Adhesion Molecule Biology in Allergic Cells

描述(申请人提供)：嗜酸性粒细胞、嗜碱性粒细胞和肥大细胞被认为是过敏反应中的关键效应细胞。它们对预先形成的和新产生的介质、颗粒蛋白和细胞因子的募集、激活和释放被认为是包括哮喘在内的过敏性疾病的病理生理学的核心。因此，更好地了解这些反应所涉及的机制对哮喘的发病机制和新疗法的开发具有重要意义。在上一次资助期间，这项建议的目的是进一步阐明哮喘患者嗜酸性粒细胞和嗜碱性粒细胞优先招募和激活的分子机制。作为正在进行的发现新的黏附分子的努力的一部分，我们最近从人嗜酸性粒细胞cDNA文库中克隆了Siglec-8。Siglecs(唾液酸结合Ig样凝集素)是一个跨膜的I型凝集素家族，其特征是N-末端的V-Set Ig结构域与唾液酸结合。我们现在知道Siglec-8只在人的嗜酸性粒细胞、嗜碱性粒细胞和肥大细胞上表达，这使得它在变态反应性疾病的效应细胞上有独特的表达模式。我们的初步数据表明，Siglec-8以两种异构体存在，其中一种包含两个假定的基于细胞质酪氨酸的信号基序，包括一个ITIM序列。由于ITIM序列，我们假设Siglec-8连接会抑制嗜酸性粒细胞、嗜碱性粒细胞和肥大细胞的功能。在交联条件下，嗜酸性粒细胞与特定的Siglec-8结合单克隆体孵育可引起caspase依赖的快速而深刻的细胞凋亡，这种反应不能被促进生存的细胞因子IL-5拯救。事实上，IL-5似乎增强了Siglec-8交联物诱导嗜酸性粒细胞凋亡的能力。对肥大细胞和嗜碱性粒细胞的平行研究也表明，抗Siglec-8具有促凋亡作用，并抑制组胺释放反应。在这次更新中，我们建议通过在体外检测人类嗜酸性粒细胞、嗜碱性粒细胞和肥大细胞对Siglec-8结扎的抑制后果来扩大这些观察结果。细胞因子启动的细胞和从晚期支气管肺泡灌洗液中分离出来的细胞也将被研究。这些反应中涉及的信号转导途径和其他机制将使用正常和激活的人类细胞，以及全长和截短的Siglec-8转染体来阐明。用Siglec-8Ig融合蛋白探测细胞和组织的初步结果已经检测到人类呼吸道上皮细胞上的潜在配体。这些研究将扩大到表征这些配体。最后，为了在体内为探索Siglec-8的生物学奠定基础，我们开始了寻找小鼠Siglec-8同源基因的努力，并确定了几个候选基因。因此，建议进行实验，以产生和表征新的试剂，以在体外和体内研究小鼠嗜酸性粒细胞和肥大细胞上的Siglec-8同系物。Siglec-8被其天然配体或药物激动剂激活，可能被用来抑制嗜酸性粒细胞、嗜碱性粒细胞和肥大细胞的功能和体内存活，为减少或抑制过敏性疾病中的这些细胞提供了一种新的策略。

DESCRIPTION (provided by applicant): Eosinophils, basophils and mast cells are considered key effector cells in allergy. Their recruitment, activation, and release of preformed and newly generated mediators, granule proteins, and cytokines are regarded as central to the pathophysiology of allergic diseases, including asthma. An improved understanding of the mechanisms involved in these responses is therefore of great relevance to asthma pathogenesis and the development of new therapeutics. The objective of this proposal during the last funding period was to further elucidate the molecular mechanisms for preferential recruitment and activation of eosinophils and basophils in asthma. As part of ongoing efforts to discover novel adhesion molecules, we recently cloned Siglec-8 from a human eosinophil cDNA library. Siglecs (sialic acid binding Ig-like lectins) are a family of transmembrane, I-type lectins characterized by an N-terminal V-set Ig domain that binds sialic acid. We now know that Siglec-8 is expressed only on human eosinophils, basophils and mast cells, giving it a unique expression pattern on effector cells of allergic disease. Our preliminary data suggests that Siglec-8 exists in two isoforms, one of which contains two putative cytoplasmic tyrosine-based signaling motifs, including an ITIM sequence. Because of the ITIM sequence, we hypothesized that Siglec-8 ligation would inhibit functions of eosinophils, basophils, and mast cells. Incubation of eosinophils with specific Siglec-8 binding monoclonals under crosslinking conditions caused rapid and profound caspase-dependent apoptosis, and this response could not be rescued by the survival-promoting cytokine IL-5. In fact, IL-5 appears to enhance the ability of Siglec-8 crosslinking to induce eosinophil apoptosis. Parallel studies with mast cells and basophils also suggest a pro-apoptotic effect of anti-Siglec-8, as well as inhibition of histamine release responses. In this renewal we propose experiments to expand these observations by examining human eosinophils, basophils, and mast cells for inhibitory consequences of Siglec-8 ligation in vitro. Cytokine-primed cells and cells isolated from late phase bronchoalveolar lavage fluids will also be studied. Signal transduction pathways and other mechanisms involved in these responses will be elucidated using normal and activated human cells, as well as full-length and truncated Siglec-8 transfectants. Preliminary results probing cells and tissues with Siglec-8 Ig fusion proteins have detected potential ligands on human respiratory epithelial cells. These studies will be expanded to characterize these ligands. Finally, in order to lay the groundwork to explore the biology of Siglec-8 in vivo, we initiated efforts to discover murine Siglec-8 homologs and have identified several candidates. Experiments are thus proposed to generate and characterize new reagents to study Siglec-8 homologs on murine eosinophils and mast cells in vitro and in vivo. Activation via Siglec-8 by its natural ligands or pharmacologic agonists could potentially be used to inhibit eosinophil, basophil and mast cell function and survival in vivo, providing a novel strategy for reducing or inhibiting these cells in allergic diseases.