North M E, Ivory K, Funauchi M, Webster A D, Lane A C, Farrant J
Department of Clinical Immunology, Royal Free Hospital School of Medicine, London, UK.
Clin Exp Immunol. 1996 Sep;105(3):517-22. doi: 10.1046/j.1365-2249.1996.d01-795.x.
Using three-colour flow cytometry, we have measured intracellular IL-2, interferon-gamma (IFN-gamma) and tumour necrosis factor-alpha (TNF-alpha) induced in human CD4+ and CD8+ T cells from normal donors and patients with common variable immunodeficiency (CVID). Since a new range of directly FITC-conjugated anti-cytokine antibodies was used, conditions were optimized for the concentration of antibody, for cell permeabilization and fixation, and for the time of exposure to monensin to retain the cytokines within the cell. Kinetics of intracellular cytokine production were measured for up to 20 h in culture with phorbol myristate acetate (PMA) and ionomycin, or with phytohaemagglutinin (PHA). Kinetic studies of activation with PMA and ionomycin show that a higher proportion of normal CD4+ cells can make IL-2 than the other two cytokines, and that there are more TNF-alpha-positive CD4+ cells than cells with IFN-gamma. For normal CD8+ cells the highest production of cytokine is of IFN-gamma (up to 50% of the cells) especially at longer times (10-20 h) of stimulation. For CD8+ cells, IL-2-positive cells exceed those with TNF-alpha. The other mitogenic stimulus used (PHA) was grossly inferior to PMA and ionomycin in its ability to induce intracellular cytokines. The time of exposure to monensin was also examined. Its continuous presence in the cultures (up to a maximum of 20 h) increased the detection of IL-2-positive cells without apparently reducing the percentage of cytokine-positive CD4+ or CD8+ cells. Finally, using optimal conditions, we compared cytokine production in cells from patients with the disease CVID and showed normal cellular levels of ability to produce IL-2 and TNF-alpha but significantly raised levels of production of IFN-gamma in both CD4+ and CD8+ lymphocytes. This suggests that the pathology of this disease may involve an excessive Th1-type response.
我们使用三色流式细胞术，检测了来自正常供体和常见可变免疫缺陷（CVID）患者的人CD4+和CD8+ T细胞中诱导产生的细胞内白细胞介素-2（IL-2）、干扰素-γ（IFN-γ）和肿瘤坏死因子-α（TNF-α）。由于使用了一系列新的直接异硫氰酸荧光素（FITC）偶联的抗细胞因子抗体，因此针对抗体浓度、细胞通透化和固定以及暴露于莫能菌素以将细胞因子保留在细胞内的时间进行了条件优化。在用佛波酯肉豆蔻酸酯乙酸酯（PMA）和离子霉素或植物血凝素（PHA）培养长达20小时的过程中，测量细胞内细胞因子产生的动力学。用PMA和离子霉素激活的动力学研究表明，正常CD4+细胞中能够产生IL-2的比例高于其他两种细胞因子，并且TNF-α阳性的CD4+细胞比IFN-γ阳性的细胞更多。对于正常CD8+细胞，细胞因子的最高产量是IFN-γ（高达50%的细胞），尤其是在刺激较长时间（10 - 20小时）时。对于CD8+细胞，IL-2阳性细胞超过TNF-α阳性细胞。所使用的另一种促有丝分裂刺激物（PHA）在诱导细胞内细胞因子的能力方面明显不如PMA和离子霉素。还研究了暴露于莫能菌素的时间。其在培养物中的持续存在（最长可达20小时）增加了IL-2阳性细胞的检测，而显然没有降低细胞因子阳性CD4+或CD8+细胞的百分比。最后，使用最佳条件，我们比较了CVID患者细胞中的细胞因子产生情况，结果显示产生IL-2和TNF-α的细胞水平正常，但CD4+和CD8+淋巴细胞中IFN-γ的产生水平显著升高。这表明该疾病的病理可能涉及过度的Th1型反应。
