Induction of IL-8 expression by bacterial flagellin is mediated through lipid raft formation and intracellular TLR5 activation in A549 cells

• Published in: Molecular immunology 2009-12, Vol.47 (2-3), p.614-622
• Main Authors: Im, Jintaek, Jeon, Jun Ho, Cho, Min Kyung, Woo, Sang Su, Kang, Seok-Seong, Yun, Cheol-Heui, Lee, Kangseok, Chung, Dae Kyun, Han, Seung Hyun
• Format: Article
• Language: English
• Subjects: A549; Alveoli; Bacillus subtilis - immunology; Bacteria; Bronchi - cytology; CCAAT-Enhancer-Binding Protein-beta - genetics; CCAAT/enhancer-binding protein; Cell Line, Tumor; Cell surface; Chemokines; Chlorpromazine; Cytoplasm - immunology; Cytoplasm - microbiology; Enzyme Activation; Epithelial cells; Epithelial Cells - immunology; Epithelial Cells - microbiology; Flagellin; Flagellin - immunology; Gene expression; Gene Expression Regulation; Humans; IL-8; Interleukin 8; Interleukin-8 - biosynthesis; Interleukin-8 - genetics; Interleukin-8 - immunology; Intracellular Space - microbiology; Lipid rafts; MAP kinase; Membrane Microdomains - immunology; Membrane Microdomains - microbiology; Mitogen-Activated Protein Kinases - metabolism; NF-B protein; NF-kappa B - genetics; Nystatin; Promoters; Protein kinase C; Protein Kinase C - metabolism; Reporter gene; RNA, Messenger - genetics; RNA, Messenger - metabolism; Salmonella typhimurium - immunology; Time Factors; TLR5 protein; Toll-like receptor 5; Toll-Like Receptor 5 - immunology; Toll-like receptors; Transcription factors; Transcription, Genetic; Transfection
• ISSN: 0161-5890; 1872-9142
• DOI: 10.1016/j.molimm.2009.09.004

We investigated the mechanism for the induction of a chemokine, IL-8, by bacterial flagellins in the human alveolar type II epithelial cell line, A549. Bacterial flagellin induced expression of IL-8 mRNA and protein in dose- and time-dependent manners. IL-8 expression was inhibited by nystatin (a lipid rafts inhibitor) but not by chlorpromazine (a clathrin-coated pits inhibitor). Interestingly, Toll-like receptor 5 (TLR5) recognizing flagellins was found in the intracellular compartment of A549 but rarely on the cell surface. Flagellin-induced IL-8 expression appears to be mediated through TLR5 as determined by in vitro transient transfection experiment in HEK-293 cells expressing TLR5 using a reporter gene construct containing IL-8 promoter. IL-8 expression was attenuated by inhibitors for protein kinase C (PKC) and mitogen-activated protein (MAP) kinases. Furthermore, NF-κB and NF-IL6 transcription factors played an important role in the flagellin-induced IL-8 gene expression in A549 cells. Collectively, these results suggest that flagellin-induced IL-8 expression requires formation of lipid rafts, intracellular TLR activation, and subsequent activation of PKC and MAP kinases leading to the activation of the transcription factors NF-κB and NF-IL6 in human alveolar type II epithelial cells.