PMA活化THP-1的巨噬细胞分化标记特征分析
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摘要
目的了解佛波酯-12-肉豆蔻酸酯-13-乙酸酯(PMA)分化的THP-1(人单核细胞白血病肿瘤细胞)上巨噬细胞标记特征,评估其作为人源巨噬细胞的替代品的可行性。方法用PMA诱导分化THP-1细胞72 h(活化组),并同步分离随机健康O型献血者的新鲜外周血单个核细胞(PBMC),其中一部分PBMC用人粒细胞-巨噬细胞集落刺激因子(GM-CSF)培养7 d(7 d PBMC),而未处理的THP-1作为未活化组,分别用流式细胞仪分析这4组细胞的CD14、CD16、HLA-Ⅰ、HLA-DR等细胞表型,并分析Fc受体CD32、CD32b的组成;并用已知含人源HLA-Ⅰ、Ⅱ类抗体、抗-E、健康献血者血浆各1份以及6种未确定抗体特异性但免疫血液学试验检出疑有不规则抗体的血浆与活化THP-1和新鲜PBMC细胞共培养,观察CD14、CD16、HLA-I、HLA-DR等变化;制备O型IgG致敏红细胞和非致敏红细胞,分别用活化THP-1和PBMC细胞与致敏、非致敏红细胞共培养,进行单核细胞单层试验(MMA)。结果观察到THP-1细胞未活化组为CD14~+CD16~-,活化组为CD14~+CD16~(+d),缺乏PBMC中CD14~-CD16~+以及非经典CD14~(+h) CD16~+,后者在7 d PBMC中明显增加;未活化THP-1表达HLA-Ⅰ类、CD32分子,但HLA-Ⅱ类和CD32b较弱,在PMA刺激后这些抗原或标记分子均升高。发现活化THP-1细胞CD14、CD16分子可被人源性血浆吸附而致下降,而6例未确定抗体特异性的血浆有4例明显降低HLA-Ⅰ类表达。活化THP-1和PBMC均可成功作为单核细胞单层试验的反应细胞,诱导红细胞调理性吞噬。结论 THP-1可替代人单核巨噬细胞作为人源PBMC来源单核巨噬细胞的替代,细胞群落比较少;但是需要注意其与PBMC来源单核巨噬细胞的不同,并具有HLA抗原特异性,因此其应用范围可能比较局限。
Objective To identify the markers of macrophage differentiation in THP-1 stimulated by PMA,and to evaluate the possibility of using the markers as an alternative to human monocyte-derived macrophages. Methods THP-1 was differentiated with PMA for 72 H( stimulated),then Flow-cytometer was used to analysis CD14,CD16,HLA-I,HLA-II,CD32,CD32 b in parallel with fresh PBMC-derived macrophages( briefly as PBMC) from unrelated O type health donors,a part of PBMC were stimulated 7 days by GM-CSF( 7 d PBMC),and un-stimulated THP-1 was treated as group 4. Then all these cells were co-cultured with plasma-source antibodies including one known both of HLA-Ⅰand Ⅱ antibodies,one antiE,one plasma from unrelated donor and other 6 unknown specificities plasma suspected as irregular reaction by immunohematology lab,plasma treated cells were re-analyzed by Flow-cytometer for CD14,CD16,HLA-Ⅰand HLA-DR distribution. In the same time,Anti-RhD opsonized red cells were prepared with cells from health O type unrelated donors,monocyte monolayer assay( MMA) was then performed to check the difference between stimulated THP-1 and PBMC in reaction with opsonized and unopsonized red cells. Results Compared with PBMC,unstimulated THP-1 was CD14~+CD16~-,while stimulated THP-1 exhibited CD14~+CD16~+d,with no CD14~-CD16~+and much less CD14~+h CD16~+,these two population were more common in PBMC and 7 d PBMC,especially significantly increased CD14~+h CD16~+in 7 d PBMC. Stimulated THP-1 exhibited more weaker HLA-Ⅱ,CD32 b than those of PBMC source,similar but still a little weaker HLA-Ⅰ and CD32 compared to PBMC.HLA-Ⅰ,HLA-DR could be strengthen by stimulation of PMA,but CD14,CD16 could be slightly weaken by most human plasma. Both of stimulated THP-1 and PBMC can induce phagocytosis of opsonized red blood cells. 4 out of 6 plasma with suspected irregular antibodies were found to decrease HLA-Ⅰ expression. Conclusions THP-1 could be used as an alternative to human resource monocytes,with less cell clusters,but needs some discretion because of the differences with those of PBMC,and the potential HLA specificities.
Objective To identify the markers of macrophage differentiation in THP-1 stimulated by PMA,and to evaluate the possibility of using the markers as an alternative to human monocyte-derived macrophages. Methods THP-1 was differentiated with PMA for 72 H( stimulated),then Flow-cytometer was used to analysis CD14,CD16,HLA-I,HLA-II,CD32,CD32 b in parallel with fresh PBMC-derived macrophages( briefly as PBMC) from unrelated O type health donors,a part of PBMC were stimulated 7 days by GM-CSF( 7 d PBMC),and un-stimulated THP-1 was treated as group 4. Then all these cells were co-cultured with plasma-source antibodies including one known both of HLA-Ⅰand Ⅱ antibodies,one antiE,one plasma from unrelated donor and other 6 unknown specificities plasma suspected as irregular reaction by immunohematology lab,plasma treated cells were re-analyzed by Flow-cytometer for CD14,CD16,HLA-Ⅰand HLA-DR distribution. In the same time,Anti-RhD opsonized red cells were prepared with cells from health O type unrelated donors,monocyte monolayer assay( MMA) was then performed to check the difference between stimulated THP-1 and PBMC in reaction with opsonized and unopsonized red cells. Results Compared with PBMC,unstimulated THP-1 was CD14~+CD16~-,while stimulated THP-1 exhibited CD14~+CD16~+d,with no CD14~-CD16~+and much less CD14~+h CD16~+,these two population were more common in PBMC and 7 d PBMC,especially significantly increased CD14~+h CD16~+in 7 d PBMC. Stimulated THP-1 exhibited more weaker HLA-Ⅱ,CD32 b than those of PBMC source,similar but still a little weaker HLA-Ⅰ and CD32 compared to PBMC.HLA-Ⅰ,HLA-DR could be strengthen by stimulation of PMA,but CD14,CD16 could be slightly weaken by most human plasma. Both of stimulated THP-1 and PBMC can induce phagocytosis of opsonized red blood cells. 4 out of 6 plasma with suspected irregular antibodies were found to decrease HLA-Ⅰ expression. Conclusions THP-1 could be used as an alternative to human resource monocytes,with less cell clusters,but needs some discretion because of the differences with those of PBMC,and the potential HLA specificities.
引文
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[10]Genin M,Clement F,Fattaccioli A,et al.M1 and M2 macrophages derived from THP-1 cells differentially modulate the response of cancer cells to etoposide.BMC Cancer,2015,15:577-591.doi:10.1186/s12885.015-1546-9.
[11]Daigneault M,Preston JA,Marriott HM,et al.The identification of markers of macrophage differentiation in PMA-stimulated THP-1cells and monocyte-derived macrophages.PLoS One,2010,13,5(1):e8668.doi:10.1371/journal.pone.0008668.
[2]Noumsi GT,Billingsley KL,Moulds JM.Successful transfusion of antigen positive blood to alloimmunised patients using a monocyte monolayer assay.Transfus Med,2015,25(2):92-100.
[3]Tong TN,Branch DR.Use of a monocyte monolayer assay to evaluate Fcγreceptor-mediated phagocytosis,J Vis Exp,2017,2(119):e55039.
[4]Gordon S,Taylor PR.Monocyte and macrophage heterogeneity.Nat Rev Immunol,2005,5(12):953-964.
[5]Mosser DM,Edwards JP.Exploring the full spectrum of macrophage activation.Nat Rev Immunol,2008,8(12):958-969.
[6]Nagelkerke SQ,Dekkers G,Kustiawan I,et al.Inhibition of FcγR-mediated phagocytosis by IVIg is independent of Ig G-Fc sialylation and FcγRIIb in human macrophages.Blood,2014,124(25):3709-3718.
[7]Zhou D,Huang C,Lin Z,et al.Macrophage polarization and function with emphasis on the evolving roles of coordinated regulation of cellular signaling pathways.Cell Signal,2014,26(2):192-197.
[8]Edwards JP,Zhang X,Frauwirth KA,et al.Biochemical and functional characterization of three activated macrophage populations.JLeukoc Biol,2006,80(6):1298-1307.
[9]Arman M,Krauel K.Human platelet Ig G Fc receptor FcγRIIA in immunity and thrombosis.J Thromb Haemos,2015,13(6):893-908.
[10]Genin M,Clement F,Fattaccioli A,et al.M1 and M2 macrophages derived from THP-1 cells differentially modulate the response of cancer cells to etoposide.BMC Cancer,2015,15:577-591.doi:10.1186/s12885.015-1546-9.
[11]Daigneault M,Preston JA,Marriott HM,et al.The identification of markers of macrophage differentiation in PMA-stimulated THP-1cells and monocyte-derived macrophages.PLoS One,2010,13,5(1):e8668.doi:10.1371/journal.pone.0008668.