PDF《Pathway》

dLPS, enzymatically deacylatedLPS lacking secondary fatty acylchains;

DTT, dithiothreitol;

EMSA,electrophoreticmobilityshiftassay;

LBP,LPS-binding protein;

LPS-LBPand dLPS-LBP,complexesofLPSand dLPS,respectively, with LBP.

485 J. Exp. Med.

9 The Rockefeller University Press 90022-1007/92/08/0485/10 $2.00 Volume

176 August

1992 485--494 on November 3,

2018 jem.rupress.org Downloaded from http://doi.org/10.1084/jem.176.2.485 Published Online:

1 August,

1992 | Supp Info: freshly drawn whole blood ex vivo (8, 12), and CD14- transfected 70Z/3 cells (13) support the contention that the LBP/CD14-dependent pathway is closely linked to initiation of cellular responses. Two models have been proposed to ex- plain how this pathway might function in LPS signaling (1, 2). In the first modal, LPS binding to CD14 directly stimu- lates the cell, whereas in the second, the CD14-LPS interac- tion facilitates the subsequent interaction of LPS with an- other signaling molecule. A direct signaling role for CD14 is suggested by reports that certain anti-CD14 mAbs can mimic some of the effects of LPS in human monocytes (14, 15). The second model is supported by the observation that increasing concentrations of LPS can overcome the ability of blockade or depletion of CD14 to inhibit TNFol production (8). The study of lipid A analogs that can function as LPS an- tagonists should provide useful information about the LPS signal pathway. One such analog is produced by the leuko- cyte enzyme, acyloxyacyl hydrolase (AOAH), which selec- tively removes secondary acyl chains from the lipid A region of LPS (16, 17). Several laboratories have reported that enzy- matically deacylated LPS (dLPS) (18-21) and its lipid A coun- terpart (known variously as synthetic analogs LA-14-PP or 406, or biosynthetic precursors Ia or IVa) (21-26) can inhibit the ability of LPS or lipid A to stimulate human cells. Al- though most investigators have assumed that these inhibi- tors compete with LPS for a common cellular target mole- cule, the site and mechanism of inhibition have not been determined. In this paper we describe the uptake of picograms of bio- synthetically radiolabeled Escherichia coli LPS and dLPS by the human monocyte-macrophage cell line, THP-1. (The term uptake is used here to refer to the association of LPS with the cell and is a measure of both membrane-bound and inter- nalized LPS.) The uptake of both of these ligands was en- hanced in the presence of LBP and blocked by a mAb to CD14. LBP also enhanced the ability of LPS to induce NF-KB and Iblf3 responses by these cells, but neither dLPS nor dLPS- LBP triggered these responses. Remarkably, dLPS-LBP and LA-14-PP-LBP inhibited responses to LPS-LBP without di- minishing LPS uptake by the cells. These observations indi- cate that CD14 can participate in the cellular uptake of both stimulatory (LPS) and nonstimulatory (dLPS) ligands. More- over, the mechanism of inhibition by dLPS does not involve inhibition of LPS binding to CD14. Our data suggest that dLPS may block the binding of LPS to a low-abundance fig- naling molecule, or that the uptake of dLPS may initiate a negative signal that counteracts or blocks the LPS signal to the cell. Materials and Methods Cell Cultur~ THIXl cellswere obtained from Dr. Dario C. AI- tieri (The ScrippsResearchInstitute, LaJolla, CA) and were grown in RPMI-1640with 7% fetalbovine serum (heat-inactivatedat 56~ for

30 rain) (Hyclone Laboratories Inc., Logan, UT),

2 mM L-glutamine,

50 U/ml penicillin G, and 50/~g/ml streptomycin in a 5% COz atmosphere at 37~ To minimize cell variability, the cells were revived from frozen stock every 2-3 too. To induce ad- herence to plastic and expression of CD14, cukures were grown in 6-wellplates (Costar,Cambridge, MA) in the presenceof0.1/~M 1,25-dihydroxy vitamin D3 (Biomol Res. Laboratories, Inc., Plymouth Meeting, PA). After