Tec Kinases ltk and RlK in Mast Cell Signaling

描述（由申请人提供）：过敏性哮喘是一种复杂的疾病，涉及淋巴细胞、嗜酸性粒细胞和肥大细胞介导的局部炎症反应。这种应答的一个关键成分是T细胞和肥大细胞产生的辅助性T细胞2型（Th 2）细胞因子，包括IL-4、IL-5、IL-9和IL-13。Tec家族酪氨酸激酶，Itk和Rlk，在激活磷脂酶C-？1下游的T细胞受体。Itk/Rlk缺陷型T细胞显示受损的TCR信号传导，导致细胞因子产生显著减少和清除病原性感染的缺陷。有趣的是，未免疫的Itk-/-和Itk-/-Rlk-/-小鼠表现出提示过量Th 2型细胞因子产生的异常，如脾脏中的生发中心增生、嗜酸性粒细胞增多和血清IgE抗体水平升高。这些数据，连同Itk和Rlk也在肥大细胞中表达的事实，提示了以下假设：Itk和/或Rlk可能在T细胞中对于IL-4产生至关重要，导致Th 2细胞分化和细胞因子产生，但也可能在肥大细胞因子产生中起作用。为了验证这一假设，我们建议确定是否在未免疫的Itk-/-和Itk-/-Rlk-/-小鼠中观察到的异常是由于T细胞内在缺陷，肥大细胞缺陷，或两者兼而有之。其次，我们发现Itk-/-骨髓来源的肥大细胞在FceRI刺激后分泌增强水平的IL-4、IL-6和IL-13。基于这些数据，我们假设Itk是SHIP 1/Dok-1通路的最佳激活所必需的，已知该通路可减弱肥大细胞反应。为了测试这一想法，我们将检查Itk-/-肥大细胞的脱粒和细胞因子的产生的变化，在刺激与IgE单独，IgE加抗原，SCF，以及FceRI和Fc？RII共聚集。生化分析将直接解决Itk在SHIP 1依赖性途径中的作用。这些研究的总体目标是进一步了解可能导致过敏性疾病发展的信号通路，并提出操纵和/或预防过敏反应的机制。

DESCRIPTION (provided by applicant): Allergic asthma is a complex disease involving a localized inflammatory response mediated by lymphocytes, eosinophils and mast cells. One critical component of this response is the T helper type 2 (Th2) cytokines, including IL-4, IL-5, IL-9, and IL-13, produced by T cells and mast cells. The Tec family tyrosine kinases, Itk and Rlk, play important roles in activating phospholipase C-?1 downstream of the T cell receptor. Itk/Rlk deficient T cells show impaired TCR signaling, leading to substantially reduced cytokine production and defects in clearing pathogenic infections. Interestingly, unimmunized Itk-/- and Itk-/-Rlk-/- mice exhibit abnormalities suggestive of excess Th2-type cytokine production, such as germinal center hyperplasia in the spleen, eosinophilia, and elevated levels of serum IgE antibodies. These data, together with the fact that Itk and Rlk are also expressed in mast cells, suggest the following hypothesis: Itk and/or Rlk may be critical in T cells for IL-4 production leading to Th2 cell differentiation and cytokine production, but may also play a role in mast cell cytokine production. To test this hypothesis, we propose to determine whether the abnormalities seen in unimmunized Itk-/- and Itk-/-Rlk-/- mice are due to a T cell-intrinsic defect, a defect in mast cells, or both. Second, we find that Itk-/- bone marrow-derived mast cells secrete enhanced levels of IL-4, IL-6, and IL-13 following FceRI stimulation. Based on these data, we hypothesize that Itk is required for optimal activation of the SHIP1/Dok-1 pathway, known to attenuate mast cell responses. To test this idea, we will examine Itk-/- mast cells for alterations in degranulation and cytokine production in response to stimulation with IgE alone, IgE plus antigen, SCF, as well as to FceRI and Fc?RII co-aggregation. Biochemical analyses will directly address the role of Itk in the SHIP1-dependent pathway. The overall goal of these studies is to further our understanding of signaling pathways that may contribute to the development of allergic diseases, and to suggest mechanisms for the manipulation and/or prevention of allergic responses.