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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 blocks γ/δ T cell and neutrophil rolling on 24 h cytokine-activated endothelial cells. Endothelial cells were grown on the internal surface of glass capillary tubes, activated with IFN-γ/TNF-α, and  integrated into the flow assay, as described in Materials and Methods. In  A, γ/δ T cell rolling was established first and then an isotype-matched  negative control mAb (control), EL-246 (anti–L- and E-selectin), and  GR113 were sequentially added. In B, the endothelial cell monolayer was  either pretreated with GR113 or a control mAb for 30 min before the addition of the γ/δ T cells. In C, the rolling of neutrophils on endothelial  cells pretreated with GR113 was compared with antibody control. These  experiments are representative of >3 other experiments showing inhibition of leukocyte rolling on the 24 h cytokine- or LPS-activated endothelial cells.
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Figure 1: GR113 blocks γ/δ T cell and neutrophil rolling on 24 h cytokine-activated endothelial cells. Endothelial cells were grown on the internal surface of glass capillary tubes, activated with IFN-γ/TNF-α, and integrated into the flow assay, as described in Materials and Methods. In A, γ/δ T cell rolling was established first and then an isotype-matched negative control mAb (control), EL-246 (anti–L- and E-selectin), and GR113 were sequentially added. In B, the endothelial cell monolayer was either pretreated with GR113 or a control mAb for 30 min before the addition of the γ/δ T cells. In C, the rolling of neutrophils on endothelial cells pretreated with GR113 was compared with antibody control. These experiments are representative of >3 other experiments showing inhibition of leukocyte rolling on the 24 h cytokine- or LPS-activated endothelial cells.

Mentions: Endothelial cells stimulated for 24 h with IFN-γ followed by LPS or TNF-α for 4–6 h avidly supported γ/δ T cell rolling under flow conditions; however, the rolling interaction was not blocked by an antibody directed against L- and E-selectin (EL-246) (Fig. 1 A). Similar rolling interactions occurred on endothelial cells treated with LPS or TNF-α alone for 24 h, showing that the IFN-γ pretreatment was not prerequisite, but the consistency of the induction of the capacity support rolling was enhanced by IFN-γ (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 blocks γ/δ T cell and neutrophil rolling on 24 h cytokine-activated endothelial cells. Endothelial cells were grown on the internal surface of glass capillary tubes, activated with IFN-γ/TNF-α, and  integrated into the flow assay, as described in Materials and Methods. In  A, γ/δ T cell rolling was established first and then an isotype-matched  negative control mAb (control), EL-246 (anti–L- and E-selectin), and  GR113 were sequentially added. In B, the endothelial cell monolayer was  either pretreated with GR113 or a control mAb for 30 min before the addition of the γ/δ T cells. In C, the rolling of neutrophils on endothelial  cells pretreated with GR113 was compared with antibody control. These  experiments are representative of >3 other experiments showing inhibition of leukocyte rolling on the 24 h cytokine- or LPS-activated endothelial cells.
© Copyright Policy
Related In: Results  -  Collection

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

Figure 1: GR113 blocks γ/δ T cell and neutrophil rolling on 24 h cytokine-activated endothelial cells. Endothelial cells were grown on the internal surface of glass capillary tubes, activated with IFN-γ/TNF-α, and integrated into the flow assay, as described in Materials and Methods. In A, γ/δ T cell rolling was established first and then an isotype-matched negative control mAb (control), EL-246 (anti–L- and E-selectin), and GR113 were sequentially added. In B, the endothelial cell monolayer was either pretreated with GR113 or a control mAb for 30 min before the addition of the γ/δ T cells. In C, the rolling of neutrophils on endothelial cells pretreated with GR113 was compared with antibody control. These experiments are representative of >3 other experiments showing inhibition of leukocyte rolling on the 24 h cytokine- or LPS-activated endothelial cells.
Mentions: Endothelial cells stimulated for 24 h with IFN-γ followed by LPS or TNF-α for 4–6 h avidly supported γ/δ T cell rolling under flow conditions; however, the rolling interaction was not blocked by an antibody directed against L- and E-selectin (EL-246) (Fig. 1 A). Similar rolling interactions occurred on endothelial cells treated with LPS or TNF-α alone for 24 h, showing that the IFN-γ pretreatment was not prerequisite, but the consistency of the induction of the capacity support rolling was enhanced by IFN-γ (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