Cytokines, asthma, and airway smooth muscle

（申请人？s摘要）现在有强有力的证据表明，Th2细胞因子 IL-4和IL-13在过敏性哮喘中起重要作用。我们假设 气道平滑肌细胞可能是这些细胞因子的重要靶点。我们 初步数据表明，IL-4和IL-13受体存在于 培养的人气道平滑肌（HASM）细胞，JAK/STAT，ERK， 和PI-3-激酶途径各自被这些细胞中的IL-13和IL-4激活。 此外，IL-4和IL-13各自诱导HASM细胞功能的变化， 包括对β-激动剂反应的降低和VCAM的增加， 和嗜酸性粒细胞趋化因子的表达，虽然IL-4和IL-13及其 信令模式不同。本提案的目的是研究 IL-13和IL-4对HASM细胞的生物学功能，并测试其潜力。 已知和新的多态性对这些功能的影响。在目标1中，我们 检验IL-4和IL-13诱导不同生物学应答的假设 在HASM细胞中。为此，我们将研究剂量和时间相关的影响， U-4和IL-13对HASM细胞对β-激动剂的反应的影响，以及它们的 对VCAM和eotaxin表达的影响。在目标2中，我们将检验假设 IL-4和IL-13信号在HASM细胞中不同。为此，我们将研究 IL-4和IL-13对STAT-6、STAT-3、ERK和 使用蛋白质印迹和体外激酶测定的PI-3-激酶活化。在 目的3，我们将检验IL-4和IL-13的差异 信号传导介导生物反应的差异。我们将使用 结合选择性ERK和PI-3激酶抑制剂的方法，以及 用显性阴性形式的MEK，PI-3激酶， 或STAT-6。结局指标将包括异丙肾上腺素（ISO）诱导的 CRE-荧光素酶活性，HASM细胞对ISO、VCAM的刚度响应 表达以及β 2受体、VCAM和嗜酸细胞活化趋化因子启动子活性。在 目的4，我们将验证EL-4受体的多态性 其与哮喘相关，影响HASM对IL-4的反应， IL-13。来自多个供体的HASM细胞的基因组DNA将被扩增。 对IL-4Ra编码区的6个多态性进行基因分型。的细胞中 对于具有信息基因型的供体，我们将检测IL-13和 IL-4对ISO诱导的HASM细胞刚度和cAMP形成、VCAM的变化的影响 以及IL-4/IL-13信号转导和分层。 基因型和单倍型的结果。了解影响和 IL-13和IL-4对HASM细胞的作用机制可能导致新的 治疗哮喘的方法。

(Applicant?s Abstract) There is now strong evidence that the Th2 cytokines IL-4 and IL-13 are important in allergic asthma. It is our hypothesis that airway smooth muscle cells may be important targets of these cytokines. Our preliminary data indicate that IL-4 and IL-13 receptors are present on cultured human airway smooth muscle (HASM) cells and that the JAK/STAT, ERK, and PI-3-kinase pathways are each activated by IL-13 and IL-4 in these cells. Furthermore, IL-4 and IL-13 each induce changes in the function of HASM cells, including reductions in the response to beta-agonists, and increases in VCAM and eotaxin expression, although the effects of IL-4 and IL-13 and their signaling patterns differ. The goal of this proposal is to study the biological functions of IL-13 and IL-4 on HASM cells and to test the potential impact of known and new polymorphisms on these functions. In Aim 1, we will test the hypothesis that IL-4 and IL-13 induce different biological responses in HASM cells. To do so, we will examine the dose and time related effects of U-4 and IL-13 on HASM cell responses to beta-agonists, as well as their effects on VCAM and eotaxin expression. In aim 2, we will test the hypothesis that IL-4 and IL-13 signaling differ in HASM cells. To do so, we will examine the dose and time related effects of IL-4 and IL-13 on STAT-6, STAT-3, ERK and PI-3-kinase activation using Western blotting and in vitro kinase assays. In aim 3, we will test the hypothesis that differences in IL-4 and IL-13 signaling mediate the differences in biological responses. We will use an approach combining selective ERK and PI-3-kinase inhibitors, as well as transfection of HASM cells with dominant negative forms of MEK, PI-3 kinase, or STAT-6. Outcome indicators will include isoproterenol (ISO) induced CRE-luciferase activity, HASM cell stiffness responses to ISO, VCAM expression, as well as beta2 receptor, VCAM, and eotaxin promoter activity. In aim 4, we will test the hypothesis that polymorphisms of the EL-4 receptor which are associated with asthma influence responses of HASM to IL-4 and IL-13. Genornic DNA from HASM cells derived from multiple donors will be genotyped for 6 polymorphisms in the coding region of IL-4Ra. In cells from donors with informative genotypes, we will examine the effects of IL-13 and IL-4 on ISO induced changes in HASM cell stiffness and cAMP formation, VCAM and eotaxin expression, as well as IL-4/IL-13 signal transduction and stratify the results by genotype and haplotype. Understanding the effects and mechanisms of action of IL-13 and IL-4 on HASM cells may lead to new modalities for the treatment of asthma.