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- and L-selectin

Blood. 1998 Jan 1;91(1):154-64.

Abstract

Interactions between P-selectin and P-selectin glycoprotein ligand-1 (PSGL-1) mediate the earliest "rolling" of leukocytes on the lumenal surface of endothelial cells at sites of inflammation. Previously, PSGL-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 mediate 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 blotting of cells transfected with human PSGL-1. This newly developed MoAb, KPL1, inhibited interactions between P-selectin expressing COS cells and either HL60 cells, neutrophils, or lymphocytes. Furthermore, KPL1 completely inhibited interactions between P-selectin and either purified CD4 T cells or neutrophils in a flow assay under physiological conditions, but had no effect on interactions of T cells or neutrophils with E-selectin. In addition, KPL1 blocked interactions between lymphoid cells transfected with L-selectin and COS cells expressing PSGL-1. The KPL1 epitope was mapped to a site within a consensus tyrosine sulfation motif of PSGL-1, previously shown to be essential for interaction 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 normal leukocytes showed that while natural killer (NK) cells (CD16(+)), monocytes, CD4 and CD8 T cells, and alpha/beta and gamma/delta T cells were uniformly positive for PSGL-1, B cells expressed low levels of the KPL1 epitope. This low level of KPL1 staining was also observed immunohistologically in germinal centers, which had no detectable KPL1 staining, 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 essentially all blood neutrophils, NK cells, B cells, T cells, and monocytes. Variation in tyrosine sulfation during B-cell differentiation may affect the ability of B cells to interact with P- and L-selectin.

Publication types

  • Comparative Study
  • Research Support, Non-U.S. Gov't
  • Research Support, U.S. Gov't, P.H.S.

MeSH terms

  • Animals
  • Antibodies, Monoclonal / immunology*
  • Antibody Specificity
  • COS Cells / metabolism
  • Cell Differentiation
  • Cell Movement / drug effects
  • Consensus Sequence
  • DNA, Complementary / genetics
  • Epitopes / immunology*
  • HL-60 Cells / metabolism
  • Humans
  • L-Selectin / metabolism*
  • Ligands
  • Lymphocyte Subsets / metabolism
  • Membrane Glycoproteins / antagonists & inhibitors
  • Membrane Glycoproteins / chemistry
  • Membrane Glycoproteins / immunology*
  • Membrane Glycoproteins / metabolism
  • Mice
  • Monocytes / metabolism
  • Neutrophils / metabolism
  • P-Selectin / metabolism*
  • Protein Processing, Post-Translational
  • Recombinant Fusion Proteins / immunology
  • Sulfates / metabolism
  • Transfection
  • Tyrosine / metabolism

Substances

  • Antibodies, Monoclonal
  • DNA, Complementary
  • Epitopes
  • Ligands
  • Membrane Glycoproteins
  • P-Selectin
  • P-selectin ligand protein
  • Recombinant Fusion Proteins
  • Sulfates
  • L-Selectin
  • Tyrosine