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D Metcalf

Publications and source records attributed to D Metcalf.

At least 271 records · Page 15Linked to original sources
PubMed1985

Clonal heterogeneity in colony stimulating factor production by murine T lymphocytes.

A panel of 55 alloreactive murine T-lymphocyte clones was screened for the production of granulocyte-macrophage colony stimulating factor (GM-CSF), multilineage CSF (multi-CSF), human-active eosinophil CSF (human-active EO-CSF), and interleukin 2 (IL-2) in response to stimulation with the lectin concanavalin A. Many clones were also characterized for cytolytic specificity and expression of the T-cell antigen receptor-associated surface markers Lyt-2 and L3T4, which reflect their specificity for Class I (H-2K, H-2D) or Class II (H-2l, Mls) histocompatibility antigens, respectively. Eighty percent of the clones secreted detectable quantities of at least one of the four factors measured. Of the factor-producing clones, all appeared to secrete GM-CSF and half also secreted multi-CSF. A subpopulation of multi-CSF producers also released human-active EO-CSF. More than half of the factor-producing clones secreted detectable IL-2; whereas the IL-2-producing clones included some that did not secrete multi-CSF, IL-2 production was always associated with concomitant synthesis of GM-CSF. Comparison of the range and quantities of factors secreted by Lyt-2+ and L3T4+ clones indicated that more L3T4+ clones produced measurable titers of the four factors; on average, this group also secreted 10- to 100-fold higher titers of both the hemopoietic regulators and IL-2 than Lyt-2+ clones. Cells of the L3T4+ phenotype would therefore be expected to account for the majority of CSF and IL-2 secretion by polyclonal populations of activated T lymphocytes.

Animals↗
PubMed1985

Binding of 125I-labeled granulocyte colony-stimulating factor to normal murine hemopoietic cells.

The binding of granulocyte colony-stimulating factor (G-CSF) to murine bone marrow cells was investigated using a radioiodinated derivative of high specific radioactivity which retained full biological activity. The binding was time- and temperature-dependent, saturable and highly specific. The apparent dissociation constant for the reaction was 60-80 pM at 37 degrees C and 90-110 pM at 4 degrees C, similar to that found for the binding of G-CSF to murine leukemic cells (WEHI-3B D+) and significantly higher than the concentration of G-CSF required to stimulate colony formation in vitro. Autoradiographic analysis confirmed the specificity of binding since granulocytic cells were labeled but lymphocytes, erythroid cells and eosinophils were not. Blast cells and monocytic cells were partially labeled, the latter at low levels. In the neutrophilic granulocyte series, grain counts increased with cell maturity, polymorphs being the most heavily labeled but all cells showed considerable heterogeneity in the degree of labeling. Combination of Scatchard analysis of binding with autoradiographic data indicated that mature granulocytes from murine bone marrow exhibited 50-500 G-CSF receptors per cell.

Animals↗
PubMed1985

Hierarchical down-modulation of hemopoietic growth factor receptors.

Granulocytes and macrophages can be produced in vitro when progenitor cells from mouse bone marrow are stimulated by any of four distinct colony stimulating factors, Multi-CSF (IL-3), GM-CSF, G-CSF, and M-CSF (CSF-1). At 0 degrees C the four CSFs do not cross-compete for binding to bone marrow cells, indicating that each has a specific cell surface receptor. However, at 21 degrees C or 37 degrees C, Multi-CSF inhibits binding of the other three CSFs and GM-CSF inhibits binding of G-CSF and M-CSF. Rather than competing directly for receptor binding, the binding of Multi-CSF, GM-CSF, or G-CSF to their own receptor induces the down-modulation (and thus activation) of other CSF receptors at 37 degrees C. The pattern and potency of down-modulation activity exhibited by each type of CSF parallels the pattern and potency of its biological activity. We propose a model in which the biological interactions of the four CSFs are explained by their ability to down-modulate and activate lineage-specific receptors.

Animals↗
PubMed1985

Expression of a hemopoietic growth factor cDNA in a factor-dependent cell line results in autonomous growth and tumorigenicity.

Production of a growth factor by a cell that responds to this factor has been termed "autocrine" stimulation of proliferation. Considerable experimental data have suggested that tumor cells often exhibit autocrine growth stimulation and that this may contribute to the process of malignant transformation. To experimentally approach the relationship of autocrine growth stimulation to the malignant transformation of hemopoietic cells, we have used a retroviral vector to express sequences encoding a hemopoietic growth factor, granulocyte-macrophage colony stimulating factor (GM-CSF) in a factor-dependent murine cell line (FDC-P1). Virally infected cells synthesized and secreted GM-CSF, grew independently of exogenous CSF, and--unlike the parental FDC-P1 cells--produced tumors in syngeneic mice. We have thus experimentally induced autocrine growth regulation in a factor-dependent hemopoietic cell line and have shown that this results in tumorigenicity.

Animals↗
PubMed1985

Synthesis by mouse peritoneal cells of G-CSF, the differentiation inducer for myeloid leukemia cells: stimulation by endotoxin, M-CSF and multi-CSF.

Normal C57BL mouse peritoneal cells were able to synthesize material with the ability to induce differentiation in colonies of the mouse myelomonocytic leukemia cell line, WEHI-3B. The active factor was provisionally identified biochemically as the normal regulator, granulocyte colony-stimulating factor, G-CSF. Thioglycollate-induced peritoneal exudate cells had little or no capacity to synthesize such material. Production of active material was elevated 10-100-fold by exposure of peritoneal cells to endotoxin, detectable elevations being observed after the addition of as little as 0.8 ng/ml. Production of G-CSF was observed using adherent peritoneal macrophages, was a radioresistant process depending on protein synthesis and was not modified by the absence or addition of T-lymphocytes. Addition of unfractionated media containing M-CSF or Multi-CSF, partially purified M-CSF or fully purified Multi-CSF elevated the production of G-CSF by peritoneal cells from both C57BL mice and mice of the endotoxin-unresponsive strain C3H/HeJ, but an involvement of endotoxin in this process could not be excluded absolutely. The experiments provide further evidence that microorganisms and perhaps hemopoietic regulators play an important role in modulating the production of G-CSF and thus have the potentiality to influence the emergence and progressive proliferation of myeloid leukemia populations.

Animals↗
PubMed1985

The proliferative effects of human GM-CSF alpha and beta and murine G-CSF in microwell cultures of fractionated human marrow cells.

Blast cell-enriched and promyelocyte-myelocyte-enriched fractions of human marrow were prepared by fluorescence-activated cell sorting using an antineutrophil monoclonal antiserum. Cells from both fractions proliferated in microwell cultures when stimulated by placental or bladder cancer cell conditioned medium containing colony stimulating factor. Semipurified preparations of both GM-CSF alpha and beta from bladder cancer cell conditioned medium were effective proliferative stimuli for both cell populations. Pure murine G-CSF, GM-CSF, M-CSF and Multi-CSF failed to stimulate detectable proliferation in blast cell fractions containing granulocyte-monocyte progenitors. However, G-CSF was an effective proliferative stimulus for human promyelocytes and myelocytes leading to the formation of differentiating granulocytic progeny. G-CSF also stimulated the proliferation of human promyelocytic leukemic cells. Promyelocyte-myelocyte-enriched fractions of human marrow appear to be useful target cells for monitoring the proliferative effects of human-active CSFs in microwell cultures.

Animals↗
PubMed1985

Malignant transformation of a growth factor-dependent myeloid cell line by Abelson virus without evidence of an autocrine mechanism.

Abelson virus has been used to transform cells of a murine, factor-dependent myeloid cell line (FD). Factor-independent (FI) cell lines were derived, which expressed the viral genome and were tumorigenic in syngeneic mice. Karyotypic analysis of FI cells before and after passage in vivo indicated that the tumorigenic cells were derived from FD cells. Northern gel analysis of mRNA, bioassay of culture supernatants, and the density-independent growth of the FI cells indicated that the transformation had not induced the synthesis of the hemopoietic growth factors normally required to support the FD cells, that is, granulocyte-macrophage CSF or Multi-CSF. The FD and FI cells displayed similar numbers of cell surface receptors for Multi-CSF (IL-3) and GM-CSF. We conclude that Abelson virus transformation of this line from factor-dependence to factor-independence and tumorigenicity did not involve autocrine stimulation.

Abelson murine leukemia virus↗
PubMed1985

Purification and partial amino acid sequence of asialo murine granulocyte-macrophage colony stimulating factor.

A procedure utilizing reversed-phase high-performance liquid chromatography is described for the purification of asialo granulocyte-macrophage colony stimulating factor (asialo-GM-CSF) from mouse lung-conditioned medium. In the purification, the partially purified factor was treated with neuraminidase to reduce charge heterogeneity due to variable degrees of sialation. Three active forms of the asialo factor were separated by the final reversed-phase liquid chromatography step. These each gave a single major band and several minor bands on polyacrylamide gel electrophoresis and had similar amino acid compositions. The specific activity of purified murine asialo-GM-CSF was approximately 8 X 10(9) colonies per mg of protein. Amino acid sequence determination of the major form gave a single amino-terminal sequence, which has been used to develop oligonucleotide probes for the isolation of two cDNA clones encoding GM-CSF. The nucleotide sequence of these two clones gave a deduced amino acid sequence almost identical with that determined for the amino terminus of asialo-GM-CSF and an amino acid composition very similar to that for asialo-GM-CSF.

Amino Acid Sequence↗
PubMed1985

Characteristics of colony-stimulating factor production by murine T-lymphocyte clones.

From a panel of cloned interleukin-2-dependent murine T-lymphocyte lines, several clones were identified that secreted high titers of one or more of the colony-stimulating factors (CSFs): granulocyte-macrophage CSF, multi-CSF (or interleukin 3), and human eosinophil CSF. These clones were used to examine the parameters of CSF production, including stimuli required, kinetics, serum concentration, and the cell dose and proliferative state of the clone. Maximal production of CSFs and interleukin 2 was stimulated by the lectin concanavalin A, which induced a burst of secretion over a 12- to 20-h period, during which all factors were released at similar rates. CSF production was not stimulated by interleukin 2 and occurred independently of clonal proliferation.

Animals↗
PubMed1985

The clonal proliferation in vitro of enriched populations of human promyelocytes and myelocytes.

The proliferative capacity of normal human promyelocytes and myelocytes was demonstrated and characterized on the basis of clonal proliferation in agar. An enriched population of normal human promyelocytes and myelocytes was obtained from bone marrow using the monoclonal antibody WEM G11 and the fluorescence-activated cell sorter (FACS). In cultures stimulated by placental-conditioned medium, these cells generated peak total clone numbers between days 3 and 5 of culture. Clones disappeared rapidly thereafter. These clones were mainly of subcolony size at day 7, although some colonies were generated by this population. The clones were primarily neutrophilic in type. These cells had a plating efficiency of up to 50%, and clonal proliferation was dependent on stimulation by colony-stimulating factor (CSF).

Antibodies, Monoclonal↗
PubMed1985

Fractionated populations of normal human marrow cells respond to both human colony-stimulating factors with granulocyte-macrophage activity.

Populations of normal human colony-forming cells (blast cells) and cluster-forming cells (promyelocytes-myelocytes) were obtained from bone marrow by using the monoclonal antibody WEM G11 and the fluorescence-activated cell sorter (FACS). Both populations were shown to be responsive to both human colony-stimulating factors (CSFs) with granulocyte-macrophage activity (CSF alpha and CSF beta), with the cluster-forming cell population being more responsive to CSF beta than the colony-forming cell population. The clonal proliferation of promyelocytes-myelocytes was transient, and the clones generated were of subcolony size (less than 40 cells) regardless of the CSF used. Clone transfer experiments demonstrated that progeny of promyelocytes-myelocytes initiated using one stimulus (CSF alpha or CSF beta) were also responsive to the other stimulus.

Animals↗
PubMed1985

Multi-CSF-dependent colony formation by cells of a murine hemopoietic cell line: specificity and action of multi-CSF.

Cells of the Multi-CSF (IL-3)-dependent hemopoietic cell line 32D c13 formed colonies of varying size in agar cultures stimulated by Multi-CSF. Colony formation was linear with respect to cultured cell numbers; colony numbers and size increased with increasing concentrations of Multi-CSF, and 32D colonies themselves contained a high frequency of clonogenic cells. Clonogenic 32D cells died in the absence of Multi-CSF (half-life six hours), and most were unable to complete cell cycles in progress at the time of withdrawal of Multi-CSF. The concentration of Multi-CSF directly influenced the length of the cell cycle of dividing 32D cells. Purified GM-CSF, G-CSF, or M-CSF had no capacity to support the survival or proliferation of 32D cells. Colonies formed by 32D cells appear to offer a useful model for analyzing the action of Multi-CSF in controlling self-renewal by clonogenic hemopoietic cells.

Animals↗
PubMed1985

Molecular control of granulocyte and macrophage production.

The four major glycoprotein colony stimulating factors (CSF's) controlling murine granulocyte-macrophage formation and function have been purified to homogeneity and some sequence data obtained. cDNA's for GM-CSF and Multi-CSF have been cloned and the full sequence of the CSF polypeptides deduced. Despite functional similarities between the two molecules, no sequence homology exists between these two regulators. Binding studies using radiolabeling M-CSF, G-CSF and GM-CSF have indicated that relatively low numbers (200-700) of specific receptors exist for G-CSF and GM-CSF on responding hemopoietic cells although up to 20,000 receptors per cell exist for M-CSF. Despite the few receptors available, biological effects of the CSF's are demonstrable with low receptor occupancy. Interactions between the four CSF's permit the production of granulocytes and macrophages to be finely controlled.

Bone Marrow Cells↗
PubMed1985

The colony-stimulating factors and myeloid leukaemia.

The production of granulocytes and macrophages is under the control of at least four well defined haemopoietic growth factors or colony-stimulating factors (CSF's) which differ in their actions within the hierarchical organization of haemopoietic progenitor cells and in the different cell lineages they affect. Multi-CSF has an extremely broad haemopoietic specificity, GM-CSF stimulates all granulocyte and macrophage progenitor cells and G-CSF and M-CSF have actions essentially restricted to the granulocyte or macrophage cell lineages, respectively. They are each, however, required for cell survival, proliferation, differentiation and mature cell activation within the cell lineages they act on. They each exert their actions through specific high-affinity cell surface receptors which show no direct cross reactivity with each other and which are structurally distinct. However, at physiological temperatures the CSF's show a specific pattern of receptor co-down-modulation which might reflect their cell lineage specificities. A major defect in myeloid leukaemias is a block to differentiation so that cell divisions result in self-renewal of the leukaemic stem cells rather than terminal differentiation to non-dividing cells. In murine models such a defect does not result in malignancy until it is accompanied by autonomy from external CSF growth control but in human myeloid leukaemias there is no evidence for autonomy from CSF growth control and CSF's may play a permissive role in the emergence of myeloid leukaemias. The possibility of using CSF's to override the differentiation block in leukaemias and cause suppression of leukaemic growth by differentiation induction has been examined for the action of G-CSF on murine leukaemic WEHI-3B D+ cells. G-CSF induces differentiation in these cells, strongly suppresses cell divisions leading to self-renewal and increases the survival time of mice injected with treated cells. The possible importance of this factor in cell differentiation is indicated by the loss of G-CSF receptors in a differentiation-defective mutant of WEHI-3B, the conservation of G-CSF and its receptor from mouse to man and the observation that all primary human myeloid leukaemias exhibit specific receptors for this factor.

Animals↗
PubMed1985

Biologic properties of molecularly cloned and expressed murine interleukin-3.

Interleukin-3 (IL-3) (multipotential colony-stimulating factor [multi-CSF] ) is an important regulator of hemopoiesis. We recently isolated a cDNA clone of this gene and describe in this manuscript the biologic properties of the expressed gene product. An SV40 expression vector carrying cDNA encoding murine interleukin-3 was constructed so that expression of the IL-3 gene was placed under the control of the SV40 early promoter. When the expression vector was transfected to COS-1 monkey cells, IL-3 activity was secreted into the medium, reaching maximal levels 72 hours after transfection. The IL-3 produced by the COS-1 cells was partially purified using diethylaminoethyl Sephacel and phenyl-Sepharose, and its chromatographic properties were the same as IL-3 produced by the WEHI-3 cell line. The biologic activities of the "expressed" IL-3 include the "induction" of 20-alpha-hydroxysteroid dehydrogenase (20-alpha-SDH) in splenic lymphocytes from nu/nu mice, proliferative activity for 32D cl-23 and FDC-P1 cell lines, and colony-stimulating activity for granulocyte-macrophage, eosinophil, megakaryocyte, natural killer-like, erythroid, and multipotential colony-forming cells from murine fetal liver and adult bone marrow.

20-Hydroxysteroid Dehydrogenases↗
PubMed1984

Activation of human and murine B cells by anti-immunoglobulin antibody: dependence of the response on the preexisting level of B-cell activation.

Previous reports of the response of B lymphocytes to soluble anti-immunoglobulin (anti-Ig) antibodies have yielded conflicting data. While most studies show activation of B cells, others have shown inhibitory effects. In the assay reported in this report, we were able to obtain widely diverse responses of human B-cell populations to anti-Ig antibody. An explanation of this variability was established by resort to an animal (murine) model. Mice maintained in a pathogen-free environment failed to respond or responded only weakly to anti-Ig antibody. Mice which had previously received heavy antigenic stimulation, but at the time of the experiment were not undergoing any known challenge, showed a marked positive response. Mice deliberately challenged with lipopolysaccharide (LPS) 24 hr prior to anti-Ig antibody exposure showed a high background mitogenesis in control cultures, which was inhibited by anti-Ig antibody. This preliminary study suggests that response to anti-Ig antibody differs in each phase of B-cell differentiation. In future studies it is hoped that this variability in response can be used to characterize different subsets of B-cell differentiation separated by physical or phenotypic parameters.

Animals↗