[CANCER RESEARCH 40. 4804-4809.
0008-5472/80/0040-OOOOS02.00
December 1980]
Production by Mouse Spleen Cells of Factors Stimulating Differentiation
of Mouse Myeloid Leukemic Cells That Differ from the Colonystimulating Factor1
Yuri Yamamoto,2 Mikio Tomida, and Motoo Hozumi
Department of Chemotherapy,
Sa/fama Cancer Center Research Institute, Ina-machi, Kita-adachi-gun,
ABSTRACT
Mouse myeloid leukemic M1 cells can be induced to differ
entiate into macrophages and granulocytes in vitro by a protein
inducer, differentiation-stimulating
factor (D-factor), and by
various other compounds. Mouse spleen cells produced Dfactors when treated with various mitogens, such as concanavalin A, phytohemagglutinin,
pokeweed mitogen, lipopolysaccharide, and synthetic double-stranded polyribonucleotide copolymer of polyinosinic and polycytidylic acids. Concanavalin
A, phytohemagglutiniry,
and pokeweed mitogen stimulated
spleen lymphocytes, but not spleen macrophages, to produce
a D-factor with an apparent molecular weight of 40,000 to
50,000. On the other hand, lipopolysaccharide and copolymer
of polyinosinic and polycytidylic acids stimulated both spleen
lymphocytes and spleen macrophages to produce D-factors.
Spleen macrophages
produced D-factors with molecular
weights of 40,000 to 50,000 and 20,000 to 25,000, whereas
spleen lymphocytes produced only the larger molecules.
In addition to D-factor, colony-stimulating factor (CSF), which
stimulates growth and differentiation of normal bone marrow
cells, and interferon, were detected in conditioned medium of
spleen cells treated with concanavalin A or lipopolysaccharide.
On gel filtration of the conditioned medium with Sephadex G100, CSF was eluted between the larger D-factor and the
smaller one. The fraction with interferon activity overlapped
that of the larger D-factor. Incubation of the conditioned me
dium at pH 2 abolished the activity of interferon but did not
affect the activity of either D-factor or CSF. The addition of
cytochalasin B suppressed the production of interferon but not
of D-factor or CSF by the spleen cells. These results indicate
that the D-factor is a different substance from CSF or type II
interferon.
INTRODUCTION
Mouse myeloid leukemic M1 cells can be induced to differ
entiate into macrophages and granulocytes in vitro by a protein
inducer, D-factor,3 and various other compounds (8, 9, 12,
26). Differentiation of the cells is accompanied by induction of
phagocytic and locomotive activities (13), lysosomal enzyme
activities (16), Fc and C3 receptors (26), and a surface glyco' Supported in part by Grants-in-Aid
for Cancer Research from the Ministry of
Health and Welfare and from the Ministry of Education. Science, and Culture of
Japan.
2 To whom requests for reprints should be addressed.
3 The abbreviations used are: D-factor. differentiation-stimulating
factor; LPS.
lipopolysaccharide;
polyd)-poly(C). copolymer of polyinosinic and polycytidylic
acids; CSF. colony-stimulating factor; PBS. phosphate-buffered saline (138 mM
sodium chloride-2.7 mM potassium chloride-8 ITIMdibasic sodium phosphate-1.5
mM monobasic potassium phosphate. pH 7.4); Con A, concanavalin A; PHA,
phytohemagglutinin;
PWM. pokeweed mitogen.
Received February 19. 1980; accepted August 27. 1980.
4804
Saitama-ken 362, Japan
protein with a molecular weight of 180,000 (27). Untreated M1
cells were leukemogenic in syngeneic mice, but on differentia
tion M1 cells lost their leukemogenicity (13). Therefore, induc
tion of normal differentiation of myeloid leukemic cells in vivo
is of potential value in therapy of myeloid leukemia.
Injection of various inducers or stimulation of the production
of D-factor in vivo may be useful for stimulating in vivo induction
of differentiation of M1 cells. LPS is known to induce differen
tiation of M1 cells (31). Injection of LPS (4) or polyd)-poly(C)
(29) into mice caused induction of D-factor activity in the
serum. Injection of these reagents enhanced induction of dif
ferentiation of M1 cells in diffusion chambers transplanted into
mice and prolonged the survival times of mice inoculated with
M1 cells (7, 29). In vivo differentiation of M1 cells in diffusion
chambers transplanted into mice was also induced by the
injection of conditioned medium containing D-factor or by the
inoculation of D-factor-producing cells (18). Macrophages and
granulocytes have been shown to play an important role in the
production of D-factor (10, 12, 18, 28) but, although wholespleen cells (12) and spleen macrophages (12) have been
found to produce D-factor, the role of lymphocytes in the
production of D-factor is unknown. Therefore, in this work, we
examined whether spleen lymphocytes produced D-factor and
found that the cells produced the factor on treatment with
mitogens or poly(l)-poly(C).
On the other hand, stimulated lymphocytes are known to
produce various lymphokines, such as CSF (2, 20, 23, 25) and
interferon (3). CSF's are glycoproteins stimulating growth and
differentiation of normal precursor cells of granulocytes and
macrophages, while protein inducers for differentiation of mye
loid leukemic M1 cells are termed D-factors. The relationship
between D-factors and CSF's is debatable. Some investigators
(22, 26) reported that the same molecules are responsible for
both activities, whereas Maeda et al. (19) reported that some
preparations of conditioned medium of mouse embryo cells
contained CSF but not D-factor; moreover, recently, Hozumi ef
al. (11) showed by DEAE-cellulose column chromatography
that some D-factors were not associated with the activity of
CSF in conditioned medium of a Yoshida sarcoma cell line
grown in serum-free medium. In the present paper, we dem
onstrate that spleen lymphocytes treated with various mitogens
or polyd).poly(C) can produce D-factor and CSF and that the
D-factor and CSF in conditioned medium of spleen cells can
be separated by gel filtration.
MATERIALS
AND METHODS
Culture of Cells
M1 Cells. Myeloid leukemic M1 cells originated from a
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Production of D-Factors by Spleen Cells
spontaneous myeloid leukemia in an SL mouse (12). The cells
used in this study were clone T-22 of the M1 cell line. The cells
were cultured in Eagle's minimal essential medium (Nissui
Assay of D-Factor
Seiyaku Co., Tokyo, Japan), supplemented with double the
usual concentrations of amino acids and vitamins and 10% (v/
v) heat-inactivated (56° for 30 min) calf serum at 37° under
phagocytic activities in M1 cells as described previously (28).
LPS present in the conditioned medium was inactivated by
incubation with polymyxin B (10 /ig/ml) for 2 hr at room
temperature (15). Similarly, poly(l)-poly(C) was digested by
incubation with RNase A (10 /ig/ml) for 1 hr at 37°. M1 cells
were incubated for 2 days at 5 x 105 cells/ml in medium
5% CO2 in air.
Spleen Cells. Cell suspensions from the spleens of adult ICR
or SL mice were prepared by gentle dispersion of the tissue by
passing it through a stainless steel mesh into cold Roswell Park
Memorial Institute Tissue Culture Medium 1640 (Nissui Seiyaku
Co., Tokyo, Japan) supplemented with 10% heat-inactivated
fetal calf serum. Erythrocytes were removed by exposing the
cell suspension to Tris-buffered 0.83% ammonium chloride,
pH 7.2, for 5 min at 4°. Viable nucleated spleen cells were
incubated at a concentration of 5 x 106 cells/ml in 60-mm
dishes for 2 days, unless otherwise noted in the text. Then, the
conditioned medium was harvested by centrifugation at 2000
x g for 10 min, filtered through a 0.45-/xm Millipore filter, and
stored at —20°until use.
Peritoneal Macrophages. As described previously (28), per
itoneal macrophages were prepared from mice given injections
of 5% soluble starch and proteose peptone. Conditioned me
dium was obtained by culturing the cells in serum-free Eagle's
minimal essential medium for 2 days.
Fractionation of Spleen Cells into Lymphocytes and Macro
phages
Spleen cells in Eagle's medium were incubated in glass
dishes for 3 hr at 37° and then adherent macrophages and
nonadherent lymphocytes were separated. The dishes were
washed vigorously with the medium to remove lymphocytes.
Morphology of the adherent cells was examined by staining the
cells with May-Griinwald-Giemsa solution. The cell number was
calculated by counting cells in a unit area under microscopy.
The adherent cells obtained amounted to 5% or less of the total
spleen cells, and more than 95% of the adherent cells were
macrophages. On the other hand, more than 98% of the
nonadherent cells were lymphocytes.
Gel Filtration
Activity of D-factor was assayed by measuring induction of
containing various concentrations of conditioned medium. The
cells were harvested by centrifugation, suspended in serumfree Eagle's medium containing 0.2% of a suspension of pol
ystyrene latex particles (average diameter, 1.099 jum; Dow
Chemical Co., Indianapolis, Ind.), and incubated for 4 hr at
37°.Then the cells were thoroughly washed 3 times with PBS,
and the percentage of phagocytic cells among more than 400
viable cells was calculated. The cells containing more than 10
latex particles were scored as positive cells. Induction of phag
ocytic activity was proportional to the concentration of the
conditioned medium containing D-factor, as described previ
ously (28). Data presently shown were obtained with a concen
tration in a range of linear dose-response relationship.
Cell Morphology
M1 cells were incubated with or without the conditioned
medium for 3 days. The percentage of cells that was morpho
logically similar to granulocytes and macrophages was deter
mined in smear preparations stained with May-GriinwaldGiemsa solution. More than 300 cells were analyzed from each
sample.
Assay of CSF
CSF in the conditioned medium was assayed by the agar
culture technique. Nucleated normal bone marrow cells (10s)
of adult ICR mice were seeded into a 35-mm Petri dish (Falcon
Plastics, Oxnard, Calif.) containing 1 ml of 0.3% agar culture
medium supplemented with 10% horse serum and the condi
tioned medium. After incubation for 7 days, the number of
colonies containing more than 50 cells was counted under a
microscope. Data presently shown were obtained with a con
centration in a range of linear dose-response relationship.
Gel filtration was carried out on a 1 x 45-cm column of
Sephadex G-75 or G-100 equilibrated with PBS. Fractions of
1 ml were collected, and the activities of D-factor, CSF, and
Assay of Interferon
Interferon in each fraction were assayed. The column was
calibrated with bovine serum albumin (M.W. 67,000), chymotrypsinogen A (M.W. 25,000), and egg white lysozyme (M.W.
14,000) as marker proteins.
L-cells by the method by Kawade ef al. (17). The titers of
Interferon were measured with NIH reference standard mouse
Interferon (Catalog No. G002-904-5111)
as a standard and
are expressed in international reference units (IU). One unit in
our system is equal to 5 IU.
Elimination of Interferon from the Conditioned Medium of
Con A-stimulated Spleen Cells
Interferon was removed by 2 methods, (a) The conditioned
medium of Con A-stimulated spleen cells was dialyzed first
against 0.05 M HCI-KCI, pH 2, for 24 hr at 4°and then against
PBS for an additional 24 hr (32). (b) The conditioned medium
was prepared by incubation of spleen cells with Con A in the
presence of cytochalasin B for 2 days and then dialyzed against
PBS to remove chytochalasin B (24).
DECEMBER
Interferon was assayed by determining the inhibition of
[3H]uridine incorporation into vesicular stomatitis virus RNA in
Chemicals
The mitogens used were Con A (Sigma Chemical Co., St.
Louis Mo.), PHA-P and LPS-W from Salmonella typhimurium
(Difco Laboratories, Detroit, Mich.), and PWM (Grand Island
Biological Co., Grand Island, N. Y.). Polyd)-poly(C) was ob
tained from Yamasa Shoyu (Choshi, Japan). Polymyxin B and
RNase A were obtained from Sigma. Cytochalasin B was pur
chased from Aldrich Chemical Co., Milwaukee, Wis.
1980
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4805
Y. Yamamoto et al.
RESULTS
Production of D-Factor by Spleen Cells Stimulated with
Mitogens or Poly(l)«Poly(C). Spleen cells were Incubated with
Con A, LPS, or polyd)-poly(C) for various times, and then the
activity of D-factor in the conditioned medium was assayed by
determining the induction of phagocytic activity in M1 cells. It
was reported that LPS is a potent inducer of differentiation of
M1 cells (31) and that polyd)-poly(C) enhances the sensitivity
of M1 cells to D-factor (33). Therefore, LPS and poly(l)poly(C) were removed from the medium before assay of Dfactor. Polymyxin B is known to bind to LPS, probably the Lipid
A portion of the molecule, and to block several actions of LPS
(15). The ability of LPS to induce differentiation of M1 cells was
also blocked completely by incubation with polymyxin B (data
not shown). These results are consistent with the observations
of Weiss and Sachs (31) that the active portion of LPS to
induce differentiation of M1 cells was Lipid A. Similarly, poly(l)poly(C) digested with RNase A had no activity to stimulate
differentiation of M1 cells (data not shown). These treatments
could eliminate the direct effect of LPS or poly(l)-poly(C) re
maining in the conditioned medium on differentiation of M1
cells. As shown in Chart 1, production of D-factor increased
from 10 hr after treatment of spleen cells with mitogens or
polyd)-poly(C) and reached a plateau in 2 days. However, no
significant production of D-factor was observed in untreated
spleen cells even after 5 days.
Then, spleen cells were incubated with various concentra
tions of Con A, LPS, or poly(l)-poly(C) for 2 days. These
reagents enhanced production of D-factor by the cells dose
dependently (Chart 2). The effect of PHA or PWM on the
production of D-factor was similar to that of Con A (data not
shown). The optimum concentrations of the compounds were
5 /ig/ml for Con A, 5 fil/ml for PHA and PWM, and 10 jug/ml
for LPS and polyd).poly(C). Although Con A, PHA, and PWM
remained in the conditioned medium, they had no effect on the
induction of phagocytosis in M1 cells.
The fact that the conditioned medium of spleen cells can
induce differentiation of M1 cells was confirmed by examining
morphological changes in M1 cells (Table 1). Untreated M1
20
Days
Chart 1. Time course of production of D-factor by stimulated spleen cells.
Spleen cells were incubated with Con A, 5/ig/ml (O), LPS, lOjÃ-g/ml (•),poly(l)poly(C), 10 /ig/ml (A), or without any addition (control) (A) for the indicated
times. The conditioned medium was harvested, and LPS and polyil)-poly(C) in
the conditioned media were inactivated by treatment with polymyxin B and RNase
A, respectively, as described in "Materials and Methods." The conditioned
medium (10%) was assayed for activity of D-factor to induce phagocytic activity
in M1 cells. The data represent the means of duplicate determinations.
4806
01
5
10
Concentration(jjg/ml)
50
Chart 2. Production of D-factor in cultured spleen cells stimulated with mito
gens and polyd)-poly(C)
Spleen cells were stimulated with various compounds
at the indicated concentrations. The conditioned media were harvested 2 days
later. LPS and polyd)-poly(C) were inactivated by treatment with polymyxin B
and RNase A, respectively. The conditioned medium (10%) was assayed for Dfactor activity to induce phagocytic activity in M1 cells. The data represent the
means of duplicate determinations. O, Con A; •.LPS; A, polyd)-poly(C).
Table 1
Induction of morphological changes in Ml cells by conditioned medium of
mitogen-stimulated spleen cells
(%)0Conditioned
Cell type
medium8NoneCon
cyte01.00
ALPSMyeloblast96.54.336.0Intermediate3.582.460.0Macrophage012.34.0Granulo-
a Prepared by incubation of spleen cells at 5 x 106 cells/ml
with 5 /ig of Con
A per ml or 10 fig of LPS per ml. LPS in the conditioned medium was inactivated
by treatment with polymyxin B.
b Morphological changes were determined 3 days after treatment of M1 cells
with or without 30% (v/v) of the conditioned medium. Morphology of the cells
was examined by staining the cells with May-Grunwald-Giemsa.
More than 300
cells were analyzed from each sample.
cells were myeloblastic cells with a large round nucleus and
little cytoplasm. On treatment with 30% (v/v) conditioned me
dium of spleen cells stimulated with Con A for 3 days, about
12% of the cells were morphologically differentiated into macrophage-like cells with a small eccentrically located nucleus,
and a few cells were differentiated into granulocyte-like cells
with a polymorphic nucleus. A large part of the residual cells
appeared to be forms intermediate between myeloblastic cells
and mature macrophages or granulocytes. The cells cultured
with the conditioned medium of LPS-stimulated spleen cells
showed similar morphological differentiation but to a lesser
extent (Table 1). These morphological changes in M1 cells
(clone T-22) were always associated with the induction of
activities of phagocytosis, locomotion, and lysozyme and the
appearance of Fc receptors on the cell surface (30). Therefore,
activity of D-factor was assayed by determining the induction
of phagocytic activity in M1 cells in the following experiments.
Cells Responsible for the Production of D-Factor in Stim
ulated Spleen Cells. For determining the type of cells respon
sible for production of D-factor, spleen cells were fractionated
into glass-adherent cells and nonadherent cells by incubation
on glass dishes for 3 hr at 37°.The adherent cells amounted
to less than 5% of the total spleen cells; more than 95% of
them were macrophages. On the other hand, more than 98%
of the nonadherent cells were lymphocytes. The 2 types of
cells were incubated with the mitogens or polyd)-poly(C). As
shown in Table 2, Con A, PHA, and PWM enhanced production
of D-factor by the nonadherent cells, but not by the adherent
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Production of D-Factors by Spleen Cells
cells, whereas LPS and poly(l)-poly(C) stimulated its produc
tion by both nonadherent and adherent cells. These results
suggest that Con A, PHA, and PWM stimulate only lympho
cytes, whereas LPS and polyd)-poly(C) stimulate both lympho
cytes and macrophages. When unstimulated spleen macro
phages were incubated at 106 cells/60-mm dish, D-factor was
not detected in the conditioned medium. However, the macro
phages at higher cell density released a significant amount of
D-factor, as reported previously (12).
Molecular Heterogeneity of D-Factor from Stimulated
Spleen Cells. The conditioned medium of unfractionated
spleen cells stimulated with mitogens or poly(l)-poly(C) was
chromatographed on Sephadex G-75. D-factor from Con Astimulated cultures showed a single peak with a molecular
weight of 40,000 to 50,000 (Chart 3a). D-factor from the
conditioned medium of spleen cells treated with PHA or PWM
had a similar molecular size. On the other hand, D-factors from
LPS-stimulated cultures were separated into 2 peaks with
molecular weights of 40,000 to 50,000 and 20,000 to 25,000
(Chart 3b). The gel filtration pattern of D-factors from the cells
treated with poly(l)- poly(C) was similar to that of D-factors from
the LPS-stimulated culture.
Since peritoneal macrophages also produce D-factor (12,
28), we compared D-factor from spleen cells with that from
peritoneal macrophages. As shown in Chart 3c, peritoneal
macrophages produced D-factor with molecular weights of
20,000 to 25,000. The molecular size of the smaller D-factor
produced by stimulated spleen cells is similar to that of peri
toneal macrophage-derived
D-factor. The smaller D-factor
might be produced by spleen macrophages. Then, we com
pared D-factor from spleen macrophages with that from peri
toneal macrophages. Spleen macrophages as well as perito
neal macrophages produced a detectable amount of D-factor
without stimulation at higher cell density than 5 x 106 cells/
60-mm dish. Enhancement of production of D-factor by LPS or
polyd)-poly(C) was observed in both macrophages. In spite of
the same culture condition, spleen macrophages produced Dfactors with molecular weights of 40,000 to 50,000 and 20,000
to 25,000, while peritoneal macrophages produced only the
smaller one. On the other hand, spleen lymphocytes treated
with LPS produced the larger D-factor but not the smaller one
(data not shown).
Separation of D-Factor from CSF and Interferon in Condi
tioned Medium of Spleen Cells Stimulated with Con A or
LPS. Lymphocytes stimulated by a specific antigen or mitogen
Table 2
Production of D-factor in cultured spleen cells stimulated with mitogens or
polyW-poly(C)
activity3TreatmentControl
D-factor
ated spleen
cells (2.5 x
10' cells/
dish)6.5
ent6 spleen
spleen cells
(0.1 x 107
cells (2. 3 x
107 cells/
cells/dish)1.3
48.2
0.9
Con A (5 fig/ml)
53.0
5.7
PHA (5 /il/ml)
25.4
3.0
PWM(5/il/ml)
22.3
29.7
LPSdO/ig/ml)
PolyCO-polyfCHIO^g/ml)Unfraction
26.4Adherent" 10.5Nonadher
a Percentage of phagocytic cells among M1 cells incubated with
conditioned medium. Values are means of duplicate determinations.
0 See "Materials and Methods."
DECEMBER
dish)7.0
41.3
41.0
17.9
32.5
33.1
10% (v/v)
10
20
25
No.
Chart 3. Gel filtration of D-factor on Sephadex G-75. Conditioned media (5
ml) of spleen cells stimulated with 5 fig of Con A per ml (a). 10 fig of LPS per ml
(b). or of peritoneal macrophages (c) were concentrated and dialyzed against
PBS. The resultant solution (1 ml) was applied to a Sephadex G-75 column
equilibrated with PBS and fractionated. Each fraction (1 ml/fraction) was assayed
for activity of D-factor to induce phagocytic activity in M1 cells.
15
Fraction
are known to release numerous substances including type II
Interferon (3) and CSF (2, 20, 23, 25). Therefore, we examined
whether the D-factor produced by spleen cells was identical
with type II interferon or with CSF.
Conditioned medium from Con A-stimulated spleen cells was
fractionated by gel filtration on Sephadex G-100, and the
activities of D-factor, CSF, and interferon in each fraction were
measured (Chart 4a). The fraction with interferon activity ov
erlapped that of D-factor, and both had a molecular weight of
40,000 to 50,000. However, CSF was smaller than D-factor or
interferon, with an activity peak at 24,000. We found previously
that type I interferon induced by vesicular stomatitis virus or
poly(l)- poly(C) was different from D-factor, but that it stimulated
induction of differentiation of leukemic M1 cells in the presence
of D-factor (30, 33). On the other hand, McNeill ef al. (21)
reported that type I interferon inhibited colony formation of
normal bone marrow cells by CSF. Although it is uncertain
whether type II interferon affects the activities of D-factor and
CSF, this seems very likely. Therefore, we tried to eliminate
interferon activity only from the conditioned medium of spleen
cells and to determine the exact relationship between D-factor
and CSF. Since type II interferon is known to be inactivated by
acidic treatment at pH 2 (3, 33), conditioned medium of spleen
cells stimulated with Con A was treated at pH 2 for 24 hr. As
shown in Table 3, this treatment inactivated interferon but not
D-factor or CSF. Moreover, when the resulting conditioned
1980
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4807
V. Yamamoto et al.
medium was subjected to gel filtration on a Sephadex G-100
column, the elution patterns of D-factor and CSF were un
changed, as shown in Chart 40. These results exclude the
possibility that CSF activity could not be detected in the frac
tions containing the D-factor and interferon because it was
inhibited by interferon. The results also show that D-factor with
a molecular weight of 40,000 to 50,000 has no CSF activity.
This conclusion was supported by experiments using cytochalasin B. As shown in Table 3, when spleen cells were treated
with Con A and Cytochalasin B simultaneously, the release of
type II interferon was inhibited completely, whereas release of
CSF was not affected, as reported previously (14, 24). It was
found that the release of D-factor was partially inhibited by
Cytochalasin B. The gel filtration patterns of D-factor and CSF
in this preparation were similar to those of the factors in
conditioned medium treated at pH 2 (data not shown).
Next, the conditioned medium from LPS-stimulated spleen
cells was fractionated by gel filtration on Sephadex G-100
column, and the activities of D-factor and CSF in each fraction
were measured. As shown in Chart 5, CSF was eluted between
the larger D-factor and the smaller one. Apparent molecular
weights of CSF and the smaller D-factor were 24,000 and
22,000, respectively.
DISCUSSION
67K
Vo
25K
4-
feoh
4
xï-\
S
208-
<ï
a
103 s
20
0
10
15
Fraction
20
No.
25
Chart 4. Fractionation of D-factor. CSF, and Interferon, a, Conditioned me
dium (5 ml) of Con A-stimulated spleen cells was concentrated and dialyzed
against PBS. The resultant solution (1 ml) was applied to a Sephadex G-100
column equilibrated with PBS and fractionated. Each fraction was assayed at a
concentration of 10% (v/v) for activities of D-factor and CSF. respectively.
Activities of D-factor (O) and CSF (•)were assayed by measuring their ability to
induce phagocytic activity in M1 cells and to stimulate colony formation of 10s
mouse bone marrow cells, respectively. The activity of interferon (A) was assayed
by measuring [3H]uridine incorporation into vesicular stomatitis virus RNA in L
cells, b. To remove interferon. the conditioned medium was treated at pH 2 and
analyzed by gel filtration on a Sephadex G-100 column.
Table 3
Elimination of interferon in the conditioned medium from Con A-stimulated
spleen cells
control)3TreatmentpH2òCytochalasin
Activity (% of
This work showed that mouse spleen lymphocytes produced
factors stimulating differentiation of mouse myeloid leukemic
M1 cells when treated with various mitogens, such as Con A,
PHA, PWM, and LPS, and synthetic double-stranded polyribonucleotide poly(l)-poly(C) and that the factors produced by
the spleen cells were different from CSF stimulating growth
and differentiation of normal bone marrow hematopoietic pre
cursor cells.
The lymphocytes responsible for production of D-factor have
not yet been characterized. Since Con A, PHA, and PWM are
known to be mitogens for T-lymphocytes while LPS and PWM
are mitogens of B-lymphocytes, it seems probable that both Tand B-lymphocytes produce D-factor. Spleen lymphocytes pro
duced D-factor with a molecular weight of 40,000 to 50,000,
whereas spleen macrophages stimulated with LPS or poly(l)poly(C) produced this D-factor and also another D-factor with
a molecular weight of 20,000 to 25,000. It is of interest that
peritoneal macrophages produce a small D-factor but not a
larger one even if the cells are stimulated with LPS or poly(l)poly(C).
Both D-factor and CSF have been found in the culture fluids
of various cells and in tissue extracts (1, 8, 9, 11, 12, 26), and
a'80
"5
Bc
1 /ig/ml
5 fig/mlD-factor10378
52CSF93107
78Interferon18397
i
Activity of D-factor was assayed by measuring the ability to induce phago
cytic activity in M1 cells. Activities of CSF and interferon were determined as
described in "Materials and Methods." Data are shown as percentage of the
activity of control. Values are means of duplicate determinations.
b The conditioned medium was treated with pH 2 for 24 hr. Untreated
conditioned medium at a concentration of 10% (v/v) induced 41% phagocytic
cells in M1 cells and 52 colonies of macrophages and granulocytes per dish ¡n
assay of CSF. Activity of interferon in the untreated conditioned medium was
1560IU/ml.
0 The conditioned medium was prepared by incubation of spleen cells with
Con A in the presence of Cytochalasin
prepared in the absence of Cytochalasin
induced 45% phagocytic cells in M1 cells
granulocytes per dish in assay of CSF.
conditioned medium was 935 Ill/ml.
4808
B for 2 days. Conditioned medium
B at a concentration of 10% (v/v)
and 57 colonies of macrophages and
Activity of interferon in the control
K>
15
Fraction
20
No.
25
30
Chart 5. Fractionation of D-factor and CSF. Conditioned medium (5 ml) of
LPS-stimulated spleen cells was concentrated and dialyzed against PBS. The
resultant solution (1 ml) was applied to a Sephadex G-100 column equilibrated
with PBS and fractionated. Each fraction was assayed at a concentration of 20%
(v/v) for activities of D-factor and CSF. respectively. Activities of D-factor (O)
and CSF (•)
were assayed by measuring their ability to induce phagocytic activity
in M1 cells and to stimulate colony formation of 105 mouse bone marrow cells,
respectively.
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Production of D-Factors by Spleen Cells
the molecules of both are heterogeneous in size and charge.
Although a protein termed MGI (macrophage and granulocyte
inducer) that was purified from conditioned medium of a mouse
fibroblast cell line had the activities of both D-factor and CSF
(6), another preparation of CSF produced by mouse embryo
cells in serum-free culture had no D-factor activity (19). With
the use of DEAE-cellulose chromatography, we also separated
several fractions with activity of D-factor but not with CSF from
conditioned medium of Yoshida sarcoma cells in serum-free
culture (11 ). Furthermore, Gallagher ef al. (5) reported that the
conditioned medium of whole human embryo celts prompted
growth and differentiation of culture of leukocytes from a
patient with acute myelogenous leukemia but had no CSF
activity. In the present work, we showed clearly that spleen
cells produced D-factors without CSF activity.
Chemical characterization of D-factor and CSF from lympho
cytes and studies on the mechanisms of their biological actions
are in progress in attempts to elucidate the factors involved in
the regulation of normal myeloid hematopoiesis and the differ
entiation of myeloid leukemic M1 cells in vivo.
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Downloaded from cancerres.aacrjournals.org on June 15, 2017. © 1980 American Association for Cancer Research.
4809
Production by Mouse Spleen Cells of Factors Stimulating
Differentiation of Mouse Myeloid Leukemic Cells That Differ
from the Colonystimulating Factor
Yuri Yamamoto, Mikio Tomida and Motoo Hozumi
Cancer Res 1980;40:4804-4809.
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