M-CSF、GM-CSF诱导人源外周血单个核细胞来源巨噬细胞的不同极化和吞噬

Differences in polarization and phagocytosis for PBMC sourced macrophage induced by M-CSF and GM-CSF

目的观察人源外周血单个核细胞(peripheral blood mononuclear cells,PBMC)在体外培养诱导单核巨噬细胞时是否因刺激剂M-CSF、GM-CSF的不同而造成极化状态差异,并衡量极化状态差异是否伴有吞噬能力的改变,并寻找可能相关的免疫表型标记。方法取健康献血员新鲜外周白膜血,分离获得PBMC后,每一份PBMC都分为MCSF、GMCSF组,分别对应M-CSF或GM-CSF作为诱导剂。培养d7时用诱导得到的单核巨噬细胞分别和致敏红细胞、未致敏红细胞、血小板(Plt)以及葡聚糖-40(dextran-40)共孵育,模拟MMA(单核细胞单层实验),利用流式细胞仪观察吞噬结果。同时用流式分析2组细胞的CD11C、CD14、CD15、CD16、DR、CD32、CD32b、CD49f、CD54、CD64、CD68、CD86、CD163等免疫表型。所有得到的结果进行配对t检验分析差异。结果 MCSF、GMCSF组CD14+CD16+细胞占比分别为(62.6±33.7)%vs(22.8±15.6)%,CD163+CD14+细胞(29.7±18.7 vs 0.64±0.42), CD14 MFI 3 829±2 091vs464±101,(P均<0.05)。同时MCSF组伴随CD64+(45.7±25)%vs(24.5±19)%、CD32b+(59.5±22.4)%vs(27.5±11.8)%细胞占比增高,CD32+、HLA-DR+细胞数虽然无明显变化,但其MFI分别较GMCSF组增高和降低(MFI CD32 1 093±380vs530±179;MFI DR 1 034±290vs1 661±367)。同时发现MCSF组具有较强的吞噬力,对被吞物的吞噬力多数为Plt> Dextran-40>致敏RBC>非致敏RBC。结论证实了人源PBMC诱导的单核巨噬细胞在体外培养时同一起源样品也可因M-CSF、GM-CSF而促成不同极化方向, M-CSF促进M2型极化, GM-CSF促进M1型极化,并由此影响对致敏红细胞、血小板(Plt)以及葡聚糖-40的吞噬能力,因此在临床使用M-CSF、GM-CSF时需注意单核巨噬细胞的极化方向,采取更有利于患者治疗和临床实验的制品。

Objective To observe different polarization for PBMC sourced macrophage induced by M-CSF or GM-CSF in vitro culture and evaluate the phagocytosis capacity under different polarization, therefore find out the possible related phenotype markers. Methods Buffy coats from healthy donors were used to isolate peripheral blood mononuclear cells(PBMC) by density centrifuge using FICOLL-paque plus. Every PBMC sample was split in half;one was cultured for GMCSF group and the other was for MCSF. On the 7 th day of culture, the phagocytosis capacity for induced macrophages were evaluated using the methods mimic MMA(monocyte monolayer assay) with dextran-40, platelets, opsonized RBC and unopsonized RBC, respectively, through Flow-cytometry, then CD11 C, CD14, CD15, CD16, DR, CD32, CD32 b, CD49 f, CD54, CD64, CD68, CD86 and CD163 in two groups were analyzed in parallel. All the results were analyzed by paired t-test.Results The percentages(%) of CD14+CD16+ cells(62.6±33.7 vs 22.8±15.6) and CD163+CD14+ cells(29.7±18.7 vs 0.64±0.42) in M-CSF group were significantly higher than those in GMCSF group, and CD14 MFI were also higher(3 829±2 091vs 464±101), along with the increased proportion(%) of CD64+(45.7±25 vs 24.5±19) and CD32 b+(59.5±22.4 vs 27.5±11.8)(P<0.05) in MCSF group, leaving CD32+ and HLA-DR+ remained almost the same between two groups,while with higher MFI CD32(1 093±380 vs 530±179) and lower MFI DR(1 034±290 vs 1 661±367) in M-CSF group vs GMCSF group. Compared to GMCSF, MCSF group exhibited more powerful phagocytosis capacity, and the strength of phagocytosis was as following: PLT> Dextran-40>opsonized RBC>unopsonized RBC. Conclusion Our study confirmed that M-CSF and GM-CSF can stimulate the same source PBMC into different polarized macrophages;M-CSF induced M2 macrophage while GM-CSF pro M1, thus affected the phagocytosis capacity of PBMC sourced macrophage for Dextran-40,platelets, opsonized RBC and unopsonized RBC,therefore we should make appropriate selection of MCSF and GMCSF in clinical experiments or patient treatments.