Georgiannakis, A; (2015) The interplay between retinal pigment epithelial cells and the C5b-9 complex. Doctoral thesis , UCL (University College London).

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Abstract

In age-related macular disease (AMD) accumulation of complement proteins in the sub-retinal space is believed to contribute to either the malfunction or apoptosis of retinal pigment epithelial (RPE) cells and ultimately the death of the associated photoreceptors. Here, the effects of basal formation of the terminal membrane attack complex, C5b-9, were examined using primary porcine RPE (pRPE) cells. Immunohistochemical analysis showed that C5b-9 complex formation appeared to stabilise at 4 h on the basal surface of pRPE cells, after which it steadily decreased and was eliminated from the cell surface by 48 h. Within 1 h of C5b-9 formation there was a significant increase in trans-epithelial resistance (TER) when compared to untreated cells. Treatment with dynasore, an endocytosis inhibitor, suggested that pRPE cells may endocytose the C5b-9 complex, as this chemical reagent blocked the removal of C5b-9 from the cell surface. Surprisingly, treatment of pRPE cells with dynasore inhibited the C5b-9-mediated increase in TER. C5b-9 did not appear to have an effect on the expression of either the negative complement regulators CD59 and DAF, or the expression of the junctional protein ZO-1. However, immunofluorescence analysis demonstrated strong junctional staining for claudin-19 in C5b-9-treated pRPE cells when compared to control conditions. Also, the prolonged presence of C5b-9 appeared to reduce both mitochondria numbers and the localisation of Tim23, a trans-membrane mitochondrial channel. We also observed that basal assembly of C5b-9 slowed the binding of photoreceptor outer segments (POS) to the apical surface of pRPE cells. However, POS internalisation and the activation levels of MerTK, FAK and Src were not affected by the presence of C5b-9. In conclusion, work in this thesis demonstrated that C5b-9 rapidly forms on the basal surface of pRPE cells, and that in doing so it modulates a number of RPE cell properties. RPE cells survive C5b-9 attack by endocytosing the complex, but when this process is blocked they exhibit signs of cellular stress. These studies provide mechanistic insight into some of the chronic cytotoxic effects of C5b-9 that could ultimately drive RPE cell loss and dysfunction in AMD.

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