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. ? ?
