PURPOSE: IL-32, a newly discovered cytokine, has been associated with a variety of inflammatory diseases. The role of innate immunity in regulation of IL-32 expression has not been elucidated. This study was to explore TLR-mediated induction of IL-32 and the inflammatory effects of IL-32 in corneal epithelium.
METHODS: Human corneal tissues and primary human corneal epithelial cells (HCECs) were treated with a variety of viral or bacterial components, as well as IL-32 without or with different TLR pathway inhibitors. The mRNA expression was determined by reverse transcription and real time PCR, and the protein levels were measured by ELISA and immunostaining.
RESULTS: IL-32 mRNA and protein were largely induced by specific microbial components, including polyinosinic-polycytidylic acid (polyI:C) and flagellin, the ligands to TLR3 and TLR5 respectively, in human corneal epithelium ex vivo and in vitro cultures. The polyI:C-induced IL-32 production was blocked by TLR3 antibody or TRIF inhibitory peptide, while flagellin-stimulated IL-32 was blocked by TLR5 antibody or MyD88 inhibitory peptide. Interestingly, IκB-α inhibitor (BAY11-7082) or nuclear factor kappa B (NF-κB) inhibitor (quinazoline) blocked NF-κB p65 protein nuclear translocation, and also suppressed IL-32 production induced by polyI:C or flagellin. When HCECs were treated with IL-32, we observed its stimulatory affects on inflammatory cytokines, TNF-α, IL-1ß and IL-8, at both mRNA and protein levels.
CONCLUSION: These findings demonstrate that IL-32 is induced by microbial ligands through TLR-mediated innate signaling pathways, suggesting an important role of corneal epithelium in inflammatory disease.
- Antibodies, Blocking/pharmacology
- Epithelium, Corneal/immunology
- Eye Banks
- Middle Aged
- NF-kappa B/immunology
- RNA, Messenger/metabolism
- Signal Transduction/immunology
- Toll-Like Receptor 3/genetics
- Toll-Like Receptor 5/genetics
- Tumor Necrosis Factor-alpha/genetics
- Young Adult