Apoptosis Of Myeloid Progenitor Cells Research Articles

Differential Expression of C/Ebpa and Its Stem Cell-Specific Targets in Congenital Neutropenias and Cyclic Neutropenias

Severe congenital neutropenias (CN) and cyclic neutropenias (CyN) are disorders of hematopoiesis that differ markedly in disease severity. Mutations in ELANE are considered responsible for most cases of CyN and CN. ELANE mutations lead to accelerated apoptosis of myeloid progenitor cells due to the production of misfolded elastase protein and subsequent elevated unfolded protein response (UPR). In CyN at the peak of the cycle up to 10 % of the CD34+ cells were immature CD49f+ HSC that did not express mutated ELANE and therefore escaped from UPR (escaper cells). The proliferation and differentiation of CD49f+ cells during the ascending arm of the cycle led to increased neutrophils at the peak of the cycle stimulated by G-CSF, C/EBPA, HLF, MLL1, HOXA9 and MEIS1. At the peak of the cycle, CD34+ cells comprising the high percentage of CD49f+ cells, expressed up to 8-fold more C/EBPA, MLL1, HOXA9, MEIS1, and HLF levels as compared to cells from untreated healthy controls. This ensured sufficient neutrophil numbers in CyN at the peak. However, the mutated neutrophil elastase protein released by the high numbers of neutrophils at the peak of the cycle caused negative feedback of granulopoiesis by proteolytic digestion of G-CSF. Since G-CSF was not available anymore, CD34+ cells including CD49f+ cells did not proliferate and differentiate anymore during the downward arm of the cycle leading to neutropenia. CD34+ cells from CN patients contained only low percentages of CD49f+ cells and there was defective or no expression of C/EBPA, HLF, MLL1, HOXA9, and MEIS1.

Exogenous cardiolipin localizes to mitochondria and prevents TAZ knockdown-induced apoptosis in myeloid progenitor cells

The concentration and composition of cardiolipin (CL) in mitochondria are altered in age-related heart disease, Barth Syndrome, and other rare genetic disorders, resulting in mitochondrial dysfunction. To explore whether exogenous CL can be delivered to cells, CL was combined with apolipoprotein A-I to generate water-soluble, nanoscale complexes termed nanodisks (ND). Mass spectrometry of HL60 myeloid progenitor cell extracts revealed a 30-fold increase in cellular CL content following incubation with CL-ND. When CL-ND containing a fluorescent CL analogue was employed, confocal microscopy revealed CL localization to mitochondria. The ability of CL-ND to elicit a physiological response was examined in an HL60 cell culture model of Barth Syndrome neutropenia. siRNA knockdown of the phospholipid transacylase, tafazzin (TAZ), induced apoptosis in these cells. When TAZ knockdown cells were incubated with CL-ND, the apoptotic response was attenuated. Thus, CL-ND represent a potential intervention strategy for replenishment of CL in Barth Syndrome, age-related heart disease, and other disorders characterized by depletion of this key mitochondrial phospholipid.

The cellular and molecular mechanisms for neutropenia in Barth syndrome

Barth syndrome (BTHS), a rare, X-linked, recessive disease, is characterized by neutropenia and cardiomyopathy. BTHS is caused by loss-of-function mutations of the tafazzin (TAZ) gene. We developed a model of BTHS by transfecting human HL60 myeloid progenitor cells with TAZ-specific shRNAs. Results demonstrate a significant downregulation in TAZ expression, mimicking the effects of naturally occurring truncation mutations in TAZ. Flow cytometry analyses of cells with TAZ-specific, but not scrambled, shRNAs demonstrate nearly twofold increase in the proportion of annexin V-positive cells and significantly increased dissipation of mitochondrial membrane potential as determined by DIOC6 staining. Transfection of TAZ-specific shRNA had similar effects in U937 myeloid cells but not in lymphoid cell lines. Further studies in HL60 myeloid progenitor cells revealed aberrant release of cytochrome c from mitochondria and significantly elevated levels of activated caspase-3 in response to TAZ knockdown. Treatment with caspase-specific inhibitor zVAD-fmk resulted in substantially reduced apoptosis to near-normal levels. These data suggest that neutropenia in BTHS is attributable to increased dissipation of mitochondrial membrane potential, aberrant release of cytochrome c, activation of caspase-3, and accelerated apoptosis of myeloid progenitor cells, and that this defect can be partially restored in vitro by treatment with caspase-specific inhibitors.

Genetic Defects in Severe Congenital Neutropenia: Emerging Insights into Life and Death of Human Neutrophil Granulocytes

The discovery of genetic defects causing congenital neutropenia has illuminated mechanisms controlling differentiation, circulation, and decay of neutrophil granulocytes. Deficiency of the mitochondrial proteins HAX1 and AK2 cause premature apoptosis of myeloid progenitor cells associated with dissipation of the mitochondrial membrane potential, whereas mutations in ELA2/ELANE and G6PC3 are associated with signs of increased endoplasmic reticulum stress. Mutations in the transcriptional repressor GFI1 and the cytoskeletal regulator WASP also lead to defective neutrophil production. This unexpected diversity of factors suggests that multiple pathways are involved in the pathogenesis of congenital neutropenia.

TNF-α–dependent loss of IKKβ-deficient myeloid progenitors triggers a cytokine loop culminating in granulocytosis

Loss of IκB kinase (IKK) β-dependent NF-κB signaling in hematopoietic cells is associated with increased granulopoiesis. Here we identify a regulatory cytokine loop that causes neutrophilia in Ikkβ-deficient mice. TNF-α-dependent apoptosis of myeloid progenitor cells leads to the release of IL-1β, which promotes Th17 polarization of peripheral CD4(+) T cells. Although the elevation of IL-17 and the consecutive induction of granulocyte colony-stimulating factor compensate for the loss of myeloid progenitor cells, the facilitated induction of Th17 cells renders Ikkβ-deficient animals more susceptible to the development of experimental autoimmune encephalitis. These results unravel so far unanticipated direct and indirect functions for IKKβ in myeloid progenitor survival and maintenance of innate and Th17 immunity and raise concerns about long-term IKKβ inhibition in IL-17-mediated diseases.

HoxA10 Regulates Myeloid Progenitor Proliferation by Activating Transcription of the Gene Encoding Transforming Growth Factor Beta 2.

AbstractAbstract 1572HoxA10 is a homeodomain transcription factor which functions as a myeloid leukemia promoter. Correlative clinical studies found that increased expression of a group of HoxA proteins, including HoxA10, in acute myeloid leukemia (AML) was associated with poor prognosis. In murine models, overexpression of HoxA10 in the bone marrow was associated with development of a myeloproliferative disease which progressed to AML with time. These results suggested that HoxA10-overexpression dysregulated cell proliferation and/or survival, and predisposed to acquisition of additional mutations which led to differentiation block and AML. Additional investigations, we and others demonstrated that HoxA10 overexpression in murine hematopoietic stem cells (HSC) expanded the granulocyte/monocyte progenitor (GMP) population in vitro and in vivo. Despite this information about the impact of HoxA10 overexpression on myeloid leukemogenesis, the mechanisms by which HoxA10 exerts this effect are largely unknown. To investigate such mechanisms, we have been identifying HoxA10 target genes. In previous studies, we identified a number of HoxA10 target genes that encode phagocyte effector proteins. HoxA10 represses transcription of these gene in myeloid progenitors, and decreased HoxA10 repression activity contributes to phenotypic differentiation as myelopoiesis proceeds. This provided a potential mechanism for HoxA10 involvement in differentiation block, but not progenitor survival or expansion. We used a chromatin immuno-precipitation based approach to identify additional HoxA10 target genes involved in these activities. Previously, we reported that HoxA10 activated the DUSP4 gene in myeloid progenitor cells. This gene encodes Mitogen Activated Protein Kinase Phosphatase 2 (Mkp2) which inhibits Jnk-induced apoptosis in myeloid progenitor cells. This provided a mechanism for increased cell survival in HoxA10-overexpressing cells. In the current studies, we identified TGFB2 as a HoxA10 target gene. This gene encodes Transforming Growth Factor Beta 2 (TgfB2) a member of the TgfB super family of cytokines. Similar to TgfB1 and 3, TgfB2 interacts with TgfB-receptors I and II. However, unlike these more classical family members, TgfB2 induces proliferation of hematopoietic stem and progenitor cells. We found that HoxA10 activated the TGFB2 promoter via tandem cis elements in the proximal promoter. This resulted in autocrine stimulation of proliferation in HoxA10-overexpressing GMP and leukemia cells in vitro. Increased proliferation in HoxA10-overexpressing cells involved activation of the MAP kinase pathway in a TgfB2 dependent manner. These studies identify autocrine production of pro-proliferative cytokines as a novel mechanism for the function of Hox proteins. These findings have implications for ex vivo expansion of HSC and myeloid progenitors for tissue engineering. These result also have implications for therapeutic approaches to poor prognosis AML characterized by increased Hox expression. Disclosures:No relevant conflicts of interest to declare.

Knock-Down of TAZ Gene Expression: A Model of Barth Syndrome with Accelerated Apoptosis of Myeloid Progenitor Cells Improved upon Treatment with Caspase-Specific Inhibitor

Barth syndrome (BTHS) is an X-linked recessive disorder characterized by neutropenia, cardiac and skeletal myopathies, and growth retardation. The mortality is high due to progressive cardiomyopathy and/or overwhelming bacterial infections. The incidence of BTHS is estimated to be as high as 1 in 100,000, but it is still a poorly recognized disease. The majority of Barth patients have mutations in the tafazzin (G4.5 or TAZ) gene, most of which appear to truncate the tafazzin protein likely resulting in the loss of its function. Tafazzin is a phospholipid acyltransferase involved in remodeling cardiolipin, the main lipid of the inner mitochondrial membrane. As a result, BTHS patients exhibit reduced levels of total CL and accumulation of monolysocardiolipin. A drosophila model of the Barth syndrome was recently reported, but cellular or mouse models of this disorder are not yet available. The link between these metabolic defects triggered by TAZ mutations and neutropenia remains largely unknown. We hypothesized that TAZ mutations lead to the loss of function of tafazzin protein causing impaired cell survival of neutrophil precursors, reduced production of neutrophils and neutropenia. To test this hypothesis, we knocked down the expression of the tafazzin gene in human myeloid progenitor HL60 cells using TAZ-specific shRNA and examined its effect on cell survival. Four shRNAs specific to exons 4 through 7 of the TAZ gene were used for transfection of human myeloid progenitor HL60 cells that were later examined by flow cytometry and Western blot analyses. At least 2 of the shRNA constructs resulted in substantial down-regulation in the expression level of the tafazzin gene in transfected human myeloid progenitor cells as determined by Western blot and confirmed by RT-PCR using gene-specific and GAPDH-specific primers. Flow cytometry analysis of DIOC6-labeled cells revealed that knock-down of the TAZ gene expression was associated with a significantly elevated dissipation of mitochondrial membrane potential compared with control cells with scrambled shRNA ((p<0.0009, n=3). Apoptosis studies using flow cytometry analyses revealed a significant increase in proportion of apoptotic annexin-positive cells compared with control cells transfected with scrambled shRNA (p<0.0002, n=6). The observed increase in apoptosis in response to TAZ knock-down was caspase3-dependent as evidenced by Western blot analysis. Treatment of the cells with caspase-specific inhibitor zVAD-fmk significantly improved cell survival characteristics to near normal level as determined by flow cytometry (p<0.02, n=4), suggesting that caspase-specific inhibitors may represent potentially therapeutic agents for patients with Barth syndrome. Thus, these data demonstrate that the loss of function of the TAZ gene is cytotoxic to hematopoietic cells and suggest that severe neutropenia is due to accelerated apoptosis of myeloid progenitor cells.

Molecular Studies of Neutropenia in Barth Syndrome.

The Barth syndrome is a rare mitochondrial disease that causes dilated cardiomyopathy, skeletal myopathy, and severe neutropenia. It has been reported that at least two patients with Barth syndrome died from septicemia attributed to neutropenia. The disease is an X-linked autosomal recessive disorder caused by mutations in the G4.5 or TAZ (tafazzin) gene, which introduce truncations, substitution, or alternative splice sites. Hematological investigations revealed normal chemotaxis, but the presence cytoplasmic vacuoles in myeloid cells and maturation arrest of neutrophil development at the myelocyte stage. Thus, a defect in neutrophil formation appears to be the primary reason for neutropenia and the susceptibility to infections in the Barth syndrome patients. Recent studies were mostly focused on cardiac, genetic and metabolic abnormalities associated with the disorder. However, the neutropenia aspect of the Barth syndrome remains unclear. The TAZ gene mutations appear to truncate the tafazzin protein likely resulting in the loss of function. Although a drosophila model of the Barth syndrome was recently reported, the cellular or mouse models of this disorder are not available yet. Thus, the link between TAZ mutations and severe neutropenia remains unknown. We hypothesized that TAZ mutations, which lead to the loss of function of tafazzin protein, trigger impaired cell survival and reduced production of neutrophils and their neutrophil precursors, thus resulting in severe neutropenia in Barth syndrome. To test this hypothesis, we attempted to knock-down the expression of the tafazzin gene in human myeloid progenitor cells using TAZ-specific shRNA. Four shRNA sequences specific to exons 4 through 7 were used for transfection of human myeloid progenitor cells that were later examined by flow cytometry and Western blot analyses. At least 2 of the shRNA constructs resulted in a substantial down-regulation in the expression level of the tafazzin protein in transfected myeloid progenitor cells as determined by Western blot. Flow cytometry analyses revealed that the knock-down of TAZ gene expression was associated with approximately 40–50% increase in proportion of apoptotic annexin-positive cells compared with cells transfected with control scrambled shRNA. These data suggest that the loss of function of TAZ gene is cytotoxic to hematopoietic cells and that severe neutropenia is due to the accelerated apoptosis of myeloid progenitor cells in patients with Barth syndrome. Further studies needed to elucidate the specific signaling pathways and to identify potentially therapeutic agents capable of controlling accelerated apoptosis of myeloid cells in Barth syndrome.

Transforming growth factor‐beta1 affects interleukin‐10 production in the bone marrow of patients with chronic idiopathic neutropenia

Chronic idiopathic neutropenia (CIN) is a bone marrow (BM) failure syndrome characterized by accelerated apoptosis of myeloid progenitor cells because of a local imbalance between pro-inflammatory and anti-inflammatory cytokines. In this study, we investigated the interplay among transforming growth factor-beta1 (TGF-beta1), interleukin-10 (IL-10), and soluble flt-3 ligand (sFL) within the BM of CIN patients and probed the role of these cytokines in the pathophysiology of CIN. We used long-term BM cultures (LTBMC) to evaluate TGF-beta1, IL-10, and sFL levels in CIN patients (n = 70) and healthy subjects (n = 35). Cytokine levels in LTBMC supernatants were correlated with the number of circulating neutrophils and the proportion of BM CD34+/CD33+ myeloid progenitor cells. CIN patients had increased TGF-beta1 and sFL levels in LTBMCs compared with controls and individual cytokine values were found to be correlated inversely with the number of neutrophils and the proportion of CD34+/CD33+ cells. Patients displayed low supernatant IL-10 levels compared with controls and cytokine values were found to be correlated positively with the number of neutrophils and the proportion of CD34+/CD33+ cells. The levels of TGF-beta1 were found to be inversely correlated with IL-10 and positively with sFL values in LTBMC, supernatants suggesting a possible interplay among these cytokines in CIN BM. Neutralization of TGF-beta1 in LTBMCs increased IL-10 levels significantly in patients but not in controls, while neutralization had no effect on sFL levels. Excessive production of TGF-beta1 within the BM microenvironment of CIN patients results in downregulation of IL-10 and reduction of myeloid progenitor cells. Overexpression of sFL probably represents a compensatory mechanism to the low myeloid progenitor cells.

Kostmann syndrome or infantile genetic agranulocytosis, part one: Celebrating 50 years of clinical and basic research on severe congenital neutropenia

Congenital neutropenia in man was first reported 50 years ago by the Swedish paediatrician Rolf Kostmann. He coined the term "infantile genetic agranulocytosis" for this condition, which is now known as Kostmann syndrome. Recent studies have demonstrated a lack of antibacterial peptides and severe periodontitis in these patients despite recombinant growth factor treatment. Moreover, an increased degree of apoptosis of myeloid progenitor cells in the bone marrow has been shown. Future studies should aim to clarify the underlying molecular genetic defect in Kostmann syndrome.

Kostmann syndrome: severe congenital neutropenia associated with defective expression of Bcl-2, constitutive mitochondrial release of cytochrome c, and excessive apoptosis of myeloid progenitor cells

AbstractKostmann syndrome, or severe congenital neutropenia (SCN), is an autosomal recessive disorder of neutrophil production. To investigate the potential role of apoptosis in SCN, bone marrow aspirates and biopsies were obtained from 4 patients belonging to the kindred originally described by Kostmann and 1 patient with SCN of unknown inheritance. An elevated degree of apoptosis was observed in the bone marrow of these patients, and a selective decrease in B-cell lymphoma-2 (Bcl-2) expression was seen in myeloid progenitor cells. Furthermore, in vitro apoptosis of bone marrow-derived Kostmann progenitor cells was increased, and mitochondrial release of cytochrome c was detected in CD34+ and CD33+ progenitors from patients, but not in controls. Administration of granulocyte colony-stimulating factor (G-CSF) restored Bcl-2 expression and improved survival of myeloid progenitor cells. In addition, cytochrome c release was partially reversed upon incubation of progenitor cells with G-CSF. In sum, these studies establish a role for mitochondria-dependent apoptosis in the pathogenesis of Kostmann syndrome and yield a tentative explanation for the beneficial effect of growth factor administration in these patients. (Blood. 2004;103:3355-3361)

Retinoic acid induces apoptosis of human CD34 + hematopoietic progenitor cells : Involvement of retinoic acid receptors and retinoid X receptors depends on lineage commitment of the hematopoietic progenitor cells

Retinoids are bifunctional regulators of growth and differentiation of hematopoietic cells. In this study we explored the effects of retinoic acid (RA) on apoptosis of human CD34 + hematopoietic progenitor cells isolated from normal bone marrow. RA (100 nM) induced an increase in the percentage of dead cells from 24% to 44% at day 6 ( p < 0.05, n = 6) as compared to control cells cultured in medium alone. The effect was dose dependent and appeared relatively late. Significant differences were observed from day 4 onward. Apoptosis, or programmed cell death, was demonstrated as the mode of cell death by using the TUNEL assay, which detects single strand nicks in DNA, or by the Nicoletti technique demonstrating a subdiploid population by DNA staining. RA previously was found to inhibit granulocyte colony-stimulating factor—and not granulocyte-macrophage colony-stimulating factor—stimulated proliferation of CD34 + cells. However, we found that RA opposed anti-apoptotic effects of G-CSF and GM-CSF on CD34 + cells (G-CSF: 8% dead cells at day 6; G-CSF + RA: 20%; GM-CSF: 12%; GM-CSF + RA: 27%). Moreover, RA induced apoptosis of CD34 + cells and CD34 + CD71 + cells stimulated with erythropoietin. To explore the receptor signaling pathways involved in RA-induced apoptosis, we used selective ligands for retinoic acid receptors (RARs; RO13-7410) and retinoid X receptors (RXRs; RO 25-6603). We found that RARs were involved in RA-mediated apoptosis of myeloid progenitor cells, whereas RARs as well as RXRs were involved in RA-mediated apoptosis of erythroid progenitor cells.