Influence of polysialic acid on leukocyte migration

项目总结 细胞表面蛋白质和脂质糖基化模式的变化越来越被认为是一种 控制细胞活动的重要因素。人们对如何监管这些变化越来越感兴趣 影响健康和疾病的生理过程。这项提案的目标是了解一个具体的 免疫系统细胞表面的碳水化合物修饰会影响这些细胞的功能 在炎症和感染状态下。作为细胞先天免疫和获得性免疫的关键成员 反应时，单核细胞被招募到肺部感染部位，在那里分化为巨噬细胞 和树突状细胞，然后通过淋巴系统迁移。这一有针对性的迁移和计划 单核细胞和单核细胞来源的细胞的成熟是由特定的、高度组织的细胞和 受体-配体相互作用，其中许多是由细胞表面受体和 黏附分子。聚唾液酸(PolySia)是至少三种这样的蛋白质的独特的糖链修饰， 神经细胞黏附分子(NCAM/CD56)、神经黏附蛋白-2(NRP-2)和E-选择素配体-1(ESL-1)，分别为 在单核细胞成熟的不同阶段均有表达。我们将检验这样的假设，即受调控的基因表达 在单核细胞分化为巨噬细胞和树突状细胞的过程中，以及在中性粒细胞上也有多聚体 是多唾液酸化的，有助于直接细胞归巢和肺组织中精心安排的免疫反应 感染病毒和细菌病原体。缺乏这种酶表达的小鼠 在白细胞(ST8SiaIV-/-)和载体蛋白(NCAM/CD56-/-和NRP-2-/-)中合成PolySIA 可用于体外和体内研究。我们的具体目标是：1)建立PolySia对体内的影响 单核细胞、巨噬细胞、树突状细胞和中性粒细胞在细菌性肺炎小鼠模型中的靶向免疫反应 和病毒性肺炎；ii)定义了表面多唾液酸化蛋白的机制(S) 人和小鼠单核细胞、巨噬细胞、树突状细胞和中性粒细胞控制黏附和跨 肺微血管单层；以及iii)定义和分析PolySia和 原代单核细胞分化为DC和巨噬细胞过程中的多唾液酸化蛋白。我们希望 确定PolySia促进或干扰的特定细胞-细胞和受体-配体相互作用 单核/巨噬细胞/树突状细胞成熟和迁移过程的不同阶段，以及 中性粒细胞募集。我们的研究结果将为工程水平的表达提供一个蓝图 多聚唾液酸和/或载体蛋白在髓系细胞中的表达，以便优化移行到和移出 感染/炎症，以改善整体免疫反应。

PROJECT SUMMARY Changes in the glycosylation pattern of cell surface proteins and lipids are increasingly being recognized as an important factor in controlling cellular activity. There is growing interest in how these changes can be regulated to affect physiologic processes in health and disease. The goal of this proposal is to understand how a specific carbohydrate modification on the surface of cells of the immune system influences the function of these cells during states of inflammation and infection. As a key member of cellular innate and adaptive immune responses, monocytes are recruited to pulmonary sites of infection where they differentiate into macrophages and dendritic cells before migrating through the lymphatic system. This targeted migration and programmed maturation of monocytes and monocyte-derived cells is guided by specific, highly-organized cell-cell and receptor-ligand interactions, many of which are modulated by glycosylation of cell surface receptors and adhesion molecules. Polysialic acid (polySia) is a unique glycan modification of at least three such proteins, neural cell adhesion molecule (NCAM/CD56), neuropilin-2 (NRP-2), and E-selectin ligand-1 (ESL-1), that are expressed at different stages of monocyte maturation. We will test the hypothesis that regulated expression of polySia on monocytes as they differentiate into macrophages and dendritic cells, and on neutrophils that also are polysialylated, helps direct cell homing and a well-orchestrated immune response during pulmonary infection with viral and bacterial pathogens. Mice that are deficient in expression of the enzyme that synthesizes polySia in leukocytes (ST8 SiaIV-/-) and of the carrier proteins (NCAM/CD56-/- and NRP-2-/-) will be used for in vitro and in vivo studies. Our Specific Aims will i) establish the impact in vivo of polySia on monocytes, macrophages, DC and neutrophils in the targeted immune response in a murine model of bacterial and viral pneumonia; ii) define the mechanism(s) through which polysialylated proteins on the surface of human and murine monocytes, macrophages, DC and neutrophils control adhesion to and migration across pulmonary microvascular monolayers; and iii) define and analyze the regulated expression of polySia and of polysialylated proteins during differentiation of primary monocytes into DC and macrophages. We expect to identify specific cell-cell and receptor-ligand interactions that polySia promotes or interferes with during different stages of the monocyte/macrophage/dendritic cell maturation and migratory processes, as well during neutrophil recruitment. The results from our studies will provide a blueprint to engineer levels of expression of polySia and/or carrier proteins in myeloid cells in order to optimize migration to and from sites of infection/inflammation to improve the overall immune response.