Expression of toll-like receptors in human limbal and conjunctival epithelial cells

Abstract

Purpose: 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.

Figure 1

Western blot analysis of toll-like receptor protein expression in cultured human limbal and conjunctival epithelial cells. Three independent samples of primary cultured limbal and conjunctival epithelial cells isolated from different donor tissues with positive identification of each TLR transcript were analyzed (n=3). Cells were lysed in RIPA buffer and 40 μg of total protein was loaded on each lane. The histogram of western blot analysis shows representative results obtained from all samples analyzed. β-Actin was used as the loading control.

Figure 2

Immunolocalization of toll-like receptors in human corneal, limbal, and conjunctival epithelium. Cryosections of human cornea and conjunctival tissues were incubated with various anti-TLR antibodies and visualized using Alex Fluor 488 conjugated secondary antibodies as described in Methods. Nuclei were stained by DAPI present in the mounting solution. The original pictures were taken at 200X magnification. The insets were taken at 400X magnification.

Figure 3

p50 and p65 activities in cultured limbal and conjunctival epithelial cells. Ligand stimulated p50 and p65 activities in cultured limbal (A) and conjunctival epithelial cells (B) are shown. Eighty percent confluent cells cultured in six well plates (positive for corresponding TLR gene expression) were incubated with different ligands at concentrations indicated in Methods for 8 h before harvesting the cells for the extraction of nuclear proteins. Equal amounts of nuclear proteins were used for the ELISA based analysis of p50 and p65 activities. p50 and p65 activities in control cells (without ligand stimulation) were set as 1 and used to normalize the activities measured in stimulated cells. The open bar represents the p65 subunit and the solid bar represents the p50 subunit. The results show the mean of three independent experiments and the error bar represents the SEM. The asterisk indicates a p<0.05 by ANOVA analysis followed with Fisher LSD test.

Figure 4

Ligand stimulated IL-6 and IL-8 secretion in cultured limbal and conjunctival epithelial cells. Eighty percent confluent conjunctival (A and B) and limbal (C and D) cells positive of corresponding TLR gene expression were incubated with different ligands at the concentrations indicated in Methods. Culture medium was collected 24 h later. IL-8 (A and C) and IL-6 (B and D) protein levels were measured by ELISA. Data represent the mean of four to five independent experiments and the error bar represents the SEM. The concentration of IL-6 and IL-8 was corrected by total protein amount in each well at the time of harvesting the supernatant. Significance compared to the controls by ANOVA and Fisher LSD analysis at a level of p<0.05 is shown by the asterisk. For CpG stimulated cells, the control refers to the CpG control DNA stimulated cells.