Institute of Pharmacology, University of Bern, CH-3010 Bern, Switzerland.
Allergy. 2013;68(5):604-13. doi: 10.1111/all.12140.
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.
A method is described to massively expand bone marrow-derived 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.
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).
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.
嗜碱性粒细胞是一种罕见的白细胞群体，以其在炎症和过敏反应中的效应功能而闻名，最近还描述了其免疫调节作用。除了频率低之外，由于其寿命短、体外分化效率低以及缺乏合适的细胞模型，因此对嗜碱性粒细胞的功能分析受到阻碍。一种在体外大量产生嗜碱性粒细胞的方法将有助于嗜碱性粒细胞的研究，并构成用于诊断和药物测试目的的一种受欢迎的工具。
本文描述了一种在体外大量扩增骨髓来源的嗜碱性粒细胞的方法。在白细胞介素-3（IL-3）存在的情况下，使用 Hoxb8 对髓样祖细胞进行条件性永生化，并且选择在 Hoxb8 表达关闭时能够分化为嗜碱性粒细胞的细胞系进行生长。
可以在体外长期扩增和维持依赖于 IL-3 的条件性 Hoxb8 永生化祖细胞系。在 Hoxb8 表达关闭后，在六天内可以在体外分化出近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的，寿命短，并表达嗜碱性粒细胞特异性表面标志物和蛋白酶。在 IgE 以及 C5a 介导的激活下，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 剥夺诱导与 BH3 仅蛋白 BCL-2 相互作用的细胞死亡介体（BIM）和 p53 上调凋亡调节剂（PUMA）的上调以及原病毒整合位点 Moloney 鼠白血病病毒 1 激酶（PIM-1）的下调相关的细胞凋亡。
本文提出了一种在体外生成定量的小鼠嗜碱性粒细胞的新方法，此外还允许对条件性永生化祖细胞进行基因操作。这种方法可能代表了分离原代嗜碱性粒细胞的有用替代方法。
