Granulocyte-macrophage colony-stimulating factor-differentiated bone marrow-derived dendritic cells were stimulated with the synthetic lipopeptide S-(2,3-bispalmitoyloxypropyl)-CGDPKHSPKSF (FSL-1) or the Escherichia coli lipopolysaccharide. FSL-1 induced the production of TNF-α and IL-12 by C57BL/6-derived bone marrow-derived dendritic cells but not by bone marrow-derived dendritic cells from Toll-like receptor 2-deficient (TLR2−/−) mice. Lipopolysaccharide induced the production of TNF-α and IL-12 by bone marrow-derived dendritic cells derived from either type of mice. FSL-1 did not induce production of IL-10 by bone marrow-derived dendritic cells from either type of mice, whereas lipopolysaccharide induced small amounts of IL-10 by bone marrow-derived dendritic cells from both types of mice. The upregulation by FSL-1 of the expression of CD80, CD86 and the MHC class II molecule IAb was dose- and time-dependent on the surfaces of C57BL/6-derived bone marrow-derived dendritic cells but not on the surface of TLR2−/−-derived bone marrow-derived dendritic cells. Lipopolysaccharide upregulated the expression of these molecules on the surfaces of bone marrow-derived dendritic cells from both types of mice. The expression of CD11c on the surfaces of C57BL/6-derived bone marrow-derived dendritic cells was upregulated by stimulation with both FSL-1 and lipopolysaccharide up to 12 h; thereafter, the expression was downregulated. The results suggest that FSL-1 can accelerate maturation of bone marrow-derived dendritic cells and this FSL-1 activity is mediated by TLR2.
Toll-like receptor 2
Mycoplasmas lack cell walls and are the smallest self-replicating micro-organisms. Mycoplasmal membrane-bound lipoproteins (LP) have been suggested to be one of the pathogenic factors because of their capability to activate macrophages, monocytes and fibroblasts (Mühlradt et al., 1996, 1997; Shibata et al., 1997, 2000; Razin et al., 1998; Dong et al., 1999). The active site of mycoplasmal LP has been shown to be the N-terminal lipopeptide moieties (Mühlradt et al., 1997; Shibata et al., 2000). We have synthesized a diacylated lipopeptide, S-(2,3-bispalmitoyloxypropyl)-CGDPKHSPKSF (FSL-1), on the basis of the structure of a 44-kDa lipoprotein of Mycoplasma salivarium that is capable of activating human gingival fibroblasts (Shibata et al., 2000). The synthesized lipopeptide FSL-1 can induce production of inflammatory cytokines such as Interleukin (IL)-6, IL-8 and monocyte chemoattractant protein-1 (MCP-1) by normal human gingival fibroblasts and production of tumor necrosis factor (TNF)-α by monocytes/macrophages (Okusawa et al., 2004).
The recognition of microbial products by the host system is mediated by members of the Toll-like receptors (TLRs) family. They are involved in the innate immune response by recognizing microbial conserved structures called pathogen-associated molecular patterns (PAMPs) (Akira et al., 2001; Takeda et al., 2003) such as lipopolysaccharide (LPS), bacterial lipoprotein, peptidoglycan, lipoteichoic acid, bacterial unmethylated CpG DNA, mycobacterial lipoarabinomannan and yeast mannans. The recognition of PAMPs leads to the activation of various intracellular signaling cascades that modulate nuclear translocation of the transcription nuclear factor NF-κB (Akira et al., 2001; Takeda et al., 2003), induction of cytokines and expression of effector molecules, such as the costimulatory molecules B7-1 (CD80) and B7-2 (CD86). Therefore, activated innate immunity leads to effective adaptive immunity.
In this study, we first focused on the biological activities of FSL-1 toward bone marrow-derived DCs (BMDCs) because the method for preparing BMDCs is well established. We found that FSL-1 is capable of activating BMDCs to induce production of the Th1-restricted cytokine IL-12 and expression of costimulatory molecules and major histocompatibility complex (MHC) class II in a TLR2-dependent manner.
Materials and methods
Sex-matched 8-week-old C57BL/6 mice (TLR2+/+) were purchased from Japan Clea (Tokyo, Japan). TLR2-deficient mice (TLR2−/−) from the same genetic background were generated by gene targeting as described previously (Takeuchi et al., 1999). All mice were maintained in specific pathogen-free conditions at our animal facility at Hokkaido University, and all experiments were carried out in accordance with the regulations of the Hokkaido University Animal Care and Use Committee.
Synthesis of the diacylated lipopeptide FSL-1
FSL-1 was synthesized according to the method described previously (Shibata et al., 2000). Briefly, side chain-protected Cys–Gly–Asp–Pro–Lys–His–Pro–Lys–Ser–Phe was built-up with an automated peptide synthesizer (model 433; PE Applied Biosystems, Foster City, CA). F-moc (9-fluorenylmethoxy)-S-(2,3-bispalmitoyloxypropyl)-cysteine (Novabiochem, Laeufelfingen, Switzerland) was manually coupled to the peptide-resin using a solvent system of 1-hydroxy-7-azabenzotriazole-1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide/CH2Cl2-N,N-dimethylformamide. The F-moc and resin were removed from the lipopeptide using trifluoroacetic acid. The lipopeptides were purified by preparative HPLC with a reverse-phase C18 column (30 × 250 mm). The purity of FSL-1 was confirmed by analytical HPLC with a reverse-phase C18 column (4.6 × 150 mm) to be 98%. The lipopeptide FSL-1 was dissolved in phosphate-buffered saline (PBS).
Preparation of BMDCs and cultures
Bone marrow-derived dendritic cells were prepared according to the method described previously (Inaba et al., 1992) with minor modifications. Briefly, bone marrow (BM) cells were obtained by flushing femurs and tibias with RPMI 1640 using a 26-gauge needle. The BM cells (1 × 106 cells mL−1) were cultured overnight in each well using 24-well plates in RPMI 1640 supplemented with 10% fetal bovine serum (FBS). Nonadherent cells were harvested and cultured in the same medium containing 10 ng mL−1 recombinant mouse granulocyte-macrophage colony-stimulating factor (GM-CSF) (Pepro Tec., Rocky Hill, NJ). On day 3, the medium was changed to a fresh medium containing 10 ng mL−1 GM-CSF. On day 6, nonadherent cells and loosely adherent cells were harvested and used for experiments as immature DCs. Immature DCs suspended in fresh RPMI 1640 were used for stimulation by FSL-1 or Escherichia coli LPS O55:B5 (Sigma, St Louis, MO).
Stimulation of BMDCs by FSL-1 or LPS
Bone marrow-derived dendritic cells (5 × 105) were seeded in a 24-well plate in RPMI 1640 containing 10% FBS. BMDCs were stimulated at 37°C for 12 h with various concentrations of FSL-1 or Escherichia coli LPS and centrifuged at 400 g for 10 min to separate the cells and culture supernatants. The cells were recultured for an additional 24 h with the same concentration of FSL-1 or LPS and centrifuged again to separate the cells and culture supernatants. This method was used to avoid secondary effects of cytokines produced and LPS- or FSL-1-induced tolerance. Pooled culture supernatants were used for measuring cytokine concentration using an enzyme-linked immunosorbent assay (ELISA) and the cells were used for analysis of the expression of cell surface antigens, costimulatory molecules and MHC class II molecules using flow cytometry as described below.
Determination of cytokines
Cell culture supernatants were assayed for various cytokines, including TNF-α, IL-12(p70) and IL-10, by using ELISA kits purchased from BD Pharmingen (San Diego, CA), Pepro Tech. and R&D Systems (Minneapolis, MN), respectively. Briefly, flat-bottomed 96-well Nunc-Immuno MaxSorp assay plates were coated overnight with the appropriate anticytokine antibodies. After blocking the plates with bovine serum albumin, the plates were incubated for 2 h with the culture supernatants and then incubated for 1 h with each of the biotin-conjugated anticytokine antibodies. Horseradish peroxidase-conjugated streptavidin was then added and developed with 3,3′,5,5′ tetramethyl benzidine (TMB) peroxidase substrate. The optical densities were measured at 405 nm using a microplate reader.
For flow cytometric analysis, BMDCs were incubated on ice (106 cells in 200-μL PBS with 1% bovine serum albumin, BSA) with antimouse CD32/CD16 (FcγII/III receptor). After 30 min, aliquots were washed and incubated in 200-μL PBS with 1% BSA with fluorescein isothiocyanate (FITC)-conjugated monoclonal antibodies (mAb) against CD11c, CD11b or CD8α and phycoerythrin (PE)-conjugated mAbs against CD80 (B7.1), CD86 (B7.2) or I-Ab (MHC class II) and appropriate isotype controls (BD Pharmingen). The cells were fixed in 0.1 M phosphate buffer containing 0.5% formaldehyde and then analyzed using a FACScan flow cytometer (BD Bioscience, Mountain View, CA). Data for 10 000 cells falling within appropriate forward and side light scatter gates were collected from each sample. Data were analyzed using CellQuest software (BD Bioscience).
Results and discussion
To determine the interaction of FSL-1 with DCs, we prepared BMDCs from TLR2+/+ and TLR2−/− mice according to the method of Inaba et al. (1992). GM-CSF-differentiated BM cells derived from TLR2+/+ (Fig. 1) and TLR2−/− (data not shown) were examined for surface expression of CD8α, CD11b and CD11c (Fig. 1, left panels) which are antigens normally used for characterization of DCs (Iwasaki & Medzhitov, 2004). The BMDCs strongly expressed CD11c and CD11b, but not CD8α (Fig. 1, right panels). This suggests that BM cells are differentiated into immature BMDCs (imBMDCs), because CD11c and CD11b are important marker antigens of mouse BMDCs (Brawand et al., 2002).
Surface expression of CD11c, CD11b and CD8α on bone marrow-derived dendritic cells differentiated by granulocyte-macrophage colony-stimulaing factor (GM-CSF). Bone marrow (BM) cells were cultured overnight in RPMI 1640 containing 10% fetal bovine serum. Nonadherent cells were harvested and cultured in the same medium supplemented with 10 ng mL−1 recombinant mouse GM-CSF. On day 3, the medium was changed to a medium containing 10 ng mL−1 GM-CSF. On day 6, nonadherent cells and loosely adherent cells were harvested and used for experiments as immature dendritic cells.
The next experiment was performed to determine whether FSL-1 or Escherichia coli LPS activates imBMDCs. The imBMDCs were stimulated for 12 h with FSL-1 or E. coli LPS and then washed and recultured for another 24 h in the presence of FSL-1 or LPS. The amounts of TNF-α, the Th1-restricted cytokine IL-12 and the Th2-restricted cytokine IL-10 released in the culture supernatant were measured using ELISA. FSL-1 induced the production of TNF-α by B6-derived BMDCs but not by TLR2KO-derived BMDCs. LPS-induced production of TNF-α by BMDCs from both types of mice (Fig. 2). Interestingly, the BMDCs were no longer capable of producing TNF-α after 12 h of stimulation (Fig. 2). LPS also induced production of IL-12 by both B6- and TLR2KO-derived BMDCs, whereas FSL-1 induced production of IL-12 only by B6-derived BMDCs (Fig. 2). In contrast to TNF-α, both FSL-1 and LPS induced IL-12 production even after 12 h of stimulation (Fig. 2). LPS also induced production of IL-10 by BMDCs derived from both types of mice, but the level of production was much lower than that of IL-12 or TNF-α (Fig. 2). FSL-1 did not induce production of IL-10 by BMDCs derived from either type of mice under the conditions used in this experiment (Fig. 2).
Differential cytokine production by Toll-like receptor (TLR)2+/+ and TLR2−/− bone marrow-derived dendritic cells (BMDCs) stimulated with S-(2,3-bispalmitoyloxypropyl)-CGDPKHSPKSF (FSL-1) and Escherichia coli lipopolysaccharide (LPS). BMDCs were stimulated at 37°C for 12 h with various concentrations of FSL-1 or Escherichia coli LPS and centrifuged at 400 g for 10 min to separate the cells and culture supernatants. The cells were recultured for an additional 24 h with the same concentration of FSL-1 or LPS and centrifuged again to separate the cells and culture supernatants. Pooled culture supernatants were used for measuring tumor necrosis factor-α, interleukin (IL)-12p70 and IL-10 by enzyme-linked immunosorbent assay. Results are representative of five experiments.
After 12-h stimulation, the culture supernatants were removed and then stimulated for another 24 h as described in Materials and methods. This method was used to avoid secondary effects of cytokines and LPS- or FSL-1-induced tolerance. At present, it is not known why BMDCs were no longer capable of producing TNF-α after being stimulated for 12 h with FSL-1 or LPS. There is a possibility that this phenomenon can be attributed to tolerance of BMDCs induced by LPS or FSL-1. If this is the case, it does not explain why IL-12 was produced even after 12 h of stimulation. It is known that both IL-12 and TNF-α are regulated by the transcription factor NF-κB (May & Ghosh, 1998). Another possibility is that the signaling pathway leading to the transcription of IL-12 upstream activation of NF-κB differs from that leading to TNF-α and that the BMDCs responsible for IL-12 production are different from those for TNF-α production because of the heterogeneity of BMDCs prepared in this study. Further studies are required to explain this phenomenon.
The mechanism of antigen presentation correlates with the ability to upregulate the expression of costimulatory molecules such as CD80 (B7.1) and CD86 (B7.2) on the surfaces of the APCs. T-cell activation generally requires a signal delivered via interaction of the T-cell receptor (TCR) with a specific antigen on MHC molecules and a costimulatory signal. Therefore, we examined the expression of costimulatory molecules such as CD80 (B7.1) and CD86 (B7.2), IAb (MHC class II) and CD11c, a DC marker, on BMDCs after stimulation with FSL-1 and LPS. The upregulation of these molecules by FSL-1 was both dose- and time-dependent on the surfaces of TLR2+/+-derived BMDCs, but not on the surfaces of TLR2−/−-derived BMDCs (Figs 3a and b). LPS, however, upregulated the expression of CD80 (B7.1), CD86 (B7.2) and IAb (MHC class II) on the surfaces of BMDCs from both types of mice (Fig. 3b). In addition, it was found that the expression of CD86 (B7.2) was upregulated more strongly than that of CD80 (B7.1) (Figs 3a and 3b). The expression of CD11c on the surfaces of TLR2+/+-derived BMDCs was upregulated by stimulation with both FSL-1 and LPS up to 12 h; thereafter, the expression was downregulated. This is in good agreement with previous findings where CD11c was downregulated as DCs mature (Pulendran, 2004).
Surface expression of CD11c, CD80 (B7.1), CD86 (B7.2) and IAb [Major histocompatibility complex (MHC) class II] on bone marrow-derived dendritic cells (BMDCs) stimulated with granulocyte-macrophage colony-stimulating factor (FSL-1) or Escherichia coli lipopolysaccharide (LPS). BMDCs were cultured for 0, 12 and 36 h in the absence or presence of FSL-1 or Escherichia coli LPS. Cells were washed and stained with anti-mouse CD11c, CD80 (B7.1), CD86 (B7.2), IAb (MHC class II) or appropriate isotype-specific antibodies, and analyzed by flow cytometry. Data profiles were obtained after analysis of 10 000 events. Results are representative of five experiments. (a) Representative histograms of CD80 (B7.1), CD86 (B7.2), IAb (MHC class II) and CD11c expression on BMDCs for 36 h with medium alone (bold line), 10 ng mL−1 FSL-1 (filled) and the isotype control (dotted line). The values in the histograms are the ratios of mean fluorescence intensities of FSL-1-stimulated BMDCs to unstimulated BMDCs. (b) BMDCs were incubated in the presence of indicated concentrations of FSL-1 or LPS for 12 or 36 h. The ratios of the mean fluorescence intensities of stimulated BMDCs to unstimulated BMDCs are shown.
In agreement with previous findings where immature DCs expressed low levels of MHC class II proteins and almost no costimulatory molecules (Pulendran, 2004), our results suggest that FSL-1 can act to accelerate the maturation of BMDCs and that this activity of FSL-1 is mediated by TLR2.
LPS upregulated the expression of CD86 and IAb more strongly in DCs derived from TLR2+/+ than from those derived from TLR2−/− (Fig. 3b). This appears to be at odds with the fact that LPS are recognized by TLR4 in combination with MD2 and CD14 (Takeda et al., 2003). However, it has previously been reported that preparations of LPS are contaminated with proteineous substances that are extremely biologically active (Morrison et al., 1976; Hogan & Vogel, 1987, 1988; Manthey et al., 1994; Manthey & Vogel, 1994; Hirschfeld et al., 2000). More recently, it was demonstrated that LPS prepared from Porphyromonas gingivalis LPS are contaminated with lipoproteins (Hashimoto et al., 2004). Therefore, the unusual upregulation of the expression of CD86 and IAb by LPS might be explained by the possibility that BMDCs were stimulated by both LPS and the contaminated lipoproteins through TLR2 and TLR4.
It has been reported that signaling via TLRs, especially TLR3, 4, 7 and 9, stimulates Th1 responses (Medzhitov & Janeway, 2000; Janeway & Medzhitov, 2002; Dillon et al., 2004; Iwasaki & Medzhitov, 2004). However, it is not clear whether TLRs can also induce Th2 responses. It has recently been reported that TLR2 ligands such as LP and lipopeptides are able to activate DCs to induce production of the Th2-restricted cytokine IL-10 and elicit Th2 responses in vivo (Rhabaoui et al., 2002; Agrawal et al., 2003; Fujita et al., 2003; Redecke et al., 2004). Although the present finding that FSL-1 is able to activate BMDCs to produce the Th1-restricted cytokine IL-12 strongly suggests that FSL-1 induces Th1 responses, the results were obtained by in vitro experiments. In vivo experiments should be carried out to conclusively determine whether FSL-1 is able to induce Th1 responses.
In conclusion, FSL-1 is capable of activating imBMDCs to produce the Th1-restricted IL-12 and to upregulate the expression of the costimulatory molecules CD80 (B7.1) and CD86 (B7.2) and IAb (MHC class II) in a TLR2-dependent manner.
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