A cell (1) docks to the vessel wall in e, moves less than 5 μm/s during the first 800 ms (e–f), speeds  up in g, and detaches in h–i. A freely flowing cell (2; velocity 1,110 μm/s) is also seen. Arrows mark a reference point at the vessel wall. (j–o) A rolling  lymphocyte. In another segment of a venule, a lymphocyte (3) that rolls along the bottom of the vessel for >2.5 s is seen (average velocity ∼24 μm/s).  Blood flow (open arrowhead) is from left to right in d–i and from top to bottom in j–o. White lines, vessel walls. Time codes in the upper right corners indicate the time elapsed (in ms) from the first frame of the series. Bar, 10 μm. (p) Anti–VAP-1 mAb 5B11 reduces interactions between human lymphocytes and rabbit vessel wall when analyzed by intravital microscopy in three independent experiments. Number of control interactions was arbitrarily set  at 100%.
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Figure 8: VAP-1 mediates initial interactions between human lymphocytes and inflamed vascular endothelium in vivo. (a) An anti–human VAP-1 mAb 5B11 recognizes VAP-1 in HEVs in frozen sections of rabbit mesenterial lymph node. (b) Negative control staining with mAb 4D7. An HEV is pointed out by an arrow. (c) Rabbit VAP-1 is expressed on the luminal surface of inflamed mesenterial vessel. After intravenous injection of mAb 5B11, the in vivo–bound mAb was immunohistochemically detected in the mesenterial section with a second-stage peroxidase-conjugated anti–mouse Ig. (d–o) Intravital microscopy was used to study the effect of a control mAb 4D7 and an anti–VAP-1 mAb 5B11 on binding of fluorescently labeled human tonsillar lymphocytes with inflamed mesenterial vessels in rabbits as detailed in Materials and Methods. (d) A micrograph of one segment of the vessel under study. L, lumen of the vessel; T, connective tissue of the mesenterium; white triangles, the walls of the vessel; white arrows, rolling rabbit granulocytes. (e–i) A tethering lymphocyte. The same segment as in d viewed under stroboscopic epiillumination after injecting fluorescently labeled human cells. A cell (1) docks to the vessel wall in e, moves less than 5 μm/s during the first 800 ms (e–f), speeds up in g, and detaches in h–i. A freely flowing cell (2; velocity 1,110 μm/s) is also seen. Arrows mark a reference point at the vessel wall. (j–o) A rolling lymphocyte. In another segment of a venule, a lymphocyte (3) that rolls along the bottom of the vessel for >2.5 s is seen (average velocity ∼24 μm/s). Blood flow (open arrowhead) is from left to right in d–i and from top to bottom in j–o. White lines, vessel walls. Time codes in the upper right corners indicate the time elapsed (in ms) from the first frame of the series. Bar, 10 μm. (p) Anti–VAP-1 mAb 5B11 reduces interactions between human lymphocytes and rabbit vessel wall when analyzed by intravital microscopy in three independent experiments. Number of control interactions was arbitrarily set at 100%.

Mentions: To confirm that VAP-1 could mediate initial interactions between PBL and endothelial lining under physiologically relevant conditions, we used intravital microscopy. Since mAb 1B2 does not cross-react with laboratory rodents, we screened a new panel of anti–human VAP-1 mAbs against different animal species and found that one mAb, 5B11, cross-reacts with rabbit. mAb 5B11 is specific against VAP-1 since it stains the same structures in human tissues as the prototype mAb 1B2, it reacts in immunoblotting with a 170-kD molecule that is absent from a lysate after preclearing with 1B2-coupled beads, and it reacts with VAP-1 transfectants (data not shown). mAb 5B11 inhibits human PBL binding to human tonsil HEVs in Stamper-Woodruff assay at least as efficiently as 1B2. Importantly, mAb 5B11 recognizes in rabbits an antigen with similar tissue and cell distribution to human VAP-1 (Fig. 8 a), and mAb 5B11 abrogated 75% of binding of rabbit mesenterial lymph node lymphocytes to rabbit mesenterial lymph node HEVs indicating conserved function of VAP-1 between humans and rabbits. This mAb also detects rabbit VAP-1 on the surface of inflamed mesenterial venules 4 h after IL-1–induced peritonitis (Fig. 8 c).


Vascular adhesion protein 1 (VAP-1) mediates lymphocyte subtype-specific, selectin-independent recognition of vascular endothelium in human lymph nodes.

Salmi M, Tohka S, Berg EL, Butcher EC, Jalkanen S - J. Exp. Med. (1997)

VAP-1 mediates initial interactions between human lymphocytes and inflamed vascular endothelium in vivo. (a) An anti–human  VAP-1 mAb 5B11 recognizes VAP-1 in HEVs in frozen sections of rabbit  mesenterial lymph node. (b) Negative control staining with mAb 4D7.  An HEV is pointed out by an arrow. (c) Rabbit VAP-1 is expressed on  the luminal surface of inflamed mesenterial vessel. After intravenous injection of mAb 5B11, the in vivo–bound mAb was immunohistochemically detected in the mesenterial section with a second-stage peroxidase-conjugated anti–mouse Ig. (d–o) Intravital microscopy was used to study  the effect of a control mAb 4D7 and an anti–VAP-1 mAb 5B11 on binding of fluorescently labeled human tonsillar lymphocytes with inflamed  mesenterial vessels in rabbits as detailed in Materials and Methods. (d) A  micrograph of one segment of the vessel under study. L, lumen of the  vessel; T, connective tissue of the mesenterium; white triangles, the walls of  the vessel; white arrows, rolling rabbit granulocytes. (e–i) A tethering lymphocyte. The same segment as in d viewed under stroboscopic epiillumination after injecting fluorescently labeled human cells. A cell (1) docks to the vessel wall in e, moves less than 5 μm/s during the first 800 ms (e–f), speeds  up in g, and detaches in h–i. A freely flowing cell (2; velocity 1,110 μm/s) is also seen. Arrows mark a reference point at the vessel wall. (j–o) A rolling  lymphocyte. In another segment of a venule, a lymphocyte (3) that rolls along the bottom of the vessel for >2.5 s is seen (average velocity ∼24 μm/s).  Blood flow (open arrowhead) is from left to right in d–i and from top to bottom in j–o. White lines, vessel walls. Time codes in the upper right corners indicate the time elapsed (in ms) from the first frame of the series. Bar, 10 μm. (p) Anti–VAP-1 mAb 5B11 reduces interactions between human lymphocytes and rabbit vessel wall when analyzed by intravital microscopy in three independent experiments. Number of control interactions was arbitrarily set  at 100%.
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Figure 8: VAP-1 mediates initial interactions between human lymphocytes and inflamed vascular endothelium in vivo. (a) An anti–human VAP-1 mAb 5B11 recognizes VAP-1 in HEVs in frozen sections of rabbit mesenterial lymph node. (b) Negative control staining with mAb 4D7. An HEV is pointed out by an arrow. (c) Rabbit VAP-1 is expressed on the luminal surface of inflamed mesenterial vessel. After intravenous injection of mAb 5B11, the in vivo–bound mAb was immunohistochemically detected in the mesenterial section with a second-stage peroxidase-conjugated anti–mouse Ig. (d–o) Intravital microscopy was used to study the effect of a control mAb 4D7 and an anti–VAP-1 mAb 5B11 on binding of fluorescently labeled human tonsillar lymphocytes with inflamed mesenterial vessels in rabbits as detailed in Materials and Methods. (d) A micrograph of one segment of the vessel under study. L, lumen of the vessel; T, connective tissue of the mesenterium; white triangles, the walls of the vessel; white arrows, rolling rabbit granulocytes. (e–i) A tethering lymphocyte. The same segment as in d viewed under stroboscopic epiillumination after injecting fluorescently labeled human cells. A cell (1) docks to the vessel wall in e, moves less than 5 μm/s during the first 800 ms (e–f), speeds up in g, and detaches in h–i. A freely flowing cell (2; velocity 1,110 μm/s) is also seen. Arrows mark a reference point at the vessel wall. (j–o) A rolling lymphocyte. In another segment of a venule, a lymphocyte (3) that rolls along the bottom of the vessel for >2.5 s is seen (average velocity ∼24 μm/s). Blood flow (open arrowhead) is from left to right in d–i and from top to bottom in j–o. White lines, vessel walls. Time codes in the upper right corners indicate the time elapsed (in ms) from the first frame of the series. Bar, 10 μm. (p) Anti–VAP-1 mAb 5B11 reduces interactions between human lymphocytes and rabbit vessel wall when analyzed by intravital microscopy in three independent experiments. Number of control interactions was arbitrarily set at 100%.
Mentions: To confirm that VAP-1 could mediate initial interactions between PBL and endothelial lining under physiologically relevant conditions, we used intravital microscopy. Since mAb 1B2 does not cross-react with laboratory rodents, we screened a new panel of anti–human VAP-1 mAbs against different animal species and found that one mAb, 5B11, cross-reacts with rabbit. mAb 5B11 is specific against VAP-1 since it stains the same structures in human tissues as the prototype mAb 1B2, it reacts in immunoblotting with a 170-kD molecule that is absent from a lysate after preclearing with 1B2-coupled beads, and it reacts with VAP-1 transfectants (data not shown). mAb 5B11 inhibits human PBL binding to human tonsil HEVs in Stamper-Woodruff assay at least as efficiently as 1B2. Importantly, mAb 5B11 recognizes in rabbits an antigen with similar tissue and cell distribution to human VAP-1 (Fig. 8 a), and mAb 5B11 abrogated 75% of binding of rabbit mesenterial lymph node lymphocytes to rabbit mesenterial lymph node HEVs indicating conserved function of VAP-1 between humans and rabbits. This mAb also detects rabbit VAP-1 on the surface of inflamed mesenterial venules 4 h after IL-1–induced peritonitis (Fig. 8 c).

Bottom Line: Distinct subsets of mononuclear cells bind to high endothelial venules (HEVs) in different lymphoid organs to a different extent, but the molecular mechanisms behind this selectivity have remained poorly characterized.Finally, intravital microscopy revealed that VAP-1 is involved in initial interactions between human lymphocytes and endothelial cells in inflamed rabbit mesenterial venules in vivo.In conclusion, VAP-1 is a novel contact-initiating ligand that discriminates between different subpopulations of mononuclear cells and is an appealing target for selective modulation of adhesion of CD8- and CD16-positive effector cells.

View Article: PubMed Central - PubMed

Affiliation: National Public Health Institute, and MediCity Research Laboratory, Turku University, 20520 Turku, Finland. marko.salmi@utu.fi

ABSTRACT
Interactions between lymphocyte surface receptors and their ligands on vascular endothelial cells regulate the exit of lymphocytes from the circulation. Distinct subsets of mononuclear cells bind to high endothelial venules (HEVs) in different lymphoid organs to a different extent, but the molecular mechanisms behind this selectivity have remained poorly characterized. Here we show that vascular adhesion protein-1 (VAP-1) mediates subtype-specific binding of CD8-positive T cells and natural killer cells to human endothelium. VAP-1-dependent, oligosaccharide-dependent peripheral lymph node (PLN) HEV adhesion under shear was independent of L-selectin, P-selectin glycoprotein ligand 1, and alpha4 integrins, the known lymphocyte receptors involved in the initial recognition of endothelial cells. PLN HEV adhesion was also critically dependent on peripheral lymph node vascular addressins (PNAds), but lymphocyte L-selectin was absolutely required for PNAd binding. Most lymphocytes relied on both PNAd and VAP-1 in HEV binding. The overlapping function of L-selectin ligands and VAP-1 in PLN introduces a new control point into the lymphocyte extravasation process. Finally, intravital microscopy revealed that VAP-1 is involved in initial interactions between human lymphocytes and endothelial cells in inflamed rabbit mesenterial venules in vivo. In conclusion, VAP-1 is a novel contact-initiating ligand that discriminates between different subpopulations of mononuclear cells and is an appealing target for selective modulation of adhesion of CD8- and CD16-positive effector cells.

Show MeSH
Related in: MedlinePlus