The cloning and expression of the cDNA for ovine stem cell factor (kit-ligand) and characterization of its in vitro haematopoietic activity.

The cDNA encoding the soluble form of ovine stem cell factor (SCF) has been cloned and expressed. The soluble protein is predicted to be 165/166 amino acids in length, one more than the human and murine SCFs with which it shares 87% and 81% identity respectively. Ovine SCF has 98.5%, 95% and 91% identity with cattle, pig and dog SCF, respectively. The recombinant ovine (rov) SCF protein has been expressed in Chinese hamster ovary (CHO) cells, purified, and its biological activity on ovine bone marrow cells compared with that of interleukin 3 (rovIL-3), granulocyte-macrophage colony-stimulating factor (rovGM-CSF), interleukin 5 (rovIL-5), human macrophage colony-stimulating factor (M-CSF) and human erythropoietin (epo). On its own rovSCF supported the development of small numbers of neutrophil, macrophage, eosinophil, granulocyte-macrophage, mixed cell phenotype, haemopoietic blast cell and basophilic granular cell colonies in a soft agar clonogenic assay. In combination with each of the above cytokines rovSCF supported an increase in the number and size of the lineage-specific colony types that were stimulated by the other cytokines on their own. In an assay for precursors of multipotential colony-forming cells (multi-CFC), rovSCF in combination with rovIL-3 (but neither cytokine alone) supported the development of these early haematopoietic progenitor cells.     

Establishment of a CSF-producing cell line from a human gastric carcinoma and characteristics of the CSF produced by that cell line

A human cell line producing colony-stimulating factor has been established in vitro from a human gastric carcinoma. The cell line was transplantable into nude mice which developed a marked neutrophilia. The cell line has been maintained for three years. The cells grew in a monolayered sheet and produced colony-stimulating factors that enhanced the formation of granulocyte and monocyte colonies in vitro with mouse bone marrow cells as the target and granulocyte colonies with human bone marrow cells as the target.     

Effect of cytokines and growth factors on the macrophage colony-stimulating factor secretion by human bone marrow stromal cells

We have investigated the effect of growth factors, inflammatory and anti-inflammatory cytokines on the macrophage colony-stimulating factor (M-CSF) secretion by cultured human bone marrow stromal cells. Their production of M-CSF cultured in serum-free medium is enhanced in a time-dependent manner in response to tumour necrosis factor (TNF-)alpha and interleukin (IL-)4 but not to IL-1, IL-3, IL-6, IL-7, IL-10, SCF, granulocyte-macrophage colony-stimulating factor (GM-CSF), G-CSF, bFGF and transforming growth factor (TGF-)beta. The co-addition of IL-4 and TNF-alpha has a greater than additive effect on the secretion of M-CSF suggesting that they act synergistically. The anti-inflammatory molecules IL-10 and TGF-beta have no effect on the TNF-alpha-induced M-CSF synthesis by marrow stromal cells. In conclusion TNF-alpha and IL-4 are potent stimulators of the M-CSF synthesis by human bone marrow stromal cells, a result of importance regarding the role of M-CSF in the proliferation/differentiation of mononuclear-phagocytic cells and the role of marrow stromal cells as regulators of marrow haematopoiesis.     

Erythroid lineage-specific activity in conditioned medium derived from cloned human marrow stromal cells (CFU-RF)

Using long-term culture techniques, it has been shown that stromal cells in the marrow microenvironment are essential for the continued production and self-renewal of hematopoietic stem cells. We previously reported the development of a methylcellulose colony assay for a population of marrow stromal progenitors called CFU-RF. In this paper, a method is described for subculturing cells from individual CFU-RF-derived colonies to allow conditioned medium production (StCM). StCM, prepared in this way, was found to possess an erythroid lineage-specific activity that stimulated the formation of macroscopic erythroid colonies in cultures containing erythropoietin (epo). Using dose-response curves, the KG1 colony assay, and antibody neutralization, it was shown that the activity could not be attributed to interleukin 3 (IL3) or granulocyte-macrophage colony-stimulating factor (GM-CSF). However, it was further shown that a monolayer of stromal cells, which had earlier been producing the erythroid activity, could be stimulated by IL1 to produce granulocytic colony-stimulating activity, but only as long as IL1 was present in the culture medium. These findings indicate a mechanism whereby the same stromal population could be modulated to promote growth and differentiation of different hematopoietic lineages.     

Purification to homogeneity of a human hematopoietic growth factor that stimulates the growth of a murine interleukin 3-dependent cell line

A human lymphokine derived from the 5637 bladder carcinoma has been purified to homogeneity by using sequential reverse-phase high pressure liquid chromatography. A high recovery of biological activity is obtained by using this purification. The NH2-terminal amino acid sequence shows no homology to human interleukin 1 (IL 1), human IL 2, murine IL 3, or human granulocyte-macrophage colony-stimulating factor. The growth-promoting properties of the 5637-derived factor can be rapidly assayed by using the murine IL 3-dependent 32D c1-23 cell line. The amino acid sequence described is identical to that recently described for a human granulocyte colony-stimulating factor.     

Effect of cytokines on the proliferation/differentiation of stroma-initiating cells

A culture system that identifies the precursor of murine bone marrow fibroblastic stromal cells (stroma-initiating cells, SIC) has been developed. In this system, mature fibroblasts are depleted by adherence to plastic dishes and the nonadherent cells are seeded at a low density, which results in the formation of colonies composed of fibroblastic cells. Macrophage colony-stimulating factor (M-CSF) has been shown to accelerate the colony formation in the system. In this study, we examined the stroma-inducing activity of a number of cytokines. Neither granulocyte-CSF, stem cell factor, interleukin (IL)-1, IL-6, transforming growth factor, epidermal growth factor, insulin-like growth factor, platelet-derived growth factor, nor fibroblast growth factor showed the activity. Similarly, tumor necrosis factor (TNF) did not show any stroma-inducing activity, but the factor inhibited the stromal colony formation induced by M-CSF. In this study, we found that granulocyte/macrophage-CSF (GM-CSF) and IL-3, as well as M-CSF had the stroma-inducing activity. Neither an additive nor synergistic effect was observed when the three factors were assayed in various combinations. The stroma-inducing activity of M-CSF, GM-CSF and IL-3 was observed even if lineage-negative bone marrow cells were used as target cells, suggesting that mature hematopoietic cells such as macrophages and granulocytes were not involved in the induction of stromal colony formation by these factors. Our results raise the possibility that GM-CSF and IL-3 as well as M-CSF stimulate the proliferation or differentiation of the precursor of bone marrow fibroblastic stromal cells.     

Properties of primary murine stroma induced by macrophage colony-stimulating factor

The ability of purified human macrophage colony-stimulating factor (M-CSF) to accelerate the formation of stromal cells from murine bone marrow cells was investigated. The liquid culture of the marrow cells with M-CSF resulted in the formation of monolayers of macrophages on day 7. When the M-CSF was removed on that day and the residual adherent cells were cultured in the absence of M-CSF for an additional 7 days, many colonies appeared with cells that were morphologically distinguishable from M-CSF-derived macrophages. The appearance of the colonies was dependent on the concentration of M-CSF used at the beginning of the culture. Each colony was isolated as a single clone and analyzed. All clones were negative for esterase staining. These cells did not express M-CSF receptor mRNA and did not show a mitogenic response to M-CSF. On the contrary, these cells could be stimulated to proliferate by fibroblast growth factor and platelet-derived growth factor. The polymerase chain reaction analysis of these cells demonstrated constitutive expression of mRNA for M-CSF, stem cell factor, and interleukin (IL)-1, but not IL-3. Some clones expressed mRNA for granulocyte/M-CSF and IL-6. We also examined the ability of the cells to maintain murine bone marrow high proliferative potential colony-forming cells (HPP-CFC) in a coculture system. Most of the clones showed a significant increase in total HPP-CFC numbers after 2 weeks of coculture, although the extent of stimulation differed among clones. These results suggested that the colonies established by M-CSF were composed of functional stromal cells that were phenotypically different from macrophages. J. Cell. Physiol. 173:1–9, 1997. © 1997 Wiley-Liss, Inc.     

Regulation of hematopoiesis

Normal hematopoiesis is a well-regulated process in which the generation of mature blood elements occurs from a primitive pluripotent stem cell in an ordered sequence of maturation and proliferation. Regulation occurs at the level of the structured microenvironment (stroma), via cell-cell interactions and by way of the generation of specific hormones and cytokines: erythropoietin, interleukin 3, granulocyte-monocyte colony-stimulating factor (GM-CSF), monocyte-macrophage colony-stimulating factor (M-CSF), granulocyte colony-stimulating factor (G-CSF), interleukin 5, interleukin 4, and other less well-defined factors, including the megakaryocyte growth factors. Understanding of this complex process has revealed insights into the pathophysiology of human disease and provided a theoretical framework for the therapeutic use of bone marrow transplantation and potential gene transfer therapy. Furthermore, ongoing clinical trials suggest that the hematopoietic growth factors may represent a significant new group of therapeutic reagents for patients with hematological and oncologic disease.     

Factors influencing in vitro production of colony-stimulating factor by mononuclear leukocytes from humans.

The purpose of this study was to identify factors that influence the production of colony-stimulating factor by leukocytes of humans. The use of nonadherent light-density bone marrow cells is semisolid agar cultures to assay the concentrations of colony-stimulating factor in the supernatant of monocyte and mononuclear leukocyte cultures made it possible to distinguish between colony-stimulating factor, which stimulates colony-forming cells directly, and monocyte-dependent stimulating activity, which acts indirectly, by increasing the monocyte production of colony-stimulating factor. Colony-stimulating factor was not detectable in the cytosol of monocytes; that detected in culture must, therefore, have been newly synthesized. Synthesis was enhanced independently by heat-inactivated human serum and by semipurified serum fractions enriched with monocyte-dependent stimulating activity. The kinetics of the production of colony-stimulating factor in the presence and absence of monocyte-dependent stimulating activity indicated that the latter facilitated monocyte production of the former. Factors released from neutrophils were shown to reduce the production of colony-stimulating factor and thr proliferation of colony-forming cells and thus may provide a feedback control mechanism limiting the proliferation of neutrophils.     

Rabbit bone marrow suppressor cells block the production or release of a soluble bone marrow growth factor

Fc gamma-receptor (Fc gamma R)-bearing cells from normal rabbit bone marrow suppressed the constitutive proliferation rates of the remaining, Fc gamma R-, cells. In the absence of the suppressive influences of Fc gamma R+ cells, cells in the Fc gamma R- population spontaneously elaborated a soluble growth factor (GF) which induced the proliferation of unseparated bone marrow cells. To examine regulation by Fc gamma R+ bone marrow cells, graded numbers of the Fc gamma R+ cells were mixed with constant numbers of the FcR- cells. At 24 hr, supernates were collected and tested for GF activity. The Fc gamma R+ suppressor cells efficiently and in a dose-dependent fashion blocked GF production or release. The GF was nondialyzable and relatively heat stable. Supernates with GF activity also had colony-stimulating factor activity, but were negative in assays modified from murine interleukin 1 (IL-1) and IL-2 assays. Regulation of GF production or release represents a new function for bone marrow suppressor cells.     

Lack of spontaneous and inducible natural killer cell activity in human bone marrow: presence of adherent suppressor cells

Human bone marrow cells collected from ribs of patients undergoing thoracotomy had low or no natural killer (NK) cell activity against K562 in a 4-hour chromium release assay. In vitro overnight treatment with interferon or interleukin 2 of bone marrow cells resulted in no induction or augmentation of NK cell activity. In the presence of adherent bone marrow cells interferon was unable to enhance NK cell activity of blood lymphocytes, although the baseline level of NK cell activity was not suppressed. These results suggest that adherent bone marrow cells regulate the development of active NK cells and that bone marrow components do not provide a favorable environment for the functional differentiation of NK cells.     

Purification and characterization of human T-lymphocyte-derived erythroid-potentiating activity

The human T-lymphoblast cell line, Mo, secretes a number of lymphokines, including erythroid-potentiating activity (EPA), an important early regulator of erythropoiesis. We report purification of EPA to homogeneity, from serum-free Mo-conditioned medium. Purification was accomplished by sequential concentration, ammonium sulfate precipitation, lentil lectin affinity chromatography, gel filtration, and reverse-phase high-performance liquid chromatography. EPA was assayed by its ability to stimulate the growth of large erythroid colonies (bursts) from normal human peripheral blood. The purified EPA has a molecular weight of 28,000 and appears as a single band when analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis under reducing or nonreducing conditions. Purified EPA stimulates the growth of both early and late erythroid precursors from human bone marrow, as well as colony formation by the K562 human erythroleukemia cell line. Purified EPA has no colony-stimulating factor activity nor does it appear to be a structural protein of the human T-cell leukemia virus subtype II which infects the Mo cells.     

Arachidonic acid and freshly isolated human bone marrow mononuclear cells

Arachidonic acid (AA), a fatty acid found in the human bone marrow plasma, is the precursor of eicosanoids that modulate bone marrow haematopoiesis. To further our understanding of the role of AA in the bone marrow physiology, we have assessed its incorporation in human bone marrow mononuclear cells. Gas chromatography analysis indicates the presence of AA in their fatty acid composition. In bone marrow mononuclear cells, [3H]-AA is incorporated into triglycerides and is later delivered into phospholipids, a result not observed with blood mononuclear cells. Prelabelling-chase experiments indicate a trafficking of labelled AA from phosphatidylcholine to phosphatidylethanolamine. Stimulation of prelabelled bone marrow mononuclear cells with granulocyte-macrophage colony-stimulating factor (GM-CSF) results in the release of a part of the incorporated labelled AA. Finally, exogenous AA (up to 1 microM) has no significant effect on cell growth. In conclusion, human bone marrow mononuclear cells participate to the control of marrow AA concentrations by incorporating AA into phospholipids and triglycerides. In turn, bone marrow mononuclear cells can release AA in response to the potent haematopoietic growth factor GM-CSF.     

Effects of recombinant human stem cell factor (SCF) on the growth of human progenitor cells in vitro.

We have studied the effect of recombinant human Stem Cell Factor (SCF) on the growth of human peripheral blood, bone marrow, and cord blood progenitor cells in semisolid medium. While SCF alone had little colony-stimulating activity under fetal bovine serum (FBS)-deprived culture conditions, SCF synergized with erythropoietin (Epo), granulocyte/macrophage colony-stimulating factor (GM-CSF), and interleukin 3 (IL-3) to stimulate colony growth. Colony morphology was determined by the late-acting growth factor added along with SCF. Of all the combinations of growth factors, SCF plus IL-3 and Epo resulted in the largest number of mixed-cell colonies--a larger number than observed with IL-3 and Epo alone even in FBS-supplemented cultures. These results suggest that SCF is a growth factor that more specifically targets early progenitor cells (mixed-cell colony-forming cells) and has the capacity to synergize with a wide variety of other hematopoietic growth factors to cause the proliferation and differentiation of committed progenitor cells. Our studies indicate that SCF may be the earliest acting growth factor described to date.     

Granulocyte colony-stimulating factor induces neutrophils to secrete macrophage colony-stimulating factor

In this work we provide evidence showing that granulocytes produce macrophage colony-stimulating factor (M-CSF) from the band cell stage and secrete this factor when induced to differentiate into polymorphonuclear cells by recombinant human granulocyte colony-stimulating factor (rhG-CSF). Using an enriched population of myeloid band cells from murine bone marrow, we identified the presence of M-CSF with a chromophore-labelled monoclonal anti-M-CSF antibody. Using ELISA we detected the secretion of M-CSF in the supernatants of cultures of enriched band cells when induced with rhG-CSF to differentiate into mature neutrophils. We also found that M-CSF is the only factor responsible for the colony forming activity in the supernatants and lysates of band cells treated with rhG-CSF.     

CSF-responsive bone marrow cells in liquid culture: characterization and screening applications

In a previous study, colony-stimulating factor (CSF) activity assayed in colony culture correlated closely with 3HTdR uptake by human marrow cells depleted of adherent cells. To use this assay for screening media for CSF and immunotoxins for marrow toxicity, cells growing in liquid culture were compared to conventional granulocyte/macrophage (CFU-gm) colony assays. CSF dose-response relationships for liquid and colony-forming assays were nearly identical. 3HTdR uptake by nonadherent marrow cells was CSF dose-related, and there was a linear relationship between number of cells cultured and 3HTdR uptake. Ricin cytotoxicity curves for liquid cultures and CFU-gm were identical on day 7 but showed some disparity with day 14 cultures. Results with all cultures showed 3HTdR uptake to be most closely correlated with CFU-gm colony, rather than cluster, growth. Myeloid cell differentiation in liquid culture was similar to colony cultures, producing mixtures of granulocytes, macrophages and eosinophils. By combining cell and differential counts, production of various myeloid cells could be quantitated. Cytotoxicity of anti-Ia for CFU-gm and liquid culture cells was compared and the majority of both cell populations expressed Ia-like antigens. Simultaneous staining for surface antigens and DNA content was used to characterize proliferating marrow cells, and the vast majority of cells expressed myeloid markers. Transferrin receptors were displayed by cells in S/G2/M and appeared after CSF stimulation on G0/G1 cells. We conclude liquid cultures can be used to screen conditioned media for human CSF and to screen for cytotoxicity to normal myeloid precursor cells. Behavior of CSF-responsive cells in liquid culture appears most closely related to that of CFU-gm colony-forming cells, and characterization of CSF-stimulated cells allows quantitative as well as qualitative estimates of myeloid cell production.     

Purification and characterization of human granulocyte colony-stimulating factor (G-CSF).

A colony-stimulating factor (CSF) has been purified to homogeneity from the serum-free medium conditioned by one of the human CSF-producing tumor cell lines, CHU-2. The molecule was a hydrophobic glycoprotein (mol. wt 19,000, pI = 6.1 as asialo form) with possible O-linked glycosides. Amino acid sequence determination of the molecule gave a single NH2-terminal sequence which had no homology to the corresponding sequence of the other CSFs previously reported. The biological activity was apparently specific for a neutrophilic granulocyte-lineage of both human and mouse bone marrow cells with a specific activity of 2.7 X 10(8) colonies/10(5) non-adherent human bone marrow cells/mg protein. The purified CSF can be regarded as a G-CSF of human origin and will become a useful material for investigation of regulatory mechanisms of human granulopoiesis.     

Bone marrow extracellular matrix induces HL-60 cells to produce an autonomous differentiation factor.

Conditioned medium from cultures of HL-60 myeloid leukemia cells grown on extracellular bone marrow matrix induces macrophage-like differentiation of fresh HL-60 cells. The active medium component is sensitive to protease treatment, indicating that it is a protein, but it is heat stable. Conditioned medium from HL-60 cells grown on protease-treated bone marrow matrix still contains the active component. Thus, it appears that the differentiation-inducing protein is produced by HL-60 cells and is not released from the bone marrow matrix. To identify this differentiation factor, RNA was isolated from HL-60 cells grown on bone marrow matrix and assayed by Northern analysis for expression of mRNA for human differentiation factor, tumor necrosis factor, and macrophage colony-stimulating factor, all inducers of monocyte/macrophage differentiation. Expression of differentiation factor, tumor necrosis factor, or macrophage colony-stimulating factor mRNA was not enhanced in HL-60 cells grown on matrix compared to cells grown on uncoated plastic flasks. Thus, the maturation factor does not appear to be differentiation factor, tumor necrosis factor, or macrophage colony-stimulating factor within the limits of detection of Northern analysis. Elution of the active conditioned medium fraction on a Sephacryl S-200 column revealed a molecular weight of approximately 40,000. The active protein eluted on a DEAE-cellulose ion-exchange column at an ionic strength of 0.3 M NaCl, indicating that it is fairly anionic. Thus, bone marrow matrix is able to induce HL-60 cells to produce a maturation-inducing 40 kilodalton protein.(ABSTRACT TRUNCATED AT 250 WORDS)     

Macrophage colony-stimulating factor is produced by human T lymphoblastoid cell line, CEM-ON: identification by amino-terminal amino acid sequence analysis

A subclone, designated CEM-ON, derived from an azaguanine-resistant human leukemic T cell line, CEM-AG(R), constitutively secretes a colony-stimulating factor (CSF) which stimulates the production of macrophages from murine bone marrow progenitor cells. This CSF has been purified from serum-free conditioned medium. Highly purified CEM-ON CSF with a specific activity of 4.7 X 10(7) units/mg protein was obtained. Amino-terminal sequence analysis showed that the first 27 amino acids were identical to the amino-terminal sequence of the M-CSF (CSF-1) based on the cDNAs for human M-CSF. On SDS-PAGE analysis, CEM-ON CSF had an apparent molecular weight of 33,000-43,000; following reduction with 2-mercaptoethanol, this migrated as a 20,000-24,000 subunit, suggesting a homodimer structure. These results show that a human T cell line, CEM-ON, secretes M-CSF into its medium.     

Role of interleukin 2 (IL 2) and hemopoietin-1 (H-1) in the generation of mouse natural killer (NK) cells from primitive bone marrow precursors

The development of natural killer (NK) cells from bone marrow (BM) precursors was studied. Recombinant interleukin 2 (IL 2) was able to induce the in vitro development of NK cells when added to cultures of mouse BM cells. Treatment of donor mice with 5-fluorouracil (150 mg/kg i.v.), which eliminates more differentiated cells but spares less differentiated cells, appears to augment NK cell development. The "NK stem cell" was found to be asialo GM1-, Thy-1+, Lyt-2-, and Lyt-1-. The cells generated in vitro had a typical phenotype of NK cells, being asialo GM1+, Lyt-5+, Thy-1+, Lyt-2-, and Lyt-1-. These effector cells also had specificity characteristics of NK cells lysing the NK-susceptible YAC-1 and K562 targets, but not the NK-resistant EL/4 or allogeneic and syngeneic blasts. Hemopoietin-1 (H-1), a factor which acts on very primitive multipotent BM cells, was able to cooperate with IL 2, increasing the development of NK cells. In contrast, other factors such as interleukin 3 or colony-stimulating factor did not cause induction of NK activity when added to cultures of BM cells, indicating that this effect, i.e., induction of NK cell development, is peculiar to IL 2. These results indicate that IL 2 can act as a differentiation as well as growth factor for NK cells, and that H-1 can promote the development of functional activity in a lymphocyte subpopulation as well as affect the differentiation of myelomonocytic and other cell lineages. This experimental system appears quite useful for characterization of BM precursors for NK cells, and should help to better understand the relationship of the NK cell lineage to the T cell or other lineages.