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
[4]. 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 [5]. However, it
also was reported that IL-3 does not induce differentiation in WEHI-3B cells
[6]. In addition, Metcalf and
Nicola [7] 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+ [8], 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
[9], 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)[10]. 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.[11] 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. [13]. 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 [14]. 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.
Results
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.
Discussion
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 [7]. Furthermore, the
cells constituting the compact colonies were undifferentiated, while those of
the diffuse colonies were differentiated [7]. 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 [1]. 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 [4]. IL-3 was purified
from WEHI-3B cell conditioned medium [16]. 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 [5]. According to Metcalf, IL-3 does not possess
differentiation-inducing factor activity on WEHI-3B cells [6].
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 [12]. 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 [2].
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 [17], 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 [21]. 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 [22]. 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.
References
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