Modulating chemokine receptors at the blood-brain barrier under flow

描述(申请人提供)：这项研究的目标是确定如何跨越血脑屏障(BBB)调节外周血单核细胞(PBMC)上趋化因子受体(PBMC)的表达，包括T细胞、B细胞和单核细胞。该研究策略利用了一种新颖而独特的体外血脑屏障模型，结合了流动条件。总体假设是，单个趋化因子受体在不同的白细胞亚群上受到不同的调节，这取决于配体的可用性、白细胞的谱系和白细胞的激活状态。这项研究的开展将从两个方面促进当前的知识：第一，我们将获得有关流动条件下趋化因子受体调节的新信息，因为PBMC与独特的脑血管内皮细胞相互作用；第二，我们将能够解释炎症的人类中枢神经系统(CNS)免疫组织化学研究揭示的组织浸润性细胞中趋化因子受体的表达。其具体目的是：1.建立鲁米那“抑制”趋化因子如何调节移行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他他珠单抗治疗的多发性硬化症患者的PBMC，在该患者中，趋化因子受体与配体的结合不受停滞和迁移的影响。2.从蛋白质和信使核糖核酸水平研究趋化因子对移行细胞趋化因子受体表达的调节作用。这一知识将确定特定的趋化因子受体作为治疗神经炎性疾病的显著治疗靶点。 与公共健康相关：血脑屏障(BBB)由专门的血管组成，这些血管直接通过大脑并为其提供营养。这些特殊的血管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，在抵御细菌和多发性硬化症(MS)等炎症性疾病的过程中，血脑屏障限制免疫细胞进入大脑。这里的“底线”是免疫细胞通过血脑屏障使用一套特殊的“规则”进入大脑。这些规则与免疫细胞表面的分子相对应，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，并在有益的情况下促进免疫细胞进入，识别这些分子是至关重要的。在本研究项目中，我们建议使用一种新开发的血脑屏障体外(组织培养)模型。该模型结合了血脑屏障的主要元素：特化的血管细胞、流动的培养液(模拟血液)和人类免疫细胞和分子。复杂的分析方法将被用来研究这些细胞，当它们进入系统的“大脑”一侧后，它们可以被恢复。我们将使用这个系统来描绘免疫细胞亚群用来穿越血脑屏障的‘规则’，我们还将测试哪些分子在穿越后仍留在细胞表面。这些结果将增加关于这一重要过程的新知识，并将有助于我们解释前几年所做的研究。随着我们朝着安全有效地阻止免疫细胞在多发性硬化症和其他疾病期间进入大脑的方向发展，这一知识是必不可少的。

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.