High proliferative potential colony-forming cells.
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Biomedical subjects
A B Kriegler
Publications and source records attributed to A B Kriegler.
Dual activity of CSF-1, produced by a murine hybrid cell line and from other sources, on the growth of bone marrow stromal cells in culture.
Medium conditioned by the cell line A.4.10 was shown to produce a factor that inhibits the growth of bone marrow (BM) stromal foci in liquid cultures. In this study we demonstrated that CSF-1, present in this conditioned medium (CM), was largely responsible for this inhibition. This was concluded from the following findings: 1) the CSF-1 activity in the CM copurified with the inhibitory activity in several biochemical purification procedures; 2) an antibody against murine CSF-1 was able to neutralize the CSF-1 activity as well as the inhibitory activity in the A.4.10 CM; and 3) immunopurified murine CSF-1 from A.4.10 CM and from another murine source and recombinant human (rh) CSF-1 were all able to inhibit stromal cell growth. In secondary stromal cultures, inhibition was demonstrated at high cell densities but not at low densities, suggesting that accessory cells may be involved in the effect. When the dose rh CSF-1 was reduced to half of that required to produce 50% inhibition, stimulation of primary stromal foci growth was demonstrated, supporting observations by others that CSF-1 can act as a growth factor for BM stromal cells. In this report we thus demonstrate both inhibitory and stimulatory effects of CSF-1 on the growth of BM stromal cells in vitro.
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Interaction between a murine myeloma cell line and bone marrow stromal cells.
Intravenous transplantation of an in vitro maintained murine myeloma cell line, 5T33, results in progressive growth in the bone marrow of C57Bl/KaLwRiJ mice. Concurrent with the growth of the tumor in vivo, the bone marrow stromal cells are inhibited, as assayed by their ability to form stromal cell foci and long-term monolayers in vitro. Inhibition of normal mouse marrow stromal cell growth also occurs when 5T33 cells are added to the marrow cells in vitro, and contact between the marrow and 5T33 cells is not necessary to achieve inhibition, indicating secretion of one or more diffusible inhibitory factors.
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Haematopoietic radioprotection by Cremophor EL: a polyethoxylated castor oil.
The polyethoxylated castor oil, Cremophor EL (Cremophor) is approved for human use as a vehicle for oral and intravenous administration of water-insoluble compounds. Cremophor has also previously been shown to reverse the multidrug resistance phenotype at clinically acceptable doses. This study demonstrates that doses of Cremophor in the range of 25-50 microliters/kg intravenously (i.v.) administered 1 day prior to near-lethal irradiation protected the regenerative capacity of the marrow, resulting in haematopoietic radioprotection and long-term survival of near-lethally-irradiated mice. In normal mice, Cremophor administration (1) markedly reduced the level of serum haematopoietic inhibitory activity 4-8 h following injection; (2) resulted in a transient decrease in femoral bone marrow cellularity and upregulated B220 (B cells), and 7/4 (neutrophils and activated macrophages), but not Thy-1 (T-cells) surface antigen expression in bone marrow cells within 24 h of injection; and (3) transiently elevated the incidence of both primitive and committed haematopoietic progenitor cells detected in clonal agar culture within 48 h of injection. Bone marrow progenitor cell content, and peripheral blood white cell, platelet and reticulocyte counts were unaffected. This suggests that the haematopoietic radioprotection and recovery observed in irradiated mice pretreated with Cremophor may be the result of accessory cell activation and/or modulation of accessory factors regulating haematopoietic progenitor cells. Our data suggest a potential clinical use of Cremophor as an adjunct to, or as a substitute for, cytokines to minimize myelosuppression following cytotoxic therapy.
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Protection of murine bone marrow by dexamethasone during cytotoxic chemotherapy.
Corticosteroids have the ability to suppress the production of growth factors and cytokines and are thus implicated in the negative regulation of hematopoiesis. We have shown that the corticosteroids, prednisolone and dexamethasone, were able to effectively protect progenitor cells in four strains of mice against cell-cycle-specific antimetabolic chemotherapy agents. The highest levels of protection against 5-fluorouracil (FU; 200 mg/kg) were achieved when two or three intraperitoneal injections of dexamethasone were administered between -7 and +3 hours at a dose of 7.5 mg/kg/injection (optimal dose) or by continuous infusion between -4 and +20 hours. This protective effect is manifested as an increase in the number of high proliferative potential colony-forming cells that survive in the bone marrow 3 days after treatment with FU from between 0.5% and 11% to between 10% and 34% of normal. The bone marrow progenitors and blood cell numbers return to normal from 3 to 5 days and 1 to 2 days earlier, respectively. Less dexamethasone than prednisolone is required to give an equivalent protective effect, which is consistent with their anti-inflammatory potency. These findings are further evidence of the negative regulatory role played by corticosteroids, and indicate that the treatment schedules of corticosteroids during cancer therapy need to be reexamined to obtain the maximum benefit from their use.
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The relationship between different high proliferative potential colony-forming cells in mouse bone marrow.
Previous work has shown that part of the hierarchical structure of the hematopoietic system can be described by HPP-CFC-1 (primitive high proliferative potential colony-forming cells responding to colony-stimulating factor-1 [CSF-1] + interleukin-3 [IL-3] + IL-1), HPP-CFC-2 (more mature HPP-CFC responding to CSF-1 + IL-3), and mature HPP-CFC responding to the single factors, CSF-1, granulocyte-macrophage colony-stimulating factor (GM-CSF), or IL-3. In this study, we have attempted to relate the murine HPP-CFC, stimulated by various combinations of growth factors (GFs)--CSF-1, GM-CSF, IL-3, IL-6, IL-1, stem cell factor (SCF), and transforming growth factor-beta (TGF-beta)--and by CSF-1, GM-CSF, and IL-3 on their own, to these known progenitors. Studies involving regeneration of the bone marrow after 5-fluorouracil (5-FU) treatment, generation of progenitors in liquid cultures in response to different GF combinations, and the HPP-CFC content of lineage-negative rhodamine-sorted bone marrow (BM) fractions have indicated that: 1. the combinations CSF-1 + IL-3 + IL-1 + SCF and CSF-1 + IL-3 + IL-1 + IL-6, and possibly CSF-1 + GM-CSF + IL-3 + IL-1, stimulate pre-HPP-CFC-1; 2. the combinations CSF-1 + IL-1 + GM-CSF, CSF-1 + IL-1 + IL-6, CSF-1 + IL-1 + SCF, CSF-1 + IL-3 + SCF, CSF-1 + IL-6 + SCF, and IL-3 + SCF, appear to overlap with the CSF-1 + IL-3 + IL-1 combination to stimulate the more mature cells of the HPP-CFC-1 compartment; 3. the combinations CSF-1 + GM-CSF, CSF-1 + IL-1, CSF-1 + IL-6, and CSF-1 + SCF may stimulate the more mature cells of the HPP-CFC-2 population, while the single factors CSF-1, GM-CSF, and IL-3, as suggested in other reports, may stimulate HPP-CFC that are more mature than the HPP-CFC-2; 4. the combinations IL-3 + IL-6 and SCF + IL-6 appear to stimulate HPP-CFC that overlap with the HPP-CFC-1 population, while those responding to the combination GM-CSF + TGF-beta overlap with the HPP-CFC-2 population within the hematopoietic hierarchy; and 5. CSF-1 and GM-CSF appear to be interchangeable in the combinations studied.
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Reversal of multidrug resistance by surfactants.
Cremophor EL, a pharmacologically inactive solubilising agent, has been shown to reverse multidrug resistance (MDR). Using flow cytometric evaluation of equilibrium intracellular levels of daunorubicin (DNR), we found that eight other surface active agents will also reverse MDR. All the active detergents contain polyethoxylated moieties but have no similarities in their hydrophobic components. The properties of three polyethoxylated surfactants that showed the lowest toxicities, Cremophor, Tween 80 and Solutol HS15, were examined in more detail. The concentrations of Tween 80 and Solutol required to reverse DNR exclusion were 10-fold lower than for Cremophor. However while concentrations greater than or equal to 1:10(2) of the former two surfactants resulted in breakdown of cells, even 1:10 of Cremophor did not lyse cells. Studies of the effects of Cremophor on the uptake and efflux of DNR in normal and MDR cell types showed that Cremophor increases intracellular DNR primarily by locking the rapid efflux from the cells. This blockage of drug efflux may be mediated by a substantial alteration in the fluidity of cell membranes induced by Cremophor, as shown by decreased fluorescence anisotropy of a membrane probe. Consistent with these data, coinjection of adriamycin plus Cremophor into mice carrying a multidrug resistant P388 transplantable tumour significantly increased the survival time of the mice compared with adriamycin treatment alone.
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Infective endocarditis--the effect of liposomes as carrier substance for alpha 1-antitrypsin and ampicillin.
Infective endocarditis has a high mortality and morbidity rate despite all available treatment. Little attention has been paid to the possible role of polymorphonuclear leucocytes in damage to the heart valves. It was postulated that if the elastases set free from these leucocytes could be neutralised, this would prevent damage to the heart valves. Alpha 1-antitrypsin (alpha 1-AT) in liposomes was used to neutralise elastases. This process on its own and in various combinations with ampicillin were compared in animal models. Evaluation was performed by measuring vegetation size, by blood and vegetation cultures, and by light microscopy of the damaged tissue. A statistically significant difference (t-test; P less than 0.005, with Bonferroni's correction for multiple comparisons) was found in vegetation size in the groups receiving ampicillin in liposomes, but the hypothesis that alpha 1-AT might reduce valvular damage was not proven.
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Colony-forming cells with high proliferative potential (HPP-CFC).
Colony-forming cells with a high proliferative potential (HPP-CFC) have been defined by their ability to form large colonies in vitro (diameters greater than 0.5 mm and containing approximately 50,000 cells) in bone marrow cell cultures. The HPP-CFC have been characterized by: 1) a relative resistance to treatment in vivo with the cytotoxic drug 5-fluorouracil, 2) a high correlation with cells capable of repopulating the bone marrow of lethally irradiated mice, 3) their multipotential ability to generate cells of the macrophage, granulocyte, megakaryocyte and erythroid lineages, and 4) their multifactor responsiveness. The HPP-CFC have been described in both mouse and human bone marrow. These properties suggest that the HPP-CFC represent an important cell type in hematopoiesis and provide a model system, particularly in the human, for studying the properties of primitive progenitor cells in vitro.
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Progenitor cells in murine bone marrow stimulated by growth factors produced by the AF1-19T rat cell line.
The AF1-19T rat cell line has been found to produce an activity that acts synergistically with colony-stimulating factor 1 (CSF-1) to stimulate primitive high proliferative potential colony-forming cells (HPP-CFC) in mouse bone marrow (BM) that appear to be the same as those stimulated by the combination of 5637-cell-conditioned medium (CM) plus CSF-1 or recombinant human (rh) interleukin 1 (IL-1) plus recombinant murine (rm) interleukin 3 (IL-3) plus CSF-1. AF1-19T also produced granulocyte-macrophage colony-stimulating factor (GM-CSF), which could be separated from this synergistic activity by gel filtration followed by hydroxylapatite chromatography. Results obtained from the mouse thymocyte costimulation assay for IL-1, the hybridoma growth factor assay for interleukin 6 (IL-6), the ability to stimulate HPP-CFC, and the ability to block this stimulation with an antibody to murine IL-1 alpha suggest that the synergistic activity in AF1-19T-CM is probably a mixture of IL-1 activity and IL-6 or an IL-6-like activity. Other workers have described a progenitor cell population in mouse BM (CFU-A) that forms large colonies in response to AF1-19T-CM plus CSF-1 or GM-CSF plus CSF-1. Experiments involving the kinetics of recovery after 5-fluorouracil treatment and generation of progenitors suggest that the GM-CSF-plus-CSF-1-responsive progenitors, and hence CFU-A, are a more mature cell type than the more primitive HPP-CFC, responsive to 5637-cell-CM plus CSF-1 or rhIL-1 plus rmIL-3 plus CSF-1.
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Studies on the myeloid synergistic factor from 5637: comparison with interleukin-1 alpha.
A synergistic factor that is produced by the human bladder carcinoma cell line 5637 (SF-1) stimulates primitive bone marrow progenitor cells, termed high proliferative-potential colony-forming cells (HPP-CFC), in the presence of an optimal dose of macrophage colony stimulating factor (CSF-1). Recent reports have demonstrated that interleukin-1 alpha (IL-1) is identical to hemopoietin 1 and have suggested that IL-1 is the synergistic factor present in 5637 conditioned medium (cm). We have compared the ability of recombinant human IL-1 alpha and partially purified preparations of SF-1 to synergize with optimal doses of CSF-1 to stimulate HPP-CFC. In all experiments performed the numbers of HPP-CFC colonies formed with IL-1 were significantly less than with SF-1. Replating experiments demonstrated that SF-1 plus CSF-1 generated HPP-CFC (responsive to IL-3 plus CSF-1); however, IL-1 plus CSF-1 resulted in no generation of HPP-CFC. Multiple factor combinations of IL-1 and SF-1 with G-CSF, GM-CSF, and CSF-1 also resulted in less HPP-CFC colony formation in cultures containing IL-1 compared with SF-1. Incubation of SF-1 with an antibody to IL-6 had no effect on HPP-CFC colony formation and IL-6 did not synergize with IL-1 plus CSF-1 or SF-1 plus CSF-1. These data suggest the presence of a factor in 5637 cm, which is distinct from G-CSF, GM-CSF, and IL-6, which synergizes with IL-1 to produce the SF-1 effect.
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In vivo effects of interleukin-1 alpha on regenerating mouse bone marrow myeloid colony-forming cells after treatment with 5-fluorouracil.
Injection of a single dose of recombinant human interleukin-1 alpha (r-hu-IL-1 alpha) into mice 24 hr after 5-fluorouracil (FU) treatment resulted in an increased rate of recovery of three types of colony-forming cells (CFCs) in the bone marrow. Myeloid progenitors with high proliferative potential (responsive to CSF-1 + IL-3 + IL-1 alpha), low proliferative potential (responsive to CSF-1), megakaryocyte progenitors, and total nucleated cells per femur increased up to 5-fold, 7-fold, 3-fold, and 3-fold, respectively, in a dose related fashion compared with the control FU treated marrows. The kinetics of FU kill and recovery of these CFCs are shown.
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Interleukin 1 plus interleukin 3 plus colony-stimulating factor 1 are essential for clonal proliferation of primitive myeloid bone marrow cells.
The clonal growth in nutrient agar at low cell densities of high-proliferative potential colony-forming cells (HPP-CFC) of bone marrow obtained from mice treated 2 days earlier with 5-fluorouracil (FU) (FU2dBM) has been shown to require a combination of three growth factors, interleukin 1 (IL-1), interleukin 3 (IL-3), and macrophage colony-stimulating factor (CSF-1). These HPP-CFC have been enriched 140-fold from FU2dBM by fluorescence-activated cell sorting of 7/4-, B220-, and L3T4-negative cells. The mean of the plating efficiencies of these enriched populations was 4.4% and no growth was observed when the factors were used singly. Similarly, enrichments of 16-fold were obtained from FU2dBM using immunomagnetic Dynabeads with anti-7/4 plus anti-B220 (meaning plating efficiency 0.5%). The further additions of human granulocyte CSF or mouse granulocyte-macrophage CSF or both to IL-1 plus IL-3 plus CSF-1 did not increase HPP-CFC colony formation, but both augmented the small colony formation with IL-1 plus IL-3, IL-3 plus CSF-1, or IL-1 plus CSF-1.
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A colorimetric liquid culture assay of a growth factor for primitive murine macrophage progenitor cells.
Synergistic factors from media conditioned (CM) by human placentas or the 5637 human bladder carcinoma cell line (SFH-HPCM and SFH-5637 respectively) have the ability to stimulate early progenitor cells in mouse bone marrow to form large colonies in agar cultures after 12-14 days, in the presence of CSF-1. Culture conditions have been examined and a quicker and more convenient liquid culture assay has been developed for this factor, using a tetrazolium salt to quantitate cell proliferation. The use of flat-bottomed vessels, high cell density, supra-optimal doses of CSF-1 or the addition of WEHI-3-CM to these cultures, all resulted in a decrease in the required incubation time. In combination, these modifications reduced the assay time to 4 days.
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Detection of murine hemopoietin-1 in media conditioned by EMT6 cells.
We have previously reported replating experiments which demonstrated the existence of subpopulations of murine high-proliferative-potential colony-forming cells (HPP-CFC). One population of HPP-CFC, termed HPP-CFC-1, is stimulated by the combination of macrophage colony-stimulating factor (CSF-1) plus hemopoietin-1 (H-1), and actively generate a second population of HPP-CFC, termed HPP-CFC-2, which is responsive to CSF-1 plus interleukin-3 (IL-3). These reclonal experiments represent an assay system that discriminates between the two types of synergistic factors, namely H-1 and IL-3. To date H-1 has only been detected in medium conditioned by human cells. In this paper we have utilized these recloning experiments to study the synergistic factor(s) present in media conditioned by the murine mammary carcinoma cell line EMT6. Colony formation in secondary cultures containing cells picked up from primary cultures incubated in CSF-1 plus EMT6-conditioned medium was identical to that seen in secondary cultures containing cells picked up from primary cultures incubated in CSF-1 plus a source of H-1. Both sets of cultures demonstrated the generation of HPP-CFC-2 in the primary cultures, indicating the presence of a molecule in EMT6-conditioned medium that is the murine equivalent of H-1.
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Generation of murine hematopoietic precursor cells from macrophage high-proliferative-potential colony-forming cells.
High-proliferative-potential colony-forming cells (HPP-CFC) have been described as primitive murine macrophage progenitors. We have previously demonstrated the existence of two populations of HPP-CFC: one population, termed HPP-CFC-1, is stimulated by the combination of macrophage colony-stimulating factor (CSF-1) plus haemopoietin-1 (H-1) and actively generates a second population of HPP-CFC, termed HPP-CFC-2. HPP-CFC-2 are stimulated by CSF-1 plus interleukin-3 and generate macrophage CFC that differentiate to form mature macrophages. In this study, we have demonstrated that HPP-CFC-1, when stimulated by CSF-1 plus H-1, generate colony-forming cells (CFC) for the megakaryocyte and granulocyte lineages in addition to HPP-CFC-2 and M-CFC. No CFC were detected with erythroid potential. In addition, HPP-CFC-1 generated cells that formed day-13 spleen colonies, cells that repopulated the bone marrow, cells with platelet-repopulating ability, and cells with erythroid-repopulating ability in lethally irradiated mice. These data support previous data that the HPP-CFC-1 represent a primitive hemopoietic cell population and demonstrate the multipotentiality but not totipotentiality of these cells.
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Isolation and analysis of primitive hemopoietic progenitor cells on the basis of differential expression of Qa-m7 antigen.
In the presence of the hemopoietic growth factor CSF-1, the later committed cells of the macrophage lineage can be detected by their ability to form small colonies in clonal agar culture (CFCCSF-1). Synergistic factors have been described that in combination with CSF-1 stimulate developmentally early hemopoietic progenitor cells of high proliferative potential (HPP-CFC). By using a monoclonal antibody to the Qa-m7 antigenic determinant, we investigated and compared the expression of Qa-m7 on CFCCSF-1 and on HPP-CFC of two types that grow in response to either 1) CSF-1 plus synergistic factor from human placenta-conditioned medium (HPP-CFCHplac+CSF-1) or 2) CSF-1 plus synergistic factor from conditioned medium of the WEHI-3 myelomonocytic cell line (HPP-CFCW+CSF-1). We have shown that HPP-CFC of both types express relatively more Qa-m7 antigen than CFCCSF-1 and can be separated and enriched on this basis by discontinuous buoyant density centrifugation and fluorescence-activated cell sorting of normal bone marrow. Significant enrichments of HPP-CFCHPlac+CSF-1 (43.5-fold) and HPP-CFCW+CSF-1 (28.8-fold) have been achieved with cloning efficiencies of HPP-CFC in the most enriched fractions reaching 4 to 5%. These results clearly illustrate the fact that there are populations of progenitor cells from normal, unperturbed bone marrow that strictly require a combination of two hemopoietic growth factors (CSF-1 plus synergistic factor) in order to be detected.
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