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72 h, cultures were fed by adding 0.5 vol of medium containing vitamin D3 and incubated for an- other

24 h. AntibodiesandReagents. Anti-CD14 mAbs, 60b (IgG1)(27)and 26ic(IgG2b) (28), werekindly providedbyRobert F. Todd(Univer- sity ofMichigan, Ann Arbor). Anti-CD18 mAb, 60.3 (IgG2a)(29), was kindly providedbyJohn M. Harlan (UniversityofWashington, Seattle). Antibodies in ascites fluid were titrated either by FACS| analysis(BectonDickinson &

Co., Mountain View,CA) ofbinding to THP-1 cells,or by their ability to block LPSuptake (mAb 60b). They were usedat 1:500dilution in the experiments reported here. Nonimmune control antibody used for FACS* analysis (Becton Dickinson &

Co.) was a mixture of

2 #g/ml each of routine IgG1, 2a, 2b, and

3 isotypes (Coulter Immunology, Hialeah, FL), and FITC-labeled F(ab'

)2fragments of goat anti-mouse IgG (H + L) were from Tago,Inc. (Burlingame, CA). LBP,purified from rabbit acute phase serum, was generously provided by Peter S. Tobias (Scripps Clinic and Research Foundation, LaJolla, CA). Optimal amounts of LBP were determined empirically by measuring LPS uptake using eachlot ofLBP.Tissueculture-tested, endotoxin-tested (0.005 ng endotoxin/mg) BSA was obtained from Sigma Chem- ical Co. (St. Louis, MO). Double-stranded poly(dI-dC) was ob- tained from Pharmacia Fine Chemicals (Piscataway, NJ). Strict pyrogen-free conditions were maintained by using sterile dispos- able plasticware and pipette tips, and pyrogen-free distilled water (Baxter Healthcare Corp., Deerfield, IL). LPS and Lipid A Preparations. E. coliLCD25, a K12 derivative with Ra or Rb LPS core structure, was biosynthetically radiola- beled with [3H]acetate as described (30). Its sp act was 4,200 3H dpm/ng, and essentially all of the radioactivity was in the fatty acylchains. The radiohbeled LPSwas deacylatedusing AOAH (19). Spact of the dLPSpreparationsusedwere 2,885 and 3,020 dpm/ng. Both deacyhted and mock-treated LPSwere suspended(100/zg/ml) in 0.9% NaC1with

5 rag/m1 BSA and stored at -70~ Nonra- dioactive dLPS was produced from E. coliJ5 LPS by using trace amounts of [3H]LPSto follow the extent of deacylation. Samples containing deacylation reaction components without LPS ( mock dLPS'

) were used ascontrols. Synthetic lipid LA-14-PPfrom ICN Biomedicals, Inc. (Cleveland, OH), was suspended in PBS, soni- cated, and stored (0.5 mg/ml) at -70~ Before use, an aliquot of each LPS or LA-14-PP preparation was diluted to 2/zg/ml in RPMI-1640 containing 0.5 mg/ml BSA and sonicated for

1 s on low power with a sonifier with a small steel probe (model 450;

Branson Ultrasonics Corp., Danbury, CT), and the radioactivity in the sample was counted. LPS-LBP complexeswere prepared by mixing aliquots of sonicated LPS with LBP immediately before use (typically a 10-foldexcessof LBP by weight was used for LPS and dLPS, and a 30-fold excesswas used for LA-14-PP), incubated 10min at 37~ diluted with RPMI/BSA to the working concen- tration, and kept at room temperature until used. Tritium counting was performedin a liquid scintillationcounter (Tricarb4000Minaxi;

Packard, Downers Grove, IL) with external standardization and quench correction. Cell Stimulation. 96-h cultures of vitamin D3-treated THP-1 cellswere prepared in 6-wellplatesasdescribedabove.Nonadherent cellswere removed by washing with RPMI, and incubations were performed in the absenceof serum using 1ml ofRPMI containing penicillin, streptomycin, glutamine, and 0.5 mg/ml BSA. Addi- tions of LPS and other reagents were made in 10-100/zl volumes. After incubation, cellswere washed at room temperature oncewith