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P. Baciu, M. Etheridge, B. Parker, E. Sung; ARPE-19 Cell Death by the Alternative Complement Cascaded. Role of Cell Surface Regulatory Proteins, Calcium, PKC and Oxidative Stress. Invest. Ophthalmol. Vis. Sci. 2010;51(13):474.
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© ARVO (1962-2015); The Authors (2016-present)
To investigate mechanisms regulating complement mediated cell death of RPE cells.
ARPE-19 cells were primed with a complement fixing antibody followed by challenge with human serum deficient in C1q. Effects of complement attack on cell lysis were monitored by measuring uptake of cell impermeant nuclear dyes. Development of membrane attack complexes (MAC) were monitored by cell swelling, C5b-9 FACS, Ca++ influx and ATP release. SiRNA’s were used to knock down either CD46, CD55 or CD59. Contribution of calcium and PKC to protect ARPE-19 cells was determined by depletion of extracellular calcium or pretreatment with PKC inhibitors chelerythrine and G06976. The effects of oxidative stress on complement mediated death was examined by pretreatment of cells with H2O2 or t-BH followed by complement challenge.
Complement driven cell death of primed ARPE-19 cells in C1q deficient serum was dependent on the alternative cascade and correlated with the formation of functional MAC on the cell surface. Down regulation of either CD46 or CD59 modestly enhanced cell death while inhibition of PKC double that observed in CD46 and CD59 knockdown studies. The greatest increase in cell death was observed after calcium depletion or pretreatment with H2O2.
We have established an in vitro assay of complement driven RPE cell death mediated by the alternative cascade. While complement regulatory proteins provided protection, cellular pathways regulated by calcium and PKC appear to play a more significant role in prevention of cell death. The most significant finding is the observed synergy between complement and oxidative stress. This synergy appears to reflect the impact of oxidative stress on cellular pathways involved in protection against the MAC and not modulation of cell surface regulatory proteins.
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