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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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Staining of GR113 on  platelets and its effect on leukocyte rolling on thrombin-activated platelets. Platelets, isolated as described in Materials and  Methods, were stained with the GR113  mAb and analyzed by flow cytometry or  they were used in rolling assays. A shows  GR113 staining of control and thrombin-activated (15 min) platelets versus  background staining (second stage alone).  B shows the effects of GR113 on the  rolling of bovine γ/δ T cells on immobilized, thrombin-activated platelets. Two  different experiments are shown in which  rolling interactions were established first,  and negative control (EL-246) and GR113  mAbs sequentially injected.
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Figure 5: Staining of GR113 on platelets and its effect on leukocyte rolling on thrombin-activated platelets. Platelets, isolated as described in Materials and Methods, were stained with the GR113 mAb and analyzed by flow cytometry or they were used in rolling assays. A shows GR113 staining of control and thrombin-activated (15 min) platelets versus background staining (second stage alone). B shows the effects of GR113 on the rolling of bovine γ/δ T cells on immobilized, thrombin-activated platelets. Two different experiments are shown in which rolling interactions were established first, and negative control (EL-246) and GR113 mAbs sequentially injected.

Mentions: Flow cytometric analysis was done to determine if GR113 reacted with any blood cells, particularly platelets. As shown in Fig. 5 A, the antibody weakly stained platelets by flow cytometric analysis. In some experiments, reactivity of GR113 on platelets was barely above background (data not shown). As shown in Fig. 5 A, thrombin activation did not lead to increased GR113 staining. The thrombin-activated platelets supported avid rolling of bovine γ/δ T cells in the capillary tube flow assay, suggesting that they did express P-selectin; however, GR113 did not block the interaction (Fig. 5 B), as seen with γ/δ T cell rolling on 24 h cytokine-stimulated endothelial cells (Fig. 1).


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)

Staining of GR113 on  platelets and its effect on leukocyte rolling on thrombin-activated platelets. Platelets, isolated as described in Materials and  Methods, were stained with the GR113  mAb and analyzed by flow cytometry or  they were used in rolling assays. A shows  GR113 staining of control and thrombin-activated (15 min) platelets versus  background staining (second stage alone).  B shows the effects of GR113 on the  rolling of bovine γ/δ T cells on immobilized, thrombin-activated platelets. Two  different experiments are shown in which  rolling interactions were established first,  and negative control (EL-246) and GR113  mAbs sequentially injected.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2199140&req=5

Figure 5: Staining of GR113 on platelets and its effect on leukocyte rolling on thrombin-activated platelets. Platelets, isolated as described in Materials and Methods, were stained with the GR113 mAb and analyzed by flow cytometry or they were used in rolling assays. A shows GR113 staining of control and thrombin-activated (15 min) platelets versus background staining (second stage alone). B shows the effects of GR113 on the rolling of bovine γ/δ T cells on immobilized, thrombin-activated platelets. Two different experiments are shown in which rolling interactions were established first, and negative control (EL-246) and GR113 mAbs sequentially injected.
Mentions: Flow cytometric analysis was done to determine if GR113 reacted with any blood cells, particularly platelets. As shown in Fig. 5 A, the antibody weakly stained platelets by flow cytometric analysis. In some experiments, reactivity of GR113 on platelets was barely above background (data not shown). As shown in Fig. 5 A, thrombin activation did not lead to increased GR113 staining. The thrombin-activated platelets supported avid rolling of bovine γ/δ T cells in the capillary tube flow assay, suggesting that they did express P-selectin; however, GR113 did not block the interaction (Fig. 5 B), as seen with γ/δ T cell rolling on 24 h cytokine-stimulated endothelial cells (Fig. 1).

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.

Show MeSH
Related in: MedlinePlus