Toll-like receptors (TLRs) are a family of mammalian homologues of Drosophila Toll and play important roles in host defense. Two of the TLRs, TLR2 and TLR4, mediate the responsiveness to LPS. Here the gene expression of TLR2 and TLR4 was analyzed in mouse macrophages. Mouse splenic macrophages responded to an intraperitoneal injection or in vitro treatment of LPS by increased gene expression of TLR2, but not TLR4. Treatment of a mouse macrophage cell line with LPS, synthetic lipid A, IL-2, IL-15, IL-1β, IFN-γ, or TNF-α significantly increased TLR2 mRNA expression, whereas TLR4 mRNA expression remained constant. TLR2 mRNA increase in response to synthetic lipid A was severely impaired in splenic macrophages isolated from TLR4-mutated C3H/HeJ mice, suggesting that TLR4 plays an essential role in the process. Specific inhibitors of mitogen-activated protein/extracellular signal-regulated kinase kinase and p38 kinase did not significantly inhibit TLR2 mRNA up-regulation by LPS. In contrast, LPS-mediated TLR2 mRNA induction was abrogated by pretreatment with a high concentration of curcumin, suggesting that NF-κB activation may be essential for the process. Taken together, our results indicate that TLR2, in contrast to TLR4, can be induced in macrophages in response to bacterial infections and may accelerate the innate immunity against pathogens.

A glycolipid known as endotoxin or LPS is the principal constituent of Gram-negative bacteria recognized by the innate immune system and often causes endotoxin shock. LPS is a complex glycolipid composed of a hydrophilic polysaccharide region and a hydrophobic domain known as lipid A responsible for most of the LPS-induced biological effects (1). LPS stimulates host cells such as monocytes, macrophages, and B cells through the activation of transcription factors and protein kinases such as NF-κB (2, 3), AP-1 (3), extracellular signal-regulated kinases (ERKs)3 (4, 5), c-Jun N-terminal kinases (JNKs) (6, 7), and p38 kinases (8, 9). CD14, a cell surface protein, and LPS binding protein, a serum factor, bind LPS with high affinity, mediating LPS responses (10, 11, 12). As CD14 is a glycosylphosphatidylinositol-anchored protein, the LPS receptor complex was assumed to contain an additional signaling component.

Toll, first identified as a protein controlling dorsoventral pattern formation in the early development of Drosophila (13), has been shown to participate in antimicrobial immune responses (14). Toll is conserved throughout various species encoding a transmembrane protein of which the intracellular domain is homologous to that of the IL-1 receptor family proteins (13). Recently, several mammalian Toll homologues have been identified and termed Toll-like receptors (TLRs) (15). In addition to their cytoplasmic tails, the newly identified receptors share repeating leucine-rich motifs in their extracellular regions. In the past few years two of the mammalian TLRs, TLR2 and TLR4, have been shown to mediate the LPS responsiveness in in vitro transfection systems (16, 17, 18, 19, 20). Although TLR2 is capable of mediating LPS signals in vitro, its role as an LPS receptor in vivo has been questioned as a result of the recent findings that two mouse strains (C3H/HeJ and C57BL10/ScCr) that exhibit impaired ability to respond to many types of LPS have different mutations in the TLR4 gene (21, 22). Also, gene-disrupted mice of TLR4, but not TLR2, demonstrate phenotypes similar to those of LPS-hyporesponsive strains (23). These findings suggest that TLR4 is the dominant receptor for at least some types of LPS, whereas TLR2 is dispensable. In contrast, TLR2 has recently been suggested to mediate signals from other bacterial components, including lipoteichoic acid, peptidoglycan, and lipoproteins/lipopeptides (24, 25, 26, 27).

In this study, we examined the gene expression of TLR2 and TLR4 in mouse macrophages. An intraperitoneal injection of LPS significantly increased the mRNA levels of TLR2, but not TLR4, in splenic macrophages. Treatment of mouse macrophage cell lines with LPS, synthetic lipid A, IL-2, IL-15, IL-1β, IFN-γ, or TNF-α significantly increased TLR2 mRNA expression, whereas TLR4 mRNA levels remained constant. The TLR2 mRNA increase in response to synthetic lipid A was severely impaired in splenic macrophages isolated from TLR4-mutated C3H/HeJ mice, suggesting that TLR4 plays an essential role in this process. Specific inhibitors of mitogen-activated protein (MAP)/ERK kinase (MEK) and p38 kinase did not inhibit TLR2 mRNA up-regulation by LPS. In contrast, LPS-mediated TLR2 mRNA induction was abrogated by pretreatment with a high concentration of an NF-κB inhibitor, curcumin, suggesting that NF-κB activation may be essential for the process. Our results indicate that TLR2, in contrast to TLR4, can be induced in macrophages in response to bacterial infections and suggest that although TLR2 is dispensable for the initial host responses against LPS, it may contribute to the accelerated macrophage responses seen at subsequent stages of infection.

Recombinant mouse IL-2, IL-1β, TNF-α, IFN-γ, and human IL-15 were purchased from Peprotech (Seattle, WA). PD98059, a specific inhibitor of ERK kinase (MEK), and SB208530, a specific inhibitor of p38 kinase, were purchased from Calbiochem (San Diego, CA). LPS from Escherichia coli serotype B6:026, curcumin, anisomycin, and PMA were obtained from Sigma (St. Louis, MO). RPMI 1640 was from Life Technologies (Rockville, MD). Synthetic E. coli-type lipid A, compound 506, was previously described (28). Anti-phospho-ERK mAb and anti-p38 MAP kinase polyclonal Ab were obtained from New England Biolabs (Beverly, MA) and Santa Cruz Biotechnology (Santa Cruz, CA), respectively.

All cell lines were grown in tissue culture flasks at 37°C in 5% CO2/95% air and passaged every 2 or 3 days to maintain logarithmic growth. A mouse macrophage cell line, RAW264.7, was obtained from The Institute of Physical and Chemical Research cell bank (Tsukuba, Japan) and maintained in DMEM with 10% FCS (Sigma).

Adherent cells from spleens of naive BALB/c, C3H/HeN, or C3H/HeJ mice were used as a source of mouse macrophages. Briefly, isolated splenocytes suspended in DMEM containing 10% FCS were cultured in plastic plates for 1 h at 37°C, nonadherent cells were removed, and the fresh complete medium was added to the adherent cells with or without stimulants.

Total cellular RNAs were extracted using TRIZOL reagent (Life Technologies, Rockville, MD) according to the manufacturer’s instructions. Aliquots (20 μg) of the total RNAs were fractionated in a 1% agarose gel containing 20 mM MOPS, 5 mM sodium acetate, 1 mM EDTA (pH 7.0), and 6% (v/v) formaldehyde and transferred to a nylon membrane. After UV cross-linking, membranes were soaked in prehybridization solution (6× SSC, 5× Denhardt’s reagent, 0.5% SDS, 100 μg/ml denatured salmon sperm DNA, and 50% formamide) for 3 h at 42°C followed by incubation with 32P-labeled probe in hybridization solution (6× SSC, 0.5% SDS, 100 μg/ml denatured salmon sperm DNA, and 50% formamide) for 14 h at 42°C. The membranes were washed in 2× SSC, 0.1% SDS for 10 min twice at room temperature and in 0.1× SSC, 0.1% SDS for 10 min twice at 50°C and were exposed to Fuji RX-U films (Fuji Film, Tokyo, Japan).

For specific DNA probes, partial cDNA fragments containing the full coding regions of mouse TLR2, TLR4, and β-actin (including no 5′ or 3′ noncoding regions) were prepared by RT-PCR using total RNA from LPS-treated RAW264.7 cells as template and labeled with [α-32P]dCTP (New England Nuclear, Boston, MA). The specific activities for the labeled probes were 1.5–5.0 × 106 cpm/ng.

Cells were lysed in PLC lysis buffer (50 mM HEPES (pH 7.0), 150 mM NaCl, 10% glycerol, 1% Triton X-100, 1.5 mM MgCl2, 1 mM EGTA, 100 mM NaF, 10 mM NaPPi, 1 mM Na3VO4, 1 mM PMSF, 10 μg/ml aprotinin, and 10 μg/ml leupeptin) at 1 × 108 cells/ml. The lysates were separated in SDS-polyacrylamide gels and then electrotransferred to Immobilon polyvinylidene difluoride membranes (Millipore, Bedford, MA). The membranes were blocked for 2 h in 5% nonfat milk-TBST (20 mM Tris-HCl (pH 7.6), 0.15 M sodium chloride, 0.1% Tween 20), incubated with primary Abs in TBST for 1 h, washed three times with TBST, and incubated for 1 h with HRP-conjugated anti-mouse or rabbit Ig (Amersham Pharmacia Biotech) diluted 1:5000 in 5% nonfat milk-TBST. After three washes in TBST, the blot was developed with the enhanced chemiluminescence system (Amersham Pharmacia Biotech) according to the manufacturer’s instructions.

The GST-ATF2(1-109) fusion protein was prepared as previously described (29). Cells were lysed in PLC lysis buffer at 1 × 108 cells/ml. p38 kinase was immunoprecipitated with anti-p38 kinase polyclonal Ab and protein A-Sepharose beads (Amersham Pharmacia Biotech) from 100 μl lysate. The beads were extensively washed with PLC lysis buffer three times followed by kinase buffer (20 mM Tris-HCl (pH 7.4), 20 mM MgCl2, and 2 mM EGTA) once. The beads were then added with 40 μl kinase buffer containing 500 ng GST-ATF2(1-109) and 0.5 μCi [γ-32P]ATP (New England Nuclear) and incubated at 30°C for 20 min. The phosphorylation of GST-ATF2(1-109) was analyzed by SDS-PAGE and autoradiography.

RAW264.7 cells were transiently transfected with 2 μg of pGL3-NF-κB/Luc (a luciferase reporter construct containing consensus NF-κB binding sequence) and 0.2 μg of pRL/SV40 (an internal control) by Lipofectamine (Life Technologies) according to the manufacturer’s instructions. Twenty-four hours after the transfection, some cells were pretreated with indicated chemicals for 30 min followed by the addition of 1 ng/ml LPS. After 8-h incubation with LPS, cells were lysed, and the luciferase activity was measured by using the Dual-Luciferase Reporter Assay System (Toyo Ink, Tokyo, Japan) according to the manufacturer’s instructions. Background luciferase activity was subtracted, and the data are presented as means ± SD of triplicate samples.

Several laboratories including our own have recently reported that both TLR2 and TLR4 are capable of mediating LPS responsiveness (16, 17, 18, 19, 20). However, the transcriptional regulation of these two proteins in response to Gram-negative bacteria has not been fully elucidated. Thus, we investigated the gene expression of TLR2 and TLR4 in mouse macrophages that play essential roles in innate immunity.

Macrophages isolated from spleens of naive BALB/c mice were treated with LPS or IL-15, a cytokine known to activate macrophages, for 2 h in vitro followed by total RNA preparation. In the Northern blot analysis, TLR2 mRNA was weakly expressed in unstimulated macrophages and significantly increased by LPS treatment (Fig. 1,A). Interestingly, IL-15, a cytokine shown to play an important role in the early phase of bacterial infection, also increased TLR2 mRNA level. In contrast, TLR4 mRNA expression remained constant after LPS or IL-15 treatment (Fig. 1 A). Multiple mRNA bands were always detected using TLR4 probes probably due to alternative splicing.

FIGURE 1.

LPS and IL-15 augment TLR2, but not TLR4, gene expression in mouse macrophages. A, Macrophages were isolated from spleens of BALB/c mice as described in Materials and Methods. The macrophages were either untreated or treated with 1 ng/ml LPS or 10 ng/ml IL-15 for 2 h. The total RNA was extracted and separated in a 1% Northern gel (5 μg/lane). The gene expression of TLR2 and TLR4 was analyzed by Northern blot using 32P-labeled specific cDNA probes. B, BALB/c mice were i.p. injected with PBS or 100 μg LPS for the indicated times. Mice were sacrificed and splenic macrophages were isolated as described in Materials and Methods. Total RNAs were prepared and examined for the expression of TLR2 by Northern blot analysis. Each lane contained 5 μg of total RNA.

FIGURE 1.

LPS and IL-15 augment TLR2, but not TLR4, gene expression in mouse macrophages. A, Macrophages were isolated from spleens of BALB/c mice as described in Materials and Methods. The macrophages were either untreated or treated with 1 ng/ml LPS or 10 ng/ml IL-15 for 2 h. The total RNA was extracted and separated in a 1% Northern gel (5 μg/lane). The gene expression of TLR2 and TLR4 was analyzed by Northern blot using 32P-labeled specific cDNA probes. B, BALB/c mice were i.p. injected with PBS or 100 μg LPS for the indicated times. Mice were sacrificed and splenic macrophages were isolated as described in Materials and Methods. Total RNAs were prepared and examined for the expression of TLR2 by Northern blot analysis. Each lane contained 5 μg of total RNA.

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Next, to investigate the TLR gene expression in a more physiological condition, mice were i.p. injected with LPS and splenic macrophages were isolated after 2 or 8 h. As shown in Fig. 1,B, splenic macrophages expressed significantly more TLR2 mRNA within 2 h after the LPS injection. The TLR2 mRNA level remained elevated for at least 8 h after the LPS injection. In contrast, there was no evident increase of TLR4 mRNA (Fig. 1 B). Taken together, these results indicate that the mRNA of two putative LPS receptors, TLR2 and TLR4, are differently regulated in macrophages by LPS.

RAW264.7 is a well-established mouse macrophage cell line. To determine the effects of LPS and various cytokines on the TLR2 mRNA expression, RAW264.7 cells were treated for 2 or 8 h with LPS, IL-15, IL-2, IL-1β, IFN-γ, or TNF-α, and total RNAs were isolated. All the cytokines examined increased the TLR2 mRNA level in <2 h (Fig. 2,A). The TLR2 mRNA increases by LPS, IL-2, IL-1β, IFN-γ, or TNF-α were rather transient, decreasing within 8 h of stimulation, whereas the effect of IL-15 remained relatively longer. In contrast, there was no significant increase of TLR4 mRNA by any of the treatments (Fig. 2,A). Similar results were obtained from J774.1, another well-established mouse macrophage cell line (data not shown). In a more detailed time course experiment, TLR2 mRNA increased by 1 h, peaked at 2 h, and decreased below the basal level by 12 h after LPS stimulation (Fig. 2,B). In contrast, TLR4 mRNA remained constant for 12 h and decreased by 24 h after LPS stimulation. TLR2 gene expression is also up-regulated in RAW264.7 cells by synthetic lipid A in a concentration-dependent manner (Fig. 2 C), suggesting that the TLR2 mRNA increase is not due to other substances possibly contaminated in the LPS and is mediated at least partly by the lipid A region of LPS.

FIGURE 2.

Increased TLR2 mRNA in a mouse macrophage cell line in response to LPS or various cytokines. A, RAW264.7 cells were treated with 1 ng/ml LPS, 10 ng/ml IL-15, 10 ng/ml IL-2, 10 ng/ml IL-1β, 10 ng/ml IFN-γ, or 10 ng/ml TNF-α for either 2 or 8 h. After the treatment, total RNA was prepared and TLR gene expression was examined by Northern blot. Each lane contained 5 μg of total RNA. B, RAW264.7 cells were stimulated with 1 ng/ml LPS for the indicated times. TLR2 and TLR4 gene expression was analyzed as above. C, RAW264.7 cells were stimulated with indicated concentrations of synthetic lipid A or LPS for 2 h. TLR2 gene expression was analyzed as above. D, RAW264.7 cells were pretreated with various concentrations of cycloheximide for 30 min followed by 1 ng/ml LPS for 2 h. TLR2 gene expression was examined as above. E, Macrophages were isolated from spleens of C3H/HeN or C3H/HeJ mice as described in Materials and Methods. The cells were untreated or treated with 1 μg/ml synthetic lipid A, 10 ng/ml TNF-α, or 10 ng/ml IL-1β for 2 h. The isolated total RNAs (5 μg each) were extracted, and TLR2 mRNA expressions were analyzed by Northern blotting analysis using 32P-labeled specific cDNA probes.

FIGURE 2.

Increased TLR2 mRNA in a mouse macrophage cell line in response to LPS or various cytokines. A, RAW264.7 cells were treated with 1 ng/ml LPS, 10 ng/ml IL-15, 10 ng/ml IL-2, 10 ng/ml IL-1β, 10 ng/ml IFN-γ, or 10 ng/ml TNF-α for either 2 or 8 h. After the treatment, total RNA was prepared and TLR gene expression was examined by Northern blot. Each lane contained 5 μg of total RNA. B, RAW264.7 cells were stimulated with 1 ng/ml LPS for the indicated times. TLR2 and TLR4 gene expression was analyzed as above. C, RAW264.7 cells were stimulated with indicated concentrations of synthetic lipid A or LPS for 2 h. TLR2 gene expression was analyzed as above. D, RAW264.7 cells were pretreated with various concentrations of cycloheximide for 30 min followed by 1 ng/ml LPS for 2 h. TLR2 gene expression was examined as above. E, Macrophages were isolated from spleens of C3H/HeN or C3H/HeJ mice as described in Materials and Methods. The cells were untreated or treated with 1 μg/ml synthetic lipid A, 10 ng/ml TNF-α, or 10 ng/ml IL-1β for 2 h. The isolated total RNAs (5 μg each) were extracted, and TLR2 mRNA expressions were analyzed by Northern blotting analysis using 32P-labeled specific cDNA probes.

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As TLR2 mRNA is responsive to cytokine stimulation (Fig. 2,A) and various cytokines are secreted from macrophages by LPS treatment, it is possible that LPS increases TLR2 mRNA indirectly by inducing cytokine secretion. To rule out this possibility, RAW264.7 cells were pretreated with a protein synthesis inhibitor, cycloheximide. As shown in Fig. 2 D, cycloheximide rather enhanced LPS-mediated increase of TLR2 mRNA, suggesting that LPS directly increases TLR2 mRNA in macrophages. The result also indicates that there may be a negative feedback mechanism for TLR2 induction that requires new protein synthesis. Additionally, cycloheximide pretreatment did not inhibit the TLR2 mRNA increase by any of the examined cytokines (data not shown), suggesting that their effects on TLR2 gene expression are also direct.

Both TLR2 and TLR4 mediate LPS signals in in vitro studies. To determine whether the LPS-induced TLR2 mRNA increase is mediated by TLR4 or TLR2, we examined macrophages from C3H/HeJ mice that contain nonfunctional mutation in the TLR4 gene. When isolated splenic macrophages from this mouse strain were stimulated with synthetic lipid A, TLR2 mRNA increase was severely impaired compared with that of macrophages isolated from the closely related C3H/HeN mice (Fig. 2 E). This finding suggests that TLR4 is essential for the lipid A-induced TLR2 mRNA increase.

LPS stimulation of macrophages is known to activate MAP kinase pathways, including ERK, JNK, and p38 kinase (4, 5, 6, 7, 8, 9). To investigate whether ERK and p38 kinase pathways are involved in mTLR2 mRNA up-regulation, RAW264.7 cells were pretreated with a specific inhibitor of ERK (PD98059) or p38 kinase (SB208530) pathway followed by LPS stimulation. Pretreatment with SB208530 only slightly inhibited the mTLR2 mRNA increase even at 50 μM (Fig. 3,A). Unexpectedly, PD98059 treatment enhanced TLR2 mRNA increase (Fig. 3,A). To further elucidate the role of MAP kinase pathways in TLR2 gene expression in macrophages, we treated RAW264.7 cells with anisomycin, a potent activator of JNK and p38 kinase (30). As shown in Fig. 3 C, no increase was observed in the TLR2 mRNA level after anisomycin treatment. Taken together, these findings suggest that neither ERK nor p38 kinase pathway is essential for LPS-mediated up-regulation of TLR2 mRNA. On the contrary, ERK activation may rather inhibit TLR2 mRNA expression.

FIGURE 3.

Neither ERK nor p38 MAP kinase pathway is essential for LPS-mediated TLR2 mRNA increase in RAW264.7 cells. A, RAW264.7 cells were pretreated with a series of concentrations of either PD98059 or SB208530 for 30 min followed by 2-h stimulation with 1 ng/ml LPS. Total RNAs (20 μg/each) were extracted for the Northern blot analysis using 32P-labeled TLR2 cDNA probe. A picture of the ethidium bromide-stained gel is shown as a control. B, Effective inhibition of the kinase activity by specific inhibitors. Upper panel, Cells were treated with PD98059 and LPS as in A. LPS-mediated ERK phosphorylation was measured by Western blot using an anti-phospho-ERK Ab. Lower panel, Cells were treated with SB208530 and LPA as in A. LPS-mediated p38 kinase activation was measured by the in vitro kinase assay using GST-ATF2(1-109) as substrate. C, RAW264.7 cells were untreated or treated with indicated concentrations of anisomycin for 2 h. Extracted total RNAs were analyzed as in A.

FIGURE 3.

Neither ERK nor p38 MAP kinase pathway is essential for LPS-mediated TLR2 mRNA increase in RAW264.7 cells. A, RAW264.7 cells were pretreated with a series of concentrations of either PD98059 or SB208530 for 30 min followed by 2-h stimulation with 1 ng/ml LPS. Total RNAs (20 μg/each) were extracted for the Northern blot analysis using 32P-labeled TLR2 cDNA probe. A picture of the ethidium bromide-stained gel is shown as a control. B, Effective inhibition of the kinase activity by specific inhibitors. Upper panel, Cells were treated with PD98059 and LPS as in A. LPS-mediated ERK phosphorylation was measured by Western blot using an anti-phospho-ERK Ab. Lower panel, Cells were treated with SB208530 and LPA as in A. LPS-mediated p38 kinase activation was measured by the in vitro kinase assay using GST-ATF2(1-109) as substrate. C, RAW264.7 cells were untreated or treated with indicated concentrations of anisomycin for 2 h. Extracted total RNAs were analyzed as in A.

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Curcumin has recently been reported to inhibit JNK activation in various cell types at a concentration of 5 or 10 μM (31). Higher concentration of curcumin (50 μM or higher), however, inhibits NF-κB activation (31). When RAW264.7 cells were treated with curcumin before LPS stimulation, it inhibited LPS-mediated TLR2 mRNA up-regulation at 50 μM, whereas lower concentrations of curcumin had no inhibitory effect (Fig. 4,A). To investigate whether this inhibitory effect correlated with the inhibition of NF-κB activity, we performed a luciferase reporter assay. RAW264.7 cells were transfected with a luciferase reporter construct containing a NF-κB recognition sequence followed by the treatment with curcumin and LPS. As shown in Fig. 4 B, concentration-dependent inhibition of NF-κB activity by curcumin correlated well with the inhibition of TLR2 gene expression, suggesting that NF-κB activation may be essential for the LPS-mediated TLR2 mRNA up-regulation.

FIGURE 4.

LPS-mediated TLR2 mRNA increase is inhibited by a high concentration of curcumin. A, RAW264.7 cells were pretreated with various concentrations of curcumin for 30 min followed by a 2-h stimulation with 1 ng/ml LPS. Total RNAs (20 μg/each) were extracted for the Northern blot analysis using the 32P-labeled TLR2 cDNA probe. A picture of the ethidium bromide-stained gel is also shown. B, Inhibition of LPS-mediated NF-κB activation by cucumin in RAW264.7 cells. RAW264.7 cells were transiently transfected with an NF-κB reporter plasmid along with an internal control. At 24 h after the transfection, the cells were pretreated with various concentrations of curcumin for 30 min followed by 1 ng/ml LPS stimulation. Cells were lysed after the 8-h LPS treatment, and the standardized luciferase activities were measured. The assay was repeated three times. The luciferase activity for LPS treatment alone was defined as 100% in each experiment. Results are presented as mean ± SD.

FIGURE 4.

LPS-mediated TLR2 mRNA increase is inhibited by a high concentration of curcumin. A, RAW264.7 cells were pretreated with various concentrations of curcumin for 30 min followed by a 2-h stimulation with 1 ng/ml LPS. Total RNAs (20 μg/each) were extracted for the Northern blot analysis using the 32P-labeled TLR2 cDNA probe. A picture of the ethidium bromide-stained gel is also shown. B, Inhibition of LPS-mediated NF-κB activation by cucumin in RAW264.7 cells. RAW264.7 cells were transiently transfected with an NF-κB reporter plasmid along with an internal control. At 24 h after the transfection, the cells were pretreated with various concentrations of curcumin for 30 min followed by 1 ng/ml LPS stimulation. Cells were lysed after the 8-h LPS treatment, and the standardized luciferase activities were measured. The assay was repeated three times. The luciferase activity for LPS treatment alone was defined as 100% in each experiment. Results are presented as mean ± SD.

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In this study, we have investigated the gene expression of putative LPS signaling receptors, TLR2 and TLR4, in mouse macrophages. The gene expression of TLR2, but not TLR4, was significantly increased in the presence of LPS in vitro and in vivo. TLR2 mRNA also significantly increased in response to various cytokine stimulations, whereas TLR4 mRNA remained constant after these stimulations.

The roles of TLR2 and TLR4 as LPS receptors are still controversial. Although both human and mouse TLR2 were shown to mediate LPS signaling in vitro (16, 17, 18), the role of TLR2 in LPS signaling in vivo is questioned due to several recent findings. Studies examining a single autosomal locus (lps) responsible for the LPS hyporesponsiveness (lpsd) of two mouse strains (C3H/HeJ and C57BL10/ScCr) (21, 22) found TLR4 gene in the lps locus and also identified mutations in the TLR4 genes of both lpsd strains, strongly suggesting that the defective TLR4 is responsible for the LPS hyporesponsiveness of these mice. More recently, both TLR2- and TLR4-lacking mice have been generated. Although TLR4-lacking mice show LPS-hyporesponsiveness very similar to that of the lpsd mouse strains (32), macrophages from TLR2-lacking mice showed TNF-α secretion similar to that of wild-type mice in response to LPS stimulation (23). These findings are consistent with the idea that TLR4 is essential in LPS signaling, whereas TLR2 is dispensable.

Our current finding may explain the discrepancies between gene-lacking mice and in vitro transfection data. In unstimulated macrophages, TLR4 mRNA is constitutively expressed, whereas TLR2 gene expression is relatively low. Thus, TLR4 may be essential to initiate the innate immunity in the first encounter with Gram-negative bacteria, whereas TLR2 is induced later by TLR4 signals and becomes the second LPS receptor to help the immune response against the bacteria. Consistent with this hypothesis, the sensitivity of lpsd mice is restored by treatment with IFN-γ or bacillus Calmette-Guérin (33, 34, 35, 36). The expression level of TLR2 in macrophages is likely to be higher after these treatments in lpsd mice as TLR2 mRNA is up-regulated by various cytokine stimulations in RAW264.7 cells (Fig. 2,A) and in C3H/HeJ mouse macrophages (Fig. 2 E). It should be noted, however, that our findings are based on the measurements of mRNA levels rather than protein levels. The surface expression of TLR2 may not necessarily correlate with the mRNA levels, and further studies are warranted on this matter.

Several other explanations are also possible. One possible explanation is that TLR2 and TLR4 respond to different types of LPS. Cells from C3H/HeJ mice are as sensitive as their normal counterparts to certain types of LPS (e.g., Porphyromonas gingivalis LPS) (37). There is divergence in LPS structure among Gram-negative bacteria, and it is reasonable to presume that TLR4 responds to certain types of LPS better than TLR2 while TLR2 responds better to others. A second possibility is that TLR2 and TLR4 may cooperate to respond to LPS. It is not known how TLRs are involved in the LPS receptor complex, and it is possible that TLR2 needs to associate with TLR4 to form a heterodimer or oligomer for proper functions, whereas TLR4 can work as a homodimer or oligomer. This seems inconsistent with the in vitro transfection data that the transfection of TLR2 alone can confer LPS responsiveness to cells (16, 17, 18). However, it cannot be excluded that the transfected TLR2 cooperates with the weakly expressed endogenous TLR4. Thirdly, it is possible that the published in vitro results that TLR2 mediates LPS signals are due to the nonphysiological TLR2 expression level, and TLR2 is not involved in LPS response in physiological conditions.

Lastly, it is also possible that TLR2 may have just responded to the other bacterial components contaminating the LPS used in the in vitro assays.

Recently, it has been reported that TLR2 mediates signals from liparabinomannan, peptidoglycan, lipoproteins, and lipoteichoic acid (24, 26, 27, 38, 39, 40). TLR2-defective mice showed unresponsiveness to these components, suggesting that TLR2 is essential for the signal transduction (23). It remains to be seen whether these bacterial components also increase the TLR2 mRNA in macrophages. However, because TNF-α up-regulates TLR2 gene expression and serum TNF-α level increases in response to these components, the same positive feedback mechanism should exist in response to bacteria containing these components, and the increased TLR2 expression will probably contribute to the accelerated immune responses by macrophages. If so, regulation of TLR2 expression may be one of the immune-regulatory mechanisms commonly involved in host defense against many bacterial strains.

Although LPS is known to activate various MAP kinase pathways (4, 5, 6, 7, 8, 9), the activation of ERK, p38 kinase, or JNK does not seem to be essential for the induction of TLR2 gene expression in RAW264.7 cells (Figs. 3 and 4). PD98059, a specific inhibitor of ERK pathway, rather enhanced TLR2 mRNA increase by LPS, suggesting that ERK activation has an inhibitory effect on TLR2 expression in macrophages. Anisomycin, a potent activator of both JNK and p38 MAP kinase, fail to induce TLR2 mRNA, suggesting that these MAP kinases are not sufficient for TLR2 gene up-regulation. Additionally, SB208530, a specific inhibitor of the p38 MAP kinase pathway, was only slightly inhibitory to LPS-mediated TLR2 induction. These results were rather unexpected, because they were in sharp contrast to those with T cells. We have previously reported that in T cells PMA potently increased TLR2 mRNA expression and both PD98059 and SB208530 effectively inhibited the increase (18). This difference clearly suggests that the regulatory roles of MAP kinase activation pathways in the regulation of TLR2 mRNA vary considerably in different cell types.

Interestingly, ERK and p38 kinase pathways were reported to play different roles in the expression of some LPS-responsive genes in macrophages. For example, P38 kinase is essential for NO synthase induction, whereas ERK plays only a minor role (41). Also, while p38 kinase promotes IL-12 (p40) expression, ERK inhibits it (41). It is of note that LPS-mediated NF-κB activation is known to play a major role in the induction of both genes (42, 43). Interestingly, phosphoglycan, a major cell wall protein of Leishmania, was suggested to suppress immune response by decreasing IL-12 production via the activation of ERK pathway (41), and this mechanism can be shared by other pathogens. Our present findings suggest that the suppression of TLR2 expression through ERK activation may be another mechanism for Leishmania to escape immune response.

Instead, in macrophages, the NF-κB activation seems to be essential for the LPS-mediated TLR2 induction. LPS is a strong activator of NF-κB (2, 3), and all of the cytokines shown here to increase TLR2 expression are known activators of NF-κB (44). Additionally, curcumin, an inhibitor of NF-κB activity at high concentrations, completely inhibited LPS-mediated TLR2 induction at 50 μM (Fig. 4). Although curcumin is not a specific inhibitor of NF-κB activation, the dose response of TLR2 mRNA inhibition correlated well with that of NF-κB activation. From these observations it is likely that LPS-induced TLR2 gene expression is mediated by NF-κB activation. In agreement with this hypothesis, there are NF-κB recognition sites in the promoter region of mouse TLR2 gene (T. Musikacharoen, manuscript in preparation).

While this paper was being reviewed, Medvedev et al. also reported the rapid increase of TLR2 mRNA in LPS-treated mouse macrophages (45). In their report, however, TLR2 mRNA elevation lasted for at least 12 h, longer than our finding with RAW264.7 cells (Fig. 2 B). The longer duration of TLR2 mRNA could be due to the effects by a small number of contaminating other types of cells, such as T cells. In fact, we also found longer TLR2 mRNA elevation in mouse macrophages than in macrophage cell lines after LPS treatment (data not shown).

In contrast to our results, TLR2 mRNA did not seem to increase in human monocytes after 3-h treatment with LPS (46). The reasons for this discrepancy are not clear. It is possible that TLR2 gene expression is differently regulated in monocytes and in tissue macrophages. Alternatively, it is also possible that the regulation varies among different species.

Recent reports have indicated that TLR4 mRNA expression in macrophages was decreased within a few hours of LPS treatment (45, 47), whereas we could not observe the obvious TLR4 mRNA decrease until 24 h after the LPS treatment (Fig. 2 B). In another report, Muzio et al. (48) found that LPS increased TLR4 mRNA in monocytes and polymorphonuclear leukocytes. While performing the time course experiments using RAW264.7 cells, we also observed a slight decrease or increase of TLR4 mRNA at 2 or 4 h of LPS treatment on several occasions. In most experiments, however, TLR4 mRNA remained constant. Thus, the discrepancies between their results and ours are most likely due to the slight differences of stimulation conditions (cell densities, etc.).

In summary, this study implies that the expression of the two putative LPS signaling receptor genes, TLR2 and TLR4, are differently regulated in mouse macrophages. TLR4 was constitutively expressed and remained constant after various stimulations, including LPS. In contrast, gene expression of TLR2 significantly increased after LPS treatment both in vitro and in vivo. This led us to suggest a model shown in Fig. 5. When Gram-negative bacteria invade the host, macrophages first recognize LPS through the constitutively expressed TLR4. Later, TLR2 is induced directly by LPS or indirectly through secondary cytokines. Through the newly synthesized TLR2, macrophages respond better to LPS or other bacterial components such as lipoproteins that are membranous components of both Gram-positive and Gram-negative bacteria. There are six mammalian TLR proteins that have been identified so far, and the number will probably increase in the future. It remains to be seen whether this inducibility is unique to TLR2 or is shared by some other members of TLRs.

FIGURE 5.

Hypothetical scheme of the roles of TLR2 and TLR4 in macrophages in response to Gram-negative bacteria. At the initial stage of infection, LPS signals are mediated by the constitutively expressed TLR4 in combination with CD14. LPS signals and signals from the secreted cytokines (such as TNF-α and IL-1β) lead to the induction of TLR2 expression. Induced TLR2 mediates signals from other bacterial components and LPS leading to accelerated immune responses.

FIGURE 5.

Hypothetical scheme of the roles of TLR2 and TLR4 in macrophages in response to Gram-negative bacteria. At the initial stage of infection, LPS signals are mediated by the constitutively expressed TLR4 in combination with CD14. LPS signals and signals from the secreted cytokines (such as TNF-α and IL-1β) lead to the induction of TLR2 expression. Induced TLR2 mediates signals from other bacterial components and LPS leading to accelerated immune responses.

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We thank K. Itano, A. Kato, and A. Nishikawa for their technical assistance.

1

This work was supported in part by grants from Ono Pharmaceutical Company, the Yokoyama Research Foundation for Clinical Pharmacology, and the Naito Foundation (to T. M.); the Ministry of Education, Science and Culture of Japan (JSPS-RFTF97L00703); and the Yakult Bioscience Foundation (to Y.Y.).

3

Abbreviations used in this paper: ERK, extracellular signal-regulated kinase; JNK, c-Jun N-terminal kinase; TLR, Toll-like receptor; MAP, mitogen-activated protein; MEK, MAP/ERK kinase.

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