ENZYMATICALLY DEACYLATED LIPOPOLYSACCHARIDE (LPS) CAN ANTAGONIZE LPS AT MULTIPLE SITES IN THE LPS RECOGNITION PATHWAY

Like other tetraacyl partial structures of lipopolysaccharide (LPS) an d lipid A, LPS that has been partially deacylated by acyloxyacyl hydro lase can inhibit LPS-induced responses in human cells, To identify the site(s) of inhibition in the LPS recognition pathway, we analyzed the apparent binding affinities and interactions of H-3-labeIed enzymatic ally deacylated LPS (dLPS) and [H-3]LPS with CD14, the LPS receptor, o n THP-1 cells. Using (i) incubation conditions that prevented ligand i nternalization and (ii) defined concentrations of LPS binding protein (LBP), which facilitates LPS and dLPS binding to CD14, we found that d LPS can antagonize LPS in at least three ways, 1) When the concentrati on of LBP in the medium was suboptimal for promoting LPS-CD14 binding, low concentrations of dLPS were able to compete with LPS for binding CD14, suggesting competition between LPS and dLPS for engaging LBP. 2) When LBP was present in excess, dLPS could compete with LPS for bindi ng CD14, but only at dLPS concentrations that were at or above its K-D for binding CD14 (100 ng/ml). 3) In contrast, substoichiometric conce ntrations of dLPS (1 ng/ml) inhibited LPS-induced (3 ng/ml) interleuki n-8 release without blocking LPS binding to CD14. Functional antagonis m was possible without competition for cell-surface binding because bo th LPS-induced interleukin-8 release and dLPS inhibition occurred at c oncentrations that were far below their respective CD14 binding K-D va lues. In addition to its expected ability to compete with-LPS far bind ing LBP and CD14, dLPS thus potently antagonizes LPS at an undiscovere d site that is distal to LPS CD14 binding in the LPS recognition pathw ay.