Peripheral blood stem cell mobilization. A role for CXC chemokines

Chemokines induce rapid hematopoietic stem and progenitor cell mobilization and synergize with hematopoietic cytokines in mobilizing stem and progenitor cells. These proteins alone and in combination offer new paradigms for autologous and allogeneic peripheral blood stem cell transplantation (PBSCT). The mechanisms responsible for hematopoietic stem cell (HSC) mobilization either with growth factors or chemokines are largely unknown, but a better understanding of these mechanisms will permit the development of novel, more rapid and efficacious regimens. Studies presented herein indicate that the CXCR2 chemokine receptor that interacts with selective chemokine ligands, particularly GRObeta/CXCL2 and GRObeta-T, may be the dominant receptor mediating hematopoietic cell mobilization, and that polymorphonuclear neutrophils may be the primary CXCR2 expressing target cell for stem and progenitor cell mobilization.     

Sulfated polysaccharides increase plasma levels of SDF-1 in monkeys and mice: involvement in mobilization of stem/progenitor cells.

It was previously reported that treatment with the sulfated polysaccharide fucoidan or the structurally similar dextran sulfate increased circulating mature white blood cells and hematopoietic progenitor/stem cells (HPCs) in mice and nonhuman primates; however, the mechanism mediating these effects was unclear. It is reported here that plasma concentrations of the highly potent chemoattractant stromal-derived factor 1 (SDF-1) increase rapidly and dramatically after treatment with fucoidan in monkeys and in mice, coinciding with decreased levels in bone marrow. In vitro and in vivo data suggest that the SDF-1 increase is due to its competitive displacement from heparan sulfate proteoglycans that sequester the chemokine on endothelial cell surfaces or extracellular matrix in bone marrow and other tissues. Although moderately increased levels of interleukin-8, MCP1, or MMP9 were also present after fucoidan treatment, studies in gene-ablated mice (GCSFR(-/-), MCP1(-/-), or MMP9(-/-)) and the use of metalloprotease inhibitors do not support their involvement in the concurrent mobilization. Instead, SDF-1 increases, uniquely associated with sulfated glycan-mobilizing treatments and not with several other mobilizing agents tested, are likely responsible. To the authors' knowledge, this is the first published report of disrupting the SDF-1 gradient between bone marrow and peripheral blood through a physiologically relevant mechanism, resulting in mobilization with kinetics similar to other mobilizing CXC chemokines. The study further underscores the importance of the biological roles of carbohydrates.     

Sulfated glycans induce rapid hematopoietic progenitor cell mobilization: evidence for selectin-dependent and independent mechanisms

The adhesive mechanisms leading to the mobilization of hematopoietic progenitor cells (HPCs) from the bone marrow into the blood are poorly understood. We report on a role for selectins and fucoidan in progenitor mobilization. Baseline levels of circulating HPCs are increased in endothelial selectin-deficient (P/E-/-) mice. Similar levels are observed when E-selectin null (E-/-) mice are treated with anti-P-selectin antibody or with fucoidan (which inhibits P- and L-selectin function). In particular, administration of 2 doses of fucoidan (25 mg/kg) over 6 hours produces profound mobilization of progenitors in wild-type mice and the response is greatly enhanced in E-/- and P/E-/- mice. Competitive reconstitution experiments reveal that fucoidan also elicits long-term (more than 6 months) repopulating stem cells. Mobilization assays using chimeric mice harboring L-selectin-deficient progenitors and wild-type progenitors expressing the green fluorescence protein suggest that L-selectin expression is not required but confers an advantage for fucoidan-induced mobilization. Sulfation is critical as desulfated fucoidan is ineffective. In addition, sulphogalactosylceramide (sulfatide) but not heparin can induce HPC mobilization. Our results indicate that administration of sulfated glycans, especially with concurrent inhibition of E-selectin function, represents a powerful novel method for rapid mobilization of long-term-repopulating stem cells. These findings may help elucidate the mechanisms of HPC trafficking during development and adult life.     

Current understanding of stem cell mobilization: the roles of chemokines,proteolytic enzymes,adhesion molecules,cytokines, and stromal cells

Mobilization of hematopoietic stem and progenitor cells from the bone marrow into the circulation by repetitive, daily stimulations with G-CSF alone, or in combination with cyclophosphamide, is increasingly used clinically; however, the mechanism is not fully understood. Moreover, following mobilization stem cells also home back to the bone marrow, suggesting that stem cell release/mobilization and homing are sequential events with physiological roles. Previously, a role for cytokines such as G-CSF and SCF, and adhesion molecules such as VLA-4 and P/E selectins, was determined for stem cell mobilization. Recent results using experimental animal models and samples from clinical mobilization protocols demonstrate major involvement of chemokines such as stromal derived factor-1 (SDF-1) and IL-8, as well as proteolytic enzymes such as elastase, cathepsin G, and various MMPs in the mobilization process. These results will be reviewed together with the central roles of SDF-1 and CXCR4 interactions in G-CSF or G-CSF in combination with cyclophosphamide-induced mobilization. Furthermore, the central role of this chemokine in stem cell homing to the bone marrow as well as retention of undifferentiated cells within this tissue will also be discussed.     

Regulation of hematopoiesis in a sea of chemokine family members with a plethora of redundant activities.

The field of chemokine biology is a rapidly advancing one, with over 50 chemokines identified that mediate their effects through one or more of 16 different chemokine receptors. Chemokines, originally identified as chemotactic cytokines, manifest a number of functions, including modulation of blood cell production at the level of hematopoietic stem/progenitor cells and the directed movement of these early blood cells. This report reviews chemokines and chemokine/receptor activities mainly in the context of hematopoietic cell regulation and the numerous chemokines that manifest suppressive activity on proliferation of stem/progenitor cells. This is contrasted with the specificity of only a few chemokines for the chemotaxis of these early cells. The large number of chemokines with suppressive activity is hypothesized to reflect the different cell, tissue, and organ sites of production of these chemokines and the need to control stem/progenitor cell proliferation in different organ sites throughout the body.     

Heparan sulfate mimetics can efficiently mobilize long-term hematopoietic stem cells

Background

Although mobilization of hematopoietic stem cells and hematopoietic progenitor cells can be achieved with a combination of granulocyte colony-stimulating factor and plerixafor (AMD3100), improving approaches for hematopoietic progenitor cell mobilization is clinically important.

Design and Methods

Heparan sulfate proteoglycans are ubiquitous macromolecules associated with the extracellular matrix that regulates biology of hematopoietic stem cells. We studied the effects of a new family of synthetic oligosaccharides mimicking heparan sulfate on hematopoietic stem cell mobilization. These oligosaccharides were administered intravenously alone or in combination with granulocyte colony-stimulating factor and/or AMD3100 in mice. Mobilized hematopoietic cells were counted and phenotyped at different times and the ability of mobilized hematopoietic stem cells to reconstitute long-term hematopoiesis was determined by competitive transplantation into syngenic lethally irradiated mice followed by secondary transplantation.

Results

Mimetics of heparan sulfate induced rapid mobilization of B-lymphocytes, T-lymphocytes, hematopoietic stem cells and hematopoietic progenitor cells. They increased the mobilization of hematopoietic stem cells and hematopoietic progenitor cells more than 3-fold when added to the granulocyte colony-stimulating factor/AMD3100 association. Hematopoietic stem cells mobilized by mimetics of heparan sulfate or by the granulocyte colony-stimulating factor/AMD3100/mimetics association were as effective as hematopoietic stem cells mobilized by the granulocyte colony-stimulating factor/AMD3100 association for primary and secondary hematopoietic reconstitution of lethally irradiated mice.

Conclusions

This new family of mobilizing agents could alone or in combination with granulocyte colony-stimulating factor and/or AMD3100 mobilize a high number of hematopoietic stem cells that were able to maintain long-term hematopoiesis. These results strengthen the role of heparan sulfates in the retention of hematopoietic stem cells in bone marrow and support the use of small glyco-drugs based on heparan sulfate in combination with granulocyte colony-stimulating factor and AMD3100 to improve high stem cell mobilization, particularly in a prospect of use in human therapeutics.     

Mobilization of hematopoietic stem and progenitor cell subpopulations from the marrow to the blood of mice following cyclophosphamide and/or granulocyte colony-stimulating factor

Committed progenitor cells and primitive stem cells mediate early and sustained engraftment, respectively, after lethal irradiation and stem cell transplantation. Peripheral blood stem cells (PBSC) from unstimulated mice are deficient in both cell types. To study techniques to mobilize both progenitor cells and primitive stem cells from the marrow to the blood, we collected peripheral blood from C57BL/6 mice 6 to 7 days after a single dose of cyclophosphamide (CY; 200 mg/kg intraperitoneally), after recombinant human granulocyte colony- stimulating factor (rhG-CSF) (250 micrograms/kg/d twice per day subcutaneously for 4 days), or after CY followed by G-CSF. Significant increases in white blood cell counts (1.6- to 2.7-fold) and circulating day 8 colony-forming unit spleen (CFU-S) (11- to 36-fold) were seen with all three mobilization methods compared with unstimulated control mice. Transplantation of mobilized blood stem cells into lethally irradiated hosts decreased the time to erythroid engraftment. Blood stem cells were analyzed for primitive stem cell content by Rs, an assay for CFU-S self-renewal, and competitive repopulation index (CRI), an assay of long-term repopulating ability. The primitive stem cell content of unstimulated blood was clearly deficient, but was significantly increased following mobilization, approaching normal bone marrow levels. These results were confirmed by an in vitro limiting dilution long-term culture assay that measures the frequency of progenitor cells and primitive stem cells. Mobilization following CY + G-CSF was accompanied by a marked loss of both progenitor cells and primitive stem cells in the marrow. In contrast, following G-CSF alone the progenitor cell and primitive stem cell content of the marrow was unchanged. Stem cell mobilization following CY + G-CSF was not affected by previous exposure of donors to cytosine arabinoside or cyclophosphamide, but was significantly reduced by previous exposure to busulfan. These data show that stem cell content in the blood may reach near-normal marrow levels after mobilization, the mobilization from the marrow to the blood is temporary and reversible, the specific technique used may mobilize different subpopulations of stem cells, and the type of prior chemotherapy may influence the ability to mobilize stem cells into the blood.     

Chemokines in hematopoiesis

PURPOSE OF REVIEW: Understanding the regulation of hematopoiesis is important for enhanced efficacy of hematopoietic stem and progenitor cell transplantation. Chemokines influence migration, survival, and other actions of hematopoietic stem and progenitor cells. This article summarizes recent progress in understanding the production and actions of chemokines and chemokine receptors, with an emphasis on the SDF-1/CXCL12-CXCR4 axis. RECENT FINDINGS: The literature from 2006 to the present is replete with information on SDF-1/CXCL12 activity, including induced intracellular signaling in hematopoietic progenitor cells, lymphocytes, other innate immune cells, breast cancer, and other tumor cells, and on production of SDF-1/CXCL12, and CXCR4, as well as on actions/production of other chemokines. Studies describing these intense research areas are discussed. SUMMARY: Chemokine-chemokine receptor interactions are important to hematopoiesis and immune cell function, two highly interactive processes. Recent studies have clarified the role of chemokines and their receptors in regulating hematopoiesis, and agents modulating chemokines are being evaluated in preclinical and clinical studies. Examples of such efforts include inhibition of CD26 for enhanced homing and engraftment of hematopoietic stem and progenitor cells, and the use of the SDF-1/CXCL12-CXCR4 antagonist, AMD3100 for mobilization of hematopoietic stem and progenitor cells and their use for stem cell transplantation.     

Fucoidan ingestion increases the expression of CXCR4 on human CD34+ cells

OBJECTIVE: Transplantation of hematopoietic progenitor stem cells (HPC) is an important treatment modality for a variety of neoplastic diseases. HPC collection for transplantation with granulocyte colony-stimulating factor may be unsuccessful in patients who have received prior chemotherapy or for other reasons. Methods to improve mobilization of HPCs are required. Disruption of the interaction between the cell surface receptor CXCR4 and its ligand stromal derived factor-1 (SDF-1) is a mechanism for HPC release from the bone marrow into the peripheral blood (PB). METHODS: We carried out a clinical trial to evaluate the effects of ingestion of a fucoidan, galactofucan sulfate (a putative HPC mobilizing agent) on circulating CD34(+) cells, CXCR4 expression, and levels of SDF-1, interferon gamma (IFN-gamma) and interleukin 12. RESULTS: Following ingestion of fucoidan, CD34(+) cells increased significantly in the PB from 1.64 to 1.84 cells/microL after 4 days. The proportion of CD34(+) cells that expressed CXCR4 increased from 45 to 90% after 12 days, the plasma level of SDF-1 increased from 1978 to 2010 pg/mL, and IFN-gamma level increased from 9.04 to 9.89 pg/mL. CONCLUSION: Oral fucoidan significantly amplified the CXCR4(+) HPC population. The ability to mobilize HPC using sulfated polysaccharides and mobilize more HPC with high levels of CXCR4 could be clinically valuable.     

The role of G-protein signaling in hematopoietic stem/progenitor cell mobilization

The directed migration of mature leukocytes to inflammatory sites and the lymphocyte trafficking in vivo are dependent on G protein-coupled receptors and delivered through pertussis toxin (Ptx)-sensitive Gi-protein signaling. In the present study, we explored the in vivo role of G-protein signaling on the redistribution or mobilization of hematopoietic stem/progenitor cells (HPCs). A single injection of Ptx in mice elicits a long-lasting leukocytosis and a progressive increase in circulating colony-forming unit-culture (CFU-C) and colony-forming unit spleen (CFU-S). We found that the prolonged effect is sustained by a continuous slow release of Ptx bound to red blood cells or other cells and is potentially enhanced by an indirect influence on cell proliferation. Plasma levels of certain cytokines (interleukin 6 [IL-6], granulocyte colony-stimulating factor [G-CSF]) increase days after Ptx treatment, but these are unlikely initiators of mobilization. In addition to normal mice, mice genetically deficient in monocyte chemotactic protein 1 (MCP-1), matrix metalloproteinase 9 (MMP-9), G-CSF receptor, beta2 integrins, or selectins responded to Ptx treatment, suggesting independence of Ptx-response from the expression of these molecules. Combined treatments of Ptx with anti-very late activation antigen (anti-VLA-4), uncovered potentially important insight in the interplay of chemokines/integrins, and the synergy of Ptx with G-CSF appeared to be dependent on MMP-9. As Ptx-mobilized kit+ cells display virtually no response to stromal-derived factor 1 (SDF-1) in vitro, our data suggest that disruption of CXCR4/SDF-1 signaling may be the underlying mechanism of Ptx-induced mobilization and indirectly reinforce the notion that active signaling through this pathway is required for continuous retention of cells within the bone marrow. Collectively, our data unveil a novel example of mobilization through pharmacologic modulation of signaling.     

Plasma elevation of stromal cell-derived factor-1 induces mobilization of mature and immature hematopoietic progenitor and stem cells

The chemokine, stromal cell-derived factor-1 (SDF1), is produced in the bone marrow and has been shown to modulate the homing of stem cells to this site by mediating chemokinesis and chemotaxis. Therefore, it was hypothesized that elevation of SDF1 level in the peripheral circulation would result in mobilization of primitive hematopoietic stem and progenitor cells. SDF1 plasma level was increased by intravenous injection of an adenoviral vector expressing SDF1alpha (AdSDF1) into severe combined immunodeficient mice. This resulted in a 10-fold increase in leukocyte count, a 3-fold increase in platelets, and mobilization of progenitors, including colony-forming units-granulocyte-macrophage to the peripheral circulation. In addition, AdSDF1 induced mobilization of cells with stem cell potential, including colony-forming units in spleen and long-term reconstituting cells. These data demonstrate that overexpression of SDF1 in the peripheral circulation results in the mobilization of hematopoietic cells with repopulating capacity, progenitor cells, and precursor cells. These studies lay the foundation for using SDF1 to induce mobilization of hematopoietic stem and progenitor cells in in vivo studies. (Blood. 2001;97:3354-3360)     

Progenitor cell mobilization by granulocyte colony-stimulating factor controlled by loci on chromosomes 2 and 11

Granulocyte colony-stimulating factor (G-CSF) can effectively mobilize hematopoietic stem and progenitor cells from bone marrow into blood, thereby allowing peripheral blood stem cells (PBSCs) to be used for transplantation. The efficiency of PBSC mobilization response to G-CSF is a multigene trait. DBA/2 (high-responder) and C57BL/6 (low-responder) mice were used for a genetic analysis of G-CSF-induced progenitor release. Significant linkages were found on chromosome 2 by analyzing segregation distortion among the high responders of 500 backcross mice and on chromosome 11 by using the quantitative trait locus analysis of 26 strains of BXD recombinant inbred mice. (Blood. 2000;95:1872-1874)     

Expression and secretion of vascular endothelial growth factor-A by cytokine-stimulated hematopoietic progenitor cells. Possible role in the hematopoietic microenvironment

In the hematopoietic microenvironment, bone marrow endothelial cells may play an important role in trafficking and maintenance of progenitor and stem cells due to adhesive interactions and paracrine secretion of hematopoietic growth factors. However, it is unknown whether progenitors in turn modulate endothelial proliferation and function.We analyzed mRNA expression (Northern blot) and release of vascular endothelial growth factor-A (VEGF-A), which specifically acts on endothelial cells, by cytokine-stimulated peripheral blood-derived CD34+ hematopoietic progenitor cells.While unstimulated CD34+ cells expressed VEGF-A mRNA weakly without cytokine release in vitro, incubation for 24 hours with a single cytokine (e.g., kit ligand [KL]) resulted in increased VEGF-A mRNA expression and significant secretion of VEGF-A into the supernatant. The amount of VEGF released was substantially augmented by incubation with a combination of cytokines (e.g., KL, IL-3, GM-CSF, G-CSF), or by exposure to hematopoietic cytokines for a longer time period. In addition, we show that VEGF induced the release of hematopoietic growth factors (GM-CSF) by bone marrow endothelial cells and that in vitro stromal cell-derived factor-1 (SDF-1) driven transendothelial progenitor cell migration was increased by the presence of VEGF, which might be due to pore formation (increased endothelial fenestration).In vivo, release of VEGF by progenitor cells may result in a paracrine loop supporting proliferation of both endothelium and progenitors and may facilitate transendothelial migration during cytokine-induced progenitor cell mobilization.     

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

The mechanisms mediating hematopoietic stem and progenitor cell (HSPC) mobilization by G-CSF are complex. We have found previously that G-CSF-enforced mobilization is controlled by peripheral sympathetic nerves via norepinephrine (NE) signaling. In the present study, we show that G-CSF likely alters sympathetic tone directly and that methods to increase adrenergic activity in the BM microenvironment enhance progenitor mobilization. Peripheral sympathetic nerve neurons express the G-CSF receptor and ex vivo stimulation of peripheral sympathetic nerve neurons with G-CSF reduced NE reuptake significantly, suggesting that G-CSF potentiates the sympathetic tone by increasing NE availability. Based on these data, we investigated the NE reuptake inhibitor desipramine in HSPC mobilization. Whereas desipramine did not by itself elicit circulating HSPCs, it increased G-CSF-triggered mobilization efficiency significantly and rescued mobilization in a model mimicking "poor mobilizers." Therefore, these data suggest that blockade of NE reuptake may be a novel therapeutic target to increase stem cell yield in patients.     

Genetically determined variation in the number of phenotypically defined hematopoietic progenitor and stem cells and in their response to early-acting cytokines

Quantitative trait analysis may shed light on mechanisms regulating hematopoiesis in vivo. Strain-dependent variation existed among C57BL/6 (B6), DBA/2, and BXD recombinant inbred mice in the responsiveness of primitive progenitor cells to the early-acting cytokines kit ligand, flt3 ligand, and thrombopoietin. A significant quantitative trait locus was found on chromosome 2 that could not be confirmed in congenic mice, however, probably because of epistasis. Because it has been shown that alleles of unknown X-linked genes confer a selective advantage to hematopoietic stem cells in vivo in humans and in cats, we also analyzed reciprocal male D2B6F1 and B6D2F1 mice, revealing an X-linked locus regulating the responsiveness of progenitor and stem cells to early-acting factors. Among DBA/2, B6, and BXD recombinant inbred mice, correlating genetic variation was found in the absolute number and frequency of Lin(-)Sca1(++)kit(+) cells, which are highly enriched in hematopoietic progenitor and stem cells, and in the number of Lin(-)Sca1(++)kit(-) cells, a population whose biologic significance is unknown, suggesting that both populations are functionally related. Suggestive quantitative trait loci (QTLs) for the number of Lin(-)Sca1(++) cells on chromosomes 2, 4, and 7 were confirmed in successive rounds of mapping. The locus on chromosome 2 was confirmed in congenic mice. We thus demonstrated genetic variation in the response to cytokines critical for hematopoiesis in vivo and in the pool size of cells belonging to a phenotype used to isolate essentially pure primitive progenitor and stem cells, and we identified loci that may be relevant to the regulation of hematopoiesis in steady state.     

Rac and Cdc42 GTPases control hematopoietic stem cell shape, adhesion, migration, and mobilization

Critical to homeostasis of blood cell production by hematopoietic stem/progenitor (HSC/P) cells is the regulation of HSC/P retention within the bone marrow microenvironment and migration between the bone marrow and the blood. Key extracellular regulatory elements for this process have been defined (cell-cell adhesion, growth factors, chemokines), but the mechanism by which HSC/P cells reconcile multiple external signals has not been elucidated. Rac and related small GTPases are candidates for this role and were studied in HSC/P deficient in Rac2, a hematopoietic cell-specific family member. Rac2 appears to be critical for HSC/P adhesion both in vitro and in vivo, whereas a compensatory increase in Cdc42 activation regulates HSC/P migration. This genetic analysis provides physiological evidence of cross-talk between GTPase proteins and suggests that a balance of these two GTPases controls HSC/P adhesion and mobilization in vivo.     

Changes in human immunodeficiency virus type 1 virus load during mobilization and harvesting of hemopoietic progenitor cells. Adult AIDS Clinical Trials Group 285 Study Team

Genetic modification of hemopoietic progenitor cells ex vivo, followed by the infusion of the genetically modified cells into the human immunodeficiency virus-1 (HIV-1) infected donor, has been proposed as a treatment for HIV-1 infection. The current study was undertaken to evaluate the effect of hemopoietic stem cell mobilization and harvesting on HIV-1 replication in persons with HIV-1 infection. Eighteen HIV-1-infected persons received recombinant granulocyte colony-stimulating factor (G-CSF; Filgrastim) 10 microg/kg per day, for 7 days. On days 4 and 5, peripheral blood mononuclear cells were harvested by leukapheresis. The CD4+ lymphocyte count at entry was >500/microL for 6 subjects, 200 to 500/microL for 6 subjects, and <200/microL for 6 subjects. For 9 of 18 subjects, plasma HIV-1 RNA levels increased 4- to 100-fold (>0.6 log(10)) above baseline between days 4 and 7 and returned to baseline by day 27. Significant increases of plasma HIV-1 RNA levels occurred in 5 subjects despite 3-drug antiretroviral therapy. Changes in CD4+ and CD34+ cells during mobilization and harvesting were similar in all subjects whether they had or did not have increased plasma HIV-1 RNA levels. Thus, mobilization and harvesting of bone marrow progenitor cells from persons infected with HIV-1 induced a transient increase in viral replication in some patients but was not associated with adverse effects. (Blood. 2000;95: 48-55)     

Development of a vascular niche platform for expansion of repopulating human cord blood stem and progenitor cells

Transplantation of ex vivo expanded human umbilical cord blood cells (hCB) only partially enhances the hematopoietic recovery after myelosuppressive therapy. Incubation of hCB with optimal combinations of cytokines and niche cells, such as endothelial cells (ECs), could augment the efficiency of hCB expansion. We have devised an approach to cultivate primary human ECs (hECs) in serum-free culture conditions. We demonstrate that coculture of CD34(+) hCB in direct cellular contact with hECs and minimal concentrations of thrombopoietin/Kit-ligand/Flt3-ligand resulted in a 400-fold expansion of total hematopoietic cells, 150-fold expansion of CD45(+)CD34(+) progenitor cells, and 23-fold expansion of CD45(+) Lin(-)CD34(hi+)CD45RA(-)CD49f(+) stem and progenitor cells over a 12-day period. Compared with cytokines alone, coculture of hCB with hECs permitted greater expansion of cells capable of multilineage engraftment and serial transplantation, hallmarks of long-term repopulating hematopoietic stem cells. Therefore, hECs establish a cellular platform for expansion of hematopoietic stem and progenitor cells and treatment of hematologic disorders.     

The coordinated action of G-CSF and ELR + CXC chemokines in neutrophil mobilization during acute inflammation

In this study, we have identified a unique combinatorial effect of the chemokines KC/MIP-2 and the cytokine granulocyte colony-stimulating factor (G-CSF) with respect to the rapid mobilization of neutrophils from the bone marrow in a model of acute peritonitis. At 2 hours following an intraperitoneal injection of thioglycollate, there was a 4.5-fold increase in blood neutrophil numbers, which was inhibited 84% and 72% by prior administration of blocking mAbs against either the chemokines KC/MIP-2 or G-CSF, respectively. An intraperitoneal injection of G-CSF acted remotely to stimulate neutrophil mobilization, but did not elicit recruitment into the peritoneum. Further, in vitro G-CSF was neither chemotactic nor chemokinetic for murine neutrophils, and had no priming effect on chemotaxis stimulated by chemokines. Here, we show that, in vitro and in vivo, G-CSF induces neutrophil mobilization by disrupting their SDF-1alpha-mediated retention in the bone marrow. Using an in situ perfusion system of the mouse femoral bone marrow to directly assess mobilization, KC and G-CSF mobilized 6.8 x 10(6) and 5.4 x 10(6) neutrophils, respectively, while the infusion of KC and G-CSF together mobilized 19.5 x 10(6) neutrophils, indicating that these factors act cooperatively with respect to neutrophil mobilization.     

Interferon-alpha overrides the deficient adhesion of chronic myeloid leukemia primitive progenitor cells to bone marrow stromal cells

Primitive blast colony-forming cells (BI-CFC) from chronic myeloid leukemia (CML) patients are defective in their attachment to bone marrow-derived stromal cells compared with normal BI-CFC. We investigated the effect of recombinant interferon-alpha 2a (IFN-alpha) on this interaction between hematopoietic progenitor cells and bone marrow-derived stromal cells by culturing normal stromal cells with IFN-alpha (50 to 5,000 U/mL). At 50 U/mL we found that: (1) the capacity of stromal cells to bind two types of CML primitive progenitor cells (BI-CFC and long-term culture-initiating cells) was increased; and (2) the amount of sulfated glycosaminoglycans (GAGs) in the stromal layer was increased. However, sulfated GAGs were not directly involved in binding CML BI-CFC, unlike binding by normal BI-CFC, which is sulfated GAG-dependent. Neuraminidase-treated control stromal cells bound an increased number of CML BI-CFC, reproducing the effect of IFN-alpha, whereas the binding to IFN-alpha-treated stromal cells was unaffected by neuraminidase treatment. Thus, the enhanced attachment by primitive CML progenitor cells to INF-alpha-treated stromal cells might be due to changes in the neuraminic acid composition in the stromal cell layer. Our in vitro evidence may provide insights into the mechanism of action of IFN-alpha in vivo. Prolonged administration may alter the marrow microenvironment in some patients such that it can restrain the aberrant proliferation of Philadelphia chromosome (Ph)-positive stem cells while permitting Ph-negative stem cells to function normally.