Anti-inflammatory effects of azithromycin in cystic fibrosis airway epithelial cells

doi:10.1016/j.bbrc.2006.09.132

Biochemical and Biophysical Research Communications

Volume 350, Issue 4, 1 December 2006, Pages 977-982

https://doi.org/10.1016/j.bbrc.2006.09.132 Get rights and content

Abstract

We aimed at identifying molecular mechanisms for anti-inflammatory effects of azithromycin (AZM) suggested by clinical evidences. IL-8 expression and DNA binding activity of two key pro-inflammatory transcription factors (TF), NF-κB and AP-1, were investigated in cystic fibrosis (CF) and isogenic non-CF airway epithelial cell lines. AZM reduced about 40% of IL-8 mRNA and protein expression (n = 9, p = 0.02, and n = 4, p = 0.00011) in CF cells reaching the levels of non-CF cells. In the presence of AZM we found about 50% and 70% reduction of NF-κB and AP-1 DNA binding, respectively (n = 3, p = 0.01, and n = 3, p = 0.0017), leading to levels of non-CF cells. The relevance of NF-κB and AP-1 in regulating IL-8 promoter transcriptional activity was demonstrated by gene reporter assays (n = 4, p = 8.54 × 10⁻⁷, and n = 4, p = 6.45 × 10⁻⁶). Our data support the anti-inflammatory effects of AZM in CF cells, indicating inhibition of transcription of pro-inflammatory genes as possible mechanism, thus providing a rationale for the possible use of specific TF inhibitors for therapy.

Section snippets

Materials and methods

Cell cultures. 16HBE14o-AS3 and 16HBE14o-S1 cells (kind gift of P. Davis, Case Western Reserve University School of Medicine, Cleveland, OH), with CF and non-CF phenotypes [19], were cultured in E-MEM supplemented with 10% fetal bovine serum in the presence of the selective agent G418 200 μg/ml (Sigma, St. Louis, MO) on coated surfaces as previously described [19]. The passage numbers of cells ranged from 18 to 37 for 16HBE14o-AS3 and from 27 to 44 for 16HBE14o-S1. The results were confirmed at

Regulation of IL-8 mRNA

We analyzed the basal levels of IL-8 mRNA in the 16HBE14o-AS3 cell line with CF phenotype and in the isogenic 16HBE14o-S1 non-CF cells in order to assess the specific constitutive pro-inflammatory state of CF airway epithelial cells.

CF cells express higher levels of IL-8 mRNA than isogenic non-CF cells (n = 7, p = 0.024) (Fig. 1A). We then investigated whether AZM was able to reduce this differential expression. In order to address this question 16HBE14o-AS3 were incubated in the presence of AZM

Discussion

Novel strategies for treatment of lung inflammation are required since no satisfactory anti-inflammatory treatment is available at present for clinical use in CF [24].

Accumulation of mistrafficked CFTR, the Cl⁻ channel mutated in CF, as well as defective glutathione transport, leading to exaggerated oxidative stress, have been proposed as possible mechanisms of NF-κB activation in CF [25] determining over-expression of IL-8.

The clinical efficacy of AZM is widely accepted [1]. Non-bactericidal

Acknowledgments

We thank Federica Quiri for excellent contribution for cell cultures and Valentino Stanzial for technical assistance. This study has been supported by the Italian Cystic Fibrosis Research Foundation (Grants FFC#03/2002; FFC#10/2005 by Istituti Scolastici Veronesi); Comitato di Vicenza dell’Associazione Veneta per la lotta contro la Fibrosi Cistica and Azienda Ospedaliera di Verona (Legge 548/93 Finanziamento Ricerca Fibrosi Cistica 2004), Italy.

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Anti-inflammatory effects of azithromycin in cystic fibrosis airway epithelial cells

Abstract

Section snippets

Materials and methods

Regulation of IL-8 mRNA

Discussion

Acknowledgments

Chest

J. Infect. Chemother.

Eur. J. Pharmacol.

J. Cyst. Fibros.

J. Surg. Res.

Pharmacol. Res.

Blood

Free Radic. Biol. Med.

Cytokine

J. Cyst. Fibros.

Lancet

J. Heart Lung Transplant.

J. Heart Lung Transplant.

Quorum-sensing antagonistic activities of azithromycin in Pseudomonas aeruginosa PAO1: a global approach

Antimicrob. Agents Chemother.

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

Antimicrob. Agents Chemother.

Long-term azithromycin in cystic fibrosis: another possible mechanism of action?

J. Chemother.

Exaggerated activation of nuclear factor-kappaB and altered IkappaB-beta processing in cystic fibrosis bronchial epithelial cells

Am. J. Respir. Cell. Mol. Biol.

Inflammatory cytokines in cystic fibrosis lungs

Am. J. Respir. Crit. Care Med.

Distinct sputum cytokine profiles in cystic fibrosis and other chronic inflammatory airway disease

Eur. Respir. J.