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* Laboratory of Angiogenesis Research, Microbiology and Tumor Biology Center, Karolinska Institute, S-171 77 Stockholm, Sweden; and
Fujisaki Institute, Hayashibara Biochemical Laboratories, Inc., Okayama 702, Japan
1Correspondence: Yihai Cao, M.D., Ph.D., Laboratory of Angiogenesis Research, Microbiology and Tumor Biology Center, Karolinska Institute, S-171 77 Stockholm, Sweden. E-mail: yihai.cao{at}mtc.ki.se
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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(IFN-)-inducing
factor, has recently been characterized as a potent IFN--inducing
cytokine. We now report that IL-18 is a novel antiangiogenic and
antitumor cytokine. In vitro, IL-18 specifically
inhibits fibroblast growth factor-2-stimulated proliferation of
capillary endothelial cells. In vivo, IL-18 is
sufficiently potent to suppress the fibroblast growth factor-induced
corneal neovascularization by systemic administration in mice. This
cytokine also inhibits embryonic angiogenesis in the chick
chorioallantoic membrane assay. Systemic and intralesional
administrations of IL-18 produce a significant suppression of the
growth of murine T241 fibrosarcoma in syngeneic C57Bl6/J and
immunodeficient SCID mice. The antitumor effect appears to be potent
because an average of >75% inhibition of primary tumor growth was
observed at a dose of 50 µg/kg/day. In cell culture, murine T241
fibrosarcoma cells are insensitive to recombinant IL-18 at
concentrations that significantly inhibit endothelial cell
proliferation. Immunohistochemical studies of tumor tissues reveal
hypovascularization of the IL-18-treated tumors. These results suggest
that IL-18 may participate in the regulation of a switch of tumor
angiogenesis.—Cao, R., Farnebo, J., Kurimoto, M., Cao, Y.
Interleukin-18 acts as an angiogenesis and tumor suppressor.
Key Words: neovascularization • antitumor • interferon-
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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2
3)
. Angiogenesis is a
complex multistep process that includes proliferation, migration, and
differentiation of endothelial cells, degradation of extracellular
matrix, microtubule formation, and sprouting of new capillary branches
(1
, 2
, 4)
. The complexity of the angiogenic processes
suggests the existence of multiple controls of the system, which can be
transiently turned on and off (5
, 6)
.
A switch to the angiogenic phenotype is believed to depend on a local
change in the balance between angiogenic stimulators and inhibitors
(1)
. Overgrowth of blood vessels may lead to development
and progression of diseases such as tumor growth and diabetic
retinopathy. Many lines of evidence support the original hypothesis
that tumor growth and metastasis are angiogenesis dependent
(7
8
9
10
11)
. Thus, suppression of tumor angiogenesis by potent
endogenous angiogenesis inhibitors can be useful for cancer treatment.
Recent examples are discoveries of two potent antitumor protein
fragments, angiostatin and endostatin, both of which specifically
target the growing microvessel compartment in tumors but have no effect
on the quiescent vasculature and tumor cells (7
, 12
13
14)
.
A few cytokines have recently been reported to participate in the
regulation of the angiogenic switch (15
, 16)
. Whereas two
of these cytokines, interleukin-8 (IL-8) and interleukin-4 (IL-4),
stimulate angiogenesis (17
18
19)
, a majority of the others,
including platelet factor 4 (PF-4) (20)
, IFN--inducing
protein 10 (IP-10) (21
22
23)
, gro-ß (24)
,
IFN-2
(25)
, IL-4 (26)
, and interleukin-12
(IL-12) (27)
inhibit neovascularization. Consequently, all
angiostatic cytokines suppress tumor growth in animal models (22
, 24
, 27
28
29
30)
. The antitumor effect of these cytokines is mediated
at least partially via the antiangiogenic pathway.
IL-18 is a newly discovered molecule with potent IFN--inducing
activity (31)
. IL-18 actually displays more potent
IFN--inducing capability than IL-12 and apparently utilizes a
separate signal transduction pathway for its action (31)
.
IL-18 has been recently suggested to inhibit angiogenesis and tumor
growth in animal models (32)
. In this study we provide
further evidence that IL-18 acts as an angiogenesis inhibitor and a
tumor suppressor. We now provide experimental data supporting this
conclusion.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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. Bovine capillary endothelial (BCE) cells were
obtained as reported (34)
and maintained in Dulbecco’s
modified Eagle’s (DME) medium with 10% heat-inactivated bovine calf
serum (BCS; Hyclone Laboratories, Logan, UT), antibiotics, and 3 ng/ml
recombinant human fibroblast growth factor-2 (FGF-2; Scios Nova Inc.,
Mountain View, CA). Murine T241 fibrosarcoma tumor cells were
maintained in culture in DME medium supplemented with 10% fetal calf
serum (FCS) and antibiotics. Male 5- to 6-week-old C57Bl6/J and SCID
mice (MTC, Karolinska Institute, Stockholm, Sweden) were acclimated and
caged in groups of six or less. Animals were anesthetized in a
methoxyflurane chamber before all procedures and killed with a lethal
dose of methoxyflurane. All animal studies were reviewed and approved
by the animal care and use committee of the Stockholm Animal Board.
Chick embryo chorioallantoic membrane (CAM) assay
The CAM assay was performed as previously described
(24)
. Three-day-old fertilized white Leghorn eggs (OVA
Production, Sörgården, Sweden) were cracked and chick embryos
with intact yolks were carefully placed in 20 x 100-mm plastic
Petri dishes. After 48 h of incubation in 4%
CO2 at 37°C, disks of methylcellulose
containing various concentrations of recombinant IL-18 dried on a nylon
mesh (4 x 4 mm) were implanted on the CAM of individual embryos.
The nylon mesh disks were made by desiccation of 20 µl of 0.45%
methylcellulose (in H2O). After 48–72 h of
incubation, embryos and CAMs were examined with a stereoscope for the
formation of avascular zones in the field of the implanted disks.
Mouse corneal micropocket assay
The mouse corneal assay was performed according to previously
described procedures (24
, 35
36
37
38)
. Corneal micropockets
were created with a modified von Graefe cataract knife in both eyes of
each male 5- to 6-week-old C57Bl6/J mouse. A micropellet (0.35 x
0.35 mm) of sucrose aluminum sulfate (Bukh Meditec, Copenhagen,
Denmark) coated with hydron polymer type NCC (IFN Sciences, New
Brunswick, NJ) containing ~80 ng of FGF-2 was implanted into each
pocket. The pellet was positioned 0.8 mm from the corneal limbus. After
implantation, erythromycin/ophthalmic ointment was applied to each eye.
One group of animals (n = 5) received daily
intraperitoneal (i.p.) injections of 50 µg/kg recombinant IL-18 in
100 µl phosphate-buffered saline (PBS), including pretreatments with
the same dose 1 day before corneal implantation. Control animals
(n = 5) received daily subcutaneous (s.c.) injections
of 100 µl PBS. The corneal neovascularization in both eyes of all
animals were examined by a slit-lamp biomicroscope on day 6 after
pellet implantation.
Proliferation assay for capillary endothelial cells and other
cells
A 72-h BCE cell proliferation assay was performed as described
previously (39
, 40)
. For tumor cell proliferation assay,
murine T241 fibrosarcoma cells (~10,000 cells/well) were seeded onto
24-well tissue culture plates and incubated in DME medium (1 ml/well)
containing 5% FCS and antibiotics for 24 h, and samples were
added to cells in triplicate. After 72 h of incubation, adherent
and nonadherent cells were dispersed in trypsin, resuspended in Isoton
solution (Kebo Lab, Sweden), and counted with a Coulter counter.
Tumor studies in mice
Male 5- to 6-week-old C57Bl6/J and SCID mice were used for tumor
studies. Approximately 1 x 106 murine T241
fibrosarcoma cells growing in log phase were harvested, resuspended in
PBS, and a single cell suspension in a volume of 100 µl was implanted
subcutaneously in the middle dorsum of each animal. Four to five mice
were used in each treatment or control group. Systemic treatments by
subcutaneous or intralesional injections with either 100 µl PBS or 50
µg/kg of IL-18 in PBS were begun shortly after implantation of tumor
cells and continued once daily for a total of 16–20 treatments.
Visible tumors were present after 72 h. Primary tumors were
measured using digital calipers on the days indicated. Tumor volumes
were calculated according to the following formula:
width2 x length x 0.52, as previously
reported (7)
.
Histology
Tumor-bearing C57Bl6/J animals were killed by an overdose of
methoxyflurane on day 20 after tumor cell implantation, and viable
tumor tissues were resected and fixed with the Carnoy’s fixative for
24 h as described previously (12
, 24)
. Tissues were
embedded in paraffin according to standard histological procedures
(12
, 24)
. The sections (5 mm thickness) were processed and
stained with a rabbit anti-human von Willebrand factor (Dako Corp,.
Carpinteria, CA) antibody as described previously (41)
.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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and
the recombinant protein was purified to homogeneity as analyzed by a
sodium dodecyl sulfate (SDS) gel (Fig. 1A
). Under non-reducing conditions, recombinant IL-18 appeared
as a single band with molecular mass of 19 kDa (Fig. 1A
,
lane 2). The molecular mass of recombinant IL-18 was increased to 20.5
kDa under reducing conditions in the presence of dithiothreitol (DTT;
lane 1). These data are compatible with the fact that the mature form
of recombinant IL-18 may contain an intramolecular disulfide bridge due
to the presence of three cysteines in its primary structure
(31)
.
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Specific inhibition of endothelial cell proliferation by IL-18
To investigate whether IL-18 could inhibit endothelial cell
proliferation, recombinant IL-18 was incubated with BCE cells
stimulated by 1 ng/ml FGF-2. At the concentration of 10 nM, IL-18
inhibited BCE cell proliferation by ~70% (P<0.0001)
(Fig. 1B
). The inhibition of endothelial cell proliferation
occurred in a dose-dependent manner (Fig. 1B
). Complete
arrest of FGF-2-stimulated endothelial cell proliferation was observed
at high concentrations (data not shown). No distinct cell morphological
changes in association with apoptotic endothelial cells including
detachment, rounding, and fragmentation could be detected, even after a
3-day incubation with a high concentration (500 nM) of IL-18.
Suppression of cell proliferation by IL-18 appeared to be endothelial
cell-selective, and proliferation of murine T241 fibrosarcoma cells,
rat smooth muscle cells, and 3T3 fibroblasts was not affected at any
concentrations used for BCE cells (data not shown). Thus, IL-18 does
not inhibit T241 fibrosarcoma tumor cell proliferation in
vitro.
Inhibition of angiogenesis in the chick embryo CAM
To study the antiangiogenic activity of IL-18 in vivo,
recombinant IL-18 was tested on the CAM (24
, 42)
. As shown
in Figure 2
, IL-18 inhibited new blood vessel growth of chick embryos in a
dose-dependent manner (P<0.0001) as measured by the
formation of avascular zones, and no obvious inflammation was detected.
No avascular zones were found in the control embryos implanted with
discs containing PBS alone. These results demonstrate that IL-18 is
able to suppress angiogenesis in embryos.
|
Suppression of mouse corneal neovascularization
To further investigate the antiangiogenic activity of IL-18
in vivo, the inhibitory effect of systemic administration of
IL-18 on FGF-2-induced neovascularization was studied. This
antiangiogenic assay requires a putative angiogenesis inhibitor to be
administrated systemically (e.g., intraperitoneal or subcutaneous
injections) to examine its capacity to suppress 80 ng of FGF-2-induced
neovascularization in a remote organ such as the cornea. Systemic
treatment of mice with IL-18 by intraperitoneal injections at a
concentration of 50 µg/kg/day significantly inhibited FGF-2-induced
corneal neovascularization (Fig. 3C, and D
). The length (Fig. 3E
) and
clock hours of corneal circumferential neovascularization (Fig. 3F
) were inhibited by >60% in 10 corneas of 5 individual
mice. The area of neovascularization in IL-18-treated mice was
suppressed by ~80% (Fig. 3G
). The density of corneal
vessels in the IL-18-treated animals (Fig. 3C, and D
) was also markedly reduced compared with that of control
animals treated with PBS (Fig. 3A, and B
). In
addition, FGF-2-induced corneal blood vessels in the IL-18-treated mice
were less dilated (Fig. 3C, and D
) than those of
control mice (Fig. 3,A and B
). The treated mice
did not experience weight loss or unusual behavior over the course of
the treatment, indicating that IL-18 was not toxic at the dose used in
our experiments.
|
Suppression of primary tumor growth by intralesional administration
of IL-18
Because IL-18 inhibited neovascularization in vivo and
tumor growth requires angiogenesis (1
2
3
4
5
6)
, we determined
the antitumor activity of IL-18. Recombinant IL-18 was used to treat
C57Bl6/J mice bearing subcutaneous implanted primary T241
fibrosarcomas. Daily intralesional injections of 1 µg of IL-18 per
20 g (50 µg/kg) mouse resulted in a significant suppression of
the growth of primary tumors during the 19-day treatment course
(Fig. 4A
). At day 19 after treatment, an average of over 75%
suppression of primary tumor growth was observed in the IL-18-treated
mice (n = 5). In contrast, tumors grew rapidly to sizes
>1200 mm3 in all saline-treated animals
(n = 4) during the same 19-day treatment period (Fig. 4A
), leading to the demise of all mice within 5 weeks after
tumor implantation. The IL-18-treated mice did not lose weight or
exhibit unusual behavior over the course of treatment.
|
Suppression of primary tumor growth by subcutaneous administration
of IL-18
To further evaluate the antitumor activity, IL-18 was
subcutaneously administered to C57Bl6/J mice bearing subcutaneous T241
tumors. IL-18 at a dose of 50 µg/kg/day was subcutaneously injected
daily in the ventral abdomen, whereas subcutaneous tumors were growing
in the midline dorsum of each mouse. In agreement with the data
obtained from the intralesional injections, systemic treatment of mice
(n = 4) with IL-18 daily produced an average >70%
inhibition of primary tumor growth after 20-day tumor implantation
(Fig. 5,A and B
). The IL-18-treated tumors appeared to
be flat and pale (Fig. 5A
), and some tumors remained at
small sizes (<50 mm3) for weeks. These are dormant primary
tumors with typical diminished neovascularization (41)
. In
contrast, tumors with central microhemorrhages grew rapidly (>750
mm3) in all saline-treated animals
(n = 4; Fig. 5A
). These results support the
notion that IL-18 displays a potent antitumor effect in C57Bl6/J
syngeneic mice. To exclude the possibility that an immune response
induced by IL-18 was involved in its antitumor activity,
immunodeficient SCID mice were also used to assess the antitumor effect
of IL-18. As shown in Figure 4B
, the growth of T241 tumors
in SCID mice (n = 4) was also significantly inhibited
by systemic administration of IL-18, although the efficacy of
inhibition was not as potent as that observed in immunocompetent mice
(Fig. 5B
). These data support our conclusion that the
antitumor effect of IL-18 is mediated by inhibition of tumor-induced
angiogenesis.
|
Suppression of tumor neovascularization
To evaluate the inhibitory effect of IL-18 on tumor angiogenesis,
tumor tissue sections that had been systemically treated with IL-18
were immunohistochemically stained with an endothelial specific
antibody against von Willebrand factor and microvessels in tumor
tissues were randomly counted. Primary tumors were resected at day 19
after IL-18 treatment. A dramatically decreased microvessel density in
tumor tissues was revealed in the IL-18-treated mice (Fig. 6B, C, and D
) as compared with control
tumor tissues treated with PBS (P<0.0001; A and
D). Figure 6D
represented an average of vessel
density of six random fields in tumors from three mice of each group.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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We should emphasize that ~50% reduction (Fig. 6D
) of
microvessel density was not directly correlated to >75% reduction of
tumor volume (Figs. 4A, 4B
, and 5B
).
This difference could be due to other immunological activities of
IL-18, which may suppress tumor growth. For example, augmentation of
natural killer (NK) cells by IL-18 could also account for its antitumor
effect. In addition, endothelial cells and tumor cells may have
different growth rates. In agreement with these findings, we found that
the antitumor effect of IL-18 was still significant in immunodeficient
SCID mice, although the effect was slightly less pronounced when
compared with that observed in immunocompetent mice.
IL-18 was first identified and isolated as an IFN-
production-inducing cytokine in mice challenged with P.
acnes and lipopolysaccharide (31)
. Coughlin et al.
(32)
showed that the antiangiogenic activity of IL-18
appeared to be specifically mediated by IFN-. Recent studies show
that IL-18 has multiple biological activities in the immune system. In
addition to IFN- production-inducing activity, IL-18 is also able to
enhance Fas ligand-mediated cytotoxicity of T helper 1 cells
(43)
, to augment the production of granulocyte-macrophage
colony-stimulating factor (44)
, to decrease synthesis of
IL-10 (33)
, to stimulate NK cell proliferation, and to
mediate inflammatory tissue damage (44)
. IL-18 was also
suggested as an IL-1-related cytokine based on their structural
similarity and biological activity (31
, 45
, 46)
. One of
the most striking common features of these two cytokines is that they
lack a classical secretory signal sequence in their primary structures
and both inactive precursors can be processed into their active forms
by the IL-1ß converting enzyme that facilitates their cellular export
(46
, 47)
. Thus, biological actions of these cytokines are
regulated by an intracellular proteolytic process.
In respect to the IFN--inducing effect, IL-18 resembles the
structurally unrelated cytokine IL-12, albeit IL-18 is a more potent
IFN- inducer than IL-12, apparently through a separate pathway
(31)
. IL-18 and IL-12 synergistically enhance IFN-
production (48)
. IL-12 was recently reported to be a
potent antitumor factor that inhibits the growth of a wide spectrum of
tumors in vivo (27
, 30)
. Similar to IL-18,
IL-12 has no direct influence on tumor cells in vitro
(27)
. It seems that the antitumor effect of IL-12 is
mediated through IFN- because an IFN--neutralizing antibody can
prevent the antitumor activity of IL-12 (27)
. Apparently
IFN- is not the last player in this cascade inhibitory event because
IP-10 has been reported to be a potent antiangiogenic cytokine in
vivo (21
22
23)
, although its direct inhibitory
activity on endothelial cells is controversial (22
, 23)
.
Although IL-12 and IL-18 have the above-mentioned overlapping
functions, the angiostatic effects of these two molecules are probably
mediated via separate pathways. Our data show that IL-18
directly inhibits the FGF-2-induced endothelial cell proliferation
in vitro but IL-12 lacks such a direct effect
(27)
. In addition, IL-18 halts new blood vessel formation
and regresses the growing blood vessels in the chick embryos. This
in vivo angiostatic effect is not likely to be due to the
induction of IFN- because 5- to 7-day-old embryos may not have an
established mature immune response. It is not yet clear whether IL-18
only blocks the FGF-2-mediated endothelial cell proliferation. During
embryonic development, FGF-2 together with other factors stimulate
angiogenesis, which is required for the chick limb bud and organ
formation (49)
. In contrast, IL-12 is unable to suppress
new blood vessel growth in embryos (27)
. Therefore, the
antiangiogenic pathway of IL-18 may not be completely mediated through
the IFN- signaling.
In addition to the above-mentioned cytokines (IL-18, IL-12, IFN-,
and IP-10), a couple of other immunocytokines also participate in
suppression of angiogenesis. These include PF-4 and gro-ß in the
-C-X-C- chemokine family and IFN-2. As a consequence, all
angiostatic cytokines exert a potent effect in suppression of tumor
growth in both immunocompetent and immunodeficient animals. Thus,
antiangiogenesis seems to be a common mechanism for these cytokines to
suppress tumor growth. These findings not only support the idea that
tumor growth is angiogenesis dependent (50)
, but also
provide evidence that the immune system and the vasculature system
cross-talk to each other. Although most cytokines inhibit angiogenesis,
some can also stimulate neovascularization. IL-8 in the -C-X-C-
chemokine family is one such example.
In conclusion, we are beginning to understand that the switch of tumor angiogenesis is controlled by a balance of angiogenic and angiostatic factors. In this study, we demonstrated that IL-18 is an important endogenous negative regulator for angiogenesis and tumor growth. The identification of endogenous angiogenesis inhibitors may become useful for cancer therapy.;1>
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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) on days indicated. Data represent the mean tumor volume (±
SE) of four to five mice in each group.
