Mechanisms mediating circadian oscillations of leukocyte migration

描述（由申请人提供）： 白细胞浸润到组织中的调节是免疫应答的关键参数。血管疾病（包括缺血性血管病和镰状细胞病）的昼夜节律表现已得到充分记录。虽然已经报道了血液白细胞计数的昼夜变化，但白细胞募集是否受昼夜节律的影响尚不清楚。此外，涉及的细胞亚群的识别和调节白细胞行为的昼夜节律振荡的机制尚不清楚。在这一领域的更多的了解将有助于解开这些内源性节律在血管生物学的生理和病理生理相关性。我们最近发现，通过由交感神经系统（SNS）经由b3肾上腺素能受体在BM微环境中局部递送的节律信号的昼夜节律调节，造血干细胞从骨髓（BM）中释放（Menvan-Ferrer等人，Nature，2008）。我们的初步研究使用实时多通道荧光活体显微镜（MECHANISM）显示，白细胞-内皮细胞的相互作用增加，在夜间在小鼠组织中导致增强白细胞募集。此外，使用化学交感神经切除术和手术去神经，我们已经发现，白细胞募集的波动被废除在小鼠与受损的SNS和依赖于BM中的内皮选择素的波动。这些研究使我们推测，外周组织白细胞募集的昼夜节律波动是由SNS控制的内皮细胞粘附分子表达的振荡调节的。这一假设将在三个具体目标中得到检验。在具体目标1中，我们将确定哪些白细胞群体在稳态条件下在BM、提睾肌和真皮组织中表现出昼夜节律波动，使用明场和MPEG4技术，通过整体离体免疫荧光成像和流式细胞术分析，对白细胞-内皮相互作用进行实时体内评价。在具体目标2中，我们将定义调节白细胞募集的昼夜节律振荡的机制。我们将确定促迁移分子，并评估实现这些昼夜节律的机制，重点是SNS和肾上腺素能受体的作用，使用手术，药理学和遗传学的方法。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中的炎症反应。从这一提议中产生的结果将增强我们对这一重要生物学现象的基本理解，并可能为炎症性疾病的干预确定新的时间靶点。(End摘要）

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)