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Open Access Research

Expression and activation of toll-like receptor 3 and toll-like receptor 4 on human corneal epithelial and conjunctival fibroblasts

Nir Erdinest1, Gal Aviel1, Eli Moallem2, Irene Anteby1, Claudia Yahalom1, Hadas Mechoulam1, Haim Ovadia3 and Abraham Solomon14*

Author Affiliations

1 Department of Ophthalmology, Hadassah-Hebrew University Medical Center, Jerusalem, Israel

2 Clinical Immunology and Allergy Unit, Hadassah-Hebrew University Medical Center, Jerusalem, Israel

3 Department of Neurology, The Agnes Ginges Center for Human Neurogenetics, Hadassah-Hebrew University Medical Center, Jerusalem, Israel

4 Cornea & Refractive Surgery Service, Department of Ophthalmology, Hadassah-Hebrew University Medical Center, Jerusalem 91120, Israel

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Journal of Inflammation 2014, 11:3  doi:10.1186/1476-9255-11-3

Published: 4 February 2014

Abstract

Background

Toll-like receptors (TLRs) are recognized as important contributors to the initiation and modulation of the inflammatory response in the eye. This study investigated the precise expression patterns and functionality of TLRs in human corneal epithelial cells (HCE) and in conjunctival fibroblasts (HCF).

Methods

The cell surface expression of TLRs 2-4, TLR7 and TLR9 in HCE and HCF was examined by flow cytometry with or without stimulation with lipopolysaccharide (LPS) or polyinosinic:polycytidylic acid (poly I:C). The mRNA expression of the TLRs was determined by real-time PCR. The protein content levels of interleukin (IL)-6, IL-8, IL-1β and tumor necrosis factor-α (TNF-α) were measured in HCE and HCF using multiplex fluorescent bead immunoassay (FBI).

Results

The surface expression of TLR3 and TLR4 was detected on both HCE and HCF. Following incubation with LPS, the percentage of HCE cells staining for TLR4 decreased from 10.18% to 0.62% (P < 0.001). Incubation with poly I:C lowered the percentage of HCE cells positive for TLR3 from 10.44% to 2.84% (P < 0.001). The mRNA expression of TLRs2, 4, 7 and 9 was detected in HCE only. Activation of HCE with LPS complex elicited protein secretion up to 4.51 ± 0.85-fold higher levels of IL-6 (P < 0.05), 2.5 ± 0.36-fold IL-8 (P > 0.05), 4.35 ± 1.12-fold IL-1β (P > 0.05) and 29.35 ± 2.3-fold TNFα (P < 0.05) compared to cells incubated in medium.

Conclusions

HCF and HCE both express TLRs that respond to specific ligands by increasing cytokine expression. Following activation, the surface expression of TLR3 and TLR4 on HCE is decreased, thus creating a negative feedback loop, mitigating the effect of TLR activation.

Keywords:
Toll-like receptor; Corneal epithelial; Conjunctival fibroblasts; Lipopolysaccharide; Polyinosinic:polycytidylic acid; Culture cells