Abstract
PurposeSystemic inflammation may be of importance in the development of AMD. RPE cells can recognize danger signals with toll-like receptors (TLR) and may react in a pro-inflammatory manner. In this study, we evaluated the basal and apical secretions of TNFα, IL-6, and IL-1β in primary RPE cells and RPE/choroid explant cells under basolateral stimulation of TLR2, 3, and 4; the effects on barrier function; and their influence on neuronal cell viability.MethodsRPE/choroid tissue explants were prepared from porcine eyes and cultivated in modified Ussing chambers; primary porcine RPE cells on transwell plates. Cells were basally stimulated with agonists Pam2CSK4 (Pam; TLR2), polyinosinic/polycytidylic acid (Poly I:C; TLR3), and lipopolysaccharide (LPS; TLR4) for 24 h. Supernatants were evaluated with ELISA for cytokines TNFα, IL-6, and IL-1β. Apical supernatants were applied to SHSY-5Y cells, and cell viability was evaluated in MTT assay. Barrier function was tested by measuring transepithelial electrical resistance (TER) and occludin immunostaining.ResultsNone of the tested TLR agonists was toxic on RPE cells after 24 h of exposure. Unstimulated RPE cells secreted hardly any cytokines. Pam induced IL-6, IL-1ß, and TNFα on the basal and apical sides at all concentrations tested. Poly I:C induced IL-6 and TNFα primarily at the basal side at lower but on both sides at higher concentrations. LPS induced IL-6, IL-1ß, and TNFα apically and basally at all concentrations tested. In the RPE/choroid, a strong difference between apical and basal secretions could be found. IL-6 was constitutively secreted basally, but not apically, but was induced by all agonists on both sides. IL-1ß and TNFα alpha were strongly induced on the basal side by all agonists. TER was reduced by all agonists, with Pam and LPS being effective in all concentrations tested. Occludin expression was unaltered, but the distribution was influenced by the agonists, with a less distinct localization at the cell borders after treatment. None of the agonists or supernatants of treated RPE and RPE/choroid organ cultures exerted any effect on viability of SHSY-5Y cells.ConclusionsDanger signals activating TLRs can induce polarized cytokine expression and contribute to the loss of barrier function in the RPE.
Highlights
Age-related macular degeneration (AMD) is the most common cause for vision loss in industrialized countries [1]
In order to investigate a potential influence of systemic inflammation on the retinal pigment epithelium (RPE) in vitro, we investigated the effect of a basal activation of TLR2, 3, and 4 of the RPE on the secretion of the cytokines IL-6, IL-1ß, and TNFα and on the barrier function of these cells
We investigated whether the treatment of RPE cells or RPE/ choroid organ culture with the toll-like receptors (TLR) agonists exerted an effect on the survival on SHSY-5Y cells
Summary
Age-related macular degeneration (AMD) is the most common cause for vision loss in industrialized countries [1]. Activation of toll-like receptors reduces the barrier function of the RPE and redistributes occludin. Activation of toll-like receptors induces a polarized secretion of cytokines with a stronger effect on the basal side discussed to be involved in AMD development. An association was shown between polymorphisms in TLR2 and 4 and AMD development [17, 18]. TLR3 activation may exert toxic effects on RPE cells and increase their VEGF secretion [12, 19]. Activation of TLR can induce the secretion of cytokines. The polarization of the secretion or the influence of TLR agonists on barrier function, has received little attention so far [11, 23, 24]
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