It has been known that melanoma cells can suppress the immune system by the Fas ligand. The present study investigated whether interleukin(IL)-18, which can enhance Fas ligand expression, is produced by B16F10 melanoma cells and is involved in immune escape of tumor cells. Immunohistology, reverse transcription-PCR, intracellular fluorescence-activated cell-sorting analysis, and immunoblotting demonstrated that melanoma cells express IL-18. C57BL/6 splenocytes cultured with culture supernatants of B16F10 melanoma cells enhanced IFN-γ production, which was blocked by anti-IL-18 antibody,indicating that IL-18 in the culture supernatants is functional. In addition to IL-18, the IL-18 receptor was also detected in B16F10 melanoma cells, suggesting a role of this cytokine in regulating the functions of B16F10 melanoma cells. The functional effect of IL-18 on B16F10 melanoma cells was shown by reduction of Fas ligand expression in cells treated with anti-IL-18 antibody or transfected with IL-18 antisense cDNA. In addition, the same treatments decreased intracellular reactive oxygen intermediate levels in B16F10 melanoma cells, indicating that IL-18 regulates reactive oxygen intermediate production, which is involved in Fas ligand expression. Furthermore,transfection of IL-18 antisense cDNA into melanoma cells increased the susceptibility of tumor cells to natural killer cells in vitro. When IL-18 antisense transfectants were implanted into syngeneic mice, severe reduction of tumor cell growth was observed with concomitant infiltrated natural killer cells in the tumor area. Taken together, these results demonstrate that IL-18 has a critical role as a survival factor for B16F10 melanoma cells.

Malignant melanoma leads to severely increased mortality, and the rate of incidence is higher than any other cancer. It has been suggested that melanoma may escape immune surveillance through several possible mechanisms, including immune suppression by soluble inhibitory factors such as IL-10,4or by down-regulation of MHC molecule expression (1, 2, 3). Recently, Fas ligand-mediated immune suppression was proposed in melanoma (4). According to the authors, melanoma cells express Fas ligand to protect themselves against tumor-infiltrating immune effector cells by killing Fas-expressing cells. Thus, it is important to define what factors involve Fas ligand expression to escape immune surveillance in melanoma.

IL-18 (IFN-γ-inducing factor) is an 18-kDa cytokine produced by lipopolysaccharide-activated macrophages or Kupffer cells(5). In addition to activated macrophages or Kupffer cells, it has been shown that epidermal keratinocytes and osteoblastic stromal cells produce IL-18 (6, 7). IL-18 affects the immune system by inducing IFN-γ secretion by T, NK, or B cells;enhancing proliferation of anti-CD3 monoclonal antibody, IL-2,or concanavalin A-stimulated T cells; augmenting Fas ligand-mediated NK cell cytotoxic activity; and inhibiting osteoclast formation in vitro(5, 7, 8, 9, 10). Furthermore, in vivo studies have demonstrated that anti-IL-18 antibodies protect against lipopolysaccharide-induced liver damage in mice,suggesting that IL-18 plays an important role in inflammatory response(11). Indeed, recent work by Puren et al.(12) showed that IL-18 can initiate a cascade of proinflammatory cytokines through the production of immediate early inflammatory cytokines such as TNF-α and IL-1β.

It has been shown that ROIs, i.e., hydrogen peroxide,hydroxyl radical, superoxide anion, and nitric oxide, frequently are produced during inflammatory response and that they regulate production of inflammatory cytokines (13, 14). In addition, it is believed that ROIs provide beneficial effects to tumor cells(15). In melanoma, it is known that ROIs play a key role in the induction of resistance to Fas-mediated cell death(16). In addition, it has been reported that ROIs regulate Fas ligand expression in NK and hepatoma cells (17, 18). Because ROIs and IL-18 are important factors in inflammatory responses and share biological functions, it is likely that ROI production may be related to IL-18 function in melanoma. Therefore, we hypothesized that IL-18 might be produced in melanoma cells and act as an autocrine factor to regulate Fas ligand expression and intracellular ROI production for immune escape. In this report, our data clearly demonstrate that melanoma cells produce IL-18 to regulate Fas ligand expression and intracellular ROI production.

Cells and Animals.

C57BL/6 mice splenocytes were prepared using a standard protocol(19). Briefly, mice were killed by cervical dislocation,and splenocytes were prepared by mechanical disruption. The murine melanoma cell lines B16F0, B16F1, and B16F10 were cultured in RPMI 1640 supplemented with 2 mml-glutamine, 100 units/ml penicillin, 100 μg/ml streptomycin, and 10%heat-inactivated fetal bovine serum, hereafter referred to as CM. These cell lines were used for experiments while in the log phase of growth. C57BL/6 mice were bred in the animal facility of the Korea Research Institute of Bioscience and Biotechnology and used at 6–8 weeks of age.

IL-18 Bioassay (IFN-γ Induction Assay).

The induction of IFN-γ from splenocytes by IL-18 was assayed to detect IL-18 bioactivity. Briefly, C57BL/6 mice splenocytes were prepared using standard protocols. Prepared splenocytes were suspended in CM at a concentration of 5 × 106 cells/ml. One ml of splenocytes (5 × 106 cells) was plated in each well of a 24-well plate, followed by addition of 1 ml of the culture supernatants in triplicate. After 72 h of incubation, cell-free supernatants were assayed for IFN-γ production using a murine IFN-γELISA kit (Endogen, Inc., Cambridge, MA). In neutralizing experiments,culture supernatants of melanoma cells were pre-incubated for 2 h with polyclonal antimouse IL-18 antibody (20 μg/ml) or an isotope control (20 μg/ml) before the IL-18 bioassay was performed.

RT-PCR.

Total RNA was extracted from B16F10 melanoma cells using RNAzol,according to the instructions of the manufacturer. After reverse transcription, the cDNA was incubated with IL-18 primers (sense,5′-ACTGTACAACCGCAGCAGTAATACGG-3′; antisense,5′-AGTGAACATTACAGATTTATCCC-3′) or IL-18 receptor primers (sense,5′-TCCTGGAGAAACAGTTTGGG-3′; antisense, 5′-CGCTGAAACTCCTGAAGTCC-3′) for PCR amplification. Cycling conditions for IL-18 were 1 min at 94°C, 1 min at 55°C, and 1 min at 72°C for 35 cycles; conditions for IL-18 receptor were 1 min at 95°C, 0.5 min at 58°C, and 1 min at 72°C.

Flow Cytometry Analysis.

Intracellular FACS analysis was performed to detect IL-18 or Fas ligand in melanoma cell lines. Briefly, cells were washed twice with ice-cold PBS containing 0.05% BSA and 0.02% sodium azide. After two washes,cells were fixed in 2% paraformaldehyde in PBS for 15 min on ice. Thereafter, the cells were washed once in cold PBS-BSA and resuspended in PBS containing 0.1% saponin and 0.05% sodium azide(permeabilization buffer) for 15 min, followed by incubation with rabbit antimouse IL-18 polyclonal antibody or antimouse Fas ligand antibody (PharMingen, San Diego, CA) for 30 min on ice. After two washes, cells were further incubated with an appropriate FITC-conjugated secondary antibody in permeabilization buffer for 30 min on ice, followed by three washes. A FACScan (Becton Dickinson,Sunnyvale, CA) flow cytometer was used for analysis. For ROI assays,melanoma cells (1 × 106 cells/ml)were incubated with 50 μm 2′,7′-dichlorofluorescein diacetate (Eastman Kodak Co., Rochester, NY) for 5 min at 37°C. After incubation, cells were analyzed using a FACScan flow cytometer.

Transfection of IL-18 Antisense cDNA.

Murine IL-18 cDNA was cloned into pcDNA3.1 vector by blunt end ligation. The orientation of inserts was confirmed by AvaI digestion. B16F10 melanoma cells were transfected with the IL-18 antisense construct by the calcium phosphate precipitation method. The stable transfectant clones were selected in the CM containing 1.4 mg/ml neomycin (G-418, Geneticin; Life Technologies, Inc.). IL-18 expression was confirmed by intracellular staining and immunoblotting for IL-18.

BLT Esterase Release.

After incubation of NK cell and melanoma cells, 50 μl of cell-free supernatant were collected and mixed with 150 μl of the reaction mixture [0.2 mm BLT and 0.22 mm5,5′-dithiobis(2-nitrobenzoic acid) in PBS] in flat-bottomed 96-well plates. After a 20-min reaction at 37°C, the absorbance was read at 412 nm in an ELISA plate reader. The percentage of BLT esterase activity was calculated using the following equation: (Experimental BLT esterase release − spontaneous BLT esterase release)/(maximum BLT esterase release − spontaneous BLT esterase release) × 100. Spontaneous BLT esterase release from NK cells was measured in the well without antibody, and maximum BLT esterase release was measured after cell lysis with 1%Triton X-100.

In Vivo Tumor Model.

C57BL/6 male mice 6 weeks of age were obtained from the Laboratory Animal Division of KRIBB. The B16F10 mouse melanoma tumors were established by s.c. injection of 1 × 105 cells in 200 μl of PBS. Tumor size was determined by measuring with calipers every 2 days, and the values were inserted into the formula: Tumor size(cm2) = 0.5 × (largest diameter) × (smallest diameter)2.

Immunohistochemistry.

The tissue samples were immersed in 25% sucrose-PBS solution for frozen sections. Each tissue was embedded in OCT compound, quick-frozen in isopentane cooled by liquid nitrogen, and sectioned into 8-μm thicknesses using a cryostat (Leica CM5060). Cryosections were dried in air and fixed in cold acetone at 4°C. These specimens were incubated with biotinylated anti-IL-18 or mouse NK cell antibody (NK1.1;PharMingen) for 1 h at 37°C in a wet chamber. Subsequently, the specimens were incubated with alkaline phosphatase-conjugated streptavidin complex. The activity was visualized by incubation with substrate solution. All specimens were counterstained with methyl green.

Detection of IL-18 mRNA and Protein in Melanoma Cells.

When tissue samples from melanoma patients were immunostained with anti-IL-18 antibody, dominant IL-18 expression was observed in tumor sites (Fig. 1,A). To examine whether murine melanoma cell lines were also able to express IL-18 protein, intracellular staining of IL-18 using FACScan analysis was performed. Fig. 1,B shows that B16F10 melanoma cells produced IL-18 protein. Three different subclones of the B16 melanoma cell lines, B16F0, B16F1, and B16F10, produced IL-18 protein, but there was no difference in the amount of IL-18 protein between subclones. In addition, constitutive expression of IL-18 mRNA was detected in B16F10 murine melanoma cells (Fig. 1,C). IL-18 protein was also detected by immunoblotting with anti-IL-18 antibody (Fig. 1 D). Collectively, these data demonstrate that melanoma cells express IL-18 mRNA and protein.

Secretion of Functional IL-18 by Murine Melanoma Cells.

To test the production of functional IL-18 protein by murine melanoma cell lines, culture supernatants of B16F10 murine melanoma cell lines were harvested and examined for their ability to induce IFN-γsecretion from splenocytes of C57BL/6 mice. Conditioned medium collected from untreated C57BL/6 splenocytes induced only a slight secretion of IFN-γ, whereas conditioned medium collected from treated C57BL/6 splenocytes induced significant secretion of IFN-γ (Table 1). To confirm that the observed enhancement of IFN-γ secretion was specifically due to IL-18, neutralizing anti-IL-18 antibody was used. Table 1 demonstrates that neutralizing antibody significantly abolished the enhancement of IFN-γ secretion in a dose-dependent manner. Thus,these data demonstrate that murine melanoma cells secrete functional IL-18. In addition, expression of IL-18 receptor in murine melanoma cells was tested. RT-PCR revealed that B16F10 murine melanoma cells express IL-18 receptor (Fig. 1 E). These results suggest that IL-18 produced by murine melanoma cells can regulate the physiological functions of tumor cells.

Effects of IL-18 Antisense cDNA and Anti-IL-18 Antibody on Expression of Fas Ligand in Melanoma Cells.

It has been reported that IL-18 up-regulates Fas ligand expression in NK cells (10) and in myelomonocytic KG-1 cells(20). Melanoma cells express Fas ligand, which is involved in tumor immune escape (4). To identify the possible roles of IL-18 in regulating Fas ligand expression and survival of melanoma cells, we established a B16F10 clone that expressed a lower level of IL-18 by transfection with IL-18 antisense cDNA (Fig. 2). FACS (Fig. 2,A) and immunoblot (Fig. 2,B)analysis showed that IL-18 expression was reduced in IL-18 antisense transfectants. MHC class I (Fig. 2,C) and intercellular adhesion molecule-1 (Fig. 2,D) expression were not altered by transfection with the IL-18 antisense construct, but interestingly CD71 (transferrin receptor), which is involved in transferrin uptake and target cell sensitivity to NK cells, was up-regulated (Fig. 2 E).

When Fas ligand expression was analyzed by intracellular FACS analysis,its expression was reduced in IL-18 antisense transfectants compared with vector transfectants (Fig. 3,A). In the same line, treatment of B16F10 melanoma cells with anti-IL-18 antibody reduced Fas ligand expression (Fig. 3,B),indicating that IL-18 is an important factor for regulating Fas ligand expression in B16F10 melanoma cells. Recent reports (17, 18) indicate that Fas ligand expression is regulated by ROIs. When B16F10 melanoma cells were treated with an antioxidant, NAC, Fas ligand expression was reduced (Fig. 3 C), suggesting that ROIs are also involved in Fas ligand expression in melanoma cells.

Effects of IL-18 Antisense cDNA and Anti-IL-18 Antibody on ROI Production in Melanoma Cells.

In addition to the regulatory roles of ROIs in Fas ligand expression, it is known that some tumor cells enhance ROI generation that is beneficial for their survival (15) compared with their normal counterparts. ROI levels are elevated in melanoma and play a key role in resistance to Fas-induced apoptosis in melanoma cells(16). We next asked whether IL-18 could regulate ROI production in melanoma cells. Transfection with IL-18 antisense cDNA reduced the intracellular ROI level compared with control cells (Fig. 4,A). In addition, B16F10 melanoma cells were incubated with anti-IL-18 polyclonal antibody for 18 h, and ROI levels were analyzed (Fig. 4,B). Anti-IL-18 antibody reduced ROI levels in B16F10 melanoma cells, confirming that IL-18 is critical for regulating ROI expression in B16F10 melanoma cells. When the cells were treated with NAC for ≥12 h, the intracellular ROI levels in B16F10 melanoma cell were also reduced, as expected (Fig. 4 C).

Effects of IL-18 Antisense cDNA on NK Susceptibility and Tumor Growth of Melanoma Cells.

Transfection with IL-18 antisense cDNA reduced Fas ligand expression and ROI production, which are related to tumor cell survival. To identify the roles of IL-18 expression in melanoma cells in the relationship between NK and tumor cells, the susceptibility of melanoma cells to NK cells were tested. Transfection with IL-18 antisense cDNA significantly increased the susceptibility to NK cells activated by incubation with IL-2 or IL-2 plus IL-18 (Fig. 5,A). In addition, BLT esterase release was elevated when NK cells were incubated with IL-18 antisense transfectants (Fig. 5,B). When IL-18 antisense transfectants were implanted into syngeneic mice, severe regression of tumor growth was observed (Fig. 6,A). In addition, the survival rate was prolonged in mice injected with IL-18 antisense transfectants compared with those injected with vector transfectants (Fig. 6,B). In immunohistological analysis by staining with anti-NK cell antibody,infiltrated NK cells were observed in the tumor sites of mice injected with IL-18 antisense transfectants (Fig. 6,C-b), but not in the mice injected with vector transfectants (Fig. 6 C-a). These in vitro and in vivo data indicate that expression of IL-18 is a key element for protecting melanoma cells from host immune cells.

Malignant transformation of melanocytes is characterized by the loss of MHC class I molecules by melanoma cells, and this abnormality may account for the immune escape of malignant melanoma cells from CTL-mediated immune surveillance (3). In addition to this mechanism, Hahne et al.(4)suggested that Fas ligand-expressing melanoma cells use Fas ligand to induce apoptosis of Fas-expressing immune cells at the tumor site,and this strategy may be a general mechanism responsible for immune privilege used by tumor cells. In vivo, this may be an explanation of why malignant melanoma cells are not susceptible to NK-cell-mediated immune surveillance although they lose the expression of MHC class I molecules.

IL-18 is a recently cloned cytokine that was primarily identified by its ability to induce IFN-γ production (5). Tsutsui et al.(10) and Dao et al.(21) additionally showed that this cytokine could enhance Fas ligand expression in T-helper 1 and NK cells. We therefore examined the production of IL-18 to investigate whether this cytokine is the regulatory factor of Fas ligand expression in melanoma cells. The present study indicated that murine melanoma cell lines produce IL-18 and express IL-18 receptors (Fig. 1). Culture supernatants derived from murine melanoma cell lines induced IFN-γ secretion by C57BL/6 splenocytes (Table 1). Blocking experiments with anti-IL-18 polyclonal antibodies showed that IFN-γ production was not totally inhibited by anti-IL-18 polyclonal antibodies. It is well known that melanoma cell lines produce IL-2 and that release of IFN-γ by splenocytes can be caused by IL-2 (22). Therefore, IL-2 might be responsible for the IFN-γ production activity found in melanoma culture supernatants after treatment with anti-IL-18 polyclonal antibodies. We performed a proliferation assay to test whether IL-18 is involved in the regulation of B16F10 cell proliferation. The data indicated that IL-18 does not affect B16F10 cell proliferation (data not shown). Because functional IL-18 protein and IL-18 receptor were expressed in B16F10 melanoma cells, we were interested in investigating whether IL-18 acts as an autocrine factor in the regulation of Fas ligand expression of these cells. Transfection with IL-18 antisense cDNA or treatment with anti-IL-18 antibody down-regulated Fas ligand expression in B16F10 melanoma cells,indicating that IL-18 commonly regulates Fas ligand expression (Fig. 3).

To our knowledge, little is known about the intracellular and molecular mechanisms that induce and regulate IL-18 production and transcription of the IL-18 gene (23, 24), whereas IL-18 signal transduction pathways are becoming clear. It has been documented that nuclear factor-κB is activated by IL-18 (25). In addition, IL-18 was able to induce activation of p561ck and mitogen-activated protein kinase, suggesting that the p561ck-mitogen-activated protein kinase pathway is involved in IL-18 signal transduction pathways (26). Recent work by Puren et al.(12) showed that IL-18 has proinflammatory properties, which are induced by IL-8 and IL-1β from CD14+ cells via direct TNF-α production by CD4+ T cells and NK cells, indicating that IL-18 may play a key role in the inflammatory cascade. In addition, it is well known that ROIs regulate the expression of inflammatory cytokines such as TNF-α through nuclear factor-κB activation and tyrosine kinase-dependent pathways (13, 14). On the basis of these studies, we hypothesized that IL-18 may act as a regulator of ROI production by B16F10 melanoma cells. To prove this hypothesis, we performed neutralizing experiments using IL-18 antisense constructs or anti-IL-18 antibody (Fig. 4). Our data indicated that IL-18 antisense cDNA or anti-IL-18 antibody reduced ROI levels in 18-h cultured B16F10 melanoma cells, demonstrating ROI regulation by IL-18. It is interesting to note that the inhibitory effect of anti-IL-18 antibody disappeared after 24-h incubation (data not shown). The possible explanation of these results is that melanoma cells re-establish normal intracellular ROI levels during prolonged exposure to anti-IL-18 antibody. It may be reasonable to predict that ROIs are essential for the survival and tumor activity of melanoma cells. Preliminary experiments showed that intracellular ROI and IL-18 expression were down-regulated in the apoptotic cells death of melanoma cells,indicating that IL-18/ROI pathways may be critically involved in the survival of melanoma cells (data not shown).

Suppression of IL-18 production by melanoma cells by transfection with IL-18 antisense constructs made the cells more susceptible to NK cells, probably because of reduced Fas ligand expression and intracellular ROI levels. As mentioned above, IL-18 activates NK cytolytic activity by up-regulating Fas ligand expression. A recent in vivo study by Osaki et al.(27)demonstrated that in vivo IL-18 administration induces antitumor effects mediated by CD4+ T cells and NK cells. On the basis of our and other observations, tumor cells and immune cells seem to use IL-18 as an effector molecule to defend them. In this context, NK cells and melanoma cells also use Fas ligand as a weapon. There are some controversial reports on the expression of Fas ligand by melanoma cells, but recent studies indicate that, in humans,expression of Fas ligand seems to be dependent on the stage of melanoma(28, 29). Fas ligand expression generally is negative in primary melanomas and positive in metastatic tumors (28, 29), suggesting that the roles of Fas ligand and IL-18 in the interaction of tumor and host immune cells are variable, depending on the progression of the melanoma. CD71 was up-regulated in IL-18 antisense transfectants (Fig. 2 E).

CD71, which regulates target cell sensitivity to NK cells, is another candidate for regulating IL-18-mediated tumor susceptibility to NK cells. More extensive studies are needed to demonstrate what factors are key modulators of immune escape in melanoma cells. For either case,IL-18 seems to be a key regulator of the expression of effector molecules. In this regard, the proper choice of immunotherapy using IL-18 and its related molecules should be considered on the basis of tumor progression and expression patterns of molecules. Collectively,it implies that IL-18 regulates the production of tumor survival factors, including Fas ligand and ROIs to escape from host effector cells and, in the case of melanoma cells, to protect themselves.

In conclusion, the data presented in this report indicate that murine melanoma cells are able to produce IL-18, which is involved in the regulation of intracellular ROI levels and Fas ligand expression,indicating that IL-18 plays a key role in the tumor activity of melanoma. The mechanisms through which factors control the regulation of IL-18 production remain to be determined.

Fig. 1.

Expression of IL-18 and IL-18 receptor in melanoma cells. A, IL-18 immunohistology of tissues from melanoma patients. The tissue samples were embedded and sectioned into 8-μm thicknesses by a cryostat (Leica CM5060). Cryosections were stained with anti-IL-18 polyclonal antibody or control serum as described in“Materials and Methods.” B, detection of IL-18 protein in B16 murine melanoma subclones B16F0 (· · · ·), B16F1(- - - - -), and B16F10 (− − −). B16 murine melanoma subclones were stained immunofluorescently with control rabbit IgG(——) or rabbit anti-IL-18 antibody (4 μg/ml) and detected by intracellular flow cytometry analysis as described in “Materials and Methods. ” A representative experiment of three performed is shown. C, IL-18 mRNA expression in B16F10 murine melanoma cell lines. Total RNA was extracted from B16F10 melanoma cells. The RNA was reverse transcribed, and PCR was performed after reverse transcription with primers for IL-18 or β-actin. PCR products were analyzed by 1% agarose gel electrophoresis. A representative experiment of five performed is shown; +, present; −,absent. D, IL-18 immunoblot (IB)of B16F10 cells. Cell lysates containing equal amounts of protein were resolved by 10% PAGE and transferred onto Immuno-Blot PVDF membrane(Bio-Rad). The blot was incubated with anti-IL-18 antibody or normal rabbit serum (NRS) followed by incubation with peroxidase-conjugated secondary antibody. The antigen-antibody complexes were detected by an enhanced chemiluminescence system(Amersham Pharmacia Biotech, Piscataway, NJ). E, RT-PCR analysis of IL-18 receptor mRNA expression. Total RNA was isolated from B16F10 murine melanoma cell lines. Reverse transcription was performed and followed by PCR with oligonucleotides specific for IL-18 receptor(IL-18R) or β-actin. PCR products were analyzed by 1%agarose gel electrophoresis. A representative experiment of five performed is shown; +, present; −, absent.

Fig. 1.

Expression of IL-18 and IL-18 receptor in melanoma cells. A, IL-18 immunohistology of tissues from melanoma patients. The tissue samples were embedded and sectioned into 8-μm thicknesses by a cryostat (Leica CM5060). Cryosections were stained with anti-IL-18 polyclonal antibody or control serum as described in“Materials and Methods.” B, detection of IL-18 protein in B16 murine melanoma subclones B16F0 (· · · ·), B16F1(- - - - -), and B16F10 (− − −). B16 murine melanoma subclones were stained immunofluorescently with control rabbit IgG(——) or rabbit anti-IL-18 antibody (4 μg/ml) and detected by intracellular flow cytometry analysis as described in “Materials and Methods. ” A representative experiment of three performed is shown. C, IL-18 mRNA expression in B16F10 murine melanoma cell lines. Total RNA was extracted from B16F10 melanoma cells. The RNA was reverse transcribed, and PCR was performed after reverse transcription with primers for IL-18 or β-actin. PCR products were analyzed by 1% agarose gel electrophoresis. A representative experiment of five performed is shown; +, present; −,absent. D, IL-18 immunoblot (IB)of B16F10 cells. Cell lysates containing equal amounts of protein were resolved by 10% PAGE and transferred onto Immuno-Blot PVDF membrane(Bio-Rad). The blot was incubated with anti-IL-18 antibody or normal rabbit serum (NRS) followed by incubation with peroxidase-conjugated secondary antibody. The antigen-antibody complexes were detected by an enhanced chemiluminescence system(Amersham Pharmacia Biotech, Piscataway, NJ). E, RT-PCR analysis of IL-18 receptor mRNA expression. Total RNA was isolated from B16F10 murine melanoma cell lines. Reverse transcription was performed and followed by PCR with oligonucleotides specific for IL-18 receptor(IL-18R) or β-actin. PCR products were analyzed by 1%agarose gel electrophoresis. A representative experiment of five performed is shown; +, present; −, absent.

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Fig. 2.

Establishment of IL-18 antisense transfectants. B16F10 melanoma cells were transfected with the IL-18 antisense construct by a calcium phosphate precipitation method, and the stable transfectant clones were selected as described in “Materials and Methods.” A, intracellular staining of IL-18 expression measured by flow cytometry. Neg. control, staining control; Vector, vector transfectants; IL-18 antisense, IL-18 antisense transfectants. B,IL-18 immunoblot of B16F10 melanoma cells (left panel). The blot was stained with Coomassie blue to normalize the protein concentrations of each lane (right panel). C, MHC class I expression. D,intercellular adhesion molecule-1 expression. E, CD71(transferrin receptor) expression.

Fig. 2.

Establishment of IL-18 antisense transfectants. B16F10 melanoma cells were transfected with the IL-18 antisense construct by a calcium phosphate precipitation method, and the stable transfectant clones were selected as described in “Materials and Methods.” A, intracellular staining of IL-18 expression measured by flow cytometry. Neg. control, staining control; Vector, vector transfectants; IL-18 antisense, IL-18 antisense transfectants. B,IL-18 immunoblot of B16F10 melanoma cells (left panel). The blot was stained with Coomassie blue to normalize the protein concentrations of each lane (right panel). C, MHC class I expression. D,intercellular adhesion molecule-1 expression. E, CD71(transferrin receptor) expression.

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Fig. 3.

Inhibitory effects of IL-18 antisense cDNA and anti-IL-18 antibody on Fas ligand expression in B16F10 melanoma cells. A, vector- (− − −) or antisense IL-18(· · · ·) cDNA-transfected cells were stained with control immunoglobulin or anti-Fas ligand antibody and detected by intracellular flow cytometric analysis as described in “Material and Methods.B, B16F10 murine melanoma cells were incubated in the absence or presence of anti-IL-18 antibody(20 μg/ml) for 18 h. After incubation, cells were collected and stained with control immunoglobulin or anti-Fas ligand antibody. C, 2 × 106 B16F10 melanoma cells were incubated for various times in the presence of 20 mm NAC before measurement of intracellular Fas ligand. One representative experiment of three is shown.

Fig. 3.

Inhibitory effects of IL-18 antisense cDNA and anti-IL-18 antibody on Fas ligand expression in B16F10 melanoma cells. A, vector- (− − −) or antisense IL-18(· · · ·) cDNA-transfected cells were stained with control immunoglobulin or anti-Fas ligand antibody and detected by intracellular flow cytometric analysis as described in “Material and Methods.B, B16F10 murine melanoma cells were incubated in the absence or presence of anti-IL-18 antibody(20 μg/ml) for 18 h. After incubation, cells were collected and stained with control immunoglobulin or anti-Fas ligand antibody. C, 2 × 106 B16F10 melanoma cells were incubated for various times in the presence of 20 mm NAC before measurement of intracellular Fas ligand. One representative experiment of three is shown.

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Fig. 4.

Inhibitory effects of IL-18 antisense cDNA and anti-IL-18 antibody on ROI levels of B16F10 melanoma cells. A,vector- or IL-18 antisense cDNA-transfected cells were stained with′,7′-dichlorofluorescein diacetate for intracellular ROI levels as described in “Material and Methods.B, cells were incubated in the presence of control rabbit immunoglobulin (· · · ·) or anti-IL-18 antibodies (20μg/ml; - - - - -) for 18 h. Melanoma cells were then harvested from each culture and tested for intracellular ROI levels. C, 2 × 106 cells were incubated for 2 h in the absence (· · · ·) or presence(- - - - -) of 20 mm NAC before measurement of intracellular ROI levels. The data are representative of three similar experiments.

Fig. 4.

Inhibitory effects of IL-18 antisense cDNA and anti-IL-18 antibody on ROI levels of B16F10 melanoma cells. A,vector- or IL-18 antisense cDNA-transfected cells were stained with′,7′-dichlorofluorescein diacetate for intracellular ROI levels as described in “Material and Methods.B, cells were incubated in the presence of control rabbit immunoglobulin (· · · ·) or anti-IL-18 antibodies (20μg/ml; - - - - -) for 18 h. Melanoma cells were then harvested from each culture and tested for intracellular ROI levels. C, 2 × 106 cells were incubated for 2 h in the absence (· · · ·) or presence(- - - - -) of 20 mm NAC before measurement of intracellular ROI levels. The data are representative of three similar experiments.

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Fig. 5.

Effects of IL-18 antisense cDNA on NK susceptibility of B16F10 melanoma cells. A, NK susceptibility of vector-or IL-18 antisense cDNA-transfected cells. 51Cr-labeled vector (filled column) or antisense IL-18 cDNA-transfected (open column) melanoma cells were incubated with IL-2 and/or IL-18-activated NK cells for 4 h at an E:T ratio of 10:1. B, after incubation of NK cell and melanoma cells, 50 μl of cell-free supernatant were collected and assayed for BLT esterase activity as described in “Materials and Methods.”

Fig. 5.

Effects of IL-18 antisense cDNA on NK susceptibility of B16F10 melanoma cells. A, NK susceptibility of vector-or IL-18 antisense cDNA-transfected cells. 51Cr-labeled vector (filled column) or antisense IL-18 cDNA-transfected (open column) melanoma cells were incubated with IL-2 and/or IL-18-activated NK cells for 4 h at an E:T ratio of 10:1. B, after incubation of NK cell and melanoma cells, 50 μl of cell-free supernatant were collected and assayed for BLT esterase activity as described in “Materials and Methods.”

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Fig. 6.

Effects of IL-18 antisense cDNA on tumor growth in vivo. The B16F10 mouse melanoma tumors was established by s.c. injection of 1 × 105 vector or IL-18 antisense transfectants into C57BL/6 male mice. A, tumor size was measured with calipers every 2 days; bars, SE. B, survival rate of tumor-injected mice. C, immunohistochemistry of NK cells. The tissue samples were immersed in 25% sucrose-PBS solution and embedded in OCT compound. Cryosections were incubated with biotinylated antimouse NK cell antibody (NK1.1; PharMingen) for 1 h. Subsequently, sections were incubated with alkaline phosphate-conjugated streptavidin complex and visualized by incubation with substrate solution. a, mice injected with vector transfectants; b, mice injected with IL-18 antisense transfectants.

Fig. 6.

Effects of IL-18 antisense cDNA on tumor growth in vivo. The B16F10 mouse melanoma tumors was established by s.c. injection of 1 × 105 vector or IL-18 antisense transfectants into C57BL/6 male mice. A, tumor size was measured with calipers every 2 days; bars, SE. B, survival rate of tumor-injected mice. C, immunohistochemistry of NK cells. The tissue samples were immersed in 25% sucrose-PBS solution and embedded in OCT compound. Cryosections were incubated with biotinylated antimouse NK cell antibody (NK1.1; PharMingen) for 1 h. Subsequently, sections were incubated with alkaline phosphate-conjugated streptavidin complex and visualized by incubation with substrate solution. a, mice injected with vector transfectants; b, mice injected with IL-18 antisense transfectants.

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

1

This work was supported by Grants HS2620(Ministry of Science and Technology) and HMP1027 (Ministry of Health and Welfare), Republic of Korea.

4

The abbreviations used are: IL-18,interleukin-18; NK, natural killer; TNF, tumor necrosis factor; ROI,reactive oxygen intermediate; CM, complete medium; RT-PCR, reverse transcription-PCR; FACS, fluorescence-activated cell sorting; BLT, N-α-carbobenzoxy-l-lysine thiobenzyl ester; NAC, N-acetyl-l-cysteine.

Table 1

Effect of B16F10 culture supernatants on IFN-γ production from C57BL/6 splenocytes

C57BL-6 splenocytes (5 × 106 cells/ml) were cultured with B16F10 culture supernatants (1:4 dilution) for 72 h at 37°C in a humidified, 5% CO2 incubator in the presence or absence of saturating amounts of rabbit anti-IL-18 Ab (5, 10, or 20μg/ml). After culture, supernatants were tested for IFN-γproduction by ELISA. These data are from a representative experiment of three independently performed experiments with C57BL/6 splenocytes from different donors.

Culture conditionIFN-γ (pg/ml)
B16F10 c.sa 153.0 ± 25.1 
C57BL/6 sp. 1,221.0 ± 68.7 
rIL-18 (100 ng/ml)+ C57BL/6 sp. 13,347.0 ± 124.1 
B16F10 c.s+ C57BL/6 sp. 11,145.0 ± 432.5 
B16F10 c.s+ C57BL/6 sp.+ Rabbit Ig (20 μg/ml) 11,560.0 ± 753.2 
B16F10 c.s+ C57BL/6 sp.+ anti-IL-18 (5 μg/ml) 9,245.0 ± 213.5 
B16F10 c.s+ C57BL/6 sp.+ anti-IL-18 (10 μg/ml) 7,755.0 ± 322.7 
B16F10 c.s+ C57BL/6 sp.+ anti-IL-18 (20 μg/ml) 4,197.0 ± 195.6 
Culture conditionIFN-γ (pg/ml)
B16F10 c.sa 153.0 ± 25.1 
C57BL/6 sp. 1,221.0 ± 68.7 
rIL-18 (100 ng/ml)+ C57BL/6 sp. 13,347.0 ± 124.1 
B16F10 c.s+ C57BL/6 sp. 11,145.0 ± 432.5 
B16F10 c.s+ C57BL/6 sp.+ Rabbit Ig (20 μg/ml) 11,560.0 ± 753.2 
B16F10 c.s+ C57BL/6 sp.+ anti-IL-18 (5 μg/ml) 9,245.0 ± 213.5 
B16F10 c.s+ C57BL/6 sp.+ anti-IL-18 (10 μg/ml) 7,755.0 ± 322.7 
B16F10 c.s+ C57BL/6 sp.+ anti-IL-18 (20 μg/ml) 4,197.0 ± 195.6 
a

c.s., culture supernatant; sp., splenocytes; Ig,immunoglobulin.

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