Yang Barbara, Pham Tuyet-Hang, Goldbach-Mansky Raphaela, Gadina Massimo
Translational Immunology Section, Office of Science and Technology, National Institute of Arthritis and Musculoskeletal and Skin Diseases.
J Vis Exp. 2011 Mar 1(49):2662. doi: 10.3791/2662.
Inflammatory processes resulting from the secretion of soluble mediators by immune cells, lead to various manifestations in skin, joints and other tissues as well as altered cytokine homeostasis. The innate immune system plays a crucial role in recognizing pathogens and other endogenous danger stimuli. One of the major cytokines released by innate immune cells is Interleukin (IL)-1. Therefore, we utilize a whole blood stimulation assay in order to measure the secretion of inflammatory cytokines and specifically of the pro-inflammatory cytokine IL-1β(1, 2, 3). Patients with genetic dysfunctions of the innate immune system causing autoinflammatory syndromes show an exaggerated release of mature IL-1β upon stimulation with LPS alone. In order to evaluate the innate immune component of patients who present with inflammatory-associated pathologies, we use a specific immunoassay to detect cellular immune responses to pathogen-associated molecular patterns (PAMPs), such as the gram-negative bacterial endotoxin, lipopolysaccharide (LPS). These PAMPs are recognized by pathogen recognition receptors (PRRs), which are found on the cells of the innate immune system (4, 5, 6, 7). A primary signal, LPS, in conjunction with a secondary signal, ATP, is necessary for the activation of the inflammasome, a multiprotein complex that processes pro-IL-1β to its mature, bioactive form (4, 5, 6, 8, 9, 10). The whole blood assay requires minimal sample manipulation to assess cytokine production when compared to other methods that require labor intensive isolation and culturing of specific cell populations. This method differs from other whole blood stimulation assays; rather than diluting samples with a ratio of RPMI media, we perform a white blood cell count directly from diluted whole blood and therefore, stimulate a known number of white blood cells in culture (2). The results of this particular whole blood assay demonstrate a novel technique useful in elucidating patient cohorts presenting with autoinflammatory pathophysiologies.
免疫细胞分泌可溶性介质所引发的炎症过程，会导致皮肤、关节和其他组织出现各种症状，同时也会改变细胞因子的稳态。先天免疫系统在识别病原体和其他内源性危险刺激方面发挥着关键作用。先天免疫细胞释放的主要细胞因子之一是白细胞介素（IL）-1。因此，我们采用全血刺激试验来测量炎性细胞因子的分泌，特别是促炎性细胞因子IL-1β（1, 2, 3）。患有导致自身炎症综合征的先天免疫系统基因功能障碍的患者，在仅用脂多糖（LPS）刺激时会过度释放成熟的IL-1β。为了评估患有炎症相关病症患者的先天免疫成分，我们使用特定的免疫测定法来检测细胞对病原体相关分子模式（PAMP）的免疫反应，例如革兰氏阴性菌内毒素脂多糖（LPS）。这些PAMP由先天免疫系统细胞上的病原体识别受体（PRR）识别（4, 5, 6, 7）。初级信号LPS与次级信号ATP共同作用，对于炎性小体的激活是必要的，炎性小体是一种多蛋白复合物，可将前体IL-1β加工成其成熟的、具有生物活性的形式（4, 5, 6, 8, 9, 10）。与其他需要对特定细胞群体进行劳动密集型分离和培养的方法相比，全血试验在评估细胞因子产生时所需的样本处理极少。该方法与其他全血刺激试验不同；我们不是用RPMI培养基按比例稀释样本，而是直接从稀释的全血中进行白细胞计数，从而在培养中刺激已知数量的白细胞（2）。这种特定全血试验的结果证明了一种新技术，该技术可用于阐明患有自身炎症病理生理学的患者群体。
