Recent studies have implicated Toll-like receptors (TLR), especially TLR2 and TLR4, as sentinel receptors that signal the interaction of macrophages with bacterial pathogens via a NF-κB-mediated pathway. The regulation of TLR gene expression, however, has not been intensively studied. Here, we report that TLR2 mRNA was induced following infection of murine macrophages with Mycobacterium avium. The changes in TLR2 mRNA correlated with an increase in TLR2 surface expression. Infection with M. avium resulted in a concomitant decrease in TLR4 mRNA. The effect of M. avium infection on TLR2 mRNA appeared to be mediated, in part, by TLR2 because the induction of the mRNA was partially blocked by preincubation of the macrophages with an anti-human TLR2 Ab. In contrast, the effect of LPS stimulation was mediated via TLR4 because infection of macrophages from LPSd mice, which do not express active TLR4, resulted in an increase in TLR2 mRNA, while treatment of macrophages from these mice with LPS failed to induce TLR2 mRNA. Several cytokines, including TNF-α, IL-1α, and GM-CSF, but not IFN-γ, induced TLR2 mRNA. M. avium infection resulted in the induction of TLR2 mRNA by macrophages from both TNFRI knockout and NF-κB p50 knockout mice.

Macrophages play a critical role in recognizing microbial pathogens and in controlling their growth (1). The interaction of bacterial pathogens with macrophages initiates a series of events that result in the production of inflammatory cytokines and the release of antimicrobial effector molecules. The mechanism of how recognition of bacterial pathogens occurs and the signals that are transmitted into the cells are not fully understood. Some bacterial constituents can be recognized by cell surface receptors on macrophages, which initiate a signaling cascade (2). The classical paradigm for this process is the response of macrophages to bacterial LPS. The molecular basis of the recognition of LPS by macrophages has been described (3). A serum protein, LPS-binding protein, can bind to LPS and transfer it to CD14. CD14, however, is a GPI-linked protein and does not have an intracellular domain that can communicate with the interior of the cell. Recent studies have shown that Toll-like receptors (TLR)3 are involved in the recognition and signaling pathway for LPS as well as other bacterial pathogens.

Toll is a type I transmembrane receptor in Drosophila that is involved in dorsal-ventral patterning in larvae and in the induction of an antifungal response in adult flies (4, 5, 6). Activation of the Toll receptor by its ligand Spatzle results in the interaction and stimulation of several signaling molecules that are homologous to proteins involved in NF-κB activation by the IL-1R in mammalian cells. Recently, Medzhitov et al. (7) cloned a human receptor, homologous to Toll, which participates in activation of cells during both innate and adaptive immune responses. Ten members of the human TLR family have been identified (8). At least some of these are involved in mediating NF-κB activation following the interaction of macrophages with different bacterial pathogens or bacterial cell wall components (9, 10, 11, 12). TLR4 has been shown to mediate the response of macrophages to LPS (13, 14). The hyporesponsiveness of C3H/HeJ mice (Lpsd) to LPS is the result of a missense mutation in TLR4 (15, 16). TLR2 has been implicated in the activation of NF-κB following interaction of macrophages with LPS, lipoarabinomannan (LAM), from mycobacterial species and lipoteichoic acids from the cell walls of Gram-positive bacteria (17, 18, 19, 20).

The purpose of this investigation was to study the regulation of TLR2 gene expression following the interaction of murine macrophages with Mycobacterium avium. We found that infection with mycobacteria resulted in an increase in TLR2 mRNA and in the expression of TLR2 on the cell surface. Infection of macrophages from Lpsd mice also resulted in an increase in TLR2 mRNA, as did treatment of the macrophages with TNF-α, IL-1α, and GM-CSF. In contrast, treatment of the cells with IFN-γ did not affect TLR2 mRNA expression. The increase in TLR2 mRNA following infection with M. avium is not mediated via the TNFRI, but appears to be the result of stimulation through TLR2 by a pathway that may be independent of NF-κB.

Male, BALB/c, and C3H/HeN mice were purchased from Harlan (Indianapolis, IN) and Charles River Laboratories (Wilmington, MA), respectively. C3H/HeJ, C57BL/10SnJ, NF-κB p50 knockout, and TNFRI knockout mice were all obtained from The Jackson Laboratory (Bar Harbor, ME). C57BL/10ScCr mice were obtained from National Institutes of Health. Mice were housed in groups of five and given food and water ad libitum. Six- to 8-wk-old mice were used as macrophage donors.

Tissue culture media as well as murine rIFN-γ and restriction enzymes PstI and HindIII were purchased from Life Technologies (Gaithersburg, MD). LPS (Escherichia coli O111:B4), cycloheximide, PGE2, and purified mouse IgG were obtained from Sigma (St. Louis, MO). Recombinant TNF-α, IL-α, and GM-CSF were purchased from Endogen (Woburn, MA). FITC-conjugated rat anti-mouse IgG2a was obtained from PharMingen (San Diego, CA). Douglas Golenbock (Boston University, Boston, MA) kindly provided a mouse anti-human TLR2 mAb (8). Gene-PAGE-denaturing gel was obtained from Amresco (Solon, OH), and cDNA probe primers were constructed by Integrated DNA Technologies (Coralville, IA). All reagents contain less than 0.1 EU/ml endotoxin, as tested by using an endotoxin detection kit from Sigma (St. Louis, MO).

Macrophages were harvested by peritoneal lavage 3–4 days following the i.p. injection of 4% sterile thioglycolate medium (Difco, Detroit, MI). The cells were washed with PBS and resuspended in complete IMDM (Life Technologies) containing 10% FBS (HyClone, Logan, UT) and 1% penicillin-streptomycin. Macrophages were purified by adding 6 × 106 cells/well in six-well culture plates and culturing the cells for 16 h at 37°C in 5% CO2 in air. Monolayers were washed with IMDM without serum to remove nonadherent cells and then infected with M. avium (ATCC 35712) at an 8:1 bacteria to macrophage ratio or stimulated with LPS for 3 h, as specified. Before use, the M. avium was grown in Middlebrook 7H9 broth cultures and aliquoted in 1-ml amounts and stored frozen at −70°C. Each vial contained 2.5 × 108 CFU, as determined by plate count on 7H11 agar plates.

Total mouse RNA was extracted using Ambion (Austin, TX) RNAqueous kit by following the manufacturer’s instructions. A total of 4–10 μg RNA was used for each RPA. Except for the template probe synthesis, all reagents used for the RPA were purchased from PharMingen and preformed as described by Chen et al. (21). The cDNA sequences for murine TLR2 and TLR 4 were obtained from the GenBank (accession AF124741 for TLR2; accession AF095353 for TLR4). The RT-PCR primers for the TLR2 are: 5′-ACAGCTACTGTGTGACTCTCCGCC-3′ and 5′-GGTCTTGGTGTTCATTATCTTGCGC-3′. The primers for the TLR4 are: 5′-GACCTCAGCTTCAATGGTGC-3′ and 5′-TATCAGAAATGCTACAGTGGATACC-3′. Briefly, the cDNA fragments of TLR2 and TLR4 were amplified by RT-PCR with 1 μg mouse peritoneal macrophage RNA using a RT-PCR kit from Roche (Indianapolis, IN). cDNA fragments of each gene were subcloned into a pGEM-T easy vector (Promega, Madison, WI). The plasmids were then transformed into DH5a competent E. coli, and selected for ampicillin resistance. Plasmid DNA was extracted using a miniprep kit from Roche, and orientation of TLR2 and TLR4 was confirmed by sequencing. For RPA, the plasmids containing the cDNA inserts were cut with PstI. The G3PDH RPA probe was created by cloning a 134-bp HindIII-ApaI fragment of G3PDH cDNA (nt 236–370) into a pBluescript SK(−) plasmid vector and the inserting plasmid DNA linearized with HindIII. The linearized plasmids were purified on 1.5% agarose gels and recovered by using a Geneclean kit (Bio 101, Vista, CA) and used as templates to create 32P-radiolabeled antisense RNA by in vitro transcription using T7 RNA polymerase for use as probes in the RPA.

Macrophages (1 × 106/ml) were fixed with 2% paraformaldehyde and washed, and 2 × 105 cells were resuspended in 200 μl of PBS containing 1% BSA and 0.01% sodium azide. The cells were preincubated with a 1 μg/ml of Fc block (PharMingen) before incubation with the mouse anti-human TLR2 Ab or an isotype control. The cells were washed and incubated with an FITC-labeled rat anti-mouse IgG2a. The cells were analyzed using a Coulter (Palo Alto, CA) Elite II flow cytometer. The data were analyzed using WinMD12.8 software. The percentage of cells expressing TLR2 was calculated by subtracting the percentage of cells stained with control IgG from the percentage of cells stained with specific anti-TLR2 Ab.

Infection of murine macrophages with M. avium resulted in an increase in TLR2 mRNA (Fig. 1). The level of TLR2 mRNA increased within 1 h following the addition of the bacteria and reached a maximum between 3 and 6 h after infection. In contrast to that observed with TLR2, the level of TLR4 mRNA, which was high initially, decreased following infection with M. avium. Identical results were observed with resident peritoneal macrophages, except that they expressed slightly higher endogenous levels of TLR2 mRNA (data not shown). The results in Fig. 2 show that expression of TLR2 on the surface of macrophages correlated with the increase in mRNA; TLR2 expression increased from 10.9% to 31.9% following the addition of M. avium. Infection of the cultures with Mycobacterium bovis (strain bacillus Calmette-Guérin) or with Mycobacterium tuberculosis (Erdman) led to similar results (data not shown).

FIGURE 1.

M. avium infection results in an increase in TLR2 mRNA and a decrease in TLR4 mRNA. Mouse peritoneal macrophages were infected with M. avium at 8:1 bacteria to macrophage ratio in serum-free media for the specified time. Samples of 6 μg of total RNA were used for RPA. The densitometric evaluation of TLR2 and TLR4 mRNA level is shown in the bottom part of the figure. The results were normalized to the expression of GAPDH. The results are representative of three independent experiments.

FIGURE 1.

M. avium infection results in an increase in TLR2 mRNA and a decrease in TLR4 mRNA. Mouse peritoneal macrophages were infected with M. avium at 8:1 bacteria to macrophage ratio in serum-free media for the specified time. Samples of 6 μg of total RNA were used for RPA. The densitometric evaluation of TLR2 and TLR4 mRNA level is shown in the bottom part of the figure. The results were normalized to the expression of GAPDH. The results are representative of three independent experiments.

Close modal
FIGURE 2.

Flow cytometric evaluation of TLR2 following infection with M. avium. The 2 × 106 thioglycolate-elicited macrophages were cultured in a six-well plate and infected with M. avium at 8:1 ratio for 18 h. Cells were removed from monolayers using a cell scraper, washed twice with PBS, and fixed with 2% paraformaldehyde. Cells were reacted with anti-TLR2 Ab or an isotype control for 30 min, washed, and stained with an FITC-conjugated anti-murine IgG. Flow cytometric analysis was carried using a Coulter Elite II flow cytometer, and data were analyzed using WinMDI2.8 software. The results are representative of four independent experiments.

FIGURE 2.

Flow cytometric evaluation of TLR2 following infection with M. avium. The 2 × 106 thioglycolate-elicited macrophages were cultured in a six-well plate and infected with M. avium at 8:1 ratio for 18 h. Cells were removed from monolayers using a cell scraper, washed twice with PBS, and fixed with 2% paraformaldehyde. Cells were reacted with anti-TLR2 Ab or an isotype control for 30 min, washed, and stained with an FITC-conjugated anti-murine IgG. Flow cytometric analysis was carried using a Coulter Elite II flow cytometer, and data were analyzed using WinMDI2.8 software. The results are representative of four independent experiments.

Close modal

Previously, it has been shown that LPS stimulates the activation of NF-κB via a TLR4-mediated pathway (14). The results in Fig. 3,A show that LPS treatment also resulted in an increase in TLR2 mRNA, but TLR4 mRNA decreased in macrophages from BALB/c mice. To further investigate the effects of M. avium or LPS on TLR2 mRNA, we also used macrophages from Lpsd mice. We found that the induction of TLR2 mRNA by low doses of LPS was impaired in C3H/HeJ (Lpsd) mice when compared with the effects of LPS on TLR2 mRNA expression in C3H/HeN (Lpsn) mice (Fig. 3,B). High doses of LPS induced TLR2 mRNA in macrophages from both strains of mice. In contrast, M. avium infection resulted in the same level of TLR2 mRNA expression by macrophages from both strains of mice (Fig. 3,C). Similar differences in the capacity of LPS and M. avium to induce TRL2 mRNA were found using macrophages from C57BL/10SnJ, Lpsn mice, and C57BL/10ScCr, Lpsd mice (data not shown). Subsequently, we found that pretreatment of the cells with an anti-TLR2 Ab blocked the increase in TLR2 mRNA following the addition of M. avium (Fig. 4).

FIGURE 3.

LPS and M. avium stimulate TLR2 mRNA expression via different receptors. A, Macrophages from BALB/c mice were treated with LPS at 50 ng/ml. After 3 h, RNA was isolated for RPA. The relative expression of TLR2 and TLR4 mRNA was plotted in the figure following normalization against the GAPDH signal. The original RPA is shown in the upper right corner of the figure. B, Thioglycolate-elicited peritoneal macrophages harvested from C3H/HeJ (Lpsd) and C3H/HeN (LpsN) mice were treated with LPS at specified concentrations for 3 h. The results of RPA as well as the densitometric evaluation of TLR2 mRNA expression are shown. C, Macrophages from C3H/HeJ and C3H/HeN were infected with M. avium for 3 h. RPA was conducted as described above. Both RPA and densitometric evaluations are shown. Similar results were observed in two independent experiments.

FIGURE 3.

LPS and M. avium stimulate TLR2 mRNA expression via different receptors. A, Macrophages from BALB/c mice were treated with LPS at 50 ng/ml. After 3 h, RNA was isolated for RPA. The relative expression of TLR2 and TLR4 mRNA was plotted in the figure following normalization against the GAPDH signal. The original RPA is shown in the upper right corner of the figure. B, Thioglycolate-elicited peritoneal macrophages harvested from C3H/HeJ (Lpsd) and C3H/HeN (LpsN) mice were treated with LPS at specified concentrations for 3 h. The results of RPA as well as the densitometric evaluation of TLR2 mRNA expression are shown. C, Macrophages from C3H/HeJ and C3H/HeN were infected with M. avium for 3 h. RPA was conducted as described above. Both RPA and densitometric evaluations are shown. Similar results were observed in two independent experiments.

Close modal
FIGURE 4.

TLR2 contributes to the induction of TLR2 mRNA following infection with M. avium. Peritoneal macrophages were preincubated with anti-human TLR2 Ab (10 μg/ml) or control IgG (10 μg/ml) for 2 h on ice before M. avium infection for 3 h. Samples were analyzed by RPA. Both RPA and densitometric analysis are presented. Lanes 1–4 are: untreated (control); M. avium treated; M. avium + anti-TLR2 Ab; M. avium + control IgG. The data shown here were representative of three independent experiments.

FIGURE 4.

TLR2 contributes to the induction of TLR2 mRNA following infection with M. avium. Peritoneal macrophages were preincubated with anti-human TLR2 Ab (10 μg/ml) or control IgG (10 μg/ml) for 2 h on ice before M. avium infection for 3 h. Samples were analyzed by RPA. Both RPA and densitometric analysis are presented. Lanes 1–4 are: untreated (control); M. avium treated; M. avium + anti-TLR2 Ab; M. avium + control IgG. The data shown here were representative of three independent experiments.

Close modal

One effect of M. avium infection of macrophages is the production of inflammatory cytokines such as IL-1 and TNF-α. Macrophages were treated with these and other cytokines, and their effect on TLR2 mRNA was evaluated. IL-α, TNF-α, and GM-CSF treatment increased TLR2 mRNA, while IFN-γ was without effect (Fig. 5,A). The addition of IL-10 before infection with M. avium did not prevent the increase in TLR2 mRNA. In contrast, the addition of PGE2 prevented the increase in TLR2 mRNA following infection with M. avium (Fig. 5 B).

FIGURE 5.

Cytokine regulation of TLR2 mRNA expression. A, Peritoneal macrophages were stimulated with TNF-α (10 ng/ml), IL-1α (50 ng/ml), and GM-CSF (50 ng/ml) for 3 h, and IFN-γ (100 U/106 cells) for 24 h. RNA was extracted and analyzed by RPA. RPA and densitometric analysis are presented. The result is representative of three experiments. B, IL-10 (50 μg/ml) or PGE2 (10−5 M) was added to macrophages for 1 or 2 h, respectively, before M. avium infection. Samples were analyzed by RPA. Lanes 1–4 are: untreated (control); M. avium (MA) alone; MA + IL-10; and MA + PGE2. The densitometric evaluation is also shown. Similar results were obtained in two independent experiments.

FIGURE 5.

Cytokine regulation of TLR2 mRNA expression. A, Peritoneal macrophages were stimulated with TNF-α (10 ng/ml), IL-1α (50 ng/ml), and GM-CSF (50 ng/ml) for 3 h, and IFN-γ (100 U/106 cells) for 24 h. RNA was extracted and analyzed by RPA. RPA and densitometric analysis are presented. The result is representative of three experiments. B, IL-10 (50 μg/ml) or PGE2 (10−5 M) was added to macrophages for 1 or 2 h, respectively, before M. avium infection. Samples were analyzed by RPA. Lanes 1–4 are: untreated (control); M. avium (MA) alone; MA + IL-10; and MA + PGE2. The densitometric evaluation is also shown. Similar results were obtained in two independent experiments.

Close modal

Infection of macrophages with M. avium results in the production of TNF-α (22). It is possible, therefore, that the effect of M. avium on TLR2 mRNA expression was the result of the production of TNF-α. To evaluate the role of TNF-α and M. avium, we used macrophages from TNFRI knockout mice. The results in Fig. 6 show that TNF-α or infection with M. avium resulted in the induction of TLR2 mRNA by macrophages from +/+ C57BL/6J mice. In contrast, M. avium infection resulted in an increase in TLR2 mRNA in macrophages from TNFRI knockout mice, while TNF-α failed to induce TLR2 mRNA. Thus, the increase in TLR2 mRNA does not appear to be the result of stimulation by TNF-α.

FIGURE 6.

Induction of TLR2 mRNA by M. avium is TNF-α independent. Macrophages from TNF RI knockout mice or control strain (C57BL/6) were treated with TNF-α (10 ng/ml) or M. avium at multiplicity of 8:1 for 3 h. Both RPA and densitometric analysis are shown. Lanes 1–3 (TNFRI−/−) or 4–6 (C57BL/6) represent unstimulated (lanes 1 and 4); M. avium (lanes 2 and 5); and TNF-α (5 ng/ml) (lanes 3 and 6). The result is representative of three independent experiments.

FIGURE 6.

Induction of TLR2 mRNA by M. avium is TNF-α independent. Macrophages from TNF RI knockout mice or control strain (C57BL/6) were treated with TNF-α (10 ng/ml) or M. avium at multiplicity of 8:1 for 3 h. Both RPA and densitometric analysis are shown. Lanes 1–3 (TNFRI−/−) or 4–6 (C57BL/6) represent unstimulated (lanes 1 and 4); M. avium (lanes 2 and 5); and TNF-α (5 ng/ml) (lanes 3 and 6). The result is representative of three independent experiments.

Close modal

The addition of the PKC inhibitor bis-indolymaleimide, before infection with M. avium, prevented the induction of TLR2 mRNA of macrophages (Fig. 7,A). Because TLR2 and TLR4 stimulation results in the activation of NF-κB, we also sought to determine whether M. avium infection activated TLR2 mRNA transcription via a NF-κB-dependent pathway. We infected macrophages from NF-κB p50 knockout mice. The results presented in Fig. 7,B show that M. avium infection resulted in the induction of TLR2 mRNA in NF-κB p50 knockout mice. Furthermore, the addition of cycloheximide prevented the increase in TLR2 mRNA. Similarly, cycloheximide also prevented the increase in TLR2 mRNA following treatment of the cells with TNF-α (Fig. 7 C). These results indicate that new protein synthesis is required for the full induction of TLR2 mRNA by M. avium or TNF-α.

FIGURE 7.

Up-regulation of TLR2 mRNA following M. avium infection is PKC dependent and NF-κB independent. A, Macrophages were treated with a specific PKC inhibitor, bis-indolymaleimide (10 μM), for 1 h, followed by M. avium infection for 3 h. Lane 1, Unstimulated (control); lane 2, M. avium (MA) alone; lane 3, MA + PKC inhibitor. B, Macrophages from NF-κB p50 knockout mice were infected with M. avium or stimulated by TNF-α (10 ng/ml) for 3 h. Samples were analyzed by RPA. Both RPA results and densitometric analysis are shown. C, Macrophages were treated with cycloheximide (10 μg/ml) for 1 h before M. avium infection. RNA was extracted after 3 h, and RPA was performed. Densitometric analysis is also shown. The results are representative of three independent experiments.

FIGURE 7.

Up-regulation of TLR2 mRNA following M. avium infection is PKC dependent and NF-κB independent. A, Macrophages were treated with a specific PKC inhibitor, bis-indolymaleimide (10 μM), for 1 h, followed by M. avium infection for 3 h. Lane 1, Unstimulated (control); lane 2, M. avium (MA) alone; lane 3, MA + PKC inhibitor. B, Macrophages from NF-κB p50 knockout mice were infected with M. avium or stimulated by TNF-α (10 ng/ml) for 3 h. Samples were analyzed by RPA. Both RPA results and densitometric analysis are shown. C, Macrophages were treated with cycloheximide (10 μg/ml) for 1 h before M. avium infection. RNA was extracted after 3 h, and RPA was performed. Densitometric analysis is also shown. The results are representative of three independent experiments.

Close modal

TLR have been shown to be sentinel receptors that trigger macrophage activation upon interaction with potential pathogens. We have studied the regulation of TLR2 and TLR4 mRNA expression following the interaction of macrophages with M. avium or LPS. Infection of macrophages with M. avium or treatment with LPS resulted in an increase in TLR2 mRNA and a decrease in TLR4 mRNA. Other reports have indicated that infection of macrophages with mycobacteria, including M. avium, resulted in the activation of NF-κB via TLR2, but have not evaluated the effect of mycobacteria interaction on TLR gene expression (8, 23). Our observations regarding an increase in TLR2 mRNA and a decrease in TLR4 mRNA following treatment of macrophages with LPS are similar to those reported by Yang et al. (18, 24).

Both TLR2 and TLR4 have been reported to mediate the signaling for LPS (14, 18). The experiments done with TLR2−/− and TLR4−/− mice have established an essential role of TLR4 in LPS signaling. TLR2 could be an LPS signal transducer in other species (17, 25). Golenbock and others (8, 26) have found that TLR2 mediates NF-κB activation following interaction of macrophages with M. avium, M. tuberculosis, Trepenoma pallidum, and some Gram-positive bacteria. In this study, we have shown that treatment of macrophages from LPS-hyporesponsive C3H/HeJ and C57BL/10ScCr mice with M. avium resulted in an increase in TLR2 mRNA, while their response to LPS remained deficient. This suggests that TLR4 does not play a role in regulating the expression of the TLR2 gene following infection with M. avium. Our results indicate, however, that TLR2 may play a significant role in regulating TLR2 gene expression upon M. avium infection. Treatment of the cells with anti-TLR2 Ab blocked the capacity of M. avium to induce TLR2 mRNA. These observations are consistent with the previous studies in which Fenton et al.(23) showed that TLR4 mediated LPS stimulation, while TLR2 mediated signaling by LAM, although both LPS and LAM are CD14 ligands. In addition, high doses of LPS were found to induce TLR2 mRNA expression in Lpsd mice. This suggests that there may be a TLR4-independent signaling pathway responsible for the LPS responsiveness in Lpsd mice.

We found that the induction of TLR2 mRNA following infection with M. avium was also dependent on PKC activation. Infection of macrophages with M. avium leads to the activation of PKC, and PKC in turn is involved in the activation of many genes, such as TNF-α, IL-1, and IL-6 (27, 28). PKC functions by regulating the activation of transcription factors such as NF-κB, c-Fos, and c-Jun (29, 30, 31). The observation that infection with M. avium results in an increase in TLR2 mRNA in NF-κB p50 knockout mice suggests that NF-κB p50 is less likely involved in the induction of TLR2 following M. avium infection. Our results do not exclude the possibility that other NF-κB family members may play a role in the induction of TLR2 mRNA. Indeed, macrophages from the NF-κB p50 knockout mice have been shown to produce both IL-1 and TNF-α, but not IL-6 following stimulation with LPS (32). The recent observation that stimulation via TLR2 leads to the activation of mitogen-activated protein kinase pathway suggests that other pathways may also be responsible for the increase in TLR2 mRNA (33, 34). The demonstration that new protein synthesis is required for the induction of TLR2 mRNA further indicates that multiple signals, other than NF-κB, may be involved in the induction of TLR2 because initial NF-κB activation does not require de novo protein synthesis (35). The observation that both LPS and M. avium induce TLR2 mRNA suggests that the TLR2 promoter contains sites for the binding of several transcription factors. LPS stimulates via TLR4 and activates a NF-κB-dependent promoter, while M. avium, which stimulates via TLR2, results in the activation of a pathway that does not depend on NF-κB, but appears to be activated via PKC.

Cytokines play an important role in regulating macrophage function (36). IL-1, TNF-α, and GM-CSF are produced by macrophages shortly after infection with M. avium. Each of these cytokines induced an increase in TLR2 mRNA. Surprisingly, in our current system, IFN-γ did not stimulate TLR2 mRNA expression; neither did IFN-γ affect TLR4 mRNA. Beutler et al. (37) reported that IFN-γ stimulation could overcome the defective production of TNF-α in C3H/HeJ (Lpsd) mice challenged with LPS. Our results suggest that IFN-γ alone does not alter the LPS receptor (TLR4) expression. Macrophages also produce deactivating factors such as PGE2 and IL-10 (38, 39). We also found that PGE2, but not IL-10, suppressed the induction of TLR2 mRNA following the addition of M. avium. Our observation with IL-10 is consistent with that of Staege et al. (40), who found that IL-4, but not IL-10, decreased human TLR2 mRNA. Although the mechanisms for the regulation of TLR2 mRNA by proinflammatory cytokines and PGE2 remain unclear, our data indicate that the TLR2 gene expression is tightly regulated by autocrine factors produced during the early stages of infection.

M. avium infection or treatment of macrophages with LPS increases TLR2 mRNA, but decreases TLR4 mRNA. This suggests that these receptors may act differently to protect the host from potential pathogens. Both receptors activate cells via the same NF-κB pathway. Thus, LPS produced by Gram-negative bacteria leads to activation of NF-κB via TLR4, cellular activation, and the immediate production of inflammatory cytokines. These cytokines result in a down-regulation of TLR4, and at the same time increase the expression of TLR2. TLR2 appears to mediate cellular activation following exposure of macrophages to pathogens whose virulence is less immediately threatening to the host.

We thank Dr. Zhong Wangjian for his help with the flow cytometric analysis.

1

This work was supported by National Institutes of Health Grants AI42901, HL 59795, and MH 54966.

3

Abbreviations used in this paper: TLR, Toll-like receptor; LAM, lipoarabinomannan; PKC, protein kinase C; RPA, RNase protection assay.

1
Adams, D. O., T. A. Hamilton.
1984
. The cell biology of macrophage activation.
Annu. Rev. Immunol.
2
:
283
2
Ulevitch, R. J., P. S. Tobias.
1995
. Receptor-dependent mechanisms of cell stimulation by bacterial endotoxin.
Annu. Rev. Immunol.
13
:
437
3
Wright, S. D., R. A. Ramos, P. S. Tobias, R. J. Ulevitch, J. C. Mathison.
1990
. CD14, a receptor for complexes of LPS and LPS binding protein.
Science
249
:
1431
4
Hashimoto, C., K. L. Hudson, K. V. Anderson.
1988
. The Toll gene of Drosophila, required for dorsal-ventral embryonic polarity, appears to encode a transmembrane protein.
Cell
52
:
269
5
Gay, N. J., F. J. Keith.
1991
. Drosophila Toll and IL-1 receptor.
Nature
351
:
355
6
Belvin, M. P., K. V. Anderson.
1996
. A conserved signaling pathway: the Drosophila Toll-dorsal pathway.
Annu. Rev. Cell. Dev. Biol.
12
:
393
7
Medzhitov, R., P. Preston-Hurlburt, C. A. Janeway, Jr.
1997
. A human homologue of the Drosophila Toll protein signals activation of adaptive immunity.
Nature
388
:
394
8
Lien, E., T. J. Sellati, A. Yoshimura, T. H. Flo, G. Rawadi, R. W. Finberg, J. D. Carrol, T. Espevik, R. R. Ingalls, J. D. Radolf, D. T. Golenbock.
1999
. Toll-like receptor 2 functions as a pattern recognition receptor for diverse bacterial products.
J. Biol. Chem.
274
:
33419
9
Anderson, K. V..
2000
. Toll signaling pathways in the innate immune response.
Curr. Opin. Immunol.
12
:
13
10
Ulevitch, R. J..
1999
. Toll gates for pathogen selection.
Nature
401
:
755
11
Kopp, E. B., R. Medzhitov.
1999
. The Toll-receptor family and control of innate immunity.
Curr. Opin. Immunol.
11
:
13
12
Wright, S. D..
1999
. Toll, a new piece in the puzzle of innate immunity.
J. Exp. Med.
189
:
605
13
Hoshino, K., O. Takeuchi, T. Kawai, H. Sanjom, T. Ogawa, Y. Takeda, K. Takeda, S. Akira.
1999
. Cutting edge: Toll-like receptor 4 (TLR4)-deficient mice are hyporesponsive to lipopolysaccharide: evidence for TLR4 as the Lps gene product.
J. Immunol.
162
:
3749
14
Chow, J. C., D. W. Young, D. T. Golenbock, W. J. Christ, F. Gusovsky.
1999
. Toll-like receptor-4 mediates lipopolysaccharide-induced signal transduction.
J. Biol. Chem.
274
:
10689
15
Poltorak, A., X. He, I. Smirnova, M. Y. Liu, C. V. Huffel, X. Du, D. Birdwell, E. Alejos, M. Silva, C. Galanos, et al
1998
. Defective LPS signaling in C3H/HeJ and C57BL/10ScCr mice: mutations in Tlr4 gene.
Science
282
:
2085
16
Qureshi, S. T., L. Lariviere, G. Leveque, S. Clermont, K. J. Moore, P. Gros, D. Malo.
1999
. Endotoxin-tolerant mice have mutations in Toll-like receptor 4 (Tlr4).
J. Exp. Med.
189
:
615
17
Kirschning, C. J., H. Wesche, A. T. Merrill, M. Rothe.
1998
. Human Toll-like receptor 2 confers responsiveness to bacterial lipopolysaccharide.
J. Exp. Med.
188
:
2091
18
Yang, R. B., M. R. Mark, A. Gray, A. Huang, M. H. Xie, M. Zhang, A. Goddard, W. I. Wood, A. L. Gurney, P. J. Godowski.
1998
. Toll-like receptor-2 mediates lipopolysaccharide-induced cellular signalling.
Nature
395
:
284
19
Yoshimura, A., E. Lien, R. R. Ingalls, E. Tuomanen, R. Dziarski, D. T. Golenbock.
1999
. Cutting edge: recognition of Gram-positive bacterial cell wall components by the innate immune system occurs via Toll-like receptor 2.
J. Immunol.
163
:
1
20
Schwandner, R., R. Dziarski, H. Wesche, M. Rothe, C. J. Kirschning.
1999
. Peptidoglycan- and lipoteichoic acid-induced cell activation is mediated by Toll-like receptor 2.
J. Biol. Chem.
274
:
17406
21
Chen, L., C. Boomershine, T. Wang, W. P. Lafuse, B. S. Zwilling.
1991
. Synergistic interaction of catecholamine hormones and Mycobacterium avium results in the induction of interleukin-10 mRNA expression by murine peritoneal macrophages.
J. Neuroimmunol.
93
:
149
22
Rudnicka, W., M. Brzychcy, M. Klink, A. G. Lopez, P. A. Fonteyne, S. Rusch-Gerdes, B. Rozalska.
1999
. The production of nitric oxide and tumor necrosis factor by murine macrophages infected with mycobacterial strains differing by hemolytic activity.
Microbiol. Immunol.
43
:
637
23
Means, T. K., S. Wang, E. Lien, A. Yoshimura, D. T. Golenbock, M. J. Fenton.
1999
. Human Toll-like receptors mediate cellular activation by Mycobacterium tuberculosis.
J. Immunol.
163
:
3920
24
Nomura, F., S. Akashi, Y. Sakao, S. Sato, T. Kawai, M. Matsumoto, K. Nakanishi, M. Kimoto, K. Miyake, K. Takeda, S. Akira.
2000
. Cutting edge: endotoxin tolerance in mouse peritoneal macrophages correlates with down-regulation of surface Toll-like receptor 4 expression.
J. Immunol.
164
:
3476
25
Takeuchi, O., K. Hoshino, T. Kawai, H. Sanjo, H. Takada, T. Ogawa, K. Takeda, S. Akira.
1999
. Differential roles of TLR2 and TLR4 in recognition of Gram-negative and Gram-positive bacterial cell wall components.
Immunity
11
:
443
26
Flo, T. H., O. Halaas, E. Lien, L. Ryan, G. Teti, D. T. Golenbock, A. Sundan, T. Espevik.
2000
. Human Toll-like receptor 2 mediates monocyte activation by Listeriamonocytogenes, but not by group B streptococci or lipopolysaccharide.
J. Immunol.
164
:
2064
27
Kontny, E., M. Zilokowska, A. Ryzewska, W. Maslinski.
1999
. Protein kinase C-dependent pathway is critical for the production of pro-inflammatory cytokines (TNF-α, IL-1β, IL-6).
Cytokine
11
:
839
28
Kontny, E., M. Kurowska, K. Szczepanska, W. Maslinski.
2000
. Rottlerin, a PKC isozyme-selective inhibitor, affects signaling events and cytokine production in human monocytes.
J. Leukocyte Biol.
67
:
249
29
Ghosh, S., D. Baltimore.
1990
. Activation in vitro of NF-κB by phosphorylation of its inhibitor IκB.
Nature
344
:
678
30
Seguin, R., K. Keller, K. Chadee.
1995
. Entamoeba histolytica stimulates the unstable transcription of c-fos and tumor necrosis factor-α mRNA by protein kinase C signal transduction in macrophages.
Immunology
86
:
49
31
Clerk, A., J. G. Harrison, C. S. Long, P. H. Sugden.
1999
. Pro-inflammatory cytokines stimulate mitogen-activated protein kinase subfamilies, increase phosphorylation of c-Jun and ATF2 and up-regulate c-Jun protein in neonatal rat ventricular myocytes.
J. Mol. Cell. Cadiol.
31
:
2087
32
Sha, W. C., H. C. Liou, E. I. Tuomanen, D. Baltimore.
1995
. Targeted disruption of the p50 subunit of NF-κB leads to multifocal defects in immune responses.
Cell
80
:
321
33
Irie, T., T. Muta, K. Takeshige.
2000
. TAK1 mediate an activation signal from Toll-like receptor(s) to nuclear factor-κB in lipopolysaccharide-stimulated macrophages.
FEBS Lett.
467
:
160
34
Matsuguchi, T., K. Takagi, T. Musikacharoen, Y. Yoshikai.
2000
. Gene expression of lipopolysaccharide receptors, Toll-like receptor 2 and 4, are differently regulated in mouse T lymphocytes.
Blood
95
:
1378
35
Hecker, M., C. Preiss, P. Klemm, R. Busse.
1996
. Inhibition by antioxidants of nitric oxide synthase expression in murine macrophages: role of nuclear factor κB and interferon regulatory factor 1.
Br. J. Pharmacol.
118
:
2178
36
Arai, K., F. Lee, A. Miyajima, S. Miyatake, N. Arai, T. Yokota.
1990
. Cytokines: coordinators of immune and inflammatory responses.
Annu. Rev. Biochem.
59
:
783
37
Beutler, B., V. Tkacenko, I. Milsark, N. Krochin, A. Cerami.
1986
. Effect of γ interferon on cachectin expression by mononuclear phagocytes: reversal of the Lpsd (endotoxin resistance) phenotype.
J. Exp. Med.
164
:
1791
38
Kunkel, S. L., M. Spengler, M. A. May, R. Spengler, J. Larrick, D. Remick.
1988
. Prostaglandin E2 regulates macrophage-derived tumor necrosis factor gene expression.
J. Biol. Chem.
263
:
5380
39
Blauer, F., P. Groscurth, M. Schneemann, G. Schoedon, A. Schaffner.
1995
. Modulation of the antilisterial activity of human blood-derived macrophages by activating and deactivating cytokines.
J. Inteferon Cytokine Res.
15
:
105
40
Staege, H., A. Schaffner, M. Schneemann.
2000
. Human Toll-like receptor 2 and 4 are targets for deactivation of mononuclear phagocytes by interleukin-4.
Immunol. Lett.
71
:
1