[The expression and functionality of Toll-like receptors in human corneal epithelium and tolerated human corneal epithelial cell line].
OBJECTIVE
To investigate the expression and functionality of Toll-like receptors (TLRs) 1 - 9 in human corneal epithelium and cell line THCE (tolerated human corneal epithelial).


METHODS
Reverse-transcriptase polymerase chain reaction (RT-PCR) analysis was used to determine the expression of TLR1 - 9 mRNA in the specimens of human corneal epithelial cells from 20 persons, 5 males and 10 females, aged 18 - 25, undergoing photorefractive keratectomy (PRK), and in cultured THCE cells. Human peripheral blood mononuclear cells (PBMCs) were used as positive control. The expression of TLR2 protein and TLR4 protein were detected with Western blotting. KEM-2 fluid containing anti-TLR4 antibody was used to block the THEC cells for 30 minutes, then KEM-2 fluid containing PolyI: C or LPS, ligands for TLR 3 and 4, was used to stimulate the THEC cells, 1, 4, and 8 hours after the concentrations of IL-8 in the supernatant were determined by ELISA.


RESULTS
The human corneal epithelial cells strongly expressed TLR1, 2, 3, 5, 6, and 9 mRNA, weakly expressed TLR7 and 8 mRNA, and very weakly expressed TLR4 mRNA. Negative expression of TLR3, 4, 6, and 8 mRNA was seen in 1 case, a female aged 22. Weak expression of TLR5 mRNA was seen in 1 case, a female aged 20. The THCE cells showed the same expression pattern as the healthy human corneal epithelial cells, except for the strong expression of TLR9. The human PBMCs, as positive controls, strongly expressed TLR1 approximately 4 and 8 mRNA, weakly expressed TLR5, 6, and 9 mRNA, and very weakly expressed TLR7 mRNA. The human corneal epithelial cells, cultured THEC cells, and PBMCs all expressed TLR2 and 4 proteins. The concentrations of IL-8 in the supernatant of the culture fluid of THEC cells increased along with the time of stimulation of LPS and Poly: C, ligands for TLR3 and 4, and reached to 297.33 pg/ml and 229.67 pg/ml respectively 8 hours after, 10 times and 7 times those of the control group (both P < 0.05). 30 minutes after the blocking of TLR4 with anti-TLR4 antibody LPS was used to stimulate the THEC, the concentration of IL-8 was 88.54 pg/ml, being 30% that of the un-blocked group (P < 0.05).


CONCLUSIONS
Human corneal epithelium expresses TLR1 approximately 9 at different levels. THCE cell line is an excellent line to study TLRs function.
Gao
Jian-lu
JL
Department of Ophthalmology, Qilu Hospital, Shandong University, Jinan 250012, China.
Wu
Xin-yi
XY
D002460:Cell Line; D002478:Cells, Cultured; D019573:Epithelium, Corneal / Q000166:cytology* / Q000378:metabolism*; D006801:Humans; D012333:RNA, Messenger / Q000096:biosynthesis / Q000235:genetics; D051194:Toll-Like Receptor 1 / Q000096:biosynthesis / Q000235:genetics / Q000502:physiology; D051195:Toll-Like Receptor 2 / Q000096:biosynthesis / Q000235:genetics / Q000502:physiology; D051196:Toll-Like Receptor 3 / Q000096:biosynthesis / Q000235:genetics / Q000502:physiology; D051193:Toll-Like Receptors / Q000096:biosynthesis* / Q000235:genetics / Q000502:physiology
D012333:RNA, Messenger; D051194:Toll-Like Receptor 1; D051195:Toll-Like Receptor 2; D051196:Toll-Like Receptor 3; D051193:Toll-Like Receptors