Inflammatory Cytokines Protect Retinal Pigment Epithelial Cells from Oxidative Stress-Induced Death

Helene B Juel,Mogens H Nissen,Signe G Svendsen,Carsten Faber,Abbe N Vallejo,Pedro Gonzalez

doi:10.1371/journal.pone.0064619

Abstract

PurposeTo investigate the effects of inflammatory factors and oxidative stress on cell survival of the human retinal pigment epithelial (RPE) cell line, ARPE-19.MethodsConfluent RPE cells were treated with peripheral blood mononuclear cells-conditioned medium (PCM), H2O2, NaIO3, interferon (IFN)-γ, tumor necrosis factor (TNF)-α, or combinations of these. Cell viability was determined by viability assays and by light microscopy. Effector molecules of cell death were investigated by immunofluorescence microscopy and flow cytometry. Microarrays were performed to screen for differential expression of anti-oxidative enzymes, and protein expression was validated by immunoblotting.ResultsViability of RPE cells was reduced by exposure to inflammatory agents (PCM, IFNγ+/-TNFα) or to oxidative agents (H2O2 or NaIO3). Unexpectedly, cells treated with either H2O2 or NaIO3 were partially protected from cell death by the addition of PCM. This protection was conferred, at least in part, by IFNγ and TNFα. Cell death induced by H2O2 or NaIO3 was preceded by mitochondrial dysfunction and by p62 upregulation, both of which were attenuated by PCM and/or by IFNγ+TNFα. RPE cells co-cultured with activated T cells, or treated with cytokines showed increased expression of anti-oxidative genes, with upregulation of superoxide dismutase 2 protein following PCM treatment.ConclusionOxidative stress-induced cell death was reduced by concomitant inflammatory stress. This is likely due to the cytokine-mediated induction of the anti-oxidative stress response, upregulating protective anti-oxidant pathway(s). These findings suggest caution for the clinical use of anti-inflammatory agents in the management of immune-associated eye diseases such as age-related macular degeneration.

Highlights

The retinal pigment epithelium (RPE) constitutes the outermost layer of the retina, and has many important functions in the homeostasis of the eye to maintain normal vision
We investigated responses of RPE cells treated with inflammatory stressors (peripheral blood mononuclear cellsconditioned medium (PCM), interferon (IFN)-c, and TNFa), oxidative stressors (H2O2 and NaIO3), or a combination of these
Reduced viability of RPE cells after treatment with peripheral blood mononuclear cells-conditioned medium (PCM) To investigate RPE cell reaction to a mixture of inflammatory cytokines from CD3/CD28-activated Peripheral blood mononuclear cells (PBMCs), RPE cells were incubated for 1–10 days with 50% PCM and 50% fresh medium to ensure adequate amounts of nutrients

Summary

Introduction

The retinal pigment epithelium (RPE) constitutes the outermost layer of the retina, and has many important functions in the homeostasis of the eye to maintain normal vision. The sub-RPE deposits that are the hallmark of AMD, contain many inflammatory proteins including complement factors [4,5,6,7,8], cytokines [9], C-reactive protein [7,10], IgG [11], and major histocompatibility class II molecules [5,7]. It has been suggested that the accumulating drusen trigger local production of inflammatory mediators, and attract leukocytes that would in turn lead to an increase in local inflammation and retinal stress [12]. Several anti-inflammatory drugs including complement inhibitors, tumor necrosis factor (TNF)-a inhibitors, and dexamethasone are currently on clinical testing for use in AMD [18,19,20,21]

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