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

148.Fc microspheres  attached and rolled on CHO-P  and -E monolayers. 148.Fc microspheres attached and rolled  on CHO-E and -P monolayers  while IgG microspheres did not  attach to either CHO monolayer. 148.Fc microsphere attachment to CHO-E and -P  monolayers was blocked by an  mAb to E-selectin (7A9) and an  mAb to P-selectin (HPDG2/3),  respectively, but was unaffected  by an isotype-matched control  mAb (W6/32). 148.Fc microspheres did not attach to the parental CHO cell line (data not  shown). (Shear stress = 2 dynes/ cm2; n = 3).
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC2139768&req=5

Figure 4: 148.Fc microspheres attached and rolled on CHO-P and -E monolayers. 148.Fc microspheres attached and rolled on CHO-E and -P monolayers while IgG microspheres did not attach to either CHO monolayer. 148.Fc microsphere attachment to CHO-E and -P monolayers was blocked by an mAb to E-selectin (7A9) and an mAb to P-selectin (HPDG2/3), respectively, but was unaffected by an isotype-matched control mAb (W6/32). 148.Fc microspheres did not attach to the parental CHO cell line (data not shown). (Shear stress = 2 dynes/ cm2; n = 3).

Mentions: At 2 dynes/cm2, 148.Fc microspheres attached to CHO-E and -P monolayers at a rate of 122 ± 50 microspheres/ mm2/min and 119 ± 43 microspheres/mm2/min, respectively (Fig. 4). Subsequent to attachment, >95% of the attached 148.Fc microspheres rolled on either CHO-E or -P monolayers. Function blocking F(ab′)2 mAbs to E- (7A9) and P-selectin (HPDG2/3) completely inhibited the attachment of 148.Fc microspheres to CHO-E and -P monolayers, respectively (Fig. 4). In contrast, control isotype-matched F(ab′)2 preparation of mAb W6/32 and non–function blocking mAbs to E- (H4/18) and P-selectin (HPDG2/1) (Fig. 4 and data not shown) had no effect on the rate of attachment of the 148.Fc microspheres to CHO-E and -P monolayers, respectively. Under identical conditions, the 148.Fc microspheres did not attach to the parental CHO cell line (n = 2, data not shown), nor did human IgG1 microspheres attach to CHO-P or -E monolayers (Fig. 4). Thus, it appears that the first 148 amino acids of PSGL-1 are sufficient to support attachment and subsequent rolling on CHO cell monolayers expressing E- or P-selectin.


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)

148.Fc microspheres  attached and rolled on CHO-P  and -E monolayers. 148.Fc microspheres attached and rolled  on CHO-E and -P monolayers  while IgG microspheres did not  attach to either CHO monolayer. 148.Fc microsphere attachment to CHO-E and -P  monolayers was blocked by an  mAb to E-selectin (7A9) and an  mAb to P-selectin (HPDG2/3),  respectively, but was unaffected  by an isotype-matched control  mAb (W6/32). 148.Fc microspheres did not attach to the parental CHO cell line (data not  shown). (Shear stress = 2 dynes/ cm2; n = 3).
© Copyright Policy
Related In: Results  -  Collection

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

Figure 4: 148.Fc microspheres attached and rolled on CHO-P and -E monolayers. 148.Fc microspheres attached and rolled on CHO-E and -P monolayers while IgG microspheres did not attach to either CHO monolayer. 148.Fc microsphere attachment to CHO-E and -P monolayers was blocked by an mAb to E-selectin (7A9) and an mAb to P-selectin (HPDG2/3), respectively, but was unaffected by an isotype-matched control mAb (W6/32). 148.Fc microspheres did not attach to the parental CHO cell line (data not shown). (Shear stress = 2 dynes/ cm2; n = 3).
Mentions: At 2 dynes/cm2, 148.Fc microspheres attached to CHO-E and -P monolayers at a rate of 122 ± 50 microspheres/ mm2/min and 119 ± 43 microspheres/mm2/min, respectively (Fig. 4). Subsequent to attachment, >95% of the attached 148.Fc microspheres rolled on either CHO-E or -P monolayers. Function blocking F(ab′)2 mAbs to E- (7A9) and P-selectin (HPDG2/3) completely inhibited the attachment of 148.Fc microspheres to CHO-E and -P monolayers, respectively (Fig. 4). In contrast, control isotype-matched F(ab′)2 preparation of mAb W6/32 and non–function blocking mAbs to E- (H4/18) and P-selectin (HPDG2/1) (Fig. 4 and data not shown) had no effect on the rate of attachment of the 148.Fc microspheres to CHO-E and -P monolayers, respectively. Under identical conditions, the 148.Fc microspheres did not attach to the parental CHO cell line (n = 2, data not shown), nor did human IgG1 microspheres attach to CHO-P or -E monolayers (Fig. 4). Thus, it appears that the first 148 amino acids of PSGL-1 are sufficient to support attachment and subsequent rolling on CHO cell monolayers expressing E- or P-selectin.

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