Effect of a protein-bound polysaccharide, PSK, on human hemopoietic progenitors

Using in vitro clonal culture assays, we investigated the effects of PSK, a protein-bound polysaccharide derived from the cultured mycelium of CM101, Coriolus versicolor (Fr.) Quél in Basidiomycetes, on human hemopoietic progenitors. PSK alone did not stimulate colony formation by human bone marrow progenitors. Although 1-100 micrograms/ml of PSK had no effects on colony formation stimulated by erythropoietin and medium conditioned by phytohemagglutinin-stimulated leukocytes, more than 1 mg/ml of PSK inhibited all types of colony formation. In contrast, medium conditioned by PSK-stimulated leukocytes significantly stimulated formation of various types of colonies including erythroid bursts, granulocyte and/or macrophage colonies, eosinophil colonies, megakaryocyte colonies and mixed hemopoietic colonies. It is speculated that administration of the optimal dose of PSK can reduce the hematological suppression of antitumor drugs.     

The unique myelopoiesis strategy of the amphibian Xenopus laevis

Myeloid progenitors reside within specific hematopoietic organs and commit to progenitor lineages bearing megakaryocyte/erythrocyte (MEP) or granulocyte/macrophage potentials (GMP) within these sites. Unlike other vertebrates, the amphibian Xenopus laevis committed macrophage precursors are absent from the hematopoietic subcapsular liver and instead reside within their bone marrow. Presently, we demonstrate that while these frogs’ liver-derived cells are unresponsive to recombinant forms of principal X. laevis macrophage (colony-stimulating factor-1; CSF-1) and granulocyte (CSF-3) growth factors, bone marrow cells cultured with CSF-1 and CSF-3 exhibit respectively archetypal macrophage and granulocyte morphology, gene expression and functionalities. Moreover, we demonstrate that liver, but not bone marrow cells possess erythropoietic capacities when stimulated with a X. laevis erythropoietin. Together, our findings indicate that X. laevis retain their MEP within the hematopoietic liver while sequestering their GMP to the bone marrow, thus marking a very novel myelopoietic strategy as compared to those seen in other jawed vertebrate species.     

骨髓基质细胞系BMSC1自发分泌多种细胞因子

骨髓基质细胞（ＢＭＳＣ）及其分泌的细胞因子在造血细胞分化发育过程中起重要作用。应用特异性细胞因子依赖株检测了自建的小鼠骨髓基质细胞株ＢＭＳＣ１的细胞因子分泌情况。实验说明ＢＭ－ＳＣ１细胞能自发分泌高水平的ＩＬ－６，中等水平的ＩＬ－７及较低水平的ＧＭ－ＣＳＦ，未检测到化学趋化因子。此细胞上清对骨髓造血干细胞有明显的促集落形成效应，所形成的集落以ＣＦＵ－ＧＭＭ及ＣＦＵ－ＧＭ为主，其促集落形成作用呈现剂量依赖关系。上述结果表明，骨髓基质细胞能够产生多种类型的细胞因子从而对于细胞骨髓内分化发育成Ｐｒｏ－Ｔ细胞可能起重要作用。     

Establishment and characterization of factor-dependent macrophage cell lines

Three macrophage cell lines from bone marrow cells of C3H/HeN mice were isolated by successive transfer of the cells in culture with L-cell-conditioned medium (LCM) or WEHI-3 cell-conditioned medium (WEHI-3CM). These cell lines, which express Fc receptors, are involved in Fc-mediated phagocytosis and possess nonspecific esterase activity. Two (BDM-1 and BDM-2) of three cell lines show dependency for growth on either macrophage colony-stimulating factor (M-CSF) (CSF-1) or granulocyte-macrophage colony-stimulating factor (GM-CSF) and do not respond to interleukin 3 (IL-3). The third clone (BDM-3) proliferates in response to IL-3 as well as to GM-CSF and weakly responds to M-CSF and to interleukin 4 (IL-4). GM-CSF, in combination with the suboptimal concentration of M-CSF, acted synergistically on the proliferation of BDM-1 cells. The tumor-promoting phorbol diester, 12-o-tetradecanoyl-phorbol-13-acetate (TPA) also acted synergistically with the three CSFs (IL-3, GM-CSF, and M-CSF) to stimulate the proliferation of BDM-1 cells. The synergistic effect was observed when cells were pretreated with TPA and subsequently stimulated with IL-3. The calcium ionophore A23187 enhanced the proliferation of BDM-1 cells costimulated with TPA and IL-3. These factor-dependent macrophage cell lines should be useful for studying signal transduction mechanisms in the regulation of cell growth.     

Effects of recombinant cytokines on murine megakaryocyte colony formation in a serum-free fibrin clot culture system.

A convenient serum-free fibrin clot culture system for murine megakaryocyte progenitor cells was developed. The culture and counting of colonies is much easier in this system, when compared with previously reported serum-free culture methods. Recombinant murine interleukin-3 (rmIL-3) stimulated megakaryocyte colony formation in a dose-dependent manner in this system. While recombinant human granulocyte colony-stimulating factor (rhG-CSF) had no effect on megakaryocytopoiesis, recombinant human erythropoietin (rhEpo) and recombinant human interleukin-6 (rhIL-6) augmented megakaryocyte colony formation stimulated by rmIL-3. The depletion of adherent cells and T cells from the cultured bone marrow did not eliminate the synergistic effect of rhEpo and rhIL-6.     

Functions of granulocyte-macrophage colony-stimulating factor

GM-CSF was originally defined by its ability to generate in vitro granulocyte and macrophage colonies from bone marrow precursor cells. Apart from its physiological role in the control of alveolar macrophage development, it now appears more likely that its major role lies in its ability to govern the properties of the more mature myeloid cells of the granulocyte and macrophage lineages, particularly during host defence and inflammatory reactions. Recent evidence is summarized below for a key role for GM-CSF in inflammatory and autoimmune diseases, making it therefore worthy of consideration for targeting. Such evidence includes disease exacerbation following its administration and amelioration of disease in animal models by GM-CSF gene targeting or by anti-GM-CSF antibody blockade. This review summarizes the evidence supporting a major role for GM-CSF in inflammation and autoimmunity and its functions as major regulator governing granulocyte and macrophage lineage populations at all stages of maturation.     

GM-CSF Biology

Granulocyte macrophage-colony stimulating factor (GM-CSF) was originally defined by its ability to generate in vitro granulocyte and macrophage colonies from bone marrow precursor cells. Apart from its physiological role in the control of alveolar macrophage development, it now appears more likely that its major role lies in its ability to govern the properties of the more mature myeloid cells of the granulocyte and macrophage lineages, particularly during host defence and inflammatory reactions. This review summarizes the in vivo evidence to support this proposition. This evidence includes both the findings obtained by administration of GM-CSF, e.g. as an adjuvant, and also includes those observed in depletion studies, e.g. during inflammatory reactions where GM-CSF can be shown to have a proinflammatory action.     

Characteristics of colony growth from normal human bone marrow

The number of granulocyte/macrophage colonies grown in vitro from bone marrow cells obtained from 90 rib segments and 30 ;normal' bone marrow aspirates was found to be highly variable. Considerable variation was also noted in the relationship between colony number and the number of cells cultured in both groups. The aspirate group was found to have a significantly greater ability to form colonies without the addition of colony-stimulating factor to the culture medium at low cell concentrations.     

Refinement and verification of test conditions for colony-forming unit assay in rat bone marrow cells with preservable colony specimens

We established test conditions for colony-forming unit assay using rat bone marrow cells in which the colony specimens can be preserved. In our test system, all colonies can be transferred from a petri dish to a slide glass with whole agar medium because the agar medium volume is small and movable, enabling the agar medium to be dried and then fixed on the slide glass. Appropriate conditions for the colony-forming unit granulocyte–macrophage assay were as follows: 10 to 30 ng/ml recombinant murine granulocyte and macrophage colony-stimulating factor, 1?×?10⁵ cells/dish, and culture periods of 3 days for neutrophil colonies and 8 days for macrophage colonies. Appropriate conditions for the colony-forming unit erythroid assay were as follows: 2 IU/ml recombinant human erythropoietin, 0.25 to 1?×?10⁵ cells/dish, and culture period of 2–3 days. We also examined responses of bone marrow toxicants 5-fluorouracil and doxorubicin in these test systems, finding that both compounds decreased the numbers of colonies in a concentration-dependent manner in both assays. These findings suggest that these test systems are useful tools in evaluating bone marrow toxicity of these particular compounds.     

Mini ReviewGM-CSF Biology

Granulocyte macrophage-colony stimulating factor (GM-CSF) was originally defined by its ability to generate in vitro granulocyte and macrophage colonies from bone marrow precursor cells. Apart from its physiological role in the control of alveolar macrophage development, it now appears more likely that its major role lies in its ability to govern the properties of the more mature myeloid cells of the granulocyte and macrophage lineages, particularly during host defence and inflammatory reactions. This review summarizes the in vivo evidence to support this proposition. This evidence includes both the findings obtained by administration of GM-CSF, e.g. as an adjuvant, and also includes those observed in depletion studies, e.g. during inflammatory reactions where GM-CSF can be shown to have a proinflammatory action.     

Reactions of the blood system and stem cells in bleomycin-induced model of lung fibrosis

On the model of toxic diffuse pulmonary fibrosis induced by intratracheal administration of bleomycin, we studied reactions of the blood system, content of stem cells, committed hemopoietic and stromal progenitor cells in the bone marrow, spleen and peripheral blood of C57Bl/6 mice. It was shown that the development of diffuse pulmonary fibrosis was accompanied by hyperplasia of bone marrow hemopoiesis and leukocytosis in the peripheral blood. Activation of the erythroid and granulocytic hemopoietic stems was related to stimulation of hemopoietic stem cells (polypotent cells, granulocyte/erythroid/macrophage/megakaryocyte precursor cells) and committed erythroid and myeloid progenitor cells in the bone marrow. At the same time, the number of stromal precursors increased. Bleomycin increased the count of hemopoietic stem cells the peripheral blood and spleen and reduced the content of mesenchymal stem cells in the spleen and bone marrow.     

Regulation of L-selectin expression on cultured bone marrow leukocytes and their precursors.

L-selectin (LECAM-1, LAM-1, MEL-14 antigen, Dreg antigen) is one of the molecules controlling lymphocyte homing from the blood to peripheral lymph nodes and granulocyte adhesion to inflamed endothelium. In this work, regulation of L-selectin expression on mouse bone marrow cells was studied. L-selectin-negative cells were isolated by panning technique, cultured for 1-7 days with cytokines and mitogens, and L-selectin expression was analyzed by immunofluorescence staining. When cultured for 3 days with interleukin (IL) 1, IL 2, IL 5, IL 6, phytohemagglutinin, pokeweed mitogen or in the medium alone, 75%-85% of L-selectin-negative large cells (including granulocytes, macrophages/monocytes, blasts and their precursors) became L-selectin positive. In contrast, IL 3, IL 4, granulocyte-macrophage colony-stimulating factor (GM-CSF) and lipopolysaccharide (LPS) prevented the induction of L-selectin in a time- and dose-dependent manner. GM-CSF was the most potent inhibitor and only 10%-15% of cells became L-selectin positive after 3 days of culture. Furthermore, L-selectin was down-regulated on cultured unselected bone marrow cells by IL 3, IL 4, GM-CSF and LPS stimulation. After culture, the relative molecular mass of L-selectin was 100 kDa, similar to the size of the granulocyte form of this antigen. Cultured cells adhered to high endothelial venules (HEV) only 10%-32% as effectively as freshly isolated bone marrow cells despite high levels of L-selectin expression. The phenotypic analysis and the HEV binding data indicate that after culturing L-selectin was almost exclusively expressed on bone marrow leukocytes of myeloid series, and on these cells it was not functional in mediating peripheral lymph node HEV binding. Overall, these results show that the expression of L-selectin can be modulated by regulating the maturation and differentiation of the cells in vitro. This supports the idea that different cytokines and mitogens may also be important in controlling migrational status of leukocytes in vivo.     

Clonal response of murine myelomonocytic leukemia cells (WEHI-3B-Y1) to purified recombinant human hemopoietic factors in serum-free culture

We tested purified recombinant hemopoietic factors for their effects on the proliferation and differentiation of murine myelomonocytic leukemia cells (WEHI-3B-Y1) in serum-free agar culture. We found that purified recombinant human G colony-stimulating factor (CSF) markedly increased the colony number of WEHI-3B-Y1 cells and differentiation-inducing activity. However, at low colony densities [( 100/dish), G-CSF did not induce the differentiation of WEHI-3B-Y1 cells. We conclude that G-CSF does not induce the differentiation of WEHI-3B-Y1 cells directly, but induce the differentiation as a result of the secondary autoinduction of differentiation. Interleukin 3 (IL-3) slightly enhanced but erythropoietin (Epo) did not alter the colony number of WEHI-3B-Y1 cells. GM-CSF or M-CSF decreased the colony number of WEHI-3B-Y1 cells. Such purified recombinant human IL-3, Epo, GM-CSF and M-CSF did not induce morphologically the distinct differentiation of WEHI-3B-Y1 cells.     

Differentiation of murine B cell precursors in agar culture. Frequency, surface marker analysis and requirements for growth of clonable pre-B cells

A semi-solid agar assay is described in which B cell progenitors, already present in day 12 fetal liver, generate colonies which contain antibody-secreting cells. Panning experiments, in which cells which initiate colony formation are depleted on plates coated with monoclonal antibodies, suggest that by the 13th day of gestation they express the antigen recognized by the monoclonal antibody AA4.1 and by day 14 they also express the B220 form of Ly-5 recognized by the monoclonal antibody 14.8. By similar criteria the precursor cells do not express mu, I-A, I-E or Lyb-2. Growth of cells in this assay is dependent upon soluble products provided by either fetal liver adherent cells, bone marrow adherent cells or colony-stimulating factor-containing conditioned media derived from placenta cells, L929 cells, WEHI-3 B(D-) cells, T helper cells or mouse lung cells. These experiments define two sets of growth conditions. In the first, when support is provided by fetal liver adherent cells, the limiting component appears to be the B cell precursor, allowing us to estimate the frequency of these cells during ontogeny. We find approximately 1 clonable pre-B cell in 300 000 fetal liver cells on day 12 of gestation and 1 in 6000 by day 16. Under the second set of growth conditions, when support is provided by bone marrow adherent cells or colony-stimulating factor-containing conditioned media, more than one cell, colony or cell product is limiting. Highly purified samples of granulocyte/macrophage colony-stimulating factor, colony-stimulating factor 1 and multilineage-hematopoietic growth factor are effective in this assay suggesting that the colony-stimulating factors are the active components under these conditions.     

Candida albicans can stimulate stromal cells resulting in enhanced granulopoiesis

Previously, we have reported that although unperturbed granulocyte colony-stimulating factor (GCSF)-deficient (G-CSF-/-) mice are neutropenic, when challenged with Candida albicans, they develop a profound neutrophilia. In an attempt to understand the basis of Candida-induced neutrophilia in G-CSF-deficient mice, we have modified the Dexter bone marrow culture system to produce an in vitro model that mimics emergency granulopoiesis in vivo. In this model, stromal cultures are overlaid with bone marrow cells in the presence or absence of heat-inactivated (HI) Candida. Irrespective of the genotype of mice used as a source of bone marrow-derived stromal cells, stimulation of these cultures with HI Candida led to a significantly greater recovery of cells compared to unstimulated stromal cultures. In addition, there was a marked increase in the number of colony-forming units granulocyte-macrophage (CFU-GM), as well as in the percentage of granulocytes in the population of nonadherent cells recovered from HI Candida-stimulated cultures. The conditioned medium generated from stromal cultures derived from either wild-type or G-CSF-/- mice exposed to HI Candida, when applied to bone marrow cells in a soft agar clonogenic assay stimulated M-, GM-, and G- type colonies. Interleukin-3 (IL-3) and GM-CSF could not be detected in the conditioned medium from either HI Candida stimulated or unstimulated stromal cultures. However, IL-6 was detected in the conditioned media from both wild-type and G-CSF-/- stromal cultures. Addition of anti-IL-6 antibody significantly impaired granulopoiesis in unstimulated and HI Candida-stimulated, wild type, and G-CSF-/- stromal cultures. Conditioned medium generated from G-CSF/IL-6-deficient stromal cells had the capacity to stimulate bone marrow cells to form colonies comprised of granulocytes and macrophages in soft agar clonogenic assay. This study demonstrates that stromal cells can be stimulated with HI Candida and gives an insight into Candida mediated granulopoiesis.     

Generation of B lymphocytes from a single hemopoietic progenitor cell in vitro

The first stages of the pathway by which lymphocytes differentiate from hemopoietic stem cells were studied at a clonal level. When 211 interleukin 3 (IL-3)-induced blast colonies shown to be capable of differentiating into a variety of hemopoietic cells were individually transferred into wells containing a monolayer of stromal cells, growth in granulocyte, macrophage, megakaryocyte, or mast cell lineages was observed in 192 wells. In seven of these 192 wells, lymphoid cell growth also was seen. The lymphoid cells were proved to be B lymphocytes by phenotype and immunoglobulin gene rearrangement analyses and by demonstration of surface expression of IgM. The clonal origin of myeloid and B lymphocyte lineage cells was further confirmed by the generation of both myeloid and B lymphoid cells in the same well following FACS clone-sorting of IL-3 induced blast cells. These results provide in vitro evidence that cells of B lymphoid and myeloid lineage can originate clonally from single primitive hemopoietic stem cells.     

The detection and initial characterization of colony-stimulating factors in synovial fluid.

In this study which included 16 patients with inflammatory or non-inflammatory arthropathies, human granulocyte-macrophage colony-stimulating activity was detected in synovial fluid. This was attributable to the presence of colony-stimulating factor(s) (CSF), as a direct action on human bone marrow progenitor cells was demonstrated using clone transfer experiments. Samples of synovial fluid also stimulated the growth of murine macrophage colonies and induced differentiation in the murine myelomonocytic leukemia cell line, WEHI-3B(D+), which are characteristic properties of human macrophage-CSF or granulocyte-CSF respectively. These findings and the results of preliminary fractionation procedures suggested that the colony-stimulating activity in synovial fluid was not explicable by the presence of any one of the well-characterized human CSF acting in isolation. This provides a new insight into the pathogenesis of inflammatory arthropathies and supports the hypothesis that CSF have important roles in vivo in addition to the regulation of haemopoiesis.     

Histamine-producing cell-stimulating activity. A biological activity shared by interleukin 3 and granulocyte-macrophage colony-stimulating factor

The histamine-producing cell-stimulating factor (HCSF) was first described as a lymphokine which is produced during secondary mixed leukocyte culture and which induces increased histamine synthesis by murine hematopoietic cells. It has been shown that it is different from interleukin 3 (IL 3), despite the fact that pure IL 3 expresses HCSF activity. Our results provide evidence that this factor (constitutively produced by the P388 D1 cell line) is identical with granulocyte-macrophage colony-stimulating factor (GM-CSF) i.e.: (a) physiochemical properties of HCSF and GM-CSF, such as molecular weight, isoelectric charge, hydrophobicity and behavior during affinity chromatography, are indistinguishable and both activities coelute during all biochemical purification procedures; (b) increased bone marrow cell histamine synthesis induced by P388 D1-derived HCSF is inhibited by anti-GM-CSF antiserum; (c) the GM-CSF cDNA probe hybridizes with a poly(A)+RNA from P388 D1 cells while no hybridizing signal was obtained with poly(A)+RNA from WEHI-3 and from P815 cells. On the other hand, the IL 3 cDNA probe hybridizes with a 1.0-kb poly(A)+RNA from WEHI-3 but not with those from P388 D1 and P815. Moreover, well known sources of GM-CSF, such as lung conditioned medium and semi-purified GM-CSF from phytohemagglutinin-induced supernatant of the murine T lymphoma LBRM-33-5 A4 (preparation devoid of IL 3), as well as recombinant murine GM-CSF, induce increased histamine synthesis by hematopoietic cells. All these results demonstrate that, in our culture conditions, the P388 D1 cell line spontaneously produces GM-CSF which is responsible for the P388 D1-induced HCS activity. Consequently, the latter is a property shared by the two distinct hematopoietic growth factors acting on the less committed cells, i.e. IL 3 and GM-CSF, whereas M-CSF or G-CSF are unable to induce histamine production. Interestingly, IL-4 which is known to support established mast cell line proliferation cannot induce HCS activity. In addition, none of the other cytokines tested, such as IL 1, IL 2, interferons or tumor necrosis factor can express HCS activity. This expression seems to be a specific property of IL 3 and GM-CSF.