Expression and function of toll-like receptor-3 and-9 in human corneal myofibroblasts
Expression and function of toll-like receptor-3 and-9 in human corneal myofibroblasts
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DOI：
10.1167/iovs.06-0968
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发表时间：
2007-07-01
影响因子：
4.4
通讯作者：
Murakami, Akira
中科院分区：
文献类型：
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作者：
Ebihara, Nobuyuki;Yamagami, Satoru;Murakami, Akira
目的.目的探讨Toll样受体（TLR）-3和-9在角膜成肌纤维细胞中的表达及功能。使用两种类型的人角膜细胞，它们是从供体角膜新鲜分离的角膜细胞和培养的角膜细胞。通过RT-PCR分析这些细胞中各种分子标记物的mRNA表达，并且还通过RT-PCR分析TLR-2、TLR-3、TLR-4和TLR-9 mRNA。通过流式细胞术评估TLR-3和TLR-9在蛋白水平的表达。此外，使用抗体阵列和ELISA检测培养的角膜细胞的上清液中的趋化因子和细胞因子，有或没有刺激的聚肌苷-聚胞苷酸（poly（I：C））或CpG-DNA。此外，进行吞噬测定以评估经由TLR-3和TLR-9的信号传导是否增强吞噬作用。培养三代的角膜细胞分化为角膜肌成纤维细胞。TLR-3和TLR-9在mRNA和蛋白水平上在角膜肌成纤维细胞中检测到，但在新鲜分离的角膜细胞中未检测到。与未处理的细胞相比，用poly（I：C）或CpG-DNA刺激角膜肌成纤维细胞增强了IL-6、IL-8、GRO、ENA-78和RANTES的产生。TLR-3和TLR-9介导的信号转导可上调肌成纤维细胞的吞噬活性。这是第一个关于TLR-3和TLR-9在角膜肌成纤维细胞中的体外表达和功能的报告。研究结果表明，角膜细胞表型决定TLR-3和TLR-9的表达，角膜肌成纤维细胞可能在细菌和病毒清除中发挥重要作用。
PURPOSE. To investigate the expression and function of toll-like receptor (TLR)-3 and -9 in corneal myofibroblasts.METHODS. Two types of human keratocytes were used, which were freshly isolated keratocytes from donor corneas and cultured keratocytes. Expression of the mRNAs for various molecular markers was analyzed in these cells by RT-PCR, and TLR-2, -3, -4, and -9 mRNAs were also analyzed by RT-PCR. Expression of TLR-3 and -9 at the protein level was assessed by flow cytometry. In addition, an antibody array and ELISA were used to detect chemokines and cytokines in the supernatant of cultured keratocytes, with or without stimulation by poly inosine-polycytidylic acid (poly (I:C)) or CpG-DNA. Furthermore, a phagocytosis assay was performed to evaluate whether signaling via TLR-3 and -9 enhances phagocytosis.RESULTS. Keratocytes cultured for three passages underwent differentiation into corneal myofibroblasts. TLR-3 and -9 were detected in corneal myofibroblasts at the mRNA and protein levels, but not in freshly isolated keratocytes. Stimulation of corneal myofibroblasts with poly (I:C) or CpG-DNA enhanced the production of IL-6, IL-8, GRO, ENA-78, and RANTES compared with that by untreated cells. Phagocytic activity of myofibroblasts was upregulated by signaling via TLR-3 and -9.CONCLUSIONS. This is the first report on the in vitro expression and function of TLR-3 and -9 in corneal myofibroblasts. The findings suggest that the keratocyte phenotype determines the expression of TLR-3 and -9 and that corneal myofibroblasts may have an important role in bacterial and viral clearance.