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Judy Hamouie, Medi Eslani, Yu Lu, Herve Sroussi, Ali R Djalilian; Endogenous Toll-like Receptor 4 (TLR4) Ligands in Human Corneal Epithelial Injury. Invest. Ophthalmol. Vis. Sci. 2015;56(7 ):707.
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© ARVO (1962-2015); The Authors (2016-present)
Human corneal epithelial cells (HCECs) are relatively hyporesponsive to lipopolysaccharide (LPS) - the ligand for Toll-Like Receptor 4 (TLR4). We previously showed that HCEC do become responsive to LPS upon injury. Since upon TLR4 inhibition, there was significant delayed wound healing in the absence of LPS, we hypothesized that endogenous TLR4 ligands (released upon injury) contribute to the activation of TLR4 in HCECs.
Growth factor starved human telomerase-immortalized HCEC were injured using multiple scratches. The supernatant was collected after 10 minutes after wounding. Supernatant was centrifuged to remove any cell debris. It then applied to HEK-Blue™-hTLR4 reporter cell line (Invivogen) to measure the amount of TLR4 activity. Polymyxin-B 100 µg/ml was used to remove any LPS contamination in the tested media. Ultrapure LPS 10 pg/ml was used as positive control whereas basic medium that was not in contact with cells was used as negative control. CLI-095 5 µg/ml was used as TLR4 internal inhibitor.
The supernatant from wounded cells demonstrated nearly 25 times (24.9 ± 4.21) more TLR4 activity compared to supernatant from unwounded cells (P <0.001). Differentiated HCECs (after injury) demonstrated 4 fold (3.8 ± 2.1) more TLR4 activity compared to undifferentiated cells (P < 0.001). Heating the supernatant to 75 C for 30 minutes significantly decreased TLR4 activity (P < 0.05). Although, inhibition of TLR4 with CLI-095 decreased the amount of TLR4 activity, it did not reach to the level of negative control (P < 0.05). Simultaneous use of a protease inhibitor cocktail and TLR4 inhibitor completely removed any TLR4 activation (P = 0.73 compared to negative control).
These results suggest that upon wounding of HCECs, endogenous ligands of TLR4 are released at the site of injury that can activate TLR4. These ligands appear to be peptides/proteins. There seems to be a cooperation between protease activated receptors and TLR4 in HCECs.
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