Modulating chemokine receptors at the blood-brain barrier under flow

描述（由申请人提供）：本研究计划的目标是确定穿越血脑屏障（BBB）如何调节外周血单核细胞（PBMC）（包括 T 细胞、B 细胞和单核细胞）上的趋化因子受体表达。该研究策略利用了一种新颖且独特的体外 BBB 模型，并结合了流动条件。总体假设是，不同的白细胞亚群上的个体趋化因子受体受到不同的调节，这取决于配体的可用性。白细胞谱系；和白细胞激活状态。这项研究的进行将从两个方面推进当前的知识：首先，我们将获得有关在流动条件下当 PBMC 与独特的脑血管内皮相互作用时趋化因子受体调节的新信息；其次，我们将能够解释组织浸润细胞的趋化因子受体表达，正如对发炎的人类中枢神经系统（CNS）的免疫组织化学研究所揭示的那样。具体目标是： 1. 确定管腔“逮捕”趋化因子如何调节迁移细胞上趋化因子受体的表达。这些研究将纳入来自健康志愿者和接受那他珠单抗的 MS 患者的 PBMC，其中趋化因子受体与配体的结合与停滞和迁移无关。 2. 确定腔外“迁移”趋化因子如何在蛋白质和 mRNA 水平上调节迁移细胞上趋化因子受体的表达。这些知识将确定特定的趋化因子受体作为治疗神经炎症疾病的显着治疗靶点。 公共健康相关性：血脑屏障 (BBB) 由直接穿过大脑并为大脑提供营养的特殊血管组成。这些专用容器限制有毒物质的进入并促进葡萄糖等有益成分的进入。此外，在防御细菌过程中以及在多发性硬化症 (MS) 等炎症性疾病的情况下，血脑屏障都会限制免疫细胞进入大脑。这里的“底线”是免疫细胞使用一套特殊的“规则”穿过血脑屏障进入大脑。这些“规则”对应于免疫细胞表面的分子，如果我们要开发安全有效的方法来减少有害的免疫细胞进入并在有益时促进免疫细胞进入，那么识别这些分子至关重要。在这个研究项目中，我们建议利用新开发的 BBB 体外（组织培养）模型。该模型融合了 BBB 的主要元素：特化血管细胞、流动培养基（模拟血液）以及人体免疫细胞和分子。复杂的分析方法将用于研究这些细胞，这些细胞在进入系统的“大脑”一侧后可以被恢复。我们将使用该系统来描绘免疫细胞亚群穿过血脑屏障的“规则”，并且我们还将测试哪些分子在穿过后仍保留在细胞表面上。这些结果将为这一重要过程增加新的知识，也将帮助我们解释前几年所做的研究。当我们在多发性硬化症和其他疾病期间安全有效地阻止免疫细胞进入大脑时，这些知识至关重要。

DESCRIPTION (provided by applicant): The goal of this research proposal is to establish how crossing the blood-brain barrier (BBB) regulates chemokine receptor expression on peripheral blood mononuclear cells (PBMC) including T cells, B cells and monocytes. The research strategy utilizes a novel and unique model of the BBB in vitro, incorporating flow conditions. The overall hypothesis is that individual chemokine receptors are regulated differentially on different subsets of leukocytes, contingent on ligand availability; leukocyte lineage; and leukocyte activation state. Performance of this research will advance current knowledge in two ways: first, we will gain new information about chemokine receptor modulation under flow conditions as PBMC interact with the unique cerebrovascular endothelium; second we will be enabled to interpret chemokine receptor expression by tissue-infiltrating cells as revealed by immunohistochemistry studies of the inflamed human central nervous system (CNS). The Specific Aims are: 1. To establish how luminal 'arrest' chemokines modulate chemokine receptor expression on transmigrated cells. These studies will incorporate both PBMC from healthy volunteers and from MS patients receiving natalizumab, in whom chemokine receptor engagement with ligand is uncoupled from arrest and transmigration. 2. To determine how abluminal 'transmigration' chemokines regulate chemokine receptor expression on transmigrated cells both at the protein and mRNA levels. This knowledge will identify specific chemokine receptors as salient therapeutic targets to treat neuroinflammatory diseases. PUBLIC HEALTH RELEVANCE: The blood-brain barrier (BBB) consists of specialized vessels which course directly through, and nourish, the brain. These specialized vessels limit the entry of noxious substances and promote access for beneficial components such as glucose. In addition, the BBB restricts entry of immune cells into the brain both during defense against germs and in the setting of inflammatory diseases such as multiple sclerosis (MS). The 'bottom line' here is that immune cells use a special set of 'rules' to enter the brain across the BBB. These 'rules' correspond to molecules on the immune cell surface and identifying these molecules is critical if we are to develop safe, effective ways to reduce harmful immune cell entry and promote immune-cell entry when it's beneficial. In this research project, we propose to utilize a newly-developed in-vitro (tissue culture) model of the BBB. This model incorporates the major elements of the BBB: specialized blood vessel cells, flowing culture medium (mimicking blood) and human immune cells and molecules. Sophisticated analytic methods will be used to study the cells, which can be recovered after they've crossed into the 'brain' side of the system. We will use this system to delineate the 'rules' which subsets of immune cells use to cross the BBB, and we will also test which molecules remain on the cell surface after crossing. These results will add new knowledge about this important process, and will also help us to interpret studies done in previous years. This knowledge is essential as we move towards safely and effectively blocking immune-cell movement into the brain during MS and other diseases.