Published ahead of print on June 26, 2003, doi:10.1164/rccm.200212-1437OC
© 2003 American Thoracic Society
In Vivo and In Vitro Effects of Macrolide Antibiotics on Mucus Secretion in Airway Epithelial CellsDepartment of Otorhinolaryngology and Third Department of Internal Medicine, Mie University School of Medicine, Edobashi, Tsu, Mie, Japan Correspondence and requests for reprints should be addressed to Takeshi Shimizu, M.D., Department of Otorhinolaryngology, Mie University School of Medicine, 2-174 Edobashi, Tsu, Mie 514, Japan. E-mail: tshimizu{at}clin.medic.mie-u.ac.jp
To examine the in vivo effects of macrolide antibiotics on mucus hypersecretion, we induced hypertrophic and metaplastic changes of goblet cells in rat nasal epithelium by intranasal instillation of ovalbumin (OVA) in OVA-sensitized rats and by intranasal LPS instillation. Oral administration of clarithromycin (CAM) (5–10 mg/kg) significantly inhibited OVA- and LPS-induced mucus production and neutrophil infiltration, whereas josamycin and ampicillin showed no effect. In vitro effects of macrolide antibiotics on airway epithelial cells were examined using NCI-H292 cells and human nasal epithelial cells cultured in air–liquid interface. Mucus secretion was evaluated by ELISA using anti-mucin monoclonal antibodies (anti-MUC5AC and HCS18). CAM and erythromycin significantly inhibited spontaneous and tumor necrosis factor–  (20 ng/ml)–induced mucus secretion from NCI-H292 cells at 10-6 to 10-7 M and from human nasal epithelial cells at 10-4 to 10-5 M. MUC5AC messenger RNA expression was also significantly inhibited. These results indicate that the 14-member macrolide antibiotics, CAM and erythromycin, exert direct inhibitory effects on mucus secretion from airway epithelial cells and that they may be useful for the treatment of mucus hypersecretion caused by allergic inflammation and LPS stimulation.
Key Words: ovalbumin • lipopolysaccharide • goblet cell • nose Clarithromycin (CAM) and erythromycin (EM) are 14-member macrolide antibiotics widely used for the treatment of airway inflammation. Low-dose, long-term macrolide therapy has recently been reported to be very effective in patients with chronic airway diseases such as diffuse panbronchiolitis (1), chronic bronchitis (2, 3), bronchial asthma (4, 5), and chronic sinusitis (6, 7). However, the mechanism of this clinical effectiveness is still unclear. It has been suggested that it depends on antiinflammatory action of macrolides rather than on their antibacterial effect because (1) treatment with low dose, in which only half of the usual dose is used, has been associated with good response; (2) it is required of long-term treatment; and (3) this treatment has been shown to be also effective in patients infected with pathogens insensitive to macrolides such as Pseudomonas aeruginosa. Many investigators have demonstrated the antiinflammatory action of macrolides, which includes their immunomodulatory effect on inflammatory cells (8–11), the modulation of cytokine production from epithelial cells (12–15), and the inhibition of bacterial functions and biofilm formation (16, 17). Mucus hypersecretion is a major consequence of airway inflammation. Therapy with macrolide results in a significant reduction in the amount of secreted mucus, sputum, and rhinorrhea. Tamaoki and coworkers (18) have reported that EM significantly inhibited LPS-induced mucus secretion in guinea pig trachea in vivo. In our previous study (19), EM also inhibited hypertrophic and metaplastic changes of goblet cells induced by LPS in rat nasal epithelium. However, the effect of macrolides on antigen-induced mucus hypersecretion is unclear, and little is known about the direct effect on mucus secretion from airway epithelial cells.
In the present study, to demonstrate the effects of macrolide antibiotics on mucus hypersecretion in airway epithelial cells, we evaluated (1) the in vivo effects of macrolides on antigen-induced mucus production in rat nasal epithelium, compared with LPS-induced changes and (2) the in vitro effects on spontaneous and tumor necrosis factor-
Mucus Hypersecretion in Rat Nasal Epithelium Sensitization and challenge of rats were performed as previously reported (21). Male Fisher-344 rats (6 weeks old) were immunized with an intraperitoneal injection of 200 µg ovalbumin (OVA) (grade V; Sigma Chemical Co., St. Louis, MO) and 10 mg of Al(OH)3 at Days 1, 2, 3, and 11. At Day 19, 0.1 ml saline containing 10 mg of OVA was instilled into the nasal cavity for 3 days. For LPS stimulation, rats (9 weeks old) were intranasally instilled with 0.1 ml saline containing 0.1 mg LPS from Escherichia coli 0111:B4 (Sigma Chemical Co.) for 3 days. CAM (Taishou Pharmaceutical, Tokyo, Japan; 1, 5, and 10 mg/kg), josamycin (JM) (Yamanouchi Pharmaceutical, Tokyo, Japan; 10 mg/kg), or ampicillin (ABPC) (Sigma Chemical Co.; 30 mg/kg) in 0.5% carboxymethyl cellulose sodium salt was given orally to rats 1 hour before the intranasal instillation of OVA or LPS for 3 days. Twenty-four hours after the last intranasal instillation of OVA or LPS, rats were killed, and the nasal cavity was transversely sectioned at the level of incisive papilla. Paraffin sections were stained with alcian blue–periodic acid–Schiff and hematoxylin. The percentage area of alcian blue-periodic acid-Schiff–stained mucosubstance in the epithelium was determined by an image analyzer (22).
Cell Cultures Human nasal epithelial cells were obtained from nasal polyps from patients with chronic sinusitis. The dissociated epithelial cells were cultured in a serum-free hormone supplement medium according to a technique described previously (23). An air–liquid interface was created when the cells became confluent, and the cultures were supplemented with medium containing 5 x 10-8 M retinoic acid. When the NCI-H292 cells become confluent, or at the 14 day culture in the air–liquid interface of nasal epithelial cells, CAM, EM (Shionogi Pharmaceutical, Osaka, Japan), or ABPC was added to the culture medium (pH 7.2) for a 24 hour period.
ELISA
Reverse Transcription–Polymerase Chain Reaction
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In Vivo Effects on Mucus Production Intranasal instillation of OVA for three consecutive days induced hypertrophic and metaplastic changes of goblet cells in nasal septal epithelium of OVA-sensitized rats (Figure 1) . Similar changes of goblet cells occurred after Day 3 of LPS instillation. Only a few goblet cells were observed in control groups (untreated control, saline-instilled, and sham-sensitized rats challenged with saline or OVA and OVA-sensitized rats challenged with saline).
The effects of macrolides on OVA-induced mucus production in OVA-sensitized allergic rats are shown in Figures 1 and 2 . Oral administration of CAM (5–10 mg/kg) significantly inhibited OVA-induced mucus production, whereas treatment with JM (16-member macrolide) or ABPC showed no significant effect. LPS-induced mucus production was also significantly inhibited by treatment with CAM (5–10 mg/kg), whereas JM or ABPC had no effect (Figures 1 and 3) .
OVA-sensitized rats, challenged with OVA, showed significant infiltration of eosinophils and neutrophils into nasal septal mucosa. Treatment with CAM, JM, or ABPC had no significant effect on eosinophil infiltration; the number of eosinophils in nasal septal mucosa/8 mm (4 mm on each side of the nasal septum x 2) was 50.8 ± 15.5, 39.5 ± 14.8, and 36.2 ± 10.7, respectively (control group, 44.2 ± 12.7). However, OVA-induced neutrophil infiltration was significantly inhibited by treatment with CAM, whereas JM or ABPC showed no effect on neutrophil infiltration (Figure 4A) . Intranasal instillation of LPS induced neutrophil infiltration in nasal septal mucosa, and CAM also inhibited LPS-induced neutrophil infiltration (Figure 4B).
In Vitro Effects on Mucin Secretion NCI-H292 cells. CAM and EM showed an inhibitory effect on mucin secretion from NCI-H292 cells at 10-5 to 10-6 M (Figure 5A) . The percentage inhibition of MUC5AC secretion was 44.7 ± 10.2% and 44.0 ± 8.6% at 10-6 M, respectively. The secretion of HCS18-reactive mucin was significantly inhibited at 10-7 M (percentage of inhibition: 44.6 ± 13.5% and 47.3 ± 5.4%, respectively, Figure 5B). ABPC (10-5 to 10-6 M) had no significant effects on mucin secretion (data not shown). TNF- (20 ng/ml) significantly stimulated mucin secretion, and CAM significantly inhibited TNF-–induced mucin secretion, whereas ABPC showed no effect (Figure 6)
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Human nasal epithelial cells. After 14 days of culture in air–liquid interface, secretory cell differentiation was induced in approximately 25% of cultured cells (21). The medium in the lower compartment did not react with HCS18. Only the samples collected from the apical side contained HCS18-reactive mucin, indicating that there was a polarity in mucin secretion. CAM had a dose-dependent (10-4 to 10-5 M) inhibitory effect on apical mucin secretion from cultured human nasal epithelial cells (Figure 7A) . EM also inhibited mucin secretion at 10-4 M; the percentage inhibition of MUC5AC secretion was 35.6 ± 2.2% and 40.8 ± 2.7% at 10-4 M, respectively. MUC5AC secretion was also significantly inhibited by 10-5 M CAM; however, 10-6 M CAM had no effect. These macrolides also showed a time-dependent (24–72 hour) inhibitory effect on secretion of HCS18-reactive mucin (Figure 7B). Lactate dehydrogenase release assay showed that this effect was not due to cytotoxicity. TNF- (20 ng/ml) significantly stimulated mucin secretion; CAM significantly inhibited TNF-–induced mucin secretion, whereas ABPC showed no effect (Figure 8)
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MUC5AC mRNA expression. Changes of MUC5AC gene expression were evaluated by reverse transcription–polymerase chain reaction. Treatment with macrolide antibiotics, CAM or EM (10-6 M), significantly inhibited MUC5AC mRNA expression of NCI-H292 cells (Figure 9) . The percentage inhibition of MUC5AC mRNA levels, as corrected by glyceraldehyde-3-phosphate dehydrogenase, was 37.1 ± 18.6% and 35.3 ± 3.1%, respectively. ABPC at 10-6 M had no significant effect on MUC5AC mRNA expression by NCI-H292 cells. MUC5AC mRNA expression of human nasal epithelial cell was also significantly inhibited by 10-4 M CAM and EM (percentage inhibition: 31.4 ± 20.1% and 35.4 ± 17.3%, respectively). CAM at 10-5 or 10-6 M and ABPC at 10-4 M had no significant effect on MUC5AC mRNA expression by human nasal epithelial cells.
In the present study, hypertrophic and metaplastic changes of goblet cells were induced in rat nasal epithelium by intranasal challenge with OVA in OVA-sensitized rats or by intranasal LPS instillation. A similar increase of epithelial mucosubstance occurred 24 hours after Day 3 of OVA or LPS instillation. Oral administration of CAM (14-member macrolide) significantly inhibited OVA- or LPS-induced mucus production, whereas JM (16-member macrolide) or ABPC showed no effect. This is the first report showing the in vivo effects of 14-member macrolide on antigen-induced mucus production. Recently, the use of macrolides has been extended to the treatment of patients with bronchial asthma; however, the clinical usefulness of 14-member macrolides on allergic airway inflammation is still controversial. Several clinical studies demonstrated that 14-member macrolides CAM or roxithromycin reduced airway hyper-responsiveness associated with eosinophilic inflammation in asthma (4, 5). Konno and coworkers (10) revealed that roxithromycin inhibited cytokine (interleukin [IL]-3, IL-4, IL-5) secretion from human monocytes and T cells in vitro. On the contrary, Iino and coworkers (24) reported that EM treatment is not effective in patients with chronic sinusitis whose subepithelial mucosa showed marked eosinophil infiltration. The results of the present study suggest that 14-member macrolides may be useful for the treatment of mucus hypersecretion caused not only by bacterial infection but also by allergic inflammation. It has been reported that macrolide antibiotics achieve higher concentrations in tissues other than blood and that they extensively diffuse into respiratory tissues such as nasal mucosa, tracheobronchus, lung, and epithelial lining fluid (25, 26). The therapeutic concentrations of macrolide antibiotics (CAM, EM, and JM) are 10-5 to 10-6 M in tissues. In our in vivo study, oral administration of 5–10 mg/kg CAM significantly inhibited epithelial mucus production, and a previous study demonstrated that this is comparable with tissue concentration of 10-5 to 10-6 M in rats (27). The mechanism by which 14-member macrolides inhibit antigen- or LPS-induced mucus production is not well understood. We found that CAM significantly inhibited neutrophil infiltration into nasal mucosa induced by intranasal challenge with OVA in OVA-sensitized rats or by intranasal LPS instillation, whereas antigen-induced eosinophil infiltration was not affected. Both in vivo and in vitro studies have demonstrated that 14-member macrolide inhibits neutrophil migration. EM treatment reduced the number of infiltrating neutrophils and neutrophil chemotactic activity of bronchoalveolar lavage fluid from patients with diffuse panbronchiolitis (12). Takizawa and coworkers (13) reported that EM and CAM suppressed secretion and mRNA expression of IL-8, a neutrophil chemoattractant, in human bronchial epithelial cells in vivo and in vitro. We showed that CAM treatment reduced the number of neutrophils and IL-8 concentration in nasal secretion from patients with chronic sinusitis (6). The inhibitory effect of macrolide on LPS-induced neutrophil infiltration is consistent with previous studies in which EM and CAM reduced LPS- or IL-8–induced neutrophil infiltration in lower airways of mice (12) and guinea pigs (18). In our previous study (21), LPS-induced mucus production was significantly inhibited in neutrophil-depleted rats, produced by intraperitoneal injection of anti-rat neutrophil antiserum. Neutrophil elastase inhibitor ONO 5046 partially inhibited LPS-induced change, and elastase-induced mucus production was not inhibited by neutrophil depletion (28). These results indicate that neutrophil, especially neutrophil elastase, is an important mediator of LPS-induced mucus production and that the in vivo effects of 14-member macrolides may be partly caused indirectly through the suppression of neutrophil infiltration. However, neutrophil infiltration is not essential for mucus production in allergic inflammation because antigen-induced mucus production was not affected by neutrophil depletion (21). These results indicate that CAM administration inhibited antigen-induced mucus production independent of neutrophil infiltration. Little is known about the direct effects of 14-member macrolide on mucus secretion from airway epithelial cells. Goswami and coworkers (29) showed that EM reduces radiolabeled glucosamine secretion from cultured explant of human bronchial epithelium and from cultured endometrial adenocarcinoma cells in vitro. This is the only article showing the direct inhibitory effects of macrolides on epithelial mucus secretion. However, the explant culture contains other cell types—fibroblast and inflammatory cells together with epithelial cells. It is difficult to study the direct effects on epithelial cells because these other cells may affect the response of epithelial cells. Anti-mucin monoclonal antibodies will be better tools for measuring secreted mucus because airway secretions contain a complex mixture of proteins. In addition, the effect on mucin gene expression has not been as yet evaluated. To elucidate the direct effects on mucus secretion from airway epithelial cells, we developed an in vitro culture system for human nasal epithelial cells with many differentiated secretory cells by using an air–liquid interface culture. After 14 days in the air–liquid interface, secretory cells occupied approximately 25% of epithelial cells, and polarized mucus secretion was observed (23). Mucus secretion in cell culture was quantitatively measured using anti-mucin monoclonal antibodies: anti-MUC5AC antibody and antibody HCS18. We have developed the antibody HCS18 against purified human nasal mucin obtained from patients with chronic sinusitis; this antibody specifically reacts with nasal mucin of epithelial secretory (goblet) cells (20).
In the present study, both CAM and EM significantly inhibited spontaneous mucin secretion, and CAM significantly inhibited TNF- In conclusion, we have induced hypertrophic and metaplastic changes of goblet cells in rat nasal epithelium by intranasal challenge with OVA in OVA-sensitized rats and by intranasal LPS instillation, and we have demonstrated in this model that 14-member macrolides significantly inhibit epithelial mucus production and mucosal neutrophil infiltration produced by allergic inflammation and by LPS stimulation. We have also demonstrated that 14-member macrolides directly inhibit mucus secretion and MUC5AC mRNA expression in NCI-H292 cells and human nasal epithelial cells. These novel findings may explain the clinical efficacy of 14-member macrolides in patients with chronic airway inflammations.
Conflict of Interest Statement: T.S. has no declared conflict of interest; S.S. has no declared conflict of interest; R.H. has no declared conflict of interest; E.C.G. has no declared conflict of interest; Y.M. has no declared conflict of interest. Received in original form December 10, 2002; accepted in final form June 19, 2003
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