Up Regulation of TLR–4 on Limbal Epithelium Does Not Drive TNF–alpha Secretion

Abstract

Abstract: : Purpose: Little is known about the expression of natural immune receptors as toll like receptors in corneal epithelium, and their function in ocular immune response is controversial.We sought to determine the extracellular expression of TLR–4 on human limbal epithelial cells cultivated in vitro and to study its cellular function after LPS stimulation. Methods: From sclera–corneal rims, limbal epithelial cells were isolated and grown in the presence of supplemented hormonal epithelial medium at 37ºC and 5% CO2 until confluence. At passages one or two, the cells were exposed to different doses of LPS from E. coli for 24 h. After stimulation, the cells were recovered and stained with PE–conjugated monoclonal antibodies against human TLR–4 and analyzed by flow citometry; mRNA was obtained and RT–PCR was performed for the identification of TLR–4, GADPH was used as an internal control. Secretion of TNF–alpha by these cells was evaluated by ELISA on the supernatant. PBMC were used as LPS activation controls. Results: Limbal epithelial cells expanded in vitro expressed constitutively low density TLR–4; after stimulation with LPS the expression of TLR4 was augmented taking into account the medium fluorescence intensity. A similar behavior was observed at the mRNA level, the expression was augmented after stimulus. When TNF–alpha was evaluated, interestingly, this cytokine was not detectable at any concentration of LPS and even at 48 h of stimulus. PBMC secreted optimal concentrations of TNF–alpha after LPS stimulation. Conclusions: Although the extra cellular expression of TLR4 on limbal epithelium stimulated in vitro up–regulates TLR4, its function seems not to be associated with the secretion of TNF–alpha on limbal epithelium.

Keywords: cornea: epithelium • cornea: basic science • inflammation