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.     

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.     

Study of early hematopoietic precursors in human cord blood.

Human cord blood is a source of transplantable stem cells. These stem cells express the antigen CD34, are resistant to treatment with 4-hydroperoxycyclophosphamide (CD34+/4-HCres), and do not give rise to colonies when plated in clonogenic assays. We studied the number of CD34+ cells present in cord blood and developed a two-step assay for early precursors (pre-colony-forming units, pre-CFU) capable of giving rise to committed progenitors. In this assay CD34+/4-HCres cord blood cells were cultured in suspension with different growth factors. After 7 days in suspension the remaining cells were plated in clonogenic assays, for granulocyte-macrophage colony-forming units (CFU-GM), erythroid burst-forming units (BFU-E), and mixed lineage colony-forming units (CFU-MIX), in the presence of pure factors or a combination of recombinant human (rh) interleukin 3 (IL-3) and medium conditioned by the PU34 primate cell line. Pre-CFU for all precursors were identified. These pre-CFU developed into committed progenitors in response to rhIL-3. The combinations of rhIL-3 plus rh interleukin 1 (IL-1) or rhIL-3 plus rh interleukin 6 (IL-6) did not enhance recovery of progenitors. The developing CFU-GM were responsive to rh granulocyte-macrophage colony-stimulating factor (GM-CSF) and rh granulocyte colony-stimulating factor (G-CSF) but much less so to rhIL-3. BFU-E and CFU-MIX developed in suspension but could only be detected when cells were replated in the presence of a combination of rhIL-3 and PU34 but not rhIL-3 alone. This assay may be useful in evaluating the number of early hematopoietic precursors present in cord blood samples and in defining growth factor combinations that could enhance hematopoietic recovery after cord blood stem cell transplants.     

Human interleukin-6 supports granulocytic differentiation of hematopoietic progenitor cells and acts synergistically with GM-CSF

Recombinant human (rh) interleukin-6 (IL-6), in a dose range of 1 to 10 U/mL, was able to induce a low number of neutrophilic-granulocytic colonies in a CFU-GM clonogenic assay, using T cells and adherent cells, depleted low density marrow cells. A synergistic increase in the number of granulocytic colonies was observed when rhGM-CSF at suboptimal doses and IL-6 at effective doses were both present in the assay; the increase was only additive when either rhIL-1 alpha or rhIL- 3 was used together with IL-6. To determine whether the increase in colony number reflects the interactions of these factors on the same hematopoietic progenitor target cells or, instead, represents activation of accessory cells, we analyzed the effect of IL-6 on the proliferation and differentiation of three growth factor-dependent leukemic cell lines that respond with continuous proliferation to the presence of GM-CSF and IL-3 in culture. One of the three cell lines (AML-193) showed limited proliferation in the presence of IL-6 followed by terminal differentiation after 14 days into basophilic-granulocytic- like cells. A synergistic proliferative response was observed on the same cells treated with both GM-CSF and IL-6. These data support the hypothesis that IL-6 may have a direct effect on myeloid hematopoietic progenitor cells, and that GM-CSF interacts synergistically with IL-6 by acting on the same target cells.     

In vitro differentiation of human granulocyte/macrophage and erythroid progenitors: comparative analysis of the influence of recombinant human erythropoietin, G-CSF, GM-CSF, and IL-3 in serum-supplemented and serum- deprived cultures

The effects of recombinant human erythropoietin (Ep), granulocyte/macrophage (GM) and granulocyte (G) colony-stimulating factors (CSF), and interleukin-3 (IL-3) on erythroid burst and GM colony growth have been studied in fetal bovine serum (FBS)- supplemented and FBS-deprived culture. Sources of progenitor cells were nonadherent or nonadherent T-lymphocyte-depleted marrow or peripheral blood cells from normal humans. G-CSF, in concentrations up to 2.3 X 10(-10) mol/L, induced only the formation of neutrophil colonies. In contrast, GM-CSF and IL-3 both induced GM colonies and sustained the formation of erythroid bursts in the presence of Ep. However, the activities of these growth factors were affected by the culture conditions. IL-3 induction of GM colonies depended on the presence of FBS, whereas the degree of GM-CSF induction of GM colonies in FBS- deprived cultures depended on the method by which adherent cells were removed. GM-CSF increased colony numbers in a concentration-dependent manner only if the cells had been prepared by overnight adherence. Both GM-CSF and IL-3 exhibited erythroid burst-promoting activity in FBS- deprived cultures. However, some lineage restriction was evident because GM-CSF was two- to threefold more active than IL-3 in inducing GM colonies but IL-3 was two- to threefold more active in promoting erythroid burst growth. Furthermore, in FBS-deprived cultures, the number of both erythroid bursts and GM colonies reached the maximum only when Ep, GM-CSF, and IL-3 or GM-CSF, IL-3, and G-CSF, respectively, were added together. These results suggest that the colonies induced by IL-3, GM-CSF, and G-CSF are derived from different progenitors.     

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.     

Interleukin-3 in vivo: kinetic of response of target cells

Human recombinant interleukin-3 (IL-3; Sandoz AG, Basel, Switzerland) was administered for 7 days to patients with neoplastic disease and normal hematopoiesis. The purpose of the study was to assess IL-3 toxicity, to identify target cells, to define their kinetics of response at different dose levels, and to determine if IL-3 in vivo increased the sensitivity of bone marrow (BM) progenitors to the action of other hematopoietic growth factors. A total of 21 patients entered the study; the dosage ranged from 0.25 to 10 micrograms/kg/d. The effect on peripheral blood cells during treatment showed no significant changes in the number of platelets, erythrocytes, neutrophils, or lymphocytes (and their subsets). A mild monocytosis and basophilia occurred. Eosinopenia, present in the first hours of treatment, was followed by a dose-and time-dependent eosinophilia. IL-3 treatment affected BM cell proliferation by increasing the percentage of BM progenitors engaged in the S-phase of the cell cycle. The effect was dose dependent, with the various progenitors showing different degrees of sensitivity. The most sensitive progenitors were the megakaryocyte progenitors (colony-forming unit-megakaryocyte), then the erythroid progenitors (burst-forming unit-erythroid), and finally the granulo- monocyte progenitors (colony-forming unit-granulocyte-macrophage) whose proliferative activity was stimulated at the higher doses of IL-3. Only a slight increase in the proliferative activity of myeloblasts, promyelocytes, and myelocytes was observed, whereas the activity of erythroblasts was unchanged. The priming effect was such that BM progenitors, purified from patients treated with IL-3, produced more colonies in vitro in the presence of granulocyte colony-stimulating factor (G-CSF; granulocyte colonies), IL-5 (eosinophil colonies), and granulocyte-macrophage CSF (GM-CSF; predominantly eosinophil colonies). These data indicate that even in vivo IL-3 acts essentially as a primer for the action of other cytokines. Therefore, optimum stimulus of myelopoiesis will require either endogenous or exogenous late-acting cytokines such as G-CSF, erythropoietin, GM-CSF, and IL-6 for achieving fully mature cells in peripheral blood. If exogenous cytokines are used with IL-3, it is likely that G-CSF will yield more neutrophils, whereas GM-CSF may enhance eosinophils, monocytes, and neutrophils. Attention to the clinical relevance of each cell type will be necessary and should determine the selection of the combination of cytokines.     

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.     

Effects of recombinant human G-CSF, GM-CSF, IL-3, and IL-1 alpha on the growth of purified human peripheral blood progenitors.

The effects of recombinant products of granulocyte colony-stimulating factors (G-CSF), granulocyte/macrophage CSF (GM-CSF), human interleukin-3 (IL-3), and interleukin-1 (IL-1) were studied using purified target cell populations from patients undergoing peripheral blood stem cell transplantation after myeloablative therapy. Cells were subjected to combined purification procedures including negative selection with a panel of monoclonal antibodies (CD2, 3, 5, 10, and 20). The purified cells were enriched for HLA-DR+ (51% to 71%) and My-10+ (CD34; 37% to 54%) and had a plating efficiency of up to 20%. In the liquid-suspension limiting dilution clonal assay (LDA), purified progenitors responded directly to IL-3 by proliferation with single-hit kinetics. The ability of GM-CSF to support progenitor growth was inferior to that of IL-3, and the cells were virtually unresponsive when cultured with G-CSF, supporting the notion that these blood-derived progenitors belong to a primitive population of hematopoietic progenitor cells. The results obtained in simultaneous methycellulose cultures (MC) showed the same trend and provided additional information on the ability of GM-CSF and IL-3 to support erythroid progenitor growth. The combination of IL-3 plus G-CSF, but not IL-3 plus GM-CSF, resulted in a synergistic increase in colony number. IL-1 alpha increased both the size and number of colonies when added to IL-3 or G-CSF. Study of this enriched progenitor cell population in MC and LDA represents an excellent model for the investigation of myeloid and erythroid differentiation and for evaluating the influence of various cytokines on human hematopoiesis.     

The effect of mast cell growth factor on peripheral blood granulocyte-macrophage colony-forming cells in methylcellulose in myeloproliferative disorders

Abstract: Clonogenic cell culture assay was used to evaluate the effect of mast cell growth factor (MGF) on peripheral blood granulocyte-macrophage (GM) progenitors in 26 patients with myeloproliferative disorders (MPDs). MGF alone had a statistically significant stimulatory effect on GM colony formation, as also did interleukin-3 (IL-3) and GM colony-stimulating factor (GM-CSF), although the progenitors could form colonies spontaneously as well. When MGF was combined with either IL-3 or GM-CSF the effect was additive and was as great as that achieved with a mixture of IL-3, GM-CSF, G-CSF and IL-6. The highest colony-forming capacity of all was seen when MGF was added to the above mixture. Within the subgroups of MPDs, the stimulatory effect of MGF was significant in polycythemia vera (PV), essential thrombocythosis (ET) and chronic myelogenous leukemia (CML). MGF was the most potent single factor in PV, while GM-CSF was most effective in idiopathic myelofibrosis and both IL-3 and GM-CSF in CML. The fact that the ability of MGF to induce colony growth varied between the subgroups of MPDs may mean that the target progenitors in these diseases are biologically different. In conclusion, MGF, either alone or with others, was a potent growth factor for GM progenitors in MPDs.     

Sequential in vivo treatment with two recombinant human hematopoietic growth factors (interleukin-3 and granulocyte-macrophage colony-stimulating factor) as a new therapeutic modality to stimulate hematopoiesis: results of a phase I study.

In a phase I study, the sequentially administered combination of recombinant human interleukin-3 (rhIL-3) and rhGM-CSF was compared with treatment with rhIL-3 alone in 15 patients with advanced tumors but normal hematopoiesis. Patients were initially treated with rhIL-3 for 15 days. After a treatment-free interval, the patients received a second 5-day cycle of rhIL-3 at an identical dosage, immediately followed by a 10-day course of rhGM-CSF, to assess the toxicity and biologic effects of this sequential rhIL-3/rhGM-CSF combination. rhIL-3 doses tested were 125, and 250 micrograms/m2, whereas rhGM-CSF was administered at a daily dosage of 250 micrograms/m2. Both cytokines were administered by subcutaneous (SC) bolus injection. rhIL-3/rhGM-CSF treatment was more effective than rhIL-3 but equally effective to each other in increasing peripheral leukocyte counts, especially neutrophilic and eosinophilic granulocyte counts. In contrast, both modes of cytokine therapy raised the platelet counts to the same degree. rhIL-3/GM-CSF treatment was more effective than rhIL-3 in increasing the number of circulating hematopoietic progenitor cells BFU-E and CFU-GM. High-dose rhIL-3, but not low-dose rhIL-3, was as effective as the rhIL-3/rhGM-CSF combinations in increasing the number of circulating CFU-GEMM. The increase in absolute neutrophil counts correlated with the increase in the number of circulating CFU-GM. Side effects, mainly fever, headache, flushing, and sweating, were generally mild, but in two patients the occurrence of chills, rigor, and dyspnea after initiation of GM-CSF treatment necessitated dose reduction and discontinuation, respectively. These results indicate that sequential treatment with rhIL-3 and rhGM-CSF is as effective as single-factor treatment with rhIL-3 in stimulating platelet counts, whereas the effect of combination therapy on neutrophil counts and circulating progenitor cells is superior.     

Activin A suppresses proliferation of interleukin-3-responsive granulocyte-macrophage colony-forming progenitors and stimulates proliferation and differentiation of interleukin-3-responsive erythroid burst-forming progenitors in the peripheral blood

We examined the effects of activin A on the proliferation and differentiation of immature hematopoietic progenitors prepared from peripheral blood (PB) using methylcellulose and liquid-suspension culture. In a kinetic analysis, colony formation by PB granulocyte- macrophage colony-forming unit (CFU-GM) was delayed in a dose-dependent manner by the addition of activin A only when stimulated with interleukin-3 (IL-3), but not when stimulated with granulocyte colony- stimulating factor (G-CSF), granulocyte-macrophage colony-stimulating factor (GM-CSF), or stem cell factor (SCF) plus G-CSF. DNA-synthesizing CFU-GM was increased by IL-3, but this effect was abolished by activin A. In contrast, PB erythroid burst-forming unit (BFU-E) was accelerated by the addition of activin A only when exposed to IL-3 plus erythropoietin (Epo), but not when exposed to Epo or Epo plus SCF. DNA- synthesizing BFU-E was increased by IL-3 and activin A, alone and additively in combination. In a mixed culture of myeloid and erythroid progenitors, activin A increased the numbers of BFU-E and CFU-Mix colonies at concentrations of 1 and 10 ng/mL and decreased the number of CFU-GM colonies in a dose-dependent manner. However, in a liquid- suspension culture of erythroid progenitors, activin A decreased total cell count and the percentage of hemoglobin-containing cells only when cells were exposed to IL-3 plus Epo. These results indicate that activin A suppresses the proliferation of IL-3-responsive CFU-GM progenitors and stimulates the proliferation and differentiation of IL- 3-responsive BFU-E progenitors, and suggest that activin A acts as a commitment factor of immature hematopoietic progenitors for erythroid differentiation.     

Lineage commitment of hemopoietic progenitor cells in developing blast cell colonies: influence of colony-stimulating factors.

In clonal cultures of normal mouse marrow cells, combination of granulocyte, granulocyte-macrophage, or multipotential colony-stimulating factor (G-CSF, GM-CSF, or multi-CSF, respectively) with stem cell factor (SCF) did not alter the number of blast colonies stimulated to develop compared with SCF alone but induced an up to 25-fold increase in their mean cell content and an up to 6-fold increase in their mean progenitor cell content. Costimulation of blast colony formation by SCF plus G-CSF did not change the relative frequency of progenitor cells of different types within the colonies compared with colonies stimulated by SCF alone. However, combination of GM-CSF or multi-CSF with SCF significantly increased the relative frequency of granulocytic progenitors and, for multi-CSF, also of eosinophil progenitor cells. These changes in the relative frequencies of progenitor cells committed to the various lineages support the hypothesis that hemopoietic regulators have some ability to induce selective lineage commitment in the progeny of multipotential cells.     

Erythropoietin alone induces erythroid burst formation by human embryonic but not adult BFU-E in unicellular serum-free culture [see comments]

Erythroid progenitors (BFU-E) from adult human peripheral blood generate erythroid bursts in semisolid culture supplemented with at least two growth factors, ie, erythropoietin (Ep) and interleukin-3 (IL- 3) or granulocyte-macrophage colony-stimulating factor (GM-CSF). We have analyzed the hematopoietin(s) requirement of human embryonic BFU- E, as compared to that of adult peripheral blood progenitors: This was basically evaluated in fetal calf serum-free (FCS-) methylcellulose culture of partially or highly purified progenitors treated with human recombinant hemopoietins. At a low seeding concentration (2 x 10(3) cells/dish) purified embryonic BFU-E generated erythroid bursts when treated only with Ep: Further addition of IL-3 or GM-CSF had no effect on BFU-E cloning efficiency, although the size of bursts was increased in a dose-dependent manner, particularly with IL-3. At a similar seeding concentration (ie, 10(3) cells/dish), purified adult BFU-E efficiently generated erythroid bursts in the presence of Ep and GM-CSF or IL-3, while only few small erythroid colonies were observed in the presence of Ep alone. In a final series of experiments, unicellular FCS- cultures of purified embryonic BFU-E gave rise to erythroid bursts in the presence of Ep alone. Furthermore, the cloning efficiency induced by Ep was unmodified by further addition of GM-CSF or IL-3. Unicellular FCS- cultures of highly purified adult peripheral blood progenitors generated no erythroid bursts in the presence of Ep alone. The addition of GM-CSF or IL-3 was required to generate BFU-E colonies. These studies indicate that in human embryonic life, BFU-E require only Ep for efficient erythroid burst formation, while IL-3 and GM-CSF essentially enhance the proliferation of early erythropoietic precursors.     

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.     

Effects of human interleukin-3 on granulocytic colony-forming cells in human bone marrow

Human multilineage colony-stimulating factor (multi-CSF)/interleukin-3 (IL-3) induces colony formation from CFU-GEMM, BFU-E, and CFU-Eo when applied to in vitro cultures of highly enriched hematopoietic progenitor cells. No granulocytic colonies are formed in response to IL- 3. However, with appropriate assays, we demonstrate that IL-3 increases the size of G-CSF-induced granulocytic colonies; these colonies contain greater proportions of immature cells as compared with colonies stimulated by G-CSF alone. Furthermore, IL-3 promotes the survival of CFU-G in vitro, whereas in cultures not supplemented with IL-3, CFU-G extinguish within seven days. We conclude that IL-3, although it does not stimulate granulocytic colony formation by itself, regulates the survival and proliferative rate of granulocytic progenitors.     

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.     

Regulation of human eosinophil precursor production by cytokines: a comparison of recombinant human interleukin-1 (rhIL-1), rhIL-3, rhIL-5, rhIL-6, and rh granulocyte-macrophage colony-stimulating factor

The effect of a panel of recombinant human (rh) cytokines on the generation of human eosinophil precursors was assessed using a two-step culture technique. Normal human bone marrow was preincubated with different cytokine combinations in liquid culture before assessment of the number of eosinophil progenitors, which give rise to eosinophil colony-forming units (CFU-Eo) on secondary semi-solid culture with either interleukin-5 (IL-5), IL-3, or granulocyte-macrophage colony-stimulating factor. rhIL-3 or rhGM-CSF, but not rhIL-5, increased the number of CFU-Eo. CFU-Eo production by rhIL-3 or rhGMCSF was maximal after 7 days' preincubation. Neither rhIL-1 or rhIL-6 acted on eosinophil precursors, either alone or in combination with rhIL-5, rhIL-3, or rhGM-CSF. A similar spectrum of activity of the cytokines was demonstrated whether rhIL-5, rhIL-3, or rhGM-CSF was used in the secondary cultures as the eosinophil CSF. However, rhIL-3 induced relatively more rhIL-5-responsive CFU-Eo than rhIL-3-responsive CFU-Eo, suggesting that rhIL-3 is pushing progenitors into an rhIL-5-responsive compartment.     

A stimulatory effect of recombinant murine interleukin-7 (IL-7) on B- cell colony formation and an inhibitory effect of IL-1 alpha

Using a clonal culture system, we investigated the lymphohematopoietic effects of recombinant interleukin-7 (IL-7) obtained from conditioned media of transfected COS 1 cells. IL-7 alone acted on murine bone marrow cells and supported the formation of B-cell colonies. These colony cells were positive for B220, and some of them were also found to have either IgM or Thy-1. B220+, IgM- cells, but not B220- cells sorted from fresh bone marrow cells were able to form B cell colonies in the presence of IL-7. Thus, IL-7 supported the differentiation of B220+, IgM- cells to B220+, IgM+ cells. B220+, IgM+ cells did not proliferate in the presence of IL-7. IL-7 did not affect the myeloid colony formation supported by IL-3, IL-5, IL-6, granulocyte macrophage colony stimulating factor (GM-CSF), and G-CSF. On the other hand, lymphocyte colony formation was not affected by IL-2, IL-3, IL-4, IL-5, IL-6, GM-CSF, or G-CSF. Interestingly, IL-1 alpha inhibited IL-7- induced B cell colony formation in a dose-dependent manner, while the same concentration of IL-1 alpha enhanced the myeloid colony formation by IL-3. This reciprocal effect of IL-1 alpha may act on hematopoietic progenitor cells without accessory cells. These data show that IL-7 is a B cell growth factor and that IL-1 alpha may play an important role in differentiation of myeloid and lymphoid lineages.     

Effect of interleukin-9 on clonogenic maturation and cell-cycle status of fetal and adult hematopoietic progenitors

We assessed the effect of interleukin-9 (IL-9) on clonogenic maturation and cell-cycle status of hematopoietic progenitors of fetal (umbilical cord blood) and adult (bone marrow) origin. As a single agent IL-9 supported, in a concentration-dependent fashion, maturation of burst-forming units-erythroid (BFU-E) of adult and fetal origin. However, only 1/3 the number of adult BFU-E colonies developed, as did in response to granulocyte-macrophage colony-stimulating factor (GM-CSF), and only 1/6 the number developed as did in response to IL-3. In contrast, the effect of IL-9 on fetal BFU-E colonies was equal to that of GM-CSF and IL-3. Synergistic effects of IL-9 with low concentrations (0.1 ng/mL) of GM-CSF and IL-3 were seen on adult BFU-E colony formation, but no effect was apparent at higher concentrations (1.0 ng/mL). In contrast, using fetal cells, synergistic effects of IL-9 with low and high concentrations of GM-CSF and IL-3 were apparent. Addition of IL-9 to plates containing fetal cells plus GM-CSF and IL-3 not only resulted in more BFU-E colonies, but also in more multicentered (greater than or equal to 10 individual centers) colonies, and more cells per colony. IL-9 had a wider spectrum of action on progenitors of fetal origin than on progenitors of adult origin, supporting the generation of fetal multipotent colony-forming unit (CFU)-Mix and CFU-GM colonies. Incubation with IL-9 did not accelerate cycling of adult or fetal BFU-E, CFU-Mix, or CFU-GM to the extent observed after incubation with IL-6. Thus, IL-9 primarily supported maturation of erythroid progenitors of adult origin, and its addition to plates containing GM-CSF and IL-3 (1.0 ng/mL) did not result in maturation of additional clones. In contrast, IL-9 had a wider spectrum of action on fetal progenitors and, when combined with IL-3 and GM-CSF, resulted in clonogenic maturation of progenitors that did not undergo maturation after stimulation with IL-3 and GM-CSF.