The Production of Differentiation-Autoinducing Activity by WEHI-3B D+ Leukemia Cells
Yasuhiko Kajigaya, et al
Abstract. We studied the differentiation-autoinducing activity in WEHI-3B D+ cell-conditioned medium (WCM). After culturing 106/ml WEHI-3B D+ cells in RPMI1640 medium without fetal calf serum (FCS) for four days, the supernatant was collected. The medium, concentrated 50-fold by YM 5 membrane filtration, was fractionated by gel exclusion on Ultrogel AcA44. We evaluated the effect of each of the four fractions on differentiation in WEHI-3B D+ cells by morphological, functional and cytochemical criteria after adding the fractions to liquid or soft-agar cultures of 103 cells in 1ml RPMI1640 containing 10% FCS: the experimental cultures contained 10% of the fractions, with a control for each without the fraction. The growth of WEHI-3B D+ cells in culture was inhibited by the addition of fraction-P only (M.W. was estimated between 10,000-20,000 daltons). In these same cultures, the cells were granulocyte-like, strongly positive for naphthol ASD chloroacetate esterase, and had phagocytic activity. Colonies grown in agar culture with fraction-P also exhibited a peripheral halo of loosely dispersed cells around a central aggregate. The fraction-P contained neither granulocyte colony-stimulating activity nor burst-promoting activity. These results suggested that fraction-P contained differentiation-autoinducing factor is different from G-CSF or interleukin-3.
Key words: WEHI-3B cells, Differentiation-autoinducing activity
A murine myelomonocytic leukemia cell line, WEHI-3B, constitutively produces interleukin-3 (IL-3) and can be induced by G-CSF to differentiate into granulocytes and macrophages [1-3]. IL-3 has various biological activities, such as mast cell growth activity, P cell-stimulating activity, colony-stimulating activity and histamine-producing cell-stimulating activity . Furthermore, it was reported recently from a study using serum-free culture, that IL-3 supports the terminal differentiation of most hemopoietic lineages, with the exception of the erythroid lineage . However, it also was reported that IL-3 does not induce differentiation in WEHI-3B cells . In addition, Metcalf and Nicola  demonstrated that when WEHI-3B cells are grown in soft agar, the proportion of differentiated colonies increases with an increase in the number of colonies in the culture dish, suggesting that WEHI-3B cells are produce a differentiation-inducing factor in addition to IL-3. We fractionated conditioned medium from WEHI-3B D+ cells by gel exclusion and found that differentiation-inducing factor activity on WEHI-3B D+ cells was present in the fraction P (molecular weight of approximately 10,000-20,000 daltons) that did not have granulocyte-macrophage colony-stimulating activity (GM-CSA), granulocyte colony-stimulating activity (G-CSA) and burst-promoting activity (BPA).
Materials and methods
Cell lines. A murine myelomonocytic leukemia cell line, WEHI-3B D+ , was kindly donated by Dr. S. Asano (Department of Medicine, Department of Pathological Pharmacology, Institute of Medical Sciences, University of Tokyo). The cells were maintained in RPMI 1640 (Nissui Pharmaceutical Company, Tokyo, Japan), supplemented with 10% fetal calf serum (FCS)(HyClone Laboratories, Logan, Utah). The cells were cultured for 4 days, collected, then aliquots of 1~106 cells/ml were transfered to RPMI 1640 without FCS. Conditioned medium (WCM) was collected after 4 days and centrifuged. The IL-3 dependent cell line, FDCP-2 , was kindly donated by Dr. T. Suda (Department of Hematology, Jichi Medical School), and maintained in RPMI 1640 with 10% FCS and 10% WCM. These two cell lines were cultured in 25 cm2 tissue culture flasks (Corning Glass Works, Corning, NY) in 8ml of medium in a humidified atmosphere of 5% CO2/95% air at 37.
Fractionation of WCM. WCM was concentrated 50-fold by ultrafiltration against an Amicon YM-5 membrane (Amicon Corporation, Denvers, MA). The retentate (1.0ml) was applied to a 1.5- ~ 80-cm Ultrogel AcA44 column that had been equilibrated in 0.03 M Tris buffer (pH 7.4); it was eluted at a flow rate of 20 ml/hr with the same buffer. Fractions (5ml) were collected and pooled into the four groups shown in Figure 1. The four fractions were collected and concentrated to 1ml as described above. The fractions were sterilized by filtration through a millipore filter (Millex-HA, Millipore, Tokyo, Japan).
Mouse-lung-conditioned medium. Mouse-lung-conditioned medium (MLCM) was prepared in vitro by lung tissue from C57BL mice (8-12 weeks old) preinjected with 5Ęg endotoxin (E. coli W, lipopolysaccharide, ,Sigma). MLCM were serially diluted using phosphate-buffered saline and millipore filtered before use.
Assays for colony-stimulating activity (CSA). We used BALB/c female mice (8-12 weeks old) for the assays. Bone marrow cells were obtained by flushing the marrow from the mouse's femurs into alpha-medium (Flow Laboratories, Irvine, Scotland) through a 23-gauge needle; the cells then were washed twice with the medium. The method described by Robinson et al. was used. The medium consisted of 1~105 of the fresh bone marrow cells, 0.3% purified agar (Difco Laboratories, Detroit, Mich), 20% FCS, 10% sample and alpha-medium. The culture was added to a 35-mm dish and incubated for 7 days in a humidified atmosphere of 5% CO2/95% air at 37. Two cultures were set up for each sample.
Soft-agar culture assays for the induction of WEHI-3B D+ differentiation. Cell cultures were maintained according to the method of Metcalf and Nicola [7,12], with some modifications. Semi-solid cultures were prepared using cloned WEHI-3B D+ cells grown in continuous liquid culture in RPMI 1640 medium with 10% FCS and containing from 0.5 to 2.0 ~ 106 cells/ml. All semi-solid cultures were prepared as 1 ml cultures in 35-mm Petri dishes using an equal volume mixture of double-strength RPMI 1640 medium and 0.6% agar. The WEHI-3B D+ cells were added to the liquid agar medium (1~103 cells/ml), and aliquots of 1 ml pipetted into each culture dish containing 0.1 ml of sample. After mixing , the cultures were allowed to gel, then incubated for 7 days in a fully humidified atmosphere of 5% CO2/95% air at 37.
Morphological and cytochemical analysis of the cells in colonies. Individual colonies were removed with a fine pipette, placed on microscope slides and allowed to dry. Cells were stained with 0.6% orcein in 60% acetic acid (Muto Pure Chemicals, Tokyo, Japan). Cytochemical analysis of the colonies was performed after double esterase staining according to the method of Kubota et al. . Colonies were classified as: chloroacetate esterase positive colony (G), with more than 90% cells with blue cytoplasmic granules after double esterase staining; butyrate esterase positive colony (M), with more than 90% cells with brown cytoplasmic granules; and granulocyte-macrophage mixed colonies (GM).
Liquid culture assays for the induction of WEHI-3B D+ differentiation. WEHI-3B D+ leukemia cells at a concentration of 1~103 cells/ml were incubated in the presence of sample for up to 7 days in a fully humidified atmosphere of 5% CO2/95% air at 37. A cytocentrifuge preparation (Cytospin Centrifuge, Shandon Southern Instrument, Sewickley, PA) of the cultured WEHI-3B D+ cells was stained with Wright-Giemsa for morphological analysis and with the double esterase staining method. For assay of phagocytosis, cultured cells were resuspended (1~104 /ml) in RPMI 1640 medium containing a 0.2% suspension of polystylene latex particles (average diameter, 0.81 Ęm; Difco Laboratories).
Assay for burst promoting activity (BPA). Bone marrow cells were obtained by flushing the marrow from the femurs of BALB/c female mice into Iscove's modified Dulbecco's medium (IMDM) (Flow Laboratories). The cells were cultured by the methylcellulose method of Iscove . BPA in fractionated WCM was assayed as follows: Bone marrow cells (2~105) were cultured in 1 ml of IMDM containing 0.9% methylcellulose (4000 cP; Fisher Scientific Company, Norcross, GA), 1% deionized bovine serum albumin (Fraction ,Sigma Chemical Company, St. Louis, Mo), 2 U/ml human urinary erythropoietin (Toyobo Company, Osaka, Japan), and 10% sample. Duplicate cultures were maintained, and they were incubated in a humidified atmosphere of 5% CO2/95% air at 37. Erythroid bursts were scored on day 11, and BPA was expressed as the number of bursts observed.
Effect of the fractions of WCM on the growth of IL-3 dependent FDCP-2 cells. FDCP-2 cells were collected by centrifugation and were resuspended in RPMI 1640 containing 10% FCS at a density of 105 cells/ml. Cultures were subsequently maintained in the presence of fractionated WCM. At the intervals of 24hr, we determined the cell number and the percentage of viable cells by trypan blue exclusion.
Granulocyte-macrophage colony-stimulating activity (GM-CSA) and differentiation-inducing activity of each fraction of WCM in agar culture
When WCM was applied on Ultrogel AcA44 column chromatography, GM-CSA eluted in fraction O, with a single peak at a molecular weight of 30,000 daltons. Differentiation-inducing activity eluted in fraction P, with a single peak at a molecular weight of 15,000 daltons (Fig. 1). When WEHI-3B D+ cells were cultured for seven days in agar containing fraction P, almost all of colonies exhibited a diffuse halo of cells surrounding a central aggregate (Table 1, Fig. 2B). Analysis of stained preparations of such colonies revealed that they were composed of differentiated granulocyte-like cells that were positive for naphthol ASD chloroacetate esterase (Table 2). In contrast, when the cells were cultured in the absence of fraction P, almost all of colonies were tightly-packed spherical aggregates (Fig. 2A).
Effect of fractions of WCM on the differentiation of WEHI-3B D+ cells in liquid culture
WEHI-3B D+ cells were cultured for 7 days in the presence or absence of fractionated WCM, then the number of cells was counted, morphological and cytochemical examinations were made. The number of cells in the control culture increased from 1~103/ml on day 0 to 2~105 /ml on day 7 (Table 3), and almost all cells were immature undifferentiated cells (Fig. 3A) which stained positively for naphthol ASD chloroacetate esterase and butyrate esterase. The addition of 10% of fraction P suppressed cell proliferation; on day 7 the number of cells was 0.3~105/ml (Table 3). Also, a large proportion of the WEHI-3B D+ cells had a morphology similar to mature myeloid cells and there were polymorphonucleated cells present (Fig. 3B). The WEHI-3B D+ cells had increased in size and they showed a decrease in the ratio of nucleus to cytoplasm. Almost all cells were strongly positive for naphthol ASD chloroacetate esterase after double esterase staining. Their capacity for phagocytosis increased tenfold by the addition of fraction P, compared with the control culture (data not shown).
BPA in each fraction of WCM
We observed the effect of each fraction of WCM on erythroid burst formation of murine bone marrow cells. Fraction O increased the number of erythroid bursts to 800% of the control, but fraction P did not enhance burst formation any more than the control (Table 4).
The growth effect of fraction of WCM on IL-3 dependent FDCP-2 cells
The dependency for growth of this cell line on fraction O of BPA and GM-CSA is illustrated in Figure 4, and cell numbers increased with a doubling time of approximately 16 hr. In the presence of fraction P, viability was rapidly lost, such that by 48 hr less than 10% of the cells were viable.
Comparison of granulocyte colony-stimulating activity (G-CSA) versus WEHI-3B D+ leukemic cell differentiation-inducing activity in dilution of MLCM and in dilution of fraction P
As shown in Figure 5, when serial dilutions of these two sources were assayed in cultures of WEHI-3B D+ cells, it was observed that a 1:256 dilution of fraction P was able to induce differentiation in significant proportion of the leukemic colonies. The percentage of differentiated colonies induced by MLCM was lower than that induced by fraction P. In parallel assays of these two sources in cultures of normal bone-marrow cells, fraction P failed to stimulate granulocyte colony formation whereas MLCM exhibited G-CSA.
WEHI-3B cells are a leukemia cell line derived from a BALB/c mouse that was injected with mineral-oil. The cells proliferate to form colonies in soft agar culture [8,15]. Metcalf et al. reported that when the number of colonies in the culture dish is 500 or fewer, 90% or more are compact colonies, but when the number of colonies increases to 1,000 or more, the proportion of diffuse colonies increases . Furthermore, the cells constituting the compact colonies were undifferentiated, while those of the diffuse colonies were differentiated . These results suggested that WEHI-3B cells produce a humoral factor that autoinduces differentiation.
The results of our study with WEHI-3B D+ cell conditioned medium demonstrated the presence in fraction P of a humoral factor with differentiation-autoinducing activity, with a molecular weight of approximately 10,000-20,000 daltons. The growth of WEHI-3B D+ cells in culture was inhibited by the addition of fraction P. Such cells exhibited increase in size, a tendency to a decrease in the ratio of nucleus to cytoplasm, polymorphonucleation, enhancement of naphthol ASD chlroacetate esterase staining and also induction of phagocytosis. In soft-agar culture, migration from the colony center was induced and similar morphological and cytochemical results were obtained as in liquid culture.
It was reported that WEHI-3B cells constitutively produce IL-3 . IL-3 is a glycoprotein with a molecular weight of 28,000-30,000 daltons, and has biological activity, namely the induction of the enzyme 20-alpha-hydroxy steroid dehydrogenase in cultures of nu/nu splenic lymphocytes, mast cell growth activity, P cell-stimulating activity, histamine-producing cell-stimulating activity and colony-stimulating activity . IL-3 was purified from WEHI-3B cell conditioned medium . It was reported recently from a study using serum-free culture, that IL-3 supports the differentiation of multilineage hemopoietic progenitors and the terminal differentiation of most hemopoietic lineages, with the exception of the erythroid lineage . According to Metcalf, IL-3 does not possess differentiation-inducing factor activity on WEHI-3B cells .
The results of our studies on fraction P of WCM demonstrated the absence of BPA and the inability to support the proliferation of IL-3 dependent FDCP-2 cells. These results indicate that the humoral factor exhibiting differentiation-inducing factor activity on the WEHI-3B D+ cells in fraction P, is distinct from IL-3.
Metcalf reported the presence of differentiation-inducing factor in mouse-lung-conditioned medium (MLCM) that acts on WEHI-3B cells . Nicola et al. purified a differentiation-inducing factor in MLCM (molecular weight approximately 25,000 daltons) which acted on WEHI-3B cells. This factor was distinct from GM-CSF, but not from G-CSF: the WEHI-3B cells differentiation-inducing factor in MLCM was identified as G-CSF . WEHI-3B cells in soft agar culture were induced to differentiate by G-CSF into granulocyte-like cells and in liquid culture into macrophage-like cells [3,12].
It was reported that WEHI-3B cells produce a humoral factor, which has G-CSF activity and mast cell growth factor activity , but Bazill and Ihle subsequently purified this factor and showed it was IL-3, different from the G-CSF that was purified by Nicola et al. [4,18].
In our study, fraction P of WCM (molecular weight 10,000-20,000 daltons) induced the differentiation of WEHI-3B D+ cells to a granulocyte-like cells in soft agar and in liquid culture. Additionally, we showed that fraction P was unable to support granulocyte colony and granulocyte-macrophage colony formation of bone marrow cells. These results suggest that the differentiation-inducing factor present in fraction P differs from G-CSF or GM-CSF.
Differentiation-inducing factors other than G-CSF that have been found to act on WEHI-3B cells are actinomycin D, adriamycin, aclacinomycin A and retinoic acid [3,19,20]. It was reported that tumor necrosis factor (TNF) retain the capacity to inhibit cloning of WEHI-3B cells . However, TNF had minimal differentiation-inducing capacity to WEHI-3B D+ leukemia cells. Gearing et al. reported that leukemia inhibitory factor (LIF) induce macrophage differentiation in established M1 leukemic colonies and inhibit colony formation . LIF had no effect on the number, size or differentiation of WEHI-3B D+ leukemic colonies, had no colony-stimulating activity for normal granulocyte-macrophage progenitor cells.
In our study, incubation with fraction P of WCM decreased total WEHI-3B D+ leukemic colony numbers and markedly increased the proportion of differentiated colonies. These results further suggested that the humoral factor exhibiting differentiation-inducing factor activity on the WEHI-3B D+ cells in fraction P, is different from TNF and LIF.
1. Lee JC, Hapel AJ, Ihle JN (1982) Constitutive production of a unique lymphokine (IL-3) by the WEHI-3 cell line. J Immunol 128:2393
2. Nicola NA, Metcalf D, Matsumoto M, Johnson GR (1983) Purification of a factor inducing differentiation in murine myelomonocytic leukemia cells:Identification as granulocyte colony-stimulating factor. J Biol Chem 258:9017
3. Cooper PC, Metcalf D, Burgess AW (1982) Biochemical and functional characterization of mature progeny purified from a myelomonocytic leukemia cell line. Leukemia Res 6:313
4. Ihle JN, Keller J, Oroszlan S, Henderson LE, Copeland TD, Fitch F, Prystowsky MB, Goldwasser E, Schrader JW, Palaszynski E, Dy M, Lebel B (1983) Biologic properties of homogeneous interleukin 3:1. Demonstration of WEHI-3 growth factor activity, mast cell growth factor activity, P cell-stimulating factor activity, colony-stimulating factor activity. J Immunol 131:282
5. Suda J, Suda T, Kubota K, Ihle JN, Saito M, Miura Y (1986) Purified interleukin-3 and erythropoietin support the terminal differentiation of hemopoietic progenitors in serum-free culture. Blood 67:1002
6. Metcalf D (1984) The hemopoietic colony stimulating factors. Amsterdam:Elsevier,p 398
7. Metcalf D, Nicola NA (1982) Autoinduction of differentiation in WEHI-3B leukemia cells. Int J Cancer 30:773
8. Warner NL, Moore MAS, Metcalf D (1969) A transplantable myelomonocytic leukemia in BALB/c mice:cytology, karyotype, and muramidase content. J Nat Cancer Inst 43:963
9. Dexter TM, Garland D, Scott E, Scolnick E, Metcalf D (1980) Growth of factor-dependent hemopoietic precursor cell lines. J Exp Med 152:1036
10. Burgess W, Camakaris J, Metcalf D (1977) Purification and properties of colony-stimulating factor from mouse lung-conditioned medium. J Biol Chem 252:1998
11. Robinson W, Metcalf D, Bradley TR (1967) Stimulation by normal and leukemic mouse sera of colony formation in vitro by mouse bone marrow cells. J Cell Physiol 69:83
12. Metcalf D (1979) Clonal analysis of the action of GM-CSF on the proliferation and differentiation of myelomonocytic leukemic cells. Int J Cancer 24:616
13. Kubota K, Mizoguchi H, Miura Y, Suda T, Takaku F (1980) A new technique for the cytochemical examination of human hemopoietic cell growth in agar gel. Exp Hematol 8:339
14. Iscove NN, Sieber F, Winterhalter KH (1974) Erythroid colony formation in cultures of mouse and human bone marrow: Analysis of the requirement of erythropoietin by gel filtration and affinity chromatography on agaroseconcanavalin A1. J Cell Physiol 83:309
15. Metcalf D, Moore MAS, Warner NL (1969) Colony formation in vitro by myelomonocytic leukemic cells. J Nat Cancer Inst 43:983
16. Ihle JN, Keller J, Henderson L, Klein F, Palaszynski E (1982) Procedures for the purification of interleukin 3 to homogeneity. J Immunol 129:2431
17. Moore MAS (1982) G-CSF: Its relationship to leukemia differentiation-inducing activity and other hemopoietic regulators. J Cell Physiol Suppl 1:53
18. Bazill GW, Haynes M, Garland J, Dexter TM (1983) Characterization and partial purification of a haemopoietic cell growth factor in WEHI-3 cell conditioned medium. Biochem J 210:747
19. Gamba-Vitalo C, Blair OC, Tritton TR, Lane PA, Carbone R, Sartorelli AC (1987) Cytotoxicity and differentiating actions of adriamycin in WEHI-3B D+ leukemia cells. Leukemia 1:188
20. Gamba-Vitalo C, Blair OC, Keyes SR, Sartorelli AC (1986) Differentiation of WEHI-3B D+ monomyelocytic leukemia cells by retinoic acid and aclacinomycin A. Cancer Res 46:1189
21. Moore MAS (1982) G-CSF: its relationship to leukemia differentiation-inducing activity and other hemopoietic regulators. J Cell Physiol Supplement 1:53
22. Gearing DP, Gough NM, King JA, Hilton DJ, Nicola NA, Simpson RJ, Nice EC, Kelso A, Metcalf D (1987) Molecular cloning and expression of cDNA encoding a murine myeloid leukaemia inhibitory factor (LIF). EMBO J 6:3995