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D Metcalf

Publications and source records attributed to D Metcalf.

At least 55 records · Page 3Linked to original sources

Cellular hematopoiesis in the twentieth century.

From work particularly In the last two decades of the century, the cell populations forming blood cells can now be purified and cultured clonally so that blood cell formation can be analyzed in vitro. A large number of specific regulators of this process have been identified and mass-produced in recombinant form. Three of these, erythropoletin, granulocyte colony-stimulating factor (G-CSF), and granulocyte-macrophage colony-stimulating factor (GM-CSF) are in extensive clinical use to stimulate hematopoiesis. Similar advances have characterized the processes by which T lymphocytes, B lymphocytes, and dendritic cells are formed and interact to mediate immune responses. In the last decade most research on blood cell formation has involved the generation of animals with inactivation of specific genes involved in hematopolesis. Major unsolved problems are the molecular control of differentiation commitment and maturation in hematopoietic cells, processes that need to be better understood to allow improvements in the management of leukemia and other disorders of hematopoiesis.

Hematology↗

The box-1 region of the leukemia inhibitory factor receptor alpha-chain cytoplasmic domain is sufficient for hemopoietic cell proliferation and differentiation.

Leukemia inhibitory factor (LIF) is a pleiotropic cytokine that acts on a variety of cell types and regulates cell proliferation and differentiation. The functional receptor for LIF is composed of LIFR alpha-chain (LIFRalpha) and gp130 both of which are shared in the functional receptors for oncostatin M, ciliary neurotrophic factor, and cardiotrophin-1. By using stable transfection of wild-type or cytoplasmic deletion mutants of LIFRalpha together with full-length gp130 into Ba/F3 cells, we found that cells expressing gp130 and an extensively deleted mutant LIFRalpha containing only the box-1 region were capable of proliferating in response to LIF, although LIF-dependent long term growth of these cells was seriously impaired. Using a similar strategy to generate WEHI-3BD+ cells expressing gp130 and wild-type or truncation mutants of LIFRalpha, studies revealed that the box-1 region of the LIFRalpha was also sufficient for LIF-dependent induction of different aspects of differentiation, including up-regulation of macrophage surface marker expression, morphological change, and cell migration in agar culture. However, the C-terminal region of the LIFRalpha, although not essential for intracellular signaling, was important for efficient receptor-mediated ligand internalization. In summary, the membrane-proximal box-1 region plays a dominant role in LIF-induced signal transduction of both proliferation and differentiation.

Cell Differentiation↗

Liver degeneration and lymphoid deficiencies in mice lacking suppressor of cytokine signaling-1.

SOCS-1, a member of the suppressor of cytokine signaling (SOCS) family, was identified in a genetic screen for inhibitors of interleukin 6 signal transduction. SOCS-1 transcription is induced by cytokines, and the protein binds and inhibits Janus kinases and reduces cytokine-stimulated tyrosine phosphorylation of signal transducers and activators of transcription 3 and the gp130 component of the interleukin 6 receptor. Thus, SOCS-1 forms part of a feedback loop that modulates signal transduction from cytokine receptors. To examine the role of SOCS-1 in vivo, we have used gene targeting to generate mice lacking this protein. SOCS-1(-/-) mice exhibited stunted growth and died before weaning with fatty degeneration of the liver and monocytic infiltration of several organs. In addition, the thymus of SOCS-1(-/-) mice was reduced markedly in size, and there was a progressive loss of maturing B lymphocytes in the bone marrow, spleen, and peripheral blood. Thus, SOCS-1 is required for in vivo regulation of multiple cell types and is indispensable for normal postnatal growth and survival.

Abnormalities, Multiple↗

Apoptosis regulator bcl-w is essential for spermatogenesis but appears otherwise redundant.

Proteins of the Bcl-2 family are important regulators of apoptosis in many tissues of the embryo and adult. The recently isolated bcl-w gene encodes a pro-survival member of the Bcl-2 family, which is widely expressed. To explore its physiological role, we have inactivated the bcl-w gene in the mouse by homologous recombination. Mice that lack Bcl-w were viable, healthy, and normal in appearance. Most tissues exhibited typical histology, and hematopoiesis was unaffected, presumably due to redundant function with other pro-survival family members. Although female reproductive function was normal, the males were infertile. The testes developed normally, and the initial, prepubertal wave of spermatogenesis was largely unaffected. The seminiferous tubules of adult males, however, were disorganized, contained numerous apoptotic cells, and produced no mature sperm. Both Sertoli cells and germ cells of all types were reduced in number, the most mature germ cells being the most severely depleted. The bcl-w-/- mouse provides a unique model of failed spermatogenesis in the adult that may be relevant to some cases of human male sterility.

Animals↗

Characterization of hematopoietic progenitor cells that express the transcription factor SCL, using a lacZ "knock-in" strategy.

Gene targeting experiments have demonstrated that the transcription factor SCL is essential for primitive and definitive hematopoiesis in the mouse. To study the functional properties of hematopoietic cells expressing SCL, we have generated mutant mice (SCLlacZ/w) in which the Escherichia coli lacZ reporter gene has been "knocked in" to the SCL locus, thereby linking beta-galactosidase expression to transcription from the SCL promoter. Bone marrow cells from heterozygous SCLlacZ/w mice were sorted into fractions expressing high, intermediate and low levels of beta-galactosidase (designated lacZhigh, lacZint, and lacZneg). Cells that were lacZhigh or lacZint were enriched for day 12 spleen colony-forming units and myeloid and erythroid colony-forming cells (CFCs). These fractions included >99% of the erythroid and >90% of the myeloid CFCs. Culture of sorted bone marrow populations on stromal cells secreting interleukin-7 or in fetal thymic organ cultures showed that B and T lymphoid progenitors were also present in the lacZhigh and lacZint fractions. These data provide a functional correlation between SCL expression and colony-forming ability in immature hematopoietic cells. Our data also suggested that expression of SCL was transient and confined to hematopoietic stem and/or progenitor cells, because the differentiated progeny of most lineages (except the erythroid) were beta-galactosidase-negative.

Animals↗

Lineage commitment in the progeny of murine hematopoietic preprogenitor cells: influence of thrombopoietin and interleukin 5.

Normal mouse marrow cells were stimulated by stem cell factor (SCF) to form dispersed or multicentric blast colonies containing progenitor cells committed to various hematopoietic lineages. Combination of the eosinophil-specific regulator interleukin 5 with SCF increased the frequency of colonies containing eosinophil-committed progenitor cells with multicentric but not dispersed blast colonies. Combination of thrombopoietin with SCF increased the frequency of colonies containing megakaryocyte-committed progenitor cells with both types of blast colony. Neither interleukin 5 nor thrombopoietin significantly altered the number or total cell content of blast colonies or progenitor cell numbers in blast colonies from those stimulated by SCF alone. No correlation was observed between total progenitor cell content and the presence or absence of either eosinophil or megakaryocyte progenitors in either type of blast colony. The data argue against a random process as being responsible for the formation of particular committed progenitor cells or the possibility that lineage-specific regulators merely enhance survival of such committed progenitor cells formed in developing blast colonies.

Animals↗

Cytokine production and function in c-mpl-deficient mice: no physiologic role for interleukin-3 in residual megakaryocyte and platelet production.

Mice lacking thrombopoietin (TPO), or its receptor c-Mpl, display defective megakaryocyte and platelet development and deficiencies in progenitor cells of multiple hematopoietic lineages. The contribution of alternative cytokines to thrombopoiesis in the absence of TPO signalling was examined in mpl-/- mice. Analysis of serum and organ-conditioned media showed no evidence of a compensatory overproduction of megakaryocytopoietic cytokines. However, consistent with a potential role in vivo, when injected into mpl-/- mice, interleukin-6 (IL-6) and leukemia inhibitory factor (LIF) retained the capacity to elevate megakaryocytes and their progenitors in hematopoietic tissues and increase circulating platelet numbers. However, double mutant mice bred to carry genetic defects both in c-Mpl and IL-3 or the alpha chain of the IL-3 receptor, displayed no greater deficiencies in megakaryocytes or platelets than mpl-deficient animals, suggesting absence of a physiologic role for IL-3 in the residual megakaryocytopoiesis and platelet production in these mice.

Animals↗

Hematopoietic stem cell deficiencies in mice lacking c-Mpl, the receptor for thrombopoietin.

Thrombopoietin (TPO) acts as a lineage-specific late-acting factor to stimulate megakaryocyte and platelet formation. However, analysis of mice lacking either the cytokine or its receptor, c-Mpl, also revealed deficiencies in progenitor cells of multiple hematopoietic lineages, suggesting that TPO signaling may play an important role in the regulation of the hematopoietic stem cell compartment. To investigate this hypothesis, we determined preprogenitor and colony forming unit-spleen (CFU-S) numbers and analyzed the long-term hematopoietic repopulating capacity of bone marrow cells from mpl-/- mice. mpl-/- mice had 4- to 12-fold fewer preprogenitor cells than wild-type mice. In irradiated normal recipients, mpl-/- bone marrow generated 8- to 10-fold fewer spleen colonies than wild-type marrow at both 8 and 12 days after transplantation. This defect was intrinsic to the transplanted hematopoietic cells, as the microenvironment of mpl-/- recipients supported similar CFU-S growth to that observed in wild-type recipients. In definitive assays of stem cell function, bone marrow cells from mpl-/- mice failed to compete effectively with normal cells for long-term reconstitution of the hematopoietic organs of irradiated recipients, even when transplanted in 10-fold excess. Serial transplantation studies further suggested that stem cell self-renewal also may be compromised in mpl-/- mice. These data imply that TPO, signaling through c-Mpl, plays a vital physiological role in the regulation of hematopoietic stem cell production and function.

Animals↗

BCR-ABL activates pathways mediating cytokine independence and protection against apoptosis in murine hematopoietic cells in a dose-dependent manner.

The hallmark of chronic myeloid leukemia (CML) is the chimeric tyrosine kinase oncogene bcr-abl. Since expression of bcr-abl mRNA frequently increases with disease progression and a duplication of the Philadelphia chromosome (harbouring the bcr-abl hybrid locus) represents the most frequent karyotypic abnormality in acute phase CML, we hypothesized that the level of BCR-ABL protein may affect the disease phenotype. Therefore, the biological effects of high and low levels of BCR-ABL expression were compared in growth factor-dependent and -independent myeloid and lymphoid cell lines. Our results demonstrated that low levels of BCR - ABL were sufficient to render these cell lines growth factor independent and tumorigenic, but higher levels were mandatory for additional protection against apoptotic stimuli. The provision of growth factor or an activated ras oncogene did not afford the same degree of protection as high levels of BCR-ABL and there were qualitative differences between the survival signals mediated by BCR-ABL and Bcl-2. These results have enabled us to establish a dose-dependent hierarchy of BCR-ABL induced biological effects, thus distinguishing the activation of pathways mediating protection from cytokine withdrawal from those protecting against other apoptotic stimuli.

Animals↗

Twenty proteins containing a C-terminal SOCS box form five structural classes.

The four members of the recently identified suppressor of cytokines signaling family (SOCS-1, SOCS-2, SOCS-3, and CIS, where CIS is cytokine-inducible SH2-containing protein) appear, by various means, to negatively regulate cytokine signal transduction. Structurally, the SOCS proteins are composed of an N-terminal region of variable length and amino acid composition, a central SH2 domain, and a previously unrecognized C-terminal motif that we have called the SOCS box. By using the SOCS box amino acid sequence consensus, we have searched DNA databases and have identified a further 16 proteins that contain this motif. These proteins fall into five classes based on the protein motifs found N-terminal of the SOCS box. In addition to four new SOCS proteins (SOCS-4 to SOCS-7) containing an SH2 domain and a SOCS box, we describe three new families of proteins that contain either WD-40 repeats (WSB-1 and -2), SPRY domains (SSB-1 to -3) or ankyrin repeats (ASB-1 to -3) N-terminal of the SOCS box. In addition, we show that a class of small GTPases also contains a SOCS box. The expression of representative members of each class of proteins differs markedly, as does the regulation of expression by cytokines. The function of the WSB, SSB, and ASB protein families remains to be determined.

Amino Acid Sequence↗

The molecular control of hematopoiesis: progress and problems with gene manipulation.

The in vitro-based discovery and characterization of hematopoietic regulators were of great value in identifying many of the agents active in controlling hematopoiesis. Subsequent in vivo studies have validated most of the information obtained from the in vitro studies, although the in vitro studies proved to be somewhat misleading in predicting which agents would exhibit the greatest quantitative effects in vivo. Establishing more clearly the actual situation in vivo has required a return to more complex, and often less satisfactory, studies on genetically manipulated whole animals. Of the two possible general approaches, gene inactivation models have proved more informative than transgenic, overexpression models. Each model has raised multiple questions in need of further resolution and the deletion studies have also indicated that other regulators must exist for various lineages, but have yet to be discovered. Of particular interest is the finding from gene inactivation studies that both G-CSF and thrombopoietin are necessary for the maintenance of normal numbers of progenitor cells in multiple lineages, suggesting that each of these lineage-dominant regulators may have broader actions when operating on cells in the stem cell and progenitor cell compartments.

Animals↗

The molecular control of hematopoiesis: progress and problems with gene manipulation.

The in vitro-based discovery and characterization of hematopoietic regulators were of great value in identifying many of the agents active in controlling hematopoiesis. Subsequent in vivo studies have validated most of the information obtained from the in vitro studies, although the in vitro studies proved to be somewhat misleading in predicting which agents would exhibit the greatest quantitative effects in vivo. Establishing more clearly the actual situation in vivo has required a return to more complex, and often less satisfactory, studies on genetically-manipulated whole animals. Of the two possible general approaches, gene inactivation models have proved more informative than transgenic, overexpression models. Each model has raised multiple questions in need of further resolution and the deletion studies have also indicated that other regulators must exist for various lineages, but have yet to be discovered. Of particular interest is the finding from gene inactivation studies that both G-CSF and thrombopoietin are necessary for the maintenance of normal numbers of progenitor cells in multiple lineages, suggesting that each of these lineage-dominant regulators may have broader actions when operating on cells in the stem cell and progenitor cell compartments.

Animals↗

Regulatory mechanisms controlling hematopoiesis: principles and problems.

Hematopoiesis is regulated by the combined action of specialized stromal cells and a consortium of hematopoietic regulatory factors. The multiplicity of these regulatory controls does result in overlapping regulator action, but multiple regulators are required to stimulate stem cell proliferation and are more efficient than single regulators when stimulation of progenitor cells is required. Gene inactivation studies have indicated that despite overlapping actions each hematopoietic regulator does have unique functions. Delayed elevations of stem and progenitor cells in the blood are a feature of enhanced hematopoiesis induced by the injection of regulators. These cells are not a random sample of marrow cells in such situations and may well be selected to rapidly amplify hematopoiesis by seeding previously inactive hematopoietic regions.

Animals↗

Pre-progenitor cells: a proposed new category of hematopoietic precursor cells.

Hematopoietic precursor cells capable of forming blast colonies in vitro appear to be a distinct transit population between stem cells and lineage-committed progenitor cells because the cells in such blast colonies are mainly lineage-committed progenitor cells. It is proposed that blast colony-forming cells be termed pre-progenitor cells (PPC) to encourage efforts to establish their biology and potential for initiating some types of leukemic populations more clearly.

Animals↗

The biological consequences of excess GM-CSF levels in transgenic mice also lacking high-affinity receptors for GM-CSF.

GM-CSF transgenic mice were crossed with mice with homozygous inactivation of the gene encoding the common beta chain (beta c) of the GM-CSF receptor to produce mice with constitutively elevated GM-CSF levels but no high-affinity GM-CSF receptors. GM-CSF transgenic beta c -/- mice had exceptionally elevated serum GM-CSF levels but failed to develop the abnormal peritoneal cell population, eye destruction or tissue lesions characteristic of GM-CSF transgenic beta c +/+ mice. The alveolar proteinosis of beta c -/- mice was not altered in GM-CSF transgenic beta c -/- mice. Levels of GM-CSF mRNA in transgenic GM-CSF beta c -/- were elevated but lower than in transgenic beta +/+ mice and the higher serum GM-CSF levels were traced in part to the longer serum half-life of GM-CSF in beta c -/- than in beta c +/+ mice although urinary loss of GM-CSF was higher in beta c -/- than in +/+ mice. The data indicate that the transgenic phenotype was due to stimulation by GM-CSF and not an insertional effect, that low-affinity receptors are not capable of initiating tissue pathology even in the presence of excess GM-CSF levels and that autocrine production of GM-CSF by GM-CSF-responsive cells also fails to induce changes in these cells. The results support current dogma that the action of polypeptide regulators is mediated exclusively by activation of high-affinity membrane receptors.

Animals↗

Cell-cell signalling in the regulation of blood cell formation and function.

Blood cell formation is continuous in adult life and requires complex regulatory control to achieve the necessary cell proliferation, differentiation commitment and maturation. Much of this control is achieved by specific glycoprotein haemopoietic regulators, more than 20 of which have now been identified and produced in recombinant form. For the granulocyte-macrophage lineage, major regulators are the colony stimulating factors (CSF) that are produced by many cell types dispersed throughout the body. The production of CSF is rapidly increased in response to induction signals, most usually of microbial origin, representing a demand-generated signalling system designed to activate and generate cells to rapidly eliminate the micro-organisms. The CSF not only control cell division but certain aspects of differentiation commitment, maturation initiation and functional activation. These multiple responses are initiated by signals arising from different regions of the cytoplasmic domains of the CSF membrane receptors. Less well understood are the signalling systems controlling the special gene activation events required to initiate the formation of the first haemopoietic cells and the specialized microenvironmental cells needed to support haemopoietic cells as they respond to haemopoietic regulators.

Animals↗

Murine hematopoietic stem cells committed to macrophage/dendritic cell formation: stimulation by Flk2-ligand with enhancement by regulators using the gp130 receptor chain.

The stimulation by Flk2-ligand (FL) of blast colony formation by murine bone marrow cells was selectively potentiated by the addition of regulators sharing in common the gp130 signaling receptor-leukemia inhibitory factor (LIF), oncostatin M, interleukin 11, or interleukin 6. Recloning of blast colony cells indicated that the majority were progenitor cells committed exclusively to macrophage formation and responding selectively to proliferative stimulation by macrophage colony-stimulating factor. Reculture of blast colony cells initiated by FL plus LIF in cultures containing granulocyte/macrophage colony-stimulating factor plus tumor necrosis factor alpha indicated that at least some of the cells were capable of maturation to dendritic cells. The cells forming blast colonies in response to FL plus LIF were unrelated to those forming blast colonies in response to stimulation by stem cell factor and appear to be a distinct subset of mature hematopoietic stem cells.

Animals↗