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-Inducing Factor/IL-18 Administration Mediates IFN-- and IL-12-Independent Antitumor Effects1
Departments of
*
Surgery and
Molecular Genetics and Biochemistry, University of Pittsburgh School of Medicine, Pittsburgh, PA 15213;
Division of Host Defenses, Institute for Advanced Medical Sciences, Hyogo College of Medicine, Nishinomiya, Hyogo; and
§
Fujisaki Institute, Hayashibara Biochemical Laboratories, Okayama, Japan
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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-inducing factor/IL-18 protein on the growth of mouse tumor
cell lines in vivo. Mice received IL-18 before or after challenge with
CL8-1, a mouse melanoma cell line. Both regimens significantly
suppressed tumor growth and reduced the number of mice with growth of
tumor from 60% (3/5) to 20% (1/5). Furthermore, IL-18 administered
before and after tumor inoculation completely abrogated the
establishment of CL8-1 in all animals. IL-18 administration also
significantly suppressed the growth of MCA205, a sarcoma cell line,
even when treatment was delayed to 7 days following tumor inoculation.
Although IL-18/IL-12 combination therapy had the most significant and
immediate antitumor effects, many mice so treated succumbed with
markedly elevated serum IFN- levels. The antitumor effects of IL-18
were abrogated almost completely when NK cells were eliminated using
anti-asialo GM1 Ab administration, but only marginally impaired in
IFN- or IL-12 gene-disrupted mice. Immunohistochemical staining
revealed that the number of the CD8+ T cells, but not
CD4+ T cells, found at the tumor site was reduced in
animals treated with IL-18. These results indicate that IL-18 has
potent antitumor effects mediated by CD4+ T cells and NK
cells, but in IFN-- and IL-12-independent pathways. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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(4, 5), IFN- (6), and TNF- (7),
have been investigated in mice bearing transplantable tumors, as well
as in patients with cancer. These cytokines have had limited antitumor
effects with significant side effects. Recently, IL-12 has been shown
to be effective in the treatment of murine tumors with minimal side
effects (8, 9, 10). In these studies, the antitumor effects of IL-12 on
cancer are mediated by NK cells, CD4+,
CD8+ T cells, as well as IFN- elaboration (9, 11, 12).
IL-12 antitumor effects have also been attributed to its potent
anti-angiogenic function that is mediated in part by
IP-10,3 in turn induced by
IFN- (13, 14, 15). Phase I clinical trials of rIL-12 administration have
been completed recently, based on the promising preclinical animal
studies (16) and phase II studies initiated.
IL-18 is a recently discovered cytokine cloned from mice with fluminant
hepatitis induced by challenge with Propionibacterium acnes
and subsequent administration of LPS (17). This cytokine was initially
termed IFN--inducing factor based on high level IFN- secretion
from NK and T cells at levels substantially greater than that observed
with IL-12, another potent IFN- inducer (17). IL-18 has many
similarities with IL-1 (12% homology with IL-1, and 19% with
IL-1ß, respectively), demonstrated in its structure and requirement
for processing to an active molecule. It, like IL-1, lacks a signal
sequence (17) and is processed into the mature form by an
IL-1ß-converting enzyme (18). Although IL-18 shares these
characteristics with IL-1, IL-18 appears to have its own receptor (19).
IL-18 is produced by cells of monocyte lineage, augments NK cytolytic
activity, and enhances proliferation of T cells (17). IL-18 also
promotes NK and T cells to secrete IFN- as well as
granulocyte-macrophage CSF (20). Although IL-18 and IL-12 synergize in
IFN- production, their receptors or signal-transduction pathways
appear to be different (21). These findings strongly suggest that IL-18
has significant immunoregulatory functions that could be modulated
further in combination with IL-12.
To examine the role of IL-18 in tumor immunity, we evaluated the effect of administration of murine rIL-18 to tumor-bearing mice. IL-18 significantly suppressed the growth of both CL8-1, a melanoma cell line, and MCA205, a fibrosarcoma cell line. Pretreatment of mice with IL-18 before tumor transplantation also significantly suppressed the subsequent establishment of tumor. Furthermore, the antitumor effects of IL-18 were augmented by combination with systemic IL-12 administration.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Murine rIL-18 was supplied by Hayashibara Biochemical Laboratories (Okayama, Japan). Murine rIL-12 was a gift from Hoffman-La Roche (Nutley, NJ).
Tumor cell lines and animals
MCA205, a methylcholanthrene-induced murine fibrosarcoma cell line, was a generous gift from National Cancer Institute (Dr. S. A. Rosenberg, Bethesda, MD). CL8-1, a BL6 melanoma cell line, was provided by Dr. E. Gorelik (University of Pittsburgh, Pittsburgh, PA). These cell lines were maintained as described previously (22) and were demonstrably free of mycoplasma contamination with the Mycoplasma PCR ELISA kit (Boehringer Mannheim Corp., Mannheim, Germany) periodically.
C57/BL6 mice were purchased from Taconic Farms (Germantown, NY) and
were used for experiments when they were 7 to 10 wk old. The IFN-
gene knockout (ko) mice were kindly provided by Dr. J. Flynn
(University of Pittsburgh, Pittsburgh, PA), and the IL-12 p40 gene ko
mice were generated and supplied by Dr. J. Magram (Hoffman-La Roche)
(23). These animals were bred in our animal facility at Center for
Biotechnology and Bioengineering, University of Pittsburgh.
Animal experiments and statistical evaluation
A quantity amounting to 3 x 104 to 1 x 106 cells of CL8-1 or MCA205 was inoculated intradermally (i.d.) in the flank of C57BL/6 mice on day 0. Each group consisted of five to six animals. Mice received once daily i.p. injections of IL-18, IL-12, or HBSS (Life Technologies, Grand Island, NY) as a control. Treatment schedule including the dosage of IL-18 and IL-12 injected into mice is specified in each experiment. The tumor size was measured every 2 to 4 days and expressed as the product of the perpendicular diameters of individual tumors. Each animal experiment was repeated at least twice. Blood samples from three to four mice per group were obtained by tail-vein phlebotomy 1 day before and 5 days after cytokine injections. Serum was separated by centrifugation and stored at -80°C until assayed. Nonparametric Wilcoxon rank test was used in the statistical analysis of the size of tumor in individual groups. The difference was considered statistically significant when p < 0.05.
Ab depletion of NK cells
To deplete NK cells in mice, 20 µl of anti-asialo GM1 antiserum (ASGM1; Wako Bioproducts, Richmond, VA) was administered i.p. 1 day prior to the tumor inoculation, and subsequently once every 5 days afterward for an additional 20 days (five times in total). This dose was confirmed to be effective in suppressing more than 95% of the lytic activity against YAC-1 cells mediated by spleen cells harvested from the animals receiving only two injections.
ELISA for cytokines
Concentration of IFN- and IL-10 in the serum of the mice
treated with IL-18 and/or IL-12 was measured by ELISA, respectively
(PharMingen, San Diego, CA). The lower limits of the detection of both
cytokines were 15.6 pg/ml.
Immunohistologic analysis
Mice received 7 days of treatment with 1 µg of IL-18 either before (pretreatment) or after (post-treatment) the i.d. inoculation of CL8-1 cells into the flank. Tumors were harvested 6 days after tumor challenge. One-half of the tumors were immediately frozen and embedded in OCT compound. Serial 5-µm sections were made from these tumors using cryostat, and underwent immunochemical staining using Abs to CD4, CD8a, and CD11b (all from PharMingen, San Diego, CA) with the Vectastain ABC kit (Vector, Burlingame, CA). The stained cells were counted in five fields at a magnification of x400 in each sample. Evaluation of the results was performed in a blinded fashion. The rest of the samples were immediately frozen, stored at -80°C, and used for extracting RNA.
Analysis of IFN- and IP-10 expressions in tumors by
reverse-transcriptase-mediated PCR
CL8-1 tumors were inoculated, treated with 1 µg of IL-18 for 6
days, and harvested. Total cellular RNA was extracted from whole tumor
tissue using RNAzol B (TEL-TEST, Friendswood, TX). Four micrograms of
total RNA were reverse transcribed using an RNase H-
reverse transcriptase (Superscript; Life Technologies). Two hundred
nanograms of RNA-equivalent cDNA were amplified by PCR. The numbers of
amplification cycles were chosen for each primer pair that shows
maximum signal intensity on the linear portion of an amplified product
(24). The sense and antisense primer sequences used, respectively
(followed in parenthesis by annealing temperature and amplification
cycles), were as follows: IP-10, 5'-ACCATGAACCAAGTGCTGCCGTC-3' and
5'-GCTTCACTCCAGTTAAGGAGCCCT-3' (22, 66°C); IFN-,
5'-TGCGGCCTAGCTGAGACAATGA-3' and 5'-TGAATGCTTGGCGCTGGACCTGTG-3'
(31, 66°C); and ß-actin, 5'-TTCTACAATGAGCTGCGTGTG-3' and
5'-CACTGTGTTGGCATAGAGGTC-3' (21, 58°C). The amplified
products were electrophoresed on 1.5% agarose gel, and the intensity
of the bands was quantified by densitometry (Molecular Dynamics,
Sunnyvale, CA).
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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To evaluate the effects of IL-18 on preventing the establishment
of tumor in vivo, 1 x 105 of CL8-1 cells were
inoculated i.d. into the right flank on day 0. Mice received daily i.p.
injections of 1 µg of IL-18 for 7 days either from day -7
(pretreatment) or from day 0 (post-treatment) (Fig. 1
A). Suppression of
tumor growth was first observed on day 6 in all IL-18-treated groups.
The mean tumor area was significantly smaller in all IL-18-treated
groups (groups II, III, IV vs I, p < 0.05 on day 8;
groups IV vs I, p < 0.05 on day 11). This growth
suppression was attained by day 13 in pretreated mice (group II) and by
day 17 in post-treated mice (group III). Furthermore, pretreatment
combined with post-treatment with IL-18 (group IV) completely
eradicated tumor in all animals (Fig. 1, A and
B).
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B). In animals rejecting the
tumor, specific immunity against CL8-1 was evaluated by rechallenge of
2 x 106 of CL8-1 cells applied to the opposite flank.
Thirteen of fifteen animals rejected the rechallenge. Of the two mice
that failed to reject the subsequent challenge, one was in group II
(pretreatment) and the other was in group IV (pre- and
post-treatment). IL-12 enhances the antitumor effects of IL-18 on established tumor in vivo
To further evaluate the antitumor effects of IL-18 in vivo, 1
x 105 of MCA205 cells were inoculated into the right
flank of mice on day 0, and treatment was begun on day 7 with daily
i.p. injections of either HBSS, IL-18 or IL-12 alone, or IL-18 combined
with IL-12 for 7 days (Fig. 2). Each
group consisted of six mice. IL-18 treatment (group II) (1 µg/day)
suppressed tumor growth through day 18 to 24 (groups II vs I,
p < 0.05 on days 18, 20, and 24). IL-12 treatment
(group III) (0.1 µg/day) was associated with more potent antitumor
effects (groups II vs III, p < 0.05 on day 16 to 29;
groups III vs I, p < 0.05 on day 12, and
p < 0.01 on day 14 to 29). Although combination
treatment with IL-18 and IL-12 (group IV) was associated with the most
marked suppression of tumor growth (groups IV vs I, p
< 0.05 on day 8, and p < 0.01 on days 12 and 14), all
mice in this group died before completion of treatment.
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).
Groups of six mice received i.d. injections with 5 x
104 of MCA205 cells in the right flanks on day 0 and were
treated with daily administration on day 7 to 13 with HBSS, IL-18, or
IL-12 i.p. Treatment with IL-18 alone (groups III and IV) or IL-18
combined with IL-12 (groups V and VI) suppressed the growth of the
tumor during treatment at both doses (0.2 and 1 µg/day) of IL-18
(groups III vs I and groups IV vs I, p < 0.05 on day
13, and p < 0.01 on day 18 to 30; groups V vs I and
groups VI vs I, p < 0.01 on day 9 to 30). IL-12 (group
II) also mediated antitumor effects at the lower dose (groups II vs I,
p < 0.05 on day 9, and p < 0.01 on
day 13 to 30). Tumor growth suppression was also observed in mice
treated with IL-18 combined with IL-12 (groups V and VI), and somewhat
better than what was observed with IL-18 alone (groups III and IV)
(groups VI vs III and groups VI vs IV, p < 0.05 on day
11; groups V vs III, p < 0.05 on days 21 and 24).
Complete abrogation of the tumor was observed in one animal treated
with the combination of 1 µg of IL-18 and 0.01 µg of IL-12 (group
VI; 1/6, 17%). However, three of the mice in this treatment group
became ill with this treatment, and it was discontinued after the
fourth injection. The animals in other groups completed the treatment
cycle without apparent deleterious effects.
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level, but
not IL-10
IL-18 and IL-12 are reported to induce the production of IFN-
in vitro (17), and administration of IL-12 induces both IFN- and
IL-10 production in vivo. To determine whether IL-18 induces
productions of IFN- and IL-10 in vivo, we analyzed the serum levels
of IFN- and IL-10 of the mice treated with IL-18 by ELISA (Table I). While five daily injections of 1 µg
of IL-18 induced measurable serum IFN- levels to 57.1 ± 64.9
pg/ml, the treatment with 0.2 µg of IL-18 did not (data not shown).
Treatment with 1 µg of IL-18 combined with 0.1 µg of IL-12 raised
serum IFN- level about 300 times higher than that observed with
IL-18 alone (17,671.5 ± 107.1 pg/ml). Serum IFN- levels in the
animals receiving 1 µg of IL-18 combined with 0.01 µg of IL-12 were
only nine times higher than IL-18 alone (data not shown). Five daily
injections of 1 µg of IL-18 did not cause significant elevation of
IL-10 above the detection level of the ELISA.
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Treatment with IL-18 inhibited the establishment of CL8-1 cells
with administration before tumor inoculation (Fig. 1). This result
suggests the involvement of NK cells in the rejection of this tumor. To
investigate the role of NK cells in the antitumor effects of IL-18, NK
cells were depleted using anti-ASGM1 administered before and during
treatment with IL-18 (Fig. 4). Mice
received 1 µg of IL-18 daily for 7 days after the inoculation of
1 x 106 of CL8-1 cells as a treatment (group
III, IL-18, and group IV, IL-18 + anti-ASGM1). The treatment
with IL-18 significantly suppressed the growth of tumor (groups III vs
I, p < 0.05 on day 15 to 29, and p <
0.01 on days 22 and 33) (Fig. 4A) and eradicated the
tumor in all mice without NK depletion (Fig. 4B).
However, depletion of NK cells with anti-ASGM1 administration
completely abrogated the growth-inhibitory effects of IL-18 (groups III
vs IV, p < 0.01 on day 12 to 33) (Fig. 4, A
and B).
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gene or in
IL-12 gene-disrupted mice
Studies using an Ab for IFN- or alternatively, using IFN-
gene ko mice, demonstrated that the antitumor effects of IL-12 require
IFN- elaboration (8, 9, 12), presumed essential for the activation
of macrophages and for enhancing the cytolytic activity of NK and T
cells (25). Since IL-18 induces IFN- from NK cells and T cells and
augments NK cell activities in a synergistic fashion with IL-12, the
IL-12 itself and/or induced IFN- expression might play an important
role in the antitumor effects of IL-18 on tumor in vivo. To test this
hypothesis, we examined the antitumor effects of IL-18 in IL-12 gene or
IFN- gene ko mice. Groups of five wild-type, IL-12, or IFN- gene
ko mice received i.p. treatment with 1 µg of IL-18 daily for 7 days
immediately after inoculation of 1 x 106 of
CL8-1 cells (Fig. 5). The tumors
inoculated in the IL-12 gene ko (Fig. 5, B and E,
HBSS) or the IFN- gene ko mice (Fig. 5, C and
F, HBSS) with HBSS treatment (control) grew more rapidly
when compared with tumors inoculated in the wild-type, immunocompetent
mice (Fig. 5, A and D, HBSS) (average tumor area
of Fig. 5A, HBSS vs that of Fig. 5B, HBSS;
p < 0.05 on day 12 to 31, average tumor area of Fig. 5A, HBSS vs that of Fig. 5C, HBSS; p
<0.01 on day 12 to 31). Treatment with IL-18 in the IL-12 gene ko mice
(Fig. 5, B and E, IL-18) effectively suppressed
tumor growth at the same order of magnitude as that in the wild-type
mice (Fig. 5, A and D, IL-18) (average tumor area
of Fig. 5B, HBSS vs that of Fig. 5B, IL-18;
p < 0.05 on day 9, and p < 0.01 on
day 12 to 31, average tumor area of Fig. 5A, IL-18 vs that
of Fig. 5B, IL-18; p = 0.114–0.837 on day 0
to 31). In the IFN- gene ko mice, the growth-suppressive effects of
IL-18 were impaired at earlier time points, but became significant
later (Fig. 5, C and F) (average tumor
area of Fig. 5C, HBSS vs that of Fig. 5C, IL-18;
p < 0.05 on day 17 to 24).
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To examine the effects of IL-18 on the characteristics of
tumor-infiltrating lymphocytes, samples of IL-18-treated CL8-1 tumor
cells were harvested from the animals and immunostained with
anti-CD4, anti-CD8a, and anti-CD11b (anti-Mac-1) Abs
(Fig. 6). Untreated tumor showed a
moderate infiltration of CD4+ (Fig. 6A) and CD8+ (Fig. 6D)
cells, and a dense penetration of Mac-1+ cells (data not
shown) into the tumor. Neither pretreatment nor post-treatment with
IL-18 affected the population of CD4+ cells (Fig. 6, B and C) or Mac-1+ cells (data
not shown). IL-18 treatment was associated with a reduced number of
CD8+ cells within tumors (Fig. 6, E and
F), and this reduction was more apparent in the
samples from the animals received post-treatment with IL-18 (Fig. 6F).
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and IP-10 in tumor were not altered
following administration of IL-18
The antitumor effects of IL-12 are attributed in part to its
ability to inhibit angiogenesis that is mediated in part by induction
of IP-10, which in turn is induced by IFN- (13, 15). Thus, we
examined the mRNA expression of IFN- and IP-10 on CL8-1 tumor in
mice treated with IL-18. CL8-1 tumors were inoculated, treated with 1
µg of IL-18 for 6 days, and harvested to analyze the expression of
IFN- and IP-10 using reverse-transcriptase PCR. IFN- and IP-10
were expressed constitutively and were not changed significantly
following treatment with IL-18 (data not shown).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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secretion from NK and T cells, and was initially termed
IFN--inducing factor (17). Although IL-18 has similarities with IL-1
in its structure and requirement for processing to an active molecule
by an IL-1ß-converting enzyme (18), IL-18 possesses its own receptor
(19) and distinctive functions. IL-18 augments NK cytolytic activity
and enhances proliferation of T cells (17). Administration of mouse
rIL-18 protein has significant antitumor activities in murine tumor
establishment and therapy models. The dose of IL-18 administered
correlated well with the induced level of serum IFN-, as well as the
magnitude of its inhibitory effects on tumor growth. The antitumor
efficacy of IL-18 was enhanced by IL-12 administration. The antitumor
effects of IL-18 were retained in IFN- or IL-12 gene-desrupted mice,
but were abrogated when NK cells were depleted following anti-ASGM1
antiserum administration. Interestingly, reduced accumulation of
CD8+ T cell was observed in regressing tumors treated
with IL-18. The CL8-1 immunogenic tumor (22) was used to evaluate the antitumor effects of IL-18. Both pre- and post-treatment with IL-18 promoted rejection of this tumor. Since IL-18 enhances the cytotoxic activity of NK cells in vitro (17), we hypothesized that augmented NK cell activity enhanced in animals by pretreatment with IL-18 was central to the antitumor effects observed. Treatment with IL-18 alone was also effective in suppressing the progression of the MCA205 tumor without apparent deleterious effects at both doses used. The ability to suppress tumor growth utilizing 1 µg of IL-18 was somewhat less than that observed with 0.1 µg of IL-12. Higher doses of IL-18 may be necessary to exert more potent antitumor effects. When the tumor is eradicated following IL-18 administration, these mice maintain a state of long lasting immunity. Thus, IL-18 possesses significant antitumor effects and induces a state of tumor-specific immunity that is mediated by activation of NK cells. NK-mediated tumor cell destruction, in turn, may play a significant role in the subsequent T cell response.
Additive antitumor effects of combination treatment with IL-18 and
IL-12 were noted. These observations are consistent with in vitro
studies that demonstrate that IL-18 cooperates with IL-12 to enhance
production of Th1-type cytokines (17) and proliferation of Th1 cells
(21). The level of serum IFN- detected in vivo was higher when
animals were treated with a combination IL-18 and IL-12, and this
appears to correlate with the antitumor effects. Moreover, NK depletion
studies support the notion that they are important cells mediating in
part the antitumor of IL-18. Nevertheless, some of the mice treated
with combined therapy died of diarrhea and weight loss. Mice treated
with both IL-12 and IL-18 developed severe hemorrhagic colitis with
diffuse mucosal erosions as well as atrophic thymi. We did not observe
diarrhea as a side effect of treatment with either IL-18 or IL-12
applied single. Levels of serum IFN- as high as 5,000 pg/ml with
rIL-12 (9) or 15,000 pg/ml with injection of the IL-12 adenovirus
vector (unpublished data) were observed in the past, but did not cause
these gastrointestinal effects. Moreover, these side effects observed
with treatment with 1 µg IL-18 and 0.01 µg IL-12 were not seen with
treatment with 0.2 µg IL-18 and 0.01 µg IL-12. Both treatments
elevated the level of the serum IFN- to the same level (500 pg/ml)
(data not shown). We believe that it is unlikely that elevated serum
IFN- is the sole cause of the side effect of combination treatment
with IL-18 and IL-12. To optimize the antitumor effects of the
combination of IL-18 with other cytokines, including IL-12, and to
minimize the side effects of combination therapy, we plan to
investigate modifications in the administration schedule and to more
carefully define the mechanisms of toxicity.
IL-12 enhances the NK cytotoxic activity (26) and regulates
differentiation of naive T cells into Th1 cells (27). IL-18 synergizes
with IL-12 to enhance IFN- production, which also correlates with
the extent of IL-18 antitumor effects. Furthermore, endogeneous
expression of IL-12 induced by tumor challenge appears to be a key
event in rejection of such a tumor in some murine tumors (Quan Cai,
manuscript in preparation). Thus, it was of interest to determine
whether intrinsic IL-12 production is necessary to mediate the
antitumor effects of IL-18. In IL-12 gene ko mice, CL8-1 tumor grow
more rapidly than in wild-type mice. Treatment with IL-18, however,
still retained significant antitumor effects in these mice. These data
suggest that endogeneous IL-12 production is not an absolute
requirement for the IL-18 antitumor effects.
To examine whether IFN- is the main mediator of IL-18 antitumor
effects or not, IL-18 treatment was performed in IFN- gene ko mice.
The antitumor effects of IL-18, represented by delayed emergence and
growth retardation, were abrogated only marginally in IFN- gene ko
mice (Fig. 5). Thus, it appears that IL-18 administration mediates
antitumor immune reaction independent of IFN- pathways, unlike
IL-12.
IP-10 (28), a potent inhibitor of angiogenesis (29, 30), is known to mediate IL-12-induced anti-angiogenesis (13, 15). However, expressions of IP-10 were not different at the tumor site following treatment with IL-18 when compared with tumors in untreated animals, suggesting that IP-10 is not directly involved in antitumor effects of IL-18.
Immunostaining of CL8-1 tumor samples revealed dense infiltration of Mac-1+ cells, which include macrophages, granulocytes, and NK cells. A significant difference in the number of Mac-1+ cells was not detected in IL-18-treated tumors. Treatment with IL-18 reduced the number of CD8+ cells found within the tumor, particularly in tumors from animals given IL-18 after establishment, but did not alter the numbers or distribution of CD4+ or Mac-1+ cells. Systemic treatment with IL-12 induces IL-10 production (31). IL-10 transduction of CL8-1 enhanced the number of CD8+ T cells found within the tumor (22). Administration of IL-18 did not induce elevation of serum IL-10 levels in mice, which may explain the apparent reduction. Local production of IL-10 is possibly associated with accumulation of CD8+ T cells within tumor. Micallef et al. prepared spleen cells from mice previously bearing the Meth A sarcoma following treatment with IL-18, and showed that CD4+ T cells, but not CD8+ T cells, were responsible for the cytotoxic activity in vitro (32). We speculate that treatment with IL-18 induced CD4+ T cell immunity to tumor cells. These characteristics of tumor-infiltrating lymphocytes in IL-18-treated animals are different from those observed in animals treated with IL-12. CD8+ T cells (8) or both CD4+ and CD8+ T cells (9) are the critical cells mediating the antitumor efficacy of IL-12. Dao et al. reported that treatment with IL-18 and IL-12 induced Fas ligand in activated murine Th1 cells in vitro (33), suggesting the possible role of Fas ligand expression on CD4+ cells in tumor immunity induced by IL-18 administration.
In conclusion, we demonstrated that the administration of mouse rIL-18
has significant antitumor effects against the growth of murine tumor
cell lines in vivo and also can induce specific immunity to the tumor.
Synergistic antitumor effects of IL-18 with IL-12 in vivo were also
observed with markedly elevated IFN- production. The antitumor
effects of IL-18 are mediated by NK cells and CD4+ cells,
but in IFN-- and IL-12-independent pathways. These results indicate
a possibility for the application of IL-18 in cancer therapy and
warrant further investigation of the role of IL-18 in immunity.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Hideaki Tahara, Department of Surgery, University of Pittsburgh Cancer Institute, 300 Kaufmann Building, 3471 Fifth Avenue, Pittsburgh, PA 15213.
3 Abbreviations used in this paper: IP-10, IFN--inducible 10-kDa protein; ASGM1, asialo GM1; i.d., intradermal; ko, knockout.
Received for publication August 22, 1997. Accepted for publication October 30, 1997.
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 M. J. Robertson, J. M. Kirkwood, T. F. Logan, K. M. Koch, S. Kathman, L. C. Kirby, W. N. Bell, L. M. Thurmond, J. Weisenbach, and M. M. Dar A Dose-Escalation Study of Recombinant Human Interleukin-18 Using Two Different Schedules of Administration in Patients with Cancer Clin. Cancer Res., June 1, 2008; 14(11): 3462 - 3469. [Abstract] [Full Text] [PDF]
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K. Yamauchi, I.-J. Choi, H. Lu, H. Ogiwara, D. Y. Graham, and Y. Yamaoka Regulation of IL-18 in Helicobacter pylori Infection J. Immunol., January 15, 2008; 180(2): 1207 - 1216. [Abstract] [Full Text] [PDF]
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 K.-H. Lee, S. S. Park, I. Kim, J. H. Kim, E. K. Ra, S.-S. Yoon, Y.-C. Hong, S. Park, and B. K. Kim P2X7 receptor polymorphism and clinical outcomes in HLA-matched sibling allogeneic hematopoietic stem cell transplantation Haematologica, May 1, 2007; 92(5): 651 - 657. [Abstract] [Full Text] [PDF]
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J. J. Subleski, V. L. Hall, T. C. Back, J. R. Ortaldo, and R. H. Wiltrout Enhanced Antitumor Response by Divergent Modulation of Natural Killer and Natural Killer T Cells in the Liver. Cancer Res., November 15, 2006; 66(22): 11005 - 11012. [Abstract] [Full Text] [PDF]
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M. J. Robertson, J. W. Mier, T. Logan, M. Atkins, H. Koon, K. M. Koch, S. Kathman, L. N. Pandite, C. Oei, L. C. Kirby, et al. Clinical and biological effects of recombinant human interleukin-18 administered by intravenous infusion to patients with advanced cancer. Clin. Cancer Res., July 15, 2006; 12(14): 4265 - 4273. [Abstract] [Full Text] [PDF]
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J. N. MacGregor, Q. Li, A. E. Chang, T. M. Braun, D. P.M. Hughes, and K. T. McDonagh Ex vivo Culture with Interleukin (IL)-12 Improves CD8+ T-Cell Adoptive Immunotherapy for Murine Leukemia Independent of IL-18 or IFN-{gamma} but Requires Perforin. Cancer Res., May 1, 2006; 66(9): 4913 - 4921. [Abstract] [Full Text] [PDF]
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 C. A Dinarello Interleukin 1 and interleukin 18 as mediators of inflammation and the aging process Am. J. Clinical Nutrition, February 1, 2006; 83(2): 447S - 455S. [Abstract] [Full Text] [PDF]
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Y. Ino, Y. Saeki, H. Fukuhara, and T. Todo Triple Combination of Oncolytic Herpes Simplex Virus-1 Vectors Armed with Interleukin-12, Interleukin-18, or Soluble B7-1 Results in Enhanced Antitumor Efficacy Clin. Cancer Res., January 15, 2006; 12(2): 643 - 652. [Abstract] [Full Text] [PDF]
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Q. Li, A. L. Carr, E. J. Donald, J. J. Skitzki, R. Okuyama, L. M. Stoolman, and A. E. Chang Synergistic Effects of IL-12 and IL-18 in Skewing Tumor-Reactive T-Cell Responses Towards a Type 1 Pattern Cancer Res., February 1, 2005; 65(3): 1063 - 1070. [Abstract] [Full Text] [PDF]
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R. Salcedo, J. K. Stauffer, E. Lincoln, T. C. Back, J. A. Hixon, C. Hahn, K. Shafer-Weaver, A. Malyguine, R. Kastelein, and J. M. Wigginton IL-27 Mediates Complete Regression of Orthotopic Primary and Metastatic Murine Neuroblastoma Tumors: Role for CD8+ T Cells J. Immunol., December 15, 2004; 173(12): 7170 - 7182. [Abstract] [Full Text] [PDF]
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S. R. Fagerlie, T. Koretsky, B. Torok-Storb, and G. C. Bagby Impaired Type I IFN-Induced Jak/STAT Signaling in FA-C Cells and Abnormal CD4+ Th Cell Subsets in Fancc-/- Mice J. Immunol., September 15, 2004; 173(6): 3863 - 3870. [Abstract] [Full Text] [PDF]
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L. Mendoza, M. Valcarcel, T. Carrascal, E. Egilegor, C. Salado, B. K. L. Sim, and F. Vidal-Vanaclocha Inhibition of Cytokine-Induced Microvascular Arrest of Tumor Cells by Recombinant Endostatin Prevents Experimental Hepatic Melanoma Metastasis Cancer Res., January 1, 2004; 64(1): 304 - 310. [Abstract] [Full Text] [PDF]
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T. Tatsumi, J. Huang, W. E. Gooding, A. Gambotto, P. D. Robbins, N. L. Vujanovic, S. M. Alber, S. C. Watkins, H. Okada, and W. J. Storkus Intratumoral Delivery of Dendritic Cells Engineered to Secrete Both Interleukin (IL)-12 and IL-18 Effectively Treats Local and Distant Disease in Association with Broadly Reactive Tc1-type Immunity Cancer Res., October 1, 2003; 63(19): 6378 - 6386. [Abstract] [Full Text] [PDF]
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C. N. Baxevanis, A. D. Gritzapis, and M. Papamichail In Vivo Antitumor Activity of NKT Cells Activated by the Combination of IL-12 and IL-18 J. Immunol., September 15, 2003; 171(6): 2953 - 2959. [Abstract] [Full Text] [PDF]
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 M. M. Gherardi, J. C. Ramirez, and M. Esteban IL-12 and IL-18 act in synergy to clear vaccinia virus infection: involvement of innate and adaptive components of the immune system J. Gen. Virol., August 1, 2003; 84(8): 1961 - 1972. [Abstract] [Full Text] [PDF]
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 D. J. Herzyk, P. J. Bugelski, T. K. Hart, and P. J. Wier Preclinical Safety of Recombinant Human Interleukin-18 Toxicol Pathol, August 1, 2003; 31(5): 554 - 561. [Abstract] [PDF]
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 Y. Luo, H. Zhou, M. Mizutani, N. Mizutani, R. A. Reisfeld, and R. Xiang Transcription factor Fos-related antigen 1 is an effective target for a breast cancer vaccine PNAS, July 22, 2003; 100(15): 8850 - 8855. [Abstract] [Full Text] [PDF]
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M. Nakamori, M. Iwahashi, M. Nakamura, K. Ueda, X. Zhang, and H. Yamaue Intensification of Antitumor Effect by T Helper 1-dominant Adoptive Immunogene Therapy for Advanced Orthotopic Colon Cancer Clin. Cancer Res., June 1, 2003; 9(6): 2357 - 2365. [Abstract] [Full Text] [PDF]
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 Y. Nishioka, H. Wen, K. Mitani, P. D. Robbins, M. T. Lotze, S. Sone, and H. Tahara Differential effects of IL-12 on the generation of alloreactive CTL mediated by murine and human dendritic cells: a critical role for nitric oxide J. Leukoc. Biol., May 1, 2003; 73(5): 621 - 629. [Abstract] [Full Text] [PDF]
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N. Suzuki, N.-J. Chen, D. G. Millar, S. Suzuki, T. Horacek, H. Hara, D. Bouchard, K. Nakanishi, J. M. Penninger, P. S. Ohashi, et al. IL-1 Receptor-Associated Kinase 4 Is Essential for IL-18-Mediated NK and Th1 Cell Responses J. Immunol., April 15, 2003; 170(8): 4031 - 4035. [Abstract] [Full Text] [PDF]
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 P. Reddy, T. Teshima, G. Hildebrandt, U. Duffner, Y. Maeda, K. R. Cooke, and J. L. M. Ferrara Interleukin 18 preserves a perforin-dependent graft-versus-leukemia effect after allogeneic bone marrow transplantation Blood, October 16, 2002; 100(9): 3429 - 3431. [Abstract] [Full Text] [PDF]
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T. Tatsumi, A. Gambotto, P. D. Robbins, and W. J. Storkus Interleukin 18 Gene Transfer Expands the Repertoire of Antitumor Th1-type Immunity Elicited by Dendritic Cell-based Vaccines in Association with Enhanced Therapeutic Efficacy Cancer Res., October 15, 2002; 62(20): 5853 - 5858. [Abstract] [Full Text] [PDF]
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J. M. Wigginton, J.-K. Lee, T. A. Wiltrout, W. G. Alvord, J. A. Hixon, J. Subleski, T. C. Back, and R. H. Wiltrout Synergistic Engagement of an Ineffective Endogenous Anti-Tumor Immune Response and Induction of IFN-{gamma} and Fas-Ligand-Dependent Tumor Eradication by Combined Administration of IL-18 and IL-2 J. Immunol., October 15, 2002; 169(8): 4467 - 4474. [Abstract] [Full Text] [PDF]
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F. N. Lauw, J. Branger, S. Florquin, P. Speelman, S. J. H. van Deventer, S. Akira, and T. van der Poll IL-18 Improves the Early Antimicrobial Host Response to Pneumococcal Pneumonia J. Immunol., January 1, 2002; 168(1): 372 - 378. [Abstract] [Full Text] [PDF]
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J. Paulukat, M. Bosmann, M. Nold, S. Garkisch, H. Kampfer, S. Frank, J. Raedle, S. Zeuzem, J. Pfeilschifter, and H. Muhl Expression and Release of IL-18 Binding Protein in Response to IFN-{gamma} J. Immunol., December 15, 2001; 167(12): 7038 - 7043. [Abstract] [Full Text] [PDF]
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 R. B. Smeltz, J. Chen, J. Hu-Li, and E. M. Shevach Regulation of Interleukin (IL)-18 Receptor {alpha} Chain Expression on CD4+ T Cells during T Helper (Th)1/Th2 Differentiation: Critical Downregulatory Role of IL-4 J. Exp. Med., July 16, 2001; 194(2): 143 - 154. [Abstract] [Full Text] [PDF]
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D. W. Ju, Q. Tao, G. Lou, M. Bai, L. He, Y. Yang, and X. Cao Interleukin 18 Transfection Enhances Antitumor Immunity Induced by Dendritic Cell-Tumor Cell Conjugates Cancer Res., May 1, 2001; 61(9): 3735 - 3740. [Abstract] [Full Text]
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Y.-I. Son, R. M. Dallal, R. B. Mailliard, S. Egawa, Z. L. Jonak, and M. T. Lotze Interleukin-18 (IL-18) Synergizes with IL-2 to Enhance Cytotoxicity, Interferon-{{gamma}} Production, and Expansion of Natural Killer Cells Cancer Res., February 1, 2001; 61(3): 884 - 888. [Abstract] [Full Text]
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M. C. Leite-de-Moraes, A. Hameg, M. Pacilio, Y. Koezuka, M. Taniguchi, L. Van Kaer, E. Schneider, M. Dy, and A. Herbelin IL-18 Enhances IL-4 Production by Ligand-Activated NKT Lymphocytes: A Pro-Th2 Effect of IL-18 Exerted Through NKT Cells J. Immunol., January 15, 2001; 166(2): 945 - 951. [Abstract] [Full Text] [PDF]
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 G. Cai, R. Kastelein, and C. A. Hunter Interleukin-18 (IL-18) Enhances Innate IL-12-Mediated Resistance to Toxoplasma gondii Infect. Immun., December 1, 2000; 68(12): 6932 - 6938. [Abstract] [Full Text] [PDF]
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C. Cahlin, A. Körner, H. Axelsson, W. Wang, K. Lundholm, and E. Svanberg Experimental Cancer Cachexia: The Role of Host-derived Cytokines Interleukin (IL)-6, IL-12, Interferon-{{gamma}}, and Tumor Necrosis Factor {{alpha}} Evaluated in Gene Knockout, Tumor-bearing Mice on C57 Bl Background and Eicosanoid-dependent Cachexia Cancer Res., October 1, 2000; 60(19): 5488 - 5493. [Abstract] [Full Text]
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F. Tanaka, W. Hashimoto, H. Okamura, P. D. Robbins, M. T. Lotze, and H. Tahara Rapid Generation of Potent and Tumor-specific Cytotoxic T Lymphocytes by Interleukin 18 Using Dendritic Cells and Natural Killer Cells Cancer Res., September 1, 2000; 60(17): 4838 - 4844. [Abstract] [Full Text]
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W. E. Carson, J. E. Dierksheide, S. Jabbour, M. Anghelina, P. Bouchard, G. Ku, H. Yu, H. Baumann, M. H. Shah, M. A. Cooper, et al. Coadministration of interleukin-18 and interleukin-12 induces a fatal inflammatory response in mice: critical role of natural killer cell interferon-gamma production and STAT-mediated signal transduction Blood, August 15, 2000; 96(4): 1465 - 1473. [Abstract] [Full Text] [PDF]
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D. Cho, H. Song, Y. M. Kim, D. Houh, D. Y. Hur, H. Park, D. Yoon, K. H. Pyun, W. J. Lee, M. Kurimoto, et al. Endogenous Interleukin-18 Modulates Immune Escape of Murine Melanoma Cells by Regulating the Expression of Fas Ligand and Reactive Oxygen Intermediates Cancer Res., May 1, 2000; 60(10): 2703 - 2709. [Abstract] [Full Text]
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S. Nakamura, T. Otani, Y. Ijiri, R. Motoda, M. Kurimoto, and K. Orita IFN-{gamma}-Dependent and -Independent Mechanisms in Adverse Effects Caused by Concomitant Administration of IL-18 and IL-12 J. Immunol., March 15, 2000; 164(6): 3330 - 3336. [Abstract] [Full Text] [PDF]
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J. Hein, V. A. J. Kempf, J. Diebold, N. Bucheler, S. Preger, I. Horak, A. Sing, U. Kramer, and I. B. Autenrieth Interferon Consensus Sequence Binding Protein Confers Resistance against Yersinia enterocolitica Infect. Immun., March 1, 2000; 68(3): 1408 - 1417. [Abstract] [Full Text] [PDF]
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J. S. Wild, A. Sigounas, N. Sur, M. S. Siddiqui, R. Alam, M. Kurimoto, and S. Sur IFN-{gamma}-Inducing Factor (IL-18) Increases Allergic Sensitization, Serum IgE, Th2 Cytokines, and Airway Eosinophilia in a Mouse Model of Allergic Asthma J. Immunol., March 1, 2000; 164(5): 2701 - 2710. [Abstract] [Full Text] [PDF]
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F. Vidal-Vanaclocha, G. Fantuzzi, L. Mendoza, A. M. Fuentes, M. J. Anasagasti, J. Martin, T. Carrascal, P. Walsh, L. L. Reznikov, S.-H. Kim, et al. IL-18 regulates IL-1beta -dependent hepatic melanoma metastasis via vascular cell adhesion molecule-1 PNAS, January 18, 2000; 97(2): 734 - 739. [Abstract] [Full Text] [PDF]
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D. L. Martin, C. L. King, E. Pearlman, E. Strine, and F. P. Heinzel IFN-{gamma} Is Necessary But Not Sufficient for Anti-CD40 Antibody-Mediated Inhibition of the Th2 Response to Schistosoma mansoni Eggs J. Immunol., January 15, 2000; 164(2): 779 - 785. [Abstract] [Full Text] [PDF]
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K. Oshikawa, F. Shi, A. L. Rakhmilevich, P. M. Sondel, D. M. Mahvi, and N.-S. Yang Synergistic inhibition of tumor growth in a murine mammary adenocarcinoma model by combinational gene therapy using IL-12, pro-IL-18, and IL-1beta converting enzyme cDNA PNAS, November 9, 1999; 96(23): 13351 - 13356. [Abstract] [Full Text] [PDF]
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D. Ito, T. C. Back, A. N. Shakhov, R. H. Wiltrout, and S. A. Nedospasov Mice with a Targeted Mutation in Lymphotoxin-{alpha} Exhibit Enhanced Tumor Growth and Metastasis: Impaired NK Cell Development and Recruitment J. Immunol., September 1, 1999; 163(5): 2809 - 2815. [Abstract] [Full Text] [PDF]
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W. Hashimoto, T. Osaki, H. Okamura, P. D. Robbins, M. Kurimoto, S. Nagata, M. T. Lotze, and H. Tahara Differential Antitumor Effects of Administration of Recombinant IL-18 or Recombinant IL-12 Are Mediated Primarily by Fas-Fas Ligand- and Perforin-Induced Tumor Apoptosis, Respectively J. Immunol., July 15, 1999; 163(2): 583 - 589. [Abstract] [Full Text] [PDF]
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H. F. Staats and F. A. Ennis Jr. IL-1 Is an Effective Adjuvant for Mucosal and Systemic Immune Responses When Coadministered with Protein Immunogens J. Immunol., May 15, 1999; 162(10): 6141 - 6147. [Abstract] [Full Text] [PDF]
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H. Kojima, Y. Aizawa, Y. Yanai, K. Nagaoka, M. Takeuchi, T. Ohta, H. Ikegami, M. Ikeda, and M. Kurimoto An Essential Role for NF-{kappa}B in IL-18-Induced IFN-{gamma} Expression in KG-1 Cells J. Immunol., May 1, 1999; 162(9): 5063 - 5069. [Abstract] [Full Text] [PDF]
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