Diphosphoryl lipid A from Rhodobacter sphaeroides inhibits complexes that form in vitro between lipopolysaccharide (LPS)-binding protein, soluble CD14, and spectrally pure LPS

An early event in septic shock is the activation of macrophages by a complex consisting of lipopolysaccharide (LPS), LPS-binding protein (LBP), and the cell surface antigen CD14, The complexes that form between [H-3]ReLPS (ReLPS is deep-rough-chemotype hexacyl LPS from B. coli D31m4), soluble CD14 (sCD14), and LBP were analyzed by two independent methods, native (nondenaturing) gel electrophoresis and size-exclusion high-performance liquid chromatography (HPLC), This is the first reported use of HPLC to purify and study LPS-protein complexes, The binding of [H-3]ReLPS to LBP and sCD14 was inhibited by preincubation with diphosphoryl lipid A from Rhodobacter sphaeroides (RsDPLA), a potent LPS antagonist, In addition, [3H]ReLPS bound to LBP and to a truncated form of sCD14 [sCD14((1-152))] that contained the LPS binding domain. Binding to both proteins was blocked by RsDPLA. Thus, RsDPLA competes in a 1:1 ratio for the same or nearby binding sites on ReLPS complexes, Analysis by sodium dodecyl sulfate-polyacrylamide gel electrophoresis of aggregated ReLPS eluting from the HPLC indicated that only LBP not sCD14, was bound to the aggregated ReLPS. This finding supports the binary model of LPS complex formation with LBP and sCD14.