Maya Makhoul

Extracellular Nucleotides and Interleukin-8 Production by ARPE Cells: Potential Role of Danger Signals in Blood–Retinal Barrier Activation

PURPOSE RPE cell activation is an important feature of autoimmune uveitis. This investigation focused on whether extracellular nucleotides could contribute to this activation, and the effects of ATPgammaS, UTP, and UDP on the production of IL-8 by RPE cells was studied in relation to their expression of functional P2Y receptors. METHODS ARPE-19 cells were cultured with ATPgammaS, UTP, UDP, and TNF. IL-8 gene transcription and protein production were measured by semiquantitative RT-PCR and ELISA. Western blot analysis and RT-PCR were used to investigate ERK 1/2 activation and P2Y expression. Changes in intracellular calcium and cAMP concentration were analyzed by spectrofluorometry and radioimmunoassay. RESULTS Stimulation of ARPE-19 cells with ATPgammaS, UTP, and UDP induced IL-8 gene transcription and protein secretion. TNFalpha induction of IL-8 secretion was also increased by ATPgammaS, UTP, and UDP. Nucleotide induction of IL-8 production was blocked by PD98059, and all nucleotides stimulated ERK 1/2 phosphorylation. P2Y(2) and P2Y(6) mRNAs were detected in ARPE-19 cells. All tested nucleotides induced a pulse of intracellular calcium. CONCLUSIONS 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.

P2Y2R Deficiency Attenuates Experimental Autoimmune Uveitis Development

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.

TNFα suppresses IFNγ-induced MHC class II expression on retinal pigmented epithelial cells cultures

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.

Characterization of retinal expression of vascular cell adhesion molecule (VCAM-1) during experimental autoimmune uveitis.

Leukocyte adhesion to the blood retinal barrier is a critical step in the pathogenesis of non-infectious uveitis and is mediated in part through the induction of adhesion molecules on retinal cells. Here, we have investigated the retinal expression of Vascular Cell Adhesion Molecule 1 (VCAM-1) in mouse experimental models of non-infectious uveitis. For each eyes, a histological score was given, and the expression of VCAM-1 analyzed by immunohistology. Co-labellings for GFAP, endoglin, aquaporin 4 and recoverin were also performed in order to determine which cell type expressed VCAM-1. In low grade uveitis, obtained after adoptive transfer of semi-purified autoreactive lymphocytes, VCAM-1 was only punctually expressed in the internal limiting membrane and epithelial cells of the ciliary body. Using the same adoptive transfer protocol, we found that, in correlation with disease severity, the staining extended to all internal limiting membranes, vasculitis lesions, Müller cell extensions, outer limiting membranes and RPE cells. VCAM-1 expression in the inner limiting membrane and Müller cell extensions co-stained with GFAP expression. In vasculitis lesions, VCAM-1 co-localized with either GFAP and endoglin expression. The labeling in the outer limiting membrane, did not exactly co-stained with AQ4 (Müller cells marker) or recoverin (photoreceptor marker) and the nature of this expression remained unexplained. Finally, VCAM-1 expression was also analyzed in classical experimental autoimmune uveitis eyes, and a similar pattern of expression was found. In conclusion VCAM-1 is expressed on all blood retinal barrier cells during experimental non-infectious uveitis and might thus play an important role in inflammatory cell recruitment during disease development.

Effect of SOCS1 overexpression on RPE cell activation by proinflammatory cytokines

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.