Relvas Lia Judice M, Bouffioux Christophe, Marcet Brice, Communi Didier, Makhoul Maya, Horckmans Michael, Blero Daniel, Bruyns Catherine, Caspers Laure, Boeynaems Jean-Marie, Willermain François
Department of Ophthalmology, CHU Centre Hospitalier, Universitaire St-Pierre and Brugmann, Université Libre de Bruxelles-Campus Erasme, Bruxelles, Belgium.
Invest Ophthalmol Vis Sci. 2009 Mar;50(3):1241-6. doi: 10.1167/iovs.08-1902. Epub 2008 Nov 21.
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.
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.
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.
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.
视网膜色素上皮（RPE）细胞活化是自身免疫性葡萄膜炎的一个重要特征。本研究聚焦于细胞外核苷酸是否会促成这种活化，并研究了三磷酸腺苷γ-硫酯（ATPγS）、尿苷三磷酸（UTP）和尿苷二磷酸（UDP）对RPE细胞白细胞介素-8（IL-8）产生的影响及其与功能性P2Y受体表达的关系。
将ARPE-19细胞与ATPγS、UTP、UDP和肿瘤坏死因子（TNF）一起培养。通过半定量逆转录-聚合酶链反应（RT-PCR）和酶联免疫吸附测定（ELISA）检测IL-8基因转录和蛋白质产生。采用蛋白质免疫印迹分析和RT-PCR研究细胞外信号调节激酶1/2（ERK 1/2）的活化及P2Y表达。通过荧光分光光度法和放射免疫测定分析细胞内钙和环磷酸腺苷（cAMP）浓度的变化。
用ATPγS、UTP和UDP刺激ARPE-19细胞可诱导IL-8基因转录和蛋白质分泌。ATPγS、UTP和UDP还增强了TNFα诱导的IL-8分泌。PD98059可阻断核苷酸诱导的IL-8产生，并且所有核苷酸均刺激ERK 1/2磷酸化。在ARPE-19细胞中检测到P2Y(2)和P2Y(6)信使核糖核酸（mRNA）。所有测试的核苷酸均诱导细胞内钙脉冲。
ATPγS、UTP和UDP通过ERK 1/2依赖性途径刺激RPE细胞基础和TNFα诱导的IL-8分泌。结果表明这些作用是由P2Y(2)和P2Y(6)受体介导的。
