Saturable CD14-dependent binding of fluorescein-labeled lipopolysaccharide to human monocytes

Abstract

We used rough lipopolysaccharide (ReLPS) to construct a fluorescein-labeled LPS (FITC-LPS) with a very high labeling efficiency that bound to isolated human monocytes in a CD14-dependent fashion and that in this respect behaved indistinctively from native LPS. The CD14-dependent binding could be inhibited either by a 1,000-fold excess of unlabeled LPS or by polymyxin B, bactericidal/permeability-increasing protein, cationic protein 18, or soluble CD14. Although this FITC-LPS preparation no longer possessed the ability to prime neutrophils for the production of reactive oxygen species or to stimulate human monocytes to produce tumor necrosis factor, activation of the Limulus amoebocyte lysate cascade was comparable to activation by native LPS. Binding to monocytes was enhanced by human pooled serum (HPS) or LPS-binding protein (LBP) for LPS concentrations up to 100 ng/ml and was completely CD14 dependent. For LPS concentrations exceeding 100 ng/ml, binding was still partially CD14 dependent, but not HPS or LBP dependent. CD14-dependent association of LPS with monocytes was shown to be totally saturable. In conclusion, we found an HPS- or LBP-dependent binding of FITC-LPS to monocytes that was CD14 dependent at up to 100 ng of LPS per ml, and saturation of binding was shown.