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(Journal of Leukocyte Biology. 2000;68:890-896.)
© 2000 by Society for Leukocyte Biology

Mechanisms mediating the effects of IL-3 gene expression on tumor growth

Yuan-Zhau Wu*, Ji-Hong Hong{dagger}, Hsin-Hong Huang*, Graeme J. Dougherty{ddagger}, William H. McBride{ddagger} and Chi-Shiun Chiang*

* Department of Atomic Science, Tsing Hua University, Hsinchu 30013, Taiwan;
{dagger} Department of Radiation Oncology, Chang Gung Memorial Hospital, Tao-Yuan 30033, Taiwan; and
{ddagger} 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


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
IL-3 gene expression within tumors leads to host-cell infiltration, particularly by macrophages, slower tumor growth, and enhanced immunogenicity. Surprisingly, tumor-associated macrophages (TAMs) from within FSAN-JmIL3 tumors had decreased expression of TNF-{alpha} and iNOS. On short-term culture, TAMs from FSAN-JmIL3 tumors regained their capacity to produce TNF-{alpha} 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-{alpha} 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-{alpha} 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


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The main host-cell population in tumors is macrophages generally, and they frequently determine tumor-growth kinetics. Macrophages can inhibit tumor growth by virtue of their cytotoxic effector function or by acting as antigen-presenting cells for tumor-directed immune responses [see review in ref. 1 ]. Conversely, macrophages can stimulate tumor growth by providing paracrine factors that directly promote tumor-cell proliferation or angiogenesis [2 ]. The resulting balance between these potentially mutually antagonistic functional activities of tumor-associated macrophages (TAMs) acting in concert with the susceptibility of the tumor cells to their effects can determine tumor growth critically.

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 {alpha} (TNF-{alpha}), 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-{alpha} and nitric oxide (NO) as indices of TAM function [16 ]. TNF-{alpha} 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-{alpha} 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-{alpha}, 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.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Mice, tumors, and tumor cell lines
Inbred female C3H/HeN mice were purchased from the National Animal Center (Taiwan) and maintained in the Department of Atomic Science, Tsing Hua University (Taiwan). They were 12 weeks old at the beginning of the experiments. A nonimmunogenic spontaneous fibrosarcoma (FSAN) that is syngeneic to C3H/Kam mice was used as the tumor model, and the generality of the findings were confirmed using a methylcholanthrene-induced immunogenic fibrosarcoma (FSAR) [2 ].

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-{alpha} 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-{alpha} (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-{alpha} 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-{alpha} 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-{alpha} 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{alpha}/ß, IL-2, IL-3, IL-4, IL-5, and IL-6, interferon (IFN)-{gamma}, TNF-{alpha}/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).


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Effects of IL-3 gene expression on the expression of TNF-{alpha} and iNOS mRNA in vivo
FSAN-JmIL3 tumors are infiltrated heavily with host cells in vivo and grow more slowly than parental FSAN or control FSAN-Jneo tumors, even though there is no difference in in vitro growth rates [5 ]. The potential role of cytokines in slowing FSAN-JmIL3 tumor growth was investigated by RPA analysis of mRNA from growing tumors. FSAN-JmIL3 tumors had higher levels of the macrophage marker Mac-1 than did parental FSAN tumors (Fig. 1A ), in keeping with our previous study that IL-3 gene-transduced tumors have a higher macrophage content [5 ]. In spite of this increase in Mac-1 expression, FSAN-JmIL3 tumors had relatively less TNF-{alpha} (41% of FSAN tumors; Fig. 1B ) and iNOS mRNA (69% of FSAN tumors; Fig. 1C ). In contrast, IFN-{gamma}, 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-{alpha} and iNOS production by TAMs in vitro and the possible role of tumors and cytokines in their regulation.



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  		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-{alpha} 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-{gamma} mRNAs but reduced levels of TNF-{alpha} 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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  		Figure 3. Analyses of TNF-{alpha}, iNOS mRNA, and Mac-1 mRNA expression by RPA (A), bioactive TNF-{alpha} 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.

 
TNF and NO production by macrophages
TAMs from FSAN tumors, immediately after isolation, expressed higher levels of TNF-{alpha} 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-{alpha} 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-{alpha} 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-{alpha} production by TAMs, in particular those isolated from FSAN-JmIL3 tumors. This is in contrast to results with the Raw macrophage cell line.



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  		Figure 2. RPA for the expression of TNF-{alpha}, iNOS, and Mac-1 mRNA expression by TAMs isolated from FSAN (I) or FSAN-JmIL3 tumors (II). (A) TAMs from FSAN tumors expressed TNF-{alpha} 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-{alpha} and iNOS than did those from FSAN tumors. Mac-1 expression was up-regulated in these TAMs following 16 h incubation. (B) TNF-{alpha} 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-{alpha} and iNOS production in vivo even if they were not expressing high levels.

 
Cells of the Raw 264.7 macrophage line were used also to explore possible differences between FSAN and FSAN-JmIL3 cells in their ability to stimulate production of TNF-{alpha} or iNOS (Fig. 3 ). FSAN and FSAN-JmIL3 cells induced similar levels of TNF-{alpha} mRNA (Fig. 3A) and bioactive TNF-{alpha} 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-{alpha} (unpublished results), but the latter were less able to induce NO (Fig. 3D) .

In vitro effects of TNF-{alpha} 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-{alpha} and NO-generating SNP was investigated in vitro. FSAN cells were resistant to rmTNF-{alpha} killing up to 1000 U/ml and to SNP up to 30 µM (Fig. 4 ). In fact, a mitogenic effect of TNF-{alpha} 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-{alpha} addition (unpublished results). In contrast to parental cells, FSAN-JmIL3 cells were very sensitive to rmTNF-{alpha} and NO, even in the absence of actinomycin D. Similar results were obtained for human TNF-{alpha}, 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-{alpha} and NO, but FSAR-JmIL3 cells were even more sensitive to both agents (unpublished results). The cytotoxicity of rmTNF-{alpha} 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-{alpha}. A TUNEL assay (Fig. 5B) showed that 100 U/ml TNF-{alpha} 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).



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  		Figure 4. The in vitro response of FSAN and FSAN-JmIL3 tumor cells to a range of doses of TNF-{alpha} 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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  		Figure 5. Toxicity of TNF-{alpha} 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-{alpha}. (B) TUNEL assay demonstrating apoptotic cells in FSAN-JmIL3 cultures but not in FSAN cultures 24 h after treatment with 100 U/ml TNF-{alpha}.

 
These results indicate that a balance, which is consistent with tumor survival and growth, is reached in vivo between FSAN-JmIL3 cells and the levels of TNF-{alpha} 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.



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  		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.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
In this study, we found that TAMs within FSAN-JmIL3 tumors produced relatively less TNF-{alpha} 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-{alpha} [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-{alpha} 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-{gamma} 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-{alpha} 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-{gamma} [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-{alpha} is found in tumors commonly. As was found in this study, tumors can stimulate TNF-{alpha} 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-{alpha} 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-{alpha} and iNOS may be reasons the TNF- and NO-sensitive FSAN-JmIL3 tumor cells survive.

In our model, tumor cells stimulated TNF-{alpha} and NO production by macrophages in vitro. FSAN-JmIL3 and FSAN were similar in their ability to activate TAMs or Raw for TNF-{alpha} 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-{alpha} and NO. This is sufficiently dramatic, suggesting that the decreased levels of TNF-{alpha} and NO in FSAN-JmIL3 tumors may be a reason that such tumors grow. Low doses of TNF-{alpha} 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-{alpha} 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-{alpha} and NO killing as well as an increase in tumor immunogenicity.


   ACKNOWLEDGEMENTS
 
This work was supported by a National Health Research Institute Grant (NHRI-GT-EX89B720C) to C-S. C. and Cancer Research Program Grant, State of California/Department of Health Services (99-00509V-10054), to G. J. D. and W. H. M. We thank Dr. I. L. Campbell, SCRIPPS Research Institute (San Diego, CA), for providing RPA templates. The technical assistance of C. J. Wu and M. Y. O-Yang is appreciated.

Received April 4, 2000; revised July 20, 2000; accepted July 21, 2000.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

  1. Elgert, K. D., Alleva, D. G., Mullins, D. W. (1998) Tumor-induced immune dysfunction: the macrophage connection J. Leukoc. Biol. 64,275-290[Abstract]
  2. Milas, L., Wike, J., Hunter, N., Volpe, J., Basic, I. (1987) Macrophage content of murine sarcomas and carcinomas: association with tumor growth parameters and tumor radiocurability Cancer Res 47,1069-1075[Abstract/Free Full Text]
  3. Blankenstein, T., Cayeux, S., Qin, Z. (1996) Genetic approaches to cancer immunotherapy Rev. Physiol. Biochem. Pharmacol. 129,1-49[Medline]
  4. Colombo, M. P., Modesti, A., Parmiani, G., Forni, G. (1992) Local cytokine availability elicits tumor rejection and systemic immunity through granulocyte-T-lymphocyte cross-talk Cancer Res 52,4853-4857[Free Full Text]
  5. McBride, W. H., Dougherty, G. D., Wallis, A. E., Economou, J. S., Chiang, C. S. (1993) Interleukin-3 in gene therapy of cancer Folia Biol 40,62-73
  6. Pulaski, B. A., McAdam, A. J., Hutter, E. K., Biggar, S., Lord, E. M., Frelinger, J. G. (1993) Interleukin 3 enhances development of tumor-reactive cytotoxic cells by a CD4-dependent mechanism Cancer Res 53,2112-2117[Abstract/Free Full Text]
  7. Pulaski, B. A., Yeh, K. Y., Shastri, N., Maltby, K. M., Penny, D. P., Lord, E. M., Frelinger, J. G. (1996) Interleukin 3 enhances cytotoxic T lymphocyte development and class_ major histocompatibility complex "re-presentation" of exogenous antigen by tumor-infiltrating antigen-presenting cells Proc. Natl. Acad. Sci. USA 93,3669-3674[Abstract/Free Full Text]
  8. Chiodoni, C., Paglia, P., Stoppacciaro, A., Rodolfo, M., Parenza, M., Colombo, M. P. (1999) Dendritic cells infiltrating tumors cotransduced with granulocyte/macrophage colony-stimulating factor (GM-CSF) and CD40 ligand genes take up and present endogenous tumor-associated antigens, and prime naive mice for a cytotoxic T lymphocyte response J. Exp. Med. 190,125-133[Abstract/Free Full Text]
  9. McBride, W. H., Economou, J. S., Kuber, N., Hong, J. H., Chiang, C. S., Syluasen, R., Dougherty, S. T., Dougherty, G. J. (1995) Modification of tumor microenvironment by cytokine gene transfer Acta Oncol 34,447-451[Medline]
  10. McBride, W. H., Economou, J. S., Syljuasen, R. G., Parrish, C., Hackman, D., Latham, V., Chiang, C. S., Dougherty, G. J. (1996) The effects of cytokine gene transfer into tumors on host cell infiltration and regression Anticancer Res 16,1139-1144[Medline]
  11. Frendl, G., Fenton, M. J., Beller, D. I. (1990) Regulation of macrophage activation by IL-3. II. IL-3 and lipopolysaccharide act synergistically in the regulation of IL-1 expression J. Immunol. 144,3400-3410[Abstract]
  12. Caux, C., Vanbervliet, B., Massacrier, C., Durand, I., Banchereau, J. (1996) Interleukin-3 cooperates with tumor necrosis factor alpha for the development of human dendritic/Langerhans cells from cord blood CD34+ hematopoietic progenitor cells Blood 87,2376-2385[Abstract/Free Full Text]
  13. Lord, E. M., Yeh, K. Y., Moran, J. A., Storozynsky, E., Frelinger, J. G. (1998) IL-3-mediated enhancement of particulate antigen presentation by macrophages J. Immunother. 21,205-210
  14. Yeh, K. Y., McAdam, A. J., Pulaski, B. A., Shastri, N., Frelinger, J. G., Lord, E. M. (1998) IL-3 enhances both presentation of exogenous particulate antigen in association with class I major histocompatibility antigen and generation of primary tumor-specific cytolytic T lymphocytes J. Immunol. 160,5773-5780[Abstract/Free Full Text]
  15. Chiang, C. S., Syljuasen, R. G., Hong, J. H., Wallis, A., Dougherty, G. J., McBride, W. H. (1997) Effects of IL-3 gene expression on tumor response to irradiation in vitro and in vivo Cancer Res 57,3899-3903[Abstract/Free Full Text]
  16. Sotomayor, E. M., Fu, Y. X., Mayra, L. C., Herbert, L., Jimenez, J. J., Albarracin, C., Lopez, D. M. (1991) Role of tumor-derived cytokines on the immune system of mice bearing a mammary adenocarcinoma II. Down-regulation of macrophage-mediated cytotoxicity by tumor-derived granulocyte-macrophage colony-stimulating factor. J. Immunol. 147,2816-2823
  17. Kirstein, M., Baglioni, C. (1988) Tumor necrosis factor stimulates proliferation of human osteosarcoma cells and accumulation of c-myc messenger RNA J. Cell. Physiol. 134,479-485[Medline]
  18. Jenkins, D. C., Charles, I. G., Thomsen, L. L., Moss, D. W., Holmes, L. S., Baylis, S. A., Rhodes, P., Westmore, K., Emson, P. C., Moncada, S. (1995) Roles of nitric oxide in tumor growth Proc. Natl. Acad. Sci. USA 92,4392-4396[Abstract/Free Full Text]
  19. Thomsen, L. L., Miles, D. W., Happerfield, L., Bobrow, L. G., Knowles, R. G., Noncada, S. (1995) Nitric oxide synthase activity in human breast cancer Br. J. Cancer 72,41-44[Medline]
  20. Hajri, A., Metzger, E., Vallat, F., Coffy, S., Flatter, E., Evrard, S., Marescaux, J., Aprahamian, M. (1998) Role of nitric oxide in pancreatic tumor growth: in vivo and in vitro studies Br. J. Cancer 78,841-849[Medline]
  21. Thomsen, L. L., Scott, J. M. J., Topley, P., Knowles, R. G., Keerie, A. J., Frend, A. J. (1997) Selective inhibition of inducible nitric oxide synthase inhibits tumor growth in vivo: studies with 1400W, a novel inhibitor Cancer Res 57,3300-3304[Abstract/Free Full Text]
  22. Janssens, M. Y., Van den Berge, D. L., Verovski, V. N., Monsaert, C., Storme, G. A. (1998) Activation of inducible nitric oxide synthase results in nitric oxide-mediated radiosensitization of hypoxic EMT-6 tumor cells Cancer Res 58,5646-5648[Abstract/Free Full Text]
  23. Chiang, C. S., McBride, W. H. (1991) Radiation-enhanced TNF production by murine brain cells Brain Res 566,265-269[Medline]
  24. Stuehr, D. J., Nathan, C. F. (1989) Nitric oxide: a macrophage product responsible for cytostasis and respiratory inhibition in tumor target cells J. Exp. Med. 169,1543-1555[Abstract/Free Full Text]
  25. Chiang, C. S., Stalder, A., Samimi, A., Campbell, I. L. (1994) Reactive gliosis as a consequence of interleukin-6 expression in the brain. Studies in transgenic mice Dev. Neurosci. 16,212-221[Medline]
  26. Hobbs, M. V., Weigle, W. O., Noonan, D. J., Torbett, B. E., McEvilly, R. J., Koch, R. J., Cardenas, G. J., Ernst, D. N. (1993) Patterns of cytokine gene expression by CD4+ T cells from young and old mice J. Immunol. 150,3602-3608[Abstract]
  27. Tartaglia, L. A., Goeddel, D. V. (1992) Two TNF-{alpha} receptors Immunol. Today 13,151-153[Medline]
  28. Beissert, S., Bergholz, M., Waase, I., Lepsien, G., Schauer, A., Pfizenmaier, K., Kronke, M. (1989) Regulation of tumor necrosis factor gene expression in colorectal adenocarcinoma: in vivo analysis by in situ hybridization Proc. Natl. Acad. Sci. USA 86,5064-5068[Abstract/Free Full Text]
  29. Mills, C. D., Shearer, J., Evans, R., Caldwell, M. D. (1992) Macrophage arginine metabolism and the inhibition or stimulation of cancer J. Immunol. 149,2709-2714[Abstract]
  30. Sotomayor, E. M., Dinapoli, M. R., Calderon, C., Colsky, A., Fu, Y. X., Lopez, D. M. (1995) Decreased macrophage-mediated cytotoxicity in mammary-tumor-bearing mice is related to alteration of nitric-oxide production and/or release Int. J. Cancer 60,660-667[Medline]
  31. Alleva, D. G., Burger, C. J., Elgert, K. D. (1994) Tumor-induced regulation of suppressor macrophage nitric oxide and TNF-alpha production. Role of tumor-derived IL-10, TGF-beta, and prostaglandin E2 J. Immunol. 153,1674-1686[Abstract]
  32. Kamdar, S. J., Fuller, J. A., Nishikawa, S. I., Evans, R. (1997) Priming of mouse macrophages with macrophage colony-stimulating factor (CSF-1) induces a variety of pathways that regulate expression of interleukin 6 (IL6) and granulocyte-macrophage colony-stimulating factor (Csfgm) genes Exp. Cell Res. 235,108-116[Medline]
  33. Alleva, D. G., Walker, T. M., Elgert, D. K. (1995) Induction of macrophage suppressor activity by fibrosarcoma-derived transforming growth factor-beta 1: contrasting effects on resting and activated macrophages J. Leukoc. Biol. 57,919-928[Abstract]
  34. Alleva, D. G., Burger, C. J., Elgert, K. D. (1993) Interferon-gamma reduces tumor-induced Ia-macrophage-mediated suppression: role of prostaglandin E2, Ia, and tumor necrosis factor-alpha Immunopharmacology 25,215-227[Medline]
  35. Shnyra, A., Brewington, R., Alipio, A., Amura, C., Morrison, D. C. (1998) Reprogramming of lipopolysaccharide-primed macrophages is controlled by a counterbalanced production of IL-10 and IL-12 J. Immunol. 160,3729-3736[Abstract/Free Full Text]
  36. Scanneli, G., Waxman, K., Kaml, G. J., Ioli, G., Gatanaga, T., Yamamoto, R., Granger, G. A. (1993) Hypoxia induces a human macrophage cell line to release tumor necrosis factor-{alpha} and its soluble receptors in vitro J. Surg. Res. 54,281-285[Medline]
  37. Lord, E. M., Lee, J., Moran, J. P., Koch, C. J., Frelinger, J. G., Fenton, B. M., Keng, P. C. (1999) IL-2 expression in a mouse mammary tumor increases vascularization, T cell infiltration and radiation sensitivity 90th Annual Meeting of AACR 422 Philadelphia, PA.




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