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Y. Garfias, J. Quevedo, Jr., J. Nieves, V. Zaga, F. Vadillo, M. Jiménez-Martínez; Effect of Denuded Amniotic Membrane on the Expression of IL-8 Cytokine Through TLR3 Pathway on Human Limbocorneal Cells. Invest. Ophthalmol. Vis. Sci. 2008;49(13):5141. doi: https://doi.org/.
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Toll like receptors (TLR) are transmembranal proteins that recognize patterns associated to molecular pathogens(PAMP), transduce signals that induce pro-immflamatory protein synthesis. It has been reported that amniotic membrane (AM) has anti immflamatory properties and its role is at the inhibition pro-inflammatory cytokines; however its role in the inhibition of TLR function is unknown. The aim of this project is at identifying whether amniotic membrane is capable to inhibit the synthesis and secretion of IL-8 using different PAMP’s in human limbocorneal cells.
Ultra frozen denuded AM was obtained from cesarean of healthy women with no evidence of active labor; AM was preserved at -80°C. Primary culture of limbal-scleral rims were performed to obtain limbocorneal cells, which were stimulated with specific PAMP’s to each TLR (1-10). To determine the function of each TLR, synthesis and secretion of IL-8 was measured by both real time PCR and ELISA, respectively. Experiments were performed in the presence or absence of AM.
All 10 specific-stimulated TLR tested synthesized IL-8. However, there was a significantly increase in IL-8 synthesis in cells incubated with Poly (I:C) and ODN2006, specific PAMP’s for TLR3 and TLR8, respectively. In order to determine the effect of AM in the function of TLR3, cells were cultured with or without AM and stimulated with poly (I:C). IL-8 synthesis and secretion were measured, using real time PCR and ELISA, respectively. A significant reduction (p<0.05) in the expression of IL-8 was observed in cells cultured in the presence of AM compared to those cells cultured in plastic. To determine whether AM downregulated protein expression of TLR3, intracellular flow cytometry on limbocorneal cells was performed, and there was no significant change in the expression of TLR3 in the cells cultured on AM compared with those cultured in plastic, taking into account the medium fluorescence intensity.
There was a decrease in the expression of IL-8 in the cells which were cultured on amniotic membrane, in contrast that there was no significant change in the expression of intracellular TLR3 expression cultured on amniotic membrane. Taking these results we can conclude that the AM did not affect the expression of the TLR3. It is possible that its mechanism of action is carried out at the signaling pathway IRF3/7 or NF-kappaB.
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