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 Previous Article | Table of Contents | Next Article 
Blood, Vol. 91 No. 6 (March 15), 1998:
pp. 2118-2125
Interleukin-18 Regulation of Interferon  Production and Cell
Proliferation as Shown in Interleukin-1 -Converting Enzyme
(Caspase-1)-Deficient Mice
By
Giamila Fantuzzi,
Adrian J. Puren,
Matthew W. Harding,
David J. Livingston, and
Charles A. Dinarello
From the Division of Infectious Diseases, University of Colorado
Health Sciences Center, Denver, CO; and the Vertex Pharmaceuticals,
Inc, Cambridge, MA.
 |
ABSTRACT |
Interleukin-18 (IL-18) is a costimulatory factor for interferon
(IFN) production. Processing of pro-IL-18 by IL-1 -converting enzyme (ICE) leads to the release of bioactive IL-18. Compared with
wild-type (WT) mice, splenocytes from ICE-deficient mice produced low
IFN after lipopolysaccharide (LPS) or zymosan (50% and 80%
reduction). In contrast, IFN production was unimpaired in
ICE-deficient mice using Concanavalin A (Con A). Comparable results
were obtained when endogenous IL-18 was blocked with a neutralizing
antibody. LPS-induced IFN was also reduced by an ICE inhibitor.
Exogenous IL-18 augmented zymosan-induced IFN production in WT mice.
In ICE-deficient cells, IFN production was only partially restored
by IL-18. The reduced levels of IFN in ICE-deficient mice were not
due to a lack of IL-12, because zymosan induced IL-12 equally in WT and
in ICE-deficient mice. IFN is an important regulator of cell
proliferation. In accordance, splenocytes from ICE-deficient mice
proliferated more when stimulated with LPS, but not with Con A. Furthermore, in ovalbumin-sensitized ICE-deficient mice, proliferation
of lymph node cells in response to the specific antigen was not
altered. Exogenous IFN inhibited, whereas blockade of endogenous
IFN or IL-18 increased, LPS induced splenocyte proliferation both in
WT and in ICE-deficient mice. Our results show that IL-18 is an
IL-12-independent regulator of IFN production and of cell
proliferation induced by microbial stimuli. However, ICE-dependent
processing of IL-18 is not needed for response to mitogens or antigens.
| |
INTRODUCTION |
INTERFERON (IFN)-inducing factor
(IGIF or interleukin-18 [IL-18]) is a recently characterized cytokine
that acts as a costimulatory factor for the production of IFN. IL-18
was initially purified and subsequently cloned from the liver of mice
conditioned with Propionibacterium acnes and challenged with
lipopolysaccharide (LPS);1,2 cloning of the human molecule
has also been recently described.3 A critical role for
IL-18 in LPS-induced toxicity has been shown. Anti-IL-18 antibodies
protect P acnes-conditioned mice from liver injury after
LPS.2 In addition, a role for IL-18 in the pathogenesis of
insulin-dependent diabetes mellitus has recently been
proposed.4
Similar to another IFN-inducing factor, IL-12, IL-18 is produced by
monocytes/macrophages, but not by B or T cells.3 However, IL-18 induction of IFN is independent of IL-12 production. In fact,
anti-IL-12 antibodies do not inhibit the increase in
anti-CD3-stimulated IFN production induced by IL-18.2
In addition to acting as a costimulus for IFN production, IL-18
enhances the production of granulocyte-macrophage colony-stimulating
factor (GM-CSF) and IL-2,5 potentiates anti-CD3-induced
T-cell proliferation,5 and increases Fas-mediated killing
of natural killer (NK) cells by augmenting the expression of Fas
ligand.6 However, unlike IL-12, IL-18 by itself, in the
absence of another stimulus, is not a strong inducer of
IFN.3
IL-18 is structurally related to IL-1, with both cytokines having a
unique, all--pleated structure.7 Also, similar to IL-1, IL-18 is synthesized as a precursor lacking a typical signal peptide.3 Pro-IL-18, as pro-IL-1, is devoid of
biological activity and precursor amino acids must be cleaved to
produce an active molecule.3 IL-1-converting enzyme
(ICE; caspase-1), which cleaves pro-IL-1 , also cleaves pro-IL-18
at aspartic acid in the P1 position, producing a mature, bioactive
peptide that is readily released from the cell.8,9 When
ICE-deficient mice are injected with LPS, with or without a
preconditioning with P acnes, only low levels of IFN are
detectable in the circulation compared with wild-type (WT)
mice.8,9 The injection of IL-18 restores the LPS-induced
IFN levels in ICE-deficient mice,8 supporting the
concept that ICE is actually involved in the production of active
IL-18.
To date, the role of endogenous IL-18 in the induction of IFN after
stimuli other than LPS remains unknown. To help elucidate the role of
IL-18 in the regulation of IFN production in response to various
stimuli, we studied the in vitro production of IFN in splenocytes
from ICE-deficient mice using two inflammatory stimuli, LPS and
zymosan, and compared it with the response to an immune stimulus, the
mitogen Concanavalin A (Con A). In addition, because IFN is an
important factor regulating cell proliferation,10,11 we
investigated the response of splenocytes obtained from WT and ICE-deficient mice to mitogenic concentrations of LPS and Con A, as
well as to specific antigen.
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MATERIALS AND METHODS |
Materials.
LPS (a phenol-extracted preparation from Escherichia coli
055:B5), zymosan, Con A, ovalbumin, and phenazine methosulfate (PMS) were purchased from Sigma Chemical Co (St Louis, MO); RPMI was from
Cellgro (Waukesha, WI); fetal bovine serum (FBS) was from GIBCO (Pascagoula, MS); MTS
[3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulphophenyl)-2H-tetrazolium, inner salt] was from Promega (Madison, WI); the reversible ICE inhibitor Ac-Tyr-Val-Ala-Asp-CHO was from Alexis (San Diego, CA); rat
recombinant IFN was from Genentech (South San Francisco, CA); human
recombinant IL-1Ra was a kind gift from Dr Daniel Tracey (Upjohn,
Kalamazoo, MI); murine recombinant IL-18 was a kind gift from Dr Y. Stabinsky (Peprotech, Inc, Princeton, NJ). The anti-IL-18 antiserum
was obtained from a New Zealand rabbit immunized by intradermal
injection of murine recombinant IL-18 (Peprotech) in the presence of
Hunter's Titermax adjuvant. After several booster injections, blood
was collected and serum obtained. The anti-IFN monoclonal antibody
(XMG1.2) was from Endogen Inc (Woburn, MA).
Animals.
The generation and genetic background of ICE- and of IL-1-deficient
mice has been previously described.12,13 Six- to
eight-week-old, sex-matched mice were used. The WT mice used were of
the same genetic background, sex, and age as that of the knock out
mice, although they were not littermates.
Isolation and culture of spleen and lymph node cells.
Spleens were aseptically removed and cell suspensions were prepared
according to standard procedures.14 Cells were washed twice
and resuspended in RPMI supplemented with 10% FBS. For cytokine measurement, spleen cells were cultured at 5 × 106/mL
in 24-well, flat-bottom culture plates in the presence or absence of
various concentrations of LPS, zymosan, or Con A. When zymosan was the
stimulus, 1% human serum was added to the culture to opsonize zymosan
particles. Cultures were incubated at 37°C in a humidified
atmosphere with 5% CO2. At the end of the incubation period, cultures were frozen at  70°C and subjected to 3 freeze-thaw cycles to obtain total cytokine levels. Before assaying,
samples were centrifuged for 10 minutes at 10,000g to remove
debris.
For proliferation assays, spleen cells were cultured in triplicate
wells at 2.5 × 106/mL in 96-well, flat-bottom
microtiter plates with increasing concentrations of either LPS (4, 20, and 100 µg/mL) or Con A (1, 3, and 10 µg/mL). Proliferation was
measured using the MTS/PMS method, as previously
described.15
For the evaluation of the proliferative response after ovalbumin, mice
were injected at the base of the tail with a suspension of 50 mg of OVA
in 100 µL complete Freund adjuvant (CFA). Fourteen days later, the
draining abdominal periaortic lymph nodes were removed, cell
suspensions were prepared, and cells were cultured at 2.5 × 106/mL in 96-well, flat-bottom microtiter plates with
increasing concentration of ovalbumin (50, 170, and 500 µg/mL).
Reverse transcription-polymerase chain reaction (RT-PCR).
Total RNA was extracted from spleen cells as previously
described.16 One microgram of RNA was reverse transcribed
using random hexamer primers (Perkin Elmer, Norwalk, CT) in a
thermocycler (42°C for 30 minutes and 99°C for 5 minutes) and
the cDNA amplification was performed as previously decribed for 30 cycles for cytokine expression and 24 cycles for GAPDH.16
Oligonucleotide primers were as follows: p40 forward,
5 -CGTGCTATGGCTGGTGCAAAG-3; p40 reverse,
5-GAACACATGCCCACTTGCTG-3; p35 forward,
5-ACCAGCACATTGAAGACCTG-3; p35 reverse,
5-GACTGCATCAGCTCATCGAT-3; GAPDH forward,
5-TGAAGGTCGGAGTCAACGGATTTGGT-3; and GAPDH
reverse, 5-CATGTGGGCCATGAGGTCCACCAC-3. The annealing temperature was 60°C. Products of PCR amplifications were separated in a 2.0% agarose gel (Sigma) containing ethidium bromide (0.5 µg/mL) and 0.5× Tris-Borate-EDTA buffer (Fisher Scientific,
Pittsburgh, PA). PCR amplification products were
illuminated with UV light and a negative image photograph taken
(Polaroid type 55 film; Polaroid, Cambridge, MA). The photographs were
scanned on a densitometer using ImageQuant software (Molecular
Dynamics, Sunnyvale, CA). Data are presented as the ratio of cytokine
densitometric units to the GAPDH units for each condition.
Cytokine measurement.
IFN was measured with an enzyme-linked immunosorbent assay (ELISA)
kit, kindly provided by Endogen, Inc. Total and p70 IL-12 were measured
with ELISA kits kindly provided by Genzyme Corp (Cambridge, MA).
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RESULTS |
Inhibition of ICE activity reduces LPS-induced IFN
production.
Splenocytes from WT mice were cultured for 24 hours with LPS (1µg/mL)
in the presence or absence of an ICE inhibitor (20 µmol/L) or of
IL-1Ra (10 µg/mL). As shown in Fig 1, the
ICE inhibitor reduced IFN production by 70% (P < .01),
whereas IL-1Ra had no significant effect.
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| Fig 1.
An ICE inhibitor, but not IL-1Ra, reduces LPS-induced
IFN production. Splenocytes from WT mice were incubated with LPS
alone (1 µg/mL) or with LPS the presence of either IL-1Ra (10 µg/mL) or of an ICE inhibitor (20 µmol/L). IFN levels were
measured 24 hours later. Data are the mean ± SEM of 6 mice per group.
**P < .01 versus LPS alone by ANOVA for repeated measures.
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Reduced production of IFN in ICE-deficient mice.
Splenocytes obtained from WT or ICE-deficient mice were cultured in
vitro for 24 hours in the presence of various concentrations of LPS,
zymosan, or Con A. Significantly lower levels of IFN were produced
in cultures from ICE-deficient splenocytes stimulated with either LPS
(Fig 2A) or zymosan (Fig 2B). However, when
Con A was used as a stimulus, the production of IFN in ICE-deficient mice did not differ from that observed in WT mice (Fig 2C). These differences between WT and ICE-deficient mice were observed at each
time point over 72 hours of culture. As shown in
Fig 3A, significantly lower levels of
IFN were found in cultures of ICE-deficient splenocytes stimulated
with LPS for 24, 48, or 72 hours. Markedly reduced levels of IFN
were also measured from ICE-deficient splenocytes stimulated with
zymosan for 12, 24, 48, or 72 hours (Fig 3B). In contrast, no
differences between WT and ICE-deficient mice were observed at any time
point when the cells were stimulated with Con A (Fig 3C).
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| Fig 2.
IFN production in splenocytes from WT and
ICE-deficient mice. Splenocytes from WT ( ) or ICE-deficient ( )
mice were incubated with the indicated concentration of LPS (A),
zymosan (B), or Con A (C) for 24 hours and levels of IFN measured.
Data are the mean ± SEM of 9 mice per group. *P < .05;
**P < .01 versus WT by factorial ANOVA.
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| Fig 3.
Time course of IFN production in splenocytes from WT
and ICE-deficient mice. Splenocytes from WT () or ICE-deficient
() mice were incubated for the indicated times with 1 µg/mL of LPS (A), 10 µg/mL of zymosan (B), or 1 µg/mL of ConA (C) and levels of
IFN were measured. Data are the mean ± SEM of 6 mice per group. *P < .05 versus WT by factorial ANOVA.
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Because the most striking differences between WT and ICE-deficient mice
for IFN production were observed after zymosan, this stimulus was
chosen for further investigation.
Effect of blockade of endogenous IL-18 on IFN production.
Splenocytes from WT mice were incubated for 24 or 72 hours with LPS (1 µg/mL), zymosan (10 µg/mL), or Con A (1 µg/mL) in the presence of
different dilutions of anti-IL-18 antiserum. As shown in
Table 1, blockade of endogenous IL-18
significantly reduced LPS- and zymosan-induced IFN at both 24 and 72 hours, but did not significantly alter IFN production when Con A was
the stimulus. At a 1:100 dilution of anti-IL-18 antiserum, 24-hour
LPS-induced IFN was inhibited by 83%, whereas zymosan-induced
IFN was inhibited by 66% compared with controls. The effect of the
anti-IL-18 antiserum was even more evident in the 72-hour culture. At
this time point, a 1:100 dilution of antiserum reduced LPS-induced
IFN production by 90% and zymosan-induced IFN levels by 75%.
Addition of normal rabbit serum did not significanlty alter IFN
production after any of the three stimuli used, thus ruling out a
possible nonspecific effect of rabbit serum on IFN levels
(data not shown). These data confirm the results obtained in
ICE-deficient mice and show that endogenous IL-18 actually plays a
critical role in the induction of IFN after LPS or zymosan.
Induction of IL-12 in ICE-deficient mice.
Because IL-12 is a potent inducer of IFN,17 we
investigated whether the reduced levels of IFN in ICE-deficient mice
are due to a deficient production of this cytokine. However, despite an
80% reduced IFN production in zymosan-stimulated ICE-deficient spleen cells, steady-state mRNA expression for both the p40 and the p35
subunits of IL-12 was induced equally in WT and in ICE-deficient mice
(Fig 4). At the protein level, no
differences were observed between WT and ICE-deficient mice for
production of the active p70 heterodimer 24 hours after stimulation
with zymosan (9.68 ± 1.39 and 10.22 ± 1.70 pg/mL in WT and
ICE-deficient mice, respectively). However, levels of
zymosan-stimulated total IL-12 (as assessed by an ELISA kit that
measures p40 monomer, p40 homodimer, and p70 heterodimer) were higher
in ICE-deficient mice compared with WT (496.72 ± 42.58 and 879.09 ± 88.46 pg/mL in WT and ICE-deficient mice, respectively; P < .01 by Student's t-test).
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| Fig 4.
Zymosan-induced IL-12 mRNA in WT and ICE-deficient mice.
Splenocytes from WT and ICE-deficient mice were incubated for 4 hours with RPMI or 10 µg/mL of zymosan. RT-PCR was performed for the p40
and p35 subunits of IL-12. Minor nonspecific amplicons were noted when
p40 was amplified. GAPDH was used as an internal control. Amplification
products for p40, p35, and GAPDH for 3 WT and 3 KO mice are shown in
(A) (C, RPMI; Z, Zymosan). (B) shows mRNA ratios (mean ± SEM, n = 3).
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Effect of IL-18 on IFN production.
To assess whether the reduced levels of IFN observed in
ICE-deficient mice could be normalized by addition of exogenous IL-18, splenocytes were stimulated with zymosan in the presence of increasing concentrations of IL-18. As shown in Fig 5,
IL-18 increased zymosan-induced IFN production both in WT and in
ICE-deficient mice. At 1 ng/mL, IL-18 added to cultures of
ICE-deficient splenocytes restored IFN production to levels no
longer significantly different from those observed in WT cells
stimulated with zymosan alone. However, this concentration of IL-18
markedly increased IFN production in WT mice. At 1 ng/mL IL-18, a
ninefold and a threefold increase in IFN production was observed in
ICE-deficient and in WT mice, respectively. The difference in IFN
production between WT and ICE-deficient mice could not be narrowed even
when higher concentrations of IL-18 were used. At 100 ng/mL of IL-18,
splenocytes from ICE-deficient mice produced 29.05 ± 10.06 ng/mL of
IFN, whereas cells from WT mice produced 59.86 ± 9.85 ng/mL of
IFN. Although the augmentation in IFN production using 100 ng/mL
of IL-18 in ICE-deficient splenocytes represents a 56-fold increase
over zymosan alone, the total IFN production was still 50% lower
than that observed in WT splenocytes.
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| Fig 5.
Effect of IL-18 on IFN production. Splenocytes from WT
( ) or ICE-deficient ( ) mice were incubated for 24 hours with 10 µg/mL of zymosan in the presence of increasing concentrations of
IL-18 and levels of IFN were measured. Data are the mean ± SEM of
5 mice per group. The unpaired Student's t-test was used for
comparison between WT and ICE-deficient mice, whereas for comparisons
within a group, ANOVA for repeated measures was used.
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These findings may reflect the lack of an additional factor involved in
IFN production in ICE-deficient mice. However, zymosan-induced IFN production was not suppressed in splenocytes obtained from IL-1-deficient mice (4.71 ± 2.51 ng/mL and 5.93 ± 1.93 ng/mL in WT and IL-1-deficient mice, respectively, with 1 µg/mL
of zymosan). Therefore, a lack of IL-1 release in splenocytes from ICE-deficient mice does not account for the reduced IFN production after zymosan stimulation.
Mitogenic responses in ICE-deficient mice.
Splenocytes from WT or ICE-deficient mice were incubated with
increasing concentrations of LPS and proliferation assessed after 72 hours. We consistently observed a greater proliferation rate in cells
obtained from ICE-deficient compared with WT mice at each of the LPS
concentrations tested (Fig 6A). The
increase in spleen cell proliferation in ICE-deficient mice ranged from 10% to 60%, depending on the experiment and the dose of LPS used. On
the other hand, when Con A was used as a mitogen, no significant difference was observed between WT and ICE-deficient mice (Fig 6B).
Comparable results were obtained when LPS- or Con A-induced proliferation was measured at either 48 or 96 hours (data not shown).
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| Fig 6.
Increased LPS-induced spleen cell proliferation in
ICE-deficient mice. Splenocytes from WT () or ICE-deficient ()
mice were incubated for 72 hours with the indicated concentrations of
LPS (A) or Con A (B) and proliferation was assessed. Lymph node cells from WT and ICE-deficient mice immunized 14 days before with ovalbumin were incubated for 72 hours with the indicated concentrations of
ovalbumin and proliferation was assessed (C). Data are the mean ± SEM
of 6 mice per group and are expressed as the percentage of change in
MTS absorbance compared with unstimulated cells (100%). ***P < .001 versus WT by factorial ANOVA.
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In addition, no significant differences between WT and ICE-deficient
mice were observed in the antigen-induced proliferation of draining
lymph node cells obtained from ovalbumin-immunized mice (Fig 6C).
Effect of IL-18 and IFN on LPS-induced spleen cell
proliferation.
To investigate the augmented LPS-induced spleen cell proliferation
observed in ICE-deficient mice, increasing concentrations of exogenous
IL-18 were added to spleen cells of WT and ICE-deficient mice in the
presence of LPS, and proliferation was assessed. IL-18 (1 to 100 ng/mL)
did not significantly alter LPS-induced spleen cell proliferation in
neither WT nor ICE-deficient mice (data not shown). When IL-18 was
added alone to spleen cells, in the absence of other stimuli, a
significant induction of cell proliferation was observed both in WT and
in ICE-deficient mice (Fig 7).
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| Fig 7.
Effect of IL-18 on spleen cell proliferation. Splenocytes
from WT () or ICE-deficient () mice were incubated with the
indicated concentrations of IL-18. Proliferation was assessed after 72 hours of incubation. Data are the mean ± SEM of 3 mice per group and are expressed as the percentage of change in MTS absorbance over unstimulated cells (100%).
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On the other hand, LPS-induced splenocyte proliferation was
significantly inhibited both in WT and in ICE-deficient mice when the
cells were cultured in the presence of increasing concentrations of
IFN. As shown in Fig 8, at 10 ng/mL of
IFN, the proliferation of ICE-deficient splenocytes was no longer
different from that of WT cells.
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| Fig 8.
Effect of IFN on LPS-induced spleen cell
proliferation. Splenocytes from WT () or ICE-deficient () mice
were incubated with 20 µg/mL of LPS in the presence of increasing
concentrations of IFN. Proliferation was assessed after 72 hours of
incubation. Data are the mean ± SEM of 3 mice per group and are
expressed as the percentage of change in MTS absorbance over
unstimulated cells (100%). *P < .05; **P < .01 versus WT by factorial ANOVA.  P < .001 versus LPS alone by ANOVA for repeated measures.
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The effect of neutralization of IL-18 or IFN on LPS-induced spleen
cell proliferation is shown in Table 2.
When the proliferation was measured 48 hours after LPS stimulation,
neutralization of either IL-18 or IFN significantly increased spleen
cell proliferation. However, when spleen cell proliferation was
measured 72 hours after LPS, only neutralization of IL-18, but not of
IFN, was effective.
Because splenocytes from ICE-deficient mice have decreased IL-1 release
after LPS stimulation, we questioned whether the lack of IL-1 in
ICE-deficient mice could account for the difference in spleen cells
proliferation. To assess a role for endogenous IL-1 activity, we
blocked IL-1 receptors with IL-1Ra (10 µg/mL). The addition of IL-1Ra
had no effect on LPS-induced splenocyte proliferation (data not shown).
Therefore, a lack of IL-1 in spleen cell cultures in ICE-deficient mice
does not account for the increased proliferation of ICE-deficient
splenocytes to LPS.
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DISCUSSION |
Because of the inability to process pro-IL-18, ICE-deficient mice
injected with LPS, with or without preconditioning with P
acnes, exhibit defective IFN production.8,9 In the
present report, we investigated whether endogenous IL-18 plays a role in the induction of IFN after various stimuli. Markedly reduced levels of IFN were present in splenocytes obtained from
ICE-deficient mice after stimulation with two inflammatory stimuli, LPS
and zymosan. In contrast, using a direct T-cell mitogen, such as Con A,
no differences in IFN production were observed between WT and
ICE-deficient mice. Two explanations are possible for this phenomenon:
(1) IL-18, similar to IL-12, is not required for IFN production
after Con A stimulation18; and/or (2) ICE is not
necessary for the cleavage of pro-IL-18 when Con A is the stimulus. We
have previously shown that ICE is not always indispensable for release
of active IL-1 and that the requirement for ICE in IL-1
processing is stimulus-dependent.19 It is therefore
possible that, similar to pro-IL-1 , ICE-independent pathways might
also exist for the cleavage of pro-IL-18. However, the observation
that inhibition of endogenous IL-18 reduces IFN production after LPS
or zymosan, but not after Con A, suggests that the first hypothesis is
probably correct.
In addition, it should be noted that T lymphocytes as well as B cells,
NK cells, and monocytes/macrophages can produce IFN when an
inflammatory stimulus such as LPS is used.20-22 When LPS is
the stimulus, the presence of monocytes/macrophages is also critical,
because these cells provide the necessary cytokines (ie, IL-12 and
IL-18) that induce IFN production from lymphocytes and NK
cells.23 By contrast, a mitogen such as Con A can react directly with T cells to induce IFN production without the need of
accessory cells, although cytokines produced by antigen-presenting cells or by bystander lymphocytes (eg, IL-2) play an important regulatory role.24
The results obtained in ICE-deficient mice were confirmed by
experiments in which endogenous IL-18 was blocked with a neutralizing antiserum. This resulted in a reduction in LPS- and zymosan-induced IFN levels, but had no significant effect on Con A-induced IFN production. These results clearly show the critical role played by
endogenous IL-18 in the production of IFN after an inflammatory stimulus and further strengthen the observation that a decreased processing of IL-18 is responsible for the reduced IFN levels observed in ICE-deficient mice. Furthermore, inhibition of ICE activity
reduced LPS-induced IFN production. However, blockade of IL-1 with
IL-1Ra had no inhibitory effect on IFN levels. These data show that
the reduced IFN levels observed in ICE-deficient mice are not due to
the lack of bioactive IL-1.
Expectedly, exogenous IL-18 increased zymosan-induced IFN production
in both WT and ICE-deficient mice. Although the relative increase in
IFN production was higher in ICE-deficient than in WT mice, the
absolute amount of IFN was consistently lower (50%) in cultures
from ICE-deficient mice, even when concentrations of exogenous IL-18 up
to 100 ng/mL were added. These findings may reflect the lack of an
additional factor involved in IFN production in ICE-deficient mice.
Because zymosan-induced IFN production was not suppressed in
splenocytes obtained from IL-1-deficient mice, it is unlikely that
the missing factor is IL-1. In addition, mRNA expression for both
subunits of IL-12, as well as protein levels for the active p70
heterodimer, was not reduced in ICE-deficient mice, thus ruling out the
possibility that defective IL-12 production might be responsible for
the reduced IFN levels in ICE-deficient mice. However, homodimers of
the p40 subunit of IL-12 can act as antagonists of the p70 heterodimer
and hence suppress IFN induction.25 Levels of total
IL-12 were increased in ICE-deficient splenocytes after stimulation
with zymosan, possibly reflecting enhanced production of the p40
homodimer. Therefore, the suppressive activity of p40 homodimers on
IFN production might be enhanced in ICE-deficient mice, thus
contributing to the low IFN levels observed in these mice.
In general, IFN inhibits proliferation of various
cells.10,11 Accordingly, IFN-deficient mice have
augmented spleen cell proliferation in response to Con A, which is
suppressed by exogenous IFN.26 Because IFN production
after Con A stimulation is normal in ICE-deficient mice, we did not
observe increased splenocyte proliferation with this stimulus. In
addition, antigen-induced proliferation of lymph node cells after
immunization with ovalbumin was not altered in ICE-deficient mice.
However, consistent with the reduced levels of IFN observed in
ICE-deficient mice after LPS stimulation, spleen cells from ICE-deficient mice underwent increased proliferation when incubated with LPS. Reconstitution with exogenous IFN inhibited LPS-induced spleen cell proliferation, and the difference between WT and
ICE-deficient mice was no longer observed. Accordingly, blockade of
endogenous IL-18 or IFN enhanced LPS-induced spleen cell
proliferation. Neutralization of IFN was effective only when
proliferation was measured at 48 hours, but not at 72 hours, after LPS
stimulation. These data suggest the role for IL-18 in regulating cell
proliferation is only partly dependent on induction of IFN. When
specifically examined, exogenous IL-18 did not influence LPS-induced
spleen cell proliferation in WT or in ICE-deficient mice. These results may be possibly confounded by the fact that IL-18, by itself, as
previously shown, can provide a proliferative signal.2,5 The lack of reduction in cell proliferation observed after coincubation with LPS and IL-18 is thus probably a result of two opposite effects of
this cytokine: induction of IFN, which inhibits cell proliferation, and a direct proliferative signal for T cells by IL-18 itself.
Inhibitors of ICE activity are effective in experimental models of
inflammatory diseases, such as pancreatitis and collagen-induced arthritis.27,28 Inhibition of ICE activity is also
effective in reducing ischemic and excitotoxic neuronal
damage29 and in delaying lethality in a model of
amyotrophic lateral sclerosis.30 In addition, ICE-deficient
mice are protected against LPS-induced toxicity.31 The
reduction in IL-1 and IL-18 release, and consequently of IFN
production, in these models should be considered to account for the
ameliorative effects of ICE inhibitors. In view of the possible use of
ICE inhibitors as therapeutic agents, it is important to identify those
disease conditions in which ICE-cleaved IL-18 plays a critical role.
From the present studies, it appears that T-cell stimuli such as
mitogens or specific antigens drive IFN production without the need
for IL-18 processing by ICE. On the other hand, microbial and
inflammatory IFN production is clearly dependent on ICE-mediated cleavage of pro-IL-18. Thus, differential IFN production reflects the stimulus and inhibiting ICE appears to spare IFN production induced by T-cell stimuli.
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FOOTNOTES |
Submitted July 25, 1997;
accepted October 30, 1997.
Supported by National Institutes of Health Grant No. AI-15614.
Address reprint requests to Charles A. Dinarello, MD, Division of
Infectious Diseases, University of Colorado Health Sciences Center,
4200 E Ninth Ave, B168, Denver, CO 80262.
The publication costs of this article were defrayed in part by page
charge payment. This article must therefore be hereby marked
"advertisement" in accordance with 18 U.S.C. section
1734 solely to indicate this fact.
| |
ACKNOWLEDGMENT |
The authors thank Drs Richard A. Flavell and K. Kuida for providing the
ICE-deficient mice and Dr H. Zheng for the IL-1-deficient mice. We
also thank J. Keutzer at Genzyme Corp for kindly providing kits for
IL-12 measurement.
| |
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Abstract
Introduction
Abstract