Ferry B, Antrobus P, Huzicka I, Farrell A, Lane A, Chapel H
Department of Immunology, The Churchill Hospital, Oxford Radcliffe Hospital, UK.
Clin Exp Immunol. 1997 Dec;110(3):410-7. doi: 10.1046/j.1365-2249.1997.4361452.x.
In recent years, the importance of characterizing the role of cytokines in a wide range of clinical conditions has resulted in development of new methods to assess cytokine expression in clinical samples. The use of anti-cytokine MoAbs and flow cytometry to detect cytokines intracellularly at the single-cell level has the potential to quantify cytokine production in different diseases. For this technique to be useful in a clinical setting, rapid throughput of clinical samples and a cheap, reliable assay would be required, therefore the development of the above technique using unseparated whole blood samples would be advantageous. Using this technique, only one study to date (Maino et al., 1996) has used unseparated whole blood as the source of cells for detecting intracellular cytokines. In clinical practice, whole blood may be optimal, since this most closely approximates conditions in vivo: as no purification of blood mononuclear cells is required, very little blood is needed to detect a number of cytokines simultaneously in various lymphocyte subpopulations, and the assay can be applied to samples from infants and children. In this study we describe an intracellular cytokine assay using unseparated whole blood from normals. In activated CD8- T cells, IL-2 and interferon-gamma (IFN-gamma) were optimally induced after 10 h stimulation with phorbol 12-myristate acetate (PMA)/ionomycin, and in CD8+ T cells IL-2 was optimally induced after 10 h and IFN-gamma after 6 h. The levels of IL-2 and IFN-gamma in CD8+ and CD8- T cells in four healthy individuals were consistent on four occasions over a 3-month period. In a large group of 34 normal subjects, there was considerable heterogeneity in CD3/IL-2+ (range 9.7-41.3) and CD3/IFN-gamma+ cells (10.1-44), expressed as a percentage of total lymphocytes. In patients with atopic dermatitis (n = 5) there was a significantly decreased percentage of CD3+/CD8+ peripheral blood T cells expressing IFN-gamma and an increased percentage of CD3+/CD8- T cells expressing IL-4 compared with non-atopic dermatitis controls (n = 5). Possible applications of this technique are discussed.
近年来，明确细胞因子在多种临床病症中的作用至关重要，这促使了评估临床样本中细胞因子表达的新方法的发展。使用抗细胞因子单克隆抗体和流式细胞术在单细胞水平检测细胞内细胞因子，有可能对不同疾病中的细胞因子产生进行定量。为使该技术在临床环境中发挥作用，需要临床样本的快速通量以及廉价、可靠的检测方法，因此开发使用未分离全血样本的上述技术将具有优势。迄今为止，仅有一项研究（Maino等人，1996年）使用未分离的全血作为检测细胞内细胞因子的细胞来源。在临床实践中，全血可能是最佳选择，因为这最接近体内情况：无需纯化血液单核细胞，只需极少的血液就能同时检测多个淋巴细胞亚群中的多种细胞因子，并且该检测方法可应用于婴儿和儿童的样本。在本研究中，我们描述了一种使用正常人未分离全血的细胞内细胞因子检测方法。在活化的CD8 - T细胞中，用佛波酯12 - 肉豆蔻酸乙酯（PMA）/离子霉素刺激10小时后，IL - 2和干扰素 - γ（IFN - γ）诱导效果最佳；在CD8 + T细胞中，IL - 2在10小时后诱导效果最佳，IFN - γ在6小时后诱导效果最佳。在3个月的时间里，4名健康个体的CD8 +和CD8 - T细胞中IL - 2和IFN - γ水平在四次检测中保持一致。在一大组34名正常受试者中，CD3/IL - 2 +（范围9.7 - 41.3）和CD3/IFN - γ +细胞（10.1 - 44），以总淋巴细胞的百分比表示，存在相当大的异质性。与非特应性皮炎对照组（n = 5）相比，特应性皮炎患者（n = 5）中表达IFN - γ的CD3 + /CD8 +外周血T细胞百分比显著降低，而表达IL - 4的CD3 + /CD8 - T细胞百分比增加。本文还讨论了该技术可能的应用。
