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Characterization of an adhesion molecule that mediates leukocyte rolling on 24 h cytokine- or lipopolysaccharide-stimulated bovine endothelial cells under flow conditions.

Jutila MA, Wilson E, Kurk S - J. Exp. Med. (1997)

Bottom Line: An antibody directed against E- and L-selectin has minimal blocking effect on this rolling interaction. mAbs were raised against the stimulated bovine endothelial cells and screened for inhibition of gamma/delta T cell rolling.Cutaneous lymphocyte-associated antigen (CLA) bright human lymphocytes exhibited the greatest capacity to bind the GR antigen, though CLA was not required for binding.Immunoprecipitation experiments showed the GR antigen to be 110-120 kD Mr. The binding of soluble GR antigen was inhibited by EDTA and O-sialoglycoprotease, but not neuraminidase treatment of the target cells.

View Article: PubMed Central - PubMed

Affiliation: Veterinary Molecular Biology, Montana State University, Bozeman 59717, USA. uvsmj@gemini.oscs.montana.edu

ABSTRACT
Bovine gamma/delta T cells and neutrophils roll on 24 h cytokine- or lipopolysaccharide-stimulated bovine fetal umbilical cord endothelial cells in assays done under physiological flow. An antibody directed against E- and L-selectin has minimal blocking effect on this rolling interaction. mAbs were raised against the stimulated bovine endothelial cells and screened for inhibition of gamma/delta T cell rolling. One mAb (GR113) was identified that recognizes an antigen (GR antigen) selectively expressed by stimulated bovine endothelial cells isolated from fetal umbilical cord, mesenteric lymph nodes, and aorta. GR113 blocked bovine gamma/delta T cell as well as neutrophil rolling on the 24 h-activated endothelial cells. The GR antigen was constitutively expressed at low levels on the cell surface of platelets and its expression was not upregulated after stimulation of these cells with thrombin or phorbol myristate acetate. However, stimulated platelets released a soluble, functionally active form of the molecule that selectively bound in solution to gamma/delta T cells in a mixed lymphocyte preparation. GR113 mAb blocked the binding of the soluble platelet molecule to the gamma/delta T cells. Soluble GR antigen also bound a subset of human lymphocytes. Cutaneous lymphocyte-associated antigen (CLA) bright human lymphocytes exhibited the greatest capacity to bind the GR antigen, though CLA was not required for binding. Subsets of both human CD4 and CD8 T cells bound the GR antigen. Immunoprecipitation experiments showed the GR antigen to be 110-120 kD Mr. The binding of soluble GR antigen was inhibited by EDTA and O-sialoglycoprotease, but not neuraminidase treatment of the target cells.

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Effect of different inhibitors on GR staining of γ/δ T cells  treated with platelet supernatant fluid. Bovine lymphocytes were treated  with neuraminidase (0.1 μg/ml for 30 min at room temperature) or  O-sialoglycoprotease (50 μg/ml for 45 min at room temperature) before  the addition of supernatant fluid from thrombin-activated platelets, or  EDTA (2 mM), GR113 antibody, or negative control antibody was  added to the platelet supernatant fluid before addition to the bovine lymphocytes. After the treatments, the lymphocytes were washed, stained  with GR antibody, and flow cytometric analysis performed. The data are  presented as histograms. Expression is shown in arbitrary fluorescence  units on a log scale. A and B show the effects of an antibody negative control  and GR113, respectively. C and D show the effects of the buffer control and EDTA, respectively. E and F show the effects of the buffer  control and neuraminidase, respectively. G and H show the effects of the  buffer control and O-sialoglycoprotease, respectively. These experiments  are representative of at least two others showing the same result.
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Figure 7: Effect of different inhibitors on GR staining of γ/δ T cells treated with platelet supernatant fluid. Bovine lymphocytes were treated with neuraminidase (0.1 μg/ml for 30 min at room temperature) or O-sialoglycoprotease (50 μg/ml for 45 min at room temperature) before the addition of supernatant fluid from thrombin-activated platelets, or EDTA (2 mM), GR113 antibody, or negative control antibody was added to the platelet supernatant fluid before addition to the bovine lymphocytes. After the treatments, the lymphocytes were washed, stained with GR antibody, and flow cytometric analysis performed. The data are presented as histograms. Expression is shown in arbitrary fluorescence units on a log scale. A and B show the effects of an antibody negative control and GR113, respectively. C and D show the effects of the buffer control and EDTA, respectively. E and F show the effects of the buffer control and neuraminidase, respectively. G and H show the effects of the buffer control and O-sialoglycoprotease, respectively. These experiments are representative of at least two others showing the same result.

Mentions: To determine the specificity of the GR antigen binding to γ/δ T cells in suspension, the platelet supernatant fluid was pretreated with the GR113 antibody before the binding assay. As shown in Fig. 7 (A and B), GR113 pretreatment prevented binding of the GR antigen to the γ/δ T cells. Importantly, antibodies directed against L-selectin or other γ/δ T cell surface antigens had no effect on the binding of the GR antigen (Fig. 7 and data not shown). Since the blocking GR113 mAb could detect bound antigen on the surface of the γ/δ T cells, this suggested that the soluble molecule was likely aggregated, creating multiple binding sites, some of which were unoccupied when bound to the leukocyte. Binding of soluble GR antigen to leukocytes was inhibited by EDTA (2 mM) and O-sialoglycoprotease (50 μg/ml), but not neuraminidase (0.1 U/ml) treatment of the γ/δ T cells (Fig. 7). EDTA and O-sialoglycoprotease treatment of γ/δ T cells also blocked their capacity to roll on the 24 h stimulated endothelial cells (data not shown).


Characterization of an adhesion molecule that mediates leukocyte rolling on 24 h cytokine- or lipopolysaccharide-stimulated bovine endothelial cells under flow conditions.

Jutila MA, Wilson E, Kurk S - J. Exp. Med. (1997)

Effect of different inhibitors on GR staining of γ/δ T cells  treated with platelet supernatant fluid. Bovine lymphocytes were treated  with neuraminidase (0.1 μg/ml for 30 min at room temperature) or  O-sialoglycoprotease (50 μg/ml for 45 min at room temperature) before  the addition of supernatant fluid from thrombin-activated platelets, or  EDTA (2 mM), GR113 antibody, or negative control antibody was  added to the platelet supernatant fluid before addition to the bovine lymphocytes. After the treatments, the lymphocytes were washed, stained  with GR antibody, and flow cytometric analysis performed. The data are  presented as histograms. Expression is shown in arbitrary fluorescence  units on a log scale. A and B show the effects of an antibody negative control  and GR113, respectively. C and D show the effects of the buffer control and EDTA, respectively. E and F show the effects of the buffer  control and neuraminidase, respectively. G and H show the effects of the  buffer control and O-sialoglycoprotease, respectively. These experiments  are representative of at least two others showing the same result.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2199140&req=5

Figure 7: Effect of different inhibitors on GR staining of γ/δ T cells treated with platelet supernatant fluid. Bovine lymphocytes were treated with neuraminidase (0.1 μg/ml for 30 min at room temperature) or O-sialoglycoprotease (50 μg/ml for 45 min at room temperature) before the addition of supernatant fluid from thrombin-activated platelets, or EDTA (2 mM), GR113 antibody, or negative control antibody was added to the platelet supernatant fluid before addition to the bovine lymphocytes. After the treatments, the lymphocytes were washed, stained with GR antibody, and flow cytometric analysis performed. The data are presented as histograms. Expression is shown in arbitrary fluorescence units on a log scale. A and B show the effects of an antibody negative control and GR113, respectively. C and D show the effects of the buffer control and EDTA, respectively. E and F show the effects of the buffer control and neuraminidase, respectively. G and H show the effects of the buffer control and O-sialoglycoprotease, respectively. These experiments are representative of at least two others showing the same result.
Mentions: To determine the specificity of the GR antigen binding to γ/δ T cells in suspension, the platelet supernatant fluid was pretreated with the GR113 antibody before the binding assay. As shown in Fig. 7 (A and B), GR113 pretreatment prevented binding of the GR antigen to the γ/δ T cells. Importantly, antibodies directed against L-selectin or other γ/δ T cell surface antigens had no effect on the binding of the GR antigen (Fig. 7 and data not shown). Since the blocking GR113 mAb could detect bound antigen on the surface of the γ/δ T cells, this suggested that the soluble molecule was likely aggregated, creating multiple binding sites, some of which were unoccupied when bound to the leukocyte. Binding of soluble GR antigen to leukocytes was inhibited by EDTA (2 mM) and O-sialoglycoprotease (50 μg/ml), but not neuraminidase (0.1 U/ml) treatment of the γ/δ T cells (Fig. 7). EDTA and O-sialoglycoprotease treatment of γ/δ T cells also blocked their capacity to roll on the 24 h stimulated endothelial cells (data not shown).

Bottom Line: An antibody directed against E- and L-selectin has minimal blocking effect on this rolling interaction. mAbs were raised against the stimulated bovine endothelial cells and screened for inhibition of gamma/delta T cell rolling.Cutaneous lymphocyte-associated antigen (CLA) bright human lymphocytes exhibited the greatest capacity to bind the GR antigen, though CLA was not required for binding.Immunoprecipitation experiments showed the GR antigen to be 110-120 kD Mr. The binding of soluble GR antigen was inhibited by EDTA and O-sialoglycoprotease, but not neuraminidase treatment of the target cells.

View Article: PubMed Central - PubMed

Affiliation: Veterinary Molecular Biology, Montana State University, Bozeman 59717, USA. uvsmj@gemini.oscs.montana.edu

ABSTRACT
Bovine gamma/delta T cells and neutrophils roll on 24 h cytokine- or lipopolysaccharide-stimulated bovine fetal umbilical cord endothelial cells in assays done under physiological flow. An antibody directed against E- and L-selectin has minimal blocking effect on this rolling interaction. mAbs were raised against the stimulated bovine endothelial cells and screened for inhibition of gamma/delta T cell rolling. One mAb (GR113) was identified that recognizes an antigen (GR antigen) selectively expressed by stimulated bovine endothelial cells isolated from fetal umbilical cord, mesenteric lymph nodes, and aorta. GR113 blocked bovine gamma/delta T cell as well as neutrophil rolling on the 24 h-activated endothelial cells. The GR antigen was constitutively expressed at low levels on the cell surface of platelets and its expression was not upregulated after stimulation of these cells with thrombin or phorbol myristate acetate. However, stimulated platelets released a soluble, functionally active form of the molecule that selectively bound in solution to gamma/delta T cells in a mixed lymphocyte preparation. GR113 mAb blocked the binding of the soluble platelet molecule to the gamma/delta T cells. Soluble GR antigen also bound a subset of human lymphocytes. Cutaneous lymphocyte-associated antigen (CLA) bright human lymphocytes exhibited the greatest capacity to bind the GR antigen, though CLA was not required for binding. Subsets of both human CD4 and CD8 T cells bound the GR antigen. Immunoprecipitation experiments showed the GR antigen to be 110-120 kD Mr. The binding of soluble GR antigen was inhibited by EDTA and O-sialoglycoprotease, but not neuraminidase treatment of the target cells.

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Related in: MedlinePlus