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Cytokine expression and barrier disruption in human corneal epithelial cells induced by alarmin released from necrotic cells

  • Laboratory Investigation
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Abstract

Purpose

Dying cells release endogenous molecules known as alarmins that signal danger to surrounding tissue. We investigated the effects of necrotic cell-derived alarmins on cytokine expression and barrier function in human corneal epithelial cells.

Methods

The release of interleukin (IL)-6 and IL-8 from immortalized human corneal epithelial (HCE) cells in culture was measured with enzyme-linked immunosorbent assays. The abundance of IL-6 and 8 mRNAs was quantitated by reverse transcription and real-time polymerase chain reaction analysis. Barrier function of HCE cells was evaluated by measurement of transepithelial electrical resistance (TER). The subcellular localization of the p65 subunit of the transcription factor NF-κB was determined by immunofluorescence analysis, and phosphorylation of the endogenous NF-κB inhibitor IκBα was examined by immunoblot analysis.

Results

A necrotic cell supernatant prepared from HCE cells induced the up-regulation of IL-6 and 8 expression at both mRNA and protein levels as well as reduced TER in intact HCE cells. Among alarmins tested, only IL-1α (not IL-33 or HMGB1) mimicked these effects of the necrotic cell supernatant. Furthermore, IL-1 receptor antagonist (IL-1RA) and neutralizing antibodies to IL-1α (but not those to IL-1β) each attenuated the effects of the necrotic cell supernatant. Exposure of HCE cells to the necrotic cell supernatant also induced the phosphorylation and degradation of IκBα as well as translocation of the p65 subunit of NF-κB to the nucleus.

Conclusion

IL-1α released from necrotic corneal epithelial cells may trigger inflammatory responses at the ocular surface, including cytokine production and barrier disruption.

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Authors and Affiliations

  1. Department of Ophthalmology and Visual Science, Kochi Medical School, Kohasu, Oko-cho, Nankoku, Kochi, 783-8505, Japan

    Ken Fukuda, Waka Ishida, Yusaku Miura, Tatsuma Kishimoto & Atsuki Fukushima

Authors
  1. Ken Fukuda
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  2. Waka Ishida
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  3. Yusaku Miura
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  4. Tatsuma Kishimoto
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  5. Atsuki Fukushima
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Corresponding author

Correspondence to Ken Fukuda.

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Conflicts of interest

K. Fukuda, Grants (Otsuka, Santen), Lecture fees (Alcon, Kissei, Novartis, Otsuka, Santen, Senju, Torii), Travel fees (Santen); W. Ishida, Grants (Otsuka, Santen), Lecture fees (Santen), Travel fees (Santen); Y. Miura, Travel fees (Alcon); T. Kishimoto, None; A. Fukushima, Grants (Alcon, AMO, Kissei, Kowa, Novartis, Otsuka, Pfizer, Santen, Senju), Lecture fees (AbbVie, Alcon, AMO, Eisai, Kissei, Kowa, Kyowa Hakko Kirin, Mitsubishi Tanabe, Nitten, Nitto Medic, Novartis, Otsuka, Pfizer, Santen, Senju, Torii), Writing assistance (Alcon).

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Fukuda, K., Ishida, W., Miura, Y. et al. Cytokine expression and barrier disruption in human corneal epithelial cells induced by alarmin released from necrotic cells. Jpn J Ophthalmol 61, 415–422 (2017). https://doi.org/10.1007/s10384-017-0528-7

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  • DOI: https://doi.org/10.1007/s10384-017-0528-7

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