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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 6 h and 24 h LPS-stimulated endothelial cells  isolated from mesenteric lymph nodes, aorta, and umbilical cords. Endothelial cell cultures in 96 well plates were stimulated with LPS for 6 and 24 h  and then stained with GR113 (denoted GR antigen) or EL-246 (anti– E-selectin, denoted ES antigen), as described in Materials and Methods.  The data represent a single comparison and are the average of duplicate  measurements. OD values reflect OD of positive staining mAb minus OD  of negative control antibody staining. The staining of the umbilical cord  endothelial cells which were used in all subsequent assays was repeated in  >3 different experiments.
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Figure 2: GR113 stains 6 h and 24 h LPS-stimulated endothelial cells isolated from mesenteric lymph nodes, aorta, and umbilical cords. Endothelial cell cultures in 96 well plates were stimulated with LPS for 6 and 24 h and then stained with GR113 (denoted GR antigen) or EL-246 (anti– E-selectin, denoted ES antigen), as described in Materials and Methods. The data represent a single comparison and are the average of duplicate measurements. OD values reflect OD of positive staining mAb minus OD of negative control antibody staining. The staining of the umbilical cord endothelial cells which were used in all subsequent assays was repeated in >3 different experiments.

Mentions: mAbs were raised against the 24 h cytokine-stimulated endothelial cells, as described in Materials and Methods. One mAb, GR113, was identified that selectively stained endothelial cells treated for 24 h. Untreated umbilical cord endothelial cells expressed little if any GR antigen on their cell surface or in intracellular pools (data not shown, also see Fig. 2). In one experiment, the expression of the GR antigen was compared with E-selectin after 6 and 24 h LPS treatment of endothelial cells isolated from mesenteric lymph nodes, aorta, and fetal umbilical cords. As shown in Fig. 2, GR113 selectively stained LPS-stimulated endothelial cells isolated from all three sources. 6 h stimulation with LPS (or TNF-α, data not shown) alone led to increased staining of endothelial cells in all three cases and, for the most part, maximal or close to maximal expression was maintained after 24 h (Fig. 2). In contrast, induction of E-selectin expression was highly variable, with endothelial cells from umbilical cords expressing significant antigen before stimulation; whereas little E-selectin was expressed on endothelial cells from mesenteric lymph nodes after either 6 or 24 h incubation in the presence of LPS (Fig. 2). The reason for the variability in E-selectin expression or the consistency of this variability was not determined, but it could have been related to the proliferative state of the cells (33). Flow cytometric analysis showed that virtually all 24 h stimulated umbilical cord endothelial cells expressed the GR antigen, though there was heterogeneity in the level of expression by different cells in the culture (Fig. 3). Overall, we have shown that TNF-α, LPS, or a combination of IFN-γ plus LPS or TNF-α, but not IFN-γ alone, induced significant levels of the GR antigen on bovine endothelial cells (data not shown). Since much of our initial analyses were on umbilical cord endothelial cells pretreated with IFN-γ for 24 h followed by LPS or TNF-α treatment, this treatment regime was used in the functional analyses reported below.


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 6 h and 24 h LPS-stimulated endothelial cells  isolated from mesenteric lymph nodes, aorta, and umbilical cords. Endothelial cell cultures in 96 well plates were stimulated with LPS for 6 and 24 h  and then stained with GR113 (denoted GR antigen) or EL-246 (anti– E-selectin, denoted ES antigen), as described in Materials and Methods.  The data represent a single comparison and are the average of duplicate  measurements. OD values reflect OD of positive staining mAb minus OD  of negative control antibody staining. The staining of the umbilical cord  endothelial cells which were used in all subsequent assays was repeated in  >3 different experiments.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2199140&req=5

Figure 2: GR113 stains 6 h and 24 h LPS-stimulated endothelial cells isolated from mesenteric lymph nodes, aorta, and umbilical cords. Endothelial cell cultures in 96 well plates were stimulated with LPS for 6 and 24 h and then stained with GR113 (denoted GR antigen) or EL-246 (anti– E-selectin, denoted ES antigen), as described in Materials and Methods. The data represent a single comparison and are the average of duplicate measurements. OD values reflect OD of positive staining mAb minus OD of negative control antibody staining. The staining of the umbilical cord endothelial cells which were used in all subsequent assays was repeated in >3 different experiments.
Mentions: mAbs were raised against the 24 h cytokine-stimulated endothelial cells, as described in Materials and Methods. One mAb, GR113, was identified that selectively stained endothelial cells treated for 24 h. Untreated umbilical cord endothelial cells expressed little if any GR antigen on their cell surface or in intracellular pools (data not shown, also see Fig. 2). In one experiment, the expression of the GR antigen was compared with E-selectin after 6 and 24 h LPS treatment of endothelial cells isolated from mesenteric lymph nodes, aorta, and fetal umbilical cords. As shown in Fig. 2, GR113 selectively stained LPS-stimulated endothelial cells isolated from all three sources. 6 h stimulation with LPS (or TNF-α, data not shown) alone led to increased staining of endothelial cells in all three cases and, for the most part, maximal or close to maximal expression was maintained after 24 h (Fig. 2). In contrast, induction of E-selectin expression was highly variable, with endothelial cells from umbilical cords expressing significant antigen before stimulation; whereas little E-selectin was expressed on endothelial cells from mesenteric lymph nodes after either 6 or 24 h incubation in the presence of LPS (Fig. 2). The reason for the variability in E-selectin expression or the consistency of this variability was not determined, but it could have been related to the proliferative state of the cells (33). Flow cytometric analysis showed that virtually all 24 h stimulated umbilical cord endothelial cells expressed the GR antigen, though there was heterogeneity in the level of expression by different cells in the culture (Fig. 3). Overall, we have shown that TNF-α, LPS, or a combination of IFN-γ plus LPS or TNF-α, but not IFN-γ alone, induced significant levels of the GR antigen on bovine endothelial cells (data not shown). Since much of our initial analyses were on umbilical cord endothelial cells pretreated with IFN-γ for 24 h followed by LPS or TNF-α treatment, this treatment regime was used in the functional analyses reported below.

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