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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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SDS-PAGE/Western blot and immunoprecipitation analysis of the platelet and endothelial cell GR antigen. In one experiment, bovine platelets were lysed in detergent lysis buffer, run on an 8% PAGE gel,  transferred to nitrocellulose, and blotted with GR113 and a polyclonal  antiserum against bovine P-selectin. A shows the results. GR113 and negative control antibody are represented in lanes 1 and 2, which were run  under nonreducing conditions. Lanes 3 and 4 represent polyclonal antiserum against bovine P-selectin and preimmune control, respectively, on  samples run under reducing conditions. In another experiment, affinity  purified GR antigen was analyzed. Lanes 5 and 6 represent affinity-purified GR antigen run under nonreducing (lane 5) and reducing (lane 6) conditions. GR113 was used to probe the nonreduced sample, whereas polyclonal anti–P-selectin was used to probe the reduced sample, as done in  lane 3. The results of the immunoprecipitation analysis of endothelial cells  are shown in B. Biotin-labeled endothelial cells were lysed in detergent  and antigens precipitated with polyclonal antiserum against either bovine  E-selectin or P-selectin, or with GR113. GR113, negative control, polyclonal antiserum against E-selectin, polyclonal antiserum against P-selectin, and preimmune rabbit sera are shown in lanes 1–5, respectively. Arrow heads point to specific bands. The GR113 band has been repeated  >3 times and a similar size E-selectin band has been repeated with a mAb  against E-selectin. The P-selectin–specific band in the endothelial cell  analysis has not been clearly seen in five other experiments.
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Figure 10: SDS-PAGE/Western blot and immunoprecipitation analysis of the platelet and endothelial cell GR antigen. In one experiment, bovine platelets were lysed in detergent lysis buffer, run on an 8% PAGE gel, transferred to nitrocellulose, and blotted with GR113 and a polyclonal antiserum against bovine P-selectin. A shows the results. GR113 and negative control antibody are represented in lanes 1 and 2, which were run under nonreducing conditions. Lanes 3 and 4 represent polyclonal antiserum against bovine P-selectin and preimmune control, respectively, on samples run under reducing conditions. In another experiment, affinity purified GR antigen was analyzed. Lanes 5 and 6 represent affinity-purified GR antigen run under nonreducing (lane 5) and reducing (lane 6) conditions. GR113 was used to probe the nonreduced sample, whereas polyclonal anti–P-selectin was used to probe the reduced sample, as done in lane 3. The results of the immunoprecipitation analysis of endothelial cells are shown in B. Biotin-labeled endothelial cells were lysed in detergent and antigens precipitated with polyclonal antiserum against either bovine E-selectin or P-selectin, or with GR113. GR113, negative control, polyclonal antiserum against E-selectin, polyclonal antiserum against P-selectin, and preimmune rabbit sera are shown in lanes 1–5, respectively. Arrow heads point to specific bands. The GR113 band has been repeated >3 times and a similar size E-selectin band has been repeated with a mAb against E-selectin. The P-selectin–specific band in the endothelial cell analysis has not been clearly seen in five other experiments.

Mentions: Though the distribution of the GR antigen is not consistent with either P- (surface of activated platelets) or E-selectin (cytokine-stimulated and/or proliferating endothelium only), most of our understanding of the selectins have come from work with human cells and it was possible that bovine P- or E-selectin were simply different and that the GR antigen represented one or the other selectin. In Western blot analysis of bovine platelet antigens, GR113 recognized a 110–120-kD glycoprotein under nonreducing conditions (Fig. 10 A, lane 1). In contrast, a polyclonal anti-bovine P-selectin antiserum recognized a band at 130 kD under reducing conditions (Fig. 10 A, lane 3), exactly as previously reported (30). The polyclonal anti–P-selectin antibody did not work as well on nonreduced samples. In most instances the band was very faint on the nonreduced gel (data not shown). The latter observation may be related to the fact that the polyclonal antiserum was raised against recombinant bovine P-selectin expressed in E. coli and not the native protein (30). GR113 did not react on reduced samples (data not shown). To determine if the anti–P-selectin polyclonal antibody reacted with reduced GR antigen, the GR antigen was affinity purified as described (26), run on a SDS-PAGE gel under nonreducing and reducing conditions, and probed with GR113 or the anti–P-selectin antiserum, respectively. As shown in Fig. 10 A, lanes 5 and 6, the anti–P-selectin antiserum did not react with reduced, purified GR antigen.


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)

SDS-PAGE/Western blot and immunoprecipitation analysis of the platelet and endothelial cell GR antigen. In one experiment, bovine platelets were lysed in detergent lysis buffer, run on an 8% PAGE gel,  transferred to nitrocellulose, and blotted with GR113 and a polyclonal  antiserum against bovine P-selectin. A shows the results. GR113 and negative control antibody are represented in lanes 1 and 2, which were run  under nonreducing conditions. Lanes 3 and 4 represent polyclonal antiserum against bovine P-selectin and preimmune control, respectively, on  samples run under reducing conditions. In another experiment, affinity  purified GR antigen was analyzed. Lanes 5 and 6 represent affinity-purified GR antigen run under nonreducing (lane 5) and reducing (lane 6) conditions. GR113 was used to probe the nonreduced sample, whereas polyclonal anti–P-selectin was used to probe the reduced sample, as done in  lane 3. The results of the immunoprecipitation analysis of endothelial cells  are shown in B. Biotin-labeled endothelial cells were lysed in detergent  and antigens precipitated with polyclonal antiserum against either bovine  E-selectin or P-selectin, or with GR113. GR113, negative control, polyclonal antiserum against E-selectin, polyclonal antiserum against P-selectin, and preimmune rabbit sera are shown in lanes 1–5, respectively. Arrow heads point to specific bands. The GR113 band has been repeated  >3 times and a similar size E-selectin band has been repeated with a mAb  against E-selectin. The P-selectin–specific band in the endothelial cell  analysis has not been clearly seen in five other experiments.
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Figure 10: SDS-PAGE/Western blot and immunoprecipitation analysis of the platelet and endothelial cell GR antigen. In one experiment, bovine platelets were lysed in detergent lysis buffer, run on an 8% PAGE gel, transferred to nitrocellulose, and blotted with GR113 and a polyclonal antiserum against bovine P-selectin. A shows the results. GR113 and negative control antibody are represented in lanes 1 and 2, which were run under nonreducing conditions. Lanes 3 and 4 represent polyclonal antiserum against bovine P-selectin and preimmune control, respectively, on samples run under reducing conditions. In another experiment, affinity purified GR antigen was analyzed. Lanes 5 and 6 represent affinity-purified GR antigen run under nonreducing (lane 5) and reducing (lane 6) conditions. GR113 was used to probe the nonreduced sample, whereas polyclonal anti–P-selectin was used to probe the reduced sample, as done in lane 3. The results of the immunoprecipitation analysis of endothelial cells are shown in B. Biotin-labeled endothelial cells were lysed in detergent and antigens precipitated with polyclonal antiserum against either bovine E-selectin or P-selectin, or with GR113. GR113, negative control, polyclonal antiserum against E-selectin, polyclonal antiserum against P-selectin, and preimmune rabbit sera are shown in lanes 1–5, respectively. Arrow heads point to specific bands. The GR113 band has been repeated >3 times and a similar size E-selectin band has been repeated with a mAb against E-selectin. The P-selectin–specific band in the endothelial cell analysis has not been clearly seen in five other experiments.
Mentions: Though the distribution of the GR antigen is not consistent with either P- (surface of activated platelets) or E-selectin (cytokine-stimulated and/or proliferating endothelium only), most of our understanding of the selectins have come from work with human cells and it was possible that bovine P- or E-selectin were simply different and that the GR antigen represented one or the other selectin. In Western blot analysis of bovine platelet antigens, GR113 recognized a 110–120-kD glycoprotein under nonreducing conditions (Fig. 10 A, lane 1). In contrast, a polyclonal anti-bovine P-selectin antiserum recognized a band at 130 kD under reducing conditions (Fig. 10 A, lane 3), exactly as previously reported (30). The polyclonal anti–P-selectin antibody did not work as well on nonreduced samples. In most instances the band was very faint on the nonreduced gel (data not shown). The latter observation may be related to the fact that the polyclonal antiserum was raised against recombinant bovine P-selectin expressed in E. coli and not the native protein (30). GR113 did not react on reduced samples (data not shown). To determine if the anti–P-selectin polyclonal antibody reacted with reduced GR antigen, the GR antigen was affinity purified as described (26), run on a SDS-PAGE gel under nonreducing and reducing conditions, and probed with GR113 or the anti–P-selectin antiserum, respectively. As shown in Fig. 10 A, lanes 5 and 6, the anti–P-selectin antiserum did not react with reduced, purified GR antigen.

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