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A Role for Double-Stranded RNA-Activated Protein Kinase PKR in Mycobacterium-Induced Cytokine Expression¹

Immunology Research Laboratory, Department of Paediatrics and Adolescent Medicine, Faculty of Medicine, The University of Hong Kong, Pokfulam, Hong Kong Special Administrative Region, People’s Republic of China

	Abstract

Top Abstract Introduction Materials and Methods Results Discussion Disclosures References

 
Following infection of the host by Mycobacterium tuberculosis, induction of cytokines is a major defense mechanism to limit the pathogen invasion. Cytokines interact with each other to form an intertwined network of pathways. For example, IFN and TNF have been shown to interact through common pathways including IFN-inducible, dsRNA-activated serine/threonine protein kinase (PKR) induction. As a signal transducer, it has been conventionally known to regulate the induction of cytokine expression in response to virus infection through NF-B. In light of the critical role of TNF in immunity and its cytotoxic effects mediated by PKR, we examined the role of the kinase in the regulation of immune response against M. tuberculosis using the interaction of bacillus Calmette-Guérin (BCG) and primary human blood monocytes as a model. Our results showed that BCG stimulates the induction of cytokine expression in human primary blood monocytes including TNF-, IL-6, and IL-10. With the suppression of PKR by using PKR-mutant gene or 2-aminopurine as PKR inhibitor, we showed that the BCG-induced cytokine expression in human monocytes is regulated by the phosphorylation and activation of PKR. We also demonstrated that downstream of PKR induction is the activation of MAPK and translocation of NF-B into the nucleus. NF-B in turn mediates the transcription of specific cytokine genes. Taken together, PKR plays a critical role in the regulation of immune responses to mycobacterial infection and may serve as an important molecule in the innate antimycobacterial defense.

	Introduction

Top Abstract Introduction Materials and Methods Results Discussion Disclosures References

 
Tuberculosis is a major cause of morbidity and mortality in the world, particularly in developing countries (1). The disease is caused by Mycobacterium tuberculosis and approximately one-third of the world’s population has been infected with this bacterium. To control this common infection, vaccines have been developed in addition to specific antibacterial treatment and public health measures. Of the vaccines, bacillus Calmette-Guérin (BCG),³ a live attenuated mycobacterium, is the most widely used human vaccine against tuberculosis. It is also known as M. bovis bacillus Calmette-Guérin, since it was derived from the parental M. bovis strain that causes bovine tuberculosis.

Following mycobacterial infection, different branches of the host immune system are mobilized to mount defense against the pathogen. One of the major defense mechanisms to limit the growth of the intracellular pathogen is through the induction of cytokines in immune cells, including macrophages (2, 3). Cytokines are pleiotropic proteins released in response to pathogens or cancer cells. They act in concert as intercellular regulatory factors responsible for the differentiation, proliferation, and function of immune cells.

Macrophages have been shown to be a major source of cytokines during mycobacterial infection (4). Mycobacterium-infected macrophages or monocytes secrete proinflammatory cytokines including TNF-, IL-1, IL-6, and IL-12 as well as anti-inflammatory cytokines including IL-4 and IL-10 (3). It was thought that the secretion of proinflammatory cytokines by macrophages is responsible for the propagation of inflammation, induction of granuloma formation, and activation of T cells leading to the development of adaptive immunity (2). Also, the complex cascades of cytokine induction appear to be beneficial for host response since interaction of different cytokines would have synergistic actions against mycobacterial infection. For instance, TNF- and IFN- interact synergistically to play a vital role in the development of protective granulomas consisting of macrophages and other immune cells to contain the mycobacteria (5).

Cytokines typically have pleiotropic properties and share some common pathways for their mechanisms of action. For example, both TNF and IFN have been demonstrated to act through common pathways, including the induction of IFN-inducible, dsRNA-activated serine/threonine protein kinase (PKR) and p53 (6, 7). PKR is well-characterized in its role in antiviral activity by inhibition of translation through the phosphorylation of eukaryotic initiation factor 2 (eIF-2) (8). In addition, PKR plays critical roles in signal transduction and transcription regulation control by activating specific MAPKs (9, 10). For example, PKR regulates the induction of IFN genes, IFN- and IFN-, in response to virus infection through the activation of nuclear factors NF-B and IFN regulatory factor 1 (8, 11, 12, 13, 14).

The NF-B family has many members, including c-Rel, NF-B1 (p50, p105), NF-B2 (p52, p100), RelA (p65), and RelB (15, 16). The activity of NF-B is suppressed by the inhibitor of NF-B, IB, which is a substrate of IB kinase (IKK) complex (17). Activation of the IKK complex by various stimuli results in phosphorylation and consequent degradation of IB, thereby leading to the release of NF-B. Upon release from IB, NF-B translocates into the nucleus and activates transcription of targeted genes by binding to specific NF-B enhancer sequences in their respective promoters. Of the cellular stress signals, it has been shown that dsRNA or virus can induce PKR to activate the IKK complex leading to the release of NF-B (9, 10, 18).

MAPKs including ERK 1 and 2, p38 MAPK, and stress-activated protein kinase/JNK are important kinases in the control of gene expression, cell proliferation, and survival (reviewed in Ref.19). For example, p38 MAPK and JNK act as significant links between PKR and its downstream target genes, including IL-6 and IL-12, in bacterial endotoxin, LPS-induced cytokine expression (20). Recently, MAPKs have been implicated as important cellular signaling molecules in mycobacterial infection (21, 22). However, the upstream regulators of MAPK activity in response to mycobacteria remain to be elucidated. We postulate that PKR is a common link in cellular response and is responsible for mediating pathogen-induced immune defense against viruses and bacteria. We used BCG as a mycobacterial model to investigate the regulation of immune response against tuberculosis. BCG is used since this nonpathogenic strain of mycobacterium allows us to investigate the normal immunity of the host against mycobacteria (23). Here we present evidence that PKR plays a critical role in the regulation of cytokine induction by BCG through the activation of MAPK and NF-B in primary human blood monocytes.

	Materials and Methods

Top Abstract Introduction Materials and Methods Results Discussion Disclosures References

 
Chemicals and Abs

Inhibitor for PKR (2-aminopurine (2-AP)) was purchased from Sigma-Aldrich, whereas specific inhibitors for ERK1/2 (PD98059) and NF-B (caffeic acid phenethyl ester (CAPE)) were purchased from Calbiochem. Specific Ab for phospho-PKR was purchased from Biosource International. Abs against actin and total PKR were purchased from Santa Cruz Biotechnology. Abs against phospho-eIF2, phospho-ERK1/2, and total ERK1/2 were purchased from Cell Signaling Technology. Anti-rabbit IgG HRP-conjugated secondary Ab was purchased from BD Transduction Laboratories, whereas peroxidase-conjugated rabbit anti-goat Ig was from DAKO. Poly(I:C) was purchased from Amersham Pharmacia Biotech.

Bacillus Calmette-Guérin

BCG vaccine strain 1077 (Aventis Pasteur) was used in all experiments unless otherwise stated. The vaccine samples have been shown to be free from any virulent mycobacteria and conform to the World Health Organization standards as stated in the quality control release provided by Aventis Pasteur.

Cell cultures and stable transformants

Promonocytic U937 cells, obtained from American Type Culture Collection, were maintained in suspension cultures in RPMI 1640 medium (Invitrogen Life Technologies) supplemented with 10% FBS (Invitrogen Life Technologies) and 1% penicillin and streptomycin (Invitrogen Life Technologies). The transdominant negative [Arg²⁹⁶]PKR mutant plasmid derived from the parental control expression vector pRC-CMV (Invitrogen Life Technologies) as well as methods to isolate and characterize clonal cell lines generated from U937 cells have been described previously (11). In brief, the mutant [Arg²⁹⁶]PKR cDNA was subcloned into pRC-CMV (Invitrogen Life Technologies) to produce the mutant plasmid. Stable transformants were generated by transfection of promonocytic U937 cells with LipofectAMINE 2000 reagent (Invitrogen Life Technologies). Cells containing pRC-CMV parental and dominant-negative [Arg²⁹⁶]PKR mutant plasmids were designated as U9KC (without PKR insert) and U9KM (with PKR mutant insert), respectively. To maintain selection pressure for the desired phenotypes, transfected cells were maintained in the same culture medium, with the addition of geneticin (0.5 mg/ml; Invitrogen Life Technologies). To characterize the mutant cell lines, transfected cells were treated with poly(I:C) (100 µg/ml) for 3 h and lysed for Western blot analysis. U9KC and U9KM cells were differentiated by treatment with 2 nM PMA overnight in the presence of medium before BCG stimulation.

Isolation of CD14⁺ monocytes

Human peripheral blood monocytes (PBMs) were isolated from buffy coats of healthy blood donors (Hong Kong Red Cross Blood Transfusion Service) by Ficoll-Paque (Amersham Pharmacia Biotech) density gradient centrifugation as described (24, 25, 26). Briefly, blood samples were centrifuged at 3000 rpm for 20 min and were separated into plasma and cell layers. For inactivation of complements, plasma was incubated at 56°C for 30 min and chilled on ice before centrifugation. The supernatant thus obtained was filtered and used as autologous plasma in subsequent cell cultures. The cell layer was diluted with PBS in the ratio of 1:1. The diluted cells were overlaid on Ficoll slowly and centrifuged at 2000 rpm for 30 min for separation of mononuclear cells from erythrocytes. The mononuclear cell layer was removed and washed with RPMI 1640 medium until the supernatant was clear. The cell pellet was resuspended in RPMI 1640 with 5% autologous plasma and plated onto petri dish for adherence of monocytes with incubation at 37°C for 1 h. Following washing with RPMI 1640, the adherent monocytes were detached by cold RPMI 1640 containing 5 mM EDTA. The monocytes were finally seeded onto tissue culture plates in RPMI 1640 supplemented with 5% autologous plasma. Cell viability was >95% as measured by trypan blue exclusion, whereas the purity of CD14⁺ monocytes was >90% as measured by flow cytometry and Ab-conjugated with FITC against CD14 (Beckman).

Induction of cytokine expression and isolation of RNA

To examine the mechanism of cytokine expression, PBMS, U9KC, and U9KM cells were treated with various doses of BCG for the indicated time periods. Cells were pretreated with inhibitors including 2-AP, PD98059, and CAPE for 1 h before the addition of BCG. A range of concentrations of each inhibitor was used to test their optimal concentrations and effects on cell viability and kinase inhibitions. Unless otherwise indicated, in subsequent experiments, concentrations of 2-AP, PD98059, and CAPE used were 5 mM, 5 µM, and 15 µg/ml, respectively. Total RNA extraction was done using TRIzol reagent (Invitrogen Life Technologies) according to the manufacturer’s instructions.

RT-PCR

The cDNA was synthesized from total RNA with oligo(dT) primers and Superscript II reverse transcriptase (Invitrogen Life Technologies). Amplification of the reverse-transcribed cDNA was performed in a 25-µl reaction containing 5 pmol of each set of upstream and downstream primers, 1 U of TaqDNA polymerase (Amersham Pharmacia Biotech), and 0.2 mM each dATP, dCTP, dTTP, dGTP, and PCR buffer (50 mM KCl, 1.5 mM MgCl₂, and 10 mM Tris-HCl (pH 9.0)). PCR were allowed to proceed for various cycles (94°C for 30 s, melting temperature (T_M) for 30 s, and 72°C for 1 min.). PCR primer sets used and assay conditions were as follows: 1) TNF-, 25 cycles (T_M = 56°C), upstream, 5'-GGCTCCAGGCGGTGCTTGTTC-3', downstream, 5'-AGACGGCGATGCGGCTGATG-3'; 2) IL-6, 25 cycles (T_M = 60°C), upstream, 5'-ATGAACTCCTTCTCCACAAGCGC-3', downstream, 5'-GAAGAGCCCTCAGGCTGGACTG-3'; 3) IL-10, 30 cycles (T_M = 60°C), upstream, 5'-ATGCCCCAAGCTGAGAACCAAG-3', downstream, 5'-TCTCAAGGGGCTGGGTCAGCTA-3'; and 4) GAPDH, 20 cycles (T_M = 60°C), upstream, 5'-ACCACAGTCCATGCCATCAC-3', downstream, 5'-TCCACCACCCTGTTGCTGTA-3'.

Measurement of TNF-, IL-6, and IL-10 in the culture supernatant

TNF-, IL-6, and IL-10 protein levels of the cell culture supernatants were measured by specific ELISA using the respective commercially available assay kits (R&D Systems). Each sample was assayed in duplicates.

Real-time RT-PCR analysis of TNF- and IL-6 mRNA

Extraction of total RNA was described as above. RNA samples were first treated with DNase I (RNase free; Roche) and then reverse transcribed by using TaqMan reverse transcription reagent kit (Applied Biosystems). To perform real-time RT-PCR, the levels of TNF- and IL-6 mRNA as well as reference gene 18S rRNA (18S) were assayed by the gene-specific Assays-on-Demand reagent kits (Applied Biosystems). All samples were run in triplicates and with no template controls on an ABI Prism 7700 Sequence Detector. The analysis method of real-time RT-PCR was the comparative cycle number to threshold (C_T) method as described in user bulletin no. 2 of the ABI Prism 7700 Sequence Detection System. The number of C_T of TNF- was normalized to that of 18S in each sample (C_T). The C_T value of the treated cells was compared with that of the untreated cells (C_T). The relative gene expression of TNF- was calculated as 2^–CT.

Preparation of cytoplasmic and nuclear proteins

Cytoplasmic and nuclear proteins were extracted as described, with modifications (27). Cells were washed with PBS and lysed by a lysis buffer containing 10 mM HEPES (pH 7.9), 10 mM KCl, 0.1 mM EDTA, 0.1 mM EGTA, 1 mM DTT, 0.5 mM PMSF, 2 µg/ml aprotinin, 1 mM sodium orthovanadate, 2 µg/ml pepstatin, 2 µg/ml leupeptin, and 50 mM sodium fluoride on ice for 15 min. Nonidet P-40 was added to a final concentration of 0.625% and the cell lysate was vigorously vortexed for 10 s. The homogenate was then centrifuged for 30 s and the supernatant was harvested as cytoplasmic proteins. The nuclear pellet was lysed with nuclear lysis buffer containing 20 mM HEPES (pH 7.9), 0.4 M NaCl, 1 mM EDTA, 1 mM EGTA, 1 mM DTT, 1 mM PMSF, 1 µg/ml aprotinin, 1 mM sodium orthovanadate, 2 µg/ml pepstatin, 2 µg/ml leupeptin, and 50 mM sodium fluoride. The sample was vigorously rocked at 4°C for 15 min on a shaking platform. The nuclear extract was centrifuged at 4°C for 5 min and the supernatant was frozen until use. Protein concentrations in cell extracts were determined by Coomassie Plus protein assay reagent kit (Pierce).

Western blot analysis

Equal amount of protein was separated by 10% SDS-PAGE, electroblotted onto nitrocellulose membranes (Schleicher & Schuell), and followed by probing with specific Abs for phospho-eIF2, actin, phospho-PKR, PKR, phospho-ERK1/2, and ERK1/2. After three washes, the membranes were incubated with the corresponding secondary Ab. The bands were detected using the Enhanced Chemiluminescence System (Amersham Pharmacia Biotech) per the manufacturer’s instructions.

EMSA

The oligodeoxynucleotides for NF-B binding (5'-GATCAGGGACTTTCCGCTGGGACTTTCC-3') were end-labeled with -³²P using the T4 Polynucleotide Kinase Labeling System (USB Corporation) and followed by separation with a G-25 purification column (Amersham Pharmacia Biotech). The radioactively labeled oligodeoxynucleotides (12 fmole) were incubated with nuclear proteins and 0.5 µg of poly(dI:dC) in DNA-protein binding buffer (5 mM HEPES (pH 7.9), 0.1 mM EDTA, 0.1 mM DTT, 0.2% Nonidet P-40, 2.4% glycerol, 10 mM NaCl). The DNA-protein complex formed was resolved in 5% nondenaturing polyacrylamide gel with Tris-glycine buffer (50 mM Tris, 0.2 M glycine, 1 mM EDTA (pH 8.0)). The gel was dried in a gel dryer (Bio-Rad) and exposed to x-ray film. An excess of competitor wild-type or mutated oligodeoxynucleotides (5'-GATCACTCACTTTCCGCTCTCACTTTCC-3') was used to examine the specificity of the binding of the DNA-protein complex.

Statistical analysis

All data were statistically analyzed by two-tailed, paired t test. A value of p < 0.05 was considered significant.

	Results

Top Abstract Introduction Materials and Methods Results Discussion Disclosures References

 
BCG induction of cytokine expression is dependent on PKR activity

As a mycobacterial vaccine, BCG has been shown to have highly immunogenic activities, including induced expression of proinflammatory cytokines in human macrophages (28). To investigate the mechanisms of immune activation, we first demonstrated the effects of BCG on the induction of cytokine mRNA in PBMs by semiquantitative RT-PCR method. Two doses of BCG were used: low dose (1 CFU/cell) or high dose (5 CFU/cell). After exposure to BCG, there were significant inductions of TNF- mRNA within 1 h of BCG treatment (Fig. 1a, lanes 6 and 10), and the effects lasted for at least 6 h (Fig. 1a, lanes 7, 8, and 11–13). The induction of IL-6 mRNA by BCG started at 3 h (Fig. 1b, lanes 7 and 11) and the mRNA levels continued to increase up to 24 h (Fig. 1b, lanes 8, 9, 12, and 13). IL-10 mRNA was slightly induced within 1 h of incubation with BCG (Fig. 1c, lanes 6 and 10) and increased significantly starting from 3 h (Fig. 1c, lanes 7–9, and 11–13).

View larger version (35K): [in this window] [in a new window]   FIGURE 1. Induction of cytokine expression in primary human monocytes treated with BCG. Human monocytes were treated with the diluent for BCG (Mock) (lanes 1–5) or were treated with a low dose of BCG (1 CFU/cell; lanes 6–9) or a high dose of BCG (5 CFU/cell; lanes 10–13) for various time points as indicated. Total RNA was extracted and cytokine mRNA levels were investigated by RT-PCR. a, TNF-; b, IL-6; c, IL-10; and d, GAPDH.

View larger version (26K): [in this window] [in a new window]   FIGURE 2. BCG induction of cytokine expression is dependent on PKR activity in primary human monocytes. A, Human monocytes were incubated with medium alone or with 2.5 mM or 5 mM 2-AP for 1 h. BCG was added and the cells were incubated for an additional 1 or 3 h before harvest. Cytokine mRNA levels were assayed by RT-PCR. Aa, TNF-; Ab, IL-10; Ac, GAPDH. B, Human monocytes were treated as in A except that the concentration of 2-AP was 10 mM and BCG was added for 3 h. Ba, IL-6; Bb, GAPDH. C, Release of TNF- from human monocytes after stimulation with BCG and/or 2-AP for 6 and 24 h. Mock, diluent for BCG; BCG, BCG (5 CFU/cell); BCG/2AP, BCG (5 CFU/cell) with 2-AP at 5 mM; 2AP, 5 mM 2-AP alone. The data for RT-PCR are representative of three independent experiments and ELISA results are shown as average ± SD of three different donors. *, p < 0.05.

To examine whether PKR regulates TNF- production in BCG-stimulated PBMs, the levels of TNF- secretion were measured by ELISA. In response to BCG treatment, a significant amount of TNF- was released at 6 and 24 h. However, the TNF- production was significantly inhibited when cells were treated with 5 mM 2-AP (Fig. 2C). Besides, BCG-induced TNF-, IL-6, and IL-10 production was progressively inhibited when cells were treated with increasing concentrations of 2-AP, ranging from 2.5 mM to 10 mM (data not shown). These results demonstrated that there is a dose-dependent effect of PKR on the induction of cytokines in response to BCG activation. Together, the results suggest that the induction of cytokines is regulated by PKR in response to BCG activation.

BCG activates the phosphorylation of PKR

To further elucidate the role of PKR in BCG-stimulated cytokine induction in PBMs, PKR expression and its kinase activity were studied by using RT-PCR and Western analysis. Our results showed that PKR mRNA levels did not change over a period of 24 h in response to BCG (data not shown), suggesting that BCG does not activate the transcription of PKR. However, BCG stimulates the specific PKR kinase activity in 30 min, as reflected by autophosphorylation of PKR (Fig. 3, lanes 1 and 2). In the presence of 2-AP, the autophosphorylation of PKR was inhibited (Fig. 3, lanes 2 and 3). Taken together, our results (Figs. 1–3) demonstrated that activated PKR plays a key role in the BCG-induced cytokine expression.

View larger version (21K): [in this window] [in a new window]   FIGURE 3. BCG activates the phosphorylation of PKR. Human monocytes were pretreated with 2-AP for 1 h and then with BCG for 30 min. Cells were lysed, and the lysate was resolved by SDS-PAGE and probed with anti-phospho-PKR and anti-PKR Abs. Mock, diluent for BCG; BCG, BCG (5 CFU/cell); BCG/2AP, BCG (5 CFU/cell) with 2-AP at 5 mM; 2AP, 5 mM 2-AP alone. The data are representative of three independent experiments.

NF-B mediates PKR regulation of cytokine expression

To delineate whether NF-B is involved in the BCG-induced and PKR-mediated cytokine expression, CAPE (a specific inhibitor of NF-B activity) was used to examine the role of NF-B in the BCG effects. CAPE is known to block the translocation of NF-B (p65 subunit) to the nucleus (32). PBMs were pretreated with CAPE for 1 h before the addition of BCG (5 CFU/cell) to the cells. Cytokine expression levels were monitored by RT-PCR. As before (Fig. 1), BCG induced TNF-, IL-6, and IL-10 mRNA expression (Fig. 4A, lane 3). The induction of cytokines was inhibited significantly in the presence of CAPE (Fig. 4A, lanes 3 and 4), whereas CAPE itself did not induce the cytokines (Fig. 4A, lanes 1 and 2). These results suggest that NF-B is involved in the cytokine induction process by BCG.

View larger version (38K): [in this window] [in a new window]   FIGURE 4. Transcription factor NF-B is involved in the induction of cytokine expression through the activation of PKR. A, Human monocytes were incubated with medium alone or with 15 µg/ml CAPE for 1 h. BCG was added, and the cells were incubated for an additional 6 h. Cells were lysed and RT-PCR was performed. Aa, TNF-; Ab, IL-6; Ac, IL-10; Ad, GAPDH. B, Human monocytes were incubated with medium alone or with 5 mM 2-AP for 1 h. BCG was added and the cells were incubated for an additional 1 h. Cells were lysed and then assayed for NF-B activation by EMSA as described. "Cold" represents the additional use of unlabeled NF-B oligodeoxynucleotides, whereas "mut." represents the use of mutated NF-B oligodeoxynucleotides. The data are representative of three independent experiments.

BCG-induced activation of MAPK phosphorylation is inhibited by 2-AP

It has been reported that PKR mediates the activation of MAPK by stress stimuli including LPS and poly(I:C) (20). To delineate the role of PKR in this process, we first investigated the involvement of MAPK in BCG-induced cytokine expression by using PD98059, a specific inhibitor of ERK1/2. This small molecule inhibitor has been widely used to study the role of ERK1/2 in cell signaling (22, 33). As shown in Fig. 5A, BCG was able to induce high levels of TNF- mRNA in PBMs (lanes 1 and 2). This induction of TNF transcription can be suppressed by pretreatment of the cells with 5 µM PD98059 for 1 h before the BCG addition (Fig. 5A, lanes 2 and 3).

View larger version (33K): [in this window] [in a new window]   FIGURE 5. BCG-induced activation of ERK1/2 phosphorylation is inhibited by 2-AP. A, Primary human monocytes were treated with medium alone or with 5 µM PD98059 for 1 h. Cells were incubated in the presence or absence of BCG for an additional 6 h. Total RNA was extracted and real-time RT-PCR for TNF- was performed as described. Mock, diluent for BCG; BCG, BCG (5 CFU/cell); BCG/PD98059, BCG (5 CFU/cell) with 5 µM PD98059; PD98059, 5 µM PD98059 alone. Results are shown as average ± SD of cells from three different donors. **, p < 0.05. B, Human monocytes were pretreated with 5 mM 2-AP or medium alone for 1 h. BCG (5 CFU/cell) was added for an additional 30 min or 2 h before proteins were harvested. Phosphorylated and total ERK1/2 were detected by Western blotting. Mock, diluent for BCG; BCG, BCG (5 CFU/cell); BCG/2AP, BCG (5 CFU/cell) with 2-AP at 5 mM; 2AP, 5 mM 2-AP alone. The data are representative of three independent experiments.

BCG-induced cytokine gene expression is impaired in PKR-deficient cells

Throughout these experiments, our primary goal was to examine the interactions of BCG with human immune cells. Thus, in examining the role of PKR, we used 2-AP as an inhibitor on the PBMs instead of cells from PKR^–/– knockout mice. To further define the requirement of PKR in BCG-induced immune response, the effects of loss of PKR activity on BCG-induced cytokine gene expression were examined. To accomplish this, human promonocytic U937 cells were stably transfected with a plasmid encoding a dominant-negative [Arg²⁹⁶]PKR mutant gene or with parental plasmid for the generation of U9KM and U9KC cells, respectively. To demonstrate that the U9KM cells are deficient in the kinase activity of PKR, we treated control and mutant cell lines with poly(I:C), a well-known inducer of PKR, for 3 h. We then investigated its effects on the activation of eIF-2, the well-defined cellular substrate of PKR. In Fig. 6A, poly(I:C) stimulated and enhanced the phosphorylation of eIF-2 in U9KC (lanes 1 and 2). In the U9KM cells, generated by transfection of a transdominant-negative PKR mutant, poly(I:C) did not enhance the phosphorylation of eIF-2 (lanes 3 and 4). The results showed that the expression of this negative mutant does abrogate the endogenous PKR activity. Also, this cellular system has been used to demonstrate the role of PKR in IFN regulation and apoptosis (11, 34, 35).

View larger version (15K): [in this window] [in a new window]   FIGURE 6. Mutant PKR inhibits BCG-induced cytokine expression. U937 cells were stably transfected with a plasmid encoding a dominant-negative [Arg296]PKR mutant gene to generate U9KM cells. Cells transfected with the parental control plasmid without PKR insert were known as U9KC cells. A, Mutant PKR abrogates the endogenous PKR activity. Stably transfected cells were treated with poly(I:C) (100 µg/ml) for 3 h. Cells were lysed, resolved by SDS-PAGE, and probed with anti-phospho-eIF2 and anti-actin Abs. UT, Untreated. B and C, Cells were pretreated with PMA (2 nM) for 24 h and then incubated with BCG (5 CFU/cell) for 3 h. Real-time RT-PCR for TNF- (B) and IL-6 (C) was performed as described. Results are shown as average ± SD of cells from three different donors.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion Disclosures References

 
In this report, we examined the mechanisms of mycobacterial activation of proinflammatory cytokine gene expression on human PBMs upon exposure to BCG. Our results demonstrated that BCG induces significant amounts of TNF- and IL-6 synthesis by human monocytes (Fig. 1). We also provided evidence that PKR mediates the cytokine induction in response to mycobacterial infection. Suppression of PKR activity by 2-AP, an inhibitor of PKR, resulted in abrogation of the cytokine induction at both mRNA and protein levels (Fig. 2). We also used stable transfectants expressing a dominant-negative mutant PKR gene to examine the role of PKR activity in cytokine expression. Cells expressing this catalytically inactive form of PKR showed defects in the production of TNF- and IL-6 after exposure to BCG, which further demonstrates the role of PKR in the mycobacterium-induced cytokine regulation processes (Fig. 6).

Proinflammatory cytokines are known to be important in the pathogenesis of mycobacterial infections. They play critical roles in the recruitment of monocytes and lymphocytes from the bloodstream to the infected area, the control of the inflammatory response, and the outcome of mycobacterial infections (3). One of the proinflammatory cytokines, TNF-, is well studied in its role as an antimycobacterial protein (3). It synergizes with IFN- to generate reactive oxygen intermediates and nitrogen oxides in macrophages to kill mycobacteria (36, 37). TNF- also plays a major role in the formation of granulomas, which control the multiplication and dissemination of the mycobacteria.

Although the exact mechanism of how TNF regulates granuloma formation is not well understood, a model hypothesizing the relationships between TNF and chemokine expression in its formation and maintenance has been suggested recently (38). When infected with M. tuberculosis, macrophages secrete significant amounts of TNF-. TNF- would in turn regulate chemokine induction in macrophages as well as other immune cells. Chemokines are chemotactic cytokines that help in chemotaxis and transendothelial migration of mononuclear cells (39, 40). More monocytes, macrophages, T lymphocytes, and B lymphocytes would migrate to the site of infection and form the granulomas, limiting the number and growth of mycobacteria. Our results above have demonstrated that the induction and production of TNF- upon BCG infection rely on PKR-dependent signaling mechanisms. These findings support the proinflammatory properties and immune defense activities of PKR, and also implicate a role for PKR in antimycobacterial activities.

Evidence was provided that there is phosphorylation and activation of PKR during BCG treatment (Fig. 3). These results draw an analogy to previous reports that PKR plays a role in antiviral response (9, 10, 18, 41). Virus-infected cells would produce large amounts of IFN. It has been proposed that such a function is to protect neighboring uninfected cells from propagating another round of infection (42, 43, 44). Following binding of IFN to its cognate receptor, IFN-stimulated genes are induced. A key protein induced to mediate the IFN effects is PKR. When cells are stimulated with IFN, the induced expression of PKR serves as an intracellular "virus detector" for the recognition of dsRNA (9, 41). Upon binding of dsRNA, PKR would dimerize and autophosphorylate to become an active protein kinase (45). This activated PKR then phosphorylates eIF-2, which forms a stable complex with eIF-2B together with GDP. This inactive complex results in inhibition of translation (46), and hence leads to apoptosis and antiviral responses (6, 9, 10). However, the exact mechanisms for PKR to elicit antimycobacterial responses remain to be elucidated.

Our results showed that transcription factor NF-B is involved in the BCG induction of cytokine transcription, and this BCG-regulated cellular event was found to be downstream of PKR by EMSA (Fig. 4). It appears that BCG would first bind to TLR (47, 48, 49) and then lead to the activation of PKR and translocation of NF-B into the nucleus, as demonstrated by the results here. TLRs are receptor molecules that are responsible for the recognition of foreign Ags and pathogens (50). For example, the specific recognition of LPS, a major component of the outer membrane of Gram-negative bacteria, by immune cells has been elucidated. LPS first binds to LPS binding protein in the serum, followed by incorporation to CD14 located on the cell surface of monocytes and macrophages. It is subsequently transferred to another signaling receptor, specifically TLR4. Activation of TLR4 triggers intracellular signaling events like the activation and phosphorylation of various MAPK pathways, which in turn activate various transcription factors. Events downstream of TLR4 also include the activation of the IKK complex through TNFR-associated factor 6 complexes. The activation of these specific transcription factors stimulates the expression of specific cytokine or chemokine genes (51). It has also been demonstrated that PKR is a part of the LPS-induced signaling pathway (52) and that 2-AP could inhibit LPS-induced cytokine expression (data not shown). Thus, BCG might induce cytokine expression in PBMs through PKR in a way similar to that of LPS. Since BCG is capable of inducing the phosphorylation of PKR and MAPK and the activation of TLR4 has been known to activate PKR activity after LPS treatment, TLR4 therefore may play an important role in the immune recognition of BCG by monocytes.

Together with our data, it appears that PKR can mediate transcriptional control of gene expression via the activation of NF-B. Our results are also in agreement with previous studies that the induction of the TNF- mRNA level was impaired in NF-B p50 knockout mice in response to M. tuberculosis (53). The incomplete abrogation of cytokine induction by CAPE may be due to incomplete inhibition of NF-B activities, or it suggests that other transcription factors might be active in the cytokine induction process by BCG (Fig. 4A). An example of these factors is the NF-IL6, which has been implicated to be involved in the activation of the IL-6 gene by M. tuberculosis (54).

Our results have also revealed that the early signal transduction events of mycobacterial activation of human monocytes involving ERK phosphorylation are regulated by PKR. Results are consistent with previous reports that the release of TNF- and IL-10 involves MAPK pathways by human monocytes infected with M. tuberculosis H37Rv (22). TNF- secretion requires ERK1/2 activation by M. avium-infected human monocyte-derived macrophages (55). The data in our report demonstrate that PKR acts as an upstream regulator of ERK1/2 because pretreatment of human monocytes with PKR inhibitors suppresses the activation of ERK1/2 after BCG infection (Fig. 5). The relationship between MAPK and PKR has been recognized not long ago through several experiments. It was first demonstrated that phosphorylation of the transcription factor STAT-1 in response to IFN is dependent on p38 (20). Moreover, phosphorylation of STAT-1 by IFN is inhibited in PKR-null cells (56). Thus, it has been hypothesized that there is a linkage between PKR and MAPK. Goh et al. (20) demonstrated that PKR is a signaling component in the activation of p38 MAPK induced by LPS. Moreover, several reports pointed out that the biological behavior of pathogenic mycobacteria is different from that of the nonpathogenic strains. Activation of MAPKs is one of the examples. Macrophages infected with nonpathogenic mycobacteria induced prolonged activation of p38 MAPK and ERK1/2, whereas pathogenic mycobacteria demonstrated earlier decreases in the activation of MAPK. As a result, there is relatively less induction of TNF- by macrophages infected with pathogenic mycobacteria compared with that of the nonpathogenic counterparts (57). It has been shown that modulation of MAPK signaling by mycobacteria could promote their own survival in the susceptible host (58). These results together with ours indicate that PKR and MAPK, through their actions as important signal transducers in cytokine regulation, may play a significant role in the innate immune response against bacteria and mycobacteria.

In summary, PKR plays a critical role in the regulation of cytokine expression upon mycobacterial infection. Up-regulation of MAPK and NF-B activities appears to be a downstream event following BCG-induced activation of PKR. The findings support the hypothesis that PKR is important in innate immunity against bacteria (20).

	Disclosures

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The authors have no financial conflict of interest.

	Footnotes

 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

¹ This work was supported by the Edward SK Hotung Paediatrics Education and Research Fund, the Research Fund for the Control of Infectious Diseases (RFCID 01031052); and the Research Grants Council of Hong Kong (HKU 7408/04M). B.K.W.C. is the recipient of a Student Travel Grant Award from the American Society for Microbiology and Studentship from The University of Hong Kong.

² Address correspondence and reprint requests to Dr. Allan S. Y. Lau, Department of Paediatrics and Adolescent Medicine, Faculty of Medicine, The University of Hong Kong, Queen Mary Hospital, 102 Pokfulam Road, Hong Kong Special Administrative Region, People’s Republic of China. E-mail address: asylau{at}hku.hk

³ Abbreviations used in this paper: BCG, bacillus Calmette-Guérin; PKR, IFN-inducible, dsRNA-activated serine/threonine protein kinase; eIF, eukaryotic initiation factor; IKK, IB kinase; 2-AP, 2-aminopurine; CAPE, caffeic acid phenethyl ester; PBM, human peripheral blood monocyte; T_M, melting temperature; C_T, cycle number to threshold.

Received for publication October 5, 2004. Accepted for publication September 2, 2005.

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