Department of Respiratory Medicine, Xinhua Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai 200092, China.
Chin Med J (Engl). 2010 Oct;123(19):2676-81.
Allergic asthma is associated with airway inflammation and hyperresponsiveness caused by dysregulated production of cytokines secreted by allergen-specific helper T-type 2 (Th2) cells. The linker for activation of T cells (LAT) is a membrane-associated adaptor protein, which has been shown to take part in regulating T cell receptor (TCR) signaling and T cell homeostasis. In this study, we established an asthmatic mouse model to examine the changes in LAT levels during allergic airway disease and the effects of LAT transgenic expression on airway inflammation.
T cells from mouse lung tissues were isolated from allergen challenged (ovalbumin (OVA)) and control mice, and the purity of these isolated T cells was examined by fluorescence-activated cell sorter (FACS). Semi-quantitative RT-PCR and Western blotting were used to detect the expression of the LAT gene and LAT protein, respectively. After an intranasally administered mixture of pCMV-HA-LAT plasmid and Lipofectamine 2000, 24 hours before and 72 hours after allergen challenge, the BALF cell count and the differential cytologies were studied. In addition, IL-4 and IFN-γ levels in the BALF were determined by ELISA, and pathological changes in lung tissues were observed.
LAT protein and mRNA expression were decreased in lung T cells in a mouse model of allergen-induced airway disease. After intranasal administration of pCMV-HA-LAT, histopathological examination of the lungs showed that intervention with LAT overexpression prevented mice from developing airway inflammation, and the number of total cells, eosinophils, neutrophils, and lymphocytes in the BALF was reduced significantly compared with the OVA sensitized and challenged group. In addition, the Th2 cytokine IL-4 decreased, while the Th1 cytokine IFN-γ increased compared to the OVA sensitized and challenged group or the OVA sensitized group plus pCMV-HA treatment.
This study demonstrates that LAT might effectively diminish Th2 cytokine responses, lung histopathological changes and lung inflammation to allergen challenge in a model of experimentally induced asthma.
过敏性哮喘与气道炎症和由过敏原特异性辅助 T 型 2（Th2）细胞产生的细胞因子失调引起的高反应性有关。衔接蛋白（LAT）是一种膜相关衔接蛋白，已被证明参与调节 T 细胞受体（TCR）信号和 T 细胞稳态。在这项研究中，我们建立了哮喘小鼠模型，以研究过敏性气道疾病过程中 LAT 水平的变化，以及 LAT 转基因表达对气道炎症的影响。
从过敏原（卵清蛋白（OVA））和对照小鼠的肺组织中分离 T 细胞，并通过荧光激活细胞分选（FACS）检查这些分离 T 细胞的纯度。半定量 RT-PCR 和 Western blot 分别用于检测 LAT 基因和 LAT 蛋白的表达。在过敏原攻击前 24 小时和后 72 小时经鼻腔给予 pCMV-HA-LAT 质粒和 Lipofectamine 2000 混合物后，研究 BALF 细胞计数和差异细胞学。此外，通过 ELISA 测定 BALF 中的 IL-4 和 IFN-γ 水平，并观察肺组织的病理变化。
在过敏原诱导的气道疾病小鼠模型中，肺 T 细胞中的 LAT 蛋白和 mRNA 表达降低。经鼻腔给予 pCMV-HA-LAT 后，肺组织的组织病理学检查显示，LAT 过表达的干预可防止小鼠发生气道炎症，BALF 中的总细胞、嗜酸性粒细胞、中性粒细胞和淋巴细胞数量明显减少与 OVA 致敏和攻击组相比。此外，与 OVA 致敏和攻击组或 OVA 致敏组加 pCMV-HA 治疗组相比，Th2 细胞因子 IL-4 减少，而 Th1 细胞因子 IFN-γ 增加。
本研究表明，LAT 可能有效抑制过敏原攻击诱导的哮喘模型中 Th2 细胞因子反应、肺组织病理变化和肺炎症。
