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. 2004 Jul;137(1):81–87. doi: 10.1111/j.1365-2249.2004.02510.x

Differential regulation of mast cell cytokines by both dexamethasone and the p38 mitogen-activated protein kinase (MAPK) inhibitor SB203580

R D KORANTENG *, E J SWINDLE *, B J DAVIS *, R J DEARMAN ‡, I KIMBER ‡, B F FLANAGAN †, J W COLEMAN *
PMCID: PMC1809098  PMID: 15196247

Abstract

Activated mast cells generate multiple cytokines but it is not known if these can be differentially regulated by pharmacological agents. We report here that the glucocorticoid dexamethasone (DEX) preferentially inhibited Ag-induced expression of IL-4 and IL-6 mRNA relative to TNF-α mRNA in RBL-2H3 cells. Likewise, the drug more readily inhibited release of IL-4 than TNF-α protein. SB203580, an inhibitor of p38 mitogen-activated protein kinase (MAPK), enhanced Ag-induced TNF-α mRNA expression without affecting IL-4 or IL-6 mRNA. At the protein level, SB203580 exerted little effect on TNF-α release but inhibited IL-4 release; notably, the ratio of TNF-α : IL-4 increased markedly with the concentration of SB203580, confirming the differential regulation of these cytokines. PD98059, an inhibitor of MAPK kinase (MEK), a component of the p44/42 MAPK pathway, partially inhibited Ag-induced expression of mRNA for all three cytokines while cyclosporin A inhibited Ag-induced IL-4 and IL-6 mRNA more readily than TNF-α mRNA. Ag activation of the cells led to phosphorylation of p38 and p44/42 MAPK but this was not influenced by DEX. In conclusion, mast cell cytokines can be differentially regulated pre- and post-translationally by DEX and SB203580 but there does not appear to be a direct mechanistic link between the actions of these two drugs.

Keywords: mast cell, cytokines, glucocorticoids, dexamethasone, mitogen-activated protein kinase (MAPK) inhibitors

INTRODUCTION

Mast cells are key effectors of IgE-mediated diseases [1,2] and play a role in immunity to bacteria [3,4] and nematodes [5,6]. Upon activation, mast cells express multiple cytokines including interleukin (IL)-4, IL-5, IL-6 and tumour necrosis factor (TNF)-α [7–11]. These products are presumed to play a role in mast cell mediated diseases and immunity. For example, mast cell-derived TNF-α is involved in cutaneous neutrophil recruitment [12], gastric inflammation [13] and bacterial immunity [3]. In order to develop strategies for preventing mast cell-mediated diseases without compromising immunity, it would be desirable to target cytokine-specific pathways. To enable such approaches, it is necessary to determine whether different mast cell cytokines are regulated co-ordinately or independently, and to begin to identify the relevant signalling pathway(s).

Cross-linkage of mast cell surface IgE by Ag triggers a concerted series of early signalling events. Key among these, in sequence, are the phosphorylation of immunoreceptor tyrosine-based activation motifs (ITAMs) located on the β and γ chains of the IgE receptor (FcɛRI), activation of the protein tyrosine kinases Syk, Lyn and Fyn, and through linker and adaptor proteins, activation of phospholipase C and mobilization of calcium ions [14,15]. Further downstream, mitogen activated protein kinases (MAPK) are activated in pathways leading to cytokine expression [16–19].

Glucocorticoids (GC) are potent and highly effective anti-inflammatory agents. In the case of mast cells, GC inhibit Ag-induced expression of cytokines such as IL-5, IL-6, TNF-α and GM-CSF [10,20,21], as well as degranulation and the associated release of prestored mediators [22–24]. FcɛRI-proximal signalling events, such as phosphorylation of receptor subunits, Lyn, Syk, or the Ras-guanine nucleotide exchange factor Vav, are not influenced by the GC dexamethasone (DEX). Rather, DEX reportedly inhibits phosphorylation of downstream components of the p44/42 MAPK (Erk1/2) pathway, namely Raf, MEK and p44/42 MAPK itself [25], the latter by increasing expression and decreasing degradation of MAPK phosphatase-1 [26]. Our current aim was to examine further the possible links between GC, MAPK and cytokine expression in mast cells. Specifically we addressed the following two related questions:

  • Are different mast cell cytokines co-ordinately or independently regulated by DEX and MAPK inhibitors?

  • are DEX effects on mast cells mimicked by MAPK inhibitors?

The findings reveal that Ag-induced mast cell expression of IL-4/IL-6 and TNF-α mRNA, release of IL-4 and TNF-α protein, and degranulation are differentially susceptible to both DEX and MAPK inhibitors, and that the actions of GC are not matched by MAPK inhibitors.

MATERIALS AND METHODS

Cells, culture conditions and activation

RBL-2H3 mast cells were grown to confluence in complete RPMI-1640 medium (cRPMI; Gibco Life Technologies, Paisley, UK) containing 10% foetal calf serum and 2·0 mm l-glutamine. For experimental use, cells were typically seeded at 1–2 × 106/well in a 6-well culture plate or 105/well in a 96-well culture plate. Cells were sensitized with 50 ng/ml of mouse monoclonal IgE anti-dinitrophenyl (DNP) (Sigma-Aldrich, Poole, UK) for 18 h before addition of DEX (Sigma-Aldrich), the p38 MAPK inhibitor SB203580 (Calbiochem, CN Biosciences, Nottingham, UK), the MEK inhibitor PD98059 (Calbiochem), or cyclosporin A (Sigma-Aldrich) over a range of concentrations and for various time periods before challenge with 10 ng/ml of DNP30-HSA Ag (Sigma-Aldrich).

Serotonin release assay

[3H]-Serotonin (5-[1,2-N-3H]hydroxytryptamine creatinine sulphate; sp. act. 27 Ci/mmol; DuPont-NEN, Dreiech, Germany) (1·0 µCi/ml) was added to RBL-2H3 cells for 3 h prior to addition of chemical agents as above. The cells were washed three times to remove unincorporated radioactivity, suspended (106/ml) in cRPMI, and added (100 µl) in duplicate to 100 µl of Ag, cRPMI (background control) or 0·05% Triton X-100 detergent (Sigma-Aldrich) for 30 min at 37°C in 96-well round bottomed plates. The plates were then centrifuged at 400 g for 2 min and 100 µl of supernatant medium removed for scintillation counting. Release of [3H]serotonin was calculated as percent of cell total and background values subtracted.

Cytokine mRNA and protein measurement

Cytokine mRNA was measured by RNase protection assay (incorporating probes for IL-1α, IL-1β, IL-2, IL-3, IL-4, IL-5, IL-6, IL-10, TNF-α, TNF-β, IFN-γ, GAPDH and L32) according to the supplier's instructions (RiboQuant rCK-1 kit, BD Biosciences, Oxford, UK). Briefly, at appropriate times after Ag challenge, RBL-2H3 cells (2·0 × 106) were solubilized in TRI reagent (1·0 ml; Sigma-Aldrich) and RNA (10–20 µg) extracted and hybridized to 32P radiolabelled antisense RNA probes at 56°C overnight. The samples were digested with RNase A and T1 (BD Biosciences) to remove single stranded RNA and the protected fragments purified by phenol/chloroform extraction and ethanol precipitation and loaded onto a 6% polyacrylamide urea gel, run at 38 mA in 0·5 × Tris-borate EDTA buffer alongside the undigested probes as size markers. Gels were exposed overnight to X-ray film and densitometric analysis carried out using the ‘Image’ programme (National Institutes of Health, USA).

Cytokines in cell supernatants harvested 20 h after Ag challenge were measured by ELISA (OptEIA rat IL-4 set, BD Biosciences; Cytoscreen rat TNF-α kit, Biosource International, Camarillo, CA, USA).

Western blots

RBL-2H3 cells were cultured (106/well) in 6-well culture plates, sensitized with IgE anti-DNP, incubated with drugs as appropriate, and challenged with Ag as described above. Five minutes after challenge the supernatant medium was aspirated and 250 µl of cell lysis buffer (1% Triton X-100 in Tris–buffered saline containing 5 mm EDTA, 1 mm DTT, 2 mm phenylmethyl sulphonyl fluoride and protease inhibitor cocktail; all from Sigma-Aldrich) at 100°C was added instantly to the adherent cells. The cell extracts were boiled for 3 min and electrophoresed on 4–12% SDS-polyacrylamine gels. The separated proteins were transferred to nitrocellulose membranes and the blots probed with rabbit antibodies to phosphorylated p38 MAPK (Thr180/Tyr182) or p44/42 MAPK (Thr202,Tyr204) (from Cell Signalling Technology, Beverley, MA, USA) followed by HRP-linked donkey anti-rabbit Ig (Amersham Biosciences, Piskataway, NJ, USA), chemiluminescent reagent (Western Lightening, PerkinElmer Life Science, Boston, MA, USA) and exposure to X-ray film.

RESULTS

Profiles of Ag-induced cytokine mRNA expression

Challenge of IgE-sensitized RBL-2H3 cells with Ag led to a time-dependent induction of mRNA for multiple cytokines (Fig. 1). IL-4, IL-6 and TNF-α mRNA were expressed constitutively at low levels in resting cells and levels increased substantially following Ag challenge. Induced expression of mRNA for several other cytokines, including IL-3, IL-5, IL-10, IL-2 and IFN-γ was also detected but at relatively low levels (Fig. 1). No resting or induced mRNA for IL-1α, IL-1β or TNF-β was detected (same probe set but not indicated in Fig. 1). IL-4, IL-6 and TNF-α were consistently the most strongly induced species of mRNA: each was up-regulated within 30 min and reached peak levels at 2–4 h post Ag challenge (Fig. 1). In subsequent experiments 2 h was selected as the routine time point for extraction of RNA.

Fig. 1.

Fig. 1

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Time course of cytokine mRNA expression by RBL-2H3 cells. IgE-sensitized cells were challenged with Ag and RNA was extracted at various time points for analysis by RNase protection. Result is representative of three experiments. Lower film exposure times confirmed constancy of L32 and GAPDH mRNA levels.

Effect of DEX on cytokine mRNA expression and serotonin release

Prior incubation of RBL-2H3 cells for 4 h with DEX resulted in a concentration-dependent and strong inhibition of Ag-induced IL-4 and IL-6 mRNA but did not inhibit Ag-induced TNF-α mRNA to a significant extent (Fig. 2a). The effect of DEX on cytokine expression was time dependent (Fig. 2b): when added to the cells simultaneously with Ag (time 0), the drug (at 10 nm) reduced Ag-induced IL-4 and IL-6 mRNA expression by 50–60%; longer incubation times progressively increased the effect such that 85–100% inhibition of IL-4 and IL-6 mRNA levels was reached at 4–24 h. Again, at each time point DEX produced only a modest effect on TNF-α mRNA expression which did not reach statistical significance (Fig. 2b).

Fig. 2.

Fig. 2

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Effect of DEX on Ag-induced cytokine mRNA expression and serotonin release. (a) Concentration-dependent effect of DEX (added for 4 h) on Ag-induced cytokine mRNA. (b) Time-dependence of the effect of DEX (10 nm) on Ag-induced cytokine mRNA. (c) Concentration-dependent effect of DEX (added for 4 h) on Ag-induced serotonin release. (d) Time-dependence of the effect of DEX (10 nm) on Ag-induced serotonin release. Results are means ± SEM for 4 experiments. All statistical analyses were by 2-way anova followed by Dunnett's test for multiple comparisons with no-drug control. *P < 0·05.

Pre-incubation of the cells for 4 h with DEX reduced Ag-induced serotonin release by up to 50% (Fig. 2c). By comparison with its effects on cytokine mRNA expression, DEX required longer incubation times (at least 4 h) with the cells to significantly inhibit degranulation (Fig. 2d). At 24 h, 10 nm DEX inhibited Ag-induced serotonin release by 85% (Fig. 2d).

Effects of MAPK inhibitors on cytokine mRNA expression and serotonin release

SB203580, added to RBL-2H3 cells for 1 h at concentrations up to 10 µm (IC50 = 0·5 µm), was without significant effect on Ag-induced expression of IL-4 and IL-6 mRNA (Fig. 3a) but enhanced Ag-induced expression of TNF-α mRNA to 170% of control values (Fig. 3a). SB203580 (1 or 24 h) was without effect on serotonin release (Fig. 3b).

Fig. 3.

Fig. 3

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Effects of MAPK inhibitors on Ag-induced cytokine mRNA expression and serotonin release. RBL-2H3 cells were preincubated with (a, b) SB203580 or (c, d) PD98059 for 1 h before Ag challenge. (a, c) Cytokine mRNA levels 2 h postchallenge. (b, d) Serotonin release 30 min postchallenge. Results are means ± SEM for five (a, c) or three (b, d) experiments. Statistical analyses were by 2-way anova followed by Dunnett's test for multiple comparisons with no-drug control. *P < 0·05.

PD98059 inhibited Ag-induced cytokine mRNA expression but this only reached statistical significance for TNF-α at a drug concentration of 30 µm (Fig. 3c). PD98059 (1 or 24 h) did not affect Ag-induced serotonin release (Fig. 3d).

Effects of DEX and SB203580 on cytokine protein release

DEX at concentrations of 1 nm or above almost completely inhibited Ag-induced IL-4 release while inhibiting TNF-α release by <50% (Fig. 4a). These cytokines were also differentially regulated by SB203580 (Fig. 4b); at low concentrations (1 µm) the drug inhibited TNF-α release by 25% but responses recovered at higher drug concentrations. Over the same range SB203580 concentration-dependently inhibited IL-4 release (Fig. 4b). This differential regulation of TNF-α and IL-4 release is best illustrated by plotting the ratio of released TNF-α : IL-4 protein against concentration of SB203580. As can be seen in Fig. 4c, this ratio increased markedly with increase in drug concentration.

Fig. 4.

Fig. 4

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Effects of DEX and SB203580 on Ag-induced IL-4 and TNF-α release. RBL-2H3 cells were incubated with DEX for 4 h or SB203580 for 1 h prior to Ag challenge. Supernatant cytokine levels were determined 20 h after Ag challenge. Results are means ± SD for triplicate assays. Similar results were obtained in each of three experiments.

Ag-induced phosphorylation of p38 and p44/42 MAPK and the effects of SB203580 and DEX

Activation of IgE-sensitized RBL-2H3 cells with Ag led to elevated levels of phosphorylated (P-)p38 and P-p44/42 MAPK (Fig. 5a). SB203580 reduced Ag-induced levels of P-p38 but not P-p44/42 MAPK confirming its selectivity for the former (Fig. 5b). The reduction in levels of Ag-induced P-p38 MAPK occurred progressively over the drug concentration range 0·03–10 µm (Fig. 5b). DEX was without effect on Ag-induced levels of P-p38 and P-p44/42 MAPK (Fig. 5C).

Fig. 5.

Fig. 5

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Western blot analysis of phosphorylated p38 and p44/42 MAPK. RBL-2H3 cells were challenged with Ag or control medium and proteins extracted 5 min later. (a) Ag activated vs. unstimulated (U) cells, duplicate samples. (b) Unstimulated cells (U) and cells stimulated with Ag alone (Ag) or Ag after incubation for 1 h with a range of concentrations of SB203580 as indicated. (c) Unstimulated cells (U) and cells stimulated with Ag alone (Ag) or Ag after incubation for 4 h with a range of concentrations of DEX as indicated.

Effect of cyclosporin on cytokine mRNA expression and serotonin release

Cyclosporin (1 h at 100 nm) approximately halved Ag-induced serotonin release (Fig. 6a) and TNF-α mRNA expression (Fig. 6b) while inhibiting Ag-induced IL-4 and IL-6 mRNA levels by 80–90% (Fig. 6b).

Fig. 6.

Fig. 6

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Effect of cyclosporin on Ag-induced cytokine mRNA expression and serotonin release. Cells were incubated with or without 10−7m cyclosporin for 1 h before Ag challenge. (a) Serotonin release 30 min postchallenge. (b) Cytokine mRNA levels 2 h postchallenge. Results are means ± SEM for four experiments. *P < 0·05 by 2-tailed paired Student's t-test.

DISCUSSION

GC are potent anti-inflammatory agents with wide-ranging effects [27,28]. In mast cells GC inhibit Ag-induced expression of cytokines [10,20,21] as well as degranulation and release of stored mediators [22–24]. In the present study we examined whether certain cytokines induced in RBL-2H3 mast cells might be differentially regulated by DEX, and, if so, whether inhibitors of MAPK produce similar profiles. We found that DEX at nanomolar concentrations almost completely blocked Ag-induced expression of IL-4 and IL-6 mRNA, while TNF-α mRNA levels were relatively unaffected. In parallel, DEX more strongly inhibited IL-4 than TNF-α protein release. DEX inhibited Ag-induced release of serotonin, a marker of degranulation, by up to 85%, but required longer incubation periods (up to 24 h) than those necessary for inhibition of cytokine expression. These results reveal that mast cell signalling cascades leading to IL-4 and IL-6 expression and degranulation are more sensitive to GC-regulated components than pathways leading to TNF-α expression. Indeed, the similarity between the concentration- and time-dependence of the effects of DEX on IL-4 and IL-6 expression are consistent with a common mechanism of GC regulation for these two cytokines. The fact that considerably longer incubation times are required for DEX to inhibit serotonin release suggests a different molecular target for GC in the coupling pathway leading to degranulation.

A number of downstream (postphospholipase C/calcium) components have been identified in the Ag-driven mast cell signalling cascade leading to cytokine expression including p44/42 MAPK, which is induced by the upstream elements Ras, Raf and MAPK kinase (MEK), and p38 MAPK [21,29]. Further downstream, the transcription factors nuclear factor of activated T cells (NF-AT) [30] and nuclear factor kappa B (NF-κB) [31,32] are involved in IL-4 and TNF-α production, respectively, and STAT6 in IL-6 and TNF-α production [33]. In the present study we confirmed that Ag stimulated phosphorylation of p38 and p44/42 MAPK but were unable to verify reports [26,34] that this is inhibited by DEX. We found that the p38 MAPK inhibitor SB203580 was without appreciable effect on Ag-induced IL-4 and IL-6 mRNA expression, but enhanced TNF-α mRNA levels in RBL-2H3 cells. At the post-translational level SB203580 produced a dual effect on TNF-α protein release (inhibition at low concentrations followed by recovery of release at concentrations above 1 µm) and inhibited IL-4 release. Nevertheless, with increasing concentration of SB203580 the ratio of TNF-α:IL-4 increased dramatically, confirming the differential regulation of these cytokines. In fact Zhang et al. [18] reported that SB203580 enhances Ag-induced release of TNF-α protein by RBL-2H3 cells, correlating with our finding that the drug, at least at higher concentrations, enhances TNF-α mRNA. In our hands the SB203580-induced elevation of TNF-α mRNA was not reflected as increased release of TNF-α protein, presumably reflecting some compensatory post-translational process. Nevertheless, at both pre- and post-translational levels the ratio of Ag-induced TNF-α: IL-4 increased with concentration of SB203580, indicating differential expression. Our results showing that the MEK inhibitor PD98059 inhibited cytokine induction are consistent with those reported previously [18,19], but unlike SB203580, PD98059 did not show cytokine selectivity.

We observed that SB203580 inhibited Ag-induced phosphorylation of p38 MAPK in BMMC. This is perhaps surprising since SB203580 is thought to inhibit the catalytic activity of activated p38 MAPK; we suggest that p38 MAPK may possess some auto-phosphorylative activity in mast cells.

In addressing the question of whether the actions of GC on mast cells could be explained by targeting of MAPK pathways, we found that DEX was without effect on Ag-induced levels of phosphorylated p38 or p44/42 MAPK. Furthermore, DEX and SB203580 each produced a distinctive profile of regulation of Ag-induced IL-4/IL-6, TNF-α and degranulation, suggesting that these agents act by unrelated mechanisms. In fact, cyclosporin, an inhibitor of NF-AT translocation, inhibited Ag-induced IL-4 and IL-6 mRNA more strongly than TNF-α mRNA, and also inhibited degranulation, indicating it is closer in pharmacological profile to DEX than either SB203580 or PD98059.

DEX and cyclosporin both inhibited Ag-induced serotonin release. Thus elements of the degranulation signalling cascade are GC- and NF-AT-dependent, possibly reflecting regulation at the gene level in resting, i.e. non-Ag-activated cells. That degranulation was not influenced by SB203580 or PD98059 confirms that these MAPK represent components of signalling leading to cytokine expression rather than degranulation.

In conclusion, we have shown that mast cell cytokines, at both pre- and post-translational levels, show distinctive profiles of regulation by DEX and SB203580. Furthermore, DEX did not influence Ag-induced levels of P-p38 and P-p44/42 MAPK. These studies appear to show that, in mast cells, cytokines can be individually regulated and DEX acts independently of MAPK.

Acknowledgments

This work was funded by The Wellcome Trust. RDK was supported by a BBSRC studentship.

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