Basic Science Investigations Received: November 18, 2014 Accepted after revision: March 12, 2015 Published online: May 12, 2015

Respiration DOI: 10.1159/000381905

Effect of Ambroxol and Beclomethasone on Lipopolysaccharide-Induced Nitrosative Stress in Bronchial Epithelial Cells Fabio L.M. Ricciardolo a Valentina Sorbello a Sabrina Benedetto a Davide Paleari b b

Department of Clinical and Biological Sciences, University of Torino, San Luigi Hospital, Orbassano, and Chiesi Farmaceutici, Parma, Italy

Key Words Ambroxol · Beclomethasone · In vitro study · Oxidative stress · Nitrosative stress · Chronic obstructive pulmonary disease

Abstract Background: Nitrosative stress is involved in different airway diseases. Lipopolysaccharide (LPS) induces neutrophilrelated cytokine release and nitrosative stress in human bronchial epithelial (BEAS-2B) cells alone or with human polymorphonuclear neutrophils (PMNs). Ambroxol protects against oxidative stress, and beclomethasone dipropionate is an anti-inflammatory drug. Objectives: We evaluated the ability of ambroxol and/or beclomethasone dipropionate to inhibit LPS-induced expression/release of RANTES, IL-8, inducible NO synthase (iNOS), myeloperoxidase (MPO) and 3-nitrotyrosine (3-NT: nitrosative stress biomarker) in BEAS2B ± PMNs stimulated with LPS (1 μg/ml). Methods: The effect of ambroxol and/or beclomethasone dipropionate on IL-8, RANTES and iNOS levels was assessed by Western blot analysis; IL-8, MPO and 3-NT levels were measured by ELISA. Cell viability was assessed by the trypan blue exclusion test. Results: In BEAS-2B alone, LPS (at 12 h) increased RANTES/ iNOS expression and IL-8 levels (p < 0.001). Ambroxol suppressed LPS-induced RANTES expression and IL-8 release

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(p < 0.001), whilst inhibiting iNOS expression (p < 0.05). Beclomethasone dipropionate had no effect on RANTES but halved iNOS expression and IL-8 release. Coculture of BEAS2B with PMNs stimulated IL-8, MPO and 3-NT production (p < 0.001), potentiated by LPS (p < 0.001). Ambroxol and beclomethasone dipropionate inhibited LPS-stimulated IL8, MPO and 3-NT release (p < 0.05). Ambroxol/beclomethasone dipropionate combination potentiated the inhibition of IL-8 and 3-NT production in BEAS-2B with PMNs (p < 0.05 and p < 0.01, respectively). Ambroxol and/or beclomethasone dipropionate inhibited nitrosative stress and the release of neutrophilic inflammatory products in vitro. Conclusion: The additive effect of ambroxol and beclomethasone dipropionate on IL-8 and 3-NT inhibition suggests new therapeutic options in the treatment of neutrophil-related respiratory diseases such as chronic obstructive pulmonary disease and respiratory infections. © 2015 S. Karger AG, Basel

Introduction

Oxidative stress is an underlying pathogenetic mechanism of many chronic inflammatory disorders. In respiratory diseases such as chronic obstructive pulmonary disease (COPD), oxidative stress and high levels of proFabio L.M. Ricciardolo Department of Clinical and Biological Sciences, University of Torino San Luigi Hospital, Regione Gonzole 10 IT–10043 Orbassano, Torino (Italy) E-Mail fabioluigimassimo.ricciardolo @ unito.it

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a

2

Respiration DOI: 10.1159/000381905

The aim of this study was to evaluate the effect of ambroxol hydrochloride and/or beclomethasone dipropionate on the LPS-induced release of neutrophil-related cytokines such as IL-8 and ‘regulated on activation, normal T lymphocyte expressed and secreted’ (RANTES/CCL5), expression of iNOS and MPO, and production of 3-NT in BEAS-2B cells alone or in coculture with human PMNs. Materials and Methods Bronchial Epithelial Cell Culture Immortalized, non-tumorigenic human bronchial epithelial cells BEAS-2B (CRL-9609; American Type Culture Collection, Manassas, Va., USA) were cultured in a serum-free medium composed of 50% LHC9 medium (Gibco, Grand Island, N.Y., USA) and 50% RPMI-1640 medium (Euroclone Ltd., Paignton, UK) supplemented with 1× non-essential amino acids (Gibco), L-glutamine (2 mM), penicillin (100 U/ml)/streptomycin (100 U/ml; Euroclone Ltd.), and incubated at 37 ° C in 100% humidity and 5% CO2 [4]. Additionally, in some experiments human PMNs were cocultured with BEAS-2B cells in 6-well plates (Falcon, BD Biosciences) equipped with porous cell culture membrane inserts with a pore diameter of 1 μm (Falcon, BD Biosciences) as previously described [24]. The study conformed to the local ethic criteria concerning the management of biological samples (PMNs from venous blood of healthy subjects). PMNs were isolated from peripheral blood by standard dextran sedimentation followed by Histopaque® (Sigma-Aldrich, St. Louis, Mo., USA) gradient centrifugation. The PMNs were resuspended in Hepes-buffered saline solution (Clonetics, Basel, Switzerland) with a final cell suspension purity average of 98%. PMN viability (assessed by the trypan blue exclusion test) was always >95%.  

 

Experimental Study Design Evaluation of neutrophil-related mediators: when 80% confluent, BEAS-2B cells were incubated for up to 12 h with 1 μg/ml LPS from Pseudomonas aeruginosa serotype 10 (Sigma-Aldrich) in order to perform either a time course (0 min, 30 min, 1, 2, 4, 8 and 12 h) or a dose-response curve (from 10–9 to 10–5 M) to assess the effect of ambroxol hydrochloride (Chiesi Farmaceutici, Parma, Italy) or beclomethasone dipropionate (Chiesi Farmaceutici) on RANTES and iNOS expression and on IL-8 release. Evaluation of neutrophil-related markers of nitrosative stress: assessment of IL-8, MPO and 3-NT production by BEAS-2B cells either alone or cocultured with human PMNs at different time points (1, 2, 4 and 8 h) with and without LPS was performed. The effect of ambroxol and/or beclomethasone on LPS-induced expression of these markers was also examined in BEAS-2B cells cocultured with human PMNs. Specifically, the 9 groups were: (1) BEAS-2B cells alone; (2) BEAS-2B cells treated with LPS (1 μg/ml); (3) BEAS-2B cells treated with LPS (1 μg/ml) and ambroxol (from 10–9 to 10–5 M);

Ricciardolo/Sorbello/Benedetto/Paleari

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inflammatory endogenous nitric oxide (NO), which plays a key role in the physiological regulation of airway function, produce strong oxidizing reactive nitrogen species [1, 2], which reinforce inflammation, damage DNA and inhibit mitochondrial respiration, causing protein dysfunction and cell damage. This, together with oxidative stress, is nitrosative stress [1]. A study investigating nitrosative stress markers, including 3-nitrotyrosine (3-NT), inducible NO synthase (iNOS), endothelial NO synthase, myeloperoxidase (MPO) and xanthine oxidase in bronchial biopsies from patients with stable COPD, showed that MPO+ and 3NT+ cells and neutrophils are markedly increased in severe COPD, indicating that MPO and neutrophilic inflammation are key features of nitrosative stress [3]. Bradykinin and lipopolysaccharide (LPS) stimulate neutrophil chemo-attractant interleukin (IL)-8 release and production of 3-NT and MPO (a marker of neutrophil activation) by human bronchial epithelial (BEAS-2B) cells cultured alone or with human polymorphonuclear neutrophils (PMNs) [4]. Furthermore, epithelial cells release IL-8 and 3-NT after stimulation by viruses [5], nitrogen dioxide [6] and acute lung injury [7]. Ambroxol hydrochloride, a mucolytic anti-inflammatory agent used to treat respiratory diseases associated with increased mucus production, has a wide range of properties including inhibition of oxidative and nitrosative stress [8–12]. Ambroxol has been shown to prevent generation of reactive oxygen species by broncho-alveolar lavage cells from COPD patients [13] suggesting that it may be useful in the management of COPD [14, 15]. Moreover, ambroxol’s antioxidant properties may be of clinical benefit in virus-induced infections. Indeed, several viruses, including influenza and respiratory syncytial virus, promote an intracellular redox state imbalance toward pro-oxidant conditions, thereby contributing to virus-induced damage to the host tissues [16]. Ambroxol improved the survival rate of mice after infection with a lethal dose of influenza virus [17] and can help prevent common acute upper respiratory diseases such as the common cold and influenza [18]. Beclomethasone dipropionate is a glucocorticoid used in maintenance therapy for persistent asthma and in the prevention of asthma and COPD exacerbations [19–21]. These drugs may inhibit nitrosative stress and neutrophilic activity [22] and this could be demonstrated using the BEAS-2B bronchial epithelial cell model which already showed a sensitivity to glucocorticoids in terms of vascular cell adhesion molecule-1 expression inhibition as glucocorticoid anti-inflammatory effect [23].

Western Blot Analysis To evaluate RANTES and iNOS expression, BEAS-2B cells were cultured alone or treated with LPS in the absence or presence of ambroxol or beclomethasone as previously described. Activation of p38 MAPK was expressed by the increase in the phosphorylated p38 (p-p38)/p38 ratio. To evaluate p-p38 and p38, BEAS-2B cells were incubated for 12 h alone, with LPS, and with LPS plus the addition of ambroxol (10–6 M). After incubation, cells were washed in PBS and lysed in 1 ml/ dish of lysis buffer (20 mM Tris HCl at pH 7.4, 150 mM NaCl, 1% Triton X-100, 0.1% sodium dodecyl sulphate, 1 mM ethylene glycol tetra-acetic acid, 1 mM ethylenediamine tetra-acetic acid, 1 mM Na orthovanadate and 1× protease inhibitor cocktail; Sigma-Aldrich). Total cell lysates were incubated in ice for 20 min, centrifuged at 9,280 g for 10 min at 4 ° C, and their protein concentration was quantified by the Bio-Rad Protein Assay (Bio-Rad Laboratories, Hercules, Calif., USA). Protein extracts (50 μg/well) were separated by 15% sodium dodecyl sulphate-polyacrylamide gel electrophoresis and transferred onto nitrocellulose membranes (Bio-Rad). Blots were incubated with goat anti-RANTES antibody (1: 1,000, R & D Systems, Abingdon, UK), mouse anti-NOS2 (iNOS) antibody (1: 200, Santa Cruz Biotechnology), and rabbit antibodies to total p38 or p-p38 (1: 1,000, New England Biolabs, Beverly, Mass., USA) and visualized using the enhanced chemiluminescence system (Amersham Biosciences, Little Chalfont, UK). Blots were then stripped using the RestoreTM Western Blot Stripping Buffer (Thermo Scientific, Waltham, Mass., USA) and reprobed with anti-β-actin antibody (clone C4; 1:1,000, Boehringer Mannheim Inc., Mannheim, Germany). The relevant band intensities were analysed using the NIH Image J 1.38x program.  

 

was evaluated in the supernatants by enzyme-linked immunosorbent assay (ELISA) following the manufacturer’s instructions. The minimum detectable concentration of each protein was 1 pg/ml, 0.1 ng/ml and 0.25 μg/ml, respectively. Results were expressed as nanograms per millilitre per 5 × 105 viable cells for IL-8 and MPO, and as micrograms per millilitre per 5 × 105 viable cells for 3-NT, respectively. Cell viability was evaluated by the trypan blue exclusion test (0.4% final dye concentration) in each experimental set. Counting the total number of cells was done by using a Burker counting chamber and a binocular microscope, at a magnification of ×200. Estimations of the percentages of non-viable cells were made by counting the percentage of blue cells. Viability values were on average higher than 98% [4]. Statistical Analysis Statistical evaluation was performed using the statistical software package GraphPad Prism 5.0 (GraphPad Software, San Diego, Calif., USA). Data are presented as means ± SEM of 3 independent experiments (each experiment was performed in duplicate). Multiple groups were tested for significance by one-way ANOVA and post hoc Dunnett’s two-sided comparison test [25]. For comparison between two groups, an unpaired t test was performed with a p value