Skip to main content
NCBI home page
Clinical and Experimental Immunology logoLink to Clinical and Experimental Immunology
. 2006 Jan;143(1):150–160. doi: 10.1111/j.1365-2249.2005.02966.x

Differential regulation of interleukin-12 and tumour necrosis factor-α by phosphatidylinositol 3-kinase and ERK 1/2 pathways during Mycobacterium tuberculosis infection

C-S Yang *, J-S Lee †, S-B Jung *, J-H Oh *, C-H Song *, H-J Kim *, J-K Park *, T-H Paik †, E-K Jo *
PMCID: PMC1809561  PMID: 16367946

Abstract

Interleukin (IL)-12 and tumour necrosis factor (TNF)-α are both thought to be critical factors in the defence against mycobacteria but are known to play different roles. In this study, we investigated the regulatory pathways for IL-12 and TNF-α expression in human monocyte-derived macrophages (MDMs) after treatment with Mycobacterium tuberculosis H37Rv or the Triton X-100 solubilized proteins (TSP) purified from M. tuberculosis. We found a rapid phosphorylation of Akt and extracellular signal-regulated kinase (ERK), albeit with differential activation kinetics, in human MDMs treated with M. tuberculosis or TSP. Studies using inhibitors selective for phosphatidylinositol 3-kinase (PI 3-K) and ERK 1/2 show that both pathway plays an essential role in the induction of TNF-α at both the transcriptional and translational levels in human MDMs. In contrast, blockade of the PI 3-K/Akt or ERK 1/2 pathways significantly increased M. tuberculosis- or TSP-induced IL-12 p40 and p35 mRNA and bioactive p70 protein. The enhancement of IL-12 levels by inhibition of PI 3-K and ERK 1/2 was not reversed by neutralization of TNF-α or addition of rhTNF-α, suggesting that the negative regulation of IL-12 is not mediated by concomitant TNF-α suppression. Further, PI 3-K activity is required for the M. tuberculosis- or TSP-induced phosphorylation of ERK 1/2 activation. TSP from M. tuberculosis shows a similar dependency on the PI 3-K and ERK 1/2 pathways to those by M. tuberculosis. Collectively, these data suggest that the Th1-driving cytokine IL-12 and proinflammatory cytokine TNF-α are differentially regulated by PI 3-K and ERK 1/2 pathways in human MDMs during mycobacterial infection. These results may provide therapeutic targets for precise and specific fine-tuning of cytokine responses.

Keywords: ERK, IL-12, Mycobacterium tuberculosis, PI 3-K, TNF-α

Introduction

Mycobacterium tuberculosis is responsible for at least 1·5 million deaths per year worldwide. The organism is a slow-growing acid-fast bacillus that is transmitted primarily by the respiratory route. Although M. tuberculosis can cause disease in most organs, pulmonary tuberculosis (TB) is the most common. Estimates are that one-third of the world's population is infected with M. tuberculosis, but the infections do not normally lead to active disease. The immune response mounted to the infection is generally successful in containing but not eliminating the pathogen. Host control of M. tuberculosis infection in both humans and mouse models has been shown to be associated with the production of interferon (IFN)-γ by CD4+ T cells [1].

Interleukin (IL)-12 is known to be a crucial cytokine in the differentiation of IFN-γ-producing Th1 cells [2]. As mycobacteria are strong inducers of IL-12, mycobacterial infection can skew the response to a secondary antigen toward a Th1 phenotype [3]. An intriguing study has indicated that the administration of IL-12 DNA could substantially reduce bacterial numbers in mice with chronic M. tuberculosis infection [4], suggesting that induction of this cytokine is an important factor in the design of a tuberculosis vaccine. Tumour necrosis factor (TNF)-α is a multi-functional cytokine that performs a variety of roles in both immune and inflammatory responses. At the cellular level, TNF-α acts in synergy with IFN-γ to enhance the expression of inducible nitric oxide synthase and the antimycobacterial activity of infected macrophages [5]. In particular, TNF is essential for the colocation of lymphocytes and macrophages within granulomas, where their close apposition facilitates the activation of mycobacterial killing and prevents dissemination of the infection [6]. The balance between pro- and anti-inflammatory signalling is likely to achieve an appropriate level of immunity that allows the host and parasite to maintain a stable interaction.

Mycobacteria trigger several intracellular signalling cascades, such as the phosphatidylinositol 3-kinase (PI 3-K) [7] and mitogen-activated protein kinases (MAPK) cascades, as well as extracellular-regulated kinase (ERK)1/2, p38 kinase and stress-activated protein kinases, such as the c-Jun-N-terminal kinase [8,9]. An increasing awareness of the significance of signal transduction mechanisms in mycobacterial infection has given rise to the development of potentially promising new strategies for antimycobacterial treatment. Recent studies indicate that the modulation of the MAPK pathways may be an important component of mycobacterial pathogenesis [10]. Our previous data and data from other researchers show the critical role of the ERK pathway in TNF-α secretion by human monocytes after M. tuberculosis H37Rv infection [11–13]. PI 3-K has been implicated in the regulation of cellular growth, and its involvement in the inhibition of apoptosis is well established [14,15]. The role of PI 3-K in mycobacterial phagocytosis was reported recently in macrophages [16]. In addition, the PI 3-K pathway plays an important role in human monocyte antimycobacterial activity [17] and up-regulates a signalling pathway involved in cell survival through lipoarabinomannan-mediated Bad phosphorylation [7].

Although previous studies have suggested the activation of various signalling enzyme cascades following mycobacterial infection, the intracellular signalling mechanisms controlling IL-12 secretion triggered by mycobacteria in human macrophages have not been elucidated. In the present study, we analysed the intracellular signalling pathways that are activated by M. tuberculosis H37Rv infection- and Triton X-114 solubilized protein (TSP) antigen-induced IL-12 and TNF-α production in human monocyte-derived macrophages (MDMs). We examined the roles of the PI 3-K and ERK 1/2 pathways involved in IL-12 and TNF-α induction by mycobacterial proteins and M. tuberculosis in human MDMs. We found that the PI 3-K/Akt and ERK pathways contribute a negative and positive regulation of M. tuberculosis- or TSP-induced IL-12 and TNF-α expression, respectively. The cross-talk between the PI 3-K and MAPK pathways was also investigated.

Materials and methods

Bacteria

M. tuberculosis H37Rv was kindly provided by Dr Richard L. Friedman, University of Arizona, Tucson. M. tuberculosis H37Ra (ATCC 25177) was grown to late log phase in Middlebrook 7H10 agar (Difco, Detroit, MI, USA) medium supplemented with 10% OADC (oleic acid, albumin, dextrose, catalase; Becton & Dickinson Immunocytometry, San Jose, CA, USA) supplemented with 0·05% Tween 80 (Sigma, St Louis, MO, USA). Batch cultures were stored at −70°C. Representative vials were thawed and enumerated for viable colony-forming units (CFU) on Middlebrook 7H10 agar (Difco). Single-cell suspensions of mycobacteria were obtained by a modification of the standard methods. Briefly, aliquots of frozen M. tuberculosis were cultured in 7H9 broth with 0·5% glycerol at 37°C and 5% CO2 for 7–10 days to reach the mid-exponential growth phase. Bacterial cultures were pelleted at 3000 g for 10 min and resuspended in 7H9. Clumped mycobacteria were dispersed with an ultrasonic cell disrupter (3–5 min, 35 kHz; Bandelin, Berlin, Germany). The bacteria were then resuspended in 1 ml of RPMI-1640, and the clumps were disrupted by multiple passages through a 25-gauge needle. Mycobacterial viability, as assessed by the number of CFU, was 60–70%. To rule out the influence of lipopolysaccharide (LPS) in the assays, the bacterial suspensions were tested in the Limulus amebocyte lysate assay (BioWhittaker, Walkersville, MD, USA). The effective LPS concentration was < 0·2 pg/ml in experiments with bacteria to cell ratios of 1: 1.

Triton X-100-solubilized protein (TSP) antigen of M. tuberculosis H37Rv. The TSP antigen of M. tuberculosis was purified as described previously [18]. In brief, M. tuberculosis H37Rv was grown for 6 weeks at 37°C as surface pellicles on Sauton's medium. Bacilli were harvested by centrifugation, and the cells (100 g wet weight) were suspended in 200 ml of 1% Triton X-100/phenylmethylsulphonyl fluoride (PMSF) and incubated with shaking at 200 r.p.m. for 16 h at 37°C. The Triton X-100 extracts were centrifuged at 10 000 g for 1 h, and the supernatants were sterilized using 0·2-µm membrane filters. The supernatants were then concentrated by ultrafiltration with dialysis tubing (Sigma). The concentrated extract was precipitated with ammonium sulphate (10–90% saturation), and precondensation with Triton X-114 was performed by repeated dilution and phase separation. The resultant aqueous phase (8·5 ml) was cleansed by the repeated addition of 25% Triton X-114 to a final concentration of 2%. The purified TSP antigen was stored in sterile aliquots at 80°C. The endotoxin content, as measured by the Limulus amebocyte lysate assay, was below 3·5 pg/ml in all of the antigen preparations.

Isolation and cultivation of human MDMs

Venous blood was drawn from donor subject into sterile blood collection tubes, and peripheral blood mononuclear cells (PBMCs) were isolated by density sedimentation over Histopaque-1077 (Sigma). The cells were incubated for 1 h at 37°C, and non-adherent cells were removed by pipetting off the supernatant. Adherent monocytes were collected as described previously [13]. The recovered cells were > 95% CD14+ cells, as determined by flow cytometry with an anti-CD14 antibody. The cells were then incubated at 37°C in a humidified 5% CO2 atmosphere until they were used in the experiments. Human MDMs were prepared by culturing peripheral blood monocytes for 5–7 days in the presence of 50 ng/ml human macrophage colony-stimulating factor (Sigma). To show that the stimulatory capacity of mycobacteria was not the result of contamination with LPS, experiments were performed that added the specific LPS-inhibiting oligopeptide polymyxin B (10·0 µg/ml) before mycobacterial stimulation. The study was approved by the bioethics committee of Chungnam University Hospital's review board overseeing studies on samples from human subjects, and all the participants gave their written consent.

Inhibitors and antibodies

Specific inhibitors of MEK (PD98059 and U0126) and PI 3-K (wortmannin and LY294002) were purchased from Calbiochem (San Diego, CA, USA). Dimethyl sulphoxide (DMSO; Sigma) was added to cultures at 0·1% (v/v) as a solvent control. MDMs were washed with phosphate-buffered saline (PBS) and pretreated with inhibitors in RPMI-1640 medium containing glutamine for 45 min before stimulation with antigens. An assessment using Trypan blue exclusion indicated that cell viability was not affected by the presence of the inhibitors (data not shown). Neutralizing rat anti-human TNF-α antibodies (IgG1; clone 1825), the appropriate IgG1 isotype control (IC; clone 11711) antibodies and the recombinant human TNF-α (rhTNF-α) were all purchased from R&D Systems (Minneapolis, MN, USA). LPS (Sigma) were used as positive controls for antigen stimulation in this study.

Determination of Akt and ERK 1/2 phosphorylation

A total of 4 × 105 human MDMs (at a concentration of 8 × 105 per ml) were treated with mycobacteria or TSP antigen for the indicated time-points. Cell lysates were prepared, and Western blot analysis was performed with specific primary antibodies [ERK1/2, phospho-ERK1/2, phospho-Akt (Thr308), Akt (Ser473), p38 and phospho-p38]. All the antibodies were purchased from Cell Signalling Technology. The membranes were developed using a chemiluminescence assay (ECL; Pharmacia-Amersham, Freiburg, Germany) and were subsequently exposed to chemiluminescence film (Pharmacia-Amersham).

Enzyme-linked immunosorbent assay (ELISA)

A sandwich ELISA was used for detecting IL-12 p70 and TNF-α (BD Biosciences) in culture supernatants, as described previously [19]. The 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 pg or ng per ml. The difference between duplicate wells was consistently less than 10% of the mean.

Reverse transcriptase-polymerase chain reaction (RT-PCR)

Human MDMs (1 × 106) were pretreated with either medium or inhibitors for 45 min in complete RPMI-1640 using a 24-well plate. The cells were then stimulated with M. tuberculosis H37Rv (MOI = 1) or TSP antigen (5·0 µg/ml) for 6 h. RNA was extracted from the cells using TRIzol (Invitrogen). Complementary DNA (cDNA) was reverse transcribed from 2 µg of total RNA using SuperScript II reverse transcriptase (Invitrogen) and oligo-dT-3′ primer in a total volume of 20 µl. Using PCR, 2 µl of cDNA was amplified. The primers and PCR conditions were as described previously [20]. The PCR products were resolved on 1% agarose gels and were stained with ethidium bromide.

Statistical analysis

The data obtained from independent experiments are presented as the mean ± s.d. Data were analysed using a paired t-test with a Bonferroni adjustment or anova for multiple comparisons and differences were considered significant at P < 0·05.

Results

M. tuberculosis H37Rv and TSP antigen strongly induced Akt and ERK 1/2 phosphorylation

We have previously found a rapid phosphorylation of ERK 1/2 in human monocytes infected with M. tuberculosis H37Rv [13]. To investigate the activation of the PI 3-K and ERK 1/2 pathways, human MDMs were treated with M. tuberculosis H37Rv (MOI = 1) or TSP antigen (5 µg/ml), and the phosphorylation profiles of Akt and ERK 1/2 were analysed (Fig. 1). As shown in Fig. 1a, M. tuberculosis or TSP induced the phosphorylation of Akt within 15 min of stimulation, and the peak activation of Akt occurred within 15 min of stimulation with M. tuberculosis H37Rv or TSP, although the time range depended on the cell donor. Akt phosphorylation was detected soon after the M. tuberculosis H37Rv or TSP was added to the MDMs, but the level of phosphorylation decreased after 4 h, with the timing of this response also being donor-dependent.

Fig. 1.

Fig. 1

Open in a new tab

Mycobacterium tuberculosis H37Rv- or Triton X-114 solubilized protein (TSP)-mediated phosphorylation of Akt and extracellular-regulated kinase (ERK) 1/2 in human monocyte-derived macrophages (MDMs). Human MDMs were stimulated by M. tuberculosis H37Rv (MOI = 1) or TSP antigen (5·0 µg/ml) for the times indicated. The cells were lysed, and aliquots of total cell lysates were separated by sodium dodecyl polyacrylamide gel electrophoresis (SDS-PAGE) and immunoblotted as described. The blots were incubated overnight with specific antiphospho-Ser473-Akt (p-Akt) or total Akt proteins (a) and antiphospho-ERK1/2 (p-ERK) or total ERK proteins (b) followed by appropriate peroxidase-coupled secondary reagents and were visualized by chemiluminescence assay (ECL). A representative experiment of five independent replicates with similar results is shown. M, media only.

We examined the ERK 1/2 activation in response to M. tuberculosis or the TSP of M. tuberculosis. As shown in Fig. 1b. M. tuberculosis or TSP led to strong phosphorylation of ERK 1/2 within 15 min after stimulation. The peak ERK 1/2 activation was always observed within 30–60 min of stimulation with M. tuberculosis or TSP in MDMs, but the timing was donor-dependent. The total inhibition of Akt or ERK 1/2 phosphorylation by their respective inhibitors at 30 min after the addition of M. tuberculosis or TSP was confirmed on Western blots (data not shown). These data indicate that the treatment of human MDMs with M. tuberculosis or TSP activates the PI 3-K/Akt and ERK pathways.

PI 3-K/Akt pathway negatively regulates M. tuberculosis- or TSP-induced IL-12 mRNA and protein production

Subsequent studies focused on the signal transduction pathways that govern the M. tuberculosis-induced expression of IL-12 and TNF-α. The peak IL-12 p40 and p35 mRNA expression, and the protein synthesis for IL-12 p70 were found at 6 h and 18 h, respectively, after stimulation with TSP or M. tuberculosis(Fig. 2). An earlier pattern of mRNA and protein expression for TNF-α was found in M. tuberculosis or TSP-induced MDMs, compared with that of IL-12 expression. We selected 6 and 18 h for the mRNA and protein expression times, respectively, in further experiments.

Fig. 2.

Fig. 2

Open in a new tab

Kinetics of Mycobacterium tuberculosis H37Rv- or Triton X-114 solubilized protein (TSP)-induced interleukin (IL)-12 expression. Human monocyte-derived macrophages (MDMs) were incubated with M. tuberculosis H37Rv (MOI = 1) or TSP (5·0 µg/ml). (a) Supernatants were harvested after the times indicated, and IL-12 p70 and tumour necrosis factor (TNF)-α formation was measured using by enzyme-linked immunosorbent assay (ELISA). Data are presented as the mean ± s.d. of five independent experiments performed in duplicate. (b) Total RNA was purified, and reverse transcription-polymerase chain reaction (RT-PCR) analysis of IL-12 p40 and p35 was performed. A representative gel of three independent replicates with similar results is shown.

To understand the functional roles of the PI 3-K/Akt pathway in IL-12 p40 and p70 secretion in human MDMs induced by M. tuberculosis H37Rv or TSP, the cells were pretreated with a PI 3-K inhibitor (LY294002 or wortmannin) for 45 min before the addition of M. tuberculosis H37Rv or TSP. The inhibitors were added to the cell cultures at a final dimethylsulphoxide (DMSO) concentration of 0·1%, and DMSO was used in the control assays. As shown in Fig. 3a, M. tuberculosis- or TSP-induced bioactive IL-12 p70 formation by human MDMs was increased significantly by LY294002 or wortmannin in a dose-dependent manner. Furthermore, M. tuberculosis- or TSP-induced IL-12 p40 and p35 mRNA production from human MDMs was up-regulated by LY294002 or wortmannin in a dose-dependent manner, although the increases of IL-12 p40 mRNA expression were more pronounced (Fig. 3b, c). The IL-12 expression was not affected by the presence of the inhibitors alone (data not shown). These data indicate that the PI 3-K/Akt pathway negatively regulates IL-12 expression in human MDMs after stimulation with M. tuberculosis H37Rv or TSP.

Fig. 3.

Fig. 3

Open in a new tab

Effects of PI 3-K inhibitors on interleukin (IL)-12 expression. (a) The PI 3-K inhibitor LY294002 (5, 10 or 20 µM) or wortmannin (100, 200 or 300 nM) was added to monocyte-derived macrophages (MDMs) at the concentrations indicated 45 min before adding Mycobacterium tuberculosis H37Rv (MOI = 1) or Triton X-114 solubilized protein (TSP) antigen (5·0 µg/ml). The supernatants were harvested after 18 h for cytokine assessment using enzyme-linked immunosorbent assay (ELISA). All experiments were performed at least five times using cells from different donors, and the qualitative effects described here were reproduced in all individuals (n = 10). One representative experiment performed in triplicate is shown. (b) Cells were preincubated in the presence of LY294002 or wortmannin for 45 min. Six hours after the addition of M. tuberculosis H37Rv (MOI = 1) or TSP antigen (5·0 µg/ml), total RNA was purified, and reverse transcription-polymerase chain reaction (RT-PCR) analysis of IL-12 p40 and p35 was performed. The experiments were performed four times using MDMs from different four donors, and the qualitative effects described here were reproduced in all individuals. (c) Densitometric analysis of data for four donors (means ± s.e.) is shown. The densitometry values for IL-12 p40 and p35 mRNA levels were normalized to the β-actin levels. Significant differences (*P < 0·05; **P < 0·01; ***P < 0·001) compared with M. tuberculosis or TSP-treated cultures. M, media only.

ERK 1/2 pathway negatively regulates M. tuberculosis- or TSP-induced IL-12 p40 and p70 production in human MDMs

To understand further the functional roles of the ERK 1/2 pathway in the activation of IL-12 in human MDMs induced by M. tuberculosis H37Rv or TSP, we added highly specific inhibitors of ERK 1/2 and assayed cytokine formation. The cells were pretreated with a MEK inhibitor (PD98059 or U0126) for 45 min before the addition of M. tuberculosis H37Rv or TSP. The inhibitors were added to the cell cultures at a final DMSO concentration of 0·1%, and DMSO was used in the control assays.

As in the case with the PI 3-K inhibitors, M. tuberculosis- or TSP-induced IL-12 p70 formation by human MDMs was increased significantly by PD98059 or U0126 in a dose-dependent manner (Fig. 4a). Furthermore, M. tuberculosis- or TSP-induced IL-12 p40 mRNA production from human MDMs was significantly up-regulated by PD98059 or U0126 in a dose-dependent manner (Fig. 4b,c). A slight, but significant, increase of IL-12 p35 mRNA synthesis was also observed by pretreatment with ERK 1/2 inhibitors. The IL-12 expression was not affected by the presence of the inhibitors alone (data not shown). These data indicate that the ERK 1/2 pathway also negatively regulates IL-12 expression in human MDMs after stimulation with M. tuberculosis or TSP.

Fig. 4.

Fig. 4

Open in a new tab

Effects of MEK inhibitors on interleukin (IL)-12 expression. (a) The MEK inhibitor PD98059 (5, 10 or 20 µM) or U0126 (5, 10 or 20 µM) was added to monocyte-derived macrophages (MDMs) at the concentrations indicated 45 min before adding Mycobacterium tuberculosis H37Rv (MOI = 1) or Triton X-114 solubilized protein (TSP) antigen (5·0 µg/ml). The supernatants were harvested after 18 h for cytokine assessment using enzyme-linked immunosorbent assay (ELISA). All experiments were performed at least five times using MDMs from different donors, and the qualitative effects described here were reproduced in all individuals (n = 12). One representative experiment performed in triplicate is shown. (b) Cells were preincubated in the presence of PD98059 or U0126 for 45 min. After 6 h following the addition of M. tuberculosis H37Rv (MOI = 1) or TSP antigen (5·0 µg/ml), total RNA was purified, and reverse transcription-polymerase chain reaction (RT-PCR) analysis of IL-12 p40 and p35 was performed. The experiments were performed four times using MDMs from different four donors, and the qualitative effects described here were reproduced in all individuals. (c) Densitometric analysis of data for four donors (means ± s.e.) is shown. The densitometry values for IL-12 p40 and p35 mRNA levels were normalized to the β-actin levels. Significant differences (*P < 0·05; **P < 0·01; ***P < 0·001) compared with M. tuberculosis or TSP-treated cultures. M, media only.

M. tuberculosis- or TSP-induced TNF-α expression is dependent on the PI 3-K/Akt and ERK 1/2 pathways

To compare the role of the PI 3-K/Akt pathway in M. tuberculosis- or TSP-induced TNF-α production with that in IL-12 secretion, LY294002 or wortmannin was used to pretreat MDMs for 45 min before the addition of M. tuberculosis or TSP to the cultures, and cytokine formation was assayed at 18 h. Figure 5a shows that TNF-α secretion in M. tuberculosis- or TSP-treated MDMs was significantly inhibited by the specific PI 3-K inhibitors in a dose-dependent manner. This inhibitory effect was observed at the transcriptional level, as M. tuberculosis- or TSP-induced TNF-α mRNA production from human MDMs was significantly down-regulated by LY294002 or wortmannin in a dose-dependent manner (Fig. 5B).

Fig. 5.

Fig. 5

Open in a new tab

Effects of PI 3-K or MEK inhibitors on tumour necrosis factor (TNF)-α expression. The PI 3-K inhibitors [LY294002 (5, 10 or 20 µM); wortmannin (100, 200, or 300 nm)] or MEK inhibitors [PD98059 (5, 10 or 20 µM); U0126 (5, 10 or 20 µM)] were added to monocyte-derived macrophages (MDMs) at the concentrations indicated 45 min before adding Mycobacterium tuberculosis H37Rv (MOI = 1) or Triton X-114 solubilized protein (TSP) antigen (5·0 µg/ml). The supernatants were harvested after 18 h for cytokine assessment using enzyme-linked immunosorbent assay (ELISA) (a,c). The total RNA was purified, and reverse transcription-polymerase chain reaction (RT-PCR) analysis was performed. The experiments were performed four times using MDMs from different four donors, and the qualitative effects described here were reproduced in all individuals (b, d). All ELISA experiments were performed at least three times using cells from different donors, and the qualitative effects described here were reproduced in all individuals (n = 10). One representative experiment performed in triplicate is shown. Significant differences (*P < 0·05; **P < 0·01; ***P < 0·001) compared with M. tuberculosis or TSP-treated cultures. M, media only.

We also used the MEK inhibitors PD98059 and U0126 to investigate the possible involvement of ERK 1/2 in the TNF-α production induced by M. tuberculosis H37Rv or TSP antigen. As shown in Fig. 5c,d, M. tuberculosis- or TSP-induced TNF-α formation (protein and mRNA) was significantly inhibited in human MDMs pretreated with specific MEK inhibitors. The TNF-α expression was not affected by the presence of the inhibitors alone (data not shown). These results show that TNF-α formation induced by M. tuberculosis or TSP is essentially regulated by PI 3-K/Akt and ERK 1/2 pathways at the transcriptional and post-transcriptional levels.

Role of TNF-α in PI 3-K or ERK 1/2-mediated enhancement of IL-12 production by M. tuberculosis- or TSP-treated MDMs

The previous studies [21] indicate that secreted TNF-α is able to down-regulate production of IL-12. Thus, we investigated whether enhanced IL-12 production induced by PI 3-K or MEK1 inhibition was the result of suppressed TNF-α production by M. tuberculosis H37Rv-infected monocytes. To test this hypothesis, human MDMs were pretreated with a neutralizing monoclonal antibody (mAb) to TNF-α or rhTNF-α in the presence or absence of LY294002 or U0126 and were then treated with M. tuberculosis H37Rv or TSP antigen.

Infection of MDMs with M. tuberculosis H37Rv that were pretreated with a neutralizing mAb to TNF-α did not induce a significant increase in the levels of IL-12 p40 or p70 in the absence or presence of LY294002 or U0126 compared to cells without treatment with anti-TNF-α (P > 0·05; Fig. 6). With pretreatment of rhTNF-α, M. tuberculosis H37Rv-induced IL-12 p40 levels by MDMs were decreased significantly in the absence (P < 0·01) or presence of LY294002 or U0126 (P < 0·05, for both), compared to those without pretreatment with rhTNF-α (Fig. 6). However, no significant differences were observed in IL-12 p70 levels between cultures pretreated with and without rhTNF-α (Fig. 6). Similar findings were observed in TSP antigen-induced IL-12 production by human MDMs, as in cases with those induced by M. tuberculosis H37Rv (data not shown). Importantly, the pretreatment of MDMs with either LY294002 or U0126 in the presence of an anti-TNF-α mAb or rhTNF-α significantly enhanced IL-12 p70 and p40 levels similar to those seen in the cells in the absence of an anti-TNF-α mAb or rhTNF-α (Fig. 6). These results reveal that the negative regulation of IL-12 levels induced in M. tuberculosis H37Rv-treated MDMs by PI 3-K and ERK 1/2 pathways is not solely attributable to inhibition of TNF-α production.

Fig. 6.

Fig. 6

Open in a new tab

Role of tumour necrosis factor (TNF)-α in PI 3-K or extracellular-regulated kinase (ERK) 1/2-mediated enhancement of interleukin (IL)-12 production by Mycobacterium tuberculosis H37Rv- or TSP-treated monocyte-derived macrophages (MDMs). Human MDMs were preincubated with a neutralizing monoclonal antibody (mAb) to TNF-α (1 µg/ml), IC antibody or rhTNF-α (10 ng/ml) in the presence or absence of LY294002 (10 µM), U0126 (10 µM) or 0·1% dimethylsulphate (DMSO) control for 45 min. MDMs were then treated with M. tuberculosis H37Rv (MOI = 1) or Triton X-114 solubilized protein (TSP) antigen (5·0 µg/ml) for 20 h. Cell-free supernatants were then collected, and the levels of IL-12 p40 and p70 were determined by enzyme-linked immunosorbent assay (ELISA). Significant differences (*P < 0·05; **P < 0·01) compared with cultures preincubated with rhTNF-α. Data are expressed as the mean of two separate experiments using cells from different five donors. M, media only.

PI 3-K inhibition attenuated M. tuberculosis H37Rv- or TSP-induced ERK 1/2 activity

Previous studies [22] have demonstrated that PI 3-K and Akt modulate the activation of ERK1/2. To determine whether the ability of M. tuberculosis to activate PI 3-K regulates the activation of ERK 1/2, human MDMs were pretreated with or without wortmannin or LY294002, stimulated with M. tuberculosis, and then assessed for ERK1/2 phosphorylation. The peak activation of ERK 1/2 was around 30 min in human MDMs with M. tuberculosis, and thus the activation status of the MAPKs was assessed at 30 min. The inhibition of PI 3-K significantly attenuated the M. tuberculosis-induced ERK 1/2 phosphorylation (by 55–70% at 10 µM LY294002) in human MDMs (Fig. 7a). A similar inhibition was found for TSP-induced ERK 1/2 phosphorylation in the presence of wortmannin or LY294002 (data not shown). These results show that PI 3-K activity is required for the M. tuberculosis- or TSP-induced phosphorylation of ERK 1/2 activation.

Fig. 7.

Fig. 7

Open in a new tab

The role of PI 3-K activity in modulating Mycobacterium tuberculosis-induced phosphorylation of extracellular-regulated kinase (ERK) 1/2 and cytokine expression. (a) Human monocyte-derived macrophages (MDMs) were preincubated with wortmannin (WM) or LY294002 (LY) for 45 min before adding M. tuberculosis H37Rv. Whole-cell lysates were prepared at given time-points, and 20 µg of total protein were analysed by immunoblotting using phospho-specific antibodies to Akt/PKB and ERK 1/2. To ensure equal protein loading, the blots were stripped and reprobed with an antibody to total Akt/PKB and ERK 1/2. A representative experiment of three independent replicates with similar results is shown (upper panel). The relative densities of the phospho-ERK1/2 band were analysed by densitometry. All densitometry values were normalized to the total ERK protein. The densitometry values are depicted as means ± s.e. of three independent experiments (lower panel). (b) The PI 3-K inhibitor [LY294002 (10 µM)], a MEK inhibitor [U0126 (10 µM)] or both were added to MDMs at the concentrations indicated 45 min before adding M. tuberculosis H37Rv (MOI = 1) or TSP antigen (5·0 µg/ml). The supernatants were harvested after 18 h for cytokine assessment using enzyme-linked immunosorbent assay (ELISA). Data are expressed as the mean of two separate experiments using cells from different five donors. Significant differences (*P < 0·05; **P < 0·01) compared with cultures pretreated with LY294002 or U0126 alone. M, media only; LY, LY294002; U, U0126.

We also assessed the effect of combination of both ERK1/2 and PI 3-K on IL-12 and TNF-α synthesis. The combined use of MEK1 and PI 3-K inhibitors reduced TNF-α levels to near background levels, whereas it significantly activated IL-12 levels compared to those pretreated with LY294002 or U0126 alone, as shown in Fig. 7b. These results suggest that co-activation of the MEK and PI 3-K pathways can modulate the amplitude of the M. tuberculosis- or TSP-induced IL-12 and TNF-α expression in human MDMs.

Discussion

In this study, we have demonstrated a differential regulation of Th1-driving (IL-12) and proinflammatory (TNF-α) cytokines by PI 3-K and ERK 1/2 pathways in human MDMs in response to M. tuberculosis or TSP. PI 3-K has been shown to regulate a wide range of physiological processes, including movement of organelle membranes, cytoskeletal rearrangement, cell proliferation and apoptosis [23,24]. Recent data suggest that the PI 3-K pathway may play an important role as a regulator that limits proinflammatory responses [25–27]. Fukao and Koyasu [26] have speculated that PI 3-K may be a negative feedback mechanism that prevents excessive innate immune responses. However, little is known about the role of the PI 3-K pathway in response to mycobacteria. We show that the PI 3-K pathway contributes a negative regulation of mycobacteria-induced IL-12 secretions in human macrophages. Our data agree partially with several previous observations by others [28,29] who found that the PI 3-K pathway negatively regulates IL-12 production. Remarkably, Fukao et al. [29] recently demonstrated the PI 3-K-mediated negative feedback regulation of IL-12 production using dendritic cells from PI 3-K–/– mice.

We also found that M. tuberculosis- or TSP-induced IL-12 production is negatively regulated by the ERK 1/2 pathway. These data partially agree with the previous findings that ERK 1/2 suppresses the production of IL-12, whereas p38 MAPK promotes IL-12 production [30]. Xia et al. demonstrated that the inhibition of ERK by PD98059 significantly increases IL-12, but suppresses IL-10, production in activated dendritic cells, suggesting that ERK activation involves the differential production of IL-10 and IL-12. Exogenous IL-10 reversed the up-regulated production of IL-12 induced by PD98059 in activated dendritic cells [31]. However, the IL-10 production was not inhibited by ERK 1/2 inhibitors in M. tuberculosis- or TSP-stimulated MDMs (data not shown). In addition, we previously reported that M. tuberculosis-induced IL-10 production was up-regulated by specific MEK inhibitors in human primary monocytes [13]. The discrepancy between previous studies [31] and our work might be owing to the different cell systems, dendritic cells versus MDMs. Human macrophages and dendritic cells are hypothesized to have different roles in promoting adaptive immune responses during infection with mycobacteria [32]. Thus, a negative regulation of M. tuberculosis- or TSP-induced IL-12 formation by ERK 1/2 inhibitors may not be associated with IL-10 decreases in our system. Taken together, our data suggest that the ERK 1/2 pathway uniquely operates a negative regulation of mycobacteria-induced IL-12 expression by human MDMs.

Contrary to IL-12 regulation, our data show that M. tuberculosis- and TSP-induced TNF-α production are positively regulated by the PI 3-K and ERK 1/2 pathways in human MDMs. Our data support the previous findings of Strassheim et al. that PI 3-K and Akt occupy central roles in inflammatory responses of Toll-like receptor 2-stimulated neutrophils [33]. However, Foey et al. reported that the inhibition of the PI 3-K/Akt pathway resulted in an elevation of TNF-α, whereas the IL-10 level was inhibited, suggesting positive and negative regulation by PI 3-K, respectively [34]. Although previous studies have shown some controversial data on the regulation of IL-10 and TNF-α, our findings demonstrate the functional significance of the PI 3-K/Akt pathway in regulating proinflammatory cytokine production by human MDMs treated with M. tuberculosis. In addition, the data in the present study agree with previous studies of mycobacteria-induced MAPK signalling: TNF-α release is dependent on ERK 1/2 or both p38 and ERK1/2 activation in M. avium-infected murine macrophages [11,12,35] or M. tuberculosis-infected human monocytes [13].

Neither a neutralizing mAb to TNF-α nor rhTNF-α pretreatment significantly affect the secretion of IL-12 p70 in the absence or presence of PI 3-K or MEK inhibitors. Our data agree partially with previous studies that TNF-α selectively inhibits IL-12 p40 steady-state mRNA, but not those of IL-12 p35, IL-1α, IL-1β or IL-6 [36]. This specific inhibitory effect is occurred at the transcriptional level for IL-12 p40 without down-regulation of the IL-12 p35 gene by nuclear run-on analysis [36]. These results reveal that the negative regulation of IL-12 levels induced in M. tuberculosis H37Rv-treated MDMs by PI 3-K and ERK 1/2 pathways is not solely attributable to inhibition of TNF-α production. Overall, our data suggest that PI 3-K or ERK 1/2 inhibition probably enhances IL-12 production via both TNF-α-dependent and -independent mechanisms.

The present studies show that the TSP from M. tuberculosis shows a comparable induction of IL-12 and TNF-α and a similar dependency on the PI 3-K and ERK 1/2 pathways to those by M. tuberculosis. TSP is a new native antigen fraction from M. tuberculosis without massive degradation of proteins by Triton X-100 and fractionated by Triton X-114 phase partitioning. The TSP contained proteins partitioned into the aqueous phase during phase fractionation because non-protein molecules and lipoproteins were recovered in the detergent phase [18]. Previously, we demonstrated significant proliferative responses produced by TSP antigen, which are higher than those produced by purified protein derivatives (PPD), in healthy tuberculin reactors, suggesting the strong T cell immunogenic properties of this antigen [18]. In the present study, we emphasize the usefulness of TSP as a total cell-wall-associated protein antigen of M. tuberculosis, in which lipids, glycoconjugates and lipoproteins have been removed by Triton X-114 partitioning [18]. Further studies should attempt to identify the nature of the unknown or poorly defined antigens in the TSP and their roles in M. tuberculosis infection.

Cross-talk between the PI 3-K/Akt and MEK-ERK pathways has been demonstrated in different cell types because this link was first reported to be involved in the signalling of G-protein coupled receptors [37]. Our studies demonstrate that PI 3-K acts as a required upstream activator for the ERK 1/2 pathway in human MDMs after treatment with M. tuberculosis or TSP. Furthermore, the ability of the PI 3-K/Akt pathway to modulate IL-12 and TNF-α production appears to be mediated by the selective suppression of ERK 1/2 activity, as the MEK1 inhibitors closely mimicked the effects of wortmannin and LY294002 to differentially regulate IL-12 and TNF-α production by M. tuberculosis- or TSP-treated MDMs. In addition, the co-activation of the MEK and PI 3-K pathways significantly modulate the amplitude of the M. tuberculosis- or TSP-induced IL-12 and TNF-α expression in MDMs. Thus, the PI 3-K-Akt-ERK 1/2 axis may affect the differential regulation of key immunoregulatory cytokines that control both Th1-driving and proinflammatory immune responses in human MDMs.

Collectively, our data first demonstrate the functional significance of the PI 3-K/Akt and ERK pathways in regulating Th1-driving IL-12 and proinflammatory cytokine TNF-α production by human macrophages stimulated with M. tuberculosis H37Rv or TSP antigen. We found distinct regulatory patterns of M. tuberculosis H37Rv-induced IL-12 and TNF-α production by PI 3-K/Akt and ERK pathways in human MDMs. In addition, these findings suggest that the signalling pathways underlying the TSP antigen-induced secretion of IL-12 and TNF-α overlap or play a major role in regulating M. tuberculosis H37Rv-induced IL-12 and TNF-α secretion.

Acknowledgments

This work was supported by grant R01-2005-000-10561-0 (2005) from the Basic Research Program of the Korea Science and Engineering Foundation.

References

  • 1.Flynn JL, Chan J. Immunology of tuberculosis. Annu Rev Immunol. 2001;19:93–129. doi: 10.1146/annurev.immunol.19.1.93. [DOI] [PubMed] [Google Scholar]
  • 2.Trinchieri G. Interleukin-12 and the regulation of innate resistance and adaptive immunity. Nat Rev Immunol. 2003;3:133–46. doi: 10.1038/nri1001. [DOI] [PubMed] [Google Scholar]
  • 3.Sano K, Handea K, Tamura G, Shirato K. Ovalbumin (OVA) and Myocbacterium tuberculosis bacilli cooperatively polarize anti-OVA T-helper cells foward a Th1-dominant phenotype and ameliorate murine tracheal eosinophilia. Am J Resp Cell Mol Biol. 1999;20:1260–7. doi: 10.1165/ajrcmb.20.6.3546. [DOI] [PubMed] [Google Scholar]
  • 4.Lowrie DB, Tascon RE, Bonato VLD, et al. Therapy of tuberculosis in mice by DNA vaccination. Nature. 1999;400:269–71. doi: 10.1038/22326. [DOI] [PubMed] [Google Scholar]
  • 5.Schaible UE, Collins HL, Kaufmann SH. Confrontation between intracellular bacteria and the immune system. Adv Immunol. 1999;71:267–377. doi: 10.1016/s0065-2776(08)60405-8. [DOI] [PubMed] [Google Scholar]
  • 6.Bean AG, Roach DR, Briscoe H, et al. Structural deficiencies in granuloma formation in TNF gene-targeted mice underlie the heightened susceptibility to aerosol Mycobacterium tuberculosis infection, which is not compensated for by lymphotoxin. J Immunol. 1999;162:3504–11. [PubMed] [Google Scholar]
  • 7.Maiti D, Bhattacharyya A, Basu J. Lipoarabinomannan from Mycobacterium tuberculosis promotes macrophage survival by phosphorylating Bad through a phosphatidylinositol 3-kinase/Akt pathway. J Biol Chem. 2001;276:329–33. doi: 10.1074/jbc.M002650200. [DOI] [PubMed] [Google Scholar]
  • 8.Chan ED, Morris KR, Belisle JT, et al. Induction of inducible nitric oxide synthase-NO* by lipoarabinomannan of Mycobacterium tuberculosis is mediated by MEK1-ERK, MKK7-JNK, and NF-kappaB signaling pathways. Infect Immun. 2001;69:2001–10. doi: 10.1128/IAI.69.4.2001-2010.2001. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 9.Jones BW, Means TK, Heldwein KA, et al. Different Toll-like receptor agonists induce distinct macrophage responses. J Leukoc Biol. 2001;69:1036–44. [PubMed] [Google Scholar]
  • 10.Schorey JS, Cooper AM. Macrophage signalling upon mycobacterial infection: the MAP kinases lead the way. Cell Microbiol. 2003;5:133–42. doi: 10.1046/j.1462-5822.2003.00263.x. [DOI] [PubMed] [Google Scholar]
  • 11.Bhattacharyya A, Pathak S, Kundu M, Basu J. Mitogen-activated protein kinases regulate Mycobacterium avium-induced tumor necrosis factor-alpha release from macrophages. FEMS Immunol Med Microbiol. 2002;34:73–80. doi: 10.1111/j.1574-695X.2002.tb00605.x. [DOI] [PubMed] [Google Scholar]
  • 12.Roach SK, Schorey JS. Differential regulation of the mitogen-activated protein kinases by pathogenic and nonpathogenic mycobacteria. Infect Immun. 2002;70:3040–52. doi: 10.1128/IAI.70.6.3040-3052.2002. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 13.Song CH, Lee JS, Lee SH, et al. Role of mitogen-activated protein kinase pathways in the production of tumor necrosis factor-alpha, interleukin-10, and monocyte chemotactic protein-1 by Mycobacterium tuberculosis H37Rv-infected human monocytes. J Clin Immunol. 2003;23:194–201. doi: 10.1023/a:1023309928879. [DOI] [PubMed] [Google Scholar]
  • 14.Fruman DA, Meyers RE, Cantley LC. Phosphoinositide kinases. Annu Rev Biochem. 1998;67:481–507. doi: 10.1146/annurev.biochem.67.1.481. [DOI] [PubMed] [Google Scholar]
  • 15.Philpott KL, McCarthy MJ, Klippel A, Rubin LL. Activated phosphatidylinositol 3-kinase and Akt kinase promote survival of superior cervical neurons. J Cell Biol. 1997;139:809–15. doi: 10.1083/jcb.139.3.809. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 16.Darieva Z, Lasunskaia EB, Campos MN, Kipnis TL, Da Silva WD. Activation of phosphatidylinositol 3-kinase and c-Jun-N-terminal kinase cascades enhances NF-kappaB-dependent gene transcription in BCG-stimulated macrophages through promotion of p65/p300 binding. J Leukoc Biol. 2004;75:689–97. doi: 10.1189/jlb.0603280. [DOI] [PubMed] [Google Scholar]
  • 17.Sly LM, Lopez M, Nauseef WM, Reiner NE. 1alpha,25-Dihydroxyvitamin D3-induced monocyte antimycobacterial activity is regulated by phosphatidylinositol 3-kinase and mediated by the NADPH-dependent phagocyte oxidase. J Biol Chem. 2001;276:35482–93. doi: 10.1074/jbc.M102876200. [DOI] [PubMed] [Google Scholar]
  • 18.Kim HJ, Jo EK, Park JK, Lim JH, Min D, Paik TH. Isolation and partial characterisation of the Triton X-100 solubilised protein antigen from Mycobacterium tuberculosis. J Med Microbiol. 1999;48:585–91. doi: 10.1099/00222615-48-6-585. [DOI] [PubMed] [Google Scholar]
  • 19.Lee JS, Song CH, Lim JH, et al. The production of tumour necrosis factor-alpha is decreased in peripheral blood mononuclear cells from multidrug-resistant tuberculosis patients following stimulation with the 30-kDa antigen of Mycobacterium tuberculosis. Clin Exp Immunol. 2003;132:443–9. doi: 10.1046/j.1365-2249.2003.02172.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 20.Song CH, Kim HJ, Park JK, et al. Depressed interleukin-12 (IL-12), but not IL-18, production in response to a 30- or 32-kilodalton mycobacterial antigen in patients with active pulmonary tuberculosis. Infect Immun. 2000;68:4477–84. doi: 10.1128/iai.68.8.4477-4484.2000. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 21.Mueller C, Corazza N, Trachsel-Loseth S, et al. Noncleavable transmembrane mouse tumor necrosis factor-alpha (TNFalpha) mediates effects distinct from those of wild-type TNFalpha in vitro and in vivo. J Biol Chem. 1999;274:38112–8. doi: 10.1074/jbc.274.53.38112. [DOI] [PubMed] [Google Scholar]
  • 22.Hirsch E, Katanaev VL, Garlanda C, et al. Central role for G protein-coupled phosphoinositide 3-kinase gamma in inflammation. Science. 2000;287:1049–53. doi: 10.1126/science.287.5455.1049. [DOI] [PubMed] [Google Scholar]
  • 23.Franke TF, Kaplan DR, Cantley LC. PI3K: downstream AKTion blocks apoptosis. Cell. 1997;88:435–7. doi: 10.1016/s0092-8674(00)81883-8. [DOI] [PubMed] [Google Scholar]
  • 24.Toker A, Cantley LC. Signalling through the lipid products of phosphoinositide-3-OH kinase. Nature. 1997;387:673–6. doi: 10.1038/42648. [DOI] [PubMed] [Google Scholar]
  • 25.Fruman DA, Cantley LC. Phosphoinositide 3-kinase in immunological systems. Semin Immunol. 2002;14:7–18. doi: 10.1006/smim.2001.0337. [DOI] [PubMed] [Google Scholar]
  • 26.Fukao T, Koyasu S. PI3K and negative regulation of TLR signaling. Trends Immunol. 2003;24:358–63. doi: 10.1016/s1471-4906(03)00139-x. [DOI] [PubMed] [Google Scholar]
  • 27.Guha M, Mackman N. The phosphatidylinositol 3-kinase-Akt pathway limits lipopolysaccharide activation of signaling pathways and expression of inflammatory mediators in human monocytic cells. J Biol Chem. 2002;277:32124–32. doi: 10.1074/jbc.M203298200. [DOI] [PubMed] [Google Scholar]
  • 28.Armant M, Avice M-N, Hermann P, et al. CD47 ligation selectively downregulates human interleukin 12 production. J Exp Med. 1999;190:1175–81. doi: 10.1084/jem.190.8.1175. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 29.Fukao T, Tanabe M, Terauchi Y, et al. PI3K-mediated negative feedback regulation of IL-12 production in DCs. Nat Immunol. 2002;3:875–81. doi: 10.1038/ni825. [DOI] [PubMed] [Google Scholar]
  • 30.Feng GJ, Goodridge HS, Harnett MM, et al. Extracellular signal-related kinase (ERK) and p38 mitogen-activated protein (MAP) kinases differentially regulate the lipopolysaccharide-mediated induction of inducible nitric oxide synthase and IL-12 in macrophages: Leishmania phosphoglycans subvert macrophage IL-12 production by targeting ERK MAP kinase. J Immunol. 1999;163:6403–12. [PubMed] [Google Scholar]
  • 31.Xia CQ, Kao KJ. Suppression of interleukin-12 production through endogenously secreted interleukin-10 in activated dendritic cells: involvement of activation of extracellular signal-regulated protein kinase. Scand J Immunol. 2003;58:23–32. doi: 10.1046/j.1365-3083.2003.01268.x. [DOI] [PubMed] [Google Scholar]
  • 32.Krutzik SR, Krutzik SR, Modlin RL. The role of Toll-like receptors in combating mycobacteria. Semin Immunol. 2004;16:35–41. doi: 10.1016/j.smim.2003.10.005. [DOI] [PubMed] [Google Scholar]
  • 33.Strassheim D, Asehnoune K, Park JS, et al. Phosphoinositide 3-kinase and Akt occupy central roles in inflammatory responses of Toll-like receptor 2-stimulated neutrophils. J Immunol. 2004;172:5727–33. doi: 10.4049/jimmunol.172.9.5727. [DOI] [PubMed] [Google Scholar]
  • 34.Foey AD, Feldmann M, Brennan FM. CD40 ligation induces macrophage IL-10 and TNF-alpha production: differential use of the PI3K and p42/44 MAPK-pathways. Cytokine. 2001;16:131–42. doi: 10.1006/cyto.2001.0954. [DOI] [PubMed] [Google Scholar]
  • 35.Reiling N, Blumenthal A, Flad HD, Ernst M, Ehlers S. Mycobacteria-induced TNF-alpha and IL-10 formation by human macrophages is differentially regulated at the level of mitogen-activated protein kinase activity. J Immunol. 2001;167:3339–45. doi: 10.4049/jimmunol.167.6.3339. [DOI] [PubMed] [Google Scholar]
  • 36.Ma X, Sun J, Papasavvas E, et al. Inhibition of IL-12 production in human monocyte-derived macrophages by TNF. J Immunol. 2000;164:1722–9. doi: 10.4049/jimmunol.164.4.1722. [DOI] [PubMed] [Google Scholar]
  • 37.Hawes BE, Luttrell LM, van Biesen T, Lefkowitz RJ. Phosphatidylinositol 3-kinase is an early intermediate in the G beta gamma-mediated mitogen-activated protein kinase signaling pathway. J Biol Chem. 1996;271:12133–6. doi: 10.1074/jbc.271.21.12133. [DOI] [PubMed] [Google Scholar]

Articles from Clinical and Experimental Immunology are provided here courtesy of British Society for Immunology

RESOURCES