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Cancer Research 67, 6717, July 15, 2007. doi: 10.1158/0008-5472.CAN-06-4263
© 2007 American Association for Cancer Research
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Cell, Tumor, and Stem Cell Biology

Effect of Dominant Negative Transforming Growth Factor-ß Receptor Type II on Cytotoxic Activity of RAW 264.7, a Murine Macrophage Cell Line

Geun Taek Lee1, Jun Hyuk Hong1,2, Cheol Kwak3, Jaesung Woo3, Victoria Liu4, Chung Lee3,4 and Isaac Yi Kim1,3

1 Division of Urologic Oncology, The Cancer Institute of New Jersey, Robert Wood Johnson Medical School, New Brunswick, New Jersey; 2 Department of Urology, University of Ulsan College of Medicine, Seoul, Korea; 3 Department of Urology, University of California, Irvine, Orange, California; and 4 Department of Urology, Northwestern University's Feinberg School of Medicine, Chicago, Illinois

Requests for reprints: Isaac Yi Kim, The Cancer Institute of New Jersey, Robert Wood Johnson Medical School, 195 Little Albany Street, 4560, New Brunswick, NJ 08901. Phone: 732-235-2043; Fax: 732-235-6596; E-mail: kimiy{at}umdnj.edu.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Transforming growth factor-ß (TGF-ß) is a potent suppressor of the immune system. In the present study, we investigated the effect of TGF-ß resistance on a murine macrophage cell line, RAW 264.7, by overexpressing a dominant negative TGF-ß receptor type II (TßRIIDN) construct. As expected, TßRIIDN-expressing RAW cells, designated as RAW-TßRIIDN, were resistant to TGF-ß signaling. When these cells were cocultured with the murine renal cell carcinoma cell line, Renca, a dramatic increase in apoptosis of Renca cells was observed. Simultaneously, elevated levels of inducible nitric oxide synthase (iNOS) and tumor necrosis factor-{alpha} (TNF-{alpha}) in association with IFN-{gamma} were detected in RAW-TßRIIDN cells. When the effects of TNF-{alpha} and iNOS were neutralized through the use of neutralizing antibody and NG-methyl-L-arginine, respectively, the enhanced cytotoxicity of TßRIIDN-RAW cells was partially reversed. Taken together, these results show that TGF-ß–resistant RAW 264.7 murine macrophage cells have increased cytotoxic activity that is in part mediated by iNOS and TNF-{alpha}. [Cancer Res 2007;67(14):6717–24]


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Transforming growth factor-ß1 (TGF-ß1) is a pleiotropic growth factor that regulates cellular proliferation, migration, differentiation, immune response, apoptosis, and angiogenesis (1–6). Based on the data obtained from knock-out mice, however, the predominant effect of TGF-ß in vivo is immune suppression (7). Because malignant cells express high levels of TGF-ß, many investigators have speculated that tumor-derived TGF-ß permits transformed cells to escape from immune surveillance. Thus, a potential therapeutic strategy for metastatic cancer is to render immune effector cells resistant to TGF-ß. In support of this view, the transplantation of TGF-ß–resistant bone marrow cells led to an eradication of metastatic prostate cancer cells in mice (8). Unfortunately, the animals eventually succumbed to a widespread autoimmune response (9). A careful examination of these animals revealed a dramatic expansion of macrophages, suggesting that TGF-ß–resistant macrophages potentially mediate antitumor and/or autoimmune response. Thus, further clarifying the phenotype associated with TGF-ß resistance in macrophages may reveal novel strategies for enhancing antitumor immune response while simultaneously decreasing the potential of a devastating autoimmune response associated with TGF-ß–based immunogene therapy.

Macrophages are key regulators of the innate immune system that originate from stem cells located in the bone marrow. The most immature form that exhibits macrophage characteristics is represented by monoblasts. Upon division, monoblasts give rise to promonocytes and monocytes in the bone marrow. In response to the appropriate stimuli mediated by cytokines, these monocytes migrate into tissues and organs where they differentiate into macrophages. The principal mediator of macrophage activation is IFN-{gamma} secreted by the armed inflammatory T (CD4) cells. Under experimental conditions, macrophages can be activated in a two-stage reaction: priming and triggering (10). Macrophages can be segregated into two broad groups: resident tissue macrophages and inflammatory macrophages. Tissue macrophages are heterogeneous, and those isolated from different tissues differ in function, possibly due to the adoptive responses to the local microenvironment (11). Inflammatory macrophages are derived largely from circulating monocytes, which infiltrate damaged tissues, but some can arise by local cell division (12). TGF-ß has been proposed to be a critical regulator of macrophages because it has been reported that TGF-ß1 suppresses inducible nitric oxide synthase (iNOS) mRNA expression induced by lipopolysaccharide (LPS) and IFN-{gamma} (13, 14), whereas TGF-ß1–/– mice exhibit dysregulation of IFN-{gamma} signaling and show high levels of iNOS expression and nitrite in the serum (15). Thus, it has been proposed that the TGF-ß–based negative autocrine loop is necessary to prevent tissue injury resulting from excessive nitric oxide (NO) produced by macrophages (16–18).

As an initial attempt to characterize the phenotype of TGF-ß–resistant macrophages, the dominant negative TGF-ß receptor type II construct (TßRIIDN) was overexpressed in RAW 264.7 murine macrophage cell line. We report that RAW 264.7 cells expressing TßIIDN have increased cytotoxic activity that is mediated, in part, by the up-regulation of iNOS and tumor necrosis factor-{alpha} (TNF-{alpha}) expression.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Cell culture. The murine macrophage cell line RAW 264.7 and the murine renal cell carcinoma cell line Renca were purchased from the American Tissue Type Collection. The packaging cell line GP293 was obtained from Invitrogen. All three cell lines were routinely maintained in DMEM supplemented with 10% fetal bovine serum (FBS). RAW 264.7 cells used in this study were from the 20th through the 25th passages, and Renca was from 165th through the 175th passages. Where indicated, cells were cultured for 2 days in DMEM supplemented with 1% FBS with or without TGF-ß1 (R&D Systems) at 10 ng/mL in the presence of IFN-{gamma} and LPS.

Transfection. The construction of the murine stem cell virus (MSCV)–based construct, MSCV-TßRIIDN-GFP, has previously been described (9). To generate the viral particles for infection, 3 x 106 GP293 packaging cells were plated in a 25-mL flask containing DMEM supplemented with 10% FBS. For transfection, 12 µL of LipofectAMINE was diluted in one tube containing 250 µL of serum-free media (Opti-Mem, Life Technologies-BRL); 1 µg of either MSCV-GFP or MSCV-TßRIIDN-GFP plus 1 µg of vesicular stomatitis virus G plasmid (VSVG) were diluted in another tube containing 250 µL of serum-free medium. The two tubes were mixed together and incubated at room temperature for 30 min. During this incubation period, the medium from GP293 cells was aspirated and washed twice with PBS, and the cells were incubated with 2 mL of Opti-Mem for 30 min. Then, the mixtures of LipofectAMINE and plasmids were added to GP293 and incubated at 37°C. Twelve hours later, transfection was stopped by adding 2.5 mL of DMEM supplemented with 20% FBS. After another 12 h, the medium was aspirated, and cells were washed twice with PBS. Next, 3 mL of DMEM supplemented with 10% FBS was added, and cells were incubated for an additional 24-h period to generate the viral particles. To infect RAW 264.7 cells, a transduction cocktail (2 mL medium, 1 mL virus, 75 µL HEPES buffer, and 4 µL polybrene) was prepared and added to 2 x 106 RAW 264.7 cells plated out in a 25-mL flask. After 24 h, fresh medium was added, and cells were sorted using flow cytometry.

Immunoblot analysis. Cells were harvested, placed in sample buffer (0.0625 mol/L Trizma base, 2% SDS, and 5% 2-mercaptoethanol), and boiled for 5 min. Electrophoresis was carried out using 50 µg of total protein in each lane. After electrophoresis, protein was transferred to a 0.2-µm nitrocellulose membrane (Bio-Rad). Subsequently, the membrane was blocked with blocking buffer (TBST–5% nonfat dry milk, TBS, and 0.1% Tween) for 1 h and incubated with the appropriate primary antibody at a concentration of 0.4 mg/mL overnight at 4°C. Anti-iNOS and anti–phospho-Smad-2 primary antibodies were obtained from Cell Signaling Technology, whereas anti–Smad-2 antibody was obtained from Abcam Inc.; anti–ß-actin and anti–IFN-{gamma} antibodies were purchased from Sigma-Aldrich and R&D Systems, respectively. Next morning, the membrane was washed with TBST (TBS and 0.1% Tween) and incubated in the presence of the goat–anti-rabbit secondary antibody (Bio-Rad Laboratories) at a dilution of 1:3,000 for 2 h at room temperature. Several washings were carried out with TBST, and immunoreactive bands were visualized with enhanced chemiluminescence (Amersham).

TNF-{alpha} and Fas Ligand immunoassays (ELISA assay). The Quantikine human TNF-{alpha} and Fas Ligand immunoassay kits (R&D Systems) were used to measure the amount of TNF-{alpha} and Fas Ligand protein produced by the cells in a 24-h period. Cells (1.0 x 105 per well) were plated on six-well plates and cultured in DMEM with 10% FBS overnight. The next day, the culture medium was replaced with DMEM with 1% FBS and incubated for an additional 24-h period. Conditioned medium was collected in 15-mL tubes and centrifuged at 1,500 rpm for 5 min. The supernatant was collected, and ELISA for TNF-{alpha} and Fas Ligand was carried out according to the manufacturer's protocol. The total number of cells in each well was counted using a hemocytometer, and levels of TNF-{alpha} and Fas Ligand were expressed as pg/105 cells/24 h. TNF-{alpha} and Fas-Ligand levels in the control medium were subtracted from those of the conditioned medium.

RNA isolation and reverse transcription-PCR. Total RNA was isolated using the TRIzol reagent (Invitrogen) according to the protocol provided by the manufacturer. Reverse transcription-PCR (RT-PCR) was done using 1 µg of total RNA and One Step RT-PCR kit (Invitrogen). The primers used were as follows: (a) iNOS: upstream primer, 5'-TAGTTTCCAGAAGCAGAATGTGACC-3'; downstream primer, 5'-CCAAGACTCTAAATCGGATCTCTC-3'; (b) TNF-{alpha}: upstream primer, 5'-TACCTTGTCTACTCCCAGGTTCTC-3'; downstream primer, 5'-AGAGCAATGACTCCAAAGTAGACC-3'; (c) TßRI: upstream primer, 5'-GAACAAAAAGGTACATGGCCCCTGA-3'; downstream primer, 5'-CCTTCTGTTCCCTCTCAGTGAGGTA-3'; (d) TßRII: upstream primer, 5'-ATGCCCATCGTGCACAGGGACCTCA-3', downstream primer, 5'-CGTTCTGCCACACACTGGGCTGTGA-3'; (e) IFN-{gamma}: upstream primer, 5'-TTCTTGGATATCTGGAGGAACTG-3'; downstream primer, 5'-GCTTCCTGAGGCTGGATTC-3'; and (f) ß-actin: upstream primer, 5'-GTGGGGCGCCCCAGGCACCA-3', downstream primer, 5'-CTTCCTTAATGTCACGCACGATTTC-3'. The condition for RT-PCR was identical for all samples. Reverse transcription was carried out at 45°C for 30 min, 95°C for 5 min, and 5°C for 5 min. After reverse transcription, PCR was done using the following condition: 94°C for 40 s, 55°C for 40 s, and 72°C for 1 min for 30 cycles, followed by a 10-min incubation at 72°C. RT-PCR products were separated using electrophoresis on a 0.9% agarose gel, and bands were visualized by ethidium bromide staining.

Apoptosis assay. The murine renal cell carcinoma cell line, Renca, was plated in triplicates at a density of 1 x 105 cells per well in six-well plates. Twenty-four hours later, 1 x 105 RAW 264.7, RAW-GFP, or RAW-TßRIIDN cells were added and cocultured for an additional 18 h. Where indicated, the two cell types were separated using the Transwell (CoStar). Cytotoxic effect was measured using flow cytometry based on the phytoerythrin-labeled anti-Annexin V antibody kit (BD Biosciences-PharMingen). To measure the magnitude of apoptosis of Renca cells only, the gate was set against RAW 264.7 cells using the FITC-labeled anti-F4/80 murine macrophage antibody (Cell Signaling Technology). When setting the gate against RAW-264.7 cells, immunoglobulin G2a (Cell Signaling Technology) was used as the isotype control. Cells were harvested using cell scrapers and centrifuged at 1,500 x g for 5 min. After washing twice with cold PBS, 100 ng of FITC-labeled anti-F4/80 antibody was added and incubated on ice for 30 min. Subsequently, samples were washed again twice with PBS and incubated with Annexin V-PE antibody and 7-AAD in binding buffer provided by the manufacturer for 15 min at room temperature. Finally, flow cytometry was done within 1 h. Renca cells treated with 600 µg/mL of G418 for 2 days were used as positive controls. All experiments were repeated at least thrice, and similar results were obtained each time.

iNOS and TNF-{alpha} neutralization. To neutralize the effect of iNOS, NG-methyl-L-arginine (NMA; Sigma-Aldrich) was added at indicated concentrations to the coculture of RAW 264.7 and Renca cells and incubated for 24 h. To block TNF-{alpha}, RAW-TßRIIDN and Renca cells were cocultured with the indicated concentrations of TNF-{alpha} neutralizing antibody (R&D Systems) for 24 h. The percentage of apoptotic Renca cells was determined using the phytoerythrin-labeled anti-Annexin V antibody-based assay described above.

Statistics. All numerical data are expressed as a mean ± SE. All experiments were done at least thrice. Differences of means among different treatments were compared by {chi}2. A value of P < 0.05 was considered statistically significant.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Expression of TßRIIDN in RAW 264.7 mouse macrophage cell line. Initially, RT-PCR was done to confirm that RAW 264.7 expressed TGF-ß1 receptors. Consistent with the published data (19–21), both TßRI and TßRII were expressed by RAW 264.7 cells (Fig. 1A ). Next, TßRIIDN was transduced into these cells using the bicistronic retroviral vector murine stem cell virus (MSCV-GFP). After sorting with flow cytometry, positive cells were designated as RAW-TßRIIDN. As the control, MSCV encoding green fluorescent protein (GFP) was used (designated as RAW-GFP). As shown Fig. 1B, fluorescence microscopy showed a high level of expression of GFP in both RAW-TßRIIDN and RAW-GFP cells. When the infected cells were treated with 10 ng/mL TGF-ß1 for 16 h, phosphorylation of Smad-2 was significantly blunted in RAW-TßRIIDN cells when compared with the parental RAW 264.7 and RAW-GFP cells (Fig. 1C). Blots stripped and reprobed with anti–Smad-2 antibody showed equal loading of samples.


Figure 1
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  		Figure 1. Profile of TGF-ß receptors and expression of TßRIIDN in RAW 264.7 mouse macrophage cell line. A, RT-PCR was carried out to confirm the expression of TGF-ß receptors in RAW 264.7 cells. RAW 264.7 cells expressed both TßRI and TßRII. B, expression of TßRIIDN in RAW 264.7 cells. TßRIIDN was transduced into RAW 264.7 cells using the bicistronic MSCV expression vector containing GFP. The control construct contained the empty MSCV-GFP vector only. Positive cells were selected using flow cytometry. C, Smad-2 phosphorylation in TßRIIDN-expressing cells (RAW-TßRIIDN). Cells were treated with 10 ng/mL TGF-ß1, and immunoblot was carried out. Compared with controls, Smad-2 phosphorylation was significantly down-regulated in TßRIIDN-expressing cells.

 
Effect of TßRIIDN on tumoricidal activity of RAW 264.7 cells. To investigate the effect of TßRIIDN expression on the cytotoxic activity of RAW 264.7 cells, cells were cocultured with the murine renal cell carcinoma cell line, Renca; Renca was chosen because this cell line, like RAW 264.7, was originally derived from a BALB/c mouse. After coculturing for 16 h, phytoerythrin-labeled anti-Annexin V antibody was used to measure the percentage of apoptotic Renca cells. To limit the apoptosis assay to Renca, the gate was set against RAW 264.7 cells using the FITC-labeled anti-F4/80 murine macrophage marker antibody. As indicated by the results of the flow cytometry (Fig. 2A and B ), the percentage of apoptotic Renca cells increased dramatically when cocultured with RAW 264.7-TßRIIDN cells. The mean percentages of apoptotic Renca cells cocultured for 16 h with parental and RAW-GFP were 6.79% and 3.45%, respectively. In comparison, 65% of Renca cells were positive for Annexin V when cocultured with RAW-TßRIIDN cells (Fig. 2B).


Figure 2
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  		Figure 2. Effect of TßRIIDN on tumoricidal activity of RAW 264.7 cells. Cells were cocultured with the murine renal cell carcinoma cell line, Renca, and the percentages of apoptotic Renca cells were determined using flow cytometry based on the phytoerythrin-labeled anti-Annexin V antibody kit. The gate was set against RAW cells using a FITC-labeled anti-F4/80 murine macrophage marker antibody. A, representative flow cytometry. B, the average of three experiments. There was a dramatic increase in the percentage of apoptotic Renca cells when cocultured with RAW-TßRIIDN cells. *, P = 0.003, statistically significant.

 
As an initial attempt to determine the mechanism of increased cytotoxic activity of RAW-TßRIIDN cells, we cocultured the two cell types in the presence of a cell separation barrier (Transwell) that permits only the diffusion of small molecules. As shown in Fig. 3A and B , flow cytometry again showed an increased percentage of apoptotic Renca cells when cocultured with RAW-TßRIIDN cells. However, the magnitude of difference between the controls and the experimental group decreased approximately 2-fold. These results suggest that both cell-cell contact and diffusible factors contribute to the increased cytotoxic effect of RAW-TßRIIDN.


Figure 3
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  		Figure 3. Effect of cell separation barrier on the cytotoxic effect of RAW-TßRIIDN cells. Cells were cocultured with the Transwell cell separation barrier, and the percentages of apoptotic Renca cells were again determined using the anti-Annexin V antibody. A, representative flow cytometry. B, the average of three experiments. RAW-TßRIIDN cells still exhibited a significant increase in cytotoxic activity. However, the magnitude of the cytotoxic activity decreased significantly when compared with the cultures incubated in the absence of Transwell. These results suggest that the enhanced cytotoxic effect of RAW-TßRIIDN cells is mediated by both cell-cell contact and diffusible factors. *, P = 0.0371, statistically significant.

 
Expression of iNOS and TNF-{alpha} in RAW-TßRIIDN cells. Because major macrophage-derived diffusible factors that mediate tumor cytotoxicity are reactive nitrous oxide intermediates and TNF-{alpha}, we next measured the levels of expression of iNOS and TNF-{alpha} in RAW-TßRIIDN cells after incubating the cells with increasing doses on IFN-{gamma} (0–10 ng/mL) and LPS (0–100 ng/mL). The expression of iNOS was investigated by immunoblot analysis and RT-PCR; ELISA and RT-PCR were carried out to measure TNF-{alpha} levels. As shown in Fig. 4A and B , the mRNA and protein of iNOS were readily detected in RAW-TßRIIDN even in the absence of stimulation with IFN-{gamma} and LPS. With regards to TNF-{alpha}, RAW-TßRIIDN cells in the absence of activation expressed a greater than 10-fold increase in the absolute level of TNF-{alpha} protein but not RNA when compared with the controls (Fig. 4C and D). These observations suggest that TNF-{alpha} along with iNOS are potential mediators of the enhanced cytotoxicity of RAW-TßRIIDN cells.


Figure 4
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  		Figure 4. Expression of iNOS and TNF-{alpha} in RAW-TßRIIDN cells. A, immunoblot analysis for iNOS. B, RT-PCR for iNOS. C, ELISA for TNF-{alpha} and FAS Ligand. *, P = 0.0096, statistically significant. D, RT-PCR for TNF-{alpha}. Cells were treated with the indicated concentrations of IFN-{gamma} and LPS for 24 h before harvest. There was a significant increase in iNOS expression both at the protein and the RNA level in RAW-TßRIIDN cells even at the basal culture conditions without activation with LPS and IFN-{gamma}. In comparison, there was a significant increase in TNF-{alpha} expression at the protein but not the RNA level in RAW-TßRIIDN cells without activation with LPS and IFN-{gamma}.

 
Neutralization of iNOS and TNF-{alpha} in RAW-TßRIIDN cells. To determine whether the enhanced cytotoxic effect of RAW-TßRIIDN cells is mediated through iNOS, the antagonist NMA was added to the coculture, and the percentage of apoptotic Renca cells was determined using flow cytometry based on the anti-Annexin V antibody. In comparison to the experiment described in Fig. 2, the coculture period was lengthened to 24 h to increase the magnitude of the apoptotic Renca cells, thereby enhancing the sensitivity of the assay. The results showed that the treatment with 2 mmol/L of NMA led to an ~30% decrease in the percentage of apoptotic Renca cells (Fig. 5A and B ). Likewise, the treatment with the TNF-{alpha} neutralizing antibody resulted in a decrease of ~20% in the percentage of apoptotic Renca cells (Fig. 5C and D). These results show that the increased cytotoxicity of RAW-TßRIIDN cells is mediated, in part, through iNOS and TNF-{alpha}.


Figure 5
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  		Figure 5. Neutralization of iNOS and TNF-{alpha} in RAW-TßRIIDN cells. A, neutralization of iNOS. Representative flow cytometry result is shown. B, the average of five experiments. The effect of iNOS was blocked using NMA at the indicated concentrations. The cytotoxic effect of RAW-TßRIIDN cells was partially reversed by NMA. C, neutralization of TNF-{alpha}. Representative flow cytometry result is shown. D, the average of five experiments. Neutralizing antibody was used to abrogate the effect of TNF-{alpha}. The cytotoxic effect of RAW-TßRIIDN cells was partially reversed when the effect of TNF-{alpha} was neutralized.

 
Expression of IFN-{gamma} in RAW-TßRIIDN cells. Because IFN-{gamma} is an important regulator of iNOS and TNF-{alpha} in macrophages, we next examined the expression of IFN-{gamma} in RAW-TßRIIDN cells. RT-PCR showed that the expression of IFN-{gamma} is increased in RAW-TßRIIDN cells at the level of mRNA (Fig. 6A ). Consistent with this observation, immunoblot also revealed a high level of IFN-{gamma} protein in TGF-ß–resistant RAW 264.7 cells (Fig. 6B).


Figure 6
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  		Figure 6. Expression of IFN-{gamma} in RAW-TßRIIDN cells. A, RT-PCR. B, immunoblot analysis. Elevated levels of IFN-{gamma} were detected in RAW-TßRIIDN cells at the RNA and protein level.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
In the present study, we have investigated the effect of TGF-ß resistance on the cytotoxic activity of the murine macrophage cell line RAW 264.7 cells using the dominant negative TßRIIDN (RAW-TßRIIDN). When these cells were cocultured with the murine renal cell carcinoma cell line Renca, an increased cytotoxicity was detected. In the presence of a cell separation barrier, the magnitude of cytotoxic activity of RAW-TßRIIDN was attenuated. Using RT-PCR, immunoblot analysis, and ELISA, increased levels of iNOS and TNF-{alpha} were detected in RAW-TßRIIDN cells. When the effects of iNOS and TNF-{alpha} were blocked using NMA and neutralizing antibody, respectively, the enhanced cytotoxicity of RAW-TßRIIDN cells was partially reversed. Taken together, these observations show that rendering RAW 264.7 murine macrophage cell line resistant to TGF-ß leads to enhanced cytotoxicity and suggest a potential mechanism for the enhanced antitumor and/or lethal autoimmunity associated with TGF-ß–based immunogene therapy.

Since its initial discovery, TGF-ß has been shown to be a critical negative regulator of the immune system because TGF-ß1 knock-out mice die of diffuse autoimmune response 3 to 4 weeks after birth (7, 22). In macrophages and monocytes, TGF-ß is a potent chemoattaractant and activates phagocytic activity (6, 23, 24). In addition, TGF-ß has been shown to deactivate macrophages by suppressing nitric oxide and reactive oxygen intermediates (13, 14). Consistent with these observations, the present study showed an increased level of cytotoxicity in TGF-ß–resistant RAW-TßRIIDN cells. Although the mechanism of enhanced cytotoxic activity of RAW-TßRIIDN cells is not entirely clear, an attenuation of the cytotoxic activity was observed when RAW-TßRIIDN cells were cocultured with Renca in the presence of a cell separation barrier. These results suggest that diffusible factors are partly involved.

The diffusible factors that mediate the enhanced cytotoxic activity of RAW-TßRIIDN likely involve nitric oxide and TNF-{alpha}. Nitric oxide (NO), synthesized by iNOS, is a short-lived gas that can diffuse freely through cells. NO has long been recognized as an important molecule involved simultaneously in the regulation of apoptotic death and cell viability. NO production from iNOS seems to be a major pathway by which TNF-{alpha} induces apoptosis as inhibition of iNOS completely abrogates the apoptotic effect of TNF-{alpha} (25). Interestingly, iNOS expression was up-regulated in RAW-TßRIIDN cells without the activation with LPS and IFN-{gamma}. Likewise, TNF-{alpha} level was also increased in RAW-TßRIIDN cells at the basal culture condition. Because RAW 264.7 cells express TGF-ß in the absence of activation (26), these observations suggest that intact TGF-ß signaling may be necessary to prevent uncontrolled activation of macrophages. This hypothesis is consistent with our observation that RAW-TßRIIDN cells have elevated levels of IFN-{gamma}.

The observation that RAW-TßRIIDN cells express elevated levels of iNOS and TNF-{alpha} in association with increased IFN-{gamma} in the absence of activation also suggests a potential mechanism for the diffuse and lethal autoimmune response seen when mice were transplanted with bone marrow stem cells expressing TßRIIDN (9). These animals, in essence, have a continuous renewal of TGF-ß–resistant macrophages which, in turn, produce excess levels of IFN-{gamma}, nitric oxide, and TNF-{alpha}; nitric oxide and TNF-{alpha} are associated with apoptosis and inflammation. Additional experiments are under way to confirm this concept.

The mechanism underlying the increased levels of expression of iNOS and TNF-{alpha} in RAW-TßRIIDN cells remains unclear. Nevertheless, our results suggest a direct effect of TGF-ß resistance on the transcriptional activity of iNOS, whereas a post-transcriptional effect is more likely for TNF-{alpha}. These observations are consistent with published data which showed that TGF-ß regulates iNOS at the transcriptional level and TNF-{alpha} at the post-transcriptional level (27). In association with elevated levels of iNOS and TNF-{alpha}, increased expression of IFN-{gamma} was detected. Previously, it has been reported that TGF-ß1 knock-out mice have abnormal IFN-{gamma} signaling (15). Because IFN-{gamma} activates macrophages and induces expression of iNOS and TNF-{alpha}, our data suggest that the dysregulated TGF-ß signaling in macrophages leads to abnormal IFN-{gamma} expression which, in turn, may result in induction of iNOS and TNF-{alpha}. Further experiments are necessary to test this hypothesis.

Results of the present study also suggest a potential therapeutic strategy using adoptive cell therapy. Macrophages represent a significant portion of leukocytic infiltrate in most malignant tumors (28–30). These macrophages, called tumor-associated macrophages (TAM), are usually derived entirely from peripheral blood monocytes recruited by hypoxia (31) and chemotactic factors such as monocyte chemoattractant protein-1 and regulated upon activation, normal T cell expressed and secreted (RANTES; ref. 32). The precise effect of TAMs on tumor cells has been controversial. However, emerging evidence suggests that TAMs are double-edged swords that can be either anti- or protumor. Antitumor effects of macrophages are divided into direct and indirect. Indirect effects are due to production of cytokines or antigen presentation that leads to the activation of cytotoxic T cells. Direct tumoricidal effects of macrophages are further subdivided into antibody-dependent and macrophage-mediated tumor cytotoxicity (MTC). Although multiple factors have been implicated, reactive nitrogen intermediates derived from nitric oxide and TNF-{alpha} have been suggested as the key mediators of MTC (33). To date, attempts to harness the potential tumoricidal effects of macrophages to treat cancers have been largely unsuccessful (34, 35). Although the reason for the failure of macrophage-based adoptive cellular therapy is uncertain, it has been suggested that macrophages are often driven to promote tumor survival by the tumor microenvironment, including hypoxia and chemotactic factors (36). For example, TAMs have been reported to secrete cytokines or growth factors with a direct mitogenic effect on malignant cells such as platelet-derived growth factor (PDGF), epidermal growth factor (EGF), TGF-ß, and TGF-{alpha} (37, 38). Furthermore, hypoxia surrounding tumor cells leads TAMs to produce proangiogenic factors such as angiogenin (39), vascular endothelial growth factor (40), and urokinase plasminogen activator (41). Thus, rendering macrophages resistant to TGF-ß may permit spontaneous activation and tip the balance toward antitumor rather than protumor effects. Further investigation is under way to verify this hypothesis.

The use of adoptive cell therapy may circumvent the devastating autoimmune response associated with TGF-ß–based immunogene therapy. When TßRIIDN expression was targeted to the bone marrow stem cells ex vivo or to the T cells transgenically, malignancies were cured in mice (8, 42). However, these animals eventually developed a profound autoimmunity. Although the mechanism for this observation remains uncertain, it is likely that the continuous renewal of TGF-ß–resistant immune effector cells contribute to the development of the autoimmune response. Consistent with this hypothesis, it has been reported that the adoptive cell therapy using TGF-ß–resistant terminally differentiated T cells led to a complete eradication of metastatic prostate cancer in mice without the lethal autoimmune effect (43). As such, the use of adoptive cell therapy using differentiated macrophages may also lead to antitumor response without the development of autoimmunity associated with anti–TGF-ß–based immunogene therapy. Additional experiments in preclinical metatstatic cancer models are under way to verify this concept.

In conclusion, we have shown that the expression of TßRIIDN in the murine macrophage cell line, RAW 264.7 cells leads to a dramatic increase in cytotoxic activity. The enhanced cytotoxicity of RAW-TßRIIDN cells requires both cell-cell contact and diffusible factors. Diffusible factors that mediate the cytotoxic effect of RAW-TßRIIDN involve, at least in part, NO and TNF-{alpha}. These observations, taken together, suggest TGF-ß–resistant macrophages as a potential anticancer therapy.


   Acknowledgments
 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Received 11/20/06. Revised 4/27/07. Accepted 5/15/07.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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