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.     

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.     

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.     

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.     

Myeloid differentiation of purified CD34+ cells after stimulation with recombinant human granulocyte-monocyte colony-stimulating factor (CSF), granulocyte-CSF, monocyte-CSF, and interleukin-3.

CD34+ cells isolated from bone marrow or umbilical cord blood from healthy donors were studied for proliferation and differentiation in liquid cultures in the presence of recombinant human granulocyte-monocyte colony-stimulating factor (GM-CSF), granulocyte CSF (G-CSF), monocyte CSF (M-CSF), and interleukin-3 (IL-3), followed by immunophenotyping for myeloid and myeloid-associated cell surface markers. IL-3, either alone or together with GM-CSF, G-CSF, or M-CSF, induced, on average, 50-fold cell multiplication, GM-CSF five fold to 10-fold, and G-CSF and M-CSF less than fivefold. Cells from cultures stimulated with GM-CSF, G-CSF, or M-CSF alone contained cells with a "broad" myeloid profile, "broader" than observed in cultures with IL-3. However, since IL-3 induced rapid cell multiplication, high numbers of cells expressing early (CD13, CD33) and late myeloid markers (CD14, CD15) were recovered. The presence of other CSFs together with IL-3 did not alter the IL-3-induced effect on the cells. When 5,000 CD34+ cells were cultured with IL-3 alone, the cultures still contained 2,000 to 5,000 CD34+ cells after 14 days of culture, while cells cultured with GM-CSF, G-CSF, or M-CSF contained less than 1,000 CD34+ cells. Furthermore, 1,000 to 3,000 cells were positive for the megakaryocytic lineage marker CD41b after cultures with GM-CSF or IL-3, while cultures with G-CSF or M-CSF did not contain detectable numbers of CD41b+ cells. Finally, erythroid cells could also be generated from purified CD34+ cells. The results show that IL-3 and GM-CSF can induce rapid proliferation of purified CD34+ cells in vitro with differentiation to multiple myeloid lineages, while certain subsets maintain expression of CD34.     

Interleukin-1 (IL-1) inhibits growth of cytomegalovirus in human marrow stromal cells: inhibition is reversed upon removal of IL-1.

A Toledo strain cytomegalovirus (CMV) containing the gene for green fluorescent protein (GFP) under the control of elongation factor-1 promoter was used to study infection of human marrow stromal cells. Two stromal cell lines were used: HS-5, which secretes copious amounts of known cytokines and interleukins; and HS-27a, which does not secrete these activities. CMV growth and spread was monitored by counting green plaques and quantitating GFP intensity. Initial studies indicated that, whereas HS-5 and 27a have similar susceptibilities to infection, as evidenced by the same number of GFP+ cells at day 2, HS-5 appears more resistant to growth and spread of CMV. Furthermore, conditioned media from HS-5 (HS-5 CM) inhibited CMV plaque formation in HS-27a, suggesting that factors secreted by HS-5 are responsible for limiting CMV growth. Neutralizing antibodies against interleukin-1alpha (IL-1alpha) and IL-1beta completely blocked the ability of HS-5 CM to limit viral growth, suggesting that IL-1, which is known to be present in HS-5 CM, is responsible for this effect. When exogenous IL-1beta was added to CMV-infected HS-27a, both the number of plaques and the intensity of GFP was significantly reduced in IL-1-treated HS-27a compared with untreated HS-27a (the number of plaques by day 18 was 20 +/- 3 v 151 +/- 12/well, respectively; GFP intensity was 535 +/- 165 v 6,516 +/- 652/well, respectively, in 4 separate experiments). At day 21, when IL-1beta-treated, CMV-infected cultures were passaged and then cultured in the absence of IL-1beta, CMV growth progressed with the kinetics of the original untreated culture, indicating that the IL-1beta effect is reversible. Because HS-27a expresses the type I IL-1 receptor, we speculate that the antiviral effects are mediated through IL-1-induced changes in cellular gene expression. DNA chip analysis of mRNA from IL-1beta-treated and nontreated HS-27a cells has identified some candidate molecules.     

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.     

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.     

Interactions among colony-stimulating factors, IL-1 beta, IL-6, and kit-ligand in the regulation of primitive murine hematopoietic cells.

We have investigated the regulation of primitive murine hematopoietic progenitors by the cytokines interleukin 1 (IL-1), interleukin 6 (IL-6), and kit-ligand (KL). Individually these cytokines have a limited ability to stimulate the growth of high proliferative potential colony-forming cells (HPP-CFC) from 5-fluorouracil (5-FU)-purged bone marrow, but in combination these cytokines demonstrate synergism in promoting the growth of HPP-CFC. Furthermore, IL-1, IL-6, and KL, alone or in combination, synergized with the colony-stimulating factors (CSFs) granulocyte CSF (G-CSF), macrophage CSF (M-CSF), granulocyte-macrophage CSF (GM-CSF), or interleukin 3 (IL-3) in clonal and liquid cultures of 5-FU-purged bone marrow. The pattern of HPP-CFC growth that was observed with 40 different cytokine combinations demonstrated the unique roles of IL-1, IL-6, and KL in the regulation of HPP-CFC proliferation. Short-term liquid cultures (delta-cultures), with secondary recloning, of 5-FU-purged bone marrow were stimulated to greatly expand the numbers of progenitor cells generated in response to cytokine stimulation. The greatest expansion, over 1800-fold, of the more mature progenitor compartments took place in delta-cultures stimulated with IL-1, IL-6, and KL plus IL-3. However, the combination of IL-1 and IL-6 plus KL was optimal in expanding HPP-CFC, increasing their numbers by 700-fold. The ability to expand early progenitor cells in delta-cultures was further demonstrated by the greater than 100-fold expansions of day-12 spleen colony-forming units (CFU-S) by the synergistic interactions of IL-1 with IL-3 or KL.     

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.     

Effects of the stem cell factor, c-kit ligand, on human megakaryocytic cells.

The kit ligand (KL), also termed stem cell factor (SCF), is a recently discovered hematopoietic growth factor that augments response of early progenitor cells to other growth factors and supports proliferation of continuous mast cell lines. Histological studies suggest that the receptor for SCF/KL, the c-kit proto-oncogene product, is present in bone marrow megakaryocytes. We studied the effects of SCF/KL on immortalized human megakaryocytic cell lines (CMK, CMK6, and CMK11-5) and on isolated human marrow megakaryocytes. Human SCF/KL alone or in combination with the hematopoietic growth factors, interleukin-3 (IL-3), granulocyte-macrophage colony-stimulating factor (GM-CSF), and IL-6, stimulated proliferation of these megakaryocytic cell lines. SCF/KL treatment did not alter expression of gpIb, gpIIb/IIIa, LFA-1, ICAM-1, or GMP-140 in CMK cells. No effect on ploidy was observed. Furthermore, human SCF/KL induced expression of IL-1 alpha, IL-1 beta, IL-2, and IL-6 in CMK cells. In a fibrin clot system, SCF/KL modestly potentiated megakaryocyte colony formation when added alone to cultures containing CD34+, DR+ bone marrow cells. Addition of SCF/KL with IL-3 or GM-CSF to these cultures resulted in a more marked marrow megakaryocytic cells. SCF/KL may directly affect megakaryocytopoiesis, as well as secondarily modulate hematopoiesis through induction of cytokines in target cells.     

Recombinant human stem cell factor enhances myeloid colony growth from human peripheral blood progenitors

Peripheral blood hematopoietic progenitors (PBHP) are capable of colony growth in vitro. The effect of stem cell factor (SCF), interleukin-6 (IL-6), and basic fibroblast growth factor (bFGF) on myeloid colony proliferation of PBHP was determined. PBHP purified by positive selection with CD34-specific antibody were plated in semisolid agarose with reported plateau doses of interleukin-3 (IL-3), granulocyte- macrophage colony-stimulating factor (GM-CSF), and granulocyte colony- stimulating factor (G-CSF) to enhance myeloid colony growth. Experiments then were done to examine colony growth in response to SCF or with SCF and bFGF and/or IL6. SCF alone in the absence of any other growth factors did not support colony growth. SCF at a determined optimum concentration of 100 ng/mL added to the combination of IL-3, GM- CSF, and G-CSF enhanced colony growth and size relative to proliferation in response to the latter three factors alone (from 78 to 188 total colonies/10(4) PBHP plated and from 10 to 93 large [> 200 cells] colonies/10(4) PBHP plated). Furthermore, addition of bFGF and/or IL-6 to the combination of optimum concentrations of SCF, IL-3, GM-CSF, and G-CSF further enhanced colony number and size in a dose- dependent fashion. Using the optimum combination of all growth factors, we determined that the number of myeloid colony-forming PBHP in whole blood was similar between individuals at about three colonies per milliliter whole blood. We conclude that progenitors capable of responding to the early-acting growth factor, SCF, are represented in PBHP and that the number of circulating myeloid colony-forming PBHP is likely a regulated parameter that may have an important biologic function.     

Characterization of the human burst-forming unit-megakaryocyte

Two classes of human marrow megakaryocyte progenitor cells are described. Colony-forming unit-megakaryocyte (CFU-MK)-derived colonies appeared in vitro after 12-day incubation; burst-forming unit-megakaryocyte (BFU-MK)-derived colonies appeared after 21 days. CFU-MK-derived colonies were primarily unifocal and composed of 11.6 +/- 1.2 cells/colony; BFU-MK-derived colonies were composed of 2.3 +/- 0.4 foci and 108.6 +/- 4.4 cells/colony. CFU-MK and BFU-MK were separable by counterflow centrifugal elutriation. CFU-MK colony formation was diminished by exposure to 5-fluorouracil (5-FU); BFU-MK colony formation was unaffected. CFU-MK and BFU-MK were immunologically phenotyped. CFU-MK expressed the human progenitor cell antigen-1 (HPCA-1, CD34, clone My10) and a major histocompatibility class II locus, HLA-DR, and BFU-MK expressed only detectable amounts of CD34. BFU-MK colony formation was entirely dependent on addition of exogenous hematopoietic growth factors. Recombinant granulocyte macrophage colony-stimulating factor (GM-CSF) and interleukin-3 (IL-3) possessed such colony-stimulating activity, whereas recombinant erythropoietin (Epo), G-CSF, IL-1 alpha, IL-4, and purified thrombocytopoiesis-stimulating factor did not. These studies indicate the existence of a human megakaryocyte progenitor cell, the BFU-MK, which has unique properties allowing it to be distinguished from the CFU-MK.     

Interleukin-6 and granulocyte-macrophage colony-stimulating factor are candidate growth factors for chronic myelomonocytic leukemia cells

Previous studies suggest that malignant cells from some patients with myeloid leukemias produce colony-stimulating factors (CSFs) that can function as autocrine growth factors in vitro. We have examined the roles of interleukin-6 (IL-6) and granulocyte-macrophage CSF (GM-CSF) in the proliferation of myeloid leukemia cells. IL-6 activity was assessed in conditioned medium (CM) from myeloid leukemia cell cultures or cell lysates using IL-6-dependent KD83 and 7TD1 murine cell lines. Media conditioned by cells from patients with chronic myelomonocytic leukemia (CMMoL), but not by normal monocytes, chronic myelogenous leukemia (CML), or acute myelogenous leukemia (AML) cells, contained substantial levels (50 to 1,000 U/10(6) cells) of IL-6. The IL-6 content of CM correlated directly with donor peripheral blood WBC count. CM from two of five CMMoL samples also contained greater than 350 pg/mL GM-CSF. Moreover, CMMoL cells spontaneously formed colonies in semisolid medium. CMMoL colony formation could be partially inhibited by antibodies to IL-6 or GM-CSF, whereas combination of these antibodies gave additive, and nearly complete (greater than 93%), inhibition of spontaneous colony formation. Cell lysates from uncultured CMMoL cells from one patient contained abundant GM-CSF protein but no detectable IL-6. These data suggest that IL-6 and GM-CSF act in vitro as autocrine growth factors for CMMoL cells, and that CMMoL cells in vivo may represent a GM-CSF-dependent autocrine growth system.     

Establishment of a human acute myeloid leukemia cell line (Kasumi-1) with 8;21 chromosome translocation

A novel leukemic cell line with an 8;21 chromosome translocation, designated as Kasumi-1, was established from the peripheral blood of a 7-year-old boy suffering from acute myeloid leukemia (AML). The Kasumi-1 cells were positive for myeloperoxidase showing a morphology of myeloid maturation. The response in proliferation assay was observed in the culture with interleukin-3 (IL-3), IL-6, granulocyte colony-stimulating factor (G-CSF), and granulocytemacrophage CSF (GM-CSF), but not with IL-1 or IL-5. Neither granulocytic nor eosinophilic maturation was observed in the liquid culture by the addition of dimethyl sulfoxide, G-CSF, or IL-5, respectively. In contrast, induction of macrophagelike cells was seen by the addition of phorbol ester. This is the first report of a human AML cell line with t(8;21) that has characteristics of myeloid and macrophage lineages. The cell line could be a useful tool for elucidating the pathophysiology of AML with t(8;21).     

Recombinant human stem cell factor enhances the formation of colonies by CD34+ and CD34+lin- cells, and the generation of colony-forming cell progeny from CD34+lin- cells cultured with interleukin-3, granulocyte colony-stimulating factor, or granulocyte-macrophage colony-stimulating factor.

We tested the ability of recombinant human stem cell factor (SCF) to stimulate isolated marrow precursor cells to form colonies in semisolid media and to generate colony-forming cells (CFC) in liquid culture. SCF, in combination with interleukin-3 (IL-3), granulocyte-macrophage colony-stimulating factor (GM-CSF), or granulocyte colony-stimulating factor (G-CSF) caused CD34+ cells to form increased numbers of granulocyte-macrophage colonies (CFU-GM), and to form macroscopic erythroid burst-forming units (BFU-E) in the presence of IL-3, erythropoietin (Epo), and SCF. We tested isolated CD34+lin- cells, a minor subset of CD34+ cells that did not display antigens associated with lymphoid or myeloid lineages, and CD34+lin+ cells, which contain the vast majority of CFC, and found that the enhanced colony growth was most dramatic within the CD34+lin- population. CD34+lin- cells cultured in liquid medium containing SCF combined with IL-3, GM-CSF, or G-CSF gave rise to increased numbers of CFC. Maximal numbers of CFU-GM were generated from CD34+lin- cells after 7 to 21 days of culture, and required the presence of SCF from the initiation of liquid culture. The addition of SCF to IL-3 and/or G-CSF in cultures of single CD34+lin- cells resulted in increased numbers of CFC due to the proliferation of otherwise quiescent precursors and an increase in the numbers of CFC generated from individual precursors. These studies demonstrate the potent synergistic interaction between SCF and other hematopoietic growth factors on a highly immature population of CD34+lin- precursor cells.     

Hematopoietic recovery following high-dose combined alkylating-agent chemotherapy and autologous bone marrow support in patients in phase-I clinical trials of colony-stimulating factors: G-CSF,GM-CSF,IL-1, IL-2, M-CSF

Summary Hematopoietic recovery in 115 patients with metastatic breast cancer or metastatic melanoma, enrolled in phase-I studies of recombinant growth factors while undergoing treatment with high-dose chemotherapy with autologous bone marrow support, was examined with assays of bone marrow progenitor cells and peripheral blood progenitor cells, and by evaluation of peripheral blood counts. Groups of patients receiving hematopoietic cytokine support [with interleukin-1 (IL-1), interleukin-2 (IL-2), granulocyte colony-stimulating factor (G-CSF), granulocyte-macrophage CSF (GM-CSF), or monocyte CSF (M-CSF)] post marrow infusion were compared with contemporaneous control patients not receiving growth factor support. Patients receiving GM-CSF demonstrated statistically significant increases in the growth of granulocyte/macrophage colony-forming units (CFU-GM) in the bone marrow and peripheral blood compared with control patients. The effect of GM-CSF was dose dependent in the early period post marrow infusion (day +6) with bone marrow CFU-GM colonies at doses 8–16 g/kg/ day 34 times those measured in controls. Significant increases in bone marrow multipotential progenitor cells (CFU-GEMM) were seen in patients receiving GMCSF day + 21 post marrow infusion. Patients receiving IL-1 demonstrated significant increases in bone marrow CFU-GM at day +21, maximal at dosages of 24–32 ng/kg/day. There were no significant increases in burst forming unit-erythroid (BFU-E) among any study group. Patients receiving G-CSF had significantly increased absolute neutrophil counts (ANC) and total white blood cell counts (WBC) by day +11 post transplant compared with control patients. Patients receiving GM-CSF demonstrated significantly increased WBC (greater than 2000/mm³) at day +11 and ANC greater than 500/mm³ at day +16. Optimal dose of GCSF and GM-CSF to stimulate neutrophil recovery post transplant was 4–8 g/kg/day and 8–16 g/kg/day, respectively. Platelet recovery did not differ among the six study groups. These data demonstrate accelerated myeloid recovery after high-dose chemotherapy and autologous bone marrow support in patients receiving either G-CSF or GM-CSF. Moreover, GM-CSF and IL-1 stimulate myelopoiesis at the level of bone marrow CFU-GM, while G-CSF causes earlier neutrophil recovery peripherally.This work has been supported in part by The National Heart, Lung, and Blood Institute, grant P01CA47741. Joanne Kurtzberg, MD is a scholar of the Leukemia Society of America     

Treatment of human bone marrow with recombinant placenta immunoregulator ferritin results in myelopoiesis and T-cell suppression through modulation of the cytokine-chemokine networks

OBJECTIVE: Placenta immunomodulator ferritin (PLIF) is a cloned human chimeric ferritin H chain with a novel non-ferritin C-terminal 48 amino acid sequence (C48). Recombinant PLIF-C48 exhibited cell-mediated immunosuppression. The aim of the current study was to investigate the regulatory effects of native placental ferritin (PLF), recombinant PLIF, and C48 on hematopoiesis of human bone marrow (BM). METHODS: BM mononuclear cells (BM-MNCs) and CD34(+) selected cells were treated in vitro with either PLF, PLIF, or C48 without and in combination with granulocyte (G)-monocyte (M) colony-stimulating factor (GM-CSF) and subjected to hematopoietic progenitor cell assay. Cytokines and chemokines secreted by the treated cells were evaluated in culture supernatant using antibody array assays to determine mechanism of action. RESULTS: In vitro treatment of BM-MNCs with PLF, PLIF, or C48 induced significant growth of myeloid colonies and when mixed with GM-CSF or Granulocyte-Colony Stimulating Factor (G-CSF) exhibited additive enhanced colony forming units-granulocyte monocyte growth. Yet, C48 treatment of selected CD34(+) cells did not yield colony formation and did not affect their response to GM-CSF. Treatment of BM-MNCs with C48 for 48 hours induced secretion of marked levels of GM-CSF, interleukin (IL)-6, IL-1, and IL-10. These cytokines were secreted primarily by C48-treated BM adherent cells and partly by nonadherent cells, whereas the CD34(+) selected cells secreted IL-6 only. CONCLUSION: C48-PLIF enhancement of myelopoiesis resulted from cross talk between BM accessory cells and progenitor cells. The differential PLIF-C48 effects (i.e., myeloid progenitor cell growth and T-cell suppression) are due to their effect on the cytokine-chemokine networks.     

Further studies on growth factor production by the TC-1 stromal cell line: pre-B stimulating activity.

Adherent murine stromal cells support long-term in vitro lymphopoiesis or myelopoiesis dependent on the culture conditions used. A cell line, TC-1, isolated from long-term liquid murine marrow cultures under conditions approaching those permissive for lymphoid growth, has been found to produce an activity that acts synergistically with interleukin-3 (IL-3) or colony-stimulating factor-1 (CSF-1) to stimulate in vitro myeloid colonies, but which has no intrinsic colony-stimulating activity. We report here the presence of multiple growth factors in conditioned medium (CM) from the TC-1 line, including granulocyte-macrophage colony-stimulating factor (GM-CSF) (bioassay with antibody blocking and messenger RNA [mRNA] analysis), granulocyte CSF (G-CSF) and IL-4 (factor-dependent cell line bioassay), and CSF-1 (radioimmunoassay, mRNA) along with a pre-B cell inducing activity, which appears separate from these CSFs and segregates with the myeloid synergizing activity through anion exchange, sizing, and Conconavalin A chromatography. Because these activities are not yet purified to homogeneity, their identity or lack of identity remains an open question. Assays of TC-1 CM or cellular mRNA analysis have given negative results for IL-1, IL-2, IL-3, IL-6, and IL-7, and IL-6 does not stimulate pre-B cells in this assay. However, IL-4 and G-CSF do stimulate in vitro induction of pre-B cells from pre-B and B-cell-depleted Balb/C marrow and are present in CM by selective cell line assay. A monoclonal antibody to IL-4 that inhibited its pre-B inducing activity did not inhibit pre-B inducing activity of TC-1 CM. These data suggest the existence of a unique synergizing and pre-B inducing factor(s) in TC-1 CM. Given the known capacity of subliminal levels of growth factors to act synergistically, an alternate possibility is that these biologic phenomena represent the actions of low concentrations of growth factors acting synergistically and possibly associated with some core protein.