Tedesco Serena, Bolego Chiara, Toniolo Alice, Nassi Alberto, Fadini Gian Paolo, Locati Massimo, Cignarella Andrea
Department of Pharmaceutical and Pharmacological Sciences, University of Padua, Italy.
Department of Pharmaceutical and Pharmacological Sciences, University of Padua, Italy.
Immunobiology. 2015 May;220(5):545-54. doi: 10.1016/j.imbio.2014.12.008. Epub 2014 Dec 23.
Macrophage activation has been observed in vivo under physiological and pathological conditions, and may represent an attractive target for pharmacological modulation. This study tested the hypothesis that human blood-derived macrophages generated in vitro in the absence of specific macrophage growth factors respond flexibly to activation stimuli and pharmacological treatment. Monocytes were differentiated to macrophages for 7 days in culture in RPMI 1640 with 10% FCS. The resulting population showed predominance of the M2 over M1 phenotype as measured by flow cytometry and the expression of M1 vs. M2 markers was not mutually exclusive. Activation with LPS/IFN-γ for 48 h significantly increased the fraction of surface CD68-expressing cells, the CD14(+)/CD16(-)/CD68(+) subset and cell-bound TNF-α levels, whereas expression of the CC chemokine receptor (CCR)-2 was unchanged. Expression of the M2 markers CD206, CD163 and CX3CR1 was down-regulated following M1 activation compared with resting and after pre-exposure to M2-triggers. By contrast, alternative activation with IL-4/IL-13 for 48 h did not increase M2 markers, while CD206 up-regulation was observed after 7 days. Both activation signals induced changes in gene expression profiles as shown by Q-PCR. Treatment with 100 nM dexamethasone enhanced the M2 morphotype and CD163 expression while preventing LPS/IFN-γ-induced CD163 down-regulation. After 1-week dexamethasone treatment, virtually all cells acquired a CD163(+)/CD206(+)/CX3CR1(+) M2 phenotype. Therefore, these protocols appear to be useful to perform screens of pharmacological agents targeting human macrophage activation.
巨噬细胞活化在生理和病理条件下的体内均有观察到，可能是药物调节的一个有吸引力的靶点。本研究检验了以下假设：在没有特定巨噬细胞生长因子的情况下体外产生的人血源性巨噬细胞对活化刺激和药物治疗具有灵活的反应。单核细胞在含有10%胎牛血清的RPMI 1640培养基中培养7天，分化为巨噬细胞。通过流式细胞术检测，所得细胞群体显示M2表型占主导地位，且M1与M2标志物的表达并非相互排斥。用LPS/IFN-γ激活48小时显著增加了表面表达CD68的细胞比例、CD14(+)/CD16(-)/CD68(+)亚群以及细胞结合的TNF-α水平，而CC趋化因子受体(CCR)-2的表达未改变。与静息状态及预先暴露于M2触发因子后相比，M1激活后M2标志物CD206、CD163和CX3CR1的表达下调。相比之下，用IL-4/IL-13进行替代激活48小时并未增加M2标志物，但在7天后观察到CD206上调。如Q-PCR所示，两种激活信号均诱导了基因表达谱的变化。用100 nM地塞米松处理可增强M2形态型和CD163表达，同时防止LPS/IFN-γ诱导的CD163下调。地塞米松处理1周后，几乎所有细胞都获得了CD163(+)/CD206(+)/CX3CR1(+) M2表型。因此，这些方案似乎有助于进行针对人巨噬细胞活化的药物筛选。
