Abstract
Purpose:
Human corneal epithelial cells (HCEC) are relatively hyporesponsive to lipopolysaccharide (LPS) -the ligand for Toll-Like Receptor 4 (TLR4). However, under certain conditions, HCEC do become responsive to LPS. We evaluated the effects of wounding on the expression and activation of TLR4 in vitro and in vivo.
Methods:
Primary HCEC from cadaveric corneoscleral rims or telomerase-immortalized HCEC were used. Ultrapure LPS 100 ng/ml was used as the TLR4 ligand whereas CLI-095 5 μM was used as its internal inhibitor. The mRNA expression was determined by RT-PCR. The TLR4/MD2 protein complex expression was assessed using Western blotting and immunostaining. Scratch assay was performed to evaluate in vitro wound closure rates using live time-lapse microscopy. Transwell migration assay and immunofluorescent staining for Ki-67 were done to evaluate migration and proliferation, respectively. HEK-Blue™-hTLR4 reporter cell line were used to measure the amount of any endogenous/exogenous TLR4 ligands activity. The expression of TLR4 during in vivo wound healing was examined in C57BL/6J mice following a 2.0-mm debridement wound of the central epithelium.
Results:
Six hours after in vitro scratch wounding, there was a 4-fold increase in the expression of the TLR4/MD2 protein complex (P<0.001) which returned to baseline after 12 hours. Confocal microscopy confirmed that TLR4 was expressed on the membrane surface. There was a significantly greater increase in the mRNA expression of TNF-α, IL-6, IL-8 and CCL5/RANTES in the wounded + LPS HCEC compared to wounding alone. One hour pretreatment with CLI-095 obviated this effect. The mean scratch closure rate in vitro was 3.12% ± 1.40 per hour in LPS treated HCEC compared to 1.96% ± 0.80 per hour in the control (P=0.03). There was a significant increase in migration and proliferation in wounded + LPS wells (P<0.05 for both experiments). The supernatant from scratch wounded HCEC demonstrated 24.9 ± 4.21 times more TLR4 activity compared to media from unwounded HCEC (P<0.001). In the murine model, TLR4 immunostaining was prominent in the wound edge 2 hours after wounding with gradual return to baseline by 24 hours.
Conclusions:
These results suggest that epithelial wounding induces the expression of functional TLR4 most prominently at the leading wound edge. TLR4 signaling appears to contribute to early corneal epithelial wound repair by enhancing migration and proliferation.
Keywords: 480 cornea: basic science •
765 wound healing •
482 cornea: epithelium