The Mycobacterial 38-Kilodalton Glycolipoprotein Antigen Activates the Mitogen-Activated Protein Kinase Pathway and Release of Proinflammato
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感染与免疫杂志 2006年第5期
Departments of Microbiology
Internal Medicine, College of Medicine, Chungnam National University, Daejeon 301-747, South Korea
Departments of Microbiology
Internal Medicine, College of Medicine, Konyang University, Nonsan, Chungnam 320-711, South Korea
Department of Pathology, Case Western Reserve University, Cleveland, Ohio 44106-7288
ABSTRACT
Although the 38-kDa glycolipoprotein of Mycobacterium tuberculosis H37Rv is known to evoke prominent cellular and humoral immune responses in human tuberculosis (TB), little information is known about intracellular regulatory mechanisms involved in 38-kDa antigen (Ag)-induced host responses. In this study, we found that purified 38-kDa glycolipoprotein activates mitogen-activated protein kinases (MAPKs; extracellular signal-regulated kinase 1/2 [ERK1/2] and p38) and induces tumor necrosis factor alpha (TNF-) and interleukin 6 (IL-6) in human monocytes. When the 38-kDa Ag was applied to monocytes from TB patients and healthy controls, the activation of ERK1/2 and p38 MAPK and the subsequent cytokine secretion were greater in the monocytes from the active pulmonary TB patients than in monocytes from the healthy controls. Additionally, neutralizing antibodies for Toll-like receptor 2 (TLR2) or TLR4 significantly reduced the ERK1/2 and p38 activation induced by the 38-kDa protein when the antibodies were applied to HEK293 cells overexpressing TLR2 or TLR4 as well as human primary monocytes. Furthermore, the inhibition of TLR2 significantly, and that of TLR4 partially, decreased the 38-kDa Ag-induced secretion of TNF- and IL-6 in human monocytes. The intact protein moieties of the 38-kDa protein were responsible for biologic activities by this Ag. These data collectively demonstrate that the 38-kDa glycolipoprotein, acting through both TLR2 and TLR4, induces the activation of the ERK1/2 and p38 MAPK pathways, which in turn play an essential role in TNF- and IL-6 expression during mycobacterial infection.
INTRODUCTION
Host immune responses are known to target proteins that are secreted by Mycobacterium tuberculosis; consequently, these proteins have been targeted for the development of vaccines and immunodiagnostics (2, 11). Among the various protein antigens (Ags) of M. tuberculosis, the 38-kDa phosphate transport protein (PstS-1) Ag is actively secreted in mycobacterial cultures (8, 21). This protein induces a strong immune response to M. tuberculosis or Mycobacterium bovis and elicits a protective immunity in animals (3, 20) and humans (15, 21, 45). The serologic reactivity of this Ag has a stronger association with latent infection or recent exposure to M. tuberculosis than with active disease (5, 41), and therefore the 38-kDa Ag is included in all serodiagnostic assays for active tuberculosis (TB). In addition, DNA vaccines encoding cytotoxic T lymphocyte and T helper (Th1) cell epitopes of the 38-kDa lipoglycoprotein were found to elicit strong CD8+ cytotoxic T lymphocyte and Th1 responses (high gamma interferon and low interleukin 4 [IL-4]) (15). Although the 38-kDa Ag has been widely used for cellular and humoral studies for TB research, little is known about the signaling mechanisms involved in the 38-kDa Ag-induced immune responses.
Mammalian Toll-like receptor (TLR) proteins comprise a family of proteins that share sequence similarities with the Drosophila Toll receptor proteins (39). The TLR proteins activate signal transduction cascades that sequentially activate the adapter protein myeloid differentiation factor 88 (MyD88) and tumor necrosis factor receptor-associated factor 6, ultimately promoting the translocation of NF-B to the nucleus. In addition, several protein kinases, such as mitogen-activated protein kinases (MAPKs) and phosphatidylinositol-3'-kinase, are also activated by the TLR signaling cascade (29). TLR signal transduction leads to the expression of several proteins with important roles in the innate immune response to pathogens; these proteins include proinflammatory cytokines, chemokines, costimulatory proteins, and inducible nitric oxide synthase (29).
Previous studies have shown that MAPK activation is essential for the mycobacterium-induced production of proinflammatory cytokines, such as tumor necrosis factor alpha (TNF-), IL-1, and monocyte chemoattractant protein 1 (4, 14, 40, 42). In addition, intracellular growth control of Mycobacterium avium was found to be dependent on the extent of MAPK phosphorylation in human monocyte-derived macrophages, which indicates an essential role for macrophage activation (4). Understanding the specificity of the human cytokine response and exploring the intracellular signaling pathways that relate to the individual mycobacterial Ags are critical for defining the mechanisms responsible for host defense and pathogenesis during TB (23).
In this study, we purified the 38-kDa glycolipoprotein from culture filtrates of M. tuberculosis H37Rv and examined the roles of MAPK signaling pathways and the subsequent production of proinflammatory cytokine-inducing activities in human primary monocytes. We found that the purified 38-kDa glycolipoprotein induces the activation of extracellular signal-regulated kinase 1/2 (ERK1/2) and p38 MAPK and subsequent production of TNF- and IL-6 in human monocytes primarily through TLR2 but also through TLR4. Furthermore, we found greater activation of ERK1/2 and p38 MAPK and cytokine secretion in monocytes from active pulmonary TB patients than in monocytes from healthy controls. The physical and chemical characterization of antigenic nature on the cytokine production was also discussed.
MATERIALS AND METHODS
Isolation of CFPs and purification of the native 38-kDa Ag. Culture filtrate proteins (CFPs) of M. tuberculosis H37Rv (ATCC 27294) were isolated by growing tubercle bacilli in Sauton's synthetic medium as a stationary pellicle culture as previously described (22). Briefly, culture supernatants were centrifuged at 15,000 x g for 1 h, filter sterilized (0.22-μm pore size), and concentrated by ultrafiltration (Amicon Ultra-15 centrifugal filter unit with a 10-kDa-molecular- mass cutoff; Millipore). All purification steps were performed with a fast-protein liquid chromatography system (Bio-Rad).
The native 38-kDa glycolipoprotein was purified by a two-step process using hydrophobic interaction chromatography (HIC) followed by anion-exchange chromatography as follows. Briefly, the 60 to 80% ammonium sulfate precipitate of CFPs was suspended in 100 mM phosphate buffer (pH 6.8) containing 1.8 M ammonium sulfate and then loaded onto an Econo-Pac methyl HIC cartridge (Bio-Rad) for HIC. The cartridge was washed with 5 column volumes of the same buffer. The 38-kDa Ag-containing fraction was eluted with a decreasing ammonium sulfate gradient from 1.35 down to 1.17 M in 100 mM phosphate buffer (pH 6.8) at a flow rate of 2 ml/min.
Further purification was performed by anion-exchange chromatography using an UNO Q6 column (Bio-Rad). The 38-kDa Ag eluted with 10 to 40 mM NaCl in 20 mM Tris-HCl (pH 8.3) was pooled, concentrated, applied to an immobilized polymyxin B column (Detoxi-Gel endotoxin removing gel; Pierce Chemical Co.) to eliminate the endotoxin level, dialyzed against phosphate-buffered saline (pH 7.2), filter sterilized, and frozen at –20°C. The protein concentration of the purified 38-kDa Ag was estimated with a bicinchoninic acid protein assay kit (Pierce, Rockford, Ill.) with bovine serum albumin as the standard. Preparations of the 38-kDa glycolipoprotein used in experiments were tested for lipopolysaccharide (LPS) contamination by a Limulus amebocyte lysate assay (BioWhittaker) and contained less than 20 pg/ml at the concentrations of the 38-kDa protein used in experiments. LPS (1 μg/ml; Sigma) or peptidoglycan (20 μg/ml; Fluka) was used as a positive control for the Ag stimulation in this study.
The 19-kDa lipoprotein. The 19-kDa lipoprotein (LpqH) was purified from M. tuberculosis H37Ra using the approach of Pai et al. (33) with modifications for LprG as described by Gehring et al. (16). In brief, M. tuberculosis was disrupted with a French press and extracted with Triton X-114; the detergent phase was subjected to acetone precipitation, phenol extraction, dialysis, and preparative electrophoresis. Electroeluted fractions containing 19-kDa lipoproteins were identified and assessed for purity by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) with silver staining and Western blotting; the 19-kDa lipoprotein was lyophilized and stored at –80°C.
Subjects. Whole blood was obtained by venipuncture from a total of 12 TB patients and 19 healthy volunteers. All TB patients and healthy volunteers gave informed consents to take part in this study. Active-pulmonary-TB patients participated in this study within 1 month of beginning first-line anti-TB drug medication at the Chungnam National University Hospital (Daejeon, Korea)10.0 and the Konyang University Hospital (Daejeon, Korea). All of the patients in this study had parenchymal TB, and none had miliary or pleural TB. All patients had a positive sputum culture for M. tuberculosis. They had no previous history of diabetes mellitus or steroid therapy, and all were negative for human immunodeficiency virus. Extensive clinical histories were obtained from the subjects and included data on age, sex, medical history, chest radiographic findings, sputum staining and culture results, drug sensitivities, treatment, and outcome.
The healthy control subjects had no previous history of clinical TB. Each of these healthy controls had received the M. bovis bacillus Calmette-Guerin (BCG) vaccinations as children. The study was approved by the bioethics committee of Chungnam University Hospital's review board overseeing studies on samples from human subjects.
Isolation and culture of human monocytes. Venous blood was drawn from healthy subjects into sterile blood collection tubes, and peripheral blood mononuclear cells were isolated by density sedimentation over Histopaque-1077 (Sigma). All healthy controls gave their informed consent before being enrolled in the study. Cells were incubated for 1 h at 37°C, and nonadherent cells were removed by pipetting off the supernatant. Adherent monocytes were collected as previously described (42). The recovered cells were >95% CD14+ cells as determined by flow cytometry with an anti-CD14 antibody (Ab). The cells were then incubated at 37°C in a humidified, 5% CO2 atmosphere until used in experiments. In order to show that the stimulatory capacity of the 38-kDa Ag was not the result of contamination with LPS, experiments were performed with the addition of the specific LPS-inhibiting oligopeptide polymyxin B (10.0 μg/ml) before mycobacterial stimulation.
Stable cell culture. Human embryonic kidney (HEK) 293 cells stably transfected with human TLR2 (HEK/TLR2) or human TLR4 (HEK 293/TLR4) were purchased from InvivoGen (San Diego, CA). The 293/TLR clones were grown in standard Dulbecco's modified Eagle's medium with 10% fetal bovine serum supplemented with blasticidin (10.0 μg/ml) and normocin (100.0 μg/ml).
Inhibitors and antibodies. Specific inhibitors of MEK (PD98059 and U0126) and the p38 MAPK inhibitor SB203580 were purchased from Calbiochem (San Diego, CA). Dimethyl sulfoxide (DMSO; Sigma) was added to cultures at 0.1% (vol/vol) as a solvent control. Monocytes were washed with phosphate-buffered saline and pretreated with inhibitors in RPMI 1640 medium containing glutamine for 30 to 45 min prior to infection with M. tuberculosis. Assessment by trypan blue exclusion indicated that monocyte viability was not affected by the presence of the inhibitors (data not shown). Mouse anti-human TLR2 monoclonal Ab (MAb; clone 2392, immunoglobulin G1 [IgG1]) was obtained from Genentech (South San Francisco, CA) and mouse anti-human TLR4 MAb (clone HTA125, IgG2a) was purchased from eBioscience (San Diego, CA). All isotype-matched control (IC) Abs (IgG1 and IgG2a) and the anti-human CD14-fluorescein isothiocyanate were purchased from eBioscience. Anti-38-kDa MAb was kindly provided by Colorado State University.
Heat inactivation and PK treatment of the 38-kDa Ag. To characterize the physical and chemical nature of the Ag, the 38-kDa Ag (5 μg/ml) was heated for 15 min at 100°C or digested for 1 h at 37°C with soluble proteinase K (PK) (Sigma) at 5 μg/ml followed by heating for 15 min at 100°C to deactivate the enzyme, as described previously (9).
Determination of MAPK phosphorylation. A total of 4 x 105 human monocytes (at a concentration of 8 x 105 per ml) were treated with the 38-kDa Ag for the times indicated. Cell lysates were prepared, and Western blot analysis was performed with specific primary antibodies (p38, phospho-p38, ERK1/2, and phospho-ERK1/2; New England Biolabs, Schwalbach, Germany) as described previously (42). Membranes were developed using a chemiluminescence assay (ECL; Pharmacia-Amersham, Freiburg, Germany) and subsequently exposed on chemiluminescence film (Pharmacia-Amersham).
Enzyme-linked immunosorbent assay. A sandwich enzyme-linked immunosorbent assay (ELISA) was used for detecting TNF- and IL-6 (PharMingen) in culture supernatants as described previously (26). Assays were performed as recommended by the manufacturers. Cytokine concentrations in the samples were calculated using standard curves generated from recombinant cytokines, and the results were expressed in picograms or nanograms per milliliter. The difference between duplicate wells was consistently less than 10% of the mean.
Statistical analysis. For statistical analysis, the data obtained from independent experiments are presented as the means ± standard deviations; they were analyzed using a paired t test with Bonferroni's adjustment or analysis of variance for multiple comparisons. Differences were considered significant at a P value of <0.05.
RESULTS
Purification of the 38-kDa glycolipoprotein. The 38-kDa protein was purified from unheated CFPs of M. tuberculosis H37Rv by ammonium sulfate precipitation, HIC, and anion- exchange chromatography. The SDS-PAGE analysis of the CFPs, 60 to 80% ammonium sulfate precipitate of CFPs, and purified 38-kDa glycolipoprotein are shown in Fig. 1A. We recovered approximately 1.8 mg of purified 38-kDa protein from the initial 985-mg quantity of CFPs. To establish the identity of the Ag, the purified 38-kDa glycolipoprotein was separated by isoelectrofocusing on an immobilized pH gradient strip (pH 4.7 to 5.9) in the first dimension and 12% SDS-PAGE in the second dimension. It had an apparent molecular mass of 38 kDa on an SDS-PAGE gel stained with Coomassie blue and was recognized on Western blots by anti-38-kDa MAb (Fig. 1B). Recognition of the purified Ag by the reference MAb confirmed the presence of the 38-kDa glycolipoprotein.
M. tuberculosis 38-kDa protein leads to the phosphorylation of MAPK and rapid TNF- and IL-6 secretion in human primary monocytes. In order to examine the signaling mechanisms involved in the 38-kDa Ag-induced proinflammatory cytokine responses, human monocytes were stimulated with various concentrations (0.5 to 10 μg/ml) or time courses (6 to 96 h) of the 38-kDa Ag. Then, cytokine ELISA was performed to assess TNF- and IL-6 secretion in culture supernatants. The 38-kDa Ag induced very low levels of TNF- and IL-6 at concentrations of 100 to 500 ng/ml (Fig. 2A). When human primary monocytes were exposed to 5.0 μg/ml of purified 38-kDa Ag, the secretion of TNF- and IL-6 significantly increased in the culture medium compared to that of monocytes exposed to 1.0 μg/ml of purified 38-kDa Ag (P < 0.01). Because the cytokine production of primary monocytes stimulated at 10.0 μg/ml was not appreciably different from that of cells stimulated at 5.0 μg/ml of Ag, we used the 5.0-μg/ml concentration in this study. In addition, the highest peak of TNF- and IL-6 production in response to the 38-kDa Ag was always detected 8 to 24 h after stimulation (Fig. 2B). Therefore, supernatants were harvested at 18 h for additional experiments.
Given that MAPKs are critical factors mediating cellular responses to many external stimuli, we examined the MAPK activation in response to the 38-kDa Ag of M. tuberculosis. Monocytes were stimulated with the 38-kDa Ag at a concentration of 5.0 μg/ml, and the phosphorylation of p38 and ERK1/2 was analyzed during various time courses (Fig. 2B). Exposure to the 38-kDa Ag produced a strong phosphorylation of p38 MAPK at 15 to 30 min after stimulation. The peak activation of ERK2 was within 30 to 60 min of stimulation with the 38-kDa Ag in monocytes (the time range was donor dependent). Control cells stimulated with LPS (0.1 μg/ml) produced a signal of comparable intensity (data not shown). The total inhibition of ERK1/2 and p38 MAPK phosphorylation by their respective inhibitors at 30 min after the addition of the 38-kDa Ag was confirmed on Western blots (data not shown).
The ERK1/2 and p38 MAPK pathways are critical for mycobacterial 38-kDa protein-induced TNF- and IL-6 formation. ERK1/2 and p38 are both pivotal to the cytokine formation induced by M. tuberculosis H37Rv (42) and to the regulation of the macrophage-antigen-presenting function by the M. tuberculosis 19-kDa lipoprotein (34). To further understand the functional roles of these kinases in the activation of human monocytes induced by the 38-kDa Ag, we used highly specific inhibitors of the kinases and measured cytokine formation. The cells were pretreated with a p38 inhibitor (SB203580) or an MEK inhibitor (PD98059 or U0126) for 45 min before being exposed to the 38-kDa Ag. Both U0126 and PD98059 were added to the cell culture at a final DMSO concentration of 0.1%, and DMSO was used as a control.
U0126 significantly decreased the formation of TNF- by human monocytes in response to the 38-kDa Ag (73.9% inhibition at 10 μM) (Fig. 3A); this decrease was confirmed using a second MEK inhibitor, PD98059 (data not shown). SB203580 reduced the TNF- secretion by human monocytes by up to 84.5% at 5 μM (Fig. 3A). Similarly, the 38-kDa Ag-induced IL-6 production was significantly reduced by both inhibitors (Fig. 3B). The inhibition was not attributable to DMSO because DMSO did not exhibit any inhibitory effects at this concentration.
Elevated 38-kDa Ag-induced MAPK activity and secretion of TNF- and IL-6 in TB patients compared with that in healthy controls. We next performed a comparative analysis of 38-kDa- or LPS-induced MAPK activation between TB patients and healthy controls. As shown in Fig. 4A, a higher phosphorylated ERK1/2 expression was observed in unstimulated and 38-kDa Ag-stimulated monocytes from TB patients than in monocytes from healthy controls (5.7- and 8.2-fold increases for unstimulated and 38-kDa Ag-stimulated cells, respectively, from TB patients). In addition, the phosphorylated-p38 expression was significantly increased in unstimulated and 38-kDa Ag-stimulated monocytes from TB patients compared with those from healthy controls (2.9- and 4.8-fold increases for unstimulated and 38-kDa Ag-stimulated cells, respectively, from TB patients) (Fig. 4B). Moreover, we found a significantly higher MAPK activation in LPS-stimulated monocytes from TB patients than in monocytes from healthy controls (P value was <0.05 for both ERK1/2 and p38).
We also compared TNF- and IL-6 production levels in peripheral blood monocytes between TB patients and healthy controls in response to stimulation with the 38-kDa Ag or LPS. The mean concentrations of TNF- and IL-6 produced were significantly increased in unstimulated and 38-kDa Ag-stimulated monocytes from TB patients compared to those in monocytes from healthy controls (Fig. 4C) (P value was <0.001 for both cytokines). Although there was a great heterogeneity in TB patients for LPS-induced proinflammatory cytokine production, LPS stimulation led to an increase in TNF- and IL-6 production by primary monocytes from TB patients compared to that of monocytes from healthy controls (P value was <0.05 for each cytokine). Moreover, we found a significantly higher MAPK activation in LPS-stimulated monocytes from TB patients than in monocytes from healthy controls (P value was <0.05 for both ERK1/2 and p38). Taken together, these data suggest that the activation status of monocytes from TB patients could be the result of the in vivo activation of ERK1/2 and p38 MAPK.
The 38-kDa Ag-induced phosphorylation of MAPKs and subsequent cytokine secretion were blocked by anti-TLR2 MAb and partially inhibited by anti-TLR4 MAb. We assessed whether the 38-kDa Ag activated the MAPK pathway through TLR2 or TLR4 signaling. The ERK1/2 phosphorylation induced by the 38-kDa Ag was measured after the preincubation of monocytes with a MAb to TLR2 or TLR4 or with an IC Ab. As shown in Fig. 5A and B, the 38-kDa-mediated phosphorylation of ERK1/2 and p38 MAPK was severely reduced in monocytes pretreated with the anti-TLR2 MAb (P < 0.001), whereas a smaller attenuation was detected by the pretreatment with anti-TLR4 MAb (P < 0.01). However, the phosphorylation of ERK1/2 and p38 MAPK was severely reduced by the pretreatment of monocytes with anti-TLR4 MAb followed by stimulation with Escherichia coli LPS (data not shown). Thus, the 38-kDa protein mediates the activation of ERK1/2 and p38 in human monocytes primarily via TLR2 but also via TLR4.
We then incubated adhered monocytes from healthy controls with or without anti-TLR2 MAb, anti-TLR4 MAb, or IC Ab for 30 min before stimulation with the 38-kDa protein to examine the extent to which TLRs are responsible for the 38-kDa Ag-induced cytokine responses. As shown in Fig. 5C, the 38-kDa Ag-induced TNF- production was prominently inhibited (by 59.4%) by anti-TLR2 Ab treatment, whereas it was inhibited to a smaller degree by anti-TLR4 MAb (by 33.0%). In a manner similar to that of the TNF- regulation, the 38-kDa Ag-induced IL-6 synthesis was inhibited to a greater extent by anti-TLR2 MAb (65.1%) than by anti-TLR4 MAb (36.1%). The cytokine responses were not changed by the pretreatment with IC Ab. These results indicate that the 38-kDa Ag-induced activation of the ERK1/2, p38, TNF-, and IL-6 responses is mainly mediated via TLR2 but is also partially mediated by TLR4.
Ectopic expression of TLRs reveals that the 38-kDa Ag activates MAPK phosphorylation in both a TLR2- and TLR4-dependent manner. To further characterize the specific interactions of the 38-kDa Ag with TLR2 and TLR4, we performed experiments with HEK/TLR2 and HEK/TLR4. In these HEK cells, the TLR2 and TLR4 expressions were confirmed in HEK/TLR2 and HEK/TLR4 cells, respectively, with Western blot, flow cytometric, and reverse transcriptase-PCR analyses. In the representative experiments (a total of three) shown in Fig. 6A and B, the 38-kDa Ag significantly activated ERK1/2 and p38 MAPK phosphorylation in HEK/TLR2 and HEK/TLR4 cells. The 38-kDa Ag-induced ERK1/2 and p38 MAPK phosphorylation was significantly inhibited by specific inhibitors in a dose-dependent manner.
Furthermore, the control experiments were conducted using anti-TLR2-MAbs on LPS-induced and anti-TLR4-MAb on peptidoglycan-induced HEK cell activation. The highly purified E. coli LPS and peptidoglycan were able to activate ERK1/2 and p38 MAPK in HEK/TLR4 and HEK/TLR2 cells, respectively (Fig. 6C). In addition, the LPS- and peptidoglycan-induced ERK1/2 and p38 MAPK activation responses in the HEK cells were specifically inhibited by pretreatment with anti-TLR4 MAb and anti-TLR2 MAb, respectively. Thus, both TLR2 and TLR4 can confer responsiveness to the 38-kDa glycolipoprotein of M. tuberculosis.
Physical and enzymatic characterization of the 38-kDa Ag. To understand whether intact protein moieties from the 38-kDa glycolipoprotein Ag are required for biologic activity, the 38-kDa Ag was treated with heat at 100°C for 30 min or incubated with PK (5 μg/ml) at 37°C for 1 h. Heat treatment of the 38-kDa Ag significantly decreased the secretion of TNF- and IL-6 in human monocytes (Fig. 7), suggesting the heat lability of the Ag. In addition, the cytokine production was completely attenuated after PK treatment of the Ag, suggesting that intact protein moieties from the 38-kDa Ag are essential for cytokine secretion. The SDS-PAGE of PK-treated 38-kDa Ag failed to show intact protein bands, although there were smaller protein bands after heat inactivation (data not shown). These data strongly suggest that heat- and PK-sensitive protein components of the 38-kDa Ag would be responsible for biologic activity by this Ag.
Comparative cytokine responses between the 38-kDa and 19-kDa Ags. The TLR2 agonist 19-kDa lipoprotein has been well characterized for immunomodulating activities, such as IL-1, IL-12 p40, and TNF- induction, by human monocyte-derived macrophages or a human monocytic cell line (6, 43). To compare the immunoregulatory functions between the 38-kDa and 19-kDa Ags, cytokine analysis for TNF-, IL-6, and IL-12 p40 secretion was undertaken in human primary monocytes after stimulation with the 38-kDa or 19-kDa Ag.
As shown in Fig. 8, the 38-kDa and 19-kDa Ags both induced significant dose-dependent cytokine secretions of TNF-, IL-6, and IL-12 p40 in human primary monocytes. The 19-kDa lipoprotein induced a large amount of TNF-, IL-6, and IL-12 p40 in human primary monocytes, with a significant difference relative to the 38-kDa Ag-induced cytokine secretion. Significant cytokine productions occurred with cells incubated with the 19-kDa lipoprotein relative to the samples that received the 38-kDa Ag (at 0.1, 0.25, and 0.5 μg/ml; P < 0.001). These data suggest that the 38-kDa Ag is less potent for inducing the proinflammatory and Th1-driving cytokine production in human primary monocytes than is the 19-kDa lipoprotein.
DISCUSSION
Research into the development of TB vaccines and immunodiagnostics has focused on the proteins released by M. tuberculosis, the causative agent of TB, because these Ags are thought to induce protective immunity and immune responses of diagnostic value (2, 10, 11). Our data demonstrate that the stimulation of human cells in vitro with the 38-kDa glycolipoprotein of M. tuberculosis induces MAPK activation primarily via TLR2 but also partially via TLR4. We also describe reproducible protocols for the purification of native 38-kDa glycolipoprotein Ag from culture filtrates of M. tuberculosis.
The purified 38-kDa protein showed a strong reactivity with antibodies in the sera of smear-positive, smear-negative, and extrapulmonary TB patients (data not shown). T-cell responses to this secreted glycolipoprotein, representing a major immunogen of M. tuberculosis, have been well characterized using human and murine lymphocytes (45-47). In addition, the immunodominant epitopes of the 38-kDa protein Ag recognized by CD4+ T cells have been characterized in TB patients and healthy controls (45, 47) as well as in murine T cells (45, 46). Zhu et al. (48) showed that the nucleic acid vaccination of C57BL/6 mice against 38-kDa mycobacterial Ag induced protection against challenge with virulent M. tuberculosis. Taken together, these findings suggest that the 38-kDa Ag is a valuable candidate for use as a target of host immune responses to M. tuberculosis in human studies and animal models.
Soluble and cell wall-associated mycobacterial factors are capable of mediating activation via distinct TLR proteins (30). Two mycobacterial cell wall glycolipids, lipoarabinomannan and mannosylated phosphatidylinositol (PIM), activate cells through TLR2 (18, 25). In addition, short-term culture filtrates of M. tuberculosis bacilli contain TLR2 agonist activity that appears to be PIM (25). Lipomannan (LM) from several mycobacterial species has been found to display dual functions by either stimulating or inhibiting proinflammatory cytokine synthesis through different pathways in murine primary macrophages. LM induces macrophage activation characterized by TNF and nitric oxide secretion through TLR2 and the adapter protein MyD88, independently of either TLR4 or TLR6 recognition (36).
One of the best-described mycobacterial TLR2 agonists is the 19-kDa lipoprotein (28, 32, 33) which induces host cell apoptosis (28) and down-regulation of major histocompatibility complex class II molecules (32, 33) and enhances the activity against mycobacteria via nitric oxide in mice (6). In addition, the 19-kDa lipoprotein elicits high levels of IL-12 from macrophages in addition to its powerful immunomodulatory properties, leading to suppression of Ag presentation signaling cascades (38). The comparative analysis between the 38-kDa and 19-kDa lipoproteins showed a greater production of TNF-, IL-6, and IL-12 p40 by the 19-kDa lipoprotein in human primary monocytes than by the 38-kDa lipoprotein. Our data are partially consistent with the previous studies (6) in which the 19-kDa lipoprotein induced more IL-12 p40 by a human monocytic cell line than was induced by the 38-kDa glycolipoprotein. Moreover, our data indicate that the biologic activities induced by the 38-kDa Ag may be dependent on the intact protein moieties from this Ag because the cytokine production was totally attenuated after PK treatment. Taken together, our data suggest that the 38-kDa Ag is less active for inducing the cytokine production than the 19-kDa lipoprotein, with a substantial difference in antigenic nature between two proteins.
Our data first demonstrate that the 38-kDa glycolipoprotein Ag has structural motifs to interact with both TLR2 and TLR4. In addition, the intact protein moieties of the 38-kDa Ag would be responsible for TNF- and IL-6 induction in human primary monocytes. TLR4 is a pattern recognition receptor not only for exogenous ligands, such as LPS of gram-negative bacteria (35), but also for endogenous ligands such as fibronectin, heat shock proteins, and hyaluronan oligosaccharides (27). Although TLR4 ligands of M. tuberculosis are poorly understood, a recent study demonstrated that mycobacterial heat shock protein 65 signals exclusively through TLR4 (7). Furthermore, BCG induced the transcription and secretion of the chemokine CXCL8 in human neutrophils by signaling through TLR2 and TLR4 in conjunction with the adapter protein MyD88 (19). In this work, immune activation induced by 38-kDa Ag through TLR4 should not be a result of contaminating LPS, as several procedures were conducted to rule out endotoxin contamination. The purified 38-kDa Ag was applied to a polymyxin B column to eliminate LPS contamination, and the remaining LPS level was determined by the Limulus amebocyte lysate assay. In our study, even 160 pg of LPS (eightfold more than the amount present in our experiments) did not activate human primary monocytes. Moreover, the heat sensitivity of 38-kDa Ag activity excludes contamination by LPS, because LPS is heat resistant. Further studies are needed to identify the individual motifs of the 38-kDa glycoprotein that are responsible for activating each pattern recognition receptor.
The N-terminally acylated lipopeptide region has been reported to be important for the immunostimulatory activity of bacterial and mycoplasmal lipoproteins. A recent study showed that cytokine expression by human and murine macrophages was induced by tri- and tetraacylated forms of LM (Ac3LM and Ac4LM, respectively) but not mono- or diacylated LM (Ac1LM and Ac2LM, respectively) (17), whereas PIM has been shown to be proinflammatory irrespective of its acylation (18). In this study, we did not assess the acylation status of the 38-kDa protein, but it is predicted to be acylated. Further studies should clarify the question of the role of fatty acids for 38-kDa Ag signaling via TLRs.
The activation of intracellular signaling pathways and subsequent inflammatory cytokines can be induced by different stimuli in different cell types (37). Our study emphasized the role of the MAPK (both ERK and p38) pathways in the 38-kDa Ag-induced TNF- and IL-6 secretion by human monocytes. The MAPK signaling pathways are highly conserved cascades that are important in diverse aspects of the immune response (13). They form a family of protein kinases, which includes ERK, p38 MAPK, and stress-activated protein kinase/c-Jun N-terminal kinase. In the immune system, MAPK activation can be directly mediated through cytokine receptors, such as the type I TNF receptor and IL-1R, as well as through microbial pattern recognition receptors, most prominently the TLR family, which functions as detectors of infection (44). The MAPK signaling cascades culminate in the induction of multiple proinflammatory genes, including the genes for IL-12 p40, TNF-, IL-6, and inducible nitric oxide synthase, that are important in the defense against microbial infection (12). Of note, the p38 MAPKs regulate the expression of many cytokines and play important roles in the activation of immune responses (24).
The importance of the MAPK signal transduction pathway in controlling many aspects of immune-mediated inflammatory responses has made it a priority for research related to many human diseases. In the present work, we demonstrated greater activation of ERK1/2 and p38 MAPK and greater secretion of cytokines in monocytes from active pulmonary TB patients than in monocytes from healthy controls. Previous studies have reported that TLR2 triggers apoptosis in polymorphonuclear neutrophils (PMNs) from TB patients, which correlates with the M. tuberculosis-induced expression of phospho-p38 in PMNs and with the increased expression of activated p38 found in circulating TB-PMNs (1). In a murine model of pulmonary inflammation, p38 activation was implicated in the recruitment of PMNs to infected lung tissue (31). Thus, our findings suggest that the high production level of proinflammatory cytokines during the early stages of TB could be attributable to the in vivo activation of MAPK. Therefore, in our system, human monocytes would detect the 38-kDa glycolipoprotein via TLR2 and TLR4, leading to activation of the ERK1/2 and p38 MAPK pathways, which in turn would induce enhanced proinflammatory cytokine responses during the early stage of human TB.
We have shown that the purified 38-kDa glycolipoprotein induces the activation of ERK1/2 and p38 MAPK via TLR2 and TLR4 and the subsequent proinflammatory cytokines TNF- and IL-6 in human primary monocytes. This study provides novel insight into the role of the TLR2- and TLR4-mediated MAPK signaling pathways induced by 38-kDa glycolipoprotein for early inflammatory responses during mycobacterial infection.
ACKNOWLEDGMENTS
This work was supported by Korea Research Foundation Grant (KRF-2004-041-E00125).
The anti-38-kDa MAb was kindly provided by Colorado State University as part of NIH, NIAID contract no. HHSN266200400091C (titled Tuberculosis Vaccine Testing and Research Materials), which was awarded to Colorado State University.
These authors contributed equally to this work.
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Internal Medicine, College of Medicine, Chungnam National University, Daejeon 301-747, South Korea
Departments of Microbiology
Internal Medicine, College of Medicine, Konyang University, Nonsan, Chungnam 320-711, South Korea
Department of Pathology, Case Western Reserve University, Cleveland, Ohio 44106-7288
ABSTRACT
Although the 38-kDa glycolipoprotein of Mycobacterium tuberculosis H37Rv is known to evoke prominent cellular and humoral immune responses in human tuberculosis (TB), little information is known about intracellular regulatory mechanisms involved in 38-kDa antigen (Ag)-induced host responses. In this study, we found that purified 38-kDa glycolipoprotein activates mitogen-activated protein kinases (MAPKs; extracellular signal-regulated kinase 1/2 [ERK1/2] and p38) and induces tumor necrosis factor alpha (TNF-) and interleukin 6 (IL-6) in human monocytes. When the 38-kDa Ag was applied to monocytes from TB patients and healthy controls, the activation of ERK1/2 and p38 MAPK and the subsequent cytokine secretion were greater in the monocytes from the active pulmonary TB patients than in monocytes from the healthy controls. Additionally, neutralizing antibodies for Toll-like receptor 2 (TLR2) or TLR4 significantly reduced the ERK1/2 and p38 activation induced by the 38-kDa protein when the antibodies were applied to HEK293 cells overexpressing TLR2 or TLR4 as well as human primary monocytes. Furthermore, the inhibition of TLR2 significantly, and that of TLR4 partially, decreased the 38-kDa Ag-induced secretion of TNF- and IL-6 in human monocytes. The intact protein moieties of the 38-kDa protein were responsible for biologic activities by this Ag. These data collectively demonstrate that the 38-kDa glycolipoprotein, acting through both TLR2 and TLR4, induces the activation of the ERK1/2 and p38 MAPK pathways, which in turn play an essential role in TNF- and IL-6 expression during mycobacterial infection.
INTRODUCTION
Host immune responses are known to target proteins that are secreted by Mycobacterium tuberculosis; consequently, these proteins have been targeted for the development of vaccines and immunodiagnostics (2, 11). Among the various protein antigens (Ags) of M. tuberculosis, the 38-kDa phosphate transport protein (PstS-1) Ag is actively secreted in mycobacterial cultures (8, 21). This protein induces a strong immune response to M. tuberculosis or Mycobacterium bovis and elicits a protective immunity in animals (3, 20) and humans (15, 21, 45). The serologic reactivity of this Ag has a stronger association with latent infection or recent exposure to M. tuberculosis than with active disease (5, 41), and therefore the 38-kDa Ag is included in all serodiagnostic assays for active tuberculosis (TB). In addition, DNA vaccines encoding cytotoxic T lymphocyte and T helper (Th1) cell epitopes of the 38-kDa lipoglycoprotein were found to elicit strong CD8+ cytotoxic T lymphocyte and Th1 responses (high gamma interferon and low interleukin 4 [IL-4]) (15). Although the 38-kDa Ag has been widely used for cellular and humoral studies for TB research, little is known about the signaling mechanisms involved in the 38-kDa Ag-induced immune responses.
Mammalian Toll-like receptor (TLR) proteins comprise a family of proteins that share sequence similarities with the Drosophila Toll receptor proteins (39). The TLR proteins activate signal transduction cascades that sequentially activate the adapter protein myeloid differentiation factor 88 (MyD88) and tumor necrosis factor receptor-associated factor 6, ultimately promoting the translocation of NF-B to the nucleus. In addition, several protein kinases, such as mitogen-activated protein kinases (MAPKs) and phosphatidylinositol-3'-kinase, are also activated by the TLR signaling cascade (29). TLR signal transduction leads to the expression of several proteins with important roles in the innate immune response to pathogens; these proteins include proinflammatory cytokines, chemokines, costimulatory proteins, and inducible nitric oxide synthase (29).
Previous studies have shown that MAPK activation is essential for the mycobacterium-induced production of proinflammatory cytokines, such as tumor necrosis factor alpha (TNF-), IL-1, and monocyte chemoattractant protein 1 (4, 14, 40, 42). In addition, intracellular growth control of Mycobacterium avium was found to be dependent on the extent of MAPK phosphorylation in human monocyte-derived macrophages, which indicates an essential role for macrophage activation (4). Understanding the specificity of the human cytokine response and exploring the intracellular signaling pathways that relate to the individual mycobacterial Ags are critical for defining the mechanisms responsible for host defense and pathogenesis during TB (23).
In this study, we purified the 38-kDa glycolipoprotein from culture filtrates of M. tuberculosis H37Rv and examined the roles of MAPK signaling pathways and the subsequent production of proinflammatory cytokine-inducing activities in human primary monocytes. We found that the purified 38-kDa glycolipoprotein induces the activation of extracellular signal-regulated kinase 1/2 (ERK1/2) and p38 MAPK and subsequent production of TNF- and IL-6 in human monocytes primarily through TLR2 but also through TLR4. Furthermore, we found greater activation of ERK1/2 and p38 MAPK and cytokine secretion in monocytes from active pulmonary TB patients than in monocytes from healthy controls. The physical and chemical characterization of antigenic nature on the cytokine production was also discussed.
MATERIALS AND METHODS
Isolation of CFPs and purification of the native 38-kDa Ag. Culture filtrate proteins (CFPs) of M. tuberculosis H37Rv (ATCC 27294) were isolated by growing tubercle bacilli in Sauton's synthetic medium as a stationary pellicle culture as previously described (22). Briefly, culture supernatants were centrifuged at 15,000 x g for 1 h, filter sterilized (0.22-μm pore size), and concentrated by ultrafiltration (Amicon Ultra-15 centrifugal filter unit with a 10-kDa-molecular- mass cutoff; Millipore). All purification steps were performed with a fast-protein liquid chromatography system (Bio-Rad).
The native 38-kDa glycolipoprotein was purified by a two-step process using hydrophobic interaction chromatography (HIC) followed by anion-exchange chromatography as follows. Briefly, the 60 to 80% ammonium sulfate precipitate of CFPs was suspended in 100 mM phosphate buffer (pH 6.8) containing 1.8 M ammonium sulfate and then loaded onto an Econo-Pac methyl HIC cartridge (Bio-Rad) for HIC. The cartridge was washed with 5 column volumes of the same buffer. The 38-kDa Ag-containing fraction was eluted with a decreasing ammonium sulfate gradient from 1.35 down to 1.17 M in 100 mM phosphate buffer (pH 6.8) at a flow rate of 2 ml/min.
Further purification was performed by anion-exchange chromatography using an UNO Q6 column (Bio-Rad). The 38-kDa Ag eluted with 10 to 40 mM NaCl in 20 mM Tris-HCl (pH 8.3) was pooled, concentrated, applied to an immobilized polymyxin B column (Detoxi-Gel endotoxin removing gel; Pierce Chemical Co.) to eliminate the endotoxin level, dialyzed against phosphate-buffered saline (pH 7.2), filter sterilized, and frozen at –20°C. The protein concentration of the purified 38-kDa Ag was estimated with a bicinchoninic acid protein assay kit (Pierce, Rockford, Ill.) with bovine serum albumin as the standard. Preparations of the 38-kDa glycolipoprotein used in experiments were tested for lipopolysaccharide (LPS) contamination by a Limulus amebocyte lysate assay (BioWhittaker) and contained less than 20 pg/ml at the concentrations of the 38-kDa protein used in experiments. LPS (1 μg/ml; Sigma) or peptidoglycan (20 μg/ml; Fluka) was used as a positive control for the Ag stimulation in this study.
The 19-kDa lipoprotein. The 19-kDa lipoprotein (LpqH) was purified from M. tuberculosis H37Ra using the approach of Pai et al. (33) with modifications for LprG as described by Gehring et al. (16). In brief, M. tuberculosis was disrupted with a French press and extracted with Triton X-114; the detergent phase was subjected to acetone precipitation, phenol extraction, dialysis, and preparative electrophoresis. Electroeluted fractions containing 19-kDa lipoproteins were identified and assessed for purity by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) with silver staining and Western blotting; the 19-kDa lipoprotein was lyophilized and stored at –80°C.
Subjects. Whole blood was obtained by venipuncture from a total of 12 TB patients and 19 healthy volunteers. All TB patients and healthy volunteers gave informed consents to take part in this study. Active-pulmonary-TB patients participated in this study within 1 month of beginning first-line anti-TB drug medication at the Chungnam National University Hospital (Daejeon, Korea)10.0 and the Konyang University Hospital (Daejeon, Korea). All of the patients in this study had parenchymal TB, and none had miliary or pleural TB. All patients had a positive sputum culture for M. tuberculosis. They had no previous history of diabetes mellitus or steroid therapy, and all were negative for human immunodeficiency virus. Extensive clinical histories were obtained from the subjects and included data on age, sex, medical history, chest radiographic findings, sputum staining and culture results, drug sensitivities, treatment, and outcome.
The healthy control subjects had no previous history of clinical TB. Each of these healthy controls had received the M. bovis bacillus Calmette-Guerin (BCG) vaccinations as children. The study was approved by the bioethics committee of Chungnam University Hospital's review board overseeing studies on samples from human subjects.
Isolation and culture of human monocytes. Venous blood was drawn from healthy subjects into sterile blood collection tubes, and peripheral blood mononuclear cells were isolated by density sedimentation over Histopaque-1077 (Sigma). All healthy controls gave their informed consent before being enrolled in the study. Cells were incubated for 1 h at 37°C, and nonadherent cells were removed by pipetting off the supernatant. Adherent monocytes were collected as previously described (42). The recovered cells were >95% CD14+ cells as determined by flow cytometry with an anti-CD14 antibody (Ab). The cells were then incubated at 37°C in a humidified, 5% CO2 atmosphere until used in experiments. In order to show that the stimulatory capacity of the 38-kDa Ag was not the result of contamination with LPS, experiments were performed with the addition of the specific LPS-inhibiting oligopeptide polymyxin B (10.0 μg/ml) before mycobacterial stimulation.
Stable cell culture. Human embryonic kidney (HEK) 293 cells stably transfected with human TLR2 (HEK/TLR2) or human TLR4 (HEK 293/TLR4) were purchased from InvivoGen (San Diego, CA). The 293/TLR clones were grown in standard Dulbecco's modified Eagle's medium with 10% fetal bovine serum supplemented with blasticidin (10.0 μg/ml) and normocin (100.0 μg/ml).
Inhibitors and antibodies. Specific inhibitors of MEK (PD98059 and U0126) and the p38 MAPK inhibitor SB203580 were purchased from Calbiochem (San Diego, CA). Dimethyl sulfoxide (DMSO; Sigma) was added to cultures at 0.1% (vol/vol) as a solvent control. Monocytes were washed with phosphate-buffered saline and pretreated with inhibitors in RPMI 1640 medium containing glutamine for 30 to 45 min prior to infection with M. tuberculosis. Assessment by trypan blue exclusion indicated that monocyte viability was not affected by the presence of the inhibitors (data not shown). Mouse anti-human TLR2 monoclonal Ab (MAb; clone 2392, immunoglobulin G1 [IgG1]) was obtained from Genentech (South San Francisco, CA) and mouse anti-human TLR4 MAb (clone HTA125, IgG2a) was purchased from eBioscience (San Diego, CA). All isotype-matched control (IC) Abs (IgG1 and IgG2a) and the anti-human CD14-fluorescein isothiocyanate were purchased from eBioscience. Anti-38-kDa MAb was kindly provided by Colorado State University.
Heat inactivation and PK treatment of the 38-kDa Ag. To characterize the physical and chemical nature of the Ag, the 38-kDa Ag (5 μg/ml) was heated for 15 min at 100°C or digested for 1 h at 37°C with soluble proteinase K (PK) (Sigma) at 5 μg/ml followed by heating for 15 min at 100°C to deactivate the enzyme, as described previously (9).
Determination of MAPK phosphorylation. A total of 4 x 105 human monocytes (at a concentration of 8 x 105 per ml) were treated with the 38-kDa Ag for the times indicated. Cell lysates were prepared, and Western blot analysis was performed with specific primary antibodies (p38, phospho-p38, ERK1/2, and phospho-ERK1/2; New England Biolabs, Schwalbach, Germany) as described previously (42). Membranes were developed using a chemiluminescence assay (ECL; Pharmacia-Amersham, Freiburg, Germany) and subsequently exposed on chemiluminescence film (Pharmacia-Amersham).
Enzyme-linked immunosorbent assay. A sandwich enzyme-linked immunosorbent assay (ELISA) was used for detecting TNF- and IL-6 (PharMingen) in culture supernatants as described previously (26). Assays were performed as recommended by the manufacturers. Cytokine concentrations in the samples were calculated using standard curves generated from recombinant cytokines, and the results were expressed in picograms or nanograms per milliliter. The difference between duplicate wells was consistently less than 10% of the mean.
Statistical analysis. For statistical analysis, the data obtained from independent experiments are presented as the means ± standard deviations; they were analyzed using a paired t test with Bonferroni's adjustment or analysis of variance for multiple comparisons. Differences were considered significant at a P value of <0.05.
RESULTS
Purification of the 38-kDa glycolipoprotein. The 38-kDa protein was purified from unheated CFPs of M. tuberculosis H37Rv by ammonium sulfate precipitation, HIC, and anion- exchange chromatography. The SDS-PAGE analysis of the CFPs, 60 to 80% ammonium sulfate precipitate of CFPs, and purified 38-kDa glycolipoprotein are shown in Fig. 1A. We recovered approximately 1.8 mg of purified 38-kDa protein from the initial 985-mg quantity of CFPs. To establish the identity of the Ag, the purified 38-kDa glycolipoprotein was separated by isoelectrofocusing on an immobilized pH gradient strip (pH 4.7 to 5.9) in the first dimension and 12% SDS-PAGE in the second dimension. It had an apparent molecular mass of 38 kDa on an SDS-PAGE gel stained with Coomassie blue and was recognized on Western blots by anti-38-kDa MAb (Fig. 1B). Recognition of the purified Ag by the reference MAb confirmed the presence of the 38-kDa glycolipoprotein.
M. tuberculosis 38-kDa protein leads to the phosphorylation of MAPK and rapid TNF- and IL-6 secretion in human primary monocytes. In order to examine the signaling mechanisms involved in the 38-kDa Ag-induced proinflammatory cytokine responses, human monocytes were stimulated with various concentrations (0.5 to 10 μg/ml) or time courses (6 to 96 h) of the 38-kDa Ag. Then, cytokine ELISA was performed to assess TNF- and IL-6 secretion in culture supernatants. The 38-kDa Ag induced very low levels of TNF- and IL-6 at concentrations of 100 to 500 ng/ml (Fig. 2A). When human primary monocytes were exposed to 5.0 μg/ml of purified 38-kDa Ag, the secretion of TNF- and IL-6 significantly increased in the culture medium compared to that of monocytes exposed to 1.0 μg/ml of purified 38-kDa Ag (P < 0.01). Because the cytokine production of primary monocytes stimulated at 10.0 μg/ml was not appreciably different from that of cells stimulated at 5.0 μg/ml of Ag, we used the 5.0-μg/ml concentration in this study. In addition, the highest peak of TNF- and IL-6 production in response to the 38-kDa Ag was always detected 8 to 24 h after stimulation (Fig. 2B). Therefore, supernatants were harvested at 18 h for additional experiments.
Given that MAPKs are critical factors mediating cellular responses to many external stimuli, we examined the MAPK activation in response to the 38-kDa Ag of M. tuberculosis. Monocytes were stimulated with the 38-kDa Ag at a concentration of 5.0 μg/ml, and the phosphorylation of p38 and ERK1/2 was analyzed during various time courses (Fig. 2B). Exposure to the 38-kDa Ag produced a strong phosphorylation of p38 MAPK at 15 to 30 min after stimulation. The peak activation of ERK2 was within 30 to 60 min of stimulation with the 38-kDa Ag in monocytes (the time range was donor dependent). Control cells stimulated with LPS (0.1 μg/ml) produced a signal of comparable intensity (data not shown). The total inhibition of ERK1/2 and p38 MAPK phosphorylation by their respective inhibitors at 30 min after the addition of the 38-kDa Ag was confirmed on Western blots (data not shown).
The ERK1/2 and p38 MAPK pathways are critical for mycobacterial 38-kDa protein-induced TNF- and IL-6 formation. ERK1/2 and p38 are both pivotal to the cytokine formation induced by M. tuberculosis H37Rv (42) and to the regulation of the macrophage-antigen-presenting function by the M. tuberculosis 19-kDa lipoprotein (34). To further understand the functional roles of these kinases in the activation of human monocytes induced by the 38-kDa Ag, we used highly specific inhibitors of the kinases and measured cytokine formation. The cells were pretreated with a p38 inhibitor (SB203580) or an MEK inhibitor (PD98059 or U0126) for 45 min before being exposed to the 38-kDa Ag. Both U0126 and PD98059 were added to the cell culture at a final DMSO concentration of 0.1%, and DMSO was used as a control.
U0126 significantly decreased the formation of TNF- by human monocytes in response to the 38-kDa Ag (73.9% inhibition at 10 μM) (Fig. 3A); this decrease was confirmed using a second MEK inhibitor, PD98059 (data not shown). SB203580 reduced the TNF- secretion by human monocytes by up to 84.5% at 5 μM (Fig. 3A). Similarly, the 38-kDa Ag-induced IL-6 production was significantly reduced by both inhibitors (Fig. 3B). The inhibition was not attributable to DMSO because DMSO did not exhibit any inhibitory effects at this concentration.
Elevated 38-kDa Ag-induced MAPK activity and secretion of TNF- and IL-6 in TB patients compared with that in healthy controls. We next performed a comparative analysis of 38-kDa- or LPS-induced MAPK activation between TB patients and healthy controls. As shown in Fig. 4A, a higher phosphorylated ERK1/2 expression was observed in unstimulated and 38-kDa Ag-stimulated monocytes from TB patients than in monocytes from healthy controls (5.7- and 8.2-fold increases for unstimulated and 38-kDa Ag-stimulated cells, respectively, from TB patients). In addition, the phosphorylated-p38 expression was significantly increased in unstimulated and 38-kDa Ag-stimulated monocytes from TB patients compared with those from healthy controls (2.9- and 4.8-fold increases for unstimulated and 38-kDa Ag-stimulated cells, respectively, from TB patients) (Fig. 4B). Moreover, we found a significantly higher MAPK activation in LPS-stimulated monocytes from TB patients than in monocytes from healthy controls (P value was <0.05 for both ERK1/2 and p38).
We also compared TNF- and IL-6 production levels in peripheral blood monocytes between TB patients and healthy controls in response to stimulation with the 38-kDa Ag or LPS. The mean concentrations of TNF- and IL-6 produced were significantly increased in unstimulated and 38-kDa Ag-stimulated monocytes from TB patients compared to those in monocytes from healthy controls (Fig. 4C) (P value was <0.001 for both cytokines). Although there was a great heterogeneity in TB patients for LPS-induced proinflammatory cytokine production, LPS stimulation led to an increase in TNF- and IL-6 production by primary monocytes from TB patients compared to that of monocytes from healthy controls (P value was <0.05 for each cytokine). Moreover, we found a significantly higher MAPK activation in LPS-stimulated monocytes from TB patients than in monocytes from healthy controls (P value was <0.05 for both ERK1/2 and p38). Taken together, these data suggest that the activation status of monocytes from TB patients could be the result of the in vivo activation of ERK1/2 and p38 MAPK.
The 38-kDa Ag-induced phosphorylation of MAPKs and subsequent cytokine secretion were blocked by anti-TLR2 MAb and partially inhibited by anti-TLR4 MAb. We assessed whether the 38-kDa Ag activated the MAPK pathway through TLR2 or TLR4 signaling. The ERK1/2 phosphorylation induced by the 38-kDa Ag was measured after the preincubation of monocytes with a MAb to TLR2 or TLR4 or with an IC Ab. As shown in Fig. 5A and B, the 38-kDa-mediated phosphorylation of ERK1/2 and p38 MAPK was severely reduced in monocytes pretreated with the anti-TLR2 MAb (P < 0.001), whereas a smaller attenuation was detected by the pretreatment with anti-TLR4 MAb (P < 0.01). However, the phosphorylation of ERK1/2 and p38 MAPK was severely reduced by the pretreatment of monocytes with anti-TLR4 MAb followed by stimulation with Escherichia coli LPS (data not shown). Thus, the 38-kDa protein mediates the activation of ERK1/2 and p38 in human monocytes primarily via TLR2 but also via TLR4.
We then incubated adhered monocytes from healthy controls with or without anti-TLR2 MAb, anti-TLR4 MAb, or IC Ab for 30 min before stimulation with the 38-kDa protein to examine the extent to which TLRs are responsible for the 38-kDa Ag-induced cytokine responses. As shown in Fig. 5C, the 38-kDa Ag-induced TNF- production was prominently inhibited (by 59.4%) by anti-TLR2 Ab treatment, whereas it was inhibited to a smaller degree by anti-TLR4 MAb (by 33.0%). In a manner similar to that of the TNF- regulation, the 38-kDa Ag-induced IL-6 synthesis was inhibited to a greater extent by anti-TLR2 MAb (65.1%) than by anti-TLR4 MAb (36.1%). The cytokine responses were not changed by the pretreatment with IC Ab. These results indicate that the 38-kDa Ag-induced activation of the ERK1/2, p38, TNF-, and IL-6 responses is mainly mediated via TLR2 but is also partially mediated by TLR4.
Ectopic expression of TLRs reveals that the 38-kDa Ag activates MAPK phosphorylation in both a TLR2- and TLR4-dependent manner. To further characterize the specific interactions of the 38-kDa Ag with TLR2 and TLR4, we performed experiments with HEK/TLR2 and HEK/TLR4. In these HEK cells, the TLR2 and TLR4 expressions were confirmed in HEK/TLR2 and HEK/TLR4 cells, respectively, with Western blot, flow cytometric, and reverse transcriptase-PCR analyses. In the representative experiments (a total of three) shown in Fig. 6A and B, the 38-kDa Ag significantly activated ERK1/2 and p38 MAPK phosphorylation in HEK/TLR2 and HEK/TLR4 cells. The 38-kDa Ag-induced ERK1/2 and p38 MAPK phosphorylation was significantly inhibited by specific inhibitors in a dose-dependent manner.
Furthermore, the control experiments were conducted using anti-TLR2-MAbs on LPS-induced and anti-TLR4-MAb on peptidoglycan-induced HEK cell activation. The highly purified E. coli LPS and peptidoglycan were able to activate ERK1/2 and p38 MAPK in HEK/TLR4 and HEK/TLR2 cells, respectively (Fig. 6C). In addition, the LPS- and peptidoglycan-induced ERK1/2 and p38 MAPK activation responses in the HEK cells were specifically inhibited by pretreatment with anti-TLR4 MAb and anti-TLR2 MAb, respectively. Thus, both TLR2 and TLR4 can confer responsiveness to the 38-kDa glycolipoprotein of M. tuberculosis.
Physical and enzymatic characterization of the 38-kDa Ag. To understand whether intact protein moieties from the 38-kDa glycolipoprotein Ag are required for biologic activity, the 38-kDa Ag was treated with heat at 100°C for 30 min or incubated with PK (5 μg/ml) at 37°C for 1 h. Heat treatment of the 38-kDa Ag significantly decreased the secretion of TNF- and IL-6 in human monocytes (Fig. 7), suggesting the heat lability of the Ag. In addition, the cytokine production was completely attenuated after PK treatment of the Ag, suggesting that intact protein moieties from the 38-kDa Ag are essential for cytokine secretion. The SDS-PAGE of PK-treated 38-kDa Ag failed to show intact protein bands, although there were smaller protein bands after heat inactivation (data not shown). These data strongly suggest that heat- and PK-sensitive protein components of the 38-kDa Ag would be responsible for biologic activity by this Ag.
Comparative cytokine responses between the 38-kDa and 19-kDa Ags. The TLR2 agonist 19-kDa lipoprotein has been well characterized for immunomodulating activities, such as IL-1, IL-12 p40, and TNF- induction, by human monocyte-derived macrophages or a human monocytic cell line (6, 43). To compare the immunoregulatory functions between the 38-kDa and 19-kDa Ags, cytokine analysis for TNF-, IL-6, and IL-12 p40 secretion was undertaken in human primary monocytes after stimulation with the 38-kDa or 19-kDa Ag.
As shown in Fig. 8, the 38-kDa and 19-kDa Ags both induced significant dose-dependent cytokine secretions of TNF-, IL-6, and IL-12 p40 in human primary monocytes. The 19-kDa lipoprotein induced a large amount of TNF-, IL-6, and IL-12 p40 in human primary monocytes, with a significant difference relative to the 38-kDa Ag-induced cytokine secretion. Significant cytokine productions occurred with cells incubated with the 19-kDa lipoprotein relative to the samples that received the 38-kDa Ag (at 0.1, 0.25, and 0.5 μg/ml; P < 0.001). These data suggest that the 38-kDa Ag is less potent for inducing the proinflammatory and Th1-driving cytokine production in human primary monocytes than is the 19-kDa lipoprotein.
DISCUSSION
Research into the development of TB vaccines and immunodiagnostics has focused on the proteins released by M. tuberculosis, the causative agent of TB, because these Ags are thought to induce protective immunity and immune responses of diagnostic value (2, 10, 11). Our data demonstrate that the stimulation of human cells in vitro with the 38-kDa glycolipoprotein of M. tuberculosis induces MAPK activation primarily via TLR2 but also partially via TLR4. We also describe reproducible protocols for the purification of native 38-kDa glycolipoprotein Ag from culture filtrates of M. tuberculosis.
The purified 38-kDa protein showed a strong reactivity with antibodies in the sera of smear-positive, smear-negative, and extrapulmonary TB patients (data not shown). T-cell responses to this secreted glycolipoprotein, representing a major immunogen of M. tuberculosis, have been well characterized using human and murine lymphocytes (45-47). In addition, the immunodominant epitopes of the 38-kDa protein Ag recognized by CD4+ T cells have been characterized in TB patients and healthy controls (45, 47) as well as in murine T cells (45, 46). Zhu et al. (48) showed that the nucleic acid vaccination of C57BL/6 mice against 38-kDa mycobacterial Ag induced protection against challenge with virulent M. tuberculosis. Taken together, these findings suggest that the 38-kDa Ag is a valuable candidate for use as a target of host immune responses to M. tuberculosis in human studies and animal models.
Soluble and cell wall-associated mycobacterial factors are capable of mediating activation via distinct TLR proteins (30). Two mycobacterial cell wall glycolipids, lipoarabinomannan and mannosylated phosphatidylinositol (PIM), activate cells through TLR2 (18, 25). In addition, short-term culture filtrates of M. tuberculosis bacilli contain TLR2 agonist activity that appears to be PIM (25). Lipomannan (LM) from several mycobacterial species has been found to display dual functions by either stimulating or inhibiting proinflammatory cytokine synthesis through different pathways in murine primary macrophages. LM induces macrophage activation characterized by TNF and nitric oxide secretion through TLR2 and the adapter protein MyD88, independently of either TLR4 or TLR6 recognition (36).
One of the best-described mycobacterial TLR2 agonists is the 19-kDa lipoprotein (28, 32, 33) which induces host cell apoptosis (28) and down-regulation of major histocompatibility complex class II molecules (32, 33) and enhances the activity against mycobacteria via nitric oxide in mice (6). In addition, the 19-kDa lipoprotein elicits high levels of IL-12 from macrophages in addition to its powerful immunomodulatory properties, leading to suppression of Ag presentation signaling cascades (38). The comparative analysis between the 38-kDa and 19-kDa lipoproteins showed a greater production of TNF-, IL-6, and IL-12 p40 by the 19-kDa lipoprotein in human primary monocytes than by the 38-kDa lipoprotein. Our data are partially consistent with the previous studies (6) in which the 19-kDa lipoprotein induced more IL-12 p40 by a human monocytic cell line than was induced by the 38-kDa glycolipoprotein. Moreover, our data indicate that the biologic activities induced by the 38-kDa Ag may be dependent on the intact protein moieties from this Ag because the cytokine production was totally attenuated after PK treatment. Taken together, our data suggest that the 38-kDa Ag is less active for inducing the cytokine production than the 19-kDa lipoprotein, with a substantial difference in antigenic nature between two proteins.
Our data first demonstrate that the 38-kDa glycolipoprotein Ag has structural motifs to interact with both TLR2 and TLR4. In addition, the intact protein moieties of the 38-kDa Ag would be responsible for TNF- and IL-6 induction in human primary monocytes. TLR4 is a pattern recognition receptor not only for exogenous ligands, such as LPS of gram-negative bacteria (35), but also for endogenous ligands such as fibronectin, heat shock proteins, and hyaluronan oligosaccharides (27). Although TLR4 ligands of M. tuberculosis are poorly understood, a recent study demonstrated that mycobacterial heat shock protein 65 signals exclusively through TLR4 (7). Furthermore, BCG induced the transcription and secretion of the chemokine CXCL8 in human neutrophils by signaling through TLR2 and TLR4 in conjunction with the adapter protein MyD88 (19). In this work, immune activation induced by 38-kDa Ag through TLR4 should not be a result of contaminating LPS, as several procedures were conducted to rule out endotoxin contamination. The purified 38-kDa Ag was applied to a polymyxin B column to eliminate LPS contamination, and the remaining LPS level was determined by the Limulus amebocyte lysate assay. In our study, even 160 pg of LPS (eightfold more than the amount present in our experiments) did not activate human primary monocytes. Moreover, the heat sensitivity of 38-kDa Ag activity excludes contamination by LPS, because LPS is heat resistant. Further studies are needed to identify the individual motifs of the 38-kDa glycoprotein that are responsible for activating each pattern recognition receptor.
The N-terminally acylated lipopeptide region has been reported to be important for the immunostimulatory activity of bacterial and mycoplasmal lipoproteins. A recent study showed that cytokine expression by human and murine macrophages was induced by tri- and tetraacylated forms of LM (Ac3LM and Ac4LM, respectively) but not mono- or diacylated LM (Ac1LM and Ac2LM, respectively) (17), whereas PIM has been shown to be proinflammatory irrespective of its acylation (18). In this study, we did not assess the acylation status of the 38-kDa protein, but it is predicted to be acylated. Further studies should clarify the question of the role of fatty acids for 38-kDa Ag signaling via TLRs.
The activation of intracellular signaling pathways and subsequent inflammatory cytokines can be induced by different stimuli in different cell types (37). Our study emphasized the role of the MAPK (both ERK and p38) pathways in the 38-kDa Ag-induced TNF- and IL-6 secretion by human monocytes. The MAPK signaling pathways are highly conserved cascades that are important in diverse aspects of the immune response (13). They form a family of protein kinases, which includes ERK, p38 MAPK, and stress-activated protein kinase/c-Jun N-terminal kinase. In the immune system, MAPK activation can be directly mediated through cytokine receptors, such as the type I TNF receptor and IL-1R, as well as through microbial pattern recognition receptors, most prominently the TLR family, which functions as detectors of infection (44). The MAPK signaling cascades culminate in the induction of multiple proinflammatory genes, including the genes for IL-12 p40, TNF-, IL-6, and inducible nitric oxide synthase, that are important in the defense against microbial infection (12). Of note, the p38 MAPKs regulate the expression of many cytokines and play important roles in the activation of immune responses (24).
The importance of the MAPK signal transduction pathway in controlling many aspects of immune-mediated inflammatory responses has made it a priority for research related to many human diseases. In the present work, we demonstrated greater activation of ERK1/2 and p38 MAPK and greater secretion of cytokines in monocytes from active pulmonary TB patients than in monocytes from healthy controls. Previous studies have reported that TLR2 triggers apoptosis in polymorphonuclear neutrophils (PMNs) from TB patients, which correlates with the M. tuberculosis-induced expression of phospho-p38 in PMNs and with the increased expression of activated p38 found in circulating TB-PMNs (1). In a murine model of pulmonary inflammation, p38 activation was implicated in the recruitment of PMNs to infected lung tissue (31). Thus, our findings suggest that the high production level of proinflammatory cytokines during the early stages of TB could be attributable to the in vivo activation of MAPK. Therefore, in our system, human monocytes would detect the 38-kDa glycolipoprotein via TLR2 and TLR4, leading to activation of the ERK1/2 and p38 MAPK pathways, which in turn would induce enhanced proinflammatory cytokine responses during the early stage of human TB.
We have shown that the purified 38-kDa glycolipoprotein induces the activation of ERK1/2 and p38 MAPK via TLR2 and TLR4 and the subsequent proinflammatory cytokines TNF- and IL-6 in human primary monocytes. This study provides novel insight into the role of the TLR2- and TLR4-mediated MAPK signaling pathways induced by 38-kDa glycolipoprotein for early inflammatory responses during mycobacterial infection.
ACKNOWLEDGMENTS
This work was supported by Korea Research Foundation Grant (KRF-2004-041-E00125).
The anti-38-kDa MAb was kindly provided by Colorado State University as part of NIH, NIAID contract no. HHSN266200400091C (titled Tuberculosis Vaccine Testing and Research Materials), which was awarded to Colorado State University.
These authors contributed equally to this work.
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