Enzymatically deacylated lipopolysaccharide (LPS) can antagonize LPS at multiple sites in the LPS recognition pathway.
Like other tetraacyl partial structures of lipopolysaccharide (LPS) and lipid A, LPS that has been partially deacylated by acyloxyacyl hydrolase 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 3H-labeled enzymatically deacylated LPS (dLPS) and [3H]LPS with CD14, the LPS receptor, on THP-1 cells. Using (i) incubation conditions that prevented ligand internalization and (ii) defined concentrations of LPS binding protein (LBP), which facilitates LPS and dLPS binding to CD14, we found that dLPS can antagonize LPS in at least three ways. 1) When the concentration 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 binding CD14, but only at dLPS concentrations that were at or above its KD for binding CD14 (100 ng/ml). 3) In contrast, substoichiometric concentrations of dLPS (1 ng/ml) inhibited LPS-induced (3 ng/ml) interleukin-8 release without blocking LPS binding to CD14. Functional antagonism was possible without competition for cell-surface binding because both LPS-induced interleukin-8 release and dLPS inhibition occurred at concentrations that were far below their respective CD14 binding KD values. In addition to its expected ability to compete with LPS for binding LBP and CD14, dLPS thus potently antagonizes LPS at an undiscovered site that is distal to LPS-CD14 binding in the LPS recognition pathway.
Kitchens
R L
RL
Department of Microbiology, University of Texas Southwestern Medical Center, Dallas 75235, USA.
Munford
R S
RS
D000215:Acylation; D015703:Antigens, CD / Q000378:metabolism; D015214:Antigens, Differentiation, Myelomonocytic / Q000378:metabolism; D001665:Binding Sites; D002021:Buffers; D002265:Carboxylic Ester Hydrolases / Q000378:metabolism*; D002460:Cell Line; D006801:Humans; D018950:Lipopolysaccharide Receptors; D008070:Lipopolysaccharides / Q000037:antagonists & inhibitors / Q000378:metabolism*; D009000:Monocytes / Q000378:metabolism
D015703:Antigens, CD; D015214:Antigens, Differentiation, Myelomonocytic; D002021:Buffers; D018950:Lipopolysaccharide Receptors; D008070:Lipopolysaccharides; D002265:Carboxylic Ester Hydrolases; C048964:acyloxyacyl hydrolase