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Activation of Airway Epithelial Cells by Toll-Like Receptor Agonists

Division of Allergy and Clinical Immunology, Department of Medicine, Johns Hopkins University School of Medicine, Asthma and Allergy Center, Baltimore, Maryland

Address correspondence to: Dr. Robert P. Schleimer, Division of Clinical Immunology, Department of Medicine, Asthma and Allergy Center, Baltimore, MD 21224. E-mail address: rschleim{at}jhmi.edu

Toll-like receptors (TLR) play an important role in pathogen recognition and innate immunity. We investigated the presence and function of TLRs in the BEAS-2B airway epithelial cell line and primary bronchial epithelial cells. Standard real-time reverse transcriptase–polymerase chain reaction (RT-PCR) analysis and Taqman RT-PCR revealed that BEAS-2B cells express mRNA for TLR1–10. Several TLR ligands were tested for their ability to activate gene expression in BEAS-2B cells using limited microarray analyses focusing on genes of the chemokine and chemokine receptor family, cytokines, and signaling pathways. While the TLR3 ligand double-stranded RNA was the most effective epithelial activator, clear responses to flagellin, lipopolysaccharide, CpG, peptidoglycan, and zymosan were also observed. RT-PCR and/or enzyme-linked immunosorbent assay were used to confirm results obtained with microarrays for five of the induced genes: interleukin-8, serum amyloid A, TLR3, macrophage inflammatory protein-3, and granulocyte-macrophage colony-stimulating factor. Stimulation of epithelial cells with double-stranded RNA induced levels of interleukin-8 exceeding 20 ng/ml and levels of serum amyloid A exceeding 80 ng/ml. Double-stranded RNA, lipopolysaccharide, zymosan A, and flagellin also induced expression of macrophage inflammatory protein-3 and granulocyte-macrophage colony-stimulating factor, which may facilitate immature dendritic cell migration and maturation. These results suggest that airway epithelial cells express several TLRs and that they are functionally active. Epithelial expression of TLRs may be of importance in inflammation and immunity in the airways in response to inhaled pathogens.

Abbreviations: third component of complement, C3 • threshold cycle, Ct • double-stranded RNA, dsRNA • glyceraldehyde phosphate dehydrogenase, GAPDH • granulocyte macrophage–colony-stimulating factor, GM-CSF • interleukin, IL • lipopolysaccharide, LPS • macrophage inflammatory protein, MIP • nuclear factor-B, NF-B • primary bronchial epithelial cells, PBEC • peptidoglycan, PGN • real-time polymerase chain reaction, RT-PCR • serum amyloid A, SAA • Toll-like receptor, TLR

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