Number Of Myeloid Progenitor Cells Research Articles

Elucidating the Importance of DOT1L Recruitment in MLL-AF9 Leukemia and Hematopoiesis.

Simple SummaryMLL-rearranged leukemia, driven by MLL-fusion proteins, is an aggressive, therapy-resistant leukemia found in >60% of infant leukemia and ~10% of adult leukemia. Studies have shown that inhibiting DOT1L enzymatic activity blocks leukemogenesis. However, DOT1L is critical for various normal cellular functions, including hematopoiesis. This study aimed to show that targeting the interaction between the MLL-AF9 fusion and DOT1L would inhibit leukemogenesis while sparing non-leukemic hematopoiesis. We found that disrupting the AF9-DOT1L interaction with a single point mutation was sufficient to impair leukemogenesis. We also demonstrate that genetic interventions that result in loss of DOT1L enzymatic activity in non-leukemic cells rapidly depletes hematopoietic stem and progenitor cells within 7–10 days; however, hematopoiesis was preserved when the AF9-DOT1L interaction was disrupted, leaving the enzymatic function intact. These studies are a proof of concept demonstrating the potential therapeutic advantage of inhibiting the AF9-DOT1L interaction and disrupting the integrity of the MLL-fusion complex.MLL1 (KMT2a) gene rearrangements underlie the pathogenesis of aggressive MLL-driven acute leukemia. AF9, one of the most common MLL-fusion partners, recruits the histone H3K79 methyltransferase DOT1L to MLL target genes, constitutively activating transcription of pro-leukemic targets. DOT1L has emerged as a therapeutic target in patients with MLL-driven leukemia. However, global DOT1L enzymatic inhibition may lead to off-target toxicities in non-leukemic cells that could decrease the therapeutic index of DOT1L inhibitors. To bypass this problem, we developed a novel approach targeting specific protein-protein interactions (PPIs) that mediate DOT1L recruitment to MLL target genes, and compared the effects of enzymatic and PPIs inhibition on leukemic and non-leukemic hematopoiesis. MLL-AF9 cell lines were engineered to carry mutant DOT1L constructs with a defective AF9 interaction site or lacking enzymatic activity. In cell lines expressing a DOT1L mutant with defective AF9 binding, we observed complete disruption of DOT1L recruitment to critical target genes and inhibition of leukemic cell growth. To evaluate the overall impact of DOT1L loss in non-leukemic hematopoiesis, we first assessed the impact of acute Dot1l inactivation in adult mouse bone marrow. We observed a rapid reduction in myeloid progenitor cell numbers within 7 days, followed by a loss of long-term hematopoietic stem cells. Furthermore, WT and PPI-deficient DOT1L mutants but not an enzymatically inactive DOT1L mutant were able to rescue sustained hematopoiesis. These data show that the AF9-DOT1L interaction is dispensable in non-leukemic hematopoiesis. Our findings support targeting of the MLL-AF9–DOT1L interaction as a promising therapeutic strategy that is selectively toxic to MLL-driven leukemic cells.

Dynamics of hematopoiesis is disrupted by impaired hematopoietic microenvironment in a mouse model of hemophagocytic lymphohistiocytosis

Hemophagocytic lymphohistiocytosis (HLH) is a life-threatening systemic hyperinflammatory disorder. We found recently that repeated lipopolysaccharide (LPS) treatment induces HLH-like features in senescence-accelerated mice (SAMP1/TA-1) but not in senescence-resistant control mice (SAMR1). In this study, we analyzed the dynamics of hematopoiesis in this mouse model of HLH. When treated repeatedly with LPS, the numbers of myeloid progenitor cells (CFU-GM) and B-lymphoid progenitor cells (CFU-preB) in the bone marrow (BM) rapidly decreased after each treatment in both strains. The number of CFU-GM in SAMP1/TA-1 and SAMR1, and of CFU-preB in SAMR1, returned to pretreatment levels by 7 days after each treatment. However, the recovery in the number of CFU-preB in SAMP1/TA-1 was limited. In both strains, the BM expression of genes encoding positive regulators of myelopoiesis (granulocyte colony-stimulating factor (G-CSF), granulocyte macrophage colony-stimulating factor (GM-CSF), and interleukin (IL)-6), and negative regulators of B lymphopoiesis (tumor necrosis factor (TNF)-α) was increased. The expression of genes encoding positive regulators of B lymphopoiesis (stromal-cell derived factor (SDF)-1, IL-7, and stem cell factor (SCF)) was persistently decreased in SAMP1/TA-1 but not in SAMR1. Expression of the gene encoding p16INK4a and the proportion of β-galactosidase-positive cells were increased in cultured stromal cells obtained from LPS-treated SAMP1/TA-1 but not in those from LPS-treated SAMR1. LPS treatment induced qualitative changes in stromal cells, which comprise the microenvironment supporting appropriate hematopoiesis, in SAMP1/TA-1; these stromal cell changes are inferred to disrupt the dynamics of hematopoiesis. Thus, hematopoietic tissue is one of the organs that suffer life-threatening damage in HLH.

PLAG alleviates chemotherapy-induced thrombocytopenia via promotion of megakaryocyte/erythrocyte progenitor differentiation in mice

Previously, PLAG (1-palmitoyl-2-linoleoyl-3-acetyl-rac-glycerol, acetylated diglyceride) was reported to have an effect on the proliferation of hematopoietic stem cells (HSCs) or to contribute to the prevention of chemotherapy-induced neutropenia. In this study, we examined the role of PLAG in the differentiation of bone marrow cells from HSCs into progenitor cells in mice. After 15days, the lineage-negative cells, especially megakaryocyte/erythrocyte progenitors (MEP), were significantly increased in mice that received daily PLAG administration compared to those in the untreated mice. Furthermore, we explored the possibility that the PLAG-induced increase in MEP will contribute to reduction of chemotherapy-induced thrombocytopenia (CIT) in a thrombocytopenia mouse model. Mice were administrated 5-fluorouracil (5-FU) and PLAG. After 7days, bone marrow cells were analyzed. Treatment with 5-FU powerfully decreased myeloid precursor populations and treatment with 5-FU/PLAG resulted in reduction of decreased myeloid progenitor cell numbers. In addition, numbers of circulating platelets were also increased by PLAG treatment. Taken together, PLAG plays a role in differentiating HSCs toward MEP and alleviating chemotherapy-induced bone marrow cell reduction. Thus PLAG shows its potential to augment the therapeutic effect of anti-cancer drugs-induced thrombocytopenia.

Clodronate Improves Survival of Transplanted Hoxb8 Myeloid Progenitors with Constitutively Active GMCSFR in Immunocompetent Mice

New methods to produce large numbers of myeloid progenitor cells, precursors to macrophages (MΦs), by maintaining Hoxb8 transcription factor activity1 has reinvigorated interest in MΦ cell therapies. We generated Hoxb8-dependent myeloid progenitors (HDPs) by transducing lineage-negative bone marrow cells with a constitutively expressed Hoxb8 flanked by loxP. HDPs proliferate indefinitely and differentiate into MΦ when Hoxb8 is removed by a tamoxifen-inducible Cre. We genetically modified HDPs with a constitutively active GMCSF receptor and the tamoxifen-induced transcription factor IRF8, which we have termed “HDP-on.” The HDP-on proliferates without GMCSF and differentiates into the MΦ upon exposure to tamoxifen and ruxolitinib (GMCSF inhibitor via JAK1/2 blockade). We quantified the biodistribution of HDPs transplanted via intraperitoneal injection into immunodeficient NCG mice with a luciferase reporter; HDPs are detected for 14 days in the peritoneal cavity, liver, spleen, kidney, bone marrow, brain, lung, heart, and blood. In immunocompetent BALB/c mice, HDP-on cells, but not HDPs, are detected 1 day post-transplantation in the peritoneal cavity. Pretreatment of BALB/c mice with liposomal clodronate significantly enhances survival at day 7 for HDPs and HDP-on cells in the peritoneal cavity, spleen, and liver, but cells are undetectable at day 14. Short-term post-transplantation survival of HDPs is significantly improved using HDP-on and liposomal clodronate, opening a path for MΦ-based therapeutics.

The relationship of clinical and haematological and immune parameters in patients with mielodisplastic syndrome

The Objective: to determine the relationship between changes in hema tology clinical condition and immunological parameters in patients with myelodysplastic syndrome (MDS).Materials and methods. There were examined 36 patients diagnosed with MDS . All patients were divided into groups depending on the response to treatment : Group 1 – patients before treatment; Group 2 – patients who have responded positively to therapy and became transfusion independent ( remission , partial remission and great hematological response ); Group 3 – patients, gave only a small hematology response and were transfusion dependent, as well as those that have not responded to treatment. The control group included 30 healthy individuals with no bad habits that can affect your blood.Results. The comparative analysis of clinical and hematological data and immunological study of peripheral blood cells and bone marrow in the initial period and in achieving clinical and hematologic compensation in MDS patients with refractory anemia with excess blasts (RANB). Established reduce the number of myeloid progenitor cells in the bone marrow origin in achieving clinical and hematologic compensation that can be considered as one of the criteria prognosis.Conclusion. Results of the study show that with a positive response to treatment of patients with MDS improved clinical and hematological parameters, patients achieved transfusion independence while they watch reducing hematopoietic cells in bone marrow. The dynamics of immunological parameters can be used in evaluating the effectiveness of treatment and prognosis of MDS.

A pharmacodynamic model of Bcr-Abl signalling in chronic myeloid leukaemia.

Chronic myeloid leukaemia (CML) is an unusual malignancy in which myeloid progenitor cells are transformed by a single chromosomal translocation where the Bcr domain of chromosome 22 is placed adjacent to the proto-oncogene c-Abl of chromosome 9, resulting in constitutive Abl tyrosine kinase activity. This has a twofold effect: it causes increased numbers of myeloid progenitor cells and circulating myeloid cells, and it causes leakage of reactive oxygen species from mitochondria. We describe a kinetic and pharmacodynamic (PD) model of Bcr-Abl signalling in myeloid cells that is used to simulate effects of four classes of drugs: Bcr-Abl signalling inhibitors, such as imatinib, cyclin-dependent kinase inhibitors, and pro- and anti-oxidants. The model also has the potential to describe the PD effects of agents acting on other sites in the Bcr-Abl signalling pathway. Having calibrated the model against dose-response curves of these drugs acting as single agents on Bcr-Abl-transformed cells in vitro, the model was used to predict effects of the agents in combination. Used in conjunction with pharmacokinetic models, our PD model enables an approach to protocol optimization: large numbers of doses and timings and (in the case of combination treatments) relative dose ratios can be simulated in silico. Predicted selectivity, as well as efficacy, can be extracted from the model. An understanding of the Bcr-Abl signalling pathway has implications for strategies to prevent acquired drug resistance, and for preventing or delaying CML progression to its blast phase.

GSK‐3β inhibition promotes early engraftment of ex vivo‐expanded haematopoietic stem cells

Umbilical cord blood (UCB) is a source of stem cells used for allogeneic transplantation, in addition to bone marrow and peripheral blood. Limited numbers of stem cells in a single UCB unit is associated with slow haematopoietic recovery and high risk of graft failure, particularly in adult patients. UCB stem cells can be expanded ex vivo; however, rapid differentiation reduces their regenerative potential. We have recently shown that Wnt/β-catenin signalling is down-regulated in ex vivo-expanded stem cells; therefore, we propose that re-activation of Wnt signalling using GSK-3β inhibition may act to improve regenerative potential of these ex vivo-expanded stem cells. Immunocompromised mice were employed in transplantation studies to determine stem-cell engraftment. Flow cytometry was used to phenotype the engrafted human cells. Retroviral gene transfer was used to examine the role of Myc gene up-regulated by GSK-3β inhibition, in ex vivo-expanded stem cells. Treatment with GSK-3β inhibitor, 6-bromoindirubin 3'-oxime (BIO) improved early human cell engraftment in the mice and elevated the numbers of myeloid progenitor cells in cytokine-stimulated culture. BIO up-regulated β-catenin and c-myc in ex vivo-expanded stem cells. Ectopic expression of Myc acted to increase clonogenic potential and to delay differentiation of haematopoietic progenitor cells, suggesting the potential mechanism to improve regenerative potential of ex vivo-expanded grafts. Pharmacological inhibition of GSK-3β provided a novel approach to improve early engraftment of ex vivo-expanded haematopoietic progenitor cells.

Acat1 Gene Ablation in Mice Increases Hematopoietic Progenitor Cell Proliferation in Bone Marrow and Causes Leukocytosis

To investigate the role of acyl-CoA:cholesterol acyltransferase 1 (ACAT1) in hematopoiesis. ACAT1 converts cellular cholesterol to cholesteryl esters for storage in multiple cell types and is a potential drug target for human diseases. In mouse models for atherosclerosis, global Acat1 knockout causes increased lesion size; bone marrow transplantation experiments suggest that the increased lesion size might be caused by ACAT1 deficiency in macrophages. However, bone marrow contains hematopoietic stem cells that give rise to cells in myeloid and lymphoid lineages; these cell types affect atherosclerosis at various stages. Here, we test the hypothesis that global Acat1(-/-) may affect hematopoiesis, rather than affecting macrophage function only, and show that Acat1(-/-) mice contain significantly higher numbers of myeloid cells and other cells than wild-type mice. Detailed analysis of bone marrow cells demonstrated that Acat1(-/-) causes a higher proportion of the stem cell-enriched Lin(-)Sca-1(+)c-Kit(+) population to proliferate, resulting in higher numbers of myeloid progenitor cells. In addition, we show that Acat1(-/-) causes higher monocytosis in Apoe(-/-) mouse during atherosclerosis development. ACAT1 plays important roles in hematopoiesis in normal mouse and in Apoe(-/-) mouse during atherosclerosis development.

Leukemia-related chromosomal loss detected in hematopoietic progenitor cells of benzene-exposed workers

Benzene exposure causes acute myeloid leukemia, and hematotoxicity, shown as suppression of mature blood and myeloid progenitor cell numbers. As the leukemia-related aneuploidies monosomy 7 and trisomy 8 previously had been detected in the mature peripheral blood cells of exposed workers, we hypothesized that benzene could cause leukemia through the induction of these aneuploidies in hematopoietic stem and progenitor cells. We measured loss and gain of chromosomes 7 and 8 by fluorescence in situ hybridization in interphase colony-forming unit-granulocyte-macrophage (CFU-GM) cells cultured from otherwise healthy benzene-exposed (n=28) and unexposed (n=14) workers. CFU-GM monosomy 7 and 8 levels (but not trisomy) were significantly increased in subjects exposed to benzene overall, compared to levels in the control subjects (p=0.0055 and p=0.0034, respectively). Levels of monosomy 7 and 8 were significantly increased in subjects exposed to <10 ppm (20%, p=0.0419 and 28%, p=0.0056, respectively) and ≥10 ppm (48%, p=0.0045 and 32%, p=0.0354) benzene, compared with controls, and significant exposure-response trends were detected (ptrend=0.0033 and 0.0057). These data show that monosomies 7 and 8 are produced in a dose-dependent fashion in the blood progenitor cells of workers exposed to benzene and may be mechanistically relevant biomarkers of early effect for benzene and other leukemogens.

The transcription factor NR4A1 (Nur77) controls bone marrow differentiation and the survival of Ly6C− monocytes

The transcription factors that regulate differentiation into the monocyte subset in bone marrow have not yet been identified. Here we found that the orphan nuclear receptor NR4A1 controlled the differentiation of Ly6C- monocytes. Ly6C- monocytes, which function in a surveillance role in circulation, were absent from Nr4a1-/- mice. Normal numbers of myeloid progenitor cells were present in Nr4a1-/- mice, which indicated that the defect occurred during later stages of monocyte development. The defect was cell intrinsic, as wild-type mice that received bone marrow from Nr4a1-/- mice developed fewer patrolling monocytes than did recipients of wild-type bone marrow. The Ly6C- monocytes remaining in the bone marrow of Nr4a1-/- mice were arrested in S phase of the cell cycle and underwent apoptosis. Thus, NR4A1 functions as a master regulator of the differentiation and survival of 'patrolling' Ly6C- monocytes.

Homeostatic Role of the Krüppel-Like Factor 4 (KLF4) in Proliferation and Differentiation of Primitive Hematopoietic Progenitor Cells.

Abstract 1497Poster Board I-520KLF4 is a tumor suppressor in the gastrointestinal tract known to induce cell cycle arrest in a cell context dependent manner. We recently reported that KLF4 maintains quiescence of T lymphocytes downstream of T-cell receptor signaling (Yamada et al., Nature Immunology, 2009). The role of KLF4 in reprogramming adult somatic cells into pluripotent stem cells along with Oct3/4, c-Myc and Sox2 suggests that KLF4 restricts proliferation of undifferentiated cells. In spite of a redundant role of KLF4 in fetal liver hematopoietic stem cells (HSC), its role in the maintenance of adult bone marrow HSCs has not been studied yet. To study the role of KLF4 in the hematopoietic system we used gain- and loss-of-function mouse models. Retroviral transfer of KLF4 into wild type bone marrow (BM) cells led to significant reduction of colony forming units (CFU) in methylcellulose cultures due to increased apoptosis and lower proliferation. Then, Mx1-Cre was used to induce deletion of Klf4-floxed mice by polyI:C administration. Analysis of peripheral blood cells up to 6-9 months post polyI:C administration showed significant reduction of monocytes, as previously reported, and expansion of CD8+CD44+ T cells due to their increased proliferative potential. BM cells from Klf4-deficient mice exhibited increased number of myeloid progenitor cells measured by flow cytometry (Lin-Sca-1-c-kit+FcRII/III+CD34+ cells), CFU and CFU-S8. Cytoablation with 5-fluorouracil (5-FU) showed lower nadir of peripheral white blood cells in Klf4-deficient mice compared to control mice. In spite of normal multilineage reconstitution in BM transplants experiments, competitive reconstitution with Klf4-deficient and normal BM cells resulted in reduced contribution of Klf4-deficient cells to peripheral blood, likely due to homing and proliferative differences. Collectively, our data shows that KLF4 has an important role in function of hematopoietic stem and progenitor cells. Disclosures:No relevant conflicts of interest to declare.

A Novel Anti-Inflammatory Function of Human Galectin-1: Inhibition of Hematopoietic Progenitor Cell Mobilization

The immunosuppressive and anti-inflammatory activity of mammalian galectin-1 (Gal-1) has been well established in experimental in vivo animal models and in vitro studies. Since the proliferation and migration of leukocytes represent a necessary and important step in response to the inflammatory insult, we have investigated whether Gal-1 affects the mobilization of hematopoietic progenitor cells (HPC) induced by cyclophosphamide (CY) and granulocyte colony-stimulating factor (G-CSF). Bone marrow HPCs were mobilized with CY/G-CSF or CY/G-CSF plus human recombinant Gal-1 in BDF1 mice. Bone marrow (BM) and blood cells were taken at different time points and analyzed for their in vivo repopulating ability in lethally irradiated syngeneic animals. The number of myeloid progenitor cells in BM and blood samples was determined by colony-forming cell assay. Expression of surface markers (Sca-1, CD3epsilon, CD45R/B220, Ter-119, GR-1, and CD11b) on nucleated marrow cells was measured by flow cytometry. The lymphocytes, granulocytes, and monocytes in blood samples were counted after Giemsa staining. Gal-1 dramatically inhibited CY/G-CSF-induced HPC migration to the periphery as well as decreased peripheral neutrophilia and monocytosis in a dose- and time-dependent manner. In contrast, Gal-1 itself stimulated HPC expansion and accumulation within the BM. The presence of the lectin for inhibition of HPC mobilization was essential during the second half of the treatment. Moreover, Gal-1 inhbited transendothelial migration of BM-derived HPCs in response to SDF-1 in vitro. Gal-1 blocked BM progenitor cell migration induced by CY/G-CSF treatment, indicating a novel anti-inflammatory function of the lectin. We suggest that the inhibition of HPC mobilization occurs mainly via obstructing the transendothelial migration of BM-derived cells including primitive hematopoietic and committed myeloid progenitor cells and mature granulocytes and monocytes.

Bone Marrow-derived Cells Require a Functional Glucose 6-Phosphate Transporter for Normal Myeloid Functions

Glycogen storage disease type Ib (GSD-Ib) is caused by a deficiency in the ubiquitously expressed glucose 6-phosphate transporter (Glc-6-PT). Glc-6-PT activity has been shown to be critical in the liver and kidney where a deficiency disrupts glucose homeostasis. GSD-Ib patients also have defects in the neutrophil respiratory burst, chemotaxis, and calcium flux. They also manifest neutropenia, but whether Glc-6-PT deficiency in the bone marrow underlies myeloid dysfunctions in GSD-Ib remains controversial. To address this, we transferred bone marrow from Glc-6-PT-deficient (Glc-6-PT(-/-)) mice to wild-type mice to generate chimeric mice (BM-Glc-6-PT(-/-)). As a control, we also transferred bone marrow between wild-type mice (BM-Glc-6-PT(+/+)). While BM-Glc-6-PT(+/+) mice have normal myeloid functions, BM-Glc-6-PT(-/-) mice manifest myeloid abnormalities characteristic of Glc-6-PT(-/-) mice. Both have impairments in their neutrophil respiratory burst, chemotaxis response, and calcium flux activities and exhibit neutropenia. In the bone marrow of BM-Glc-6-PT(-/-) and Glc-6-PT(-/-) mice, the numbers of myeloid progenitor cells are increased, while in the serum there is an increase in granulocyte colony-stimulating factor and chemokine KC levels. Moreover, in an experimental model of peritoneal inflammation, local production of KC and the related chemokine macrophage inflammatory protein-2 is decreased in both BM-Glc-6-PT(-/-) and Glc-6-PT(-/-) mice along with depressed peritoneal neutrophil accumulation. The neutrophil recruitment defect was less severe in BM-Glc-6-PT(-/-) mice than in Glc-6-PT(-/-) mice. These findings demonstrate that Glc-6-PT expression in bone marrow and neutrophils is required for normal myeloid functions and that non-marrow Glc-6-PT activity also influences some myeloid functions.

Uncaria tomentosa extract increases the number of myeloid progenitor cells in the bone marrow of mice infected with Listeria monocytogenes

In this study, we demonstrated that Uncaria tomentosa extract (UTE) protects mice from a lethal dose of Listeria monocytogenes when administered prophylactically at 50, 100, 150 and 200 mg/kg for 7 days, with survival rates up to 35%. These doses also prevented the myelosuppression and the splenomegaly caused by a sublethal infection with L. monocytogenes, due to increased numbers of granulocyte-macrophage progenitors (CFU-GM) in the bone marrow. Non-infected mice treated with 100 mg/kg UTE also presented higher numbers of CFU-GM in the bone marrow than the controls. Investigation of the production of colony-stimulating factors revealed increased colony-stimulating activity (CSA) in the serum of normal and infected mice pre-treated with UTE. Moreover, stimulation of myelopoiesis and CSA occurred in a dose-dependent manner, a plateaux being reached with the dose of 100 mg/kg. Further studies to investigate the levels of factors such as IL-1 and IL-6 were undertaken. We observed increases in the levels of IL-1 and IL-6 in mice infected with L. monocytogenes and treated with 100 mg/kg of UTE. White blood cells (WBC) and differential counting were also performed, and our results demonstrated no significant changes in these data, when infected mice were pre-treated with 100 mg/kg of UTE. All together, our results suggest that UTE indirectly modulates immune activity and probably disengages Listeria-induced supression of these responses by inducing a higher reserve of myeloid progenitors in the bone marrow in consequence of biologically active cytokine release (CSFs, IL-1 and IL-6).

Impaired granulocytopoiesis in patients with chronic idiopathic neutropenia is associated with increased apoptosis of bone marrow myeloid progenitor cells

To probe the pathophysiologic mechanisms underlying neutropenia in patients with chronic idiopathic neutropenia (CIN) with hypoplastic and left-shifted granulocytic series in the bone marrow (BM), we have studied granulocytopoiesis in 32 adults with CIN by evaluating the number and survival characteristics of cells in several stages of granulocyte differentiation using flow cytometry and BM culture assays. We found that patients with CIN displayed a low percentage of CD34(+)/CD33(+) cells, defective granulocyte colony-forming unit (CFU-G) growth potential of BM mononuclear or purified CD34(+) cells, and low CFU-G recovery in long-term BM cultures (LTBMCs), compared with controls (n = 46). A low percentage of CD34(+)/CD33(+) cells in patients was associated with accelerated apoptosis and Fas overexpression within this cell compartment compared with controls. No significant difference was documented in the percentage of apoptotic cells or the Fas(+) cells within the fractionated CD34(+)/CD33(-), CD34(-)/CD33(+), and CD34(-)/CD33(-)/CD15(+) BM subpopulations or the peripheral blood neutrophils, suggesting that the underlying cellular defect in CIN probably concerns the committed granulocyte progenitors. LTBMC stromal layers from patients produced abnormally high amounts of tumor necrosis factor alpha and cytokine levels in culture supernatants inversely correlated with the number of myeloid progenitor cells and positively with the proportion of apoptotic CD34(+) cells. Patient LTBMC stromal layers displayed pathologic interferon gamma and Fas-ligand mRNA expression and failed to support normal myelopoiesis. These data suggest that impaired granulocytopoiesis in CIN is probably due to overproduction of inflammatory cytokines by immune cells within the BM microenvironment that may exert an inhibitory effect on myelopoiesis by inducing Fas-mediated apoptosis in the granulocyte progenitors.

Effect of leukotactin-1 on the protection in vivo of myeloid progenitor cells against cytotoxic chemotherapeutics.

Leukotactin-1 (Lkn-1) is a human CC chemokine that induces chemotaxis of neutrophils, monocytes, eosinophils, and lymphocytes and suppresses colony formation of myeloid progenitor cells in vitro. Present studies evaluated the myeloprotective capabilities of Lkn-1 in vivo against Ara-C and 5-fluorouracil (5-FU). The effect of Lkn-1 on myelopoiesis was first assessed in vivo by injecting recombinant Lkn-1 in C3H/HeJ mice. Lkn-1 rapidly decreased cycling rates and absolute numbers of myeloid progenitor cells in marrow. Lkn-1 administration prior to and during the chemotherapeutics treatment resulted in increased progenitors for colony-forming units-granulocyte/macrophage (CFU-GM), colony-forming units-granulocyte/erythroid/megakaryocyte/macrophage (CFU-GEMM), and burst-forming units-erythroid (BFU-E) compared with a saline-treated group. The protective effects lasted until day 3 after the termination of Ara-C administration and until day 7 after the termination of 5-FU administration. The results indicate that Lkn-1 protects bone marrow myeloid progenitor cells when cytotoxic chemotherapeutics are used in a preclinical setting. These results may be of use in clinical treatment for myeloprotection.

ICSBP/IRF-8: its regulatory roles in the development of myeloid cells.

Interferon (IFN) consensus sequence binding protein (ICSBP)/IFN regulatory factor (IRF)-8 is an IFNgamma-inducible transcription factor of the IRF family and regulates transcription through multiple target DNA elements, such as IFN-stimulated response element (ISRE), Ets/IRF composite element, and IFN-gamma activation site (GAS). ICSBP(-/-) mice are immunodeficient and susceptible to various pathogens. They have defects in the macrophage function, including the ability to induce interleukin-12 (IL-12) p40 and some IFN-gamma-responsible genes. In addition, ICSBP(-/-) mice develop a chronic myelogenous leukemia (CML)-like syndrome, where a systemic expansion of granulocytes is followed by a fatal blast crisis. ICSBP(-/-) mice harbor an increased number of myeloid progenitor cells, and the -/- progenitors preferentially give rise to granulocytes, although they cannot efficiently generate another descendant of the myeloid lineage, macrophages. Studies with myeloid progenitor cells have shown that ICSBP drives their differentiation toward macrophage, whereas it inhibits granulocyte differentiation. Furthermore, myeloid cells from ICSBP(-/-) mice are resistant to apoptosis. These results illustrate the mechanism by which the loss of ICSBP leads to immunodeficiency and CML-like syndrome and suggest ICSBP's critical role in the development of myeloid cells.

Effect of rhGM-CSF on the Kinetics of Hematopoiesis in Long-term Marrow Cultures from Patients with Acute Myelogenous Leukemia

In the present study, we have assessed the effects of recombinant human Granulocyte-Macrophage Colony-Stimulating Factor (rhGM-CSF) in Dexter-type long-term marrow cultures (LTMC) from patients with acute myelogenous leukemia (AML). Addition of rhGM-CSF to AML LTMC resulted in a significant increase in the number of total nucleated cells (1.3-4.3-fold, as compared to untreated cultures). However, a simultaneous decrease in the numbers of myeloid progenitor cells (CFU) was observed. Interestingly, there was a selective stimulation of the growth of leukemic progenitors (AML-CFU). Indeed, whereas on day 0 these cells were detected in only 2 patients, between weeks 1 and 5 they were detected in 10 of the 14 patients included in the study. It is noteworthy that around 50% of the cells detected in the non-adherent fraction of rhGM-CSF-treated AML LTMC were blasts, whereas in untreated cultures, blasts corresponded to only 23% of the non-adherent cells, and the majority corresponded to cells of the monocyte-macrophage lineage. These results indicate that rhGM-CSF is a cytokine with a significant stimulatory activity for the in vitro growth of AML progenitor and blast cells, and, together with previous reports in the literature, suggest that the use of rhGM-CSF in clinical settings must be taken with caution since this cytokine, although beneficial in reducing the risk of infections after chemotherapy, may induce the reappearance of the disease after treatment. Further studies should be encouraged to understand in greater detail the effects of rhGM-CSF, and other cytokines, on the hematopoietic system of AML patients.

A distinct role for apoptosis in the changes in lymphopoiesis and myelopoiesis created by deficiencies in zinc.

Reduced numbers of lymphocytes in the peripheral immune system appeared to be a significant cause of the loss in host defense capacity in humans and animals that are zinc deficient (ZD). A series of studies verified that ZD substantially reduced the lymphocyte compartment of both the marrow and thymus in young adult mice, with large losses noted among the pre-B and pre-T cells. Suboptimal nutriture along with chronic production of glucocorticoids generated during ZD had accelerated apoptosis among these precursor lymphocytes two- to threefold. Thus, the primary cause of the lymphopenia created by ZD was reduced production of lymphocytes and heightened cell death among precursor cells. The data will also show that myelopoiesis in the marrow was protected and enhanced numbers of myeloid progenitor cells were found in S and G2/M. Thus, as zinc became limiting the second line of defense appeared to be down-regulated via reduction of lymphopoiesis while cells of the myeloid lineage were protected to maintain the first line of defense that provides innate immunity. This may represent an important adaptation of the immune system to suboptimal nutriture that deserves further exploration.

Increased peripheral stem cell pool in MDS: an indication of disease progression?

The colony-forming capacity of the peripheral blood stem/progenitor cells (PBSC) in different forms of myelodysplastic syndrome (MDS) was investigated. In most cases of refractory anemia (RA) the colony growth of PBSC was definitely reduced as compared to the controls. However, in RA with unfavorable chromosomal aberrations, in refractory anemia with ringed sideroblasts (RARS) and in advanced stages of MDS such as refractory anemia with excess blasts (RAEB) and refractory anemia in transformation (RAEB-t), the number of myeloid progenitor cells increased up to 100-fold. In chronic myelomonocytic leukemia (CMML), the increase was even more marked, up to 350-fold. Although the number of PBSC was strongly elevated, these cells were not able to restore hematopoiesis in vivo. In conclusion, the increase of circulating colony-forming cells (CFC) seems to be associated with disease progression, and thus, the evaluation of PBSC could be an important parameter in the diagnosis of MDS.