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G. Shi, S. Jalickee, T. Banzon, J. Hammer, A. Maminishkis, S.S. Miller; Inflammatory Cytokines Alter Polarized Chemokine Secretion and Fluid Transport by Human Fetal RPE in vitro . Invest. Ophthalmol. Vis. Sci. 2006;47(13):2066.
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© ARVO (1962-2015); The Authors (2016-present)
To determine the physiological effects and polarity of chemokine secretion induced by inflammatory cytokine addition to the solutions bathing intact monolayers of human fetal retinal pigment epithelial (hfRPE) primary cultures.
HfRPE monolayers were grown on transwell filters coated with ECM. A capacitance probe technique was used to measure transepithelial fluid flow (JV), transepithelial potential (TEP) and resistance (RT) in standard bicarbonate Ringers (Maminishkis et al., IOVS,2002;43:3555). The inflammatory cytokines were added as a cocktail consisting of interleukin–1ß (IL–1ß), tumor necrosis factor–α (TNF–α) and interferon–γ (IFN–γ) to the apical or the basal bath or to both baths. Western blots and immunofluorescence were used to determine the expression and polarity of the cytokine receptors (R). Polarized secretion of chemokines including CCL–2,3,4,5,17,19 and CXCL–1,8,9,10,11,12 was measured using Searchlight technology.
All data are represented as mean ± SEM. Steady state TEP, RT and JV in control Ringers was 2.0 ± 0.6mV, 642.9 ± 81.8 Ω·cm2 and 9.2 ± 2.1 µl·cm–2·hr–1 (n = 9), respectively. Apical addition of the cocktail produced no changes in Jv. In contrast, basal addition increased net fluid absorption (retina to choroid) by 8.6 + 2.9 µl·cm–2·hr–1 (n=3). Jv increased by 5.5 + 1.3 µl·cm–2·hr–1 (n=3) after addition to both sides. RT and TEP were unchanged in all experiments. Addition of cocktail to both bathing solutions preferentially increased chemokine secretion to the apical bath. Expression of IL–1R, TNF–R and IFN–γR was verified by Western blot. Immunolocalization showed apical expression of IL–1R and basolateral expression of TNF–R and IFN–γR.
These data show that inflammatory cytokines can activate basolateral membrane receptors and significantly alter RPE physiology in vitro. The cytokine–induced activation in JV and the preferential secretion of chemokines to the apical bath suggests a critical role of the RPE in mediating an inflammatory environment of the extracellular spaces adjacent to the distal retina and Bruch’s membrane.
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