Rough and smooth forms of fluorescein-labelled bacterial endotoxin exhibit CD14/LBP dependent and independent binding that is influencedby endotoxin concentration

Lipopolysaccharide (LPS, or endotoxin), is a major constituent of the outer membrane of Gram-negative bacteria. Bacteria express either smooth LPS, which is composed of O-antigen (O-Ag), complete core oligosaccharides, and the lipid A, or rough LPS which lack O-Ag but possess lipid A and progressively shorter core oligosaccharides. CD14 has been described as the receptor for complexes of LPS with LPS-binding protein (LBP). Using flow cytometry we have compared the binding of Salmonella minnesota rough LPS (ReLPS) and Escherichia coli smooth LPS labelled with fluorescein isothiocyanate (FITC-LPS) to Chinese hamster ovary (CHO) cells transfected with human CD14 gene (hCD14-CHO), to MonoMac 6 cells and to endothelial cells. Our results showed that both forms of LPS display the same binding characteristics, and that the binding of FITC-LPS to cells was both CD14- and LBP-dependent for LPS concentrations up to 100 ng.mL-1. At LPS concentrations higher than 100 ng.mL-1 we observed CD14/LBP-independent binding. CD14/LBP-dependent binding was dose dependent, saturable, and enhanced in the presence of human pooled serum (HPS), and the monoclonal anti-CD14 antibody (MY4) or unlabelled LPS could outcompete it.