Mice Deficient in the Fifth Complement Cascade Protein (C5) Do Not Display Circadian Changes in the Number of Circulating Hematopoietic Stem/Progenitor Cells (HSPCs) in Peripheral Blood (PB)—evidence for the Pivotal Role of the Distal Part of the Complement Cascade in Inducing the Circadian Release of HSPCs into PB

Introduction. Activation of three ancient serum proteolytic cascades, the complement cascade (ComC), the coagulation cascade (CoaC), and the fibrynolytic cascade (FibC), is essential for release of hematopoietic stem progenitor cells (HSPCs) from bone marrow (BM) into peripheral blood (PB) during stress- or pharmacology-induced mobilization (Leukemia 2014,doi:10.1038/leu.2014.115). On the other hand, it has been convincingly demonstrated that there is a circadian oscillation in the number of circulating HSPCs in PB, with the peak occurring in the early morning hours and the nadir at night (Nature 2008, 452, 442-447 ). The timing of this peak has been attributed to the enhanced tonus of the vegetative nervous system in the early morning hours. In support of such a role for the vegetative nervous system, it has been shown that UDP-galactose:ceramide galactosyltransferase-deficient mice, which exhibit aberrant nerve conduction and do not release norepinephrine (NE) into the BM microenvironment, do not mobilize HSPCs. However, by contrast, modification of the sympathetic output, as seen in normal human HSPC volunteer donors receiving NE reuptake inhibitors (NRI) for depression or β2-blockers for hypertension, induces mobilization in a similar manner as normal controls (Leukemia 2013, 27, 24–31). Mobilization in these patients was neither enhanced by NRI administration nor suppressed by β2-blockers, as one would expect based on the murine data reported in the literature.

Aim of the study. Since it is known that the ComC, CoaC, and FibC show circadian activation at late/night early morning hours due to deep sleep hypoxia, we became interested in the role of these proteolytic cascades in the circadian release of HSPCs from the BM into PB.

Materials and Methods. To address this important question, we studied the circadian oscillation in the number of circulating HSPCs in C5-deficient (C5^–/–) mice, which do not activate the distal part of the ComC, unlike their wild type (WT) littermates. Mice were accustomed to alternating periods of 12 hours light and 12 hours darkness. Light was turned on at 6 AM (T0), and the number of circulating white blood cells (WBC), Sca-1⁺kit⁺Lin^– HSCs, Sca-1⁺Lin^–CD45⁺ HSCs, clonogenic CFU-GM progenitors, and the number of non-hematopoietic Sca-1⁺Lin^–CD45^– (VSELs) were measured at 7 AM (T1), 11 AM (T5), 7 PM (T13), and 3 AM (T21). At the same time points, we evaluated activation of the ComC (by C5a ELISA), the CoaC (by thrombin/antithrombin ELISA), and the FibC (by plasmin/antiplasmin complex ELISA).

Results. We observed circadian changes in the number of circulating WBCs, HSCs, and non-HSCs at T5 in WT but not in C5^–/– animals. This increase in the number of circulating cells in WT animals was preceeded by an increase in C5a concentration in PB at T1 as well as activation of the CoaC and FibC at T21. As expected, C5^–/– mice did not have measurable levels of C5a; however, they displayed an increase in activation of the CoaC and FibC at T21 that was similar to WT.

Conclusions. Our study confirms circadian activation of the ComC, CoaC, and FibC in WT animals at late night/early morning hours preceding the release of HSPCs from BM into PB. The fact that we did not observe circadian changes in the number of circulating cells in PB in C5a^–/– mice confirms the pivotal role of the ComC in executing circadian release of HSPCs from BM into PB. Moreover, the fact that C5a^–/– mice show normal activation of the CoaC and FibC indicates that, of the ancient proteolytic cascades tested, the ComC is the major player regulating circadian egress of HSPCs.