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Biomedical subjects

D Metcalf

Publications and source records attributed to D Metcalf.

At least 343 records · Page 19Linked to original sources
PubMed1980

Detection of a new type of mouse eosinophil colony by Luxol-fast-blue staining.

Staining with Luxol-fast-blue was shown to be a satisfactory method for identifying mouse eosinophils. IN agar cultures of mouse marrow stimulated by pokeweed mitogen-stimulated spleen conditioned medium (SCM), 2-10 compact Luxol-fast-blue positive eosinophil colonies varied according to the batch of human plasma used in the culture medium. Similar colony-forming cells were detected in the spleen and peripheral blood but not in the thymus or lymph nodes. Some Luxol-fast-blue positive eosinophil colonies were stimulated to develop by crude mouse lung conditioned medium and by high concentrations of GM-CSF purified from this source. The cells forming Luxol-fast-blue positive eosinophil colonies sedimented more rapidly (5.5-6.5 mm/h) than the cells forming dispersed-eosinophil colonies 94.5 mm/h). Transfer studies using intact colonies or redispersed colony cells failed to demonstrate an interrelationship between the two types of eosinophil colonies and the cells forming Luxol-fast-blue positive eosinophil colonies appear to be a distinct subset of eosinophil percursors.

Animals↗
PubMed1980

The presence of mast cells in agar cultures.

Using a specific stain and electron microscopy, small numbers of mast cells were detected in human bone marrow cultures. However, they were not detected in agar cultures containing murine bone marrow cells. In bone marrow cultures from three patients with acute myeloid leukemia the number of mast cells was elevated.

Agar↗
PubMed1980

Purification of hemopoietic progenitor cells from human marrow using a fucose-binding lectin and cell sorting.

Human peripheral blood granulocytes, but not lymphocytes, erythrocytes, or monocytes, bound the fucose-binding lectin from Lotus tetragonolobus (FBP), and this binding was competitively inhibited by the sugar alpha-L-fucose. The fluorescence-activated cell sorter was used to study the appearance of this receptor on human marrow cells during granulocyte differentiation and to prepare fractions enriched for granulocyte-macrophage progenitor cells (granulocyte-macrophage colony-forming cells--GM-CFC). Cell binding of fluoresceinated FBP increased for bone marrow cells in the sequence--lymphocytes, blast cells, promyelocytes and myelocytes, monocytes, and polymorphonuclear cells. Selection of cells with appropriate low-angle or high-angle light scatter characteristics achieved a 10-fold or 2-3-fold enrichment of progenitor cells, respectively. By selecting cells with intermediate fluorescence intensity, a further 2-3-fold enrichment for GM-CFC was obtained. Cell sorting using the optimal selection of these three parameters produced up to 36-fold enrichment of the progenitor cells from human bone marrow. The most enriched fraction was composed of 23% progenitor cells (colony- and cluster-forming cells) with a yield of 36%. In populations most highly enriched by GM-CFC, immature cells (blast cells, promyelocytes, and myelocytes) made up 95% of the cells present.

Binding Sites↗
PubMed1980

Use of spleen organ cultures to monitor hemopoietic progenitor cell regeneration following irradiation and marrow transplantation.

After lethal irradiation of C57BL mice followed by the injection of 10(7) marrow cells, total cellularity and progenitor cell levels exceeded pretreatment levels within 12 days in the spleen, but regeneration remained incomplete in the marrow. The exceptional regenerative capacity of progenitor populations in the spleen was observed in organ cultures of spleen slices prepared 24 hr after irradiation and transplantation, excluding continuous repopulation from the marrow as a significant factor in splenic regeneration.

Animals↗
PubMed1980

The nature and action of granulocyte-macrophage colony stimulating factors.

Granulocyte-macrophage colony stimulating factor (GM-CSF) stimulates the in vitro proliferation and differentiation of granulocytic and macrophage cells. This regulator is now known to act at other levels of hemopoietic regulation. The heterogeneity of GM-CSFs is not only related to the tissue of origin and the in vitro production method, but also to functional subclasses of the molecule that have distinct biologic specificities. Most adult mouse organs produce GM-CSF (mol wt 23,000), but a macrophage (M)-CSF has been detected in fetal conditioned medium (CM) and isolated from L-cell CM. Murine endotoxin serum appears to contain a M-CSF, GM-CSF, and G-CSF, the last of which cofractionates with a differentiation factor active on leukemic cells. Human GM-CSFs, G-CSF, and EO-CSFs active on human cells have been detected in a variety of CM, but as yet none have been purified. Again, there are subclasses of progenitor cells that respond to particular forms of human active CSFs. GM-CSF isolated from mouse lung CM stimulates multipotential progenitor cells, the initial proliferatin of progenitors in the erythroid, eosinophil, and megakaryocyte series, as well as mature cells in the GM series. While GM-CSF is also able to stimulate the differentiation of myeloid leukemic cells, other factors appear to be more potent in this respect. Information on the regulation of GM-CSF production, on the modulators of its action on specific target cells, and on its role in vivo will be required before the physiologic function of this molecule can be properly assessed.

Amino Acid Sequence↗
PubMed1980

Humoral regulation of splenic hemopoiesis in mice.

The effects on splenic hemopoiesis were investigated of injecting the bacterial cell wall component, lipid A, or post-lipid A serum (PLAS) into mice. Both lipid A and syngeneic PLAS caused an increase in splenic numbers of multipotential hematopoietic stem cells (CFUS), committed hemopoietic progenitor cells (CFC) of different hemopoietic lineages and morphologically recognizable hemopoietic cells of various lineages. C57BL/6 Sld/Sld PLAS had a lower stimulating effect on hemopoiesis than C57BL/6 +/+ PLAS. PLAS from pre-irradiated mice was as active as normal PLAS in elevating splenic CFC levels. Serum from mice which received irradiation only, had no stimulating effect on splenic hemopoiesis. Medium conditioned by the myelomonocytic leukemia cell line WEHI-3B elevated splenic CFC numbers of similarly to PLAS, but supernatants from long-term marrow cultures or serum of mice treated with latex or phenylhydrazine did not have such an effect. Despite its marked effect on splenic numbers of nucleated erythroid cell precursors, PLAS did not contain elevated levels of erythropoietin.

Animals↗
PubMed1980

The effects of acute thrombocytopenia on megakaryocyte-CFC and granulocyte-macrophage-CFC in mice: studies of bone marrow and spleen.

The effects of acute thrombocytopenia, produced by platelet antiserum (PAS), on both megakaryocyte colony-forming cells (Meg-CFC) and granulocyte-macrophage colony-forming cells (GM-CFC) were studied. During the 1-hr to 14-day period following acute thrombocytopenia (platelet counts < 5% of normal), bone marrow and splenic cells of C57BL/6J mice were obtained and cultured for 7 days in 0.3% agar. Numbers of GM and Meg colonies were determined. At no times were alterations in frequency of GM-CFC and Meg-CFC detected in femoral bone marrow. In contrast, GM-CFC in spleen were increased from 3 to 7 days after PAS and from 4 to 7 days after normal serum (NS). Increase in Meg-CFC in the spleen occurred from 3 to 5 days after PAS with a lesser, not significant increase after NS. Alterations in white blood cells and hematocrit values were not detected. Similar responses were observed in germ-free mice and after rechallenge of animals that had received PAS or NS 14 days previously. The delayed increase in Meg-CFC indicates that they are unlikely to be responsible for the altered megakaryopoiesis previously reported in bone marrow after acute thrombocytopenia and was not due to inhibition by PAS. The increase in GM-CFC may reflect stimulation of the reticuloendothelial system by heterologous proteins.

Acute Disease↗
PubMed1980

Lectin receptors on human blood and bone marrow cells and their use in cell separation.

A series of fluorescein-conjugated lectins (Sophora japonica agglutinin, Helix pomatia agglutinin, peanut agglutinin, the erythroagglutinin from Phaseolus vulgaris, pokeweed mitogen, wheat germ agglutinin and the fucose-binding lectin from Lotus tetragonolobus) were analyzed for their binding to human peripheral blood cells with a fluorescence-activated cell sorter. Most of the lectins showed increasing cell binding in the order erythrocytes less than lymphocytes less than monocytes less than neutrophils and the degree of fluorescence was not related to the major blood groups. The fucose-binding lectin (FBP) was the main exception in that it appeared to bind only to blood neutrophils. From an analysis of the binding of this lectin to human bone marrow cells it was found that the degree of binding within the granulocytic series increased with progressive differentiation. Marrow monocytes and nucleated erythroid cells bound to FBP in contrast to monocytes and non-nucleated red cells in the peripheral blood which showed negligible binding. Lymphocytes both in the marrow and blood displayed negligible binding of FBP. These properties allowed an enrichment of haemopoietic progenitor (colony-forming) cells from human marrow cell suspensions and a depletion of colony-inhibiting cells, when these were present, by selection of cells with the appropriate fluorescence intensity.

Blood Cells↗
PubMed1979

Clonal analysis of the action of GM-CSF on the proliferation and differentiation of myelomonocytic leukemic cells.

With 214 subclones of the BALB/c myelomonocytic leukemia WEHI-3B, the granulocyte-macrophage colony-stimulating factor (GM-CSF) in impure or purified form, consistently increased the proportion of colonies exhibiting partial or complete differentiation in agar cultures. GM-CSF also increased colony size and content of daughter colony-forming cells. Serial recloning of WEHI-3B colonies in the presence of GM-CSF showed that when colonies differentiated completely, self-replication of the colony-forming cell was suppressed (clonal extinction). However, WEHI-3B cells exhibited clonal instability and even in the continuous presence of GM-CSF many colony-forming cells still generated cells able to form undifferentiated colonies. It appears unlikely that GM-CSF can completely suppress the progressive proliferation of a myeloid leukemic population of the WEHI-3B type.

Animals↗
PubMed1979

Similar molecular properties of granulocyte-macrophage colony-stimulating factors produced by different mouse organs in vitro and in vivo.

Granulocyte-macrophage colony-stimulating factor (GM-CSF) was partially purified from post-endotoxin serum and conditioned media produced by organs from both normal and endotoxin-injected C57BL mice. The organs used to condition medium were heart, thigh muscle, salivary gland, thymus, spleen, kidney, brain, and femur shaft. The charge properties, molecular weights, and concanavalin A binding profiles of these GM-CSFs were analyzed and compared to purified mouse lung GM-CSF. All the GM-CSFs examined were shown to be gycoproteins since a proportion of the activity (80 to 100%) bound to concanavalin A-Sepharose. The organ-conditioned medium GM-CSFs were purified (3- to 13-fold) by absorption to calcium phosphate gel and chromatography on DEAE-Sepharose (further 2- to 10-fold). Analysis of the DEAE-Sepharose elution profiles indicated that there were two major charge species of GM-CSF eluting at conductivities of 10 and 14 mmho. These partially purified GM-CSFs showed considerable differences in their apparent molecular weights on Sephacryl S-200 (37,000 to 200,000). However, these differences could be eliminated by treating the GM-CSFs with neuraminidase and performing molecular sizing experiments under dissociating conditions (Sepharose CL-6B, 6 M guanidine hydrochloride). Although some of the GM-CSFs showed anomalously high molecular weights (40,000) on gel filtration columns, even under dissociating conditions, this appeared to be due to properties of the sialic acid residues. After neuraminidase treatment all of the conditioned medium GM-CSFs eluted from DEAE-Sepharose as a single peak of biological activity at a conductivity of 10 mmho and from gel filtration columns in the presence of 6 M guanidine hydrochloride as a single molecular weight species of approximately 23,000. GM-CSF from post-endotoxin serum (produced in vivo) eluted from the gel filtration column with an apparent molecular weight of 39,000, but analysis using polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate indicated that this GM-CSF also had an apparent molecular weight of 23,000.

Animals↗
PubMed1979

Regulation of hemopoiesis.

The development of in vitro cloning systems for populations of hemopoietic and lymphoid cells has enabled a family of specific regulatory macromolecules to be detected and characterized. These macromolecules control proliferation and differentiation in hemopoietic cell populations. Studies have demonstrated the complexity of the spectrum of molecules which are involved in the regulation of each group of hemopoietic cells. Heterogeneous subpopulations of molecules exist for each regulatory function and more than one molecular form exists for the various regulator molecules. While the cellular origin of many of these regulators has not been clearly demonstrated, it is apparent that hemopoietic populations themselves can be significant sources of both stimulatory and inhibitory regulators. In particular, there are now clear examples of regulatory interactions which occur in both directions between hemopoietic and lymphoid populations. While in no case a complete analysis of the control systems has been achieved, maintenance of homeostasis in hemopoietic systems is now better understood than for most other cell populations in the body. Further, with existing techniques, and in particular, the exploitation of the full potential of the semi-solid cloning systems, it is realistic to expect that a complete analysis of hemopoietic regulation can now be accomplished.

Cell Communication↗
PubMed1979

Colony formation in agar by multipotential hemopoietic cells.

Agar cultures of CBA fetal liver, peripheral blood, yolk sac and adult marrow cells were stimulated by pokeweed mitogen-stimulated spleen conditioned medium. Two to ten percent of the colonies developing were mixed colonies, documented by light or electron microscopy to contain erythroid, neutrophil, macrophage, eosinophil and megakaryocytic cells. No lymphoid cells were detected. Mean size for 7-day mixed colonies was 1,800-7,300 cells. When 7-day mixed colonies were recloned in agar, low levels of colony-forming cells were detected in 10% of the colonies but most daughter colonies formed were small neutrophil and/or macrophage colonies. Injection of pooled 7-day mixed colony cells to irradiated CBA mice produced low numbers of spleen colonies, mainly erythroid in composition. Karyotypic analysis using the T6T6 marker chromosome showed that some of these colonies were of donor origin. With an assumed f factor of 0.2, the mean content of spleen colony-forming cells per 7-day mixed colony was calculated to vary from 0.09 to 0.76 according to the type of mixed colony assayed. The fetal and adult multipotential hemopoietic cells forming mixed colonies in agar may be hemopoietic stem cells perhaps of a special or fetal type.

Animals↗
PubMed1979

Interactions between purified GM-CSF, purified erythropoietin and spleen conditioned medium on hemopoietic colony formation in vitro.

Preincubation of C57BL adult marrow cells or CBA fetal liver cells with a 250-fold excess concentration of purified GM-CSF failed to reduce the frequency of cells forming eosinophil, megakaryocyte or erythroid colonies in subsequent agar cultures. When excess concentrations of purified GM-CSF were added to agar cultures stimulated by pokeweed mitogen-stimulated spleen conditioned medium (SCM), no reduction was observed in the frequency of eosinophil, megakaryocyte or erythroid colonies. Addition of 4 units of purified erythropoietin (EPO) to cultures of fetal liver or adult marrow cells stimulated by SCM increased the number of erythroid colonies but did not reduce the number of non-erythroid colonies or the non-erythroid content of mixed erythroid colonies. Although neither GM-CSF nor EPO alone was able to stimulate erythroid colony formation in agar cultures of fetal liver cells, small numbers of large erythroid colonies were stimulated to develop in cultures containing both purified regulators. Purified GM-CSF was also able to support the survival in vitro of a small proportion of erythroid colony-forming cells in fetal liver populations cultured initially in the absence of SCM and the survival of some eosinophil and megakaryocyte colony-forming cells in similar cultures of adult marrow cells. The results do not support the hypothesis that GM-CSF and EPO compete for a common pool of uncommitted progenitor cells. On the contrary, the data indicate that GM-CSF und EPO are able to collaborate in stimulating the proliferation of some erythropoietic cells. Furthermore, purified GM-CSF appears to be able to support temporarily the survival and/or initial proliferation of at least some cells forming erythroid, eosinophil and megakaryocyte colonies, even though GM-CSF is unable to stimulate the formation of colonies of these types.

Animals↗
PubMed1979

Isolation and surface labeling of murine polymorphonuclear neutrophils.

Methods for the induction of an exudate of polymorphonuclear neutrophilic leukocytes (PMN) in the peritoneal cavity of C57BL, BALB/c, SJL and CBA mice were analysed. Peritoneal exudates in male mice were highly enriched for PMN (80-90%) three hours after a single injection of calcium caseinate whereas eosinophils comprised less than 1% of the exudate population. Female mice were a less satisfactory source of PMN because the proportion of eosinophilis in the exudate was variable. Purification of PMN from peritoneal exudate cells was performed on the basis of light scattering using a Becton-Dickinson cell sorter or by density gradient centrifugation with graded polyvinylpyrrolidone-coated silica particles (Percoll). Both techniques yielded approximately 97% pure PMN preparations. Electrophoretic analysis of the PMN proteins revealed an abundance of lactoferrin and actin, but several other proteins were also present in high concentrations. Proteolytic degradation of several high molecular weight proteins (greater than 90,000) was prevented by the addition of phenylmethylsulphonyl fluoride (PMSF) and ethylene diamine tetracetic acid (EDTA). Surface iodination, using diphenyl, tetrachloroglycouril (IODO-DEN), indicated that there were six tyrosine-containing proteins present on the external cell membrane. The apparent molecular weights of these surface proteins ranged from 185,000 to 90,000 and the major 125I-labeled protein had an apparent molecular weight of 90,000. Neither actin nor lactoferrin was labeled with 125I unless cell viability was lost during the iodination procedure. Standard conditions for labeling the cell surface only, required low iodide and IODO-GEN concentrations. Biosynthetic labeling of PMN using S-methionine increased the sensitivity of detection for most of the proteins, but some of the granule storage proteins (such as lactoferrin) were not effectively labeled within three hours.

Animals↗