Expression of toll-like receptors in human limbal and conjunctival epithelial cells.
OBJECTIVE
To determine the expression and function of toll-like receptors (TLRs) in human conjunctival, limbal and corneal epithelial cells.


METHODS
Expression of TLRs was examined by real-time polymerase chain reaction, immunohistochemistry, and western blot analysis in human conjunctival, corneal and limbal epithelial cells and tissues. Ligand-stimulated nuclear factor kappaB activation; interleukin 6 and interleukin 8 protein secretion was measured in the cultured conjunctival and limbal epithelial cells by ELISA analysis.


RESULTS
Expression of TLR1, 2, 3, 5, and 6 was found in all conjunctival and limbal epithelial cell samples analyzed by real time PCR and western blot. TLR4 and TLR9 transcripts were undetectable in some samples by real-time PCR. TLR7, 8 and 10 transcripts were not detected by real time PCR in any of the samples tested. TLR1, 2, 3, 4, and 5 proteins were found in conjunctival, limbal and corneal epithelium by immunohistochemistry. Cultured conjunctival epithelial cells expressed significantly lower levels of TLRs than uncultured conjunctival cells obtained by applying nitrocellulose paper to the bulbar conjunctival surface. Cultured limbal and conjunctival cells responded to stimulation by polyriboinosinic polyribocytidylic acid (poly[I:C]), palmitoyl-3-cysteine-serine-lysine-4 (Pam3CSK) and flagellin with increased secretion of IL-6 and IL-8 and the activation of NFkappaB. Peptidoglycans (PGN) and CpG DNA caused increased NFkappaB activity; however, only conjunctival epithelial cells showed increased cytokine secretion. Lipoteichoic acid (LTA) or lipopolysacchride (LPS) did not change cytokine secretion or NFkappaB levels in either cell type.


CONCLUSIONS
The TLRs found in human conjunctival and limbal epithelial cells provide a basis for responses to many common ocular pathogens. Although the mRNA and protein for TLR4 and TLR2 was found, neither conjunctival or limbal cells in culture responded to LPS or LTA stimulation.
Li
Jing
J
Singapore Eye Research Institute, Department of Ophthalmology, Yong Loo Lin School of Medicine, National University of Singapore, Singapore. li.jing@seri.com.sg <li.jing@seri.com.sg>
Shen
JiangBo
J
Beuerman
Roger W
RW
D000209:Acute-Phase Proteins / Q000378:metabolism; D000368:Aged; D015153:Blotting, Western; D002352:Carrier Proteins / Q000378:metabolism; D002478:Cells, Cultured; D003228:Conjunctiva / Q000166:cytology* / Q000378:metabolism*; D004847:Epithelial Cells / Q000378:metabolism*; D005260:Female; D005786:Gene Expression Regulation; D006801:Humans; D007150:Immunohistochemistry; D015850:Interleukin-6 / Q000378:metabolism; D016209:Interleukin-8 / Q000378:metabolism; D008024:Ligands; D016850:Limbus Corneae / Q000166:cytology* / Q000378:metabolism*; D018950:Lipopolysaccharide Receptors / Q000378:metabolism; D051218:Lymphocyte Antigen 96 / Q000378:metabolism; D008297:Male; D008562:Membrane Glycoproteins / Q000378:metabolism; D008875:Middle Aged; D052002:NF-kappa B p50 Subunit / Q000378:metabolism; D051193:Toll-Like Receptors / Q000235:genetics* / Q000378:metabolism*; D051996:Transcription Factor RelA / Q000378:metabolism; D000099231:Lipopolysaccharide-Binding Protein
D000209:Acute-Phase Proteins; D002352:Carrier Proteins; D015850:Interleukin-6; D016209:Interleukin-8; D008024:Ligands; D018950:Lipopolysaccharide Receptors; D051218:Lymphocyte Antigen 96; D008562:Membrane Glycoproteins; D052002:NF-kappa B p50 Subunit; D051193:Toll-Like Receptors; D051996:Transcription Factor RelA; D000099231:Lipopolysaccharide-Binding Protein; C495377:LY96 protein, human