Insights into the mechanism of enhanced mobilization of hematopoietic progenitor cells and release of CXCL12 by a combination of AMD3100 and aminoglycoside-polyarginine conjugates

Mobilization of hematopoietic stem and progenitor cells (HSPCs) from the bone marrow to the peripheral blood is utilized in clinical HSPC transplantation protocols. Retention of HSPCs in the bone marrow is determined by relationships between the chemokine chemokine (C-X-C motif) ligand 12 (CXCL12) and its major receptor C-X-C chemokine receptor type 4 (CXCR4), and disruption of this retention by CXCR4 antagonists such as AMD3100 induces rapid HSPC mobilization. Here, we report that aminoglycoside-polyarginine conjugates (APACs) and N-α-acetyl-nona-D-arginine (r9) induce mobilization of white blood cells and, preferentially, immature hematopoietic progenitor cells (HPCs) in mice, similarly to AMD3100. Remarkably, administration of AMD3100 with each one of the APACs or r9 caused additional HPC mobilization. The mobilizing activity of APACs and r9 was accompanied by a significant elevation in plasma CXCL12 levels. To further understand how APACs, r9 and their combinations with AMD3100 compete with CXCL12 binding to CXCR4, as well with antibody against CXCR4 for CXCR4 binding, we have undertaken an approach combining experimental validation and docking to determine plausible binding modes for these ligands. On the basis of our biological and docking findings, and recently published NMR data, we suggest that combination of pairs of compounds such as APACs (or r9) with AMD3100 induces more efficient disruption of the CXCL12-CXCR4 interaction than AMD3100 alone, resulting in enhanced HPC mobilization.     

Neutrophil mobilization from the bone marrow during polymicrobial sepsis is dependent on CXCL12 signaling

Neutrophils are essential for successful host eradication of bacterial pathogens and for survival to polymicrobial sepsis. During inflammation, the bone marrow provides a large reserve of neutrophils that are released into the peripheral circulation where they traverse to sites of infection. Although neutrophils are essential for survival, few studies have investigated the mechanisms responsible for neutrophil mobilization from the bone marrow during polymicrobial sepsis. Using a cecal ligation and puncture model of polymicrobial sepsis, we demonstrated that neutrophil mobilization from the bone marrow is not dependent on TLR4, MyD88, TRIF, IFNARα/β, or CXCR2 pathway signaling during sepsis. In contrast, we observed that bone marrow CXCL12 mRNA abundance and specific CXCL12 levels are sharply reduced, whereas splenic CXCR4 mRNA and cell surface expression are increased during sepsis. Blocking CXCL12 activity significantly reduced blood neutrophilia by inhibiting bone marrow release of granulocytes during sepsis. However, CXCL12 inhibition had no impact on the expansion of bone marrow neutrophil precursors and hematopoietic progenitors. Bone marrow neutrophil retention by CXCL12 blockade prevented blood neutrophilia, inhibited peritoneal neutrophil accumulation, allowed significant peritoneal bacterial invasion, and increased polymicrobial sepsis mortality. We concluded that changes in the pattern of CXCL12 signaling during sepsis are essential for neutrophil bone marrow mobilization and host survival but have little impact on bone marrow granulopoiesis.     

Transgene expression study of CXCR4 active mutants: Potential prospects in up-modulation of homing and engraftment efficiency of hematopoietic stem/progenitor cells

Homing and engraftment, a determining factor in hematopoietic stem cell transplantation success is defined as a process through which hematopoietic stem/progenitor cells (HSPCs) lodge recipient bone marrow. SDF-1/CXCR4 axis acts as a principle regulator in homing and engraftment, however, CXCR4 signaling is dependent upon expression of CXCR4 and its ligand SDF-1, which is highly dynamic. Hence, present investigation was aimed to explore the potential of CXCR4 constitutive active mutants (CXCR4-CAMs) in overcoming the limitation of CXCR4 signaling and up-modulate its efficiency in homing and engraftment. Regulated transgene expression study of these mutants revealed their significantly enhanced cell adhesion efficiency to endothelium and extracellular matrix protein. This altogether indicates promising prospects of CXCR4-CAMs in research aimed to improve HSPCs engraftment efficiency.     

Transgene expression study of CXCR4 active mutants

Homing and engraftment, a determining factor in hematopoietic stem cell transplantation success is defined as a process through which hematopoietic stem/progenitor cells (HSPCs) lodge recipient bone marrow. SDF-1/CXCR4 axis acts as a principle regulator in homing and engraftment, however, CXCR4 signaling is dependent upon expression of CXCR4 and its ligand SDF-1, which is highly dynamic. Hence, present investigation was aimed to explore the potential of CXCR4 constitutive active mutants (CXCR4-CAMs) in overcoming the limitation of CXCR4 signaling and up-modulate its efficiency in homing and engraftment. Regulated transgene expression study of these mutants revealed their significantly enhanced cell adhesion efficiency to endothelium and extracellular matrix protein. This altogether indicates promising prospects of CXCR4-CAMs in research aimed to improve HSPCs engraftment efficiency.     

BIGH3 modulates adhesion and migration of hematopoietic stem and progenitor cells

Cell adhesion and migration are important determinants of homing and development of hematopoietic stem and progenitor cells (HSPCs) in bone marrow (BM) niches. The extracellular matrix protein transforming growth factor-β (TGF-β) inducible gene H3 (BIGH3) is involved in adhesion and migration, although the effect of BIGH3 is highly cell type-dependent. BIGH3 is abundantly expressed by mesenchymal stromal cells, while its expression in HSPCs is relatively low unless induced by certain BM stressors. Here, we set out to determine how BIGH3 modulates HSPC adhesion and migration. We show that primary HSPCs adhere to BIGH3-coated substrates, which is, in part, integrin-dependent. Overexpression of BIGH3 in HSPCs and HL60 cells reduced the adhesion to the substrate fibronectin in adhesion assays, which was even more profound in electrical cell-substrate impedance sensing (ECIS) assays. Accordingly, the CXCL12 induced migration over fibronectin-coated surface was reduced in BIGH3-expressing HSPCs. The integrin expression profile of HSPCs was not altered upon BIGH3 expression. Although expression of BIGH3 did not alter actin polymerization in response to CXCL12, it inhibited the PMA-induced activation of the small GTPase RAC1 as well as the phosphorylation and activation of extracellular-regulated kinases (ERKs). Reduced activation of ERK and RAC1 may be responsible for the inhibition of cell adhesion and migration by BIGH3 in HSPCs. Induced BIGH3 expression upon BM stress may contribute to the regulation of BM homeostasis.     

CXC chemokine ligand 12-induced focal adhesion kinase activation and segregation into membrane domains is modulated by regulator of G protein signaling 1 in pro-B cells

CXCL12-induced chemotaxis and adhesion to VCAM-1 decrease as B cells differentiate in the bone marrow. However, the mechanisms that regulate CXCL12/CXCR4-mediated signaling are poorly understood. We report that after CXCL12 stimulation of progenitor B cells, focal adhesion kinase (FAK) and PI3K are inducibly recruited to raft-associated membrane domains. After CXCL12 stimulation, phosphorylated FAK is also localized in membrane domains. The CXCL12/CXCR4-FAK pathway is membrane cholesterol dependent and impaired by metabolic inhibitors of G(i), Src family, and the GTPase-activating protein, regulator of G protein signaling 1 (RGS1). In the bone marrow, RGS1 mRNA expression is low in progenitor B cells and high in mature B cells, implying developmental regulation of CXCL12/CXCR4 signaling by RGS1. CXCL12-induced chemotaxis and adhesion are impaired when FAK recruitment and phosphorylation are inhibited by either membrane cholesterol depletion or overexpression of RGS1 in progenitor B cells. We conclude that the recruitment of signaling molecules to specific membrane domains plays an important role in CXCL12/CXCR4-induced cellular responses.     

Signals from the sympathetic nervous system regulate hematopoietic stem cell egress from bone marrow

Hematopoietic stem and progenitor cells (HSPC), attracted by the chemokine CXCL12, reside in specific niches in the bone marrow (BM). HSPC migration out of the BM is a critical process that underlies modern clinical stem cell transplantation. Here we demonstrate that enforced HSPC egress from BM niches depends critically on the nervous system. UDP-galactose ceramide galactosyltransferase-deficient (Cgt(-/-)) mice exhibit aberrant nerve conduction and display virtually no HSPC egress from BM following granulocyte colony-stimulating factor (G-CSF) or fucoidan administration. Adrenergic tone, osteoblast function, and bone CXCL12 are dysregulated in Cgt(-/-) mice. Pharmacological or genetic ablation of adrenergic neurotransmission indicates that norepinephrine (NE) signaling controls G-CSF-induced osteoblast suppression, bone CXCL12 downregulation, and HSPC mobilization. Further, administration of a beta(2) adrenergic agonist enhances mobilization in both control and NE-deficient mice. Thus, these results indicate that the sympathetic nervous system regulates the attraction of stem cells to their niche.     

SDF-1 and CXCR4 in normal and malignant hematopoiesis

Over recent years it has become apparent that the chemokine SDF-1 and its receptor CXCR4 play pivotal roles in normal hematopoiesis. They are essential for the normal ontogeny of hematopoiesis during embryogenesis and continue to play a key role in retaining hematopoietic progenitors within the bone marrow microenvironment in the adult. As a result of this role disruption of SDF-1/CXCR4 interactions results in mobilization of hematopoietic progenitors and standard mobilization protocols disrupt this axis. Similarly SDF-1/CXCR4 interactions are required for homing and engraftment of hematopoietic stem cells during transplantation. SDF-1 regulates the localisation of leukemic cells and like their normal counterparts most leukemic cells respond to SDF-1 with increased adhesion, survival and proliferation. However in some instances leukemic cell responses to SDF-1 can be disregulated, the impact of which on the progression of disease in not known. In this review we discuss the pleiotropic roles of SDF-1/CXCR4 interactions in human hematopoietic stem cell ontogeny, bone marrow homing and engraftment, mobilization and how these interactions impact on malignant hematopoiesis.     

Hyaluronan-based polymer scaffold modulates the expression of inflammatory and degradative factors in mesenchymal stem cells: Involvement of Cd44 and Cd54

Hyaluronan (HA), in the bone marrow stroma, is the major non-protein glycosaminoglycan component of extracellular matrix (ECM) involved in cell positioning, proliferation, differentiation as well as in receptor-mediated changes in gene expression. Repair of bone and regeneration of bone marrow is dependent on ECM, inflammatory factors, like chemokines and degradative factors, like metalloproteinases. We analyzed the interaction between human mesenchymal stem cells (h-MSCs) and a three-dimensional (3-D) HA-based scaffold in vitro. The expression of CXC chemokines/receptors, CXCL8 (IL-8)/CXCR1-2, CXCL10 (IP-10)/CXCR3, CXCL12 (SDF-1)/CXCR4, and CXCL13 (BCA-1)/CXCR5, and metalloproteinases/inhibitors MMP-1, MMP-3, MMP-13/TIMP-1 were evaluated in h-MSCs grown on plastic or on HA-based scaffold by Real-time PCR, ELISA, and immunocytochemical techniques. Moreover, the expression of two HA receptors, CD44 and CD54, was analyzed. We found both at mRNA and protein levels that HA-based scaffold induced the expression of CXCR4, CXCL13, and MMP-3 and downmodulated the expression of CXCL12, CXCR5, MMP-13, and TIMP-1 while HA-based scaffold induced CD54 expression but not CD44. We found that these two HA receptors were directly involved in the modulation of CXCL12, CXCL13, and CXCR5. This study demonstrates a direct action of a 3-D HA-based scaffold, widely used for cartilage and bone repair, in modulating both h-MSCs inflammatory and degradative factors directly involved in the engraftment of specific cell types in a damaged area. Our data clearly demonstrate that HA in this 3-D conformation acts as a signaling molecule for h-MSCs.     

To stay or to leave: Stem cells and progenitor cells navigating the S1P gradient

Most hematopoietic stem progenitor cells (HSPCs) reside in bone marrow (BM), but a small amount of HSPCs have been found to circulate between BM and tissues through blood and lymph. Several lines of evidence suggest that sphingosine-1-phosphate (S1P) gradient triggers HSPC egression to blood circulation after mobilization from BM stem cell niches. Stem cells also visit certain tissues. After a temporary 36 h short stay in local tissues, HSPCs go to lymph in response to S1P gradient between lymph and tissue and eventually enter the blood circulation. S1P also has a role in the guidance of the primitive HSPCs homing to BM in vivo, as S1P analogue FTY720 treatment can improve HSPC BM homing and engraftment. In stress conditions, various stem cells or progenitor cells can be attracted to local injured tissues and participate in local tissue cell differentiation and tissue rebuilding through modulation the expression level of S1P1, S1P2 or S1P3 receptors. Hence, S1P is important for stem cells circulation in blood system to accomplish its role in body surveillance and injury recovery.     

ZAP-70 Promotes the Infiltration of Malignant B-Lymphocytes into the Bone Marrow by Enhancing Signaling and Migration after CXCR4 Stimulation

ZAP-70 in chronic lymphocytic leukemia (CLL) is associated with enhanced response to microenvironmental stimuli. We analyzed the functional consequences of ZAP-70 ectopic expression in malignant B-cells in a xenograft mouse model of disseminated B-cell leukemia. Mice injected with B-cells expressing ZAP-70 showed a prominently higher infiltration of the bone marrow. In vitro analysis of the response of malignant B-cells to CXCL12, the main attracting chemokine regulating trafficking of lymphocytes to the bone marrow, or to bone marrow stromal cells, revealed that ZAP-70 induces an increased response in terms of signaling and migration. These effects are probably mediated by direct participation of ZAP-70 in CXCL12-CXCR4 signaling since CXCR4 stimulation led to activation of ZAP-70 and downstream signaling pathways, such as MAPK and Akt, whereas ZAP-70 did not alter the expression of the CXCR4 receptor. In addition, subclones of primary CLL cells with high expression of ZAP-70 also showed increased migrative capacity toward CXCL12. Neutralization of CXCR4 with a monoclonal antibody resulted in impaired in vitro responses to CXCL12 and bone marrow stromal cells. We conclude that ZAP-70 enhances the migration of malignant B-cells into the supportive microenvironment found in the bone marrow mainly by enhancing signaling and migration after CXCR4 stimulation.     

The role of monocytosis and neutrophilia in atherosclerosis

Monocytosis and neutrophilia are frequent events in atherosclerosis. These phenomena arise from the increased proliferation of hematopoietic stem and multipotential progenitor cells (HSPCs) and HSPC mobilization from the bone marrow to other immune organs and circulation. High cholesterol and inflammatory signals promote HSPC proliferation and preferential differentiation to the myeloid precursors (i.e., myelopoiesis) that than give rise to pro‐inflammatory immune cells. These cells accumulate in the plaques thereby enhancing vascular inflammation and contributing to further lesion progression. Studies in animal models of atherosclerosis showed that manipulation with HSPC proliferation and differentiation through the activation of LXR‐dependent mechanisms and restoration of cholesterol efflux may have a significant therapeutic potential.     

CXCL12/CXCR4 transactivates HER2 in lipid rafts of prostate cancer cells and promotes growth of metastatic deposits in bone

Chemokines and their receptors function in migration and homing of cells to target tissues. Recent evidence suggests that cancer cells use a chemokine receptor axis for metastasis formation at secondary sites. Previously, we showed that binding of the chemokine CXCL12 to its receptor CXCR4 mediated signaling events resulting in matrix metalloproteinase-9 expression in prostate cancer bone metastasis. A variety of methods, including lipid raft isolation, stable overexpression of CXCR4, cellular adhesion, invasion assays, and the severe combined immunodeficient-human bone tumor growth model were used. We found that (a) CXCR4 and HER2 coexist in lipid rafts of prostate cancer cells; (b) the CXCL12/CXCR4 axis results in transactivation of the HER2 receptor in lipid rafts of prostate cancer cells; (c) Src kinase mediates CXCL12/CXCR4 transactivation of HER2 in prostate cancer cells; (d) a pan-HER inhibitor desensitizes CXCR4-induced transactivation and subsequent matrix metalloproteinase-9 secretion and invasion; (e) lipid raft-disrupting agents inhibited raft-associated CXCL12/CXCR4 transactivation of the HER2 and cellular invasion; (f) overexpression of CXCR4 in prostate cancer cells leads to increased HER2 phosphorylation and migratory properties of prostate cancer cells; and (g) CXCR4 overexpression enhances bone tumor growth and osteolysis. These data suggest that lipid rafts on the cell membrane are the key site for CXCL12/CXCR4-induced HER2 receptor transactivation. This transactivation contributes to enhanced invasive signals and metastatic growth in the bone microenvironment.     

A genetic approach to inactivating chemokine receptors using a modified viral protein

We have developed a genetic system, called degrakine, that specifically and stably inactivates chemokine receptors (CKR) by redirecting the ability of the HIV-1 protein, Vpu, to degrade CD4 in the endoplasmic reticulum (ER) via the host proteasome machinery. To harness Vpu's proteolytic targeting capability to degrade new receptors, we fused a chemokine with the C terminal region of Vpu. The fusion protein, or degrakine, accumulates in the ER, trapping and functionally inactivating its target CKR. We have demonstrated that degrakines based on SDF-1 (CXCL12), MDC (CCL22) and RANTES (CCL5) specifically inactivate their respective receptor functions. Using a retroviral vector expressing the SDF-1 degrakine, we have established that CXCR4 is required for the homing of hematopoietic stem/progenitor cells (HSPC) to the bone marrow immediately after transplantation. Thus the degrakine provides an effective genetic tool to dissect receptor functions in a number of biological systems in vitro and in vivo.     

Cutting edge: profile of chemokine receptor expression on human plasma cells accounts for their efficient recruitment to target tissues

We systematically examined the repertoire of chemokine receptors expressed by human plasma cells. Fresh bone marrow plasma cells and myeloma cells consistently expressed CXCR4, CXCR6, CCR10, and CCR3. Accordingly, plasma cells responded to their respective ligands in chemotaxis and very late Ag-4-dependent cell adhesion to fibronectin. Immobilized CXC chemokine ligand (CXCL)16, a novel transmembrane-type chemokine and CXCR6 ligand, also directly induced adhesion of plasma cells without requiring G(alpha i) signaling or divalent cations. Furthermore, we revealed consistent expression of CXCL12 (CXCR4 ligand), CXCL16 (CXCR6 ligand), and CC chemokine ligand 28 (CCR10 and CCR3 ligand) in tissues enriched with plasma cells including bone marrow, and constitutive expression of CXCL12, CXCL16, and CC chemokine ligand 28 by cultured human bone marrow stromal cells. Collectively, plasma cells are likely to be recruited to bone marrow and other target tissues via CXCR4, CXCR6, CCR10, and CCR3. CXCR6 may also contribute to tissue localization of plasma cells through its direct binding to membrane-anchored CXCL16.     

The cholinergic system is involved in regulation of the development of the hematopoietic system

Gene expression profiling demonstrated that components of the cholinergic system, including choline acetyltransferase, acetylcholinesterase and nicotinic acetylcholine receptors (nAChRs), are expressed in embryonic stem cells and differentiating embryoid bodies (EBs). Triggering of nAChRs expressed in EBs by nicotine resulted in activation of MAPK and shifts of spontaneous differentiation toward hemangioblast. In vivo, non-neural nAChRs are detected early during development in fetal sites of hematopoiesis. Similarly, in vivo exposure of the developing embryo to nicotine resulted in higher numbers of hematopoietic progenitors in fetal liver. However postpartum, the number of hematopoietic stem/progenitor cells (HSPC) was decreased, suggesting an impaired colonization of the fetal bone marrow with HSPCs. This correlated with increased number of circulating HSPC and decreased expression of CXCR4 that mediates migration of circulating cells into the bone marrow regulatory niche. In addition, protein microarrays demonstrated that nicotine changed the profile of cytokines produced in the niche. While the levels of IL1alpha, IL1beta, IL2, IL9 and IL10 were not changed, the production of hematopoiesis-supportive cytokines including G-CSF, GM-CSF, IL3, IL6 and IGFBP-3 was decreased. This correlated with the decreased repopulating ability of HSPC in vivo and diminished hematopoietic activity in bone marrow cultures treated with nicotine. Interestingly, nicotine stimulated the production of IL4 and IL5, implying a possible role of the cholinergic system in pathogenesis of allergic diseases. Our data provide evidence that the nicotine-induced imbalance of the cholinergic system during gestation interferes with normal development and provides the basis for negative health outcomes postpartum in active and passive smokers.     

Human osteoclasts express different CXC chemokines depending on cell culture substrate: molecular and immunocytochemical evidence of high levels of CXCL10 and CXCL12

Chemokines are important mediators of chemotaxis, cell adherence, and proliferation and exert specific functions in bone remodeling. Despite the potential intriguing role of chemokines in the regulation of osteoclast (OC) functions, little is known about the expression of chemokines and their receptors in human OCs at different stages of differentiation. Therefore, we analyzed the expression of CXC chemokine receptors (CXCR1, CXCR2, CXCR3, CXCR4 and CXCR5) and ligands (CXCL8, CXCL10, CXCL12 and CXCL13) both at molecular and protein levels, in human OCs grown on plastic or calcium phosphate-coated slides at different stages of differentiation. Real-time PCR showed that CXCR1, CXCR2, CXCR3, CXCR4, CXCR5 and CXCL8 were expressed in undifferentiated cells and significantly decreased during OC differentiation. By contrast, CXCL10 and CXCL12 were strongly upregulated from day 0 to day 8 in cells grown on calcium phosphate-coated slides. Immunocytochemistry showed that OCs grown on plastic expressed CXCR3, CXCR4, CXCR5, CXCL8 and CXCL12, while they were negative for CXCR1, CXCR2 and CXCL10. Interestingly, both at molecular and protein levels CXCL10 and CXCL12 significantly increased only when cells were differentiated on calcium phosphate-coated slides. These data suggest that the selection of a substrate that better mimics the tridimensional structure of bone tissue, thus favoring OC maturation and differentiation, may be necessary when studying osteoclastogenesis in vitro.     

Cellular characteristics of neuroblastoma cells: regulation by the ELR--CXC chemokine CXCL10 and expression of a CXCR3-like receptor

Bone marrow stroma cells secrete the chemokine CXCL12 that may support bone marrow metastasis formation by neuroblastoma cells. The present study demonstrates that bone marrow stroma cell lines also secrete CXCL10, a chemokine that was shown in the past to have anti-malignancy functions. A receptor recognized by antibodies against CXCR3 was shown to be expressed by six neuroblastoma cell lines. Further detailed analysis was performed on the NUB6 and SK-NMC neuroblastoma cells, showing that CXCL10 induced potent Erk phosphorylation in a G(alpha)i-dependent manner. The role of a CXCR3-like receptor in Erk phosphorylation was substantiated by the ability of CXCL11, another potent CXCR3 ligand, to induce Erk phosphorylation in the NUB6 and SK-NMC cells. Further characterization of CXCL10 activities indicated that CXCL10 partly inhibited the growth of the NUB6 and SK-NMC cells. Both NUB6 and SK-NMC cells did not migrate to CXCL10, although their migratory machinery was intact, as evidenced by their migration to bone marrow constituents. Altogether, these results suggest that CXCL10 interacts with a CXCR3-like receptor in neuroblastoma cell lines, raising the possibility that following the homing of the tumor cells to the bone marrow (through a CXCL10-independent mechanism), CXCL10 may partly inhibit neuroblastoma cell growth at this site.