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The Journal of Immunology, 2001, 166: 5407-5415.
Copyright © 2001 by The American Association of Immunologists

The Involvement of TNF-{alpha}-Related Apoptosis-Inducing Ligand in the Enhanced Cytotoxicity of IFN-{beta}-Stimulated Human Dendritic Cells to Tumor Cells1

Shuxun Liu, Yizhi Yu, Minghui Zhang, Wenya Wang and Xuetao Cao2

Department of Immunology, Second Military Medical University, Shanghei, People’s Republic of China


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
TNF-{alpha}-related apoptosis-inducing ligand (TRAIL) is characterized by its preferential induction of apoptosis of tumor cells but not normal cells. Dendritic cells (DCs), besides their role as APCs, now have been demonstrated to exert cytotoxicity or cytostasis on some tumor cells. Here, we report that both human CD34+ stem cell-derived DCs (CD34DCs) and human CD14+ monocyte-derived DCs (MoDCs) express TRAIL and exhibit cytotoxicity to some types of tumor cells partially through TRAIL. Moderate expression of TRAIL appeared on CD34DCs from the 8th day of culture and was also seen on freshly isolated monocytes. The level of TRAIL expression remained constant until DC maturation. TRAIL expression on immature CD34DCs or MoDCs was greatly up-regulated after IFN-{beta} stimulation. Moreover, IFN-{beta} could strikingly enhance the ability of CD34DCs or MoDCs to kill TRAIL-sensitive tumor cells, but LPS did not have such an effect. The up-regulation of TRAIL on IFN-{beta}-stimulated DCs partially contributed to the increased cytotoxicity of DCs. Pretreatment of TRAIL-sensitive tumor cells with caspase-3 inhibitor could significantly increase their resistance to the cytotoxicity of IFN-{beta}-stimulated DCs. In contrast, NF-{kappa}B inhibitor could significantly increase the sensitivity of tumor cells to the killing by nonstimulated or LPS-stimulated DCs. Our studies demonstrate that IFN-{beta}-stimulated DCs are functionally cytotoxic. Thus, an innate mechanism of DC-mediated antitumor immunity might exist in vivo in which DCs act as effectors to directly kill tumor cells partially via TRAIL. Subsequently, DCs act as APCs involved in the uptake, processing, and presentation of apoptotic tumor Ags to cross-prime CD8+ CTL cells.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Dendritic cells (DCs),3 as highly specialized Ag APCs, are responsible for the induction of primary immune responses (1). DCs play critical roles in antitumor or antivirus immune responses, autoimmune diseases, and graft rejection. The participation of CD8+ CTL cells that are induced by cross-priming by DCs is considered a major mechanism involved in such immune responses. Exogenous antigenic peptides are cross-presented by DCs to T cells in the form of peptide-MHC class I complexes, thus stimulating Ag-specific CTL responses.

The induction of specific CD8+ CTL is a major possibility in the treatment of cancer (2, 3, 4). To date, DC-based tumor vaccinations in the treatment of cancer patients have shown promising results from clinical trials (5, 6, 7, 8). Apoptotic tumor cells are considered more effective than tumor debris or tumor cells alone at supplying host DCs with a source of antigenic epitopes for cross-priming autologous CD8+ CTLs (9, 10). Bystander apoptotic cells are able to trigger DC maturation to Ag presentation function even without exogenous "danger" signals (11). In the capturing of Ags, the {alpha}v{beta}5 and CD36 molecules on DCs possibly mediate phagocytosis of apoptotic cells; whereas in processing and presentation, heat shock protein 70 and other maturation factors released from apoptotic cells are likely to help induction of DC maturation and direct antigenic peptides for MHC class I presentation (12, 13). Optimal cross-priming of CD8+ CTLs by DCs requires both apoptotic bodies for Ags and necrotic cells for delivering maturation signals (14). Nevertheless, a critical prerequisite is the existence of apoptotic bodies, but little is known about their origin. Innate defense mechanisms are comprised of macrophages, NK cells, complement, and interferons, which can directly delete pathogens or unwanted cells. Although it is well known that DCs are widely distributed in peripheral tissues, are important as a first line of defense, and make regular encounters with tumor cells, there is no evidence that DCs also belong to the natural defense arm of the immune response, directly eliminating deleterious factors. Some reports suggest that DCs may have a direct role in the elimination of malignant cells. In murine tumor models, injection of DCs can inhibit growth of tumors to some degree (15, 16). In rats, NKR-P1+ spleen DCs possess cytotoxic activity toward NK-sensitive YAC-1 cells (17). In humans, activated DCs appear to suppress growth of a variety spectrum of tumor cell lines in vitro (18). Furthermore, Fanger et al. (19) have shown that a peripheral blood subset of CD11c+ DCs, but not IL-3R{alpha}+ pre-DCs, stimulated by IFN-{gamma} or IFN-{alpha} can induce cellular apoptosis in several tumor cells.

Recently, numerous apoptosis-inducing ligands have been discovered, most of them belonging to the TNF family (20, 21, 22, 23, 24) TNF-{alpha}-related apoptosis-inducing ligand (TRAIL), a newly discovered member of the TNF family, together with CD95 ligand (CD95L) and TNF-{alpha}, belongs to subgroup of apoptosis-inducing ligands (25, 26, 27). The corresponding death receptors are members of the TNFR family, the cytoplasmic regions of which all contain a crucial domain for mediating apoptotic signals, called the death domain. Upon the oligomerization of these receptors triggered by their ligands on the surface, the death domains can recruit intracellular signal molecules, such as Fas-associated death domain-containing protein (FADD), which can activate downstream caspase cascades leading to a series of apoptotic events (28).

Unlike CD95L and TNF-{alpha}, TRAIL was characterized by selective induction of apoptotic cell death in a variety of tumor cells or transformed cells, but not in normal cells. The TRAIL-resistant property of normal cells and some tumor cells may be due to the expression of its two decoy receptors and some intracellularly resistant molecules, such as cellular FADD-like IL-1{beta}-converting enzyme protease-inhibitory protein (c-FLIP); and to the activation of antiapoptotic mechanisms, e.g., NF-{kappa}B activation (29, 30, 31, 32, 33, 34, 35, 36). TRAIL has been shown to participate in the tumoricidal activity of CD4+ T cells, NK cells, monocytes, and CD11c+ DCs under certain conditions (19, 26, 37, 38). However, there have been no reports concerning the killing of tumor cells by in vitro cultured human CD34+ stem cell-derived DCs (CD34DCs) and human CD14+ monocyte-derived DCs (MoDCs) via TRAIL.

The major aim of this study was to investigate whether human CD34DCs and MoDCs cultured in vitro are able to kill TRAIL-sensitive tumor cells via TRAIL and to dissect relevant intracellular mechanisms. Our studies revealed that IFN-{beta} could enhance the cytotoxicity of CD34DCs or MoDCs to TRAIL-sensitive tumor cells, which was partially related to the up-regulation of TRAIL expression. However, a maturation factor, LPS, did not have such an effect. The cytotoxicity of DCs toward tumor cells was regulated by activation of both the caspase cascade and the NF-{kappa}B pathway in tumor cells. The physiological and pathological significance of the phenomenon is discussed.


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

Human recombinant GM-CSF, IL-4, TNF-{alpha}, IFN-{beta}, and the fusion protein of the extracellular region of TRAIL-R3 and the constant region of human IgG1 were obtained from R&D Systems (Minneapolis, MN). TRAIL and TRAIL enhancer were purchased from Alexis (San Diego, CA). Human IgG1 protein was from Biodesign International (Saco, ME). LPS, NF-{kappa}B inhibitor (pyrrolidinedithiocarbamate), propidium iodide, and MTT were obtained from Sigma (St. Louis, MO). Caspase-3 inhibitor (N-benzyloxycarbonyl-Asp-Glu-Val-Asp-fluromethyl ketone) was purchased from Calbiochem (San Diego, CA). The reagents mentioned above were dissolved with serum-free RPMI 1640 (Life Technologies, Gaithersburg, MD) to 10x working concentration, aliquoted, and stored at -20°C or -80°C. Before use, cytokines and other agents were diluted with fresh complete medium to working concentrations.

FITC- or PE-Abs mouse anti-human CD40, CD86, CD83, HLA-DR, CD14, and isotype control Abs; mouse anti-human TRAIL mAb (IgG2b), mouse IgG2b and FITC-secondary Ab were obtained from PharMingen (San Diego, CA). Goat anti-human polyclonal Ab against caspase-3 p20 or caspase-8 p20, and donkey anti-goat HRP-conjugated secondary Ab were obtained from Santa Cruz Biotechnology (Santa Cruz, CA).

Cell lines

The following human tumor cell lines were obtained from American Type Culture Collection (ATCC; Manassas, VA): HL60 (promyelocytic leukemia, ATCC CCL 240); U937 (histiocytic lymphoma, ATCC CRL 1593); Daudi (Burkitt’s lymphoma, ATCC CRL 213); Jurkat (acute T cell leukemia, ATCC TIB 152); Reh (acute B cell leukemia, ATCC CRL-8283). These cell lines were cultured in RPMI 1640, supplemented with 10% FCS (Life Technologies), 2 mM glutamine, 100 U/ml penicillin, 100 µg/ml streptomycin at 37°C in 5% CO2.

Generation of human bone marrow CD34DCs

The generation of DCs from human bone marrow CD34+ stem cell was as described previously with some modification (39). Briefly, bone marrow suspensions were obtained from healthy donors for allogeneic transplantation under the permission of institutional guidelines. CD34-positive cells (purity, >90%) were isolated with a CD34+ cell immunomagnetic bead system (Miltenyi Biotec, Bergische Aladbach, Germany). CD34+ stem cells were cultured in complete medium supplemented with GM-CSF (100 ng/ml) and TNF-{alpha} (50 ng/ml). Every 4 days, one-half of the medium was refreshed with complete medium containing a double concentration of GM-CSF and TNF-{alpha} as indicated above. Cultured cells were harvested for analysis of surface phenotype at different stages of development. Up to the 12th day of culture, CD34DC suspensions (>95% pure as confirmed by analysis of relatively DC-specific phenotype and with a typical DC morphology) were collected for experimental purposes.

Generation of human peripheral blood MoDCs

DCs were generated from human peripheral blood monocytes as described previously with minor modifications (40). Briefly, PBMC were obtained from buffy coats from heparinized whole blood of healthy donors (the Blood Bank of Shanghai, Shanghai, China) by density gradient centrifugation on Histopaque 1077 (Sigma). These cells were resuspended in serum-free RPMI 1640 and allowed to adhere to six-well plates (Costar, Cambridge, MA) at a final concentration of 1 x 107 cells/3 ml/well. After 2 h at 37°C, nonadherent cells were gently removed with warm medium. The resulting adherent cells were cultured in medium supplemented with GM-CSF (100 ng/ml) and IL-4 (100 ng/ml) in 5% CO2 at 37°C. Every 2 days, one-half of the medium was replaced by fresh medium containing double concentration of GM-CSF and IL-4 as indicated above. Cell suspensions were collected for analysis of surface phenotype at different stages of development. After 5 days of culture, MoDCs were harvested for subsequent experiments (>90% pure as confirmed by analysis of relatively DC-specific phenotype and with a typical DC morphology).

Flow cytometry

Phenotypic analysis was performed by FACS as previously described (41). DCs (5 x 105/ml) were washed, resuspended in cold PBS containing 0.1% sodium azide (Sigma) and 10% mouse serum, and then incubated for 10 min at 4°C. Subsequently, they were incubated with FITC- or PE-labeled mAbs specific for human CD14, CD86, CD40, CD83, HLA-DR, or isotype-matched controls (5 µg/ml) for 30 min at 4°C in PBS. For analysis of TRAIL expression, DCs were labeled with antihuman TRAIL mAb (5 µg/ml) or antihuman IgG2b (5 µg/ml) for 30 min at 4°C. After 2 washes, cells were incubated with FITC-labeled secondary Ab (5 µg/ml) for another 30 min at 4°C. Stained cells were analyzed by FACSCaliber flow cytometry (Becton Dickinson, Mountain View, CA) and CellQuest software (Becton Dickinson). Dead cells were excluded by gating out propidium iodide-positive cells.

MTT reduction assay for growth inhibition of tumor cells by TRAIL

Tumor cells were plated in 96-well plates at 5 x 104 cells/well. TRAIL (100 ng/ml) and TRAIL enhancer (500 ng/ml) were added at the indicated concentrations. Each concentration was performed in triplicate. After culture at 37°C for 24 h, 20 µl MTT (5 mg/ml) were added to each well for another 4 h. Plates were centrifuged at 1000 rpm for 5 min, and then supernatants were gently removed. DMSO (100 µl; Sigma) was added to each well to dissolve formazan. The OD of each well was read using a microplate reader (model 550; Bio-Rad, Hercules, CA) at 570 nm. The percent of growth inhibition was calculated as: growth inhibition (%) = [1 - (experimental group (OD)/control group (OD))] x 100. Data were expressed as the mean ± SD of triplicate wells.

Assay for DC-mediated cytotoxicity

DC-mediated cytotoxicity to tumor cells was measured by an 18-h 51Cr release assay. This method was as previously described with some modification (42). Human MoDCs (cultured for 5 days from CD14+ monocytes) and CD34DCs (cultured for 12 days from CD34+ stem cells) were incubated in the presence of LPS (100 ng/ml) or IFN-{beta} (100 ng/ml) or in medium alone for 12 h and then washed. Tumor cells (1 x 107 cells/ml) were labeled with 100 µCi Na251CrO4 (Amersham, Arlington Heights, IL) for 1 h at 37°C. 51Cr-labeled tumor cells were cocultured with CD34+DCs or MoDCs as described at the indicated E:T ratios for 18 h. In some experiments, stimulated and unstimulated CD34DCs or MoDCs were pretreated with TRAILR3-Fc (1 µg/ml) or isotype control human IgG1 (1 µg/ml) 30 min before coincubation with labeled tumor cells. In other experiments, 51Cr-labeled tumor cells were pretreated with caspase-3 inhibitor (50 µM) or NF-{kappa}B inhibitor (15 µM) for 30 min before coculture with stimulated or unstimulated DCs. Each well contained a total volume of 200 µl medium in 96-well round-bottom plates (Costar). After coculture, 100 µl of supernatants were collected, and their radioactivity was measured in a gamma counter (1275; Wallac, Turku, Finland). Total and spontaneous 51Cr release values were obtained as cpm from supernatants of target cells in 1% Nonidet P-40 or in medium alone, respectively. Spontaneous release was <15% of maximum. TRAIL-R3-Fc and IgG1 were not toxic to target cells. NF-{kappa}B inhibitor (15 µM) and caspase-3 inhibitor (50 µM) also had no effect on the spontaneous release of radioisotope from target cells. Caspase-3 inhibitor did not decrease the viability of DCs. NF-{kappa}B inhibitor slightly decreased the viability of DCs but had no effect on the cytotoxicity of DCs. All experiments were performed in triplicate or sextuplicate wells. The percentage of cytotoxicity was calculated as: cytotoxicity (%) = [(experimental group cpm - spontaneous cpm)/(total cpm - spontaneous cpm)] x 100.

Analysis of caspase activation by Western blotting

HL60 cells or Reh cells (1 x 107 cells/3 ml/group) were cultured in the presence of TRAIL (100 ng/ml) plus TRAIL enhancer (500 ng/ml) for the indicated time. The cells were then harvested, and media were removed by two rinsings with ice-cold PBS. Cells were lysed in 0.5 ml ice-cold lysis buffer (1% Nonidet P-40, 20 mMTris-HCl (pH 8.0), 10% glycerol, 137 mM NaCl, 2 mM EDTA, 10 µg/ml aprotinin, 10 µg/ml leupeptin, 1 mM PMSF, and 1 mM sodium o-vanadate) for 5 min on ice. Cell lysates were transferred to a new tube after centrifugation at 10,000 x g for 10 min at 4°C and if necessary stored at -80°C. Equal volumes of cell lysates in electrophoresis sample buffer were boiled for 3 min and then resolved on a 12% separating polyacrylamide slab gel. After electrophoresis, the proteins were electrotransferred to nitrocellulose membranes. After blocking with 5% nonfat dried milk in 0.1% Tween 20 + PBS for 1 h at room temperature, the membrane was incubated with primary Ab (specific for caspase-3 p20 or caspase-8 p20) at a 1/500 dilution overnight at 4°C. Then the membrane was incubated with HRP-conjugated secondary Ab and detected by chemiluminescence (Santa Cruz Biotechnology).

Statistics

The Student t test was used for analyzing whether the differences between the values of the test groups and those of the relevant controls were significant. p < 0.05 was regarded as statistically significance. Data are shown as the mean ± SD of triplicate wells.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
TRAIL expression on human DCs derived from CD34+ stem cells or peripheral blood monocytes

We detected the kinetic expression of TRAIL on two kinds of distinct precursor-derived DCs, CD34DCs and MoDCs, during their differentiation and development in vitro. CD34DCs were generated from CD34+ hemopoietic progenitors in bone marrow in the presence of GM-CSF plus TNF-{alpha} for 14 days. During this period, we examined TRAIL expression and DC-specific phenotype on the cultured cells at different stages. As shown in Table IGo, CD34+ stem cells (purity up to 94%) did not express TRAIL. After 4 days of culture, these cells still did not express TRAIL, the costimulatory molecules CD86 and CD40, the monocyte marker CD14, or the DC maturity marker CD83; but did express moderate levels of HLA-DR. Expression of TRAIL was evident on the 8th day of culture, along with CD14 and CD40. HLA-DR expression was also up-regulated at this time, but CD86 and CD83 molecules were not yet expressed. In addition, adherent DC-like clusters could be seen (data not shown). By the 10th day of culture, levels of TRAIL remained constant, and the phenotype revealed that the DCs were still immature, although they showed the appearance of typical veils or dendritic processes (data not shown). On the 14th day of culture, TRAIL expression was still stable. HLA-DR was expressed on up to 98% of cells, the costimulatory molecules CD86 and CD40 were expressed on more than one-half of cells, CD14 was down-regulated to control level, and the CD83 maturation marker was expressed, which suggested that at this stage mature DCs were present. In addition, <5% of cultured cells expressed CD3, CD16, or CD19 after 8 days of culture, showing that there was very little contamination with T cells, NK cells, or B cells (data not shown). In summary, TRAIL expression appeared in the middle of CD34DC differentiation and the level was maintained until the mature stage.


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  		Table I. Relative DC-specific phenotypes and TRAIL expression on the CD34DCs during differentiation and development1

 
MoDCs were induced from peripheral blood CD14+ monocytes cultured in the presence of GM-CSF plus IL-4 for 7 days. As shown in Table IIGo, TRAIL expression was detected on freshly isolated monocytes (purity up to 99%), and the level of TRAIL expression was moderate. After 7 days of culture, the cells expressed high levels of HLA-DR, moderate levels of the costimulatory molecules CD86 and CD40 and the maturation marker CD83, but no CD14. Moreover, they showed veiled or dendritic morphology, indicating that they were mature MoDCs. In addition, contamination with cells of other lineages (CD3 T cells, CD16 NK cells, and CD19 B cells) was <5% (data not shown). During the time spanning maturation from CD14+ monocytes to mature MoDCs, the level of TRAIL expression was slightly down-regulated.


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  		Table II. Relative DC-specific phenotypes and TRAIL expression on the MoDCs at different developmental stages1

 
Up-regulation of TRAIL expression on the surface of MoDCs and CD34DCs by IFN-{beta} stimulation

Many studies have shown that different subpopulations of PBMC can express TRAIL under certain conditions. TRAIL expression has been shown to be up-regulated on CD3-NK1.1+ NK cells after IL-2 or IL-15 stimulation; on activated T cells after PHA or anti-CD3 mAb stimulation; and on macrophages or CD11c+ DCs after IFN-{gamma} or IFN-{alpha} stimulation (19, 26, 27, 37, 38). To date there have been no data concerning the change of TRAIL expression on CD34DCs or MoDCs. Considering that IFN-{gamma} or IFN-{alpha} have been shown to stimulate TRAIL expression on CD11c+ DCs, we evaluated whether IFN-{beta} could also produce the same effect on CD34DCs or MoDCs. In addition, we examined the effect of LPS (which can induce DC into maturation) on the regulation of TRAIL expression of DCs. As shown in Fig. 1Go, the level of TRAIL on the 12th day of culture of CD34DCs was increased by IFN-{beta} stimulation, compared with levels seen in unstimulated CD34DCs. However, TRAIL expression was downregulated after LPS stimulation. When MoDCs cultured for 5 days were examined, IFN-{beta} was seen to significantly up-regulate TRAIL expression, whereas LPS had no effect.



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  		FIGURE 1. TRAIL expression was up-regulated on the human MoDCs and CD34DCs stimulated with IFN-{beta} but not with LPS. Human MoDCs (cultured for 5 days) or CD34DCs (cultured for 12 days) were incubated in the absence or presence of IFN-{beta} (100 ng/ml) or LPS (100 ng/ml) for 12 h and then analyzed for TRAIL expression. Dotted line, Staining by isotype control mAb; solid line, TRAIL staining on unstimulated DCs, LPS-stimulated DCs, or IFN-{beta}-stimulated DCs. The percentage of gated cells expressing TRAIL is shown in each panel. Results represent one consistent result from at least three independent experiments. FL, fluorescence; H, height.

 
Growth inhibition of hemopoietic origin tumor cells by TRAIL

Many tumor cells of hemopoietic-origin are sensitive to TRAIL-mediated apoptosis. To ascertain whether DCs can kill tumor cells via TRAIL, we first screened for TRAIL-sensitive tumor cell lines in a variety of tumor cells of hemopoietic origin as measured by the MTT reduction assay. As shown in Fig. 2Go, HL60 cells and Reh cells were highly sensitive to TRAIL-mediated growth inhibition. Obvious cellular debris of HL60 cells or Reh cells was observed under the microscope (data not shown). However, no obvious growth inhibition of other tumor cells, including Jurkat cells, Daudi cells, and U937 cells, was observed under the indicated range of TRAIL concentrations, which was inconsistent with previous reports (33). Thus, in our system, HL60 cells and Reh cells were determined as the TRAIL-sensitive tumor cells for the following experiments.



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  		FIGURE 2. Growth inhibition of tumor cells of hemopoietic origin by TRAIL. HL60, Reh, Jurkat, Daudi, and U937 cells were incubated in the presence of TRAIL at the indicated concentration for 24 h, and then their viability was measured by MTT assay. Percent growth inhibition was quantitated as previously described in Materials and Methods. Data represent one consistent result from three independent experiments.

 
Involvement of TRAIL in the cytotoxicity of IFN-{beta}-stimulated DCs against HL60 cells and Reh cells

CD4+T cells, NK cells, macrophages, and CD11c+ DCs can kill some tumor cells via TRAIL under certain conditions (19, 26, 27, 37, 38). To investigate whether CD34DCs or MoDCs cultured in vitro have similar cytotoxic capacity, and if so whether they use TRAIL to perform this function, we first evaluated the susceptibility of the two TRAIL-sensitive tumor cells to the cytotoxic effects of CD34DCs or MoDCs. As shown in Fig. 3Go A, unstimulated MoDCs or LPS-stimulated MoDCs did not have obvious cytotoxicity to HL60 cells at the E:T ratios used. However, IFN-{beta} could markedly potentiate MoDCs to kill HL60 cells. Unstimulated and LPS-stimulated MoDCs could kill Reh cells only at a high E:T ratio (20:1), whereas IFN-{beta}-stimulated MoDCs could induce obvious death of Reh cells at all E:T ratios (Fig. 3GoB). Similarly, only after IFN-{beta} stimulation could CD34DCs efficiently kill HL60 cells at an E:T ratio of 20:1 and Reh cells at all E:T ratios, as compared with unstimulated and LPS-stimulated CD34DCs (Fig. 3Go, C and D). Thus, IFN-{beta} could significantly enhance the cytotoxicity of CD34DCs or MoDCs.



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  		FIGURE 3. Cytotoxicity of DCs to TRAIL-sensitive tumor cells. Human MoDCs (cultured for 5 days) or CD34DCs (cultured for 12 days) were cultured with IFN-{beta} (100 ng/ml), with LPS (100 ng/ml), or in medium alone for 12 h. They were then cocultured with 51Cr-labeled TRAIL-sensitive tumor cells at the indicated E:T ratios for another 18 h. The cytotoxicity of DCs to TRAIL-sensitive tumor cells was detected using the 51Cr release assays as described in Materials and Methods. A, Cytotoxicity of MoDCs to HL60 cells; B, cytotoxicity of MoDCs to Reh cells; C, cytotoxicity of CD34DCs to HL60 cells at an E:T ratio of 20:1; D, cytotoxicity of CD34DCs to Reh cells. Data at each E:T ratio are expressed as mean ± SD of triplicate wells. Similar results were obtained in three independent experiments.

 
Having ascertained that IFN-{beta} can up-regulate TRAIL expression on DCs, and to confirm whether up-regulation of TRAIL was responsible for the enhanced cytotoxicity of CD34DCs or MoDCs induced by IFN-{beta} stimulation, we pretreated IFN-{beta}-stimulated CD34DCs or MoDCs with TRAIL-R3-Fc to block TRAIL. We then observed their cytotoxicity against HL60 cells or Reh cells. As shown in Fig. 4Go, A and B, TRAIL-R3-Fc but not the isotype-matched control could partially reduce the cytotoxicity of IFN-{beta}-stimulated MoDCs or CD34DCs against HL60 cells as compared with the control (p < 0.05). Similarly, TRAIL-R3-Fc (but not human IgG1) could incompletely inhibit IFN-{beta}-stimulated MoDCs or CD34DCs to kill Reh cells (Fig. 4Go, C and D). Therefore, IFN-{beta}-stimulated CD34DCs or MoDCs could effectively kill TRAIL-sensitive tumor cells partially via TRAIL.



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  		FIGURE 4. Involvement of TRAIL in the cytotoxicity of IFN-{beta}-stimulated DCs to TRAIL-sensitive tumor cells. IFN-{beta}-stimulated MoDCs or CD34DCs were pretreated with TRAIL-R3-Fc (1 µg/ml) or human IgG1 (1 µg/ml) for 30 min. They were then cocultured with 51Cr-labeled TRAIL-sensitive tumor cells for another 18 h. The effect of TRAIL-R3-Fc on the cytotoxicity of IFN-{beta}-stimulated DCs to TRAIL-sensitive tumor cells was detected by 51Cr release assays. A, Cytotoxicity of IFN-{beta}-stimulated MoDCs to HL60 cells; B, cytotoxicity of IFN-{beta}-stimulated CD34DCs to HL60 cells; C, cytotoxicity of IFN-{beta}-stimulated MoDCs to Reh cells; D, cytotoxicity of IFN-{beta}-stimulated CD34DCs to Reh cells. Data at each E:T ratio are shown as mean ± SD of triplicate wells. Similar results were obtained in three independent experiments.

 
Involvement of caspase-3 activation in the sensitivity of tumor cells to the cytotoxicity of IFN-{beta}-stimulated DCs

Next, we investigated the intracellular mechanism of the cytotoxicity of IFN-{beta}-stimulated DCs toward tumor cells. Most apoptosis-inducing ligands (CD95L, TRAIL, TNF-{alpha}) can induce apoptosis through activation of the caspase cascade. Caspase-8 is an apical member of the caspase cascade, the activation of which can induce activation of downstream caspases. Caspase-3 activation is thought to contribute to cleavage of downstream substrates such as poly(ADP-ribose)polymerase, lamins, and actins, leading to a series of apoptotic events, such as DNA fragmentation, cellular skeleton collapse, etc. (28). Before we investigated whether the activation of caspases was involved in the killing of HL60 cells or Reh cells by IFN-{beta}-stimulated CD34DCs or MoDCs, we examined the kinetic activation of caspase-3 and caspase-8 in HL60 cells and Reh cells induced by TRAIL. As shown in Fig. 5Go, activated subunits were released from procaspase 3 and procaspase 8 after incubating for 2 h. Maximal levels of activated subunits were seen after 6 h and were maintained until 12 h in the presence of TRAIL. After incubation with TRAIL for 24 h, activated subunits in HL60 cells or Reh cells were still detectable but at a low level. We then preincubated HL60 cells and Reh cells with caspase-3 inhibitor for 30 min before coculture with IFN-{beta}-stimulated CD34DCs or MoDCs and observed its effect on cytotoxicity. As shown in Fig. 6Go, A and B, the sensitivity of HL60 cells and Reh cells to the cytotoxicity of IFN-{beta}-stimulated MoDCs or CD34DCs was significantly reduced (p < 0.05). Thus, caspase-3 activation was clearly involved in the killing of TRAIL-sensitive tumor cells by IFN-{beta}-stimulated MoDCs or CD34DCs.



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  		FIGURE 5. The activation of caspase-3 and caspase-8 in the TRAIL-sensitive tumor cells induced by TRAIL. HL60 cells or Reh cells were cultured in the presence of TRAIL (100 ng/ml) plus TRAIL enhancer (500 ng/ml) for 0, 2, 6, 12, and 24 h (lanes 1–5), and then the activated subunits of caspase-3 or caspase-8 were examined by Western blot assay. kd, Kilodaltons.

 


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  		FIGURE 6. Involvement of caspase-3 activation in the sensitivity of tumor cells to the cytotoxicity of IFN-{beta}-stimulated DCs. 51Cr-labeled TRAIL-sensitive tumor cells (HL60 or Reh cells) were pretreated with caspase-3 inhibitor (50 µM) for 30 min and then cocultured with IFN-{beta}-stimulated DCs for another 18 h. The effect of caspase-3 inhibitor on the susceptibility of TRAIL-sensitive tumor cells to the cytotoxicity of IFN-{beta}-stimulated DCs at an E:T ratio of 10:1 was detected by 51Cr release assays. A, Cytotoxicity of IFN-{beta}-stimulated MoDCs to TRAIL-sensitive tumor cells pretreated with caspase-3 inhibitor; B, cytotoxicity of IFN-{beta}-stimulated CD34DCs to TRAIL-sensitive tumor cells pretreated with caspase-3 inhibitor. Data are shown as the mean ± SD of triplicate wells. Similar results were obtained in three independent experiments.

 
Involvement of NF-{kappa}B activation in the sensitivity of tumor cells to the cytotoxicity of DCs

TRAIL can induce not only the activation of the caspase cascade leading to apoptotic death events but also the activation of the NF-{kappa}B pathway, which is associated with the resistance of some tumor cells to TRAIL-mediated apoptosis. NF-{kappa}B inhibitor can efficiently change some TRAIL-resistant tumor cells into TRAIL-sensitive cells (30, 32). Because CD34DCs and LPS-stimulated CD34DCs did not kill HL60 cells or Reh cells efficiently, we evaluated whether they could induce death of HL60 cells or Reh cells pretreated with NF-{kappa}B inhibitor. In this case, CD34DCs and LPS-stimulated CD34DCs or MoDCs could easily kill HL60 cells or Reh cells after pretreatment with NF-{kappa}B inhibitor as compared to untreated groups (p < 0.05). (Fig. 7Go). Therefore, the sensitivity of tumor cells to the cytotoxicity of DCs could be up-regulated by inhibition of NF-{kappa}B activation.



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  		FIGURE 7. Involvement of NF-{kappa}B activation in the TRAIL-sensitive tumor cells to the cytotoxicity of DCs. 51Cr-labeled TRAIL-sensitive tumor cells were pretreated with NF-{kappa}B inhibitor (15 µM) for 30 min, and then cocultured with stimulated or unstimulated CD34DCs or MoDCs at an E:T ratio of 10:1 for another 18 h. The effect of NF-{kappa}B inhibitor on the cytotoxicity of stimulated or unstimulated DCs to TRAIL-sensitive tumor cells was detected by 51Cr release assays. A, Cytotoxicity of stimulated or unstimulated MoDCs to HL60 cells; B, cytotoxicity of stimulated or unstimulated MoDCs to Reh cells; C, cytotoxicity of stimulated or unstimulated CD34DCs to HL-60 cells; D, cytotoxicity of stimulated or unstimulated CD34DCs to Reh cells. Data are expressed as the mean ± SD of triplicate wells. Similar results were obtained in three independent experiments.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Our studies show that CD34DCs or MoDCs stimulated by IFN-{beta} can efficiently kill TRAIL-sensitive HL60 cells and Reh cells. The cytotoxicity was partially related to the up-regulation of TRAIL expression on the DCs by IFN-{beta} stimulation. Both caspase activation and NF-{kappa}B activation were involved in the control of the sensitivity of HL60 cells and Reh cells to IFN-{beta}-stimulated DC-mediated killing.

The tumoricidal activity of DCs was enhanced more markedly by stimulation of IFN-{beta} than LPS, which was partially associated with the up-regulation of TRAIL expression. We do not exclude the possibility that other mechanisms also contribute to the antitumor effect. Fas ligand, TNF-{alpha}, and nitric oxide expressed or secreted by activated DCs may play a role in the tumoricidal activity (15, 43, 44). Our data showing that TRAIL-R3-Fc is unable to completely block the killing of tumor cells induced by DCs confirm the existence of other mechanisms. In addition, we were unable to observe whether CD34DCs or MoDCs also have the ability to kill TRAIL-resistant tumor cells, which would supply more convincing evidence that DCs use TRAIL, or other toxic molecules, or both, to kill tumor cells. The study of Fanger et al. revealed that the tumoricidal activity of CD11c+DCs was TRAIL dependent, whereas that of Chapoval et al. demonstrated that the cytostatic effect mediated by MoDCs was partially membrane-bound TNF-{alpha} specific (18, 19).

IFNs were discovered through their antiviral activity. In addition, they also have far-ranging immunomodulatory effects on several kinds of immune cells such as NK cells, T cells, and macrophages to regulate host defense against infectious agents and tumor cells. IFNs induce apoptotic cell death by up-regulating the expression of a variety of apoptosis-inducing molecules (e.g., FasL, TRAIL) (19, 45, 46). Moreover, type I IFNs are able to rapidly induce CD14+ monocytes into short-lived and TRAIL-expressing DCs with potent functional activities (47). These previous data are somewhat consistent with our results, where we have shown that IFN-{beta} is capable of up-regulating TRAIL expression on immature CD34DCs and MoDCs. There has been no relevant report of the effect of IFN-{beta} on DC-mediated tumoricidal activities to date. Our study is the first to report that IFN-{beta} can enhance DC-mediated tumoricidal activity partially via up-regulation of TRAIL. The fact that type I IFNs stimulate DCs to utilize TRAIL for generating apoptotic bodies might enable subsequent cross-priming of CD8+ CTLs, indicating that type I IFNs may be powerful adjuvants to bridge innate and adoptive immunity.

In our study, we examined the killing function of immature CD34DCs and MoDCs. To determine whether the degree of maturity correlates with cytotoxicity, we analyzed the cytotoxicity of mature DCs induced by exogenous stimuli simultaneously. It has been confirmed that LPS, TNF-{alpha}, CD40 ligand, and IFN-{gamma} are powerful maturation factors that induce DC maturation (48). We selected LPS to stimulate immature DCs into mature cells. After 12 h incubation of immature CD34DCs and MoDCs with LPS, a mature phenotype and potent functionality (in an MLR assay) were observed, which suggested that the immature DC were indeed induced into mature DCs (data not shown). However, in cytotoxicity experiments, we only observed minimal DC-mediated tumoricidal activity in the presence of LPS compared with IFN-{beta}. Thus, distinct effects of LPS and IFN-{beta} on DC were suggested. IFN-{beta} seems to enhance killing function, which allows for DCs to be innate "defense soldiers" able to induce apoptosis, and then to take up and process apoptotic Ags. Alternatively, LPS, as an inflammatory factor, helps to stimulate DCs into maturation and then to play conventional Ag-presenting roles.

A question remains that we were unable to answer in our system, whether the cytotoxicity of IFN-{beta}-stimulated DCs via TRAIL was due to soluble TRAIL either secreted from DCs or cleaved from DCs by protease in the supernatants or due to the membrane-bound TRAIL on DCs. One report has shown that DC-mediated inhibition of growth of tumors requires a cell-to-cell interaction through the use of a Transwell cell culture system (Millipore, Bedford, MA) (18).

Caspases are a family of intracellular cysteine proteases that contribute to apoptosis. Under most circumstances, the heterotetramer complex is in an inactive form but can be cleaved to release activated subunits of ~20 kDa. The mechanisms of TRAIL-mediated apoptosis are unknown. At least two pathways have been confirmed as regulating sensitivity to TRAIL. One pathway participates in an apoptotic event that involves the activation of FADD-caspase cascades triggered by DR4 or DR5. The other pathway counteracts apoptosis and may be correlated with activation of NF-{kappa}B by DR4, DR5, and DCR2. Certain molecules have been shown to control cellular survival or death. Among these, the most implicated is c-FLIP the gene of which is homologous to that of caspase-8. c-FLIP prevents caspase-8 from entering the death-inducing signal complex by binding to itself, but it is unable to perform protease activity, thus failing to deliver downstream death signals (28, 34, 35, 36). The high levels of expression of c-FLIP in some TRAIL-resistant tumor cells and normal cells may explain their TRAIL-resistant properties. In summary, whether cells are sensitive to TRAIL or not is determined not only by the net abundance of death receptors vs decoy receptors exposed to TRAIL but also by the balance of intracellular apoptotic and antiapoptotic mechanisms. Our studies have shown that DC-mediated cytotoxicity to tumor cells via TRAIL involves the activation of caspase-3 in tumor cells and that the sensitivity of tumor cells to DC-mediated cytotoxicity was increased by inhibition of NF-{kappa}B activation in tumor cells. Therefore, whether tumor cells in vivo are eliminated by effector arms such as T cells, NK cells, macrophages, and DC is dependent on both the function of activated effector cells under given circumstances and the genetic properties of tumor cells.

In conclusion, DCs were effective at killing TRAIL-sensitive tumor cells partially via TRAIL in an IFN-{beta}-stimulated microenvironment. To date, both type I and type II IFNs have been confirmed as enabling DCs to remove tumor cells via up-regulation of TRAIL expression. In addition, type II DC precursors (pDC2s) have been confirmed to be "natural IFN-producing cells," which generate 200–1000 times more IFN than any other subset of PBMC on pathogenic challenge (49). Thus, it is possible that an innate antitumor mechanism mediated by DCs is active in vivo. DC precursors may be stimulated to secret IFNs when they encounter spontaneous or metastatic tumor cells. The resulting IFNs may trigger DCs to efficiently induce apoptosis of tumor cells for subsequent uptake and processing of apoptotic bodies and presentation of antigenic peptides to CD8+ CTLs. The mechanism may also be extended to antiviral immunity, rejection of transplantation, and autoimmunity, because DCs could induce apoptosis of, and then capture apoptotic Ags from, infectious cells, allogeneic transplants, or even self-tissues, which would be used to cross-prime CD8+ CTLs. Thus, DCs may be viewed as a bridge linking the innate and the adaptive immune systems.


   Acknowledgments
 
We thank the Blood Bank of Shanghai for gifts of healthy whole blood; Changhai Hospital for gifts of bone marrow from allogeneic transplantation; Dr. W. Zhang, Dr. X. Huang, Dr. Z. Yuan, Dr. T. Y. Chen, and Dr. W. G. Song for their excellent technical assistance and discussion of this work. We also thank Prof. Frances Gotch for helpful review of the manuscript.


   Footnotes
 
1 This work was supported by Grants 39825123 and 39730420 from the National Natural Science Foundation of China. Back

2 Address correspondence and reprint requests to Dr. Xuetao Cao, Department of Immunology, Second Military Medical University, 800 Xiangyin Road, Shanghai 200433, People’s Republic of China. Back

3 Abbreviations used in this paper: DC, dendritic cell; TRAIL, TNF-{alpha}-related apoptosis-inducing ligand; MoDC, monocyte-derived DC; FADD, Fas-associated death domain-containging protein; c-FLIP, cellular FADD-like IL-1{beta} converting enzyme protease-inhibitory protein; CD34DC, human CD34+ stem cell-drived DCs; CD95L, C95 ligand. Back

Received for publication August 11, 2000. Accepted for publication February 21, 2001.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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