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.
Triantafilou
M
M
Department of Biological Sciences, University of Essex, Colchester, Essex, UK.
Triantafilou
K
K
Fernandez
N
N
D000818:Animals; D000954:Antigens, Surface / Q000276:immunology / Q000378:metabolism; D001426:Bacterial Proteins / Q000378:metabolism; D016466:CHO Cells; D002460:Cell Line; D006224:Cricetinae; D004731:Endotoxins / Q000235:genetics / Q000378:metabolism*; D004926:Escherichia coli; D005434:Flow Cytometry; D016650:Fluorescein-5-isothiocyanate; D006801:Humans; D018950:Lipopolysaccharide Receptors / Q000235:genetics* / Q000276:immunology; D008070:Lipopolysaccharides / Q000378:metabolism*; D011485:Protein Binding; D012475:Salmonella / Q000737:chemistry; D014162:Transfection
D000954:Antigens, Surface; D001426:Bacterial Proteins; D004731:Endotoxins; D018950:Lipopolysaccharide Receptors; D008070:Lipopolysaccharides; C044101:endotoxin, Escherichia coli; D016650:Fluorescein-5-isothiocyanate