Biomedical subjects

D Metcalf

Publications and source records attributed to D Metcalf.

At least 217 records · Page 12Linked to original sources

PubMed1988

Myeloid leukaemia inhibitory factor maintains the developmental potential of embryonic stem cells.

Embryonic stem (ES) cells, the totipotent outgrowths of blastocysts, can be cultured and manipulated in vitro and then returned to the embryonic environment where they develop normally and can contribute to all cell lineages. Maintenance of the stem-cell phenotype in vitro requires the presence of a feeder layer of fibroblasts or of a soluble factor, differentiation inhibitory activity (DIA) produced by a number of sources; in the absence of DIA the ES cells differentiate into a wide variety of cell types. We recently noted several similarities between partially purified DIA and a haemopoietic regulator, myeloid leukaemia inhibitory factor (LIF), a molecule which induces differentiation in M1 myeloid leukaemic cells and which we have recently purified, cloned and characterized. We demonstrate here that purified, recombinant LIF can substitute for DIA in the maintenance of totipotent ES cell lines that retain the potential to form chimaeric mice.

Animals↗

PubMed1988

Structural characterization of a murine myeloid leukaemia inhibitory factor.

A leukaemia inhibitory factor (LIF) which induces macrophage differentiation in M1 murine myeloid leukaemia cells and suppresses their proliferation in vitro has been isolated in sufficient quantities (30 micrograms) from Krebs ascites tumour cell conditioned medium to permit its partial characterization by amino acid sequence analysis. The combination of sensitive microbore column (1.0 and 2.1 mm internal diameter) HPLC technology and microsequence analysis has enabled the positive identification of 125 of the total 179 amino acid residues (70%) in the molecule. The amino acid sequence data reported here permitted the isolation of a partial cDNA clone encoding LIF [Gearing et al. (1987) EMBO J. 6, 3995-4002]. A candidate C-terminus of the LIF molecule predicted from the amino acid sequence was confirmed by subsequent isolation of a cDNA clone corresponding to the C-terminus of the protein. No strong similarity was revealed when the amino acid sequence of LIF was compared with other haemopoietic growth factors, in particular granulocyte-macrophage colony-stimulating factor, granulocyte colony-stimulating factor and tumour necrosis factor-alpha or interleukins. The protein sequence data reported here indicate three sites of post-translational modification (N-linked glycosylation).

Amino Acid Sequence↗

PubMed1988

Resolution and purification of three distinct factors produced by Krebs ascites cells which have differentiation-inducing activity on murine myeloid leukemic cell lines.

The use of different myeloid leukemic cell lines (WEHI-3B D+ and M1) and different sources of factors has led to discrepancies concerning the identity of factors capable of inducing differentiation in leukemic cells. We have biochemically fractionated medium conditioned by one such source (Krebs II ascites cells) and assayed fractions for their bone marrow colony-stimulating activity as well as their differentiation-inducing activity for WEHI-3B D+ and M1 cells. This resulted in the resolution of four distinct molecular species with differentiation-inducing activity. One activity was purified to homogeneity and shown by a variety of biochemical, biological, and receptor-binding criteria to be authentic granulocyte colony-stimulating factor (G-CSF). A second activity was identified as granulocyte-macrophage colony-stimulating factor (GM-CSF). Two other activities termed LIF-A and LIF-B (leukemia inhibitory factor) were shown to probably be different glycosylation variants of the same protein and one of these (LIF-A) was purified 12,000-fold to homogeneity. G-CSF induced differentiation in both WEHI-3B D+ and at higher concentrations M1 cells while GM-CSF weakly induced differentiation in WEHI-3B D+ cells. LIF-A had no colony-stimulating activity and induced differentiation in and inhibited the proliferation of only M1 cells. Each factor bound to a unique cell surface receptor with no evidence of direct cross-reactivity.

Animals↗

PubMed1988

Haemopoietic growth factors.

Erythropoietin, GM-colony-stimulating factor and G-colony-stimulating factor are the first recombinant haemopoietic growth factors to reach clinical use. There are a number of additional haemopoietic regulators that have now been cloned and are being mass-produced with a view to clinical use. The next decade should witness exciting advances in the clinical treatment of haematological diseases and infections that will be comparable with those that were seen last with the introduction of effective treatments for pernicious anaemia.

Animals↗

PubMed1988

Effect of granulocyte colony stimulating factor on neutropenia induced by cytotoxic chemotherapy.

A phase I/II study of granulocyte colony stimulating factor (G-CSF) was undertaken in patients with advanced malignancy receiving melphalan to determine the granulocyte response, side-effects, and pharmacokinetics. Patients received doses of 1-60 micrograms/kg intravenously. There were 3 patients at each dose level. Before chemotherapy the immediate effect of G-CSF was a transient depression in circulating neutrophils followed by a dose-dependent rise. Neutrophil counts up to 80 X 10(9)/l were achieved. G-CSF administration following melphalan reduced the period of neutropenia caused by melphalan. G-CSF was well tolerated and the only clinical observation that appeared related to G-CSF administration was slight bone pain during some infusions. G-CSF was rapidly cleared from the blood with a mean half-life of 110 min for the second phase. Reductions in the number of days of neutropenia following cytotoxic chemotherapy may reduce the morbidity and mortality of chemotherapy.

Aged↗

PubMed1988

Purification of a murine leukemia inhibitory factor from Krebs ascites cells.

A factor capable of inducing terminal differentiation in the murine myeloid leukemia cell line M1 has been purified to apparent homogeneity from the medium conditioned by Krebs II ascites tumor cells. The factor, termed leukemia inhibitory factor (LIF) is a single chain glycoprotein of apparent Mr 58,000 which induces differentiation and inhibits proliferation of the M1 cell line but not the WEHI-3B D+ murine myeloid leukemic cell line and has no detectable proliferative activity on normal myeloid progenitor cells. It was purified using four successive high-efficiency purification steps--anion-exchange chromatography on DEAE-Sepharose; cation-exchange chromatography on CM-Sepharose; affinity chromatography on lentil lectin-Sepharose; and reverse-phase high-performance liquid chromatography on a phenyl-silica matrix--to a specific biological activity of approximately 1.25 X 10(8) units/mg with an overall purification of 12,000-fold and a yield of 73% for the activity failing to bind to DEAE-Sepharose. Sufficient quantities of the factor (12 micrograms, 200 pmol) have been purified to allow structural and functional analysis of the molecule and comparison with other know differentiation inducers.

Animals↗

PubMed1988

Specific binding of murine leukemia inhibitory factor to normal and leukemic monocytic cells.

Leukemia inhibitory factor (LIF), a glycoprotein capable of suppressing the clonogenicity and inducing the differentiation of the murine myeloid leukemia cell line M1, was radioiodinated to a high specific radioactivity with retention of full biological activity. Binding of 125I-labeled LIF to M1 cells reached a steady state at 37 degrees C after approximately equal to 40 min and was in competition with unlabeled LIF but not granulocyte colony-stimulating factor or a range of other cytokines or differentiation-inducing agents. Specific binding was demonstrable to cells from a range of murine hemopoietic tissues including the bone marrow, the spleen, and the peritoneal cavity. Autoradiography revealed macrophages, monocytes, and their precursors to be the major cell types responsible for 125I-labeled LIF binding within these tissues. Receptors on M1 cells were of high affinity (apparent Kd, 100-200 pM) and few in number (300-500 per cell).

Animals↗

PubMed1988

Divergent disease patterns in granulocyte-macrophage colony-stimulating factor transgenic mice associated with different transgene insertion sites.

A comparison was made of disease development in two lines of transgenic mice in which the granulocyte-macrophage colony-stimulating factor (GM-CSF) transgene was inserted in different chromosomal locations. Female-line mice (X chromosome insertion) had equivalent elevations of serum GM-CSF levels to those in male-line mice (autosomal insertion) but a shorter survival (median survival, 95 versus 145 days) and a significantly higher incidence of large inflammatory foci in skeletal muscle and gut congestion. Male-line transgenic mice had higher levels of cells in the peritoneal cavity and a higher frequency of spleen enlargement with excess erythropoiesis than female-line mice and uniquely developed fibrotic nodules in the abdominal and pleural cavities. The various diseases in GM-CSF transgenic mice are likely to have been induced by GM-CSF-stimulated products of macrophages, and in the two transgenic lines the macrophages exhibit characteristic differences in morphology and possibly functional activity.

Animals↗

PubMed1988

Molecular cloning and expression of the human homologue of the murine gene encoding myeloid leukemia-inhibitory factor.

A human homologue of the recently cloned murine leukemia-inhibitory factor (LIF) gene was isolated from a genomic library by using the murine cDNA as a hybridization probe. The nucleotide sequence of the human gene indicated that human LIF has 78% amino acid sequence identity with murine LIF, with no insertions or deletions, and that the region of the human gene encoding the mature protein has one intervening sequence. After oligonucleotide-mediated mutagenesis, the mature protein-coding region of the LIF gene was introduced into the yeast expression vector YEpsec1. Yeast cells transformed with the resulting recombinant could be induced with galactose to produce high levels of a factor that induced the differentiation of murine M1 leukemic cells in a manner analogous to murine LIF. This factor competed with 125I-labeled native murine LIF for binding to specific cellular receptors on murine cells, compatible with a high degree of structural similarity between the murine and human factors.

Amino Acid Sequence↗

PubMed1988

Tissue localization and fate in mice of injected multipotential colony-stimulating factor.

The hemopoietic regulator multipotential colony-stimulating factor [Multi-CSF (interleukin 3)] has proliferative effects on a wide range of hemopoietic cells in vitro and in vivo. Native or recombinant Multi-CSF injected intravenously into adult mice had an initial half-life of 3-5 min and a second phase of 50 min. Clear labeling of hemopoietic cells was observed in the bone marrow and spleen of mice injected intravenously with recombinant 125I-labeled Multi-CSF showing that injected Multi-CSF can obtain access to such cells in situ. A high proportion of injected 125I-labeled Multi-CSF of both types became localized in the liver and in the kidney (in cells of the Bowman's capsule and proximal renal tubules). The kidney appeared to be an active site of degradation of Multi-CSF with the early appearance of low molecular weight labeled material in the urine.

Animals↗

PubMed1988

Production of colony-stimulating factors (CSFs) during infection: separate determinations of macrophage-, granulocyte-, granulocyte-macrophage-, and multi-CSFs.

After infection of mice with Listeria monocytogenes, elevated levels of colony-stimulating factors (CSFs) in the serum were quantitated by six different assays: ability to stimulate colony formation, the proliferation of 2 suspension of bone marrow cells (both measuring total colony-stimulating activity), a radioimmunoassay for macrophage-CSF (CSF-1), the WEHI-3B differentiation assay for granulocyte-CSF, and proliferation of 32D-c1-3 and FDC-P1 cell lines (specific for multi-CSF and either multi- or granulocyte-macrophage-CSFs, respectively). The great bulk of serum colony-stimulating activity represented macrophage- and granulocyte-CSFs, with small but measurable amounts of granulocyte-macrophage-CSF. The degree of elevation of serum CSF depended on the infecting dose used and the numbers of bacteria growing in the spleens and livers of the two mouse strains compared, i.e., L. monocytogenes-resistant C57BL/10 and susceptible BALB/cJ. The increase in serum CSFs occurred before the peak in bone marrow granulocyte-macrophage progenitors and before the reduction in bacterial numbers which follows the onset of specific cell-mediated immunity.

Animals↗

PubMed1988

Binding, internalization, and degradation of 125I-multipotential colony-stimulating factor (interleukin-3) by FDCP-1 cells.

The kinetic parameters involved in determining the steady-state interaction of Multi-CSF with FDCP-1 cells at 37 degrees C have been determined by kinetic analysis under steady-state conditions and by curve-fitting the rate of approach to steady-state conditions. The two methods are in substantial agreement and yield values of Vr = 28 receptors/cell/min for the rate of appearance of receptors at the cell surface, ke and kt = 0.061 min-1 and 0.0044 min-1 for the rate constants of internalization of occupied and unoccupied receptors, respectively, kh = 0.008 min-1 for the rate constant of degradation of internalized ligand, ka = 2.9 X 10(8) M-1 min-1 for the rate constant of association and kd = 0.11 min-1 for the rate constant of dissociation of ligand with receptor. Analysis of steady-state conditions indicated that Multi-CSF caused substantial down-regulation of surface receptors and that considerably more Multi-CSF was inside the cell than at the cell surface. The implications of these results for utilization rates of Multi-CSF by FDCP-1 cells and the relationship of receptor occupancy to biological activity are discussed.

Biological Transport↗

PubMed1988

Cellular processing of murine colony-stimulating factor (Multi-CSF, GM-CSF, G-CSF) receptors by normal hemopoietic cells and cell lines.

The binding, internalization and degradation rates of three different murine colony-stimulating factors (Multi-CSF or interleukin-3, GM-CSF and G-CSF) and their receptor turnover rates were determined for normal bone marrow cells and a number of different cell lines at 37 degrees C. The kinetic parameters were extracted from a curve-fitting analysis of the approach to steady-state of surface-bound and internalized CSFs by methods described by Myers et al. (1987). The primary binding kinetic constants (association and dissociation) for each CSF on different cell types were similar, suggesting a single type of receptor for each CSF. In all cases, CSF binding induced a faster rate of internalization of occupied receptors than unoccupied receptors and resulted in significant accumulation of CSF inside the cell under steady-state conditions. The steady-state constant, determining the relationship between CSF concentration and receptor occupancy, indicated that, in all cases, more receptors were occupied at a given CSF concentration under steady-state conditions than would be under equilibrium conditions. Nevertheless, the data predicted that maximal biological effects of the CSFs were exerted at concentrations that did not result in full receptor occupancy. Comparison of the kinetic constants derived for the same CSF interacting with different types of cells or different CSFs interacting with the same cell type indicated that CSF and receptor processing resulted from a dynamic interplay of receptor-determined and cell-determined events. This resulted in a flexibility of the kinetic parameters that matched the variety of biological responses elicited by CSFs in different cell types.

Animals↗

PubMed1988

Binding characteristics and proliferative action of purified granulocyte colony-stimulating factor (G-CSF) on normal and leukemic human promyelocytes.

The binding of purified 125I-labeled murine granulocyte colony stimulating factor (125I-G-CSF) to normal and leukemic human cells was examined. Normal neutrophils and their precursors demonstrated specific labeling with 125I-G-CSF, whereas eosinophils, lymphocytes, and erythroid cells did not. Normal human promyelocytes demonstrated the highest binding among hemopoietic cells. Human myeloid leukemic cells also demonstrated consistent specific labeling with 125I-G-CSF. Normal promyelocytes and chronic myeloid leukemia promyelocytes demonstrated only transient clonal proliferation in vitro when stimulated by G-CSF, but this was not always the case with acute promyelocytic leukemic cells. The qualitative responsiveness of normal and leukemic cells to G-CSF was very similar despite heterogeneity in receptor numbers on individual cells. A subset of acute promyelocytic leukemic cells appeared unresponsive to stimulation by GM-CSF.

Antibodies, Monoclonal↗

PubMed1988

Proliferation of normal human promyelocytes and myelocytes after a single pulse stimulation by purified GM-CSF or G-CSF.

Enriched populations of either normal human promyelocytes and myelocytes or blast cells were obtained by fluorescence-activated cell sorting with the monoclonal antibody WEM-G11. These populations were used to study the effect of pulse stimulation by purified recombinant human granulocyte-macrophage colony-stimulating factor (GM-CSF) or cross-reacting purified murine granulocyte colony-stimulating factor (G-CSF). Maximal clone formation by promyelocytes and myelocytes was observed in 1-mL agar cultures stimulated continuously with 400 units of either CSF and in cultures of cells that were pulse stimulated by 3,200 units (or greater) of either CSF. Pulse stimulation by 800 units of GM-CSF or G-CSF generated 75% clone formation, and pulse stimulation by 200 units CSF gave 50% clone formation. The majority of clones formed by pulse-stimulated cells were only two cells in size; however, some clones were up to 15 cells in size after a single exposure to CSF. Clone formation was not observed in cultures of blast cell populations after a single pulse stimulation with GM-CSF or G-CSF.

Bone Marrow Cells↗

PubMed1988

Clonal analysis of the actions of the murine leukemia inhibitory factor on leukemic and normal murine hemopoietic cells.

The in vitro actions of leukemia inhibitory factor (LIF) purified from Krebs tumor conditioned medium, were analyzed on murine leukemic M1 and WEHI-3B D+ cells and on normal hemopoietic progenitor cells. LIF has no observable effects on WEHI-3B D+ cells but rapidly induced macrophage differentiation and loss of clonogenicity in M1 cells, resulting in the formation of abortive clones or differentiating colonies of reduced size and number. These effects were observable within one to two cell divisions in the presence of LIF and were irreversible. Addition of macrophage-colony-stimulating factor (CSF) but not granulocyte/macrophage-CSF, granulocyte-CSF, or multi-CSF reduced the LIF-induced suppression of colony numbers and size. G-CSF had a slower differentiation-inducing action on M1 cells than LIF but potentiated the differentiation-inducing effects of low concentrations of LIF. LIF had no colony-stimulating activity for normal granulocyte-macrophage progenitor cells and did not alter their quantitative responsiveness to CSF. However, culture of normal progenitor cells in the presence of LIF, but initial absence of CSF, reduced the survival of these cells. The differing actions of LIF and G-CSF on M1 leukemic cells suggest the existence of distinct mechanisms for inducing macrophage differentiation in these leukemic cells.

Animals↗

PubMed1988

Activation and proliferation signals in murine macrophages: synergistic interactions between the hematopoietic growth factors and with phorbol ester for DNA synthesis.

There has been recent interest in the synergistic interactions between the growth factors involved in the in vitro control of hematopoiesis and other cell lineages. As a convenient model system, such interactions governing the DNA synthesis in murine bone marrow-derived macrophages (BMMs) were studied. By themselves, murine colony-stimulating factor-1 (CSF-1) and recombinant murine granulocyte-macrophage CSF (GM-CSF) were stimulators of DNA synthesis in quiescent or noncycling BMMs, whereas recombinant murine interleukin-3 (IL-3) and the phorbol ester, 12-O-tetradecanoyl-phorbol-13-acetate (TPA), were weak mitogens. On the other hand, murine granulocyte CSF (G-CSF), concanavalin A (Con A), and lipopolysaccharide (LPS) were inactive on their own. When the quiescent BMMs were exposed to combinations of the CSFs, there were striking synergistic effects for both GM-CSF and IL-3 with suboptimal doses of CSF-1, with a smaller effect for GM-CSF with IL-3 and little or no effect for CSF-1 with G-CSF. CSF-1, GM-CSF, and IL-3 could also synergize with TPA; CSF-1 cooperated with 1-oleoyl-2-acetylglycerol (OAG), both sets of results pointing to an interaction with protein kinase C. LPS completely abolished the CSF-1-mediated stimulation of DNA synthesis. We propose that BMMs are suitable normal cells in which to examine in depth the various mechanistic possibilities for these interactions.

Animals↗

PubMed1988

Colony stimulating factors and hemopoiesis.

Work in the past twenty years has led to the discovery of multiple families of hemopoietic growth factors that regulate the production and functional activity of the various subsets of blood cells. For granulocyte-monocyte populations, four such regulators (the colony stimulating factors GM-CSF, G-CSF, M-CSF and Multi-CSF) have been shown to interact to regulate these populations. Each has been purified, cDNA's for each have been cloned and mass-produced recombinant CSF's are becoming available for clinical use. Initial trials indicate that the CSF's will be valuable agents in stimulating hemopoiesis either following chemotherapy or marrow transplants or in stimulating resistance to life-threatening infections. CSF's play an important role in the development of myeloid leukemia but curiously can suppress myeloid leukemic populations because of their differentiation-inducing actions. However, it is still unclear whether the CSF's will prove of value in the management of particular myeloid leukemias.

Colony-Stimulating Factors↗