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Biomedical subjects

C A Sieff

Publications and source records attributed to C A Sieff.

At least 37 records · Page 2Linked to original sources
PubMed1994

Expression and function of the human granulocyte-macrophage colony-stimulating factor receptor alpha subunit.

To determine the expression and function of the granulocyte-macrophage colony-stimulating factor (GM-CSF) receptor alpha chain (GMR alpha) during hematopoiesis and on leukemic cells, monoclonal antibodies were raised by immunizing mice with cells expressing high levels of human GMR alpha. A pool of five antibodies isolated from three different mice was used to characterize GMR alpha. This antibody pool (anti-GMR alpha) immunoprecipitated a protein with the expected molecular weight of GMR alpha from COS cells transiently transfected with the GMR alpha gene. In factor-dependent cells, GMR alpha existed as a phosphoprotein. However, its phosphorylation was not stimulated by the presence of GM-CSF. Anti-GMR alpha inhibited the GM-CSF-dependent growth of cell lines and normal bone marrow cells and inhibited the binding of iodinated GM-CSF to its receptor. Cell surface expression of GMR alpha was examined using anti-GMR alpha and flow cytometry. GMR alpha was readily detectable on both blood monocytes and neutrophils. In adherence-depleted normal bone marrow, two separate populations expressed GMR alpha. The most positive cells were predominantly macrophages, whereas the cells that expressed less GMR alpha were largely myelocytes and metamyelocytes. A small population of lin-CD34+ or CD34+CD38- cells also expressed GMR alpha, but they were not capable of significant growth in colony-forming assays. In contrast, the majority of lin-CD34+ and CD34+CD38- cells were GMR alpha-, yet they produced large numbers of myeloid and erythroid colonies in the same assay. Malignant cells from patients with leukemia were also tested for GMR alpha expression. All of the myeloid leukemias and only rare lymphoid leukemias surveyed tested positive for GMR alpha. These results show that anti-GMR alpha is useful for the functional characterization of the GMR alpha and for the detection of myeloid leukemia and that GMR alpha is expressed on certain lineages throughout hematopoietic development; however, progenitors that express the receptor may have a reduced capacity to proliferate in response to hematopoietic growth factors.

Animals↗
PubMed1994

Hematopoietic cell proliferation and differentiation.

Most hematopoietic stem cells are quiescent in the G0 stage of the cell cycle. Certain combinations of hematopoietic growth factors can bind to their cognate receptors on the stem cell surface, shorten the G0 stage of the cell cycle, and stimulate cell division. Hematopoietic growth factors also act on committed progenitors to increase their survival and to amplify maturing populations by stimulating proliferation. Although differentiation is probably determined largely by an intrinsic program, there is evidence that certain receptors expressed on more mature cells can also direct differentiation. This view is still controversial, as is the view that hematopoietic growth factors act merely to prevent apoptosis. Different members of the hematopoietic growth factor family of receptors recruit overlapping sets of tyrosine kinases and share common signal transduction pathways such as Ras. They also directly activate a cytoplasmic class of transcription factors that then translocate to the nucleus. The development of committed hematopoietic progenitor cells into mature effector cells is regulated by "master" transcription factors that recognize motifs in the promoters of many genes that encode lineage-specific proteins.

Animals↗
PubMed1993

Human granulocyte-macrophage colony-stimulating factor receptor signal transduction requires the proximal cytoplasmic domains of the alpha and beta subunits.

Human granulocyte-macrophage colony-stimulating factor (GM-CSF) controls the production, maturation, and function of cells in multiple hematopoietic lineages. These effects are mediated by a cell-surface receptor (GM-R) composed of alpha and beta subunits, each containing 378 and 881 amino acids, respectively. Whereas the alpha subunit exists as several isoforms that bind GM-CSF with low affinity, the beta common subunit (beta c) does not bind GM-CSF itself, but acts as a high-affinity converter for GM-CSF, interleukin-3 (IL-3), and IL-5 receptor alpha subunits. The cytoplasmic region of GM-R alpha consists of a membrane-proximal conserved region shared by the alpha 1 and alpha 2 isoforms and a C-terminal variable region that is divergent between alpha 1 and alpha 2. The cytoplasmic region of beta c contains membrane proximal serine and acidic domains. To investigate the amino acid sequences that influence signal transduction by this receptor complex, we constructed a series of cytoplasmic truncation mutants of the alpha 2 and beta subunits. To study these truncations, we stably transfected the IL-3-dependent murine cell line Ba/F3 with wild-type or mutant cDNAs. We found that the wild-type and mutant alpha subunits conferred similar low-affinity binding sites for human GM-CSF to Ba/F3, and the wild-type or mutant beta subunit converted some of these sites to high-affinity; the cytoplasmic domain of beta was unnecessary for this high-affinity conversion. Proliferation assays showed that the membrane-proximal conserved region of GM-R alpha and the serine-acidic domain of beta c are required for both cell proliferation and ligand-dependent phosphorylation of a 93-kD cytoplasmic protein. We suggest that these regions may represent an important signal transduction motif present in several cytokine receptors.

Amino Acid Sequence↗
PubMed1993

A low-affinity human granulocyte-macrophage colony-stimulating factor/murine erythropoietin hybrid receptor functions in murine cell lines.

To identify domains in hematopoietic growth factor receptors that are important for signal transduction, a hybrid receptor (GMER) was constructed by splicing the DNA of the entire extracellular and transmembrane domains of the human granulocyte-macrophage colony-stimulating factor (GM-CSF) receptor alpha 2 subunit (GMR) to the cytoplasmic domain of the murine erythropoietin receptor (mEpoR). The hybrid receptor was introduced into the interleukin-3 factor-dependent murine hematopoietic cell line Ba/F3. Cells that expressed high receptor numbers were selected by cell sorting using phycoerythrin-labeled human GM-CSF. Immunoprecipitation of GMER from Ba/F3 cells showed a band with an Mr of 105,000 daltons. Human GM-CSF binding to Ba/F3 cells that expressed GMER showed a kd of 3.0 nmol/L and 475 binding sites/cell, while the same cells that expressed GMR had 300 sites/cell and a kd of 3.5 nmol/L. The proliferative response to GM-CSF of Ba/F3 cells that expressed GMER showed 1/2 maximal cell growth (as measured by 3H-thymidine incorporation) at a GM-CSF concentration of 2.5 x 10(-8) mol/L. When cultured in human GM-CSF, Ba/F3-GMER cells expressed cell surface glycophorin. Similar results were obtained with Ba/F3 cells transfected with the mEpoR and cultured in erythropoietin. Expression of GMR plus the human GM-CSF receptor beta chain in the same cell line also resulted in human GM-CSF stimulated proliferation; however, cell surface glycophorin was not detected. These data show that a low-affinity GM-CSF/Epo hybrid receptor can promote GM-CSF-dependent proliferation and can induce the expression of glycophorin, an erythroid-specific protein.

Animals↗
PubMed1992

The production of steel factor mRNA in Diamond-Blackfan anaemia long-term cultures and interactions of steel factor with erythropoietin and interleukin-3.

Diamond-Blackfan anaemia (DBA) is a congenital macrocytic anaemia. To investigate whether DBA is due to hyporesponsiveness to or hypoproduction of Steel factor (SF), we compared the in vitro responsiveness of the BFU-E contained in the Ficoll-Hypaque non-adherent cell fraction of six DBA marrows with that of four normal marrows and one transient erythroblastopenia of childhood (TEC) marrow. In addition, we studied the effect of soluble SF on long-term marrow cultures (LTMC) and analysed the stromal cells from these cultures for SF mRNA transcripts. All the patients showed an erythropoietin dose-related increase of small BFU-E. The number and size of BFU-E was increased with the addition to the epo of IL-3 or SF; IL-3+SF was not synergistic. The addition of soluble SF to LTMC of DBA patients was associated with a small but consistent increase in non-adherent cell production and an increase in the number of progenitors. Messenger RNA from immortalized stromal cell lines of three patients and from primary bone marrow stromal cells of one patient showed the presence of expected SF transcripts by PCR analysis. These results demonstrate that this group of DBA patients responds to SF and produces SF mRNA normally, indicating that SF itself is not involved in DBA pathophysiology. The effects observed suggest that, despite the lack of evidence for a causative role, SF may prove to be effective treatment for such patients.

Adult↗
PubMed1991

A functional isoform of the human granulocyte/macrophage colony-stimulating factor receptor has an unusual cytoplasmic domain.

The granulocyte/macrophage colony-stimulating factor (GM-CSF) receptor (GMR) transduces a signal that results in the proliferation, differentiation, and functional activation of hematopoietic cells. This study sought to determine whether functional isoforms of the receptor exist that may be important in generating this diversity of cellular response. We have isolated a cDNA encoding an isoform of the low-affinity human GMR that is a product of alternative splicing of the GMR gene and results in a predicted 410-amino acid protein with a cytoplasmic domain that is rich in serine residues, a feature of regions critical in signal transduction for other receptors of the hematopoietin receptor superfamily. This receptor bound ligand and was functionally active when introduced into a murine factor-dependent cell line; mRNA transcripts representative of this isoform were coexpressed with those for a previously isolated 400-amino acid isoform of the GMR in normal hematopoietic and leukemic cells. In view of the recent isolation of a cDNA, designated GM-CSF R beta, that confers high-affinity binding of GM-CSF in cotransfection experiments with the low-affinity receptor, we suggest that the previously isolated low-affinity receptor be designated GM-CSF R alpha 1 and the one described in this report be designated GM-CSF R alpha 2.

Amino Acid Sequence↗
PubMed1990

Restoration of phagocyte function by interferon-gamma in X-linked chronic granulomatous disease occurs at the level of a progenitor cell.

Phagocytes from X-linked chronic granulomatous disease (X-CGD) patients are deficient in their ability to generate superoxide because of a defective gene that encodes a heavy chain of cytochrome b, a critical component in the superoxide-generating pathway. Previously we have shown that a single in vivo treatment of selected X-CGD patients with interferon-gamma (INF-gamma) resulted 14 days later in near-normal levels of superoxide generation by phagocytes. The effect persisted for 28 days. This prolonged effect suggested that the lymphokine affected progenitor cells. In this study, we examined progenitor-derived colonies from the peripheral blood from this unusual X-CGD kindred. Progenitor-derived colonies examined before treatment were unable to generate superoxide as visualized by lack of nitro blue tetrazolium (NBT) reduction compared with normal controls. By contrast, colonies derived 7 days after a single INF-gamma injection were able to generate superoxide as shown by increased NBT reduction. Colonies harvested 21 days after treatment contained only rare cells capable of NBT reduction. Our results indicate that INF-gamma can reprogram the myeloid progenitor cells to express a partially corrected phenotype. This corrected phenotype is later expressed in daughter cells.

Cell Differentiation↗
PubMed1990

A phase I/II trial of recombinant granulocyte-macrophage colony-stimulating factor for children with aplastic anemia.

Nine pediatric patients (median age, 8 years; range, 0.7 to 19 years), eight with refractory aplastic anemia and one with newly diagnosed aplasia, were enrolled in a phase I/II trial of recombinant human granulocyte-macrophage colony-stimulating factor (rhGM-CSF) administered via continuous intravenous infusion. Doses ranged from 8 to 32 micrograms/kg/d. Six of eight evaluable patients responded with a significant rise in neutrophil count (median fourfold increase; range, 2.5- to 31-fold) during the 28-day induction period. Five patients completed 2 further months of therapy (maintenance) with persistent or improved neutrophil responses. Three patients had bone marrow aspirates suggestive of increased erythropoiesis, although only one patient had improvement in peripheral hematocrit and platelet count. In the five patients completing maintenance, three experienced a rapid return to baseline counts after rhGM-CSF was discontinued, one maintained a neutrophil response for 2 months after drug discontinuation, and one has maintained a trilineage response for greater than 1 year off study. Drug therapy was well tolerated. Toxicity was minimal at doses from 8 to 16 micrograms/kg/d. Fever and rash were more commonly seen at 32 micrograms/kg/d. No patient developed an infection during the course of rhGM-CSF administration. These results demonstrate that rhGM-CSF increases peripheral neutrophil counts in children with refractory and newly diagnosed aplastic anemia and may be able to stimulate a multilineage response in a more limited number. Randomized, prospective trials are necessary to determine if rhGM-CSF administration will impact favorably on the morbidity and mortality of severe aplastic anemia.

Adolescent↗
PubMed1990

Biology and clinical aspects of the hematopoietic growth factors.

Molecular cloning of the genes for several members of a family of glycoproteins, the hematopoietic growth factors (HGFs), has transformed experimental hematology. Molecular probes have been used to investigate the cellular source of the HGFs and the mechanism by which their genes are regulated. Recombinant purified HGFs have been used to investigate their functions in vitro, the binding characteristics of their receptors, and the isolation of certain receptor cDNAs. This increase in knowledge of the cellular biology of the HGFs has led to clinical trials of their efficacy. Specific HGFs or HGF combinations are likely to find a place in the management of several different bone marrow failure states.

Animals↗
PubMed1989

Hematopoiesis in vitro coexists with natural killer lymphocytes.

The role of natural killer (NK) lymphocytes in the regulation of human hematopoiesis is controversial. NK-mediated inhibition of colony formation of hematopoietic progenitor cells has been irregularly reported for various cell lineages. In an effort to clarify such disparate findings, we studied the interaction of clearly defined NK and partially purified progenitor cell populations. Cell sorter purified CD16 positive blood NK cells and enriched autologous marrow progenitors were co-incubated at various lymphocyte to marrow cell ratios and then cultured in methylcellulose. There was no inhibition of myeloid, erythroid, or mixed colony formation. Similarly, activation of CD16 positive lymphocytes by interleukin-2 (IL-2) before co-incubation and co-culture did not result in inhibition of colony formation. Furthermore, in a newly designed assay system, we demonstrated that NK cells, which did not modulate colony-formation, remained capable of recognizing and killing rare K562 target cells seeded within the marrow cell population. Our results indicate that unstimulated and IL-2 activated isolated blood NK cells coexist with functioning autologous marrow progenitors in vitro.

Antigens, Differentiation↗
PubMed1989

Combinations of recombinant colony-stimulating factors are required for optimal hematopoietic differentiation in serum-deprived culture.

Previous in vitro investigations on enriched human hematopoietic progenitors have led to the conclusion that the purified recombinant multipoietins, interleukin 3 (IL-3) and granulocyte-macrophage colony-stimulating factor (GM-CSF) can alone induce the formation of colonies from a variety of multipotent and lineage committed progenitors. Since fetal calf serum was included in these cultures and itself might contain growth factors or other cofactors, we re-examined the actions of the CSFs in serum-deprived conditions. Results show that both the multipoietins are inadequate stimuli of colony formation. At maximal concentrations IL-3 alone induces only 25% of the granulocyte and macrophage colony-forming units (CFU-G and CFU-M) produced by a T-cell conditioned medium that contains a mixture of CSFs. When IL-3 was added at the initiation of the cultures and erythropoietin (ep), G-CSF, or M-CSF added on day 3, almost full recovery of erythroid, granulocytic, and monocytic colonies, respectively, was obtained. Similar results were obtained with GM-CSF except that fewer erythroid colonies were recovered at high concentrations, and almost maximal CFU-M proliferation could be induced. These results show that in serum-deprived conditions, the multipoietins must be combined with lineage specific CSFs for full progenitor expression.

Colony-Forming Units Assay↗
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

Granulocyte-macrophage colony-stimulating factor and interleukin-3 mRNAs are produced by a small fraction of blood mononuclear cells.

Northern blot analysis has identified granulocyte macrophage colony stimulating factor (GM-CSF) mRNA in monocytes and both GM-CSF and interleukin-3 (IL-3) mRNA in lymphocytes. However, these results have not addressed whether all cells or a subset of the population is capable of hematopoietic growth factor (HGF) production. To resolve this question, we applied in situ hybridization of radiolabeled antisense RNA probes to centrifuged preparations of total blood mononuclear cells (BMCs) and fractionated lymphocyte subpopulations. Without stimulation, no circulating cells expressed detectable levels of GM-CSF or IL-3 mRNA. On stimulation of BMCs with phorbol myristate acetate (PMA) and phytohemagglutinin or PMA and the calcium ionophore ionomycin, approximately 5% expressed GM-CSF mRNA and approximately 1% IL-3 mRNA. Control sense probes produced no labeled cells. To determine the subsets of lymphocytes capable of GM-CSF and IL-3 expression, BMCs were fractionated by FACS into CD8+ and CD4+ lymphocyte subsets and CD16+ (NK) cells. The unfractionated cells and cell fractions were then stimulated with PMA and ionomycin. Results demonstrated that 3% to 5% of the CD16+, CD8+, and CD4+ lymphocytes produced GM-CSF mRNA. However, the number of IL-3 mRNA-positive cells in the FACS-sorted subsets was greatly reduced (0.02% to 0.05%) as compared with the unseparated cells (1%). Treatment of BMCs with high-dose interleukin-2 (IL-2) for 1 week followed by PMA plus ionomycin resulted in a lymphocyte population in which 50% and 3% of cells expressed GM-CSF and IL-3 mRNA, respectively. Thus, GM-CSF and IL-3 mRNA expression in T cells and NK cells is restricted to a small fraction of cells that can be greatly expanded by IL-2 stimulation. These results suggest a possible physiologic mechanism for increasing HGF production by circulating lymphocytes.

Blotting, Northern↗
PubMed1988

Interleukin-1, tumor necrosis factor, and the production of colony-stimulating factors by cultured mesenchymal cells.

Although the genes for four hematopoietic colony-stimulating factors (CSFs) have been cloned, neither the mechanism of the regulation of their production nor their cellular origins have been established with certainty. Monocytes are known to produce colony-stimulating and burst-promoting activities, as well as several monokines such as interleukin-1 (IL-1) and tumor necrosis factor (TNF). These monokines indirectly stimulate other mesenchymal cells to produce certain colony-stimulating factors such as granulocyte-macrophage CSF (GM-CSF). To determine whether monocytes produce other CSFs and if so, to compare the mechanism of regulation of production with that of endothelial cells and fibroblasts, we investigated the synthesis of CSFs by monocytes, human umbilical vein endothelial cells, and fibroblasts. We used total cellular RNA blot analysis to determine interleukin-3 (IL-3), GM-CSF, granulocyte CSF (G-CSF), and monocyte CSF (M-CSF) messenger RNA (mRNA) content and immunoprecipitation or bioassay to confirm the presence of the specific secreted proteins. The results indicate that M-CSF mRNA and protein are produced constitutively by all three cell types and their level of expression does not increase after induction. In contrast, GM-CSF and G-CSF mRNAs are barely detectable in uninduced monocytes and show an increase in expression after lipopolysaccharide treatment. Retrovirus-immortalized endothelial cells, unlike primary endothelial cells or both primary and immortalized fibroblasts, produce IL-1 constitutively; this correlates with their constitutive production of GM-CSF and G-CSF. IL-3 mRNA was not detectable in any of these cells either before or after induction. The results indicate that these mesenchymal cells can produce three CSFs: GM-CSF, G-CSF, and M-CSF; furthermore, the data suggest that the mechanism of regulation of M-CSF production is different from that of GM-CSF and G-CSF, and that the latter two inducible CSFs are regulated by different factors in monocytes compared with the other mesenchymal cells.

Adult↗
PubMed1988

Human colony-stimulating factors and stromal cell function.

Hematopoiesis and the function of mature blood cells are linked by the CSFs, a class of distinct glycoproteins that have considerable overlap in their actions, tissue sources, and production by cells in response to different inducing agents. These relationships are most evident when the system is stimulated in response to exogenous agents. Monocytes may lie at the center of this response, as LPS induces the release of GM-CSF and G-CSF, as well as the monokines IL-1 and TNF. GM-CSF and G-CSF act on mature cells at the site of inflammation, increasing their functional capacity by concentration-dependent effects on chemotaxis, increased phagocytosis, enhanced superoxide production, and increased antibody-dependent cellular cytotoxicity (Vadas et al., 1983a, b; Gasson et al., 1984; Weisbart et al., 1985). The release by monocytes of IL-1 and TNF may amplify this response by inducing local fibroblasts and endothelial cells to produce more GM-CSF and G-CSF. The release of GM-CSF and G-CSF into the circulation could increase bone marrow granulocyte production. Increased circulating levels of IL-1 and/or TNF would also be expected to increase bone marrow stromal cell production of GM-CSF and G-CSF. In conjunction with foreign antigen, IL-1 may also induce T lymphocytes to produce both GM-CSF and IL-3, and these two factors would be expected to increase bone marrow hematopoiesis. Erythroid-specific interactions with fibroblastoid cells is suggested by in vitro studies showing binding of erythroid progenitors to a fibroblastoid cell strain and to fibronectin.(ABSTRACT TRUNCATED AT 250 WORDS)

Bone Marrow↗