TGF-b1 Activation of Monocytes

描述(由申请人提供)： 转化生长因子-β1是一种强有力的免疫调节剂，其促炎和免疫抑制活性似乎同等重要。数据表明，对纤维连接蛋白的黏附在调节转化生长因子-β1依赖的单核细胞IL-8基因表达的激活中起关键作用。拟议的实验使用体外和人体模型来检验这一假设，即转录和转录后机制在炎症过程中控制单核细胞IL-8的时空表达方面发挥着同样关键的作用。具体地说：1)为了验证这一假说，即转化生长因子-β1通过Smad依赖的信号通路来刺激IL-8基因转录的延迟和持续增加，我们将使用核运行检测来检验IL-8基因转录的时间依赖性开始。Western blotting和ELLSA将被用来检测Smad蛋白和NF-kB的时间依赖性激活。2)为了验证对纤维连接蛋白的黏附作为第二信号刺激转化生长因子β1诱导的单核细胞中IL-8转录本翻译的假说，将通过测量黏附FN前后IL-8和CXC趋化因子在转化生长因子β1诱导的单核细胞中的多聚体分布来研究它们的翻译。还将通过测量不同时间点的[35S]蛋氨酸的总掺入速率来评估转化生长因子-β1和纤维连接蛋白对从头开始的IL-8和全球蛋白质合成的影响。3)利用荧光素酶报告基因构建法研究IL-8mRNA的5‘-非翻译区和3’-非翻译区在转录后调控IL-8基因表达中的相对作用。4)我们推测，限制IL-8蛋白表达到黏附性单核细胞的机制丢失是导致AR DS的原因。为了验证是否存在细胞特异性缺陷导致ARDS的发生，我们将使用实时动态聚合酶链式反应和ELISAs来比较转化生长因子-β1对急性肺损伤患者和非急性肺损伤患者肺血栓内膜剥脱术后单核细胞IL-8和其他介质表达的影响。这些研究的结果可能导致确定使某些患者容易发生肺损伤的危险因素。阐明调节转化生长因子β1净活性的因素对于了解炎症反应的演变和消退至关重要，也将有助于开发和使用新的治疗药物来治疗这种高度致命的疾病。该研究计划与教学研讨会和会议相结合，旨在促进PI作为肺部内科科学家的持续增长和生产力。

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.