In vitro differentiation of near-unlimited numbers of functional mouse basophils using conditional Hoxb8
In vitro differentiation of near-unlimited numbers of functional mouse basophils using conditional Hoxb8
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DOI：
10.1111/all.12140
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发表时间：
2013-05-01
期刊：
影响因子：
12.4
通讯作者：
Kaufmann, T.
中科院分区：
文献类型：
--
作者：
Gurzeler, U.;Rabachini, T.;Kaufmann, T.
背景嗜碱性粒细胞构成了一种罕见的白细胞群体，以其在炎症和过敏中的效应器功能以及最近描述的免疫调节作用而闻名。除了频率低之外，嗜碱性粒细胞的功能分析还受到寿命短、体外分化方案效率低以及缺乏合适的细胞模型的阻碍。一种在体外产生大量嗜碱性粒细胞的方法将促进嗜碱性粒细胞研究，并成为诊断和药物测试目的的抢手工具。方法描述了一种在体外大规模扩增骨髓来源的嗜碱性粒细胞的方法。在白介素 3 (IL-3) 存在的情况下，使用 Hoxb8 对骨髓祖细胞进行条件永生化，并根据 Hoxb8 表达关闭后分化为嗜碱性粒细胞的潜力来选择生长的细胞系。结果 IL-3 依赖性、条件性 Hoxb8 永生化祖细胞系可以在培养物中扩增并维持较长时间。 Hoxb8表达关闭后，六天内可在体外分化出几乎无限数量的成熟功能性嗜碱性粒细胞。这些细胞是终末分化且寿命短的，并表达嗜碱性粒细胞特异性表面标记物和蛋白酶。在 IgE 和 C5a 介导的激活后，分化的嗜碱性粒细胞释放颗粒酶和组胺，并分泌 Th2 型细胞因子（IL-4、IL-13）和白三烯 C4。 IL-3 剥夺诱导细胞凋亡，与 BH3 蛋白、BCL-2 相互作用细胞死亡介质 (BIM) 和 p53 上调细胞凋亡调节剂 (PUMA) 上调以及莫洛尼鼠白血病病毒 1 激酶 (PIM-1) 前病毒整合位点下调相关。结论提出了一种在体外定量产生小鼠嗜碱性粒细胞的新方法，此外该方法还允许对条件永生化祖细胞进行遗传操作。这种方法可能是分离原代嗜碱性粒细胞的一种有用的替代方法。
Background Basophils constitute a rare leukocyte population known for their effector functions in inflammation and allergy, as well as more recently described immunoregulatory roles. Besides their low frequency, functional analysis of basophils is hindered by a short life span, inefficient ex vivo differentiation protocols, and lack of suitable cell models. A method to produce large quantities of basophils in vitro would facilitate basophil research and constitute a sought-after tool for diagnostic and drug testing purposes. Methods A method is described to massively expand bone marrowderived basophils in vitro. Myeloid progenitors are conditionally immortalized using Hoxb8 in the presence of interleukin-3 (IL-3) and outgrowing cell lines selected for their potential to differentiate into basophils upon shutdown of Hoxb8 expression. Results IL-3-dependent, conditional Hoxb8-immortalized progenitor cell lines can be expanded and maintained in culture for prolonged periods. Upon shutdown of Hoxb8 expression, near-unlimited numbers of mature functional basophils can be differentiated in vitro within six days. The cells are end-differentiated and short-lived and express basophil-specific surface markers and proteases. Upon IgE- as well as C5a-mediated activation, differentiated basophils release granule enzymes and histamine and secrete Th2-type cytokines (IL-4, IL-13) and leukotriene C4. IL-3-deprivation induces apoptosis correlating with upregulation of the BH3-only proteins BCL-2-interacting mediator of cell death (BIM) and p53 upregulated modulator of apoptosis (PUMA) and downregulation of proviral integration site for Moloney murine leukemia virus 1 kinase (PIM-1). Conclusion A novel method is presented to generate quantitative amounts of mouse basophils in vitro, which moreover allows genetic manipulation of conditionally immortalized progenitors. This approach may represent a useful alternative method to isolating primary basophils.