Abstract
Purpose: :
Cytokines secreted by retinal pigment epithelium (RPE) may induce local retinal inflammation, an important factor in the pathogenesis of age related macular degeneration (AMD). Little is known of the constitutive expression of cytokines from human RPE, or whether RPE respond to stress factors by changes in cytokine expression. Given that oxidative stress and chronologic age are two risk factors for AMD, we sought to identify the key cytokines secreted by cultured human RPE after oxidant injury due to hydroquinone (HQ) stimulation or after multiple passages of RPE, an in vitro model of aging.
Methods: :
We used xMAP technology (BioPlex), a panel of antibodies against 27 cytokines and 3 growth factors to detect their secreted levels in culture medium after stimulation with HQ (75uM, 24hrs) on primary culture of hRPE. We also tested the constitutive levels of cytokine and growth factors secreted from early (P5) versus late (P21) passage hRPE cells. Experiments were repeated three times, significance set at p<0.05 and fold change criterion set at 2.0 fold or greater. RPE cell cultures were genotyped to determine which CFH allelic variants were present.
Results: :
HQ stimulation (75uM for 24 hours) resulted in five upregulated genes that reached significance. VEGF, an angiogenic growth factor, was 5 fold higher in supernatant of HQ stimulated RPE cells. Secreted levels of interleukins IL12, IL10, IL13 and CXCL12 were also higher after HQ stimulation. Comparisons of secreted cytokines of RPE cells at high passage number, P21, compared to P5, revealed one upregulated and six downregulated molecules that reached significance. Downregulated genes were MCP3, PDGFbb, IL6, IL15, IL8 and CXCL12. The single upregulated gene, IL1ra, an inhibitor of IL1, demonstrated increased secretion in P21 cultures compared to P5.
Conclusions: :
Our data show that HQ stimulation provokes secretion of VEGF and selected interleukins in RPE in vitro. These results suggest that oxidant injury by HQ may provoke angiogenic and pro-inflammatory pathways in RPE. Results from early versus late passage cells, an in vitro model of aging, suggest that constitutive gene expression in RPE (P21) supports antiinflammatory, but not angiogenic or pro-inflammatory pathways. Future studies Future studies are needed to assess age- and oxidative stress-related cytokine secretion by RPE in vivo.
Keywords: age-related macular degeneration • cytokines/chemokines • retinal pigment epithelium