IL-1β induces eotaxin gene transcription in A549 airway epithelial cells through NF-κB

Eotaxin is an asthma-related C-C chemokine that is produced in response to interleukin-1 beta (IL-1 beta). We detected an increase in newly transcribed eotaxin mRNA in IL-1 beta -stimulated airway epithelial cells. Transient transfection assays using promoter-reporter constructs identified a region as essential for IL-1 beta -induced increases in eotaxin transcription. Using site-directed mutagenesis, we found that a nuclear factor-kappaB (NF-kappaB) site located 46 bp upstream from the transcriptional start site was both necessary and sufficient for IL-1 beta induction of reporter construct activity. Electrophoretic mobility shift assay demonstrated that IL-1 beta -stimulated airway epithelial cells produced p50 and p65 protein that bound this site in a sequence-specific manner. The functional importance of the NF-kappaB site was demonstrated by coexpression experiments in which increasing doses of p65 expression vector were directly associated with reporter activity exclusively in constructs with an intact NF-kappaB site (r(2) = 0.97, P = 0.002). Moreover, IL-1 beta -induced increases in eotaxin mRNA expression are inhibited by inhibitors of NF-kappaB. Our findings implicate NF-kappaB and its binding sequence in IL-1 beta -induced transcriptional activation of the eotaxin gene.