title: 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.
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.
authors.affiliation: Department of Microbiology/Immunology, Northwestern University Medical School, Chicago, IL 60611, USA.
authors.forename: K R
authors.initials: KR
authors.lastname: Snapp
authors.forename: H
authors.initials: H
authors.lastname: Ding
authors.forename: K
authors.initials: K
authors.lastname: Atkins
authors.forename: R
authors.initials: R
authors.lastname: Warnke
authors.forename: F W
authors.initials: FW
authors.lastname: Luscinskas
authors.forename: G S
authors.initials: GS
authors.lastname: Kansas
mesh_terms: D000818:Animals; D000911:Antibodies, Monoclonal / Q000276:immunology*; D000918:Antibody Specificity; D019556:COS Cells / Q000378:metabolism; D002454:Cell Differentiation; D002465:Cell Movement / Q000187:drug effects; D016384:Consensus Sequence; D018076:DNA, Complementary / Q000235:genetics; D000939:Epitopes / Q000276:immunology*; D018922:HL-60 Cells / Q000378:metabolism; D006801:Humans; D019041:L-Selectin / Q000378:metabolism*; D008024:Ligands; D016131:Lymphocyte Subsets / Q000378:metabolism; D008562:Membrane Glycoproteins / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000276:immunology* / Q000378:metabolism; D051379:Mice; D009000:Monocytes / Q000378:metabolism; D009504:Neutrophils / Q000378:metabolism; D019007:P-Selectin / Q000378:metabolism*; D011499:Protein Processing, Post-Translational; D011993:Recombinant Fusion Proteins / Q000276:immunology; D013431:Sulfates / Q000378:metabolism; D014162:Transfection; D014443:Tyrosine / Q000378:metabolism
chemical_list: D000911:Antibodies, Monoclonal; D018076:DNA, Complementary; D000939:Epitopes; D008024:Ligands; D008562:Membrane Glycoproteins; D019007:P-Selectin; C084882:P-selectin ligand protein; D011993:Recombinant Fusion Proteins; D013431:Sulfates; D019041:L-Selectin; D014443:Tyrosine