ANALYSIS OF LIPOPOLYSACCHARIDE-BINDING BY CD14

The cell surface protein CD14 binds bacterial lipopolysaccharide (LPS) in the presence of the serum protein, LPS-binding protein (LBP). This interaction is important for LPS-induced activation of mammalian myel oid cells. We performed quantitative studies of H-3-labeled LPS bindin g to human CD14 expressed on Chinese hamster ovary cells and on a huma n macrophage cell line (THP-1). At the concentrations studied (20-100 nM) LPS binding required the expression of CD14 and could be inhibited by a subset of anti-CD14 monoclonal antibodies. LBP was required for LPS binding to CD14. The binding occurred within 10 min and was relati vely unaffected by temperature over the range of 4-37-degrees-C. Quant itative binding assays were performed at 10-degrees-C, or at 37-degree s-C, using Chinese hamster ovary cells depleted of ATP. In both cases, 75-90% of the LPS could be released by treatment with phosphatidylino sitol-specific phospholipase C, suggesting that it remains associated with the glycosyl phosphatidylinostol-anchored CD14. The apparent diss ociation constant of recombinant human CD14 expressed on Chinese hamst er ovary cells for LPS at 10-degrees-C was 2.74 (+/- 0.99) x 10(-8) M; the apparent dissociation constant of CD14 expressed on THP-1 cells a t 10-degrees-C was 4.89 (+/- 1.42) x 10(-8) M. In both cell lines, at saturating LPS concentrations, the molar ratio of LPS bound per surfac e CD14 was approximately 20:1. At 37-degrees-C the apparent dissociati on constant of recombinant human CD14 for LPS at 37-degrees-C was 2.7 (+/- 1.2) x 10(-8) M, and the molar ratio of LPS bound per surface CD1 4 was approximately 8:1. Although the difference in molar ratio of LPS bound per surface CD14 at the two temperatures is difficult to interp ret, it is clear that at both temperatures the molar ratio is not 1:1. The basis of this phenomenon is unclear, but may involve the repeated leucine-rich motifs, which are found within CD14.