巨噬细胞活化因子(GcMAF-RF)对巨噬细胞离体极化、树突状细胞活化和人全血细胞产生细胞因子的影响

S. Kirikovich, E. Levites, E. Dolgova, A. Proskurina, G. Ritter, V. Ruzanova, O. Leplina, E. Shevela, A. Ostanin, T. Ryabicheva, S. Ryzhikova, Yu. G. Druzhinina, N. Varaksin, E. Chernykh, S. Bogachev
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The stimulating effect of the obtained Mph and DC upon cell proliferation was assessed in allogeneic mixed culture of leukocytes (alloMLC) using radiometric technique, by 3 H-thymidine incorporation. The influence index (IR) of Mph or DC upon allo-SCL was calculated as the ratio of the proliferative response of MNCs in the presence of Mph, or DC to the level of spontaneous MNC proliferation. To determine the cytokine production by human whole blood cells ex vivo, peripheral blood samples from 3 donors with two replicate GcMAF-RF preparations were used, at a total of 6 points. All variants of the study were carried out with mitogen-activated and non-activated blood cells. The cytokine content was determined by the ELISA assays. The effects of GcMAF-RF were quantified as a fold increase (FI), i.e., the ratio of cytokine production in the presence of GcMAF-RF to the level of their spontaneous production. 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引用次数: 0

摘要

这篇文章是一系列文章中的第二篇,这些文章致力于巨噬细胞活化因子(GcMAF-RF)的国产制剂的作用及其生物学特性的评估。这项工作的目的是研究GcMAF RF对M0的影响→ 巨噬细胞(Mph)的M1极化,体外产生的抗原呈递树突状细胞(DC)的专业特性的激活,以及促炎细胞因子(TNFα、IL-1β、IL-6、IFNγ、IL-17、IL-18)和抗炎细胞因子(TGF-β、IL-4、IL-10)的体外产生,生长因子(IL-2、GM-CSF、G-CSF、VEGF)和趋化因子(MCP、IL-8)。Mph和DC由健康供体的外周血单核细胞(MNC)的粘附部分衍生的单核细胞产生(3至5×106/ml)。粒细胞/巨噬细胞集落刺激因子(rhGM-CSF)用于获得Mph,而GM-CSF和干扰素-α诱导DC产生。为了提供M1极化信号,在对照中使用细菌脂多糖(来自大肠杆菌0114:B4的LPS)。在实验系列中,在培养结束前48小时加入GcMAF RF。在异基因白细胞混合培养物(alloMLC)中,使用放射技术,通过3H-胸苷掺入,评估获得的Mph和DC对细胞增殖的刺激作用。Mph或DC对allo SCL的影响指数(IR)计算为存在Mph或直流时MNC的增殖反应与自发MNC增殖水平的比率。为了测定人全血细胞体外产生的细胞因子,使用了来自3名捐献者的外周血样本,共6个点,这些捐献者具有两种重复的GcMAF RF制剂。研究的所有变体都是用促有丝分裂原活化和非活化的血细胞进行的。细胞因子含量通过ELISA测定法测定。GcMAF RF的作用被量化为倍数增加(FI),即在GcMAFRF存在下细胞因子产生与其自发产生水平的比率。结果表明,GcMAF RF制剂与脂多糖(LPS)一样有效,脂多糖是标准的Mph和DC激活剂,可诱导分化的M0巨噬细胞极化为M1细胞并最终成熟DC,表现为在混合白细胞培养物(allo-MLC)中其allo刺激活性显著增加。此外,GcMAF RF刺激血细胞(粒细胞、淋巴细胞、单核细胞)产生大量细胞因子和趋化因子(TNFα、IL-1β、IL-6、IL-18、IL-4、IL-10、GM-CSF、G-CSF、VEGF、IL-8),从而表明巨噬细胞激活剂GcMAF-RF直接参与各种免疫过程。国产GcMAF射频药物诱导巨噬细胞M0极化→ M1,DC的最终成熟以及Mf和DC的异刺激活性,并且还能够有效刺激循环血细胞合成具有促炎和免疫调节活性的细胞因子/趋化因子。
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Effect of macrophage-activating factor (GcMAF-RF) upon ex vivo polarization of macrophages, activation of dendritic cells and production of cytokines by human whole blood cells
This article is the second communication in a series of articles devoted to the effects of a domestic preparation of macrophage-activating factor (GcMAF-RF) and assessment of its biological properties. The aim of this work was to study the effect of the GcMAF-RF upon M0 → M1 polarization of macrophages (Mph), and activation of the professional properties of ex vivo generated antigen-presenting dendritic cells (DC), as well as on ex vivo production of pro-inflammatory (TNFα, IL-1β, IL-6, IFNγ, IL-17, IL-18) and anti-inflammatory (TGF-β, IL-4, IL-10) cytokines, growth factors (IL-2, GM-CSF, G-CSF, VEGF) and chemokines (MCP, IL-8) by the whole blood cells from healthy donors. Mph and DC were generated from the monocytes (3 to 5×106 /ml) derived from adherent fraction of peripheral blood mononuclear cells (MNC) of healthy donors. Granulocyte/macrophage colony-stimulating factor (rhGM-CSF) was used to obtain Mph, whereas DC production was induced by GM-CSF and interferon-α. To provide M1 polarizing signals, bacterial lipopolysaccharide (LPS from E. coli 0114:B4) was used in controls. In experimental series, GcMAF-RF was added 48 h before the end of culture. The stimulating effect of the obtained Mph and DC upon cell proliferation was assessed in allogeneic mixed culture of leukocytes (alloMLC) using radiometric technique, by 3 H-thymidine incorporation. The influence index (IR) of Mph or DC upon allo-SCL was calculated as the ratio of the proliferative response of MNCs in the presence of Mph, or DC to the level of spontaneous MNC proliferation. To determine the cytokine production by human whole blood cells ex vivo, peripheral blood samples from 3 donors with two replicate GcMAF-RF preparations were used, at a total of 6 points. All variants of the study were carried out with mitogen-activated and non-activated blood cells. The cytokine content was determined by the ELISA assays. The effects of GcMAF-RF were quantified as a fold increase (FI), i.e., the ratio of cytokine production in the presence of GcMAF-RF to the level of their spontaneous production. It was shown that the GcMAF-RF preparation was as effective, as lipopolysaccharide (LPS), the standard Mph and DC activator which induces polarization of differentiated M0-macrophages into M1 cells and final maturation of DCs, manifesting by a significant increase in their allo-stimulatory activity in a mixed leukocyte culture (allo-MLC). Moreover, GcMAF-RF stimulates production of numerous cytokines and chemokines (TNFα, IL-1β, IL-6, IL-18, IL-4, IL-10, GM-CSF, G-CSF, VEGF, IL-8), by blood cells (granulocytes, lymphocytes, monocytes), thus indicating direct participation of the macrophage activator GcMAF-RF in various immune processes. The domestic GcMAF-RF drug induces polarization of macrophages M0 → M1, final maturation of DCs and allostimulating activity of Mf and DCs, and is also able to effectively stimulate circulating blood cells to synthesize cytokines/chemokines with pro-inflammatory and immunoregulatory activities. 
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