Human siglec ligands control mast cell and eosinophil mediated inflammation

炎症在一定程度上是通过具有细胞外的白细胞上的细胞表面信号分子来解决的。 与细胞内免疫抑制区域相连的配基结合域。这些人中有 在其短胞内域上具有ITIM和ITSM抑制序列的Siglec家族 当它们以高度特异性与细胞外唾液酸聚糖(Siglec配体)结合时被激活 炎性靶组织。项目3基于这样的假设，即内源性人类Siglec配体 是由呼吸道、皮肤和肠道等炎症靶组织产生的多价唾液酸聚糖。 在过敏性炎症方面，Siglec-8在人嗜酸性粒细胞上表达，而CD33(Siglec-3)， Siglec-6和Siglec-8由肥大细胞表达。当这些粒细胞上的Siglecs遇到他们的 唾液酸聚糖配体，炎症被阻止；Siglec-8与嗜酸性粒细胞结合导致其 Siglecs与肥大细胞的结合抑制了免疫介质的释放。我们建议 免疫抑制唾液酸聚糖结构的鉴定及其如何表达的知识 对健康和疾病的调控将为免疫调控的分子基础提供新的见解 也许还有治疗干预人类过敏性炎症的新靶点。 为了实现这些目标，我们提出了三个目标。目标1是基于我们发现的Siglec-8配体 在人的呼吸道上，这些配体在粘膜下腺和导管中大量表达，这些配体是 分泌到呼吸道粘液层上。这一目标将比较粘液中的Siglec-8唾液聚糖配体 从发炎和非发炎的人的呼吸道分泌物，揭示了数量和质量 (蛋白质载体，多糖结构)信息。我们对呼吸道Siglec-8配体的研究将扩展到 其他易受过敏性炎症影响的人体组织，使用健康和发炎的人体皮肤和胃肠道。 其中将比较Siglec-8配体的组织分布和分子特征 纸巾。Aim 2将探索嗜酸性粒细胞与Siglec-8最佳结合所需的糖链结构 和肥大细胞。根据我们的发现，特别是硫酸盐化和唾液酸化的角蛋白硫酸酯链是 负责Siglec-8与人呼吸道的结合，我们将使用化学酶方法产生 修饰的多聚糖结构以测试结合要求。我们将创建多价形式的这些 与人类嗜酸性粒细胞和肥大细胞上的Siglec-8发生功能性结合的配体。所获得的知识将 被用来鉴定当转染到人粘膜下腺细胞时，糖链生物合成酶 导致Siglec-8配体的表达。同时，在目标3中，我们将应用我们 建立识别Siglec-8配体，以寻找CD33和Siglec-6的内源性人类配体。 通过这些目标，我们预计将极大地提高我们对涉及 调节过敏性炎症及其在健康和疾病中的表达。

Inflammation is resolved, in part, by cell surface signaling molecules on leukocytes that have extracellular ligand binding domains linked to intracellular immune inhibitory domains. Among these are members of the siglec family that have ITIM and ITSM inhibitory sequences on their short intracellular domains that are actuated when they bind, with high specificity, to extracellular sialoglycans (siglec ligands) expressed on inflammatory target tissues. Project 3 is based on the hypothesis that endogenous human siglec ligands are multivalent sialoglycans produced by inflammatory target tissues including airway, skin and intestine. In terms of allergic inflammation, Siglec-8 is expressed on human eosinophils, whereas CD33 (Siglec-3), Siglec-6 and Siglec-8 are expressed by mast cells. When siglecs on these granulocytes encounter their sialoglycan ligands, inflammation is halted; engagement of Siglec-8 on eosinophils results in their apoptosis whereas engagement of siglecs on mast cells inhibits immune mediator release. We propose that identification of immune inhibitory sialoglycan structures and knowledge of how their expression is regulated in health and disease will provide new insights into the molecular basis of immune regulation and perhaps new targets for therapeutic intervention in human allergic inflammation. Three aims are proposed to accomplish these goals. Aim 1 is based on our finding that Siglec-8 ligands on human airway are robustly expressed in submucosal glands and ducts, and that these ligands are secreted onto the airway mucus layer. This aim will compare Siglec-8 sialoglycan ligands in mucus secretions from inflamed and non-inflamed human airways, revealing quantitative as well as qualitative (protein carrier, glycan structure) information. Our studies on airway Siglec-8 ligands will be extended to other human tissues subject to allergic inflammation, using healthy and inflamed human skin and GI tract. Tissue distributions and molecular characteristics of Siglec-8 ligands will be compared among these tissues. Aim 2 will explore the glycan structures required for optimal Siglec-8 engagement on eosinophils and mast cells. Based on our finding that specifically sulfated and sialylated keratan sulfate chains are responsible for Siglec-8 binding on human airways, we will use chemoenzymatic methods to generate modified glycan structures to test for binding requirements. We will create multivalent forms of these ligands to functionally engage Siglec-8 on human eosinophils and mast cells. The knowledge gained will be used to identify glycan biosynthetic enzymes that, when transfected into human submucosal gland cell lines, result in expression of Siglec-8 ligands. Concurrently, in Aim 3, we will apply the technologies we established to identify Siglec-8 ligands to the search for endogenous human ligands for CD33 and Siglec-6. Through these aims we anticipate significantly enhancing our understanding of glycans involved in regulating allergic inflammation and their expression in health and disease.