Inflammatory Cytokines Decrease the Expression of RDH5 and RDH10 Genes in Human Retinal Pigment Epithelial Cells in Culture

Abstract

Purpose: : Atrophy of the retinal pigment epithelium (RPE) which is often associated with age-related macular degeneration (AMD) could stem from the impaired response of the RPE to the inflammatory mediators produced by infiltrating lymphocytes and macrophages. The inflammatory mediators could elicit their effect by perturbing the expression of important genes such as those involved in the retinoid metabolism in the RPE cells. The objective of the present study is to identify genes whose expression in the human RPE cells is modulated by the inflammatory cytokines IFN-γ, TNF-α and IL-1β.

Methods: : Confluent cultures of adult human RPE cells established from donor eyes were treated with the inflammatory cytokine mixture (IFN-γ + TNF-α + IL-1β) in a serum free medium for various time intervals. The total RNA fraction was then isolated for microarray analysis of gene expression with Affymetrix GeneChip arrays. Real-time PCR analysis was performed using TaqMan reagents (Applied Biosystems) with GAPDH as an endogenous control.

Results: : Microarray analysis showed that the human RPE cells in culture respond to the inflammatory cytokines (IFN-γ, TNF-α and IL-1β) by markedly decreasing the expression of retinol dehydrogenase genes RDH5 and RDH10. Real-time PCR analysis showed that the cytokine treatment resulted in more than 90% reduction in the expression of both RDH5 and RDH10. The decrease in the expression of these genes was both time- and concentration-dependent, and was observed even when the cells were treated for 16 hours with concentrations of IFN-γ, TNF-α and IL-1β as low as 1 unit/ml, 0.1 ng/ml and 0.1 ng/ml, respectively. IFN-γ, TNF-α or IL-1β when tested individually showed similar ability to decrease the RDH5 and RDH10 expression in the RPE cells, however, the combination of IFN-γ with either TNF-α or IL-1β elicited the maximum response. The human RPE cell line ARPE-19 also responded to the cytokine mixture by drastically decreasing the expression of RDH5 and RDH10.

Conclusions: : We have shown that the inflammatory cytokines could adversely affect the expression of retinol dehydrogenase genes RDH5 and RDH10 in the human RPE cells. The enzyme encoded by the RDH5 gene is thought to catalyze the conversion of 11-cis-retinol into 11-cis-retinal in the RPE cell, a key step in the regeneration of the visual pigment, and mutations in this gene are known to cause fundus albipunctatus. Thus, the inflammatory cytokines could cause RPE dysfunction by their ability to potentially impair visual function by targeting the RDH5 gene.

Keywords: retinal pigment epithelium • cytokines/chemokines • retinoids/retinoid binding proteins