Glycosaminoglycans and Cell Traffic in the Lymphatic Microenvironment

描述(由申请人提供)： 在炎症反应和肿瘤形成过程中，淋巴管细胞从外周到淋巴结的运输都会发生。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)在驱动免疫细胞或肿瘤细胞各自的转运中发挥着核心作用。这项建议研究了糖胺多聚糖在调节淋巴微环境中趋化因子依赖的细胞交通中的遗传重要性。糖胺聚糖是一类重要的复杂碳水化合物，包括硫酸肝素(HS)和硫酸软骨素(CS)。这些硫酸化的糖链与独特的细胞表面结合和分泌的蛋白多糖核心蛋白相连，它们与几种趋化因子的富含氨基酸的碱性结构域相互作用。本文的重点是淋巴管内皮细胞产生的糖胺多聚糖，因为初步的工作表明，淋巴管HS(可能还有CS)介导两个重要的趋化因子功能：(1)主要淋巴管趋化因子(如CCL21和CXCL12)在淋巴管周围空间梯度中的支架，似乎由多糖保持；(2)趋化因子独特地呈现给运输细胞上的同源受体，其中分泌的淋巴糖胺多聚糖可能作为趋化因子辅助受体。这项工作将集中在肿瘤的背景下，淋巴趋化因子依赖的运输既涉及癌细胞，也涉及免疫细胞(这里的重点是树突状细胞的迁移)。这一建议解决了这样的假设，即淋巴糖胺聚糖生物合成的遗传干扰将改变肿瘤以及树突状细胞以趋化因子依赖的方式向淋巴管系统迁移和向淋巴结运输的能力。我们的目标是：(1)鉴定淋巴管内皮细胞产生的蛋白多糖核心蛋白，并表征糖胺多糖链在建立淋巴管趋化因子梯度中的作用。一旦确定了连接淋巴管内皮细胞HS和CS链的蛋白多糖核心蛋白，将对这些糖链进行纯化，并测试它们与主要淋巴管内皮细胞趋化因子的结合能力。在基于淋巴管内皮细胞和基质的检测中，将检测在静止和流动条件下，基因改变糖胺多聚糖生物合成对趋化因子梯度形成的影响。(2)评估淋巴糖胺聚糖在肿瘤和树突状细胞趋化因子依赖的迁移中的遗传学重要性。淋巴糖胺多糖的生物合成将在小鼠Cre-loxP模型和siRNA改变的人淋巴管内皮细胞中进行靶向，并将确定肿瘤和树突状细胞向目标内皮细胞迁移的能力。此外，还将检测从突变细胞中获得的条件培养液寡聚(集群)淋巴趋化因子以及支持趋化因子介导的肿瘤激活和树突状细胞迁移信号通路的能力。(3)在以淋巴微环境中的糖胺聚糖精细结构为目标的遗传改变的背景下，表征实时细胞运输的特征。肿瘤和树突状细胞从外周淋巴管到区域淋巴结的运输将在携带淋巴糖胺聚糖生物合成基因缺陷的小鼠中进行检测。在基因靶向小鼠的不同癌症模型中，也将检查通过运输肿瘤和树突状细胞进行的淋巴定植。其他机制研究将检查趋化因子在体内的分布和受体结合。这项工作可能揭示一种新的机制范式，即多种趋化因子的分布和作用如何由淋巴微环境中的多糖控制。这也可能为新的治疗药物的发现奠定合理的基础。 公共卫生相关性： 趋化因子是“感应”分子，驱动特定细胞向趋化因子来源迁移。在通常困扰退伍军人的炎症或癌症等疾病状态下，由血管或淋巴管产生的趋化因子可能会驱动炎症或肿瘤细胞的转移，从而产生有害后果。肺癌是退伍军人痛苦和死亡的主要原因，其后果可能包括癌症扩散到淋巴结，以及由趋化因子驱动的特化(树突状)免疫细胞向淋巴结移动，这反过来可以促进对肿瘤的免疫“耐受性”。初步工作表明，一类复杂的糖分子(糖胺多聚糖)可能是这种淋巴趋化因子的基础或中介作用。在这里，我们使用最先进的基因工具阻止淋巴管细胞中这种复合糖的产生，并检测其对树突状细胞迁移和淋巴结转移的影响。这项工作可能导致新的治疗方法，以抑制癌症的扩散，发病率和死亡率在我们的退伍军人群体中。

DESCRIPTION (provided by applicant): Lymphatic vascular cell traffic from the periphery to lymph nodes occurs during both inflammatory responses as well as neoplasia. In both cases, unique chemokines (chemotactic cytokines) produced by the lymphatic endothelium play central roles in driving the respective transit of either immune cells or tumor cells. This proposal examines the genetic importance of glycosaminoglycans, an important class of complex carbohydrates that includes heparan sulfate (HS) and chondroitin sulfate (CS), in mediating chemokine- dependent cell traffic in the lymphatic microenvironment. These sulfated glycan chains are tethered to unique cell-surface bound as well as secreted proteoglycan core proteins, and they interact with basic amino acid-rich domains of several chemokines. The specific focus herein is on glycosaminoglycans produced by the lymphatic endothelium, as a preliminary body of work suggests that lymphatic HS (and possibly CS) mediate two critical chemokine functions: (i) the scaffolding of major lymphatic chemokines (such as CCL21 and CXCL12) in peri-lymphatic spatial gradients that appear to be held by the glycans, and (2) the unique presentation of chemokines to their cognate receptors on trafficking cells, wherein secreted lymphatic glycosaminoglycans may serve as chemokine co-receptors. This work will focus on the setting of neoplasia, where lymphatic chemokine-dependent trafficking involves both carcinoma cells as well as immune cells (with focus herein on dendritic cell migration). This proposal addresses the hypothesis that genetic disruption of lymphatic glycosaminoglycan biosynthesis will alter the ability of tumor as well as dendritic cells to migrate toward lymphatic vasculature and traffic to lymph nodes in a chemokine-dependent manner. The goals are to: (1) Identify proteoglycan core proteins produced by the lymphatic endothelium, and characterize the roles of glycosaminoglycan chains in establishing lymphatic chemokine gradients. Upon identifying the repertoire of proteoglycan core proteins that tether lymphatic endothelial HS and CS chains, the glycan chains will be purified and tested for their ability to bind to major lymphatic endothelial chemokines. In lymphatic endothelial cell- and matrix-based assays, the effects of genetically altering glycosaminoglycan biosynthesis on chemokine gradient formation in static as well as flow conditions will be examined. (2) Assess the genetic importance of lymphatic glycosaminoglycans in chemokine-dependent migration of tumor and dendritic cells. Lymphatic glycosaminoglycan biosynthesis will be targeted in murine Cre-LoxP models and in siRNA-altered human lymphatic endothelium, and the ability of tumor and dendritic cells to migrate toward the targeted endothelium will be determined. In addition, the ability of conditioned medium harvested from the mutant cells to oligomerize (cluster) lymphatic chemokines as well as support chemokine-mediated activation of tumor and dendritic-cell migration signaling pathways will be examined. (3) Characterize real-time cell trafficking in the setting of genetic alterations that target glycosaminoglycan fine structure in the lymphatic microenvironment in vivo. Trafficking of tumor and dendritic cells from peripheral lymphatic vessels to regional lymph nodes will be examined in mice bearing gene defects in lymphatic glycosaminoglycan biosynthesis. In separate carcinoma models in the gene-targeted mice, lymph node colonization by trafficking tumor and dendritic cells will also be examined. Additional mechanistic studies will examine chemokine distribution and receptor binding in vivo. This work may uncover a novel mechanistic paradigm for how the distribution and actions of multiple chemokines may be controlled by glycans in the lymphatic microenvironment. It may also establish a rational basis for novel therapeutic drug discovery. PUBLIC HEALTH RELEVANCE: Chemokines are "sensing" molecules that drive migration of specific cells toward a chemokine source. In states of disease such as inflammation or cancer that commonly afflict the Veteran population, chemokines produced by blood or lymph vessels may drive the transit of inflammatory or tumor cells with deleterious consequences. In the case of lung cancer, a leading cause of Veteran suffering and death, the consequences may include the spread of cancer to lymph nodes as well as chemokine-driven movement of specialized ("dendritic") immune cells to the nodes, which in turn can promote immune "tolerance" to the tumor. Preliminary work shows that a class of complex sugar molecules (glycosaminoglycans) may underlie or mediate the actions of such lymphatic chemokines. Herein, we block the production of such complex sugars in lymphatic vessel cells using state-of- the-art genetic tools, and examine the effects on dendritic cell migration and lymph node metastasis. This work may lead to novel therapy to inhibit the spread, morbidity, and mortality of cancer in our Veteran population.