Anchored and soluble gangliosides contribute to myelosupportivity of stromal cells

Stroma-mediated myelopoiesis depends upon growth factors and an appropriate intercellular microenvironment. Previous studies have demonstrated that gangliosides, produced by hepatic stromal cell types, are required for optimal myelosupportive function. Here, we compared the mielossuportive functions of a bone marrow stroma (S17) and skin fibroblasts (SF) regarding their ganglioside pattern of synthesis and shedding. The survival and proliferation of a myeloid precursor cell (FDC-P1) were used as reporter. Although the ganglioside synthesis of the two stromal cells was similar, their relative content and shedding were distinct. The ganglioside requirement for mielossuportive function was confirmed by the decreased proliferation of FDC-P1 cells in ganglioside synthesis-inhibited cultures and in presence of an antibody to GM3 ganglioside. The distinct mielossuportive activities of the S17 and SF stromata may be related to differences on plasma membrane ganglioside concentrations or to differences on the gangliosides shed and their subsequent uptake by myeloid cells, specially, GM3 ganglioside.     

Gangliosides of the stroma layer participate in the interferon-gamma receptor-dependent controls of myelopoiesis

Stroma-mediated myelopoiesis depends upon growth-factors and an appropriate intercellular microenvironment, whose polarity is relevant for granulocyte-macrophage colony stimulating factor (GM-CSF) mediated myeloid cell proliferation. Here we have studied qualitative and quantitative aspects of ganglioside participation in controls of the microenvironment required to sustain myelopoiesis. We analysed ganglioside synthesis, expression and shedding by two primary liver stromal cell cultures isolated from wild type and interferon-gamma (IFNgamma) receptor knockout mice. The latter one has a higher capacity to sustain myelopoiesis. FDC-P1 myeloid growth factor-dependent cell line was used as the reporter system, monitoring the cell survival and proliferation that reflect the bio-availability and the activity of GM-CSF. Although the two stromal cells synthesised the same gangliosides their relative content was quite different. FDC-P1 proliferation decreased in cultures in which ganglioside synthesis was inhibited in the stroma, as well as in presence of stroma cell supernatants in which GM3 was neutralised by the anti-GM3 monoclonal antibody. Addition of exogenous GM3 reverted the inhibition and sustained proliferation of FDC-P1 cells. FDC-P1 cells do not accumulate GM3, but they are able to take up the stroma-produced sphingolipids. Thus, stroma has a double role in sustaining myelopoiesis, providing both growth factor(s) and ganglioside(s) required for the optimal stimulation of the myeloid cell proliferation, and the IFNgamma mediated stroma-dependent controls of myelopoiesis are determinant for this cell interaction.     

Stroma-mediated granulocyte-macrophage colony-stimulating factor (GM-CSF) control of myelopoiesis: spatial organisation of intercellular interactions

Granulocyte-macrophage colony-stimulating factor (GM-CSF) is one of the major cytokines involved in control of haemopoiesis both in bone marrow and in extramedullar sites. Its biological activity depends upon the composition and physicochemical properties of the microenvironment provided by the supporting stroma. GM-CSF activity is modulated and controlled by the stromal heparan-sulphate proteoglycans, but their optimal interaction occurs only at low pH. We questioned whether the microenvironment organisation of the interface between stroma and haemopoietic cells provides such conditions. We studied myeloid progenitor proliferation in contact with bone marrow-derived and extramedullar stromas using electron microscopy and selective labelling of pericellular components. We present evidence that, upon interaction, the two cell types reorganise their interface both in shape and molecular composition. Haemopoietic cells extend projections that considerably increase the area of intercellular contact, and stromal cells form lamellipodia and carry out a redistribution of membrane-associated sialylated glycoconjugates and proteoglycans. Such rearrangements lead to extensive capping of negatively charged molecules at the interface between the supporting stroma and the haemopoietic cells, leading potentially to a local decrease in pH. Our results indicate that the distribution of negative charges at the cellular interface may be responsible for the selectivity of cell response to GM-CSF.Publication of the Millennium Institute for Tissue Bioengineering. The study was supported by PRONEX, CNPq and FINEP grants from the Brazilian Ministry of Science and Technology and a FAPERJ grant from the Rio de Janeiro State Government.     

The regulation of haemopoiesis in long-term bone marrow cultures: I. Role of L-cell CSF

The effects of L-cell conditioned medium which contains granulocyte/macrophage colony stimulating factor (CSF); of highly purified L-cell CSF; and the antiserum directed against L-cell CSF, have been investigated in long-term murine bone marrow cultures. Treatment of cultures with CSF containing conditioned medium led to a rapid decline in haemopoiesis. However, this inhibition of in vitro haemopoiesis is probably caused by materials other than CSF, since the addition of highly purified L-cell CSF had no appreciable effect upon long-term haemopoietic cell proliferation or differentiation. Furthermore, the inhibitory activity of L-cell conditioned medium was not abrogated following neutralization of the CSF activity by CSF antiserum. The direct addition of CSF antiserum did not inhibit granulocyte or macrophage formation. These results suggest that long-term cultures of murine marrow cells may show extensive interactions with stromal cells which are not influenced by exogenous stimulatory or inhibitory factors.     

Bovine milk kininogen fragment 1.2 promotes the proliferation of osteoblastic MC3T3-E1 cells

The active component on the proliferation of osteoblastic MC3T3-E1 cells was purified and identified from bovine milk. The growth-promoting activity was measured by [(3)H]thymidine incorporation on the cell. The purified protein showed a molecular size of 17 kDa on SDS-PAGE. Its amino-terminal amino acid sequence was very similar to the internal sequence of bovine high molecular weight (HMW) kininogen, which comprises fragment 1.2. The promotion of proliferation was specific for osteoblastic MC3T3-E1 cells, not for fibroblast BALB/3T3 cells. In blood coagulation, HMW kininogen is considered to be cleaved by a specific enzyme kallikrein. HMW kininogen then releases two peptides, a biologically active peptide bradykinin and fragment 1.2, but the fate of fragment 1.2 is unknown. This milk-derived protein that comprises to fragment 1.2 showed a growth-promoting activity of osteoblasts. We propose the possibility that milk plays an important role in bone formation by supplying the active agent for osteoblasts as well as supplying calcium.     

The role of cells and their products in the regulation of in vitro stem cell proliferation and granulocyte development

In long-term marrow cultures haemopoiesis can be maintained in vitro for up to 6 months. Critical analysis of the cell populations produced has shown that the stem cells and their committed progeny have characteristics in common with the corresponding cell types in vivo. The maintenance of haemopoiesis in vitro is associated with the development of an appropriate inductive environment provided by bone marrow derived adherent cells. Analysis of the interactions between environmental and haemopoietic cells has been facilitated by the development of in vitro systems reproducing the naturally occurring genetic environmental defects and other systems where the development of a competent inductive environment shows a dependency upon corticosteroid hormones. Investigations have shown that stem cell proliferation may be controlled by production of opposing activities, one stimulatory for DNA synthesis, the other inhibitory. A model is proposed whereby modulation in the production of these factors is determined by the physical presence of stem cells in a proposed cellular milieu, within the adherent layer. The adherent layer, apart from acting at the level of stem cell proliferation, can also modify the response of differentiating cells (eg, GM-CFC) to exogenous stimulatory activities. Addition of GM-CSF or of CSF-antiserum has no effect on haemopoiesis in long-term cultures.     

The granulocyte-macrophage colony stimulating factor and the radiation protective effect of yeast mannan

The effect of mannan polysaccharide on the haemopoiesis recovery in irradiated mice has been investigated. Mannan has been shown to exert both the protective and the stimulatory effect: it accelerates restoration of femur bone marrow cellularity and nucleate cell number in the peripheral blood and causes a larger initial yield and subsequent more rapid postirradiation dynamics of pluripotent haemopoietic stem cells and precursor cells of granulocytes and macrophages.     

Bone marrow stromal culture from patients with myelodysplastic syndromes and patients at different stages of acute nonlymphocytic leukaemia

The haematopoietic microenvironment or stroma plays a decisive role for the proliferation and differentiation of haemopoietic cells. We studied if bone marrow cells from patients with myelodysplastic syndromes (MDS) and acute nonlymphocytic leukaemias (ANLL) are altered in their ability to form adherent stromal layer with active haemopoiesis in the Dexter liquid culture. Bone marrow cells were obtained from 24 normal volunteers, 28 patients with ANLL in different stages of the disease and 9 patients with MDS. There are no differences between the stromal layers of patients with ANLL in complete remission and those of normal volunteers after two weeks of cultivation. However, bone marrow cells from patients with ANLL before treatment and from patients in relapse formed a poor adherent stromal layer in most cases. In 6 of 9 cases we found the normal stromal grade of bone marrow cells from patients with MDS. There were qualitative differences in the nonadherent cell population between normal and ANLL patients in complete remission. In most cases we found morphologically recognizable erythroid cells after two-weeks Dexter liquid culture of bone marrow cells from patients with ANLL in complete remission, which were not seen with normal volunteers. This could be an indication of harmful effects on the balance of haematopoiesis caused by previous infiltration with leukaemic cells or/and high-dose chemotherapy.     

A rapid and efficient protocol to purify biologically active recombinant proteins from mammalian cells

Here, we describe a simple and efficient method for the expression and purification of active recombinant proteins in mammalian cells. This method uses the expression of T7 epitope-tagged proteins in transiently transfected 293T cells grown in monolayer, followed by anti-T7-agarose affinity chromatography. This procedure yields approximately between 75 and 100 microg of biologically active protein/150 cm(2) flask that can be used for biochemical studies. We have tested this protocol for the expression of the prototype SR protein, SF2/ASF, which is a member of the SR protein family with a role in constitutive and alternative splicing. We show that SF2/ASF purified using this protocol is able to complement an S100 HeLa extract, demonstrating that is biologically active. Moreover, expression of a novel SR-related protein that it is required for the second step of pre-mRNA splicing also rendered an active protein. In summary, we present a protocol based on transient transfection of mammalian cells that results in easy purification of significant amounts of biologically active proteins.     

Macrophages in haemopoietic and other tissues of the developing mouse detected by the monoclonal antibody F4/80.

Macrophages are widely distributed in lymphohaemopoietic and other tissues of the normal and diseased adult, where they play an important role in host defence and repair. Although the development of haemopoiesis has been well studied in several species, the ontogeny of the mononuclear phagocyte system remains poorly understood. We have used a highly specific mAb, F4/80, to examine the distribution of mature macrophages in the developing mouse, with special reference to their presence in the haemopoietic microenvironment. Monocytes and macrophages were first seen in embryos on day 10 in the yolk sac and liver as well as in mesenchyme. In liver, spleen and bone marrow, there was expansion of this population associated with the initiation of haemopoiesis on days 11, 15 and 17, respectively. Macrophages in these sites formed part of the haemopoietic stroma and their extensively spread plasma membrane processes could be seen making intimate contacts with clusters of differentiating haemopoietic cells. F4/80+ cells were widely dispersed in undifferentiated mesenchymal tissue in organs such as lung, kidney and gut. Numbers of F4/80-labelled cells increased concomitantly with organ growth and local mitoses were evident, as well as actively phagocytic macrophages. Our studies establish that macrophages are among the earliest haemopoietic cells to be produced during development and that they are relatively abundant in fetal tissues in the absence of overt inflammatory stimuli. Their distribution is correlated with the sequential migration of haemopoiesis and they constitute a prominent component of the stroma in fetal liver, spleen red pulp and bone marrow. Apart from a role in haemopoietic cellular interactions, their highly developed endocytic and biosynthetic activities suggest that macrophages contribute major undefined functions during growth, turnover and modelling of fetal tissues.     

Combined biochemical and electron microscopic analyses reveal the architecture of the mammalian U2 snRNP.

The 17S U2 small nuclear ribonucleoprotein particle (snRNP) represents the active form of U2 snRNP that binds to the pre-mRNA during spliceosome assembly. This particle forms by sequential interactions of splicing factors SF3b and SF3a with the 12S U2 snRNP. We have purified SF3b and the 15S U2 snRNP, an intermediate in the assembly pathway, from HeLa cell nuclear extracts and show that SF3b consists of four subunits of 49, 130, 145, and 155 kD. Biochemical analysis indicates that both SF3b and the 12S U2 snRNP are required for the incorporation of SF3a into the 17S U2 snRNP. Nuclease protection studies demonstrate interactions of SF3b with the 5' half of U2 small nuclear RNA, whereas SF3a associates with the 3' portion of the U2 snRNP and possibly also interacts with SF3b. Electron microscopy of the 15S U2 snRNP shows that it consists of two domains in which the characteristic features of isolated SF3b and the 12S U2 snRNP are conserved. Comparison to the two-domain structure of the 17S U2 snRNP corroborates the biochemical results in that binding of SF3a contributes to an increase in size of the 12S U2 domain and possibly induces a structural change in the SF3b domain.     

Haemopoietic cell growth factor mediates cell survival via its action on glucose transport

A number of haematopoietic precursor cell lines have been established which exhibit an absolute dependence on haematopoietic cell growth factor (HCGF) which is secreted by WEHI-3 myelomonocytic leukaemia cells. In the presence of HCGF, ATP levels are maintained in these factor-dependent cells (FDC-P cells); in the absence of HCGF, intracellular ATP levels undergo a steady depletion. The cell death that follows this ATP depletion can be prevented by supplying exogenous ATP suggesting that HCGF maintains these cells via its effects on energy metabolism. We have investigated the effect of HCGF on FDC-P cells further and found that: (i) HCGF markedly and rapidly increases lactate production; (ii) high extracellular glucose or glycolytic intermediate concentrations can maintain FDC-P cell viability to some extent whilst stimulating lactate production; (iii) the uptake of 2-deoxyglucose by FDC-P2 cells is stimulated by HCGF in a dose-dependent fashion. This uptake is inhibited by cytochalasin B; (iv) HCGF does not stimulate L-glucose uptake by FDC-P cells. These results suggest that HCGF acts to maintain FDC-P cells via its action on glucose transport. The significance of these results to haemopoiesis and leukaemogenesis is discussed.     

Functional characterization of two stromal cell lines that support B lymphopoiesis.

Bone marrow stromal cells have well documented effects on the production of B lymphocytes, but whether or not stromal cell signals are involved in the pre-B to B cell transition is unclear. The potential of two stromal cell lines, S10 and S17, in this process was examined. Initial experiments, using a short term liquid culture, indicated that S10 and S17 stroma efficiently supported the generation of clonable B cells (B lymphocyte CFU) from their immediate precursors in fresh bone marrow. The contribution of macrophages and other accessory cells in those experiments was minimized through use of a colony assay system that permits the direct effects of stromal cell signals on single B cell progenitors to be evaluated. The results indicated that soluble mediators from the S10 and S17 lines could support colony formation from fresh or cultured surface Ig- bone marrow cells. Colonies supported by S17 stroma appeared on day 15 and contained cells that expressed the B220 Ag; surface IgM expression was never observed. S10 supported colonies appeared on day 7 and routinely included surface IgM+ cells. Individual colonies were capable of undergoing additional growth when picked and replated directly onto the different stroma. Those colonies replated onto S10 stroma generated surface IgM expressing cells in up to 60% of experiments, but colonies transferred onto the S17 cell line included B cells only 10% of the time. These data demonstrate that stromal cells alone can provide the signals necessary for generating a surface IgM+ B cell from precursors but that not all stromal cell lines are equally efficient at doing so.     

Age-related characteristics of postradiation regeneration of the blood system

This study was pursued on hybrid (CBA X C57Bl)F1 mice at the ages of 17 and 27 months irradiated at a dose of 4 Gy. It was shown that the postirradiation recovery of haemopoiesis in old mice was the same as in the young (2-3 month-old), and in some respects (for instance, granulocytic and erythroid cells of the bone marrow, and CFUc) the recovery was even more active exhibiting a period of a pronounced hyperregeneration.     

Human spreading factor: synthesis and response by HepG2 hepatoma cells in culture

Human serum spreading factor (SF) is a cell adhesion and spreading-promoting glycoprotein purified from serum or plasma that mediates effects in a wide variety of animal cell culture systems. HepG2 human hepatoma cells were found to synthesize and secrete SF into culture medium. Quantitative immunoassay of the protein indicated a concentration of about 1 microgram/ml in 48 hr-conditioned medium from confluent cultures. Although fibronectin also was synthesized and secreted into the culture medium, HepG2 cell spreading was observed in response to human serum SF, but not in response to human plasma fibronectin. Immunoprecipitation of SF from culture medium of cells metabolically-labeled with leucine, fucose or glucosamine identified a single form of the molecule of approximately 70,000 daltons. Treatment of cultures with tunicamycin inhibited incorporation of fucose and glucosamine into immunoprecipitated SF, but did not prevent synthesis and secretion of the protein. Electrophoretic analysis and cell spreading assays showed that SF secreted by tunicamycin-treated HepG2 cells was of molecular weight (mw) approximately 60,000, and was biologically active.     

The biological effects in animals related to the accident at the Chernobyl Atomic Electric Power Station. 2. The state of bone marrow hematopoiesis in rats]

It has been shown that rats born during the first months after the Chernobyl A.P.S. disaster exhibit essential changes in the peripheral blood and bone marrow haemopoiesis throughout the entire lifetime. Rats brought up in Chernobyl from the age of three months on display even more pronounced changes. It is assumed that the changes in the haemopoiesis develop due to the continuous influence of low-level radiation of different quality and are attributed to the effect of the incorporated radionuclides.     

Myelopoiesis in the omentum of normal mice and during abdominal inflammatory processes

Coelomic cavities are relatively isolated from the systemic circulation of blood cells. Resident cell populations have a proper phenotype and kinetics, maintaining their steady-state populations and their responsiveness to local inflammatory reactions, in which the number and quality of coelomic cells can be greatly increased and modified. We have addressed the question of whether the increase in cell infiltrate in the inflamed abdominal cavity is sustained by the proliferation of myeloid cells in the omentum, and if so what are the characteristics of the progenitor cells involved and how the omentum controls their proliferation and differentiation. In the omentum under normal conditions and with inflammation due to schistosomal infection we found that pluripotent early myeloid progenitors were capable of giving rise to all the myeloid lineages in clonogenic assays, but not to the totipotent blood stem cells. Besides the major haemopoietins (GM-CSF, M-CSF, G-CSF, IL-5), the omentum stroma constitutively expressed SDF-1 alpha, the chemokine which elicits homing of circulating early haemopoietic progenitors. While normal omentum stroma produced LIF, its expression was substituted by SCF in inflamed tissues. In the first situation a slow steady-state renewal of progenitors is potentially favoured, while their intense expansion may be predominant in the latter one. We propose that the increase in cells in the abdominal cavity in inflammatory reactions is due to the enhanced input and expansion of early myeloid progenitors sustaining the in situ production of abdominal cell populations, rather than to the input of systemic circulating inflammatory cells.     

Blast colony-forming cell binding from CML bone marrow, or blood, on stromal layers pretreated with G-CSF or SCF

Blast colony-forming cells (CFU-BL) represent a specific subpopulation of special primitive progenitors characterized by colony formation only in close contact with a preformed stromal layer. CFU-BL derived from bone marrow of chronic myeloid leukaemia (CML) patients have been proved to adhere poorly to bone marrow derived stromal layers suggesting that the appearance of progenitors and precursors in the circulation is due to a defective adhesion of these cells to the bone marrow microenvironment. In the present experiments the effect of short-term incubation of preformed normal bone marrow stroma on the adherence of CML derived CFU-BL was studied. For stroma cultures bone marrow cells were cultured in microplates in the presence of hydrocortisone. Cultures were used when stromal layers became confluent and no sign of haemopoiesis could be observed. CFU-BL were studied by panning plastic non-adherent mononuclear (PNAMNC) bone marrow or blood cells. 8.9 +/- 2.4 colonies/103 PNAMNC (six experiments) were formed from normal bone marrow on stromal layers and 4.8 +/- 2.1 colonies/103 PNAMNC (five experiments) from CML bone marrow. Colony formation from normal bone marrow was not increased if stromal layers were incubated with 100 ng/mL granulocyte colony-stimulating factor (G-CSF) or stem cell factor (SCF). Incubation of stroma with G-CSF or SCF, however, increased the colony formation of PNAMNC from CML bone marrow or blood significantly. These findings suggest that local concentration of haemopoietic growth factors at the time of panning may influence the attachment of CML progenitors to the stroma.     

c-Myc is required for transformation of FDC-P1 cells by EGFRvIII

In contrast to wtEGFR, its truncated version EGFRvIII transformed non-tumorigenic FDC-P1 cells only when c-Myc was coexpressed. In nude mice, EGFRvIII/c-Myc coexpressing cells induced tumors, whereas wtEGFR-expressing EGF-dependent FDC-P1 cells did not. EGFRvIII function was required for both the induction and maintenance of tumor growth. Cellular proliferation was inhibited by a selective EGFR tyrosine kinase inhibitor indicating intrinsic tyrosine kinase activities for both receptors. Unlike wtEGFR, constitutive signaling by EGFRvIII was refractory to stimulation by the EGFR ligands EGF and TGF-alpha. Summarized, EGFRvIII is a constitutively active receptor tyrosine kinase whose transforming capacity is lower than that of EGF-stimulated wtEGFR.