Cytokines, asthma, and airway smooth muscle

S摘要：现在有强有力的证据表明Th2型细胞因子 IL-4和IL-13在过敏性哮喘中起重要作用。我们的假设是 气道平滑肌细胞可能是这些细胞因子的重要靶点。我们的 初步数据显示，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， 在这些细胞中，PI-3-激酶通路分别被IL-13和IL-4激活。 此外，IL-4和IL-13分别诱导HASM细胞功能的改变， 包括对β-激动剂反应的减少和VCAM的增加 和嗜酸性粒细胞趋化因子的表达，尽管IL-4和IL-13及其受体的作用 信令模式不同。这项建议的目标是研究 IL-13和IL-4对HASM细胞的生物学功能及其潜能的检测 已知的和新的多态对这些功能的影响。在目标1中，我们将 检验IL-4和IL-13引起不同生物反应的假设 在HASM细胞中。为此，我们将研究与剂量和时间相关的影响 U-4和IL-13对HASM细胞对β-激动剂的反应及其机制 对血管细胞黏附分子和嗜酸性粒细胞趋化因子表达的影响。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 用Western blotting和体外激活法检测PI-3-激酶的活性。在……里面 目的3，我们将检验假设，IL-4和IL-13的差异 信号调节生物反应的差异。我们将使用 结合选择性ERK和PI-3-激酶抑制剂以及 以MEK、PI-3激酶为显性阴性形式转导HASM细胞， 或者STAT-6。结果指标将包括异丙肾上腺素(ISO)诱导 Cre-荧光素酶活性、HASM细胞硬度对ISO、VCAM的响应 表达，以及Beta2受体、血管细胞黏附分子和嗜酸性粒细胞趋化因子启动子活性。在……里面 目的4，我们将检验EL-4受体基因多态性的假设 它们影响HASM对IL-4和IL-4的反应 IL-13。来自多个捐赠者的HASM细胞的基因组DNA将是 IL-4ra编码区6个多态性的基因分型。在来自的单元格中 ，我们将检查IL-13和 IL-4对ISO诱导的HASM细胞僵硬和cAMP形成、VCAM的影响 和嗜酸性粒细胞趋化因子的表达，以及IL-4/IL-13的信号转导和分层 结果由基因分型和单倍型决定。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.