Colony-forming cell proliferation: a rapid and sensitive assay system for murine granulocyte and macrophage colony-stimulating factors

A microculture assay for murine granulocyte-macrophage colony- stimulating factor (GM-CSF) has been developed using fetal liver GM colony-forming cells (CFC) isolated by fluorescence-activated cell sorting. These GM-CFC are free of mature hemopoietic cells, such as granulocytes and macrophages, which may interfere with direct assays for GM-CSF. The assay procedure allows the quantitation of GM-CSF within 48 hr by measuring the number of cells produced from 50 GM-CFC in microcultures (15 microliter). The assay is particularly simple to set up and score and yet, because of the reduced volumes, this assay is still capable of detecting 0.2 pg (i.e., 0.2 U) of GM-CSF within 48 hr, i.e., 100 times less GM-CSF than the conventional soft agar assay. By allowing the microcultures to develop for 7 days, the extra proliferation allows a further tenfold increase in the sensitivity of CSF detection. The time and cost of setting up hundreds of GM-CSF assays for fractions from chromatographic columns, e.g., reverse phase high performance liquid chromatography, is reduced by at least five- fold. Enough GM-CFC can be isolated and stored frozen in one afternoon to provide sufficient cells for the daily assay of 200 samples of GM- CSF for several months. Microassay results for several sources of GM- CSF at different stages of purification are compared to the results obtained from the soft agar assay.     

Abnormal responses of myeloid progenitor cells to recombinant human colony-stimulating factors in congenital neutropenia.

The effects of recombinant human interleukin-3 (IL-3) and recombinant human granulocyte colony-stimulating factor (G-CSF) on the growth of myeloid progenitor cells (CFU-C) in semisolid agar culture were studied in two patients with Kostmann-type congenital neutropenia. CFU-C growth in bone marrow cells from patients was significantly reduced in response to various concentrations of either IL-3 or G-CSF alone, compared with that from normal subjects. There was no inhibitory effect of bone marrow cells from patients on normal CFU-C formation supported by IL-3 or G-CSF. However, the simultaneous stimulation with IL-3 and G-CSF induced the increase of CFU-C formation in patients with congenital neutropenia. Furthermore, CFU-C growth in both patients was supported when bone marrow cells were preincubated with IL-3 in liquid culture followed by the stimulation with G-CSF in semisolid agar culture. In contrast, that was not supported by the preincubation with G-CSF and the subsequent stimulation with IL-3. This evidence suggests that the hematopoietic progenitor cells in patients with congenital neutropenia have the potential for developing CFU-C in the combined stimulation with IL-3 and G-CSF, and that this growth may be dependent on the priming of IL-3 followed by the stimulation with G-CSF. The level of mature neutrophils in peripheral blood was not fully restored to normal levels by the daily administration of G-CSF in doses of 100 to 200 micrograms/m2 of body surface area for 20 to 25 days in both patients. These observations raise the possibility that the combination of IL-3 and G-CSF might have a potential role for the increase of neutrophil counts in patients with congenital neutropenia.     

Abnormalities of primitive myeloid progenitor cells expressing granulocyte colony-stimulating factor receptor in patients with severe congenital neutropenia

To define the basis for faulty granulopoiesis in patients with severe congenital neutropenia (SCN), the expression of granulocyte colony-stimulating factor receptor (G-CSFR) in primitive myeloid progenitor cells and their responsiveness to hematopoietic factors were studied. Flow cytometric analysis of bone marrow cells based on the expression of CD34, Kit receptor, and G-CSFR demonstrated a reduced frequency of CD34(+)/Kit(+)/ G-CSFR(+) cells in patients with SCN. The granulocyte-macrophage colony formation of CD34(+)/Kit(+)/G-CSFR(+) cells in patients was markedly decreased in response to G-CSF alone and to the combination of stem cell factor, the ligand for flk2/flt3, and IL-3 with or without G-CSF in serum-deprived semisolid culture. In contrast, no difference in the responsiveness of CD34(+)/Kit(+)/G-CSFR(-) cells was noted between patients with SCN and subjects without SCN. These results demonstrate that the presence of qualitative and quantitative abnormalities of primitive myeloid progenitor cells expressing G-CSFR may play an important role in the impairment of granulopoiesis in patients with SCN. (Blood. 2000;96:4366-4369)     

Effects of recombinant human granulocyte colony-stimulating factor on hematopoietic progenitor cells in cancer patients

Hematopoietic progenitor cell levels were monitored in the peripheral blood and bone marrow of 30 cancer patients receiving recombinant human granulocyte-colony stimulating-factor (rG-CSF) in a phase I/II clinical trial. The absolute number of circulating progenitor cells of granulocyte-macrophage, erythroid, and megakaryocyte lineages showed a dose-related increase up to 100-fold after four days of treatment with rG-CSF and often remained elevated two days after the cessation of therapy. The relative frequency of different types of progenitor cells in peripheral blood remained unchanged. The frequency of progenitor cells in the marrow was variable after rG-CSF treatment but in most patients was slightly decreased. The responsiveness of bone marrow progenitor cells to stimulation in vitro by rG-CSF and granulocyte- macrophage colony-stimulating factor did not change significantly during rG-CSF treatment. In patients nine days after treatment with melphalan and then rG-CSF, progenitor cell levels were very low with doses of rG-CSF at or below 10 micrograms/kg/d, but equaled or exceeded pretreatment values when 30 or 60 micrograms/kg/d of rG-CSF was given.     

Expression of granulocyte colony-stimulating factor receptor increases with differentiation in myeloid cells by a newly-devised quantitative flow-cytometric assay

Summary. In order to develop a non-isotopic quantitative assay of granulocyte colony-stimulating factor (G-CSF) receptors on human or murine cells, we devised a flow-cytometric assay using cells stained with biotin-labelled G-CSF (b-G-CSF) and a streptavidin-RED670 conjugate. For quantification, we applied the Kolmogorov-Smirnov test and calculated the D value. The D value was evaluated from the degree of shift in two fluorescence profiles according to the increase of fluorescence intensity due to the specific binding of b-G-CSF to G-CSF receptors. A good correlation was observed between the number of G-CSF receptors obtained by the radioisotopic binding assay and the number calculated from the D value by the flow-cytometric assay. Then, expression of G-CSF receptors on human bone marrow cells, peripheral blood granulocytes and blast cells from patients with acute myeloid leukaemia (AML) were studied. G-CSF receptors was expressed on CD34⁺CD33⁻, CD34+CD33+ and CD34⁻CD33+ cells in the following order: CD34 CD33⁺CD34⁺CD33⁺CD34⁺CD33 cells, indicating that the receptors increased with maturation. The receptor levels of CD34⁻CD33⁺ cells in bone marrow were apparently lower than those of CD34⁻CD33⁺ cells in peripheral blood granulocytes. On the other hand, an abnormal expression pattern of G-CSF receptors was noted in AML blast cells.     

Growth response of human myeloid leukemia cells to colony-stimulating factors

The effects of human recombinant granulocyte and granulocyte-macrophage- (G- and GM-CSF), and of purified macrophage-stimulating factors (CSF-1), were tested on populations of leukemia cells isolated from 18 patients with different types of acute myeloid leukemia. Cell proliferation and differentiation were studied by culturing the cells in suspension for 7 days in the presence of CSF or medium alone. Spontaneous cell proliferation, as assessed by tritiated thymidine uptake, was observed in 9 of the 18 cases. GM-CSF induced proliferation in seven of the nine cases without spontaneous growth and increased spontaneous proliferation in nine cases. G-CSF added alone was also found to strongly stimulate leukemic blast cell proliferation, in which a translocation involving the long arm of chromosome 17 was observed. Low levels of CSF-1 stimulation were also observed in some cases. No clear morphological modification supporting evidence of terminal differentiation was observed, whereas modulation of some cell surface antigens was detected by flow cytometry. Thus, most leukemia cells still depend on growth factors for their proliferation, GM-CSF appearing the most effective. On the other hand these factors were not able to induce terminal differentiation.     

Decreased levels of myeloid progenitor cells associated with long-term administration of recombinant human granulocyte colony-stimulating factor in patients with autoimmune neutropenia

Summary. We studied the long-term in vivo effect of recombinant human granulocyte colony stimulating factor (rhG-CSF) on in vitro growth of granulocyte/macrophage colony forming cells (GM-CFC) in bone marrow and peripheral blood obtained from two patients with autoimmune neutropenia, who received rhG-CSF. Along with rhG-CSF treatment for more than 40 d, numbers of GM-CFC-derived colonies from both bone marrow and peripheral blood gradually decreased to a significant level though white blood cells in peripheral blood and nucleated cells in bone marow were increased in number. This observation suggests that long-term administration of rhG-CSF may preferentially activate a differentiation pathway for granulopoiesis while proliferation of GM-CFC is not induced as expected in response to rhG-CSF.     

Heterogeneous expression pattern of pro- and anti-apoptotic factors in myeloid progenitor cells of patients with severe congenital neutropenia treated with granulocyte colony-stimulating factor

Apoptosis is accelerated in the myeloid progenitor cells of patients with severe congenital neutropenia (CN). Granulocyte colony-stimulating factor (G-CSF) increases neutrophil numbers in most CN patients. The effect of G-CSF on apoptosis in CN was analysed by apoptosis rate and expression of anti- and pro-apoptotic factors. G-CSF-treated patients showed higher apoptosis frequency, lower expression of bcl-2 and bcl-xL, but higher expression of bfl-1/A1 and mcl-1. Caspase 9 was highly expressed in patients and controls after G-CSF administration. Thus, G-CSF acts on apoptosis regulation, but additional mechanisms leading to the increase of neutrophil numbers must be assumed.     

Induction of granulocyte and granulocyte-macrophage colony-stimulating factors from human monocytes stimulated by Fc fragments of human IgG

The effect of human IgG on human haemopoiesis has been studied in vitro. Dialysed purified IgG stimulated haemopoietic colony growth by bone marrow mononuclear cells (MNC) but not by monocyte-depleted MNC. Culture media, conditioned by IgG-stimulated peripheral blood MNC, augmented formation of neutrophil-macrophage, eosinophil, and megakaryocyte colonies by monocyte-depleted marrow MNC. Serum-free IgG-conditioned media also contained colony-stimulating activity (CSA). IgG Fc fragments and heat-aggregated IgG promoted the secretion of CSA, but F(ab')2 fragments, Fab fragments or ultracentrifuged IgG did not. In the cell-selection studies, CSA was produced by highly enriched monocytes following stimulation with Fc fragments. The antiserum against human granulocyte colony-stimulating factor (G-CSF) and/or granulocyte-macrophage CSF (GM-CSF) neutralized the CSA produced by Fc fragment-activated monocytes. Enzyme immunoassays showed G-CSF and GM-CSF in media conditioned by monocytes stimulated with the Fc fragments, heat-aggregated IgG and anti-D-sensitized red blood cells (RBC). Northern hybridization analysis showed mRNA encoding G-CSF and GM-CSF in RNA extracted from MNC and monocytes cultured with the Fc fragments, but not in the RNA from unstimulated cells or monocyte-depleted MNC. These results indicate that IgG Fc fragments, aggregated IgG and antigen-antibody complexes induce monocytes to produce G-CSF and GM-CSF in vitro. The CSFs release induced by IgG may be involved in the in vivo regulatory network in haemopoiesis.     

Purified murine granulocyte/macrophage progenitor cells express a high-affinity receptor for recombinant murine granulocyte/macrophage colony-stimulating factor

Purified recombinant murine granulocyte/macrophage colony-stimulating factor (GM-CSF) was labeled with 125I and used to examine the GM-CSF receptor on unfractionated normal murine bone marrow cells, casein-induced peritoneal exudate cells, and highly purified murine granulocyte/macrophage progenitor cells (CFU-GM). CFU-GM were isolated from cyclophosphamide-treated mice by Ficoll-Hypaque density centrifugation followed by counterflow centrifugal elutriation. The resulting population had a cloning efficiency of 62-99% in cultures containing conditioned medium from pokeweed mitogen-stimulated spleen cells and 55-86% in the presence of a plateau concentration of purified recombinant murine GM-CSF. Equilibrium binding studies with 125I-labeled GM-CSF showed that normal bone marrow cells, casein-induced peritoneal exudate cells, and purified CFU-GM had a single class of high-affinity receptor with an approximate Ka of 10(8)-10(9) M-1. CFU-GM expressed an average of 3783 +/- 4 receptors per cell; normal bone marrow cells, 1518 +/- 242 receptors per cell; and peritoneal exudate cells, 2025 +/- 216 receptors per cell. Affinity crosslinking studies demonstrated that 125I-labeled GM-CSF bound specifically to two species of Mr 180,000 and 70,000 on CFU-GM, normal bone marrow cells, and peritoneal exudate cells. The Mr 70,000 species is thought to be a proteolytic fragment of the intact Mr 180,000 receptor. The present studies indicate that the GM-CSF receptor expressed on CFU-GM and mature myeloid cells are structurally similar. In addition, the number of GM-CSF receptors on CFU-GM is twice the average number of receptors on casein-induced mature myeloid cells, suggesting that receptor number may decrease as CFU-GM mature.     

Stimulation of human hematopoietic progenitor cell proliferation and differentiation by recombinant human interleukin 3. Comparison and interactions with recombinant human granulocyte-macrophage and granulocyte colony-stimulating factors

Recombinant human interleukin 3 (IL3) produced in Escherichia coli was purified and its activities examined in cultures of highly enriched human bone marrow progenitor cells. Human IL3 stimulated multipotential (CFU-GEMM) and erythroid (BFU-E) progenitor cells, generating 95% more BFU-E than recombinant human granulocyte-macrophage colony-stimulating factor (GM-CSF). No further enhancement of BFU-E or CFU-GEMM occurred when IL3 and GM-CSF were used in combination. Human IL3 was more effective than GM-CSF in stimulating granulocyte-macrophage colony-forming cells (CFU-GM) in short-term suspension cultures, but did not induce an increase of CFU-GM, BFU-E, or CFU-GEMM above input levels. IL3 was more active on day-14 (d14) than on d7 CFU-GM, similar to GM-CSF, but generated fewer and smaller CFU-GM-derived clones than either GM-CSF or granulocyte CSF (CI-CSF). The simultaneous addition of plateau levels of IL3 and GM-CSF resulted in an infra-additive augmentation of d7 and d14 CFU-GM-derived clones, whereas IL3 and G-CSF enhanced the number and cellularity predominantly of d14 CFU-GM. In liquid cultures, IL3 induced a greater than 100-fold increase in the number of basophil-mast-like cells and eosinophils and allowed maintenance of these cultures for up to 7 weeks. Human GM-CSF was an almost equally potent, stimulus of eosinophil development but had only a marginal effect on basophilic precursors, whereas G-CSF lacked both activities. Therefore, human IL3 is a multilineage hemopoietic growth factor whose activities appear to encompass and extend beyond those of GM-CSF.     

Putative involvement of protein kinase C in proliferation of human myeloid progenitor cells

The effects of two different potent inhibitors of protein kinase C, 1- (5-isoquinolinylsulfonyl)-2-methylpiperazine (H-7) and staurosporine on human myeloid (CFU-C) and late erythroid progenitor cells (CFU-E) were studied using an in vitro clonal assay. Our objective was to determine whether protein kinase C has a role in signal transduction related to proliferation of these committed progenitor cells. The presence of H-7 or staurosporine led to an inhibition of colony formation stimulated by crude colony-stimulating factor (CSF), interleukin-3 (IL-3), granulocyte-macrophage CSF (GM-CSF), granulocyte CSF (G-CSF), or macrophage CSF (M-CSF) in a dose-dependent manner. N-(2-guanidinoethyl)- 5-isoquinolinesulfonamide (HA-1004), a weaker analog of H-7, did not inhibit proliferation of CFU-C. Neither H-7 nor staurosporine had any effect on CFU-E formation. H-7 and staurosporine dose-dependently inhibited the protein kinase C from K562 cells. The potential of these compounds to inhibit proliferation of CFU-C correlated well with the magnitude of their inhibition of protein kinase C from K562 cells. The inhibition of proliferation of CFU-C appears to relate to the potential of these compounds to inhibit protein kinase C. Thus, activation of protein kinase C is presumably involved in the proliferation of CFU-C, and the regulatory system of CFU-E appears to differ from that of CFU-C.     

Effects of purified human native granulocyte colony-stimulating factor (G-CSF) on proliferation of blast progenitors in acute myeloblastic leukemia

Blast progenitors in acute myeloblastic leukemia (AML) grow in methylcellulose and suspension cultures. Blast colony formation in methylcellulose culture reflects the terminal divisions of blast progenitors, while secondary colony formation, by replating in methylcellulose and recovering clonogenic cells in suspension culture, reflects the self-renewal of blast progenitors. To analyze the regulatory mechanisms of the proliferation of leukemic blast progenitors, the effects of highly purified native granulocyte colony-stimulating factor (G-CSF) obtained from human squamous cell carcinoma line (CHU-2) on blast progenitors in AML patients were studied in methylcellulose and suspension cultures. Purified G-CSF stimulated the growth of blast progenitors in both culture systems, although sensitivity to G-CSF varied from patient to patient. No obvious maturation of leukemic blasts was noted in suspension culture in the presence of G-CSF. The data suggest that a normal hemopoietic regulator may play a role in the growth of blast progenitors in AML patients.     

粒细胞集落刺激因子与动脉粥样硬化对外周血内皮祖细胞数量的影响

目的观察粒细胞集落刺激因子（granuloeyte colony-stimulating factor,G-CSF）与动脉粥样硬化（atherosclerosis,AS）对外周血内皮祖细胞（EPC）的影响。方法将32只雄性新西兰白兔随机分为G组（重组人粒细胞集落刺激因子rhG-CSF50彬d）、G＋AS组（rhG-CSF 50μg/d、高脂饲料）、AS组（高脂饲料）及对照组,每组各8只。4组动物分别于实验前及第1、4、8、12周采血,培养7天后,用荧光显微镜观察鉴定FITC-UEA-1和Dil-acLDL双染色阳性细胞为正在分化的EPC;细胞培养3天后,通过流式细胞仪计数各组PE-CD34、FITC-CD133双阳性细胞为EPC;第12周测血清一氧化氮、血脂水平并做主动脉斑块分析。结果实验前,各组EPC含量均很低;用G-CSF治疗1周,G组及G＋AS组EPC迅速升高（与用药前比较增加了约13倍,P〈0．001）,继续给药G组EPC维持在一个较高水平（第1、4、8、12周比较P〉0．05）;给予高脂饮食后,G＋AS组EPC数量逐渐下降（第4、8周与对照组比较P〈0．001、P〈0．001;第12周与对照组比较P=0．326）;对照组EPC一直处于低水平,AS组EPC数量较对照组低,但两组各周比较P〉0．05。经过12周的高脂喂养,G＋AS组及AS组主动脉均形成了动脉粥样硬化斑块,但G＋AS组斑块面积、斑块／内膜面积均低于AS组（P〈0．01）。第12周G＋AS组及AS组一氧化氮含量均低于G组和对照组（P〈0．001）。结论EPC与动脉粥样硬化关系密切,AS损害内皮,减少EPC,G-CSF对EPC有动员作用,能够增加外周血EPC数量,因而对血管有保护作用,抑制AS进展。     

Activities of four purified growth factors on highly enriched human hematopoietic progenitor cells

The activities of four purified human growth factors: biosynthetic (recombinant) granulocyte-macrophage colony-stimulating factor (GM- CSF); recombinant erythroid-potentiating activity (EPA); natural and recombinant pluripoietin (Ppo); and natural pluripoietin alpha (Ppo alpha), were compared on the growth of hematopoietic colonies from enriched populations of human marrow and blood progenitor cells. Conditioned medium from the Mo T cell line (MoCM) was used as a standard positive control. We found that activities of GM-CSF and Ppo alpha on the growth of hematopoietic colonies were indistinguishable; Ppo alpha is now believed to be identical to GM-CSF. Both factors were able to promote the growth of colonies derived from subpopulations of CFU-GM, BFU-E, and CFU-GEM. Colonies derived from CFU-GM and CFU-GEM in cultures stimulated by GM-CSF and Ppo alpha were much smaller than in cultures stimulated by MoCM. In contrast to previous reports in which less highly enriched progenitors were used as target cells, Ppo had no detectable activity on the growth of colonies derived from BFU-E or CFU- GEM but promoted the growth of a subpopulation of CFU-GM derived colonies. Ppo is now recognized to be identical to G-CSF. The GM colonies in cultures stimulated by G-CSF (Ppo) were much smaller than in cultures stimulated by MoCM. EPA had no detectable activity on either the size or number of colonies derived from CFU-GM, BFU-E, or CFU-GEM. Results from experiments using target cell populations of marrow fractions separated by velocity sedimentation and marrow populations following freezing suggested that GM-CSF (Ppo alpha) and G- CSF (Ppo) primarily affect the growth of relatively mature subpopulations of progenitor cells. It is clear from these results that additional factor(s) are present in MoCM that are necessary to stimulate CFU-GM, BFU-E, and CFU-GEM maximally in vitro.     

Quantitative and cell-cycle differences in progenitor cells mobilized by recombinant human interleukin-7 and recombinant human granulocyte colony-stimulating factor

Administration of recombinant human interleukin-7 (rhIL-7) to mice increases the exportation of myeloid progenitors (colony-forming unit [CFU]-c and CFU-granulocyte erythroid megakaryocyte macrophage [CFU- GEMM]) from the bone marrow (BM) to peripheral organs, including blood, and also increases the number of primitive progenitor and stem cells in the peripheral blood (PB). We now report that combined treatment of mice with rhIL-7 and recombinant human granulocyte-colony stimulating factor (rhG-CSF) stimulates a twofold to 10-fold increase in the total number of PB CFU-c, and a twofold to fivefold increase in the total number of PB CFU-spleen at day 8 (CFU-S8) over the increase stimulated by rhIL-7 or rhG-CSF alone. In addition, the quality of mobilized cells with trilineage, long-term marrow-repopulating activity is maintained or increased in mice treated with rhIL-7 and rhG-CSF compared with rhIL- 7 or rhG-CSF alone. These differences in mobilizing efficiency suggest qualitative differences in the mechanisms by which rhIL-7 and rhG-CSF mobilize progenitor cells, in fact, the functional status of progenitor cells mobilized by rhIL-7 differs from that of cells mobilized by rhG- CSF in that the incidence of actively cycling (S-phase) progenitors obtained from the PB is about 20-fold higher for rhIL-7-treated mice than for mice treated with rhG-CSF. These results suggest the use of rhIL-7-mobilized progenitor/stem cells for gene-modification and tracking studies, and highlight different functions and rates of repopulation after reconstitution with PB leukocytes obtained from mice treated with rhIL-7 versus rhG-CSF.     

Recombinant granulocyte colony-stimulating factor administration to healthy volunteers: induction of immunophenotypically and functionally altered neutrophils via an effect on myeloid progenitor cells

We performed a detailed kinetic study on the in vivo effect of a single subcutaneous dose of granulocyte colony-stimulating factor (G-CSF; 300 micrograms) in four healthy individuals on the expression and function of neutrophil Fc gamma receptors (Fc gamma R). G-CSF did not induce Fc gamma RI (CD64) on circulating neutrophils. However, neutrophils newly formed in response to G-CSF were Fc gamma RI positive and were able to perform antibody-dependent cellular cytotoxicity in an Fc gamma RI- dependent way. Fc gamma RII (CD32) expression was not changed significantly. Fc gamma RIII (CD16, phosphatidylinositol-linked) expression, slightly increased immediately (30 minutes) postinjection, was found to be strongly decreased on the newly formed population. For comparison, we studied the expression of the PI-linked proteins leukocyte alkaline phosphatase (LAP) and CD14. Intracellular levels of LAP mirrored the biphasic expression pattern as membrane-bound Fc gamma RIII. In contrast, CD14 expression on neutrophils was initially constant, followed by high levels on the newly formed neutrophils. Soluble CD14 levels were found to be elevated transiently, whereas peak levels of soluble Fc gamma III were observed as late as 6 days postinjection. In conclusion, we have shown that G-CSF results in an immunophenotypically and functionally altered neutrophil population for an important part as a result of its effect on myeloid precursor cells.