Triggering of TLR3 by polyI:C in human corneal epithelial cells to induce inflammatory cytokines
Section snippets
Materials and methods
All experimental procedures were conducted in accordance with the principles set forth in the Helsinki Declaration. The purpose of the research and the experimental protocols were explained to all participants and their prior written informed consent was obtained.
Human corneal epithelial cells. For RT-PCR, human corneal epithelial cells (HCEC) were obtained from corneal buttons of patients undergoing corneal transplantation for early-stage bullous keratopathy (one eye) or keratoconus (two eyes)
Expression of TLR3-specific mRNA in human corneal epithelium
We first examined whether human corneal epithelium expresses specific mRNA for TLRs 1–10. TLR-specific RT-PCR showed that mRNA from all but TLR8 was present in normal human corneal epithelium (Fig. 1). Among TLR-specific mRNA tested, TLR3 was expressed most intensely. When, as a positive control, we also subjected mRNA isolated from HPMC to RT-PCR, we found that these cells expressed TLRs 1–10. We then isolated, subcloned, and sequenced the PCR products. The obtained sequences were >95%
Discussion
We provide evidence for the gene and surface expression of TLR3 in human corneal epithelium and suggest that expressed TLR3 is functionally active in the secretion of the inflammatory mediators IL-6, IL-8, and IFN-β. We thus documented that polyI:C can induce the secretion of inflammatory mediators by primary HCEC. The ability of IFN-β to prevent the death of anergic cells, in addition to its anti-proliferative effect, may be one way in which the immune system regains a quiescent state after
Acknowledgments
This work was supported in part by Grants-in-Aid for scientific research from the Japanese Ministry of Health, Labour and Welfare, the Japanese Ministry of Education, Culture, Sports, Science and Technology, CREST from JST, a research grant from the Kyoto Foundation for the Promotion of Medical Science, and the Intramural Research Fund of Kyoto Prefectural University of Medicine. We thank Dr. T. Seya and Dr. J. Yamada for the gift of MRC5, HeLa cells, and HCFB, respectively, and Ms. C. Mochida
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