[CANCER
RESEARCH
38, 1414-1419,
May 1978]
Induction of Myeloid Colony-stimulating
Tumor Cell Lines by Macrophage
Concanavalin A1
Peter Ralph, Hal E. Broxmeyer,2
Sloan-Kettering
Institute
for Cancer Research,
Activators and in a T-Cell Line by
Malcolm A. S. Moore, and Ilona Nakoinz
Rye, New York 10580
activating
ABSTRACT
Certain fibrosarcoma lines in culture and the WEHI-3
myelomonocytic leukemia cell line have previously been
shown to secrete myeloid colony-stimulating
activity
(CSA) spontaneously. We describe here other hematopoietic tumor cell lines in which CSA is either produced
constitutively or inducible by immunostimulators.
CSA
production in macrophage and monocyte tumor lines is
induced by lipopolysaccharide, zymosan, Mycobacterium
strain Bacillus Calmette-Guerin,
tuberculin purified pro
tein-derivative preparation from mycobacteria, and dextran sulfate. Myeloma, mastocytoma, and T-lymphoma
lines do not produce CSA with or without these agents. In
contrast, the T-lymphocyte mitogen concanavalin A (but
not phytohemagglutinin) induces CSA synthesis in one of
seven T-lymphomas tested. In most cases induction of
CSA is correlated with conditions of cell growth inhibition
by the immunomodulators.
However, other drugs that
cause cytostasis or cytotoxicity do not lead to CSA pro
duction. Leukemic cells thus may retain sensitivity to
normal regulatory events with resultant effects on host
hematopoietic cell functions.
INTRODUCTION
Elevated numbers of granulocytes and macrophages are
associated with immunological
reactions and inflammatory
conditions. Marrow and blood monocytes have been iden
tified as producers of CSA,3 necessary for the development
of granulocytes and macrophages in vitro (6,8, 25). Bacte
rial LPS stimulates macrophages to increased CSA produc
tion (8, 10, 35). However, CSA synthesis is also induced
during T-lymphocyte-dependent
mitogen or antigen stim
ulation of spleen or blood cells (7, 22, 26, 37, 38).
We have described constitutive
CSA production
in the
mouse myelomonocyte
tumor cell line WEHI-3 (30) and
induction of CSA in a macrophage tumor cell line PU5-1.8
by LPS, BCG, PPD, yeast zymosan, and phorbol myristate
(29). This study investigates the induction of CSA in other
monocyte-macrophage
tumor cell lines by macrophage-
1 Supported
by National
Science
Foundation
Grant PCM 75-19734. Na
tional Cancer Institute Grants CA 17353 and CA 17085, and the Gar Reichman
Foundation.
2 Special Fellow of the Leukemia Society of America
3 The abbreviations
used are: CSA, myeloid colony-stimulating
activity;
LPS, lipopolysaccharide;
BCG, Mycobacterium
strain Bacillus CalmetteGuerin; PPD, tuberculin purified protein derivative; PHA, phytohemaggluti
nin; Con A, concanavalin A; T-cell, thymus-derived lymphocyte; B-cell, bone
marrow-derived
lymphocyte.
Received November 7. 1977; accepted February 2. 1978.
1414
Activity in Murine Monocyte
agents
and in T-lymphomas
by T-lymphocyte
mitogens.
MATERIALS
AND METHODS
Murine Tumor Cell Lines. Monocyte and macrophage
tumor cell lines are described in Ref. 32, except for Abelson
leukemia virus-induced line RAW264 (33). T-lymphoma lines
EL4, RBL-5, BW5147,
and S49; myelomas
P3 and
MOPC315; mastocytoma P815; lymphoma P388; and Abelson line R8 are described in Ref. 31. Rauscher leukemia
virus line RBL-3 and chemically induced leukemia L1210
(39) were obtained from K. Chang (NIH, Bethesda, Md.);
fibrosarcoma
L929 (5) was obtained from B. Williams
(Sloan-Kettering
Institute, Rye, N. Y.); bone marrow fibroblast JLSV9 and Rauscher leukemia virus-infected
JLSV9RLV (9) were obtained from A. Demsey (Sloan-Kettering
Institute); Abelson lymphoma RAW309.1 was obtained from
W. Raschke (Salk Institute for Biological
Studies, San
Diego, Calif.); F22.WC-2 T-lymphoma was obtained from T.
Watanabe (Osaka University Hospital, Osaka, Japan); mesothelioma BALTNMS201, T-lymphomas
P1798 and BALENTL5, and Abelson lymphomas ABPL1 and ABPL2 were
obtained from M. Potter (National Cancer Institute, Be
thesda, Md.).
Substances Tested for Induction of CSA. Dialyzed latex
beads 0.81 ¿¿m
in diameter, PHA P, and LPS (Salmonella
typhosa W0901; Difco Laboratories,
Detroit, Mich.); PPD
(gift from Dr. A. Gray, Merck Sharp & Dohme, West Point,
Pa.); BCG (Tice strain; University of Illinois, Chicago, III.);
zymosan and dextran sulfate (M.W., 500,000; Sigma Chem
ical Co., St. Louis, Mo.); and Con A (Pharmacia
Fine
Chemicals, Inc., Piscataway, N. J.) were dissolved or sus
pended in phosphate-buffered
saline, pH 7.4 (8 g NaCI, 0.2
g KCI, 1.15 g Na, HPO,, 0.2 g KH,PO4> 0.1 g CaCL, and 0.1 g
MgCL-6H,O per liter), at 20 to 100 times the final concentra
tion.
Assay for CSA (3). Mouse bone marrow cells (7.5 x 104)
were suspended in 1 ml 0.3% Difco agar culture medium
containing enriched McCoy's medium plus 10% fetal calf
serum. Medium conditioned
by cell lines (0.1 ml) was
placed on the bottom of Petri dishes prior to addition of
agar cell suspensions. Cultures were incubated in 5% CO2humidified
air, and colonies (>40 cells/aggregate)
and
clusters (3 to 40 cells/aggregate)
were scored after 5 to 7
days. Under these conditions a maximum of 100 to 200
colonies of granulocytes and macrophages can be obtained
with optimal concentrations
of CSA. For intracellular CSA,
cell pellets were suspended in phosphate-buffered
saline to
the original volume and lysed by 2 cycles of freeze-thaw. No
CANCER
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Induction of Monocyte and J-Lymphoma Cell Lines
additional CSA was detected in producing lines following
further cycles of freeze-thaw.
RESULTS
Spontaneous
Production of CSA by Tumor Cell Lines. A
number of murine cell lines adapted to culture were inves
tigated for spontaneous production of CSA. Besides the
previously described L-cell fibrosarcoma (2) and myelomonocytic leukemia WEHI-3 (30), 2 lymphoid lines secreted
CSA constitutively (Table 1). Large amounts of CSA were
found in supernatants of RBL-3 and, to a lesser extent, of
L1210 lymphoid cell lines. A fibroblastoid cell line derived
from normal mouse bone marrow (JLSV9) and its Moloney
leukemia virus-infected counterpart, JLSV9-RLV, also pro
duced CSA spontaneously, as described for the similar
JLSV5 virus-transformed line (44). The fibroblastoid lines
produced only or predominately macrophage CSA. CSA
from the other constitutive lines caused bone marrow
colonies of granulocyte and macrophage morphology (not
shown). In the CSA-secreting lines studied by us, undiluted
supernatants and 1:10 dilutions in most cases produced
greater than 100 colonies/7.5 x 104bone marrow cells, and
dilutions greater than 1:10 were required before titrating
out the activity. No CSA was found in supernatants of
Table 1
Spontaneous CSA production in tumor cell lines
untreated cultures of a variety of macrophage, monocyte,
T-lymphoma, myeloma, or mastocytoma cell lines (Table 1);
in a number of these lines tested, an inhibitor of the CSA
assay was also undetected, indicating the absence of the
molecule(s).
Induction of CSA by LPS and Other Macrophage Acti
vators in Monocyte-related Lines. Since LPS will induce
CSA synthesis in the macrophage line PU5-1.8 (29), this
agent was tested in the other hematopoietic lines. We use
the term induction to denote the stimulation of CSA produc
tion in experimental cultures when control cultures do not
contain detectable activity. As shown in Table 2, LPS
Table 2
Induced CSA production and drug toxicity in hematopoietic tumor
cell lines
CSA"Cell
neous
LPS''1210000000000000014024000001305556786"0000000000
A*1170000108000000000132300000ND
lineMyelomonocyteWEHI-3MacrophagePU5-1
(20)0
.8J774P388D1RAW264T-lymphomaEL4S49RBL-5P1798F22.WC-2BALENTL5BW514
(0)0
(39)0
(16)0
(41)0
lineT-lymphomaEL4S49RBL-5P1798F22.WC-2
lineMyelomonocyteWEHI-3Macrophage
Cell
8*0000068
±
(66)0
(K)e0
(K)0
(71)0
(K)0
(K)0
(16)0
.8P388D1J774RAW264MastocytomaP815MesotheliomaBALTNMS201FibrosarcomaL929Marrow
PU5-1
BALENTL5BW5147MyelomaP3MOPC315LymphomaRBL-3L1210R8RAW309.1C1498ABPL1ABPL2CSA00000
0000140
(0)0
(0)0
(0)129
496±
2160±
1498ABPL1ABPL2RAW309.1Sponta
1624
±
200000
±
fibroblastJLSV9JLSV9-RLVCSA«121
11170
±
±7Cell
" Colonies/7.5 x 10" mouse bone marrow cells stimulated by 0.1
ml tumor cell line supernatant in 1-ml agar cultures. Cell line
cultures were initiated at 2 x 105 cells/ml, and supernatants were
collected when cultures reached 10Vml (2 to 3 days). WEHI-3,
L929, JLSV9, JLSV9-RLV, and RBL-3 supernatants stimulated 90 to
150 colonies also when tested at 1:10 dilution. The other constitu
tive producers and inducible lines (described below) showed less
activity when their supernatants were diluted more than 1:2.
6 Mean ±S.E.
MAY 1978
(51)26
(57)0
(20)0
(60)0
(3)0
(55)ND
" Colonies/7.5 x 104mouse bone marrow cells stimulated by 0.1
ml tumor cell line supernatant in 1-ml agar cultures. Cell line
cultures were initiated at 2 x 105cells/ml and incubated with drugs
for 3 days. Viable cells were then counted, percentage of inhibition
of tumor cell growth was compared to control cultures calculated,
and supernatants were tested for CSA.
* LPS, 1 ¿ig/ml,and Con A and PHA, 10 /¿g/ml,were incubated
with tumor cell lines for 3 days. Control solutions of these mitogens
alone had no effect on the bone marrow colony assay.
c Numbers in parentheses, percentage of tumor cell growth
inhibition.
'' LPS, 0.01 /¿g/ml.
e K, killed, fewer viable cells after 3 days than initial concentra
tion.
f ND, not done.
1415
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P. Ralph et al.
induced CSA to varying degrees in all of the monocyte and
macrophage
lines except for the constitutive
producer
WEHI-3. Results are shown for a 3-day incubation with LPS,
1 /xg/ml. This concentration
of LPS inhibits the growth of
the monocyte-macrophage
lines (except P388D1) more than
50% but not of the other cells shown in Table 1 (32). In
some experiments, control WEHI-3 cultures had lower activ
ity (40 to 80 colonies/0.1 ml supernatants) than was shown
in Table 2, and then LPS stimulated CSA production up to
2-fold (4). Cell lysates (at 106 cells/ml) of control cultures of
inducible tumor cell lines neither contained CSA nor in
hibited the activity of preformed CSA (0.1 ml tested in
assay), except for slight inhibition with J774.
Production
of CSA by L1210 cells was augmented by
incubation with LPS, and induction of CSA occurred in an
Abelson lymphoma line, R8. These 2 lines and RBL-3 lack Tlymphocyte Thy-1 antigen, surface ¡mmunoglobulin, and
myeloid markers of phagocytosis and lysozyme but bear Fc
receptors and possess an alkaline phosphatase
activity
characteristic
of lymphoid tumors (unpublished
observa
tions). LPS did not induce CSA in a number of T-lymphoma,
myeloma, mastocytoma, or other lymphoma lines (Table 2).
The monocyte-related
tumor lines were also tested with
other macrophage-active
agents known to induce CSA in
PU5-1.8 (29) and to inhibit specifically
growth of these
tumor types (32). As shown in Chart 1, the immunopotentiators LPS, BCG, PPD, zymosan, and dextran sulfate induced
CSA in monocyte and macrophage tumor lines at concen
trations generally correlated with tumor cell cytostasis. The
decline in CSA activity in supernatants of several lines as
LPS concentration
is increased to 10 pig/ml may be due to
inhibition of the assay by LPS (32). However, the suboptimal
activity in cultures of the most sensitive line, RAW264, at
PU5-I.8
0.1 and 1 us LPS per ml must be due to high-dose inhibition
of CSA production,
as these concentrations
do not affect
the assay system. Latex beads, which are actively phagocytosed by the cell lines without interfering with growth
(32), did not induce CSA.
Induction of CSA by T-Lymphocyte Mitogens. Since
mitogen stimulation of normal T-lymphocytes induces CSA
production,
these agents were tested on a number of Tlymphoma cell lines. PHA and Con A are preferentially toxic
to murine (27) and human (28) T tumor cell lines compared
to myelomas; however, many other hematopoietic cell lines
are as sensitive as T lines (Ref. 27; unpublished observa
tions). Con A was very effective in stimulating granulocytemacrophage CSA in the T-lymphoma
EL4 (Table 2). Six
other T-lymphomas and other types of hematopoietic
cell
lines were not induced by Con A. Dose titration (Chart 2)
shows that the threshold for CSA induction and tumor cell
growth inhibition occurred at 2 to 5 ^g Con A per ml. Con
A, up to 50 /ng/ml, strongly induced CSA production despite
the fact that no viable EL4 cells remained after 3 days of
incubation in this high toxic concentration.
PHA showed a toxicity curve almost identical with that of
Con A, but no CSA was induced in EL4 by this lectin.
Controls showed that PHA did not inhibit the activity of
preformed CSA or the induction of CSA in EL4 cells by Con
A. PHA did not induce CSA in any cell line tested (Table 1).
Kinetics of CSA Production. Chart 3 shows that small
amounts of CSA were detected in supernatants
of EL4
cultures incubated for 16 hr with Con A, 10 or 30 ¿ig/ml. At
this time 30% of EL4 cells were killed by the higher Con A
concentration.
There was a great increase in supernatant
CSA during the subsequent 2 days of incubation. At 30 ¿ig
Con A per ml, viable cells decreased to 5% of initial
numbers at Day 2 and 0% at Day 3, and total live and dead
IOO
50
-IOO
ó
50
25
I
S?
J774
50
IOO
25
50
I80
S
i«
RAW264
50
IOO
25
50
20
I05 IO6
LPS
BCG
I 10 100
10 roo
PPD
Z
i ra loo
DS
Chart 1. Induction of CSA and inhibition of growth in macrophage cell
lines by macrophage activators. Cultures were initiated at 2 x 105 cells/ml
plus final concentrations of LPS, PPD, zymosan (Z), and dextran sulfate (OS)
in ng/m\ or BCG in bacteria/ml as shown. After 3 days of incubation, viable
tumor cells were counted (percentage of growth inhibition equals 100 times
the increase in experimental-increase in control culture), and supernatants
were tested for CSA (colonies stimulated per 7.5 x 1Q.4bone marrow cells
plated in agar with 0.1 ml cell line supernatant). For several lines tested the
product induced was granulocyte-macrophage CSA.
1416
30
on A or PHA
50
Chart 2. Induction of CSA in T-lymphoma EL4 by Con A. EL4 cultures
were initiated at 10s cells/ml and incubated with Con A or PHA, 0 to 50 ¿ig/
ml. After 3 days viable cells were counted, and the supernatant was assayed
for CSA. Increase in viable cells in experimental cultures was compared to
the increase in controls to determine the percentage of control growth.
Control cultures contained 28.6 x 105 EL4 cells/ml at 3 days. *, high
concentrations of Con A and PHA were toxic to EL4. No viable cells were
found after 3 days with 30-^g/ml doses of either lectin. Supernatants of
control EL4 cultures to which Con A was added at the end of 3 days of
incubation contained no detectable CSA. Con A and PHA added to pre
formed WEHI-3 CSA did not inhibit the activity of the latter in the assay
system. PHA, 5 ng/ml, did not affect the induction of CSA in EL4 cells by Con
A, 5 pglm\.
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Induction of Monocyte and T-Lymphoma Cell Lines
0
8 16 20 30 4O 48
Hours
Chart 3. Kinetics of CSA induction. EL4 cultures with Con A, 10
(
) or 30 ng/m\ (
), and PU5-1.8 cultures with LPS, 1 ^g/ml, were
initiated at 2 x 10scells/ml. At the times shown viable cells in duplicate 2-ml
cultures were counted, and the supernatant was assayed for CSA (colonies/
7.5 x 10* bone marrow cells/0.1 ml supernatant). Con A, 10 /¿g/ml,inhibited
EL4 growth 58%, and 30 pg/ml was toxic, while LPS inhibited PU5-1.8
growth more than 95% when measured at 48 hr.
cell numbers never exceeded initial concentrations by more
than 10%. Thus, CSA induction can occur under conditions
in which the inducing agent almost completely inhibits cell
growth. The induction of CSA in the monocyte line PU5-1.8
by LPS was also a slow process, taking at least 3 days for
maximal expression (Chart 3). In contrast to lectin induction
of EL4, LPS stimulation of CSA in PU5-1.8 cells was accom
panied by reversible cytostasis of the tumor cells rather
than by cytotoxicity (32). DNA synthesis is not required for
induction of CSA in normal lymphocytes by Con A (36) nor
in PU5-1.8by LPS (29).
DISCUSSION
Several facts are apparent from this study of CSA induc
tion in different types of hematopoietic tumor cell lines, (a)
Only cell type-specific agents (macrophage activators for
monocyte-related lines; Con A for the T line) can induce
CSA production, (b) All inductions of CSA are accompanied
by drug effects strong enough to block tumor cell prolifer
ation. Other inhibitors of cell growth [dibutyryl cyclic adenosine 5'-phosphate, high levels of thymidine, dimethyl sulfoxide (29), PHA, and Con A in most cell lines] do not induce
CSA. (c) The macrophage lines differ in sensitivity to induc
ing agents, presumably because of differences in quantity
or affinity of specific receptors for each kind of activating
molecule, (d) The inducible T-cell line EL4 must share with
the other T lines binding sites for PHA and Con A leading to
toxicity but has a unique Con A-binding site for functional
activation of CSA production.
The LPS induction of CSA in the monocyte-related tumor
lines described here is similar to the results of experiments
of Eaves and Bruce (10) with normal mouse macrophages
and of Ruscelli and Chervenick (35) wilh human monocytes
in the dose tilralion and kinelics requiring several days for
maximal stimulation.
MAY 1978
Stimulation of CSA by dextran sulfate in bone marrow
cells, probably macrophages, has been described by Gronowiczef al. (14). In addition to these agents and BCG, PPD,
and zymosan used in the present paper, we previously
showed that phorbol myristate induces CSA in the mono
cyte cell line PU5-1.8 (29) and in normal adherent bone
marrow cells (unpublished observations). Phorbol myristate
is a potent stimulator of enzyme release and other activities
of granulocytes (11, 34), induces plasminogen activator
production in macrophages (43), and is a T-lymphocyte
mitogen (42). Other macrophage properties stimulated by
activators in the macrophage tumor cell lines include anti
body-dependent cellular immunity against erythrocyte (33)
and tumor targets," production of T-lymphocyte-activating
factor (21, 24), prostaglandin release (Ref. 20; E. Rietschel,
personal communication), /3-glucuronidase, elastase, plas
minogen activator, collagenase, endogenous pyrogen, and
factors required for induction of murine T killer cells (16),
murine antibody-producing cells,5 and human B-lymphocytes (17) not replaceable by mercaptoethanol. A summary
of macrophage properties retained by murine and human
monocyte-related cell lines is shown in Table 3.
The constitutive production of CSA by the least differen
tiated myelomonocytic line, WEHI-3, but not by other lines
with more properties of mature macrophages (33) deserves
comment. Normal, immature myeloid cells or their progen
itors are dependent on CSA for growth. There must be
stringent controls to prevent self-stimulation of the progen
itor by premature synthesis of CSA, which is a function of
the mature cells of the monocyte branch of the myeloid
series. Production of CSA associated with murine (24) and
human (13) myelomonocytic leukemias suggests that 1 path
to leukemogenesis in this early cell type is the inappropriate
activation of genes for the molecule that stimulates its own
growth. There is evidence that the murine WEHI-3 tumor
(24) and WEHI-3 culture line cells (4) are still dependent on
CSA for growth. A granulocyte inhibitor of CSA production
blocks WEHI-3 colony formation in agar but does not block
colony formation of other tumor lines, and this growth
inhibition is overcome by adding an external source of CSA
or by stimulating endogenous synthesis of CSA (4).
Induction of CSA in spleen and peripheral blood cells by
Con A and PHA (and perhaps by pokeweed mitogen) is a
property of T-lymphocytes (26, 27). However, there is evi
dence for separate subsets of T-cells responding to PHA
and Con A, in terms of induction of DNA synthesis (41) and
immune functions of helper, suppressor, and killer cells.
The induction of CSA in the T-cell line EL4 by Con A but not
by PHA suggests that these cells belong to a Con A-responsive, PHA-unresponsive subset and that other T lines may
be found to be activated by the other T mitogens. Pokeweed
mitogen did not induce CSA in EL4 cells. However, adher
ent cells are required for activation of some normal T-cell
1 P. Ralph, M. Ito, H. E. Broxmeyer, and I. Nakoinz. Corticosteroids Block
Newly-induced but Not Constitutive Functions of Macrophage Cell Lines:
CSA Production, Latex Phagocytosis, Antibody-dependent Lysis of RBC and
Tumor Targets, submitted for publication.
* M. K. Hoffmann, C. Galanos, S. Koenig, R. S. Mittler, P. Ralph, H. F.
Oettgen, and U. Hämmerling. Differentiation of B Lymphocytes: Factor
Released by LPS-stimulated Macrophages Induces Functional and Phenotypic Differentiation, submitted for publication.
1417
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P. Ralph et al.
Table 3
General properties of monocyte-related
immunity''RBCLyso-
tumor cell lines in culture
genouspyro-gen'++++NT'"NTNT+--NTPr
Tumorzyme" PhagophagoRBC
lysis+
cytosis*
CSA''
cytosis
lysis
teases''+++++NTNTNT---Endo
LineWEHI-3P388D1J774PU5-1.8RAW264RAW309CrWR19MU937T-lymphomasMyelomasMastocytomaP815StrainC"DBA/2CCBAB/14CXC.BCHumanDBA/2Eti
ologyOilS'?OilSVirusVirusVirusDHLCell-mediated
(-)+
+
+
(-)
(-)j+
+
+
+
+
(-)•*+
+
+
+
+
+k+
+
+k
+
+
(-)
+++
+
+
+
+'
NT+
+
NT+
+
+
+
+
(-)_
NT
NT
+
+
+
+
n-----(-)Neutralpro
0 Lysozyme synthesis and predominate secretion (30).
h Phagocytosis of zymosan and latex beads; P815 has slight ingestive activity against latex beads (32).
'' Granulocyte-monocyte
CSA, constitutive
in WEHI-3 and inducible in the other macrophage lines by LPS, BCG, PPD, zymosan,
or
dextran sulfate (Chart 1).
'' Antibody-dependent
phagocytosis
or lysis of sheep RBC and tumor targets (33).
*' Plasminogen activator, collagenase, elastase (J. Hamilton and Z. Werb, personal communication).
fP. Bodel. Endogenous Pyrogen Production by Murine Macrophage Cell Lines, submitted for publication.
" Prostaglandin
E production,
especially in response to LPS or CSA (20).
'' Mouse strain: C, BALB/c.
' S, spontaneous; virus, Abelson leukemia virus (W. C. Raschke, S. Baird, I. Nakoinz, and P. Ralph. Functional
Macrophage
Cell Lines
Transformed by Abelson Leukemia Virus, submitted for publication);
DHL, patient with diffuse histiocytic lymphoma (30).
1 Ascites preparations are very active in lysis of RBC targets (Ref. 33; unpublished observations).
* Latex bead phagocytosis
and antibody-dependent
tumor lysis by PU5-1.8 line is stimulated by preincubation
with LPS or PPD.4
' Stimulated further by preincubation
with LPS or PPD (33).
'" NT, not tested.
" One (EL4) of 7 T-lymphomas produces CSA in response to Con A (Table 2).
functions, and this is being tested with the cell line. Other
functions of T-cell lines have been described, such as
inhibition (12, 40) and stimulation (T. Watanabe, personal
communication) of in vitro antibody production, but the
present experiments are the first examples of an induced
function in a lymphoid cell line.
In addition to macrophage and T-cell sources of CSA,
there are suggestions that B-lymphocytes can produce
myeloid CSA. Since LPS-induced B-cell mitogenesis is
correlated with CSA production by mouse spleen cultures
in kinetics, genetics, and dose response, Apte ef al. (1)
have implied that B-cells are the source of CSA. However,
spleen macrophages produce CSA in response to LPS, and
no attempt was made to exclude this possibility. We find
that spleen cell production of CSA in response to LPS is
macrophage dependent, whereas LPS-induced prolifera
tion of B-lymphocytes is independent of macrophages,
implying that B-cells are not a major contributor to LPSinduced CSA (unpublished observations).
What uses can be made of these hematopoietic tumor
line systems? Due to their homogeneity, they are ideally
suited for biochemical analysis of induction and regulation,
from surface membrane receptors for external stimuli to
cytoplasmic transduction of activating signals to nuclear
events. For example, LPS at CSA-inducing concentrations
stimulates intracellular cyclic adenosine 5'-phosphate lev
els and phosphorylation of nonhistone nuclear protein in
the J774 macrophage line, similar to that described for
1418
antiimmunoglobulin
activation of rabbit B-lymphocytes
(Ref. 18 and Footnote 6). Study of macrophage lines differ
ing in expression of receptors for microbial immunostimulating agents (Chart 1) and differing in functional properties
of antibody-dependent cellular cytotoxicity to RBC and
tumor targets will aid investigations of similar diversity in
normal macrophage subsets. Defining tumor cell retention
of sensitivity to physiological controls, as in this paper or in
the inhibition of growth and cancer accompanying stimula
tion of differentiated properties in myeloblastic leukemia
cell lines by CSA (15, 19), is a continuing goal in these
studies.
ACKNOWLEDGMENT
The authors thank Sa Schrader for excellent technical assistance.
REFERENCES
1. Apte. R. N., Hertogs, C. F., and Pluznik, D. H. Regulation of Lipopolysaccharide-lnduced Granulopoiesis and Macrophage Formation by Spleen
Cells. I. Relationship between Colony-Stimulating Factor Release and
Lymphocyte Activation in Vitro. J. Immunol., 118: 1435-1440, 1977.
2. Austin, P. E., McCulloch, E. A., and Till, J. E. Characterization of the
Factor in L-Cell Conditioned Medium Capable of Stimulating Colony
Formation by Mouse Marrow Cells in Culture. J. Cellular Physiol., 77:
121-133, 1971.
•¿
Y. Nishizawa, T. Kishimoto, H. Kikutani, Y. Yamamura, and P. Ralph,
manuscript in preparation.
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Induction
3. Broxmeyer, H. E., Moore, M. A. S., and Ralph, P. Cell-Free Granulocyte
Colony Inhibiting Activity Derived from Human Polymorphonuclear
Neutrophils. Exptl. Hematology, 5: 87-102, 1977.
4. Broxmeyer, H. E., and Ralph, P. Regulation of a Mouse Myelomonocytic
Leukemia Cell Line in Culture. Cancer Res., 37: 3578-3584, 1977.
5. Carswell, E. A., Old, L. J., Kassel, R. L., Green, S.. Fiore, N., and
Williamson, B. An Endotoxin-lnduced
Serum Factor That Causes Necro
sis of Tumors. Proc. Nati. Acad. Sei. U. S., 72: 3666-3670, 1975.
6. Chervenick,
P. A., and LoBuglio,
A. F. Human Blood Monocytes:
Stimulators of Granulocyte and Mononuclear Colony Formation in Vitro.
Science, 178: 164-166, 1972.
7. Cline, M. J., and Golde, D. W. Production of Colony-Stimulating
Activity
by Human Lymphocytes. Nature, 248: 703-704, 1974.
8. Cline, M. J., Rothman, B., and Golde, D. W. Effect of Endotoxin on the
Production
of Colony-Stimulating
Factor by Human Monocytes and
Macrophages. J. Cellular Physiol., 84: 193-196, 1974.
9. Demsey, A., Kawka. D., and Stackpole, C. W. Application
of FreezeDrying Intact Cells to Studies of Murine Oncornavirus
Morphogenesis.
J. Virol., 21: 358-365, 1977.
10. Eaves, A. C., and Bruce, W. R. In Vitro Production of Colony-Stimulating
Activity. I. Exposure of Mouse Peritoneal Cells to Endotoxin. Cell Tissue
Kinet., 7: 19-30, 1974.
11. Estensen, R. D., White, J. G., and Holmes, B. Specific Degranulation of
Human Polymorphonuclear
Leukocytes. Nature, 248: 347-348, 1974.
12. Feldman, M., Boylston, A., and Hogg, N. M. Immunological
Effects of
IgT Synthesized by Thêta-Positive Cell Lines. European J. Immunol., 5.
426-431, 1975.
13. Goldman, J. M.. Th'ng, K. H., Catovsky, D., and Galton, D. A. G.
Production
of Colony-Stimulating
Factor by Leukemic Leukocytes.
Blood, 47: 381-388, 1976.
14. Gronowicz, E., Biberfeld, P., Wahren, B., and Coutinho, A. Characteri
zation of Dextran Sulfate-Sensitive
Cells. Scand. J. Immunol., 5: 573582, 1976.
15. Ichikawa, Y., Maeda, M., and Horiuchi, M. Induction of Differentiated
Functions Which Are Reversibly Suppressed by Cytochalasin B. Exptl.
Cell Res., 90: 20-30, 1975.
16. Igarashi, T., Okada, M., Kishimoto,
T., and Yamamura, Y. In Vitro
Induction of Polyclonal Killer T Cells with 2-Mercaptoethanol
and the
Essential Role of Macrophages in This Process. J. Immunol., 118, 16971703, 1977.
17. Kishimoto, T., Hirano, T., Kuritani, T., Yamamura, Y., Ralph, P.. and
Good, R. A. Induction of IgG Production in Human B Lymphoblastoid
Cell Lines with Normal Human T-Cells. Nature, 277. 756-758, 1978.
18. Kishimoto, T., Nishizawa, Y., Kikutani, H.. and Yamamura, Y. Biophasic
Effect of Cyclic AMP on IgG Production and on the Changes of NonHistone Nuclear Proteins Induced with Anti-lmmunoglobulin
and En
hancing Soluble Factor. J. Immunol., 118: 2027-2033, 1977.
19. Krystosek, A., and Sachs, L. Control of Lysozyme Induction in the
Differentiation
of Myeloid Leukemic Cells. Cell, 9: 675-684, 1976.
20. Kurland, J. I., and Bookman, R. Prostaglandin
E Production by Human
Blood Monocytes and Mouse Peritoneal Macrophages. J. Exptl. Med.,
in press, 1978.
21. Lachman, L. B., Hacker, M. P., Blyden, G. T., and Handschumacher,
R.
E. Preparation of Lymphocyte-Activating
Factorfrom Continuous Murine
Macrophage Cell Lines. Cellular Immunol., 34: 416-419, 1977.
22. McNeill, T. A. Release of Bone Marrow Colony Stimulating
Activity
during Immunological
Reactions in Vitro. Nature New Biol., 244: 175176,1973.
23. Meltzer, M. S., and Oppenheim, J. J. Bidirectional
Amplification
of
Macrophage-Lymphocyte
Interactions:
Enhanced Lymphocyte Activa
tion Factor Production by Activated Adherent Mouse Peritoneal Cells. J.
Immunol., 118: 77-82, 1977.
24. Metcalf, D., Moore, M. A. S., and Warner, N. L. Colony Formation in
Vitro by Myelomonocytic
Leukemic Cells. J. Nati. Cancer Inst.. 43: 983-
MAY
of Monocyte
and T-Lymphoma
Cell Lines
1001,1969.
25. Moore, M. A. S., Williams, N., and Metcalf, D. In Vitro Colony Formation
by Normal and Leukemic Human Hematopoietic
Cells: Interaction be
tween Colony Forming and Colony-Stimulating
Cells. J. Nati. Cancer
Inst.,50. 591-602, 1973.
26. Parker, J. W., and Metcalf, D. Production of Colony-Stimulating
Factor
in Mitogen-Stimulated
Lymphocyte Cultures. J. Immunol.. 112: 502-510,
1974.
27. Ralph, P. Retention of Lymphocyte Characteristics
by Myelomas and V
Lymphomas: Sensitivity to Cortisol and Phytohemagglutinin.
J. Immu
nol., 110: 1470-1475, 1973.
28. Ralph, P. Differential Toxicity of Con A and PHA on Murine and Human
Hematopoietic
Cell Lines. In: H. Bittiger and H. P. Schnebli (eds.),
Concanavalin A as a Tool, pp. 613-621. New York: John Wiley and Sons,
Ltd., 1976.
29. Ralph, R., Broxmeyer, H. E., and Nakoinz. I. Immunostimulators
Induce
Granulocyte/Macrophage
Colony-Stimulating
Activity and Block Prolif
eration in a Monocyte Tumor Cell Line. J. Exptl. Med.. 746. 611-616,
1977.
30. Ralph, P., Moore. M. A. S., and Nilsson. K. Lysozyme Synthesis by
Established Human and Murine Histiocytic
Lymphoma Cell Lines. J.
Exptl. Med., 743: 1528-1533, 1976.
31. Ralph, P., and Nakoinz, I. lipopolysaccharides
Inhibit Lymphosarcomas
of Bone Marrow Origin. Nature, 249: 49-51, 1974.
32. Ralph, P., and Nakoinz. I. Direct Toxic Effects of Immunopotentiators
on
Monocytic,
Myelomonocytic,
and Histiocytic
or Macrophage
Tumor
Cells in Culture. Cancer Res., 37: 546-550, 1977.
33. Ralph, P.. and Nakoinz. I. Antibody-Dependent
Killing of Erythrocyte
and Tumor Targets by Monocyte-Related
Cell Lines: Enhancement by
LPS and PPD. J. Immunol., 779: 950-954, 1977.
34. Repine, J. E., White, J. G., Clawson, C. C., and Holmes. B. M. Effects of
Phorbol Myristate Acetate on the Metabolism
and Ultrastructure
of
Neutrophils in Chronic Granulomatous
Disease. J. Clin. Invest., 54: 8390, 1974.
35. Ruscetti, F. A., and Chervenick, P. A. Release of Colony-Stimulating
Activity from Monocytes by Endotoxin and Potyinosinic-Polycytidylic
Acid. J. Lab. Clin. Med.,83: 64-72, 1974
36. Ruscetti, F. W., and Chervenick, P. A. Regulation of the Release of
Colony-Stimulating
Activity from Mitogen-Stimulated
Lymphocytes.
J.
Immunol., 774: 1513-1517. 1975.
37. Ruscetti, F. W.. and Chervenick, P. A. Release of Colony-Stimulating
Activity from Thymus-Derived Lymphocytes. J. Clin. Invest., 55: 520-526,
1975.
38. Ruscetti, F. W., Cypress, R. H., and Chervenick, P. A. Specific Release
of Neutrophilicand Eosinophilic-Stimulating
Factors from Sensitized
Lymphocytes. Blood. 47: 757-762. 1976.
39. Shevach, E. M., Stobo, J. D., and Green, I. Immunoglobulin
and HBearing Murine Leukemias and Lymphomas. J. Immunol . 708: 11461151,1972.
40. Stocker, J. W., Marchalonis, J. J., and Harris, A. W. Inhibition of a T
Cell-Dependent
Immune Response in Vitro by Thymoma Cell Immuno
globulin. J. Exptl. Med., 739: 785-790, 1974.
41. Strobo, J. D., Paul, W. E., and Henney, C. S. Allogeneic Mixed Lympho
cyte Reactivity and Cytolytic Activity as Functions of Distinct T Cell
Subsets. J. Immunol.. 770: 652-660, 1973.
42. Touraine, J.-L., Hadden, J. W.. Touraine. F., Hadden. E. M , Estensen.
R., and Good, R. A. Phorbol Myristate Acetate: A Mitogen Selective for
a T-Lymphocyte Subpopulation.
J. Exptl. Med , 745 460-465. 1977.
43. Vassalli, J.-D., Hamilton, J.. and Reich, E. Macrophage Plasminogen
Activator: Induction by Concanavalin A and Phorbol Myristate Acetate.
Cell, 77: 695-705, 1977.
44. Watson, J., and Prichard, J. Characterization
of a Factor Required for
the Differentiation
of Myeloid and Lymphoid Cells in Vitro. J. Immunol..
708: 1209-1217, 1972.
1978
Downloaded from cancerres.aacrjournals.org on June 17, 2017. © 1978 American Association for Cancer Research.
1419
Induction of Myeloid Colony-stimulating Activity in Murine
Monocyte Tumor Cell Lines by Macrophage Activators and in a
T-Cell Line by Concanavalin A
Peter Ralph, Hal E. Broxmeyer, Malcolm A. S. Moore, et al.
Cancer Res 1978;38:1414-1419.
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