Molecular mechanisms that regulate eosinophil cytokine production

（申请人的摘要）外周血嗜酸性粒细胞通过其调节细胞增殖的机制。 （PBEos）分化为基于气道的效应细胞，负责 哮喘的病理生理学知之甚少。一个候选调解人 过程是粒细胞巨噬细胞集落刺激因子（GM-CSF），一种有效的 嗜酸性粒细胞产生的细胞因子。GM-CSF通常在BAL液中升高 有症状的哮喘患者体外激活的PBEo分泌 免疫学可检测GM-CSF和表达GM-CSF mRNA。PBEos还表示， 细胞表面GM-CSF受体，表明GM-CSF作为一种关键的 自分泌生长和存活因子。尽管 GM-CSF可能的功能意义，很少有人知道， 通过以下方式控制其产生和释放的分子机制 嗜酸性粒细胞最近，我们已经表明，GM-CSF mRNA的稳定性， 在用肿瘤坏死因子α（TNF）治疗的PBE中显著增强， 和纤连蛋白或来自过敏原激发的BAL来源的嗜酸性粒细胞 志愿者使用酵母3杂交筛选，我们鉴定了YB-1，一种已知的核酸， 酸结合蛋白作为GM-CSF mRNA结合蛋白。重组YB-1 在体外特异性结合富含AU的3' UTR不稳定决定子， GM-CSF mRNA。当转染到外周血嗜酸性粒细胞中时，YB-1增强了 体外存活率提高3-5倍，可被抗GM-CSF完全阻断 抗体的最后，在初步研究中，转染的YB-1稳定 PBEOS中GM-CSF mRNA。因此，我们假设YB-1介导了 活化的嗜酸性粒细胞中GM-CSF mRNA的转录后调节。因此，在本发明中， 该项目的目标是：（1）。表征YB-1如何增加GM-CSF 2）表征TNF α诱导的信号级联，和 纤连蛋白，其使YB-1能够与GM-CSF mRNA相互作用并调节GM-CSF mRNA，3）。 确定YB-1是否是该系统中的唯一效应器或与 调节GM-CSF mRNA的额外蛋白组分，4）。确定哪些 YB-1的结构域是GM-CSF转录后基因所必需的 调控总的来说，这些研究将阐明 活化的嗜酸性粒细胞中潜在的GM-CSF mRNA调节，因此， 为预防和治疗提供了额外的新的治疗靶点， 哮喘

(Applicant's Abstract) The mechanisms by which peripheral blood eosinophils (PBEos) differentiate into airway based, effector cells responsible for the pathophysiology of asthma are poorly understood. A candidate mediator for this process is granulocyte macrophage colony stimulating factor (GM-CSF), a potent cytokine produced by eosinophils. GM-CSF is commonly elevated in the BAL fluid of symptomatic asthmatics. PBEos which are activated in vitro secrete immunologically detectable GM-CSF and express GM-CSF mRNA. PBEos also express cell surface GM-CSF receptors, suggesting GM-CSF functions as a critical autocrine growth and survival factor both in vitro and in vivo. Despite the likely functional significance of GM-CSF, very little is known about the molecular mechanism(s) which controls its production and release by eosinophils. Recently we have shown that GM-CSF mRNA stability was significantly enhanced in PBEos treated with tumor necrosis factor alpha (TNF) and fibronectin or in BAL derived eosinophils from allergen challenged volunteers. Using a yeast 3 hybrid screen, we identified YB-1, a known nucleic acid binding protein as a GM-CSF mRNA binding protein. Recombinant YB-1 specifically bound in vitro to the AU-rich, 3' UTR instability determinants of GM-CSF mRNA. When transfected into peripheral blood eosinophils, YB-1 enhanced in vitro survival by 3-5 fold, which was completely blocked by anti-GM-CSF antibodies. Finally, in preliminary studies, transfected YB-1 stabilized GM-CSF mRNA in PBEos. Therefore, we hypothesize that YB-1 mediates the post-transcriptional regulation of GM-CSF mRNA in activated eosinophils. Thus, the aims of this project are to 1). Characterize how YB-1 increases GM-CSF mRNA in PBEos, 2).Characterize the signaling cascades induced by TNFalpha, and fibronectin which enable YB-1 to interact with and regulate GM-CSF mRNA, 3). Determine if YB-1 is the sole effector in this system or interacts with additional protein components to regulate GM-CSF mRNA, 4). Determine which domain(s) of YB-1 is/are required for GM-CSF post-transcriptional gene regulation. In aggregate these studies will clarify the molecular mechanisms underlying GM-CSF mRNA regulation in activated eosinophils, and as such, provide additional, novel therapeutic targets for the prevention and treatment of asthma.