Mechanisms mediating circadian oscillations of leukocyte migration

描述(由申请人提供)： 白细胞渗入组织的调节是免疫反应的重要参数。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，已经被很好地记录下来。尽管已有血液白细胞计数的昼夜变化的报道，但白细胞募集是否受昼夜节律的影响尚不清楚。此外，对所涉及的细胞亚群的识别和调节白细胞行为的昼夜振荡的机制尚不清楚。对这一领域的进一步了解将有助于揭示这些内源性节律在血管生物学中的生理学和病理生理学相关性。我们最近发现，通过交感神经系统(SNS)通过b3肾上腺素能受体(Mendez-Ferrer等人)在骨髓微环境局部传递节律信号，造血干细胞通过昼夜节律调节从骨髓(BM)释放出来。《自然》，2008)。我们使用实时多通道荧光活体显微镜(MFIM)进行的初步研究表明，在夜间，小鼠白细胞与内皮细胞的相互作用增加，导致白细胞在组织中的募集增加。此外，使用化学交感神经切除和外科手术去神经，我们发现在SNS受损的小鼠中，白细胞募集的波动被消除，并依赖于骨髓中内皮细胞选择素的波动。这些研究使我们假设，周围组织中白细胞募集的昼夜波动是由SNS控制的内皮细胞黏附分子表达的振荡调节的。这一假设将在三个具体目标上得到检验。在具体目标1中，我们将使用Brightfield和MFIM技术，通过整体体外免疫荧光成像和流式细胞术分析，确定在动态平衡条件下，哪些白细胞群体在骨髓、提睾肌和真皮组织中表现出昼夜波动，用于实时评估白细胞与内皮的相互作用。在特定的目标2中，我们将定义白细胞募集中昼夜节律振荡的调节机制。我们将利用外科、药理学和遗传学的方法，识别前迁移分子并评估执行这些昼夜节律的机制，重点是SNS和肾上腺素能受体的作用。在具体目标3中，我们将研究白细胞运输的昼夜节律是否影响急性和慢性炎症模型的炎症反应。这一提议产生的结果将增强我们对这一重要生物现象的基本理解，并可能确定干预炎症性疾病的新的时间治疗靶点。(摘要结束)

DESCRIPTION (provided by applicant): 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 b3 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 implementing 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. (End of Abstract)