Bone Marrow CXCL12 Research Articles

Bone marrow CD169+ macrophages promote the retention of hematopoietic stem and progenitor cells in the mesenchymal stem cell niche

Hematopoietic stem cells (HSCs) reside in specialized bone marrow (BM) niches regulated by the sympathetic nervous system (SNS). Here, we have examined whether mononuclear phagocytes modulate the HSC niche. We defined three populations of BM mononuclear phagocytes that include Gr-1hi monocytes (MOs), Gr-1lo MOs, and macrophages (MΦ) based on differential expression of Gr-1, CD115, F4/80, and CD169. Using MO and MΦ conditional depletion models, we found that reductions in BM mononuclear phagocytes led to reduced BM CXCL12 levels, the selective down-regulation of HSC retention genes in Nestin+ niche cells, and egress of HSCs/progenitors to the bloodstream. Furthermore, specific depletion of CD169+ MΦ, which spares BM MOs, was sufficient to induce HSC/progenitor egress. MΦ depletion also enhanced mobilization induced by a CXCR4 antagonist or granulocyte colony-stimulating factor. These results highlight two antagonistic, tightly balanced pathways that regulate maintenance of HSCs/progenitors in the niche during homeostasis, in which MΦ cross talk with the Nestin+ niche cell promotes retention, and in contrast, SNS signals enhance egress. Thus, strategies that target BM MΦ hold the potential to augment stem cell yields in patients that mobilize HSCs/progenitors poorly.

Mobilization efficiency of granulocyte colony-stimulating factor and stem cell factor to bone marrow mononuclear cells and mechanisms

The mobilization efficiency of granulocyte colony-stimulating factor (G-CSF) and stem cell factor (SCF) to bone marrow mononuclear cells (MNCs) in mice was observed, and the changes of CXCL12/CXCR4 signal were detected in order to find out the mobilization mechanism of stem cells. Kunming mice were randomly divided into two groups. The mice in treatment group were subjected to subcutaneous injection of G-CSF at a dose of 100 microg/kg and SCF at a dose of 25 microg/kg every day for 5 days, and those in control group were given isodose physiological saline. The MNCs were separated, counted and cultured, and the colony-forming unit-fibroblast (CFU-F) was evaluated. CD34+CXCR4+ MNCs were sorted by flow cytometry. The expression of CXCL12 protein in bone marrow extracellular fluid was detected by ELISA, and that of CXCL12 mRNA in bone marrow was measured by RT-PCR. The results showed that the counts of MNCs in peripheral blood and bone marrow were increased after administration of G-CSF/SCF (P<0.01). The factors had a dramatic effect on the expansion capability of CFU-F (P<0.05). Flow cytometric of bone marrow MNCs surface markers revealed that CD34+CXCR4+ cells accounted for 44.6%+/-8.7% of the total CD34+ MNCs. Moreover, G-CSF/SCF treatment induced a decrease in bone marrow CXCL12 mRNA that closely mirrored the fall in CXCL12 protein. In this study, it is evidenced that G-CSF/SCF can effectively induce MNCs mobilization by disrupting the balance of CXCL12/CXCR4 signaling pathway in the bone marrow and down-regulating the interaction of CXCL12/CXCR4.

Experimental study on the mobilization effects and mechanisms of granulocyte colony-stimulating factor and stem cell factor to bone marrow mononuclear cells

Objective To observe the mobilization effects of granulocyte colony-stimulating factor (G-CSF) and stem cell factor (SCF) to bone marrow mononuclear cells ( MNCs) in mice, and detect the changes of CXCL12/CXCR4 signal, so as to find out the mobilization mechanism of stem cells. Methods Kunming mice were randomly divided into two groups: the treatment group subject to subcutaneous injections of G-CSF at a dose of 100μg/kg and SCF at a dose of 25μg/kg per day for 5 days; the control group subject to isodose physiological saline. The MNCs were separated, counted and cultured, and the colony-forming unit-fibroblast (CFU-F) was evaluated. CD34+ CXCR4+ MNCs were sorted by flow cytometry. The CXCL12 protein in bone marrow extracellular fluid was detected by enzyme-linked immunosorbent assay (ELISA), and bone marrow CXCL12 mRNA by reverse transcription-polymerase chain reaction (RT-PCR). Results The counts of MNCs in peripheral blood and bone marrow were increased after administration of G-CSF/SCF (P<0. 01). The factors had a dramatic effect on the expansion capability of CFU-F (P<0.05). Flow cytometic detection of BM-MNCs surface marks showed CD34+ CXCR4+ cells of (44. 6 ± 8.7)% of the total CD34+ MNCs. G-CSF treatment induced a decrease in bone marrow CXCL12 mRNA that closely mirrored the fall in CXCL12 protein. Conclusion In this study, we characterize G-CSF/SCF-induced BM-MNCs mobilization and CXCL12 expression in the bone marrow in a series of tests. We provide further evidence that disruption of CXCL12/CXCR4 signaling in the bone marrow is a key step in BM-MNC mobilization. The consequent attenuation of CXCR4 signaling ultimately leads to BM-MNCs mobilization. Key words: Granulocyte colony-stimulating factor; Stem cell factor; Mononuclear cell

Cooperation of β2‐ and β3‐adrenergic receptors in hematopoietic progenitor cell mobilization

CXCL12/SDF‐1 dynamically regulates hematopoietic stem cell (HSC) attraction in the bone marrow (BM). Circadian regulation of bone formation and HSC traffic is relayed in bone and BM by β‐adrenergic receptors (β‐AR) expressed on HSCs, osteoblasts, and mesenchymal stem/progenitor cells. Circadian HSC release from the BM follows rhythmic secretion of norepinephrine from nerve terminals, β3‐AR activation, and Cxcl12 downregulation, possibly from reduced Sp1 nuclear content. Here, we show that β‐AR stimulation in stromal cells causes Sp1 degradation, partially mediated by the 26S proteasome. Inverted trends of circulating hematopoietic progenitors and BM Cxcl12 mRNA levels change acutely after light onset, shown to induce sympathetic efferent activity. In BM stromal cells, activation of β3‐AR downregulates Cxcl12, whereas β2‐AR stimulation induces clock gene expression. Double deficiency in β2‐ and β3‐ARs compromises enforced mobilization. Therefore, β2‐ and β3‐ARs have specific roles in stromal cells and cooperate during progenitor mobilization.

Infection withAnaplasma phagocytophilumInduces Multilineage Alterations in Hematopoietic Progenitor Cells and Peripheral Blood Cells

Infection with Anaplasma phagocytophilum, a gram-negative, lipopolysaccharide (LPS)-negative, obligate intracellular bacterium, results in multiple peripheral blood cytopenias. We hypothesized that infection with this organism would result in decreased bone marrow (BM) function and shifts in hematopoietic progenitor cells (HPCs) and lineage-committed cells in a well-established murine model of infection. HPCs and lineage-committed progenitors were enumerated in the BM and spleen during acute infection. BM cytokine production and BM CXCL12 expression were determined. Infection resulted in peripheral blood bicytopenia, marked decreases in the number of lineage-committed HPCs in the BM along with concurrent increases in the number of lineage-committed HPCs in the spleen, and a mixed, predominantly myelosuppressive BM cytokine environment. There was significant downregulation of CXCL12 in BM cells that may have been partially responsible for changes in HPC trafficking observed. Changes occurred in the absence of direct pathogen infection of BM cells. Hematopoietic lineage assessment demonstrated that there was loss of erythrocytes and B lymphocytes from the BM along with increased granulopoiesis. These changes were accompanied by splenomegaly due to lymphoid hyperplasia and increased hematopoiesis, most notably erythropoiesis. These changes largely mimic well-described inflammation and endotoxin-mediated effects on the BM and spleen; however, the numbers of peripheral blood neutrophils appear to be independently modulated as granulocytic hyperplasia does not result in neutrophilia. Our findings highlight a well-conserved series of events that we demonstrate can be instigated by an LPS-negative pathogen in the absence of an endotoxin-mediated acute proinflammatory response.

Suppression of CXCL12 Production by Bone Marrow Osteoblasts Is a Common and Critical Pathway for Cytokine-Induced Mobilization.

There is strong evidence that CXCL12 (stromal-derived factor-1)/CXCR4 signaling is a key regulator of hematopoietic stem and progenitor cell (HSPC) trafficking in the bone marrow. CXCL12 protein and mRNA expression in the bone marrow are markedly reduced with G-CSF treatment. We and others recently showed that G-CSF treatment results in a marked loss of mature endosteal and trabecular osteoblasts. Since osteoblasts are a major source of CXCL12, this observation provides a potential mechanism by which G-CSF downregulates CXCL12 expression in the bone marrow. To test this hypothesis, we performed RNA in situ studies for CXCL12. These studies show that CXCL12 is highly expressed in endosteal osteoblasts as well as in scattered cells in the bone marrow in untreated mice. In mice treated with G-CSF, there is a near complete loss of CXCL12 signal along the endosteum. Together, these data suggest a model in which G-CSF induced suppression of osteoblasts leads to a decrease in CXCL12 expression in the bone marrow, disrupting CXCR4 signaling and ultimately leading to HSPC mobilization. This model raises several important questions. Many different hematopoietic cytokines can induce HSPC mobilization. Is loss of CXCL12 expression by osteoblasts a common mechanism by which cytokines induce HSPC mobilization? To address this question, we studied HSPC mobilization by Flt3 ligand (Flt3L) and stem cell factor (SCF), two potent mobilizing hematopoietic cytokines. Treatment with Flt3L or SCF resulted in a significant decrease in bone marrow CXCL12 protein and mRNA expression. Moreover, the decrease in CXCL12 expression was accompanied by a loss in trabecular osteoblasts [number osteoblasts per mm bone ± SEM: 5.1 ± 0.1 (control), 3.1 ± 0.4 (Flt3L), 1.8 ± 0.8 (SCF); n=2, p<0.05]. There is considerable evidence implicating other pathways in HSPC mobilization, including upregulation of protease activity in the bone marrow and downregulation of critical adhesion molecules on HSPC and their supporting stromal cells. What is the relative importance of each of these pathways to mobilization in vivo? To address this question, we established chimeras containing CXCR4−/− hematopoietic cells by transplanting CXCR4−/− fetal liver cells into irradiated syngeneic recipients. Consistent with previous reports, CXCR4−/− chimeras display constitutive mobilization of HSPC (number CFU-C/ml blood ± SEM: 12,080 ± 2,904 compared with 120 ± 25 for control chimeras; n=7–9). Surprisingly, G-CSF treatment of these mice did not result in an increase in circulating HSPC (9,511 ± 3,044; n=7–9). This lack of response to G-CSF was not simply due to a loss of mobilizable HSPC in the bone marrow, as treatment with AMD15057, a specific VLA4 antagonist, induced robust mobilization in CXCR4−/− chimeras (30,600 ± 3,790; n=6–9, p<0.01 compared with G-CSF-treated chimeras). Together, these findings suggest that:1.suppression of CXCL12 expression by osteoblasts is a common mechanism of cytokine-induced HSPC mobilization;2.this mechanism represents the principal pathway by which G-CSF mobilizes HSPC, with other pathways (e.g. protease activation) operating upstream or downstream from this pathway; and3.VLA-4 antagonism, in contrast, provides a distinct mechanism by which HSPC may be mobilized from the bone marrow.

Developmental stage–specific shift in responsiveness to chemokines during human B-cell development

To better understand the role of chemokines during human B-cell development in bone marrow. Differentiation stage-specific B cells (pro-B, pre-B, immature, and mature) were analyzed for chemokine receptor expression and for migration to corresponding ligands. We also hypothesized that inflammatory conditions may cause the upregulation of certain chemokine receptors on early B cells, rendering them sensitive to extramedullary chemotactic cues. To test this hypothesis, we used human pre-B 697 cells to investigate whether various inflammatory agents could modify chemokine receptor expression and function. Chemotaxis to CXCL12 was observed for all B cell subsets. However, chemotactic responses to CCL19, CCL21, CXCL13, and CCL20 were limited to late-stage, IgM+ bone marrow B cells (immature B and mature B). Chemotactic responses to corresponding ligands correlated with the pattern of chemokine receptor expression. The expression of CCR7, however, was low on early (pro-B and pre-B) B cells and did not induce chemotaxis. Interestingly, both CCL19 and CCL21 could trigger ERK1/2 phosphorylation in early B cells. Exposure of pre-B 697 cells to TNF-alpha upregulated CCR7 and CXCR5 expression, whereas it had no effect on CCR6 surface expression. Correspondingly, TNF-alpha-stimulated pre-B cells chemotaxed towards CCL19 and CXCL13, in contrast to non-TNF-alpha-stimulated controls. We postulate that CXCR5, CCR7, and CCR6 participate in bone marrow trafficking and/or bone marrow egress of late-stage B cells under steady-state conditions, whereas inflammation-induced expression of CCR7 and CXCR5 may facilitate early B-cell emigration out of the bone marrow and their positioning in secondary lymphoid organs.

G-CSF potently inhibits osteoblast activity and CXCL12 mRNA expression in the bone marrow

AbstractAccumulating evidence indicates that interaction of stromal cell-derived factor 1 (SDF-1/CXCL12 [CXC motif, ligand 12]) with its cognate receptor, CXCR4 (CXC motif, receptor 4), generates signals that regulate hematopoietic progenitor cell (HPC) trafficking in the bone marrow. During granulocyte colony-stimulating factor (G-CSF)–induced HPC mobilization, CXCL12 protein expression in the bone marrow decreases. Herein, we show that in a series of transgenic mice carrying targeted mutations of their G-CSF receptor and displaying markedly different G-CSF–induced HPC mobilization responses, the decrease in bone marrow CXCL12 protein expression closely correlates with the degree of HPC mobilization. G-CSF treatment induced a decrease in bone marrow CXCL12 mRNA that closely mirrored the fall in CXCL12 protein. Cell sorting experiments showed that osteoblasts and to a lesser degree endothelial cells are the major sources of CXCL12 production in the bone marrow. Interestingly, osteoblast activity, as measured by histomorphometry and osteocalcin expression, is strongly down-regulated during G-CSF treatment. However, the G-CSF receptor is not expressed on osteoblasts; accordingly, G-CSF had no direct effect on osteoblast function. Collectively, these data suggest a model in which G-CSF, through an indirect mechanism, potently inhibits osteoblast activity resulting in decreased CXCL12 expression in the bone marrow. The consequent attenuation of CXCR4 signaling ultimately leads to HPC mobilization.

Bone Marrow Is a Reservoir for CD4+CD25+ Regulatory T Cells that Traffic through CXCL12/CXCR4 Signals

CD4(+)CD25(+) regulatory T cells (Tregs) mediate peripheral T-cell homeostasis and contribute to self-tolerance. Their homeostatic and pathologic trafficking is poorly understood. Under homeostatic conditions, we show a relatively high prevalence of functional Tregs in human bone marrow. Bone marrow strongly expresses functional stromal-derived factor (CXCL12), the ligand for CXCR4. Human Tregs traffic to and are retained in bone marrow through CXCR4/CXCL12 signals as shown in chimeric nonobese diabetic/severe combined immunodeficient mice. Granulocyte colony-stimulating factor (G-CSF) reduces human bone marrow CXCL12 expression in vivo, associated with mobilization of marrow Tregs to peripheral blood in human volunteers. These findings show a mechanism for homeostatic Treg trafficking and indicate that bone marrow is a significant reservoir for Tregs. These data also suggest a novel mechanism explaining reduced acute graft-versus-host disease and improvement in autoimmune diseases following G-CSF treatment.

Sustained activation of cell adhesion is a differentially regulated process in B lymphopoiesis.

It is largely unknown how hematopoietic progenitors are positioned within specialized niches of the bone marrow microenvironment during development. Chemokines such as CXCL12, previously called stromal cell–derived factor 1, are known to activate cell integrins of circulating leukocytes resulting in transient adhesion before extravasation into tissues. However, this short-term effect does not explain the mechanism by which progenitor cells are retained for prolonged periods in the bone marrow. Here we show that in human bone marrow CXCL12 triggers a sustained adhesion response specifically in progenitor (pro- and pre-) B cells. This sustained adhesion diminishes during B cell maturation in the bone marrow and, strikingly, is absent in circulating mature B cells, which exhibit only transient CXCL12-induced adhesion. The duration of adhesion is tightly correlated with CXCL12-induced activation of focal adhesion kinase (FAK), a known molecule involved in integrin-mediated signaling. Sustained adhesion of progenitor B cells is associated with prolonged FAK activation, whereas transient adhesion in circulating B cells is associated with short-lived FAK activation. Moreover, sustained and transient adhesion responses are differentially affected by pharmacological inhibitors of protein kinase C and phosphatidylinositol 3-kinase. These results provide a developmental cell stage–specific mechanism by which chemokines orchestrate hematopoiesis through sustained rather than transient activation of adhesion and cell survival pathways.