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Marina Mesquida, David A Copland, Philippa J P Lait, Victor Llorens, Maite Sainz De La Maza, Alfredo Adan Civera, Andrew D Dick, Richard W J Lee, Blanca Molins; In vitro effects of IL-6 and IL-6R blockade on the blood-retinal barrier. Invest. Ophthalmol. Vis. Sci. 2017;58(8):5728. 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 disruption of the blood-retinal barrier (BRB). IL-6 is a pro-inflammatory cytokine that has been implicated in ME pathogenesis, and clinical cohort studies showed that IL-6R blockade may be beneficial in the treatment of uveitic ME. The aim of this study was to interrogate the effect of IL-6 and its blockade with the IL-6R inhibitor tocilizumab (TCZ) on the barrier properties of an in vitro model of the inner and outer BRB.
The paracellular permeability of human retinal pigment epithelial cell (ARPE-19) and human retinal microvascular endothelial cell (HRMEC) monolayers was assessed by measuring the passive permeation of 40 kDa FITC-dextran across confluent cells grown on transwell filters. At day 19 cells were treated with IL-6 (200, 400 ng/mL) for 48h. In some cases TCZ (200 ng/mL) was added at day 20. At day 21, FITC-dextran was added to the apical compartment of the chamber and samples from the basal medium were collected 120 min later. Transepithelial/endothelial electrical resistance (TEER) was also measured in both cell monolayers grown on filters.The distribution of the tight junction (TJ) protein ZO-1 in ARPE-19 and HRMEC monolayers was then examined by immunofluorescence. Cells grown on coverslips were incubated with or without IL-6 (200 ng/ml or 400 ng/ml) for 48 hours. When indicated, TCZ was added 24h after IL-6 stimulation. Then cells were fixed and immunolabeled with anti-ZO-1 and imaged by confocal microscopy.
Treatment with IL-6 for 48h significantly increased the diffusion rate of FITC-dextran and decreased TEER in both ARPE-19 cells and HRMEC compared to untreated cells. TCZ restored the diffusion rate of FITC-dextran and TEER in IL-6-treated cells to the level observed in untreated cells. Immunofluorescence showed a normal distribution of ZO-1 in untreated ARPE-19 and HRMEC monolayers, although ZO-1 expression was markedly disrupted following exposure to IL-6 for 2 days. TCZ restored ZO-1 distribution in IL-6-treated cells.
These in vitro data support the hypothesis that IL-6 disrupts the BRB and contributes to the pathogenesis of ME. IL-6-induced disrupted ZO-1 expression was accompanied by an increase in paracellular permeability, and decreased TEER of ARPE-19 and HRMEC monolayers, whereas TCZ restored IL-6-induced BRB breakdown.
This is an abstract that was submitted for the 2017 ARVO Annual Meeting, held in Baltimore, MD, May 7-11, 2017.
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