ANALYSIS OF THE CD14 RECEPTOR-ASSOCIATED WITH BOVINE ALVEOLAR MACROPHAGES

Previous studies have suggested the existence of a bovine homolog of t he membrane-associated CD14 receptor (mCD14) on macrophages, and funct ional similarity of bovine mCD14 receptor activity to that reported fo r other species. Bovine alveolar macrophages (bAM) reportedly possess two mRNA transcripts of 1.5 and 3.1 kb for CD14, rather than a single 1.5 kb transcript as reported for other species. The purpose of this s tudy was to determine the molecular mass of the bovine CD14 receptor, and to determine if the two mRNA transcripts for bovine CD14 yield eit her a single or two different gene products. Culture supernatant from I-125-surface-labeled bAM was examined for the existence of bovine CD1 4 using SDS-PAGE and autoradiography. A single protein band of 49 kD w as immunoprecipitated from the supernatant using anti-CD14 monoclonal antibodies (MAb). Macrophage-derived mRNA was subjected to hybrid-sele ction using a human CD14 cDNA probe immobilized on a nitrocellulose fi lter. The resultant, selected bovine mRNA was then utilized for in vit ro translation, and protein of 38-40 kD was synthesized. This size is consistent with an unglycosylated CD14 receptor protein. Protein was a lso synthesized from total RNA by in vitro translation, and was immuno precipitated with anti-CD14 monoclonal antibodies. A doublet-band of p rotein was seen at 38 kD using SDS-PAGE and autoradiography. Anti-CD14 antibodies were also used to inhibit serum-and LPS-dependent bovine m acrophage activation as measured by tissue factor expression, which is compatible with the presence and function of CD14 receptors on macrop hages. These results collectively demonstrate that a receptor consiste nt with CD14 is present on bovine macrophages, the form of the recepto r released into supernatants is 49 kD, and that it functions as an LPS receptor on these cells.