Interleukin-8 up-regulation by neutrophil elastase is mediated by MyD88/IRAK/TRAF-6 in human bronchial epithelium

Cystic fibrosis is characterized in the lungs by neutrophil-dominated inflammation mediated significantly by neutrophil elastase (NE). Previous work has shown that NE induces interleukin-8 (IL-8) gene expression and protein secretion in bronchial epithelial cells. We sought to determine the intracellular mechanisms by which NE up-regulates IL-8 in bronchial epithelial cells. The data show that stimulation of 16HBE14o(-) cells with NE induced IL-8 protein production and gene expression. Both responses were abrogated by actinomycin D, indicating that regulation is at the transcriptional level. Electrophoretic mobility shift assays demonstrated that nuclear factor kappaB (NF kappaB) was activated in 16HBE14o(-) cells stimulated with NE. Western blot analysis demonstrated that activation of NF kappaB by NE was preceded by phosphorylation and degradation of I kappaB proteins, principally I kappaB beta. In addition, we observed that interleukin-1 receptor-associated kinase (IRAK) was degraded in 16HBE14o(-) cells stimulated with NE. Quantification of IL-8 reporter gene activity by luminometry demonstrated that dominant negative MyD88 (MyD88 Delta) or TRAF-6 (TRAF-6 Delta) inhibited IL-8 reporter gene expression in response to NE. Furthermore, MyD88 Delta inhibited NE-induced IRAK degradation. These results show that NE induces IL-8 gene up-regulation in bronchial epithelial cells through an IRAK signaling pathway involving both MyD88 and TRAF-6, resulting in degradation of I kappaB beta and nuclear translocation of NF kappaB. These findings may have implications for therapeutic treatments in the cystic fibrosis condition.