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Jing Z Cui, Sijia Cao, Jiangyuan Gao, Joanne A Matsubara; Drusen component amyloid beta promotes membrane attack complex formation on ARPE-19 via classical pathway and upregulates pro-inflammatory cytokines . Invest. Ophthalmol. Vis. Sci. 2017;58(8):2280.
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The deposition of drusen is associated with development and progression of age-related macular degeneration (AMD). Amyloid beta (Aβ) and membrane attack complex (MAC) have been both reported in drusen. In our previous study we have shown that Aβ promotes MAC formation on RPE cells by downregulating the expression of the complement regulator CD55. However, it is not yet known through which pathway Aβ activates complements and what effect complement activation has on RPE cells.
ARPE-19 cells were stimulated with fibrillar Aβ in the presence of 10% normal human serum (NHS), heat-inactivated (HI)-NHS and serum depleted of pathway-specific components for 6 or 12 hours. Negative controls included NHS, Aβ or serum-free medium alone. Next, ARPE-19 cells were fixed and immunostained for MAC (1:50, Clone aE11, Dako) for flow cytometry analysis. The MAC formation was quantified as the percentage of MAC immunoreactive cells over the total number of cells. The gene expression changes were measured by reverse transcription PCR.
The percentage of MAC positive cells after treatment with Aβ combined with C1q or complement factor B (CFB) depleted (Dpl) serum was significantly lower than that with Aβ combined with NHS (Mean ± standard error of mean (SEM), 0.38±0.14 or 8.91±0.21 vs. 12.36±0.21, p<0.01). The level treated with Aβ combined with C1q-Dpl serum is equivalent to treatment with NHS alone (p>0.05), while CFB-Dpl serum is significantly higher than that treated with NHS alone (p<0.01). The C7-Dpl serum reduced the MAC positive cells to the background levels. The trend was similar at both time points tested. The Aβ-induced MAC formation induced IL-6, IL-8 upregulation in RPE cells, which was abolished by C9-Dpl or HI-NHS treatment (p<0.05).
The drusen component, Aβ, promoted MAC formation on ARPE-19 cells mainly via classical pathway and partially via alternative pathway. The action is also associated with the downregulation of the complement regulator CD55. Combined with other risk factors such as CFH Y402H polymorphism, Aβ may have a synergistic effect to activate complement and exacerbate MAC formation leading to a pro-inflammatory environment and RPE dysfunction in the outer retina.
This is an abstract that was submitted for the 2017 ARVO Annual Meeting, held in Baltimore, MD, May 7-11, 2017.
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