title: CD14 and tissue factor expression by bacterial lipopolysaccharide-stimulated bovine alveolar macrophages in vitro.
abstract: The membrane-associated CD14 receptor (mCD14) is a monocyte/macrophage differentiation antigen, and it has been demonstrated to serve as a receptor for bacterial lipopolysaccharide (LPS; endotoxin). Binding of LPS to mCD14 has been shown to be associated with LPS-induced macrophage, monocyte, and neutrophil activation in humans. In this report, we describe the presence and function of an mCD14-like receptor on bovine alveolar macrophages (bAM). An immunofluorescence technique and flow cytometric analysis indicated binding of anti-human CD14 monoclonal antibodies (MAb) My4, 3C10, and 60bd to bAM. Binding of anti-CD14 MAb (3C10 and MY4) was reduced over 20% by pretreatment of bAM with phosphatidylinositol-specific phospholipase C (0.5 to 1.0 U/ml), indicating that bovine mCD14 is a glycosyl phosphatidylinositol-anchored protein. In addition, pretreatment of bAM with anti-CD14 MAb decreased binding of 125I-labeled LPS to macrophages, suggesting that bovine mCD14 serves as a receptor for LPS. A cDNA probe based on the human sequence for CD14 was used in Northern (RNA) blot analysis, and hybridization to human monocyte CD14 yielded the expected 1.5-kb band. Hybridization to bovine mRNA yielded a 1.5-kb band plus an unexpected 3.1-kb band. Constitutive expression of bovine CD14 mRNA was observed, and the expression level was modestly elevated in bAM stimulated for 24 h with LPS (1 ng/ml) in the presence of bovine serum. The function and activation of bAM were assessed by quantitation of tissue factor (TF) expression on the cells using an activated factor X-related chromogenic assay and S-2222 substrate. LPS (1 ng/ml)-mediated upregulation of TF expression on bAM was dependent on the presence of bovine serum components, and TF expression was inhibited by anti-CD14 MAb. In addition, TF mRNA levels in LPS-stimulated bAM were decreased by pretreatment of cells with anti-CD14 MAb (MAb 60bd, 10 micrograms/ml).
authors.affiliation: Department of Pathology, University of Tennessee College of Veterinary Medicine, Knoxville 37901-1071.
authors.forename: Z
authors.initials: Z
authors.lastname: Yang
authors.forename: C D
authors.initials: CD
authors.lastname: Carter
authors.forename: M S
authors.initials: MS
authors.lastname: Miller
authors.forename: P N
authors.initials: PN
authors.lastname: Bochsler
mesh_terms: D000818:Animals; D000911:Antibodies, Monoclonal; D015703:Antigens, CD / Q000096:biosynthesis* / Q000235:genetics / Q000276:immunology; D015214:Antigens, Differentiation, Myelomonocytic / Q000096:biosynthesis* / Q000235:genetics / Q000276:immunology; D015152:Blotting, Northern; D002417:Cattle; D005434:Flow Cytometry; D017261:Glycosylphosphatidylinositols; D018950:Lipopolysaccharide Receptors; D008070:Lipopolysaccharides / Q000494:pharmacology*; D008262:Macrophage Activation; D016676:Macrophages, Alveolar / Q000187:drug effects*; D012333:RNA, Messenger / Q000032:analysis; D013925:Thromboplastin / Q000096:biosynthesis* / Q000235:genetics; D015854:Up-Regulation
chemical_list: D000911:Antibodies, Monoclonal; D015703:Antigens, CD; D015214:Antigens, Differentiation, Myelomonocytic; D017261:Glycosylphosphatidylinositols; D018950:Lipopolysaccharide Receptors; D008070:Lipopolysaccharides; D012333:RNA, Messenger; D013925:Thromboplastin