Limits...
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.

Show MeSH

Related in: MedlinePlus

GR113 stains venules in a site of chronic inflammation. A  chronic inflammatory lesion was induced by potassium permanganate, as  described in Materials and Methods, which was characterized by an infiltration of mononuclear leukocytes with many of the inflammatory cells  being lymphocytes (data not shown). Frozen sections were stained with  GR113 by standard immunoperoxidase techniques. A shows a low power  (125× final magnification) field demonstrating many GR113 positive  vessels in the site of leukocyte infiltration (arrowheads). B shows a high  power field (250× final magnification) of the same tissue. The arrow head  points to a GR113 positive vessel containing leukocytes bound to its lumenal surface.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC2199140&req=5

Figure 4: GR113 stains venules in a site of chronic inflammation. A chronic inflammatory lesion was induced by potassium permanganate, as described in Materials and Methods, which was characterized by an infiltration of mononuclear leukocytes with many of the inflammatory cells being lymphocytes (data not shown). Frozen sections were stained with GR113 by standard immunoperoxidase techniques. A shows a low power (125× final magnification) field demonstrating many GR113 positive vessels in the site of leukocyte infiltration (arrowheads). B shows a high power field (250× final magnification) of the same tissue. The arrow head points to a GR113 positive vessel containing leukocytes bound to its lumenal surface.

Mentions: Since GR113 selectively stained cytokine-stimulated endothelial cells in vitro, the staining pattern in chronic inflammatory lesions in vivo was tested. As shown in Fig. 4 A, GR113 intensely stained vessels associated with leukocytes in a chronic inflammatory lesion induced by potassium permanganate. Upon closer examination of the GR113 positive vessels it was noted that not only did the antibody stain endothelial cells, scattered granular platelet-like particles (also see below) were also stained (Fig. 4 B). Note in Fig. 4 B that leukocytes were adhered to the lumenal surface of the large GR113 positive vessel. We detected variable, low level of staining of GR113 on vessels in uninflamed skin. 4 h LPS-inflamed skin exhibited positive vessels, but much of the staining was associated with platelet-like particles. In contrast, E-selectin was expressed on many vessels in the 4 h inflamed tissues, whereas minimal expression was seen in the chronic lesion (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 venules in a site of chronic inflammation. A  chronic inflammatory lesion was induced by potassium permanganate, as  described in Materials and Methods, which was characterized by an infiltration of mononuclear leukocytes with many of the inflammatory cells  being lymphocytes (data not shown). Frozen sections were stained with  GR113 by standard immunoperoxidase techniques. A shows a low power  (125× final magnification) field demonstrating many GR113 positive  vessels in the site of leukocyte infiltration (arrowheads). B shows a high  power field (250× final magnification) of the same tissue. The arrow head  points to a GR113 positive vessel containing leukocytes bound to its lumenal surface.
© Copyright Policy
Related In: Results  -  Collection

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

Figure 4: GR113 stains venules in a site of chronic inflammation. A chronic inflammatory lesion was induced by potassium permanganate, as described in Materials and Methods, which was characterized by an infiltration of mononuclear leukocytes with many of the inflammatory cells being lymphocytes (data not shown). Frozen sections were stained with GR113 by standard immunoperoxidase techniques. A shows a low power (125× final magnification) field demonstrating many GR113 positive vessels in the site of leukocyte infiltration (arrowheads). B shows a high power field (250× final magnification) of the same tissue. The arrow head points to a GR113 positive vessel containing leukocytes bound to its lumenal surface.
Mentions: Since GR113 selectively stained cytokine-stimulated endothelial cells in vitro, the staining pattern in chronic inflammatory lesions in vivo was tested. As shown in Fig. 4 A, GR113 intensely stained vessels associated with leukocytes in a chronic inflammatory lesion induced by potassium permanganate. Upon closer examination of the GR113 positive vessels it was noted that not only did the antibody stain endothelial cells, scattered granular platelet-like particles (also see below) were also stained (Fig. 4 B). Note in Fig. 4 B that leukocytes were adhered to the lumenal surface of the large GR113 positive vessel. We detected variable, low level of staining of GR113 on vessels in uninflamed skin. 4 h LPS-inflamed skin exhibited positive vessels, but much of the staining was associated with platelet-like particles. In contrast, E-selectin was expressed on many vessels in the 4 h inflamed tissues, whereas minimal expression was seen in the chronic lesion (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