JAC Advance Access originally published online on April 5, 2007
Journal of Antimicrobial Chemotherapy 2007 59(6):1096-1101; doi:10.1093/jac/dkm084
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Clarithromycin has an immunomodulatory effect on ERK-mediated inflammation induced by Pseudomonas aeruginosa flagellin
1 Department of Pediatrics, Wake Forest University School of Medicine, Medical Center Boulevard, Winston-Salem, NC 27157-1081, USA 2 Department of Molecular Medicine, Wake Forest University School of Medicine, Medical Center Boulevard, Winston-Salem, NC 27157-1081, USA
* Corresponding author. Tel: +1-336-716-0262; Fax: +1-336-716-9229; E-mail: brubin{at}wfubmc.edu
Received 19 September 2006; returned 28 January 2006; revised 9 January 2007; accepted 22 February 2007
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Abstract |
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Objectives: Pseudomonas aeruginosa exoproducts are potent triggers of immune responses in eukaryotic cells. Clarithromycin initially decreases, then increases and finally produces a sustained suppression of interleukin (IL)-8 secretion from normal human bronchial epithelial (NHBE) cells through inhibition and activation of extracellular signal-regulated kinase (ERK). This polyphasic immune response is referred to as immunomodulation.
Methods: We studied the effects of P. aeruginosa flagellin and alginate on IL-8 secretion from NHBE cells and how this was affected by clarithromycin or dexamethasone. We also assessed the upstream kinase cell signalling intermediates that appear to be responsible for flagellin-induced IL-8 secretion. ELISA was used to measure IL-8 in culture supernatants, and western blots were used to measure kinase and phosphokinase levels.
Results: Flagellin dose-dependently increased IL-8 secretion in NHBE cells at 24 h, whereas alginate had no effect on IL-8. Clarithromycin significantly decreased flagellin-induced IL-8 over the first 9 h but not at 24 h. A 60 min exposure to clarithromycin decreased flagellin-induced ERK phosphorylation, but at 24 h, clarithromycin increased phospho-ERK1/2 beyond the effect of flagellin alone. Pre-treatment with PD98059 (MEK inhibitor) decreased flagellin-induced IL-8 secretion by 47.7% (P < 0.0001) compared with control flagellin exposure and decreased basal IL-8 in the absence of flagellin by 27.9% compared with untreated control cells (P < 0.0001). Dexamethasone and PD98059 together had an additive suppressive effect on flagellin-induced IL-8 secretion.
Conclusions: P. aeruginosa flagellin, but not alginate, stimulates IL-8 secretion in NHBE cells in part through ERK1/2. This effect is modulated by clarithromycin, whereas suppression of IL-8 secretion by dexamethasone probably occurs through different pathways.
Keywords: interleukin-8 , bronchial epithelial cells , extracellular signal-regulated MAP kinases
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Introduction |
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Pseudomonas aeruginosa is an opportunistic pathogen that accounts for much of the morbidity in persons with cystic fibrosis (CF) and diffuse panbronchiolitis (DPB). A number of bacterial virulence factors, including flagellin, alginate and lipopolysaccharide (LPS), can increase the production of inflammatory mediators by airway epithelial cells. Flagellin, the major structural protein of the bacterial flagellum, is a potent trigger of innate immune responses in eukaryotic cells1 and is important in initiating inflammatory responses during pulmonary infection in vivo and in vitro.2,3
Interleukin (IL)-8, a potent neutrophil-activating chemokine, is produced in airway cells after stimulation by Pseudomonas gene products.4 Increased IL-8 in sputum and bronchoalveolar lavage fluid is associated with the severity of airway inflammation in CF and DPB. The extracellular signal-regulated kinase (ERK), p38 mitogen-activated protein kinase (MAPK) and c-jun N-terminal kinase (JNK) activation can increase IL-8 expression.
Long-term use of the 14- and 15-member macrolide antibiotics such as clarithromycin, erythromycin and azithromycin can decrease IL-8 production from airway cells5 and are routinely used to treat CF and DPB, but their mechanism(s) of action are not fully elucidated. We have reported that macrolide antibiotics have a significant and non-linear (or chaotic, in mathematical terms) effect on IL-8 secretion from normal human bronchial epithelial (NHBE) cells. This suppression, followed by increased activation and finally a sustained suppression of IL-8 secretion in response to clarithromycin, has been termed immunomodulation and is probably dependent on the ERK pathway.6
Alginate is an exopolysaccharide produced by mucoid P. aeruginosa isolates. Although both flagellin and alginate are thought to induce IL-8 secretion, these stimuli have not been directly compared in primary NHBE cultures. We compared these stimuli and evaluated whether stimulated IL-8 secretion was modulated by clarithromycin or dexamethasone. We also assessed the upstream kinase cell signalling intermediates that appear to be responsible for flagellin-induced IL-8 secretion.
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Reagents
Clarithromycin was donated by Abbott Labs (Abbott Park, IL, USA). LPS (10 mg/L, Escherichia coli serotype 0111: B4), dimethyl sulphoxide (DMSO), anti-ß-actin, anti-mouse-IgG-HRP and dexamethasone were purchased from Sigma Chemical Co. (St Louis, MO, USA). SP600125, a JNK-II inhibitor, PD98059, an MAP kinase/ERK kinase (MEK: an upstream kinase of ERK1/2) inhibitor, and SB203580, a p38 inhibitor, were purchased from Calbiochem (La Jolla, CA, USA). Phospho- and non-phospho-specific anti-p42/44 MAPK, and anti-I-
B-
as well as anti-rabbit-IgG horseradish peroxidase (HRP) antibodies, were purchased from Cell Signaling Technology Inc. (Beverly, MA, USA). Kinase inhibitors were dissolved in DMSO before use.
NHBE cells (Cambrex Bio Science; Walkersville, MD, USA) were plated at 3500 cells/cm2 in culture dishes in bronchial epithelial cell growth medium (BEGM) supplemented with 52 mg/L bovine pituitary extract, 0.5 mg/L hydrocortisone, 0.5 pg/mL human recombinant epidermal growth factor, 0.5 mg/L epinephrine, 10 mg/L transferrin, 5 mg/L insulin, 0.1 ng/mL retinoic acid and 6.5 ng/mL triiodothyronine without antibiotics and cultured at 37°C in a 5% CO2 incubator. Endotoxin-free media were used (<0.005 EU/mL). We used second passage cells for all experiments. NHBE cells were routinely cultured on 25 cm2 culture flasks coated with Type 1 rat-tail collagen (Sigma). The medium was changed at day 1 and subsequently every 48 h. The cells were grown to confluence for 6 days. Cultures without antibiotics were then transferred to 6-well or 35 mm dishes and seeded at 2 x 105 cells/well in order to reach confluence at 24 h. The medium was changed every 24 h. As growth factors can stimulate IL-8, cells were grown in supplement-free BEGM medium for 24 h before drug exposure. Culture supernatants were harvested, centrifuged and stored at –70°C until assayed. Cells were also cultured for 2 or 24 h following stimulation for western blot analysis. The cell number was counted using a haemocytometer and cell viability was assessed by Trypan Blue dye exclusion. Cell morphology was evaluated using a phase-contrast microscope (Olympus, Tokyo, Japan).
Flagellin was purified from P. aeruginosa ATCC 10145. P. aeruginosa flagellin was purified from overnight culture supernatants and then was further purified by using polymyxin B beads (Sigma-Aldrich) according to the manufacturer's instructions. Removal of endotoxin to <0.06 endotoxin U/mL was verified by using the Limulus amebocyte lysate detection kit (BioWhittaker, Walkersville, MD, USA).
The Pseudomonas flagellin preparations used in this work were endotoxin-free. We have performed control experiments using Pseudomonas flagellin to stimulate HEK293 cells stably transfected with human TLR-2, and we observed no activation of this receptor using our different flagellin preparations. We have compared the effect of flagellin from different bacterial species, including P. aeruginosa and Salmonella typhimurium. The activity of flagellin from these two species, as measured by the capacity to induce pro-inflammatory responses and activation of TLR-5 receptor, is similar.3
Alginate was purified from the Pseudomonas strain FRD1. Briefly, FRD1 was grown overnight at 37°C in 3% tryptic soy broth medium. Following the addition of 1 volume of saline, alginate was precipitated from the supernatant of this mucoid culture by the addition of an equal volume of 2% cetylpyridinium chloride (Sigma-Aldrich). After centrifugation at 25 000 g for 30 min, the pellet was re-suspended in the initial volume of 1 M NaCl. Finally, alginate was precipitated by the addition of 1 volume of chilled isopropanol, re-suspended in PBS and quantified in a colorimetric assay using alginic acid (Sigma-Aldrich) to plot a standard curve.
Cell stimulation and inhibition
Clarithromycin was dissolved in DMSO at a final concentration of 3 mg/mL; dexamethasone was dissolved in DMSO media at 1:49 (DMSO:BEGM) at a final concentration of 5 x 10–5 M. Solutions were stored at –20°C. The cells were exposed to clarithromycin (10 mg/L) or dexamethasone (10–6 M) once daily. These concentrations were chosen to match the reported mean therapeutic concentration in lung tissue. Flagellin or other test compounds were added to cultures 24 h before harvesting. The MEK inhibitor, PD98059 (10 µM), the p38 MAPK inhibitor, SB203580 (10 µM), or the JNK inhibitor II, SP600125 (10 µM), was added 30 min before exposure to flagellin.
Preliminary experiments showed that flagellin, alginate, LPS, clarithromycin, dexamethasone, DMSO and the inhibitors had no significant effect on cell counts or cell viability for up to 24 h at the concentrations used here (data not shown).
Following stimulation, culture supernatants were collected and centrifuged for 5 min at 200 g and stored at –20°C. Cytokine immunoreactivity was measured in culture supernatants by ELISA for IL-8 (Immunotech, France) with a detection limit of 8 pg/mL, according to the manufacturer's instructions. Optical density was measured at 450 nm on a microtitre plate reader (Spectra Max Plus; Molecular Devices Corporation, Sunnyvale, CA, USA). Software (Soft Max Pro version 2.0; Molecular Devices Corporation) was used to determine the concentrations by interpolation from standard curves. Final concentrations in each sample were calculated as the mean of the results at the sample dilution, yielding optical densities in the linear portion of the calibration curves.
The cells were washed twice with 2 mL cold PBS. After the supernatants were completely aspirated, the cells were lysed on ice in a modified radioimmunoprecipitation buffer (1% NP-40, 1% sodium deoxycholate, 150 mM NaCl, 10 mM Tris pH 7.5, 5 mM sodium pyrophosphate, 1 mM NaVO4, 5 mM NaF, 1 mg/L aprotinin, 1 mg/L leupeptin, 0.1 mM PMSF) for 15 min and then scraped from the dishes and collected into a tube. DNA was sheared by passing the lysate through a 27G needle. Insoluble material was removed by centrifugation at 20 000 g for 15 min at 4°C. The protein concentration of the resulting supernatant was quantified by the DC protein assay (Bio-Rad, Hercules, CA, USA), and the lysate was stored at –70°C until used. Equal amounts of protein extracts were loaded on a 12% SDS–PAGE mini gel and transferred to a nitrocellulose membrane (Bio-Rad) by electroblotting overnight. Membranes were rinsed with distilled water, incubated for 1 h at room temperature in Tris-buffered saline (0.8% NaCl and 20 mM Tris pH 7.6) with 0.1% Tween 20 (TBS-T) with 5% non-fat dry milk to block non-specific interactions, rinsed twice and washed three times for 10 min with TBS-T. After washing, membranes were exposed overnight to the primary antibodies 1 mg/L phospho- (p-) p44/42 MAPK (Thr-202/Tyr-204) rabbit polyclonal IgG (Cell Signaling Technology Inc.) at 4°C in TBS-T, with 5% milk. The blots were then washed and incubated at room temperature for 2 h with the anti-rabbit IgG HRP secondary antibody. Subsequently, the membranes were washed again and antibody binding was detected using LumiGLO chemiluminescent substrate peroxide (Cell Signaling Technology Inc.).
Membranes were stripped with a stripping buffer (100 mM ß-mercaptoethanol, 2% SDS, 62.5 mM Tris–HCl pH 6.7) for 30 min at 60°C. The blots were washed twice with TBS-T and re-probed with anti-p44/42 MAPK antibodies, followed by anti-rabbit-IgG HRP secondary antibody. Blots were stripped again, and equivalent protein loading was confirmed by western blot using anti-human ß-actin antibody, followed by anti-mouse IgG and HRP secondary antibody and detected by ECL (Amersham, Piscataway, NJ, USA). Western blot images were scanned and analysed using NIH Image J software (http://rsb.info.nih.gov/ij/, as accessed in December 2005).
Results are expressed as the mean values ± SD or SEM as indicated. Statistical analysis of data was performed using the StatView 5 statistics package (SAS Institute, Cary, NC, USA). Parametric testing was conducted after confirming that raw data were normally distributed. Data were analysed temporally and by drug exposure using ANOVA, and in comparison to a specific control using a two-sided, unpaired t-test. Conventionally, P < 0.05 was considered significant.
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Effects of flagellin, alginate and endotoxin (LPS) on IL-8 release
To evaluate the effect of P. aeruginosa flagellin and alginate on IL-8 release, NHBE cell supernatants were harvested 24 h after exposure to 10–9, 10–8 or 10–7 M flagellin or 40 or 80 mg/L alginate. We used LPS as a positive control for IL-8 secretion. Flagellin dose-dependently increased IL-8 secretion at 24 h (Figure 1a), whereas alginate did not stimulate IL-8 over control.
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Temporal effects of clarithromycin on flagellin-induced IL-8 release over 24 h
We evaluated the time-dependent effect of clarithromycin 10 mg/L on flagellin-induced IL-8 secretion during the initial 24 h of exposure. Clarithromycin decreased flagellin-induced IL-8 at 9 h (P < 0.05), but not at 24 h compared with flagellin alone (Figure 1b, top panel). By itself, clarithromycin initially decreased IL-8 secretion at 4 and 9 h (P < 0.05) and then increased IL-8 secretion above baseline at 18 and 24 h (P < 0.0001, Figure 1b, bottom panel).
Effect of clarithromycin, dexamethasone and inhibiting ERK1/2, p38MAPK or JNK on flagellin-induced IL-8 release
To evaluate the effect of clarithromycin and dexamethasone on flagellin-induced IL-8 secretion, NHBE cell supernatants were harvested 24 h after exposure to clarithromycin 10 mg/L, dexamethasone 10–6 M or both clarithromycin and dexamethasone, added to the media at the same time flagellin 10–8 M was added. At 24 h, dexamethasone inhibited IL-8 secretion in unstimulated cells by 27.7% (P < 0.001) and in flagellin-stimulated cells by 57.9% (P < 0.0001), whereas clarithromycin had no effect (Figure 1c).
We examined the role of intracellular kinase signalling pathways in cells treated with flagellin by using the MEK inhibitor PD98059, p38 MAPK inhibitor SB203580 or the JNK inhibitor SP600125, each at 10 µM. Cells were treated with a kinase inhibitor 30 min before adding flagellin 10–8 M, and cell supernatants were harvested after 24 h. Pre-treatment with PD98059 (MEK inhibitor) decreased flagellin-induced IL-8 secretion by 47.7% (P < 0.0001) compared with control flagellin exposure and decreased basal IL-8 in the absence of flagellin by 27.9% compared with untreated control cells (P < 0.0001, Figure 1c). SB203580 (p38 MAPK inhibitor) slightly increased IL-8 secretion both with (P = 0.23) and without flagellin (P < 0.001), and SP600125 (a selective inhibitor of JNK) also slightly increased IL-8 secretion both with (P = 0.05) and without flagellin (P = 0.21).
The combination of PD98059 and dexamethasone further decreased flagellin-induced IL-8 secretion by 81.8%. The combination significantly decreased IL-8 compared with PD98059 or dexamethasone alone (P < 0.001 for each) (Figure 1c).
Clarithromycin inhibits flagellin-induced phospho-ERK at 60 min but induces ERK phosphorylation at 24 h
We have reported that clarithromycin initially inhibits phospho-ERK (pERK) in unstimulated NHBE cells.6 We therefore tested whether a 60 min exposure to clarithromycin would decrease flagellin-induced phosphorylation of ERK. Flagellin 10–8 M increased pERK at 2 h, but a 60 min exposure to clarithromycin decreased pERK induced by flagellin by 78.0%, as shown in Figure 2a.
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After 24 h, ERK phosphorylation increased after flagellin exposure compared with control and clarithromycin further increased flagellin-induced pERK (Figure 2b). Dexamethasone had no effect on ERK phosphorylation.
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Discussion |
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We report here that P. aeruginosa flagellin dose-dependently increases IL-8 release from NHBE cells, but P. aeruginosa alginate does not affect IL-8 secretion. Consistent with previous reports,4 flagellin appears to be a potent IL-8 stimulator in lung epithelial cells. The importance of flagellin as a soluble virulence factor has been demonstrated by studies showing its potent pro-inflammatory effects.1 Bacterial flagellin elicits a strong inflammatory programme in epithelial cells, including the production of IL-8,4,7 iNOS expression and activity and NF-
B activation.8 Furthermore, flagellin plays a role in triggering adaptive immune responses by stimulating chemokine secretion by epithelial cells and subsequent migration and maturation of dendritic cells9 and by modulating T cell activation in vivo.
Here we show that clarithromycin decreases flagellin-induced IL-8 secretion at 9 h but enhances secretion at 24 h, whereas dexamethasone suppression of IL-8 was still present at 24 h. IL-8 secretion is increased by activation of NF-B. Dexamethasone decreases cytokine production, in part, through the Ikk-NF-B pathway. We show here that dexamethasone decreases flagellin-induced IL-8 at 24 h by 57.9% and baseline IL-8 secretion by 27.7% in the absence of flagellin. This effect of dexamethasone is consistent with NF-B inhibition.
IL-8 secretion is also increased by activation of MAPK. Flagellin increased ERK phosphorylation over 24 h, and PD98059, a specific inhibitor of MEK-1, inhibited flagellin-induced IL-8 secretion by 47.7%. PD98059 decreased baseline IL-8 secretion by 27.9% in the absence of flagellin and decreased IL-8 secretion by 17.5% from baseline in the presence of flagellin, although this was not statistically significant (P = 0.12). These results suggest that P. aeruginosa flagellin stimulates IL-8 secretion in NHBE cells in part through ERK1/2. Both SB203580 (p38 inhibitor) and SP600125 (JNK inhibitor) slightly increased IL-8 secretion with or without flagellin.
ERK can regulate IL-8 independently of NF-B. As well, ERK can directly affect the phosphorylation of IB- through IKK/ß and thus activate NF-B in human airway epithelial cells. We show here that the ERK inhibitor combined with dexamethasone additively decreased flagellin-induced IL-8 secretion compared with PD98059 or dexamethasone alone (P < 0.001), suggesting that both NF-B and ERK pathways are independently involved in flagellin-induced IL-8 secretion in NHBE cells.
A 60 min exposure to clarithromycin initially decreased flagellin-stimulated ERK phosphorylation, but at 24 h, clarithromycin increased ERK phosphorylation by 154% above that induced by flagellin alone. Clarithromycin also initially decreased flagellin-induced IL-8 secretion over 9 h but increased IL-8 at 24 h, and this was inhibited by PD98059. The time lag between the changes in MAPK and IL-8 protein levels is consistent with studies showing that pulmonary epithelial cells exposed to crystalline silica increase MAPK to a maximum at 30 min, whereas IL-8 release occurs 4–8 h later.10 We propose that clarithromycin initially decreases pro-inflammatory cytokine secretion via ERK inhibition, but that clarithromycin later increases ERK signalling, perhaps because of cross-talk among signal transduction pathways or through negative feedback, and suppression of IL-8 secretion by dexamethasone probably occurs through different pathways.
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None to declare.
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Footnotes |
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Present address. Third Department of Internal Medicine, National Defense Medical College, Tokorozawa-city, Japan. 
Present address. RI-CEDD, GlaxoSmithKline, Philadelphia, PA 19406, USA.
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Acknowledgements |
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We thank Drs Jun Tamaoki, Hajime Takizawa and Samir A. Shah for helpful discussions and Lauren Vannoy for technical assistance. This work was funded in part by the Japan Defense Agency (salary support for M. S.) and the Cystic Fibrosis Foundation (Research Grant SANCHE04G0, to Y. S. L.-B.).
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References |
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1 Liaudet L, Szabo C, Evgenov OV, et al. Flagellin from gram-negative bacteria is a potent mediator of acute pulmonary inflammation in sepsis. Shock (2003) 19:131–7.[CrossRef][ISI][Medline]
2
Zhang Z, Louboutin JP, Weiner DJ, et al. Human airway epithelial cells sense Pseudomonas aeruginosa infection via recognition of flagellin by Toll-like receptor 5. Infect Immun (2005) 73:7151–60.
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Lopez-Boado YS, Cobb LM, Deora R. Bordetella bronchiseptica flagellin is a proinflammatory determinant for airway epithelial cells. Infect Immun (2005) 73:7525–34.
4 DiMango E, Zar HJ, Bryan R, et al. Diverse Pseudomonas aeruginosa gene products stimulate respiratory epithelial cells to produce interleukin-8. J Clin Invest (1995) 96:2204–10.[ISI][Medline]
5
Oishi K, Sonoda F, Kobayashi S, et al. Role of interleukin-8 (IL-8) and an inhibitory effect of erythromycin on IL-8 release in the airways of patients with chronic airway diseases. Infect Immun (1994) 62:4145–52.
6
Shinkai M, Foster GH, Rubin BK. Macrolide antibiotics modulate ERK phosphorylation and IL-8 and GM-CSF production by human bronchial epithelial cells. Am J Physiol Lung Cell Mol Physiol (2006) 290:L75–L85.
7
Gewirtz AT, Navas TA, Lyons S, et al. Cutting edge: bacterial flagellin activates basolaterally expressed TLR5 to induce epithelial proinflammatory gene expression. J Immunol (2001) 167:1882–5.
8
Eaves-Pyles T, Murthy K, Liaudet L, et al. Flagellin, a novel mediator of Salmonella-induced epithelial activation and systemic inflammation: IB degradation, induction of nitric oxide synthase, induction of proinflammatory mediators, and cardiovascular dysfunction. J Immunol (2001) 166:1248–60.
9
Means TK, Hayashi F, Smith KD, et al. The Toll-like receptor 5 stimulus bacterial flagellin induces maturation and chemokine production in human dendritic cells. J Immunol (2003) 170:5165–75.
10
Ovrevik J, Lag M, Schwarze P, et al. p38 and Src-ERK1/2 pathways regulate crystalline silica-induced chemokine release in pulmonary epithelial cells. Toxicol Sci (2004) 81:480–90.
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