Mechanisms mediating circadian oscillations of leukocyte migration

白细胞在组织中的渗透调节是肿瘤发生发展的重要参数。 免疫反应。血管疾病的昼夜节律表现，包括 缺血性血管病变和镰状细胞病，已经有了很好的文献记载。 尽管已经报道了血液白细胞计数的昼夜变化， 白细胞募集是否受昼夜节律的影响尚不清楚。在……里面 此外，所涉及的细胞亚集的识别以及 调节白细胞行为的昼夜节律尚不清楚。 对这一领域的更多了解将有助于解开生理学和 这些内源性节律在血管生物学中的病理生理学相关性。 我们最近发现，造血干细胞从骨骼中释放出来 通过节律信号局部传递的昼夜节律调节骨髓(BM) 交感神经系统(SNS)通过交感神经系统(SNS)影响骨髓微环境 肾上腺素能受体(Mendez-Ferrer等人)《自然》，2008)。我们的初步研究 使用实时多通道荧光活体显微镜(MFIM) 发现小鼠的白细胞和内皮细胞的相互作用在夜间增加 从而增强了组织中白细胞的募集。此外，使用 化学交感神经切除和外科去神经手术后，我们发现 受损的小鼠白细胞募集的波动被消除 并依赖于骨髓内内皮细胞选择素的变化。 这些研究让我们假设，地球的昼夜波动 白细胞向外周组织的募集受细胞内信号的振荡调节 内皮细胞黏附分子的表达受SNS调控。这 假设将在三个具体目标中得到检验。在具体目标1中，我们将确定 在BM中，哪些白细胞群体表现出昼夜节律波动 在动态平衡条件下的肌肉和真皮组织 MFIM技术在实时评价白细胞-内皮细胞中的应用 与整体安装的体外免疫荧光成像的相互作用和流动 细胞学分析。在具体目标2中，我们将定义监管机制 白细胞募集的昼夜节律波动。我们将确定 前游走分子和评估实现这些的机制 昼夜节律，关注SNS和肾上腺素能受体的作用 使用外科、药理学和遗传学方法。在具体目标3中，我们 将调查白细胞运输的昼夜节律是否会影响 急性和慢性炎症模型的炎症反应。结果是 从这一提案中产生的将加强我们对此的基本理解 重要的生物现象，可能会发现新的时间疗法 炎症性疾病干预的目标。

The regulation of leukocyte infiltration into tissues is a crucial parameter of the immune response. Circadian manifestations of vascular diseases, including ischemic vasculopathies and sickle cell disease, have been well documented. Although diurnal variations in blood leukocyte counts have been reported, whether leukocyte recruitment is influenced by circadian rhythms is unclear. In addition, the identification of the cell subsets involved and the mechanisms that regulate the circadian oscillations in leukocyte behavior is not understood. Greater understanding in this area will help to unravel the physiological and pathophysiological relevance of these endogenous rhythms in vascular biology. We have recently found that hematopoietic stem cells are released from the bone marrow (BM) through circadian regulation by rhythmic signals delivered locally in the BM microenvironment by the sympathetic nervous system (SNS) via the ¿3 adrenergic receptor (Mendez-Ferrer et al. Nature, 2008). Our preliminary studies using real-time multichannel fluorescence intravital microscopy (MFIM) have revealed that leukocyte-endothelial cell interactions are increased at night in mice resulting in enhanced leukocyte recruitment in tissues. Furthermore, using chemical sympathectomy and surgical denervation, we have found that the fluctuations of leukocyte recruitment were abolished in mice with an impaired SNS and were dependent on the fluctuations of endothelial selectins in the BM. These studies have led us to hypothesize that the circadian fluctuations in leukocyte recruitment to peripheral tissues are regulated by oscillations in endothelial cell adhesion molecule expression that is controlled by the SNS. This hypothesis will be tested in three Specific Aims. In Specific Aim 1, we will identify which leukocyte populations exhibit circadian fluctuations in the BM, cremaster muscle and dermal tissues under homeostatic conditions using brightfield and MFIM techniques for the real-time in vivo evaluation of leukocyte-endothelial interactions, with whole-mount ex vivo immunofluorescence imaging, and flow cytometry analyses. In Specific Aim 2, we will define the mechanisms regulating the circadian oscillations in leukocyte recruitment. We will identify the promigratory molecules and assess the mechanisms implementating these circadian rhythms, focusing on the role of the SNS and adrenergic receptors using surgical, pharmacological and genetic approaches. In Specific Aim 3, we will investigate whether circadian rhythms of leukocyte trafficking influence the inflammatory response in models of acute and chronic inflammation. The results generated from this proposal will enhance our basic understanding of this important biological phenomenon, and will likely identify novel chronotherapeutic targets for interventions in inflammatory diseases.