Using Microfluidics to Investigate Mechanisms of Leukocyte Intravascular Crawling

利用微流控技术研究白细胞血管内蠕动的机制

• 批准号: 9259097
• 负责人: Bryan Lauck Benson
• 金额: $ 3.72万
• 依托单位: CASE WESTERN RESERVE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2016
• 资助国家: 美国
• 起止时间: 2016-12-05 至 2018-12-04
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9259097
• 关键词: Address; Adhesions; Adverse effects; Affect; Affinity; Alzheimer' s Disease; American; Antibodies; Apical; Autoimmune Diseases; Behavior; Binding; Biological Assay; Blood - brain barrier anatomy; Blood Vessels; Blood flow; Brain; Cell Culture System; Cell Culture Techniques; Cells; Chelating Agents; Data; Detergents; Devices; Diffuse; Dimensions; Disease; Dissection; Drug usage; Endothelial Cells; Endothelium; Event; Exhibits; Future; Glycosaminoglycans; Golgi Apparatus; Human; Immobilization; Immune; In Vitro; Individual; Infiltration; Inflammation; Inflammatory; Injury; Intercellular Junctions; Knowledge; Lead; Leukocytes; Ligation; Light; Mediating; Microfluidic Microchips; Microfluidics; Microscopy; Modeling; Multiple Sclerosis; Neuraxis; Neurodegenerative Disorders; Pathway interactions; Patients; Pattern; Peripheral Blood Mononuclear Cell; Pertussis Toxin; Pharmaceutical Preparations; Play; Process; Production; Receptor Signaling; Resolution; Role; Side; Signal Transduction; Signaling Molecule; Site; Small Interfering RNA; Stimulus; Stroke; Structure; Surface; System; Temperature; Testing; Time; Tissues; Vascular Dementia; Vesicle; Video Microscopy; brain endothelial cell; brain parenchyma; chemokine; chemokine receptor; cytokine; design; effective therapy; experimental study; fluid flow; genetic manipulation; human imaging; in vitro Model; in vivo; migration; multiple sclerosis treatment; nervous system disorder; neutrophil; new therapeutic target; novel therapeutics; peripheral blood; postcapillary venule; prevent; response; small molecule; spatiotemporal; temporal measurement; trafficking; two-dimensional; white matter

Abstract Multiple sclerosis (MS) is an autoimmune disease of the central nervous system (CNS) that affects 400 thousand Americans. In MS attacks, peripheral blood leukocytes gain access to the brain parenchyma and attack white matter structures. Effective therapies for MS target the steps by which these leukocytes gain access to the brain, called the leukocyte adhesion cascade. These therapies, while effective, can cause rare infectious complications and exhibit variability in response between individuals. A more complete understanding of the leukocyte adhesion cascade may lead to therapies that are safer and effectively treat disease in patients that do not respond to current treatment. Crawling of leukocytes within the blood vessels towards sites of inflammation is an important part of the leukocyte adhesion cascade and is the least understood of its steps. Chemokines may guide the directed intravascular crawling of leukocytes, but proof and mechanistic detail are lacking. Most current knowledge about crawling comes from in vivo studies which are limited in scope due to low throughput. An in vitro model to enable the dissection of this process in fine detail and with high throughput would advance the field. To address this, I designed a microfluidic device that uses principles of laminar flow to allow careful studies of the mechanisms underlying directed leukocyte crawling for the first time in vitro. This device allows high resolution live video imaging of human peripheral blood mononuclear cells (PBMC) and their adhesion cascade interactions with human brain microvascular endothelial cells (hBMEC). Production of this device and cell culture within it are now routine, allowing us to pursue the objective of establishing whether endothelial cells guide leukocyte intravascular crawling via chemokines. The project has two specific aims. Aim 1 will answer whether intraluminal chemokine gradients can plausibly direct intravascular crawling by testing whether an acellular chemokine gradient is sufficient to induce directed crawling of leukocytes. This will be extended by quantifying leukocyte crawling behavior on a directionally-stimulated endothelium in the device. Finally, the contribution of chemokine receptors to these phenomena will be assessed, using drugs to block downstream signaling and antibodies to block ligation. Aim 2 seeks to shed more light on an alternate pathway that has been proposed: direct signaling of endothelial cells to leukocytes via Golgi-derived vesicles laden with chemokine. To do so, we will use lattice light sheet microscopy (LLSM) to quantify chemokine-laden vesicle trafficking with a high degree of spatiotemporal resolution. We will use this information to test whether the trafficking of these vesicles is influenced by fluid flow, proximity to endothelial-endothelial boundaries, and the presence of adherent leukocytes. The proposed experiments will significantly advance our understanding of leukocyte intravascular crawling, as well as provide refined models for future studies of this phenomenon, which has implications for cell-mediated autoimmune diseases such as MS.

摘要 多发性硬化症（MS）是中枢神经系统（CNS）的自身免疫性疾病， 影响了40万美国人在多发性硬化症发作时，外周血白细胞进入大脑 薄壁组织和攻击白色物质结构。MS的有效疗法针对以下步骤： 这些白细胞进入大脑，称为白细胞粘附级联。这些疗法， 虽然有效，但可引起罕见的感染性并发症， 个体对白细胞粘附级联反应的更全面的了解可能会导致治疗 更安全有效地治疗对当前治疗无反应的患者的疾病。 白细胞在血管内向炎症部位爬行是一种重要的免疫调节机制。 白细胞粘附级联的一部分，是其步骤中了解最少的。趋化因子可以引导 白细胞在血管内定向爬行，但缺乏证据和机制细节。最 目前关于爬行的知识来自于体内研究，由于低的生物活性， 吞吐量一个体外模型，使解剖这一过程的细节和高 吞吐量将推动该领域的发展。 为了解决这个问题，我设计了一个微流体装置，它使用层流原理， 首次在体外对定向白细胞爬行的机制进行了仔细研究。这 设备允许高分辨率的人外周血单核细胞（PBMC）的实时视频成像 以及它们与人脑微血管内皮细胞（hBMEC）的粘附级联相互作用。 这种设备的生产和细胞培养现在已经成为常规，使我们能够实现目标 内皮细胞是否通过趋化因子引导白细胞在血管内爬行。 该项目有两个具体目标。目的1将回答是否管腔内趋化因子梯度 可以通过检测无细胞趋化因子梯度是否 足以诱导白细胞的定向爬行。这将通过定量白细胞来扩展 装置中定向刺激的内皮上的爬行行为。最后，贡献 这些现象的趋化因子受体将被评估，使用药物阻断下游信号传导 和阻断连接的抗体。 目标2旨在进一步阐明已提出的替代途径：直接 内皮细胞通过高尔基体衍生的载有趋化因子的囊泡向白细胞发出信号。为此， 我们将使用点阵光片显微镜（LLSM）来量化载有趋化因子的囊泡运输， 高时空分辨率。我们将使用这些信息来测试是否贩运 这些囊泡受到流体流动、内皮-内皮边界的接近性以及 存在粘附的白细胞。 这些实验将极大地促进我们对白细胞的理解 血管内爬行，并为未来对这种现象的研究提供精确的模型， 对细胞介导的自身免疫性疾病如多发性硬化症有影响。