Multiparameter phospho-flow analysis of lymphocytes in early rheumatoid arthritis: implications for diagnosis and monitoring drug therapy.
Multiparameter phospho-flow analysis of lymphocytes in early rheumatoid arthritis: implications for diagnosis and monitoring drug therapy.
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DOI：
10.1371/journal.pone.0006703
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发表时间：
2009-08-20
期刊：
影响因子：
3.7
通讯作者：
Fish EN
中科院分区：
文献类型：
--
作者：
Galligan CL;Siebert JC;Siminovitch KA;Keystone EC;Bykerk V;Perez OD;Fish EN
类风湿关节炎（RA）发生和进展的确切机制尚不清楚。 RA 的早期阶段通常有非特异性症状，从而延误诊断和治疗。此外，目前还没有确定的方法来预测临床对治疗的反应。免疫细胞激活是一个关键组成部分，因此我们检查了 RA 早期阶段外周血单核细胞 (PBMC) 的细胞激活情况，以开发一种新的诊断方式。从诊断为早期 RA (ERA) (n = 38)、长期 RA (n = 10)、骨关节炎 (OA) (n = 19) 的个体和健康个体 (n = 10) 中分离出 PBMC。通过流式细胞术（磷酸化流），使用磷酸化状态作为免疫表型细胞激活的量度，检查 PBMC 的 15 个信号传导效应器的激活。从 ERA、RA 和 OA 患者中分离的 CD3+CD4+、CD3+CD8+ 和 CD20+ 细胞表现出多个磷酸表位的激活。与 OA PBMC 相比，ERA 患者 PBMC 在 CD4+ 和 CD20+ 区室中表现出磷酸化激活的倾向，其中磷酸化激活主要在 CD8+ 细胞中观察到。 ERA 患者中磷酸 (p)-AKT/p-p38 的比率显着升高，可能具有诊断潜力。 CD4+、CD8+ 和 CD20+ T 细胞中 p-AKT 和 p-H3 的平均荧光强度 (MFI) 水平与医师总体评估评分 (MDGA) 和 DAS（疾病活动评分）直接相关。按药物分层显示，接受来氟米特、全身类固醇或抗 TNF 治疗的患者与未接受这些治疗的患者相比，磷酸特异性激活显着减少。观察到药物相关的特定信号效应物磷酸化水平的降低与疾病活动性降低之间的相关趋势。磷酸流分析鉴定了 ERA 患者 PB 中特定信号传导效应器的磷酸化激活。值得注意的是，这些信号传导效应子的磷酸化并不能区分 ERA 和晚期 RA，这表明离散细胞群的激活状态在疾病早期就已经确立。然而，当 p-AKT 和 p-p38 的 MFI 值之比 >1.5 时，诊断 RA 的可能性很高。我们的结果表明，对接受治疗的患者进行纵向采样可能会产生可预测药物反应的磷酸化特征。
The precise mechanisms involved in the initiation and progression of rheumatoid arthritis (RA) are not known. Early stages of RA often have non-specific symptoms, delaying diagnosis and therapy. Additionally, there are currently no established means to predict clinical responsiveness to therapy. Immune cell activation is a critical component therefore we examined the cellular activation of peripheral blood mononuclear cells (PBMCs) in the early stages of RA, in order to develop a novel diagnostic modality. PBMCs were isolated from individuals diagnosed with early RA (ERA) (n = 38), longstanding RA (n = 10), osteoarthritis (OA) (n = 19) and from healthy individuals (n = 10). PBMCs were examined for activation of 15 signaling effectors, using phosphorylation status as a measure of activation in immunophenotyped cells, by flow cytometry (phospho-flow). CD3+CD4+, CD3+CD8+ and CD20+ cells isolated from patients with ERA, RA and OA exhibited activation of multiple phospho-epitopes. ERA patient PBMCs showed a bias towards phosphorylation-activation in the CD4+ and CD20+ compartments compared to OA PBMCs, where phospho-activation was primarily observed in CD8+ cells. The ratio of phospho (p)-AKT/p-p38 was significantly elevated in patients with ERA and may have diagnostic potential. The mean fluorescent intensity (MFI) levels for p-AKT and p-H3 in CD4+, CD8+ and CD20+ T cells correlated directly with physician global assessment scores (MDGA) and DAS (disease activity score). Stratification by medications revealed that patients receiving leflunomide, systemic steroids or anti-TNF therapy had significant reductions in phospho-specific activation compared with patients not receiving these therapies. Correlative trends between medication-associated reductions in the levels of phosphorylation of specific signaling effectors and lower disease activity were observed. Phospho-flow analysis identified phosphorylation-activation of specific signaling effectors in the PB from patients with ERA. Notably, phosphorylation of these signaling effectors did not distinguish ERA from late RA, suggesting that the activation status of discrete cell populations is already established early in disease. However, when the ratio of MFI values for p-AKT and p-p38 is >1.5, there is a high likelihood of having a diagnosis of RA. Our results suggest that longitudinal sampling of patients undergoing therapy may result in phospho-signatures that are predictive of drug responsiveness.
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