Mechanisms mediating circadian oscillations of leukocyte migration

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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.