TGF-b1 Activation of Monocytes

描述（由申请人提供）： TGF-β 1是一种有效的免疫调节剂，其促炎和免疫抑制活性似乎同样重要。数据表明，粘附到纤连蛋白在调节单核细胞中IL-8基因表达的TGF-β 1依赖性活化中起关键作用。所提出的实验使用体外和人体模型来测试转录和转录后机制在炎症期间控制单核细胞中IL-8的空间和时间表达中发挥同等关键作用的假设。具体而言：1）为了验证TGF-β 1通过Smad依赖性信号通路刺激IL-8 mRNA的延迟和持续增加的假设，我们将使用Nuclear run on测定来检查IL-8基因转录的时间依赖性起始。Western印迹和ELISA将用于检查Smad蛋白和NF-κ B的时间依赖性活化。2)为了检验粘附于纤连蛋白作为刺激TGF-β 1致敏单核细胞中IL-8转录物翻译的第二信号的假设，将通过测量粘附于Fn之前和之后TGF-β 1致敏细胞中IL-8和CXC趋化因子的多聚体分布来研究IL-8和CXC趋化因子的翻译。还将通过在不同时间点测量[35 S]甲硫氨酸的总掺入率来评估TGF-β 1和Fn对从头IL-8和整体蛋白质合成的影响。3)用荧光素酶报告基因构建体检测IL-8 mRNA的5 ′-非翻译区（UTR）和3 ′-非翻译区（UTR）在IL-8基因表达的转录后调控中所起的相对作用。4)我们假设，限制IL-8蛋白表达粘附单核细胞的机制的损失有助于AR DS。为了检测是否存在导致ARDS发生的细胞特异性缺陷，我们将使用实时动态PCR和ELISA比较TGF-β 1对肺血栓内膜切除术后有和无急性肺损伤患者单核细胞中IL-8和其他介质表达的影响。这些研究的结果可能会导致识别风险因素，使某些患者容易发生肺损伤。阐明调节净TGF-β 1活性的因素对于理解炎症反应的演变和解决至关重要，也将促进这种高度致命疾病的新型治疗药物的开发和使用。该研究计划与教学研讨会和会议相结合，旨在促进PI作为肺科医生科学家的持续成长和生产力。

DESCRIPTION (provided by applicant): TGF-b1 is a potent immunomodulator whose pro-inflammatory and immunosuppressive activities appear to be equally vital. Data indicate that adherence to fibronectin plays a key role in modulating the TGF-b1-dependent activation of IL-8 gene expression in monocytes. The proposed experiments use in vitro and human models to test the hypothesis that transcriptional and post-transcriptional mechanisms play an equally critic al role in controlling the spatial and temporal expression of IL-8 in monocytes during inflammation. Specifically: 1) To test the hypothesis that TGF-b1 acts through Smad dependent signaling pathways to stimulate the delayed and sustained increase in IL-8 mRNA, we will examine the time-dependent onset of IL-8 gene transcription using Nuclear run on assays. Western blotting and ELlSAs will be used to examine the time-dependent activation of Smad proteins and NF-kB. 2) To test the hypothesis that adherence to fibronectin acts a second signal to stimulate the translation of IL-8 transcripts in TGF-b1-primed monocytes, IL-8 and CXC chemokine translation will be studied by measuring their polysomal distribution in TGF-b1-primed cells prior to and following adherence to Fn. The effect of TGF-b1 and Fn on de novo IL-8 and global protein synthesis will also be assessed by measuring the overall rate of incorporation of [35S] methionine at various timepoints. 3) The relative roles played by the 5'-untranslated region (UTR) and 3'-UTR of the IL-8 mRNA in the post-transcriptional control of IL-8 gene expression wiII be examined using luciferase reporter constructs. 4) We hypothesize that the loss of mechanisms restricting IL-8 protein expression to adherent monocytes contributes to AR DS. To test whether there is a cell-specific defect that contributes to the development of ARDS, we will use real-time kinetic PCR and ELISAs to compare the effects of TGF-b1 on the expression of IL-8 and other mediators in monocytes harvested from patients with and without acute lung injury following pulmonary thromboendarterectomy. The results of these studies may lead to the identification of risk factors that render certain patients susceptible to developing lung injury. Elucidating the factors that modulate net TGF-b1 activity is critical to understanding the evolution and resolution of the inflammatory response and will also facilitate the development and usage of novel therapeutic agents for this highly fatal illness. The research plan, in conjunction with didactic seminars and conferences, is designed to foster the continued growth and productivity of the PI as a pulmonary physician scientist.