L-selectin ligands expressed by human leukocytes are HECA-452 antibody-defined carbohydrate epitopes preferentially displayed by P-selectin glycoprotein ligand-1

Leukocytes express L-selectin ligands critical for leukocyte-leukocyte inte ractions at sites of inflammation, The predominant leukocyte L-selectin lig and is P-selectin glycoprotein ligand-l (PSGL-1), which displays appropriat e sialyl Lewis x (sLe(x))-like carbohydrate determinants for L-selectin rec ognition, Among the sLe(x)-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 endo thelial cells. However, HECA-452 Ag expression on human leukocyte L-selecti n 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 additi on, the HECA-452 and anti-PSGL-1 mAbs blocked the majority of P-selectin bi nding to neutrophils, Western blot analysis revealed that PSGL-1 immunoprec ipitated from neutrophils displayed MECA-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 li gand-bearing cells with NaClO3 abrogated L-selectin binding. Consistent wit h this, human neutrophils expressed mRNA encoding five different sulfotrans ferases associated with the generation of selectin ligands: CHST1, CHST2, C HST3, TPST1, and HEC-GlcNAc6ST. Therefore, the HECA-452-defined carbohydrat e determinant displayed on PSGL-1 represented the predominant L-selectin an d P-selectin ligand expressed by neutrophils.