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Antimicrobial Agents and Chemotherapy, September 2004, p . 3457-3461, Vol . 48, No . 9

Azithromycin Inhibits MUC5AC Production Induced by the Pseudomonas aeruginosa Autoinducer N-(3-Oxododecanoyl) Homoserine Lactone in NCI-H292 Cells

Yoshifumi Imamura, Katsunori Yanagihara,* Yohei Mizuta, Masafumi Seki, Hideaki Ohno, Yasuhito Higashiyama, Yoshitsugu Miyazaki, Kazuhiro Tsukamoto, Yoichi Hirakata, Kazunori Tomono, Jun-ichi Kadota, and Shigeru Kohno

Second Department of Internal Medicine, School of Medicine, Nagasaki University, Nagasaki, Japan

Received 16 December 2003/ Returned for modification 25 April 2004/ Accepted 7 May 2004


   ABSTRACT

 
The features of chronic airway diseases, including chronic bronchitis, cystic fibrosis, bronchiectasis, and diffuse panbronchiolitis, include chronic bacterial infection and airway obstruction by mucus . Pseudomonas aeruginosa is one of the most common pathogens in chronic lung infection, and quorum-sensing systems contribute to the pathogenesis of this disease . The quorum-sensing signal molecule [N-(3-oxododecanoyl) homoserine lactone (3O-C12-HSL)] not only regulates bacterial virulence but also is associated with the immune response . In this study, we investigated whether 3O-C12-HSL could stimulate the production of a major mucin core protein, MUC5AC . The effect of a macrolide on MUC5AC production was also studied . 3O-C12-HSL induced NCI-H292 cells to express MUC5AC at both the mRNA and the protein levels in time- and dose-dependent manners . A 15-membered macrolide, azithromycin, inhibited MUC5AC production that was activated by 3O-C12-HSL . 3O-C12-HSL induced extracellular signal-regulated kinase (ERK) 1/2 and I-{kappa}B phosphorylation in cells, and this induction was suppressed by azithromycin . 3O-C12-HSL-induced MUC5AC production was blocked by the ERK pathway inhibitor PD98059 . Our findings suggest that the P . aeruginosa autoinducer 3O-C12-HSL contributes to excessive mucin production in chronic bacterial infection . Azithromycin seems to reduce this mucin production by interfering with intracellular signal transduction .


   INTRODUCTION

 
Mucus secretion is useful for host protection of mucosal surfaces against pathogens and irritants . However, in chronic airway diseases, mucus hyperproduction is an important hallmark of pathogenesis because excessive mucus secretion causes airway obstruction and impairment of gas exchange . Thus, preventing mucus overproduction is beneficial in chronic airway diseases . The major macromolecular component of mucus is mucin protein . Of the 14 currently identified human MUC genes, the MUC5AC gene encodes the major core protein of mucin secreted from the airway surface epithelium (3, 8, 10) .

Among many factors that contribute to mucin hypersecretion, bacterial infection is one of the most important in chronic airway infection, such as diffuse panbronchiolitis (DPB) and cystic fibrosis (CF) . Pseudomonas aeruginosa is a major pathogen causing chronic respiratory infection in DPB and CF . Previous studies showed that P . aeruginosa airway infection stimulates mucin production (6, 13, 17, 35) . These organisms use cell-to-cell communication, called a quorum-sensing system, to regulate the expression of their virulence factors (4) . This communication relies on the production of small, diffusible signal molecules called acyl homoserine lactones . These molecules activate transcriptional regulators and induce the transcription of many genes . There are at least two quorum-sensing systems, Las and Rhl (7, 20) . Their signal molecules are N-(3-oxododecanoyl) homoserine lactone (3O-C12-HSL) (22) and N-butyryl homoserine lactone (23), respectively . Recently, several studies suggested that 3O-C12-HSL is a signal transfer molecule and also has immunomodulatory activity (5, 27-29, 33) . 3O-C12-HSL has been shown to be a potent activator of transcription factor NF-{kappa}B (27) . In nonactivated cells, NF-{kappa}B is kept in the cytoplasm by the inhibitory protein I-{kappa}B . In stimulated cells, I-{kappa}B is phosphorylated and degraded by proteolysis, allowing NF-{kappa}B to enter the nucleus and activate the transcription of multiple genes . Because NF-{kappa}B activation is considered to be important in mucin production, we expected that 3O-C12-HSL may be a potent activator for mucin production .

Macrolide antibiotics have been shown to be effective for the treatment of chronic airway diseases, especially DPB (16) . Because macrolides have no bactericidal activities against P . aeruginosa, their effectiveness in chronic airway diseases has been considered to depend on other actions .

In this study, we investigated whether 3O-C12-HSL-induced mucin secretion from airway epithelial cells activates airway epithelial cells to produce the mucin core protein MUC5AC .


   MATERIALS AND METHODS

 
Materials. 3O-C12-HSL was kindly provided by B . H . Iglewski (School of Medicine and Dentistry, University of Rochester, Rochester, N.Y.) . PD98059, an extracellular signal-regulated kinase (ERK) pathway inhibitor, was obtained from Promega (Madison, Wis.) . Azithromycin was obtained from Pfizer (Tokyo, Japan) . RPMI 1640 medium, penicillin-streptomycin, fetal bovine serum, and a reverse transcription (RT)-PCR kit were obtained from Invitrogen (Carlsbad, Calif.) . A mouse MUC5AC monoclonal antibody (clone 45M1) was obtained from Neo Markers (Fremont, Calif.) . Goat anti-mouse and goat anti-rabbit horseradish peroxidase (HRP)-conjugated secondary antibodies were obtained from Bio-Rad (Hercules, Calif.) . Anti-ERK1/2, anti-phospho-ERK1/2, and anti-phospho-I-{kappa}B antibodies were obtained from Cell Signaling Technology (Beverly, Mass.) .

Cell culture. The NCI-H292 epithelial cell line was obtained from the American Type Culture Collection (Manassas, Va.) . The cells were cultured in RPMI 1640 medium with 10% fetal bovine serum, 100 U of penicillin/ml, and 100 µg of streptomycin/ml . The cells were grown at 37°C with 5% CO2 in fully humidified air and were subcultured twice weekly . The cells were seeded in a 12-well plate at 5 x 105 cells/well . When confluent, the cells were incubated in RPMI 1640 medium containing 0.5% fetal bovine serum for 24 h . The cells then were rinsed with serum-free RPMI 1640 medium and exposed to 1, 10, or 100 µM 3O-C12-HSL for 4, 8, 12, or 24 h . For inhibition studies, cells were pretreated with 10 or 100 µg of azithromycin/ml and 50 µM PD98059 for 30 min before exposure to 3O-C12-HSL . For controls, the cells were incubated with medium alone .

RT-PCR. The NCI-H292 cells were cultured, harvested, and subsequently washed three times with phosphate-buffered saline (PBS) containing 2% bovine serum albumin . RNA was extracted with Isogen (Nippon Gene, Toyama, Japan), and RT-PCR was performed to determine the mRNA level . Oligonucleotide primers for PCR were designed according to the published sequence for human MUC5AC (sense, ATC ACC GAA GGC TGC TTC TGT C; antisense, GTT GAT GCT GCA CAC TGT CCA G) (15) . PCR products were separated by electrophoresis through a 1% agarose gel containing ethidium bromide, and the signal intensity was analyzed with NIH Image software . ß-Actin controls were used to standardize the quantification of RNA samples . The ß-actin bands for each condition were measured, and MUC5AC/ß-actin ratios were compared .

ELISA. The NCI-H292 cells were plated in a 12-well plate, and MUC5AC protein was measured by an enzyme-linked immunosorbent assay (ELISA) (31) . The cells then were washed with cold PBS, exposed to trypsin, and formed into pellets at 700 x g at 4°C . The pellets were resuspended in lysis buffer (20 mM Tris-HCl [pH 8.0], 133 mM NaCl, 1% NP-40, 10% glycerol) . The preparation then was cleared by centrifugation, and the supernatant was saved as a whole-cell lysate . Protein concentrations in the lysates were measured, and equal amounts of total protein were incubated at 40°C in a 96-well plate until dry . The plates then were washed three times with PBS, blocked with 2% bovine serum albumin for 1 h at room temperature, washed again three times with PBS, and incubated with MUC5AC antibody diluted in PBS containing 0.05% Tween 20 for 1 h . The wells were washed three times with PBS, HRP-conjugated anti-goat immunoglobulin G was dispensed into each well and, after 4 h, the plates were washed three times with PBS . Color was developed with 3,3',5,5'-tetramethylbenzidine-peroxidase solution, and the reaction was stopped with 2 N H2SO4 . The absorbance was read at 450 nm .

Western blot analysis. Proteins were separated by reducing sodium dodecyl sulfate-12% polyacrylamide gel electrophoresis and transferred to nitrocellulose membranes (Amersham Pharmacia Biotech, Piscataway, N.J.) in 20% methanol-25 mM Tris-HCl-0.2 M glycine . Nonspecific binding was blocked by incubating the membranes with 5% skim milk in Tris-buffered saline-0.1% Tween 20 for 1 h at room temperature . Immunoreactive proteins were detected by incubating the membranes with rabbit anti-human ERK1/2, anti-phospho-ERK1/2, or anti-phospho-I-{kappa}B antibodies (each at 1:1,000) overnight at 4°C . Between each step, the membranes were washed three times for 5 min each time with Tris-buffered saline-0.1% Tween 20 . Subsequently, the membranes were incubated for 1 h with anti-rabbit immunoglobulin G conjugated to HRP (1:10,000), rewashed, and developed with enhanced chemiluminescence reagents (Amersham Pharmacia Biotech) .

Statistical analysis. All data were expressed as the mean and standard error of the mean (SEM) . Differences were examined for statistical significance by using one-way analysis of variance for comparisons involving more than two groups and Student's t test for comparisons between two groups . A P value of less than 0.05 denoted the presence of a statistically significant difference .


   RESULTS

 
3O-C12-HSL up-regulates MUC5AC gene and protein expression. To determine whether 3O-C12-HSL can induce mucin production in NCI-H292 cells, we evaluated MUC5AC expression at both the mRNA and the protein levels after the addition of 3O-C12-HSL to the cells . We found that 3O-C12-HSL activated the cells to express MUC5AC . The level of mRNA expression (Fig . 1A) and the protein level (Fig . 1B) were maximal at 8 and 12 h after the addition of 3O-C12-HSL, respectively . The up-regulation of MUC5AC by the addition of 3O-C12-HSL occurred in a dose-dependent manner, and the level of mRNA expression (Fig . 2A) and the protein level (Fig . 2B) were maximal at 100 µM 3O-C12-HSL .


FIG . 1 . Time-dependent effect of 3O-C12-HSL on MUC5AC synthesis . NCI-H292 cells were stimulated with 100 µM 3O-C12-HSL . (A) Total RNA was isolated, and the MUC5AC mRNA level was analyzed by RT-PCR . (B) MUC5AC protein was measured by an ELISA . Data are expressed as the mean and SEM for three experiments . A dagger indicates a P value of <0.01 for a comparison with culture medium alone.

 

FIG . 2 . Dose-dependent effect of 3O-C12-HSL on MUC5AC synthesis . Cells were stimulated with 1, 10, or 100 µM 3O-C12-HSL . (A) The mRNA level of MUC5AC expression at 8 h after the addition of 3O-C12-HSL was analyzed by RT-PCR . (B) MUC5AC protein was measured by an ELISA at 12 h after the addition of 3O-C12-HSL . Data are expressed as the mean and SEM for three experiments . An asterisk and a dagger indicate P values of <0.05 and <0.01, respectively, for comparisons with culture medium alone.

 
Azithromycin inhibits MUC5AC production by 3O-C12-HSL-activated cells. Certain macrolide antibiotics are considered to reduce mucus hypersecretion . We evaluated the effect of azithromycin on MUC5AC expression induced by 3O-C12-HSL . The cells were pretreated with azithromycin at 10 or 100 µg/ml for 30 min before the addition of 3O-C12-HSL . Azithromycin at 10 µg/ml reduced MUC5AC expression by more than 50% and azithromycin at 100 µg/ml reduced MUC5AC expression by more than 90% at both the mRNA (Fig . 3A) and the protein (Fig . 3B) levels . Azithromycin had no inhibitory effect on the basal expression of MUC5AC (data not shown) .


FIG . 3 . Effect of azithromycin on MUC5AC production in cells activated by 3O-C12-HSL . Cells were pretreated with 10 or 100 µg of azithromycin/ml for 30 min before exposure to 3O-C12-HSL . (A) MUC5AC mRNA expression at 8 h after the addition of 3O-C12-HSL was determined by RT-PCR . (B) MUC5AC protein was measured by an ELISA at 12 h after the addition of 3O-C12-HSL . Data are expressed as the mean and SEM for three experiments . An asterisk indicates a P value of <0.05 for a comparison with culture medium alone.

 
Increased I-{kappa}B and ERK1/2 phosphorylation in 3O-C12-HSL-activated cells. To study the involvement of NF-{kappa}B activation in MUC5AC production induced by 3O-C12-HSL stimulation of NCI-H292 cells, we treated the cells with 3O-C12-HSL for 10 min and evaluated I-{kappa}B phosphorylation by Western blotting . As shown in Fig . 4A, I-{kappa}B phosphorylation was stimulated in 3O-C12-HSL-treated cells but not in untreated cells . To better understand the activation of NF-{kappa}B and the subsequent production of MUC5AC, we examined a potential role for mitogen-activated protein kinases . When the cells were stimulated with 3O-C12-HSL, the phosphorylated forms of ERK1/2 were induced . Next, we examined the effect of azithromycin on ERK1/2 phosphorylation . Pretreatment with azithromycin exerted an inhibitory effect on the phosphorylation of ERK1/2 (Fig . 4B) .


FIG . 4 . Phosphorylation of I-{kappa}B and ERK1/2 induced by 3O-C12-HSL . Cells were treated with 3O-C12-HSL for 10 min and evaluated by Western blotting . (A) I-{kappa}B phosphorylation was induced in 3O-C12-HSL-treated cells but not in untreated cells . (B) ERK1/2 phosphorylation was induced in 3O-C12-HSL-treated cells . This phosphorylation was blocked by the addition of azithromycin . Data are representative of three separate experiments . p, phospho.

 
PD98059, an ERK1/2 inhibitor, blocks 3O-C12-HSL-induced MUC5AC production. To confirm the involvement of mitogen-activated protein kinases in MUC5AC production, we examined the effect of the ERK pathway inhibitor PD98059 on MUC5AC production induced by 3O-C12-HSL . PD98059 (50 µM) blocked the synthesis of MUC5AC from 3O-C12-HSL-treated cells . This result was observed at both the mRNA (Fig . 5A; treated after 8 h and analyzed by RT-PCR) and the protein (Fig . 5B; treated after 12 h and analyzed by ELISA) levels . PD98059 had no inhibitory effect on the basal expression of MUC5AC (data not shown) .


FIG . 5 . Effect of PD98059 on MUC5AC production in cells activated by 3O-C12-HSL . Cells were pretreated with 50 µM PD98059 for 30 min prior to 3O-C12-HSL stimulation . (A) MUC5AC mRNA expression was determined by RT-PCR . (B) MUC5AC protein was measured by an ELISA . Data are expressed as the mean and SEM for three experiments . An asterisk indicates a P value of <0.05 for a comparison with culture medium alone.

 

   DISCUSSION

 
Chronic airway diseases, such as diffuse DPB and CF, are characterized by inflammation of the airways and mucus hypersecretion (11, 12) . MUC5AC is the major core protein of mucin secreted from the airway surface epithelium . MUC5AC expression is up-regulated by various factors, such as inflammatory mediators (30) and cytokines (2, 19, 25, 34, 36) .

Clinically, P . aeruginosa infection in the lungs is often accompanied by mucus overproduction . The supernatant of P . aeruginosa contains an activity that up-regulates transcription of the mucin genes (17) . Lipopolysaccharide has already been confirmed to activate MUC5AC in the supernatant (6, 35) .

In CF patients, P . aeruginosa has been found to grow to high concentrations (108 CFU/ml of sputum) and to live in microcolonies or biofilms (26) . 3O-C12-HSL in the biofilm of P . aeruginosa was detected at ~600 µM in an in vitro model (1) . It was also reported that 3O-C12-HSL could be detected in the sputum of CF patients colonized with P . aeruginosa (26) . In recent years, it has been revealed that 3O-C12-HSL acts not only as a regulator of virulence factor genes but also as a potent activator of the immune response in eukaryotic cells (5, 27, 29, 33) . In this study, we showed that 3O-C12-HSL induced MUC5AC expression in airway epithelial cells (Fig . 1) . These data suggest that a quorum-sensing system may play an important role in mucus hypersecretion .

Long-term treatment with macrolide antibiotics is considered to be effective in DPB (16) and CF (24) . Although the reason why macrolides are effective in such diseases remains unclear, one possible mechanism is their inhibitory effect on mucus . Some studies reported that erythromycin reduced mucus production in vitro (9, 32) . Kaneko et al . recently reported that clarithromycin could directly inhibit MUC5AC expression in a murine model of chronic respiratory P . aeruginosa infection (13) . In the present study, we found that 10 and 100 µg of azithromycin/ml inhibited MUC5AC production induced by 3O-C12-HSL . The concentration of azithromycin was reported to reach more than 3 and 400 µg/ml in epithelial lining fluid and in alveolar macrophages after oral administration, respectively (21) . Therefore, 10 and 100 µg of azithromycin/ml may be attainable concentrations in the lungs . Because DPB patients are usually treated with macrolide antibiotics at concentrations below the MICs, it has been considered that macrolides may affect the host defense mechanism . Our data support this hypothesis from the aspect of inhibitory efficacy against mucus hypersecretion . We also examined the effect of N-butyryl homoserine lactone on MUC5AC production, but hyperexpression of MUC5AC was not detected (data not shown) .

Among a variety of transcriptional regulators, NF-{kappa}B has been shown to play an important role in mucin production (18) . 3O-C12-HSL is reported to be a potent activator of NF-{kappa}B (27) . The activation of NF-{kappa}B is controlled by an inhibitory subunit, I-{kappa}B, which retains NF-{kappa}B in the cytoplasm . Phosphorylation of I-{kappa}B leads to the nuclear translocation of NF-{kappa}B, which activates the expression of target genes in the nucleus . We showed that phosphorylation of I-{kappa}B was induced in 3O-C12-HSL-treated cells, suggesting that NF-{kappa}B was activated by the addition of 3O-C12-HSL . To determine which signal pathway is involved upstream of I-{kappa}B phosphorylation, we examined the ERK pathway . We found that ERK1/2 was phosphorylated by 3O-C12-HSL and that PD98059, an ERK pathway inhibitor, inhibited MUC5AC expression . These data indicate that the ERK pathway is important in 3O-C12-HSL-induced MUC5AC production and suggest that this pathway should be an attractive target for anti-inflammatory therapy .

Macrolide antibiotics have been shown to affect NF-{kappa}B activation (14) . In our experiments, azithromycin inhibited the ERK1/2 phosphorylation induced by 3O-C12-HSL . These data indicate that azithromycin reduces MUC5AC production through an inhibitory effect on the ERK pathway . In addition, we also found that clarithromycin could inhibit the phosphorylation of ERK1/2 (data not shown), suggesting that clarithromycin should decrease MUC5AC production induced by 3O-C12-HSL .

In summary, the P . aeruginosa autoinducer 3O-C12-HSL was capable of up-regulating major mucin core protein MUC5AC . Azithromycin seemed to reduce MUC5AC production by interfering with intracellular signal transduction . Our results provide a possible explanation for the clinical efficacy of macrolides in chronic Pseudomonas airway infection .

 


   ACKNOWLEDGMENTS

 
We thank B . H . Iglewski for providing 3O-C12-HSL and F . G . Issa (Word-Medex, Sydney, Australia) for assistance with editing the manuscript .


   FOOTNOTES

 
* Corresponding author . Mailing address: Second Department of Internal Medicine, School of Medicine, Nagasaki University, Nagasaki 852-8501, Japan . Phone: 81-95-849-7273 . Fax: 81-95-849-7285 . E-mail: kyana-ngs{at}umin.ac.jp .


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