Laboratory of Cellular and Molecular Cardiology, University of Antwerp, Wilrijk, Antwerp, Belgium.
PLoS One. 2012;7(8):e43357. doi: 10.1371/journal.pone.0043357. Epub 2012 Aug 15.
Dendritic cells (DCs), professional antigen-presenting cells with the unique ability to initiate primary T-cell responses, are present in atherosclerotic lesions where they are exposed to oxidative stress that generates cytotoxic reactive oxygen species (ROS). A large body of evidence indicates that cell death is a major modulating factor of atherogenesis. We examined antioxidant defence systems of human monocyte-derived (mo)DCs and monocytes in response to oxidative stress.
Oxidative stress was induced by addition of tertiary-butylhydroperoxide (tert-BHP, 30 min). Cellular responses were evaluated using flow cytometry and confocal live cell imaging (both using 5-(and-6)-chloromethyl-2,7-dichlorodihydrofluorescein diacetate, CM-H(2)DCFDA). Viability was assessed by the neutral red assay. Total RNA was extracted for a PCR profiler array. Five genes were selected for confirmation by Taqman gene expression assays, and by immunoblotting or immunohistochemistry for protein levels.
Tert-BHP increased CM-H(2)DCFDA fluorescence and caused cell death. Interestingly, all processes occurred more slowly in moDCs than in monocytes. The mRNA profiler array showed more than 2-fold differential expression of 32 oxidative stress-related genes in unstimulated moDCs, including peroxiredoxin-2 (PRDX2), an enzyme reducing hydrogen peroxide and lipid peroxides. PRDX2 upregulation was confirmed by Taqman assays, immunoblotting and immunohistochemistry. Silencing PRDX2 in moDCs by means of siRNA significantly increased CM-DCF fluorescence and cell death upon tert-BHP-stimulation.
Our results indicate that moDCs exhibit higher intracellular antioxidant capacities, making them better equipped to resist oxidative stress than monocytes. Upregulation of PRDX2 is involved in the neutralization of ROS in moDCs. Taken together, this points to better survival skills of DCs in oxidative stress environments, such as atherosclerotic plaques.
树突状细胞（DCs）是一种具有独特能力的专业抗原呈递细胞，能够启动初始 T 细胞反应，存在于动脉粥样硬化病变中，在那里它们会暴露于产生细胞毒性活性氧（ROS）的氧化应激下。大量证据表明，细胞死亡是动脉粥样硬化形成的主要调节因素。我们研究了人类单核细胞来源的（mo）DCs 和单核细胞在氧化应激下的抗氧化防御系统。
通过添加叔丁基过氧化氢（tert-BHP，30 分钟）诱导氧化应激。使用流式细胞术和共聚焦活细胞成像（均使用 5-（和-6）-氯甲基-2,7-二氯二氢荧光素二乙酸酯，CM-H（2）DCFDA）评估细胞反应。通过中性红测定法评估细胞活力。提取总 RNA 进行 PCR 分析。通过 Taqman 基因表达测定、免疫印迹或免疫组化法选择 5 个基因来验证蛋白水平。
tert-BHP 增加了 CM-H（2）DCFDA 荧光并导致细胞死亡。有趣的是，moDCs 中的所有过程都比单核细胞慢。mRNA 分析显示，未刺激的 moDCs 中有 32 个与氧化应激相关的基因的表达差异超过 2 倍，包括过氧化物还原酶-2（PRDX2），这是一种还原过氧化氢和脂质过氧化物的酶。通过 Taqman 测定、免疫印迹和免疫组化证实了 PRDX2 的上调。用 siRNA 沉默 moDCs 中的 PRDX2 显著增加了 tert-BHP 刺激后的 CM-DCF 荧光和细胞死亡。
我们的结果表明，moDCs 表现出更高的细胞内抗氧化能力，使它们比单核细胞更能抵抗氧化应激。PRDX2 的上调参与了 moDCs 中 ROS 的中和。综上所述，这表明 DCs 在氧化应激环境（如动脉粥样硬化斑块）中具有更好的生存能力。
