ATPgammaS, UTP, and UDP stimulate both basal and TNFalpha-induced IL-8 secretion in RPE cells through an ERK 1/2-dependent pathway. The results suggest that those effects are mediated by P2Y(2) and P2Y(6) receptors.

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. Purpose:  One major consequence of retinal pigment epithelium (RPE) cell activation during autoimmune uveitis is the induction of MHC II molecules expression at their surface. IFNγ is regarded as the main cytokine involved in this induction. As TNFα plays a central role in autoimmune uveitis, we investigated its effects on IFNγ‐mediated MHC II induction on RPE cells. Methods:  Retinal pigment epithelium cells (ARPE‐19) were stimulated with IFNγ, TNFα and the anti‐TNFα antibody infliximab. The expression of MHCII and ICAM‐1 was analysed by flow cytometry. The activation and expression of IRF‐1 and STAT‐1, two proteins involved in IFNγ‐signalling pathway, were analysed by WB. Class II transactivator (CIITA) expression was monitored by qRT‐PCR and immunoprecipitation. Results:  TNFα inhibits IFNγ‐induced MHC II expression on ARPE cells in a dose‐dependent manner. Infliximab completely reverses the inhibitory effect of TNFα. We did not observe an inhibitory effect of TNFα on the expression of ICAM‐1 induced by IFNγ. Similarly, IFNγ‐induced STAT1 phosphorylation and IRF1 expression were not affected by TNFα. On the contrary, we found that TNFα suppresses IFNγ‐induced CIITA mRNA accumulation and protein expression. Conclusion:  TNFα inhibits IFNγ‐induced MHC II expression in RPE cells. This inhibitory effect was reversed by infliximab and was not because of a global inhibition of IFNγ ‐mediated RPE cell activation but rather to a specific down‐regulation of CIITA expression. Those findings are consistent with the role of TNFα in the resolution of inflammation and might help to elucidate the complex development of autoimmune uveitis.

We aimed to study the role of the nucleotide receptor P2Y2R in the development of experimental autoimmune uveitis (EAU). EAU was induced in P2Y2 +/+ and P2Y2 -/- mice by immunization with IRBP peptide or by adoptive transfer of in vitro restimulated semi-purified IRBP-specific enriched T lymphocytes from spleens and lymph nodes isolated from native C57Bl/6 or P2Y2 +/+ and P2Y2 -/- immunized mice. Clinical and histological scores were used to grade disease severity. Splenocytes and lymph node cell phenotypes were analyzed using flow cytometry. Semi-purified lymphocytes and MACS-purified CD4+ T lymphocytes from P2Y2 +/+ and P2Y2 -/- immunized mice were tested for proliferation and cytokine secretion. Our data show that clinical and histological scores were significantly decreased in IRBP-immunized P2Y2 -/- mice as in P2Y2 -/- mice adoptively transfered with enriched T lymphocytes from C57Bl/6 IRBP-immunized mice. In parallel, naïve C57Bl/6 mice adoptively transferred with T lymphocytes from P2Y2 -/- IRBP-immunized mice also showed significantly less disease. No differences in term of spleen and lymph node cell recruitment or phenotype appeared between P2Y2 -/- and P2Y2 +/+ immunized mice. However, once restimulated in vitro with IRBP, P2Y2 -/- T cells proliferate less and secrete less cytokines than the P2Y2 +/+ one. We further found that antigen-presenting cells of P2Y2 -/- immunized mice were responsible for this proliferation defect. Together our data show that P2Y2 -/- mice are less susceptible to mount an autoimmune response against IRBP. Those results are in accordance with the danger model, which makes a link between autoreactive lymphocyte activation, cell migration and the release of danger signals such as extracellular nucleotides.

The purpose of this study was to investigate the in vitro effect of Suppressor Of Cytokine Signaling 1 (SOCS1) overexpression in retinal pigment epithelium (RPE) cells on their activation by pro-inflammatory cytokines IFNγ, TNFα and IL-17. Retinal pigment epithelium cells (ARPE-19) were stably transfected with the control plasmid pIRES2-AcGFP1 or the plasmid pSOCS1-IRES2-AcGFP1. They were stimulated by IFNγ (150ng/ml), TNFα (30ng/ml) or IL-17 (100ng/ml). The levels of SOCS1 mRNA were measured by real-time PCR. Signal Transducer and Activator of Transcription 1 (STAT1) phosphorylation and IκBα expression were analysed by western Blot (WB). IL-8 secretion was analysed by ELISA and expression of MHCII molecules and ICAM-1/CD54 by flow cytometry. Our data show that SOCS1 mRNA overexpression in RPE cells prevents IFNγ-induced SOCS1 mRNA increase and IFNγ-mediated STAT1 phosphorylation. Moreover, SOCS1 overexpression in RPE cells inhibits IFNγ-induced decrease of IL-8 secretion and prevents IFNγ-induced MHC II and ICAM1/CD54 upregulation. However, SOCS1 overexpression does not affect TNFα-induced IκBα degradation nor block TNFα-induced or IL-17-induced IL-8 secretion. On the contrary, IL-17-induced secretion is increased by SOCS1 overexpression. In conclusion, SOCS1 overexpression in RPE cells inhibits some IFNγ-mediated responses that lead to uveitis development. This notion raises the possibility that SOCS1 overexpression could be a novel target for treating non-infectious uveitis. However, some proinflammatory effects of TNFα and IL-17 stimulation on RPE are not blocked by SOCS1 overexpression.