CD14 AND TISSUE FACTOR EXPRESSION BY BACTERIAL LIPOPOLYSACCHARIDE-STIMULATED BOVINE ALVEOLAR MACROPHAGES IN-VITRO

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 2O% 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 h Wb decreased binding of I-125-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,vas 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 mu g/ml).