Here, we show that the emigration of leukocytes to tissues is regulated by signals from the autonomic nervous system where the peak recruitment occurs at night in rodents, during a period of activity. Circadian hematopoietic cell recruitment is synchronized by the molecular clock via sympathetic nerves, which induce through β-adrenoreceptors oscillations in endothelial cell expression of ICAM-1 and CCL2 in skeletal muscle and endothelial selectins, VCAM-1 as well as CXCL12 (Mendez-Ferrer et al., 2008
) in the BM. In addition, our data suggest that rhythmic leukocyte recruitment is not restricted to these tissues but occurs in many vital organs. The difference in the molecular signature of circadian expression of endothelial cell adhesion molecules and chemokines in tissues is likely responsible for the diurnal preference in hematopoietic cell populations recruited to various organs.
Changes in the light cycle, a major zeitgeber
for circadian rhythms (Golombek and Rosenstein, 2010
), were sufficient to ablate oscillations in hematopoietic cell recruitment. This finding, along with the involvement of BMAL-1, strongly indicates bona fide
circadian rhythms and suggests their central orchestration by the molecular clock (Dibner et al., 2010
; Green et al., 2008
). Additionally, the surprising anti-inflammatory effect seen after acute jetlag suggests a potent and broad interference of light in the normally rhythmically generated inflammatory response. It is possible that chronic jetlag may perturb circadian immune-surveillance mechanisms and contribute to the higher incidence of cancer in shift workers (Filipski et al., 2004
). How jetlag exerts these broad changes in the inflammatory response should be the subject of future investigations.
Most prior studies on the regulation of circadian rhythms have focused on the involvement of humoral factors, notably of glucocorticoid (GC) levels, which peak at night in mice and reach trough values during the day (Dickmeis, 2009
). Since GCs exhibit anti-inflammatory properties, we would expect that GC hormones might antagonize the rhythms described herein which depend on local innervation. It is possible however, that neural and hormonal pathways complement each other to fine tune the physiological and inflammatory responses (Elenkov et al., 2000
). Thus, higher levels of GCs at night in mice may in fact keep the night surge in leukocyte infiltration in check.
Although catecholamine treatment of donor progenitors in vitro
enhanced their engraftment ability through the β2-adrenoreceptor (Spiegel et al., 2007
), we have excluded a role for adrenergic receptors on transplantable hematopoietic cells in our studies using radiation chimeras and adoptive transfer experiments. Therefore, endothelial cells in BM and skeletal muscle, which can express both the β2- and β3-receptors (Steinle et al., 2003
), likely represent a logical cellular target since they also express the necessary adhesion molecules to mediate hematopoietic cell/endothelial cell interactions. Our results indicate that BM transplantation performed after stimulation with β3-adrenergic agonists in the recipient may improve clinical outcomes through enhanced engraftment efficiency. Since adrenergic stimulation can also induce the mobilization of HSPCs (Mendez-Ferrer et al., 2008
), it remains unclear as to how the cells targeted by adrenergic signals interpret these signals at different times. The fact that the same adrenergic receptors can lead to distinct adhesion molecule expression in different vascular beds, suggests tissue-specific signaling responses that remain undefined. The present findings are consistent with previous studies in which circadian oscillations of BM CXCL12 with trough expression levels of the chemokine occurring in the morning were associated with the peak of HSC egress (Mendez-Ferrer et al., 2008
). It is likely that the nightly increase of CXCL12 in the BM cooperates with endothelial upregulation of adhesion molecules for maximal hematopoietic cell homing in the evening. In the bone marrow, β3-adrenoreceptor expression is enriched in rare Nestin+
perivascular mesenchymal stem cells, which express high amounts of CXCL12 and are targeted by the SNS in the stem cell niche (Mendez-Ferrer et al., 2010
). How microvascular pericytes collaborate with endothelial cells to entrain rhythms in extramedullary tissues will be the subject of future studies in the laboratory.
he rhythmic pattern of leukocyte recruitment may have evolved for the benefit of having readily available tissue phagocytes to enhance the response to pathogens during the period of activity, when injuries and encounters with microorganisms are more likely to occur. Although macrophages represent the major leukocyte subset oscillating under homeostasis, we show that neutrophil recruitment after trauma or in the setting of inflammatory diseases is significantly influenced by endogenous rhythms of endothelial cell adhesion. It is conceivable that the diurnal clearance of senescent neutrophils by the BM and liver is mediated by these circadian mechanisms. Whether the HSPC pool is rejuvenated by diurnal migratory incentives is possible. We have presented clear evidence, however, that circadian timing can influence the engraftment efficiency when HSPC counts are sub-optimal, demonstrating the significance of circadian hematopoietic cell recruitment to the BM. As such, given the inverted rhythms between rodents and humans (Lucas et al., 2008
), we would predict that human hematopoietic stem cell transplantation is optimal early in the morning, rather than later in the day.
We postulate that the diurnal mechanisms of keeping neutrophil numbers in check may affect cardiovascular diseases – which are influenced by circadian time – as their levels are known to correlate positively with the degree of severity (Coller, 2005
; Muller et al., 1985
). When exaggerated, these mechanisms may exert detrimental effects. Indeed, in models of TNF-α-induced vaso-occlusion in SCD mice and LPS-induced lethality, we have found that circadian rhythms modulate the robustness of the inflammatory response with implications on survival. While we recognize potential mechanistic differences between atherosclerosis and inflammatory models studied herein, these data are in line with prior studies that have shown persistent oscillations in the susceptibility of mice to bacterial infection and inflammatory leukocyte trafficking (Castanon-Cervantes et al., 2010
; Feigin et al., 1969
; Gibbs et al., 2011
; Halberg et al., 1960
; House et al., 1997
; Keller et al., 2009
; Liu et al., 2006
; Shackelford and Feigin, 1973
; Silver et al., 2012
). Sensitivity to inflammatory stimuli at times of higher SNS tone likely interacts at multiple levels encompassing local neural input as we describe herein, endogenous rhythms within tissues as well as interactions with humoral factors and the parasympathetic nervous system.
Given the reported critical role of blood leukocytes in ischemic vascular diseases, the present results argue that circadian leukocyte adhesion or an exaggeration thereof might contribute to triggering acute vascular diseases in the morning. Our own as well as published studies have clearly shown that leukocyte recruitment promotes LPS-induced lethality (Xu et al., 1994
) and SCD vaso-occlusion (Hidalgo et al., 2009
; Turhan et al., 2002
), which led us to evaluate experimentally the effect of circadian time in inflammatory diseases. Our results provide the proof-of-principle that time-dependent differences in leukocyte recruitment translate into significant changes in survival. Thus, a better understanding of rhythms in leukocyte migration will allow the design of targeted chronotherapy that may lead to meaningful clinical impact on disease outcome.