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

描述（由申请方提供）：化学引诱剂在嗜酸性粒细胞活化和定向迁移至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，血清素（5-羟色胺[5-HT]）可以直接诱导嗜酸性粒细胞的趋化性，但不是中性粒细胞，并在过敏性炎症小鼠模型中的嗜酸性粒细胞的招募中发挥重要作用。5-HT与嗜酸性粒细胞趋化因子在体外和体内均能单独或相加诱导嗜酸性粒细胞的趋化性。初步研究表明，嗜酸性粒细胞从过敏的捐助者，但不控制对象表达5-HT 2A受体。此外，5-HT诱导的嗜酸性粒细胞体外趋化性和过敏原诱导的嗜酸性粒细胞募集的野生型和eotaxin-/-小鼠可以抑制5-HT 2A受体拮抗剂赛庚啶和MDL-100907。基于这些发现，我们假设5-HT，通过5-HT 2A受体发挥作用，是一种新的嗜酸性粒细胞活性的化学引诱剂，在嗜酸性粒细胞的招募和过敏性炎症的整体发病机制中起着重要作用。为了评估我们的假设，在特定目标1中，我们将评估5-HT和5-HT 2A受体通路在介导过敏性炎症的各个方面中所起的作用，包括嗜酸性粒细胞募集、气道高反应性（AHR）、Th 1/Th 2细胞因子和气道重塑。我们将在野生型和T-bet-/-小鼠以及5-HT 2A-/-和CCR 3-/-小鼠的过敏性气道炎症的鼠模型中，评价在存在和不存在CCR 3拮抗剂的情况下5-HT 2A受体拮抗剂MDL-100907的作用。通过产生5-HT 2A-/-/7 CCR 3-/-双敲除小鼠，将更详细地检查5-HT 2A和CCR 3的累加效应的重要性。在具体目标2中，我们将研究5-HT 2A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），以研究如何鼠嗜酸性粒细胞分离IL-5 trg小鼠交通内5-HT刺激的肺微血管使用一种新的鼠肺同种异体移植模型。为了进一步研究5-HT对血管内皮介导的嗜酸性粒细胞运输的作用，将来自OVA攻击的5-HT 2A-/-小鼠的肺组织用于肺同种异体移植物模型。此外，我们的研究已经描绘了重要的差异和相似性的信号转导途径，介导5-HT（p160 ROCK）与嗜酸性粒细胞趋化因子（Gai）诱导的趋化性。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-HT 2A和CCR 3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-HT介导的过敏性炎症的细胞和分子机制，并假设单独阻断5-HT 2A受体或与CCR 3受体联合阻断可能导致减轻气道过敏性炎症，包括嗜酸性粒细胞募集，AHR，Th 1/Th 2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.