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S. Peng, V. Rao, R. A. Adelman, L. J. Rizzolo; Effects of TNF and IL-1β on the Tight Junctions of Human Fetal RPE. Invest. Ophthalmol. Vis. Sci. 2010;51(13):2796.
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Age-related macular degeneration (AMD) is a chronic disease with a low-grade inflammatory component. Proinflammatory cytokines affect the permeability of tight junctions in a variety of epithelia. This study investigates how tumor necrosis factor (TNF)-α and interleukin (IL)-1β, affect the permeability and selectivity of tight junctions in the retinal pigment epithelium (RPE).
Human fetal RPE (hfRPE) was cultured on filters. TNFα (20ng/ml) or IL-1β (20ng/ml) was added to the apical and basal media chambers for 48 hours. Transepithelial electrical resistance (TER) was used to monitor the permeability and selectivity of RPE tight junctions. The relative conductance of Na+ and K+ was estimated from the TER in buffered NaCl or KCL solutions. The claudin composition of RPE tight junctions was examined using real-time RT-PCR to estimate relative mRNA levels, immunoblotting to monitor protein expression, and immunofluorescence to monitor protein distribution.
TNFα decreased the TER from 675 Ω-cm2 to 102 Ω-cm2. The ratio of the conductance for Na+ /K+ increased from 0.54 to 0.71. By contrast, IL-1β had little effect on TER or relative conductance. The expression of claudin 19 mRNA was more than 20X greater than the other claudin, but was minimally affected by TNFα. TNFα increased the expression of claudin 1, 2, and 3 mRNAs and decreased the expression of claudin 10 mRNA. Other claudins were only slightly affected. Corresponding changes in protein expression were observed by immunoblotting. Although the effects on protein distribution were minimal, TNFα affected structure, giving the tight junctions a jagged appearance in the plane of the monolayer and increasing the number of apical stress fibers. IL-1β had minimal effect on claudin mRNA or protein expression, claudin subcellular localization, or junctional structure.
Rather than compromise the outer blood-retinal barrier by disrupting RPE tight junctions, TNFα altered the properties of the junctions. The junctions became more permeable to ions and less selective for K+ over Na+. These changes correlated with increased expression of minor claudins and altered tight junction morphology.
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