title: L-selectin ligands expressed by human leukocytes are HECA-452 antibody-defined carbohydrate epitopes preferentially displayed by P-selectin glycoprotein ligand-1.
abstract: Leukocytes express L-selectin ligands critical for leukocyte-leukocyte interactions at sites of inflammation. The predominant leukocyte L-selectin ligand is P-selectin glycoprotein ligand-1 (PSGL-1), which displays appropriate sialyl Lewis x (sLex)-like carbohydrate determinants for L-selectin recognition. Among the sLex-like determinants expressed by human leukocytes is a unique carbohydrate epitope defined by the HECA-452 mAb. The HECA-452 Ag is a critical component of L-selectin ligands expressed by vascular endothelial cells. However, HECA-452 Ag expression on human leukocyte L-selectin ligands has not been assessed. In this study, the HECA-452 mAb blocked 88-99% of neutrophil rolling on, or attachment to, adherent cells expressing L-selectin in multiple experimental systems. A function-blocking anti-PSGL-1 mAb also inhibited L-selectin binding to neutrophils by 89-98%. In addition, the HECA-452 and anti-PSGL-1 mAbs blocked the majority of P-selectin binding to neutrophils. Western blot analysis revealed that PSGL-1 immunoprecipitated from neutrophils displayed HECA-452 mAb-reactive determinants and that PSGL-1 was the predominant scaffold for HECA-452 Ag display. Leukocyte L-selectin ligands also contained sulfated determinants since culturing ligand-bearing cells with NaClO3 abrogated L-selectin binding. Consistent with this, human neutrophils expressed mRNA encoding five different sulfotransferases associated with the generation of selectin ligands: CHST1, CHST2, CHST3, TPST1, and HEC-GlcNAc6ST. Therefore, the HECA-452-defined carbohydrate determinant displayed on PSGL-1 represented the predominant L-selectin and P-selectin ligand expressed by neutrophils.
authors.affiliation: Department of Immunology, Duke University Medical Center, Durham, NC 27710, USA.
authors.forename: L
authors.initials: L
authors.lastname: Tu
authors.forename: P G
authors.initials: PG
authors.lastname: Murphy
authors.forename: X
authors.initials: X
authors.lastname: Li
authors.forename: T F
authors.initials: TF
authors.lastname: Tedder
mesh_terms: D000818:Animals; D019138:Antibodies, Blocking / Q000494:pharmacology; D000911:Antibodies, Monoclonal / Q000378:metabolism* / Q000494:pharmacology; D000945:Antigens, Differentiation, T-Lymphocyte; D000951:Antigens, Neoplasm; D001775:Blood Circulation / Q000276:immunology / Q000502:physiology; D019556:COS Cells; D050260:Carbohydrate Metabolism; D002241:Carbohydrates / Q000276:immunology; D002465:Cell Movement / Q000276:immunology / Q000502:physiology; D000939:Epitopes / Q000378:metabolism*; D005647:Fucosyltransferases / Q000235:genetics; D018922:HL-60 Cells; D006801:Humans; D019041:L-Selectin / Q000096:biosynthesis* / Q000378:metabolism; D007962:Leukocytes / Q000378:metabolism*; D008024:Ligands; D008562:Membrane Glycoproteins / Q000276:immunology / Q000378:metabolism*; D051379:Mice; D009504:Neutrophils / Q000276:immunology / Q000378:metabolism / Q000502:physiology; D019007:P-Selectin / Q000276:immunology / Q000378:metabolism*; D015238:Sulfotransferases / Q000096:biosynthesis / Q000235:genetics; D014162:Transfection
chemical_list: D019138:Antibodies, Blocking; D000911:Antibodies, Monoclonal; D000945:Antigens, Differentiation, T-Lymphocyte; D000951:Antigens, Neoplasm; C063419:CTAGE1 protein, human; D002241:Carbohydrates; D000939:Epitopes; D008024:Ligands; D008562:Membrane Glycoproteins; D019007:P-Selectin; C084882:P-selectin ligand protein; D019041:L-Selectin; D005647:Fucosyltransferases; C031692:galactoside 3-fucosyltransferase; D015238:Sulfotransferases