Abstract
Purpose: :
To determine the effects of photoreceptor oxidation in the immunoglobulin production of human RPE in vitro.
Methods: :
Three human donor eyes (41-88 years old) were obtained from the eye bank within 24 hours of death. POSs were isolated and exposed to 0.5 mM hydrogen peroxide for protein oxidation. ARPE-19 cells were incubated with oxidized POSs for 6 days with and without the presence of LPS (10 µg/ml). At the end of the incubation period, ARPE-19 cell proteins were extracted and cross-blotted with native and oxidized POS proteins. Mass spectrometry (MALDI-TOF-MS) was used to identify POS proteins recognized by RPE. Native POS, ARPE-19 and human lymphoblast (RPMI 6666) protein extracts were used as internal controls.
Results: :
ARPE-19 cytoplasm contains antibodies against proteins within POS, such as ATP synthase, creatine kinase, porin 31HM and solute carrier family 25. Incubation of ARPE-19 cells with oxidized POS proteins resulted in antibody production to additional proteins, such as phospholipase D2 and voltage-dependent anion channel 2. ARPE-19 cells not exposed to oxidized POS proteins revealed no quantitative or qualitative changes in their antibody repertoire, although the presence of LPS increased antibody production.
Conclusions: :
ARPE-19 cells produce immunoglobulins recognizing proteins within POS. Oxidative damage of POS can unmask hidden epitopes that can prime ARPE-19 cells to produce immunoglobulins against proteins that play important roles in transmembrane transport, mitochondrial function, endocytosis and cell death.
Keywords: age-related macular degeneration • retinal pigment epithelium • oxidation/oxidative or free radical damage