[Immunological characteristics of limbal epithelial cells: in vitro analysis of TLR4 function].
OBJECTIVE
The aim of the present study was to determine the expression of TLR4 on human limbal epithelial cells cultivated in vitro, and to determine its cellular function after stimulation with lipopolysaccharide (LPS).


METHODS
Limbal epithelial cells were isolated from sclera-corneal rims and stimulated for 24 hours with different doses of LPS from E. coli and from Pseudomonas. After stimulation, the cells were harvested, stained with antibodies against human TLR4 and analysed by flow cytometry. mRNA was obtained and RT-PCR was performed for the identification of TLR4. Secretion of TNF-alpha by these cells was evaluated by ELISA of the supernatant.


RESULTS
Limbal epithelial cells expanded in vitro constitutively expressed the TLR4 molecule. After stimulation of cells with LPS the average fluorescence intensity increased, indicating that the expression of extracellular TLR4 was augmented. The expression of TLR4 mRNA was also increased with LPS stimulation, with maximum expression measured at 10 ng/ml LPS. The level of TNF-alpha in the supernatant was not different between the stimulated and the non-stimulated cells.


CONCLUSIONS
Although stimulation of in vitro limbal epithelial cells with LPS up-regulates the extracellular expression of TLR4, the function of TLR4 does not seem to be associated with the secretion of TNF-alpha by these cells. These results are consistent with the proposal that the corneal epithelium is an immunosilent site in the eye.
Garfias
Y
Y
Instituto de Oftalmología, Fundación Conde de Valenciana, Unidad de Investigación, Departamento de Córnea, Ciudad de México, México. yogarfias@yahoo.com
Linares
M
M
Suárez
R
R
Sánchez-Navarro
A
A
Jiménez-Martínez
M C
MC
D002478:Cells, Cultured; D004847:Epithelial Cells / Q000276:immunology*; D006801:Humans; D016850:Limbus Corneae / Q000166:cytology*; D051197:Toll-Like Receptor 4 / Q000096:biosynthesis / Q000502:physiology*
C495345:TLR4 protein, human; D051197:Toll-Like Receptor 4