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Lipopolysaccharide (LPS) Partial Structures Inhibit Responses to LPS in a Human Macrophage Cell Line without Inhibiting LPS Uptake by a CD14-mediated Pathway By Richard L.

Kitchens,* Richard J. Ulevitch,~ and RobertS. Munford* From the *Departments of Microbiology and Internal Medicine and the Immunology Graduate Program, University of Texas Southwestern Medical Center, Dallas, Texas 75235;

and the *Department of Immunology, Research Institute of Scripps Clinic, La Jolla, California

92037 Sllmmary Lipopolysaccharides (LPS) that lack acyloxyacylgroups can antagonize responses to LPS in human cells. Although the site and mechanism of inhibition are not known, it has been proposed that these inhibitory molecules compete with LPS for a common cellular target such as a cell-surface binding receptor. In the present study, we used an in vitro model system to test this hypothesis and to evaluatethe role of CD14 in cellular responses to LPS. Cells of the THP-1 human monocyte- macrophage cell line were exposed to 1,25 dihydroxyvitamin D3 to induce adherence to plastic and expressionof CD14, abinding receptor for LPS complexedwith LPS-bindingprotein (LBP). The uptake of picograms of [3H]LPS (agonist) and enzymatically deacylated LPS [3H]dLPS (antagonist) was measured by exposing the cells to the radiolabeled ligands for short incubation periods. The amounts of cell-associatedLPS and dLPSwere then correlated with cellular responses by measuring the induction of nuclear NF-KBbinding activity and the productionof cell-assodated interleukin (IL)-IB. We found that similar amounts of [3H]LPS or [3H]dLPS were taken up by the cells. The rate of cellular accumulation of the ligands was greatly enhanced by LBP and blocked by a monoclonal antibody to CD14 (mAb 60b), yet no cellular responses were induced by dLPS or dLPS-LBP complexes. In contrast, LPS stimulatedmarkedincreasesof NF-rB binding activity and Ibl~. These responses were enhanced by LBP and inhibited by mAb 60b. dLPS and its synthetic lipid A counterpart, LA-14-PP (also known as lipid Ia, lipid IVa, or compound 406) strongly inhibited LPS-inducedNF-KBand IL1B, yet neither antagonist inhibited the uptake of LPS via CD14. dLPS did not inhibit NF-KB responses to tumor necrosis factor (TNF)c~ or phorbol ester. Our results indicate that (a) both stimulatory and nonstimulatory ligands can bind to CD14 in the presence of LBP;

(b) the mechanism of inhibition by dLPS is LPS-specific, yet does not involve blockade of LPS binding to CD14;

and (c) in keeping with previous results of others, large concentrations of LPS can stimulate the cells in the absence of detectable binding to CD14. The findings indicate that the site of dLPS inhibition is distal to CD14 binding in the LPS signal pathway in THP-1 cells, and suggest that molecules other than CD14 are important in LPS signaling. nimals have sensitive mechanismsfor recognizing and re- sponding to gram-negativebacterial LPS (also called en- dotoxin). Severalcellular mechanisms for binding LPS have been described (1-8) and recent evidence suggests that the uptake of LPS by cells of monocytic origin (macrophages, monocytes) or neutrophils is linked to LPS recognition and cellular responses. However, the relationships between LPS- binding proteins in the cellmembrane and signalingpathways are not understood. CD14, a glycosylphosphatidylinositol-anchored protein ex- pressed on the surface of monocytes, macrophages, and acti- vated neutrophils (9-11), has been shown to be a receptor for complexes of LPS and LPS-binding protein (LBP)1 (8, 12). Laboratory studies with isolated monocytes, neutrophils, 1Abbreviationsusedin thispaper:AOAH, acyloxyacylhydrohse;