Theophylline restores histone deacetylase activity and steroid responses in COPD macrophages

Abstract

Chronic obstructive pulmonary disease (COPD) is a common chronic inflammatory disease of the lungs with little or no response to glucocorticoids and a high level of oxidative stress. Histone deacetylase (HDAC) activity is reduced in cells of cigarette smokers, and low concentrations of theophylline can increase HDAC activity. We measured the effect of theophylline on HDAC activity and inflammatory gene expression in alveolar macrophages (AM) from patients with COPD. AM from normal smokers showed a decrease in HDAC activity compared with normal control subjects, and this was further reduced in COPD patients (51% decrease, P < 0.01). COPD AMs also showed increased basal release of IL-8 and TNF-alpha, which was poorly suppressed by dexamethasone. Theophylline induced a sixfold increase in HDAC activity in COPD AM lysates and significantly enhanced dexamethasone suppression of induced IL-8 release, an effect that was blocked by the HDAC inhibitor trichostatin A. Therefore, theophylline might restore steroid responsiveness in COPD patients.

Figure 1.

Effect of dexamethasone on cytokine release from AM. AM from normal subjects, healthy smokers, and patients with COPD were incubated with dexamethasone (Dex; 10⁻⁶ M) for 30 min before being stimulated with LPS (10 μg/ml). LPS-induced IL-8 release (A) and TNFα release (B) after 18 h were measured. *P < 0.05 versus control subjects; ^#P < 0.05 versus unstimulated cells; ^¶P < 0.05 versus LPS-stimulated cells; n ≥ 6 in each group.

Figure 2.

HDAC activity and expression in AM. (A) HDAC activity was measured in protein extracts from AM obtained from normal subjects, healthy smokers, and COPD patients and expressed according to protein content. ^##P < 0.01 versus nonsmoker; *P < 0.05 versus smoker; n ≥ 6 in each group. (B) Immunocytochemical analysis of HDAC2 expression in AM from normal subjects and patients with COPD. Results are representative of at least six subjects in each group. (C) Western blot analysis of HDAC2 expression in AM from normal subjects, healthy smokers, and COPD patients and expressed according to protein content. *P < 0.05 versus nonsmoker, n = 3 in each group.

Figure 3.

Effect of theophylline effect on HDAC activity and dexamethasone sensitivity in AM. (A) Nuclear extracts obtained from AM of heavy smokers and patients with COPD were incubated with theophylline (10⁻⁵ and 10⁻⁶ M) for 20 min, and HDAC activity was measured. **P < 0.01, n = 3. (B) Effect of theophylline (Theo; 10⁻⁶ M) and dexamethasone (Dex; 10⁻⁶ M) alone and in combination on LPS-stimulated IL-8 release from AM obtained from COPD patients cultured overnight. ^#P < 0.05 versus LPS; ^##P < 0.01 versus LPS; ^*P < 0.05 versus LPS plus dexamethasone (n = 6).

Figure 4.

Theophylline effect on HDAC activity and steroid sensitivity in oxidant-stressed U937 cells and AM. (A) Effect of theophylline (Theo, 10⁻⁶ M) and dexamethasone (Dex; 10⁻⁸ M) alone and in combination on IL-1β (1 ng/ml) and IL-1β + hydrogen peroxide (H₂O₂; 100 μM)–stimulated IL-8 release from U937 cells. Theophylline (10⁻⁶ M) enhanced sensitivity compared with cells treated with IL-1β and H₂O₂. *P < 0.05 versus IL-1β; ^#P < 0.01 versus IL-1β + H₂O₂· (B) Effect of theophylline (Theo; 10⁻⁶ M) on cigarette smoke extract (CSE; 0.15 dilution)–induced reduction in HDAC activity. ***P < 0.001 versus control; ^##P < 0.01 versus CSE in U937 cells. (C) Effect of theophylline (Theo; 10⁻⁵ M) and dexamethasone (Dex; 10⁻¹⁰ M) alone and in combination on LPS (10 ng/ml)-induced IL-8 release from AM obtained from smokers. Theophylline enhanced Dex sensitivity, an effect that was blocked by addition of the HDAC inhibitor trichostatin A (TSA; 10 ng/ml). *P < 0.05 versus LPS, n = 3. (D) Specific HDAC isoforms were immunoprecipitated from U937 cells and incubated with theophylline (10⁻⁶ M) for 20 min before HDAC activity was measured. ***P < 0.001 (n = 4). (E) Specific HDAC isoforms were immunoprecipitated from AM from smokers and incubated with theophylline (10⁻⁶ M) for 20 min before HDAC activity was measured. n = 3.