Rapid flow cytometric measurement of cytokine-induced phosphorylation pathways [CIPP] in human peripheral blood leukocytes
Rapid flow cytometric measurement of cytokine-induced phosphorylation pathways [CIPP] in human peripheral blood leukocytes
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DOI：
10.1016/j.clim.2006.06.013
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发表时间：
2006-11-01
影响因子：
8.6
通讯作者：
Lotze, Michael T.
中科院分区：
文献类型：
--
作者：
Montag, David T.;Lotze, Michael T.
目前设计用于评估外周血细胞的策略仅限于评估表型或延迟测量[> 6 h]功能，通常定量细胞因子产生、细胞溶解活性或对抗原的应答。我们推断，信号通路中可测量的异常可以反映细胞在炎症状态/癌症背景下经历的病理环境，并且可以在外周血中表现出来。调节免疫应答的两个主要途径是JAK/STAT和MAPK/ERK途径。这些途径由配体-受体结合启动，并通过随后的蛋白质磷酸化级联反应快速传播。我们评估了细胞因子在体外的简单应用，以询问这些信号通路在正常外周血单核细胞（PBMC）的早期磷酸化事件。评估个体细胞因子剂量和治疗时间间隔，以确定在临床实验室中有用的条件，并作为诱导最大磷酸化的初始目标。令人惊讶的是，所有评估的STAT蛋白质和ERK 1/2在人PBMC中仅在加入细胞因子后15分钟内最大限度地磷酸化，而不预活化细胞。在2小时时，细胞通常恢复到其基础磷酸化状态。对于大多数测试的细胞因子，增加的磷酸化与增加的单个细胞因子浓度直接相关。这些策略将使简单血液分析的稳健发展成为可能，以确定STAT和MAPK/ERK信号通路中的正常水平以及与各种人类疾病状态相关的损伤，包括整个临床免疫学中的急性和慢性炎症状况。(c)2006年爱思唯尔公司All rights reserved.
Current strategies designed to assess cells in the peripheral blood are limited to evaluation of phenotype or delayed measurement [> 6 h] of function, usually quantifying cytokine production, cytolytic activity, or response to antigens. We reasoned that measurable abnormalities in signaling pathways could reflect pathological environs that cells experience in the setting of inflammatory states/cancer and could be represented in the peripheral blood. Two major pathways regulating the immune response are the JAK/STAT and MAPK/ERK pathways. These pathways are initiated by ligand-receptor binding and are rapidly propagated by subsequent protein phosphorylation cascades. We evaluated the brief application of cytokines in vitro to interrogate the early phosphorylation events of these signaling pathways in normal peripheral blood mononuclear cells (PBMC). Individual cytokine doses and time intervals of treatment were assessed to identify conditions useful in a clinical laboratory and as an initial goal to induce maximal phosphorylation. Surprisingly, all of the STAT proteins assessed and ERK1/2 are maximally phosphorylated within 15 min in human PBMC simply following addition of cytokines without preactivation of the cells. At 2 h, cells typically return to their basal phosphorylation states. For most of the cytokines tested, increased phosphorylation directly correlated with increased concentrations of the individual cytokines. These strategies will enable robust development of simple blood analyses to identify normal Levels as well as impairments in STAT and MAPK/ERK signaling pathways associated with various human disease states including acute and chronic inflammatory conditions throughout clinical immunology. (c) 2006 Elsevier Inc. All rights reserved.