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S C Clark

Publications and source records attributed to S C Clark.

At least 127 records · Page 7Linked to original sources
PubMed1989

The murine Il-6 gene maps to the proximal region of chromosome 5.

Murine Il-6 cDNAs were isolated by cross-hybridization with a human IL-6 cDNA from an IL-1 activated bone marrow stromal cell line (W20). Mouse-hamster somatic cell hybrids were utilized to localize murine Il-6 to chromosome 5. Genetic mapping with respect to En-2, AlbH, and Gus in backcross progeny from an interspecific mating between C57BL/6J and Mus spretus positioned Il-6 3 cM distal to En-2. The syntenic relationships of Il-6 and En-2 in mouse and man, as well as the potential role of IL-6 in tumorigenesis, are discussed.

Amino Acid Sequence↗
PubMed1989

Human interleukin-6 supports granulocytic differentiation of hematopoietic progenitor cells and acts synergistically with GM-CSF.

Recombinant human (rh) interleukin-6 (IL-6), in a dose range of 1 to 10 U/mL, was able to induce a low number of neutrophilic-granulocytic colonies in a CFU-GM clonogenic assay, using T cells and adherent cells, depleted low density marrow cells. A synergistic increase in the number of granulocytic colonies was observed when rhGM-CSF at suboptimal doses and IL-6 at effective doses were both present in the assay; the increase was only additive when either rhIL-1 alpha or rhIL-3 was used together with IL-6. To determine whether the increase in colony number reflects the interactions of these factors on the same hematopoietic progenitor target cells or, instead, represents activation of accessory cells, we analyzed the effect of IL-6 on the proliferation and differentiation of three growth factor-dependent leukemic cell lines that respond with continuous proliferation to the presence of GM-CSF and IL-3 in culture. One of the three cell lines (AML-193) showed limited proliferation in the presence of IL-6 followed by terminal differentiation after 14 days into basophilic-granulocytic-like cells. A synergistic proliferative response was observed on the same cells treated with both GM-CSF and IL-6. These data support the hypothesis that IL-6 may have a direct effect on myeloid hematopoietic progenitor cells, and that GM-CSF interacts synergistically with IL-6 by acting on the same target cells.

Biological Factors↗
PubMed1989

Production of hematopoietic colony-stimulating factors by human natural killer cells.

We have analyzed the ability of highly purified preparations of human NK cells to produce CSF. NK cells, purified by negative selection from 10-d cultures of PBMC incubated with irradiated B-lymphoblastoid cell lines, were stimulated with rIL-2, FcR(CD16) ligands (particulate immune complexes or anti-CD16 antibodies bound to Sepharose), a combination of CD16 ligands and rIL-2, or the phorbol diester phorbol dibutyrate (PDBu) together with the Ca2+ ionophore A23187. Both rIL-2 and CD16 ligands induce accumulation of GM-CSF mRNA in NK cells and the combined effect of the two stimuli is synergistic. Maximal accumulation of GM-CSF mRNA is observed after PDBu/A23187 stimulation. The participation of contaminant T cells in the observed expression of the GM-CSF gene is excluded because CD16 ligands do not stimulate T cells and CD3 ligands, powerful stimulators of T cells, are inactive on NK cells. Accumulation of CSF-1 mRNA is observed only in NK cells stimulated with both CD16 ligands and rIL-2, whereas accumulation of IL-3 mRNA is observed only in NK cells stimulated with PDBu/A23187. Transcripts of the G-CSF, IL-1 alpha, and IL-1 beta genes were never detected in NK cells in these experiments. The kinetics of accumulation of GM-CSF and CSF-1 mRNA in NK cells stimulated with CD16 ligands and rIL-2 peaked at 2-4 h and was slower than that of TNF and IFN-gamma mRNA, which peak at 1 h. GM-CSF was precipitated from the supernatant fluids of NK cells stimulated with PDBu/A23187 and its biological activity was demonstrated by the ability of the supernatants to sustain proliferation of the TALL-101 cell line or CML blasts. Biological activity of IL-3 and CSF-1 was demonstrable in supernatant fluids of NK cells stimulated with PDBu/A23187 and CD16 ligands/rIL-2, respectively.

Antigens, Differentiation↗
PubMed1989

Human B cell proliferation in response to IL-4 is associated with enhanced production of B cell-derived growth factors.

To investigate the capacity of human IL-4 to function as a B cell growth factor (BCGF), we studied its ability to promote proliferation of a selected B cell line. We show that the cell line, designated A4, proliferated in response to IL-4 in a dose-dependent manner. The A4 cells also proliferated in response to their own B cell derived growth factor (B. BCGF), suggesting autocrine-mediated growth. The ability of IL-4 to induce proliferation of the A4 cell line was dependent on the level of autocrine growth. At low cell density, IL-4 induced marked dose-dependent proliferation. However, as A4 cell density increased, the ability of IL-4 to induce proliferation was diminished. The possibility that IL-4 may be mediating the autocrine growth of A4 cells was ruled out, because A4 cell-derived BCGF failed to induce CD23/low affinity receptors for the Fc region of IgE on activated tonsillar B cells and anti-IL-4 antibody did not block B. BCGF activity. We found that IL-4 stimulation of A4 cells and activated tonsillar B cells is associated with enhanced production of B. BCGF. These data indicate that human IL-4 has the capacity to promote proliferation of the B cell line A4, and that the ability of IL-4 to function as BCGF is associated with enhanced autocrine growth of activated B cells.

Adjuvants, Immunologic↗
PubMed1989

Recombinant interleukin (IL) 2-induced human B cell differentiation is mediated by autocrine IL6.

The molecular mechanism of the interleukin (IL) 2-induced differentiation of human B cells has been investigated. The experimental results show that Staphylococcus aureus Cowan strain I (SAC) activation alone induces IL6 secretion from B cells. When B cells were activated by SAC, there was an increased transcription of the IL6 mRNA. It reached the peak level by 6 h and rapidly decreased to an undetectable level within 24 h. The IL6 concentration in the culture supernatants reached the peak at 24-48 h and decreased slightly in the following culture periods. Since IL 2 alone could induce IgG secretion, whether exogenous IL6 was added or not, and IL2 did not increase autocrine IL6 synthesis, it appears that IL2 induces the IL6 responsiveness of SAC-activated B cells to differentiate in the later stage of the culture. The addition of polyclonal anti-IL6 antibody inhibited IgG secretion. The antibody still efficiently blocked IgG secretion up to day 5, indicating an important role of autocrine IL6 in the IL2-driven B cell differentiation. However, the saturation dose of anti-IL6 antibody inhibited 50%-70% of IgG secretion, suggesting that IL2-induced B cell differentiation appears to be mediated by other factors besides IL6.

B-Lymphocytes↗
PubMed1989

Biological and molecular evidence for the production of IL-6 by human natural killer cells in culture.

As several of the biological functions of NK cells are similar to the biological effects of IL-6, we tested for the production of this cytokine by cultured NK cells. Conditioned medium from the NK cells supported the proliferation of an IL-6 dependent human leukemic plasma cell line in dose-dependent fashion and this response was abolished by a neutralizing anti-IL-6 serum. Analysis of the mRNA from the NK cell cultures by RNA blotting demonstrated a specific transcript for IL-6 providing further confirmation that these cells elaborate this factor in culture.

Blotting, Northern↗
PubMed1989

Survival of hemopoietic progenitors in the G0 period of the cell cycle does not require early hemopoietic regulators.

Although it is generally held that hemopoietic stem cells in steady-state marrow are dormant in the cell cycle, the direct proof for this concept has been lacking. In the present study, we have documented the development of human multipotential blast cell colonies from single cells by daily observation of the growth of candidate progenitors. The results clearly demonstrated that early hemopoietic progenitors may remain as single cells for more than 2 weeks of incubation. Once the progenitors began proliferation, the subsequent growth was characterized by steady cell doubling. Next, we tested the survival of blast cell colony progenitors in the presence of neutralizing antibodies prepared against early acting hemopoietic factors including interleukin (IL) 1 alpha, IL-1 beta, IL-3, IL-6, and granulocyte colony-stimulating factor. Cultures were initiated with individual antibodies, and, on day 14, IL-3 and the corresponding growth factor in concentrations that neutralize the antibodies were added. On days 18-27 of culture, blast cell colonies containing 25 or more cells were identified and replated for analysis of their ability to form secondary colonies. The cumulative frequency of the blast cell colonies in cultures containing antibody did not differ significantly from that of the control group containing rabbit IgG. A combination of anti-IL-1 alpha, anti-IL-1 beta, anti-IL-6, and anti-granulocyte colony-stimulating factor did not affect the survival of dormant blast cell colony-forming cells. These results indicate that survival of hemopoietic stem cells in the G0 period of the cell cycle is independent of early hemopoietic regulators.

Adult↗
PubMed1989

Molecular cloning of a cDNA encoding a human macrophage migration inhibitory factor.

A cDNA encoding a human macrophage migration inhibitory factor (MIF) was isolated, through functional expression cloning in COS-1 cells, from a cDNA library prepared from a lectin-stimulated T-cell hybridoma, T-CEMB. The 115-amino acid polypeptide encoded by the MIF cDNA (p7-1) was effectively released from the transfected COS-1 cells and yielded readily detectable MIF activity in the culture supernatant despite the apparent lack of a classical protein secretory sequence. Insertional mutational analysis and elution of MIF activity from polyacrylamide gel slices demonstrated that the Mr 12,000 protein with MIF activity released by the COS-1 cells is encoded by p7-1. The p7-1 cDNA hybridized with a 700-base mRNA expressed by Con-A-stimulated lymphocytes but not unstimulated lymphocytes. The availability of the MIF cDNA clone and recombinant MIF will facilitate the analysis of the role of this lymphokine in cell-mediated immunity, immunoregulation, and inflammation.

Amino Acid Sequence↗
PubMed1989

Identification through chemical cross-linking of distinct granulocyte-macrophage colony-stimulating factor and interleukin-3 receptors on myeloid leukemic cells, KG-1.

Granulocyte/macrophage colony-stimulating factor (GM-CSF) and interleukin-3 (IL-3) each bind specifically to a small number of high-affinity receptors present on the surface of the cells of the acute myelogenous leukemia line, KG-1. Through chemical cross-linking of IL-3 and GM-CSF to KG-1 cells, we identified distinct binding proteins for each of these cytokines with approximate molecular masses of 69 and 93 Kd, respectively. Although these two binding proteins are distinct, GM-CSF and IL-3 compete with each other for binding to KG-1 cells. Other cell lines, which express receptors for either factor but not for both do not display this cross-competition for binding with IL-3 and GM-CSF. These findings imply that distinct IL-3 and GM-CSF binding proteins are expressed on the cell surface and that an association exists between these proteins on KG-1 cells.

Binding, Competitive↗
PubMed1989

Expression of human interleukin-3 (multi-CSF) is restricted to human lymphocytes and T-cell tumor lines.

While the cellular sources for granulocyte-macrophage colony-stimulating factor (GM-CSF) are known to be widely distributed among several cell types, interleukin-3 (IL-3) gene expression has been demonstrated in only certain T-cell clones and in blood mononuclear cells stimulated with phytohemagglutinin (PHA) and phorbol-myristate-acetate (PMA). To determine which blood cells were responsible for this expression, we fractionated PHA/PMA-stimulated mononuclear cells and identified T lymphocytes as the source of IL-3 mRNA. Low-level IL-3 expression was detected as well in several stimulated human T-cell lines. Hematopoietic stromal cells such as fibroblasts and endothelial cells could not be induced to express IL-3 mRNA. The kinetics of IL-3 mRNA induction in mononuclear cells and lymphocytes stimulated with PHA/PMA or anti-CD3 monoclonal antibody (MoAb) and interleukin-1 (IL-1) were similar to those observed for GM-CSF expression.

Colony-Stimulating Factors↗
PubMed1989

In polycythemia vera human interleukin 3 and granulocyte-macrophage colony-stimulating factor enhance erythroid colony growth in the absence of erythropoietin.

To further define the growth factors required for the in vitro proliferation of erythroid progenitors in polycythemia vera (PV), we have compared the ability of interleukin 3 (IL-3) and granulocyte-macrophage colony-stimulating factor (GM-CSF) to support the growth of erythropoietin (Epo)-dependent and -independent erythroid colony formation. By using nonadherent mononuclear cells from peripheral blood, Epo-dependent colony formation was enhanced by IL-3 and GM-CSF in PV patients. Comparable results were obtained with normal erythroid progenitors. Augmenting effects of IL-3 and GM-CSF were observed on spontaneous erythroid colony formation, i.e., erythroid colony formation in the absence of exogenous supplied Epo. This was not due to a small amount of Epo in the culture media because an anti-Epo antibody did not prevent endogenous colony formation, nor did it prevent the enhancing effects of IL-3. Finally it was observed that in contrast to IL-3, monocyte depletion was required for the enhancing effects of GM-CSF on erythroid colony formation. These results provide evidence that endogenous colony formation in PV is independent of Epo but can be augmented by IL-3 or GM-CSF.

Cells, Cultured↗
PubMed1989

Interleukin-3: molecular biology and biologic activities.

IL-3 is one of the primary factors capable of supporting the growth and development of hematopoietic cells in culture. In comparison with the other hematopoietic growth factors, IL-3 preferentially supports the proliferation of early multilineage progenitors or progenitors at early stages of development within the different lineages. Subsequently, the developing cells lose responsiveness to IL-3 while acquiring dependence on the later acting factors: GM-CSF, G-CSF, M-CSF, or Ep. In addition, IL-3 has been demonstrated to exert biologic effects with other target cell populations. These activities include the potentiation of the IL-2-dependent growth of normal T cells; the potentiation of the IL-2-dependent secretion of IgG by activated B cells; and the potentiation of the activities of eosinophils, basophils, and monocytes. The importance of any of these activities of IL-3 in vivo in either normal or stressed animals remains to be determined. Initial experiments in primates with IL-3 have yielded results consistent with its role as a regulator of early hematopoietic cell development. Although administration of IL-3 alone has relatively little effect on the levels of circulating blood cells, this treatment primes the animals to become hyper-responsive to subsequent administration of the later acting factors GM-CSF and Ep. Thus combinations of factors, at least in some situations, can provide a more potent stimulation of hematopoiesis than provided by the individual molecules, a finding that should greatly expand the utility of the different hematopoietins to more indications in the clinic.

Animals↗
PubMed1988

Leukaemia inhibitory factor is identical to the myeloid growth factor human interleukin for DA cells.

Leukaemia inhibitory factor (LIF) is a cytokine that induces macrophage differentiation of the murine M1 myeloid leukaemia cell line. We have isolated a cDNA clone encoding a novel human haemopoietic growth factor, human interleukin for DA cells (HILDA) that supports the proliferation of the murine interleukin-3-dependent leukaemic cell line, DA-la (refs 3-5). HILDA proved to be identical to LIF. The demonstration that the differentiation factor LIF will also serve as a growth factor for at least one myeloid leukaemic cell line provides further evidence that the distinction between growth-promoting and differentiation-inducing activities are largely determined by the target cell type.

Animals↗
PubMed1988

Interleukin 1 enhances growth factor-dependent proliferation of the clonogenic cells in acute myeloblastic leukemia and of normal human primitive hemopoietic precursors.

IL-1 is released by activated monocytes and is thought to be a key mediator of the host immune response. The availability of the purified and, more recently, recombinant IL-1 has allowed the characterization of other biological properties of this molecule. Thus, IL-1 is thought to have the same properties as hemopoietic 1, a growth factor that has been shown to act on primitive murine hemopoietic cells. Here we report that rIL-1 acts synergistically with granulocyte/macrophage CSF (GM-CSF) or granulocyte CSF in the stimulation of clonogenic cells from many patients with acute myeloblastic leukemia (AML). Although IL-1 by itself has no effect on AML blasts, it can support colony formation under conditions where there is detectable production of endogenous GM-CSF. IL-1 also promotes the growth of multipotential progenitors from normal human bone marrow cells in the presence of GM-CSF. These observations support the hypothesis that in the hemopoietic system, IL-1 has a selective effect on primitive precursors.

Bone Marrow Cells↗
PubMed1988

Amplification of IL-2-driven T cell proliferation by recombinant human IL-3 and granulocyte-macrophage colony-stimulating factor.

Two recombinant human preparations of CSF, namely granulocyte-macrophage-CSF (GM-CSF) and IL-3 (multi-CSF), were tested for their ability to stimulate the growth of human freshly separated and in vitro activated lymphocytes. Both CSF independently induced short term proliferation in unfractionated PBL and lectin-stimulated T cells. Despite the great variability among different donors in the magnitude of lymphocyte response to the two growth factors, IL-3 at suboptimal concentrations (10 U/ml) consistently induced a higher proliferative response than did GM-CSF at suboptimal concentrations (5 ng/ml) in all of the preparations tested. When used in combination with IL-2, GM-CSF and, especially, IL-3 significantly potentiated the proliferative responses induced by IL-2 in both unstimulated and mitogen-activated lymphocytes. Dose-response curves using increasing concentrations of IL-2 and IL-3 and isobologram analysis of these interactions revealed a clear synergy of action between the two growth factors in inducing proliferation of unfractionated PBL, purified T cells, mitogen-activated lymphocytes, and alloantigen-stimulated T cells. In addition to enhancing the short term responsiveness to IL-2, GM-CSF and, especially, IL-3 drastically potentiated the long term growth of non-activated human lymphocytes and of lectin- or Ag-activated T cells in the presence of IL-2. Immunofluorescence analysis indicated a higher expression of activation Ag (anti-Tac receptors and HLA class II Ag) in cultures incubated in the presence of IL-3 either alone or in conjunction with IL-2. The overall data indicate that human GM-CSF and IL-3 can support the growth of cells within the lymphoid lineage and exert potent amplifying effects on IL-2-induced T cell growth in vitro.

Adjuvants, Immunologic↗
PubMed1988

Stimulation of murine hemopoietic colony formation by human IL-6.

A novel hemopoietic CSF has been identified in the medium conditioned by lectin-stimulated human T cells. The cDNA clone encoding this factor, isolated by functional expression cloning in monkey cos-1 cells, proved to be identical with the cDNA encoding the cytokine B cell stimulatory factor-2/IFN-beta 2, a factor now known as IL-6. In the murine system, IL-6 indirectly supports the formation of several different types of hemopoietic colonies, including those derived from early blast cells, and directly supports the proliferation of granulocyte/macrophage progenitors. These results expand the range of known target cells of IL-6 to include hemopoietic progenitors in addition to B cells, T cells, and fibroblasts and provide further evidence that this cytokine plays an important role within a network of interacting cytokines that regulates many different biologic responses.

Animals↗