A molecular mechanism of action of theophylline: Induction of histone deacetylase activity to decrease inflammatory gene expression Kazuhiro Ito, Sam Lim, Gaetano Caramori, Borja Cosio, K. Fan Chung, Ian M. Adcock*, and Peter J. Barnes Thoracic Medicine, Imperial College School of Science, Technology, and Medicine, National Heart and Lung Institute, Dovehouse Street, London SW3 6LY, United Kingdom Edited by Joseph A. Beavo, University of Washington School of Medicine, Seattle, WA, and approved May 1, 2002 (received for review October 18, 2001) The molecular mechanism for the anti-inflammatory action of low-dose theophylline gives a greater improvement in asthma theophylline is currently unknown, but low-dose theophylline is an control, measured as lung function, symptoms, and rescue ␤ effective add-on therapy to corticosteroids in controlling asthma. 2-agonist use, than that achieved by doubling the dose of Corticosteroids act, at least in part, by recruitment of histone inhaled corticosteroid (5, 14, 15). deacetylases (HDACs) to the site of active inflammatory gene The molecular mechanisms for the anti-inflammatory action transcription. They thereby inhibit the acetylation of core histones of theophylline are unclear. The bronchodilator action of the- that is necessary for inflammatory gene transcription. We show ophylline can be explained by the inhibition of phosphodiester- both in vitro and in vivo that low-dose theophylline enhances ases (PDEs) in airway smooth muscle, but this occurs at con- HDAC activity in epithelial cells and macrophages. This increased centrations of Ͼ50 ␮M (16). In addition, the common side HDAC activity is then available for corticosteroid recruitment and effects of theophylline, nausea and vomiting, are probably predicts a cooperative interaction between corticosteroids and because of PDE4 inhibition (13, 17). Another proposed mech- theophylline. This mechanism occurs at therapeutic concentrations anism involves the antagonism of the bronchoconstrictor aden- of theophylline and is dissociated from phosphodiesterase inhibi- osine, which may also account for some of the serious side effects tion (the mechanism of bronchodilation) or the blockade of aden- of theophylline, including cardiac arrhythmias and seizures. osine receptors, which are partially responsible for its side effects. There is also evidence for other anti-inflammatory mechanisms Thus we have shown that low-dose theophylline exerts an anti- that cannot be accounted for by either PDE inhibition or asthma effect through increasing activation of HDAC which is adenosine-receptor antagonism, including the inhibition of nu- subsequently recruited by corticosteroids to suppress inflamma- clear factor ␬B (NF-␬B) (18) and the inhibition of IL-5- and tory genes. granulocyte-macrophage colony stimulating factor (GM-CSF)- induced eosinophil survival (19, 20). macrophages ͉ corticosteroids ͉ histone deacetylation ͉ granulocyte– Acetylation of core histones by coactivator proteins, such as macrophage colony-stimulating factor CREB-binding protein (CBP), facilitates transcription (21). We have recently demonstrated that the activation of GM-CSF by sthma is a chronic inflammatory disease of the airways IL-1␤ results from NF-␬B activation and increased histone Acharacterized by reduced airway patency, which is regulated acetyltransferase (HAT) activity, leading to increased inflam- ␤ by bronchodilators such as -agonists, and by the infiltration of matory gene transcription (22). Corticosteroids inhibit the ex- inflammatory and immune cells, which is treated by corticoste- pression of GM-CSF by reversing the activation of HAT through roids (1). Theophylline has been used in the treatment of asthma the activated glucocorticoid receptor recruiting corepressor for over 70 years, but its use has recently declined, as inhaled proteins that have histone deacetylase (HDAC) activity (22). corticosteroids have become the mainstay of asthma control and ␬ ␤ HDACs then deacetylate the histones acetylated by NF- B inhaled 2-agonists are more effective bronchodilators. Further- activation, thereby suppressing inflammatory gene expression. more, side effects, such as nausea and headaches, commonly Because theophylline affects gene transcription in low concen- occur at previously recommended doses of theophylline. Orig- trations and appears to interact beneficially with corticosteroids, inally, theophylline was used as a bronchodilator and the optimal we studied the effect of theophylline alone, and in combination plasma concentration that gave maximal bronchodilation with with dexamethasone, on histone acetylation and deacetylation in the lowest risk of side effects was found to be 10–20 mg͞liter vitro by using bronchoalveolar lavage (BAL) macrophages and in (55–110 ␮M) (2). bronchial biopsies of asthmatic patients treated with low-dose There is increasing evidence that theophylline has anti- MEDICAL SCIENCES theophylline. inflammatory or immunomodulatory actions in asthma (2). Low Ͻ doses of theophylline, which give a plasma concentration of 5 Materials and Methods mg͞liter, may achieve control of asthma comparable to a low Effect of Theophylline on Clinical Parameters. We examined the dose of inhaled corticosteroids in both children and adults (3, 4). effect of 4 weeks of treatment with low-dose theophylline In asthmatic patients low-dose theophylline reduces eosinophils and other inflammatory markers (5–7), inhibits the eosinophilia (Euphylong, 250 mg twice daily) on HDAC activity in 14 mild induced by an inhaled allergen (8), and reduces the expression stable asthmatics by using a double-blind crossover controlled study. Blood concentrations of theophylline were elevated in of cytokines, such as interleukin (IL)-5 (9). Long-term treatment Ϯ ͞ with theophylline reduces airway hyperresponsiveness to metha- treated subjects (4.3 0.85 mg liter) as compared with placebo choline challenge (10). In addition, in patients with severe asthma who are withdrawn from theophylline, there is a dete- This paper was submitted directly (Track II) to the PNAS office. rioration of asthma control, despite the fact that patients are Abbreviations: HDAC, histone deacetylase; PDE, phosphodiesterase; IL, interleukin; GM- maintained on high does of inhaled corticosteroids (11, 12). CSF, granulocyte–macrophage colony-stimulating factor; HAT, histone acetyltransferase; Several studies have demonstrated an interaction with corti- BAL, bronchoalveolar lavage; LPS, lipopolysaccharide; TSA, trichostatin A; IBMX, 3-isobutyl- costeroid therapy and the steroid-sparing effects of theophylline 1-methylxanthine; MAPK, mitogen-activated protein kinase. (13). In patients with mild and moderate asthma, the addition of *To whom reprint requests should be addressed. E-mail: [email protected]. www.pnas.org͞cgi͞doi͞10.1073͞pnas.132556899 PNAS ͉ June 25, 2002 ͉ vol. 99 ͉ no. 13 ͉ 8921–8926 Downloaded by guest on September 25, 2021 (Ͻ1mg͞liter). The clinical characteristics and effects on eosin- acrylamide gels. Gels were stained with Coomassie brilliant blue, ophilia have been published elsewhere (5). and the core histones (H2A, H2B, H3, and H4) were excised. The radioactivity in extracted core histones was determined by liquid Fiberoptic Bronchoscopy and Isolation of BAL Macrophages. Subjects scintillation counting and normalized to protein level. attended our bronchoscopy suite at 8.30 a.m. after having fasted from midnight and were pretreated with atropine (0.6 mg i.v.) Histone Deacetylation Activity. Radiolabeled histones were pre- ͞ and midazolam (5–10 mg i.v.). Oxygen (3 liters min) was pared from A549 cells after incubation with the HDAC inhibitor administered with nasal prongs throughout the procedure and trichostatin A (TSA), at 100 ng͞ml for 6 h, in the presence of 0.1 oxygen saturation was monitored with a digital oximeter. While mCi͞ml [3H]acetate. Histones were dried and resuspended in the subject received local anesthesia with lidocaine (4%) to the distilled water. Crude HDAC preparations were extracted from upper airways and larynx, a fiberoptic bronchoscope (Olympus total cellular homogenates with Tris-based high-salt buffer (10 BF10, Key-Med, Southall, U.K.) was passed through the nasal mM Tris⅐HCl, pH 8.0͞500 mM NaCl͞0.25 mM EDTA͞10 mM passages into the trachea. BAL was performed from the right middle lobe by using warmed 0.9% NaCl with four successive 2-mercaptoethanol) as reported (25). The crude HDAC prep- aliquots of 60 ml. BAL cells were spun (500 ϫ g; 10 min) and aration or immunoprecipitates were resuspended in the Tris- based low-salt buffer (10 mM Tris⅐HCl, pH 8.0͞20 mM washed twice with Hanks’ buffered salt solution (HBSS) (12). ͞ ͞ Cytospins were prepared and stained with May–Grunwald stain NaCl 0.25 mM EDTA 10 mM 2-mercaptoethanol), and incu- 3 for differential cell counts. Cell viability was assessed by using bated with H-labeled histone for 30 min at 30°C before the ͞ trypan blue exclusion. In some experiments macrophages were reaction was stopped by the addition of1MHCl 0.4 M acetic isolated by plastic adhesion and cells (1 ϫ 106) incubated in acid. Released 3H-labeled acetic acid was extracted by ethyl 24-well plates in the presence of theophylline, dexamethasone, or acetate, and the radioactivity of the supernatant was determined Salmonella enteritidis lipopolysaccharide (LPS; 100 ng͞ml). In by liquid scintillation counting. In some experiments the pH and addition, we studied BAL macrophages isolated from six normal substrate concentrations were altered. Experiments