Ebo D G, Dombrecht E J, Bridts C H, Aerts N E, de Clerck L S, Stevens W J
Department of Immunology, Allergology, Rheumatology, Faculty of Medicine, University of Antwerp, Antwerp, Belgium.
Clin Exp Allergy. 2007 Nov;37(11):1668-75. doi: 10.1111/j.1365-2222.2007.02819.x. Epub 2007 Sep 14.
The signal transduction pathways and control mechanisms involved in IgE-mediated basophil activation remain incompletely understood.
To investigate whether basophilic intracellular signal transduction and immunophenotype can be analysed simultaneously by flow cytometry.
Basophils in whole blood were stimulated with anti-IgE and latex antigen at various concentrations and during different time courses. Phosphorylation of p38 mitogen-activated protein kinase (MAPK) as a representative of the intracellular signal transduction pathway and surface expression of CD63 was assessed simultaneously flow cytometrically. The effect of pre-incubation with IL-3 was assessed.
Stimulation of the basophils with anti-IgE and allergen induces a rapid phosphorylation of p38 MAPK that peaks between 1 and 5 min and returns to baseline levels after 60 min. In contrast, CD63 up-regulation demonstrates a maximal but more continuous expression that peaks approximately 5 min later than phosphorylation of p38 MAPK. Specific inhibition of p38 MAPK reduced or almost completely abrogated up-regulation of CD63. Pre-incubation of the basophils with IL-3 produces a rapid p38 MAPK phosphorylation over basal levels, but this was weaker and shorter than for anti-IgE stimulation. Pre-incubation of the basophils with IL-3 did not potentiate anti-IgE-induced phosphorylation of p38 MAPK and did affect spontaneous or IgE-mediated CD63 up-regulation.
This study provides the proof that the flow cytometer allows an integrated analysis of basophilic intracellular signalling and immunophenotyping. Owing to its technical simplicity, the low number of cells required and rapid analysis, the technique seems promising for use in the clinic as a diagnostic tool or to monitor therapy.
This study is the first to provide evidence for a combined analysis of basophilic intracellular signalling and immunophenotyping by flow cytometry. Owing to its technical simplicity, the low number of cells required and rapid analysis, the technique seems promising for use in the clinic as a diagnostic tool or to monitor therapy.
IgE介导的嗜碱性粒细胞激活所涉及的信号转导途径和调控机制仍未完全明确。
探讨是否可通过流式细胞术同时分析嗜碱性粒细胞的细胞内信号转导和免疫表型。
用不同浓度的抗IgE和乳胶抗原在不同时间进程刺激全血中的嗜碱性粒细胞。同时通过流式细胞术评估作为细胞内信号转导途径代表的p38丝裂原活化蛋白激酶（MAPK）的磷酸化以及CD63的表面表达。评估白细胞介素-3预孵育的作用。
用抗IgE和变应原刺激嗜碱性粒细胞可诱导p38 MAPK快速磷酸化，在1至5分钟达到峰值，并在60分钟后恢复至基线水平。相比之下，CD63上调表现为最大但更持续的表达，其峰值比p38 MAPK磷酸化晚约5分钟。p38 MAPK的特异性抑制减少或几乎完全消除了CD63的上调。用白细胞介素-3预孵育嗜碱性粒细胞会导致p38 MAPK磷酸化迅速超过基础水平，但比抗IgE刺激更弱且持续时间更短。用白细胞介素-3预孵育嗜碱性粒细胞不会增强抗IgE诱导的p38 MAPK磷酸化，也不影响自发或IgE介导的CD63上调。
本研究证明流式细胞仪可对嗜碱性粒细胞的细胞内信号传导和免疫表型进行综合分析。由于其技术简单、所需细胞数量少且分析快速，该技术有望作为诊断工具或监测治疗手段应用于临床。
本研究首次提供了通过流式细胞术对嗜碱性粒细胞细胞内信号传导和免疫表型进行联合分析的证据。由于其技术简单、所需细胞数量少且分析快速，该技术有望作为诊断工具或监测治疗手段应用于临床。
