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T. H. Tezel, L. Geng, E. Bodek, S. Schaal, H. J. Kaplan; Immunoglobulin Biosynthesis by Human Retinal Pigment Epithelium (RPE). Invest. Ophthalmol. Vis. Sci. 2008;49(13):208.
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To characterize immunoglobulins (Igs) produced by human RPE and to determine the effect of age on RPE-Ig synthesis.
Human donor eyes (25-73 years old) were obtained from the eye bank within 24 hours of death. RPE proteins were extracted and focused using non-liner (pH 3-10) 7.7cm IEF strips. Proteins were further separated in the second dimension on 4-12% Bis-Tris gels. Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) was used to identify Ig components. Results were confirmed with Western blotting and RT-PCR. Presence of T-(CD5) and B-lymphocyte (CD20) markers were also investigated with Western blotting, RT-PCR and immunohistochemistry to ascertain RPE as the source of Igs. The effect of age on Ig production in vivo was determined by plotting cytosolic RPE-Ig amount against the donor’s age. LPS (10 µgr/ml) was used to induce Ig production of cultured human RPE and ARPE-19 cells in vitro.
Human RPE proteome reveals several components of Igs including variable regions of heavy and light chains. RT-PCR and Western blot showed that IgG is the main Ig type within RPE cytosol. Absence of other lymphocyte markers (CD5 and CD20) indicated the source of Igs as RPE itself. Ig synthesis increases by age (r=0.73, p<0.05). Human RPE and ARPE-19 cells continued to produce Ig in vitro. Addition of LPS increased RPE-Ig production 2.3 ± 0.4 times.
Human RPE produces Igs. These Igs may play a role in initiating or augmenting the cellular damage in several outer retinal pathologies including age-related macular degeneration.
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