MECHANISMS OF AIRWAY HYPERRESPONSIVENESS

拟议研究的主要目标是测试 假设嗜酸性粒细胞和中性粒细胞之间的双向相互作用 C纤维感觉神经增强呼吸道炎症并促进 呼吸道高反应性。我们假设每种细胞类型 招募和/或刺激他人释放其生物活性 调解人，这种积极的反馈循环对于 变应原或辣椒素诱导的呼吸道的全面表达 高反应性。在这一应用中，提出了实验 为了评估嗜酸性粒细胞的机制和功能相关性- 感觉性C纤维的相互作用。为了实现这一广泛目标， 研究人员计划进行体外研究，首先定义 这些细胞物种各自招募和/或 刺激另一方释放其生物活性介体。然后，他们 将在体内测试这些机制的重要性，使用两种 气道缩窄器高反应性的动物模型 所提议的循环相互作用由以下两种方式启动：(I)主要 刺激呼吸道感觉C纤维(辣椒素气雾剂 吸入)，或(2)初级招募和激活 嗜酸性粒细胞(免疫后吸入变应原气雾剂- 敏化)。提出了三个具体目标：(1)确定 嗜酸性粒细胞促进感觉C-纤维的机制 神经肽在呼吸道内释放。新生大鼠背根 神经节细胞，在原代培养中，将暴露在嗜酸性粒细胞- 条件培养液或单个嗜酸性粒细胞介体，以进行测试 嗜酸性粒细胞衍生物质作用于感觉的假说 纤维可：(I)直接刺激感觉神经肽的释放； (Ii)促进非-氨基丁酸诱导的感觉神经肽的释放 刺激嗜酸性粒细胞；以及(Iii)促进速激肽的合成。(2) 识别感觉C纤维促进嗜酸性粒细胞的机制 渗透和活化。静止的人外周血 嗜酸性粒细胞将暴露于背根神经节细胞- 条件培养液或个别感觉神经肽，以测试 C纤维促进气道嗜酸性粒细胞募集和 通过以下方式激活：(I)上调该基因的表达和活性 嗜酸性粒细胞整合素LFA-1、VLA-4和Mac-1(使用 免疫荧光流式细胞术与新型嗜酸性粒细胞黏附 检测)；(Ii)增加嗜酸性粒细胞黏附配体的表达 (ICAM-1、VCAM-1和E-选择素[ELAM])对全身血管的影响 内皮细胞；及(Iii)直接刺激释放 生物活性介体。(3)界定嗜酸性粒细胞的角色和 变应原和辣椒素诱导的呼吸道中的感觉C纤维 体内的高反应性。豚鼠将被用于测试 嗜酸性粒细胞和感觉C纤维都是必需的假说 过敏原和辣椒素诱导的呼吸道参与者 高反应性；任一种细胞的功能抑制 (即，使用单抗或 药物干预)减少呼吸道嗜酸性粒细胞浸润 过敏原或辣椒素后的呼吸道高反应性 气雾剂暴露。从这些研究中，调查人员应该 了解嗜酸性粒细胞和感觉c-纤维是否以及如何相互作用 在体内促进呼吸道炎症和高反应性。 从这项工作中获得的洞察力可能会对并联机构有所帮助 这可能在人类哮喘中起作用。

The major objective of the proposed research is to test the hypothesis that bidirectional interactions between eosinophils and sensory C-fiber nerves enhance airway inflammation and promote airway hyperresponsiveness. We postulate that each cell type recruits and/or stimulates the other to release its bioactive mediators, and that this positive feedback cycle is necessary for the full expression of allergen- or capsaicin-induced airway hyperresponsiveness. In this application, experiments are proposed to evaluate the mechanisms and functional relevance of eosinophil- sensory C-fiber interactions. To achieve this broad objective, the investigators plan studies in vitro that will first define the mechanisms by which these cell species each recruit and/or stimulate the other to release its bioactive mediators. Then, they will test the importance of these mechanisms in vivo, using two animal models of airway constrictor hyperresponsiveness, in which the proposed cyclic interaction is initiated either by: (i) primary stimulation of airway sensory C-fibers (capsaicin aerosol inhalation), or (ii) primary recruitment and activation of eosinophils (allergen aerosol inhalation after immune- sensitization). Three specific aims are put forth: (1) Identify mechanisms by which eosinophils promote sensory C-fiber neuropeptide release within the airways. Neonatal rat dorsal root ganglion cells, in primary culture, will be exposed to eosinophil- conditioned media or to individual eosinophil mediators, to test the hypotheses that eosinophil-derived substances act on sensory C- fibers to: (i) directly stimulate sensory neuropeptide release; (ii) potentiate release of sensory neuropeptides induced by non- eosinophil stimuli; and (iii) promote tachykinin synthesis. (2) Identify mechanisms by which sensory C-fibers promote eosinophil infiltration and activation. Quiescent human peripheral blood eosinophils will be exposed to dorsal root ganglion cell- conditioned medium or individual sensory neuropeptides, to test the hypotheses that C-fibers promote airway eosinophil recruitment and activation by: (i) upregulating the expression and activity of the eosinophil integrins LFA-1, VLA-4, and Mac-1 (using immunofluorescent flow cytometry and a novel eosinophil adhesion assay); (ii) increasing expression of eosinophil adhesion ligands (ICAM-1, VCAM-1, and E-selectin [ELAM]) on systemic vascular endothelium; and (iii) directly stimulating the release of bioactive mediators. (3) Delineate the roles of eosinophils and sensory C-fibers in allergen-and capsaicin-induced airway hyperresponsiveness in vivo. Guinea pigs will be used to test the hypothesis that both eosinophils and sensory C-fibers are necessary participants in both allergen- and capsaicin-induced airway hyperresponsiveness; functional inhibition of either cell type (i.e., of recruitment or activation, using monoclonal antibodies or pharmacologic interventions) reduces airway eosinophil infiltration and airway hyperresponsiveness after either allergen or capsaicin aerosol exposure. From these studies, the investigators should learn whether and how eosinophils and sensory c-fibers interact to promote airway inflammation and hyperresponsiveness in vivo. Insight gained from this work may shed light on parallel mechanisms that may operate in human asthma.