Effect of erythromycin on cigarette-induced histone deacetylase protein expression and nuclear factor-κB activity in human macrophages in vitro

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Abstract

Histone deacetylases (HDACs) are families of enzymes that regulate chromatin structure and thus affect inflammatory gene expression. The anti-inflammatory properties of macrolides are well documented. However, the effects of macrolides on HDAC protein expression have not been studied. This study aimed to examine the molecular mechanism of the inflammatory responses caused by cigarette smoke extract (CSE) and the effects of erythromycin (EM) on CSE-induced HDAC protein expression in human macrophages in vitro. The cells were preincubated with EM and were then exposed to CSE. Levels of interleukin-8 (IL-8) and tumor necrosis factor-a (TNF-a) were assayed by enzyme linked immunosorbent assay (ELISA). Nuclear factor-κB (NF-κB) activity was assessed by an electrophoretic mobility shift assay. HDAC activity was measured with a colorimetric assay kit, and Western blotting was used for HDAC1, -2, -3 and NF-κB protein expression assays. The results showed that CSE causes decreases in HDAC activity and HDAC1, -2, -3 levels and upregulates NF-κB activity, resulting in increased NF-κB-dependent proinflammatory cytokine release in human macrophage cells. Moreover, EM was able to reverse the CSE-induced decline in HDAC1, -2, -3 protein expression, which was most prominent for HDAC2; these changes were associated with the suppression of both NF-κB protein expression and the production of inflammatory mediators. These results suggest that relieving inflammation with EM can be useful in therapeutic approaches for modulating intracellular nuclear signaling in chronic airway inflammatory diseases such as chronic obstructive pulmonary disease (COPD).

Highlights

► EM increases cigarette-induced declines in HDAC1, 2, 3 protein expression. ► EM inhibits cigarette-increased NF-κB activity and protein expression. ► EM suppresses cigarette-mediated IL-8 and TNF-a protein release. ► EM increases HDAC expression which may be associated with inhibition of NF-κB and cytokine.

Introduction

In addition to their well-known antimicrobial activity, macrolide antibiotics possess anti-inflammatory and immunomodulatory properties that may confer beneficial effects to patients with respiratory diseases associated with chronic inflammation, including diffuse panbronchiolitis (DPB), cystic fibrosis (CF), asthma and chronic obstructive pulmonary disease (COPD) [1], [2]. Recently, macrolides have been proposed for the treatment of patients with moderate to severe stable COPD and acute exacerbations of COPD [3], [4]. The anti-inflammatory and immunomodulatory properties of macrolides are well documented; they have been postulated to reduce airway inflammation via several mechanisms [5], [6]. These effects include regulation of leukocyte function and the production of inflammatory mediators, control of mucus hypersecretion, resolution of inflammation, and modulation of host defense mechanisms. However, the molecular mechanism of the anti-inflammatory action of macrolides remains unclear.

COPD is a common and debilitating chronic inflammatory disease characterized by progressive airflow limitation that is poorly reversible [7]. In COPD, oxidative stress due to cigarette smoke exposure is considered to be the main etiologic factor in disease pathogenesis [8]. Cigarette smoking is the major factor for the ongoing inflammation in the airways and lung parenchyma, and the severity of airflow limitation is correlated with the degree of pulmonary inflammation [9]. Cigarette smoke causes airway inflammation by activating macrophages, neutrophils, and T lymphocytes, which release proteases and reactive oxygen species (ROS), leading to cellular injury [10], [11]. Alveolar macrophages are considered to be an important component in perpetuating the inflammatory responses to cigarette smoke; following exposure, they secrete many inflammatory mediators, oxidants, proteins and proteinases [12]. Thus, macrophages are thought to be the main orchestrators of the chronic inflammatory response and tissue destruction observed in patients with COPD [13]. IL-8 is a multifunctional cytokine that has significant neutrophil chemoattractant and activating properties. It is produced by a variety of inflammatory and pulmonary cell types in response to oxidative stress. IL-8 and proinflammatory cytokines such as TNF-a are important inflammatory mediators in COPD; both mediators are increased in the sputum of patients with COPD [14]. It has also been shown that alveolar macrophages from patients with COPD release increased levels of IL-8 and TNF-a [15].

Histone acetyltransferases (HATs) and histone deacetylases (HDACs) are families of enzymes that regulate chromatin structure and thus affect inflammatory gene expression [16], [17]. The acetylation of core histones by coactivator proteins that possess intrinsic HAT activity leads to the unwinding of chromatin, which subsequently allows transcription factors and RNA polymerase II to switch on gene transcription. Conversely, the deacetylation of core histones is generally associated with transcriptional repression [18]. HDACs are key molecules for repressing the production of proinflammatory cytokines [19]. The HDAC enzyme family consists of 18 isoforms grouped into three classes. Various members of the class I HDAC family (HDAC1, -2, -3, -8, and -11) have been shown to play roles in regulating cell proliferation and inflammatory responses [20]. Recently, HDAC2 has been reported to be required for corticosteroid-mediated anti-inflammatory activity [21]. Corticosteroid resistance is known to occur in COPD; inflammation becomes unresponsive to treatment [22]. Even high doses of inhaled and oral glucocorticoids have no effect on the inflammatory cell and cytokine profile and fail to reverse the protease–antiprotease imbalance [23]. Bronchoalveolar lavage (BAL) macrophages isolated from patients with COPD also display resistance to corticosteroid-mediated suppression of inflammation [24]. It has been shown that there are decreases in HDAC activity and HDAC2 expression in peripheral lung and bronchial biopsy specimens and in alveolar macrophages from COPD patients; this is correlated with disease severity and with increased gene expression levels of IL-8. This may account for the amplified inflammation and resistance to corticosteroids that occur as COPD progresses [25].

COPD is characterized by the increased expression of multiple inflammatory genes that are regulated by proinflammatory transcription factors, such as NF-κB, that bind to and activate coactivator molecules, which then acetylate core histones to switch on gene transcription. Conversely, gene repression is mediated via HDACs and other corepressors [26]. Previous studies have shown that cigarette smoke causes a decrease in HDAC activity and in HDAC1, -2, -3 protein levels and upregulates NF-κB-dependent proinflammatory cytokine release in MonoMac6 cells [27]. Therefore, in this report, we evaluated the effects of erythromycin on CSE-induced HDAC activity and HDAC1, -2, -3 protein expression, the correlation with NF-κB activity, and proinflammatory cytokine synthesis in human macrophages.

Section snippets

U937 cell culture

The human monocytic cell line (U937) [28], [29], obtained from the ATCC (CRL-1593.2; Manassas, VA, USA), was grown in RPMI 1640 medium (Life Technologies, Gaithersburg, MD) supplemented with 10% fetal bovine serum (HyClone Laboratories, Logan, UT), 2 mM l-glutamine, 100 μg/ml penicillin, 100 U/ml streptomycin, 1% nonessential amino acids, 1% sodium pyruvate, 1 μg/ml human holotransferrin, and 1 mM oxaloacetic acid. The cells were cultured at 37 °C in a humidified atmosphere containing 5% CO2. The

Effects of CSE and EM on the growth of human macrophages

Because CSE can be toxic to cells, we confirmed cell survival rates under the various conditions using MTT assays. The MTT assay demonstrated that neither CSE (0.1% and 1%) nor EM (0.1, 1 and 10 μg/ml) affected the proliferation of human macrophage cells (p > 0.05). Microscopically, no morphological changes were visible in either control cells or cells treated with CSE (0.1% and 1%) or EM (0.1, 1 and 10 μg/ml). However, 2.5% CSE affected the proliferation of human macrophage cells (p < 0.05).

Discussion

Macrolides have anti-inflammatory and immunomodulatory effects that appear to be the reason for the clinical benefits of DPB. A literature search was conducted for studies on the clinical effectiveness of macrolides in other chronic lung conditions [37]. Preliminary data from studies of patients with COPD have shown improvements in symptom scores and the forced expiratory volume in 1 s (FEV1) after macrolide treatment. It was recently reported that low-dose, long-term treatment with the

Acknowledgment

This study was supported by grants from the National Nature Science Foundation of China (30760085).

References (54)

  • L. Wu et al.

    In vitro effects of erythromycin on RANKL and nuclear factor-kappa B by human TNF-alpha stimulated Jurkat cells

    Int Immunopharmacol

    (2009)
  • K. Ito et al.

    Oxidative stress reduces histone deacetylase 2 activity and enhances IL-8 gene expression: role of tyrosine nitration

    Biochem Biophys Res Commun

    (2004)
  • H. Zhong et al.

    The phosphorylation status of nuclear NF-kappa B determines its association with CBP/p300 or HDAC-1

    Mol Cell

    (2002)
  • M.H. Gotfried

    Macrolides for the treatment of chronic sinusitis, asthma, and COPD

    Chest

    (2004)
  • D. Banerjee et al.

    The effect of oral clarithromycin on bronchial airway inflammation in moderate-to-severe stable COPD: a randomized controlled trial

    Treat Respir Med

    (2004)
  • P.J. Barnes

    Chronic obstructive pulmonary disease

    N Engl J Med

    (2000)
  • P. Rytila et al.

    Increased oxidative stress in asymptomatic current chronic smokers and GOLD stage 0 COPD

    Respir Res

    (2006)
  • M. Saetta et al.

    Cellular and structural bases of chronic obstructive pulmonary disease

    Am J Respir Crit Care Med

    (2001)
  • J.C. Hogg et al.

    The nature of small-airway obstruction in chronic obstructive pulmonary disease

    N Engl J Med

    (2004)
  • P.J. Barnes

    Alveolar macrophages in chronic obstructive pulmonary disease (COPD)

    Cell Mol Biol (Noisy-le-grand)

    (2004)
  • P.J. Barnes et al.

    Chronic obstructive pulmonary disease: molecular and cellular mechanisms

    Eur Respir J

    (2003)
  • V.M. Keatings et al.

    Differences in interleukin-8 and tumor necrosis factor-alpha in induced sputum from patients with chronic obstructive pulmonary disease or asthma

    Am J Respir Crit Care Med

    (1996)
  • B.G. Cosio et al.

    Theophylline restores histone deacetylase activity and steroid responses in COPD macrophages

    J Exp Med

    (2004)
  • F.D. Urnov et al.

    Chromatin remodeling and transcriptional activation: the cast (in order of appearance)

    Oncogene

    (2001)
  • K. Ito et al.

    Decreased histone deacetylase activity in chronic obstructive pulmonary disease

    N Engl J Med

    (2005)
  • A.J. de Ruijter et al.

    Histone deacetylases (HDACs): characterization of the classical HDAC family

    Biochem J

    (2003)
  • K. Ito et al.

    Glucocorticoid receptor recruitment of histone deacetylase 2 inhibits interleukin-1beta-induced histone H4 acetylation on lysines 8 and 12

    Mol Cell Biol

    (2000)
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