Search PubMed⌕ Search

Biomedical subjects

D Metcalf

Publications and source records attributed to D Metcalf.

At least 73 records · Page 4Linked to original sources
PubMed1997

The unusual species cross-reactivity of the leukemia inhibitory factor receptor alpha-chain is determined primarily by the immunoglobulin-like domain.

Human leukemia inhibitory factor (hLIF) binds to both human and mouse LIF receptors (LIFRs), while mouse LIF (mLIF) binds only to mouse LIFRs. Furthermore, hLIF binds with much higher affinity to the mouse LIFR (mLIFR) alpha-chain than does mLIF itself. To define the structural elements of the mLIFR alpha-chain conferring high affinity binding of hLIF and the species-specific interaction with mLIF, we first constructed C-terminally truncated extracellular domains of both the mLIFR and the human LIFR (hLIFR) alpha-chains, which contained only the two hemopoietin domains separated by an immunoglobulin-like domain. These recombinant truncated LIFR alpha-chains had identical binding and biological characteristics to either their naturally occurring or transfected counterparts. On the basis of this, we have generated eight interspecies receptor chimeras by combining different regions of the mouse and human LIFR sequence. Surprisingly, the immunoglobulin-like domain of the mLIFR alpha-chain played the predominant role in receptor-ligand interactions. Moreover, both high affinity binding for hLIF and the species-specific binding for mLIF mapped to the same domain of mLIFR molecule. These findings should enable the development of a "humanized" mouse LIFR that could act as a potent antagonist of hLIF biological activities in vivo.

Animals↗
PubMed1997

Distinct roles for leukemia inhibitory factor receptor alpha-chain and gp130 in cell type-specific signal transduction.

Leukemia inhibitory factor (LIF) induces a variety of disparate biological responses in different cell types. These responses are thought to be mediated through the functional LIF receptor (LIFR), consisting of a heterodimeric complex of LIFR alpha-chain (LIFRalpha) and gp130. The present study investigated the relative capacity of the cytoplasmic domains of each receptor subunit to signal particular responses in several cell types. To monitor the signaling potential of LIFRalpha and gp130 individually, we constructed chimeric receptors by linking the extracellular domain of granulocyte colony-stimulating factor receptor (GCSFR) to the transmembrane and cytoplasmic regions of either LIFRalpha or gp130. Both chimeric receptors and the full-length GCSFR in expressed in M1 myeloid leukemic cells to measure differentiation induction, in embryonic stem cells to measure differentiation inhibition, and in Ba/F3 cells to measure cell proliferation. Our results demonstrated that whereas GCSFR-gp130 receptor homodimer mediated a GCSF-induced signal in all three cell types, the GCSFR-LIFRalpha receptor homodimer was only functional in embryonic stem cells. These findings suggest that the signaling potential of gp130 and LIFRalpha cytoplasmic domains may differ depending upon the tissue and cellular response initiated.

Antigens, CD↗
PubMed1997

A family of cytokine-inducible inhibitors of signalling.

Cytokines are secreted proteins that regulate important cellular responses such as proliferation and differentiation. Key events in cytokine signal transduction are well defined: cytokines induce receptor aggregation, leading to activation of members of the JAK family of cytoplasmic tyrosine kinases. In turn, members of the STAT family of transcription factors are phosphorylated, dimerize and increase the transcription of genes with STAT recognition sites in their promoters. Less is known of how cytokine signal transduction is switched off. We have cloned a complementary DNA encoding a protein SOCS-1, containing an SH2-domain, by its ability to inhibit the macrophage differentiation of M1 cells in response to interleukin-6. Expression of SOCS-1 inhibited both interleukin-6-induced receptor phosphorylation and STAT activation. We have also cloned two relatives of SOCS-1, named SOCS-2 and SOCS-3, which together with the previously described CIS form a new family of proteins. Transcription of all four SOCS genes is increased rapidly in response to interleukin-6, in vitro and in vivo, suggesting they may act in a classic negative feedback loop to regulate cytokine signal transduction.

Amino Acid Sequence↗
PubMed1997

Genetic influences determining progenitor cell mobilization and leukocytosis induced by granulocyte colony-stimulating factor.

The mechanisms involved in the mobilization of progenitor cells into the blood by granulocyte colony-stimulating factor (G-CSF) and other cytokines are poorly understood. To identify important influences on this complex process, in vivo murine models were used. Granulocyte-macrophage colony-stimulating factor (GM-CSF) transgenic, Max41 transgenic, W/Wv, Mpl-null, GM-CSF receptor (beta chain)-null mice, wild-type littermate controls, and six inbred strains of mice were injected with 200 microg/kg/d G-CSF for 5 days. Three parameters of response were monitored: white blood cell count (WCC), peripheral blood progenitor cell (PBPC) numbers, and spleen weight. In all genotypes studied, G-CSF induced increases in these three parameters. However, PBPC mobilization in W/Wv and Mpl-null mice was only 30% and 9%, respectively, of that observed in wild-type mice. In contrast, perturbations of GM-CSF signalling had no demonstrable effect on in vivo responses to G-CSF. Broad variability was evident between inbred strains for each parameter of the response to G-CSF. A 10-fold range in response was observed for circulating progenitor cell numbers, similar to that observed for normal human subjects receiving G-CSF. The interstrain differences were in the distribution of mature and progenitor cells between peripheral blood, bone marrow, and spleen rather than in the total numbers of these cells in the body. Results of an F2 intercross of low-responding C57BL/6 and intermediate-responding SJL mice indicated that regulation of progenitor cell mobilization is a complex genetic trait, that there is a correlation between this trait and WCC response (r2 = .5), and that this approach may serve as a useful model for the identification of genes involved in the mobilization process.

Animals↗
PubMed1997

Identification, purification, and characterization of a soluble interleukin (IL)-13-binding protein. Evidence that it is distinct from the cloned Il-13 receptor and Il-4 receptor alpha-chains.

Interleukin-4 (IL-4) and interleukin-13 (IL-13) are structurally and functionally related cytokines which play an important role in the regulation of the immune response to infection. The functional similarity of IL-4 and IL-13 can be explained, at least in part, by the common components that form their cell surface receptors, namely the IL-4 receptor alpha-chain (IL-4Ralpha) and the IL-13 receptor alpha-chain (IL-13Ralpha). Soluble forms of the IL-4Ralpha have also been described and implicated in modulating the effect of IL-4. In this paper we describe the presence of a 45,000-50,000 Mr IL-13-binding protein (IL-13BP) in the serum and urine of mice. This protein binds IL-13 with a 100-300-fold higher affinity (KD = 20-90 pM) than does the cloned IL-13Ralpha (KD = 3-10 nM). In addition to this functional difference, the IL-13BP appears to be structurally and antigenically distinct from the IL-13Ralpha. Finally, unlike the cloned receptor, the IL-13BP acts as a potent inhibitor of IL-13 binding to its cell surface receptor, raising the possibility that it may be used to modulate the effects of IL-13 in vivo.

Animals↗
PubMed1997

Cytoplasmic domains of the common beta-chain of the GM-CSF/IL-3/IL-5 receptors that are required for inducing differentiation or clonal suppression in myeloid leukaemic cell lines.

Granulocyte-macrophage colony stimulating factor (GM-CSF) is a cytokine that controls the production and function of myeloid cells by interaction with a cell surface receptor composed of a specific ligand-binding alpha-chain (hGMRalpha) and a shared signal-transducing beta-chain (beta c). Co-expression of human GMR alpha-chain and wild-type human beta c in two murine leukaemic cell lines (M1 and WEHI-3B D+) conferred the ability to terminally differentiate into macrophages when stimulated with human GM-CSF. Analysis of cytoplasmic truncation mutants of beta c showed that residues to amino acid 783 (numbering from the first amino acid of the leader sequence) were sufficient for the GM-CSF-dependent induction of all aspects of differentiation in both cell types. However, shorter truncations selectively lost, in a cell-specific manner, first the capacity to induce macrophage migration in agar and then cell surface differentiation antigens and clonal suppression of proliferative potential. The data suggest that different aspects of the differentiated phenotype can be dissociated with the required signalling pathways originating from distinct regions of the receptor cytoplasmic domain and cooperating to produce a fully differentiated macrophage. The cooperativity of these pathways and limiting cell signalling intermediate pool sizes could explain the observed cell line differences and may have implications for normal haemopoiesis.

Animals↗
PubMed1997

Cytokine receptor expression on hematopoietic stem and progenitor cells.

Hematopoietic stem and progenitor cell populations were obtained by fluorescence activated cell sorting of murine bone marrow (BM) cells into Rhodamine-123io lineage-Ly6A/ E+ c-kit+ (primitive stem cells highly enriched for long-term BM repopulating activity), Rhodamine-123med/hl lineage- Ly6A/E+ c-kit+ (mature stem cells highly enriched for shortterm BM repopulating activity and day 13 spleen colony-forming activity) and lineage- Ly6A/E- c-kit+ (enriched for in vitro colony forming cells) populations. Neither stem cell population responds to single cytokines in vitro and each requires the synergistic action of two or more cytokines for proliferation, whereas the progenitor cell population proliferates in response to single cytokines. Since each of these cell populations was sorted as c-kit+, they express receptors for stem cell factor. Cell populations were also analyzed by autoradiography for their ability to specifically bind iodinated cytokines and this revealed that both stem cell populations expressed receptors for interleukin-1 alpha (IL-1 alpha), IL-3, IL-6, and granulocyte colony-stimulating factor (G-CSF), but lacked receptors for macrophage colony-stimulating factor (M-CSF), granulocyte-macrophage colony stimulating factor (GM-CSF), and leukemia inhibitory factor (LIF). Cells within the progenitor cell population specifically bound IL-3, GM-CSF, G-CSF, IL-6, and IL-1 alpha, whereas no receptors were detected for M-CSF and LIF. Within each cell population examined, heterogeneity was observed in the percentage of cells labeled and the number of receptors per cell. These results suggest that stem cell populations can be further subdivided according to their cytokine receptor profile and it will be of interest to determine if such subpopulations have distinctive functional properties.

Animals↗
PubMed1997

Differentiation commitment in normal hemopoiesis and leukemic transformation.

Differentiation commitment events are essential for the initiation of hemopoiesis and, in one form or another, occur continuously during adult hemopoiesis. The most studied type of differentiation commitment decision a hemopoietic cell can make involves the alternative choice of self-renewal versus the formation of progeny destined for maturation. Aberration in this commitment choice is a key abnormality necessary for the formation of a leukemic population.

Animals↗
PubMed1997

The structural basis of the biological actions of the GM-CSF receptor.

The receptor for granulocyte/macrophage colony-stimulating factor (GM-CSF) consists of a ligand-specific low-affinity binding chain (GM-CSFR alpha) and a second chain that is required for high-affinity binding and signal transduction. This second chain is shared by the ligand-specific alpha-chains for the interleukin 3 (IL-3) and IL-5 receptors and is therefore called beta common (beta c). In mice but not humans the IL-3 receptor can also use a closely related but IL-3-specific beta-chain (beta IL-3). In order to define the contributions of each chain to receptor signalling we generated mice in which either beta c or beta IL-3 expression was deleted. beta IL-3 null mice were phenotypically normal but displayed a decreased responsiveness to IL-3 in vitro. beta c null mice, on the other hand, were unresponsive to GM-CSF or IL-5 but still responded to IL-3. These data demonstrated that GM-CSF and IL-5 receptors can use only one beta-chain for signalling (beta c) while IL-3 can effectively use either beta-chain. The hierarchical basis of receptor transmodulation was shown to result from this differential usage of beta-chains. To define the regions required for different types of cell signalling, we constructed human beta c mutants with successive cytoplasmic truncation. By the use of appropriate biological read-out systems we found that the cytoplasmic region of the receptor has a modular design with distinct domains required for cell proliferation, cell survival, differentiation and growth suppression. Appropriate targeting of these domains and the signalling pathways they initiate may provide highly specific cell therapies in the future.

Animals↗
PubMed1997

The molecular control of granulocytes and macrophages.

The proliferation in vitro of granulocytes and macrophages can be regulated by the four colony stimulating factors (CSFs), stem cell factor and interleukin 6, with Flk ligand having a weaker action. Combinations of these glycoprotein regulators produce superadditive proliferative responses. The CSFs also influence commitment, maturation and mature cell functional activity and these various responses are initiated by distinct regions of the individual receptors. The injection of single CSFs into experimental animals or patients reproducibly enhances granulocyte or monocyte formation or function despite the existence of complex interacting networks of regulatory molecules. Verification of the importance of the CSFs for the regulation of basal haemopoiesis has been obtained by analysis of mice in which the genes encoding the CSFs or their receptors have been inactivated. In a casein-induced model of acute inflammatory responses, the migration of neutrophils from the marrow and localization of these cells to the inflammatory site appear not to be CSF-dependent processes even though major increases occur in CSF levels at the inflammatory site.

Animals↗
PubMed1997

The influence of granulocyte/macrophage colony-stimulating factor on dendritic cell levels in mouse lymphoid organs.

To ascertain whether the development of dendritic cells (DC) in mouse lymphoid organs is dependent on granulocyte/macrophage colony-stimulating factor (GM-CSF), we determined the number of DC in the thymus, spleen and lymph nodes of normal mice, of mice with the genes coding for GM-CSF or its receptor inactivated, and of transgenic mice with excessive levels of GM-CSE DC were extracted from the tissues and enriched prior to flow cytometric analysis. The total DC level and the incidence of DC expressing lymphoid-related markers (CD8(hi) CD11b(lo)) and myeloid-related markers (CD8(lo) CD11b(hi)) were monitored. Both in GM-CSF null mice, and GM-CSF receptor null mice, DC of all surface phenotypes were present in all lymphoid organs; only small decreases in DC levels were recorded, except for the lymph nodes of GM-CSF receptor null mice which showed a more pronounced (threefold) decrease in DC numbers. Since the GM-CSF receptor null mice lacked the beta chain common to the GM-CSF, interleukin (IL)-3 and IL-5 receptors, the development of DC in the absence of GM-CSF was not due to common beta chain mediated developmental signals elicited by IL-3 or IL-5. In GM-CSF transgenic mice, there was only a 50 % increase in DC numbers in thymus and spleen, paralleling an increase in overall cellularity, but a more pronounced (threefold) increase in DC numbers in lymph nodes. There was no evidence that GM-CSF had a selective effect on any particular DC subpopulation defined by CD8 or CD11b expression. We conclude that the development of most lymphoid tissue DC can proceed in the absence of GM-CSF, although this cytokine can produce some elevation of DC levels. It is not clear whether the enhancing effect of GM-CSF is direct or an indirect effect mediated by other cytokines.

Animals↗
PubMed1997

Suppression or overexpression of genes encoding myeloid growth factors or their receptors.

Gene inactivation data have documented that erythropoietin, G-CSF, M-CSF and thrombopoietin are major regulators in vivo respectively of red cell, neutrophil, macrophage and platelet production. Transgenic mice can provide valuable models for observing the consequences of excessive stimulation by a particular growth factor but are subject to variation based on the use of differing promoters for the inserted gene.

Animals↗
PubMed1997

Establishment of multipotential and antigen presenting cell lines derived from myeloid leukemias in GM-CSF transgenic mice.

In this study neonatal mice expressing a GM-CSF transgene (GMT mice), and their normal littermate controls, were infected with Moloney murine leukemia virus (MoMLV) to examine in vivo tumorigenesis. By 200 days, all of the GMT mice had died whereas median survival had not been reached in the littermates (P < 0.0003). Thymomas developed in 32% of GMT mice and were more frequently CD4+CD8+ (83%) compared to the CD4+CD8- phenotype seen in 90% of thymomas developing in MoMLV-infected littermate mice. A primitive myeloid leukemia was induced in 21% of GMT mice, but none of the littermates. To characterize further the nature of the leukemic cells, a factor-dependent cell line (DGM36) was derived. DGM36 cells were tumorigenic, capable of differentiation to neutrophils, macrophages and eosinophils, and contained a partial deletion of chromosome 2. A subline arose spontaneously that was factor-independent and produced GM-CSF in an autocrine manner (IGM36 cells). Stimulation of the IGM36 cells with TNF alpha and IFNgamma resulted in increased expression of B7-1, class I MHC and class II MHC and consequent presentation of antigen in allogeneic MLRs. IGM36 cells thereby satisfy many of the criteria of dendritic cells and consequently may be used to examine antigen presentation by leukemic cells. This is the first report of primary myeloid leukemias arising in GMT mice and documents the derivation of a multipotential, autocrine leukemic cell line with dendritic cell characteristics.

Animals↗
PubMed1997

The Charlotte Friend Memorial Lecture. The role of hematopoietic growth factors in the development and suppression of myeloid leukemias.

The transformation of normal hematopoietic cells to leukemic cells requires cells to acquire two intrinsic changes. These are the acquisition by some method of autocrine growth stimulation and a perturbation of differentiation commitment leading to abnormal levels of self-generation. Hematopoietic regulator action can be involved to produce or facilitate both these changes but the same regulators can also suppress some leukemic populations by enforced differentiation commitment.

Animals↗
PubMed1996

The Rel subunit of NF-kappaB-like transcription factors is a positive and negative regulator of macrophage gene expression: distinct roles for Rel in different macrophage populations.

The role of Rel in the monocyte/macrophage lineage was examined in mice with an inactivated c-rel gene. Although the frequency of monocytic cells was normal in Rel-/- mice, we show that Rel serves distinct roles in regulating gene expression and immune effector function in different mature macrophage populations. Stimulated Rel-/- resident peritoneal macrophages produced higher than normal levels of granulocyte-macrophage colony-stimulating factor (GM-CSF), granulocyte colony-stimulating factor (G-CSF) and interleukin-6 (IL-6), but tumour necrosis factor-alpha (TNF-alpha) production was not induced. Diminished cytotoxic activity exhibited by resident Rel-/- macrophages was consistent with reduced nitric oxide production resulting from impaired up-regulation of inducible nitric oxide synthase expression. While a similar altered pattern of IL-6 and TNF-alpha expression was observed in stimulated Rel-/- peritoneal effusion macrophages, cytotoxic activity, nitric oxide, GM-CSF and G-CSF production by these cells was normal. The alternate regulation of certain genes in the two macrophage populations coincided with different patterns of nuclear Rel/NF-kappaB complexes expressed in normal resident and elicited cells. Collectively, these results establish that Rel is a positive or negative regulator of transcription in macrophages and that Rel has distinct roles in different macrophage populations.

Animals↗
PubMed1996

Leptin can induce proliferation, differentiation, and functional activation of hemopoietic cells.

Many cytokines exert their biological effect through members of the hemopoietin receptor family. Using degenerate oligonucleotides to the common WSXWS motif, we have cloned from human hemopoietic cell cDNA libraries various forms of the receptor that was recently shown to bind the obesity hormone, leptin. mRNAs encoding long and short forms of the human leptin receptor were found to be coexpressed in a range of human and murine hemopoietic organs, and a subset of cells from these tissues bound leptin at the cell surface. Ectopic expression in murine Ba/F3 and M1 cell lines revealed that the long, but not the short, form of the leptin receptor can signal proliferation and differentiation, respectively. In cultures of murine or human marrow cells, human leptin exhibited no capacity to stimulate cell survival or proliferation, but it enhanced cytokine production and phagocytosis of Leishmania parasites by murine peritoneal macrophages. Our data provide evidence that, in addition to its role in fat regulation, leptin may also be able to regulate aspects of hemopoiesis and macrophage function.

Amino Acid Sequence↗
PubMed1996

Role of granulocyte-macrophage colony-stimulating factor and granulocyte colony-stimulating factor in the development of an acute neutrophil inflammatory response in mice.

The intraperitoneal injection into mice of casein preparations containing bacteria induced a rapid accumulation of neutrophils within 3 hours due to selective release of mature cells from the bone marrow. Significant increases in the concentrations of granulocyte-macrophage colony-stimulating factor (GM-CSF) and granulocyte colony-stimulating factor (G-CSF) occurred in the peritoneal cavity during the process, but the intraperitoneal injection of neither CSF induced a significant accumulation of neutrophils and the coinjection of G-CSF and casein failed to enhance the neutrophil response. The lack of involvement of either CSF in the neutrophil migration was confirmed by the development of typical neutrophil exudates when casein was injected into mice with inactivation of the genes encoding GM-CSF, G-CSF, or the beta-common chain of the GM-CSF receptor. However, preinjection of G-CSF increased the number of marrow neutrophils available for migration and did result in increased numbers of neutrophils in the peritoneal cavity after casein injection. Typical eosinophil inflammatory responses to the injection of casein or thioglycollate occurred in GM-CSF -/ -mice but not in beta c -/- mice, suggesting that interleukin-5 was necessary for this response.

Animals↗