Functional and structural characterization of the eosinophil P-selectin ligand.
Our recent studies have indicated an important role for P-selectin in eosinophil adhesion. We have therefore compared eosinophil and neutrophil binding with nasal polyp endothelium as well as purified P-selectin. We have also compared the structure and expression of the eosinophil and neutrophil P-selectin ligands. Using the frozen section assay, eosinophils bound to 2-fold more blood vessels within the nasal polyp tissue than neutrophils. Up to 10-fold more eosinophils than neutrophils bound per unit length of endothelium. Neutrophil and eosinophil binding was inhibited by a mAb against P-selectin and a P-selectin chimera which binds to the P-selectin ligand. Eosinophils bound with approximately 2-fold greater avidity to purified P-selectin under flow conditions. Using SDS-PAGE we characterized the eosinophil P-selectin ligand as a sialylated, homodimeric glycoprotein consistent with the known structure of PSGL-1. However, expression of PSGL-1 by eosinophils was significantly greater than on neutrophils. The eosinophil ligand had a calculated molecular mass by SDS-PAGE of approximately 10 kDa greater than the neutrophil ligand, which was not due to differences in N-glycosylation. Eosinophils expressed the 15-decapeptide repeat form of PSGL-1 compared with neutrophils that have the 16-decapeptide repeat form. The increased binding of eosinophils, compared with neutrophils, to P-selectin in both an ex-vivo and in vitro assay suggests that P-selectin may have a role directing the specific migration of eosinophils in diseases such as asthma. The increased avidity may be due to increased expression of PSGL-1 by eosinophils, differences in the peptide backbone, or post-translational modifications.
Symon
F A
FA
Department of Respiratory Medicine, Leicester University Medical School, Glenfield Hospital, United Kingdom.
Lawrence
M B
MB
Williamson
M L
ML
Walsh
G M
GM
Watson
S R
SR
Wardlaw
A J
AJ
D000911:Antibodies, Monoclonal / Q000276:immunology; D001483:Base Sequence; D002448:Cell Adhesion; D004727:Endothelium / Q000378:metabolism; D004802:Eosinophilia / Q000209:etiology; D004804:Eosinophils / Q000737:chemistry*; D005629:Frozen Sections; D006031:Glycosylation; D008562:Membrane Glycoproteins / Q000737:chemistry / Q000276:immunology / Q000502:physiology*; D008969:Molecular Sequence Data; D008970:Molecular Weight; D009298:Nasal Polyps / Q000737:chemistry*; D009363:Neoplasm Proteins / Q000737:chemistry / Q000276:immunology / Q000502:physiology; D009504:Neutrophils / Q000737:chemistry; D009928:Organ Specificity; D019007:P-Selectin / Q000378:metabolism*; D011499:Protein Processing, Post-Translational; D011993:Recombinant Fusion Proteins / Q000276:immunology; D013329:Structure-Activity Relationship
D000911:Antibodies, Monoclonal; D008562:Membrane Glycoproteins; D009363:Neoplasm Proteins; D019007:P-Selectin; C084882:P-selectin ligand protein; D011993:Recombinant Fusion Proteins