A NOVEL P-SELECTIN GLYCOPROTEIN LIGAND-1 MONOCLONAL-ANTIBODY RECOGNIZES AN EPITOPE WITHIN THE TYROSINE SULFATE MOTIF OF HUMAN PSGL-1 AND BLOCKS RECOGNITION OF BOTH P-SELECTIN AND L-SELECTIN

Interactions between P-selectin and P-selectin glycoprotein ligand-l ( PSGL-1) mediate the earliest ''rolling'' of leukocytes on the lumenal surface of endothelial cells at sites of inflammation. Previously, PSG L-1 has been shown to be the primary mediator of interactions between neutrophils and P-selectin, but studies on the ability of PSGL-1 to me diate interactions between P-selectin and other subsets of leukocytes have yielded variable and conflicting results, A novel IgG monoclonal antibody (MoAb) to human PSGL-1 was generated, and the specificity of this MoAb was confirmed by both flow cytometric analysis and Western b lotting of cells transfected with human PSGL-1. This newly developed M oAb, KPL1, inhibited interactions between P-selectin expressing COS ce lls and either HL60 cells, neutrophils, or lymphocytes. Furthermore, K PL1 completely inhibited interactions between P-selectin and either pu rified CD4 T cells or neutrophils in a flow assay under physiological conditions, but had no effect on interactions of T cells or neutrophil s with E-selectin. In addition, KPL1 blocked interactions between lymp hoid cells transfected with L-selectin and COS cells expressing PSGL-1 . The KPL1 epitope was mapped to a site within a consensus tyrosine su lfation motif of PSGL-1, previously shown to be essential for interact ion with P-selectin and now shown to be essential for interaction with L-selectin, and to be distinct from the epitope identified by the PL1 function blocking anti-PSGL-1 MoAb. Two-color flow cytometry of norma l leukocytes showed that while natural killer (NK) cells (CD16(+)), mo nocytes, CD4 and CD8 T cells, and alpha/beta and gamma/delta T cells w ere uniformly positive for PSGL-1, B cells expressed low levels of the KPL1 epitope. This low level of KPL1 staining was also observed immun ohistologically in germinal centers, which had no detectable KPL1 stai ning, whereas T-cell areas (interfollicular region) were positive for KPL1. Interestingly, plasma cells in situ and interleukin-6-dependent myeloma cell lines were KPL1(+), Thus, PSGL-1 is expressed on essentia lly ail blood neutrophils, NK cells, B cells, T cells, and monocytes. Variation in tyrosine sulfation during B-cell differentiation may affe ct the ability of B cells to interact with P- and L-selectin. (C) 1998 by The American Society of Hematology.