SEROTONIN (5-HT) AND 5-HT2A IN ALLERGIC INFLAMMATION

描述(由申请人提供)：化学诱导剂在嗜酸性粒细胞的激活和定向迁移到过敏性炎症的血管外部位中起着关键作用。我们实验室最近发表的研究首次表明，5-羟色胺(5-羟色胺[5-HT])可以直接诱导嗜酸性粒细胞趋化，但不能诱导中性粒细胞趋化，并在变态反应性炎症小鼠模型中重新募集嗜酸性粒细胞发挥重要作用。5-羟色胺单独或联合嗜酸性粒细胞趋化因子在体外和体内均可诱导嗜酸性粒细胞趋化。初步研究表明，来自过敏捐赠者而不是对照组的嗜酸性粒细胞表达5-HT2a受体。体外5-羟色胺诱导的嗜酸性粒细胞趋化和变应原诱导的嗜酸性粒细胞在野生型和嗜酸性粒细胞趋化中的募集均可被5-羟色胺受体拮抗剂赛庚定和mdl-100907所抑制。基于这些发现，我们推测5-羟色胺通过5-HT2a受体发挥作用，是一种新的嗜酸性粒细胞活性趋化因子，在嗜酸性粒细胞募集和变态反应性炎症的整体发病机制中发挥重要作用。为了评估我们的假设，在特定的目标1中，我们将评估5-羟色胺和5-羟色胺2 A受体通路在介导变态反应性炎症的各个方面所起的作用，包括嗜酸性粒细胞聚集、气道高反应性(AHR)、Th1/Th2细胞因子和气道重塑。我们将在存在和不存在CCR3拮抗剂的情况下评估5-HT2A受体拮抗剂MDL-100907在野生型和T-bet-/-小鼠以及5-HT2A-/-和CCR3-/-小鼠过敏性呼吸道炎症模型中的作用。5-HT2A和CCR3相加效应的重要性将通过产生5-HT2A-/-/7CCR3-/-双重敲击小鼠来更详细地检验。在具体目标2中，我们将研究5-HT2A受体拮抗剂在流动条件下对嗜酸性粒细胞迁移的影响。此外，我们将利用活体显微镜(IVM)研究从IL-5trg小鼠中分离的小鼠嗜酸性粒细胞如何在5-羟色胺内运输刺激肺微血管，使用一种新的小鼠肺移植模型。为了进一步研究5-羟色胺在血管内皮细胞介导的嗜酸性粒细胞转运中的作用，将使用OVA攻击的5-HT2A-/-小鼠的肺组织作为肺移植模型。此外，我们的研究还描述了介导5-羟色胺(P160 ROCK)和嗜酸性粒细胞趋化蛋白(GAI)诱导趋化的信号通路的重要区别和相似之处。作为第二个目标的一部分，我们将研究这些信号分子的作用，以确定5-HT2A和CCR3介导的通路之间的分歧点和汇聚点。总之，我们建议详细研究参与5-羟色胺介导的过敏性炎症的细胞和分子机制，并假设单独阻断5-HT2A受体或与CCR3受体联合阻断可能导致气道过敏性炎症的缓解，包括嗜酸性粒细胞聚集、AHR、Th1/Th2细胞因子和气道重塑。

DESCRIPTION (provided by applicant): Chemoattractants play a critical role in the activation and directed migration of eosinophils to extravascular sites of allergic inflammation. Recently published studies from our laboratory have shown for the first time that serotonin (5-hydroxytryptamine [5-HT]) can directly induce chemotaxis of eosinophils, but not neutrophils, and play an important role in the recruitment of eosinophils in a murine model of allergic inflammation. 5-HT both independently and additively with eotaxin induces eosinophil chemotaxis in vitro and in vivo. Preliminary studies suggest that eosinophils from allergic donors but not control subjects express the 5-HT2A receptor. Furthermore, both 5-HT induced eosinophil chemotaxis in vitro and allergen induced eosinophil recruitment in wild type and eotaxin-/- mice could be inhibited by the 5-HT2A receptor antagonists cyproheptadine and MDL-100907. Based on these findings, we hypothesize that 5-HT, functioning via the 5-HT2A receptor, is a novel eosinophil-active chemoattractant that plays an important role in eosinophil recruitment and overall pathogenesis of allergic inflammation. To evaluate our hypotheses, in Specific Aim 1, we will evaluate the role played by the 5-HT and 5-HT2A receptor pathway in mediating various aspects of allergic inflammation including eosinophil recruitment, airway hyperreactivity (AHR), Th1/Th2 cytokines and airway remodeling. We will evaluate the effect of the 5-HT2A receptor antagonist MDL-100907 in the presence and absence of a CCR3 antagonist in a murine model of allergic airway inflammation in wild type and T-bet-/- mice as well as 5-HT2A-/- and CCR3-/- mice. The importance of the additive effect of 5-HT2A and CCR3 will be examined in greater detail by generating a 5-HT2A-/-/7CCR3-/- double knock mouse. In Specific Aim 2, we will study the effect of the 5-HT2A receptor antagonist on eosinophil trafficking under condition of flow. In addition, we will utilize intravital microscopy (IVM) to examine how murine eosinophils isolated from IL-5trg mice traffic within 5-HT stimulated lung microvessels using a novel murine lung allograft model. To further examine the role of 5-HT on vascular endothelium mediated eosinophil trafficking, lung tissue from OVA-challenged 5-HT2A-/- mice will be used in the lung allograft model. Moreover, our studies have delineated important differences and similarities in the signaling pathways that mediate 5-HT (p160 ROCK) versus eotaxin (Gai) induced chemotaxis. As part of the second aim, we will examine the role of these signaling molecules to determine the points of divergence and convergence between the 5-HT2A- and CCR3-mediated pathways. Overall, we propose to examine in detail the cellular and molecular mechanisms that participate in 5-HT-mediated allergic inflammation and hypothesize that blockade of 5-HT2A receptor individually or in combination with the CCR3 receptor might lead to alleviation of airway allergic inflammation including eosinophil recruitment, AHR, Th1/Th2 cytokines and airway remodeling.