Transferrin receptor-mediated suppression of in vitro hematopoiesis by transferrin-gallium

The expression of transferrin receptors on cells is felt to reflect iron requirements for proliferation or for hemoglobin production. We have recently shown that transferrin-gallium (Tf-Ga) complexes bind to cellular transferrin receptors and inhibit cellular iron incorporation. In this study, Tf-Ga in a dose-dependent manner inhibited the growth of erythroid (erythroid burst-forming units [BFU-E]-derived), granulocyte-macrophage (granulocyte-macrophage colony-forming units [CFU-GM]-derived) and mixed (mixed CFU [CFU-GEMM]-derived) hematopoietic colonies. Although major differences in the response of the different progenitor cells to Tf-Ga were not seen, CFU-GEMM-derived colonies appeared to be more sensitive to growth inhibition by Tf-Ga. The inhibitory effects on colony growth were reversible after 48 h of exposure of marrow cells to Tf-Ga, suggesting that the initial effects of Tf-Ga were mainly cytostatic and that continuous exposure of cells to Tf-Ga was required for maximal growth inhibition. Transferrin-iron (Tf-Fe) added to the Tf-Ga-containing cultures restored colony growth; however, this effect was best seen when Tf-Fe was added at day 0 of incubation. Tf-Fe added on days 3 or 7 failed to restore GEMM colonies and restored only a fraction of BFU-E and GM colonies. Tf-Ga appears to inhibit hematopoietic progenitor cell growth by interfering with cellular iron utilization during an early phase of progenitor cell proliferation. The use of Tf-Ga may allow further exploration of the role of iron and the Tf receptor in the regulation of hematopoietic progenitor cell growth.     

Bovine erythroid (CFU-E, BFU-E) and granulocyte-macrophage colony formation in culture

Progenitor cells of bovine erythrocytes, megakaryocytes, and granulocyte-macrophages were cultured in agar or methylcellulose media. Colony formation was supported by cell-free conditioned medium from short-term cultures of concanavalin A-stimulated bovine peripheral blood leukocytes. Granulocyte-macrophage progenitors proliferated well in both types of semisolid culture media, giving rise to neutrophils (from granulocyte colony-forming units, CFU-G), eosinophils (from eosinophil CFU, CFU-Eo), monocyte-macrophages (from macrophage CFU, CFU-M), and mixed granulocyte-macrophages (from granulocyte-macrophage CFU, CFU-GM). Better growth of megakaryocytes, as well as late (erythroid CFU, CFU-E) and early (erythroid burst-forming units, BFU-E) erythroid progenitors was obtained with methylcellulose. Despite considerable variation in the numbers and types of colonies formed from different aspirates of bovine marrow, the numbers observed were generally comparable to those obtained from human and mouse bone marrow cells. The proliferation of bovine BFU-E and eosinophil progenitors herein described is the first successful report of the culture of these cell types.     

Human peripheral blood erythroid burst forming unit (BFU(E)): evidence against T-lymphocyte requirement for proliferation in vitro

Erythroid burst-forming units (BFU(E)) are proliferative cells which may be precursors of the erythroid colony-forming unit (CFU(E)). To examine the role of T lymphocytes in the proliferation and/or differentiation of human blood BFU(E), the effect of purified T lymphocytes on erythroid colony (EC) formation by purified null cells was examined in vitro. Lymphocyte subpopulations were prepared by Ficoll-Hypaque centrifugation, immunoadsorbent chromatography, and sheep red blood cell rosetting after removal of monocytes by adherence to plastic. Cultures of isolated B, T, or null lymphocytes alone revealed that BFU(E) were present in the null cell fraction. Addition of isolated B and/or T lymphocytes in various ratios to null cells failed to influence the number or size of EC formed. These results indicate that normal human circulating BFU(E) are contained in the null cell fraction of peripheral blood lymphocytes and do not require T lymphocytes for normal growth and differentiation in vitro.     

Myelosuppression in HCL: Role of hairy cells,T cells and haematopoietic growth factors

Abstract: To elucidate mechanisms which may be responsible for the haematopoietic insufficiency in hairy cell leukaemia (HCL), we investigated in an autologous in vitro system the influence of haematopoietic growth factors (CSFs) and the effects of hairy cells (HCs) as well as T cells on the formation of haematopoietic colonies (CFU). Colony forming assays were performed using peripheral blood mononuclear cells (PBMC) of 6 HCL patients. To remove HCs, PBMCs were subjected to complement-mediated lysis, T cells were removed by E-rosette formation. Assays were done with and without recombinant human (rh) interleukin-3 (IL-3) and rh granulocyte-macrophage-colony-stimulating factor (GM-CSF). All 6 patients exhibited a severe reduction of their circulating progenitor cell (CPC) compartment. There was no correlation between the degree of colony reduction and the number of HCs. However, a correlation was found between the numbers of CPCs of HCL patients and healthy donors and the monocyte counts in these groups (r = 0.8573, p<0.001). The removal of autologous HCs, but also of T cells, resulted in a significant increase in colony formation (BFU-E, CFU-GM, CFU-mix). In none of the experiments, however, did colony numbers come close to the normal range. This was only achieved by supplementation of the culture medium with rh IL-3 and rh GM-CSF. The results suggest that the haematopoietic failure observed in HCL patients is probably due to an inadequate supply of CSFs as well as to an inhibitory activity of HCs and T cells which might exert their effects in a synergistic fashion. There is also evidence that the lack of monocytes plays a role in the development of the haematopoietic insufficiency in HCL.     

Very low density lipoprotein hematopoiesis inhibitor from rat plasma

Although the stimulatory effect of specific glycoproteins on bone marrow cell proliferation is acknowledged, little attention has been directed toward growth inhibitors. In this report we have explored the role of plasma lipoproteins in regulating the proliferation of hematopoietic cells. Lipoproteins were isolated from the plasma of normal rats and rats with cancer by density gradient ultracentrifugation. Lipoprotein fractions were then added to cell cultures to assess their effect on: 1) erythropoietin (Ep) stimulated rat marrow DNA and protein synthesis, 2) Ep and colony stimulating factor induced marrow colony formation (CFU(E), CFU(C)), and 3) phytohemagglutinin (PHA) stimulated lymphocyte DNA synthesis. The results indicated that very low density lipoproteins (VLDL) completely inhibited CFU(E) and CFU(C) formation. VLDL inhibited (> 80%) the synthesis of DNA by marrow cells cultured with Ep and lymphocytes cultured with PHA. VLDL from rats with Walker-256 cancer had a greater inhibitory effect than normal rat VLDL. Chylomicrons had moderate growth inhibitory effect, and plasma LDL and HDL were inactive. VLDL, however, did not inhibit the proliferation of rat fibroblasts. We conclude that physiologic concentrations of plasma VLDL have a significant inhibitory effect on the proliferation of erythroid, granulocytic and lymphocytic cells. A pathophysiologic role for VLDL in the impairment of erythropoiesis and immune function in cancer is suggested.     

Modulation of erythropoiesis and myelopoiesis by exogenous erythropoietin in human long-term marrow cultures

In spite of their ability to support myelopoiesis for several months, human long-term marrow cultures (LTMC) are unable to sustain the production of mature erythroid cells for greater than 4 weeks. Because this preference correlates with the presence of myeloid growth factors and possible absence of erythroid factors in LTMC, we studied the effects of the erythroid growth and differentiation factor erythropoietin (Epo) on both erythropoiesis and myelopoiesis in human LTMC. Either natural or recombinant Epo was added weekly to LTMC for 10 weeks, and total cell number, numbers of hemopoietic progenitors (mixed lineage colony-forming units, CFU-MIX; erythroid burst-forming units, BFU-E; erythroid CFU, CFU-E; granulocyte-macrophage CFU (CFU-GM); granulocyte CFU, CFU-G; and macrophage CFU, CFU-M), erythroblasts (early and late), granulocytes, and macrophages were quantitated separately in the adherent and nonadherent layers of the cultures. In the absence of Epo, mature erythroid cells disappeared within the first 3-4 weeks, whereas in cultures supplemented with Epo, erythropoiesis was supported for up to 8 weeks. Results indicate that erythroid maturation is blocked at the BFU-E stage and that exogenous Epo may act on a mature subpopulation of BFU-E located in the nonadherent fraction of the cultures, promoting its maturation into CFU-E, which in turn develop into erythroblasts. However, despite Epo supplementation, erythropoiesis was not restored to in vivo proportions, suggesting that additional factors or conditions necessary for erythropoiesis are lacking in LTMC. Interestingly, we found that exogenous Epo reduced the numbers of presumably more mature (nonadherent) myeloid CFU (CFU-C), granulocytes, and macrophages compared to controls and did not alter the levels of any of the most primitive hemopoietic progenitors measured (CFU-MIX, adherent BFU-E, and adherent CFU-C). Thus the data show that exogenous Epo modulates hemopoiesis in human LTMC, enhancing erythropoiesis and suppressing myelopoiesis, but that its effects appear limited to modulating levels of the nonadherent (more mature) progenitors, leaving the numbers of the adherent (immature) progenitor cells unchanged.     

Adiponectin, a new member of the family of soluble defense collagens, negatively regulates the growth of myelomonocytic progenitors and the functions of macrophages

We investigated the functions of adiponectin, an adipocyte-specific secretory protein and a new member of the family of soluble defense collagens, in hematopoiesis and immune responses. Adiponectin suppressed colony formation from colony-forming units (CFU)-granulocyte-macrophage, CFU-macrophage, and CFU-granulocyte, whereas it had no effect on that of burst-forming units-erythroid or mixed erythroid-myeloid CFU. In addition, adiponectin inhibited proliferation of 4 of 9 myeloid cell lines but did not suppress proliferation of erythroid or lymphoid cell lines except for one cell line. These results suggest that adiponectin predominantly inhibits proliferation of myelomonocytic lineage cells. At least one mechanism of the growth inhibition is induction of apoptosis because treatment of acute myelomonocytic leukemia lines with adiponectin induced the appearance of subdiploid peaks and oligonucleosomal DNA fragmentation. Aside from inhibiting growth of myelomonocytic progenitors, adiponectin suppressed mature macrophage functions. Treatment of cultured macrophages with adiponectin significantly inhibited their phagocytic activity and their lipopolysaccharide-induced production of tumor necrosis factor alpha. Suppression of phagocytosis by adiponectin is mediated by one of the complement C1q receptors, C1qRp, because this function was completely abrogated by the addition of an anti-C1qRp monoclonal antibody. These observations suggest that adiponectin is an important negative regulator in hematopoiesis and immune systems and raise the possibility that it may be involved in ending inflammatory responses through its inhibitory functions. (Blood. 2000;96:1723-1732)     

Apoptosis in refractory anaemia with ringed sideroblasts is initiated at the stem cell level and associated with increased activation of caspases

Treatment with granulocyte colony-stimulating factor plus erythropoietin may improve haemoglobin levels in patients with ringsideroblastic anaemia (RARS) and reduce bone marrow apoptosis. We studied bone marrow from 10 RARS patients, two of whom were also investigated after successful treatment. Mononuclear, erythroid and CD34+ cells were analysed with regard to proliferation, apoptosis, clonogenic capacity and oncoprotein expression, in the presence or absence of Fas-agonist, Fas-blocking antibody 2 and caspase-3 inhibitor. During culture, RARS bone marrow cells showed higher spontaneous apoptosis (P < 0.05) and caspase activity (P < 0.05)) than bone marrow cells from healthy donors. Eight out of nine patients had reduced growth of erythroid colony-forming units (CFU-E) (< 10% of control) and granulocyte-macrophage CFU (CFU-GM) (< 50% of control) from CD34+ cells. Fas ligation increased apoptosis and decreased colony growth equally in RARS and controls, but caused significantly more caspase activation in RARS (P < 0.01). Fas-blocking antibody showed no significant inhibitory effect on spontaneous apoptosis or ineffective haematopoiesis, as measured using phosphatidylserine exposure, the terminal deoxynucleotide transferase-mediated dUTP-biotin nick-end labelling technique, caspase activity or clonogenic growth. Caspase inhibition reduced apoptosis, increased proliferation and enhanced erythroid colony growth from CD34+ cells in RARS, but showed no effect on normal cells. CFU-E improved > 1000% after successful treatment. Thus, erythroid apoptosis in RARS is initiated at the CD34+ level and growth factor treatment may improve stem cell function. Enhanced caspase activation at the stem cell level, albeit not mediated through endogenous activation of the Fas receptor, contributes to the erythroid apoptosis in RARS.     

The role of methylcellulose on colony growth of human myeloid leukemic progenitors (AML-CFU)

The use of a semisolid support like methylcellulose (MC) in a clonogenic assay prevents cell migration and nonspecific aggregation. However, the inhibitory effect of MC on myeloid cell lines has been reported. To assess the effect of MC on human leukemic progenitor cell growth (acute myeloblastic leukemia colony-forming units, AML-CFU), increasing concentrations of MC (0.36%, 0.72%, and 1.44%) were added in a double-feeder culture system. T-lymphocyte-depleted leukemic cells from 12 patients with AML were cultured in the presence of 2.5% phytohemagglutinin (PHA) in a liquid and a semisolid (MC) medium over a leukocyte feeder layer. The leukemic nature of the colonies was confirmed by cytogenetic studies. The median cloning efficiency in the optimal MC assay system was significantly higher (217 leukemic colony-forming units [CFU-L]/5 x 10(4) cells) than the one obtained in the liquid assay system (72.5 CFU-L/5 x 10(4) cells). However, three patterns of growth were observed: 1) colony formation was significantly better in MC than in the liquid assay system (seven of ten cases), 2) there was no difference in growth response (three of ten cases), and 3) colony formation was significantly better in the liquid assay system (one of ten cases). In the semisolid assay system, colony growth was dependent on MC concentration and varied among individual patients. A striking feature was the partial reduction of AML-CFU growth at 1.44% MC, with complete inhibition in 4/11 cases. This phenomenon was not observed for normal progenitors cultured under the same conditions. Cytological evaluation of AML-CFU showed an incomplete maturation to the myelocyte state, accompanied occasionally by macrophagic differentiation. In contrast, maturation of the granulocyte-macrophage colony-forming unit (CFU-GM) clones was harmonious, resulting in greater than 40% polynuclear cells, even from a 7-day culture. Despite a variable clonal response of leukemic progenitors from individual patients, we conclude that 0.72% MC is the optimal concentration of MC in our system, allowing clonal growth of AML-CFU.     

Tumor necrosis factor-alpha and hematopoietic progenitors: effects of tumor necrosis factor on the growth of erythroid progenitors CFU-E and BFU-E and the hematopoietic cell lines K562, HL60, and HEL cells

Macrophages can modulate the growth of hematopoietic progenitors. We have examined the effects of tumor necrosis factor-alpha, a product of activated macrophages, on human erythroid progenitors (CFU-E, BFU-E) and the hematopoietic cell lines K562, HL60, and HEL cells. Tumor necrosis factor (TNF) significantly inhibited CFU-E and BFU-E growth at concentrations as low as 10(-11)-10(-12) M (0.2 U/ml), although erythroid colony and burst formation were not totally ablated. Preincubation of marrow samples with TNF for 15 min was sufficient to suppress erythroid colony and burst formation. Addition of TNF after the start of culture inhibited CFU-E- and BFU-E-derived colony formation if TNF was added within the first 48 h of culture. Additionally, TNF inhibited the growth of highly purified erythroid progenitors harvested from day 5 BFU-E. The colonies which formed in cultures treated with TNF were significantly smaller than those formed in control cultures. TNF (10(-8)-10(-10) M) also suppressed the growth of the hematopoietic cell lines K562, HL60, and HEL cells, with 40%-60% of the cells being sensitive to TNF. Preincubation of HL60 cells with TNF for 15 min significantly inhibited their growth. K562, HL60, and HEL cells expressed high-affinity receptors for TNF in low numbers (6000-10,000 receptors per cell). Fluorescence-activated cell sorter analysis of TNF binding to HEL cells demonstrated that the majority of these cells expressed TNF receptors. These data suggest that: (1) TNF is a rapid irreversible and extremely potent inhibitor of CFU-E, BFU-E, and hematopoietic cell lines K562, HL60, and HEL cells; (2) TNF appears to be acting on a subpopulation of erythroid cells, predominantly CFU-E, BFU-E, and possibly proerythroblasts; (3) TNF appears not to require accessory cells such as lymphocytes or macrophages to inhibit erythroid progenitors; and (4) the presence of TNF receptors on hematopoietic cells is not sufficient to confer sensitivity to TNF since the majority (80%-95%) of HEL cells express TNF receptors while only 40%-60% are inhibited by TNF.     

Human granulocyte-macrophage colony-stimulating factor (GM-CSF) stimulates immature marrow precursors but no CFU-GM, CFU-G, or CFU-M

Human granulocyte-macrophage colony-stimulating factor (GM-CSF) has been described as a multilineage growth factor that induces in vitro colony formation from erythroid burst-forming units (BFU-E), eosinophil colony-forming units (CFU-Eo), and multipotential CFU (CFU-GEMM) as well as from granulocyte-macrophage CFU (CFU-GM), granulocyte CFU (CFU-G), and macrophage CFU (CFU-M). In this paper we provide evidence indicating that GM-CSF, when tested for its stimulating capacities expressed upon highly enriched hematopoietic progenitor cells (CD34+/monocyte-depleted), is unable to induce colonies from CFU-GM, CFU-G, or CFU-M. Only BFU-E, CFU-Eo, and CFU-GEMM were stimulated, and thus GM-CSF induces a similarly restricted spectrum of progenitor cells as does recombinant human interleukin 3 (IL-3). We then compared the relative stimulating potencies of GM-CSF and IL-3 by measuring colony numbers of CFU-GEMM, BFU-E, and CFU-Eo generated from CD34+ progenitor cells. IL-3 and GM-CSF as single factors were equally active in stimulating CFU-GEMM, but the combination of both factors produced additive stimulative effects upon CFU-GEMM. IL-3 was a more potent stimulus of BFU-E, and GM-CSF was the more active stimulating factor for CFU-Eo. We conclude that GM-CSF and IL-3, although stimulating the outgrowth of identical types of progenitor cells, particularly differ as regards their comparative quantitative efficiency of stimulation.     

Pure red cell aplasia in a patient with systemic lupus erythematosus

Pure red cell aplasia developed in a female patient with systemic lupus erythematosus (SLE). Erythroid colony growth was assessed in semisolid medium culture of bone marrow obtained from a normal donor and cultured in the presence of normal and patient sera. Colony forming units of erythropoiesis and burst forming units of erythropoiesis obtained from a normal donor were inhibited in the presence of patient sera. Our findings support the concept that circulating inhibitors might influence the proliferation of erythroid progenitor cells and erythroid aplasia may be an immunologically mediated syndrome.     

Hemangiopoietin supports animal survival and accelerates hematopoietic recovery of chemotherapy-suppressed mice

Objective: To determine if recombinant human hemangiopoietin (HAPO), a novel growth factor for primitive cells of hematopoietic and endothelial cell lineages, accelerates hematopoietic reconstitution after high‐dose chemotherapy in vivo in mice. Methods: Male Balb/c mice after treatment of 5‐flourouracil were subcutaneously injected with HAPO or its dilution for consecutive 10 d. Their survival and body weight together with peripheral blood were routinely tested. At day 7 and 14, the numbers of bone marrow (BM) cells as well as colony‐forming units (CFU) after in vitro colony culture were counted. The peripheral blood CFU and the percentage of CD34⁺CD117⁺ cells in BM were analyzed. Transwell chamber was used for cell migratory assay. Results: HAPO at different doses significantly increased the survival rate and body weight, with an optimal effect in the HAPO 10 μg/d group. The number of BM cells and the percentage of CD34⁺CD117⁺ cells were also increased after HAPO administration. The number of granulocyte/macrophage CFU and granulocyte, erythroid, macrophage and megakaryocyte CFU in BM after HAPO treatment was greater than that from the HAPO dilution group. More circulating CFU could be observed after injection of HAPO. In addition, this novel cytokine had a chemotactic effect on the hematopoietic stem/progenitor cells. Conclusion: HAPO improves animal survival and accelerates hematopoietic reconstitution of mice after high‐dose chemotherapy.     

The hematopoietic stem cell compartments in mice during and after long-term inhalation of three doses of benzene

Female BDF1 mice were exposed for 16 weeks to airborne concentrations of 100, 300, and 900 ppm of benzene, 6 h per day, 5 days per week. Bone marrow hemopoietic stem cell compartments and peripheral blood cell counts were studied using clonal assays and standard methods. Dose-dependent depressive effects were observed on all stem cell compartments. Only the erythroid colony-forming units (CFU-E) compartment was depressed during exposures to 100 ppm; CFU-E were more sensitive than the erythroid burst-forming units (BFU-E), spleen CFU (CFU-S), or G-M CFU (CFU-C) during exposure to 300 ppm or 900 ppm. Lymphocytopenia was observed in the peripheral blood. After benzene-free intervals, a regeneration of lymphocyte numbers and slow normalization of stem cell numbers was seen. Complete recovery from the 16 weeks exposure to 300 ppm was seen between 73 and 185 days.     

Effect of murine mast cell growth factor (c-kit proto-oncogene ligand) on colony formation by human marrow hematopoietic progenitor cells.

Purified natural (n) and recombinant (r) murine (mu) mast cell growth factor (MGF, a c-kit ligand) were evaluated alone and in combination with r human (hu) erythropoietin (Epo), rhu granulocyte-macrophage colony-stimulating factor (rhuGM-CSF), rhuG-CSF, and/or rhuM-CSF for effects in vitro on colony formation by multipotential (colony-forming unit-granulocyte, erythroid, monocyte, megakaryocyte [CFU-GEMM]), erythroid (burst-forming unit erythroid [BFU-E]) and granulocyte-macrophage (CFU-GM) progenitor cells from normal human bone marrow. MGF was a potent enhancing cytokine for Epo-dependent CFU-GEMM and BFU-E colony formation, stimulating more colonies and of a larger size than either rhu interleukin-3 (rhuIL-3) or rhuGM-CSF. MGF, especially at lower concentrations, also acted with rhuIL-3 or rhuGM-CSF to enhance Epo-dependent CFU-GEMM and BFU-E colony formation. MGF had little stimulating activity for CFU-GM colonies by itself, but in combination with suboptimal to optimal amounts of rhuGM-CSF enhanced the numbers and the size of CFU-GM colonies in an additive to greater than additive manner. While we did not detect an effect of MGF on CFU-G colony numbers stimulated by maximal concentrations of rhuG-CSF, MGF did enhance the size of CFU-G-derived colonies. MGF did not enhance the activity of rhuM-CSF. In a comparative assay, maximal concentrations of rmu and rhuMGF were equally effective in the enhancement of human bone marrow colony formation, but rhuMGF, in contrast to rmuMGF, did not at the concentrations tested enhance colony formation by mouse bone marrow cells. MGF effects on BFU-E, CFU-GM, and CFU-GEMM may be direct acting ones as MGF-enhanced colony formation by these cells in highly enriched progenitor cell populations of CD34 HLA-DR+ and CD34 HLA-DR+CD33- sorted cells in which greater than or equal to 1 of 2 cells was a BFU-E plus CFU-GM plus CFU-GEMM. MGF appears to be an early acting cytokine that preferentially stimulates the growth of immature hematopoietic progenitor cells.     

Erythroid differentiation of fetal, newborn and adult haemopoietic stem cells.

Erythroid regeneration was studied in lethally irradiated mice given transplants containing equivalent numbers of haemopoietic stem cells (i.e. CFU) from fetal liver, neonatal marrow or adult marrow. Adult marrow was taken from normal control mice, whose CFU for the most part were not in active cell cycle, as well as from phenylhydrazine-treated groups whose CFU were in similar state of proliferation (i.e. approximately 40-50% in DNA SYNTHESIS) AS THOSE DERIVED FROM FETAL LIVER AND NEONATAL MARROW. Splenic and femoral radioiron (59Fe) incorporation were measured at intervals after transplantation and were found to begin earliest in mice given fetal liver, then in animals given neonatal marrow and latest in recipients of adult marrow. Peripheral reticulocytes showed a similar pattern of recovery. The data reported herein suggest that the differences in erythroid regeneration evoked by transplants of fetal liver, neonatal marrow or adult marrow, are not solely attributed to the degree of proliferation in the pluripotential stem cell compartment. These data may, however, suggest a shorter doubling time for cells comprising the fetal and newborn committed erythroid compartments.     

Transforming growth factor beta selectively inhibits normal and leukemic human bone marrow cell growth in vitro

The effects of transforming growth factor beta 1 or beta 2 (TGF-beta 1 or -beta 2) on the in vitro proliferation and differentiation of normal and malignant human hematopoietic cells were studied. Both forms of TGF- beta suppressed both the normal cellular proliferation and colony formation induced by recombinant human interleukin-3 (IL-3) and granulocyte-macrophage colony-stimulating factor (GM-CSF). In the presence of GM-CSF or IL-3, optimal concentrations of TGF-beta (400 pmol/L) inhibited colony formation by erythroid (BFU-E), multipotential (CFU-GEMM), and granulocyte-macrophage (CFU-GM) progenitor cells by 90% to 100%, whereas granulocyte or monocyte cluster formation was not inhibited. In contrast, neither form of TGF-beta had any effect on G- CSF-induced hematopoiesis. The suppressive action appeared to be mediated directly by TGF-beta since antiproliferative responses were also observed in accessory cell-depleted bone marrow cells. In contrast to normal bone marrow cells, both GM- and G-CSF-induced proliferation of cells from patients with chronic myelogenous leukemia were suppressed in a dose-dependent manner by TGF-beta. Differential effects of TGF-beta on the proliferation of established leukemic lines were also observed since most cell lines of myelomonocytic nature studied were strongly inhibited where erythroid cell lines were either insensitive or poorly inhibited by TGF-beta. These results suggest that TGF-beta is an important modulator of human hematopoiesis that selectively regulates the growth of less mature hematopoietic cell populations with a high proliferative capacity as opposed to more differentiated cells, which are not affected by TGF-beta.     

Erythroid Differentiation of Fetal,Newborn and Adult Haemopoietic Stem Cells

Erythroid regeneration was studied in lethally irradiated mice given transplants containing equivalent numbers of haemopoietic stem cells (i.e. CFU) from fetal liver, neonatal marrow or adult marrow. Adult marrow was taken from normal control mice, whose CFU for the most part were not in active cell cycle, as well as from phenylhydrazine-treated groups whose CFU were in similar state of proliferation (i.e. ?40–50% in DNA synthesis) as those derived from fetal liver and neonatal marrow. Splenic and femoral radioiron (⁵⁹Fe) incorporation were measured at intervals after transplantation and were found to begin earliest in mice given fetal liver, then in animals given neonatal marrow and latest in recipients of adult marrow. Peripheral reticulocytes showed a similar pattern of recovery. The data reported herein suggest that the differences in erythroid regeneration evoked by transplants of fetal liver, neonatal marrow or adult marrow, are not solely attributed to the degree of proliferation in the pluripotential stem cell compartment. These data may, however, suggest a shorter doubling time for cells comprising the fetal and newborn committed erythroid compartments.     

Phosphatidylinositol 3-kinase is involved in the protection of primary cultured human erythroid precursor cells from apoptosis.

Little is known about the physiologic role of phosphatidylinositol 3-kinase (PI-3K) in the development of erythrocytes. Previous studies have shown that the effects of the PI-3K inhibitor wortmannin on erythropoietin (EPO)-dependent cell lines differed depending on the cell type used. Wortmannin inhibited EPO-induced differentiation of some cell lines without affecting their proliferation; however, the EPO-induced proliferation of other cell lines was inhibited by wortmannin. In neither case were signs of apoptosis observed. We have previously reported that signaling in highly purified human colony forming units-erythroid (CFU-E), generated in vitro from CD34(+) cells, differed from that in EPO-dependent cell lines. In the current study, we examined the effects of a more specific PI-3K inhibitor (LY294002) on human CFU-E. We found that LY294002 dose-dependently inhibits the proliferation of erythroid progenitor cells with a half-maximal effect at 10 micromol/L LY294002. LY294002 at similar concentrations also induces apoptosis of these cells, as evidenced by the appearance of annexin V-binding cells and DNA fragmentation. The steady-state phosphorylation of AKT at Ser-473 that occurs as a result of PI-3K activation was also inhibited by LY294002 at similar concentrations, suggesting that the effects of LY294002 are specific. Interestingly, the acceleration of apoptosis by LY294002 was observed in the presence or absence of EPO. Further, deprivation of EPO resulted in accelerated apoptosis irrespective of the presence of LY294002. Our study confirms and extends the finding that signaling in human primary cultured erythroid cells is significantly different from that in EPO-dependent cell lines. These data suggest that PI-3K has an antiapoptotic role in erythroid progenitor cells. In addition, 2 different pathways for the protection of primary erythroid cells from apoptosis likely exist: 1 independent of EPO that is LY294002-sensitive and one that is EPO-dependent and at least partly insensitive to LY294002.     

Influence of murine mast cell growth factor (c-kit ligand) on colony formation by mouse marrow hematopoietic progenitor cells.

Purified natural and recombinant murine mast cell growth factor (MGF, a c-kit ligand) were evaluated alone and in combination with other cytokines for effects in vitro on colony formation by multipotential (CFU-GEMM), erythroid (BFU-E) and granulocyte-macrophage (CFU-GM) progenitor cells from BDF1 mouse bone marrow. Both preparations stimulated Epo-dependent CFU-GEMM and enhanced Epo-dependent BFU-E colony numbers and size. MGF had some stimulating activity for CFU-GM. When used in combination with plateau concentrations of pokeweed mitogen mouse spleen cell conditioned medium or granulocyte-macrophage colony stimulating factor (CSF), MGF enhanced in greater than additive fashion colony formation by CFU-GM. MGF also enhanced the size of colonies formed, an enhancement greatest for colonies containing granulocytes and macrophages. MGF did not enhance Macrophage-CSF stimulated colony numbers or size. MGF seems to be an early acting cytokine with preferential effects on the growth of more immature hematopoietic progenitor cells.