Neutrophils Modulate Osteoblastic Niche for Hematopoietic Progenitors Via Prostaglandin E2 Production Triggered By Sympathetic Nervous System

The mobilization of hematopoietic stem/progenitor cells (HSC/HPCs) from the bone marrow (BM) to peripheral blood by granulocyte colony-stimulating factor (G-CSF) is an essential method in clinic. We have shown that the suppression of osteoblastic niche by β-adrenergic signal is critical for this phenomenon (Cell 2006; Cell Stem Cell 2013). Because G-CSF administration causes fever and back pain and these symptoms are ameliorated by non-steroidal anti-inflammatory drugs, we investigated the role of prostaglandin E2 (PGE2) in the BM microenvironment during G-CSF treatment. First, HPC (CFU-C) mobilization by G-CSF (125μg/kg/dose, every 12 hours, 8 divided doses) was significantly augmented in microsomal PGE syntase-1-deficient (mPGES-1-/-) mice (42% increase, n=16, p<0.05), and strongly inhibited by exogenous administration of PGE2 (6mg/kg/day for 2 weeks) to wild-type (WT) mice (52%, n=8-9 p<0.05). These data suggest that G-CSF induces mPGES-1-mediated PGE2 production, which suppresses the HPC mobilization. In the early phase (at 4 doses) of G-CSF administration, mPGES-1 mRNA in BM cells was upregulated (34% increase, n=5, p<0.05). Furthermore, WT mice reconstituted with mPGES-1-/- BM showed higher HPC mobilization than control mice reconstituted with mPGES-1+/+ BM (2.2-fold increase, n=4, p<0.05), which indicated that blood cells might be responsible for additional PGE2 synthesis. To address this, we examined the PGE2 production by ELISA from various lineage cell lines, such as neutrophil precursor 32D, macrophage RAW264.7, B cell Ba/F3, and T cell EL4. No increase was observed by G-CSF in any cell lines; whereas, isoproterenol induced PGE2 production significantly only in 32D culture supernatant (2.4-fold increase compared to vehicle treatment, n=4, p<0.05) accompanied with drastic increase of mPGES-1 mRNA in the cells and norepinephrine showed a similar effect. Primary neutrophils sorted from the BM also demonstrated prompt PGE2 production by isoproterenol (3-fold increase compared to vehicle treatment, n=4, p<0.05) but not by G-CSF. These data suggest that G-CSF-triggered high sympathetic tone stimulates the BM neutrophils to lead PGE2 production. We next assessed the exact roles of PGE2 in HPC mobilization. The inhibitory effect of PGE2 on HPC mobilization was completely abrogated in PGE2 receptor EP4-deficient (EP4-/-) mice, and the chimeric model generated by the reciprocal BM transplantation revealed that it was EP4 in microenvironment, but not in hematopoietic cells, that was critical for this effect. Since PGE2 did not change the CXCL12 behavior, we speculated that PGE2 increased another anchor in the niche, osteopontin (OPN). Immunofluorescence staining demonstrated upregulation of OPN by PGE2 and/or G-CSF in the endosteum, which was abolished in EP4-/- mice. Indeed, the inhibitory effect of PGE2 on HPC mobilization was canceled partially in OPN-/- mice and almost completely in anti-OPN antibody-treated WT mice. PGE2 also inhibited AMD3100-induced HPC mobilization, and this effect was canceled by anti-OPN antibody, which confirmed that PGE2-mediated niche modulation was independent of CXCL12 axis. To assess the induction of OPN by PGE2 directly, we fractionated non-hematopoietic (CD45-CD31-Ter119-) cells isolated from adult femur into three populations, i.e. Sca-1+ALCAM- immature mesenchymal cells, Sca-1-ALCAM- preosteoblasts that favorably support HPCs, and Sca-1-ALCAM+ mature osteoblasts that are most potent to maintain quiescent HSCs in vitro. PGE2 upregulated the OPN protein 2-fold in Sca-1+ALCAM- immature mesenchymal cells in cultures and more dramatically (6-fold) in Sca-1-ALCAM- preosteoblasts as assessed by flow cytometry. In sharp contrast, no OPN induction was observed in Sca-1-ALCAM+ mature osteoblasts. PGE2 failed to induce OPN in all three fractions from EP4-/- mice. In contrast to HPCs, the mobilization of long-term (6 months) repopulating HSCs was not altered in mPGES-1-/- and in PGE2-treated WT mice. This was consistent with the OPN induction profile in fractionated osteoblasts. These results suggest that PGE2 selectively regulates the osteoblastic niche for hematopoietic progenitors, but not for stem cells, by the induction of OPN via EP4 receptor. Collectively, we propose the inter-communication between the mature hematopoietic cells and the niche for their immature progenitors governed by the sympathetic nervous system.