A molecular mechanism of action of theophylline: Induction of histone deacetylase activity to decrease inflammatory gene expression

Abstract

The molecular mechanism for the anti-inflammatory action of theophylline is currently unknown, but low-dose theophylline is an effective add-on therapy to corticosteroids in controlling asthma. Corticosteroids act, at least in part, by recruitment of histone deacetylases (HDACs) to the site of active inflammatory gene transcription. They thereby inhibit the acetylation of core histones that is necessary for inflammatory gene transcription. We show both in vitro and in vivo that low-dose theophylline enhances HDAC activity in epithelial cells and macrophages. This increased HDAC activity is then available for corticosteroid recruitment and predicts a cooperative interaction between corticosteroids and theophylline. This mechanism occurs at therapeutic concentrations of theophylline and is dissociated from phosphodiesterase inhibition (the mechanism of bronchodilation) or the blockade of adenosine receptors, which are partially responsible for its side effects. Thus we have shown that low-dose theophylline exerts an anti-asthma effect through increasing activation of HDAC which is subsequently recruited by corticosteroids to suppress inflammatory genes.

Figure 1

Effect of theophylline on HDAC expression and activity in vivo. (a and b Upper) Western blot analysis of HDAC1 (a) and HDAC2 (b) expression in bronchial biopsies from mild asthmatic subjects treated with low-dose theophylline (T) or placebo (P) (20). (a and b Lower) Graphical expression of the effect of low-dose theophylline (T) and placebo (P) on HDAC1 and HDAC2 expression relative to β-actin. (c) Effect of low-dose theophylline (T) and placebo (P) on HDAC activity in bronchial biopsies. Mean values are given by bars. n = 14 for HDAC1, HDAC2, and HDAC activity assays.

Figure 2

Correlation between theophylline actions on HDAC activity and clinical parameters. (a and b) Correlation between changes in HDAC activity induced by theophylline and theophylline-induced changes in PC₂₀ (concentration that provokes a 20% change in FEV) for methacholine (a) and inhibition of sputum eosinophils (b). (c) Correlation between HDAC activity and sputum eosinophils in normal and asthmatic subjects. (d) No correlation between FEV₁ (forced expiratory volume in 1 s) and theophylline-induced HDAC activity. Dotted lines represent 95% confidence limits.

Figure 3

Effect of theophylline on HDAC activity. BAL macrophages were incubated for 12 h with LPS after treatment with theophylline for 10 min or dexamethasone for 30 min. HDAC activity (a) and IL-8 secretion (b) were measured in normal nonsmoking subjects as described in Materials and Methods. Results are expressed as mean ± SEM (n = 5; *, P < 0.05; **, P < 0.01 vs. LPS control; ***, P < 0.05 vs. unstimulated cells.

Figure 4

Theophylline synergizes with dexamethasone through the induction of HDAC activity in A549 cells. (a) Direct effect of theophylline and dexamethasone on HDAC activity in A549 cells. Nuclear proteins containing HDAC activity were isolated from untreated cells and incubated with ³H-labeled histones for 45 min in the presence of theophylline or dexamethasone. (b) Effect of theophylline (T, 10⁻⁵ M) on immunoprecipitated HDAC1, HDAC2, and HDAC3 in A549 cells. Nuclear protein was extracted and immunoprecipitated with anti-HDAC1, anti-HDAC2, and anti-HDAC3 antibody. The immunoprecipitates were stimulated with theophylline for 30 min. HDAC assays were performed and the effect of theophylline on each HDAC was examined. Results are expressed as mean ± SEM (n = 3–5; **, P < 0.01). (c) GM-CSF release into the culture medium of IL-1β-stimulated cells in the presence of theophylline, dexamethasone, or combined treatment was determined by ELISA after 24 h. In additional experiments the effect of pretreatment with TSA (10 ng/ml) on these actions was examined. Results are expressed as mean ± SEM (n = 3–5; *, P < 0.05; **, P < 0.01). (d) Theophylline and dexamethasone in combination inhibit IL-1β-stimulated association of acetylated histone 4 with the GM-CSF promoter. A549 cells pretreated with theophylline (10⁻⁵ M) or dexamethasone (10⁻¹⁰ M or 10⁻⁶ M) for 30 min were incubated with IL-1β (1 ng/ml) for 4 h. Proteins and DNA were crosslinked by formaldehyde treatment, and chromatin pellets were extracted. After sonication, acetylated histone H4 was immunoprecipitated and the associated DNA was amplified by PCR. Results are representative of three independent experiments.

Figure 5

Theophylline effects on HDAC activity are not mediated through inhibition of PDEs in A549 cells. (a) Direct effect of theophylline (Theo, 10⁻¹¹ to 10⁻³ M), the nonspecific PDE inhibitor IBMX (10⁻¹¹ to 10⁻³ μM), the PDE3 inhibitor motapizone (10⁻¹¹ to 10⁻³ M), the PDE4 inhibitor rolipram (10⁻¹¹ to 10⁻³ M), and the adenosine receptor antagonist CGS-15943 (CGS) (10⁻¹¹ to 10⁻³ M) on HDAC activity. Nuclear proteins containing HDAC activity were isolated from untreated cells and incubated with ³H-labeled histones for 45 min. Results are expressed as mean ± SEM (n = 3–5; *, P < 0.05). (b) Direct effect of pH on theophylline (Theo, 10⁻⁵ M) induction of HDAC activity. Nuclear proteins containing HDAC activity were isolated from untreated cells and incubated with ³H-labeled histones for 45 min in differing assay pH conditions. Results are expressed as mean ± SEM (n = 3; *, P < 0.05). (c) Direct effect of inhibition of p38 MAPK (SB203580) and MEK (PD098059) on theophylline (Theo, 10⁻⁵ M) induced HDAC activity. Results are expressed as mean ± SEM (n = 3; *, P < 0.05).