Interleukin-8 gene repression by clarithromycin is mediated by the activator protein-1 binding site in human bronchial epithelial cells

Macrolide antibiotics are known to be effective for the treatment of chroni c inflammatory airway diseases including diffuse panbronchiolitis, chronic bronchitis, and bronchial asthma. Other than having antimicrobial activitie s, macrolides have antiinflammatory effects, such as the inhibition of cyto kine production. In the present study we investigated the effects of clarit hromycin (CAM) on interleukin (IL)-8 gene expression and protein levels, us ing the human bronchial epithelial cell Line BET-IA, Northern blot analyses showed that CAM inhibited tumor necrosis factor (TNF)-alpha-induced IL-8 g ene expression in a dose- and incubation time-dependent manner. The half-li fe of IL-8 messenger RNA transcripts in TNF-alpha-treated BET-1A cells did not change with CAM. Transfection studies with BET-1A cells, using fusion g enes com posed of the 5'-flanking sequences of the IL-8 gene and a lucifera se reporter gene, demonstrated potent promoter activity in a 174-bp segment (-130 to +44 bp relative to the transcription start site). This segment in cludes activator protein (AP)-1 and nuclear factor (NF)-kappa B-like sites, and exhibited its strongest response to TNF-alpha. TNF-alpha-induced promo ter activity in this segment showed a significant repression by CAM. Howeve r, a 156-bp segment(-112 to +44 bp) that does not include an AP-1 site but includes an NF-kappa B-like site did not show a significant repression of T NF-alpha-induced promoter activity by CAM. Mutation of the AP-1 binding sit e abrogated the suppression by CAM of TNF-alpha-induced enhancement of luci ferase activity. In accord with promoter analyses, an electrophoretic mobil ity shift assay showed that CAM repressed AP-1 binding in TNF-alpha-treated BET-1A cells; however, TNF-alpha induced both AP-1 and NF-kappa B binding activities in BET-1A cells. These data suggest that macrolides such as CAM repress IL-8 gene transcription mainly via the AP-1 binding site in human b ronchial epithelial cells. Our findings provide a novel mechanism for the a ntiinflammatory function of macrolides, implicating a target for the develo pment of new drugs for treating chronic airway inflammation.