Bueno C, Almeida J, Alguero M C, Sánchez M L, Vaquero J M, Laso F J, San Miguel J F, Escribano L, Orfao A
Servicio General de Citometría, Departamento de Medicina, Universidad de Salamanca, Salamanca, Spain.
Cytometry. 2001 Feb 15;46(1):33-40.
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 I (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, CD33(-/+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 SAC, especially within the CD33(hi) DC subset; interestingly, the addition of IFN-gamma enhanced the response of monocytes to both LPS and SAC. 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.
在本文中，我们比较分析了以下不同刺激因素对正常人外周血（PB）树突状细胞（DC）亚群和单核细胞中白细胞介素（IL）-1β、IL-6、IL-12、肿瘤坏死因子-α（TNF-α）和IL-8等炎性细胞因子产生及细胞内蓄积的影响：脂多糖（LPS）与考恩I型金黄色葡萄球菌（SAC），存在或不存在干扰素-γ（IFN-γ）、细胞因子分泌阻断剂（单独使用布雷菲德菌素A与布雷菲德菌素A加莫能菌素）以及孵育时间（6、12和24小时）。为此，在所有实验（n = 19）中系统地使用了通过流式细胞术分析的四色多重染色直接免疫荧光技术。我们的结果表明，刺激后，两个CD33(+)髓样DC亚群以及单核细胞中的重要比例在每种测定的实验条件下都产生了大量分析的细胞因子。相比之下，在相同刺激条件下，CD33(-/+lo)淋巴浆细胞样DC未能产生可检测水平的上述任何细胞因子。在比较所使用的不同刺激因素时，与SAC相比，LPS与产生细胞因子的细胞百分比更高相关，特别是在CD33(hi) DC亚群中；有趣的是，添加IFN-γ增强了单核细胞对LPS和SAC的反应。关于分泌阻断剂，单独使用布雷菲德菌素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以及每个细胞中更多可检测到的细胞因子相关。对细胞培养6、12和24小时后PB DC和单核细胞产生细胞因子的顺序分析表明，6小时后，DC中的细胞死亡率增加，在24小时时甚至变得无法检测到，而细胞因子分泌没有显著增加。总之，我们的结果表明，从本文测定的实验条件来看，为了诱导正常人DC和单核细胞产生细胞因子，在单独存在布雷菲德菌素A的情况下，用LPS（有或没有IFN-γ）刺激PB样本6小时可获得最大反应。
