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

描述（由申请人提供）：趋化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-hydroxytryptamine [5-HT]）可以直接诱导嗜酸性粒细胞趋化，而不是中性粒细胞，并且在小鼠变应性炎症模型中嗜酸性粒细胞的募集中发挥重要作用。在体外和体内，5-羟色胺单独或与eotaxin共同诱导嗜酸性粒细胞趋化。初步研究表明，来自过敏供体的嗜酸性粒细胞表达5-HT2A受体，而对照组不表达。此外，5-HT2A受体拮抗剂赛庚啶和MDL-100907均可抑制5-HT诱导的体外嗜酸性粒细胞趋化性和过敏原诱导的野生型和eotaxin-/-小鼠嗜酸性粒细胞募集。基于这些发现，我们假设5-HT通过5-HT2A受体发挥作用，是一种新的嗜酸性粒细胞活性化学引诱剂，在嗜酸性粒细胞募集和过敏性炎症的整体发病机制中起重要作用。为了评估我们的假设，在Specific Aim 1中，我们将评估5-HT和5-HT2A受体通路在介导过敏性炎症的各个方面所起的作用，包括嗜酸性粒细胞募集、气道高反应性（AHR）、Th1/Th2细胞因子和气道重塑。我们将在野生型和T-bet-/-小鼠以及5-HT2A-/-和CCR3-/-小鼠变应性气道炎症小鼠模型中评估5-HT2A受体拮抗剂MDL-100907在存在和不存在CCR3拮抗剂的情况下的作用。通过产生5-HT2A-/-/7CCR3-/-双敲小鼠，我们将更详细地研究5-HT2A和CCR3加性效应的重要性。在Specific Aim 2中，我们将研究5-HT2A受体拮抗剂对流动条件下嗜酸性粒细胞运输的影响。此外，我们将利用活体显微镜（IVM）检查从IL-5trg小鼠中分离的嗜酸性粒细胞如何在5-HT刺激的肺微血管内运输，并使用一种新的小鼠肺同种异体移植模型。为了进一步研究5-HT在血管内皮介导的嗜酸性粒细胞运输中的作用，我们将ova激发的5-HT2A-/-小鼠肺组织用于肺移植模型。此外，我们的研究已经描述了介导5-HT （p160 ROCK）和eotaxin （Gai）诱导趋化的信号通路的重要差异和相似之处。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介导的途径之间的分歧和收敛点。总之，我们建议详细研究参与5-HT2A介导的变应性炎症的细胞和分子机制，并假设单独或与CCR3受体联合阻断5-HT2A受体可能导致气道变应性炎症的缓解，包括嗜酸性粒细胞募集、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.