Department of Biochemistry & Molecular Biology, University of Texas Medical Branch , Galveston, Texas 77555, United States.
Sealy Center for Molecular Medicine, University of Texas Medical Branch , Galveston, Texas 77555, United States.
J Proteome Res. 2017 Aug 4;16(8):2663-2679. doi: 10.1021/acs.jproteome.6b00367. Epub 2017 Jul 21.
Activated eosinophils contribute to airway dysfunction and tissue remodeling in asthma and thus are considered to be important factors in asthma pathology. We report here comparative proteomic and phosphoproteomic changes upon activation of eosinophils using eight cytokines individually and in selected cytokine combinations in time-course reactions. Differential protein and phosphoprotein expressions were determined by mass spectrometry after 2-dimensional gel electrophoresis (2DGE) and by LC-MS/MS. We found that each cytokine-stimulation produced significantly different changes in the eosinophil proteome and phosphoproteome, with phosphoproteomic changes being more pronounced and having an earlier onset. Furthermore, we observed that IL-5, GM-CSF, and IL-3 showed the greatest change in protein expression and phosphorylation, and this expression differed markedly from those of the other five cytokines evaluated. Comprehensive univariate and multivariate statistical analyses were employed to evaluate the comparative results. We also monitored eosinophil activation using flow cytometry (FC) analysis of CD69. In agreement with our proteomic studies, FC indicated that IL-5, GM-CSF, and IL-3 were more effective than the other five cytokines studied in stimulating a cell surface CD69 increase indicative of eosinophil activation. Moreover, selected combinations of cytokines revealed proteomic patterns with many proteins in common with single cytokine expression patterns but also showed a greater effect of the two cytokines employed, indicating a more complex signaling pathway that was reflective of a more typical inflammatory pathology.
活化的嗜酸性粒细胞有助于哮喘中的气道功能障碍和组织重塑，因此被认为是哮喘病理学中的重要因素。我们在此报告了使用八种细胞因子单独和在选定的细胞因子组合中在时间反应中激活嗜酸性粒细胞时的比较蛋白质组学和磷酸蛋白质组学变化。通过二维凝胶电泳（2DGE）和 LC-MS/MS 确定了差异蛋白和磷酸蛋白表达。我们发现，每种细胞因子刺激都会导致嗜酸性粒细胞蛋白质组和磷酸蛋白质组发生明显不同的变化，磷酸蛋白质组的变化更为明显，且更早发生。此外，我们观察到 IL-5、GM-CSF 和 IL-3 在蛋白质表达和磷酸化方面表现出最大的变化，并且与评估的其他五种细胞因子的表达明显不同。综合使用单变量和多变量统计分析来评估比较结果。我们还使用 CD69 的流式细胞术（FC）分析监测嗜酸性粒细胞的激活。与我们的蛋白质组学研究一致，FC 表明 IL-5、GM-CSF 和 IL-3 比研究的其他五种细胞因子更有效地刺激细胞表面 CD69 增加，表明嗜酸性粒细胞激活。此外，选定的细胞因子组合显示出与单个细胞因子表达模式具有许多共同蛋白质的蛋白质组学模式，但也显示出两种细胞因子的作用更大，表明更复杂的信号通路反映了更典型的炎症病理学。
