Adhesion Molecule Biology in Allergic Cells

描述（由申请方提供）：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。它们的募集、激活和释放预先形成的和新产生的介质、颗粒蛋白和细胞因子被认为是过敏性疾病（包括哮喘）的病理生理学的中心。因此，对这些反应中所涉及的机制的更好理解对于哮喘发病机制和新疗法的开发具有重要意义。在上一个资助期间，该提案的目的是进一步阐明哮喘中嗜酸性粒细胞和嗜碱性粒细胞优先募集和激活的分子机制。作为正在进行的发现新的粘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）是跨膜I型凝集素家族，其特征在于结合唾液酸的N-末端V-组IG结构域。我们现在知道Siglec-8仅在人嗜酸性粒细胞、嗜碱性粒细胞和肥大细胞上表达，使其在过敏性疾病的效应细胞上具有独特的表达模式。我们的初步数据表明Siglec-8以两种同种型存在，其中一种含有两个推定的基于细胞质酪氨酸的信号传导基序，包括ITIM序列。由于ITIM序列，我们假设Siglec-8连接将抑制嗜酸性粒细胞、嗜碱性粒细胞和肥大细胞的功能。在交联条件下将嗜酸性粒细胞与特异性Siglec-8结合单克隆抗体孵育引起快速且显著的半胱天冬酶依赖性细胞凋亡，并且该应答不能被促进存活的细胞因子IL-5拯救。事实上，IL-5增强Siglec-8交联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的促凋亡作用以及组胺释放反应的抑制。在这次更新中，我们提出了通过检查人嗜酸性粒细胞、嗜碱性粒细胞和肥大细胞的体外Siglec-8连接的抑制后果来扩展这些观察结果的实验。还将研究细胞因子引发的细胞、从晚期支气管肺泡灌洗液中分离的细胞和组织驻留细胞。将使用正常和活化的人细胞以及全长和截短的Siglec-8转染子来阐明这些应答中涉及的信号转导途径和其他机制。最后，为了为探索Siglec-8的体内生物学奠定基础，我们开始努力发现鼠Siglec-8直系同源物，并已鉴定出先导候选物。因此，提出了实验来产生和表征新试剂，以在体外和体内研究Siglec-8直系同源物对鼠嗜酸性粒细胞和肥大细胞的作用。通过Siglec-8的激活可能潜在地用于抑制体内嗜酸性粒细胞、嗜碱性粒细胞和肥大细胞功能和存活，从而提供用于减少或抑制过敏性疾病和其他疾病中的这些细胞的新策略。

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 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 cross linking 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 enhances the ability of Siglec-8 cross linking 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, cells isolated from late phase bronchoalveolar lavage fluids, and tissue resident cells 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. Finally, in order to lay the groundwork to explore the biology of Siglec-8 in vivo, we initiated efforts to discover murine Siglec-8 orthologs and have identified lead candidates. Experiments are thus proposed to generate and characterize new reagents to study Siglec-8 orthologs on murine eosinophils and mast cells in vitro and in vivo. Activation via Siglec-8 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 and other diseases.