Azithromycin selectively reduces tumor necrosis factor alpha levels in cystic fibrosis airway epithelial cells

Abstract

Azithromycin (AZM) ameliorates lung function in cystic fibrosis (CF) patients. This macrolide has been suggested to have anti-inflammatory properties as well as other effects potentially relevant for therapy of CF. In this study, we utilized three CF (IB3-1, 16HBE14o- AS3, and 2CFSMEo-) and two isogenic non-CF (C38 and 16HBE14o- S1) airway epithelial cell lines to investigate whether AZM could reduce tumor necrosis factor alpha (TNF-alpha) mRNA and protein levels by real-time quantitative PCR analysis and an enzyme-linked immunosorbent assay (ELISA), respectively. We studied the effects on the DNA binding of NF-kappaB and specificity protein 1 (Sp1) by an ELISA. Non-CF cells express significantly lower TNF-alpha mRNA and protein levels than an isogenic CF cell line. In CF cells, AZM treatment causes a 30% reduction of TNF-alpha mRNA levels (P < 0.05) and a 45% decrease in TNF-alpha secretion (P < 0.05), reaching approximately the levels of the untreated isogenic non-CF cells. In CF cells, NF-kappaB and Sp1 DNA binding activities were also significantly decreased (about 45 and 60%, respectively; P < 0.05) after AZM treatment. Josamycin, a macrolide lacking clinically described anti-inflammatory effects, was ineffective. Finally, AZM did not alter the mRNA expression levels of interleukin-6, a proinflammatory molecule not differentially expressed in CF and isogenic non-CF cells. The results of our study support the anti-inflammatory activities of this macrolide, since we show that AZM reduced the levels of TNF-alpha and propose inhibitions of NF-kappaB and Sp1 DNA binding as possible mechanisms of this effect.

FIG. 1.

TNF-α mRNA expression. Expression of TNF-α mRNA in (A) non-CF C38 cells and an isogenic CF IB3-1 cell line, (B) non-CF 16HBE14o- S1 cells and an isogenic CF 16HBE14o- AS3 cell line, and (C) 2CFSMEo- cells at a constitutive level and after treatment with AZM. Total RNA was extracted and retrotranscribed. The values of TNF-α mRNA are based on real-time PCR analysis. The values represent the expression levels relative to those of untreated (A) IB3-1, (B) 16HBE14o- AS3, and (C) 2CFSMEo- cells (means ± SD). The experiment was repeated five times. *, P < 0.05; **, P < 0.01; ***, P < 0.001.

FIG. 2.

IL-6 mRNA expression. Expression of IL-6 mRNA in (A) non-CF C38 cells and an isogenic CF IB3-1 cell line, (B) non-CF 16HBE14o- S1 cells and an isogenic CF 16HBE14o- AS3 cell line, and (C) 2CFSMEo- cells at a constitutive level and after treatment with AZM. Total RNA was extracted and retrotranscribed. The values of IL-6 mRNA are based on real-time PCR analysis. The values represent the expression levels relative to those of untreated (A) IB3-1, (B) 16HBE14o- AS3, and (C) 2CFSMEo- cells (means ± SD). The experiment was repeated five times.

FIG. 3.

TNF-α protein release. Secretion of TNF-α in (A) non-CF C38 cells and an isogenic CF IB3-1 cell line, (B) non-CF 16HBE14o- S1 cells and an isogenic CF 16HBE14o- AS3 cell line, and (C) 2CFSMEo- cells at a constitutive level and after treatment with AZM and JM. TNF-α protein levels were measured by a commercial enzyme-linked immunosorbent assay kit. The values represent the secretion levels relative to those of untreated (A) IB3-1, (B) 16HBE14o- AS3, and (C) 2CFSMEo- cells (means ± SD). The experiment was repeated three times. *, P < 0.05; **, P < 0.01.

FIG. 4.

DNA binding of NF-κB. (A) Constitutive binding to the DNA of NF-κB in non-CF C38 cells and an isogenic CF IB3-1 cell line and in non-CF 16HBE14o- S1 cells and an isogenic CF 16HBE14o- AS3 cell line. (B) Effect of the treatment with AZM on the DNA binding of NF-κB in CF cells (IB3-1, 16HBE14o- AS3, and 2CFSMEo-). NF-κB DNA binding activity was analyzed using a commercial kit following the manufacturer's instructions. The experiment was repeated three times (means ± SD). *, P < 0.05; **, P < 0.01. OD, optical density.

FIG. 5.

DNA binding of Sp1. (A) Constitutive binding to the DNA of Sp1 in non-CF C38 cells and an isogenic CF IB3-1 cell line and in non-CF 16HBE14o- S1 cells and an isogenic CF 16HBE14o- AS3 cell line. (B) Effect of the treatment with AZM on the DNA binding of Sp1 in CF cells (IB3-1, 16HBE14o- AS3, and 2CFSMEo-). Sp1 DNA binding activity was analyzed using a commercial kit following the manufacturer's instructions. The experiment was repeated three times (means ± SD). *, P < 0.05; **, P < 0.01. OD, optical density.