Circadian Adrenergic Regulation of Bone Marrow Endothelial Adhesion Molecule Expression Impacts Progenitor Recruitment and Engraftment Efficiency

Abstract 398

Previous studies have revealed that hematopoietic stem cells (HSCs) are released into peripheral blood in a circadian manner in a process 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). Here, we have evaluated the constitutive recruitment of hematopoietic cells back to the BM. We have observed using high-speed multichannel fluorescence intravital microscopy (MFIM) significant circadian oscillations in the number of adherent BM cells in sinusoids with a nadir in the morning (Zeitgeber time, ZT5: 0.97 ± 0.17 adherent cells/ 100 μm² vessel area) and a peak at night (ZT13: 2.54 ± 0.53 adherent cells/100 μm², p = 0.007) after adoptive transfer on a 12 hour light-12 hour darkness cycle. Flow cytometric analyses revealed that the majority (∼70 %) of homed BM cells were Gr-1+ Mac-1+ myeloid cells. To investigate the underlying mechanisms, we have examined the expression levels of P- and E-selectins and VCAM-1, essential homing receptors for progenitor cells in the BM, and found that their mRNA and protein expression on BM endothelium oscillated over the course of a day with the peak expression overlapping the time of the highest cell adhesion numbers (ZT13). To examine the role of the SNS in this process, we surgically sympathectomized mice by unilateral section of the superior cervical ganglion (SCGx) whose neurons project into the calvarial vasculature, while performing sham surgery on the contralateral side. Sympathectomy abolished circadian fluctuations in the number of adoptively transferred adherent cells to the denervated calvarial BM compared to the control side in the same animals (nerve-intact side: ZT5 / ZT13: 1.66 ± 0.10 / 2.41 ± 0.08 cells / 100 μm², p<0.0001; SCGx: ZT5 / ZT13: 1.65 ± 0.09 vs 1.63 ± 0.09 / 100 μm² vessel area, p=0.90). We then ascertained further the role of adrenergic signals by evaluating mice deficient in b-adrenergic receptors. We found that the oscillations in cell adhesion molecule expression were markedly reduced in β2 (Adrb2-/-) and β3 (Adrb3-/-) adrenergic receptor deficient mice. These results suggest that hematopoietic cell recruitment to the BM is under circadian control, which is dependent on oscillating expression of endothelial selectins and VCAM-1, and regulated by the SNS. To test the relevance of circadian leukocyte recruitment, we investigated whether isoproterenol, a pan-b-adrenergic agonist commonly used in the clinic, could promote hematopoietic progenitor recruitment and thus BM reconstitution after BM transplantation (BMT). Treatment with isoproterenol (5 mg/kg) for 5 days significantly up-regulated expression of P-selectin (1.2-fold increase; p = 0.027), E-selectin (1.5-fold increase; P = 0.003) and VCAM-1 (2.3-fold increase; P=0.006) on BM endothelium in irradiated recipients as determined by flow cytometry of Tie-2+ PECAM-1+ endothelial cells. Consequently, homing of BM cells was dramatically increased (control / isoproterenol: 2.4 ± 0.2 ×10⁴/4.9 ± 0.4 × 10⁴ donor cells/femur; p = 0.0002) as was the number of recruited hematopoietic progenitors (17.0 ± 3.5/74.1 ± 18.8 CFU-C/femur; p = 0.017). In addition, the recovery of mature myeloid cells in peripheral blood was significantly accelerated in 3 weeks after transplantation of 5 × 10⁴ BM cells (0.38 ± 0.21 × 10³/1.64 ± 0.50 ×10³/μl; p = 0.024). Of importance, using limiting numbers of BM cells (2.5 × 10⁴) for transplantation, isoproterenol treatment markedly improved the survival (median survival time 10 vs 18 days, percent survival at 4 weeks post-BMT 5.8 vs 35.2%; p = 0.0097). These results indicate that the circadian timing of donor cell infusion and/or manipulation of adrenergic signals in the BM microenvironment may improve transplantation outcome through enhanced engraftment efficiency.