Functional and structural characterization of the eosinophil P-selectin ligand.

Abstract

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.