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Apostolos Georgiannakis, Katharina Lueck, John Greenwood, Stephen E Moss; How RPE cells respond to C5b-9 assembly in vitro. Invest. Ophthalmol. Vis. Sci. 2014;55(13):5993. doi: https://doi.org/.
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In age-related macular disease accumulation of complement proteins in the sub-retinal space is believed to contribute to the malfunction or apoptosis of retinal pigment epithelial (RPE) cells and ultimately the death of adjacent photoreceptors. Here, the effects of basal formation of the terminal membrane attack complex, C5b-9, was examined using primary porcine RPE (pRPE) cells.
pRPE cells were cultured on transwell filters with DMEM containing 10% fetal bovine serum. At two weeks, formation of an intact monolayer was confirmed by measuring the trans-epithelial resistance (TER). Formation of C5b-9 was accomplished by incubating cells with purified complement proteins in serum-free DMEM. Basal deposition of C5b-9 and the effects on expression of the complement negative regulators CD59 and DAF were examined by immunohistochemistry. Also, by treating pRPE cells with 200μM/ml of Dynasore, we investigated whether pRPE cells eliminate C5b-9 via endocytosis.
C5b-9 formation developed gradually and stabilised at 4h on the basal surface of pRPE cells. Immunohistochemical analysis of pRPE cells at different times following C5b-9 assembly showed that basal C5b-9 staining started to decrease after 8h and was eliminated by 48h. Within 1h of C5b-9 formation there was a significant increase in TER when compared to untreated cells. In addition, immunofluorescence data and western blots demonstrated increased expression of CD59 and DAF proteins, which act as negative regulators of the complement system. Also, treatment of pRPE cells with Dynasore blocked the ability of C5b-9 to increase the trans-epithelial resistance and also resulted in the accumulation of C5b-9 on the basal surface of pRPE cells at 24h.
At 24h following formation, C5b-9 levels on the basal aspect of the pRPE declined significantly compared to levels observed at 4h. Dynasore treatment suggests that C5b-9 complex loss from the basal side of pRPE cells may be due to endocytosis, as this inhibitor resulted in the sustained expression on the cell surface at 24h. The results also indicate that C5b-9 might alter the properties of cell junctions in pRPE cells but also have an effect on the expression of some of the negative regulators of the complement system. The C5b-9-induced TER increase was further supported by Dynasore treatment, suggesting that endocytosis of C5b-9 is necessary both for clearance of the complex, and the transient rise in TER.
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