外周血培养肥大细胞：不同培养方案的表型和功能结果。
Peripheral blood cultured mast cells: Phenotypic and functional outcomes of different culture protocols.
机构信息
University of Antwerp, Faculty of Medicine and Health Sciences, Department of Immunology, Allergology, Rheumatology and the Infla-Med Centre of Excellence, Antwerp (Belgium) and Immunology, Allergology, Rheumatology, Antwerp University Hospital, Antwerp, Belgium.
University of Antwerp, Faculty of Medicine and Health Sciences, Department of Immunology, Allergology, Rheumatology and the Infla-Med Centre of Excellence, Antwerp (Belgium) and Immunology, Allergology, Rheumatology, Antwerp University Hospital, Antwerp, Belgium; Department of Immunology and Allergology, AZ Jan Palfijn Gent, Ghent, Belgium.
出版信息
J Immunol Methods. 2021 May;492:113003. doi: 10.1016/j.jim.2021.113003. Epub 2021 Feb 26.
BACKGROUND
Mast cells (MCs) play a pivotal role in innate and adaptive immune responses. However, MCs are also involved in different pathologic conditions. Studies on the mechanisms that govern human MC functions are impeded by their limited and difficult recovery. Therefore, several research groups have developed protocols to culture human MCs from progenitor cells. These protocols vary with respect to culture duration and used maturation cytokines. How MCs obtained by different protocols differ in phenotype and functionality is currently unknown.
OBJECTIVE
To compare different protocols for the generation of human MCs from peripheral blood progenitors.
METHODS
Thirteen paired human MC cultures were investigated. MCs were cultured form CD34 progenitors cells for 4 or 8 weeks and with or without the addition of IL-6. Phenotyping comprised staining for CD117, CD203c, FcεRI, MRGPRX2, CD300a and CD32. Functional studies included measurements of the up-regulation of CD63 and CD203c after allergen-specific cross-linking of sIgE/FcεRI complexes or ligation of MRGPRX2 with substance P and different drugs.
RESULTS
Cell cultures for 4 weeks in the presence of IL-6 consistently yielded the highest numbers of MCs. MCs cultured for 8 weeks with IL-6 showed more autofluorescence significantly impeding correct analyses of FcεRI and CD32. The density of FcεRI and CD32 was comparable between the different culture conditions. MRGPRX2 expression was significantly higher in the 8 week cultures. The density of CD300a was increased in the cultures with IL-6. Cells cultured for 8 weeks were more responsive to MRGPRX2 activation. In contrast, the 4-weeks cultures with IL-6 showed significantly higher allergen-specific activation.
CONCLUSION
Four weeks of culture with IL-6 are sufficient to generate sizeable numbers of human mast cells from blood progenitors, thereby enabling simultaneous exploration of allergen-specific sIgE/FcεRI cross-linking and non-specific activation via MRGPRX2.
背景
肥大细胞（MCs）在先天和适应性免疫反应中发挥关键作用。然而，MCs 也参与了不同的病理状况。由于 MC 功能的调控机制难以捉摸且恢复有限，因此研究人员难以进行相关研究。因此，一些研究小组已经开发了从祖细胞培养人 MC 的方案。这些方案在培养时间和使用的成熟细胞因子方面有所不同。目前尚不清楚通过不同方案获得的 MC 在表型和功能上有何不同。
目的
比较从外周血祖细胞生成人 MC 的不同方案。
方法
研究了 13 对配对的人 MC 培养物。将 MC 从 CD34 祖细胞培养 4 或 8 周，并添加或不添加 IL-6。表型鉴定包括对 CD117、CD203c、FcεRI、MRGPRX2、CD300a 和 CD32 进行染色。功能研究包括测量过敏原特异性交联 sIgE/FcεRI 复合物或 MRGPRX2 与 P 物质和不同药物结合后 CD63 和 CD203c 的上调。
结果
在存在 IL-6 的情况下培养 4 周可始终获得最多数量的 MC。用 IL-6 培养 8 周的 MC 显示出更高的自发荧光，显著阻碍了对 FcεRI 和 CD32 的正确分析。不同培养条件下 FcεRI 和 CD32 的密度相当。MRGPRX2 的表达在 8 周培养物中明显更高。在有 IL-6 的培养物中，CD300a 的密度增加。培养 8 周的细胞对 MRGPRX2 激活的反应性更高。相反，用 IL-6 培养 4 周的细胞表现出明显更高的过敏原特异性激活。
结论
用 IL-6 培养 4 周足以从血液祖细胞中生成大量的人肥大细胞，从而能够同时探索过敏原特异性 sIgE/FcεRI 交联和通过 MRGPRX2 的非特异性激活。
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