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The Journal of Immunology, 2001, 167: 5583-5593.
Copyright © 2001 by The American Association of Immunologists

Tumor-Infiltrating Macrophages Induce Apoptosis in Activated CD8+ T Cells by a Mechanism Requiring Cell Contact and Mediated by Both the Cell-Associated Form of TNF and Nitric Oxide1

Masanao Saio*,{dagger}, Sasa Radoja*,{ddagger}, Mike Marino§ and Alan B. Frey*,2

* Department of Cell Biology and Kaplan Cancer Center, New York University School of Medicine, New York, NY 10016; {dagger} Second Department of Pathology, Gifu University School of Medicine, Gifu, Japan; {ddagger} Institute of Molecular Genetics and Genetic Engineering, Belgrade, Yugoslavia; and § Ludwig Institute for Cancer Research, Memorial Sloan-Kettering Cancer Center, New York, NY 10021


   Abstract
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 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
We have investigated the ability of different cells present in murine tumors to induce apoptosis of activated CD8+ T cells in vitro. Tumor cells do not induce apoptosis of T cells; however, macrophages that infiltrate tumors are potent inducers of apoptosis. Tumor macrophages express cell surface-associated TNF, TNF type I (CD120a) and II (CD120b) receptors, and, upon contact with T cells which induces release of IFN-{gamma} from T cells, secrete nitric oxide. Killing of T cells in vitro is blocked by Abs to IFN-{gamma}, TNF, CD120a, or CD120b, or N-methyl-L-arginine. In concert with that finding, tumor macrophages isolated from either TNF type I or type II receptor -/- mice are not proapoptotic and do not produce nitric oxide upon contact with activated T cells. Control macrophages do not express TNF receptors or release nitric oxide. Tumor cells or tumor-derived macrophages do not express FasL, and blocking Abs to either Fas or FasL have no effect on macrophage-mediated T cell killing. These results demonstrate that macrophages which infiltrate tumors are highly proapoptotic and may be responsible for elimination of activated antitumor T cells within the tumor bed.


   Introduction
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 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
An essential role for T cells in elimination of murine tumors has been demonstrated in adoptive transfer experiments and active immunization of tumor-bearing hosts (1, 2, 3, 4, 5). In addition, experimental adoptive T cell immunotherapy of cancer patients has, in a limited number of cases, shown dramatic tumor remission (6). For adoptive therapy CD8+ T cells reactive with Ags expressed on tumors are sufficient for tumor elimination, but both CD4+ and CD8+ T cells are required for tumor killing after immunization. The mechanisms by which T cells cause tumor elimination include direct lysis of tumor (7, 8), recruitment of host cells which have cytotoxic function (9), or secretion of cytokines that are either cytotoxic toward tumor or affect the tumor vasculature (10, 11, 12, 13, 14). The goal of experimental immunotherapy using rodent cancers is to define principals which can guide human immunotherapy trials, some of which have begun (15, 16).

Despite findings that activation of enhanced antitumor T cell immune response by immunization or adoptive transfer of antitumor T cells can kill tumors in situ (16), the antitumor T cell immune response in nonmanipulated hosts is frequently ineffective in eliminating tumor. In addition, while experimental immunotherapy of humans has produced some dramatic examples of remission (17), most trials have been disappointing in that after initial tumoricidal or tumoristatic effects, tumor growth often continues. Several nonmutually exclusive possibilities have been postulated to explain the failure of antitumor immune response to eliminate antigenic tumors, and this subject has been recently reviewed (18, 19).

Recently several laboratories have reported that various human tumor cell lines or primary tumor cells express FasL, an observation that suggests yet another potential mechanism for tumors to escape immune destruction (20, 21, 22, 23). Compelling contradictory data have been published concerning FasL expression in human melanoma (24), but several other tumor types have been reported to both express FasL and induce apoptosis in model Fas+ targets in vitro (21). Tumor-induced apoptosis of T cells is a conceptually attractive mechanism for tumors to avoid immune killing, because only activated antitumor T cells which are recruited to the tumor site would be induced to undergo apoptosis, thereby explaining the observation that cancer patients (25) and rodents bearing tumors (26) have normal systemic T cell functions.

We have investigated the mechanism of induction of apoptosis in activated CD8+ T cells and find that macrophages which infiltrate tumors are potent inducers of apoptosis. T cell death is caused by release of NO from tumor macrophages, which is dependent on macrophage cell surface TNF and initiated by secretion of IFN-{gamma} from activated T cells. Experiments using macrophages from tumors grown in TNF receptor -/- mice demonstrated that expression of both type I and II receptors on macrophages is required to induce both NO production and T cell death. Collectively these data suggest that NO produced by macrophages within the tumor microenvironment may be the basis for apoptotic death of tumor-infiltrating T cells in situ.


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

C57BL/6 and TNF receptor -/- male mice were obtained from The Jackson Laboratory (Bar Harbor, ME). TNF receptor -/- mice were back-crossed to C57BL/6 by The Jackson Laboratory 10–13 times (n = 10–13) and familial-crossed seven times before purchase. They were bred by brother-sister cross at New York University School of Medicine (New York, NY) for more than six generations before use in experiments described in this work (F > 13). TNF -/- mice have been described (27). Mice were housed four per cage in a barrier facility and maintained on a 12-h light/dark cycle (7 a.m. to 7 p.m.) with ad libitum access to food and water. A sentinel program revealed that the mice were mouse hepatitis virus-negative and the tumor cell lines are mouse hepatitis virus-negative as assessed by MAPS testing. Experiments involving animals were conducted with the approval of the New York University School of Medicine Committee on Animal Research.

Tumors

3-Methylcholanthrene-38 (MCA-38)3 adenocarcinoma was the gift of Y. Liu (Ohio State University, Columbus, OH). Tumor cell lines were removed from tissue culture plastic by incubation in HBSS containing 2 mM EDTA and washed three times in HBSS. Viability of cell lines was determined by trypan blue dye exclusion, and 2 x 106 cells were injected s.c. in a volume of 0.1 ml of HBSS for tumor induction.

Tissue culture

RPMI 1640 medium (BioWhittaker, Walkersville, MD) was used for isolation and culture of macrophages and T cells and was supplemented with 100 U/ml penicillin, 0.1 mg/ml streptomycin, 0.002 mM L-glutamine, and 10% FBS (Intergen, Purchase, NY). Tissue culture supplements were supplied by Grand Island Biological (Grand Island, NY).

Isolation of macrophages from tumor or PEC

Peritoneal exudate cells (PEC) were isolated by lavage with HBSS 5 days after i.p. injection of 4 ml of thioglycolate broth. Conditions for tumor digestion were optimized to permit maximal recovery of viable cells. Tumors were dissected and chopped into small pieces using a razor blade before incubation with a mixture of enzymes dissolved in HBSS (0.05 mg/ml collagenase type I, 0.05 mg/ml collagenase type IV, 0.025 mg/ml hyaluronidase, all from Sigma-Aldrich, St. Louis, MO; and 0.01 mg/ml DNase I and 0.2 TIU/ml soybean trypsin inhibitor, both from Boehringer Mannheim, Indianapolis, IN) for 15 min at 37°C. Cells were recovered by centrifugation and resuspended in a fresh aliquot of enzymes for a second 15-min incubation at 37°C. Undigested material was settled for 2 min at 1 x g and liberated cells were recovered and washed by centrifugation in complete medium. T cells were depleted using immunomagnetic separation using type MS+ or VS+ columns and anti-CD4 and anti-CD8 conjugated magnetic beads according to the manufacturer’s instructions (MACS; Miltenyi Biotec, Bergish-Gladbach, Germany) as described previously (26). Briefly, 0.01 ml of bead suspension was added to 107 cells in a final volume of 0.1 ml of cold HBSS (containing 0.5% BSA). After incubation for 15 min on ice, cells were washed and passed through the separation column. Cells not retained were collected and incubated with biotinylated anti-F4/80 (Caltag Laboratories, Burlingame, CA) for 30 min and then with streptavidin-conjugated magnetic beads before macrophage isolation by magnetic adsorption. F4/80+ macrophages were isolated from PEC by magnetic immunobeading in an identical manner. More than 95% of F4/80+ macrophages express CD11b.

Generation of activated CD8+ T cells from control mouse spleens

Spleens were isolated and single-cell suspensions were prepared. Following lysis of RBC in hypotonic buffer cells were filtered through a nylon mesh and washed by centrifugation. CD8+ T cells were enriched by negative selection using magnetic immunobeads reactive with CD4, CD11b, and B220 Ags. Isolated CD8+ T cells (typically ~90% pure) were plated at 3 x 106 cells/well in 24-well dishes containing 5 ng/ml phorbol 12-myristate 13-acetate (Sigma-Aldrich) plus 500 ng/ml ionomycin (Sigma-Aldrich) or plate-bound anti-TCR (H57). After 24 h of stimulation viable cells were recovered on a 30% Percoll step gradient and washed three times in complete medium. Activation of CD8+ T cells by incubation with plate-bound anti-TCR Ab (H57) produced identical results in all experiments.

Fluorocytometry

For single-color analysis, splenocytes (106) from control or tumor-bearing mice or tumor-infiltrating lymphocyte (TIL) (2–3 x 105) were washed once with FACS buffer (HBSS without phenol red, 1% BSA (Sigma-Aldrich), and 0.1% sodium azide). Cells were incubated for 45 min on ice with 0.0005 mg of fluorochrome-conjugated Abs in a volume of 0.1 ml including 0.01 mg/ml human IgG (Baxter, Chicago, IL) and 0.002 mg/ml anti-murine CD16/32 to block nonspecific binding (clone CT-17.1; Caltag Laboratories). Following washing with FACS buffer, cells were fixed with 1% paraformaldehyde before analysis using a FACScan Flow Cytometer (BD Biosciences, Mountain View, CA).

For use in fluorocytometry analyses the following Abs were used: CD11b (clone M1/70.15; Caltag Laboratories), CD14 (clone rmC5-3; BD PharMingen, San Diego, CA), CD25 (clone PC61.5.3; Caltag Laboratories), CD69 (clone H1.2F3; Caltag Laboratories), F4/80 (clone CI:A3-1; Caltag Laboratories), TNF (clone MP6-XT22; BD PharMingen), FasL (clone MFL3; BD PharMingen), Ly6C (clone RB6-8C5; Caltag Laboratories), TNF receptor type I (clone HM104; Caltag Laboratories), and TNF receptor type II (clone HM104; Caltag Laboratories).

For use in Ab blocking experiments the following reagents were used: TNF receptor type I (clone 55R-170; BD PharMingen), TNF receptor type II (clone TK75-54; BD PharMingen), nonimmune hamster IgG (clone G235-2356; BD PharMingen), IFN-{gamma} (clone R4-6A2; BD PharMingen), and TNF (clone MP6-XT3; BD PharMingen).

Coculture of macrophage and activated T cells

A total of 3 x 106 macrophages were incubated for 4 h in 24-well plates before T cells were added at various cell ratios (ranging from 1:1 to 0.1:1, macrophage:T cell). Cocultures were performed in triplicate using identical numbers of macrophages, and recovery data are expressed as the percentage of input cells showing standard deviations for triplicate wells. At different times following coculture, T cells were recovered by collection of culture supernatants and washing of tissue culture plates with medium. The recovery of T cells was determined by enumeration of trypan blue stained cells and also by fluorocytometry after staining with 7-amino-actinomycin D, PE-conjugated anti-CD8 (Caltag Laboratories), and annexin V-FITC (BD PharMingen).

All T cell recovery experiments were performed more than three times and representative data are shown.

Nitrite assay

NO production was assayed in supernatants collected from triplicate cocultures of T cells and macrophages by the method of Griess (28). Supernatant (0.05 ml) was mixed with 0.050 ml of Griess reagent (Sigma-Aldrich) and A550 nm was recorded. Nitrite concentration was determined using NaNO2 to prepare a standard curve.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
TAM, but not control F4/80+ peritoneal macrophages, induce T cell apoptosis

To study the ability of different cells within tumors to induce apoptosis in activated T cells, single-cell suspensions of MCA-38 adenocarcinoma were prepared by enzymatic digestion, and different cells were isolated and tested for apoptosis induction in coculture experiments with activated CD8+ T cells. Primary tumor cells, and also tumor cell lines carried in culture, were unable to induce death of either naive (data not shown) or activated T cells (Fig. 1GoA). However, recovery of CD8+ T cells activated by either PMA plus ionomycin or plate-bound anti-TCR was dramatically reduced by coculture with highly purified F4/80+ tumor-associated macrophages (TAM) (3) isolated from primary tumors (Fig. 1GoB). As a control, F4/80+ macrophages were purified from PEC and tested in parallel with TAM for T cell killing.



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  		FIGURE 1. Tumor macrophages induce apoptosis in activated CD8+ T cells. CD8+ T cells were isolated from spleens of mice (by negative selection as described in Materials and Methods) and cocultured in 24-well assay plates with either syngeneic PEC or TAM from MCA-38 tumors. F4/80+ macrophages from either source were purified by positive selection as described in Materials and Methods and were used at a ratio of 3:2 (macrophage:T cell). A, Tumor cells were used at a ratio of 1:1. At the indicated times wells were harvested, disaggregated (by treatment with collagenase and hyaluronidase for 1 h at room temperature), washed with PBS, and then stained with trypan blue and enumerated. Shown in A–C and F are the percentage of cell recovery at each time point. D, Cells were labeled with annexin V-FITC and CD8b-PE, stained with 7-amino actinomycin D, and analyzed by flow cytometry instead of purification by magnetic immunobeading. E, An agarose gel of activated CD8+ T cell total cellular DNA after 14 h of incubation with tumor macrophages as indicated. Control T cells and TAM alone were also incubated for 14 h. F, TAM and T cells were cocultured in transwell plates for the indicated times before harvesting of T cells, enumeration, and flow cytometry. A, CD8+ T cells prepared from control mice after activation in vitro were cultured with tumor cells isolated from primary tumor after magnetic immunobead depletion of F4/80+, CD4+, CD8+, and B220+ host cells. B, CD8+ T cells prepared from control mice after activation in vitro (with either PMA plus ionomycin or plate-bound anti-TCR) were cultured with TAM. C, CD8+ T cells without activation in vitro. D, CD8+ T cells after activation in vitro. E, CD8+ T cells after activation in vitro and control mouse thymocytes (after treatment with dexamethasone for 24 h in vitro). F, CD8+ T cells after activation in vitro and then coculture in transwell plates.

 
Activated T cells grow without the requirement of feeder cells (Fig. 1GoA) and, in contrast to the cytotoxic phenotype of TAM, equivalent numbers of PEC F4/80+ macrophages dramatically stimulated the growth of activated T cells in vitro (Fig. 1GoB). The effect of TAM coculture on control (unactivated) T cells was very different from the effect on activated T cells: control T cells were not eliminated from cultures with TAM until the latest time points of the assay, and then not in significant numbers (Fig. 1GoC). Coculture with PEC did not stimulate expansion of control T cells, but cells were not lysed.

To characterize the mechanism of T cell killing by TAM in vitro we asked whether macrophages induced apoptosis of T cells. Because activated T cells express annexin V binding without prior contact with TAM, to detect a difference between coculture with PEC and TAM we compared the relative mean fluorescence intensity of activated T cells after culture with PEC or TAM and found that annexin V labeling was enhanced by TAM (Fig. 1GoD). In addition, total cellular DNA prepared from either TAM alone (Fig. 1GoE, lane 3) or activated CD8+ T cells alone (Fig. 1GoE, lane 5) is not fragmented. However, DNA from T cell-tumor macrophage cocultures is fragmented into a ladder pattern characteristic of apoptosis (Fig. 1GoE, lane 4).

We then asked whether TAM secrete a factor or factors which might induce T cell apoptosis. Conditioned medium of macrophage cultures did not induce apoptosis in activated T cells (data not shown), so we determined whether cell-to-cell contact was required for T cell death. Transwell cultures showed that cell contact was required for induction of T cell apoptosis: when separated by porous filters growth of T cells is stimulated (Fig. 1GoF). We conclude from these experiments that TAM, but not macrophages isolated from PEC, induce apoptosis in activated CD8+ T cells in a contact-dependent manner.

CD8+ T cells must be activated to be susceptible to TAM-induced apoptosis

We asked whether activation Ags are expressed on susceptible T cells. Activated and control T cells were prepared and analyzed for cell surface expression of CD69, an early activation marker (data not shown), and other molecules characteristic of cellular activation (see below). Control cells express very low levels of CD69 in concert with their nonproliferating phenotype characterized by very low levels of both thymidine incorporation and low percentage of cells in S phase (data not shown). In contrast, activated T cells express higher levels of CD69 in keeping with their high proliferation index (data not shown). Importantly, at early times following coculture with TAM, control T cells rapidly up-regulate expression of CD69. Incubation of nonproapoptotic PEC with control T cells does not cause up-regulation of CD69 expression. Collectively these data suggest that to be susceptible to TAM-induced apoptosis T cells must be activated and in a proliferative state.

Characterization of macrophage cell surface activation Ags

TAM were purified by magnetic immunobeading and analyzed by flow cytometry (data not shown). F4/80+ TAM do not express CD80, CD86, the neutrophil marker Ly6C, FasL, or Fas. TAM express low levels of the LPS receptor CD14 and high levels of CD11b and MHC class I and class II. Importantly, TAM express cell surface TNF (see below) which is not shed upon culture in vitro either in the presence or absence of cognate tumor cells (data not shown).

Blocking Abs to TNF or TNF type I or type II receptor inhibits TAM-induced T cell death in vitro

We considered that induction of T cell apoptotic cell death uses a mechanism dependent upon TAM cell surface-associated TNF, because TAM express cell surface TNF and induction of apoptosis required cell-to-cell contact. To directly measure this we performed cell lysis coculture experiments in the presence or absence of selected blocking Abs (Fig. 2Go). Inclusion of anti-TNF Ab resulted in dramatic recovery of T cells: nearly 190% of the input T cells were recovered after 48 h of coculture. Anti-TNF type I receptor Ab was nearly as potent in reversing T cell death: ~140% of input T cells were recovered. Blocking Ab to the type II TNF receptor was able to partially recover T cell lysis (~75% of input cells). Anti-FasL Ab had only a marginal effect and control Ab had no effect on cell recovery (~25% of input cells recovered). These data strongly suggest a role for macrophage cell surface TNF in apoptotic T cell death.



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  		FIGURE 2. Tumor macrophages induce apoptosis in activated T cells via TNF. TAM were isolated from MCA-38 tumors and cocultured with syngeneic activated CD8+ T cells in 24-well plates in triplicate as described in Materials and Methods. Wells contained the following Abs during the 48-h coculture: nonimmune hamster IgG (control), anti-FasL, anti-TNF, anti-TNF receptor type I, or anti-TNF receptor type II. Abs were used at final concentration of 0.01 mg/ml and were added to cultures at the time of cell mixing. At the end of the coculture wells were harvested and cell recovery was determined as described in Materials and Methods.

 
Expression of cell surface TNF and TNF receptor on TAM, PEC-derived macrophages, and CD8+ T cells

Activated T cells express the TNF receptor but do not express cell surface TNF, corroborating the contention that T cells cannot be the source of TNF in this system (Fig. 3Go). In addition, although PEC-derived F4/80+ macrophages express cell surface TNF, they do not express either CD120a or CD120b, an observation which we suggest is the basis for their nonproapoptotic phenotype (see below). A role for TNF receptor signaling in TAM was additionally confirmed by flow cytometric analysis of TAM, which showed that TAM from TNF receptor +/+ mice express CD120a and CD120b. The pattern of expression of TNF receptor correlates with the proapoptotic character of TAM or PEC derived from wild-type or TNF receptor -/- mice. These findings collectively demonstrate a requirement for both TNF type I and type II receptor signaling in TAM to manifest the proapoptotic phenotype.



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  		FIGURE 3. Flow cytometric analysis of T cells, F4/80+ PEC, or F4/80+TAM. F4/80+ cells were isolated by magnetic immunobeading from either a representative MCA-38 tumor 3–4 wk after tumor seeding (~3 cm2) or PEC. Activated T cells were isolated from splenocytes as described previously. Immediately after isolation cells were analyzed by flow cytometry after labeling with the indicated fluorochrome-conjugated Abs. The filled portion of the histograms shows staining achieved with isotype-matched control Ab. This analysis shows that control TAM express TNF type I and TNF type II receptor and that nonproapoptotic PEC do not express TNF type I or type II receptor.

 
Activated T cells from TNF receptor -/- mice are susceptible to TAM-induced apoptosis

Blockade of apoptosis with anti-TNF or anti-TNF receptor Abs demonstrated a role for TNF signaling in TAM-induced T cell apoptosis but did not identify which cell, macrophage or T cell, uses TNF signaling in this process. To address this point we performed the following experiments. First, we prepared activated T cells or TAM from mice having targeted disruption in either the TNF type I or type II receptor genes (CD120a or CD120b, respectively), which are syngeneic with MCA-38 adenocarcinoma, and performed several experiments. The first experiment was to coculture activated T cells (TNF receptor -/-) with TAM from wild-type control mice (TNF receptor +/+) and assess recovery (Fig. 4GoA). T cells from either TNF receptor -/- mice were killed by coculture with TAM to the same extent as control T cells (~20–30% of T cells remain after 34 h of coculture) although there was a slight difference between recovery of T cells from TNF receptor I -/- mice at early times of coculture compared with wild-type T cells (180% of input vs 105% at 10 h; see Discussion).



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  		FIGURE 4. A, Recovery of CD8+ T cells obtained from TNF receptor I or II -/- mice after coculture with TAM. Coculture experiments were performed exactly as described previously using activated CD8+ T cells obtained from either normal (control) or TNF receptor type I or II -/- mice, and TAM were obtained from MCA-38 tumors grown in normal mice. This experiment shows that activated T cells lacking TNF receptor type I or II are susceptible to killing by TAM obtained from wild-type mice. B, Recovery of CD8+ T cells from control mice after coculture with TAM obtained from either normal, TNF receptor type I, or TNF receptor type II -/- mice. Coculture experiments were performed exactly as described previously using activated T cells obtained from normal mice and TAM obtained from MCA-38 tumors grown in either normal, TNF receptor I, or TNF receptor II -/- mice. C, TAM obtained from TNF receptor -/- mice do not induce apoptosis in activated T cells. Flow cytometric analysis of T cells cocultured for 16 h before TUNEL assay for apoptosis. This experiment shows that activated T cells expand significantly in number after coculture with TAM obtained from TNF receptor I -/- mice and that apoptotic T cell death is enhanced by TAM obtained from TNF receptor +/+ mice. D, Recovery of CD8+ T cells obtained from TNF -/- mice after coculture with TAM. Coculture experiments were performed exactly as described previously using activated CD8+ T cells obtained from TNF -/- mice. TAM were obtained from MCA-38 tumors grown in control mice and PEC were obtained from control mice. This experiment shows that activated T cells lacking TNF expression are susceptible to killing by TAM obtained from wild-type mice.

 
TAM obtained from TNF type I or type II receptor -/- mice are not proapoptotic

Because T cells derived from TNF receptor -/- mice resemble T cells from wild-type mice in terms of sensitivity to TAM-induced apoptosis, we next asked whether TNF receptor function in TAM was required for the proapoptotic phenotype. TAM were prepared from TNF receptor -/- mice and cocultured with activated T cells from wild-type mice (Fig. 4GoB). TAM from both TNF type I and type II receptor -/- mice were not only not proapoptotic, but stimulated T cell growth. T cell apoptosis was confirmed by TUNEL assay after coculture (Fig. 4GoC).

T cells obtained from TNF -/- mice are susceptible to TAM-induced apoptosis

We were unable to detect secretion of TNF by TAM or activated T cells (either alone or after coculture with tumor cells; data not shown), and we showed that TAM express cell surface TNF, implying strongly that TAM are the source of TNF which induce T cell apoptosis. However, it was conceivable that activated T cells may secrete TNF upon contact with TAM, which may contribute to cell death, but that we failed to detect the secreted TNF for unknown reasons. We investigated this possibility by using activated T cells prepared from TNF -/- mice in coculture experiments with TAM isolated from control mice (Fig. 4GoD). We found that T cells isolated from TNF -/- mice are susceptible to TAM-induced apoptosis (with similar kinetics as wild-type mice), thereby corroborating the observation that there is no detectable TNF secreted by T cells and eliminating the possibility that T cell-derived TNF is responsible for induction of apoptosis.

Recovery of T cells is enhanced by inhibition of iNOS

Coculture experiments showed that induction of T cell death by proapoptotic TAM required extended contact (~18 h). T cell death was shown to involve DNA fragmentation (Fig. 1Go) and caspase function (data not shown). To further investigate the mechanism of T cell death, we asked whether NO played a role in cell death (Fig. 5GoA). Inclusion of N-methyl-L-arginine, a competitive inhibitor of the rate-limiting enzyme in NO production, in cocultures of TAM and T cells resulted in dramatic enhancement of T cell recovery, strongly suggesting that NO production from TAM was responsible for T cell death.



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  		FIGURE 5. A, Recovery of T cells is enhanced by inhibition of iNOS. Coculture experiments were performed exactly as described previously using activated T cells and TAM obtained from normal mice and included 1 mM N-methyl-L-arginine in cultures as indicated. This experiment shows that inhibition of NO production significantly enhances T cell recovery. B, Production of nitric oxide by TAM obtained from control or TNF type I receptor and TNF type II receptor -/- mice. Coculture experiments were performed exactly as described previously using activated CD8+ T cells obtained from normal mice and TAM obtained from either control, TNF type I receptor, or TNF type II receptor -/- mice. PEC were prepared in normal mice. Supernatants were collected from cocultures and nitrite concentration was determined. This experiment shows that both nonproapoptotic PEC and TAM prepared from both TNF type I or type II receptor -/- mice do not produce significant levels of NO, whereas TAM from control mice secrete significant NO upon coculture with activated T cells.

 
To corroborate this finding, direct assessment of NO levels in cocultures was made (Fig. 5GoB). Nonproapoptotic PEC and TAM isolated from TNF type I receptor -/- mice produce low levels of NO upon coculture with activated T cells. In contrast, TAM isolated from wild-type TNF receptor +/+ mice produce almost 10 times more NO upon coculture (at 34 h). The requirement for TAM contact with T cells to kill as noted above is also true for NO production: TAM cultured without T cells produce significantly less NO than after coculture with T cells. The kinetics of NO production showed that greater levels of NO were produced upon extended cell contact, an observation in keeping with the kinetics of T cell death.

NO production from TAM is stimulated by coculture with activated T cells and inhibited by anti-IFN-{gamma} Ab

Stimulation of NO production from TAM after coculture with activated T cells suggested the possibility that T cell contact initiated NO production. Because NO production has been shown to be augmented by IFN-{gamma} (29, 30, 31, 32, 33), we tested the effect of anti-IFN-{gamma} Ab on NO production from T cell-TAM cocultures (Fig. 6GoA). As previously shown, TAM cultured with activated T cells produce significantly more NO than TAM cultured alone, and TNF receptor -/- TAM produce very low levels of NO. Inclusion of anti-IFN-{gamma} Ab in cocultures significantly reduced, but did not eliminate, nitrite accumulation. The inability to reduce NO levels to background by inclusion of blocking Ab (i.e., to that of TNF type I receptor -/- levels) suggests that IFN-{gamma} initiates or augments NO production but TNF is the major stimulus. In addition, as shown below, very high levels of IFN-{gamma} are secreted by T cells upon contact with TAM which may be difficult to inhibit completely in vitro (see Fig. 6GoC).



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  		FIGURE 6. A, Nitrite production from TAM is stimulated by coculture with activated T cells and inhibited by anti-IFN-{gamma} Ab. Cocultures using activated CD8+ T cells and TAM from either control or TNF type I receptor -/- mice were prepared as described previously. Cultures included blocking anti-IFN-{gamma} Ab or not as indicated (0.04 mg/ml). Supernatants were collected at the indicated times of culture and nitrite concentration was determined as described previously. This experiment shows that TAM prepared from control mice produce high levels of NO upon culture with activated T cells which is inhibited by anti-IFN-{gamma} Ab. NO production is also dependent upon TNF type I receptor signaling because TAM obtained from TNF type I receptor -/- mice produce low levels of NO. B, Neutralizing anti-IFN-{gamma} Ab partially restores recovery of T cells after coculture with TAM. Cocultures using activated CD8+ T cells and TAM from control mice were prepared as described previously. Cultures included blocking anti-IFN-{gamma} Ab at the concentrations indicated or isotype-matched control Ab (0.02 mg/ml). Recovery of viable cells was determined as described previously. This experiment shows that IFN-{gamma} plays a role in macrophage-mediated induction of T cell apoptosis. C, Coculture of TAM with activated T cells induces IFN-{gamma} secretion. Cocultures using activated CD8+ T cells and TAM from either control or TNF receptor type I -/- mice were prepared as described previously. Cultures included blocking anti-IFN-{gamma} Ab or not as indicated. Supernatants were collected at the indicated times of culture and IFN-{gamma} concentration was determined by ELISA using Ab pairs R4-6A2 and XMG1.2 (BD PharMingen) as described previously (47 ). This experiment shows that secretion of IFN-{gamma} from activated CD8+ T cells is enhanced after coculture with TAM.

 
Neutralizing anti-IFN-{gamma} Ab restores recovery of T cells after coculture with TAM

Because coculture NO levels were significantly reduced by anti-IFN-{gamma} Ab, we asked whether anti-IFN-{gamma} Ab could inhibit T cell apoptosis (Fig. 6GoB). In agreement with the finding that anti-IFN-{gamma} Ab reduced NO levels in cocultures, inclusion of anti-IFN-{gamma} Ab reduced T cell killing in a dose-dependent manner.

Coculture of TAM with activated T cells induces IFN-{gamma} secretion

To corroborate the finding that anti-IFN-{gamma} Ab reduced T cell death, the levels of IFN-{gamma} produced by activated T cells were determined by ELISA of conditioned medium (Fig. 6GoC). T cells cultured without TAM secreted ~1.8 µg/ml/2 x 106 cells (by 6 h) and, as expected, TAM secreted no IFN-{gamma}. However, cocultures of T cells plus TAM secreted ~5-fold more, showing that contact between T cells and TAM stimulated IFN-{gamma} release. Production of high levels of IFN-{gamma} by activated T cells upon contact with TAM correlates with the observation that inclusion into the coculture assay system of anti-IFN-{gamma} Ab reduces NO production and increases T cell recovery.

Nonproapoptotic F4/80+ PEC cannot be activated by LPS or IFN-{gamma} to induce T cell apoptosis

In these experiments we used F4/80+ macrophages obtained from PEC as controls, and these cells are not proapoptotic. Because PEC macrophages do not express TNF receptor, which may indicate that PEC are not as fully activated as TAM, we asked whether the inability of PEC to induce T cell apoptosis may reflect a relatively inactivated phenotype compared with TAM. To test this notion PEC were isolated and treated with IFN-{gamma} or LPS overnight in vitro before use in coculture experiments with activated T cells (Fig. 7Go). PEC activated in vitro do not become proapoptotic (or release NO; data not shown) after culture with T cells despite this treatment, suggesting that expression of the proapoptotic phenotype by TAM requires some developmental stimulus other than that provided by LPS or IFN-{gamma}. In comparison to TAM, the nonproapoptotic phenotype of PEC after activation in vitro correlates with cell surface expression of TNF receptor and release of NO, implying that TAM are more activated than PEC, although requiring a final activation event (IFN-{gamma}) to release NO.



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  		FIGURE 7. PEC activated with LPS or IFN-{gamma} in vitro are not proapoptotic. Cocultures using activated CD8+ T cells and F4/80+ PEC from control mice were prepared as described in Materials and Methods and used in coculture experiments. T cells were activated with plate-bound anti-TCR (24 h). PEC were pretreated for 24 h as follows: plated in medium only, plated in the presence of LPS (0.020 mg/ml), or plated in the presence of IFN-{gamma} (100 ng/ml). At the indicated times (0 or 30 h) T cells were purified by immunobeading, analyzed by flow cytometry, and enumerated. Cocultures were plated in triplicate in 48-well plates and the data were averaged for the respective experimental conditions for two experiments.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Apoptosis of infiltrating T cells in primary human tumors has been reported for several tumor types: ovarian (34, 35), lung (36), breast (37), renal cell (38), and squamous cell carcinomas (39). FasL expression by tumor cells has recently been postulated to be the basis for TIL apoptosis because of the observations by several laboratories that primary tumors can express FasL (21). Tumor-induced death of antitumor T cells is conceptually appealing because only antitumor T cells in contact with tumor cells would be expected to be eliminated, leaving systemic T cell functions intact. The observation of intact systemic immune function has been made with both animal tumor models (26) and human patients (25), thereby indirectly supporting the possibility of tumor-expressed, FasL-mediated TIL destruction. However, the notion of melanoma expression of FasL has recently been questioned (20). Therefore, the conclusion that human melanoma express FasL may be premature. Whether primary human tumors other than melanoma can be definitively shown to express FasL remains to be determined and is under investigation.

Irrespective of whether tumors express FasL or not, TIL in primary human tumors have been reported to be apoptotic, although the percentage of TIL undergoing apoptosis in situ for any given tumor type is variable and may depend upon several factors, including tumor stage and size (40). Additionally, activation-induced cell death of T cells occurs upon recognition of cognate Ag, so at least some TIL are possibly undergoing apoptosis in the tumor microenvironment because of recognition of tumor Ag expressed on tumor cells (25). Our results suggest an additional potential mechanism for induction of apoptosis in TIL in situ. We show that a FasL-negative murine tumor elicits host TAM which are highly proapoptotic toward activated CD8+ T cells. Susceptible T cells express various activation Ags indicative of proliferation (ca. CD25 and CD69), and nonactivated CD8+ T cells have a low mitotic index in concert with their resistance to killing by tumor macrophages.

The mechanism by which TAM induce T cell apoptosis is dependent upon production of NO as shown by several results. First, proapoptotic TAM produce high levels of NO upon contact with activated T cells, whereas nonproapoptotic PEC-derived macrophages do not. In addition, inhibition of inducible NO synthase (iNOS) in T cell-TAM cocultures prevents T cell killing. Finally, NO production from both TAM and T cell apoptosis is inhibited in cocultures by blocking anti-IFN-{gamma} Ab. A role for IFN-{gamma} in induction of NO has been shown in several models of immune suppression, including infection with Mycobacterium bovis (41), high-dose IL-12 therapy (42), and infection with Trypanosoma cruzi (43). IFN-{gamma} is involved in TAM-induced T cell apoptosis because high levels of IFN-{gamma} are secreted from activated T cells upon contact with TAM, which is in turn responsible for augmenting NO production from TAM. However, a cooperative effect of IFN-{gamma} with TNF in tumor macrophage-induced T cell apoptosis is shown by several findings: proapoptotic TAM express cell surface TNF and the TNF receptor, lysis of susceptible target T cells in vitro is blocked by Ab to TNF, T cells isolated from TNF -/- mice are susceptible to TAM-induced apoptosis, and TAM isolated from mice bearing disruption in either the TNF type I and type II receptor genes are unable to induce apoptosis.

The cooperative nature of IFN-{gamma} and TNF in induction of NO by TAM has a precedent in that initiation of divergent macrophage responses to treatment with poly(I:C) was shown by Riches and colleagues (44, 45, 46) to be dependent upon TNF stimulation of macrophages but required IFN for initiation and maintenance of the response. The role of IFN-{gamma} in initiating the TAM proapoptotic phenotype may be to increase the number or activity of TNF receptors (47, 48) or to provide a second signal required for full implementation of TNF receptor-mediated signaling. Alternatively, IFN-{gamma} may permit divergence of TNF receptor I signals from induction of apoptosis in TAM to a different response, that of NO production.

The work of Riches and colleagues, like our results, showed a requirement for both TNF receptors expressed on macrophages in activation of macrophages mediated by TNF. Each of the TNF receptors may generate different downstream signals, both of which are required for NO production. Alternatively, the type II receptor may recruit or accumulate cell surface TNF, which can then interact with the type I receptor, which in turn initiates NO synthesis. This putative mechanism of killing by TNF has been postulated previously by Goeddel and colleagues (49) and termed ligand passing. Because type I receptor signaling has been shown to increase expression of phospholipase C and protein kinase C activation (50), perhaps type II signals potentiate type I effects either by recruiting additional components of the signaling cascade or by ligand passing.

Induction of apoptosis in murine CD8+ T cells has been shown previously by Zheng et al. (51) to be mediated by TNF expressed by T cells, which was responsible for fratricidal cell death of activated peripheral CD8+ T cells. Using mice with homozygous deficiencies in CD120a or CD120b (prepared by targeted gene disruption) it was concluded that the TNF type II receptor was required and sufficient for TNF-mediated cell death. There are some differences between our findings and those of Zheng and colleagues (51). The most important difference is that TAM express cell surface-associated TNF and do not secrete detectable soluble TNF in vitro, whereas, although cell surface TNF expression was not directly examined in the work of Zheng et al. (51), expression of TNF by T cells is presumably the secreted form. In addition, inclusion of blocking TNF Ab had only minor effect on T cell recovery in the work of Zheng et al. (51), whereas a dramatic effect on T cell recovery in coculture with TAM is seen. Finally, use of T cells obtained from TNF receptor -/- mice showed a requirement for both CD120a and CD120b expression on TAM. We consider it likely that the different requirements for TNF in the different model systems studied reflects the fact that T cell killing is initiated by different forms of TNF, secreted and cell surface, which use different receptors in induction of cell death.

Collectively these experiments suggest the following mechanism for TAM-induced apoptosis of activated CD8+ T cells (Fig. 8Go). Upon contact with TAM, T cells are activated to secrete high levels of IFN-{gamma}, which in turn augments production of NO by TAM. Enhanced NO release is mediated via the TAM TNF type I and type II receptors because NO secretion by either TNF type I or type II receptor -/- TAM is very low. NO release in turn induces T cell apoptosis. In concert to being deficient in production of NO in response to coculture with T cells, TAM in TNF receptor -/- mice, either type I or type II, are unable to induce T cell apoptosis. TAM differ from PEC in expression of TNF receptor and are, as such, in a more activated state, but the requirement for IFN-{gamma} to secrete NO suggests that TAM, although activated relative to control PEC-derived F4/80+ macrophages, are incompletely activated to express the proapoptotic phenotype.



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  		FIGURE 8. A model of TAM-induced T cell apoptosis.

 
There may be a differential requirement for the two TNF receptors in TAM-mediated killing of activated T cells, implying perhaps a role for direct TNF receptor signaling in T cell death because, at very early times points after TAM coculture with T cells obtained from TNF receptor -/- mice (6–10 h), type II -/- T cells were more sensitive to killing, suggesting that TNF receptor I may mediate direct T cell killing and that this is independent of the type II receptor. However, this was the only time point which demonstrated such an effect (T cell recovery was identical to that of T cells from wild-type mice at 16 h of coculture), and we consider unlikely the possibility that T cell killing by TAM in vivo may have two phases mediated possibly via different mechanisms, one directly caused by cell surface TNF and the other NO-mediated. That conclusion is supported by the observation that cell surface TNF is expressed by PEC-derived F4/80+ macrophages but activated T cells proliferate vigorously upon coculture in vitro.

Our model of T cell killing by TAM, proposed to explain the relationship between IFN-{gamma}/TNF and NO production leading to T cell apoptosis, is supported by the observations of others in several systems. A causal role for TNF in induction of NO is clearly shown, although the exclusive involvement of the cell-associated and not the secreted form of TNF has not been addressed in the work of others. In all examples referenced herein, IFN-{gamma} synergizes with TNF to induce NO, which is the mediator of target cell death, including murine cardiac cells (52), rat mesangial cells (53), rat (54) and murine (55) endothelial cells, human (56) and murine (57) tumor cell lines, murine splenocytes in vivo after infection of host macrophages with T. cruzi (58), and macrophage cell lines when infected with various pathogens (Pneumocystis carinii (59), Leishmania major (60), and Klebsiella pneumonia (61)). The role of NO in induction of target cell apoptosis is likely species-specific in that, in general, normal human cells in culture are protected from cell TNF-mediated cell death by NO including endothelia (62), smooth muscle (63), and liver (64). As mentioned above, human transformed cells in culture may be an exception to the notion of species selectivity of NO protection from TNF killing, an observation taken to support the notion of attempts to augment macrophage production of NO in tumor immunotherapy. Our data suggest that expression of NO at the site of the tumor, while potentially able to kill tumor cells, may impact negatively on antitumor T cells in the tumor microenvironment.

Our findings show that proapoptotic macrophages which infiltrate tumors are able to induce apoptosis in activated T cells and are relevant to recent studies which suggest that human TIL are apoptotic in situ. If activated tumor macrophages kill T cells in human tumors, it may be possible to regulate TIL death by blocking NO expression from tumor macrophage by inhibiting either IFN-{gamma} activation of TAM or NO production by TAM at the tumor site. Disruption of macrophage-induced TIL death in situ would be expected to have beneficial effects on antitumor T cell survival and possibly enhance T cell-mediated tumor killing.


   Acknowledgments
 
We thank David Levy for anti-IFN-{gamma} Ab and John Hirst for flow cytometry analyses.


   Footnotes
 
1 This work was partially supported by National Institutes of Health Grant CA 16087 to the Kaplan Cancer Center in support of flow cytometric facilities. Back

2 Address correspondence and reprint requests to Dr. Alan B. Frey, Department of Cell Biology, New York University School of Medicine, Room MSB 690, 550 First Avenue, New York, NY 10016. E-mail address: freya01{at}popmail.med.nyu.edu Back

3 Abbreviations used in this paper: MCA-38, 3-methylcholanthrene-38; PEC, peritoneal exudate cell; TIL, tumor-infiltrating lymphocyte; TAM, tumor-associated macrophages; iNOS, inducible NO synthase. Back

Received for publication January 29, 2001. Accepted for publication September 7, 2001.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

  1. Burger, U. L., M. P. Chang, M. Nagoshi, P. S. Goedegebuure, T. J. Eberlein. 1996. Improved in vivo efficacy of tumor-infiltrating lymphocytes after restimulation with irradiated tumor cells in vitro. Ann. Surg. Oncol. 3:580.[Abstract]
  2. Cheever, M. A., W. Chen. 1997. Therapy with cultured T cells: principles revisited. Immunol. Rev. 157:177.[Medline]
  3. Lipshy, K. A., P. J. Kostuchenko, G. G. Hamad, C. E. Bland, S. K. Barrett, H. D. Bear. 1997. Sensitizing T-lymphocytes for adoptive immunotherapy by vaccination with wild-type or cytokine gene-transduced melanoma. Ann. Surg. Oncol. 4:334.[Abstract]
  4. Mukai, S., J. Kjaergaard, S. Shu, G. E. Plautz. 1999. Infiltration of tumors by systemically transferred tumor-reactive T lymphocytes is required for antitumor efficacy. Cancer Res. 59:5245.[Abstract/Free Full Text]
  5. Winter, H., H. M. Hu, W. J. Urba, B. A. Fox. 1999. Tumor regression after adoptive transfer of effector T cells is independent of perforin or Fas ligand (APO-1L/CD95L). J. Immunol. 163:4462.[Abstract/Free Full Text]
  6. Li, Q., A. E. Chang. 1999. Adoptive T-cell immunotherapy of cancer. Cytokines Cell. Mol. Ther. 5:105.[Medline]
  7. Frey, A. B.. 1996. Role of host antigen receptor-bearing and antigen receptor-negative cells in immune response to rat adenocarcinoma 13762. J. Immunol. 156:3841.[Abstract]
  8. Frey, A. B., S. Cestari. 1997. Killing of rat adenocarcinoma 13762 in situ by adoptive transfer of CD4+ anti-tumor T cells requires tumor expression of cell surface MHC class II molecules. Cell. Immunol. 178:79.[Medline]
  9. Yu, J. S., J. A. Burwick, G. Dranoff, X. O. Breakefield. 1997. Gene therapy for metastatic brain tumors by vaccination with granulocyte-macrophage colony-stimulating factor-transduced tumor cells. Hum. Gene Ther. 8:1065.[Medline]
  10. Duda, D. G., M. Sunamura, L. Lozonschi, T. Kodama, S. Egawa, G. Matsumoto, H. Shimamura, K. Shibuya, K. Takeda, S. Matsuno. 2000. Direct in vitro evidence and in vivo analysis of the antiangiogenesis effects of interleukin 12. Cancer Res. 60:1111.[Abstract/Free Full Text]
  11. Fidler, I. J.. 2000. Angiogenesis and cancer metastasis. Cancer J. Sci. Am. 6:(Suppl 2):S134.
  12. Gee, M. S., C. J. Koch, S. M. Evans, W. T. Jenkins, Jr C. H. Pletcher, J. S. Moore, H. K. Koblish, J. Lee, E. M. Lord, G. Trinchieri, W. M. Lee. 1999. Hypoxia-mediated apoptosis from angiogenesis inhibition underlies tumor control by recombinant interleukin 12. Cancer Res. 59:4882.[Abstract/Free Full Text]
  13. Gilboa, E.. 1999. How tumors escape immune destruction and what we can do about it. Cancer Immunol. Immunother. 48:382.[Medline]
  14. Tasaki, K., Y. Yoshida, T. Maeda, M. Miyauchi, K. Kawamura, K. Takenaga, H. Yamamoto, T. Kouzu, T. Asano, T. Ochiai, et al 2000. Protective immunity is induced in murine colon carcinoma cells by the expression of interleukin-12 or interleukin-18, which activate type 1 helper T cells. Cancer Gene Ther. 7:247.[Medline]
  15. Restifo, N. P., S. A. Rosenberg. 1999. Developing recombinant and synthetic vaccines for the treatment of melanoma. Curr. Opin. Oncol. 11:50.[Medline]
  16. Rosenberg, S. A.. 1998. New opportunities for the development of cancer immunotherapies. Cancer J. Sci. Am. 4:(Suppl 1):S1.
  17. Pardoll, D. M.. 1998. Cancer vaccines. Nat. Med. 4:525.[Medline]
  18. Pawelec, G., J. Zeuthen, R. Kiessling. 1997. Escape from host-antitumor immunity. Crit. Rev. Oncog. 8:111.[Medline]
  19. Radoja, S., A. B. Frey. 2000. Cancer-induced defective cytotoxic T lymphocyte effector function: another mechanism how antigenic tumors escape immune-mediated killing. Mol. Med. 6:465.[Medline]
  20. Chappell, D. B., N. P. Restifo. 1998. T cell-tumor cell: a fatal interaction?. Cancer Immunol. Immunother. 47:65.[Medline]
  21. O’Connell, J., M. W. Bennett, G. C. O’Sullivan, J. K. Collins, F. Shanahan. 1999. The Fas counterattack: cancer as a site of immune privilege. Immunol. Today 20:46.[Medline]
  22. Walker, P. R., P. Saas, P. Y. Dietrich. 1998. Tumor expression of Fas ligand (CD95L) and the consequences. Curr. Opin. Immunol. 10:564.[Medline]
  23. Whiteside, T. L., H. Rabinowich. 1998. The role of Fas/FasL in immunosuppression induced by human tumors. Cancer Immunol. Immunother. 46:175.[Medline]
  24. Chappell, D. B., T. Z. Zaks, S. A. Rosenberg, N. P. Restifo. 1999. Human melanoma cells do not express Fas (Apo-1/CD95) ligand. Cancer Res. 59:59.[Abstract/Free Full Text]
  25. Levey, D., P. Srivastava. 1996. Alterations in T cells of cancer-bearers: whence specificity?. Immunol. Today 17:365.[Medline]
  26. Radoja, S., T. D. Rao, D. Hillman, A. B. Frey. 2000. Mice bearing late-stage tumors have normal functional systemic T cell responses in vitro and in vivo. J. Immunol. 164:2619.[Abstract/Free Full Text]
  27. Marino, M. W., A. Dunn, D. Grail, M. Inglese, Y. Noguchi, E. Richards, A. Jungbluth, H. Wada, M. Moore, B. Williamson, et al 1997. Characterization of tumor necrosis factor-deficient mice. Proc. Natl. Acad. Sci. USA 94:8093.[Abstract/Free Full Text]
  28. Misko, T. P., R. J. Schilling, D. Salvemini, W. M. Moore, M. G. Currie. 1993. A fluorometric assay for the measurement of nitrite in biological samples. Anal. Biochem. 214:11.[Medline]
  29. Chan, E. D., D. W. Riches. 2001. IFN-{gamma} + LPS induction of iNOS is modulated by ERK, JNK/SAPK, and p38(mapk) in a mouse macrophage cell line. Am. J. Physiol. 280:C441.
  30. Kang, J., M. Yang, I. Jou, E. Joe. 2001. Identification of protein kinase C isoforms involved in interferon-{gamma}-induced expression of inducible nitric oxide synthase in murine BV2 microglia. Neurosci. Lett. 299:205.[Medline]
  31. Paludan, S. R., L. Malmgaard, S. Ellermann-Eriksen, L. Bosca, S. C. Mogensen. 2001. Interferon (IFN)-{gamma} and herpes simplex virus/tumor necrosis factor-{alpha} synergistically induce nitric oxide synthase 2 in macrophages through cooperative action of nuclear factor-{kappa}B and IFN regulatory factor-1. Eur. Cytokine Network 12:297.[Medline]
  32. Reis, D., M. Souza, J. Mineo, F. Espindola. 2001. Myosin V and iNOS expression is enhanced in J774 murine macrophages treated with IFN-{gamma}. Braz. J. Med. Biol. Res. 34:221.[Medline]
  33. Samardzic, T., V. Jankovic, S. Stosic-Grujicic, V. Trajkovic. 2001. STAT1 is required for iNOS activation, but not IL-6 production in murine fibroblasts. Cytokine 13:179.[Medline]
  34. Ben-Hur, H., P. Gurevich, M. Huszar, A. Ben-Arie, V. Berman, Y. Tendler, O. Zinder, R. Tchanishev, S. Gershon, G. Mor, et al 2000. Apoptosis and apoptosis-related proteins (Fas, Fas ligand, Blc-2, p53) in lymphoid elements of human ovarian tumors. Eur. J. Gynaecol. Oncol. 21:53.[Medline]
  35. Munakata, S., T. Enomoto, M. Tsujimoto, Y. Otsuki, H. Miwa, H. Kanno, K. Aozasa. 2000. Expressions of Fas ligand and other apoptosis-related genes and their prognostic significance in epithelial ovarian neoplasms. Br. J. Cancer 82:1446.[Medline]
  36. Eerola, A. K., Y. Soini, P. Paakko. 1999. Tumour infiltrating lymphocytes in relation to tumour angiogenesis, apoptosis and prognosis in patients with large cell lung carcinoma. Lung Cancer 26:73.[Medline]
  37. O’Connell, J., M. W. Bennett, G. C. O’Sullivan, J. O’Callaghan, J. K. Collins, F. Shanahan. 1999. Expression of Fas (CD95/APO-1) ligand by human breast cancers: significance for tumor immune privilege. Clin. Diagn. Lab. Immunol. 6:457.[Abstract/Free Full Text]
  38. Uzzo, R. G., P. Rayman, V. Kolenko, P. E. Clark, T. Bloom, A. M. Ward, L. Molto, C. Tannenbaum, L. J. Worford, R. Bukowski, et al 1999. Mechanisms of apoptosis in T cells from patients with renal cell carcinoma. Clin. Cancer Res. 5:1219.[Abstract/Free Full Text]
  39. Gastman, B. R., Y. Atarshi, T. E. Reichert, T. Saito, L. Balkir, H. Rabinowich, T. L. Whiteside. 1999. Fas ligand is expressed on human squamous cell carcinomas of the head and neck, and it promotes apoptosis of T lymphocytes. Cancer Res. 59:5356.[Abstract/Free Full Text]
  40. Whiteside, T. L.. 1998. Immune cells in the tumor microenvironment: mechanisms responsible for functional and signaling defects. Adv. Exp. Med. Biol. 451:167.[Medline]
  41. Dalton, D. K., L. Haynes, C. Q. Chu, S. L. Swain, S. Wittmer. 2000. Interferon {gamma} eliminates responding CD4 T cells during mycobacterial infection by inducing apoptosis of activated CD4 T cells. J. Exp. Med. 192:117.[Abstract/Free Full Text]
  42. Koblish, H. K., C. A. Hunter, M. Wysocka, G. Trinchieri, W. M. Lee. 1998. Immune suppression by recombinant interleukin (rIL)-12 involves interferon {gamma} induction of nitric oxide synthase 2 (iNOS) activity: inhibitors of NO generation reveal the extent of rIL-12 vaccine adjuvant effect. J. Exp. Med. 188:1603.[Abstract/Free Full Text]
  43. Martins, G. A., L. Q. Vieira, F. Q. Cunha, J. S. Silva. 1999. {gamma} interferon modulates CD95 (Fas) and CD95 ligand (Fas-L) expression and nitric oxide-induced apoptosis during the acute phase of Trypanosoma cruzi infection: a possible role in immune response control. Infect. Immun. 67:3864.[Abstract/Free Full Text]
  44. Lake, F. R., P. W. Noble, P. M. Henson, D. W. Riches. 1994. Functional switching of macrophage responses to tumor necrosis factor-{alpha} (TNF {alpha}) by interferons: implications for the pleiotropic activities of TNF {alpha}. J. Clin. Invest. 93:1661.
  45. Riches, D. W., P. M. Henson, L. K. Remigio, J. F. Catterall, R. C. Strunk. 1988. Differential regulation of gene expression during macrophage activation with a polyribonucleotide: the role of endogenously derived IFN. J. Immunol. 141:180.[Abstract]
  46. Riches, D. W., G. A. Underwood. 1991. Expression of interferon-{beta} during the triggering phase of macrophage cytocidal activation: evidence for an autocrine/paracrine role in the regulation of this state. J. Biol. Chem. 266:24785.[Abstract/Free Full Text]
  47. Ruggiero, V., J. Tavernier, W. Fiers, C. Baglioni. 1986. Induction of the synthesis of TNF receptors by interferon-{gamma}. J. Immunol. 136:2445.[Abstract]
  48. Tsujimoto, M., R. Feinman, J. Vilcek. 1986. Differential effects of type I IFN and IFN-{gamma} on the binding of TNF to receptors in two human cell lines. J. Immunol. 137:2272.[Abstract]
  49. Tartaglia, L. A., D. Pennica, D. V. Goeddel. 1993. Ligand passing: the 75-kDa tumor necrosis factor (TNF) receptor recruits TNF for signaling by the 55-kDa TNF receptor. J. Biol. Chem. 268:18542.[Abstract/Free Full Text]
  50. Schutze, S., D. Berkovic, O. Tomsing, C. Unger, M. Kronke. 1991. Tumor necrosis factor induces rapid production of 1'2'diacylglycerol by a phosphatidylcholine-specific phospholipase C. J. Exp. Med. 174:975.[Abstract/Free Full Text]
  51. Zheng, L., G. Fisher, R. E. Miller, J. Peschon, D. H. Lynch, M. J. Lenardo. 1995. Induction of apoptosis in mature T cells by tumour necrosis factor. Nature 377:348.[Medline]
  52. Sanders, D. B., D. F. Larson, K. Hunter, M. Gorman, B. Yang. 2001. Comparison of tumor necrosis factor-{alpha} effect on the expression of iNOS in macrophage and cardiac myocytes. Perfusion 16:67.[Abstract/Free Full Text]
  53. Duffield, J. S., L. P. Erwig, X. Wei, F. Y. Liew, A. J. Rees, J. S. Savill. 2000. Activated macrophages direct apoptosis and suppress mitosis of mesangial cells. J. Immunol. 164:2110.[Abstract/Free Full Text]
  54. Smith, J. D., S. D. McLean, D. K. Nakayama. 1998. Nitric oxide causes apoptosis in pulmonary vascular smooth muscle cells. J. Surg. Res. 79:121.[Medline]
  55. Walker, D. H., V. L. Popov, P. A. Crocquet-Valdes, C. J. Welsh, H. M. Feng. 1997. Cytokine-induced, nitric oxide-dependent, intracellular antirickettsial activity of mouse endothelial cells. Lab. Invest. 76:129.[Medline]
  56. Garban, H. J., B. Bonavida. 1999. Nitric oxide sensitizes ovarian tumor cells to Fas-induced apoptosis. Gynecol. Oncol. 73:257.[Medline]
  57. Higuchi, M., N. Higashi, H. Taki, T. Osawa. 1990. Cytolytic mechanisms of activated macrophages: TNF and L-arginine-dependent mechanisms act synergistically as the major cytolytic mechanisms of activated macrophages. J. Immunol. 144:1425.[Abstract]
  58. Martins, G. A., M. A. Cardoso, J. C. Aliberti, J. S. Silva. 1998. Nitric oxide-induced apoptotic cell death in the acute phase of Trypanosoma cruzi infection in mice. Immunol. Lett. 63:113.[Medline]
  59. Downing, J. F., D. L. Kachel, R. Pasula, 2nd. W. J. Martin. 1999. {gamma} interferon stimulates rat alveolar macrophages to kill Pneumocystis carinii by L-arginine- and tumor necrosis factor-dependent mechanisms. Infect. Immun. 67:1347.[Abstract/Free Full Text]
  60. Liew, F. Y., Y. Li, S. Millott. 1990. TNF-{alpha} synergizes with IFN-{gamma} in mediating killing of Leishmania major through the induction of nitric oxide. J. Immunol. 145:4306.[Abstract]
  61. Tsai, W. C., R. M. Strieter, D. A. Zisman, J. M. Wilkowski, K. A. Bucknell, G. H. Chen, T. J. Standiford. 1997. Nitric oxide is required for effective innate immunity against Klebsiella pneumoniae. Infect. Immun. 65:1870.[Abstract]
  62. Dimmeler, S., J. Haendeler, M. Nehls, A. M. Zeiher. 1997. Suppression of apoptosis by nitric oxide via inhibition of interleukin-1{beta}-converting enzyme (ICE)-like and cysteine protease protein (CPP)-32-like proteases. J. Exp. Med. 185:601.[Abstract/Free Full Text]
  63. Geng, Y. J., Q. Wu, M. Muszynski, G. K. Hansson, P. Libby. 1996. Apoptosis of vascular smooth muscle cells induced by in vitro stimulation with interferon-{gamma}, tumor necrosis factor-{alpha}, and interleukin-1{beta}. Arterioscler. Thromb. Vasc. Biol. 16:19.[Abstract/Free Full Text]
  64. Li, J., C. A. Bombeck, S. Yang, Y. M. Kim, T. R. Billiar. 1999. Nitric oxide suppresses apoptosis via interrupting caspase activation and mitochondrial dysfunction in cultured hepatocytes. J. Biol. Chem. 274:17325.[Abstract/Free Full Text]



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