September 2016
Volume 57, Issue 12
Open Access
ARVO Annual Meeting Abstract  |   September 2016
In vitro effects of interleukin-6 on the outer blood-retinal barrier
Author Affiliations & Notes
  • Marina Mesquida
    Fundació Clínic per a la Recerca Biomèdica, Barcelona, Spain
    Ophthalmology, Hospital Clinic de Barcelona, Barcelona, Spain
  • Philippa J P Lait
    School of Clinical Sciences, University of Bristol, Bristol, United Kingdom
  • David Alexander Copland
    School of Clinical Sciences, University of Bristol, Bristol, United Kingdom
  • Jian Liu
    School of Clinical Sciences, University of Bristol, Bristol, United Kingdom
  • Victor Llorens
    Ophthalmology, Hospital Clinic de Barcelona, Barcelona, Spain
  • Maite Sainz De La Maza
    Ophthalmology, Hospital Clinic de Barcelona, Barcelona, Spain
  • Alfredo Adan Civera
    Ophthalmology, Hospital Clinic de Barcelona, Barcelona, Spain
  • Andrew D Dick
    School of Clinical Sciences, University of Bristol, Bristol, United Kingdom
  • Richard W J Lee
    School of Clinical Sciences, University of Bristol, Bristol, United Kingdom
    Biomedical Research Centre at Moorfields Eye Hospital, National Institute for Health Research, London, United Kingdom
  • Blanca Molins
    Fundació Clínic per a la Recerca Biomèdica, Barcelona, Spain
  • Footnotes
    Commercial Relationships   Marina Mesquida, Roche (F); Philippa Lait, None; David Copland, None; Jian Liu, None; Victor Llorens, None; Maite Sainz De La Maza, None; Alfredo Adan Civera, Roche (F); Andrew Dick, None; Richard Lee, None; Blanca Molins, None
  • Footnotes
    Support  None
Investigative Ophthalmology & Visual Science September 2016, Vol.57, 4184. doi:
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    • Get Citation

      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.

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      © ARVO (1962-2015); The Authors (2016-present)

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Abstract

Purpose : 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.

Methods : 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).

Results : 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.

Conclusions : 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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