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Immunology |
Laboratory of Immunology, Korea Research Institute of Bioscience and Biotechnology, Yusong, Taejon 305-333, Republic of Korea [D. C., H. S., Y. M. K., D. Y., K. H. P., I. C.]; Department of Dermatology, Kangdong Sacred Heart Hospital, College of Medicine, Hallym University, Seoul 134-701, Republic of Korea [D. H.]; Department of Anatomy, Inje University College of Medicine, Pusan 614-735, Republic of Korea [D. Y. H.]; Department of Dermatology, Holy Family Hospital, College of Medicine, The Catholic University of Korea, Pucheon 422-717, Republic of Korea [H. P.]; Department of Anatomy, Seoul National University, Seoul 110-799, Republic of Korea [W. J. L.]; Department of Biochemistry, College of Natural Sciences, Chungnam National University, Taejon 305-764, Republic of Korea [H. S., Y. S. K.]; Fujisaki Institute, Hayashibara Biochemical Laboratories, Okayama, Japan [M. K.]; Department of Microbiology and Immunology, Finch University of Health Science/The Chicago Medical School, Illinois 60064, [Y. B. K.]
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ABSTRACT |
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 Top ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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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. |
INTRODUCTION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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IL-18 (IFN--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- 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-
and IL-1ß.
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.
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MATERIALS AND METHODS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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IL-18 Bioassay (IFN- Induction Assay).
The induction of IFN- 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 x 106 cells/ml. One ml of splenocytes (5 x 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- production using a murine IFN-
ELISA 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 x 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 mM
5,5'-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) x 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 x 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 x (largest diameter) x (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.
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RESULTS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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). 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. 1B
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. 1C
).
IL-18 protein was also detected by immunoblotting with anti-IL-18
antibody (Fig. 1D
). Collectively, these data demonstrate
that melanoma cells express IL-18 mRNA and protein.
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secretion from splenocytes of C57BL/6 mice. Conditioned medium
collected from untreated C57BL/6 splenocytes induced only a slight
secretion of IFN-, whereas conditioned medium collected from treated
C57BL/6 splenocytes induced significant secretion of IFN- (Table 1)
. To confirm that the observed enhancement of IFN- 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- 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. 1E
). These results suggest that
IL-18 produced by murine melanoma cells can regulate the physiological
functions of tumor cells.
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). FACS (Fig. 2A
) and immunoblot (Fig. 2B
)
analysis showed that IL-18 expression was reduced in IL-18 antisense
transfectants. MHC class I (Fig. 2C
) and intercellular
adhesion molecule-1 (Fig. 2D
) 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. 2E
).
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). In the same line, treatment of B16F10 melanoma cells with
anti-IL-18 antibody reduced Fas ligand expression (Fig. 3B
),
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. 3C
), suggesting that
ROIs are also involved in Fas ligand expression in melanoma cells.
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). In addition, B16F10 melanoma cells were incubated with
anti-IL-18 polyclonal antibody for 18 h, and ROI levels were
analyzed (Fig. 4B
). 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. 4C
).
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). In addition, BLT esterase release was elevated when NK
cells were incubated with IL-18 antisense transfectants (Fig. 5B
). When IL-18 antisense transfectants were implanted into
syngeneic mice, severe regression of tumor growth was observed (Fig. 6A
). In addition, the survival rate was prolonged in mice
injected with IL-18 antisense transfectants compared with those
injected with vector transfectants (Fig. 6B
). 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. 6C-b
), but not in
the mice injected with vector transfectants (Fig. 6C-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.
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DISCUSSION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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IL-18 is a recently cloned cytokine that was primarily identified
by its ability to induce IFN- 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- secretion by C57BL/6
splenocytes (Table 1)
. Blocking experiments with anti-IL-18 polyclonal
antibodies showed that IFN- 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- by
splenocytes can be caused by IL-2 (22)
. Therefore, IL-2
might be responsible for the IFN- 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-
B 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-1ß from
CD14+ cells via direct TNF- 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- through nuclear factor-B 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. 2E
).
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.
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FOOTNOTES |
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1 This work was supported by Grants HS2620
(Ministry of Science and Technology) and HMP1027 (Ministry of Health
and Welfare), Republic of Korea. 
2 These authors have contributed equally to this
work.
3 To whom requests for reprints should be
addressed, at Laboratory of Immunology, Korea Research Institute of
Bioscience and Biotechnology, Eoun-Dong 52, Yusong, Taejon 305-333,
Republic of Korea. Phone: 82-42-860-4223; Fax: 82-42-860-4593; E-mail: ipchoi{at}kribb4680.kribb.re.kr
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--carbobenzoxy-L-lysine thiobenzyl
ester; NAC, N-acetyl-L-cysteine.
Received 11/22/99. Accepted 3/20/00.
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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