The JI
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     
 

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow Request Permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Wu, T. H.
Right arrow Articles by Schreiber, H.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Wu, T. H.
Right arrow Articles by Schreiber, H.
The Journal of Immunology, 2004, 172: 3243-3251.
Copyright © 2004 by The American Association of Immunologists

Long-Term Suppression of Tumor Growth by TNF Requires a Stat1- and IFN Regulatory Factor 1-Dependent IFN-{gamma} Pathway but Not IL-12 or IL-181

Terry H. Wu2,*, Christine N. Pabin*, Zhihai Qin§, Thomas Blankenstein§,¶, Mary Philip{dagger}, James Dignam{ddagger}, Karin Schreiber* and Hans Schreiber*,{dagger}

* Department of Pathology, {dagger} Committee on Cancer Biology, {ddagger} Department of Health Studies, University of Chicago, Chicago, IL 60637; § Institute of Immunology, Free University of Berlin, Berlin, Germany; and Max-Delbrück-Center for Molecular Medicine, Berlin, Germany


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Tumor cells engineered to secrete TNF were used as a model to examine how persistently high local concentrations of TNF suppress tumor growth. TNF secretion had no effect on tumor cell proliferation in vitro but caused a very impressive growth arrest in vivo that was dependent on both bone marrow- and non-bone marrow-derived host cells expressing TNFR. Suppression also required an endogenous IFN-{gamma} pathway consisting minimally of IFN-{gamma}, IFN-{gamma} receptor, Stat1, and IFN regulatory factor 1 since mice with targeted disruption of any of the four genes failed to arrest tumor growth. The ability of these mice to suppress tumor growth was restored after they were reconstituted with bone marrow cells from Wt mice. Interestingly, mice lacking the major IFN-{gamma}-inducing cytokines IL-12 and IL-18 or T cells, B cells, and the majority of NK cells that are potential sources of IFN-{gamma} nevertheless inhibited tumor development. Moreover, multiple lines of evidence indicated that local release of IFN-{gamma} was not required to inhibit tumor formation. These results strongly suggest a novel function for the endogenous IFN-{gamma} pathway that without measurable IFN-{gamma} production or activity affects the ability of TNF to suppress tumor development.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Tumornecrosis factor released locally by immune effector cells such as lymphocytes and macrophages can play an essential role in the rejection of cancer cells (1). The local effects of TNF on tumor development have been modeled extensively in mice using tumor cells engineered to secrete TNF (2, 3, 4, 5, 6, 7). TNF secretion consistently causes a strong long-term suppression of tumor formation in vivo regardless of the genetic background of the host and origin of the tumor cells. As in natural immune responses, the suppression is localized near the TNF-secreting cells and does not extend to tumor cells implanted at distant sites because the TNF concentration rarely increases systemically. The powerful antitumor effect of TNF is thought to be triggered through TNF-RI even though almost all cell types express two TNFRs, TNF-RI and TNF-RII, because murine TNF and human TNF, which binds only murine TNF-RI (8), have similar antitumor effects in mice (5). The mechanism also appears to be indirect (4, 9, 10) and requires radiation-sensitive host cells (2, 3) using the B2 integrins ICAM-1 (7) and/or MAC-1 (4). Granulocytes (6) and T cells (2, 4, 7) have been implicated in suppression. CD4 and CD8 T cells may initiate suppression by releasing TNF at the tumor site, but once TNF is produced, they are clearly not essential for suppression (3, 4, 5) and only contribute to a stronger suppressive effect and occasionally the complete rejection of antigenic tumor cells (1, 2, 7). The contribution of radiation-resistant, non-bone marrow (BM)3-derived cells constituting the tumor bed that supports tumor growth has largely been neglected even though they also express both TNFRs. Moreover, the involvement of additional cytokines that may help TNF to orchestrate the appropriate immune response has not been examined.

The aim of this study was to determine whether TNFR expression by non-BM-derived host cells and IFN-{gamma}, a cytokine known to enhance synergistically the antitumor activities of TNF, are required for locally released TNF to suppress tumor development. We report herein that tumor suppression required TNFR expression by both BM- and non-BM-derived host cells. It also required that the BM-derived cells have intact IFN-{gamma}, IFN-{gamma}R, Stat1, and IFN regulatory factor 1 (IRF-1) genes, but the active production of IFN-{gamma} and its activity could not be detected. These results point to a novel function of the endogenous IFN-{gamma} pathway that affects the BM cells essential for locally released TNF to suppress tumor growth.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Mice and tumor lines

B6.129-Tnfrsf1atm1Mak (TNF-RI-/-), B6.129S7-Ifngtm1Ts (IFN-{gamma}-/-), B6.129S2-Irf1tm1Mak (IRF-1-/-), B6.129S1-Il12btm1Jm (IL-12-/-), B6.129S7-Rag1tm1Mom (Rag1-/-), and the 129-Ifngrtm1Agt (IFN-{gamma}R-/-) mice originally generated in the 129 background (11) were bred from breeder pairs initially obtained from The Jackson Laboratory (Bar Harbor, ME). B6.129P2-Il18tm1Aki (IL-18-/-) mice, backcrossed to C57BL/6 for eight generations, were kindly provided by Dr. A. Zychlinsky (New York University School of Medicine, New York, NY) and were intercrossed with IL-12-/- mice to generate IL-12-/-IL-18-/- mice. C57BL/6 mice were purchased from the National Cancer Institute, Frederick Cancer Research Facility (Frederick, MD). 129S6/SvEv and 129S6/SvEv-Stat1tm1 (Stat1-/-) mice were obtained from Taconic Farms (Germantown, NY). All mice were maintained in a specific pathogen-free barrier facility at the University of Chicago (Chicago, IL). The 8101 and the 9203 cell lines were established from UV-induced tumors in a C57BL/6 mouse and a 129S6/SvEv-Stat1tm1 mouse, respectively. The PRO4L.7 progressor tumor cell line was derived from a UV-induced regressor tumor in a C3H mouse (12). The Mc51-9 tumor cell line was established from a tumor induced by methylcholanthrene in a 129-Ifngrtm1Agt mouse (13). The J558L-IFN-{gamma}, 6132A-IFN-{gamma} cell lines and the XMG1.2 hybridoma secreting anti-IFN-{gamma} Ab have been described previously (14, 15, 16). All tumor cell lines caused progressive tumors when 5 x 106 cells were injected s.c. into wild-type (Wt) syngeneic mice. The tumor cell lines were cultured in DMEM with 5% FCS (cDMEM).

Transfection and retroviral infections

Transfection of PRO4L.7 cells with the human TNF gene has been described elsewhere (5). 8101 was transfected with the pCMVIEAK1-DHFR plasmid encoding human TNF (17) using Superfect (Qiagen, Valencia, CA). A clone, 8101-TNF, that secreted up to 12,000 U (216 ng)/105 cells/day into 1 ml of medium and proliferated at a rate similar to the parental 8101 cells was expanded from G418-resistant transfectants. 9203 and Mc51-9 tumor cells and XMG1.2 hybridoma cells were made to secrete TNF by retroviral transduction. Infectious retroviruses were generated by transfecting the Phoenix ecotropic or amphotropic packaging cell lines (Dr. G. Nolan; Stanford University, Palo Alto, CA) with the SFG-hTNF construct (Dr. G. Dranoff; Dana-Farber Cancer Institute, Boston, MA). Culture supernatants were collected 48 h after transfection, filtered through a 0.45-µm Millex-HA syringe filter (Millipore, Bedford, MA), and incubated with 9203, Mc51-9, and XMG1.2 cells in the presence of 8 µg/ml Polybrene (Sigma-Aldrich, St. Louis, MO) for 24 h. Flow cytometric analyses for intracellular TNF in the bulk infectants 9203-TNF, Mc51-9-TNF, and XMG-TNF using PE-conjugated anti-Hu-TNF-{alpha} (BD Immunocytometry Systems, San Jose, CA) showed that the majority of infectants produced TNF. The high infection efficiency of this system allowed the use of bulk infectants for experiments. 9203-TNF and Mc51-9-TNF secreted up to 10,600 U (189 ng) and 7000 U (125 ng)/105 cells/day into 1 ml of medium, respectively.

Tumor proliferation in culture and growth in vivo

Tumor cell proliferation was measured over several days using the MTT assay. Briefly, 103 tumor cells in 100 µl of cDMEM were plated into the wells of a 96-well tissue culture plate (Costar, Corning, NY) and maintained in a 10% CO2 humidified incubator. At specified times, 20 µl of 5 mg/ml MTT (Sigma-Aldrich) in PBS was added to each well and the cultures were incubated for 4–24 h at 37°C. After addition of 100 µl of acidified SDS (10% SDS and 0.02 N HCl), plates were incubated overnight at 37°C. The absorbance at 570 and 650 nm was measured in a VERSAmax 96-well plate reader (Molecular Devices, Sunnyvale, CA). The value obtained by subtracting the absorbance at 650 nm from 570 nm reflects tumor cell density and was used as a measure of tumor cell proliferation over time. For growth in vivo, mice were injected s.c. in the flanks with 5 x 106 tumor cells. Tumor volume was calculated using the formula V = {pi}abc/6, where a, b, and c are the three orthogonal diameters of the tumor.

TNF assay

The TNF assay is based on the cytotoxicity of TNF against the TNF-sensitive cell line 1591-RE3.5. Briefly, 105 TNF-secreting tumor cells were cultured in 1 ml of cDMEM in a 24-well tissue culture plate (Costar). Culture supernatants were collected after 24 h and serially diluted 2-fold with cDMEM in 96-well plates. Recombinant human TNF (Genentech, South San Francisco, CA) was serially diluted 10-fold, starting at 5 x 105 U/ml. Pipette tips were changed with every dilution to prevent carryover. Five x 103 1591-RE3.5 cells were then added to each well and incubated at 37°C for 48 h. The cytotoxicity of TNF against 1591-RE3.5 cells was measured using the MTT assay described in the previous section. A standard curve was generated by plotting the percent cytotoxicity, calculated using the formula (1 - AbsTNF/Absmedium) x 100 against the concentration of rTNF, and the amount of TNF secreted by the tumor cells was extrapolated from the amount of rTNF required to achieve 50% cytotoxicity.

Treatment of mice with Ab in vivo

Thirty milligrams of lyophilized gammaglobin fraction of rabbit anti-asialo GM1 antiserum precipitated with 50% ammonium sulfate (Wako Chemicals, Dallas, TX) was reconstituted with 1 ml of PBS. Rag-1-/- mice were depleted of NK cells by i.p. injection of 22 µl of the reconstituted antiserum 3 days before tumor challenge and every 3 days thereafter until the end of the experiment. Depletion was confirmed at the end of the experiment by flow cytometric analyses of peripheral blood cells stained for the pan-NK-reactive DX5 Ab (BD PharMingen, San Diego, CA). Ascites used for neutralizing IFN-{gamma} in vivo was generated in athymic nude mice primed with pristane (Sigma-Aldrich) and inoculated with the XMG1.2 hybridoma cells. Endogenous IFN-{gamma} was neutralized by i.p. injection of ascites fluid at indicated times before tumor inoculation and weekly thereafter until the end of the experiment.

Tumor reisolation

Tumor-bearing mice were killed by overexposure to CO2. Mice were sprayed with 70% ethanol before the skin was peeled away to expose the tumors. Fragments of the tumors were removed and minced using sterilized razor blades. The minced tumor fragments were transferred to 25-cm2 tissue culture flasks containing 5 ml of cDMEM supplemented with 50 µg/ml gentamicin (Life Technologies, Rockville, MD) and 50 U/ml penicillin/streptomycin (Life Technologies) and cultured at 37°C in a humidified incubator. The culture medium was refreshed when tumor cells from the fragments had adhered to the flask and begun to proliferate.

Analysis of IFN-{gamma} message by RT-PCR

Total RNA was isolated from tumor fragments using an RNeasy kit according to the manufacturer’s instructions (Qiagen). Total RNA was treated with DNase I (Life Technologies) and then reverse transcribed into cDNA. First-round PCR was performed with the primers 5'-AAC GCT ACA CAC TGC ATC TTG G-3' and 5'-GAC TCC TTT TCC GCT TCC TG-3' and nested PCR was performed with the primers 5'-GAC TTC AAA GAG TCT GAG G-3' and 5'-GCT GTT TCT GGC TGT TAC TG-3'. The primers were purchased from Life Technologies.

IFN-{gamma} production by splenocytes and BM cells

Briefly, 2 x 105 BM cells or 5 x 105 splenocytes from Wt C57BL/6 mice were incubated with 100, 500, or 2500 tumor cells in 1 ml of cDMEM in the wells of 24-well tissue culture plates. After 24 or 48 h, culture supernatants were collected and measured for IFN-{gamma} production by ELISA (Endogen, Woburn, MA). As a positive control for IFN-{gamma} release, BM cells and splenocytes were primed with 20 U/ml recombinant human IL-2 for 4 h and then stimulated with 10 µg/ml LPS (18).

Generation of BM chimeric mice

Recipient mice were medicated with Bactrim (Hoffman-LaRoche, Nutley, NJ) in drinking water (5 ml per 500-ml drinking bottle) 3 days before BM transfer. Recipient mice were irradiated on the day of transfer. In preliminary experiments, mice were irradiated with 10 Gy; however, because the 8101 tumor grew significantly slower or not at all in some recipient mice, the radiation dose was reduced to 3 and 6 Gy. Since the lower radiation doses did not significantly affect the growth rate of the 8101 tumor when it was transplanted 3–4 wk after irradiation, these radiation doses were used in all of the presented experiments. The BM from the femurs of one donor mouse was used for four recipient mice. To isolate the BM, the ends of the femurs were cut off and the BM was flushed from the femur with serum-free DMEM using a 25-gauge needle attached to a 1-ml syringe. The BM was dispersed into a single cell suspension, and debris and cell clumps were removed by filtration through a Nytex membrane (TETKO, Kansas City, MO). Typical yield from two femurs was between 1 and 1.5 x 107 cells. The BM cells were resuspended at 107 cells/ml and 0.2 ml was injected either in the tail vein or through the retro-orbital capillary plexus. The extent of reconstitution was determined 3–4 wk after reconstitution by flow cytometric analyses of peripheral blood from chimeric mice for donor-derived cells.

Statistical analysis

Mean tumor volume at the specified time after tumor challenge was compared between groups using the Wilcoxon rank sum test. Time to tumor distribution was computed using the Kaplan-Meier estimator and compared between groups by the log rank test.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Growth arrest of tumor cells transfected to secrete TNF

When injected s.c., tumor cells secreting TNF caused a poorly vascularized, pale, flat, and granular lesion that persisted for weeks without changing or only very slowly increasing in size (Fig. 1, E and F). In contrast, tumor cells not secreting TNF rapidly caused a well-vascularized tumor (Fig. 1, I and J). Histologically, individual TNF-secreting tumor cells were interspersed among a dense mononuclear and polymorphonuclear infiltrate (Fig. 1, G and H) while non-TNF-secreting tumor cells formed a densely packed mass with occasional cellular infiltrate (Fig. 1, K and L). Similar results were obtained with TNF-secreting tumor cells from different strains (data not shown). This histologic appearance agrees with previous results demonstrating that suppression of TNF-secreting tumor cells requires inflammatory cells (3, 4).



View larger version (95K):
[in this window]
[in a new window]
  		FIGURE 1. Example of the macroscopic and histological appearance of the injection site with TNF-secreting tumor cells. A total of 2.5 x 105 PRO4L.7 tumor cells transfected to secrete TNF or untransfected was injected into the pinnae of nude mice. A–D, Macroscopic and histological appearance of control uninjected ears. A thin, well-discernable vascular tree was visible in the pale transparent skin. Microscopically, no infiltrates were found. E–H, Appearance of ear with TNF-secreting tumor cells 10 days after injection. TNF-secreting tumor cells caused a persistent lesion with a flat, dull, brownish, and indurated appearance. The arrest of tumor growth by TNF occurred in the absence of T cell immunity, since the cancer cells were injected into nude mice. There was no visible tumor but the inflammatory infiltrate obscured the view of the vascular tree. The injection site was characterized histologically by a heavy inflammatory infiltrate consisting of polymorphonuclear neutrophils and mononuclear leukocytes interspersed with a few viable cancer cells. The arrows point to patent capillaries in the areas of dense cellular infiltrates. I–L, Appearance of ear injected with untransfected cancer cells. Untransfected cancer cells generated a sizable tumor with engorged vessels within 10 days of injection. In contrast to the ear injected with TNF-secreting cancer cells, the rapidly growing tumor caused by the untransfected Wt cells contained mostly viable cancer cells and very few inflammatory cells. The results are representative of similar observations in multiple mice. Bottom rows, H&E staining; magnification, x220 and x500, respectively.

 
To take advantage of the various knockout strains available in the C57BL/6 genetic background, a C57BL/6 tumor model was established. The C57BL/6 tumor cell line 8101 caused progressively growing tumors in Wt C57BL/6 mice (Fig. 2, left). 8101 was transfected to secrete human TNF; the resulting tumor cell line was referred to as 8101-TNF. The decision to use human TNF in a murine model was based on the previous finding that secretion of human TNF suppressed the growth of a murine tumor in nude mice (5). In addition, since human TNF binds only murine TNF-RI (8), TNF-RI-/- mice can be used to show that suppression of tumor growth was caused by human TNF secreted by the tumor cells. The growth of 8101-TNF tumor cells was significantly suppressed in Wt mice (Fig. 2, left). The suppression was caused by TNF secreted by the tumor cells since it was abrogated in TNF-RI-/- mice (Fig. 2, left) but not due to a direct effect of TNF on the tumor cells because 8101-TNF and 8101 grew at a similar rate in culture (Fig. 2, right). Growing tumors from TNF-RI-/- mice were reisolated and found to secrete as much TNF as cells that had not been injected into mice (data not shown). Thus, TNF secretion suppressed the growth of the otherwise progressively growing C57BL/6 tumor 8101.



View larger version (27K):
[in this window]
[in a new window]
  		FIGURE 2. TNF secretion by tumor cells suppressed tumor growth in vivo but not in vitro. Left panel, Wt or TNF-RI-/- C57BL/6 mice were injected s.c. with 5 x 106 8101 or 8101-TNF tumor cells. TNF secretion suppressed tumor growth in Wt mice (Wilcoxon, p = 0.01) but not in TNF-RI-/- mice (p = 0.01). The growth curves show the mean tumor volume ± SEM from five mice per group pooled from three independent experiments. Right panel, 103 tumor cells were plated in triplicate in 96-well flat-bottom plates and proliferation was measured on indicated days using the MTT assay.

 
Suppression of tumor growth by TNF required TNF-RI on nonhemopoietic cells

The fact that TNF-secreting tumor cells are resistant to the direct cytotoxic effects of TNF (9, 10) suggested that TNF must act on host cells. To determine whether suppression requires TNF-RI expression on non-BM-derived cells, BM chimeric mice were generated by transferring Wt BM cells into sublethally (3 or 6 Gy) irradiated TNF-RI-/- mice. Control chimeric mice were generated by reconstituting irradiated Wt mice with Wt BM cells (Wt -> Wt) and irradiated TNF-RI-/- mice with TNF-RI-/- BM cells (TNF-RI-/- -> TNF-RI-/-). One month after reconstitution, 40% of the peripheral blood cells in the chimeric mice were found to have originated from donor BM cells (data not shown). Fig. 3 shows that 8101-TNF caused tumors with similar kinetics in Wt -> TNF-RI-/- and TNF-RI-/- -> TNF-RI-/- mice and that the Wt -> Wt mice were significantly more resistant to 8101-TNF, developing tumors after prolonged delay. These results show that suppression required TNF-RI expression on non-BM-derived host cells.



View larger version (21K):
[in this window]
[in a new window]
  		FIGURE 3. TNF-induced suppression required TNF-RI expression on non-BM-derived cells. TNF-RI-/- mice were irradiated with 3 or 6 Gy and reconstituted with Wt BM cells. At least 1 mo after reconstitution, the chimeric mice were injected s.c. with 5 x 106 8101-TNF cells. Mice with no tumors or tumors <0.1 cm3 were considered tumor free. Analysis of four TNF-RI-/- -> TNF-RI-/- mice, seven Wt -> TNF-RI-/- mice, and seven Wt -> Wt mice showed that Wt BM cells did not delay tumor development in TNF-RI-/- mice (log rank, p = 0.57).

 
Suppression of tumor growth by TNF required a Stat1- and IRF-1-dependent IFN-{gamma} pathway in host cells but not in the tumor cells

IFN-{gamma}-/- mice were used to determine whether IFN-{gamma} was required for the suppression of TNF-secreting tumor cells. 8101-TNF tumor cells were suppressed in Wt mice as previously observed but grew progressively and at similar rates in IFN-{gamma}-/- mice and TNF-RI-/- mice (Fig. 4A), indicating a strict dependence on IFN-{gamma} for TNF-induced suppression. To confirm the requirement for IFN-{gamma}, IFN-{gamma}R-/- mice were also tested for the ability to suppress TNF-secreting tumor cells. Since the IFN-{gamma}R-/- mice were maintained in the 129 instead of the C57BL/6 genetic background, the Mc51-9 tumor cell line, derived from a syngeneic IFN-{gamma}R-/- mouse, was infected to secrete TNF. Fig. 4B shows that the resulting tumor cell line Mc51-9-TNF was suppressed in Wt mice but grew progressively in IFN-{gamma}R-/- mice.



View larger version (38K):
[in this window]
[in a new window]
  		FIGURE 4. TNF-induced suppression required the Stat1/IRF-1-dependent IFN-{gamma} pathway in host cells but not in the tumor cells. IFN-{gamma}-/- (A), IFN-{gamma}R-/- (B), Stat1-/- (C), and IRF-1-/- (D) mice were injected s.c. with 5 x 106 TNF-secreting tumor cells. The cell lines indicated above each panel were fully syngeneic to the mice strain used. The results of a representative experiment are shown as the mean tumor volume ± SEM and similar results were obtained in several independent experiments. The mean tumor volume was significantly larger in the IFN-{gamma}-/- mice (p = 0.01), the IFN-{gamma}R-/- mice (p = 0.06), the Stat1-/- mice (p = 0.02), and the IRF-1-/- mice (p = 0.01) than in Wt mice, showing abrogation of suppression in mice with a defect in the IFN-{gamma} pathway. The fact that Mc51-TNF cells lack IFN-{gamma}R and that 9203-TNF cells lack Stat1 but were nevertheless suppressed in Wt mice indicated that the IFN-{gamma}R on the tumor cells was not required for suppression to occur.

 
The IFN-{gamma} signaling pathway diverges at the transcription factor Stat1 (19) and again at the downstream transcription factor IRF-1. Differential activation of these transcription factors leads to induction of different subsets of IFN-{gamma}-regulated genes. To determine whether pathways involving these two transcription factors were required for TNF-induced suppression, Stat1-/- mice and IRF-1-/- mice were challenged with TNF-secreting tumor cells. The 9203 tumor cell line, derived from a Stat1-/- mouse, was infected to secrete TNF, and the resulting 9203-TNF cells were suppressed in Wt mice but grew progressively in Stat1-/- mice (Fig. 4C). Similarly, 8101-TNF cells were suppressed in Wt mice but grew progressively in IRF-1-/- mice (Fig. 4D).

These results indicated that an intact host IFN-{gamma} pathway was required for TNF to suppress tumor growth. The fact that Wt mice suppressed both the IFN-{gamma}R-deficient Mc51-9-TNF cells (Fig. 4B) and the Stat1-deficient 9203-TNF cells (Fig. 4C) showed that the suppression was due to IFN-{gamma} acting on host cells but not on the TNF-secreting tumor cells. Moreover, the effects of IFN-{gamma} must be mediated through Stat1 and IRF-1 and the subset of IFN-{gamma}-inducible genes they regulate.

IFN-{gamma} secretion at the tumor site suppressed tumor growth independent of TNF

It is possible that the only function for TNF was to induce IFN-{gamma} and that once induced IFN-{gamma} may cause suppression without requiring the TNF pathway. To address this possibility, mice lacking both TNFRs were challenged with J558L tumor cells secreting IFN-{gamma} (J558L-IFN-{gamma}). In Wt mice, J558L grew progressively (Fig. 5A) while J558L-IFN-{gamma} was rejected (Fig. 5B). In TNF-RI/II-/- mice, J558L grew progressively in four of eight mice (Fig. 5C) while J558L-IFN-{gamma} was rejected by all seven mice (Fig. 5D). The reason that some TNF-RI/II-/- but not Wt mice rejected J558L is not clear, but this difference was also observed with three other independently generated tumor cell lines (data now shown). These results showed that locally released IFN-{gamma} can suppress tumor growth in the absence of the TNF pathway.



View larger version (36K):
[in this window]
[in a new window]
  		FIGURE 5. Tumor suppression induced by IFN-{gamma} did not require TNF. Briefly, 5 x 106 J558L (A and C) or IFN-{gamma}-secreting J558L-IFN-{gamma} (B and D) tumor cells were injected s.c. into Wt (A and B) or TNF-RI/II-/- (C and D) BALB/c mice. Each curve follows tumor growth in a single mouse. The reason that J558L was rejected by some TNF-RI/II-/- mice but not Wt mice is not clear; however, three other tumors also showed reduced tumorigenicity in TNF-RI/II-/- mice.

 
BM cells from Wt mice restored the ability of TNF to suppress tumor growth in IFN-{gamma}-/- and IFN-{gamma}R-/- mice

IFN-{gamma} is known to be produced only by hemopoietic cells such as NK cells, T cells, macrophages, B cells, and CD8{alpha}+ dendritic cells. To verify that BM cells are the source of IFN-{gamma}, sublethally irradiated IFN-{gamma}-/- mice were reconstituted with Wt BM cells (Wt -> IFN-{gamma}-/-) and tested for the ability to suppress TNF-secreting tumor cells. One month after reconstitution, flow cytometric analyses showed that 45–60% of peripheral blood cells were derived from donor BM cells (data not shown). Wt BM cells enabled IFN-{gamma}-/- mice to suppress the growth of 8101-TNF, delaying tumor development like the Wt -> Wt mice (Fig. 6A). Since 8101-TNF grew progressively in the IFN-{gamma}-/- -> IFN-{gamma}-/- mice, it is unlikely that the suppression in the Wt -> IFN-{gamma}-/- mice resulted from radiation damage, also called "tumor bed effect" (20).



View larger version (23K):
[in this window]
[in a new window]
  		FIGURE 6. TNF-mediated suppression of tumor growth required BM cells with an intact IFN-{gamma} pathway and TNF-RI expression. A, BM cells from Wt but not IFN-{gamma}-/- mice restored the ability of IFN-{gamma}-/- mice to suppress tumor growth. Sublethally irradiated IFN-{gamma}-/- mice were reconstituted with Wt BM cells and after a minimum of 1 mo were challenged with 5 x 106 8101-TNF cells. Mice with no tumors or tumors <0.1 cm3 were considered tumor free. Analysis of 11 IFN-{gamma}-/- -> IFN-{gamma}-/- mice, 12 Wt -> IFN-{gamma}-/- mice, and 10 Wt -> Wt mice indicated that Wt BM cells significantly delayed tumor development in the IFN-{gamma}-/- mice (p = 0.004). B, BM cells from Wt but not TNF-RI-/- mice restored the ability of IFN-{gamma}-/- mice to suppress tumor growth. Sublethally irradiated IFN-{gamma}-/- mice were reconstituted with TNF-RI-/- BM cells and after a minimum of 1 mo were challenged with 5 x 106 8101-TNF cells. Mice with no tumors or tumors <0.1 cm3 were considered tumor free. Analysis of seven IFN-{gamma}-/- -> IFN-{gamma}-/- mice, seven Wt -> IFN-{gamma}-/- mice, and eight TNF-RI-/- -> Wt mice indicated that the TNF-RI-/- -> IFN-{gamma}-/- mice developed tumors significantly faster than the Wt -> IFN-{gamma}-/- mice (p = 0.01), showing that TNF-RI on BM cells was required for suppression. C, BM cells from Wt mice restored the ability of IFN-{gamma}R-/- mice to suppress tumor growth. Sublethally irradiated IFN-{gamma}R-/- mice were reconstituted with Wt BM cells and after a minimum of 1 mo were challenged with 5 x 106 Mc51–9-TNF cells. Mice with no tumors or tumors <0.1 cm3 were considered tumor free. Analysis of eight IFN-{gamma}R-/--> IFN-{gamma}R-/- mice, eight Wt -> IFN-{gamma}R-/- mice, and four Wt -> Wt mice showed that Wt BM cells significantly delayed tumor development in IFN-{gamma}R-/- mice (p = 0.02). However, five of eight Wt -> IFN-{gamma}R-/- mice eventually developed tumors, whereas all of the Wt -> Wt mice completely eliminated the tumor challenge.

 
To determine whether TNF directly activated BM cells to produce IFN-{gamma}, sublethally irradiated IFN-{gamma}-/- mice were reconstituted with BM cells from TNF-RI-/- mice (TNF-RI-/- -> IFN-{gamma}-/-) and challenged with 8101-TNF. BM-derived cells in these chimeric mice have the ability to produce IFN-{gamma} but cannot be activated by TNF. The TNF-RI-/- -> IFN-{gamma}-/- mice developed tumors at the same rate as the IFN-{gamma}-/- -> IFN-{gamma}-/- mice and significantly faster than the Wt -> IFN-{gamma}-/- mice (Fig. 6B), indicating that TNF acted directly on the BM cells. However, it appears that TNF did not induce the BM cells to secrete IFN-{gamma} because when BM cells or splenocytes were cultured with 8101-TNF as a source of TNF for 2 days, there was no detectable IFN-{gamma} in the culture supernatant (data not shown).

To identify the target of IFN-{gamma}, sublethally irradiated IFN-{gamma}R-/- mice were reconstituted with Wt BM cells (Wt -> IFN-{gamma}R-/-) and then challenged with Mc51-9-TNF cells. Flow cytometric analyses showed that up to 70% of the peripheral blood cells in the chimeric mice expressed IFN-{gamma}R (data not shown). Tumor development was significantly delayed in the Wt -> IFN-{gamma}R-/- mice compared with the IFN-{gamma}R-/- -> IFN-{gamma}R-/- mice (Fig. 6C). However, unlike the Wt -> Wt mice that all rejected the tumor challenge (Fig. 6C), five of eight Wt -> IFN-{gamma}R-/- mice developed tumors after a significant delay. This difference suggested that the number of Wt BM cells in the reconstituted IFN-{gamma}R-/- mice may not have been enough to reject the tumor challenge and that a higher degree of reconstitution would have led to tumor rejection. It is also possible that IFN-{gamma} acting on BM cells was sufficient to reduce the rate of tumor growth, but IFN-{gamma} acting on both BM and non-BM cells was required for tumor rejection.

Suppression of tumor growth occurred in mice lacking T cells, B cells, and the majority of NK cells

Rag-1-/- and NK-depleted Rag-1-/- mice were used to determine whether T, B, or NK cells produced the IFN-{gamma} required for growth suppression. Rag-1-/- mice were depleted of NK cells by treatment with rabbit anti-asialo GM1 antiserum 3 days before tumor challenge and every 3 days thereafter. Flow cytometric analyses of peripheral blood from treated mice indicated that the number of cells expressing the pan-NK marker DX-5 was reduced from 50 to 60% to 2 to 10% (data not shown). The cytolytic activity of splenocytes from the treated mice against the NK-sensitive target YAC-1 cell line was also significantly reduced (data not shown). Fig. 7 shows that mice lacking T cells, B cells, and the majority of NK cells remained capable of suppressing the growth of 8101-TNF. This result suggested that another cell type, such as macrophages or CD8{alpha}+ dendritic cells, may be the source of IFN-{gamma} required for the suppression.



View larger version (27K):
[in this window]
[in a new window]
  		FIGURE 7. TNF-mediated suppression occurred in the absence of T cells and B cells and the majority of NK cells. Rag-1-/- mice were treated 3 days before challenge with 8101-TNF and every 3 days thereafter with PBS or rabbit anti-asialo GM1 serum (anti-NK) to deplete NK cells. NK cell depletion reduced the number of circulating DX-5-positive cells from 50 to 60% to 2 to 10% and the cytolysis of the NK-sensitive YAC-1 cells by splenocytes. Treated mice were injected s.c. with 5 x 106 8101-TNF cells. The growth curves shown are from a representative experiment and show the mean tumor volumes ± SEM from three PBS-treated mice, three NK-depleted mice, and one TNF-RI-/- mouse. Tumor suppression in Rag-1-/- mice has been shown previously. Depletion of NK did not abrogate suppression (p = 0.51).

 
Suppression of tumor growth did not require IL-12 and IL-18

Earlier results suggested that TNF did not directly induce BM cells or splenocytes to produce IFN-{gamma}. TNF may have instead activated stromal cells or inflammatory cells to produce secondary factors such as IL-12 and IL-18 that in turn induced IFN-{gamma} production. To determine whether these two major IFN-{gamma}-inducing cytokines were necessary for TNF to suppress tumor growth, IL-12-/- (21) and IL-18-/- (22) mice were challenged with 8101-TNF. Since IFN-{gamma} production is further reduced in the absence of both IL-12 and IL-18 (22), IL-12-/-IL-18-/- mice were also generated and then challenged with 8101-TNF. Fig. 8 shows that mice lacking IL-12, IL-18, or both IL-12 and IL-18 remained fully capable of suppressing the TNF-secreting tumor cells.



View larger version (24K):
[in this window]
[in a new window]
  		FIGURE 8. Mice lacking both IL-12 and IL-18 suppressed the growth of 8101-TNF. IL-12-/- mice (left), IL-18-/- mice (middle), and IL-12-/-/IL-18-/- mice (right) were injected s.c. with 5 x 106 8101-TNF cells. The growth curves show the mean tumor volume ± SEM from a representative experiment containing two to three mice per group. The mean tumor volume was considerably smaller in the IL-12-/-, IL-18-/-, or IL-12-/-IL-18-/- mice than in the TNF-RI-/- mice.

 
Suppression of tumor growth occurred in the absence of IFN-{gamma} message in the tumor and Ab-neutralizable IFN-{gamma}

The fact that TNF-mediated suppression was abrogated in mice lacking IFN-{gamma}, IFN-{gamma}R, Stat1, or IRF-1suggested that IFN-{gamma} was produced in response to the TNF-secreting tumor cells. To determine whether the IFN-{gamma} message was produced within the lesion caused by 8101-TNF, total RNA was isolated from the small lesion produced at the site of tumor injection in Rag-1-/- mice and from the large tumor in TNF-RI-/- mice 26 days after tumor injection. The IFN-{gamma} message was readily detected by RT-PCR in the 8101-TNF tumor isolated from TNF-RI-/- mice and in an IFN-{gamma}-secreting tumor isolated from Rag-1-/- mice treated with anti-IFN-{gamma} Ab (Fig. 9A). Surprisingly, there was no detectable IFN-{gamma} message in the nodule of the 8101-TNF tumor remaining in the Rag-1-/- mice (Fig. 9A); in some samples, no product was generated even after an additional 35 cycles with nested PCR primers (data not shown).



View larger version (40K):
[in this window]
[in a new window]
  		FIGURE 9. Suppression occurred in the absence of detectable IFN-{gamma} message and protein. A, IFN-{gamma} message was not detected in tumors under suppression by TNF. RT-PCR analysis for IFN-{gamma} message was performed on 8101-TNF tumors reisolated from Rag-1-/- mice and TNF-RI-/- mice and on 6132A-IFN-{gamma} reisolated from Rag-1-/- mice treated systemically with IFN-{gamma}-neutralizing Ab. The results show products generated after 35 amplification cycles with IFN-{gamma}-specific primers. Similar results were obtained when the first-round products were diluted 1/100 and subjected to 35 additional cycles of amplification using nested, IFN-{gamma}-specific PCR primers. B, TNF-secreting cancer cells were prevented from forming a tumor even when they produce large amounts of IFN-{gamma}-neutralizing Abs. The XMG1.2 (XMG) hybridoma cells were transduced with the TNF gene to generate the XMG-TNF hybridoma cells secreting both TNF- and IFN-{gamma}-neutralizing Ab. Mice were injected s.c. with 2–3 x 106 of XMG or XMG-TNF hybridoma cells. Similar results were obtained in an additional experiment using four more mice. C, Treatment of mice with IFN-{gamma}-neutralizing Ab prevented the growth suppression of IFN-{gamma}-secreting tumor cells but not of TNF-secreting tumor cells. Upper right and left, Athymic nude mice were treated weekly with IFN-{gamma}-neutralizing Ab or PBS starting 3 days before challenge with 3 x 105 IFN-{gamma}-secreting (6132-IFN-{gamma}) tumor cells or 5 x 106 TNF-secreting (8101-TNF) tumor cells. The data are expressed as the mean tumor volume ± SEM from five mice treated with anti-IFN-{gamma} Ab or PBS and from four TNF-RI-/-Rag-1-/- mice. The anti-IFN-{gamma} Ab treatment increased the growth of 6132-IFN-{gamma} significantly over PBS treatment (p = 0.01). IFN-{gamma} neutralization also increased the growth of 8101-TNF significantly over PBS treatment (p = 0.02), but all of the tumors were still smaller than the tumors from three of four TNF-RI-/-Rag-1-/- mice by day 26. Lower right and left, Weekly treatment with IFN-{gamma}-neutralizing Ab or PBS was started 30 days before tumor challenge. With this treatment schedule, there was no significant difference in tumor volume between mice treated with PBS (n = 5) or anti-IFN-{gamma} Ab (n = 4).

 
To confirm that TNF-mediated suppression did not require IFN-{gamma} protein within the lesion, the XMG1.2 hybridoma cells producing neutralizing anti-IFN-{gamma} Ab were transduced to secrete TNF (XMG-TNF). This provided the means to focus a high concentration of IFN-{gamma}-neutralizing Ab in the vicinity of the TNF-secreting tumor cells. The amount of anti-IFN-{gamma} Ab produced in 1 ml of culture medium by 106 XMG-TNF hybridoma cells within 24 h was sufficient to neutralize 62 ng of recombinant murine IFN-{gamma} (data not shown). Fig. 9B shows that while the XMG1.2 hybridoma cells developed large tumors, the XMG-TNF hybridoma cells were strongly inhibited. These results along with the absence of the IFN-{gamma} message in the suppressed tumor lesion firmly established that IFN-{gamma} was not produced within the tumor.

To determine whether IFN-{gamma} produced away from the tumor cells is essential for tumor inhibition, athymic nude mice were treated systemically with anti-IFN-{gamma} Ab 3 days before tumor inoculation and weekly thereafter. We expected that systemic neutralization of IFN-{gamma} would reproduce the phenotype of the IFN-{gamma}-/- mice and eliminate growth suppression, but treatment with IFN-{gamma}-neutralizing Ab had no measurable effect over PBS treatment on tumor suppression 2 wk after tumor challenge (Fig. 9C, upper right). The Ab treatment effectively neutralized IFN-{gamma} because in the same mouse it enabled the progressive growth of IFN-{gamma}-secreting 6132A tumor cells (Fig. 9C, upper left), which is usually suppressed unless the secreted IFN-{gamma} was neutralized (16). IFN-{gamma} neutralization also enabled 8101 tumor cells engineered to secrete IFN-{gamma} to grow as progressively as the parental 8101 tumor cells (data not shown). Interestingly, with continued treatment, 8101-TNF began to grow considerably faster in anti-IFN-{gamma} Ab-treated mice than in PBS-treated mice, but still much slower than in TNF-RI-/- mice. Since it was possible that extended Ab treatment was required to completely neutralize IFN-{gamma}, the anti-IFN-{gamma} Ab treatment was initiated 30 days before tumor challenge. However, this treatment failed to accelerate the growth rate of 8101-TNF in nude mice to that in the TNF-RI-/- mice (Fig. 9C, lower right).


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
In this study, the local effects of TNF on cancer cells were modeled using cancer cells secreting TNF. TNF-secreting cancer cells were insensitive to the cytotoxicity of TNF in vitro (10) and therefore unlikely to be the direct targets for TNF in vivo. Instead, TNF arrested tumor development through an indirect mechanism involving both BM- and non-BM-derived nonmalignant host cells. We propose that TNF stimulated non-BM-derived cells to release chemokines (23, 24) that attracted BM-derived cells to the tumor cells (Fig. 10). When the recruited BM cells, most likely monocytes, were then exposed to TNF in situ, an inflammatory environment was created that arrested tumor cell growth (25). BM-derived cells may also have been directly recruited by TNF to the tumor cells (26) and then activated in situ by factors produced by TNF-stimulated non-BM-derived cells.



View larger version (25K):
[in this window]
[in a new window]
  		FIGURE 10. Proposed model for the suppression of tumor development by cancer cells secreting TNF. Locally released TNF suppressed tumor growth through an indirect mechanism that involved activation of BM-derived cells at the tumor site. Since the endogenous IFN-{gamma} pathway was required but active IFN-{gamma} production and activity could not be detected, the most likely function of the endogenous IFN-{gamma} pathway was to facilitate the development and/or differentiation of a BM-derived cell essential for suppression. TNF recruited these BM-derived cells to the tumor cells by stimulating the non-BM-derived stromal cells surrounding tumor cells to produce chemokines. Once recruited, the BM-derived cells were activated in situ by TNF to kill the tumor cells or the stromal cells supporting their growth. Alternatively, BM-derived cells were directly recruited by TNF to the tumor cells and then activated in situ by cytokines produced by TNF-stimulated stromal cells. It must be emphasized that although only single cells are depicted, the BM and non-BM cells may in fact include multiple cell types that are required for TNF to suppress tumor growth.

 
TNFR expression on non-BM-derived stroma was necessary for TNF-induced tumor suppression, but the type of stromal cells targeted by TNF was not identified in this study. Because of the known antiangiogenic effects of TNF, it is tempting to suggest that TNF directly targeted endothelial cells and prevented tumor development by inhibiting tumor angiogenesis (27). Indeed, lesions containing TNF-producing tumor cells showed much less prominent vessel formation than tumors arising from Wt tumor cells (Fig. 1, E and F compared with Fig. 1, I and J). However, abundant patent capillaries were clearly visible in areas of dense cellular infiltrates surrounding the growth-inhibited TNF-secreting tumor cells (arrows in Fig. 1, G and H), suggesting that destruction of capillaries may not be the primary event preventing tumor development. It is important to consider the alternative possibility that the absence of a proangiogenic environment suppressed tumor development. Inhibition of tumor cells that produce large amounts of angiogenic factors such as vascular endothelial growth factor may limit the extent of angiogenesis by reducing the availability of angiogenic factors. The inflammatory environment associated with the TNF-secreting tumor cells may also prevent macrophage/monocytes from performing their essential role in normal and pathological angiogenesis (27, 28). Thus, TNF-induced tumor suppression may have resulted from an inability to induce tumor angiogenesis rather than active vessel destruction. In addition to the mechanisms outlined above, suppression also required an endogenous IFN-{gamma} pathway involving IFN-{gamma}, IFN-{gamma}R, Stat1, and IRF-1. We showed specifically that the BM-derived cells must originate from mice that have the IFN-{gamma} and IFN-{gamma}R genes and most likely the Stat1 and IRF-1 genes. Interestingly, a connection between IFN-{gamma} production or activity with TNF-induced tumor suppression could not be established by the various assays that were performed. First, TNF-mediated suppression occurred in the absence of detectable IFN-{gamma} message in the growth-arrested lesions. Second, systemic treatment with IFN-{gamma}-neutralizing Abs failed to block TNF-mediated suppression while effectively abrogating the suppression of three independently generated IFN-{gamma}-producing cancer cell lines 6123A-IFN-{gamma}, J558L-IFN-{gamma}, and 8101-IFN-{gamma}. It could be argued that the anti-IFN-{gamma} Abs had not penetrated into the diffusion-limited space between TNF-secreting tumor cells, but this argument becomes untenable for explaining the continued growth arrest of TNF-producing hybridoma cells that also produced large amounts of neutralizing anti-IFN-{gamma} Abs. However, we cannot exclude the possibility that a more complete neutralization of IFN-{gamma} produced and functioning outside of the tumor site is required to block the inhibitory effects of TNF. Third, there was no detectable IFN-{gamma} protein when 8101-TNF was cultured with BM cells and splenocytes as potential sources of IFN-{gamma} (data not shown), suggesting that TNF cannot directly stimulate BM cells to produce IFN-{gamma}. Fourth, tumor suppression occurred in the absence of T cells and B cells and the majority of NK cells that are potential sources of IFN-{gamma} as well as the two major IFN-{gamma}-inducing cytokines IL-12 and IL-18. Other cytokines such as IFN{alpha}{beta} (29) and IL-15 (30) can also induce IFN-{gamma}, but were not examined in this study. Finally, TNF-mediated suppression did not require IFN-{gamma} acting on or through the tumor cells (31) since tumor cells lacking IFN-{gamma}R or Stat1 were nevertheless suppressed. These results show that despite the requirement for an intact IFN-{gamma} pathway, IFN-{gamma} production or activity could not be measured. This is clearly different from tumor suppression resulting from measurable IFN-{gamma} production at the tumor site that can be effectively blocked by treatment with anti-IFN-{gamma} Ab, as modeled in this study by the IFN-{gamma}-secreting tumor cells (Figs. 5 and 9). Thus, different mechanisms must be involved in the growth inhibition of the IFN-{gamma}-secreting tumor cells and the TNF-secreting tumor cells. It appears that TNF does not mediate all of its antitumor activities through IFN-{gamma} but rather utilize alternative mechanisms which facilitate the essential effects of IFN-{gamma}.

This is the only study that we are aware of that definitively shows that an intact endogenous IFN-{gamma} pathway is required for TNF to suppress tumor growth. Previously, Keller et al. (32) had shown that treatment with anti-IFN-{gamma} antiserum abrogated the increased resistance of rats inoculated with heat-killed Corynebacterium parvum or Listeria monocytogenes against transplantable tumors. Although C. parvum and L. monocytogenes can prime macrophages to produce TNF, this study did not show that the enhanced resistance was strictly due to TNF production or that IFN-{gamma} neutralization blocked an antitumor response induced by TNF. Doherty et al. (33) had shown that treatment with anti-IFN-{gamma} antiserum blocked the ability of TNF to induce necrosis of established tumors in T cell-competent mice. However, IFN-{gamma} neutralization also accelerated tumor growth when mice were not treated with exogenous TNF, thereby raising the possibility that IFN-{gamma} neutralization did not block the antitumor effects of TNF but only increased the rate of tumor growth by interfering with a T cell-dependent antitumor response. It is also important to emphasize that the necrosis of established tumors studied by Doherty et al. (33) is quite different from the arrest of tumor development in our study. Although central hemorrhagic necrosis can be induced by TNF only when established tumors are at least 1 cm in average diameter (34, 35, 36) and the effectiveness of repeated injection is limited by development of tolerance (37), the persistent tumor suppression caused by sustained TNF release is not affected by these limitations.

The exact timing when the IFN-{gamma} pathway was utilized could not be established in this study. Nevertheless, the results may be reconciled by proposing that the IFN-{gamma} pathway is important long before tumor challenge for the differentiation/development of a BM-derived cell that later responds to TNF or factors induced by TNF and mediates growth arrest. IFN-{gamma} neutralization may have had little or no effect on growth suppression in Wt mice because the putative cell had already differentiated and/or developed and become fully capable of mediating tumor growth arrest. That IFN-{gamma} may play such a role is consistent with the recent observation that IFN-{gamma} drives human monocyte differentiation into macrophages instead of dendritic cells (38). The putative cell is likely to have a long life span because continuous treatment with IFN-{gamma}-neutralizing Ab for nearly 2 mo did not completely abrogate TNF-induced suppression. Macrophages are BM-derived cells that have powerful antitumor activities (39). So far, however, there has been no reported defect in macrophage development in IFN-{gamma}-/-, IFN-{gamma}R-/-, Stat1-/-, and IRF-1-/- mice. IFN-{gamma} may direct macrophages to polarize into specific functional subsets analogous to IFN-{gamma} directing CD4+ T cell polarization to the Th1 subset. Mills et al. (40) recently showed that mice with a tendency to develop Th1 or Th2 T cells also develop macrophages with a corresponding profile characterized by NO and TGF-{beta}1 production. A transcription factor that may be critical for determining the macrophage phenotype is IFN consensus sequence binding protein (ICSBP), an IFN-{gamma}-inducible transcription factor specifically expressed in hemopoietic cells. ICSBP is required for the differentiation of myeloid progenitors into mature macrophages (41) and, in combination with IRF-1 and PU.1, regulates a number of IFN-{gamma}-responsive genes. It is tempting to speculate that ICSBP may direct mature macrophages to differentiate further into a cell type that is required for TNF to suppress tumor growth. Additional studies using the ICSBP-/- mice and other knockout mice with defects in the endogenous IFN-{gamma} pathway may elucidate the role of the endogenous IFN-{gamma} pathway and possibly help to define a novel type or characteristic of effector cells needed for TNF to suppress tumor development.


   Acknowledgments
 
We thank Dr. Arturo Zychlinsky for providing the IL-18-deficient mice and Drs. Donald A. Rowley and Yang-Xin Fu for critical reading of this manuscript.


   Footnotes
 
1 This work was supported by National Institutes of Health Grants RO1-CA22677, RO1-CA37516, and PO1-CA097296 and by University of Chicago Cancer Center Grant CA-14599. Back

2 Address correspondence and reprint requests to his current address: Dr. Terry H. Wu, Department of Internal Medicine, MSC10 5550 University of New Mexico, 1 University of New Mexico, Albuquerque, NM 87131. E-mail address: terrywu{at}unm.edu Back

3 Abbreviations used in this paper: BM, bone marrow; IRF-1, IFN regulatory factor 1; cDMEM, complete DMEM; Wt, wild type; ICSBP, IFN consensus sequence binding protein. Back

Received for publication August 21, 2003. Accepted for publication December 22, 2003.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

  1. Poehlein, C. H., H. Hu, J. Yamada, I. Assmann, W. G. Alvord, W. J. Urba, B. A. Fox. 2003. TNF plays an essential role in tumor regression after adoptive transfer of perforin/IFN-{gamma} double knockout effector T cells. J. Immunol. 170:2004.[Abstract/Free Full Text]
  2. Asher, A. L., J. J. Mule, A. Kasid, N. P. Restifo, J. C. Salo, C. M. Reichert, G. Jaffe, B. Fendly, M. Kriegler, S. A. Rosenberg. 1991. Murine tumor cells transduced with the gene for tumor necrosis factor-{alpha}: evidence for paracrine immune effects of tumor necrosis factor against tumors. J. Immunol. 146:3227.[Abstract]
  3. Vanhaesebroeck, B., M. Mareel, F. Van Roy, J. Grooten, W. Fiers. 1991. Expression of the tumor necrosis factor gene in tumor cells correlates with reduced tumorigenicity and reduced invasiveness in vivo. Cancer Res. 51:2229.[Abstract/Free Full Text]
  4. Blankenstein, T., Z. Qin, K. Uberla, W. Muller, H. Rosen, H. Volk, T. Diamantstein. 1991. Tumor suppression after tumor cell-targeted tumor necrosis factor-{alpha} gene transfer. J. Exp. Med. 173:1047.[Abstract/Free Full Text]
  5. Teng, M. N., B. H. Park, H. K. W. Koeppen, K. J. Tracey, B. M. Fendly, H. Schreiber. 1991. Long-term inhibition of tumor growth by tumor necrosis factor in the absence of cachexia or T cell immunity. Proc. Natl. Acad. Sci. USA 88:3535.[Abstract/Free Full Text]
  6. Musiani, P., A. Allione, A. Modica, P. L. Lollini, M. Giovarelli, F. Cavallo, F. Belardelli, G. Forni, A. Modesti. 1996. Role of neutrophils and lymphocytes in inhibition of a mouse mammary adenocarcinoma engineered to release IL-2, IL-4, IL-7, IL-10, IFN-{alpha}, IFN-{gamma} and TNF-{alpha}. Lab. Invest. 74:146.[Medline]
  7. Matory, Y. L., D. M. Dorfman, L. Wu, M. Chen, P. Goedegebuure, T. J. Eberlein. 1999. Treatment of established tumor is associated with ICAM-1 upregulation and reversed by CD8 depletion in a tumor necrosis factor-{alpha} gene transfected mammary tumor. Pathobiology 67:186.[Medline]
  8. Lewis, M., L. A. Tartaglia, A. Lee, G. L. Bennett, G. C. Rice, G. H. W. Wong, E. Y. Chen, D. V. Goeddel. 1991. Cloning and expression of cDNAs for two distinct murine tumor necrosis factor receptors demonstrate one receptor is species specific. Proc. Natl. Acad. Sci. USA 88:2830.[Abstract/Free Full Text]
  9. Himeno, T., N. Watanabe, N. Yamauchi, M. Maeda, Y. Tsuji, T. Okamoto, H. Neda, Y. Niitsu. 1990. Expression of endogenous tumor necrosis factor as a protective protein against the cytotoxicity of exogenous tumor necrosis factor. Cancer Res. 50:4941.[Abstract/Free Full Text]
  10. Vanhaesebroeck, B., E. Decoster, X. Van Ostade, S. Van Bladel, A. Lenaerts, F. Van Roy, W. Fiers. 1992. Expression of an exogenous tumor necrosis factor (TNF) gene in TNF-sensitive cell lines confers resistance to TNF-mediated cell lysis. J. Immunol. 148:2785.[Abstract]
  11. Huang, S., W. Hendriks, A. Althage, S. Hemmi, H. Blüthmann, R. Kamijo, J. Vilcek, R. M. Zinkernagel, M. Aguet. 1993. Immune response in mice that lack the interferon-{gamma} receptor. Science 259:1742.[Abstract/Free Full Text]
  12. Stauss, H. J., C. Van Waes, M. A. Fink, B. Starr, H. Schreiber. 1986. Identification of a unique tumor antigen as rejection antigen by molecular cloning and gene transfer. J. Exp. Med. 164:1516.[Abstract/Free Full Text]
  13. Qin, Z., T. Blankenstein. 2000. CD4+ T cell-mediated tumor rejection involves inhibition of angiogenesis that is dependent on IFN{gamma} receptor expression by nonhematopoietic cells. Immunity 12:677.[Medline]
  14. Cherwinski, H. M., J. H. Schumacher, K. D. Brown, T. R. Mosmann. 1987. Two types of mouse helper T cell clone. III. Further differences in lymphokine synthesis between Th1 and Th2 clones revealed by RNA hybridization, functionally monospecific bioassays, and monoclonal antibodies. J. Exp. Med. 166:1229.[Abstract/Free Full Text]
  15. Hock, H., M. Dorsch, U. Kunzendorf, Z. Qin, T. Diamantstein, T. Blankenstein. 1993. Mechanism of rejection induced by tumor cell-targeted gene transfer of interleukin 2, interleukin 4, interleukin 7, tumor necrosis factor, or interferon {gamma}. Proc. Natl. Acad. Sci. USA 90:2774.[Abstract/Free Full Text]
  16. Mumberg, D., P. A. Monach, S. Wanderling, M. Philip, A. Y. Toledano, R. D. Schreiber, H. Schreiber. 1999. CD4+ T cells eliminate MHC class II-negative cancer cells in vivo by indirect effects of IFN-{gamma}. Proc. Natl. Acad. Sci. USA 96:8633.[Abstract/Free Full Text]
  17. Oliff, A., D. Defeo-Jones, M. Boyer, D. Martinez, D. Kiefer, G. Vuocolo, A. Wolfe, S. H. Socher. 1987. Tumors secreting human TNF/cachectin induce cachexia in mice. Cell 50:555.[Medline]
  18. Blanchard, D. K., J. Y. Djeu, T. W. Klein, H. Friedman, W. E. Stewart II. 1986. Interferon-{gamma} induction by lipopolysaccharide: dependence on interleukin 2 and macrophages. J. Immunol. 136:963.[Abstract]
  19. Ramana, C. V., M. P. Gil, Y. Han, R. M. Ransohoff, R. D. Schreiber, G. R. Stark. 2001. Stat1-independent regulation of gene expression in response to IFN-{gamma}. Proc. Natl. Acad. Sci. USA 98:6674.[Abstract/Free Full Text]
  20. Seung, L. P., R. R. Weichselbaum, A. Toledano, K. Schreiber, H. Schreiber. 1996. Radiation can inhibit tumor growth indirectly while depleting circulating leukocytes. Radiat. Res. 146:612.[Medline]
  21. Magram, J., S. E. Connaughton, R. R. Warrier, D. M. Carvajal, C. Y. Wu, J. Ferrante, C. Stewart, U. Sarmiento, D. A. Faherty, M. K. Gately. 1996. IL-12-deficient mice are defective in IFN{gamma} production and type 1 cytokine responses. Immunity 4:471.[Medline]
  22. Takeda, K., H. Tsutsui, T. Yoshimoto, O. Adachi, N. Yoshida, T. Kishimoto, H. Okamura, K. Nakanishi, S. Akira. 1998. Defective NK cell activity and Th1 response in IL-18-deficient mice. Immunity 8:383.[Medline]
  23. Lukacs, N. W., S. L. Kunkel, R. Allen, H. L. Evanoff, C. L. Shaklee, J. S. Sherman, M. D. Burdick, R. M. Strieter. 1995. Stimulus and cell-specific expression of C-X-C and C-C chemokines by pulmonary stromal cell populations. Am. J. Physiol. 268:L856.
  24. Ngo, V. N., H. Korner, M. D. Gunn, K. N. Schmidt, D. S. Riminton, M. D. Cooper, J. L. Browning, J. D. Sedgwick, J. G. Cyster. 1999. Lymphotoxin {alpha}/{beta} and tumor necrosis factor are required for stromal cell expression of homing chemokines in B and T cell areas of the spleen. J. Exp. Med. 189:403.[Abstract/Free Full Text]
  25. Pinhal-Enfield, G., M. Ramanathan, G. Hasko, S. N. Vogel, A. L. Salzman, G. J. Boons, S. J. Leibovich. 2003. An angiogenic switch in macrophages involving synergy between Toll-like receptors 2, 4, 7, and 9 and adenosine A(2A) receptors. Am. J. Pathol. 163:711.[Abstract/Free Full Text]
  26. Ming, W. J., L. Bersani, A. J. Mantovani. 1987. Tumor necrosis factor is chemotactic for monocytes and polymorphonuclear leukocytes. J. Immunol. 138:1469.[Abstract]
  27. Mano-Hirano, Y., N. Sato, Y. Sawasaki, K. Haranaka, N. Satomi, H. Nariuchi, T. Goto. 1987. Inhibition of tumor-induced migration of bovine capillary endothelial cells by mouse and rabbit tumor necrosis factor. J. Natl. Cancer Inst. 78:115.
  28. Moldovan, N. I.. 2002. Role of monocytes and macrophages in adult angiogenesis: a light at the tunnel’s end. J. Hematother. Stem Cell Res. 11:179.[Medline]
  29. Cousens, L. P., R. Peterson, S. Hsu, A. Dorner, J. D. Altman, R. Ahmed, C. A. Biron. 1999. Two roads diverged: interferon {alpha}/{beta}- and interleukin 12-mediated pathways in promoting T cell interferon {gamma} responses during viral infection. J. Exp. Med. 189:1315.[Abstract/Free Full Text]
  30. Fehniger, T. A., H. Yu, M. A. Cooper, K. Suzuki, M. H. Shah, M. A. Caligiuri. 2000. IL-15 costimulates the generalized Shwartzman reaction and innate immune IFN-{gamma} production in vivo. J. Immunol. 164:1643.[Abstract/Free Full Text]
  31. Coughlin, C. M., K. E. Salhany, M. S. Gee, D. C. LaTemple, S. Kotenko, X. J. Ma, G. Gri, M. Wysocka, J. E. Kim, L. Liu, et al 1998. Tumor cell responses to IFN{gamma} affect tumorigenicity and response to IL-12 therapy and antiangiogenesis. Immunity 9:25.[Medline]
  32. Keller, R., R. Keist, T. P. Leist, P. H. van der Meide. 1990. Resistance to a non-immunogenic tumor, induced by Corynebacterium parvum or Listeria monocytogenes, is abrogated by anti-interferon {gamma}. Int. J. Cancer 46:687.[Medline]
  33. Doherty, G. M., H. R. Alexander, M. J. Merino, D. J. Venzon, J. A. Norton. 1996. Role of endogenous interferon {gamma} in murine tumor growth and tumor necrosis factor {alpha} antitumor efficacy. Ann. Surg. Oncol. 3:198.[Medline]
  34. Carswell, E. A., L. J. Old, R. L. Kassel, S. Green, N. Fiore, B. Williamson. 1975. An endotoxin-induced serum factor that causes necrosis of tumor. Proc. Natl. Acad. Sci. USA 72:3666.[Abstract/Free Full Text]
  35. Haranaka, K., N. Satomi, A. Sakurai. 1984. Antitumor activity of murine tumor necrosis factor (TNF) against transplanted murine tumors and heterotransplanted human tumors in nude mice. Int. J. Cancer 34:263.[Medline]
  36. Havell, E. A., W. Fiers, R. J. North. 1988. The antitumor function of tumor necrosis factor (TNF) I. Therapeutic action of TNF against an established murine sarcoma is indirect, immunologically dependent, and limited by severe toxicity. J. Exp. Med. 167:1067.[Abstract/Free Full Text]
  37. Fraker, D. L., B. C. Sheppard, J. A. Norton. 1990. Impact of tolerance on antitumor efficacy of tumor necrosis factor in mice. Cancer Res. 50:2261.[Abstract/Free Full Text]
  38. Delneste, Y., P. Charbonnier, N. Herbault, G. Magistrelli, G. Caron, J. Y. Bonnefoy, P. Jeannin. 2003. Interferon-{gamma} switches monocyte differentiation from dendritic cells to macrophages. Blood 101:143.[Abstract/Free Full Text]
  39. Klimp, A. H., E. G. de Vries, G. L. Scherphof, T. Daemen. 2002. A potential role of macrophage activation in the treatment of cancer. Crit. Rev. Oncol. Hematol. 44:143.[Medline]
  40. Mills, C. D., K. Kincaid, J. M. Alt, M. J. Heilman, A. M. Hill. 2000. M-1/M-2 macrophages and the Th1/Th2 paradigm. J. Immunol. 164:6166.[Abstract/Free Full Text]
  41. Tamura, T., T. Nagamura-Inoue, Z. Shmeltzer, T. Kuwata, K. Ozato. 2000. ICSBP directs bipotential myeloid progenitor cells to differentiate into mature macrophages. Immunity 13:155.[Medline]



This article has been cited by other articles:


Home page
 J. Immunol.Home page
Y. Lu, W. Yang, C. Qin, L. Zhang, J. Deng, S. Liu, and Z. Qin
Responsiveness of Stromal Fibroblasts to IFN-{gamma} Blocks Tumor Growth via Angiostasis
J. Immunol., November 15, 2009; 183(10): 6413 - 6421.
[Abstract] [Full Text] [PDF]

Home page
 BloodHome page
T. Hagemann, S. K. Biswas, T. Lawrence, A. Sica, and C. E. Lewis
Regulation of macrophage function in tumors: the multifaceted role of NF-{kappa}B
Blood, April 2, 2009; 113(14): 3139 - 3146.
[Abstract] [Full Text] [PDF]

Home page
 J. Immunol.Home page
D. Li, Y. Li, X. Wu, Q. Li, J. Yu, J. Gen, and X.-L. Zhang
Knockdown of Mgat5 Inhibits Breast Cancer Cell Growth with Activation of CD4+ T Cells and Macrophages
J. Immunol., March 1, 2008; 180(5): 3158 - 3165.
[Abstract] [Full Text] [PDF]

Home page
 Clin. Cancer Res.Home page
S. Zhu, M. Waguespack, S. A. Barker, and S. Li
Doxorubicin Directs the Accumulation of Interleukin-12 Induced IFN{gamma} into Tumors for Enhancing STAT1 Dependent Antitumor Effect
Clin. Cancer Res., July 15, 2007; 13(14): 4252 - 4260.
[Abstract] [Full Text] [PDF]

Home page
 Cancer Res.Home page
X. Zhao, M. Mohaupt, J. Jiang, S. Liu, B. Li, and Z. Qin
Tumor Necrosis Factor Receptor 2-Mediated Tumor Suppression Is Nitric Oxide Dependent and Involves Angiostasis
Cancer Res., May 1, 2007; 67(9): 4443 - 4450.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow Request Permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Wu, T. H.
Right arrow Articles by Schreiber, H.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Wu, T. H.
Right arrow Articles by Schreiber, H.

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS