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Biomedical subjects

D Metcalf

Publications and source records attributed to D Metcalf.

At least 289 records · Page 16Linked to original sources
PubMed1984

Induction of differentiation in HL60 leukaemic cells: a cell cycle dependent all-or-none event.

The human leukaemia cell line (HL60) shows a limited capacity to differentiate spontaneously, but this property can be greatly enhanced by chemical inducers. Sodium butyrate induced differentiation in virtually 100% of HL60 cells over a four-day interval to cells with multiple phenotypic markers of monocytes. Clonogenic analysis in agar demonstrated that differentiated cells (either spontaneous or induced) irreversibly lost clonogenic potential. This appeared to be an all-or-none process with unaffected cells exhibiting unaltered clonogeneity. A study of the kinetics of colony formation showed that most, if not all, cells completed one division in the presence of butyrate and sometimes several divisions before loss of proliferative potential. Despite the uniform spectrum of cell cycle states present in HL60 cultures when butyrate was added, all differentiated cells were shown to be arrested in G1. Evidence was obtained suggesting that the 'switch' into the differentiation pathway occurred during a restricted stage of the cell cycle, either late in the cycle (G2-M) or early in G1.

Animals↗
PubMed1984

Binding of the differentiation-inducer, granulocyte-colony-stimulating factor, to responsive but not unresponsive leukemic cell lines.

Granulocyte-colony-stimulating factor (G-CSF) is a tissue-derived 25,000 Mr glycoprotein that stimulates neutrophilic granulocyte colony formation from murine bone marrow progenitor cells in vitro. It is also a potent inducer of terminal differentiation and suppressor of stem cell renewal in the murine myelomonocytic leukemic cell line WEHI-3B. Purified G-CSF was radioiodinated to high specific radioactivity with retention of full biological activity. Iodinated G-CSF bound specifically to WEHI-3B cells, J774 macrophage tumor cells, and normal murine bone marrow cells but not to a variety of other tumor cell lines or murine thymocytes. WEHI-3B cells showed a high affinity for 125I-labeled G-CSF (Kd = 90 pM) but displayed only a small number of specific receptors (300-700 per cell) at 37 degrees C. Other purified colony-stimulating factors showed no competition for binding to these receptors. WEHI-3B (D-), a subline of WEHI-3B that cannot be induced to differentiate by G-CSF, showed no specific binding of this factor, indicating that it is deficient in receptor presentation.

Animals↗
PubMed1984

The regulatory factors controlling murine erythropoiesis in vitro.

In the mouse, control of the proliferation of erythroid precursors is complex and probably based on the sequential expression of membrane receptors for different regulator molecules during erythroid differentiation. Purified GM-CSF, G-CSF and multi-CSF are all able to initiate proliferation of the earlier erythroid precursors, the multi-CFC and BFU-E, and multi-CSF can stimulate the proliferation of erythroid precursors through all subsequent stages of differentiation. Erythropoietin acts as proliferative stimulus only for the terminal stages of erythropoiesis. There is as yet no evidence for the existence of a distinct BPA regulator with proliferative effects restricted to early erythroid precursors.

Anemia, Aplastic↗
PubMed1984

Mononuclear cell-mediated enhancement of granulocyte function in man.

Human monocyte-enriched mononuclear cells (MNC) (greater than 95% monocytes) powerfully stimulated the antibody-dependent killing of tumor cells by purified human neutrophils (NE) or eosinophils (EO). The enhancement was observed when the mononuclear cell to granulocyte ratio was as low as 1:100. Media conditioned by MNC also stimulated antibody-dependent killing by NE and EO as well as the uptake and killing of Candida albicans by NE. There was a heterogeneity amongst individuals in the capacity of their MNC to elaborate this factor, and approximately 15% of individuals did not produce detectable factor. Different factors in the MNC supernatant were involved in NE and EO activation. The EO-activating factor (AF) had a m.w. of approximately 27,000 and migrated as a single band on phenyl-Sepharose chromatography. The NE-AF had a m.w. of 22,000 and migrated as two distinct bands on phenyl-Sepharose chromatography. EO-colony-stimulating factor (CSF) co-purified with EO-AF, and granulocyte-macrophage-CSF co-purified with both bands of NE-AF. It is concluded that MNC elaborate CSF-like molecules that powerfully stimulate human granulocyte function.

Antibody-Dependent Cell Cytotoxicity↗
PubMed1984

Biological and biochemical properties of a serum factor that stimulates splenic hemopoiesis in mice.

Some biological and biochemical properties of a distinct hemopoietic factor that stimulates splenic hemopoiesis in mice are described. This factor can be detected by measuring the increase in the number of in vitro hemopoietic colony-forming cells (CFCs) in the spleens of mice after transfer of serum from syngeneic donors that have been treated previously with the bacterial cell-wall components: lipid A or lipoprotein. Serum collected 5 min after the IV injection of lipid A contained almost no splenic hemopoiesis-stimulating factor (SHSF). The highest serum levels of the factor were found between 30 min and 3 h after lipid A was injected IV. The residual levels of lipid A or lipoprotein in the serum of treated mice were too low to account for their splenic hemopoiesis-stimulating effects. A component of SHSF in both post-lipid-A serum (PLAS) and postlipoprotein serum (PLPS) bound to concanavalin A (Con A)-Sepharose and could be eluted by alpha-methyglucopyranoside (0.05 M). Partial fractionation of PLAS using Con A-Sepharose and gel filtration (Sephacryl S-200) indicated that the SHSF glycoprotein had an apparent molecular weight of 30,000 daltons. SHSF was detected in serum in response to lipid A and lipoprotein, but this was separable (by gel filtration) from the major form of granulocyte-macrophage colony-stimulating factor (GM-CSF) in PLAS.

Animals↗
PubMed1983

Purification of a factor inducing differentiation in murine myelomonocytic leukemia cells. Identification as granulocyte colony-stimulating factor.

A naturally occurring inducer of terminal differentiation in a murine myelomonocytic leukemia cell line (WEHI-3B) was purified to apparent homogeneity from medium conditioned by lungs from mice injected with bacterial endotoxin. The factor was purified over 400,000-fold by sequential fractionation using salting out chromatography, chromatography on phenyl-Sepharose, gel filtration on Bio-Gel P-60 in 1 M acetic acid, reverse-phase high performance liquid chromatography on a phenyl-silica column, and high performance liquid chromatography on a gel filtration column. During the first two steps, the differentiation-inducing factor was separated completely from a known proliferative regulator for normal myeloid cells, granulocyte-macrophage colony-stimulating factor, but it co-purified through all remaining steps with a distinct granulocyte-specific colony-stimulating factor. The purified factor showed a single protein band of Mr = 24,000-25,000 on sodium dodecyl sulfate-polyacrylamide gels coincident with both differentiation-inducing and granulocyte colony-stimulating activity. The granulocyte-specific colony-stimulating factor was active on WEHI-3B cells and normal granulocytic progenitor cells in vitro at the same half-maximally active concentration of 3 X 10(-12) M.

Animals↗
PubMed1983

Proliferative effects of purified granulocyte colony-stimulating factor (G-CSF) on normal mouse hemopoietic cells.

When granulocyte colony-stimulating factor (G-CSF), purified to homogeneity from mouse lung-conditioned medium, was added to agar cultures of mouse bone marrow cells, it stimulated the formation of small numbers of granulocytic colonies. At high concentrations of G-CSF, a small proportion of macrophage and granulocyte-macrophage colonies also developed. G-CSF stimulated colony formation by highly enriched progenitor cell populations obtained by fractionation of mouse fetal liver cells using a fluorescence-activated cell sorter, indicating that G-CSF probably acts directly on target progenitor cells. Granulocytic colonies stimulated by G-CSF were small and uniform in size, and at 7 days of culture were composed of highly differentiated cells. Studies using clonal transfer and the delayed addition of other regulators showed that G-CSF could directly stimulate the initial proliferation of a large proportion of the granulocyte-macrophage progenitors in adult marrow and also the survival and/or proliferation of some multipotential, erythroid, and eosinophil progenitors in fetal liver. However, G-CSF was unable to sustain continued proliferation of these cells to result in colony formation. When G-CSF was mixed with purified granulocyte-macrophage colony-stimulating factor (GM-CSF) or macrophage colony-stimulating factor (M-CSF), the combination stimulated the formation by adult marrow cells of more granulocyte-macrophage colonies than either stimulus alone and an overall size increase in all colonies. G-CSF behaves as a predominantly granulopoietic stimulating factor but has some capacity to stimulate the initial proliferation of the same wide range of progenitor cells as that stimulated by GM-CSF.

Animals↗
PubMed1983

Early events in the suppression of myeloid leukemic cells by biological regulators.

Differentiation of mouse and human myeloid leukemic cells in vitro can be induced by some members of the granulocytemacrophage family of colony-stimulating factors. In the mouse, the most active molecule (G-CSF) is able to suppress leukemic stem cell self-generation in an irreversible, asymmetric process, suggesting that the factor permanently modifies newly synthesized one or more daughter chromatids in dividing leukemic stem cells.

Animals↗
PubMed1983

Clonal analysis of the response of HL60 human myeloid leukemia cells to biological regulators.

Human myeloid leukemia (HL60) cells formed colonies in semi-solid agar cultures with a cloning efficiency of 20-90%. Addition of unfractionated human placental conditioned medium (HPCM) or the two semi-purified granulocyte-macrophage colony stimulating factors from HPCM (GM-CSF alpha and beta) increased colony size and the frequency of colonies exhibiting differentiation of colony cells. Differentiation induction using GM-CSF alpha or beta did not reduce the total number of clonogenic cells per colony but did reduce the proportion of clonogenic cells within colonies. Sera from some patients with acute infections exhibited an elevated capacity to induce differentiation both in HL60 colonies and in colonies of the mouse myelomonocytic leukemia cell line, WEHI-3B. A low percentage of HL60 colonies contained maturing eosinophils but the frequency was not influenced by human-active eosinophil colony stimulating factors or by sequential recloning of HL60 colonies. The studies suggest that, as is true for mouse myeloid leukemia cell lines, the granulocyte-macrophage colony stimulating factors are able to induce significant differentiation in human HL60 myeloid leukemia cells.

Cell Differentiation↗
PubMed1983

Selective stimulation by mouse spleen cell conditioned medium of human eosinophil colony formation.

Stimulation of unfractionated or nonadherent human marrow cells in agar culture by pokeweed-mitogen-stimulated BALB/c mouse spleen cell conditioned medium (SCM) led, in most cultures, to the exclusive formation of eosinophil colonies. The culture system exhibited linearity of eosinophil colony formation with varying numbers of cells cultured, and the absolute numbers and size of SCM-stimulated eosinophil colonies approximated those in cultures stimulated by human placental conditioned medium. The active factor in SCM for human eosinophil colony formation was not clearly separable from the factors stimulating granulocyte-macrophage and eosinophil colony formation by mouse marrow cells on ammonium sulfate and phenyl boronate chromatography, but was of larger size than the mouse-active factors and separable from them by phenyl sepharose chromatography. This selective culture system for eosinophil colony formation should be of value for studies on human eosinophil progenitor and maturing cell populations in a variety of disease states.

Animals↗
PubMed1983

Eosinophil activation by colony-stimulating factor in man: metabolic effects and analysis by flow cytometry.

Substantial increases in the killing capacity of human eosinophils after in vitro incubation with human placental conditioned medium (HPCM), a standard source of colony-stimulating factor (CSF), have recently been described. In this article, the interaction between HPCM and purified human eosinophils is analyzed by flow cytometry and by effects on iodination, superoxide production, and protein synthesis. HPCM increased the intensity of natural eosinophil autofluorescence (aFlu) (460 nm) after the absorption of ultraviolet light (360 nm) in a manner that was both time and dose dependent. Measured in arbitrary units, eosinophil aFlu was 72 +/- 7.3 (arithmetic mean +/- SEM) and 121 +/- 3.2 after 18-hr incubations in the absence or presence of HPCM, respectively. The activity in HPCM responsible for these changes cochromatographed on Ultrogel AcA44 columns with CSF and with the less hydrophobic variant of CSF (CSF-alpha) on phenyl Sepharose. Mouse spleen, but not mouse lung, conditioned medium was also active on human eosinophils in this assay. Both CSF-alpha and mouse spleen conditioned medium also contain eosinophil colony-stimulating activity (CSA), whereas inactive CSFs with no effect on mature eosinophils, CSF-beta, and mouse lung conditioned medium also lack eosinophil CSA. CSF-alpha stimulated superoxide production of resting eosinophils (from 0.03 +/- 0.03 to 0.47 +/- 0.08 nmole cytochrome-c reduced/10(5) eosinophils) and of eosinophils incubated with preopsonized zymosan (from 0.15 +/- 0.06 to 0.73 +/- 0.07). It also stimulated iodination by resting eosinophils (from 0.76 +/- 0.16 to 2.60 +/- 0.72 nmoles l/10(7) eosinophils/hr) and of eosinophils incubated with preopsonized zymosan (from 7.52 +/- 2.08 to 29.8 +/- 1.32). In contrast, CSF-beta was inactive in these assays. CSF-alpha also stimulated, between 2- and 15-fold, the new protein synthesis of eosinophils. Thus, substances that stimulate the differentiation of progenitor cells into eosinophils also interact with peripheral mature eosinophils, and the activation of postmitotic cells may be a physiologic role of CSF-like molecules.

Cell Separation↗
PubMed1983

Separation of hemopoietic cells from adult mouse marrow by use of monoclonal antibodies.

Primitive hemopoietic progenitor cells from adult mouse marrow have been substantially enriched by virtue of a negative selection procedure with monoclonal antibodies. It has been possible to segregate erythroid progenitor cells at distinct stages of differentiation on the basis of their cell surface antigens. This has been achieved with two monoclonal antibodies reactive with the mature elements of bone marrow. YBM 34.3 binds to a heat-stable antigen expressed on B lymphocytes, neutrophils, and cells of the erythroid lineage. YBM 6.1 reacts with cells of the neutrophil, eosinophil, and monocyte series but does not bind to colony-forming cells. Separation is achieved by indirect immunoadsorption (panning) with YBM 34.3 on Protein-A-coated plastic plates followed by FACS II cell sorting with YBM 6.1. The combined procedures yield a marrow population containing 58% immature cells (blasts, promyelocytes, and myelocytes) and 9.5% clonogenic cells. In addition, differential binding of YBM 34.3 can be used to segregate erythroid progenitor cells at distinct stages of differentiation (day 7 BFU-E, day 5 BFU-E and CFU-E) either by cell sorting or panning. It is shown that both techniques give a comparable degree of resolution of the different cell types with, however, an appreciable advantage of panning over cell sorting in allowing the rapid handling of large numbers of cells.

Animals↗
PubMed1983

Removal of T cells from bone marrow for transplantation: a monoclonal antilymphocyte antibody that fixes human complement.

Graft-versus-host disease is one of the major problems in clinical bone marrow transplantation. Many experiments in animals have shown that it could be greatly reduced if mature T lymphocytes were removed from the donor marrow. Here we describe a new rat monoclonal antibody, CAMPATH 1, which is suitable for depleting lymphocytes from human marrow grafts. CAMPATH 1 is an IgM that fixes human complement. It binds to both T and B lymphocytes and some monocytes but not to other hemopoietic cells. When peripheral blood mononuclear cells were treated with CAMPATH 1 and complement, more than 99% of lymphocytes were killed and viable T cells could no longer be detected. Under these conditions, in vitro multipotential erythroid and myeloid colony-forming cells were unaffected. As well as being used for in vitro treatment of bone marrow to remove T cells, CAMPATH 1 could potentially be applied to other experimental and clinical situations where depletion of lymphoid cells is required, including serotherapy to achieve immunosuppression for organ transplants or to treat lymphocytic leukemias.

Antibodies, Monoclonal↗
PubMed1983

Activation of antibody-dependent cell-mediated cytotoxicity of human neutrophils and eosinophils by separate colony-stimulating factors.

Semi-purified human colony-stimulating factors (CSF) powerfully enhanced the antibody-dependent cell-mediated cytotoxicity (ADCC) by metrizamide gradient-purified human neutrophils and eosinophils. The stimulation was observed on three different tumor targets, was rapid (less than 1 hr) in onset, and CSF-stimulated cells needed direct contact with targets for killing. A subspecies of human CSF, CSF-alpha, with eosinophil and granulocyte-macrophage (GM) colony-stimulating activity enhanced both eosinophil and neutrophil killing. In contrast, another subspecies of human CSF, CSF-beta, having only GM colony-stimulating activity, only enhanced neutrophil-mediated ADCC. These results support the notion that human CSF have two sites of action: i) the progenitor cell, where they stimulate a relatively slow process of differentiation, and ii) the mature cell, where they have a rapid action of increasing functional capacity. Furthermore, it seems the pattern of CSF receptors on progenitor cells is maintained throughout the lineage of such cells and serves to regulate the function of mature cells.

Antibody Formation↗
PubMed1983

Activation of granulocyte cytotoxic function by purified mouse colony-stimulating factors.

Highly purified mouse colony-stimulating factors (CSF) were tested for their effect on neutrophil cytotoxic function in a homologous antibody-dependent cell-mediated cytotoxicity (ADCC) assay in which TNP-coupled mouse thymoma cells coated with mouse anti-TNP antibodies were used as targets, and purified normal mouse bone marrow neutrophils or induced peritoneal neutrophils were used as effector cells. Biochemically pure granulocyte-macrophage (GM)- and granulocyte (G)-CSF enhanced the cytotoxic activity of neutrophils obtained from both sources, allowing them to kill target cells at low antibody concentrations. Furthermore, GM- and G-CSF showed an additive effect, suggesting either the presence of separate receptors for GM- and G-CSF or of separate subsets of neutrophils. Induced peritoneal neutrophils showed a higher level of basal cytotoxic activity than did bone marrow neutrophils, suggesting neutrophil activation in vivo, but both reached similar levels of cytotoxicity upon maximal stimulation with CSF. In addition, CSF was found to be cross-reactive between mouse and human species in their enhancement of neutrophil cytotoxicity. By testing purified mouse CSF on human neutrophils, it could be shown that G-CSF and GM-CSF are functionally distinct molecules, because only G-CSF enhanced ADCC by human neutrophils. These experiments show that the purified factors that control the production of neutrophils by progenitor cells in vitro also activate differentiated neutrophils to carry out their cytotoxic activity in a more effective manner.

Animals↗