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Studies on the immunological and genetic properties of sensitive hosts for the pathogenic mycobacteria

病原分枝杆菌敏感宿主的免疫学和遗传特性研究

基本信息

批准号：
15580274
负责人：
GOTO Yoshitaka
金额：
$ 2.24万
依托单位：
University of Miyazaki
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (C)
财政年份：
2003
资助国家：
日本
起止时间：
2003 至 2004
项目状态：
已结题

项目摘要

The murine natural resistance-associated macrophage protein, NRAMP1 has multiple pleiotropic effects on macrophage activation and regulates survival of intracellular pathogens. Genetic analyses have now linked polymorphisms in and around the NRAMP1 locus with susceptibility to some mycobacterial species in humans. In this time, the expression of NRAMP-1 proteins and mRNAs in the human macrophage cell line THP-1 was investigated by western blot analysis and real time-polymerase chain reaction(RT-PCR), respectively. Phorbol myristate acetate(PMA)- differentiated THP-1 cells were used in these studies. Unstimulated (without PMA) THP-1 cells constitutively expressed a low level of NRAMP1 mRNA and proteins. Both PMA and live bacteria stimulation (M.avium) could slightly accelerate the mRNA synthesis. The amount of NRAMP1 mRNA dramatically increased (>10 times) if THP-1 cells were stimulated with recombinant human IFN-γ(200U/ml). The growth of M.avium in THP-1 cells was inhibited if cells had been stimulated with PMA, and IFN-γ did not affect the growth of M.avium. This in vitro infection model seems to be a useful tool for studying the function of human NRAMP1. Next we examined the NRAMP-1 function in Mycobacterium lepraemurium(MLM) infected mice. Although NRAMP1 did not affect the susceptibility/resistance to the MLM infection among mouse strains, female mice were more sensitive than male mice if they were received with 10^8 bacteria intravenously. Finally, we tried to clarify the relationship between NRAMP1 and NK cells and their roles in M.avium infected mice. NK cell deficient beige mice increased their susceptibility to M.avium. Anti-NK cell antibody treatment in vivo decreased total number of NK cells in the mouse and reduced the ability to produce IFN-γ dramatically. However no relationship was observed between natural resistance to the infection by the NRAMP1 and NK cell function.

鼠天然抗性相关巨噬细胞蛋白NRAMP 1对巨噬细胞活化具有多种多效性作用，并调节细胞内病原体的存活。遗传分析现在已经将NRAMP 1基因座及其周围的多态性与人类对某些分枝杆菌物种的易感性联系起来。在此期间，分别通过蛋白质印迹分析和真实的时间-聚合酶链反应（RT-PCR）来研究NRAMP-1蛋白和mRNA在人巨噬细胞系THP-1中的表达。在这些研究中使用佛波醇肉豆蔻酸酯乙酸酯（PMA）分化的THP-1细胞。未刺激的THP-1细胞组成型表达低水平的NRAMP 1 mRNA和蛋白。PMA和活菌刺激（鸟分枝杆菌）都能轻微促进mRNA的合成。重组人IFN-γ（200 U/ml）刺激THP-1细胞后，NRAMP 1 mRNA表达量明显增加（>10倍）。鸟分枝杆菌在THP-1细胞中的生长受到PMA刺激的抑制，IFN-γ对鸟分枝杆菌的生长无影响。这种体外感染模型似乎是研究人NRAMP 1功能的有用工具。接下来，我们检查了NRAMP-1在鼠麻风分枝杆菌（MLM）感染的小鼠中的功能。尽管NRAMP 1不影响小鼠品系对MLM感染的易感性/抗性，但如果雌性小鼠静脉注射10^8个细菌，则它们比雄性小鼠更敏感。最后，我们试图阐明NRAMP 1和NK细胞之间的关系及其在鸟支原体感染小鼠中的作用。NK细胞缺陷的米色小鼠增加了他们的易感性M.鸟。体内抗NK细胞抗体处理降低了小鼠中NK细胞的总数，并显著降低了产生IFN-γ的能力。然而，未观察到对NRAMP 1感染的天然抗性与NK细胞功能之间的关系。