Novel effects of azithromycin on tight junction proteins in human airway epithelia

Abstract

The macrolide antibiotic azithromycin improves lung function and prognosis among patients with cystic fibrosis or diffuse panbronchiolitis, independently of bacterial eradication. Anti-inflammatory effects have been implicated, but data from in vivo studies are scarce, and the link between abnormal electrolyte content in airway surface liquid and bronchial infections remains uncertain. In the present study, we treated human airway epithelia on filter supports with azithromycin and monitored transepithelial electrical resistance. We found that azithromycin increased transepithelial electrical resistance of airway epithelia in a dose-dependent manner. Immunocytochemistry and Western blotting showed that addition of azithromycin changed the locations of proteins in cell cultures and induced processing of the tight junction proteins claudin-1 and claudin-4, occludin, and junctional adhesion molecule-A. These effects were reversible, and no effect was seen when cells were treated with penicillin or erythromycin. The data indicate that azithromycin increases the transepithelial electrical resistance of human airway epithelia by changing the processing of tight junction proteins. The results are novel and may help explain the beneficial effects of azithromycin in patients with cystic fibrosis, diffuse panbronchiolitis, and community-acquired pneumonia.

FIG. 1.

(A) Effect of azithromycin on TER of human airway epithelia in vitro. Human airway epithelial cells were cultured on Transwell filters. After reaching confluence, azithromycin (0.4, 4.0, and 40 μg/ml) was added to the basolateral side of epithelia every 48 h for 8 days. TER was measured using a Millicell-ERS electrical resistance system. Data are given as means ± SEM (n = 3). Azithromycin increased TER in a dose-dependent manner. (B) Azithromycin increases TER in human airway epithelia. Measurements were made at day 0 (open bars), before any treatment, and at day 4 (solid bars), after four doses of 40 μg/ml azithromycin. Data are given as means ± SEM (n = 6). Forty micrograms per milliliter of azithromycin added daily increased TER significantly (P < 0.0001). (C) Growth curve. Human airway epithelial cells were cultured on 24-well plates and treated continuously with 40 μg/ml azithromycin. Data are given as means ± SEM (n = 3). Azithromycin (40 μg/ml) decreases cell proliferation. AZM, azithromycin.

FIG. 2.

(A) Immunocytochemical analyses of the effects of azithromycin on the expression of claudin-1 and -4. Human airway epithelial cells were cultured on chamber slides. Green indicates expression of claudin-1 or -4. The left row is the control. The right row shows cells after treatment with 40 μg/ml azithromycin. (B) Western blot of the effects of azithromycin on the expression of claudin-1 and -4. Equal amounts of protein from cells treated with different concentrations of azithromycin were subjected to Western blotting. Blotting for claudin-1 and -4 revealed a rapidly migrating band in lysates from cells treated with 40 μg/ml azithromycin. (C) Effect of penicillin and erythromycin on the expression of claudin-1. Equal amounts of protein from human airway epithelial cells treated with penicillin or erythromycin were subjected to Western blotting. Unlike azithromycin, neither penicillin nor erythromycin produced a rapidly migrating band. CLDN, claudin; PCN, penicillin; EM, erythromycin.

FIG.3.

Effect of azithromycin on the expression of occludin, JAM-A, and E-cadherin. (A) Immunocytochemistry. Human airway epithelial cells were cultured on chamber slides. Green indicates expression of junctional molecules. The left row is the control. The right row shows cells after treatment with 40 μg/ml azithromycin. (B) Equal amounts of protein from cells treated with different concentrations of azithromycin were subjected to Western blotting. Blotting for occludin and JAM-A revealed a rapidly migrating band in lysates from cells treated with 40 μg/ml azithromycin.

FIG.3.

Effect of azithromycin on the expression of occludin, JAM-A, and E-cadherin. (A) Immunocytochemistry. Human airway epithelial cells were cultured on chamber slides. Green indicates expression of junctional molecules. The left row is the control. The right row shows cells after treatment with 40 μg/ml azithromycin. (B) Equal amounts of protein from cells treated with different concentrations of azithromycin were subjected to Western blotting. Blotting for occludin and JAM-A revealed a rapidly migrating band in lysates from cells treated with 40 μg/ml azithromycin.

FIG. 4.

Reversible effect of azithromycin on claudin-1 and occludin. Confluent cells were treated with 40 μg/ml azithromycin daily for 4 days. After day 4, cells were cultured with medium alone. Equal amounts of protein were subjected to Western blotting. Protein was extracted before addition of azithromycin and then daily for 7 days and again on day 9. CLDN1, claudin-1.