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Sijia Cao, Aikun Wang, Jing Z Cui, Joanne A Matsubara; Upregulation of complement components and chemokines by interferon gamma (IFN-γ) stimulation of retinal pigment epithelium (RPE) in vitro. Invest. Ophthalmol. Vis. Sci. 2014;55(13):3436. doi: https://doi.org/.
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Drusen are hallmark deposits of age-related macular degeneration (AMD) and are hypothesized to mediate RPE dysfunction. Our previous studies demonstrated that two drusen components, advanced glycation end products and amyloid-beta, upregulated IFN-γ and IFN-γ signaling pathways in RPE cells in vitro. IFN-γ is a soluble cytokine associated with innate and adaptive immunity, and recent studies point towards an emerging relationship between IFN-γ and mechanisms underlying the pathogenesis of AMD. Here we focus on understanding effects of IFN-γ on RPE gene expression.
Primary fetal RPE cells were exposed to 12.5 or 25ng/ml IFN-γ in serum-free DMEM culture medium for 12h and 24h, respectively. The IFN-γ induced cytotoxicity was evaluated by lactate dehydrogenase (LDH) assay. Cells were lysed to collect mRNA and the RNA expression of complement components, inflammatory cytokines and chemokines were analyzed by quantitative PCR (q-PCR).
Cell death was unchanged for both 12.5 and 25ng/ml IFN-γ at 24h compared to untreated cells. IFN-γ treatment upregulated complement factor H (CFH), complement factor B (CFB) and C3 (fold change > 2, p < 0.05), but did not affect the expression level of complement regulators CD46/55/59, complement factor I (CFI), complement factor D (CFD) or C5. IFN-γ also upregulated CX3CL1 (fractalkine) (>10) and MCP-1 (>2) in RPE cells at 12h and 24h (p < 0.01). IFN-γ at the tested concentration did not change the expression level of IL-1β, IL-18, IL-6, IL-8, TNF-α, IL-1ra and VEGF-A.
IFN-γ at the concentration tested does not have an adverse effect on survival of RPE cells, but it can affect the gene expression of RPE related to pro-inflammatory mediators. Our data demonstrated that several complement components are upregulated by IFN-γ. In addition, IFN-γ stimulation promoted chemokine upregulation, especially CX3CL1 from RPE cells. The significant upregulation of fractalkine, CX3CL1, a membrane-bound chemokine, may play an important role in the early AMD by promoting immune cell chemotaxis in outer retina in response to drusen components. Therefore, targeting the release of cleaved CX3CL1 from RPE origin may be an important strategy to suppress immune cell chemotaxis into outer retina.
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