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首页> 外文期刊>Cytometry: The Journal of the Society for Analytical Cytology >Flow cytometric analysis of cytokine production by normal human peripheral blood dendritic cells and monocytes: Comparative analysis of different stimuli, secretion-blocking agents and incubation periods
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Flow cytometric analysis of cytokine production by normal human peripheral blood dendritic cells and monocytes: Comparative analysis of different stimuli, secretion-blocking agents and incubation periods

机译：正常人外周血树突状细胞和单核细胞产生细胞因子的流式细胞术分析：不同刺激，分泌阻断剂和潜伏期的比较分析

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摘要

In this paper, we comparatively analyze the effects of the following different stimuli on the production and intracellular accumulation of the interleukin (IL)-1 beta, IL-6, IL-12, tumor necrosis factor-alpha (TNF-alpha), and IL-8 inflammatory cytokines in both normal human peripheral blood (PB) dendritic cell (DC) subsets and monocytes: lipopolysaccharide (LPS) versus Staphylococcus aureus cowan 1 (SAC) in the presence or absence of interferon(IFN)-gamma-, cytokine secretion-blocking agents (brefeldin A alone versus brefeldin A plus monensin), and incubation periods (6, 12, and 24 h), For this purpose, a four-color multiple-staining direct immunofluorescence technique analyzed by flow cytometry was systematically used in all experiments (n = 19), Our results show that after stimulation, an important proportion of each of the two CD33(+) myeloid DC subsets as well as the monocytes produce significant amounts of all cytokines analyzed under each of the experimental conditions assayed. In contrast, CC33(-/+lo) lymphoplasmocytoid DC failed to produce detectable levels of any of the above-mentioned cytokines under the same stimulatory conditions. Upon comparing the different stimuli used, LPS was associated with higher percentages of cytokine-producing cells compared with SAG, especially within the CD33(hi) DC subset; interestingly, the addition of IFN-gamma enhanced the response of monocytes to both LPS and SAG. As regards the secretion-blocking agents, brefeldin A alone was superior to the combination of brefeldin A and monensin, This is because it was frequently associated with both a higher percentage of cytokine-positive cells and greater amounts of detectable cytokines per cell. Sequential analysis of cytokine production by PB DC and monocytes after 6, 12, and 24 h of cell culture showed that after 6 h, an increased cell death rate existed among DC, which became even undetectable at 24 h, in the absence of a significant increase in cytokine secretion. In summary, our results show that from the experimental conditions assayed in this paper, to induce cytokine production by normal human DC and monocytes, maximum response is obtained once PB samples are stimulated for 6 h with LPS (with or without IFN-gamma) in the presence of brefeldin A alone. Cytometry (Comm, Clin. Cytometry) 46:33-40, 2001, (C) 2001 Wiley-Liss, Inc. [References: 38]
机译：在本文中，我们比较分析了以下不同刺激对白介素（IL）-1 beta，IL-6，IL-12，肿瘤坏死因子-α（TNF-alpha）和正常人外周血（PB）树突状细胞（DC）亚组和单核细胞中的IL-8炎性细胞因子：在存在或不存在干扰素（IFN）-γ-，细胞因子的情况下，脂多糖（LPS）与金黄色葡萄球菌Cowan 1（SAC）分泌阻滞剂（单独的布雷菲德菌素A相对于布雷菲德菌素A加莫能菌素）和潜伏期（6、12和24小时），为此目的，系统地使用了流式细胞仪分析的四色多色直接免疫荧光技术所有实验（n = 19），我们的结果表明，刺激后，两个CD33（+）髓样DC子集以及单核细胞中的每一个的重要比例都会在每个实验条件下产生大量的所有细胞因子。相反，CC33（-/ + lo）淋巴浆细胞样DC在相同的刺激条件下无法产生任何可检测水平的上述细胞因子。通过比较所使用的不同刺激，与SAG相比，LPS与产生细胞因子的细胞百分比更高有关，尤其是在CD33（hi）DC子集中;有趣的是，添加IFN-γ增强了单核细胞对LPS和SAG的反应。关于分泌阻滞剂，单独的布雷菲德菌素A优于布雷菲德菌素A和莫能菌素的组合，这是因为它经常与更高百分比的细胞因子阳性细胞和每细胞大量可检测的细胞因子有关。 PB DC和单核细胞在培养6、12和24 h后对细胞因子产生的顺序分析表明，在6 h之后，DC中存在较高的细胞死亡率，在24 h时甚至无法检测到，而没有明显的死亡。细胞因子分泌增加。总而言之，我们的结果表明，根据本文测试的实验条件，通过正常人DC和单核细胞诱导细胞因子的产生，在PB样品中用LPS（有或无IFN-γ）刺激PB样品6小时后，可获得最大的响应。仅存在布雷菲德菌素A。细胞计数法（Comm，临床细胞计数法）46：33-40，2001，（C）2001 Wiley-Liss，Inc. [参考：38]

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