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.     

Growth regulation of human acute myeloid leukemia: effects of five recombinant hematopoietic factors in a serum-free culture system

The response of human acute myeloid leukemia (AML) cells to the distinct hematopoietic growth factors (HGFs), ie, recombinant interleukin-3 (IL-3), granulocyte-macrophage-CSF (GM-CSF), granulocyte-CSF (G-CSF), macrophage-CSF (M-CSF), and erythropoietin (Epo) was investigated under well-defined serum-free conditions. Proliferative responses to these factors, when added separately as well as in combinations, were analyzed in 25 cases of human AML using 3H-thymidine incorporation and colony assays. The 3H-thymidine uptake data revealed that IL-3, GM-CSF, G-CSF, and M-CSF were stimulators of AML proliferation in 19, 15, 13, and 4 cases, respectively. Epo only stimulated DNA synthesis in the cells of the single erythroleukemia case. GM-CSF stimulation was seen only in IL-3 reactive cases and GM-CSF, when combined with IL-3, could not further elevate the DNA synthesis evoked by IL-3 alone. On the other hand, in six cases, G-CSF enhanced the IL-3- or GM-CSF-stimulated thymidine uptake. These results suggest that subpopulations of AML cells that are activated by distinct CSFs (eg, IL-3/GM-CSF-responsive cells and G-CSF-responsive cells) coexist. The 3H-thymidine incorporation assay was more sensitive for measuring CSF responses than methylcellulose colony cultures, since activation of DNA synthesis was more frequently seen than induction of colony formation. DNA synthesis experiments revealed eight different CSF response patterns among these 25 cases. CSF phenotyping may be a useful addition to the morphologic classification of AML, since these patterns directly reflect the ability of the proliferating subsets of AML cells to respond to the CSFs.     

Mechanism of synergy between granulocyte-macrophage colony-stimulating factor and granulocyte colony-stimulating factor in colony formation from human marrow cells in vitro

The synergy of human granulocyte-macrophage colony-stimulating factor (GM-CSF) and human granulocyte colony-stimulating factor (G-CSF) in the colony formation derived from human marrow cells was studied. The colony formation stimulated by GM-CSF plus G-CSF was dependent on the dose of each CSF, with the plateau for the number of GM colonies being higher than the sum of the individual plateaus by GM-CSF or G-CSF. Analysis of the colonies formed by GM-CSF plus G-CSF revealed efficient formation of neutrophil and monocyte colonies. To study the effect of GM-CSF and G-CSF on the maintenance of the progenitors that respond to the synergy of the CSFs, addition of each CSF to the medium of clonal cell culture was delayed. The progenitors that formed colonies on day 7 due to synergy of the CSFs were perfectly maintained by GM-CSF for at least 72 h and the progenitors that formed colonies on day 14 due to synergy of the CSFs were partly maintained by G-CSF or GM-CSF. The DNA synthetic rate of the progenitor cells that respond to GM-CSF plus G-CSF was significantly lower than those that respond to GM-CSF or G-CSF. According to light scatter analysis of phagocyte-depleted marrow mononuclear cells (PD-MMCs) using a flow cytometer, the peak population of progenitors that respond to GM-CSF plus G-CSF was in the smaller part of the PD-MMCs than those to GM-CSF or G-CSF. These results indicated that the progenitors to the synergy of GM-CSF and G-CSF are in a different proliferative state than those to each CSF. The synergy of GM-CSF and G-CSF depends on each CSF maintaining the viability of a different population of GM progenitors that can form GM colonies by both CSFs together.     

Combinations of recombinant colony-stimulating factors are required for optimal hematopoietic differentiation in serum-deprived culture

Previous in vitro investigations on enriched human hematopoietic progenitors have led to the conclusion that the purified recombinant multipoietins, interleukin 3 (IL-3) and granulocyte-macrophage colony- stimulating factor (GM-CSF) can alone induce the formation of colonies from a variety of multipotent and lineage committed progenitors. Since fetal calf serum was included in these cultures and itself might contain growth factors or other cofactors, we re-examined the actions of the CSFs in serum-deprived conditions. Results show that both the multipoietins are inadequate stimuli of colony formation. At maximal concentrations IL-3 alone induces only 25% of the granulocyte and macrophage colony-forming units (CFU-G and CFU-M) produced by a T-cell conditioned medium that contains a mixture of CSFs. When IL-3 was added at the initiation of the cultures and erythropoietin (ep), G-CSF, or M- CSF added on day 3, almost full recovery of erythroid, granulocytic, and monocytic colonies, respectively, was obtained. Similar results were obtained with GM-CSF except that fewer erythroid colonies were recovered at high concentrations, and almost maximal CFU-M proliferation could be induced. These results show that in serum- deprived conditions, the multipoietins must be combined with lineage specific CSFs for full progenitor expression.     

Regulation of differentiation of murine progenitor cells derived from blast cell colonies under serum-deprived conditions

We have examined the effect of interleukin 3 (IL-3), granulocyte-macrophage (GM)-, granulocyte (G)-, and macrophage (M)-colony-stimulating factors (CSFs) on the induction of GM colonies from highly enriched murine hematopoietic progenitor cells under serum-deprived conditions. Each growth factor was tested alone or in combination with suboptimal concentrations of the others. The effect of each CSF on GM colony growth in fetal bovine serum (FBS)-supplemented cultures of unfractionated marrow cells is reported for comparison. GM-CSF induced GM colony growth in serum-deprived cultures of purified progenitor cells to the same extent as in FBS-supplemented cultures of unfractionated marrow cells. In contrast, IL-3 was only one-tenth as active in promoting the growth of enriched progenitor cells under serum-deprived conditions when compared with its effect on colony growth from unfractionated marrow. M-CSF and G-CSF were almost completely ineffective in both cases. G-CSF induction of GM colony growth from purified progenitor cells was restored by addition of suboptimal concentrations of IL-3 or GM-CSF, suggesting that either IL-3 or GM-CSF is required to observe the effect of G-CSF. Addition of G-CSF to GM-CSF-stimulated cultures did not increase the maximal number of colonies detected, indicating that these two growth factors may act on the same subset of progenitor cells. Addition of GM-CSF or IL-3 to IL-3- or GM-CSF-stimulated cultures, respectively, increased by 40% the maximal number of colonies detected, suggesting that these two factors act on at least partially separate subsets of GM progenitors. These data parallel the recent observations on the control of human GM colony formation under FBS-deprived conditions and support a model for the control of myeloid differentiation that requires the interplay of different growth factors.     

Cytokine regulation of colony-stimulating factor (CSF) production in cultured human synovial fibroblasts. II. Similarities and differences in the control of interleukin-1 induction of granulocyte-macrophage CSF and granulocyte-CSF production.

Synovial fibroblasts are likely to be a significant source of granulocyte-macrophage colony-stimulating factor (GM-CSF) and granulocyte-CSF (G-CSF), which could be crucial to the pathogenesis of rheumatoid arthritis. Using specific enzyme-linked immunosorbent assays (ELISAs) and Northern analysis, GM-CSF and G-CSF expression were followed in human synovial fibroblast-like cells in response to a number of agents, either alone or in the presence of an optimal stimulatory concentration of interleukin-1 (IL-1). For both CSFs, interferon-gamma (100 U/mL) did not increase their levels but dramatically suppressed the stimulatory action of IL-1, while basic fibroblast growth factor (10(-8) mol/L), although nonstimulatory by itself, potentiated IL-1 action. The glucocorticoid, dexamethasone (10(-7) mol/L), inhibited IL-1-stimulated CSF production. However, evidence was obtained for noncoordinated CSF regulation. Cyclooxygenase inhibitors potentiated the action of IL-1 on GM-CSF synthesis but suppressed G-CSF synthesis, suggesting that endogenous cyclooxygenase products can have opposite effects in modulating the levels of each CSF. Also, the lymphokine, IL-4 (250 pmol/L), slightly inhibited GM-CSF formation in the presence of IL-1 but elevated the G-CSF levels in these cultures without having an effect by itself. Transforming growth factor beta (less than or equal to 20 ng/mL) did not modulate levels of either CSF. Mesenchymal cell production of both GM-CSF and G-CSF is generally viewed as being under coordinate control; our findings suggest that their synthesis in IL-1-stimulated human synoviocytes can be modulated by a number of agents, in some cases with divergent actions depending on which CSF is examined.     

Interleukin-3, GM-CSF, and G-CSF receptor expression on cell lines and primary leukemia cells: receptor heterogeneity and relationship to growth factor responsiveness

Recombinant human granulocyte-macrophage (GM) colony-stimulating factor (GM-CSF), G-CSF, and interleukin-3 (IL-3) labeled with 125I were used to study the characteristics and distribution of receptors for these factors on in vitro cell lines and on cells from patients with acute nonlymphocytic leukemia (ANL) and acute lymphocytic leukemia (ALL). Receptors for GM-CSF and G-CSF were restricted to a subset of myelomonocytic cell lines whereas IL-3 receptors were also found on pre- B- or early B-cell lines. Receptors for all three CSFs were broadly distributed on ANL cells, with considerable variability in levels of expression. Measurement of the colony-forming ability of ANL cells in response to the CSFs showed that there was no direct correlation between the ability of the cells to respond to a growth factor and the absolute number of receptors expressed for that growth factor. Binding of radiolabeled IL-3 and GM-CSF to ANL cells produced complex biphasic curves. Further analysis showed that both IL-3 and GM-CSF were able to partially compete for specific binding of the heterologous radiolabeled ligand to cells from several ANL patients, suggesting that heterogeneity may exist in human CSF receptors. These results provide new insights into the complex role that CSFs may play in ANL.     

Initiation in DNA synthesis by colony-stimulating factors in subsets of human CD34+ marrow cells.

Initiation of DNA synthesis by recombinant colony-stimulating factors (CSFs) was assessed in normal human marrow blast cells isolated by expression of CD34 antigen (tritiated thymidine incorporation). Continuous exposure to CSF was required. A mild increase in DNA synthesis was initiated by granulocyte CSF (G-CSF; greater than or equal to 1 ng/ml), to approximately 1.5 times control levels. A greater increase was initiated by granulocyte-macrophage CSF (GM-CSF), with a threshold of approximately 0.1 ng/ml and a plateau increment 2.5 times control levels. CD34+ cells were stimulated by interleukin 3 (IL-3) over a wide concentration range: two times control at 0.1/ml, three times control at 1 ng/ml, and four times control at 10 ng/ml. Overlap between responding populations was analyzed. G-CSF plus GM-CSF induced DNA synthesis greater than GM-CSF alone and supported the growth of much larger granulocyte-monocyte colonies. At saturating IL-3 concentrations, neither G-CSF nor GM-CSF induced additional DNA synthesis; at lower concentrations of IL-3, however, GM-CSF recruited additional cells into DNA synthesis. Using CD10 and CD19 antibodies to separate B-lineage cells, the CD34+ cells responding to CSF were observed to be in the non-B-lineage subset. Therefore 1) the response of CD34+ cell subsets CSFs is IL-3 greater than GM-CSF greater than G-CSF, and the IL-3-responsive population is heterogeneous for dose requirement; 2) a CD34+ subpopulation responding to concurrent G-CSF and GM-CSF includes increased proliferative potential cells; 3) IL-3-responsive cells include GM-CSF- and G-CSF-responsive cells, but cells responding to lower IL-3 concentration do not respond to GM-CSF; and 4) B-cell precursors do not respond to GM-CSF or IL-3 in this assay.     

Interleukin-3 is significantly more effective than other colony- stimulating factors in long-term maintenance of human bone marrow- derived colony-forming cells in vitro

Human bone marrow cells cultured for 21 days in the presence of recombinant human interleukin-3 (IL-3) produced up to 28 times more colony-forming cells (CFC) than could be obtained from cultures stimulated with granulocyte colony stimulating factor (G-CSF) or granulocyte-macrophage CSF (GM-CSF). IL-3-cultured cells retained a multipotent response to IL-3 in colony assays but were restricted to formation of granulocyte colonies in G-CSF and granulocyte or macrophage colonies in GM-CSF. Culture of bone marrow cells in IL-3 also led to accumulation of large numbers of eosinophils and basophils. These data contrast with the effects of G-CSF, GM-CSF, and IL-3 in seven-day cultures. Here both GM-CSF and IL-3 amplified total CFC that had similar multipotential colony-forming capability in either factor. G-CSF, on the other hand, depleted IL-3-responsive colony-forming cells dramatically, apparently by causing these cells to mature into granulocytes. The data suggest that a large proportion of IL-3- responsive cells in human bone marrow express receptors for G-CSF and can respond to this factor, the majority becoming neutrophils. Furthermore, the CFC maintained for 21 days in IL-3 may be a functionally distinct population from that produced after seven days culture of bone marrow cells in either IL-3 or GM-CSF.     

Interleukin-6 reduces the optimal growth in vitro of leukemic blast progenitors from acute myeloblastic leukemia patients.

The combined effects of five cytokines; recombinant human (rHu) granulocyte colony-stimulating factor (G-CSF), rHu granulocyte-macrophage colony-stimulating factor (GM-CSF), rHu interleukin-1 beta (IL-1 beta), rHu interleukin-3 (IL-3), and rHu interleukin-6 (IL-6) on blast colony formation in methylcellulose by leukemic blast progenitors from 10 patients with acute myeloblastic leukemia (AML) were studied. Combination of G-CSF, GM-CSF, IL-1 beta, and IL-3 stimulated maximum blast colony formation in 9 patients. Further addition of IL-6 reduced the combined effect of the four cytokines on blast colony formation. IL-6 regulates the proliferation of leukemic blast progenitors and may play an important role in the regulation of hematopoiesis.     

Growth of human hemopoietic colonies in response to recombinant gibbon interleukin 3: comparison with human recombinant granulocyte and granulocyte-macrophage colony-stimulating factor.

Supernatants of COS-1 cells transfected with gibbon cDNA encoding interleukin 3 (IL-3) with homology to sequences for human IL-3 were tested for ability to promote growth of various human hemopoietic progenitors. The effect of these supernatants as a source of recombinant IL-3 was compared to that of recombinant human granulocyte-macrophage colony-stimulating factor (GM-CSF) and granulocyte colony-stimulating factor (G-CSF) as well as to that of medium conditioned by phytohemagglutinin-stimulated leukocytes. The frequency of multilineage colonies, erythroid bursts, and megakaryocyte colonies in cultures containing the COS-1 cell supernatant was equivalent to the frequency observed in the controls and significantly higher than found in cultures plated with recombinant GM-CSF. G-CSF did not support the formation of multilineage colonies, erythroid bursts, and megakaryocyte colonies. In contrast, growth of granulocyte-macrophage colonies was best supported with GM-CSF, while recombinant IL-3 yielded colonies at lower or at best equivalent frequency. The simultaneous addition of higher concentrations of GM-CSF to cultures containing IL-3 in optimal amounts did not enhance the formation of multilineage colonies, erythroid bursts, and megakaryocyte colonies. However, the frequency of such colonies and bursts increased with GM-CSF when cultures were plated with suboptimal concentrations of IL-3. Growth of colonies within the granulocyte-macrophage lineage is optimally supported by GM-CSF and does not increase with further addition of IL-3.     

Modulation of tartrate-resistant acid phosphatase expression by calcitriol in CSF-induced macrophage colonies

The osteoclast is thought to be a hemopoietically derived cell, but questions exist about which hemopoietic growth factors are responsible for proliferation of osteoclast precursors. Experiments were thus performed to see if recombinant human colony-stimulating factors (CSFs) influenced the expression of tartrate-resistant acid phosphatase (TRAP), an osteoclast marker enzyme, by monkey bone marrow colonies in vitro. In addition, the effect of 1,25-dihydroxyvitamin D3 (calcitriol) on CSF-induced colony growth and TRAP expression was also determined. Bone marrow was obtained from a single Macaca nemestrina monkey, kept frozen in liquid nitrogen, and aliquots of frozen cells were thawed and placed at 10(5) cells per plate in a standard cell colony-forming unit (CFU-C) assay. The recombinant human CSFs (granulocyte-macrophage CSF, GM-CSF; macrophage CSF, M-CSF; interleukin 3, IL-3; and granulocyte CSF, G-CSF) were added to the cultures at 50 U/ml, and calcitriol was titrated for each CSF from 0.1 to 100 nM. Day-14 colonies were stained to demonstrate TRAP-positive cells in individual colonies. GM-CSF caused an increase (193%, p less than 0.0004) in total colony numbers that was only partially inhibited by calcitriol. IL-3 and M-CSF had less effect, and G-CSF had no effect. GM-CSF also caused a large increase in TRAP-positive macrophage (M) colonies (326%, p less than 0.0001) and changed the relative proportion of TRAP-positive M colonies from 39% to 62% of all M colonies. M-CSF caused less increase in numbers of TRAP-positive M colonies and had no effect on the proportion of TRAP-positive colonies. When GM-CSF was present, calcitriol caused a maximum number of TRAP-positive colonies to appear at 1 nM, and it caused a drastic decrease in TRAP-positive colonies at higher doses. Calcitriol at 10 nM caused TRAP-negative colonies to increase in number and proportion when GM-CSF was present, but in the presence of M-CSF, the same dose of calcitriol caused a decline in numbers of TRAP-negative colonies. These results suggest that GM-CSF may be important in the replication of TRAP-positive mononuclear cells that resemble osteoclast precursors and that myeloid cell development may be weighted toward TRAP-positive or TRAP-negative progeny depending on whether GM-CSF or M-CSF predominates. They further suggest that calcitriol concentration may be critical in this process.     

Interactions between purified murine colony-stimulating factors (natural CSF-1, recombinant GM-CSF, and recombinant IL-3) on the in vitro proliferation of purified murine granulocyte-macrophage progenitor cells

Purified preparations of natural CSF-1 (nCSF-1), recombinant GM-CSF (rGM-CSF), and recombinant IL-3 (rIL-3), alone and in combination, were investigated for their proliferative effects on highly enriched murine granulocyte-macrophage progenitor cells (CFU-GM). These CFU-GM had cloning efficiencies of 62%-95% in the presence of 10% (vol/vol) pokeweed mitogen-stimulated spleen cell-conditioned medium, and contained few, if any (less than or equal to 3%), contaminating morphologically recognizable monocytes or lymphocytes. The combination of low concentrations of nCSF-1 plus rIL-3, or nCSF-1 plus rGM-CSF, increased colony number greater than additively compared to the sum of colony formation with each factor alone, whereas total aggregate (colony plus cluster) number increased additively. At plateau concentrations, the previous CSF combinations increased colony number additively. Colony size was increased when nCSF-1 plus either rGM-CSF or rIL-3 were added simultaneously at either low or plateau concentrations, when compared to the size of colonies with any of the CSFs alone. Addition of rGM-CSF plus rIL-3 demonstrated no cooperative proliferative effect on either colony number or size. It is likely that these effects are mediated at the progenitor cell level and do not require accessory cell participation.     

Differential effects of IL-3, GM-CSF and G-CSF in an adult with congenital neutropenia.

Using a methylcellulose culture system, we studied the effects of recombinant human interleukin-3 (IL-3), recombinant human granulocyte-macrophage colony-stimulating factor (GM-CSF), and recombinant human granulocyte colony-stimulating factor (G-CSF) on the growth of myeloid progenitor cells (CFU-C) from an adult patient with congenital neutropenia. The moderate clinical course and the maturation arrest at blast-promyelocyte stage in the marrow differentiated this patient from those described as having Kostmann-type congenital neutropenia. CFU-C growth in bone marrow cells from the patient responded to IL-3 normally in a dose-dependent manner. GM-CSF stimulated only macrophage colony formation in a dose-dependent manner comparable to that in normal subjects. Neither GM-CSF nor G-CSF stimulated any significant granulocyte colony formation. This evidence suggests that the hematopoietic progenitor cells in this patient had the potential for developing CFU-C with IL-3, and that the neutropenia in this patient could be a result of an intrinsic defect in myelopoiesis along a granulocytic pathway responsive to GM-CSF or G-CSF.     

Interleukin-1, tumor necrosis factor, and the production of colony- stimulating factors by cultured mesenchymal cells

Although the genes for four hematopoietic colony-stimulating factors (CSFs) have been cloned, neither the mechanism of the regulation of their production nor their cellular origins have been established with certainty. Monocytes are known to produce colony-stimulating and burst- promoting activities, as well as several monokines such as interleukin- 1 (IL-1) and tumor necrosis factor (TNF). These monokines indirectly stimulate other mesenchymal cells to produce certain colony-stimulating factors such as granulocyte-macrophage CSF (GM-CSF). To determine whether monocytes produce other CSFs and if so, to compare the mechanism of regulation of production with that of endothelial cells and fibroblasts, we investigated the synthesis of CSFs by monocytes, human umbilical vein endothelial cells, and fibroblasts. We used total cellular RNA blot analysis to determine interleukin-3 (IL-3), GM-CSF, granulocyte CSF (G-CSF), and monocyte CSF (M-CSF) messenger RNA (mRNA) content and immunoprecipitation or bioassay to confirm the presence of the specific secreted proteins. The results indicate that M-CSF mRNA and protein are produced constitutively by all three cell types and their level of expression does not increase after induction. In contrast, GM-CSF and G-CSF mRNAs are barely detectable in uninduced monocytes and show an increase in expression after lipopolysaccharide treatment. Retrovirus-immortalized endothelial cells, unlike primary endothelial cells or both primary and immortalized fibroblasts, produce IL-1 constitutively; this correlates with their constitutive production of GM-CSF and G-CSF. IL-3 mRNA was not detectable in any of these cells either before or after induction. The results indicate that these mesenchymal cells can produce three CSFs: GM-CSF, G-CSF, and M-CSF; furthermore, the data suggest that the mechanism of regulation of M-CSF production is different from that of GM-CSF and G-CSF, and that the latter two inducible CSFs are regulated by different factors in monocytes compared with the other mesenchymal cells.     

A role for calmodulin in the growth of human hematopoietic progenitor cells.

A possible role for calmodulin in the colony growth of human hematopoietic progenitor cells was investigated using pharmacologic approaches. We obtained evidence for a dose-dependent inhibition of colony formation of myeloid progenitor cells (CFU-C) stimulated by interleukin-3 (IL-3), granulocyte-macrophage colony-stimulating factor (GM-CSF), or granulocyte CSF (G-CSF) by three calmodulin antagonists, N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide hydrochloride (W-7), N-(4-aminobutyl)-5-chloro-2-naphthalenesulfonamide hydrochloride (W-13), and trifluoperazine. Chlorine-deficient analogs of W-7 and W-13, with a lower affinity for calmodulin, did not inhibit the growth of CFU-C colonies. W-7, W-13, and trifluoperazine inhibited the colony formation of immature erythroid progenitor cells (BFU-E) stimulated by IL-3 plus erythropoietin (Ep) or GM-CSF plus Ep, in a dose-dependent manner, while they did not affect the colony formation of mature erythroid progenitor cells (CFU-E) induced by Ep. W-7, W-13, and trifluoperazine also led to a dose-dependent inhibition of GM-CSF-induced colony formation of KG-1 cells. Calmodulin-dependent kinase activity derived from the KG-1 cells was inhibited by these three calmodulin antagonists in a dose-dependent manner. These data suggest that calmodulin may play an important regulatory role via a common process in the growth of hematopoietic progenitor cells stimulated by IL-3, GM-CSF, and G-CSF. Mechanisms related to the growth signal of Ep apparently are not associated with calmodulin-mediated systems.     

Different haematopoietic growth factors have different capacity in overcoming the in vitro interferon gamma-induced suppression of bone marrow progenitor cells

Interferon gamma (IFN gamma) inhibits haematopoiesis in vitro and an in vivo role in bone marrow suppression has been implied from clinical studies. We investigated the capacity of three recombinant (r), human (h), haematopoietic growth factors to overcome the in vitro IFN gamma inhibition of bone marrow progenitor cells in a methylcellulose culture system. Granulocyte macrophage-colony stimulating factor (GM-CSF) partially reversed IFN gamma-induced suppression of granulocyte-macrophage colony formation, by increasing colony forming units-granulocyte macrophage (CFU-GM) in a proportion ranging from 54-101%. Interleukin-3 (IL-3) and granulocyte-colony stimulating factor (G-CSF) were much less effective. For erythropoiesis, IL-3 was much more effective and partially reversed IFN gamma-mediated inhibition by increasing burst forming units-erythroid (BFU-E) in a proportion ranging from 52-138%. GM-CSF and G-CSF had no significant effect on IFN gamma-induced suppression of BFU-E. In conclusion, haematopoietic growth factors have different capacity to overcome IFN gamma-induced suppression of marrow progenitor cells in vitro. The findings may have therapeutic implications, as combinations of growth factors may be more effective in treating bone marrow failure syndromes.     

Interleukin-1 synergizes with granulocyte-macrophage colony-stimulating factor on granulocytic colony formation by intermediate production of granulocyte colony-stimulating factor

Interleukin-1 (IL-1) was found to act synergistically with granulocyte-macrophage colony-stimulating factor (GM-CSF) on granulocytic colony growth of normal human bone marrow cells, depleted of mononuclear phagocytes and T lymphocytes. Using CD34/HLA-DR-enriched bone marrow cells we demonstrated that this activity of IL-1 was not a direct action on hematopoietic progenitor cells, but an effect of an intermediate factor produced by residual accessory cells in response to IL-1. Neutralization experiments using an anti-IL-6 antiserum showed that IL-1-induced IL-6 did not contribute to the observed synergy. Furthermore, IL-6 by itself had neither a direct stimulatory effect on CFU-GM colony growth, nor did it act synergistically with GM-CSF on granulocytic or monocytic colony formation. Neutralization experiments with an anti-G-CSF monoclonal antibody showed that IL-1-induced G-CSF production was responsible for the synergy with GM-CSF. Using combinations of G-CSF and GM-CSF this synergistic activity could be detected at concentrations of G-CSF as low as 0.1 ng/mL (10 U/mL). Our results indicate that IL-1, but not IL-6, stimulates the GM-CSF-dependent proliferation of relatively mature myeloid progenitor cells in the presence of small numbers of accessory cells.     

Effect of recombinant and purified human haematopoietic growth factors on in vitro colony formation by enriched populations of human megakaryocyte progenitor cells

Nonadherent low density T-lymphocyte depleted (NALT-) marrow cells from normal donors were sorted on a Coulter Epics 753 Dye Laser System using Texas Red labelled My10 and phycoerythrin conjugated anti HLA-DR monoclonal antibodies in order to obtain enriched populations of colony forming unit-megakaryocyte (CFU-MK). The CFU-MK cloning efficiency (CE) was 1.1 +/- 0.5% for cells expressing both high densities of My10 and low densities of HLA-DR (My10 DR+). This procedure resulted in an 18-fold increase in CE over NALT- cells. The effect of purified or recombinant human haematopoietic growth factors including erythropoietin (Epo), thrombocytopoiesis stimulating factor (TSF), interleukin 1 alpha (IL-1 alpha), granulocyte colony stimulating factor (G-CSF), granulocyte-macrophage colony stimulating factor (GM-CSF), macrophage colony stimulating factor (M-CSF or CSF-1) and interleukin MK colony formation by My10 DR+ cells was determined utilizing a serum depleted assay system. Neither Epo, TSF, CSF-1, IL-1 alpha nor G-CSF alone augmented MK colony formation above baseline (2.5 +/- 0.8/5 x 10(3) My10 DR+ cells plated). In contrast, the addition of GM-CSF and IL-3 each increased both CFU-MK colony formation and the size of colonies with maximal stimulation occurring following the addition of 200 units/ml of IL-3 and 25 units/ml of GM-CSF. At maximal concentration, IL-3 had a greater ability to promote megakaryocyte colony formation than GM-CSF. The stimulatory effects of GM-CSF and IL-3 were also additive in that the effects of a combination of the two factors approximated the sum of colony formation in the presence of each factor alone. The CFU-MK appears, therefore, to express HPCA-1 and HLA-DR antigens. These studies also indicate that GM-CSF and IL-3 are important in vitro regulators of megakaryocytopoiesis, and that these growth factors are not dependent on the presence of large numbers of macrophages or T cells for their activity since the My10 DR+ cells are largely devoid of these accessory cells.