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Tomohito Sato, Yoko Karasawa, Masataka Ito, Masaru Takeuchi; Simultaneous analysis of multiple cytokines from cultured human retinal epithelial cells under continuous fluorescent lamp illumination. Invest. Ophthalmol. Vis. Sci. 2014;55(13):64.
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To investigate cytokines secreted from cultured human retinal pigment epithelial (RPE) cells under continuous fluorescent lamp illumination.
Human RPE line cells (ARPE-19) were cultured to confluence. The cells were incubated in 0.21 mL/cm2 of colorless medium in the dark or under 2000 lx illumination from a daylight-colored (6500 K) fluorescent lamp mimicking ambient light for 24 hours. After the culture, the levels of 27 proinflammatory cytokines in culture supernatants were measured with a multiplex beads array system. To confirm whether the illumination induced oxidative stress, total levels of intracellular reactive oxygen species (ROS) were evaluated by a fluorescent molecular probe using the ROS/RNS Detection Kit. For detection of lipid peroxide and protein oxidization, immunostaining of malondialdehyde (MDA) and western blot of protein carbonyls were performed.
Fourteen out of 27 cytokines were detected both in the cultures in the dark and under illumination. The levels of IL-1ra, IL-9, IL-15, Il-17, basic FGF and G-CSF were significantly higher in the culture under illumination than in the culture in the dark. The levels of basic FGF increased 3.6-fold. Conversely, the levels of IL-8 and MCP-1 were significantly lower in the culture under illumination than in the culture in the dark. There were not significant differences in the levels of IL-6, IL-7, IL-10, Il-12, IP-10 and VEGF between the cultures in the dark and under illumination. The intensity of ROS signals increased in the culture under illumination compared to the culture in the dark. MDA-positive cells and protein carbonyls with diffuse bands over 240 kDa on the western blot were detected in the cultures under illumination.
These findings suggest that human RPE cells secrete proinflammatory cytokines, and that photic stimulation which induces oxidative stress modifies production of some of these cytokines by human RPE cells.
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