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. 2001 Jul;125(1):64–70. doi: 10.1046/j.1365-2249.2001.01574.x

Inhibition of p38 MAP kinase during cellular activation results in IFN-γ-dependent augmentation of IL-12 production by human monocytes/macrophages

J B Marriott 1, I A Clarke 1, A G Dalgleish 1
PMCID: PMC1906092  PMID: 11472427

Abstract

Interleukin-12 (IL-12) is a key immunomodulatory cytokine produced by antigen-presenting cells that promotes cellular immunity and enables the generation of protective immunity against intracellular pathogens and tumours. Therefore, modulation of IL-12 activity is a primary immunotherapeutic goal. However, little is known about its regulation. Signalling via p38 MAPK has been implicated in the control of inflammatory responses and is therefore a potential therapeutic target. We have used the highly selective p38 MAPK inhibitor (SB203580) to examine the effect of this pathway on the production of IL-12. Surprisingly, we found that SB203580 strongly up-regulated LPS induced IL-12p40 at the protein (intracellular and secreted) and mRNA levels in PBMC cultures. The effect on IL-12 was apparent using both T cell-independent and T cell-dependent stimuli but not in unstimulated cultures, indicating that activation signals are required. Furthermore, the production of IFN-γ by T cells is crucial as production was not increased in LPS-stimulated, purified adherent monocytes/macrophages without the addition of exogenous IFN-γ. These results provide evidence that p38 MAPK has an unexpected suppressive effect on IL-12p40 gene transcription, and suggests interplay between p38 MAPK- and IFN-γ -mediated signals in the regulation of IL-12 production by monocytes/macrophages. Furthermore, the importance of IL-12 as a key immunoregulatory cytokine suggests that the clinical application of pyrinidyl imidazole inhibitors, such as SB203580, may need to be reassessed.

Keywords: monocytes/macrophages, immunoregulation, IL-12, immunomodulators, p38 MAPK

Introduction

Interleukin-12 (IL-12) is produced by antigen-presenting cells via exposure to pathogens and their products (T cell independent), and via APC interaction with activated T cells via CD40/CD40L (T cell dependent). The bioactive heterodimer (p70) acts on T cells and NK cells to promote Th1-type cellular immunity whilst actively suppressing Th2-type immunity. Defects in the production of IL-12 are associated with human disease, including HIV infection [1,2], immunological paralysis following toxic shock [3], allergic reactions [4], recurrent infections [5] and advanced colorectal cancer [6]. In this respect, the administration of recombinant IL-12 has been shown to be efficacious in murine models of cancer [7,8] and infectious disease [9,10], and may also be an effective Th1-inducing adjuvant in the treatment of human disease. Conversely, the overproduction of IL-12 is associated with a range of Th1 mediated autoimmune pathologies.

There is little information concerning the regulation of IL-12 considering its key role in the generation of cellular immune responses, although the expression of p40 appears to regulate the level of bioactive IL-12p70. There is evidence to suggest that NF-κB [11–14] and Ets [12,15] transcription factors are involved in p40 transcription, which appears to be regulated differently to other pro-inflammatory cytokines and requires de novo protein synthesis [16].

The p38 mitogen-activated protein kinase (p38 MAPK) pathway is one of three distinct mammalian MAPK pathways that transduce a variety of extracellular (mainly stressful and inflammatory) signals, and is activated by at least two specific MAPK kinases, MKK-3 and MKK-6 [17,18]. Selective inhibition of this pathway can be achieved using pyrinidyl imidazole compounds, which prevent activation of the downstream effector (MAPK-activating protein kinase-2). The use of these highly specific inhibitors has shown that p38 MAPK is crucial for the production of inflammatory cytokines, such as TNF-α and IFN-γ [19,20]. Thus, there is considerable interest in the development of p38 MAPK inhibitors as immunotherapeutic agents.

We have utilized SB203580 in order to examine the effect of this pathway on the production of IL-12. Surprisingly, we found that the inhibition of p38 MAPK by SB203580 during cell activation of peripheral blood mononuclear cells (PBMC) and whole blood leads to increased levels of IL-12p40 protein and mRNA, an effect that is IFN-γ dependent.

Materials and methods

Preparation of whole blood, PBMC and monocyte cultures

Heparinized venous whole blood was diluted (1:4 for ELISA; 1:1 for PCR) in RPMI-1640 medium (Sigma). PBMC were prepared by density centrifugation of whole blood on ficoll-hypaque (Sigma), washing, and resuspension at 1 × 106/ml in complete RPMI medium +10% fetal calf serum (FCS; Life Techologies, Paisley, UK).

Monocytes/macrophages were purified by adherence of PBMC to plastic tissue culture flasks (Falcon) at 37°C for 2 h. Non-adherent cells were removed, and the remaining cells were washed three times in PBS and resuspended at 1 × 106/ml in complete RPMI medium +10% fetal calf serum. Phenotypic analysis of isolated monocytes/macrophages was performed in order to confirm purity by surface-staining cells with anti-CD14 FITC (clone UCHMI; Serotec, Oxford, UK); an appropriate isotype-matched control was included. Cellular events gated on forward scatter (FSC) versus side scatter (SSC) properties were acquired on a Becton Dickinson FACScan using CellQuest™ software.

Stimulation and treatment of cultures

PBMC and whole blood cultures were stimulated with either lipopolysaccharide (LPS; 1 µg/ml, Sigma, Escherichia coli serotype 0127:B8), Staphylococcus aureus Cowan Strain I (SAC, Pansorbin; 0·01%, Calbiochem) or phytohaemagglutinin (PHA; 1 µg/ml, Murex Diagnostics, Dartford, UK). For intracellular detection of p70, PBMC were first primed with rhIFN-γ (100 U/ml) prior to LPS stimulation. Isolated monocytes were stimulated with LPS ± rhIFN-γ (100 U/ml)

Cultures were incubated in the presence of SB203580 (0·1–5 µm) or the MEKK inhibitor PD98059 (up to 25 µm) (both Calbiochem, Nottingham, UK) dissolved in dimethyl sulphoxide (DMSO, Sigma, Poole, Dorset; <0·04% v/v final concentration of all cultures). Control cultures without inhibitor were incubated in the presence of 0·04% v/v DMSO. All cultures were incubated at 37°C in 5% CO2 for between 4 and 96 h, depending on the stimulus and the assay.

Enzyme-linked immunosorbent assay (ELISA)

Cell-free supernatant fluids were collected by microcentrifugation and stored in aliquots at −70°C until assayed by ELISA. Supernatant fluids were assayed for IL-12p40, IL-12p70, TNF-α, IFN-γ and IL-10, using an assay procedure and reagents (anti-cytokine capture mAb, biotinylated anti-cytokine detecting mAb and recombinant cytokine) provided by Pharmingen (Becton Dickinson, Oxford, UK). In each case, the manufacturers instructions were followed exactly. Standard absorbances (405 nm) of duplicate wells (minus control zero standard) were plotted as absorbance versus concentration. Cytokine levels were calculated and corrected for any dilution factor necessary to ensure that cytokines derived from the various cell types during different stimuli were within the assay range.

Competitive polymerase chain reaction (PCR)

PBMC were prepared from pre-stimulated whole blood cultures and frozen at −70°C in RNA isolator (Genosys, Cambridge, UK). Total RNA was prepared with the inclusion of a step incorporating treatment with RNase-free DNase (Promega, Madison, WI). RNA was reverse-transcribed at 42°C for 1 h using Reverse Transcriptor (R & D Systems, Abingdon, Oxon, UK). Each sample was assayed for β-actin and cytokine mRNAs by competitive PCR. For each PCR amplification, cDNA was co-amplified with a known amount of specific competitor ‘mimic’ fragment (Clontech, Palo Alto, CA) designed to give a product 150 base pairs larger than the target product. For each sample, cDNA was co-amplified in a series of four reactions with a fivefold dilution series of mimic. The products were separated on 1·5% agarose gels, and the relative amount of target and mimic in each reaction was determined by densitometry (UV Products, Cambridge, UK) and plotted to enable the determination of equimolarity and thus, the amount of target in sample. The conditions for amplification were as follows: β-actin, 5 min at 96°C, then 40 cycles of 96°C for 45 s, 60°C for 45 s and 72°C for 45 s; TNF-α, 34 cycles of 94°C for 45 s, 65°C for 45 s and 72°C for 90 s; for IL-12p40, 5 min at 94°C, then 40 cycles of 94°C for 60 s, 58°C for 60 s and 72°C for 60 s.

Flow cytometric analysis

Detection of intracellular IL-12p70 and IL-12p40/p70 was achieved by priming PBMC (at 2 × 106/ml in 15 ml polypropylene tubes) for 2 h with rhIFN-γ (100 U/ml, R & D Systems), then activating with LPS (1 µg/ml) for an additional 22 h in the presence of the protein transport inhibitor, monensin (2 µm; Sigma) ± SB203580 (1 µm). For the determination of LPS-induced p70, priming with IFN-γ is required prior to the addition of LPS, as LPS alone is a poor inducer of p70. Cells were harvested with EDTA (2 mm) in PBS for 15 min at RT, then fixed and permeabilized (at 5 × 105 cells per condition) using the Pharmingen Cytofix/Cytoperm kit, as per manufacturers instructions.

For analysis of monocytes/macrophages within the PBMC population, cells were stained with anti-IL-12p40/p70 FITC (C11·5; Pharmingen) and anti-IL-12p70 PE (20C2; Pharmingen), with appropriate isotype-matched and compensation controls. Upon flow cytometric analysis, monocytes/macrophages were gated according to their FSC versus SSC properties and displayed as two colour dot-plots, with quadrants set according to isotype-matched controls. For each sample, 5000 gated monocytes/macrophages were acquired and analysed.

Analysis of B cells was performed by staining with a cocktail of lineage-specific mAbs (consisting of anti-CD4 PE, clone SK3 BDIS; anti-CD8 PE, clone SK1 BDIS; and anti-CD56 PE, clone MY31 BDIS) and anti-IL-12p40/p70 FITC. Lymphocytes were gated according to their FSC versus SSC properties, and B cells were identified as T cell marker/NK cell marker negative lymphocytes. Appropriate isotype-matched controls were again used to set quadrants in two colour dot-plots, and 10 000 lymphocytes were acquired and analysed per sample.

For the determination of CD40 (monocytes/macrophages) and CD40L (T cells) expression on PBMC, stimulated for 6 h with PHA (1 µg/ml) ± SB203580 (1 µm), cells were surface-stained with anti-CD4 PE (SK3; BDIS) and anti-CD154 FITC (TRAP1; Pharmingen), or anti-CD14 FITC and anti-CD40 PE plus appropriate isotype-matched controls; 5000 CD4+ lymphocytes and 5000 CD14+ monocytes were acquired and analysed as two colour dot-plots, with quadrants set according to isotype-matched controls.

Results

SB203580 strongly augments the production of IL-12 during T cell independent- and T cell-dependent stimulation of PBMC and whole blood cultures

The production of p40 by LPS-stimulated whole blood cultures was strongly up-regulated by SB203580 in a consistent and dose-dependent fashion (Fig. 1). A twofold increase in p40 levels was observed at approximately 0·1–0·2 µm. There was no effect on unstimulated cultures (even in the presence of exogenous IFN-γ), nor was there any effect of PD98059, a specific inhibitor of another MAP kinase (MEK1) on cultures stimulated with LPS (not shown). The effect of SB203580 on p40 was concomitant with a strong, dose-dependent inhibition of TNF-α, IFN-γ and IL-10 protein. The IC50s for these cytokines were approximately 0·1–0·3 µm, in accordance with previously published work with SB203580.

Fig. 1.

Fig. 1

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Effect of SB203580 on the production of p40 (▪), TNF-α (▵), IFN-γ (◊) and IL-10 (○) protein by whole blood cultures stimulated with LPS. Cultures were co-incubated for 48 h in the presence or absence of SB203580 (0–5 µm) as detailed in the methods section. Culture supernatant fluids were assayed by ELISA and results expressed as cytokine levels relative to DMSO controls without SB203580 (denoted as 100%). Control levels were 9548 pg/ml for p40, 1928 pg/ml for TNF-α, 1137pg/ml for IL-10 and 1646 pg/ml for IFN-γ. The data presented are from one experiment using blood from three normal volunteers, and are representative of over five separate experiments. Error bars represent the range of percentage values relative to DMSO controls without SB203580.

SB203580 increased the production of IL-12 during both T cell-dependent (PHA) and T cell-independent (SAC) stimulation of human PBMC/whole blood (Fig. 2 shows production at 48 h). Production of both p40 and p70 was increased by SB203580 (two- to threefold) using both stimuli, and this effect was prolonged during at least 3 days of culture (data not shown). However, in some donors (in which p40 levels were also relatively low), p70 was not detected and induction of IL-12 was not seen.

Fig. 2.

Fig. 2

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Effect of SB203580 on the production of (a) p40 and (b) p70 protein by PBMC stimulated with SAC (white and black bars) and PHA (light grey and dark grey bars). PBMC were co-incubated for 48 h in the presence of SB203580 (1 µm) (black and dark grey) or DMSO in control cultures (white and light grey). The results shown are from PBMC derived from two normal donors and are representative of two (SAC) and three (PHA) separate experiments. Culture supernatant fluids were assayed by ELISA and results expressed as pg/ml.

IFN-γ primed PBMC stimulated with LPS in the presence of SB203580 show augmentation of IL-12p40 and IL-12p70 production from monocytes/macrophages, but not B cells

In order to establish the source of IL-12 production induced by SB203580, we used a method for the detection of intracellular p70 and p40/p70 in PBMC cultures. For p70 detection in this assay, it is necessary to prime PBMC with IFN-γ 2 h prior to stimulation with LPS (as LPS alone is a poor inducer of p70). Figure 3 shows that gated monocytes/macrophages were the exclusive source of p40 and p70 in the PBMC population, and that intracellular levels of both were strongly augmented in the presence of SB203580. There was little (<1%) production of p70 and p40/p70 from B cells, indicating that this is not the source of SB203580-induced IL-12.

Fig. 3.

Fig. 3

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Effect of SB203580 on the production of LPS-induced intracellular p40 and p70 protein by PBMC from two normal donors. In order to enable the detection of p70 protein, PBMC were primed with rhIFN-γ prior to activation with LPS in the presence of monensin. Cells were fixed, permeabilized and then stained with antip40/p70 FITC and antip70 PE. Isotype-matched controls were used to set the quadrants for two colour dot-plot analyses. The dot-plots represent cells stimulated (a) in DMSO control cultures and (b) with SB203580 (1 µm) from each donor. The percentage of p40 (▪)- and p70 (□)-positive cells from the plots are also represented from duplicate samples (±s.e.m.).

SB203580 increased the level of IL-12p40 mRNA in PBMC derived from LPS stimulated whole blood cultures

We generated cDNA from PBMC stimulated with or without SB203580. The level of β-actin transcripts was quantified by competitive PCR, and each sample was then equilibrated prior to the determination of p40 mRNA. Figure 4a shows a fourfold increase in p40 transcripts in the presence of SB203580 (5 µm) compared with LPS alone. This increase closely corresponds to the increase in p40 protein seen in the LPS-stimulated culture supernatant fluids (approximately fivefold), and indicates that SB203580 is able to increase the production of p40 at the mRNA level. In contrast to the effect on p40, the level of TNF-α mRNA was not affected by the addition of SB203580 at the same concentration (Fig. 4b).

Fig. 4.

Fig. 4

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Effect of SB203580 on the LPS-induced expression of (a) p40 and (b) TNF-α by whole blood cultures (▪, DMSO control; □, + 5 µm SB203580) determined by competitive PCR. From each culture condition cDNA was prepared as described in the methods section. From a series of PCR reactions, plots are obtained from ethidium bromide-stained gels showing the ratio of competitor fragment to target versus competitor fragment concentration. Each gel (and graph line) represents a single sample, and each lane on a gel represents one PCR fraction with the highest competitor ‘mimic’ concentration at the left (cDNA is constant), descending in a fivefold reaction series to the right. The target (cytokine) band is represented by the lower band. For each cytokine, cDNA concentration (in attamole) corresponds to the x-value where y = 1. The data presented are representative of two separate experiments.

SB203580-induced augmentation of p40 production by PHA-stimulated PBMC cultures is not due to increased CD40L expression by activated T cells or increased CD40 expression by antigen-presenting cells

We next determined whether the increase in p40 production in PHA-stimulated PBMC in the presence of SB203580 correlates with an increased expression of CD4+ T cell surface CD40L. The expression of CD40L on activated T cells leads to T cell-dependent activation via interaction with monocyte/macrophage CD40. However, surface staining of CD4+ T cells and CD14+ monocytes showed that although PHA induced expression of both molecules, there was no effect of SB203580 on either molecule during PHA stimulation (data not shown).

The ability of SB203580 to augment LPS-induced p40 production by adherent monocytes/macrophages is dependent on IFN-γ produced by non-adherent cells

We tested whether SB203580 was able to enhance IL-12 production in isolated adherent monocytes/macrophages in a similar manner to that shown with PBMC and whole blood cultures. Using PBMC and whole blood cultures, and isolated monocytes/macrophages derived from the same donors, we found that although p40 was still produced, the ability of SB203580 to augment LPS-induced p40 production was not apparent in the isolated monocyte/macrophage population (Fig. 5). SB203580-treated PBMC and whole blood cultures showed a similar degree of p40 augmentation (approximately 2·5-fold and 2·8-fold, respectively), whereas cultures of adherent cells stimulated in exactly the same manner led to only a marginal increase in p40 production (1·09-fold). However, in contrast to the PBMC and whole blood cultures (which contain T cells), these cultures did not produce any detectable IFN-γ (data not shown). When exogenous recombinant IFN-γ was added to the monocyte/macrophage cultures. the ability of these cells to produce increased levels of p40 when treated with SB203580 was totally restored (Fig. 5).

Fig. 5.

Fig. 5

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Differential effect of SB203580 on p40 production by PBMC/whole blood and isolated adherent monocytes/macrophages is reversed by IFN-γ. SB203580 (1 µm) augments LPS-induced p40 production in 48 h PBMC and whole blood cultures but has very little effect on isolated adherent monocytes/macrophages which do not produce detectable IFN-γ. The addition of exogenous rhIFN-γ (100 U/ml) during LPS treatment restores the ability of SB203580 to augment p40 by isolated adherent monocytes/macrophages. Data are from two normal donors, and are representative of two separate experiments. Error bars represent the range of percentage p40 values relative to DMSO control samples without SB203580. Control (100%) values are 1432 pg/ml for whole blood cultures, 5530 pg/ml for PBMC and 4870 pg/ml for adherent cells.

Discussion

In this study, we have used a pharmacological approach which has enabled us to study the effect of the p38 MAPK pathway on normal human cells. SB203580 is known to inhibit p38 MAPK effectively at between 0·1 and 0·3 µm, and the inhibitory effects that we observed on TNF-α, IFN-γ and IL-10 are entirely consistent with this [21]. It should be noted that even highly specific compounds such as SB203580 may have effects on unknown targets at the cellular level. In this respect, there is evidence that SB203580 inhibits T cell proliferation via a p38 MAPK-independent pathway at higher concentrations than we used in this study (>5–10 µm) [22]. However, in our hands, SB203580 at up to 5 µm does not effect PBMC cellular proliferation (unpublished observation). Taken together, our results suggest that the effect on p40 at 0·1–1 µm is most likely to be due to inhibition of p38 MAPK alone.

The effect of SB203580 on p40 mRNA is seen within four hours of LPS activation. This would appear to rule out an effect due to concomitant inhibition of IL-10, a known inhibitor of IL-12 [16]. Specific p38 MAPK inhibitors generally block cytokine synthesis at the translational level. In this respect, we have confirmed previous reports that although SB203580 inhibits LPS-induced TNF-α protein production, it has no effect on TNF-α transcription [23,24].

We have shown that the effect on p40 is apparent using both T cell-dependent and T cell-independent stimuli, and is applicable to the production of heterodimeric p70. We have also shown that monocytes/macrophages, but not B cells, are the source of both p40 and p70 during LPS stimulation of PBMC. The lack of IL-12 production by B cells during these cultures may relate to differential activation pathways as B cells do not express CD14, a key LPS receptor.

We have observed a definite requirement for the presence of IFN-γ during augmentation of IL-12 by SB203580. Even though the production of p40 by isolated human monocytes was at a similar level to that produced by whole blood or PBMC cultures, p40 production was unaffected by SB203580. However, in contrast to whole blood/PBMC cultures, isolated monocyte cultures produce undetectable levels of IFN-γ. We found that on addition of exogenous IFN-γ, the level of p40 produced by isolated monoytes increased to a similar extent to that seen with the PBMC/whole blood cultures from which these cells were originally derived. Furthermore, the addition of exogenous IFN-γ plus SB203580 has no effect on p40 production by unstimulated PBMC, indicating that both cellular activation and IFN-γ signalling are required.

IFN-γ-mediated macrophage activation is required to provide IL-12 critical to the expansion of Th1 T cells during infection with intracellular pathogens [25]. We therefore suggest that during cellular activation of monocytes/macrophages, IFN-γ receptor-mediated activation of the p40 promoter is partially inhibited by signals generated by the activation of p38 MAPK. Although such a signal pathway was not investigated and remains undefined, this would prevent IL-12 overproduction and dampen down an on-going immune response, thereby minimizing the possibility of autoimmunity.

Our findings appear to contrast with those of Lu et al., who used knockout mice deficient in MAPKK3 (Mkk-3) expression [23]. Their work, the first to implicate p38 MAPK in the regulation of IL-12, indicates that production of IL-12 by macrophages requires Mkk-3-driven activation of downstream p38 MAPK. Also, Häcker et al. have reported that murine macrophages do not produce IL-12p40 in the presence of SB202580 [26]. However, it is worth noting that Shafer et al. have shown that although mouse and human p38 MAPK were equivalently sensitive to inhibition by SB203580, IL-4 production by mouse T cells was about 20-fold less sensitive than human T cell IL-4 production to inhibition by this compound [27]. Furthermore, our own studies have shown that the augmentary effect of SB203580 (and also the other p38 inhibitors SB202190 and SB220025; data not shown) on p40 production is also apparent in LPS-stimulated mouse whole blood cultures. Interestingly, the effect in mouse cultures is also less sensitive to p38 inhibition than equivalent human cultures (unpublished observations).

There is other evidence that p38 MAPK inhibitors may promote cellular immunity. For example, SB203580 has been shown to inhibit production of IL-4, but not IL-2, in human CD4+ T cells [27]. Furthermore, ethanol has been shown to both inhibit p38 activation in human mononuclear cells activated by LPS [28], and up-regulate IL-12 production in human monocytes [29,30]. Indeed, increased serum IL-12 levels have been associated with chronic alcoholism [31]. It is therefore possible that the effect of ethanol on IL-12 is due to inhibition of p38 MAPK.

In conclusion, our results suggest that transcriptional regulation of p40 by IFN-γ is influenced by signals generated by the p38 MAPK pathway. They also suggest that activation of p38 MAPK is not required for IL-12 production by human monocytes/macrophages. However, p38 MAPK does appear to have a role in preventing excess IL-12 production induced by IFN-γ-mediated signals. They further suggest that the clinical use of p38 MAPK inhibitors may need to be reassessed, and that caution is merited concerning their use in the treatment of inflammatory and immunopathological disorders in general, and those associated with IL-12 overproduction in particular.

Acknowledgments

The authors are indebted to Dr Steve Goodbourn, Dr Mike Westby, Dr Steve Todryk and Dr Peter Schafer for helpful discussions during the preparation of the manuscript. This work was supported by Celgene Corporation, NJ, USA

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