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Laboratory science
Gene-expression analysis of polyI:C-stimulated primary human conjunctival epithelial cells
  1. Mayumi Ueta1,2,
  2. Katsura Mizushima3,
  3. Norihiko Yokoi2,
  4. Yuji Naito3,
  5. Shigeru Kinoshita2
  1. 1Research Center for Inflammation and Regenerative Medicine, Faculty of Life and Medical Sciences, Doshisha University, Doshisha, Kyoto, Japan
  2. 2Department of Ophthalmology, Kyoto Prefectural University of Medicine, Kyoto, Japan
  3. 3Department of Molecular Gastroenterology and Hepatology, Kyoto Prefectural University of Medicine, Kyoto, Japan
  1. Correspondence to Dr Mayumi Ueta, Department of Ophthalmology, Kyoto Prefectural University of Medicine, 465 Kajiicho, Hirokoji, Kawaramachi, Kamigyoku, Kyoto 602-0841, Japan; mueta{at}koto.kpu-m.ac.jp

Abstract

Background The authors previously reported that human ocular surface epithelium expressed TLR3 and that its ligand polyI:C stimulated the secretion of IL-6, IL-8 and IFN-β. In this study, to examine comprehensive effects of polyI:C stimulation of primary human conjunctival epithelial cells (PHCjECs), the authors performed a gene-expression analysis of the polyI:C-stimulated PHCjECs using oligonucleotide microarrays, GeneChip.

Methods The transcripts upregulated upon polyI:C stimulation in PHCjECs from two individuals were examined using GeneChip. Eleven new upregulated transcripts of interest were confirmed by quantitative real-time PCR (RT-PCR), and seven proteins produced by those transcripts were examined by ELISA or immunoblot analysis in PHCjECs from three other individuals, respectively.

Results GeneChip analysis showed that 150 transcripts were upregulated more than threefold and that 47 transcripts were upregulated more than 10-fold upon polyI:C stimulation in the PHCjECs. Eleven of the 47 upregulated transcripts (CXCL11, RIG-I, IL28A, CXCL10, CCL5, CCL4, MDA5, IL7R, TSLP, CCL20 and ICAM-1) were significantly upregulated upon polyI:C stimulation by quantitative RT-PCR, and the levels of seven proteins of the transcripts CXCL11, CXCL10, CCL5, CCL20, TSLP, RIG-I and MDA5 were confirmed by ELISA or immunoblot analysis to increase significantly in polyI:C-stimulated PHCjECs.

Conclusions Our results might show that TLR3 of conjunctival epithelium could not only induce antiviral innate immune responses but also regulate the allergic reactions.

  • Toll like receptor 3 (TLR3)
  • polyI:C
  • human conjunctival epithelial cells (HCjEC)
  • gene expression analysis
  • ocular surface
  • conjunctiva

https://doi.org/10.1136/bjo.2010.180554

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    Footnotes

    • Funding 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, the Intramural Research Fund of Kyoto Prefectural University of Medicine and a research grant from the Japan Allergy Foundation.

    • Competing interests None.

    • Ethics approval Ethics approval was provided by the institutional review board at Kyoto Prefectural University of Medicine, Kyoto, Japan, and all experimental procedures were conducted in accordance with the tenets of the Declaration of Helsinki.

    • Provenance and peer review Not commissioned; externally peer reviewed.