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Heat stable antigen (mouse CD24) supports myeloid cell binding to endothelial and platelet P-selectin
1Tumor Immunology Programme, German Cancer Research Center, Heidelberg, Germany.
Insights
Heat stable antigen (HSA) acts as a P-selectin ligand, mediating myeloid cell and neutrophil adhesion to endothelial cells and platelets. This interaction involves sialylated N-linked glycans on HSA.
Area of Science:
- Immunology
- Cell Biology
- Biochemistry
Background:
- P-selectin mediates leukocyte adhesion to endothelium and platelets.
- Heat stable antigen (HSA/mouse CD24) is a heavily glycosylated cell surface molecule expressed on various mouse cells.
- Previous studies suggested HSA might be a ligand for P-selectin.
Purpose of the Study:
- To investigate whether Heat Stable Antigen (HSA) functions as a ligand for P-selectin.
- To determine the role of HSA in the adhesion of monocytic cells and neutrophils to P-selectin.
- To characterize the specific glycans on HSA recognized by P-selectin.
Main Methods:
- Utilized cell adhesion assays under shear forces involving monocytic cell lines, neutrophils, and activated endothelial cells.
- Employed monoclonal antibodies (mAbs) against P-selectin and HSA to block cell binding.
- Used purified HSA-coated latex beads to assess direct binding to activated cells and platelets.
- Analyzed binding with P-selectin-IgG and other selectin-IgG fusion proteins.
- Investigated the role of divalent cations and enzymatic deglycosylation (endoglycosidase F, neuraminidase) on binding.
- Confirmed glycan presence using biosynthetic labeling studies.
Main Results:
- HSA mediates the binding of monocytic cells and neutrophils to P-selectin expressed on activated endothelial cells and platelets.
- Inhibition of binding was observed using mAbs against P-selectin and HSA.
- Adhesion was more efficient at 4°C, suggesting reduced integrin involvement.
- HSA-coated beads bound to activated cells and platelets, with binding blocked by specific mAbs.
- Binding of HSA-coated beads to P-selectin was confirmed, while binding to L- and E-selectin was minimal.
- Sialylated N-linked glycans on HSA were identified as the recognition structures for P-selectin.
Conclusions:
- Heat Stable Antigen (HSA) is a functional P-selectin ligand mediating myeloid cell and neutrophil adhesion.
- HSA belongs to a class of monospecific P-selectin ligands on myeloid cells.
- Sialylated N-linked glycans on HSA are crucial for P-selectin recognition.
Abstract:
P-selectin is a Ca(2+)-dependent lectin that participates in leukocyte adhesion to vascular endothelium and platelets. Myeloid cells and a subset of T lymphocytes express carbohydrate ligands at the cell surface. Previously, we suggested that heat stable antigen (HSA/mouse CD24), an extensively glycosylated cell surface molecule on many mouse cells, is a ligand for P-selectin. Here we show that HSA mediates the binding of monocytic cells and neutrophils to P-selectin. The monocytic cell lines ESb-MP and J774, peritoneal exudate cells, and bone marrow neutrophils could bind to lipopolysaccharide-activated bend3 endothelioma cells under rotation-induced shear forces and this binding was inhibited by mAb to P-selectin and HSA. Blocking was weak at room temperature but more efficient at 4 degrees C when integrin-mediated binding was decreased. Also the adhesion of neutrophils to stimulated platelets expressing P-selectin was blocked by HSA- and P-selectin-specific mAb. Latex beads coated with purified HSA from myeloid cells bound to activated endothelioma cells or platelets, and the binding was similarly blocked by mAb to P-selectin and HSA respectively. The HSA-coated beads were stained with P-selectin-IgG, very weakly with L-selectin-IgG but not with E-selectin-IgG. The staining was dependent on divalent cations and treatment with endoglycosidase F or neuraminidase indicated that sialylated N-linked glycans were recognized. The presence of these glycans was confirmed by biosynthetic labeling studies. Our data suggest that HSA, in addition to the recently identified 160 kDa glycoprotein ligand on mouse neutrophils, belongs to a group of monospecific P-selectin ligands on myeloid cells.