1. Art and Diseño

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Macrophages Can Recognize and Kill Tumor Cells Bearing the Membrane
Isoform of Macrophage Colony-Stimulating Factor
By Martin R. Jadus, Melanie C.N. Irwin, Michael R. Irwin, Robert D. Horansky, Sant Sekhon, Karen A. Pepper,
Donald B. Kohn, and H. Terry Wepsic
NBXFO hybridoma cells produced both the membrane and
secreted isoforms of macrophage colony-stimulating factor
(M-CSF).Murine bonemarrow cells stimulated by thesecreted
Mac1+, Mae+, Ma&+, and
form of M-CSF (sM-CSF) became
F4/80+macrophages that inhibited the growth of NBXFO
cells, but not L1210 or P815 tumor cells. In cytotoxicity studies,
M-CSF activated macrophages and freshly isolated macrophages killed NBXFO cells in the presence of polymyxin B,
eliminating the possibility that contaminating lipopolysaccharide (LPS) was responsible for the delivery of the cytotoxic
signal. Retroviral-mediatedtransfection of T9 glioma cells with
the gene for the membrane isoform of M-CSF (mM-CSF), but
not for the secreted isoform of M-CSF, transferred the ability
of macrophages t o kill these transfected T9 cells in a mM-CSF
dose-dependent manner. Macrophage-mediated killing of the
mM-CSF transfected clone was blocked by using a 100-fold
excessof recombinant M-CSF.Catalase, superoxide dismutase, and the nitric oxide inhibitor, Nunitro-arginine methyl
ester (NAME), did not effect macrophage cytotoxicity against
the mM-CSF transfectant T9 clones. T9 parental cells when
cultured in thepresence of an equal number of the mM-CSF
transfectant cells were not killed, indicating specific target cell
cytotoxicity by themacrophages.Electron microscopy showed
that macrophages were capable of phagocytosizing mM-CSF
bearing T9 tumor cells and NBXFO hybridoma cells; this suggested a possible mechanism of this cytotoxicity. This study
indicates that mM-CSF provides the necessary binding and
triggering molecules through which macrophages can initiate
direct tumor cell cytotoxicity.
0 1996 by The American Society of Hematology.
M
ACROPHAGES PLAY a complex role in tumor biolisoform of M-CSF that stays attachedto the membrane (m"
ogy; their presence within a tumor can either correCSF) forextended periods of time. Transfection experiments
late with tumor destruction or tumor growth.'-4 Macrophages withthe mM-CSF gene by Stein et al" showed thatthis
because paraformaldebecome cytotoxic for tumor cells
in a two-step p r o ~ e s s . ~ . ~isoformwasafunctionalmolecule
hyde-fixed cells stimulated macrophage colonyformation
Cytokines such as interferon-y (IFN-Y),~ granulocyte-macwhen coincubated with bone marrowstemcells. The true
rophage colony-stimulating factor (GM-CSF),' tumornecrophysiologicalsignificance of this isoform isnotcurrently
sis factor (TNF),9 and macrophage
colony stimulating factor
known. Alternatively, if the mRNA is processed into either
(M-CSF also known as colony
stimulating factor-l, CSFthe 4 kbor 2.3 kbmRNA, theproteinhasretainedthe
initially primethe macrophages.Asecondarytrigproteolytic sensitive site that will be cut within the secretory
gering signal is supplied either by an antibody or lipopolyvesicle. This cut M-CSF protein will be released from the
saccharide (LPS) so that the macrophages cankill the tumor
cell when the secretoryvesicle fuses withthe membrane.
cell in a process that takes between 12 to 24 hours. Possible
This secreted form of M-CSF (sM-CSF)then stimulates cells
mediators of tumor cytotoxicity include: TNF, oncostatin-M,
in either autocrine, paracrine, or endocrine manners. Many
hydrogen peroxide, reactive oxygen intermediates, reactive
different cells including several tumor types are known to
nitrogen intermediates, direct phagocytosis or a combination
produce M-CSF" and this cytokine may be responsible for
of all the above.""'
the presence of macrophages within the tumor.
M-CSF is coded by one gene, but due to alternative splicIn preliminarystudies, we discovered that NBXFO hying routes, different forms of M-CSF are produced.'",2" The
bridoma cells (16 X 10' cells injected intraperitoneally (IP)
first 5 exons are common to
all forms of M-CSF. Within
per syngeneic mouse) did not develop tumors, even if the
exon 6, an alternative splicing segment allows a region that
micewere first immunosuppressed with high-dose cyclocodesfor a proteasesensitive protein to be deleted. This
phosphamide (300 mgkg), whichinducedpotentmacro1.6 kbmRNAis
translatedinto the
alternativelyspliced
phage
suppressor
We
previously
identified
that
NBXFOcells producedboththesecreted
and membrane
forms of M-CSF." Bone marrow cells stimulated with the
From the Department of Laborator?, Service, Veterans Affairs
Medical Center, Long Beach; the Pathology Department. Universit>,
secretedform of M-CSF (sM-CSF) became macrophages
of California, Irvine; and the Department of Bone Murrow Transthat directly killed theNBXFO cellswithout the use ofexogplantation, Children's Hospital of Los Angeles, Los Angeles, CA.
enous LPS. We tested the hypothesis that the membrane
Submitted August 22, 1995; accepted Februan 12, 1996.
isoform of M-CSF (mM-CSF) present on tumor cells could
Supported in part from grants obtained from the University qf
provide a recognition molecule for macrophages to induce
California, Irvine and the Long Beach Research b'oundation
direct tumor cytotoxicity. By using retroviral gene transfer
(M.R.J.).
technology, we provide evidence that macrophages kill tuAddress reprint requests to Martin R. Jadus, PhD, Box 113 h b
mor
cells expressing the membrane isoform of M-CSF.
Services, VeteransAffairs Medical Center, 5901 E 7th St. Long
Beach, CA 90822.
The publicationcosts of this article were defrayed in part by page
charge payment.This article muSt therefore be hereby marked
"advertisement" in accordance with 18 U.S.C. section 1734 solel! to
indicate this fact.
0 I996 by The American Society of Hematology.
0006-4971/96/8712-0014$3.00/0
5232
MATERIALS AND METHODS
Anirnrds. Male DBA/2J mice (4 to 6 weeks old) were purchased
from Jackson Labs (Bar Harbor. ME). Mice housed
in out facility
for 6 monthshave tested negativeforvariousvirusesandmycoplasma on routinescreening.Sprague-Dawley
rdtS were obtained
from either Dr A. Tarnawski or Dr S. Szabo (VAMC, Long Beach,
Blood, Vol 87, No 12 (June 15). 1996:pp 5232-5241
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MM-CSF INDUCES NOVEL TUMORICIDAL MACROPHAGES
CA) who purchased these animals from Harlan Sprague-Dawley
(San Diego, CA).
Cell lines. Mycoplasma free cells as determined using the GenProbe assay (Fisher Scientific, Tustin, CA) were grown either in
RPM-1640 media supplemented with 5% fetal bovine serum
(Hyclone, Logan, UT) or in a macrophage serum free media (endotoxin levels were undetectable; GIBCO, Grand Island, NY) for 2 to
4 days as a monolayer until confluence, when they were passaged
1:6. The conditioned media were saved and filter-sterilized through
0.22 pm filters. The NBXFO cells were obtained from Dr Beverly
Barton (Schering-Plough, Kenilworth, NJ); while the L1210 cells
were obtained from Dr Lewis Slater (Department of Pathology, Universityof California, Irvine, CA). T9 glioma cells were obtained
from M. Graf and Dr J. Hiserodt (Departments of Molecular Biology
and Pathology, University of California, Irvine, CA). The P815,
PA317, and WEHI-3 cells were purchased from the American Type
Culture Collection (ATCC, Rockville, MD).
The hybridomas producing monoclonal antibodies against murine
Mac l a (TIB 128), Mac 2 (TIB 166), Mac 3 (TIB 168), and F4180
(HB 198) were purchased from the American Type Culture Collection. The supernates derived from NBXFO and M-CSF transfected
clones were filtered through 0.22 p filters and were used at 25% to
33% concentrations to stimulate the growth of either murine macrophages or rat macrophages.
Bone marrow macrophage cultures. Bone marrow cells were
cultured in 33% M-CSF containing conditioned media for 1 week
Initial work with the
at 37°C in a humidified 5% COz atm~sphere.'~
NBXFO cells was done in RPMI-1640 media with 5% fetal bovine
serum (Hyclone) and using NBXFO supernate as the source of MCSF. The work using rat macrophages was done with macrophage
serum-free media (GIBCO) using M-CSF transfectant supernate as
the source of M-CSF. After 1 week, the media was replaced with
fresh 33% conditioned media. All culture materials were disposable
plastics and free of endotoxin. Macrophages were removed by washing off the tissue culture media, and then incubating the cells in
clinical grade imgation saline (Kendal McGaw Inc, Irvine, CA) for
30 minutes to 1 hour at4°C. The cells were scraped using a cell
scraper. This procedure results in >95% viability of the macrophages.
Construction of amphotropic retroviruses to transfer the M-CSF
isoforms. The production of amphotropic retroviruses has been previously described in detail in Nolta et al.25. The cDNA genes
encoding for functional human M-CSF both the membrane-isoform
and the secreted-isoform contained in pBR325 plasmids were obtained from Dr Carl Rettenmier (Children's Hospital of Los Angeles,
Los Angeles, CA)" after Materials Transfer Agreements were signed
with Chiron Corporation (Emoryville, CA). The M-CSF genes were
excised using Xho I and then ligated into the Xho I site of the pLXSN
shuttle vector.27 These plasmids transformed DH5a bacteria and
were selected in 50 pg/mL ampicillin. Aliquots of various plasmid
clonal isolates were then digested with various restriction enzymes
to insure that the M-CSF genes were oriented in the sense position.
Plasmids containing the proper gene orientation were then used to
transfect GPE cells via DOTAP (Boehringer-Mannhiem, Indianapolis, IN) to produce ecotropic retrovirus?' After 2 days, the supernates
from these retroviruses were used to infect PA317 cells.29 Cells
were selected in 1 mg/mL G418 (Geneticin, GIBCO) for 1 week.
Afterwards, the PA3 17 cells that were resistant to G418 were cloned.
Clones of PA3 17 producing high titers of retrovirus ( IO5 to lo6
infectious unitslml) were selected and tested for functional retroviral
activity.
Transfection of M-CSF genes intotumor cells. Rat T9 glioma
cells were infected in six-well cluster dishes (Coming, Corning,
NY). One hundred thousand exponentially growing cells were incu-
5233
bated either in the presence or absence of the supernates of the
retroviruses overnight. The cells were refed with fresh media containing 1 mg/mL G418. After 2 weeks of G418 selection, cells that
were not infected with any retrovirus died, whereas, the infected
cells continued to grow. Cells were selected based on production
of human M-CSF using the human M-CSF Quantikine kits (R&D
Systems, Minneapolis, MN). For sM-CSF production, cells were
grown at IO5 cells/mL for 3 days and then tested. For m"CSF
detection, cells were tested by flow cytometric analysis as described
below.
Antibodies and flow cyzornetty. Cells to be phenotyped were
first incubated in phosphate-buffered saline (PBS) for 10 minutes,
1 mg/mL
followed by a 5 to IO-minute incubation at37°Cwith
collagenase (Sigma Chemical, St Louis, MO). After cells detached
from the plastic, they were centrifuged and resuspended in PBS and
counted. One-half million cells in 50 pL were first incubated with
25 pL of normal rabbit serum for 5 minutes on ice to saturate all
membrane bound Fc receptors followed by an incubation with 2.5
p L of the anti-M-CSF antibody or 2.5 pL of an isotypic IgGl antibody on ice for 1 hour. Rat antimouse M-CSF (IgGI) antibody (0.1
pg/mL) was purchased from Oncogene Sciences (Manhasset, NY)."
The cells were washed once and then incubated in a 1:lO dilution
of a fluorescein isothiocyanate (F1TC)-labeled rabbit antirat antibody
(Vector Laboratories, Burlingame, CA) for an additional hour on
ice. The cells were washed three times with ice coldPBSin
a
refrigerated centrifuge. Ten thousand cells were analyzed on the
EPICS Profile. Data was collected and then analyzed on the Multi2D program (Phoenix Flow Systems, San Diego, CA).
Hybridoma cells producing monoclonal antibodies against the murine Mac-l, Mac-2, Mac-3, and F4/80 determinants were purchased
from ATCC. These cells were grown in vitro, and then the antibodies
were isolated using the affinity purification reagents available from
Sigma Chemical. Monoclonal antibodies against rat macrophage determinant ED1 was purchased from Harlan Bioproducts for Science
(Indianapolis, IN).
Cytostasis and cytotoxicity studies. Macrophages used for both
types of experiments were first treated with 100 pg/mL of mitomycin-C (Sigma Chemical CO) for 1 hour at 37°C to prevent macrophage-mediated division in these 'H-thymidine-based assays. Macrophage-mediated cytostasis experiments were performed using the
procedure of Krahenbuhl andRernington?' in 96 flatwell plates.
Here macrophages were incubated at various ratios with the tumor
cells starting at 2.1 and finishing at 0.25:l. Fifty thousand tumor
cells were plated with the macrophages in a final volume of 200 pL
of macrophage serum-free media. On the next day, 8 hours before
harvesting the cells, the individual wells were pulsed with 1 pCi of
'H-thymidine ('H-TdR; New England Nuclear, NET-O27A, 74 GBq/
m o l ) in a volume of 25 pL. Immediately before the cultures were
harvested, cultures were viewedunderan inverted microscope to
confirm whether tumor cells were present or absent under the various
experimental conditions. Cells were then aspirated through a glass
wool fiber filter with a multiple sample harvester (PhD Harvester,
Cambridge, MA), and total 'H-TdR incorporation was determined
by liquid scintillation procedures using Bio-Safe I1 (Research Products Int, Mount Prospect, IL). Data are expressed as the mean counts
per minute (CPM) 2 standard deviation (SD) per triplicate culture.
Visual observations from each experiment confirmed the cytostasis
results.
Macrophage mediated cytotoxicity studies were performed according to the method of Meltzer.'' Target tumor cells were labeled
with 4 pCi of 'H-TdR overnight in the media. The next morning,
tissue culture media was replaced with fresh media and allowed to
incubate a further 1 to 3 hours to reduce spontaneous release by the
tumor cells. Ten thousand target cells were incubated in 200 pL
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JADUS ET AL
5234
of macrophage serum-free media overnight with graded doses of
macrophages ranging from IO: 1 to 0.75:I at 37°C in a humidified 5%
CO2 incubator. Immediately before the supernates were harvested,
cultures were viewed under an inverted microscope to confirm
whether tumor cells were present or absent under the various experimental conditions. Afterwards, 100 pL of supernate was removed
and placed into 2 mL of scintillation fluid. Spontaneous release after
24 hours was about 10% of maximum release. Maximum release is
calculated by taking IO4 target cells and freeze-thawing them three
times in liquid nitrogen. Specific release is calculated using the
standard equation for cytotoxicity reactions.'2,'3 Visual observations
from each experiment confirmed the cytotoxicity results. Cytotoxicity data from multiple experiments were pooled together at each
macrophage: tumor cell ratio and is then presented as the mean 2
standard of the error of the means. Cytotoxicity is not considered
specific release.
relevant if values are ~ 1 0 %
Data from the cytostasis and cytotoxicity assays were analyzed
using Student's t tests on the Sigma Plot Version 5.0(Jandel Scientific, San Rafael, CA) computer program. Values were considered
significantly different at the P < 0.05 levels.
Clinical grade Cetus M-CSF (activity: 6.94 X IO' unitslmg; endotoxin content <0.08 EUlmL) was kindly provided by Chiron Corporation (Emoryville, CA).
Electronmicroscopicstudies.
Cells were gently scraped from
monolayer cultures and then centrifuged (1.OOOg) in a 15-mL centrifuge tube for 10 minutes. The cells were then prepared the same
The grids were examined
way as described in detail in Jadus et al.24.32
with a Joel Electron microscope (Peabody, MA).
RESULTS
M-CSF-activated bone marrow-derived macrophages inhibit the growth of NBXFO cells. NBXFO cells expressed
the membrane isoform of M-CSF (mM-CSF) in Fig 1; the
supernate from these cells supported the growth of murine
bone marrow macrophages. These M-CSF stimulated cells
are >90% positive for Macl, Mac2, Mac3, and F4/80antigens, consistent with a macrophage phenotype. When these
macrophages were cocultured with various tumor cells in a
cytostasis experiment, the NBXFO cells were significantly
(P < .05)inhibited in their growth at2:1 to 0.5:l macrophage: tumor ratios (Fig 2). These same macrophages failed
to inhibit the growth of L1210 and P815 tumor cells ( P >
0
52
04
W
l26
l60
l92
224
ZOO
L
o
g
-
Fig 1. NBXFO cellspossess mM-CSF. NBXFOcells were stained
with either an lgGl isotypic antibody or a monoclonal anti-M-CSF
antibody (IgG1). Ten thousand cells were analyzed on an EPICS profile. The isotypic controls were subtracted from the M-CSF fluorescent values usingthe Multi-2D computer program and isrepresented
by the shaded area. The computed positive cells are labeled 97%
positive in upper right corner.
350000
I
t
I
f
300000
I
250000
200000
150000
4
3
400000
300000
a
I 200000
4
I
I
I
0
"
400000
300000
200000
100000
C9"4
t
I
l
I
I
Control 2:l
I
P8 15
I
I
1
1:1 0.6:l
0.26:l
Yacrophage:tumor ratio
Fig 2. Macrophages inhibit the growth ofNBXFOcells. Murine
bone marrow-derived cells were cultured in M-CSF containing media
for the first week. These cells were refed with a change of fresh
media containing M-CSF.These cellswere then incubatedfor another
week. These macrophages were cultured concurrently with either
NBXFOcells (top panel), L1210cells (middle panel) orP815cells
(bottom panel). Cells were pulsed with H3-TdR on the next day for
the last 8 hours of the incubation.Data is presentedas countslminute
(CPM) Hl-TdR incorporated ? standard deviation of triplicate cultures.
.05)when assayed concurrently. Similar results were found
in a repeated experiment. These studies suggested that macrophages specifically inhibited the growth of NBXFO cells.
Bone marrow-derived macrophages kill NBXFO cells, but
not NIH 3T3-transfected cells with mM-CSF. Figure 3
demonstrated that the bone marrow derived macrophages
killed NBXFO cells in cytotoxicity assays. We used 30 pg/
mL polymyxin-B tobindany
endotoxin that could have
contaminated the media or the cells. This data was pooled
together at each effector:target ratio from eight independent
assays. M-CSF-activated macrophages from two separate experiments didnot kill NIH 3T3 cells transfected with the
human mM-CSF gene. Byflow cytometric analyses, these
mM-CSF-transfected 3T3 cells were >90% for mM-CSF.
This indicates that onlytumor cells with themM-CSF phenotype are killed by these macrophages.
Freshly isolated adherent cells also kill NBXFO cells.
We took adherent cells obtained from murine bone marrow
and spleen and assayed them to determine if freshly isolated
macrophages could kill NBXFO cells. Table 1 shows that
these adherent cells lysed the NBXFO cells after 24 hours.
Thus, freshly isolated macrophages without prior in vitro
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5235
MM-CSF INDUCES NOVEL TUMORICIDAL MACROPHAGES
50 r
40
Lt
1
50
-
20
.
NBXFO
v 3T3 mM-CSF
0
&\T
lot
I
0
-
1O:l
6:l
%.5:1 1.8:l 0.6:l .3:1
Macrophage:tumor ratio
Fig 3. Macrophages kill NBXFOcells. Murine bone marrow-derived cells were cultured in M-CSF containing media for the first
week. These cellswere refed with a changeof fresh media containing
M-CSF for another week. These macrophageswere cultured with H
'TdR labeled NBXFO cells in the presence of 30 pglmL polymyxin B.
After 1 day, the wpernates were harvested. Data is presented as
percent specific release C standard arror of means from eight separate experiments. Data is also presented from two separate experiments where macrophages were tested against NIH 3T3 cells
transfected with mM-CSF. Data is represented as percent specific
release ? standard deviation.
exposure to M-CSF are capable of killing these target cells.
Thymocytes did not kill the NBXFO cells, eliminating the
trivial possibility that physical overcrowding was responsible for the death of the NBXFO cells.
Transfected tumor cells displaying mM-CSF are killed
by M-CSF activated macrophages. The previous studies
suggested that macrophages killed NBXFO cells by recognizing the mM-CSF found on NBXFO cells. Because
NBXFO cells are hybridoma cells and may have other molecules that could provide macrophages with other ligands for
binding, we performed more definitive experiments using
T9 glioma cells expressing m"CSF.
Rat T9 glioma cells were infected with retroviruses constructed to transfect the genes for either the membrane isoform or the secreted isoform of human M-CSF (sM-CSF).
Transfected cells were selected in G418 for 2 weeks and
subsequently cloned. Figure 4 shows the cell surface m"
CSF phenotype of three randomly chosen T9 mM-CSF
transfectant clones (C2, F2, F6) along with 1 sM-CSF
transfected clone (Hl, by culturing 10' cells/mL for 3 days
these cells produced >2,000 pg/mL). Byflow cytometry,
the T9 parental cells and the H1 clone showed negligible
mM-CSF positivity, 0.29% and 0.46%, respectively. The
m"CSF transfectant clones all expressed m"CSF, but to
variable amounts. The C2 clone was the most fluorescent,
while the F6 clone was the least fluorescent.
Rat bone marrow derived macrophages (>90% ED1 i)
were tested for their ability to kill these transfected T9 cells
inFig S. The macrophages killed the mM-CSF infected
clones in a m"CSF concentration-dependent manner. The
C2 clone was killed the best, followed by the F2 clone. The
F6 clone was not killed any better than were H1 or the T9
parental cells. Thus, there is a required threshold amount of
mM-CSF present on the target cell before the macrophages
will kill that target cell. We selected the C2 and H1 clones
for all further work. The macrophages have reproducibly
killed only the C2 cells and not H1 or the parental cells.
We modified the M-CSF enzyme-linked immunosorbent
assay (ELISA) assay to quantitate the amount of mM-CSF
present on C2 cells (Jadus et al, manuscript submitted) We
found that 10,000 C2 cells express 1,002 pgof mM-CSF,
while an equivalent number ofH1 clones or parental T9
cells were negative for m"CSF. We used a 100-fold excess
of M-CSF (100,000 pp) to completely block macrophage
cytotoxicity against the C2 clones as shown in Table 2. This
experiment has been successfully reproduced one more time.
The mechanism of cytotoxicity displayed by the macrophages against the mM-CSF transfectant C2 clone does not
involve a soluble factor, but may include phagocytosis. Tumoricidal macrophages can kill through the production of
short-lived soluble factors such as superoxide radicals, hydrogen peroxide, and nitric oxide. We tested whether inhibitors of these cytotoxins could prevent macrophage-mediated
killing of the C2 clones. Figure 6 shows that SO U/mL of
catalase, 20 U/mL superoxide dismutase, and 20 pmoVL
NAME failed to prevent macrophage-mediated killing of the
C2 clones (upper panel). All experimental results at each
macrophage:tumor ratio were not significantlydifferent ( P >
.OS) from the untreated control cells. None of these reagents
affected macrophage cytotoxicity of the parental T9 cells or
H1 clones (lower two panels).
To eliminate the possibility that other unknown soluble
cytotoxins are responsible for this macrophage-mediated cytotoxicity against the m"CSF clones, we performed mixing
experiments. Here labeled T9 or H1 clones were mixed with
an equal number of unlabeled m"CSF C2 cells in the presence of the bone marrow-derived macrophages. In Table 3,
the macrophages did kill the m"CSF C2 clone, butthe
macrophages did notkill the labeled parental T9 or H1 clone,
either alone or in the presence of the unlabeled C2 clone.
This study eliminates that any soluble cytotoxic factor such
Table 1. Freshly Isolated Adherent Splenocytes and Bone Marrow
Cells Can Lyse NBXFO Cells
% Specific Release 2
Effector:Target Ratio
27.5 1O:l
5: 1
2.51
SD*
Bone Marrowt
Spleent
Thymus*
2 0.7
11.0 5 4.2
5.0 2 0.0
23.0 c 2.8
22.5 2 4.9
14.5 2 0.5
2.8 2 2.0
4.9 2 1.0
4.9 2 0.0
* Macrophage mediated cytotoxicity was measured after a 24-hour
incubation in the presence of 20 pg/mL polymyxin B.
t Freshly isolated mouse adherent spleen and bone marrow cells
were isolated.
Freshly isolated mouse thymocytes were used.
*
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JADUS ET AL
5236
280
240
200
160
120
80
40
0
200
o T9 Parental
sM-CSF H 1
v mM-CSF F6
0
mM-CSF F2
v mM-CSF C2
I
Y\
C2 96.11%
Po8
1
T
160
120
1O:l
2.5:l 1.2:l
Macrophage:TS ratio
80
40
0
F2 91.87% Pos
200
160
5:l
Fig 5. Macrophages kill cloned mM-CSF transfectant T9 glioma
cells. Rat bone marrow-derived cells were cultured in M-CSF containing media for the first week. The cells were refed with fresh
media containing M-CSF for another week. These M-CSF activated
macrophages were cultured with H3-TdR labeled target cells in the
presence of 30 pg/mL polymyxinB.The target cells included parental
T9, one sM-CSF transfectant clone: H1; three randomly picked mMCSF transfected clones: C2, F2, F6. After 1 day, the supernates were
harvested. Data is presented as percent specific release 2 standard
deviation of triplicate cultures.
120
80
40
0
F6 63.42% POS
200
160
as TNF, oncostatin-M, or interferon could be released by
the cytotoxic macrophages.
This last experiment suggested that cell-to-cell contact
was required for this specific cytotoxicity. Weperformed
electron microscopy to determine whether phagocytosis
could be responsible for this cytotoxicity. Figure 7A shows
a typical T9 cell, while Fig 7B shows a typical rat bone
marrow-derived macrophage cultured in macrophage serumfree media in the presence of M-CSF derived form HI cells
120
Table 2. A 100-fold Excess of Recombinant M-CSF Will Prevent
Bone Marrow Macrophages From Killing themM-CSF
Transfectant C2 Clone
80
40
% Specific Release 2 SD'
-
0
32
64
96
LOG
128 160 192
R 1
EffectocTarget Ratio
Without M-CSF
2O:l
25 2 3
2 4
18 z 9
With M-CSFt
l21
23
1O:l
120
Fig 4. mM-CSF flow cytometric profile of cloned transfected T9
5:1
220
glioma cells. Various cloned transfectant T9 glioma cells IH1: sM8+1
620
2.5:l
CSF; C2, F2, F 6 mM-CSF) were incubatedwith either an lgGl isotypic
c Macrophage mediated cytotoxicitywas measured after a 24-hour
or an anti-M-CSF antibody. The surface fluorescence of 10,000 cells
were collected. The isotypic controls were subtracted from the anti- incubation in the presence of 20 pglmL polymyxin B.
M-CSF fluorescence values and are labeled percent positivein upper
t Ten thousand C2 cells possess 1,002 pg mM-CSF; recombinant
right corner of each graph.
M-CSF (100.000 pg) was added to achieve a 100-fold excess.
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5237
MM-CSF INDUCES NOVEL TUMORICIDAL MACROPHAGES
DISCUSSION
:i
I
c2
x0
..
.rl
0
a
,
a
l K1
1O:l 5:l 2.5:l 1.2:l .6:1
Macrophage:Tumor ratio
Fig 6. Macrophage-mediated killing of mM-CSF
transfected
clones are unaffectedby catalase, superoxide dismutase, and
NAME.
Rat M-CSF activated macrophages grown in the presence of M-CSF
for 2 weeks were cocultured with either H'-TdR-labeled parentalTS,
mM-CSF transfected C2 clone, or sM-CSF T9 H1 clone for 1 day in
the presenceof 20 pmol/L NAME, 50 UlmL catalase or 20 UlmL
superoxidedismutase.Data is presented as percent specific release
k standard deviation of triplicate cultures.
for 2 weeks. The macrophage contained numerous granules.
Additionally, these cells possessed several lipid droplets.
These fat droplets provided uswitha
fortunate internal
marker for the macrophages so we can positively identify
the macrophage when we mixed these cells with the T9
tumor cells. Figure 7C shows a macrophage that was incubated with the m"CSF transfectant cells after 24 hours.
Here a macrophage has ingested several m " C S F transfected T9 tumor cells (labeled T). The macrophage is identified because the macrophage is the bigger cell, and the lipid
droplets are present. These lipid droplets have coalesced into
bigger ones. The numerous granules found in the normal
macrophage have disappeared in these phagocytic macrophages. Presumably these granules fused with the m " C S F
bearing tumor cells when they formed the phagolysosome.
To determine whether phagocytosis could be responsible
for the cytotoxicity observed against NBXFO cells. We also
performed electron microscopy with the murine macrophages and NBXFO cells. Again, we found macrophages
that ingested NBXFO cells, one such cell is shown in Fig
8. These murine macrophages were previously cultured in
complete RPMI-I640 media along with the NBXFO supernate and did not display any fat droplets as seen in the
rat macrophages when cultured in the macrophage serumfree media. This study illustrates that the presence of fat
droplets within the-macrophages are not necessary for macrophage cytotoxicity.
In tumor biology, macrophages may be considered a
''double edged sword". Macrophages frequently associate
within breast and ovarian tumors in response to M-CSF produced by these tumor^?^-^' Macrophages may induce tumor
growth by releasing stimulatory or angiogenic factors or by
acting as immunosuppressor cells?943 Other studies have
concluded that macrophages were beneficial
for the host.449
In several cytokine transfection models, macrophages were
one effector cell when the tumor cells were expressing interleukin-2 (L-2):'
L-4:' L-6:'
IL-7,53
and
TNF?5 For macrophages to become tumoricidal in vitro, they
must be stimulated in two ways?*6First, cytokines prime the
macrophages, while secondary signals allow the macrophage
to kill the tumor cell. It is tempting to speculate that this
"double edged sword" effect could be explained by the twosignal model. When macrophages only receive the priming
signal, these macrophages promote tumor growth and metastases. Whereas, when both signals are received the macrophages mediated tumor regression. One possible mechanism
to tip the balance toward a favorable prognostic response is
to devise a molecule that delivers to the macrophage both
cytotoxic delivery signals simultaneously. In studies presented here, macrophage cytotoxicity against tumor cells
may be accomplished by asingle molecule, namely the membrane isoform of macrophage colony stimulating factor.
In this report, we found that M-CSF-activated macrophages inhibited the growth of m"CSF expressing NBXFO
cells (Fig 2) and killed NBXFO cells in an endotoxin-free
environment (Fig 3). NBXFO cells are hybridoma cells and
may possess other cell surface molecules that may induce
immune responsiveness as shown by Guo et al.56 Wecreated
retroviral vectors to transfer the m"CSF gene into a defined
Table 3. Macrophages Will Not Kill T9 or the S"CSF Clone, H1, in
the Presence of the mM-CSF Clone, C2
% Specific Release ? SD'
Addition of Unlabeled Targett
Macrophage:Target
Ratio
None
T9
H1
c2
Labeled T9 target
1O:l
51
2.51
Labeled H1 target
1O:l
5: 1
251
Labeled C2 target
1O:l
1222
1020
6 2 3
1252
1323
8 2 3
51
34 2 3
28 5 2
251
22 -c 2
* Macrophage
922
10t6
9 2 3
4 2 3
4 2 2
1151
at2
124
5 t 2
953
822
7+2
1025
1356
7 2 1
6 2 1
at2
4 t 3
mediatedcytotoxicity was measured after a 24-hour
incubation in the presence of 20 yglmL polymyxin B.
t Labeled target cells were cultured in the presence of an equal
number of unlabeled target cells throughout the course of this experiment.
From www.bloodjournal.org by guest on February 6, 2015. For personal use only.
5238
JADUS ET AL
.
tumor cell to prove our hypothesis that macrophages can kill
mM-CSF bearing tumor cells.
Rat T9 glioma clones transfected with the mM-CSF retrovirus, did express mM-CSF (Fig 4) and were killed by MCSF activated macrophages in a dose-dependent juxtacrine
manner (Fig 5). Parental T9 cells and sM-CSF transfected
HI clones were notkilledby
these macrophages. This
macrophage-mediated cytotoxicity against the m"CSF
transfectant was prevented by using a 100-fold excess of
recombinant M-CSF (Table 2). NIH 3T3 fibroblasts expressing mM-CSF were not killed bymacrophages (Fig 3). Membrane M-CSF by itself is insufficient to allow macrophages
to kill nontransformed cells bearing this molecule. Therefore,
another tumor specific molecule allows the macrophages to
distinguish the tumor cell from a normal cell. This finding
Fig 7. Rat macrophages can phagacytosize m"CSF T9 transfecx magnification. (B1Shows
a typical rat macrophage cultured in macrophage serum-free media
for 2 w w k s at 1,500 x magnification. Arrows point to lipid droplets.
(C) Shows a rat macrophage that has ingested several tumor cells
(labeled TI at 1,000 x magnification. Arrows point to coalesced lipid
droplets. All cells were cultured 24 hours at 37°C before being processed for electron microscopy.
tants. (AI Shows a T9 glioma cell at 2,000
is consistent with previous work5' showing that macrophages
only kill transformed tumor cells and not rapidly growing
fibroblasts.
Most M-CSF producing cells only make the secreted form
of M-CSF. When sM-CSF transfected myeloma cells grew
as a tumor, macrophages were found within the tumor bed.5R
This work showed that M-CSF acted as a strong chemoattractant for macrophages, but probably did not induce any
direct tumoricidal activity, perhaps by not allowing the macrophage to physically contact the tumor cell. When we injected NBXFO cells IP into mice (up to 16 million cells/
syngeneic mouse), no tumors ever developed, even in mice
firsttreatedwith
high-dose cyclophosphamide, which induced potent macrophage suppressor cells.22.23
Freshly isolated adherent cells did killmM-CSF positive T9 cells (Table
From www.bloodjournal.org by guest on February 6, 2015. For personal use only.
5239
MM-CSFINDUCESNOVELTUMORICIDALMACROPHAGES
2. Fidler IJ, Barnes Z, Fogler WE, Kirsch R, Bugelski P, Poste G:
Involvement of macrophages in eradication of established metastases
following intravenous injection of liposomes containing macrophage
activators. Cancer Res 42:496, 1982
3. Mantovani A, Bottazzi B, Colotta F, Sozzani S, Ruco L The
origin and function of tumor-associated macrophages. Immunol Today 13:265,1992
4. KleinE, Mantovani A: Actionofnatural
killer andmacrophages in cancer. Cum Opin Immunol 5:714, 1993
5. Russell SW, Doe WF, McIntosh AT: Functional characterization of a stable nonlytic stage of macrophage activation in tumors.
J Exp Med 146:1511, 1977
6. Adam DO, Marino PA: Evidence for a multistep mechanism
of cytolysis by BCG activated macrophages: The interrelationship
between the capacity for cytolysis, target binding and secretion of
cytolytic factor. J Immunol 126:981, 1981
7. Pace JL, Russell SW, Torres BA, Johnson HM, Gray PW:
Recombinant mouse interferon induces the priming step in macrophage activation for tumor cell killing. J Immunol 130201 1, 1983
Fig 8. Mouse macrophagehas phagocytosized 1 NBXFO cell. Mu8. Young DA, Lowe LD, Clark SC: Comparison of the effects
rine bone marrow-derived macrophages were cultured with NBXFO
of IL3, granulocyte-macrophage colony stimulating factor and maccells for7 hours at 37°C before being processing for electron microsrophage colony stimulating factor in supporting monocyte differenticopy. Magnification is 3,000 x . Macrophage is labeled M, while the
ation in culture. J Immunol 145:607, 1990
killed NBXFO hybridoma is labeled H.
9. Hori K, Ehrke MJ, Mace K. Maccubbin D, Doyle MJ, Otsuka
Y, Mihich E: Effect of recombinant human tumor necrosis factor
on the induction of murine macrophage tumoricidal activity. Cancer
1). Thus, killing of mM-CSF tumor cells is not just restricted
Res 41:2793, 1987
to M-CSF activated macrophages. If this is true in vivo, the
IO. Mufson RA, Aghajanian J, Wong G, Woodhouse C, Morgan
injection of the mM-CSF retrovirus directly into
a tumor
with a high macrophage content may induce the endogenous AC: Macrophage colony stimulating factor enhances monocyte and
macrophage antibody dependent cell mediated cytotoxicity. Cell Immacrophages to kill those infected tumor cells and perhaps
munol 119:182,1989
reduce the tumor burden. Another therapeutic approach may
1 I . Wing W,Ampel NM, Waheed A,Shadduck RK: Macrophage
involve allowing the mM-CSF transfected tumor cell to be
colony-stimulating factor (M-CSF) enhances the capacity of murine
killed by the macrophages in vivo and then allowing these
macrophages to secrete oxygen reduction products. J lmmunol
macrophages to actas antigen presenting cells and then stim- 135:2052, 1985
12. Sum S, Yokota H, Yamada M,Yanai N. Saito M, Kawashima
ulating systemic immune responses. The exact physiological
T, Saito M. Takaku F, Motoyoshi K: Enhancing effect ofhuman
role of mM-CSF is unknown, but we believe this unique
molecule may representa novel way of targeting tumor cells monocytic colony stimulating factor on monocyte tumoricidal activity. Cancer Res 49:5013, 1989
to macrophages to stimulate an immune response.
13. Feinman R, Henriksen-De Stephano D, Tsujimoto M, Vilcek
In summary, we have found that M-CSF activated macroJ: Tumor necrosis factor is an important mediator of tumour cell
phages inhibited the growth and killed NBXFO hybridoma
killing by human monocytes. J lmmunol 138:635, 1987
cells that express the membrane isoform of macrophage col- 14. MunnDH, Cheung NKV: Phagocytosis of tumor cells by
ony-stimulating factor. This cytotoxic activity was mediated human macrophages cultured in recombinant macrophage colony
bybothM-CSF-activatedbonemarrow-derivedmacrostimulating factor. J Exp Med 172:231, 1990
phages and freshly isolated macrophages. When T9 glioma
15. Green SJ, Chen TY, Crawford RM, Nacy CA, Morrison D C ,
cells were transfected with retroviruses containing the gene
Meltzer MS: Cytotoxic activity and production of toxic nitrogen
oxides by macrophages treated with IFN-y and monoclonal antibodfor mM-CSF, macrophages killed thoseT9 glioma cells that
ies against the 73-kda lipopolysaccharide receptor. J lmmunol
only expressed the mM-CSF. This killing was inhibited by
using a 100-fold excess of recombinant M-CSF. One putative 149:2069,1992
16. Martin JHJ, Edwards SW: Changes in mechanisms of monomechanism of macrophage mediated killing may include dicytelmacrophage-mediatedcytotoxicity during culture. J Immunol
rect phagocytosis of the mM-CSF tumor cells, because no
150:3478,1993
evidence that a soluble cytotoxic mediator was found.
17. Drapier JC, Hibbs JB: Differentiation of murine macrophages
We thank Dr D. Forrest for excising the M-CSF genes from the
bacterial plasmids. We thank Dr Stanley Rubin for his help in interpreting the data. We also thank Nora Tang for doing the electron
microscopy preparatory work.
to express nonspecific cytotoxicity for tumor cells results in L-arginine dependent inhibition of mitochondrial iron-sulfur enzymes in
the macrophage effector cells. J Immunol 1402829, 1988
18. Zarling JM, Shoyab M, Marquardt H, Hanson MB, Lioubin
MN, Todaro GJ: Oncostatin M: A growth regulator produced by
differentiated histiocytic lymphoma cells. Proc Natl Acad Sci USA
REFERENCES
83:9739, 1986
19. Cerreti DP, Wignall J, Anderson D: Human macrophage col-
1. Fidler IJ: Macrophages and metastasis- a biological approach
to cancer therapy: Presidential address. Cancer Res 45:4714, 1985
ony stimulating factor: Alternative RNA and proteinprocessing from
a single gene. Mol Immunol 25:277, 1988
ACKNOWLEDGMENT
.
From www.bloodjournal.org by guest on February 6, 2015. For personal use only.
5240
20. Stanley ER: Colony stimulating factor-l, in Aggrawal BB,
Gutterman GU (eds): Human cytokines. Boston, MA, Blackwell
Scientific, 1991, p 196
21. Stein J, Borzillo GV, Rettenmier CW: Direct stimulation of
cells expressing receptors for macrophage colony-stimulating factor
(CSF-l) by plasma membrane-bound precursor ofhuman CSF-l.
Blood 76:1308, 1990
22. Nikcevich DA, Young MR, Ellis NK, Newby M, Wepsic HT:
Stimulation of hematopoiesis in untreated and cyclophosphamide
treated mice by the inhibition of prostaglandin synthesis. J Immunopharmacology 8:299, 1986
23. Nikcevich DA, Duffie GP, Young MR, Ellis NK, Kaufman
GE, Wepsic HT: Stimulation of suppressor cells in the bone marrow
and spleens of high dose cyclophosphamide-treated C57BL/6 mice.
Cell Immunol 109:349, 1986
24. Jadus MR, IrwinMR, Barton BE, Irwin MN, Wepsic HT:
The identification of the neonatal NBXFO hybridoma cell and its
mediator. Dev Comp Immunol 19:261, 1995
25. Nolta JA, Hanle MB, Kohn DB: Sustained human hematopoiesis in immunodeficient mice by cotransplantation of marrow stroma
expressing human interleukin-3: Analysis of gene transduction of
long lived progenitors. Blood 83:3041, 1994
26. Nolta JA, Yu XJ, Bahner I, Kohn DB: Retroviral mediated
transfer of the human glucocerebrosidase gene into cultured Gaucher
bone marrow. J Clin Invest 90:343, 1992
27. Miller AD, Rosman GJ: Improved retroviral vectors for gene
transfer and expression. Biotechniques 7:980, 1989
28. Markowitz D, Goff S, Bank A: A safe packaging line for
gene transfer: Separating viral genes on two different plasmids. J
Virol 62:1120, 1988
29. Miller AD, Buttimore C: Redesign of retrovirus packaging
cell lines to avoid recombination leading to helper virus production.
Mol Cell Biol 6:2895, 1986
30. Krahenbuhl JL, Remington JS: The role of activated macrophages in specific and non-specific cytostasis of tumor cells. J Immuno1 113:507, 1983
3 I . Meltzer MS: Macrophage activation-quantitation of cytotoxicity by 'H-thymidine release, in Herscowitz HB, Holden HT, Bellanti JA, Ghaffar (eds): Manual of Macrophage Methodology. New
York, NY, Marcel Dekker, 1981, p 329
32. Jadus MR, Schmunk G, Djeu J, Parkman R: Morphology and
lytic mechanism of interleukin-3 dependent natural cytotoxic cells:
Tumor necrosis factor as a possible mediator. J Immunol 137:2774,
1986
33. Jadus MR, Good RW, Crumpacker DB, Yannelli JR: The
effects of interleukin 4 upon human tumoricidal cells obtained from
patients bearing solid tumors. J Leukoc Biol 49:139, 1991
34. Ramakrishnan S, Xu FJ, Brandt J, Niedel JE, Bast RC, Brown
EL: Constitutive production of macrophage colony stimulating factor
by human ovarian and breast cancer cell lines. J Clin Invest 83:921,
1989
35. Kacinski BM: CSF-l and its receptor in ovarian, endometrial
and breast cancer. Ann Med 27:79, 1995
36. Chambers SK, Wang Y, Gertz RE, Kacinski BM: Macrophage
colony stimulating factor mediates invasion of ovarian cancer cells
through urokinase. Cancer Res 55:1578, 1995
37. Scholl SM, Pallud C, Beuvon F, Hacene K, Stanley ER,
Rohrschneider L, Tang R, Pouillart P, Lidereau R: Anti-colony stimulating factor- 1 antibody staining in primary breast adenocarcinomas
correlated with marked inflammatory cell infiltrates and prognosis.
J Natl Cancer Inst 86:120, 1994
38. Kacinski BM, Scata KA, Carter D, Yee LD, Sapi E, King
BL, Chambers SK, Jones MA, Pirro MH, Stanley ER, Rohrschneider
LR: FMS (CSF-l receptor) and CSF-I transcripts andprotein are
JADUS ET AL
expressed by human breast carcinoma in vivo and in v i m . Oncogene
6:941, 1991
39. Bottazzi B, Erba E, Nobili N, Fazioli F, Rambaldi A, Mantovani A: A paracrine circuit in the regulation of the proliferation
of macrophages infiltrating murine sarcomas. J Immunol 144:2409,
1990
40. Erroi A, Sironi M, Chiaffarino F, Zhen-Guo C, Mengozzi M,
Mantovani A: ILl and IL6 release by tumor associated macrophages
from human ovarian carcinoma. Int J Cancer 44:795, 1989
41. Brocker EB, Zwadlo G, Suter L, Brune M, Sorg C: Infiltration
of primary and metastatic melanomas with macrophages of the 25F9positive phenotype. Cancer Immunol Immunother 25:81, 1987
42. Mussoni L, Riganti M, Acero R, Erroi A, ConfortiG , Mantovani A, Donati MB: Macrophages associated with murine tumours express plasminogen activatoractivity.
Int J Cancer
41:227,1988
43. Jadus MR, Sekhon S, Barton BE, Wepsic HT: Macrophage
colony stimulatingfactoractivatedbone
marrow macrophages
suppress lymphocytic responses through phagocytosis: A tentative in vitro model of Rosai-Dorfman Disease. J Leukoc Biol
57:936, 1995
44. Allen C, Hogg N: Association of colorectal tumor epithelium
expressing HLA-D/DR with CD8 positive T cells and mononuclear
phagocytes. Cancer Res 47:2919, 1987
45. Gottlinger HG, Rieber P, Gokel JM, Lohe KJ, Riethmuller
G: Infiltrating mononuclear cells in human breast carcinoma: Predominance of T4+ mononuclear cells inthe tumor stroma. Int J
Cancer 35: 199, 1985
46. Brocker EB, Zwadlo G, Holzmann B, Macher E, Sorg C:
Inflammatory cell infiltrates in human melanoma at different stages
of tumor progression. Int J Cancer 41:562, 1988
47. Hibbs JB, Lambert LH, Remington JS: Possible role of macrophage mediated nonspecific cytotoxicity in tumor resistance. Nature 235:48, 1972
48. Fidler IJ, Barnes 2, Fogler WE, Kirsch R, Bugelski P, Poste
G: Involvement of macrophages in eradication of established metastases following intravenous injection of liposomes containing macrophage activators. Cancer Res 42:496, 1982
49. Hume DA, Donahue RE, Fidler IJ: The therapeutic effect of
human recombinant macrophage colony stimulating factor (CSF-l)
in experimental murine metastatic melanoma. Lymphokine Cytokine
Res 8:69, 1989
50. Cavallo F, Giovarello M, Gulino A, Vacca A, Stoppacciaro
A, Modesti A, Forni G: Role of neutrophils and CD4+ T lymphocytes in the primary and memory response to nonimmunogenic murine mammary adenocarcinoma made immunogenic by the L 2 gene.
J Immunol 149:3627, 1992
51. Pericle F, Giovarello M, Gulino A, Colombo M, Ferrari G,
Musiani P, Modesti A, Cavallo F, Forni G: An efficient Th2-type
memory follows CD8+ lymphocyte driven and eosinophil mediated
rejection of a spontaneous mouse mammary adenocarcinoma engineered to release IL4. J Immunol 153:5659, 1994
52. Allione A, Consalvo M, Nanni P, Lollini PL, Cavallo F,
Giovarelli M, Forni M, Gulno A, Colombo MP, Dellabona P, Hock
H, Blankenstein T, Rosenthal FM, Gansbacher B, Bosco MC, Muss0
T, Gusella L, Forni G: Immunizing and curative potential of replicating andnonreplicating murine mammary adenocarcinoma cells engineered with interleukin-2, IL4, IL6, IL7, ILIO, GM-CSF, and y-IFN
gene or admixed with conventional adjuvants. Cancer Res 54:6022,
1994
53. Hock H, Dorsch M, Diamantstein T, Blankenstein T: Interleukin-7 induces CD4+ T cell-dependent tumor rejection. J Exp Med
174:1291, 1991
54. Lollini PL, Bosco MC, Cavallo F, De Giovanni C, Giovarelli
From www.bloodjournal.org by guest on February 6, 2015. For personal use only.
MM-CSFINDUCESNOVELTUMORICIDALMACROPHAGES
M, Landuzzi L, Musiani P, Modesti A, Nicoletti G, Palmieri G,
Santoni A, Young HA, Forni G , Nanni P: Inhibition of tumor growth
and enhancement of metastasis after transfection of the y-interferon
gene. Int J Cancer 55:320, 1993
55. Blankenstein T, Qin Z, Uberla K, Muller W, Rosen H,
Volk HD, Diamantstein T: Tumor suppression after tumor celltargetedtumornecrosisfactor
(Y gene transfer.
J Exp Med
173:1047, 1991
56. Guo Y, Wu M, Chen H,Wang X, Liu G , Li G , Ma J, Sy
5241
MS: Effective tumor vaccine generated by fusion of hepatoma cells
with activated B cells. Science 263:518, 1994
57. Fidler U,Schroit AJ: Recognition and destruction of neoplastic cells by activated macrophages: Discrimination of altered self.
Biochim Biophys Acta 948:151, 1988
58. Dorsch M, Hock H,Kunzendorf U, Diamantstein T, Blackenstein T: Macrophage colony stimulating factor gene transfer into
tumor cells induces macrophage infiltration but not tumor suppression. Eur J Immunol 23:186, 1993
From www.bloodjournal.org by guest on February 6, 2015. For personal use only.
1996 87: 5232-5241
Macrophages can recognize and kill tumor cells bearing the
membrane isoform of macrophage colony-stimulating factor
MR Jadus, MC Irwin, MR Irwin, RD Horansky, S Sekhon, KA Pepper, DB Kohn and HT Wepsic
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