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(Journal of Leukocyte Biology. 2001;69:1036-1044.)
© 2001 by Society for Leukocyte Biology

Different Toll-like receptor agonists induce distinct macrophage responses

Bryan W. Jones*, Terry K. Means*, Kurt A. Heldwein*, Marc A. Keen{dagger}, Preston J. Hill{dagger}, John T. Belisle{dagger} and Matthew J. Fenton*

* The Pulmonary Center, Boston University School of Medicine, Boston Massachusetts, and
{dagger} Mycobacteria Research Laboratories, Department of Microbiology, Colorado State University, Ft. Collins, Colorado

Correspondence: Dr. Matthew J. Fenton, Pulmonary Center R-220, Boston University School of Medicine, 80 East Concord St., Boston MA 02118-2394. E-mail: mfenton{at}bu.edu


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
We previously reported that gram-negative bacterial lipopolysaccharide (LPS) activates cells via Toll-like receptor (TLR) 4, whereas the mycobacterial cell wall glycolipid lipoarabinomannan (LAM) activates cells via TLR2. We also identified a secreted TLR2 agonist activity in short-term culture filtrates of Mycobacterium tuberculosis bacilli, termed soluble tuberculosis factor (STF). Here we show that STF contains mannosylated phosphatidylinositol (PIM) and that purified PIM possesses TLR2 agonist activity. Stimulation of RAW 264.7 macrophages by LPS, LAM, STF, and PIM rapidly activated nuclear factor (NF)-{kappa}B, activator protein-1 (AP-1), and mitogen-activated protein (MAP) kinases. These TLR agonists induced similar levels of NF-{kappa}B and AP-1 DNA-binding activity, as well as trans-activation function. Unexpectedly, these TLR agonists induced tumor necrosis factor {alpha} secretion, whereas only LPS was capable of inducing interleukin-1ß and nitric oxide secretion. Thus, different TLR proteins are still capable of activating distinct cellular responses, in spite of their shared capacities to activate NF-{kappa}B, AP-1, and MAP kinases.

Key Words: signal transduction • tuberculosis • innate immunity


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Mammalian Toll-like receptor (TLR) proteins have recently been shown to play important roles in host cell responses to bacteria and bacterial products in vitro. Several published reports have shown that the TLR2 and TLR4 proteins mediate cellular activation by a variety of bacterial products, including gram-negative bacterial lipopolysaccharide (LPS), the mycobacterial glycolipid lipoarabinomannan (LAM), bacterial lipoproteins, peptidoglycan, and zymosan [reviewed in ref. 1 ]. In vivo, knockout mice that lack functional TLR2 and TLR4 genes exhibit selective defects in their responses to both bacteria and bacterial products [2 3 4 ]. More recently, TLR4 was implicated in the cellular recognition of respiratory syncytial virus [5 ], suggesting that TLR proteins also participate in the innate immune response to viral pathogens. Activation of TLR-dependent signaling pathways leads to the activation of genes that participate in innate immune responses. These responses include the expression of cytokines [tumor necrosis factor (TNF)-{alpha}, Interleukin (IL)-1ß, IL-6, and IL-12], coactivator molecules (B7.1), and nitric oxide (NO) [6 , 7 ]. Currently, nine distinct TLR proteins have been identified [8 9 10 11 ], although the specific roles that these receptors play in regulating host innate immune responses remain poorly understood.

Different TLR proteins recognize a variety of chemically diverse bacterial products. LPS is predominantly recognized by TLR4 [4 , 12 , 13 ], whereas LAM, peptidoglycan, and bacterial lipoproteins are recognized by TLR2 [7 , 14 15 16 ]. Although it is clear that purified bacterial products can initiate TLR-dependent signaling, the relative contributions of different TLR proteins, as well as TLR-independent signaling, to cellular responses induced by whole bacteria have only recently been examined. Takeuchi et al. reported that TLR4-deficient murine peritoneal macrophages exhibit normal production of TNF-{alpha} and IL-6 after stimulation with heat-killed Staphylococcus aureus in vitro, compared with that of wild-type cells [17 ]. In contrast, TLR2-deficient macrophages exhibited a moderate reduction in S. aureus-induced cytokine production compared with that of wild-type cells. We recently compared the in vitro responses of normal and TLR-deficient macrophages to live Mycobacterium tuberculosis bacilli. These studies revealed that TNF-{alpha} production is largely dependent on cellular activation via TLR4, whereas M. tuberculosis-induced NO production is independent of TLR proteins [18 ]. Although cellular responses to purified bacterial products often provide little insight into responses initiated by challenge with whole bacteria, purified TLR agonists are still essential tools for dissecting the signal transduction pathways that mediate TLR-dependent cellular responses.

TLR proteins are members of a larger family of receptors, which includes the type I IL-1 receptor, the IL-18 receptor, and the cytosolic adapter protein (AP) MyD88 [19 ]. The engagement of some TLR proteins by bacterial products sometimes requires coreceptors, such as CD14 and MD-2 [20 , 21 ]. Intracellular signaling triggered by engagement of the IL-1 receptor, the IL-18 receptor, and the TLR proteins involves a shared cascade of APs and kinases [reviewed in ref. 22 ]. These include MyD88, IL-1 receptor-associated kinase (IRAK), TNF receptor-associated factor 6 (TRAF6), and several mitogen-activated protein (MAP) kinase kinase kinases. This signaling pathway ultimately leads to the activation of transcription nuclear factor (NF)-{kappa}B and phosphorylation of MAP kinases, although these events are not the only consequences of TLR-mediated signaling. For example, both IL-1ß and LPS have been reported to activate several additional protein kinases [23 24 25 ], lipid kinases [26 , 27 ], and phospholipases [28 , 29 ]. To date, no studies have directly compared the signaling pathways activated by different TLR agonists.

We have now compared the capacities of different TLR agonists to trigger several signal transduction pathways and ultimately to activate selected cellular responses. Here we report that two mycobacterial TLR2 agonists, soluble tuberculosis factor (STF) and LAM, induce a different pattern of cellular responses than that induced by the TLR4 agonist LPS.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Cell lines and reagents
The RAW 264.7 murine macrophage (TIB-71) and Chinese hamster ovary cell (CHO)-K1 fibroblast (CCL-61) cell lines were obtained from the American Type Culture Collection (ATCC; Manassas, VA). RAW 264.7 macrophages, CHO fibroblasts, and CHO-derived cell lines were cultured as we have previously described [14 ]. The CHO-derived cell lines 3E10 [CHO/CD14/endothelial leukocyte adhesion molecule (ELAM)-CD25], 3E10/TLR2, and 3E10/TLR4 were generated and cultured as previously described [15 , 16 , 30 ]. These 3E10/TLR cells expressed FLAG epitope-tagged human TLR proteins. Clones that expressed similar levels of TLR proteins, measured by flow cytometry using an anti-FLAG antibody, were selected for further study. Furthermore, the TLR2- and TLR4-expressing cell lines were generated from the same CHO/CD14 parental clones; therefore, each line also expressed the same levels of CD14. All medium components contained a <10-pg/mL final concentration of LPS as measured with a Limulus amoebocyte lysate kit (BioWhittaker, Walkersville, MD).

In experiments using 3E10 cells, which contain a stably transfected CD25 reporter gene under the control of the NF-{kappa}B-dependent ELAM-1 promoter, CD25 expression was measured by flow cytometry as previously described [14 ]. Data were collected using CellQuest software (Becton Dickinson, Bedford, MA) and expressed as the ratio (fold activation) of the percent of CD25+ cells in unstimulated and stimulated cell populations (gated to exclude the lowest 5% of cells based on mean fluorescence). The 95% confidence limit for nonspecific fluorescence was established using isotype control antibodies. Fluorescein isothiocyanate and phosphatidylethanolamine-conjugated anti-human CD25 monoclonal antibodies were purchased from Becton Dickinson.

LPS (purified from Eschericia coli 055:B5) was purchased from Sigma (St. Louis, MO) and repurified by the method of Hirschfeld et al. [31 ]. Mycobacterial arabinose-capped LAM, purified from a rapidly growing avirulent Mycobacterium species, and mannosylated phosphatidylinositol (PIM) were purified as previously described [32 , 33 ]. The levels of contaminating LPS in the LAM and PIM preparations were determined using a quantitative Limulus lysate assay (BioWhittaker) and were <1-pg/mL final concentrations in all experiments.

Preparation of mycobacterial STF and culture filtrate factors
M. tuberculosis strain H37Ra (ATCC 25177) was purchased from the ATCC. Bacterial cultures were grown in LPS-free Middlebrook 7H9 medium supplemented with Tween 80 and albumin-deoxycholate medium supplement (Difco, Detroit, MI) at 37°C in LPS-free flasks under biosafety-level-3 conditions to mid-logarithmic phase [optical density at 620 nm, 0.4). Bacteria were then removed by centrifugation followed by two rounds of filtration through a 0.22-µm-pore-size membrane. This short-term culture filtrate was digested with proteinase K (100 µg/mL) for 18 h at 56°C and then used as a crude source of STF. Further purification of STF was accomplished by extraction with Triton X-114 as previously described [34 ]. Triton X-114 was added to crude ice-cold STF preparations until a final concentration of 4% was achieved. This mixture was incubated overnight at 4°C on a rotating wheel and then warmed to 37°C to promote the formation of two phases. The lower phase containing the Triton X-114 and glycolipids was removed, and the glycolipids were recovered by acetone precipitation. Culture filtrates were also subjected to size fractionation using preparative sodium dodecyl sulfate (SDS)-15% polyacrylamide gels [35 ]. The resolved products were recovered by electroelution using a Whole Gel Eluter (Bio-Rad Laboratories, Hercules, CA) [35 ]. Individual fractions were lyophilized, dissolved in LPS-free phosphate-buffered saline, and adjusted to 1 µg of total protein per µL with phosphate-buffered saline.

Measurement of cytokine and NO levels
TNF-{alpha} and IL-1ß protein levels in culture supernatants were determined using a specific enzyme-linked immunosorbent assay (ELISA) (R&D Systems, Minneapolis, MN), as recommended by the manufacturer. Levels of NO catabolite nitrite in the culture supernatants were measured using the Griess reagent assay as previously described [36 ]. All assays were performed in triplicate, and data are expressed as mean values ± SD. The data were subsequently analyzed using an analysis of variance to determine statistical significance.

Measurement of TNF-{alpha}, IL-1ß, and NOS2 mRNA levels by reverse transcription-PCR
Total RNA from RAW 264.7 murine macrophages was purified using RNeasy (Qiagen, Valencia, CA) as recommended by the manufacturer. Reverse transcriptase (RT) reactions to generate cDNA were performed using AMV RT (Promega, Madison, WI). PCR was performed using 0.5 µg of cDNA, 0.12 µM oligonucleotide primers (each), 2 mM MgCl2, 0.2 mM deoxynucleotide triphosphatase, and 1 U of Taq polymerase (Promega) in a final reaction volume of 25 µL. Thirty amplification cycles were performed (each cycle consisting of 1 min of 95°C denaturation, 1 min of 55°C annealing, and 1 min of 72°C extension). PCR primers used in this study are listed below and were purchased from Gibco-BRL (Frederick, MD): sense strand inducible nitric oxide synthase (iNOS) primer, 5'-AAT GGC AAC ATC AGG TCG GCC ATC ACT-3'; anti-sense strand iNOS primer, 5'-GCT GTG TGT CAC AGA AGT CTC GAA CTC-3'; sense strand TNF-{alpha} primer, 5'-ATG AGC ACA GAA AGC ATG ATC-3'; anti-sense strand TNF-{alpha} primer, 5'-TAC AGG CTT GTC ACT CGA ATT-3'; sense strand IL-1ß primer, 5'-TAC AGG CTC CGA GAT GAA CAA CAA-3'; anti-sense strand IL-1ß primer, 5'-TGG GGA AGG CAT TAG AAA CAG TCC-3'; sense-strand ß actin primer, 5'-TCA TGA AGT GTG ACG TTG ACA TCC GT-3'; anti-sense strand ß actin primer, 5'-CCT AGA AGC ATT TGC GGT GCA CGA TG-3'. As a control for contaminating genomic DNA, parallel PCR reactions were performed in which the template nucleic acids were not reverse transcribed. After amplification, portions of the PCR reactions were electrophoresed on a 2% agarose gel and visualized using ethidium bromide.

Western blot analysis
Whole-cell lysates were prepared from unstimulated RAW 264.7 macrophages, and from cells stimulated with different TLR agonists for various times as we have previously described [37 ]. Aliquots containing 20–100 µg of total protein per lane were fractionated on SDS-12% polyacrylamide gels and then electrophoretically transferred to nitrocellulose membranes (Bio-Rad). Activated MAP kinases were detected using specific antisera against the phosphorylated forms of extracellular regulated kinase (ERK) and c-Jun kinase (JNK) (New England Biolabs, Beverly, MA), according to the manufacturer’s instructions. Membranes were developed using a donkey anti-rabbit antiserum linked to horseradish peroxidase (Amersham Pharmacia Biotech, Piscataway, NJ) and then visualized using an enhanced chemiluminesence reagent (chemiluminescence-horseradish peroxidase substrate system; Pierce Corp., Rockford, IL).

Electrophoretic mobility shift assays
Nuclear extracts were prepared from RAW 264.7 cells as described by Schreiber et al. [38 ] and then analyzed as we have previously described [14 ]. Double-stranded oligonucleotides containing a single consensus NF-{kappa}B (5'-AGT TGA GGG GAC TTT CCC AGG C-3')- or AP-1 (5'-CGC TTG ATG ACT CAG CCG GAA-3')-binding site were used as electrophoretic mobility shift assay (EMSA) probes. DNA probes were labeled with {alpha}-32P-labeled deoxynucleoside triphosphates (DuPont-NEN, Boston, MA) using E. coli DNA polymerase Klenow fragment (U.S. Biochemicals, Cleveland, OH), and unincorporated nucleotides were removed using Sephadex G-25 columns (5 Prime-3 Prime Inc., Boulder, CO). Nuclear extracts (typically 3 µg) were incubated with radiolabeled-probe DNA in the presence of 2 µg of poly dI-dC (Pharmacia), 1.0 mM ethylenediaminetetraacetate, 10 mM Tris-HCl (pH 7.9), 25 mM glycerol, and 0.5 mM dithiothreitol in a final volume of 20 µL. Binding reactions were then incubated at room temperature for 30 min. After incubation, a portion of each binding reaction was loaded onto 7% nondenaturing low-ionic-strength polyacrylamide gel. The gels were then dried and visualized by autoradiography.

Plasmids
The NF-{kappa}B-dependent ELAM-luciferase reporter plasmid was obtained from Douglas Golenbock and was previously described [30 ]. The AP-1-luciferase reporter plasmid, containing four copies of a consensus AP-1 site, was previously described [39 ]. All plasmids were prepared using Qiagen plasmid DNA purification columns, DNA was eluted from the columns using LPS-free buffers, and contaminating LPS levels were found to be less than 10 pg/mL. Furthermore, all plasmid preparations were unable to activate the LPS-sensitive RAW 264.7 macrophages at the concentrations used for transfection, demonstrating that the plasmids were not contaminated with LPS (data not shown).

Transfection and reporter assays
Transient transfections were performed using Super-Fect reagent (Qiagen) according to the manufacturer’s instructions, with minor modifications that we previously described [14 ]. Transfection mixtures containing a total of 3 µg of plasmid DNA were incubated with the RAW 264.7 cells for 2–3 h, whereupon the reaction was removed from the cells, and fresh medium containing serum was added. On the following day, individual wells were left untreated or were stimulated with TLR agonists for various times as indicated in the figures. Cells were then incubated for an additional 5–18 h prior to harvesting. Luciferase assays were performed using the Luciferase Assay System (Promega), according to manufacturer’s instructions. All transfection experiments were performed in triplicate, repeated at least three times using different plasmid preparations, and a single representative experiment is shown. Each single experiment represents triplicate independent transfections, and data are expressed as average values ± SD.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
The mycobacterial glycolipid PIM is a TLR2 agonist
We previously reported that the live M. tuberculosis cultures secreted a heat- and protease-insensitive TLR2 agonist, which we termed STF [40 ]. To determine the identity of the TLR2 agonists present in STF, we used preparative SDS-polyacrylamide gel electrophoresis (PAGE) to isolate various culture filtrate fractions on the basis of molecular size. As shown in Figure 1A , the bulk of the TLR2 agonist activity was located within a single size fraction (fraction 27). Other fractions (e.g., 2 and 13) also possessed some TLR2 agonist activity, although the identities of these agonists are currently unknown. Fraction 27 had an apparent molecular size of 6 kDa, raising the possibility that the TLR2 agonist was phosphatidylinositol dimannoside (PIM), a small mycobacterial glycolipid with a similar mobility on SDS-PAGE [41 ].



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  		Figure 1. PIM is a TLR2 agonist that is present in STF. Size-fractionated mycobacterial filtrate fractions were prepared as described in Materials and Methods and tested for TLR2 agonist activity using 3E10/TLR2 cells. Aliquots of size-fractionated STF were added to 3E10/TLR2 cells at a final concentration of 20 µg/mL. Cells were also stimulated with crude STF (20 µL/mL) as a positive control. Twenty-four hours later, the cells were stained with a fluorescein isothiocyanate-labeled anti-CD25 monoclonal antibody and subjected to flow cytometry analysis to measure the expression of the CD25. Data are expressed as the ratio (fold activation) of the percents of CD25+ cells in unstimulated and stimulated cell populations that were gated to exclude the lowest 95% of FL1 fluorescence obtained using the isotype control antibody (A). 3E10/TLR2 cells were stimulated with STF (20 µL/mL), purified PIM (1 µg/mL), and purified AraLAM (1 µg/mL), and then analyzed 24 h later as described in the text (B). STF was extracted with Triton X-114 to yield Triton-soluble and aqueous STF fractions as described in Materials and Methods. The ability of these fractions and crude STF to activate 3E10/TLR2 cells was analyzed as described in the text (C). In B and C, assays were performed in triplicate, and repeated on three separate occasions. A single representative experiment is shown, and the data are expressed as mean values ± SE.

 
We then proceeded to measure the ability of purified PIM in order to activate cells via TLR2. This activity was measured using CHO cell lines that contain an integrated CD25 reporter gene under the control of an NF-{kappa}B-dependent promoter (3E10 cells), as well as lines that overexpress TLR2 (3E10/TLR2) and TLR4 (3E10/TLR4). As shown in Figure 1B , purified PIM was capable of activating the 3E10/TLR2 cells. PIM could not activate the 3E10 or 3E10/TLR4 cells (data not shown), demonstrating that PIM is a specific TLR2 agonist. Because PIM can be extracted from aqueous solutions using Triton X-114 [34 ], we then sought to determine whether the TLR2 agonist activity could also be extracted from crude STF with Triton X-114. As shown in Figure 1C , the TLR2 agonist activity could be completely removed from crude STF by Triton X-114 extraction (aqueous STF) and subsequently recovered from the Triton-soluble layer (Triton-soluble STF). This finding also suggests that additional minor TLR2 agonists are present in STF (e.g. fractions 2 and 13) and are lipid conjugates such as glycolipids or lipoproteins. Last, analysis of this Triton-extracted STF using thin-layer chromatography revealed the presence of two major species that shared identical mobilities with monophosphatidylinositol and PIM (PIM1 and PIM2, respectively; data not shown). It should be noted that PIM1 and PIM2 have similar mobilities on SDS-PAGE (~6 kDa) and thus are not likely to be the TLR2 agonists contained in fractions 2 and 13 (Fig. 1A) . Taken together, these data indicate that the mycobacterial glycolipid PIM is a TLR2 agonist and is likely to contribute to the total TLR2 agonist activity present in STF. Our findings do not exclude the possibility that STF and even a single size fraction purified from crude STF contain multiple TLR2 agonists.

LPS, LAM, and STF induce distinct cellular responses in macrophages
We subsequently sought to compare the cellular responses induced by these various TLR2 and TLR4 agonists. RAW 264.7 murine macrophages were stimulated with LPS, LAM, and STF for 24 h, and culture supernatants were then collected. The levels of cytokines (TNF-{alpha} and IL-1ß) and nitrite, a stable catabolite of NO, were measured by ELISA and the Greiss reagent assay, respectively. As shown in Figure 2 , LPS was capable of inducing RAW 264.7 macrophages to secrete TNF-{alpha}, IL-1ß, and NO. In contrast, LAM and STF were capable of inducing only TNF-{alpha} secretion. Higher concentrations of LAM, PIM, and STF failed to induce IL-1ß and NO secretion (data not shown). Thus, the cellular responses of macrophages induced by stimulation with these TLR2 and TLR4 agonists were qualitatively distinct.



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  		Figure 2. Macrophage responses induced by LPS differ from those induced by AraLAM, PIM, and STF. RAW 264.7 macrophages (15 x 105 cells/mL) were stimulated for 24 h with LPS (100 ng/mL), STF (10 µL/mL), PIM (1 µg/mL), or AraLAM (1 µg/mL). Levels of TNF-{alpha} and IL-1ß protein in the supernatants were then measured by ELISA. Nitrite levels were also measured in these supernatants using the Greiss reagent assay. All assays were performed in triplicate and repeated on four separate occasions. A single representative experiment is shown, and the data are expressed as mean values ± SEM.

 
LAM and STF fail to activate iNOS gene expression and IL-1ß mRNA translation
We next sought to determine whether the inability of LAM and STF to induce IL-1ß and NO secretion was due to the inability of these TLR2 agonists to induce IL-1ß and iNOS mRNA expression. RT-PCR analysis was performed using total RNA prepared from RAW 264.7 cells that had been stimulated with the various TLR agonists in vitro for 4 h. As shown in Figure 3 , unstimulated macrophages did not express IL-1ß and iNOS mRNA. TNF-{alpha} mRNA was constitutively expressed by the RAW 264.7 cells. Previous studies have reported that the TNF-{alpha} gene is constitutively transcribed in unstimulated RAW 264.7 cells, although this mRNA is not translated in the absence of stimulation [42 ]. As expected, LPS stimulated the expression of both IL-1ß and iNOS mRNA in the RAW 264.7 cells. Similarly, LAM and STF induced IL-1ß mRNA expression in the macrophages but failed to induce substantial iNOS mRNA expression.



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  		Figure 3. AraLAM, PIM, and STF induce TNF-{alpha} and IL-1ß but not iNOS transcription in RAW 264.7 macrophages. RAW 264.7 macrophages were stimulated with LPS (100 ng/mL), STF (10 µL/mL), PIM (4 µg/mL), or AraLAM (4 µg/mL) for 4 h. Total RNA was then prepared from the cells and analyzed by semiquantitative RT-PCR for iNOS, IL-1ß, and TNF-{alpha} mRNA as described in Material and Methods. PCR products were fractionated on a 2% agarose gel and visualized using ethidium bromide. PCR reactions that did not include RT RNA failed to generate a product (data not shown), demonstrating the absence of contaminating genomic DNA in the RNA preparations.

 
The finding that LAM and STF induced IL-1ß mRNA expression in the macrophages but not IL-1ß secretion prompted us to determine whether this was due to the absence of IL-1ß mRNA translation or to proteolytic processing and secretion of mature IL-1ß protein. To discriminate between these possibilities we prepared cellular lysates from unstimulated and stimulated RAW 264.7 cells by repeated freeze/thaw cycles and then measured the levels of intracellular IL-1ß protein production by ELISA. As shown in Figure 4 , LPS was capable of inducing the synthesis of intracellular IL-1ß protein and its subsequent secretion, whereas LAM and STF stimulation did not result in the production of intracellular IL-1ß protein. The finding that IL-1ß mRNA was expressed by macrophages stimulated with LAM and STF, although no intracellular protein was synthesized, suggests that LAM and STF fail to activate the intracellular signals necessary for IL-1ß mRNA translation. Similarly, primary mouse peritoneal macrophages also failed to synthesize intracellular IL-1ß protein following stimulation in vitro with Triton-extracted STF, although crude STF was capable of inducing secretion of this cytokine (data not shown).



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  		Figure 4. AraLAM, PIM, and STF fail to induce translation of IL-1ß mRNA in RAW 264.7 macrophages. RAW 264.7 macrophages were stimulated for 6, 12, and 24 h with LPS (100 ng/mL), STF (10 µL/mL), PIM (1 µg/mL), or AraLAM (1 µg/mL). Culture supernatants were then collected and saved. Adherent macrophages were removed by scraping in ice-cold sterile phosphate-buffered saline, freeze-thawed three times, and centrifuged at 14,000 g for 10 min. These cell-free supernatants were recovered and used as a source of intracellular lysate. IL-1ß protein levels in the lysates and culture supernatants were measured by ELISA. These assays were performed in triplicate and repeated on four separate occasions. A single representative experiment is shown, and the data are expressed as mean values ± SEM.

 
Comparison of MAP kinase activation by LPS, LAM, and STF
The finding that TLR agonists LPS, LAM, and STF differ in their capacities to induce IL-1ß and NO secretion suggests that the intracellular signal transduction pathways induced in macrophages by LPS must differ from those triggered by LAM and STF. We first sought to determine whether LPS, LAM, PIM, and STF differed in their capacities to activate the ERK and JNK MAP kinases. For these experiments, RAW 264.7 cells were stimulated for various times using concentrations of TLR agonists that generated similar levels of TNF-{alpha} secretion, as measured by ELISA (Figure 5A ). Using these concentrations, the levels of activated ERK and JNK were measured by Western blotting using antibodies that specifically recognize the phosphorylated forms of these MAP kinases. Antibodies that recognize both phosphorylated and nonphosphorylated MAP kinases were used as controls for equal loading. As shown in Figure 5B , LPS was a potent activator of both ERK and JNK. Multiple isoforms of ERK (p42 and p44) and JNK (JNK1 and JNK2) were detected and became phosphorylated within 15 min after LPS stimulation. The TLR2 agonists STF, PIM, and LAM also induced ERK phosphorylation, with maximal activation observed within 35 min after stimulation. In contrast, these TLR2 agonists were markedly less potent activators of JNK compared with LPS. These TLR2 agonists were not wholly incapable of activating JNK, as demonstrated by the finding that higher concentrations of these agonists did activate this MAP kinase (Figure 5C) . Even at higher concentrations, the TLR2 agonists were poor inducers of JNK2 phosphorylation compared with LPS (Figure 5C) . These studies indicate that LPS, STF, LAM, and PIM induce similar levels of ERK activation in the macrophages, whereas they differ in their capacities to activate JNK in these cells.



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  		Figure 5. Differential activation of MAP kinase in RAW 264.7 cells by LPS and mycobacterial glycolipids. RAW 264.7 cells were stimulated for 6 h using the indicated amounts of LPS, STF, PIM, and AraLAM. Secreted TNF-{alpha} levels were then measured by ELISA (A). RAW 264.7 cells were stimulated for various times with amounts of LPS, STF, PIM, and AraLAM that induced similar levels of TNF{alpha} secretion. After stimulation, cell lysates were prepared, fractionated by SDS-PAGE (100 µg/lane for JNK and 20 µg/lane for ERK), and probed with antibodies that specifically recognize the phosphorylated forms of JNK and ERK (B). Membranes were subsequently stripped and reprobed with antibodies that bound both phosphorylated and nonphosphorylated forms of JNK and ERK. Arrows indicate the positions of various JNK and ERK isoforms, based on published data [29 ]. RAW 264.7 cells were stimulated for 28 min with the indicated concentrations of LPS, STF, and AraLAM. Cell lysates were then prepared, fractionated on SDS-PAGE (100 µg total protein/lane), and probed for JNK as described above (C).

 
LPS and STF induce similar levels of AP-1 and NF-{kappa}B DNA-binding activities
The finding that the TLR2 agonist STF was less capable of inducing JNK activation compared with the TLR4 agonist LPS prompted us to determine whether this resulted in differential activation of the transcription factor AP-1. Because phosphorylation of the c-Jun subunit of AP-1 by JNK is required for the DNA-binding and trans-activation function of this factor [43 ], we hypothesized that STF might be a relatively poor inducer of AP-1 compared with LPS. EMSA analysis was performed to compare the activation of AP-1 DNA-binding activity by macrophages stimulated with LPS versus STF. As shown in Figure 6 , AP-1 DNA-binding activity was observed in nuclear extracts prepared from macrophages stimulated with either LPS or STF. The specificity of this DNA-protein complex was demonstrated by the ability of excess unlabeled probe DNA to compete for complex formation, whereas a nonspecific DNA probe could not compete for complex formation. Notably, both TLR agonists rapidly induced similar levels of AP-1 DNA-binding activity (even under these conditions of EMSA probe excess). Thus, AP-1 DNA-binding activity in the RAW 264.7 macrophages was induced to similar levels by LPS and STF, even though these TLR agonists differed in their capacities to activate JNK (see Fig. 5B ). We subsequently compared the activation of NF-{kappa}B DNA-binding activity by macrophages stimulated with LPS versus STF. As shown in Figure 6 , NF-{kappa}B DNA-binding activity was observed in nuclear extracts prepared from macrophages stimulated with either LPS or STF. Two major DNA-protein complexes were observed, and the specificity of these complexes was demonstrated by the ability of excess unlabeled probe DNA to compete for complex formation, whereas a nonspecific DNA probe could not compete for complex formation. Like AP-1, both LPS and STF rapidly induced similar levels of NF-{kappa}B DNA-binding activity.



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  		Figure 6. NF-{kappa}B and AP-1 activation by LPS and STF. RAW 264.7 macrophages were stimulated with LPS (100 ng/mL) or STF (10 µl/mL) for 0.5, 1, 2, or 4 h. Nuclear extracts from stimulated and unstimulated cells were prepared as described in Materials and Methods, and then assayed for AP-1 (A) or NF-{kappa}B (B) DNA-binding activities by EMSA. The positions of specific DNA-protein complexes containing AP-1 and NF-{kappa}B are indicated by arrows.

 
LPS and STF induce similar levels of AP-1- and NF-{kappa}B-dependent promoter activation
It is well known for transcription factors that induction of DNA-binding activity does not always correlate with trans-activation function, the latter activity often being controlled by posttranslational mechanisms that are distinct from those that control DNA-binding activity [37 ]. Thus, we sought to determine whether LPS and STF differed in their capacities to induce AP-1- and NF-{kappa}B-dependent promoter activation. Luciferase reporter plasmids containing AP-1- and NF-{kappa}B-dependent promoters were transiently transfected into RAW 264.7 cells, and a portion of the cells was then stimulated with either LPS or STF for 4 h. Both unstimulated and stimulated transfected cells were harvested, and luciferase activity was measured as we previously described [14 ]. As shown in Figure 7 , similar levels of inducible AP-1- and NF-{kappa}B-dependent reporter gene activities were observed when transfected cells were stimulated with either LPS or STF. Similar results were obtained when transfected cells were stimulated with purified PIM (data not shown). Thus, like the DNA-binding activities reported above, LPS and STF activated similar levels of AP-1- and NF-{kappa}B-dependent transcription, even though these TLR agonists differed in their capacities to activate JNK (see Fig. 5B ).



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  		Figure 7. NF-{kappa}B and AP-1 luciferase reporter gene activation by LPS and STF. RAW 264.7 macrophages were transiently transfected with one of the following luciferase reporters: an NF-{kappa}B-dependent ELAM-Luc promoter (A) or a synthetic AP-1-Luc promoter (B). A portion of the cells was then stimulated with LPS (100 ng/mL) or STF (10 µl/mL) for 2 or 4 h. Cells were then harvested, and luciferase activity was measured as described in Materials and Methods. All transfection experiments were performed in triplicate and repeated three times using different plasmid preparations, and a single representative experiment is shown. Data are expressed as average luciferase values from a single experiment (subtracted for background) ± SD.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
Here we have provided evidence that STF contains the mycobacterial glycolipid PIM and that purified PIM is a TLR2 agonist. Like the related glycolipid LAM, PIM can induce TNF-{alpha} secretion by the RAW 264.7 murine macrophage cell line. Unlike the TLR4 agonist LPS, these glycolipid TLR2 agonists failed to induce IL-1ß and NO secretion by these macrophages. Although LAM, STF, and PIM failed to induce iNOS transcription, these TLR2 agonists were fully capable of inducing de novo IL-1ß gene expression. In spite of the presence of abundant IL-1ß mRNA in the LAM-, STF-, and PIM-stimulated cells, there was no detectable intracellular IL-1ß protein, suggesting that the newly transcribed mRNA was not being translated. These findings suggest that LPS and the glycolipid TLR2 agonists trigger nonredundant signal transduction pathways and that only LPS is capable of triggering the pathway(s) necessary to activate IL-1ß mRNA translation. We subsequently attempted to identify differences in intracellular signaling in macrophages stimulated with LPS, LAM, STF, and PIM. These studies revealed that LPS was a more potent activator of JNK compared with LAM, STF, and PIM. In contrast, all of these TLR agonists were capable of activating similar levels of ERK phosphorylation. In spite of relative quantitative differences in the levels of JNK phosphorylation induced by the different TLR agonists, there were no significant differences in the levels of AP-1 DNA-binding activity and trans-activation function observed in the stimulated RAW 264.7 macrophages. Moreover, NF-{kappa}B DNA-binding activity and trans-activation function were also found to be similar in macrophages stimulated with LPS and STF, with similar results having been obtained using LAM and PIM (data not shown). Thus, it is unlikely that differences in the relative capacities of these TLR agonists to induce JNK phosphorylation are sufficient to explain the inability of LAM and STF/PIM to induce IL-1ß and NO secretion.

To date, a number of published studies have described a variety of cellular responses that can be induced by different purified TLR agonists [reviewed in ref. 1 22 44 ]. All known TLR agonists have been shown to induce TNF-{alpha} secretion in monocytic cells [14 , 45 46 47 48 ]. Similarly, both TLR2 and TLR4 agonists have been previously reported to trigger rapid NF-{kappa}B and MAP kinase activation. Here, we have provided some of the first evidence to demonstrate that, in spite of their similarities, different TLR agonists can stimulate distinct cellular responses in macrophages. Very recent studies by Hirschfeld et al. [49 ] have reported similar differences in cellular responses using primary murine macrophages stimulated in vitro with LPS versus the TLR2 agonist Porphyromonas gingivalis LPS.

It should be noted that the inability of the glycolipid TLR2 agonists to induce IL-1ß and NO secretion is a characteristic that is not shared by all TLR2 agonists. For example, IL-1ß secretion can be induced in macrophages stimulated with a synthetic mycoplasmal lipopeptide, a TLR2 agonist [50 ]. Similarly, NO production can be induced in macrophages by peptidoglycan [2 ], mycoplasmal lipopeptides [3 ], when cells were primed with IFN-{gamma} and Treponema glycolipids [47 ]. Also, Brightbill et al. [7 ] reported that the Borellia OspA and M. tuberculosis 19-kDa lipoproteins could activate a reporter gene under control of the iNOS promoter in transiently transfected RAW 264.7 cells [7 ]. Using this same iNOS reporter plasmid, we found that STF could activate reporter gene expression in transiently transfected RAW 264.7 cells [18 ], even though the endogenous iNOS gene was not expressed (see Fig. 3 ). Thus, care must be taken to assure that reporter gene responses accurately reflect endogenous gene expression.

It is reasonable to assume that the differential induction of IL-1ß and NO by LPS and the glycolipid TLR2 agonists is the result of differential intracellular signaling triggered by these stimuli. If JNK-, AP-1-, and NF-{kappa}B-dependent responses are not responsible for these functional differences, then additional signaling pathways must be considered. Activation of the TLR signal transduction pathway involves sequential recruitment of MyD88, IRAK, and TRAF6 [51 52 53 ]. This signaling pathway is largely shared by the IL-1 and IL-18 receptors [reviewed in ref. 54 ]. The intermediate steps between TRAF6 and the I{kappa}B kinase complex have not been clearly identified, although several investigators have proposed that pathways leading to MAP kinase activation are initiated at this level in the signaling cascade [51 , 55 , 56 ]. Earlier studies using LPS and IL-1ß have implicated several additional protein and lipid kinases in signaling initiated by TLR and IL-1 receptor proteins. These include Src family protein tyrosine kinases, protein kinase C {zeta} [29 ], phosphatidylinositol 3' kinase [27 , 57 ], and phosphatidylcholine-specific phospholipase C [28 ]. Additional studies will be needed to determine whether the differential capacities of LPS and the glycolipid TLR2 agonists to induce IL-1ß and NO secretion arise from differences in the activation of any of these parallel signaling pathways.

During the course of our studies, we obtained evidence demonstrating that PIM is a TLR2 agonist and that it is likely to be present in crude STF. PIM is a biosynthetic precursor of the larger glycolipid LAM and may be one of the simplest structures known to have TLR agonist activity. We are also unaware of any other studies demonstrating that PIM is present in short-term culture filtrates from live M. tuberculosis cultures. Recently, glycolipids with putative TLR2 agonist activity have been isolated from culture filtrates of Treponema spirochetes [47 ]. Thus, glycolipids with TLR agonist activity may be secreted by pathogens present in vivo at the sites of infection. These glycolipids are likely to be immunomodulatory and act of neighboring uninfected cells in a paracrine manner. This is consistent with previous studies showing that LAM can be exported from M. tuberculosis-infected cells [58 ] and that exogenous LAM can be passively inserted into the plasma membranes of uninfected cells [59 ]. It is tempting to speculate that glycolipids or other bacterial products secreted by pathogens might be capable of affecting the development of an acquired immune response. Support for this possibility comes from recent studies by Mokuno et al. [60 ] showing that infection of mice with E. coli induces the recruitment of {gamma}/{delta} T cells to the site of infection. These T cells express TLR2 but not TLR4 mRNA and can be directly activated by bacterial products in vitro in a TLR2-dependent manner. If, like macrophages, T cells also differ in their response to different TLR agonists, it is possible that these agonists could selectively skew the development of normal Th1 and Th2 responses. In conclusion, TLR2 agonists released by pathogens in vivo are likely to affect the function of both early innate and acquired immune responses.

Received December 18, 2000; revised January 25, 2001; accepted January 26, 2001.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

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