Limits...
Isolated P-selectin glycoprotein ligand-1 dynamic adhesion to P- and E-selectin.

Goetz DJ, Greif DM, Ding H, Camphausen RT, Howes S, Comess KM, Snapp KR, Kansas GS, Luscinskas FW - J. Cell Biol. (1997)

Bottom Line: To study, in isolation, the interaction of PSGL-1 with P- and E-selectin under flow, we developed an in vitro model in which various recombinant regions of extracellular PSGL-1 were coupled to 10-microm-diameter microspheres.In a parallel plate chamber with well defined flow conditions, live time video microscopy analyses revealed that microspheres coated with PSGL-1 attached and rolled on 4-h tumor necrosis factor-alpha-activated endothelial cell monolayers, which express high levels of E-selectin, and CHO monolayers stably expressing E- or P-selectin.In addition, the results suggest that PSGL-1 has more than one binding site for E-selectin: one site located within the first 19 amino acids of PSGL-1 and one or more sites located between amino acids 19 through 148.

View Article: PubMed Central - PubMed

Affiliation: Department of Pathology, Brigham and Women's Hospital, Harvard Medical School, Boston, Massachusetts 02115, USA. dgoetz@cc.memphis.edu

ABSTRACT
Leukocyte adhesion to vascular endothelium under flow involves an adhesion cascade consisting of multiple receptor pairs that may function in an overlapping fashion. P-selectin glycoprotein ligand-1 (PSGL-1) and L-selectin have been implicated in neutrophil adhesion to P- and E-selectin under flow conditions. To study, in isolation, the interaction of PSGL-1 with P- and E-selectin under flow, we developed an in vitro model in which various recombinant regions of extracellular PSGL-1 were coupled to 10-microm-diameter microspheres. In a parallel plate chamber with well defined flow conditions, live time video microscopy analyses revealed that microspheres coated with PSGL-1 attached and rolled on 4-h tumor necrosis factor-alpha-activated endothelial cell monolayers, which express high levels of E-selectin, and CHO monolayers stably expressing E- or P-selectin. Further studies using CHO-E and -P monolayers demonstrate that the first 19 amino acids of PSGL-1 are sufficient for attachment and rolling on both E- and P-selectin and suggest that a sialyl Lewis x-containing glycan at Threonine-16 is critical for this sequence of amino acids to mediate attachment to E- and P-selectin. The data also demonstrate that a sulfated, anionic polypeptide segment within the amino terminus of PSGL-1 is necessary for PSGL-1-mediated attachment to P- but not to E-selectin. In addition, the results suggest that PSGL-1 has more than one binding site for E-selectin: one site located within the first 19 amino acids of PSGL-1 and one or more sites located between amino acids 19 through 148.

Show MeSH

Related in: MedlinePlus

OSGE abolishes 148.Fc microsphere attachment to CHO-P or -E monolayers. (A) 148.Fc microspheres were incubated with  buffer (a and c) or OSGE (b and d) at 37°C for 30 min. Untreated (a) or OSGE-treated (b) 148.Fc microspheres were incubated with an  mAb to PSGL-1 (PSL-275) (open histograms) or an isotype-matched control mAb to ICAM-1 (Hu5/3) (shaded histograms) and subsequently an FITC-labeled secondary antibody. As a control for the specificity of OSGE, the presence of the human Fc region of the  148.Fc chimera was detected on the 148.Fc microspheres. Untreated (c) or OSGE-treated (d) 148.Fc microspheres were incubated with  an FITC-labeled polyclonal antibody to human Fc (open histograms) or control, mouse IgG (shaded histograms). Results shown are representative of n = 2–4 separate experiments. (B) Treatment of 148.Fc microspheres with OSGE before use in the in vitro flow assay  abolished 148.Fc microsphere attachment to CHO-E and -P monolayers. (Shear stress = 2 dynes/cm2; P < 0.05; n = 3).
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC2139768&req=5

Figure 5: OSGE abolishes 148.Fc microsphere attachment to CHO-P or -E monolayers. (A) 148.Fc microspheres were incubated with buffer (a and c) or OSGE (b and d) at 37°C for 30 min. Untreated (a) or OSGE-treated (b) 148.Fc microspheres were incubated with an mAb to PSGL-1 (PSL-275) (open histograms) or an isotype-matched control mAb to ICAM-1 (Hu5/3) (shaded histograms) and subsequently an FITC-labeled secondary antibody. As a control for the specificity of OSGE, the presence of the human Fc region of the 148.Fc chimera was detected on the 148.Fc microspheres. Untreated (c) or OSGE-treated (d) 148.Fc microspheres were incubated with an FITC-labeled polyclonal antibody to human Fc (open histograms) or control, mouse IgG (shaded histograms). Results shown are representative of n = 2–4 separate experiments. (B) Treatment of 148.Fc microspheres with OSGE before use in the in vitro flow assay abolished 148.Fc microsphere attachment to CHO-E and -P monolayers. (Shear stress = 2 dynes/cm2; P < 0.05; n = 3).

Mentions: Previous studies have demonstrated that PSGL-1 is cleaved by the metalloprotease OSGE (37). To investigate the specificity of the 148.Fc microsphere attachment events, we treated 148.Fc microspheres with OSGE before use in our flow assay. Treatment of the 148.Fc microspheres with OSGE significantly reduced (>95%) the presence of the epitope recognized by the PSGL-1 mAb PSL-275 (Fig. 5 A). However, the level of human IgG Fc present on the microsphere was unchanged indicating that the PSGL-1 peptide of the 148.Fc chimera was specifically cleaved by OSGE (Fig. 5 A). Treatment of 148.Fc microspheres with OSGE before use in the flow assay eliminated attachment to both CHO-P and -E monolayers (Fig. 5 B). From these data, we conclude that 148.Fc microsphere attachment to CHO-E and -P monolayers is specifically mediated by the PSGL-1 peptide of the 148.Fc chimera.


Isolated P-selectin glycoprotein ligand-1 dynamic adhesion to P- and E-selectin.

Goetz DJ, Greif DM, Ding H, Camphausen RT, Howes S, Comess KM, Snapp KR, Kansas GS, Luscinskas FW - J. Cell Biol. (1997)

OSGE abolishes 148.Fc microsphere attachment to CHO-P or -E monolayers. (A) 148.Fc microspheres were incubated with  buffer (a and c) or OSGE (b and d) at 37°C for 30 min. Untreated (a) or OSGE-treated (b) 148.Fc microspheres were incubated with an  mAb to PSGL-1 (PSL-275) (open histograms) or an isotype-matched control mAb to ICAM-1 (Hu5/3) (shaded histograms) and subsequently an FITC-labeled secondary antibody. As a control for the specificity of OSGE, the presence of the human Fc region of the  148.Fc chimera was detected on the 148.Fc microspheres. Untreated (c) or OSGE-treated (d) 148.Fc microspheres were incubated with  an FITC-labeled polyclonal antibody to human Fc (open histograms) or control, mouse IgG (shaded histograms). Results shown are representative of n = 2–4 separate experiments. (B) Treatment of 148.Fc microspheres with OSGE before use in the in vitro flow assay  abolished 148.Fc microsphere attachment to CHO-E and -P monolayers. (Shear stress = 2 dynes/cm2; P < 0.05; n = 3).
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2139768&req=5

Figure 5: OSGE abolishes 148.Fc microsphere attachment to CHO-P or -E monolayers. (A) 148.Fc microspheres were incubated with buffer (a and c) or OSGE (b and d) at 37°C for 30 min. Untreated (a) or OSGE-treated (b) 148.Fc microspheres were incubated with an mAb to PSGL-1 (PSL-275) (open histograms) or an isotype-matched control mAb to ICAM-1 (Hu5/3) (shaded histograms) and subsequently an FITC-labeled secondary antibody. As a control for the specificity of OSGE, the presence of the human Fc region of the 148.Fc chimera was detected on the 148.Fc microspheres. Untreated (c) or OSGE-treated (d) 148.Fc microspheres were incubated with an FITC-labeled polyclonal antibody to human Fc (open histograms) or control, mouse IgG (shaded histograms). Results shown are representative of n = 2–4 separate experiments. (B) Treatment of 148.Fc microspheres with OSGE before use in the in vitro flow assay abolished 148.Fc microsphere attachment to CHO-E and -P monolayers. (Shear stress = 2 dynes/cm2; P < 0.05; n = 3).
Mentions: Previous studies have demonstrated that PSGL-1 is cleaved by the metalloprotease OSGE (37). To investigate the specificity of the 148.Fc microsphere attachment events, we treated 148.Fc microspheres with OSGE before use in our flow assay. Treatment of the 148.Fc microspheres with OSGE significantly reduced (>95%) the presence of the epitope recognized by the PSGL-1 mAb PSL-275 (Fig. 5 A). However, the level of human IgG Fc present on the microsphere was unchanged indicating that the PSGL-1 peptide of the 148.Fc chimera was specifically cleaved by OSGE (Fig. 5 A). Treatment of 148.Fc microspheres with OSGE before use in the flow assay eliminated attachment to both CHO-P and -E monolayers (Fig. 5 B). From these data, we conclude that 148.Fc microsphere attachment to CHO-E and -P monolayers is specifically mediated by the PSGL-1 peptide of the 148.Fc chimera.

Bottom Line: To study, in isolation, the interaction of PSGL-1 with P- and E-selectin under flow, we developed an in vitro model in which various recombinant regions of extracellular PSGL-1 were coupled to 10-microm-diameter microspheres.In a parallel plate chamber with well defined flow conditions, live time video microscopy analyses revealed that microspheres coated with PSGL-1 attached and rolled on 4-h tumor necrosis factor-alpha-activated endothelial cell monolayers, which express high levels of E-selectin, and CHO monolayers stably expressing E- or P-selectin.In addition, the results suggest that PSGL-1 has more than one binding site for E-selectin: one site located within the first 19 amino acids of PSGL-1 and one or more sites located between amino acids 19 through 148.

View Article: PubMed Central - PubMed

Affiliation: Department of Pathology, Brigham and Women's Hospital, Harvard Medical School, Boston, Massachusetts 02115, USA. dgoetz@cc.memphis.edu

ABSTRACT
Leukocyte adhesion to vascular endothelium under flow involves an adhesion cascade consisting of multiple receptor pairs that may function in an overlapping fashion. P-selectin glycoprotein ligand-1 (PSGL-1) and L-selectin have been implicated in neutrophil adhesion to P- and E-selectin under flow conditions. To study, in isolation, the interaction of PSGL-1 with P- and E-selectin under flow, we developed an in vitro model in which various recombinant regions of extracellular PSGL-1 were coupled to 10-microm-diameter microspheres. In a parallel plate chamber with well defined flow conditions, live time video microscopy analyses revealed that microspheres coated with PSGL-1 attached and rolled on 4-h tumor necrosis factor-alpha-activated endothelial cell monolayers, which express high levels of E-selectin, and CHO monolayers stably expressing E- or P-selectin. Further studies using CHO-E and -P monolayers demonstrate that the first 19 amino acids of PSGL-1 are sufficient for attachment and rolling on both E- and P-selectin and suggest that a sialyl Lewis x-containing glycan at Threonine-16 is critical for this sequence of amino acids to mediate attachment to E- and P-selectin. The data also demonstrate that a sulfated, anionic polypeptide segment within the amino terminus of PSGL-1 is necessary for PSGL-1-mediated attachment to P- but not to E-selectin. In addition, the results suggest that PSGL-1 has more than one binding site for E-selectin: one site located within the first 19 amino acids of PSGL-1 and one or more sites located between amino acids 19 through 148.

Show MeSH
Related in: MedlinePlus