Macrolide antibiotics broadly and distinctively inhibit cytokine and chemokine production by COPD sputum cells in vitro

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Abstract

Macrolide antibiotics are known to exert anti-inflammatory actions in vivo, including certain effects in COPD patients. In order to investigate the immunomodulatory profile of activity of macrolide antibiotics, we have studied the effects of azithromycin, clarithromycin, erythromycin and roxithromycin on the in vitro production of a panel of inflammatory mediators from cells isolated from human, steroid-naïve, COPD sputum samples. Macrolide effects were compared to three other commonly used anti-inflammatory compounds, the corticosteroid dexamethasone, the PDE4 inhibitor, roflumilast and the p38 kinase inhibitor, SB203580. Three of the four tested macrolides, azithromycin, clarithromycin and roxithromycin, exhibited pronounced, concentration-related reduction of IL-1β, IL-6, IL-10, TNF-α, CCL3, CCL5, CCL20, CCL22, CXCL1, CXCL5, and G-CSF release. Further slight inhibitory effects on IL-1α, CXCL8, GM-CSF, and PAI-1 production were also observed. Erythromycin was very weakly active. Qualitatively and quantitatively, macrolides exerted distinctive and, compared to other tested classes of compounds, more pronounced immunomodulatory effects, particularly in terms of chemokine (CCL3, CCL5, CCL20, CCL22, and CXCL5), IL-1β, G-CSF and PAI-1 release. The described modulation of inflammatory mediators could potentially contribute to further definition of biomarkers of macrolide anti-inflammatory activity in COPD.

Introduction

Chronic obstructive pulmonary disease (COPD) is a progressive inflammatory condition, which is punctuated by frequent exacerbations in association with pulmonary infection. It is characterized by emphysema, bronchitis, breathlessness, coughing and progressive and irreversible loss of lung function [1]. Systemic features include cachexia, skeletal muscle wasting, and increased risk of cardiovascular disease, associated with elevated C-reactive protein level in the systemic circulation [2]. While cigarette smoking is a common etiologic factor, other injurious stimuli also contribute to the deterioration of airway function. Cell infiltration into the lungs of COPD patients is dominated by neutrophils. Release by these cells of elastase, as well as their production of reactive oxygen species have long been considered to play important roles in disease development, particularly in response to cigarette smoke [3]. More recently, a regulatory function of mononuclear phagocytes has been suggested to underlie the chronic inflammatory process in the lungs of COPD patients [4]. COPD is also characterized by airway infiltration of T-cells which further increase the inflammation and development of emphysema [5]. Such infiltrated inflammatory cells are the sources of a wide spectrum of mediators which are highly increased in sputum and/or airways of COPD patients and often correlate with disease severity. Pro-inflammatory cytokines, like IL-1β, IL-6, and TNF-α, amplify the inflammatory response, while growth factors, such as GM-CSF and G-CSF, increase the survival of neutrophils and macrophages in the airways. Several chemokines (CCL2, CCL3, CCL5, CCL20, CCL22, CXCL1, CXCL5, and CXCL8) are crucial for recruitment of inflammatory cells (neutrophils, monocytes, dendritic cells or T-cells) into the lungs [6], [7].

Therapy of COPD, apart from cessation of smoking, consists essentially of symptomatic relief, particularly with cholinergic bronchodilator drugs. Corticosteroids provide some benefit in the treatment of acute exacerbations, but their effects on the chronic inflammatory process are modest [8]. This ineffectiveness may be related to loss of inhibitory effects on histone acetylation [4]. On the other hand, the oral phosphodiesterase 4 (PDE4) inhibitor, roflumilast, has shown efficacy in COPD patients, but this is often accompanied by adverse effects, such as nausea, diarrhea and weight loss (reviewed in [9]). Inhibitors of p38 kinase demonstrated anti-inflammatory activity in lung macrophages in vitro as well as in a smoking model of COPD in vivo. Several of these compounds have entered clinical trials, but there have been problems with side effects and toxicity after oral administration (reviewed in [10]).

Acute COPD exacerbations are also treated with antibiotics [8]. Among these drugs, macrolide antibacterials exert anti-inflammatory or immunomodulatory effects [11], [12]. Actions observed with macrolides include inhibition of neutrophil accumulation, chemokine and cytokine production, as well as inhibition of adhesion molecule expression. Such effects account, at least partially, for the essential role of macrolide antibacterials in the treatment of diffuse panbronchiolitis [13], bronchiolitis obliterans syndrome and of a subpopulation of cystic fibrosis patients [12], [14]. Beneficial clinical effects have also been observed in chronic sinusitis, bronchiectasis and asthma, as well as in COPD patients [15], [16], [17], [18].

The mechanism(s) of the anti-inflammatory/immunomodulatory actions of macrolides remain unclear. Several different targets have been proposed, including NF-κB, AP-1 and ERK1/2 (for review see [19]). Recent data suggest that their ability to inhibit neutrophil-dominated pulmonary inflammation may be indirect, through modulatory effects on mononuclear phagocytes. Azithromycin, for instance, has been shown to restore the deficient phagocytosis of apoptotic cells (efferocytosis) in vitro by alveolar macrophages from COPD patients [20]. This restorative effect on defective efferocytosis has also been observed following 12 weeks treatment of COPD patients with azithromycin [21]. The concept of inhibition of neutrophil-dominated lung inflammation through modulatory effects of macrolides on macrophage function is also supported by our findings in bacterial lipopolysaccharide (LPS)-induced lung inflammation in mice [22]. Oral treatment with azithromycin and clarithromycin inhibited the pulmonary inflammatory response, reducing lung homogenate concentrations of inflammatory mediators, including IL-1β and granulocyte-macrophage colony stimulating factor (GM-CSF). Inhibition of LPS-induced IL-1β and GM-CSF production from J774 macrophage-derived cells, but not by epithelial-derived MLE-12 cells, was further observed on incubation with the macrolides in vitro.

In order to investigate the profile of activity of macrolide antibacterials on human lung cell populations, we have studied the effects of azithromycin, clarithromycin, erythromycin and roxithromycin on the in vitro production of a panel of inflammatory mediators from cells isolated from human, steroid-naïve COPD sputum samples. The spontaneous production of inflammatory mediators from the cells isolated from clinical samples was high, and further stimulation with LPS in vitro was not effective (results not shown and in accordance with [23]). The effects of the macrolides were compared with those of the corticosteroid, dexamethasone, the p38 kinase inhibitor, 4-{4-(4-fluorophenyl)-2-[4-(methylsulfinyl)phenyl]-1H-imidazol-5-yl}pyridine (SB203580) and the PDE4 inhibitor, roflumilast.

Section snippets

Patients

Subjects diagnosed with COPD (GOLD stages II–IV) [1], who had not received any corticosteroid treatment within the last 3 months, provided spontaneously produced sputum samples. Patients with a known, pre-existing diagnosis of asthma, alpha-1-anti-trypsin deficiency, uncontrolled diabetes, inflammatory bowel disease, rheumatoid arthritis or any other chronic inflammatory disorder were excluded from the study. Additional exclusion criteria were treatment with macrolide, tetracycline or quinolone

Analysis of cell populations

According to differential cell counts, determined in a total of 60 patient samples included in the study, the highest average proportion of cells was found for neutrophils (around 82%), followed by monocytes/macrophages (around 13%). The proportional counts of eosinophils and especially lymphocytes were much lower, as shown in Table 2.

Spontaneous release of inflammatory mediators

Cells isolated from COPD sputum spontaneously released measurable amounts of IL-1α, IL-1β, IL-1ra, IL-6, IL-10, G-CSF, GM-CSF, TNF-α, CCL2, CCL3, CCL5, CCL20,

Discussion

Despite intensive research efforts, no significant breakthrough has been made in the development of novel drugs for COPD. In several small scale trials, macrolide antibiotics reduced COPD exacerbations and improved quality of life [15], [18], [25]. In order to investigate the immunomodulatory profile of activity of macrolides we have studied the effects of azithromycin, clarithromycin, erythromycin and roxithromycin on the in vitro production of a panel of inflammatory mediators in a clinically

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    Both authors made equal contribution to the study.

    2

    Present address: Galapagos istraživački centar d.o.o., Zagreb, Croatia.

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    Present address: Modara Pelessa Magama, Sri Lanka.

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    Present address: University Hospital for Infectious Diseases, Zagreb, Croatia.

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