Excess Heparan Sulphate Inhibits CXCL12-Mediated Hematopoietic Cell Migration and Engraftment After Bone Marrow Transplant in Mice with Mucopolysaccharidosis Type I,

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 4010-4010
Author(s):  
H. Angharad Watson ◽  
Rebecca J Holley ◽  
Kia J Langford-Smith ◽  
Fiona L Wilkinson ◽  
Toin H van Kuppevelt ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Cell Migration ◽  
Peripheral Blood ◽  
Progenitor Cell ◽  
Conflicts Of Interest ◽  
Heparan Sulphate ◽  
Type I ◽  
Lysosomal Storage ◽  
Hematopoietic Stem ◽  
Haematopoietic Progenitor Cell

Abstract Abstract 4010 The primary axis of migration for transplanted hematopoietic stem and progenitor cells (HSPC) is CXCL12/CXCR4. Heparan sulphate (HS) is required for CXCL12 presentation and receptor binding, but the functional role of HS is poorly defined. The alpha-L-iduronidase knockout mouse (Idua−/−) accumulates HS and dermatan sulphate, recapitulating the neurodegenerative lysosomal storage disease Mucopolysaccharidosis I Hurler (MPSIH). MPSIH is primarily treated with HSPC transplant, but clinical experience suggests a historical engraftment defect in patients. We show significantly reduced HSPC migration in Idua−/− recipients and under limiting engraftment conditions we show a significant haematopoietic engraftment defect in Idua−/− recipients. No significant donor cell effect was observed. Bone marrow but not peripheral blood CXCL12 levels are slightly elevated in Idua−/− mice. CFU frequency in BM is unchanged between genotypes but reduced significantly in peripheral blood of Idua−/− mice. In whole bone marrow, and on mesenchymal stem cells from Idua−/− mice, HS is present in significant excess, particularly in extracellular matrix, and cell surface locations, with significant increases in all sulphation modifications, especially 2-O-sulphation. Finally we show that excess HS, and particularly HS with increased 2-O -sulphation, functionally inhibit haematopoietic progenitor cell migration in vitro. These data provide novel insight into the influence of highly sulphated HS in CXCL12 mediated haematopoietic progenitor cell migration and help to explain why HSCT engraftment has been historically low in MPSIH. Disclosures: No relevant conflicts of interest to declare.

Peripheral Blood Dendritic Cell Subsets of Patients with Type I Gaucher’s Disease Are Decreased in Number but Functionally Normal.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 3810-3810 ◽  
Author(s):  
Argiris Symeonidis ◽  
Ilina Mitseva ◽  
Theodoros Marinakis ◽  
Constantina Repa ◽  
Alexandra Kouraklis ◽  
...  
Keyword(s):  
Bone Marrow ◽  
T Cells ◽  
Peripheral Blood ◽  
Type I ◽  
Lysosomal Storage ◽  
Gaucher's Disease ◽  
Gaucher’S Disease ◽  
Hematopoietic Stem ◽  
Gm Csf ◽  
Stimulatory Activity

Abstract Introduction - Aims: Gaucher’s disease is a lysosomal storage disorder, in which undigested glucocerebroside is deposited in the cytoplasm of mature macrophages, which accumulate in the bone marrow and the reticuloendothelial system. Dendritic cells (DC) are bone-marrow-derived leukocytes, originating from the pluripotent hematopoietic stem cell, via different developmental pathways, related to myeloid or lymphoid lineage. They belong to the monocyte-macrophage system and are specialized for the uptake, processing, transport and presentation of antigens to T-cells. There is no information about the functional capacity of DC among patients with lysosomal storage disorders. We therefore, investigated in the status of blood DC precursor populations as well as the potential of bone marrow (BM)-derived progenitor cells to produce mature DC in patients with Gaucher’s disease. Patients and methods: Samples of heparinized PB and/or BM were obtained from 11 patients with type I Gaucher’s disease and 15 healthy volunteers, after informed consent. Nine patients were studied before any kind of treatment and the remaining 2 had been treated with imiglucerase 40 IU/kg of body weight at monthly intervals, for 24 months. All patients were anemic and thrombocytopenic, but none had severe bone disease. The myeloid DC (MDC) precursors and the more specialized cell type, the plasmacytoid DC (PDC) were detected in the peripheral blood by flow cytometry, based on the expression of immunoglobulin-like transcript (ILT)-3, together with lineage-specific markers CD3, CD56, CD14, CD16 and CD11c. The potential of PB monocytes, as DC precursors, and of BM CD34+ progenitors to differentiate into mature DC was studied in culture systems. Generated mature DC were immunophenotyped, and tested for their dextran-endocytic capacity, as well as for their stimulatory activity against allogeneic T-cells in mixed cultures. Results: Both, MDC and PDC from peripheral blood of patients with Gaucher’s disease were decreased, when compared to controls (MDC 0.20±0.11% vs. 0.33±0.14%, p=0.02 and PDC 0.19±0.15% vs. 0.40±0.17%, p=0.005). The yield of monocyte-derived DC (MoDC), obtained after GM-CSF and IL-4 stimulation, was lower than in controls (4.9±3.5% vs. 8.2±4%, p=0.012), although no difference was found in the percentage of monocytes initiating the culture. However, the immunophenotype profile, estimated by CD1a, CD40, CD54, CD80, CD83, and HLA-DR expression, the endocytic capacity, and the allo-stimulatory capacity of immature, and of TNFα- or LPS-stimulated mature MoDC were similar to those obtained by healthy controls. In addition, BM-derived CD34+ cells, differentiated in the presence of GM-CSF, SCF, TNF-α and IL-4 into mature DC, did not differ in number, phenotype and allo-stimulatory activity from those of controls. Conclusive remarks: Our findings suggest that in patients with type I Gaucher’s disease, mainly quantitative defects of DC system are present, demonstrated by decreased circulating DC precursors of both, MDC and PDC type. Moreover, with the exception of decreased MoDC production, no additional functional/qualitative defects of both, CD34-DC and MoDC, concerning their membrane immunophenotype, endocytic and allostimulatory capacity were detected.

Neutropenia Does Not Delay Lymphopoietic Regeneration in NODSCIDcγ−/− Mice

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 4831-4831
Author(s):  
Stefanie Bugl ◽  
Stefan Wirths ◽  
R Müller Martin ◽  
Märklin Melanie ◽  
Tina Wiesner ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Peripheral Blood ◽  
Nk Cell ◽  
Conflicts Of Interest ◽  
Control Group ◽  
Early Event ◽  
Wild Type ◽  
Hematopoietic Stem ◽  
Lymphoid Progenitors ◽  
Fate Decision

Abstract Abstract 4831 Introduction: Previously it was demonstrated that lymphopoiesis is rapidly established after transplantation of wild type stem cells into lymphopenic NODSCIDcγ−/− mice. These data were interpreted as evidence for an “empty” preformed lymphopoietic niche being replenished by lymphoid progenitors. We hypothesized that antibody-induced neutropenia might influence early post transplant fate decision to myeloid rather than lymphoid differentiation resulting in delayed lymphoid reconstitution. Materials and Methods: 25,000 flow sorted CD45.2-expressing wild type Lin-/Sca1+/c-Kit+ (LSK) cells from C57BL/6 mice were transplanted into sublethally irradiated B-/T-/NK-cell deficient NODSCIDcγ−/− mice (CD45.1). Three groups of n = 7 mice received anti-Gr1 or anti-1A8 i.p. every 48 h to induce continuous antibody-mediated neutropenia vs. PBS as control. Blood was harvested at regular intervals to monitor the engraftment. After 16, 22, and 34 days, animals were sacrificed and underwent blood and bone marrow analysis. Results: Hematopoietic regeneration started with the emergence of donor-derived monocytes in all groups as well as neutrophils in the control group as early as 9 days after transplantation. On day 14, B cells were to be detected for the first time, followed by T lymphocytes approximately 20 days after transplantation. Besides the fact that neutrophils were undetectable in the antibody treated groups, the peripheral blood revealed no significant changes between the neutropenic mice and the control group at any point of time. At the bone marrow level, an increase of LSK and granulocyte-macrophage progenitors (GMPs) at the expense of megakaryocyte erythrocyte progenitor cells (MEPs) was found in neutropenic mice. Common lymphoid progenitors (CLPs), however, were not significantly different. Conclusions: The engraftment of wild type donor cells after hematopoietic stem cell transplantation into NODSCIDcγ−/− mice started with the production of monocytes and neutrophils. B-lymphocytes were detectable by day 14 after transplantation. The production of T-cells started around day 20. Continuous antibody-mediated neutropenia did not significantly delay lymphoid regeneration. Although the marrow of neutropenic mice displayed increased proliferation of granulocyte progenitors, CLPs were unchanged. We conclude that the detection of donor-derived lymphocytes in the host peripheral blood is a relatively early event after LSK transplantation. Moreover, antibody induced neutropenia is not sufficient to induce sustainable changes in early hematopoietic fate decisions on the bone marrow level. Disclosures: No relevant conflicts of interest to declare.

scholarly journals Administration of interleukin-6 stimulates multilineage hematopoiesis and accelerates recovery from radiation-induced hematopoietic depression

Blood ◽  
1991 ◽  
Vol 77 (3) ◽  
pp. 472-480 ◽  
Author(s):  
ML Patchen ◽  
TJ MacVittie ◽  
JL Williams ◽  
GN Schwartz ◽  
LM Souza
Keyword(s):  
Bone Marrow ◽  
Interleukin 6 ◽  
Peripheral Blood ◽  
Progenitor Cell ◽  
White Cell ◽  
Red Cell ◽  
Hematopoietic Stem ◽  
Hematopoietic Recovery ◽  
Radiation Induced ◽  
Peripheral Blood White Cell

Abstract Hematopoietic depression and subsequent susceptibility to potentially lethal opportunistic infections are well-documented phenomena following radiotherapy. Methods to therapeutically mitigate radiation-induced myelosuppression could offer great clinical value. In vivo studies in our laboratory have demonstrated that interleukin-6 (IL-6) stimulates pluripotent hematopoietic stem cell (CFU-s), granulocyte-macrophage progenitor cell (GM-CFC), and erythroid progenitor cell (CFU-e) proliferation in normal mice. Based on these results, the ability of IL- 6 to stimulate hematopoietic regeneration following radiation-induced hematopoietic injury was also evaluated. C3H/HeN female mice were exposed to 6.5 Gy 60Co radiation and subcutaneously administered either saline or IL-6 (1,000 micrograms/kg) on days 1 through 3 or 1 through 6 postexposure. On days 7, 10, 14, 17, and 22, femoral and splenic CFU-s, GM-CFC, and CFU-e contents and peripheral blood white cell, red cell, and platelet counts were determined. Compared with saline treatment, both 3-day and 6-day IL-6 treatments accelerated hematopoietic recovery; 6-day treatment produced the greater effects. For example, compared with normal control values (N), femoral and splenic CFU-s numbers in IL-6-treated mice 17 days postirradiation were 27% N and 136% N versus 2% N and 10% N in saline-treated mice. At the same time, bone marrow and splenic GM-CFC values were 58% N and 473% N versus 6% N and 196% N in saline-treated mice; bone marrow and splenic CFU-e numbers were 91% N and 250% N versus 31% N and 130% N in saline-treated mice; and peripheral blood white cell, red cell, and platelet values were 210% N, 60% N, and 24% N versus 18% N, 39% N, and 7% N in saline- treated mice. These studies demonstrate that therapeutically administered IL-6 can effectively accelerate multilineage hematopoietic recovery following radiation-induced hematopoietic injury.

Erythroid Lineage Cells Are Found In Close Association With Bone In The Marrow Microenvironment

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 945-945
Author(s):  
Rialnat Adebisi Lawal ◽  
Kathleen E. McGrath ◽  
Laura M. Calvi
Keyword(s):  
Bone Marrow ◽  
Peripheral Blood ◽  
Conflicts Of Interest ◽  
Flow Cytometric Analysis ◽  
Erythroid Differentiation ◽  
Enzymatic Digestion ◽  
Erythroid Progenitors ◽  
Hematopoietic Stem ◽  
Flow Cytometric ◽  
Osteolineage Cells

Abstract Osteolineage cells within the bone marrow microenvironment have been implicated in support and regulation of hematopoietic stem cells (HSCs). Recently, augmented hypoxia-inducible factor (HIF) signaling in osteoprogenitors has been shown to expand the HSC niche, and surprisingly these cells have also been demonstrated to express erythropoietin, the critical cytokine stimulating erythropoiesis. We therefore hypothesize that endosteal cells may represent an additional regulatory site for erythropoiesis. To further delineate the role of the osteolineage cells in the support of erythropoiesis, we isolated bone associated cells (BACs) with enzymatic digestion of adult C57bl/6 mice hind limbs after bone marrow flushing and depleted the BACs of CD45+ cells to enrich for osteogenic cells. We suspected some contribution of erythroid cells to CD45- BACs, however we were surprised to find that ter119+ cells represented a large percentage of BACs after enzymatic digestion. After CD45 depletion, ter119+ cells constituted about 30% percent compared to approximately 0.85% of CD45+ cells (33 ± 4.4vs. 0.85 ± 0.26, p= 0.0018) by flow cytometric analysis. Additionally, CD45 depleted BACs had approximately 46 fold higher osteocalcin expression than CD45+ cells (1300 ± 120 vs. 28 ± 9.5, p < 0.0001), while CD45/Ter119/CD31 depleted BACs had approximately 2000 fold higher osteocalcin expression than CD45/Ter119/CD31 (+) cells (2000 ± 520 vs. 0.98 ± 0.02, p= 0.0044) by qRT-PCR, confirming enrichment of the osteoblastic lineage by this immunophenotypic panel. These data suggest that there are a large number of erythroid lineage cells associated with the BACs along the endosteum. In the bone marrow of adult mice, ter119 + cells represented approximately 85% in the CD45- pool as compared to 5% in the CD45+ cell pool. To determine if the endosteum is an active site of erythropoiesis, we quantified erythroid progenitors and precursors in the BAC pool compared to whole bone marrow (wbm) and peripheral blood (pb) by both flow cytometric analysis and colony forming assays. Flow cytometric analysis demonstrated the presence of every phase of erythroid differentiation in the BAC pool, including the presence of phenotypic MEPs (wbm vs bac vs pb: 250 ± 30 vs 84 ± 22 vs 0), BFU-E (wbm vs bac vs pb: 300 ± 14 vs 110 ± 36 vs 0 ), CFU-E (wbm vs bac vs pb: 2900 ± 2 vs 430 ± 23 vs 1 ± 0.8) and proerythroblasts (wbm vs bac vs pb: 11000 ± 2500 vs 7600 ± 1600 vs 2300 ± 920) per million cells. The phenotypic frequency of CFU-E was particularly remarkable in the BACs (430 ± 23) as compared to peripheral blood (1 ± 0.8) , demonstrating that all stages of erythroid differentiation are found in tight association with the endosteum and are not due to contamination from circulating erythroid progenitors. Colony assays were performed for CFU-E (wbm vs. bac 108 ± 16 vs 6.3 ± 2 colonies per 20,000cells plated), BFU-E (wbm vs. bac 55 ±1.0 vs 2 ±1.0; colonies per 40,000 cells plated) and myeloid progenitors (wbm vs. bac 66 ± 28 vs 11 ± 2.5 ; colonies per 10,000 cells plated) also confirmed the presence of erythroid progenitors at endosteal sites. Together these results identify the endosteal surface as a site for erythroid differentiation. The presence of all phases of erythroid lineage differentiation in the BACs suggests a potential role for osteolineage cells for maintenance and regulation of erythropoiesis. Whether osteolineage cells contribute to erythroid lineage homeostasis and/or stress response, and whether activation or damage to osteolineage cells alters local erythroid differentiation remains to be demonstrated. However our data suggest further study of the endosteum and osteolineage cells as a potential and unexpected site of erythroid regulation, which could potentially be targeted to accelerate erythropoiesis and treat anemia. Disclosures: No relevant conflicts of interest to declare.

scholarly journals TNF-α-Induced Bone Marrow Stromal Progenitor Alterations Enhance Leukemic Stem Cell Growth and Treatment Resistance Via Increased CXCL1-CXCR2 Signaling

Blood ◽  
2018 ◽  
Vol 132 (Supplement 1) ◽  
pp. 875-875
Author(s):  
Puneet Agarwal ◽  
Hui Li ◽  
Kwangmin Choi ◽  
Robert S. Welner ◽  
Jianbo He ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Stem Cell ◽  
Peripheral Blood ◽  
Conflicts Of Interest ◽  
Critical Role ◽  
Mesenchymal Cell ◽  
Osteogenic Potential ◽  
Pcr Analysis ◽  
Hematopoietic Stem ◽  
Cxcl1 Expression

Abstract Mesenchymal cells within the bone marrow (BM) microenvironment play an important role in regulation of hematopoietic stem cell (HSC) fate. However, the effects of leukemia development on distribution and function of mesenchymal cell subpopulations are not well understood. Here we used the SCL-tTA-BCR-ABL transgenic CML mouse model to examine how CML development affected murine BM mesenchymal subpopulations within a recently delineated skeletal stem cell (SSC) hierarchy, and evaluate how mesenchymal subpopulations affected HSC and leukemia stem cell (LSC) growth. We observed a significant increase in bone-forming "Thy" and stroma-forming "6C3" progenitor subsets in CML compared to normal BM. CML Thy and 6C3 progenitors demonstrated increased proliferation and CFU-F potential. In addition, CML Thy cells exhibiting increased osteogenic potential whereas CML 6C3 cells showed increased adipogenic potential compared to their normal counterparts. CML LSC and normal HSC were cocultured with Thy and 6C3 cells, purified from CML and normal mice for 3 days and transplanted into irradiated normal recipients to evaluate long-term engraftment. Normal HSC engraftment was enhanced by coculture with normal Thy and 6C3 cells but not by their CML counterparts. On the other hand, LSC engraftment was enhanced by CML 6C3 cells compared to normal 6C3 cells, but not by CML Thy cells compared to normal Thy cells. These results indicate that CML stromal progenitors demonstrate enhanced support of LSC and reduced support of normal HSC. Q-PCR analysis showed that expression of major hematopoietic regulatory molecules, including CXCL12, G-CSF, SCF, IL-1, IL-6, and IGF-1, was significantly reduced in CML 6C3 and Thy progenitors.RNA-Seq analysis demonstrated that expression of TNFaand NF-kbrelated gene sets was significantly increased in CML compared with normal 6C3 cells. Ligand-receptor interactome analysis, based on differential gene expression in LSC and normal HSC, and CML and normal 6C3 cells, revealed upregulation of the chemokines CXCL1 and CXCL5 in CML 6C3 cells, and of their cognate receptor CXCR2 in LSC. We have previously shown that TNFalevels are increased in CML compared to normal BM (Zhang et al., 2012). Here we found that treatment of WT mice with TNFα led to expansion of 6C3 cells and increased CXCL1 expression on 6C3 cells. In contrast, treatment of CML mice with anti-TNFα antibodies led to reduction in 6C3 cell numbers and reduced CXCL1 expression in 6C3 cells. These results support a critical role for TNFα signaling in expansion and increased CXCL1 expression by stromal progenitors in CML BM. We evaluated the role of paracrine CXCL1-CXCR2 signaling in growth and TKI resistance of LSC. Treatment with CXCL1 and CXCL5 resulted in expansion of LSC and leukemic progenitors respectively, and this effect was blocked by the CXCR2 inhibitor SB225002. Treatment with SB225002 significantly reduced proliferation of LSC cocultured with CML 6C3 cells. SB225002 administration to mice engrafted with CML cells resulted in significant reduction in peripheral blood WBC counts, neutrophil percentage, and leukemic short-term HSC (STHSC) and granulocyte-macrophage progenitors (GMP) in the BM. The combination of SB225002 and the TKI Nilotinib (50mg/kg) resulted in significantly greater reduction in peripheral blood WBC and neutrophils, and in BM LSC compared to Nilotinib or SB225002 alone.SB225002 treatment also significantly reduced proliferation and enhanced apoptosis of human CML CD34+CD38- cells cocultured with human CML BM mesenchymal stromal cells, and that the combination of SB225002 and Nilotinib significantly enhanced apoptosis and inhibited proliferation of CML CD34+CD38- cells compared to Nilotinib alone. We conclude that increased TNFα signaling results in expansion of stromal progenitors in CML BM, which differentially support LSC compared to normal HSC. TNFα-signaling leads to overexpression of CXCL1 by stromal progenitors, which interacts with CXCR2 overexpressed on LSC to enhance their growth. Inhibition of CXCR2 signaling reduces LSC proliferation and survival, and enhances LSC elimination in combination with TKI. These observations support further exploration of targeting of CXCL1-CXCR2 interactions as a novel and effective strategy to target BM microenvironment-protected TKI-resistant LSC. Disclosures No relevant conflicts of interest to declare.

The Sympathetic Nervous System Regulates Hematopoietic Stem and Progenitor Cell Homing and Engraftment.

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 1387-1387
Author(s):  
Daniel Lucas ◽  
Paul S. Frenette
Keyword(s):  
Bone Marrow ◽  
Nervous System ◽  
Stem Cell ◽  
Sympathetic Nervous System ◽  
Peripheral Blood ◽  
Progenitor Cell ◽  
Recovery Phase ◽  
Logrank Test ◽  
Hematopoietic Stem ◽  
Sympathetic Nervous

Abstract The sympathetic nervous system (SNS) controls hematopoietic stem and progenitor cell (HSPC) release from their niches in the bone marrow (BM) by acting on specific niche cells: bone-lining osteoblasts during mobilization (Cell2006;124:407) and CXCL12-secreting BM stromal cells (Nature2008;452:442) in homeostasis. However, it remains unknown whether the SNS controls other processes in HSC physiology. Here, we show that an intact SNS is necessary for bone marrow reconstitution. Transplantation of lethally-irradiated recipient mice, in which the SNS was lesioned using 6-hydroxydopamine (6OHDA), with 105 healthy BM nucleated cells resulted in 65% survival of the sympathectomized mice compared to 100% survival in the control groups (6OHDA, not irradiated; Saline, not irradiated; Saline, irradiated and transplanted; p&lt;0.01, Kaplan-Meyer survival and Logrank test). Peripheral blood analyses revealed hematopoietic failure and a severe reduction in trilineage cell counts during the recovery phase. These results indicated that HSPCs engrafted with diminished efficiency in sympathectomized mice. Since donor HSPCs were obtained from intact wild-type mice the observed phenotype suggested that the SNS regulated HSPC engraftment by acting on niche cells. The observed phenotype could be due to reduced homing (i.e. the ability of HSPCs to migrate and enter the bone marrow) or to reduced proliferation of HSPC after appropriate homing. To evaluate these two possibilities, we first injected HSPCs into lethally irradiated 6OHDA-treated or control recipients and allowed the cells to migrate to the BM for 3 hours (homing assays). We found that 2.7-fold fewer donor clonogenic progenitors (CFU-C) could be detected in the BM of sympathectomized mice compared to control animals (p&lt;0.001), indicating that an intact SNS is required for HSPC homing to the bone marrow. To determine whether SNS signals are required for HSPC proliferation after homing, we evaluated the proliferative capacity of HSPCs following the administration of the cytotoxic drug 5-fluorouracyl (5FU). Mice treated with 6OHDA or control recipient mice received a single dose of 5FU (150 mg/kg) and their hematopoietic parameters and survival were monitored for 16 days. Peripheral blood analysis revealed reductions in both WBC (1.6 fold; p&lt;0.05) and RBC (1.4 fold; p&lt;0.01) in 6OHDA-treated compared to SNS-intact mice. Further analyses revealed a strong reduction (1.8 fold; p&lt;0.01) in the BM cellular content suggesting that HSPC failed to proliferate normally in 6OHDA-treated mice. The number of CFU-C and stem cell-enriched Lin−Sca1+c-kit+ (LSK) cells in the recovery phase (12 days after 5FU) were reduced 1.9-fold (p&lt;0.01). Moreover, 5FU produced a high mortality rate in the 6OHDA-lesioned group (76% survival at the end of the study; p&lt;0.05 Kaplan- Meyer survival and Logrank test) when compared to 5FU-treated SNS-intact mice (100% survival). These results suggest that HSPC require SNS signals to proliferate in response to stress to restore a functional hematopoiesis. Taken together these data indicate that the sympathetic nervous system acts on the hematopoietic stem cell niche to control both homing and proliferation in the bone marrow, two critical steps that determine the success of a bone marrow transplantation procedure.

A Novel Observation That Heme Oxygenase-1 (HO-1) Deficient Mice Are Easy Mobilizers and That HO-1 Plays An Important Role in Maintaining Expression of SDF-1 in Bone Marrow (BM) Stroma and Promotes Retention of Hematopoietic Stem/Progenitor Cells (HSPCs) in the Bone Marrow Microenvironment

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 315-315
Author(s):  
Marcin Wysoczynski ◽  
Janina Ratajczak ◽  
Gregg Rokosh ◽  
Roberto Bolli ◽  
Mariusz Z Ratajczak
Keyword(s):  
Bone Marrow ◽  
Progenitor Cells ◽  
Heme Oxygenase ◽  
Peripheral Blood ◽  
Conflicts Of Interest ◽  
Mrna Level ◽  
Heme Oxygenase 1 ◽  
Hematopoietic Stem ◽  
Normal Peripheral Blood ◽  
Cell Counts

Abstract Abstract 315 Background. Heme oxygenase (HO) is an enzyme that catalyzes the degradation of heme. Two distinct HO isoforms have been identified: HO-2, which is constitutively expressed, and HO-1, which is stress-responsive and plays an important function in various physiological and pathophysiological states associated with cellular stress. HO-1 plays a role in ischemic/reperfusion injury, atherosclerosis, and cancer. It has also been reported that HO-1 regulates expression of a-chemokine stromal derived factor-1 (SDF-1) in myocardium (J Mol Cell Cardiol.2008;45:44–55). Aim of study. Since SDF-1 plays a crucial role in retention and survival of hematopoietic stem cell/progenitor cells (HSPCs) in BM, we become interested in whether deficiency of HO-1 affects normal hematopoiesis and retention of HSPCs in BM. Experimental approach. To address this issue, we employed several complementary strategies to investigate HO-1−/−, HO+/–, and wild type (wt) mouse littermates for i) the expression level of SDF-1 in BM, ii) the number of clonogenic progenitors from major hematopoietic lineages in BM, iii) peripheral blood (PB) cell counts, iv) chemotactic responsiveness of HSPCs to an SDF-1 gradient, iv) adhesiveness of clonogenic progenitors, v) the number of circulating HSPCs in PB, and vi) the degree of mobilization in response to granulocyte-colony stimulating factor (G-CSF) or AMD3100 assessed by enumerating the number of CD34–SKL cells and clonogeneic progenitors (CFU-GM) circulating in PB. Results: Our data indicate that under normal, steady-state conditions, HO-1−/− and HO+/– mice have normal peripheral blood cell counts and numbers of circulating CFU-GM. Interestingly, lack of HO-1 leads to an increase in the number of erythroid (BFU-E) and megakaryocytic (CFU-GM) progenitors in BM. Next, BMMNCs from HO-1−/−have normal expression of the SDF-1-binding receptor, CXCR4, but a 5-times lower level of CXCR7, which is another SDF-1-binding receptor. Of note, we observed that the mRNA level for SDF-1 in BM-derived fibroblasts was ∼4 times lower. This corresponded with the observation in vitro that HSPCs from HO-1−/−animals responded more robustly to an SDF-1 gradient, and HO-1−/−animals mobilized a higher number of CD34–SKL cells and CFU-GM progenitors into peripheral blood in response to G-CSF and AMD3100. Conclusions: Our data demonstrate for the first time that heme oxygenase plays an important and underappreciated role in BM retention of HSPCs and may affect their trafficking. Since small non-toxic molecular inhibitors of HO-1 have been developed for clinical use (e.g., metaloporhirins), blockage of HO-1 could be a novel strategy for mobilizing HSPCs. Our recent in vivo mobilization studies lend support to this hypothesis. Disclosures: No relevant conflicts of interest to declare.

A Novel View of Paroxysmal Nocturnal Hemoglobinuria (PNH) Pathogenesis: Do Pathologic PNH Hematopoietic Stem/Progenitor Cells (HSPCs) Displace Normal HSPCs From Their Niches in Bone Marrow Because They Are More Motile Due to Defective Adhesion and Enhanced Migratory Properties?

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 732-732
Author(s):  
Janina Ratajczak ◽  
Rui Liu ◽  
Nagendra Natarajan ◽  
Jaroslaw P. Maciejewski ◽  
Vivek R. Sharma ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Progenitor Cells ◽  
Lipid Rafts ◽  
Lipid Raft ◽  
Peripheral Blood ◽  
Paroxysmal Nocturnal Hemoglobinuria ◽  
Conflicts Of Interest ◽  
Gpi Anchor ◽  
Hematopoietic Stem ◽  
Cd34 Antigen

Abstract Abstract 732 Background . Paroxysmal nocturnal hemoglobinuria (PNH) is an uncommon acquired hemolytic anemia that results from the expansion of hematopoietic stem cells with a mutation in one of the enzymes (PIG-A) responsible for glycosylphosphatidylinositol (GPI anchor) biosynthesis, which is a post-translation modification of proteins associated with lipid rafts on the cell membrane surface. Some of these proteins are involved in the resistance of erythrocytes to lysis by the final product of complement cascade (CC) activation, C5b-C9, also known as the membrane attack complex (MAC). As we reported, the CXCR4 receptor, which binds a-chemokine stromal derived factor-1 (SDF-1) in regulating the trafficking of hematopoietic stem/progenitor cells (HSPCs), is also associated with lipid rafts (Blood 2005;105:40-8). In addition, we recently demonstrated that the bioactive lipid sphingosine-1-phosphate (S1P), which is a major chemoattractant directing egress of HSPCs from bone marrow (BM) into peripheral blood (PB) during mobilization, is released from erythrocytes by C5b-C9/MAC (Leukemia 2010;24:976-85). Hypothesis. Based on this finding, we hypothesized that HSPCs are continuously mobilized from the BM of PNH patients due to the susceptibility of PIG-A-deficient erythrocytes to CC activation, which elevates the free S1P level in plasma, as well as to their defective adhesion in the BM microenvironment due to impaired lipid raft formation. Experimental strategies. To address this hypothesis, peripheral blood mononuclear cells (PBMNC) were isolated from 6 PNH patients and stained with the fluorescent variant of aerolysin (FLAER), which binds GPI anchor and thus identifies normal, but not PNH, cells in FACS analysis. PNH patient-derived cells were tested for i) the level of CD34 antigen expression, ii) chemotaxis in response to SDF-1 and S1P, and iii) adhesion to fibronectin and bone marrow stromal cells. Results. We observed in PNH patients ∼3-fold higher expression of CD34 antigen on FLAER– cells circulating in PB than FLAER+ cells, which suggests that PNH-mutated HSPCs are preferentially released/mobilized into PB. Next, in Transwell chemotaxis assays followed by in vitro clonogenic assays with cells collected from the lower Transwell chambers, we observed that FLAER– cells responding to SDF-1 are ∼20 times more enriched in migrating clonogenic BFU-E and CFU-GM progenitors than their normal FLAER+ counterparts. Moreover, in parallel experiments, FLAER– CFU-GM that were plated over BM-derived fibroblasts or fibronectin in the presence of SDF-1 and S1P (known activators of VLA-4–VCAM-1-mediated cell adhesion) exhibited impaired adhesion in comparison to normal FLAER+ CFU-GM cells. Conclusions. Based on these observations, we propose a novel view of the pathogenesis of PNH and the expansion of PNH-affected cells in BM. Accordingly, the lack of PIG-A protein, which plays an important role in lipid raft formation, confers an advantage to PNH-affected HSPCs, which become more mobile. These cells are preferentially mobilized into PB in response to S1P released from C5b-C9/MAC-lysed erythrocytes. Thus, PNH-mutated HSPCs over time may outcompete normal HSPCs for their niches in BM, due to their increased motility, and contribute to the PNH type of hematopoiesis. Disclosures: No relevant conflicts of interest to declare.

Lmo2 Overexpression and Arf Loss Induce Myeloid Differentiation in Primitive Thymocytes

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 47-47
Author(s):  
Louise M. Treanor ◽  
Sheng Zhou ◽  
Taihe Lu ◽  
Charles G Mullighan ◽  
Brian P. Sorrentino
Keyword(s):  
Bone Marrow ◽  
T Cell ◽  
Peripheral Blood ◽  
Conflicts Of Interest ◽  
Myelogenous Leukemia ◽  
Myeloid Differentiation ◽  
Differentiation Potential ◽  
Hematopoietic Stem ◽  
Blast Cells

Abstract Abstract 47 Lmo2 overexpression and Arf loss induce myeloid differentiation in primitive thymocytes LMO2 is a hematopoietic transcription factor that is deregulated as a consequence of chromosomal translocations in T-cell leukemia. Recently we reported that Lmo2 overexpression collaborates with loss of the p19Arf tumor suppressor to induce central T cell leukemias in mice, in part by conferring an increase in self-renewal and engraftment potential in thymic repopulating cells (Treanor LM et al, Blood 2011). Primary recipients were transplanted with Lmo2-transduced, Arf−/− DN2 (CD4−CD8−CD44+CD25+) thymocytes that were cultured on OP9-DL1 cells for 20 days before transplant. Primary recipients engrafted and secondary transplants were later performed. Several secondary recipients developed acute myelogenous leukemia originating from the thymocyte graft. These mice had elevated white blood counts between 200–500×103/μl and their spleen, thymus, bone marrow and peripheral blood contained 90% mCherry+, Gr1+, Mac1+ cells. Both the spleen and liver were infiltrated with myeloperoxidase positive blast cells and pathological review confirmed acute myeloid leukemia. Tertiary irradiated hosts transplanted with these cells developed a Gr1+ tumor with the same phenotype as the secondary animal. All of the tumor cells observed had a high expression level of the mCherry vector indicating that Lmo2 was expressed in the blast cells. Vector integration site clonality analyses confirmed that the vector was present in the blast cells and was from the same clone in the primary, secondary and tertiary recipient. This data led to the hypothesis that enforced Lmo2 expression and Arf loss may reprogram DN2 thymocytes to obtain myeloid differentiation potential. Transduced DN2 thymocytes were then assayed for myeloid colony formation in semisolid cultures containing cytokines that are specific for myeloid differentiation. Initially CD4−CD8− thymocytes were selected from Arf+/+ and Arf−/− thymi, transduced with either control vector or Lmo2 and cultured on OP9-DL1 stromal cells for 20 days. At day 20 the thymocytes were sorted for the vector positive DN2 population and 5×104 of these DN2 thymocytes were plated into these semisolid cultures. Only thymocytes that contained the Lmo2 vector were able to form myeloid colonies and this colony forming ability was greatly enhanced by the absence of Arf (n=3) as shown in figure 1. Moving to an in vivo assay, sublethally irradiated Rag2−/−γc−/−were transplanted with 2×105 transduced DN2 thymocytes. Three weeks after transplant the spleen, bone marrow and peripheral blood contained greater than 50% mCherry+ cells and of these cells between 3%-10% were Mac1+Gr1+ double positive. Vector+ (mCherry+) cells were sorted from the bone marrow and plated in semisolid culture with myeloid cytokines. After seven days the cultures were positive for myeloid colonies that were mCherry+, Gr1+, Mac1+. These in vivo and in vitro assays demonstrate that Lmo2 induces myeloid potential in DN2 thymocytes. These data indicate that Lmo2 expression combined with loss of the Arf locus may recapitulate a hematopoietic stem cell (HSC) “state” in the DN2 thymocytes as HSCs express relatively high levels of Lmo2 and do not express p19Arf due to Bmi1-mediated epigenetic suppression. The novel reprogramming events that we now report could have relevance to early thymic precursor leukemia, in which various degrees of myeloid conversion are noted. We recently documented high amounts of Lmo2 mRNA expression in pediatric early thymic precursor leukemia by expression array analysis in 11/12 cases. Disclosures: No relevant conflicts of interest to declare.

The Ryk Receptor Regulates Hematopoietic Stem and Progenitor Cell Proliferation and Their Sensitivity To Myeloablative Agents

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 796-796
Author(s):  
Benjamin Povinelli ◽  
Michael Nemeth
Keyword(s):  
Bone Marrow ◽  
Peripheral Blood ◽  
Blood Cells ◽  
Molecular Mechanisms ◽  
Bone Marrow Cells ◽  
Conflicts Of Interest ◽  
Peripheral Blood Cells ◽  
Hematopoietic Stem ◽  
Marrow Cells

Abstract The molecular mechanisms that control the balance between quiescence and proliferation of hematopoietic stem and progenitor cells (HSPCs) are critical for maintaining life-long hematopoiesis. In a recent study (Povinelli, et al. Stem Cells, In Press, 2013) we demonstrated that the Wnt5a ligand inhibits HSPC proliferation through a functional interaction with a non-canonical Wnt ligand receptor termed Related to Receptor Tyrosine Kinase (Ryk). Expression of Ryk on HSPCs in vivo was associated with a decreased rate of proliferation. Following treatment with fluorouracil (5-FU), the percentage of Ryk+ HSPCs increased at the expense of Ryk-/low HSPCs. Based on these data, we hypothesized that one function of the Ryk receptor is to protect HSPCs from the effects of myeloablative agents. To test this hypothesis, we injected 6-8 week old C57BL/6 mice with 150 mg/kg of 5-FU and analyzed bone marrow 48 hours later for the presence of apoptotic HSPCs, defined as lineage negative (Lin-), Sca-1+, CD48- cells positive for active caspase-3. There was a 2.5-fold decrease in the percentage of apoptotic Ryk+ HSPCs (12.9 ± 1.7%) compared to Ryk-/low HSPCs (32.4 ± 5.3%, p < 0.001, n = 3). To test whether this effect was limited to 5-FU, we performed a similar study in which we irradiated C57BL/6 mice with 3 cGy of total body irradiation (TBI) and analyzed bone marrow 72 hours later for apoptotic HSPCs (for this experiment, defined by a Lin-, c-kit+, Sca-1+, CD150+, CD48- immunophenotype or LSK, SLAM). Comparable to the effects of 5-FU, there was a significant 3.0-fold reduction in the percentage of apoptotic Ryk+ HSPCs (3.1 ± 0.2%) compared to Ryk-/low HSPCs (9.2 ± 1.5%, p < 0. 001, n = 3) in mice receiving 3 cGy TBI. These results demonstrated an association between Ryk expression and survival of HSPCs following myeloablative injury. To determine whether in vivo targeting of the Ryk receptor would increase the sensitivity of HSPCs to myeloablative injury, we utilized a neutralizing rabbit anti-Ryk antibody (α-Ryk). We injected C57BL/6 mice with 5 mg/kg α-Ryk or rabbit IgG isotype for 2 consecutive days. Twenty-four hours after the second dose, we determined the frequency and cell cycle status of LSK SLAM cells. Treatment with α-Ryk significantly increased the percentage of LSK SLAM cells in the S/G2/M phases compared to control (α-Ryk: 17.8 ± 2.2%; isotype IgG: 11.6 ± 2.7%, p < 0.05, n = 3). This was associated with a decrease in the percentage of LSK, SLAM cells in G1 following treatment with α-Ryk (α-Ryk: 40.5 ± 3.2%, isotype IgG: 51.3 ± 2.2; p < 0.01, n = 3). The percentage of G0 LSK SLAM cells was unchanged (α-Ryk: 37.9 ± 2.6, isotype IgG: 35.7 ± 3.1% n = 3) indicating that inhibiting Ryk promoted the exit of LSK SLAM cells from G1. Treatment with α-Ryk also increased the percentage of whole bone marrow cells expressing the LSK SLAM phenotype by 1.4-fold compared to controls (p < 0.05, n = 3). To determine if α-Ryk treatment altered HSPC function, we transplanted whole bone marrow cells from C57BL/6 mice treated with two days of α-Ryk or isotype IgG at a 1:1 ratio with whole bone marrow from untreated Ubc-GFP transgenic mice into lethally irradiated B6.SJL mice. Four weeks after transplant, we analyzed peripheral blood cells for the percentage of CD45.2+ GFP- cells. There was no difference in engraftment by transplanted bone marrow cells from mice treated with α-Ryk or isotype IgG (α-Ryk: 61.6 ± 6.1% n = 4, isotype IgG: 52.8 ± 13.6%, n = 5), indicating that the neutralizing antibody does not inhibit short-term HSPC function on its own. We then tested whether blocking Ryk function resulted in greater sensitivity of HSPCs to 5-FU. We treated B6.SJL mice with 5 mg/kg α-Ryk or isotype IgG for 2 consecutive days, followed by 150 mg/kg of 5-FU. Forty-eight hours after 5-FU treatment, we transplanted 2x106 C57BL/6 whole bone marrow cells into treated B6.SJL mice without additional conditioning. Four weeks after transplant, we determined the percentage of donor-derived CD45.2+ peripheral blood cells. Treatment of recipient mice with α-Ryk prior to 5-FU treatment resulted in increased engraftment of donor bone marrow by 3.6-fold compared to isotype (p < 0.05, n = 5), suggesting that inhibition of Ryk resulted in increased elimination of host HSPCs by 5-FU. Collectively, these data suggest a model in which inhibition of the Ryk receptor results in increased proliferation of HSPCs, rendering them more sensitive to the effects of myeloablative agents such as chemotherapy or TBI. Disclosures: No relevant conflicts of interest to declare.

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