Cytometric evaluation of intracellular IFN-γ and IL-4 levels in thyroid follicular cells from patients with autoimmune thyroid diseases.
Cytometric evaluation of intracellular IFN-γ and IL-4 levels in thyroid follicular cells from patients with autoimmune thyroid diseases.
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DOI：
10.1186/1756-6614-4-13
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发表时间：
2011-09-23
期刊：
影响因子：
2.2
通讯作者：
Ratomski K
中科院分区：
文献类型：
--
作者：
Bossowski A;Harasymczuk J;Moniuszko A;Bossowska A;Hilczer M;Ratomski K
近年来，人们强调促炎细胞因子和抗炎细胞因子的平衡改变在 AITD 的发病机制中发挥着重要作用。本研究的目的是评估 54 名 8-21 岁青少年患者的甲状腺浸润淋巴细胞和甲状腺细胞中的细胞内 INF-γ 和 IL-4 水平，这些患者患有格雷夫斯病 (GD; n = 18)、桥本甲状腺炎 (HT; n = 18) 和非毒性多结节性甲状腺肿 (NTMG; n = 18)。取新鲜甲状腺组织于RPMI -1640培养基上，进行机械制备。在下一步中添加细胞活化剂-12-肉豆蔻酸13-乙酸盐(PMA)和离子霉素以及蛋白质运输抑制剂-Breferdin A。将它们在50ml烧瓶中在37°C、5-95%CO2-空气水饱和气氛中培养24小时。之后，通过与 FITC 标记的兔抗小鼠抗体 IgG (Fab')2 缀合的针对人 TPO 表位 64 的小鼠单克隆抗体来鉴定甲状腺细胞。在室温下孵育后，向每个样品添加试剂A固定剂和细胞膜。在下一步中，向细胞悬浮液中添加试剂 B 以透化细胞膜和特异性抗 IL-4-PE 或抗 IFN-γ-PE mAb。使用 T 淋巴细胞特异性抗 CD4-PerCP 和抗 CD8-PerCP mAb 以相同的程序进行 T 淋巴细胞中细胞内细胞因子的鉴定。在流式细胞仪（Coulter EPICS XL）中分析细胞。在 GD 患者检查组中，与 HT 和 NTMG 患者相比，我们观察到表型 CD4+IL-4 (p < 0.05; p < 0.025)、CD8+IL-4 (p < 0.033; p < 0.01) 和 TFCs-IL-4+ (p < 0.05; p < 0.01) 的细胞平均百分比具有统计学显着更高的百分比。对 HT 患者细胞内 INF-g 水平阳性 TIL 和 TFC 平均百分比的分析显示，与来自 HT 患者的阳性细胞百分比相比，CD4+INF-γ (p < 0.04; p < 0.001)、CD8+ INF-γ (NS; p < 0.025)、TFC+INF-γ (p < 0.03; p < 0.001) 细胞的百分比显着增加。 GD 和 NTMG 患者。我们得出的结论是，自身免疫性甲状腺疾病中的人类甲状腺细胞可能是细胞因子产生的来源，并且它们的激活影响与流入甲状腺的 T 淋巴细胞的局部相互作用。
In recent few years is underlined that altered balance of pro- and anti-inflammatory cytokines play an important role in the pathogenesis of AITD. The aim of this study was to estimate intracellular INF-γ and IL-4 levels in thyroid-infiltrating lymphocytes and thyrocytes isolated from thyroid tissues in 54 adolescent patients aged 8-21 years, with Graves' disease (GD; n = 18), Hashimoto's thyroiditis (HT; n = 18) and non-toxic multinodular goiter (NTMG; n = 18). Fresh thyroid tissues were taken on culture medium RPMI -1640, it was mechanically prepared. In next step were added cell activators -12- myristate 13- the acetate (PMA) and Ionomycin as well as the inhibitor of transportation of proteins - Breferdin A. They were cultured 24 hours in 50 ml flasks at 37°C in a 5-95% CO2-air water-saturated atmosphere. After that, thyrocytes were identified by mouse mAb directed against human TPO epitope 64 conjugated with rabbit anti-mouse antibodies IgG (Fab')2 labeled by FITC. After incubation at room temperature to each of samples added reagent A fixative the cellular membrane. In next step into the cell suspensions were added reagent B to permeabilization of cellular membrane and specific anti-IL-4-PE or anti-IFN-γ-PE mAbs. Identification of intracellular cytokines in T lymphocytes was performed in the same procedure with application of anti-CD4-PerCP and anti-CD8-PerCP mAbs specific for T lymphocytes. The cells were analyzed in a flow cytometry (Coulter EPICS XL). In examined group of patients with GD we observed statistically significant higher mean percentage of cells with phenotype CD4+IL-4 (p < 0.05; p < 0.025), CD8+IL-4 (p < 0.033; p < 0.01) and TFCs-IL-4+ (p < 0.05; p < 0.01) in comparison to patients with HT and NTMG. The analysis of mean percentages of positive TILs and TFCs with intracellular INF-g levels in patients with HT revealed statistically significant increase percentage of CD4+INF-γ (p < 0.04; p < 0.001), CD8+ INF-γ (NS; p < 0.025), TFCs+INF-γ (p < 0.03; p < 0.001) cells in comparison to the percentage of positive cells from patients with GD and NTMG. We conclude that human thyrocytes in autoimmune thyroid disorders could be a source of cytokine production and that their activation influences local interaction with T lymphocytes inflowing to the thyroid gland.