Wei Liu, Hong Zhou, Jiang Zheng, Guofu Ding, Hongwei Cao, Liangxi Wang, Yongling Lu, Bin Li, Ping Luo
Department of Pharmacology, College of Medicine, The Third Military Medical University, Chongqing 400038, PR China.
Int Immunopharmacol. 2006 Jun;6(6):935-46. doi: 10.1016/j.intimp.2006.01.004. Epub 2006 Feb 3.
Low-dose CpG ODN pretreatment is known to induce effective protective immunity against acute infectious diseases. In the present study, using primary murine peritoneal macrophages and the macrophage-like cell line, RAW264.7, we investigated whether low-dose CpG ODN pretreatment would induce hyporesponsiveness in response to a subsequent high-dose CpG ODN challenge and further investigated the molecular mechanisms underlying this event. Our results showed that pretreatment with a low dose of CpG ODN inhibits TNF-alpha production stimulated by later high-dose CpG ODN stimulation in a dose- and time-dependent manner. Interestingly, anti-mouse TLR9 blocking antibody added prior to CpG ODN pretreatment did not affect TNF-alpha release, but antibody added after CpG ODN pretreatment augmented the pretreatment effect of CpG ODN. This difference suggests that cell-surface TLR9 is indeed functional on activated cells. Flow cytometry revealed that low-dose CpG ODN pretreatment decreased cell-surface binding and internalization of a subsequent high-dose stimulation, suggesting that decreased internalization of succeeding CpG ODN is associated with reduced TNF-alpha release. Although both intracellular and cell-surface TLR9 expression are observed, low dose of CpG ODN pretreatment increased only cell-surface TLR9 levels. Importantly, low-dose CpG ODN pretreatment also significantly inhibited the activation of NF-kappaB, an important downstream regulator of various proinflammatory cytokines. In summary, our results demonstrate that suppression of TNF-alpha production by low dose of CpG ODN pretreatment correlates with decreased binding and internalization of subsequent CpG ODN, decreased intracellular content of TLR9, and inhibition of NF-kappaB activation.
已知低剂量的CpG ODN预处理可诱导针对急性传染病的有效保护性免疫。在本研究中，我们使用原代小鼠腹腔巨噬细胞和巨噬细胞样细胞系RAW264.7，研究了低剂量CpG ODN预处理是否会导致对随后高剂量CpG ODN刺激的反应性降低，并进一步研究了这一现象背后的分子机制。我们的结果表明，低剂量的CpG ODN预处理以剂量和时间依赖性方式抑制了随后高剂量CpG ODN刺激所诱导的TNF-α产生。有趣的是，在CpG ODN预处理之前添加抗小鼠TLR9阻断抗体并不影响TNF-α的释放，但在CpG ODN预处理之后添加抗体则增强了CpG ODN的预处理效果。这种差异表明细胞表面的TLR9在活化细胞上确实具有功能。流式细胞术显示，低剂量的CpG ODN预处理降低了随后高剂量刺激的细胞表面结合和内化，这表明后续CpG ODN内化的减少与TNF-α释放的减少相关。尽管观察到细胞内和细胞表面均有TLR9表达，但低剂量的CpG ODN预处理仅增加了细胞表面TLR9的水平。重要的是，低剂量的CpG ODN预处理还显著抑制了NF-κB的激活，NF-κB是多种促炎细胞因子的重要下游调节因子。总之，我们的结果表明，低剂量的CpG ODN预处理对TNF-α产生的抑制作用与随后CpG ODN的结合和内化减少、TLR9的细胞内含量降低以及NF-κB激活的抑制有关。
