Department of Genetics and Molecular Biology, Xi'an Jiaotong University School of Medicine, Yanta West Road, Xi'an, Shaanxi 710061, PR China.
Arthritis Res Ther. 2010;12(3):R103. doi: 10.1186/ar3034. Epub 2010 May 25.
Toll-like receptors (TLRs) are involved in both innate and adaptive immune responses and are likely to play a complex role in the pathogenesis of human rheumatoid arthritis (RA) and experimental arthritis. The objective of this study was to identify the key TLR in pristane-induced arthritis (PIA), a rat model for RA, and to clarify its roles in the initiation and maintenance of arthritis.
Arthritis in DA rats was induced by pristane and the severity was evaluated by macroscopic and microscopic score systems. Spleen TLR and cytokine expression was detected at different time points by real-time polymerase chain reaction (PCR) and flow cytometry. Polyinosine-polycytidylic acid (polyI:C, a ligand of TLR3) or TLR3 specific short-hairpin RNA plasmid for RNA interference was administrated to PIA rats in vivo. Serum nitrogen oxide concentration was determined by Griess method, and tumor necrosis factor alpha (TNF-alpha) was determined by L929 biotest. In splenic macrophages, TLR3 expression was measured by flow cytometry. A rat macrophage cell line (NR8383) was stimulated by pristane, and anti-TLR3 antibody were used to block TLR3 pathway. TLR3 and cytokine expression in NR8383 were detected by real-time PCR.
By screening the TLR expression profile in spleen of DA rats after pristane injection, we found that TLR3 was the most early and prominently upregulated TLR. Both TLR3 mRNA and protein expression of spleen were upregulated at 6 and 26 days after pristane injection. Furthermore, administration of polyI:C exacerbated, whereas RNA interference targeting TLR3 ameliorated, the arthritis. Particularly, TLR3 expression was induced in splenic macrophages of PIA rats, and also in the NR8383 cell line after pristane stimulation in a dose- and time- dependent manner. Upregulation of interferon beta (IFN-beta) and TNF-alpha by pristane stimulation was blocked by anti-TLR3 antibody in NR8383.
TLR3 plays a pivotal role in the initiation and development of PIA which may dependent on macrophage. These findings are useful to understand the pathogenesis of RA and may provide an intriguing therapeutic opportunity for RA.
Toll 样受体（TLR）参与固有和适应性免疫反应，并且可能在人类类风湿关节炎（RA）和实验性关节炎的发病机制中发挥复杂作用。本研究的目的是鉴定 pristane 诱导的关节炎（PIA），即 RA 大鼠模型中的关键 TLR，并阐明其在关节炎的起始和维持中的作用。
通过 pristane 诱导 DA 大鼠关节炎，并通过宏观和微观评分系统评估严重程度。通过实时聚合酶链反应（PCR）和流式细胞术检测不同时间点脾脏 TLR 和细胞因子的表达。体内给予 PIA 大鼠聚肌苷酸-聚胞苷酸（polyI:C，TLR3 的配体）或 TLR3 特异性短发夹 RNA 质粒。通过格里斯法测定血清氮氧化物浓度，通过 L929 生物测定法测定肿瘤坏死因子-α（TNF-α）。通过流式细胞术测定脾巨噬细胞中 TLR3 的表达。用 pristane 刺激大鼠巨噬细胞系（NR8383），并用抗 TLR3 抗体阻断 TLR3 途径。通过实时 PCR 检测 NR8383 中的 TLR3 和细胞因子表达。
通过筛选 pristane 注射后 DA 大鼠脾脏中 TLR 表达谱，我们发现 TLR3 是最早且最显著上调的 TLR。TLR3 基因和蛋白表达在 pristane 注射后 6 和 26 天均上调。此外，polyI:C 的给药加重了关节炎，而针对 TLR3 的 RNA 干扰则改善了关节炎。特别地，TLR3 在 PIA 大鼠的脾巨噬细胞中表达，并且在 NR8383 细胞系中也在剂量和时间依赖性方式下在 pristane 刺激后被诱导。NR8383 中，抗 TLR3 抗体阻断了 pristane 刺激引起的干扰素-β（IFN-β）和 TNF-α的上调。
TLR3 在 PIA 的起始和发展中起关键作用，这可能依赖于巨噬细胞。这些发现有助于理解 RA 的发病机制，并为 RA 提供了一种有吸引力的治疗机会。
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