Azithromycin and Clarithromycin Inhibit Lipopolysaccharide- Induced Murine Pulmonary Neutrophilia Mainly through Effects on Macrophage-Derived Granulocyte-Macrophage Colony- Stimulating Factor and Interleukin-1

Macrolide antibiotics possess immunomodulatory/anti-inflammatory properties. These properties are considered fundamental for the efficacy of macrolide antibiotics in the treatment of chronic inflammatory diseases like diffuse panbronchiolitis and cystic fibrosis. However, the molecular mechanisms and cellular targets of anti-inflammatory/immunomodulatory macrolide activity are still not fully understood. To describe anti-inflammatory effects of macrolides in more detail and to identify potential biomarkers of their activity, we have investigated the influence of azithromycin and clarithromycin on the inflammatory cascade leading to neutrophil infiltration into lungs after intranasal lipopolysaccharide challenge in mice. Azithromycin and clarithromycin pretreatment reduced total cell and neutrophil numbers in bronchoalveolar lavage fluid and myeloperoxidase concentration in lung tissue. In addition, concentrations of several inflammatory mediators, including CCL2, granulocyte-macrophage colony stimulating factor (GM-CSF), interleukin-1 (IL-1 ), tumor necrosis factor , and sE-selectin in lung homogenates were decreased after macrolide treatment. Inhibition of cytokine production observed in vivo was also corroborated in vitro in lipopolysaccharide-stimulated monocytes/ macrophages, but not in an epithelial cell line. In summary, results presented in this article confirm that macrolides can suppress neutrophil-dominated pulmonary inflammation and suggest that the effect is mediated through inhibition of GMCSF and IL-1 production by alveolar macrophages. Besides GM-CSF and IL-1 , CCL2 and sE-selectin are also identified as potential biomarkers of macrolide anti-inflammatory activity in the lungs. Macrolide antibiotics (macrolides) are a well established class of antimicrobial agents characterized by the presence of a highly substituted macrocyclic lactone ring. Erythromycin, a natural product isolated from Saccharopolyspora erythraea, was the first macrolide to be introduced to clinical use over 50 years ago. Afterward, several semisynthetic derivatives of erythromycin, like clarithromycin (6-O-methylerythromycin A) and azithromycin (9-deoxy-9a-aza-9a-methyl-9ahomoerythromycin A), were designed to broaden the antimicrobial spectrum, reduce gastrointestinal side effects, and increase acid stability and bioavailability in this class of antibiotics (Whitman and Tunkel, 1992). Nowadays, macrolides are widely used in the treatment of respiratory tract and soft tissue infections. In addition to their efficacy in treatment of bacterial infections, many studies over the past 20 years have demonstrated that macrolides are effective in the treatment of various chronic inflammatory disorders of the respiratory tract. Introduction of erythromycin to the treatment of diffuse panbronchiolitis (DPB) in the 1980s drastically increased the 10-year survival rate, decreased frequency of exacerbations, and restored lung function (Kudoh et al., 1998). Afterward, macrolides were successfully used in the treatment of cystic fibrosis (CF), which shares a number of This work was supported by GlaxoSmithKline Research Centre Zagreb Limited. M.B. and B.B. contributed equally to this work. Article, publication date, and citation information can be found at http://jpet.aspetjournals.org. doi:10.1124/jpet.109.155838. ABBREVIATIONS: DPB, diffuse panbronchiolitis; BALF, bronchoalveolar lavage fluid; BEBM, bronchial epithelial basal medium; CF, cystic fibrosis; DMSO, dimethyl sulfoxide; ELISA, enzyme-linked immunosorbent assay; FBS, fetal bovine serum; GM-CSF, granulocyte-macrophage colony stimulating factor; siCAM-1, soluble intracellular adhesion molecule 1; IL, interleukin; LPS, lipopolysaccharide; macrolides, macrolide antibiotics; MPO, myeloperoxidase; PBS, phosphate-buffered saline; TNF, tumor necrosis factor ; sVCAM-1, soluble vascular adhesion molecule 1; ANOVA, analysis of variance. 0022-3565/09/3311-104–113$20.00 THE JOURNAL OF PHARMACOLOGY AND EXPERIMENTAL THERAPEUTICS Vol. 331, No. 1 Copyright © 2009 by The American Society for Pharmacology and Experimental Therapeutics 155838/3518330 JPET 331:104–113, 2009 Printed in U.S.A. 104 at A PE T Jornals on O cber 2, 2017 jpet.asjournals.org D ow nladed from similarities in clinical and pathological characteristics with DPB. In CF patients, macrolide treatment was shown to significantly improve lung function and reduce frequency of exacerbations. Consequently, macrolides are now first-line therapy for DPB and recommended for patients with CF, and they are also being evaluated in the therapy of chronic obstructive lung disease, chronic sinusitis, asthma, bronchiectasis, and bronchiolitis obliterans (reviewed in Crosbie and Woodhead, 2009). Beneficial effects of macrolide treatment in chronic inflammatory lung diseases can not be attributed solely to their antimicrobial activity, because the doses used are lower than those used in standard antibiotic therapy and thus below the minimum inhibitory concentration for the most common respiratory pathogens. In addition, numerous in vitro and in vivo studies have shown that macrolides also possess a number of anti-inflammatory and immunomodulatory properties. They were shown to inhibit expression of proinflammatory cytokines including TNF, IL-1 and IL-6, chemokines, and adhesion molecules. Moreover, macrolides inhibit recruitment and migration of inflammatory cells and mucus hypersecretion (reviewed in Culić et al., 2001; Shinkai et al., 2008). Each or a combination of these anti-inflammatory or immunomodulatory properties could, therefore, account for the efficacy of macrolides in chronic inflammatory lung diseases. Development of novel macrolide compounds that have antiinflammatory properties, but are devoid of antimicrobial activity, has been hindered by the lack of knowledge of molecular mechanisms and cellular targets of their anti-inflammatory/immunomodulatory activity. Because several groups, including ours, have reported that various macrolides inhibit the lipopolysaccharide-induced pulmonary inflammatory response (Kadota et al., 1993; Tamaoki et al., 1995; Sanz et al., 2005; Ivetić Tkalcević et al., 2006; Leiva et al., 2008; Ou et al., 2008), we have used this model to perform an in-depth investigation of the effect of azithromycin and clarithromycin on the inflammatory cascade leading to neutrophil infiltration into lungs after intranasal lipopolysaccharide (LPS) challenge in mice. The effect of macrolides on various cytokines, chemokines, and adhesion molecules induced by LPS challenge was carefully assessed to identify potential biomarkers of their anti-inflammatory activity. The data gathered from the model allowed us to establish a simple in vitro test by which the anti-inflammatory activity of novel macrolide compounds could be measured. Finally, based on in vitro and in vivo experiments, a hypothesis for the possible mechanism of inhibition of lung neutrophil infiltration was proposed. Materials and Methods Mice. Studies were performed on 10-week-old male BALB/cJ mice (Charles River Laboratories, Lyon, France). Mice were kept on wire mesh floors with irradiated maize granulate bedding (Scobis Due, Mucedola, Settimo Milanese, Italy) and maintained under standard laboratory conditions (temperature, 23–24°C; relative humidity, 60 5%; 15 air changes per hour; artificial lighting with circadian cycle of 12 h). Pelleted food (VRF-1, Altromin; Charles River, Isaszag, Hungary) and tap water were provided ad libitum. All procedures on animals were approved by the ethics committee of GlaxoSmithKline Research Centre Zagreb Limited, and performed in accordance with the European Economic Community Council Directive 86/609. Materials: Chemicals, Antibodies, and Drugs. LPS from Escherichia coli serotype 0111:B4 was obtained from Sigma-Aldrich (St Louis, MO). Luminex kits and antibodies for enzyme-linked immunosorbent assay (ELISA) were purchased from R&D Systems (Minneapolis, MN). Azithromycin was from PLIVA Inc. (Zagreb, Croatia) and clarithromycin was from Spectrum Chemical Mfg. Corp. (Gardena, CA). All other reagents, if not indicated otherwise, were from Sigma-Aldrich. LPS-Induced Pulmonary Neutrophilia. Experimental pulmonary neutrophilia was induced as described earlier (Ivetić Tkalcević et al., 2006). In brief, mice, under light anesthesia, were instilled intranasally with 2 g of LPS from E. coli/60 l of phosphatebuffered saline (PBS). Vehicle, clarithromycin, and azithromycin were administered orally by gavage 4 h before intranasal challenge with LPS. For administration, macrolides were first dissolved in dimethyl sulfoxide (DMSO) and then diluted with 0.5% (w/v) methylcellulose [final concentration of DMSO was 5% (v/v)]. Azithromycin was further solubilized by addition of an equimolar quantity of citric acid. Solutions obtained were applied orally in a volume of 20 ml/kg (body weight). First, macrolides were tested at doses of 150, 300, and 600 mg/kg to determine the lowest effective dose at which compounds statistically significantly decreased total cell and neutrophil numbers in bronchoalveolar lavage fluid 24 h after challenge with LPS. Based on the results obtained, a dose of 600 mg/kg was used in subsequent time course experiments. Bronchoalveolar Lavage and Determination of Total and Relative Cell Number in Bronchoalveolar Lavage Fluid. Immediately before (0 h) or at various time points after LPS application, the animals were euthanized by an intraperitoneal overdose of thiopental (Inresa Arzneimittel GmbH, Freiburg, Germany). After preparation and cannulation of tracheas, bronchoalveolar lavage was performed with PBS in a total volume of 1 ml (0.4, 0.3 and 0.3 ml). After bronchoalveolar lavage, lungs were excised and fixed in 10% buffered neutral formalin fixative. The bronchoalveolar lavage samples were centrifuged (4°C, 100g, 5 min), the pellet of cells resuspended in an equal volume of fresh PBS and used for total and differential cell counts. Total number of cells in bronchoalveolar lavage fluid (BALF) was counted with a hematological analyzer (Sysmex SF 3000; Sysmex Corp., Kobe, Japan). Percentages of neutrophils and macrophages were determined by morphological examination of at least 200 cells on smears prepared by cytocentrifugation (Cytospin-3, Thermo Fisher Scientific, Waltham, MA) and stained with Kwik-Diff staining set (Thermo Fisher Scientific). The number of neutrophils (and macrophages) in BALF was calculated for each sample according to the formula: Number of neutrophils total number of cells (neutrophil percentage/100%). Histopathological Examination of Lungs. After collection of BALF, lungs from all animals were formalin-fixed, paraffin-embedded in toto, cut at 3 m, and stained routinely with hematoxylin and eosin. For each lung specimen, neutrophilic granulocyte infiltration into peribronchial, periarterial, and perivenular areas, in the interstitium and alveolar space, was examined by an observer blinded to the experimental design and was graded according to the following criteria: 0. No granulocytes 1. Few scattered granulocytes 2. Larger aggregates of granulocytes 3. Marked accumulation of granulocytes. In borderline cases, an intermediate grade was used (0–1, 1–2, 2–3), extending the scoring to a total of seven grades. Preparation of Lung Homogenates for Determination of Inflammatory Mediators and Adhesion Molecules. In separate groups of mice, nonlavaged lungs were collected for determination of the concentrations of inflammatory mediators. Lungs were homogenized on ice in PBS with protease inhibitors (1 g/ml leupeptin, 2 Azithromycin and Clarithromycin in Pulmonary Neutrophilia 105 at A PE T Jornals on O cber 2, 2017 jpet.asjournals.org D ow nladed from g/ml aprotinin, 1 g/ml pepstatin, and 17 g/ml phenylmethylsulfonyl fluoride); 4 ml of PBS with protease inhibitors was added per gram of lung tissue. Homogenates were centrifuged (4°C, 2500g, 15 min) and stored at 80°C until analysis. Determination of Protein Concentration. Protein concentration in lung homogenates was determined by BCA Protein Assay (Thermo Fisher Scientific) according to the manufacturer’s recommendation. Measurement of Inflammatory Mediators in Lungs. Samples were analyzed by use of xMAP technology (Luminex, Austin, TX), which enables simultaneous measurement of multiple biomarkers. Concentrations of GM-CSF, interferon , IL-1 , IL-2, IL-4, IL-5, IL-6, IL-10, IL-12p70, IL-13, IL-17, CCL2 (JE), CXCL1 (KC), CXCL2 (MIP-2), and TNFwere determined by use of fluorokine MAP multiplex kit (R&D Systems) according to the manufacturer’s protocol. In brief, 50 l of samples were incubated with antibody-coated microparticles for 3 h at room temperature. Afterward, washed beads were incubated with biotinylated detection antibody cocktail for 1 h at room temperature, washed, and incubated for 30 min with streptavidin-phycoerythrin conjugate. After the final wash, the microparticles were resuspended in buffer and analyzed with the Luminex 200 (Luminex) and STarStation software v2.3 (Applied Cytometry Systems, Sheffield, UK) with use of a five-parameter, logistic-curve fitting. Myeloperoxidase (MPO) concentration in lung homogenates was determined by Mouse MPO ELISA kit (Hycult Biotechnology, Uden, The Netherlands) according to the manufacturer’s recommendations. Optical density was measured at 450 nm by use of a microplate reader (SpectraMax 190; Molecular Devices, Sunnyvale, CA). MPO concentration was determined by interpolation from standard curves with SoftMax Pro v 4.3.1 software (Molecular Devices). Concentration of analytes in lung homogenates was further normalized to protein concentration in the samples and expressed as picograms of analyte per milligram of protein. Measurement of Adhesion Molecules in Lungs. Concentrations of sE-selectin, soluble intracellular adhesion molecule 1 (sICAM-1) and soluble vascular adhesion molecule 1 (sVCAM-1) were determined by use of a mouse cardiovascular disease panel LINCOplex kit (Linco Research, St. Charles, MO) according to the manufacturer’s protocol. In brief, 25 l of sample was incubated with antibody-coated microparticles overnight at 4°C. Afterward, washed beads were incubated with biotinylated detection antibody cocktail for 1 h at room temperature followed by a 30-min incubation with streptavidin-phycoerythrin conjugate. After the final wash the microparticles were resuspended in sheath fluid and analyzed with the Luminex 200 (Luminex) and STarStation software v2.3 (Applied Cytometry Systems) with use of a five-parameter, logistic-curve fitting. Concentration of analytes in lung homogenates was further normalized to protein concentration in the samples and expressed as picograms or nanograms of analyte per milligram of protein. Cells. The murine monocyte/macrophage cell line, J774.2 was obtained from the European Collection of Cell Cultures (Porton Down, Wiltshire, UK), whereas murine lung epithelial cells, MLE 12, were from American Type Culture Collection (Manassas, VA). J774.2 cells were grown in Dulbecco’s modified Eagle’s medium (Invitrogen, Carlsbad, CA) supplemented with 10% heat-inactivated fetal bovine serum (FBS) (BioWest, Nuaillé, France). MLE 12 cells were maintained in bronchial epithelial basal medium (BEBM; Lonza Walkersville, Walkersville, MD) supplemented with growth factors (bovine pituitary extract, hydrocortisone, human epidermal growth factor, insulin, and transferrin), gentamicin, and amphotericin-B (all provided as BEGM SingleQuots; Lonza Walkersville), and 2% FBS. Cell Stimulation and Inhibition. For all in vitro experiments macrolides were dissolved in DMSO at a concentration of 50 mM and a series of 2-fold dilutions in DMSO were prepared. Macrolide DMSO stock solutions were diluted 1000-fold in cell culture medium to the desired concentrations. Therefore, DMSO concentration was 0.1% in all samples. J774.2 cells were seeded in a 24-well plates in Dulbecco’s modified Eagle’s medium with 10% FBS at a concentration of 3 10 cells per well. The next day, cells were preincubated with macrolides for 2 h and stimulated overnight with 1 g/ml LPS from E. coli serotype 0111:B4. At the end of the incubation period, supernatants were collected and stored at 20°C until assayed. MLE 12 cells were seeded in 24-well plates in supplemented BEBM at a concentration of 2.5 10 cells per well and grown to confluence. Afterward, cells were preincubated with macrolides in BEBM supplemented only with 2% FBS for 2 h and stimulated overnight with 10 ng/ml LPS from E. coli serotype 0111:B4. At the end of the incubation period, supernatants were collected and stored at 20°C until assayed. Optimal concentrations of LPS for stimulation of J774.2 and MLE were established in preliminary experiments. ELISA. Cytokine concentrations were determined in cell culture supernatants by sandwich ELISA using capture and detection antibodies according to the manufacturer’s instructions. Sensitivity of the assay was 0.1 pg/ml for CCL2, 12.2 pg/ml for CXCL1, 154.1 pg/ml for CXCL2, 4.6 pg/ml for GM-CSF, 20.2 pg/ml for IL-1 , 5.5 pg/ml for IL-6, and 25.5 pg/ml for TNF. Optical density was measured at 450 nm by use of a microplate reader (SpectraMax 190, Molecular Devices). Concentration of cytokines was determined by interpolation from standard curves with SoftMax Pro v4.3.1 software (Molecular Devices). Statistical Analysis. All values are presented as means S.E.M. To define statistically significant differences among vehicleand macrolide-treated mice after LPS challenge, the data were subjected to two-way ANOVA followed by a Bonferroni post test using GraphPad Prism version 5.00 for Windows (GraphPad Software, San Diego, CA). To define statistically significant differences between vehicle and macrolide-exposed cells in vitro, the data were subjected to one-way ANOVA followed by Dunnett’s post-test using GraphPad Prism (GraphPad Software). The level of significance was set at p 0.05 in all cases.