* Department of Atomic Science, Tsing Hua University, Hsinchu 30013, Taiwan;
Department of Radiation Oncology, Chang Gung Memorial Hospital, Tao-Yuan 30033, Taiwan; and
Roy E. Coats Laboratories, Department of Radiation Oncology, University of California, Los Angeles, California
Correspondence: Chi-Shiun Chiang, Ph.D., Department of Atomic Science, Tsing Hua University, 101 Sec. 2, Kuang-Fu Road, Hsinchu 30013, Taiwan. E-mail: cschiang{at}mx.nthu.edu.tw
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and iNOS.
On short-term culture, TAMs from FSAN-JmIL3 tumors regained their
capacity to produce TNF- and NO, indicating that they were primed
in vivo. In vitro experiments were unable to
demonstrate differences between FSAN-JmIL3 and FSAN tumor cells in
their ability to stimulate TNF- production by TAMs. In the absence
of evidence that TAM activation was responsible for the slower growth
of FSAN-JmIL3 tumors, the response of tumor cells to these effector
molecules was studied. TNF- and NO were cytotoxic for FSAN-JmIL3
cells but growth stimulatory for FSAN. These tumor-related phenotypic
changes may contribute as much if not more than functional changes in
host infiltrating cells to the slower growth of FSAN-JmIL3 tumors
in vivo.
Key Words: cytokine • TNF • NO • iNOS • immunotherapy
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Tumor-directed cytokine gene therapy has shown promise as a component of tumor-cell vaccines for cancer therapy. Many studies have shown that expression of cytokine genes introduced into tumor cells often reduces their tumorigenicity and, in some cases, increases their immunogenicity [see review in ref. 3 ]. The mechanisms underlying these effects are often not identified but are generally presumed to be mediated by infiltrating host cells. The nature of the infiltrating host cells is generally predictable, reflecting the biological properties of the specific cytokine. Some cytokines, such as interleukin (IL)-2, IL-4, and IL-12, target T cells. Others, such as granulocyte-macrophage colony-stimulating factor (GM-CSF) [4 ] and IL-3 [5 , 6 ], target macrophages or dendritic antigen-presenting cells (APC) preferentially [7 , 8 ].
Our previous studies, and those by others, have shown that transduction
of tumor cells with the murine IL-3 (mIL-3) gene alters their in
vivo tumorigenicity and immunogenicity [5
6
7
,
9
, 10
]. Analysis of the cellular composition
of IL-3-transduced tumors demonstrated infiltration by host
macrophages, polymorphs, and, to a lesser extent, T cells. The effects
of IL-3 on TAMs are of particular interest because this cytokine is
known to participate in the development and functional behavior of
macrophages [11
]. Also, in cooperation with tumor
necrosis factor (TNF-), it can induce growth of dendritic cells
[12
]. Lord and colleagues [13
,
14
] have shown recently that IL-3 can enhance
presentation of ovalbumin (OVA) antigen in the context of major
histocompatibility complex (MHC) class I molecules to CD8+ cytolytic T
lymphocyte (CTL). The predominance of macrophages in the murine
fibrosarcoma FSAN [2
] and the detailed characterization
of the effects of IL-3 gene transfer into this tumor [5
,
15
] make it a good model for studying the interaction
between tumors and TAMs.
We chose to explore the effect of IL-3 gene expression on the
relationship between tumors and TAMs by first examining production of
the effector molecules TNF- and nitric oxide (NO) as indices of TAM
function [16
]. TNF- is produced in significant
amounts by TAMs and is one of the most important factors known to
affect tumor growth. Among its many diverse functions, TNF- can be
directly cytotoxic for certain tumor cells [17
], and
enhance proliferation of others [17
]. Although the level
of NO within tumors has been correlated with the degree of tumor
malignancy [18
, 19
], like TNF-, the
effects of NO on tumor growth are ambivalent [18
,
20
]. Altering inducible NO synthase (iNOS) levels can
have inhibitory [21
] and stimulatory [20
,
22
] effects on tumor growth.
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Cell lines obtained from long-term culture of FSAN or FSAR were transduced with the Moloney murine leukemia virus-based vector Jzen.1 to express the full-length mIL-3 cDNA, as described previously [5 ]. IL-3 gene expression was examined by RNase protection assay (RPA) analysis, as described below, and protein production was measured by enzyme-linked immunosorbent assay (ELISA; ELISA kit from Oncogene Science, Uniondale, NY). Cells (5x105) produced 34 ± 5 ng/ml (FSAN-JmIL3) or 38 ± 4 ng/ml (FSAR-JmIL3) IL-3 in 24 h.
Isolation of TAMs
Tumors were generated by inoculating 5 x 105
FSAN or 2 x 106-viable FSAN-JmIL3 cells into the
right thighs of mice; the different inocula sizes help compensate for
the reduced tumorigenicity associated with IL-3 gene expression. TAMs
were isolated from tumors 
8 mm in diameter. Cell suspensions were
prepared by disaggregation in 1% (w/v) Dispase (Boehringer Mannheim,
Indianapolis, IN) at room temperature for 1 h, as described
previously [15
]. Single-cell suspensions were
resuspended at 1 x 106 cells/ml in Hanks’ buffered
salt solution with 20% low endotoxin (<1 ng/ml) fetal calf serum
(FCS; Gibco, Grand Island, NY) and 0.5% (w/v) Dispase. Aliquots (25
ml) of each cell suspension were then added to 100 mm diameter
tissue-culture dishes (Costar, Cambridge, MA) and incubated at 37°C
for 30 min. Nonadherent cells were removed by vigorous pipetting.
Cell culture and TNF- bioassay
Tumor cells were maintained in 100 mm tissue culture plates
(Costar) with Dulbecco’s modified Eagle’s medium (DMEM; Gibco)
containing antibiotics and 10% low-endotoxin (<1 ng/ml) FCS. They
were seeded (2000/well) into 96-well plates (Costar) in culture medium
to which graded concentrations of recombinant murine TNF- (spec.
act. 1.8x107 U/mg; Genetec, San Francisco, CA) or culture
supernatants were added. The plates were incubated at 37°C for 3
days. After incubation, 10 µl of 10 mg/ml of
3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl- tetrazolium bromide (MTT;
Sigma Chemical Co., St. Louis, MO) in phosphate-buffered saline (PBS)
was added to each well to measure cell viability, as described
previously [23
]. The reaction was terminated after
4 h incubation at 37°C by removing the medium. The crystals that
formed were dissolved in 200 µl dimethyl sulfoxide (DMSO), and the
optical density (OD) was read at 550 nm using an ELISA plate reader.
Data from 3–5 separate experiments, with each assay performed in
triplicate within each experiment, were pooled. In some experiments,
the amount of TNF- produced by Raw cells or TAMs was measured by its
cytotoxic effects on L929 cells in the presence or absence of 5 µg/ml
of actinomycin D [23
]. The surviving L929 cells were
assayed after overnight incubation by the MTT method described above.
TNF- production assay
Raw 264.7 cells (8x105) were seeded in 0.5 ml
medium into 24-well plates and incubated at 37°C overnight. Medium
was removed and 1 x 104 tumor cells in 0.5 ml medium
was added. At the indicated time, 100 µl supernatant was collected
into a 96-well plate and stored at -70°C for TNF- bioassay using
L929 cells.
NO production assay
Cells (2x105; Raw 264.7 or TAM) in 500 µl culture
medium were seeded into 24-well plates (Costar). After 12 h
incubation at 37°C, medium was removed, and 2 x 105
FSAN or FSAN-JmIL3 cells in 500 µl culture medium were added. After
24 h incubation at 37°C, 100 µl supernatant was collected into
a 96-well plate and stored at -70°C pending measurement of NO as the
sum of the nitrite (NO2-) and the nitrate
(NO3-) content using a colorimetric assay
[24
]. For NO2- determination, a
100 µl aliquot of sample was mixed with 100 µl Griess reagent (1
vol 0.1% naphthylethylenediamine dihydrochloride in distilled water
plus 1 vol 1% p-aminobenzene-sulfonamide in 5% phosphoric acid). The
mixture was incubated in 96-well plates for 10 min and shaken at room
temperature. ODs were measured at 550 nm and 690 nm. Nitrate was
measured by reducing NO3- to
NO2- with nitrate reductase (Boehringer
Mannheim) in the presence of reduced nicotinamide adenine dinucleotide
phosphate (NADPH). The level of NO2- was then
estimated as described above.
NO response assay
Parental or IL-3-producing FSAN or FSAR tumor cells were seeded
(2000/well) into 96-well plates (Costar) in culture medium to which
graded concentrations of sodium nitroprussate (SNP; Sigma) were added.
The plates were incubated at 37°C for 3 days, and an MTT assay was
performed as described above. Data are the result of 3–5 separate
experiments with each assay performed in triplicate within each
experiment. We have found that 72 h are required for cell death to
occur.
Lung colony assay
For the lung tumor colony assay, 1 x 105 FSAN
or FSAN-JmIL3 cells in 200 µl saline were injected into the tail
vein. Mice were euthanized 12 days later, and the lungs were removed
and fixed in Bouin’s solution (Sigma). The tumor colonies were counted
under microscope.
RPA
Total RNA was isolated from cells or tumors using single-step
acid guanidium thiocyanate-phenol-chloroform extraction, as described
before [15
]. Expression of IL-1/ß, IL-2, IL-3,
IL-4, IL-5, and IL-6, interferon (IFN)-
, TNF-/ß, iNOS, and
Mac-1 mRNA was measured [25
, 26
]. The
templates for making these probes were kindly provided by Dr I. L.
Campbell, SCRIPPS Research Institute (San Diego,CA). Expression of
apoptosis-associated genes was assessed using RPA templates (m-APO-3
set) from Pharmingen (San Diego, CA), according to the manufacturer’s
protocol. Autoradiograms were scanned and analyzed by National
Institutes of Health image software. The intensity of each gene was
normalized against expression of the ribosomal protein RPL32 or
glyceraldehyde 3-phosphate dehydrogenase (GAPHD). To get more accurate
quantification, gels were exposed to films for varying time periods.
Apoptosis assay
At the indicated times, cells were fixed in 4% paraformaldehyde
(Sigma) for 10 min. Apoptotic cells were detected using TUNEL (terminal
deoxynucleotidyl transferase-mediated dUTP nick end-labeling) assay
(Boehringer Mannheim). Expression of apoptosis-associated mRNA was
assayed by RPA using the mAPO-3 RPA template set (Pharmingen).
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and
iNOS mRNA in vivo (41% of FSAN tumors; Fig. 1B
) and iNOS mRNA (69% of FSAN
tumors; Fig. 1C
). In contrast, IFN-, IL-1ß, and IL-6 mRNA
expression was up-regulated (Fig. 1B)
. Because FSAN and FSAN-JmIL3
tumor cells grown in vitro do not express proinflammatory
cytokines (Fig. 1D)
or iNOS (see Fig. 3A
), the expression profiles
obtained from in vivo tumors are associated most likely with
tumor-infiltrating cells, in particular TAMs. A similar altered
gene-expression pattern was seen in FSAR and FSAR-JmIL3 tumors
(unpublished results), indicating that the profile was a result of the
influence of IL-3 rather than some other more trivial cause. In light
of these observations, in vitro experiments were performed
to examine TNF- and iNOS production by TAMs in vitro and
the possible role of tumors and cytokines in their regulation.
 View larger version (57K): [in a new window] |
Figure 1. ;45 RPA for the expression of Mac-1 (A), cytokines (B), and iNOS (C) in
FSAN and FSAN-JmIL3 tumors growing in vivo. Tumors were
harvested when they reached 8 mm in diameter, and RNA was extracted
according to the detailed methods. The main cytokine mRNAs expressed in
FSAN tumors are TNF- and IL-1ß. A weak IL-6 mRNA signal can be
observed also. FSAN-JmIL3 tumors have obvious IL-3 gene expression and
enhanced levels of IL-6, IL-1ß, and IFN- mRNAs but reduced levels
of TNF- mRNA; Mac-1 mRNA expression was increased also. The only
cytokine expressed by FSAN and FSAN-JmIL3 tumor cells cultured in
vitro was IL-3 by the transduced cells (D).
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 View larger version (42K): [in a new window] |
Figure 3. Analyses of TNF-, iNOS mRNA, and Mac-1 mRNA expression by RPA (A),
bioactive TNF- protein production (B), and nitrate/nitrite
production (C and D) by Raw 264.7 cells. Raw cells were stimulated
in vitro with FSAN or FSAN-JmIL3 tumor cells (A, B, and C)
or by cell supernatant from a 24 h culture of FSAN or FSAN-JmIL3
tumor cells (D). (A) Total RNA was isolated 12 h after
stimulation. (B) Supernatant (100 µl) was collected at the indicated
times and assayed using L929 cells as target cells, as described in
Materials and Methods. (C) Supernatant (100 µl) was collected after
24 h incubation of indicated tumor cells or/and AG at 37°C and
assayed by Griess reagent for NO levels, as described in Materials and
Methods. AG was used as a potent and somewhat specific inhibitor of
iNOS (C, lanes 4 and 5). FSAN and FSAN-JmIL3 cells did not produce NO,
as shown in lanes 7 and 8 (C), and in lanes 4 and 5 (D), in which Raw
264.7 cells were absent.
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and iNOS mRNA than did those from FSAN-JmIL3
tumors, reflecting the in vivo situation. After 16 h
in vitro incubation, these levels subsided (Fig. 2A
), but on restimulation with FSAN or FSAN-JIL3 tumor cells (Fig. 2B)
, TAMs from FSAN-JmIL3 tumors responded strongly with TNF- and
iNOS mRNA production. Responses were as good, and in the case of iNOS,
better than those from FSAN tumors, indicating that they had been
primed for TNF- and NO production as opposed to being rendered
unresponsive. It is interesting that FSAN-JmIL3 cells appeared superior
to FSAN cells in stimulating iNOS and TNF- production by TAMs, in
particular those isolated from FSAN-JmIL3 tumors. This is in contrast
to results with the Raw macrophage cell line.
 View larger version (46K): [in a new window] |
Figure 2. RPA for the expression of TNF-, iNOS, and Mac-1 mRNA expression by
TAMs isolated from FSAN (I) or FSAN-JmIL3 tumors (II). (A) TAMs from
FSAN tumors expressed TNF- and iNOS immediately after isolation, but
this was down-regulated by 16 h in vitro incubation.
TAMs from FSAN-IL-3 tumors expressed less TNF- and iNOS than did
those from FSAN tumors. Mac-1 expression was up-regulated in these TAMs
following 16 h incubation. (B) TNF- and iNOS responses could be
generated in TAMs from FSAN-JmIL3 tumors by in vitro
incubation with FSAN (lane 3) or FSAN-JmIL3 (lane 4) tumor cells for
12 h. The responses were considerably greater than those for TAMs
from FSAN tumors that were similarly treated (lane 1, FSAN stimulation;
lane 2, FSAN-JmIL3 stimulation), indicating that TAMs from FSAN-JmIL3
tumors were better primed for TNF- and iNOS production in
vivo even if they were not expressing high levels.
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or iNOS (Fig. 3
). FSAN and FSAN-JmIL3 cells induced similar levels of TNF- mRNA
(Fig. 3A)
and bioactive TNF- production in Raw 264.7 cells (Fig. 3B)
but less iNOS (Fig. 3A)
and NO (Fig. 3C)
production in contrast to
TAMs. These differences may be because the TAMs were coming from an
environment where they had been exposed to tumor already. Tumor-cell
culture supernatants gave the same responses as whole cells, indicating
that tumor-associated soluble factors could regulate the macrophage
responses. FSAN and FSAN-JmIL3 cell supernatants were equivalent at
inducing TNF- (unpublished results), but the latter were less able
to induce NO (Fig. 3D)
.
In vitro effects of TNF- and NO on tumor survival
In the absence of any data to support the hypothesis that
increased functional activation of TAM was responsible for the slowed
in vivo growth of FSAN-JmIL3 tumors, the response of the
tumor cells to TNF- and NO-generating SNP was investigated in
vitro. FSAN cells were resistant to rmTNF- killing up to 1000
U/ml and to SNP up to 30 µM (Fig. 4
). In fact, a mitogenic effect of TNF- was seen in the lower
dose range. This result was confirmed by demonstrating an increase in
cell number and in 3H-thymidine incorporation after
rmTNF- addition (unpublished results). In contrast to parental
cells, FSAN-JmIL3 cells were very sensitive to rmTNF- and NO, even
in the absence of actinomycin D. Similar results were obtained for
human TNF-, which binds only to the p55 TNF receptor of
murine macrophages [27
], confirming the primary role of
this receptor in these responses (unpublished results). The experiment
was repeated using FSAR and FSAR-JmIL3 cell lines to determine if the
response was unique to FSAN. FSAR cells, unlike FSAN, were sensitive to
the cytotoxic effect of rmTNF- and NO, but FSAR-JmIL3 cells were
even more sensitive to both agents (unpublished results). The
cytotoxicity of rmTNF- and NO for FSAN-JmIL3 resulted in apoptosis.
An RPA assay for apoptosis-associated genes (Fig. 5A
) demonstrated that caspase-8 mRNA was activated significantly in
FSAN-JmIL3 cells within 6 h of addition of TNF-. A TUNEL assay
(Fig. 5B)
showed that 100 U/ml TNF- induced apoptosis in FSAN-JmIL3
also but not in FSAN cells within 24 h. Similar results were seen
in the response to 30 µM SNP also (unpublished results).
 View larger version (27K): [in a new window] |
Figure 4. The in vitro response of FSAN and FSAN-JmIL3 tumor cells to
a range of doses of TNF- and SNP (B). Cell survival was assessed
after 3 days incubation by MTT assay, as described in Materials and
Methods. Data were obtained from three separate experiments, with each
assay performed in triplicate within each experiment.
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 View larger version (65K): [in a new window] |
Figure 5. Toxicity of TNF- toward FSAN-JmIL3 cells. (A) RPA assay for
expression of apoptosis-associated mRNAs by FSAN-JmIL3 cells at varying
times after treatment with 100 U/ml TNF-. (B) TUNEL assay
demonstrating apoptotic cells in FSAN-JmIL3 cultures but not in FSAN
cultures 24 h after treatment with 100 U/ml TNF-.
|
and NO expressed by TAM. The complexity
of this relationship is indicated by attempts to inhibit iNOS activity
by daily injection of an inhibitor, aminoguanidine (AG). This increased
the number of FSAN-JmIL3 colony formation in the lung initially
(Fig. 6
), as would be expected. However, the tumor colonies regressed
subsequently, and all mice survived at least up to 2 months
(unpublished results); presumably the result of the generation of an
IL-3-elicited, T cell-mediated, antitumor-immune response.
 View larger version (20K): [in a new window] |
Figure 6. Influence of the iNOS inhibitor of AG on the formation of lung tumor
colonies by FSAN and FSAN-JmIL3 tumor cells. Twelve days after i.v.
injection of 1 x 105 tumor cells, mice were killed,
lungs were fixed, and tumor colonies were counted, as described in
Materials and Methods. AG (10 mg/mouse) was injected i.p. daily
following tumor injections. Administration of AG increased the number
of FSAN-JmIL3 lung tumors significantly but not that of FSAN tumors.
Data were obtained from three separate experiments with each assay
performed in five mice. *P < 0.01, Student’s
t-test.
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and NO than did those from FSAN, in
spite of the fact that they were numerically better represented
[5
]. It should be noted that the conclusions drawn from
this study are unlikely to be influenced by the gene-transduction
process. Retroviral vectors with high-transduction efficiency were used
to generate the IL-3-producing lines that were used [5
].
No differences were found between control FSAN-Jneo cells and parental
FSAN cells in their in vivo or in vitro behavior
[5
]. Also, the same results have been obtained
essentially with another IL-3-transduced murine fibrosarcoma (FSAR).
Many studies have described suppression of macrophage cytotoxicity by
tumors [1
]. Suppression of TNF- [16
,
28
29
30
] and NO production [1
,
31
] has been demonstrated also. The mechanisms of
suppression are multiple probably and include tumor-derived cytokines
such as GM-CSF [16
]. The decrease in TNF- and iNOS
production in FSAN-IL-3 tumors may be part of a coordinated change in
cytokine production, because IL-6, IL-1ß, and IFN- were
up-regulated simultaneously. The number of TAMs within a tumor, as well
as their phenotypic and functional properties, is dictated in part by
cytokines such as macrophage (M)-CSF, GM-CSF [32
], and
TGF-ß [33
], which are produced by the tumor.
Infiltrating host cells play a role also. In contrast to responses
observed in situ and immediately after isolation, TAMs from
FSAN-JmIL3 tumors were primed for TNF- and iNOS production in
response to stimulation by tumor cells, indicating that they were not
functionally inert. This primed state has been observed by others
[34
] and may involve T cell interactions and/or IFN-
[34
]. This would be consistent with the observed greater
responsiveness of TAMs from FSAN-JmIL3 tumors, which are more
immunogenic than FSAN [5
]. It has been hypothesized
further [35
] that preexisting TAMs/APCs can
"educate" newly recruited macrophages in the cytokine profile they
express, which provides a mechanism for "locking" TAMs into an
expressed profile.
Conversely, TNF- is found in tumors commonly. As was found in this
study, tumors can stimulate TNF- production, as can hypoxia and
reoxygenation [36
]. Alterations in the hypoxic status of
tumors resulting from cytokine expression could have profound
implications for tumor growth and therapy. Alteration in the extent of
hypoxia has been suggested as one reason for increased
radioresponsiveness of IL-2-expressing tumors [37
]. We
have demonstrated less hypoxia and greater radiation-responsiveness
[15
] in FSAN-JmIL3 and FSAR-JmIL3 tumors compared with
controls (unpublished results). The size of the hypoxic fraction in
FSAN and FSAN-JmIL3 tumors could dictate the levels of TNF- and NO
production by TAMs and explain why those from FSAN-JmIL3 tumors were
less endowed. Ironically, a lack of hypoxia and decreased levels of
intratumoral TNF- and iNOS may be reasons the TNF- and NO-sensitive
FSAN-JmIL3 tumor cells survive.
In our model, tumor cells stimulated TNF- and NO production by
macrophages in vitro. FSAN-JmIL3 and FSAN were similar in
their ability to activate TAMs or Raw for TNF- production, but
FSAN-JmIL3 cells were less able to generate NO production. Soluble
factors produced by the tumor cells gave the same responses as intact
tumor cells. Differences in the ability of FSAN-JmIL3 and FSAN cells to
stimulate iNOS could not be attributed to direct effects of IL-3 on
macrophage function (unpublished results), but FSAN-JmIL3 cells lose
the ability presumably to activate iNOS production as a result of
phenotypic changes induced by IL-3 expression. We [5
]
and others [6
] have demonstrated phenotypic changes in
tumor cells as a result of IL-3 gene transduction. These include
increased expression of TNF-Rp75, IL-1Rp80, intercellular adhesion
molecule-1 (ICAM-1), CD44, and MHC-I [5
,
6
]. The change responsible for alteration in the ability
to stimulate iNOS production is not known.
Perhaps the most interesting phenotypic change in tumor cells
resulting from IL-3 gene expression is the increased sensitivity to
TNF- and NO. This is sufficiently dramatic, suggesting that the
decreased levels of TNF- and NO in FSAN-JmIL3 tumors may be a reason
that such tumors grow. Low doses of TNF- and NO stimulated growth of
FSAN cells but were cytotoxic for FSAN-JmIL3. An early study by Milas
et al. [2
] showed that TAMs from FSAN tumors
could stimulate proliferation of FSAN, and TAMs from FSAR tumors were
cytotoxic to FSAR—findings that are consistent with those described
here. It is possible that lower TNF- and NO production by TAMs
within FSAN-JmIL3 tumors is the result of the establishment of a
balance between tumor and host that allows survival and growth of
FSAN-JmIL-IL3 cells. The complexity of this relationship is indicated
by the result of attempts to examine the effect of inhibiting NO
production on the growth of FSAN-JmIL3 tumor colonies in the lung. The
initial increase in the number of FSAN-JmIL3 lung colonies might have
been predicted, but these regressed subsequently, suggesting that NO
may be involved in the generation of suppression of T cell-mediated
responses.
In conclusion, the interactions wrought by cytokine gene
expression in tumor-host interactions are multiple and diverse. In this
study, the slowing of tumor growth associated with IL-3 gene expression
in murine fibrosarcomas could not be ascribed to an increased effector
function of TAMs but is probably more a result of phenotypic changes in
the tumor cells themselves, which results in increased sensitivity to
TNF- and NO killing as well as an increase in tumor immunogenicity.
Received April 4, 2000; revised July 20, 2000; accepted July 21, 2000.
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receptors Immunol. Today 13,151-153[Medline] and its soluble receptors in vitro J. Surg. Res. 54,281-285[Medline]
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