Mechanisms mediating circadian oscillations of leukocyte migration

白细胞浸润到组织中的调节是免疫调节的关键参数。 免疫反应血管疾病的昼夜节律表现，包括 缺血性血管病和镰状细胞病已经被很好地记录。 虽然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还不清楚。在 此外，涉及的细胞亚群的鉴定和 调节白细胞行为的昼夜节律振荡还不清楚。 在这一领域的更多了解将有助于解开生理和 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 骨髓（BM）通过昼夜节律调节的节奏信号提供局部 BM微环境由交感神经系统（SNS）通过？3 肾上腺素能受体（Menal-Ferrer et al. Nature，2008）。我们的初步研究 使用实时多通道荧光活体显微镜（MPEG4）， 显示白细胞-内皮细胞相互作用在小鼠的夜间增加， 导致组织中白细胞募集增强。此外，使用 化学交感神经切除术和手术去神经，我们发现， 白细胞募集的波动在受损的小鼠中被消除， SNS和依赖于BM中内皮选择素的波动。 这些研究使我们假设， 白细胞向外周组织的募集受以下因素的振荡调节： 内皮细胞粘附分子表达受SNS控制。这 假设将在三个具体目标中进行检验。在具体目标1中，我们将确定 哪些白细胞群体在BM、提睾肌、 肌肉和皮肤组织在稳态条件下使用明场和 用于实时体内评价白细胞-内皮细胞的MPEG 1技术 相互作用，与整体安装离体免疫荧光成像和流动 流式细胞术分析。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。我们将确定 前迁移分子，并评估实现这些机制 昼夜节律，侧重于SNS和肾上腺素能受体的作用 通过手术、药理学和遗传学方法。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。结果 从这个建议中产生的结果将加强我们对这一问题的基本认识， 重要的生物学现象，并可能确定新的计时 炎症性疾病的干预目标。

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.