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Biomedical subjects

D Metcalf

Publications and source records attributed to D Metcalf.

At least 127 records · Page 7Linked to original sources
PubMed1994

Leukaemia inhibitory factor and interleukin 6 are expressed at very low levels in the normal adult mouse and are induced by inflammation.

We have assessed the limitations of the polymerase chain reaction (PCR) as a semiquantitative technique for assessing very low level gene expression. Using PCR, the in vivo expression patterns of the cytokines Leukaemia Inhibitory Factor (LIF) and Interleukin 6 (IL-6) in the normal adult mouse, have been examined. We show that both LIF and IL-6 mRNA are constitutively expressed, albeit at extremely low levels, in most tissues. While it is unclear whether this low level of expression is of biological significance, it is possible that it reflects a local mode of action of these potent polyfunctional molecules. Lipopolysaccharide, the bacterial cell wall product responsible for endotoxic shock, when administered in vivo, was capable of inducing the expression of both LIF and IL-6 in all of the tissues examined. In addition, LIF and IL-6 expression was induced in lung tissue by in vitro culturing in serum-free media. This induction of LIF and IL-6, by LPS and culturing, may reflect the role of these molecules as mediators of the acute phase response to tissue damage.

Animals↗
PubMed1994

Circulating leukemia inhibitory factor levels correlate with disease severity in meningococcemia.

Circulating concentrations of the proinflammatory cytokine leukemia inhibitory factor (LIF) were prospectively determined by radioreceptor competition assay (sensitivity, 1 ng/mL) in 33 subjects with meningococcemia. LIF was detected in the plasma of 13 subjects and was associated with development of septic shock (P < .01), disseminated intravascular coagulation (P < .05), multiorgan failure (P < .05), and death (P < .01). Plasma LIF concentrations were highest (1-1772 ng/mL) at hospital admission and became undetectable within 36 h, and the peak levels correlated inversely with systolic blood pressure (r, -.70, P < .001), peripheral blood leukocyte count (r, -.58, P < .01), and prodromal interval (r, -.60, P < .001). Plasma LIF concentrations > 400 ng/mL were present only in subjects with fatal fulminant infection. LIF concentrations in plasma collected within 12 h of hospital admission correlated with disease severity in patients with meningococcemia. It is likely that LIF participates in the host response to infection, and it may contribute to the pathogenesis of septic shock.

Adolescent↗
PubMed1994

Receptor insertion into factor-dependent murine cell lines to develop specific bioassays for murine G-CSF and M-CSF and human GM-CSF.

cDNAs encoding the receptors for murine G-CSF, M-CSF or human GM-CSF were inserted into the murine hemopoietic continuous cell lines Ba/F3 or FDC-P1 and sublines selected that were then responsive to proliferative stimulation by these growth factors. When used in microwell assays the Ba/F3 G-CSF receptor-expressing cell line was able to detect 100 pg G-CSF per ml, the Ba/F3 M-CSF receptor-expressing cell line 100-400 pg M-CSF per ml and the FDC-P1 line expressing the alpha- and beta-chains of the human GM-CSF receptor detected 5-10 pg/ml of GM-CSF in test material. These cell lines appear satisfactory for use as selective bioassays for these colony stimulating factors in material potentially containing a mixture of growth factors.

Animals↗
PubMed1994

Complex binding of leukemia inhibitory factor to its membrane-expressed and soluble receptors.

The complex interaction of leukemia inhibitory factor (LIF) with its specific receptor present on the cell surface, in isolated membranes and in solution, has been examined in detail. Several aspects of this complexity have been highlighted, including the presence of high- and low-affinity murine LIF receptors, biphasic dissociation of human LIF from apparently homogeneous high- or low-affinity human LIF receptors, and unusual species cross-reactivity. The unusual species cross-reactivity observed between murine and human LIF has also been exploited to map an important receptor binding epitope on human LIF.

Animals↗
PubMed1994

Granulocyte colony-stimulating factor induces selective elevations of progenitor cells in the peripheral blood of mice.

The absolute numbers and relative frequencies of progenitor cells in six nonerythroid lineages were monitored in the peripheral blood (PB), bone marrow (BM), and spleen of Balb/c mice during 8 days of granulocyte colony-stimulating factor (G-CSF) injections. G-CSF induced a dose-related increase, up to 570-fold, in progenitor cell numbers in the blood and up to 620-fold rise of these cells in the spleen. The relative frequency of megakaryocyte progenitor cells was significantly increased in the blood compared with values in the BM or spleen. Time-dependent variations were also observed in the relative frequencies of three lineages of progenitor cells in the blood (megakaryocyte, granulocyte, and macrophage), but not in the BM or spleen. The consistent differences induced in the relative frequencies of various progenitor cell types between the blood, marrow, and spleen were independent of G-CSF dose. These data suggest that the increase in progenitor cells in the blood induced by G-CSF cannot simply be explained by a nonselective release of progenitor cells from the marrow or spleen.

Animals↗
PubMed1994

Distribution of IL-6 receptors on murine hemopoietic and lymphoid cells and the anomalous action of IL-6 in stimulating granulocytic proliferation.

Autoradiographic analysis of the binding of radiolabeled murine IL-6 to murine cells revealed that blast cells, megakaryocytic and monocytic cells in the marrow expressed receptors but not erythroid, eosinophilic or neutrophilic granulocytic cells. In the thymus, spleen and lymph nodes, most cells expressing receptors were single positive CD4+ and CD8+ T lymphocytes while relatively few B220+ slg+ B lymphocytes expressed receptors. The absence of receptors on granulocytes raised a problem with the observed ability of IL-6 to stimulate granulocyte colony formation in mouse bone marrow cultures. Analysis showed that this colony formation was linear with respect to cultured cell numbers and that IL-6 was able to stimulate granulocyte colony formation by at least some transferred isolated clones or individual blast colony cells recultured in the absence of other cell types. Some of the proliferative effects of IL-6 on unfractionated cells may be indirectly mediated, but the data suggest that IL-6 can have direct effects on granulocyte proliferation despite the absence of detectable IL-6 receptors on granulocytic cells.

Animals↗
PubMed1994

Fms-like tyrosine kinase 3 catalytic domain can transduce a proliferative signal in FDC-P1 cells that is qualitatively similar to the signal delivered by c-Fms.

A full length clone of murine fms-like tyrosine kinase 3 [flt3, also known as fetal liver kinase 2 (flk2)] was constructed from sequences obtained from a brain complementary DNA (cDNA) library and from cDNA prepared from the cell line Tikaut. In the absence of a ligand to study the function of Flt3, a chimeric molecule was constructed comprising the extracellular domain of murine c-Fms and the transmembrane and cytoplasmic domains of Flt3. A plasmid encoding the chimeric receptor was cotransfected along with a plasmid conferring neomycin resistance into FDC-P1 cells that do not normally express c-fms or flt3 and require granulocyte-macrophage colony-stimulating factor (GM-CSF) or interleukin 3 for growth. Two types of clones were obtained following selection in GM-CSF and G418. Two of seven clones had the capacity for M-CSF-dependent colony formation in semisolid medium, indicating that the cytoplasmic domain of Flt3 can transduce a proliferative signal. From the remaining clones, M-CSF-dependent clonogenic cells could be selected by prior bulk liquid culture in M-CSF. It has been shown previously that the GM-CSF-dependent proliferative capacity is strongly inhibited by M-CSF in FDC-P1 cells engineered to express full length c-fms. This phenomenon was also observed with FD/fms-flt3 cells that were clonogenic in M-CSF. Stimulation of FD/fms or FD/fms-flt3 cells in liquid culture by M-CSF caused differentiation of a small proportion of cells along the myelomonocytic pathway which was enhanced by the combination of M-CSF and GM-CSF.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1993

Neutralizing and nonneutralizing monoclonal antibodies to the human granulocyte-macrophage colony-stimulating factor receptor alpha-chain.

A panel of monoclonal antibodies was raised against the low-affinity human granulocyte-macrophage colony-stimulating factor (hGM-CSF) receptor alpha-chain expressed as recombinant protein on murine FDC-P1 cells. All the selected antibodies were of the IgG2A isotype and bound to protein A. They each recognized both native and recombinant receptors by indirect surface immunofluorescence and by immunoprecipitation. Several of the antibodies also recognized presumably denatured receptors as detected by immunoblotting of sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Three different epitopes on the extracellular domain of the GM-CSF receptor alpha-chain were defined by these antibodies, and two of the epitopes did not appear to be involved in binding hGM-CSF or in interactions with the beta-chain of the GM-CSF receptor that are required for high-affinity binding of GM-CSF. On the other hand, the epitope recognized by antibody 2B7-17-A appeared to be critically involved in the binding of GM-CSF because this antibody completely abrogated both high- and low-affinity binding of GM-CSF to native and recombinant receptors. Antibody 2B7-17-A had a relatively high affinity for the GM-CSF receptor alpha-chain (kd = 3 nmol/L) and slow dissociation kinetics (kd = 0.002 min-1). These properties made the 2B7-17-A antibody a potent inhibitor of hGM-CSF biologic action in several different bioassays, with a half-maximal inhibitory dose of about 6 nmol/L (1 microgram/mL). This antibody could prove useful in alleviating any pathologic states mediated by excess GM-CSF levels and in defining the domains of the GM-CSF receptor required for ligand binding.

Animals↗
PubMed1993

The SCL gene product is regulated by and differentially regulates cytokine responses during myeloid leukemic cell differentiation.

Differentiation induction in murine M1 leukemia cells by interleukin 6 (IL-6), leukemia inhibitory factor (LIF), and oncostatin M (OSM) is postulated to occur via a common receptor chain, gp130. In this study, growth factor-induced differentiation of M1 cells was accompanied by a late and persistent decrease in levels of mRNA and protein for a helix-loop-helix transcription factor, the SCL gene product. To evaluate whether reduced SCL expression was instrumental in monocyte differentiation, an SCL cDNA expression vector was introduced into M1 cells to obtain cell lines in which overexpression of SCL mRNA and protein was enforced. This resulted in a reduction in cells differentiating in response to LIF and OSM but not in response to IL-6. Scatchard analysis indicated that both parental and SCL-transfected cell lines exhibited similar receptor numbers and receptor affinities for LIF, OSM, and IL-6, suggesting that the differential responsiveness was not due to selective receptor down-modulation. Thus, these data implicate SCL in monocytic differentiation and provide evidence for differential receptor signaling pathways despite utilization of a common gp130 subunit by all three receptors.

Animals↗
PubMed1993

Recombinant human leukemia inhibitory factor induces acute phase proteins and raises the blood platelet counts in nonhuman primates.

Recombinant human leukemia inhibitory factor (rhLIF) produced by Escherichia coli was administered subcutaneously (sc) to rhesus monkeys at doses of 2, 10, and 50 micrograms/kg body weight/d for 14 days to assess its biologic activities in vivo. Serum levels of positively regulated acute phase proteins (APP) (C-reactive protein, alpha 1-antitrypsin, haptoglobin, and ceruloplasmin) were increased, whereas the negatively regulated APP prealbumin decreased in response to rhLIF treatment. During the second week of treatment, blood platelet counts began to increase, resulting in a maximum of a twofold increase above normal levels a week after termination of the rhLIF treatment. No changes were seen in total and differential white blood cell counts in blood progenitor levels and in red blood cell numbers. The low- and medium-dose rhLIF treatments were tolerated without significant side effects. The animals treated with the high dose showed a reduction in body weight of approximately 10%. In conclusion, rhLIF was shown to stimulate APP and to increase the number of platelets in circulation in nonhuman primates.

Acute-Phase Proteins↗
PubMed1993

Increased levels of leukemia inhibitory factor in synovial fluid from patients with rheumatoid arthritis and other inflammatory arthritides.

OBJECTIVE: To examine synovial fluid (SF) from patients with arthritis, for the presence of the cytokine leukemia inhibitory factor (LIF). METHODS: SF from 152 subjects was examined for LIF, using a radioreceptor competition assay. RESULTS: LIF was present at concentrations of 1-43 ng/ml in the SF of 23% of patients with rheumatoid arthritis (RA) or other inflammatory or infectious arthritides but in only 1 of 29 patients with osteoarthritis (P < 0.01). In the RA patients, the SF LIF concentration correlated significantly with the peripheral blood white blood cell count (WBC) (P < 0.05) and the SF WBC count (P < 0.01), but not with other clinical or radiologic parameters of disease activity or progression. CONCLUSION: LIF is implicated as a potential mediator of the local or systemic inflammatory response or the joint destruction seen in inflammatory arthritis.

Arthritis↗
PubMed1993

Inter-species chimeras of leukaemia inhibitory factor define a major human receptor-binding determinant.

Human leukaemia inhibitory factor (hLIF) binds to both human and mouse LIF receptors (LIF-R), while mouse LIF (mLIF) binds only to mouse LIF-R. Moreover, hLIF binds with higher affinity to the mLIF-R than does mLIF. In order to define the regions of the hLIF molecule responsible for species-specific interaction with the hLIF-R and for the unusual high-affinity binding to the mLIF-R, a series of 15 mouse/human LIF hybrids has been generated. Perhaps surprisingly, both of these properties mapped to the same region of the hLIF molecule. The predominant contribution was from residues in the loop linking the third and fourth helices, with lesser contributions from residues in the third helix and the loop connecting the second and third helices in the predicted three-dimensional structure. Since all chimeras retained full biological activity and receptor-binding activity on mouse cells, and there was little variation in the specific biological activity of the purified proteins, it can be concluded that the overall secondary and tertiary structures of each chimera were intact. This observation also implied that the primary binding sites on mLIF and hLIF for the mLIF-R were unaltered by inter-species domain swapping. Consequently, the site on the hLIF molecule that confers species-specific binding to the hLIF-R and higher affinity binding to the mLIF-R, must constitute an additional interaction site to that used by both mLIF and hLIF to bind to the mLIF-R. These studies define a maximum of 15 amino acid differences between hLIF and mLIF that are responsible for the different properties of these proteins.

Amino Acid Sequence↗
PubMed1993

The cellular basis for enhancement interactions between stem cell factor and the colony stimulating factors.

In cultures of normal mouse fetal liver cells, combination of stem cell factor (SCF) with erythropoietin enhanced erythroid colony formation and, in bone marrow cultures, combination of SCF with interleukin 6 (IL-6) enhanced megakaryocyte colony formation. Combination in marrow cultures of SCF and granulocyte colony stimulating factor (G-CSF) increased cell numbers more than tenfold in developing granulocytic and blast cell colonies. Combination with G-CSF enhanced progenitor cell numbers five-fold in developing blast colonies, but a majority of these were committed macrophage progenitor cells not able to proliferate further with SCF plus G-CSF. Similarly, many of the granulocyte progenitor cells could not proliferate further with this combination of stimuli. This process of amplified but abortive formation of progenitor cells was also noted with use of the combination of SCF plus IL-6 but not with combination of SCF with granulocyte-macrophage CSF (GM-CSF) or Multi-CSF (IL-3). Analysis indicated that where colony size was amplified by combination of a factor with SCF, quantitatively the more important process was amplification of progenitor cell formation rather than amplification of the number of progeny formed by individual committed progenitor cells.

Animals↗
PubMed1993

The effects of leukaemia inhibitory factor on platelet function.

Leukaemia inhibitory factor (LIF) is able to promote megakaryocytopoiesis in vitro and elevate platelet counts in vivo, and is a potential new therapeutic agent for the treatment of thrombocytopenia. To determine whether platelets released under conditions of LIF-stimulated megakaryocytopoiesis have intact function, we compared aggregation responses of platelets from mice with constitutively elevated LIF levels (FD/LIF mice) and mice injected with recombinant murine LIF (rmLIF mice) with their respective control mice. We report that ex vivo platelet aggregability and thromboxane B2 release were intact in the LIF-treated mice, and were significantly enhanced in some situations. LIF-treated mice also had significantly increased platelet counts (FD/LIF mice: 1302 +/- 173 x 10(9)/l compared to 1012 +/- 99 x 10(9)/l for FD mice; rmLIF mice: 1460 +/- 193 x 10(9)/l compared to 985 +/- 67 x 10(9)/l for FCS/NS mice), increased platelet volumes and elevated plasma fibrinogen and calcium levels. The platelet hyperreactivity seen in the LIF-treated mice is likely to reflect the larger platelet volumes and/or the effect of plasma components such as fibrinogen, elevated levels of which were due to the concomitant action of LIF as a stimulant of acute phase protein synthesis.

Animals↗
PubMed1993

The molecular control of proliferation and differentiation in hemopoietic cells.

The control of granulocyte and macrophage formation and function offers many insights into the developmental processes involved in hemopoiesis. Regulation of granulocyte and macrophage populations is mainly controlled by the actions of four glycoprotein regulators--the colony stimulating factors. These factors exhibit some redundancy in their actions but each is capable of regulating multiple events in the production, differentiation commitment, maturation and functional activities of the cells. The design of this regulatory system is such that it can ensure precise cell production under basal conditions but is also able to achieve large variations in cell production in response to emergency situations.

Animals↗
PubMed1993

SCL, the gene implicated in human T-cell leukaemia, is oncogenic in a murine T-lymphocyte cell line.

SCL (TAL-1) is implicated in the generation of human T-cell acute lymphoblastic leukaemia. To directly examine the role of this putative oncogene, an SCL retrovirus was constructed and used to infect a v-ABL transformed T-lymphocyte cell line. Thirteen independent SCL-infected and four control cell lines were established and injected subcutaneously into syngeneic mice. Mice injected with SCL-infected clonal cell lines died significantly more rapidly than control animals. By day 200 46% (40/87) of animals injected with SCL-infected cell lines had died due to disseminated transplantable lymphoid tumours. In contrast only 22% of control mice were dead by day 200 (P < 0.0015). Of possible relevance to the enhanced tumourigenesis, some SCL-infected cell lines displayed increased clonogenicity in agar. Increased cell growth was even more striking when ex-vivo tumour-derived cell lines were studied. Thus, SCL can co-operate with v-ABL to hasten T-cell tumourigenesis. This is the first direct evidence demonstrating that SCL can behave as an oncogene.

Amino Acid Sequence↗