Control of normal differentiation of myeloid leukemic cells. IV. Induction of differentiation by serum from endotoxin treated mice

Sera from different strains of mice injected with endotoxin induced clones (D⁺) from a cultured line of myeloid leukemic cells to undergo normal differentiation to mature granulocytes and macrophages. Other clones (D^?) derived from the same cell line were not inducible by these sera to undergo normal cell differentiation. Sera from the same strains of mice that had not been injected with endotoxin, increased the cloning efficiency of D⁺ and D ^? clones but did not induce differentiation. Endotoxin serum induced differentiation in D⁺ cells at dilutions up to 1:64, but increased the cloning efficiency of these cells at dilutions up to 1:2048. The end point of the dilution of endotoxin serum that induced differentiation in D⁺ cells, was also the end point that induced the formation of colonies with differentiation from normal bone marrow cells. The results indicate that serum from endotoxin treated animals can serve as a good in vivo source to induce normal differentiation in D⁺ myeloid leukemic cells; that the progeny of a single leukemic cell was induced to undergo differentiation to both macrophages and granulocytes; that endotoxin serum contained two activities, one that increased cloning efficiency and the other that induced cell differentiation; and that the same material in endotoxin serum induced cell differentiation in normal and leukemic cells.     

The in vitro differentiation of density sub-populations of colony-forming cells under the influence of different types of colony-stimulating factor.

The in vitro proliferation and differentiation of myeloid progenitor cells (CFU-c) in agar culture from CBA/Ca mouse bone marrow cells was studied. Density subpopulations of marrow cells were obtained by equilibrium centrifugation in continuous albumin density gradients. The formation of colonies of granulocytes and/or macrophages was studied under the influence of three types of colony-stimulating factor (CSF) from mouse lung conditioned medium CSFMLCM), post-endotoxin mouse serum (CSFES) and from human urine (CSFHu). The effect of the sulphydryl reagent mercaptoethanol on colony development was also examined. The density distribution of CFU-c was dependent on the type of CSF. Functional heterogeneity was found among CFU-c with partial discrimination between progenitor cells forming pure granulocytic colonies and those forming pure macrophage colonies. Mercaptoethanol increased colony incidence but had no apparent effect on colony morphology or the density distribution of CFU-c.     

Purification of a murine leukemia inhibitory factor from Krebs ascites cells

A factor capable of inducing terminal differentiation in the murine myeloid leukemia cell line M1 has been purified to apparent homogeneity from the medium conditioned by Krebs II ascites tumor cells. The factor, termed leukemia inhibitory factor (LIF) is a single chain glycoprotein of apparent Mr 58,000 which induces differentiation and inhibits proliferation of the M1 cell line but not the WEHI-3B D+ murine myeloid leukemic cell line and has no detectable proliferative activity on normal myeloid progenitor cells. It was purified using four successive high-efficiency purification steps--anion-exchange chromatography on DEAE-Sepharose; cation-exchange chromatography on CM-Sepharose; affinity chromatography on lentil lectin-Sepharose; and reverse-phase high-performance liquid chromatography on a phenyl-silica matrix--to a specific biological activity of approximately 1.25 X 10(8) units/mg with an overall purification of 12,000-fold and a yield of 73% for the activity failing to bind to DEAE-Sepharose. Sufficient quantities of the factor (12 micrograms, 200 pmol) have been purified to allow structural and functional analysis of the molecule and comparison with other know differentiation inducers.     

The role of humoral factors in the regression of leukemia in chickens as measured by in vitro colony formation

Leukemic myeloblasts induced by avian myeloblastosis virus in the chicken formed small compact (type II) colonies in semi-solid agar medium. Normal yolk sac cells from 12-day old embryos formed large diffuse (type I) colonies under the same conditions. Type I colony formation (but not type II) was strictly dependent upon the presence in the medium of a colony stimulating factor (CSF) present in fresh chicken serum or conditioned medium. Serum CSF levels were determined for normal, leukemic, and birds which had spontaneously regressed from myeloblastic leukemia. When type I colony formation was used as the assay, serum CSF levels of leukemic birds were found to be significantly lower than levels in either normal or regressed birds. When the same sera were tested for their ability to induce type II colonies, leukemic birds demonstrated a significantly higher CSF level than either normal or regressed sera. Regressed chickens had serum CSF levels similar to normal birds.     

THE IN VITRO DIFFERENTIATION OF DENSITY SUB-POPULATIONS OF COLONY-FORMING CELLS UNDER THE INFLUENCE OF DIFFERENT TYPES OF COLONY-STIMULATING FACTOR

The in vitro proliferation and differentiation of myeloid progenitor cells (CFU-c) in agar culture from CBA/Ca mouse bone marrow cells was studied. Density sub-populations of marrow cells were obtained by equilibrium centrifugation in continuous albumin density gradients. The formation of colonies of granulocytes and/or macrophages was studied under the influence of three types of colony-stimulating factor (CSF) from mouse lung conditioned medium CSF_MLCM), post-endotoxin mouse serum (CSF_ES) and from human urine (CSF_Hu). The effect of the sulphydryl reagent mercaptoethanol on colony development was also examined. The density distribution of CFU-c was dependent on the type of CSF. Functional heterogeneity was found among CFU-c with partial discrimination between progenitor cells forming pure granulocytic colonies and those forming pure macro-phage colonies. Mercaptoethanol increased colony incidence but had no apparent effect on colony morphology or the density distribution of CFU-c.     

Low-dose Ara-C in the treatment of acute leukemia. Cytotoxicity or differentiation induction?

The differentiation inducing effect of low-dose Ara-C on human myeloid leukemic cells was studied in two patients with subacute myelocytic and subacute myelomonocytic leukemia in vivo and in vitro. By continuous i.v. administration of 10 mg Ara-C/m2 over 12 h daily for 12 or 20 days complete remissions were obtained in both patients with normalization of the incidence of the committed progenitor cells BFU-E and CFU-C in the marrow while the incidence of pluripotent CFU-GEMM remained subnormal. Parallel cultures of the patients' bone marrow cells in diffusion chambers (DC) implanted in mice demonstrated a clear cytotoxic effect of low-dose Ara-C. The greater increase of granulopoietic cells within DC in the Ara-C exposed group than in control mice after the end of drug administration is, in addition, an indication for differentiation induction by this kind of Ara-C therapy.     

Effect of AML serum on normal human myeloid differentiation and hexamethylene bisacetamide-induced granulocytic differentiation of the human HL-60 promyelocytic leukaemic cell line.

The effect of serum from 32 AML patients on the normal human myeloid differentiation and the hexamethylene-bisacetamide induced granulocytic differentiation of HL-60 promyelocytic leukaemic cell line was studied. Nonadherent normal mononuclear marrow cells were cultured in vitro at a concentration of 5 x 10(5) cells/ml for 6 days with each of the 32 AML sera. Ten normal human AB sera were used as control. The results showed an inhibitory activity on both morphological and functional differentiation of normal human myeloid immature marrow cells by 29 out of the 32 AML sera tested. These 29 AML sera were added to cultures of HL-60 (2.5 x 10(5)/ml) leukaemia cell line which incorporated 2 mM hexamethylene-bisacetamide for 6 days. The results showed no significant inhibition of hexamethylene-bisacetamide induced granulocytic differentiation by any of the 29 AML sera. The efficacy of hexamethylene-bisacetamide in inducing differentiation in the presence of inhibitory factors suggests a possible role in the treatment of AML patients.     

牙髓紫卟啉菌内毒素对炎症性细胞因子的介导作用

牙髓紫叶琳菌ＡＴＣＣ３５４０６是近年来新发现的重要致病性专性厌氧菌,采用改良酚－氯仿－石油醚法提取牙髓紫卟啉菌ＡＴＣＣ３５４０６内毒素脂多糖,通过Ｋｒａｍｅｒ测定法、软琼脂细胞培养法以及胸腺细胞增殖法测定脂多糖的细胞生物学活性。结果显示：纯化脂多糖可不同程度地诱导小鼠模型生成肿瘤坏死因子（ＴＮＦ）、集落刺激因子（ＣＳＦ）和白介素－１（ＩＬ－１）,并在一定范围内呈剂量依赖型,提示牙髓紫卟啉菌内毒素在动物模型和细胞模型中具有显著的细胞生物学和免疫学活性．     

Recombinant human interferon sensitizes resistant myeloid leukemic cells to induction of terminal differentiation

Recombinant human leukocyte interferon (IFN-alpha A) inhibits growth of the human promyelocytic leukemic cell line HL-60 without inducing these cells to differentiate terminally. When IFN-alpha A is combined with agents capable of inducing differentiation in HL-60 cells, such as 12-O-tetradecanoyl-phorbol-13-acetate (TPA), cis or trans retinoic acid (RA) or dimethylsulfoxide (DMSO), growth suppression and induction of differentiation are dramatically increased. By growing HL-60 cells in increasing concentrations of TPA, RA, or DMSO, a series of sublines have been developed which are resistant to the usual growth inhibition and induction of differentiation seen when wild type HL-60 cells are exposed to these agents. Treatment of these resistant HL-60 cells with the combination of IFN-alpha A and the appropriate inducer results, however, in a synergistic suppression in cell growth and a concomitant induction of terminal differentiation. The ability of interferon to interact synergistically with agents which promote leukemic cell maturation may represents a novel means of reducing resistant leukemic cell populations.     

Stromal cell regulation of lymphoid and myeloid differentiation

In vitro microenvironmental influences seem to be critical for both B lymphocyte and myeloid differentiation. Studies on murine Dexter cultures and Whitlock-Witte lymphocyte cultures suggest the presence of two critical stromal regulatory cells: an alkaline-phosphatase-positive epithelioid cell and a macrophage. Further data suggest that these cells are capable of producing colony stimulating factor-1, granulocyte-macrophage CSF, a myeloid synergizing activity, and probably separate B cell growth factors. Isolation of a cell line from Dexter stroma was accomplished and this line produced CSF-1, GM-CSF, a pre-B cell and myeloid synergizing activity, and an activity acting on differentiated B cells. We speculate that the Dexter and Whitlock-Witte in vitro culture systems are regulated by factors produced by the two adherent cell types. A lineage nonspecific factor capable of inducing cells into the B lineage or synergizing with interleukin-3, GM-CSF, and CSF-1 is produced, which presumably acts on early stem cells. In addition, the cell line produces GM-CSF, CSF-1, and a factor acting on differentiated B cells. We speculate that in these culture systems, these "terminal differentiating hormones" regulate the final pathway of differentiation, whereas the pre-B-synergizing activity supports early stem cells that can then respond to the other differentiating hormones.     

The network of hemopoietic regulatory proteins in myeloid cell differentiation.

There are clones of myeloid leukemic cells that can be induced to undergo terminal cell differentiation to macrophages by normal hemopoietic regulatory proteins. Induction of differentiation in two different clones of myeloid leukemic cells with interleukin 6 (IL-6) or granulocyte-macrophage colony-stimulating factor (GM-CSF) resulted in induction of mRNA for the hemopoietic regulatory proteins IL-6, GM-CSF, interleukin 1 alpha and interleukin 1 beta, tumor necrosis factor, and transforming growth factor beta 1. In one of these clones, induction of differentiation with GM-CSF was also associated with induction of mRNA for macrophage colony-stimulating factor (M-CSF) but not for the receptor for M-CSF (c-fms), whereas in the other clone, induction of differentiation with IL-6 was associated with induction of mRNA for both c-fms and M-CSF. The clones also differed in their responsiveness to these regulators. There was no induction of mRNA for granulocyte colony-stimulating factor or interleukin 3 during differentiation of either clone. The results indicate that the genes for a nearly normal network of positive and negative hemopoietic regulatory proteins are induced during differentiation of these myeloid leukemic cells and that there are leukemic clones with specific defects in this network.     

Colony formation in agar by murine plasmacytoma cells: potentiation by hemopoietic cells and serum

Clonal growth in semisolid agar medium was obtained using cells from 19 of 25 transplanted murine plasmacytomas when the medium was supplemented by whole mouse blood or washed red cells. With different tumors cloning efficiency ranged from 0.01% to 21.6%. With two exceptions, mouse blood did not potentiate colony formation in agar by cells from transplantable myelomonocytic, myeloid, and lymphoid leukemias, reticulum cell sarcomas and fibrosarcomas. The clonal growth of some plasmacytomas was also potentiated by syngeneic thymic, spleen or bone marrow cells. Plasmacytoma colony growth was not stimulated by normal mouse serum but serum from mice injected with endotoxin or polymerised flagellin stimulated colony growth by some plasmacytomas. The active serum factor was not the colony stimulating factor (CSF) and its appearance after antigenic stimulation was not T cell-dependent. Preimmunised mice failed tq respond to antigenic stimulation. Whole body irradiation did not induce a rise in the capacity of serum to stimulate colony formation by plasmacytoma cells.     

IL-6 is a differentiation factor for M1 and WEHI-3B myeloid leukemic cells

IL-6 has multiple biologic activities in different cell systems including both the ability to support cell proliferation and to induce differentiation. We reported previously the isolation and functional expression of a mouse IL-6 (mIL-6) cDNA clone derived from bone marrow stromal cells. In this paper, we show that mIL-6 is a potent inducer of terminal macrophage differentiation for a mouse myeloid leukemic cell line, M1. Addition of mIL-6 to cultures of M1 cells rapidly inhibits their proliferation and induces phagocytic activity and morphologic changes characteristic of mature macrophages. These phenotypic changes are accompanied at the molecular level by a decrease in proto-oncogene c-myc mRNA accumulation and increases in Fc gamma R, proto-oncogenes c-fos and c-fms (CSF-1R) mRNA expression. Furthermore, IL-6 enhances the expression of Fc gamma R and c-fms in differentiation-responsive D+, but not unresponsive D- sublines of mouse myelomonocytic leukemic WEHI-3B cells. Together with our previous observation that IL-6 stimulates colony formation by normal myeloid progenitors, these results strongly suggest an important regulatory role for IL-6 in myeloid cell growth and differentiation.     

Macrophage differentiation inducing factor from human monocytic cells is equivalent to murine leukemia inhibitory factor

Human macrophage differentiation inducing factor (DIF) can induce differentiation of human myeloid leukemic cells into macrophage-like cells in vitro. A procedure is described for purification of DIF from serum-free human monocytic leukemia THP-1 cell-conditioned medium. The procedure included concentration of a conditioned medium by ultrafiltration, lentil lectin-Sepharose affinity chromatography, and fast protein liquid chromatography using Mono S and Mono Q. DIF-A of pI 9.0 and DIF-B of pI 8.8 were obtained after a final purification with a Mono Q column, and both DIF gave a single peak with a molecular weight of approximately 51,000 determined by gel chromatography. NH2-terminal amino acid analysis of DIF-A showed a noticeable homology with murine leukemia inhibitory factor. Human DIF-A was found to induce maturation of human and murine leukemic cells into both macrophage-like cells with nitro blue tetrazolium reducing activity and phagocytic cells, but was found to suppress proliferation of these leukemic cells.     

Purification of a factor inducing differentiation in murine myelomonocytic leukemia cells. Identification as granulocyte colony-stimulating factor

A naturally occurring inducer of terminal differentiation in a murine myelomonocytic leukemia cell line (WEHI-3B) was purified to apparent homogeneity from medium conditioned by lungs from mice injected with bacterial endotoxin. The factor was purified over 400,000-fold by sequential fractionation using salting out chromatography, chromatography on phenyl-Sepharose, gel filtration on Bio-Gel P-60 in 1 M acetic acid, reverse-phase high performance liquid chromatography on a phenyl-silica column, and high performance liquid chromatography on a gel filtration column. During the first two steps, the differentiation-inducing factor was separated completely from a known proliferative regulator for normal myeloid cells, granulocyte-macrophage colony-stimulating factor, but it co-purified through all remaining steps with a distinct granulocyte-specific colony-stimulating factor. The purified factor showed a single protein band of Mr = 24,000-25,000 on sodium dodecyl sulfate-polyacrylamide gels coincident with both differentiation-inducing and granulocyte colony-stimulating activity. The granulocyte-specific colony-stimulating factor was active on WEHI-3B cells and normal granulocytic progenitor cells in vitro at the same half-maximally active concentration of 3 X 10(-12) M.     

Studies of inhibitors of bone marrow colony formation in normal human sera and during a viral infection

Various methods are compared for removing inhibitors of bone marrow colony stimulating activity from the serum of normal individuals. The best method proved to be chloroform treatment of sera, which is easily performed on a large scale. Chloroform treatment had no effect in itself on the colony-stimulating factor (CSF) and no additional inhibitors were detected after such treatment. Inhibitors were characterized by preparative ultracentrifugation. After fractionation, most of the inhibitory activity was recovered in the light (LDL) and very light density lipoprotein (VLDL) fractions. Treatment with specific anti human β-lipoprotein serum removed most of the inhibitory activity. Increased CSF levels have been reported to accompany the acute phase of infectious diseases. Application of the chloroform treatment to sera from patients with mumps showed that the mean CSF in these sera did not differ significantly from that found in sera from normal individuals. High CSF levels in acute infections therefore seem to reflect variations in inhibitor levels rater than a true increase in CSF.     

Further studies on the factor in lung-conditioned medium stimulating granulocyte and monocyte colony formation in vitro

The CSF's extracted from 6-hour “in vivo” post-endotoxin mouse lung tissue and 48-hour “in vitro” lung-conditioned medium appear to be glyco-proteins, for like human urinary CSF they were inactivated by proteases and altered in charge but not inactivated by sialidase. Neither endotoxin injection nor length of in vitro incubation significantly affected the apparent S_{20 W} (approximately 2.OS) of the bulk of the CSF in mouse lung-conditioned medium. The lower apparent molecular weight of CSF in mouse lung-conditioned medium contrasted with that of monkey lung-conditioned medium (S_{20 W} approximately 3.3S). CSF's in both monkey and mouse lung-conditioned media exhibited a much higher apparent MW on gel nitration than one would expect from their sedimentation behavior, a characteristic generally observed with most unpurified CSF preparations. Mouse lung-conditioned medium CSF was antigenically distinguishable from other CSF's. Failure to demonstrate this form of CSF in the tissue serum or urine of normal or endotoxin-injected mice suggests that this type of CSF might normally lack in vivo significance.     

Interleukin-6- and leukemia inhibitory factor-induced terminal differentiation of myeloid leukemia cells is blocked at an intermediate stage by constitutive c-myc.

Interleukin-6 (IL-6) and leukemia inhibitory factor (LIF), two multifunctional cytokines, recently have been identified as physiological inducers of hematopoietic cell differentiation which also induce terminal differentiation and growth arrest of the myeloblastic leukemic M1 cell line. In this work, it is shown that c-myc exhibited a unique pattern of expression upon induction of M1 terminal differentiation by LIF or IL-6, with an early transient increase followed by a decrease to control levels by 12 h and no detectable c-myc mRNA by 1 day; in contrast, c-myb expression was rapidly suppressed, with no detectable c-myb mRNA by 12 h. Vectors containing the c-myc gene under control of the beta-actin gene promoter were transfected into M1 cells to obtain M1myc cell lines which constitutively synthesized c-myc. Deregulated and continued expression of c-myc blocked terminal differentiation induced by IL-6 or LIF at an intermediate stage in the progression from immature blasts to mature macrophages, precisely at the point in time when c-myc is normally suppressed, leading to intermediate-stage myeloid cells which continued to proliferate in the absence of c-myb expression.     

Preparation and neutralization characteristics of an anti-CSF antibody

Nine of ten rabbits immunized with a partially purified L-cell CSF had demonstrable titers of anti-CSF activity. In vitro the antibody was markedly inhibitory to both post-endotoxin mouse sera and several mouse tissue extracts. CSF containing conditioned media prepared from a number of sources showed variable inhibition suggesting that murine CSF's may be characterized by marked antigenic differences. Human sources of CSF were also inhibited thus indicating a degree of cross-reactivity between murine and human factors. These studies may provide the initial steps toward definition of the role of CSF in vivo.     

Dissociation of c-fos induction from macrophage differentiation in human myeloid leukemic cell lines.

Treatment of five human myeloid leukemic cell lines (KG1, ML3, HL-60, U-937, and HEL) with TPA was followed by macrophage differentiation and was accompanied by an early and transient increase in the mRNA level of c-fos proto-oncogene. The induction of c-fos was also observed in human cell lines K562 and K-Gla that did not respond to TPA with terminal macrophage differentiation. The treatment of HL-60 and U-937 cell lines with 1-oleoyl-2-acetylglycerol, a synthetic analog of diacylglycerol that, like TPA, stimulates protein kinase C activity, was followed by early and transient induction of c-fos mRNA in the absence of terminal macrophage differentiation. Finally, treatment of HL-60 with TPA in the presence of retinal, an inhibitor of protein kinase C, drastically reduced the induction of c-fos mRNA but had no effect on the terminal macrophage differentiation that is induced in this cell line by TPA. These results indicate that the induction of c-fos and terminal macrophage differentiation in response to TPA treatment can be dissociated in the in vitro models provided by human myeloid leukemic cell lines. Moreover, these findings suggest that the induction of c-fos is not only insufficient but may also be unnecessary for the differentiation along the monocyte-macrophage pathway.