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. 2011 Jul;301(1):G91-9.
doi: 10.1152/ajpgi.00471.2010. Epub 2011 Apr 7.

Toll-like receptor 3 signaling enables human esophageal epithelial cells to sense endogenous danger signals released by necrotic cells

Diana M Lim  1 , Mei-Lun Wang
Affiliations

Toll-like receptor 3 signaling enables human esophageal epithelial cells to sense endogenous danger signals released by necrotic cells

Diana M Lim et al. Am J Physiol Gastrointest Liver Physiol. 2011 Jul.

Abstract

The mechanisms by which gastroesophageal reflux disease esophagitis develops are controversial. Although many support the notion that caustic injury leads to reflux esophagitis, others have proposed that reflux esophagitis is caused by esophageal epithelial cytokine-mediated inflammation. We previously demonstrated that Toll-like receptor 3 (TLR3) is highly expressed and functional in the nontransformed human esophageal epithelial cell line EPC2-hTERT. In addition to activation by viral double-stranded RNA, TLR3 can be activated by endogenous mRNA released by necrotic cells. In the present study, we investigated the role of esophageal epithelial TLR3 to sense danger signals released by necrotic esophageal epithelial cells in vitro. Following induction of freeze-thaw necrosis, necrotic EPC2-hTERT cell supernatants (NCS) were used to stimulate EPC2-hTERT monolayers, leading to NF-κB-dependent induction of IL-8 mRNA expression. Responses to self-derived NCS were not observed in transformed gastrointestinal epithelial cell lines, including TE-1 and Caco-2 cells, suggesting that the ability to sense endogenous danger signals is unique to nontransformed esophageal epithelial cells. To determine the immunostimulatory role of epithelial RNA, EPC2-hTERT cells were stimulated with self-derived mRNA, which significantly induced IL-8 mRNA expression. Finally, suppression of TLR3 signaling in a DN-TLR3 cell line, hTERT-ΔTIR-TLR3, led to reduced NCS-induced IL-8 induction by both NCS and mRNA stimulation. Our results demonstrate that human esophageal epithelial cells can sense endogenous danger signals, in part through TLR3 signaling. This supports the concept that epithelial injury plays an inciting role in the pathogenesis of reflux-induced esophagitis, providing important insights into the mechanisms by which epithelial injury leads to inflammation.

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Figures

Fig. 1.
Fig. 1.
Necrotic cell supernatant (NCS) stimulation of EPC2-hTERT cells leads to the inducible expression of IL-8. EPC2-hTERT cells were suspended in fresh keratinocyte serum-free medium (KSFM), subjected to 5 cycles of freeze-thaw necrosis, then centrifuged and separated into NCS and necrotic cell pellets (NCP). Fresh medium (control) and freeze-thawed medium (FT-KSFM) were used as controls. A: time course of IL-8 mRNA expression by live EPC2-hTERT monolayers following stimulation with NCS for various time points. B: quantification of IL-8 protein in medium (KSFM), freeze-thawed KSFM (FT-KSFM), and NCS before (fresh) and after (stimulated) being used to stimulate EPC2-hTERT cells for 3 h. C: expression of IL-8 mRNA by EPC2-hTERT cells following stimulation with fresh medium (control), FT-KSFM, NCP, and NCS at the 3-h time point. D: IL-8 mRNA expression in EPC2-hTERT cells following stimulation with neutral HBSS (control), acidified HBSS (acid-HBSS), and NCS made from acid-killed EPC2-hTERT cells (acid-NCS). Results are representative of 3 separate experiments. *P < 0.05, **P < 0.005, ***P < 0.001; NS, not significant.
Fig. 2.
Fig. 2.
NCS-induced activation of NF-κB signaling in EPC2-hTERT cells. A: Western blot analysis of the cytoplasmic NF-κB inhibitor IκBα, using proteins isolated from EPC2-hTERT cells following NCS stimulation for various time points. B: IL-8 mRNA expression by EPC2-hTERT cells following NCS stimulation in the presence or absence of the NF-κB inhibitor, Bay11-7082 (Bay11), compared with vehicle control (DMSO). Results are representative of 2 separate experiments. *P < 0.05, **P < 0.005.
Fig. 3.
Fig. 3.
Reduced responses to self-derived NCS by transformed gastrointestinal epithelial cell lines. Esophageal TE-1 squamous cell carcinoma cells and Caco-2 colon cancer cells were subjected to 5 cycles of freeze-thaw necrosis. A: IL-8 expression by live TE-1 and Caco-2 monolayers following stimulation with self-derived NCS for 3 h. TE-1- and Caco-2-derived NCS was generated in DMEM cell culture medium. B: IL-8 expression by EPC2-hTERT monolayers following stimulation with NCS derived from TE-1 and Caco-2 cells. TE-1 and Caco-2 NCS was generated in KSFM. Freeze-thawed KSFM was used as a control. Results are representative of 3 separate experiments. *P < 0.05, ***P < 0.001.
Fig. 4.
Fig. 4.
Induction of IL-8 expression by stimulation with purified epithelial mRNA. mRNA was isolated from EPC2-hTERT and TE-1 cells and was used to stimulate live EPC2-hTERT cells (10 μg/ml). Results are representative of 3 separate experiments. *P < 0.05, ***P < 0.001.
Fig. 5.
Fig. 5.
Expression of Toll-like receptor (TLR) 3 by hTERT-puro and dominant-negative TLR3 expressing hTERT-ΔTIR-TLR3 cells. Immunofluorescence was performed to determine the localization of TLR3 by both cell lines. For intracellular staining, cells were fixed and permeabilized, followed by incubation with anti-TLR3 MAb. For cell surface staining, cells were fixed and incubated with anti-TLR3 MAb. DAPI, 4,6-diamidino-2-phenylindole; ×63 magnification.
Fig. 6.
Fig. 6.
Suppression of NCS and mRNA-induced IL-8 induction in dominant-negative TLR3 expressing human esophageal epithelial cells. A: IL-8 mRNA expression in hTERT-puro and hTERT-ΔTIR-TLR3 cells following poly(I:C)-stimulation (1 μg/ml for 3 h). B: effect of NCS stimulation on IL-8 expression in hTERT-puro and hTERT-ΔTIR-TLR3 cells (3 h). C: change in IL-8 mRNA induction by hTERT-puro and hTERT-ΔTIR-TLR3 cells stimulated with EPC2-hTERT mRNA (open bars) compared with TE-1 mRNA (solid bars). Results represent fold induction in IL-8 compared with unstimulated hTERT-puro and hTERT-ΔTIR-TLR3 cells (10 μg/ml for 2 h). All results are representative of 3 separate experiments. ***P < 0.001.
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References

    1. Abraham E, Arcaroli J, Carmody A, Wang H, Tracey K. Cutting edge: HMG-1 as a mediator of acute lung inflammation. J Immunol 165: 2950–2954, 2000 - PubMed
    1. Aimanianda V, Haensler J, Lacroix-Desmazes S, Kaveri SV, Bayry J. Novel cellular and molecular mechanisms of induction of immune responses by aluminum adjuvants. Trends Pharmacol Sci 30: 287–295, 2009 - PubMed
    1. Andl CD, Mizushima T, Nakagawa H, Oyama K, Harada H, Chruma K, Herlyn M, Rustgi AK. Epidermal growth factor receptor mediates increased cell proliferation, migration, and aggregation in esophageal keratinocytes in vitro and in vivo. J Biol Chem 278: 1824–1830, 2003 - PubMed
    1. Arenzana-Seisdedos F, Thompson J, Rodriguez MS, Bachelerie F. Inducible nuclear expression of newly synthesized I kappa B alpha negatively regulates DNA binding and transcriptional activities of NF-kappa B. Mol Cell Biol 15: 2689–2696, 1995 - PMC - PubMed
    1. Basu S, Binder RJ, Suto R, Anderson KM, Srivastava PK. Necrotic but not apoptotic cell death releases heat shock proteins, which deliver a partial maturation signal to dendritic cells and activate the NF-κB pathway. Int Immunol 12: 1539–1546, 2000 - PubMed

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