Soriano-Romaní Laura, García-Posadas Laura, López-García Antonio, Paraoan Luminita, Diebold Yolanda
Ocular Surface Group-IOBA, University of Valladolid, Valladolid, Spain.
Department of Eye and Vision Science, University of Liverpool, Liverpool, UK.
Exp Eye Res. 2015 May;134:1-14. doi: 10.1016/j.exer.2015.03.004. Epub 2015 Mar 6.
Recently, thrombospondin-1 (TSP-1) has been reported to be critical for maintaining a healthy ocular surface. The purpose of the study was to characterize the expression of TSP-1 and of its receptors CD36 and CD47 in corneal and conjunctival epithelial cells and determine the effect of exogenous TSP-1 treatment on these cells, following the induction of inflammation- and apoptosis-related changes. The expression of TSP-1, CD36 and CD47 by corneal and conjunctival cell lines was firstly characterized by ELISA, immunofluorescence analysis, Western blotting and reverse transcription polymerase chain reaction (RT-PCR). Benzalkonium chloride (BAC) exposure for 5 or 15 min was used as pro-inflammatory and pro-apoptotic stimulus for corneal or conjunctival epithelial cells, respectively. To analyze inflammation and apoptosis-related changes, IL-6 and TGF-β2 secretion determined by ELISA was used as inflammatory markers, while activated caspase-3/7 levels and cell viability, determined by CellEvent™ Caspase-3/7 Green Detection Reagent and XTT cytotoxicity assay, respectively, were used as apoptotic markers. Changes in CD36 and CD47 mRNA expression were quantified by real time RT-PCR. Corneal epithelial cells secreted and expressed higher protein levels of TSP-1 than conjunctival epithelial cells, although TSP-1 mRNA expression levels were similar and had lower CD36 and CD47, both at protein and mRNA levels. Both cell lines responded to exogenous TSP-1 treatment increasing CD36 at protein and mRNA levels. Blocking experiments revealed a predominance of TSP-1/CD47 rather than TSP-1/CD36 interactions to up-regulate CD36 levels in conjunctival epithelial cells, but not in corneal epithelial cells. BAC exposure increased IL-6 secretion and caspase-3/7 levels and decreased cell viability in both, corneal and conjunctival epithelial cells. Moreover, BAC exposure increased latent TGF-β2 levels in conjunctival epithelial cells. Interestingly, CD36 mRNA expression was down-regulated after BAC exposure in both cell lines. Exogenous TSP-1 treatment reduced TGF-β2 up-regulated levels by BAC exposure in conjunctival epithelial cells and less pronounced reduced IL-6 in BAC-exposed corneal epithelial cells. The effect on CD36 and CD47 regulation was less pronounced or even opposite depending on the inflammation- and apoptosis-related markers tested. Our results show evidence of the capacity of corneal and conjunctival epithelial cells to respond to TSP-1 via CD36 or CD47. Experimental simulation of inflammation- and apoptosis-related conditions changed the effects differentially elicited by TSP-1 on corneal and conjunctival epithelial cells, suggesting an unexpected and relevant contribution of TSP-1 on ocular surface homeostasis regulation.
最近，有报道称血小板反应蛋白-1（TSP-1）对维持健康的眼表至关重要。本研究的目的是表征TSP-1及其受体CD36和CD47在角膜和结膜上皮细胞中的表达，并确定外源性TSP-1处理对这些细胞的影响，以及诱导炎症和凋亡相关变化后的影响。首先通过酶联免疫吸附测定（ELISA）、免疫荧光分析、蛋白质印迹法和逆转录聚合酶链反应（RT-PCR）来表征角膜和结膜细胞系中TSP-1、CD36和CD47的表达。分别用苯扎氯铵（BAC）处理5分钟或15分钟作为角膜或结膜上皮细胞的促炎和促凋亡刺激。为了分析炎症和凋亡相关变化，将ELISA测定的IL-6和TGF-β2分泌用作炎症标志物，而分别用CellEvent™ Caspase-3/7绿色检测试剂和XTT细胞毒性测定法测定的活化的caspase-3/7水平和细胞活力用作凋亡标志物。通过实时RT-PCR对CD36和CD47 mRNA表达的变化进行定量分析。角膜上皮细胞分泌并表达的TSP-1蛋白水平高于结膜上皮细胞，尽管TSP-1 mRNA表达水平相似，且在蛋白质和mRNA水平上CD36和CD47的表达较低。两种细胞系对外源性TSP-1处理均有反应，在蛋白质和mRNA水平上CD36表达增加。阻断实验表明，在结膜上皮细胞中，TSP-1/CD47相互作用而非TSP-1/CD36相互作用在上调CD36水平方面占主导地位，但在角膜上皮细胞中并非如此。BAC处理可增加角膜和结膜上皮细胞中IL-6的分泌和caspase-3/7水平，并降低细胞活力。此外，BAC处理可增加结膜上皮细胞中潜伏性TGF-β2的水平。有趣的是，在两种细胞系中，BAC处理后CD36 mRNA表达均下调。外源性TSP-1处理可降低BAC处理引起的结膜上皮细胞中TGF-β2上调水平，并在BAC处理的角膜上皮细胞中较不明显地降低IL-6水平。根据所测试的炎症和凋亡相关标志物，对CD36和CD47调节的影响较不明显甚至相反。我们的数据表明角膜和结膜上皮细胞能够通过CD36或CD47对TSP-1作出反应。炎症和凋亡相关条件的实验模拟改变了TSP-1对角膜和结膜上皮细胞的不同影响，这表明TSP-1在眼表稳态调节中具有意想不到的重要作用。
