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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.

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148.Fc microspheres attached and rolled on TNF-α–activated HUVEC monolayers under flow. 148.Fc or IgG microspheres were perfused across TNF-α– (4 h, 25 ng/ml) activated or  –unactivated HUVEC monolayers. 148.Fc microspheres attached  and rolled on TNF-α–activated HUVEC monolayers while IgG  microspheres did not attach to the TNF-α–activated HUVEC  monolayers. 148.Fc microsphere attachment was blocked by an  mAb to E-selectin (7A9) but unaffected by a negative isotypematched control endothelial cell binding mAb to Class I (W6/32).  148.Fc microspheres did not attach to unactivated HUVEC  monolayers. (Shear stress = 2 dynes/cm2; n = 3).
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Figure 2: 148.Fc microspheres attached and rolled on TNF-α–activated HUVEC monolayers under flow. 148.Fc or IgG microspheres were perfused across TNF-α– (4 h, 25 ng/ml) activated or –unactivated HUVEC monolayers. 148.Fc microspheres attached and rolled on TNF-α–activated HUVEC monolayers while IgG microspheres did not attach to the TNF-α–activated HUVEC monolayers. 148.Fc microsphere attachment was blocked by an mAb to E-selectin (7A9) but unaffected by a negative isotypematched control endothelial cell binding mAb to Class I (W6/32). 148.Fc microspheres did not attach to unactivated HUVEC monolayers. (Shear stress = 2 dynes/cm2; n = 3).

Mentions: At 2 dynes/cm2, 148.Fc microspheres attached to 4-h TNF-α–activated HUVEC at a rate of 101 ± 14 microspheres/mm2/min (Fig. 2). The attachment events were inhibited by F(ab′)2 fragments of a blocking anti–E-selectin mAb, 7A9, but unaffected by F(ab′)2 fragments of an endothelial cell binding control mAb anti–Class I, W6/32, indicating that the attachment was mediated by E-selectin (Fig. 2). Unstimulated HUVEC, which do not express E-selectin (7), did not support attachment of 148.Fc microspheres (Fig. 2). Microspheres coated with human IgG1 did not attach to TNF-α–stimulated HUVEC (Fig. 2). Subsequent to attachment, >95% of the attached 148.Fc microspheres rolled in a manner similar to that described for leukocytes (Fig. 3), i.e., a low-velocity, high-variance translation (12). Thus, it appears that the first 148 amino acids of PSGL-1 are sufficient to support initial attachment and subsequent rolling on TNF-α–activated endothelium and that the attachment step is mediated by E-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 TNF-α–activated HUVEC monolayers under flow. 148.Fc or IgG microspheres were perfused across TNF-α– (4 h, 25 ng/ml) activated or  –unactivated HUVEC monolayers. 148.Fc microspheres attached  and rolled on TNF-α–activated HUVEC monolayers while IgG  microspheres did not attach to the TNF-α–activated HUVEC  monolayers. 148.Fc microsphere attachment was blocked by an  mAb to E-selectin (7A9) but unaffected by a negative isotypematched control endothelial cell binding mAb to Class I (W6/32).  148.Fc microspheres did not attach to unactivated HUVEC  monolayers. (Shear stress = 2 dynes/cm2; n = 3).
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Related In: Results  -  Collection

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Figure 2: 148.Fc microspheres attached and rolled on TNF-α–activated HUVEC monolayers under flow. 148.Fc or IgG microspheres were perfused across TNF-α– (4 h, 25 ng/ml) activated or –unactivated HUVEC monolayers. 148.Fc microspheres attached and rolled on TNF-α–activated HUVEC monolayers while IgG microspheres did not attach to the TNF-α–activated HUVEC monolayers. 148.Fc microsphere attachment was blocked by an mAb to E-selectin (7A9) but unaffected by a negative isotypematched control endothelial cell binding mAb to Class I (W6/32). 148.Fc microspheres did not attach to unactivated HUVEC monolayers. (Shear stress = 2 dynes/cm2; n = 3).
Mentions: At 2 dynes/cm2, 148.Fc microspheres attached to 4-h TNF-α–activated HUVEC at a rate of 101 ± 14 microspheres/mm2/min (Fig. 2). The attachment events were inhibited by F(ab′)2 fragments of a blocking anti–E-selectin mAb, 7A9, but unaffected by F(ab′)2 fragments of an endothelial cell binding control mAb anti–Class I, W6/32, indicating that the attachment was mediated by E-selectin (Fig. 2). Unstimulated HUVEC, which do not express E-selectin (7), did not support attachment of 148.Fc microspheres (Fig. 2). Microspheres coated with human IgG1 did not attach to TNF-α–stimulated HUVEC (Fig. 2). Subsequent to attachment, >95% of the attached 148.Fc microspheres rolled in a manner similar to that described for leukocytes (Fig. 3), i.e., a low-velocity, high-variance translation (12). Thus, it appears that the first 148 amino acids of PSGL-1 are sufficient to support initial attachment and subsequent rolling on TNF-α–activated endothelium and that the attachment step is mediated by E-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