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 (NFkappaB) was activated in 16HBE14o(-) cells stimulated with NE. Western blot analysis demonstrated that activation of NFkappaB by NE was preceded by phosphorylation and degradation of IkappaB proteins, principally IkappaBbeta. 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 (MyD88Delta) or TRAF-6 (TRAF-6Delta) inhibited IL-8 reporter gene expression in response to NE. Furthermore, MyD88Delta 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 IkappaBbeta and nuclear translocation of NFkappaB. These findings may have implications for therapeutic treatments in the cystic fibrosis condition.
Walsh
D E
DE
Respiratory Research Division, Royal College of Surgeons in Ireland, Education and Research Centre, Beaumont Hospital, Dublin 9, Ireland.
Greene
C M
CM
Carroll
T P
TP
Taggart
C C
CC
Gallagher
P M
PM
O'Neill
S J
SJ
McElvaney
N G
NG
D048868:Adaptor Proteins, Signal Transducing; D000943:Antigens, Differentiation / Q000378:metabolism*; D001483:Base Sequence; D015153:Blotting, Western; D001980:Bronchi / Q000187:drug effects / Q000378:metabolism*; D002461:Cell Line, Transformed; D017931:DNA Primers; D004847:Epithelial Cells / Q000187:drug effects / Q000378:metabolism; D006801:Humans; D006868:Hydrolysis; D053592:Interleukin-1 Receptor-Associated Kinases; D016209:Interleukin-8 / Q000096:biosynthesis / Q000235:genetics*; D019272:Leukocyte Elastase / Q000378:metabolism*; D053594:Myeloid Differentiation Factor 88; D016328:NF-kappa B / Q000378:metabolism; D009638:Norepinephrine / Q000494:pharmacology; D011494:Protein Kinases / Q000378:metabolism*; D011506:Proteins / Q000378:metabolism*; D011971:Receptors, Immunologic; D048029:TNF Receptor-Associated Factor 6; D015854:Up-Regulation / Q000187:drug effects
D048868:Adaptor Proteins, Signal Transducing; D000943:Antigens, Differentiation; D017931:DNA Primers; D016209:Interleukin-8; C507779:MYD88 protein, human; D053594:Myeloid Differentiation Factor 88; D016328:NF-kappa B; D011506:Proteins; D011971:Receptors, Immunologic; D048029:TNF Receptor-Associated Factor 6; D011494:Protein Kinases; D053592:Interleukin-1 Receptor-Associated Kinases; D019272:Leukocyte Elastase; D009638:Norepinephrine