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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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GR113 stains HECA 452 bright human lymphocytes. Human lymphocytes were treated with supernatant fluid from thrombin-activated bovine platelets for 1.5 h, as described in Materials and Methods,  washed, and then analyzed by two-color flow cytometry using the  GR113 and HECA 452 mAb. A and B show the staining of the platelet  supernatant fluid treated cells in the absence or in the presence of the  blocking GR antibody, respectively. The quadrants reflect the upper level  of staining with a negative control antibody. These flow cytometric plots  are representative of at least three different experiments showing the same  result.
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Figure 8: GR113 stains HECA 452 bright human lymphocytes. Human lymphocytes were treated with supernatant fluid from thrombin-activated bovine platelets for 1.5 h, as described in Materials and Methods, washed, and then analyzed by two-color flow cytometry using the GR113 and HECA 452 mAb. A and B show the staining of the platelet supernatant fluid treated cells in the absence or in the presence of the blocking GR antibody, respectively. The quadrants reflect the upper level of staining with a negative control antibody. These flow cytometric plots are representative of at least three different experiments showing the same result.

Mentions: The GR113 antibody did not crossreact with human cells (data not shown). To determine if human leukocytes express receptors for the GR antigen, the binding of soluble bovine GR antigen to human mononuclear cells was tested. GR antigen containing supernatant fluid from bovine platelets was used to treat human mononuclear cells. The cells were washed and then analyzed by two-color analysis using GR113 and HECA 452 to stain the E-selectin binding lymphocyte population (CLA) (27), as a comparison. These analyses showed that most HECA 452 bright cells bound the GR antigen (GR antigen also bound some HECA 452-negative cells, Fig. 8). Importantly, the binding of the soluble GR antigen to human lymphocytes was completely blocked by pretreatment of the platelet supernatant fluid with GR113 (Fig. 8). As with bovine cells, neuraminidase treatment had no effect on GR antigen binding, though HECA 452 reactivity was greatly reduced (data not shown). Human monocytes and neutrophils also bound the GR antigen and rolled on 24 h activated bovine endothelial cells, which was blocked by the GR113 mAb (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)

GR113 stains HECA 452 bright human lymphocytes. Human lymphocytes were treated with supernatant fluid from thrombin-activated bovine platelets for 1.5 h, as described in Materials and Methods,  washed, and then analyzed by two-color flow cytometry using the  GR113 and HECA 452 mAb. A and B show the staining of the platelet  supernatant fluid treated cells in the absence or in the presence of the  blocking GR antibody, respectively. The quadrants reflect the upper level  of staining with a negative control antibody. These flow cytometric plots  are representative of at least three different experiments showing the same  result.
© Copyright Policy
Related In: Results  -  Collection

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

Figure 8: GR113 stains HECA 452 bright human lymphocytes. Human lymphocytes were treated with supernatant fluid from thrombin-activated bovine platelets for 1.5 h, as described in Materials and Methods, washed, and then analyzed by two-color flow cytometry using the GR113 and HECA 452 mAb. A and B show the staining of the platelet supernatant fluid treated cells in the absence or in the presence of the blocking GR antibody, respectively. The quadrants reflect the upper level of staining with a negative control antibody. These flow cytometric plots are representative of at least three different experiments showing the same result.
Mentions: The GR113 antibody did not crossreact with human cells (data not shown). To determine if human leukocytes express receptors for the GR antigen, the binding of soluble bovine GR antigen to human mononuclear cells was tested. GR antigen containing supernatant fluid from bovine platelets was used to treat human mononuclear cells. The cells were washed and then analyzed by two-color analysis using GR113 and HECA 452 to stain the E-selectin binding lymphocyte population (CLA) (27), as a comparison. These analyses showed that most HECA 452 bright cells bound the GR antigen (GR antigen also bound some HECA 452-negative cells, Fig. 8). Importantly, the binding of the soluble GR antigen to human lymphocytes was completely blocked by pretreatment of the platelet supernatant fluid with GR113 (Fig. 8). As with bovine cells, neuraminidase treatment had no effect on GR antigen binding, though HECA 452 reactivity was greatly reduced (data not shown). Human monocytes and neutrophils also bound the GR antigen and rolled on 24 h activated bovine endothelial cells, which was blocked by the GR113 mAb (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.

Show MeSH
Related in: MedlinePlus