Response of equine monocytes to lipopolysaccharide from enteric and non-enteric bacteria
Lohmann, Katharina Luise
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Endotoxin (lipopolysaccharide, LPS) activates inflammatory cells by interacting with a cellular receptor complex consisting of cluster differentiation antigen 14 (CD14), Toll-like receptor 4 (TLR4) and MD-2. LPS antagonists are structurally atypical LPS compounds that do not activate inflammatory cells and in fact inhibit cellular activation by endotoxin. LPS antagonists may be of therapeutic value and offer a tool for investigation of ligand-receptor interactions and species-specific differences in the response to different LPS compounds. This report describes the results of an investigation of the biological activities of three structurally atypical LPS compounds in equine and human cells. LPS from Rhodobacter sphaeroides (RsLPS), Rhizobium galegae (R. galegae) and Rhizobium Sin-1 (R. Sin-1) stimulated tumor necrosis factor ? (TNF?) production in equine monocytes while they inhibited the response to enteric LPS in a human monocyte cell line, Mono Mac 6. Using transfection experiments, it was determined that RsLPS stimulated nuclear factor ?B (NF-?B) activation in equine cells but not in human cells, and that stimulation of equine cells occurred independent of CD14. It was further determined that the TLR4/MD-2 complex determined the equine-specific response to RsLPS. Using transfection experiments and binding assays, it was determined that LPS from R. galegae and R. Sin-1 activated equine cells via CD14, TLR4 and MD-2 and that these compounds competed with enteric LPS for binding to equine monocytes. Contrary to their effect on TNF? production, LPS from R. galegae and R. Sin-1 also stimulated NF-?B activation in cells expressing human receptor proteins, thereby putting into question a competitive mechanism of antagonism for these compounds. In an additional study, binding assays were used to estimate the number of binding sites for LPS on Mono Mac 6 cells and the affinity of LPS binding to these cells. Comparison to cell stimulation assays suggested the presence of spare receptors. Competition experiments further suggested an allosteric effect of certain LPS compounds on LPS binding to Mono Mac 6 cells.