Norepinephrine reuptake inhibition promotes mobilization in mice: potential impact to rescue low stem cell yields

Hematopoietic stem and progenitor cells (HSPCs) traffic continuously between the blood and BM under homeostasis.^1,2  G-CSF can enhance this phenomenon and therefore has emerged as one of the most potent and safe agents to mobilize HSPCs for stem cell transplantation, but its mechanisms are complex. Studies have attributed its mobilizing activity to the release of neutrophil-derived proteolytic enzymes that leads to the cleavage of CXCL12 and other retention signals.^3,4  However, this model has been challenged by the normal mobilization found in the absence of serine protease and metalloproteinase activity,⁵  suggesting functional redundancy with other proteases or the involvement of additional mechanisms. Adrenergic signals play a key role in the release of HSPCs either when enforced by G-CSF⁶  or during steady state.¹  Egress is associated with the down-regulation in Nestin⁺ niche cells of Cxcl12 mRNA expression and other genes (eg, Kitl, Angpt1, and Vcam1) that contribute to HSPC retention in the BM.⁷  However, expression of the G-CSF receptor (encoded by Csf3r) on transplantable hematopoietic cells has been reported to be required for mobilization,⁸  and recent studies suggest that this cell may be a macrophage^9-11  with activity that promotes hematopoietic stem cell (HSC) retention by the niche.⁹  Because the administration of either G-CSF or β3 adrenergic agonists has the same effect on the expression of these retention factors by the niche,⁷  it suggests that increased sympathetic tone may enhance mobilization.

Eight-week-old male C57BL/6J mice were purchased from the National Cancer Institute (Frederick Cancer Research Center, Frederick, MD). All mice were housed at the Center for Comparative Medicine and Surgery at Mount Sinai School of Medicine or the institute for Animal Studies at Albert Einstein College of Medicine, and the animal care and use committees of both institutions approved the experimental procedures performed on mice. For HSPC mobilization, mice received 8 consecutive injections of G-CSF (125 μg/kg twice a day subcutaneously) or saline. Desipramine (10 mg/kg intraperitoneally), reboxetine (5 mg/kg intraperitoneally), or saline treatment was started 4 days before G-CSF treatment and continued throughout the experiment. For AMD3100-induced mobilization, mice were treated with desipramine (10 mg/kg intraperitoneally) or saline. On day 8, mice were injected with AMD3100 (5 mg/kg subcutaneously) 1 hour before blood was harvested by retroorbital bleeding. All other experimental methods are described in supplemental Methods (available on the Blood Web site; see the Supplemental Materials link at the top of the online article).

We performed immunofluorescence analyses in murine superior cervical sympathetic ganglia (SCSG) to assess the expression of Csf3r in the peripheral sympathetic nerves. SCSG neurons have large cell bodies and express the catecholaminergic enzyme tyrosine hydroxylase (Figure 1A). These SCSG neurons also expressed Csf3r (Figure 1A) and Csf3r mRNA specifically (Figure 1B). This is consistent with other studies that have shown Csf3r expression in neocortical neurons and that G-CSF promotes neuronal survival after ischemic brain injury.¹² 

The activity of the natural neurotransmitter of the sympathetic nervous system, norepinephrine (NE), is tightly regulated by a balance of secretion and reuptake by nerve terminals.¹³  To determine whether Csf3r on sympathetic neurons was functional, we evaluated whether G-CSF could influence NE release. In these studies, isolated SCSGs were loaded with tritiated NE ([3H]-NE), and the ability of G-CSF to increase the release of radiolabeled NE in these organ cultures was assessed. Whereas control KCl induced profound NE release, there was no difference between unstimulated and G-CSF–treated cultures in [3H]-NE content in the medium (supplemental Figure 1). We then evaluated the influence of G-CSF on the reuptake of NE by the peripheral sympathetic nerves by culturing SCSGs in medium containing [3H]-NE in the presence or absence of G-CSF. We found that incubation with G-CSF reduced [3H]-NE reuptake significantly compared with the unstimulated control (Figure 1C). Preincubation with an Ab that blocked Csf3r abrogated this effect, indicating that the effect was specific (Figure 1C). These data therefore suggest that G-CSF enhances the sympathetic tone by altering the ability of sympathetic neurons to take up NE, thereby increasing catecholamines available to target cells. The drug desipramine, an NE reuptake inhibitor, completely blocked NE uptake in the SCSG organ culture (Figure 1C), which led us to investigate whether in vivo blockade of NE reuptake could enhance G-CSF mobilization efficiency.

Mice were treated with desipramine and G-CSF, as shown in Figure 2A. Desipramine alone did not mobilize HSPCs (supplemental Figure 2A-B), an observation consistent with previous studies indicating that β-adrenergic agonists are not sufficient to induce robust mobilization⁶  and that other G-CSF–mediated signals cooperate with adrenergic receptors.^1,6,9  When administered in conjunction with G-CSF, desipramine increased the number of clonogenic progenitors (CFU-Cs) and Lin⁻Sca-1⁺c-kit⁺Flt3⁻ (LSKF) cells (which are highly enriched in HSCs) in the peripheral blood significantly compared with mice treated with G-CSF alone (Figure 2B-D). The combination of G-CSF and desipramine increased the number of functional HSCs, as measured by the number of long-term culture-initiating cells in the blood (Figure 2E and supplemental Table 1). We detected no difference in BM cellularity, CFU-Cs, or LSKF cell numbers in the BM (supplemental Figure 2C-E), indicating that the increase in progenitor mobilization does not result from increased numbers in the BM. Consistent with the fact that desipramine treatment enhanced G-CSF–induced sympathetic outflow, it did not affect AMD3100-induced mobilization (supplemental Figure 2F-G). This notion is further supported by the fact that another selective NE reuptake blocker, reboxetine, enhanced G-CSF–induced mobilization to a similar extent (supplemental Figure 2H-I).

We reasoned that patients who mobilize poorly might benefit from increased sympathetic tone more than healthy individuals. To test this idea in a preclinical setting, we modeled “poor mobilizers,” which commonly result from prior cytotoxic therapy,¹⁴  by subjecting mice to a lethal dose of irradiation and BM transplantation. After a 4-month recovery period, mice were treated with desipramine and mobilized with G-CSF (Figure 2F). Desipramine treatment did not change the number of HSPCs in the BM (Figure 2G); however, transplanted animals exhibited lower numbers of G-CSF–mobilized progenitors and LSKF cells than did healthy mice (Figure 2H-I). Remarkably, desipramine approximately doubled HSPC mobilization in these mice (Figure 2H-I) to a level closer to the values of healthy mice (compare “D+G” with dashed red lines in Figure 2H-I). Because a 2-fold increase in mobilization is sufficient to obtain adequate HSPC yields in approximately 50% of poor mobilizers,¹⁵  this approach has the potential to be clinically useful in this setting. Although desipramine may not be as potent as AMD3100, which induces an approximately 3-fold increase in mobilization when administered together with G-CSF,¹⁶  it provides an inexpensive alternative that should be investigated further in a clinical trial. Inhibition of NE reuptake might be used not only to reduce the number of patients with poor HSPC yield, but also to increase HSPC yields in healthy individuals. Transplantation with increased numbers of HSPCs may accelerate engraftment¹⁷  and reduce transplantation-related morbidity. Further, because AMD3100 and desipramine seem to act through different pathways (supplemental Figure 2F-G), it is possible that the combination of G-CSF, desipramine, and AMD3100 may enhance mobilization even further.

The data from the present study indicate that G-CSF likely increases the sympathetic tone in the BM microenvironment by engaging neurally expressed Csf3r directly and by inhibiting NE reuptake. Modulation of the sympathetic outflow by desipramine increases the efficiency of HSPC harvests and may thus provide a therapeutic alternative.

The authors thank Colette Prophete for mouse husbandry and technical assistance.

D.L. was a fellow of the Fundación Ramón Areces. I.B. is the recipient of a 2010 American Society of Hematology–European Hematology Association research-exchange award. This work was supported by the National Institutes of Health (R01DK056638 and R01HL097819 to P.S.F.).

National Institutes of Health

Contribution: D.L. and I.B. performed the experiments, analyzed the data, and wrote the manuscript; M.B. and Y.K. performed the experiments; C.M. performed the experiments and analyzed the data; S.M.-F. contributed to experiments on norepinephrine reuptake and release; and P.S.F. designed the research, supervised the study, and wrote the manuscript.

Conflict-of-interest disclosure: The authors declare no competing financial interests.

The current affiliation for S.M.-F. is Centro Nacional de Investigaciones Cardiovasculares Carlos CNIC, Madrid, Spain.

Correspondence: Paul S. Frenette, Ruth L. and David S. Gottesman Institute for Stem Cell Biology and Regenerative Medicine, Albert Einstein College of Medicine, 1301 Morris Park Ave, Rm 101B, Bronx, NY 10461; e-mail: paul.frenette@einstein.yu.edu.