Lymphatic Microenvironment: Altering Cell Traffic by Targeting Glycans

描述(申请人提供)：淋巴管细胞从外周到淋巴结的运输发生在炎症反应和肿瘤形成过程中。在这两种情况下，淋巴管内皮细胞产生的独特的趋化因子(趋化细胞因子)在驱动先天免疫细胞(如树突状细胞)或肿瘤细胞的转运中发挥核心作用。这项建议检查了独特的复杂碳水化合物(多糖)在调节淋巴微环境中依赖趋化因子的细胞交通中的遗传重要性。我们的重点是硫酸乙酰肝素(HS)，它由与独特的细胞表面结合的硫酸多糖以及分泌的蛋白多糖核心蛋白组成。HS与几种趋化因子的碱性氨基酸富集区相互作用。本文的重点是淋巴管内皮细胞产生的HS，因为初步的工作表明淋巴管HS可能介导两个重要的趋化因子功能：(1)主要的淋巴管趋化因子(如CCL21和CXCL12)在淋巴管周围的空间梯度上的支架；(2)趋化因子的独特聚集呈现给运输细胞上的同源受体，其中分泌的淋巴管HS蛋白多糖可能作为趋化因子的辅受体。这一建议解决了以淋巴多糖生物合成为靶点的假设，即以趋化因子依赖的方式改变树突状细胞或肿瘤细胞向淋巴管迁移和向淋巴结运输的能力。我们的目标是：(1)检测基因改变的淋巴管HS对体内淋巴管微环境中细胞运输的影响。树突状细胞和肿瘤细胞从外周组织到区域淋巴结的迁移将在携带淋巴HS生物合成基因缺陷的小鼠中进行检测。还将研究抗原驱动的贩运研究中的T细胞反应。突变体的特征是淋巴HS结构和蛋白多糖的表达。(2)确定HS在建立淋巴趋化因子梯度中的作用。HS链将被提纯并测试其结合主要淋巴趋化因子的能力。在基于淋巴管内皮细胞基质的检测中，我们将研究在静止和流动条件下，HS生物合成基因改变对趋化因子梯度形成的影响。突变状态下的趋化因子分布也将在体内进行检测。(3)表征淋巴管HS作为趋化运动依赖性细胞迁移和信号转导的辅助受体的重要性。将测定携带HS生物合成突变的树突状细胞向淋巴管内皮细胞迁移的能力，并将测试来自突变的淋巴管内皮细胞的条件培养液对淋巴管趋化因子的寡聚化(集群)和激活树突状细胞中依赖于趋化因子的迁移信号的能力。最后，我们将在体内检测淋巴管HS突变对淋巴管趋化因子和同源受体在运输细胞上的关联的影响。总而言之，这项工作可能会揭示多种趋化因子的作用如何被淋巴微环境中的多糖控制的新机制。这也可能为新的治疗发现奠定基础。

DESCRIPTION (provided by applicant): Lymphatic vascular cell traffic from the periphery to lymph nodes occurs during inflammatory responses as well as neoplasia. In both cases, unique chemokines (chemotactic cytokines) produced by the lymphatic endothelium play central roles in driving the transit of either innate immune cells, such as dendritic cells, or tumor cells. This proposal examines the genetic importance of unique complex carbohydrates (glycans) in mediating chemokine-dependent cell traffic in the lymphatic microenvironment. We focus on heparan sulfate (HS), composed of sulfated glycans tethered to unique cell-surface bound as well as secreted proteoglycan core proteins. HS interacts with basic amino acid-rich domains of several chemokines. The focus herein is on HS produced by the lymphatic endothelium, as preliminary work suggests that lymphatic HS may mediate two critical chemokine functions: (1) scaffolding of major lymphatic chemokines (such as CCL21 and CXCL12) onto peri-lymphatic spatial gradients, and (2) unique clustered presentation of chemokines to cognate receptors on trafficking cells, wherein secreted lymphatic HS proteoglycans may serve as chemokine co-receptors. This proposal addresses the hypothesis that targeting lymphatic glycan biosynthesis will alter the ability of dendritic cells or tumor cells to migrate toward lymphatic vessels and traffic to lymph nodes in a chemokine-dependent manner. The goals are to: (1) Examine the effects of genetically altering lymphatic HS on cell trafficking in the lymphatic microenvironment in vivo. Migration of dendritic cells and tumor cells from peripheral tissue to regional lymph nodes will be examined in mice bearing gene defects in lymphatic HS biosynthesis. T cell responses in antigen-driven trafficking studies will also be examined. Mutants will be characterized for lymphatic HS structure and proteoglycan expression. (2) Determine the role of HS in establishing lymphatic chemokine gradients. HS chains will be purified and tested for their ability to bind major lymphatic chemokines. In lymphatic endothelial matrix-based assays, the effects of genetically altering HS biosynthesis on chemokine gradient formation in static and flow conditions will be examined. Chemokine distribution in the mutant state will also be examined in vivo. (3) Characterize the importance of lymphatic HS as a co-receptor for chemokinedependent cell migration and signaling. The ability of dendritic cells to migrate toward lymphatic endothelium bearing mutations in HS biosynthesis will be determined, and conditioned medium from mutant lymphatic endothelia will be tested for its ability to oligomerize (cluster) lymphatic chemokines and activate chemokine-dependent migration signaling in dendritic cells. Finally, the effect of lymphatic HS mutation on the association between lymphatic chemokines and cognate receptors on trafficking cells will be examined in vivo. Collectively, this work may uncover novel mechanisms for how the actions of multiple chemokines may be controlled by glycans in the lymphatic microenvironment. It may also establish a basis for novel therapeutic discovery.