Open AccessRough and smooth forms of fluorescein‐labelled bacterial endotoxin exhibit CD14/LBP dependent and independent binding that is influencedby endotoxin concentration
Author(s) -
Triantafilou Martha,
Triantafilou Kathy,
Fernandez Nelson
Publication year - 2000
Publication title -
european journal of biochemistry
Language(s) - English
Resource type - Journals
eISSN - 1432-1033
pISSN - 0014-2956
DOI - 10.1046/j.1432-1327.2000.01222.x
Subject(s) - chinese hamster ovary cell , lipopolysaccharide , cd14 , lipopolysaccharide binding protein , lipid a , fluorescein isothiocyanate , microbiology and biotechnology , tlr4 , monoclonal antibody , chemistry , flow cytometry , fluorescein , receptor , antibody , biology , biochemistry , endocrinology , immunology , fluorescence , physics , quantum mechanics
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.