Steroids and hematopoiesis. III. The response of granulocytic and erythroid colony-forming cells to steroids of different classes. (1/5038)

Selected androgenic and nonandrogenic steroids enhance in vitro granulocytic and erythroid colony formation by mouse marrow cells, but do so by influencing either different target cells or cells in different states of cell cycle. Etiocholanolone, a naturally occurring nonandrogenic testosterone metabolite, permits cells not in active cycle to respond to colony-stimulating factor or erythropoietin. Fluoxymesterone, a synthetic androgen, appears to enhance colony growth by increasing the responsiveness of target cells to tropic stimuli. The majority of cells responding to this androgen are in active DNA synthesis. Direct comparison, however, of etiocholanolone-dependent erythroid or granulocytic colony-forming cells demonstrates nonidentity of the target cells. Thus colony-forming units responding to different classes of steroids are in different states of cell cycle and are physically separable. The enhancement of the in vitro response of colony-forming cells to regulating hormones by steroids such as etiocholanolane suggests a mechanism by which such agents may be therapeutically effective in certain cases of marrow failure in man.  (+info)

The cardiac homeobox gene Csx/Nkx2.5 lies genetically upstream of multiple genes essential for heart development. (2/5038)

Csx/Nkx2.5 is a vertebrate homeobox gene with a sequence homology to the Drosophila tinman, which is required for the dorsal mesoderm specification. Recently, heterozygous mutations of this gene were found to cause human congenital heart disease (Schott, J.-J., Benson, D. W., Basson, C. T., Pease, W., Silberbach, G. M., Moak, J. P., Maron, B. J., Seidman, C. E. and Seidman, J. G. (1998) Science 281, 108-111). To investigate the functions of Csx/Nkx2.5 in cardiac and extracardiac development in the vertebrate, we have generated and analyzed mutant mice completely null for Csx/Nkx2.5. Homozygous null embryos showed arrest of cardiac development after looping and poor development of blood vessels. Moreover, there were severe defects in vascular formation and hematopoiesis in the mutant yolk sac. Interestingly, TUNEL staining and PCNA staining showed neither enhanced apoptosis nor reduced cell proliferation in the mutant myocardium. In situ hybridization studies demonstrated that, among 20 candidate genes examined, expression of ANF, BNP, MLC2V, N-myc, MEF2C, HAND1 and Msx2 was disturbed in the mutant heart. Moreover, in the heart of adult chimeric mice generated from Csx/Nkx2.5 null ES cells, there were almost no ES cell-derived cardiac myocytes, while there were substantial contributions of Csx /Nkx2.5-deficient cells in other organs. Whole-mount &bgr;-gal staining of chimeric embryos showed that more than 20% contribution of Csx/Nkx2. 5-deficient cells in the heart arrested cardiac development. These results indicate that (1) the complete null mutation of Csx/Nkx2.5 did not abolish initial heart looping, (2) there was no enhanced apoptosis or defective cell cycle entry in Csx/Nkx2.5 null cardiac myocytes, (3) Csx/Nkx2.5 regulates expression of several essential transcription factors in the developing heart, (4) Csx/Nkx2.5 is required for later differentiation of cardiac myocytes, (5) Csx/Nkx2. 5 null cells exert dominant interfering effects on cardiac development, and (6) there were severe defects in yolk sac angiogenesis and hematopoiesis in the Csx/Nkx2.5 null embryos.  (+info)

Phenotypic and functional evidence for the expression of CXCR4 receptor during megakaryocytopoiesis. (3/5038)

The identification of stromal cell-derived factor (SDF)-1alpha as a chemoattractant for human progenitor cells suggests that this chemokine and its receptor might represent critical determinants for the homing, retention, and exit of precursor cells from hematopoietic organs. In this study, we investigated the expression profile of CXCR4 receptor and the biological activity of SDF-1alpha during megakaryocytopoiesis. CD34(+) cells from bone marrow and cord blood were purified and induced to differentiate toward the megakaryocyte lineage by a combination of stem-cell factor (SCF) and recombinant human pegylated megakaryocyte growth and development factor (PEG-rhuMGDF). After 6 days of culture, a time where mature and immature megakaryocytes were present, CD41(+) cells were immunopurified and CXCR4mRNA expression was studied. High transcript levels were detected by a RNase protection assay in cultured megakaryocytes derived from cord blood CD34(+) cells as well as in peripheral blood platelets. The transcript levels were about equivalent to that found in activated T cells. By flow cytometry, a large fraction (ranging from 30% to 100%) of CD41(+) cells showed high levels of CXCR4 antigen on their surface, its expression increasing in parallel with the CD41 antigen during megakaryocytic differentiation. CXCR4 protein was also detected on peripheral blood platelets. SDF-1alpha acts on megakaryocytes by inducing intracellular calcium mobilization and actin polymerization. In addition, in in vitro transmigration experiments, a significant proportion of megakaryocytes was observed to respond to this chemokine. This cell migration was inhibited by pertussis toxin, indicating coupling of this signal to heterotrimeric guanine nucleotide binding proteins. Although a close correlation between CD41a and CXCR4 expession was observed, cell surface markers as well as morphological criteria indicate a preferential attraction of immature megakaryocytes (low level of CD41a and CD42a), suggesting that SDF-1alpha is a potent attractant for immature megakaryocytic cells but is less active on fully mature megakaryocytes. This hypothesis was further supported by the observation that SDF-1alpha induced the migration of colony forming unit-megakaryocyte progenitors (CFU-MK) and the expression of activation-dependent P-selectin (CD62P) surface antigen on early megakaryocytes, although no effect was observed on mature megakaryocytes and platelets. These results indicate that CXCR4 is expressed by human megakaryocytes and platelets. Furthermore, based on the lower responses of mature megakaryocytes and platelets to SDF-1alpha as compared with early precursors, these data suggest a role for this chemokine in the maintenance and homing during early stages of megakaryocyte development. Moreover, because megakaryocytes are also reported to express CD4, it becomes important to reevaluate the role of direct infection of these cells by the human immunodeficiency virus (HIV)-1 in HIV-1-related thrombocytopenia.  (+info)

Organ-selective homing defines engraftment kinetics of murine hematopoietic stem cells and is compromised by Ex vivo expansion. (4/5038)

Hematopoietic reconstitution of ablated recipients requires that intravenously (IV) transplanted stem and progenitor cells "home" to organs that support their proliferation and differentiation. To examine the possible relationship between homing properties and subsequent engraftment potential, murine bone marrow (BM) cells were labeled with fluorescent PKH26 dye and injected into lethally irradiated hosts. PKH26(+) cells homing to marrow or spleen were then isolated by fluorescence-activated cell sorting and assayed for in vitro colony-forming cells (CFCs). Progenitors accumulated rapidly in the spleen, but declined to only 6% of input numbers after 24 hours. Although egress from this organ was accompanied by a simultaneous accumulation of CFCs in the BM (plateauing at 6% to 8% of input after 3 hours), spleen cells remained enriched in donor CFCs compared with marrow during this time. To determine whether this differential homing of clonogenic cells to the marrow and spleen influenced their contribution to short-term or long-term hematopoiesis in vivo, PKH26(+) cells were sorted from each organ 3 hours after transplantation and injected into lethally irradiated Ly-5 congenic mice. Cells that had homed initially to the spleen regenerated circulating leukocytes (20% of normal counts) approximately 2 weeks faster than cells that had homed to the marrow, or PKH26-labeled cells that had not been selected by a prior homing step. Both primary (17 weeks) and secondary (10 weeks) recipients of "spleen-homed" cells also contained approximately 50% higher numbers of CFCs per femur than recipients of "BM-homed" cells. To examine whether progenitor homing was altered upon ex vivo expansion, highly enriched Sca-1(+)c-kit+Lin- cells were cultured for 9 days in serum-free medium containing interleukin (IL)-6, IL-11, granulocyte colony-stimulating factor, stem cell factor, flk-2/flt3 ligand, and thrombopoietin. Expanded cells were then stained with PKH26 and assayed as above. Strikingly, CFCs generated in vitro exhibited a 10-fold reduction in homing capacity compared with fresh progenitors. These studies demonstrate that clonogenic cells with differential homing properties contribute variably to early and late hematopoiesis in vivo. The dramatic decline in the homing capacity of progenitors generated in vitro underscores critical qualitative changes that may compromise their biologic function and potential clinical utility, despite their efficient numerical expansion.  (+info)

Increase of hematopoietic responses by triple or single helical conformer of an antitumor (1-->3)-beta-D-glucan preparation, Sonifilan, in cyclophosphamide-induced leukopenic mice. (5/5038)

It has been suggested that the immunopharmacological activity of soluble (1-->3)-beta-D-glucan depends on its conformation in mice. In this study, we examined the relationship between the conformation of Sonifilan (SPG) and hematopietic responses in cyclophosphamide (Cy)-induced leukopenic mice. SPG, a high molecular weight (1-->3)-beta-D-glucan, has a triple helical conformation in water, and it was changed by treatment with aqueous sodium hydroxide to the single helical conformer (SPG-OH). The effects of SPG or SPG-OH on hematopoietic responses in cyclophosphamide induced leukopenic mice were investigated by monitoring i) gene expression of cytokines by RT-PCR, ii) protein synthesis of interleukin 6 (IL-6) by ELISA and iii) colony formation of bone marrow cells (BMC). The mice administered Cy and SPG or SPG-OH expressed and produced higher levels of IL-6 mRNA and protein than the mice administered only Cy. Gene expression of NK1.1 was also induced by Cy/SPG (or SPG-OH) treatment. Induced gene expression of stem cell factor (SCF) and macrophage-colony stimulating factor (M-CSF) by SPG/SPG-OH were also found in in vitro culture of BMC from Cy treated mice. These results strongly suggested that conformation of the glucans, single and triple helix, are independent of the hematopietic response.  (+info)

Influence of monoclonal antiplatelet glycoprotein antibodies on in vitro human megakaryocyte colony formation and proplatelet formation. (6/5038)

The influence of antiplatelet glycoprotein (GP) antibodies on megakaryocytopoiesis in patients with idiopathic or immune thrombocytopenic purpura (ITP) has been well studied. However, the influence of GP antibodies on proplatelet formation is poorly understood. Here we investigated whether in vitro human megakaryocyte colony formation and proplatelet formation are affected by various monoclonal antiplatelet GP antibodies (MoAb). The megakaryocyte colony formation inhibition assay was performed by methylcellulose culture with modifications, using peripheral blood nonadherent mononuclear cells. The proplatelet formation inhibition assay was performed by megakaryocytes derived from CD34(+) cells, stimulated with thrombopoietin + stem cell factor, which were then incubated with antiplatelet GP MoAb for 24 or 48 hours. Anti-GP-Ibalpha MoAb (CD42b; HIP1) slightly inhibited megakaryocyte colony formation (P < .05). and strongly inhibited proplatelet formation (after 24 hours incubation, P < .0002; after 48 hours incubation, P < .0007). Anti-GP-IIb MoAb (CD41; 5B12) inhibited only proplatelet formation (only after 24 hours incubation, P <. 03). Anti-integrin alphavbeta3 MoAb (CD51/CD61; 23C6) only slightly inhibited colony size (P < .05). However, anti-GP-IIIa MoAb (CD61; Y2/51) did not inhibit either colony formation or proplatelet formation. These results suggest that antiplatelet GP MoAbs have differing effects on in vitro megakaryocyte colony formation and proplatelet formation.  (+info)

Mutant N-ras induces myeloproliferative disorders and apoptosis in bone marrow repopulated mice. (7/5038)

Mutations that activate the N-ras oncogene are among the most frequently detected genetic alterations in human acute myeloid leukemias (AMLs), Philadelphia chromosome-negative myeloproliferative disorders (MPDs), and myelodysplastic syndromes (MDSs). However, because N-ras has not been shown to induce these disorders in an in vivo model, the role of N-ras in the evolution of myeloid leukemia is unclear. To investigate the potential of N-ras to induce myeloid leukemia, lethally irradiated mice were reconstituted with bone marrow (BM) cells infected with a retroviral vector carrying activated N-ras. Approximately 60% of these mice developed hematopoietic disorders, including severe MPDs resembling human chronic myelogenous leukemia (CML) or AML with differentiation (French-American-British [FAB] classification M2). Other reconstituted mice succumbed to hematopoietic defects that were pathologically similar to human MDSs. The latter disorders appeared to be due to a myeloid impairment that was demonstrated by enumeration of day-12 colony-forming units-spleen (CFU-S) and by in vitro colony assays. A high level of apoptosis associated with thymic atrophy and peripheral blood (PB) lymphopenia was also evident in N-ras reconstituted mice. Our results are consistent with a model in which antiproliferative effects are a primary consequence of N-ras mutations and secondary transforming events are necessary for the development of myeloid leukemia. This is the first report of an in vivo model for N-ras induced MPD and leukemia.  (+info)

Deficiency of the hematopoietic cell-specific Rho family GTPase Rac2 is characterized by abnormalities in neutrophil function and host defense. (8/5038)

In mammals, the Rho family GTPase Rac2 is restricted in expression to hematopoietic cells, where it is coexpressed with Rac1. Rac2-deficient mice were created to define the physiological requirement for two near-identical Rac proteins in hematopoietic cells. rac2-/- neutrophils displayed significant defects in chemotaxis, in shear-dependent L-selectin-mediated capture on the endothelial substrate Glycam-1, and in both F-actin generation and p38 and, unexpectedly, p42/p44 MAP kinase activation induced by chemoattractants. Superoxide production by rac2-/- bone marrow neutrophils was significantly reduced compared to wild type, but it was normal in activated peritoneal exudate neutrophils. These defects were reflected in vivo by baseline neutrophilia, reduced inflammatory peritoneal exudate formation, and increased mortality when challenged with Aspergillus fumigatus. Rac2 is an essential regulator of multiple specialized neutrophil functions.  (+info)