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Matthew Trese, Yue Li, Hua Gao, Xiaoxi Qiao; Inflammation Exacerbates Hydroxychloroquine-Induced Retinotoxicity in Human Retinal Pigment Epithelial Cells. Invest. Ophthalmol. Vis. Sci. 2013;54(15):6090.
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© ARVO (1962-2015); The Authors (2016-present)
It is well known that a subset of patients treated with hydroxychloroquine (HCQ) for autoimmunue diseases, such as systemic lupus erythematosus or rheumatoid arthritis, may develop a vision threatening maculopathy due to HCQ toxicity. This study attempts to investigate whether inflammation contributes to the toxic effect of HCQ on retinal pigment epithelial (RPE) cell morphology, viability and survival.
A human RPE cell line, ARPE19, was seeded at sub-confluent densities and cultured in standard conditions (20% O2, 5% CO2, 37°C). The cultured cells were exposed to HCQ, inflammatory cytokine mixture (ICM), or HCQ plus ICM. An 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium (MTS) assay was used to determine an HCQ exposure range. In the HCQ group, ARPE19 cells were exposed to an increasing gradient of concentrations from 10µM to 500µM of HCQ. In the ICM group, the cells were exposed to a sublethal dosage of ICM containing 10ng/mL interleukin 1-β, 10ng/mL tumor necrosis factor α and 100 units/mL interferon γ. In the HCQ+ICM group, the cells were treated with HCQ and the ICM cocktail. Finally, terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) was used to detect apoptosis in HCQ, ICM and HCQ+ICM treated groups.
HCQ reduced ARPE19 cell viability by nearly 50% at a concentration of 50µM and demonstrated complete cellular inhibition at 500µM. Phase contrast microscopy showed that all 3 groups (HCQ, ICM and HCQ+ICM) induced unique morphologic changes in ARPE19 cultures. The ICM treated cells presented with mild fibroblast-like morphologic change but otherwise appeared healthy. HCQ alone did not induce morphological changes at 10µM, but caused a significant increase in cell size at 25µM. Furthermore perinuclear and cytoplasmic vacuoles were observed at 50 to 500 µM. The cells in HCQ+ICM group displayed progressive cellular disruption at a low dosage 10µm HCQ. These changes included an increase in cell size and a prominent cellular ultrastructure. Despite these changes, apoptosis was not observed in any group of treatments.
The combination of HCQ and ICM induced more pathologic changes in ARPE19 cells than HCQ alone. This suggests that inflammation in patients with autoimmune disorders may contribute HCQ toxicity to their RPE, and exacerbate toxic maculopathy.
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