Chair of Physiology, Department of Veterinary Sciences, LMU Munich, Munich, Germany.
Research Unit Protein Science, Helmholtz Center Munich, German Research Center for Environmental Health GmbH, Munich, Germany.
Front Immunol. 2020 Jan 14;10:3064. doi: 10.3389/fimmu.2019.03064. eCollection 2019.
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 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.
迄今为止，驱动中性粒细胞特异性调节的分子机制尚不完全清楚。为了在蛋白质水平上描述基本的粒细胞特征，我们分析了细胞激活过程应激反应导致的蛋白质组组成变化。为此，我们通过密度梯度离心从马全血中分离出原代粒细胞，然后用氯化钠裂解，并用白细胞介素-8（IL8）刺激细胞 30 分钟，因为它作为一种趋化因子对中性粒细胞具有作用。我们还使用佛波醇 12-肉豆蔻酸 13-乙酸酯（PMA）和脂多糖（LPS），它们主要与中性粒细胞胞外陷阱的形成和活性氧的释放有关。通过质谱分析，我们共鉴定出 2032 种蛋白质，描述了整个粒细胞蛋白质组，其中包括 245 种（鉴定蛋白质组的 12%）与马原代粒细胞中 表达新相关的蛋白质（假设蛋白）。我们还发现，用不同的刺激物短暂刺激细胞后，蛋白质丰度（比值≥2）有明显而不同的变化，表明存在快速而分化的反应模式。IL8 刺激导致 58 种蛋白质的丰度增加（蛋白质组的 3%），而 PMA 诱导 207 种（蛋白质组的 10%）和 LPS 诱导 46 种（蛋白质组的 2%）蛋白质丰度变化。富集分析清楚地显示了刺激物之间的基本差异，IL8 刺激的主要关联是免疫反应、受体信号和信号转导过程。另一方面，PMA 的顶级富集指向囊泡介导的运输和胞吐作用。LPS 刺激没有导致任何显著的富集。尽管我们检测到 IL8 和 PMA 刺激的富集类别有 43%的重叠，表明用不同的刺激物激活中性粒细胞部分诱导了一些相似的生物学过程和途径，但层次聚类显示了所选刺激物之间聚类的分布和生物学相关性的明显差异。我们的研究提供了粒细胞蛋白质组的新信息，并深入了解了粒细胞对刺激的早期、分化反应，有助于更好地理解炎症刺激下中性粒细胞的激活和募集的分子机制。
