Antagonistic effects of interleukin 6 and G-CSF in the later stage of human granulopoiesis in vitro.

The effect of recombinant human interleukin 6 (rhIL-6) on the in vitro growth of human bone marrow myeloid progenitors (granulocyte-macrophage colony-forming units, CFU-GM) was investigated. Recombinant human IL-6 by itself did not induce colony formation. When rhIL-6 at various concentrations was added to the CFU-GM colony cultures containing recombinant human granulocyte colony-stimulating factor (rhG-CSF) or recombinant human granulocyte-monocyte/macrophage colony-stimulating factor (rhGM-CSF), rhIL-6 significantly suppressed the colony formation induced by rhG-CSF, but not by rhGM-CSF. This suppressive effect of rhIL-6 on rhG-CSF-induced, but not rhGM-CSF-induced colony formation was confirmed by using an MY10(+)-cell-enriched population. Neither interleukin 3 nor interleukin 1 alpha suppressed the growth of myeloid progenitors. The preincubation of bone marrow cells with rhIL-6 for a short time (30 min) resulted in a reduction of colonies induced by rhG-CSF, but not by rhGM-CSF. The suppressive effect of rhIL-6 on rhG-CSF-induced colony formation was not observed when the cells were preincubated together with rhG-CSF at a high ratio of rhG-CSF to rhIL-6. The rhIL-6-mediated suppressive effect was further confirmed by blocking the effect by the anti-IL-6 antibody. These results suggest antagonistic interaction between IL-6 and G-CSF in the later differentiation of myeloid progenitors.     

Various human hematopoietic growth factors (interleukin-3, GM-CSF, G-CSF) stimulate clonal growth of nonhematopoietic tumor cells

We have studied the effect of recombinant human hematopoietic growth factors (interleukin-3 [rhIL-3], granulocyte-macrophage colony-stimulating factor [rhGM-CSF], and granulocyte CSF [rhG-CSF]) on the clonal growth of human colon adenocarcinoma cell lines HTB-38, CCL 187, and WiDr (CCL 218). The factors stimulated clonal growth of HTB-38 and CCL 187 in a capillary modification of the human tumor clonogenic assay in agar up to twofold. There were dose-response correlations over a range of 1 to 10,000 U/mL for rhIL-3, rhGM-CSF, and rhG-CSF. Incubation with neutralizing monoclonal antibodies abolished the stimulation of clonal growth by rhGM-CSF. The WiDr cell line was nonresponsive to rhIL-3 and rhGM-CSF. These results represent the first evidence that a variety of hematopoietic growth factors can stimulate the growth of clonogenic cells of some nonhematopoietic malignant cell lines in vitro.     

In vivo synergy between recombinant human stem cell factor and recombinant human granulocyte colony-stimulating factor in baboons enhanced circulation of progenitor cells

Recombinant human stem cell factor (rhSCF) and recombinant human granulocyte colony-stimulating factor (rhG-CSF) are synergistic in vitro in stimulating the proliferation of hematopoietic progenitor cells and their precursors. We examined the in vivo synergy of rhSCF with rhG-CSF for stimulating hematopoiesis in vivo in baboons. Administration of low-dose (LD) rhSCF (25 micrograms/kg) alone did not stimulate changes in circulating WBCs. In comparison, administration of LD rhSCF in combination with rhG-CSF at 10 micrograms/kg or 100 micrograms/kg stimulated increases in circulating WBCs of multiple types up to twofold higher than was stimulated by administration of the same dose of rhG-CSF alone. When the dose of rhG-CSF is increased to 250 micrograms/kg, the administration of LD rhSCF does not further increase the circulating WBC counts. Administration of LD rhSCF in combination with rhG-CSF also stimulated increased circulation of hematopoietic progenitors. LD rhSCF alone stimulated less of an increase in circulating progenitors, per milliliter of blood, than did administration of rhG-CSF alone at 100 micrograms/kg. Baboons administered LD rhSCF together with rhG-CSF at 10, 100, or 250 micrograms/kg had 3.5- to 16-fold higher numbers per milliliter of blood of progenitors cells of multiple types, including colony-forming units granulocyte/macrophage (CFU-GM), burst-forming unit-erythroid (BFU-E), and colony-forming and burst-forming units-megakaryocyte (CFU- MK and BFU-MK) compared with animals given the same dose of rhG-CSF without rhSCF, regardless of the rhG-CSF dose. The increased circulation of progenitor cells stimulated by the combination of rhSCF plus rhG-CSF was not necessarily directly related to the increase in WBCs, as this effect on peripheral blood progenitors was observed even at an rhG-CSF dose of 250 micrograms/kg, where coadministration of LD rhSCF did not further increase WBC counts. Administration of very-low- dose rhSCF (2.5 micrograms/kg) with rhG-CSF, 10 micrograms/kg, did not stimulate increases in circulating WBCs, but did increase the number of megakaryocyte progenitor cells in blood compared with rhG-CSF alone. LD rhSCF administered alone for 7 days before rhG-CSF did not result in increased levels of circulating WBCs or progenitors compared with rhG- CSF alone. Thus, the synergistic effects of rhSCF with rhG-CSF were both dose- and time-dependent. The doses of rhSCF used in these studies have been tolerated in vivo in humans.(ABSTRACT TRUNCATED AT 400 WORDS)     

Differential activity of recombinant colony-stimulating factors in supporting proliferation of human peripheral blood and bone marrow myeloid progenitors in culture

Unlike bone marrow progenitor cells, human myeloid progenitors isolated from peripheral blood do not form colonies in semi-solid medium in the presence of rhG-CSF, rhM-CSF or rhIL-6, but do form colonies containing neutrophils, macrophages, eosinophils, basophils or mixed neutrophilic-macrophages colonies in the presence of rhIL-3 or rhGM-CSF. Priming of blood progenitors by culturing them for several days in the presence of rhGM-CSF resulted in a dramatic increase in the frequency of cells that proliferate in response to G-CSF and IL-6 and form neutrophilic granulocytic colonies. Suspension cultures maintained in the presence of IL-3 yielded increased numbers of clonogenic cells responsive to GM-CSF and G-CSF, but not to M-CSF or IL-6. rhIL-6 did not directly stimulate colony formation of peripheral blood progenitors but did prime them to respond to G-CSF. These results are consistent with a hierarchical model of granulocytic differentiation in which circulating progenitors proceed sequentially through a programme of changing growth factor sensitivity with the following sequence: IL-3, GM-CSF, IL-6 and/or G-CSF.     

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.     

Effect of granulocyte-macrophage colony-stimulating factor and interleukin-3 on interleukin-8 production by human neutrophils and monocytes

Interleukin-8 (IL-8) is a major neutrophil chemoattractant and functional stimulant that is induced by IL-1, tumor necrosis factor alpha (TNF alpha), and lipopolysaccharide (LPS). We report that recombinant human (rh) granulocyte-macrophage colony-stimulating factor (GM-CSF) and rhIL-3 are also potent inducers of IL-8 messenger RNA (mRNA) accumulation and protein secretion by normal peripheral blood monocytes. Neutrophils produce IL-8 in response to GM-CSF but not to IL- 3. In contrast, recombinant human granulocyte-CSF (rhG-CSF), at concentrations as high as 100 ng/mL, does not induce IL-8 in either cell type. rhGM-CSF also induces IL-8 mRNA expression and IL-8 protein in the promonocytic cell line, U-937, whereas rhG-CSF does not. IL-8 secretion by monocytes was stimulated within 2 hours after incubation with rhGM-CSF or rhIL-3. Stimulation of neutrophils with rhGM-CSF resulted in an increase in cell-associated IL-8 at 4 hours. At 24 hours, cell-associated IL-8 levels declined, whereas secreted IL-8 levels increased. In contrast, virtually all IL-8 induced in monocytes appeared as secreted protein. Neither rhGM-CSF nor rhIL-3 induced detectable secretion of IL-1, TNF alpha, or IL-6 protein by monocytes. rhGM-CSF, and to a lesser degree rhIL-3, potently stimulated IL-8 secretion in cultures of heparinized whole blood, whereas rhG-CSF had no significant effect on IL-8 secretion. Induction of IL-8 by GM-CSF may be physiologically important in enhancing the acute inflammatory response.     

Synergism of interleukin-12 and interleukin-3 on development of hematopoietic progenitors

Abstract: The recently cloned cytotoxic lymphocyte maturation factor [CLMF] also called NK cell stimulatory factor [NKSF] or interleukin-12 [IL-12] has been described as a growth factor for mature lymphoid cells. The present study investigated whether purified recombinant human IL-12 could stimulate CFU colony growth. Source of progenitor cells were peripheral blood cells depleted of adherent, CD2- and CD56-positive cells. RhIL-12 was investigated either alone or in combination with rhIL-3, rhIL-6 and rhGM-CSF. RhIL-12 alone did not support colony formation of myeloid or erythroid progenitors. RhIL-12 in combination with rhIL-3 increased the numbers of BFU-E and CFU-GM. No synergism or additive effect was seen with the combination of rhIL-12 and rhGM-CSF or rhIL-12 and rhIL-6. An additive increase in the number of granulocytic colonies was observed when rhIL-3, rhIL-6 and rhGM-CSF were used together with rhIL-12. Our result therefore suggest that, in addition to being a potent lymphopoietic stimulator, IL-12 acts synergistically with IL-3 in enhancing the sensitivity of hemopoietic progenitors to IL-3.     

Rapid engraftment by peripheral blood progenitor cells mobilized by recombinant human stem cell factor and recombinant human granulocyte colony-stimulating factor in nonhuman primates

We have previously shown that administration of low-dose recombinant human stem cell factor (rhSCF) plus recombinant human granulocyte colony-stimulating factor (rhG-CSF) to baboons mobilizes greater numbers of progenitor cells in the blood than does administration of rhG-CSF alone. The purpose of the present study was to determine whether marrow repopulating cells are present in the blood of nonhuman primates administered low-dose rhSCF plus rhG-CSF, and if present, whether these cells engraft lethally irradiated recipients as rapidly as blood cells mobilized by treatment with rhG-CSF alone. One group of baboons was administered low-dose rhSCF (25 micrograms/kg/d) plus rhG- CSF (100 micrograms/kg/d) while a second group received rhG-CSF alone (100 micrograms/kg/d). Each animal underwent a single 2-hour leukapheresis occurring the day when the number of progenitor cells per volume of blood was maximal. For baboons administered low-dose rhSCF plus rhG-CSF, the leukapheresis products contained 1.8-fold more mononuclear cells and 14.0-fold more progenitor cells compared to the leukapheresis products from animals treated with rhG-CSF alone. All animals successfully engrafted after transplantation of cryopreserved autologous blood cells. In animals transplanted with low-dose rhSCF plus rhG-CSF mobilized blood cells, we observed a time to a platelet count of > 20,000 was 8 days +/- 0, to a white blood cell count (WBC) of > 1,000 was 11 +/- 1 days, and to an absolute neutrophil count (ANC) of > 500 was 12 +/- 1 days. These results compared with 42 +/- 12, 16 +/- 1, and 24 +/- 4 days to achieve platelets > 20,000, WBC > 1,000, and ANC > 500, respectively, for baboons transplanted with rhG-CSF mobilized blood cells. Animals transplanted with low-dose rhSCF plus rhG-CSF mobilized blood cells had blood counts equivalent to pretransplant values within 3 weeks after transplant. The results suggest that the combination of low-dose rhSCF plus rhG-CSF mobilizes greater numbers of progenitor cells that can be collected by leukapheresis than does rhG-CSF alone, that blood cells mobilized by low-dose rhSCF plus rhG-CSF contain marrow repopulating cells, and finally that using a single 2-hour leukapheresis to collect cells, the blood cells mobilized by low-dose rhSCF plus rhG-CSF engraft lethally irradiated recipients more rapidly than do blood cells mobilized by rhG- CSF alone.     

Clonal growth of functionally normal and deficient neutrophils from the bone marrow of a patient with variant chronic granulomatous disease

Summary To evaluate the effect of colony-stimulating factors and interferon on the oxidative burst capacity of neutrophils in chronic granulomatous disease (CGD) we studied the neutrophils of a patient with variant CGD both from peripheral blood and from bone marrow culture on day 7 and 14. The results revealed that preincubation of peripheral neutrophils for 24 h in medium containing recombinant human granulocyte colony-stimulating factor (rhG-CSF), recombinant human granulocytemacrophage colony-stimulating factor (rhGM-CSF), and recombinant human interferon gamma (rhIFN-) alone or in combination did not improve the maximal oxidative burst activity measured by MTT assay. The colonies of this patient formed in agar assay were either composed of predominantly nitroblue tetrazolium (NBT)-positive cells or completely unable to reduce NBT. Despite variable colony numbers in the presence of different cytokines, the rate of NBT-positive colonies was less than 17% of the total number of colonies. However, more than 72% of the colonies were NBT positive in controls. In liquid culture, bone marrow cells yielded a comparable rate of NBT-positive and -negative populations at day 7. These data indicate that rhG-CSF, rhGM-CSF, and rhIFN- alone or rhG-CSF and rhGM-CSF in combination with rhIFN- are not able to reconstitute the oxidative burst defect in CGD in vitro. Indeed, regarding colony-forming capacity, the bone marrow cells from the patient responded to CSFs as well as those from control donors did. This fact may warrant the administration of hematopoietic growth factors, at least in variant CGD, in order to enhance the absolute number of functionally normal neutrophils.     

A comparison of treatment of canine cyclic hematopoiesis with recombinant human granulocyte-macrophage colony-stimulating factor (GM-CSF), G-CSF interleukin-3, and canine G-CSF.

Cyclic hematopoiesis in gray collie dogs is a stem cell disease in which abnormal regulation of cell production in the bone marrow causes cyclic fluctuations of blood cell counts. In vitro studies demonstrated that recombinant human granulocyte-macrophage colony-stimulating factor (GM-CSF), interleukin-3 (IL-3), and granulocyte colony stimulating factor (G-CSF) all stimulated increases in colony formation by canine bone marrow progenitor cells. Based on these results, gray collie dogs were then treated with recombinant human (rh) GM-CSF, IL-3, or G-CSF subcutaneously to test the hypothesis that pharmacologic doses of one of these hematopoietic growth factors could alter cyclic production of cells. When recombinant canine G-CSF became available, it was tested over a range of doses. In vivo rhIL-3 had no effect on the recurrent neutropenia but was associated with eosinophilia, rhGM-CSF caused neutrophilia and eosinophilia but cycling of hematopoiesis persisted. However, rhG-CSF caused neutrophilia, prevented the recurrent neutropenia and, in the two animals not developing antibodies to rhG-CSF, obliterated periodic fluctuation of monocyte, eosinophil, reticulocyte, and platelet counts. Recombinant canine G-CSF increased the nadir neutrophil counts and amplitude of fluctuations at low doses (1 micrograms/kg/d) and eliminated all cycling of cell counts at high doses (5 and 10 micrograms/kg/d). These data suggest significant differences in the actions of these growth factors and imply a critical role for G-CSF in the homeostatic regulation of hematopoiesis.     

The polysaccharide, PGG-glucan, enhances human myelopoiesis by direct action independent of and additive to early-acting cytokines.

beta-Glucans stimulate leukocyte anti-infective activity, enhance murine hematopoietic recovery following bone marrow injury and mobilize murine progenitor cells from bone marrow. This study evaluated the in vitro hematopoietic potential of the beta-glucan, PGG-glucan, on human bone marrow mononuclear cells (BMMC) and CD34+ BMMC compared with protein cytokines. In the presence of submaximal concentrations of recombinant human granulocyte-macrophage colony-stimulating factor (rhGM-CSF; 0.5 ng/ml), PGG-glucan significantly increased BMMC myeloid colony formation comparable to the increase observed with either interleukin-3 (rhIL-3) or stem cell factor (rhSCF). Moreover, the addition of PGG-glucan to cultures containing GM-CSF + IL-3 or GM-CSF + SCF significantly augmented granulocyte-macrophage colony production above baseline, demonstrating that PGG-glucan acts independently of those early-acting cytokines and can enhance their activity in an additive manner. Anti-PGG-glucan monoclonal antibody specifically abrogated the growth-enhancing effect of added PGG-glucan in a saturable manner and other control carbohydrate polymers failed to affect colony formation. Further, PGG-glucan was not associated with induction of IL-6, GM-CSF production and removal of accessory cells by CD34+ cell isolation did not alter the PGG-glucan effect. These data demonstrate that PGG-glucan acts on committed myeloid progenitors to enhance human hematopoietic activity by a mechanism of direct action independent of IL-3 or SCF and independent of secondary cytokine stimulation.     

Demonstration that human mast cells arise from a progenitor cell population that is CD34(+), c-kit(+), and expresses aminopeptidase N (CD13).

Human mast cells are known to arise from a CD34(+)/c-kit(+) progenitor cell population that also gives rise to neutrophils, eosinophils, basophils, and monocytes. To further characterize cells within the CD34(+)/c-kit(+) population that yield mast cells, this progenitor was additionally sorted for CD13, a myeloid marker known to appear early on rodent mast cells and cultured human mast cells, but not expressed or expressed at low levels on human tissue mast cells; and cultured in recombinant human (rh) stem cell factor (rhSCF), rh interleukin-3 (rhIL-3; first week only), and rhIL-6. Initial sorts revealed that although the majority of cells in culture arose from the CD34(+)/c-kit(+)/CD13(-) cell population, mast cells arose from a CD34(+)/c-kit(+)/CD13(+) progenitor cell that also gave rise to a population of monocytes. Sequential sorting confirmed that CD34(+)/c-kit(+)/CD13(+) cells in CD34(+)/c-kit(+)/CD13(-) sorts gave rise to the few mast cells observed in CD13(-) sorted cells. CD34(+)/c-kit(+)/CD13(+) cells plated as single cells in the presence of various cytokine combinations gave rise to pure mast cell, monocyte, or mixed mast cell/monocyte progeny. Addition of either rh granulocyte-macrophage colony-stimulating factor (rhGM-CSF) or rhIL-5 to the CD34(+)/c-kit(+)/CD13(+) progenitor cell population cultured in rhSCF, rhIL-3, and rhIL-6 did increase the number of total cells cultured and in the case of rhIL-5, did increase total mast cell numbers. Neither rhGM-CSF or rhIL-5 led to additional cell populations, ie, even with the addition of rhGM-CSF or rhIL-5, only mast cells and monocytes grew from CD34(+)/c-kit(+)/CD13(+) cells. Thus, human mast cells and a population of monocytes arise from precursor cells that express CD34, c-kit, and CD13; and within which, are mast cell, monocyte, and mast/monocyte (bipotential) precursors.     

Enhancement of release from MHC class II antigen-positive monocytes of hematopoietic colony stimulating factors CSF-1 and G-CSF by recombinant human tumor necrosis factor-alpha: synergism with recombinant human interferon-gamma

The influence of purified recombinant human tumor necrosis factor-alpha (rhuTNF-alpha) was assessed alone and in combination with purified recombinant human interferon gamma (rhuIFN-gamma) for its effects on enhancing release from human monocytes of activities that stimulate colony formation by granulocyte-macrophage (CFU-GM), erythroid (BFU-E), and multipotential (CFU-GEMM) progenitor cells. RhuTNF-alpha or rhuIFN-gamma enhanced release of colony stimulating factors (CSFs), which were determined by a combination of human and mouse colony assays, morphological assessment of colony types and neutralization studies with anti-human macrophage CSF (CSF-1) and anti-human granulocyte (G)-CSF to be CSF-1 and G-CSF. The activity in the uninduced and induced monocyte conditioned media (CM) for CFU-GM-type colonies and clusters was attributed to the presence of both CSF-1 and G-CSF, while the activity in the monocyte CM for BFU-E and CFU-GEMM colonies was attributed to the presence of G-CSF. Monocytes were separated by two-color fluorescence using a dye laser flow cytometry system with cells labeled with anti-leu M3 conjugated with fluorescein isothiocyanate and anti-HLA-DR conjugated with phycoerythrin. While "constitutive" release of CSFs from monocytes was apparent from both the leu M3+, HLA-DR+ and the leu M3+, HLA-DR- (low density or negative DR) fractions, enhanced release of CSFs in response to rhuTNF-alpha or rhuIFN-gamma was confined to the leu M3+, HLA-DR+ population of cells. RhuTNF-alpha and rhuIFN-gamma synergized to enhance release of CSFs such that low concentrations of each molecule, which were inactive when used alone, were active when the two molecules were used together. These studies suggest a role, at least in vitro, for TNF-alpha and IFN-gamma in the release of CSFs from cells of the mononuclear phagocytic lineage.     

Stimulation of canine hematopoiesis by recombinant human granulocyte-macrophage colony-stimulating factor

The effect of recombinant human granulocyte-macrophage colony-stimulating factor (rhGM-CSF) on canine hematopoiesis was evaluated. rhGM-CSF stimulated granulocyte-macrophage colony formation of canine marrow depleted of accessory cells up to tenfold. Stimulation of colony formation was abrogated by anti-rhGM-CSF antiserum or heat inactivation. rhGM-CSF also stimulated in vivo canine hematopoiesis both when given as continuous i.v. infusion and as intermittent s.c. injections. Neutrophil, monocyte, and lymphocyte counts were increased three- to eightfold above controls, whereas values for eosinophils, reticulocytes, and hematocrits were not changed. Bone marrow histology after 2 weeks of treatment with rhGM-CSF showed hypercellularity with myeloid hyperplasia and left-shifted granulocytopoiesis. After discontinuation of rhGM-CSF, peripheral leukocyte counts returned to control level within 3-7 days. Platelet counts decreased rapidly after starting rhGM-CSF, to 5000-15,000 platelets/mm3, and increased within 24 h after stopping rhGM-CSF treatment, whereas marrow histology after 2 weeks of rhGM-CSF application showed the normal number and morphology of megakaryocytes.     

Action of interleukin-3, G-CSF, and GM-CSF on highly enriched human hematopoietic progenitor cells: synergistic interaction of GM-CSF plus G-CSF

Purified preparations of recombinant human granulocyte-macrophage colony-stimulating factor (GM-CSF), granulocyte CSF (G-CSF), and interleukin 3 (IL-3 or multi-CSF) alone and in combination, have been compared for their stimulatory effects on human granulocyte-macrophage colony forming cells (GM-CFC). In cultures of unseparated normal human bone marrow, the combinations of G-CSF plus IL-3 and GM-CSF plus IL-3 stimulated additive numbers of GM colonies, while GM-CSF plus G-CSF stimulated greater than additive numbers of GM colonies, compared with the sum of the colony formation obtained with each factor alone. Cultures of unseparated bone marrow, harvested from patients four to six days after administration of 5-fluorouracil (5-FU), resulted in additive GM colony formation with GM-CSF plus G-CSF, GM-CSF plus IL-3, and G-CSF plus IL-3. In order to address the possibility of secondary factor involvement in the synergistic interaction of GM-CSF and G-CSF, CD33+/CD34+ colony forming cells were separated from normal and post FU marrow by two color fluorescence activated cell sorting. In cultures of CD33+/CD34+ cells the combination of GM-CSF plus G-CSF stimulated a synergistic increase in GM colonies while GM-CSF plus IL-3 stimulated additive numbers of colonies. These results suggest that GM-CSF, G-CSF, and IL-3 stimulate distinct populations of GM-CFC. Furthermore GM-CSF and G-CSF interact synergistically and this action is a direct effect on progenitor cells not stimulated by GM-CSF or G-CSF alone.     

Monocyte-mediated killing of human leukaemia is enhanced by administration of granulocyte-macrophage colony stimulating factor following chemotherapy

We studied the capacity of recombinant human granulocyte-macrophage colony stimulating factor (rhGM-CSF) to modulate monocyte anti-leukaemic activity when administered to patients following myelosuppressive chemotherapy. The leukaemic cell lines K562, U937 and KG-1 were used as models of human leukaemia as they exhibit differential sensitivity to cell-mediated or TNF-mediated cytotoxicity. Monocyte tumouricidal activity was augmented by rhGM-CSF or lipopolysaccharide (LPS) alone in vitro against leukaemic blasts, whereas granulocyte-colony stimulating factor (rhG-CSF) was without effect. rhGM-CSF and LPS exhibited an additive effect in stimulating the cytotoxic effect of monocytes against K562 blasts compared with either agent alone ( P  < 0.001). Both cell-mediated and soluble TNF-mediated killing of leukaemic blasts was augmented by rhGM-CSF administration to patients following chemotherapy. This effect persisted for up to 4 weeks after cessation of GM-CSF therapy. The administration of rhGM-CSF significantly increased the anti-leukaemic activity of monocytes against leukaemic targets that were resistant to secreted TNF, probably via a transmembrane TNF-dependent mechanism. Therapy with rhG-CSF exhibited a minimal effect. We conclude that administration of rhGM-CSF, but not rhG-CSF, augments the tumouricidal properties of the monocyte-macrophage system, particularly during recovery from myelosuppressive chemotherapy. Moreover, the killing mechanism is direct and not mediated by an antibody-dependent cellular cytotoxic (ADCC) mechanism. Killing of TNF-resistant leukaemic cells in particular may be augmented via cell-to-cell contact.     

Prevalence of the primitive megakaryocyte progenitors (BFU-meg) in adult human peripheral blood.

The in vitro growth of early (megakaryocyte burst-forming units, BFU-meg) and late (megakaryocyte colony-forming units, CFU-meg) megakaryocyte (meg) progenitors has been evaluated in normal adult human peripheral blood (PB). All the experiments were carried out using CD34+ cells, which were assayed in a serum-free fibrinclot assay. PB BFU-meg were morphologically characterized as plurifocal aggregates containing greater than 50 cells/colony, distinct from unifocal CFU-meg, in a limiting dilution assay. At variance with PB CFU-meg, PB BFU-meg were unaffected by the complement-mediated cytotoxicity with anti-HLA-DR. The optimal source of colony-stimulating activity for PB BFU-meg growth was recombinant human interleukin 3 (rhIL-3; 100 U/ml), which supported a significantly higher number of BFU-meg in comparison with recombinant human granulocyte-macrophage colony-stimulating factor (rhGM-CSF; 200 U/ml, p = 0.043). Combinations of rhIL-3 (100 U/ml) plus rhGM-CSF (200 U/ml), rhIL-3 plus recombinant human interleukin 6 (rhIL-6; 100 U plus 100 U/ml) or rhIL-3 plus rhGM-CSF plus rhIL-6 (100 U plus 200 U/ml plus 100 U/ml) failed to further increase the number of PB BFU-meg with respect to rhIL-3 (100 U/ml) alone. Both PB BFU-meg and CFU-meg were markedly inhibited, in a dose-dependent fashion, by increasing doses of human purified transforming growth factor-beta 1 (TGF-beta 1) (from 0.001 to 10 ng/ml). Finally, the CFU-meg/BFU-meg ratio in PB (0.52) was significantly different from that of normal bone marrow (2.3), clearly indicating that adult human peripheral blood predominantly carries primitive megakaryocytic progenitors.     

Proliferative properties of unfractionated, purified, and single cell human progenitor populations stimulated by recombinant human interleukin-3

In this report, the biological properties of human recombinant interleukin-3 (rhIL-3) were studied. We investigated the range of unfractionated, purified and single cell human progenitors responsive to IL-3; compared the colony types observed with those obtained in the presence of recombinant human granulocyte-macrophage colony-stimulating factor (GM-CSF) or granulocyte-CSF (G-CSF). The results show that IL-3 directly stimulates the formation of colonies derived from eosinophil and, to a lesser degree, granulocyte and macrophage progenitors. In combination with erythropoietin, it supports the development of erythroid and mixed-erythroid colonies. Furthermore, the data show that IL-3 is a more potent stimulus for both erythroid and eosinophil progenitors than GM-CSF. Interleukin-3 stimulates the formation of both compact and dispersed colonies derived from eosinophil progenitors, whereas GM-CSF stimulates the formation of only the compact type. We conclude that some of the proliferative effects of IL-3 observed on unfractionated and semipurified bone marrow populations are indirect and most likely involve accessory cell interactions.     

Interleukin-3 and interleukin-1 alpha allow earlier bone marrow progenitors to respond to human colony-stimulating factor 1

By using human bone marrow cells enriched for early progenitors by selective immunoadsorption and plated at low cell density (10(3) to 10(4) cells/mL/9.6 cm2) in semisolid methylcellulose culture, we have analyzed the cooperative effects of human colony-stimulating factor 1 (CSF-1), granulocyte-macrophage-CSF (GM-CSF), interleukin-1 alpha (IL-1 alpha), and gibbon as well as human recombinant IL-3 on the formation of monocytic colonies. CSF-1 alone stimulated mature monocytic colony formation by human CFU-M. However, in the presence of IL-3 and erythropoietin, CSF-1 stimulated maximal immature monocytic colony formation at low concentrations and inhibited the formation of granulomonocytic, erythrocytic, and mixed colonies. Cultures with CSF-1 and IL-3 contained more immature monocytic colonies than did cultures with CSF-1 alone. IL-1 alpha alone had little effect. However, IL-1 alpha in combination with optimal concentrations of either CSF-1, GM- CSF, or IL-3 increased the number of colonies containing immature or mature monocytic colonies.     

Recombinant gibbon interleukin-3 acts synergistically with recombinant human G-CSF and GM-CSF in vitro

Recombinant gibbon interleukin-3 (IL-3) is a multilineage hematopoietic colony-stimulating factor (CSF) that recently was cloned and found to be highly homologous with human IL-3. Gibbon IL-3, as well as human granulocyte-CSF (G-CSF) and human granulocyte-macrophage CSF (GM-CSF), stimulated normal human bone marrow cells to form myeloid colonies in soft agar in a sigmoidal dose-response manner. When IL-3 was added to increasing concentrations of G-CSF or GM-CSF, synergistic colony formation occurred as compared with the effects of each CSF alone. Synergism was also noted when G-CSF was added with GM-CSF and when all the CSFs were added simultaneously. The combination of IL-3 and GM-CSF was less stimulatory than all the other CSF combinations. At day 11 of culture, IL-3 induced granulocyte-macrophage (38%), eosinophil (30%), granulocyte (18%), and macrophage (14%) colony formation. In summary, gibbon IL-3 is a growth factor that can synergize with other CSFs to enhance proliferation of myeloid-committed progenitors, suggesting that combinations of CSFs may have clinical utility in patients with neutropenia of various etiologies.