Circadian Expression of Endothelial Selectins, Regulated by the Sympathetic Nervous System, Controls Peripheral Leukocyte Homeostasis

Abstract

We have recently shown that hematopoietic stem cells (HSCs) are released into peripheral blood in a circadian manner, controlled by the sympathetic nervous system (SNS) through the regulation of CXCL12 levels in the bone marrow (BM) (Mendez-Ferrer et. al. Nature 2008; 452:442). In addition to the oscillations in circulating HSC numbers, we have also observed circadian fluctuations in the numbers of blood leukocytes in mice maintained on a standard 12h light - 12h dark cycle. Systemic leukocyte counts (4129 ± 521/μl (12 pm) vs 2000 ± 233/μl (8 pm); p=0.003) and their subsets, neutrophils (2762 ± 118 vs 732 ± 45/μl; p=0.02) and lymphocytes (5997 ± 151 vs 1867 ± 661/μl; p=0.03), were significantly reduced at night compared to the morning. We hypothesized that peripheral leukocyte counts are regulated by their interactions with endothelial cells in the BM microcirculation. To test this hypothesis we analyzed the constitutive interactions of leukocytes with BM microvessels using intravital microscopy to evaluate whether the trafficking of leukocytes in the BM microvasculature is also subjected to circadian regulation. We found that the absolute number of rolling leukocytes (18.0 ± 0.2 (12 pm) vs 31.1 ± 3.5 (8 pm) x103/mm2;p=0.004) and the rolling flux fraction (RFF, i.e. rolling/systemic leukocyte counts)(6.5 ± 0.6 vs 20.7 ± 3.1 %; p=0.0001) were increased three-fold at night, inversely correlating with the number of circulating leukocytes. Adoptive transfer experiments of fluorescently labeled BM leukocytes also revealed a two-fold increase in the number of recruited adherent leukocytes at night compared to when cells were injected in the morning (0.97 ± 0.17 (12 pm) vs 2.54 ± 0.53 (8 pm)/vessel area (μm2);p=0.007). Flow cytometry analyses revealed that the majority of these recruited cells were Mac-1+/Gr-1+ myeloid cells. This suggests that the capacity of the BM to recruit leukocytes fluctuates over the course of a day. Moreover, adoptively transferred cells formed clusters at specific sites in the BM at night. To investigate the mechanisms, we first subjected mice lacking both endothelial selectins (P-and E-selectins) to the same experimental protocols. Double deficient mice did not exhibit circadian variations in PB (9725 ± 1185/μl (12 pm) vs 8271 ± 1394/μl (8 pm); p=0.50) or BM (RFF 0.28 ± 0.05 vs 0.34 ± 0.05 %; p=0.48), and did not show clusters of adoptively transferred cells in the BM. To investigate the role of the SNS, we sympathectomized mice with 6-hydroxydopamine. Chemical sympathectomy significantly reduced the circadian differences of leukocyte rolling on BM endothelium (RFF 7.8 ± 1.1% (12 pm) vs 14.9 ± 2.8% (8 pm); p=0.01) and leukocyte numbers in PB (2750 ± 322 vs 2225 ± 363/μl; p=0.32). In addition, the number of adherent adoptively transferred cells in the BM did not show significant fluctuation (1.66 ± 0.26 vs 1.54 ± 0.20/vessel area(μm2);p=0.72). These results suggest that the process of homing/cluster formation is dependent on constitutive, oscillatory expression of P- and/or E-selectins and regulated by the SNS.