IL8 and PMA Trigger the Regulation of Different Biological Processes in Granulocyte Activation
IL8 and PMA Trigger the Regulation of Different Biological Processes in Granulocyte Activation
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DOI：
10.3389/fimmu.2019.03064
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发表时间：
2020-01-28
影响因子：
7.3
通讯作者：
Deeg, Cornelia A.
中科院分区：
文献类型：
--
作者：
Degroote, Roxane L.;Weigand, Maria;Deeg, Cornelia A.
到目前为止，驱动嗜中性粒细胞特异性调节的分子机制还不完全清楚。为了表征蛋白质水平上的基本粒细胞特征，我们分析了蛋白质组组成的变化作为对细胞活化过程中的应激的反应。为了这个目的，我们通过密度梯度离心分离马全血中的初级粒细胞，然后用氯化钠裂解并用白细胞介素-8（IL-8）刺激细胞30分钟，因为IL-8是中性粒细胞的趋化因子。我们另外使用佛波醇12-肉豆蔻酸酯13-乙酸酯（PMA）和脂多糖（LPS），其主要与中性粒细胞胞外陷阱形成和活性氧释放相关。从质谱分析中，我们鉴定了总共2，032种描述整个粒细胞蛋白质组的蛋白质，包括245种蛋白质（占鉴定的蛋白质组的12%），这些蛋白质新与原代马粒细胞中的体内表达相关（假设蛋白质）。我们还发现，在用各种刺激物对细胞进行短刺激后，蛋白质丰度（比率>= 2）发生了明显和不同的变化，这表明反应模式是快速和分化的。IL 8刺激导致58种蛋白质（蛋白质组的3%）的蛋白质丰度增加，而PMA诱导207种蛋白质（蛋白质组的10%）和46种蛋白质（蛋白质组的2%）的LPS改变的蛋白质丰度。富集分析清楚地显示了刺激之间的根本差异，其中IL 8刺激与免疫应答、受体信号传导和信号转导过程的主要关联。另一方面，PMA的顶部富集指向囊泡介导的运输和胞吐作用。用LPS刺激没有导致任何显著的富集。尽管我们检测到IL 8和PMA刺激的富集类别的43%重叠，表明用不同刺激激活中性粒细胞部分诱导一些相似的生物过程和途径，但分层聚类显示所选刺激之间簇的分布和生物相关性存在明显差异。我们的研究提供了新的信息，粒细胞蛋白质组，并提供洞察早期，分化粒细胞反应的刺激，这有助于更好地了解参与激活和招聘的中性粒细胞的分子机制，通过炎症刺激。
The molecular mechanisms driving specific regulation of neutrophils are not completely understood to date. In order to characterize fundamental granulocyte features on protein level, we analyzed changes in proteome composition as reaction to stress from cell activation processes. For this purpose, we isolated primary granulocytes from equine whole blood through density gradient centrifugation followed by sodium chloride lysis and stimulated cells for 30 min with interleukin-8 (IL8) due to its role as a chemotactic factor for neutrophils. We additionally used phorbol 12-myristate 13-acetate (PMA) and lipopolysaccharide (LPS), which are primarily associated to neutrophil extracellular trap formation and release of reactive oxygen species. From mass spectrometry analysis, we identified a total of 2,032 proteins describing the whole granulocyte proteome, including 245 proteins (12% of identified proteome) newly associated to in vivo expression in primary equine granulocytes (hypothetical proteins). We also found distinct and different changes in protein abundance (ratio >= 2) after short stimulation of cells with various stimuli, pointing to rapid and differentiated reaction pattern. IL8 stimulation resulted in increased protein abundance of 58 proteins (3% of proteome), whereas PMA induced changed protein abundance of 207 (10 % of proteome) and LPS of 46 proteins (2% of proteome). Enrichment analyses clearly showed fundamental differences between stimuli, with primary association of IL8 stimulation to processes in immune response, receptor signaling and signal transduction. Top enrichment for PMA on the other hand pointed to vesicle mediated transport and exocytosis. Stimulation with LPS did not result in any significant enrichment. Although we detected 43% overlap of enrichment categories for IL8 and PMA stimulation, indicating that activation of neutrophils with different stimuli partly induces some similar biological processes and pathways, hierarchical clustering showed clear differences in distribution and biological relevance of clusters between the chosen stimuli. Our studies provide novel information on the granulocyte proteome and offer insights into early, differentiated granulocyte reaction to stimuli, which contribute to a better understanding of molecular mechanisms involved in activation and recruitment of neutrophils, through inflammatory stimuli.