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Marina Mesquida, Philippa J P Lait, David Alexander Copland, Jian Liu, Victor Llorens, Maite Sainz De La Maza, Alfredo Adan Civera, Andrew D Dick, Richard W J Lee, Blanca Molins; In vitro effects of interleukin-6 on the outer blood-retinal barrier. Invest. Ophthalmol. Vis. Sci. 2016;57(12):4184. doi: https://doi.org/.
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© ARVO (1962-2015); The Authors (2016-present)
Macular edema (ME) is a leading cause of visual loss in uveitis patients. The pathogenesis of uveitic ME includes, in part, disruption of tight junctions (TJ) of the blood-retinal barrier (BRB). A consequence of this is both intracellular and extracellular fluid accumulation. Excessive IL-6 production is a pro-inflammatory cytokine driving many tissue inflammatory responses, including uveitis and it has also been implicated in uveitic ME. Such a notion is supported by clinical cohort studies showing that IL-6R blockade is beneficial in the treatment of uveitic ME.The aim of this study is to validate these clinical findings by interrogating the effect of IL-6 on an in vitro model of the outer blood retinal barrier (BRB) by measuring the paracellular permeability and the distribution of the tight junction ZO-1 in ARPE-19 cells.
The paracellular permeability of ARPE-19 monolayers was assessed by measuring the passive permeation of FITC-dextran (40 kDa) across confluent cells grown on filters. At day 19, the ARPE-19 cells were treated with IL-6 for 48h and at day 21, 500 μg/ml FITC-dextran was added to the apical compartment of the chambers and samples from the basal and apical compartments were collected at different time points. The absorbance of basal and apical medium samples was measured in a microplate reader (SpectraMax Gemini, Molecular Devices). The diffusion rate was expressed as a percentage and calculated as follows: (amount of dextran lower chamber)x100/(amount of dextran upper chamber).ARPE-19 cells grown on coverslips were incubated with or without IL-6 or IL-17A (100 ng/ml, 200 ng/ml or 400 ng/ml ) for 48 hours, then fixed and immunolabeled with rabbit anti-ZO-1 (Life Technologies) and analysed following image capture via confocal microscope (TCSSP2; Leica, Germany).
In the paracellular permeability assays, ARPE-19 cells treated with IL-6 (200 and 400 ng/mL) showed an increased diffusion rate of FITC-dextran across the monolayer compared to untreated cells. Although the expression of ZO-1 was uniform in untreated ARPE-19 monolayer cultures, it was markedly disrupted following exposure of these cells to IL-6 or IL-17A for 2 days the distribution of ZO-1.
These in vitro data support the notion that IL-6 disrupts the RPE barrier and contributes to ME pathogenesis. IL-6 reduced ZO-1 immunostaining and significantly increased the paracellular permeability of ARPE-19 monolayer
This is an abstract that was submitted for the 2016 ARVO Annual Meeting, held in Seattle, Wash., May 1-5, 2016.
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