SATURABLE CD14-DEPENDENT BINDING OF FLUORESCEIN-LABELED LIPOPOLYSACCHARIDE TO HUMAN MONOCYTES

We used rough lipopolysaccharide (ReLPS) to construct a fluorescein-la beled LPS (FITC-LPS) with a very high labeling efficiency that bound t o isolated human monocytes in a CD14-dependent fashion and that in thi s respect behaved indistinctively from native LPS, The CD14-dependent binding could be inhibited either bg a 1,000-fold excess of unlabeled LPS or by polymyxin B, bactericidal/permeability-increasing protein, c ationic protein 18, or soluble CD14. Although this FITC-LPS preparatio n no longer possessed the ability to prime neutrophils for the product ion of reactive oxygen species or to stimulate human monocytes to prod uce tumor necrosis factor, activation of the Limulus amoebocyte lysate cascade was comparable to activation bg native LPS, Binding to monocy tes was enhanced by human pooled serum (HPS) or LPS-binding protein (L BP) for LPS concentrations up to 100 ng/ml and was completely CD14 dep endent, For LPS concentrations exceeding 100 ng/ml, binding was still partially CD14 dependent, bat 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 t o monocytes that was CD14 dependent at up to 100 ng of LPS per ml, and saturation of binding was shown.