C-C趋化因子受体5（CCR5）抑制剂对CCR5动态变化及C-C趋化因子与CCR5相互作用的影响。
Effects of CC chemokine receptor 5 (CCR5) inhibitors on the dynamics of CCR5 and CC-chemokine-CCR5 interactions.
作者信息
Nakata Hirotomo, Kruhlak Michael, Kamata Wakako, Ogata-Aoki Hiromi, Li Jianfeng, Maeda Kenji, Ghosh Arun K, Mitsuya Hiroaki
机构信息
Experimental Retrovirology Section, HIV and AIDS Malignancy Branch, Center for Cancer Research, National Cancer Institute, National Institutes of Health, Bethesda, MD, USA.
出版信息
Antivir Ther. 2010;15(3):321-31. doi: 10.3851/IMP1529.
BACKGROUND
This study aimed to examine how CC chemokine receptor 5 (CCR5) inhibitors (aplaviroc [APL], TAK779 and maraviroc [MVC]) interact with CCR5 and affect its dynamics and physiological CC-chemokine-CCR5 interactions.
METHODS
A yellow fluorescent protein (YFP)-tagged CCR5-expressing U373-MAGI cell line was generated and a stable CCR5-expressing clonal population, (YFP)CCR5-UM16, was prepared. (YFP)CCR5-UM16 cells were exposed to RANTES, macrophage inflammatory protein (MIP)-1alpha or MIP-1beta (all 100 ng/ml) with or without CCR5 inhibitors and (YFP)CCR5 internalization was visualized with real-time by laser scanning confocal microscopy. The mobility of (YFP)CCR5 was also examined in the presence of CCR5 inhibitors with fluorescence recovery after photobleaching (FRAP) imaging.
RESULTS
Following the addition of each CC chemokine, intracellular fluorescence intensity increased whereas membranous fluorescence decreased, signifying (YFP)CCR5 internalization. All three CCR5 inhibitors failed to induce (YFP)CCR5 internalization. All three CCR5 inhibitors blocked the CC-chemokine-induced internalization at a high concentration of 1 microM; however, the ratio of APL concentration that blocked RANTES-induced internalization by 50% over APL concentration that blocked HIV type-1 (HIV-1) replication by 50% was 16.4, indicating that APL permits CC-chemokine-induced internalization to a much greater extent compared with TAK779 and MVC, having ratios of 1.1 and 0.9, respectively. The examination of (YFP)CCR5 mobility with FRAP imaging revealed that (YFP)CCR5 continuously underwent rapid redistribution, which none of the three inhibitors blocked.
CONCLUSIONS
The finding that APL moderately blocked the RANTES-triggered (YFP)CCR5 internalization despite the highly potent anti-HIV-1 activity of APL strongly suggests that development of CCR5 inhibitors, which do not overly inhibit physiological CC-chemokine-CCR5 interactions, is practically feasible.
背景
本研究旨在探讨C-C趋化因子受体5（CCR5）抑制剂（阿普韦罗克[APL]、TAK779和马拉维罗克[MVC]）如何与CCR5相互作用，并影响其动力学以及生理性C-C趋化因子与CCR5的相互作用。
方法
构建了一种表达黄色荧光蛋白（YFP）标记的CCR5的U373-MAGI细胞系，并制备了稳定表达CCR5的克隆群体（YFP）CCR5-UM16。将（YFP）CCR5-UM16细胞暴露于RANTES、巨噬细胞炎性蛋白（MIP）-1α或MIP-1β（均为100 ng/ml），同时加入或不加入CCR5抑制剂，通过激光扫描共聚焦显微镜实时观察（YFP）CCR5的内化情况。还利用光漂白后荧光恢复（FRAP）成像技术在存在CCR5抑制剂的情况下检测（YFP）CCR5的流动性。
结果
添加每种C-C趋化因子后，细胞内荧光强度增加而膜荧光强度降低，表明（YFP）CCR5发生了内化。所有三种CCR5抑制剂均未能诱导（YFP）CCR5内化。所有三种CCR5抑制剂在1 μM的高浓度下均能阻断C-C趋化因子诱导的内化；然而，阻断RANTES诱导内化50%的APL浓度与阻断1型人类免疫缺陷病毒（HIV-1）复制50%的APL浓度之比为16.4，这表明与TAK779和MVC相比，APL在更大程度上允许C-C趋化因子诱导的内化，TAK779和MVC的该比值分别为1.1和0.9。通过FRAP成像对（YFP）CCR5流动性的检测显示，（YFP）CCR5持续进行快速重新分布，而这三种抑制剂均未阻断这种重新分布。
结论
尽管APL具有高效的抗HIV-1活性，但它能适度阻断RANTES触发的（YFP）CCR5内化，这一发现强烈表明，开发不过度抑制生理性C-C趋化因子与CCR5相互作用的CCR5抑制剂在实际中是可行的。
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