Host Defense Mechanisms Triggered by Microbial Lipoproteins Through Toll-Like Receptors

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THP-1 cells [American Type Culture Collection (ATCC), Manassas, VA] were stimulated with M. tuberculosis lysate and various subcellular fractions in a range of concentrations from 0.1 to 10 μg/ml. Cell-free supernatants were collected at 20 hours and assayed for IL-12 p40 by enzyme-linked immunosorbent assay (ELISA) (Pharmingen, San Diego, CA). THP-1 cells were plated at a concentration of 2 × 10⁵ cells/well in 96-well plates. The cells were cultured in the presence of various M. tuberculosispreparations at 37°C in a CO₂ incubator with RPMI 1640 and 10% heat-inactivated fetal bovine serum (FBS). Cell-free supernatants were harvested 20 hours later and stored at −20°C until assayed for cytokine concentrations by ELISA as previously described (38, 39). The amount of lipopolysaccharide in all preparations was measured quantitatively with a Limulus Amoebocyte Lysate assay (Whittaker Bioproducts, Walkersville, MD) and found to be <40 pg per microgram of M. tuberculosis fraction or protein, an amount that did not stimulate IL-12 activity by itself.

The mycolyl arabinogalactan peptidoglycan complex (mAGP) was also derived from the cell wall pellet after SDS detergent extraction. Most of the IL-12 p40–inducing activity was associated with the SCWP fraction compared with mAGP (see supplementary material available at www.sciencemag.org/feature/data/1040444.shl). Because macrophage phagocytosis of protein-adsorbed particulate suspensions has been reported to induce IL-12 release, we filtered both fractions through a 0.2-μm sterile filter. The SCWP fraction retained most of its IL-12 p40–inducing activity after filtration, whereas the mAGP suspension did not induce IL-12 p40.

Cell wall–associated proteins were obtained by suspending the cell wall pellet at a concentration of 1 mg of protein per milliliter of phosphate-buffered saline (PBS) (pH 7.4) containing 2% SDS. The proteins were extracted for 2 hours at room temperature followed by centrifugation at 27,000g for 30 min. The supernatant was applied to a Sephacryl S-200 column, and proteins were eluted at a flow rate of 0.5 ml/min with PBS (pH 7.4), 2% SDS. Fractions were pooled on the basis of the relative molecular weight of the products as determined by SDS–polyacrylamide gel electrophoresis (SDS-PAGE), and SDS was removed. Specific pools of size-fractionated SCWP were further purified by preparative isoelectric focusing. Specifically, the desired fractions were dialyzed against 4 M urea, 10% glycerol and brought to a final volume of 50 ml with the same. NP-40 was then added to a final concentration of 1%, and Pharmalytes pH 5–7 and pH 3–10 were added to a final concentration of 4 and 1%, respectively. This suspension was loaded into a 50-ml Rotofor cell (BioRad Laboratories, Hercules, CA) and electrophoresed with 15-W constant power for 4 hours. Fractions were collected, dialyzed against 100 mM NH₄HCO₃, and stored at −20°C. The fractions were loaded onto a 13.5% preparative SDS-polyacrylamide gel (16 cm by 20 cm by 1.5 mm) and electrophoresed at 20 mA for 1.5 hours. The resolved proteins were visualized by using Coomassie Brilliant Blue R250, excised from the gel, and electroeluted with a BioRad 422 Electro-Eluter with 50 mM NH₄HCO₃. At each step, fractions were tested for their ability to induce IL-12 p40, and the fractions with potent activity were further purified. Rotofor fraction number 4 was electrophoresed under denaturing conditions on a 4 to 20% tris-glycine minigel and transferred to nitrocellulose. Areas containing the four protein bands noted in Fig. 1B were isolated and solubilized as described previously (11) and used to stimulate THP-1 cells.

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Peripheral blood mononuclear cells were isolated on Ficoll-Paque gradients (Pharmacia LKB Biotechnology, Piscataway, NJ) and cultured (10⁶ cells/well) for 2 hours in 96-well plates at 37°C in a CO₂ incubator with RPMI 1640 and 10% heat-inactivated FBS. Nonadherent cells were removed and the medium replaced with the addition of 19-kD lipoprotein at 1, 10, and 100 ng/ml. Neutralizing monoclonal antibody to human TNF-α (Endogen, Cambridge, MA; Biosource, Camarillo, CA) and a mouse isotype control [immunoglobulin G1 (IgG1)] were added to some cultures at 5 and 10 μg/ml. Supernatants were harvested 16 hours later and assayed by IL-12 p40 ELISA (Pharmingen, San Diego, CA). All samples were assayed in duplicate. Previously, it was demonstrated that mycobacteria infection does not induce IL-12 in TNFR1 knockout animals [I. E. Flesch et al., J. Exp. Med. 181, 1615 (1995)].

Adherent monocytes were isolated from 11 tuberculin-negative healthy donors (13). Cells were stimulated with the 19-kD lipoprotein at a concentration of 10 ng/ml for 20 hours, and IL-12 p70 amounts were determined by ELISA with monoclonal anti–IL-12 p70 and antibodies 2OC2 and POD-4D6 as described (39). Under these conditions the monocytes from 8 out of 11 donors were able to produce measurable IL-12 p70 amounts 3 to 13 times above background levels in the range of 10 to 40 pg/ml. All samples were assayed in duplicate.

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All RAW 264.7 cell (ATCC, Manassas, VA) transfections were carried out with the Superfect protocol (Qiagen, Valencia, CA) at a ratio of 1 μg of DNA to 3 μl of Superfect for 2.5 hours. After a PBS wash, cells were divided into two wells and left unactivated or stimulated with LPS or lipoproteins for 24 hours and then harvested for the chloramphenicol acetyltransferase (CAT) assay. In IL-12 p40 and inducible nitric oxide synthase (iNOS) promoter induction studies, RAW 264.7 cells were transiently transfected with either IL-12 p40 (15) or iNOS promoter (36), CAT reporter plasmid (2 μg), and HSP70–β-Gal construct (0.5 μg) as an internal control. In the TLR-2 dn1 overexpression experiments, increasing doses of TLR-2 dn1 expression plasmid (100 ng, 500 ng, 1 μg, and 2 μg) (19) were transfected with the IL-12 p40 promoter construct and β-Gal as an internal control. The amount of expression plasmid was normalized to 2 μg with expression plasmid lacking the TLR-2 dn1 coding sequence (pCDNA3). IL-12 p40 promoter transfectants were either left unactivated or stimulated with LPS, 19-kD lipoprotein, or OspA at 50 ng/ml or Tp47 lipopeptide (24) at 10 μM for 24 hours. iNOS promoter transfectants were stimulated with LPS, 19-kD lipoprotein, OspA, or deacylated OspA (d-OspA) from 0.01 to 100 ng/ml, or left unstimulated for 24 hours. The iNOS promoter was also cotransfected with the TLR-2 dn1 construct (1 μg) or vector control (1 μg) and stimulated with LPS at 1 ng/ml, 19-kD lipoprotein and OspA at 5 ng/ml, or Tp47 lipopeptide at 10 μM for 24 hours. IL-12 p40 and iNOS promoter activities were measured according to CAT activity (percent chloramphenicol acetylation) with a phosphorimager. Data were normalized to a cotransfected β-Gal construct for transfection efficiency.

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THP-1 cells were stimulated with LPS, OspA, or d-OspA at 1 μg/ml (31). Supernatants were then assayed for IL-12 p40 by ELISA. LPS and OspA induced IL-12 p40 from THP-1 cells in a range of 1000 to 3500 pg/ml, whereas the amount of IL-12 p40 induced by d-OspA was below the level of detection.

HEK 293 cells not expressing human TLR-2 (detected with monoclonal anti–TLR-2 or reverse-transcriptase polymerase chain reaction) or HEK 293 clones stably transfected to express human TLR-2 were then further transfected with CD14 and reporter constructs as previously described (19). We plated 10⁵ cells/well in six-well plates and transiently transfected them the following day with the NF-κB responsive E-selectin (ELAM) gene enhancer luciferase (pGL3) reporter gene (0.5 μg) and a β-galactosidase reporter plasmid (0.5 μg) as an internal control by the Superfect protocol at a 1:3 ratio of DNA (micrograms to Superfect (microliters). Cells were then incubated with the DNA-Superfect mixture for 2 hours and washed, and multiple transfectants were pooled and divided into separate wells for activation by a titration of LPS or lipoprotein stimuli (19-kD lipoprotein, Tp47, and OspA). Twenty-four hours later cells were stimulated for 6 hours then lysed in 200 μl of reporter lysis buffer (Promega, Madison, WI), and 20 μl was used in the luciferase assay. HEK 293 control cells and TLR-2 stable clones were also cotransfected with a CD14 expression plasmid (19) (1 μg) or vector control (pCDNA3, 1 μg) with the same protocol.

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Adherent monocytes were isolated as described previously (14). Cells were treated with no antibody, mouse anti–human TLR-2 neutralizing monoclonal antibody, or an isotype control mouse IgG1 (10 μg/ml) for 30 min before stimulation with LPS or 19-kD lipoprotein (50 ng/ml) and incubated for 16 hours. Supernatants were then harvested and assayed for IL-12 p40 by ELISA. CD40L- and interferon-γ (IFN-γ)−stimulated adherent monocytes were used to control for the anti–TLR-2 blocking of lipoprotein-induced IL-12. Adherent monocytes were treated with human IFN-γ (100 units/ml; Endogen, Cambridge, MA) for 16 hours to up-regulate CD40 expression. Cells were then treated with antibodies as stated above, then stimulated with soluble CD40L trimer (250 ng/ml; Immunex, Seattle, WA) for 16 hours. Neither antibody nor IFN-γ alone induced IL-12 production.

We thank P. Sieling, G. Cheng, M. Mark, C. Nathan, and H. Herschman for scientific discussion; M. Gately (Hoffman-La Roche) for POD-46D; and C. Nathan (Cornell Medical College, New York, NY) for the iNOS promoter. Supported by the NIH (to R.L.M., P.J.B., B.R.B., M.V.N.), the Howard Hughes Medical Institute (to B.R.B. and S.T.S.), the United Nations Development Programme/World Bank/World Health Organization Special Program for Research and Training in Tropical Diseases (IMMLEP), and the Dermatology Research Foundation of California, Incorporated.