Recent progress in the discovery and invention of novel hematopoietic cytokines.
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Animals↗
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Cytokine production by murine cells activated by erythrogenic toxin type A superantigen of Streptococcus pyogenes.
The mode of pathogenic action of the Steptococcus pyogenes superantigen erythrogenic toxin type A (ETA) in causing toxic shock-like syndrome in humans is thought to be mediated by massive release of cytokines by patients immune cells. The cytokine-inducing capacity of ETA as an extracellular protein was compared with that of lipopolysaccharide (LPS), a component of cell wall of gram-negative bacteria. Peritoneal macrophages and splenocytes of BALB/c and C3H/HeJ mice were stimulated by ETA and LPS. Tumor necrosis factor (TNF), interleukin 3 (IL-3) and interleukin 6 (IL-6) activities in the supernatants of stimulated cells were evaluated. In contrast to LPS, ETA induced only low amounts of IL-6 and no detectable TNF activities in peritoneal macrophage supernatants. ETA-triggered BALB/c and C3H/HeJ splenocytes produced great amounts of IL-6. ETA triggered the production of IL-3 by both mice strains splenocytes in a dose dependent manner. The amounts of IL-3 in supernatants were comparable to those induced by concanavalin A. The simultaneous presence of ETA and LPS in macrophage and splenocyte cultures induced a slight enhancement above an additive value after 72-96 h. Challenge of BALB/c mice with ETA 6 h before the harvest of peritoneal macrophages led to an enhanced production of IL-6 upon stimulation with ETA as well as with LPS. Splenocytes of nude BALB/c mice did not produce IL-6 upon stimulation with ETA, whereas LPS-induced IL-6 production was similar in these mice and in their littermates. The pathogenic effect of ETA on host's immune cells could most likely be explained as a consequence of T cell activation. The results confirm also that LPS- and ETA-induced shock is mediated by different cell types.
Animals↗
Myeloid cell kinetics in response to haemopoietic growth factors.
The morphologically recognizable cells of haemopoietic tissue comprise some 95% of the total and their kinetic performance is both flexible and adaptable to stress conditions. The effects of the common myeloid growth factors on these cells have been examined. Using the classically developed analyses of cell kinetics with tritiated thymidine labelling autoradiography, it is shown that continuously infused or repeated injections of G-CSF increases the proliferative activity of marrow haemopoietic tissue in both mouse and man, shortening the average cell cycle times by 35 and 65% respectively, amplifying neutrophil production so that levels in the peripheral blood rise 10-15-fold. This amplified production, amounting to 3-4 extra proliferation divisions, is mostly confined to the proliferatively active maturing neutrophil cell compartments. In addition, the neutrophil maturation time is also reduced, leading to a rapid and sustained release of postmitotic cells within 1-2 days, compared with the normal time of 4-6 days. Neither GM-CSF nor IL-3 generates any significant amplification of neutrophil production in mice but, in humans, GM-CSF stimulates cell proliferation in the bone marrow to a similar degree as doses G-CSF. Studies on the granulocyte-macrophage progenitor population show that the proliferation stimulus in response to GM-CSF is not confined to the maturing populations. In the maturing neutrophil precursor population, average cell cycle times are shortened by 60%, but in this case the overall maturation times are unaffected and their time of release into the circulation is normal. The response to GM-CSF, however, is not so straightforward as that to G-CSF. The peripheral half-life of the mature cells is considerably prolonged and this is consistent with some suggestion of functional impairment. In addition, a significant release of immature cells and eosinophils (also expanded in the bone marrow in response to GM-CSF) dilutes the neutrophilic response. Monocyte production is also stimulated by G- and GM-CSF and, though no direct measurement of proliferation has been made, stimulation at all stages of their proliferation, maturation and release are implied.
Agranulocytosis↗
Formation of haematopoietic microenvironment and haematopoietic stem cells from single human bone marrow stem cells.
Haematopoietic stem cells are a population of cells capable both of self renewal and of differentiation into a variety of haematopoietic lineages. Enrichment techniques of human haematopoietic stem cells have used the expression of CD34, present on bone marrow progenitor cells. But most CD34+ bone marrow cells are committed to their lineage, and more recent efforts have focused on the precise characterization of the pluripotent subset of CD34+ cells. Here we report the characterization of two distinct subsets of pluripotent stem cells from human fetal bone marrow, a CD34+, HLA-DR+, CD38- subset that can differentiate into all haematopoietic lineages, and a distinct more primitive subset, that is CD34+, HLA-DR-, CD38-, that can differentiate into haematopoietic precursors and stromal cells capable of supporting the differentiation of these precursors. These data represent, to our knowledge, the first identification of a single cell capable of reconstituting the haematopoietic cells and their associated bone marrow microenvironment.
ADP-ribosyl Cyclase↗
[Cellular immunity in pregnancy-induced hypertensive diseases].
Cytokines (IL-1, sIL-2R, IL-3, IL-6, TNF-alpha, IFN-gamma, GM-CSF and neopterin) were measured in sera of 37 patients with hypertensive disorders of pregnancy, 10 healthy pregnant and 10 healthy non-pregnant controls. With the exception of neopterin (p = 0.004) there were no statistically significant differences in cytokine concentrations between healthy pregnant and non-pregnant controls. No statistically relevant differences between healthy pregnant women and hypertensive patients could be found in cytokines of T-lymphocytic origin except GM-CSF in patients with HELLP syndrome (p = 0.02). Elevated levels of IL-6, TNF-alpha and neopterin were observed in hypertensive women. Differences to healthy pregnant controls were statistically significant for IL-6 (p = 0.008), TNF-alpha (p = 0.009) and neopterin (p = 0.04) and were more pronounced in severe forms of the disease. These 3 parameters of monocytic origin showed significant positive correlations amongst each other. A participation of cell-mediated immunity (especially monocytes/macrophages) in the pathomechanism of hypertensive disorders of pregnancy can thus be assumed.
Biopterins↗
Protooncogenes in the regulation of normal hematopoiesis. An antisense approach.
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Antigens, CD↗
Capability of various cytokines to induce quiescent myeloid leukemia cells to the proliferative stage.
In order to investigate the capability of cytokines to induce myeloid leukemia cells from G0 phase to the proliferative stage, blasts from 9 patients with AML and 1 patient with CML-MC were cultured with various cytokines (IL-3, GM-CSF, IL-3 + GM-CSF, G-CSF) for 48 hours or 96 hours in a serum-free culture system. Cells were analyzed by two-color flow cytometry, using PI and the monoclonal antibody Ki-67. The percentage of cells in G0 phase was reduced significantly when the cells were cultured with IL-3 (p < 0.01), GM-CSF (p < 0.01), and IL-3 + GM-CSF (p < 0.01) for 48 hours, as compared with the percentage of cells in G0 phase before culture. Moreover, the percentage of cells in S phase increased significantly when the cells were cultured with IL-3 (p < 0.01), GM-CSF (p < 0.02), and IL-3 + GM-CSF (p < 0.01) for 48 hours, as compared with the percentage of cells in S phase before culture. It is well known that many drugs which are widely used in the treatment of acute leukemia are cytotoxic mainly to proliferating cells, so that if quiescent G0 phase cells can be induced to the proliferative stage, the treatment of acute leukemia would become more effective. The present findings showed that a considerable variation was observed among individual patients in the induction of the G0 component to the proliferative stage.(ABSTRACT TRUNCATED AT 250 WORDS)
Acute Disease↗
[Clinical application of hematopoietic growth factor (IL-3, G-CSF, GM-CSF, and EPO)].
Hematopoietic growth factors were found as factors stimulating hematopoietic colony formation in in vitro culture system using bone marrow cells as a source of hematopoietic progenitor cells. Erythropoietin, a growth factor stimulating erythroid lineage has now been clinically used as an therapeutic agent for anemia of chronic renal failure. Macrophage colony-stimulating factor (M-CSF), a growth factor stimulating the production of leukocytes including monocytes and neutrophils has been clinically used as an agent for leukopenic patients after anti-cancer therapy. M-CSF improves a survival rate after bone marrow transplantation (BMT) through the reduction of mortality rate associated with BMT such as bleeding, engraftment failure and GVHD. M-CSF accelerated platelet production when injected to thrombopenic patients with solid tumor after anticancer therapy. Granulocyte CSF (G-CSF) is a most powerful agent for various kinds of neutropenia such as neutropenia after anti cancer therapy, neutropenia after BMT, aplastic anemia, chronic neutropenia of children and myelodysplastic syndrome. However, since G-CSF stimulates growth of leukemic cells in vitro, careful observations should be required when clinically used on leukemic patients. Clinical studies of granulocyte-macrophage CSF (GM-CSF) and interleukin 3 (IL-3) are now in progress, in which a promoting activity of leukocyte production of these factors is evaluated.
Erythropoietin↗
[Clinical use of hematopoietic growth factors].
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Acquired Immunodeficiency Syndrome↗
The effect of PIXY-321 on 4-hydroperoxycyclophosphamide purged and CD34+ positively selected bone marrow.
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Antigens, CD↗
In vivo effects of interleukin-6 on thrombopoiesis in healthy and irradiated primates.
We have studied the in vivo effects of recombinant human interleukin-6 (rhIL-6) on hematopoiesis in eight healthy and nine irradiated cynomolgus monkeys. Of the healthy animals, three received rhIL-6 alone (10 micrograms/kg/d, subcutaneously [SC]), one received rhIL-6 in combination with rhIL-3 (10 micrograms/kg/d, SC), one received rhIL-6 in combination with recombinant cynomolgus granulocyte-macrophage colony-stimulating factor (rcGM-CSF; 10 micrograms/kg/d, SC), two received rhIL-6 in combination with recombinant human granulocyte-CSF (rhG-CSF; 10 micrograms/kg/d, SC), and one received rhIL-6 in combination with recombinant human leukemia inhibitory factor (rhLIF; 10 micrograms/kg/d, SC). All animals were treated for at least 2 weeks with rhIL-6 or the above mentioned combinations. rhIL-6 alone significantly increased the peripheral blood platelet counts (2- to 3.5-fold). The platelets reached a plateau between days 10 and 15 of treatment. No synergistic effects on platelet numbers were observed when rhIL-6 was combined with rhIL-3, rcGM-CSF, rhG-CSF, or rhLIF. In addition to rhIL-6, only rhLIF increased the platelet numbers when administered alone. To test whether rhIL-6 might also protect the animal from thrombocytopenia or shorten the time of thrombocytopenia after irradiation, we treated nine animals with total body irradiation (3.8 Gy). Six of the animals were additional treated with rhIL-6 (4 with 10 micrograms/kg/d; and 2 with 100 micrograms/kg/d) from day -1 or +1 to day 28 post irradiation. In these animals, rhIL-6 at the same dose effective in healthy animals (10 micrograms/kg/d) was not capable of protecting the animals from platelet nadir. However, when pegylated rhIL-6 was used at a dosage of 100 micrograms/kg/d post irradiation, the mean of the nadirs was 71,000/microL as compared with 39,000/microL in control animals and the time of thrombocytopenia was shorter (3 v 5 days). In all animals (healthy and irradiated), rhIL-6 did not increase the number of bone marrow megakaryocytes but induced a right shift of DNA ploidy in megakaryocytes. These data suggest that IL-6 acts as "thrombopoietin"-like activity, but not as "megakaryocyte-CSF"-like activity.
Animals↗
Prolongation of survival of human polymorphonuclear neutrophils by granulocyte-macrophage colony-stimulating factor is caused by inhibition of programmed cell death.
In the absence of appropriate stimuli, polymorphonuclear neutrophils (PMN) undergo programmed cell death (PCD), also termed apoptosis. We show that granulocyte-macrophage colony-stimulating factor (GM-CSF), but not the chemotactic factors formyl-methionyl-leucyl-phenylalanine (FMLP), recombinant human (rh) C5a, transforming growth factor (TGF)-beta, and interleukin-8 (IL-8), or other cytokines including IL-3, IL-4, IL-6, and G-CSF, maintains viability of PMN in culture by preventing these cells from undergoing PCD. Prevention from PCD by GM-CSF was associated with induction of RNA and protein synthesis in PMN. Inhibition of RNA and protein synthesis by actinomycin-D and cycloheximide impeded the protection of apoptosis by GM-CSF. Similarly, neutralization of GM-CSF biologic activity by a specific antiserum abrogated GM-CSF-mediated inhibition of PCD.
Apoptosis↗
Measurement of serum cytokine levels in patients with myelodysplastic syndromes.
Sera of 25 healthy controls and 75 patients suffering from myelodysplastic syndromes (MDS) were investigated for serum concentration of interleukin-1 alpha (IL-1 alpha), IL-3, IL-6, granulocyte-colony-stimulating factor (G-CSF), granulocyte-macrophage-CSF (GM-CSF), erythropoietin (Epo), and tumor necrosis factor-alpha (TNF-alpha). According to French-American-British (FAB) classification, 21 refractory anemia (RA), seven refractory anemia with ring sideroblasts (RARS), 15 chronic myelomonocytic leukemia (CMML), 12 refractory anemia with excess of blasts (RAEB), and 20 RAEB in transformation (RAEBt) were examined. TNF-alpha levels were inversely correlated with lower levels of hemoglobin concentration (r = -0.31, p = 0.005), irrespective of the requirements for transfusion in anemic MDS patients. Significant differences in TNF-alpha levels between CMML (26.2 +/- 5.9 pg/ml) and the FAB subgroups (16.1 +/- 1.6 pg/ml) were detected. There was an overall inverse relationship between the level of erythropoietin and the degree of anemia, but a wide range of Epo response between patients with similar hemoglobin concentrations. Serum levels of IL-1 alpha and GM-CSF were undetected in most of the patients. In 57% of the samples there were detectable levels of G-CSF, without a correlation of the serum levels with blood cell counts, nor with any of the FAB subcategories. Overall, 29% and 25% of the patient sera exhibited elevated IL-3 and IL-6 levels, respectively. There was no correlation of the serum levels with any of the blood counts, other cytokines, nor FAB subcategories. In conclusion, simple negative feedback mechanism between a specific cytokine and the production of blood cells seems not to be the case in MDS, except for red cell production and erythropoietin concentration. Our data may suggest the involvement of TNF-alpha in the pathogenesis of anemia in MDS.
Adolescent↗
tat protein stimulates production of transforming growth factor-beta 1 by marrow macrophages: a potential mechanism for human immunodeficiency virus-1-induced hematopoietic suppression.
In this study, we examined the potential role of the human immunodeficiency virus (HIV) tat protein in causing the hematopoietic abnormalities frequently observed in HIV-infected individuals. Recombinant tat (r-tat) protein, at concentrations up to 10 micrograms/mL, did not display any stimulatory or inhibitory effect on the survival/proliferative capacity of CD34+ hematopoietic progenitor cells, purified from normal bone marrow (BM). However, exposure of r-tat protein (at concentrations between 10 ng/mL and 10 micrograms/mL) to enriched normal BM macrophages induced the production of a factor(s) in conditioned media that inhibited the in vitro growth of CD34+ cells in liquid cultures and of immature hematopoietic progenitors (day 14 colony-forming unit-granulocyte-macrophage, burst-forming unit-erythroid, and colony-forming unit-megakaryocyte) in semisolid assays. Pre-exposure of r-tat protein with a monoclonal neutralizing anti-tat antibody completely abrogated the inhibitory activity present in BM macrophage culture supernatants. The main factor responsible for this suppressive activity was transforming growth factor-beta 1 (TGF-beta 1), as shown by the ability of a polyclonal anti-TGF-beta 1 neutralizing antibody to almost completely reverse the suppressive effect of BM macrophage supernatants on CD34+ cells. TGF-beta 1 bioassays showed that exposure of r-tat protein to BM macrophages significantly increased the levels of both active and latent forms of TGF-beta 1. These results indicate that the production of TGF-beta 1, one of the most potent negative regulator of hematopoiesis, is increased by HIV tat protein and that such increase could contribute to the derangement of the hematopoietic system in HIV-infected individuals.
Antibodies↗
In vivo and in vitro stem cell function of c-kit- and Sca-1-positive murine hematopoietic cells.
c-kit is expressed on hematopoietic stem cells and progenitor cells, but not on lymphohematopoietic differentiated cells. Lineage marker-negative, c-kit-positive (Lin-c-kit+) bone marrow cells were fractionated by means of Ly6A/E or Sca-1 expression. Lin-c-kit+Sca-1+ cells, which consisted of 0.08% of bone marrow nucleated cells, did not contain day-8 colony-forming units-spleen (CFU-S), but 80% were day-12 CFU-S. One hundred cells rescued the lethally irradiated mice and reconstituted hematopoiesis. On the other hand, 2 x 10(3) of Lin-c-kit+Sca-1- cells formed 20 day-8 and 11 day-12 spleen colonies, but they could not rescue the lethally irradiated mice. These data indicate that Lin-c-kit+Sca-1+ cells are primitive hematopoietic stem cells and that Sca-1-cells do not contain stem cells that reconstitute hematopoiesis. Lin-c-kit+Sca-1+ cells formed no colonies in the presence of stem cell factor (SCF) or interleukin-6 (IL-6), and only 10% of them formed colonies in the presence of IL-3. However, approximately 50% of them formed large colonies in the presence of IL-3, IL-6, and SCF. Moreover, when single cells were deposited into culture medium by fluorescence-activated cell sorter clone sorting system, 40% of them proliferated on a stromal cell line (PA-6) and proliferated for more than 2 weeks. In contrast, 15% of the Lin-c-kit+Sca-1-cells formed colonies in the presence of IL-3, but no synergistic effects were observed in combination with SCF plus IL-6 and/or IL-3. Approximately 10% proliferated on PA-6, but most of them degenerated within 2 weeks. The population ratio of c-kit+Sca-1+ to c-kit+Sca-1- increased 2 and 4 days after exposure to 5-fluorouracil (5-FU). These results are consistent with the relative enrichment of highly proliferative colony-forming cells by 5-FU. These data show that, although c-kit is found both on the primitive hematopoietic stem cells and progenitors, Sca-1+ cells are more primitive and respond better than Sca-1- cells to a combination of hematopoietic factors, including SCF and stromal cells.
Animals↗
Study of early hematopoietic precursors in human cord blood.
Human cord blood is a source of transplantable stem cells. These stem cells express the antigen CD34, are resistant to treatment with 4-hydroperoxycyclophosphamide (CD34+/4-HCres), and do not give rise to colonies when plated in clonogenic assays. We studied the number of CD34+ cells present in cord blood and developed a two-step assay for early precursors (pre-colony-forming units, pre-CFU) capable of giving rise to committed progenitors. In this assay CD34+/4-HCres cord blood cells were cultured in suspension with different growth factors. After 7 days in suspension the remaining cells were plated in clonogenic assays, for granulocyte-macrophage colony-forming units (CFU-GM), erythroid burst-forming units (BFU-E), and mixed lineage colony-forming units (CFU-MIX), in the presence of pure factors or a combination of recombinant human (rh) interleukin 3 (IL-3) and medium conditioned by the PU34 primate cell line. Pre-CFU for all precursors were identified. These pre-CFU developed into committed progenitors in response to rhIL-3. The combinations of rhIL-3 plus rh interleukin 1 (IL-1) or rhIL-3 plus rh interleukin 6 (IL-6) did not enhance recovery of progenitors. The developing CFU-GM were responsive to rh granulocyte-macrophage colony-stimulating factor (GM-CSF) and rh granulocyte colony-stimulating factor (G-CSF) but much less so to rhIL-3. BFU-E and CFU-MIX developed in suspension but could only be detected when cells were replated in the presence of a combination of rhIL-3 and PU34 but not rhIL-3 alone. This assay may be useful in evaluating the number of early hematopoietic precursors present in cord blood samples and in defining growth factor combinations that could enhance hematopoietic recovery after cord blood stem cell transplants.
Antigens, CD↗
Vanadate mimics the effect of stem cell factor on highly purified human erythroid burst-forming units in vitro, but not the effect of erythropoietin.
When orthovanadate, an inhibitor of protein tyrosine phosphatase activity, was added to highly purified human blood erythroid burst-forming units (BFU-E) a marked increase in the number and size of erythroid bursts was evident at an optimum concentration of 4 microM. Because BFU-E are stimulated by stem cell factor (SCF), interleukin 3 (IL-3), and erythropoietin (EP), this effect could occur through an enhancement of any one of these pathways. However, no effect was observed on human erythroid colony-forming units (CFU-E), indicating that vanadate was not potentiating the effect of EP. The time course of decline in BFU-E, when vanadate was added in vitro on successive days, followed the time course produced by delayed addition of SCF but not IL-3. In addition, vanadate markedly enhanced the effect of an optimal concentration of IL-3, but it could only enhance the effect of SCF when SCF concentrations were less than optimum. These experiments demonstrate that vanadate markedly stimulates the number and size of human BFU-E in vitro and that it mimics the effect of SCF. Vanadate may be acting as a phosphatase inhibitor that potentiates the kinase activity induced by SCF, but elucidation of its specific biochemical effects on these cells awaits further investigation.
Cells, Cultured↗
Retroviral vector-mediated gene transfer into primitive human hematopoietic progenitor cells: effects of mast cell growth factor (MGF) combined with other cytokines.
Retroviral vector-mediated gene transfer into human hematopoietic stem cells may permit gene therapy of numerous genetic diseases. Stimulation of marrow with hematopoietic growth factors (HGFs) has been shown to increase the level of retroviral transduction. We have examined the effects of recombinant human mast cell growth factor (MGF), alone and in combination with other HGFs, on the efficiency of gene transfer into human hematopoietic progenitor cells. MGF acts in concert with interleukin 3 (IL-3) and interleukin 6 (IL-6) to increase the percentage of CD34+ progenitors transduced with a retroviral vector expressing the neo gene. The most potent combination of growth factors that we examined, interleukin 1 (IL-1)/IL-3/IL-6/MGF, resulted in the conferral of G418 resistance to 45% of progenitors and long-term culture-initiating cells. Extending the time of cocultivation of the marrow cells with the vector-producing cells did not further increase gene transfer frequency, suggesting that the amount of available vector is not limiting. To analyze the effects of the HGF on gene transfer into more primitive hematopoietic progenitors, CD34+ cells were isolated from marrow samples that were purged of committed progenitor cells by treatment with 4-hydroperoxycyclophosphamide (4-HC). Preculturing the CD34+ 4-HC-treated cells with the combination of four HGF (IL-1/IL-3/IL-6/MGF) permitted transduction of 20%-28% of the progenitors that formed colonies after 30 days in culture. These results demonstrate that MGF in combination with other HGFs enhances gene transduction of human hematopoietic progenitor cells.
Antigens, CD↗