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J. A. Williams, E. Longbottom, J. Greenwood, S. E. Moss; Regulation of Complement Factor H Secretion by RPE. Invest. Ophthalmol. Vis. Sci. 2009;50(13):705.
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The Y402H mutation in Complement Factor H (CFH) is associated with increased susceptibility to Age-related Macular Degeneration (AMD). Currently the contribution of this variant of CFH to the pathology of AMD is not understood. This study proposes to investigate the role CFH has within the retina by examining how CFH secretion is regulated from retinal pigment epithelium, its trafficking pathway and whether this is influenced in an inflammatory setting.
Human ARPE19 cells or primary porcine RPE were used to measure CFH secretion. Experiments were carried out in the absence of serum. Cells were exposed to inflammatory cytokines IFNγ (25-200U/ml), IL-1β (25-200U/ml) or TNF (5-40ng/ml) for 2-8 hours after serum removal. Culture media were collected and TCA precipitated. Total protein was analysed by SDS-PAGE and blotted with a polyclonal antibody to huCFH.
After serum removal, CFH secretion peaks after 4-6 hours in both ARPE19 and primary porcine RPE. After 8 hours in the absence of serum, cells secrete very little CFH. In both cases IFNγ stimulated an increase in production whilst the effect of TNF and IL-1β was insignificant. Initial data from experiments using RPE cells grown in culture well inserts suggest that CFH is secreted both apically and basally.
The peak in CFH secretion after 4-6 hours may be a stress response due to the removal of serum. Cessation of secretion after 8 hours is transient since CFH secretion is detectable in further assays once the cells have been cultured in complete medium overnight. Although IFNγ was shown to increase CFH mRNA and protein expression in RPE, it is not known whether the increase in secretion was due to de novo synthesis or stimulation of secretion. Although other inflammatory cytokines did not have an effect alone, they may have additive/synergistic effects when applied in combination. Our data suggest that CFH synthesis by RPE cells is a regulated activity that may be modulated by inflammatory stimuli.
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