Abstract
Purpose: :
Studies have identified the alternative complement pathway as an important component of AMD. Our lab has shown that oxidized lipoproteins accumulate in drusen in early AMD and promote complement activation and apoptosis in RPE cells. This study was conducted to determine the mechanisms by which the CD46 and CD59 decrease and how these changes apply to AMD.
Methods: :
ARPE-19 cells were cultured and exposed to 100 µg/ml oxLDL for 24 h. Protein was extracted. The supernatant was lyophilized. Western blot analysis was performed using mouse monoclonal anti-human CD59, CD46, CD63. Smears of the supernatant were prepared and sequential double immunofluorescent staining for CD46 and EO6 was performed. Immunohistochemistry was performed on age-matched normal (n=9) and early-stage AMD (n=9) autopsy eyes. Sections were deparaffinized, antigens were retrieved, and incubated with mouse anti-human CD59 monoclonal antibody and isotype control.
Results: :
Previously we demonstrated that treatment of ARPE-19 cells decreases transmembrane CD46 with increased CD46 protein in the supernatant. Oxidized phosphorycholine is recognized by the E06 antibody and decorates apoptotic blebs. To demonstrate that the CD46 release is associated with apoptotic blebs, using sequential double immunofluorescence labeling for CD46 and EO6, we identified CD46 positive membrane bound particles in the supernatant. The EO6 bound to these particles along with apoptotic cells in a punctuate pattern. OxLDL treatment also decreased cellular CD59, but increased CD59 in the supernatant. The tetraspanin CD63, is highly abundant on exosomes, was also increased in the supernatant after oxLDL treatment. This suggests that decreased CD59 may be related in part, to the release of CD63 containing exosomes. Macular and peripheral sections of unaffected normal individuals did not show detectable CD59 immunostaining. In peripheral sections of early AMD patients, minimal CD59 labeling was seen in the RPE. In contrast, maculas from patients with early AMD showed strong CD59 labeling apically throughout the RPE with occasional basolateral staining.When overlying drusen, the RPE displayed weaker CD59 immunolabeling.
Conclusions: :
In early AMD, the identification of CD59 at the RPE-photoreceptor junction suggests heightened complement activity. Our data support a novel hypothesis that reduced CD59 through exosomal release leads to complement activation in the RPE. We also suggest that the decreased CD46 was released in subapoptotic blebs after an oxLDL stimulus. Like CD59, this not only reduces complement control of the RPE, but also releases CD46 into BrM and drusen.
Keywords: age-related macular degeneration • lipids • retinal pigment epithelium